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Chapter 5

R&D TECHNOLOGICAL RESULTS

OVERVIEW Most of the R&D effort in the area of petroleum biorefining has been concentrated on biodesulfurization (BDS), with a few scattered reports on bioconversion (BCK) and more recently, new findings being reported on biodenitrogenation (BDN) and biodemetallization (BDM). Enchira Biotechnology Corporation and formerly Energy BioSystems Corporation (EBC) spent the most money worldwide on microbial desulfurization research since 1992 until about 2002. The other major spender has been the Petroleum Energy Center (PEC) in Japan. EBC spent more than $50 million isolating, characterizing, and manipulating the desulfurization genes from a variety of microorganisms and developing and testing the reactor, separations, and recovery technology that is required to commercialize BDS. PEC has also been active in this area and, in 1994, pledged $50 million of its own for fundamental research and technology development. While for EBC, as a publicly traded company in the United States, its expenditures are easy to calculate based on annual reports, for PEC, which is a Japanese government–industry consortium, it is difficult to determine any actual spending amount. The multitude of collaborative efforts, exemplified in patents and papers that have appeared over the past 10 years from PEC and its collaborators indicates a substantial and ambitious program. The U.S. government, through the ongoing support of several academic and government labs, has spent about $15 million since 1990. This effort includes: • a $3-million Advanced Technology Program (ATP) grant to EBC for crude oil desulfurization • steady participation of DOE in cooperative R&D arrangements at the Oak Ridge and Brookhaven National Laboratories, and • small sponsored programs at the Institute of Gas Technology and other academic labs. DOE awarded EBC a $2.4-million grant to pursue gasoline biodesulfurization, which was completed in about 2001. However, the funding from the DOE program appears to have reduced significantly since 2003. The oil companies by themselves have not invested much on biotechnology development. However, there are exceptions: • Total Raffinage Distribution, S.A., which was working with EBC on diesel biodesulfurization;

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• Texaco Exploration and Production Technology Division, which was working with EBC on crude oil biodesulfurization; • Koch Refining Company, which was working with EBC on gasoline biodesulfurization; and • Exxon had its own effort and had sponsored work in Canada, which ended by 2003, and no further work seems to have been carried out since then.

The total expenditure over the past five years by the oil companies is probably only $5–10 million. As might be expected with such a competitive and segregated effort (within each oil company), a lot of money has probably been spent on repetitive efforts, targeted to develop in-house technology by each company. The analysis of the patents issued worldwide reflects that situation. To have a clear picture, it is necessary to exclude equivalent patents and leave only those claiming different inventions or different parts of the same invention (biocatalyst/bioprocess or genetic engineering of microorganisms, or in the biocatalyst manufacture, etc.). So far, the most advanced work has only reached the pilot plant scale. No commercial bioprocess or biocatalyst is available to the oil refining industry. There is a vast amount of knowledge reported in the open literature; however, there is still a long way to go before that knowledge is converted to a technological solution. For this reason, the following analysis is based on the issued patents, which might be closer to represent a potential technology. It should also be noted that the technology described in patents and the claims made within it may at times be extended to obtain wider coverage, but not proven at the larger scale or under the extended conditions. The patented results have been organized in alphabetical order of the name of the entity owning the patent or the assignee. For each company, their own patent packages will be cited chronologically as they were issued, to reveal any possible link to their development strategy. Any technical or technological connection among the different patents would be discussed separately. The fact that a given company has changed its name during the considered development has been taken into account and all those patents are discussed together. Some difficulties were found with Asian (Chinese or Korean) and European (Rumanian, Russian, etc.) patents, particularly those which do not have a published or any English equivalent. More than 50 organizations and individuals were found to have been involved in the development of biotechnologies related to refining. The largest package (or the most widely assigned intellectual property) corresponds to EBC. As could be derived from the sequence of patents, it seems that the Institute of Gas Technology discovered the biocatalyst, which was licensed to EBC, which then developed the biocatalyst and related technologies further. Indeed, the genus Rhodococcus received the most attention and the progress made with this biocatalyst served as inspiration for many research groups. Second in productivity are the national organizations in Japan, which have been working in an integrated, cooperative manner. Three Japanese organizations hold a significant number of shared patents: Agency of Industrial Science & Technology, which later became the National Institute of Advanced Industrial Science & Technology, Japan Cooperation Center Petroleum (JCCP), and Petroleum Energy Center (PEC).

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1. AGIP PETROLI (ITALY), ENICHEM ANIC SPA (ITALY), AND ENI TECNOLOGIE SPA (ITALY) EniTecnologie, the Corporate Technology Company of Eni, is in charge of technological innovation to support the competitiveness of the Group business units, in all sectors. By the end of 2000, the potential of biotechnology in oil exploration and production (E&P) and the ability to synergize it with the traditional physical and geochemical methods was ascertained via biosurvey campaigns, in which documentation and information regarding exploration and determination of new hydrocarbon reserves were collected. The biosurvey methods include propane and butane oxidation induced by microorganisms present in the ground. A thermophilic and hyperthermophilic microbial consortium was isolated from the reservoirs. Those strains were found to be active for anaerobic oxidation (hydrocarbons degradation) at extreme conditions (high pressure and salinity). Other activities in the E&P Sector include a simulation apparatus for control techniques of microbial acidification and corrosion phenomena in oil production fields. Strains, which produce hydrogen sulfide, were isolated and identified and the reaction conditions were established for a crude oil production system. Further, the biocompetition mechanisms were exploited to promote inhibition of sulfate reducing processes. The biocatalytic activities and the knowledge derived from the E&P research on H2 S was then applied to sweetening of natural gas. Thus, an experimental plant was set up to operate continuously on simulated gaseous streams with the aim of developing a hybrid (chemical + biological) H2 S removal process. Closer to the preparation of this book, they were working on the area of fuel manufacturing, pursuing activities for developing a biological approach to the desulfurization of gas oils. Both natural and engineered biocatalysts were studied by means of a continuous fermentation process. Genetic engineering techniques were used to improve performance and lower inhibition towards inorganic sulfur with respect to the first generation catalysts. A conceptual design of the biocatalyst production process was developed based on an economic analysis, and with identification of features, barriers and difficulties to make it more competitive. A significant catalytic activity for the removal of sulfur from the most common organosulfur compounds present in gas oil was confirmed and the process conditions were determined. The first mutants obtained by means of genetic modification of the microorganism showed improved catalytic characteristics, leading research into the construction of new strains. Several advantages influenced not only the final process costs but also from a patenting point of view. Five relevant patents have been awarded to Agip Petroli/Enichem Anic, which more recently integrated operations in Italy as Eni, of which, EniTecnologie is the Research and Technology affiliate. Most of these patents were issued in Europe with one in US (as equivalent to a European patent): 1. 2. 3. 4.

Anaerobic desulfurization process for crude oil and petroleum products [1]. Arthrobacter sp. and its use for the desulfurization of fossil fuels [2]. Desulfurization of gases containing hydrogen sulfide [3]. Means and methods for the expression of homologous and heterologous proteins in strains of Rhodococcus [4]. 5. Promoter and expression vector for Rhodococcus [5].

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The last patent that was awarded was dated 2003, indicating current activity in the area. The H2 S removal project was partially funded by the Ministry for Universities, Scientific, and Technological Research (MURST). The investigations were oriented to minimizing costs for both, initial investment/construction and operations, leading to the definition of an optimum reactor configuration. Immobilization was a special feature of that arrangement, particularly because the immobilization support was inert and allowed cell replication, without causing corrosion. Pilot plant and industrial scale reactors were designed and preliminary techno-economic evaluations demonstrated the competitiveness of the technology. A 10 kg/d H2 S removal capacity pilot plant was constructed. An Eni Agip Division demonstration unit is currently being set up in Italy. In addition to sulfur removal, nitrogen removal is also being investigated for the denitrogenation of field gas. An anaerobic biological hydrodesulfurization (BHDS) route to sulfur removal was among their first patents. It must follow a completely different pathway from those classically reported in the literature, which are typically oxidative pathways (e.g., which is followed in EP0795603). In fact, an anaerobic desulfurization process for crude oil and petroleum products [1] is described in an invention, which is equivalent to those registered under DE69000315D, DE69000315T, ES2036086T, GR3005977T, and IT1229852. It relates to a new mixed culture of obligative anaerobic microorganisms and facultative anaerobes, which was isolated from industrial effluents and has been filed as DSM 5307, in the microbial desulfurization of crude oil, petroleum fractions, and petroleum products. The use of the new culture in this process, which can be conducted either continuously or batch wise, results in a high degree of desulfurization of the substrate, with simultaneous controlled demolition of the high molecular weight structure. In this anaerobic case, hydrogen is used instead, in the presence of a consortium of Desulfovibrio desulfuricans, Clostridium, and an anaerobic facultative Coccus. The Desulfovibrio desulfuricans is known to produce iron hydrogenases. The process is carried out with the oil feedstock emulsified in water, in the total absence of any surfactant. However, the aqueous biocatalyst concentrations was very high (15–99%) and the residence time lasted from few days up to a week. The biocatalyst solution contains the two microorganisms in culture medium 161 (DSM). Conversion to light volatiles was observed but no product distribution was reported. Also interesting is their hybrid desulfurization process (for gases), in which a chemical reaction is coupled to a biochemical step (EP0811416). The second patent described a strain of Arthrobacter sp. useful for the desulfurization of fossil fuels [2]. The BDS activity of that strain was developed by successive enrichment and purification phases in a medium containing a carbon source and DBT as the only sulfur source. The invention [2] relates to a strain of Arthrobacter sp. CBS 208.96 (DS7 strain) capable of selectively effecting the opening of the C−S bond of sulfur containing organic molecules present in carbonaceous materials and its use in a process for the selective removal of organic sulfur from fossil fuels contained therein. Equivalent patents are: BR9701250, CA2198921, CN1167147, EA970021, ITMI960477, IT1283233, JP10094389, DE69722990D, and US 5874295. Among the advantages provided by this strain are: • The bacterium acts through a 4S metabolic pathway and it has been found that it expresses its maximum enzymatic activity after over halfway of the exponential growth phase (i.e., approximately 28 hours),

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• Activity is maintained for long periods biomass during/storage (7 days at 4 C) proving the stability of the enzymatic system, • The DS7 strain is characterized for its activity on the representative sulfur groups of the molecules present in fuel producing cuts, both gasoline and diesel. Examples given include straight-run gas oils, gas oils from hydrodesulfurization and the main streams coming from the atmospheric distillation of petroleum (cuts: 70–160 C 160–230 C and 230–350 C.), • The DS7 strain does not have rigorous nutritional demands, • No need for the addition of external agents, such as surfactants, the strain DS7 is capable of producing certain emulsifying compounds, • Formed microemulsions/microdispersions can be easily separated. Due to the oleophilous nature of the strain DS7, the reaction can take place without surfactants using reduced quantities of water. Therefore, the feedstock will acquire a state of emulsion or suspension without surfactants or having to assist it with mechanical equipments mixers with moving parts. Then, the mix is kept at room temperature and atmospheric pressure, with a substrate to be desulfurated/aqueous phase volumetric ratio of about 3:1, while reaction proceeds. The oil phase and the aqueous phase are fed counter-currently, thus producing the desirable mixing and separation effects simultaneously. Therefore, the mixing allows the biocatalyst to diffuse from the aqueous phase to the oil phase and the formed sulfates into the aqueous phase. A final separation is required to yield the desulfurized oil, a large aqueous phase and a mucilaginous phase consisting mainly of the biocatalyst together with some oil and water. This stream, depending on the content of sulfates, is recycled directly to the desulfurization reactor or subjected to removal of the sulfates by centrifugation in additional water (amended with precipitants). The fresh catalyst can be produced using the water coming from the removal of the sulfates. This company also undertook some biological initiatives for developing a process for the desulfurization of gases containing hydrogen sulfide. They end up with a hybrid process [3] effected by a chemical step, in which the separation of the sulfur compound is achieved by transforming it into elemental sulfur by oxidation with ferric sulfate. The second step is a biological step, where ferrous sulfate produced in the chemical step is reoxidized into ferric sulfate. The recommended strain belongs to the genus Thiobacillus. Equivalent patents were awarded in Germany, Spain, Italy, and Portugal (DE69704450D, DE69704450T, ES2155639T, ITMI961131, IT1282787, and PT811416T, respectively). Further work at EniTecnologies was conducted with Rhodococcus strains. Rhodococcus was selected for its metabolical versatility, easy availability in soils and water, and remarkable solvent tolerance. Its capabilities for catalyzing diverse transformation reactions of crude oils, such as sulfur removal, alkanes and aromatics oxidation and catabolism caught their attention. Hence, genetic tools for the engineering of Rhodococcus strains have been applied to improve its biotransformation performance and its tolerance to certain common contaminants of the crude oil, such as cadmium. The development of active biomolecules led to the isolation and characterization of plasmid vectors and promoters. Strains have been constructed in which the careful over-expression of selected components of the desulfurization pathway leads to the enhancement of the sulfur removal activity in model systems. Rhodococcus, Gordona, and Nocardia were transformed in this way trying to improve their catalytic performance in BDS. In a

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first patent [4] (equivalent to IT1317849 B1), they describe means and methods for the expression of homologous and heterologous proteins in strains of Rhodococcus. The means include sequencing, cutting, deleting, insertion, construction, cloning, etc. A plasmid vector of Rhodococcus, a new constitutive promoter, an expression vector containing such promoter and the microorganisms transformed with the expression vector were included in that patent. The expression vector has a high stability in the absence of selective pressure in the transformed strains of Rhodococcus and is particularly useful for the production of proteins of interest. The expression vector according to that invention comprises: a. the rep genes, ORF81 and trbA, which encode proteins, involved in replication in Rhodococcus; b. a gene called parA whose product is necessary for maintaining the plasmid in the absence of selective pressure and is characterized by the sequence identified in the document as SEQ ID. Nr. 1; c. a constitutive promoter of Rhodococcus having the sequence identified in the document SEQ. ID. Nr. 2; d. a multiple cloning site downstream the promoter, and. e. at least one gene encoding a genetic marker selected, e.g., from genes of the cad operon (identified in the document as SEQ. ID. Nr. 3), which provide resistance to cadmium or genes, which encode resistance to an antibiotic. Improving activity involves following steps, which consists of: i. transforming a strain of Rhodococcus directly with a gene reporter without its promoter or with a multicopy plasmid of E. coli containing such gene and linearized upstream the gene reporter; ii. selecting the clones which express the gene; iii. digesting the chromosomal DNA of the selected clones with restriction enzymes which cut upstream and downstream of the gene; iv. amplifying the DNA obtained in step (iii); and v. sequencing the promoter upstream of the gene reporter. A gene reporter refers to a fragment of DNA, which encodes a product that allows the selection of the clones, which express it. Examples of gene reporters useful for this purpose are those, which encode resistance to antibiotics or heavy metals or enzymes such as xylE or the same sox proteins. In a subsequent patent [5], the characteristics of a constitutive promoter of Rhodococcus, the vectors expressing this promoter and the cloned microorganisms were provided. The objectives are to overcome the drawbacks created by the inhibition of sulfate. The construction of a shuttle vector (pSM73) which can be replicated both in Rhodococcus and in E. coli containing a gene reporter without its own promoter and on the cloning, before such gene, of random fragments of chromosomal DNA of Rhodococcus consists of: a. rep and par genes, which encode proteins implicated in the replication and maintenance of the plasmid in Rhodococcus; b. a gene reporter;

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c. a multiple cloning site before the gene reporter; d. at least one gene, which encodes a genetic marker selected, e.g., from genes of the cad operon, which confer resistance to cadmium or genes which encode resistance to an antibiotic; and. e. the replication origin in E. coli. The shuttle vector also comprises: i. a multiple cloning site after the promoter, ii. the genes rep and para, which encode proteins involved in the replication and maintenance of the vector in Rhodococcus, iii. a genetic marker selected from an antibiotic resistance gene or a cadmium resistance gene (from the cad operon), and iv. the replication origin of E. coli. The vector is used to express the genes of the sox operon (soxA, soxB, and soxC) from the constitutive promoter and so, Rhodococcus, Gordona, and Nocardia may be transformed with this vector and used in BDS of fossil fuels. Equivalent patents: GB0311395D, ITMI20021217.

2. ARCHAEUS TECHNOLOGY GROUP LTD. (GREAT BRITAIN) In Chapter 1, Microbial Enhanced Oil Recovery (MEOR) was defined as the use of microbes in the oil wells, in situ to enhance production of oil and prolong their active life cycle. Most conventional oil recovery processes are able to retrieve only, approximately 50% of the oil at the well. Theoretically, microbes are supposed to act by either: i. physically displacing the oil due to biomass growth between the oil and the rock. ii. oxidizing the oil to fatty acids, which act as detergents. iii. producing CO2 via -oxidation of the fatty acids, which then acts as a gas drive. MEOR is an active research area in the oil industry but is not specifically detailed in this book. The patent (‘Enhanced oil recovery’) discussed below, WO9215771, directly concerns oil production by MEOR, rather than refining. However, as will be described below, the organisms works by excreting lactic acid, which chemically affect certain compounds in oil and most particularly metal compounds. So far, the concept of applying metabolic excretions has not been exploited for oil related chemical reactions. Their invention [6] (with the equivalent patents AU1353792 and CA2105288) relates to the use of lactic acid-producing bacteria in oil recovery operations. The invention provides a method of stimulating oil recovery from an oil reservoir which comprises: (i) injecting an inoculum of a lactic acid-producing bacterium compatible with the reservoir conditions into a well bore drilled into the reservoir; (ii) injecting a source of nutrients for the bacterium into the reservoir; (iii) allowing the bacterium to ferment thereby producing lactic acid; and (iv) recovering oil from the reservoir. The selected bacteria

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are those of the genera Lactobacillus or Pediococcus. Lactic acid produced by bacteria may also be used for removal of carbonate or iron scale in oilfield equipment.

3. ARCTECH INC/ATLANTIC RESEARCH CORPORATION (UNITED STATES) Atlantic Research Corporation, ARC (a unit of Sequa Corporation) devoted to R&D for the Defense Sector, is a leading developer and producer of solid propellant rocket motors, gas generators, advanced composite materials, and liquid propulsion systems. ARCTECH, Inc., a spin-off company from the Environmental Science and Technology division of ARC, provides cost-effective solutions for the energy, environmental, and agriculture market sectors. The ARCTECH group through 25 years of experience in energy, energetics, environment, and agriculture, has created holistic solutions in these interrelated market sectors. In ARC, BDS R&D seems to have been devoted to the chemical mutagenesis of microorganisms (Pseudomonas and Acinetobacter) to improve their biocatalytic activity. The scope of their work seems to be limited to assessing the effect of the mutation in improving BDS activity but not in studying strain stability or process development. Atlantic Research Corporation has four patents related to fossil fuel, which have resulted from its R&D work: 1. A novel mutant microorganism and its use in removing organic sulfur compounds [7], 2. Acinetobacter sp. and its use in removing organic sulfur compounds [8], 3. Biological production of humic acid and clean fuels from coal (2 patents) [9,10]. In the first patent [7], a novel mutant microorganism was claimed for its use in removing organic sulfur compounds (equivalent patents: AU566157 B2; AU2959784 A; CA1211394; EP0218734; FI842762; FI842762D D0; IL72274; IL72274D D0; JP60041479; and ZA8404621). The mutant microorganism Pseudomonas sp. CB1, ATCC 39381 was produced by chemical mutagenesis and was found to be effective in removing organic sulfur compounds from carbonaceous materials such as fossil fuels, e.g., coal, petroleum, and petroleum products. Similarly, in the second patent, an Acinetobacter species (ATCC # 53515), obtained by chemical mutagenesis, was claimed for its use in removing organic sulfur compounds. Both strains, CB1 and CB2 were obtained by isolation, adaptation, and mutagenesis. Original strains were isolated from soil samples of coal mines in Maryland, Virginia, and Pennsylvania, and adapted to grow on specific sulfur molecules in trypticase soy broth, and then on minimal salts medium, using benzoate as the carbon source. The surviving cultures were evaluated for oxidation of the specific sulfur molecules in the aqueous medium. Three active cultures were isolated and treated both, individually and as a ‘combo’. These four cultures and the Pennsylvania coal mine cultures were subjected to diethyl sulfate (DES) mutagenesis. The strains surviving mutagenesis were ‘rescued’ by serial dilution (1/100) into fresh nutrient broth and grown successively in minimal salts medium with benzoate as sole C-source and then with the sulfur molecule. Only the

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treated ‘combo’ showed significant growth in the minimal medium and a single colony was finally sub-cultured from it. The selectivity toward sulfur compound structure was differently induced for the CB1 culture, than that of the CB2 culture. In the first case DBT was used as the sole sulfur substrate, while in the second case, biphenyl sulfide was employed. One might deduce then, the application of the CB1 strain for oil and CB2 strain for coal and heavy oils. Plasmid characterization and strain catalytic evaluation results indicated that oxidation of DBT by CB1 is not plasmid mediated and that inorganic sulfur forms cannot be oxidized by CB1. Their following two patents (see Ref. [9,10]), although dealing with coal and gas, are related to the scope of this book and will be discussed here. These patents deal with a microbial consortium capable of converting aromatics, which because of the chemistry involved, could be applied for diesel cetane improvement. Thus, coal is treated aerobically or anaerobically to produce a wide range of organic compounds, such as humic acid, volatile fatty acids, lower alcohols, and/or methane, using a consortium of bacteria designated Mic-1 or KSARC56. This process can also be used to convert aromatic compounds, such as phenols and derivatives, to methane and carbon dioxide.

4. ASS UNIVERSITIES INC/BROOKHAVEN SCIENCE (UNITED STATES) The technology developed by Brookhaven National Laboratory has been jointly protected with ASS Universities Inc. It includes five patents dealing with the upgrading of heavy crudes: 1. Biochemically enhanced oil recovery and oil treatment [11], 2. Process for producing modified microorganisms for oil treatment at high temperatures, pressures and salinity [12], 3. Biochemical upgrading of oils [13], 4. Biochemical transformation of coals [14], 5. Biochemical transformation of solid carbonaceous material [15]. The protection strategy involves not only the use (or application) of the biocatalyst in a given process, but also the method of adapting the microorganism to perform the desired function. A wide range of microorganisms have been claimed within each patent (including their preparation and use). From their first patent, it was clear that they had envisioned using the same biocatalysts for down-hole as well as surface (refining) applications. Besides, the same development strategy was followed to extrapolate the application to solid carbonaceous materials, such as coal as well as bitumen. In the invention regarding the biochemically enhanced oil recovery and oil treatment [11], the preparation of new, modified organisms through a so-called challenge growth process was claimed. The claim included adaptation of the organisms to the extreme temperature, pressure and pH conditions, and salt concentrations of an oil reservoir. The challenge growth process leads to the selection of a thermophilic microorganism or a mixture of thermophilic microorganisms, which are also oil-tolerant and capable of growth in limited nutrients. These nutrients can be available in reservoir or can be supplied under reservoir conditions. A stepwise adaptation process is reported, which

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follows the order: nutrients, then temperature, then pressure, and then oil tolerance. The adaptation to nutrient conditions is reported via a gradual removal of nutrients to a 10% level, while maintaining the growth of the microorganisms. Then, a temperature and pressure adaptation process is carried out, in which the microorganisms are first adapted to the desired reservoir temperature, up to about 85 C. Next, the pressure is increased from atmospheric to the desired value (in the range of 200 psi up to about 2,500 psi). The selected microorganisms are then grown to a high density before injecting them for pursuing the oil biotreatment in the reservoir. A set of 67 strains were selected via the so-called challenge growth treatment. These included Achromobacter sp. BNL-423 (ATCC 55021), Sulfolobus solfataricus BNL-TH-29 (ATCC 55022), S. solfataricus BNL-TH-31 (ATCC 55023), S. acidocaldarius BNL-TH-1, Pseudomonas sp. BNL-4-24 (ATCC 55024), Leptospirillum ferrooxidans BNL-5-30 (ATCC 53992), Leptospirillum ferrooxidans BNL-5-31 (ATCC 53993), Acinetobacter calcoaceticus BNL-4-21 (ATCC 53996) and Arthrobacter sp. BNL-4-22 (ATCC 53997). These were mentioned as the preferred organisms for microbial enhanced oil recovery (MEOR) applications. However, they categorized the strains: BNL-TH-1, BNL-TH-29, and BNL-TH-31, obtained from the parent strains S. acidocaldarius (ATCC # 33909) and S. solfataricus (ATCC # 35091 and 35092), respectively, as the best strains. The main application was MEOR; however, other applications such as BDS and BDM were covered as well. When employed in the reservoir, an aqueous solution containing the microorganism or a mixture of them is injected into the reservoir directly. A special bioreactor to withstand the temperature and pressure conditions of the growth was designed using glass and metal concentric tubes. The reactor is said to hold a gradient temperature of 4 C at the bottom and 100 C at the top. Stainless steal was used for the higher-pressure range. The range of temperature used in the reactor raises interesting questions as to the reasons for employing such extreme temperatures (especially the low temperatures) and also the purpose of using a gradient. The answers are not evident from the patent and therefore raise intriguing questions. The effective production of the biocatalyst was as an important objective for ASS during its initial stages of development. They reported a process for producing modified microorganisms for oil treatment at high temperatures, pressures, and salinity [12], and filed a patent in 1993 that was awarded in 1996. This improvement to the previous patent relates to adapting the challenge growth processes to the salinity conditions found in oil wells. Therefore, the preparation of new, modified organisms, through the challenge growth processes, besides the extreme temperature, pressure and pH conditions, and salt concentrations typical of an oil reservoir were included. Again, the applications considered were MEOR, BDS, and metals removal from crude oils. The salinity range was claimed to vary from about 1.3 up to 35%; however, the patent document does not describe how this variation is carried out. There is also a mention of pH change from 1.5 to 7.5 in the document. Microbial strains were isolated from a series of cultures that survive growth in a medium containing crude oil supplemented with carbon sources other than the crude oil and nutrients. A great diversity of effects in the crude oil was produced by the modified organisms, under the biotreatment disclosed in this patent. The overall changes of the content of the different hydrocarbon types of heavy crudes comprised: decrease of the organic sulfur compounds, of thionaphthalene organic compounds, of the C20–C40 alkanes; increase of the
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A biochemical upgrading of oils [13] involving a very wide range of biocatalysts, obtained by the modifying adaptative method already described, was patented in the third patent. In the context of this invention, the term ‘induced biochemical conversion’ was defined as the multiple, simultaneous, or concurrent chemical reactions taking place over a period of 24 to 50 hours. Heavy crude oil could be upgraded by exposing it to a bacterial strain selected from the group consisting of Thiobacillus thiooxidans BNL-3-26 (ATCC 55009), T. ferrooxidans BNL-2-44 (ATCC 53982), T. ferrooxidans BNL-2-45 (ATCC 53983), T. ferrooxidans BNL-2-46 (ATCC 53984), T. ferrooxidans BNL-2-47 (ATCC 53985), T. ferrooxidans BNL-2-48 (ATCC 53986), T. ferrooxidans BNL-2-49 (ATCC 53987), T. thiooxidans BNL-3-25 (ATCC 53990), Leptospirillum ferrooxidans BNL-5-30 (ATCC 53992), L. ferrooxidans BNL-5-31 (ATCC 53993), Arthrobacter sp. BNL-4-22s (ATCC 55490), Achrmobacter sp. BNL-423s (ATCC 55491), Pseudomonas sp. BNL-4-24s (ATCC 55492), Mixed Culture R.I.-1 (ATCC 55501), Acinetobacter calcoaceticus BNL-4-21s (ATCC 55489), Arthrobacter sp. BNL-4-22 (ATCC 53997), A. calcoaceticus BNL-4-21 (ATCC 53996), Pseudomonas sp. BNL-4-24 (ATCC 55024), S. solfataricus BNL-TH-31 (ATCC 55023), S. solfataricus BNL-TH-29 (ATCC 55022), Achromobacter sp. BNL-4-23 (ATCC 55021), Mixed culture R.I.-10 (ATCC 55510), T. thioooxidans BNL-3-24 (ATCC 55020), T. thiooxidans BNL-3-23 (ATCC 55019), T. thiooxidans BNL-3-23 (ATCC 55007), Arthrobacter sp. BNL-4-22s (ATCC 55520), Mixed culture R.I.-9 (ATCC 55509), Mixed culture R.I.-8 (ATCC 55508), Mixed culture R.I.-7 (ATCC 55507), Mixed culture R.I.-6 (ATCC 55506), Mixed culture R.I.-5 (ATCC 55505), Mixed culture R.I.-4 (ATCC 55504), Mixed culture R.I.-3 (ATCC 55503), Mixed culture R.I.-2 (ATCC 55502), Unknown NZ-3 BNL-NZ-3 (ATCC 55488), Mixed culture R.I.-14 (ATCC 55514), Mixed culture R.I.-13 (ATCC 55513), Mixed culture- R.I.-12 (ATCC 55512), and their mixtures. From these wide variety of biocatalysts, a preferred set for the upgrading of heavy crudes included only the strains of T. ferrooxidans BNL-2-49s (ATCC 55530), T. ferrooxidans BNL-2-48s (ATCC 55529), T. ferrooxidans BNL-2-47s (ATCC 55528), T. ferrooxidans BNL-2-46s (ATCC 55527), T. ferrooxidans BNL-2-45s (ATCC 55526), T. ferrooxidans BNL-2-44s (ATCC 55525), L. ferrooxidans NL-5-31s (ATCC 55524), and L. ferrooxidans BNL-5-30s (ATCC 55523). The process yield upgraded oil in which the content of saturated hydrocarbons increased, but the level of organic sulfur, organic nitrogen, and metal compounds decreased, compared to that of the untreated heavy crude oil. The resulting upgraded oil also shows production of emulsifiers and oxygenates. The removal of sulfur, nitrogen and metals ranged from 20% to 50%, 15% to 45%, and 20% to 45%, respectively. Since the quantitative and qualitative chemical and physical effects observed in the crude oil depend upon the particular modified and adapted extremophilic microorganism employed, one may be inclined to think that mixed cultures or a biocatalytic ‘cocktail’ would be used when a wide functionality is desired. The first three inventions [11–13] concerned applications principally to heavy crude oils, while the latter two [14,15] are more applicable to coal. In the biochemical transformation of coals patent [14], a method of biochemically transforming macromolecular compounds found in solid carbonaceous materials, such as coal (lignite or bituminous coal) was described. The challenge growth method, already disclosed in the previous patents, was again applied for obtaining the biocatalysts. The process is carried out in a slurry phase at temperatures between 40 C and 85 C, pressures in the range from atmospheric to 2500 psi, pH values of 2–10 and a salinity range from about 1.5 wt. %

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to 35 wt. %. A wide variety of biocatalysts were claimed to have been developed, including Achromobacter sp. BNL-4-23 (ATCC 55021), S. solfataricus BNL-TH-29 (ATCC 55022), S. solfataricus BNL-TH-31 (ATCC 55023), S. acidocaldarius BNL-TH-1 (ATCC 35091), Pseudomonas sp. BNL-4-24 (ATCC 55024), L. ferrooxidans BNL-5-30 (ATCC 53992), L. ferrooxidans BNL-5-31 (ATCC 53993), A. calcoaceticus BNL4-21 (ATCC 53996), Arthrobacter sp. BNL-4-22 (ATCC 53997), A. calcoaceticus BNL-4-21s (ATCC 55489), Arthrobacter sp. BNL-4-22s (ATCC 55490), Achromobacter sp. BNL4-23s (ATCC 55491), Pseudomonas sp. BNL4-24s (ATCC 55492), Mixed culture R.I.-1 (ATCC 55501), L. ferrooxidans BNL-5-30s (ATCC 55523), L. ferrooxidans BNL-5-31s (ATCC 55524), T. ferroxidans BNL-2-44s (ATCC 55525), T. ferrooxidans BNL-2-45s (ATCC 55526), T. ferrooxidans BNL-2-46s (ATCC 55527), T. ferrooxidans BNL-2-47s (ATCC 55528), T. ferrooxidans BNL-2-48s (ATCC 55529), T. ferrooxidans BNL-249s (ATCC 55530), Unknown BNL-NZ-3 (ATCC 55488), Mixed culture R.I.-2 (ATCC 55502), Mixed culture R.I.-3 (ATCC 55503), Mixed culture R.I.-4 (ATCC 55504), Mixed culture R.I-5 (ATCC 55505), Mixed culture R.I.-6 (ATCC 55506), Mixed culture R.I.-7 (ATCC 55507), Mixed culture R.I.-8 (ATCC 55508), Mixed culture R.I.-9 (ATCC 55509), Mixed culture R.I.-10 (ATCC 55510), Mixed culture R.I.-11 (ATCC 55511), Mixed culture R.I.-12 (ATCC 55512), Mixed culture R.I.-13 (ATCC 55513), and Mixed culture R.I-14 (ATCC 5554). The biocatalyst preparation method was separately patented, in the document entitled ‘Biochemical transformation of solid carbonaceous material’ [15]. The method itself is not more than a summary of the development achieved in the above described patents [11–14], but with coal application in mind. The metabolically selected preparation obtained through the challenge growth process was nutritionally stressed via the same methodology of passing through the gradually increasing severity of environmental parameters. The method defined originally for the preparation of heavy crude oil biocatalyst was extrapolated to coal materials. So, the previously modified microorganisms were subjected to a medium in which the crude oil concentration is decreased and the coal concentration gradually increased until coal is essentially the sole carbon source. Besides, the salinity and trace metal concentration is also stepwise increased. The adaptation process ends when the thermophilic bacterial strain is capable of growing on coal as essentially the sole carbon source and at conditions of salinity level between 1.5 wt.% and 35 wt.%, trace metal concentration from about 0.01 wt.% to about 10 wt.%, and pressure from about atmospheric to about 2500 psi. In this patent, the pH range was widened to a range from 2 to 10. In summary, Premusic et al.’s findings indicate that the upgrading of carbonaceous materials and crude oils, most likely, requires a complex biocatalytic system, in which more than a modified bacterial strain is involved. The preparation of the catalytic microorganisms includes an adaptation stage at conditions including extreme temperatures, pressures, pH, and salt and toxic metal concentrations. It should be noted that while several patents have been obtained claiming sulfur, nitrogen, and metals removal, the specific biochemical mechanisms, were not described. Most of the information published by this group in journal articles is also similar in nature with little justification of the claims from an academician’s point of view, which results in an uncertainty about the application potential of the biocatalysts reported. A close examination shows that the biocatalysts belong to the Thiobacillus, Leptospirillum, Sulfolobus, Pseudomonas and Arthrobacter genus. The first three are known for their potential in transformation of inorganic sulfur while the Thiobacillus and the Pseudomonas are known for nitrogen

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conversion, which is again mostly inorganic nitrogen related, except for certain Pseudomonas, which can degrade organonitrogen compounds. The latter two are known to be involved in breakdown of hydrocarbons. In absence of definite bioconversion mechanisms and inaccessibility to the biocatalysts, the potential to investigate these processes further for commercial applicability or better mechanistic understanding becomes difficult. Additional information is available in Chapter 3 (see Section 2, and more specifically Section 2.2.3).

5. ATLANTIC RICHFIELD CO (UNITED STATES) Atlantic Richfield Company, Arco has developed several conventional technologies for sulfur removal from coal and ashes and for manufacturing oil-coal mixed fuels, prior to becoming involved in BDS. Their patent entitled, ‘Biodesulfurization of carbonaceous materials’ [16] was developed for the coal application, but nevertheless was extrapolated for application to petroleum desulfurization as well. The process claimed for reducing the sulfur content of a sulfur-containing carbonaceous material is carried out by contacting it, in an aqueous medium with the organism Bacillus sulfasportare per (ATCC 39909). The microorganism culture was isolated from soil samples collected from an abandoned coal refuse site in Braidwood, III, within a depth, which did not exceed one inch. The strain was adapted to a medium containing DBT as the sole sulfur source. The mutant microorganism is a Gram-positive rod, mesophilic, salt tolerant, and it grows on a mixture of amino acids, but not on sugars. The organism reverts to the wild type when grown through several generations in yeast extract. Sulfur removal from the carbonaceous materials was supposed to occur by an aerobic metabolic pathway.

6. ATLAS, RONALD/SOUTHERN PACIFIC PETROLEUM (UNITED STATES) Professor Ronald Atlas, from the University of Louisville holds three patents, one of which was assigned to Southern Pacific Petroleum. Most of his research was shared with Prof. Aislabe and was initially related to shale oil rather than crude oil. The first two patents claimed oil upgrading; however, the claims were limited to nitrogen removal and did not include viscosity reduction or conversion of high molecular weight hydrocarbons and condensed aromatics present in heavy oils. In the first patent, ‘Process for biotechnological upgrading of shale oil’ [17], special microbial cultures capable of degrading nitrogen compounds were developed by a sequential enrichment technique. The shale oil, as a whole, was used as the sole nitrogen source when culturing the selected strain. The biocatalyst is especially active for compounds such as amines, nitriles, quinolines, and pyridines. Specimens were collected from nature and identified as belonging to the Pseudomonas genus. The mutants produced were identified (and deposited with ATCC) as Pseudomonas aeruginosa ATCC 55081, P. aeruginosa ATCC 55083, and P. fluorescens ATCC 55084. Among them, P. aeruginosa was the most active for the removal of heterocyclic nitrogen.

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The second patent was also entitled ‘Process for biotechnological upgrading of shale oil’ [18], but only dealt with nitrogen removal. Most of the patent description covering biocatalyst development and the methodology corresponds to the previous patent [17]. The exceptions in this patent were the inclusion of Acinetobacter sp. (ATCC 55082), the selectivity of P. fluorescens and P. alcaligenes strains for the degradation of nitriles and addition of isoquinolines as substrates for nitrogen conversion. Mixed bacterial cultures (not completely identified) were claimed to be best for conversion of the isoquinolines. The third invention reported by the authors [19] was related to sulfur oxidation, but the invention was not patented. It was only registered with the Statutory Invention Registration (SIR). What this means is that, it is not a utility patent as the rest of the documents considered in this Chapter, rather a SIR registered document containing the specification and drawings of a provisional application filed for a patent, which has not been subjected to examination yet. It is issued by the PTO, if the applicant, among other requirements, waives the right to receive a patent on the invention within such period as may be prescribed by the Commissioner. The invention such as this, on which a statutory invention certificate is published, is not a patented invention for most purposes. The desulfurization process reported by the authors was a hybrid process, with a biooxidation step followed by a FCC step. The desulfurization apparently occurs in the second step. Thus, the process seems of no value, since it does not remove sulfur prior to the FCC step, but only oxidizes it to sulfoxides, sulfones, or sulfonic acids. The benefit of such an approach is not clearly outlined. The benefit of sulfur conversion can be realized only after its removal, and not via a partial oxidation. Most of the hydrotreatment is carried out prior to the FCC units, partially due to the detrimental effect that sulfur compounds exert on the cracking catalyst. It is widely accepted that the presence of sulfur, during the regeneration stage of the FCC units, causes catalyst deactivation associated with zeolite decay. In general terms, the subject matter of this document has apparent drawbacks.

7. BABCOCK & WILCOX CO (UNITED STATES) The Babcock & Wilcox Company is an operating unit of McDermott International, which has been supplying solutions to meet the world’s growing energy needs. Recently, a bankruptcy voluntary petition was filed (2000) in the U.S. Bankruptcy Court, Eastern District of Louisiana in New Orleans to reorganize under Chapter 11 of the U.S. Bankruptcy Code. McDermott announced Bankruptcy Court Action on B&W’s Chapter 11 Plan of Reorganization, by the end 2004. However, on February 2006, the Company informed that it had exited from Chapter 11 bankruptcy and entered into its previously announced settlement. Only one related invention (patent EP0643987 equivalent to CA2132372, DE69407905D, DE69407905T, and ES2111250T) has been developed by this company. Its relevant feature is the use of biotechnology for the regeneration stage of a sorption/removal process for the desulfurization of flue gas. Still, it is not clear which technological advantages or improvements can be achieved by its use in comparison to what exists today. In the bioregenerative flue gas desulfurization [20], the desulfurization reagent is bioregenerated. In a first stage, the flue gas, the reagent and water are mixed in an absorber, in which the reagent reacts with the sulfur compound, to provide desulfurized flue gas and spent reagent. This spent reagent is then biotreated in a digester reactor

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using sulfur-reducing bacteria, which will convert it into H2 S. The gases produced in the digester are collected and processed by conventional mechanisms. The regenerated reagent is fed back into the absorber. Bacterial growth could be promoted by a nutrient source, such as organic waste sludge from treatment plants of municipal waste water. Critical parameters affecting bacterial growth were pointed out to be temperature, pH, oxygen content, and flow rate of the nutrient.

8. BHP MINERALS INTERNATIONAL INC. (UNITED STATES) BHP Minerals International Inc. is a subsidiary of Australia-based BHP Billiton Limited, which in turn is the result of a merger between two complementary companies: BHP and Billiton. BHP is a global natural resources company, with a wide spread of products including minerals, oil, gas and steel and is one of Australia’s oldest and largest companies. Billiton, on the other hand is one of the world’s leading mining companies. Technology development, within the scope considered, was concentrated on bitumen froth. The processing of bitumen froth, described in US5968349 and US6074558, could be possibly extrapolated to other crude froths as well. The results make a technoeconomical feasibility study worth undertaking. The first patent considers the extraction of bitumen from bitumen froth and a biotreatment of bitumen froth tailings generated from tar sands [21] to produce paraffin-diluted bitumen. In this process, bitumen froth is extracted from tar sands using a water process without requiring the use of caustic soda. The froth is treated in a counter-current decantation circuit with a paraffinic solvent to remove precipitated asphaltenes, water, and solids from the bitumen froth. The bitumen might be considered a mixture of light hydrocarbons and aromatics acting as a solvent of the heavy hydrocarbons, such as the asphaltenes, in which the role of the aromatics is holding the asphaltenes in solution with the lighter hydrocarbons. Addition of a paraffinic solvent forces the asphaltic materials to precipitate out of solution by diminishing the peptizing action of the aromatics. The diluted bitumen produced has final water and solids contents of about 0.01 to about 1% by weight. Therefore, the process consists of: a. decanting the bitumen froth concentrate in counter-current with an organic solvent and producing two phases: a diluted bitumen and a bitumen froth tailings containing some residual bitumen, solvent, water, solids, and precipitated asphaltenes; b. separating the bitumen froth tailings by gravity to obtain a residual bitumen, a second phase containing the solvent, the precipitated asphaltenes, and water, and a third phase with some water and solids; c. recycling the residual bitumen phase to the counter-current decantation system; d. producing a bacterial inoculum from the solvent, precipitated asphaltenes, and water phase by mixing it with growing media or nutrient and incubating in an isothermal environment for an amount of time sufficient to produce a solid–liquid bioliquor mixture, containing biosurfactants, solvent, and water, and a solids phase, containing a reduced amount of precipitated asphaltenes and biomass; e. separating bioliquor product and a solid residue as tailings; f. filtering the water and solids phase to produce filtered solids, which are discarded as tails, and a water filtrate, which is recycled to the tar sands treatment process.

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This renders the dilute bitumen product amenable to direct hydrocracking. The process provides an alternative route to the conventional process of utilizing centrifuges to separate bitumen from precipitated asphaltenes, water, and solids and thus avoids the high capital and operating costs associated with the conventional bitumen froth treatment by centrifugation. The invention utilizes bitumen froth produced from a water process in which the use of caustic soda is not required. The process advantageously avoids the production of tailings sludge caused by clay dispersions. The present invention also teaches a novel process for the biotreatment of bitumen froth tailings resulting in a reduced amount of waste products and waste byproducts. Furthermore, the bacterial inocula and the biosurfactant produced might be an interesting byproduct for applications in some other bioprocesses. In the present invention, the biosurfactants enable the bitumen contained in the tar sands to be more efficiently separated from the solids, but its compatibility with the hydrocarbon phase make them worth trying for API gravity enhancement. Although the previous patent introduced the idea of producing bacterial inocula for the treatment of bitumen froth tailings, it is better detailed in the ‘Biochemical treatment of bitumen froth tailings’ patent [22]. In fact, the protected material is concentrated in the production of a bioliquor from bitumen froth tailings, which are characterized by the presence of bacterial cultures of microorganisms, though the addition of a non-indigenous bacterial culture is also considered. In this process, the native hydrocarbon-metabolizing microorganisms, originally present in the bitumen froth tailings, are cultured in an appropriate growth media to form an inoculum. An aqueous phase of a bioliquor is obtained by incubation under isothermal conditions. Initial applications of this bioliquor include tar sands conditioning process and direct injection into the tar sands formation. Degradation of the asphaltenes is also envisioned as a possible application for the bioliquor. Pseudomonas sp., Carynebacterium sp., Flavobacterium sp., Nocardia sp., Arthrobacter sp., Micrococcus sp., Mycabacterium sp., Streplamyces sp., and Achromobacter sp. were the selected microorganisms (it is not clear whether they have been identified) for this invention. Thus, as said above, the adaptation process those strains have been through make them potential candidates for other biocatalytic applications. The presence of biosurfactants in that bioliqour appear to render favorable results in terms of decreasing the amount of wastes and conditions employed in conventional processes, such as caustic soda treatment. In comparison, lower temperatures and no need of caustic soda are clear advantages, and consequently the present invention is said to avoid the production of clay dispersion sludges.

9. BIOSTAR BV (NETHERLANDS) Biostar is a leading company in the field of biological flue gas scrubbing. Biostar regards it as its task to develop and exploit the best technology for flue gas cleaning. In this respect, Biostar gives preference to biotechnology because it has been proven that flue gas can be treated in the most efficient and effective way by using biological processes. Biostar was established in 1993 as a result of a joint venture between Paques B.V. and Corus Consulting & Technical Services B.V. (formerly Hoogovens Technical Services). Besides Corus and Paques, the current shareholders of Biostar are Nuon Milieubedrijf N.V. and NOM N.V. (Noordelijke Ontwikkelings Maatschappij).

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Biostar holds intellectual property on a sulfur-reducing bacterium and its use in biological desulfurization processes [23] (with equivalent patents: WO9802524 A1, US6217766 B1, SK282687B B6, BG63467 B1, and AU713615 B2). This patent has been added here as an example of the treatment of highly sulfur-contaminated waters, particularly those from the flue gas processing. Inorganic sulfur compounds typically present in such waters are known to attack the sewer pipes, etc., and to cause acidification, eutrophication, and silting. Although, there are a large variety of microorganisms, which have been recognized for the treatment of waste waters, only a few of them have been found to be of interest for conforming as BDS consortium biocatalysts. The genus Desulfotomaculum claimed in patent EP0819756 has not been mentioned before for such application. The new sulfur-reducing bacterium was denoted as KT7 and it is characterized as being Gram-positive and has low-GC content. The new strain was isolated from a coal pile outside a power plant in Sweden. The strain reduces sulfite and sulfate to sulfide under anaerobic conditions at temperatures between 48 C and 70 C pH = 5–9 and at a conductivity of the liquid medium between 0 and 40 mS/cm. The process does require the supplementation of an electron donor. It is clear, that numerous technologies exist for these purposes. The BDS process has been shown to treat a highly sulfur-contaminated aqueous phase, allowing its downstream integration into the ‘sulfur management’ units. Such units are usually designed to handle sulfur gaseous compounds, such as SOx or H2 S by the use of heterogeneous catalysts. The present invention involves reduction in an anaerobic step to give sulfide, nevertheless potential for further biological processing to oxidize the sulfides to elementary sulfur also exists.

10. BWN LIVE OIL (AUSTRALIA) The work of Sheehy concentrated on the enhancement of oil recovery from a reservoir using microorganisms, and led to the patent, ‘Oil Recovery Using Microorganisms’ [24]. The main idea was to use a biocide to selectively kill certain microorganisms and then induce the growth of others by carefully controlled nutritional conditions. This idea has potential for some other applications and could be extrapolated to in situ refining. We have touched the topic of MEOR in earlier patents, particularly those related to oil upgrading and refining. The MEOR techniques referred to in the Sheehy patent involve the assistance of oil production by the action of endogenous microbial strains in an oil well. The nutrients such as sugars, sources of nitrogen, and mineral salts have to be supplied as well. However, other hydrocarbon substrates can also serve as carbon sources, fermentable sugars are economically advantageous. The parameters prevailing in the oil reservoir control the microorganism’s development, similar to those reported already for the thermophile development, where extremely high temperatures, salinity/ionic strengths, and pressures limited the type, range and number of microorganisms that can be selected. A disadvantage of injecting microorganisms employed in this patent is that they may occlude the reservoir pores. Further, these microbial cells may also find it difficult to diffuse into the small pores of the rock. The method involved in the present invention deals with the native bacterial population of the well. A biocide is applied so that

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to cause limited microbial death. Subsequently, the discharge of an effective carbon source would induce microbial growth. To facilitate microbial growth, the addition of non-carbonaceous nutrients to the reservoir could be performed before and/or after biocide use. The invention relies on the results of growth limiting nutrients, obtained by removing and analyzing a sample of liquid from the reservoir, in which microorganisms are resident. The given example considered an assessment of microbial growth made by defining the numbers of organisms in culture upon incubation with different nutrients. The production of surface active compounds, which facilitate oil recovery, has been commonly accepted as an action mechanism in MEOR. However, the present invention taught that microbial growth following the nutrient addition stage and after the depletion of at least one of the added nutrients resulted in microorganisms having reduced cell volume.

11. CLEAN DIESEL TECHNOLOGIES, INC. (UNITED STATES & INTERNATIONAL) Clean Diesel Technologies is a specialty chemical company devoted to technology development, which has many of its products and procedures patented. Its main products are fuel additives and systems technology, and its orientation is toward reduction of polluting emissions from diesel combustion, with the exception of a patent on a microbial catalyst for desulfurization of fossil fuels ([25], with equivalent patents: WO0009761, EP1112386, CA2339579, and JP2002522098T). This patent describes sulfur removal from fossil fuels by microbial biocatalyst. The microorganism was isolated and purified from soil and shown to be selective for extracting the sulfur without utilizing the carbon from the fuel. The naturally collected soil sample was incubated in a bacterial culture medium containing inorganic nutrients, an assimilable carbon source devoid of sulfur, and dibenzothiophene as a sole source of sulfur. This incubation is typically carried out for more than three weeks, while measuring growth. Intermediate weekly dilutions into fresh culture to levels of 1:1000 were conducted to enrich the strains that were growing. Three dilutions were made to obtain an ‘enrichment culture’ capable of growth on DBT as a sole source of sulfur. Two strains were isolated and purified, Pseudomonas sp. CDT-4, and Nocardia asteroides, CDT-4b (ATCC 202160 and 202161, respectively). The microbes were passed through a multiple screen, first to allow growth on dibenzothiophene (DBT) as a sole source of sulfur, and then on fossil fuels, to identify organisms capable of desulfurization without metabolizing the DBT phenyl ring structures. N. asteroides sp. CDT-4b was found to metabolize DBT. The Pseudomonas species was found to utilize trace levels of sulfate from media and was found to be incapable of growth on DBT as a sole source of sulfur. However, the co-culture could remove more than 20% sulfur, with supplementation of a second sulfur-free carbon source. A former President and COO of Clean Diesel Technologies, on behalf of Energy & Environmental Partners, in May 2006, announced a licensing program for US and Canadian BDS patents. A patent family is being offered in this licensing program and includes those mentioned in the above paragraphs [25], together with those which will be considered in Section 49. Valentine disclosed the use of microorganisms for the desulfurization of emulsified high-sulfur crude and bitumen petroleum products [26,27].

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It seems that a cross-licensing agreement has taken place between Clean Diesel Technologies and Energy & Environmental Partners, for the joint commercialization of these three patents. Now under the trade mark of EMULSOx, this patent family is offering a way to desulfurize high-sulfur crudes. Technical details of these patents were given in Chapter 3, and discussion of Valentine’s patents will be incorporated in the corresponding section below. The technology covered the methods for bitumens BDS and has been proved to remove 47% of the sulfur compounds present in the feed, using low-temperature bioreactors. The licensing offer clearly states the need for investing in further development, not only in the biocatalyst, but also in the process, starting even at process design level.

12. COMBUSTION ENGINEERING (UNITED STATES) Combustion Engineering is a North American company devoted to consulting and process evaluation services to a variety of industries operating combustion systems and furnace controls. The presence of contaminants in the fuel might lead to the formation of sulfides during its burning process. Thus, this company concentrated its development efforts in the treatment of gases produced during combustion of contaminated fuels. A significant amount of work was carried out by Kerry Sublette on microbial oxidation of inorganic sulfides. This has led to development of biocatalysts based on genus Thiobacillus and a patent entitled Microbiological desulfurization of gases (equivalent patents: CA1278761 and DE3677875D) [28]. There are several features characteristics of the genera Thiobacilli, which make them suitable for oil refining or gas processing applications, some of which are listed below: i. T. denitrificans is strictly dependent upon elemental sulfur and reduced sulfur compounds for its energy needs and on carbon dioxide for its carbon needs (obligate autotroph), ii. Optimal conditions for growth require: a. Nitrate or Ammonia as nitrogen source, b. Iron and, c. pH in the range of 6.2–7.0. iii. T. denitrificans is a facultative anaerobe that uses oxygen under aerobic conditions, and nitrate under anaerobic conditions. The biocatalyst is used in a microbial process for the removal and oxidation of sulfides. Feedstock ranges among natural gas, sour water (water co-produced with petroleum), and even spent sulfides containing caustics (from refinery wastes). This last feed has a pH greater than 13 but they reckon it can be biotreated. Recently, it has been announced that the process for natural gas has been successfully tested at the bench and pilot scales. The patent disclosed a method for desulfurizing gases by microbiological techniques, which involve the use of chemoautotrophic bacteria of the Thiobacillus genus to convert sulfides to sulfates either as a sulfide removal process or as a process for producing biomass. The term ‘chemoautotrophic mode of metabolism’ refers to a mechanism for deriving energy from the oxidation of inorganic compounds by microorganism with carbon dioxide serving as the carbon source.

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More specifically, the invention involves the use of Thiobacillus denitrificans under anaerobic conditions to oxidize sulfur compounds such as hydrogen sulfide to sulfate. The process may be carried out in various ways such as in a batch or a continuous bioreactor system using a suspended or an immobilized biocatalyst. The method is particularly applicable to treating natural gas containing hydrogen sulfide and producing a biomass byproduct.

13. ENVIRONMENTAL BIOSCIENCE CORPORATION/ ENERGY BIOSYSTEMS CORP./ENCHIRA BIOTECHNOLOGY CORP. (UNITED STATES) The biodesulfurization process which has achieved the most advanced stage, is the one developed by EBC under the leadership of Daniel Monticello. During this process, the company changed name two times, first, being Environmental BioScience Corporation (EBC1), followed by Energy BioSystems Corporation (EBC2) and finally, Enchira Biotechnology Corporation (EBC3). Most of the advancements made by the company were related to biocatalyst development. However, it should be noted that the organism was discovered by Kilbane team at Institute of Gas Technology (IGT). In fact, the pioneering research in this field was conducted at the IGT (see Section 20) on two microorganisms, Rhodococcus rhodochrous strain ATCC 53968 (IGTS8) and Bacillus sphaericus strain ATCC 53969, which were protected in eight patents (see also Chapter 3). In addition to the overlap of intellectual property between IGT (Kilbane’s group) and EBC, there is also an overlap with Valentine (see Section 49 of this Chapter) on patents awarded in 1992 and 1997 for the use of such strains and enzymes for petroleum desulfurization and emulsified bitumens. This overlap might suggest either cross-licensing agreements (between the parties) or infringement problems, when the technologies become commercial. In the case of IGT and EBC, some activities seem to have been carried out by the two companies together at the beginning of the 1990s. Two jointly assigned patents (between Monticello, EBC and Kilbane, IGT) were awarded, which may indicate licensing of the IGTS8 biocatalyst from IGT. For comparison purposes and as a quick reference guide, Table 1 collects all the inventions in chronological order. Not all the patents are shown in Table 1, since in most cases global coverage was attempted, instead only the representative patents of each patent family are included. The complete intellectual package is considered in the next sections, organized by assignee namely EBC1, EBC2 and EBC3, the discussion that follows, in some instances, is kept thematical rather than chronological. Before giving details on the patent portfolio and the chronology, let us mention that its first invention (patented by EBC1) was described as a microbial process for reducing the viscosity of a petroleum feedstock by using a sulfur bioremoval method [29]. The developmental work initiated by Monticello continued after the first change in the name of the company, incorporating numerous new inventors. As can be seen down below, most of the development carried out by EBC was based on the strain ATCC No. 53968 and included improvements in both, catalyst and process. An analysis of the intellectual property package awarded before 1994 might suggest a completely developed process. However, no demonstration plant was built. So, certain

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Table 1. EBC intellectual property package First inventor Patent No. (Ref.)

Title

Summary of main claim

Haney, W. H. WO9211343 [29]

Microbial Process for Reduction of Petroleum Viscosity.

A viscosity reducing method based on the selective cleavage of carbon sulfur bonds of aromatic heterocyclic molecules containing a sulfur heteroatom. The physicochemical properties of these heterocycles contribute to the liquid viscosity. The method comprises contacting the liquid with an effective amount of a biocatalytic agent.

Monticello, D. J. WO9216602 [30]

Multistage System for Deep Desulfurization of Fossil Fuels

Deep desulfurization method of fossil fuels, comprising a first step of HDS and a BDS step for the removal of HDS sulfur refractory compounds, using an effective amount of a biocatalyst. The fuel is incubated in the presence of one or more BDS-active microorganisms, which converts the organic sulfur compounds into water-soluble inorganic sulfur. Then, in a separation stage, the products of the incubation are separated into a deeply desulfurized liquid fossil fuel, and the water-soluble inorganic sulfur.

Monticello, D. J. US5232854 [31]

Multistage System for Deep Desulfurization of Fossil Fuels

Deep desulfurization method of fossil fuels, comprising a first step of HDS and a BDS step for the removal of HDS sulfur refractory compounds, using an effective amount of a biocatalyst. The fuel is incubated in the presence of one or more BDS-active microorganisms, which converts the organic sulfur compounds into water-soluble inorganic sulfur. Then, in a separation stage, the products of the incubation are separated into a deeply desulfurized liquid fossil fuel, and the water-soluble inorganic sulfur.

Monticello, D. J. US5387523 [32]

Multistage System for Deep Desulfurization of Fossil Fuels

Deep desulfurization method of fossil fuels, comprising a first step of HDS and a BDS step for the removal of HDS sulfur refractory compounds, using an effective amount of a biocatalyst. The fuel is incubated in the presence of enzymes obtained as a lysate, extract, fraction or subfraction of one or more microorganisms. The organic sulfur compounds are converted into water-soluble inorganic sulfur. Then, the desulfurized liquid fossil fuel is separated from the aqueous medium. (Continued)

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Table 1. (Continued) First inventor Patent No. (Ref.)

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Summary of main claim

Monticello, D. J. US5510265 [33]

Multistage System for Deep Desulfurization of Fossil Fuels

Deep desulfurization method of fossil fuels, comprising a first step of HDS and a BDS step for the removal of HDS sulfur refractory compounds, using an effective amount of a biocatalyst. The fuel is incubated in the presence bacteria or a substantially cell-free preparation, for converting the organic sulfur compounds into water-soluble inorganic sulfur. Then, the desulfurized liquid fossil fuel is separated from the aqueous medium.

Monticello, D. J. WO9219700 [34]

Continuous process for biocatalytic desulfurization of sulfur-bearing heterocyclic molecules.

A BDS continuous process in which the petroleum liquid is contacted with an oxygen source. The oxygenated feed is introduced, simultaneously with the aqueous biocatalytic solution, in a vertically elongated reaction vessel, from which it will be decanted after reaction. The reaction occurred during the incubation of the reacting mixture. Then, the aqueous phase is removed through the lower zone of the vessel, and treated for substantial removing the inorganic sulfate from the biocatalyst, whereby the biocatalytic activity of the agent is regenerated. The regenerated aqueous biocatalytic agent is recycled to the reaction vessel.

Monticello, D. J. WO9325637 [35]

Biocatalytic Desulfurization of Organosulfur Molecules.

A BDS method in which the feed has been emulsified with the aqueous solution of the biocatalytic agent and subjected to incubation for carrying out the reaction.

Monticello, D. J. US5358870 [36]

Biocatalytic Desulfurization of Organosulfur Molecules.

A BDS method in which the feed has been emulsified with the aqueous solution of the biocatalytic agent and subjected to incubation for carrying out the reaction. The biocatalytic agent is a cell-free enzyme preparation from Rhodococcus sp. ATCC 53969, B. sphaericus ATCC 53969, or any of their mutant derivations.

Rambosek, J. US5356801 [37]

Recombinant DNA Encoding a Desulfurization Biocatalyst.

A recombinant DNA molecule from Rhodococcus origin encoding a biocatalyst capable of desulfurizing a fossil fuel.

Rambosek, J. WO9401563 [38]

Recombinant DNA Encoding a Desulfurization Biocatalyst.

A recombinant DNA molecule containing a gene, which encodes a biocatalyst capable of desulfurizing a fossil fuel, which contains organic sulfur molecules.

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Table 1. (Continued) First inventor Patent No. (Ref.)

Title

Summary of main claim

Rambosek, J. US5578478 [39]

Recombinant DNA Encoding a Desulfurization Biocatalyst.

A purified protein having the amino acid sequence for ORF-1 (SEQ ID No:2)1 or an enzymatically active fragment thereof.

Rambosek, J. US5879914 [40]

Recombinant DNA Encoding a Desulfurization Biocatalyst.

A BDS method using a transformed microorganism containing a recombinant DNA molecule of Rhodococcus origin. This transformed microorganism expresses a BDS-active biocatalyst. (Use in BDS of the biocatalyst protected in Refs. [37–39])

Monticello, D. J. US5496729 [41]

Process for the desulfurization and the desalting of a fossil fuel.

A method for simultaneous desulfurization and desalting of fossil fuels by mixing the aqueous biocatalytic solution with the feed under conditions for both processes to occur. Both inorganic salts, the originally present in the fuel and the produced from the conversion of the organic sulfur compounds are solubilized by the added water, resulting in an aqueous phase with a salt concentration greater than 0.5 wt%. The biocatalyst comprises a bacterial organism or bacterial cell-free fractions.

Monticello, D. J. US5356813 [42]

Process for the desulfurization and the desalting of a fossil fuel.

A method for simultaneous desulfurization and desalting of fossil fuels by mixing the aqueous biocatalytic solution with the feed under conditions for both processes to occur. Both inorganic salts, the originally present in the fuel and the produced from the conversion of the organic sulfur compounds are solubilized by the added water, resulting in an aqueous phase with a salt concentration greater than 0.5 wt%. The biocatalyst consists of Rhodococcus sp. ATCC 53968, its mutants or cell-free fractions.

Johnson, S. W. US5468626 [43]

Method for separating a sulfur compound from carbonaceous materials.

A biosorption method for the separation of sulfur compounds from fossil fuels, by using a sulfur-biosorption agent and followed by the oxidation of the biosorbed complex. The oxidation is carried out in an aqueous phase containing an effective amount of oxygen and, optionally a biocatalyst, in which case an incubating stage is incorporated for the reaction to take place.

1

See original document for details on the sequence (Continued)

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Table 1. (Continued)

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Title

Monticello, D. J. US5525235 [44]

Method for separating a petroleum containing emulsion

A method of separating an intermixed multiple phase liquid mixture, using wet filters. The liquid mixture contains a fossil fuel, an aqueous phase and a biocatalyst. The first filter is wetted with an agent miscible with the fossil fuel but immiscible with the aqueous phase. The second filter is wetted with a wetting agent miscible with the aqueous phase but immiscible with the fossil fuel. The mixture is then passed sequentially for each filter. The fossil fuel is recovered from the final filtrate and the biocatalyst is retained in the aqueous phase of the final retentate.

Ortego, B. C. WO9617940 [45]

Method of desulfurization of fossil fuel with flavoprotein.

A method for enhancing the rate of biodesulfurization of a fossil fuel by adding an amount of a flavoprotein to the biocatalytic reaction mixture. Incubation and separation complete the process scheme.

Ortego, B. C. US5733773 [46]

Method of desulfurization of fossil fuel with flavoprotein.

An isolated and purified DNA molecule comprising DNA which encodes a flavoprotein and DNA of Rhodococcal origin which encodes a protein biocatalyst active for BDS. The DNA biomolecule is not a Rhodococcus genome.

Ortego, B. C. US5985650 [47]

Method of desulfurization of fossil fuel with flavoprotein.

A method for enhancing the rate of biodesulfurization of a fossil fuel by adding an amount of a flavoprotein to the biocatalytic reaction mixture. The flavoprotein amount enhances the biodesulfurization rate by at least 25% in comparison to the rate employing the biocatalyst alone. Incubation and separation complete the process scheme.

Monticello, D. J. US5846813 [48]

DszD utilization in desulfurization of DBT by Rhodococcus Sp. IGTS8.

A method for enhancing the rate of biodesulfurization of a fossil fuel by adding to the biocatalytic aqueous phase a nicotinamide adenosine dinucleotide and an additional amount of a group III alcohol dehydrogenase. Incubation and separation follows the mixing step.

Monticello, D. J. WO9711185 [49]

DszD utilization in desulfurization of DBT by Rhodococcus Sp. IGTS8.

A method for enhancing the rate of biodesulfurization of a fossil fuel by adding an amount of oxidoreductase, to the biocatalytic aqueous phase. Incubation and separation follows the mixing step.

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Table 1. (Continued) First inventor Patent No. (Ref.)

Title

Monticello, D. J. US5811285 [50]

DszD utilization in desulfurization of DBT by Rhodococcus Sp. IGTS8

An isolated DNA molecule comprising DNA which encodes a group III alcohol dehydrogenase and DNA which encodes a BDS-active biocatalyst via nicotinamide adenosine dinucleotide-dependent manner.

Xu, G.-W. US5624844 [51]

Process for demetallizing a fossil fuel.

A method of removing metals from a fossil fuel using an oxygenase which degrades porphyrin molecules, under suitable conditions.

Xu, G.-W. US5726056 [52]

Process for demetallizing a fossil fuel.

A demetallized fossil fuel produced by a method of removing metals from a fossil fuel using an oxygenase which degrades porphyrin molecules, under suitable conditions.

Yu, L.-Q. US5772901 [53]

Oil/water/biocatalyst three phase separation process.

An emulsion separation method using hydrocyclones. The emulsion comprises a continuous phase, a discontinuous phase and fine solid particles. In the first step, the original emulsion is separated into an overflow emulsion and an underflow emulsion, in a first hydrocyclone. The overflow emulsion comprises portions of the continuous phase, the discontinuous phase and the fine solid particles. The overflow emulsion is inverted in which the continuous phase of the overflow emulsion is now a second discontinuous phase and the original discontinuous phase becomes a second continuous phase. Then, the inverted emulsion is directed to one or more subsequent hydrocyclones and the second continuous and discontinuous phases are collected. The fine solid particles remain in the second discontinuous phase.

Squires, C. H. WO9817787 [54]

Rhodococcus flavinreductase complementing DszA and DszC activity.

An isolated nucleotide sequence2 encoding the enzyme set forth in SEQ. ID No. 2, a mutant or a homologue thereof.

Squires, C. H. US5804433 [55]

Rhodococcus flavinreductase complementing DszA and DszC activity.

An isolated nucleotide sequence2 encoding the enzyme of SEQ. ID No. 2, or an enzymatically active mutant thereof.

2

Summary of main claim

See original document for details on the sequence (Continued)

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Table 1. (Continued) First inventor Patent No. (Ref.)

Title

Summary of main claim

Squires, C. H. US5919683 [56]

Rhodococcus flavin reductase complementing DszA and DszC activity

An isolated protein having NADH:FMN oxidoreductase activity having the amino acid sequence2 set forth in SEQ ID No: 2 or an enzymatically active mutant thereof.

Squires, C. H. US6274372 [57]

Rhodococcus flavinreductase complementing DszA and DszC activity

A BDS method consisting in the incubation of a mixture formed by a fossil fuel and an aqueous phase containing a biocatalyst and a rate-enhancing amount of a protein having NADH:FMN oxidoreductase activity or enzymatically active mutant thereof. The oxidoreductase has the amino acid sequence set forth in SEQ ID No. 2, described in the original document (see Ref. [57]). A separation stage is also claimed.

Monticello, D. J. US5952208 [58]

Dsz gene expression in pseudomonas hosts.

A recombinant pseudomonad comprising a heterologous nucleic acid molecule which encodes one or more desulfurization enzymes.

Mrachko, G. T. US5968812 [59]

Removal of sulfinic acids.

A desulfurizing method consisting of a first step for the bioconversion of the organosulfur compounds to organosulfinates. The organosulfinate compounds are contacted with an effective amount of a copper(II) or a soft Lewis acid in the presence of a protic solvent, thereby desulfinating the organosulfinate compound and producing a carbonaceous material of reduced sulfur content.

Lange, E. A. US5973195 [60] WO9947496 [61]

Surfactants derived from 2-(2hydroxiyphenyl)benzene-sulfinate and alkyl-substituted derivatives.

A compound of Formula I: R8

R9

R1

R2

R7

R3

R6

S(O)nO– R5

R4

wherein R2 -R9 are each, independently, hydrogen, a substituted or unsubstituted, normal, branched or cyclic alkyl group, a substituted or unsubstituted aryl group, an hydroxyl group, a cyano group, a nitro group or a halogen atom; R1 is a moiety of the formula YC(O)O– or YO–, wherein Y is a hydrophobic group; and n is 1 or 2.

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Table 1. (Continued) First inventor Patent No. (Ref.)

Title

Summary of main claim

Colin, J.-M. US6071738 [62]

Conversion of organosulfur compounds to oxyorganosulfur compounds for desulfurization of fossil fuels

A desulfurizing method, in which the organosulfur compounds are microbially converted in organosulfinate or organosulfonate compounds. Reaction is followed by the separation of the organosulfinate or organosulfonate compounds from the fossil fuels.

Folsom, B. R. WO0042122 [63]

Growth of biocatalyst within biodesulfurization system.

A desulfurizing method, in which the biocatalytic aqueous phase also contains sufficient nutrients for viability of the microorganism. The reacting mixture is incubated and the products are then separated.

Mrachko, G. T. US6133016 [64]

Sphingomonas biodesulfurization catalyst.

A nucleotide molecule encoding an enzyme having an amino acid sequence3 set forth in SEQ ID No. 2; or an enzymatically active fragment thereof.

Squires, C. H. US6235519 [65]

Gene involved in thiophene biotransformation from N. asteroides KGB1.

A nucleic acid molecule encoding an enzyme having the amino acid sequence3 set forth in SEQ ID No. 2; or an enzymatically active fragment thereof.

Lange, E. A. US6303562 [66]

Compositions comprising 2-(2-hydroxyphenyl)benzene-sulfinate and alkyl-substituted derivatives thereof.

A nucleic acid molecule encoding an enzyme having the amino acid sequence3 set forth in SEQ ID No. 2; or an enzymatically active fragment thereof.

3

See original document for details of the sequence

problems must have been realized during the scaling up which hindered the transition towards commercial scale. It would be interesting to speculate what problems were uncovered at that early time during the development process. Nevertheless, the second half of the 1990s incorporated molecular biology and genetic engineering techniques to make long strides in the biocatalyst development process. Furthermore, innovative ideas were used to create value by identifying useful secondary products. Process development received the least attention, although two research targets could be identified. These seemed to be (1) identifying mass transfer limitations and mitigating them via use of process modifications and (2) developing separation strategies for batch/continuous operation of BDS. The IP strategy involved patenting any discovered active biomolecules, together with its corresponding use or BDS application. Therefore, a broad set of biomolecules,

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including not only enzymes but also any pertinent genetic material, composed the intellectual property of EBC. The use and application of those biomolecules is also part of the IP package. Finally, some process units, their combination with the BDS process and the integration of the BDS process into refining operations was also protected. Most of the equivalent patents are incorporated in the discussion, to give a sense of the international coverage to the reader. A summary of the main claim is included as a definition of the property subject matter. As could be seen the property subject matter of a World Intellectual Property Organization (WIPO) patent generally, is identical to one of the corresponding US patent (from the US Patent and Trademark Office, USPTO). However, in some instances a WIPO patent has more than one US equivalent patent. The equivalence between WIPO and US patents will be discussed for each particular case wherever relevant.

13.1. Environmental Bioscience Corp (EBC1) The first known entrepreneurial invention devoted to BDS development was awarded to Environmental Bioscience Corp., which led to two inventions protected with five patents: 1. Microbial process for reduction of petroleum viscosity [29]. 2. Multi-stage system for deep desulfurization of fossil fuels [30–33]. Although, the patents are referred as processes, apart from mentioning ambient temperature, mechanical stirring, and a need for an oxygen supply, no further process details and conditions are included in the documents. The first patent protected in 1992 relates to a viscosity reduction process and was awarded worldwide coverage [29], and extended with numerous equivalent patents in individual countries (EP0563142, AU651164, AU9103591, CA2098836, CN1062765, DE69115507D, DE69115507T, EP0563142, JP6507649T, MX9102704, US5529930, and KR176994), with filing dates up to 1999. Although the claimed matter is basically the same in all the equivalent patents, the number of allowed claims differs from one another, but mainly for dependent claims. The biocatalyst employed was claimed to selectively cleave C−S bonds and the viscosity reduction was claimed to be a consequence of the ring opening of the sulfur containing heterocyclic compounds. The biocatalyst was described, as any microorganism (such as R. rhodochrous ATCC 53968, or its mutants) capable of expressing the enzymes involved in the 4S pathway or the enzymes themselves (with or without the support of a co-factor). The mode of application of the biocatalyst was quite broad and ranged from above ground application via a simple contact between the biocatalyst and the oil feedstock to application in the well, at the top of the well (at surface level), in the pipelines, in the storage tank or any desulfurizing plant or a refinery plant. No process conditions were claimed. The method is based on the macromolecular structure created by the mutual interaction of the aromatic heterocyclic sulfur compounds, which are present in the crude oil liquids. Such heterocycles are thought to be very rigid and planar rings, which condense in organized stable macrostructures. Haney and Monticello suggested the strength of hydrophobic interactions between adjacent molecules as responsible for such molecular arrangement. Certain crude oils contain heterocyclic aromatic sulfur

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compounds, in a relative proportion so as to believe that their physicochemical properties would contribute significantly to the viscosity of the liquid. In the approach followed in this invention [29], a biocatalytic agent converts the sulfur heterocycles into different molecules that do not exhibit the hydrophobic interactions. This is achieved by selectively cleaving carbon–sulfur bonds. The selectivity of the biocatalytic agent employed is limited to the carbon–sulfur bonds and no attack to the carbon–carbon skeleton was reported. Thus, it is expected that the proposed biocatalytic reduction of viscosity would not diminish the fuel value of the treated petroleum liquids. The biocatalyst employed consisted of the strain ATCC No. 53968 (see Section 20 and references therein), in an aqueous culture conventionally prepared by fermentation under aerobic conditions. The fermenting bioreactor is fed with a suitable nutrient medium, which comprises a conventional carbon source (dextrose and glycerol are recommended carbon sources. To confer maximal biocatalytic activity for the desired cleavage of organic C−S bonds, the bacteria was kept in a state of sulfur deprivation. The search for integration schemes for refinery operations led to an invention entitled: ‘Multistage system for deep desulfurization of fossil fuels’ [30–33], which was first published worldwide (assigned to EBC1) [30], and then its equivalent US patent awarded to EBC2 [31], who remained as assignee for the equivalent patents: AU1643992, AU656962, BR9205746, CA2105779, CN1032483B, CN1064880, DE69201131D, DE69201131T, EP0576557 (B1, A3), ES2066615T, HK68997, JP6506016T, and KR188615. The world patent contained 19 claims, while the sum of the three US patents give a total of 32 claims; however, basically the intellectual property awarded is not significantly different. The reason is that the main US patent contains some claims which were incorporated in the ‘continuation-in-part’ issued as the second patent [32] and also in the ‘divisional’ patent awarded in 1996 [33]. Subsequent to realizing the conversion levels achieved by typical BDS biocatalysts, its integration with HDS operations seemed beneficial due to following two reasons. First, BDS could convert sulfur compounds which cannot be converted under ordinary HDS conditions and second, BDS employs conditions milder than those required by HDS. Hence, integration results in a complementary synergetic effect. Thus, the method disclosed in this invention [30–33] may achieve deeper desulfurization via a two-step process namely, HDS followed by BDS. The HDS stage will remove sulfur from labile compounds sensitive to HDS conditions, while some of the HDS refractory compounds will be exposed to a biocatalyst, which selectively cleaves the carbon–sulfur bonds. In this manner, during the HDS stage, milder conditions would be needed, compared to the severity required for a similar overall desulfurization, which would be detrimental to the fuel value of the products. In general, the BDS method developed is recommended wherever a sulfur-containing stream exists in the refinery, i.e., atmospheric and vacuum residues, hydrotreated heavy vacuum gas oil previously to FCC, hydrotreated gasoline, etc. Once more, the preferred biocatalyst is a culture of R. rhodochrous bacteria, ATCC No. 53968. Cell extracts, enzyme preparation and DNA shuffling were incorporated in the late US equivalent awarded patents, but some of these issues will be considered in the next section, where other patents are also considered.

13.2. Energy Biosystems Corporation (EBC2) Most of the BDS development was carried out by EBC2 (see list of awarded patents [34–65] in Table 1), which continued the work initiated by EBC1. EBC’s second

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patent [34], under EBC2 targeted biocatalyst regeneration so as to establish a continuous/cyclic treatment. Its intellectual property assets began with this continuous process for biocatalytic desulfurization of sulfur-bearing heterocyclic molecules present in petroleum liquids [34] (equivalent patents: EP0584281, AU2233992, AU659480, BR9205954, CA2109091, CN1066285, DE69201792D, JP6507436T, MX9202062, and US5472875). The process is particularly recommended for petroleum liquids, which contain organic sulfur molecules, especially sulfur-bearing heterocycles, such as dibenzothiophene. The desulfurization reaction involves oxygenating the petroleum liquid and treating it with a biocatalyst capable of catalyzing the sulfur-specific oxidative cleavage of organic carbon–sulfur bonds. Although, the process is no more than the exposure of the feedstock to the biocatalyst, it is described through seven steps, which are included here for completeness: a. increase the oxygen tension in the liquid fossil fuel by contacting the liquid fossil fuel with a source of oxygen under certain conditions; b. introducing simultaneously, to a reaction vessel: the oxygenated liquid fossil fuel and an aqueous, sulfur-deprived biocatalytic agent, through different feeding pipelines, as to create a counter-current flow system within the vessel; c. incubating the reacting mixture under conditions sufficient for the selective biocatalytic oxidative cleavage of the carbon–sulfur bonds, and so become converted to water-soluble inorganic sulfur ions; d. removing the desulfurized liquid fossil fuel from the reaction vessel by decanting; e. removing the spent aqueous biocatalytic agent from the bottom of the reaction vessel; f. regenerating the biocatalytic agent by treating and removing the inorganic sulfur from the aqueous solution obtained in the previous step, and, g. recycling the regenerated aqueous biocatalytic agent to the reaction vessel. The regeneration step described above indicates that deactivation seems to be only caused by the presence of inorganic sulfur and its removal allows many recycles of the spent-regenerated biocatalyst. Obviously, such features are rarely observed and consequently the quality of such regeneration may be judged. The biocatalytic agent comprises one or more bacterial organisms expressing an enzyme or enzymes that catalyze the sulfur-specific oxidative cleavage of carbon– sulfur bonds in sulfur-bearing heterocycles to produce desulfurized organic molecules and inorganic sulfur ions. A particularly preferred biocatalyst is a culture of mutant R. rhodochrous bacteria, ATCC No. 53968. Furthermore, cell-free extract biocatalysts were proposed, for which some introductory descriptions were given in this patent. The preparation methods consider standard techniques (centrifugal fractionation, ammonium sulfate fractionation, filtration, bioaffinity or immunoaffinity precipitation, gel permeation chromatography, liquid chromatography, high pressure liquid chromatography, reverse phase liquid chromatography, preparative electrophoresis, isoelectric focusing, etc.). The US equivalent patent (US5472875) included some GE manipulation on the DNA molecules, which was not awarded in the World Patent [34] probably due to the three-year period that separates these two patents. Such manipulation (DNA shuffling) constitutes the subject matter of other patents, and will be discussed later in this section. The next awarded patents were jointly awarded to EBC and the Institute of Gas Technology namely, Biocatalytic Desulfurization of Organosulfur Molecules (also named as

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Microemulsion process for direct biocatalytic desulfurization of organosulfur molecules for the US PTO and in some other countries where equivalent patents were issued: AU4406593, CN1082094, and MX9303465) [35,36]. These patents refer to improvements on both, process and catalyst. Regarding the process, the improvement consists in forming a microemulsion oil-water, between the feedstock and the biocatalyst solution, as a mean of overcoming the mass transfer limitations typically found in organic bioprocessing. To ensure a suitable partial pressure of oxygen in the emulsion, the feedstock is previously stirred, mixed, or bubbled with the oxygen source. The oxygen sources include air, oxygen-enriched air, pure oxygen, or oxygen-saturated perfluorocarbons. The feedstock (any petroleum liquid) and the biocatalytic solution are mixed as to form an emulsion, preferably a microemulsion. For this water-in-oil emulsion, aqueous droplets are dispersed within the organic phase, the latter being the continuous phase. Reversible microemulsions are recommended to facilitate the biocatalyst recovery from the desulfurized petroleum liquid at the conclusion of the treatment. Although, several physical means of preparing the microemulsion are described in the patent document, careful manipulation is emphasized to avoid damaging the biocatalyst and the use of surfactants may facilitate this step. The microemulsion is incubated for a period of time, during which sulfur content is monitored. A period of 16 hours was mentioned as a minimum time span. With respect to biocatalyst modifications, an aqueous solution of a cell extract derived from ATCC No. 53968 replaced the former whole cells or pure enzymes. In the World patent [35], the disclosed method of desulfurizing a petroleum liquid containing organosulfur molecules is described as a catalytic process, for specifically distinguishing it from those relying on microbial metabolism. The main difference made by the biocatalyst is breaking the C−S bonds from a variety of compounds, unlike conventional microbial desulfurization agents which are only capable of cleaving carbon–sulfur bonds from a limited type and stereospecific compounds. The method uses an aqueous biocatalytic agent comprising a substantially cell-free extract of a microorganism. The biocatalyst, though regarded as ‘an enzyme’, includes one or more proteinaceous catalysts together with such co-enzymes, co-factors, or co-reactants as may be required to bring about the selective liberation of sulfur from organic molecules through the cleavage of organic carbon–sulfur bonds. The selected microorganism functionally expresses the ‘enzyme’ capable of selectively cleaving organic carbon–sulfur bonds, and the referred extract contains a substantial proportion of the total activity of said enzyme expressed by the microorganism. The preferred microorganism for the present invention is a strain of R. rhodochrous ATCC No. 53968, which is realized as a source of biocatalytic activity (see Section 20, Ref. [67]). The biocatalyst is prepared initially under suitable conditions from the intact ATCC No. 53968 microorganisms, by using it to obtain either a lysate or a substantially cell-free extract. A fraction of the extract containing a substantial proportion of the total BDS reactivity expressed by said microorganisms could also be used. Suitable preparation conditions for ATCC No. 53968 microorganisms comprise aerobic fermentation in the presence of a sulfur-free mineral salts medium (e.g., 4 g/L K2 PO4  4 g/L Na2 HPO4  2 g/L NH4 C1 02 g/L MgCl2 ∗ 6H2 O 0001 g/L CaCl2 ∗ 2H2 O, and 0001 g/L FeCl3 ∗ 6H2 O), together with a source of assimilable carbon such as glycerol, benzoate, acetate, glucose, ethanol, isobutanol, or sucrose. Typical organosulfur compounds present in the petroleum feedstocks are employed as the sole source of sulfur provided to the ATCC No. 53968

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bacteria. Examples of the sulfur source are thiophene, benzothiophene, DBT or its derivatives. Prior to fermentation, pH is adjusted between 6 and 7, and temperature is kept at ambient temperature 28–32 C. The bacterial culture is separated from the medium, re-suspended in fresh medium and a BDS-active suspension of lysed microorganisms, substantially free of intact cells is prepared. Any lysis process can be used, provided that the enzyme responsible for BDS reactivity remains functional. Methods such as freeze– thaw cycles, treatment with a detergent and/or chaotropic agent, lysis using French press, or sonication were mentioned in the document. This last technique is preferred since it is capable of yielding microemulsions, which can directly be used. The cell-free aqueous extract of ATCC No. 53968 is considered to contained the BDS reactive enzymes, which may be functionally expressed as a cell envelope associated enzyme (see Section 20, Ref. [68] this Chapter). Thus, a substantially cell-free extract of ATCC No. 53968, comprising cell membranes and cell membrane fragments can be used as biocatalyst for the purposes of the present invention. Any standard technique could be followed for preparing the cell-free extract, such as centrifugal fractionation, ammonium sulfate fractionation, filtration, bioaffinity or immunoaffinity precipitation, gel permeation chromatography, liquid chromatography, high pressure liquid chromatography, reverse-phase liquid chromatography, preparative electrophoresis, isoelectric focusing, etc. From this invention, a detour for biocatalytic development clearly appeared from the whole cell strategy to a MB plan, where enzyme isolation or preparations for the like were targeted. From this patent, the enzyme responsible for biocatalytic cleavage of carbon–sulfur bonds was believed to be present on the exterior surface (the cell envelope) of the microorganism, which is not the case as we know now, since the BDS enzymes are all soluble enzymes and are present in the cytoplasm (see Chapter 3). Other useful enzyme preparations, suggested in the patent document, include microbial lysates, extracts, fractions, subfractions, or purified products obtained by conventional means and capable of carrying out the desired biocatalytic function. Such enzyme preparations are free of intact microbial cells (and their derived handling problems). Kilbane (in his own patents, see Section 20) and together with Monticello (described down below in this section) disclosed suitable enzyme preparations, as well. The EBC starting point for involving MB and GE techniques, in the claimed subject matter began with an invention entitled ‘Recombinant DNA Encoding a Desulfurization Biocatalyst’ [37–40], which received awarded patents in USA, Europe and six other countries (AU4671893, AU5835398, AU684253, BR9306856, CA2139876, CN1085254, EP0651808-A1, JP2979178B2, JP7507691T, MX9304135, and NO950081D). This invention concerns biomolecules, directly and indirectly related to catalytic activity. Particularly, it considers a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of R. rhodochrous. The first world patent that was issued [38] contained 50 claims. This included 39 claims contained in three US patents [37,39,40] which were issued after the world patent. The world patent widely protects the biocatalytic system, which can be described as one comprising of a recombinant DNA molecule, containing a gene or genes, which encode a biocatalyst capable of desulfurizing a fossil fuel. In this case, the recombinant DNA molecule is the gene or genes of a strain of R. erythropolis. The patent also includes other genetic components and associated microorganisms, and their use in BDS. The patent includes not just the

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desulfurization genes and their application, but a variety of diverse molecular biology components including the recombinant DNA molecule and microorganisms containing it, its fragments or vectors therein, plasmids and ORF sequences. The protein sequences of different catalytic biomolecules, including ORFs were included in the North American patent (US5879914) [40]. The application of all possible biocatalytic systems for BDS was claimed in this patent, as well as in the other two US patents [37,39]. Fundamental constituents of biomolecules present in active microorganisms were deeply characterized, to associate them with the exhibit catalytic features. The BDS biocatalyst is defined as comprising one or more microorganisms (referred as CS+ or Dsz+) that functionally express one or more enzymes that direct, alone or in concert with each other remove sulfur from organosulfur compounds. Furthermore, the biocatalyst might be also one or more enzymes obtained from such microorganisms or a mixture of such microorganisms and enzymes. The GE process requires similar organisms that lack biocatalytic activity (CS- or Dsz-). Mutant CS- or Dsz- strains of R. rhodochrous were obtained by the action of mutagen agents over a CS+ or Dsz+ strain of R. rhodochrous, e.g., ATCC No. 53968, under appropriate conditions. Suitable mutagens include radiation and chemical mutagens. A DNA library was made up by fragments of various kilobase lengths taken from DNA extracted from a CS+ strain of R. rhodochrous. The various DNA fragments are inserted into several CS- mutant clones of R. rhodochrous and observing whether there is any transformation of a previously CS- mutant cell to a CS+ transformed cell is considered as evidence that the inserted DNA fragment encodes a biocatalyst. The next step was identification and isolation of DNA fragments encoding the biocatalyst. The same procedure can be applied to any active microorganism. DNA plasmid (Plasmid pTOXI-1 and Plasmid pTOXI-2) and vectors (four different nucleic acid sequences were included, namely SEQ ID No. 1 to 4; see original reference for details [38]) were constructed based on the identified and isolated fragments and constitute part of the claimed subject matter. The described expression of the Dsz+ trait in both a related and non-related heterologous host detailed in the patent, seems to indicate that pTOXI-1 (see patent document [38]) carries all of the genetic information required for conversion of DBT to 2-HBP. In general, any nuclear material capable of hybridizing to at least a portion of the DNA was included in the claims (three ORFs). The functions of the ORFs present within the pTOXI-1 sequence were tentatively assigned: ORF 3 was responsible for expressing an oxidase capable of conversion of DBT to DBT-sulfone. ORFs 1 and 2 appear to be responsible for conversion of DBT-sulfone to 2-HBP. However, it was indicated that neither ORF 1 nor ORF 2 alone was capable of converting DBT-sulfone. In summary, the GE improvements were based on the genomic information underlying the enzymes present in the R. rhodochrous bacteria (ATCC 53968). Some of the techniques developed were novel and were included in the patent by EBC2 [37–40]. These GE tools described in the document were depicted in Fig. 11 of Chapter 3. This description within the patent document might become somehow, part of the ‘know how’ property package of the assignee (EBC). In a way, describing the GE methodology indicates that they have the related knowledge; however, the GE tool was not included in the awarded claims indicating the lack of intellectual property rights over it. In the other hand, this public disclosure stops others from obtaining the property. Indeed, in 2001, Maxygen was allowed a patent [69] for a similar methodology (filed in June 1999)

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only after a long arbitration process with EBC. This patent covers recombination-based methods using single-stand templates to produce novel genes and proteins. Such methods include the RACHITT™ technology [70,71] that Enchira Biotechnology claimed to have developed. In the arbitration, Maxygen was determined to be the exclusive owner of the RACHITT™ technology, and Enchira was barred from using, licensing or otherwise commercializing the RACHITT™ technology until 2017. As mentioned above, EBC used a modified RACHITT method on chemostat mutants, in 2002, with no success on improving BDS of DBT, though new functionality was attained [72]. In an effort to conceptualize how a BDS unit could be integrated into the refining operations a patent for a ‘Process for the desulfurization and the desalting of a fossil fuel’ [41,42] was awarded in several countries (AU4115493, BR9306370, CN1079771, DE69312703D, EP0638114, B1, HK1001005, JP7506388T, MX9302528, RU2093543, and WO9322403). The basis of this integration is that both processes need water to be fed. The method was claimed comprising three steps: a. contacting a fossil fuel with: i. a sufficient amount of an aqueous solution capable of depleting the fossil fuel of forms of water soluble salt contaminants; and ii. an effective amount of a biocatalyst capable of depleting the fossil fuel of forms of sulfur-bearing organic molecules; b. incubating the above mixture whereby; i. the fossil fuel is significantly depleted of forms of water soluble salt contaminants; and ii. the biocatalytic agent selectively catalyzes carbon–sulfur bonds in sulfur-bearing organic molecules generating a significant amount of water-soluble inorganic sulfur molecules; both reactions occurring without depleting the fossil fuel of combustible organic molecules; and c. separating the aqueous component from the fossil fuel component, the fossil fuel now being significantly reduced in sulfur and salt contamination, and the aqueous component now being significantly enriched with inorganic salts and inorganic sulfur molecules. The selected biocatalyst is any of the already described alternatives based on R. rhodochrous bacteria ATCC No. 53968. Its concentration or proportion to the fossil fuel feedstock was neither reported nor claimed; only a slight comment is made on the proportion between the crude oil and the biocatalytic aqueous solution, which states that it will not exceed one half the total incubation volumes. In addition, an additional amount of water, enough for desalting, is simultaneously added with the biocatalytic solution. The process is carried out by feeding the crude oil and water into a CSTR reaction vessel and stirred until an emulsion is formed. The mixture is incubated under stirring at temperature and pressure conditions, for a period of time adequate for both to occur, desalting and desulfurization. The well-known refining practitioner knows that desalting is an operation commonly carried out at its very limits under extreme conditions. Yield improvements typically require multi-stage severe processes. A great deal of technologies are known and usually involved severe stirring, intense electrostatic fields, acid pH, high crude throughput, high temperatures (compare to those used in biocatalytic processes), long residence time

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under high voltage AC coalescing field, etc. Such a process looks enormously aggressive and harsh for a biocatalytic process to sustain. The inclusion of a sorption step prior to the desulfurization stage was considered in the application for a ‘Method for separating a sulfur compound from carbonaceous materials’ [43] (the equivalent patents are AU1261695 and WO9516762). This method involves contacting the fossil fuel with a biosorption agent, which binds sulfur compounds by a complexing action. The biosorption agent is said to be encountered in the same microorganisms, which show activity towards desulfurization. So, the biosorption agents might be either the same or a different biomaterial from the biocatalysts already described in the literature. Hence, enzyme preparations could also be useful, then in general, microbial lysates, extracts, fractions, subfractions, or purified products are examples for both biosorption and biocatalytic agents. While the biocatalyst is preferably used in solution, the biosorption agent would rather be immobilized. Recommended carriers for immobilization include non-viable microorganism, membranes, filters, polymeric resins, diatomaceous materials, glass particles or beads, ceramic particles or beads, etc. A characteristic feature of the biosorption step is that can be achieved in the absence of oxygen and of water. The biosorption agent and fossil fuel streams are contacted in counter-current mode. The biosorption agent could be recovered by first separating the oil–agent complex and subjecting it to BDS. If the biosorption agent carries BDS activity, then the oil–agent complex is simply introduced to a reaction medium, under BDS conditions. If the biosorption agent does not exhibit BDS activity, a biocatalyst capable of desulfurization has to be added. The reaction medium should contain the oxygen and water needed for the biocatalytic reaction to occur. That invention also relates to the preparation of the products of the oxidation reaction of organic sulfur compounds by a biocatalyst, such as 2-hydroxybiphenyl compounds. Although, BDN or BDM are not explicitly included in the claims, there is a mention that biosorption agents could be developed for such purposes. The use of emulsions, and more particularly microemulsions represents an attempt for sorting out the mass transfer limitations encountered by the fact of reacting an oily phase on a catalyst contained in an aqueous phase. However, when mass transfer problem is resolved by emulsifying the reaction media, a new separation challenge appears downstream. The emulsion post-treatment received much attention, since recovering the sulfur-free products call for a cost-effective solution. A counter-current liquid filtering method was developed for separating a petroleum containing emulsion [44] (also published as AU2398495, CN1150445, GB2302333, JP10500168T, and WO9531516). A multiple phase liquid target medium is contacted with two different filters comprising two liquid phases. The targeted multi-phase liquid medium might be composed by a fossil fuel, water, and a biocatalytic system. In that case, only two phases would be present, namely, the oil and the aqueous phases. The first filter is a solid material wetted by a wetting agent, which is miscible with one of the liquid phases but immiscible with the second liquid phase. When the targeted medium passes through the first filter, the wetting liquid phase agent will retain one of the phases, thus obtaining a filtrate substantially free of the second liquid phase. The retentate will then flow to the second filter which will collect the component not removed before, as the filtrate. The remaining retentate, containing the biocatalyst, can then, preferably, be recycled. The pore size of the solid material selected for the filters has to be appropriately designed to avoid diffusion limitations or excessive pressure drop through the system. A capillary cross-flow

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membrane of sintered metal with pore sizes in the range of about 02–1 m and a porosity of 30% by volume was recommended. The wetting agent for retaining the oil phase might be chosen from the treated liquid fossil fuel itself, an aliphatic or aromatic hydrocarbon, synthetic oils, tall oils, vegetable oils, modified vegetable oils, liquid animal fats or modified liquid animal fats, non-polar solvents immiscible with water (e.g., ethers, carbon tetrachloride, alkyl esters, etc.). On the other hand, a hydrophilic polar solvent (such as water, alcohol, or dimethylformamide) was suggested for retaining the aqueous phase. The order or a filtering sequence was not specifically mentioned. The process can be used to resolve an emulsion or microemulsion of the liquid fossil fuel and aqueous phase resulting from a BDS process. The inventors suggested this method might be more effective and efficient resolving such BDS emulsions or microemulsions, than that accomplished by conventional equipment, such as conventional separators, coalescors, or electrical precipitators. Also described in that patent is a method for controlling the reaction parameters of a mixed phase reaction process. Measuring involves passing the separated phases, either the aqueous or the oil phase, through a corresponding analyzer. Properties and parameters such as oxygen content, chemical quantitative analysis (sulfur content, pH, for instance) could be monitored. The signals from the analyzer can be fed back to a given instrument, equipment or mechanical device to control the specific parameters within the reaction medium. The advanced results, obtained by employing MB and GE methods, set a different platform, in this case, incorporation of biomolecular descriptions of the biocatalyst, such as shown in the patents that follow. An invention regarding the use of flavoprotein as an enhancer of BDS performance [45–47] was filed in the USPTO in December 1994 [46,47] and in the WIPO one year later [45]. While the World Patent was issued in 1996, it was divided in two in US and issued in 1998 and 1999. The patent family includes AU4375596, AU693633, CA2206987, CN1169159, EP0797668, and JP10510423T. The claims allowed by WIPO focused more on the BDS method than the biocatalyst, like those included in the 1999 US issued patent. Nevertheless, the 1998 US issued patent [45] granted intellectual property rights to EBC on catalytic biomolecules and catalytic precursor’s biomolecules. Thus, a patent covering isolated and purified DNA molecules was awarded to EBC. The main claim describes these molecules as ‘comprising a DNA which encodes a flavoprotein and DNA of Rhodococcal origin which encodes a protein biocatalyst capable of catalyzing the selective cleavage of carbon– sulfur bonds in organosulfur compounds’ (providing such DNA is derived from an BDS-active microorganism, such as Rhodococcus genome). This patent introduces a new EBC concept for the biocatalyst developmental strategy. At this stage, a move toward an enzyme biocatalyst definition seemed to emerge. So, living microorganisms biocatalysts are put aside this time, instead biocatalytic enzyme preparations were targeted. By enzyme preparations, the inventors mean to include microbial lysates, extracts, fractions, subfractions, or purified products obtained by conventional means. Several patents filed before 1992, include such preparations [37–40,68,73]. The immobilization of the enzyme biocatalyst on a solid carrier was also recommended. The source of flavoproteins might be the nature directly, but also they might be obtained commercially or can be made recombinantly. Flavins, such as flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) and Flavoproteins, as flavin reductase or FMN reductase can be used. The Flavoprotein can be over expressed in a genetically engineered microorganism. The fragments encoding the flavoproteins were identified and isolated by the methodology

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described previously [38]. Those fragments were inserted in a plasmid, so as to obtain a recombinant DNA molecule of the present invention including a gene encoding a flavoprotein not originally present in the Rhodococcus genome chain. The plasmids which included a replication region and were recombination compatible (i.e., capable of having foreign or exogenous DNA inserted into it and, capable of expressing the product of the foreign or exogenous DNA subsequently after transformation into another host) were constructed using foreign flavoprotein DNA exhibiting higher activity. The characterized structure of the constructed plasmids is showed in the patent document [38]. The discovery that the rate of reaction of the desulfurization of fossil fuels is enhanced by the addition of a flavoprotein to the biocatalyst was then claimed in the other two family patents. So, the patents are related to the use of a flavoprotein, particularly FMN reductase, in addition to the biocatalytic material for increasing the rate of desulfurization. In the World patent, ten more claims were allowed, compared to the US issued patent. The excess claims include a set of dependant claims in which the microorganism containing the recombinant DNA molecule is considered. However, in the invention a two-step process is stated, it is just the contact between the fossil fuel with an aqueous phase containing a biocatalyst and a rate-enhancing amount of a flavoprotein. There is no indication whatsoever on how much that amount could be. Along the same ideas, another patent family, ‘DszD utilization in desulfurization of DBT by Rhodococcus sp. IGTS8’ [48–50] was filed. The invention refers to a DNA fragment encoding a group III alcohol dehydrogenase claimed to enhance fossil fuel desulfurization when use along with a biocatalyst and a nicotinamide adenine dinucleotidedependent co-factor. The AU4375496, AU698977, CN1198187, and EP0851928 patents complete the coverage of this patent family composed of three other patents, including a World patent [49] and two US patents [48,50]. In the World patent, 39 claims were awarded including both, the biocatalytic system and the method of use. The claims in the US issued patents are essentially the same as those in WIPO patent, although it is four claims short. The biocatalytic system is based on the same Rhodococcus sp. ATCC 53968 or any previously awarded derivatives. This time the inventors discovered that the rate of reaction of the desulfurization of fossil fuels was enhanced by the addition of an oxidoreductase to the biocatalytic reacting mixture. The claimed enzyme is a type III alcohol dehydrogenase (N N -dimethyl-4-nitrosoaniline-dependent alcohol oxidoreductase) and the patented subject matter also includes the addition of the co-factor additives such as NADH or NADPH. The critical finding on which this patent is based is that neither the DszC nor DszA enzymes are catalytically active when purified to homogeneity, but are activated upon the addition of DszD protein. It was suggested that the function of this protein is to couple the oxidation of NADH with the oxidation of the substrate molecule, which has been proven now. The detailed sequence of the DszD gene is included in the patent document. Regarding the biomolecular description of the improved catalysts, it seems to have followed a MB and GE strategy, in which the detailed sequence was the objective of the intellectual property. Thus, the patent family entitled ‘Rhodococcus flavin reductase for complementing DszA and DszC activity’ describes the detailed sequencing of a series of nucleotides, which encode the NADH:FMN oxidoreductase, DszA, DszB, and DszC. The family was awarded with a WIPO patent [54], which was divided in three equivalent patents in USA [55–57] (the family also contains the Australian Patent No. AU4990097). The WIPO patent included 44 claims, while the US patents as a whole only have

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37 claims, with the subject matter being essentially the same. The catalytic biomolecules considered here include a series of nucleotides, which encode the NADH:FMN oxidoreductase, DszA, DszB, and DszC individually or all together; the plasmid comprising the nucleotides, the recombinant DNA molecule encoding the claimed sequences, the recombinant microorganisms containing the claimed DNA molecules, the transformed microorganism including any of the claimed biomolecules, the corresponding proteins and enzymes. The BDS usage method, which includes any biocatalytic material or combinations of the above catalytic biomolecules, was also claimed. The focus of these patents consists in a molecular description of the novel flavin reductase, which was isolated from Rhodococcus strain sp. IGTS8. The flavin reductase is a naturally occurring homologue (containing at least 50% of the reported sequence) isolated from a source other than IGTS8 and also a non-naturally occurring homologue obtained by modification (deletion, insertion, or substitution of amino acid residues in the amino acid sequence) of the amino acid sequence of the native enzyme. The flavin reductase, characterized by a molecular weight of 25 kDa, is considered to be particularly useful for biocatalytic desulfurization of fossil fuels. The DNA sequence and the corresponding amino acid sequence of the flavin reductase gene are given in the patent document (see SEQ ID No. 1 and 2 of Fig. 1 in Ref. [54–57], respectively). The role of NADH:FMN oxidoreductase in activating the two main monooxygenases, DszA and DszC, was incorporated in these patents. The details of the reaction mechanisms and enzymatic action are given in Section 2.2.8 of Chapter 3. The method is described in three steps, namely contacting, incubation, and separation. As in the previously described similar inventions, the additive is added in a ‘rate-enhancing amount’, but no value or range is given. Although, it is well-known that the refiner could adjust the relative proportions of biocatalyst, additives and feedstock to suit particular conditions, or to produce a particular level of desulfurization, usually the PTOs require from the inventors, to indicate or estimate at least, certain values or ranges. An invention not related to the desulfurization effort, namely, ‘Process for demetallizing a fossil fuel’ ([51,52], also published as AU5968296, CA2221377, JP11506490T, and WO9638517), was awarded to EBC2. This may have been an isolated effort, since there is no other evidence of continuing work in this area. The invention relates to a degradation method for removing metals from fossil fuels. Similar to the previously described BDS methods, this is merely the use of a biocatalyst in a given application. The biocatalyst consists of an oxygenase enzyme which degrades porphyrin molecules. This degrading metabolic pathway leads to the removal of metals present in the fuel. The preferred oxygenase includes heme oxygenase and cytochrome C reductase, which can be employed as a substantially cell-free preparation or a whole cell preparation. The capabilities of that enzyme for demetallizing crude oil was exemplified in the document [51] and although claimed as a significant reduction of the nickel and vanadium content, the reported values were only 9.7% and 21.1% removal, respectively. In this case, the second patent [52] was awarded for the fuel obtained from the actual demetallization method. This second patent document is exactly the same as the first one and do not include any further description of such a fuel or how the BDM method makes it different of any other equivalent fuel. Research on the development of separation methods for the multiphase reacting system led to the development of a three phase (oil, water, and a solid) separation process. A patent was awarded in 1998 in US [53] and subsequently in some other countries

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(AU2993697, AU716922, CA2253213, CN1216478, EP0900113, JP2000509325T, and WO9740903). The chosen apparatus for this separation was capable of handling particles with a size of 50 m or less. The solid can be a biocatalyst, such as an intact bacterial, fungal, or yeast cell. The organic phase is a liquid material substantially comprising carbon, which is substantially immiscible with water, such as petroleum. The inventors specifically stated that the considered emulsion might be the effluent of a BDS reactor. The apparatus of the present invention comprised a series of (two or more) hydrocyclones. The document exemplified the case of two hydrocyclones, in which the intake tube of the first hydrocyclone is connected by a line to the emulsion source (the BDS reactor); and a means for inverting the phase of the emulsion, interposed before the second hydrocyclone. As it is claimed, the method for separating an original emulsion containing a first continuous phase, a first discontinuous phase, and fine solid particles comprised four steps: a. directing the original emulsion through a first hydrocyclone, in which the first emulsion is separated into an overflow emulsion and an underflow emulsion. The overflow emulsion (the phase that emerges from the output tube at the top or the wide end of a hydrocyclone) contains a portion of the continuous phase of the original emulsion, another portion the discontinuous phase, and another one of the fine solid particles; b. inverting the phases of said overflow emulsion so as the continuous phase of the overflow emulsion becomes a discontinuous phase in the inverted emulsion and the discontinuous phase of the overflow emulsion becomes the continuous phase in the inverted emulsion; c. directing the inverted emulsion through one or more subsequent hydrocyclones arranged in series; and. d. collecting the continuous phase and the discontinuous phase of the inverted emulsion, the fine solid particles are collected within this discontinuous phase of the emulsion. Prior to step (b), the overflow emulsion might be pressurized, to at least 120 psig. The means for inverting the emulsion was not specified but any suitable method employing agitation, etc., may be used. The first hydrocyclone is considered to be a de-oiling hydrocyclone, so, the overflow emulsion is an oil-enriched emulsion. The inverting stirred tank is operated in a pressure range of about 400–600 psig to invert the oil-enriched emulsion from water-continuous to oil-continuous. After inversion, a dehydrating hydrocyclone series, with a minimum of three hydrocyclones were recommended. All the oil-separated phases are collected in a pool, which is subjected to electrostatic precipitation to recover substantially pure treated oil. Up to this stage, EBC seemed to have identified, characterized, and isolated a series of biomolecules, all of them playing very important roles in the BDS reaction mechanism. Furthermore, relevant microorganisms were discovered and genetically engineered. Still, certain catalytic parameters (probably activity, selectivity, functionality, or stability) were lacking the desired values. Therefore, the development strategy had to be oriented to find better biocatalysts or improving what they had until that moment. One approach was to use Pseudomonas hosts to express the Dsz genes [58] (also published as US5952208 and AU6950698). The nucleotide sequences of the Rhodococcus desulfurization genes

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and the predicted amino acid sequences of the proteins encoded by these nucleotide sequences were included in this document. Also a correction for the possibility that amino acid 56 has been misidentified as an alanine versus a glycine, in SEQ ID No. 6, (and, thus, the corresponding codon) was stated. The SEQ ID No. 6 corresponds to the protein encoded by the nucleotide ORF-3(R). The selected Pseudomonas microorganisms have to be resistant to the reacting media and conditions encountered in a BDS reaction, such as compounds present in petroleum, high salt concentrations, and elevated temperatures. The GE procedures and materials are given in the patent document; the reader is referred to the original document for additional details. In line with their typical protection strategy, the patented material comprises a method for use of the biocatalyst. The three typical steps of contacting, incubating, and separation were included. The earlier kinetic studies showed that the first two steps of the 4S pathway (oxidation of DBT and its analogs to DBTS and subsequently to HPBS) were the faster (see Chapter 3 and references thereon). The developmental stages demonstrated that they could be improved by inclusion or overexpression of the NADH:FMN oxidoreductase. However, the rate limitation of the pathway, the actual cleavage of the last C−S bond (third step) was difficult to overcome. They dedicated significant time and effort to try to improve it, without much fruit. Measuring the advance of the reaction by the disappearance of a given signal obtained from the reacting molecule, certainly indicates a transformation; however, for a multiple step reaction, the only definite means is to follow the appearance of a signal due to the desired product. In the case of BDS, the final response is given by the sulfur concentration prior and after the reaction had taken place. As a consequence, it seems EBC2 followed two divergent developmental strategies. One strategy considered using other means (biological or not) to enhance the rate of reaction for the C−S bond cleavage (we name this as Strategy S1). The other was to find new cost-effective applications of the intermediate products to add value without realizing complete desulfurization (named Strategy S2). In the following discussion, we will refer to S1 and S2, regarding these two developmental strategies, respectively. So, in S1 strategy, the removal of sulfinic acids [59] was carried out by using Lewis acids. Although, the patent is related to generic organosulfinate compounds, it actually concerns the HPBS removal. Therefore, the application of S1 consists here in the incorporation of a chemical reaction for the substitution of the biocatalytic last step. This chemical reaction involves the substitution of the sulfinate group by a hydrogen atom. The referred sulfinic acid is the acid form of an organosulfinate compound, which consists of an organic compound comprising the protonated functional group –S(O)OH. The basic form, the deprotonated group –SOO− is usually accompanied by a cation in the desulfurization reaction medium. This method was described as a three step process: a. contacting the feedstock with a biocatalytic aqueous solution for the conversion of the organosulfur compound to organosulfinate compounds, which probably would remain in the oil phase; b. maintaining the mixture of step (a) under conditions sufficient for conversion of the organosulfur compound to an organosulfinate compound; and c. contacting the organosulfinate compound with an effective amount of a copper(II) compound or a soft Lewis acid in the presence of a protic solvent, thereby desulfinating the organosulfinate compound and producing a carbonaceous material having a reduced sulfur content.

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The amount of Lewis acid to be used is depicted as ‘an effective amount’ and a minimum limit of 0.5 mole equivalent with respect to the sulfinated compound concentration was mentioned. A wide variety of Lewis acids was mentioned to be useful for the present invention in the patent document, but only copper (II) compounds were claimed. The way in which the Lewis acid is used (either as a homogeneous or a heterogeneous phase), was reported to be irrelevant. So, it could be employed in solution in the reaction medium or insoluble as powders or on a solid support, such as alumina or a zeolite. The Lewis acid is supposed to be acting as a catalyst in the desulfination process. The temperature and pressure conditions for this reaction are substantially higher than the microbial conditions. The temperature and pressure conditions did not form part of any claim, but the document stipulates values between 50 C and 100 C, and 10 and 15 psi, respectively. The quantitative effectiveness or conversion values of this reaction were not given, but it looks like it would diminish the advantages of a biocatalytic process. The approach using the S2 strategy of finding value-added products from HPBS led to a patent on surfactants. A surfactant derived from 2-(2-hydroxyphenyl)-benzenesulfinate and alkyl-substituted derivatives was patented in 1999 ([60,61], also published as AU3010799 A). The discovery is based on the feature that the organosulfur intermediate compounds produced in petroleum biodesulfurization exhibit surfactant properties. The invention generally described the intermediates as acyloxybiphenylsulfinates, acyloxybiphenylsulfonates, alkyl sulfinatobiphenyl ethers, and alkyl sulfonatobiphenyl ethers. The sulfinates defined in the previous referred patent were considered as starting materials in this one. Besides, compounds such as sulfonates and sulfonic acids, comprising the de-protonated functional group –SO2 O− or the protonated –SO2 OH, respectively, were also considered. The claimed method defined a surfactant preparation method for which the organosulfur compounds from the incomplete BDS reaction medium are converted to molecules with amphiphilic characteristics. Such a molecule is obtained by the reaction of the starting compounds with a carboxylic acid, under acylation conditions to yield a product generally defined as an acyloxybiphenylsulfonate compounds. Another reaction considered was the sulfonylation of a phenolic hydroxyl group, for preparing sulfonoxybiphenylsulfinate and sulfonoxybiphenylsulfonate compounds. Similarly, alkyl sulfonatobiphenyl ether could be produced by alkylating a hydroxybiphenylsulfonate at the hydroxyl oxygen atom. Alternately, other oxidation reactions, apart from the microbial desulfurization, were mentioned as a means for producing or modifying the starting molecules. In the patent entitled ‘Conversion of organosulfur compounds to oxyorganosulfur compounds’ [62] (also awarded as WO9914291), the S2 strategy implies the possibility of stopping the 4S reaction pathway at an intermediate stage and extracting these formed compounds from the reaction medium. It appeared that a case was being made for not pursuing complete desulfurization, confirming the inability of improving rates for the last rate limiting step. The present invention deals with the removal of those intermediate metabolites by exploiting their physical and chemical properties. The removal is simply the separation of the oxyorganosulfur compound from the biocatalytic reacting fossil fuel medium. Separation is perform by physical or chemical processes, such as extraction (with a polar compound), precipitation, adsorption (on a polar solid), or reaction with a suitable agent to form a complex compound, which can be easier to separate from the fossil fuel. It is also suggested that the isolated oxyorganosulfur compound could

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be further processed via conventional desulfurization. The microbial conversion could therefore, by this approach, be regarded as a pretreatment for desulfurization. From this turn in the development, one might thus conclude that the biological conversion of sulfur compounds cannot be concreted into an effective BDS process and its viability rests on whether improvements can be made in the last step of the 4S pathway. In an attempt to compensate for the lack of activity, the addition of nutrients to the aqueous solution was considered as an alternative to improve desulfurization. The method included addition of sufficient nutrients for growth and/or reproduction of the microorganism within the reaction medium, during the contacting stage [63]. Applicability of this nutrient supplementation method to almost any biodesulfurization process, including aerobic and anaerobic pathways has been included in the patent. The method is also recommended for the partial oxidative conversion of sulfur compounds present in fossil fuels, probably recognizing the idea that complete desulfurization does not occur in a measurable way. Actually, the inventors explicitly inferred that previous desulfurization methods were carried out under conditions which compromised biocatalyst viability requiring continuous addition of fresh biocatalyst to the system, to maintain satisfactory activity. In this new method, it is the nutrients that should be continuously incorporated to the reacting system. Nutrients would comprise assimilable sources of carbon, nitrogen, phosphorus, potassium, magnesium, calcium, iron, and sodium, as well as trace elements such as cobalt, zinc, molybdenum, copper, manganese, nickel, tungsten, and selenium. The amount of each of the nutrients added should take into account the nutritional requirements of the microorganism used as biocatalyst. Living cells, which might incorporate the advances in GE and the discovered catalytic biomolecules, were also included under the embodiment of the present invention. The full characterization attained on the active genomic material conferred to this company the possibility of protecting specifically described biomolecules with catalytic capabilities. Such is the case, in which biodesulfurization catalysts were derived from a novel Sphingomonas microorganism [64], the designated Sphingomonas sp. strain AD109. The invention includes isolated proteins and nucleic acid sequences obtained from this microorganism. In fact, nucleotides encoding specifically sequenced enzymes and the corresponding enzyme preparations were claimed. In addition, plasmids and derived microorganisms were included in the protected subject matter. The associated sequences are given in the referred patent document (and in its equivalent World Patent WO9845446). In this invention, the enrichment procedure used 2-(2-phenyl)benzenesulfinate (HPBS) as the sole sulfur source, probably as a way of developing the poorest activity of previous catalysts for the last step of the 4S biocatalytic desulfurization. That reaction step consists of the conversion of HPBS into 2-hydroxybiphenyl (2-HBP). For this reason, the biologically pure isolated Sphingomonas sp. strain AD109 was designated as ATCC Deposit No. 55954. The microorganism was originally obtained from soil samples, at sites contaminated with petroleum and petroleum by-products. Its capabilities for expressing a collection of enzymes which all together catalyze the conversion of DBT to 2-HBP and inorganic sulfur were attained by cloning the nucleic acid sequence (as it was previously identified in Rhodococcus sp. IGTS8) required for this overall process, by the general method described in Ref. [38]. The nucleic acid sequence (‘Sphingomonas dsz sequence’) was depicted as comprising three open reading frames, designated as ORF-1 (base pairs 442–1800), ORF-2 (base pairs 1800–2909), and ORF-3 (base pairs 2906–4141) and were identified in the patent

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document as SEQ ID NO.: 1, SEQ ID NO.: 3, and SEQ ID NO.: 5, respectively. Thus, the core of this patent is the isolated nucleic acid molecule, which has been characterized as comprising one or more nucleotide sequences which encode one or more of the biodesulfurization enzymes of Sphingomonas sp. strain AD109. The set of 46 claims embraces biological material derived from the manipulation of Sphingomonas sp. strain AD109, in which the whole nucleic acid biomolecule with the three ORFs, but also biomolecules including only one or two of the ORFs. In the same way, other biomolecules were incorporated in the proprietary subject matter, namely, nucleic acid molecules for hybridization probe uses, or chemically synthesized, or as a recombinant DNA molecule; plasmids or vectors comprising the recombinant DNA sequence, or fragment containing the claimed genes or nucleotide sequence; isolated enzymes with the claimed sequence and homologous enzymes from desulfurizing microorganisms or non-naturally occurring or chemically synthesized. The regulatory or promoter sequences were derived from the native Sphingomonas operon containing the considered genes. The proprietary oxidoreductase co-catalysts, such as flavoprotein or flavin reductase were mentioned within the text but not included in the claims. The fossil fuel desulfurization biocatalysts were limited to the defined term of ‘Sphingomonas-derived biocatalyst’ which includes one or more of the enzymes encoded by the SEQ ID NO.: 1, SEQ ID NO.: 3, and SEQ ID NO.: 5; or their derived mutants. Furthermore and in line of previous Rhodococcus findings, the role and activity of each of the enzymes were explained as: • The DszC enzyme encoded by the nucleotide sequence of ORF-3 catalyzes the oxidation of dibenzothiophene to dibenzothiophene-5,5-dioxide (dibenzothiophene sulfone), • The DszA enzyme encoded by the nucleotide sequence of ORF-1 catalyzes the oxidation of dibenzothiophene-5,5-dioxide to HPBS • The DszB enzyme encoded by the nucleotide sequence of ORF-2 catalyzes the conversion of HPBS to 2-hydroxybiphenyl and inorganic sulfur. In the likelihood of processing gasoline or widen up the functionality of the developed biocatalysts a patent for thiophene biotransformation employing the genes from N. asteroides KGB1 [65] was issued in 2001. The same MB patentability approach followed in the closely awarded patents was pursued for this biological material in the awarded patents (including the AU2779399 and WO9943826 equivalents). In this case, it seems that only one enzyme was isolated and characterized (referred as SEQ ID NO.: 2 in the patent document), also the nucleic acid molecule encoding this enzyme was sequenced (referred as SEQ ID NO.: 1 in the patent document). Therefore, the claimed matter includes the enzyme, the nucleic acid molecule, the nucleotides sequence corresponding to relevant parts of the DNA molecule, and plasmids containing the DNA sequence linked to a promoter and the transformed microorganism with the recombinant DNA plasmid comprising the claimed DNA molecule. The isolation of a microorganism, which uses thiophene and substituted thiophenes as a sole source of sulfur was accomplished by the enrichment method using benzothiophene as the sole sulfur source. The resulted N. asteroides strain KGB1 was obtained after cloning of genes from the original organism. The isolated and characterized genes encode one or more enzymes which catalyze the desulfurization of thiophene and substituted thiophenes. The gene

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expressing thiophene desulfurization activity was confined to a 2.4 kb PstI fragment. This nucleotide sequence contains an ORF, which encodes a 455 aminoacid protein with an estimated molecular weight of 51,800. A sample of this microorganism was deposited with the American Type Culture Collection and assigned deposit No. ATCC 202089.

13.3. Enchira Biotechnology Corporation (EBC3) The EBC development ended with a final patent awarded to the third Company (EBC3). This last patent can be envisioned as part of the so-named S2 strategy, as well as in US Patent 5973195 [60]. The search consists in adding value by seeking applications of the HPBS, the chemical transformation and uses of the HPBS. Enchira may have thought in the S2 strategy as a mean for somehow improving the economics of a BDS process, as well as integrating two approaches into a single objective. The core compound of this invention [66] is the sulfinate molecule represented with the formula shown in Fig. 1. The referred compound is useful as hydrotropes and starting material for chemicals manufacturing (surfactants have been mentioned in the above referenced patent, but it is also useful for the preparation of polymers and resins, solvents, detergents, adhesives, and biocides). So, certain compositions comprising 2-(2-hydroxyphenyl)benzene-sulfinate and its alkyl-substituted derivatives were considered as protected matter in that last awarded patent [66]. The related compounds, which can be directly or indirectly synthesized from HPBS, include alkylated 2-(2-hydroxyphenyl) benzenesulfinic acid, 2-(2-hydroxyphenyl)-benzenesulfonic acid compounds and compositions which consist essentially of 2-(2-hydroxyphenyl) benzenesulfinic acid, 2-(2-hydroxyphenyl)benzenesulfonic acid and/or their substituted derivatives. Different transformation mechanisms involve one or more of the R-substituents. Some of the included reactions were: alkylations with an alkene or substituted alkene and any other substitutions leading to converting any R-substituent for an alkyl, aryl, or arylalkyl group, a halogen atom, an amino group or a cyano group. It is clear that these chemical transformations are carried out in the absence of biological catalysts, but in presence of organic solvents and inorganic acids. Although, the reactions considered are not biocatalytic, they are intended to support the BDS process and improve its economics. Indeed, it is explicitly stated that the disclosed compositions are preferably derived from a petroleum biodesulfurization process (most of their own patents are included in the list of recommended BDS processes). The water-soluble HPBS (and its substituted

R4

R3

HO

R5

R2

R6 R1

O R8

S HO

R7

O

Figure 1. Sulfinate molecule.

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derivatives) accumulate in the aqueous phase when fossil fuel is subjected to BDS. These compounds have to be isolated and purified for the application of the stated invention.

14. EXXON RESEARCH AND ENGINEERING (ER&E) CO (UNITED STATES) ER&E, the S&T organization of the World largest oil company, ExxonMobil, was not very prolific in R&D activities related to biocatalytic refining, but was focused on addressing a few key problems identified in BDS. They began in early 1990, with works on gas treatment; however, the developments took an oil twist towards the end of the decade when they became involved with heavier hydrocarbon feedstocks. This company possesses inventions, which are protected with six patents: • Biological process for conversion of hydrogen sulfide [74]. • Microbial desulfurization of organic compounds [75]. • Rhodococcus species for removing sulfur from organic carbonaceous fuel substrates (LAW295) [76]. • Solvent-resistant microorganisms [77]. • Method for the removal of organic sulfur from carbonaceous materials [78]. • Biological activation of aromatics for chemical processing and/or upgrading of aromatic compounds, petroleum, coal, resids, bitumen and other petrochemical streams [79]. Information regarding their first Canadian patent could not be found; however, inventors and priority numbers coincided for another most likely equivalent patents. ER&E focused their effort on identification of microorganisms capable of desulfurization of substituted DBTs and genetically engineering of the microorganisms to improve their activity towards substituted-DBT. The goal was to broaden the substrate specificity to enable desulfurization of HDS-recalcitrant substrates. Addressing the reactivity towards substituted-DBTs helps in developing BDS as a finishing stage for obtaining lower sulfur diesel. ER&E also investigated organic tolerance thus improving processability characteristics of the microorganisms. Improving organic tolerance will decrease mass transfer limitations and minimize the water to be used. Additionally, different reactor types and configurations, as well as immobilization and membrane designs were investigated to address process improvements. The first patent awarded to ER&E was a Canadian patent titled: ‘Biological Process for the Conversion of Hydrogen Sulfide’ [74]. It described a microbiological method for desulfurizing gases (e.g., natural gas). Chemoautotrophic bacteria of the Thiobacillus genus (T. thioxidans) were used to remove sulfides from gases. The main sulfur contaminant in natural gas is H2 S, which is oxidized by the bacteria to produce sulfuric acid. Thus, these microorganisms are aerobic and tolerant of low pH, with optimum pH between 2.0 and 2.5. The process was described to take place in a column, by contacting the gas counter-currently with an aqueous biocatalytic medium, resulting in oxidation of hydrogen sulfide to sulfur or sulfate. The gas flow is previously oxygenated before feeding it to the biotreating column. Part of the gas is recycled to increase efficiency and multiple contactors are used to achieve continuity. The bacterial concentration used

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is in a range from 108 to 1010 cells/ml. Conversion values of up to 0.38 mmole/h were reported. In the case of petroleum desulfurization, the microorganism search criteria focused on identifying organisms capable of sulfur-specific cleavage and avoid C−C bond cleavage leading to two different strains that were disclosed, ATCC 55309 and ATCC 55310 [75,76]. These were identified as Rhodococcus species differing slightly in their morphology and further characterized as non-spore forming Gram-positive, irregular rodshaped bacteria. The strains were activated by employing a growth medium containing 4,6-diethyldibenzothiophene (DEDBT) as the sole source of sulfur. The biocatalysts were active towards sterically hindered sulfur compounds, particularly those with restricted reactivity under hydrotreatment conditions. The desulfurization mechanism seems to be different than the traditional 4S pathway, since monohydroxylated products (with no-sulfur) are yielded from the sulfur source compounds. For instance, 2-hydroxy-3,3diethyl biphenyl is produced from DEDBT. The same chemistry takes place with other sulfur-hindered compounds when treated with the referred biocatalysts. The claims state that the BDS process can be carried out using whole cells, or prepared cell fractions containing the desulfurization enzymes or the isolated enzymes themselves. Five different configurations were described, in which the system operates in a batch, semi-batch, or continuous mode: 1. A slurry bioreactor with an aqueous biocatalytic solution, containing the mineral nutrients and an assimilable source of carbon. 2. A slurry bioreactor where carbonaceous feedstock is directly contacted with the biocatalyst. 3. A fixed bed or slurry bioreactor incorporates the biocatalyst immobilized on a solid support in an aqueous solution, mineral nutrients and an assimilable source of carbon. 4. As in configuration (3) but essentially free of non-added water. 5. A membrane bioreactor in which the aqueous biocatalyst solution, with mineral nutrients and an assimilable source of carbon is separated from the feedstock by a membrane which provides the active contact surface for desulfurization. Although, soil microorganisms develop tolerance to organics and even catabolize them, the physical nature of that resistance remains unknown. The most accepted reason for the sensitivity to organic solvents of most bacterial species is the solvents partitioning into the membrane and consequently producing a proton leak, which leads to uncoupling of oxidative phosphorylation [80]. ER&E in one of their development strategies involved the improvements of the microorganisms to make them resistant to non-aqueous solvents. Furthermore, those organisms are intended to be able to grow and/or carry out hydrocarbon transformations in aqueous/non-aqueous systems. That is the case of some solvent-resistant microorganisms quoted in Ref. [77]. The mechanism of solvent resistance involves enzymatic reduction of hydroperoxides. Thus, that invention includes a gene encoding the hydroperoxide reductase enzyme, responsible for the organic solvent resistance of the host microorganisms. Following the teachings of this invention, a tetralin-resistant mutant E. Coli KLE400 was obtained by growing a wild type E. coli K12 (strain CAG18492) to late-logarithmic state in LB medium, concentrated 10-fold. The broadness of the conferred resistance to solvents was confirmed using an overlay

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assay. This assay consisted in plating approximately 109 cells on a medium A plate, overlaid with 20 ml of tetralin, and sealed and incubated at 37 C. The mutant resistance to cyclohexane, propylbenzene, and 1,2-dihydronaphthalene was confirmed. DNA from strain KLE400 was isolated, digested with Sau3A1 to process and render purified fragments of 4–6 kB, which were then linked into the plasmid vector pACYC184. The created library was transformed into E. coli DH5  cells and plated on LB medium with chloramphenicol to select for transformants. Among nearly 2,200 colonies tested, the two resistant clones were used to obtain the recombinant plasmid, pAF1. Deletion mapping and the sequencing led to the identification of ahpC gene and ahpCF operon encoding alkylhydroperoxide reductase (AHPR), an enzyme that detoxifies organic hydroperoxides. The AhpC protein is a small subunit (21 kDa) containing the catalytic site, while an AhpF protein, also identified, is the NAD(P)H dehydrogenase. Therefore, the invention includes the operon which includes the mutant gene, the ahpF gene which encodes an NAD(P)H dehydrogenase, a plasmid vehicle, and a host microorganism containing these genes. Furthermore, cloning the genes encoding for solvent-resistance into other organisms rendering them solvent-resistant was claimed as well. Alternatively, the genes encoding for a specific organic or hydrocarbon transformation, (such as BDS) are placed into the solvent-resistant microorganism. Other reactions of interest suitable for applications of the solvent-resistant strains (in addition to BDS) include aromatics oxidation, and xylene tolerance. Following the development of the solvent resistant strains, ER&E developed a sulfur removal method [78]. The steps in the desulfurization comprised of biocatalytic oxidation of the sulfur species to the sulfone and/or sulfoxide form, and a hydride transfer to a reducing agent from the sulfone and/or the sulfoxide in an aqueous media. The reducing step is a chemical reaction involving sodium formate as reduction agent. Meanwhile, the oxidizing agent was Rhodococcus species ATCC 55309/or 55310. This is another incidence where alternatives to employment of the desulfinase enzyme for biological sulfur removal have been investigated. Therefore, the context of this patent seems to indicate that biocatalysis is probably not effective enough for sulfur removal, therefore eliciting the need for alternate methods to achieve complete sulfur removal. The oxidative activity of the biocatalyst was enhanced via genetic manipulations. Actually, the gene(s) encoding the enzyme(s) responsible for the selective oxidation of sulfur were cloned, from the organism originally possessing these genes, into a recipient E. coli under the control of a genetic promoter, which allows production of the enzyme(s) at some desired levels. Several options are also included for using the responsible enzymes directly, namely: i. as free enzymes, ii. organic-soluble enzymes obtained by modification with chemical groups (e.g., fatty acids), iii. immobilized enzymes (e.g., in gels such as polyacrylamide, agarose, and alginate; or on solid support such as diatomaceous earth, glass beads, or ion-exchange resins). The oxidation reaction is carried out by a simple contact between the feed and the biocatalyst, followed by separation prior to the aqueous chemical desulfurization reaction, which details can be seen in Ref. [78].

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Following the same orientation and to facilitate the processing of petrochemical and other heavy hydrocarbon streams containing aromatic compounds, biological hydroxylation was used prior to hydrogenation and/or hydrogenolysis. Therefore, the activation of aromatic rings and their subsequent reactions result in cracked and ring-opened products. An invention provides a process for the biological activation of aromatics and the subsequent chemical processing and/or upgrading of the hydroxylated aromatic compounds, which can be applied to petroleum, coal, resids, bitumen, and other petrochemical streams [79]. The specific application for which this invention was developed was not explicitly stated; however, one may think of its usefulness for cetane improving of the diesel cut. The biological process was just described as a contacting between the organic material with a microorganism or enzymes. The target organics not only include single and/or multi-ring aromatic compounds and alkylaromatic compounds but also their heteroatom-containing analogs, crude oil, petroleum, petrochemical streams, coals, shales, coal liquids, shale oils, heavy oils, and bitumens. The biocatalytic reaction consists of an aromatic dihydroxylation by the action of a dioxygenase enzyme. The cis-dihydroxy non-aromatic compound produced in this reaction is subsequently converted to a 1,2dihydroxyaromatic in another enzymatic step catalyzed by a dehydrogenase enzyme.

15. GAS RESEARCH INSTITUTE (UNITED STATES) The Gas Research Institute (GRI) is an organization located in Chicago, USA, which was established in 1976, to develop gas technologies by carrying out co-operative research programs. Its mission was defined as ‘the conduction of research, development, and demonstration (RD&D) programs that benefit the entire gas industry and its customers’. It was largely funded by a consumer surcharge, approved by the Federal Energy Regulatory Commission (FERC), and since consumers were paying for the research, its focus had to be primarily to provide benefits to them. Part of the budget came from the private industry in the form of partnerships or R&D consortia. In fact, GRI became one of a number of powerful US consortia formed to manage co-operative research programs. These organizations do not carry out research themselves but act as managers for research programs that are sub-contracted to commercial companies. This model of research funding makes sense for utility industries operating in a given territory, but which do not compete with each other. Furthermore, the pooling research funds into common objectives results in a more effective R&D investment, a better approach to environmental issues and for the promotion of government alliances. However, the changes in the gas sector, particularly liberalization, progressively introduced competition among the industry inducing its annual budget to peak in 1987, reaching $187M. Since then, private support began to decline steeply and the co-operative research programs began to wane. Since 1993, efforts have been concentrated in restructuring the research program and in 1999, the GRI and FERC agreed to transition from a research program funded by consumers to an entirely voluntarily funded program. This was to be phased in over a seven-year period, to be ended by 2005. During this period, there was a four-year post-transition period during which FERC-approved funding was to be used only for the core program. In April 2000, GRI merged with the Institute of Gas Technology (IGT) to form the Gas Technology Institute (GTI) geared to meet the needs of a more competitive,

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deregulated energy industry and continuing to provide new technologies with benefits for gas consumers. Both GRI and IGT continue to exist as separate and distinct corporate entities within the overall GTI organization. Since their biocatalytic developments were individually attained, the two companies will be discussed in separate sections of this Chapter (this section for GRI and Section 20 for IGT). GTI’s activities include research and deployment, exploration and production services, and education. Major customers are gas-industry companies and government agencies with interests in energy and the environment. GTI maintains a large portfolio of intellectual property (IP), which includes more than 800 patents, 250 licensing arrangements, and equity positions in several portfolio companies. GTI is currently active in biorefining research for both, US and international clients, and has established itself as a pioneering leader in the development of biocatalysts acting at high-temperature enabling new bioprocess applications. The R&D activities of GRI led to a group of two patents, for gas sweetening (and also useful for flue gas treatment), based on biocatalytic processes for the selective removal of sulfur compounds in the presence of other reactive gases. • Microbial process for the mitigation of sulfur compounds from natural gas [81]. • Microbiological desulfurization of sulfur containing gases [82]. Although the first patent was designed for application to natural gas, two aspects of the patent make it interesting for the oil industry. The reactions result in conversion of anionic sulfur into elemental sulfur. From the process point of view, the anaerobic conditions and the high-operating pressure and temperature are of interest. In fact, the microbiological treatment of sour natural gas removes H2 S, carbon disulfide, methyl mercaptan, ethyl mercaptan, and dimethyl sulfide, and recovers the sulfur as elemental sulfur [81]. The reaction is carried out using a consortium of chemoautotrophic bacteria (named SSII) at pressures lower than 1000 psi and temperatures not higher than 60 C. It is expected to reduce sulfur from streams containing up to 10,000 ppm H2 S to less than 4 ppm. The SSII microbial consortium used in this process has been deposited in the American Type Culture Collection (ATCC 202177) and comprises at least four morphologically distinct microbes. The amount of biomass in the reactor does not exceed 10%. The reactor vessel is loaded with an organic source of nitrogen. In the second patent entitled ‘Microbiological desulfurization of sulfur containing gases’, the consortium, ATCC 202177, was further developed to desulfurize sulfurcontaining gases in the presence of other highly reactive contaminants [82]. The microbial consortium, ATCC 202177, was chemically enriched to remove target sulfur compounds from gases in the presence of ammonia, cyanide, carbon oxides, hydrogen, nitrogen, and other toxic gases and their mixtures. The microorganisms were grown in vessels containing a mixture of target sulfur compounds, such as hydrogen sulfide, carbon disulfide, carbonyl sulfide, dimethyl, methyl, and ethyl mercaptans. The enrichment process was carried out at a pH near neutral, with pressures ranging from 1 to 80 atm and temperatures in the range of 10–60 C. The ATCC culture was grown in an anaerobic or aerobic nutrient medium until sufficient cells were obtained for the desulfurization process. The growth was monitored at 460 nm and also by direct microscopic counts. The cultures showing appreciable growth were transferred into fresh nutrient medium (TSN, see full composition in Table 1 of Ref. [82]) for at least three passages to ensure

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growth stability. The most rapidly growing culture was designated SSII (also known as DSC2). To carry out the sulfur removal reaction, the active biocatalyst is suspended in an aqueous solution, while the gas feedstock fills the headspace of the anaerobic batch reactor up to the designated pressure and incubated at the given temperature and pH conditions of the process. While the temperature and pressure conditions and inoculum usage may be practical, the reaction time, the investigated scale and the discontinuous nature of the process contribute to the difficulty of using this process for pretreatment operations. However, in cases where low throughput is needed, such as in the post-treatment of exhaust gases, this process would probably become a candidate to be considered.

16. HOUSTON INDUSTRIES INC. (UNITED STATES) Originally, a gas supplier named Houston Gas Light Company was created to supply gas (made from oyster shells and coal) for the street lights of southeast Texas. This company evolved through a series of processes to become Houston Gas and Fuel by 1912, after creating Houston Lighting & Power Company in 1905. A great deal of acquisitions, mergers, and diversification ended as Houston Industries Inc. in 1984. Growth and changes did not stop there and currently Center Point Energy is the holding corporation devoted to providing electricity transmission and distribution service for the Houston metropolitan area and natural gas distribution service in Arkansas, Louisiana, Minnesota, Mississippi, Oklahoma, and Texas. At the beginning of the 1990s, Houston Industries developed an enzymatic process (‘Enzymatic Coal Desulfurization’) protected in Canada and US [83,84]. Although, the application was focused to coal desulfurization it may also be applicable to crude oil and fossil fuel-derived liquids. The processes claim the removal of both, organic as well as inorganic sulfur species. The process was described as using ground coal 10–50 m slurried with water, while the oil was treated in an aqueous emulsion. The enzymes used belong to two classes: oxidases and hydrolases. Use of enzymes immobilized on support particles was also included as an alternative. Feedstocks were first treated with the oxidase enzyme to convert organosulfur moieties to organic sulfates and then treated with a sulfatase, which cleaves the sulfates from the carbonaceous material. In cases where the organic sulfur is predominantly in the form of organic sulfates the first oxidation stage may not be necessary. The oxidase enzymes referred in the patent include peroxidase and laccase (e.g., horseradish peroxidase [E.C. number 1.11.1.7] and Pyricularis oxyzae laccase [E.C. number 1.10.3.2]). Use of a mild alkaline or acidic treatment was also suggested as an alternative to the use of enzymatic oxidation. The process described rapid heating (within 3–5 min) of the acidic or basic slurry/emulsion to temperatures between 150–180 C. The final step is a hydrolyzing step with sulfatase enzymes (E.C. number 3.1.6.1), such as limpet sulfatase, Aerobacter aerogenes sulfatase, Abalone entrail sulfatase, or Helix pomatia sulfatase. This step was suggested to be carried out in a CSTR or fluidized bed reactors, with counter-current flow between the aqueous and the oil phase. A more efficient removal of the sulfate into the aqueous stream is expected to occur in this cross-flow manner. A final separation of the reacting mixture was suggested to obtain sulfur-free product and aqueous enzyme solution for recycle.

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17. IMPERATRIX (UNITED STATES) An interesting process for converting organic ring compounds present in the crude oils via in situ (in the well) anaerobic treatment (Process for producing product from fossil fuel, 1974) has been disclosed [85]. The process is designed to deal with most ring compounds including cycloparaffins, cycloalkenes, aromatics and heterocyclic ring compounds. Four different types of microorganisms were considered: ring-converting microorganisms, paraffin-converting microorganisms, organic acid-converting microorganisms, and/or carbohydrate-converting microorganisms. The microorganisms considered for organic ring conversion include various Pseudomonads: P. arvilla, P. dacunhae, P. desmolytica, P. rathones, P. salopia, P. crucivial, P. indoloxidans, P. pictorum, and P. lacunogens; various Vibrios: V. neocistes, V. cyclosites, and V. cuneaties; Agarbacterium, Beneckea, and Bacteroides; two Achromobacter species: A. iophagus and A. cyloclastes; fungi: Actinomyces, Myrothecium, Mycobacterium, Myococcus, Nocardia, Sporocytophaga, Streptomyces, Trichoderma, Oscillospira, and Aspergillus; and yeasts: Gospora, Candida, Debarejomyces, Pichia, Saccharomyces, Dekkera, and Hauseniaspora. The paraffin-converting microorganisms include Mycobacterium, Cornebacterium, and Pseudomonas and yeasts: Rhodotorula, Lipomyces, Candida, Debaryomyces, Hansenula, Schizoblastosporion, Trichosporon, Torulopsis, Saccharomyces, Gospora, and Pichia. The organic acid-converting microorganisms include the bacteria P. riboflavin, Xanthomonas, Spirillum, Selenomonas, Rhodomicrobium, and Propionibacterium and yeasts: Candida, Torulopsis, Rhodotorula, Lipomyces, and Saccharomyces. Finally, the microorganisms reported for carbohydrate conversion include the bacteria P. tralucida, P. lacia, Cellfalcicula, Flavobacterium ferrugineum, Cellulomonas, and Clostridium. The organic ring compounds are converted to organic acids and/or paraffins under anaerobic fermentation conditions. The in situ contact is preferably performed at a depth of at least 500 ft. The aqueous bioactive solution is added in a 10–30% proportion to the fossil fuel. The anaerobic fermenting microorganism may be introduced to a reservoir deposit through a rubble chimney, created by action of explosives. Alternatively, the treatment can be carried out co-currently or counter-currently through the chimney. Therefore, each type of microorganism can be used separately or mixed in a single culture; in this last case the mixed culture is incorporated through a single chimney and all the microorganisms used should be anaerobic (in situ fermentation of either organic acid or the simultaneous conversion of the organic ring compound and the paraffin to organic acids). Only when the treatments are performed sequentially, there would be choice for aerobic microorganisms. Operating temperatures are typically below 60 C and pH between 3 and 9. This process is claimed to yield a product lighter than the original fossil fuel and with decreased sulfur content. This product reportedly has a lower ring content, larger paraffin content, and increased organic acids content. The product would be more water-soluble than the starting ring compounds. To further convert the organic acids/paraffins, they could be contacted either with the paraffin-converting microorganism or the organic acidconverting organisms. The process may be done in multiple steps (one class of organisms at a time) or simultaneously using several classes or organisms together. Finally, a carbohydrate-conversion step may be included. This could be done either aerobically or anaerobically, in which case use of hydrogen-rich atmosphere was recommended. The

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various steps are claimed to produce value-added products, such as gaseous hydrocarbon or mixture of hydrocarbons (e.g., methane, ethane, propane, and butane). Besides fossil fuels, a process for conversion of sewage sludge was also suggested. Lastly, chlorination and/or the addition of heavy metals were suggested as a means of containing microorganisms.

18. INSTITUTE FRANCAIS DU PETROL (FRANCE) IFP is an organization devoted to R&D, training, and information services for the oil & natural gas industries. Main working fields include vehicle uses and new energy and environmental technologies (fuels from biomass, biofuels, hydrogen, CO2 capture, and storage, etc.). Its role is to invigorate the economic competitiveness of supporting industries by generating technologies, which provide growth and increase the number of jobs. The actual IFP’s patent portfolio includes more than 12,000 patents. The department of Biotechnology and Biomass Chemistry is devoted to the use of microorganisms for industrial purposes, focused on three areas: • Hydrocarbon Microbiology: biodegradation mechanisms of oil products (gasoline, kerosene, diesel, etc.), pyrolysis, polycyclic aromatic hydrocarbons, chlorinated solvents, and ether fuels; refining processes (e.g., oil product microbial desulfurization) and oil production processes (e.g., bacterial corrosion). • Upgrading Biomass: enzymatic work for non-food uses of the biomass (new lubricants, emulsifiers (biosurfactants), and viscosity agents (polysaccharides)). • Water and Soil Protection Only two patents have been issued to this company indicating their recent venture in the BDS research area: • New cultures of Rhodococcus strains, useful for desulfurization of petroleum and its products, comprises high catalytic stability which allows culture recycling [86]. • Biological culture containing R. erythropolis and/or R. rhodnii and process for desulfurization of petroleum fraction [87]. The patents, however, protected the microorganisms (biocatalysts/biocatalytic systems) [86,87] as well as process to use the microorganism [87]. So far, there are no records of any other international protection. The patent reports new cultures of Rhodococcus strains, and a method to improve biocatalyst stability, which allows recycling. The bacterial strains were submitted to the National Collection of Microorganism Cultures of the Pasteur Institute (CNCM) and included R. erythropolis CNCM I-2204, -2205, -2207, and -2208, and/or R. rhodnii I-2206. The activity of the microorganisms includes a selective attack on the carbon–sulfur bonds in the organic molecules without significant alteration of the carbon atom structure. As usual, strains were isolated and subjected to successive enrichment phases, in media containing various sources of carbon and DBT as the only sulfur source, then a purification of the enhanced culture led to the fifteen DBT specific strains. From those strains, a subset of ten strains was selected

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based on hydroxybiphenyl (HBP) production. An additional criterion based on activity was applied and only strains exhibiting activity greater than 1 mg of DBT degraded per gram of cells (dry weight) per hour were selected. Further stability treatments were conducted on those ten strains, and only those retaining 70% of their original activity were kept. The biocatalyst (whole cells, derivatives, and isolated enzymes) and their use in BDS were protected. The patented subject matter includes the microorganism culturing, biocatalyst preparation, reaction, separation, and sulfate elimination. The main difference probably lies in the gradual culturing method. The inoculum to total media ratio was targeted to be below 10%.

19. INSTITUTO MEXICANO DE PETROLEO/UNIVERSIDAD NACIONAL AUTONOMA DE MEXICO (MEXICO) The Instituto Mexicano de Petróleo (IMP) is a Petroleum R&D Center created in 1965. It is mainly devoted to support the operational activities of Petróleos Mexicanos (Pemex), by developing new technologies and pursuing special technical services. During its history, IMP has been characterized by its closed relationship with academia in several cooperative interactions. Particularly, in biorefining a collaborative work with the Universidad Nacional Autónoma de México (UNAM) was focused into the development of enzymatic catalysts. The Instituto de Biotecnología (IBT) of UNAM is the result of the evolution of the Centro de Investigación sobre Ingeniería Genética y Biotecnología (CIIGB). Since 1982, has been working to become a symbol of Mexican biotechnology efforts. IBT is constituted by 93 researchers, 70 research staff and more than 180 graduate students. All scientific and technological initiatives are centered on research with proteins and nucleic acids. There are five departments within IBT including Bioengineering, Molecular Biology, Plants Molecular Genetics and Physiology, Molecular Microbiology, and Molecular Identification and Biostructure. The IMP/UNAM invention [88] is based on enzymatic desulfurization of fossil fuels. It uses the catalytic properties of hemoproteins (proteins containing a heme prosthetic group) to catalyze oxidative transformations of sulfur-containing compounds to sulfoxides and sulfones. The patent includes chemical and genetic modification of the enzymes, to improve tolerance to and activity in organic environments. The hemoproteins referenced in the patent include chloroperoxidase (EC 1.11.1.10) from Caldariomyces fumago, lignin peroxidase (EC 1.11.1), and manganese peroxidase (EC 1.11.1.7) from lignolytic fungi, and cytochromes from animal, plant or microbial cells. The biocatalytic system is also broadly defined so that to include microbial lysates, cell-free extracts, cell extracts, fractions, subfractions or purified products comprising the proteins capable of carrying out the desired biocatalytic function. Co-enzymes, co-factors, or co-reactants are mentioned as optional additives. Although, the protein preparation methods are wellknown in the literature, some examples were included in the patent document (more details can be seen in Section 2.2 in Chapter 3). The patent described a method for the removal of thiophenic compounds from fossil fuels, in which the reacting media might contain organic solvents. Additionally, the biocatalyst may be contacted with the fuel directly either as free enzyme or in its immobilized form. The process could, therefore, be performed either in a batch reactor or in a semi-continuous or continuous manner. Further, it may be performed either as a stand

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alone process or in a combination with one or more refining processes. The separation of the reaction product was carried out by distillation, cutting off at 50 C lower than the distillation temperature of the original cut. The reaction can be carried out in the presence of the fuel alone or with addition of any organic solvent. The biocatalytically oxidized fuel is then distilled to eliminate the heavy fraction which contains most of oxidized organosulfur compounds. The light distillate contains significantly lower concentrations of sulfur when compared with the starting fossil fuel.

20. INSTITUTE OF GAS TECHNOLOGY (UNITED STATES) The IGT was founded in 1941 as a not-for-profit R&D organization funded by membership contributions from energy industries in the USA and Canada. In the early 21st century, IGT was merged with the GRI to form the GTI. As already mentioned, GRI manages research programs, which are contracted externally. Therefore, the research activities in GTI are carried out by IGT. Most of the funding is received from the participating members. Membership has grown from about 200 by the end of the 1990s to nearly 350 members and international associate members, by 2006. The current members include most of the major oil industries. Apart from direct contributions from the companies, IGT receives funding from the GRI, the US Department of Energy, the Environmental Protection Agency and industry. The members elect a Board of Trustees that provides the overall direction for the Institute. Most of the reputation of GTI comes from the achievements of IGT, accumulated for nearly 70 years. Its technology developmental focus is determined for US’s energy and environmental challenges. GTI maintains an IP portfolio of more than 800 patents and 250 licensing agreements. Its biotechnology history includes isolating novel microorganisms that perform industrially relevant biochemical reactions and gene expression systems in various microbial species to facilitate the use of high-temperature bioprocesses and industrial bioprocesses. Biorefining research has been funded by U.S. and international clients for more than 20 years. Specifically, GTI has isolated several bacterial cultures that can selectively cleave carbon–sulfur and carbon–nitrogen bonds, which can be used to process fossil fuels. Currently, the focus has been shifted to high-temperature bioprocesses. GTI is currently collaborating with Petrobras, Suncor Energy, Inc., and the University of Illinois to develop biocatalysts active for carbon-nitrogen cleavage through selective pathways. The first bioconcept patented by the Institute of Gas Technology (IGT) was related to an oxidative improvement of the thermal liquefaction process, namely, a hybrid bio-thermal liquefaction [89]. The carbonaceous material is subjected to fermentation to produce alcohols and then, the resid from this process is subjected to a thermochemical conversion. Although, the process is suggested for handling biomass and agricultural waste feedstocks, rather than refinery streams, its products could be integrated to any refinery throughput. By the time, that development was carried out, the oxygenated products found importance as reformulated fuel additives and certainly, the biofuels idea was still emerging. In this hybrid process, liquefaction was improved to produce alcoholcontaining fuels. The organic carbonaceous feed is subjected to fermentation producing alcohols plus a residue, which is processed in a thermochemical converter under elevated temperature conditions. A portion of the converted residue is recycled to the

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fermentation reactor. It provides high overall process and energy efficiency and utilizes the total agricultural biomass crop thereby greatly reducing waste disposal problems. The alcohol content (ethanol + methanol) can be increased by emphasizing thermochemical gas production followed by catalytic synthesis that suggests integration to petrochemical processes downstream, while on the other hand, fuel oil and gasoline content of the liquid fuel products can be increased by emphasizing thermochemical liquefaction. The second development for which IGT is known world-wide is their work on biodesulfurization. The IGT intellectual property package, developed by Kilbane’s group, includes two microorganisms, R. rhodochrous strain ATCC 53968 (IGTS8) and B. sphaericus strain ATCC 53969 as well as the enzymes derived from them and cellfree extracts. The biocatalysts and their use were protected in a series of eight patents (plus one US equivalent) and though in some patents a process is claimed, the main emphasis is on biocatalyst. A summary of these patents and the comparison with the early patents of EBC was given in Chapter 3. The patents were entitled: • • • • • •

Mutant microorganisms useful for cleavage of organic C−S bonds [67,90,91]. Bacterial produced extracts and enzymes for cleavage of organic C−S bonds [68]. Biochemical cleavage of organic C−S bonds [92]. Useful for cleavage of organic C−S bonds B. sphaericus microorganism [93] Microbial cleavage of organic C−S bonds [73]. Process for enzymatic cleavage of C−S bonds and process for reducing the sulfur content of sulfur-containing organic carbonaceous material [94]. • Enzyme from R. rhodochrous ATCC 53968, B. sphaericus ATCC 53969 or a mutant thereof for cleavage of organic C−S bonds [95]. The two organisms were capable of cleaving the C−S bond in molecules found in oil fractions in a specific manner. Kilbane’s intellectual property (specifically, EP0441462 [67] and EP0445896 [68]) focused on the following: a. a mutant R. rhodochrous strain ATCC No. 53968 (which has the property of sulfur removal and sulfur metabolism by selective cleavage of C−S bonds in organic carbonaceous materials), b. a derived extract of membrane fragments from that strain, c. an enzyme and a composition of enzymes associated with cell membranes of R. rhodochrous strain ATCC No. 53968 (which have the ability to selectively react with organic sulfur of sulfur-containing organic carbonaceous material by cleavage or organic C−S bonds). The invented biocatalysts based on R. rhodochrous strain ATCC No. 53968 and on B. sphaericus strain ATCC No. 53969, were protected not only as whole cell biocatalysts, but also their derivatives. Biocatalyst definition includes in addition to whole cells; cell membranes, cell extracts and enzymes from those microorganisms. It should be noted that the first six patents are actually sets of similar patents with the first one providing coverage in Europe and the second one in US. This strategy involves coverage in US as well as Europe (the total number of patents is higher than the number of inventions); however, the allowed claims in US were always smaller than that allowed in Europe. IGT’s last patent (May 1996) was filed in July 1994, when they already had begun

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working with EBC on process development. It can be said that by the end of 1996, IGT did not see itself commercializing the BDS process or complete full development of the biocatalyst and initiated collaboration with EBC. In summary, the IP strategy for the eight inventions protected: first, the mutant microorganisms in two patents, then the second two expanded the biocatalyst concept, and finally the use or application of each of these biocatalytic concepts produced the other four patents. These two strains, B. sphaericus strain ATCC 53969 and R. rhodochrous strain ATCC 53968 discovered by Kilbane as being capable of dibenzothiophene desulfurization were patented as two separate (European) patents [67,91], respectively. These two patents issued by 1991 also described the use of the enzymes derived from these organisms and their cell-free extracts for desulfurization applications. Both strains were reported to carry out selective cleavage of C−S bonds in organic carbonaceous materials. The organism, Bacillus sphaericus strain ATCC 53969, was, however, reported in Exxon patents, to be capable of C−C bond cleavage as well and therefore its ability to perform desulfurization without loss of fuel value is questionable. These pioneering discoveries were possible due to the screening protocols employed by the Kilbane group. The protocol consisted of screening environmental cultures having a known history of exposure to organosulfur compounds using culture medium free of sulfur compounds except the organosulfur compound of interest. The cultures were subjected to enrichment processes using carbon sources such as acetate, benzene, benzoic acid, ethanol, glucose, glycerol, nutrient broth, succinate, and toluene in the presence of various organic sulfur compounds including benzothiophene, dibenzothiophene, thiophene, and trithiane. The bacterial enrichments obtained were found to be capable of metabolizing the specific organic sulfur compounds included in the screen. However, most of the environmental isolates and enrichment cultures tested were found to metabolize the target compounds by biodegradation at the carbon–carbon skeleton. It was realized by Kilbane and co-workers that to develop any microorganism having the desired sulfur metabolism, the mutation process besides being selective, seemed to be unnatural. The sulfur-specific mutation of the strains could be accelerated by mutagenesis, either exposing them to 1-methyl-3-nitro-1-nitrosoguanidine or to ultraviolet irradiation. Therefore, microorganisms showing the ability of sulfur-specific metabolisms were pursued for further examination and development by mutagenesis and enrichment techniques. A specially designed bioreactor/selectostat allowed continuous liquid flow for feeding and the use of organosulfur nutrients which are not normally found in living cells and were fed as the solely supplied sulfur. The recommended organosulfur compounds (part of the documented knowledge of the patent) included some of the most commonly found in crude oil, but also some which are rarely mentioned: benzothiophene, benzyldisulfide, dibenzothiophene, dibenzothiophene sulfone, phenyldisulfide, thianthrene, thioxanthene, dibenzothiophene sulfoxide and trithiane, its concentration must support microbial growth, so about 20 mM should be enough. Gas and vapors such as thiophene could also be used. The two patents described above were particularly important in the initiation of the developments of biodesulfurization catalysts. The bioreactor arrays required for operation and growth method constituted key elements in the following developments of the area, which would condition viability and successful path to industrialization. A sulfur bioavailability assay was incorporated into the screen for monitoring the sulfur uptake by the microorganisms, and the concept formed a claim in the patents [67,91]. The objective

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of the assay was to verify whether bacterial growth occurred at the expense of sulfur obtained from the target organosulfur compound. A mixed bacterial culture capable of utilizing a range of organosulfur compounds could be obtained from the selectostats after several months of operation. The sulfur bioavailability assay was used to verify that the targeted compound was the sole source of sulfur for microbial growth. In this way, the two organisms, B. sphaericus strain ATCC No. 53969 [91] and R. rhodochrous strain ATCC No. 53968 [67], were confirmed to selectively cleave the C−S bonds. The next patent awarded to IGT (in 1993) [93] was related to the organism, B. sphaericus ATCC 53969, and included extended protection for its capabilities to cleave C−S bonds from organosulfur molecules. That full protection also involved any known possible derivative, which might directly or indirectly carry or enhance the observed capabilities. Following these earlier patents, the latter patents introduced additional concepts and practices into the oil-refining biocatalysis arena, by broadening the traditional (whole cell or enzyme) concept of a biocatalyst. Later, in 1994, the use of the derivatives from the original strains for BDS was awarded to IGT (‘Bacterial produced extracts and enzymes for cleavage of organic C−S bonds’) [68]. The same intellectual property was previously awarded to EBC2 on 1992–93, as mentioned in Section 13 of this Chapter. This patent appeared to represent a shift in the development, by which the inventors seems to try to effectively isolate the active species from the living cells. The protected subject matter regards extracts and enzymes of R. rhodochrous ATCC No. 53968 and B. sphaericus ATCC No. 53969 and the patent document prove how they work in the removal of organic sulfur from fossil fuels (coal and oils). Such biocatalysts were supposed to work either in organic or aqueous media. Use of some new terms in the invention, including ‘sulfur-specific reactant’ referring to the active material, and ‘desulfurizing degradation’ to the sulfur removal, are interesting and one wonders why such a choice of words would entail. This patent can also be considered as pioneer in the use of non-living biocatalysts and having induced the idea that BDS enzymes are related to membrane material. The referred extracts from the above microorganisms were prepared by ordinary known methods, such as by lysis processes, e.g., sonication, use of detergents, or use of a French press. Furthermore, the enzyme extraction methods were also routine techniques, such as ammonium sulfate precipitation, fractionation, gel permeation chromatography, electrophoresis, isoelectric focusing, high pressure liquid chromatography, liquid chromatography, affinity chromatography, or immunoprecipitation, etc. The biological membranes are normally hydrophobic, so the use of non-aqueous liquids might be preferred. Their findings suggest that higher catalytic rates may be achieved, with increased stability and expanded range of substrate utilization, when using this type of biocatalysts. Besides, operational temperatures could be slightly higher than those normally employed with living microorganisms. From these newly introduced features, several other improvements were mentioned to have been achieved, like the removal of both organic and inorganic sulfur and the possibility of performing the process in a continuous manner. Removal values as high as 90% were reported in the patent document. The application of the microorganisms for the biochemical cleavage of organic C−S bonds was awarded in separate patents, with the first cited in Ref. [92], also being awarded in Japan and Mexico (JP6054695 and MX9301323). The second one, awarded in US (Process for enzymatic cleavage of C−S bonds and process for reducing the sulfur

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content of sulfur-containing organic carbonaceous material), is partially equivalent to the first, but only includes the BDS use of the microorganisms and their derivatives [94]. In both patents, a microbial process for selective cleavage of organic C−S bonds consisted in contacting the feedstock with at least one ‘sulfur-specific reactant agent’. The biocatalyst, again referred as the ‘sulfur-specific reactant agent’ was described as at least one species of the claimed microorganisms under growth conditions or the already mentioned derivatives (an extract of microorganism cell membrane fragments or at least one enzyme associated with a microorganism cell membrane). The patent documents include all the disclosed details about the biocatalysts, i.e., the microorganisms (R. rhodochrous strain ATCC 53968 and B. sphaericus strain ATCC 53969) and their derivatives. The feedstocks involve coal or hydrocarbon oils. The first European patent [92] differs from the US patent [94] in that the European additionally includes the use of the microorganisms in the biochemical inactivation and modification of defensins. In this application the selective cleavage of the organic C−S bond of the disulfide bonding in the defensin molecule takes place in vivo and/or in vitro. Defensin is a polypeptide having several cysteine residues and multiple disulfide bonds. Defensins are natural peptide antibiotics from neutrophils present in humans and other mammals as well as in insects and appear to be fundamental components of innate host defense systems. Additionally, defensins act as antifungal, antiviral, and cytotoxic agents. Enzymatic cleavage of the C−S bonds of the defensins will inactivate them and so alleviate auto-immune diseases caused by the inappropriate production of defensins. Two additional patents were allowed for the BDS processes, (Microbial cleavage of organic C−S bonds [73] and Enzyme from R. rhodochrous ATCC 53968, B. sphaericus ATCC 53969 or a mutant thereof for cleavage of organic C−S bonds [95]), which only refer to the use or application of the different biocatalytic concepts in BDS. The first [73] considers the use of whole cells, while the second refers to the membrane fragments, extracts and enzymes [95]. As such, the patent did not describe an out of the ordinary use of a biocatalyst under BDS conditions. As previously mentioned, R. rhodochrous and B. sphaericus and their derivatives have been found to have the ability of selective cleavage of organic C−S bonds. The process, of course, was targeted for reduction of the organically bound sulfur content in coal or hydrocarbon oils. However, real process information is not given in either of the two documents. Both of them collect the information already given in the previous documents, (related to the microorganisms and their derivatives). The biocatalysts stability and the possibility of using freeze-drying, to prepare them at one location, freeze-dried for shipping at ambient temperature to other locations, was added in the new documents. For instance, when the freeze-dried cells were maintained in a screw capped bottle wrapped with aluminum foil for two and a half weeks at room temperature, exhibited a 23% loss of their original activity. However, the use of an alternate freezing method via flash-freezing in liquid nitrogen, followed by freeze-drying and storage at room temperature for 24 hours, the activity remained essentially unchanged. Another way of preserving activity while preparing the biocatalyst for transportation is to freeze the cells in 10% glycerol at –70 C. With this method, the cells exhibited their original C−S bond cleavage ability after 7 days of storage. The membrane-based biocatalyst supposedly had the ability to selectively react by cleaving the organic C−S bonds, with organic sulfur of sulfur-containing organic

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hydrocarbonaceous compounds. However, as it was later discovered the desulfurizing enzymes are all soluble enzymes, not associated with the membrane fraction [96]. From the intellectual property point of view, IGT must have been interested in protecting all possible catalytic biomolecules or components. The patent document itself did not show enough evidence on which component was exclusively responsible for the exhibited activity.

21. INSTITUTE OF PROCESS ENGINEERING (CHINA) The Institute of Process Engineering (IPE) is an educational organization of the Chinese Academy of Sciences. It was founded in 1958 as the Institute of Chemical Metallurgy. By 1970s, its R&D extended into new areas including biochemical technology among others. This broadened horizon pushed the original ICM into the Institute of Process Engineering (officially created on April 7, 2001). During the more than 40 years of activities, the Institute has accumulated a variety of technological results, some of them commercially implemented: utilization of (straw) biomass, organic fertilizer from lignin, polysaccharide bio-calcium, etc. The Laboratory of Biochemical Engineering (SKLBE) was created in 1988, but its R&D activities were initiated in 1992. Biochemical engineering activities support the downstream research needed for commercialization of new biotechnological inventions. When a breakthrough appears, the corresponding bioprocesses are scaled up. Thus, the IPE deals with all the stages involved in the manufacture, separation, and purification of biological products at large scale. The current R&D projects concerns: • • • • • •

Large-scale plant and animal cell culture Preparation process optimization for sea biological products Novel solid-state fermentation and biomass conversion Integration of bio-desulfurization, bio-reaction, and bio-separation Separation and purification for biological products Chemical modifications of proteins

The patent held by the Chinese Institute discloses a new organism, Gordona nitida capable of removing sulfur from organosulfur compounds [97]. The strain Gordona nitida LSSEJ-1, deposited as CGMCC No.0700 was claimed along with its application in removing sulfur. This Chinese patent has no English written equivalent and only a small summary is accessible. The characteristics of the organism were reported as follows: 2–3 m in length, rod-shaped, Gram-positive, contains meso diaminopimelic acid, arabinose galactose, nocardomycolic acid, and MK-9 (H2).

22. INTEVEP S. A. (VENEZUELA) Intevep S. A. is the R&D Center of Petróleos de Venezuela (PDVSA). It was created in February, 1974 and started operations in 1976, soon after the nationalization of the oil industry in that country. It took some years before it became an affiliate company

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of PDVSA. All the oil industry segments are attended and its activities involve R&D and specialized technical services. Its technological portfolio includes more than 1100 patents. In the biotechnological area it holds two patents (‘Biodesulfurization of hydrocarbons’) on BDS, one for a microorganism Alcaligenes xyloxidans capable of desulfurization [98] and its use in hydrocarbon desulfurization [99]. The organism was isolated from a sample of Venezuelan soil and enriched using DBT as the sole sulfur source in an otherwise sulfur-free growth medium. The biocatalyst is prepared by breaking the cells from a cell suspension using a Braun homogenizer. This biocatalyst is said to consist of an enzymatic extract, exhibiting hydrophilic and lipophobic properties, for what an emulsion formation was recommended. The process description involves exposing the liquid hydrocarbon to the bioactive material under specific conditions. Such conditions include 1–5 mg/ml of total protein extract per ml of hydrocarbon, a temperature between 30 C and 50 C and a pH between 7.0 and 8.0. Although, the analysis of the reaction products showed the substantial transformation of DBT and other sulfur compounds, it failed to verify whether they were converted into inorganic sulfur (sulfite/sulfate) or existed as solubilized partially oxidized organosulfur compounds. Besides, there was no evidence given for the formation of 2-hydroxybiphenyl, or actual sulfur removal. The only suggestion of a probable desulfurization was presence of minute amounts of biphenyl, which could not account for all the transformed compounds.

23. JAPANESE COOPERATING ORGANIZATIONS (JAPAN) Science and Technology (S&T) in Japan is integrated in a very complex network, in which cooperation seems to be taking place throughout the system, bilaterally as well as between multiple organizations. The Science Council of Japan is regarded as the representative organization of Japanese scientists and is structured in seven divisions. Pure Sciences and Engineering Division holds 63 of the 210 institutional members. The Council coordinates the actions of 180 Liaison Committees composed of 2370 members and has affiliations to 1481 Registered Academic Societies composed by about 760,000 scientists. The Ministry of International Trade and Industry is in charge of the execution of the S&T policies, designed by the National Institute of Advanced Industrial Science and Technology (NIAIST), which evolved from the previously known Agency of Industrial Science and Technology (AIST). NIAIST is headquartered in Tokyo and includes 15 research institutes. NIAIST holds approximately 11000 patents, from which more than 7000 were awarded to the joint efforts with the research institutes. Within the activities leading to the development of bioprocess technologies, three organizations were identified to be working in close collaboration: The Agency of Industrial Science & Technology/National Institute of Advanced Industrial Science & Technology, the Japan Cooperation Center Petroleum (JCCP) and the Petroleum Energy Center (PEC). Their evolution and strong links through collaborative work led to merges and creation of new organizations. Those organizations relevant to the scope of this book will be included here.

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23.1. Agency of Industrial Science & Technology/National Institute of Advanced Industrial Science & Technology The AIST was created by the Ministry of International Trade and Industry (MITI) in 1948. Its main activities involve planning industrial S&T policies and national R&D projects, conducting R&D in AIST laboratories, promotion of R&D in the private sector, promoting international cooperation and developing national standardization policies. By 1995, AIST had accumulated 10888 patents, from which 1666 were foreign patents. AIST seems to have protected its petroleum related inventions only in Japan, for that reason only the abstracts were accessible for reporting here. They were awarded four patents, between 1994 and 1999, so it is reasonable to think that they were still active in the area, before merging into the National Institute of Advanced Industrial Science & Technology. The Japan National Oil Corp. (Sekiyu Kodan Japan) was a participant in the collaborative work leading to the four patents. Both, BDS and BDN of hydrocarbonaceous materials (oil, coal, etc.) have been reported. Although, the patent law in Japan requires the microorganisms to be deposited prior to filing the patent, the reported abstracts did not show the registration records for any of them. The awarded patents are: • Biological desulfurization method [100]. • Denitrification by microorganism [101]. • Method for biodenitrification of hardly removable aromatic organic nitrogen compound [102]. • Method for obtaining organic solvent-resistant microorganisms and organic solventresistant microorganisms obtainable by the method [103]. • Method for oxidation of organic sulfur compound included in organic compound and method for oxidative desulfurization of fuel oil [104]. • Recombinant Desulfurizing Bacterium [105]. • Method of Desulfurization by Using Microorganisms which Decompose Dibenzothiophenes [106]. • Method for Specifying Desulfurizing Enzyme Expression – Inhibiting Gene and Desulfurizing Microorganism whose Desulfurizing Enzyme Expression Inhibition is Terminated and Method for Producing the Desulfurizing Microorganism [107]. In a patent on biological desulfurization [100] of petroleum/coal, only the use of whole cell biocatalysts was claimed. The biocatalysts included microorganisms belonging to the genus Pseudomonas, Flavobacterium, Enterobacter, Aeromonas, Bacillus or Corynebacterium. The desulfurization pathway (sulfur-specific vs. destructive) was not specified. The Japanese patents No. JP2071936C and JP7103379B seem to be equivalent patents. A similar method (described as just the contact between biocatalyst and feed) was applied for denitrification of organonitrogen compounds, by using microorganisms belonging to Pseudomonas, Bacillus, Escherichia or Serratia [101]. The details of isolation of the Bacillus species were described. The bacterium was isolated from the environment by selective enrichment under low-oxygen conditions (oxygen partial pressure near the explosion limit) and using carbazole as a sole nitrogen source and carbon source. The process was carried out at ambient temperature and atmospheric pressure on petroleum or coal as feedstock. An equivalent patent is registered as JP2636175B2.

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A two phase process, in which the feedstock (e.g., petroleum) was mixed with water and an organic solvent to improve denitrogenation of aromatic nitrogen compounds [102], led to an improvement of the process. Additionally, a surfactant was used to increase the interfacial area. Carbazole and quinoline and their alkyl derivatives were used as primary compounds for demonstration. The biocatalyst is used in resting stage and is continuously fed to the system to keep the reaction rate at an acceptable level. It was observed that quinoline was hardly removed under the conditions at which carbazole was decomposed and assimilated. A method for providing resistance towards organic solvents for microorganisms was claimed in a following patent [103].The parent strain was subjected to mutagenesis and then to selective cultivation in the presence of 0.1% to 10% (v/v) of a toxic organic solvent. Various BDS mutant bacteria capable of decomposing recalcitrant organosulfur compounds, under microaerobic conditions, in the presence of organic solvents were treated by the current method. The parent strain was P. putida, and the mutated strains were P. putida No. 69-1 (FERM BP-4519), P. putida No. 69-2 (FERM BP-4520), and P. putida No. 69-3 (FERM BP-4521). The objectives of the mutagenesis are to provide the bacteria strain with properties such as: a. high ratio of free fatty acids to total fatty acids; b. reduction of the high molecular weight portions and/or oxygenation of lipopolysaccharides; c. increasing concentration of fatty acids with odd carbon numbers via oxidation of straight chain fatty alcohols such as heptanol, nonanol and the like; and, d. intracellular fatty acids with odd carbon numbers are increased by other mechanisms due to contact with a highly toxic organic solvent. It is believed that the toxicity of an organic solvent is related to one or all of the above mentioned properties. Additionally, high toxicity involves an organic solvent having a small log P value, namely readiness to water solubility. Hence, a bacterium has to become hydrophobic to exclude such a highly toxic organic solvent. Since a free fatty acid does not contain polar groups such as glyceryl phosphate, typically present on lipidintegrated fatty acids, its hydrophobicity is higher. The absolute amount of free fatty acids is not as important as its ratio to total fatty acids. Besides, in lipopolysaccharides the deletion of the side branches and substituents of high molecular weight eliminates linkage vehicles for hydrophilic sugars; and so results in hydrophobicity. The selected mutants were assessed to verify that the desulfurization activity was retained after the mutagenesis to improve solvent tolerance. The National Institute of Advanced Industrial Science & Technology (NIAIST) began operations on April 1, 2001. The NIAIST included the Weights and Measures Training Institute in addition to the 15 other institutions already existing within AIST. The NIAIST is Japan’s largest public research organization. Some of the patents awarded to NIAIST were in collaboration with the JCCP and some others were also shared with MITI. The first patent, issued in 2001, describes a method for oxidative removal of sulfur compounds (DBTs) with inclusion of photo and chemical catalytic processes to assist in the desulfurization [104]. A BDS process in the presence of a recombinant bacterium [105], which was not identified in the patent abstract, was awarded in 2002 to NIAIST in cooperation with

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PEC. The patent includes the production method for the biocatalyst, with a characteristic inhibitory effect on certain enzyme expression. The expression recombinant vector contains a promoter free from manifesting inhibition due to an inorganic sulfur compound or a sulfur-containing amino acid. The recombinant microorganism also contains a gene for desulfurizing the sulfur-containing heterocyclic compound. Another BDS process comprises microorganisms for the decomposition of benzothiophenes [106]. A vector capable of co-expressing a gene which codes a dibenzothiophenedecomposing enzyme and another gene which codes an oxidation–reduction enzyme was introduced into the microorganisms. The gene which codes the dibenzothiophenedecomposing enzyme is derived from microorganisms belonging to Rhodococcus, Sphingomonas, Paenibacillus, Agrobacterium, Mycobacterium, Gordona, Bacillus or Arthrobacter genus, or its mutant. The gene which codes the oxidation–reduction enzyme is derived from Paenibacillus polymyxa A-1 strain. Finally, a more MB-oriented patent concerns a gene which participates in desulfurizing enzyme expression inhibition [107]. The production of the microorganism which highly expresses the desulfurizing enzyme, even when cultured in the presence of a sulfate as only one sulfur source was included. The application was focused towards the decomposition of thiophene compounds.

23.2. Japan Cooperation Center, Petroleum (JCCP) JCCP was created in November 1981 with the aim of promoting technical cooperation and scientific and technological exchanges within oil-producing countries, particularly for the downstream sectors. Its programs include training, lecturing and research. In April 2001, tasks related to the promotion of information exchange and international collaborations were assigned by the Petroleum Energy Center (PEC) to the JCCP. During the last few years, JCCP has continued to promote cooperation and collaboration among the research community interested in oil-related activities including the private companies. Now, JCCP functions under the auspices of the Ministry of Economy, Trade and Industry of Japan. The following four patents awarded to JCCP represent its internal effort in the BDS area (no collaboration with any other organization): • • • •

Method of Desulfurizing Heterocyclic Sulfur Compound Using Bacterium [108]. Desulfurization Method Using Recombinant Microorganism [109]. Recombinant Microorganism and Desulfurization Method Utilizing the Same [110]. High-Quality Desulfurization Method by Using Recombinant Microorganism [111].

The research was oriented towards the development of biocatalysts for removal of recalcitrant sulfur heterocyclic compounds including benzothiophenes, naphthothiophenes, and alkylbenzothiophenes. To begin with, they focused on asymmetric sulfur compounds in this class and developed a method for desulfurization of these compounds present in petroleum products [108]. The identity of the microorganisms was not disclosed in the abstract but they do claim use of the enzymes as well in the application. Further studies led to the development of a recombinant microorganism containing a desulfurization enzyme gene at the tail end of a promoter composed of (a) a DNA characterized by a claimed sequence (No. 1 in the referred application) and having

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promoter activity or (b) a DNA having promoter activity and hybridizable under a stringent condition with a DNA having a base sequence complimentary to the DNA expressed by the given sequence No. 1 [109]. A desulfurization method and the recombinant microorganism employed were protected in a single patent [110]. The recombinant microorganisms were produced by functionalizing the DNA of microorganisms belonging to the genus Mycobacterium, especially those having the ability to methylate hydroxy compounds, with a promoter inert to sulfate ions inhibition. The microorganism also encodes desulfurization genes capable of expressing active enzymes for the selective cleavage of C−S bonds. The production of a virtually colorless desulfurized petroleum fraction could be accomplished by a desulfurization method, which uses a recombinant microorganism from the Nocardia family (genus Gordonia, genus Rhodococcus, or genus Mycobacterium) [111]. In the biocatalytic microorganism, a carotenoid-producing gene group was destroyed to prevent coloration of fuel due to release of carotenoids during the desulfurization process. This mutation did not change the desulfurization capability of the organism.

23.3. Petroleum Energy Center (PEC) The Petroleum Energy Center (PEC) was founded in May 1986. PEC was established to promote comprehensive programs for deep renovations and to contribute to the viability of the petroleum industry in Japan. The following patents were registered by PEC: • • • • • • • • • • • • • • • • • • •

Oxidoreductase Gene [112]. Gene coding for desulfurizing enzyme [113]. Method for Desulfurizing with Microorganism at High Temperatures [114]. Method for Decomposing Heterocyclic Sulfur Compound Using Bacterium [115]. Thermally Stable Desulfurization Enzyme and Gene Encoding the Same [116]. High-temperature desulfurization with microorganism [117]. Production of desulfurization-active microorganism [118]. Gene encoding desulfurases [119]. Gene encoding desulfurases [120]. High-temperature desulfurization by microorganisms [121]. High-temperature desulfurization by microorganism [122]. Desulfurization by microorganism [123]. Desulfurization using microorganism capable of degrading alkylbenzothiophene and alkyldibenzothiophene [124]. Method for Decomposing Porphyrin-Based Compound [125]. Method for Culturing Microorganism having Ability in Desulfurization [126]. Biological desulfurization [127]. Gene encoding desulfurases [128] Decomposition of porphyrin [129]. Gene encoding desulfurases [130].

Some of the patents reported earlier in Section 23.1 were awarded jointly with PEC. Some of these patents have been filed in the US and others in Japan and Europe.

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Since temperature can reduce oil viscosity, operation at higher temperatures can be beneficial in terms of oil-water contacting, potentially improving mass transfer and possibly improving reaction rates as well. Thus, PEC concentrated its work on development of high temperature biocatalysts [112–128,130]. A themostable Paenibacillus for BDS was disclosed in patent number US5925560 [121], its adaptation, culturing and growth in JP11181446 [118], and its use in BDS application in US6130081 [122]. This biocatalyst has been reported to be active for S removal from benzothiophene, dibenzothiophene and their derivatives contained in fossil fuels. As described in Section Chapter 32.2.3 of Chapter 3, Konishi et al. defined a method for degrading organic sulfur compounds, in which the heterocyclic sulfur compounds were decomposed by a microorganism belonging to the genus Paenibacillus [121]. In fact, Paenibacillus sp. A11-1 and Paenibacillus sp. A11-2 have been reported to desulfurize heterocyclic sulfur compounds at high temperatures via selective cleavage of the C−S bond. In a further development, a biocatalyst having excellent desulfurization activity was prepared by culturing the microorganism in a medium supplemented with water-soluble sulfonic acid compounds as inducers of the bds genes. The induction with soluble sulfonic acids resulted in higher growth rate as well as improved enzyme expression, resulting in a biocatalyst preparation with high cell density and high specific activity [118]. Details of this preparation and the biocatalyst activity obtained are given in Section 2.2.5 The method for degrading organic sulfur compounds, by using these microorganisms was disclosed in a separate patent [122]. The desulfurizing medium containing the microorganism Paenibacillus sp. A11-1 and A11-2 were described in detail (Section 2.2.3 of Chapter 3). The inventors showed examples involving both, model compounds and real feedstocks as substrates. In the first case, 5 ml of the desulfurization medium was inoculated with the specific colony in a test tube with a stopper and cultured for 1 day, at 50 C. The resting cell reaction biocatalyst was prepared by inoculating 2 ml of the previous stock in 100 ml desulfurization medium containing 20 ppm DBT, with stirring until the OD660 of the culture reached 0.5 (approximately 15 h). The culture was then centrifuged at 8000 g for 5 min and the bacterial cells were recovered and washed. The bacterial cells were suspended again in 0.1 M phosphate buffer (pH 7.0) such that the OD660 gets close to 20. An ethanol solution 75 l of the reacting substrate, DBT (10,000 ppm) was added to 500 l of that resting cell suspension, preheated (at the reaction temperature), in a 7 ml test tube with a stopper. The test tube was subjected to rotation in an inverted position and let the reaction to proceed. BDS activity was measure as the 2-hydroxybiphenyl formed. Degradation of organic sulfur compounds in light gas oil by the high-temperature desulfurizing microorganism was carried out at the same conditions. In this case, the proportion of reacting feedstock to desulfurization medium is 1:99. The conversion in the case of the model feedstock was always less than 30%, while for the real gas oil the highest value was 11%. Two aspects should be noticed: the clear evidences of very small laboratory scale and the explicit statement on monitoring true desulfurization, by following the formed HBP. A second thermophile belonging to the genus Bacillus was discovered and patented for desulfurization of organosulfur compounds [117]. The microorganism was isolated from environmental samples and was found to grow and desulfurize over a wide temperature range. The microbial cells as well as cellular fractions were included as biocatalysts in the patent claims. The microorganism was found to be active for cleaving the C−S bonds of naphthothiophene and benzonaphthothiophene. The process using this biocatalyst was protected in a separate patent [115].

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A third thermophile, M. phlei WU-F1 also capable of desulfurization over a wide temperature range was patented in 2001 [114]. The patent also protects any variant of the strain or a transformant derived from M. phlei WU-F1. To improve process conditions and to obtain higher reaction rate, the water/oil ratio was studied. The influence of the oil/water ratio on separation and on the separation cost was studied employing R. erythropolis KA2-5-1 (deposited as FERN P-16277). The use of an oil/water ratio within the range of (3:1) to (8:1) was found to be optimum and was patented [127]. To broaden the substrate specificity of biocatalysts, the presence of multiple genes capable of desulfurization of different substrates was described. A strain capable of desulfurizing both benzothiophenes and DBTs was developed by transferring the dsz genes involved in the DBT desulfurization into microorganisms capable of desulfurizing benzothiophenes. Two strains, Rhodococcus pRKPP/T09 and Rhodococcus pRKPPR/T09 were prepared from Rhodococcus T09 strain (FERM P-17268) in this manner by transferring the genes derived from R. erythropolis KA2-5-1 (FERM P-16277) [124]. Some biocatalysts based on dormant cells were developed from microorganisms of the genus of Rhodococcus or genus Corynebacterium [123]. The objective was to decompose recalcitrant alkylated benzothiophenes contained in fossil fuels such as petroleum. The targeted sulfur heterocyclic compounds included benzothiophene, 2-methylbenzothiophene, 3-methylbenzothiophene, 5-methylbenzothiophene, and 2-ethylbenzothiophene. The Rhodococcus strains might be T09 strain, but also T14 strain of the genus Corynebacterium, which were cultured in a medium containing the above mentioned sulfur-containing heterocyclic compounds. An improving in the culturing technique was conceived as feeding the sulfur solely source compound as a rate equal to that of the microorganism intake so as needed for growth [126]. This optimization technique is believed to act in two ways, for the proliferation efficiency of the microorganism in the culturing medium and for the desulfurization activity and substrate specificity. Their Intellectual Property (IP) in Japan includes a number of MB/GE related patents. Some examples regard the gene encoding flavin oxidoreductase for the redox cycle NADH/FMN (JP00245478 [112]) and encoding the desulfurizing enzymes (JP00245477 [113], EP1069186 [119], US6420158 [130], and US6479271 [128]). As can be derived from what is mentioned in the presiding Section 13, the corresponding IP in United States for the enzymes and encoding genes belongs to Enchira (EBC). The sequencing of novel gene-encoding enzymes which decompose recalcitrant thiophene compounds was first protected in Japan, then in US and Europe [119,120,128,130]. The environmental benefits of using such enzymatic catalyst for sulfur removal from fossil fuels were explicitly described in the patent. A new gene, which encodes a protein with a specific amino acid sequence and has a function for oxidizing NADH and reducing FMN, when it acts in cooperation with another enzyme active for degrading thiophene-based compounds, can be used for desulfurization of fossil fuels and so on [112]. The new gene encoding such specific oxidoreductase or its modifications (obtained by deleting, substituting, adding, or inserting one or more amino acid (s) from, in, to, or into the amino acid sequence, and also has a function of oxidizing NADH and reducing FMN) was protected in the same patent. That oxidoreductase is particularly useful in biodesulfurization, since its cooperation would maintain the desulfurization activity with no need for NADH or FMN make up. This

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gene is obtained from DNA derived from P. polymyxa strain A-1 as a template and primers consisting of its partial sequences by PCR. The desulfurizing enzymes were found to be encoded by a new gene, which specific amino acid sequence was characterized [113]. The enzymes function as a ferredoxin reductase subunit of DBT dioxygenase, converting the organic sulfur from thiophene compounds into water-soluble compounds. The enzyme was obtained by screening a DNA library derived from Burkholderia sp. strain C1 for a DBT-degrading activity. The new gene coding the desulfurizing enzymes or any of its modifications (prepared by deleting, substituting, adding, or inserting one or more amino acid (s) from, in, to, or into the amino acid sequence, and has a function as a ferredoxin reductase subunit of dibenzothiophene (DBT) dioxygenase) was protected in the same patent. Since, it catalyzes the conversion from a thiophene-based compound to a water-soluble compound, and allows the removal of sulfur components from fossil fuels and so on, its application for BDS was included in the patent. The biomolecules derived from the thermophiles, genes encoding enzymes, enzymes, vector including the gene, and the transformant were all protected as thermally stable biocatalysts [116]. Besides BDS, pioneering work at PEC included biodemetallization as well. A method for decomposing porphyrin-type compounds was defined. The method uses P. azelaica YA-1(FERM P-15356) [125,129], which was first cultured in an (unspecified) medium. Following, the microbial strain is cultured in a meat extract-containing medium doped with vanadium containing porphyrin, vanadium octaethylporphyrin or one of its metal complexes. The bacterial cells were used as spawns, for decomposing the porphyrin-based compounds under mild conditions. Whether the actual removal of the porphyrin metals (nickel or vanadium) from fossil fuel occurs by the microbial action or whether it is removed in a subsequent stage, is not clear from the available patent document. Moreover, even though the porphyrin complex may be converted, there is no evidence given on actual demetallization.

24. KANSAI ELECTRIC POWER (JAPAN) Kansai Electric Power of Japan was created in 1951 from a reorganization of the Energy Sector in Japan. The power generation in Japan is achieved using diversified energy sources, so the infrastructure consists of thermal, hydro, and nuclear stations. R&D portfolio consists of areas supporting their actual operations, but also emerging and future technologies related to energy and environment. Topics such as hydrogen-based energy, revolutionary nanotechnologies for new generation of energy sources, chemical absorbents of CO2 are part of the new technology pipeline. A microorganism for the conversion of hydrocarbons and the method of use for that application was awarded to the Kansai Electric Power [131]. The Vibrio furnissii M1 (FERM P-18382) is said to possess a rather unique proportion of cracking activity in comparison to degradation activity, which could consequently be employed in manufacturing synthetic crudes. The microorganism has been isolated and cultivated using adequate carbon sources, such as lower fatty acids (acetic acid, maleic acid, succinic acid, propionic acid, and starch, cane sugar, etc). The use involves direct contact under anaerobic conditions, followed by separation. The conversion of organic waste is reported to produce n-alkanes, such as C12 H32  C21 H44  C22 H46 , and the C24 H50 , all in the kerosene/diesel range.

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25. KOREA ADVANCED INSTITUTE OF SCIENCE AND TECHNOLOGY (KOREA) The Korea Advanced Institute of Science (KAIS) was founded in February 1971, which 10 years later merged into the Korea Advanced Institute of Science and Technology (KAIST). Since then, its growth has been driven by the incorporation of new groups and organizations from different origins. By 2006, its staff was up to 700, including faculty members along with a registry of nearly 8000 students, national and international in all levels (undergraduate and graduate schools). The productivity of the research results has been continuously increasing in time and in average is about four international articles per faculty. That is the consequence of a very aggressive international cooperative academic program with France, US, Germany, Japan, UK, Russia, China, Australia and Poland; and a close relationship with industry. The biotechnology activities focus on (fundamental and applied) research in genomics, structure and function of genes, nanobio area, design and evolution of organisms, study of diversity in animals and plants, cell signal transmission, cell cycle control and carcinogenesis mechanisms, medicinal chemistry, human disease research, physiology of nerve cells and related phenomena, cell culture engineering, medical engineering, transmission of medicine, environmental science, fermentation and fermenting processes for precision biochemicals production (antibiotics and biological macromolecules), foods and organic active substances, and evaluation of safety. In the area of petroleum refining, they hold five patents: • A novel Klebisiella oxytoca which removes sulfur from fossil fuel that contains organic sulfur compound and desulfurizing method [132]. • Recombinant coliform bacillus expressing desulfurizing enzyme and biological desulfurization method using thereof [133]. • Gordona sp. CYKS1 (KCTC 0431BP) capable of desulfurizing fossil fuel containing organic sulfur compounds [134]. • Norcardia sp. CKYS2 (KCTC 0432BP) capable of desulfurizing fossil fuel containing organic sulfur compounds [135]. • Bioelectrochemical desulfurization of petroleum [136]. Several microorganisms for use in BDS have been discovered by the Korea Institute of Science and Technology, among them, Klebisiella Oxytoca [132], a recombinant coliform Bacillus [133], Norcardia sp. CKYS2 (US6197570) [134] and Gordona sp. CYKS1 (US6204046) [135]. The first two were awarded by the Korean Patent Office and there was not possible to extract any information from those Korean patents. However, information related to the latter three has been published in open literature and was described in Sections 2.2.2 and 2.5 in Chapter 3. From their patent title, the first one is supposedly use as whole cell catalyst, while the Bacillus expresses a BDS enzyme, which could be used separately. The last two microorganisms, patented in US, are claimed to be active towards a wide spectrum of organic sulfur compounds: methyl sulfide, thiophene, thiazole, 2-methyl thiophene, 3-methyl thiophene, 4,5-dimethyl thiophene, thianaphthene, phenyl sulfide, benzyl sulfide, dibenzothiosulfone. The biocatalytic material protected in the patent includes whole cells, cell extracts from the claimed strains, recombinant microorganism harboring genes related to desulfurization and enzymes isolated from these organisms.

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Their intellectual property also included a hybrid bioelectrochemical desulfurization process, which was awarded in their first refining biotechnology patent [136]. This bioelectrochemical process is a method for removing sulfur from fuels. The sulfur compounds are converted into hydrogen sulfide by subjecting the fuel to an electrochemical reaction in the presence of a bacterium. The claimed bacterium has the ability to catalyze the reduction of sulfur compounds. The intellectual property does not include the cultures, which were obtained from the National Collection of Industrial and Marine Bacteria (NCIMB). Two species were considered, namely Desulfovibrio vulgaris NCIMB 8303 and Desulfovibrio desulfuricans NCIMB 8310. The anaerobic procedures described by Kim et al. [137] were used in all experiments. The electrochemical process components used were conventional. A two compartment type of cell held together by a U-shape agar bridge. The electrodes could be made from platinum wires or graphite. The reaction mixture comprised a sulfate-free medium C with 2 mM methyl viologen, the biocatalyst suspension (whole cell of sulfate reducing bacteria) and petroleum in the cathode compartment, maintained anaerobically. Methyl viologen was used as electron transfer between electrodes and bacterial cells.

26. KURASHOV, VIKTOR MIKHAJLOVICH (RUSSIA) Viktor Kurashov holds two patents awarded individually in his name: • Microbiological method of sulfur and nitrogen content decrease in petroleum and hydrogen sulfide in deposit waters and casing-head gases [138]. • Microbiological method for enriching petrol and petroleum products by isoparaffin and aromatic hydrocarbons with simultaneous removal of injurious additives and means for carrying out said method [139]. He discovered microorganisms, which have the capability for working in situ, anaerobically, in the well. These discovered microorganisms seem to decrease S and N content from oil and convert also H2 S, resulting of potential interest for both, gas and oil wells. Unfortunately, both of his patents are in Russian and only the abstract is accessible. A microbiological method, which involves contact of an aqueous suspension of bacteria with petroleum for performing sulfur and nitrogen removal, uses bacteria T. denitrificans, T. aquaesulis, or Thiosphaera pantotropha [138]. The previous adaptation of bacteria of these species to hydrocarbons is carried out preliminary by culturing bacterium strain on petroleum-containing media. The process is carried out in situ under anaerobic conditions directly in the oil wells. The process conditions are a temperature of about 25 C to 50 C and pH between 6 and 9. However, in salted layers and at temperature above 50 C the treatment is also possible. These bacteria may oxidize the sulfur and nitrogen heterocycles to carbonyls or phenolic compounds, without breaking the C−S or C−N bonds. The new polar compounds formed might easily solubilize into the aqueous phase, thereby removing them from the oil phase. The second patent [139], also in Russian, with only an English abstract, regards a microbiological conversion method, which results in an enrichment on isoparaffin and aromatic hydrocarbons of the petroleum products. It also states an unclear fact of simultaneously removing ‘inhibitory additives’. The abstract specifies the nature of

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these additives as being mainly composed of sulfur, nitrogen, and oxygen, therefore the term ‘inhibitory additives’ is probable due to a mistranslation, rather than to a real addition of other compounds. It is interesting that the conversion process leads to an isoparaffin and aromatic enrichment, which could have a potential application for diesel cetane enhancement. The described reactions include isomerization and aromatization of lineal hydrocarbons with boiling points equal to or less than 200 C. The conversion reactions also involve the breaking up of high-boiling polycyclic, polyaromatic and hybrid petroleum hydrocarbons to yield lighter low-boiling cyclic, aromatic, isoparaffin, and methane hydrocarbons. The biocatalysts are microorganisms of the T. aquaesulis species suspended in a biologically acceptable medium. Other claimed microorganisms include T. thioparus and/or T. denitrificans in any possible combination. The process is carried out in a reactive medium at a temperature ranging approximately from 4 C to 60 C and a pH ranging approximately from 6.0 to 9.0 under stirring.

27. KYUSHU KANKYO KANRI KYOKAI (JAPAN) The Kyushu Kankyo Kanri Kyokai or Kyushu Environmental Control Association is a consortium type of organization supported by industry and government. It is devoted to the study, monitoring, management and assessment of the environmental emissions and conditions, and to the design of correcting and prevention plans related to global warming. The nuclear area comprises the management and processing of ecosystems and environmental radioactivity. There is only one patent awarded to Kyushu Kankyo Kanri Kyokay related to biocatalyst manufacturing process. A method for culturing and growth of microorganisms with oxidizing and decomposing sulfur compounds capabilities was described [140]. However, the sole novelty provided by this patent is the inclusion of an inorganic sulfur source during the growth and culturing of the microbiological catalyst. The method efficiently renders a microorganism culture useful for the decomposition of sulfur compounds contained in petroleum, particularly heavy oils. The microorganism has been isolated from sludge of industrial wastewaters containing sewage and sulfur compounds. The growth takes place by adding a water-soluble inorganic sulfur compound to a biological reaction tank. The mentioned inorganic sulfur compound can be a thiosulfate compound or a thiocyanogen compound but also water-soluble organosulfur compounds such as thiourea can be employed. The removal of sulfur from petroleum products is performed under aerobic conditions, using the developed cultures (T. perometabolis and T. rubellus) in aqueous media.

28. LAMBDA GROUP INC. (UNITED STATES) The Lambda Group Inc. was an industrial organization involved in coal business, which supported the development of a coal cleaning process led by Jo Davison, during the second half of the 1980s. The developed process aimed for the removal of iron and sulfur from coal. While several patents for microbial removal of pyrite from coal exist, this one is unique because of the microbial mixture developed by the group for this purpose, which is partially

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self-sustaining in terms of the nutrient requirements and also supports product separation in addition to conversion of the iron and sulfur. The process requires a specific sequence of microbe deployment, which includes T. ferrooxidans, T. thiooxidans, T. thioparus, T. neopolitanus, T. acidophilus, Euglenoids, and Cyanochlorophyta; which is done in two sequential stages using mixtures of microbial species as described below [141]. The overall process is a five-stage process which includes separation steps as well. Removal of iron can be regarded as a BDM process and it should be noted that metals other than iron present in coal can also be removed in this process. Iron pyrite is the highest metal contaminant in coal along with organic sulfur compounds. This process removes the inorganic sulfur and iron contaminants via an oxidative mechanism which may be recovered at the end of the process. In an open report, this company informed how the process evolved into three different applications, depending on where the coal was cleaned and what part of the coal was being cleaned. Only the latter is fully based in the awarded patent. The process was presented internationally as the ‘Lambda Coal Cleaning Process’ [142] in 1987, but after that announcement there is no additional information on whether it reached commercial demonstration.

29. MARINE BIOTECHNOLOGY INSTITUTE CO LTD (JAPAN) The Marine Biotechnology Institute (MBI) was founded in 1987 for the study and applications of biodiversity in marine environments. Currently, and within the scope of the present book, the research area of relevance is ‘industrial applications of microbiology’. The institute also performs research in CO2 fixation. The Applied Microbiology Laboratory is searching cost-effective utilization of microbes for environmental conservation and restoration. In particular, the decontamination of areas polluted with petroleumrelated compounds has been addressed. Attention has been paid to removal of polycyclic hydrocarbons. They are also developing processing methods for the conversion of biomass and organic wastes by methane fermentation. Their activities in desulfurization lead to a hybrid process, protected under the title of: ‘Method for electrobiologically desulfurizing petroleum’ [143], awarded to the Marine Biotechnology Institute by the Japanese Patent Office [143]. This method is based on contacting anaerobic sulfur-oxidizing bacteria with petroleum under anaerobic condition or microaerophilic conditions. The bacteria used belonged to Proteobacteria or Thiomicrospira bacteria.

30. MICROBES INC. (UNITED STATES) Microbes, Inc. founded in 1988, is a Delaware corporation, whose business includes two areas: increasing yields from (1) oilfields and (2) food crops. The oil-related business entails the development and supply of microbial products and services for enhancing oil recovery from existing reservoirs. National Parakleen Company, Inc. is a subsidiary, which since 1988, have applied their proprietary technology in oil wells and reservoirs, jointly with Microbes, Inc. Their enhanced recovery technology (Migration Microbial

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Enhanced Oil Recovery, MMEOR) was successfully demonstrated at pilot scale in China, Argentina, Indonesia, and Former Soviet Union oilfields. A commercially application trial in the U.S. demonstrated its industrial viability. The technology is based upon cultivating microorganisms and introducing these bacteria into petroleum reservoirs through production and injection wells. The strains colonize and migrate outward, living at oil–water interfaces in the water. The acquisition of Advanced Microbial Systems, Inc., brought about a complementary technology for increasing the microbial concentration to be economically employed in their applications. The basic concept is a process to convert oil bearing rock formations into bioreactors. This oil reservoir bioreactor is used to sustain microbial life and thrive under high temperatures and pressures in the absence of oxygen. The microbes are supposed to metabolize portions of the crude oil resulting in an improved oil viscosity, which facilitates recovery. Two different unproved hypotheses are used to explain microbial action: surfactant production and molecular conversion (C−C bond cleavage). A produced surfactant in the oil-and-water interface may reduce the viscosity of heavy oils into thinner, less viscous components, as would a conversion of heavy molecules into lighter compounds. An invention regarding the biocatalytic chemical transformation of the crude oil was disclosed in their only patent directly concerning refining processes: ‘Microbial-Induced Controllable Cracking of Normal and Branched Alkanes in Oils’ [144]. This different approach was claimed for upstream and downstream applications, including reservoir, oil transportation, surface storage oil and refining, was based purely in chemical transformation. Here we will briefly consider the potential refining application. Biocracking is a process, which has not received much attention and very little has been practically done and so achieved. The cracking of normal and branched alkanes in oils was microbially induced via a mechanism referred to as Repetitive Alternating Carboxylation-Decarboxylation Cycle (RACDC) [144] yielding, in consequence, a volume enhancement. The treatment results in intermediate, products such as crude oil emulsions and refining oil cuts.

31. NI AOOT; VATEL SKIJ INST NEFTEPROMYSLOV (RUSSIA) A strain of Pseudomonas Sp. 45 was isolated from a soil contaminated with gasoline and adapted for the desulfurization of oil and petroleum products [145]. The strain was able to grow on mineral medium containing oil or petroleum products as a carbon source at pH 6.8 and temperatures of about 25 C. The decrease of sulfur content in oil achieved is in the order of 30–50%. Sulfur is said to be removed from oil as sulfates and hydrogen sulfide, which requires the simultaneous or sequential occurrence of oxidation and reduction reactions. It would be interesting to know the sequence of events leading to such product formation. Self-disproportionation reactions are unknown in sulfur compounds. One could imagine oxidation and reduction reactions taking place in a single process but in different stages, under different set of conditions. Probably, this is what the inventors explain in the Russian document but we were unable to verify.

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32. NIPPON OIL CO LTD (JAPAN) Nippon Oil Corporation (NOC) created in 1888 is now part of the Eneos Group, a transnational corporation devoted to the energy business. Technology development in the group is decentralized and is carried out in NOC’s Central Technical Research Laboratory, Research & Development Department, and in other departments. The R&D programs develop and improve petroleum products and refining technologies, carbon fibers, cogeneration systems, biotechnologies, high-performance polymers, petrochemical-related technologies, and construction and environmental protection technologies. The biotechnologies developed by the NOC Group are currently used in remediation of contaminated soil and water. Those technologies have been commercially proven in on-site tests. In the context of this book, Nippon Oil holds only one patent for the bioproduction of (high cetane/high lubricity) low-sulfur diesel. A process for biocatalytic production of low-sulfur gas oil [146] was patented to Nippon Oil. This was based on microbial oxidation of naphthalene rings, which apparently reduced the sulfur content, while increasing the alkali extractables of the gas oil. The final product was a low-sulfur, high-lubricity diesel oil. The bacterial approach is thought to achieve this goal by selectively converting the di-aromatic rings, into high cetane compounds, avoiding a drastic increase in paraffinic content. The targeted specification was then set in terms of the alkali extractables (sulfur and oxygen containing aromatics), particularly, because the biocatalyst was selected from the naphthaleneconverting microorganisms. The naphthalene oxidization products include 1,2-dihydroxy naphthalene, 2-naphthoic acid, a salicylic acid, 4-methylsalicylic acid, a 6-methyl-2naphthoic acid, etc. Setting the alkali extractable to a maximum will result in the highest achievable cetane improvement, while the minimum value would allow monitoring the advance in desulfurization. Some of the naphthalene oxidation strains mentioned, e.g., belong to the Pseudomonas genera, such as P. putida and P. aeruginosa; Bacillus, Corynebacterium, Rhodococcus genera, Nocardia genera, Streptomyces, and Sphingomonas genera, etc. The most preferred strains were P. putida, P. aeruginosa and R. erythropolis for their combined capacity for oxidizing naphthalene and decomposing dibenzothiophenes.

33. OLDFIELD, CHRISTOPHER, COURT OF NAPIER UNIVERSITY Oldfield discovered microorganisms capable of desulfurizing benzothiophenes and DBTs [147]. The strains were isolated from oil shale spoil heap by repeated culturing in a mineral salts, glycerol, and Rhodococcus medium (GlyRM) containing benzothiophene as sole sulfur source. Single colonies were picked by streaking agar plates overlaid with benzothiophene. The strains were identified as Rhodococcus sp. NUE213E and NUE213F and deposited with NCIMB under Accession No 40816 and 40817, respectively. Both organisms were Gram-positive, aerobic, non-motile, partially acid-fast, catalase positive, and oxidase positive. The strains were characterized as being V-shaped rods of irregular length when grown in both FruRM/sulfate and FruRM/BTH and the physiological characteristics did not change during growth cycle. The Fructose-Rhodococcus medium (FruRM, pH = 72) contained fructose (10 g/l), together with the following components

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S

(2) BTO

(5)

O

(3) BTO2 O

O

O

S

(9) Unknown product

S

S

O

S

O

(4) O

O

(6)

O

OH

O

(8)

O

(7)

Figure 2. Benzothiophene metabolic pathway.

(per liter): Na2 HPO4 , 4.33 g; KH2 PO4 , 2.65 g; glucose, 20 g; NH4 Cl, 2 g; MgCl2 6H2 O, 0.64 g; nitrilotriacetic acid, 0.1 g; CaCl2 2H2 O, 33 mg; ZnCl2 , 2.6 mg; FeCl2  4H2 O, 2.6 mg; EDTA, 1.25 mg; MnCl2 4H2 O, 1.0 mg; CuCl2 2H2 O, 0.15 mg; CoNO3 2 6H2 O, 0.125 mg; NaB4 O7 10H2 O, 0.10 mg; NH4 6 Mo7 O24 4H2 O, 0.09 mg. Fructose was added as a filter-sterilized solution following autoclaving (121 C, 20 min). The genes corresponding to the enzymes involved in conversion of benzothiophene from the claimed organisms as well as the vectors carrying the genes were protected in the patent. The details of the metabolic pathway were given in Section 2.2.3, the proposed metabolic pathway is shown here for completion purposes (Fig. 2). This pathway is not an exact mirror of the DBT 4S pathway. Here, the sulfinate group of HBESi, which is an alkylsulfinic acid, may not be removed by hydrolysis. Hence, there does not seem to be a DszB equivalent enzyme, in the BTH desulfurization pathway. Instead, the sulfinate group seems to be removed by hydroxylation of the C−S carbon bond, yielding HEP. Two hypothesis were suggested regarding this mechanism: (i) a flavin-dependent hydroxylase enzyme catalyzing the final ‘oxidative desulfination’ converting HBESi into HEP; (ii) the conversion of HEP to BFU is probably a noncatalyzed reaction. Although, HEP can exist in both cis- and trans-isomeric forms, the present reaction is expected to yield the cis-isomer mainly.

34. PAQUES BIOLOGICAL SYSTEMS BV (NETHERLANDS) Paques is a medium-sized company operating on an international basis, which has been developing and producing purification biotechnologies for gas (and water), since the beginning of the 1980s. Technology development includes research and development, pilot testing, engineering design progressing to project management and

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contracting. Their commercial experience is extended through more than twenty countries. Paques offers two products to the petrochemical industry since 1996: THIOPAQ® and BIODESOX® process. The University of Wageningen, the Technical University of Delft, Shell Global Solutions and UOP participated in the development and commercialization of THIOPAQ® process. The first biological purification plant was initiated early 2000, in cooperation with UOP and Shell Global Solutions. THIOPAQ® scrubber for biological desulfurization of various gas streams. In the removal of H2 S from methanerich streams, first the stream is washed out in an absorber and then the H2 S is removed from the wash water by anaerobically converting it into elemental sulfur. The removal of SO2 from flue gas is carried out with BIODESOX® process. Similarly, the SO2 is washed out from the contaminated stream by the scrubbing liquid in an absorber. The biological process is used to convert SO2 to elemental sulfur in two steps. The only one patent that has been awarded to Paques Bio Systems b.v. to protect the above described technologies, globally practiced nowadays: ‘Process for Biological Removal of Sulfide Process for Biological Removal of Sulfide’. The sulfur compounds sensitive for such process include: sulfides, such as hydrogen sulfide, carbonyl sulfide, and carbon disulfide [148]. It consists of scrubbing the gas with an aqueous washing liquid and treating the washing liquid with sulfide-oxidizing bacteria. Other sulfur compounds (in which the S oxidation state is −2) that can also be treated include lower alkyl mercaptans (methane-thiol), lower alkyl sulfides and disulfides (dimethyl sulfide). The treated gases may also contain carbon dioxide, which is known to contribute to the H2 S loading capacity of the scrubbing liquid, especially at high pressures. The presence of an electron acceptor (nitrate) is required and the treated liquid could be reused as a washing liquid. A single reactor is employed for both stages. The genus Thiobacillus, especially the species T. denitrificans catalyzed the oxidation reactions of hydrogen sulfide yielding soluble hydrosulfide compounds, elemental sulfur, and sulfuric acid. Carbonyl sulfide and carbon disulfide are converted to hydrogen sulfide by hydrolysis. Additionally, they are oxidized to SOx and sulfates via microbial action. The reported oxidation reactions of thiosulfate using nitrate as electron acceptor are: 5S2 O3 2− + 8NO3 − + 2OH− → 10SO4 2− + 4N2 + H2 O 5S2 O3 2− + 2NO3 − + H2 O → 5S + 5SO4 2− + N2 + 2OH−  Regarding the conditions for the biological oxidation, the optimum pH is in the range of 7–9, temperature of about 30 C, nutrients supply and the electric conductivity of the washing liquid must be kept between 30 and 100 mS/cm. In general, neutrophilic sulfide-oxidizing cultures of Thiobacillus species can be used, especially T. denitrificans.

35. PETROLEO BRASILEIRO SA (BRAZIL) Petrobras is a public corporation dealing with the energy business, with operations in various countries, but mainly in South America. Cenpes is its Research and Development Center, whose purpose is to meet Petrobras’ technological demands. Petrobras has been using the technology as its basis for consolidation and expansion in the world energy

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scene. Cenpes has more than 1500 employees and its IP portfolio contains nearly 500 patents. A Strategic Refining Technologies Program (PROTER) was initiated in 1994 focused in the processing of heavy oil. The advance technological solutions have to consider integration and optimization of the entire productive chain. The objective of the developed technologies is to optimize the existing facilities by increasing the residual fraction conversion and maximizing the volume of premium fuels. This program contains projects in the Biorefining area: such as the biodesulfurization, bio-denitrogenation and metal removal from oils and refining fractions. They have already disclosed four inventions, which have been protected through: • • • •

Microbial cleavage of organic C−N bonds [149]. Pseudomonas ayucida useful for cleavage of organic C−N bonds [150]. Microorganisms useful for cleavage of organic C−N bonds [151]. Bacterial cleavage of only organic C−N bonds of carbonaceous materials to reduce nitrogen content [152].

Petrobras has been working with IGT on development of BDN and has reported three patents in this area [149–152]. They have discovered a mutant P. ayucida strain (ATCC N PTA-806) which can selectively remove nitrogen from organosulfur compounds present in petroleum. Based on the patents and the published literature, the laboratory stage of this development seems to be complete with respect to identification of a nitrogen-selective strain. The details of this strain has been discussed in Chapter 3 along with the other BDN strains including pseudomonads which forms the majority of the organisms involved in BDN. Most of these organisms and their application for BDN have been protected in various patents. The three patents awarded to Petrobras/IGT contain a total of 27 claims. The intellectual subject matter has been also patented in Europe, Mexico, and Brazil. The first two patents [150,151] protect the biocatalytic system and its use in the form of whole cells or cell-free extracts or enzymes purified from the organisms. The other claimed organisms in the patent include the thermophilic culture Aneurinibacillus sp. IGTN4T (ATCC N PTA-4581), P. stutzeri, Yokenella sp. and P. nitroreducens. The following text gives the details of the strain PTA-806, since the others have not been reported in much detail. The patent documents indicated a nitrogen removal of only 5%, which most likely corresponded to the quinoline conversion of 68%. However, quinoline was thought to be denitrogenated through the coumarin pathway, but instead its total degradation was reported.

36. PETROZYME TECHNOLOGIES INC (CANADA) CVF Technologies Corporation, founded in 1989, is a technology development company, which fully owns Petrozyme Technologies Inc. The main technological product of Petrozyme is a fermentation method for the safe on-line remediation of sludge. The technology can be employed in the treatment of streams in oil refineries and chemical plants. The only patent they hold in the scope of this book, does not refer to a refining process itself, but rather to a complementary method, which could enable the feasibility of the other refining bioprocesses.

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A complementary process for breaking an oil-water emulsion is needed in conjunction with any other biological process, which uses an emulsified medium to diminish the mass transfer limitations commonly found in microbial treatments. Besides, the same need exists in several oil exploration and production activities. A biological process for breaking oil-water emulsions has been developed [153]. The biological approach comprises contacting the oil-water emulsion with a bacterial culture produced by growth in a liquid medium containing hydrocarbons under non-sterile conditions. The bacterial culture is claimed to cause minimal degradation of the oil. The mechanism of emulsion breakage appears to be degradation or modification of the emulsion stabilizing agents. The form of application may be whole bacterial cells or culture extracts. Very little is said regarding the strain itself. The only characteristic mentioned is its capacity to degrade or to transform one or more components of oil, chemicals, and nutrients added to oil. The microbial culture is also capable of oil sludge biodegradation. The oil-water emulsion is contacted with the bacterial culture, under non-sterile conditions, at temperatures between 20 C and 35 C, for about 5 days. Under these conditions, an oil layer and a water layer are formed permitting their separation. Shaking or aeration may not be required.

37. PETROLEUM INDUSTRY DEVELOPMENT CENTER (SEKIYU SANGYO KASSEIKA CENTER); MITSUBISHI OIL CO LTD (JAPAN) Three biotechnological patents have been awarded to Sekiyu Sangyo Kasseika Center and Mitsubishi Oil, which are focused on three different application areas, namely separation, demetallization, and desulfurization: • Separation of oil and water in microbial reaction of oily and aqueous two-phase system [154]. • Microorganism capable of degrading alkylated heterocyclic sulfur compound [155]. • Microbial desulfurization of sulfur-containing hetero-cyclic compound [156]. The separation method targets recovery of the aqueous phase from oil/water mixtures of microbial reactions by filtration through a ceramic filter module [154]. The invention particularly referred to a two-phase system resulting from a process used for production of 2,6-naphthalenedicarboxylic acid using S. paucimobilis AK2M16 (FERM P-13996). The aqueous phase is said to be recovered free of microbial cells and oil. Although, it is mentioned that the reaction product can be recovered readily in high yield, the need for an additional unit operation looks obvious. Two patents were awarded on microbial desulfurization of sulfur-containing heterocyclic compound [155,156], the first targeting DBT and alkylated DBTs and the other benzothiophenes and alkylated benzothiophenes. In both cases, the selective cleavage of the C−S bonds is reported as the main mechanism. The claimed bacteria strains are Mycobacterium G3 strain (FERM P-16105) and R. erythropolis KA2-5-1 strain (FERM P-16277), respectively. Special emphasis was made to the desulfurization of the recalcitrant 4,6-dimethyl-dibenzothiophene. The main product from DBT

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was 2-hydroxybiphenyl. The new strains were obtained by screening soil samples collected throughout Japan and testing the ability of the strain for degrading the mentioned compounds. The isolation technique and adaptation methods were not reported.

38. PLUMMER, MARK A (UNITED STATES) Plummer invented a process for the biodesulfurization of hydrocarbons [157], in which organic sulfur compounds contained in liquid hydrocarbons are converted to elemental sulfur. The reaction is carried out in the presence of a biocatalyst and hydrogen, by dissolving completely the liquid hydrocarbons in an organic solvent, such as a nucleophilic and/or electrophilic solvent(s). The nucleophilic solvent should have a pKa greater than 2, and the electrophilic solvent more negative than –2. Recommended nucleophilic solvents include n-butylamine, diethylamine, butanediamine, ethylenimine, toluene, pyridine, aniline, and acetophenone. The electrophilic solvents could be methylethylketone, pyrrole, or benzaldehyde. The liquid hydrocarbon stream to be treated may be a crude oil, heavy crude oil, bitumen, or a refined fraction of the crude oil. The hydrogen gas stream is added to the mixture of the hydrocarbon stream with the organic solvent. The reactor, which is fed upflow, is a packed bed of biocatalyst dispersed on a support and is operated at about 74 C. Alternatively, the reactor can also be a batch reactor under stirring conditions. The biocatalyst used in this invention is a microorganism such as that available from Finnerty Enterprises Inc., Athens, Ga. Two different conversion reactions occur, one exhibited by organic sulfides and another by organic thiophenes, namely; R1 − S − R2 + H2 → R1 H + R2 H + S R1

R2 S

+ H2 → HR1 R2 H + S

In cases where both types of compounds are present in significant amount, the use of a mixture of solvents, nucleophilic and electrophilic, was suggested. In such cases, an amphiprotic solvent could also be suitable (an amphiprotic solvent contains simultaneously an electron donating and electron accepting groups in a single molecule), for instance ethanolamine. The biocatalyst may be supported on a Lewis acid. Elemental sulfur is removed from the liquid hydrocarbons and the recovered solvent is counter-currently washed with water in a separate unit. Prior to reuse, the solvent is distilled to decontaminate it from remaining water or sulfur slurry. The treated product not only has a reduced concentration of organic sulfur compounds, but also its viscosity is reduced.

39. SHELL OIL CO (NETHERLANDS) Research and technology development in Shell Oil, one of the major companies of the energy sector, is organized in a decentralized organization. The best known is Shell Global Solutions, which besides R&D, also takes over consultancy and technology

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services to the petrochemical and processing industries. It’s refining experience and reputation is also very broadly realized; however, in biotechnology they have not been as prolific as in other technologies. Only one invention, within our scope could be found and it is not directly associated with oil refining. Shell’s microbiological desulfurization process is carried out by mixing coal with an aqueous biocatalyst solution [158]. The coal considered in this invention concerns bituminous coal containing inorganic sulfur (pyritic).This process seems to be applicable to refinery pet-coke, which contains sulfur in the form of inorganic sulfides. Nowadays, when coke has become one of the major products of heavy oil and bitumens refining, such desulfurization processes might have potential uses. The process involves two stages: grinding and desulfurization. The objective of the grinding stage is to pulverize the coal to a fine particle size (70–150 microns) in a wet grinding device, to form slurry. The wet operation needs less energy and produces better size distribution even from shear resistant coals. Water content and pH have to be adjusted prior to the desulfurization step. A 1:1 or 1:1.5 water to coal proportion is recommended. The bacterial strain is selected among species of the genera Thiobacilli, Pseudomonas, Alcaligenes, Bacillus, Desulfovibrio, Arthrobacter, Flavobacterium, Beijerinckia, Rhizobium, Acinetobacter, etc. Specifically recommended were: T. thiooxidans, T. ferrooxidans, T. acidophilus, and T. denitrificans. The process takes place by contacting the coal-water mixture with an aqueous solution of the selected microorganism, at temperatures lower than 90 C and during a time shorter than 170 h. Time and temperature are interdependent and have to be adjusted relative to each other. After desulfurization, the reacting mixture is separated by centrifugation, filtration, decantation or a combination of these techniques, but the coal recovered retains less than 15 wt% of water and no further drying is performed. A separation step capable of recovering the aqueous medium containing the bacteria would be desirable. This would allow the aqueous bacterial solution to be recycled to the desulfurization step. However, in this invention the bacteria are not recycled, since the coal–water mixture can be directly used for combustion purposes using specially designed power plants/furnaces available at Shell. If these were not available, energy for thermally drying the desulfurized coal might result in undesired inefficiencies. The coal-water mixture fed to the boilers has to be formulated to a certain specification. This is done with the proper inclusion of additives to facilitate combustion. The recommended additives include a petroleum-based surfactant (polysulfones or polycarboxylates), a stabilizing (e.g. xanthan gum), a biocide and an anti-foaming agent (such as a silicone base).

40. STANDARD OIL CO (UNITED STATES) Standard Oil was large, integrated, oil producing, transporting, refining, and marketing organization, which operated in the period of time from 1870 to 1911. Legal regulations (anti-trust laws) compelled the dissolution of the trust in 1892. Different laws in the State of New Jersey allows the reborn of the Standard Oil Company of New Jersey, but several other processes of the same nature lead to the Supreme Court ruling the reorganization into 34 different companies, among them Amoco (Standard of Indiana), Atlantic Richfield (the Atlantic side), Conoco (Standard Oil company in the Rocky

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Mountain states), Chevron (Standard of California), Exxon (from Standard of New Jersey), Sohio (Standard of Ohio), Marathon (covering western Ohio and other parts of Ohio not covered by Sohio), and Mobil (Standard of New York). These companies, derived from the Standard Oil Co., formed an original oil industry map in US, but that map no longer exist, rather the merging and acquisition processes reduced all them into four of the seven majors (ExxonMobil, ChevronTexaco, ConocoPhillips, and BP America). Standard Oil Co. early activities on coal desulfurization were awarded with two patents. These patents include novel microorganisms, such as Hansenula sydowiorum, Hansenula ciferrii, Hansenula lynferdii, and/or Cryptococcus albidus. The first invention [159] concerned the organic sulfur, but since the content of inorganic, pyritic sulfur, is larger than the organic, a pretreatment is needed when removal has to be above than 75%. The preparation method of the microorganisms was described in the patent document. The mixed culture of microorganisms identified as ATCC No. 39327, used in the study was capable of reproducible reduction of sulfur and was prepared in situ from normal soil by the enrichment technique using organic sulfur compounds. The in situ enrichment takes place firstly with sulfur compounds and subsequently in the presence of a coal substrate and nutrient medium, both of them at neutral pH. The sulfur compounds for the first adaptation period include cystine, cysteine, methionine, thiophene, thianapthene, beta-mercaptoethanol, methionine sulfoximine, methionine sulfoxide, and dibenzothiophene. The second patent describes the use of a microbial mixed culture (Hansenula sydowiorum, Hansenula ciferrii, Hansenula lynferdii, and/or Cryptococcus albidus) in coal desulfurization [160]. In this process, the raw mined coal is ground to a particle size smaller than 200 mesh forming a slurry with water, at a solids concentration of less than 40 wt%. The bacterial cultures are then inoculated into the feedstock slurry. An incubation step is carried out at a temperature near 25 C and at a pH close to neutral. The highest removal achieved was in the range of 46% S removal.

41. TECHNOLOGY LICENSING ORGANIZATION In Japan, a Technology Licensing Organization (TLO) was created as a corporation, for dealing with IP and licensing of academic inventions. This Corporation is then an ‘intermediary’ between industry and universities. The legal basis lays on a law promoting technology transfer from universities to industry. The academia-industry cooperation results are managed dynamically in a ‘cycle of intellectual creation’ as embryos for new businesses. Part of the profit is returned to the universities as research funds to close the cycle. A patent on microorganisms capable of heavy oil conversion was disclosed by Technology Licensing Organization (TLO) [161]. The claimed subject matter comprises the heavy oil degrading bacteria and their mixtures, the nurturing composition for such bacteria, the inexpensively preparation method and the simplified degradation method and removal operations. The document also mentioned the preparation procedure for storage and shipping. The method of degrading heavy oil using such bacteria, and materials containing a substance obtained from the heavy oil degradation treatment was also disclosed. These byproduct materials were described to be useful as building and

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construction materials. For refinery operations, a BCK type of process is desired, in which the extent of conversion could be controlled. However, for spill incidents a full and complete degradation scheme might be the preferred alternative; i.e., cases in which the bacteria mixture degrades the high and low molecular weight hydrocarbons. In this last case, a concentration between 106 –109 bacteria/g of heavy oil was found to be required. The ability of the organisms to grow in a nutrient-poor media was regarded as an advantage in terms of the cost of biocatalyst production and ability to transport them to field after their preparation. The invention also considered the manufacture of road paving material or construction material using the hydrocarbon-free sand produced when treating heavy oil contaminated soils. Production of a paving asphalt component by this treatment of heavy oil-contaminated sands might be of potential interest for increasing and diversifying the asphalt pool; however, the reported process took 56 days for treatment to obtain the recycling sand, thus making it of low practical value.

42. TONEN CORP (JAPAN) Tonen Corporation was originally established in 1939 and grew into the oil sector of Japan, particularly in the refining area. It merged with General Sekiyu K.K. (created in 1947) forming Tonen General Sekiyu K.K. on July 1, 2000. Each former company had a long history in the oil business of Japan and established several alliances with Exxon (Esso Japan) and Mobil, separately before these two companies also merged. Currently, ExxonMobil holds 50.02% of the share of TonenGeneral. Tonen Corp has targeted development of organic-resistant microorganisms via genetically engineering and using P. putida as the host organism. The biocatalyst has been also optimized for BDS reactions via genetic changes obtained by mutagenesis (irradiation with ultraviolet light). In Section 23.1 of this Chapter, the method for obtaining organic solvent-resistant microorganisms was described. This invention was awarded to AIST [103] and was shared with Tonen Corp. The microorganisms capable of desulfurization were mutated without jeopardizing the BDS catalytic properties leading to development of three P. putida strains reported in Section 23.1. First, the microbial parent strain is subjected to UV radiation mutagenesis and then to selective cultivation in the presence of 0.1% to 10 v/v% organic solvent concentration. A second patent related to BDS gene regulation has been awarded to Tonen. The specificity of the amino acid sequence of a protein participating in the regulation of the genes involved in the oxidation reaction of organic sulfur compounds was reported [162]. Particularly, the regulation of the expression of benzothiophene oxidase group gene was found to be useful for the desulfurization of petroleum products. The formula included in the patent represents the amino acid sequence responsible for the gene regulating expression of benzothiophene oxidase. It can be obtain from the modification of another amino acid sequence by deleting, adding and/or substituting one or plural amino acids while keeping the function for regulating the expression of benzothiophene oxidase. The genes from microorganism strains belonging to the genus Pseudomonas can be employed to obtain this protein. Ultraviolet irradiation treatment

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of the genes and protein separation were recommended as the means for producing the protein. The third patent is related to demulsification of oil–water emulsions. Microbial demulsification can be applied on a variety of situations, among them conventional petroleum refining emulsions (e.g., desalter emulsions), separation of oil components, bacteria and moisture from petroleum bio-processing emulsions (e.g., bio-desulfurization processing emulsions, bio-demetallization processing emulsions, and bio-chemical conversion processing emulsions) fall into the scope of the present book, and are particularly interesting when along with the separation, the efficient recovery of the microorganism is also attained [163]. Bacteria belonging to the genera Aeromonas, Alteromonas, or Rhodococcus were found to be capable of breaking W/O emulsions. They were identified, isolated and cultured from soil sludge samples, just by their emulsion breaking ability. Ordinary culturing methods are employed, such as nutrient liquid medium, containing a carbon source and nitrogen source, under aerobic conditions, agitation, or shaking, etc. The emulsion breaking mechanism includes hydrolysis of the surfactant (or the surface activating substances of the emulsion) by lipase enzymes. This is the case for the separation of kerosene and desalter emulsions by Aeromonas and Alteromonas. Lipase can either be secreted externally by the microorganism or stay on the surface of the bacterial cells. The method is carried out using 100 to 200 mg of bacterial cells per kg of oil in the emulsion, at temperatures lower than 35 C, with stirring for about a day. Viscosity changes immediately, and if the process is used for this purpose, it can be stop when achieving the desired viscosity. The separating method leads to the formation of a bi-layer, namely an aqueous layer and oil layer, which can be easily separated by decanting, or any other physical means.

43. UNITIKA LTD (JAPAN) Unitika is a Petrochemical company created in 1889 and which has grown especially into the textile business. Today, their business covers a wide range of economical areas, including financial activities. Its environmental division provides facilities for water treatment, incinerators, air pollution prevention facilities and heavy metal fixing agents. Their R&D center conducts research to develop new business areas and products in four segments: polymers, environment, advanced materials, and health. The patent held by this company (awarded in Japan) describes a Burkholderia strain for desulfurizing petroleum [164]. The English translation of the patent retrieved only one claim that protected the use of the bacteria in the desulfurization of crude oils. The preferred biocatalyst is a strain of Burkholderia PHN-5, which has been deposited under the ascension No. FERMP-15024. Certain degradative activity is retained by the microorganism, since it is explicitly mentioned that the desulfurization of petroleum is carried out ‘almost’ without causing the decomposition of aliphatic hydrocarbons. Therefore, the heating value of the oil is lost in the process due to degradation of aliphatic hydrocarbons. The process conditions for an effective contact between the oil with the microorganism include stirring, pH control, and aeration. Immobilization on a fibrous support or on a ceramic material was also mentioned in the document. Although the method claimed to decrease the sulfur in petroleum to 0.001–0.05%, no

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direct evidence for treatment of oil feedstocks are provided in the document, or are the products identified. The desulfurization of DBT was described without much detail.

44. UNIVERSIDAD DE ALCALÁ, UNIVERSIDAD COMPLUTENSE DE MADRID, AND CONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS (SPAIN) Use of an alternate flavin:NADH reductase, encoded by hpaC gene from an E. coli strain W ATCC 11105, for improving BDS conversion has been patented by CONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS (CSIC) [165]. The hpaC gene was cloned into a BDS biocatalyst, P. Putida KT2442 carrying the dsz gene cassette from R. erythropolis IGTS8 ATCC53968. The recombinant strain is the result of constructing a pBG8 plasmid with a broad host spectrum. As explained in detail in Chapter 3, the flavin:NADH reductase catalyzes the NADH reduction reaction, i.e.: FMN + NADH + H+ → FMNH2 + NAD+  Under BDS conditions, the HpaC enzyme transfers reducing equivalents, FMNH2 from NADH to DszA and DszC monooxygenases. After proving the role of HpaC enzyme, in vitro, a pBG1 plasmid was constructed for cloning into the Pseudomonas strain. The hpaC gene was cloned in the broad spectrum Ptac promoter of pVLT31 host. The pBG1 was then constructed, by extracting a DNA fragment containing the hpaC gene from pAJ28, using EcoR1 and Patl restriction enzymes and linking it to the pVLT31 plasmid. The enhanced activity of the flavin:NADH reductase in the newly created recombinant Pseudomonad was confirmed prior to further development. The next step was to combine the reductase activity with the BDS activity in the recombinant strain. The process began with the amplification of the hpaC gene, from the pAJ28 plasmid. The DNA fragment from the amplification reaction was digested with EcoR1 and Xtol restriction enzymes and linked to the pESOX1 plasmid and pUC18 plasmid derivative, which contains dszABC and confers ampicillin resistance. The result was a plasmid named pBG7. This plasmid already contains all the desired genes in an operon form, but can only be replicated in E. coli. Thus, subcloning in the pVLT31 plasmid was necessary and the pBG8 plasmid was obtained. Finally, the ability of the recombinant strain, with enhanced DBT conversion was demonstrated.

45. UNIVERSITY OF OSAKA (JAPAN) The University of Osaka is the holder of two patents regarding the least studied biorefining processes, demetallization, and bioconversion. The metals are removed from the fossil fuel, under mild conditions (room temperature and atmospheric pressure) by the microbial oxidation action and a UV-photochemical reaction [166]. The bioconversion refers to conversion of high molecular weight alkanes by the action of B. thermoleovorans B23 and B. thermoleovorans H41 strains to lower molecular weight molecules [167].

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46. UNIVERSITY OF SHANDONG (CHINA) A process for deep desulfurization of fossil fuels using Mycobacteria [168] has been developed by the University of Shandong. The given information is limited to the steps they have taken so far, such as isolating, identification, culturing, and testing the Mycobacteria. They also claimed to provide a simple process, operated at room temperature, with low energy consumption and obtaining high desulfurizing rate.

47. UNIVERSITY OF WASEDA (JAPAN) The researchers from Waseda University have been following the most recent GE strategy for developing improved biocatalysts with desulfurization activity. They hold two patents, the first concerns with the use of Mycobacterium frei WU-0103 strain in a method for decomposing heterocyclic sulfur compounds [169]. This bacterium has the ability for desulfurizing DBTs, benzothiophenes, naphthothiophene, and their alkyl derivatives, at 50 C. Details about this strain and the method can be found in Section 2.2.3 in Chapter 3. In the second patent, the gene encoding the NADH-FMN oxidoreductase enzymes and the method for constructing it was disclosed [170]. The gene sequence is included in the document. The gene encoding an enzyme oxidizing NADH and reducing FMN was separated from thermophilic desulfurization bacteria, namely B. subtilis WU-S2B or M. phlei WU-F1. The oxidation reactions promoted by those enzymes occur in the range of 30–55 C. Details on this strain and the method can also be found in Section 2.2.3 in Chapter 3.

48. UOP LLC (UNITED STATES)/PAQUES BIOLOGICAL SYSTEMS BV (NETHERLANDS) UOP LLC is now part of the Honeywell company. It was founded in 1914 as the National Hydrocarbon Company and it is dedicated to the development and delivering of technologies for more than 90 years. Processes for the petroleum refining, gas processing, petrochemical production and major manufacturing industries are disclosed in the thousands of patents they hold. Activities regard engineering and technical services, operational support services and the licensing and technology transfer of a full portfolio of process technology, adsorbents and catalysts, and specialized equipment. Some of the most widely used refining technologies have been either developed or improved by UOP (Isomerization, Reforming, FCC, etc.). Presently, licensing activities include more than 50 processes, for which more than 90 different catalysts and adsorbents are supplied to the refining industry. In the biotechnological arena, a process for removing H2 S and mercaptans from a hydrocarbon stream, such as a LPG, was disclosed [171]. Sulfide oxidizing microorganisms, such as those from the genera Thiobacillus and Thiomicrospira, are employed to convert H2 S to sulfur and mercaptans to disulfides. First a weakly basic stream, (e.g., a sodium bicarbonate solution) is used to extract the sulfur molecules from the hydrocarbon stream using an ordinary extraction column. The extracted sulfur molecules are then

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treated in a reactor vessel containing the microbial strain. Oxygen is fed to the reactor with the oxidation reaction lasting for about 5 h operated at temperatures between 20 and 50 C and a pH of 7–10 under atmospheric pressure. The sulfide oxidation consumes about 0.4 to about 2.0 moles O2 /moles S (total sulfur). Care has to be exercised to keep the hydrocarbon content in the basic stream below 500 ppm by weight. The disulfides formed in the reaction will float at the top of the aqueous solution, when the concentration is high, or will be dispersed as droplets throughout the solution. Finally, the sulfur and disulfides can be separated from the basic aqueous stream which can be recycled to the washing device. This hydrocarbon cycle treatment can be repeated until purification level reaches the point that it reaches specifications. The purified basic stream can be reused only if it contains less than 0.08 g/l of elemental sulfur.

49. VALENTINE, JAMES M (UNITED STATES) The two patents awarded to Valentine [26,27] concern with desulfurization and both are applicable to the biotreatment of bitumen fuels. The first one deals with desulfurization of Orimulsion®, which is a bitumen derived fuel in an O/W emulsion form. Therefore, it seems than the inventor wanted to take the advantage of having the water already incorporated in the feedstock and alleviate the mass transfer limitations of the biotreatment. The second one deals with bitumens in general. In these inventions, R. rhodochrous IGTS8 ATCC 53968 [67] can be used, as well as a list of some other patented biocatalysts, such as Pseudomonas sp. CB1 ATCC No. 39381 [7], Acinetobacter sp. CB2 ATCC No. 53515 [8], B. Sulfasportare [16], B. sphaericus IGTS9 ATCC No. 53969 [91], the mixed cultures ATCC No. 39327 [159] and those described in Ref. [160]. Valentine discloses the use of all possible biocatalyst types, namely cell cultures (free, immobilized or fragmented), cell extracts, enzyme mixtures (extracted or synthesized) or mixtures of all of them. The innovating features of his disclosure are the incorporation of SOx sorbent within the catalytic system to separate the oxisulfides from the reaction medium and the inclusion of more than one biocatalytic stages with intermediate emulsion breaking/making steps. The biodesulfurization of bitumen fuels is described as a simple and effective biochemical process for solving the problems associated with sulfur in bitumen. In one aspect, an emulsion of bitumen and water (e.g., Orimulsion, which contains about 70% Venezuelan Orinoco crude and 30% water) is contacted with a microbiological desulfurization agent in a liquid–liquid reactor, for a specified time and under conditions effective to reduce the oxidizable sulfur content of the bitumen. The advantage of using a preformed emulsion is that strong stirring, which would require substantial energy input is not needed, instead only small agitation is needed for mixing the air, nutrients, substrates, and desulfurization agent. An emulsion with droplets size between 10 to 100 m is preferred. The preferred agents do not affect the heating value of the fuel, but selectively oxidize organic sulfur to water-soluble sulfates which can either be physically removed or chemically bound so that they do not cause SOx production. The biocatalytic material was broadly defined as being any biological organism, living or dead, any biologically produced enzyme or sequence of enzymes, cellular matter of an organism, or chemically

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synthesized equivalent and capable of converting the sulfur in the bitumen, by oxidizing it to sulfate. Under this definition falls any cell cultures, immobilized cell masses, fragmented cells, cell extracts, enzyme mixtures, and synthetically prepared copies of active enzyme sequences or components. The used cell culture can be saved for re-use in a subsequent reaction or reaction stage, but the need to be treated so as to obtain a cell extract, which is the form for re-use. The desulfurization process can be carried out either, in a dedicated reactor, or within a simple storage vessel, or during transportation (in pipelines) or intermediate processing vessels. Nutrients addition, pH, and aeration are adjusted as necessary. Multiple stages can be added to the reaction to enhance the sulfur removal process and decrease the reaction time below the probable 300 h required. The produced sulfates are removed by the addition of agents such as alkaline calcium, magnesium, aluminum, barium, and metal compounds such as oxides, hydroxides, and carbonates. So, Valentine [27] disclosed the use of R. rhodochrous ATCC 53968 for a desulfurization process; however, as it is known the biocatalyst property was divided between IGT and EBC. Through a US patent US5593889, Valentine received the rights of using such strain for desulfurizing emulsified bitumens, which are as a matter of fact, a carbonaceous material for which, priority rights seems to belong to IGT and EBC (whose emulsified process was allowed in 1994 [35,36]). Surprisingly enough, Valentine’s 17th claim discloses a variety of possible biocatalyst types, namely cell cultures (free, immobilized, or fragmented), cell extracts, enzyme mixtures (extracted or synthesized) or mixtures of all of them, derived from R. rhodochrous ATCC 53968. This fact does give rise to overlap between the claims related to biocatalyst uses between the three organizations and unveils concerns on the legal feasibility of processes using this biocatalyst.

GENERAL DISCUSSION At this point, we can conclude that the biotechnology portfolio relevant to the oil refining industry is composed by nearly 170 inventions, created by more than 50 organizations and individuals, acting in an isolated or cooperative manner. The biological treatment of crude oil, its fractions and products, and natural gas have received the major attention; however, separation processes, in situ (in the well) transformation and processing schemes are also part of that portfolio. The efforts on process development are the least investigated among the entire R&D conducted so far. The largest investment has been in desulfurization, and in most instances it has been proven that the sulfur compounds have been transformed into oxidized moieties, but the actual cleavage of the last C−S bond in most cases does not take place to the extent desired or to levels needed for implementing BDS. Other processes such as demetallization and upgrading are just starting to be studied. Collateral technologies, for gas treatment and reducing viscosity by emulsification (“in well” treatments) are commercially available. Despite all the intellectual property generated in this field, the application has not reached commercial scale. It does not mean that there has not been any progress. In fact, the biocatalyst development has greatly advanced, much of it due to the advancements in the techniques, methods and tools related to MB and GE. MB techniques raised the understanding of the biocatalyst from the level of whole cells to clearly defined

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biomolecules. From the IP point of view, the biocatalyst definition now covers a wider spectrum of biological materials, and not just microorganisms. In the past, the success in developing a biocatalyst for a process relied on the ability of microbiologist to identify and classify the organism and certain mutagenesis techniques to make improvements, but the MB evolution has taken biocatalyst development to the next stage via a molecular understanding of biocatalyst. In this process, the enzymes were isolated, identified and characterized, and so were the corresponding genes. The kinetics was investigated and once the metabolic pathway was defined, the need for genetic promoters and inducers was established. At this point, the fundamental biocatalytic knowledge and the mechanism of action were known, but the process was not economically feasible. The reason was mainly insufficient biocatalyst activity. The incorporation of GE tools was seen as a means for improving the catalytic properties including activity, selectivity, and stability. Converting the microbial cell into an efficient catalytic machine responsible for production and operation of the enzymes became the working strategy. Thus, genetic modifications via the available techniques were used to create recombinant DNA molecules, to modify existing genes, to construct plasmids, vectors, ORFs, etc., to clone host microorganisms, and as a whole to try to obtain an improved biocatalyst. All these approaches have been implemented and have worked to improve the catalyst; however, the developments have not been enough to move towards commercialization. In terms of the process, very little has been achieved. The mass transfer limitations still exist although emulsification has solved the problem partially, but not without creating another problem downstream in separation of the product from the rest of the stream and the issue still needs further work. The IP portfolio contains very few real process concepts. The patented material refers to a BDS process several times, but the process referred to, is no more than a simple description of the pH, temperature, etc., and the particular use of a given biocatalyst in an application. Some protected subject matter concerns the integration of a bioprocess into the flow sheet of the refinery, but again those are no more than theoretical scheme proposed for implementation, with no actual evidence with real feedstocks. In an ‘in depth’ comparison of the cumulative knowledge discussed in Chapter 3, with what one could extract from the technological results reported in this Chapter, perhaps the first observation that one can make is the difference between the content of the biocatalyst development vs. process development results. The results on biocatalyst improvements constitute the majority of the open literature reports. The most important bottleneck holding advancement of the biodesulfurization technology is the ability to break the second C−S bond, releasing the sulfur from the organosulfur molecules. The IP portfolio does not provide a real solution for that problem. The IP issues surrounding the use of the Rhodococcus genera in a commercial BDS application are complex and appear difficult to assess. For a technology based on the best known biocatalysts to become commercial, all those uncertain issues will have to be addressed. Obviously, each company has their own IP and development strategies, but in general terms, the trend observed in the IP strategy indicates that the organizations were engaged in obtaining property rights to a biocatalyst capable of BDS. A systemic development to yield a biorefining process does not appear to be the IP strategy. Instead, the development strategy has been concentrated on the biocatalyst, and some isolated

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efforts related to the process issues have been observed. Phase separation and conceptual schemes for the integration of BDS into a refinery were considered. It may be time to pay more attention to the process, although the biocatalyst activity still needs more work to achieve levels at least 10 fold of the best biocatalyst available as of 2005.

REFERENCES 1. Pifferi, P. G.; Lanzarini, G.; Matteuzzi, D., et al., Anaerobic desulfurization process for crude oil and petroleum products. Patent No. EP0401922, B1. 1990, Dec. 12. 2. Sacceddu, P., D Addario, E., Gianna, R., et al., Arthrobacter sp. and its use for the desulfurization of fossil fuels. Patent No. EP 0795603, A3 1997, Sep. 17. 3. Gianna, R., and Sisto, R., Desulfurization of gases containing hydrogen sulfide. Patent No. EP0811416. Dec.10, 1997. 4. Margarit Ros, Y. I.; Rodriguez, F.; Serbolisca, L. P., and De Ferra, F., Means and methods for the expression of homologous and heterologous proteins in strains of Rhodococcus. Patent No. EP1127943. 5. Rodriguez, F.; Serbolisca, L. P.; De Ferra, F., and Franchi, E., Promoter and expression vector for Rhodococcus. Patent No. GB2389363. 2003, Dec. 10. 6. Coleman, J. K.; Brown, M. J.; Moses, V., and Burton, C. C., Enhanced oil recovery Patent No. WO9215771. 7. Isbister, J. D., and Doyle, R. C., A novel mutant microorganism and its use in removing organic sulfur compounds. Patent No. US4562156. 1987, April 22. 8. Isbister, J. D., Acinetobacter species and its use in removing organic sulfur compounds. Patent No. US4808535. 1989, Feb. 28. 9. Walia, D. S., and Srivastava, K. C., Biological production of humic acid and clean fuels from coal. Patent No. US5670345. 1997, Sep. 23. 10. Walia, D. S., and Srivastava, K. C., Biological production of humic acid and clean fuels from coal. Patent No. US5854032. 1998, Dec. 29. 11. Premusic, E. T., and Lin, M. S., Biochemically enhanced oil recovery and oil treatment. Patent No. US5297625 1999, March 29. 12. Premusic, E. T., and Lin, M. S., Process for producing modified microorganisms for oil treatment at high temperatures, pressures and salinity. Patent No. US5492828. 1996, Feb. 20. 13. Premusic, E. T., and Lin, M. S., Biochemical upgrading of oils. Patent No. US5858766. 1999, Jan.12. 14. Premusic, E. T., and Lin, M. S., Biochemical transformation of coals. Patent No. US5885825 1999, March 23. 15. Premuzic, E. T., and Lin, M. S., Biochemical transformation of solid carbonaceous material. Patent No. US6294351. 2001, Sep. 25. 16. Kopacz, E. P., Biodesulfurization of carbonaceous materials. Patent No. US4632906. 17. Atlas, R. M., and Aislabie, J., Process for biotechnological upgrading of shale oil. Patent No. US5049499. 18. Atlas, R. M., and Aislabie, J., Process for biotechnological upgrading of shale oil. Patent No. US5143827. 19. Boron, D. J.; Atlas, R. M.; Johnson, A. R., et al., Method for removing organic sulfur from heterocyclic sulfur containing organic compounds. Patent No. USH1986H. 20. Bhat, P. A.; Johnson, D. W.; Murphy, D. W., and Myers, R. B., Bioregenerative flue gas desulfurization. Patent No. EP0643987. 21. Duyvesteyn, W. P. C.; Budden, J. R., and Picavet, M. A., Extraction of bitumen from bitumen froth and biotreatment of bitumen froth tailings generated from tar sands. Patent No. US5968349.

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22. Budden, J. R.; Duyvesteyn, W. P. C., and Huls, B. J., Biochemical treatment of bitumen froth tailings. Patent No. US6074558. 23. Stetter, K. O.; Huber, H.; Buisman, C. J. N., et al., Sulfur reducing bacterium and its use in biological desulfurization processes. Patent No. EP0819756. 1998, Jan. 21. 24. Sheehy, A., Oil Recovery Using Microorganisms. Patent No. WO9201780. 1992, Feb. 06. 25. Olson, G., Microbial catalyst for desulfurization of fossil fuels. Patent No. US6124130. 2000, Sep. 26. 26. Valentine, J. M., Biodesulfurization of bitumen fuels. Patent No. US5593889. 1992, June 11. 27. Valentine, J. M., Biodesulfurization of fuels. Patent No. US5874294. 1997, Jan. 14. 28. Sublette, K. L., Microbiological desulfurization of gases. Patent No. EP0218958. 1987, April 22. 29. Haney III, W. H., and Monticello, D. J., Microbial Process for Reduction of Petroleum Viscosity. Patent No. WO9211343. 1992, July 09. 30. Monticello, D. J., Multistage System for Deep Desulfurization of Fossil Fuels. Patent No. WO9216602. 1992, Oct. 01. 31. Monticello, D. J., Multistage System for Deep Desulfurization of Fossil Fuels. Patent No. US5232854. 1993, Aug. 03. 32. Monticello, D. J., Multistage System for Deep Desulfurization of Fossil Fuels. Patent No. US5387523. 1995, Feb. 07. 33. Monticello, D. J., Multistage System for Deep Desulfurization of Fossil Fuels. Patent No. US5510265. 1996, April 23. 34. Monticello, D. J., Continuous process for biocatalytic desulfurization of sulfur-bearing heterocyclic molecules. Patent No. WO9219700. 1992, Nov. 12. 35. Monticello, D. J., and Kilbane II, J. J., Biocatalytic Desulfurization of Organosulfur Molecules. Patent No. WO9325637. 1993, Dec. 23. 36. Monticello, D. J., and Kilbane II, J. J., Biocatalytic Desulfurization of Organosulfur Molecules. Patent No. US5358870. 1994, Oct. 25. 37. Rambosek, J.; Kovacevich, B. R.; Piddington, C. S., et al., Recombinant DNA Encoding A Desulfurization Biocatalyst. Patent No. US5356801. 1994, Oct. 20. 38. Rambosek, J.; Piddington, C. S.; Kovacevich, B. R., et al., Recombinant DNA Encoding A Desulfurization Biocatalyst. Patent No. WO9401563. 1994, Jan. 20. 39. Rambosek, J.; Piddington, C. S.; Kovacevich, B. R., et al., Recombinant DNA Encoding A Desulfurization Biocatalyst. Patent No. US5578478. 1996, Nov. 26. 40. Rambosek, J.; Piddington, C. S.; Kovacevich, B. R., et al., Recombinant DNA Encoding A Desulfurization Biocatalyst. Patent No. US5879914. 1999, March 09. 41. Monticello, D. J., Process for the desulfurization and the desalting of a fossil fuel. Patent No. US5496729. 1996, March 05. 42. Monticello, D. J., Process for the desulfurization and the desalting of a fossil fuel. Patent No. US5356813. 1994, Oct. 18. 43. Johnson, S. W.; Monticello, D. J.; Gibbs, P. R., and Kulpa, C. F., Method for separating a sulfur compound from carbonaceous materials. Patent No. US5468626. 1995, Nov. 21. 44. Monticello, D. J., and Chen, J. C. T., Method for separating a petroleum containing emulsion. Patent No. US5525235. 1996, June 11. 45. Ortego, B. C.; Squires, C. H.; Childs, J. D., et al., Method of desulfurization of fossil fuel with flavoprotein. Patent No. WO9617940. 1996, June 13. 46. Ortego, B. C.; Squires, C. H.; Childs, J. D., et al., Method of desulfurization of fossil fuel with flavoprotein. Patent No. US5733773. 1998, March 31. 47. Ortego, B. C.; Squires, C. H.; Childs, J. D., et al., Method of desulfurization of fossil fuel with flavoprotein. Patent No. US5985650. 1999, Nov. 16. 48. Monticello, D. J.; Gray, K. A., and Squires, C. H., Dszd utilization in desulfurization of DBT by Rhodococcus sp. IGTS8. Patent No. US5846813. 1998, Feb. 08.

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49. Monticello, D. J.; Squires, C. H., and Gray, K. A., Dszd utilization in desulfurization of DBT by Rhodococcus sp. IGTS8. Patent No. WO9711185 1997, March 27. 50. Monticello, D. J.; Squires, C. H., and Gray, K. A., Dszd utilization in desulfurization of DBT by Rhodococcus sp. IGTS8. Patent No. US5811285. 1998, Sep. 22. 51. Xu, G.-W.; Mitchell, K. W., and Monticello, D. J., Process for demetalizing a fossil fuel. Patent No. US5624844. 1997, April 29. 52. Xu, G.-W.; Mitchell, K. W., and Monticello, D. J., Process for demetalizing a fossil fuel. Patent No. US5726056. 1998, March 10. 53. Yu, L.-Q.; Folsom, B. R., and Meyer, T. A., Oil/water/biocatalyst three phase separation process. Patent No. US5772901. 1998, June 30. 54. Squires, C. H.; Childs, J. D., and Gray, K. A., Rhodococcus flavin reductase complementing DszA and DszC activity. Patent No. WO9817787. 1998, April 30. 55. Squires, C. H.; Childs, J. D., and Gray, K. A., Rhodococcus flavin reductase complementing DszA and DszC activity. Patent No. US5804433. 1998, Sep. 08. 56. Squires, C. H.; Childs, J. D., and Gray, K. A., Rhodococcus flavin reductase complementing DszA and DszC activity. Patent No. US5919683. 1999, July 06. 57. Squires, C. H.; Childs, J. D., and Gray, K. A., Rhodococcus flavin reductase complementing DszA and DszC activity. Patent No. US6274372. 2001, Aug. 14. 58. Monticello, D. J.; Squires, C. H.; Childs, J. D., et al., Dsz gene expression in pseudomonas hosts. Patent No. US5952208. 1999, Sep. 14. 59. Mrachko, G. T., Removal of sulfinic acids. Patent No. US5968812. 1999, Oct. 19. 60. Lange, E. A.; Lin, R. N. K., and Dooyema, C. C., Surfactants derived from 2-(2hydroxiyphenyl)-benzene-sulfinate and alkyl-substituted derivatives. Patent No. US5973195. 1999, Oct. 26. 61. Lange, E. A.; Lin, R. N. K., and Dooyema, C. C., Surfactants derived from 2-(2-hydroxiyphenyl)-benzene-sulfinate and alkyl-substituted derivatives. Patent No. WO9947496. 1999, March 18. 62. Colin, J.-M.; Hazan, C.; Monticello, D. J., and Johnson, S. W., Conversion of organosulfur compounds to oxyorganosulfur compounds for desulfurization of fossil fuels. Patent No. US6071738. 2000, June 06. 63. Folsom, B. R., and Hoggard, K. D., Growth of biocatalyst within biodesulfurization system. Patent No. WO0042122. 2000, July 20. 64. Mrachko, G. T., and Darzins, A., Sphingomonas biodesulfurization catalyst. Patent No. US6133016. 2000, Oct. 17. 65. Squires, C. H.; Childs, J. D., and Wang, Y., Gene involved in thiophene biotransformation from Nocardia asteroides KGB1. Patent No. US6235519, 2001 May 22. 66. Lange, E. A., and Lin, Q., Compositions comprising 2-(2-hydroxyphenyl)-benzene-sulfinate and alkyl-substituted derivatives thereof. Patent No. US6303562. 2001, Oct.16. 67. Kilbane II, J. J., Mutant microorganisms useful for cleavage of organic C−S bonds. Patent No. EP0441462. 1991, Aug 14. (Also published as US5104801). 68. Kilbane II, J. J., Bacterial produced extracts and enzymes for cleavage of organic C−S bonds. Patent No. EP0445896. 1991, Sep. 11. (Also published as US5132219). 69. Patten, P. A., and Stemmer, W. P. C., Methods and compositions for polypeptide engineering. Patent No. US6319713. 2001, Nov. 20. 70. Coco, W. M., and, R. A. C. H. I. T. T., Gene Family Shuffling by Random Chimeragenesis on Transient Templates. Methods. Mol. Biol., 2003. 231: pp. 111–127. 71. Coco, W. M.; Levinson, W. E.; Crist, M. J., et al., DNA Shuffling Method for Generating Highly Recombined Genes and Evolved Enzymes. Nature Biotechnology, 2001. 19(4): pp. 354–359. 72. Arensdorf, J. J.; Loomis, A. K.; DiGrazia, P. M., et al., Chemostat approach for the directed evolution of biodesulfurization gain-of-function mutants. Applied and Environmental Microbiology, 2002. 68(2): pp. 691–698.

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73. Kilbane II, J. J., Microbial cleavage of organic C−S bonds. Patent No. US5358869. 1994, Oct. 25. 74. Lizama, H. M., and Sankey, B. M., Biological Process for Conversion of Hydrogen Sulphide. Patent No. CA2039863. 1992, Oct. 06. 75. Grossman, M. J.; Lee, M. K.; Senius, J. D., et al., Microbial Desulfurization of Organic Compounds. Patent No. CA2097217. 1993, Dec. 16. 76. Grossman, M. J.; Lee, M. K.; Senius, J. D., et al., Rhodococcus species for removing sulfur from organic carbonaceous fuel substrates-(LAW295). Patent No. US5607857. 1997, March 04. 77. Coyle, C. L.; Logan, M. S. P.; Lewis, K., et al., Solvent-resistant microorganisms. Patent No. US5807735. 1998, Sep. 15. 78. Ferrughelli, D. T.; Lee, M. K.; Senius, J. D., et al., Method for the removal of organic sulfur from carbonaceous materials. Patent No. US5910440. 1999, June 08. 79. Coyle, C. L.; Ferrughelli, D. T.; Logan, M. S. P., et al., Biological activation of aromatics for chemical processing and/or upgrading of aromatic compounds, petroleum, coal, resid, bitumen and other petrochemical streams. Patent No. US6156946. 2000, Dec. 05. 80. Sikkema, J.; de Bont, J. A. M.; and Poolman, B., Interactions of cyclic hydrocarbons with biological membranes. J. Biol. Chem., 269: pp. 8022–8028. 1994. 81. Walia, D. S., and Srivastava, K. C., Microbial process for the mitigation of sulfur compounds from natural gas. Patent No. US5981266. 1999, Nov. 09. 82. Walia, D. S.; Garg, S., and Srivastava, K. C., Microbiological desulfurization of sulfur containing gases. Patent No. US6287873. 2001, Sep. 11. 83. Sinskey, A. S.; Kern, E. E.; Wise, D. L., et al., Enzymatic Coal Desulfurization. Patent No. CA1334947. 1992, March 10. 84. Sinskey, A. S.; Kern, E. E.; Wise, D. L., et al., Enzymatic Coal Desulfurization. Patent No. US5094668. 1995, March 28. 85. Compere, W. A., Process for producing product from fossil fuel. Patent No. US3826308. 1974, July 30. 86. Monot, F.; Abbad, A. S., and Warzywoda, M., New cultures of Rhodococcus strains, useful for desulfurization of petroleum and its products, comprises high catalytic stability which allows culture recycling. Patent No. FR2795090. 2000, Dec. 22. 87. Monot, F.; Abbad, A. S., and Warzywoda, M., Biological culture containing Rhodococcus erythropolis and/or Rhodococcus rhodnii and process for desulfurization of petroleum fraction. Patent No. US6337204. 2002, Jan. 08. 88. Vazquez-Duhalt, R.; Bremauntz, M. D. P.; Barzana, E., and Tinoco, R., Enzymatic oxidation process for desulfurization of fossil fuels. Patent No. US6461859. 2002, Oct. 08. 89. Chynoweth, D. P., and Tarman, P. B., Hybrid bio-thermal liquefaction. Patent No. US4334026. 1982, June 08. 90. Kilbane II, J. J., Mutant microorganisms useful for cleavage of organic C−S bonds. Patent No. US5002888. 1991, March 26. 91. Kilbane II, J. J., Mutant microorganisms useful for cleavage of organic C−S bonds. Patent No. EP0436508. 1991, July 10. 92. Kilbane II, J. J., Biochemical cleavage of organic C−S bonds. Patent No. EP0562313. 1993, Sep. 29. 93. Kilbane II, J. J., Useful for cleavage of organic C−S bonds Bacillus sphaericus microorganism Patent No. US5198341. 1993, March 30. 94. Kilbane II, J. J., Process for enzymatic cleavage of C−S bonds and process for reducing the sulfur content of sulfur-containing organic carbonaceous material. Patent No. US5344778. 1994, Sep. 06. 95. Kilbane II, J. J., Enzyme from Rhodococcus rhodochrous ATCC 53968, Bacillus sphaericus ATCC 53969 or a mutant thereof for cleavage of organic C−S bonds. Patent No. US5516677. 1996, May 14.

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96. Gray, K. A.; Pogrebinsky, O. S.; Mrachko, G. T., et al., Molecular mechanisms of biocatalytic desulfurization of fossil fuels. Nature Biotechnology, 14(13): pp. 1705–1709. 1996. 97. Gou, Z.; Liu, Y., and Xing, J., Gordona nitida and application of removing sulfur element from sulfur compound. Patent No. CN1445361. 2003, Oct. 01. 98. Ranson, I., and Rivas, C. M., Biodesulfurization of hydrocarbons. Patent No. US6808919 2003, Sep. 11. 99. Ranson, I., and Rivas, C. M., Biodesulfurization of hydrocarbons. Patent No. US2003170874. 2003, Sep. 11. 100. Kurane, R.; Tomizuka, N.; Nakahara, T., and Makino, K., Biological desulfurization method. Patent No. JP6184557. 1994, Jul. 05. 101. Kurane, R.; Nakamura, Y.; Nakajima, K., et al., Denitrification by microorganism. Patent No. JP8034981. 1996, Feb. 06. 102. Kurane, R.; Usami, S.; Kirimura, K., and Tsuji, K., Method for biodenitrification of hardly removable aromatic organic nitrogen compound. Patent No. JP10244294. 1998, Sep. 14. 103. Kurane, R., and Tsubata, T., Method for obtaining organic solvent-resistant. microorganisms and organic solvent-resistant microorganisms obtainable by the method. Patent No. US5804435. 1998, Sep. 08. 104. Kurane, R.; Miki, K.; Ukekawa, K., and Yamamoto, J., Method for oxidation of organic sulfur compound included in organic compound and method for oxidative desulfurization of fuel oil. Patent No. JP2001151748. 2001, June 05. 105. Matsui, T.; Tanaka, Y.; Noda, K., et al., Recombinant Desulfurizing Bacterium. Patent No. JP2002223767. 2002, Aug. 13. 106. Kurane, R.; Ota, Y.; Maruhashi, K., et al., Method of Desulfurization by Using Microorganisms which Decompose Dibenzothiophenes. Patent No. JP2003018995. 2003, Jan. 21. 107. Kurane, R.; Tanaka, Y., and Maruhashi, K., Method for Specifying Desulfurizing Enzyme Expression- Inhibiting Gene and Desulfurizing Microorganism whose Desulfurizing Enzyme Expression Inhibition is Terminated and Method for Producing the Desulfurizing Microorganism. Patent No. JP2003061669. 2003 March 04. 108. Kirimura, K.; Furuya, T.; Ishii, Y., et al., Method of Desulfurizing Heterocyclic Sulfur Compound Using Bacterium. Patent No. JP2003135054. 2003, May 13. 109. Watanabe, K.; Noda, K., and Maruhashi, K., Desulfurization Method Using Recombinant Microorganism. Patent No. JP2003144167. 2003, May 20. 110. Watanabe, K.; Noda, K., and Maruhashi, K., Recombinant Microorganism and Desulfurization Method Utilizing the Same. Patent No. JP2004049116. 2004, Feb. 19. 111. Matsui, T., and Maruhashi, K., High-Quality Desulfurization Method by Using Recombinant Microorganism. Patent No. JP2004113117. 2004, April 15. 112. Ishii, Y.; Suzuki, M.; Oshiro, T., and Izumi, Y., Oxidoreductase Gene. Patent No. JP2000245478. 2000, Sep. 12. 113. Kato, N., and Yanase, E., Gene coding for desulfurizing enzyme. Patent No. JP2000245477. 2000, Sep.12. 114. Kirimura, K.; Furuya, T.; Kino, K., et al., Method for Desulfurizing with Microorganism at High Temperatures. Patent No. JP2001231546. 2001, Aug. 28. 115. Kirimura, K.; Furuya, T.; Kino, K., et al., Method for Decomposing Heterocyclic Sulfur Compound Using Bacterium. Patent No. JP2002000259. 2002. Jan. 08. 116. Kirimura, K.; Izumi, Y.; Oshiro, T., et al., Thermally Stable Desulfurization Enzyme and Gene Encoding the Same. Patent No. JP2002253247. 2002, Sep. 10. 117. Kirimura, K., and Nishii, Y., High-temperature desulfurization with microorganism. Patent No. JP2000139450. 2000, May 23. 118. Koizumi, K.; Takagi, M., and Suzuki, M., Production of desulfurization-active microorganism. Patent No. JP11181446. 1999, July 06. 119. Konishi, J.; Ishii, Y.; Okada, H., et al., Gene encoding desulfurases. Patent No. JP11341987. 1999, Dec. 14.

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120. Konishi, J.; Ishii, Y.; Okada, H., et al., Gene encoding desulfurases. Patent No. US6607903 2003 Aug. 19. 121. Konishi, J.; Ishii, Y.; Okumura, K., and Suzuki, M., High-temperature desulfurization by microorganisms. Patent No. US5925560. 1998, Feb. 04. 122. Konishi, J.; Ishii, Y.; Okumura, K., and Suzuki, M., High-temperature desulfurization by microorganisms. Patent No. US6130081. 2000, Oct. 10. 123. Matsui, T.; Hirasawa, K.; Suzuki, M., and Kurane, R., Desulfurization by microorganism. Patent No. JP2000245446. 2000, Sep. 12. 124. Matsui, T.; Hirasawa, K.; Suzuki, M., and Kurane, R., Desulfurization using microorganism capable of degrading alkylbenzothiophene and alkyldibenzothiophene. Patent No. JP2001029065. 2001, Feb. 06. 125. Tomita, F.; Yokota, A.; Saito, K., and Abe, A., Method for Decomposing Porphyrin-Based Compound. Patent No. JP11187890. 1999, July 13. 126. Yoshikawa, O.; Kishimoto, M.; Okumura, K., et al., Method for Culturing Microorganism having Ability in Desulfurization. Patent No. JP2001186875. 2001, July 10. 127. Yoshikawa, O.; Kobayashi, M.; Sugiyama, H., and Sugaya, K., Biological desulfurization. Patent No. JP2000144149. 2000, May 26. 128. Konishi, J.; Ishii, Y.; Okada, H., et al., Gene encoding desulfurases. Patent No. US6479271. 2002, Nov. 12. 129. Tomita, F.; Yokota, A.; Saito, K., et al., Decomposition of porphyrin Patent No. JP9176168. 1997, July 08. 130. Konishi, J.; Ishii, Y.; Okada, H., et al., Gene encoding desulfurases. Patent No. US6420158. 2001, Jan. 17. 131. Miyamoto, K.; Hirata, K.; Boku, M., and Miyasaka, H., Microorganism and Method for Producing Alternative Oil for Petroleum Using the Same. Patent No. JP2003000229. 2003, Jan. 07. 132. Jang, H.-N.; Son, H.-Y.; Jang, J.-H., et al., A novel klebisiella oxytoca which removes sulfur from fossil fuel that contains organic sulfur compound and desulfurizing method. Patent No. KR205255. 1999, July 01. 133. Lee, S. Y.; Park, S. J., and Jang, Y. K., Recombinant Coliform Bacillus Expressing Desulfurizing Enzyme and Biological Desulfurization Method Using Thereof. Patent No. KR2000051096. 2000, Aug. 16. 134. Chang, J. H.; Chang, H. N.; Chang, Y. K., et al., Gordona sp. CYKS1 (KCTC 0431BP) capable of desulfurizing fossil fuel containing organic sulfur compounds. Patent No. US6204046. 2001, March 20. 135. Chang, J. H.; Chang, H. N.; Chang, Y. K., et al., Norcardia sp. CKYS2 (KCTC 0432BP) capable of desulfurizing fossil fuel containing organic sulfur compounds. Patent No. US6197570. 2001, March 06. 136. Kim, B.-H.; Kim, T.-S., and Kim, H.-Y., Bioelectrochemical desulfurization of petroleum. Patent No. US4954229. 1989, July 12. 137. Kim, B. H.; Bellows, P.; Datta, R., and Zeikus, J. G., Control of carbon and electron flow in Clostridium acetobutylicum fermentations: Utilization of carbon monoxide to inhibit hydrogen production and to enhance butanol yields. Appl. Environ. Microbiol., 1984. 48: pp. 764–770. 138. Kurashov, V. M., Microbiological method of sulfur and nitrogen content decrease in petroleum and hydrogen sulfide in deposit waters and casing-head gases. Patent No. RU2137839. 1999, Sep. 20. 139. Kurashov, V. M., Microbiological method for enriching petrol and petroleum products by isoparaffin and aromatic hydrocarbons with simultaneous removal of injurious additives and means for carrying out said method. Patent No. WO0246445. 2002, June 13.

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Biocatalysis in Oil Refining

140. Ishio, M.; Fujii, M., and Matsubara, S., Culture and growth of microorganism capable of oxidizing and decomposing sulfur compound contained in petroleum and biological desulfurization Patent No. JP7111900. 1995, May 02. 141. Davison, M. J., Microbiological method for the removal of contaminants from coal. Patent No. WO8601820. 1986, March 27. 142. Davison, J., The lambda process – for desulfurization of slurry coal fines prior to combustion. in Eighth Annual Surface Mine Drainage Task Force Symposium. 1987. Morgantown, West Virginia, April 7-8. 143. Kodama, Y.; Watanabe, K., and Harayama, S., Method for electrobiologically desulfurizing petroleum Patent No. JP2003201484. 2003, July 18. 144. Maure, A.; Warren, A., and Brown, F., Microbial-induced controllable cracking of normal and branched alkanes in oils. Patent No. US2002119557. 2002, August 01. 145. Lebedev, N. A.; Akhmetshina, S. M.; Ariskina, E. V., et al., Strain Pseudomonas Species45 Used for Desulfurization of Oil and Petroleum Products. Patent No. RU2189391, 2002 Sep. 20. 146. Enomoto, T.; Sekimoto, M.; Nasuno K., and Yoshimura, T., Low-Sulfur Gas Oil Patent No. JP10017874. 1998, Jan. 20. 147. Oldfield, C., Microorganism which can desulfurize benzothiophenes. Patent No. EP0917563. 2001, July 19. 148. Buisman, C. J. N., and Janssen, A. J. H., Process for biological removal of sulphide Patent No. US6221652 1998, June 04. 149. Linhares, M. O. N. M.; Ribeiro, C. M. S., and Kilbane II, J. J., Microbial cleavage of organic C-N bonds. Patent No. EP1106700. 2001, June 13. 150. Linhares, M. O. N. M.; Ribeiro, C. M. S., and Kilbane II, J. J., Pseudomonas ayucida useful for cleavage of organic C−N bonds. Patent No. US6221651. 2001, April 24. 151. Linhares, M. O. N. M.; Ribeiro, C. M. S., and Kilbane II, J. J., Microorganisms useful for cleavage of organic C−N bonds. Patent No. US6204048. 2001, March 20. 152. Linhares, M. O. N. M.; Ribeiro, C. M. S., and Kilbane II, J. J., Bacterial cleavage of only organic C−N bonds of carbonaceous materials to reduce nitrogen content. Patent No. US6541240. 2003, April 01. 153. Ward, O. P., and Singh, A., Biological process for breaking oil-water emulsions. Patent No. US6171500. 1999, Oct. 22. 154. Kobayashi, T., and Kamimura, N., Separation of oil and water in microbial reaction of oily and aqueous two-phase system Patent No. JP9009981. 1997, Jan. 14. 155. Ishii, Y.; Okumura, K.; Kobayashi, M., and Suzuki, M., Microorganism capable of degrading alkylated heterocyclic sulfur compound Patent No. JP11009293. 1999, Jan. 19. 156. Nakahara, T.; Nakajima, T.; Nomura, N., and Nekotsuka, S., Microbial desulfurization of sulfur-containing hetero-cyclic compound. Patent No. JP10243791. 1998, Sep. 14. 157. Plummer, M. A., Biodesulfurization of hydrocarbons. Patent No. US2003092169. 2003, May 15. 158. Madgavkar, A. M., Microbiological desulfurization of coal and coal water admixture to provide a desulfurized fuel. Patent No. US4861723. 1989, August 29. 159. Stevens, S. E. Jr., and D. B. W., Biological desulfurization of coal. Patent No. US4659670. 1987, April 21. 160. Stevens, S. E. Jr., and Burgess, W. D., Microbial desulfurization of coal. Patent No. US4851350. 1989, July 25. 161. Fujita, T., Bacteria mixture having heavy oil degrading ability and method of treating oil components. Patent No. US6649400. 2003, Nov. 18. 162. Hayano, T.; Hino, S., and Kajie, S., Protein participating in oxidation reaction of organic sulfur compound and gene coding for the same Patent No. JP2000093180. 2000, April 04. 163. Tomohiko, T.; Kazuya, W.; Sanae, H., et al., Demulsification by microorganisms Patent No. US5989892. 2002, June 20.

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164. Kawanaka, S., and Kitahata, K., Method for desulfurizing petroleum Patent No. JP10025482. 1998, Jan. 27. 165. Galan Sicilia, B.; Garcia Calvo, E.; Diaz Fernandez, E., et al., Method for desulfurization of dibenzothiophene using a recombinant Pseudomonas putida strain as biocatalyst Patent No. WO0170996. 2002, June 16. 166. Komazawa, I., and Hirai, T., Method for demetalyzing fossil fuel. Patent No. JP2001172647. 2001, June 26. 167. Morikawa, M., and Kato, T., New microorganism decomposing alkanes and method for decomposing alkanes. Patent No. JP2001224360. 2001, Aug. 21. 168. Li, F.; Xu, P., and Ma, C., Process for deeply removing organosulfur from fossil fuel by mycobacteria. Patent No. CN1379084. 2002, Nov. 13. 169. Ishii, Y.; Ozaki, S.; Kirimura, K., and Kino, K., Method for decomposing heterocyclic sulfur compound. Patent No. JP2004089131. 2004, March 25. 170. Kirimura, K.; Ishii, Y.; Tsuji, H., et al., Gene encoding desulfurization-relating oxidoreductase and method for preparing the same Patent No. JP2004283120. 2004, Oct. 14. 171. Janssen, A. J.; Pittman, R., and Arena, B. J., A process for removing sulfur compounds from hydrocarbon streams Patent No. US6306288. 2001, Oct. 23.

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