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Mixed culture biotechnology for bioenergy production
Mixed culture biotechnology for bioenergy production Robbert Kleerebezem and Mark CM van Loosdrecht Mixed culture biotechnology (MCB) could become an attractive addition or alternative to traditional pure culture based biotechnology for the production of chemicals and/or bioenergy. On the basis of ecological selection principles, MCB-based processes can be established that generate a narrow product spectrum from a mixed substrate. Three example processes are briefly discussed in this paper: anaerobic digestion aimed at the production of methanecontaining biogas, mixed culture fermentation for the production of solvents or biohydrogen, and a two-step process for the production of polyhydroxyalkanoates. These examples give an idea of the potential contribution of mixed culture biotechnology to sustainable production of bioenergy from waste. Addresses Delft University of Technology, Department of Biotechnology, Julianalaan 67, 2628 BC Delft, The Netherlands Corresponding author: Kleerebezem, Robbert ([email protected])
Current Opinion in Biotechnology 2007, 18:207–212 This review comes from a themed issue on Energy biotechnology Edited by Largus T Angenent
both disciplines could be defined as ‘mixed culture biotechnology’ (MCB), as the application of mixed cultures is a specific characteristic of biotechnologies originating from the waste treatment field. Owing to the use of undefined mixed cultures, process development in MCB can only be based on natural/ecological selection by manipulating the operation of the bioprocess or by varying the source of the natural inoculum. Using this approach, the required metabolic capacities and the corresponding microbial population can be effectively enriched from a natural environment. Compared with pure culture based industrial biotechnology, specific advantages of MCB include: no sterilization requirements, adaptive capacity owing to microbial diversity, the capacity to use mixed substrates, and the possibility of a continuous process. This paper will give a short description of three types of MCB processes that are potentially interesting for the production of energy carriers, while treating a waste stream. The first classical example of a highly effective MCB process is the anaerobic digestion process. Alternatively one could aim for the production of solvents by fermentation of organic substrates or the production of bioplastics using a sequencing batch process. In the description of these processes emphasis will be given to the ecological selection criteria that form the basis of the process.
Available online 16th May 2007 0958-1669/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2007.05.001
Introduction The traditional treatment objective of environmental biotechnological processes is the elimination of polluting compounds to generate a liquid, gaseous or solid residue that can be reused in a natural environment without detrimental ecological effects. Given the increasing interest in the effective use of natural resources, a second treatment objective can be suggested: the recovery of specific nutrients (e.g. nitrogen, phosphorous or sulfur) or the production of specific organic products (e.g. methane, organic solvents, bioplastics). The addition of the second treatment objective changes the status of several streams generated in the agro-industry from waste to raw material for the production of specific chemicals or energy carriers. Herewith a biotechnological research field has been created that combines traditional elements from environmental biotechnology in terms of cleaning of waste streams, with industrial biotechnology that is aiming for product maximization (see Table 1). The combination of www.sciencedirect.com
Selective pressure for product formation Anaerobic digestion
Anaerobic digestion is the classical example of a process that combines the objectives of elimination of organic compounds from a waste stream with the generation of a valuable product in the form of methane-containing biogas. Different bioreactor configurations have been developed for the treatment of liquid and solid waste streams [1–4]. For wastewater treatment, the application potential of anaerobic digestion has been extended from medium to highly concentrated wastewaters of agro-industrial origin, to more complex applications like those generated in petrochemical industries [5,6], paper industries [6,7] and even sewage [8]. There are three clear advantages of the anaerobic treatment over aerobic degradation of organic substrates: the high product and low biomass yield resulting in a limited generation of waste sludge as an unwanted side product; the in situ separation of the product as biogas, limiting costs for product separation; and the use of simple technology, as mixing by the biogas produced circumvents the need for other mixing requirements. The selective pressure required to induce methane production from a waste stream containing organic compounds is very simple: avoid the presence of an electron acceptor Current Opinion in Biotechnology 2007, 18:207–212
208 Energy biotechnology
Table 1 Historic differences between environmental and industrial biotechnology. Variable
Environmental biotechnology
Industrial biotechnology
History Basis Biomass Process type Process models Process objectives Substrates Process establishment
Wastewater treatment Catabolism Mixed culture (sludge) Continuous Lumped black box models Minimize effluent substrate concentrations Mixed substrates (waste) Ecological selection by process operation
Product formation Anabolism Specific strains of microorganisms (Fed) batch Omics-based metabolic network models Maximize productivity Pure and well-defined substrates Specific microorganisms and genetic engineering
(e.g. oxygen, nitrate or sulfate) or an external energy source (e.g. light). In the absence of an external electron acceptor, organic substrates can only be fermented; a process where the organic substrate is both electron donor and acceptor. The final end products of the organic substrate fermentation are methane, carbon dioxide and ammonia. Methane is the organic compound with the lowest free energy content per electron upon oxidation to carbon dioxide (see Figure 1) [9]. This indicates that in a thermodynamically closed system substrates will eventually be converted to methane and carbon dioxide. Microorganisms can obtain the energy required for growth by (stepwise) catalyzing the conversion of organic substrates to methane and carbon dioxide. From a bioenergy concept point of view, the biotechnological production of methane has limitations owing to the low price of natural gas (US$ 0.5 per kg). In cases where a network for natural gas is available, biogas can be distributed through this existing infrastructure; however, carbon dioxide and hydrogen sulfide need to be removed from the biogas [10]. Figure 1
Anaerobic digestion for bioenergy production is generally based on the treatment of substrates like activated sludge, manure and concentrated industrial waste [11]. Process advances in the field of anaerobic digestion of complex substrates are limited, owing to the rate-limiting hydrolysis process for solubilization of the particulate substrates. Enhanced degradation rates can only be established using generally expensive pretreatment methods. Consequently, most interesting developments are expected to occur in the field of post-treatment of the nutrient-rich solid and liquid residues obtained after digestion, and the effective incorporation of anaerobic digestion in biorefinery concepts. Solvent and/or biohydrogen production by mixed culture fermentation
Biohydrogen production by fermentation of carbohydrates has received significant attention in recent years. The main driving force for investigating the production of hydrogen instead of methane is the higher economic value of hydrogen, owing to its wider range of applications in the chemical industry [12]. The main limitation of hydrogen production by fermentative bacteria is the maximum electron-based yield that can be established. Owing to biochemical and thermodynamic limitations, the maximum theoretical number of moles of hydrogen that can be generated per mole glucose is four, and can be obtained by glucose oxidation to two moles acetate :C H O þ 2H O ! 2C H O 1 þ 2Hþ1 þ 2CO þ 4H 6 12 6 2 2 3 2 2 2 DG01 ¼ 145 k j mol 1
The substrate degradation sequence in the anaerobic digestion process. The free energy content per electron (upon oxidation to carbon dioxide) of the substrates is given together with the intermediates and products involved. Current Opinion in Biotechnology 2007, 18:207–212
However, the measured hydrogen production per mole glucose, as observed in mixed culture fermentation studies, is much lower and will normally not exceed two moles [12,13–15,16]. This practical limitation seems to be related to a biochemical restriction associated with the electron carriers utilized in the different fermentation pathways (Figure 2) [15]. Because of thermodynamic limitations, hydrogen can only be produced in oxidation reactions in the metabolic pathways coupled to formate www.sciencedirect.com
Mixed culture biotechnology Kleerebezem and van Loosdrecht 209
Figure 2
pure culture based processes can be used for the conversion of mixed substrates like municipal solid waste for solvent production is a subject of debate [25]. Development of an MCB-based continuous process for bioethanol production from waste would decrease the production costs significantly. However, as yet, no clear selection criterion for an ethanol-producing microorganism has been established. Some suggestions have been made on how to direct a fermentation process towards ethanol [21], but experimental evidence is lacking. Bioethanol production using MCB could also aim for combined processes based on the fermentation of carbohydrates to volatile fatty acids, and subsequent (biological) reduction of the products formed to alcohols using molecular hydrogen.
The dominant catabolic pathways of anaerobic glucose fermentation.
Polyhydroxyalkanoate production
production or ferrodoxin reduction, and not in the NADdependent steps. Fermentative hydrogen production provides only a partial oxidation of the organic substrate and is therefore normally integrated in a two-step process. In such a process, the fermentation products (volatile fatty acids) are either converted to methane-containing biogas [17] or converted to hydrogen and carbon dioxide in a phototrophic process [18,19]. A main limitation of the fermentative hydrogen production process is that no generally accepted selection criterion for the most favorable fermentative hydrogen production route is available [12,13,14,20]. Rodriquez et al. [21] have proposed that thermodynamics determine the product spectrum in a mixed culture fermentation process. The model is based on generalized biochemical information of fermentation pathways and an inventory of all free energy producing and consuming steps in the process. Optimization of the process is subsequently assumed to occur by maximization of the free energy yield in the catabolic pathway catalyzed, and consequently the growth rate in an energy-limited system. Owing to the lack of experimental evidence for the selection criteria proposed, this model should be regarded as a first step in an effort to define the metabolic selection criteria in a mixed culture fermentation system. Future experiments should elucidate whether the approach chosen can be related to experimental evidence. Alternative end products from a mixed culture fermentation process for bioenergy production are solvents (e.g. ethanol, butanol or acetone). These compounds are promising substitutes for gasoline in the transportation sector. Production of ethanol from different types of biomass is generally based on genetically engineered bacteria and yeasts [22,23,24]. To what extent these www.sciencedirect.com
Polyhydroxyalkanoates (PHAs) are the raw material for the production of biodegradable plastics. Polyhydroxybutyrate (PHB), the dominant PHA produced in bacteria, has similar properties to polypropylene. The commercial production of PHB currently employs genetically modified Escherichia coli and Alkaligenes species. Disadvantages of the pure culture production of PHB include the high costs for the pure substrates utilized, the costs for sterile precultivation of the bacteria utilized, and the sterile operation of the final production process. We are investigating the potential use of mixed microbial cultures for the production of PHA from waste streams that are rich in organic compounds [26]. The most successful method for the natural selection of microorganisms with the capacity to store PHAs is based on cultivation in an aerobic sequencing batch reactor. The batch-wise feeding of this type of bioreactor results in periods with substrate (feast phase) and without substrate (famine phase). Microorganisms that have the capacity to store the substrates in the form of PHA and subsequently grow by degradation of PHA will have a more equilibrated growth rate in such a process when compared with microorganisms that are only capable of growth in the presence of substrate [27] (Figure 3). Furthermore, the biochemical route of substrate (e.g. acetate) conversion to PHA is much shorter and less energy-demanding than the production of biomass. This results in higher specific substrate uptake rates when storing substrate as PHA, allowing for effective selection of PHA-storing microorganisms from a mixed population [26,28,29]. In an industrial PHA-production process the cultivation phase of PHA-producing biomass will be followed by an accumulation phase, where the microorganisms are saturated with PHA by supplying an excess of substrate. In this way 70–80% of the dry matter formed in the process can be recovered as PHA. This is only slightly worse than the genetically modified E. coli-based process (http://www.metabolix.com), and potentially suffiCurrent Opinion in Biotechnology 2007, 18:207–212
210 Energy biotechnology
Figure 3
PHA production in mixed microbial cultures. (a) Graph showing the sequence of reactions that form the basis of the selection of PHA-producing cultures in sequencing batch reactors. Initially, acetate is rapidly taken up by the cell and stored in the form of PHB; after acetate depletion, PHA is degraded and microbial growth continues as reflected by the ammonium (NH4) curve. (b) PHA-containing cells as demonstrated by Nile-blue staining.
cient for establishing an economically feasible process [26]. The principle objective of the PHA production process we are working on is the production of bioplastics. Evidently, the production of chemicals for the chemical industry is more profitable than the production of energy carriers, owing to the low price of fossil fuels. However, the production of PHA as energy carrier might also be of interest in cases where no complex infrastructure for methane compression or gas engines for electricity generation are available or cannot be established in an economically viable way. In this case nutrient-deficient wastewaters rich in soluble organic material could effectively be treated in a PHA production process where up to 70% of the organic material is concentrated in a solid product with a relatively low nitrogen content. Furthermore, the oxygen requirements of such an aerobic process are minimized compared with traditional aerobic wastewater treatment. After (partial) drying, the PHA biomass mixture will form a concentrated pool of readily degradable organic material with a relatively low nitrogen content. Herewith, it becomes a highly suitable raw material for energy generation by direct combustion in a power plant or for conversion to methane-containing biogas in an anaerobic (co)digestion process.
Conclusions The examples described above indicate the possibilities and limitations of using selective pressure in mixed microbial cultures to establish stable biotechnological processes for product formation. Numerous other examples Current Opinion in Biotechnology 2007, 18:207–212
can be found in the field of environmental biotechnology and are used, for example, for the removal and/or recovery of phosphate, sulfur and nitrogen from wastewaters. Other MCB-based processes for sustainable energy production, like electricity or molecular hydrogen production in microbial fuel cells, are discussed elsewhere [30–32]. Combinations of physical/chemical and biological processes could provide additional possibilities for the sustainable production of biofuels. Poorly degradable biomass (e.g. lignin-based substrates) can be converted by chemical pretreatment to syngas, which can subsequently be converted into biodiesel in the chemical Fisher-Tropsch process at elevated temperature and pressure. Alternatively, synthesis gas can be biologically converted to organic acids and alcohols by subsequent homoacetogenesis and reduction of the volatile fatty acids formed into alcohols [33,34]. Moreover, molecular hydrogen from syngas could be used to reduce the volatile fatty acids formed in a mixed culture fermentation process. To date, little attention has been given to these processes, but they could represent interesting additions or alternatives to existing processes. We feel that we have explored only a fraction of the potential available in nature for the sustainable production of specific chemicals and energy carriers. Major challenges that remain in this field include the identification of novel selection criteria for specific production processes, and the development of stable processes with less stringent selection criteria, like the solvent-producing fermentation process described above. www.sciencedirect.com
Mixed culture biotechnology Kleerebezem and van Loosdrecht 211
Acknowledgements This work was supported by the Dutch Technology Foundation (STW), project no. DPC5904.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Driessen W, Yspeert P: Anaerobic treatment of low, medium and high strength effluent in the agro-industry. Water Sci Technol 1999, 40:221-228.
2.
Hartmann H, Ahring BK: Status of ADSW 2005. Water Sci Technol 2006, 53:1-5.
3.
Lettinga G, Pol LWH, Koster IW, Wiegant WM, Dezeeuw WJ, Rinzema A, Grin PC, Roersma RE, Hobma SW: High-rate anaerobic wastewater-treatment using the UASB reactor under a wide-range of temperature conditions. Biotechnol Genet Eng Rev 1984, 2:253-284.
4.
Gonzalez-Gil G, Kleerebezem R, van Aelst A, Zoutberg GR, Versprille AI, Lettinga G: Toxicity effects of formaldehyde on methanol degrading sludge and its anaerobic conversion in W Biobed expanded granular sludge bed (EGSB) reactors. Water Sci Technol 1999, 40:195-202.
5.
Kleerebezem R, Macarie H: Treating industrial wastewater: Anaerobic digestion comes of age. Chem Eng 2003, 110:56-64.
6.
van Lier JB, Lens PNL, Pol LWH: Anaerobic treatment for C and S removal in ‘zero-discharge’ paper mills: effects of process design on S removal efficiencies. Water Sci Technol 2001, 44:189-195.
7.
Kortekaas S, Vidal G, He YL, Lettinga G, Field JA: Anaerobicaerobic treatment of toxic pulping black liquor with upfront effluent recirculation. J Ferment Bioeng 1998, 86:97-110.
8.
Seghezzo L, Cuevas CM, Trupiano AP, Guerra RG, Gonzalez SM, Zeeman G, Lettinga G: Stability and activity of anaerobic sludge from UASB reactors treating sewage in subtropical regions. Water Sci Technol 2006, 54:223-229.
9. Hanselmann KW: Microbial energetics applied to waste repositories. Experientia 1991, 47:645-687. Excellent, but generally forgotten, overview paper on the thermodynamics of environmental systems. 10. Janssen AJH, Meijer S, Bontsema J, Lettinga G: Application of the redox potential for controlling a sulfide oxidizing bioreactor. Biotechnol Bioeng 1998, 60:147-155. 11. Alatriste-Mondragon F, Samar P, Cox HHJ, Ahring BK, Iranpour R: Anaerobic codigestion of municipal, farm, and industrial organic wastes: a survey of recent literature. Water Environ Res 2006, 78:607-636. 12. Li CL, Fang HHP: Fermentative hydrogen production from wastewater and solid wastes by mixed cultures. Crit Rev Environ Sci Technol 2007, 37:1-39. Review on the use of anaerobic microbial fermentation for biohydrogen production. The paper presents extensive data tables containing product yield values, as measured in numerous studies. 13. Zoetemeyer RJ, Vandenheuvel JC, Cohen A: Ph influence on acidogenic dissimilation of glucose in an anaerobic digester. Water Res 1982, 16:303-311. 14. Zoetemeyer RJ, Arnoldy P, Cohen A, Boelhouwer C: Influence of temperature on the anaerobic acidification of glucose in a mixed culture forming part of a 2-stage digestion process. Water Res 1982, 16:313-321. 15. Temudo MF, Kleerebezem R, van Loosdrecht MCM: Influence of the pH on (open) mixed culture fermentation of glucose, a chemostat study. Biotechnol Bioeng 2007. Early view DOI:10.1002/bit.21412. www.sciencedirect.com
16. Liu D, Liu DP, Zeng RJ, Angelidaki I: Hydrogen and methane production from household solid waste in the two-stage fermentation process. Water Res 2006, 40:2230-2236. A paper that proves the concept of separate hydrogen and methane production from household solid waste. 17. Gomez X, Moran A, Cuetos MJ, Sanchez ME: The production of hydrogen by dark fermentation of municipal solid wastes and slaughterhouse waste: a two-phase process. J Power Sources 2006, 157:727-732. 18. Akkerman I, Janssen M, Rocha J, Wijffels RH: Photobiological hydrogen production: photochemical efficiency and bioreactor design. Int J Hydrogen Energy 2002, 27:1195-1208. 19. Barbosa MJ, Rocha JMS, Tramper J, Wijffels RH: Acetate as a carbon source for hydrogen production by photosynthetic bacteria. J Biotechnol 2001, 85:25-33. 20. Wangnai C, Zeng RJ, Keller J: Characterisation of high-rate acidogenesis processes using a titration and off-gas analysis sensor. Water Sci Technol 2005, 52:413-418. 21. Rodriguez J, Kleerebezem R, Lema JM, van Loosdrecht MCM: Modeling product formation in anaerobic mixed culture fermentations. Biotechnol Bioeng 2006, 93:592-606. Conceptual model that describes the product pattern in a mixed culture fermentation process as a function of the operational variables established. 22. Dien BS, Cotta MA, Jeffries TW: Bacteria engineered for fuel ethanol production: current status. Appl Microbiol Biotechnol 2003, 63:258-266. 23. Himmel ME, Adney WS, Baker JO, Elander R, McMillan JD, Nieves RA, Sheehan JJ, Thomas SR, Vinzant TB, Zhang M: Advanced bioethanol production technologies: A perspective. In Fuels and Chemicals from Biomass. 1997:2-45. [Acs Symposium Series, vol 666.] 24. van Maris AJA, Abbott DA, Bellissimi E, van den Brink J, Kuyper M, Luttik MAH, Wisselink HW, Scheffers WA, van Dijken JP, Pronk JT: Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status. Antonie Van Leeuwenhoek Int J Gen Mol Microbiol 2006, 90:391-418. Review describing the recent advances made in bioethanol production from different monosaccharides using Saccharomyces cerevisiae. 25. Kalogo Y, Habibi S, Maclean HL, Joshi SV: Environmental implications of municipal solid waste-derived ethanol. Environ Sci Technol 2007, 41:35-41. 26. Reis MAM, Serafim LS, Lemos PC, Ramos AM, Aguiar FR, Van Loosdrecht MCM: Production of polyhydroxyalkanoates by mixed microbial cultures. Bioprocess Biosyst Eng 2003, 25:377-385. Extensive review on the production of bioplastics using mixed microbial cultures. The paper provides biochemical PHA production and degradation pathways together with product yields established in numerous investigations. 27. van Loosdrecht MCM, Heijnen JJ: Modelling of activated sludge processes with structured biomass. Water Sci Technol 2002, 45:13-23. 28. Beun JJ, Paletta F, Van Loosdrecht MCM, Heijnen JJ: Stoichiometry and kinetics of poly-b-hydroxybutyrate metabolism in aerobic, slow growing, activated sludge cultures. Biotechnol Bioeng 2000, 67:379-389. 29. Beun JJ, Dircks K, Van Loosdrecht MCM, Heijnen JJ: Poly-b-hydroxybutyrate metabolism in dynamically fed mixed microbial cultures. Water Res 2002, 36:1167-1180. 30. Logan BE, Hamelers B, Rozendal R, Schrorder U, Keller J, Freguia S, Aelterman P, Verstraete W, Rabaey K: Microbial fuel cells: methodology and technology. Environ Sci Technol 2006, 40:5181-5192. 31. Rozendal RA, Hamelers HVM, Euverink GJW, Metz SJ, Buisman CJN: Principle and perspectives of hydrogen Current Opinion in Biotechnology 2007, 18:207–212
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production through biocatalyzed electrolysis. Int J Hydrogen Energy 2006, 31:1632-1640. 32. Logan BE, Liu H, Heilmann J, Oh SE, Cheng S, Grot S.: Electricity and hydrogen production using different types of microbial fuel cell technologies. Abstracts of Papers of the American Chemical Society 2005, 230:U1680-U1680.
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33. Smith DP, McCarty PL: Reduced product formation following perturbation of ethanol-fed and propionate-fed methanogenic CSTRs. Biotechnol Bioeng 1989, 34:885-895. 34. Smith DP, McCarty PL: Energetic and rate effects on methanogenesis of ethanol and propionate in perturbed CSTRs. Biotechnol Bioeng 1989, 34:39-54.
www.sciencedirect.com
Current Opinion in Biotechnology 2007, 18:207–212 This review comes from a themed issue on Energy biotechnology Edited by Largus T Angenent
both disciplines could be defined as ‘mixed culture biotechnology’ (MCB), as the application of mixed cultures is a specific characteristic of biotechnologies originating from the waste treatment field. Owing to the use of undefined mixed cultures, process development in MCB can only be based on natural/ecological selection by manipulating the operation of the bioprocess or by varying the source of the natural inoculum. Using this approach, the required metabolic capacities and the corresponding microbial population can be effectively enriched from a natural environment. Compared with pure culture based industrial biotechnology, specific advantages of MCB include: no sterilization requirements, adaptive capacity owing to microbial diversity, the capacity to use mixed substrates, and the possibility of a continuous process. This paper will give a short description of three types of MCB processes that are potentially interesting for the production of energy carriers, while treating a waste stream. The first classical example of a highly effective MCB process is the anaerobic digestion process. Alternatively one could aim for the production of solvents by fermentation of organic substrates or the production of bioplastics using a sequencing batch process. In the description of these processes emphasis will be given to the ecological selection criteria that form the basis of the process.
Available online 16th May 2007 0958-1669/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2007.05.001
Introduction The traditional treatment objective of environmental biotechnological processes is the elimination of polluting compounds to generate a liquid, gaseous or solid residue that can be reused in a natural environment without detrimental ecological effects. Given the increasing interest in the effective use of natural resources, a second treatment objective can be suggested: the recovery of specific nutrients (e.g. nitrogen, phosphorous or sulfur) or the production of specific organic products (e.g. methane, organic solvents, bioplastics). The addition of the second treatment objective changes the status of several streams generated in the agro-industry from waste to raw material for the production of specific chemicals or energy carriers. Herewith a biotechnological research field has been created that combines traditional elements from environmental biotechnology in terms of cleaning of waste streams, with industrial biotechnology that is aiming for product maximization (see Table 1). The combination of www.sciencedirect.com
Selective pressure for product formation Anaerobic digestion
Anaerobic digestion is the classical example of a process that combines the objectives of elimination of organic compounds from a waste stream with the generation of a valuable product in the form of methane-containing biogas. Different bioreactor configurations have been developed for the treatment of liquid and solid waste streams [1–4]. For wastewater treatment, the application potential of anaerobic digestion has been extended from medium to highly concentrated wastewaters of agro-industrial origin, to more complex applications like those generated in petrochemical industries [5,6], paper industries [6,7] and even sewage [8]. There are three clear advantages of the anaerobic treatment over aerobic degradation of organic substrates: the high product and low biomass yield resulting in a limited generation of waste sludge as an unwanted side product; the in situ separation of the product as biogas, limiting costs for product separation; and the use of simple technology, as mixing by the biogas produced circumvents the need for other mixing requirements. The selective pressure required to induce methane production from a waste stream containing organic compounds is very simple: avoid the presence of an electron acceptor Current Opinion in Biotechnology 2007, 18:207–212
208 Energy biotechnology
Table 1 Historic differences between environmental and industrial biotechnology. Variable
Environmental biotechnology
Industrial biotechnology
History Basis Biomass Process type Process models Process objectives Substrates Process establishment
Wastewater treatment Catabolism Mixed culture (sludge) Continuous Lumped black box models Minimize effluent substrate concentrations Mixed substrates (waste) Ecological selection by process operation
Product formation Anabolism Specific strains of microorganisms (Fed) batch Omics-based metabolic network models Maximize productivity Pure and well-defined substrates Specific microorganisms and genetic engineering
(e.g. oxygen, nitrate or sulfate) or an external energy source (e.g. light). In the absence of an external electron acceptor, organic substrates can only be fermented; a process where the organic substrate is both electron donor and acceptor. The final end products of the organic substrate fermentation are methane, carbon dioxide and ammonia. Methane is the organic compound with the lowest free energy content per electron upon oxidation to carbon dioxide (see Figure 1) [9]. This indicates that in a thermodynamically closed system substrates will eventually be converted to methane and carbon dioxide. Microorganisms can obtain the energy required for growth by (stepwise) catalyzing the conversion of organic substrates to methane and carbon dioxide. From a bioenergy concept point of view, the biotechnological production of methane has limitations owing to the low price of natural gas (US$ 0.5 per kg). In cases where a network for natural gas is available, biogas can be distributed through this existing infrastructure; however, carbon dioxide and hydrogen sulfide need to be removed from the biogas [10]. Figure 1
Anaerobic digestion for bioenergy production is generally based on the treatment of substrates like activated sludge, manure and concentrated industrial waste [11]. Process advances in the field of anaerobic digestion of complex substrates are limited, owing to the rate-limiting hydrolysis process for solubilization of the particulate substrates. Enhanced degradation rates can only be established using generally expensive pretreatment methods. Consequently, most interesting developments are expected to occur in the field of post-treatment of the nutrient-rich solid and liquid residues obtained after digestion, and the effective incorporation of anaerobic digestion in biorefinery concepts. Solvent and/or biohydrogen production by mixed culture fermentation
Biohydrogen production by fermentation of carbohydrates has received significant attention in recent years. The main driving force for investigating the production of hydrogen instead of methane is the higher economic value of hydrogen, owing to its wider range of applications in the chemical industry [12]. The main limitation of hydrogen production by fermentative bacteria is the maximum electron-based yield that can be established. Owing to biochemical and thermodynamic limitations, the maximum theoretical number of moles of hydrogen that can be generated per mole glucose is four, and can be obtained by glucose oxidation to two moles acetate :C H O þ 2H O ! 2C H O 1 þ 2Hþ1 þ 2CO þ 4H 6 12 6 2 2 3 2 2 2 DG01 ¼ 145 k j mol 1
The substrate degradation sequence in the anaerobic digestion process. The free energy content per electron (upon oxidation to carbon dioxide) of the substrates is given together with the intermediates and products involved. Current Opinion in Biotechnology 2007, 18:207–212
However, the measured hydrogen production per mole glucose, as observed in mixed culture fermentation studies, is much lower and will normally not exceed two moles [12,13–15,16]. This practical limitation seems to be related to a biochemical restriction associated with the electron carriers utilized in the different fermentation pathways (Figure 2) [15]. Because of thermodynamic limitations, hydrogen can only be produced in oxidation reactions in the metabolic pathways coupled to formate www.sciencedirect.com
Mixed culture biotechnology Kleerebezem and van Loosdrecht 209
Figure 2
pure culture based processes can be used for the conversion of mixed substrates like municipal solid waste for solvent production is a subject of debate [25]. Development of an MCB-based continuous process for bioethanol production from waste would decrease the production costs significantly. However, as yet, no clear selection criterion for an ethanol-producing microorganism has been established. Some suggestions have been made on how to direct a fermentation process towards ethanol [21], but experimental evidence is lacking. Bioethanol production using MCB could also aim for combined processes based on the fermentation of carbohydrates to volatile fatty acids, and subsequent (biological) reduction of the products formed to alcohols using molecular hydrogen.
The dominant catabolic pathways of anaerobic glucose fermentation.
Polyhydroxyalkanoate production
production or ferrodoxin reduction, and not in the NADdependent steps. Fermentative hydrogen production provides only a partial oxidation of the organic substrate and is therefore normally integrated in a two-step process. In such a process, the fermentation products (volatile fatty acids) are either converted to methane-containing biogas [17] or converted to hydrogen and carbon dioxide in a phototrophic process [18,19]. A main limitation of the fermentative hydrogen production process is that no generally accepted selection criterion for the most favorable fermentative hydrogen production route is available [12,13,14,20]. Rodriquez et al. [21] have proposed that thermodynamics determine the product spectrum in a mixed culture fermentation process. The model is based on generalized biochemical information of fermentation pathways and an inventory of all free energy producing and consuming steps in the process. Optimization of the process is subsequently assumed to occur by maximization of the free energy yield in the catabolic pathway catalyzed, and consequently the growth rate in an energy-limited system. Owing to the lack of experimental evidence for the selection criteria proposed, this model should be regarded as a first step in an effort to define the metabolic selection criteria in a mixed culture fermentation system. Future experiments should elucidate whether the approach chosen can be related to experimental evidence. Alternative end products from a mixed culture fermentation process for bioenergy production are solvents (e.g. ethanol, butanol or acetone). These compounds are promising substitutes for gasoline in the transportation sector. Production of ethanol from different types of biomass is generally based on genetically engineered bacteria and yeasts [22,23,24]. To what extent these www.sciencedirect.com
Polyhydroxyalkanoates (PHAs) are the raw material for the production of biodegradable plastics. Polyhydroxybutyrate (PHB), the dominant PHA produced in bacteria, has similar properties to polypropylene. The commercial production of PHB currently employs genetically modified Escherichia coli and Alkaligenes species. Disadvantages of the pure culture production of PHB include the high costs for the pure substrates utilized, the costs for sterile precultivation of the bacteria utilized, and the sterile operation of the final production process. We are investigating the potential use of mixed microbial cultures for the production of PHA from waste streams that are rich in organic compounds [26]. The most successful method for the natural selection of microorganisms with the capacity to store PHAs is based on cultivation in an aerobic sequencing batch reactor. The batch-wise feeding of this type of bioreactor results in periods with substrate (feast phase) and without substrate (famine phase). Microorganisms that have the capacity to store the substrates in the form of PHA and subsequently grow by degradation of PHA will have a more equilibrated growth rate in such a process when compared with microorganisms that are only capable of growth in the presence of substrate [27] (Figure 3). Furthermore, the biochemical route of substrate (e.g. acetate) conversion to PHA is much shorter and less energy-demanding than the production of biomass. This results in higher specific substrate uptake rates when storing substrate as PHA, allowing for effective selection of PHA-storing microorganisms from a mixed population [26,28,29]. In an industrial PHA-production process the cultivation phase of PHA-producing biomass will be followed by an accumulation phase, where the microorganisms are saturated with PHA by supplying an excess of substrate. In this way 70–80% of the dry matter formed in the process can be recovered as PHA. This is only slightly worse than the genetically modified E. coli-based process (http://www.metabolix.com), and potentially suffiCurrent Opinion in Biotechnology 2007, 18:207–212
210 Energy biotechnology
Figure 3
PHA production in mixed microbial cultures. (a) Graph showing the sequence of reactions that form the basis of the selection of PHA-producing cultures in sequencing batch reactors. Initially, acetate is rapidly taken up by the cell and stored in the form of PHB; after acetate depletion, PHA is degraded and microbial growth continues as reflected by the ammonium (NH4) curve. (b) PHA-containing cells as demonstrated by Nile-blue staining.
cient for establishing an economically feasible process [26]. The principle objective of the PHA production process we are working on is the production of bioplastics. Evidently, the production of chemicals for the chemical industry is more profitable than the production of energy carriers, owing to the low price of fossil fuels. However, the production of PHA as energy carrier might also be of interest in cases where no complex infrastructure for methane compression or gas engines for electricity generation are available or cannot be established in an economically viable way. In this case nutrient-deficient wastewaters rich in soluble organic material could effectively be treated in a PHA production process where up to 70% of the organic material is concentrated in a solid product with a relatively low nitrogen content. Furthermore, the oxygen requirements of such an aerobic process are minimized compared with traditional aerobic wastewater treatment. After (partial) drying, the PHA biomass mixture will form a concentrated pool of readily degradable organic material with a relatively low nitrogen content. Herewith, it becomes a highly suitable raw material for energy generation by direct combustion in a power plant or for conversion to methane-containing biogas in an anaerobic (co)digestion process.
Conclusions The examples described above indicate the possibilities and limitations of using selective pressure in mixed microbial cultures to establish stable biotechnological processes for product formation. Numerous other examples Current Opinion in Biotechnology 2007, 18:207–212
can be found in the field of environmental biotechnology and are used, for example, for the removal and/or recovery of phosphate, sulfur and nitrogen from wastewaters. Other MCB-based processes for sustainable energy production, like electricity or molecular hydrogen production in microbial fuel cells, are discussed elsewhere [30–32]. Combinations of physical/chemical and biological processes could provide additional possibilities for the sustainable production of biofuels. Poorly degradable biomass (e.g. lignin-based substrates) can be converted by chemical pretreatment to syngas, which can subsequently be converted into biodiesel in the chemical Fisher-Tropsch process at elevated temperature and pressure. Alternatively, synthesis gas can be biologically converted to organic acids and alcohols by subsequent homoacetogenesis and reduction of the volatile fatty acids formed into alcohols [33,34]. Moreover, molecular hydrogen from syngas could be used to reduce the volatile fatty acids formed in a mixed culture fermentation process. To date, little attention has been given to these processes, but they could represent interesting additions or alternatives to existing processes. We feel that we have explored only a fraction of the potential available in nature for the sustainable production of specific chemicals and energy carriers. Major challenges that remain in this field include the identification of novel selection criteria for specific production processes, and the development of stable processes with less stringent selection criteria, like the solvent-producing fermentation process described above. www.sciencedirect.com
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Acknowledgements This work was supported by the Dutch Technology Foundation (STW), project no. DPC5904.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Driessen W, Yspeert P: Anaerobic treatment of low, medium and high strength effluent in the agro-industry. Water Sci Technol 1999, 40:221-228.
2.
Hartmann H, Ahring BK: Status of ADSW 2005. Water Sci Technol 2006, 53:1-5.
3.
Lettinga G, Pol LWH, Koster IW, Wiegant WM, Dezeeuw WJ, Rinzema A, Grin PC, Roersma RE, Hobma SW: High-rate anaerobic wastewater-treatment using the UASB reactor under a wide-range of temperature conditions. Biotechnol Genet Eng Rev 1984, 2:253-284.
4.
Gonzalez-Gil G, Kleerebezem R, van Aelst A, Zoutberg GR, Versprille AI, Lettinga G: Toxicity effects of formaldehyde on methanol degrading sludge and its anaerobic conversion in W Biobed expanded granular sludge bed (EGSB) reactors. Water Sci Technol 1999, 40:195-202.
5.
Kleerebezem R, Macarie H: Treating industrial wastewater: Anaerobic digestion comes of age. Chem Eng 2003, 110:56-64.
6.
van Lier JB, Lens PNL, Pol LWH: Anaerobic treatment for C and S removal in ‘zero-discharge’ paper mills: effects of process design on S removal efficiencies. Water Sci Technol 2001, 44:189-195.
7.
Kortekaas S, Vidal G, He YL, Lettinga G, Field JA: Anaerobicaerobic treatment of toxic pulping black liquor with upfront effluent recirculation. J Ferment Bioeng 1998, 86:97-110.
8.
Seghezzo L, Cuevas CM, Trupiano AP, Guerra RG, Gonzalez SM, Zeeman G, Lettinga G: Stability and activity of anaerobic sludge from UASB reactors treating sewage in subtropical regions. Water Sci Technol 2006, 54:223-229.
9. Hanselmann KW: Microbial energetics applied to waste repositories. Experientia 1991, 47:645-687. Excellent, but generally forgotten, overview paper on the thermodynamics of environmental systems. 10. Janssen AJH, Meijer S, Bontsema J, Lettinga G: Application of the redox potential for controlling a sulfide oxidizing bioreactor. Biotechnol Bioeng 1998, 60:147-155. 11. Alatriste-Mondragon F, Samar P, Cox HHJ, Ahring BK, Iranpour R: Anaerobic codigestion of municipal, farm, and industrial organic wastes: a survey of recent literature. Water Environ Res 2006, 78:607-636. 12. Li CL, Fang HHP: Fermentative hydrogen production from wastewater and solid wastes by mixed cultures. Crit Rev Environ Sci Technol 2007, 37:1-39. Review on the use of anaerobic microbial fermentation for biohydrogen production. The paper presents extensive data tables containing product yield values, as measured in numerous studies. 13. Zoetemeyer RJ, Vandenheuvel JC, Cohen A: Ph influence on acidogenic dissimilation of glucose in an anaerobic digester. Water Res 1982, 16:303-311. 14. Zoetemeyer RJ, Arnoldy P, Cohen A, Boelhouwer C: Influence of temperature on the anaerobic acidification of glucose in a mixed culture forming part of a 2-stage digestion process. Water Res 1982, 16:313-321. 15. Temudo MF, Kleerebezem R, van Loosdrecht MCM: Influence of the pH on (open) mixed culture fermentation of glucose, a chemostat study. Biotechnol Bioeng 2007. Early view DOI:10.1002/bit.21412. www.sciencedirect.com
16. Liu D, Liu DP, Zeng RJ, Angelidaki I: Hydrogen and methane production from household solid waste in the two-stage fermentation process. Water Res 2006, 40:2230-2236. A paper that proves the concept of separate hydrogen and methane production from household solid waste. 17. Gomez X, Moran A, Cuetos MJ, Sanchez ME: The production of hydrogen by dark fermentation of municipal solid wastes and slaughterhouse waste: a two-phase process. J Power Sources 2006, 157:727-732. 18. Akkerman I, Janssen M, Rocha J, Wijffels RH: Photobiological hydrogen production: photochemical efficiency and bioreactor design. Int J Hydrogen Energy 2002, 27:1195-1208. 19. Barbosa MJ, Rocha JMS, Tramper J, Wijffels RH: Acetate as a carbon source for hydrogen production by photosynthetic bacteria. J Biotechnol 2001, 85:25-33. 20. Wangnai C, Zeng RJ, Keller J: Characterisation of high-rate acidogenesis processes using a titration and off-gas analysis sensor. Water Sci Technol 2005, 52:413-418. 21. Rodriguez J, Kleerebezem R, Lema JM, van Loosdrecht MCM: Modeling product formation in anaerobic mixed culture fermentations. Biotechnol Bioeng 2006, 93:592-606. Conceptual model that describes the product pattern in a mixed culture fermentation process as a function of the operational variables established. 22. Dien BS, Cotta MA, Jeffries TW: Bacteria engineered for fuel ethanol production: current status. Appl Microbiol Biotechnol 2003, 63:258-266. 23. Himmel ME, Adney WS, Baker JO, Elander R, McMillan JD, Nieves RA, Sheehan JJ, Thomas SR, Vinzant TB, Zhang M: Advanced bioethanol production technologies: A perspective. In Fuels and Chemicals from Biomass. 1997:2-45. [Acs Symposium Series, vol 666.] 24. van Maris AJA, Abbott DA, Bellissimi E, van den Brink J, Kuyper M, Luttik MAH, Wisselink HW, Scheffers WA, van Dijken JP, Pronk JT: Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status. Antonie Van Leeuwenhoek Int J Gen Mol Microbiol 2006, 90:391-418. Review describing the recent advances made in bioethanol production from different monosaccharides using Saccharomyces cerevisiae. 25. Kalogo Y, Habibi S, Maclean HL, Joshi SV: Environmental implications of municipal solid waste-derived ethanol. Environ Sci Technol 2007, 41:35-41. 26. Reis MAM, Serafim LS, Lemos PC, Ramos AM, Aguiar FR, Van Loosdrecht MCM: Production of polyhydroxyalkanoates by mixed microbial cultures. Bioprocess Biosyst Eng 2003, 25:377-385. Extensive review on the production of bioplastics using mixed microbial cultures. The paper provides biochemical PHA production and degradation pathways together with product yields established in numerous investigations. 27. van Loosdrecht MCM, Heijnen JJ: Modelling of activated sludge processes with structured biomass. Water Sci Technol 2002, 45:13-23. 28. Beun JJ, Paletta F, Van Loosdrecht MCM, Heijnen JJ: Stoichiometry and kinetics of poly-b-hydroxybutyrate metabolism in aerobic, slow growing, activated sludge cultures. Biotechnol Bioeng 2000, 67:379-389. 29. Beun JJ, Dircks K, Van Loosdrecht MCM, Heijnen JJ: Poly-b-hydroxybutyrate metabolism in dynamically fed mixed microbial cultures. Water Res 2002, 36:1167-1180. 30. Logan BE, Hamelers B, Rozendal R, Schrorder U, Keller J, Freguia S, Aelterman P, Verstraete W, Rabaey K: Microbial fuel cells: methodology and technology. Environ Sci Technol 2006, 40:5181-5192. 31. Rozendal RA, Hamelers HVM, Euverink GJW, Metz SJ, Buisman CJN: Principle and perspectives of hydrogen Current Opinion in Biotechnology 2007, 18:207–212
212 Energy biotechnology
production through biocatalyzed electrolysis. Int J Hydrogen Energy 2006, 31:1632-1640. 32. Logan BE, Liu H, Heilmann J, Oh SE, Cheng S, Grot S.: Electricity and hydrogen production using different types of microbial fuel cell technologies. Abstracts of Papers of the American Chemical Society 2005, 230:U1680-U1680.
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33. Smith DP, McCarty PL: Reduced product formation following perturbation of ethanol-fed and propionate-fed methanogenic CSTRs. Biotechnol Bioeng 1989, 34:885-895. 34. Smith DP, McCarty PL: Energetic and rate effects on methanogenesis of ethanol and propionate in perturbed CSTRs. Biotechnol Bioeng 1989, 34:39-54.
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