Biotech Advs vol.l,pp 47-58, 1983
0734-9750/83 $0.00.+.50 Printed in Great Britain. All Rights Reserved. Copyright © Pergamon Press Ltd
FUELS AND INDUSTRIAL CHEMICALS P. LINKO He/sinki University of Technology, Department of Chemistry, Laboratory of Biochemistry ond Food Technology, SF-02150 Espoo 15, F/n/and
ABSTRACT The favorite subject of recent literature on biotechnical processes has been ethanol fermentation. This review covers a number of new technics developed, including immobilized biocatalyst technology and bacterial fermentations. Reference is also made to recent work on whey, starch, inulin, and cellulosic materials as substrates for ethanol production. Renewed interest in acetonebutanol fermentation for solvent and liquid fuel production has also been clearly evident during the last two years. Biotechnical production of organic acids has been considered as an alternative route to chemical feedstocks. New developments in amino acid, methane, hydrogen, and hydrocarbon production, and on hydrocarbon oxidation are also briefly covered. INTRODUCTION A number of major events in the area of biotechnical production of fuel and chemicals took place during the period of 1981 to 1982. The 2nd European Congress of Biotechnology was held in Eastbourne, England in April 1981 (67), International Symposium "Cereals: A Renewable Resource, Theory and Practice" was held in Copenhagen in August with the Proceedings published late 1981 (59), and the 6th Enzyme Engineering Conference was held in Kashikojima, Japan in September with Proceedings published a year later (9). Among major events in 1982 were the 13th International Congress in Microbiology in Boston in August, the 3rd Biochemical Engineering Conference in Santa Barbara in September, and the 2nd E.C. Conference "Energy from Biomass" in Berlin also in September with Proceedings to be published in 1983. Several books, reports and reviews in the subject area have appeared during the two year period, and only a few can be mentioned here. The 3-volume report (52,53,77) on the 6th International Fermentation Symposium held in London, Ontario in July 1980 has now become available. In 1981 a special volume of the Advances in Biochemical Engineering on "Bioenergy" was published (20), Goldstein (26) edited a book on "Organic Chemicals from Biomass," and Sofer and Zaborsky (68) a book on "Biomass Conversion Processes for Energy and Fuels." Finally, a book on "Energy: The Biomass Options" by Bungay (7) should be mentioned. In 1982 the 4th edition of the Prescott and Dunn's Industrial Microbiology (62) appeared, Wise (83) edited a book on "Fuels and Organic Chemicals from Biomass," and a very comprehensive report on the Japanese 47
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biotechnical industry was published by the Yano Research Institute Ltd, Japan (85). Of the several general overview articles papers on "Microbiological production of industrial chemicals" by Eveleigh (19), on "Chemicals from biomass: Petrochemical substitution options" by Lipinsky (44), and on "Immobilized cells" by Fukui and Tanaka (21) should be mentioned. LIQUID FUEL AND SOLVENTS Ethanol Ethanol is both a valuable fuel and solvent, and a potentially important feedstock for the chemical industry. The alcohol fuels program has been recently reviewed (69). Rudd et al. (64) published an extensive resum~ on petrochemical technology assesment, including a survey on ethanol as an alternative feedstock with comparision of petrochemical and biotechnical processes in chemical production. Ethanol can be converted to ethylene with relative ease, and the renewed interest in biotechnical ethanol production also has catalyzed research on ethanol to ethylene conversion process (36,78). As an example, only a new process based on a new catalyst for simple, nearly complete dehydration of ethanol, developed by the Scientific Design Company, a subsidiary of Halcon International, Inc., is mentioned here. It has been shown that the initial rate of ethanol fermentation may be significantly increased by raising the temperature up to about 40°C but the overall productivity decreases owing to increased inhibition by ethanol (32). Prince and Barford (60) showed recently that sugar cane juice can be rapidly fermented to ethanol in a tower fermentor with flocculent yeast. In a so called Ex-Ferm solid-liquid mixed-phase fermentation process sucrose extraction and fermentation are carried out simultaneously (15,63). A rotating drum fermentor, providing efficient mixing at high raw-susbstrate/water ratios has also been suggested (18). Multi-organism Fermentations. Jones and Greenfield (31) proposed the simultaneous utilization of two different organisms, one which would tolerate high initial sugar concentration and the other which would tolerate high product ethanol level. Cellulosic substrates have been succesfully fermented by employing cocultures with pentose utilizing Clostridium thermohydrosulfuricum (55), although it has also been shown that cellulose may be directly fermented to ethanol by Monilia sp. isolated from bagasse compost (28). A new semicontinuous dilute acid hydrolysis process has been reported which in combination with continuous ethanol fermentation would produce about 140 L of ethanol from one metric ton of wood (50). Another, a so called Emert process developed at the University of Arkansas is based on simultaneous enzymatic hydrolysis of cellulose with Trichoderma reesei and ethanol fermentation with a mixed yeast culture (84). A production of 300 L of ethanol per day from one metric ton of communal waste of 55% cellulose has been reported. U e d a e t a L (75) obtained ethanol yields of up to 99.6% of theoretical in 5 days by fermenting uncooked raw cassava starch with a mixture of Asp~rgillus niger glucoamylase and Sacc~2ro~ryc~s cerevisiae yeast. Acid or enzyme liquefied corn mash has been first inoculated with Aspergillus oryzae wheat bran koji for one hour to initiate saccharification, followed by simultaneous fermentation with S. cerevisiae yeast (88). The authors also suggested that a high solids corn medium would be suitable for extrusion cooking pretreatment followed by a continuous plug flow bioreactor, although no such experiments were reported. The liquefaction of starch by extrusion cooking with or without thermostable aamylase, followed by saccharification and continuous ethanol fermentation has been recently demonstrated (39). Fermentation of Pentoses. Many hydrolysates of lignocellulosic materials contain high levels of pentoses which are not normally fermented by S. cerevisi-
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49
~ . Gong et a~. (27) suggested, however, that if D-xylose is first enzymatically isomerized to D-xylulose ethanol fermentation may be subsequently carried out with S. cerevis~e. They also reported on direct conversion of Dxylose to ethanol by a pentose fermenting mutant strain C ~ i ~ sp. XF 217. Pachysolen t ~ p h i l u 8 yeast has recently attracted greatest attention as an organism able to convert pentoses to ethanol, and volumetric productivities of 2.1 to 2.2 g/Lh at 2.5 hour residence time with 5% (w/v) initial D-xylose concentration, and at 8 hour residence time with 10% (w/v) initial D-xylose has been reported (66). Immobilized Yeast Bioreactors. According to Holceberg and Margallth (29) approximately 20 to 25% increase in ethanol production may be obtained by immobilizing yeast cells to gel structure. Linko and Linko (43) employed calcium alginate (6-8%) gel bead entrapped living S. cerevisiae yeast cells in a packed-bed column reactor for continuous ethanol production from sugar cane molasses (17.5% w/v sugar), and were able to maintain about 7% (w/v) product ethanol level at about 5 hour residence time for several months. Chibata and coworkers (79) developed a method to immobilize yeast cells in ~-carrageenan, and with a stepwise increase in feed glucose concentration at 6 day intervals up to 25% were able to obtain product ethanol concentration of 114 g/L at >95% yield and 2.6 hour residence time. An increase in ethanol productivity with K-carrageenan entrapped yeast cells by colmmobilizlng with trlcalcium phosphate has been later reported (81). Among others, whey lactose has also been shown to be suitable substrate for ethanol fermentation, and a constant about 2% (w/v) product ethanol level with 5% (w/v) initial lactose level could be maintained for at least one month, employing electrodialytlcally demineralized whey as substrate for calcium alginate gel immobilized lactose fermenting Kluyvero~yces fragills yeast (42). Alternatively, a good conversion of whey lactose to ethanol was obtained with a continuous column bioreactor of mixed (50/50) calcium alginate gel entrapped S. cerev~siae baker's yeast and Duolite S-761 phenolformaldehyde resin adsorbed and glutaraldehyde cross-linked Aspergillus niger Bgalactosidase (38). Chen and Zall (8) employed an expanded-bed attached film reactor with cellulose acetate as support medium to obtain a volumetric ethanol productivity of up to 6.9 g/Lh at I.i hour residence time and effluent ethanol concentration of 7.6 g/L from reconstituted acid whey of 10% lactose, supplemented with 0.7% yeast extract and I0 mg/L ergosterol. A new reactor design for ethanol fermentation was recently presented by Fukushima and Hanai (22). They employed aluminium alginate gel entrapped mutant strain of S. formosensis M-Ill in a specially constructed 3-stage fluidizedbed reactor with rhombus-shaped units, and a 150 ml working volume. Contamination was controlled by feeding sugar cane molasses at pH 2.8 to 3.6. Product ethanol concentration of about 78 g/L was reported with 162 g/L sugar feed, and 96 g/L with 198 g/L sugar feed. According to Day and Sarkar (12) aseptic conditions do not appear to be necessary in laboratory scale ethanol fermentations with immobilized cell bioreactors, and a small 500 ml/h pilot plant was reported to be under construction at the Louisiana State University to further investigate the feasibility of the process. This observation is in accordance with our own experience at the Helsinkl University of Technology, although some contamination problems were encountered when calcium alginate gel immobilized Kluyveromyces fragilis cells were used for continuous fermentation of whey ultrafiltrate at Valio Laboratory's Lapinlahti, Finland 50-L bed-volume pilot column reactor. Contamination problems could, however, be controlled by decreasing the pH to about 3.5. Occasional contaminations at the Hofu Laboratory, Kyowa Hakko Kogyo Co Ltd, Japan, pilot plant operated for the Research Association of Petroleum Alternatives (RAPAD) could be eliminated either by acidifying with sulfuric acid or by bacterlcidical agents
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(54). The complete pilot ~lant consisting of 5 reactors in two channels with a total bed-volume of 4 m J, and capable of producing 2.4 m 3 of ethanol per day has been in operation since April 1982. The reactors were shown to operate in a fluidized state owing to the rapidly evolving carbon dioxide, and best results were obtained with two reactors in series. Employing a new yeast strain T-620, about 10% (v/v) product ethanol level could be maintained in continuous operation, and at 8.5% (v/v) ethanol level, 95% conversion, and at 2.5 hour residence time the volumetric productivity with calcium alginate entrapped yeast was about 20 g/Lh. Another RAPAD sponsored ethanol pilot plant of 250 L daily capacity, based on yeast immobilized on photocross-linkable resin, has been constructed at the Yatsushiro distillery of Sanraku-Ocean Co Ltd (3). Ethanol Production from Inulin. Jerusalem artichoke tubers of one of the highest carbohydrate contents of any known crop represent a potential feedstock for ethanol fermentation. A two-step juice expression process followed by 2 hour hydrolysis of the inulin obtained and by subsequent batch ethanol fermentation by a special inulase active yeast has been reported (17). Margaritis and Bajpai (47) employed calcium alginate gel entrapped Kluyveromyces marxiamus UCD 55-82 cells for continuous ethanol fermentation of 10% inulin sugars supplemented with 0.05% Tween 80, 0.01% oleic acid, and 0.01% cornsteep liquor. Volumetric productivities from about 30 g/Lh with 94% yield at 1.5 hour residence time to a maximum of 104 g/Lh with 86% yield at 20 min residence time were reported. Et__hanol Production with Zymomonas mobilis. Considerable amount of work on ethanol fermentation with ZB~omonas mobilis has been done during the last two years. Z. mobilis bacterial cells have been immobilized in <-carrageenan/ locust bean gum mixed gel to obtain mean volumetric productivity in continuous operation from 10% (w/v) glucose feed of about 40 g/Lh with 82% yield at I hour residence time (40,41). Typical biocatalyst cell count was about i0 I0 cells/g, and the effluent cell count 107 to 108 cells/g, sufficient for a rapid after fermentation of any sugar remaining. It should be noted that if the volumetric productivity is reported on the basis of the reactor void volume instead of the biocatalyst bed volume, values over i00 g/Lh are obtained. Similar observations have been reported by Margaritis et al. (48). The problems encountered in extended continuous operations owing to the very rapid carbon dioxide evolution have been overcome either by a conical reactor design (41) or by a slightly inclined horizontal reactor (48). Another type of approach was reported by Arcuri (4), who allowed the bacterial cells to grow on glass-fiber pads in a vertical column reactor to obtain about 1012 cells/g of biocatalyst, and a maximum volumetric productivity of 152 g/Lh (based on reactor volume) at 10-15 min residence time and 6.4% maximum effluent ethanol concentration. Novel Developments. Much attention has been also devoted to themophilic bacteria which, owing to high catabolic activity at elevated temperatures, allow shorter fermentation times and an increased productivity. Other potential advantages include a decreased contamination risk and improved ethanol recovery. A new extreme thermophile Thermoe~naerobacter ethanolicus gen. nov., spec. nov., isolated from hot springs, is of special interest (82). The bacterium grows between 37°-78°C, pH 4.4-9.8, and is able to ferment both hexoses and pentoses. Zertuche and Zall (86) demonstrated that direct continuous fermentation of cellulosic materials with up to 3% of cellulose is possible with another thermophilic bacterium Clostridium thermocellum. Finally, new achievements may be expected through novel genetic engineering techniques. Panchal et al. (57) have recently reported on preliminary experiments to construct new yeast strains by hybridization and protoplast fusion to improve both fermentation rate and ethanol tolerance.
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Glycerol Bisping and Rehm (5) produced glycerol with K-carrageenan immobilized S a c c ~ -
romyces cerevisiae yeast by reacting the intermediate acetaldehyde with sulfite which results in an excess of NADH 2 to reduce dihydroxyacetone phosphate to glycerol phosphate. Sulfite addition increased the effluent glycerol level from about 4-5 g/L to about 25-27 g/L. Acetone and Butanol Acetone-Butanol fermentation has recently gained interest because of the potential solvent and liquid fuel value of n-butanol which, unlike ethanol, is water tolerant in gashol mixtures (56). France has recently announced a program for supplementing super gasoline either by 3% methanol or 5% ethanol together with an additional 2% of acetone-butanol-ethanol mixture obtained from fermentation of either sugar beet or Jerusalem artichoke. Sony et al. (70) hydrolyzed bagasse of rice straw by mixed culture filtrates of T r ~ c ~ d e ~ reese~ and Aspergillus wentii, and employed Clostridium saccharoper butyloacetonlcum ATCC 27022 to ferment sugars obtained to n-butanol. Maximum effluent butanol content of 16 g/L was claimed. Maddox (45) reported on a two-stage fermentation system of molasses (5% solids) supplemented with pentoses (30 g/L) as a model substrate, where hexoses were fermented to ethanol by Sacc~or~jce8 cer ~ v i ~ a e in the first stage, and pentoses to n-butanol after adjusting of the pH to 6.5 by C1. acetobutyllcum in the second stage. The product contained 6,6 g/L n-butanol from L-arabinose or 3.7 g/L from L-xylose, with 22 g/L of ethanol from the first fermentation stage. Mattlasson et al. (49) recently proposed an aqueous 2-phase system for continuous production of acetone and n-butanol by calcium alginate gel immobilized CI. acetobutyllcum. The phase system consisted of a medium supplemented with 6% (w/w) Dextrane T-40 (Pharmacia Fine Chemicals, Uppsala, Sweden) and 25% (w/w) Carbowax peg 8000 (Union Carbide, New York), resulting in top to bottom phase ratio of 6 to I. The microorganisms were totally partitioned in the bottom phase. The mean productivity of the system was estimated at 0.24 g/Lh, with about 13 g/L n-butanol produced from 40 g of glucose in 50 hours. ORGANIC AND AMINO ACIDS Organic Acids Acetic Acid. Acid fermentations offer an interesting alternative to ethanol production, and acetic and lactic acids in particular have been considered potentially important feedstocks for chemical industry. Ghommidth et al. (2~) recently demonstrated an improvement in mass transport in immobilized biocatalyst systems by employing a fixed-bed reactor with pulsed flow, and they used the oxidation of ethanol to acetic acid by Cordlerite adsorbed Aceto1:~=cter cells as a model system. Acetic acid productivities of up to 10.4 g/Lh with pure oxygen and 4.6 g/Lh with air at 5 hour residence time were reported, and the authors believed that technique based on this principle could be competitive with current acetic acid production technology. Goldberg and Cooney (25) showed that acetic acid can also be formed from hydrogen and carbon dioxide by Acetobacter woodil and Clostr~d~m acet~c~m. Lactic Acid. Stenroos et al. (7~,7Z) proposed the use of immobilized whole cells for the production of lactic acid. When living ~ctobac~l~u8 de~b~¢cki~ cells were entrapped in calcium alglnate (6%) gel beads and employed in recycle batch or in continuous column reactors, the maximum lactic acid yield from 4.8% (w/v) glucose was 97% with more than 90% of L-lactic acid. The biocatalyst activity half-life was about I00 days. More recently Tipayang and Kozaki (74) employed calcium alginate (3%) gel entrapped L. vacc~nostercu8 Kozaki and Okada sp. nov. for lactic acid production from 2% xylose.
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Citric Acid. Citric acid is one of the few bulk chemicals that is produced biotechnically today. Kristiansen and Charley (37) reported on a continuous signle-stage process for citric acid production by Aspergillus niger 2270 (Peoria). In comparision with batch processing a significant increase in productivity from less than I g/Lh to about 2 g/Lh at dilution rate of 0.0075 h -1, pH 1.7 and with a final citric acid concentration of 26.5 g/L was realized. Vaija et al. (76) obtained a maximum yield of 12 g/L of citric acid from 10% (w/v) sucrose medium with about 40% overall fermentation efficiency, employing an air-lift type completely mixed continuous fermentor and calcium alginate gel immobilized Aspergillu8 niger ATCC 9142. The maximum production rate of about 70 mg/gh was about 5 times that obtained in conventional batch fermentation. Malie Acid. Chibata and coworkers (73) reported an improvement in the production method used since 1974 by Tanabe Seiyaku Company Ltd, Japan for L-malic acid by immobilizing Brevibacterium ammoniagene8 cells in a mixture of 3%
glutc~t~c~ ATCC 13058 cells in K-carrageenan
Alanine. Chibata and his coworkers (65) have recently reported on an elegant method for L-alanine production, combining the previously described industrialized process for L-aspartic acid from ammonium fumarate by <-carrageenan immo bilized E 8 c ~ c i a aoli cells with the decarboxylation of aspartic acid by similarly entrapped P s e ~ m o r ~ 8 ~ c u n ~ e cells. The interfering rasemase and fumarase activities were selectively eliminated prior to immobilization. A similar method was recently presented by Jandel et al. (30), who used two membrane reactors in series. Tryptophan. Wagner et al. (80) reported on continuous production of L-tryptophan by polyacrylamide of chitosan entrapped Esche~cia coli B I0 cells of high tryptophan synthesize activity. A steady state could be maintained in a CSTR at 1.3 hour residence time, and about 80% of the biocatalyst activity remained after 50 days. A maximum productivity of about 1.3 g/Lh from 2 g indole and 2 g serine in the presence of i0 mg pyridoxalphosphate in I L at pH 8 was obtained using i00 g of biocatalyst at 16% (w/v) cell loading.
FUELS AND INDUSTRIAL
CHEMICALS
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Aspartame. It is likely that the demand for both L-aspartic acid and L-phenylalanine will increase during the coming few years owing to the new dipeptide sweetener aspartmae. According to Parkinson (58) Genex Corporation process for L-aspartic acid would produce about 5000 kg annually with a i L continuous reactor, in comparision with an I00 L fermentation tank requirement by conventional batch fermentation for same production. Toyo Soda Manufacturing Co, Japan (2) has been reported to have developed a process based on enzyme technology to produce aspartame from L-aspartic acid and L-phenylalanine. METHANE AND HYDROGEN Methane Considerable interest in anaerobic digestion has been witnessed during the last decade, and Bungay (7) and Mahin (46) have recently reviewed the biogas situation. China has the largest anaerobic digestion utilization program today with more than 7 million village level units in operation, and several larger ones for electricity generation. Several thouthands of units have been built elsewhere in the Far East and in Africa. The suitability of immobilized biocatalyst technology for methane production has been investigated recently. Messing (51) designed a laboratory scale two-stage, anaerobic, im~nobilized microbial cell reactor based on porous ceramic as carrier, which could convert organic sewage waste continuously to methane at 2 to 5.5 hour residence times for periods of up to 2 years. The gas contained over 90% of methane and less than 5% of carbon dioxide. A horizontal reactor assembly for continuous removal of methane was employed. Hydrogen Hydrogen has been considered as one of the fuels of the future, although many problems remain to be solved before economic large scale biotechnical production can be realized. Klibanov et al. (35) have recently shown that immobilized whole cells of Alcaligenes eutrophu~ can be employed both for the production of hydrogen from formate, and for the synthesis of formate from hydrogen and carbon dioxide. Yields of formate of up to 30% were reported. Karube et al. (33) demonstrated on the other hand that C l o s t ~ d i ~ b u t y ~ c ~ IFO 3847 cells immobilized on acetylcellulose filter with agar could produce hydrogen, and Brosseau and Zajic (6) demonstrated hydrogen production by Citrobacter intermedius and Clostridium pasteurianum. OIL AND HYDROCARBONS Oil Recently an interest in microbial oil production has been expressed (61). For example a species of Arthrobacter has been reported to produce biomass of about 70% lipid content in 7 days, and some algae may contain as much as 50% lipid material of the biomass. Hydrocarbons A novel approach to obtain hydrocarbons via ethanol fermentation has recently been reported by de Boks #t al. (13). They developed an integrated two-stage system for continuous ethanol fermentation with Z ~ o ~ s mobili~ , followed by continuous catalytic conversion of the obtained ethanol-water-gas mixture to hydrocarbons at 350°C, I atm over Zeolite H-ZSM-5. Continuous removal of ethanol from the first stage could result in high productivity even at high glucose feeds. A two-stage biogasification process, Biothermgas process, has been developed at the Institute of Gas Technology, Chicago (I0). Biomass is first degraded to methane, carbon dioxide, and bacterial biomass by anaerobic
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digestion, and a mixture of hydrocarbons, hydrogen, carbon monoxide, carbon dioxide, and ammonia are then obtained by thermal gasification. Hydrocarbon oxidation Alkene oxides are important intermediates in chemical synthesis. Furuhashi
et al. (23) reported that glucose grown polyacrylamide gel immobilized living Nocardia corallina cells could produce propylene oxide continuously for a week in a bubble tower reactor. The production of both ethylene and propylene oxides by calcium alginate gel immobilized Mycobacterium sp. was reported by de Bont et al. (14). Furthermore, de Smet et al. (16) showed that 1,2-epoxioctane can be produced with growing cells of Pseudomonas oleovorans. REFERENCES i.
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