6 Microbial Production of Butanol

6 Microbial Production of AcetoneD3utanoVIsopropanol
PETERDURRE
Ulm, Federal Republic of Germany
HUBERT BAHL
Rostock, Federal Republic of Germany
1 Introduction 230 2 History 230 3 Biochemistry and Genetics of Solvent-Producing Clostridia 233 3.1 Microorganisms 233 3.2 Utilization of Substrates 235 3.2.1 Degradation of Polymers 235 3.2.2 Uptake of Mono- and Disaccharides 237 3.2.3 Intracellular Sugar Metabolism to Pyruvate 237 3.3 Formation of Products 239 3.3.1 Formation of Acids 239 3.3.2 Formation of Solvents 243 3.3.3 Regulation of Product Formation 245 3.4 Strain Improvement 246 4 New Developments of the Fermentation Process 247 4.1 Continuous Culture 248 4.2 Cell Immobilization and Cell Recycling 252 4.3 Product Recovery 253 4.4 Alternative Fermentation Substrates 255 5 Conclusions 256 6 References 257
230
6 Microbial Production of Acetone/ButanoWIsopropanol
1 Introduction
The solvents acetone, 1-butanol, and 2-propanol are natural products of a few anaerobic bacteria. During the first half of this century, their synthesis was mainly achieved by fermentation. Acetone-butanol fermentation became in volume the second largest fermentation process in the world, only exceeded by ethanol fermentation of yeast. After 1950, the importance of the process declined rapidly, because the production of acetone and butano1 from oil became economically more favorable. However, the oil crisis in 1973 revived the interest in this fermentation and in the bacteria performing it. This chapter tries to review the enormous progress made in the last few years with respect to biochemistry, genetics, and physiology of the respective microorganisms and new developments in process technology. A number of reviews on various aspects of this topic have been published in the last decade (AWANG al., 1988; BAHLand GOTTet SCHALK, 1988; DURREet al., 1992; ENNISet al., 1986a; JONESand WOODS, 1986; LENZ and MOREIRA, 1980; MCNEIL and KRISTIANSEN, 1986; MOREIRA,1983; ROGERS, 1986); they should be consulted for additional information. Some of the physical properties of acetone, 1-butanol, and 2-propanol (isopropanol) are summarized in Tab. 1. Acetone is an important intermediate in the manufacture of methacrylates and methyl isobutyl ketone and a solvent for resins, paints, varnishes, laquers, and cellulose acetate; it is miscible in all pro-
portions with water. 1-Butanol is a precursor of butyl acetate and dibutyl phthalate and like acetone a good solvent. Its solubility in water is 8% (wlw). 2-Propanol or isopropanol is used in antifreeze composition, as a solvent, e.g., in quick-drying oils and ink, and in cosmetics such as hand lotions and after-shave lotions. Like acetone, it is completely miscible with water.
2 History
Butanol formation by fermentation was first noticed by PASTEUR connection with in his discovery of the butyrate fermentation (PASTEUR,1861a, 1862). The causative organism was named vibrion butyrique, and the term "anaerobic" was coined (PASTEUR, 1861b, 1863), when it was realized that the bacteria responsible for this fermentation were killed in air. A more detailed study on butanol producHe ing bacteria was then conducted by FITZ. described an organism fermenting glycerol to butanol, butyric acid, COz, HZ, and small amounts of acetic acid, ethanol, lactate, and propanediol (FITZ, 1876, 1878, 1882). It was an anaerobic, sporulating, rod-shaped bacterium, and a drawing showed typical clostridial forms (FITZ, 1878). Lactose and starch did not lead to active fermentation, but mannitol and sucrose could serve as additional substrates. FITZ already observed that from the products formed, butanol was the most toxic compound, and he finally described the isola-
Tab. 1. Physical Properties of Acetone, Butanol, and Isopropanol
Property Molecular weight Melting point at 101.3 kPa Boiling point at 101.3 kPa Specific gravity at 20C Heat of vaporization Heat of combustion Vapor pressure at 20C
Acetone
58.08 - 94.6"C 56.1 "C 0.807 29.1 kJ mol-I 1787 kJ mol-l 24.7 kPa
1-Butanol
74.12 -90.2"C 117.7 "C 0.813 43.8 kJ rno1-l 198.2 kJ mol-' 0.63 kPa
2-Propanol (Isopropanol)
60.1 -88.5"C 82.3 "C 0.786 39.8 kJ mol 2005.8 kJ mol-' 4.4 kPa
-'
Data taken from NELSON and WEBB,1978; SHERMAN, 1978; PAPA,1982
2 History Tab. 2. History of Description and Isolation of Solvent-Forming Bacteria

Designation of Microorganism Vibrion butyrique Bacillus butylicus Clostridium butyricum I, 11, I11 Bacille amylozyme Bacillus butyricus Bacillus orthobutylicus Granulobacter butylicum Granulobacter saccharobutyricum Amylobacter butylicus Bacillus butylicus (Fitz) Granulobacillus saccharobutyricus immobilis liquefaciens Clostridium pastorianum Bacillus macerans Clostridium americanum Clostridium sp. Bacillus butylicus (Fitz) Bacillus amylobacter A.M. et Bredemann Clostridium acetobutylicum (Weizmann) Solvents Formed Butanol Butanol Butanol Butanol" Butanol Butanol Butanol Butanol Butanol Butanol Butanol Butanol, isobutanol Acetone Butanol, isopropanol Butanol Butanol Butanol, propanol Acetone. butanol Year of Publication 1861a, b; 1862 1878; 1882 1887 1891 1892 1893 1893 1893 1895 1897 1899; 1900 1902 1905 1906a, b 1907 1908 1909 1926 Author( s) PASTEUR FITZ GRUBER PERDRIX BOTKIN GRIMBERT BEIJERINCK BEIJERINCK DUCLAUX EMMERLING SCHATENFROH and GRASSBERGER WINOGRADSKY SCHARDINGER PRINGSHEIM SCHARDINGER BUCHNER and MEISENHEIMER BREDEMANN McCoy et al.
231
a The original description reported amylalcohol as a product, but due to the determination procedure butanol is much more likely (PRINGSHEIM, 1906a).
The biological formation of isopropanol tion of pure cultures of Bacillus butylicus from cow feces and hay (FITZ, 1877, 1878, (together with butanol) was first reported in 1906a, 1882). This pioneering work stimulated many 1906 for C. americanum (PRINGSHEIM, other scientists to investigations on butanol b). Acetone was discovered as a product of (1905). producing anaerobic bacteria. Among them microbial activity by SCHARDINGER were a number of famous microbiologists The responsible organism was named Bacillus macerans, and ethanol, acetate, and formate (Tab. 2). BEIJERINCK (1893) described two different were produced in addition. Thus, the two strains yielding appreciable quantities of bu- products butanol and acetone were found in tanol. One of them, producing butyrate addi- rather different microorganisms. The discovetionally, was designated Granulobacter sac- ry of their common formation by a single specharobutyricum and was probably identical to cies was connected with the efforts of the Bacillus butylicus. The other species, Granu- chemical industry to produce synthetic rublobacter butylicum, produced isopropanol ber. Butanol was considered a precursor of along with butanol which, however, had not butadiene, the starting material for synthetic been discovered before 1920 (FOLPMERS, rubber production. The British company cited in OSBURN WERKMAN, and 1935). This Strange and Graham Ltd. got interested in and bacterium was the later Clostridium butyli- such a project and contracted PERKINS (University of Manchester) and cum which was shown to belong to the species WEIZMANN and (Institut Pasteur) in C. beiierinckii (GEORGEet al., 1983). Pure FERNBACH SCHOEN cultures of butyrate producers additionally 1910 to study the formation of butanol by mi1928). forming small amounts of butanol were ob- crobial fermentation (see GABRIEL, FERNBACH isolated an acetone-butanol protained by WINOGRADSKY1902. in
232
6 Microbial Production of AcetondButanoWlsopropanol
ducer in 1911; WEIZMANN terminated his cooperation with the company in 1912, but continued his work at the University of Manchester. He succeeded in isolating an organism, later named Clostridium acetobutylicum, that produced acetone and butanol from starchy materials in better yields than the organism of FERNBACH. Patent applications were filed for the Fernbach process in 1911 and 1912 (FERNBACH STRANGE, and 1911, 1912) and for the Weizmann process in 1915 (WEIZMANN, 1915). Production of acetone and butanol by Strange and Graham Ltd. based on the Fernbach process began in 1913. Potatoes were used as raw material. Following the outbreak of World War I the interest turned from butanol to acetone which was required in large amounts for the production of smokeless powder. Strange and Graham Ltd. supplied the British government with acetone produced in their plant at Kings Lynn. WEIZMANN continued research work on his process; pilot-scale studies were performed and the results convinced the government of the superiority of this process. Finally, a production plant was built at the Royal Naval Cordite Factory at Poole. There and in some other distilleries acetone was produced. In addition, the Weizmann process was also introduced into the Strange and Graham Ltd. plant. Because of the German blockade grain and maize could not be imported to Great Britain in the quantities required. Starchy materials could not be supplied to the fermentation industry anymore, and acetone-butanol production had to be abandoned in Great Britain. However, the know-how was transferred to Canada, and a fermentation plant went into operation in Toronto in August 1916. There and in a plant at Terre Haute (Indiana, USA) acetone was produced until the end of the war when all plants were closed. There was little use for butanol produced at that time, so it was stored in large vats. However, as a result of the rapid growth of the automobile industry, increasing amounts of lacquers were needed for which butanol and its ester, butyl acetate, are excellent solvents. In addition, Prohibition endangered the supply of amyl alcohol, which had been used as a solvent for lacquers and which had been ob-
tained as a by-product of the manufacture of spirits (WALTON,1945). Soon the situation was reversed as compared to war times: l-butanol was the product wanted, and acetone became less useful. Commercial Solvents Corp. of Maryland (USA) was founded, they bought the Terre Haute plant in 1919 and started butanol production in 1920. Difficulties with the process emerged in 1923, when the solvent yield went down considerably due to bacteriophage infection (OGATA and HONGO,1979). It took almost one year to overcome these problems. A second plant was opened by Commercial Solvents Corp. at Peoria in 1923 with 32 fermenters with a volume of about 190000 L each. It was extended to 96 fermenters in the following years. Production was in the range of 100 tons of solvents per day (JONESand WOODS,1986). Strange and Graham Ltd. started production of solvents at Kings Lynn again in 1923. The factory, however, was soon destroyed by an explosion, and Strange and Graham Ltd. went into liquidation. The story of the acetone-butanol process in Great Britain continued in 1935 when Commercial Solvent Corp. and the British Distillors Co. built a plant at Brombrough. The plant was operated with molasses, the substrate that also had replaced grain as raw material in the American plants in the early 1930s. By this replacement the acetone-butanol process was saved at that time, because molasses was much cheaper than grain. In addition, strains of Clostridium acetobutylicum had been developed fermenting up to 6.5% sugar and producing 2% solvents. This resulted in a considerable reduction of the costs of product recovery. Until 1936, the patents of WEIZMANN (1915, 1919) prevented a further extension of the acetonebutanol process. These patents expired in 1936, and new fermentation plants were built in the United States, in the former USSR, in Japan, India, Australia, and South Africa. These plants operated through World War 11. In 1945,66% of the l-butanol and 10% of the acetone produced in the United States came from the fermentation industry. Starting around 1950, the fermentation process went into sharp decline because of the severe competition of the growing petrochemical industry and of steeply rising prices of molasses
3 Biochemistry and Genetics of Solvent-Producing Clostridia
233
and grain. Soon all plants in the Western countries were closed; only the plant in South Africa operated until 1982 (JONES and WOODS, 1986); a few plants may still be operating in Russia and in the Peoples Republic of China. A critical parameter in fermentations using C. acetobutylicum is the tendency of the organism to degenerate, i. e., to lose the ability to produce solvents and to sporulate (KUTZENOK and ASCHNER, 1952; FINNand NowREY, 1959). Therefore, sporulation is used as a convenient method to store the organism and to maintain its solvent-forming ability. Different methods of spore preservation have been reported. Spores can be kept in sterile dry sand or soil (SPIVEY, 1978; DAVIES and STEPHENSON, 1941; BEESCH,1952), lyophilized (LAPAGE al., 1970), or stored in milk et medium (BAHLet al., 1982a) or fermentation broth (MONOT et al., 1982; LIN and BLASCHEK, 1983). A heat shock for 90s at 70C of a freshly inoculated culture helps to induce sporulation. A similar procedure (1 min at 90C) will ensure germination of a spore preparation. The mechanisms responsible for clostridial degeneration are not yet understood. However, a recent publication has described the isolation of transposon-induced degeneration-resistant mutants (KASHKET and CAO, 1993) which will allow an analysis of this phenomenon at the molecular level. Batch culture fermentation with C. acetobutylicum became economically unfavorable because of high substrate costs, low solvent yields, large volumes of waste, high amounts of energy required for product recovery (distillation), and the low butanol tolerance of the organism. Starting at concentrations of 200 mM growth is completely inhibited by butanol, whereas acetone and ethanol in similar concentrations show no effect (MOREIRA et al., 1981). The sudden increase of crude oil prices in 1973 triggered a revival of the interest in the biotechnological production of 1butanol and acetone. So far, it has not led to a process able to compete with the oil-based production of solvents. Acetone is currently produced by the cumene hydroperoxide process or by catalytic dehydrogenation of isopropanol (NELSON WEBB,1978). 1-Butaand no1 is synthesized from propylene by the 0x0-
process or from acetaldehyde by the aldol process (SHERMAN, 1978). 2-Propanol or isopropanol is generally manufactured from propene, either by the indirect hydration process or by catalytic hydration PAPA, 1982).
3 Biochemistry and Genetics of SolventProducing Clostridia

3.1 Microorganisms
Solvents such as acetone, butanol, and isopropanol are produced in more than trace amounts only by few bacterial species. Most of them are members of the genus Clostridium (BAHLand DURRE,1993). However, butanol as a major fermentation product has also been detected with Butyribacterium methylotrophicum (GRETHLEIN al., 1991) and et Hyperthermus butylicus (ZILLIG al., 1991). et The most prominent species of clostridia, of course, is C. acetobutylicum. In times when dozens of patents were obtained for certain aspects of the acetone-butanol fermentation process, a variety of partly exotic names was given to the many production strains included in these patents. Some of the names are depicted in Tab. 3; they are now invalid. Clostridia recognized of producing solvents are summarized in Tab. 4. In addition to C. acetobutylicum, the species C. aurantibutyricum, C. beijerinckii, C. butyricum, C. cadaveris, C. chauvoei, C. felsineum, C. pasteurianum, C. puniceum, C. roseum, C. sporogenes, C. tetani, C. tetanomorphum, and C. thermosaccharolyticum are shown. The products formed by these species vary slightly (Tab. 5). C. aurantibutyricum and strains of C. beijerinckii (GEORGE al., 1983; CHENand HIU, 1986) et as well as an aggregate-forming variant of C. butyricum (ZOUTBERG al., 1989b) also et form isopropanol which is not produced by the other species. With glucose as substrate, butanol and acetone are recovered in a ratio of 2:l from the fermentation broth of C. acetobutylicum and in a ratio in the order of 1O:l
234
6 Microbial Production of AcetondButanoVlsopropanol
Tab. 3. A Small Collection of Names of Solvent-Producing Bacteria Mentioned in Patents

Name of Organism
Bacillus butylaceticum Bacillus acetobutylicum Clostridium saccharobutylicum gamma Clostridium saccharobutylacetonicum Clostridium saccharoacetobutylicum beta, gamma Clostridium saccharoacetobutylicum (Y Clostridium invertoacetobutylicum Bacillus tetryl P-bacillus Clostridium propylbutylicum alpha Clostridium saccharobutylacetonicum liquefaciens Clostridium saccharobutylacetonicum liquefaciens gamma, delta Clostridium madisonii Clostridium saccharoperbutylicum Clostridium saccharoperbutylacetonicum
Author(s) FREIBERG FUNK IZSAK and FUNK LOUGHLIN ARZBERGER WOODRUFF al. et LEGGand STILES ARROYO MULLER MULLER ARZBERGER CARNARIUS and MCCUTCH AN McCoy BEESCH HONGO
Year 1925 1925 1933 1935 1936 1937 1937 1938 1938b 1938a 1938 1938 1946 1948 1960
u. s.
Patent No. 1 537 597 1 538 516 1 908 361 1 992 921 2 050 219 2 089 522 2 089 562 2 113 471 2 123 078 2 132 039 2 139 108 2 139 111 2 398 837 2 439 791 2 945 786
in the broth of C. puniceum and C. beijerinckii (HOLTet al., 1988; CHENand HIU, 1986). Butanol and equal amounts of ethanol, but no acetone or isopropanol, are produced by C. tetanomorphum (GOTTWALD al., 1984). et Cultures of C. acetobutylicum continue to produce solvents after growth has ceased, while with C. puniceum and C. tetanomorphum solvent production is only observed during growth. When C. pasteurianum is grown in media of high sugar content or with glycerol as a substrate under phosphate limitation, high butanol concentrations are yielded (130 and 45 mmol L-', respectively) (HARRIS al., 1986; DABROCK al., 1992). et et Small amounts of butanol are among the products of a number of other Clostridium species (CATOet al., 1986; HIPPE al., 1992). et Enrichment and isolation of solvent producing clostridia is relatively easy. Soil samples from potatoes, other root crops, or roots of beans are good sources (CALAM, 1979). Enrichment can be done in stabbed potato tubers (VELDKAMP, 1965) or maize mash medium (WEIZMANN, 1919). Isolation of pure cultures can be achieved by applying techniques commonly used with strictly anaerobic bacteria (BREZNAK COSTILOW, and 1994). Since C. acetobutylicum produces and tolerates the highest solvent concentrations and
since it has been employed in the industrial process, the further discussions will primarily deal with this microorganism. C. beiierinckii will be included as a model organism for isopropanol formation. C. acetobutylicum is a gram-positive, straight rod measuring 0.6-0.9 by 2.44.7 pm. The vegetative cells are motile with peritrichous flagella. Subterminal ovoid spores are formed (Fig. 1). The optimum growth temperature is 37"C, and biotin and p-aminobenzoate are required as growth factors (RUBBO al., 1941). Further physiologiet cal properties have been summarized in other review articles (CATO et al., 1986; HIPPEet al., 1992). A note of caution must be added as to comparison of experimental data obtained with different strains of C. acetobutylicum and C. beijerinckii. About 50 of such strains are currently in use or available from the various culture collections. Although some of them were supposed to be identical, this has been disproven by studying their physiological characteristics and by hybridization analyses of cloned genes (SAUERand DURRE, 1993; WOOLLEY MORRIS, and 1990; YOUNGet al., 1989). A recent investigation has provided unequivocal evidence that of the mostly used strains only ATCC 824, DSM 792, and DSM 1731 are true C. acetobutylicum. Strain
3 Biochemistry and Genetics of Solvent-Producing Clostridia Tab. 4. Some Properties of Butanol-Producing Clostridia C. acetobutylicum C. aurantibutyricum C. beijerinckii C. butyricum C. cadaveris C. chauvoei
235
C. felsineum
Gelatin hydrolyzed Motility Lipase produced Starch hydrolyzed Sugars utilized Glucose Fructose Lactose Maltose Sucrose
+ + + + + + +
+ + + + + + + + +
+
f
+ + + + +
+ + + + + +
f -
+
f
+ +
-
+ +
+
f
+ + + +
+ + + f +
C. thermosaccharolyticum
Tab. 4. (continued)
C. pasteurianum C. puniceum C. roseum C. sporogenes C. tetani C. tetanomorphum
Gelatin hydrolyzed Motility Lipase produced Starch hydrolyzed Sugars utilized Glucose Fructose Lactose Maltose Sucrose
+ + + +
+ + + +
+
f -
+ f +
+
k
*
+ +
+ + + +
+ + +
+ + + + +
Data taken from CATOet al., 1986; CHENand Hiu, 1986; FREIER-SCHRODERal., 1989; GEORGE al., et et 1983; GOTTWALD al., 1984; HARRIS al., 1986; HOLT et a]., 1988; MCCLUNGand McCoy, 1935; et et McCoy and MCCLUNG, 1935; NAKAMURA al., 1979; PETERSEN, et 1991; WILDE al., 1989. et
NCIMB 8052 is a member of the C. beiierinckii group, whereas strain P 262 belongs to a different species (KEIS et al., 1994; JOHNSON and JONES, personal communication).
3.2 Utilization of Substrates

A great variety of mono- and disaccharides and related compounds are used by C. acetobutylicurn for growth. In addition to the substrates listed in Tab. 3, these are: cellobiose, D-mannose, D-galactose, D-gluconate, D-galacturonate, D-glucosamine, D-ribose, D-xylose, L-arabinose, L-rhamnose, and glycerol. Furthermore, polysaccharides as starch and xylan can be fermented. The process of sub-
strate acquisition can be considered in two or three stages: (1) degradation of polymeric substrates by secreted enzymes, (2) uptake of the degradation products or other low-molecular weight substrates, and (3) intracellular metabolism of the carbohydrates.
3.2.1 Degradation of Polymers

Despite the fact that starch was the first industrial substrate for the production of acetone-butanol by fermentation, only little information is available on the extracellular enzymes involved in the degradation of this polymer. During growth of C. acefobufylicurnon starch an a-amylase and a glucoamylase, ori-
236
Glucose" Organism
Tab. 5. Solvent Production by Some Clostridial Species Grown in Complex Media Contaning 2% (w/v)
Products Formed [mmol L-'1 Acetone Butanol Ethanol Isopropanol
C. acetobutylicum C. aurantibutyricum C. beijerinckii C. beijerinckii C. butyricum" C. cadaveris C. pasteurianum C. puniceurn C. sporogenes C. tetanomorphum C. thermosaccharolyticum
14.0 20.5 6.0
16.8
30.2 45.4 67.9 44.8 17 11.2 22 75.6 11.2 47.1 4. 00
5.0
45 .
9.8 7
42.7 85.0
Data taken from DABROCK al., 1992;FREIER-SCHRODERal., 1989;GEORGE al., 1983;GOTTWALD et et et et et al., 1984,HOLTet al., 1988 ZOUTBERG al., 1989a. " An aggregate-forming variant with 2.7% glucose has been used.
Fig. 1 Phase-contrast photomicrograph of Clostridium acetobutylicum (courtesy by H. HIPPE). .
237
ginally referred to as a maltase, are produced 3.2.2 Uptake of Mono- and (HOCKENHULL HERBERT, and 1945; FRENCH and KNAPP, 1950; Scorr and HEDRICK, 1952; Disaccharides ENSLEY al., 1975). The synthesis of these et enzymes is generally subjected to catabolite Very little is known on substrate transport repression by glucose and induced by starch in C. acetobutylicum. Phosphotransferase sysor its degradation products (HOCKENHULLtems (PTS) are apparently responsible for upand HERBERT,1945; CHOJECKI and BLA- take of glucose and fructose (VONHUGOand SCHEK, 1986). Recently, the 0-amylase from GOTTSCHALK, 1974; HUTKINS KASHKET, and C. acetobutylicum ATCC 824 has been puri- 1986): fied and characterized (PAQUET al., 1991). et This enzyme has a molecular weight of 84 Glucose P-HPr Glucose-6kDa, an isoelectric point of 4.7, an optimal phosphate HPr pH of 5.6, and is very sensitive to thermal Fructose-1inactivation. Higher activities were found Fructose + P-HPr phosphate + HPr with high-molecular weight substrates as compared to low-molecular weight maltooligosaccharides. Glycogen and pullulan were slowly P-HPr is a phosphorylated protein generated hydrolyzed, whereas dextrans and cyclodex- from HPr with phosphoenolpyruvate (PEP) trins were not attacked. Interestingly, the as the source of energy-rich phosphate: product of an amylase gene of C. acetobutylicum cloned by VERHASSELT al. (1989) has PEP + HPr et P-HPr + Pyruvate a molecular mass of only 53.9 kDa. This gene is not identical to a truncated open reading A detailed biochemical study of the glucose frame with significant similarity to the a- PTS of C. acetobutylicum has been provided et amylase gene of Bacillus subtilis identified by recently (MITCHELL al., 1991). The presGERISCHER DURRE(1990). Therefore, it ence of the four components of the system, and is likely that C. acetobutylicum produces dif- enzyme I, HPr, enzyme IIAgl",and enzyme IIglu, was demonstrated. The C. acetobutyliferent amylases for the utilization of starch. Xylan can serve as sole carbon source for cum PTS therefore displays the same archiC. acetobutylicum (LEEet al., 1985a; LEMMEL tecture as in other bacteria. Other substrates et al., 1986). However, growth is slow, and of C. acetobutylicum might be taken up either larch wood xylan is only partially hydrolyzed. also by PTS as in the case of glucitol (MITTwo endoxylanases and a p-D-xylosidase of CHELL, 1992), by ABC transport sytems 1992), or by symport mechanisms C. acetobutylicum were purified and charac- (HIGGINS, terized (LEE et al., 1985a, 1987). The smallest driven by the transmembrane proton graoligosaccharides degraded by xylanase A and dient. Disaccharides such as sucrose or malB are xylohexaose and xylotetraose, respec- tose might then be cleaved by appropriate tively. Xylanase A also exhibits carboxyme- phosphorylases, e.g., thy1 cellulase activity. C. acetobutylicum does not grow on cellu- ~~l~~~~ + phosphate Maltose Phosphorylase lose; nevertheless, two activities of the cellulGlucose + Glucose-1-phosphate ase complex can be detected in certain strains of this organism (ALLCOCKand WOODS, Free glucose can be converted to glucose-61981). LEE et al. (1985b) showed that two phosphate by hexokinase. strains out of 21 tested produced extracellular endo- 1,4-p-glucanase and cellobiase (p-Dglucosidase) activities during growth on cello- 3.2.3 Intracellular Sugar biose. Metabolism to Pyruvate
Clostridium species generally employ the Embden-Meyerhof-Parnas (EMP) pathway
238
6 Microbial Production of Acetone/Butanol/lsopropanol

Hexose
1
BytyrylJAcetyl-CoA
ButyrnWAcelate
Acetoahtyl-CoA
&
Pool
ATP Y P
Butyryl-CoA
F;;L!m+N,L+H+
NAD+
,! & &
. b d F
Fig. 2. Carbon (thick arrows) and electron (thin arrows) flow during hexose fermentation in Clostridium acetobutylicum or C. beijerinckii. Enzymes involved in oxidation-reduction reactions are numbered as follows: (1) glyceraldehyde-3-phosphatedehydrogenase; (2) lactate dehydrogenase; (3) pyruvate: ferredoxin oxidoreductase; (4) hydrogenase; (5) NADH:ferredoxin oxidoreductase; (6) acetaldehyde dehydrogenase; (7) ethanol dehydrogenase; ( 8 ) P-hydroxybutyryl-CoA dehydrogenase; (9) isopropanol dehydrogenase; (10) butyryl-CoA dehydrogenase; (11) butyraldehyde dehydrogenase; (12) butanol dehydrogenase. In certain alcohol dehydrogenease reactions some strains use NADP +/NADPH H + as electron acceptorldonor instead of NAD +/NADH H + , which are generally shown.
for the degradation of hexose phosphates (THAUER al., 1977; GOITSCHALK, et 1986; ROGERS GOTTSCHALK, and 1993). This is apparently also true for C. acetobutylicum. A corresponding scheme is shown in Fig. 2. Sugar acids such as gluconate are degraded via a modified Entner-Doudoroff pathway (ANDREESEN and GOTTSCHALK, 1969). D-G~uconate is dehydrated to yield 2-keto-3-deoxy-
gluconate which subsequently is phosphorylated and cleaved to yield pyruvate and 3-phosphoglyceraldehyde (Fig. 3). Pentoses are converted into ribose-5-phosphate and xylulose-5-phosphate which are fed into the pentose phosphate cycle; the resulting hexose phosphates are subsequently catabolized by the EMP pathway. Enzymes such as transketolase and transaldolase have been
239
I c=o I
COOH
I HC-OH I
HO-CH
COOH HZO
I c=o I
CHI
COOH
ADP
COOH
CH3
I c=o I
HC-OH CHz I HC-OH
HC-OH
I I HC-OH I
jW
CHzOH
I I HC-OH I
HC-OH
I
I
<
Pyruvate
CHzOH
CHzO@
2-Keto-3-deoxy6-phosphogluconate
HC =O HC -OH CHzO@
Glyceraldehyde-3-phosphate
Gluconate
2-Keto-3-deoxygluconate
I I
Fig. 3. Degradation of gluconate by Clostridium species via the modified Entner-Doudoroff pathway. (1) gluconate dehydratase; (2) 2-keto-3-deoxygluconate kinase; (3) 2-keto-3-deoxy-6-phosphogluconate aldolase.
1987). detected in species closely related to C. aceto- phate (FREIER and GOTTSCHALK, butylicum (C. butylicum). The results of Lactate might be taken up again and subsetracer studies were in agreement with the op- quently consumed, if the pH decreases to valeration of the pentose phosphate cycle and ues below 4.5 and if the iron concentration is the EMP pathway (CYNKIN DELWICHE, no longer growth-limiting. For consumption, and 1958; CYNKIN and GIBBS,1958; VOLESKY however, acetate is needed as a co-substrate. Conversion of lactate into pyruvate seems to and SZCZESNY, 1983). be mediated by a different lactate dehydrogenase (DIEZ-GONZALEZ al., 1994). A lacet tate dehydrogenase gene has been cloned 3.3 Formation of Products from C. acetobutylicum strain B643. The recombinant plasmid encoded a 38 kDa enzyme 3.3.1 Formation of Acids that was activated by addition of fructose-1,6diphosphate (CONTAG al., 1990). et The central enzyme for the breakdown of Lactate becomes a major fermentation product of C. acetobutylicum under condi- pyruvate is pyruvate :ferredoxin oxidoreducttions of growth limiting iron concentrations ase (Pfo) (Fig. 2). The enzyme from Clostrid(10 p,M) and pH values higher than 5 (BAHL ium acetobutylicum was purified and characet et al., 1986). Inhibition of the hydrogenase by terized (MEINECKE al., 1989). The molecucarbon monoxide or potassium cyanide lar mass was found to be 123 kDa per monocauses a similar effect (HANSON and ROD- mer in SDS polyacrylamide gel electrophoreGERS,1946; KATAGIRI al., 1960; KEMPNER sis. The subunit composition of the native enet and KUBOWITZ, 1933; KUBOWITZ, 1934). The zyme remained undetermined due to the high lactate dehydrogenase (Ldh) responsible for oxygen sensitivity (50% inactivation in 1h). reduction of pyruvate to lactate has been pu- The protein contained 2.9 mol sulfur, 4.1 mol rified. It consists of 4 identical subunits with a iron, and 0.4 mol thiamine pyrophosphate per molecular mass of 36 kDa, has a pH optimum mol monomer, suggesting the presence of one of 5.8, uses NADH as a coenzyme, and is 4Fe-4S cluster and one thiamine pyrophosspecifically activated by fructose-l,6-diphosphate in this subunit. The apparent K , values
240
for pyruvate and coenzyme were determined these different thiolases might be alternativeto be 322 p M and 3.7 pM, respectively. Prod- ly active during acidogenic resp. solventogenucts of the reaction are acetyl-CoA, COz, and ic growth phases. The presence of two different thiolases has also been described for anreduced ferredoxin. Acetyl-CoA is partly converted to acetate other solvent forming Clostridiurn, C. pasteurand 1975). A reand partly to butyrate. The ratio in which ianurn (BERNDT SCHLEGEL, these products are formed varies to some ex- port by a Japanese group might indicate that tent. Acetate formation involves the enzymes one of the thiolases from C. acetobutylicurn phosphotransacetylase and acetate kinase, ATCC 824 exhibits coenzyme A transferase et whereas butyrate production is catalyzed by activity in addition (NAKAMURA al., 1990). subsequent action of thiolase, L( )-3-hy- A thiolase with a native molecular mass of droxybutyryl-CoA dehydrogenase, crotonase, 100-120 kDa has also been purified from two butyryl-CoA dehydrogenase, phosphotrans- C. beijerinckii strains (CHEN,1993). Reduction of acetoacetyl-CoA to butyrylbutyrylase, and butyrate kinase (GAVARD et al., 1957; TWAROG and WOLFE, 1962; VAL- CoA is catalyzed by the subsequent action of 3-hydroxybutyryl-CoA dehydrogenase ENTINE and WOLFE,1960; ANDERSCH al., et 1983; HARTMANIS GATENBECK, and 1984). (Hbd), crotonase (Cch), and butyryl-CoA deProduction of both acids is associated with hydrogenase (Bcd). Hbd has not been puriATP synthesis in the kinase reactions. In Tab. fied from C. acetobutylicurn. Determination 6, some characteristics of enzymes involved in of its activity in crude extracts showed a rapid pyruvate breakdown by C. acetobutylicurn are decrease several hours after the onset of solventogenesis (HARTMANIS GATENBECK, and summarized. Both, phosphotransacetylase (Pta) and 1984). The enzyme has been purified from acetate kinase (Ack), have not been purified C. beijerinckii NRRL B593. The subunit and from C. acetobutylicurn. Their activity has native molecular masses were reported to be been measured in crude extracts. Strong ex- 31 and 213 kDa, respectively (COLBYand pression could only be found during the aci- CHEN,1992). The amino terminus of the prodogenic growth phase (ANDERSCHet al., tein proved to be almost identical to the de1983; BALLONGUE al., 1986; HARTMANIS duced amino acid sequence of a hbd structuret and GATENBECK, 1984). However, in another al gene that had been cloned and sequenced report such a strict activity pattern is not de- from strain P262 (COLBYand CHEN,1992; et noted (HUSEMANN PAPOUTSAKIS, and 1989). YOUNGLESON al., 1989b). Crotonase (croPhosphotransacetylase from C. beijerinckii tonyl-CoA hydratase) has been obtained in has been partially purified. Its molecular mass homogenous form from an unspecified C. acetobutylicurn strain (WATERSON al., 1972), et is 56-57 kDa (CHEN,1993). Formation of C4-compounds starts by con- possibly strain NRRL B528 (CHEN, 1993). densation of two acetyl-CoA molecules to The enzyme is a tetramer, consisting of 4 yield acetoacetyl-CoA. This reaction is cata- identical subunits with a molecular mass of lyzed by thiolase (Thl). The respective en- 40 kDa. It has a limited substrate specificity, zyme has been purified from C. acetobutyli- being active only with C4- and C6-enoyl CoA, .cum and consists of 4 identical subunits with a and shows a remarkable sensitivity towards molecular mass of 44 kDa (WIESENBORN high concentrations of crotonyl-CoA (WAet al., 1988). The apparent K,,, values for acetyl- TERSON et al., 1972). Its activity pattern durCoA, sulfhydryl-CoA, and acetoacetyl-CoA ing growth is almost identical to that of Hbd and 1984). Little are 270 p M ,4.8 pM, and 32 pM, respectively. (HARTMANIS GATENBECK, The structural gene of this enzyme has been information is available on the last enzyme of cloned (PETERSEN BENNETT,1991). Re- the reaction sequence, butyryl-CoA dehydroand cently, a different gene for a second thiolase genase. Enzymatic determinations in crude from C. acetobutylicurn as well as C. beijer- extracts of C. acetobutylicurn were of poor reinckii has been detected and sequenced producibility, which might indicate a high and (WINZER, MINTON, DURRE,unpublished oxygen sensitivity (HARTMANIS GATENand observations). It is tempting to speculate that BECK, 1984).
Tab. 6. Enzymes of C. acetobutylicum and C. beijerinckii Involved in Pyruvate Degradation

Coenzyme NADH TPPd, CoASH CoASH Purified Native Molecular Subunit Size Mass [kDa] WaI Composition Cloned Sequenced
Enzyme
Host
Lactate DH" Pyruvate:ferredoxin OR' Phosphotransacetylase
+ +
+'
NR NR 100-120 213
NRb NR NR
36 123 56-57
44 44 NR 31 40 31 33 39 63
ADP NADH NADPH CoASH CoASH NADH NADH
158 264 205 85
NR CoASH CoASH ADP NADH NADPH NADPH, FAD
+ + + + + + + + NR 93 85 330 200-230
NR NADH NADH NADH NADH NADPH
115 100 NR 82 100 80
41 23, 24 23, 28 28 NR 96 56 55 43 43 38 40, 43.5
C. acetobutylicum C. acetobutylicum C. beijerinckii C. acetobutylicum C. acetobutylicum Acetate kinase Acetaldehyde DH C. acetobutylicurn Alcohol DH C. acetobutylicum Thiolase C. acetobutylicum C. beijerinckii P-Hydroxybutyryl-CoA DH C. beijerinckii C. acetobutylicum Crotonase C. acetobutylicum Butyryl-CoA DH C. acetobutylicum Phosphotransbutyrylase C. acetobutylicum C. beijerinckii Butyrate kinase C. acetobutylicum Hydrogenase C. acetobutylicum NADH:ferredoxin OR C. acetobutylicum NADPH:ferredoxin OR C. acetobutylicum NADH: rubredoxin OR C. acetobutylicum CoA transferase C. acetobutylicum C. beijerinckii Acetoacetate decarboxylase C. acetobutylicum C. beijerinckii Aldehyde/alcohol DH (E) C. acetobutylicum Butyraldehyde DH C. acetobutylicum C. beijerinckii Butanol DH (I) C. acetobutylicum Butanol DH (11) C. acetobutylicum Primarykecondary C. beijerinckii alcohol DH Alcohol DH C. beijerinckii C. beijerinckii Alcohol DH
NAD(P)H NADPH
+ + + + + + + + + + + -
Dehydrogenase Not reported Oxidoreductase
Thiamine pyrophosphate Partially purified
242
6 Microbial Production of Acetone/ButanoUIsopropanol
Glucose
However, not only 2 HJglucose but approximately 2.3 H,/glucose are produced during acid formation. The additional 0.3 H2 originate from NADH; it is oxidized by NADH:ferredoxin oxidoreductase which has been studied in C. acetobutylicum to some extent (PETITDEMANGEal., 1976, 1977). The et enzyme is activated by acetyl-CoA and inhibited by NADH. Hydrogenase in turn utilizes the electrons carried by reduced ferredoxin and, together with protons, forms molecular hydrogen. Since part of the NADH of the above equation is oxidized by H2 evolution, not all acetyl-CoA is converted to butyrate. This is the reason why acetate is a fermentation product in addition to butyrate. There are thermodynamic limitations as to the extent of H2 evolution from NADH, because the redox potential of the couple NADH/ N A D H + + H + is more positive than that of HJ2 H + (THAUER al., 1977). Another enet zyme, NADPH:ferredoxin oxidoreductase, can also utilize reduced ferredoxin in the controlled production of NADPH. This may be the only route for the production of NADPH required for biosynthetic reactions, since most clostridia appear to lack the enzymes necessary for the NADPH-yielding oxidation of glucosed-phosphate (JUNGERMANNal., et 1973). A NADH:rubredoxin oxidoreductase with a molecular mass of 41 kDa and FAD as a prosthetic group has also been purified from C. acetobutylicum ATCC 824 (PETITDEMANGE et al., 1979). The physiological function of this enzyme is still unknown. It is induced by acetate (and, to a lower extent, by butyrate) only at low pH values. Maximal activity has been observed at pH 4.8 (BALLONG U E et al., 1986). After the onset of solventogenesis the enzymatic activity rapidly decreases (MARCZAK al., 1983, 1984). The et level of rubredoxin in the cells fluctuates accordingly, while the concentration of ferredoxin remains almost constant throughout
-+
+ Butyrate + 2 C 0 2+ 2 H2
+
The final steps of butyrate formation are catalyzed by phosphotransbutyrylase (Ptb) and butyrate kinase (Buk). Ptb activity is mainly found during the acidogenic fermentation phase (ANDERSCH al., 1983; HARTet MANIS and GATENBECK, 1984). The enzyme has been purified from C. acetobutylicum strain ATCC 824 and from C. beijerinckii (WIESENBORN al., 1989a; THOMPSON et and CHEN,1990). Both proteins consist of identical subunits with a molecular mass of 31 and 33 kDa, respectively. However, sizes of the native enzymes differ considerably (264 vs. 205 kDa). This might be due to variations in the determination procedures. Ptb is very sensitive to pH changes and almost completely inactive in the butyryl phosphate-forming direction at a pH of about 6. The enzyme from C. beijerinckii also reacts with acetoacetyl-CoA in the presence of phosphate, and acetoacetyl phosphate might be a product (THOMPSON CHEN,1990). Whether this and reaction is of physiological relevance is not yet known. Butyrate kinase has been purified from C. acetobutylicum ATCC 824. The native enzyme is a dimer of two apparently identical subunits that have a molecular mass of 39 kDa (HARTMANIS, 1987). Its relative activity with acetate is only 6% of that with butyrate. The genes for both enzymes have been cloned and sequenced from C. acetobucylicum ATCC 824 (CARYet al., 1988; WALTER et al., 1993) and NCIMB 8052 (now grouped with C. beijerinckii, as mentioned above) (OULTRAM al., 1993). The two et genes are contiguous on the chromosome and most likely form an operon (WALTER al., et 1993). During acid production, reduced ferredoxin is oxidized by hydrogenase and H2 is produced. Hydrogenase activity has been demonstrated in cell extracts of C. acetobutylicum (ANDERSCH al., 1983; KIM and ZEIKUS, et 1985). The respective gene has recently been cloned and sequenced from strain P 262 (SANTANGELO al., 1994). The NADH genet erated in the 3-phosphoglyceraldehyde dehydrogenase reaction is oxidized in the P-hydroxybutyryl-CoA and butyryl-CoA dehydrogenase reactions. This results in the following redox balance:
Glucose 2 NAD 2 Pyruvate +2NADH +2 H 2 Acetyl-CoA 2 C 0 2 2 H 2 Pyruvate 2 Acetyl-CoA + 2 NADH + 2 H Butyrate 2 NAD
243
the fermentation (MARCZAKet al., 1985). both subunits in strain DSM 792 (GERISCHER Under iron limitation ferredoxin can no long- and DURRE,1990; FISCHER al., 1993). Simet er be synthesized. Instead, flavodoxin is ilar data have been determined with purified formed, the gene of which has been cloned CoA transferase from C. beijerinckii NRRL from C. acetobutylicum P 262 (SANTANGELO B.593 (CHEN, 1993). CoA transferase plays a et al., 1991). major role in the uptake of acids during solventogenesis (preferentially butyrate) that are subsequently converted to acetone and 3.3.2 Formation of Solvents butanol (ANDERSCH al., 1983; HARTMANIS et et al., 1984). To some extent, butyrate can be Several conditions must be met to ensure a taken up in batch cultures via the reversed reproducible metabolic shift from acidogene- reactions of butyrate kinase and phosphosis to solventogenesis in C. acetobutylicum. transbutyrylase (HUSEMANN PAPOUTSAand These are a low pH, excess of substrate, KIS, 1989). A surprising feature of CoA transthreshold concentrations of butyrate and/or ferase are the high K , values for acetate and acetate, and a suitable growth-limiting com- butyrate (1.2 mol L- and 0.6 mol L-I, repound such as phosphate or sulfate (BAHL spectively). They have been suggested to reand GOTTSCHALK, 1984). In strain NCIMB flect a gradational response to the progressive 8052 (now considered to be a member of the toxic effects of increasing levels of these acids C. beijerinckii group) formation of acetone (WIESENBORN al., 1989b). K , values for et and butanol could be initiated at neutral pH the corresponding enzyme from C. beijerinckby adding high concentrations (100 mmol ii are significantly lower (CHEN,1993). L- each) of acetate and butyrate (HOLT et Acetoacetate decarboxylase (Adc) of al., 1984). Before the onset of solventogenesis C. acetobutylicum has been the subject of exa new set of enzymes is synthesized. Acetone tensive investigations in the 1960s aiming to production is catalyzed by acetoacetyl- elucidate its reaction mechanism (FRIDOCoA:acetate/butyrate coenzyme A transfer- VICH, 1972; WESTHEIMER, 1969). Purification ase (CoA transferase or Ctf) and acetoacetate of the native enzyme revealed that it had a decarboxylase. In C. beijerinckii strains able molecular mass of 340 kDa and consisted of to form isopropanol, a primary/secondary al- 12 identical subunits with a molecular mass of cohol dehydrogenase subsequently converts 29 kDa (TAGAKI and WESTHEIMER, 1968). acetone into isopropanol. Butanol is made Later investigations showed the respective from butyryl-CoA by action of various butyr- data to be rather 330 kDa and 28 kDa (GERIaldehyde and butanol dehydrogenase activi- SCHER and DURRE, 1990; PETERSENand 1990), confirmed by the deduced ties. Ethanol on the other hand is constitu- BENNETT, tively produced by C. acetobutylicum amino acid sequence after cloning and sethroughout the fermentation (GERISCHER quencing the structural gene, which forms a and DURRE, 1992). Its synthesis is brought monocistronic operon (GERISCHER and about by a NADPH-dependent alcohol dehy- DURRE, 1990; 1992; PETERSENand BENdrogenase and a specific acetaldehyde dehy- NETT, 1990; PETERSEN al., 1993). This opeet drogenase that does not react with butyryl- ron is adjacent, but convergently arranged to CoA (BERTRAM al., 1990). et the ctf genes that form a common transcripCoA transferase of C. acetobutylicum tion unit together with the gene for an aldeet ATCC 824 has been purified to homogeneity hyde/alcohol dehydrogenase (FISCHER al., (WIESENBORN al., 1989b). The native en- 1993; NAIRet al., 1994). The reaction mechaet zyme was a heterotetramer of two different nism of Adc involves the intermediate formasubunits with a molecular mass of 23 kDa and tion of a Schiff base between the substrate 28 kDa, respectively. However, cloning and acetoacetate and a lysine residue at the active et sequencing of the respective genes (ctfA and site (WARREN al., 1966). Native acetoacectfB) led to deduced values of 22.7 kDa and tate decarboxylase from C. beijerinckii NRRL 23.7 kDa for strain ATCC 824 (CARYet al., B592 and NRRL B593 has a considerably 1990; PETERSEN al., 1993) and 23.6 kDa for smaller molecular mass of 200-230 kDa et
244
(CHEN,1993). The latter strain belongs to the few clostridia able to further reduce acetone to isopropanol (GEORGEet al., 1983). The enzyme catalyzing this reaction is a primary/ secondary alcohol dehydrogenase with a native molecular mass of 100 kDa, consisting of identical subunits with a molecular mass of 38 kDa (CHEN,1993; HIU et al., 1987). It is NADPH dependent and, although it converts acetaldehyde to ethanol in vitro, reduction of acetone to isopropanol is the preferred reaction. The structural gene has been cloned and sequenced. The deduced amino acid sequence shows 75% identity to the thermostable alcohol dehydrogenase of Thermoanaerobium brockii, an anaerobe producing ethanol (CHEN,1993). Butanol formation is catalyzed by butyraldehyde (Bad) and butanol dehydrogenase (Bdh) activities. A butyraldehyde dehydrogenase has been purified from C. acetobutylicum NRRL B643 and from C. beijerinckii NRRL B592 (PALOSAARIand ROGERS, 1988; YAN and CHEN,. 1990). Molecular size and subunit composition were similar for the two enzymes. Bad from C. acetobutylicum was a homodimer of two 56 kDa subunits, yielding a native enzyme with a molecular mass of 115 kDa. The respective data for C. beijerinckii are 55 kDa and 100 kDa. Both enzymes prefer NADH as a coenzyme over NADPH, and butyryl-CoA is a better substrate than acetyl-CoA. Recently, in C. acetobutylicum a gene has been cloned and sequenced that has high homology with the adhE gene of Escherichia coli known to encode a protein with aldehyde and alcohol dehydrogenase domains. The respective clostridial enzyme is believed to play a role in butanol synthesis at the onset of solvent formation (FISCHERet al., 1993; NAIR et al., 1994). For a long time, conflicting data have been reported on alcohol dehydrogenase in C. acetobutylicum. The situation was clarified in 1987 by a report providing evidence for at least two different enzyme activities in this organism (DURRE et al., 1987). An NADPHdependent enzyme was purified from strain DSM 792. It consists of identical subunits with a molecular mass of 44 kDa, it is very unstable and has a pH optimum between 7.8
and 8.5 (MICHELS,1990). The physiological role of this alcohol dehydrogenase is probably in ethanol formation in cooperation with a specific acetaldehyde dehydrogenase as suggested from studies with transposon-induced mutants (BERTRAM al., 1990). Activity of et the NADPH-dependent alcohol dehydrogenase is found throughout the fermentation (DURREet al., 1987) confirming the constitutive nature of the enzyme which is in agreement with the constitutive formation of low ethanol levels (GERISCHERand DURRE, 1992). This alcohol dehydrogenase probably regulates the pool of reduced nicotinamide adenine dinucleotide phosphate which is supplied by NADPH:ferredoxin oxidoreductase. From C. acetobutylicum P 262 a gene encoding a NADPH-dependent alcohol dehydrogenase has been cloned and sequenced (YOUNGLESON al., 1988, 1989a). However, et due to the different taxonomic grouping of P 262 it is questionable whether the respective enzyme has the same physiological function as in strain DSM 792. Two primarily NADH-dependent butanol dehydrogenase isozymes have been purified from C. acetobutylicum ATCC 824 (PETERSEN et al., 1991; WELCH et al., 1989). Both native enzymes are homodimers with subunits of molecular masses of about 42 kDa. Bdh I had only a 2-fold higher activity with butyraldehyde than with acetaldehyde, whereas Bdh I1 was reported to have a 46-fold higher activity with butyraldehyde than with acetaldehyde. Bdh I1 required Zn2+ for activity and had a pH optimum of 5.5, a value around which the cells switch from acidogenesis to solventogenesis. Thus, Bdh I1 is probably the major alcohol dehydrogenase involved in butanol production. The genes of both isozymes (bdhA and bdhB) have been cloned and sequenced (PETERSEN al., 1991; WALTERet et al., 1992). They are arranged in monocistronic operons each, that are contiguous on the chromosome and are controlled by different promoters. Recently, another gene of C. acetobutylicum with high homology to the E. coli adhE gene has been cloned and sequenced (FISCHER al., 1993; NAIRet al., 1994). The et respective E. coli gene product has aldehyde and alcohol dehydrogenase domains (GOODLOVE et al., 1989) and its clostridial analog is
245
believed to be involved in butanol synthesis (FISCHER al., 1993; NAIRet al., 1994). The et clostridial adhE (or aad) gene is part of an operon additionally containing the ctfA and ctfB genes. This transcription unit (sol operon) is controlled by two different promoters. Thus, at least three different butanol dehydrogenases might be involved in butanol production in C. acetobutylicum. A similar situation has been found with C. beijerinckii. Strain NRRL B592 producing acetone, butanol, and ethanol, but not isopropanol, expresses two types of alcohol dehydrogenases with distinct coenzyme specificity. One of them is NADPH-dependent whereas the other can use either NADH or NADPH (CHEN, 1993). This latter enzyme has a native molecular mass of 80 kDa, consists of two subunits with molecular masses of 40 kDa and 43.5 kDa, and yields three distinct activity bands after electrophoresis of purified protein under non-denaturing conditions. These three species might be dimers of the composition a2, and pZ CHEN,1992). ap,
I
6
I
5
L
._
Fig. 4. Course of the acetone-butanol fermentation in batch culture. Butanol, 0;acetone, A; ethanol, W; butyrate, 0; acetate, A; pH, @ (BAHLet al., 1982b).
3.3.3 Regulation of Product Formation

A batch culture of C. acetobutylicum classically shifts from acid to solvent formation towards the end of growth at pH values below 5 (Fig. 4). The shift can reproducibly be achieved in complex media (OXFORDet al., 1940) and is often associated with sporulation (LONGet al., 1984a, b). The ability to initiate and complete the process of sporulation is not a prerequisite of solvent production. In agreement with this an asporogenous mutant is selected in solvent producing continuous cultures (MEINECKE al., 1984). Although the et signal for initiating sporulation is not nutrient limitation as in Bacillus species (LONGet al., 1984b) the process of spore formation seems to be identical since analogs of the sporulation-specific Bacillus sigma factor genes have been cloned and sequenced from C. acetobutylicum (SAUER al., 1994). et The shift to solventogenesis in C. acetobutylicum and C. beijerinckii is characterized by a decrease of the activity of some acidogenic
enzymes (ANDERSCH al., 1983; HARTMAet NIS and GATENBECK, 1984). At about the same time the enzymes needed for solventogenesis are induced or derepressed (ANDERSCH et al., 1983; DURRE et al., 1987; HARTMANIS GATENBECK, and 1984; YANet al., 1988). Induction requires synthesis of new mRNA and protein as shown by experiments with C. acetobutylicum using the transcription or translation inhibitors rifampicin and chloramphenicol (BALLONGUE al., 1985; PALOet SAARI and ROGERS, 1988; WELCH et al., 1992). mRNA analyses provided evidence that induction or derepression started several hours before solvents could be detected in the medium. Induction of the adc, bdhA, bdhB, and sol operons at the mRNA level showed a rapid increase up to a maximum shortly before the production of acetone and butanol followed by a massive decrease of transcripts (FISCHERet al., 1993; GERISCHERand DURRE,1992; WALTER al., 1992). During et the shift from acidogenesis to solventogenesis also the synthesis of five heat shock proteins including GroEL and DnaK is induced (PICH et al., 1990; BAHL,1993). This stress response
246
is linked to onset of solventogenesis and spo- of expression (SAUER and DURRE, 1992). rulation in a yet unknown way. Genes encod- This tRNA recognizes the rarely used ACG ing several of these proteins have been cloned codon which, however, is present in all genes and sequenced (BEHRENS al., 1993; NAR- required for acetone and butanol synthesis. In et BERHAUS and BAHL,1992; NARBERHAUS et addition, it has been found in inducible genes for sporulation, autolysis, and uptake or meal., 1992; SAUER and DURRE, 1993). Despite the enormous progress made in tabolism of minor C or N substrates (SAUER elucidation of gene structure and mRNA reg- and DURRE, 1992). This codon distribution ulation, little is known on the signal(s) that could either reflect an evolutionary process initiate solventogenesis (and possibly sporula- with low mutational pressure on weakly extion and stress response). Involvement of al- pressed genes or represent a novel translaternate sigma factors in transcription of genes tional control mechanism. Such a system has encoding solventogenic enzymes is unlikely, been found in the genus Streptomyces where since the RNA polymerase of C. acetobutyli- a rare tRNA controls production of aerial cum has been purified (PICH and BAHL, mycelium and antibiotics (LESKIW et al., 1991) and, after isolation from acidogenic and 1991). solventogenic cells, showed the same composition including only the vegetative sigma factor (BAHL,1993). Recently, a model has been 3.4 Strain Improvement proposed that postulates the ATP pool to be Various research activities are devoted to a signal for acetone formation and the NAD(P)H pool to be a signal for butanol the solution of cultivation and product recovproduction (GRUPE and GOTTSCHALK, ery problems from which the acetone-buta1992). However, since CoA transferase (re- no1 fermentation suffers most. In addition, quired for acetone production) and AdhE knowledge on the physiology and genetics of (needed for butanol formation) are encoded C. acetobutylicum and C. beijerinckii is rapin a common transcription unit (FISCHER et idly increasing. Besides the work concerned al., 1993), this model must at least be modi- with correlation of culture conditions to the fied, although the pools of ATP and reducing physiology of the organism and with the reguequivalents certainly play a major role in reg- latory mechanisms controlling the key enulation. On the other hand it might be possi- zymes of the different metabolic pathways, ble that DNA topology directly responds to there is great interest in removing some limitchanges in the environment (pH, salt concen- ing features of C. acetobutylicum. An imtration, osmotic pressure, temperature) and provement of the microorganisms could inthus provokes transcription of certain genes. clude: increased resistance to butanol, fewer The degree of supercoiling of DNA isolated end products (e.g., no acetone), and broader from acidogenic and solventogenic cells was substrate range (e.g., cellulose). Butanol toxicity is the limiting factor with different (WONG and BENNETT, 1994). Supercoiling is controlled by topoisomerase I respect to the maximum amount of solvents and DNA gyrase, the genes of which have (acetone, butanol, ethanol) to be achieved in been cloned and sequenced from C. acetobu- the fermentation broth (about 20 g L-'). Imtylicum DSM 792 (ULLMANN and DURRE, provement in this area is critical for the eco1994). Transcription analysis and targeted nomics of the process (reduction of product inactivation of the gyr genes should provide a recovery costs). It is generally accepted that the cell membrane is one target of alcohol atconclusive answer to this problem. 1986). Owing to their amphiAn additional level of regulation might be tack (INGRAM, present as indicated by studies with a trans- phatic character, alcohols dissolve in the poson-induced, acetone- and butanol-nega- membrane lipids, increase their permeability tive mutant of C. acetobutylicum (BERTRAM (DOMBEKand INGRAM,1984), and affect et al., 1990). Genetic analysis revealed that membrane fluidity (VOLLHERBST-SCHNECK the transposon Tn916 had inserted in front of et al., 1984). Thus, high concentrations of bua tRNA gene thus causing massive reduction tanol lead to a complete abolition of the pH
4 New Developments of the Fermentation Process
247
gradient, lower the intracellular level of ATP, BALD, 1993), DNA transfer by conjugation is cause the release of intracellular metabolites, easily performable (OULTRAM and YOUNG, and inhibit sugar uptake (MOREIRA al., 1985; OULTRAM al., 1987,1988b; REYSSET et et 1981; GOTTWALDand GOTTSCHALK, 1985; and SEBALD, 1985; WILKINSON YOUNG, and BOWLES and ELLEFSON, 1985; HUTKINS and 1994; WILLIAMS al., 1990; YOUNG,1993; et KASHKET,1986). These effects clearly de- Y u and PEARCE,1986), the isolation of a filmonstrate the importance of the cell mem- amentous phage led to the construction of a brane with respect to butanol tolerance. Oth- phagemid (KIMand BLASCHEK, 1991, 1993), er sites of interference, e.g., inhibition of gly- and transposon mutagenesis is well estabcolytic enzymes (HERRERO, 1983), cannot be lished (BERTRAM and DURRE, 1989; BERTexcluded. In view of the complex effects of RAM et al., 1990 DURRE,1993; WOOLLEY et butanol, it might be very difficult to isolate al., 1989; YOUNG,1993). Very important was strains of C. acetobutylicum with a substantial the finding that DNA to be transformed into higher butanol tolerance. Chemical mutagen- C. acetobutylicum must be methylated to esis has been used to obtain butanol-tolerant avoid restriction (MERMELSTEIN and PAstrains that, however, produced only little POUTSAKIS, 1993). Protease-deficient mutants more butanol (if at all) than the wild type will be helpful for the study of protease-labile (HERMANN al., 1985; LEMMEL, et 1985; LIN proteins (SASSet al., 1993) and DNase-negaand BLASCHEK, 1983). tive mutants might be helpful in genetic maNon-production of the less desirable sol- nipulations (BURCHHARDTand DURRE, vent acetone can be achieved by selecting for 1990). mutants in the presence of 2-bromobutyrate Recently, an artificial operon (ace operon) (JANATI-IDRISSI al., 1987). Low acetone has been constructed containing the adc, ctfA, et yields also result from growth on whey or un- and ctfB genes, all transcribed from the adc der iron limitation (BAHLet al., 1986). Im- promoter. After transformation into a aceprovements in substrate utilization have been tone-butanol-negative mutant of C. acetobuattempted by isolating mutants with enhanced tylicum, the recombinant strain regained the amylolytic activity (ANNOUS BLASCHEK, ability to form acetone (MERMELSTEIN al., and et 1991) or by transformation of endoglucanase 1993). When introduced into the wild type the genes from C. cellulovorans or C. thermocel- ace-containing plasmid also led to an increase lum into C. acetobutylicum (KIM et al., 1994; in solvent formation, although plasmid withOULTRAM al., 1990). A class of sponta- out insert caused a significant (but somewhat et neous mutants of C. acetobutylicum NRRL lower) stimulation of acetone, butanol, and B643 resistant to ally1 alcohol produced con- ethanol production. Thus, genetic manipulasiderable quantities of butyraldehyde in addi- tions to alter the product formation capabilition to acetone and butanol which could be of ties are now available and might lead to a interest for chemical syntheses (ROGERS and reintroduction of this fermentation at an industrial scale. PALOSAARI, 1987). Meanwhile, the repertoire of genetic and recombinant DNA techniques to be used with C. acetobutylicum has increased impressively. A variety of shuttle vectors (with either E. coli or B. subtilis as alternative host) are available (AZEDDOUG al., 1992; LEE et al., et 1992; MINTONet al., 1993; STRATZ et al., 1994; TRUFFAUT and SEBALD,1988; TRUFFAUT et al., 1989; YOON et al., 1991; YOSHIThe industrial acetone-butanol fermentation process, which was in use for over forty NO et al., 1990), transformation and electroporation protocols have been worked out years, is documented well and in detail in the (LINand BLASCHEK, 1984; MERMELSTEIN literature (BEESCH, 1952; PRESCOTT and et 1978; al., 1992; OULTRAM al., 1988a; REIDet al., DUNN,1959; Ross, 1961; HASTINGS, et 1983; REYSSET al., 1988; REYSSET SE- SPIVEY, 1978; JONES and WOODS, 1986; et and
248
6 Microbial Production of Acetone/Butanol/lsopropanol
BAHL and GOTISCHALK, 1988). This traditional commercial batch process of acetonebutanol fermentation is no longer in use. The main factors influencing the unfavorable economics were high raw material and transportation costs and intrinsic limitations (low solvent yields and low final concentrations, undesirable solvent ratios) resulting in high processing costs and waste disposal problems. The substrate caused about 60% of the overall production costs (ROSS, 1961). The price for maize starch and molasses increased in the years after World War I1 to a level at which the fermentation could no longer compete with the synthetic route that used cheap oil as a feedstock. Although in some cases alternative substrates were available, transportation costs of the bulky material prevented its use. In addition, a theoretical calculation taking the biochemical pathways of C. acetobutylicum into account showed that the maximum possible yield of solvents is 0.38 g per g of glucose converted (LEUNGand WANG, 1981). In practice, this yield was not always reached, particularly with molasses as substrate. Also, the productivity of a typical batch fermentation process was low (0.24.6 kg m P 3 h - solvents). The solvents represented a mixture of acetone, butanol, and ethanol, whereas often only one product, e.g., butanol as desired. Another major drawback is the low butano1 tolerance of C. acetobutylicum. A maximum total solvent concentration of 20 kg m-3 can be achieved in the fermentation broth. The recovery of solvents from this dilute solution (normally by distillation) resulted in substantial costs. The fuel expenses for steam generation amounted to about 15-20% of the production costs; 65% of the steam was used for distillation. Due to the low product concentrations the acetone-butanol fermentation generates especially large volumes of waste. The disposal of such waste often was a problem (SPIVEY, 1978). After the era of cheap oil has gone - made obvious through the oil crisis of 1973174 there is a renewed interest in fermentation processes for the production of fuels and chemicals from biomass. This holds also for the acetone-butanol fermentation. The fol-
lowing section will emphasize recent research related to the improvement of the fermentation process. Included are topics such as alternative substrates, continuous culture, immobilized cells, cell recycling, and product recovery. Progress in several of these areas will be crucial for renewed economic viability of acetone-butanol fermentation. Three parameters are important for the evaluation of new processes in comparison to the traditional batch process: (1) the final solvent concentration obtained [g solvents L-1, (2) the yield [kg solvents kg- sugar], and (3) the productivity [g solvents L- h-I.
4.1 Continuous Culture

A fermentation process that can be operated continuously has some advantages compared to a batch process. Only one series of inoculum cultures is needed for a long production period. A dead season necessary for filling, sterilization, cooling, and cleaning of the equipment is largely decreased, and the volume of the fermenter vessel can be reduced without a loss of production capacity (higher productivities). The use of a continuous process for acetone-butanol fermentation might provide an additional advantage. The fermentation time can be shortened by eliminating the first acidogenic phase when the parameters are known to keep the cells in the solventogenic phase constantly for a long time. Despite all potential advantages over a batch process there are only few reports about commercial continuous production of solvents by C. acetobutylicum. DYR et al. (1958) reported a continuous acetone-butano1 production in a three to five vessel flow system. Although the productivity was three times higher compared to the batch process the substrate concentration and the solvent yields were low, and the acids formed in the first stage were not converted into solvents in the following stages. YAROVENKO (1964) described a similar fermentation carried out in a pilot plant consisting of a chain of eleven fermenters. Detailed information of the conditions of these continuous fermentations was not given.
249
To be economically attractive a continuous process must at least achieve the final concentration and yield of solvents that can be obtained in batch culture. In addition, significantly higher productivities are necessary. In recent years, the continuous culture of C. acetobutylicum has been mainly used as a research tool to define parameters responsible for changes in the physiology and the activity of this microorganism. Under steadystate conditions a constant environment is provided and the influence of a single parameter and its interactions with other factors can be determined. The following fundamental areas of acetone-butanol fermentation were examined: the effect of medium components and acidic fermentation products on solvent production, the influence of temperature, culture pH, dilution rate ( D ) , maximum attainable solvent concentration and yield, and the stability of a continuous culture with respect to the ability of solvent production. Tab. 7 gives a summary of results obtained with chemostat cultures of C. acetobutylicum. A direct comparison of the results is difficult because of differences in the strains used, in medium composition, and in fermentation conditions. Generally, it can be concluded that no single growth-limiting factor specifically induces solvent production in a chemostat. However, some nutrients have been shown to be more suitable for growth limitation and production of solvents in high yields than others. In glucose-, nitrogen-, or magnesium-limited chemostats steady-state solvent production was low or difficult to maintain, and an application of these kinds of limitations to an industrial process seems unlikely (GOTTSCHAL and MORRIS,1981b; BAHL et al., 1982a; ANDERSCH et al., 1982; MONOT and ENGGASSER, 1983; JOBSES and ROELS,1983; STEPHENS et al., 1985; R o o s et al., 1984; MONOT et al., 1983; BAHL and GOTTSCHALK, 1984). Phosphate and sulfate belong to the group of suitable growth-limiting factors (BAHLet al., 1982b; BAHLand GOTTSCHALK, 1984). The pH value had an important influence on the product pattern of C. acetobutylicum in continuous culture. Significant solvent production was only observed at pH values of 5 and below. The optimal pH may vary with re-
spect to the strain used, e.g., C. acetobutylicum DSM 1731: pH 4.3 (BAHLet al., 1982a, b); ATCC 824: pH 4.8-5.0 (MONOTand ENGASSER, 1983). The temperature applied was in the same range as in the batch process (33-37C) and had minor influence. Increased levels of butyrate and/or acetate are able to induce solvent production in batch culture (GO~TSCHAL MORRIS, and 1981a). In agreement with these results, acids have to be present in continuous culture at threshold concentrations (about 10 mmol L-I) to make solvent production possible (BAHL and GOTTSCHALK, 1984). All experiments in continuous culture to test the influence of the dilution rate showed that high solvent concentrations could be obtained at a low dilution rate ( D s 0 . 0 5 h-I) (LEUNG and WANG, 1981; BAHL et al., 1982b). Although higher dilution rates resulted in improved productivity, this was at the expense of reduced substrate turnover and solvent concentration. Using a two-stage phosphate-limited chemostat BAHLet al. (1982b) reported solvent concentrations of 18.2 g L - ' with a yield of 0.34 g solvents per g glucose and a productivity of 0.44 g L - ' h - ' (Tab. 8). The first stage was run at D =0.125 h - I (pH 4.3, 37"C), and the second stage was operated at D =0.03 h - ' (pH 4.3, 33C). Compared to a batch culture using the same strain (C. acetobutylicum DSM 1731) and medium, the productivity doubled without a loss of substrate utilization, solvent concentration, or yield (BAHLet al., 1982b). Although the economics remain to be established this two-stage process seems to be promising. Performance of the fermentation in this way has the advantage that the conditions at the stages (e.g., temperature, dilution rate) can be optimized with respect to either growth or solvent formation. At the first stage the cells are growing under conditions under which solvent formation is induced. The second stage is primarily devoted to the conversion of the residual sugars to solvents. Since the limiting growth factor is exhausted, growth is not possible here. This principle seems to have an additional advantage with respect to culture stability. Culture degeneration and difficulties in maintaining steady-
Tab. 7. Production of Solvents by Clostridium acetobutykum in Chemostats

N VI 0
Comments Yield
References
rn
Fermentation Process Nutrient Strain Limitation D ["CI 35

-
Substrate Temper- pH Concen- ature tration
Results Solvents Productivity

[g L - 7 [g L-I h-ll
[g L-II
W'I
5.7 No solvent production 4.3 0.01 0.02 Induction of solvent formation by lowering the pH below 5.0; optimum: pH 4.3 0.13 0.07 0.08
[&I
-
S'
0.
Glucose 2.7 g L-'
NCIB 8052 2.7 37
a R
3.4 g L-l
DSM 1731 3.4
E 3
3
GOTTSCHAL and MORRIS(1981b) BAHLet al. (1982a)
%
cs
NCIB 8052 2.7

-
35
5.7
0.08
8
GOTTSCHAL and MORRIS(1981b) ANDERSCH al. et (1982) MONOTand ENGASSER (1983)
Nitrogen 0.4 g L-' Ammonium chloride

37 0.95 0.08 5.2 0.22 2.50
$
E
2gL-I Ammonium . sulfate 35 0.30
DSM 1731 54
s %
0.2 g L-' Ammonium acetate
ATCC 824 45.5
5.0
0.04
8.00
0.29
No solvent production at relatively high pH values and low sugar concentrations Solvent production at pH values from 5.2 to 4.3 and at high glucose concentrations Solvent production at low dilution rate, low pH, and high glucose concentrations 0.32 Yield comparable to batch process; higher solvent concentration possible at lower dilution rates
: 2
3
Sulfate 0.05 g L Magnesium sulfate 0.01 g L-' Manganese sulfate

37 4.3 0.10 4.3
DSM 1731 54
0.43
BAHLand GOTTSCHALK (1984)
stage I
0.125 18.2 0.44 0.34
Phosphate Two-stage chemostat 0.1 g L-' DSM 1731 54 Monopotassium phosphate
37 4.3 stage I1 33 4.3
0.03
Successful laboratoryBAHLet al. scale two-stage system for (1982b) continuous solvent production, no culture degeneration over a period of one year Poor solvent production at low pH, low dilution rate, and excess of substrate BAHLand GorrSCHALK (1984)
Magnesium 0.02 g L - ' Magnesium sulfate

4.3
DSM 1731 54
0.8
37
0.06
0.05
0.07
252
6 Microbial Production of Acetone/ButanoUIsopropanol
Tab. 8. Concentration of Products and Consumption of Substrate in a Two-Stage Continuous Culture of Clostridium acetobutylicum under Phosphate Limitation (BAHL al., 1982b) et Stage 1
~
Stage 2
4.3 33 0.03 12.6 4.8 0.8 0.8 0.5 99.7 0.34 0.44"
Process Parameter PH Temperature ["C] Dilution rate [h-'1 Products [g L-'1 Butanol Acetone Ethanol Butyrate Acetate Consumption of substrate [%] Yield [g solventlg glucose] Productivity [g solvent L - ' h -'I
a
4.3 37 0.125 3.6 1.6 0.3 1.3 0.4

40
0.25 0.08
Productivity of the overall process
state solvent production over a longer period (i. e., >200 h) have often been encountered (STEPHENSet al., 1985; GOTTSCHAL and MORRIS, 1981a). High solvent concentrations seem to be responsible for this effect (FICK et al., 1985). On the other hand, long-term solvent production with C. acetobutylicum in the two-stage phosphate-limited chemostat has been reported. Running the first stage under conditions of low solvent concentrations (high dilution rate) but solvent formation is absolutely required because of the effect of acids at a low pH (4.3), the selection of such non-producing strains is apparently prevented. It is interesting that under these conditions an asporogenous strain of C. acetobutylicum was selected with an unchanged capability of producing solvents (MEINECKE al., et 1984). Other continuous flow fermentation processes (excess substrates, LEUNG and WANG, 1981; turbidostat, GOTTSCHALand MORRIS, 1982; pH-auxostat, STEPHENS al., et 1985) were also used, and the results support the view that a continuous acetone-butanol fermentation is feasible.
4.2 Cell Immobilization and Cell Recycling

The low productivity and the low final concentration of solvents in the fermentation broth are the major drawbacks. Improvement in this field is necessary before a reintroduction of the acetone-butanol fermentation process can be envisaged. Cell immobilization is a technique to confine the biocatalysts within the fermenter system. Thereby higher cell densities are possible resulting in higher productivities (smaller reactor volumes possible). Other advantages include the use of simpler non-growth media and easier separation of the cells from the products. On the other hand. the activity of the cells may be affected due to immobilization conditions. Immobilization of vegetative cells and spores of C. acetobutylicum has been described. Calcium alginate (spherical beads or spiral wound flat sheets) was used to entrap the organism for a continuous solvent production in a glucose medium (HAGGSTROM and ENFORS, 1982; FORERGet al., 1983). Packedbed reactors, continuous stirred tank reactors, and fluidized column reactors served as reaction vessels.
253
Generally, higher solvent productivities (2.4-2.8 g L - ' h-') have been obtained. However, substrate utilization and solvent concentrations were low (1.54.5g L-I). Another problem is the rapid loss of activity of C. acetobutylicum in non-growth media. The pulsewise addition of nutrients to the fermentation medium resulted in some improvement (FORBERG al., 1983). et In addition to the entrapment technique, adsorption of cells to a solid surface can be used for immobilization. The successful adsorption of C. acetobutylicum to beech wood shavings (FORBERG HAGGSTROM, and 1985), arranged as parallel sheets, is promising since it is a simple, cheap, and non-toxic immobilization method. This process using the intermittent nutrient dosage method was run for over one month with low cell leakage, but again the solvent concentrations were low (5-6 g L-I). To overcome this problem the use of an immobilized sporulation-deficient strain of C. acetobutylicum P 262 has been reported (LARGIER al., 1985). At a dilution et rate of 0.42 h - ' the solvent concentration reached 15 g L - ' with a productivity of 3.02 g L - ' h - ' and a yield of 0.44 kg solvents per kg sucrose. The 5-fold increase in solvent concentration at a similar productivity represents major progress as compared to immobilized wild type cells. These results clearly show that solvent production with immobilized cells is principally possible. Advances in other fields, e.g., strain improvement are necessary for the application of such a process. Cell recycling is an alternative to increase productivity by the reuse of productive cells in a continuous culture system. Using this method the solvent productivity of C. acetobutylicum could be increased considerably (up to 3 g L - ' h-I) without a decrease in the concentration of solvents (up to 22 g L - ' ) (AFSCHAR al., 1985; SCHLOTE et and GOTTSCHALK, 1986). Instability of the cells with respect to solvent formation was again observed after a long operation period at high solvent concentrations. AFSCHAR al. (1985) could et maintain a long-term cultivation under stable conditions by combining cell recycling with a two-stage fermentation. At the first stage the growing cells were not exposed to high sol-
vent concentrations which otherwise favor the selection of non-producing strains. SCHLOTE and COTECHALK (1986), on the other hand, observed no change in the activity of the cells over a period of three months in a phosphatelimited chemostat with cell recycling. The special conditions of phosphate limitation and the low pH of 4.3 might have been responsible for this stability of the culture. Industrial application of this technique will depend on the availability of filtration modules that are easy to handle and not affecting the activity of the C. acetobutylicum cells. A wide variety of new membrane types in different configurations (hollow fiber, tubular, flat sheet) are available now which might lead to a more practicable process, in conjunction with a better knowledge of the physiology of C. acetobutylicum in high density suspensions. In Tab. 9 the solvent concentrations, yields, and productivities obtained in different continuous acetone-butanol fermentations with cell recycle or immobilized cells are summarized.
4.3 Product Recovery

The traditional recovery by distillation of the accumulated solvents present in the fermentation broth in relatively low concentrations results in high costs and is one of the major drawbacks to the acetone-butanol fermentation. As long as strains of C. acetobutylicum tolerating higher concentrations of butanol are not available improvement of this fermentation might come from a more economic recovery process. Furthermore, as described above, substantial progress has been made in the development of continuous processes. However, the high productivities of such processes were often accompanied by reduced substrate utilization and low product concentration. These problems might be solved by recycling the fermenter effluent to allow further substrate turnover, and then to avoid product inhibition by an effective product removal technique. Such techniques could include the use of membranes (reverse osmosis, pervaporation), adsorbents, liquid-liquid extraction, and chemical recovery methods. A
254
Tab. 9. Solvent Concentration, Yield, and Productivity in Continuous Acetone-Butanol Fermentations by Clostridium acefobutylicum with Cell Recycle or Immobilization Operation Mode Concentration [g L-'I
1 .00 15.42 3.94 4.10 12.00 13.00 21.71
Yield
[g g-'I
Productivity tg L-I h - l l
0.70 3.02 4.02 4.10 3.00 6.50 2.17
Reference
Immobilized cells Single-stage, complex medium, intermittent feeding Immobilized cells Single-stage, complex medium, steady state Immobilized cells Two-stage, complex medium, steady state Immobilized cells Single-stage, complex medium, steady state Cell recycle Two-stage, synthetic medium, steady state Cell recycle Single-stage, synthetic medium, steady state Cell recycle Single-stage, synthetic medium, phosphate-limited, steady state
0.20 0.34 0.21 0.23 0.30 0.29 0.32
FORBERG et al., 1983
LARGIER et al., 1985 FRICK and SCHUGERL, 1986 QURESHI and MADDOX, 1987 AFSCHAR et al., 1985 PIERROT et al., 1986 SCHLOTE and GOTTSCHALK, 1986
detailed overview of these technologies and their future prospects is given by ENNIS al. et (1986a). Although most of the work aimed at the possible use of alternative solvent recovery processes and is related to ethanol fermentation, there is great interest to apply the results to the acetone-butanol fermentation as well (ENNIS et al., 1986b; DADGAR and FOUTCH, 1985; TAYA al., 1985; GARCIA et I11 et al., 1984, 1986; GROOTet al., 1992). Solvent extraction can be performed either in situ in the fermentation vessel or in a bypass outside the fermenter in a recycle stream of the fermentation broth (LINDEN et al., 1986). Several in situ adsorption systems have been tried in acetone-butanol fermentation. One prerequisite of the extractants used is that they do not affect cell growth and activity. Corn oil, paraffin oil, kerosene, dibutyl phthalate (WANG al., 1979), and oleyl alcoet hol (cis-9-octadecene-1-01) (TAYA et al., 1985) proved to be good extractants for butanol. By automatic withdrawing and feeding
operations of oleyl alcohol regulated by the volume of gas evolved, the glucose concentration used in a fed-batch extractive fermentation system of C. acetobutylicum was 120 g L- ' and the solvent concentration was held below 2 g L-'. The total amount of butanol produced was 20.4 g L-'. Other adsorbents used were activated carbon or silicalite (MADDOX, 1983). 85 mg of butanol could be adsorbed per g silicalite and later released by thermal desorption. The use of a pervaporation system in batch culture resulted in an increase in glucose utilization and in continuous culture with immobilized cells in higher glucose conversion and higher productivity (GROOT et al., 1984a, b). GARCIA et al. (1986) examined the sepI11 aration of butanol from the fermentation broth by reverse osmosis. They concluded that the integration of membrane technology could overcome the problems of low productivity and dilute product concentration associated with the acetone-butanol fermentation. Development in membrane technology
255
sal problem to the dairy industry if it is not further used, e. g., for the manufacture of lactose. The lactose content of whey is 4 5 % (w/v) and C. acetobutylicum is capable of fermenting lactose directly. Depending on the strain, the metabolism of lactose involves either a /?-galactosidase, a phospho-/?-galactosidase, or both enzymes ( Y u et al., 1987;HANCOCK et al., 1991). The relatively low sugar content is unsuitable for many other fermentation processes without prior concentration, but almost optimal for the acetone-butanol fermentation. The amount of sugar that can be utilized by C. acetobutylicum is limited by the product toxicity, especially of butanol. C. acetobutylicum yields the maximum possible solvent concentration with about 6% (w/v) initial fermentable carbohydrate in the medium. Furthermore, it should be mentioned that butanol/acetone ratios after fermentation of 4.4 Alternative Fermentation whey are higher (e.g., 1O:l) as compared to those obtained from starch or molasses (2:l). Substrates For an industrial process this preponderance Starch and molasses were the traditional of butanol is useful not only from the viewraw materials for the biological production of point of the desired product, but it would also acetone and butanol. However, a wide variety simplify the product recovery process. Alof other fermentable sugars from other though not all factors responsible for the shift sources, including wastes, are potential sub- of the butanol/acetone ratio are known, it was strates for this fermentation process. Exam- shown that iron limitation had the greatest efples of alternative resources are apple po- fect on this ratio (BAHLet al., 1986). On the mace containing fructose, glucose, sucrose as other hand, whey is a relatively poor medium. fermentable sugars (VOGET et al., 1985), Reduced productivities compared to molasses whey (lactose) (MADDOXet al., 1994), Jeru- as substrate and incomplete utilization of lacsalem artichokes (fructan) (MARCHAL al., tose are the major problems (MADDOX,1980 et 1984;ENNIS MADand 1985), and lignocellulose (xylan and cellu- WELSHand VELIKY, et lose) (MADDOXand MURRAY, 1983;Y u and DOX, 1985;LINDEN al., 1986). However, an SADDLER, 1983;Yu et al., 1985;FONDet al., optimized fermentation process for the pro1983). Although lignocellulose as raw materi- duction of butanol from whey has been deal is potentially cheap and abundant more scribed recently (MADDOXet al., 1994). The fundamental physiological, biochemical, and improved process includes a fluidized bed genetic research has to be done before it can reactor of bonechar-immobilized cells coube used in an industrial acetone-butanol fer- pled with pervaporation to remove and conmentation process (BAHLand GOTTSCHALK, centrate the solvents. With respect to eco1988). A strain converting these polymers di- nomics the use of such a process would result rectly to solvents, e.g., butanol, would be in a product price of $0.62(US.) per liter for a plant capacity of 900 m3 whey permeate per highly desirable. Whey, on the other hand, seems to be an day based on a whey permeate price of $ 116 (U.S.). In the case that whey permeate can be ideal alternative substrate for acetone-butano1 fermentation and will be considered in obtained at zero cost, the price drops to $0.21 more detail (MADDOXet al., 1994).Whey is a (U.S.). Thus the acetone-butanol fermentaby-product during the manufacture of cheese tion may be one economically viable way of or casein and represents a major waste dispo- producing a useful product from whey.
is one factor influencing the economic feasibility of alternative product recovery processes for large-scale solvent production. Which of the different solvent recovery methods will finally be the most suitable for acetone-butanol fermentation is still an open question. Recently, five technologies for in situ product recovery (stripping, adsorption, liquid-liquid extraction, pervaporation, membrane solvent extraction) have been directly compared using otherwise identical fermentation conditions (GROOT et al., 1992). From these, pervaporation and liquid-liquid extraction were found to have the greatest potentials. MADDOXet al. (1994)investigated four product removal techniques and showed that gas stripping allowed the highest solvent production and productivity.
256
6 Microbial Production of Acetone/ButanoWlsopropanol

)
HzandCOz
Efnuent :ell Concentrate

I
Reverse Osmosis Pervaporation Liquid-LiquidExtraction AdsorptiOll Stripping
Growth
Production
Initiation of Solvent Formation
of
Solvents
4
I
Cell Recyle
Ultrnliltration
+Excess Cell Mass

Reservoir
ultrafillrale Containing
k-Pmjuction
4-
Recovery
Fig. 5. Flow diagram of a continous acetone-butanol fermentation with cell recycle and integrated product recovery.
A combination of the different improved technologies described above will be necessary for an optimized acetone-butanol fermentation. A promising example was given by FRIEDL al. (1991). A stable high solvent et productivity of 3.5 g L-' h-' was obtained using immobilized cells of C. acetobutylicum coupled with product removal by pervaporation. A lactose utilization value of 97.9% was observed at a concentration of 130g lactose L --I in the feed solution, and the solvent yield was remarkedly high (0.39 g solvents g -' lactose). In Fig. 5, a possible flow diagram of an optimized acetone-butanol fermentation is shown taking the recent developments of fermentation technology into account. In most cases the new developments were tested on a laboratory scale. Thus, it will be necessary to determine the economic viability of such an integrated acetone-butanol fermentation process as shown in Fig. 5 on a pilot and production scale.
5 Conclusions
Acetone-butanol fermentation was applied at a large industrial scale for about 40 years. After the last fermentation plants were closed, considerable progress was made regarding the physiology, biochemistry and genetics of Clostridium acetobutylicum. Furthermore, recent developments in technology of continuous fermentation using free or immobilized cells are promising and the integration of sophisticated product removal techniques into these highly productive systems can overcome the severe problem of product inhibition by butanol. Thus, it is possible now to achieve high fermenter productivity, high substrate utilization, high solvent yield, and a high solvent removal rate using continuous acetone-butanol fermentation with an integrated product removal. In addition, based on the progress made in recent years with respect to basic research on C. acetobutylicum it seems to be possible now to construct strains with improved features for a commercial application. In general, the results obtained to
6 References
257
tion, CRC Crit. Rev. Microbiol. 15 (Suppl. l), S33-S67. AZEDDOUG, HUBERT, REYSSET, (1992), H., J., G. Stable inheritance of shuttle vectors based on plasmid pIMl3 in a mutant strain of Clostridium acetobutylicum, J. Gen. Microbiol. 138, 1371Acknowledgement 1378. Work in the authors laboratories has been BAHL,H. (1993), Heat shock response and onset of solvent formation in Clostridium acetobutylicum, supported by grants from the Deutsche Forin: The Clostridia and Biotechnology (WOODS, schungsgemeinschaft and the Bundesminister D. R., Ed.), pp. 247-259, Stoneham: Butterfur Forschung und Technologie. worth-Heinemann. BAHL, H., DURRE, P. (1993), Clostridia, in: Biotechnology, 2nd Edn., Vol. 1 (REHM, H.-J., REED, G., Eds.), pp. 285-323, Weinheim: VCH. G. BAHL,H., GOITSCHALK, (1984), Parameters affecting solvent production by Clostridium acetobutylicum in continuous culture, Biotechnol. Bioeng. Symp. 14, 215-223. AFSCHAR, S., BIEBL,H., SCHALLER, SCHUA. K., G. BAHL,H., GOTTSCHALK, (1988), Microbial proGERL, K. (1985), Production of acetone and buduction of butanollacetone, in: Biotechnology 1st tanol by Clostridium acetobutylicum in continEdn., Vol. 6b (REHM, H.-J., REED,G., Eds.), pp. uous culture with cell recycle, Appl. Microbiol. 1-30, Weinheim: VCH. Biotechnol. 22, 394-398. ALLCOCK, R., WOODS, D. R. (1981), Carboxy- BAHL, H., ANDERSCH,W., BRAUN,K., GOTTE. SCHALK, (1982a), Effect of pH and butyrate G. methyl cellulase and cellobiase production by concentration on the production of acetone and Clostridium acetobutylicum in an industrial ferbutanol by Clostridium acetobutylicum grown in mentation medium, Appl. Environ. Microbiol. continuous culture, Eur. J. Appl. Microbiol. Bio41, 539-541. technol. 14, 17-20. ANDERSCH,W., BAHL, H., GOTTSCHALK,G. (1982), Acetone-butanol production by Clos- BAHL, H., ANDERSCH,W., GOTTSCHALK,G. (1982b), Continuous production of acetone and tridium acetobutylicum in an ammonium-limited butanol by Clostridium acetobutylicum in a twochemostat at low pH values, Biotechnol. Lett. 4, stage phosphate limited chemostat, Eur. J. Appl. 29-32. Microbiol. Biotechnol. 15, 201-205. ANDERSCH,W., BAHL, H., GOTTSCHALK, G. M., (1983), Level of enzymes involved in acetate, bu- BAHL,H., GOTTWALD, KUHN,A., RALE, V., ANDERSCH, GOTTSCHALK, (1986), NuW., G. tyrate, acetone and butanol formation by Clostritional factors affecting the ratio of solvents tridium acetobutylicum, Appl. Microbiol. Bioproduced by Clostridium acetobutylicum, Appl. technol. 18, 327-332. Environ. Microbiol. 52, 169-172. ANDREESEN, R., GOTTSCHALK, (1969), The J. G. J., J., E., occurrence of a modified Entner-Doudoroff BALLONGUE, AMINE, MASION, PETITDEMANGE, GAY, R. (1985), Induction of aceH., pathway in Clostridium aceticum, Arch. Microtoacetate decarboxylase in Clostridium acetobubiol. 69, 16G170. tylicum, FEMS Microbiol. Lett. 29, 273-277. ANNOUS, A., BLASCHEK, P. (1991), Isolation B. H. J., J., E., and characterization of Clostridium acetobutyli- BALLONGUE, AMINE, MASION, PETITDEMANGE, H., GAY, R. (1986), R61e de lacktate et cum mutants with enhanced amylolytic activity, du butyrate dans Iinduction de la NADH:rubreAppl. Environ. MicrobioI. 57, 2544-2548. doxine oxydorCductase chez Clostridium acetoARROYO, (1938), Art of producing butanol and R. butylicum, Biochimic 68,575-580. acetone by fermentation of molasses, U. S. PaBEESCH,S. C. (1948), Process for fermenting cartent 2113471. bohydrates to butanol and acetone, U. s. Patent ARZBERGER, F. (1936), Process for the producC. 2439791. tion of butyl alcohol by fermentation, U. S. PaBEESCH,S. C. (1952), Acetone-butanol fermentatent 2 050 219. tion of sugars, Eng. Proc. Dev. 44,1677-1682. ARZBERGER, F. (1938), Process for the producC. tion of butyl alcohol by fermentation, U. S. Pa- BEHRENS, NARBERHAUS, BAHL,H. (1993), S., F., tent 2 139 108. Cloning, nucleotide sequence and structural AWANG, M., JONES,G. A., INGLEDEW, M. G. W. analysis of the Clostridium acetobutylicum d n d (1988), The acetone-butanol ethanol fermentagene, FEMS Microbiol. Lett. 114, 53-60.
date suggest that an economically viable acetone-butanol fermentation is at least technically possible and could be reintroduced on an industrial scale.
6 References
258
BEIJERINCK, W. (1893), Ueber die ButylalkoM. holgahrung und das Butylferment, Verhand. Kon. Akad. Wetenschappen Amsterdam 2. Sect., Teil 1, Nr. 10, 1-51. BERNDT,H., SCHLEGEL,H. G. (1975). Kinetics and properties of p-ketothiolase from Clostridium pasteurianum, Arch. Microbiol. 103, 21-30. J., BERTRAM, DURRE,P. (1989), Conjugal transfer and expression of streptococcal transposons in Clostridium acetobutylicum, Arch. Microbiol. 151,551-557. BERTRAM, KUHN,A., DURRE,P. (1990), Tn916J., induced mutants of Clostridium acetobutylicum defective in regulation of solvent formation, Arch. Microbiol. 153,373-377. S. BOTKIN, (1892), Ueber einen Bacillus butyricus, Z. Hyg. Infektionskrankh. 1 ,421-434. 1 BOWLES, K., ELLEFSON, L. (1985), Effects of L. W. butanol on Clostridium acetobutylicum, Appl. Environ. Microbiol. 50, 1165-1170. BREDEMANN, (1909), Bacillus amylobacter G. A.M. et Bredemann in morphologischer, physiologischer und systematischer Beziehung. Mit besonderer Berucksichtigung des Stickstoffbindungsvermogens dieser Spezies, Cbl. Bakteriol. Parasitenkd. Infektionskrankh., 11. Abt. 23, 385568. BREZNAK, A,, COSTILOW, N. (1994), PhysicoJ. R. chemical factors in growth, in: Methods for General and Molecular Bacteriology (GERHARDT, P., MURRAY, G. E., WOOD, W. A,, KRIEG, R. N. R., Eds.), pp. 137-154, Washington: Am. SOC. Microbiol. BUCHNER, E., MEISENHEIMER, (1908), Uber J. Buttersauregarung, Ber. Dtsch. Chem. Ges. 41, 1410-1419. BURCHHARDT, DURRE, P. (1990), Isolation G., and characterization of DNase-deficient mutants of Clostridium acetobutylicum, Curr. Microbiol. 21, 307-311. CALAM, T. (1979), Isolation of Clostridium aceC. tobutylicum strains producing butanol and acetone, in: Proc. 4th Symp. Technische Mikrobiologie (DELLWEG, Ed.), pp. 233-242, Berlin: H., Verlag Versuchs- und Lehranstalt fur Spiritusfabrikation und Fermentationstechnologie im Institut fur Garungsgewerbe und Biotechnologie. E. W. CARNARIUS, H., MCCUTCHAN, N. (1938), Process for the production of butyl alcohol by fermentation, U.S. Patent 2139111. D. E. CARY,J. W., PETERSEN, J., PAPOUTSAKIS, T., BENNETT,G. N. (1988), Cloning and expression of Clostridium acetobutylicum phosphotransbutyrylase and butyrate kinase genes in Escherichia coli, J. Bacteriol. 170, 46134618. D. E. CARY,J. W., PETERSEN, J., PAPOUTSAKIS, T., BENNETT, N. (1990), Cloning and expresG.
sion of Clostridium acetobutylicum ATCC 824 acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase in Escherichia coli, Appl. Environ. Microbiol. 56, 15761583. S. CATO, E. P., GEORGE,W. L., FINEGOLD, M. (1986), Genus Clostridium Prazmowski 1880, 23AL,in: Bergeys Manual of Systematic Bacteriology (SNEATH,P. H. A., MAIR,N. S., SHARPE, M. E., HOLT,J. G., Eds.), Vol. 11, pp. 1141-1200, Baltimore: Williams & Wilkins. CHEN,J . 4 . (1993), Properties of acid- and solventforming enzymes of clostridia, in: The Clostridia and Biotechnology (WOODS,D. R., Ed.), pp. 5176, Stoneham: Butterworth-Heinemann. CHEN,J.-S., Hru, S. F. (1986), Acetone-butanolisopropanol production by Clostridium beijerinckii (synonym, Clostridium butylicum), Biotechnol. Lett. 8, 371-376. CHOJECKI, BLASCHEK, P. (1986), Effect of A., H. carbohydrate source on a-amylase and glucoamylase formation by Clostridium acetobutylicum SA1, J. Ind. Microbiol. 1, 63-67. COLBY,G. D., CHEN,J.-S. (1992), Purification and properties of 3-hydroxybutyryl-coenzyme A dehydrogenase from Clostridium beijerinckii (Clostridium butylicum) NRRL B593, Appl. Environ. Microbiol. 58, 3297-3302. CONTAG,P. R., WILLIAMS, G., ROGERS,P. M. (1990), Cloning of a lactate dehydrogenase gene from Clostridium acetobutylicum B643 and expression in Escherichia coli, Appl. Envrron. Microbiol. 56,3760-3765. CYNKIN, A., DELWICHE, A. (1958), MetabolM. E. ism of pentoses by clostridia. I. Enzymes of ribose dissimilation in extracts of Clostridium perfringens, J. Bacteriol. 75, 331-334. CYNKIN, A,, GIBBS,M. (1958), Metabolism of M. pentoses by clostridia. 11. The fermentation of C I4-labeled pentoses by Clostridium perfringens, Clostridium beijerinckii, and Clostridium butylicum, J. Bacteriol. 75, 335-338. DABROCK, BAHL,H., GOTISCHALK, G. (1992), B., Parameters affecting solvent production by Clostridium pasteurianum, Appl. Environ. Microbiol. 58, 1233-1239. DADGAR, M., FOUTCH,G. L. (1985), EvaluaA. tion of solvents for the recovery of Clostridium fermentation products by liquid-liquid extraction, Biotechnol. Bioeng. Symp. 15,611420. M. DAVIES, STEPHENSON, (1941), Studies on R., the acetone-butyl alcohol fermentation. I. Nutritional and other factors involved in the preparation of active suspensions of CI. acetobutylicum (Weizmann), Biochem. J. 35, 132CL1331. J. J. DIEZ-GONZALEZ, HUNTER, B., RUSSELL. F., B. (1994), The acetilactic fermentation of Clostridium acetobutylicum P262, Abstr. Cen.
6 References Meet. Am. SOC. Microbiol., Las Vegas, May 2327, 0-52. DOMBECK, M., INGRAM, 0. (1984), Effects of K. L. ethanol on the Escherichia coli plasma membrane, J. Bacteriol. 157, 233-239. DUCLAUX, (1895), Sur la nutrition intra-celluE. laire, Ann. Inst. Pasteur 9,811-839. DURRE, P. (1993), Transposons in clostridia, in: The Clostridia and Biotechnology (WOODS, D. R., Ed.), pp. 227-246, Stoneham: ButterworthHeinemann. DURRE, P., KUHN, A., GOTTWALD,M., GOTTSCHALK, (1987), Enzymatic investigations on G. butanol dehydrogenase in extracts of Clostridium acetobutylicum, Appl. Microbiol. Biotechnol. 26,268-272. DURRE, P., BAHL, H., GOTTSCHALK, (1992), G. Die Aceton-Butanol-Garung: Grundlage fur einen modernen biotechnologischen ProzeB? Chem. Ing. Tech. 64,491-498. J., R. DYR,J., PROTIVA, PRAUS, (1958), Formation of neutral solvents in continuous fermentation by means of Clostridium acetobutylicum, in: Continuous Cultivation of Microorganisms. A Symposium (MALEK, I., Ed.), pp. 21CL226, Prague: Cechoslovakian Academy of Sciences. EMMERLING, (1897), Butylalkoholische Gah0. rung, Ber. Dtsch. Chem. Ges. 30,451-453. I. ENNIS, M., MADDOX, S. (1985), Use of ClosB. tridium acetobutylicum P 262 for production of solvents from whey permeate, Biotechnol. Lett. 7,503-508. ENNIS,B. M., GUTIERREZ, A,, MADDOX, S. N. I. (1986a), The acetone-butanol-ethanol fermentation: A current assessment, Process Biochem. 21, 131-147. ENNIS,B. M., MARSHALL, T., MADDOX,I. S., C. PATERSON, H. J. (1986b), Continuous prodA. uct recovery by in situ gas strippinglcondensation during solvent production from whey permeate using Clostridium acetobutylicum, Biotechnol. Lett. 8, 725-730. J. L. ENSLEY, MCHUGH, J., BARTON, L. (1975), B., Effect of carbon sources on formation of aamylase and glucoamylase by Clostridium acetobutylicum, J. Gen. Appl. Microbiol. 21, 51-59. FERNBACH, STRANGE, H. (1911), ImproveA,, E. ments in the manufacture of higher alcohols, British Patent 15204. FERNBACH, STRANGE, H. (1912), ImproveA,, E. ments connected with fermentation processes for the production of acetone, and higher alcohols, from starch, sugars, and other carbohydrate materials, British Patent 21 073. FICK,M., PIERROT,P., ENGASSER, M. (1985), J. Optimal conditions for long-term stability of acetone-butanol production by continuous cul-
259
tures of Clostridium acetobutylicum, Biotechnol. Lett. 7, 503-508. FINN, K, NOWREY, E. (1959), A note on the R. J. stability of clostridia when held in continuous culture, Appl. Microbiol. 7, 29-32. FISCHER, J., HELMS, DURRE,P. (1993), ClonR. J., ing, sequencing, and molecular analysis of the sol operon of Clostridium acetobutylicum, a chromosomal locus involved in solventogenesis, J. Bacteriol. 175, 6959-6969. FITZ,A. (1876), Ueber die Gahrung des Glycerins, Ber. Dtsch. Chem. Ges. 9,1348-1352. FITZ, A. (1877), Ueber Schizomyceten-Gahrungen I1 [Glycerin, Mannit, Starke, Dextrin], Ber. Dtsch. Chem. Ges. 10, 276-283. FITZ,A. (1878), Ueber Schizomyceten-Gahrungen 111, Ber. Dtsch. Chem. Ges. 11, 42-55. FITZ, A. (1882), Ueber Spaltpilzgahrungen. VII. Mittheilung, Ber. Dtsch. Chem. Ges. 15, 867880. FOND, O.,PETITDEMANGE, PETITDEMANGE, E., H., ENGASSER, M. (1983), Cellulose fermentaJ. tion by a coculture of a mesophilic Clostridium and Clostridium acetobutylicum, Biotechnol. Bioeng. Symp. 13, 217-224. FORBERG, HAGGSTROM, (1985), Control of C., L. cell adhesion and activity during continuous production of acetone and butanol with adsorbed cells, Enzyme Microb. Techno/. 7, 230-234. FORBERG,C., ENFORS,S. 0.. HAGGSTROM,L. (1983), Control of immobilized, non-growing cells for continuous production of metabolites, Eur. J. Appl. Microbiol. Biotechnol. 17, 143147. FREIBERG, W. (1925), Process for producing G. acetone and butyl alcohol, U. S. Patent 1537597. FREIER, GOTTSCHALK, (1987), L(+)-lactate D., G. dehydrogenase of Clostridium acetobutylicurn is activated by fructose-1,6-bisphosphate,FEMS Microbiol. Lett. 43, 229-233. FREIER-SCHRODER, D., WIEGEL, J., C o n SCHALK, G. (1989), Butanol formation by Clostridium thermosaccharolyticum at neutral pH, Biotechnol. Lett. 11, 831-836. FRENCH, KNAPP, W. (1950), The maltase of D., D. Clostridium acetobutylicum. Its specificity range and mode of action, J. Biol. Chem. 187, 463467. FRICK, SCHUGERL, (1986), Continuous aceC., K. tone-butanol production with free and immobilized Clostridium acetobutylicum, Appl. Microbiol. Biotechnol. 25, 186-193. FRIDOVICH, (1972), Acetoacetate decarboxylase, I. in: The Enzymes (BOYER,P. D., Ed.,), Vol. VI, pp. 255-270, New York: Academic Press.
260
FRIEDL,A., QURESHI, MADDOX,I. S. (1991), GOTTSCHALK,G. (1986). Bacterial Metabolism, N., 2nd Edn., New York: Springer-Verlag. Continuous acetone-butanol-ethanol (ABE) fermentation using immobilized cells of Clostrid- GOTTWALD, GOTTSCHALK, (1985), The inM., G. ium acetobutylicum in a packed bed reactor and ternal pH of Clostridium acetobutylicum and its integration with product removal by pervaporaeffect on the shift from acid to solvent formation, Biotechnol. Bioeng. 38, 518-527. tion, Arch. Microbiol. 143, 42-46. FUNK,F. J. (1925), Butyl alcohol and acetone fer- GOTTWALD,M., HIPPE, H., GOTTSCHALK,G. (1984), Formation of n-butanol from o-glucose mentation process, U.S. Patent 1538516. by strains of the Clostridium tetanomorphunz GABRIEL, L. (1928). Butanol fermentation proC. group, Appl. Environ. Microbiol. 48, 573-576. cess, Ind. Eng. Chem. 28, 1063-1067. A. GARCIA111, A., IANNOTTI, L., FISHER,J. R. GRETHLEIN, J., WORDEN,R. M., JAIN,M. K., E. DATTA, R. (1991), Evidence for production of (1984), Reverse osmosis application for acen-butanol from carbon monoxide by Butyribactone-butanol fermentation, Biotechnol. Bioeng. terium methylotrophicum, J. Ferment. Bioeng. 72, Symp. 14,543-552. 58-60, E. GARCIA111, A., IANNOTTI, L., FISHER,J. R. GRIMBERT, (1893), Fermentation anakrobie proL. (1986). Butanol fermentation liquor production duite par le Bacillus orthobutylicus. ses variaand separation by reverse osmosis, Biotechnol. tions sous certaines influences biologiques, Ann. Bioeng. 28,785-791. Inst. Pasteur 7, 353-402. GAVARD, HAUTECOEUR, DESCOURTIEUX, R., B., G. H. (1957), Phosphotransbutyrylase de Clostrid- GROOT, W. J., SCHOUTENS, H., VAN BEELEN, P. N., VAN DEN OEVER,c. E., KOSSEN, W. F. N. ium acetobutylilcum, C. R. Acad. Sci. 244, 2323(1984a), Increase of substrate conversion by per2326. vaporation in the continuous butanol fermentaGEORGE, A., JOHNSON, L, MOORE,W. E. C., H. J. tion, Biotechnol. Lett. 6, 789-792. HOLDEMAN, V., CHEN,J. S. (1983), Acetone, GROOT,w. J., VAN DEN OEVER, c. E., KOSSEN, L. N. isopropanol, and butanol production by ClosW. F. (1984b), Pervaporation for simultaneous tridium bejierinckii (syn. Clostridium butylicum) product recovery in the butanol/isopropanol and Clostridium aurantibutyricum, Appl. Envibatch fermentation, Biotechnol. Lett. 6, 709ron. Microbiol. 45, 1160-1163. 714. GERISCHER, DURRE, P. (1990), Cloning, se- GROOT,w. VAN DER LANS,R. G. J. M., LUYBU., J., quencing, and molecular analysis of the acetoEN, CH. A. M. (1992), Technologies for butanol acetate decarboxylase gene region from Closrecovery integrated with fermentations, Process tridium acetobutylicum, J. Bacteriol. 172, 6907Biochem. 27, 61-75. 6918. GRUBER,M. (1887), Eine Methode der Cultur GERISCHER, DURRE,P. (1992). mRNA analyU., anaerobischer Bacterien nebst Bemerkungen sis of the adc gene region of Clostridium acetozur Morphologie der Buttersauregahrung, Cbl. butylicum during the shift to solventogenesis, J. Bacteriol. Parasitenkd. 1,367-372. Bacteriol. 174, 426433. GRUPE,H., GOTTSCHALK, (1992), Physiological G. GOODLOVE, E., CUNNINGHAM,R., PARKER, P. P. events in Clostridium acetobutylicum during the J., CLARK, P. (1989), Cloning and sequence D. shift from acidogenesis to solventogenesis in analysis of the fermentative alcohol-dehydrocontinuous culture and presentation of a model genase-encoding gene of Escherichia coli, Gene for shift induction, Appl. Environ. Microbiol. 58, 85,209-214. 38963902. GOTTSCHAL, C., MORRIS, G. (1981a), The in- HAGGSTROM,L., ENFORS,S. 0. (1982), ContinJ. J. duction of acetone and butanol production in uous production of butanol with immobilized cultures of Clostridium acetobutylicum by elecells of Clostridium acetobutylicum, Appl. Biovated concentrations of acetate and butyrate, chem. Biotechnol. 7, 35-37. FEMS Microbiol. Lett. U , 385-389. HANCOCK, R., ROCKMAN, YOUNG,C. A., K. E., GOTTSCHAL, C., MORRIS, G. (1981b), NonproJ. J. PEARCE, L., MADDOX, I. S., Scorn, D. B. duction of acetone and butanol by Clostridium (1991), Expression and nucleotide sequence of acetobutylicum during glucose- and ammoniumthe Clostridium acetobutylicum P-galactosidase limitation in continuous culture, Biotechnol. gene cloned in Escherichia coli, J. Bacteriol. 173, Lett. 3, 525-530. 3084-3095. .. J. GOTTSCHAL, IC., MORRIS, G. (1982), Contin- HANSON, M., RODGERS, E. (1946), Influence N. A. uous production of acetone and butanol by Closof iron concentration and attenuation on the metridium acetobutylicum growing in turbidostat tabolism of Clostridium acetobutylicum, J. Bacteculture, Biotechnol. Lett. 4, 477-482. riol. 51, 568-569.
6 References
261
HARRIS, MULDER,R., KELL, D. B., WALTER, HUSEMANN, H. W., PAPOUTSAKIS, T. (1989), J., M. E. R. P., MORRIS, G. (1986), Solvent production J. Comparison between in vivo and in vitro enzyme by Clostridium pasteurianum in media of high activities in continuous and batch fermentations sugar content, Biotechnol. Lett. 8,889-892. of Clostridium acetobutylicum, Appl. Microbiol. HARTMANIS, G. N. (1987), Butyrate kinase M. Biotechnol. 30, 585-595. from Clostridium acetobutylicum, J. Biol. Chem. HUTKINS, W., KASHKET, R. (1986), PhosphoR. E. 262,617421. transferase activity in Clostridium acetobutyliS. HARTMANIS, G. N., GATENBECK, (1984), InM. cum from acidogenic to solventogenic phases of termediary metabolism in Clostridium acetobugrowth, Appl. Environ. Microbiol. 51, 11211123. tylicum: Levels of enzymes involved in the formation of acetate and butyrate, Appl. Environ. INGRAM, 0. (1986), Microbial tolerance to alcoL. Microbiol. 47, 1277-1283. hols: Role of the cell membrane, Trends BiotechHARTMANIS, G. N., KLASON, GATENBECK, M. T., nol. 4, 40-44. S. (1984), Uptake and activation of acetate and IZSAK, FUNK, J. (1933), Process of producing A., F. butyrate in Clostridium acetobutylicum, Appl. butyl alcohol, U. S. Patent 1908361. Microbiol. Biotechnol. 20, 66-71. JANATI-IDRISSI, JUNELLES, M., EL KANOUR., A. J. HASTINGS, J. H. (1978). Acetone-butyl alcohol NI,A., PETITDEMANGE, GAY,R. (1987), SeH., fermentation, in: Economic Microbiology, Vol. lection de mutants de Clostridium acetobutyli2. Primary Products of Metabolism (ROSE, A. cum defectifs dans la production dacktone, Ann. H., Ed.), pp. 3145, New York: Academic Inst. Pasteur/Microbiol. 138, 313-323. Press. JOBSES,I. M. L., ROELS,J. A. (1983), Experience HERMANN, FAYOLLE, MARCHAL, PODM., F., R., with solvent production by Clostridium beijerVIN, L., SEBALD,M., VANDECASTEELE, J.-P. inckii in continuous culture, Biotechnol. Bioeng. (1985), Isolation and characterization of buta25, 1187-1194. nol-resistant mutants of Clostridium acetobutyli- JONES,D. T., WOODS,D. R. (1986), Acetone-butacum, Appl. Environ. Microbiol. 50, 1238-1243. no1 fermentation revisited, Microbiol. Rev. 50, HERRERO, A. (1983), End-product inhibition in A. 484-524. anaerobic fermentations, Trends Biotechnol. 1, JUNGERMANN, THAUER, K., LEIMENSTOLL, K., R. 49-53. G., DECKER, (1973), Function of reduced pyK. HIGGINS, F. (1992), ABC transporters from miC. ridine nucleotide-ferredoxin oxidoreductases in croorganisms to man, Annu. Rev. Cell Biol. 8, saccharolytic clostridia, Biochim. Biophys. Acta 67-113. 305, 268-280. E. HIPPE, H., ANDREESEN, R., GOTTSCHALK, J. G. KASHKET, R., CAO, Z.-Y. (1993), Isolation of a (1992), The genus Clostridium - nonmedical, in: degeneration-resistant mutant of Clostridium The Prokaryotes, 2nd Edn. (BALOWS, TRUA., acetobutylicum NCIMB 8052, Appl. Environ. PER, H. G., DWORKIN, M., HARDER, w . , Microbiol. 59, 41984202. SCHLEIFER, K.-H., Eds.), Vol. 11, pp. 18W1866, KATAGIRI, IMAI,K., SUGIMARI, (1960), On H., T. New York: Springer-Verlag. the metabolism of organic acids by Clostridium HIU, S. F., ZHU, C.-Z., YAN, R.-T., CHEN, J . 4 . acetobutylicum. Part I. Formation of lactic acid and racemase, Bull. Agr. Chem. SOC. Jpn. 24, (1987), Butanol-ethanol dehydrogenase and butanol-ethanol-isopropanoldehydrogenase: Dif163-172. ferent alcohol dehydrogenases in two strains of KEIS,S., BENNETT, JONES,D. T. (1994), DifferC., Clostridium beijerinckii (Clostridium butylicum), entiation of solvent producing clostridial strains Appl. Environ. Microbiol. 53, 697-703. by restriction analysis typing, ribotyping and HOCKENHULL, J., HERBERT,D. (1945), The D. phage typing, Abstr. 7th Int. Symp. Genet. Inn. amylase and maltase of Clostridium acetobutyliMicroorg., Montreal, June 26-July 1, p. 201. cum, Biochem. J. 39, 102-106. KEMPNER, KUBOWITZ, (1933), Wirkung des W., F. Lichtes auf die Kohlenoxydhemmung der ButHOLT, R. A., STEPHENS, M., MORRIS,J. G. G. tersauregarung, Biochem. Z. 265, 245-252. (1984), Production of solvents by Clostridium acetobutylicum cultures maintained at neutral KIM,A. Y., BLASCHEK, P. (1991), Isolation and H. pH, Appl. Environ. Microbiol. 48, 1166-1170. characterization of a filamentous viruslike parHOLT, R. A,, CAIRNS, J., MORRIS, G. (1988), A. J. ticle from Clostridium acetobutylicum NCIB Production of butanol by Clostridium puniceum 6444, J. Bacteriol. 173, 530-535. in batch and continuous culture, Appl. Micro- KIM,A. Y., BLASCHEK, P. (1993), Construction H. biol: Biotechnol. 27, 319-324. and characterization of a phage-plasmid hybrid (phagemid), pCAK1, containing the replicative HONGO,M. (1960), Process for producing butanol by fermentation, U. S. Patent 2945786. form of viruslike particle CAKl isolated from
262
evaluation of the acetone-butanol fermentation, Clostridium acetobutylicum NCIB 6444, J. BacteInd. Eng. Chem. Prod. Res. Dev. 19,478-483. riol. 175, 3838-3843. B. K. KIM,B. H., ZEIKUS, G. (1985), Importance of hy- LESKIW, K., BIBB,M. J., CHATER, F. (1991), J. The use of a rare codon specifically during dedrogen metabolism in regulation of solventogenvelopment? Mol. Microbiol. 5, 2861-2867. esis by Clostridium acetobutylicum, Dev. Ind. LEUNG, C. Y., WANG,D. I. C. (1981), Production J. Microbiol. 26, 549-556. of acetone and butanol by Clostridium acetobuKIM,A. Y., ATTWOOD, T., HOLT,S. M., WHITE, G. tylicum in continuous culture using free and imB. A,, BLASCHEK, P. (1994), Heterologous H. mobilized cells, Proc. 2nd World Congr. Cham. expression of endo-@1,4-~-ghcanase from closEng. 1,348-352. tridium cellulovorans in Clostridium acetobutyliH. cum ATCC 824 following transformation of the LIN,Y.-L., BLASCHEK, P. (1983), Butanol production by a butanol-tolerant strain of ClostridengB gene, Appl. Environ. Microbiol. 60,337ium acetobutylicum in extruded corn broth, 340. Appl. Environ. Microbiol. 45, 966-973. KUBOWITZ, (1934), Uber die Hemmung der ButF. H. tersauregarung durch Kohlenoxyd, Biochem. 2. LIN,Y.-L., BLASCHEK, P. (1984), Transformation of heat-treated Clostridium acetobutylicum 274,285-298. protoplasts with pUBllO plasmid DNA, Appl. KUTZENOK, ASCHNER, (1952), DegeneraA., M. Environ. Microbiol. 48, 737-742. tive processes in a strain of Clostridium butyliLINDEN, C., MOREIRA, R., LENZ, T. G. J. A. cum, J. Bacteriol. 64, 829-836. (1986), Acetone and butanol, in: Comprehensive S. J. T. LAPAGE, P., SHELTON, E., MITCHELL, G., Biotechnology. The Principles of Biotechnology; MACKENZIE, R. (1970), Culture collections A. Engineering Consideration (COONEY, C. L., and the preservation of bacteria, in: Methods in HUMPHREY, E., Eds.), pp. 915-931, Oxford: A. Microbiology (NORRIS, R., RIBBONS, W., J. D. Pergamon Press. Eds.), Vol. 3 A, pp. 125-228, London: Academic LONG,S., JONES, D. T., WOODS, D. R. (1984a), Press. The relationship between sporulation and solLARGIER, T., LONG, S., SANTANGELO, D., S. J. vent production in Clostridium acetobutylicum JONES,D. T., WOODS,D. R. (1985), ImmobilP262, Biotechnol. Lett. 6, 529-534. ized Clostridium acetobutylicum P 262 mutants for solvent production, Appl. Environ. Micro- LONG,S., JONES,D. T., WOODS,D. R. (1984b), Initiation of solvent production, clostridial stage biol. 50, 477-481. and endospore formation in Clostridium acetoLEE, S. F., FORSBERG, W., GIBBINS,L. N. C. butylicum P262, Appl. Microbiol. Biotechnol. 20, (1985a), Xylanolytic activity of Clostridium ace256261. tobutylicum, Appl. Environ. Microbiol. 50,10681076. LOUGHLIN, F. (1935), Production of butyl alcoJ. hol and acetone by fermentation, U. S. Patent LEE, S. F., FORSBERG, W., GIBBINS, N. C. L. 1992 921. (1985b), Cellulytic activity of Clostridium acetobutylicum, Appl. Environ. Microbiol. 50, 220MADDOX,I. S. (1980), Production of n-butanol from whey filtrate using Clostridium acetobutyli228. cum NCIB 2951, Biotechnol. Lett. 2,493498. LEE, S. F., FORSBERG, W., RATTRAY, B. C. J. (1987), Purification and characterization of two MADDOX, S. (1983), Use of silicalite for the adI. sorption of n-butanol from fermentation liquors, endoxylanases from Clostridium acetobutylicum ATCC 824, Appl. Environ. Microbiol. 53, 664Biotechnol. Lett. 5, 89-94. 650. I. A. MADDOX, S., MURRAY, E. (1983), Production LEE, S. Y., BENNETT, N., PAPOUTSAKIS, T. G. E. of n-butanol by fermentation of wood hydroly(1992), Construction of Escherichia coli-Clossate, Biotechnol. Lett. 5, 175-178. tridium acetobutylicum shuttle vectors and trans- MADDOX, S., QURESHI, GUTIERREZ, A. I. N., N. formation of Clostridium acetobutylicum strains, (1994), Utilization of whey by clostridia and Biotechnol. Lett. 14, 427432. process technology, in: The Clostridia and BioLEGG,D. A., STILES,H. R. (1937), Process of protechnology (WOODS, D. R., Ed.), pp. 343-369, ducing butyl alcohol, U. s. Patent 2089562. Stoneham: Butterworth-Heinemann. S R., D., LEMMEL, . A. (1985), Mutagenesis in Clostridium MARCHAL, BLANCHET, VANDECASTEELE, acetobutylicum, Biotechnol. Lett. 7, 711-716. J. P. (1985), Industrial optimization of acetonebutanol fermentation: A study of the utilization S. J. LEMMEL, A., DATTA, R., FRANKIEWICZ, R. of Jerusalem artichokes, Appl. Microbiol. Bio(1986), Fermentation of xylan by Clostridium technol. 23,92-98. acetobutylicum, Enzyme Microb. Technol. 8, 217-221. MARCZAK,R., PETITDEMANGE, ALIMI, F., H., BALLONGUE, GAY, R. (1983), Influence de R., LENZ,T. G., MOREIRA, R. (1980), Economic A.
6 References
263
la phase de croissance et de la composition du ATCC 824 for increased solvent production by milieu sur le taux de biosynthkse de la enhancement of acetone formation enzyme acNADH:rubrCdoxine oxydorCductase chez Clostivities using a synthetic acetone operon, Biotridium acetolbutylicum, C. R. Acad. Sci., Ser. 3 technol. Bioeng. 42, 1053-1060. 296,469474. MICHELS, (1990), Reinigung der NADP +-abhanJ. MARCZAK, BALLONGUE, PETITDEMANGE, gigen Butanol-Dehydrogenase aus Clostridium R., R., H., GAY,R. (1984), Regulation of the biosyntheacetobutylicum iiber Triazinyl-Farbstoffe, Thesis, sis of NADH-rubredoxin oxidoreductase in University of Gottingen. Clostridium acetobutylicum, Curr. Microbiol. 10, MINTON,N. P., BREHM,J. K., SWINFIELD, T.-J., 165-1 68. M. WHELAN,S. M., MAUCHLINE, L., BODSMARCZAK, BALLONGUE, PETITDEMANGE, WORTH, N., OULTRAM, D. (1993), Clostridial R., J., J. H., GAY, R. (1985), Differential levels of ferrecloning vectors, in: The Clostridia and Biotechdoxin and rubredoxin in Clostridium acetobutylinology (WOODS, D. R., Ed.), pp. 119-150, cum, Biochimie 67,241-248. Stoneham: Butterworth-Heinemann. MCCLUNG, S., McCoy, E. (1935), Studies on MITCHELL, J. (1992), Carbohydrate assimilation L. W. by saccharolytic clostridia, Res. Microbiol. 143, anaerobic bacteria. VII. The serological relations of Clostridium acetobutylicum, Cl. felsi245-250. neum and Cl. roseurn, Arch. Mikrobiol. 6, 239MITCHELL, W. J., SHAW, J. E., ANDREWS,L. 249. (1991), Properties of the glucose phosphotransferase system of Clostridium acetobutylicum McCoy, E. F. (1946), Production of butyl alcohol NCIB 8052, Appl. Environ. Microbiol. 57,2534and acetone by fermentation, U. S Patent . 2539. 2398837. J. McCoy, E., MCCLUNG, S. (1935), Studies on MONOT,F., ENGASSER, M. (1983), Production of L. acetone and butanol by batch and continuous anaerobic bacteria. VI. The nature and systematculture of Clostridium acetobutylicum under niic position of a new chromogenic Clostridium, trogen limitation, Biotechnol. Lett. 5, 213-218. Arch. Mikrobiol. 6, 230-238. H., McCoy, E., FRED,E. B., PETERSON, H., HAST- MONOT, F., MARTIN,J. R., PETITDEMANGE, W. GAY, R. (1982), Acetone and butanol producI N G ~ , G. (1926), A cultural study of the aceE. tion by Clostridium acetobutylicum in a synthetic tone butyl alcohol organism, J. Infect. Dis. 39, medium, Appl. Environ. Microbiol. 44, 1318457483. 1324. MCNEIL,B., KRISTIANSEN, (1986), The acetone B. J. H. butanol fermentation, Adv. Appl. Microbiol. 31, MONOT,F., ENGASSER, M., PETITDEMANGE, (1983), Regulation of acetone butanol produc61-92. tion in batch and continuous cultures of ClosMEINECKE,B., BAHL, H., GOTTSCHALK,G. tridium acetobutylicum, Biotechnol. Bioeng. (1984). Selection of an asporogenous strain of Symp. 13, 207-216. Clostridium acetobutylicum in continuous culA. ture under phosphate limitation, Appl. Environ. MOREIRA, R. (1983), Acetone-butanol fermentation, in: Organic Chemicals from Biomass Microbiol. 48, 1064-1065. (WISE, D. L., Ed.), pp. 385-406, Menlo Park: MEINECKE, BERTRAM, GOTTSCHALK, B., J., G. BenjaminlCummins Publ. Co., Inc. (1989), Purification and characterization of the A. J. pyruvate-ferredoxin oxidoreductase from Clos- MOREIRA, R., ULMER,D. C., LINDEN, C. (1981), Butanol toxicity in the butylic fermentatridium acetobutylicum, Arch. Microbiol. 152, tion, Biotechnol. Bioeng. Symp. 11, 567-579. 244-250. J. MERMELSTEIN, D., PAPOUTSAKIS, T. (1993), MULLER, (1938a), Production of neutral solvents L. E. by fermentation, U. S Patent 2132039. . In vivo methylation in Escherichia coli by the MULLER,J. (1938b). Fermentation mash, ( . S Pa1 . Bacillus subtilis phage +3T I methyltransferase tent 2 123078. to protect plasmids from restriction upon transG. E. formation of Clostridium acetobutylicum ATCC NAIR,R. V., BENNETT, N., PAPOUTSAKIS, T. (1994), Molecular characterization of an alde824, Appl. Environ. Microbiol. 59, 1077-1081. hydelalcohol dehydrogenase gene from ClostridMERMELSTEIN, D., WELKER, E., BENNETT, L. N. ium acetobutylicum ATCC 824, J. Bacteriol. 176, G. N., PAPOUTSAKIS, T. (1992), Expression E. 87 1-885. of cloned homologous fermentative genes in S., S. Clostridium acetobutylicum ATCC 824, Bio/ NAKAMURA, OKADO,I., ABE, T., NISHIDA, (1979), Taxonomy of Clostridium tetani and reTechnology 10, 190-195. lated species, J. Gen. Microbiol. 113, 29-35. MERMELSTEIN, D., PAPOUTSAKIS, T., PEL. E. T., IGARASHI,Y., KODAMA, T. TERSEN, J., BENNETT, N. (1993), Meta- NAKAMURA, D. G. (1990), Acetoacetyl-CoA transferase and thiolbolic engineering of Clostridium acetobutylicum
264
acetobutylicum NCIMB 8052, Gene 131, 107ase reactions are catalyzed by a single enzyme in 112. Clostridium acetobutylicum ATCC824, Agr. Biol. Chem. 54,276-268. W. OXFORD, E., LAMPEN, O., PETERSON, H. A. J. (1940), 190. Growth factor and other nutritional NARBERHAUS, BAHL,H. (1992), Cloning, seF., requirements of the acetone butanol organism, quencing, and molecular analysis of the groESL Cl. acetobutylicum, Biochem. J. 34, 1588-1597. operon of Clostridium acetobutylicum, J. Bacteri01. 174, 3282-3289. PALOSAARI, R, ROGERS, (1988), Purification N. P. NARBERHAUS, GIEBELER, BAHL, (1992), F., K., H. and properties of the inducible coenzyme AMolecular characterization of the dnaK gene relinked butyraldehyde dehydrogenase from Closgion of Clostridiuin acetobutylicum, including tridium acetobutylicum, J. Bacteriol. 170, 2971grpE, d n d , and a new heat shock gene, J. Bacte2976. rial. 174, 3290-3299. PAPA,A. J. (1982), Isopropyl alcohol, in: KirkNELSON, L., WEBB,B. P. (1978), Acetone, in: D. Othmer Encyclopedia of Chemical Technology, Kirk-Othmer Encyclopedia of Chemical Tech3rd Edn. (MARK, F., OTHMER, F., OVERH. D. nology, 3rd Edn. (MARK, F., OTHMER, F., H. D. M., BERGER, G., SEABORG, T., GRAYSON, C. G. OVERBERGER, G., SEABORG, T., GRAYC. G. ECKROTH, Eds.), Vol. 19, pp. 198-220, New D., SON, M., ECKROTH, Eds.), Vol. 1, pp. 179D., York: John Wiley & Sons. 192, New York: John Wiley & Sons. PAQUET, CROUX, GOMA,G., SOUCAILLE, V., C., OGATA, HONGO, (1979), Bacteriophages of S., M. P. (1991), Purification and characterization of the genus Clostridium, Adv. Appl. Microbiol. 25, the extracellular cr-amylase from Clostridium 241-273. acetobutylicum, Appl. Environ. Microbiol. 57, 0. C. OSBURN, L., WERKMAN, H. (1935), Utiliza212-218. tion of agricultural wastes. 11. Influence of nitro- PASTEUR, (1861a), Animacules infusoires vivant L. genous substrate on production of butyl and isosans gaz oxygkne libre et determinant des ferpropyl alcohols by Clostridium butylicum, Ind. mentations, C. R. Hebd. Stances Acad. Sci. 52, Eng. Chem. 27,416419. 344-347. OULTRAM, D., YOUNG,M. (1985), Conjugal PASTEUR, (1861b). Expiriences et vues nouJ. L. transfer of plasmid pAMbl from Streptococcus velles sur la nature des fermentations, C. I?. lactis and Bacillus subtilis to Clostridium acetoHebd. Stances Acad. Sci. 52, 1260-1264. butylicum, FEMS Microbiol. Lett. 27, 129-134. L. PASTEUR, (1862), Quelques rksultats nouveaux OULTRAM, D., DAVIES, YOUNG, (1987), J. A., M. relatifs aux fermentations acetique et butyrique, Conjugal transfer of a small plasmid from BacilBull. SOC. Chim. Paris Mai 1862,52-53. lus subtilis to Clostridium acetobutylicum by L. cointegrate formation with plasmid pAMP1, PASTEUR, (1863), Recherches sur le putrkfaction, C. R. Hebd. Stances Acad. Sci. 56, 1189FEMS Microbiol. Lett. 42, 113-119. 1194. OULTRAM,D., LOUGHLIN, SWINFIELD, J. M., T.-J., PERDRIX, (1891), Sur les fermentations proL. D. N. BREHM, K., THOMPSON, E., MINTON, P. J. duites par un microbe anaerobie de leau, Ann. (1988a), Introduction of plasmids into whole Inst. Pasteur 5, 287-311. cells of Clostridium acetobutylicum by electropoPETERSEN, (1991), Untersuchungen zur LipaseM. ration, FEMS Microbiol. Lett. 56, 83-88. Sekretion von Clostridium tetanomorphum und OULTRAM, D., PECK,H., BREHM, K., THOMPJ. J. Reinigung des lipolytisch aktiven Enzyme, TheSON, D. E., SWINFIELD, J., MINTON, T. N. P. sis, University of Gottingen. (1988b), Introduction of genes for leucine bioD. G. synthesis from Clostridium pasteurianum into C. PETERSEN, J., BENNETT, N. (1990), Purification of acetoacetate decarboxylase from Closacetobutylicum by cointegrate conjugal transfer, tridium acetobutylicum ATCC 824 and cloning Mol. Gen. Genet. 214, 177-179. of the acetoacetate decarboxylase gene in OULTRAM, D., SCHIMMING, WALMSLEY, J. S., R., LOUGHLIN, M., SCHWARTZ, W., STAUDEN- Escherichia coli, Appl. Environ. Microbiol. 56, 3491-3498. BAUER, W. L., MINTON, P. (1990), IntroducN. D. G. tion of cellulase (cel) genes from Clostridium PETERSEN, J., BENNETT, N. (1991), Cloning of the Clostridium acetobutylicum ATCC 824 thermocellum into Clostridium acetobutylicum, acetyl coenzyme A acetyltransferase (thiolase; Abstr. 6th Int. Symp. Genet. Ind. Microorg., EC 2.3.1.9) gene, Appl. Environ. Microbiol. 57, Strasbourg, August 12-18, C 54. 2735-2741. OULTRAM, D., BURR,I. A., ELMORE, J., J. M. D. MINTON,N. P. (1993), Cloning and sequence PETERSEN, J., WELCH, W., RUDOLPH, B., R. F. analysis of the genes encoding phosphotransbuBENNETT, N. (1991), Molecular cloning of an G. tyrylase and butyrate kinase from Clostridium alcohol (butanol) dehydrogenase gene cluster
6 References
265
from Clostridium acetobutylicum ATCC 824, J. from streptococci to Clostridium acetobutylicum, Bacteriol. 173, 1831-1834. Ann. Inst. Pasteur/Microbiol. lMB, 275-282. PETERSEN, J., CARY, W., VANDERLEYDEN, D. J. REYSSET, SEBALD, (1993), Transformation/ G., M. J., BENNETT, N. (1993), Sequence and arG. electrotransformation of clostridia, in: The Closrangement of genes encoding enzymes of the tridia and Biotechnology (WOODS,D. R., Ed.), acetone-production pathway of Clostridium acepp. 151-156, Stoneham: Butterworth-Heinetobutylicum ATCC 824, Gene 123, 93-97. mann. PETITDEMANGE, CHERRIER, RAVAL,G., REYSSET, HUBERT, PODVIN, SEBALD, H., C., G., J., L., M. GAY,R. (1976), Regulation of the NADH and (1988), Transfection and transformation of ClosNADPH-ferredoxin oxidoreductases in clostridtridium acetobutylicum strain NI-4081 protoia of the butyric group, Biochim. Biophys. Acta plasts, Biotechnol. Techniques 2, 199-204. 421,334-347. ROGERS, (1986), Genetics and biochemistry of P. PETITDEMANGE, CHERRIER, BENGONE, H., C., J. Clostridium relevant to development of fermenM., GAY,R. (1977), Etude des activitks NADH tation processes, Adv. Appl. Microbiol. 31, 1et NADPH-ferrkdoxine oxydorkductasiques 60. chez Clostridium acetobutylicum, Can. J. Micro- ROGERS, GOTISCHALK, (1993), BiochemisP., G. biol. 23, 152-160. try and regulation of acid and solvent producPETITDEMANGE, MARCZAK, BLUSSON, H., R, H., tion in clostridia, in: The Clostridia and BiotechGAY,R. (1979), Isolation and properties of renology (WOODS, R., Ed.), pp. 25-50, StoneD. duced nicotinamide adenine dinucleotide-rubreham: Butterworth-Heinemann. doxin oxidoreductase of Clostridium acetobutyli- ROGERS, PALOSAARI, (1987), Clostridium P., N. cum, Biochem. Biophys. Res. Commun. 4, 1258acetobutylicum mutants that produce butyralde1265. hyde and altered quantities of solvents, Appl. PICH, BAHL, (1991), Purification and characA., H. Environ. Microbiol. 53, 2761-2766. terization of the DNA-dependent RNA poly- Roos, J. W., MCLAUGHLIN, PAPOUTSAKIS, E. J., merase from Clostridium acetobutylicum, J. BacT. (1984), The effect of pH on nitrogen supply, teriol. 173, 2120-2124. cell lysis, and solvent production in fermentaPICH,A., NARBERHAUS, BAHL,H. (1990), InF., tions of Clostridium acetobutylicum, Biotechnol. duction of heat shock proteins during initiation Bioeng. 27, 681-694. of solvent formation in Clostridium acetobutyli- Ross, D. (1961), The acetone-butanol fermentacum, Appl. Microbiol. Biotechnol. 33,697-704. tion, Prog. Ind. Microbiol. 3, 73-85. PIERROT, FICK,M., ENGASSER, M. (1986). RUBBO, D., MAXWELL, FAIRBRIDGE, A,, P., J. S. M., R. Continuous acetone-butanol fermentation with GILLESPIE, M. (1941), The bacteriology, J. high productivity by cell ultrafiltration and cell growth factor requirements and fermentation recycling, Biotechnol. Lett. 8, 253-256. reactions of Clostridium acetobutylicum (WeizS. C. PRESCOTT, G., DUNN, G. (1959). Industrial mann), Aust. J. Exp. Biol. Med. 19, 185-197. Microbiology, 3rd Edn., New York: McGraw- SANTANGELO, JONES, T., WOODS, R. J. D., D. D. Hill Book Co. (1991), Metronidazole activation and isolation of PRINGSHEIM, H. (1906a), Ueber den Ursprung H. Clostridium acetobutylicum electron transport des Fuselols und eine Alkohole bildende Bakgenes, J. Bacteriol. 173, 1088-1095. terienform, Cbl. Bakteriol. Parasitenkd. Infek- SANTANGELO, D., DURRE,P., WOODS,D. R. J. tionskrankh., 11. Abt. 15, 3W321. (1994), Characterization and expression of the PRINGSHEIM, (1906b), Ueber ein Stickstoff asH. hydrogenase I gene from Clostridium acetobutysimilierendes Clostridium, Cbl. Bakteriol. Parasilicum P262, Abstr. 7th Int. Symp. Genet. Ind. Mitenkd. Infektionskrankh., 11. Abt. 15, 795-800. croorg., Montreal, June 26-July l, p. 96. J., G. I. QURESHI, MADDOX, S. (1987), Continuous SASS,C., WALTER, BENNETT, N. (1993), IsoN., solvent production from whey permeate using lation of mutants of Clostridium acetobutylicum cells of Clostridium acetobutylicum immobilized ATCC 824 deficient in protease activity, Curr. by adsorption onto bonechar, Enzyme Microb. Microbiol. 26, 151-154. Technol. 9, 668-671. SAUER, DURRE,P. (1992), Possible function of U., tRNAT& in regulation of solvent formation in REID, S. J., ALLCOCK,E. R., JONES, D. T., WOODS, R. (1983), Transformation of ClosD. Clostridium acetobutylicum, FEMS Microbiol. tridium acetobutylicum protoplasts with bacteLett. 100, 147-154. riophage DNA, Appl. Environ. Microbiol. 45, SAUER, DURRE,P. (1993), Sequence and moU., 305-307. lecular characterization of a DNA region encodREYSSET, SEBALD, (1985), Conjugal transG., M. ing a small heat shock protein of Clostridium fer of plasmid-mediated antibiotic resistance acetobutylicum, J. Bacteriol. 175, 3394-3400.
266

Clostridium acetobutylicum, J. Ferment. Technol. 63, 181-187. THAUER,R. K., JUNGERMANN, DECKER,K. K., (1977), Energy conservation in chemotrophic anaerobic bacteria, Bacteriol. Rev. 41, 100-180. D. THOMPSON, K., CHEN,J.3. (1990), Purification and properties of an acetoacetyl coenzyme Areacting phosphotransbutyrylase from Clostridium beijerinckii (Clostridium butylicum) NRRL B593, Appl. Environ. Microbiol. 56, 607613. TRUFFAUT, SEBALD, (1988), Study of plasN., M. mids isolated from saccharolytic clostridia: Construction of hybrid plasmids and transfer to Escherichia coli and Bacillus subtilis, Biotechnol. Lett. 10, 1-6. TRUFFAUT, HUBERT,J., REYSSET,G. (1989), N., Construction of shuttle vectors useful for transforming Clostridium acetobutylicum, FEMS Microbiol. Lett. 58, 15-20. TWAROG,R., WOLFE, R. S. (1962), Enzymatic phosphorylation of butyrate, J. Biol. Chem. 237, 2474-2477. ULLMANN, DURRE,P. (1994), Cloning and seS., quencing of the DNA gyrase genes of Clostridium acetobutylicum DSM 792, Abstr. 7th Int. Symp. Genet. Ind. Microorg., Montreal, June 26July 1, p. 23. VALENTINE, C., WOLFE, R. S. (1960), PurificaR. tion and role of phosphotransbutyrylase, J. Biol. Chem. 235, 1948-1952. VELDKAMP, (1965), Enrichment cultures of proH. karyotic organisms, in: Methods in Microbiology (NORRIS, R., RIBBONS, W., Eds.), Vol. 3A, J. D. pp. 305-361, London: Academic Press. VERHASSELT, PONCELET,F., VITS, K., VAN P., GOOL, A., VANDERLEYDEN, (1989), Cloning J. and expression of a Clostridium acetobutylicum a-amylase gene in Escherichia coli, FEMS Microbiol. Lett. 59, 135-140. VOGET, C. E., MIGNONE, F., ERTOLA,R. J. C. (1985), Butanol production from apple pomace, Biotechnol. Lett. 7, 4346. VOLESKY, SZCZESNY, (1983), Bacterial conB., T. version of pentose sugars to acetone and butanol, Adv. Biochem. Eng. Biotechnol. 27, 101117. VOLLHERBST-SCHNECK, SANDS,J. A., MONK., TENECOURT, B. s. (1984), Effect of butanol o n lipid composition and fluidity of Clostridium acetobutylicum ATCC 824, Appl. Environ. Microbiol. 47, 193-194. VON HUGO,H., GOTTSCHALK, (1974), DistribuG. tion of 1-phosphofructokinase and PEP:fructose phosphotransferase activity in clostridia, FEBS iett. -46, 106-108.
SAUER,U., TREUNER, SANTANGELO, D., A., J. BUCHHOLZ, DURRE,P. (1994), Sporulation M., and primary u factor homologous genes in Clostridium acetobutylicum, Abstr. 7th Int. Symp. Genet. Ind. Microorg., Montreal, June 26-July l , p. 249. SCHARDINGER, (1905), Bacillus macerans, ein F. Aceton bildender Rottebacillus, Cbl. Bakteriol. Parasitenkd. Infektionskrankh., 11. Abt. 14, 772781. SCHARDINGER, (1907), Verhalten von WeizenF. und Roggenmehl zu Methylenblau und zu Starkekleister, nebst einem Anhange uber die Bildung hoherer Alkohole durch hitzebestandige Mikroorganismen aus Weizenmehl, Cbl. Bakteriol. Parasitenkd. Infektionskrankh., 11. Abt. 18, 748-767. SCHATTENFROH, GRASSBERGER, (1899), A., R. Weitere Mitteilungen uber Buttersauregarung, Cbl. Bakteriol. Parasitenkd. Infektionskrankh., 11. Abt. 5, 697-702. SCHATTENFROH, GRASSBERGER, (1900), A., R. Ueber Buttersauregahrung. I. Abhandlung, Arch. Hyg. 37, 54-103. SCHLOTE,D., GOTTSCHALK, (1986), Effect of G. cell recycle on continuous butanol-acetone fermentation with Clostridium acetobutylicum under phosphate limitation, Appl. Microbiol. Biotechnol. 24, 1-6. L. SCOTT,D., HEDRICK, R. (1952), The amylase of Clostridium acetobutylicum, J. Bacteriol. 63, 795803. SHERMAN, D. (1978), Butyl alcohols, in: KirkP. Othmer Encyclopedia of Chemical Technology, 3rd Edn. (MARK,H. F., OTHMER, F., OVERD. BERGER, G., SEABORG, T., GRAYSON, C. G. M., ECKROTH, Eds.), Vol. 4, pp. 338-345, New D. York: John Wiley & Sons. SPIVEY, J. (1978), The acetone/butanol/ethanol M. fermentation, Process Biochem. 13, 2-5. STEPHENS, M., HOLT,R. A., GOTTSCHAL, C., G. J. MORRIS, G. (1985), Studies on the stability of J. solvent production by Clostridium acetobutylicum in continuous culture, J. Appl. Bacteriol. 59, 597-605. STRATZ, M., SAUER,U., KUHN,A., DURRE, P. (1994), Plasmid transfer into the homoacetogen Acetobacterium woodii by electroporation and conjugation, Appl. Environ. Microbiol. 60, 10331037. TAGAKI, WESTHEIMER, H. (1968), AcetoW., F. acetate decarboxylase. The molecular weight of the enzyme and subunits, Biochemistry 7, 895900. TAYA, ISHII,S., KOBAYASHI, (1985), MoniM., T. toring and control for extractive fermentation of
6 References WALTER,K. A., BENNETT, N., PAPOUTSAKIS, G. E. T. (1992), Molecular characterization of two Clostridium acetobutylicum ATCC 824 butanol dehydrogenase isozyme genes, J. Bacteriol. 174, 7149-7158. WALTER,K. A., NAIR,R. V., CARY,J. W., BENNET^, G. N., PAPOUTSAKIS, T. (1993), SeE. quence and arrangement of two genes of the butyrate-synthesis pathway of Clostridium acetobutylicum ATCC 824, Gene 134, 107-111. WALTON, T. (1945), Process for the production M. of riboflavin by butyl alcohol producing bacteria, U.S. Patent 2368074. WANG,D. I. C., COONEY,C. L., DEMAIN. L., A. DUNHILL, (1979), Fermentation and Enzyme P. Technology, New York: John Wiley & Sons. F. WARREN,S., ZERNER,B., WESTHEIMER, H. (1966), Acetoacetate decarboxylase. Identification of lysine at the active site, Biochemistry 5, 817-823. WATERSON, M., CASTELLINO, J., HASS, G. R. F. M., HILL, L. (1972), Purification and characR. terization of crotonase from Clostridium acetobutylicum, J. Biol. Chem. 247, 52665271. WEIZMANN, (1915), Improvements in the bacteC. rial fermentation of carbohydrates and in bacterial cultures for the same, British Patent 4845. WEIZMANN, (1919), Production of acetone and C. alcohol by bacteriological processes, U.S. Patent 1315585. F. E. WELCH,R. W., RUDOLPH, B., PAPOUTSAKIS, T. (1989), Purification and characterization of the NADH-dependent butanol dehydrogenase from Clostridium acetobutylicurn (ATCC 824), Arch. Biochem. Biophys. 273, 309-318. WELCH, R. W., CLARK,S. W., BENNETT,G. N., RUDOLPH, B. (1992). Effects of rifampicin F. and chloramphenicol on product and enzyme levels of the acid- and solvent-producing pathways of Clostridium acetobutylicum (ATCC 824), Enzyme Microb. Technol. 14,277-283. WELSH,F. W., VELIKY, A. (1984), The producI. tion of acetone-butanol from acid whey, Biotechnol. Lett. 6 , 61-64. F. WESTHEIMER, H. (1969), Acetoacetate decarboxylase from Clostridium acetobutylicum, Methods Enzyrnol. 14, 231-241. F. WIESENBORN, P., RUDOLPH, B., PAPOUTSAD. KIS, E. T. (1988), Thiolase from Clostridium acetobutylicum ATCC 824 and its role in the synthesis of acids and solvents, Appl. Environ. Microbiol. 54, 2717-2722. D. F. WIESENBORN, P., RUDOLPH, B., PAPOUTSAKIS,E. T. (1989a), Phosphotransbutyrylase from Clostridium acetobutylicum ATCC 824 and its role in acidogenesis, Appl. Environ. Microbiol. 55,317-322.
I
267
WIESENBORN, P., RUDOLPH, B., PAPOUTSAD. F. KIS,E. T. (1989b), Coenzyme A transferase from Clostridium acetobutylicum ATCC 824 and its role in the uptake of acids, Appl. Environ. Microbiol. 55, 323-329. WILDE,E., HIPPE,H., TOSUNOGLU, SCHALN., LEHN, G., HERWIG, K., GOTTSCHALK,G. (1989), Clostridium tetanomorphum sp. nov., nom. rev., Int. J. Syst. Bacteriol. 39, 127-134. S. M. WILKINSON, R., YOUNG, (1994), Targeted integration of genes into the Clostridium acetobutylicum chromosome, Microbiology 140, 89-95. WILLIAMS, R., YOUNG, D. I., YOUNG, M. D. (1990), Conjugative plasmid transfer from Escherichia coli to Clostridium acetobutylicum, J. Gen. Microbiol. 136, 819-826. WINOGRADSKY, (1902), Clostridium pastorianS. urn, seine Morphologie und,seine Eigenschaften als Buttersaureferment, Cbl. Bakteriol. Parasitenkd. Infektionskrankh., 11. Abt. 9, 43-54, 107112. WONG,J., BENNETT, N. (1994), Studies of DNA G. supercoiling in Clostridium acetobutylicum ATCC824, Abstr. Gen. Meet. Am. SOC. Microbiol., Las Vegas, May 23-27, 0-40. WOODRUFF,J. C., STILES, H. R., LEGG, D. A. (1937). Process of producing butyl alcohol, U.S. Patent 2 089 522. WOOLLEY, C., MORRIS, G. (1990), Stability of R. J. solvent production by Clostridium acetobutylicum in continuous culture: strain differences, J. Appl. Bacteriol. 69, 718-728. WOOLLEY, C., PENNOCK, ASHTON,R. J., R. A., DAVIES,A,, YOUNG, M. (1989), Transfer of TnI545 and Tn916 to Clostridium acetobutylicum, Plasmid 22, 169-174. YAN,R.-T., CHEN,J.-S. (1990), Coenzyme A-acylating aldehyde dehydrogenase from Clostridium beijerinckii NRRL B592, Appl. Environ. Microbiol. 56, 2591-2599. YAN,R.-T., ZHU, C.-X., GOLEMBOSKI, CHEN, C., J . 4 . (1988), Expression of solvent-forming enzymes and onset of solvent production in batch cultures of Clostridium beijerinckii (Clostridium butylicum ), Appl. Environ. Microbiol. 54, 642-648. YAROVENKO, L. (1964), Principles of the conV. tinuous alcohol and butanol-acetone fermentation processes, in: Continuous Cultivation of Microorganisms, 2nd Symp. (MALEK,I., Ed.), pp. 205-217, Prague: Cechoslovakian Academy of Sciences. YOON,K.-H., LEE, J.-K., KIM,B. H. (1991), Construction of a Clostridium acetobutylicum-Escherichia coli shuttle vector, Biotechnol. Lett. 13, 14.
268
YOSHINO, YOSHINO, HARA,S., OGATA,S., S., T., HAYASHIDA, (1990), Construction of shuttle S. vector plasmid between Clostridium acetobutylicum and Escherichia coli, Agr. Biol. Chem. 54, 437441. YOUNG,M. (1993), Development and exploitation of conjugative gene transfer in clostridia, in: The Clostridia and Biotechnology (WOODS, D. R., Ed.), pp. 99-117, Stoneham: Butterworth-Heinemann. YOUNG, M., MINTON, P., STAUDENBAUER, N. W. L. (1989), Recent advances in the genetics of the clostridia, FEMS Microbiol. Rev. 63, 301-326. YOUNGLESON, S., SANTANGELO, D., JONES, J. J. D. T., WOODS, D. R. (1988). Cloning and expression of a Clostridium acetobutylicum alcohol dehydrogenase gene in Escherichia coli, Appl. Environ. Microbiol. 54, 676-682. YOUNGLESON, S., JONES, W. A., JONES, D. T., J. WOODS, D. R. (1989a), Molecular analysis and nucleotide sequence of the adhl gene encoding an NADPH-dependent butanol dehydrogenase in the Gram-positive anaerobe Clostridium acetobutylicum, Gene 78, 355-364. YOUNGLESON, S., JONES, D. T., WOODS, D. R. J. (1989b), Homology between hydroxybutyryl and hydroxyacyl coenzyme A dehydrogenase enzymes from Clostridium acetobutylicum fermentation and vertebrate fatty acid P-oxidation pathways, J. Bacteriol. 171, 68004807. Yu, P.-L., PEARCE, E. (1986), Conjugal transfer L. of streptococcal antibiotic resistance plasmids
into Clostridium acetobutylicum, Biotechnol. Lett. 8, 469-474. Yu, E. K. C., SADDLER,J. N. (1983), Enhanced acetone-butanol fermentation by Clostridium, acetobutylicum grown on D-xylose in the presence of acetic or butyric acid, FEMS Microbiol. Lett. 18, 103-107. Yu, E. K. C., CHAN, M. K.-H., SADDLER, N. J. (1985), Butanol production from cellulosic substrates by sequential co-culture of Clostridium thermocellum and C. acetobutylicum, Biotechnol. Lett. 7, 509-514. Yu, P. L., SMART, B., ENNIS,B. M. (1987), DifJ. ferential induction of P-galactosidase activities in the fermentation of whey permeate by Clostridium acetobutylicum, Appl. Microbiol. Biotechnol. 26,254-257. ZILLIG, HOLZ,I., WUNDERL, (1991), HyperW., S. thermus butylicus gen. nov., sp. nov., a hyperthermophilic, anaerobic, peptide-fermenting, facultatively H2S-generating archaebacterium, Int. J. Syst. Bacteriol. 41, 169-170. ZOUTBERG, R., WILLEMSBERG, SMIT,G., G. R., TEIXEIRA MATTOS,M. J., NEIJSSEL., M. DE 0. (1989a), Aggregate-formation by Clostridium butyricum, Appl. Microbiol. Biotechnol. 32, 1721. ZOUTBERG, R., WILLEMSBERG, SMIT,G., G. R., TEIXEIRA MATTOS,M. J., NEIJSSEL, M. DE 0. (1989b), Solvent production by an aggregateforming variant of Clostridium butyricum, Appl. Microbiol. Biotechnol. 32, 22-26.

6 Microbial Production of Butanol

Hochgeladen von

Dokumentinformationen

Originalbeschreibung:

Copyright

Verfügbare Formate

Dieses Dokument teilen

Dokument teilen oder einbetten

Freigabeoptionen

Stufen Sie dieses Dokument als nützlich ein?

Sind diese Inhalte unangemessen?

Copyright:

Verfügbare Formate

6 Microbial Production of Butanol

Hochgeladen von

Copyright:

Verfügbare Formate

6 Microbial Production of AcetoneD3utanoVIsopropanol

6 Microbial Production of Acetone/ButanoWIsopropanol

Tab. 1. Physical Properties of Acetone, Butanol, and Isopropanol

Data taken from NELSON and WEBB,1978; SHERMAN, 1978; PAPA,1982

2 History Tab. 2. History of Description and Isolation of Solvent-Forming Bacteria

6 Microbial Production of AcetondButanoWlsopropanol

3 Biochemistry and Genetics of Solvent-Producing Clostridia

3 Biochemistry and Genetics of SolventProducing Clostridia

6 Microbial Production of AcetondButanoVlsopropanol

Tab. 3. A Small Collection of Names of Solvent-Producing Bacteria Mentioned in Patents

3.2 Utilization of Substrates

3.2.1 Degradation of Polymers

6 Microbial Production of AcetondButanoWlsopropanol

Products Formed [mmol L-'1 Acetone Butanol Ethanol Isopropanol

14.0 20.5 6.0

30.2 45.4 67.9 44.8 17 11.2 22 75.6 11.2 47.1 4. 00

Fig. 1 Phase-contrast photomicrograph of Clostridium acetobutylicum (courtesy by H. HIPPE). .

3 Biochemistry and Genetics of Solvent-Producing Clostridia

Clostridium species generally employ the Embden-Meyerhof-Parnas (EMP) pathway

6 Microbial Production of Acetone/Butanol/lsopropanol

3 Biochemistry and Genetics of Solvent-Producing Clostridia

6 Microbial Production of AcetondButanoWlsopropanol

Tab. 6. Enzymes of C. acetobutylicum and C. beijerinckii Involved in Pyruvate Degradation

Lactate DH" Pyruvate:ferredoxin OR' Phosphotransacetylase

ADP NADH NADPH CoASH CoASH NADH NADH

158 264 205 85

NR CoASH CoASH ADP NADH NADPH NADPH, FAD

NR NADH NADH NADH NADH NADPH

115 100 NR 82 100 80

41 23, 24 23, 28 28 NR 96 56 55 43 43 38 40, 43.5

Dehydrogenase Not reported Oxidoreductase

Thiamine pyrophosphate Partially purified

6 Microbial Production of Acetone/ButanoUIsopropanol

3 Biochemistry and Genetics of Solvent-Producing Clostridia

6 Microbial Production of AcetondButanoWlsopropanol

3 Biochemistry and Genetics of Solvent-Producing Clostridia

3.3.3 Regulation of Product Formation

6 Microbial Production of Acetone/ButanoWIsopropanol

4 New Developments of the Fermentation Process

4 New Developments of the Fermentation Process

6 Microbial Production of Acetone/Butanol/lsopropanol

4.1 Continuous Culture

4 New Developments of the Fermentation Process

Tab. 7. Production of Solvents by Clostridium acetobutykum in Chemostats

Fermentation Process Nutrient Strain Limitation D ["CI 35

Substrate Temper- pH Concen- ature tration

Results Solvents Productivity

Glucose 2.7 g L-'

NCIB 8052 2.7 37

DSM 1731 3.4

GOTTSCHAL and MORRIS(1981b) BAHLet al. (1982a)

NCIB 8052 2.7

Nitrogen 0.4 g L-' Ammonium chloride

2gL-I Ammonium . sulfate 35 0.30

0.2 g L-' Ammonium acetate

ATCC 824 45.5

Sulfate 0.05 g L Magnesium sulfate 0.01 g L-' Manganese sulfate

BAHLand GOTTSCHALK (1984)

Phosphate Two-stage chemostat 0.1 g L-' DSM 1731 54 Monopotassium phosphate

37 4.3 stage I1 33 4.3

Magnesium 0.02 g L - ' Magnesium sulfate

6 Microbial Production of Acetone/ButanoUIsopropanol

4.3 37 0.125 3.6 1.6 0.3 1.3 0.4