6 Microbial Production of AcetoneD3utanoVIsopropanol PETER DURRE 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 3.3.1 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 4.1 Continuous Culture 248 4.2 Cell Immobilization and Cell Recycling 252 4.3 Product Recovery 253 4.4 Alternative Fermentation Substrates 255 3.3 Formation of Products 239 247 5 Conclusions 256 6 References 257
Transcript
6 Microbial Production of AcetoneD3utanoVIsopropanol
PETER DURRE 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
3.3.1 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
4.1 Continuous Culture 248 4.2 Cell Immobilization and Cell Recycling 252 4.3 Product Recovery 253 4.4 Alternative Fermentation Substrates 255
3.3 Formation of Products 239
247
5 Conclusions 256 6 References 257
230 6 Microbial Production of Acetone/ButanoWIsopropanol
1 Introduction
The solvents acetone, 1-butanol, and 2-pro- panol are natural products of a few anaerobic bacteria. During the first half of this century, their synthesis was mainly achieved by fer- mentation. Acetone-butanol fermentation became in volume the second largest fermen- tation 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 buta- no1 from oil became economically more fa- vorable. However, the oil crisis in 1973 re- vived 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 mi- croorganisms and new developments in pro- cess technology.
A number of reviews on various aspects of this topic have been published in the last de- cade (AWANG et al., 1988; BAHL and GOTT- SCHALK, 1988; DURRE et al., 1992; ENNIS et al., 1986a; JONES and WOODS, 1986; LENZ and MOREIRA, 1980; MCNEIL and KRIS- TIANSEN, 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 impor- tant intermediate in the manufacture of meth- acrylates 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 cos- metics 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 in connection with his discovery of the butyrate fermentation (PASTEUR, 1861a, 1862). The causative or- ganism 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 produc- ing bacteria was then conducted by FITZ. He 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 bacteri- um, 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 sub- strates. 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
GRASSBERGER WINOGRADSKY SCHARDINGER PRINGSHEIM SCHARDINGER BUCHNER and
BREDEMANN MEISENHEIMER
McCoy et al.
a The original description reported amylalcohol as a product, but due to the determination procedure butanol is much more likely (PRINGSHEIM, 1906a).
tion of pure cultures of Bacillus butylicus from cow feces and hay (FITZ, 1877, 1878, 1882). This pioneering work stimulated many other scientists to investigations on butanol producing anaerobic bacteria. Among them were a number of famous microbiologists (Tab. 2).
BEIJERINCK (1893) described two different strains yielding appreciable quantities of bu- tanol. One of them, producing butyrate addi- tionally, was designated Granulobacter sac- charobutyricum and was probably identical to Bacillus butylicus. The other species, Granu- lobacter butylicum, produced isopropanol along with butanol which, however, had not been discovered before 1920 (FOLPMERS, cited in OSBURN and WERKMAN, 1935). This bacterium was the later Clostridium butyli- cum which was shown to belong to the species C. beiierinckii (GEORGE et al., 1983). Pure cultures of butyrate producers additionally forming small amounts of butanol were ob- tained by WINOGRADSKY in 1902.
The biological formation of isopropanol (together with butanol) was first reported in 1906 for C. americanum (PRINGSHEIM, 1906a, b). Acetone was discovered as a product of microbial activity by SCHARDINGER (1905). The responsible organism was named Bacillus macerans, and ethanol, acetate, and formate were produced in addition. Thus, the two products butanol and acetone were found in rather different microorganisms. The discove- ry of their common formation by a single spe- cies was connected with the efforts of the chemical industry to produce synthetic rub- ber. Butanol was considered a precursor of butadiene, the starting material for synthetic rubber production. The British company Strange and Graham Ltd. got interested in such a project and contracted PERKINS and WEIZMANN (University of Manchester) and FERNBACH and SCHOEN (Institut Pasteur) in 1910 to study the formation of butanol by mi- crobial fermentation (see GABRIEL, 1928). FERNBACH isolated an acetone-butanol pro-
232 6 Microbial Production of AcetondButanoWlsopropanol
ducer in 1911; WEIZMANN terminated his co- operation with the company in 1912, but con- tinued his work at the University of Manches- ter. 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 and STRANGE, 1911, 1912) and for the Weizmann process in 1915 (WEIZ- MANN, 1915). Production of acetone and bu- tanol by Strange and Graham Ltd. based on the Fernbach process began in 1913. Potatoes were used as raw material. Following the out- break 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 King’s Lynn. WEIZ- MANN continued research work on his pro- cess; pilot-scale studies were performed and the results convinced the government of the superiority of this process. Finally, a produc- tion plant was built at the Royal Naval Cor- dite Factory at Poole. There and in some oth- er distilleries acetone was produced. In addi- tion, the Weizmann process was also intro- duced 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 in- dustry anymore, and acetone-butanol pro- duction had to be abandoned in Great Brit- ain. 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 automo- bile industry, increasing amounts of lacquers were needed for which butanol and its ester, butyl acetate, are excellent solvents. In addi- tion, Prohibition endangered the supply of amyl alcohol, which had been used as a sol- vent 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-bu- tanol 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. Difficul- ties 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 vol- ume of about 190000 L each. It was extended to 96 fermenters in the following years. Pro- duction was in the range of 100 tons of sol- vents per day (JONES and WOODS, 1986).
Strange and Graham Ltd. started produc- tion of solvents at King’s 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 ace- tone-butanol process in Great Britain contin- ued in 1935 when Commercial Solvent Corp. and the British Distillor’s 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 ferment- ing up to 6.5% sugar and producing 2% sol- vents. This resulted in a considerable reduc- tion of the costs of product recovery. Until 1936, the patents of WEIZMANN (1915, 1919) prevented a further extension of the acetone- butanol 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 com- petition of the growing petrochemical indus- try 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 oper- ating in Russia and in the People’s Republic of China.
A critical parameter in fermentations using C. acetobutylicum is the tendency of the or- ganism to degenerate, i. e., to lose the ability to produce solvents and to sporulate (KUTZE- NOK and ASCHNER, 1952; FINN and Now- REY, 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), lyophil- ized (LAPAGE et al., 1970), or stored in milk medium (BAHL et al., 1982a) or fermentation broth (MONOT et al., 1982; LIN and BLA- SCHEK, 1983). A heat shock for 90s at 70°C of a freshly inoculated culture helps to induce sporulation. A similar procedure (1 min at 90°C) will ensure germination of a spore pre- paration. The mechanisms responsible for clostridial degeneration are not yet under- stood. However, a recent publication has de- scribed 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. aceto- butylicum became economically unfavorable because of high substrate costs, low solvent yields, large volumes of waste, high amounts of energy required for product recovery (dis- tillation), and the low butanol tolerance of the organism. Starting at concentrations of 200 mM growth is completely inhibited by bu- tanol, 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 inter- est in the biotechnological production of 1- butanol 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 pro- cess or by catalytic dehydrogenation of iso- propanol (NELSON and WEBB, 1978). 1-Buta- no1 is synthesized from propylene by the 0x0-
process or from acetaldehyde by the aldol process (SHERMAN, 1978). 2-Propanol or iso- propanol is generally manufactured from pro- pene, either by the indirect hydration process or by catalytic hydration PAPA, 1982).
3 Biochemistry and Genetics of Solvent- Producing Clostridia
3.1 Microorganisms
Solvents such as acetone, butanol, and iso- propanol are produced in more than trace amounts only by few bacterial species. Most of them are members of the genus Clostrid- ium (BAHL and DURRE, 1993). However, bu- tanol as a major fermentation product has also been detected with Butyribacterium me- thylotrophicum (GRETHLEIN et al., 1991) and Hyperthermus butylicus (ZILLIG et al., 1991). 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 de- picted in Tab. 3; they are now invalid. Clos- tridia recognized of producing solvents are summarized in Tab. 4. In addition to C. aceto- butylicum, the species C. aurantibutyricum, C. beijerinckii, C. butyricum, C. cadaveris, C. chauvoei, C. felsineum, C. pasteurianum, C. puniceum, C. roseum, C. sporogenes, C. te- tani, C. tetanomorphum, and C. thermosac- charolyticum are shown. The products formed by these species vary slightly (Tab. 5). C. au- rantibutyricum and strains of C. beijerinckii (GEORGE et al., 1983; CHEN and HIU, 1986) as well as an aggregate-forming variant of C. butyricum (ZOUTBERG et al., 1989b) also 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. ace- tobutylicum and in a ratio in the order of 1O:l
234
Tab. 3. A Small Collection of Names of Solvent-Producing Bacteria Mentioned in Patents
6 Microbial Production of AcetondButanoVlsopropanol
in the broth of C. puniceum and C. beijerinck- ii (HOLT et al., 1988; CHEN and HIU, 1986). Butanol and equal amounts of ethanol, but no acetone or isopropanol, are produced by C. tetanomorphum (GOTTWALD et al., 1984). Cultures of C. acetobutylicum continue to produce solvents after growth has ceased, while with C. puniceum and C. tetanomor- phum 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 limi- tation, high butanol concentrations are yielded (130 and 45 mmol L-', respectively) (HARRIS et al., 1986; DABROCK et al., 1992). Small amounts of butanol are among the products of a number of other Clostridium species (CATO et al., 1986; HIPPE et al., 1992). Enrichment and isolation of solvent produc- ing clostridia is relatively easy. Soil samples from potatoes, other root crops, or roots of beans are good sources (CALAM, 1979). En- richment can be done in stabbed potato tu- bers (VELDKAMP, 1965) or maize mash me- dium (WEIZMANN, 1919). Isolation of pure cultures can be achieved by applying tech- niques commonly used with strictly anaerobic bacteria (BREZNAK and COSTILOW, 1994).
Since C. acetobutylicum produces and tol- erates 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 iso- propanol 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 fac- tors (RUBBO et al., 1941). Further physiologi- cal properties have been summarized in other review articles (CATO et al., 1986; HIPPE et 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 (SAUER and DURRE, 1993; WOOLLEY and MORRIS, 1990; YOUNG et 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 235
Tab. 4. Some Properties of Butanol-Producing Clostridia
C. aceto- C. auranti- C. beijer- C. buty- C. cada- C. chau- C. fel- butylicum butyricum inckii ricum veris voei sineum
+ + + - - Gelatin hydrolyzed - + Motility + + + f f + + Lipase produced - + Starch hydrolyzed + + f +
C. pasteur- C. puni- C. roseum C. sporo- C. tetani C. tetano- C. thermo- ianum ceum genes morphum saccharo-
lyticum
Gelatin hydrolyzed Motility Lipase produced Starch hydrolyzed Sugars utilized
Glucose Fructose Lactose Maltose Sucrose
+ + + -
+ + + + -
+ - + * + +
+ f -
+ f +
+ + + + -
+ k
+ + + -
+ + + + +
Data taken from CATO et al., 1986; CHEN and Hiu, 1986; FREIER-SCHRODER et al., 1989; GEORGE et al., 1983; GOTTWALD et al., 1984; HARRIS et al., 1986; HOLT et a]., 1988; MCCLUNG and McCoy, 1935; McCoy and MCCLUNG, 1935; NAKAMURA et al., 1979; PETERSEN, 1991; WILDE et al., 1989.
NCIMB 8052 is a member of the C. beiier- inckii group, whereas strain P 262 belongs to a different species (KEIS et al., 1994; JOHN- SON and JONES, personal communication).
3.2 Utilization of Substrates
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-molec- ular weight substrates, and (3) intracellular metabolism of the carbohydrates.
A great variety of mono- and disaccharides and related compounds are used by C. aceto- butylicurn for growth. In addition to the sub- strates listed in Tab. 3, these are: cellobiose, D-mannose, D-galactose, D-gluconate, D-ga- lacturonate, D-glucosamine, D-ribose, D-xy- lose, L-arabinose, L-rhamnose, and glycerol. Furthermore, polysaccharides as starch and xylan can be fermented. The process of sub-
3.2.1 Degradation of Polymers
Despite the fact that starch was the first in- dustrial substrate for the production of ace- tone-butanol by fermentation, only little in- formation is available on the extracellular en- zymes involved in the degradation of this po- lymer. During growth of C. acefobufylicurn on starch an a-amylase and a glucoamylase, ori-
236 6 Microbial Production of AcetondButanoWlsopropanol
Tab. 5. Solvent Production by Some Clostridial Species Grown in Complex Media Contaning 2% (w/v) Glucose"
Organism 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
Data taken from DABROCK et al., 1992; FREIER-SCHRODER et al., 1989; GEORGE et al., 1983; GOTTWALD et al., 1984, HOLT et al., 1988 ZOUTBERG et 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).
3 Biochemistry and Genetics of Solvent-Producing Clostridia 237
ginally referred to as a maltase, are produced (HOCKENHULL and HERBERT, 1945; FRENCH and KNAPP, 1950; Scorr and HEDRICK, 1952; ENSLEY et al., 1975). The synthesis of these enzymes is generally subjected to catabolite repression by glucose and induced by starch or its degradation products (HOCKENHULL and HERBERT, 1945; CHOJECKI and BLA- SCHEK, 1986). Recently, the 0-amylase from C. acetobutylicum ATCC 824 has been puri- fied and characterized (PAQUET et al., 1991). This enzyme has a molecular weight of 84 kDa, an isoelectric point of 4.7, an optimal pH of 5.6, and is very sensitive to thermal inactivation. Higher activities were found with high-molecular weight substrates as com- pared to low-molecular weight maltooligosac- charides. Glycogen and pullulan were slowly hydrolyzed, whereas dextrans and cyclodex- trins were not attacked. Interestingly, the product of an amylase gene of C. acetobutyli- cum cloned by VERHASSELT et al. (1989) has a molecular mass of only 53.9 kDa. This gene is not identical to a truncated open reading frame with significant similarity to the a- amylase gene of Bacillus subtilis identified by GERISCHER and DURRE (1990). Therefore, it is likely that C. acetobutylicum produces dif- ferent amylases for the utilization of starch.
Xylan can serve as sole carbon source for C. acetobutylicum (LEE et al., 1985a; LEMMEL et al., 1986). However, growth is slow, and larch wood xylan is only partially hydrolyzed. Two endoxylanases and a p-D-xylosidase of C. acetobutylicum were purified and charac- terized (LEE et al., 1985a, 1987). The smallest oligosaccharides degraded by xylanase A and B are xylohexaose and xylotetraose, respec- tively. Xylanase A also exhibits carboxyme- thy1 cellulase activity.
C. acetobutylicum does not grow on cellu- lose; nevertheless, two activities of the cellul- ase complex can be detected in certain strains of this organism (ALLCOCK and WOODS, 1981). LEE et al. (1985b) showed that two strains out of 21 tested produced extracellular endo- 1 ,4-p-glucanase and cellobiase ( p-D- glucosidase) activities during growth on cello- biose.
3.2.2 Uptake of Mono- and Disaccharides
Very little is known on substrate transport in C. acetobutylicum. Phosphotransferase sys- tems (PTS) are apparently responsible for up- take of glucose and fructose (VON HUGO and GOTTSCHALK, 1974; HUTKINS and KASHKET, 1986):
Glucose + P-HPr Glucose-6- phosphate + HPr
phosphate + HPr Fructose + P-HPr Fructose-1-
P-HPr is a phosphorylated protein generated from HPr with phosphoenolpyruvate (PEP) as the source of energy-rich phosphate:
PEP + HPr P-HPr + Pyruvate
A detailed biochemical study of the glucose PTS of C. acetobutylicum has been provided recently (MITCHELL et al., 1991). The pres- ence of the four components of the system, enzyme I, HPr, enzyme IIAgl", and enzyme IIglu, was demonstrated. The C. acetobutyli- cum PTS therefore displays the same archi- tecture as in other bacteria. Other substrates of C. acetobutylicum might be taken up either also by PTS as in the case of glucitol (MIT- CHELL, 1992), by ABC transport sytems (HIGGINS, 1992), or by symport mechanisms driven by the transmembrane proton gra- dient. Disaccharides such as sucrose or mal- tose might then be cleaved by appropriate phosphorylases, e.g.,
~~l~~~~ + phosphate Maltose Phosphorylase
Glucose + Glucose-1-phosphate
Free glucose can be converted to glucose-6- phosphate by hexokinase.
3.2.3 Intracellular Sugar Metabolism to Pyruvate
Clostridium species generally employ the Embden-Meyerhof-Parnas (EMP) pathway
238 6 Microbial Production of Acetone/Butanol/lsopropanol
Hexose
I 1 1 ButyrnWAcelate BytyrylJAcetyl-CoA
Acetoahtyl-CoA & I Pool
ATP 4 YP F;;L!m+N,L+H+ NAD+
Butyryl-CoA ,&!& .db F I 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 fol- lows: (1) glyceraldehyde-3-phosphate dehydrogenase; (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 cer- tain alcohol dehydrogenease reactions some strains use NADP +/NADPH + H + as electron acceptorldo- nor instead of NAD +/NADH + H + , which are generally shown.
for the degradation of hexose phosphates (THAUER et al., 1977; GOITSCHALK, 1986; ROGERS and GOTTSCHALK, 1993). This is ap- parently also true for C. acetobutylicum. A corresponding scheme is shown in Fig. 2. Su- gar acids such as gluconate are degraded via a modified Entner-Doudoroff pathway (AN- DREESEN and GOTTSCHALK, 1969). D-G~u- conate is dehydrated to yield 2-keto-3-deoxy-
gluconate which subsequently is phosphoryl- ated and cleaved to yield pyruvate and 3-phosphoglyceraldehyde (Fig. 3).
Pentoses are converted into ribose-5-phos- phate and xylulose-5-phosphate which are fed into the pentose phosphate cycle; the result- ing hexose phosphates are subsequently cata- bolized by the EMP pathway. Enzymes such as transketolase and transaldolase have been
3 Biochemistry and Genetics of Solvent-Producing Clostridia
COOH
c=o
239
I I
COOH COOH COOH CH3 I
I I I
I Pyruvate c=o I c=o HC-OH I HZO I ADP I
I I I
HC-OH
HC-OH
2
HC =O
CHz < HO-CH j W CHI I I I
HC-OH
HC-OH
CHzOH
HC-OH
HC-OH
CHzOH I
gluconate 6-phosphogluconate I
CHzO@
Gluconate 2-Keto-3-deoxy- 2-Keto-3-deoxy- HC -OH
CHzO@
Glyceraldehyde-3-phosphate
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.
detected in species closely related to C. aceto- butylicum (“C. butylicum”). The results of tracer studies were in agreement with the op- eration of the pentose phosphate cycle and the EMP pathway (CYNKIN and DELWICHE, 1958; CYNKIN and GIBBS, 1958; VOLESKY and SZCZESNY, 1983).
3.3 Formation of Products
3.3.1 Formation of Acids
Lactate becomes a major fermentation product of C. acetobutylicum under condi- tions of growth limiting iron concentrations (10 p,M) and pH values higher than 5 (BAHL et al., 1986). Inhibition of the hydrogenase by carbon monoxide or potassium cyanide causes a similar effect (HANSON and ROD- GERS, 1946; KATAGIRI et al., 1960; KEMPNER and KUBOWITZ, 1933; KUBOWITZ, 1934). The lactate dehydrogenase (Ldh) responsible for reduction of pyruvate to lactate has been pu- rified. It consists of 4 identical subunits with a molecular mass of 36 kDa, has a pH optimum of 5.8, uses NADH as a coenzyme, and is specifically activated by fructose-l,6-diphos-
phate (FREIER and GOTTSCHALK, 1987). Lactate might be taken up again and subse- quently consumed, if the pH decreases to val- ues below 4.5 and if the iron concentration is no longer growth-limiting. For consumption, however, acetate is needed as a co-substrate. Conversion of lactate into pyruvate seems to be mediated by a different lactate dehydro- genase (DIEZ-GONZALEZ et al., 1994). A lac- tate dehydrogenase gene has been cloned from C. acetobutylicum strain B643. The re- combinant plasmid encoded a 38 kDa enzyme that was activated by addition of fructose-1,6- diphosphate (CONTAG et al., 1990).
The central enzyme for the breakdown of pyruvate is pyruvate : ferredoxin oxidoreduct- ase (Pfo) (Fig. 2). The enzyme from Clostrid- ium acetobutylicum was purified and charac- terized (MEINECKE et al., 1989). The molecu- lar mass was found to be 123 kDa per mono- mer in SDS polyacrylamide gel electrophore- sis. The subunit composition of the native en- zyme remained undetermined due to the high oxygen sensitivity (50% inactivation in 1 h). The protein contained 2.9 mol sulfur, 4.1 mol iron, and 0.4 mol thiamine pyrophosphate per mol monomer, suggesting the presence of one 4Fe-4S cluster and one thiamine pyrophos- phate in this subunit. The apparent K , values
240 6 Microbial Production of AcetondButanoWlsopropanol
for pyruvate and coenzyme were determined to be 322 p M and 3.7 pM, respectively. Prod- ucts of the reaction are acetyl-CoA, COz, and reduced ferredoxin.
Acetyl-CoA is partly converted to acetate and partly to butyrate. The ratio in which these products are formed varies to some ex- tent. Acetate formation involves the enzymes phosphotransacetylase and acetate kinase, whereas butyrate production is catalyzed by subsequent action of thiolase, L( + )-3-hy- droxybutyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, phosphotrans- butyrylase, and butyrate kinase (GAVARD et al., 1957; TWAROG and WOLFE, 1962; VAL- ENTINE and WOLFE, 1960; ANDERSCH et al., 1983; HARTMANIS and GATENBECK, 1984). Production of both acids is associated with ATP synthesis in the kinase reactions. In Tab. 6, some characteristics of enzymes involved in pyruvate breakdown by C. acetobutylicurn are summarized.
Both, phosphotransacetylase (Pta) and acetate kinase (Ack), have not been purified from C. acetobutylicurn. Their activity has been measured in crude extracts. Strong ex- pression could only be found during the aci- dogenic growth phase (ANDERSCH et al., 1983; BALLONGUE et al., 1986; HARTMANIS and GATENBECK, 1984). However, in another report such a strict activity pattern is not de- noted (HUSEMANN and PAPOUTSAKIS, 1989). Phosphotransacetylase from C. beijerinckii has been partially purified. Its molecular mass is 56-57 kDa (CHEN, 1993).
Formation of C4-compounds starts by con- densation of two acetyl-CoA molecules to yield acetoacetyl-CoA. This reaction is cata- lyzed by thiolase (Thl). The respective en- zyme has been purified from C. acetobutyli- .cum and consists of 4 identical subunits with a molecular mass of 44 kDa (WIESENBORN et al., 1988). The apparent K,,, values for acetyl- CoA, sulfhydryl-CoA, and acetoacetyl-CoA are 270 pM, 4.8 pM, and 32 pM, respectively. The structural gene of this enzyme has been cloned (PETERSEN and BENNETT, 1991). Re- cently, a different gene for a second thiolase from C. acetobutylicurn as well as C. beijer- inckii has been detected and sequenced (WINZER, MINTON, and DURRE, unpublished observations). It is tempting to speculate that
these different thiolases might be alternative- ly active during acidogenic resp. solventogen- ic growth phases. The presence of two differ- ent thiolases has also been described for an- other solvent forming Clostridiurn, C. pasteur- ianurn (BERNDT and SCHLEGEL, 1975). A re- port by a Japanese group might indicate that one of the thiolases from C. acetobutylicurn ATCC 824 exhibits coenzyme A transferase activity in addition (NAKAMURA et al., 1990). A thiolase with a native molecular mass of 100-120 kDa has also been purified from two C. beijerinckii strains (CHEN, 1993).
Reduction of acetoacetyl-CoA to butyryl- CoA is catalyzed by the subsequent action of 3-hydroxybutyryl-CoA dehydrogenase (Hbd), crotonase (Cch), and butyryl-CoA de- hydrogenase (Bcd). Hbd has not been puri- fied from C. acetobutylicurn. Determination of its activity in crude extracts showed a rapid decrease several hours after the onset of sol- ventogenesis (HARTMANIS and GATENBECK, 1984). The enzyme has been purified from C. beijerinckii NRRL B593. The subunit and native molecular masses were reported to be 31 and 213 kDa, respectively (COLBY and CHEN, 1992). The amino terminus of the pro- tein proved to be almost identical to the de- duced amino acid sequence of a hbd structur- al gene that had been cloned and sequenced from strain P262 (COLBY and CHEN, 1992; YOUNGLESON et al., 1989b). Crotonase (cro- tonyl-CoA hydratase) has been obtained in homogenous form from an unspecified C. ace- tobutylicurn strain (WATERSON et al., 1972), possibly strain NRRL B528 (CHEN, 1993). The enzyme is a tetramer, consisting of 4 identical subunits with a molecular mass of 40 kDa. It has a limited substrate specificity, being active only with C4- and C6-enoyl CoA, and shows a remarkable sensitivity towards high concentrations of crotonyl-CoA (WA- TERSON et al., 1972). Its activity pattern dur- ing growth is almost identical to that of Hbd (HARTMANIS and GATENBECK, 1984). Little information is available on the last enzyme of the reaction sequence, butyryl-CoA dehydro- genase. Enzymatic determinations in crude extracts of C. acetobutylicurn were of poor re- producibility, which might indicate a high oxygen sensitivity (HARTMANIS and GATEN- BECK, 1984).
Tab.
6. E
nzym
es o
f C.
ace
tobu
tylic
um a
nd C
. bei
jeri
ncki
i Inv
olve
d in
Pyr
uvat
e D
egra
datio
n
Enz
yme
Hos
t C
oenz
yme
Purif
ied
Nat
ive
Mol
ecul
ar
Subu
nit S
ize
Com
posi
tion
Clo
ned
Sequ
ence
d M
ass
[kD
a]
Wa
I
Lac
tate
DH
" C.
ace
tobu
tylic
um
NA
DH
Py
ruva
te:f
erre
doxi
n O
R'
C. a
ceto
buty
licum
T
PPd,
CoA
SH
Phos
phot
rans
acet
ylas
e C.
bei
jeri
ncki
i C
oASH
Ace
tate
kin
ase
C. a
ceto
buty
licum
A
DP
Ace
tald
ehyd
e D
H
C. a
ceto
buty
licur
n N
AD
H
Alc
ohol
DH
C.
ace
tobu
tylic
um
NA
DPH
T
hiol
ase
C. a
ceto
buty
licum
C
oASH
C.
bei
jeri
ncki
i C
oASH
P-
Hyd
roxy
buty
ryl-C
oA D
H C
. bei
jeri
ncki
i N
AD
H
C. a
ceto
buty
licum
N
AD
H
Cro
tona
se
C. a
ceto
buty
licum
B
utyr
yl-C
oA D
H
C. a
ceto
buty
licum
N
R
Phos
phot
rans
buty
ryla
se
C. a
ceto
buty
licum
C
oASH
C
. bei
jeri
ncki
i C
oASH
B
utyr
ate
kina
se
C. a
ceto
buty
licum
A
DP
Hyd
roge
nase
C.
ace
tobu
tylic
um
NA
DH
:fer
redo
xin
OR
C.
ace
tobu
tylic
um
NA
DH
N
AD
PH:f
erre
doxi
n O
R
C. a
ceto
buty
licum
N
AD
PH
NA
DH
: rub
redo
xin
OR
C.
ace
tobu
tylic
um
NA
DPH
, FA
D
CoA
tran
sfer
ase
C. a
ceto
buty
licum
Ace
toac
etat
e de
carb
oxyl
ase
C. a
ceto
buty
licum
Ald
ehyd
e/al
coho
l DH
(E
) C
. ace
tobu
tylic
um
NR
B
utyr
alde
hyde
DH
C
. ace
tobu
tylic
um
NA
DH
C
. bei
jeri
ncki
i N
AD
H
But
anol
DH
(I)
C.
ace
tobu
tylic
um
NA
DH
B
utan
ol D
H (
11)
C. a
ceto
buty
licum
N
AD
H
Prim
aryk
econ
dary
C.
bei
jeri
ncki
i N
AD
PH
alco
hol D
H
Alc
ohol
DH
C
. bei
jeri
ncki
i N
AD
(P)H
A
lcoh
ol D
H
C. b
eije
rinc
kii
NA
DPH
a D
ehyd
roge
nase
T
hiam
ine
pyro
phos
phat
e
C. a
ceto
buty
licum
C. b
eije
rinc
kii
C. b
eije
rinc
kii
Not
rep
orte
d Pa
rtia
lly p
urifi
ed
Oxi
dore
duct
ase
+ + +' - + + + + + + + + - -
- - + + + + + + + + + + - + -
NR
b N
R
NR
NR
N
R
100-
120
213
158
264
205 85
NR
93
85
33
0 20
0-23
0
115
100
NR
82
10
0 80
36
123
56-5
7
44
44
NR
31
40
31
33
39
63
41
23, 2
4 23
, 28
28
NR
96
56
55
43
43
38
40, 4
3.5
242 6 Microbial Production of Acetone/ButanoUIsopropanol
The final steps of butyrate formation are catalyzed by phosphotransbutyrylase (Ptb) and butyrate kinase (Buk). Ptb activity is mainly found during the acidogenic fermenta- tion phase (ANDERSCH et al., 1983; HART- MANIS and GATENBECK, 1984). The enzyme has been purified from C. acetobutylicum strain ATCC 824 and from C. beijerinckii (WIESENBORN et al., 1989a; THOMPSON and CHEN, 1990). Both proteins consist of identi- cal 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 complete- ly inactive in the butyryl phosphate-forming direction at a pH of about 6. The enzyme from C. beijerinckii also reacts with acetoace- tyl-CoA in the presence of phosphate, and acetoacetyl phosphate might be a product (THOMPSON and CHEN, 1990). Whether this reaction is of physiological relevance is not yet known. Butyrate kinase has been purified from C. acetobutylicum ATCC 824. The na- tive 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. acetobu- cylicum ATCC 824 (CARY et al., 1988; WAL- TER et al., 1993) and NCIMB 8052 (now grouped with C. beijerinckii, as mentioned above) (OULTRAM et al., 1993). The two genes are contiguous on the chromosome and most likely form an operon (WALTER et al., 1993).
During acid production, reduced ferredox- in is oxidized by hydrogenase and H2 is pro- duced. Hydrogenase activity has been demon- strated in cell extracts of C. acetobutylicum (ANDERSCH et al., 1983; KIM and ZEIKUS, 1985). The respective gene has recently been cloned and sequenced from strain P 262 (SANTANGELO et al., 1994). The NADH gen- erated in the 3-phosphoglyceraldehyde dehy- drogenase reaction is oxidized in the P-hy- droxybutyryl-CoA and butyryl-CoA dehydro- genase reactions. This results in the following redox balance:
Glucose + 2 NAD +
2 Pyruvate 2 Acetyl-CoA + 2 NADH + 2 H +
-+ 2 Pyruvate + 2NADH + 2 H +
-+ 2 Acetyl-CoA + 2 C 0 2 + 2 H
-+ Butyrate + 2 NAD +
Glucose + Butyrate + 2 C 0 2 + 2 H2
However, not only 2 HJglucose but ap- proximately 2.3 H,/glucose are produced dur- ing 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 ex- tent (PETITDEMANGE et al., 1976, 1977). The enzyme is activated by acetyl-CoA and inhi- bited 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 fermenta- tion product in addition to butyrate. There are thermodynamic limitations as to the ex- tent 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 et al., 1977). Another en- zyme, NADPH:ferredoxin oxidoreductase, can also utilize reduced ferredoxin in the con- trolled 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 (JUNGERMANN et al., 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 (PETITDE- MANGE et al., 1979). The physiological func- tion of this enzyme is still unknown. It is in- duced by acetate (and, to a lower extent, by butyrate) only at low pH values. Maximal ac- tivity has been observed at pH 4.8 (BALLON- GUE et al., 1986). After the onset of solvento- genesis the enzymatic activity rapidly de- creases (MARCZAK et al., 1983, 1984). The level of rubredoxin in the cells fluctuates ac- cordingly, while the concentration of ferre- doxin remains almost constant throughout
3 Biochemistry and Genetics of Solvent-Producing Clostridia 243
the fermentation (MARCZAK et al., 1985). Under iron limitation ferredoxin can no long- er be synthesized. Instead, flavodoxin is formed, the gene of which has been cloned from C. acetobutylicum P 262 (SANTANGELO et al., 1991).
3.3.2 Formation of Solvents
Several conditions must be met to ensure a reproducible metabolic shift from acidogene- sis to solventogenesis in C. acetobutylicum. These are a low pH, excess of substrate, threshold concentrations of butyrate and/or acetate, and a suitable growth-limiting com- pound such as phosphate or sulfate (BAHL and GOTTSCHALK, 1984). In strain NCIMB 8052 (now considered to be a member of the C. beijerinckii group) formation of acetone and butanol could be initiated at neutral pH by adding high concentrations (100 mmol L-’ each) of acetate and butyrate (HOLT et al., 1984). Before the onset of solventogenesis a new set of enzymes is synthesized. Acetone production is catalyzed by acetoacetyl- CoA:acetate/butyrate coenzyme A transfer- ase (CoA transferase or Ctf) and acetoacetate decarboxylase. In C. beijerinckii strains able to form isopropanol, a primary/secondary al- cohol dehydrogenase subsequently converts acetone into isopropanol. Butanol is made from butyryl-CoA by action of various butyr- aldehyde and butanol dehydrogenase activi- ties. Ethanol on the other hand is constitu- tively produced by C. acetobutylicum throughout the fermentation (GERISCHER and DURRE, 1992). Its synthesis is brought about by a NADPH-dependent alcohol dehy- drogenase and a specific acetaldehyde dehy- drogenase that does not react with butyryl- CoA (BERTRAM et al., 1990).
CoA transferase of C. acetobutylicum ATCC 824 has been purified to homogeneity (WIESENBORN et al., 1989b). The native en- zyme was a heterotetramer of two different subunits with a molecular mass of 23 kDa and 28 kDa, respectively. However, cloning and sequencing of the respective genes (ctfA and ctfB) led to deduced values of 22.7 kDa and 23.7 kDa for strain ATCC 824 (CARY et al., 1990; PETERSEN et al., 1993) and 23.6 kDa for
both subunits in strain DSM 792 (GERISCHER and DURRE, 1990; FISCHER et al., 1993). Sim- ilar data have been determined with purified CoA transferase from C. beijerinckii NRRL B.593 (CHEN, 1993). CoA transferase plays a major role in the uptake of acids during sol- ventogenesis (preferentially butyrate) that are subsequently converted to acetone and butanol (ANDERSCH et al., 1983; HARTMANIS et al., 1984). To some extent, butyrate can be taken up in batch cultures via the reversed reactions of butyrate kinase and phospho- transbutyrylase (HUSEMANN and PAPOUTSA- KIS, 1989). A surprising feature of CoA trans- ferase are the high K , values for acetate and butyrate (1.2 mol L-’ and 0.6 mol L-I, re- spectively). They have been suggested to re- flect a gradational response to the progressive toxic effects of increasing levels of these acids (WIESENBORN et al., 1989b). K , values for the corresponding enzyme from C. beijerinck- ii are significantly lower (CHEN, 1993).
Acetoacetate decarboxylase (Adc) of C. acetobutylicum has been the subject of ex- tensive investigations in the 1960s aiming to elucidate its reaction mechanism (FRIDO- VICH, 1972; WESTHEIMER, 1969). Purification of the native enzyme revealed that it had a molecular mass of 340 kDa and consisted of 12 identical subunits with a molecular mass of 29 kDa (TAGAKI and WESTHEIMER, 1968). Later investigations showed the respective data to be rather 330 kDa and 28 kDa (GERI- SCHER and DURRE, 1990; PETERSEN and BENNETT, 1990), confirmed by the deduced amino acid sequence after cloning and se- quencing the structural gene, which forms a monocistronic operon (GERISCHER and DURRE, 1990; 1992; PETERSEN and BEN- NETT, 1990; PETERSEN et al., 1993). This ope- ron is adjacent, but convergently arranged to the ctf genes that form a common transcrip- tion unit together with the gene for an alde- hyde/alcohol dehydrogenase (FISCHER et al., 1993; NAIR et al., 1994). The reaction mecha- nism of Adc involves the intermediate forma- tion of a Schiff base between the substrate acetoacetate and a lysine residue at the active site (WARREN et al., 1966). Native acetoace- tate decarboxylase from C. beijerinckii NRRL B592 and NRRL B593 has a considerably smaller molecular mass of 200-230 kDa
244 6 Microbial Production of AcetondButanoWlsopropanol
(CHEN, 1993). The latter strain belongs to the few clostridia able to further reduce acetone to isopropanol (GEORGE et al., 1983). The enzyme catalyzing this reaction is a primary/ secondary alcohol dehydrogenase with a na- tive 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 reac- tion. The structural gene has been cloned and sequenced. The deduced amino acid sequence shows 75% identity to the thermostable alco- hol dehydrogenase of Thermoanaerobium brockii, an anaerobe producing ethanol (CHEN, 1993).
Butanol formation is catalyzed by butyral- dehyde (Bad) and butanol dehydrogenase (Bdh) activities. A butyraldehyde dehydro- genase has been purified from C. acetobutyli- cum NRRL B643 and from C. beijerinckii NRRL B592 (PALOSAARI and 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 sub- strate than acetyl-CoA. Recently, in C. aceto- butylicum a gene has been cloned and se- quenced that has high homology with the adhE gene of Escherichia coli known to en- code a protein with aldehyde and alcohol de- hydrogenase domains. The respective clos- tridial enzyme is believed to play a role in bu- tanol synthesis at the onset of solvent forma- tion (FISCHER et al., 1993; NAIR et al., 1994).
For a long time, conflicting data have been reported on alcohol dehydrogenase in C. ace- tobutylicum. The situation was clarified in 1987 by a report providing evidence for at least two different enzyme activities in this or- ganism (DURRE et al., 1987). An NADPH- dependent 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 proba- bly in ethanol formation in cooperation with a specific acetaldehyde dehydrogenase as sug- gested from studies with transposon-induced mutants (BERTRAM et al., 1990). Activity of the NADPH-dependent alcohol dehydrogen- ase is found throughout the fermentation (DURRE et al., 1987) confirming the constitu- tive nature of the enzyme which is in agree- ment with the constitutive formation of low ethanol levels (GERISCHER and DURRE, 1992). This alcohol dehydrogenase probably regulates the pool of reduced nicotinamide adenine dinucleotide phosphate which is sup- plied by NADPH: ferredoxin oxidoreductase. From C. acetobutylicum P 262 a gene encod- ing a NADPH-dependent alcohol dehydro- genase has been cloned and sequenced (YOUNGLESON et al., 1988, 1989a). However, 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 (PETER- SEN et al., 1991; WELCH et al., 1989). Both na- tive enzymes are homodimers with subunits of molecular masses of about 42 kDa. Bdh I had only a 2-fold higher activity with butyral- dehyde than with acetaldehyde, whereas Bdh I1 was reported to have a 46-fold higher activ- ity with butyraldehyde than with acetalde- hyde. 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 bu- tanol production. The genes of both isozymes (bdhA and bdhB) have been cloned and se- quenced (PETERSEN et al., 1991; WALTER 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. ace- tobutylicum with high homology to the E. coli adhE gene has been cloned and sequenced (FISCHER et al., 1993; NAIR et al., 1994). The respective E. coli gene product has aldehyde and alcohol dehydrogenase domains (GOOD- LOVE et al., 1989) and its clostridial analog is
3 Biochemistry and Genetics of Solvent-Producing Clostridia
believed to be involved in butanol synthesis (FISCHER et al., 1993; NAIR et al., 1994). The clostridial adhE (or aad) gene is part of an operon additionally containing the ctfA and ctfB genes. This transcription unit (sol ope- ron) is controlled by two different promoters. Thus, at least three different butanol dehy- drogenases might be involved in butanol pro- duction in C. acetobutylicum.
A similar situation has been found with C. beijerinckii. Strain NRRL B592 producing acetone, butanol, and ethanol, but not isopro- panol, expresses two types of alcohol dehy- drogenases 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 pro- tein under non-denaturing conditions. These three species might be dimers of the composi- tion a2, ap, and pZ CHEN, 1992).
3.3.3 Regulation of Product Formation
A batch culture of C. acetobutylicum classi- cally shifts from acid to solvent formation to- wards the end of growth at pH values below 5 (Fig. 4). The shift can reproducibly be achieved in complex media (OXFORD et al., 1940) and is often associated with sporulation (LONG et al., 1984a, b). The ability to initiate and complete the process of sporulation is not a prerequisite of solvent production. In agree- ment with this an asporogenous mutant is se- lected in solvent producing continuous cul- tures (MEINECKE et al., 1984). Although the signal for initiating sporulation is not nutrient limitation as in Bacillus species (LONG et al., 1984b) the process of spore formation seems to be identical since analogs of the sporula- tion-specific Bacillus sigma factor genes have been cloned and sequenced from C. acetobu- tylicum (SAUER et al., 1994).
The shift to solventogenesis in C. acetobu- tylicum and C. beijerinckii is characterized by a decrease of the activity of some acidogenic
245
I
6
5
L
I n
. _
Fig. 4. Course of the acetone-butanol fermentation in batch culture. Butanol, 0; acetone, A; ethanol, W; butyrate, 0; acetate, A; pH, @ (BAHL et al., 1982b).
enzymes (ANDERSCH et al., 1983; HARTMA- NIS and GATENBECK, 1984). At about the same time the enzymes needed for solvento- genesis are induced or derepressed (AN- DERSCH et al., 1983; DURRE et al., 1987; HARTMANIS and GATENBECK, 1984; YAN et 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 chlor- amphenicol (BALLONGUE et al., 1985; PALO- 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 be- fore the production of acetone and butanol followed by a massive decrease of transcripts (FISCHER et al., 1993; GERISCHER and DURRE, 1992; WALTER et al., 1992). During 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 6 Microbial Production of Acetone/ButanoWIsopropanol
is linked to onset of solventogenesis and spo- rulation in a yet unknown way. Genes encod- ing several of these proteins have been cloned and sequenced (BEHRENS et al., 1993; NAR- BERHAUS and BAHL, 1992; NARBERHAUS et al., 1992; SAUER and DURRE, 1993).
Despite the enormous progress made in elucidation of gene structure and mRNA reg- ulation, little is known on the signal(s) that initiate solventogenesis (and possibly sporula- tion and stress response). Involvement of al- ternate sigma factors in transcription of genes encoding solventogenic enzymes is unlikely, since the RNA polymerase of C. acetobutyli- cum has been purified (PICH and BAHL, 1991) and, after isolation from acidogenic and solventogenic cells, showed the same compo- sition including only the vegetative sigma fac- tor (BAHL, 1993). Recently, a model has been proposed that postulates the ATP pool to be a signal for acetone formation and the NAD(P)H pool to be a signal for butanol production (GRUPE and GOTTSCHALK, 1992). However, since CoA transferase (re- quired for acetone production) and AdhE (needed for butanol formation) are encoded in a common transcription unit (FISCHER et al., 1993), this model must at least be modi- fied, although the pools of ATP and reducing equivalents certainly play a major role in reg- ulation. On the other hand it might be possi- ble that DNA topology directly responds to changes in the environment (pH, salt concen- tration, osmotic pressure, temperature) and thus provokes transcription of certain genes. The degree of supercoiling of DNA isolated from acidogenic and solventogenic cells was different (WONG and BENNETT, 1994). Su- percoiling is controlled by topoisomerase I and DNA gyrase, the genes of which have been cloned and sequenced from C. acetobu- tylicum DSM 792 (ULLMANN and DURRE, 1994). Transcription analysis and targeted inactivation of the gyr genes should provide a conclusive answer to this problem.
An additional level of regulation might be present as indicated by studies with a trans- poson-induced, acetone- and butanol-nega- tive mutant of C. acetobutylicum (BERTRAM et al., 1990). Genetic analysis revealed that the transposon Tn916 had inserted in front of a tRNA gene thus causing massive reduction
of expression (SAUER and DURRE, 1992). This tRNA recognizes the rarely used ACG codon which, however, is present in all genes required for acetone and butanol synthesis. In addition, it has been found in inducible genes for sporulation, autolysis, and uptake or me- tabolism of minor C or N substrates (SAUER and DURRE, 1992). This codon distribution could either reflect an evolutionary process with low mutational pressure on weakly ex- pressed genes or represent a novel transla- tional control mechanism. Such a system has been found in the genus Streptomyces where a rare tRNA controls production of aerial mycelium and antibiotics (LESKIW et al., 1991).
3.4 Strain Improvement
Various research activities are devoted to the solution of cultivation and product recov- ery problems from which the acetone-buta- no1 fermentation suffers most. In addition, knowledge on the physiology and genetics of C. acetobutylicum and C. beijerinckii is rap- idly increasing. Besides the work concerned with correlation of culture conditions to the physiology of the organism and with the regu- latory mechanisms controlling the key en- zymes of the different metabolic pathways, there is great interest in removing some limit- ing features of C. acetobutylicum. An im- provement of the microorganisms could in- clude: increased resistance to butanol, fewer end products (e.g., no acetone), and broader substrate range (e.g., cellulose).
Butanol toxicity is the limiting factor with respect to the maximum amount of solvents (acetone, butanol, ethanol) to be achieved in the fermentation broth (about 20 g L- ') . Im- provement in this area is critical for the eco- nomics of the process (reduction of product recovery costs). It is generally accepted that the cell membrane is one target of alcohol at- tack (INGRAM, 1986). Owing to their amphi- phatic character, alcohols dissolve in the membrane lipids, increase their permeability (DOMBEK and INGRAM, 1984), and affect membrane fluidity (VOLLHERBST-SCHNECK et al., 1984). Thus, high concentrations of bu- tanol lead to a complete abolition of the pH
4 New Developments of the Fermentation Process 247
gradient, lower the intracellular level of ATP, cause the release of intracellular metabolites, and inhibit sugar uptake (MOREIRA et al., 1981; GOTTWALD and GOTTSCHALK, 1985; BOWLES and ELLEFSON, 1985; HUTKINS and KASHKET, 1986). These effects clearly de- monstrate the importance of the cell mem- brane with respect to butanol tolerance. Oth- er sites of interference, e.g., inhibition of gly- colytic enzymes (HERRERO, 1983), cannot be excluded. In view of the complex effects of butanol, it might be very difficult to isolate strains of C. acetobutylicum with a substantial higher butanol tolerance. Chemical mutagen- esis has been used to obtain butanol-tolerant strains that, however, produced only little more butanol (if at all) than the wild type (HERMANN et al., 1985; LEMMEL, 1985; LIN and BLASCHEK, 1983).
Non-production of the less desirable sol- vent acetone can be achieved by selecting for mutants in the presence of 2-bromobutyrate (JANATI-IDRISSI et al., 1987). Low acetone yields also result from growth on whey or un- der iron limitation (BAHL et al., 1986). Im- provements in substrate utilization have been attempted by isolating mutants with enhanced amylolytic activity (ANNOUS and BLASCHEK, 1991) or by transformation of endoglucanase genes from C. cellulovorans or C. thermocel- lum into C. acetobutylicum (KIM et al., 1994; OULTRAM et al., 1990). A class of sponta- neous mutants of C. acetobutylicum NRRL B643 resistant to ally1 alcohol produced con- siderable quantities of butyraldehyde in addi- tion to acetone and butanol which could be of interest for chemical syntheses (ROGERS and 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 et al., 1992; LEE et al., 1992; MINTON et al., 1993; STRATZ et al., 1994; TRUFFAUT and SEBALD, 1988; TRUF- FAUT et al., 1989; YOON et al., 1991; YOSHI- NO et al., 1990), transformation and electro- poration protocols have been worked out (LIN and BLASCHEK, 1984; MERMELSTEIN et al., 1992; OULTRAM et al., 1988a; REID et al., 1983; REYSSET et al., 1988; REYSSET and SE-
BALD, 1993), DNA transfer by conjugation is easily performable (OULTRAM and YOUNG, 1985; OULTRAM et al., 1987,1988b; REYSSET and SEBALD, 1985; WILKINSON and YOUNG, 1994; WILLIAMS et al., 1990; YOUNG, 1993; Yu and PEARCE, 1986), the isolation of a fil- amentous phage led to the construction of a phagemid (KIM and BLASCHEK, 1991, 1993), and transposon mutagenesis is well estab- lished (BERTRAM and DURRE, 1989; BERT-
al., 1989; YOUNG, 1993). Very important was the finding that DNA to be transformed into C. acetobutylicum must be methylated to avoid restriction (MERMELSTEIN and PA- POUTSAKIS, 1993). Protease-deficient mutants will be helpful for the study of protease-labile proteins (SASS et al., 1993) and DNase-nega- tive mutants might be helpful in genetic ma- nipulations (BURCHHARDT and DURRE, 1990).
Recently, an artificial operon (ace operon) has been constructed containing the adc, ctfA, and ctfB genes, all transcribed from the adc promoter. After transformation into a ace- tone-butanol-negative mutant of C. acetobu- tylicum, the recombinant strain regained the ability to form acetone (MERMELSTEIN et al., 1993). When introduced into the wild type the ace-containing plasmid also led to an increase in solvent formation, although plasmid with- out insert caused a significant (but somewhat lower) stimulation of acetone, butanol, and ethanol production. Thus, genetic manipula- tions to alter the product formation capabili- ties are now available and might lead to a reintroduction of this fermentation at an in- dustrial scale.
RAM et al., 1990 DURRE, 1993; WOOLLEY et
4 New Developments of the Fermentation Process
The industrial acetone-butanol fermenta- tion process, which was in use for over forty years, is documented well and in detail in the literature (BEESCH, 1952; PRESCOTT and DUNN, 1959; Ross, 1961; HASTINGS, 1978; SPIVEY, 1978; JONES and WOODS, 1986;
248 6 Microbial Production of Acetone/Butanol/lsopropanol
BAHL and GOTISCHALK, 1988). This tradi- tional commercial batch process of acetone- butanol fermentation is no longer in use. The main factors influencing the unfavorable eco- nomics were high raw material and transpor- tation costs and intrinsic limitations (low sol- vent yields and low final concentrations, un- desirable solvent ratios) resulting in high processing costs and waste disposal prob- lems.
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 com- pete with the synthetic route that used cheap oil as a feedstock. Although in some cases al- ternative substrates were available, transpor- tation costs of the bulky material prevented its use. In addition, a theoretical calculation taking the biochemical pathways of C. aceto- butylicum into account showed that the maxi- mum possible yield of solvents is 0.38 g per g of glucose converted (LEUNG and WANG, 1981). In practice, this yield was not always reached, particularly with molasses as sub- strate. Also, the productivity of a typical batch fermentation process was low (0.24.6 kg m P 3 h- ’ solvents). The solvents repre- sented a mixture of acetone, butanol, and ethanol, whereas often only one product, e.g., butanol as desired.
Another major drawback is the low buta- no1 tolerance of C. acetobutylicum. A maxi- mum total solvent concentration of 20 kg m-3 can be achieved in the fermentation broth. The recovery of solvents from this dilute solu- tion (normally by distillation) resulted in sub- stantial 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 con- centrations the acetone-butanol fermentation generates especially large volumes of waste. The disposal of such waste often was a prob- lem (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 fermenta- tion process. Included are topics such as alter- native substrates, continuous culture, immo- bilized cells, cell recycling, and product recov- ery. Progress in several of these areas will be crucial for renewed economic viability of ace- tone-butanol fermentation. Three parameters are important for the evaluation of new pro- cesses 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 produc- tivity [g solvents L-’ h-’I.
4.1 Continuous Culture
A fermentation process that can be oper- ated continuously has some advantages com- pared to a batch process. Only one series of inoculum cultures is needed for a long pro- duction 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 re- duced without a loss of production capacity (higher productivities). The use of a contin- uous process for acetone-butanol fermenta- tion 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-buta- no1 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) de- scribed a similar fermentation carried out in a pilot plant consisting of a chain of eleven fer- menters. Detailed information of the condi- tions of these continuous fermentations was not given.
4 New Developments of the Fermentation Process 249
To be economically attractive a continuous process must at least achieve the final concen- tration and yield of solvents that can be ob- tained in batch culture. In addition, signifi- cantly higher productivities are necessary.
In recent years, the continuous culture of C. acetobutylicum has been mainly used as a research tool to define parameters responsi- ble for changes in the physiology and the ac- tivity of this microorganism. Under steady- state conditions a constant environment is provided and the influence of a single param- eter 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, cul- ture pH, dilution rate ( D ) , maximum attaina- ble 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 sin- gle 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 glu- cose-, nitrogen-, or magnesium-limited che- mostats steady-state solvent production was low or difficult to maintain, and an applica- tion of these kinds of limitations to an indus- trial process seems unlikely (GOTTSCHAL and MORRIS, 1981b; BAHL et al., 1982a; AN- DERSCH et al., 1982; MONOT and ENGGAS- SER, 1983; JOBSES and ROELS, 1983; STEPH- ENS 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 (BAHL et al., 1982b; BAHL and GOTTSCHALK, 1984).
The pH value had an important influence on the product pattern of C. acetobutylicum in continuous culture. Significant solvent pro- duction 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. acetobutyli- cum DSM 1731: pH 4.3 (BAHL et al., 1982a, b); ATCC 824: pH 4.8-5.0 (MONOT and EN- GASSER, 1983). The temperature applied was in the same range as in the batch process (33-37°C) and had minor influence. In- creased levels of butyrate and/or acetate are able to induce solvent production in batch culture (GO~TSCHAL and MORRIS, 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 ob- tained at a low dilution rate (Ds0 .05 h-I) (LEUNG and WANG, 1981; BAHL et al., 1982b). Although higher dilution rates re- sulted in improved productivity, this was at the expense of reduced substrate turnover and solvent concentration.
Using a two-stage phosphate-limited che- mostat BAHL et al. (1982b) reported solvent concentrations of 18.2 g L - ' with a yield of 0.34 g solvents per g glucose and a productivi- ty 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, 33°C). Compared to a batch culture using the same strain (C. acetobutylicum DSM 1731) and medium, the productivity doubled without a loss of substrate utiliza- tion, solvent concentration, or yield (BAHL et al., 1982b).
Although the economics remain to be es- tablished this two-stage process seems to be promising. Performance of the fermentation in this way has the advantage that the condi- tions 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 sec- ond stage is primarily devoted to the conver- sion 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 degenera- tion and difficulties in maintaining steady-
N
VI
0
Tab
. 7. P
rodu
ctio
n of
Sol
vent
s by
Clo
stri
dium
ace
tobu
tyku
m i
n C
hem
osta
ts
Ferm
enta
tion
Proc
ess
Res
ults
C
omm
ents
R
efer
ence
s N
utrie
nt
Stra
in
Subs
trat
e T
empe
r- p
H
D
Solv
ents
Pr
oduc
- Y
ield
Li
mita
tion
Con
cen-
at
ure
tivity
rn
trat
ion
% g S
' [g
L-I
I ["C
I W
'I [g
L-7
[g
L-I
h-l
l [&I
a 0.
Glu
cose
2.
7 g
L-'
3.4
g L
-l
Nitr
ogen
0.
4 g
L-'
A
mm
oniu
m
chlo
ride
2g
L-I
A
mm
oniu
m
. sul
fate
0.2
g L
-'
Am
mon
ium
ac
etat
e
Sulf
ate
Mag
nesi
um
sulfa
te
Man
gane
se
sulfa
te
0.05
g L
-
0.01
g L
-'
NC
IB 8
052
2.7
35
5.7
0.08
-
DSM
173
1 3.
4 37
4.
3 0.
13
0.07
NC
IB 8
052
2.7
35
5.7
0.08
-
DSM
173
1 54
37
5.
2 0.
22
2.50
AT
CC
824
45
.5
35
5.0
0.04
8.
00
DSM
173
1 54
37
4.
3 0.
10
4.3
- -
0.01
0.
02
-
-
0.95
0.
08
0.30
0.
29
0.43
0.
32
No
solv
ent p
rodu
ctio
n
Indu
ctio
n of
sol
vent
for
- m
atio
n by
low
erin
g th
e pH
bel
ow 5
.0; o
pti-
mum
: pH
4.3
No
solv
ent p
rodu
ctio
n at
re
lativ
ely
high
pH
val
ues
and
low
sug
ar c
once
ntra
- tio
ns
Solv
ent p
rodu
ctio
n at
pH
va
lues
fro
m 5
.2 t
o 4.
3 an
d at
hig
h gl
ucos
e co
ncen
- tr
atio
ns
Solv
ent
prod
uctio
n at
low
di
lutio
n ra
te, l
ow p
H,
and
high
glu
cose
con
cen-
tr
atio
ns
3 a
MO
RR
IS (1
981b
) R
E B
AH
L et
al.
3 (1
982a
) 3
% 8
MO
RR
IS (1
981b
) E s %
(198
2)
5 :
GO
TTSC
HA
L and
b
cs $
GO
TTSC
HA
L and
AN
DE
RSC
H et a
l. 2
3
MO
NO
T and
2
ENG
ASS
ER
(198
3)
Yie
ld c
ompa
rabl
e to
B
AH
L an
d ba
tch
proc
ess;
hig
her
sol-
GO
TTSC
HA
LK
vent
con
cent
ratio
n po
ssi-
(198
4)
ble
at lo
wer
dilu
tion
rate
s
Phos
phat
e Tw
o-st
age
stag
e I
chem
osta
t 0.
1 g
L-'
D
SM 1
731
54
37
4.3
0.12
5 18
.2
Mon
opot
assi
um
stag
e I1
phos
phat
e 33
4.
3 0.
03
Mag
nesi
um
0.02
g L
- '
DSM
173
1 54
37
4.
3 0.
06
0.8
Mag
nesi
um
sulfa
te
0.44
0.
34
Succ
essf
ul l
abor
ator
y-
BA
HL
et a
l. sc
ale
two-
stag
e sy
stem
for
(1
982b
) co
ntin
uous
sol
vent
pro
- du
ctio
n, n
o cu
lture
de-
ge
nera
tion
over
a p
erio
d of
one
yea
r
0.05
0.
07
Poor
sol
vent
pro
duct
ion
BA
HL
and
Gor
r-
at lo
w p
H, l
ow d
ilutio
n ra
te, a
nd e
xces
s of
sub
- st
rate
SCH
AL
K (
1984
)
252
Tab. 8. Concentration of Products and Consumption of Substrate in a Two-Stage Continuous Culture of Clostridium acetobutylicum under Phosphate Limitation (BAHL et al., 1982b)
6 Microbial Production of Acetone/ButanoUIsopropanol
Stage 1 Stage 2 ~
Process Parameter PH Temperature ["C] Dilution rate [h-'1
Butanol Acetone Ethanol Butyrate Acetate
Products [g L-'1
Consumption of substrate [%]
Yield [g solventlg glucose] Productivity [g solvent L - ' h - ' I
4.3
0.125
3.6 1.6 0.3 1.3 0.4
37
40
0.25 0.08
4.3
0.03 33
12.6 4.8 0.8 0.8 0.5
99.7 0.34 0.44"
a Productivity of the overall process
state solvent production over a longer period (i. e., >200 h) have often been encountered (STEPHENS et 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 sol- vent 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 pre- vented. It is interesting that under these con- ditions an asporogenous strain of C. acetobu- tylicum was selected with an unchanged capa- bility of producing solvents (MEINECKE et al., 1984). Other continuous flow fermentation processes (excess substrates, LEUNG and WANG, 1981; turbidostat, GOTTSCHAL and MORRIS, 1982; pH-auxostat, STEPHENS et al., 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 con- centration of solvents in the fermentation broth are the major drawbacks. Improvement in this field is necessary before a reintroduc- tion of the acetone-butanol fermentation process can be envisaged.
Cell immobilization is a technique to con- fine the biocatalysts within the fermenter sys- tem. Thereby higher cell densities are possi- ble 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 immobiliza- tion conditions.
Immobilization of vegetative cells and spores of C. acetobutylicum has been de- scribed. Calcium alginate (spherical beads or spiral wound flat sheets) was used to entrap the organism for a continuous solvent produc- tion in a glucose medium (HAGGSTROM and ENFORS, 1982; FORERG et al., 1983). Packed- bed reactors, continuous stirred tank reactors, and fluidized column reactors served as reac- tion vessels.
4 New Developments of the Fermentation Process 253
Generally, higher solvent productivities (2.4-2.8 g L - ' h- ') have been obtained. However, substrate utilization and solvent concentrations were low (1.54.5 g L-I). An- other problem is the rapid loss of activity of C. acetobutylicum in non-growth media. The pulsewise addition of nutrients to the fermen- tation medium resulted in some improvement (FORBERG et al., 1983).
In addition to the entrapment technique, adsorption of cells to a solid surface can be used for immobilization. The successful ad- sorption of C. acetobutylicum to beech wood shavings (FORBERG and HAGGSTROM, 1985), arranged as parallel sheets, is promising since it is a simple, cheap, and non-toxic immobili- zation method. This process using the inter- mittent 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 re- ported (LARGIER et al., 1985). At a dilution 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 repre- sents major progress as compared to immobil- ized wild type cells.
These results clearly show that solvent pro- duction with immobilized cells is principally possible. Advances in other fields, e.g., strain improvement are necessary for the applica- tion 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. aceto- butylicum 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 et al., 1985; SCHLOTE and GOTT- SCHALK, 1986). Instability of the cells with re- spect to solvent formation was again observed after a long operation period at high solvent concentrations. AFSCHAR et al. (1985) could 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 phosphate- limited chemostat with cell recycling. The special conditions of phosphate limitation and the low pH of 4.3 might have been responsi- ble for this stability of the culture.
Industrial application of this technique will depend on the availability of filtration mod- ules that are easy to handle and not affecting the activity of the C. acetobutylicum cells. A wide variety of new membrane types in differ- ent 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 suspen- sions.
In Tab. 9 the solvent concentrations, yields, and productivities obtained in different con- tinuous acetone-butanol fermentations with cell recycle or immobilized cells are summa- rized.
4.3 Product Recovery
The traditional recovery by distillation of the accumulated solvents present in the fer- mentation broth in relatively low concentra- tions results in high costs and is one of the major drawbacks to the acetone-butanol fer- mentation. As long as strains of C. acetobuty- licum tolerating higher concentrations of bu- tanol are not available improvement of this fermentation might come from a more eco- nomic recovery process. Furthermore, as de- scribed above, substantial progress has been made in the development of continuous pro- cesses. However, the high productivities of such processes were often accompanied by re- duced 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 prod- uct removal technique. Such techniques could include the use of membranes (reverse osmo- sis, 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 Concen- Yield Prod u c - Reference
6 Microbial Production of AcetondButanoWlsopropanol
tration tivity [g L-'I [g g-'I tg L-I h- l l
Immobilized cells Single-stage, complex medium,
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,
Cell recycle Single-stage, synthetic medium,
Cell recycle Single-stage, synthetic medium,
intermittent feeding
steady state
steady state
phosphate-limited, steady state
1 .00 0.20 0.70 FORBERG et al., 1983
15.42 0.34 3.02 LARGIER et al., 1985
3.94 0.21 4.02 FRICK and SCHUGERL, 1986
4.10 0.23 4.10 QURESHI and MADDOX, 1987
12.00 0.30 3.00 AFSCHAR et al., 1985
13.00 0.29 6.50 PIERROT et al., 1986
21.71 0.32 2.17 SCHLOTE and GOTTSCHALK, 1986
detailed overview of these technologies and their future prospects is given by ENNIS et al. (1986a). Although most of the work aimed at the possible use of alternative solvent recove- ry processes and is related to ethanol fermen- tation, there is great interest to apply the re- sults to the acetone-butanol fermentation as well (ENNIS et al., 1986b; DADGAR and FOUTCH, 1985; TAYA et al., 1985; GARCIA I11 et al., 1984, 1986; GROOT et al., 1992). Sol- vent 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 activi- ty. Corn oil, paraffin oil, kerosene, dibutyl phthalate (WANG et al., 1979), and oleyl alco- hol (cis-9-octadecene-1-01) (TAYA et al., 1985) proved to be good extractants for buta- nol. By automatic withdrawing and feeding
operations of oleyl alcohol regulated by the volume of gas evolved, the glucose concentra- tion used in a fed-batch extractive fermenta- tion system of C. acetobutylicum was 120 g L- ' and the solvent concentration was held below 2 g L-'. The total amount of butanol pro- duced 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 sys- tem in batch culture resulted in an increase in glucose utilization and in continuous culture with immobilized cells in higher glucose con- version and higher productivity (GROOT et al., 1984a, b).
GARCIA I11 et al. (1986) examined the sep- aration of butanol from the fermentation broth by reverse osmosis. They concluded that the integration of membrane technology could overcome the problems of low produc- tivity and dilute product concentration asso- ciated with the acetone-butanol fermenta- tion. Development in membrane technology
4 New Developments of the Fermentation Process 255
is one factor influencing the economic feasi- bility of alternative product recovery pro- cesses 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, mem- brane solvent extraction) have been directly compared using otherwise identical fermenta- tion conditions (GROOT et al., 1992). From these, pervaporation and liquid-liquid extrac- tion were found to have the greatest poten- tials. MADDOX et al. (1994) investigated four product removal techniques and showed that gas stripping allowed the highest solvent pro- duction and productivity.
4.4 Alternative Fermentation Substrates
Starch and molasses were the traditional raw materials for the biological production of acetone and butanol. However, a wide variety of other fermentable sugars from other sources, including wastes, are potential sub- strates for this fermentation process. Exam- ples of alternative resources are apple po- mace containing fructose, glucose, sucrose as fermentable sugars (VOGET et al., 1985), whey (lactose) (MADDOX et al., 1994), Jeru- salem artichokes (fructan) (MARCHAL et al., 1985), and lignocellulose (xylan and cellu- lose) (MADDOX and MURRAY, 1983; Y u and SADDLER, 1983; Yu et al., 1985; FOND et al., 1983). Although lignocellulose as raw materi- al is potentially cheap and abundant more fundamental physiological, biochemical, and genetic research has to be done before it can be used in an industrial acetone-butanol fer- mentation process (BAHL and GOTTSCHALK, 1988). A strain converting these polymers di- rectly to solvents, e.g., butanol, would be highly desirable.
Whey, on the other hand, seems to be an ideal alternative substrate for acetone-buta- no1 fermentation and will be considered in more detail (MADDOX et al., 1994). Whey is a by-product during the manufacture of cheese or casein and represents a major waste dispo-
sal problem to the dairy industry if it is not further used, e. g., for the manufacture of lac- tose. The lactose content of whey is 4 5 % (w/v) and C. acetobutylicum is capable of ferment- ing 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 al- most optimal for the acetone-butanol fer- mentation. The amount of sugar that can be utilized by C. acetobutylicum is limited by the product toxicity, especially of butanol. C. ace- tobutylicum yields the maximum possible sol- vent concentration with about 6% (w/v) ini- tial fermentable carbohydrate in the medium. Furthermore, it should be mentioned that bu- tanol/acetone ratios after fermentation of whey are higher (e.g., 1O:l) as compared to those obtained from starch or molasses (2:l). For an industrial process this preponderance of butanol is useful not only from the view- point of the desired product, but it would also simplify the product recovery process. Al- though not all factors responsible for the shift of the butanol/acetone ratio are known, it was shown that iron limitation had the greatest ef- fect on this ratio (BAHL et al., 1986). On the other hand, whey is a relatively poor medium. Reduced productivities compared to molasses as substrate and incomplete utilization of lac- tose are the major problems (MADDOX, 1980 WELSH and VELIKY, 1984; ENNIS and MAD- DOX, 1985; LINDEN et al., 1986). However, an optimized fermentation process for the pro- duction of butanol from whey has been de- scribed recently (MADDOX et al., 1994). The improved process includes a fluidized bed reactor of bonechar-immobilized cells cou- pled with pervaporation to remove and con- centrate the solvents. With respect to eco- nomics the use of such a process would result in a product price of $0.62 (US.) per liter for a plant capacity of 900 m3 whey permeate per day based on a whey permeate price of $ 116 (U.S.). In the case that whey permeate can be obtained at zero cost, the price drops to $0.21 (U.S.). Thus the acetone-butanol fermenta- tion may be one economically viable way of producing a useful product from whey.
256 6 Microbial Production of Acetone/ButanoWlsopropanol
) HzandCOz
Efnuent
:ell Concentrate I
Reverse Osmosis Pervaporation
Liquid-Liquid Extraction
Stripping AdsorptiOll
+ Excess Cell Mass I
4 Growth Initiation of Solvent
Formation
Production of
Solvents
Reservoir I
ultrafillrale Containing
Cell Recyle I Ultrnliltration
k-Pmjuction 4- Recovery w
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 neces- sary for an optimized acetone-butanol fer- mentation. A promising example was given by FRIEDL et al. (1991). A stable high solvent productivity of 3.5 g L- ' h- ' was obtained using immobilized cells of C. acetobutylicum coupled with product removal by pervapora- tion. 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 -' lac- tose). In Fig. 5, a possible flow diagram of an optimized acetone-butanol fermentation is shown taking the recent developments of fer- mentation 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 pro- duction 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 re- garding the physiology, biochemistry and ge- netics of Clostridium acetobutylicum. Further- more, recent developments in technology of continuous fermentation using free or immo- bilized cells are promising and the integration of sophisticated product removal techniques into these highly productive systems can over- come the severe problem of product inhibi- tion 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 inte- grated product removal. In addition, based on the progress made in recent years with re- spect to basic research on C. acetobutylicum it seems to be possible now to construct strains with improved features for a commercial ap- plication. In general, the results obtained to
6 References 257
tion, CRC Crit. Rev. Microbiol. 15 (Suppl. l),
AZEDDOUG, H., HUBERT, J., REYSSET, G. (1992), Stable inheritance of shuttle vectors based on plasmid pIMl3 in a mutant strain of Clostridium acetobutylicum, J. Gen. Microbiol. 138, 1371- 1378.
BAHL, H. (1993), Heat shock response and onset of solvent formation in Clostridium acetobutylicum, in: The Clostridia and Biotechnology (WOODS, D. R., Ed.), pp. 247-259, Stoneham: Butter- worth-Heinemann.
BAHL, H., GOITSCHALK, G. (1984), Parameters af- fecting solvent production by Clostridium aceto- butylicum in continuous culture, Biotechnol. Bioeng. Symp. 14, 215-223.
BAHL, H., GOTTSCHALK, G. (1988), Microbial pro- duction of butanollacetone, in: Biotechnology 1st Edn., Vol. 6b (REHM, H.-J., REED, G., Eds.), pp. 1-30, Weinheim: VCH.
BAHL, H., ANDERSCH, W., BRAUN, K., GOTT- SCHALK, G. (1982a), Effect of pH and butyrate concentration on the production of acetone and butanol by Clostridium acetobutylicum grown in continuous culture, Eur. J. Appl. Microbiol. Bio- technol. 14, 17-20.
BAHL, H., ANDERSCH, W., GOTTSCHALK, G. (1982b), Continuous production of acetone and butanol by Clostridium acetobutylicum in a two- stage phosphate limited chemostat, Eur. J. Appl. Microbiol. Biotechnol. 15, 201-205.
BAHL, H., GOTTWALD, M., KUHN, A., RALE, V., ANDERSCH, W., GOTTSCHALK, G. (1986), Nu- tritional factors affecting the ratio of solvents produced by Clostridium acetobutylicum, Appl. Environ. Microbiol. 52, 169-172.
BALLONGUE, J., AMINE, J., MASION, E., PETITDE- MANGE, H., GAY, R. (1985), Induction of ace- toacetate decarboxylase in Clostridium acetobu- tylicum, FEMS Microbiol. Lett. 29, 273-277.
BALLONGUE, J., AMINE, J., MASION, E., PETITDE- MANGE, H., GAY, R. (1986), R61e de l’acktate et du butyrate dans I’induction de la NADH:rubre- doxine oxydorCductase chez Clostridium aceto- butylicum, Biochimic 68, 575-580.
BEESCH, S. C. (1948), Process for fermenting car- bohydrates to butanol and acetone, U. s. Patent 2439791.
BEESCH, S. C. (1952), Acetone-butanol fermenta- tion of sugars, Eng. Proc. Dev. 44, 1677-1682.
BEHRENS, S., NARBERHAUS, F., BAHL, H. (1993), Cloning, nucleotide sequence and structural analysis of the Clostridium acetobutylicum d n d gene, FEMS Microbiol. Lett. 114, 53-60.
S33-S67. date suggest that an economically viable ace- tone-butanol fermentation is at least techni- cally possible and could be reintroduced on an industrial scale.
Acknowledgement
Work in the authors’ laboratories has been supported by grants from the Deutsche For- schungsgemeinschaft and the Bundesminister fur Forschung und Technologie.
6 References AFSCHAR, A. S., BIEBL, H., SCHALLER, K., SCHU-
GERL, K. (1985), Production of acetone and bu- tanol by Clostridium acetobutylicum in contin- uous culture with cell recycle, Appl. Microbiol. Biotechnol. 22, 394-398.
ALLCOCK, E. R., WOODS, D. R. (1981), Carboxy- methyl cellulase and cellobiase production by Clostridium acetobutylicum in an industrial fer- mentation medium, Appl. Environ. Microbiol.
ANDERSCH, W., BAHL, H., GOTTSCHALK, G. (1982), Acetone-butanol production by Clos- tridium acetobutylicum in an ammonium-limited chemostat at low pH values, Biotechnol. Lett. 4,
ANDERSCH, W., BAHL, H., GOTTSCHALK, G. (1983), Level of enzymes involved in acetate, bu- tyrate, acetone and butanol formation by Clos- tridium acetobutylicum, Appl. Microbiol. Bio- technol. 18, 327-332.
ANDREESEN, J. R., GOTTSCHALK, G. (1969), The occurrence of a modified Entner-Doudoroff pathway in Clostridium aceticum, Arch. Micro- biol. 69, 16G170.
ANNOUS, B. A., BLASCHEK, H. P. (1991), Isolation and characterization of Clostridium acetobutyli- cum mutants with enhanced amylolytic activity, Appl. Environ. MicrobioI. 57, 2544-2548.
ARROYO, R. (1938), Art of producing butanol and acetone by fermentation of molasses, U. S. Pa- tent 2113471.
ARZBERGER, C. F. (1936), Process for the produc- tion of butyl alcohol by fermentation, U. S. Pa- tent 2 050 21 9.
ARZBERGER, C. F. (1938), Process for the produc- tion of butyl alcohol by fermentation, U. S. Pa- tent 2 139 108.
AWANG, G. M., JONES, G. A., INGLEDEW, W. M. (1988), The acetone-butanol ethanol fermenta-
41, 539-541.
29-32.
258 6 Microbial Production of Acetone/ButanoWlsopropanol
BEIJERINCK, M. W. (1893), Ueber die Butylalko- 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 Clostrid- ium pasteurianum, Arch. Microbiol. 103, 21-30.
BERTRAM, J., DURRE, P. (1989), Conjugal transfer and expression of streptococcal transposons in Clostridium acetobutylicum, Arch. Microbiol.
BERTRAM, J., KUHN, A., DURRE, P. (1990), Tn916- induced mutants of Clostridium acetobutylicum defective in regulation of solvent formation, Arch. Microbiol. 153, 373-377.
BOTKIN, S. (1892), Ueber einen Bacillus butyricus, Z. Hyg. Infektionskrankh. 11, 421-434.
BOWLES, L. K., ELLEFSON, W. L. (1985), Effects of butanol on Clostridium acetobutylicum, Appl. Environ. Microbiol. 50, 1165-1170.
BREDEMANN, G. (1909), Bacillus amylobacter A.M. et Bredemann in morphologischer, physio- logischer und systematischer Beziehung. Mit be- sonderer Berucksichtigung des Stickstoffbin- dungsvermogens dieser Spezies, Cbl. Bakteriol. Parasitenkd. Infektionskrankh., 11. Abt. 23, 385- 568.
BREZNAK, J. A,, COSTILOW, R. N. (1994), Physico- chemical factors in growth, in: Methods for Gen- eral and Molecular Bacteriology ( GERHARDT, P., MURRAY, R. G. E., WOOD, W. A,, KRIEG, N. R., Eds.), pp. 137-154, Washington: Am. SOC. Microbiol.
BUCHNER, E., MEISENHEIMER, J. (1908), Uber Buttersauregarung, Ber. Dtsch. Chem. Ges. 41, 1410-1419.
BURCHHARDT, G., DURRE, P. (1990), Isolation and characterization of DNase-deficient mutants of Clostridium acetobutylicum, Curr. Microbiol.
CALAM, C. T. (1979), Isolation of Clostridium ace- tobutylicum strains producing butanol and ace- tone, in: Proc. 4th Symp. “Technische Mikrobio- logie” (DELLWEG, H., Ed.), pp. 233-242, Berlin: Verlag Versuchs- und Lehranstalt fur Spiritusfa- brikation und Fermentationstechnologie im In- stitut fur Garungsgewerbe und Biotechnologie.
CARNARIUS, E. H., MCCUTCHAN, W. N. (1938), Process for the production of butyl alcohol by fermentation, U. S. Patent 2139111.
CARY, J. W., PETERSEN, D. J., PAPOUTSAKIS, E. T., BENNETT, G. N. (1988), Cloning and expres- sion of Clostridium acetobutylicum phospho- transbutyrylase and butyrate kinase genes in Escherichia coli, J. Bacteriol. 170, 46134618.
CARY, J. W., PETERSEN, D. J., PAPOUTSAKIS, E. T., BENNETT, G. N. (1990), Cloning and expres-
151,551-557.
21, 307-311.
sion of Clostridium acetobutylicum ATCC 824 acetoacetyl-coenzyme A:acetate/butyrate:coen- zyme A-transferase in Escherichia coli, Appl. Environ. Microbiol. 56, 15761583.
CATO, E. P., GEORGE, W. L., FINEGOLD, S. M. (1986), Genus Clostridium Prazmowski 1880, 23AL, in: Bergey’s Manual of Systematic Bacteri- ology (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 solvent- forming enzymes of clostridia, in: The C‘lostridia and Biotechnology (WOODS, D. R., Ed.), pp. 51- 76, Stoneham: Butterworth-Heinemann.
CHEN, J.-S., Hru, S. F. (1986), Acetone-butanol- isopropanol production by Clostridium beijer- inckii (synonym, Clostridium butylicum), Bio- technol. Lett. 8, 371-376.
CHOJECKI, A., BLASCHEK, H. P. (1986), Effect of carbohydrate source on a-amylase and gluco- amylase formation by Clostridium acetobutyli- cum SA1, J. Ind. Microbiol. 1, 63-67.
COLBY, G. D., CHEN, J.-S. (1992), Purification and properties of 3-hydroxybutyryl-coenzyme A de- hydrogenase from Clostridium beijerinckii (“Clostridium butylicum”) NRRL B593, Appl. Environ. Microbiol. 58, 3297-3302.
CONTAG, P. R., WILLIAMS, M. G., ROGERS, P. (1990), Cloning of a lactate dehydrogenase gene from Clostridium acetobutylicum B643 and ex- pression in Escherichia coli, Appl. Envrron. Mi- crobiol. 56,3760-3765.
CYNKIN, M. A., DELWICHE, E. A. (1958), Metabol- ism of pentoses by clostridia. I. Enzymes of ri- bose dissimilation in extracts of Clostridium per- fringens, J. Bacteriol. 75, 331-334.
CYNKIN, M. A,, GIBBS, M. (1958), Metabolism of pentoses by clostridia. 11. The fermentation of C I4-labeled pentoses by Clostridium perfringens, Clostridium beijerinckii, and Clostridium butyli- cum, J. Bacteriol. 75, 335-338.
DABROCK, B., BAHL, H., GOTISCHALK, G. (1992), Parameters affecting solvent production by Clos- tridium pasteurianum, Appl. Environ. Microbiol.
DADGAR, A. M., FOUTCH, G. L. (1985), Evalua- tion of solvents for the recovery of Clostridium fermentation products by liquid-liquid extrac- tion, Biotechnol. Bioeng. Symp. 15, 611420.
DAVIES, R., STEPHENSON, M. (1941), Studies on the acetone-butyl alcohol fermentation. I. Nutri- tional and other factors involved in the prepara- tion of active suspensions of CI. acetobutylicum (Weizmann), Biochem. J. 35, 132CL1331.
DIEZ-GONZALEZ, F., HUNTER, J. B., RUSSELL. J. B. (1994), The “acetilactic” fermentation of Clostridium acetobutylicum P262, Abstr. Cen.
58, 1233-1239.
6 References 259
Meet. Am. SOC. Microbiol., Las Vegas, May 23-
DOMBECK, K. M., INGRAM, L. 0. (1984), Effects of ethanol on the Escherichia coli plasma mem- brane, J. Bacteriol. 157, 233-239.
DUCLAUX, E. (1895), Sur la nutrition intra-cellu- 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: Butterworth- Heinemann.
DURRE, P., KUHN, A., GOTTWALD, M., GOTT- SCHALK, G. (1987), Enzymatic investigations on butanol dehydrogenase in extracts of Clostrid- ium acetobutylicum, Appl. Microbiol. Biotech- nol. 26,268-272.
DURRE, P., BAHL, H., GOTTSCHALK, G. (1992), Die Aceton-Butanol-Garung: Grundlage fur ei- nen modernen biotechnologischen ProzeB? Chem. Ing. Tech. 64,491-498.
DYR, J., PROTIVA, J., PRAUS, R. (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.
ENNIS, B. M., MADDOX, I. S. (1985), Use of Clos- tridium acetobutylicum P 262 for production of solvents from whey permeate, Biotechnol. Lett.
ENNIS, B. M., GUTIERREZ, N. A,, MADDOX, I. S. (1986a), The acetone-butanol-ethanol fermen- tation: A current assessment, Process Biochem.
ENNIS, B. M., MARSHALL, C. T., MADDOX, I. S., PATERSON, A. H. J. (1986b), Continuous prod- uct recovery by in situ gas strippinglcondensa- tion during solvent production from whey per- meate using Clostridium acetobutylicum, Bio- technol. Lett. 8, 725-730.
ENSLEY, B., MCHUGH, J. J., BARTON, L. L. (1975), Effect of carbon sources on formation of a- amylase and glucoamylase by Clostridium aceto- butylicum, J. Gen. Appl. Microbiol. 21, 51-59.
FERNBACH, A,, STRANGE, E. H. (1911), Improve- ments in the manufacture of higher alcohols, British Patent 15 204.
FERNBACH, A,, STRANGE, E. H. (1912), Improve- ments connected with fermentation processes for the production of acetone, and higher alco- hols, from starch, sugars, and other carbohydrate materials, British Patent 21 073.
FICK, M., PIERROT, P., ENGASSER, J. M. (1985), Optimal conditions for long-term stability of acetone-butanol production by continuous cul-
27, 0-52.
7,503-508.
21, 131-147.
tures of Clostridium acetobutylicum, Biotechnol. Lett. 7, 503-508.
FINN, R. K, NOWREY, J. E. (1959), A note on the stability of clostridia when held in continuous culture, Appl. Microbiol. 7, 29-32.
FISCHER, R. J., HELMS, J., DURRE, P. (1993), Clon- 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. (1882), Ueber Spaltpilzgahrungen. VII. Mittheilung, Ber. Dtsch. Chem. Ges. 15, 867- 880.
FOND, O., PETITDEMANGE, E., PETITDEMANGE, H., ENGASSER, J. M. (1983), Cellulose fermenta- tion by a coculture of a mesophilic Clostridium and Clostridium acetobutylicum, Biotechnol. Bioeng. Symp. 13, 217-224.
FORBERG, C., HAGGSTROM, L. (1985), Control of cell adhesion and activity during continuous pro- duction 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, 143- 147.
FREIBERG, G. W. (1925), Process for producing acetone and butyl alcohol, U. S. Patent 1537597.
FREIER, D., GOTTSCHALK, G. (1987), L( +)-lactate 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 Clos- tridium thermosaccharolyticum at neutral pH, Biotechnol. Lett. 11, 831-836.
FRENCH, D., KNAPP, D. W. (1950), The maltase of Clostridium acetobutylicum. Its specificity range and mode of action, J. Biol. Chem. 187, 463- 467.
FRICK, C., SCHUGERL, K. (1986), Continuous ace- tone-butanol production with free and immobil- ized Clostridium acetobutylicum, Appl. Micro- biol. Biotechnol. 25, 186-193.
FRIDOVICH, I. (1972), Acetoacetate decarboxylase, in: The Enzymes (BOYER, P. D., Ed.,), Vol. VI, pp. 255-270, New York: Academic Press.
260 6 Microbial Production of Acetone/ButanoWlsopropanol
FRIEDL, A., QURESHI, N., MADDOX, I. S. (1991), Continuous acetone-butanol-ethanol (ABE) fermentation using immobilized cells of Clostrid- ium acetobutylicum in a packed bed reactor and integration with product removal by pervapora- tion, Biotechnol. Bioeng. 38, 518-527.
FUNK, F. J. (1925), Butyl alcohol and acetone fer- mentation process, U. S. Patent 1538516.
GABRIEL, C. L. (1928). Butanol fermentation pro- cess, Ind. Eng. Chem. 28, 1063-1067.
GARCIA 111, A., IANNOTTI, E. L., FISHER, J. R. (1984), Reverse osmosis application for ace- tone-butanol fermentation, Biotechnol. Bioeng. Symp. 14,543-552.
GARCIA 111, A., IANNOTTI, E. L., FISHER, J. R. (1986). Butanol fermentation liquor production and separation by reverse osmosis, Biotechnol. Bioeng. 28,785-791.
GAVARD, R., HAUTECOEUR, B., DESCOURTIEUX, H. (1957), Phosphotransbutyrylase de Clostrid- ium acetobutylilcum, C. R. Acad. Sci. 244, 2323- 2326.
GEORGE, H. A., JOHNSON, J. L, MOORE, W. E. C., HOLDEMAN, L. V., CHEN, J. S. (1983), Acetone, isopropanol, and butanol production by Clos- tridium bejierinckii (syn. Clostridium butylicum) and Clostridium aurantibutyricum, Appl. Envi- ron. Microbiol. 45, 1160-1163.
GERISCHER, U., DURRE, P. (1990), Cloning, se- quencing, and molecular analysis of the aceto- acetate decarboxylase gene region from Clos- tridium acetobutylicum, J. Bacteriol. 172, 6907- 6918.
GERISCHER, U., DURRE, P. (1992). mRNA analy- sis of the adc gene region of Clostridium aceto- butylicum during the shift to solventogenesis, J. Bacteriol. 174, 426433.
GOODLOVE, P. E., CUNNINGHAM, P. R., PARKER, J., CLARK, D. P. (1989), Cloning and sequence analysis of the fermentative alcohol-dehydro- genase-encoding gene of Escherichia coli, Gene
GOTTSCHAL, J. C., MORRIS, J. G. (1981a), The in- duction of acetone and butanol production in cultures of Clostridium acetobutylicum by ele- vated concentrations of acetate and butyrate, FEMS Microbiol. Lett. U, 385-389.
GOTTSCHAL, J. C., MORRIS, J. G. (1981b), Nonpro- duction of acetone and butanol by Clostridium acetobutylicum during glucose- and ammonium- limitation in continuous culture, Biotechnol. Lett. 3, 525-530.
GOTTSCHAL, .I. C., MORRIS, J. G. (1982), Contin- uous production of acetone and butanol by Clos- tridium acetobutylicum growing in turbidostat culture, Biotechnol. Lett. 4, 477-482.
85,209-214.
GOTTSCHALK, G. (1986). Bacterial Metabolism, 2nd Edn., New York: Springer-Verlag.
GOTTWALD, M., GOTTSCHALK, G. (1985), The in- ternal pH of Clostridium acetobutylicum and its effect on the shift from acid to solvent forma- tion, Arch. Microbiol. 143, 42-46.
GOTTWALD, M., HIPPE, H., GOTTSCHALK, G. (1984), Formation of n-butanol from o-glucose by strains of the “Clostridium tetanomorphunz ” group, Appl. Environ. Microbiol. 48, 573-576.
GRETHLEIN, A. J., WORDEN, R. M., JAIN, M. K., DATTA, R. (1991), Evidence for production of n-butanol from carbon monoxide by Butyribac- terium methylotrophicum, J . Ferment. Bioeng. 72, 58-60,
GRIMBERT, L. (1893), Fermentation anakrobie pro- duite par le Bacillus orthobutylicus. ses varia- tions sous certaines influences biologiques, Ann. Inst. Pasteur 7, 353-402.
GROOT, W. J., SCHOUTENS, G. H., VAN BEELEN, P. N., VAN DEN OEVER, c. E., KOSSEN, N. W. F. (1984a), Increase of substrate conversion by per- vaporation in the continuous butanol fermenta- tion, Biotechnol. Lett. 6, 789-792.
W. F. (1984b), Pervaporation for simultaneous product recovery in the butanol/isopropanol batch fermentation, Biotechnol. Lett. 6, 709- 714.
GROOT, w. J., VAN DER LANS, R. G. J. M., LUYB- EN, CH. A. M. (1992), Technologies for butanol recovery integrated with fermentations, Process Biochem. 27, 61-75.
GRUBER, M. (1887), Eine Methode der Cultur anaerobischer Bacterien nebst Bemerkungen zur Morphologie der Buttersauregahrung, Cbl. Bacteriol. Parasitenkd. 1, 367-372.
GRUPE, H., GOTTSCHALK, G. (1992), Physiological events in Clostridium acetobutylicum during the shift from acidogenesis to solventogenesis in continuous culture and presentation of a model for shift induction, Appl. Environ. Microbiol. 58, 38963902.
HAGGSTROM, L., ENFORS, S. 0. (1982), Contin- uous production of butanol with immobilized cells of Clostridium acetobutylicum, Appl. Bio- chem. Biotechnol. 7, 35-37.
HANCOCK, K. R., ROCKMAN, E., YOUNG, C. A., PEARCE, L., MADDOX, I. S., Scorn, D. B. (1991), Expression and nucleotide sequence of the Clostridium acetobutylicum P-galactosidase gene cloned in Escherichia coli, J. Bacteriol. 173, 3084-3095.
HANSON, A. M., RODGERS, N. E. (1946), Influence of iron concentration and attenuation on the me- tabolism of Clostridium acetobutylicum, J. Bacte- riol. 51, 568-569.
GROOT, w. J., VAN DEN OEVER, c. E., KOSSEN, N.
6 References 261
HUSEMANN, M. H. W., PAPOUTSAKIS, E. T. (1989), Comparison between in vivo and in vitro enzyme activities in continuous and batch fermentations of Clostridium acetobutylicum, Appl. Microbiol. Biotechnol. 30, 585-595.
HUTKINS, R. W., KASHKET, E. R. (1986), Phospho- transferase activity in Clostridium acetobutyli- cum from acidogenic to solventogenic phases of growth, Appl. Environ. Microbiol. 51, 1121- 1123.
INGRAM, L. 0. (1986), Microbial tolerance to alco- hols: Role of the cell membrane, Trends Biotech- nol. 4, 40-44.
IZSAK, A., FUNK, F. J. (1933), Process of producing butyl alcohol, U. S. Patent 1908361.
JANATI-IDRISSI, R., JUNELLES, A. M., EL KANOU- NI, A., PETITDEMANGE, H., GAY, R. (1987), Se- lection de mutants de Clostridium acetobutyli- cum defectifs dans la production d’acktone, Ann. Inst. Pasteur/Microbiol. 138, 313-323.
JOBSES, I. M. L., ROELS, J. A. (1983), Experience with solvent production by Clostridium beijer- inckii in continuous culture, Biotechnol. Bioeng.
JONES, D. T., WOODS, D. R. (1986), Acetone-buta- no1 fermentation revisited, Microbiol. Rev. 50, 484-524.
JUNGERMANN, K., THAUER, R. K., LEIMENSTOLL, G., DECKER, K. (1973), Function of reduced py- ridine nucleotide-ferredoxin oxidoreductases in saccharolytic clostridia, Biochim. Biophys. Acta
KASHKET, E. R., CAO, Z.-Y. (1993), Isolation of a degeneration-resistant mutant of Clostridium acetobutylicum NCIMB 8052, Appl. Environ. Microbiol. 59, 41984202.
KATAGIRI, H., IMAI, K., SUGIMARI, T. (1960), On the metabolism of organic acids by Clostridium acetobutylicum. Part I. Formation of lactic acid and racemase, Bull. Agr. Chem. SOC. Jpn. 24,
KEIS, S., BENNETT, C., JONES, D. T. (1994), Differ- entiation of solvent producing clostridial strains by restriction analysis typing, ribotyping and phage typing, Abstr. 7th Int. Symp. Genet. Inn. Microorg., Montreal, June 26-July 1, p. 201.
KEMPNER, W., KUBOWITZ, F. (1933), Wirkung des Lichtes auf die Kohlenoxydhemmung der But- tersauregarung, Biochem. Z. 265, 245-252.
KIM, A. Y., BLASCHEK, H. P. (1991), Isolation and characterization of a filamentous viruslike par- ticle from Clostridium acetobutylicum NCIB 6444, J. Bacteriol. 173, 530-535.
KIM, A. Y., BLASCHEK, H. P. (1993), Construction and characterization of a phage-plasmid hybrid (phagemid), pCAK1, containing the replicative form of viruslike particle CAKl isolated from
25, 1187-1194.
305, 268-280.
163-172.
HARRIS, J., MULDER, R., KELL, D. B., WALTER, R. P., MORRIS, J. G. (1986), Solvent production by Clostridium pasteurianum in media of high sugar content, Biotechnol. Lett. 8,889-892.
HARTMANIS, M. G. N. (1987), Butyrate kinase from Clostridium acetobutylicum, J . Biol. Chem. 262,617421.
HARTMANIS, M. G. N., GATENBECK, S. (1984), In- termediary metabolism in Clostridium acetobu- tylicum: Levels of enzymes involved in the for- mation of acetate and butyrate, Appl. Environ. Microbiol. 47, 1277-1283.
HARTMANIS, M. G. N., KLASON, T., GATENBECK, S. (1984), Uptake and activation of acetate and butyrate in Clostridium acetobutylicum, Appl. Microbiol. Biotechnol. 20, 66-71.
HASTINGS, J. J. H. (1978). Acetone-butyl alcohol fermentation, in: Economic Microbiology, Vol. 2. Primary Products of Metabolism (ROSE, A. H., Ed.), pp. 3145, New York: Academic Press.
HERMANN, M., FAYOLLE, F., MARCHAL, R., POD- VIN, L., SEBALD, M., VANDECASTEELE, J.-P. (1985), Isolation and characterization of buta- nol-resistant mutants of Clostridium acetobutyli- cum, Appl. Environ. Microbiol. 50, 1238-1243.
HERRERO, A. A. (1983), End-product inhibition in anaerobic fermentations, Trends Biotechnol. 1, 49-53.
HIGGINS, C. F. (1992), ABC transporters from mi- croorganisms to man, Annu. Rev. Cell Biol. 8,
HIPPE, H., ANDREESEN, J. R., GOTTSCHALK, G. (1992), The genus Clostridium - nonmedical, in: The Prokaryotes, 2nd Edn. (BALOWS, A., TRU- PER, H. G., DWORKIN, M., HARDER, w. , SCHLEIFER, K.-H., Eds.), Vol. 11, pp. 18W1866, New York: Springer-Verlag.
HIU, S. F., ZHU, C.-Z., YAN, R.-T., CHEN, J . 4 . (1987), Butanol-ethanol dehydrogenase and bu- tanol-ethanol-isopropanol dehydrogenase: Dif- ferent alcohol dehydrogenases in two strains of Clostridium beijerinckii (Clostridium butylicum), Appl. Environ. Microbiol. 53, 697-703.
HOCKENHULL, D. J., HERBERT, D. (1945), The amylase and maltase of Clostridium acetobutyli- cum, Biochem. J . 39, 102-106.
HOLT, R. A., STEPHENS, G. M., MORRIS, J. G. (1984), Production of solvents by Clostridium acetobutylicum cultures maintained at neutral pH, Appl. Environ. Microbiol. 48, 1166-1170.
HOLT, R. A,, CAIRNS, A. J., MORRIS, J. G. (1988), Production of butanol by Clostridium puniceum in batch and continuous culture, Appl. Micro- biol: Biotechnol. 27, 319-324.
HONGO, M. (1960), Process for producing butanol by fermentation, U. S. Patent 2945786.
67-113.
262 6 Microbial Production of Acetone/ButanoWIsopropanol
Clostridium acetobutylicum NCIB 6444, J. Bacte- riol. 175, 3838-3843.
KIM, B. H., ZEIKUS, J. G. (1985), Importance of hy- drogen metabolism in regulation of solventogen- esis by Clostridium acetobutylicum, Dev. Ind. Microbiol. 26, 549-556.
KIM, A. Y., ATTWOOD, G. T., HOLT, S. M., WHITE, B. A,, BLASCHEK, H. P. (1994), Heterologous expression of endo-@1,4-~-ghcanase from clos- tridium cellulovorans in Clostridium acetobutyli- cum ATCC 824 following transformation of the engB gene, Appl. Environ. Microbiol. 60, 337- 340.
KUBOWITZ, F. (1934), Uber die Hemmung der But- tersauregarung durch Kohlenoxyd, Biochem. 2.
KUTZENOK, A., ASCHNER, M. (1952), Degenera- tive processes in a strain of Clostridium butyli- cum, J. Bacteriol. 64, 829-836.
LAPAGE, S. P., SHELTON, J. E., MITCHELL, T. G., MACKENZIE, A. R. (1970), Culture collections and the preservation of bacteria, in: Methods in Microbiology (NORRIS, J. R., RIBBONS, D. W., Eds.), Vol. 3 A, pp. 125-228, London: Academic Press.
LARGIER, S. T., LONG, S., SANTANGELO, J. D., JONES, D. T., WOODS, D. R. (1985), Immobil- ized Clostridium acetobutylicum P 262 mutants for solvent production, Appl. Environ. Micro- biol. 50, 477-481.
LEE, S. F., FORSBERG, C. W., GIBBINS, L. N. (1985a), Xylanolytic activity of Clostridium ace- tobutylicum, Appl. Environ. Microbiol. 50,1068- 1076.
LEE, S. F., FORSBERG, C. W., GIBBINS, L. N. (1985b), Cellulytic activity of Clostridium aceto- butylicum, Appl. Environ. Microbiol. 50, 220- 228.
LEE, S. F., FORSBERG, C. W., RATTRAY, J. B. (1987), Purification and characterization of two endoxylanases from Clostridium acetobutylicum ATCC 824, Appl. Environ. Microbiol. 53, 664- 650.
LEE, S. Y., BENNETT, G. N., PAPOUTSAKIS, E. T. (1992), Construction of Escherichia coli-Clos- tridium acetobutylicum shuttle vectors and trans- formation of Clostridium acetobutylicum strains, Biotechnol. Lett. 14, 427432.
LEGG, D. A., STILES, H. R. (1937), Process of pro- ducing butyl alcohol, U. s. Patent 2089562.
LEMMEL, S . A. (1985), Mutagenesis in Clostridium acetobutylicum, Biotechnol. Lett. 7, 711-716.
LEMMEL, S. A., DATTA, R., FRANKIEWICZ, J. R. (1986), Fermentation of xylan by Clostridium acetobutylicum, Enzyme Microb. Technol. 8,
LENZ, T. G., MOREIRA, A. R. (1980), Economic
274,285-298.
217-221.
evaluation of the acetone-butanol fermentation, Ind. Eng. Chem. Prod. Res. Dev. 19,478-483.
LESKIW, B. K., BIBB, M. J., CHATER, K. F. (1991), The use of a rare codon specifically during de- velopment? Mol. Microbiol. 5, 2861-2867.
LEUNG, J. C. Y., WANG, D. I. C. (1981), Production of acetone and butanol by Clostridium acetobu- tylicum in continuous culture using free and im- mobilized cells, Proc. 2nd World Congr. Cham. Eng. 1, 348-352.
LIN, Y.-L., BLASCHEK, H. P. (1983), Butanol pro- duction by a butanol-tolerant strain of Clostrid- ium acetobutylicum in extruded corn broth, Appl. Environ. Microbiol. 45, 966-973.
LIN, Y.-L., BLASCHEK, H. P. (1984), Transforma- tion of heat-treated Clostridium acetobutylicum protoplasts with pUBllO plasmid DNA, Appl. Environ. Microbiol. 48, 737-742.
LINDEN, J. C., MOREIRA, A. R., LENZ, T. G. (1986), Acetone and butanol, in: Comprehensive Biotechnology. The Principles of Biotechnology; Engineering Consideration ( COONEY, C. L., HUMPHREY, A. E., Eds.), pp. 915-931, Oxford: Pergamon Press.
LONG, S., JONES, D. T., WOODS, D. R. (1984a), The relationship between sporulation and sol- vent production in Clostridium acetobutylicum P262, Biotechnol. Lett. 6, 529-534.
LONG, S., JONES, D. T., WOODS, D. R. (1984b), In- itiation of solvent production, clostridial stage and endospore formation in Clostridium aceto- butylicum P262, Appl. Microbiol. Biotechnol. 20, 256261.
LOUGHLIN, J. F. (1935), Production of butyl alco- hol and acetone by fermentation, U. S. Patent 1 992 921.
MADDOX, I. S. (1980), Production of n-butanol from whey filtrate using Clostridium acetobutyli- cum NCIB 2951, Biotechnol. Lett. 2,493498.
MADDOX, I. S. (1983), Use of silicalite for the ad- sorption of n-butanol from fermentation liquors, Biotechnol. Lett. 5, 89-94.
MADDOX, I. S., MURRAY, A. E. (1983), Production of n-butanol by fermentation of wood hydroly- sate, Biotechnol. Lett. 5, 175-178.
MADDOX, I. S., QURESHI, N., GUTIERREZ, N. A. (1994), Utilization of whey by clostridia and process technology, in: The Clostridia and Bio- technology (WOODS, D. R., Ed.), pp. 343-369, Stoneham: Butterworth-Heinemann.
MARCHAL, R., BLANCHET, D., VANDECASTEELE, J. P. (1985), Industrial optimization of acetone- butanol fermentation: A study of the utilization of Jerusalem artichokes, Appl. Microbiol. Bio- technol. 23,92-98.
MARCZAK, R., PETITDEMANGE, H., ALIMI, F., BALLONGUE, R., GAY, R. (1983), Influence de
6 References 263
ATCC 824 for increased solvent production by enhancement of acetone formation enzyme ac- tivities using a synthetic acetone operon, Bio- technol. Bioeng. 42, 1053-1060.
MICHELS, J. (1990), Reinigung der NADP +-abhan- gigen Butanol-Dehydrogenase aus Clostridium acetobutylicum iiber Triazinyl-Farbstoffe, Thesis, University of Gottingen.
MINTON, N. P., BREHM, J. K., SWINFIELD, T.-J., WHELAN, S. M., MAUCHLINE, M. L., BODS- WORTH, N., OULTRAM, J. D. (1993), Clostridial cloning vectors, in: The Clostridia and Biotech- nology (WOODS, D. R., Ed.), pp. 119-150, Stoneham: Butterworth-Heinemann.
MITCHELL, W. J. (1992), Carbohydrate assimilation by saccharolytic clostridia, Res. Microbiol. 143,
MITCHELL, W. J., SHAW, J. E., ANDREWS, L. (1991), Properties of the glucose phosphotrans- ferase system of Clostridium acetobutylicum NCIB 8052, Appl. Environ. Microbiol. 57,2534- 2539.
MONOT, F., ENGASSER, J. M. (1983), Production of acetone and butanol by batch and continuous culture of Clostridium acetobutylicum under ni- trogen limitation, Biotechnol. Lett. 5, 213-218.
MONOT, F., MARTIN, J. R., PETITDEMANGE, H., GAY, R. (1982), Acetone and butanol produc- tion by Clostridium acetobutylicum in a synthetic medium, Appl. Environ. Microbiol. 44, 1318- 1324.
MONOT, F., ENGASSER, J. M., PETITDEMANGE, H. (1983), Regulation of acetone butanol produc- tion in batch and continuous cultures of Clos- tridium acetobutylicum, Biotechnol. Bioeng. Symp. 13, 207-216.
MOREIRA, A. R. (1983), Acetone-butanol fermen- tation, in: Organic Chemicals from Biomass (WISE, D. L., Ed.), pp. 385-406, Menlo Park: BenjaminlCummins Publ. Co., Inc.
MOREIRA, A. R., ULMER, D. C., LINDEN, J. C. (1981), Butanol toxicity in the butylic fermenta- tion, Biotechnol. Bioeng. Symp. 11, 567-579.
MULLER, J. (1938a), Production of neutral solvents by fermentation, U. S. Patent 2132039.
MULLER, J. (1938b). Fermentation mash, (1. S. Pa- tent 2 123078.
NAIR, R. V., BENNETT, G. N., PAPOUTSAKIS, E. T. (1994), Molecular characterization of an alde- hydelalcohol dehydrogenase gene from Clostrid- ium acetobutylicum ATCC 824, J. Bacteriol. 176,
NAKAMURA, S., OKADO, I., ABE, T., NISHIDA, S. (1979), Taxonomy of Clostridium tetani and re- lated species, J. Gen. Microbiol. 113, 29-35.
NAKAMURA, T., IGARASHI, Y., KODAMA, T. (1990), Acetoacetyl-CoA transferase and thiol-
245-250.
87 1-885.
la phase de croissance et de la composition du milieu sur le taux de biosynthkse de la NADH:rubrCdoxine oxydorCductase chez Clos- tridium acetolbutylicum, C. R. Acad. Sci., Ser. 3 296,469474.
MARCZAK, R., BALLONGUE, R., PETITDEMANGE, H., GAY, R. (1984), Regulation of the biosynthe- sis of NADH-rubredoxin oxidoreductase in Clostridium acetobutylicum, Curr. Microbiol. 10,
MARCZAK, R., BALLONGUE, J., PETITDEMANGE, H., GAY, R. (1985), Differential levels of ferre- doxin and rubredoxin in Clostridium acetobutyli- cum, Biochimie 67,241-248.
MCCLUNG, L. S., McCoy, E. (1935), Studies on anaerobic bacteria. VII. The serological rela- tions of Clostridium acetobutylicum, Cl. felsi- neum and Cl. roseurn, Arch. Mikrobiol. 6, 239- 249.
McCoy, E. F. (1946), Production of butyl alcohol and acetone by fermentation, U. S. Patent 2398837.
McCoy, E., MCCLUNG, L. S. (1935), Studies on anaerobic bacteria. VI. The nature and systemat- ic position of a new chromogenic Clostridium, Arch. Mikrobiol. 6, 230-238.
McCoy, E., FRED, E. B., PETERSON, W. H., HAST- I N G ~ , E. G. (1926), A cultural study of the ace- tone butyl alcohol organism, J. Infect. Dis. 39, 457483.
MCNEIL, B., KRISTIANSEN, B. (1986), The acetone butanol fermentation, Adv. Appl. Microbiol. 31,
MEINECKE, B., BAHL, H., GOTTSCHALK, G. (1984). Selection of an asporogenous strain of Clostridium acetobutylicum in continuous cul- ture under phosphate limitation, Appl. Environ. Microbiol. 48, 1064-1065.
MEINECKE, B., BERTRAM, J., GOTTSCHALK, G. (1989), Purification and characterization of the pyruvate-ferredoxin oxidoreductase from Clos- tridium acetobutylicum, Arch. Microbiol. 152, 244-250.
MERMELSTEIN, L. D., PAPOUTSAKIS, E. T. (1993), In vivo methylation in Escherichia coli by the Bacillus subtilis phage +3T I methyltransferase to protect plasmids from restriction upon trans- formation of Clostridium acetobutylicum ATCC 824, Appl. Environ. Microbiol. 59, 1077-1081.
MERMELSTEIN, L. D., WELKER, N. E., BENNETT, G. N., PAPOUTSAKIS, E. T. (1992), Expression of cloned homologous fermentative genes in Clostridium acetobutylicum ATCC 824, Bio/ Technology 10, 190-195.
MERMELSTEIN, L. D., PAPOUTSAKIS, E. T., PE- TERSEN, D. J., BENNETT, G. N. (1993), Meta- bolic engineering of Clostridium acetobutylicum
165-1 68.
61-92.
264 6 Microbial Production of Acetone/ButanoWlsopropanol
ase reactions are catalyzed by a single enzyme in Clostridium acetobutylicum ATCC824, Agr. Biol. Chem. 54,276-268.
NARBERHAUS, F., BAHL, H. (1992), Cloning, se- quencing, and molecular analysis of the groESL operon of Clostridium acetobutylicum, J. Bacteri- 01. 174, 3282-3289.
NARBERHAUS, F., GIEBELER, K., BAHL, H. (1992), Molecular characterization of the dnaK gene re- gion of Clostridiuin acetobutylicum, including grpE, d n d , and a new heat shock gene, J. Bacte- rial. 174, 3290-3299.
NELSON, D. L., WEBB, B. P. (1978), Acetone, in: Kirk-Othmer Encyclopedia of Chemical Tech- nology, 3rd Edn. (MARK, H. F., OTHMER, D. F., OVERBERGER, C. G., SEABORG, G. T., GRAY- SON, M., ECKROTH, D., Eds.), Vol. 1, pp. 179- 192, New York: John Wiley & Sons.
OGATA, S., HONGO, M. (1979), Bacteriophages of the genus Clostridium, Adv. Appl. Microbiol. 25,
OSBURN, 0. L., WERKMAN, C. H. (1935), Utiliza- tion of agricultural wastes. 11. Influence of nitro- genous substrate on production of butyl and iso- propyl alcohols by Clostridium butylicum, Ind. Eng. Chem. 27,416419.
OULTRAM, J. D., YOUNG, M. (1985), Conjugal transfer of plasmid pAMbl from Streptococcus lactis and Bacillus subtilis to Clostridium aceto- butylicum, FEMS Microbiol. Lett. 27, 129-134.
OULTRAM, J. D., DAVIES, A., YOUNG, M. (1987), Conjugal transfer of a small plasmid from Bacil- lus subtilis to Clostridium acetobutylicum by cointegrate formation with plasmid pAMP1, FEMS Microbiol. Lett. 42, 113-119.
OULTRAM, J. D., LOUGHLIN, M., SWINFIELD, T.-J., BREHM, J. K., THOMPSON, D. E., MINTON, N. P. (1988a), Introduction of plasmids into whole cells of Clostridium acetobutylicum by electropo- ration, FEMS Microbiol. Lett. 56, 83-88.
OULTRAM, J. D., PECK, H., BREHM, J. K., THOMP- SON, D. E., SWINFIELD, T. J., MINTON, N. P. (1988b), Introduction of genes for leucine bio- synthesis from Clostridium pasteurianum into C. acetobutylicum by cointegrate conjugal transfer, Mol. Gen. Genet. 214, 177-179.
OULTRAM, J. D., SCHIMMING, S., WALMSLEY, R., LOUGHLIN, M., SCHWARTZ, W., STAUDEN-
tion of cellulase (cel) genes from Clostridium thermocellum into Clostridium acetobutylicum, Abstr. 6th Int. Symp. Genet. Ind. Microorg., Strasbourg, August 12-18, C 54.
OULTRAM, J. D., BURR, I. A., ELMORE, M. J., MINTON, N. P. (1993), Cloning and sequence analysis of the genes encoding phosphotransbu- tyrylase and butyrate kinase from Clostridium
241-273.
BAUER, W. L., MINTON, N. P. (1990), Introduc-
acetobutylicum NCIMB 8052, Gene 131, 107- 112.
OXFORD, A. E., LAMPEN, J. O., PETERSON, W. H. (1940), 190. Growth factor and other nutritional requirements of the acetone butanol organism, Cl. acetobutylicum, Biochem. J. 34, 1588-1597.
PALOSAARI, N. R, ROGERS, P. (1988), Purification and properties of the inducible coenzyme A- linked butyraldehyde dehydrogenase from Clos- tridium acetobutylicum, J. Bacteriol. 170, 2971- 2976.
PAPA, A. J. (1982), Isopropyl alcohol, in: Kirk- Othmer Encyclopedia of Chemical Technology, 3rd Edn. (MARK, H. F., OTHMER, D. F., OVER- BERGER, C. G., SEABORG, G. T., GRAYSON, M., ECKROTH, D., Eds.), Vol. 19, pp. 198-220, New York: John Wiley & Sons.
PAQUET, V., CROUX, C., GOMA, G., SOUCAILLE, P. (1991), Purification and characterization of the extracellular cr-amylase from Clostridium acetobutylicum, Appl. Environ. Microbiol. 57,
PASTEUR, L. (1861a), Animacules infusoires vivant sans gaz oxygkne libre et determinant des fer- mentations, C. R. Hebd. Stances Acad. Sci. 52, 344-347.
PASTEUR, L. (1861b). Expiriences et vues nou- velles sur la nature des fermentations, C. I?. Hebd. Stances Acad. Sci. 52, 1260-1264.
PASTEUR, L. (1862), Quelques rksultats nouveaux relatifs aux fermentations acetique et butyrique, Bull. SOC. Chim. Paris Mai 1862,52-53.
PASTEUR, L. (1863), Recherches sur le putrkfac- tion, C. R. Hebd. Stances Acad. Sci. 56, 1189- 1194.
PERDRIX, L. (1891), Sur les fermentations pro- duites par un microbe anaerobie de l’eau, Ann. Inst. Pasteur 5, 287-311.
PETERSEN, M. (1991), Untersuchungen zur Lipase- Sekretion von Clostridium tetanomorphum und Reinigung des lipolytisch aktiven Enzyme, The- sis, University of Gottingen.
PETERSEN, D. J., BENNETT, G. N. (1990), Purifica- tion of acetoacetate decarboxylase from Clos- tridium acetobutylicum ATCC 824 and cloning of the acetoacetate decarboxylase gene in Escherichia coli, Appl. Environ. Microbiol. 56,
PETERSEN, D. J., BENNETT, G. N. (1991), Cloning of the Clostridium acetobutylicum ATCC 824 acetyl coenzyme A acetyltransferase (thiolase; EC 2.3.1.9) gene, Appl. Environ. Microbiol. 57,
PETERSEN, D. J., WELCH, R. W., RUDOLPH, F. B., BENNETT, G. N. (1991), Molecular cloning of an alcohol (butanol) dehydrogenase gene cluster
212-218.
3491-3498.
2735-2741.
6 References 265
from streptococci to Clostridium acetobutylicum, Ann. Inst. Pasteur/Microbiol. lMB, 275-282.
REYSSET, G., SEBALD, M. (1993), Transformation/ electrotransformation of clostridia, in: The Clos- tridia and Biotechnology (WOODS, D. R., Ed.), pp. 151-156, Stoneham: Butterworth-Heine- mann.
REYSSET, G., HUBERT, J., PODVIN, L., SEBALD, M. (1988), Transfection and transformation of Clos- tridium acetobutylicum strain NI-4081 proto- plasts, Biotechnol. Techniques 2, 199-204.
ROGERS, P. (1986), Genetics and biochemistry of Clostridium relevant to development of fermen- tation processes, Adv. Appl. Microbiol. 31, 1- 60.
ROGERS, P., GOTISCHALK, G. (1993), Biochemis- try and regulation of acid and solvent produc- tion in clostridia, in: The Clostridia and Biotech- nology (WOODS, D. R., Ed.), pp. 25-50, Stone- ham: Butterworth-Heinemann.
ROGERS, P., PALOSAARI, N. (1987), Clostridium acetobutylicum mutants that produce butyralde- hyde and altered quantities of solvents, Appl. Environ. Microbiol. 53, 2761-2766.
Roos, J. W., MCLAUGHLIN, J., PAPOUTSAKIS, E. T. (1984), The effect of pH on nitrogen supply, cell lysis, and solvent production in fermenta- tions of Clostridium acetobutylicum, Biotechnol. Bioeng. 27, 681-694.
Ross, D. (1961), The acetone-butanol fermenta- tion, Prog. Ind. Microbiol. 3, 73-85.
RUBBO, S. D., MAXWELL, M., FAIRBRIDGE, R. A,, GILLESPIE, J. M. (1941), The bacteriology, growth factor requirements and fermentation reactions of Clostridium acetobutylicum (Weiz- mann), Aust. J . Exp. Biol. Med. 19, 185-197.
SANTANGELO, J. D., JONES, D. T., WOODS, D. R. (1991), Metronidazole activation and isolation of Clostridium acetobutylicum electron transport genes, J. Bacteriol. 173, 1088-1095.
SANTANGELO, J. D., DURRE, P., WOODS, D. R. (1994), Characterization and expression of the hydrogenase I gene from Clostridium acetobuty- licum P262, Abstr. 7th Int. Symp. Genet. Ind. Mi- croorg., Montreal, June 26-July l, p. 96.
SASS, C., WALTER, J., BENNETT, G. N. (1993), Iso- lation of mutants of Clostridium acetobutylicum ATCC 824 deficient in protease activity, Curr. Microbiol. 26, 151-154.
SAUER, U., DURRE, P. (1992), Possible function of tRNAT& in regulation of solvent formation in Clostridium acetobutylicum, FEMS Microbiol. Lett. 100, 147-154.
SAUER, U., DURRE, P. (1993), Sequence and mo- lecular characterization of a DNA region encod- ing a small heat shock protein of Clostridium acetobutylicum, J. Bacteriol. 175, 3394-3400.
from Clostridium acetobutylicum ATCC 824, J. Bacteriol. 173, 1831-1834.
PETERSEN, D. J., CARY, J. W., VANDERLEYDEN, J., BENNETT, G. N. (1993), Sequence and ar- rangement of genes encoding enzymes of the acetone-production pathway of Clostridium ace- tobutylicum ATCC 824, Gene 123, 93-97.
PETITDEMANGE, H., CHERRIER, C., RAVAL, G., GAY, R. (1976), Regulation of the NADH and NADPH-ferredoxin oxidoreductases in clostrid- ia of the butyric group, Biochim. Biophys. Acta 421,334-347.
PETITDEMANGE, H., CHERRIER, C., BENGONE, J. M., GAY, R. (1977), Etude des activitks NADH et NADPH-ferrkdoxine oxydorkductasiques chez Clostridium acetobutylicum, Can. J. Micro- biol. 23, 152-160.
PETITDEMANGE, H., MARCZAK, R, BLUSSON, H., GAY, R. (1979), Isolation and properties of re- duced nicotinamide adenine dinucleotide-rubre- doxin oxidoreductase of Clostridium acetobutyli- cum, Biochem. Biophys. Res. Commun. 4, 1258- 1265.
PICH, A., BAHL, H. (1991), Purification and charac- terization of the DNA-dependent RNA poly- merase from Clostridium acetobutylicum, J. Bac- teriol. 173, 2120-2124.
PICH, A., NARBERHAUS, F., BAHL, H. (1990), In- duction of heat shock proteins during initiation of solvent formation in Clostridium acetobutyli- cum, Appl. Microbiol. Biotechnol. 33,697-704.
PIERROT, P., FICK, M., ENGASSER, J. M. (1986). Continuous acetone-butanol fermentation with high productivity by cell ultrafiltration and cell recycling, Biotechnol. Lett. 8, 253-256.
PRESCOTT, S. G., DUNN, C. G. (1959). Industrial Microbiology, 3rd Edn., New York: McGraw- Hill Book Co.
PRINGSHEIM, H. H. (1906a), Ueber den Ursprung des Fuselols und eine Alkohole bildende Bak- terienform, Cbl. Bakteriol. Parasitenkd. Infek- tionskrankh., 11. Abt. 15, 3W321.
PRINGSHEIM, H. (1906b), Ueber ein Stickstoff as- similierendes Clostridium, Cbl. Bakteriol. Parasi- tenkd. Infektionskrankh., 11. Abt. 15, 795-800.
QURESHI, N., MADDOX, I. S. (1987), Continuous solvent production from whey permeate using cells of Clostridium acetobutylicum immobilized by adsorption onto bonechar, Enzyme Microb. Technol. 9, 668-671.
REID, S. J., ALLCOCK, E. R., JONES, D. T., WOODS, D. R. (1983), Transformation of Clos- tridium acetobutylicum protoplasts with bacte- riophage DNA, Appl. Environ. Microbiol. 45,
REYSSET, G., SEBALD, M. (1985), Conjugal trans- fer of plasmid-mediated antibiotic resistance
305-307.
266 6 Microbial Production of Acetone/ButanoWIsopropanol
SAUER, U., TREUNER, A., SANTANGELO, J. D., BUCHHOLZ, M., DURRE, P. (1994), Sporulation and primary u factor homologous genes in Clos- tridium acetobutylicum, Abstr. 7th Int. Symp. Ge- net. Ind. Microorg., Montreal, June 26-July l , p. 249.
SCHARDINGER, F. (1905), Bacillus macerans, ein Aceton bildender Rottebacillus, Cbl. Bakteriol. Parasitenkd. Infektionskrankh., 11. Abt. 14, 772- 781.
SCHARDINGER, F. (1907), Verhalten von Weizen- und Roggenmehl zu Methylenblau und zu Star- kekleister, nebst einem Anhange uber die Bil- dung hoherer Alkohole durch hitzebestandige Mikroorganismen aus Weizenmehl, Cbl. Bakter- iol. Parasitenkd. Infektionskrankh., 11. Abt. 18, 748-767.
SCHATTENFROH, A., GRASSBERGER, R. (1899), Weitere Mitteilungen uber Buttersauregarung, Cbl. Bakteriol. Parasitenkd. Infektionskrankh.,
SCHATTENFROH, A., GRASSBERGER, R. (1900), Ueber Buttersauregahrung. I. Abhandlung, Arch. Hyg. 37, 54-103.
SCHLOTE, D., GOTTSCHALK, G. (1986), Effect of cell recycle on continuous butanol-acetone fer- mentation with Clostridium acetobutylicum un- der phosphate limitation, Appl. Microbiol. Bio- technol. 24, 1-6.
SCOTT, D., HEDRICK, L. R. (1952), The amylase of Clostridium acetobutylicum, J . Bacteriol. 63, 795- 803.
SHERMAN, P. D. (1978), Butyl alcohols, in: Kirk- Othmer Encyclopedia of Chemical Technology, 3rd Edn. (MARK, H. F., OTHMER, D. F., OVER- BERGER, C. G., SEABORG, G. T., GRAYSON, M., ECKROTH, D. Eds.), Vol. 4, pp. 338-345, New York: John Wiley & Sons.
SPIVEY, M. J. (1978), The acetone/butanol/ethanol fermentation, Process Biochem. 13, 2-5.
STEPHENS, G. M., HOLT, R. A., GOTTSCHAL, J. C., MORRIS, J. G. (1985), Studies on the stability of solvent production by Clostridium acetobutyli- cum 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, 1033- 1037.
TAGAKI, W., WESTHEIMER, F. H. (1968), Aceto- acetate decarboxylase. The molecular weight of the enzyme and subunits, Biochemistry 7, 895- 900.
TAYA, M., ISHII, S., KOBAYASHI, T. (1985), Moni-
11. Abt. 5, 697-702.
Clostridium acetobutylicum, J. Ferment. Technol.
THAUER, R. K., JUNGERMANN, K., DECKER, K. (1977), Energy conservation in chemotrophic anaerobic bacteria, Bacteriol. Rev. 41, 100-180.
THOMPSON, D. K., CHEN, J.3. (1990), Purification and properties of an acetoacetyl coenzyme A- reacting phosphotransbutyrylase from Clostrid- ium beijerinckii (“Clostridium butylicum”) NRRL B593, Appl. Environ. Microbiol. 56, 607- 613.
TRUFFAUT, N., SEBALD, M. (1988), Study of plas- mids isolated from saccharolytic clostridia: Con- struction of hybrid plasmids and transfer to Escherichia coli and Bacillus subtilis, Biotechnol. Lett. 10, 1-6.
TRUFFAUT, N., HUBERT, J., REYSSET, G. (1989), Construction of shuttle vectors useful for trans- forming Clostridium acetobutylicum, FEMS Mi- crobiol. Lett. 58, 15-20.
TWAROG, R., WOLFE, R. S. (1962), Enzymatic phosphorylation of butyrate, J. Biol. Chem. 237, 2474-2477.
ULLMANN, S., DURRE, P. (1994), Cloning and se- quencing of the DNA gyrase genes of Clostrid- ium acetobutylicum DSM 792, Abstr. 7th Int. Symp. Genet. Ind. Microorg., Montreal, June 26- July 1, p. 23.
VALENTINE, R. C., WOLFE, R. S. (1960), Purifica- tion and role of phosphotransbutyrylase, J. Biol. Chem. 235, 1948-1952.
VELDKAMP, H. (1965), Enrichment cultures of pro- karyotic organisms, in: Methods in Microbiology (NORRIS, J. R., RIBBONS, D. W., Eds.), Vol. 3A, pp. 305-361, London: Academic Press.
VERHASSELT, P., PONCELET, F., VITS, K., VAN GOOL, A., VANDERLEYDEN, J. (1989), Cloning and expression of a Clostridium acetobutylicum a-amylase gene in Escherichia coli, FEMS Mi- crobiol. Lett. 59, 135-140.
VOGET, C. E., MIGNONE, C. F., ERTOLA, R. J. (1985), Butanol production from apple pomace, Biotechnol. Lett. 7, 4346.
VOLESKY, B., SZCZESNY, T. (1983), Bacterial con- version of pentose sugars to acetone and buta- nol, Adv. Biochem. Eng. Biotechnol. 27, 101- 117.
VOLLHERBST-SCHNECK, K., SANDS, J. A., MON- TENECOURT, B. s. (1984), Effect of butanol on lipid composition and fluidity of Clostridium acetobutylicum ATCC 824, Appl. Environ. Mi- crobiol. 47, 193-194.
VON HUGO, H., GOTTSCHALK, G. (1974), Distribu- tion of 1-phosphofructokinase and PEP:fructose phosphotransferase activity in clostridia, FEBS
63, 181-187.
toring and control for extractive fermentation of ie t t . -46, 106-108.
6 References 267
WALTER, K. A., BENNETT, G. N., PAPOUTSAKIS, E. T. (1992), Molecular characterization of two Clostridium acetobutylicum ATCC 824 butanol dehydrogenase isozyme genes, J. Bacteriol. 174,
WALTER, K. A., NAIR, R. V., CARY, J. W., BEN- NET^, G. N., PAPOUTSAKIS, E. T. (1993), Se- quence and arrangement of two genes of the bu- tyrate-synthesis pathway of Clostridium acetobu- tylicum ATCC 824, Gene 134, 107-111.
WALTON, M. T. (1945), Process for the production of riboflavin by butyl alcohol producing bacte- ria, U. S. Patent 2368074.
WANG, D. I. C., COONEY, C. L., DEMAIN. A. L., DUNHILL, P. (1979), Fermentation and Enzyme Technology, New York: John Wiley & Sons.
WARREN, S., ZERNER, B., WESTHEIMER, F. H. (1966), Acetoacetate decarboxylase. Identifica- tion of lysine at the active site, Biochemistry 5,
WATERSON, R. M., CASTELLINO, F. J., HASS, G. M., HILL, R. L. (1972), Purification and charac- terization of crotonase from Clostridium aceto- butylicum, J. Biol. Chem. 247, 52665271.
WEIZMANN, C. (1915), Improvements in the bacte- rial fermentation of carbohydrates and in bacte- rial cultures for the same, British Patent 4845.
WEIZMANN, C. (1919), Production of acetone and alcohol by bacteriological processes, U. S. Patent 1315585.
WELCH, R. W., RUDOLPH, F. B., PAPOUTSAKIS, E. 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, F. B. (1992). Effects of rifampicin and chloramphenicol on product and enzyme levels of the acid- and solvent-producing path- ways of Clostridium acetobutylicum (ATCC 824), Enzyme Microb. Technol. 14,277-283.
WELSH, F. W., VELIKY, I. A. (1984), The produc- tion of acetone-butanol from acid whey, Biotech- nol. Lett. 6 , 61-64.
WESTHEIMER, F. H. (1969), Acetoacetate decar- boxylase from Clostridium acetobutylicum, Methods Enzyrnol. 14, 231-241.
WIESENBORN, D. P., RUDOLPH, F. B., PAPOUTSA- KIS, E. T. (1988), Thiolase from Clostridium ace- I
tobutylicum ATCC 824 and its role in the syn- thesis of acids and solvents, Appl. Environ. Mi- crobiol. 54, 2717-2722.
WIESENBORN, D. P., RUDOLPH, F. B., PAPOUTSA- KIS, E. T. (1989a), Phosphotransbutyrylase from Clostridium acetobutylicum ATCC 824 and its role in acidogenesis, Appl. Environ. Microbiol.
7149-7158.
81 7-823.
55,317-322.
WIESENBORN, D. P., RUDOLPH, F. B., PAPOUTSA- KIS, E. T. (1989b), Coenzyme A transferase from Clostridium acetobutylicum ATCC 824 and its role in the uptake of acids, Appl. Environ. Mi- crobiol. 55, 323-329.
WILKINSON, S. R., YOUNG, M. (1994), Targeted in- tegration of genes into the Clostridium acetobu- tylicum chromosome, Microbiology 140, 89-95.
WILLIAMS, D. R., YOUNG, D. I., YOUNG, M. (1990), Conjugative plasmid transfer from Escherichia coli to Clostridium acetobutylicum, J. Gen. Microbiol. 136, 819-826.
WINOGRADSKY, S. (1902), Clostridium pastorian- urn, seine Morphologie und,seine Eigenschaften als Buttersaureferment, Cbl. Bakteriol. Parasi- tenkd. Infektionskrankh., 11. Abt. 9, 43-54, 107- 112.
WONG, J., BENNETT, G. N. (1994), Studies of DNA supercoiling in Clostridium acetobutylicum ATCC824, Abstr. Gen. Meet. Am. SOC. Micro- biol., 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, R. C., MORRIS, J. G. (1990), Stability of solvent production by Clostridium acetobutyli- cum in continuous culture: strain differences, J. Appl. Bacteriol. 69, 718-728.
WOOLLEY, R. C., PENNOCK, A., ASHTON, R. J., DAVIES, A,, YOUNG, M. (1989), Transfer of TnI545 and Tn916 to Clostridium acetobutyli- cum, Plasmid 22, 169-174.
YAN, R.-T., ZHU, C.-X., GOLEMBOSKI, C., CHEN, J . 4 . (1988), Expression of solvent-forming en- zymes and onset of solvent production in batch cultures of Clostridium beijerinckii (“Clostrid- ium butylicum ”), Appl. Environ. Microbiol. 54, 642-648.
YAROVENKO, V. L. (1964), Principles of the con- tinuous alcohol and butanol-acetone fermenta- tion processes, in: Continuous Cultivation of Mi- croorganisms, 2nd Symp. (MALEK, I., Ed.), pp. 205-217, Prague: Cechoslovakian Academy of Sciences.
struction of a Clostridium acetobutylicum-Esch- erichia coli shuttle vector, Biotechnol. Lett. 13, 14 .
LEHN, G., HERWIG, K., GOTTSCHALK, G.
YOON, K.-H., LEE, J.-K., KIM, B. H. (1991), Con-
268 6 Microbial Production of AcetondButanoWlsopropanol
YOSHINO, S., YOSHINO, T., HARA, S., OGATA, S., HAYASHIDA, S. (1990), Construction of shuttle vector plasmid between Clostridium acetobutyli- cum 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-Heine- mann.
YOUNG, M., MINTON, N. P., STAUDENBAUER, W. L. (1989), Recent advances in the genetics of the clostridia, FEMS Microbiol. Rev. 63, 301-326.
YOUNGLESON, J. S., SANTANGELO, J. D., JONES, D. T., WOODS, D. R. (1988). Cloning and ex- pression of a Clostridium acetobutylicum alcohol dehydrogenase gene in Escherichia coli, Appl. Environ. Microbiol. 54, 676-682.
YOUNGLESON, J. S., JONES, W. A., JONES, D. T., 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 ace- tobutylicum, Gene 78, 355-364.
YOUNGLESON, J. S., JONES, D. T., WOODS, D. R. (1989b), Homology between hydroxybutyryl and hydroxyacyl coenzyme A dehydrogenase en- zymes from Clostridium acetobutylicum fermen- tation and vertebrate fatty acid P-oxidation pathways, J. Bacteriol. 171, 68004807.
Yu, P.-L., PEARCE, L. E. (1986), Conjugal transfer 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 pres- ence of acetic or butyric acid, FEMS Microbiol. Lett. 18, 103-107.
Yu, E. K. C., CHAN, M. K.-H., SADDLER, J. N. (1985), Butanol production from cellulosic sub- strates by sequential co-culture of Clostridium thermocellum and C. acetobutylicum, Biotechnol. Lett. 7, 509-514.
Yu, P. L., SMART, J. B., ENNIS, B. M. (1987), Dif- ferential induction of P-galactosidase activities in the fermentation of whey permeate by Clos- tridium acetobutylicum, Appl. Microbiol. Bio- technol. 26,254-257.
ZILLIG, W., HOLZ, I., WUNDERL, S. (1991), Hyper- thermus butylicus gen. nov., sp. nov., a hyper- thermophilic, anaerobic, peptide-fermenting, facultatively H2S-generating archaebacterium, Int. J. Syst. Bacteriol. 41, 169-170.
ZOUTBERG, G. R., WILLEMSBERG, R., SMIT, G., TEIXEIRA DE MATTOS, M. J., NEIJSSEL., 0. M. (1989a), Aggregate-formation by Clostridium butyricum, Appl. Microbiol. Biotechnol. 32, 17- 21.
ZOUTBERG, G. R., WILLEMSBERG, R., SMIT, G., TEIXEIRA DE MATTOS, M. J., NEIJSSEL, 0. M. (1989b), Solvent production by an aggregate- forming variant of Clostridium butyricum, Appl. Microbiol. Biotechnol. 32, 22-26.
