Enhanced Production of 2,3-Butanediol by Klebsiella pneumoniae ...

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Jun 25, 1982 - crucial in increasing the efficiency of biocon- version processes, most of which currently emphasize cellulose utilization (3, 4). Interest in.
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ENVIRONMENTAL MICROBIOLOGY, Oct. 1982,

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Vol. 44, No. 4

0099-2240/82/100777-08$02.00/0 Copyright C 1982, American Society for Microbiology

Enhanced Production of 2,3-Butanediol by Klebsiella pneumoniae Grown on High Sugar Concentrations in the Presence of Acetic Acid ERNEST K. C. YU AND JOHN N. SADDLER* Biotechnology Group, Forintek Canada Corporation, Eastern Laboratory, Ottawa, Ontario, Canada KIG

3ZS Received 25 April 1982/Accepted 25 June 1982

The bioconversion of sugars present in wood hemicellulose to 2,3-butanediol (hereafter referred to as butanediol) by Klebsiella pneumoniae grown on high initial concentrations (up to 10%) of sugars was investigated. Initial fermentation studies with a chemically defined medium suggested that sugar levels in excess of 2% could not be utlized even when a higher inoculum size (5 to 10%) was used. The addition of nutrient supplements, viz., yeast extract, urea, ammonium sulfate, and trace elements resulted in a 10 to 50% increase in butanediol yields, although sugar utilization remained incomplete. The concentration of end products normally found at the termination of fermentation was shown to be noninhibitory to growth and substrate utilization. Acetic acid was inhibitory at concentrations above 1%, although growth and butanediol yield were stimulated in cultures supplemented with lower levels of acetic acid. The efficient utilization of 4% substrate concentrations of D-glucose and D-xylose was achieved, resulting in butanediol yields of 19.6 and 22.0 g/liter, respectively.

The efficient utilization of lignocellulosic hemicellulose can be achieved by adding nutriwastes requires that most or all of the constitu- ent supplements, particularly acetic acid, to the ents of biomass substrates be converted to use- culture medium. ful products (3, 4, 18). This is particularly apparent in the field of energy from renewable MATERIALS AND METHODS resources if the products, liquid fuels, are to Organisms. K. pneumoniae (formerly Aerobacter be economically competitive with fossil fuels. (NRRL B-199; ATCC 8724) was obtained Research in hemicellulose utilization is therefore aerogenes) from National Research Council of Canada Culture crucial in increasing the efficiency of biocon- Collection, Ottawa, Ontario, Canada (strain NRCC version processes, most of which currently 3006). Cultures were maintained on slants of nutrient emphasize cellulose utilization (3, 4). Interest in agar (Difco Laboratories, Detroit, Mich.) at -20°C. hemicellulose utilization has recently increased Medium preparation. The medium used in this study considerably, but most work to date has concen- was a chemically modified medium (Kp 1) modified trated on D-xylose fermentation (5, 6, 19), even from Anderson and Wood (2), containing per liter though other hexoses and pentoses can account 11,400 mg of K2HPO4 * 3H20, 1,500 mg of KH2PO4, 400 mg of EDTA, 240 mg of for a significant portion of the total carbohydrate 3,000 mg of (NH4)2SO4, * 4H20, 100 mg of NaCl, 14 mg of content of hemicelluloses (23). We initiated a MgSO4 * 2H20, 10 mg of FeSO4 * 7H20, 2.8 mg of program to screen for microorganisms capable CaC12 MnSO4 * 4H20, 7.5 mg of ZnSO4 7H20, and 10,000 of utilizing most of these sugars and converting mg of 2-(N-morpholino)ethanesulfonic acid. The initial them to possible liquid fuels. Klebsiella pneumo- pH of the medium was set at 6.5. All sugars were niae was selected because of its nutritional ver- prepared separately in 20 and 40% solutions and were satility and its ability to produce 2,3-butanediol sterilized either by autoclaving (121°C, 15 min) or by (hereafter referred to as butanediol), a valuable passage through a 0.22-,um filter (Millipore Ltd. Misfuel additive. After initial studies to define the sissauga, Ontario, Canada). Samples of the sugar solutions were then transferred to presterilized medioptimum conditions for butanediol accumulation um appropriate final concentrations. For studies (25), we have attempted to improve the econom- withforinitial concentrations above 4%, media ic feasibility of the bioconversion process by were preparedsugar at double strength and then diluted (1:1) the utilization of levels of studying higher sugars after sterilization with concentrated sugar solutions to by K. pneumoniae. The work described in this the desired sugar levels. report indicates that efficient fermentation of Media for aerobic cultures were prepared as 10-ml high concentrations of sugars present in wood aliquots in 50-mi Erlenmeyer flasks and were covered -

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with sponge stoppers. Media for cultures grown under finite air conditions were prepared in Wheaton serum bottles and sealed with butyl rubber stoppers and aluminum caps. Studies with nutrient supplements and various acetic acid concentrations were carried out with 10-ml portions of medium in 60-ml vials. All other studies were done with 50-ml portions of medium in 160-ml vials. Supplementary components to the medium were used at the following concentrations: yeast extract, 10 g/liter; urea, 10 g/liter; (NH4)2SO4, 3 g/liter; trace elements (viz., CaCl2 * 2H20, 126 mg/liter; FeSO4 * 7H20, 90 mg/liter; MnSO4 * 4H20, 25.2 mg/liter; and ZnSO4 * 7H20, 67.5 mg/liter); ethanol and acetic acid, 0.5 to 1.0% (wt/vol); and butanediol, 1.0 to 2.5% (wt/vol). Supplementary components were all added to the medium before pH adjustment and sterilization. Additions of extra components during fermentation were carried out by transferring small samples of concentrated solutions to the fermentation media to achieve the desired levels of the particular supplements. Culture conditions. Working cultures at -20°C were thawed at 37°C for 30 min and streaked onto nutrient agar plates to be incubated aerobically at 37°C for 24 h. Colonies were checked for purity and then were used to inoculate culture broths containing 1.0% D-glucose. These cultures were incubated aerobically at 37°C with shaking (120 rpm) for 24 h and then were used to inoculate fresh test media (routinely with a 2% inoculum) which were incubated under finite air conditions at 30°C (120 rpm shaking) for specified time periods. For cultures grown in 50-ml broths in 160-ml vials, sampling was carried out by withdrawing 2-ml samples periodically for analysis. Studies with end-product inhibitors were carried out in media with initial sugar concentrations of 0.2%. Additional 1.6% sugars were added after 1 and 2 days of incubation. End products were added either before inoculation or after 1 day of incubation. All studies were carried out in duplicate and were repeated at least twice. Analytical methods. Culture growth, after appropriate dilution, was monitored by reading optical density at 540 nm (OD540) in a 1-cm light-path cuvette with a Spectronic 710 spectrophotometer (Bausch & Lomb, Inc., Rochester, N.Y.). Cells were harvested by centrifuging (40C) at 10,000 x g for 15 min, and the supernatant fluids were analyzed for volatile fermentation products by gas chromatography (1) with a model 4600 gas chromatograph (Varian Associates, Inc., Palo Alto, Calif.) equipped with an autosampler and a Varian CDS 401 data system. Chromosorb 101 column (6 ft by 1/8 in [183 by 0.32 cm], 100/120 mesh; Chromatographic Specialties Ltd., Brockville, Ontario, Canada) was used with a flame ionization detector. The injection and detector temperatures were set at 200 and 250°C, respectively. The column oven was operated isothermally at 190°C. Helium was used as the carrier gas, and the flow rate was set at 30 ml/min. Reducing sugars were assayed with dinitrosalicylic acid (14) by using the corresponding sugars as standards. Chemicals. D-(+)-Cellobiose, 2-(N-morpholino)ethanesulfonic acid, and D-(+)-xylose were purchased from Sigma Chemical Co., St. Louis, Mo. (--)-2,3Butanediol was obtained from Aldrich Chemical Co.,

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Milwaukee, Wis. 3,5-Dinitrosalicylic acid was purchased from Eastman Kodak Co., Rochester, N.Y. All other reagents were obtained from Fisher Chemical Co., Fairlawn, N.J.

RESULTS

K. pneumoniae organisms could efficently produce butanediol when they were grown in a chemically defined medium (Kp 1) containing an initial concentration of 1 to 2% D-glucose or Dxylose as substrates (Fig. 1). Solvent production at high sugar concentrations, however, was less efficient. Substrate concentrations of D-glucose and D-xylose in excess of 2% were not efficiently utilized even after prolonged incubation. Although growth and butanediol accumulation were slower in cultures containing higher substrate concentrations, the final yields obtained were not significantly different. Butanediol levels all declined with prolonged incubation, presumably due to utilization of butanediol by the microorganisms. Attempts were made to improve the rate and efficiency of fermentation by studying the effect of the inoculum size on cultures grown at various initial sugar concentrations (Fig. 2). The inoculum size had no effect on butanediol production when D-glucose was used as the substrate, and the effect of the inoculum size on Dxylose fermentation was apparent only at the highest D-xylose concentration tested (10%). Even in this last case, the inoculum size only influenced the initial rate of fermentation and did not affect the final solvent yields. Similar profiles for growth and sugar utilization were found under these conditions, with the larger inocula failing to result in complete utilization of the substrate after prolonged incubation. Since cell lysis, as indicated by a decline in the absorbance of the culture, was apparent at later stages of incubation, we tested the possibility that the cessation of bioconversion of the substrates was due to the lack of viable cells during prolonged fermentation. The finding of large numbers of viable cells obtained from 30-day-old cultures streaked onto nutrient agar plates argued against this hypothesis. So did the fact that reinoculation with fresh, healthy cultures also failed to revitalize the fermentation process. We next examined the possibility that high initial sugar concentrations might be detrimental to bioconversion, and we attempted to circumvent the problem by a fed batch approach. However, after the complete consumption of an initial 2% concentration of D-glucose or D-Xylose in the medium, supplementary sugars later added to the cultures were not utilized. This led us to suspect that the medium could have become deficient in certain essential nutrients, or the end products of fermentation may have

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FIG. 1. Effect of initial sugar concentration on butanediol fermentation by K. pneumoniae. Cultures were incubated until maximal solvent yields were obtained. (a) D-Glucose, 1 to 5%, 1 day of incubation; 8 to 10%o, 5 days. (b) D-Xylose, 1 to 5%, 2 days; 8 to 10%o, 5 days. EtoH, Ethanol; HAc, acetic acid; Diol, butanediol.

Since efficient bioconversion was not achieved, possible inhibition owing to major fermentation end products was investigated (Fig. 3 and 4). When cultures were grown on Dglucose in the presence of added end products at levels above those normally attained from the fermentation of 2% sugars, no apparent inhibition of growth or butanediol production was observed (Fig. 3). On the contrary, growth and butanediol yields were significantly enhanced in the presence of acetic acid (Fig. 3c). An improvement in butanediol formation was also noted with the addition of ethanol and butanediol, though to a lesser extent (Fig. 3b and d). Similar trends were obtained in cultures with

reached an inhibitory level and prevented further utilization of the substrates. Both of these possibilities were tested. The inclusion of yeast extract, a supplementary source of nitrogen and growth factors, enhanced growth, sugar utilization, and butanediol production (Table 1). The addition of other nitrogen supplements or trace elements also improved sugar utilization and butanediol yields, although the effect on the growth of K. pneumoniae was not well defined. All such supplements, however, failed to result in the completion of the bioconversion process, even when the fermentation was allowed to proceed for up to 7 days.

TABLE 1. Effect of nutrient supplement on solvent production of K. pneumoniaea Nutrient

C source

supplement

D-Glucose

None (control) 1% Yeast extract 1% Urea 2x (NH4)2SO4 lOx Trace

OD540

%C

utilized

Ethanol

Solvent (g/liter)

Acetic acid

Butanediol

4.97 6.00

55.9 68.4

1.87 2.35

0.20 0.64

8.21 12.40

4.24 4.95 (4.35)b 4.83

81.7 66.3 (63.2) 63.2

2.52 1.56 (1.89) 1.78

0.75 0.60 (0.82) 0.48

10.49 6.91 (10.45) 9.64

elements 7.55 1.84 0.59 50.6 3.96 None (control) 11.30 0.42 2.59 66.3 5.96 1% Yeast extract 9.78 2.38 0.40 53.0 4.44 1% Urea 10.91 0.42 57.8 1.96 3.50 2x (NH4)2SO4 8.36 0.58 1.76 44.6 lOx Trace 4.50 elements a Cultures were grown in Kp 1 medium (pH 6.5) with an initial sugar concentration of 4% and were incubated under finite air conditions at 30°C with shaking (120 rpm) for 2 days. b Values in parentheses were obtained from cultures incubated for 1 day under the same conditions as the other cultures.

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D-Xylose

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INCUBATION TIME (d)

FIG. 2. Butanediol production of K. pneumoniae in cultures with various sugar concentrations (as shown) and inoculum sizes of 2 (U), 5 (0), and 10% (A).

end products added after 1 day of growth. One notable exception was the initial inhibition observed upon the addition of 0.2% unneutralized acetic acid (Fig. 4b). Even in this case, however, the organisms were capable of overcoming the initial combined effects of pH and acetic acid, and they restored growth to the level of the control culture (Fig. 3a) while increasing the butanediol yield (Fig. 4b). Cultures were then grown on 2% D-glucose supplemented with high levels of end products. Inhibitions of butanediol production of 45 and 75% were observed after 24 h of growth in the

presence of 1% ethanol and acetic acid, respectively. These inhibitions, however, were completely overcome by the microorganisms. The addition of 0.5% ethanol or acetic acid (pH 6.5) to the culture after 8 h, 24 h, or 48 h of growth did not result in any apparent inhibition. When 2.5% butanediol was added, no adverse effects on the bioconversion process were apparent in any case. Similar results were obtained with cultures grown on D-xylose. The significant increase in butanediol production in the presence of acetic acid (Fig. 3c, 4b) prompted us to investigate the effects of various

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FIG. 3. Glucose fermentation of K. pneumoniae in the Abbreviations are explained in the legend to Fig. 1.

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FIG. 4. Glucose fermentation of K. pneumoniae in the presence of end products added after 1 day of fermentation. Abbreviations are explained in the legend to Fig. 1.

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initial acetic acid concentrations on the overall fermentation process. Complete inhibition of growth occurred in the presence of 2% acetic acid (Fig. 5). Initial inhibition of butanediol production by the addition of 1% acetic acid on the first day of growth was subsequently replaced by an enhancement resulting in the accumulation of twice as much butanediol after 5 days of incubation. The addition of lower concentrations of acetic acid resulted in a more complete utilization of D-xylose as well as higher butanediol production (Fig. 5). Similar results were obtained when D-glucose was used as the substrate. The highest butanediol production values were obtained after growth on both of these substrates containing 0.5% acetic acid in the culture media. A 4% concentration of Dglucose was completely utilized within 24 h, resulting in a butanediol yield of 19.6 g/liter. A 4% concentration of D-xylose was completely utilized with 48 h, and a butanediol yield of 22 g/liter was obtained. DISCUSSION Production of butanediol from hemicellulosederived sugars of wood and agricultural residues has recently been viewed as an alternative approach in the utilization of lignocellulosics for conversion to liquid fuels and chemical feed stocks (3, 4, 18). For the process to be economically feasible, the end products should be at high concentrations to minimize the solvent recovery cost, which is a major expense in butanediol fermentation (4, 18). We therefore investigated the practicality of increasing butanediol yields by carrying out fermentations with higher initial sugar concentrations. Earlier researchers reported that K. pneumoniae is capable of utilizing 5 to 15% concentrations of sugars in complex fermentation media

containing various types of raw materials (8, 11, 12, 15, 16). In our study with commercially available sugars in a chemically defined medium, bioconversion by the organism was efficient only at relatively low sugar concentrations (up to 2.0%). To ensure that this inability to utilize higher sugar levels was not due to a deficiency in the medium, various nutrients were added as supplements. Since the increase in sugar content could foreseeably disrupt the ratio of carbon to nitrogen, nitrogen supplements which have previously been successfully applied in butanediol fermentations (9-11, 15, 16) were tested. Although a marked improvement in butanediol production was obtained when these supplements were used, the overall utilization and bioconversion remained low. Earlier workers suggested that the fermentation process could be enhanced in the presence of certain cations (24) which may be involved in the active sites of enzymes necessary for butanediol formation. Our attempts to provide supplementary cation requirements by increasing the levels of trace elements in the medium resulted in only marginal improvements in butanediol yields and failed to lead to complete sugar utilization. Supplementing the culture media with end products at levels above those normally attained in fermentation also failed to reveal any prolonged inhibition. These results also confirmed earlier statements by other workers (4) that butanediols are less toxic to microbes than other solvents are. This resistance could be of considerable importance since the major end product could be accumulated at much higher concentrations without affecting the fermentation process. Although ethanol and acetic acid are also formed as by-products in fermentation, they are usually present at subinhibitory levels. Consequently, butanediol fermentation will have a competitive loor 7.Or- 10

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FIG. 5. Effect of initial acetic acid concentration on butanediol fermentation by K. pneumoniae grown on 4% D-xylose for 2 days. Acetic acid levels reported were the total amounts detected (including the acetic acid added in the medium). Abbreviations are explained in the legend to Fig. 1.

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advantage over ethanol fermentation or acetonebutanol fermentation, in which the end products are considerably more toxic. Sugar utilization and butanediol production of K. pneumoniae at high initial substrate concentrations was considerably enhanced by the addition of 0.5% acetic acid to the media. Earlier workers on Aerobacter indologenes (13, 17) and Bacillus polymyxa (formerly Aerobacillus polymyxa) (20) reported an increase in butanediol production in glucose-grown cells concomitant with a decrease in the level of added acetate in the media and suggested that such an effect was due to the reduction and subsequent condensation of acetate with pyruvate to form butanediol. In view of the two- to threefold increase in butanediol yields obtained in our study as a result of the relatively low levels of acetate added, it appears unlikely that acetate assimilation alone can account for such an increase in butanediol production. More recent work on cell-free extracts of Aerobacter aerogenes (7, 21, 22) has demonstrated that acetate at low pH (i.e., in the form of acetic acid) serves as an effective inducer for the three enzymes involved in the formation of butanediol from pyruvate, viz., pH 6 acetolactate-forming enzyme (21, 22), acetolactate decarboxylase (22), and diacetyl (acetoin) reductase (formerly butyleneglycol dehydrogenase) (7, 22). Such an induction mechanism probably plays a major role in the enhanced butanediol production of our study, even though the exact extent of stimulation is not known. Most previous work with wood and agricultural hydrolysates has been carried out with substrate concentrations of 5 to 10% (8, 11). In work with spent sulphite liquor and wood hydrolysates as the substrates, the initial sugar concentration was around 4.0% (6, 11). In this work we have been able to show that K. pneumoniae can efficiently utilize sugars at this substrate concentration. A 4% substrate concentration was completely utilized within 24 to 48 h, and this resulted in butanediol yields which were 82 to 92% of the theoretical maximum. In other work (J. N. Saddler, E. Yu, M. Mes-Hartree, N. Levitin, and H. H. Brownell, Proc. Am. Inst. Chem. Eng. Meet., Orlando, Fla., March 1982, section 43, paper 43G), we have shown that K. pneumoniae can also use hemicellulose-derived sugars successfully and produce butanediol yields of 0.2 to 0.35 g/g of the sugar utilized, giving values which were comparable to those obtained by other workers (16). These results indicate that butanediol fermentation of lignocellulosic-derived sugars may be one alternative to obtaining liquid fuels from biomass. We are currently studying the scaling up of this process in fermentors, and efforts will

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be made to improve butanediol yields by using fed batch or continuous culture systems. ACKNOWLEDGMENTS We thank Joan P. Torrie for excellent technical assistance. This worl was supported financially by the ENFOR (Energy from Forest) program of the Canadian Forestry Service (Project No. C-187). E.K.C.Y. is the recipient of a Natural Sciences and Engineering Research Council (Canada) Industrial Research Fellowship. LITERATURE CITED 1. Ackman, R. G. 1972. Porous polymer bead packings and formic acid vapour in the GLC of volatile free fatty acids. J. Chromatogr. Sci. 10:560-565. 2. Anderson, R. L., and W. A. Wood. 1962. Pathway of Lxylose and L-lyxose degradation in Aerobacter aerogenes. J. Biol. Chem. 237:296-303. 3. BLsaria, V. S., and T. K. Ghose. 1981. Biodegradation of cellulosic materials: substrates, microorganisms, enzymes and products. Enzyme Microb. Technol. 3:90-104. 4. Flickinger, M. C. 1980. Current biological research in conversion of cellulosic carbohydrates into liquid fuels: how far have we come? Biotechnol. Bioeng. 22(Suppl.):27-48. 5. Gong, C. S., L. D. McCraken, and G. T. Tsao. 1981. Direct fermentation of D-Xylose to ethanol by a xylosefermenting yeast mutant, Candida SP XF 217. Biotechnol. Lett. 3:245-250. 6. Jeffries, T. W. 1981. Conversion of xylose to ethanol under aerobic conditions by Candida tropicalis. Biotechnol. Lett. 3:213-218. 7. Larsen, S. H., and F. C. Stormer. 1973. Diacetyl (acetoin) reductase from Aerobacter aerogenes kinetic mechanism and regulation by acetate of the reversible reduction of acetoin to 2,3-butanediol. Eur. J. Biochem. 34:100-106. 8. Ledingham, G. A., and A. C. Neish. 1954. Fermentative production of 2,3-butanediol, p. 27-93. In L. A. Underkofler and R. J. Hickey (ed.), Industrial fermentations, vol. 2. Chemical Publishing Co., Inc., New York. 9. Long, S. K., and R. Patrick. 1960. 2,3-Butylene glycol as a fermentation by-product from citrus wastes. Proc. Fla. State Hortic. Soc. 73:241-246. 10. Long, S. K., and R. Patrick. 1961. Production of 2,3butylene glycol from citrus wastes. I. The Aerobacter aerogenes fermentation. Appl. Microbiol. 9:244-248. 11. Long, S. K., and R. Patrick. 1963. The present status of the 2,3-butylene glycol fermentation, p. 135-155. In W. W. Umbreit (ed.), Advances in applied microbiology, vol. 5. Academic Press, Inc., New York. 12. McCall, K. B., and C. E. Georgi. 1954. The production of 2,3-butanediol by fermentation of sugar beet molasses. Appl. Microbiol. 2:355-359. 13. Mickelson, M., and C. H. Werkman. 1938. Influence of pH on the dissimilation of glucose by Aerobacter indologenes. J. Bacteriol. 36:67-76. 14. Miller, G. L. 1959. Use of dinitrosalicylic reagent for the determination of reducing sugars. Anal. Chem. 31:426428. 15. Olson, B. H., and M. J. Johnson. 1948. The production of 2,3-butylene glycol by Aerobacter aerogenes 199. J. Bacteriol. 55:209-222. 16. Perlman, D. 1944. Production of 2,3-butylene glycol from wood hydrolyzates. Ind. Agric. Chem. 36:803-804. 17. Reynolds, H., B. J. Jacobsson, and C. H. Werkman. 1937. The dissimilation of organic acids by Aerobacter indologenes. J. Bacteriol. 34:15-20. 18. Rosenberg, S. L. 1980. Fermentation of pentose sugars to ethanol and other neutral products by microorganisms. Enzyme Microb. Technol. 2:185-193. 19. Schneider, H., P. Y. Wang, Y. K. Chan, and R. Maleszka. 1981. Conversion of D-xylose into ethanol by the yeast Pachysolen tannophilus. Biotechnol. Lett. 3:89-92.

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20. Stahyl, G. L., and C. H. Werkman. 1942. Origin and relationship of acetylmethylcarbinol to 2,3-butylene glycol in bacterial fermentations. Biochem. J. 36:575-581. 21. Stormer, F. C. 1968. The pH 6 acetolactate-forming enzyme from Aerobacter aerogenes. J. Biol. Chem. 243:3735-3739. 22. Stormer, F. C. 1968. Evidence for induction of the 2,3butanediol-forming enzymes in Aerobacter aerogenes. FEBS Lett. 2:36-38.

APPL. ENVIRON. MICROBIOL. 23. Timell, T. E. 1967. Recent progress in the chemistry of wood hemicelluloses. Wood Sci. Technol. 1:45-70. 24. Ward, G. E., 0. G. Pettljohn, and R. D. Coghill. 1945. Production of 2,3-butanediol from acid-hydrolyzed starch. Ind. Eng. Chem. 37:1189-1194. 25. Yu, E. K. C., and J. N. Saddler. 1982. Power solvent production by Klebsiella pneumoniae grown on sugars present in wood hemicellulose. Biotechnol. Lett. 4:121126.