Klebsiella Planticola JEFFREY S

Fermentation of D-Xylose to Ethanol by Genetically Modified Klebsiella planticola JEFFREY S. TOLAN AND R. K. FINN* School of Chemical Engineering, Cornell University, Ithaca, New York 14853 Received 19 November 1986/Accepted 1 June 1987

D-Xylose is a plentiful pentose sugar derived from agricultural or forest residues. Enteric bacteria such as Klebsiella spp. ferment D-xylose to form mixed acids and butanediol in addition to ethanol. Thus the ethanol yield is normally low. Zymomonas spp. and most yeasts are unable to ferment xylose, but they do ferment hexose sugars to ethanol in high yield because they contain pyruvate decarboxylase (EC 4.1.1.1), a key enzyme that is absent from enteric bacteria. This report describes the fermentation of D-xylose by Klebsiella planticola ATCC 33531 bearing multicopy plasmids containing the pdc gene inserted from Zymomonas mobilis. Expression of the gene markedly increased the yield of ethanol to 1.3 mol/mol of xylose, or 25.1 g/liter. Concurrently, there were significant decreases in the yields of formate, acetate, lactate, and butanediol. Transconjugant Klebsiella spp. grew almost as fast as the wild type and tolerated up to 4% ethanol. The plasmid was retained by the cells during at least one batch culture, even in the absence of selective pressure by antibiotics to maintain the plasmid. Ethanol production was 31.6 g/liter from 79.6 g of mixed substrate per liter chosen to simulate hydrolyzed hemicellulose. The physiology of the wild-type of K. planticola is described in more detail than in the original report of its isolation.

Hemicellulose, a major constituent of plant cell wall (7; see also Fig. 1 of reference 35). The ethanol yield from materials, makes up 30 to 40% of many agricultural residues these pathways is low, because ethanol is produced only (20). Upon hydrolysis with acids or enzymes, hemicellulose from the phosphoroclastic split of pyruvate to ethanol- is converted to a mixture of hexose sugars and the pentose acetate-formate (or H2 plus C02) in the molar ratio 1:1:2. sugars D-xylose and L-arabinose (11, 21). The microbial The key enzyme for ethanol production in the obligately conversion of these pentose sugars to ethanol for use as a fermentative bacterium Z. mobilis, PDC, was implanted into fuel additive has received considerable attention (31). the Klebsiella sp. to increase the ethanol yield. PDC Current research is focused on fermentation of xylose by catalyzes the conversion of pyruvate to CO2 and acetalde- yeasts (10, 18, 34) or by both mesophilic and thermophilic hyde, which is subsequently reduced to ethanol with con- clostridia (1, 22, 24, 37). Yeasts produce ethanol efficiently current oxidation of NADH by alcohol dehydrogenase, a from hexoses by the pyruvate decarboxylase (EC 4.1.1.1)- common enzyme present in many organisms including Kleb- alcohol dehydrogenase (EC 1.1.1.1) (PDC-ADH) system. siella spp. The structural gene which codes for PDC has However, during xylose fermentation the by-product xylitol been isolated in Z. mobilis and cloned into Escherichia coli accumulates, thereby reducing the yield of ethanol (6, 18). (3) and Erwinia chrysanthemi (35). In both cases the amount Furthermore, yeasts are reported to ferment L-arabinose of ethanol produced increased markedly relative to untrans- only very weakly (11). In contrast to yeasts, the clostridia conjugated cells, with concurrent decreases in acetate and can rapidly catabolize a variety of pentoses. However, they formate levels. However, cell growth rates and tolerance to do not possess the PDC-ADH system for dissimilation of ethanol were sharply diminished. The reason for these pyruvate; rather, pyruvate undergoes a thioclastic cleavage effects was not clear. By using Klebsiella sp. as the recipient to yield acetyl coenzyme A, C02, and H2. Clostridia are of the Zymomonas pdc gene, we hoped to avoid these therefore limited in ethanol yield by production of unwanted problems; the Klebsiellae have a higher growth rate on metabolites such as acetate, lactate, butanol, and butyrate. xylose and a higher alcohol tolerance than many other Only a handful of bacterial species are known which do enteric bacteria have (Tolan, unpublished data). possess the important PDC-ADH pathway to ethanol (8, 32). This paper describes the improvement in the production of Among these, Zymomonas mobilis has the most active PDC ethanol from D-xylose by a recombinant Klebsiella sp. over system, although it is incapable of dissimilating pentose production by the wild type. Klebsiella planticola was sugars. chosen as the recipient of the pdc gene from Z. mobilis on We chose to study the fermentation of xylose to ethanol the basis of its lack of pathogenicity. The effect of the by the enteric bacterium Klebsiella sp., which contains the implanted pdc gene on growth, ethanol tolerance, and fer- gene that codes for pyruvate decarboxylase from Z. mobilis. mentation products is described here. Klebsiellae are gram-negative facultative anaerobes. Of the enteric bacteria, they ferment the broadest range of sugars, MATERIALS AND METHODS including all of the pentoses (23, 26). Klebsiellae ferment Cultures. The cultures used were K. planticola ATCC both hexoses and pentoses by the Embden-Meyerhof path- 33531 (2) and, in some experiments, Klebsiella oxytoca way to yield pyruvate, which is dissimilated to a coliform- NRRL B-199, provided by L. Nakamura. Monthly subcul- type mix of acidic, neutral, and gaseous products including tures were made on nutrient agar slants and stored under ethanol, acetate, formate, lactate, butanediol, C02, and H2 refrigeration. Escherichia coli S17-1(pZM15) was kindly supplied by B. * Corresponding author. Brau (3). Plasmid pZM15 comprises the pdc gene from Z. 2039 2040 TOLAN AND FINN APPL. ENVIRON. MICROBIOL.

at 3.5% ethanol, growth was limited to one or two doublings Anaerobic Growth with much less than 2 g of xylose consumed per liter, and at 5% ethanol, no growth was observed. A somewhat similar pattern of inhibition has been reported for E. coli (9). The 0.45 F decline in growth rate at 3% ethanol was accompanied by the appearance of filamentous cells, 25-fold longer than cells in OD an ethanol-free medium. Such filaments have been observed in cultures of Z. mobilis and E. coli in excess ethanol and 0.30 indicate irregularities of cell replication (16). Growth and ethanol tolerance were stimulated by the addition of yeast extract to the growth medium. More than 2

0 g of xylose per liter was utilized in up to 4% ethanol in the 0.15 0- presence of 2.5 g of yeast extract per liter. The factor(s) responsible for growth stimulation are not known. Addition of Casamino Acids (Difco) to the growth medium stimulated growth only in the absence of ethanol (data not shown).

0 2 4 6 Ashing of the yeast extract or treatment with activated Time (hr) charcoal to leave mainly inorganic salts completely removed the stimulatory effect, in contrast to the findings of Osman FIG. 1. Anaerobic growth (optical density [OD] at 600 nm) of K. and Ingram, who studied the role of yeast extract in promot- planticola and K. oxytoca at initial pH 7 in minimal xylose medium ing alcohol tolerance in Zymomonas spp. (28). Extraction of plus 1 g of yeast extract per liter. Symbols: 0, A, 37°C; 0, A, 30°C; yeast extract with diethyl ether to remove membrane com- 0, 0, K. oxytoca; A, A, K. planticola. K. planticola grows best at 30°C, and K. oxytoca grows best at 37°C. ponents (33) had no effect on yeast extract activity. The anaerobic growth rate of transconjugant cells was within 17 to 28% of that of the wild type in the absence of mobilis and its promoter and the chloramphenicol resistance ethanol, and consumption of 2 g of sugar per liter was gene. E. coli S17-1 is a histidine auxotroph. Plasmid pZM15 attained in up to 3.5 to 4% ethanol (Fig. 2). Aerobically, the was conjugated into K. planticola by filter mating by G. transconjugants grew 20% more slowly than the wild type. Schatz and S. V. Beer, Department of Plant Pathology, Cell growth and xylose consumption follow Monod kinet- Cornell University. ics, with a saturation constant (K, value) measured from Cultivations and analysis. Composition of the media, cul- steady-state chemostat conditions (22) of 1.2 g/liter. This tivation methods for cultures in broth tubes or jar fer- relatively high K, value for xylose (several hundredfold mentors, and analytical methods were the same as those higher than that for glucose) is similar to the K, vlaues for described in the companion paper (35). The conjugated K. growth of several other bacteria on xylose (17, 29, 30, 35). planticola cells were cultivated as the wild type, with the Fermentation products. Batch fermentations were run at addition of 20 mg of chloramphenicol per liter unless other- 30°C and several controlled pH values for cells with and wise indicated. Sugars were autoclaved as 41% (wt/vol) without the pdc plasmid (Fig. 3). The fermentation products concentrates and injected into the fermentor, or added by did not vary with temperature over the range 22 to 37°C (data Masterflex pump (Cole-Parmer Instrument Co.) during fed- not shown). The mass balances accounted for 85 to 108% of batch runs. For cultures with more than 10 g of carbon source per liter, the basal salt medium was supplemented with 5 g of yeast extract (BBL Microbiology System) per liter, 5.7 g of (NH4)2SO4 per liter, and 0.2 ml of Antifoam C emulsion (Sigma Chemical Co.) per liter. Unless otherwise stated, cultures were grown at 30°C and the initial pH was 7.0. In the mixed-substrate experiments, total pentoses were 0.20F determined by the orcinol assay (13) and glucose was determined by an enzymatic assay (Sigma). 0.15 RESULTS Strain selection. K. planticola grew optimally at 30°C, (hrl) while K. oxytoca grew best at 37°C (Fig. 1). The difference in o.1 oF optimum growth temperature was somewhat surprising, since both K. planticola and K. oxytoca give negative fecal coliform reactions (fermentation of lactose at 44.5°C) and grow at 10°C (2). The lower temperature for optimum growth 0.05 of K. planticola provides a further indication of its nonpath- ogenic, saprophytic nature, in contrast to the common clinical isolates of K. pneumoniae and K. oxytoca. K. planticola was therefore used for all subsequent experi- 0 1 2 3 4 ments. Ethanol (Wt.%) Growth and ethanol tolerance. The anaerobic growth rate FIG. 2. Growth of K. planticola wild type (0) and PDC trans- of K. planticola on xylose plus salts medium supplemented conjugants (0) at several ethanol concentrations at 30°C and initial with 0.5 g of yeast extract per liter was 0.21/h (Fig. 2). pH 7 in 5 g of xylose per liter plus salts medium supplemented with Growth rate decreased as ethanol was added to the medium; yeast extract at 0.5 g/liter (slash through points) or 2.5 g/liter. VOL. 53, 1987 FERMENTATION OF PENTOSES TO ETHANOL BY KLEBSIELLA SPP. 2041

CELLS 1.0 E Co2cE CELLS E CELLS CELLS 0.8 Co2 E Product Co2cE Co2cE Co2 g/gxyIose F F F 0.6 CELLS CELLS CELLS 23-8 IHA CELLS F F HAC 0.4 HAC HAC HAC HAC HAC HAC HAC 0.2 EtOH EtOH EtOH EtOH EtOH EtOH EtOH EtOH 0 pH 5.4 6.2 7.8 5.4 6.2 7 7.8 WILD TYPE PDC TRANSCONJUGANTS FIG. 3. Batch fermentation products of K. planticola grown on 5 g of xylose per liter at 30°C and controlled pH. Abbreviations: 2,3-B, 2,3-butanediol; L, lactate; HAC, acetate; EtOH, ethanol; F, formate; CELLS, cell dry weight; CO2E, carbon dioxide estimated from equation 1. The PDC transconjugants produce significantly more ethanol than the wild type does at low pH but not at neutral to high pH. the sugar used, including the following estimate for CO2 transconjugants might have resulted from their diversion of production (7): carbon away from acetate toward ethanol. The rate of Moles of CO2 = moles of acetate + moles of ethanol fermentation was not affected by pZM15 over the range of - pH 5.4 to 7.8 (Fig. 4B). + 2 x (moles of butanediol) moles of formate (1) Ethanol production. Fermentations were run with wild- The estimate of CO2 includes a small amount of hydrogen. type and conjugated K. planticola organisms to determine The cells without pZM15 produced ethanol, acetate, and the maximum attainable ethanol production. Xylose was formate in the molar ratio 1:1:2 during fermentation at added in 25-g/liter increments until product accumulation neutral to high pH. This product distribution is characteristic stopped growth and sugar consumption. These experiments of the phosphoroclastic cleavage of pyruvate by pyruvate were done at pH 5.8 to obtain a high ethanol yield (Fig. 3), formate-lyase (7) and resulted in the formation of0.59 to 0.72 little formate, which had been found to be growth inhibitory moles of ethanol per mol of xylose. When the fermentation environment was acidic, ethanol and acetate yields were unchanged, but the amount of gassing was noticeably higher at the expense of formate production. Formic hydrogen lyase catalyzes the conversion of formate to CO2 plus H2 and is particularly active at low pH (39). Lactate and butanediol 0.081 Yield were produced at low pH, which is also typical of enteric 0 bacteria. However, the amounts (less than 0.05 g/g of xylose) Yx/s .0. 0/ were much lower than have been observed with K. oxytoca 0.041 fermenting xylose (17) or with Klebsiella aerogenes fermenting glucose (25). LA The transconjugants produced much more ethanol than acetate at pH 5.4; the ethanol yield of 1.3 mol/mol of xylose, p) and the ratio 8.7 mol of ethanol per mol of acetate demon- 0 strated that the PDC-ADH system was more active than the 0 pyruvate formate-lyase system. At neutral and high pH, 4110 however, the distribution of products was quite similar to Fermentation that expected for the wild type, with equimolar production of Time ethanol and acetate. Analysis by polyacrylamide gel electro- phoresis revealed that the cells made more PDC at pH 5.6 (hr) 2.0I than 7.8 (G. Schatz, unpublished data). The maximum B activity of Zymomonas PDC is detected at pH 6 (15). The L lower ethanol yield at pH 7.8 was therefore caused by both 01 I I I I a smaller amount of PDC present and less PDC activity than 5 6 7 8 at pH 5.6. pH The presence of pZM15 decreased the cell yield (Fig. 4A). FIG. 4. Cell yield (A) (grams [dry weight] per gram of xylose) Since each mole of acetate produced affords the cell an and fermentation time (B) (time for exhaustion of 5 g of xylose per additional mole of ATP (7), the lower cell yield of the liter) for PDC transconjugants (0) and wild-type (O) K. planticola. 2042 TOLAN AND FINN APPL. ENVIRON. MICROBIOL.

4 batch culture in the absence of chloramphenicol, as measured by colony formation with and without the antibiotic (Table 1). Sugar consumption and ethanol production were 3 only slightly decreased by the absence of chloramphenicol. To test whether the yield of ethanol depended on sugar Optical concentration, three schemes for feeding the xylose were Density compared. In the batch fermentation an initial 75 g of xylose 2 per liter was added; in pulsed-feed mode the same total amount was added but in three increments of 25 g/liter. During fed-batch operation a continuous feed stream was used to maintain a low level (0.3 to 3.0 g/liter) of xylose during fermentation. From such experiments it was found that the yield of ethanol is essentially independent of the fermentation 0 xylose concentration (data not shown). The time was 1.5 times longer during fed-batch operation, however, reflecting the high K, value. 60 Ethanol production from a pentose-hexose mixture. Since a typical hydrolysate of hemicellulose would contain other Xylose d45 sugars besides xylose, it was of interest to know how the K. Consumed planticola PDC transconjugants would ferment a sugar mix- (g/L) ; I30 ture. Figure 7 shows the time course of batch and fed-batch fermentations of a mixture of D-glucose, D-xylose, and L-arabinose. By operating the fermentor in a fed-batch mode at pH 5.3, we obtained the highest ethanol concentration, 31.6 g/liter, from 79.6 g of total sugar per liter. It was evident from the batch runs that much more 2,3-butanediol and Time (hr) acetate were produced from the mixed substrate than from xylose alone (Fig. 3). During the fed-batch run, we sup- FIG. 5. Growth rate (A) and xylose consumption (B) by K. the production of butanediol by using a slow feed cells. Arrows indicate pressed planticola wild-type (0) and conjugated (0) rate to prevent glucose accumulation in the fermentor. The addition of 25 g of xylose per liter. Wild-type cells grew well and fermented xylose for 43 h before extensive lysis. The PDC trans- use of pH 5.3 rather than pH 5.8 reduced the rate of acetate conjugants continued to grow for 75 h and used 67% more xylose accumulation, as has been previously reported for K. aero- than the wild type did. genes fermenting glucose (25). The toxic effect that limited the fermentations shown in Fig. 7 was apparently the accumulation of acetate. (data not shown), and a reasonably short fermentation time (Fig. 4B). The transconjugated cells fermented 67 g of xylose per 32 liter, compared with just 40.4 g/liter for the wild type (Fig. 24 0-.....o---O - 5B). The growth curves and rates of sugar consumption (a Ethanol maximum of 1.6 glliter per h) were similar for wild-type and (g/L) 16 conjugated cells for the first 40 h (Fig. SA). The transconjugants continued to grow and consume xylose, but the wild 8 type lysed, as reflected by the sharp drop in the optical moo== I I density after 43 h. Figure 6 shows the time course of product formation during this experiment. The transconjugants pro- 6 Acetate duced 24 g of ethanol per liter, but the wild type produced or 4 only 9.4 g/liter. The toxic effect that limited fermentation for Formate both types of cells was apparently the accumulation of 2 formic and acetic acids rather than the accumulation of (g/L) ethanol. According to the earlier experiments (Fig. 2), eth- 0 anol could be tolerated at higher levels than 24 g/liter. In separate experiments (data not shown), acetate had a low - threshold of toxicity, particularly at low pH; 50% inhibition 2 -101~ Butanediol I of growth was observed with 20 g/liter at pH 6.8 and with 8 F only 3 g/liter at pH 6. The free acid probably decreases the (g/L) proton gradient across the membrane and uncouples the 4-~~~~~~~ proton motive force, a mechanism that has been established in other organisms (14). Thus the transconjugants attained a 20 40 60 80 higher ethanol concentration because of both higher ethanol Time (hr) yield and lower acetate production. Significant amounts of FIG. 6. Fermentation products (in grams per liter) at pH 5.8 of butanediol were produced, especially by the wild-type cells cells in xylose and 5 g of yeast extract per liter until accumulation of (Fig. 6). Separate experiments (data not shown) showed that end products stopped the fermentation (see Fig. 6). The transcon- growth is not inhibited even at levels up to at least 40 g of jugants (0) consumed more xylose and produced more ethanol than butanediol per liter. the wild type (0) did because of higher ethanol yield and less The plasmid was retained by 65 to 73% of the cells after acetate. VOL. 53, 1987 FERMENTATION OF PENTOSES TO ETHANOL BY KLEBSIELLA SPP. 2043

TABLE 1. Effect of chloramphenicol on fermentationa Cell density (109/ml)b Fermentor condition Chloramphenicol Chloramphenicol % Plasmid retained (g/liter) Ethanol level present absent gitr(gler Chloramphenicol added (20 mg/i) Expt 1 9.2 9.3 99 67 24 Expt 2 8.8 8.2 >100 69.1 25.1 No chloramphenicol added Expt 1 6.4 8.7 73 64 22.5 Expt 2 5.5 8.4 65 59.9 20 a pH 5.8, 300C. b Plates poured 60 h after inoculation; total fermentation time was 70 to 85 h or roughly 10 generations of growth.

The sugar mixture was chosen to simullate the acid pyruvate, accumulated, which activated the enzymes of the hydrolysate of sugarcane bagasse (21). In addiition, 2.4 g of butanediol pathway. furfural per liter (36) and some dissolved calc:ium salts are Jeffries (19) has identified the following requirements for a likely to be present in an actual hydrolysate6. Such com- commercial process to produce ethanol from pentose sugars: pounds inhibit the fermentation of bagasse h3ydrolysate by (i) an ethanol yield of at least 0.4 g/g of pentose, (ii) an yeast cells. We found that dissolved calcium vvas not inhib- ethanol concentration of at least 50 g/liter, (iii) a fermenta- itory to K. planticola but that 0.8 g of furfFural per liter tion time of less than 36 h, and (iv) complete utilization of the caused a 50% growth inhibition (data not shvown). Others sugars. The K. planticola PDC transconjugants attain the using Candida spp. (21) have reduced the problem offurfural required ethanol yield. The ethanol concentration is limited toxicity by selection of tolerant mutants. by accumulation of acetate to 25.1 g/liter from xylose and 31.6 g/liter from the glucose-xylose-arabinose mixture. At- DISCUSSION tempts to select for mutants deficient in acetate production by resistance to fluoroacetate (5) were unsuccessful. Perhaps K. planticola has several desirable features ftcr the fermen- the gene which codes for the key enzyme in acetate produc- tation of xylose to ethanol. The 30°C optiimum growth tion, phosphotransacetylase, could be deleted (4, 12). Alter- suggests a lack of human pathogenicity. The r;ates of anaer- natively, PDC transconjugants with low acetate yield at pH obic growth (0.21/h) and xylose fermentation (1.6 g/liter per 7 would be desirable, because acetate is less toxic at neutral h) are high. The 4% native ethanol tolerance is;as high as any pH than at low pH. However, even in the absence of acetate, reported for enteric bacteria (9). Little butanediol was pro- K. planticola does not tolerate 50 g of ethanol per liter. Yeast duced from xylose; the use of glucose or high 14evels ofyeast extract stimulates ethanol tolerance, but the key growth extract stimulated butanediol formation. Gluci ose and yeast factor(s) have not been identified. extract caused rapid growth and sugar uptake; perhaps high The measured fermentation time of 70 to 85 h is longer intracellular concentrations of key intermedi;ates, such as than suggested by Jeffries (19). Attempts were made to grow a cell crop rapidly by sparging with air. However, the subsequent anaerobic fermentation of xylose resulted in the 52 dismutation of pyruvate to lactate, acetate, and CO2 (27; :~~~~~~~~ R. P. Mortlock, personal communication), which inhibited Ethanol 2>4 fermentation and resulted in a low ethanol yield. It might, (g/L) I16 -0 however, be possible to obtain rapid cell growth and fermen- - pH5.8 tation with a controlled low level of oxygen addition. A 8 _ { -*. o pH 5.3(E3atch) continuous fermentation could be envisioned, with the addi- L*pH5.3 (FFed Batch) tion of flocculents or the use of a filter to achieve a high cell 0 density. However, the low rate of xylose uptake, as indi- 6 cated by the high (1.2 g/liter) K, value, might result in a high residual sugar level in a single-stage continuous system. 4 Acetate Among the broader implications of this work are the (g/L) 0I-A pleiotropic effects of plasmid pZM15 when inserted into K. 2 0 A z -- planticola and into E. chrysanthemi, two closely related 0 0 I - --- I I genera. The implant of the pdc gene markedly enhanced the 0 - I~ I ethanol production by K. planticola, with little loss of o ~o growth rate or ethanol tolerance, and the plasmid was 12 - maintained without the use of antibiotic-selective medium. Butanediol E. chrysanthemi pdc transconjugants (35) also produced (g/L) 8 I- ethanol in high yield, but grew only one-fourth as fast as the wild type and tolerated only 2% ethanol instead of 4%. The plasmid was not maintained, even for one batch growth OL I^/ A I -A I A cycle, in the absence of chloramphenicol. 0 20 40 60 80 100 The enhanced ethanol yield of the pdc transconjugants is a Time (hr) rare example of the redirection of metabolic pathways by FIG. 7. Ethanol, acetate, and butanediol produiction by PDC gene insertion. Perhaps the best known previous example of transconjugants from a glucose-pentose mixture (se(e text). such altered pathways is the expression of glutamic dehy- 2044 TOLAN AND FINN APPL. ENVIRON. MICROBIOL. drogenase from E. coli in Methylophilus methylotrophus so ATCC 8724. Biotechnol. Bioeng. 26:362-369. as to enhance the cell yield (38). 18. Jeffries, T. W. 1983. Utilization of xylose by bacteria, yeasts, and fungi. Adv. Biochem. Eng. 27:1-32. 19. Jeffries, T. W. 1985. Emerging technology for fermenting D- ACKNOWLEDGMENTS xylose. Trends Biotechnol. 3:208-212. We thank S. Bringer-Meyer and B. Brau, Nuclear Research 20. Krull, L. H., and G. I. Inglet. 1980. Analysis of neutral carbo- Center, Julich, Federal Republic of Germany, who provided the hydrates in agricultural residues by gas-liquid chromatography. initiative for this work and participated in many helpful discussions. J. Agric. Food Chem. 28:917-919. The collaborative effort of S. Beer and G. Schatz, Cornell Univer- 21. Lodics, T. A., and C.-S. Gong. 1984. Isolation of sugarcane sity, is also gratefully acknowledged. bagasse hydrolyzate-tolerant yeast mutants by continuous se- This work was supported by a grant from the Cornell Biotechnol- lection. Biotechnol. Bioeng. Symp. 14:303-307. ogy Program, which is sponsored by the New York State Science 22. Lovitt, R. W., R. Longin, and J. G. Zeikus. 1984. 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