Clostridium formicoaceticum

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1987, p /87/ $02.OO/O Copyright 1987, American Society for Microbiology Vol. 53, No. 4 Kinetics of Homoacetic Fermentation of Lactate by Clostridium formicoaceticum SHANG-TIAN YANG,'* I-CHING TANG,2 AND MARTIN R. OKOS2 Department of Chemical Engineering, The Ohio State University, Columbus, Ohio 43210,' and Department of Agricultural Engineering, Purdue University, West Lafayette, Indiana Received 14 October 1986/Accepted 20 December 1986 Clostridiumformicoaceticum homofermentatively converted lactate to acetate at mesophilic temperatures (30 to 42 C) and at phs between 6.6 and 9.6. The production of acetate was found to be growth associated. Approximately 0.96 g of acetic acid and g of cells were formed from each gram of lactic acid consumed at 3rC. The concentration of the substrate (lactate) had little or no effect on the growth rate; however, the fermentation was inhibited by acetic acid. The bacterium grew at an optimal ph of 7.6 and an optimal temperature of 37C. Small amounts of bicarbonate were stimulatory to bacterial growth. Bacterial growth was enhanced, however, by the use of higher concentrations of bicarbonate in the media, only because higher buffer capacities were obtained and proper medium ph could be maintained for growth. Based on its ability to convert lactate to acetate, this homoacetic bacterium may be important in the anaerobic methanogenic process when lactate is a major intermediary metabolite. In the past decade, growing concern about environmental pollution and energy shortages have generated interest in the production of methane through anaerobic digestion of organic wastes. The anaerobic methanogenic process degrades organic matter to the gaseous products CH4 and CO2 (8), which occurs as a result of three distinct but simultaneous metabolic phases (9). First, in the hydrolytic phase, large, complex organic matter is hydrolyzed and fermented to C, to C5 organic acids, alcohols, H2, and CO2. In the second (acetogenic) phase, C3 to C5 organic acids and alcohols are further reduced to acetate, formate, methanol, C02, and H2. These products are then used for methanogenesis in the last phase. The complete process involves at least four metabolically distinct groups of bacteria: hydrolytic, H2-producing acetogenic, homoacetogenic, and methanogenic (9, 10). Acetic acid is the major immediate precursor for methane formation (8, 9). Acetic acid could be derived from carbohydrates by the following methods: (i) homoacetic fermentation of glucose to acetate; (ii) heteroacetogenic fermentation of glucose in the presence of H2-utilizing methanogens to shift the metabolism to favor the acetate formation; (iii) homolactic fermentation of glucose or lactose to lactate and then to acetate by homoacetic fermentation or by H2- producing acetogenic fermentation with the coproductions of H2, CO2, and formate; and (iv) ethanol fermentation followed by H2-producing acetogenic fermentation. The exact fermentation kinetics is not known yet and could be very complicated and highly dependent on the environmental conditions (S. T. Yang, M. R. Okos, and J. C. Nye, Meet. Am. Soc. Agric. Eng., paper , 1982). Recently, results of 14C tracer studies (6) on the intermediary metabolism during anaerobic degradation of whey lactose have shown that lactate is the major intermediary metabolite of lactose fermentation in a chemostat operated under lactose-limited conditions. Some 82% of the lactose was transformed into lactate during the hydrolytic phase. Lactate and other intermediary products were then transformed into acetate and H2-CO2, which were the substrates for methanogens. Although lactate is usually not found in * Corresponding author. 823 large amounts in a normal anaerobic digester under steadystate conditions, large amounts of lactate are accumulated during the anaerobic fermentation when the digester ph is below 6 (11), indicating that lactate may have been produced and converted to acetate or other compounds during the digestion process. Homoacetogenic bacteria can ferment a wide spectrum of multi- or one-carbon compounds to produce acetic acid (1, 3, 4, 7). Because more than 70% of the methane formed in the anaerobic digester is directly derived from acetic acid (8, 9), the homoacetic bacteria may play an important role in the anaerobic digestion process. Although several homoacetic bacteria have been isolated from anaerobic digesters (1, 3, 4, 7), in contrast to the methanogens, very little has been done on characterizing the homoacetic bacteria. To our knowledge, no homoacetic bacteria can ferment lactose. However, results of previous research (1, 3) have shown that two homoacetic bacteria, Acetobacterium woodii and Clostridium formicoaceticum, can grow on lactate. In addition, the sulfate-reducing bacteria Desulfovibrio spp. can produce acetate from lactate in the presence of sulfate or H2-utilizing bacteria (5). These bacteria may be responsible for the conversion of lactate to acetate in anaerobic digestion. The growth of homoacetic bacteria on sugars and H2-CO2 has been relatively well studied (1, 3), but very little is known about the growth of these bacteria on lactate. C. formicoaceticum is a gram-negative rod, strictly anaerobic, and mesophilic with a reported optimum temperature of about 37 C and a ph of about 7.2 when grown on fructose (1). This bacterium can neither use nor produce hydrogen gas because of a lack of hydrogenase. About 3 mol of acetate was produced from each mole of fructose that was fermented during the exponential phase. However, formate was also produced at the expense of acetate in the stationary phase. This bacterium is also known to grow on lactate, but no work has been reported on the growth kinetics. In this study the fermentation kinetics of C. formicoaceticum grown on lactate was investigated. The effects of temperature, ph, sodium chloride, lactate, acetate, and bicarbonate on bacterial growth were studied and are reported here. The possible functional importance of this bacterium in

2 824 YANG ET AL. APPL. ENVIRON. MICROBIOL. anaerobic digestion is also discussed, based on its ability to convert lactate to acetate. MATERIALS AND METHODS Culture. C. formicoaceticum ATCC was isolated from sewage sludge by Andreesen et al. (1) and was used throughout this study. Medium. The medium was prepared by the procedures described by Balch et al. (2). Unless otherwise noted, the medium contained the following, in grams per liter: K2HPO4. 3H20, 0.31; KH2PO4, 0.24; (NH4)2SO4, 0.24; MgSO4 7H20, 0.1; NaCl, 0.48; CaCl2-2H20, ; FeSO4.7H20, 0.002; resazurin, ; Trypticase (BBL Microbiology Systems, Cockeysville, Md.), 1.0; yeast extract (Difco Laboratories, Detroit, Mich.), 1.0; L-cysteine hydrochloride H20, 0.5; sodium lactate, 7.4; NaHCO3, 6.0. A vitamin solution and a trace mineral solution were each added at 10 ml per liter of medium. The gas phase was 40% C02-60% N2 at 24.7 lb/in2, and the final ph was 7.0. In the experiments for determining the growth yield, concentrations of sodium lactate in the medium varied from to M. To study the ph range for growth, the medium ph was adjusted to the desired ph range (between ph 6 and 10) by adding 4 N HCl or 4 N NaOH. To evaluate the effect of sodium ion, NaCl solution was added to the medium to a final concentration of 0.05 to 3.5%. To study the effect of NaHCO3 on bacterial growth, 0.5 M potassium phosphate buffer (ph 7.6) was used for media containing various amounts of NaHCO3 (0 to 16 g/liter). Initial concentrations of sodium acetate of between 0 and 0.3 M were used to study the effect of acetate on bacterial growth. Culture techniques. The anaerobic syringe techniques described by Balch et al. (2) were used in this study. Cells were cultivated in serum tubes (18 by 150 mm) and 125-ml serum bottles containing 7.5 and 50.0 ml of the medium, respectively. The culture was maintained in an active state by transferring 2% of the culture volume to fresh medium every week. All cultures were incubated at 37 C without shaking in the dark. Kinetic studies. Batch fermentations were performed in serum tubes, serum bottles, or both under uncontrolled ph conditions to evaluate effects of ph, sodium ion, lactate, acetate, and bicarbonate. A liquid sample (1 ml) was taken for optical density (OD) reading, ph measurement, and high-performance liquid chromatographic (HPLC) assay. Fermentation kinetics was also studied under controlled constant ph conditions with 5-liter fermentors (New Brunswick Scientific Co., Inc., Edison, N.J.). The fermentation broth was automatically titrated with membranefiltered (pore size, 0.2,um) 4 N NaOH to maintain a desired ph (±0.01). A total of 3 liters of medium was used for each batch fermentation, and 50 ml of active culture was inoculated into the medium. The anaerobiosis was maintained by keeping a positive N2-CO2 gas pressure (19.7 lb/in2 or 1.34 atm) inside the fermentor. At proper time intervals (depending on the fermentation rate), 5 ml of liquid sample was withdrawn from the sampling port for OD reading and then frozen for future HPLC analysis. Analytical techniques. Lactate, acetate, and other organic compounds (if present) were identified and quantitated by the HPLC method. A system (Waters Associates, Inc., Milford, Mass.) consisting of a pump (6000A; Waters), injector (U6K; Waters), and differential refractometer (R401; Waters) was used. A 10-pd portion of supernatant from centrifuged samples was injected onto an organic acid i pho -70t Calf.4a -6.8 Bacterial wt ~ nm ectropotomete TIME (HOURS) cf 0. lmn airto ure o tnad fec FIG. 1. Batch fermentation of C. formicoaceticum grown at an initial ph of The medium contained 8 g of NaHCO3 per liter, and the gas phase contained 16 lb/in2 Of CO2 and 14.7 lb/in2 of N2. column (HPX-87H; Bio-Rad Laboratories, Richmond, Calif.) at 46aC. The eluant was N H2S04 at a flow rate of 0.6 ml/min. Calibration curves for standards of each compound were determined, and the organic content of each sample was determined by peak height measurements. Bacterial growth was monitored by measuring the gd at 660(OD6l) nm on a spectrophotometer (model 350; Turner). A direct measurement of GD in the growth tube was made for cultures grown in the serum tubes. Otherwise, GD was measured in a 1.5-ml polystyrene cuvette with a path length of 10 mm. Samples were diluted with water if GD readings were greater than 0.4. Cell density was found to be proportional to the OD60 when it was smaller than 0.4. One unit of ODw0 was found to be equivalent to 0.55 g (dry weight) of cell per liter. The specific (ps) growth rate was determined from the slope of semi-logarithmic plot of OD6w versus time. The fermentation product yield was determined from the negative slope of the plot of acetic acid concentration versus lactic acid concentration. The cell growth yield was determined from the negative slope of the plot of cell density (OD660) versus lactic acid concentration in the exponential growth phase. RESULTS AND DISCUSSION Fermentation products. From cultures grown on lactate at various phs (6.6 to 9.6), NaHCO3 concentrations (2 to 16 g/liter), acetate concentrations (0 to 18 g/liter), or temperatures (25 to 420C), it was determined that acetic acid is the only fermentation product from lactic acid. Figure 1 shows typical time course data of the fermentation under uncontrolled ph conditions. The ph of the fermentation broth decreased with the production of acetic acid. No formic acid or any other organic compounds were formed throughout the fermentation, suggesting a homoacetic fermentation for this bacterium when it is grown on lactate. This is different from growth on fructose, in which both acetic acid and formic acid were formed during the stationary phase (1). Growth yield. Plots from seven batch fermentations with different initial lactate concentrations (0.027 to 0.16 M) indicate that the acetate yield from lactate was constant at g of acetic acid per gram of lactic acid consumed, or about 1.5 mol of acetate produced per mole of lactate fermented. The cell growth yield at ph 7.0 and 37 C was found to be g of cell (dry weight) per gram of lactic acid consumed. In general, about 50% of the bacterial

3 VOL. 53, 1987 cell mass was carbon. Thus, nearly 100% of the carbon and hydrogen in the substrate (lactic acid) was recovered in the fermentation product (acetic acid) and the cells. Both product formation and cell growth were proportional to the substrate consumption, suggesting that the formation of acetate from lactate is growth associated. Effect of temperature. It was found that the growth rate decreased when the temperature was increased from 37 to 42 C in a controlled, constant ph experiment. Temperature did not affect the acetate yield, but only about 60% of the cell growth yield was obtained at 42 C as compared with that at 37 C (Fig. 2). The observed lower fermentation rate at 42 C might be due largely to the lower cell growth yield. This finding was consistent with the reported optimal temperature of about 37 C for C. formicoaceticum when grown on fructose (1). Effect of NaCl. NaCl in the concentration range of 0.05 to 3.0% (85 to 510 mm) did not significantly affect the specific growth rate. However, a longer lag phase and a slower growth rate were found with 3.5% NaCl. Effect of lactate. The concentration of lactate in the range of to 0.16 M did not affect the specific growth rate. However, a longer lag phase was found for those cultures grown in a medium with a higher sodium concentration when cells in the stationary phase were used to initiate the fermentation. This effect was not found for experiments with active cultures. Because a concentration of NaCl up to 0.51 M did not affect bacterial growth, we conclude that substrate (lactate) concentrations between and 0.16 M are neither stimulatory nor inhibitory to this fermentation. ph range for growth. Neither bacterial growth nor change in lactate or acetate concentration was found for cells cultivated at a ph below 6.6 or above 9.6 for a period of 30 days, suggesting a growth ph range of between 6.6 and 9.6. Effects of ph. By using data from the early exponential growth phase, the specific growth rates at various ph values were determined, and the effect of the ph on the specific growth rate is shown in Fig. 3. Apparently, this bacterium has an optimal ph for growth at about 7.6. The fermentation rate was relatively insensitive to ph changes between 7.5 and 8.8 but decreased dramatically at a ph below 7.5 or above 8.8. The medium ph also affected the lag phase In the ph HOMOACETIC FERMENTATION OF LACTATE _ FIG. 3. Effect of ph on specific growth rate.,u, Specific growth rate. range of 6.6 to 7.6, the bacterium had a longer lag phase at a lower ph. However, the length of lag phase was also greatly affected by the viability of the inoculum and other environmental factors such as salt concentration.; Although the growth of C. formicoaceticurn was sensitive to ph, the acetate yield was not dependent on ph. This might be because the production of acetic acid during the fermentation was always growth associated. However, higher ultimate cell densities were observed for cultures grown at higher ph values. This could be due to either the higher cell yield or the lower cell decay rate at a higher ph. Effect of acetate. The specific growth rate was lower when the concentration of sodium acetate was higher at the same ph. This effect was mainly due to the acetic acid because concentrations of NaCl up to 0.5 M did not affect the fermentation. The specific growth rate decreased from 0.14 to h-1 when the concentration of acetic acid increased from 0 to 18 g/liter (0.3 M) at ph 7.0 (Fig. 4). However, concentrations of acetate up to 0.6 M did not completely inhibit bacterial growth at ph 7.0 or higher. The effect of acetate was found to be stronger at a lower medium ph, indicating that the undissociated acid may be more inhibitory than the dissociated acid. Effect of bicarbonate. The concentration of bicarbonate may affect bacterial growth either directly or indirectly through its buffering effect on the ph. Andreesen et al. (1) ph 0.16 I r-i I 1 T,--I- T ' 1.00 (D d 0.75 Ct) Z 0.50 C] -J w c Is. LACTIC ACID (G/L) Effect of temperature on cell yield. In a batch fermenta- FIG. 2. tion under controlled constant ph conditions, the cell growth yield dropped significantly when the temperature was increased from 37 to 42 C. I n C: o ph 7.0, a ACETIC ACID (G/L) FIG. 4. Effect of acetic acid on specific growth rate at ph 7.0.

4 826 YANG ET AL. TABLE 1. Effects of NaHCO3 and ph on homoacetic fermentation Pco2 NaHCO3 concn b pc[hlactatelf (lb/in2)a (g/liter) (hr'1) (g/fiter) a pc02, Partial pressure of CO2 in the gas phase; all culture bottles also contained 14.7 lb/in2 of N2 in the gas phase. b phi, Initial medium ph value. c.i., Initial specific growth rate. d phf, final medium ph value. e [Lactatelf, Final lactate concentration; all media initially contained about 9.2 g of lactic acid per liter. have reported that the growth of C. formicoaceticum on fructose is enhanced by NaHCO3 and is faster when the medium contains more NaHCO3. However, the observed increased growth at highter NaHCO3 concentrations might actually have been due to the ph. Cultures grown in media having 0.5 M potassium phosphate buffer (ph 7.6) and various concentrations of NaHCO3 (0, 4, 6, 8, 12, 16 g/liter) were studied. The gas phase contained only N2, and the intial ph for these cultures was between 7.6 and 8.9. Because the buffer capacity was high and all media had a ph within or close to the ph-insensitive range (ph 7.5 to 8.8), the effect of ph on bacterial growth would be negligible under the experimental conditions used in this study. The growth in this high concentration of phosphate buffer started after an abnormally long lag phase of 50 h. However, it was found that C. formicoaceticum grew about equally well in all media, except for the one not containing NaHCO3. An extremely long lag phase of 150 h and much slower growth were found for the culture in the medium without NaHCO3. Cultures grown in all other NaHCO3 concentrations had about the same specific growth rate with slight differences; this followed the trend that was due to the ph effect. This suggests that a sodium bicarbonate concentration of between 4 and 16 g/liter is not important in affecting the fermentation rate if the medium ph is about the same, although the presence of sodium bicarbonate may stimulate cell growth. Further kinetic studies were performed with the buffer system C02-NaHCO3, and the media ph were adjusted between 6.2 and 7.7 by varying the concentration of NaHCO3 (2 to 8 gfliter) and the partial pressure of CO 2 (2 to 16 lb/in2). The performance of 12 batch fermentations is given in Table 1. No bacterial growth was found for three batches at an initial ph of below 6.6. However, for other batches with a ph higher than 6.6, bacterial growth proceeded until the medium ph was below 6.3 (Fig. 5) or the substrate (lactate) was comnpletely consumed (Fig. 1). The difference in the lower ph limit for growth might imply that either active cells can tolerate more acidic phs than dormant cells or that some acidic products are released into the medium after the cells stop growth. One exception was observed. Growth stopped at ph 6.56 when the initial ph s 6.0 TIME (HOURS) FIG. 5. Batch fermentation of C. formicoaceticum grown at an initial ph of The medium contained 4 g of NaHCO3 per liter, and the gas phase contained 2 lb/in2 of CO2 and 14.7 lb/in2 of N2. was 6.6. This was probably because the ph was too close to the lower limit to allow the fermentation to proceed. Four batch fermentations stopped before all substrates were depleted (Table 1). This was because the bicarbonate content (buffer capacity) was low and the acetic acid production caused the ph to drop rapidly to the lower limit and stopped the fermentation. Therefore, at the same initial medium ph, fermentation with a higher bicarbonate content has a faster fermentation rate and higher acetic acid production because the medium ph is better buffered. This explains the better growth in the medium containing more bicarbonate. However, the specific growth rate was found to be dependent only on the ph but not on the NaHCO3 concentration or the partial pressure of CO2 (Table 1). This is consistent with the results from other experiments in this study. The growth yield and the ph effect on the growth rate found in this study were also consistent with earlier findings (Fig. 3). Batch fermentation at controlled constant phs of between 6.9 and 7.6 were also studied. Figure 6 shows a batch fermentation at a constant ph of 7.2. Results from these experiments were consistent with those of previous uncontrolled ph experiments. As mentioned above, large amounts of lactate are accumulated in a digester when the medium ph is below 6 (11). We also have detected significant amounts of lactate as an important intermediary metabolite, along with other organic acids, in the ananerobic degradation of lactose and glucose Lactic Acid.I 1.0' _"_se Acetic Acid ph T (u7.0.5 '04 4%0 FIG. 6. Kinetics of aceticum at ph ~ APPL. ENVIRON. MICROBIOL. 0>. 5 C' A0-0 C'. I-. TIME (Hours) homoacetic fermentation of C. formico

5 VOL. 53, 1987 (S. T. Yang, Ph.D. thesis, Purdue University, West Lafayette, Ind., 1984). While no significant amounts of lactate could be detected in a normal anaerobic digester at the steady-state operation, lactate may have been produced and immediately converted to acetate in the digester by homoacetic and sulfate-reducing bacteria. Because the growth rate of C. formicoaceticum is not dependent on the concentration of lactate, all the lactate produced during the anaerobic digestion can be readily converted to acetate by this bacterium at a ph above 6.3. This may partially explain why lactate is only present in the sour digester with a ph below 6. Results of a study with mixed cultures of homolactic, homoacetic, and methanogenic bacteria has shown that the steady-state lactate concentration in this mixed culture fermentation could be close to 0 if growth was balanced (Yang, Ph.D. thesis). About 340 ml of methane was produced from each gram of lactose consumed in this fermentation. Populations of 105 to 106 homoacetogenic bacteria per ml of sewage sludge have been reported (10). Clostridium and Acetobacterium are two well-recognized genera. The importance of homoacetogenic bacteria in anaerobic digestion has been ascribed to their ability to ferment multicarbon compounds (mainly sugars) to acetate. In this study we showed that C. formicoaceticum can effectively convert lactate to acetate at a ph of between 6.3 and 9.6 and acetate concentrations of up to 0.6 M. While A. woodii also ferments lactate to acetate at a ph of between 5 and 7, it only grows at temperatures lower than 32 C and acetate concentrations below M (Yang, Ph.D. thesis). Because most anaerobic digesters are operated at a ph of about 7.0 and acetate concentrations of about 0.1 M, C. formicoaceticum would be more important than A. woodii for converting lactate to acetate in the anaerobic digestion process with lactate as a major intermediary metabolite. Populations of 104 of the sulfate-reducing bacteria Desulfovibrio spp. have also been reported (10). In the absence of sulfate, these bacteria convert lactate to acetate, H2, and CO2 when H2-utilizing methanogens (e.g., Methanobacterium formicicum) are present (5). However, the fermentation rate of a coculture of Desulfovibrio desulfuricans and M. formicicum was slow and was strongly inhibited by acetate, even at a low acetate concentration, as compared with that of C. formicoaceticum (Yang, Ph.D. thesis). Nevertheless, one cannot overlook the importance of sulfate-reducing bacteria in anaerobic digestion because they have been inevitably found in the anaerobic digesters. HOMOACETIC FERMENTATION OF LACTATE 827 The potential importance of C. formicoaceticum in anaerobic digestion may be ascribed to its ability to convert lactate to acetate. However, further work is needed to prove this hypothesis because most of the methanogenic ecosystems are very complex and the fermentation kinetics is highly dependent on the environmental conditions. ACKNOWLEDGMENTS This study was supported in part by grant 82-CRSR from the U.S. Department of Agriculture and a seed grant from the Office of Research and Graduate Studies, The Ohio State University. LITERATURE CITED 1. Andreesen, J. R., G. Gottschalk, and H. G. Schlegel Clostridium formicoaceticum nov. spec. Isolation, description and distinction from C. aceticum and C. thermoaceticum. Arch. Microbiol. 72: Balch, W. E., G. E. Fox, L. G. Magrum, C. R. Woese, and R. S. Wolfe Methanogens: reevaluation of a unique biological group. Microbiol. Rev. 43: Balch, W. E., S. Schoberth, R. S. Tanner, and R. S. Wolfe Acetobacterium, a new genus of hydrogen-oxidizing, carbon dioxide-reducing, anaerobic bacteria. Int. J. Syst. Bacteriol. 27: Braun, M., F. Mayer, and G. Gottschalk Clostridium aceticum (Wieringa), a microorganism producing acetic acid from molecular hydrogen and carbon dioxide. Arch. Microbiol. 128: Bryant, M. P., L. L. Campbell, C. A. Reddy, and M. R. Crabill Growth of Desulfovibrio in lactate or ethanol media low in sulfate in association with H2-utilizing methanogenic bacteria. Appl. Environ. Microbiol. 33: Chartrain, M., and J. G. Zeikus Microbial ecophysiology of whey biomethanation: intermediary metabolism of lactose degradation in continuous culture. Appl. Environ. Microbiol. 51: Leigh, J. A., F. Mayer, and R. S. Wolfe Acetogenium kivui, a new thermophilic hydrogen-oxidizing, acetogenic bacterium. Arch. Microbiol. 129: McCarty, P. L Anaerobic waste treatment fundamentals. I. Chemistry and microbiology. Public Works 95: Winfrey, M. R Microbial production of methane, p In R. M. Atlas (ed.), Petroleum microbiology. Macmillan Publishing Co., New York. 10. Zeikus, J. G Microbial populations in digesters, p In D. A. Stafford, B. I. Wheatley, and D. E. Hughes (ed.), Anaerobic digestion. Applied Science Publishers Ltd., London. 11. Zoetemeyer, R. J., A. J. C. M. Matthijsen, J. C. Van den Heuvel, A. Cohen, and C. Boelhouwer Anaerobic acidification of glucose in an upflow reactor. Biomass 2: