Abstract
The influence of a number of environmental parameters on the fermentation of glucose, and on the energetics of growth of Clostridium butyricum in chemostat culture, have been studied. With cultures that were continuously sparged with nitrogen gas, glucose was fermented primarily to acetate and butyrate with a fixed stoichiometry. Thus, irrespective of the growth rate, input glucose concentration specific nutrient limitation and, within limits, the culture pH value, the acetate/butyrate molar ratio in the culture extracellular fluids was uniformly 0.74±0.07. Thus, the efficiency with which ATP was generated from glucose catabolism also was constant at 3.27±0.02 mol ATP/mol glucose fermented. However, the rate of glucose fermentation at a fixed growth rate, and hence the rate of ATP generation, varied markedly under some conditions leading to changes in the Y glucose and Y ATP values. In general, glucose-sufficient cultures expressed lower yield values than a correponding glucose-limited culture, and this was particularly marked with a potassium-limited culture. However, with a glucose-limited culture increasing the input glucose concentration above 40g glucose·l-1 also led to a significant decrease in the yield values that could be partially reversed by increasing the sparging rate of the nitrogen gas. Finally glucose-limited cultures immediately expressed an increased rate of glucose fermentation when relieved of their growth limitation. Since the rate of cell synthesis did not increase instantaneously, again the yield values with respect to glucose consumed and ATP generated transiently decreased.
Two conditions were found to effect a change in the fermentation pattern with a lowering of the acetate/butyrate molar ratio. First, a significant decrease in this ratio was observed when a glucose-limited culture was not sparged with nitrogen gas; and second, a substantial (and progressive) decrease was observed to follow addition of increasing amounts of mannitol to a glucose-limited culture. In both cases, however, there was no apparent change in the Y ATP value.
These results are discussed with respect to two imponder-ables, namely the mechanism(s) by which C. butyricum might partially or totally dissociate catabolism from anabolism, and how it might dispose of the excess reductant [as NAD(P)H] that attends both the formation of acetate from glucose and the fermentation of mannitol. With regards to the latter, evidence is presented that supports the conclusion that the ferredoxin-mediated oxidation of NAD(P)H, generating H2, is neither coupled to, nor driven by, an energy-yielding reaction.
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References
Bahl H, Andersch W, Braun K, Gottschalk G (1982) Effect of pH and butyrate concentration on the production of acetone and butanol by Clostridium acetobutylicum. Eur J Appl Microbiol Biotechnol 14:17–20
Bernt E, Gutmann I (1974) Äthanol. Bestimmung mit Alkoholdehydrogenase und NAD. In: Bergmeyer HU (ed) Methoden der enzymatischen Analyse, 3 Aufl, Bd II. Verlag Chemie, Weinheim, pp 1545–1548
Buchanan BB, Arnon DI (1970) Ferredoxins: chemistry and function in photosynthesis, nitrogen fixation and fermentative metabolism. Adv. Enzymol 33:119–176
Cohen A, Breure AM, Andel JG van, Deursen A van (1980) Influence of phase separation on the anaerobic digestion of glucose. I. Maximum COD-turnover rate during continuous operation. Water Res 14:1439–1448
Dawes EA, McGill DJ, Midgley M (1971) Analysis of fermentation products. In: Norris JR, Ribbons DW (eds) Methods in microbiology, vol 6A. Academic Press, London New York, pp 53–216
Evans CGT, Herbert D, Tempest DW (1970) The continuous cultivation of micro-organisms. 2. Construction, of a chemostat. In: Norris JR, Ribbons DW (eds) Methods in microbiology, vol 2. Academic Press, London New York, pp 277–327
Gottschal JC, Morris JG (1981) The induction of acetone and butanol production in cultures of Clostridium acetobutylicum by elevated concentrations of acetate and butyrate. FEMS Microbiol Lett 12:385–389
Gottschal JC, Morris JG (1982) Continuous production of acetone and butanol by Clostridium acetobutylicum growing in turbidostat culture. Biotechnol Lett 4:477–482
Gottschalk G (1979) Bacterial metabolism. In: Starr MP (ed) Springer series in microbiology. Springer, Berlin Heidelberg New York, pp 1–281
Gutmann I, Wahlefeld AW (1974) l-Lactat. Bestimmung mit Lactatdehydrogenase and NAD. In: Bergmeyer HU (ed) Methoden der enzymatischen Analyse, 3. Aufl, Bd II. Verlag Chemie, Weinheim, pp 1510–1514
Harrison DEF (1976) The regulation of respiration rate in growing bacteria. In: Rose AH, Tempest DW (eds) Advances in microbial physiology, vol 14. Academic Press London New York San Francisco, pp 243–313
Herbert D, Phipps PJ, Tempest DW (1965) The chemostat: Design and instrumentation. Lab Pract 14:1150–1161
Herbert D, Phipps PJ, Strange RE (1971) Chemical analysis of microbial cells. In: Norris JR, Ribbons DW (eds) Methods in microbiology, vol 5B. Academic Press, London New York, pp 209–344
Hollaender A (1981) Trends in the biology of fermentations for fuels and chemicals. In: Hollaender A (ed) Basic life sciences, vol 18. Plenum Press, New York London, pp 1–591
Jungermann K, Thauer RK, Leimenstoll G, Decker K (1973) Function of reduced pyridine nucleotide-ferredoxin oxidoreductases in saccharolytic Clostridia. Biochim Biophys Acta 305:268–290
Jungermann K, Kirchniawy FH, Katz N, Thauer RK (1974) NADH a physiological electron donor in clostridial nitrogen fixation. FEBS Lett 43:203–206
Jungermann K, Kern M, Riebeling V, Thauer RK (1976) Function and regulation of ferredoxin reduction with NADH in Clostridia. In: Schlegel HG, Gottschalk G, Pfennig N (eds) Microbial production and utilization of gases. Goltze, Göttingen, pp 85–96
Leegwater MPM (1983) Microbial reactivity: its relevance to growth in natural and artificial environments. PhD thesis. University of Amsterdam, pp 1–196
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193:165–175
Stucki JW (1980) The optimal efficiency and its economic degree of coupling of oxidative phosphorylation. Eur J Biochem 109:269–283
Teixeira de Mattos MJ, Tempest DW (1983) Metabolic and energetic aspects of the growth of Klebsiella aerogenes NCTC 418 on glucose in anaerobic chemostat culture. Arch Microbiol 134:80–85
Teixeira de Mattos MJ, Plomp PJAM, Neijssel OM, Tempest DW (1984a) Influence of metabolic end-products on the growth efficiency of Klebsiella aerogenes in anaerobic chemostat culture. Antonie van Leeuwenhoek J Microbiol Serol 50:461–472
Teixeira de Mattos MJ, Streekstra H, Tempest DW (1984b) Metabolic uncoupling of substrate level phosphorylation in anaerobic glucose-limited chemostat cultures of Klebsiella aerogenes NCTC 418. Arch Microbiol 139:260–264
Tempest DW, Neijssel OM (1984) The status of Y ATP and maintenance energy as biologically interpretable phenomena. Ann Rev Microbiol 38:459–486
Thauer RK, Jungermann K, Decker K (1977) Energy conservation in chemotrophic anaerobic bacteria. Bacteriol Rev 41:100–180
Thomas TD, Ellwood DC, Longyear VMC (1979) Change from homo- to heterolactic fermentation by Streptococcus lactis resulting from glucose limitation in anaerobic chemostat culture. J Bacteriol 138:109–117
Westerhoff AV (1983) Mosaic non-equilibrium thermodynamics and its control of biological free energy transduction. PhD thesis. University of Amsterdam, pp 1–367
Yamada T, Carlsson J (1975) Regulation of lactate dehydrogenase and change of fermentation products in Streptococci. J Bacteriol 124:55–61
Zeikus JG (1980) Chemical and fuel production by anaerobic bacteria. Ann Rev Microbiol 34:423–464
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Crabbendam, P.M., Neijssel, O.M. & Tempest, D.W. Metabolic and energetic aspects of the growth of Clostridium butyricum on glucose in chemostat culture. Arch. Microbiol. 142, 375–382 (1985). https://doi.org/10.1007/BF00491907
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DOI: https://doi.org/10.1007/BF00491907