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Volume 147, Issue 6

Metabolic flux in cellulose batch and cellulose-fed continuous cultures of in response to acidic environment Free

Abstract

, a nonruminal cellulolytic mesophilic bacterium, was grown in batch and continuous cultures on cellulose using a chemically defined medium. In batch culture with unregulated pH, less cellulose degradation and higher accumulation of soluble glucides were obtained compared to a culture with the pH controlled at 72. The gain in cellulose degradation achieved with pH control was offset by catabolite production rather than soluble sugar accumulation. The pH-controlled condition improved biomass, ethanol and acetate production, whereas maximum lactate and extracellular pyruvate concentrations were lower than in the non-pH-controlled condition. In a cellulose-fed chemostat at constant dilution rate and pH values ranging from 74 to 62, maximum cell density was obtained at pH 70. Environmental acidification chiefly influenced biomass formation, since at pH 64 the dry weight of cells was more than fourfold lower compared to that at pH 70, whereas the specific rate of cellulose assimilation decreased only from 1174 to 1013 milliequivalents of carbon (g cells) h. The molar growth yield and the energetic growth yield did not decline as pH was lowered, and an abrupt transition to washout was observed. Decreasing the pH induced a shift from an acetate-ethanol fermentation to a lactate-ethanol fermentation. The acetate/ethanol ratio decreased as the pH declined, reaching close to 1 at pH 64. Whatever the pH conditions, lactate dehydrogenase was always greatly in excess. As pH decreased, both the biosynthesis and the catabolic efficiency of the pyruvate-ferredoxin oxidoreductase declined, as indicated by the ratio of the specific enzyme activity to the specific metabolic rate, which fell from 98 to 18. Thus a change of only 1 pH unit induced considerable metabolic change and ended by washout at around pH 62. appeared to be similar to rumen cellulolytic bacteria in its sensitivity to acidic conditions. Apparently, the cellulolytic anaerobes studied thus far do not thrive when the pH drops below 60, suggesting that they evolved in environments where acid tolerance was not required for successful competition with other microbes.

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Keyword(s): AADH, acetaldehyde dehydrogenase , ADH, alcohol dehydrogenase , AK, acetate kinase , ATP-Eff, efficiency of ATP generation , cellulolytic bacteria , cellulose degradation , chemostat , environmental pH , Fd, ferredoxin , flux analysis , G1P, glucose 1-phosphate , G6P, glucose 6-phosphate , LDH, lactate dehydrogenase , meq C, milliequivalent of carbon , PFO, pyruvate–ferredoxin oxidoreductase , PTA, phosphotransacetylase and R, ratio of specific enzyme activity to metabolic flux
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2001-06-01
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References

  1. Bayer E. A., Lamed R. 1992; The cellulose paradox: pollutant par excellence and/or a reclaimable natural resource?. Biodegradation 3:171–188 [CrossRef]
     [Google Scholar]
  2. Bayer E. A., Shoham Y., Tormo J., Lamed R. 1996; The cellulosome: a cell surface organelle for the adhesion to and degradation of cellulose. In Bacterial Adhesion: Molecular and Ecological Diversity pp 155–182 Edited by Fletcher M. New York: Wiley-Liss;
     [Google Scholar]
  3. Bayer E. A., Chanzy H., Lamed R., Shoham Y. 1998; Cellulose, cellulases and cellulosomes. Curr Opin Struct Biol 8:548–557 [CrossRef]
     [Google Scholar]
  4. Béguin P., Lemaire M. 1996; The cellulosome: an exocellular, multiprotein complex specialized in cellulose degradation. Crit Rev Biochem Mol Biol 31:201–236 [CrossRef]
     [Google Scholar]
  5. Boisset C., Chanzy H., Henrissat B., Lamed R., Shoham Y., Bayer E. A. 1999; Digestion of crystalline cellulose substrates by Clostridium thermocellum cellulosome: structural and morphological aspects. Biochem J 340:829–835 [CrossRef]
     [Google Scholar]
  6. Collins M. D., Lawson P. A., Willems A., Cordoba J. J., Fernandez-Garayzabal J., Garcia P., Cai J., Hippe H., Farrow J. A. E. 1994; The phylogeny of the genus Clostridium : proposal of five new genera and eleven species combinations. Int J Syst Bacteriol 44:812–826 [CrossRef]
     [Google Scholar]
  7. Costerton J. W., Lewandowski Z., Caldwell D. E., Korber D. R., Lappin-Scott H. M. 1995; Microbial biofilms. Annu Rev Microbiol 49:711–745 [CrossRef]
     [Google Scholar]
  8. Decker K., Kreusch J., Rössle M. 1976; The role of nucleotides in the regulation of the energy metabolism of Clostridium kluyveri . In Microbial Production and Utilization of Gases pp 75–83 Edited by Schlegel H. G. Gottschalk G., Pfennig N. Göttingen: Akademie der Wissenshaften zu Göttingen;
     [Google Scholar]
  9. Desai R. P., Harris L. M., Welker N. E., Papoutsakis E. T. 1999a; Metabolic flux analysis elucidates the importance of acid-formation pathways in regulating solvent production by Clostridium acetobutylicum.. Metab Eng 1:206–213 [CrossRef]
     [Google Scholar]
  10. Desai R. P., Nielsen L. K., Papoutsakis E. T. 1999b; Metabolic flux analysis elucidates the importance of acid-formation pathways in regulating solvent production by Clostridium acetobutylicum fermentations with non-linear constraints. J Biotechnol 71:191–205 [CrossRef]
     [Google Scholar]
  11. Desvaux M., Guedon E., Petitdemange H. 2000; Cellulose catabolism by Clostridium cellulolyticum growing in batch culture on defined medium. Appl Environ Microbiol 66:2461–2470 [CrossRef]
     [Google Scholar]
  12. Desvaux M., Guedon E., Petitdemange H. 2001; Carbon flux distribution and kinetics of cellulose fermentation in steady-state continuous cultures of Clostridium cellulolyticum on a chemically defined medium. J Bacteriol 183:119–130 [CrossRef]
     [Google Scholar]
  13. Duong T. V. C., Johnson E. A., Demain A. L. 1983; Thermophilic, anaerobic and cellulolytic bacteria. In Topics in Enzyme and Fermentation Biotechnology pp 156–195 Edited by Weisman A. New York: Wiley;
     [Google Scholar]
  14. Fields M. W., Russell J. B. 2000; Fibrobacter succinogenes S85 ferments ball-milled cellulose as fast as cellobiose until cellulose surface area is limiting. Appl Microbiol Biotechnol 54:570–574 [CrossRef]
     [Google Scholar]
  15. Gelhaye E., Gehin A., Petitdemange H. 1993a; Colonization of crystalline cellulose by Clostridium cellulolyticum ATCC 35319. Appl Environ Microbiol 59:3154–3156
     [Google Scholar]
  16. Gelhaye E., Petitdemange H., Gay R. 1993b; Adhesion and growth rate of Clostridium cellulolyticum ATCC 35319 on crystalline cellulose. J Bacteriol 175:3452–3458
     [Google Scholar]
  17. Giallo J., Gaudin C., Belaich J. P., Petitdemange E., Caillet-Mangin F. 1983; Metabolism of glucose and cellobiose by cellulolytic mesophilic Clostridium sp. strain H10. Appl Environ Microbiol 45:843–849
     [Google Scholar]
  18. Giallo J., Gaudin C., Belaich J. P. 1985; Metabolism and solubilization of cellulose by Clostridium cellulolyticum H10. Appl Environ Microbiol 49:1216–1221
     [Google Scholar]
  19. Goodwin S., Zeikus J. G. 1987; Ecophysiological adaptations of anaerobic bacteria to low pH: analysis of anaerobic digestion in acidic bog sediments. Appl Environ Microbiol 53:57–64
     [Google Scholar]
  20. Guedon E., Desvaux M., Payot S., Petitdemange H. 1999a; Growth inhibition of Clostridium cellulolyticum by an inefficiently regulated carbon flow. Microbiology 145:1831–1838 [CrossRef]
     [Google Scholar]
  21. Guedon E., Payot S., Desvaux M., Petitdemange H. 1999b; Carbon and electron flow in Clostridium cellulolyticum grown in chemostat culture on synthetic medium. J Bacteriol 181:3262–3269
     [Google Scholar]
  22. Guedon E., Desvaux M., Petitdemange H. 2000a; Kinetic analysis of Clostridium cellulolyticum carbohydrate metabolism: importance of glucose 1-phosphate and glucose 6-phosphate branch points for distribution of carbon fluxes inside and outside cells as revealed by steady-state continuous culture. J Bacteriol 182:2010–2017 [CrossRef]
     [Google Scholar]
  23. Guedon E., Payot S., Desvaux M., Petitdemange H. 2000b; Relationships between cellobiose catabolism, enzyme levels and metabolic intermediates in Clostridium cellulolyticum grown in a synthetic medium. Biotechnol Bioeng 67:327–335 [CrossRef]
     [Google Scholar]
  24. Holms H. 1986; The central metabolic pathways of Escherichia coli : relationship between flux and control at a branch point, efficiency of conversion to biomass, and excretion of acetate. Curr Top Cell Regul 28:69–105
     [Google Scholar]
  25. Holms H. 1996; Flux analysis and control of the central metabolic pathways in Escherichia coli. FEMS. Microbiol Rev 19:85–116 [CrossRef]
     [Google Scholar]
  26. Huang L., Gibbins L. N., Forsberg C. W. 1985; Transmembrane pH gradient and membrane potential in Clostridium acetobutylicum during growth under acetogenic and solventogenic conditions. Appl Environ Microbiol 50:1043–1047
     [Google Scholar]
  27. Kalachniuk H. I., Marounek M., Kalachniuk L. H., Savka O. H. 1994; Rumen bacterial metabolism as affected by extracellular redox potential. Ukr Biokhim Zh 66:30–40
     [Google Scholar]
  28. Kovárová-Kovar K., Egli T. 1998; Growth kinetics of suspended microbial cells: from single-substrate-controlled growth to mixed-substrate kinetics. Microbiol Mol Biol Rev 62:646–666
     [Google Scholar]
  29. Kuznetsov S. I., Dubinina G. A., Lapteva N. A. 1979; Biology of oligotrophic bacteria. Annu Rev Microbiol 33:377–387 [CrossRef]
     [Google Scholar]
  30. Leschine S. B. 1995; Cellulose degradation in anaerobic environments. Annu Rev Microbiol 8:237–299
     [Google Scholar]
  31. Ljungdahl L. G., Eriksson K. E. 1985; Ecology of microbial cellulose degradation. Adv Microb Ecol 8:237–299
     [Google Scholar]
  32. Mitchell W. J. 1998; Physiology of carbohydrate to solvent conversion by clostridia. Adv Microb Physiol 39:31–130
     [Google Scholar]
  33. Miyagi A., Ohta H., Kodama T., Fukui K., Kato K., Shimono T. 1994; Metabolic and energetic aspects of the growth response of Streptococcus rattus to environmental acidification in anaerobic continuous culture. Microbiology 140:1945–1952 [CrossRef]
     [Google Scholar]
  34. Mokrasch L. C. 1967; Use of 2,4,6-trinitrobenzenesulfonic acid for the coestimation of amines, amino acids, and proteins in mixtures. Anal Biochem 18:64–71 [CrossRef]
     [Google Scholar]
  35. O’Sullivan E., Condon S. 1999; Relationship between acid tolerance, cytoplasmic pH, and ATP and H+-ATPase levels in chemostat cultures of Lactococcus lactis. . Appl Environ Microbiol 65:2287–2293
     [Google Scholar]
  36. Papoutsakis E. T. 1984; Equations and calculations for fermentations of butyric acid bacteria. Biotechnol Bioeng 26:174–187 [CrossRef]
     [Google Scholar]
  37. Payot S., Guedon E., Cailliez C., Gelhaye E., Petitdemange H. 1998; Metabolism of cellobiose by Clostridium cellulolyticum growing in continuous culture: evidence for decreased NADH reoxidation as a factor limiting growth. Microbiology 144:375–384 [CrossRef]
     [Google Scholar]
  38. Petitdemange E., Caillet F., Giallo J., Gaudin C. 1984; Clostridium cellulolyticum sp. nov., a cellulolytic mesophilic species from decayed grass. Int J Syst Bacteriol 34:155–159 [CrossRef]
     [Google Scholar]
  39. Russell J. B., Diez-Gonzalez F. 1998; The effects of fermentation acids on bacterial growth. Adv Microb Physiol 39:205–234
     [Google Scholar]
  40. Russell J. B., Dombrowski D. B. 1980; Effect of pH on the efficiency of growth by pure cultures of rumen bacteria in continuous culture. Appl Environ Microbiol 39:604–610
     [Google Scholar]
  41. Russell J. B., Wilson D. B. 1996; Why are ruminal cellulolytic bacteria unable to digest cellulose at low pH?. J Dairy Sci 79:1503–1509 [CrossRef]
     [Google Scholar]
  42. Russell J. B., Bond D. R., Cook G. M. 1996; The fructose diphosphate/phosphate regulation of carbohydrate metabolism in low G+C Gram positive anaerobes. Res Microbiol 147:528–534 [CrossRef]
     [Google Scholar]
  43. Stewart C. S. 1977; Factors affecting the cellulolytic activity of rumen contents. Appl Environ Microbiol 33:497–502
     [Google Scholar]
  44. Tomme P., Warren R. A. J., Gilkes N. R. 1995; Cellulose hydrolysis by bacteria and fungi. Adv Microb Physiol 37:1–81
     [Google Scholar]
  45. Weimer P. J., Lopez-Guisa J. M., French A. D. 1990; Effect of cellulose fine structure on kinetics of its digestion by mixed ruminal microorganisms in vitro. Appl Environ Microbiol 56:2421–2429
     [Google Scholar]
  46. Weimer P. J., French A. D., Calamari T. A. 1991a; Differential fermentation of cellulose allomorphs by ruminal cellulolytic bacteria. Appl Environ Microbiol 57:3101–3106
     [Google Scholar]
  47. Weimer P. J., Shi Y., Odt C. L. 1991b; A segmented gas/liquid delivery system for continuous culture of microorganisms on insoluble substrates and its use for growth of Ruminococcus flavefaciens on cellulose. Appl Microbiol Biotechnol 36:178–183 [CrossRef]
     [Google Scholar]
  48. Wolin M. J., Miller T. L. 1987; Bioconversion of organic carbon to CH4 and CO2. Geomicrobiol J 5:239–259 [CrossRef]
     [Google Scholar]
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Metabolic flux in cellulose batch and cellulose-fed continuous cultures of Clostridium cellulolyticum in response to acidic environment
Microbiology 147, 1461 (2001); https://doi.org/10.1099/00221287-147-6-1461
/content/journal/micro/10.1099/00221287-147-6-1461
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