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

Abstract

Clostridium cellulolyticum, 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) N1 h N1 . 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. C. cellulolyticum 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.

Production of oligosaccharides and cellobionic acid by Fibrobacter succinogenes S85 growing on sugars, cellulose and wheat straw

“…Growth on cellulose or straw differs from that on soluble sugars by several factors: (a) the necessity for bacteria first to adhere to the substrate and second to degrade it, (b) different growth rate, (c) different physiological state of the bacteria; the carbon flow is consequently different in cellulose and/or wheat straw grown cells. The metabolism of another anaerobic cellulolytic bacterium Clostridium cellulolyticum was studied in details in batch and continuous cultures on cellobiose or cellulose, and a modification of metabolic fluxes was also observed between the two substrates (Desvaux et al 2000;Desvaux et al 2001). Accumulation of phosphorylated sugars in the extracellular medium of growing bacteria (present work) but also of resting cells (Nouaille et al 2004) is quite surprising.…”

“…For example, there could be a need of more energy in the first hours of growth on solid substrate for induction of synthesis of specific proteins involved in adhesion (Roger et al 1990) or cellulolysis (Béra-Maillet et al 2000). In addition, acetate and succinate production may be affected differently by several factors such as pH, as shown previously on continuous cultures of S85 (Weimer 1993) or on other bacteria (Desvaux 2001). As F. succinogenes also produces formate as a minor metabolite (Miller 1978;Matheron et al 1997), it would have been interesting to monitor its concentration during the growth on the two substrates to see the evolution of its production with time.…”

Continuous Cellulosic Bioethanol Fermentation by Cyclic Fed-Batch Cocultivation

“…Corresponding to the accumulation of sol- uble sugars under high initial cellulose concentrations, lactate was produced by the coculture. In fact, it was found that lactate was produced by Clostridium cellulolyticum (28) and C. thermocellum ATCC 27405 (29) with an increase in initial cellulose concentrations. Therefore, initial cellulose concentrations might play a role in influencing metabolism pathways in fermentative thermophilic bacteria.…”

Impact of Substrate Glycoside Linkage and Elemental Sulfur on Bioenergetics of and Hydrogen Production by the Hyperthermophilic Archaeon Pyrococcus furiosus

“…A longer-term option being considered is the production of molecular hydrogen from biomass using fermentative, anaerobic microorganisms (38). For example, many mesophilic Clostridium and Enterobacter species can grow on fermentable sugars and produce hydrogen as a by-product of energy metabolism (8,11,16,28,38).Studies suggest that biohydrogen production rates may be enhanced at higher temperatures (9, 23). In fact, the production and consumption of molecular hydrogen drive the microbial physiology and bioenergetics of many hyperthermophilic bacteria and archaea inhabiting hydrothermal environments (1).…”