Conditions for Ethanol Production during Bioconversion of Cellulose-Containing Raw Material

Several natural associations composed of thermophilic anaerobic bacteria capable of utilizing various cellulose materials at 60 ± 2°C and pH 6.0–7.0 were isolated from the sludge of Kamchatka geothermal springs. The rate of ethanol production (up to 1.7 g/l per day) and the concentration of ethanol in the medium (up to 1.2%), as well as the fermentation period (10–15 days), were determined under anaerobic conditions in the presence of cellulose, coniferous sawdust, newsprint, or paper pulp as a carbon source. Microorganisms were found that inhibited the production of ethanol. The initial pH value was found to influence both the ethanol production rate and ethanol/acetate ratio. A pH decrease from 7.0 to 5.0 led to a 6.7-fold increase in ethanol production and caused a 23.8-fold increase in the ethanol/acetate ratio.     

Selection of a Mixed Culture of Cellulolytic Thermophilic Anaerobes from Various Natural Sources

Microbial associations capable of converting cellulose-containing substrates to ethanol and organic acids were isolated from natural sources. The resulting mixed cultures utilized cellulose, cellobiose, glucose, maize residue, cotton, and flax boon producing ethanol (up to 0.9 g/l) and acetic acid (up to 0.8 g/l). The most complete conversion of cellulose-containing substrates occurred at 60°C and pH 7.0. The selected association of thermophilic anaerobic bacteria produced 0.64 g of ethanol per g substrate utilized at the ethanol/acetate ratio 4.7 : 1.     

Selection of a mixed culture of cellulosolytic thermophilic anaerobes from various natural sources

Microbial associations capable of converting cellulose-containing substrates to ethanol and organic acids were isolated from natural sources. The resulting mixed cultures utilized cellulose, cellobiose, glucose, maize residue, cotton, and flax boon producing ethanol (up to 0.9 g/l) and acetic acid (up to 0.8 g/l). The most complete conversion of cellulose-containing substrates occurred at 60 degrees C, pH 7.0. The selected association of thermophilic anaerobic bacteria produced 0.64 g ethanol per g substrate utilized at the ethanol/acetate ratio 4.7:1.     

A study of producing ethanol from cellulose using Clostridium thermocellum

The feasibility of producing ethanol in a continuous system from cellulose using Clostridirrrn thermocellum was investigated. This anaerobic and therniophilic bacterium was able to degrade cellulose directly into ethanol with acetic acid, hydrogen. and carbon dioxide as by-products of this fermentation. The fermentation was first carried out in a batch mode to study the effects of buffers, temperature, and agitation on microbial growth and ethanol production. From the compounds used to control pH. sodium bicarbonate had the most preferred effects on generation time and ethanol production. As expected, there was a positive relationship between temperature and growth rate. On the other hand, agitation did not benefit from ethanol production or microbial growth. The possibility of noncompetitive inhibition within such a system was deduced from the calculation of the kinetic constants K(m) and V(max). Continuous fermentations were carried out at 60 degrees C and pH 7.0 using 1.5 and 3% pure cellulose as a limiting substrate. The maximum ethanol concentration reached during the 1.5% cellulose fermentation was 0.3%. and 0.9% during the 3% cellulose fermentation. The yield of ethanol was about 0.3 grams per gram of consumed cellulose. The overall yield in both schemes was around 0.45 and 0.75 grams per gram of cellulose degraded. It was concluded that cellulose could be degraded continuously in a system with C. thermocellum for production of ethanol. While the continuous system like the batch method is feasible, it may not be promising as yet because of the slow generation time of this microorganism.     

Purification and characterization of an endo-1,4-beta-glucanase from Neisseria sicca SB that hydrolyzes beta-1,4 linkages in cellulose acetate

An enzyme catalyzing hydrolysis of beta-1,4 bonds in cellulose acetate was purified 18.3-fold to electrophoretic homogeneity from a culture supernatant of Neisseria sicca SB, which can assimilate cellulose acetate as the sole carbon and energy source. The molecular mass of the enzyme was 41 kDa and the isoelectric point was 4.8. The pH and temperature optima of the enzyme were 6.0-7.0 and 60 degrees C. The enzyme catalyzed hydrolysis of water-soluble cellulose acetate (degree of substitution, 0.88) and carboxymethyl cellulose. The Km and Vmax for water-soluble cellulose acetate and carboxymethyl cellulose were 0.242% and 2.24 micromol/min/mg, and 2.28% and 12.8 micromol/min/mg, respectively. It is estimated that the enzyme is a kind of endo-1,4-beta-glucanase (EC 3.2.1.4) from the substrate specificity and hydrolysis products of cellooligosaccharides. The enzyme and cellulose acetate esterase from Neisseria sicca SB degraded water-insoluble cellulose acetate by synergistic action.     

Improvement of biohydrogen production by Enterobacter cloacae IIT-BT 08 under regulated pH

The present study investigates the effect of pH and intermediate products formation on biological hydrogen production using Enterobacter cloacae IIT-BT 08. Initial pH was found to have a profound effect on hydrogen production potential, while regulating the pH 6.5 throughout the fermentation was found to increase the cumulative hydrogen production rate and yield significantly. Modified Gompertz equation was used to fit the cumulative hydrogen production curves to obtain the hydrogen production potential P, the hydrogen production rate R and lag phase λ. At regulated pH 6.5, higher H(2) yield (3.1molH(2)mol(-1) glucose), specific hydrogen production potential (798.1mL/g) and specific rate of H(2) production (72.1mLL(-1)h(-1)g(-1)) were obtained. The volatile fatty acid profile showed butyrate, ethanol and acetate as the major end metabolites of fermentation under the operating pH conditions tested; however, their pattern of distribution was pH dependent. At the optimum pH of 6.5, the acetate to butyrate ratio (A/B ratio) was found to be higher than that at any other pH. The study also investigates the effect of sodium ions on biohydrogen production potential. It was also found that sodium ion concentration up to 250mM enhanced the hydrogen production potential; however, any further increase in the metal ion concentration had an inhibitory effect.     

Production of ethyl acetate from dilute ethanol solutions by Candida utilis

The conversion of ethanol to ethyl acetate has an advantage as a method of ethanol recovery since ethyl acetate is amenable to simple solvent extraction. The potential of Candida utilis in this conversion was studied. The kinetics of accumulation of ethanol and ethyl acetate in glucose-grown C. utilis showed that ester formation resulted from ethanol utilization under appropriate aeration and was inhibited by Fe(3+) supplementation. Candida utilis converted ethanol to ethyl acetate optimally at pH 5.0-7.0. The five-hour rate of ester production increased as the ethanol concentration increased to 10 g/L, and rapidly declined to zero at concentrations exceeding 35 g/L. Thus, C. utilis has potential to recover dilute ethanol in the form of ethyl acetate.     

果蔬废弃物两相厌氧发酵产己酸的效能研究

利用厌氧菌群生物合成己酸被认为是一种非常有潜力的新型废弃物资源化技术,但是其合成效能的提高是目前亟待解决的关键问题。本研究以实际果蔬废弃物为原料,对两相厌氧发酵产己酸的效能进行了研究。首先优化接种比以提高酸化相的水解转化效率;在此基础上通过调控醇酸比和pH以强化产己酸相的发酵效能。结果显示,果蔬废弃物厌氧产酸的最佳接种比为2∶1,此时水解率和酸化率分别可达到98.1%和83.2%,乙酸和丁酸产量分别达到5.4 g/L和3.3 g/L。合理控制醇酸比和pH对提高产己酸相的发酵效能非常关键。当醇酸比和pH控制为4∶1和7.5时,己酸生成量可达14.9 g/L,约占液相总COD的80.84%;而低醇酸比和低pH易造成丁酸的累积,从而降低了己酸产量。己酸发酵过程属于非生长偶联型,己酸菌(Clostridium kluyveri)指数增长期伴随着丁酸的生成,而己酸合成主要发生在生长中后期。此外,己酸菌对于pH变化较为敏感,适当提高pH有助于减轻有机酸毒性,提高生物量;但是碱性环境会严重抑制己酸菌的生长繁殖。研究表明,通过分别对酸化相和产己酸相进行优化和调控,两相发酵策略更有利于提高己酸合成效能。     

Ethanol formation from cellulose by thermophilic bacteria

Summary A wild coculture of obligately thermophilic bacteria, including only a single cellulolytic species Clostridium, ferments 2% crystalline cellulose and produces 4.6–5.1 g·l^–1 of ethanol at 55°–60° C; that is, 0.96–1.1 moles of ethanol from 1 mole of glucose equivalent of cellulose degraded. However, the ethanol yield decreases with increasing cellulose concentration. Ethanolacetic acid ratio varies around 1 and cannot be influenced by substrate concentration. However, this ratio can be influenced by changing pH and temperature. For the ethanol production from cellulose, neutral and weekly alkaline media with a pH of 7.0–8.0 and a temperature of 55° C are optimal. Experiments in which the coculture was subjected to high ethanol concentrations showed that higher concentrations of added ethanol (up to 20 g·l^–1) suppress cellulose degradation by 50% and inhibit the actual production of ethanol.     

Anaerobic 2-ketogluconate metabolism of Klebsiella pneumoniae NCTC 418 grown in chemostat culture: involvement of the pentose phosphate pathway.

Under anaerobic 2-ketogluconate-limited growth conditions (D = 0.1 h-1), Klebsiella pneumoniae NCTC 418 was found to convert this carbon source to biomass, acetate, formate, CO2, ethanol and succinate. The observed fermentation pattern is in agreement with the simultaneous functioning of the pentose phosphate pathway and the Entner-Doudoroff pathway in 2-ketogluconate catabolism. When cultured at pH 8.0 apparent YATP values were lower than those found at culture pH 6.5. This difference can be explained by assuming that at high culture pH values approximately 0.5 mol ATP was invested in the uptake of 1 mol 2-ketogluconate. Sudden relief of 2-ketogluconate-limited conditions led to lowering of the intracellular NADPH/NADP ratio and (possibly as a result of this) to inhibition of biosynthesis. Whereas production of ethanol stopped, lactate was produced at high rate. This product was formed, at least partly, via the methylglyoxal bypass.     

嗜热厌氧纤维素分解菌的分离、鉴定及其酶学特性

【目的】分离高效降解纤维素的嗜热厌氧菌,通过与嗜热产乙醇菌株联合培养的方式,为生产纤维素乙醇提供微生物资源。【方法】利用厌氧分离技术从降解纤维素的马粪富集物中分离到一株嗜热厌氧细菌HCp。采用形态学观察、生理生化鉴定、结合16S rDNA序列的系统发育学分析确定该菌株的分类地位,利用DNS酶活分析方法测定此分离菌株的酶学性质。【结果】分离菌株HCp革兰氏染色阴性,直杆,细胞单个或成对出现,菌体大小为(0.35-0.50)μm×(2.42-6.40)μm,严格厌氧,形成芽胞,能运动,对新霉素有一定的抗性。此菌能利用滤纸纤维素、纤维素粉、微晶纤维素、脱脂棉和水稻秸秆、明胶等,还可以利用葡萄糖、纤维二糖、木糖、木聚糖、果糖、蔗糖、核糖、半乳糖、麦芽糖、山梨糖、海藻糖、蜜二糖、甘露糖等。该菌株在pH6.5-8.5、温度35-70℃、盐浓度0%-1.0%范围内利用纤维素生长,最适pH为6.85,最适温度为60℃,最适NaCl浓度为0.2%,最佳生长条件下,在10 d内滤纸纤维素降解率可达90.40%。在HCp的纤维小体中,滤纸酶、羧甲基纤维素酶、β-葡萄糖苷酶、木聚糖酶的最适作用温度分别为70℃、70℃、70℃、60℃,并且羧甲基纤维素酶具有较高的热稳定性。部分长度的16S rDNA序列分析表明,分离菌株HCp与Acetivibrio cellulolyticus、A.cellulosolvens相似性为97.5%。【结论】分离菌株HCp是从马粪富集物中分离到的一株嗜热厌氧细菌,该菌具有较强的降解纤维素能力,生长温度范围广,酶的热稳定性好,纤维素底物利用广泛等特性,为纤维素降解产乙醇提供了良好的材料。     

Bioconversion of cellulose into ethanol by Clostridium thermocellum--product inhibition

Direct anaerobic bioconversion of cellulosic substances into ethanol by Clostridium thermocellum ATCC 27405 has been carried out at 60 degrees C and pH 7.0 (initial for 100 L) under continuous sparging of oxygen free nitrogen in a culture vessel. Raw bagasse, mild alkali-treated bagasse, and solka floc were used as substrates. The extent of conversion of raw bagasse (cellulose, 50%; hemicellulose, 25%; lignin, 19%) was observed as 52% (w/w) and 79% (w/w) in the case of mild alkali and steam-treated bagasse (cellulose, 72%; hemicellulose, 11%; lignin, 12%), respectively. Use of bagasse concentration above 10 g/L showed a decreased rate in ethanol production. An inoculum age between 28-30 h and cell mass content of 0.027-0.036 g/L (dry basis) were used. The results obtained with raw and pretreated bagasse have been compared with those of highly pure Solka Floc (hemicellulose, 10%). Studies on the product inhibition indicated a linear fall of the percent of survivors with time. An Arrhenius type correlation between the cell decay rate constant and the product concentration was predicted. Even at low levels, the inhibitory effects of products on cell viability, the specific growth rate, and extracellular cellulase enzyme were observed.     

Kinetics and metabolism of cellulose degradation at high substrate concentrations in steady-state continuous cultures of Clostridium cellulolyticum on a chemically defined medium

The hydrolysis and fermentation of insoluble cellulose were investigated using continuous cultures of Clostridium cellulolyticum with increasing amounts of carbon substrate. At a dilution rate (D) of 0.048 h(-1), biomass formation increased proportionately to the cellulose concentration provided by the feed reservoir, but at and above 7.6 g of cellulose x liter(-1) the cell density at steady state leveled off. The percentage of cellulose degradation declined from 32.3 to 8.3 with 1.9 and 27.0 g of cellulose x liter(-1), respectively, while cellodextrin accumulation rose and represented up to 4.0% of the original carbon consumed. The shift from cellulose-limited to cellulose-sufficient conditions was accompanied by an increase of both the acetate/ethanol ratio and lactate biosynthesis. A kinetics study of C. cellulolyticum metabolism in cellulose saturation was performed by varying D with 18.1 g of cellulose x liter(-1). Compared to cellulose limitation (M. Desvaux, E. Guedon, and H. Petitdemange, J. Bacteriol. 183:119-130, 2001), in cellulose-sufficient continuous culture (i) the ATP/ADP, NADH/NAD+, and q(NADH produced)/q(NADH used) ratios were higher and were related to a more active catabolism, (ii) the acetate/ethanol ratio increased while the lactate production decreased as D rose, and (iii) the maximum growth yield (Y(max)X/S) (40.6 g of biomass per mol of hexose equivalent) and the maximum energetic yield (Y(max)ATP) (19.4 g of biomass per mol of ATP) were lowered. C. cellulolyticum was then able to regulate and optimize carbon metabolism under cellulose-saturated conditions. However, the facts that some catabolized hexose and hence ATP were no longer associated with biomass production with a cellulose excess and that concomitantly lactate production and pyruvate leakage rose suggest the accumulation of an intracellular inhibitory compound(s), which could further explain the establishment of steady-state continuous cultures under conditions of excesses of all nutrients. The following differences were found between growth on cellulose in this study and growth under cellobiose-sufficient conditions (E. Guedon, S. Payot, M. Desvaux, and H. Petitdemange, Biotechnol. Bioeng. 67:327-335, 2000): (i) while with cellobiose, a carbon flow into the cell of as high as 5.14 mmol of hexose equivalent g of cells(-1) x h(-1) could be reached, the maximum entering carbon flow obtained here on cellulose was 2.91 mmol of hexose equivalent g of cells(-1) x h(-1); (ii) while the NADH/NAD+ ratio could reach 1.51 on cellobiose, it was always lower than 1 on cellulose; and (iii) while a high proportion of cellobiose was directed towards exopolysaccharide, extracellular protein, and free amino acid excretions, these overflows were more limited under cellulose-excess conditions. Such differences were related to the carbon consumption rate, which was higher on cellobiose than on cellulose.     

Changes in product formation and bacterial community by dilution rate on carbohydrate fermentation by methanogenic microflora in continuous flow stirred tank reactor

Changes in product formation during carbohydrate fermentation by anaerobic microflora in a continuous flow stirred tank reactor were investigated with respect to the dilution rate in the reactor. In the fermentation by methanogenic microflora, stable methane fermentation, producing methane and carbon dioxide, was observed at relatively low dilution rates (less than 0.33 d(-1) on glucose and 0.20 d(-1) on cellulose). Decomposition of cellulose in the medium was a rate-limiting step in the reaction, because glucose was easily consumed at all applied dilution rates (0.07-4.81 d(-1)). Intermediate metabolites of methane fermentation, such as lactate, ethanol, acetate, butyrate, formate, hydrogen, and carbon dioxide, were accumulated as dilution rate increased. Maximum yield of hydrogen was obtained at 4.81 d(-1) of dilution rate (0.1 mol/mol glucose on glucose or 0.7 mol/mol hexose on cellulose). Lactate was the major product on glucose (1.2 mol/mol glucose), whereas ethanol was predominant on cellulose (0.7 mol/mol hexose). An analysis by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified bacterial 16S rDNA of the microflora indicated that changes in the microbial community took place at various dilution rates, and these changes appeared to correspond to the changes in product distributions. Sequence analyses of the DGGE fragments revealed the probable major population of the microflora. A band closely related to the microorganisms of thermophilic anaerobic bacteria was detected with strong intensity on both glucose and cellulose. Differences in the production yield of hydrogen could have been caused by different populations of microorganisms in each microflora. In the case of cellulose, increasing the dilution rate brought about an accumulation of microorganisms related to Clostridia species that have cellulolytic activity, this being in accordance with the notion of cellulose decomposition being the rate-limiting reaction.     

初始pH值对产氢产乙酸/耗氢产乙酸两段耦合工艺定向生产乙酸的影响

以葡萄糖为底物,以经加热预处理并活化过的厌氧污泥为种泥,研究了初始pH值对产氢产乙酸/耗氢产乙酸两段耦合工艺厌氧发酵定向生产乙酸的影响。实验考察了7个初始pH值(5、6、7、8、9、10、11)条件下的底物降解、产物产生和发酵过程pH值的变化。结果表明:产氢产乙酸段初始pH值的变化不仅影响本阶段产酸,而且影响耗氢产乙酸段产酸。初始pH=5时主要进行乙醇型发酵;pH=6和7时主要进行丁酸型发酵;pH=8时混合酸型发酵类型逐渐占优势,pH=8~11时均以乙酸为主要产物,耦合系统生产乙酸最优初始pH值为10。在初始pH=8~11范围内,产氢产乙酸段初期的乙醇浓度一般较高,但到后期因乙醇被微生物进一步代谢转化成乙酸而使其含量下降。     

Influence of particle size and pH on anaerobic degradation of cellulose by ruminal microbes

Batch experiments were performed to investigate the influence of cellulose particle size and pH on the anaerobic degradation of crystalline cellulose by ruminal microbes. At a particle size of 50 μm there was a higher hydrolysis and acidogenesis rate, and a reduced degradation time, than for 100-μm particles. Reduction in cellulose particle size resulted in decreased methane production, but an increase of soluble products. Cellulose degradation increased with pH from pH 6.0 to 7.5, whereas at pH⩽5.5 there was no degradation. The inhibitory effect of low pH (⩽5.5) on ruminal microbes was not completely remedied even when the pH of the medium was adjusted to a neutral range. In an anaerobic cellulosic waste degrading system inoculated with ruminal microbes the fermentation system should therefore be maintained above pH 6.0. In all cases, volatile fatty acids were the major water-soluble products of cellulose degradation; acetate and propionate accounted for more than 90% of the volatile fatty acid total.     

Physiological adaptations of anaerobic bacteria to low pH: metabolic control of proton motive force in Sarcina ventriculi.

Detailed physiological studies were done to compare the influence of environmental pH and fermentation end product formation on metabolism, growth, and proton motive force in Sarcina ventriculi. The kinetics of end product formation during glucose fermentation in unbuffered batch cultures shifted from hydrogen-acetate production to ethanol production as the medium pH dropped from 7.0 to 3.3. At a constant pH of 3.0, the production of acetate ceased when the accumulation of acetate in the medium reached 40 mmol/liter. At a constant pH of 7.0, acetate production continued throughout the entire growth time course. The in vivo hydrogenase activity was much higher in cells grown at pH 7.0 than at pH 3.0. The magnitude of the proton motive force increased in relation to a decrease of the medium pH from 7.5 to 3.0. When the organism was grown at pH 3.0, the cytoplasmic pH was 4.25 and the organism was unable to exclude acetic acid or butyric acid from the cytoplasm. Addition of acetic acid, but not hydrogen or ethanol, inhibited growth and resulted in proton motive force dissipation and the accumulation of acetic acid in the cytoplasm. The results indicate that S. ventriculi is an acidophile that can continue to produce ethanol at low cytoplasmic pH values. Both the ability to shift to ethanol production and the ability to continue to ferment glucose while cytoplasmic pH values are low adapt S. ventriculi for growth at low pH.     

Calorimetric studies of the elongation of Avena coleoptile segments

Elongation rate and heat produced by Avena coleoptile segments suspended in sucrose buffer solutions were measured at pH values from 3.5 to 8.5. The caloric efficiency of elongation (CEE) was defined as the ratio of the rate of elongation to the rate of heat production. Elongation and CEE were greatest at intermediate pH values, but heat production (about 1 cal/g.hr) was insensitive to pH within the limits of experimental error (+/-20%). Quantitative agreement was found between the results of previous respiration studies and the rate of heat production in an aerobic atmosphere, which indicates that oxidative metabolism accounts for essentially all energy changes in the cell, so matter flow is a significant component of the bioenergetics of cell function. Indole-3-acetic acid up to 1 mm, produced about a 10-fold increase in elongation rate, a 5-fold increase of the CEE, and a 25% increase in heat production. Above this concentration, sharp drops in both elongation and heat production occurred, without altering the CEE at pH 6.5, but greatly decreasing the CEE at pH 4.5. Elongation and CEE showed marked decreases after 4 hours in an anaerobic atmosphere, but heat production did not exhibit a proportional decrease. These studies indicate that rate of cell elongation in the presence and absence of auxin is not directly proportional to the overall metabolism of the cell.     

The characteristics of a new non-spore-forming cellulolytic mesophilic anaerobe strain CM126 isolated from municipal sewage sludge

A new mesophilic anaerobic cellulolytic bacterium, CM126, was isolated from an anaerobic sewage sludge digester. The organism was non-spore-forming, rod-shaped, Gram-negative and motile with peritrichous flagella. It fermented microcrystalline Avicel cellulose, xylan, Solka floc cellulose, filter paper, L-arabinose, D-xylose, beta-methyl xyloside, D-glucose, cellobiose and xylitol and produced indole. The % G + C content was 36. Acetic acid, ethanol, lactic acid, pyruvic acid, carbon dioxide and hydrogen were produced as metabolic products. This strain could grow at 20-44.5 degrees C and at pH values 5.2-7.4 with optimal growth at 37-41.5 degrees C and pH 7. Both endoglucanase and xylanase were detected in the supernatant fluid of a culture grown on medium containing Avicel cellulose and cellobiose. Exoglucanase could not be found in either supernatant fluid or the cell lysate. When cellulose and cellobiose fermentation were compared, the enzyme production rate in cellobiose fermentation was higher than in cellulose fermentation. The optimum pH for both enzyme activities was 5.0, the optimum temperature was 40 degrees C for the endoglucanase and 50 degrees C for the xylanase. Both enzyme activities were inhibited at 70 degrees C Co-culture of this organism with a Methanosarcina sp. (A145) had no effect on cellulose degradation and both endoglucanase and xylanase were stable in the co-culture.     

Carbon flux distribution and kinetics of cellulose fermentation in steady-state continuous cultures of Clostridium cellulolyticum on a chemically defined medium

The metabolic characteristics of Clostridium cellulolyticum, a mesophilic cellulolytic nonruminal bacterium, were investigated and characterized kinetically for the fermentation of cellulose by using chemostat culture analysis. Since with C. cellulolyticum (i) the ATP/ADP ratio is lower than 1, (ii) the production of lactate at low specific growth rate (mu) is low, and (iii) there is a decrease of the NADH/NAD(+) ratio and q(NADH produced)/ q(NADH used) ratio as the dilution rate (D) increases in carbon-limited conditions, the chemostats used were cellulose-limited continuously fed cultures. Under all conditions, ethanol and acetate were the main end products of catabolism. There was no shift from an acetate-ethanol fermentation to a lactate-ethanol fermentation as previously observed on cellobiose as mu increased (E. Guedon, S. Payot, M. Desvaux, and H. Petitdemange, J. Bacteriol. 181:3262-3269, 1999). The acetate/ethanol ratio was always higher than 1 but decreased with D. On cellulose, glucose 6-phosphate and glucose 1-phosphate are important branch points since the longer the soluble beta-glucan uptake is, the more glucose 1-phosphate will be generated. The proportion of carbon flowing toward phosphoglucomutase remained constant (around 59.0%), while the carbon surplus was dissipated through exopolysaccharide and glycogen synthesis. The percentage of carbon metabolized via pyruvate-ferredoxin oxidoreductase decreased with D. Acetyl coenzyme A was mainly directed toward the acetate formation pathway, which represented a minimum of 27.1% of the carbon substrate. Yet the proportion of carbon directed through biosynthesis (i.e., biomass, extracellular proteins, and free amino acids) and ethanol increased with D, reaching 27.3 and 16.8%, respectively, at 0.083 h(-1). Lactate and extracellular pyruvate remained low, representing up to 1.5 and 0.2%, respectively, of the original carbon uptake. The true growth yield obtained on cellulose was higher, [50.5 g of cells (mol of hexose eq)(-1)] than on cellobiose, a soluble cellodextrin [36.2 g of cells (mol of hexose eq)(-1)]. The rate of cellulose utilization depended on the solid retention time and was first order, with a rate constant of 0.05 h(-1). Compared to cellobiose, substrate hydrolysis by cellulosome when bacteria are grown on cellulose fibers introduces an extra means for regulation of the entering carbon flow. This led to a lower mu, and so metabolism was not as distorted as previously observed with a soluble substrate. From these results, C. cellulolyticum appeared well adapted and even restricted to a cellulolytic lifestyle.