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 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 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 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) (40.6 g of biomass per mol of hexose equivalent) and the maximum energetic yield (Y) (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 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 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.     

Cellulose catabolism by Clostridium cellulolyticum growing in batch culture on defined medium

A reinvestigation of cellulose degradation by Clostridium cellulolyticum in a bioreactor with pH control of the batch culture and using a defined medium was performed. Depending on cellulose concentration, the carbon flow distribution was affected, showing the high flexibility of the metabolism. With less than 6.7 g of cellulose liter(-1), acetate, ethanol, H(2), and CO(2) were the main end products of the fermentation and cellulose degradation reached more than 85% in 5 days. The electron flow from the glycolysis was balanced by the production of H(2) and ethanol, the latter increasing with increasing initial cellulose concentration. From 6.7 to 29.1 g of cellulose liter(-1), the percentage of cellulose degradation declined; most of the cellulase activity remained on the cellulose fibers, the maximum cell density leveled off, and the carbon flow was reoriented from ethanol to acetate. In addition to that of previously indicated end products, lactate production rose, and, surprisingly enough, pyruvate overflow occurred. Concomitantly the molar growth yield and the energetic yield of the biomass decreased. Growth arrest may be linked to sufficiently high carbon flow, leading to the accumulation of an intracellular inhibitory compound(s), as observed on cellobiose (E. Guedon, M. Desvaux, S. Payot, and H. Petitdemange, Microbiology 145:1831-1838, 1999). These results indicated that bacterial metabolism exhibited on cellobiose was distorted compared to that exhibited on a substrate more closely related to the natural ecosystem of C. cellulolyticum. To overcome growth arrest and to improve degradation at high cellulose concentrations (29.1 g liter(-1)), a reinoculation mode was evaluated. This procedure resulted in an increase in the maximum dry weight of cells (2,175 mg liter(-1)), cellulose solubilization (95%), and end product concentrations compared to a classical batch fermentation with a final dry weight of cells of 580 mg liter(-1) and 45% cellulose degradation within 18 days.     

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.     

Flux analysis of the metabolism of Clostridium cellulolyticum grown in cellulose-fed continuous culture on a chemically defined medium under ammonium-limited conditions

An investigation of cellulose degradation by the nonruminal, cellulolytic, mesophilic bacterium Clostridium cellulolyticum was performed in cellulose-fed chemostat cultures with ammonium as the growth-limiting nutrient. At any dilution rate (D), acetate was always the main product of the catabolism, with a yield of product from substrate ranging between 37.7 and 51.5 g per mol of hexose equivalent fermented and an acetate/ethanol ratio always higher than 1. As D rose, the acetyl coenzyme A was rerouted in favor of ethanol pathways, and ethanol production could represent up to 17.7% of the carbon consumed. Lactate was significantly produced, but with increasing D, the specific lactate production rate declined, as did the specific rate of production of extracellular pyruvate. The proportion of the original carbon directed towards phosphoglucomutase remained constant, and the carbon surplus was balanced mainly by exopolysaccharide and glycogen biosyntheses at high D values, while cellodextrin excretion occurred mainly at lower ones. With increasing D, the specific rate of carbon flowing down catabolites increased as well, but when expressed as a percentage of carbon it declined, while the percentage of carbon directed through biosynthesis pathways was enhanced. The maximum growth and energetic yields were lower than those obtained in cellulose-limited chemostats and were related to an uncoupling between catabolism and anabolism leading to an excess of energy. Compared to growth on cellobiose in ammonium-limited chemostats (E. Guedon, M. Desvaux, and H. Petitdemange, J. Bacteriol. 182:2010-2017, 2000), (i) a specific consumption rate of carbon of as high as 26.72 mmol of hexose equivalent g of cells(-1) x h(-1) could not be reached and (ii) the proportions of carbon directed towards cellodextrin, glycogen, and exopolysaccharide pathways were not as high as first determined on cellobiose. While the use of cellobiose allows highlighting of metabolic limitation and regulation of C. cellulolyticum under ammonium-limited conditions, some of these events should then rather be interpreted as distortions of the metabolism. Growth of cellulolytic bacteria on easily available carbon and nitrogen sources represents conditions far different from those of the natural lignocellulosic compounds.     

Flux Analysis of the Metabolism of Clostridium cellulolyticum Grown in Cellulose-Fed Continuous Culture on a Chemically Defined Medium under Ammonium-Limited Conditions

An investigation of cellulose degradation by the nonruminal, cellulolytic, mesophilic bacterium Clostridium cellulolyticum was performed in cellulose-fed chemostat cultures with ammonium as the growth-limiting nutrient. At any dilution rate (D), acetate was always the main product of the catabolism, with a yield of product from substrate ranging between 37.7 and 51.5 g per mol of hexose equivalent fermented and an acetate/ethanol ratio always higher than 1. As D rose, the acetyl coenzyme A was rerouted in favor of ethanol pathways, and ethanol production could represent up to 17.7% of the carbon consumed. Lactate was significantly produced, but with increasing D, the specific lactate production rate declined, as did the specific rate of production of extracellular pyruvate. The proportion of the original carbon directed towards phosphoglucomutase remained constant, and the carbon surplus was balanced mainly by exopolysaccharide and glycogen biosyntheses at high D values, while cellodextrin excretion occurred mainly at lower ones. With increasing D, the specific rate of carbon flowing down catabolites increased as well, but when expressed as a percentage of carbon it declined, while the percentage of carbon directed through biosynthesis pathways was enhanced. The maximum growth and energetic yields were lower than those obtained in cellulose-limited chemostats and were related to an uncoupling between catabolism and anabolism leading to an excess of energy. Compared to growth on cellobiose in ammonium-limited chemostats (E. Guedon, M. Desvaux, and H. Petitdemange, J. Bacteriol. 182:2010–2017, 2000), (i) a specific consumption rate of carbon of as high as 26.72 mmol of hexose equivalent g of cells⁻¹ h⁻¹ could not be reached and (ii) the proportions of carbon directed towards cellodextrin, glycogen, and exopolysaccharide pathways were not as high as first determined on cellobiose. While the use of cellobiose allows highlighting of metabolic limitation and regulation of C. cellulolyticum under ammonium-limited conditions, some of these events should then rather be interpreted as distortions of the metabolism. Growth of cellulolytic bacteria on easily available carbon and nitrogen sources represents conditions far different from those of the natural lignocellulosic compounds.     

Characterization of the cellulolytic and hydrogen-producing activities of six mesophilic Clostridium species

AIMS: To characterize cellulolytic, hydrogen-producing clostridia on a comparable basis. METHODS AND RESULTS: H(2) production from cellulose by six mesophilic clostridia was characterized in standardized batch experiments using MN301 cellulose, Avicel and cellobiose. Daily H(2) production, substrate degradation, biomass production and the end-point distribution of soluble fermentation products varied with species and substrates. All species produced a significant amount of H(2) from cellobiose, with Clostridium acetobutylicum achieving the highest H(2) yield of 2.3 mol H(2) mol(-1) hexose, but it did not degrade cellulose. Clostridium cellulolyticum and Clostridium populeti catalysed the highest H(2) production from cellulose, with yields of 1.7 and 1.6 mol H(2 )mol(-1) hexose from MN301 and 1.6 and 1.4 mol H(2) mol(-1) hexose from Avicel, respectively. These species also achieved 25-100% higher H(2) production rates from cellulose than the other species. CONCLUSIONS: These cellulolytic, hydrogen-producing clostridia varied in H(2) production, with Cl. cellulolyticum and Cl. populeti achieving the highest H(2) yields and cellulose degradation. SIGNIFICANCE AND IMPACT OF THE STUDY: The fermentation of cellulosic materials presents a means of H(2) production from renewable resources. This standardized comparison provides a quantitative baseline for improving H(2) production from cellulose through medium and process optimization and metabolic engineering.     

Studies of Clostridium cellulolyticum ATCC 35319 under dialysis and co-culture conditions

A. Gehin, C. Cailliez, E. Petitdemange And L. Benoit. 1996. The degradation of cellulose by Clostridium celulolyticum has been studied in several ways; (1) in batch fermentation in 50-ml sealed-cap flasks, referred to as the control; (2) in batch fermentation with pH at 7.2; (3) fermentation in dialysis which permits elimination of all the products of metabolism; (4) fermentation in dialysis with a constant bubbling of nitrogen; (5) in co-culture with Clostridium A22 in batch with and without pH regulation and with dialysis. H₂, CO₂, acetate, ethanol and lactate were the major end-products of cellobiose and cellulose fermentation. Compared to batch culture, growth of CI. cellulolyticum on cellobiose increased by a factor of 10 in dialysed culture. The end products from the dialysed culture were detected in a small range compared to the concentration for the batch culture. Related to the biomass, CMCase activities were of the same level, showing a direct relation between the biomass formation and the cellulase production. The percentage of cellulose degradation (50%) by CI. cellulolyticum was greater when dialysis of end products with a constant bubbling of nitrogen took place during the course of fermentation (6 d) in comparison with cultures in 50-ml sealedcap flasks (23%), in a fermentor (36%) or using dialysis without N₂ bubbling (40%). The presence of two micro-organisms produced no further enzyme activities and hence the percentage of cellulose degradation was quite similar in mono- and co-culture. No synergistic action was found between two cellulolytic strains.     

Relationships between cellobiose catabolism, enzyme levels, and metabolic intermediates in Clostridium cellulolyticum grown in a synthetic medium

Continuous cultures, under cellobiose sufficient concentrations (14. 62 mM) using a chemically defined medium, were examined to determine the carbon regulation selected by Clostridium cellulolyticum. Using a synthetic medium, a q(cellobiose) of 2.57 mmol g cells(-1) h(-1) was attained whereas the highest value obtained on complex media was 0.68 mmol g cells(-1) h(-1) (Payot et al. 1998. Microbiology 144:375-384). On a synthetic medium at D = 0.035 h(-1) under cellobiose excess, lactate and ethanol biosynthesis were able to use the reducing equivalents supplied by acetic acid formation and the H(2)/CO(2) ratio was found equal to 1. At a higher dilution rate (D = 0.115 h(-1)), there was no lactate production and the pathways toward ethanol and NADH-ferredoxin-hydrogenase contributed to balance the reducing equivalents; in this case a H(2)/CO(2) ratio of 1.54 was found. With increasing D, there was a progressive increase (i) in the steady-state concentration of NADH and NAD(+) pools from 11.8 to 22.1 micromol (g cells) (-1), (ii) in the intracellular NADH/NAD(+) ratios from 0.43 to 1.51. On synthetic media, under cellobiose excess the carbon flow was also equilibrated by three overflows: exopolysaccharide, extracellular protein, and amino acid excretions. At D = 0.115 h(-1), 34% of the cellobiose consumed was converted into exopolysaccharides; this deviation of the carbon flow and the increase of the phosphoroclastic activity decreased dramatically the pyruvate excretion and explained the break in lactate production. Whatever the dilution rate, C. cellulolyticum, using ammonium and cellobiose excess, always spilled usual amino acids accompanied by other amino compounds. In vitro, GAPDH, phosphoroclastic reaction, alcohol dehydrogenase, and acetate kinase activities were high under conditions giving high in vivo specific production rates. There were also correlations between the in vitro lactate dehydrogenase activity and in vivo lactate production, but in contrast with the preceding activities, these two parameters decreased with D. All the results demonstrate that C. cellulolyticum was able to optimize carbon catabolism from cellulosic substrates in a synthetic medium.     

Carbon and Electron Flow in Clostridium cellulolyticum Grown in Chemostat Culture on Synthetic Medium

Previous results indicated poor sugar consumption and early inhibition of metabolism and growth when Clostridium cellulolyticum was cultured on medium containing cellobiose and yeast extract. Changing from complex medium to a synthetic medium had a strong effect on (i) the specific cellobiose consumption, which was increased threefold; and (ii) the electron flow, since the NADH/NAD+ ratios ranged from 0.29 to 2.08 on synthetic medium whereas ratios as high as 42 to 57 on complex medium were observed. These data indicate a better control of the carbon flow on mineral salts medium than on complex medium. By continuous culture, it was shown that the electron flow from glycolysis was balanced by the production of hydrogen gas, ethanol, and lactate. At low levels of carbon flow, pyruvate was preferentially cleaved to acetate and ethanol, enabling the bacteria to maximize ATP formation. A high catabolic rate led to pyruvate overflow and to increased ethanol and lactate production. In vitro, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and ethanol dehydrogenase levels were higher under conditions giving higher in vivo specific production rates. Redox balance is essentially maintained by NADH-ferredoxin reductase-hydrogenase at low levels of carbon flow and by ethanol dehydrogenase and lactate dehydrogenase at high levels of carbon flow. The same maximum growth rate (0.150 h-1) was found in both mineral salts and complex media, proving that the uptake of nutrients or the generation of biosynthetic precursors occurred faster than their utilization. On synthetic medium, cellobiose carbon was converted into cell mass and catabolized to produce ATP, while on complex medium, it served mainly as an energy supply and, if present in excess, led to an accumulation of intracellular metabolites as demonstrated for NADH. Cells grown on synthetic medium and at high levels of carbon flow were able to induce regulatory responses such as the production of ethanol and lactate dehydrogenase.     

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

During the growth of Clostridium cellulolyticum in chemostat cultures with ammonia as the growth-limiting nutrient, as much as 30% of the original cellobiose consumed by C. cellulolyticum was converted to cellotriose, glycogen, and polysaccharides regardless of the specific growth rates. Whereas the specific consumption rate of cellobiose and of the carbon flux through glycolysis increased, the carbon flux through the phosphoglucomutase slowed. The limitation of the path through the phosphoglucomutase had a great effect on the accumulation of glucose 1-phosphate (G1P), the precursor of cellotriose, exopolysaccharides, and glycogen. The specific rates of biosynthesis of these compounds are important since as much as 16.7, 16.0, and 21.4% of the specific rate of cellobiose consumed by the cells could be converted to cellotriose, exopolysaccharides, and glycogen, respectively. With the increase of the carbon flux through glycolysis, the glucose 6-phosphate (G6P) pool decreased, whereas the G1P pool increased. Continuous culture experiments showed that glycogen biosynthesis was associated with rapid growth. The same result was obtained in batch culture, where glycogen biosynthesis reached a maximum during the exponential growth phase. Glycogen synthesis in C. cellulolyticum was also not subject to stimulation by nutrient limitation. Flux analyses demonstrate that G1P and G6P, connected by the phosphoglucomutase reaction, constitute important branch points for the distribution of carbon fluxes inside and outside cells. From this study it appears that the properties of the G1P-G6P branch points have been selected to control excretion of carbon surplus and to dissipate excess energy, whereas the pyruvate-acetyl coenzyme A branch points chiefly regulate the redox balance of the carbon catabolism as was shown previously (E. Guedon et al., J. Bacteriol. 181:3262-3269, 1999).     

Metabolism of cellobiose and cellulose byBacteroides cellulosolvens

Summary The metabolism ofBacteroides cellulosolvens was studied on cellobiose and cellulose as energy and carbon sources. The growth rate was faster on cellobiose; however, growth on cellulose resulted in consumption of 55% more hexose equivalents, and in production of 49% more biomass, and 30% more metabolites (ethanol, acetate, and lactate). On each substrateB. cellulosolvens exhibited two distinct ranges of molar growth yields (Y _H g cells/mol hexose). At low substrate concentrations (less than 30 mmol) hexoseY _H values were 25.5 for cellulose and 28.5 for cellobiose, while at hexose levels greater than 30 mmolY _H values were 13.5 and 15, respectively. Shifts in metabolism towards greater lactic acid production resulted in decreased ATP production; however, this did not cause early growth cessation, as these shifts occurred after the drop inY _H.Issued as NRCC No. 27409.     

Clostridium cellulolyticum: model organism of mesophilic cellulolytic clostridia

Clostridium cellulolyticum ATCC 35319 is a non-ruminal mesophilic cellulolytic bacterium originally isolated from decayed grass. As with most truly cellulolytic clostridia, C. cellulolyticum possesses an extracellular multi-enzymatic complex, the cellulosome. The catalytic components of the cellulosome release soluble cello-oligosaccharides from cellulose providing the primary carbon substrates to support bacterial growth. As most cellulolytic bacteria, C. cellulolyticum was initially characterised by limited carbon consumption and subsequent limited growth in comparison to other saccharolytic clostridia. The first metabolic studies performed in batch cultures suggested nutrient(s) limitation and/or by-product(s) inhibition as the reasons for this limited growth. In most recent investigations using chemostat cultures, metabolic flux analysis suggests a self-intoxication of bacterial metabolism resulting from an inefficiently regulated carbon flow. The investigation of C. cellulolyticum physiology with cellobiose, as a model of soluble cellodextrin, and with pure cellulose, as a carbon source more closely related to lignocellulosic compounds, strengthen the idea of a bacterium particularly well adapted, and even restricted, to a cellulolytic lifestyle. The metabolic flux analysis from continuous cultures revealed that (i) in comparison to cellobiose, the cellulose hydrolysis by the cellulosome introduces an extra regulation of entering carbon flow resulting in globally lower metabolic fluxes on cellulose than on cellobiose, (ii) the glucose 1-phosphate/glucose 6-phosphate branch point controls the carbon flow directed towards glycolysis and dissipates carbon excess towards the formation of cellodextrins, glycogen and exopolysaccharides, (iii) the pyruvate/acetyl-CoA metabolic node is essential to the regulation of electronic and energetic fluxes. This in-depth analysis of C. cellulolyticum metabolism has permitted the first attempt to engineer metabolically a cellulolytic microorganism.     

Physiological properties of Cellulomonas fermentans,a mesophilic cellulolytic bacterium

Summary The fermentation of cellobiose, glucose and cellulose MN 300 by Cellulomonas fermentans was studied. The molar growth yields (i.e. grams of cells per mole of hexose equivalent) were similar on cellobiose and cellulose at low sugar consumption levels (47.8 and 46.5 respectively), but was lower on glucose (38.0). The occurrence of cellobiose phosphorylase activity, detected in cellobiose- and cellulose-grown cells, might explain this result. The specific growth rates measured in cultures on cellobiose, glucose and cellulose were 0.055 h^-1, 0.040 h^-1 and 0.013 h^-1 respectively. Growth inhibition was observed, and a drop in Y_H occurred after relatively low but different quantities of hexose were consumed (2.2 mM, 5 mM and 8 mM hexose equivalent with cellulose, glucose and cellobiose respectively), which coincided with a change in the fermentative metabolism from a typical mixed acid metabolism (1 ethanol, 1 acetate and 2 formate synthesized by consumed hexose) to a more ethanolic fermentation. When growth ceased in cellulose cultures, consumption of cellulose continued, as did production of ethanol.Molar growth yields of C. fermentans were similar in anaerobic and aerobic cellobiose cultures (47.8 g/mol and 42.2 g/mol respectively). Specific growth rates were also quite similar under both culture conditions (0.055±0.013 h^-1 and 0.070±0.007 h^-1 respectively). Aerobic metabolism was studied using ¹⁴C glucose. During the exponential growth phase, acetate, succinate and nonidentified compound(s) accumulated in the supernatant, but no ¹⁴CO₂ was produced. During the stationary phase, acetate was oxidized and ¹⁴CO₂ produced, but without any further biomass synthesis. It seems that a blocking of metabolite oxidation may have occurred in C. fermentans except in the case of acetate, but acetate oxidation was apparently not coupled with production of energy utilizable in biosynthesis.     

Adhesion and growth rate of Clostridium cellulolyticum ATCC 35319 on crystalline cellulose.

The rate of tritiated-thymidine incorporation into DNA was used to estimate Clostridium cellulolyticum H10 growth rates on Avicel cellulose, taking into consideration both the unattached cells and the cells adhered to the substrate. The generation time on cellobiose calculated from the data on cell density (4.5 h) agreed well with the generation time calculated by tritiated-thymidine incorporation (3.8 h). Growth on Avicel cellulose occurred when bacteria were adhered to their substrate; 80% of the biomass was detected on the cellulose. Taking into consideration attached and free bacteria, the generation time as determined by thymidine incorporation was about 8 h, whereas by bacterial-protein estimation it was about 13 h. In addition to the growth rate of the bacteria on the cellulose, the release of adhered cells constituted an important factor in the efficiency of the cellulolysis. The stage of growth influenced adhesion of C. cellulolyticum; maximum adhesion was found during the exponential phase. Under the conditions used, the end of growth was characterized by an acute release of biomass and cellulase activity from the cellulose. An exhaustion of the accessible cellulose could be responsible for this release.     

Unravelling carbon metabolism in anaerobic cellulolytic bacteria

Carbon metabolism in anaerobic cellulolytic bacteria has been investigated essentially in Clostridium thermocellum, Clostridium cellulolyticum, Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminococcus albus. While cellulose depolymerization into soluble sugars by various cellulases is undoubtedly the first step in bacterial metabolisation of cellulose, it is not the only one to consider. Among anaerobic cellulolytic bacteria, C. cellulolyticum has been investigated metabolically the most in the past few years. Summarizing metabolic flux analyses in continuous culture using either cellobiose (a soluble cellodextrin resulting from cellulose hydrolysis) or cellulose (an insoluble biopolymer), this review aims to stress the importance of the insoluble nature of a carbon source on bacterial metabolism. Furthermore, some general and specific traits of anaerobic cellulolytic bacteria trends, namely, the importance and benefits of (i) cellodextrins with degree of polymerization higher than 2, (ii) intracellular phosphorolytic cleavage, (iii) glycogen cycling on cell bioenergetics, and (iv) carbon overflows in regulation of carbon metabolism, as well as detrimental effects of (i) soluble sugars and (ii) acidic environment on bacterial growth. Future directions for improving bacterial cellulose degradation are discussed.     

Metabolism and Solubilization of Cellulose by Clostridium cellulolyticum H10

When Clostridium cellulolyticum was grown with cellulose MN300 as the substrate, the rates of growth and metabolite production were found to be lower than those observed with soluble sugars as the substrate. At low cellulose concentrations, the growth yields were equal to those obtained with cellobiose. The main fermentation products from cellulose and soluble sugars were the same. Up to 15 mM of consumed hexose, a change in the metabolic pathway favoring lactate production similar to that observed with soluble sugars was found to occur concomitantly with a decrease in molar growth yield. With cellulose concentrations above 5 g/liter, accumulation of soluble sugars occurred once growth had ceased. Glucose accounted for 30% of these sugars. A kinetic analysis of cellulose solubilization revealed that cellulolysis by C. cellulolyticum involved three stages whatever cellulose concentration was used. Analysis of these kinetics showed three consecutive enzymatic activity levels having the same K_m (0.8 g of cellulose per liter, i.e., 5 mM hexose equivalent) but decreasing values of V_max. The hypothesis is suggested that each step corresponds to differences in cellulose structure.     

Induction of Cellulases in chaetomium cellulolyticum by cellobiose

The production of extracellular cellulases by Chaetomium cellulolyticum could be induced by slow feeding of cellobiose to the cultures. Both the rate of production and the amount of activity were comparable to that obtained in batch cultivation on cellulose. The specific filter paper activity of 2.06 U per mg protein was almost two times higher than that obtained in cellulose medium. Cellulases were not induced when glucose was slowly fed to the cultures. Changing the feed stream from glucose to cellobiose resulted in a rapid accumulation of cellulases. Thus cellobiose has a similar role in cellulase induction in C. cellulolyticum, as earlier shown for Trichoderma reesei.     

Influence of readily metabolizable carbon on pentachlorophenol metabolism by a pentachlorophenol-degrading Flavobacterium sp.

The influence of high concentrations of pentachlorophenol (PCP) and readily metabolizable carbon on the activity and viability of a PCP-degrading Flavobacterium sp. was examined in a mineral salts medium. Lags preceding PCP removal by glutamate-grown Flavobacterium cells were greatly attenuated by the addition of glutamate, aspartate, succinate, acetate, glucose, or cellobiose. The effect of these supplementary carbon sources on the apparent lag was not mediated entirely through the stimulation of growth since PCP metabolism accompanied the onset of growth. The specific activity of PCP-degrading cells in the absence of supplementary carbon was 1.51 x 10(-13) +/- 0.08 x 10(-13) g of PCP per cell per h and in the presence of supplementary carbon was 0.92 x 10(-13) +/- 0.09 x 10(-13) g of PCP per cell per h. Glutamate in combination with glucose or cellobiose partially repressed PCP metabolism. PCP removal by PCP-induced, glutamate-grown cells suspended in the presence of 4 g of sodium glutamate per liter was sensitive to shock loads of PCP, with a Ki of about 86.8 micrograms/ml. Subsequent removal rates, however, were more resistant to PCP. Optimal stimulation of PCP removal by sodium glutamate required 3.0 g/liter, about the same concentration as that which saturated growth in the absence of PCP. PCP removal rates decayed within minutes following the transfer of PCP-induced, glutamate-grown cells to media containing PCP without supplementary carbon, and increasing PCP concentrations accelerated the decay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of readily metabolizable carbon on pentachlorophenol metabolism by a pentachlorophenol-degrading Flavobacterium sp

The influence of high concentrations of pentachlorophenol (PCP) and readily metabolizable carbon on the activity and viability of a PCP-degrading Flavobacterium sp. was examined in a mineral salts medium. Lags preceding PCP removal by glutamate-grown Flavobacterium cells were greatly attenuated by the addition of glutamate, aspartate, succinate, acetate, glucose, or cellobiose. The effect of these supplementary carbon sources on the apparent lag was not mediated entirely through the stimulation of growth since PCP metabolism accompanied the onset of growth. The specific activity of PCP-degrading cells in the absence of supplementary carbon was 1.51 x 10(-13) +/- 0.08 x 10(-13) g of PCP per cell per h and in the presence of supplementary carbon was 0.92 x 10(-13) +/- 0.09 x 10(-13) g of PCP per cell per h. Glutamate in combination with glucose or cellobiose partially repressed PCP metabolism. PCP removal by PCP-induced, glutamate-grown cells suspended in the presence of 4 g of sodium glutamate per liter was sensitive to shock loads of PCP, with a Ki of about 86.8 micrograms/ml. Subsequent removal rates, however, were more resistant to PCP. Optimal stimulation of PCP removal by sodium glutamate required 3.0 g/liter, about the same concentration as that which saturated growth in the absence of PCP. PCP removal rates decayed within minutes following the transfer of PCP-induced, glutamate-grown cells to media containing PCP without supplementary carbon, and increasing PCP concentrations accelerated the decay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic engineering of glycerol production in Saccharomyces cerevisiae

Inactivation of TPI1, the Saccharomyces cerevisiae structural gene encoding triose phosphate isomerase, completely eliminates growth on glucose as the sole carbon source. In tpi1-null mutants, intracellular accumulation of dihydroxyacetone phosphate might be prevented if the cytosolic NADH generated in glycolysis by glyceraldehyde-3-phosphate dehydrogenase were quantitatively used to reduce dihydroxyacetone phosphate to glycerol. We hypothesize that the growth defect of tpi1-null mutants is caused by mitochondrial reoxidation of cytosolic NADH, thus rendering it unavailable for dihydroxyacetone-phosphate reduction. To test this hypothesis, a tpi1delta nde1delta nde2delta gut2delta quadruple mutant was constructed. NDE1 and NDE2 encode isoenzymes of mitochondrial external NADH dehydrogenase; GUT2 encodes a key enzyme of the glycerol-3-phosphate shuttle. It has recently been demonstrated that these two systems are primarily responsible for mitochondrial oxidation of cytosolic NADH in S. cerevisiae. Consistent with the hypothesis, the quadruple mutant grew on glucose as the sole carbon source. The growth on glucose, which was accompanied by glycerol production, was inhibited at high-glucose concentrations. This inhibition was attributed to glucose repression of respiratory enzymes as, in the quadruple mutant, respiratory pyruvate dissimilation is essential for ATP synthesis and growth. Serial transfer of the quadruple mutant on high-glucose media yielded a spontaneous mutant with much higher specific growth rates in high-glucose media (up to 0.10 h(-1) at 100 g of glucose. liter(-1)). In aerated batch cultures grown on 400 g of glucose. liter(-1), this engineered S. cerevisiae strain produced over 200 g of glycerol. liter(-1), corresponding to a molar yield of glycerol on glucose close to unity.