Emmanuel Guedon

Improvement of Cellulolytic Properties of Clostridium cellulolyticum by Metabolic Engineering

ABSTRACT Cellulolytic clostridia have evolved to catabolize lignocellulosic materials at a seasonal biorhythm, so their biotechnological exploitation requires genetic improvements. As high carbon flux leads to pyruvate accumulation, which is responsible for the cessation of growth of Clostridium cellulolyticum, this accumulation is decreased by heterologous expression of pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis. In comparison with that of the wild strain, growth of the recombinant strain at the same specific rate but for 145 h instead of 80 h led to a 150% increase in cellulose consumption and a 180% increase in cell dry weight. The fermentation pattern was shifted significantly: lactate production decreased by 48%, whereas the concentrations of acetate and ethanol increased by 93 and 53%, respectively. This study demonstrates that the fermentation of cellulose, the most abundant and renewable polymer on earth, can be greatly improved by using genetically engineered C. cellulolyticum.

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

ABSTRACT 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, H2, and CO2 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 H2 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.

Metabolism of cellobiose by Clostridium cellulolyticum growing in continuous culture: evidence for decreased NADH reoxidation as a factor limiting growth

Previous results indicated that molar growth yields are reduced when Clostridium cellulolyticum is cultured in media containing cellobiose concentrations greater than 1 g I-1. Continuous cultures were examined to determine the physiological basis of these poor growth yields. Acetate was the main product of C. cellulolyticum metabolism, whereas the production of reduced compounds such as ethanol or lactate was low. Such patterns of product formation were accompanied by a 12-fold increase in intracellular NADH concentration when the cellobiose flow was increased. Catabolic enzymic activities were measured in vitro. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), acetate kinase and phosphoroclastic activities were found at similar levels as in cells metabolizing higher substrate concentrations. In contrast, lactate dehydrogenase activity was low and correlated with the rate of lactate production. Furthermore, an inhibition of GAPDH activity by high NADH/NAD+ ratios was established. These results suggested that a decreased NADH reoxidation could be responsible for limiting C. cellulolyticum growth. Lactate and ethanol production were not sufficient to balance out the NADH produced in the GAPDH step of glycolysis. One consequence of poor NADH reoxidation would be an increase in intracellular concentration of NADH, which in turn could inhibit GAPDH activity.

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).

Growth inhibition of Clostridium cellulolyticum by an inefficiently regulated carbon flow.

Carbon flow in Clostridium cellulolyticum was investigated either in batch or continuous culture using a synthetic medium with cellobiose as the sole source of carbon and energy. Previous experiments carried out using a complex growth medium led to the conclusion that the carbon flow was stopped by intracellular NADH. In this study, results showed that cells cultured in a synthetic medium were better able to control electron flow since the NADH/NAD+ ratios were in the range 0.3-0.7, whereas a ratio as high as 57 was previously found in cells cultured on a complex medium. Furthermore, a specific rate of cellobiose consumption of 2.13 mmol (g cells)-1 h-1 was observed on synthetic medium whereas the highest value obtained on complex medium was 0.68 mmol (g cells)-1 h-1. When C. cellulolyticum was grown in continuous culture and cellobiose in the feed medium was increased from 5.84 to 17.57 mM in stepwise fashion, there was an increase in cellobiose utilization without growth inhibition. In contrast, when the reactor was fed directly with 14.62 mM cellobiose, residual cellobiose was observed (4.24 mM) and growth was limited. These data indicate that C. cellulolyticum is not able to optimize its growth and carbon flow in response to a sudden increase in the concentration of growth substrate cellobiose. This interpretation was confirmed (i) by the study of cellobiose batch fermentation where it was demonstrated that growth inhibition was not due to nutritional limitation or inhibition by fermentation products but was associated with carbon excess and (ii) by the growth of C. cellulolyticum in dialysis culture where no growth inhibition was observed due to the limitation of carbon flow by the low rate of cellobiose diffusion through the dialysis tubing.

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.

Kinetics and Metabolism of Cellulose Degradation at High Substrate Concentrations in Steady-State Continuous Cultures of Clostridium cellulolyticum on a Chemically Defined Medium

ABSTRACT 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 qNADH produced/qNADH 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 ( YX/Smax) (40.6 g of biomass per mol of hexose equivalent) and the maximum energetic yield ( YATPmax) (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−1h−1 could be reached, the maximum entering carbon flow obtained here on cellulose was 2.91 mmol of hexose equivalent g of cells−1h−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.

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

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 7.2. 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 7.4 to 6.2, maximum cell density was obtained at pH 7.0. Environmental acidification chiefly influenced biomass formation, since at pH 6.4 the dry weight of cells was more than fourfold lower compared to that at pH 7.0, whereas the specific rate of cellulose assimilation decreased only from 11.74 to 10.13 milliequivalents of carbon (g cells)(-1) h(-1). 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 6.4. 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 9.8 to 1.8. Thus a change of only 1 pH unit induced considerable metabolic change and ended by washout at around pH 6.2. 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 6.0, suggesting that they evolved in environments where acid tolerance was not required for successful competition with other microbes.

Induction of lactate production associated with a decrease in NADH cell content enables growth resumption of Clostridium cellulolyticum in batch cultures on cellobiose

When grown in batch cultures in fermentors with 23.4 mM cellobiose, Clostridium cellulolyticum displayed biphasic growth kinetics not associated with sequential substrate consumption and which led to a twofold higher production of biomass than previously reported. In the first growth phase, acetate was the major product of cellobiose metabolism, since lactate and ethanol productions remained low. Furthermore, an accumulation of intracellular NADH was observed. The transition towards the second growth phase was accompanied by an induction of lactate production, in such a way that lactate became the major product of C. cellulolyticum metabolism. In addition, a decrease in NADH concentration was measured, concomitant with this induction of lactate production and with the growth resumption. During both growth phases, the NADH-ferredoxin reductase-hydrogenase system played a major function in NADH regeneration, since H2 production was 1.4- to 1.5-fold higher than that of CO2. Thus, we found that lactate production serves as an additional catabolic pathway enabling C. cellulolyticum to cope with excesses of carbon and NADH produced. Growth experiments on C. cellulolyticum under an atmosphere of carbon monoxide mimicked this phenomenon and confirmed that a high intracellular level of NADH can provide a barrier to bacterial growth.

Effect of dilution rate, cellobiose and ammonium availabilities on Clostridium cellulolyticum sporulation

Abstract The nutritional and physiological factors affecting sporulation of Clostridium cellulolyticum were studied using steady-state continuous cultures grown in both complex and synthetic media. Under cellobiose limitation, the probability that cells will sporulate appears to be directly related to the growth rate. In complex medium, the highest percentage of sporulation was 20% at a dilution rate of 0.015 h−1 whereas in synthetic medium it was 10% at 0.035 h−1. In both media, when the dilution rate was either higher or lower the percentage of sporulation decreased by between 2% and 4%. At low dilution rates, endospore formation was repressed under cellobiose-sufficient concentrations, suggesting catabolite repression by cellobiose. Furthermore, the concentration of ammonium was important in determining the percentage of sporulation, as ammonium limitation induced extensive sporulation at low growth rates even in an excess of cellobiose. The sporulation process is not triggered when cells are cellobiose-exhausted both in complex and synthetic media. These data suggest that, in C. cellulolyticum, an exogenous supply of carbon is required throughout the sporulation process. In the experimental conditions used in this work, no relationship between glycogen accumulation or glycogen mobilization and endospore formation was detected in C. cellulolyticum.