INTRODUCTION

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Cellulose is the most abundantly produced biopolymer on earth (5, 28). Due to its recalcitrant, durable nature, cellulose accumulates in terrestrial environments, where a variety of cellulolytic microorganism, existing in virtually every niche and clime, decompose it (4, 28, 29). Around 5 to 10% of cellulasic materials are degraded anaerobically, and the final products released during fermentation are methane and carbon dioxide (28, 51); among cellulolytic bacteria, clostridia play an important role in such processes (28).

Clostridium cellulolyticum, a nonruminal, strictly anaerobic, cellulolytic bacterium (45), digests cellulose through the cellulosome (43). This extracellular multienzymatic complex is composed of a variety of cellulases organized around a scaffolding protein called CipC (6, 41, 42). The cellulosomes are found at the surface of the cells and allow both adhesion and efficient degradative activity against the cellulose fibers (3, 8).

Using cellobiose, which is one of the soluble cellodextrins released during cellulolysis, major differences in the regulation of the carbon flow between carbon-limited and carbon-sufficient continuous cultures have been reported (19, 20). As the dilution rate increases, in cellobiose-limited chemostats the metabolic pathways towards ethanol and lactate contribute to balance the reducing equivalents supplied by acetate formation (19), while under cellobiose-saturated conditions (20) the redox balance is essentially maintained by NADH-ferredoxin (NADH-Fd) reductase-hydrogenase and ethanol dehydrogenase activities and the carbon flow is equilibrated by three overflows, i.e., exopolysaccharide, extracellular protein, and amino acid excretions.

Using a substrate more closely related to the natural ecosystem of the bacterium, recent investigations with cellulose-limited chemostats (11) have indicated that there is neither a shift from an acetate-ethanol fermentation to a lactate-ethanol fermentation nor pyruvate overflow at high catabolic rates as previously observed on cellobiose (18, 19). Thus, with this culture condition, C. cellulolyticum appeared well adapted and even restricted to a cellulolytic lifestyle (11, 12). In its natural biota, however, growth under carbon-sufficient conditions is undoubtedly experienced by bacteria and probably more frequently than carbon limitation, since cellulose accumulates in environments (4, 5).

The aim of the present study, then, was to investigate kinetically the C. cellulolyticum fermentation under carbon-saturated conditions by using continuous culture analysis with cellulose and a mineral salt-based medium, which are more closely related to the natural ecosystem of the bacterium (11, 19).

	MATERIALS AND METHODS

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Organism and growth conditions. C. cellulolyticum ATCC 35319 (45) was cultured as previously reported (10) on a defined medium (19) containing cellulose MN301 (Macherey-Nagel, Düren, Germany) at various concentrations as specified in Results. All experiments in segmented gas-liquid continuous culture (49) were performed in a 1.5-liter-working-volume fermentor (LSL Biolafitte, St. Germain en Laye, France) at 34°C and pH 7.2 and monitored as previously indicated (11).

Analytical procedures. Biomass, cellulose concentration, gas analysis, extracellular protein, amino acid, glucose, soluble cellodextrins, glycogen, acetate, ethanol, lactate, and extracellular pyruvate were determined as described previously (10, 11, 17).

Calculations. The metabolic pathways and equations for cellulose fermentation by C. cellulolyticum (expressed as n hexose equivalents [hexose eq], corresponding to n glucose residues of the cellulose chain) were reported previously (10, 11).

View larger version (23K): [in this window] [in a new window]   FIG. 1.   Carbon flow within the central metabolic pathways of C. cellulolyticum grown in cellulose excess. n, number of hexose residues inside the biopolymer. The specific consumption and production rates (q) correspond to the equations given in Table 1.

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	RESULTS

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C. cellulolyticum continuous culture with increasing concentrations of cellulose. C. cellulolyticum was grown on cellulose in independent runs using a segmented gas-liquid continuous culture device at a dilution rate of 0.048 h¹ with substrate concentrations ranging from 1.9 to 27.0 g liter¹ (Table 2). With increasing amounts of substrate, the concentration of consumed cellulose rose, but at above 7.6 g liter¹ (i.e., 47.0 mM hexose eq) the concentration of consumed cellulose stagnated at 13.9 to 14.3 mM hexose eq (Table 2). The biomass concentration at each steady state increased with the cellulose concentration in the feed medium reservoir, but at above 7.6 g liter¹ it remained quite constant at around 0.296 g liter¹ (Table 2), and thus the production of biomass paralleled the amount of digested cellulose. Microscopic examination indicated that at low cellulose concentrations unattached cells were observable and that almost all of the cellulose fibers were colonized by bacteria. All of these results indicated that at above 7.6 g of cellulose liter¹, continuous cultures were carried out under cellulose-sufficient conditions.

View this table: [in this window] [in a new window]   TABLE 2.   Fermentation parameters from continuous culture of C. cellulolyticum with increasing concentrations of cellulose at a D value of 0.048 h1

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Cellulose degradation in continuous culture at high substrate concentrations. C. cellulolyticum was cultivated in cellulose excess with 18.1 g of cellulose liter¹ at different D values, which ranged from 0.026 to 0.080 h¹ (Table 3). From the lowest to the highest D value tested, the cell density at steady state decreased while the observed cell yield (Y_X/S) increased (Table 3). The Pirt plot of these data (r² = 0.992) permitted determination of a Y of 40.6 g of biomass mol of hexose eq consumed¹ and a maintenance coefficient (m) of 1.0 mmol of hexose eq g of cells¹ h¹. The percentage of nonadherent cells remained very low, ranging from 7.2 to 2.7% (Fig. 2a). In a cellulose-limited chemostat (i.e., 3.7 g of cellulose liter¹), however, the percentage of planktonic cells decreased from 59.6 to 21.0% as D increased from 0.027 to 0.083 h¹ and was always much higher than in cellulose saturation (Fig. 2a). The proportion of undegraded cellulose was much lower in cellulose limitation than under cellulose-sufficient conditions; with increasing D it rose from 49.6 to 79.0% and from 78.6 to 94.7%, respectively (Fig. 2b). With this culture condition, C. cellulolyticum always left undigested cellulose. Plots of S_r/S₀ versus t_R (t_R = 1/D) were linear, with a first-order rate constant of 0.008 h¹ determined from linear regression of the data (r² = 0.996) (Fig. 2b).

View larger version (16K): [in this window] [in a new window]   FIG. 2.   Percentage of nonadherent cells (a) and proportion of undigested cellulose (b) in cellulose-limited (i.e., 3.7 g liter1) (, ) and in cellulose-excess (i.e., 18.1 g liter1) (, ) continuous culture of C. cellulolyticum. Error bars indicated standard deviations. Inset, correlation between SR/S0 and tR under cellulose-sufficient conditions.

Kinetics analysis of microbial cellulose conversion under cellulose-sufficient conditions. The carbon flow in the central metabolic pathway of C. cellulolyticum (Fig. 1) grown in cellulose-sufficient continuous culture is compiled in Table 4. With increasing D, the proportion of carbon flowing down the catabolite declined from 81.2 to 69.1%, while it was enhanced through biosynthesis pathways from 16.3 to 23.8%. In parallel, q_G6P gradually rose, but this increase represented a decreasing proportion of the original carbon. As a result, the G6P pool slowly declined (Fig. 3) as D rose, and when expressed in term of turnover this pool increased from 22.1 to 41.7 h¹. The proportion of carbon directed towards exopolysaccharide and glycogen was low at a D of 0.026 h¹ and reached 5.7 and 1.4%, respectively, with the highest D tested (Table 4). In contrast, the q_{G1P towards cellodextrin} declined from 2.0% to nil at a D of 0.080 h¹, since no cellodextrin could then be detected (Table 4). The G1P flux through phosphoglucomutase varied from 60.4 to 55.9% (Table 4). G1P then accumulated with increasing D (Fig. 3), which resulted in the turnover decreasing from 46.4 to 15.4 h¹. The proportion of the carbon flux towards phosphoglucomutase declined, and the G1P, which was directed towards cellodextrin at low D values, was rerouted towards glycogen and exopolysaccharide at higher D values. The percentage of carbon directed towards the fermentative end products declined as D rose (Table 4). One part of the flux was converted to acetyl coenzyme A (acetyl-CoA), i.e., from 50.9 to 45.2%. In the same time, q_acetate and q_ethanol increased, but when expressed as a percentage of q_cellulose, the two fluxes declined (Table 4). Another part of the carbon flowing down glycolysis was oriented towards the lactate production pathway. As D was enhanced, lactate production decreased, as did the pyruvate leak.

View larger version (16K): [in this window] [in a new window]   FIG. 3.   G1P (), G6P (), and G6P/G1P ratio () as a function of dilution rate in cellulose-sufficient continuous culture of C. cellulolyticum.

Relationships between carbon flow and enzymatic activities, energetic balance, and redox balance. In vitro GAPDH, PFO, ADH, and AK activities were higher under growth conditions giving higher in vivo specific production rates (Table 5). For LDH, however, the specific enzyme activities decreased with D, which was correlated with the in vivo lactate production rate. A ratio of specific enzyme activity to metabolic flux (R) (11, 27) was then calculated; R was higher than 1 for all enzymes tested. Therefore, these enzymes were not limiting with respect to the carbon flow, and thus fluxes were determined more by the concentration of substrate available than by the enzyme activity (12, 26).

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	DISCUSSION

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A continuous culture system in which the feed rate is set externally is generally regarded as a chemostat if cell growth is also limited by a selected nutrient(s) (21, 52). Upon increasing the carbon substrate concentration in the feed reservoir, cellulose was no longer the growth-limiting nutrient at and above 7.6 g of cellulose liter¹, and all other nutrients appeared in excess (10, 20). Growth under substrate excess generally results in oscillations and hysteresis (23, 24, 37, 52), but such phenomena did not occur, since steady states of both residual cellulose concentration and biomass monitored during cell culture could be maintained (39). Then, as previously observed with cellobiose (20), a stable carbon excess continuous culture could be imposed on C. cellulolyticum. From studies of growth of C. cellulolyticum in cellobiose-fed continuous culture in a stepwise fashion (18) and in cellulose batch culture with a reinoculation mode (10), it was demonstrated that growth arrest was not associated with the production of extracellular toxic compounds, and thus it is unlikely that the steady state of the present continuous culture could be maintained by such growth inhibition. Yet, comparing the maximum growth yields and maximum energetic yield obtained under cellulose-sufficient conditions (i.e., Y = 40.6 g of biomass mol of hexose eq consumed¹ and Y = 19.4 g of cells mol of ATP¹) to those resulting from cellulose limitation (i.e., Y = 50.5 g of biomass mol of hexose eq consumed¹ and Y = 30.3 g of cells mol of ATP¹ [11]), it could be observed that both maximum yields were lowered in the presence of a cellulose excess. The decline of these yields indicated that an uncoupling growth phenomenon had occurred; it also took place with the rise in lactate production accompanied by a pyruvate leak. Thus, the growth stagnation was certainly related to an accumulation of an intracellular inhibitory compound(s) (18); intracellular inhibition could furthermore explain the establishment of a steady state under the condition of an excess of all nutrients (52).

The understanding of microbial cellulose metabolism is of both ecological and biotechnological interest. Cellulose degradation plays a key role in the global carbon cycle (28, 29, 51) and is a promising strategy in consolidated bioprocessing for the production of biochemical compounds (25, 31-34). So far, however, very few studies have been devoted to cellulose digestion by cellulolytic bacteria under substrate-saturated conditions (38, 40, 48). Under cellulose-sufficient culture conditions, cellulose digestion always follows first-order kinetics, where k (0.008 h¹) was much lower than under cellulose-limited conditions (0.046 h¹) (11). Once cellulose-saturated conditions were attained, approximately the same amount of cellulose was digested, since the biomass concentration at steady state stagnated. Therefore, k will vary for each cellulose concentration used with this growth condition, since cellulose degradation will follow first-order kinetics with respect to the remaining cellulose concentration (44). Opposite to what was observed with cellulose limitation (11), the lactate-ethanol production was lowered as D rose and had to be balanced by dihydrogen production via the NADH-Fd reductase, which led to an increased H₂/CO₂ ratio. The acetate/ethanol ratio was always higher than 1, but in contrast to the case with cellulose limitation, it increased with D (11). The specific lactate production rate as well as the pyruvate leak decreased with increasing D but always remained higher than in a cellulose-limited chemostat (11). The R values for LDH were around 80.0 and were not as high as with cellulose limitation, where R could reach 434.2 (11). From the G1P metabolic node, cellodextrins were produced and represented up to 3.5% of the carbon uptake at the lowest D value tested. G1P was rerouted towards exopolysaccharide and glycogen as D rose; as in cellulose limitation, these biosyntheses could be adjusted as a function of carbon flux. Such glycogen turnover was recently observed with Fibrobacter succinogenes on cellulose (7, 14). A concomitant decrease of the percentage of carbon flowing through q_{phosphoglucomutase}, q_G6P, and q_pyruvate in favor of biosynthesis pathways could explain the relative drop in lactate production as D increased. Compared to that in cellulose-limited chemostats, the proportion of unattached cells was low. With cellulose limitation the available surface area is saturated by bacteria, while under cellulose-sufficient conditions the cellulose surface area is largely accessible for bacterial adhesion (10, 13). This was correlated with a higher cellulose digestion rate reflected by higher q_cellulose under substrate excess conditions, since most of the cells adhered to cellulose fibers and thus participated directly in cellulose digestion. While cellodextrin was undetectable with cellulose limitation (11), cellobiose and cellotriose were detected here in the supernatant; such a finding was certainly related to a reversible phosphorylase reaction (1, 2, 30, 35, 36, 47, 50). Under cellobiose-sufficient conditions (17, 20), only cellotriose was detected, but the present results suggest that cellobiose could also be synthesized de novo during cell growth on cellobiose.

Previous reports on experiments with cellobiose stated that the adhesion-colonization phase of the process of cellulose digestion by C. cellulolyticum (15, 16) corresponded to a carbon-sufficient period (20). It was thus argued that a carbon flow of as high as 5.14 mmol of hexose eq g of cells¹ could be attained with cellulose as a substrate (20). With cellulose saturation, however, the entering carbon flow remained lower than expected, i.e., 2.91 mmol of hexose eq g of cells¹ h¹. The NADH/NAD⁺ ratio was always lower than 1 on cellulose, whereas a ratio of as high as 1.51 was obtained with cellobiose excess (20); this result was most probably related to a higher carbon consumption rate which led to rate-limiting fluxes through ethanol and dihydrogen production pathways on cellobiose. Thus, the proper NADH/NAD⁺ ratio was maintained only when ethanol and lactate production complemented the path towards H₂ production via NADH-Fd reductase activity. With a carbon excess, free amino acid could account for 15.4% of the cellobiose fermented (20), against a maximum of 5.8% on cellulose, while exopolysaccharide represented up to 38.1% of the cellobiose consumed (20) and there was a maximum of only 5.7% with cellulose as the substrate. It thus appeared that some of the general metabolic trends associated with carbon-sufficient conditions, such as (i) the ATP/ADP ratio always being higher than 1, (ii) the elevated production of lactate at a low D, and (iii) the concomitant increase of q_ethanol, q_NADH-Fd, q_{NADH produced}/q_{NADH used}, NADH/NAD⁺, and H₂/CO₂ as D rose and some other regulations of bacterial metabolism, were not observed on cellulose compared to cellobiose. Even if cellulose degradation must be considered as a microbial process rather than a purely enzymatic event, the strong influence of the cellulosome on the entering carbon flow must be taken into account (10, 11). The study of C. cellulolyticum catabolism with soluble glucide allowed the demonstration of bacterial metabolic limitation, but this response should be interpreted as deregulation of the metabolism. All of these results demonstrate that C. cellulolyticum was able to correctly regulate and optimize carbon metabolism in limited and saturated conditions using a substrate more representative of its natural environment, i.e., cellulose (12).