INTRODUCTION

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Cellulolytic clostridia play a major role in cellulose decomposition, which is a key process in carbon cycling (29). Clostridium cellulolyticum is a nonruminal cellulolytic mesophilic bacterium isolated from decayed grass and capable of degrading crystalline cellulose (36). The biotechnological exploitation of this microorganism as well as the understanding of the role it plays in its own ecosystem requires knowledge of its metabolism and of its behavior when developed on cellulose.

C. cellulolyticum is a low-G+C gram-positive anaerobe belonging to clostridial group III (39, 40); it is also placed in family 4, genus 2, in a new proposed-hierarchical structure for clostridia (7). Recent metabolic investigations with this bacterium indicated that (i) compared to a complex medium previously used, mineral salt medium clearly produced a different regulatory response and permitted better control of the carbon flow (19, 34), (ii) early growth inhibition was associated with a carbon excess (18), and (iii) carbon-limited and carbon-saturated chemostats displayed major discrepancies in the regulation of carbon flow (20). These studies were performed using cellobiose, which is considered the most important end product of the enzymatic cellulose hydrolysis (31, 44, 45), and it was assumed that growth on cellulosic material was rather difficult to monitor and that metabolism changes would be more observable with soluble sugar (14, 17).

Concerning the behavior of C. cellulolyticum towards cellulose substrate, earlier studies (i) suggested that the release of soluble sugars inhibited both cell growth and cellulase production (37) and (ii) described the growth of the bacteria on cellulose in terms of adhesion, colonization, release, and readhesion processes (13). These experiments, however, were conducted systematically in complex media without pH regulation, and many of the effects observed may have been due to a decrease in the pH of these cultures (47). In fact, compared with other low-G+C gram-positive anaerobes, particularly with bacteria defined as lactic acid bacteria, the bacteria of the clostridial type are generally considered to be restricted to a less acidic ecological niche due to their particular pattern of intracellular pH regulation (21, 41).

Bacterial growth on cellulose differs from that on cellobiose by the necessity for bacteria first to adhere on the substrate and second to degrade it into soluble sugars. Thus both substrate limitation and substrate-sufficient periods could be encountered by bacteria, and so the carbon flow on a cellulose batch culture could be somewhere between that associated with carbon limitation when bacteria are released and that associated with carbon-sufficient conditions when bacteria have adhered to cellulose fibers.

The aim of the present work was to investigate how C. cellulolyticum managed the abundance of insoluble substrate and to see whether or not the previous observations of cellobiose (15, 17-20, 34) were typical of bacterial behavior on cellulose. Taking into account previous considerations, this investigation on the metabolism and cellulolytic performance of C. cellulolyticum in batch fermentations was performed with controlled pH and using a mineral salt-based medium, which is more representative of the natural ecosystem of the bacterium.

	MATERIALS AND METHODS

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Chemicals. All chemicals were of highest-purity analytical grade. Unless mentioned otherwise, commercial reagents, enzymes, and coenzymes were supplied from Sigma Chemical Co., St. Louis, Mo. All gases used were purchased from Air Liquide, Paris, France.

Organism and medium. C. cellulolyticum ATCC 35319 was isolated from decayed grass by Petitdemange et al. (36). Stocks of spores from the original isolation, stored at 4°C, were heated to 80°C for 10 min and inoculated on cellulose medium (28, 51). Cells were subcultured once on cellobiose or cellulose before transfer and growth in a bioreactor as previously described (18). The anaerobic culture technique used was that proposed by Hungate (23) as modified by Bryant (6).

Growth conditions. C. cellulolyticum was grown in batch culture with cellobiose or cellulose as the sole carbon and energy source as previously described by Guedon et al. (18). Bacteria were cultured aseptically in a 2-liter bioreactor (LSL Biolafitte, St. Germain en Laye, France) with a 1.5-liter working volume. The temperature was maintained at 34°C, and the pH was controlled at 7.2 by automatic addition of 3 N NaOH. Agitation was kept constant at 50 rpm. The inoculum was 10% by volume from an exponentially growing culture. Cellulose bioreactors were connected to a gasometer filled with a saturated NaCl-water solution and acidified at pH 1.0 with H₂SO₄ to prevent gas dissolution as described by Pollack (38). All tubing was made of Viton to preserve the anoxic condition of the cell culture.

Analytical procedures. Cell growth on cellobiose was monitored spectrophotometrically at 600 nm and calibrated against cell dry weight measurement as previously described (18). On cellulose, biomass was estimated by bacterial protein measurement (33). From bacteria growing on cellobiose a cell dry weight-protein correlation was established, and this correlation was assumed to be the same for cells grown on particulate cellulose. Protein was measured by a modification of the Bradford dye method (5) as follows. A sample (30 ml) was centrifuged (8,000 × g for 15 min at 4°C) and washed twice with 0.9% (wt/vol) NaCl. The pellet was resuspended in 2 ml of 0.2 N NaOH, and this suspension was placed in a boiling water bath for 10 min (50). After cooling, the hydrolyzed sample was centrifuged as described above and the supernatant was diluted in 0.2 N NaOH as well as crystalline bovine serum albumin, which was used as the standard. The protein concentration was then estimated using the Coomassie brilliant blue reagent and reading the absorbance at 595 nm.

Calculations. The main products of cellulose fermentation by C. cellulolyticum were acetate, ethanol, lactate, H₂, and CO₂ (see Results). Balance equations were established taking into account previous investigations (20, 34) and compiled in Fig. 1. Since cellodextrins are water-soluble -1,4 oligomers of glucose with degrees of polymerization (n) between 2 and 7 (35) it was assumed that carbohydrates from glucose to celloheptaose could potentially be incorporated by bacteria. The cellulose fermented into products that can then be expressed as n hexose equivalents (hexose eq), which correspond to the glucose residue inside the cellulose chain. As specified in the scheme of cellulose catabolism (Fig. 1), it was assumed that (i) two ATP molecules were consumed for the transport system (42), (ii) one molecule of glucose and (n  1) molecules of glucose-1-phosphate were formed from one molecule of soluble -glucan (n) (1  n  7) according to the model of Strobel (44), (iii) the net ATP formation from glucose to pyruvate via the Embden-Meyerhof-Parnas (EMP) pathway was two molecules of ATP, (iv) a balance of three molecules of ATP was produced by the EMP pathway from glucose-1-phosphate, (v) there was production of two extra ATP molecules per hexose equivalent by acetate kinase, (vi) two NAD⁺ molecules per hexose eq were reduced by GAPDH (glyceraldehyde-3-phosphate dehydrogenase), (vii) two molecules of NAD⁺ per hexose eq were formed by the lactate dehydrogenase, and (viii) four molecules of NADH per hexose eq were reoxidized by acetaldehyde dehydrogenase and alcohol dehydrogenase. Then the cellulose conversion can be written as a general equation as follows. The equation for the conversion to acetate is

(1)

The equation for the conversion to ethanol is

(2)

And the equation for the conversion to lactate is

(3)

The energetic yield of biomass (Y_ATP) was estimated from acetate, ethanol, and lactate concentrations. From equation 1, when a molecule of acetate was formed, (5n  3)/2n molecules of ATP were produced. For example, if n = 1 (glucose), then 2 acetate molecules and 2 ATP molecules are produced, i.e., 1 ATP molecule per acetate molecule; if n = 2 (cellobiose), 4 acetate molecules and 7 ATP molecules are produced, i.e., 1.75 ATP molecules per acetate molecule; if n = 3 (cellotriose), 6 acetate molecules and 12 ATP molecules are produced, i.e., 2 ATP molecules per acetate molecule. From equations 2 and 3, when a molecule of ethanol or lactate was formed, (3n  3)/2n ATP molecules were produced. For example, if n = 1, no ATP molecules and either 2 ethanol molecules or 2 lactate molecules are produced, i.e., 0 ATP molecules per ethanol or lactate molecule; if n = 2, 3 ATP molecules and either 4 ethanol molecules or 4 lactate molecules are produced, i.e., 0.75 molecules of ATP per ethanol or lactate molecule; if n = 3, 6 ATP molecules and either 6 ethanol molecules or 6 lactate molecules are produced, i.e., 1 ATP molecule per ethanol or lactate molecule. So from n = 1 to 7 averages of 1.94 ATP molecules per acetate molecule and 0.94 ATP molecules per ethanol or lactate molecule were found and Y_ATP was estimated as follows: Y_ATP = biomass concn/(1.94 concn_acetate + 0.94 concn_ethanol + 0.94 concn_lactate) where concn stands for concentration and Y_ATP is expressed in grams of cells per mole of ATP produced.

View larger version (24K): [in this window] [in a new window]   FIG. 1.   Scheme of the catabolism of cellulose by C. cellulolyticum. Total cellulase activity liberated soluble -glucans (n, the number of hexose residues inside the polymer) (i.e., 1  n  7), which were then incorporated and metabolized by bacteria. 1, cellodextrin phosphorylase (EC 2.4.1.49); 2, cellobiose phosphorylase (EC 2.4.1.20); 3, glucokinase (EC 2.7.1.2); 4, phosphoglucomutase (EC 5.4.2.2); 5, L-lactate dehydrogenase (EC 1.1.1.27); 6, pyruvate-fd oxidoreductase (EC 1.2.7.1); 7, hydrogenase (EC 1.18.99.1); 8, NADH-fd reductase (EC 1.18.1.3); 9, phosphotransacetylase (EC 2.3.1.8); 10, acetate kinase (EC 2.7.2.1); 11, acetaldehyde dehydrogenase (EC 1.2.1.10); 12, Alcohol dehydrogenase (EC 1.1.1.1). CoA-SH, coenzyme A; ox, oxidized; red, reduced. Fd, ferredoxin.

	RESULTS

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Cellulose degradation by C. cellulolyticum in batch cultures. C. cellulolyticum was grown in batch culture on synthetic medium at pH 7.2 with seven concentrations of cellulose MN301: 5.6, 14.8, 24.1, 41.4, 78.4, 116.7, and 179.6 mM, expressed as hexose eq, i.e., 0.9, 2.4, 3.9, 6.7, 12.7, 18.9, and 29.1 g of cellulose liter¹, respectively. The percentage of solubilized cellulose within 120 h as a function of the initial cellulose concentration was determined (Fig. 2), and it was found that around 91% degradation was achieved with less than 3.9 g of initial cellulose liter¹, but this percentage dropped and reached 21% with the highest cellulose concentration. Such a decrease indicated that approximately the same amount of cellulose was hydrolyzed so that cellulolysis performances were close to their maximum at and above 3.9 g of cellulose added liter¹. In fact, the maximum dry weight of cells increased with initial cellulose amount, and above 6.7 g liter¹ it remained quite constant around 575 mg liter¹ (Fig. 2). The maximum rate of cellulose degradation measured in the course of each fermentation paralleled the maximum cell dry weight and reached about 1.5 × 10³ g liter¹ min¹ at and above 6.7 g of cellulose added liter¹. The rate of cellulose hydrolysis appeared therefore to be related to biomass production, and this partly explained why the percentage of degradation was restricted above 3.9 g of cellulose added liter¹.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   The percentage of solubilized cellulose , maximum biomass , and maximum rate of cellulose degradation  reached within 120 h of batch fermentation of C. cellulolyticum as a function of the initial cellulose concentration.

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View larger version (21K): [in this window] [in a new window]   FIG. 3.   Kinetics of cellulose consumption (a) and cellulase activity in the pellet (b) and in the culture supernatant (c) during batch fermentation of C. cellulolyticum on cellulose at various initial concentrations.

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Metabolite production during batch cellulose fermentation. With increasing amounts of substrate and within 120 h of fermentation, the consumed cellulose concentration, expressed as millimolar hexose eq, rose (Fig. 4a). Above 41.4 mM initial cellulose (i.e., 6.7 g liter¹), the increase in the quantity of consumed cellulose slowed down and the concentration reached 36.8 mM with 179.6 mM initial cellulose (i.e., 29.1 g liter¹). The production of biomass paralleled the amount of hexose eq consumed. Substrate limitation or inhibition by fermentation products could not explain growth arrest since the same culture, when reinoculated, allowed further growth of a new cell inoculum (see Fig. 6aII).

View larger version (15K): [in this window] [in a new window]   FIG. 4.   Maximum biomass and cellulose consumption (a), metabolite concentrations (b and c), and product ratio (d) obtained within 120 h of batch culture of C. cellulolyticum as a function of the initial cellulose concentration. , biomass; , consumed cellulose as hexose eq; , ethanol; , acetate; , H2; , CO2; , lactate; , extracellular pyruvate; , ethanol/acetate ratio; , H2/CO2 ratio.

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Kinetic analysis of fermentation products in batch culture on cellulose. When C. cellulolyticum was grown in batch culture on a defined medium with 179.6 mM initial cellulose (i.e., 29.1 g of cellulose liter¹), the specific production rates of acetate, ethanol, CO₂, and H₂ accelerated during the first 60 h after inoculation, and this acceleration was followed by decreasing specific production rates (Fig. 5a). During the first 60-h period, the specific rate of lactate production was almost constant and low: around 0.07 mmol (g of cells)¹ h¹ (Fig. 5b). The peak of extracellular pyruvate formation, i.e., 78.60 µmol (g of cells)¹ h¹, coincided with the start of lactate production. The extracellular q_pyruvate decreased continuously, and pyruvate was consumed about 50 h after its maximum production rate was reached. The increasing q_lactate, with a maximum of 1.17 mmol (g of cells)¹ h¹, corresponded to the decrease in the specific rates of acetate, ethanol, CO₂, and H₂ production.

View larger version (27K): [in this window] [in a new window]   FIG. 5.   Specific rates of product formation during batch fermentations of C. cellulolyticum. The initial cellulose concentrations were 179.6 mM (i.e., 29.1 g liter1) (a and b) and 24.1 mM (i.e., 3.9 g liter1) (c and d). , , , , , and , specific rates of ethanol, acetate, lactate, H2, CO2, and extracellular pyruvate formation, respectively.

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Cellulose degradation in a bioreactor reinoculated with C. cellulolyticum. With substrate concentrations higher than 6.7 g of cellulose liter¹, cell growth was not limited by available cellulose sites and the growth arrest of the seeding could be explained by a carbon flow which led to an accumulation of an intracellular inhibitory compound(s) (18). Indeed the rate of cellulose catabolism apparently exceeded the rate of pyruvate consumption, since pyruvate accumulated. Taking into account this hypothesis, a reinoculation process to improve the degradation of high cellulose concentrations was attempted. By this process, when the cells entered the stationary phase (i.e., about every 96 h), a new inoculum of C. cellulolyticum was introduced into the bioreactor (Fig. 6). In this way, with 29.1 g of initial cellulose liter¹ (i.e., 179.6 mM cellulose) more than 95% degradation occurred within 18 days (i.e., four reinoculations) against 45% in classical batch fermentation. In the reinoculated culture, the final concentrations of ethanol and acetate were 65.6 and 90.5 mM, respectively (Fig. 6bII) compared to 11.4 and 40.6 mM, respectively, in classical culture (Fig. 6bI). The final lactate concentration (77.3 mM), however, was lower than that in classical batch culture (93.1 mM). A final cell dry weight of 2,175 mg liter¹ was reached with the reinoculation process compared to about 580 mg liter¹ with the classical procedure (Fig. 6aI and 6aII). In classical batch fermentation, once the stationary phase was reached, bacteria acted as resting cells (Fig. 6aI): cellulose was hydrolyzed, cell lysis did not occur within the remaining 14 days, and bacteria were catabolically active since metabolites, mainly lactate, were produced. These data indicated that acetate and ethanol production was associated with cell growth whereas lactate formation paralleled growth inhibition. With the fourth inoculum, the cellulose was not degraded further (Fig. 6aII). In fact, no growth was observed, and, at this time, the cellulose concentration was less than 2 g liter¹; the remaining cellulose was certainly saturated with bacteria coming from the preceding inocula, which were not able to initiate a new cell division and which did not allow adhesion and growth of cells from a new inoculum. Thus such a reinoculation process allowed improved maximum dry weight of cells, cellulose solubilization, and end product concentrations.

View larger version (23K): [in this window] [in a new window]   FIG. 6.   Growth and residual cellulose concentration (a) and product concentrations (b) during classical batch fermentation (I) and reinoculated culture (II) of C. cellulolyticum with 29.1 g of cellulose liter1. Arrows, reinoculation times. , biomass; , cellulose; , ethanol; , acetate; , lactate.

	DISCUSSION

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Since the early studies of cellulose hydrolysis by C. cellulolyticum, this microorganism has been considered a sluggish cellulolytic bacterium (16, 37). It was described as taking half a month to attain about 70% degradation with initial cellulose concentrations lower than 7.6 g liter¹ (16). Moreover, limitation of cellulolysis was attributed to a change in cellulose structure, such as a progressive increase in the lattice crystallinity due to the initial degradation through the cellulasic system. As a result, the kinetics of cellulose degradation could be divided into three distinct periods corresponding to three consecutive enzymatic activity levels.

In the present study, cellulolytic performance of C. cellulolyticum was improved, since with initial cellulose concentrations less than 6.7 g liter¹ more than 85% degradation occurred in 5 days. A modification of the crystalline structure of the substrate could not account for the slowdown in the cellulose degradation rate. Changes in the distribution of the cellulase activity (i.e., present on the cells or as a free cellulasic system) between the cellulose fibers and the supernatant explain the slowing down in the cellulose solubilization during the culture. Studies of the fermentation of different cellulose structures by ruminal cellulolytic bacteria indicated that the available surface area was a more important determinant of the digestion rate than the crystallinity of the cellulose (48, 49). Recent investigations of crystalline cellulose degradation by the Clostridium thermocellum cellulosome showed that the various cellulase factors acted in unison with a very efficient synergism in which the influence of digestion time on the crystallinity of the substrate was limited (4). In the present investigation, the maximum generation time was greatly reduced (i.e., 12.4 h versus the 24.0 h found by Giallo et al. [16]) and primary metabolite production, i.e., of acetate, ethanol, and lactate, evolved differently (16). These differences could be attributed to several modifications made in the present study such as the use of a synthetic medium (19, 34), the pH regulation in the course of fermentation (11), the continuous stirring of the medium (12, 31), and the fact that the cultures were carried out at atmospheric pressure without accumulation of the fermentation gases inside the bioreactor (12, 27).

With initial cellulose concentrations lower than 41.4 mM (i.e., 6.7 g liter¹), the more the cellulose was hydrolyzed the less the cellulasic system could find new adherence sites on the cellulose fibers. As the initial cellulose amount increased, the biomass production increased and in turn the total culture cellulase activity rose globally but more activity was measured in the supernatant at the end of the fermentation. Since the stationary phase occurred before cellulose was depleted, biomass limitation was more probably due to a lack of available adhesion sites on cellulose as bacteria grew (13, 14). Thus, with less than 41.4 mM cellulose (i.e., 6.7 g liter¹), cellulolysis was limited by both the accessibility of the cellulasic system to cellulose fibers and, as a result, the biomass concentration, which depends on the quantity of soluble cellodextrins released. In this range of cellulose concentrations, the ethanol-to-acetate ratio increased continuously towards 1 and concomitantly less H₂ was produced. So H₂ and ethanol production may regulate the intracellular NADH/NAD⁺ ratio as a result of a good control of the electron flow (18, 19). Likewise, carbon flow appeared to be correctly regulated since pyruvate overflow did not occur. Moreover, with increasing substrate concentration the flux, which was first mainly directed to acetate production, was partly reoriented to the ethanol pathway, while little lactate was formed. Y_X/S and Y_ATP values were comparable to those obtained on cellobiose even though q_hexose was at least five times lower. In these conditions, cells managed to optimize their growth on cellulose.

With higher initial cellulose concentrations, above 41.4 mM (i.e., 6.7 g liter¹), the increase of lactate production concomitant with the decrease of acetate and (mainly) ethanol biosynthesis showed the clear reorientation of the catabolism. The change was paralleled by an increase in the H₂/CO₂ ratio, which suggested that the intracellular NADH/NAD⁺ ratio was equilibrated through NADH-fd reductase and hydrogenase activities, which may compensate for the decrease of ethanol production (18-20). Moreover, cell dry weight did not increase, and most of the total cellulase activity remained on cellulose fibers. This biomass limitation could not be due to nutritional restriction or inhibitory fermentation metabolites since new cells reinoculated in the same culture were able to grow. The presence of extracellular pyruvate, though, means that the rate of cellulose catabolism exceeded the rate of pyruvate consumption. In this range of cellulose concentrations, as previously observed for cellobiose, these results suggest that the rate of hexose catabolism could exceed the rate of pyruvate consumption via pyruvate-fd oxidoreductase as well as anabolic pathways and may indicate that these enzymes are the rate-limiting step (19, 20). Values of Y_X/S and Y_ATP were both lower than values obtained with less than 41.4 mM initial cellulose (i.e., 6.7 g liter¹), indicating that an uncoupling growth phenomenon had occurred. Since this decrease also came with pyruvate overflow accompanied by lactate production, growth inhibition was certainly related to an accumulation of an intracellular inhibitory compound(s) due to a deficient regulation of the entering carbon, as suggested by results with cellobiose (18). Even at high cellulose concentrations, however, lower specific rates of product formation and consumption indicated that the metabolism was not deregulated as much as it was on cellobiose (Table 1).

Whatever the initial cellulose concentrations, low soluble sugar concentrations were detected in the supernatant. These results are in agreement with the concept that the depolymerization of insoluble substrate to soluble cellodextrins limits the cellulose fermentation (33). By estimating and comparing q_hexose as well as specific rates of product formation to those obtained with cellobiose batch cultures, the present investigation provides further evidence that this concept is well founded. As expected, the entering carbon flow and, as a result, the specific production rates with insoluble cellulose were lower than those with cellobiose, a soluble sugar. In addition, extracellular pyruvate was reconsumed earlier at low initial cellulose concentrations than at higher ones. These data showed that the conversion of insoluble carbohydrate to soluble cello-oligosaccharides was the rate-limiting step in cellulose fermentation, but even so, pyruvate overflow could occur.

The results from the reinoculated culture clearly indicate that the growth arrest observed in classical batch culture was not due to high end product concentrations and was not induced by signal molecules excreted by bacteria at a particular cell density or growth phase (1, 10). The reinoculation process may allow the bypassing of the cell density limitation, a priori, due to self-intoxication of the cell with time, and may then permit the further colonization of available sites on the cellulose fibers and, as a result, an increased cellulose solubilization.

Even if at high carbohydrate concentrations cellulose catabolism was similar to that observed on cellobiose in some previously described aspects, it remained clearly different especially at low cellulose concentrations. In accordance with the initial substrate concentrations used, great differences of product concentrations as well as of specific production rates were observed. In cellulose batch fermentations, the cells managed, therefore, to efficiently regulate their metabolism by reorienting the carbon and electron flows. The higher values of specific rates of product formation and consumption obtained in cellobiose batch cultures showed that metabolism was distorted compared to that for a substrate more closely related to the natural ecosystem of the bacterium.

Since the situation of a microorganism under natural conditions is most probably somewhere between the closed batch culture and the open continuous-culture system (24, 25), further investigations with chemostats should allow a better understanding of the C. cellulolyticum behavior in its ecological niche with its natural insoluble substrate, the cellulose.