INTRODUCTION

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Cellulolytic clostridia are of cardinal importance in anaerobic environments rich in plant material (4, 23, 42). For many years, the cellulase complex of cellulolytic clostridia and genes encoding cellulases have been the subjects of considerable research, which has led to the cellulosome concept (1-3, 12). The cellulosomes found at the surface of the cells, where they form protuberances, are responsible for the specific adherence of numerous cellulolytic clostridia to cellulose. They contain a multiplicity of enzyme components showing a marked synergism against cellulosic compounds. Thus, enzymes involved in degradation of cellulose and hemicellulose have been well characterized, while few studies have focused on the carbon metabolic pathway. Poor sugar consumption and an early inhibition of metabolism and growth have been documented (7, 10, 15, 18, 19, 27, 37, 38), and Clostridium cellulolyticum, a mesophilic cellulolytic bacterium isolated from compost (35), shows this behavior. Using yeast extract, Casamino Acids, and vitamin supplements, Giallo et al. (15) tried to improve C. cellulolyticum growth, but without success; nevertheless, complex media were used systematically for the cultivation of this organism (4, 7, 13-16, 34). However, the complex metabolism associated with the use of the numerous compounds included in the rich media is such that analyses of metabolism and energy use are difficult to undertake. Moreover, many natural ecosystems are oligotrophic and rarely contain all nutrients in high quantity (22). In light of these considerations, during this investigation a synthetic medium was used to study the behavior of C. cellulolyticum under conditions of nutrient limitation. To permit identification of regulatory responses occasioned by low nutrient concentrations, studies were conducted in chemostats, which can maintain low steady-state nutrient concentrations, using cellobiose as the carbon source, since this disaccharide is the major end product of the cellulose degradation process (32, 39).

	MATERIALS AND METHODS

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

Organism and medium. The bacterium used in this study, C. cellulolyticum ATCC 35319, was originally isolated by Petitdemange et al. from decayed grass (35). Stock cultures of C. cellulolyticum were maintained on cellulose and were grown for one transfer in cellobiose before initiation of growth experiments. The anaerobic culture technique used was that proposed by Hungate (20) as modified by Bryant (6).

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Growth conditions. C. cellulolyticum ATCC 35319 was grown in chemostat culture at varying dilution rates on cellobiose (5.84 mM) as carbon and energy source. Cultures were maintained 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 the automatic addition of 1 M NaOH. All tubing used was made of Viton (Du Pont Co., Wilmington, Del.) to eliminate oxygen entry. Agitation was kept constant at 50 rpm. Medium was pumped into the fermentor at the appropriate dilution rate. The volume was kept constant at 1.5 liters by automatic regulation of the culture level. The inoculum was 10% by volume and was in the exponential growth phase. The culture was grown in batch for 15 h before the medium flow was started.

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Analytical procedures. Bacterial growth was measured spectrophotometrically at 600 nm and calibrated against cell dry weight measurements. Samples (30 ml) were centrifuged for 10 min at 8,000 × g, washed with 0.9% (wt/vol) NaCl, and dried at 65°C to constant weight (48 h). A mean biomass formula of C₄H₇O₂N and an average extracellular protein formula of C₁₆H₂₅O₉N₆ were determined by elemental analysis (Service Central d'Analyses, CNRS, Vernaison, France) and were used for elemental recovery calculations.

Assay of glycolytic intermediates in cell extracts. Dihydroxyacetone phosphate (DHAP), glyceraldehyde-3-phosphate (GAP) and fructose-1,6-biphosphate (FBP) were extracted from a culture broth sample by HClO₄ by using the rapid system described by Thomas et al. (40). The average time between cells leaving the fermentor and mixing with HClO₄ was ca. 30 ms, and the final HClO₄ concentration was 0.6 M. Samples were held in the syringe with the needle closed by a septum for 2 min at room temperature. Extracts were placed in ice for 10 min under N₂ atmosphere before addition of 230 mg of K₂CO₃ and were finally neutralized with 3 M KOH. Extracts were centrifuged (10,000 × g, 4°C, 10 min), and supernatants were stored at 80°C until assayed.

Determination of nucleotide pools. Levels of nucleotides, ATP, ADP, NAD(P)⁺, and NAD(P)H in the biomass were measured by first extracting the nucleotides from a sample of culture. ATP and ADP were extracted with perchloric acid as described above for glycolytic intermediates.

Assay of extracellular pyruvate. The NADH fluorimetric assay (see above) was adapted for the measurement of extracellular pyruvate by using 0.1 M triethanolamine buffer (pH 7.6) and 12.5 µM NADH. Enzymatic determination of intracellular pyruvate levels was not possible due to significant interference with extracellular pyruvate, which lead to erroneous estimates of intracellular concentrations.

Preparation of cell extracts. Cell extracts were obtained as described previously (34). Protein concentrations of cell extracts were determined by the Lowry method (26) by using crystalline bovine serum albumin as the standard.

Enzyme assays. Fd-NAD(P)⁺ reductase, NADH-fd reductase, hydrogenase (EC 1.12.7.1), glyceraldehyde-3-P-dehydrogenase (GAPDH) (EC 1.2.1.12), acetate kinase (EC 2.7.2.1), alcohol dehydrogenase (EC 1.1.1.1) (ADH), and lactate dehydrogenase (EC 1.1.1.27) (LDH), activities were assayed at 34°C as described previously (34). Endo-1,4--glucanase (EC 3.2.1.4) activity was determined by the method of Miller et al. (30) with carboxymethylcellulose as the substrate.

Determination of the K_m values of LDH and PFO for pyruvate. Crude extract (5 ml) was dialyzed anaerobically against 5 liters of phosphate buffer (5 mM, pH 7.4) changed three times at 4°C to remove intracellular pyruvate and FBP. LDH activity was measured as described previously (34). To determine K_m and V_max of the LDH, the NADH concentration was held constant at 0.4 mM and the pyruvate concentrations were varied from 0.25 to 30.00 mM. Pyruvate-fd oxidoreductase (PFO) (EC 1.2.7.1) was assayed as described by Meinecke et al. (28) except that 1 mM methyl viologen was used as the artificial electron acceptor. The reduction of methyl viologen was measured at 570 nm, and to determine K_m and V_max of the PFO, the pyruvate concentrations were varied from 0.05 to 2.00 mM.

Calculations. According to Papoutsakis and Meyer (33), a stoichiometric balance equation for biomass formation from cellobiose can be written in the form:
where C₄H₇O₂N denotes the elemental composition of the biomass.

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	RESULTS

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Effect of the dilution rate on biomass and metabolite formation. C. cellulolyticum was grown under cellobiose limitation at proportions of culture volume replaced per hour (D) of 0.016 to 0.138 h¹, the highest D value at which a steady state could be attained (Table 1), since a maximum growth rate (µ_max) of 0.150 h¹ was calculated during the early exponential phase of batch growth. At a cellobiose concentration in the feed medium of 5.84 mM and over a wide range of low dilution rates, the residual cellobiose concentrations were low and increased only at a D of 0.138 h¹. These data are typical of a continuous culture carried out under carbon limitation. The dry weight increased between 0.016 and 0.120 h¹ and decreased markedly at 0.138 h¹, corresponding to cellobiose accumulation.

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View larger version (19K): [in this window] [in a new window]   FIG. 1.   Influence of the specific rates of pyruvate production in a cellobiose-limited continuous culture of C. cellulolyticum on specific rates of lactate production () and in vitro specific LDH activities () (a) and on specific rates of acetate () and ethanol production () (b).

Redox balances. The formation of biomass from cellobiose is stoichiometrically written as follows:
where C₄H₇O₂N denotes the elemental biomass composition and corresponds to a molecular weight of 101 g mol¹.

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Enzyme activities. Specific activities in extracts of pelleted cells were measured at each steady state, and the influence of the growth rate on the specific activities of the enzymes is shown in Table 4. In vitro, GAPDH, lactate dehydrogenase, ethanol dehydrogenase, and acetate kinase activities were higher under conditions giving higher in vivo specific production rates. From the lowest (0.016 h¹) to the highest (0.138 h¹) D values, GAPDH increased 4.4-fold, LDH increased 5.3-fold, ADH increased 7.9-fold, and acetate kinase increased 1.8-fold.

View larger version (16K): [in this window] [in a new window]   FIG. 2.   Relation between the specific rates of extracellular pyruvate diffusion and the specific rates of pyruvate production () coming from cellobiose catabolism and the specific rates of lactate production ().

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	DISCUSSION

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The results presented in this article give consistent information on the mechanisms used by C. cellulolyticum to regulate cellobiose catabolism. Previous work carried out by using complex medium have led to a bottleneck, since carbon flow was stopped by a high level of intracellular NADH (34). This study shows that changing from complex to synthetic medium had strong effects on the following: (i) specific cellobiose consumption, which was increased from 0.68 (for the complex medium) to 1.98 mmol (g of cells)¹ h¹ (for the synthetic medium), whereas the highest dilution rate at which a steady state could be attained was increased from 0.120 (for the complex medium) to 0.138 h¹ (for the synthetic medium) (in complex medium at a growth rate [µ] of 0.120 h¹, the dilution rate approaches the washout point since the dry weight of cells decreased by 78% of the maximal biomass [34] whereas in synthetic medium at a µ of 0.138 h¹, the biomass decreased by 35%); (ii) electron flow, since the NADH/NAD⁺ ratios were in the range 0.29 to 2.08 whereas ratios as high as 42 to 57 were observed on complex media (34). Clearly, these data indicate a better control of cellobiose catabolism by C. cellulolyticum on mineral salts medium than on complex medium.

When C. cellulolyticum was grown on complex medium, regardless of the dilution rate, cellobiose catabolism showed the same pattern, i.e., acetate was the main product, whereas the biosynthesis of ethanol and lactate was low (34). Conversely, on synthetic medium, this study reveals that formation of end products and their ratios can change within broad limits. These changes were not due to iron-limited culture media, which could have effects on the biosynthesis of iron proteins such as ferredoxin (25), PFO (43), and hydrogenase (9) and hence on the cellobiose metabolism, since we found that the growth of C. cellulolyticum was limited when the iron added to the medium was less than 0.2 mg liter¹ (data not shown).

With low D values (Fig. 3), when cellobiose flow and hence ATP synthesis were limited, pyruvate was preferentially cleaved to acetate and ethanol. This enabled the bacteria to maximize formation of ATP. The high percentage of acetate formation versus the low ethanol production found at low growth rates under cellobiose limitation proved that C. cellulolyticum maintained its redox balance via the efficient NADH-fd reductase activity, as corroborated by an H₂/CO₂ ratio greater than 1. At increased D values, there was a large effect on ethanol and lactate production, which approximately coincided with pyruvate accumulation (Fig. 4). The level of lactate production increased sharply when the q_cellobiose reached approximately 1.34 mmol (g of cells)¹ h¹, values which were never obtained on the complex medium, since the highest value reported was 0.68 mmol (g of cells)¹ h¹ (34).

View larger version (27K): [in this window] [in a new window]   FIG. 3.   Scheme of the carbon and electron flow distribution at a D of 0.033 h1. Only the major flows are indicated in the direction of the arrows. Carbon and electron flows are symbolized by thick and thin lines, respectively. Enzymes (indicated by circled numbers): 1, GAPDH; 2, PFO; 3, phosphotransacetylase; 4, acetate kinase; 5, acetaldehyde dehydrogenase; 6, ADH; 7, hydrogenase; 8, Fd-NADP+ reductase; 9, NADH-fd reductase.

View larger version (30K): [in this window] [in a new window]   FIG. 4.   Scheme of the carbon and electron flows distribution at a D of 0.138 h1. Notation is the same as in the legend for Fig. 3, except that the lactate dehydrogenase is numbered 10.

Pyruvate overflow means that PFO could no longer support the flow arriving at pyruvate from the catabolism of cellobiose. Under these conditions, electron flow from glycolysis was balanced chiefly by ethanol and lactate production; the NADH-fd reductase was not active since the H₂/CO₂ ratio was near 1. This indicates that the NADH-fd reductase activity under low rates of cellobiose consumption (Fig. 3) and ADH and LDH activities under high rates of cellobiose consumption (Fig. 4) were able to maintain the redox balance of C. cellulolyticum; the increase in the NAD⁺ and NADH content with the D values could be correlated with the increase in the levels of the three NAD⁺ oxidoreductases GAPDH, ADH, and LDH.

We must also take into account the fact that on synthetic medium, cellobiose carbon can be converted into cell mass and used in catabolism to produce ATP, while on complex medium, yeast extract carried many cell building blocks and cellobiose carbon served mainly as an energy supply. Since the same µ_max of 0.150 h¹ was found in both media, it is clear that the uptake of nutrients or the generation of biosynthetic precursors occurs faster than the utilization of these precursors for biomass production. This interpretation is confirmed by the fact that numerous precursors were found in the synthetic culture medium. The fact that lactate production occurred only in conditions of pyruvate overflow avoids competition between the lactate pathway and (i) PFO, which maximizes ATP formation via acetate production, and (ii) anabolism, since pyruvate is also the precursor of compounds such as alanine, valine, and leucine.

We can suppose that in natural environments, C. cellulolyticum will rarely find nutrient substances in high concentrations and that the complex medium with high concentrations of substrate may be unfavorable to C. cellulolyticum, which was unable to deal with a surfeit of substrates; under these conditions, the nutrients or products of its metabolism, such as were demonstrated for NADH (34), may accumulate intracellularly to toxic levels. It can be argued that during the course of evolution, C. cellulolyticum has evolved to optimize cellobiose catabolism and nitrogen anabolism under nutrient-poor conditions.

This study of synthetic medium with cellobiose has permitted the identification of regulatory responses of C. cellulolyticum and serves as a basis for additional work using bacteria growing on cellulose. Much remains to be learned about the physiology of these bacteria on insoluble substrates.