INTRODUCTION

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Clostridium cellulolyticum, a strictly anaerobic cellulolytic bacterium, was isolated from decayed grass (18) and degrades cellulose by using a complex cellulolytic system called cellulosome (11). The cellulosome, wherein the cellulases were found to be organized into a high-molecular-weight, cellulolytic complex, has been extensively investigated (2, 27), while few studies have focused on the carbon metabolic pathway, mainly in C. cellulolyticum (5). Most studies of the carbon metabolism in cellulolytic clostridias have been performed with cellobiose, the major end product of the degradation process, which is taken up and assimilated by the cells (5, 16, 28). Our recent study (7) demonstrated that when C. cellulolyticum was grown in continuous cellobiose-limited culture, there was a shift from an acetate-ethanol fermentation at low levels of carbon flow to a lactate-ethanol fermentation at high catabolic rates; in addition, increasing levels of pyruvate in the extracellular medium were detected as the dilution rate increased. The pyruvate overflow suggested that the carbon flow through glycolysis was higher than the rate of processing by pyruvate-ferredoxin oxidoreductase (7) (Fig. 1). Consequently, under such conditions, the rates of energy production in the catabolic pathway were not correlated with the anabolic energy requirements, i.e., more ATP was produced than was needed by the biosynthetic and maintenance demands (25). Furthermore, because lignocellulosic compounds usually contain high levels of carbon and a low levels of nitrogen, growth of C. cellulolyticum on these compounds would lead to an excess of energy, and energy-spilling reactions must be utilized. In addition, it is also necessary to overcome the potentially deleterious osmotic effects of the accumulation of surplus intracellular metabolites (19). There are different types of energy-consuming reactions selected by the bacteria during the course of evolution; for instance, (i) an overflow metabolism, wherein bacteria excrete or leak partially oxidized metabolites (29); (ii) metabolic shifts, where bacteria can change their end products and alter ATP production (30); (iii) futile cycles (15); and (iv) the synthesis of intra- and extracellular polysaccharides, which are energy-consuming processes and limit the carbon flow toward glycolysis and ATP production (20-22). In addition to pyruvate, extracellular polysaccharides were previously found to be secreted by C. cellulolyticum at high rates of cellobiose consumption (7), leading to the belief that carbon flow was regulated at the branch point of the glucose phosphate pools (Fig. 1). This kind of regulation is further suggested by the fact that growth of C. cellulolyticum was inhibited in the presence of excess carbon (8). The aim of the present study was to investigate how changes in catabolic flux affected (i) the intracellular turnover of glucose 1-phosphate (G1P) and glucose 6-phosphate (G6P) and (ii) the distribution of the carbon fluxes inside and outside the cells.

View larger version (26K): [in this window] [in a new window]   FIG. 1.   Estimation of flux distribution within the central metabolic pathways based on the steady-state kinetic data from continuous culture of C. cellulolyticum grown on cellobiose at four dilution rates (0.013, 0.036, 0.066, and 0.115 h1). Fluxes are calculated according to the values in Table 1 and Table 2. All fluxes are expressed as meqC per gram of cells per hour and are indicated by a thin line. Specific rates: 1, cellobiose consumption; 2, cellobiose toward cellotriose formation; 3, cellobiose toward G1P and G6P formations; 4, G1P formation; 5, cellobiose toward G6P formation; 6, G1P toward cellotriose formation; 7, G1P toward exopolysaccharide formation; 8, pyruvate formation; 9, G6P toward biosyntheses; 10, G6P formation; 11, G1P through phosphoglucomutase activity; 12, G1P toward glycogen cycle; and 13, cellotriose formation.

	MATERIALS AND METHODS

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Chemicals. All chemicals were of highest-purity analytical grade. Unless stated 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, C. cellulolyticum ATCC 35319 used in this study was originally isolated by Petitdemange et al. (18) from decayed grass. 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 of Hungate (10), as modified by Bryant (3), was used.

Growth conditions. C. cellulolyticum ATCC 35319 was batch grown and grown in chemostat culture at various dilution rates as described previously (7, 8).

Analytical procedures. Bacterial growth, biomass, extracellular proteins, amino acid composition, ammonia, acetate, lactate, ethanol, and exopolysaccharides were determined as described previously (7).

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Assay of metabolic intermediates in cell extracts. G1P, G6P, fructose 6-phosphate (F6P), 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. (30) and Guedon et al. (7).

Determination of adenylate pools. ATP, ADP, and AMP were extracted with perchloric acid as described above for metabolic intermediates. ATP levels were measured by a luminescence assay by using a luciferin-luciferase system (Microbiol Biomass Test Kit; Celsis Lumac, Landgraaf, The Netherlands). ADP was converted to ATP in a reaction mixture containing 2 ml of 14 mM phosphocreatine in glycine buffer (0.1 M, pH 9.0), 0.4 mM MgSO₄, and 4 U of creatine phosphokinase from rabbit muscle (EC 2.7.3.2); after 15 min at 38°C for the conversion of AMP to ATP, 5 U of myokinase from rabbit muscle (EC 2.7.4.3) was added in the same mixture. Reactions were stopped by heating (100°C) for 3 min, and the mixtures were centrifuged (8,000 × g, 4°C, 15 min). ADP and AMP were determined by calculating the difference.

Preparation of cell extracts. Cells were centrifuged (12,000 × g, 15 min, 0°C), and pellets were rapidly frozen with liquid nitrogen and stored at 80°C.

Enzyme assays. All assays were performed at 34°C. The specific activities were determined in a range in which linearity with protein concentration was established.

Calculations and nomenclature. The main products of cellobiose fermentation by C. cellulolyticum were acetate, ethanol, lactate, H₂, and CO₂ (5).

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	RESULTS

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Glycogen synthesis in C. cellulolyticum grown in batch culture in the presence of carbon and nitrogen excess. After about 20 h of incubation in batch culture, cells entered the stationary phase, although neither carbohydrate nor NH₄⁺ was exhausted (Fig. 2a). Intracellular polysaccharide biosynthesis appeared to be associated with the vegetative growth (Fig. 2b); it reached a maximum of almost 40 mg g of cells¹ during the exponential growth phase. Then, after about 9 h, a gradual utilization of this polysaccharide took place even in the medium which contained cellobiose. This intracellular polysaccharide was obtained after KOH-ethanol extraction and was characterized by use of enzymatic digestion and comparison with several commercial preparations. Only glucose appeared on a thin-layer chromatography plate after the polysaccharide was digested with amyloglucosidase (data not shown). Partial hydrolysis occurred with -amylase or isoamylase, indicating a polyglucan containing -1,4 and -1,6 glucosidic linkages. Thus, it appeared to be glycogen.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Glycogen synthesis in C. cellulolyticum grown in batch culture in the presence of carbon and nitrogen excess [cellobiose, 23.4 mM; (NH4)2SO4, 26.5 mM]. (a) Optical density at 600 nm (), residual cellobiose (), and ammonia () concentration. (b) Glycogen biosynthesis.

Glycogen synthesis in C. cellulolyticum grown in a chemostat. In order to determine the precise conditions where glycogen was stored in C. cellulolyticum, its production was measured during continuous cultures. Figure 3a shows the effect of dilution rate on glycogen formation in cells grown under nitrogen limitation (NH₄⁺, 4.00 mM) and cellobiose excess (14.62 mM), which is usually the best condition for glycogen storage. Glycogen was synthesized during all growth rates, ranging from 20.6 to 53.4 mg of glucose eq g of cells¹. Similar results were obtained both under carbon limitation and nitrogen excess (Fig. 3b) or carbon and nitrogen excess (Fig. 3c). Further, the usual condition, i.e., carbon excess (14.62 mM) and nitrogen limitation (4.00 mM) was used to study the glycogen function in the distribution of the carbon flux in C. cellulolyticum.

View larger version (13K): [in this window] [in a new window]   FIG. 3.   Effect of dilution rate on glycogen formation by C. cellulolyticum. Growth conditions: a, cellobiose excess (14.6 mM) and ammonia limitation (4.0 mM); b, cellobiose limitation (5.8 mM) and ammonia excess (15.1 mM); c, cellobiose excess (14.6 mM) and ammonia excess (15.1 mM). Symbols represent the averages from three different chemostats, and the values were determined with an average accuracy of ±10%.

Effect of dilution rate on biomass and metabolite formation. C. cellulolyticum was grown in continuous culture over a wide range of dilution rates (D = 0.013 to 0.115 h¹) with 14.62 mM cellobiose as the carbon source and 4.0 mM ammonia as the nitrogen source (Table 2). At this concentration, cellobiose was always in excess, and residual cellobiose concentrations were in the range of 6.44 to 12.11 mM. Ammonia was limiting since, over a wide range (0.013 to 0.082 h¹) of low dilution rates, the ammonia concentrations were less than 0.03 mM; at >0.082 h¹, the concentration increased and was found to be 2.20 mM at a D value of 0.115 h¹. These data are typical of a continuous culture carried out under nitrogen limitation.

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Specific consumption and production rates. Data on the effect of D on cellobiose consumption and product formation are shown in Fig. 4. The rate of cellobiose consumption varied from 7.01 to 26.72 meqC g of cells¹ h¹ with increasing growth rate (Fig. 4a), while 0.36 to 1.95 meqC g of cells¹ h¹ were converted into biomass, proteins, and amino compounds (Fig. 4b). Cellobiose catabolism, leading to the end product formation via pyruvate, increased from 4.64 to 15.12 meqC g of cells¹ h¹ with increasing growth rate (Fig. 4c). Below a D of 0.052 h¹, the specific rate of cellotriose was stable and at levels above this value decreased sharply (Fig. 4d), concomitantly with the increase of the extracellular polysaccharide formation (Fig. 4e), indicating a switch in the carbon flux. The specific production rate of glycogen increased with dilution rate from 0.48 to 5.32 meqC g of cells¹ h¹ (Fig. 4f).

View larger version (20K): [in this window] [in a new window]   FIG. 4.   Effect of dilution rate on specific rates: a, rate of cellobiose consumption (qcellobiose); b, rate of biosynthesis (qbiosynthesis); c, rate of pyruvate formation (qpyruvate); d, rate of cellotriose formation (qcellotriose); e, rate of polysaccharide formation (qpolysaccharides); and f, rate of glycogen formation (qglycogen). Values are calculated as explained in Table 1 and Fig. 1 and are expressed as the meqC per gram of cells per hour.

Intracellular hexose-phosphate and adenylate pools of continuous steady-state cultures of C. cellulolyticum. Previous work indicated that C. cellulolyticum was unable to regulate its cellobiose consumption (8); this explains the large amounts of G1P and G6P detected inside the cells (Table 3). G1P concentrations were found to be higher at high dilution rates, whereas the highest values of G6P were found at low dilution rates (Table 3). This could result from the decrease of the carbon flux through the phosphoglucomutase from 27 to 14.3% of the original carbon with the increase in D (Fig. 1). Moreover, no significant variation between the dilution rate and in vitro intracellular levels of F6P and FBP could be detected (data not shown).

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View larger version (17K): [in this window] [in a new window]   FIG. 5.   Effect of dilution rate on G6P turnover () and G1P turnover () (a) and on glycogen turnover (b). The flux values are calculated as explained in Table 1 and Fig. 1; the pools of G1P and G6P are given in Table 3, and the glycogen pools are obtained from the intracellular concentrations reported in Table 2. Symbols represent the averages from three different chemostats, and the values are determined with an average accuracy of ±10%.

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Synthesis of the key enzymes of glycogen biosynthesis and mobilization. The measurements of the total cellobiose cleavage activities (cellobiose phosphorylase and -glucosidase) were not significantly different from the cellobiose phosphorylase activity itself. On the basis of these results, cellobiose phosphorylase and -glucosidase activities were found at ca. 28 and 2 nmol min¹ mg of proteins¹, respectively, and it appears that C. cellulolyticum uses the phosphorylase for the cellobiose catabolism.

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	DISCUSSION

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This study describing the carbon fluxes in C. cellulolyticum growing on cellobiose in chemostat cultures with ammonia as the growth-limiting nutrient shows that whatever the specific growth rate, carbon flux into the cells exceeds the fluxes needed for biosynthesis of cell monomers and to sustain reactions leading to energy production. Consequently, ca. 30% of the original specific rate of cellobiose uptake by C. cellulolyticum was converted to cellotriose, glycogen, and polysaccharides rather than being used for generation of metabolic energy and biosynthetic precursors.

The data from the flux analyses are reported as a function of the growth rate in Fig. 1. It is clear that G1P and G6P connected by phosphoglucomutase constitute important branch points for distributing the carbon fluxes inside and outside the cells.

The regular increase of the fluxes through steps 1, 3, 5, and 10 (Fig. 1) contrasts with the flux through the phosphoglucomutase (step 11), which reached a limitation level at D = 0.066 h¹. The steady-state concentrations of the G6P and G1P corroborate the analysis of the carbon flux, since at low dilution rates the ratio G6P/G1P is 8.2, whereas at the high dilution rate the ratio is 0.08. This suggests that the enzyme is limiting at high dilution rates but not at low ones. The limitation of the path through the phosphoglucomutase had a great effect on the accumulation of G1P, the precursor of cellotriose, exopolysaccharides, and glycogen. The biosynthesis of these compounds are important since as much as 16.7, 19.9, and 14.9% of the specific rates of cellobiose consumed were used to supply specific rates of cellotriose, glycogen, and polysaccharide production, respectively.

One of the features of microbial phosphorylases is their ability to synthesize oligosaccharides (1); cellobiose serves as a glucosyl acceptor, and G1P serves as a glucosyl donor. In C. cellulolyticum at low growth rates, when the flux through glycolysis was low, only 88.8% of the intracellular cellobiose was used through the glycolysis and, hence, 11.2% was used to supply the specific rate of cellotriose biosynthesis. This biosynthesis was efficient and led to a high value of the G1P turnover. With the increase of the dilution rate, the flux of cellobiose through step 3 increased from 88.8 to 99.14%, whereas its flux through the step 2 decreased, thus explaining the drop in the cellotriose excretion.

In batch culture, glycogen in C. cellulolyticum reached a maximum during the exponential growth phase. This result is in contrast to many bacterial species which synthesize glycogen during a limited growth period at the outset of the stationary phase (20-22). Our chemostat studies demonstrate that glycogen was synthesized during all growth rates in carbon- and nitrogen-sufficient media, in carbon excess and nitrogen limitation, as well as in carbon limitation and ammonia excess; in these three cases, similar quantities of glycogen were detected. Similarly, the synthesis of glycogen in C. cellulolyticum was not subject to stimulation by nutrient limitation. In contrast, in many bacterial species glycogen accumulates when growth is limited, e.g., by phosphorus, sulfur, or nitrogen in the presence of an excess of a carbon source (20-22). These data, along with the fact that glycogen phosphorylase activity and glycogen content were measured throughout the growth cycle, suggest that glycogen was simultaneously synthesized and degraded during cell growth. Continuous culture experiments confirmed that high glycogen turnover in C. cellulolyticum, i.e., glycogen accumulation and degradation, was associated with rapid growth and hence with a high carbon flux. Such glycogen turnover would limit the flux of carbon through glycolysis, avoiding the potentially deleterious effects of generating high concentrations of intracellular metabolites.

Whatever the dilution rate, the values of the adenylate energy charge were high, indicating that the inefficiently regulated carbon flow also led to an excess of energy, which is consumed by the biosynthesis of cellotriose and exopolysaccharides, as well as by the turnover of the intracellular glycogen.

From this study and previous results it appears that pyruvate-acetyl coenzyme A are important branch points for regulating the redox balance of the carbon catabolism, whereas the properties of the G1P-G6P branch points have been selected to control excretion of carbon surplus and to dissipate excess energy.

Regulations at these last branch points are needed since C. cellulolyticum is not able to regulate cellobiose consumption (8); nevertheless, they are imperfect since overflow of pyruvate occurred at the second branch point. In other words, the G1P-G6P branch points carry out a coarse control of the carbon flux and the pyruvate-acetyl coenzyme A branch points to a fine control beside the regulation of the redox balance.