INTRODUCTION

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Cellulose is of cardinal importance in the global carbon cycle: it accumulates in the environment due to its durable nature (5), and the main final products released during its fermentation are CH₄ and CO₂ (76). Bacteria are the major cellulose hydrolyzers in anaerobic cellulosic microbiota (35, 67), where cellulolytic clostridia play a key role (34).

The cellulose degradation process which occurs through cellulases has been studied extensively on cellulolytic clostridia, leading to the cellulosome concept (4, 6). The multienzymatic complexes found at the surface of the cells are responsible for adhesion of bacteria to cellulose fibers and allow a very efficient synergism of action of the different enzyme components (8). Genes encoding cellulases as well as the mechanism of action of the cellulosome are the subject of considerable research, while few studies have focused on the metabolic aspects of cellulose digestion by clostridia (27, 40).

Recent characterization of the carbohydrate catabolism of Clostridium cellulolyticum, a nonruminal mesophilic bacterium able to degrade crystalline cellulose, showed that (i) better control of catabolism occurred on a mineral salt-based medium (24, 48), (ii) carbon-limited and carbon-sufficient chemostats displayed major differences in regulatory responses of the carbon flow (25), and (iii) in nitrogen-limited conditions, glucose 6-phosphate (G6P) and glucose 1-phosphate (G1P) branch points play an important role in carbon flux divergence (22). These investigations, however, were performed with cellobiose, which is one of the soluble cellodextrins released during cellulolysis (56). In such investigations, the use of soluble sugars obviated the bacterial metabolic analysis on cellulose that was assumed difficult to undertake. Metabolic regulation processes found using cellobiose could differ or even be distorted from those with insoluble substrates.

While the first studies of cellulose focused mainly on C. cellulolyticum behavior, such as colonization or degradation with an insoluble substrate (19-21), recent investigations of cellulose fermentation in batch culture (12) have indicated that (i) metabolite yields depend strongly on the initial cellulose concentration and (ii) early growth arrest is linked to pyruvate overflow as in cellobiose batch culture (23).

In the last decade, efficient continuous-culture devices for growth on insoluble compounds have been developed (30, 31, 33, 37, 46, 63, 74) and used mainly to estimate the kinetics of cellulose degradation or colonization by various bacteria (1, 43, 58, 59, 71). Continuous culture is also a particularly useful and powerful tool for analyzing the physiology of microorganisms (42, 64).

The aim of this study was to investigate the carbon flow distribution and degradative characteristics of C. cellulolyticum when grown in mineral salt-based medium with cellulose, its natural substrate, in chemostat culture.

	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 obtained 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 originally isolated from decayed grass (52). Stocks of spores, stored at 4°C, were transferred to cellulose medium and heat shocked at 80°C for 10 min (12). Anaerobic cell cultures were subcultured once on cellulose before inoculation and growth in a bioreactor (12, 24). The defined medium used in all experiments was a modified CM3 medium (24) containing 0.37% cellulose MN301 (Macherey-Nagel, Düren, Germany).

Growth conditions. C. cellulolyticum was grown on cellulose as the sole carbon and energy source in a mineral salt-based medium. All experiments were performed in a 1.5-liter-working-volume fermentor (LSL Biolafitte, St. Germain en Laye, France). 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. Cells were grown in chemostat at various dilution rates, and each run was independent.

Analytical procedures. Biomass was estimated by bacterial protein measurement (46) using the Bradford dye method (10) as previously described (12).

Enzyme assays. Cells were centrifuged (12,000 × g, 15 min, 0°C), and pellets were rapidly frozen with liquid nitrogen and stored at 80°C. Cells were resuspended in Tris-HCl buffer (50 mM, 2 mM dithiothreitol [DTT] [pH 7.4]) and then sonicated four times for 20 s each with a break of 60 s at a frequency of 20 kilocycles s¹. The supernatant was collected from the cell lysate following centrifugation (12,000 × g, 20 min, 4°C). The protein content of extracts was determined by the method of Bradford (10), using crystalline bovine serum albumin as the standard. Anaerobic conditions were maintained throughout the entire procedure, and all manipulations were performed under oxygen-free nitrogen atmosphere. All enzyme assays were performed at 34°C. Specific activity was determined in a range where linearity with protein concentration was established. For calculation of enzymatic activity, the molar extinction coefficients used for 5,5'-dithiobis-(2-nitrobenzoic acid), methyl viologen, and NAD(P)H were 13.6 (15), 7.71 (50), and 6.22 mM¹ cm¹ (55), respectively.

Assay of intracellular compounds. NAD(P)⁺ and NAD(P)H were first rapidly extracted with HCl and KOH, respectively, as described by Wimpenny and Firth (75). Levels of coenzymes were determined by fluorimetric measurements as previously described (24). Other intracellular compounds were extracted from a broth sample by HClO₄, using a rapid extraction system (24).

Calculations. The metabolic pathways and equations for cellulose fermentation by C. cellulolyticum, expressed as n hexose equivalents corresponding to n glucose residues of the cellulose chain, were previously reported (12).

Mapping of the carbon flow. Distribution of the carbon flow was determined by adapting the model developed by Holms (28) to C. cellulolyticum metabolism. In the steady state, the flux through each enzyme of the known metabolic pathway was determined as specified in Table 1. Carbon fluxes were expressed in milliequivalents of carbon per gram of cells per hour.

Statistics. Statistical analysis of the data was performed following analysis of variance and Student t test (9) with Excel.

	RESULTS

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Cellulose digestion and biomass formation. Preliminary results in chemostats indicated that with between 2 and 6 g of cellulose liter¹ and at a dilution rate of 0.025 h¹, biomass formation increased proportionately to cellulose concentration (data not shown). C. cellulolyticum was then grown with 3.7 g of cellulose liter¹ at different steady-state combinations of Dequal to the microbial specific growth rate (µ)which ranged from 0.014 to 0.083 h¹ (Table 2); steady-state growth on cellulose was attained neither at D < 0.01 h¹ nor at D > 0.09 h¹ since washout then occurred. The latter value corroborated the maximum growth rate (µ_max) of 0.087 h¹ (i.e., a generation time of 8 h) estimated by following growth with the rate of tritiated thymidine incorporation into DNA during the exponential phase of batch growth on cellulose (20). Cell density was maximum at low D, i.e., 0.207 g liter¹ at 0.014 h¹, but slowly decreased with increasing D to reach 0.154 g liter¹ at 0.083 h¹ (Table 2). In all of the runs, microscopic examinations of the cultures revealed that almost all of the cellulose fibers were colonized by bacteria; the few particles that were not were most likely those that had been recently introduced into the bioreactor (74). Unattached cells were mostly observable under conditions of low D.

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View larger version (18K): [in this window] [in a new window]   FIG. 1.   Effect of solid tR on cellulose digestion by C. cellulolyticum. Inset, correlation between SR/S0 and tR.

Metabolite production. Acetate, ethanol, lactate, H₂, and CO₂ were the primary metabolic end products, and no succinate accumulation was observed (Table 2). The percentage of carbon flow toward fermentative metabolites, given by the ratio q_pyruvate/q_cellulose, indicated that 67.0 to 81.5% of the consumed cellulose was converted to extracellular pyruvate, lactate, CO₂, acetate, and ethanol. The remaining carbon flow was oriented toward amino acid, protein, biomass, and exopolysaccharide formation. Exopolysaccharides could not be measured as previously described (48) because of significant interference with cellulose fibers leading to erroneous estimation of their concentrations, but they were readily observable by microscopic examination. The carbon balance, calculated by taking into account amino acids, proteins, fermentative end products, and biomass, was then in a range between 95.8 and 98.7%.

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View larger version (16K): [in this window] [in a new window]   FIG. 2.   Influence of D on qacetate (), qethanol (), and qlactate ().

Fermentation balance. Formulation of a reducing equivalent balance equation requires a fair knowledge of the biochemistry of carbon assimilation for a particular substrate (44, 45). The reactions leading to the formation of catabolites during the fermentation of cellulose by C. cellulolyticum were previously described (12). Cellulose hydrolysis liberates soluble sugars, which are then metabolized by bacteria and allow cell growth. The hexose residues of a -1,4 polymer of glucose have a mean
polymerization, i.e., the number ofglucose residues inside the biopolymer. From cellulose degradation, it was assumed that soluble cellodextrins with a degree of polymerization n between 1 and 7 could potentially be incorporated and fermented by the cells (12, 51, 56). This corresponds to an average formula of hexose equivalents from glucose to celloheptaose utilized by bacteria of C_6.0H_10.7O_5.4.

Energetic and redox balance. The stoichiometry of ATP generated over hexose equivalents fermented, i.e., ATP-Eff, was higher at low D since it was 2.72 and declined to 2.07 with the highest D value (Table 3) due to the decrease of the percentage of acetate production (Table 2). As D increased, q_ATP rose from 3.24 to 5.25 mmol (g of cells)¹ h¹ (Table 3). The apparent energetic yield increased with increasing D as well (Table 3). The low Y_ATP obtained at low µ reflected an expenditure of energy due to the more pronounced maintenance requirement at low µ. The Pirt plot (r² = 0.984) permitted determining a true energetic yield (Y_ATP^max) of 30.3 g of cells (mol of ATP)¹ and a maintenance energy (m_ATP) estimated at 2.9 mmol of ATP (g of cells)¹ h¹.

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Kinetic analysis of cellulose fermentation. The effects of D on cellulose consumption and product formation are summarized in Fig. 3. The rate of cellulose consumption varied from 7.14 to 15.20 meq of C (g of cells)¹ h¹ with increasing D.

View larger version (33K): [in this window] [in a new window]   FIG. 3.   Distribution of carbon flux within the central metabolic pathways based on steady-state kinetics from continuous cultures of C. cellulolyticum grown on cellulose at D = 0.014, 0.035, 0.064, and 0.083 h1. Carbon flow () and turnover () were calculated as specified in Materials and Methods and according to the values in Tables 1 and 2. Fluxes through metabolic pathways are expressed as millequivalents of C per gram of cells per hour and are indicated by thin lines where numbers in parentheses refer to step numbers in Table 1. Turnover per hour is indicated by a thin line connected to a box with a rolling circle symbolizing the turnover of the pool.

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Intracellular pool of hexose phosphate and CoA derivative. The G6P pool was fueled by the carbon flow from glucokinase activity and by PGM activity from the G1P pool (Fig. 3). At this metabolic node, the G6P pool decreased with increasing growth rate (Table 5), indicating that the turnover of the pool increased and actually reached 212.3 h¹ at µ = 0.083 h¹ (Fig. 3). This variation was correlated with the increase of the carbon flow through glycolysis and biosynthesis metabolic pathways with higher µ. The q_G6P increased from 6.86 to 14.32 meq of C (g of cells)¹ h¹ with higher D (Fig. 3); whatever the D, it represented a mean of 96.0% of the carbon uptake. The G1P pool rose from 5.71 to 20.12 µmol (g of cells)¹ with D (Table 5) and thus corresponded to a decrease in the turnover of the pool (Fig. 3). As a result, the G6P/G1P ratio ranged from 6.48 to 0.56 with increasing µ (Table 5).

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Enzymatic activities. The effects of µ on specific activities of the enzymes are compiled in Table 6. In vitro enzyme activities were higher under conditions giving higher in vivo specific production rates. The level of GAPDH rose continuously with increasing carbon flow, indicating efficient hexose catabolism during glycolysis; from the lowest to the highest D, the GAPDH level increased 3.3-fold. From 0.014 to 0.083 h¹, PFO increased 1.7-fold, PTA increased 1.4-fold, AK increased 7.1-fold, AADH increased 3.2-fold, and ADH increased 2.9-fold.

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	DISCUSSION

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Based on microscopic observations, cellulose fibers were found to be covered by the microbial cell; such continuous cultures are generally regarded as cellulose limited (59, 71, 74). Yet cells which adhere to cellulose can also be considered to be in substrate-sufficient conditions. In fact, soluble sugars liberated from cellulose are the real substrate for growth and are incorporated by cells as rapidly as they are formed, since no glucose or cellodextrins could be detected in the supernatant. It may therefore be possible that bacteria display a metabolism under or near carbon-sufficient conditions and not carbon-limited conditions as previously suggested by microscopic examination. In cellobiose-sufficient conditions, the ATP/ADP ratios were as high as 7.21 and always higher than 1 (25), while in cellobiose limitation the ATP/ADP ratios ranged between 0.21 and 0.69 (E. Guedon, M. Desvaux, and H. Petitdemange, unpublished data). In the cellulose chemostat cultures carried out, the ATP/ADP ratios ranged from 0.37 to 0.46 and were always lower than 1 as in carbon-limited chemostats. In cellobiose limitation, few lactate molecules were produced at low D, contrary to what was observed in cellobiose-sufficient continuous culture (25). Moreover, on cellulose continuous culture, the ratios NADH/NAD⁺ and q_{NADH produced}/q_{NADH used} dropped as D increased, while in cellobiose-sufficient conditions these ratios rose (25). All of these results lead to the conclusion that the cellulose continuous cultures described here were performed under carbon limitation; it can thus be maintained that the chemostats were cellulose-limited continuous cultures.

In such conditions, the cellulose catabolism of C. cellulolyticum led to an acetate-ethanol fermentation maximizing ATP production. Concerning the distribution of the fluxes throughout the metabolic network, at least 94.2% of the carbon flow was used for generation of energy and biosynthetic precursors; the remaining carbon was converted to glycogen and exopolysaccharides. The proportion of carbon flowing through G6P remained quite constant regardless of D but differed between q_biosynthesis and q_pyruvate as µ increased; more of the carbon uptake was directed toward biosynthesis pathways at high D values than at lower ones. Regardless of D, extracellular proteins and free amino acids represented around 7.5% of the entering carbon. The carbon flow was always directed mainly toward acetate production, which represented a minimum of 27.1% of the original carbon at the highest µ. Yet the carbon from glycolysis directed through biosynthesis and ethanol increased to 27.3 and 16.8%, respectively, at D = 0.083 h¹. Theorical calculation suggest on the one hand that with Trichoderma reesei used as a model, optimal growth of a cellulolytic anaerobe requires major allocation of ATP toward cellulase biosynthesis (70) and on the other hand that with low ATP production per mole of hexose equivalent, most of the carbon source is used for the generation of energy (57). From lowest to highest D, C. cellulolyticum diverted 7.8 to 21.6% of the cellulose to cell carbon, whereas 81.3 to 66.7% was used for ATP production.

On cellulose, G6P-G1P was an important branch point since the longer the soluble -glucan uptake period is, the more G1P will be generated. The ratio of PGM specific activity to q_PGM (close to 1) reflected the precision of the control exerted by this enzyme on the partition of the flux at this junction (28), where the conversion of G1P to G6P feeds further the Embden-Meyerhof pathway. A high R indicates that the fluxes are determined by the concentration of substrate available in the pool rather than the enzyme activity (28). However, intracellular concentrations of substrates, products, cofactors, or effector molecules as well as intracellular ionic strength, redox potential, or pH could influence the partition and regulation of flux at each step in the central metabolic pathways (28). In vitro enzyme assays could then differ from in vivo conditions, and the significance of a low R could be more difficult to establish. Control of the PGM pathway by the amount of PGM was reinforced by the finding that K_m was more than 10-fold lower than the lowest intracellular G1P concentration determined. With higher D, the flow through glycolysis and biosynthesis increased, as did G6P turnover. The G1P pool, however, increased because the proportion of carbon flowing via PGM did not vary as much. G1P was thus directed toward exopolysaccharide formation, which reached a maximum of 4.2% of the original carbon uptake. The proportion of the flow through glycogen synthesis increased as well and represented up to 1.6% of the entering carbon flow. Here, glycogen turnover increased with D but remained low (maximum of 0.09 h¹), while the glycogen pool increased up to 108.7 mg (g of cells)¹ at 0.035 h¹ and then slowly decreased; glycogen biosynthesis could be adjusted as a function of the carbon flow. At the G1P-G6P branch point, the flux partitioning was then stabilized via PGM while the carbon surplus was dissipated by exopolysaccharide and intracellular glycogen synthesis. In fact, the percentage of G1P converted to G6P via PGM remained constant at around 93.7%, whereas the proportion of G1P converted to glycogen increased from 0.7 to 2.6% between D = 0.014 and 0.083 h¹. In the same time, exopolysaccharide represented around 5.3% of the G1P produced. These productions seem to buffer the increasing carbon flow from G1P, which could not be metabolized via PGM. On cellulose-limited chemostat cultures, carbon excess at this branch point was limited compared to ammonium-limited chemostat culture performed on cellobiose (which corresponded to an uncoupling between catabolism and anabolism), where exopolysaccharides and glycogen could represent up to 16.0 and 21.4%, respectively, of the specific rate of carbon consumed and where cellotriose was detected extracellularly (22). Glycogen was synthesized even in carbon-limited conditions and was present at all dilution rates. Such observations parallel those for Fibrobacter succinogenes, for which futile cycling of glycogen was reported (18, 38). These results could suggest that glycogen biosynthesis in cellulolytic bacteria in involved in carbon flow regulation (7) rather than in prolonging cell viability by providing energy storage (54) as for sporogenesis (49). Such a cycle could also be considered an energy-wasting reaction, as suggested by use of the term "futile cycle" for glycogen metabolism in Fibrobacter; however, here cellulose-fed continuous cultures were energy-limited cultures which do not normally waste ATP (57).

The flow of carbon was facilitated by the increase of specific enzymatic activity as µ increased. The fact that metabolic fluxes were much less than the available enzyme activities (except for PGM as specified above) indicated that specific enzyme activities were limited by the supply of substrate. The decline of acetyl-CoA turnover was corroborated by the analysis of metabolic flux distribution; the percentage of the carbon flowing through PFO diminished as D increased and the flux was directed away from acetate production. The acetyl-CoA/CoA ratio decreased and paralleled the decline of the ratios H₂/CO₂ and q_{NADH produced}/q_{NADH used}. These results are in agreement with the model of Decker et al. (11, 66) for the regulation of NADH-fd reductase activities which, through the consumption of NADH, direct the fluxes of metabolites in the direction of acetate for ATP production. On cellulose, the cells manage to maintain an equilibrated electron balance through the hydrogenase NADH-fd reductase activities and the ethanol pathway as indicated by the O/R index (23, 24). The pyruvate and acetyl-CoA branch points were parts of an independent two-node metabolic network (60). From acetyl-CoA, the main end product formed was acetate, but partitioning of the flux was modified in favor of ethanol production as D increased. Pyruvate was catabolized mainly via PFO. Whatever the D, the extracellular pyruvate formation rate was low and was correlated with the low lactate production which, moreover, represented less and less of the q_pyruvate; the specific formation rates of both lactate and extracellular pyruvate represented a maximum of 1.8% of the original carbon uptake. Thus, whatever the D, there was no competition between PFO and LDH for the carbon flowing from glycolysis, contrary to what was observed on soluble sugar (24, 25). On cellobiose, lactate and extracellular pyruvate levels rose with increasing specific rates of consumed hexose higher than 2.82 mmol (g of cells)¹ h¹ (24), while on cellulose q_hexose reached a maximum of 2.53 mmol (g of cells)¹ h¹.

The m_ATP obtained on cellulose, i.e., 2.9 mmol of ATP/g of cells/h, was very close to that obtained on cellobiose (r² = 0.923), i.e., 2.2 mmol of ATP/g of cells/h; as well, the true energetic yield on cellobiose was 28.4 g of biomass per mol of ATP, near the Y_ATP^max of 30.3 g of biomass per mol of ATP obtained on cellulose (values for the cellobiose-limited chemostat calculation were taken from a previous report 24). As for the true growth yield, the Y_X/S^max determined on cellobiose was lower than that on cellulose (Table 7). From statistical analysis of the Pirt plots, following regression analysis and Student t test (9) of the data, the maximum true growth yields were significantly different. This indicated a benefit for cells when grown on cellulose since a higher biomass could be reached for the same hexose equivalent quantity consumed. Such a result is consistent with the mechanism of carbohydrate uptake described by Strobel et al. (62), which is an energy-efficient transport system of soluble cellodextrins released from cellulolysis.

This investigation has also allowed characterization of the cellulose degradation properties of C. cellulolyticum from the consistent data summarized in Table 7. Our data were compiled along with those of other chemostat cultures performed with cellulose as described in the current literature. R. albus and C. cellulolyticum appear to be the bacteria with the lowest first-order cellulose hydrolysis rate constant; it is 3.4 times lower than that of C. thermocellum, which is the highest one so far determined, 0.17 h¹. Although a high cellulose degradation rate is often regarded as a general feature of thermophilic microorganisms relative to mesophilic species (37), such a difference in the rate constant of cellulose degradation could also result from the difference in cellulasic substrates used; in fact, the relative crystallinity index, porosity, allomorphs, capillary or gross surface area of the cellulose used could result in different kinetics of cellulose degradation (72, 73). Thus, the comparison of rate constant of cellulose digestion should not be overinterpreted, a caveat which points out the necessity of standardization of cellulose substrates used in such studies for further direct comparison. The true growth yield is among the highest reported, i.e., 50.5 g of cells/mol of hexose eq (Table 7). The growth maintenance coefficient reported in the literature could not be truly comparable since the rate of consumed hexose required for maintenance is related to the metabolic pathway which allowed the ATP production (57, 61, 65). Since the catabolic networks vary among the reported bacterial species (Table 7), differences in m values could be attributed to ATP generating a more or less efficient pathway that thus requires more or less hexose equivalents but can generate the same quantity of ATP per gram of cells per hour. Thus, comparison of m_ATP between cellulolytic bacteria would be more informative for evaluating the energy maintenance requirement.

The first step in investigating C. cellulolyticum metabolism was the use of a chemostat technique which leads to the accumulation of NADH (48). The use of a mineral salt-based medium permitted the induction of different metabolic regulatory responses and better control of the carbon and electron flows (24). Such findings led to the hypothesis of growth adapted to nutrient-poor conditions (23). These studies, however, were performed with soluble sugars, which facilitated study of the bacterial metabolism of cellulolytic bacteria. The next step was therefore investigation of the physiology of this microorganism on an insoluble substrate, more closely related to the natural ecological niche. In the present work with cellulose-limited continuous culture, no accumulation of NADH was observed and pyruvate overflow as high as on cellobiose chemostats did not occur (24). The insolubility and resistance of the cellulose to enzymatic hydrolysis physically prevents pyruvate overflow since µ_max and carbon flow are inevitably lower than with a soluble substrate (24). Thus, on cellobiose-limited chemostats, some metabolic regulation such as the shift from acetate-ethanol fermentation to lactate-ethanol fermentation at high catabolic rates should be interpreted as a deregulation of the metabolism attributed to the growth of C. cellulolyticum on soluble sugars which represent conditions far from the physical nature of the cellulose. In cellulose continuous cultures, compared to cellobiose chemostats, a second regulation of the entering carbon was introduced by the depolymerization of the insoluble substrate into soluble sugars readily metabolized by bacteria for growth. This limitation led to a lower maximum specific growth rate reached on cellulose than on cellobiose; in turn, pyruvate leakage was limited. The observations with cellobiose chemostats should be interpreted as laboratory artifacts due to culture conditions far from those in which this bacterium has evolved in nature and emphasizes that the efficiency of catabolism is related to the degradative property of the bacterial cellulosome. In the course of evolution, the catabolic pathways are optimized as a function of the carbon flowing from cellulase activities and are not adapted to higher catabolic rates as in other clostridial bacteria (43). C. cellulolyticum appeared, therefore, well adapted and even restricted to a low carbon flow, which is characteristic of growth on its natural substrate, cellulose.