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Carbon and Electron Flow in Clostridium cellulolyticum Grown in Chemostat Culture on Synthetic Medium Downloaded from

Abstract : Previous results indicated poor sugar consumption and early inhibition of metabolism and growth when Clostridium cellulolyticum was cultured on medium containing cellobiose and yeast extract. Changing from complex medium to a synthetic medium had a strong effect on (i) the specific cellobiose consumption, which was increased threefold; and (ii) the electron flow, since the NADH/NAD ؉ ratios ranged from 0.29 to 2.08 on synthetic medium whereas ratios as high as 42 to 57 on complex medium were observed. These data indicate a better control of the carbon flow on mineral salts medium than on complex medium. By continuous culture, it was shown that the electron flow from glycolysis was balanced by the production of hydrogen gas, ethanol, and lactate. At low levels of carbon flow, pyruvate was preferentially cleaved to acetate and ethanol, enabling the bacteria to maximize ATP formation. A high catabolic rate led to pyruvate overflow and to increased ethanol and lactate production. In vitro, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and ethanol dehydrogenase levels were higher under conditions giving higher in vivo specific production rates. Redox balance is essentially maintained by NADH-ferredoxin reductase-hydrogenase at low levels of carbon flow and by ethanol dehydrogenase and lactate dehydrogenase at high levels of carbon flow. The same maximum growth rate (0.150 h ؊1) was found in both mineral salts and complex media, proving that the uptake of nutrients or the generation of biosynthetic precursors occurred faster than their utilization. On synthetic medium, cellobiose carbon was converted into cell mass and catabolized to produce ATP, while on complex medium, it served mainly as an energy supply and, if present in excess, led to an accumulation of intracellular metabolites as demonstrated for NADH. Cells grown on synthetic medium and at high levels of carbon flow were able to induce regulatory responses such as the production of ethanol and lactate dehydrogenase. Cellulolytic clostridia are of cardinal importance in anaero-bic 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 syner-gism 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 chemo-stats, which can maintain low steady-state nutrient concentrations , using cellobiose as the carbon source, since this disac-charide is the major end product of the cellulose degradation process (32, 39). MATERIALS AND METHODS 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). The defined medium used in all experiments was a modification of the CM3 medium described by Weimer and Zeikus (44), in which 5 g of yeast extract per liter is replaced by oligoelement and vitamin solutions. The composition was (in grams liter Ϫ1 unless otherwise indicated): KH 2 PO 4 , 1.40; K 2 HPO 4 ⅐ 3H 2 O, 2.90; (NH 4) 2 SO 4 , 1.00; MgCl 2 ⅐ 6H 2 O, 0.10; CaCl 2 , 0.02; FeSO 4 ⅐ 7H 2 O, 9.15% (wt/ vol) in 50 mM H 2 SO 4 , 25 l; oligoelement solution, 1.0 ml; vitamin solution, 10 ml; Na 2 S, 0.50; and resazurin at 0.2% (wt/vol), 0.5 ml. In addition, a constant limited cellobiose concentration (5.84 mM) was added to the feed medium. The oligoelement solution contained (in grams liter Ϫ1 unless otherwise indicated): FeSO 4 ⅐ 7H 2 O, 5.00; ZnSO 4 ⅐ 7H 2 O, 1.44; MnSO 4 ⅐ 7H 2 O, 1.12; CuSO 4 ⅐ 5H 2 O, 0.25; Na 2 B 4 O 7 , 0.20; (Mo) 7 (NH 4) 6 O 24 ⅐ 4H 2 O, 1.00; NiCl 2 , 0.04; CoCl 2 , 0.02; HBO 3 , 0.03; Na 2 SeO 3 , 0.02; HCl (10 M), 50.0 ml. The composition of the vitamin solution was (in milligrams per 100 ml of distilled water): d-biotin, 10; para-aminobenzoic acid, 25; nicotinic acid, 15; riboflavin, 25; pantothenic acid, 25; thiamine, 25; and cyanocobalamin, 10. The vitamin in solution was sterilized by filtration through a 0.2-m-pore-size filter.
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https://hal.inrae.fr/hal-02910828
Déposant : Stéphanie Dutilloy <>
Soumis le : lundi 3 août 2020 - 10:16:50
Dernière modification le : vendredi 11 septembre 2020 - 10:08:02

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Emmanuel Guedon, S. Payot, M. Desvaux, H. Petitdemange. Carbon and Electron Flow in Clostridium cellulolyticum Grown in Chemostat Culture on Synthetic Medium Downloaded from. Journal of Bacteriology, American Society for Microbiology, 1999, 181 (10), pp.3262 - 3269. ⟨10.1128/JB.181.10.3262-3269.1999⟩. ⟨hal-02910828⟩

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