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Conversion of cellulose to methane and carbon dioxide by anaerobic, nitrogen dioxide-fixing bacterial communities

Esteban Monserrate, University of Massachusetts Amherst

Abstract

Two strains of Clostridium hungatei sp. nov., a cellulolytic, N$\sb2$-fixing bacterium were isolated from samples of soil rich in decaying plant material. On the basis of comparisons of their morphological, physiological and phylogenetic characteristics, and of their G + C mol % content, with those of other Clostridium species, it was concluded that the two strains (designated strain AD and strain B3B) were representatives of a novel species of Clostridium. C. hungatei produces an extracellular cellulase complex which exhibits cellulase (i.e., Avicelase, carboxymethylcellulase) and xylanase activities. The cellulase-xylanase complex biosynthesis, in C. hungatei, was induced when cells of strain AD were grown with soluble products of cellulose hydrolysis (e.g., cellobiose) as the carbon and energy source. Induction of cellulase-xylanase complex, above a low constitutive level, occurred when the cellobiose concentration was 0.1% (wt/vol) or lower, but not at higher concentrations. Induction above the constitutive level was not observed when other soluble sugars (e.g., D-mannose) served as carbon and energy source. Cellulase activity of the cellulase-xylanase complex produced by C. hungatei is inhibited by cellobiose (at concentrations as low as 0.02%, wt/vol). Growth kinetics, cellulose degradation studies and enzyme assays revealed that supernatant fluid samples of C. hungatei cultures growing under N$\sb2$-fixing conditions, or at low dilution rates (in a chemostat limited by carbon and energy source) in the presence of NH$\sb4$Cl showed higher cellulase activity per cell mass produced as compared to cultures growing in the presence of combined nitrogen or at higher dilution rates. In nature, the ability to regulate the cellulase-xylanase complex synthesis, and the ability to enhance cellulase activity per cell mass produced under conditions of higher energy demand (e.g., N$\sb2$ fixation) may give C. hungatei an advantage over other bacteria living in the same environment. A N$\sb2$-fixing stable coculture, consisting of C. hungatei strain AD and a facultatively anaerobic, non-cellulolytic bacterium (strain CU-1), was used as a model to determine whether extracellular cellulases produced by cellulolytic bacteria may serve as nitrogen sources for non-N$\sb2$-fixing bacteria growing in heterogeneous microbial communities. Inasmuch as supernatant fluids of cocultures showed lower cellulase and xylanase activities as compared to clostridial monoculture supernatants, and supernatant fluids of strain CU-1 showed proteolytic activity when grown in the presence of sources of amino acids (e.g., C. hungatei's cellulase system), and of the basis of other evidence it was concluded that, in anaerobic environments deficient in combined nitrogen, extracellular cellulases may serve as nitrogen sources for non-N$\sb2$-fixing bacteria. In addition, strain CU-1 grew in monoculture in a chemically-defined cellobiose-containing medium to which a gel-filtration purified strain AD cellulase preparation was added as the only nitrogen source. Using a cellulolytic methanogenic coculture, composed of C. hungatei strain AD, and a strain of H$\sb2$-consuming methanogen (e.g., Methanobacterium formicicum strain WH), the effects of interspecies H$\sb2$ transfer on growth, cellulose degradation and product formation were studied. The results indicated that interspecies H$\sb2$ transfer enhances growth rate and affects product formation by the cellulolytic clostridium, and indirectly stimulates cellulose degradation.

Subject Area

Microbiology|Environmental science

Recommended Citation

Monserrate, Esteban, "Conversion of cellulose to methane and carbon dioxide by anaerobic, nitrogen dioxide-fixing bacterial communities" (1994). Doctoral Dissertations Available from Proquest. AAI9510509.
https://scholarworks.umass.edu/dissertations/AAI9510509

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