Cellulose- and xylan-degrading thermophilic anaerobic bacteria from biocompost

Abstract

Nine thermophilic cellulolytic clostridial isolates and four other noncellulolytic bacterial isolates were isolated from self-heated biocompost via preliminary enrichment culture on microcrystalline cellulose. All cellulolytic isolates grew vigorously on cellulose, with the formation of either ethanol and acetate or acetate and formate as principal fermentation products as well as lactate and glycerol as minor products. In addition, two out of nine cellulolytic strains were able to utilize xylan and pretreated wood with roughly the same efficiency as for cellulose. The major products of xylan fermentation were acetate and formate, with minor contributions of lactate and ethanol. Phylogenetic analyses of 16S rRNA and glycosyl hydrolase family 48 (GH48) gene sequences revealed that two xylan-utilizing isolates were related to a Clostridium clariflavum strain and represent a distinct novel branch within the GH48 family. Both isolates possessed high cellulase and xylanase activity induced independently by either cellulose or xylan. Enzymatic activity decayed after growth cessation, with more-rapid disappearance of cellulase activity than of xylanase activity. A mixture of xylan and cellulose was utilized simultaneously, with a significant synergistic effect observed as a reduction of lag phase in cellulose degradation.

FIG. 1.

Phylogenetic tree of anaerobic thermophilic cellulolytic (⋄), cellulolytic/xylanolytic (♦), and noncellulolytic (○) isolates from biocompost based on 16S rRNA gene sequence comparisons.

FIG. 2.

Phylogenetic tree of thermophilic anaerobic cellulolytic (⋄) and cellulolytic and xylanolytic (♦) isolates from biocompost based on GH48 gene sequence comparisons.

FIG. 3.

Fermentation of cellulose (left column) and xylan (right column) by isolate 4-2a. Top panels, total pellet carbon (TPC; C substrate plus C cell) and total pellet nitrogen (TPN; measure of cell mass concentration); bottom panels, fermentation products and depolymerization intermediates.

FIG. 4.

Growth dynamics of isolate 4-2a on medium with cellulose (open symbols, continuous lines), xylan (gray-filled symbols, continuous lines), or a mixture of cellulose and xylan (black-filled symbols, dotted lines). Top, cell biomass concentration (A) and sum of fermentation products (B) (see Fig. 3 for individual products); middle, cellulase (C) and xylanase (D) activity; bottom, pH (E) and intermediate degradation products (F).

FIG. 5.

Relationship between cellulase and xylanase activities. The pooled xylanase activity is plotted versus respective cellulase activity. The shaded areas indicate three domains with different relationships between the activities of two enzymes, allowing rejection of the hypothesis that degradation of xylan and cellulose is catalyzed by the same enzyme.