Synergism of Glycoside Hydrolase Secretomes from Two Thermophilic Bacteria Cocultivated on Lignocellulose

Two cellulolytic thermophilic bacterial strains, CS-3-2 and CS-4-4, were isolated from decayed cornstalk by the addition of growth-supporting factors to the medium. According to 16S rRNA gene-sequencing results, these strains belonged to the genus Clostridium and showed 98.87% and 98.86% identity with Clostridium stercorarium subsp. leptospartum ATCC 35414(T) and Clostridium cellulosi AS 1.1777(T), respectively. The endoglucanase and exoglucanase activities of strain CS-4-4 were approximately 3 to 5 times those of strain CS-3-2, whereas the β-glucosidase activity of strain CS-3-2 was 18 times higher than that of strain CS-4-4. The xylanase activity of strain CS-3-2 was 9 times that of strain CS-4-4, whereas the β-xylosidase activity of strain CS-4-4 was 27 times that of strain CS-3-2. The enzyme activities in spent cultures following cocultivation of the two strains with cornstalk as the substrate were much greater than those in pure cultures or an artificial mixture of samples, indicating synergism of glycoside hydrolase secretomes between the two strains. Quantitative measurement of the two strains in the cocultivation system indicated that strain CS-3-2 grew robustly during the initial stages, whereas strain CS-4-4 dominated the system in the late-exponential phase. Liquid chromatography-tandem mass spectrometry analysis of protein bands appearing in the native zymograms showed that ORF3880 and ORF3883 from strain CS-4-4 played key roles in the lignocellulose degradation process. Both these open reading frames (ORFs) exhibited endoglucanase and xylanase activities, but ORF3880 showed tighter adhesion to insoluble substrates at 4, 25, and 60°C owing to its five carbohydrate-binding modules (CBMs).