Raw Starch-utilizing Anaerobe from Mesophilic Methane Sludge and Its Production of Amylase

Biogas production from various agro-wastes has been carried out by means of methane fermentation using both mesophilic and thermophilic methane sludge.1>2) Several kinds of Clostridium sp. were found to be involved in the acidogenic processes during methane fermentation.3) In this paper, we report the isolation and characterization of an anaerobe involved in the acidogenic digestion of raw starch granules during mesophilic methane fermentation. The sludge sample1} was streaked on a PY-S agar plate supplemented with 0.5% sweet potato starch without heating, and then incubated at 37°C inside an anaerobic glove box according to our previous method.4) Strain T-7 was isolated as the bacterium forming the largest halo on the mediumdue to the digestion of the raw sweet potato starch. Physiological and biochemical properties of strain T-7 were investigated according to the descriptions in the "Anaerobic Laboratory Manual"5* and "The Prokaryotes".6) Utilization of carbohydrates by strain T-7 was also examined by the rriethod previously reported.4) Strain T-7 was a rod (0.7~0.9/mi in diameter by 3~4.5/mi in length) with rounded ends and no flagella, and usually formed terminal spores (oval to spherical). The bacterium was absolutely anaerobic and gram positive, but becamegram negative on prolonged cultivation in PY-S broth. Catalase was not produced. These facts clearly indicated that strain T-7 was a species of Clostridium. Its biochemical properties were the same as those of Clostridium butyricum6J) except for that strain T7 did not require biotin as a growth factor. Strain T-7 utilized many of the carbohydrates tested but not eel-, lulose, xylan or sugar alcohols. Its carbohydrate utilization pattern was almost identical to that reported for C. butyricum^ utilization of carbohydrates such as larabinose, L-rhamnose, D-mannitol, inositol and esclin being indicated to depend on the strain of C. butyricum. It is quite reasonable to identify strain T-7 as a strain of C. butyricum, although typical C. butyricum strains reportedly have phenotypic characteristics such as subterminal spore formation and flagellation as well as a nutritional requirement of biotin.7) Raw starch utilization and production of amylase by strain T-7 were examined using PY-broth containing 1% raw starch at 37°C under anaerobic conditions.4) The bacterial suspension was taken up with a sterilized syringe, and then the bacterial growth (turbidity at 660nm), and remaining raw starch and soluble sugars in the super-