Clostridium akagii sp. nov. and Clostridium acidisoli sp. nov.: acid-tolerant, N2-fixing clostridia isolated from acidic forest soil and litter

Two anaerobic acid-tolerant bacteria, CK58T and CK74T, were isolated from acidic beech litter and acidic peat-bog soil, respectively. Both bacteria were spore-forming, motile rods with peritrichous flagella. The capacity to sporulate decreased with prolonged cultivation. Cells of CK58T formed chains or aggregates and were linked by a connecting filament that consisted of a core and a surrounding sheath. Cellobiose, glucose, xylose, arabinose, maltose, mannose and salicin supported growth of CK58T. These substrates, as well as mannitol, lactose, sucrose, glycerol, melezitose, raffinose and rhamnose, supported growth of CK74T. Sorbitol, trehalose, H2/CO2, CO/CO2, vanillate, Casamino acids, peptone, and various purines and pyrimidines did not support the growth of either organism. Growth of CK58T and CK74T on glucose yielded butyrate, lactate, acetate, formate, H2 and CO2 as end products. Growth of CK58T and CK74T was observed at pH 3.7-7.1 and 3.6-6.9, respectively. CK58T and CK74T grew in nitrogen-free medium at pH 3.7 under an N2 atmosphere and reduced acetylene at rates approximating 1 nmol min-1 (mg protein)-1. CK58T and CK74T did not contain carbon monoxide dehydrogenase or cytochromes, produce methane, or dissimilate nitrate or sulfate. Thus, CK58T and CK74T were characterized as nonacetogenic, N2-fixing, fermentative chemo-organotrophs. The G + C contents of CK58T and CK74T were 31.4 and 30.7 mol%, respectively. CK58T and CK74T were phylogenetically most closely related to Clostridium pasteurianum. The 16S rRNA gene sequence similarity values of CK58T and CK74T to C. pasteurianum and each other did not exceed 96.5%, and it is proposed that strains CK58T and CK74T be named Clostridium akagii CK58T (DSM 12554T) and Clostridium acidisoli CK74T (DSM 12555T), respectively. These results suggest that previously uncharacterized clostridial species reside and might fix N2 in the annoxic microzones of acidic forest soil and litter.