Clostridium akagii sp nov and Clostridium acidisoli sp nov.: acid-tolerant, N-2-fixing clostridia isolated from acidic forest soil and litter

Two anaerobic acid-tolerant bacteria, CK58(T) and CK74(T), 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 CK58(T) 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 CK58(T). These substrates, as well as mannitol, lactose, sucrose, glycerol, melezitose. raffinose and rhamnose, supported growth of CK74(T). Sorbitol, trehalose, H-2/CO2 CO/CO2, vanillate, Casamino acids, peptone, and various purines and pyrimidines did not support the growth of either organism. Growth of CK58(T) and CK74(T) on glucose yielded butyrate, lactate, acetate, formate, H-2 and CO2 as end products. Growth of CK58(T) and CK74(T) was observed at ph 3.7-7.1 and 3.6-6.9, respectively. CK58(T) and CK74(T) grew in nitrogen-free medium at ph 3.7 under an N-2 atmosphere and reduced acetylene at rates approximating 1 nmol min(-1) (mg protein)(-1). CK58(T) and CK74(T) did not contain carbon monoxide dehydrogenase or cytochromes, produce methane, or dissimilate nitrate or sulfate. Thus, CK58(T) and CK74(T) were characterized as nonacetogenic, N-2-fixing, fermentative chemo-organotrophs. The G+C contents of CK58(T) and CK74(T) were 31.4 and 30.7 mol%, respectively. CK58(T) and CK74(T) were phylogenetically most closely related to Clostridium pasteurianum, The 16S rRNA gene sequence similarity values of CK58(T) and CK74(T) to C. pasteurianum and each other did not exceed 96.5%, and it is proposed that strains CK58(T) and CK74(T) be named Clostridium akagii CK58T (DSM 12554(T)) and Clostridium acidisoli CK74(T) (DSM 12555(T)), respectively. These results suggest that previously uncharacterized clostridial species reside and might fix N-2 in the annoxic microzones of acidic forest soil and litter.