Physiological Ecology of Clostridium glycolicum RD-1, an Aerotolerant Acetogen Isolated from Sea Grass Roots

Abstract

ABSTRACT An anaerobic, H 2 -utilizing bacterium, strain RD-1, was isolated from the highest growth-positive dilution series of a root homogenate prepared from the sea grass Halodule wrightii . Cells of RD-1 were gram-positive, spore-forming, motile rods that were linked by connecting filaments. Acetate was produced in stoichiometries indicative of an acetyl coenzyme A (acetyl-CoA) pathway-dependent metabolism when RD-1 utilized H 2 -CO 2 , formate, lactate, or pyruvate. Growth on sugars or ethylene glycol yielded acetate and ethanol as end products. RD-1 grew at the expense of glucose in the presence of low initial concentrations (up to 6% [vol/vol]) of O 2 in the headspace of static, horizontally incubated culture tubes; the concentration of O 2 decreased during growth in such cultures. Peroxidase, NADH oxidase, and superoxide dismutase activities were detected in the cytoplasmic fraction of cells grown in the presence of O 2 . In comparison to cultures incubated under strictly anoxic conditions, acetate production decreased, higher amounts of ethanol were produced, and lactate and H 2 became significant end products when RD-1 was grown on glucose in the presence of O 2 . Similarly, when RD-1 was grown on fructose in the presence of elevated salt concentrations, lower amounts of acetate and higher amounts of ethanol and H 2 were produced. When the concentration of O 2 in the headspace exceeded 1% (vol/vol), supplemental H 2 was not utilized. The 16S rRNA gene of RD-1 had a 99.7% sequence similarity to that of Clostridium glycolicum DSM 1288 T , an organism characterized as a fermentative anaerobe. Comparative experiments with C . glycolicum DSM 1288 T demonstrated that it had negligible H 2 - and formate-utilizing capacities. However, carbon monoxide dehydrogenase was detected in both RD-1 and C . glycolicum DSM 1288 T . A 91.4% DNA-DNA hybridization between the genomic DNA of RD-1 and that of C . glycolicum DSM 1288 T confirmed that RD-1 was a strain of C . glycolicum . These results indicate that (i) RD-1 metabolizes certain substrates via the acetyl-CoA pathway, (ii) RD-1 can tolerate and consume limited amounts of O 2 , (iii) oxic conditions favor the production of ethanol, lactate, and H 2 by RD-1, and (iv) the ability of RD-1 to cope with limited amounts of O 2 might contribute to its survival in a habitat subject to daily gradients of photosynthesis-derived O 2 .