Excessive Extracellular Ammonium Production by a Free-Living Nitrogen-Fixing Soil Clostridium sp. Strain

A Gram-positive, rod-shaped, and obligate anaerobic bacterial strain OS1-26 was isolated from apple orchard soil in Iksan, South Korea. Interestingly, strain OS1-26 was observed to possess the functional genes involved in biological nitrogen fixation (BNF), including <i>nifH</i>, which was actively transcribed during the anaerobic cultivation with excessive production of extracellular NH₄⁺ despite of presence of other fixed N nutrients. The BNF of strain OS1-26 was distinguished from the other well-known <i>Clostridium</i> diazotrophs, such as <i>C. pasteurianum</i> and <i>C. acetobutylicum</i>. The altruistic N-fixing ability of the strain may play a pivotal role in providing N nutrients to the microbial community and plants in the soil ecosystem. The microorganism grew at 25-35 °C (optimum 30-35 °C) and pH 5.0-8.0 (optimum 6.0-8.0) but was not able to grow in the presence of >0.5% NaCl. The major cellular fatty acids of strain OS1-26 were C_16:0, C_14:0, and the summed feature consisted of C_16:1 ω7c and C_16:1 ω6c (35.63%, 25.29%, and 18.84%, respectively). The 16S rRNA phylogeny indicated that strain OS1-26 is a member of the genus <i>Clostridium</i>, and the closest species are <i>C. aciditolerans</i>, <i>C. nitrophenolicum,</i> and <i>C. thailandense,</i> with 16S rRNA sequence similarities such as 99.71%, 98.52%, and 98.45%, respectively. In spite of the high 16S rRNA sequence similarity, strain OS1-26 showed overall genomic relatedness, such as the average nucleotide identity (ANI), and phenotypical features distinctly different from <i>Clostridium aciditolerans</i>. Although the species taxonomy of strain OS1-26 is undetermined within the genus <i>Clostridium</i> based on overall genomic and phenotypic properties, further studies on the soil bacterial strain would enhance our understanding of its taxonomic identity, ecological roles for the terrestrial soil N cycle, and the potential to be developed as a biological N fertilizer.