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

Abstract

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 nifH, 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 Clostridium diazotrophs, such as C. pasteurianum and C. acetobutylicum. 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 Clostridium, and the closest species are C. aciditolerans, C. nitrophenolicum, and C. thailandense, 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 Clostridium aciditolerans. Although the species taxonomy of strain OS1-26 is undetermined within the genus Clostridium 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.

Keywords: biological N fixation; free-living soil diazotroph; soil Clostridium species.

Figure 1

Phylogenetic relationship of Clostridium species based on the 16S rRNA gene sequences. The tree was constructed by the maximum-likelihood method using MEGA X software. GenBank accession numbers are shown in square brackets. Bootstrap values (%) were generated from 1000 replicates are shown. Branch lengths correspond to sequence differences as indicated by the scale bar.

Figure 2

Changes of extracellular NH₄⁺ concentration and biomass (OD₆₀₀) during the anaerobic cultivation of three Clostridium strains, such as OS1-26, KCTC 1674^T, and KCTC 1790^T, by incubation time in R2A medium.

Figure 3

Transcription levels of nifH gene of Clostridium sp. OS1-26 during the anaerobic cultivation in R2A medium. Transcription levels were normalized by the number of 16S rRNA copies. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. p > 0.05 (ns), p < 0.01 (**), p < 0.001 (***).