Semester of Graduation

Fall 2020


Master of Civil Engineering (MCE)


Civil and Environmental Engineering

Document Type



In an effort to better understand the role that various microbes may play in toluene production, bacteria from a toluene-producing enrichment culture derived from contaminated groundwater at a Superfund site were cultivated and isolated on low nutrient solid media. A total of 14 solid medium formulations containing varying pH ranges, carbon sources, solidifying agents, and incubation gas headspaces were used to obtain 278 isolates in pure culture. Isolated bacteria, identified using partial 16S rRNA gene sequences, were most closely related with the genera Anoxybacillus, Azospira, Bacillus, Bradyrhizobium, Cellulosimicrobium, Micrococcus, and Propionicimonas.

Further attempts to target putative toluene-producing organisms made use of a screening approach involving a plate washing technique in conjunction with PCR amplification employing primers specifically designed to target 16S rRNA gene sequences unique to a subset of bacteria clustering within the phylum Acidobacteria. Experiments resulted in the recovery of an additional 56 bacterial isolates related to the genera Agrobacterium, Bacillus and Rhizobium.

Three isolates were selected for comparative testing to clarify their taxonomic positions. Nearly complete 16S rRNA gene sequences determined for the three isolates were identical to one another and were most closely related to but clearly different from strains of the related species Azospira oryzae and Azospira restricta. Comparative testing revealed multiple of phenotypic differences between the new isolates and the reference strains. Collectively, data suggest that the new isolates represent a novel species.

Committee Chair

Moe, William