A moderately thermophilic Mycobacterium strain (denoted Myco-U) was isolated in our lab as a contaminant during the cultivation of another... Show moreA moderately thermophilic Mycobacterium strain (denoted Myco-U) was isolated in our lab as a contaminant during the cultivation of another thermophilic Paenibacillus strain, and has been proved to harbor the genes that encode the enzymes for dibenzothiophene (DBT) biodegradation via the 4S pathway; this makes it possible to efficiently remove organosulfur compounds (e.g. DBT and DBTO2) from the crude petroleum. Through directed evolution (continuous passage and selection with increasing temperature), the Myco-U strain has shown moderate capacity of metabolizing DBT to 2-hydroxybiphenyl (2-HBP) by the 4S pathway at a thermophilic temperature (~53 ℃). In the middle-to-late stages of the selections, a stubborn contaminant (denoted Bacillus X) arose and has remained difficult to get rid of from our medium and cultures ever since, leading to a persistent contamination and repetition of our experiments. The reason why Bacillus X is regarded as a contaminant is that it does not obtain sulfur by the expected means (e.g., DBT desulfurization), but utilizes the tiny amount of sulfate from the vitamin mix and 50 μg/ml yeast extract in our medium to grow. This is confirmed by the facts that Bacillus X does not yield any detectable 2-HBP, and a significant difference of growth has been detected between cultures with DBT as the sole sulfur source and the positive controls, which employs Na2SO4 as sulfur source. Through 16S rDNA amplification, cloning, and sequencing, Bacillus X was identified as a Bacillus species, and shows the highest homology (99 % identical) to a specific uncultured bacterium clone (NCBI #Accession: JN882111.1). The chromogenic mechanism and impacts of pH and carbonate upon the Gibbs assay, by which 2-HBP is quantified, were investigated. When DBT is absent or at undetectably low levels, cultures measured by the assay display a yellowish-to-brownish background after adding the Gibbs reagent (2,6-dichloroquinone-4-chloroimide). According to our results, the Gibbs reaction prefers alkaline conditions and pH 8.0 ranks the optimal (compared with pH 7.0, 7.5, 8.5 and 9.0) at which the maximum A610nm is detected. M.S. in Biology, May 2016 Show less
