Isolation and characterization of a novel bacterial strain from a Tris-Acetate-Phosphate agar medium plate of the green micro-alga Chlamydomonas reinhardtii that can utilize common environmental pollutants as a carbon source

Abstract

Background: Chlamydomonas reinhardtii, a green micro-alga can be grown at the lab heterotrophically or photo-heterotrophically in Tris-Phosphate-Acetate (TAP) medium which contains acetate as the sole carbon source. When grown in TAP medium, Chlamydomonas can utilize the exogenous acetate in the medium for gluconeogenesis using the glyoxylate cycle, which is also present in many bacteria and higher plants. A novel bacterial strain, LMJ, was isolated from a contaminated TAP medium plate of Chlamydomonas. We present our work on the isolation and physiological and biochemical characterizations of LMJ. Methods: Several microbiological tests were conducted to characterize LMJ, including its sensitivity to four antibiotics. We amplified and sequenced partially the 16S rRNA gene of LMJ. We tested if LMJ can utilize cyclic alkanes, aromatic hydrocarbons, poly-hydroxyalkanoates, and fresh and combusted car motor oil as the sole carbon source on Tris-Phosphate (TP) agar medium plates for growth. Results: LMJ is a gram-negative rod, oxidase-positive, mesophilic, non-enteric, pigmented, salt-sensitive bacterium. LMJ can ferment glucose, is starch hydrolysis-negative, and is very sensitive to penicillin and chloramphenicol. Preliminary spectrophotometric analyses indicate LMJ produces pyomelanin. NCBI-BLAST analyses of the partial 16S rRNA gene sequence of LMJ showed that it matched to that of an uncultured bacterium clone LIB091_C05_1243. The nearest genus relative of LMJ is an Acidovorax sp. strain. LMJ was able to use alkane hydrocarbons, fresh and combusted car motor oil, poly-hydroxybutyrate, phenanthrene, naphthalene, benzoic acid and phenyl acetate as the sole carbon source for growth on TP-agar medium plates. Conclusions: LMJ has 99.14% sequence identity with the Acidovorax sp. strain A16OP12 whose genome has not been sequenced yet. LMJ’s ability to use chemicals that are common environmental pollutants makes it a promising candidate for further investigation for its use in bioremediation and, provides us with an incentive to sequence its genome.

Isolation and characterization of a novel Sphingobium yanoikuyae strain variant that uses biohazardous saturated hydrocarbons and aromatic compounds as sole carbon sources

“…TP-sugar medium plates were imaged after 5 days of growth at 22°C. Results were interpreted as described in Mitra et al 2020 41 .…”

“…16S rRNA gene forward (16SF) and reverse (16SR) PCR primers were designed based on primer sequences given in article by Klindworth et al (2013) 51 . Primer sequences, details about PCR cycling conditions, specific DNA polymerase used in the PCR, separation and visualization of PCR samples can be found in the article by Mitra et al 2020 41 .…”

“…PCR product cloning and DNA sequencing The partial 16S rRNA genomic PCR product was extracted and purified from the DNA agarose gel using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). The purified PCR product was cloned as described in Mitra et al 2020 41…”

“…We performed starch hydrolysis test on CC4533 and E. coli on Mueller-Hinton agar medium, which contains 0.15% starch (Figure 9). Background and scientific basis of the starch hydrolysis test can be found in Mitra et al 2020 41 . Both CC4533 ( Figure 9B) and E. coli ( Figure 9D) cannot hydrolyze starch as there was no visible clear zone around the growth on the Mueller Hinton agar.…”

“…Hence, we tested if CC4533 can utilize saturated hydrocarbons, PAH, other biohazardous aromatic compounds and PHB as carbon sources for growth. These growth analyses were performed as described in Mitra et al 2020 41 . Different doses of the chemicals that were tested can be also found in Mitra et al 2020 and are also available as Underlying data 48 .…”