Isolation and characterization of a heavy metal- and antibiotic-tolerant novel bacterial strain from a contaminated culture plate of Chlamydomonas reinhardtii, a green micro-alga.

Background:Chlamydomonas reinhardtii, a green micro-alga, is normally cultured in laboratories in Tris-Acetate Phosphate (TAP), a medium which contains acetate as the sole carbon source. Acetate in TAP can lead to occasional bacterial and fungal contamination. We isolated a yellow-pigmented bacterium from a Chlamydomonas TAP plate. It was named Clip185 based on the Chlamydomonas strain plate it was isolated from. In this article we present our work on the isolation, taxonomic identification and physiological and biochemical characterizations of Clip185. Methods: We measured sensitivities of Clip185 to five antibiotics and performed standard microbiological tests to characterize it. We partially sequenced the 16S rRNA gene of Clip185. We identified the yellow pigment of Clip185 by spectrophotometric analyses. We tested tolerance of Clip185 to six heavy metals by monitoring its growth on Lysogeny Broth (LB) media plates containing 0.5 mM -10 mM concentrations of six different heavy metals. Results: Clip185 is an aerobic, gram-positive rod, oxidase-negative, mesophilic, alpha-hemolytic bacterium. It can ferment glucose, sucrose and mannitol. It is starch hydrolysis-positive. It is very sensitive to vancomycin but resistant to penicillin and other bacterial cell membrane- and protein synthesis-disrupting antibiotics. Clip185 produces a C50 carotenoid, decaprenoxanthin, which is a powerful anti-oxidant with a commercial demand. Decaprenoxanthin production is induced in Clip185 under light. NCBI-BLAST analyses of the partial 16S rRNA gene sequence of Clip185 revealed a 99% sequence identity to that of Microbacterium binotii strain PK1-12M and Microbacterium sp. strain MDP6. Clip185 is able to tolerate toxic concentrations of six heavy metals. Conclusions: Our results show that Clip185 belongs to the genus Microbacterium. In the future, whole genome sequencing of Clip185 will clarify if Clip185 is a new Microbacterium species or a novel strain of Microbacterium binotii, and will reveal its genes involved in antibiotic-resistance, heavy-metal tolerance and regulation of decaprenoxanthin biosynthesis.

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

Background: Green micro-alga, Chlamydomonas reinhardtii (a Chlorophyte), can be cultured in the laboratory heterotrophically or photo-heterotrophically in Tris- Phosphate- Acetate (TAP) medium, which contains acetate as the carbon source. Chlamydomonas can convert acetate in the TAP medium to glucose via the glyoxylate cycle, a pathway present in many microbes and higher plants. A novel bacterial strain, CC4533, was isolated from a contaminated TAP agar medium culture plate of a Chlamydomonas wild type strain. In this article, we present our research on the isolation, and biochemical and molecular characterizations of CC4533. Methods: We conducted several microbiological tests and spectrophotometric analyses to biochemically characterize CC4533. The 16S rRNA gene of CC4533 was partially sequenced for taxonomic identification. We monitored the growth of CC4533 on Tris-Phosphate (TP) agar medium (lacks a carbon source) containing different sugars, aromatic compounds and saturated hydrocarbons, to see if CC4533 can use these chemicals as the sole source of carbon. Results: CC4533 is a Gram-negative, non-enteric yellow pigmented, aerobic, mesophilic bacillus. It is alpha-hemolytic and oxidase-positive. CC4533 can ferment glucose, sucrose and lactose, is starch hydrolysis-negative, resistant to penicillin, polymyxin B and chloramphenicol. CC4533 is sensitive to neomycin. Preliminary spectrophotometric analyses indicate that CC4533 produces b-carotenes. NCBI-BLAST analyses of the partial 16S rRNA gene sequence of CC4533 show 99.55% DNA sequence identity to that of Sphingobium yanoikuyae strain PR86 and S. yanoikuyae strain NRB095. CC4533 can use cyclo-chloroalkanes, saturated hydrocarbons present in car motor oil, polyhydroxyalkanoate, and mono- and poly-cyclic aromatic compounds, as sole carbon sources for growth. Conclusions: Taxonomically, CC4533 is very closely related to the alpha-proteobacterium S. yanoikuyae, whose genome has been sequenced. Future research is needed to probe the potential of CC4533 for environmental bioremediation. Whole genome sequencing of CC4533 will confirm if it is a novel strain of S. yanoikuyae or a new Sphingobium species.

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.

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.