F1000Research 2020, 9:656 Last updated: 01 JUN 2021 RESEARCH ARTICLE 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 [version 1; peer review: 3 approved] Mautusi Mitra 1, Kevin Manoap-Anh-Khoa Nguyen1,2, Taylor Wayland Box1, Jesse Scott Gilpin1, Seth Ryan Hamby1, Taylor Lynne Berry3,4, Erin Harper Duckett1 1Department of Biology, University of West Georgia, Carrollton, Georgia, 30118, USA 2Department of Mechanical Engineering, Kennesaw State University, Marietta, Georgia, 30060, USA 3Carrollton High School, Carrollton, Georgia, 30117, USA 4Department of Chemistry and Biochemistry, University of North Georgia, Dahlonega, Georgia, 30597, USA v1 First published: 29 Jun 2020, 9:656 Open Peer Review https://doi.org/10.12688/f1000research.24680.1 Latest published: 29 Jun 2020, 9:656 https://doi.org/10.12688/f1000research.24680.1 Reviewer Status Invited Reviewers Abstract Background: Chlamydomonas reinhardtii, a green micro-alga can be 1 2 3 grown at the lab heterotrophically or photo-heterotrophically in Tris- Phosphate-Acetate (TAP) medium which contains acetate as the sole version 1 carbon source. When grown in TAP medium, Chlamydomonas can 29 Jun 2020 report report report utilize the exogenous acetate in the medium for gluconeogenesis using the glyoxylate cycle, which is also present in many bacteria and 1. Dickson Aruhomukama , Makerere higher plants. A novel bacterial strain, LMJ, was isolated from a contaminated TAP medium plate of Chlamydomonas. We present our University, Kampala, Uganda work on the isolation and physiological and biochemical characterizations of LMJ. 2. Ruby A Ynalvez , Texas A&M International Methods: Several microbiological tests were conducted to University, Laredo, TX, USA characterize LMJ, including its sensitivity to four antibiotics. We amplified and sequenced partially the 16S rRNA gene of LMJ. We 3. Juan Campos Guillén , Universidad tested if LMJ can utilize cyclic alkanes, aromatic hydrocarbons, poly- Autónoma de Querétaro, Santiago de hydroxyalkanoates, and fresh and combusted car motor oil as the sole Querétaro, Mexico carbon source on Tris-Phosphate (TP) agar medium plates for growth. Results: LMJ is a gram-negative rod, oxidase-positive, mesophilic, Any reports and responses or comments on the non-enteric, pigmented, salt-sensitive bacterium. LMJ can ferment article can be found at the end of the article. 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 Page 1 of 26 F1000Research 2020, 9:656 Last updated: 01 JUN 2021 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. Keywords Chlamydomonas, Acidovorax sp., TAP medium, LMJ, bioremediation, 16S rRNA gene, penicillin-sensitivity, pyomelanin Corresponding author: Mautusi Mitra ([email protected]) Author roles: Mitra M: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing; Nguyen KMAK: Formal Analysis, Investigation, Methodology, Software, Supervision, Validation, Visualization; Box TW: Formal Analysis, Investigation, Methodology, Supervision, Visualization; Gilpin JS: Formal Analysis, Investigation, Methodology, Validation, Visualization; Hamby SR: Formal Analysis, Investigation, Methodology, Validation, Visualization; Berry TL: Formal Analysis, Investigation, Methodology, Visualization; Duckett EH: Methodology, Resources, Supervision Competing interests: No competing interests were disclosed. Grant information: The project was supported by the University of West Georgia, College of Science & Mathematics Faculty Research Grant (2018). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2020 Mitra M et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite this article: Mitra M, Nguyen KMAK, Box TW et al. 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 [version 1; peer review: 3 approved] F1000Research 2020, 9:656 https://doi.org/10.12688/f1000research.24680.1 First published: 29 Jun 2020, 9:656 https://doi.org/10.12688/f1000research.24680.1 Page 2 of 26 F1000Research 2020, 9:656 Last updated: 01 JUN 2021 Introduction JX086489.1). We have submitted the partial 16S rRNA Our research laboratory primarily focuses on the functional sequence of LMJ to the GenBank with the definition: Bacterium genomics of eukaryotic oxygenic photosynthesis using the strain clone LIB091_C05_1243 variant 16S ribosomal RNA gene, model green micro-alga, Chlamydomonas reinhardtii. The work partial sequence (Accession number: MN633292.1). The in this article stemmed from a side research project of four nearest relative of LMJ with a genus name is Acidovorax sp. undergraduates from the University of West Georgia and, one strain A16OP12 (Accession #: MN519578.1). high school student from the Carrollton High School in Georgia. This undergraduate/high school student research Betaproteobacteria are highly metabolically diverse and con- project was centered on minimizing bacterial contamination of tain chemolithoautotrophs, photoautotrophs, generalist het- C. reinhardtii in our research lab. erotrophs and opportunistic pathogens. Acidovorax is a genus of betaproteobacteria of the order Burkholderiales and family Chlamydomonas can be grown at the lab heterotrophi- Comamonadaceae18,19. Strains classified as Acidovorax have cally or photo-heterotrophically at room temperature in been associated with soils, wastewater treatment plants, plants, Tris-Phosphate-Acetate (TAP) medium, which contains ace- and clinical samples20–28. Reflecting the diversity inherent in this tate as the sole carbon source1. When grown in TAP medium, widespread distribution, isolates within this diverse genus Chlamydomonas can utilize the exogenous acetate to synthe- have been linked to a variety of phenotypes ranging from size glucose using the glyoxylate cycle, without being depend- plant pathogenicity, to denitrification, to the biodegradation of ent on photosynthesis for glucose biosynthesis2. Aerobic bacteria contaminants29. can also utilize glyoxylate cycle to metabolize fatty-acids or two carbon-compounds such as acetate to synthesize oxaloac- Acidovorax sp. can degrade alkanes, polyhydroxyalkanoates etate, which can be used for gluconeogenesis3,4. The acetate in (PHA) and polycyclic aromatic hydrocarbons (PAH)30–38. We the TAP medium can be substituted with alternative carbon tested the growth of LMJ on Tris-Phosphate (TP)-agar medium sources to test if bacteria can use the tested chemicals as the containing PAH, PHA, other aromatic compounds, cyclic sole alternative carbon and energy source. alkanes and 10W30 car motor oil to determine if LMJ can use these compounds as the sole carbon source. LMJ was able to We isolated a bacterial strain from a contaminated Chlamydomonas use cyclohexyl chloride, phenanthrene, naphthalene, benzoate, TAP-agar medium culture plate. This bacterium was named phenyl acetate and fresh and combusted 10W30 car motor oil. LMJ, after the abbreviated name of the Chlamydomonas strain The abovementioned chemicals occur as common environmental it contaminated: LMJ.SG0182. We performed growth analyses pollutants in soil and water. In future, whole genome sequenc- and several microbiological tests on LMJ. LMJ grows faster ing of LMJ and proper quantitative assays to determine LMJ’s on TAP medium than on lysogeny broth (LB) medium, is a ability to degrade (and remove) toxic chemicals from the mesophilic, non-enteric, gram-negative bacillus. LMJ does not TP-growth medium are needed to probe its full potential for grow on Mannitol Salt Agar (MSA) and MacConkey agar usage in environmental bioremediation. In this primarily (MAC) and is sensitive to 1% NaCl. LMJ is starch hydrolysis- undergraduate and high school student-driven research project, negative, gamma hemolytic and is oxidase-positive. we are presenting our research on the isolation and physi- ological and biochemical characterizations of the novel bacterial LMJ has a light pinkish-brown color on LB agar and strain, LMJ. creamy white color on TAP agar. We have found that on TAP + 1%