{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,30]],"date-time":"2026-06-30T01:19:39Z","timestamp":1782782379011,"version":"3.54.5"},"update-to":[{"DOI":"10.12688\/f1000research.53779.1","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2021,9,7]],"date-time":"2021-09-07T00:00:00Z","timestamp":1630972800000}}],"reference-count":75,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2021,9,7]],"date-time":"2021-09-07T00:00:00Z","timestamp":1630972800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["f1000research.com"],"crossmark-restriction":false},"short-container-title":["F1000Res"],"abstract":"\n Background:<\/ns3:bold>\n Chlamydomonas reinhardtii<\/ns3:italic>\n , 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\n Chlamydomonas<\/ns3:italic>\n TAP plate. It was named Clip185 based on the\n Chlamydomonas<\/ns3:italic>\n 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.\n <\/ns3:p>\n \n Methods:<\/ns3:bold>\n 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.\n <\/ns3:p>\n \n Results:<\/ns3:bold>\n 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\n Microbacterium binotii<\/ns3:italic>\n strain PK1-12M and\n Microbacterium sp.<\/ns3:italic>\n strain MDP6. Clip185 is able to tolerate toxic concentrations of six heavy metals.\n <\/ns3:p>\n \n Conclusions:<\/ns3:bold>\n Our results show that Clip185 belongs to the genus\n Microbacterium<\/ns3:italic>\n . In the future, whole genome sequencing of Clip185 will clarify if Clip185 is a new\n Microbacterium<\/ns3:italic>\n species or a novel strain of\n Microbacterium binotii<\/ns3:italic>\n , and will reveal its genes involved in antibiotic-resistance, heavy-metal tolerance and regulation of decaprenoxanthin biosynthesis.\n <\/ns3:p>","DOI":"10.12688\/f1000research.53779.2","type":"journal-article","created":{"date-parts":[[2021,9,7]],"date-time":"2021-09-07T08:25:08Z","timestamp":1631003108000},"page":"533","update-policy":"https:\/\/doi.org\/10.12688\/f1000research.crossmark-policy","source":"Crossref","is-referenced-by-count":5,"title":["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."],"prefix":"10.12688","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8391-8375","authenticated-orcid":false,"given":"Mautusi","family":"Mitra","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kevin Manoap-Anh-Khoa","family":"Nguyen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Taylor Wayland","family":"Box","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Taylor Lynne","family":"Berry","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Megumi","family":"Fujita","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"2560","published-online":{"date-parts":[[2021,9,7]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"245-250","DOI":"10.1126\/science.1143609","article-title":"The Chlamydomonas genome reveals the evolution of key animal and plant functions.","volume":"318","author":"S Merchant","year":"2007 Oct 12","journal-title":"Science."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"523-531","DOI":"10.1111\/tpj.12780","article-title":"Chlamydomonas as a model for biofuels and bio-products production.","volume":"82","author":"M Scranton","year":"2015 May 1","journal-title":"Plant J."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"486-501","DOI":"10.1128\/EC.00364-09","article-title":"Genetic engineering of algae for enhanced biofuel production. 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Ynalvez, Department of Biology and Chemistry, Texas A&M International University, Laredo, TX, USA, 12 Aug 2021, version 1, 1 approved, 1 approved with reservations","URL":"https:\/\/f1000research.com\/articles\/10-533\/v1#referee-response-89082","order":1,"name":"referee-response-89082","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"Mautusi Mitra<\/b>; \nPosted: 26 Aug 2021<\/i>; Please see our point-by-point responses to your queries given below. Thanks for investing your time and energy to review our manuscript and providing us valuable inputs to improve the manuscript quality. Hope you will be willing to review our version 2 of the article that has addressed your comments. \n1. Reviewer\u2019s query\/comments for Keywords: <\/b>Italicize "Chlamydomonas", "Microbacterium binotii". \nOur answer:<\/b> We had the above-stated two words in italics when we submitted our article in version 1. The italicization was not retained after publication as the italics function is not yet implemented into this section of the F1000Research article management system. Hence we are unable to implement this change. \n2. Reviewer\u2019s query\/comments for Introduction section: <\/b> a.\n Reviewer\u2019s query\/comments:<\/b>  Tris-Phosphate-Acetate (TAP) change to Tris- Acetate- Phosphate (TAP). \nOur answer:<\/b> We have made the change. b.  \nReviewer\u2019s query\/comments:<\/b> Clip185 could grow in the presence of 6 mM chromium, 2 mM of nickel, cadmium and zinc, and 0.5 mM of copper and cobalt in the LB medium. As a reader, I was wondering what will be the bacteria\u2019s minimum tolerable concentration of each of these heavy metals? It will be helpful to have these tolerable concentrations as reported in the literature. It does not have to be Microbacterium sp., but if there were reports for Microbacterium sp. then the better. \nOur answer: <\/b>We agree that this is a very good suggestion.\n <\/b>We could not find any literature record of minimum concentrations of different heavy metals tolerated by \nMicrobacterium sp.<\/i> or by any species of bacteria. There is a report of heavy metal tolerance of 16 \nMicrobacterium sp.<\/i> strains isolated from metal-contaminated soil in Indiana, USA. We have compared the heavy metal tolerance of Clip185 with these 16 \nMicrobacterium sp.<\/i> isolates and have added the information with citations under Introduction and also in paragraph 5 under the Discussion section. 3. \nReviewer\u2019s query\/comments for the <\/b>\nMethods section:<\/b> a. \nReviewer\u2019s query\/comments<\/b>: Antibiotic plates were incubated at 22°C. Clip185 and \nChlamydomonas<\/i> plates were imaged after 7 days of incubation. On the other hand, streaked TAP + vancomycin plates were imaged after incubation at 22°C for 2.5 weeks. What is the reason there is a difference in incubation, 7 days versus 2.5 weeks between the other antibiotics and that of vancomycin? \nOur answer:<\/b> We have added the explanation for monitoring Clip185 growth for 3 days on LB plates vs. monitoring Clip185 growth for 7 days on TAP plates under the Methods section sub-section: Testing antibiotic sensitivity of Clip185. We have also revised the Results section sub-section: Clip185 antibiotic susceptibility tests accordingly. We wanted to see if the selected amount of vancomycin was bactericidal or bacteriostatic. In other words, we wanted to see if we will see re-growth of Clip185 on the antibiotic-containing plate if we have a longer incubation of 2.5 weeks compared to 7-days incubation. b. \nReviewer\u2019s query\/comments<\/b>: In Clip 185 growth analysis: The same thing, it will be clearer to all readers if the authors will add why LB-agar and TAP agar culture plates were imaged after 3 days and 7 days of growth, respectively. \nOur answer: <\/b> We have added the explanation in the Methods section sub-section: Clip185 growth analyses and also have revised the result section sub-section: Clip185 growth analyses on TAP- and LB-agar medium plates at different temperatures. c. \nReviewer\u2019s query\/comments<\/b>: The authors specified why \nS. aureus<\/i> was used in MSA. Thus, also specify why \nAcidovorax sp.<\/i> was used on TSBA. \nOur answer:<\/b> We have added the explanation under the Methods section sub-section: Growth assays on Tryptic Soy Blood Agar (TSBA) and also have revised the Results section sub-section: Testing for Clip185\u2019s ability to lyse red blood cells. d. \nReviewer\u2019s query\/comments<\/b>: Please indicate why \nCorynebacterium glutamicum<\/i> strain ATCC13032 and \nKocuria rhizophila<\/i> ATCC?? were used along with Clip 185 testing light-regulation of decaprenoxanthin production? Although it was mentioned later in the article why, I think it\u2019s better to clarify this already in the Methods section, again, just like how it was clear why \nS. aureus<\/i> was used in MSA. \nOur answer: <\/b>We have added the explanation under the Methods section sub-section: Decaprenoxanthin extractions and spectrophotometric analyses. We also added the \nK. rhizophila<\/i> ATCC strain number (ATCC 9341) in version 2. e. \nReviewer\u2019s query\/comments<\/b>: It was mentioned that there were three replicates per metal concentration in the growth assays on LB media containing heavy metals experiment. On the other hand, how many trials were there per replicate? Same thing, three replicates per strain were used in the decaprenoxanthin extractions and spectrophotometric analyses experiment. On the other hand, how many trials were there per replicate? \nOur answer: <\/b>We had three biological replicates. Each biological replicate had three internal replicates. This information has been added under the Methods section sub-sections: Growth assays on LB media containing heavy metals and Decaprenoxanthin extractions and spectrophotometric analyses. f. \nReviewer\u2019s query\/comments<\/b>: There was no mention of how many replicates and trials were performed in physiological and biochemical experiments. I recommend mentioning the number of replicates and trials within replicates for the other experiments. \nOur answer:<\/b> We have added the number of biological replicates and technical replicates for all experiments under the Methods section. 4. \nReviewer\u2019s query\/comments for the Result section:<\/b> a. \nReviewer\u2019s query\/comments<\/b>: Grey and white rows represent 50 µg and 100 µg of each antibiotic applied on the filter paper discs \u2013 please double-check Table 1. \nOur answer:<\/b> We have double-checked Table 1. The Table 1 legend is correct. Grey and white rows represent 50 µg and 100 µg of each antibiotic applied on the filter paper discs, respectively. Table 1 captions are correct in version 1 of the article. The numerical values in each row denote the diameters of zones of antibiotic-induced growth inhibitions. The diameter of the zone of growth inhibition is directly proportional to the sensitivity of the bacterium to the applied antibiotic. Hence, the larger the diameter of the zone of growth inhibition is, the more sensitive is the bacterium to the applied antibiotic and vice versa.  But we did find contradictory wordings and phrases in the text that describes Table 1 which might have confused the reviewer. We have revised the text description of Table 1. We hope this clarifies the concern of the reviewer. b. \nReviewer\u2019s query\/comments<\/b>: Authors stated: \u201cWe used polymyxin B amounts that were 1.4- fold to 2.8-fold higher than what is used for KB tests, and growth inhibition zones of Chlamydomonas and Clip185 were less than 13 mm (Table 1).\u201d Why not use less than 12 mm instead of less than 13 mm to be consistent with a previous statement that used less than 12 mm as a reference? \nOur answer:<\/b> We have rectified the typographic error and have changed the growth inhibition zone diameter from 13 mm to 12 mm. Thanks for catching that mistake. c. \nReviewer\u2019s query\/comments<\/b>: In Figure 9, it could be helpful to add: Both Clip185 (B) and (D) were tested positive in the starch hydrolysis test as shown by a visible clear zone indicated by the arrows surrounding the growth (or if not an arrow perhaps encircle that area, so that the clear zone will be more easily seen by the readers). \nOur answer:<\/b> We have added black arrows in Figure 9B and Figure 9D to highlight the clear starch-hydrolyzed zone in these two figures. We have revised the Figure 9 legend and the respective result section accordingly. d. \nReviewer\u2019s query\/comments<\/b>: I recommend a revision to: This result strongly suggests the presence of decaprenoxanthin in Clip185. HPLC and\/or LC\/MS analyses could confirm the presence of decaprenoxanthin in Clip185. However, we do not have access to such analytical tools at our institution. Original statements were: This result very strongly indicates the presence of decaprenoxanthin in Clip185. We do not have access to such analytical tools at our institution. \nOur answer:<\/b>  We have accepted the reviewer\u2019s suggestion and have revised the sentences under the Results section sub-section: Spectrophotometric analyses of pigments synthesized by Clip185. e. \nReviewer\u2019s query\/comments<\/b>: Please double-check the figures and the corresponding figure titles for Figures 15, 16, and 17. For example, Figure 15 has plates labelled Zinc but the title is Figure 15. Growth of Clip185 on LB media containing Cobalt. \nOur answer: <\/b> We have uploaded correct Figures 15, 16, and 17 which match the respective figure legends in version 2. We did submit the correct figures\/legends when we submitted version 1 of the article but during the publication process, for some reason the figures and legends got switched.","URL":"https:\/\/f1000research.com\/articles\/10-533\/v2#referee-comment-7085","order":2,"name":"referee-comment-7085","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"This project was supported by the University of West Georgia (UWG), College of Science & Mathematics Faculty Research Grant (2018) and UWG Student Research Assistant Program Grants (2018 - 2020). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.","order":3,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}