Evaluating the microcystin-LR-degrading potential of bacteria growing in extreme and polluted environments

{"Background": "Microcystins are cyclic heptapeptide toxins produced by freshwater cyanobacteria, with microcystin-LR (MC-LR) being the most toxic congener (LD50 of 50 \u00b5g/kg in mice). These toxins are hepatotoxic and carcinogenic, and due to their cyclic structure, they are highly stable in aquatic environments. Microbial degradation is the primary natural mechanism for microcystin removal and detoxification. While microcystin-degrading bacteria have been isolated from various environments, extreme and polluted environments\u2014which harbor specialized microbial communities with unique metabolic capabilities\u2014remain largely unexplored as sources of MC-LR degraders. This study aimed to evaluate the MC-LR-degrading potential of bacteria isolated from saltpans, hot springs, car wash effluents, oil refinery effluents, and tannery dumpsite soils.", "Methods": "Bacterial isolates were obtained from five sources: saltpans, hot springs, car wash effluents (previously isolated), and oil refinery plant effluents and tannery dumpsite soils (isolated in this study). For the latter two, samples were collected aseptically, transported on ice, and processed within 12 hours. Bacterial communities were enriched in MC-LR (3 mg/L) for 21 days at 25\u00b0C in minimal salts media (MSM). Surviving bacteria were isolated on nutrient agar. Isolates were characterized by MALDI-TOF-MS biotyping (formic acid extraction method, Bruker ultrafleXtreme MALDI Biotyper 2.0) and 16S rRNA gene sequencing (27F/1492R primers, Sanger sequencing). The Biolog MT2 microplate colorimetric assay was used to screen 32 bacterial strains for MC-LR-degrading ability. Cells were washed, resuspended in MSM (OD590 adjusted to 1.5), and inoculated into wells containing MC-LR at 1, 10, and 100 \u00b5g/L. Plates were incubated at 30\u00b0C in the dark for up to 120 hours, with OD590 measurements taken at 0, 3, 6, 9, 18, 21, 24, 27, 30, 33, 48, 60, 72, and 96 hours. The effect of pH was tested at pH 6, 8, and 9 with 10 \u00b5g/L MC-LR. Positive growth was defined as a significant increase (ANOVA, p < 0.05) in OD590 compared to controls. All isolates were screened by PCR for the mlr gene cluster (mlrA, mlrB, mlrC, mlrD) using published primers and cycling conditions.", "Key Results": "Seventeen isolates were obtained from oil refinery effluents (10 isolates) and tannery dumpsite soil (7 isolates), belonging to genera Bacillus, Paenibacillus, and Lysinibacillus (phylum Firmicutes). Of the 32 total bacterial strains screened, 10 showed significant growth (ANOVA, p < 0.05) in the presence of MC-LR: Arthrobacter sp. B105, Arthrobacter junii, Plantibacter sp. PDD-56b-14, Acinetobacter sp. DUT-2, Salinivibrio sp. YH4, Pseudomonas protegens, Bacillus sp. (TL2), Bacillus thuringiensis, Lysinibacillus boronitolerans, and Bacillus sp. (TL17). Most bacteria grew optimally at 10 \u00b5g/L MC-LR. Arthrobacter sp. and Bacillus sp. APB SML B20 showed highest growth at 100 \u00b5g/L. For Acinetobacter sp. and Bacillus sp. (TL2), growth was minimal at 1 \u00b5g/L with no significant difference between 10 and 100 \u00b5g/L. Acinetobacter junii showed minimal growth at 100 \u00b5g/L. Regarding pH effects: Pseudomonas protegens, Arthrobacter sp., and Plantibacter sp. grew optimally at alkaline pH (8 and 9); Bacillus sp. (TL2) and Lysinibacillus boronitolerans P2IIIb grew optimally at pH 6; for Salinivibrio sp., Bacillus sp. (TL17), Bacillus thuringiensis, and Acinetobacter junii, pH had no significant effect. PCR screening for mlrA, mlrB, mlrC, and mlrD genes was negative for all 32 isolates, indicating absence of the known mlr gene cluster.", "Clinical Implications": "This study identifies several novel potential MC-LR-degrading bacteria from underexplored extreme and polluted environments, including first reports of Pseudomonas protegens, Bacillus thuringiensis, Plantibacter sp., Salinivibrio sp., and Lysinibacillus boronitolerans as tentative MC-LR degraders. The ability of these bacteria to metabolize MC-LR at environmentally relevant concentrations (1\u2013100 \u00b5g/L) and under alkaline pH conditions (common during cyanobacterial blooms) suggests practical applicability for water treatment and bioremediation. The absence of the mlr gene cluster in all positive isolates implies a potentially novel degradation pathway, which warrants further investigation. These findings expand the known taxonomic and environmental distribution of MC-LR-degrading microorganisms and highlight the value of extreme environments as reservoirs of bioremediation-relevant microbes."}