Isolation of ethidium bromide degrading bacteria

Abstract

Ethidium bromide (EtBr), a widely used fluorescent dye in molecular biology for DNA visualization in gel electrophoresis, poses significant environmental and health risks due to its mutagenic and carcinogenic properties. Traditional disposal methods, such as incineration, charcoal filtration, chemical neutralization, and direct burial or drainage, often lead to contamination of groundwater and ecosystems through various pathways. This study aimed to address these challenges by isolating and evaluating bacteria capable of biologically degrading EtBr, offering a safer, environmentally friendly alternative. The general objective was to isolate EtBr-degrading bacteria, with specific goals to source them from gel electrophoresis waste and rubbish soil at Makerere University's College of Natural Sciences, and to assess their degradation efficiency. Samples were collected from laboratory waste buckets and a rubbish pit, diluted serially (up to 10^7 for soil and 10^4 for gel waste), and plated on nutrient agar (NA) for initial colony isolation. Five distinct bacterial isolates (EtBr1–EtBr5) were obtained: four from rubbish soil and one (EtBr3) from gel waste. Pure subcultures were streaked onto Bushnell-Haas (BH) media amended with 0%, 0.5%, 1%, and 1.5% EtBr to evaluate growth and degradation. Gram staining revealed EtBr1 and EtBr5 as Gram-negative rods, while EtBr2, EtBr3, and EtBr4 were Grampositive (rods or concave/round shapes). Cultural characteristics included variations in color (greyish white to greenish blue), margin (smooth or rough), transmittance (opaque or translucent), and appearance (moist or glistening). Degradation was quantified by measuring halo zones under UV illumination (00s08 exposure) using ImageJ software, expressed as mean areas in mm². At 1.5% EtBr, degradation ranged from 0.001 mm² (EtBr1) to 0.222 mm² (EtBr3), with EtBr3 showing the highest overall efficiency across concentrations. Statistical analysis via Shapiro-Wilk normality test confirmed normal distribution (p > 0.05), and one-way ANOVA (n=15, DF=14, F=3.56 > F-critical=3.48) indicated significant differences in degradation among isolates at 1% EtBr, rejecting the null hypothesis of no difference. All isolates demonstrated EtBr degradation by thriving on amended media, likely utilizing it as a carbon source, with EtBr3's superior performance attributed to its direct exposure in gel waste. The study concludes that gel electrophoresis waste is the optimal source for potent EtBr-degrading bacteria. Recommendations include molecular characterization of EtBr3 for potential application in bioremediation systems or disposal plants, promoting sustainable laboratory waste management to mitigate environmental contamination. This research highlights the viability of microbial degradation as a non-toxic, cost-effective solution over conventional methods.