Optimizing conditions for carbofuran degrading isolates: a pathway to sustainable bioremediation in agricultural settings

Agriculture, driven by the escalating global demand for food, heavily relies on pesticides to ensure high yields and pest-free produce. The carbamate group of pesticides, including the widely used carbofuran, presents advantages such as low retention, high efficacy, and broad application. However, the associated health concerns categorize these pesticides as hidden threats. In response, biological remediation emerges as a more cost-efficient and environmentally friendly alternative to chemical or physical approaches. Our prior investigation identified 17 highly potential carbofuran degraders, forming the basis for this research. The focus lies on optimizing the isolates’ growth conditions, exploring temperatures (30 °C and 37 °C) and pH levels (7 and 9) in Bushnell Haas Agar. It was observed that all four pesticide doses (250, 500, 750, and 1000 mg/L) in Bushnell Haas Agar medium were supported by the majority of isolates at 30 °C and pH 9, whereas 750 and 1000 mg/L were favored at pH 7 (30 °C) and at both pH 7 and 9 (37 °C). Despite limited biofilm-forming ability rendered due to presence of ompA along with or with not bap gene, the isolates’ extracellular polymeric substance (EPS) composition indicated a polymeric nature. Also 13 of the isolates’ EPS production accelerates at 250 mg/L pesticide concentration. Metabolic pathway analyses revealed a preference for hydrolytic degradation, while molecular examinations confirmed the presence of plasmid and the absence of specific genes mcd and cehA responsible for carbamate – hydrolase on the chromosome. Concern is raised by the presence of colistin and tetracycline resistant genes (mcr2 (47.05%), tetA (29.41%), and qnrs (23.53%) in the genomic content of some isolates, in addition to ESBL resistance genes found in varying frequencies, such as blaIMP3 (5.89%), blaVIM (70.59%), blaKPC (88.25%), blaOXA-1 (11.76%), bla-SHV (23.53%) & blaOXA1 (11.76%), bla-CTMX15 (17.65%), necessitating alternative mechanisms. The findings demonstrate the isolates’ dual function as repositories of antibiotic resistance and prospective bioremediation agents, requiring careful handling and application. Developing safe and efficient bioremediation systems will need addressing resistance issues while maximizing degrading efficiency.

受全球食品需求持续攀升驱动，农业生产高度依赖农药以保障高产及无虫害的农产品。包括广泛使用的克百威（carbofuran）在内的氨基甲酸酯类农药（carbamate pesticides）具有残留量低、药效高、应用范围广等优势，但伴随的健康风险将此类农药列为隐性威胁。对此，生物修复（biological remediation）相较于化学或物理修复手段，是一种更具成本效益且环境友好的替代方案。我们此前的研究已筛选出17株具有高降解潜力的克百威降解菌，为本研究奠定了基础。本研究聚焦于优化分离菌株的生长条件，在布什内尔-哈斯琼脂（Bushnell Haas Agar）培养基中探究温度（30℃与37℃）及pH值（7与9）对其生长的影响。实验结果显示，在30℃、pH9的条件下，多数分离菌株可耐受布什内尔-哈斯琼脂培养基中的全部4种农药浓度（250、500、750及1000mg/L）；而在30℃、pH7的条件下，以及37℃、pH7与pH9的条件下，菌株更偏好750与1000mg/L的农药浓度。尽管携带ompA基因且伴随/不携带bap基因会限制菌株的生物膜（biofilm）形成能力，但分离菌株的胞外聚合物（extracellular polymeric substance, EPS）组分仍呈现出聚合物特性。此外，在250mg/L的农药浓度下，13株分离菌株的EPS合成量显著提升。代谢通路分析显示菌株偏好采用水解降解（hydrolytic degradation）途径；分子检测结果证实，菌株携带质粒（plasmid），但染色体上不存在负责编码氨基甲酸酯水解酶（carbamate hydrolase）的特定基因mcd与cehA。部分分离菌株的基因组中携带黏菌素（colistin）与四环素（tetracycline）耐药基因：mcr2（47.05%）、tetA（29.41%）及qnrs（23.53%）；同时还存在不同频率的超广谱β-内酰胺酶（extended-spectrum beta-lactamases, ESBL）耐药基因，包括blaIMP3（5.89%）、blaVIM（70.59%）、blaKPC（88.25%）、blaOXA-1（11.76%）、bla-SHV（23.53%）、blaOXA1（11.76%）及bla-CTMX15（17.65%），这提示需采用替代防控策略。研究结果表明，这些分离菌株兼具抗生素耐药基因储存库与潜在生物修复剂的双重属性，因此需谨慎管控其使用与应用。开发安全高效的生物修复系统，需在最大化降解效率的同时解决耐药性问题。