海水养殖生境中磺胺甲恶唑降解菌的筛选及降解条件优化

Screening of sulfamethoxazole-degrading bacteria in mariculture habitats and optimization of degradation conditions

摘要:

在海水养殖过程中，磺胺甲恶唑(sulfamethoxazole, SMX)等抗生素类药物的大量残留加速了抗性细菌和抗性基因的传播，严重威胁生态环境健康。生物法控制抗生素废水是解决其环境危害的重要途径。目的 从近海养殖池底泥中筛选出一株耐盐且对SMX具有高效降解能力的菌株LS-1，分析环境因素对其降解能力的影响，优化菌株对SMX的降解性能，并通过产物类型解析其降解途径，最终对降解产物进行毒性分析。方法 通过对分离菌株进行16S rRNA基因序列测序与系统发育树分析进行鉴定，采用单因素和响应面试验对降解条件进行优化，利用气质色谱法及发光细菌水质急性毒性试验检测分析其降解产物及产物毒性。结果 分离获得的菌株LS-1与产碱杆菌属(Alcaligenes)中的水生产碱菌(Alcaligenes aquatilis) AS1序列相似度达99.79%。单因素试验确定胰蛋白胨是菌株生长和降解SMX时的最佳外源碳源。菌株在温度20-35 ℃、盐度15‰-35‰、SMX浓度10-100 mg/L、pH 7.0-9.0的条件下时生长良好。响应面分析表明，对SMX降解率有显著影响的因素依次为：SMX浓度>初始pH>环境温度。在SMX浓度为33 mg/L、pH 7.4和30 ℃的条件下，该菌株在48 h内的最高降解率达60.17%。质谱检测分析推测菌株LS-1通过乙酰化和羟基化等途径降解SMX，发光细菌急性毒性试验表明在SMX降解过程中生物毒性逐渐降低。结论 本研究分离的SMX降解菌能够很好地适应海洋环境条件，降低SMX的水质毒性，对海水养殖废水中抗生素污染的防治具有重要的应用前景。

关键词:海水养殖;磺胺甲恶唑;Alcaligenes sp.;微生物降解;降解产物

Abstract:

In marine aquaculture, the accumulation of antibiotics such as sulfamethoxazole (SMX) has contributed to the spread of antibiotic-resistant bacteria and genes, posing a serious threat to ecological health. Biological treatment of antibiotic-contaminated wastewater is an essential approach to mitigate these environmental risks.Objective To isolate a salt-tolerant strain LS-1 with high SMX degradation efficiency from the sediment of an inshore aquaculture pond, examine the effects of environmental factors on the degradation capacity of this strain, optimize the SMX degradation conditions, elucidate the degradation pathway through product analysis, and evaluate the toxicity of the degradation products.Methods The isolated strain was identified by 16S rRNA gene sequencing and phylogenetic analysis. Single factor experiments and response surface methodology were employed to optimize the degradation conditions. GC-MS and the luminescent bacteria test for acute toxicity were adopted to analyze the degradation products and their toxicity.Results Strain LS-1 showed 99.79% sequence similarity with Alcaligenes aquatilis strain AS1. Tryptone was determined to be the optimal exogenous carbon source for both growth and SMX degradation. The strain exhibited robust growth across a temperature range of 20?35 ℃, salinities of 15‰?35‰, SMX concentrations from 10 to 100 mg/L, and pH 7.0?9.0. Response surface analysis revealed that SMX concentration, initial pH, and temperature significantly influenced the SMX degradation rate, in descending order of importance. Under optimal conditions (SMX concentration of 33 mg/L, pH 7.4, and 30 ℃), the strain achieved a maximum degradation rate of 60.17% within 48 h. MS results indicated that LS-1 degraded SMX via acetylation and hydroxylation pathways. The results of the luminescent bacteria test for acute toxicity demonstrated a progressive reduction in biological toxicity during the SMX degradation process.Conclusion The SMX-degrading strain LS-1 can effectively adapt to marine environmental conditions, reducing SMX-induced toxicity in water. This study highlights the potential of LS-1 for controlling antibiotic pollution in marine aquaculture wastewater.

Key words:marine aquaculture;sulfamethoxazole;Alcaligenes sp.;microbial degradation;degradation products