一株对乙氧基苯胺降解菌的筛选及降解特性

Screening and degradation characterization of a para-ethoxyaniline-degrading strain

摘要:

对乙氧基苯胺(para-ethoxyaniline, ETH)作为一种广泛应用的工业原料和中间体，其在环境中的残留可能具有持久性，从而对生态环境和人类健康构成潜在风险。目的 从活性污泥中筛选对ETH具有高效降解能力的菌株，优化其降解条件，并基于转录组学和质谱分析揭示该菌株在ETH胁迫下的基因表达调控机制，识别关键降解相关基因及代谢通路。方法 以ETH为唯一碳源，从活性污泥中筛选和分离高效降解菌株，结合形态学观察、生理生化实验，以及基于16S rRNA基因序列构建系统发育树分析，对目标菌株进行分类鉴定。研究不同温度、pH和ETH初始浓度对菌株生长及ETH降解效率的影响。进一步利用转录组测序技术鉴定相关差异表达基因(differentially expressed genes, DEGs)，并使用实时荧光定量逆转录PCR (real-time reverse transcription quantitative PCR, RT-qPCR)验证部分上调基因，结合质谱分析结果深入解析相关代谢通路。结果 成功分离获得1株ETH高效降解菌DQ78，经形态学观察及分子生物学鉴定为假单胞菌(Pseudomonas sp.)。降解条件优化试验结果表明，菌株DQ78在温度28 ℃、pH 8.0、ETH初始浓度4 mmol/L、接种量1%的条件下，40 h内对ETH的降解率达到100%。对其降解产物进行鉴定，共鉴定出3种中间产物，初步提出了ETH的降解途径。转录组分析显示，ETH诱导组检测到3 380个DEGs，其中1 609个基因上调，1 771个基因下调。GO富集分析表明，在ETH胁迫条件下上调基因显著富集的GO terms有57个，主要涉及氨基酸代谢和合成、细胞运动、三价铁结合、物质转运等过程，这些通路可激活ETH降解相关酶的合成，增强底物摄取及中间产物跨膜代谢。下调的GO terms有58个，主要包括肽的代谢与合成、核糖体结构及细胞组成等通路，反映菌株为优先保障ETH降解相关功能蛋白合成，对非核心代谢进行能量重分配。KEGG富集结果显示，在ETH胁迫条件下上调的通路有183个，下调的有184个，主要涉及鞭毛组装、硫代谢及胞外生物合成等，表明菌株通过增强底物趋化捕获，促进酶分泌与抗逆物质合成等方式协同响应ETH胁迫。结论 菌株DQ78在40 h内完全降解ETH，为环境中ETH污染的生物修复提供了优良菌种和理论支持。转录组分析揭示了该菌响应ETH的分子调控机制，为深入探索微生物降解有机污染物的遗传基础提供了理论支持。

Abstract:

Para-ethoxyaniline (ETH), a widely used industrial raw material and intermediate, persists in the environment, posing potential risks to ecosystems and human health.Objective To isolate an efficient ETH-degrading strain from activated sludge, optimize its degradation conditions, and elucidate the gene regulatory mechanisms and metabolic pathways under ETH stress by transcriptomic and mass spectrometric analyses.Methods A strain capable of utilizing ETH as the sole carbon source was isolated from activated sludge and identified through morphological observation, physiological and biochemical tests, and phylogenetic tree construction based on 16S rRNA gene sequences. The effects of temperature, pH, and initial ETH concentration on bacterial growth and degradation efficiency were examined. Transcriptome sequencing was employed to identify differentially expressed genes (DEGs), with selected up-regulated DEGs validated by real-time reverse transcription quantitative (RT-qPCR). Furthermore, mass spectrometry was employed to investigate the metabolic pathways.Results A highly efficient ETH-degrading strain, designated DQ78 and identified as Pseudomonas sp., was isolated. Under optimal conditions (28 ℃, pH 8.0, 4 mmol/L ETH, and 1% inoculum), it completely degraded ETH within 40 h. Three metabolic intermediates were identified, allowing the proposal of a preliminary degradation pathway. Transcriptomic analysis revealed 3 380 DEGs under ETH stress, including 1 609 up-regulated and 1 771 down-regulated genes. GO enrichment indicated up-regulated genes were primarily involved in 57 GO terms such as amino acid metabolism, cell motility, iron binding, and transport, which might activate the synthesis of ETF-degrading enzymes and enhance substrate uptake and transmembrane metabolism of intermediates. The down-regulated genes were enriched in 58 GO terms such as peptide metabolism and synthesis, ribosomal structure, and cellular components, suggesting a metabolic reallocation toward stress adaptation. KEGG analysis predicted 183 up-regulated pathways and 184 down-regulated pathways such as flagellar assembly, sulfur metabolism, and extracellular biosynthesis under ETH stress, indicating enhanced chemotaxis, enzyme secretion, and stress-resistant substance synthesis.Conclusion Strain DQ78 achieved complete degradation of ETH within 40 h, being a promising candidate for the bioremediation of ETH-contaminated environments. Transcriptomic analysis reveals the molecular regulatory mechanism of this strain in response to ETH, which lays a theoretical foundation for further exploring the genetic foundation of microbial degradation of organic pollutants.