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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.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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