梯度驯化法定向培育高效石油烃降解菌群及功能菌资源挖掘

Directed cultivation of efficient petroleum hydrocarbon-degrading microbial consortia via gradient domestication and exploitation of functional bacterial resources

摘要:

目的 探究不同浓度石油烃条件下微生物群落结构特征，定向培育高效石油烃降解微生物菌群，并挖掘具有石油烃降解功能的菌株资源。方法 以0#柴油作为唯一碳源，通过逐级提高其浓度对石油污染土壤样品进行逐级驯化，共进行5代驯化。基于16S rRNA基因扩增子测序技术揭示微生物群落结构变化，利用稀释涂布和平板划线法分离纯化具有石油烃降解潜力的菌株。通过改进2,6-二氯靛酚培养体系验证实验筛选高效降解菌株。结果 随着驯化过程中0#柴油浓度的升高，在7 000 mg/L条件下拟杆菌门(Bacteroidota)、芽孢杆菌门(Bacillota)等具有石油烃降解功能的细菌门相对丰度显著升高。共分离得到58株细菌，分属于4门22科25属。其中，假单胞菌门(Pseudomonadota) 31株，占比53.45%；放线菌门(Actinomycetota) 13株，占比22.41%；芽孢杆菌门(Bacillota) 11株，占比18.97%；拟杆菌门(Bacteroidota) 3株，占比5.17%。同时，筛选获得18株具有潜在石油烃降解功能的菌株。结论 通过逐级驯化培养，成功富集获得7个在7 000 mg/L 0#柴油浓度下石油烃降解率超过70%的驯化菌群。扩增子测序表明，不同浓度的0#柴油明显改变了微生物群落结构，并筛选得到18株能以0#柴油作为唯一碳源进行生长的细菌，为微生物修复石油污染土壤提供了潜在的菌种资源。

Abstract:

Objective To investigate the structural characteristics of microbial consortia in different concentrations of petroleum hydrocarbons, cultivate efficient petroleum hydrocarbon-degrading microbial consortia, and mine the strain resources capable of degrading petroleum hydrocarbons.Methods We used 0# diesel as the sole carbon source to domesticate oil-contaminated soil samples through five successive generations by gradually increasing the 0# diesel concentration. The structural changes of microbial consortia were uncovered by 16S rRNA gene amplicon sequencing. The strains with petroleum hydrocarbon-degrading potential were isolated and purified via dilution plating and streaking. Finally, the improved 2,6-dichlorophenol indophenol (DCPIP) cultivation system was employed to identify efficient degrading strains.Results During domestication, when the concentration of 0# diesel was raised to 7 000 mg/L, the relative abundance of petroleum hydrocarbon-degrading bacteria including Bacteroidota and Bacillota significantly increased. A total of 58 bacterial strains belonging to 25 genera, 22 families of 4 phyla were isolated, including 31 (53.45%) strains of Pseudomonadota, 13 (22.41%) strains of Actinomycetota, 11 (18.97%) strains of Bacillota, and 3 (5.17%) strains of Bacteroidota. From the isolated strains, 18 petroleum hydrocarbon-degrading strains were screened out.Conclusion Through gradient domestication, seven natural microbial consortia were successfully enriched, achieving over 70% degradation of petroleum hydrocarbons at 7 000 mg/L of 0# diesel. Amplicon sequencing revealed that varying 0# diesel concentrations altered the microbial consortium structure. Additionally, 18 strains capable of using 0# diesel as the sole carbon source were identified, providing potential microbial resources for the bioremediation of oil-contaminated soil.