混合菌群DBFC对二苯并呋喃的降解特性及其代谢途径

Characterization of dibenzofuran biodegradation by a bacterial consortium DBFC

摘要:

[目的] 二苯并呋喃（DBF）是研究多环芳烃降解过程的模式化合物，研究其代谢过程和代谢途径对于阐明多环芳烃的代谢机制有重要意义。[方法] 从辽河河口区石油污染土壤中筛选到1个高效降解DBF的混合菌群DBFC。提取总DNA对菌群的生物多样性进行分析，通过稀释涂布平板法对菌株进行分离纯化。通过测定OD₆₀₀的吸收值对混合菌群的最适生长条件进行研究。在最适生长条件下研究底物浓度、底物谱、营养物质及表面活性剂对菌群降解效率的影响。利用超高分辨质谱检测混合菌群降解DBF的中间代谢物质，并推测其代谢途径。[结果] 生物多样性分析表明该混合菌群的组成为类芽孢杆菌（84.06%）、无色杆菌（8.17%）、假单胞菌（0.77%）、其他菌株（2.13%）。分离得到苍白杆菌、无色杆菌、寡养单胞菌和细杆菌。生长测定结果显示苍白杆菌、无色杆菌、寡养单胞菌和细杆菌均不能在DBF培养基中生长。混合菌群DBFC的最适生长条件为30℃、pH 8.0。在该条件下，混合菌群DBFC能将1.0 g/L的DBF在8 d内完全降解。在DBF浓度1.0 g/L条件下，混合菌群DBFC的最大降解速率为0.031 mmol/（L·h）。在培养基中添加葡萄糖、酵母粉和蛋白胨能将菌群降解DBF的效率分别提高1.38倍、1.14倍和1.24倍。在培养基中添加十二烷基磺酸钠或Triton-X-100能够抑制混合菌群降解DBF的效率。利用超高分辨质谱检测到4种中间代谢物质（2，2'，3-三羟基联苯、2，4-已二烯酸、龙胆酸和水杨酸），并推测了DBF代谢途径。[结论] 本研究分离到高效DBF降解菌群，该菌群能在碱性（pH 8.0）条件下完全降解DBF，为该类污染物的原位修复提供优良菌系；利用超高分辨质谱分析得到了DBF降解途径，为该类物质的混合菌群代谢研究提供了参考。

Abstract:

[Objective] Dibenzofuran (DBF) is a highly toxic compounds and widely spread in environment, used as a model compound in studying the microbial degradation of polycyclic aromatic compounds. [Methods] A bacterial consortium DBFC was isolated from petroleum contaminated soil in Liao River China. The total DNA was extracted to analyze the biodiversity of DBFC, and the strain was isolated and purified by plate dilution coating method. The optimum growth conditions of mixed bacteria were studied through measuring the absorption value of OD₆₀₀. The effects of substrate concentration, substrate, adding nutrient and surface-active substance on the degradation efficiency were studied under the optimum growth conditions. The intermediate metabolites of DBF degradation were detected by ultra-high resolution mass spectrometry, and the metabolic pathway was speculated. [Results] Microbial diversity analysis showed 84.06% of the strains identified in DBFC were Paenibacillus sp., 8.17% were Achromobacter sp., 0.77% were Pseudmononas sp., other strains contain only 2.13%. Four strains, belong to Ochrobactrum sp., Achromobacter sp., Stenotrophomonas sp. and Microbacterium sp., were isolated. Through cultivated in mineral-NH₄Cl salt medium adding DBF as the carbon source, the four stains could not grow. The optimal growth temperature and pH value was 30℃ and 8.0, respectively. Under optimal conditions, DBFC could completely degrade up to 1000 g/L of DBF in 8 days. The maximum degradation rate of DBFC was 0.031 mmol/(L·h) under 1.0 g/L of DBF. Adding glucose, yeast and peptone, the degradation rate of DBF were increased by 1.38, 1.14, and 1.24 fold, respectively. Adding sodium dodecyl sulfonate and Triton-X-100, the degradation rate were inhibited; while adding Tween-80, the degradation rate was decreased. Metabolites 2,2',3-trihydroxybiphenyl, 2,4-hexadienoic acid, gentisic acid and salicylic acid were identified by ultra-high performance liquid chromatography (UPLC). The bacterial consortium DBFC could also use gentisic acid and salicylic acid as sole carbon and energy source, which were respondence with the previous results. [Conclusion] The bacterial consortium DBFC had high efficient in degrading of DBF under pH 8.0 condition, which could be further exploited in situ remediation of polycyclic aromatic compounds contaminated sites. Through UPLC-MS/MS analysis, the metabolites were identified, which provides methods and directions for the study of metabolic process of the polycyclic aromatic compounds.