枯草芽孢杆菌G1与烟曲霉Z5共培养对蜡质油的协同降解效应

Synergistic degradation of waxy crude oil by co-culture of Bacillus subtilis G1 and Aspergillus fumigatus Z5

摘要:

背景 蜡质沉积严重影响油田开采与集输效率。传统清防蜡方法存在污染环境与高能耗问题，微生物技术有望提供绿色替代途径，但单一菌株降解能力有限，亟需构建功能互补的共培养体系，用以提升降解效率。目的 为了提高油田生物清防蜡效率，探究一株产表面活性剂的细菌枯草芽孢杆菌(Bacillus subtilis) G1与一株高分子多环芳烃降解真菌烟曲霉(Aspergillus fumigatus) Z5在共培养条件下对蜡质油的协同降解效应。方法 通过气相色谱-质谱联用技术(gas chromatography-mass spectrometry, GC-MS)分析降解前后蜡质油组分变化，利用差示扫描量热法(differential scanning calorimetry, DSC)和偏光显微镜测定蜡质油的结晶温度并观察晶体形态，最后通过基因组学分析初步解析协同降解路径。结果 共培养体系对蜡质油的降解率达73.33%，显著高于单菌培养降解率。GC-MS分析显示，C24-C36区间的峰面积较对照组减少60%以上，C40-C44重质烷烃仅残留微弱信号，整体峰面积降低超过70%。DSC结果表明，经菌株G1与Z5共培养处理后，蜡析温度由41.08 ℃降至6.97 ℃，结晶温度由33.87 ℃降至2.86 ℃。偏光显微镜观察显示，蜡晶体由聚集态转变为分散微晶体，平均粒径下降至约30-50 μm，形态趋于圆润。基因组分析结果显示，菌株G1携带完整的长链烷烃降解与脂肪酸β-氧化代谢模块，其中乙醇脱氢酶基因yahK和醛脱氢酶基因gene3962构成了将烷烃逐步氧化为脂肪酸的关键酶系。然后，典型的β-氧化相关基因fadA、fadN、fadD与atoB促进脂肪酸活化与逐步裂解。菌株Z5携带的catA、hppD和hmgA等关键酶可能参与芳香环加氧与开环反应，其代谢产物或可为菌株G1提供交叉喂养底物。结论 B. subtilis G1与A. fumigatus Z5共培养可显著提升蜡质油的生物降解效率，为油田高效清防蜡技术提供了理论依据与菌种资源。

Abstract:

Background Wax deposition severely impacts the efficiency of oilfield extraction and gathering/transportation processes. Traditional wax removal and inhibition methods face issues such as environmental pollution and high energy consumption. Microbial technology offers a promising green alternative; however, the degradation capability of single bacterial strains is limited. This limitation necessitates the construction of a functionally complementary co-culture system to enhance degradation efficiency.Objective To enhance the microbial efficiency of wax removal and inhibition in oilfields, this study investigated the synergistic degradation effect of waxy crude oil by co-culturing a surfactant-producing bacterium (Bacillus subtilis G1) and a fungus (Aspergillus fumigatus Z5) capable of degrading high-molecular-weight polycyclic aromatic hydrocarbons.Methods Changes in waxy crude oil composition before and after degradation were analyzed via gas chromatography-mass spectrometry (GC-MS). The crystallization temperature and crystal morphology of waxy crude oil were determined by differential scanning calorimetry (DSC) and polarized light microscopy, respectively. Genomic analysis was employed to explore the synergistic degradation pathways.Results The co-culture system achieved a degradation rate of 73.33% for wax crude oil, which was significantly higher than those of monocultures. GC-MS analysis indicated that the co-culture group had a decrease of over 60% in the peak area in the C24-C36 range compared with the control group. Only faint signals were detected for heavy alkanes (C40-C44), and the overall peak area decreased by more than 70% in the co-culture group. DSC results demonstrated that after treatment with both G1 and Z5 strains, the wax precipitation temperature decreased from 41.08 ℃ to 6.97 ℃, and the crystallization temperature dropped from 33.87 ℃ to 2.86 ℃. Polarized light microscopy observations indicated that wax crystals transitioned from an aggregated state to dispersed microcrystals, with an average particle size of 30-50 μm and more rounded morphology. Genome sequencing analysis reveals that G1 harbored complete metabolic modules for long-chain alkane degradation and fatty acid β-oxidation. The metabolic modules included the key enzyme system comprising the alcohol dehydrogenase gene yahK and aldehyde dehydrogenase gene 3962, which oxidizes alkanes into fatty acids. Subsequently, typical β-oxidation-associated genes fadA, fadN, fadD, and atoB promote fatty acid activation and subsequent cleavage. The key enzymes catA, hppD, and hmgA carried by Z5 may participate in aromatic ring oxygenation and ring-opening reactions, with their metabolites potentially providing cross-feeding substrates for G1.Conclusion Co-culture of B. subtilis G1 and A. fumigatus Z5 significantly enhances the biodegradation efficiency of waxy crude oil, providing both theoretical support and microbial resources for efficient wax removal and inhibition in oilfields.