Abstract:

This study focuses on benzo[a]pyrene (BaP), a highly hazardous pollutant that belongs to the class of polycyclic aromatic hydrocarbons (PAHs). BaP is highly stable and is considered the most carcinogenic PAHs. It is widely distributed in the atmosphere, soil, and aquatic environments and poses a serious threat to ecosystems and human health. The development of efficient methods for its degradation through bioremediation has emerged as a critical challenge in environmental science. Owing to the complex structure, hydrophobicity, and low bioavailability of BaP, only a limited number of microbial species can degrade it, which significantly restricts the application of bioremediation strategies for BaP pollution control. Therefore, there is an urgent need to identify efficient degradation pathways for these compounds. The primary aim of this study was to isolate highly efficient BaP-degrading bacteria from naturally contaminated soil and enhance BaP biodegradation during bioremediation by constructing a bacterial consortium, thereby offering an innovative solution for BaP pollution. An enrichment culture method was employed. Soil samples containing 5.7 mg∙kg⁻¹ of BaP, collected from an oilfield in Taizhou, Jiangsu Province, were inoculated into a selective medium containing BaP as the sole carbon source and incubated at 37 ℃ and 150 rpm under shaking conditions. After multiple rounds of enrichment, a highly efficient BaP-degrading strain, TB1, was isolated from a solid selective medium using the spread plate method. Strain TB1 was identified as the Gram-positive Bacillus cereus TB1 based on morphological characteristics and 16S rRNA gene sequencing. Blood agar plates were used to assess the ability of the strain to produce extracellular surfactants. Hemolysis provided preliminary evidence of the surfactant-producing capacity of the isolate. To further elucidate the structural and compositional characteristics of the surfactant, Fourier-transform Infrared spectroscopy (FTIR) and Pyrolysis Gas Chromatography-Mass Spectrometry (Py-GC/MS) were employed. The results identified this compound as a lipopeptide. This finding represents a notable advancement. The lipopeptide surfactant offers unique advantages by enhancing the water solubility and bioavailability of BaP, providing a crucial foundation for the subsequent development of bacterial consortia. A series of BaP degradation experiments and strain antagonism assays were conducted to screen for compatible strains. In the BaP degradation experiments, different candidate strains were individually inoculated into media containing 10 mg∙L⁻¹ of BaP as the sole carbon source. Changes in BaP concentration were measured using High-Performance Liquid Chromatography (HPLC) to accurately calculate the degradation rates. In the antagonism assays, candidate strains and strain TB1 were streaked onto agar plates, and their interactions were assessed based on observable growth inhibition. Following a comprehensive evaluation, the Gram-negative Acinetobacter sp. TX3 was selected for consortium construction with strain TB1. This screening approach, grounded in both degradation efficiency and strain compatibility, ensures the scientific rigor and effectiveness of the consortium construction process. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) was used to analyze the degradation products of BaP by strains TB1 and TX3. The two strains were individually cultured in media containing BaP as the sole carbon source. After 7 days, the culture broth was collected, processed, and analyzed using LC-MS/MS. To optimize the BaP degradation rate of the bacterial consortium, three different composite ratios of TB1꞉TX3 (1꞉1, 1꞉2, and 2꞉1) were tested in this study. These combinations were inoculated into media containing 10 mg∙L⁻¹ BaP and incubated for seven days. By comparing the BaP degradation rates across different ratios, the optimal ratio was identified, and a bacterial consortium was constructed from it. Single-factor experiments were conducted to investigate the effects of environmental conditions on BaP degradation by a microbial consortium. The variables included initial BaP concentrations (5‒100 mg∙L⁻¹), pH levels (5‒9), salt concentrations (4‒20 g∙L⁻¹), and the addition of various supplementary carbon and nitrogen sources. The bacterial consortium was inoculated, and BaP degradation was measured using HPLC, enabling a systematic analysis of environmental influences and providing comprehensive data to support practical applications. When strain TB1 was cultured alone, the BaP biodegradation rate was 20.17%. In contrast, the strain TX3 alone achieved a degradation rate of 15.35%. LC-MS/MS analysis revealed that the degradation products of strain TB1 included BaP−4,5−diol and BaP quinone, whereas those produced by strain TX3 included BaP−4,5−dihydrodiol and BaP−4,5−diol, respectively. When the bacterial consortium was constructed using a TB1꞉TX3 ratio of 2꞉1, the degradation rate increased to 31.98%, which was significantly higher than that of the individual strains or other ratios. These results indicate that the rational construction of the bacterial consortium led to synergistic interactions between the strains, significantly enhancing the degradation efficiency. In experiments investigating the effect of initial BaP concentration, the consortium achieved a degradation rate of 38.52% at 5 mg∙L⁻¹. As the concentration increased to 100 mg∙L⁻¹, the degradation rate gradually declined to 18.23%, although a degree of tolerance was still demonstrated. In the pH variation experiments, the consortium maintained degradation rates above 24.00% across the pH range of 5‒9, indicating stable performance under varying pH conditions. Across varying salinity levels, the degradation rate remained above 23.60% within a salt concentration range of 4‒20 g∙L⁻¹. The optimal salt concentration was identified as 12 g∙L⁻¹, at which the BaP degradation rate reached 32.77%. In experiments involving nutrient supplementation, the addition of 1 g∙L⁻¹ of soybean meal enhanced the degradation rate to 34.67%. However, other carbon or nitrogen sources resulted in relatively unchanged or reduced degradation efficiencies. The bacterial consortium composed of Bacillus and Acinetobacter demonstrated effective degradation capability at low to medium BaP concentrations and exhibited strong environmental adaptability, thereby establishing a theoretical basis for its potential application in BaP remediation in aquatic or soil environments, particularly saline-alkaline soils.

Key words: bioremediation, PAHs, biosurfactants, bacterial consortium, BaP, environmental adaptability

摘要：

苯并[a]芘（BaP）是多环芳烃污染物中稳定且致癌性最强的污染物，广泛存在于大气、土壤和水环境中。通过筛选苯并[a]芘降解菌，构建复合菌群，提升生物修复降解苯并[a]芘性能是控制苯并[a]芘降解的有效途径。采用富集培养方法从泰州油田土壤中分离筛选出1株苯并[a]芘高效降解菌株TB1，经形态特征观察及16S rRNA基因序列分析鉴定菌株TB1为革兰氏阳性蜡样芽孢杆菌（Bacillus cereus TB1）。经傅里叶红外光谱和热裂解气相质谱鉴定菌株TB1所产胞外表面活性剂为脂肽类物质。通过降解实验与拮抗实验，选择革兰氏阴性不动杆菌（Acinetobacter sp. TX3）与菌株TB1复配。LC-MS/MS分析表明，菌株TB1、菌株TX3均检出苯并[a]芘降解中间产物。为进一步提高复合菌群苯并[a]芘降解率，测定不同复合比例（TB1꞉TX3分别为1꞉1、1꞉2、2꞉1）菌株培养7 d后对10 mg∙L⁻¹苯并[a]芘的降解率。选择苯并[a]芘降解最佳复合比例构建菌株TB1与菌株TX3的复合菌群，通过单因素实验探究初始苯并[a]芘质量浓度、pH、盐浓度、外加碳源、外加氮源对复合菌群降解苯并[a]芘的影响。 结果表明，当复合比例TB1꞉TX3=2꞉1时复合菌群较单一菌株对苯并[a]芘的降解率显著提高36.93%-52.00%。复合菌群在5-100 mg∙L⁻¹苯并[a]芘质量浓度范围内对苯并[a]芘具有耐受性，苯并[a]芘质量浓度为5 mg∙L⁻¹时降解率最高；复合菌群在pH 5-9、NaCl质量浓度4-20 g∙L⁻¹范围内保持对苯并[a]芘降解的稳定性；外加1 g∙L⁻¹豆粕可显著提升复合菌群对苯并[a]芘的降解率。芽孢杆菌与不动杆菌复合菌群对中低浓度苯并[a]芘有较好的降解性能和环境适应能力，这表明复合菌群在水环境、土壤（尤其是盐碱地）的苯并[a]芘污染治理中具有潜在应用价值。

关键词: 生物修复, 多环芳烃, 生物表面活性剂, 复合菌群, 苯并[a]芘, 环境适应能力