固定化混合菌修复冻融土壤PAHs污染的研究

从石油污染冻融土壤中筛选出1株细菌(Pseudomonas sp.)和1株真菌(Mortierella alpina),以玉米芯为载体对混合菌进行固定化,研究低温冻融环境下,固定化混合菌对菲(Phe)和苯并b]荧恩(BbF)污染土壤的生物强化修复作用。通过高效液相色谱法(HPLC)分析Phe和BbF的降解动态,用Michaelis-Menton与Monod动力学方程将结果进行拟合,采用高通测序分析修复过程中微生物群落的变化。结果表明,处理前,冻融土壤中Phe、BbF的浓度分别为(105.4±4.8)、(6.12±1.1)mg·kg~(-1),60 d修复试验后,固定化混合菌可降解土壤中(56.62±3.21)%的Phe和(38.21±1.82)%的BbF,固定化混合菌对冻融环境有较好的抗性,其降解能力优于游离菌。修复试验中,稳定前期降解速率均高于稳定期降解速率。固定化混合菌的投加,提高了Phe、BbF的降解速率,缩短了Phe、BbF降解的半衰期,反应速率分别提高至2.02、0.65 d-1,半衰期分别缩短至50.17 d和82.12 d;改变了土壤中微生物的群落结构及多样性,其中细菌的多样性和均匀度均降低,多环芳烃(PAHs)的降解与细菌的群落多样性和均匀度呈现负相关;细菌变形杆菌门(Proteobacteria)和真菌鞭毛菌门(Mortierellomycota)成为主要的优势菌门,相对丰富度分别为88.72%和81.15%;细菌假单胞菌(Pseudomonas sp. SDR4)和真菌高山被孢霉菌(Mortierella alpina. JDR7)相对丰度分别上升至80.03%和81.15%,形成了显著的降解真菌-细菌共生优势菌株体系,明显提高了低温土壤中的PAHs污染的修复效果。固定化混合菌可广泛应用于冻融环境下土壤PAHs污染的生物强化修复。

Application of immobilization technology of cold tolerance microorganisms in the remediation of polycyclic aromatic hydrocarbons in freeze-thaw soil

A bacterial(Pseudomonas sp.)and fungal(Mortierella alpina)strain were screened from oil-contaminated frozen-thawed soil, and the mixture of microorganisms was immobilized using corncobs as carriers to study the bio-augmented remediation of phenanthrene (Phe)and benzob]fluoran(BbF)-contaminated soil by the immobilized mixed bacteria under a low temperature freeze-thaw environment. The degradation dynamics of Phe and BbF in soil were analyzed using a high-performance liquid chromatography(HPLC)method, and the results were fitted to the Michaelis-Menton and Monod dynamic models, while changes of the microbial community were analyzed using high-throughput sequencing technology. The results showed that before the bio-augmented treatment, the concentrations of Phe and BbF in freeze-thaw soil were(105.4±4.8)and(6.12±1.1)mg·kg^-1, respectively. After a 60 d remediation experiment,(56.62±3.21)% and (38.21±1.82)% of Phe and BbF, respectively, in the soil could be removed by the immobilized mixed bacteria, which displayed a better resistance and degradation ability to the freeze-thaw environment than did the free bacteria. In the remediation test, the pre-stabilization degradation rate was higher than the stable degradation rate was. The addition of immobilized mixed bacteria significantly increased the degradation rate of Phe and BbF to 2.02 d^-1 and 0.65 d^-1, respectively, while the half-life of degradation was shortened to 50.17 d^-1 and 82.12 d^-1, respectively. In the bio-augmented remediation process, the microbial community composition and diversity in soil were changed, and the diversity and uniformity of bacteria decreased. The degradation rate of polycyclic aromatic hydrocarbons(PAHs)was negatively correlated with community diversity and uniformity of bacteria. Proteobacteria and Mortierellomycota became the dominant phyla with a relative abundance of 80.03% and 81.15%, respectively, while Pseudomonas SDR4 and Mortierella alpina JDR7 became the dominant genera with a relative abundance of 80.03% and 81.15%, respectively, forming a degradation system consisting of a significant fungal-bacterial symbiotic dominant strain, which significantly improved the remediation efficiency of PAH pollution in low temperature soil. The immobilized strains could be widely used in bioremediation of soil PAH pollution in freeze-thaw environments.