盐生植物内生细菌协同宿主修复石油污染盐渍化土壤的作用及机制

Environmental risk from crude oil pollution was enhanced by soil salinization, and the remediation of saline soil pollution was particularly urgent. Phytoremediation has been becoming a hot spot, however, till today, little is known about the role of endophytic bacteria from halophytes in remediation of petroleum-contaminated saline soil. Based on the pilot studies, five species of halophytes from Yellow River Delta, i.e., Paspalum thunbergii Steud., Chloris virgata Sw., Bothriochloa ischaemum L., Digitaria sanguinalis L., Phragmites australis Cav. were employed in this study. Using traditional culture method and molecular ecological techniques, petroleum-degrading endophytic bacteria diversity would be thus evaluated in this study, in order to discover the role of endophytic bacteria in soil remediation. The endophytic bacteria strains with high-efficient degrading and plant growth-promoting effects will be screened, and the mechanism of petroleum-degrading will also be determined by measuring the cell surface hydrophobicity, substrate range, producing biosurfactant. Based on the above studies, the synergetic effects of endophytic bacteria and halophytes on remediation of petroleum-contaminated saline soil will be evaluated and its mechanism will also be discussed, in order to discover the role of endophytic bacteria in soil remediation. The results are of the great significance to the development of petroleum-degrading bacteria repository and gene pool, and illuminating the interaction between endophytic bacteria and halophytes under the condition of crude oil pollution. Simultaneously, our results can provide theoretic bases and technical supports for petroleum-contaminated saline soil remediation by halophytes.

土壤盐渍化导致石油污染物环境风险加大，因此对石油污染盐渍化土壤治理尤为迫切。植物修复是近年来环境污染治理研究的活跃领域，对于内生细菌在盐生植物修复石油污染土壤方面的研究几乎无人涉足。本项目以黄河三角洲盐生植物雀稗、虎尾草、白羊草、马唐、芦苇为材料，利用传统培养方法并结合现代分子生态手段，分析植物内生解烃细菌资源多样性；筛选高效解烃兼植物促生效应的内生细菌，通过测定菌体细胞表面疏水性，分析菌株产表面活性剂特性和可降解的底物范围，研究该菌株的解烃机理；在此基础上，采用盆栽试验研究内生细菌协同盐生植物修复石油污染盐渍化土壤的过程和机理，从而明确内生细菌在植物修复石油污染盐渍化土壤中所发挥的作用及其主要机制。预期研究结果对丰富石油降解细菌资源库、基因库以及深入阐明内生解烃细菌与盐生植物之间的作用关系具有重要的理论意义，同时可为发展盐生植物修复石油污染盐渍化土壤新途径提供科学依据和技术支持。

利用内生细菌协同宿主植物修复石油污染土壤是一新兴方向，国内外在这方面的研究尚处于起步阶段。目前对于石油污染胁迫下盐生植物功能内生细菌多样性特征以及高盐环境下内生细菌对植物修复石油污染土壤的作用尚不明确。本项目以黄河三角洲耐盐/盐生植物为材料，研究石油污染胁迫下盐生植物功能内生细菌多样性，筛选高效解烃兼植物促生效应的内生细菌，明确内生细菌协同植物修复作用。结果表明，（1）4种耐盐/盐生植物虎尾草、白羊草、芦苇和马唐内生解烃细菌主要分布于Firmicutes、Actinobacteria、Proteobacteria 3个门，Bacilli、Actinobacteria、Alphaproteobacteria和Gammaproteobacteria 4个纲。Bacillus属和Pseudomonas属植物内生解烃细菌种类最多；（2）筛选得到16株具有解烃兼促生作用内生细菌，其中Pseudomonas aeruginosa L10对柴油降解率达到79.3%（培养7 d），L10在4% NaCl浓度无机盐液体培养基中对烃类物质仍具有较高的降解能力。L10同时具有固氮、解磷、产生1-氨基环丙烷-1-羧酸( ACC)脱氨酶和吲哚乙酸( IAA)等促生特性和产生生物表面活性剂能力，L10合成生物表面活性剂与其利用的碳氮源类型有关。L10的基因组中存在多个与烃类物质降解、产生生物表面活性剂和促生功能相关的关键基因（簇）；（3）Bacillus pumilus BF04具有固氮、解磷、产生1-氨基环丙烷-1-羧酸( ACC)脱氨酶和吲哚乙酸( IAA)等促生特性，对直链烷烃具有较强的代谢能力，其次是支链烷烃，能代谢环烷烃和单环芳烃，不能代谢的苯酚和多环芳烃菲、萘；（4）内生细菌L10协同宿主植物芦苇能显著提高植物修复石油污染盐渍化土壤效率，其主要作用是促进宿主植物在高盐和重石油污染土壤环境的生长，为微生物降解石油烃类物质提供良好环境，另一方面提高植物内和根际土壤中降解基因的丰度和表达量，促进石油烃污染物质的降解。