Neorhizobium petrolearium OS53联合紫花苜蓿协同修复石油污染土壤研究

Neorhizobium petrolearium OS53 combined with alfalfa (Medicago sativa L.) for remediation of petroleum-contaminated soil

摘要:

【目的】探究Neorhizobium petrolearium OS53与紫花苜蓿协同修复石油污染土壤的机制。【方法】使用Illumina和Nanopore平台对菌株OS53进行全基因组测序,构建菌株基因组完成图,并进行基因预测及功能注释,分析其中与结瘤促生及石油降解相关基因,并通过实验测定菌株OS53产吲哚乙酸(indole acetic acid, IAA)、铁载体、溶磷和解钾等与促生相关的能力。使用试剂盒对联合修复前后土壤中土壤脲酶、脱氢酶、多酚氧化酶和脂肪酶活性及紫花苜蓿的叶绿素、丙二醛、脯氨酸、可溶性蛋白、可溶性糖和超氧化物歧化酶等生理指标进行测定。【结果】菌株OS53的基因组由一个5.56 Mb的环形染色体和2个大小分别为0.92 Mb和0.38 Mb的质粒组成,基因组G+C含量为60.2%,共编码6 968个基因。菌株OS53与N. petrolearium DSM 26482^T的16S rRNA基因序列相似性最高,为99.86%,且在系统发育树上形成稳定分支,表明菌株OS53与N. petrolearium为同一种,因此将OS53命名为N. petrolearium OS53。试验结果表明,菌株OS53具有产IAA能力,并在其基因组中也发现相关基因。在初始石油含量为(4 403.30±222.10) mg/kg时,经过120 d修复,OS53与紫花苜蓿协同修复效率能够达到57.53%,比不接种OS53、仅接种OS53和仅种植苜蓿分别提高了44.26%、41.69%和8.84%。在联合修复体系中,紫花苜蓿叶绿素、可溶性蛋白和可溶性糖的含量有所提高,丙二醛和脯氨酸的含量以及超氧化物歧化酶的活性有所降低,同时土壤中多酚氧化酶、脱氢酶、脂肪酶和脲酶的活性都有所提高。【结论】菌株OS53具有产IAA的能力,并且能够促进紫花苜蓿在石油污染土壤中的生长,进而提高土壤中与石油降解相关部分酶活,最终提高联合修复体系对石油污染土壤的修复效果。

关键词:Neorhizobium petrolearium;全基因组测序;紫花苜蓿;石油;联合修复

Abstract:

【Objective】 To explore the mechanism of Neorhizobium petrolearium OS53 combined with alfalfa (Medicago sativa L.) in the remediation of petroleum-contaminated soil. 【Methods】 Illumina and Nanopore were employed to sequence the whole genome of N. petrolearium OS53, and the complete genome map of the strain was constructed. Gene prediction and functional annotation were carried out to analyze the genes involved in nodulation and oil degradation. The abilities of strain OS53 to produce indole acetic acid (IAA), secrete siderophore, and solubilize phosphorus and potassium were tested. The activities of urease, dehydrogenase, polyphenol oxidase, and lipase in soil and the levels of chlorophyll, malondialdehyde, proline, soluble protein, soluble sugar, and superoxide dismutase in alfalfa were measured by kits. 【Results】 The genome of strain OS53 consisted of a circular chromosome of 5.56 Mb and two plasmids of 0.92 Mb and 0.38 Mb, respectively, with the G+C content of 60.2%. The genome encoded a total of 6 968 genes. The strain OS53 and N. petrolearium DSM 26482^T showed the 16S rRNA gene sequence similarity of 99.86%, and formed stable branches on the phylogenetic tree, indicating that strain OS53 and N. petrolearium were the same species. Therefore, OS53 was named as N. petrolearium OS53. The strain OS53 had the ability to produce IAA, and the related genes were identified in the genome. After 120 days of remediation of the soil with the initial oil content of (4 403.30±222.10) mg/kg, OS53 and alfalfa showed the remediation efficiency up to 57.53%, which was 44.26%, 41.69%, and 8.84% higher than that of no inoculation of strain OS53, inoculation of OS53 only, and planting alfalfa only, respectively. In the combined remediation system, alfalfa showed elevated the levels of chlorophyll, soluble protein, and soluble sugar and lowered levels of malondialdehyde, proline, and superoxide dismutase, and the soil showed increased activities of polyphenol oxidase, dehydrogenase, lipase, and urease. 【Conclusion】 The strain OS53 had the ability to produce IAA to promote the growth of alfalfa in the petroleum-contaminated soil, which increased the activity of enzymes involved in oil degradation in the soil. Finally, the combined system improved the remediation efficiency of the soil.

Key words:Neorhizobium petrolearium;whole genome sequencing;Medicago sativa L.;petroleum;combined system for remediation