土壤微生物群落及Cd/Cu抗性基因对重金属钝化剂MHA/NHA响应机制研究

Immobilization of heavy metals in soil is a widely used method for heavy metal polluted-soil remediation. Micro-/nano- hydroxyapatite (MHA/NHA) has been used as a soil heavy metal immobilization agent with significant remediation effect. The current research on MHA/NHA immobilization effect mainly focused on the change of soil heavy metals chemical form after remediation. Microorganisms as the important components of soil ecosystem, the response mechanism research is important for ecological evaluation of immobilization effect on remediation. This project is based on the cadmium/copper contaminated soil as the research object, select MHA and NHA as the soil heavy metals immobilization agent, at the same time with the conventional immobilization agent of apatite, lime and alkaline residue as the control material. Quantitative PCR technique was used to study the effects of different immobilization agents on the abundance of Cd/Cu-resistance genes in soil. Illumina Miseq high throughput sequencing technology was used to study the effects of soil microbial communities caused by different immobilization agents. At the same time combined with soil environmental factors, explains the response mechanism of cadmium and copper contaminated soil microbial community and the Cd/Cu-resistance gene after application of micro-/nano- and conventional immobilization agent, the driving factor and mechanism of environmental factors caused differences of resistance genes and microbial community structure. The aim was to provide scientific basis for the soil microorganism ecological evaluation after application of MHA/NHA.

土壤重金属生物有效态钝化技术是当前土壤重金属污染治理广泛应用的方法。微/纳米羟基磷灰石（MHA/NHA）已作为具有显著修复效果的钝化剂而被应用。目前关于MHA/NHA钝化修复效果的研究主要集中于修复后土壤重金属化学形态的改变，而微生物作为土壤生态系统的重要组分，其响应机制的研究对于钝化修复效果的生态评价具有重要意义。本项目以镉铜污染的农田土壤为研究对象，选择MHA和NHA为土壤重金属钝化剂，同时以常规钝化剂磷灰石、石灰和碱渣为作对照，采用定量PCR技术对比研究不同钝化剂对土壤Cd/Cu抗性基因丰度的影响；采用Illumina Miseq高通量测序技术研究不同钝化剂对土壤微生物群落结构的影响，同时结合土壤环境因子，阐释施用不同钝化剂后，镉铜污染土壤微生物群落及Cd/Cu抗性基因的响应机制，驱动抗性基因和微生物群落结构差异的环境因子及机制，为MHA/NHA修复后的土壤微生物生态评价提供科学依据。

本研究以长期受到铜污染的农田土壤为研究对象，选择包括MHA和NHA在内的五种材料作为土壤重金属钝化材料，研究土壤微生物对其的响应机制。结果表明，MHA/NHA相比其它三种经典钝化材料，能够显著降低土壤重金属有效态含量，缓解土壤酸性，并使得修复植物海州香薷能够正常生长。基因芯片结果表面，MHA和NHA的施加显著降低了Cd/Cu抗性基因的丰度。定量PCR结果表明，钝化材料的施用显著提高了土壤细菌和真菌的丰度，说明土壤性质的改变有利于土壤微生物的生长和繁殖。高通量测序结果表明，随着钝化材料的施加，土壤细菌和真菌的群落结构发生显著改变，且显著提高了土壤细菌和真菌（NHA除外）群落的多样性。差异物种分析结果表明，MHA和NHA能显著降低土壤中嗜酸菌、土壤肥力退化指示种、淋溶菌和植物病原菌的相对丰度，而显著增加嗜碱菌、吸附菌和潜在的植物促生菌的相对丰度。这些物种可以作为钝化修复过程中的指示性物种，以表征土壤生态质量的回复。而驱动细菌种群结构发生变化的环境因子主要是土壤pH。功能预测结果表明钝化材料的加入减少了土壤重金属溶出的风险，以及土壤有效态重金属对微生物的胁迫，一定程度使得土壤生态系统回复健康，同时提高土壤有机质含量和营养循环速率，有利于植物的生长。以上研究结果不仅有助于阐明MHA和NHA施加后土壤微生物的响应机制，还未修复后的土壤微生物生态评价提供科学依据。