铬、铜和镍对人工湿地硝化细菌与反硝化细菌群落结构分布的影响

在铬、铜和镍复合污染的3段波形潜流人工湿地中采集0~15 cm土壤样品，分别以单氨加氧酶的编码基因amoA和亚硝酸还原酶的编码基因nirK的部分序列作为硝化细菌和反硝化细菌的指示基因，采用实时聚合酶链式反应（实时PCR）与PCR-变性梯度凝胶电泳（PCR-DGGE）结合克隆和测序的方法，研究了硝化细菌与反硝化细菌群落结构的空间分布。结果表明，在人工湿地中，amoA和nirK基因丰度均在第3段最高，分别为（8.60×2.75）×106拷贝·g-1和（137.76×42.16）×10^6拷贝·g-1。回收的15个amoA基因序列中有7个属于簇3a，而8个nirK基因序列中有3个属于簇III。相关性分析表明，amoA基因拷贝数与全量铜、全量镍和铬的活化率呈显著负相关（r分别为-0.870、-0.884和-0.898），但有效铜与amoA香农-维纳指数和辛普森指数之间均呈显著正相关（r分别为0.892和0.940），铜的活化率与amoA基因均匀度呈显著正相关（r=0.887）；nirK基因拷贝数与全量铜、全量镍和铬的活化率也均呈显著负相关（r分别为-0.818、-0.820和-0.860），但有效铜与nirK基因均匀度呈显著正相关（r=0.917）。以上结果表明，硝化细菌和反硝化细菌在人工湿地各个阶段的变化规律基本相同，这二者的丰度与多样性指数对重金属污染的反应总体上呈相反的变化。

Effect of Cr,Cu and Ni on distributions of nitrifying and denitrifying bacteria communities in a constructed wetland

The topsoil samples （0-15 cm） were collected in a three-stage wavy subsurface constructed wetland （W-SFCW） contaminated with heavy metals （Cr, Cu and Ni）. The partial encoding genes of ammonia monooxygenase （amoA） and nitrite reductase （nirK） were used as the indicators of nitrifying and denitrifying bacteria, respectively. Spatial distributions of community abundance, composition and diversity of both bacteria were investigated through real-time polymerase chain reaction （real-time PCR） and PCR-denaturing gradinent electrophoresis （PCR-DGGE） combining cloning and sequencing. The abundance of amoA ranged from（1.32×0.43）×106 copies·g-1 to（8.60×2.75）×106 copies·g-1and nirK ranged from（5.78×1.15）×106 copies·g-1 to （137.75×42.16）×106 copies·g-1. The 7 of 15 amoA bands retrieved from DGGE gels were grouped in cluster 3a and 3 of 8 nirK bands were grouped in cluster III. A negative correlation between the total content of Cu and Ni and the activation rate of Cr and the amoA abundance （r=-0.870,-0.884 and-0.898）,but a positive correlation between the Shannon-Weaver indices and Simpson indices of amoA and the available Cu （r=0.892 and 0.940）, as well as the Evenness of amoA and the activation rate of Cu （r=0.887）. A negative correlation between the total content of Cu and Ni and the activation rate of Cr and nirK abundance （r=-0.818,-0.820 and-0.860） was also detected, but a positive correlation between the nirK Evenness and the available Cu （r= 0.917）. The above results indicated that nitrifying bacteria community responsed similar with the denitrifying bacteria community and microbial abundances changed generally contrary to diversities in wetland contanminated with heavy metals.