鸡粪菌渣好氧堆肥过程中氨氧化古菌及氨氧化细菌群落的动态变化

Dynamics of ammonia-oxidizing archaea and ammonia-oxidizing bacteria during composting of chicken manure and mushroom cultural waste

以amoA基因为标记,通过Real-Time PCR和限制性片段长度多态性（Restriction fragment length polymorphism,RFLP）法对鸡粪菌渣好氧堆肥过程中的氨氧化古菌（Ammonia-oxidizing archaea,AOA）和氨氧化细菌（Ammonia-oxidizing bacteria,AOB）进行了丰度及群落结构的分析。结果表明,在堆制初期、好氧发酵高温期及后熟期,AOB的amoA基因丰度均占主导优势,是AOA的38-992倍。进入好氧发酵高温期,AOA amoA基因丰度下降至发酵前的0.9%,AOB下降至17.6%,后熟期AOA与AOB的amoA基因丰度与好氧发酵高温期相当。在上述3个阶段AOA与AOB各自存在一个绝对优势菌群,分别为Cluster 3和Nitrosomonas europaea,其中Cluster 3克隆子数目分别占整个克隆文库的70.73%、54.28%、72.45%,Nitrosomonas europaea克隆子数目分别占整个克隆文库的78.44%、93.20%、94.27%。堆肥3个阶段AOA的多样性指数变化不大,Shannon-Wiener值维持在1.53-1.60,但群落结构发生明显演替,随着堆肥温度升高,堆肥前期的一些菌群（Cluster 4、Cluster 5、Cluster 6）逐渐消失,新的菌群Cluster 1出现并成为堆肥中后期的第二大优势菌群。AOB无论是多样性指数还是群落组成,都发生剧烈的变化。AOB在堆肥前期Shannon-Wiener指数值最大（1.47）,种群数最多（6个基因簇,分别为Nitrosomonas europaea Cluster,Nitrosomonas halophila Cluster,Nitrosomonas communis Cluster,Nitrosomonas nitrosa Cluster,Nitrosospira briensis Cluster,Nitrosospira multiformis Cluster）;进入高温发酵期,Shannon-Wiener下降至0.45,群落结构单一,只有Nitrosomonas europaea Cluster和Nitrosomonas halophila Cluster;进入后熟期,AOB多样性及种群数得到一定程度的回升。

The transformation and loss of nitrogen in composting materials were affected profoundly by ammonia oxidizers during the composting process. The abundance and composition of ammonia-oxidizing bacteria（AOB）and ammonia-oxidizing archaea（AOA）during composting of chicken manure and mushroom cultural waste were examined employing Real-Time PCR and PCR-restriction fragment length polymorphism（RFLP）techniques with the monooxygenase gene（amoA）as biomarker of ammonia oxidizers. Samples were collected at three representative stages, including mesophilic, thermophilic, and maturing stages. Results showed that the amoA gene abundance of AOB dominated and the gene copy numbers were about 38 to 992 times those of AOA at all three stages of composting. At the thermophilic stage, the amoA abundance of both AOA and AOB dropped to 0.9% and 17.6% of that at the mesophilic stage, respectively. The numbers of AOA and AOB at the maturing stage were similar to those at the thermophilic stage. Cluster 3 was the most dominant group of AOA at the three stages,and its amount of clones accounted for 70.73% of the clone library at the mesophilic stage of composting, 54.28% at the thermophilic stage,and 72.45% at the maturing stage. Nitrosomonas europaea was the most dominant species of AOB at the three stages, with clone amount representing 78.44%, 93.20% and 94.27% of all clones at mesophilic, thermophilic, and maturing stage, respectively. For AOA, the Shannon-Wiener index ranged from 1.53 to 1.60 at these three stages, showing no obvious changes, but the community composition revealed obvious succession. With increase in compost temperature, Cluster 4, Cluster 5 and Cluster 6 of AOA disappeared, but a novel member（Cluster 1） appeared and became the second dominant group. For AOB, both diversity and community structure showed dramatic changes. At the mesophilic stage, AOB gave the highest Shannon-Wiener index（1.47） and the richest number of species, with 6 clusters including Nitrosomonas europaea,Nitrosomonas halophila,Nitrosomonas communis,Nitrosomonas nitrosa,Nitrosospira briensis,and Nitrosospira multiformis clusters. However, with arrival of thermophilic stage, the Shannon-Wiener index of AOB sharply reduced to 0.45, and the community structure became simple, including Nitrosomonas europaea and Nitrosomonas halophila clusters. At the maturing stage, the diversity and species number of AOB recovered to some extent.