Diversity Of β-Proteobacterial Ammonia-oxidizing Bacteria And Ammonia-oxidizing Archaea In Shrimp Farm Sediment

The oxidation of ammonia to nitrate via nitrie, i.e. nitrification, is of fundamental importance in the global nitrogen (N) cycle. The ammonia-oxdizing bacteria (AOB) are responsible for the oxidation of ammonia to nitrite,which is the first rate-limiting step in nitrification. AOB are generally related to the members ofβ-subdivision of class Proteobacteria excepet for the marine genus Nitrosococcus, which belongs to theγ-subdivison. Recently, a combination of metagenomic and genomic analyses and the cultivation of a novel ammonia-oxidizing marine crenarchaeote revealed strong evdidence for nitrification within the domain Archaea, including identification of genes putatively encoding archaeal ammonia monooxygenase subunits.Excessive accumulation of ammonia and nitrite endanger the healthy development of the aquaculture industy. Biological nitrification is a useful method for the degradation of these toxic nitrogen compounds. As the discovery of ammonia-oxidation archaea (AOA) in kinds of environment, AOA may be playing an important role in the processes of ammonia oxidation. In this paper, the diversity of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) in shrimp farm sediment are studied by using the AOA and AOB-AMO-amoA genes based clone library and qRT-PCR-DGGE technology. This study lays the foundation of the methods and provides reference date for the fuure study on diversity of AOA and AOB.The main results are as follows:a. The amoA genes clone libraries were constructed withβ-Proteobacterial-AOB and AOA specific primers, respectively. The libraries were screened by PCR-restriction fragment length polymorphism (RFLP) analysis and clones from unique RFLP patterns were sequenced. Phylogenetic analyses of the amoA gene fragments showed that all AOB sequences from shrimp farm sediment were affiliated with Nitrosomonas (61.54%) or Nitrosomonas-like (38.46%) species, which were grouped into Nitrosomonas communis cluster,Nitrosomonas sp.Nm148 cluster,Nitrosomonas oligotropha cluster. All AOA sequences belonged to the kingdom Crenarchaeote except an Operational Taxa Uni(tOTU)sequence, which was related to the Unclassified-Archaea and fell within cluster S (soil origin).b. Diversity changes of AOA and AOB communities in shrimp farm sediment after time were studied by qRT-PCR-DGGE technology.AOA-amoA-DGGE sequencing results showed that all the sequences belonged to the kingdom Crenarchaeote. The gene sequences of Cluster A1 and Cluster A2 gathered together with the amoA gene sequences from the paddy soil, protected green soil. Cluster C gene sequences with freshwater, hot spring water. Cluster B sequences with seawater and marine sediment together.AOB-amoA-DGGE sequencing results indicated that Nitrosomonas or Nitrosomonas-like pecies and grouped into Nitrosomonas communis cluster,Nitrosomonas oligotropha cluster,except Nitrosomonas sp.Nm148 cluster, which is different from the results of AOB-amoA clone library. The diversity changes between AOB-amoA-DGGE sequences and AOB-amoA-clone library sequences were according to the changes of the physical and chemical properties during the whole period of shrmp farm. Proportion of Nitrosomonas-like pecies sequences from AOB-moA-DGGE is greater than the sequences from AOB-amoA clone library.AOA-amoA gene qRT-PCR results showed the copy numbers ranged from 1.34 x 10⁵ to 7.96 x 10⁵ copies per microgranm DNA and from 1.09 x 10⁷ to 6. 63 x 10⁷ copies per garm of sediment (wet weight). The total archaea copy numbers accounted from 4.49 x 10⁸ to 5.45 x 10⁹ copies per garm of sediment (wet weight). The ratios of AOA to total archaea ranged from 0.24% to 1.12%.AOB-amoA gene qRT-PCR results indicated the copy numbers ranged from 2.35 x 10³ to 2.20 x 10⁴ copies per microgranm DNA and from 1.08 x 10⁵ to 1.01 x 10⁶ copies per garm of sediment (wet weight). The total bacteria copy numbers accounted from 1.75 x 10⁹ to 2.61 x 10¹⁰ copies per garm of sediment (wet weight). The ratios of AOB to total bacteria ranged from 0.012% to 0.074%.c. Results of Principal Component Analysis (PCA) and Canonical Correspondence Analysis (CCA) showed that:AOA-CCA Analysis showed the most relevance of these indicators as pH, ammonium, and nitrite associated with AOA community, followed the second indicators as COD, temperature, nitrate.AOB-CCA Analysis showed the most relevance of these indicators as COD, temperature nitrate, ammonium associated with AOB community, followed by the second indicators as pH, nitrite.The number of bacterial 16S rRNA gene copies was significant positive correlation with temperature(r=0.835, P<0.05). The number of AOA-amoA gene copies was a significant positive correlation with ammonium concentration (r=0.971, P<0.01).The number of archaeal 16S rRNA gene copies was significant positive correlation with temperature(r=0.828, P<0.05). The number of AOB-amoA gene copies was a significant positive correlation with ammonium (0.894, P<0.05)å’Œand pH (0.845, P<0.05).d. For the frist time, we studied the amino acids, hydrophobic, physical and chemical properties, secondary structure, and three-dimensional structure of AOA-AMO by bioinformatics analysis.