Diversity Of Nitrogen Cycling Microorganisms In Aquaculture Water Based On The Stable Isotope Probing

Nitrogen cycle is an important element cycle in the biogeochemistry. Microbial activitiesdrive the nitrogen cycle in shrimp farming environment. The excessive accumulation ofnitrogen compounds, like ammonia and nitrite, have toxic effects on fish and shrimp, andendanger the healthy development of aquaculture industy.In this paper, the diversity of nitrogen cycling microorganisms in the typical shrimpaquaculture water of the Pearl River Delta was studied. Method basing on15N-stable isotopeprobing （15N-SIP） was established and optimized. Two microcosms were set up for thelabeling experiments by two kinds of labeling substrates, respectively. Combined withPCR-DGGE, changes of three community structures of typical nitrogen cyclingmicroorganisms during the labeling processes were analysed. By the¹⁵N-DNA from the twomicrocosms, the bacterial and archaeal16S rRNA gene clone libraries were constructedrespectively, on which the diversity of nitrogen cycling microorganisms were studied.The main results are as follows:The labeling and separation experiment results of14N-DNA and¹⁵N-DNA proved that¹⁵N-DNA-SIP was feasible to the available experimental conditions. Based on the¹⁵N-DNA-SIP, three typical microorganisms in nitrogen cycle were analysed by PCR-DGGE.The results revealed almost all of the community structures and distributions of SIP labelingsamples were changing coherently with the enrich cultural time, and relating to the change ofnitrogen compounds. According to these results, day12is the most suitable labeling time.With the¹⁵N-DNA of day12in the¹⁵NH₄⁺-SIP microcosm,16S rRNA gene clonelibraries (¹⁵NH₄⁺-SIP clone libraries) of bacteria and archaea were constructed. The BLASTand RFLP analyses showed that bacterial clone library were consisted of β-proteobacteria（53.7%）, γ-proteobacteria （32.2%）, α-proteobacteria （13.3%）, and Planctomycetes（0.8%）. Andarchaeal library were consisted of Thaumarchaeota （53.8%）, Crenarchaeota （34.7%）, andEuryarchaeota （11.5%）.With the¹⁵N-DNA of day12in the15NO2--SIP microcosm,16S rRNA gene clonelibraries （15NO2--SIP clone libraries） of bacteria and archaea were constructed. Analysisresults showed that bacterial clone library were consisted of β-proteobacteria（39.7%）,γ-proteobacteria （37.2%）, α-proteobacteria （17.4%）, Actinobacteria （4.1%） and Firmicutes（1.6%）. And archaeal library were consisted of Thaumarchaeota （44.9%）, Crenarchaeota（30.8%） and Euryarchaeota （24.4%）. Some sequences were not found in¹⁵NH₄⁺-SIP clonelibraries. Comprehensive analysis of the four16S rRNA gene clone libraries based on15N-SIPindicated that, the clone numbers of ammonia-oxdizing bacteria, nitrite-oxidizing bacteria,and denitrifying bacteria were similar, while nitrogen-fixing bacteria was the least. Thecombination of¹⁵NH₄⁺-SIP artificial media labeling and15NO2--SIP and semi-in-site labelingwas proved to be more effective to study the diversity of the nitrogen cycling microorganisms.