Assessment Of Abundance And Composition Of Denitrifiers In The Surface Sediment Of Aquaculture Ponds

Nitrogen is one of the basic elements that all aquatic organisms need in aquaculture ponds. Generally, the accumulation of nitrogen in ponds is high, which has toxic effects on water and cultivation objects. As denitrification is of major importance to the ecosystem nitrogen cycle, the study of denitrifying bacteria is important to control the nitrogen balance of ecosystem. Due to large part of the microorganisms in environment are non-culturable, we can not use the conventional culture methods to investigate the abundance and composition of microorganism.The development of molecular biology techniques brings a breakthrough. Investigation was made on the abundance and composition of dentirifiers and the relationship between the abundance and composition of denitrifiers and environmental factors in the surface sediments using the functional gene nirS as a molecular marker, in Chonghu fishing ground, Gong’an County, Hubei Province, China.We used RT-PCR and denaturing gradient gel electrophoresis (DGGE) to assess the spatio-temporal patterns of denitrifier diversity and composition as well as the quantity of denitrifying bacteria among three types of aquaculture ponds.The results can provide basic data to the controlling of N in ponds. The results are as follws:1. Results showed that the annual abundance of denitrifiers was ranged from6.71х106～6.01х107copies/g. The abundance of denitrifying bacteria do not have significant difference among cultivation types(p>0.05, p=0.357).The abundance of denitrifiers in type1ponds was ranged from1.08х107～6.83х107copies/g,8.60х106～6.64х107copies/g in type2ponds, and6.42х106～8.75хl07copies/g in type3ponds.2. The abundance of nirS denitrifier exhibited a monthly difference (p<0.01, p=0.000). The abundance of denitrifiers was the highest in September in three type ponds [(6.27±0.59)х107copies/g,(5.58±1.03)х107copies/g,(7.25±2.12)х107copies/g, respectively], and then in August [(4.37±0.95)х107copies/g,(3.78±1.55)х107copies/g,(5.53±1.19)х107copies/g, respectively], and the lowest in November [(1.66±0.07)х107copies/g,(1.32±0.22)х107copies/g,(2.39±0.56) х107copies/g, respectively].3. Correlation analysis demonstrated that the abundance of nriS-denitrifiers was positively correlated with temperature and ammonia, but negatively correlated with DO. Principal component analysis showed that inorganic N, temperature and DO were the three major factors affecting the abundance of denitrifiers.4. Based on DGGE fingerprint bands analysis,54bands were detected. The number of band in different lanes was ranged from25-36. Most of the bands were common in different samples, and the intensities of common bands were high; The intensities of non-common bands were different. The results suggested the dominant population remained relatively stable in three cultivation type ponds among different seasons.5. According to the sequencing results of54representative bands, more than70%of the sequences obtained belonged to Uncultured bacterium or Unidentified bacterium, the rest of the sequences were affiliated with Proteobacteria、Firmate. Proteobacteria was dominant, Betaproteobacteria was the dominant sub-groups, then Alphproteobacteria and Gamaproteobacteria.6. In principal component analysis, denitrifiers of different samples were distinctly grouped according to the cultivation type, which represented the similarity of their community composition in each type measured by Chi-square distance. However, the diversity did not exhibit in three cultivation type ponds among different seasons. In Monte Carlo permutation test within RDA, the type of cultivation, temperature and time affected the nirS-type denitrifier communities significantly.