| Nitrite-dependent anaerobic methane oxidation(n-damo) is a recently discovered process coupled to nitrite reduction, which is performed by Candidatus Methylomirabilis oxyfera’’(M. oxyfera) being affiliated with NC10. The n-damo process is believed to alleviate the greenhouse effect by converting methane to CO2 under anaerobic conditions. In addition, it can convert nitrite to dinitrogen gas(N2) coupled to anaerobic oxidation of methane(AOM) and thus constitutes a unique link between the two major global nutrient cycles of carbon and nitrogen. However, the distribution and diversity of these bacteria is not well understood. In addition, the other important nitrite dependent anaerobic process is anaerobic ammonium oxidation, anammox, which directly uses ammonium and nitrite as electronic donor and acceptor to produce N2 under anaerobic conditions. The reaction is considered to be the most economic and efficient process for removing ammonia from wastewater and play an important role in nitrogen cycle under anaerobic conditions. Although distribution and functions of anammox in many natural environments have been widely investigated, the co-occurrence of M. oxyfera-like and anammox bacteria in the natural environment was rarely reported. The water-level-fluctuation zone(WLFZ) in The Three Gorges Reservoir Dam was a special aquatic ecosystem, where the land is regularly exposed and submerged by the periodic water storage and drainage. The methane emission has attracted great attention since the dam was completed. In this study: firstly, the co-occurrence of M. oxyfera-like bacteria and anammox bacteria was detected. The possible relationship between their distribution, diversity and abundance and the main environmental factors were discussed in the WLFZ soil of the Three Gorges Reservoir. Next, the sequencing batch bioreactor nitration reactor(SBR) and complete autotrophic denitrification reactor(CANON reactor) were constructed to explored hydrazine related functional microbes in the process of denitrification. The following results were obtained:1. The samples were collected in six sample sites named XXH(Xiangxihe), WS(Wushan), WJG(Wangjiagou), XJ(Xiaojiang), ZXH(Zhuxihe) and PS(Paddy soil) along WLFZ of the Three Gorges Reservoir in March 2014 when the water level dropped down to 165 m. The analysis of physical-chemical characteristics indicated that the content of ammonia and nitrite was below the detection limit. The methaneemission flux were 0.34 mg/m2 h in sampling sites XXH 、 WS and XJ and 4.86 mg/m2 h in paddy soil according previous reports.2. The genome DNA was extracted from the soil cores. The M. oxyfera-like, anammox and total bacteria were quantified through a real time PCR technique using primer pairs specific to their 16 S r RNA genes, respectively. The results of real-time PCR indicated that M. oxyfera-like bacteria can co-ocurr with anammox bacteria in all samples. The abundance of M. oxyfera-like and anammox bacteria had significant variation among samples, especially in different sites(p<0.01). The copies of M. oxyfera-like bacteria in samples of WLFZ ranged from 1.15 ± 0.2 × 105 g-1 to 1.48 ± 0.16 × 107 copies g-1(dry weight soil), which were much higher than 1.20 ± 0.19 × 104 to 2.53 ± 0.39 × 104 g-1 in the paddy field samples(p<0.01).However, the copies of anammox bacteria in samples of WLFZ ranged from 1.07±0.42×103 to7.77±0.87×104 copies g-1(dry weight), which were much lower than 9.78±0.78×103 to 3.65±0.76×106copies g-1 in the paddy field samples.3. Based on the above results, three sampling sites(WS, WJG and ZXH) in WLFZ which reflected the highest, middle and lowest abundance of M. oxyfera-like and anammox bacteria, were selected to research the diversity and compositions of M. oxyfera-like and anammox bacteria. 16 S r RNA and pmo A genes were used to analyze the diversity and compositions of M. oxyfera-like bacteria, the hzs B gene was used to analyze the diversity and compositions of anammox bacteria. Clone library sequencing analysis indicated that a total of 134 positive 16 S r RNA gene sequences were obtained from the three samples, and 128 of them were related to M. oxyfera(similarity 84.38-98.92%) and clustered into 4 groups; 134 of the 141 sequences related to pmo A gene of M. oxyfera(similarity 84.62-95.38%) were binned to 10 OTUs and fell into the six clusters in the phylogenetic analysis. Further phylogenetic analysis showed that all 103 sequences of hzs B gene were clustered into 4 groups, Group A, B, C and D, which had the identity of88.02%-89.06%, 84.11%-86.58% and 89.58%-98.44% with the hzs B gene from the known anammox species, Candidatus ‘Brocadia fulgida’, Candidatus ‘Brocadia anammoxdianas’, and Candidatus‘Kuenenia’, respectively.4. Successful construction of SBR nitrifying reactor and qualitative and diversity analysis of functional genes related to nitrogen transformation. During the run of SBR nitrifying reactor, the concentration of NH4+-N in inflow is from 51 mg/L increased to 505 mg/L; nitrogen load rate is from thebeginning of 102.9 mg/L * d increase gradually to the end of 1056.2 mg/L * d. The addition of 2 mg/L N2H4 resulted in a sharp decline in the running efficiency of the reactor. Finally, ammonia oxidation rate ruduced to 2.45%. Quantitative PCR analysis showed that amo A gene copy number increased to 1.0 x 109copies/g from 2.01 x 106 copies/g during enrichment; nxr B gene copy number is increased to1.28×107copies/g from 4.20×105copies/g. After adding trace N2H4, the abundance of amo A and nxr B genes decreased to 2.09×104 copies/g and 2.56×105 copies/g, respectively, suggesting that N2H4 can significantly inhibit the growth of the AOB and NOB. Library cloning sequencing and evolutionary analysis showed that the diversity of AOB amo A genes and NOB nxr B genes was very high. For amo A gene, 52.63%, 31.58%and 15.79% of total 19 sequences belonged to genus Nitrosococcu, Nitrosomonas and Nitrosospira,respectively. For nxr B gene, about 55%, 20% and 15% in 20 sequences were distributed in Nitrobacter winogradskyi, Nitrobacter vulgaris and Nitrobacter hamburgensis,respectively.5. Successful construction of CANON reactor, and qualitative and diversity analysis of functional genes related to nitrogen transformation. In the stable operation of CANON reactor, NH4+-N and total nitrogen removal rate were 68% and 65% and NLR is about 269 mg/L*d prior to adding N2H4. After adding trace N2H4, the removal rate of the NH4+-N and total nitrogen is 82.13% and 72.65%, respectively.The nitrogen load rate is as high as 456.80 mg/L * d, increaseing by about 76.74%. Quantitative PCR analysis showed that the copies of amo A and nxr B genes significantly reduced from 1.03×107 copies/g and1.30 x 106copies/g to 6.26 x 104 copies/g and 1.66 x 105 copies/g, respectively, under the condition adding hydrazine treatment. However, the abundance of hzs A gene which specific to anammox increased to 5.84 x1010 copies/g from 3.14x109 copies/g. Analysis of cloning library indicated that 66.67% and 33.33%sequences of amo A gene belonged to genus Nitrosomonas and Nitrosococcus, respectively. 26.67%,53.33% and 20% of 15 nxr B gene sequences belonged to Nitrobacter vulgaris, Nitrobacter winogradskyi and Nitrobacter hamburgensis, respectively. 38.46% and 15.38% of 13 nxr B gene sequences fall into Candidatus Scalindua and Candidatus Anammoxoglobus propionicus, respectivley. In addition, the species of 46.15% in total sequences were unclear because of the absence of reference sequences in the phylogenetic analysis. |
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