| In metal processing industries and production of fertilizer, steel, explosives, fodder, pectin, electronic component, and nuclear fuel, high nitrate concentration of wastewater containing a lot of salt (Cl-、Na+, et al) and some toxic substances (phenol, chlorobenzene, heavy metal chromium and cadmium, et al) are generated. Moreover, physical and chemical treatment processes of low nitrate concentration of wastewater often produce nitrate-highly concentrated liquid that contains toxic substances like phenol, chlorobenzene, heavy metal chromium and cadmium, et al, in turn inhibits the growth of microorganisms, and increases the difficulty of the biological treatment. Thus, effective treatment of high-nitrate wastewater has become a major problem that has to be solved in the world. In this study, the simulated high-nitrate wastewater was treated in an expanded granular sludge bed (EGSB) reactor. The operation parameters of the EGSB reactor a re expected to be optimized and the effect the different stressed condition on denitrification are supposed to be analyzed in detail in order to treat the high-nitrate wastewater effectively. At the same time, the community structure and functional genes in an EGSB reactor have been investigated by means of the molecular biotechnology such as PCR-DGGE,16S rDNA sequencing, high-throughput sequencing technologies and real-time fluorescent quantitative PCR and so on. Internal connection between macro variation in properties and micro structure has been illustrated too. Therefore, certain theoretical basis and technical support will be provided for effective treatment of high nitrate wastewater in the EGSB reactor. The major research work and conclusions are described as follows:1) The EGSB reactor was successfully started up. And the optimal parameters were achieved with C/N mole ratio of2.0, liquid up-flow velocity (Vup) of3.0m/h and pH of6.2-8.2. Under the optimal operating conditions, the effects of the different stressed condition on dinitrificaiton in the EGSB reactor have been investigated, and microbial community structure and functional genes have been analyzed. Here the stress condition refers to nitrate load, salinity of NaCl, temperature, phenol, heavy metal chromium and cadmium.2) Complete denitrification can be achieved with nitrate nitrogen concentration reaching as high as14000mg/L in an EGSB reactor while nitrite nitrogen content of the effluent was a little. However, nitrite nitrogen concentration of the effluent obviously increased, and the highest concentration reached2909mg/L when the optimal operating conditions was changed, that is HRT decreased from24to16hr. DGGE fingerprint indicated that the evolution and novation of the total bacterial community were obvious with the change of the influent nitrate nitrogen concentration. The comparison between stripe-shaped cloning sequencing and NCBI showed that bacteria in an EGSB reactor are mostly salt resistant, alkali resistant, and denitrifying. Meanwhile, those bacteria had to do with the degradation and the largest proportion belongs to Gammaproteobacteria class. The analysis of454sequencing results showed that although the microbial diversity of the highest nitrate concentration is obviously lower than that of the inoculated sludge, it remains relatively abundant. The values of OTUs were448and shannon index was4.83at the similarity of97%. There were five phylogenetic groups, including Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes and Chloroflexi. Proteobacteria (83.72%) was the dominant microbial population, followed by Firmicutes (13.12%). The Sporolactobcillaceae incertae sedis, Anaerobranca, Alkalibacterium, Marinimicrobium, Nitrincola and Halomonas genus had played an important role in denitrification.3) The results of continuous experiments showed complete denitrification was achieved when nitrate concentration was as high as6000mg N/L and the salinity of influent reached11%NaCl under optimal operation condition. Furthermore The results of batch experiments indicated that denitrification degradation process accorded with zero order kinetics reaction when NaCl content of the influent was less than or equal to5%, corresponding linear correlation coefficient R2more than0.93. DGGE fingerprint indicated that the evolution and novation of the total bacterial community were obvious as the change of the influent NaCl content. The stripe cloning sequencing and compared results with NCBI showed the bacteria in an EGSB reactor had the features of the degradation, salt resistance, alkali resistance and denitrifying. And the bacteria of the largest proportion was Gammaproteobacteria class. The analysis of454sequencing results suggested that the abundance and diversity of microbial communities of the anaerobic sludge in the EGSB reactor declined with the increase of NaCl content of the influent. There were six phylogenetic groups, including Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, Tenericutes and Deinococcus-Thermus. Proteobacteria, accounting for90%, was the dominant microbial population, followed by Firmicutes. Abundance of Marinobacter and Halomonas genus accounted for more than75%of the total sequences which had played a main role in denitrification in the EGSB reactor. The analysis of qPCR indicated that the restrained degree of microoganisms containing the denitrification functional gens narG, nirK, nirS and nosZ increased with the increase of the NaCl salt content. And microoganisms containing the denitrification functional gens narG, nirK, nirS were more strongly inhibited. However, microoganisms containing genes nosZ can adapt to NaCl salt environment, and the environment of high NaCl salt content may promote its growth.4) The analysis of continuous and batch experiment indicated that the suitable reaction temperature of the EGSB reactor for the treatment of high nitrate wastewater was room temperature and moderated temperature. There was the nitrite nitrogen accumulation in the effluent when the reaction temperature was below10℃or higher than52℃. The analysis of high-throughput sequencing results indicated that microbial community structure and diversity in the EGSB reactor was greatly impacted by reaction temperature. Microbial diversity struck the highest point at35℃and the lowest at52℃. There were7phyla and84genera, and the dominant phylum was Proteobacteria, Firmicutes and Bacteroidetes, accounting for89-96%,1.90-6.00%and2.20-3.30%in each sample respectively. Furthermore, the dominant genus was Halomonas, Azoarcus and Marinobacter, accounting for41.62-81.59%,2.61-29.13%, and0.86-2.89%in each sample respectively. They played a main role in denitrification for high-nitrate in the EGSB reactor. The analysis of qPCR indicated that the diversity of microbial containing the denitrification functional gens narG, nirK, nirS and nosZ had different richness at the different reaction temperature. The abundance of nosZ gene was the highest in the four genes, and the diversity of corresponding microorganisms was the most abundant. The abundance of nirK gene was the lowest and the diversity of corresponding microorganisms was the most abundant at35℃.5) The results of the continuous and batch experiment indicated that when the concentration of phenol in the influent was less than or equal to600mg/L which also meant that the substitute sodium acetate as carbon source was26.8%, nitrate nitrogen was almost entirely denitrificated and its degradation dynamics accorded with the non-linear equations and corresponding correlation coefficient over0.97. While the effect of denitrification began to deteriorate, and the correlation coefficient of the corresponding nonlinear equation decreased to0.79when the concentration of phenol in the influent was more than600mg/L. Furthermore, Nitrate nitrogen and COD removal efficiency decreased obviously, and there was a lot of the nitrate accumulation, when chlorobenzene concentration outnumbered200mg/L. When the hexavalent chromium concentration of the influent was more than80mg/L, nitrite nitrogen was accumulated in large amount. The removal rate of TOC is obviously reduced and nitrate removal rate fell slightly as well. In addition, hexavalent chromium removal rate reached about99%. With the increase of cadmium concentration, the effect of denitrification was not very obvious, and cadmium removal rate was over80%. The analysis of high-flux sequencing results suggested that the order of rich microbial diversity degree in the samples was T35>B600>Cd16. Toxic substances inhibited the growth of microorganisms in an EGSB reactor, and the inhibitory effect of metal cadmium was stronger than that of phenol. There were7phyla, including Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, Tenericutes, Synergistetes and Deinococcus-Thermus. The dominant phylum was Proteobacteria, Firmicutes and Bacteroidetes, accounting for88.42-97.26%,2.00-5.30%and0.17-3.79%in each sample, respectively. Inhibitory effect of Phenol on Proteobacteria was stronger than of metal cadmium. Firmicutes was obviously inhibited by Phenol and cadmium ion. Actually, Cadmium ion of bacteroidetes’ inhibitory effect on Bacteroidetes was very obvious, while phenol slightly promoted its growth. Deinococcus-Thermus adapted to nitrate wastewater containing phenol, and it didn’t adapt to nitrate wastewater containing cadmium ion. The abundance of three samples was the same of the top five genera, which were Halomonas, Azoarcus, Pseudomonas, Wandonia, Marinobacter. While their proportion in the three samples was different, Halomonas adapted to grow in the nitrate wastewater containing cadmium ion more than in one containing phenol. However, Azoarcus adapted to growth in the high-nitrate wastewater containing phenol. The analysis of qPCR showed that the denitrification functional gens narG, nirK, nirS and nosZ were inhibited by phenol and cadmium ion, the phenol environment maybe promote the growth of microorganisms containing gene narG, this presented the corresponding relationship with the increase of the abundance of Azoarcus in the sample B600, which concluded that Azoarcus contained gene narG. |
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