| Since wastewater discharges contain excessive nitrogen compounds that cause oxygen depletion and eutrophication in the receiving water. Therefore, reducing nitrogen levels from the discharges has become one of the most important concerns in water pollution control. In municipal treatment systems, the most widely used method for nitrogen removal from wastewater is achieved by the combined process of aerobic autotrophic nitrification and anaerobic heterotrophic denitrification. However, this process tends to be long procedure, time-consuming, cost-intensive, and vulnerable to high loads of ammonium and organic matter due to the low rate of nitrification and the complexity of separating aerobic and anoxic tanks. To overcome these limitations, several novel nitrogen removal processes have been developed in the past few years, especially in heterotrophic nitrification. Recently, more studies have highlighted the existence of bacteria that are capable of performing heterotrophic nitrification and have a marked ability to denitrify their nitrification products under aerobic conditions. These special heterotrophic bacteria exhibit higher growth rates than autotrophs and can use organic substrates as sources of energy for denitrification. Therefore, simultaneous nitrification and denitrification(SND) could be achieved for low operation costs and highly accelerated rates of nitrogen removal in a single reactor. In this study, aerotolerant nitrogen removal sludge was acclimated in a laboratory-scale sequencing batch reactor(SBR), which high simultaneous nitrification and denitrification was demonstrated. Three novel heterotrophic nitrifying-aerobic denitrifying bacteria were isolated from the activated sludge, and the characteristics of heterotrophic nitrification and aerobic denitrification, the nitrogen removal mechanism and the application potential of the isolated strain in wastewater treatment were investigated. The main research results are as follows:(1) The aerotolerant nitrogen removal sludge was successfully acclimated in a SBR, which exhibited high efficient nitrogen removal ability. The removal percentages of COD, NH4+–N and TN were as high as 96%, 99% and 70%, respectively. During the culture period, the flocculation and settleability of sludge were gradually increased,and the sludge gradually changed to regular and dense structure. The analysis of extracellular polymeric substances(EPS) showed that the contents of proteins, DNA and TOC were decreased, but polysaccharides were increased after sludge acclimation. Comparing with the three-dimensional fluorescence peaks of EPS, the intensity of humic acid fluorescence peak was significantly decreased.(2) DO and COD/N have significant influence on the nitrogen removal efficiency and flocs flocculation properities. The results indicated that the sludge system exhibited poor nitrogen removal and flocculation performance under grester or lesser degree of DO condition, as well as an overload of organic substrates. The sludge system could achieve desired nutrient removal and flocculation-settleability performance under the operation conditions of aeration rate 160 L/h and COD/N 12. Based on the effects of DO and COD/N on SND and the results of microbial count, it can be speculated that the microenvironment theory and biology theory should be the main reasons of SND.(3) The three strains were all Gram-negative and appeared as short rods. On the basis of physiological-biochemical test and phylogenetic analysis of the 16 S rRNA gene sequence, strain YB, YH and YL were identified as Acinetobacter junii YB, Pseudomonas putida YH and Pseudomonas aeruginosa YL, respectively. The partial 16 S rRNA sequences of the isolates(YB, YH and YL) were deposited in the GenBank database(GenBank ID: # KJ623740, KJ765710 and KJ765709).(4) Single-factor experiments indicated that the optimal conditions for all the three heterotrophic ammonium oxidation strains were succinate as the carbon source, C/N ratio of 10, pH of 7.0, temperature of 37 °C, and shaking speed of 200 rpm. All three strains exhibited efficient heterotrophic nitrification-aerobic denitrification abilities with a low accumulation of nitrification products at a wide range of ammonium loads. At an initial NH4+–N concentrations of 100 mg/L, the maximum specific growth rates of strains YB, YL and YH were about 0.34 h-1, 0.49 h-1 and 0.26 h-1, respectively. The removal of nutrient was accompanied by the growth of isolates. After 48 h cultivation, the TOC removal efficiencies were all higher than 90%, and the removal efficiencies of NH4+–N and TN were all reached to 95%. The nitrogen balance showed that intracellular nitrogen concentrations accounted for about 51–57% of removed TN.(5) Strain YB, YH and YL all had the capability to metabolize hydroxylamine, nitrite and nitrateas a sole nitrogen source, and the mutual transformation of differnet nitrogen sources were observed during the culture period. Ammonium was preferential removal by the isolates in the ammonium-nitrite and ammonium-nitrate mixed N-source, and the nitrite and nitrate started to decline gradually after the complete removal of ammonium. The addition of nitrite and nitrate did not inhibit the ammonium nitrogen removal activity, but promoted the removal of ammonium.(6) The PCR amplification of five potential enzymes genes involved in this heterotrophic nitrogen removal process were carried out. The results showed that amoA hao and nap A genes were successfully amplified in all the isolates, the nir S gene was detected in strain YB, while nirK gene was expressed in strain YH and YL. The maximum activity of HAO was found in YH with a specific activity of 0.0431U/mg proteins, followed by 0.0314 and 0.0195 U/mg proteins in the case of YL and YB, respectively. The NiR activities of YB, YL and YH were 0.0386, 0.0439 and 0.0489 U/mg proteins, respectively. And the corresponding specific activities of NR were 0.0095, 0.0117 and 0.0186 U/mg proteins, respectively. The utilization of different nitrogen sources and the successful amplification of the nitrifying and denitrifying enzymes genes provided additional evidence of heterotrophic nitrification and aerobic denitrification by the isolates, and further proved the nitrogen removal pathway of the isolate could be achieved through heterotrophic nitrification coupled with aerobic denitrification.(7) The auto-aggregation experiment demonstrated that strain YB had superior auto-aggregation ability in heterotrophic nitrification and denitrification media, the change of visual aggregation degree, FA, RH, Zeta potential and floc size of the isolate showed regular variation trends during the whole cultivation. The aggregation of microbial cells was determined to elucidate the flocculating properties and subsequent biomass enrichment of the isolate for future practical applications. In addition, stable nitrogen and organics removal were achieved by one-time dosing of enriched bacteria in a SBR. The inoculation of strain YB significantly improved the denitrification efficiency with minimal accumulation of nitrified products, which demonstrated high potential of the isolate for future practical applications. Moreover, heterotrophic nitrogen removal sludge formed by strain YB exhibited high nitrogen and COD removal ability under aerobic conditions. After the SBR system had reached steady state of 60 d, the respective removal efficiencies of TOC, NH4+–N and TN were stable at 95%, 99% and 94%, and the number of strain YB account for about 50 percent of total bacterial after two months. During the culture period of heterotrophic nitrogen removal sludge, the sludge gradually changed to regular and dense structure, and the flocculation and settleability of sludge were increased gradually. DO and COD/N had significant influence on the nitrogen removal efficiency and flocs flocculation properity. The desired nutrient removal and flocculation-settleability performance could be achieved under the operation conditions of aeration rate 100 L/h and C/N 12, which demonstrated high potential of the isolate for future practical applications in the treatment of high-strength carbon and nitrogen wastewater. |
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