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Purification of aquaculture wastewater is a key process for aquaculture process, in particular, circulation aquaculture systems, because water quality and aquaculture system sustained, stable and healthy operated are closely related. Many researches have found that the use of plants or microorganisms to treat aquaculture wastewater is a cost-effective method. So, in this thesis, some highly efficient heterotrophic nitrifiers were isolated from the sewage outfall of the circulation aquaculture system, and then, which were immobilized with ceramic and artificial plant. Finally, the immobilized microorganisms were integrated with the summer and autumn vegetables Ipomoea aquatica Forsk and the winter and spring forage grass Lolium multiflorum Lam. respectively. And the effect of purifying aquaculture wastewater by the integrated system was tested. The results summarized as follows:1. Eight strains of heterotrophic nitrifers were isolated from the sewage outfall of the circulation aquaculture system through the enrichment, separation, initial screening and re-screening. The sterile aquaculture wastewater was treated by these eight strains, and X1, X2, X3 and X4 strains were selected for the high removal rate of various forms of nitrogen. Within 24h, the removal rate of ammonia with X1, X2 and X3 strains were 80.01%, 67.65% and 66.49% in sterile aquaculture wastewater, and within 72h the removal rate of ammonia with X4 strain was 75.01%. Within 96h, the removal rate of TN by X1, X2, X3 and X4 strains were 32.63%, 31.77%, 12.03% and 21.48%, and in the process, the all treatments were without accumulation of nitrite and nitrate. By morphological, physiological and biochemical tests and 16S rDNA sequence analysis initially identified X1, X2, X3 and X4 strains were Pseudomonas sp., Bacillus megaterium, Bacillus flexus and Ochrobactrum intermedium respectively.2. Through the aerobic/anaerobic denitrification testing, it was found that under aerobic condition, glucose and sodium nitrate as the sole source of carbon and nitrogen source, X1, X2 and X3 strains, with aerobic denitrification capacity, had the high removal of nitrate, and without accumulation of ammonia and nitrite, and which the strongest of aerobic denitrification was X1 strain. But, under the same condition, the X4 strain could not grow, without the ability of aerobic denitrification, and was no removal of nitrate. Under anaerobic or facultative anaerobic condition, glucose and sodium nitrate as the sole source of carbon and nitrogen source, X1, X2, X3 and X4 strains, without the capacity of anaerobic denitrification, can not grow, and were disability to remove nitrate. Through refering to large amounts of material, I found that only X2 (B.megaterium) and X3 (B.flexus) are non-pathogenic strains, and can be directly used for in situ treatment of aquaculture wastewater. The effects of removing nitrogen by X2 and X3 were tested preliminary with different culture temperature and initial pH values in the laboratory condition. It was confirmed that the removal rate of TN by X2 strain was the highest, when the pH value was neutral, and the temperature was 30℃. The removal rate of TN by X3 strain was the highest, when the pH value was neutral and alkaline, and the temperature is 30~37℃. Comparing the effects of aquaculture wastewater treatment by the X2, X3 separated and the X2, X3 mixed, it was found that the TN removal in wastewater by the mixed bacteria was better than by a single species of bacteria.3. By adsorption the mixed microorganisms(X2 and X3) were immobilized used ceramic and artificial aquatic plants as carrier. Comparing the effect of immobilized microorganisms under the different immobilization time, and the removal rate of ammonia and TN by the ceramic and artificial plant immobilized microorganisms in 1d,3d,5d,7d,10d. It was found that the immobilization time had the remarkable influence to the immobilization effect and the removal rete of ammonia and TN. The effect of ceramic was the best by immobilized 5d, and the ammonia and TN removal of ceramic immobilized by 5d and 7d were the best in the artificial wastewater. The immobilization time has slight effect to the artificial plant. In addition to the immobilization time of 1d, the difference of the total number of bacteria was not obvious in artificial plant with the other immobilization time. Moreover, the ammonia and TN removal of the artificial plant immobilized with 3d and 5d was highest in the artificial wastewater.There was no significant difference between the removal of ammonia and TN by the ceramic and artificial plant immobilized microorganisms in the artificial wastewater. However, the ceramic was easier to stay microorganisms, on account of the pore diameter of the ceramic is smaller than artificial plant, and the ceramic can impact the great water power for its higher strength, and aerobic microbial more easily grow and reproduce for its floating is large, so immobilized ceramic was more suitable for aquaculture wastewater treatment.Compared with the immobilized microorganism ceramic(IM) and unimmobilized microorganism ceramic(UIM), IM was better for removing nitrogen in the artificial wastewater, because the microorganisms in the immobilized ceramic could use the organic matter included wastewater to grow and reproduce, and then removed nitrogen through the nitrification and denitrification.4. Comparing the immobilized microorganisms with plants, free microorganisms with plants, plants, immobilized microorganisms, free microorganisms, as well as the control group treating the aquaculture wastewater separately, the results showed:⑴under the high temperature condition, the effects of purifying the aquaculture wastewater by the vegatale I.aquatica Forsks and microorganisms united or separated were tested. The effects of removing TN were immobilized microorganisms + I.aquatica(IB+I)>free microorganisms+ I.aquatica(FB+I) > I.aquatica(I) > immobilized microorganisms(IB) > free microorganisms(FB) , and the control group (CK) was not distinctly different with the FB. All treatment groups had the effect of removing ammonia, nitrite, nitrate and CODMn in the aquaculture wastewater, and the removal rate of ammonia was the highest. Along with the extension of treatment time, the combined effect of the I.aquatica and microorganisms was more obvious. Compared with free microorganisms, the superiority of immobilized microorganisms had been demonstrated unceasingly. When the experiment ended, the removal rate of TN by IB+I, FB+I, I, IB, FB and the CK treatment groups were respectively 74.94%, 59.78%, 49.18%, 17.91%, 1.45% and 1.21%, and the differences between various treatment groups were remarkable (p<0.05)⑵under the low temperature condition, the effect of purifying the aquaculture wastewater by L.multiflorum Lam and microorganisms united or separated was tested, but the effect was inferior to use the I.aquatica Forsks and microorganism jointed or alone in the hot condition. The effects of removing TN were immobilized microorganisms + L.multiflorum Lam(IB+L) = free microorganisms+ L.multiflorum Lam(FB+L) > L.multiflorum Lam(L) > immobilized microorganisms(IB) > free microorganisms(FB), and the control group (CK) was not obviously different with the FB. The elimination effect of ammonia was remarkablely different among all treatment groups, and the removal rate of ammonia was the greatest by various treatment groups. The effect of removing nitrite and nitrate by the treatment groups included L.multiflorum Lam were better than the independent microorganism treatment groups. This may be due to low temperature, the function of microorganisms was normally displayed, and the role of plant been highlighted⑶From the dynamic change of microorganism’s total quantity in the plant rhizosphere analysis, It is discovered that no matter is the high temperature or the low temperature, the total quantity of microorganism in the plant and microorganisms union treatment groups were higher than that in the plant independent treatments, moreover under the high temperature condition, the difference was more obvious among various treatment groups. During the experiment, the total number of microorganisms in the plant rhizosphere rose first, and then drops. Along with time extension, the advantages of X2(B.megaterium) and X3(B.flexus) gradually disappeared in all treatment groups⑷I n the same period of time, the removal rate of TN, ammonia, nitrite, nitrate, CODMn was positive related with the total quantity of rhizosphere microorganism by the correlation analysis, and the correlation was more remarkable in high temperature condition. Either high or low temperature, the relevance between the removal rate of TN, ammonia, nitrite, nitrate, CODMn and the total number of rhizosphere bacteria was higher than the total number of rhizosphere actinomycetes. As well as, the relevance between the removal rate and rhizosphere fungus was the smallest.
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