【作者】 谢丽凤；

【导师】 陆开宏；

【作者基本信息】 宁波大学 ， 海洋生物学， 2011， 硕士

【摘要】 水质与水产养殖系统持续、稳定、健康运行密切相关,养殖废水净化成为养殖过程(特别是闭合循环养殖系统)的一个重要环节。许多研究发现利用植物或微生物对养殖废水进行处理是一种经济有效的方法。本论文从水产养殖系统排污口筛选出脱氮效果较好的异养硝化菌,之后将筛选出的异养硝化菌固定化于陶粒或人工水草中,最后将固定化微生物分别与夏秋季蔬菜植物—水蕹菜(Ipomoea aquatica Forsk)和冬春季牧草—多花黑麦草(Lolium multiflorum Lam. )联合净化养殖废水,研究结果概况如下:1、通过富集、分离、初筛选和复筛选,从水产养殖闭合循环系统的排污口原位筛选出8株异养硝化菌,将这8株菌分别处理灭菌的水产养殖废水,从中挑选出脱氮效果较好的X1、X2、X3和X4菌株。24h内,X1、X2和X3对养殖废水中氨氮的去除率分别为80.01%、67.65%和66.49%,120h内,X4对氨氮的去除率也达到75.01%;96h内,X1、X2、X3和X4对TN的去除率分别为32.63%、31.77%、12.03%和21.48%,并且在处理过程中各处理组均无亚硝态氮和硝态氮积累现象。通过形态观察、生理生化试验和16S rDNA序列分析初步确定X1、X2、X3和X4分别为假单胞菌(Pseudomonas sp.)、巨大芽孢杆菌(Bacillus megaterium)、弯曲芽孢杆菌(Bacillus flexus)和中间苍白杆菌(Ochrobactrum intermedium)。2、在好氧条件下,以葡萄糖和硝酸钠为唯一碳源和氮源时,X1、X2和X3去除硝态氮的能力强,并且氨氮和亚硝态氮基本无积累,具有好氧反硝化能力,其中X1的能力最强;而X4不能生长,无去除硝态氮的能力,不具有好氧反硝化能力。在厌氧或兼性厌氧条件下,以葡萄糖和硝酸钠为唯一碳源和氮源时, X1、X2、X3和X4均不能生长,无去除硝态氮的能力,四株菌不具有厌氧反硝化能力。通过查阅大量资料发现只有X2(巨大芽孢杆菌)和X3(弯曲芽孢杆菌)是无致病性菌,可直接用于水产养殖废水的原位处理。在实验室条件下研究发现:在中性环境中,温度为30℃时,菌株X2的脱氮效果最佳;在中性偏碱性环境中,温度为30～37℃时,菌株X3的脱氮效果最佳。并且X2和X3混合菌对废水的脱氮效果好于X2或X3单种菌。3、以陶粒和人工水草为载体,采用吸附法对混合菌(X2、X3)进行固定化,比较了固定化时间(1、3、5、7、10d)对固定化效果的影响,以及在处理人工模拟废水时,固定化时间对固定化微生物陶粒和固定化微生物人工水草的氨氮去除和脱氮效果的影响。结果显示:固定化时间对固定化效果和固定化微生物的氨氮去除和脱氮效果有显著影响。固定化5d的陶粒的效果最好,并且固定化5d和7d的陶粒对废水的氨氮去除和脱氮效果最好;固定化时间对人工水草固定化效果影响较小,除了固定化时间为1d外,其他固定时间处理之间的固定化效果差异不明显,而且固定化3d和5d的人工水草对废水的氨氮去除和脱氮效果最好。固定化微生物陶粒和固定化微生物人工水草对废水中氨氮的去除率以及脱氮效果之间比较差异不明显。但由于陶粒孔隙的孔径比人工水草小,更容易滞留微生物;强度大,耐水力冲击;浮性大,好氧微生物易生长繁殖,因此固定化微生物陶粒更适合于水产养殖废水处理。固定化微生物陶粒与陶粒相比,固定化微生物陶粒对人工模拟废水的脱氮效果更好,固定化陶粒中的微生物可以利用废水中的有机物生长繁殖,再通过硝化反硝化作用进行脱氮。4、用固定化微生物与植物联合、游离微生物与植物联合、植物、固定化微生物、游离微生物以及对照组分别处理水产养殖废水,结果显示:⑴较高温度条件下,水蕹菜与微生物联合以及水蕹菜或微生物单独处理养殖废水均有一定的净化效果。从对TN的去除效果分析,总体效果为固定化菌+水蕹菜(IB+I)>游离菌+水蕹菜(FB+I)>水蕹菜(I)>固定化菌(IB)>游离菌(FB),其中游离菌组净化效果与对照组(CK)没有显著差异。各处理组对氨氮、亚硝态氮、硝态氮和CODMn均有去除效果,对氨氮的去除效果最好。随着处理时间的延长,水蕹菜与微生物联合作用的效果更加明显;并且与游离微生物相比,固定化微生物的优势也不断显示出来。试验结束时,IB+I、FB+I、I、IB、FB和CK对TN的去除率分别为74.94%、59.78%、49.18%、17.91%、1.45%和1.21%,各处理组之间的差异显著(p<0.05)。⑵在较低温度条件下,多花黑麦草与微生物联合以及多花黑麦草或微生物单独处理水产养殖废水也具有一定的效果,但效果不如高温条件下采用水蕹菜和微生物联合作用或者单独作用。从对TN的去除效果分析,总体效果为固定化菌+多花黑麦草(IB+L)=游离菌+多花黑麦草(FB+L)>多花黑麦草(L)>固定化菌(IB)>游离菌(FB),其中游离菌组净化效果与对照组(CK)没有显著差异。各处理组对氨氮的去除效果明显,去除速率最快;而从对亚硝态氮和硝态氮的去除效果上看,含有多花黑麦草的处理效果好,单独微生物处理组效果不明显。这可能是低温时,微生物的作用未正常发挥,植物的作用被凸显。⑶从植物根际微生物数量动态变化分析发现:不论是高温还是低温,植物与微生物联合处理组的植物根际微生物的数量高于植物单独处理组,且高温时差异更明显。试验过程中,植物根际微生物的数量先升后降,从细菌平板上可观察到X2(巨大芽孢杆菌)和X3(弯曲芽孢杆菌)的优势性随处理时间的延长逐渐消失。⑷通过相关性分析发现:同一时间段内,各处理组对TN、氨氮、亚硝态氮、硝态氮和CODMn的去除率与植物根际微生物总量之间成正相关,高温条件下,相关性显著;不论高温还是低温,植物对各氮素和CODMn的去除率与植物根际细菌总量之间的正相关性高于放线菌,且真菌的正相关性最小。更多还原

【Abstract】 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.更多还原