【作者】 汪彩华；

【导师】 郑平；

【作者基本信息】 浙江大学 ， 环境工程， 2010， 硕士

【摘要】 菌种是国家的重要资源,菌种保藏是贮备良种、满足应用之需的重要手段。在废水生物处理领域,涌现了以短程硝化-厌氧氨氧化工艺为代表的一批新型生物脱氮技术,迫切需要高效生物脱氮菌种提供支持。相对于常规菌种,硝化细菌和厌氧氨氧化菌生长十分缓慢,对环境异常敏感,难以获得优质菌源,获得的优质菌源难以保藏。鉴于此,本文对硝化细菌混培物和厌氧氨氧化菌混培物的保藏技术进行了有益探索,主要结论如下:1、研究了硝化细菌混培物保藏特性的变化规律,提出了硝化细菌混培物大量保藏的有效方法。①比较了硝化细菌混培物保藏过程中硝化活性和总菌体量的变化规律,研究证明硝化活性更适宜用作保藏效果的评价指标。采用不同保藏方法保藏硝化细菌,保藏5个月后,总菌体量保留率是硝化活性保留率的2.5～3.9倍,以总菌体量作为保藏效果评价指标会产生虚高现象。②考察了硝化细菌混培物保藏过程中血红素c含量、ATP含量以及颜色的变化规律,研究证明血红素c含量和ATP含量可用于定性表征硝化活性,但颜色与硝化活性间没有直接相关性。在缺氧保藏中,硝化细菌混培物颜色变黑,其主要致因是硫酸盐还原生成硫化物,而低温或添加硝化产物可降低硫化物的生成速度,减缓硝化细菌混培物变黑速度。③试验了氧气对硝化细菌混培物保藏性状的影响,研究证明缺氧保藏优于好氧保藏。缺氧保藏和好氧保藏的硝化活性半衰期分别为1.71个月和1.60个月,饥饿保藏5个月后缺氧保藏和好氧保藏的活性保留率分别为15.9%和5.6%。④试验了硝化产物对硝化细菌混培物保藏性状的影响,研究证明添加亚硝酸盐可以改善硝化细菌的保藏效果。在硝化细菌保藏过程中,亚硝氮可明显降低硝化活性的衰减速率。在缺氧条件下,采用4℃加5 mmol·L^-1亚硝氮、4℃加5 mmol·L^-1硝氮、4℃三种方法保藏,硝化活性衰减速率分别为0.010 d^-1、0.020 d^-1、0.021 d^-1,半衰期分别为2.41、1.13、1.09个月,饥饿保藏5个月后三种保藏方法的硝化活性保留率分别为13.1%、14.4%、13.9%。⑤试验了温度对硝化细菌混培物保藏性状的影响,研究证明自然温度保藏适用于硝化细菌保藏。在缺氧条件下,采用自然温度、4℃、-20℃、-20℃加15%体积甘油四种方法保藏,硝化活性衰减速率分别为0.012 d^-1、0.021 d^-1、0.019 d^-1、0.013 d^-1,半衰期分别为1.71、1.09、1.22、1.62个月,饥饿保藏5个月后四种保藏方法的硝化活性保留率分别为16.0%、13.9%、20.8%、20.4%。以自然温度保藏硝化细菌,活性保留率居中,但保藏成本较低。2、研究了厌氧氨氧化菌混培物保藏特性的变化规律,填补了厌氧氨氧化菌混培物保藏技术的空白。①考察了厌氧氨氧化菌混培物保藏过程中血红素c含量、硫化物含量、外源硫酸盐含量及颜色的变化规律,研究证明血红素c含量可定性表征厌氧氨氧化活性,但受硫化物影响,缺氧保藏过程中混培物颜色变黑,可干扰颜色与活性间的关系。②试验了间歇性饥饿对厌氧氨氧化菌混培物保藏性能的影响,研究表明在厌氧氨氧化菌混培物保藏过程中,间歇性饥饿较长期性饥饿损害更大。缺氧条件下,采用4℃加（NH₄）₂SO₄、4℃加每10 d换一次NO₂^--N/NH₄⁺-N浓度比为1.32的基质、4℃加每10 d换一次NO₂^--N/NH₄⁺-N浓度比为1.63的基质三种方法保藏,保藏5个月后,厌氧氨氧化活性保留率分别为74.7%、68.0%、60.3%。③试验了温度对厌氧氨氧化菌混培物保藏性能的影响,研究表明4℃加（NH₄）₂SO₄的缺氧保藏法最为有效,但自然温度加（NH₄）₂SO₄的缺氧保藏法最为经济。在缺氧条件下,采用4℃加（NH₄）₂SO₄、4℃加NH₄Cl、自然温度加（NH₄）₂SO₄三种方法保藏,饥饿保藏5个月后,厌氧氨氧化活性保留率分别为74.7%、45.0%、64.2%。更多还原

【Abstract】 Microorganisms are a kind of important national resource, and their preservation is a means of storing good strain and meeting application requirements. In the field of wastewater treatment, a lot of novel biotechnologies such as short-cut nitrification and anammox （anaerobic ammonium oxidation） process for nitrogen removal were developed over the past decades, so a large amount of quality microorganisms for nitrogen removal are needed to promote the application of novel biotechnologies. Compared with other microorganisms, it is difficult to obtain and store quality nitrifying bacteria and anammox bacteria because of their slow growth and high sensitivity to environment. Therefore, the preservation methods of nitrifying bacteria and anammox bacteria were studied, and main results are as follows:1. The preservation characteristics were studied and some efficient preservation methods were suggested for mixed nitrifying culture.①As preservation evaluation index, nitrifying activity is better than total biomass. After preservation for 5 months, the survival biomass percentage was 2.5 to 3.9 times larger than the survival activity percentage with different preservation methods, so it is easy to mislead the evaluation results by using total biomass as evaluation index.②The content of heme c and ATP could qualitatively describe nitrifying activity, but the color had no correlation with nitrifying activity. Under anaerobic conditions, the color of preserved mixed nitrifying culture was changed from orange yellow to black due to sulfate reduction, but low temperature and nitrite/nitrate could decrease the sulfide formation and the color change.③Anaerobic preservation was better than aerobic preservation for mixed nitrifying culture. Under anaerobic and aerobic conditions, the half decay times of survival activity were 1.71 months and 1.60 months, and the survival activity percentages were 15.9% and 5.6% respectively after preservation for five months.④Nitrite could improve the preservation of mixed nitrifying culture, and could decrease the decay rate of nitrifying activity. Under anaerobic conditions, the decay rates of nitrifying activity were 0.010 d^-1, 0.020 d^-1 and 0.021 d^-1, the half decay times of survival activity were 2.41, 1.13 and 1.09 months, and the survival activity percentages were 13.1%, 14.4% and 13.9% after preservation for 5 months by means of 4℃with 5 mmol·L^-1 nitrite, 4℃with 5 mmol·L^-1 nitrate and 4°C, respectively.⑤Natural temperature was suitable for storing mixed nitrifying culture. Under anaerobic conditions, the activity decay rates were 0.012 d^-1, 0.021 d^-1, 0.019 d^-1, 0.013 d^-1, the half decay times of survival activity were 1.71, 1.09, 1.22 , 1.62 months, and the survival activity percentages were 16.0%, 13.9%, 20.8%, 20.4% after preservation for 5 months by means of natural temperature, 4℃, -20℃and -20℃with 15% glycerol respectively. The survival activity percentage at natural temperature was not the highest, but the cost was the lowest of all preservation methods.2. The preservation characteristics of anaerobic ammonium oxidation were studied, and some efficient preservation methods were suggested for mixed anammox culture.①The content of heme c could qualitatively describe anammox activity, but the color had no correlation with anammox activity because of sulfide production which led to the change of color from red to black during the preservation of mixed anammox culture.②Intermittent starvation was worse than persistent starvation during the preservation of mixed anammox culture. Under anaerobic conditions, three preservation methods, 4℃with substrate of （NH₄）₂SO₄, 4℃with addition of substrate （NO₂^--N/NH₄⁺-N ratio of 1.32） every ten days and 4℃with addition of substrate （NO₂^--N/NH₄⁺-N ratio of 1.63） every ten days, were carried out. After preservation for 5 months, their survival activity percentages were 74.7%, 68.0% and 60.3% respectively.③The preservation at 4℃with （NH₄）₂SO₄ was the best, but that at natural temperature with （NH₄）₂SO₄ was the cheapest. Under anaerobic conditions, the survival activity 4℃with （NH₄）₂SO₄, 4℃with NH₄Cl and natural temperature with （NH₄）₂SO₄ were 74.7%, 45.0% and 64.2% respectively after preservation for 5 months.更多还原