【作者】 曾庆武；

【导师】 葛向阳； 梁运祥；

【作者基本信息】 华中农业大学 ， 微生物学， 2008， 硕士

【摘要】 越来越严重的富营养化,尤其是亚硝酸盐污染已成为水产养殖业常见且难以预防的危害因素之一。微生物的反硝化脱氮法是解决上述问题的有效手段之一。本课题通过广泛取样,分离出202株反硝化细菌。用灭菌的LB培养基调节控制水样的COD_Mn约为25 mg/L,以此水样为基础,通过定量测定菌株反硝化能力的方法,同时结合实验室保存的反硝化芽孢杆菌DNF409的反硝化特点,筛选出一株同等条件下不会严重积累亚硝酸盐的菌株A13。通过生理生化试验和16S rDNA序列分析,菌株A13鉴定为地衣芽孢杆菌。研究表明:在影响反硝化效率的因素中,COD_Mn是关键因素。在COD_Mn低于10.5 mg/L时,两株菌A13和DNF409都只能进行微弱的反硝化作用。添加碳源后能显著提高反硝化作用,其中以添加乙醇效果最明显,向水样中添加0.2%（v/v）的乙醇后,反硝化脱氮率达99%以上。在pH为6.0-8.0的范围内,反硝化效率没有明显的差别,即在自然水体中,pH不会成为反硝化脱氮的限制因素。在温度超过20℃时,反硝化作用能进行得比较完全。投菌量对反硝化作用也有明显的影响,在COD_Mn为19 mg/L时,一次投菌量需要达到10⁷ cfu/mL才会有明显效果。由于反硝化作用的进行,两株菌对降低水样的COD_Mn也能起到一定的作用。研究过程中发现,不灭菌水样中,两株菌的反硝化效率明显优于灭菌水样;相同COD_Mn的野芷湖水样和蒸馏水样,前者的反硝化作用明显优于后者。分析可能是微生物菌群的协同反硝化作用。因此将两株菌协同联合应用。为了确定它们的联合应用方式,分别跟踪检测了它们各自的反硝化特点。研究结果显示:在前12 h内,DNF409降解硝酸盐的速度明显优于A13,但同时迅速积累大量的亚硝酸盐;在前24 h内,它们都能将硝酸盐降解到很低的水平,但DNF409积累的亚硝酸盐比A13严重,随着时间的进一步延长,达到156 h时,亚硝酸盐均会上升。根据两株菌的不同的作用特点及协同作用原理,通过试验建立了组合应用模式,首先使用DNF4`09菌株,24 h后再向水样中投加A13,比仅仅应用单株菌脱氮率提高近30%。以此组合应用模式为基础运用响应面分析法,得出最佳反硝化作用条件为:COD_Mn为35.1 mg/L,温度为32.5℃,投菌量为6.2×10⁶ cfu/mL,反硝化时间为114.2 h,此时脱氮率达99%以上。本课题研究了在特殊条件下的反硝化作用。在有机氮如酵母粉存在的条件下,能促进反硝化过程的进行;在无机氮如硫酸铵存在的情况下,仍能进行反硝化作用;如果溶氧较高,将会显著的抑制反硝化作用。更多还原

【Abstract】 The deterioration of aquarium water,especially nitrite pollution which was difficult to prevent,had being becoming an usual harmful factor of national aquiculture development in our country. Biodenitrification is one of the effective methods to remove nitrite from water at present.202 denitrifying strains were separated from numbers of samples. Their respective denitrifying activities were idetified quantitively in the condition that the CODMn of water sample controlled by sterilized LB medium was about 25 mg/L.At last,thinking the characters of denitrification about DNF409, A13 was obtained. Physiological and biochemical tests besides the analysis of 16SrDNA revealed that the A13 stain was Bacillus Licheniformis.The research result indicated that CODMn was the critical facters which affected denitrification efficiency. when CODMn was below 10.5 mg/L,they could denitrify slenderly.Adding carbon source,especial ethanol,could enhance denitrification obviously.In the range of pH from 6.0 to 8.0,the denitrification efficiency had no difference.In other words, the value of pH would not be a limited facter in actual water.When temperature exceeded 20℃, they could denitrify smoothly.The bacterial concentration also affected denitrification clearly.And they could play the role of eliminating the CODMn .The result showed that denitrification efficiency in actual water was much higher than that in sterilized water.Comparing Yezhi lake water with distilled water,the former advanced the latter obviously.Maybe it was the cooperation of microorganisms in denitrification.So the two strains were applied.Their respective denitrification characters were checked .The research revealed that DNF409 advanced A13 for the degradation of nitrate before 12 hrs,while DNF409 accelerated accumulation of nitrite heavily.They could both degradate nitrate to the low level,but DNF409 also exceeded A13 in the terms of the accumulation of nitrite.With time extending to 156 hrs,nitrite would both elevate.According to this result and the principle of cooperation,the combination application model was confirmed that DNF409 strain was used first,then 24 hrs later A13 strain was applied. Compared with the use of single strain, the combination application of DNF409 and A13 increased the percentage of denitrification about 30%.On the base of this model, response surface analysis was implemented. The result showed that the optimum denitrification condition included as follows: COD_Mn 35.1 mg/L, temperature 32.5℃, bacterial concentration 6.2×10⁶ cfu/mL, time 114.2 hrs.This article studied the dinitrification in the outstanding condition.For example,organic nitrogen existing,it could accelerate denitrification.Too much oxygen would inhibit denitrification.更多还原