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1、合肥工业大学 硕士学位论文 新型脱氮菌株的筛选、鉴定及其固定化研究 姓名：司文玫 申请学位级别：硕士 专业：食品科学 指导教师：曾庆梅 20100401 新型脱氮菌株的筛选、鉴定及其固定化研究新型脱氮菌株的筛选、鉴定及其固定化研究 摘摘 要要 水体富营养化是我国目前水环境面临的重大问题，而氮素是主要的污染因 子。废水脱氮的方法有多种，生物脱氮被认为是目前最经济有效的方法之一。 传统生物脱氮理论认为脱氮主要由一群自养好氧型硝化菌和一群异养厌氧型反 硝化菌完成。然而在实践中人们发现这两种菌用在脱氮工艺中缺陷较多，所以 近年来对异养硝化菌和好氧反硝化菌的研究和应用成为热点，并有望克服传统 脱氮工艺的
2、缺陷，丰富生物脱氮理论。因固定化微生物在废水脱氮上的优势， 许多脱氮菌株已被固定化。在固定化方法上，微胶囊固定化因其良好的稳定性、 生物相容性和传质性而备受关注，但目前对异养硝化菌和好氧反硝化菌的同步 微胶囊固定化还未见报道。 本文从合肥市内多家污水处理厂活性污泥、化肥厂土壤及市郊农田土壤中 取得 26 个土样，通过一系列步骤，从中筛得一株高效的异养硝化菌和一株高效 的好氧反硝化菌，鉴定了它们的种属，考察了它们的脱氮条件和脱氮特征，将 它们优化组合，对组合菌株脱氮条件进行了优化，考察了组合菌株对实际污水 的脱氮效果，并将组合菌株进行了微胶囊同步固定化研究。 本研究所得结果如下： 1. 采用污泥
3、驯化、驯化过程中驯化液连续梯度稀释、平板划线分离及颜色 指示剂快速硝化、反硝化效果检测等步骤，筛得一株高效的异养硝化菌 HN-S 和一株高效的好氧反硝化菌 DN-S，鉴定其种属分别属产碱菌属和鲁氏不动杆 菌，并命名为 Alcaligenes sp.HN-S 和 Acinetobacter lwoffii DN-S。 2. HN-S 为专性异养硝化菌，无自养硝化能力。其在处理初始氨氮浓度为 182.30 mg/L 的废水时，30 h 后氨氮去除率为 99.8%，指数期降解氨氮的平均 速率为 9.61 mg-N/L/h，高于目前所筛得的大多数异养硝化菌株，且在降解氨 氮的过程中无亚硝酸盐氮和硝酸盐
4、氮的积累；最佳碳源为柠檬酸钠；高的溶氧 量和高的 C/N 比有利于其降解氨氮， 当 C/N 比为 12 时即可达到最好的脱氮效果； 该菌株在温度为 3035，pH 为 5.09.0 范围内均能较彻底地降解氨氮； 能利用亚硝酸盐氮作为氮源反硝化，但不能利用硝酸盐氮；能将氨氮直接转变 为以氮气为主的含氮气体。后两点与目前报道的大多数异养硝化菌不同。 3. 好氧反硝化菌 DN-S 在处理初始硝酸盐氮浓度为 166.05 mg/L 的废水时， 72 h 后硝酸盐氮去除率为 99.8%，总氮去除率为 48.3%，指数期降解硝酸盐氮 的平均速率为 8.2 mg-N/L/h；其在反硝化过程中未出现亚硝酸盐氮
5、的积累；高 的溶氧量有利于其反硝化作用，实验中最佳溶氧量为 4.2 mg/L；最佳 C/N 比为 912；能将硝酸盐氮转变为含氮气体，能利用氨氮，也能利用亚硝酸盐氮反硝 化，是一株同步硝化反硝化菌。 4. 以总氮去除率为指标， 通过正交试验确定了HN-S和DN-S混合培养时的最 佳脱氮条件：两菌接种量比例为 1:2，C/N比为12.5，温度为35，pH为9.0， 溶氧量为2.5 mg/L。将组合菌株接种于实际污水中，与活性污泥相比，发现组 合菌株对不同的污水适应能力更强， 其对不同来源的污水都有较好的脱氮效果， 尤其对氮素含量相对较低的生活污水脱氮效果更好。 组合菌株在脱氮时对碳 源有一定的依
6、赖性，柠檬酸钠的加入可以明显促进其脱氮能力。同时两菌的组 合使用也加强了其脱氮能力。 5. 用微胶囊固定化组合菌株处理人工模拟废水结果显示，相比游离，固定 化菌株脱氮速率稍低，但脱氮持续时间长，产生的菌泥少，同化的氮量低，从 而提高了总氮去除率；固定化也提高了菌株抵抗不良环境的能力。 关键词：关键词：异养硝化；好氧反硝化；同步硝化反硝化；固定化技术；微胶囊 Isolation, identification and immobilization of novel denitrification strains ABSTRACT Water eutrophication is one of ma
7、jor problems of water environment in our country, and the nitrogen is the main pollution factor. There are several ways for wastewater denitrification, and the biological denitrification is considered as one of most cost-effective methods. Traditional biological denitrification theory suggests that
8、the wastewater denitrification is mainly completed by a group of autotrophic, aerobic nitrifying bacteria and a group of heterotrophic, anaerobic denitrifying bacteria. However, in practice, it is discovered that there are many defects for these two kinds of bacteria used in the denitrifying process
9、. So, in recent years the research and application of heterotrophic nitrifier and aerobic denitrifier being a hot spot, and it is expected to overcome the shortcomings of the traditional nitrogen removal process and enrich the biological denitrification theory. During the applied study of biology de
10、nitrifying technology, immobilization technology has been attracted attention beacuse of the superiority of itself. As one of immobilization methods, microcapsule immobilized has been concerned because of its good stabiliy, biocompatibility and mass trasfer. But the synchronous microcapsule immobili
11、zation of the heterotrophic nitrifier and aerobic denitrifier has not been repoted. We obtained 26 activated sludged samples from sewage plants, chemical fertilizer plants and farmland. After performing a series of stemps, finally a high-efficient heterotrophic nitrifier and a high-efficient aerobic
12、 denitrifier were obtained. We identified the strains and studied their denitrification conditions and characteristics. Then we mixed the two kinds of strains, and optimized the mixed strains denitrification conditions, investgated the denitrification effects in real wasrewater, synchronous immobili
13、zed the mixed strains by microcapsules. The results of this study are as follows. 1. After sludge domestication, gradient dilution of domestication liquid, isolation from streaking plate, utilization of color indicator as rapid denitrification detection method, finally a high-efficient heterotrophic
14、 nitrifier and a high-efficient aerobic denitrifier were obtained. The two strains were identified as Alcaligenes sp. and Acinetobacter lwoffii respectively, and named as Alcaligenes sp. HN-S and Acinetobacter lwoffii DN-S. 2. HN-S was a obligate heterotrophic nitrifying bacteria, no autotrophic nit
15、rification capacity. In the artificial wastewater with 182.30 mg/L ammonia nitrogen as initial concentration, the removal efficiency by the strain was 99.8% after 30 h cultivation. The average nitrogen removal rate was 9.61 mg-N/L/h in its exponential phase, faster than the most heterotrophic nitrif
16、iers that screened before. It produced almost no NO2-N and NO3-N in the entire nitrification process. The optimal carbon source is sodium citrate. Higher dissolved oxygen and C/N ratio favored its nitrification. When temperature is ranged from 30 to 35 and pH is ranged from 5.0 to 9.0, it can completely remove ammonia nitrogen. It was able to denitrify when nitrite nitrogen was used as
