鸡粪中氨氮降解菌的分离鉴定及除氨适宜条件研究

【目的】氨气是畜禽养殖业中最常见、危害最大的有害气体之一。为了获得用于治理畜禽粪便氨气污染的微生物,从发酵3 d的鸡粪中分离筛选高效氨氮降解菌,研究其对鸡粪的除氨效果。【方法】以硫酸铵为唯一氮源,对鸡粪中具有氨氮降解能力的微生物进行连续10代的富集培养,将获得的富集培养液按10-1梯度稀释后进行分离纯化。分离得到的单菌落接种至富集培养基中,培养24h后,测定培养基中剩余的氨氮含量,比较各菌株之间的氨氮降解率,筛选出具有高效氨氮降解能力的菌株。通过形态观察、分子生物学以及生物化学的方法进行菌株鉴定。研究不同温度(20℃、25℃、30℃、35℃、40℃)和p H(3.0、4.0、5.0、6.0、7.0、8.0、9.0)对菌株生长的影响,探索不同碳源(淀粉、甘露醇、柠檬酸钠、葡萄糖、乙酸钠、碳酸氢钠)、C/N(5、10、20、40)以及初始氨氮浓度(100、300、600、1 200mg·L~(-1))对菌株氨氮降解性能的影响。最后将得到的目标菌株制成菌悬液,按10%的接种量接种到鸡粪中,同时以等量无菌生理盐水作为对照组,分别培养24 h、48 h、72 h和96 h,测定鸡粪的氨气散发量以及不同形态氮素的变化情况,评价目标菌株对鸡粪的除氨效果。【结果】通过富集培养,从鸡粪中共分离出15株能够降解氨氮的菌株,进一步筛选得到1株氨氮高效降解菌LSA,经鉴定为克柔假丝酵母(Candida krusei),与Candida krusei isolate EM12(JF274497.1)的相似性达到99%,Gen Bank中的登录号为KT025851。该菌株的对数生长期为6-12h,可在p H 3-7,20-40℃条件下生长,能够分别利用葡萄糖、乙酸钠、淀粉、柠檬酸钠、甘露醇作为碳源,不能利用无机碳,当培养基的C/N为20时氨氮去除效果最佳。随着培养基中初始氨氮浓度的升高,菌株LSA对氨氮的降解率呈现下降趋势;与之相反,氨氮降解速率则随着初始浓度的升高呈现升高趋势。当氨氮初始浓度为327.20mg·L~(-1)时,60h内菌株LSA对氨氮的去除率达到71.88%,菌体含量为OD6002.45;当氨氮初始浓度为1 105.26mg·L~(-1)时,96h内菌株LSA对氨氮的去除率达到57.44%,菌体含量达到OD600 2.96。将其接种到鸡粪中可以显著降低鸡粪中氨氮的含量,最高可降低22.30%;减少粪中氨气的挥发量,最高可降低15.92%;增加粪便总氮含量;降低粪便氨氮占总氮的比重。【结论】克柔假丝酵母(Candida krusei)LSA菌株具有高效氨氮降解能力和较强的环境适应性,可有效减少鸡粪中的氨氮含量,降低氨气的挥发量。

Isolation and Characterization of Ammonia Nitrogen-Degrading Microbe from Chicken Manure

【Objective】Ammonia is one of the most harmful gases inanimal production. In order to inhibit the production of ammonia, we planned to screen microbes with the ability of ammonia removal and investigate its functional features.【Method】Ammonium sulfate was used as the sole nitrogen source to isolate the ammonia nitrogen-degrading microbe from chicken manure. Enrichment cultivation was conducted ten times. The final enrichment culture was streaked on the isolation agar after a ten-time step dilution. The colonies with different morphological characters were isolated and purified. The strain with the highest efficiency on ammonia nitrogen-degrading is our choice. The morphology, molecular biology and physiological and biochemical identification method was used to identified the target strain. Single factor experiments were conducted to identify its optimum degradation conditions, such as temperature (20, 25, 30, 35, 40℃), pH (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0), carbon source (starch, mannitol, Na-Citrate, glucose, sodium acetate, sodium bicarbonate), C/N (5, 10, 20, 40) and initial ammonium concentrations (100, 300, 600, 1 200 mg·L^-1). The bacterial suspension was inoculated into chicken manure with the inoculation of 10%. The ammonia emissions and nitrogen forms from chicken manure were studied.【Results】Fifteen strains with the ability of ammonium-degrading were isolated from the chicken manure. One strain named LSA, showed high efficient degradation ability. The strain was identified as Candida krusei and 99% sequence similarity with 18S rDNA of Candida krusei isolate EM12（JF274497.1）was revealed compared with homologous genes in GenBank. The GenBank accession number of the strain LSA was KT025851. Further degradation studies showed that the optimal conditions for the ammonium degrading of the strain LSA were pH 3.0–7.0, 20–40°C, C/N 20 with glucose as carbon source. The removal ef?ciency increased with increasing initial NH₄⁺-N concentrations. When the initial NH₄⁺-N concentration was 327.20 mg·L^-1, the NH₄⁺-N removal rate was 71.88% and the OD₆₀₀ was 2.45 within 60h. When the initial NH₄⁺-N concentration was 1 105.26 mg·L^-1, the NH₄⁺-N removal rate was 57.44% and the OD₆₀₀ was 2.96 within 96h. The ammonium concentration and ammonia emissions from chicken manure with LSA were lower than that in the control group. The reduction rates in the treatment groups were 22.30% and 15.92%. The total nitrogen in chicken manure inoculated with strain LSA is higher than that in the control group, while the ammonia nitrogen/ total nitrogen is lower. 【Conclusion】Strain LSA has good performance in ammonium degrading and displays good potential for future applications of ammonia emission control from chicken manure.