鸡粪好氧堆肥氨氧化霉菌的筛选及生物学特性研究
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摘要
异养氨氧化作用是异养型的细菌或霉菌等将铵态氮或其他负三价氮氧化为羟胺、亚硝态氮或硝态氮的过程。作为氮素循环的重要环节,异养氨氧化作用对高有机物、高氮环境中的氮素转化具有重要意义。鸡粪等固体废弃物的好氧堆肥是高有机物、高氮的好氧体系,因其中的氨挥发致使氮素严重损失,影响堆肥肥效。在该体系中强化异养氨氧化,即将铵态氮转化为亚硝态氮和硝态氮,具有遏制其中氮素挥发、保持肥效的意义。
本研究以鸡粪好氧堆肥中分离的10株霉菌为对象,测定受试菌株的氮转化能力,筛选高效氨氧化菌株;测定高效氨氧化菌株不同生长时期的氮转化能力,明确菌体生长与氮转化作用的关系;改变培养条件,明确各环境因素对高效氨氧化菌株氮转化作用的影响;将高效菌株回归至鸡粪好氧堆肥,验证其实际效果。该研究旨在明确鸡粪好氧堆肥体系中氨氧化霉菌的存在情况、作用特性及实际效果,为降低该体系的氮素损失提供依据。
主要研究结果:
(1)受试的10株霉菌均能将铵态氮氧化为亚硝态氮和硝态氮,即均为氨氧化霉菌,提示该类霉菌在鸡粪好氧堆肥中普遍存在。确定的两株高效氨氧化霉菌菌株M25-22(Penicillium sp.)与M40-4(Aspergillus sp.)在氨氧化霉菌培养基中培养5d后,均能使铵态氮降低0.3mg·mL-1以上,生成亚硝态氮和硝态氮总量分别约在1.1×10-3mg·mL-1、1.5×10-3mg·mL-1。
(2)M25-22菌体生长量及铵态氮利用率在1～5 d迅速增加;硝态氮浓度3～5d上升迅速,以后维持在1.1～1.2μg·mL-1;亚硝态氮在2d后开始有微量生成,4d后呈降低趋势。M40-4菌体生长量及铵态氮利用率在1～3d迅速增加;硝态氮3～5d迅速增加;亚硝态氮在2d后开始增加,4d后趋于稳定。提示两株菌的氨氧化作用均属于次级代谢。
(3)M25-22与M40-4均能在以葡萄糖、蔗糖、淀粉或纤维素为唯一碳源的培养基中进行氨氧化作用,能氧化硫酸铵、蛋白胨、乙酰胺、尿素或L-天冬氨酸中的负三价氮,其中可溶性淀粉、纤维素、蛋白胨等缓效碳、氮源对该作用更有利。在以蔗糖为唯一碳源、铵盐为唯一氮源的培养基中,M25-22氨氧化的最适条件为:蔗糖浓度12g·L-1、铵态氮浓度2.438 mg·mL-1、pH 7.5和培养温度30℃;M40-4氨氧化的最适条件为:蔗糖浓度10g·L-1、铵态氮浓度2.014 mg·mL-1、pH 7.0和培养温度45℃。
(4)M25-22与M40-4分别回归堆肥后,均能明显降低堆肥氮素损失,促进堆肥腐熟,在鸡粪等固体废弃物的好氧堆肥中具有应用价值。
Heterotrophic ammonium oxidation is a process that ammonium nitrogen or negative trivalent organic nitrogen are oxidized into hydroxylamine, nitrite nitrogen or nitrate nitrogen by heterotrophic bacteria or molds. As an important part of global nitrogen cycle, heterotrophic ammonium oxidation plays a significant role in nitrogen transformation in systems containing high contents of organic compounds and nitrogen. Aerobic composting of solid waste, such as aerobic chicken manure composting is such a system in which ammonium volatilization resulted in serious nitrogen loss. The enhancement of heterotrophic ammonium oxidation is of significance in preventing nitrogen loss.
In the study, nitrogen transformation activities of 10 strains of molds isolated from aerobic chicken manure composting were determined to screen ammonia-oxidizing molds and define high-effective strains. Culture conditions were changed to evaluate effects of environmental factors on the nitrogen transformation activity of high-effective strains. Finally, the high-effective strains were added into aerobic chicken manure composting to verify application effects. The objective of the study is to make clear the existence, characteristics of nitrogen transformation and application effects of ammonia-oxidizing molds in aerobic chicken manure composting, and to provide evidence for the inhibition of ammonium volatilization in the process of aerobic composting. The main results were as follows.
(1) All of the 10 strains could oxidize ammonium nitrogen into nitrite and nitrate nitrogen, showing that they are ammonia-oxidizing molds, and indicating that ammonia-oxidizing molds are widely distributed in aerobic chicken manure composting. Both of the two defined high-effective strains, named M25-22 and M40-4, could decrease ammonium nitrogen by above 0.3mg·mL-1, and produce nitrite and nitrate nitrogen with a total concentration of 1.1×10-3mg·mL-1, 1.5×10-3mg·mL-1, respectively.
(2) In media containing (NH4)2SO4 at a concentration of 5mg·mL-1, mycelium weight of M25-22 and its utilization ratio of ammonium nitrogen increased markedly during the first 5 days; nitrate concentration increased markedly from the 3rd to 5th day, and remained constant thereafter; nitrite concentration remained at a low level. Mycelium weight of M40-4 and its utilization ratio of ammonium nitrogen increased markedly during the first 3 days; nitrate concentration increased markedly from the 3rd to 5th day; nitrite was produced after 2 days, and remained constant after 4 days.
(3) Both of the strain M25-22 and M40-4 showed the activity to oxidize ammonium nitrogen growing in media containing glucose, sucrose, starch or cellulose as the sole carbon source, and to oxidize the negative trivalent nitrogen of ammonium sulfate, peptone, acetamide, urea or L-aspartate. Slowly available carbon or nitrogen sources, such as starch, cellulose and peptone, were beneficial to ammonium oxidation by them. In media containing sucrose as the sole carbon source and ammonium sulfate as the sole nitrogen source, nitrate formation was the most pronounced with sucrose present at a concentration of 12g·L-1 and ammonium nitrogen present at a concentration of 2.438mg·mL-1, at the initial pH level of 7.5 and at 30℃by the strain M25-22, and with sucrose present at a concentration of 10g·L-1 and ammonium nitrogen present at a concentration of 2.014mg mL·-1, at the initial pH level of 7.0 and at 45℃.
(4) After added into aerobic chicken manure composting, both of the two high-effective strains could decrease nitrogen loss and promote the maturity of compost, indicating that they have significant application value in aerobic composting of solid wastes such as chicken manure.
本研究以鸡粪好氧堆肥中分离的10株霉菌为对象,测定受试菌株的氮转化能力,筛选高效氨氧化菌株;测定高效氨氧化菌株不同生长时期的氮转化能力,明确菌体生长与氮转化作用的关系;改变培养条件,明确各环境因素对高效氨氧化菌株氮转化作用的影响;将高效菌株回归至鸡粪好氧堆肥,验证其实际效果。该研究旨在明确鸡粪好氧堆肥体系中氨氧化霉菌的存在情况、作用特性及实际效果,为降低该体系的氮素损失提供依据。
主要研究结果:
(1)受试的10株霉菌均能将铵态氮氧化为亚硝态氮和硝态氮,即均为氨氧化霉菌,提示该类霉菌在鸡粪好氧堆肥中普遍存在。确定的两株高效氨氧化霉菌菌株M25-22(Penicillium sp.)与M40-4(Aspergillus sp.)在氨氧化霉菌培养基中培养5d后,均能使铵态氮降低0.3mg·mL-1以上,生成亚硝态氮和硝态氮总量分别约在1.1×10-3mg·mL-1、1.5×10-3mg·mL-1。
(2)M25-22菌体生长量及铵态氮利用率在1～5 d迅速增加;硝态氮浓度3～5d上升迅速,以后维持在1.1～1.2μg·mL-1;亚硝态氮在2d后开始有微量生成,4d后呈降低趋势。M40-4菌体生长量及铵态氮利用率在1～3d迅速增加;硝态氮3～5d迅速增加;亚硝态氮在2d后开始增加,4d后趋于稳定。提示两株菌的氨氧化作用均属于次级代谢。
(3)M25-22与M40-4均能在以葡萄糖、蔗糖、淀粉或纤维素为唯一碳源的培养基中进行氨氧化作用,能氧化硫酸铵、蛋白胨、乙酰胺、尿素或L-天冬氨酸中的负三价氮,其中可溶性淀粉、纤维素、蛋白胨等缓效碳、氮源对该作用更有利。在以蔗糖为唯一碳源、铵盐为唯一氮源的培养基中,M25-22氨氧化的最适条件为:蔗糖浓度12g·L-1、铵态氮浓度2.438 mg·mL-1、pH 7.5和培养温度30℃;M40-4氨氧化的最适条件为:蔗糖浓度10g·L-1、铵态氮浓度2.014 mg·mL-1、pH 7.0和培养温度45℃。
(4)M25-22与M40-4分别回归堆肥后,均能明显降低堆肥氮素损失,促进堆肥腐熟,在鸡粪等固体废弃物的好氧堆肥中具有应用价值。
Heterotrophic ammonium oxidation is a process that ammonium nitrogen or negative trivalent organic nitrogen are oxidized into hydroxylamine, nitrite nitrogen or nitrate nitrogen by heterotrophic bacteria or molds. As an important part of global nitrogen cycle, heterotrophic ammonium oxidation plays a significant role in nitrogen transformation in systems containing high contents of organic compounds and nitrogen. Aerobic composting of solid waste, such as aerobic chicken manure composting is such a system in which ammonium volatilization resulted in serious nitrogen loss. The enhancement of heterotrophic ammonium oxidation is of significance in preventing nitrogen loss.
In the study, nitrogen transformation activities of 10 strains of molds isolated from aerobic chicken manure composting were determined to screen ammonia-oxidizing molds and define high-effective strains. Culture conditions were changed to evaluate effects of environmental factors on the nitrogen transformation activity of high-effective strains. Finally, the high-effective strains were added into aerobic chicken manure composting to verify application effects. The objective of the study is to make clear the existence, characteristics of nitrogen transformation and application effects of ammonia-oxidizing molds in aerobic chicken manure composting, and to provide evidence for the inhibition of ammonium volatilization in the process of aerobic composting. The main results were as follows.
(1) All of the 10 strains could oxidize ammonium nitrogen into nitrite and nitrate nitrogen, showing that they are ammonia-oxidizing molds, and indicating that ammonia-oxidizing molds are widely distributed in aerobic chicken manure composting. Both of the two defined high-effective strains, named M25-22 and M40-4, could decrease ammonium nitrogen by above 0.3mg·mL-1, and produce nitrite and nitrate nitrogen with a total concentration of 1.1×10-3mg·mL-1, 1.5×10-3mg·mL-1, respectively.
(2) In media containing (NH4)2SO4 at a concentration of 5mg·mL-1, mycelium weight of M25-22 and its utilization ratio of ammonium nitrogen increased markedly during the first 5 days; nitrate concentration increased markedly from the 3rd to 5th day, and remained constant thereafter; nitrite concentration remained at a low level. Mycelium weight of M40-4 and its utilization ratio of ammonium nitrogen increased markedly during the first 3 days; nitrate concentration increased markedly from the 3rd to 5th day; nitrite was produced after 2 days, and remained constant after 4 days.
(3) Both of the strain M25-22 and M40-4 showed the activity to oxidize ammonium nitrogen growing in media containing glucose, sucrose, starch or cellulose as the sole carbon source, and to oxidize the negative trivalent nitrogen of ammonium sulfate, peptone, acetamide, urea or L-aspartate. Slowly available carbon or nitrogen sources, such as starch, cellulose and peptone, were beneficial to ammonium oxidation by them. In media containing sucrose as the sole carbon source and ammonium sulfate as the sole nitrogen source, nitrate formation was the most pronounced with sucrose present at a concentration of 12g·L-1 and ammonium nitrogen present at a concentration of 2.438mg·mL-1, at the initial pH level of 7.5 and at 30℃by the strain M25-22, and with sucrose present at a concentration of 10g·L-1 and ammonium nitrogen present at a concentration of 2.014mg mL·-1, at the initial pH level of 7.0 and at 45℃.
(4) After added into aerobic chicken manure composting, both of the two high-effective strains could decrease nitrogen loss and promote the maturity of compost, indicating that they have significant application value in aerobic composting of solid wastes such as chicken manure.
引文
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