两株高效好氧反硝化细菌的分离鉴定及其好氧反硝化特性研究
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摘要
好氧反硝化作用一直是国外的研究热点,近年来在国内也备受关注。目前对于好氧反硝化作用的机理解释还不清晰,但是已有的研究表明好氧反硝化细菌在污水脱氮处理中有着很强的应用潜力,为克服传统的脱氮工艺中出现的不足提供了的解决方法。因此本试验着手于分离和鉴定出高效的好氧反硝化细菌,对细菌的好氧反硝化特性展开研究,以丰富和发展对好氧反硝化细菌的认识,并为将来的在水处理中的应用提供依据。
本试验从水稻土和活性污泥中各分离到一株可以在好氧条件下进行反硝化作用的细菌ZW23和ZW27。通过对这两株细菌的生理生化特征研究,以及16SrDNA序列测定认为菌株ZW23和ZW27分别与假单胞菌属类产碱杆菌(Pseudomonaspseudoalcaligenes)和假单胞菌属门多萨菌(Pseudomonas mendocina)亲缘关系最近。好氧培养条件下,在初始氮源约为280 mg·l~(-1)的反应体系中,37℃时,两株细菌在12h内均引起体系中总氮显著下降,削减率均达66%,其余总氮几乎全部转化为内源氮。脱氮速率分别达到约21.7mg·l~(-1)·h~(-1)和22.3 mg·l~(-1)·h~(-1),比现已分离出的兼性好氧反硝化细菌的脱氮速率要快得多。反应过程中没有检测到亚硝态氮和氨态氮的积累。
对细菌进行纯培养,研究发现细菌ZW23和ZW27在好氧条件下的反硝化作用比缺氧条件下进行得快,细菌数量也较缺氧条件下的细菌数量大,但是反硝化率(66%和62%)比缺氧条件下的反硝化率(90%和92%)低。细菌ZW23可以NH_4~+-N为唯一氮源,利用率达到96%以上,但是反应过程中没有检测到硝酸盐和亚硝酸盐的积累,也没有检测到总氮的削减。研究发现,菌株ZW23的好氧反硝化率与硝酸盐浓度在一定范围内呈正相关关系,随着硝酸盐浓度在一定范围内(50-400 mg·l~(-1))地不断提高,细菌的好氧反硝化率也逐渐提高(0%-65%),硝酸盐浓度400-500 mg·l~(-1)之间时,好氧反硝化率即维持稳定,不再变化。细菌ZW23和ZW27都可以利用NO_2~--N作为唯一氮源生长并进行好氧反硝化作用。NH_4~+-N和NO_3~--N复合氮源更有利于菌株ZW23进行好氧反硝化作用,好氧反硝化速度随着NH_4~+-N浓度在一定范围内(0-300 mg·l~(-1))的提高而不断加快。研究发现琥珀酸盐是细菌ZW23和ZW27进行好氧反硝化作用最理想的碳源。
好氧条件下,对细菌进行纯培养,研究了培养介质在不同C/N比,起始pH,培养温度条件下,菌株ZW23和ZW27的好氧反硝化活性。研究表明,菌株ZW23和ZW27最适好氧反硝化温度在32℃-37℃之间。在pH为6.0-8.0范围内,pH对细菌ZW23的好氧反硝化作用影响不大,好氧反硝化率均可达到57%-60%。pH为5.0时细菌没有生长迹象。碳源是细菌好氧反硝化作用的重要影响因素,碳源过低(C/N比2),菌株不进行好氧反硝化作用;碳源不足(C/N比5),菌株的好氧反硝化率仅30%左右;细菌进行好氧反硝化作用最适的C/N比是10-15之间,此时的好氧反硝化率在60%左右。本试验同时研究了细菌在废水处理中的潜力,菌株ZW23和ZW27接入经过高浓度氨氮人工废水驯化的好氧活性污泥中后,发现活性污泥体系对人工合成的高浓度氨氮污水的TN的去除率有一定程度的改善(提高10-20%)作用。
Aerobic denitrification has been paid more and more attention in recent years, however,there is limited information on the mechanism of aerobic denitrification.Recently, aerobic denitrification bacteria such as Thiosphaera pantotropha and Alcaligenes faecalis have been intensively studied as potential microorganisms that may be used to overcome problems inherent in the conventional method.For obtaining more aerobic denitrifers,the isolation and characteristic of new strains capable of high aerobic denitrification were conducted in the study.
Two strains of facultative and gram negative aerobic denitriflers,coded as ZW23 and ZW27,were isolated from flooded paddy soil and active sludge,respectively.In the light of their morphological and physiological features as well as the 16S rDNA sequences,they were identified as Pseudomonas pseudoalcaligenes and Pseudomonas mendocina.Under the aerobic incubation condition at 37℃,N loss through denitrification in the liquid culture medium inoculated with ZW23 and ZW27 both reached to 66%within12 hours, respectively.Correspondingly,N removal rates were 21.7 mg·l~(-1)9h~(-1) and 22.3 mg·l~(-1)·h~(-1), respectively,which was much higher than those reported by other researchers.It was found that a part of total nitrogen was assimilated completely by the denitrifiers.Nitrite and ammonia weren't observed during the incubation.Obviously,strains ZW23 and ZW27 are two typical facultative aerobic denitrifiers of high efficiency in the removal of N in cultural medium.
The strains ZW23 and ZW27 displayed denitridication under both aerobic and anaerobic conditions,but denitrification efficiency under aerobic condition(66%and 62%) was much lower than that under anaerobic condition(90%)..Strain ZW23 growing in the medium containing NH_4~+-N as the sole nitrogen source could remove more than 95%of NH_4~+-N in 36h.However,strain ZW23 had not the function of heterotrophic nitrification. Aerobic denitrifacation rate increased from 0%to 65%with the increase of the nitrate concentration for 50 to 400 mg·l~(-1) in cultural medium.Strains ZW23 and ZW27 could also grow and showed aerobic denitrification ability in the medium containing NO_2~--N as the sole nitrogen source.In addition,strain ZW23 could simultaneously utilize both NH_4~+-N and NO_3~--N.Moreover,the addition of NH_4~+-N could accelerate aerobic denitrification speed of strain ZW23.
For aerobic denitrification,optimum growth temperature of strains ZW23 and ZW27 was 32℃to 37℃.When initial pH of the mediums was 6.0-8.0,pH didn't affect the efficiency of aerobic denitrificaion of ZW23 strain with about 57%-60%of aerobic denitrification rate.The strain ZW23 could not grow under the initial pH 5.0.Carbon source could affect aerobic denitrification seriously,and the optimum C/N ratio for aerobic denitrificaion was between 10-15.In this study,we also investigated the application potential in wastewater treatment by the two aerobic denitrifiers ZW23 and ZW27.It was found that the two strains could improve total nitrogen removal efficiency of the wastewater by 10-20%.
本试验从水稻土和活性污泥中各分离到一株可以在好氧条件下进行反硝化作用的细菌ZW23和ZW27。通过对这两株细菌的生理生化特征研究,以及16SrDNA序列测定认为菌株ZW23和ZW27分别与假单胞菌属类产碱杆菌(Pseudomonaspseudoalcaligenes)和假单胞菌属门多萨菌(Pseudomonas mendocina)亲缘关系最近。好氧培养条件下,在初始氮源约为280 mg·l~(-1)的反应体系中,37℃时,两株细菌在12h内均引起体系中总氮显著下降,削减率均达66%,其余总氮几乎全部转化为内源氮。脱氮速率分别达到约21.7mg·l~(-1)·h~(-1)和22.3 mg·l~(-1)·h~(-1),比现已分离出的兼性好氧反硝化细菌的脱氮速率要快得多。反应过程中没有检测到亚硝态氮和氨态氮的积累。
对细菌进行纯培养,研究发现细菌ZW23和ZW27在好氧条件下的反硝化作用比缺氧条件下进行得快,细菌数量也较缺氧条件下的细菌数量大,但是反硝化率(66%和62%)比缺氧条件下的反硝化率(90%和92%)低。细菌ZW23可以NH_4~+-N为唯一氮源,利用率达到96%以上,但是反应过程中没有检测到硝酸盐和亚硝酸盐的积累,也没有检测到总氮的削减。研究发现,菌株ZW23的好氧反硝化率与硝酸盐浓度在一定范围内呈正相关关系,随着硝酸盐浓度在一定范围内(50-400 mg·l~(-1))地不断提高,细菌的好氧反硝化率也逐渐提高(0%-65%),硝酸盐浓度400-500 mg·l~(-1)之间时,好氧反硝化率即维持稳定,不再变化。细菌ZW23和ZW27都可以利用NO_2~--N作为唯一氮源生长并进行好氧反硝化作用。NH_4~+-N和NO_3~--N复合氮源更有利于菌株ZW23进行好氧反硝化作用,好氧反硝化速度随着NH_4~+-N浓度在一定范围内(0-300 mg·l~(-1))的提高而不断加快。研究发现琥珀酸盐是细菌ZW23和ZW27进行好氧反硝化作用最理想的碳源。
好氧条件下,对细菌进行纯培养,研究了培养介质在不同C/N比,起始pH,培养温度条件下,菌株ZW23和ZW27的好氧反硝化活性。研究表明,菌株ZW23和ZW27最适好氧反硝化温度在32℃-37℃之间。在pH为6.0-8.0范围内,pH对细菌ZW23的好氧反硝化作用影响不大,好氧反硝化率均可达到57%-60%。pH为5.0时细菌没有生长迹象。碳源是细菌好氧反硝化作用的重要影响因素,碳源过低(C/N比2),菌株不进行好氧反硝化作用;碳源不足(C/N比5),菌株的好氧反硝化率仅30%左右;细菌进行好氧反硝化作用最适的C/N比是10-15之间,此时的好氧反硝化率在60%左右。本试验同时研究了细菌在废水处理中的潜力,菌株ZW23和ZW27接入经过高浓度氨氮人工废水驯化的好氧活性污泥中后,发现活性污泥体系对人工合成的高浓度氨氮污水的TN的去除率有一定程度的改善(提高10-20%)作用。
Aerobic denitrification has been paid more and more attention in recent years, however,there is limited information on the mechanism of aerobic denitrification.Recently, aerobic denitrification bacteria such as Thiosphaera pantotropha and Alcaligenes faecalis have been intensively studied as potential microorganisms that may be used to overcome problems inherent in the conventional method.For obtaining more aerobic denitrifers,the isolation and characteristic of new strains capable of high aerobic denitrification were conducted in the study.
Two strains of facultative and gram negative aerobic denitriflers,coded as ZW23 and ZW27,were isolated from flooded paddy soil and active sludge,respectively.In the light of their morphological and physiological features as well as the 16S rDNA sequences,they were identified as Pseudomonas pseudoalcaligenes and Pseudomonas mendocina.Under the aerobic incubation condition at 37℃,N loss through denitrification in the liquid culture medium inoculated with ZW23 and ZW27 both reached to 66%within12 hours, respectively.Correspondingly,N removal rates were 21.7 mg·l~(-1)9h~(-1) and 22.3 mg·l~(-1)·h~(-1), respectively,which was much higher than those reported by other researchers.It was found that a part of total nitrogen was assimilated completely by the denitrifiers.Nitrite and ammonia weren't observed during the incubation.Obviously,strains ZW23 and ZW27 are two typical facultative aerobic denitrifiers of high efficiency in the removal of N in cultural medium.
The strains ZW23 and ZW27 displayed denitridication under both aerobic and anaerobic conditions,but denitrification efficiency under aerobic condition(66%and 62%) was much lower than that under anaerobic condition(90%)..Strain ZW23 growing in the medium containing NH_4~+-N as the sole nitrogen source could remove more than 95%of NH_4~+-N in 36h.However,strain ZW23 had not the function of heterotrophic nitrification. Aerobic denitrifacation rate increased from 0%to 65%with the increase of the nitrate concentration for 50 to 400 mg·l~(-1) in cultural medium.Strains ZW23 and ZW27 could also grow and showed aerobic denitrification ability in the medium containing NO_2~--N as the sole nitrogen source.In addition,strain ZW23 could simultaneously utilize both NH_4~+-N and NO_3~--N.Moreover,the addition of NH_4~+-N could accelerate aerobic denitrification speed of strain ZW23.
For aerobic denitrification,optimum growth temperature of strains ZW23 and ZW27 was 32℃to 37℃.When initial pH of the mediums was 6.0-8.0,pH didn't affect the efficiency of aerobic denitrificaion of ZW23 strain with about 57%-60%of aerobic denitrification rate.The strain ZW23 could not grow under the initial pH 5.0.Carbon source could affect aerobic denitrification seriously,and the optimum C/N ratio for aerobic denitrificaion was between 10-15.In this study,we also investigated the application potential in wastewater treatment by the two aerobic denitrifiers ZW23 and ZW27.It was found that the two strains could improve total nitrogen removal efficiency of the wastewater by 10-20%.
引文
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