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Article(id=1241049970770497876, tenantId=1146029695717560320, journalId=1234093305789726721, issueId=1241049962679694215, articleNumber=null, orderNo=null, doi=null, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1719331200000, receivedDateStr=2024-06-26, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1773818970625, onlineDateStr=2026-03-18, pubDate=1737302400000, pubDateStr=2025-01-20, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1773818970625, onlineIssueDateStr=2026-03-18, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1773818970625, creator=13701087609, updateTime=1773818970625, updator=13701087609, issue=Issue{id=1241049962679694215, tenantId=1146029695717560320, journalId=1234093305789726721, year='2025', volume='45', issue='1', pageStart='1', pageEnd='592', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1773818968696, creator=13701087609, updateTime=1773819749443, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1241053237428671382, tenantId=1146029695717560320, journalId=1234093305789726721, issueId=1241049962679694215, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1241053237428671383, tenantId=1146029695717560320, journalId=1234093305789726721, issueId=1241049962679694215, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=500, endPage=507, ext={EN=ArticleExt(id=1241049971298980187, articleId=1241049970770497876, tenantId=1146029695717560320, journalId=1234093305789726721, language=EN, title=Isolation and degradation kinetic analysis of a heterotrophic nitrification-aerobic denitrification strain tolerant to high-strength ammonium, columnId=1240689595814302661, journalTitle=China Environmental Science, columnName=Environmental Microbiology, runingTitle=null, highlight=null, articleAbstract=

A heterotrophic nitrification and aerobic denitrification (HNAD) strain WN1 with significant tolerance against high ammonium concentrations was isolated from a reactor treating high-strength ammonium organic wastewater. Based on colony morphology and 16S rDNA analysis, it was identified as Corynebacterium glutamicum. The growth and metabolic characteristics of WN1 were studied under different initial ammonium concentrations of 500, 1000, 1500, 2000, 2500 and 3000mg/L, respectively. The results showed that strain WN1 could still grow well (48h, OD600=3.264) at an initial ammonium concentration of 3000mg/L, corresponding to a free ammonia concentration of 590.2mg/L. As initial ammonium concentrations increased from 500 to 3000mg/L, the maximum ammonium conversion rate (Rm) of strain WN1increased from 15.93 to 43.29mg/(L·h). Additionally, the average conversion rates of ammonium and COD were 7.06~9.36mg/(L·h) and 95.63~199.13mg/(L·h), respectively. Furthermore, the Haldane model was used to characterize the growth and substrate degradation properties of strain WN1under different initial ammonium concentrations (R2 = 0.99). Strain WN1had a maximum specific growth rate of umax = 0.36h-1, a maximum specific ammonium degradation rate of rmax = 6.45gN/(gDCW·d), and a maximum specific COD degradation rate of rmax = 122.85gCOD/(gDCW·d). The ammonium inhibition constant Ki of strain WN1was 3749.49mg/L, indicating greater resistance to high ammonium concentrations compared with other autotrophic or heterotrophic ammonium-oxidizing bacteria. The results suggest that strain WN1is promising for treating high-strength ammonium organic wastewater.

, correspAuthors=Xin-ying LIU, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Nuo WANG, Yu DAO, Xing-yu QIN, De-zhi SUN, Xin-ying LIU), CN=ArticleExt(id=1241049985173738316, articleId=1241049970770497876, tenantId=1146029695717560320, journalId=1234093305789726721, language=CN, title=一株耐高氨氮异养硝化-好氧反硝化菌的筛选及降解动力学分析, columnId=1240689596959346705, journalTitle=中国环境科学, columnName=环境微生物, runingTitle=null, highlight=null, articleAbstract=

从处理含高碳、氮浓度废水的反应器中筛选出一株耐高氨氮的异养硝化-好氧反硝化菌WN1，通过菌落形态以及16S rDNA分析鉴定为谷氨酸棒杆菌Corynebacterium glutamicum.对该菌在初始氨氮浓度分别为500，1000，1500，2000，2500，3000mg/L的生长及代谢特性进行研究.结果表明，菌株WN1在氨氮浓度高达3000mg/L，对应游离氨浓度为590.2mg/L时仍能较好生长(48h，OD600=3.264).随着初始氨氮浓度由500提升至3000mg/L，菌株WN1的最大氨氮转化速率Rm由15.93增加至43.29mg/(L·h).同时，反应过程中氨氮和COD的平均转化速率分别为7.06~9.36mg/(L·h)和95.63~199.13mg/(L·h).进一步，采用Haldane模型拟合不同初始氨氮浓度下菌株的生长及基质降解特性(R2=0.99)，菌株WN1的最大比生长速率、最大氨氮比降解速率和最大COD比降解速率分别为0.36h-1、6.45gN/(gDCW·d)，122.85gCOD/(gDCW·d).菌株WN1的氨氮抑制常数Ki为3749.49mg/L，比其它自养或异养的氨氧化菌对高浓度氨氮具有更强的抗抑制能力，结果表明，菌株WN1在处理高浓度含氮有机废水中具有很好的应用前景.

, correspAuthors=刘新颖, authorNote=null, correspAuthorsNote=
*责任作者，讲师，
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汪诺(2000-)，女，安徽安庆人，北京林业大学硕士研究生，主要从事生物脱氮及废水处理研究..

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汪诺(2000-)，女，安徽安庆人，北京林业大学硕士研究生，主要从事生物脱氮及废水处理研究..

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汪诺(2000-)，女，安徽安庆人，北京林业大学硕士研究生，主要从事生物脱氮及废水处理研究..

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China Environmental Science，2016，36(5):1409-1416., articleTitle=Nitrogen removal and inhibition kinetics of ANAMMOX reactor fed with the mature landfill leachate, refAbstract=null)], funds=[Fund(id=1241049999371457008, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1241049970770497876, awardId=52300075, language=CN, fundingSource=国家自然科学基金青年科学基金项目(52300075), fundOrder=null, country=null), Fund(id=1241050000969486853, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1241049970770497876, awardId=642201053, language=CN, fundingSource=国家自然科学基金项目(642201053), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1241049985471533934, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1241049970770497876, xref=null, ext=[AuthorCompanyExt(id=1241049985475728241, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1241049970770497876, companyId=1241049985471533934, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=Beijing Key Laboratory of Water Pollution Source Control Technology, College of Environmental Science and Engineering, Beijing Forestry University, Beijing 100083, China), AuthorCompanyExt(id=1241049985484116848, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1241049970770497876, companyId=1241049985471533934, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=北京林业大学环境科学与工程学院，北京市水体污染源控制技术重点实验室，北京 100083)])], figs=[ArticleFig(id=1241049994275377411, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1241049970770497876, language=EN, label=Fig.1, caption=Colony morphology and phylogenetic tree of strain WN1, figureFileSmall=jGXse3g3ajUgAjl6MC2WvA==, figureFileBig=a4ROJpg3DkssyavJVfqZlA==, tableContent=null), ArticleFig(id=1241049994434760976, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1241049970770497876, language=CN, label=图1, caption=菌株WN1的菌落形态和系统发育树, figureFileSmall=jGXse3g3ajUgAjl6MC2WvA==, figureFileBig=a4ROJpg3DkssyavJVfqZlA==, tableContent=null), ArticleFig(id=1241049994845802795, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1241049970770497876, language=EN, label=Fig.2, caption=The growth and substrate degradation properties of strain WN1 under different initial ammonium concentrations, figureFileSmall=RMT4ty0EqV8HR21Ev9BB5Q==, figureFileBig=hAj2DUkop8pZdeAHba0I4Q==, tableContent=null), ArticleFig(id=1241049996531913022, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1241049970770497876, language=CN, label=图2, caption=不同初始氨氮浓度下菌株WN1的生长与基质降解特性, figureFileSmall=RMT4ty0EqV8HR21Ev9BB5Q==, figureFileBig=hAj2DUkop8pZdeAHba0I4Q==, tableContent=null), ArticleFig(id=1241049997077172555, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1241049970770497876, language=EN, label=Fig.3, caption=The inhibition kinetic model for the growth of strain WN1under different initial ammonium concentrations, figureFileSmall=yH+/1IFs1xWPJ9TO7ivQ8w==, figureFileBig=xM1EZazwc/hSctZbPlLKeA==, tableContent=null), ArticleFig(id=1241049997236556120, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1241049970770497876, language=CN, label=图3, caption=不同初始氨氮浓度菌株WN1生长的抑制动力学模型, figureFileSmall=yH+/1IFs1xWPJ9TO7ivQ8w==, figureFileBig=xM1EZazwc/hSctZbPlLKeA==, tableContent=null), ArticleFig(id=1241049997479825766, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1241049970770497876, language=EN, label=Fig.4, caption=The inhibition kinetic model for ammonium degradation by strain WN1under different initial ammonium concentrations, figureFileSmall=YCp2KHFG08QQcFxJnPNCwQ==, figureFileBig=CF1ddWuEfaj7fHNzMoppQg==, tableContent=null), ArticleFig(id=1241049997672763764, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1241049970770497876, language=CN, label=图4, caption=初始氨氮浓度菌株WN1降解氨氮的抑制动力学模型, figureFileSmall=YCp2KHFG08QQcFxJnPNCwQ==, figureFileBig=CF1ddWuEfaj7fHNzMoppQg==, tableContent=null), ArticleFig(id=1241049997802787203, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1241049970770497876, language=EN, label=Fig.5, caption=The inhibition kinetic model for COD degradation by strain WN1under different initial ammonium concentrations, figureFileSmall=jGdoE4yE3ACq0t0ofrzMHg==, figureFileBig=tuAgsABQyfwOkt60yHaesg==, tableContent=null), ArticleFig(id=1241049997890867597, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1241049970770497876, language=CN, label=图5, caption=初始氨氮浓度菌株WN1降解COD的抑制动力学模型, figureFileSmall=jGdoE4yE3ACq0t0ofrzMHg==, figureFileBig=tuAgsABQyfwOkt60yHaesg==, tableContent=null), ArticleFig(id=1241049998108971422, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1241049970770497876, language=EN, label=Table 1, caption=

Kinetic parameters for ammonium degradation of strain WN1under different initial ammonium concentrations

, figureFileSmall=null, figureFileBig=null, tableContent=
起始浓度(mg/L)Rm(mg/(L·h))t0(h)R2
50015.933.440.96
100030.265.990.98
150028.2916.860.97
200041.2421.400.98
250045.8531.850.99
300043.2934.300.96
), ArticleFig(id=1241049998360629669, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1241049970770497876, language=CN, label=表1, caption=

不同初始氨氮浓度下菌株WN1的氨氮降解动力学参数

, figureFileSmall=null, figureFileBig=null, tableContent=
起始浓度(mg/L)Rm(mg/(L·h))t0(h)R2
50015.933.440.96
100030.265.990.98
150028.2916.860.97
200041.2421.400.98
250045.8531.850.99
300043.2934.300.96
), ArticleFig(id=1241049998582927799, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1241049970770497876, language=EN, label=Table 2, caption=

Specific cell growth rate and substrate degradation rate of strain WN1under different initial ammonium concentrations

, figureFileSmall=null, figureFileBig=null, tableContent=
初始氨氮
浓度
(mg/L)		FA
浓度(mg/L)		开始结束q氨氮max
(gN/(gDCW·
d))		qCODmax
(gCOD/(g
DCW·d))		umax(h-1)Y(gDCW
/gN)
t0时刻氨氮浓度S0(mg/L)COD浓度M0(mg/L)细胞浓度X0(mg/L)t时刻氨氮浓度S(mg/L)COD浓度M(mg/L)细胞浓度X(mg/L)
50097.064514815130.35123772985919.692.25556.1550.32610.739
1000188.969669665140.34128637505910.552.94361.5660.3127.460
1500284.66148014625120.9312138112978778.033.27554.4770.3106.637
2000412.56209519385102.0012201018080586.113.33351.1760.2915.695
2500470.37252623855133.6812240822558560.983.14246.5730.2874.883
3000590.27308429070107.1512299228135407.903.08741.8850.2674.365
), ArticleFig(id=1241049998687785407, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1241049970770497876, language=CN, label=表2, caption=

不同初始氨氮浓度下菌株WN1的比生长速率与基质比降解速率

, figureFileSmall=null, figureFileBig=null, tableContent=
初始氨氮
浓度
(mg/L)		FA
浓度(mg/L)		开始结束q氨氮max
(gN/(gDCW·
d))		qCODmax
(gCOD/(g
DCW·d))		umax(h-1)Y(gDCW
/gN)
t0时刻氨氮浓度S0(mg/L)COD浓度M0(mg/L)细胞浓度X0(mg/L)t时刻氨氮浓度S(mg/L)COD浓度M(mg/L)细胞浓度X(mg/L)
50097.064514815130.35123772985919.692.25556.1550.32610.739
1000188.969669665140.34128637505910.552.94361.5660.3127.460
1500284.66148014625120.9312138112978778.033.27554.4770.3106.637
2000412.56209519385102.0012201018080586.113.33351.1760.2915.695
2500470.37252623855133.6812240822558560.983.14246.5730.2874.883
3000590.27308429070107.1512299228135407.903.08741.8850.2674.365
), ArticleFig(id=1241049998792643024, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1241049970770497876, language=EN, label=Table 3, caption=

Estimated parameters from the Haldane kinetics model

, figureFileSmall=null, figureFileBig=null, tableContent=
Haldane模型rmaxKs(mg/L)Ki(mg/L)R2
比生长速率0.3623.029754.770.99
氨氮比降解速率6.45880.403749.490.99
COD比降解速率122.85439.881677.950.99
), ArticleFig(id=1241049999090438625, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1241049970770497876, language=CN, label=表3, caption=

Haldane动力学模型参数的拟合值

, figureFileSmall=null, figureFileBig=null, tableContent=
Haldane模型rmaxKs(mg/L)Ki(mg/L)R2
比生长速率0.3623.029754.770.99
氨氮比降解速率6.45880.403749.490.99
COD比降解速率122.85439.881677.950.99
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一株耐高氨氮异养硝化-好氧反硝化菌的筛选及降解动力学分析
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汪诺 , 刀禹 , 覃星雨 , 孙德智 , 刘新颖 *
中国环境科学 | 环境微生物 2025,45(1): 500-507
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中国环境科学 | 环境微生物 2025, 45(1): 500-507
一株耐高氨氮异养硝化-好氧反硝化菌的筛选及降解动力学分析
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汪诺 , 刀禹, 覃星雨, 孙德智, 刘新颖*
作者信息
  • 北京林业大学环境科学与工程学院，北京市水体污染源控制技术重点实验室，北京 100083

    • 汪诺(2000-)，女，安徽安庆人，北京林业大学硕士研究生，主要从事生物脱氮及废水处理研究..

通讯作者:

*责任作者，讲师，
Isolation and degradation kinetic analysis of a heterotrophic nitrification-aerobic denitrification strain tolerant to high-strength ammonium
Nuo WANG , Yu DAO, Xing-yu QIN, De-zhi SUN, Xin-ying LIU*
Affiliations
  • Beijing Key Laboratory of Water Pollution Source Control Technology, College of Environmental Science and Engineering, Beijing Forestry University, Beijing 100083, China
出版时间: 2025-01-20
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摘要
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从处理含高碳、氮浓度废水的反应器中筛选出一株耐高氨氮的异养硝化-好氧反硝化菌WN1，通过菌落形态以及16S rDNA分析鉴定为谷氨酸棒杆菌Corynebacterium glutamicum.对该菌在初始氨氮浓度分别为500，1000，1500，2000，2500，3000mg/L的生长及代谢特性进行研究.结果表明，菌株WN1在氨氮浓度高达3000mg/L，对应游离氨浓度为590.2mg/L时仍能较好生长(48h，OD600=3.264).随着初始氨氮浓度由500提升至3000mg/L，菌株WN1的最大氨氮转化速率Rm由15.93增加至43.29mg/(L·h).同时，反应过程中氨氮和COD的平均转化速率分别为7.06~9.36mg/(L·h)和95.63~199.13mg/(L·h).进一步，采用Haldane模型拟合不同初始氨氮浓度下菌株的生长及基质降解特性(R2=0.99)，菌株WN1的最大比生长速率、最大氨氮比降解速率和最大COD比降解速率分别为0.36h-1、6.45gN/(gDCW·d)，122.85gCOD/(gDCW·d).菌株WN1的氨氮抑制常数Ki为3749.49mg/L，比其它自养或异养的氨氧化菌对高浓度氨氮具有更强的抗抑制能力，结果表明，菌株WN1在处理高浓度含氮有机废水中具有很好的应用前景.

异养硝化-好氧反硝化  /  动力学  /  高氨氮  /  游离氨  /  比降解速率

A heterotrophic nitrification and aerobic denitrification (HNAD) strain WN1 with significant tolerance against high ammonium concentrations was isolated from a reactor treating high-strength ammonium organic wastewater. Based on colony morphology and 16S rDNA analysis, it was identified as Corynebacterium glutamicum. The growth and metabolic characteristics of WN1 were studied under different initial ammonium concentrations of 500, 1000, 1500, 2000, 2500 and 3000mg/L, respectively. The results showed that strain WN1 could still grow well (48h, OD600=3.264) at an initial ammonium concentration of 3000mg/L, corresponding to a free ammonia concentration of 590.2mg/L. As initial ammonium concentrations increased from 500 to 3000mg/L, the maximum ammonium conversion rate (Rm) of strain WN1increased from 15.93 to 43.29mg/(L·h). Additionally, the average conversion rates of ammonium and COD were 7.06~9.36mg/(L·h) and 95.63~199.13mg/(L·h), respectively. Furthermore, the Haldane model was used to characterize the growth and substrate degradation properties of strain WN1under different initial ammonium concentrations (R2 = 0.99). Strain WN1had a maximum specific growth rate of umax = 0.36h-1, a maximum specific ammonium degradation rate of rmax = 6.45gN/(gDCW·d), and a maximum specific COD degradation rate of rmax = 122.85gCOD/(gDCW·d). The ammonium inhibition constant Ki of strain WN1was 3749.49mg/L, indicating greater resistance to high ammonium concentrations compared with other autotrophic or heterotrophic ammonium-oxidizing bacteria. The results suggest that strain WN1is promising for treating high-strength ammonium organic wastewater.

HNAD  /  kinetics  /  high-strength ammonium  /  free ammonia  /  specific degradation rate
汪诺, 刀禹, 覃星雨, 孙德智, 刘新颖. 一株耐高氨氮异养硝化-好氧反硝化菌的筛选及降解动力学分析. 中国环境科学, 2025 , 45 (1) : 500 -507 .
Nuo WANG, Yu DAO, Xing-yu QIN, De-zhi SUN, Xin-ying LIU. Isolation and degradation kinetic analysis of a heterotrophic nitrification-aerobic denitrification strain tolerant to high-strength ammonium[J]. China Environmental Science, 2025 , 45 (1) : 500 -507 .
正文
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含氮有机废水如养殖废水、污泥脱水液和垃圾渗沥液等[1]在经过厌氧消化处理后含有高浓度的氨氮（>1000mg/L），需要得到妥善处理.厌氧处理阶段的甲烷化和氨化过程会产生碱度导致pH值不断升高（>8.0），碱性条件下氨氮从离子态水解转化为分子态，厌氧出水中的游离氨（FA）浓度常常高达上百mg/L.相比于高成本且易产生二次污染的物化处理技术，生物脱氮技术具有经济、绿色的优点[2].然而，FA的生物毒害性较强[3]，会影响传统生物氨氧化过程（如自养硝化和厌氧氨氧化）.研究发现当FA浓度为20~50mg/L时，绝大多数自养细菌的生长和氨氧化性能受到抑制[4-5]，从而造成传统生物脱氮过程不稳定和处理效果差等问题，成为了高氨氮废水生物脱氮处理的瓶颈.
Robertson等[6]首次从脱硫脱硝废水处理系统中分离得到了一株兼有异养硝化和好氧反硝化功能（HNAD）的菌株Paracoccus pantotrophus.近年来，越来越多的HNAD菌被分离出来，分布在Alcaligenes[7]、Pseudomonas[8]、Acinetobacter[9]等不同菌属.HNAD菌具有世代周期短，生理活性高和环境适应力强等特性，其脱氮同时能够降解转化废水中的有机物，在处理含氮有机废水中具有很好的应用前景[10]. HNAD菌在耐受较高浓度氨氮方面展现出良好潜力[11-12]，然而，目前关于HNAD菌的筛选和脱氮特性研究所用的氨氮浓度仍然有限，通常小于2000mg/L且主要为离子态氨氮，高浓度氨氮尤其是高FA下对HNAD菌生长代谢的影响应得到关注.
本文从处理含高碳、氮浓度废水的反应器中筛选出一株对高浓度氨氮（FA浓度也高）具有较好耐受和脱除能力的HNAD菌WN1，研究菌株在不同初始氨氮浓度下的生长和代谢特性，并通过动力学模型进行拟合，以期为HNAD菌在处理高浓度含氮有机废水中的应用提供理论支持.
1 材料与方法
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1.1 培养基
对常规异养硝化培养基[13]改进后用于本实验，其配方为（1L）：NH4Cl 3.821g，CH3COONa 12.82g，K2HPO4 0.25g，新维氏盐溶液10mL，NB微量元素10mL，维他命溶液10mL.其中新维氏盐溶液配方为（1L）：KCl 20.0g，MgSO4·7H2O 12.5g，CaCl2·2H2O 2.0g；NB微量元素配方为（1L）：FeSO4·7H2O 0.3g，CuCl2·2H2O 0.03g，ZnSO4·7H2O 0.2g，MnCl2·4H2O 0.1g，CoCl2·6H2O 0.17g，NiSO4·6H2O 0.11g.通过改变加入NH4Cl的质量调整培养基初始氨氮浓度，并相应改变加入CH3COONa量固定C/N为10.培养基在使用前用1mol/L NaOH或1mol/L HCl调节初始pH为8.4±0.1，121℃高压蒸汽灭菌30min.固体平板培养基需加入1.5%~2%的琼脂粉.
1.2 实验方法
1.2.1 异养硝化-好氧反硝化菌株的筛选
实验室长期运行的SBR反应器（5.0L）对含高碳、氮浓度的垃圾焚烧厂渗沥液厌氧出水（COD 4000~6000mg/L、）具有较好的处理效果[14]，COD去除率>95%，总氮去除率>99%.从反应器中取出2.0mL活性污泥并涡旋振荡混匀，采用梯度稀释法稀释样品后，取0.1mL的10-4、10-5和10-6稀释液分别涂布于初始氨氮浓度为1000mg/L的HNAD固体培养基，pH值为8.4±0.1，对应FA浓度为210.4~ 302.8mg/L.于30℃恒温培养箱培养至长出菌落（3d），选取成长茁壮且不同形态的单菌落进行编号并挑取至HNAD液体培养基，于30℃、150r/min条件下培养2d.分别测定12h、24h、36h和48h培养基中氨氮浓度与COD浓度，选出其中氨氮和COD转化率高且经过几代分离纯化后效果稳定的菌株.
1.2.2 菌株鉴定
采用DNA提取试剂盒（QIAGEN）提取菌株DNA，对16S rDNA进行PCR采用引物27F（5’-AGAGTTT-GATCCTGGCTCAG-3’）/1492R（5’-TACGGYTACCTTGTTACGACTT-3’）. PCR反应体系为（50μL）：DNA模板1μL，Taq DNA聚合酶（PCR Master Mix）46 μL，前后引物各1.5μL.PCR反应条件为：98℃预变性2min，98℃变性10s，54℃退火15s，72℃延伸30s，30个循环，72℃延伸10min.将获得的PCR产物交由中美泰和生物技术有限公司（北京）进行测序，测序结果提交GenBank获得序列号.使用NCBI网站的BLAST在线程序将结果序列与Genbank中已有菌株序列进行比对，选择同源性高的菌株序列下载，将测序结果在NCBI里进行Blast分析，利用MEGA11.0软件将筛选菌株与其他已报道的典型HNAD菌进行比对，并采用近邻结合法（Neighbour-joining）构建该菌株的系统发育树.
1.2.3 不同初始氨氮浓度条件下菌株脱氮性能研究
向密闭小瓶（250mL）装入HNAD培养基（50mL），上方连通空气气袋（2L）提供菌株所需氧气，并每隔4h向气袋中补充纯氧.设置HNAD培养基（pH值约8.4）初始氨氮浓度分别为500，1000，1500，2000，2500，3000mg/L.将长至对数生长期的菌液于8000r/min离心收集菌体，用PBS溶液洗涤3次后重悬于无菌水中作为接种液，固定接种液OD600值约为0.6，并接种（2%）至上述培养基，同时设置不加接种液的空白对照组.在30℃，150r/min恒温摇床中进行培养，定时取样测定氨氮、硝氮、亚硝氮、COD浓度以及菌悬液OD600值.取样同时抽取气袋中气体（100mL），通入吸收液（0.005mol/L稀硫酸，50mL）后测定氨氮浓度，并通过公式（1）计算物理逸散的氨氮量.
式中：ρ为逸散的氨氮（mg/L）；A为顶空总体积与抽取气体量的比（22）；C吸为吸收液氨氮（mg/L）；V吸为吸收液体积（50mL）；V培为培养基体积（50mL）.
1.2.4 细胞浓度与OD600的标准曲线
以去离子水做空白，通过分光光度计（600nm）测定一系列不同生长状态的菌悬液吸光度（OD600），通过干燥法测定对应菌株细胞浓度（mg/L），即细胞干重（DCW），将细胞干重同OD600建立标准曲线，所得方程为：Y=571.19X-57.05（R2=0.99）.
1.2.5 菌株比生长速率、基质比降解速率、产率系数计算
式中：µ为菌株的比生长速率（h-1）；X为t时刻时菌株的细胞浓度（mg/L）；X0为t0时刻细胞浓度（mg/L）；t0为菌株培养任一阶段的初始时刻（h）；t为菌株培养任一阶段的结束时刻（h）.
式中：q为比降解速率（g/（gDCW·d））；S0为初始时刻t0氨氮浓度（mg/L）；S为t时刻氨氮浓度（mg/L）；X0为t0时刻菌株的细胞浓度（mg/L）.
式中：Y为产率系数（g/（DCW·gN））；X0为初始时刻细胞浓度（mg/L）；X为结束时刻细胞浓度（mg/L）；S0为初始时刻氨氮浓度（mg/L）；S为结束时氨氮浓度（mg/L）.
1.3 Compertz降解动力学模型
菌株WN1的氨氮转化过程可以采用修饰过的Compertz模型[15]进行拟合，见公式（5）：
式中：S0为初始时刻氨氮浓度（mg/L）；S为t时刻氨氮浓度（mg/L）；Rm为最大转化速率（mg/（L·h））；t0为迟滞时间（h）；t为反应时间（h）；e为常数.
1.4 Haldane抑制动力学模型
当氨氮浓度过高对硝化菌的活性产生抑制时，氨氮浓度对其降解过程的影响可以用Haldane模型拟合[16]，见公式（6）：
式中：S0为底物浓度（mg/L）；r为底物的比降解速率[g/（gDCW·d）]；rmax为微生物未被抑制时的最大比降解速率[g/（gDCW·d）]；KS为饱和常数（mg/L）；Ki为抑制常数（mg/L）.
1.5 分析项目与方法
pH值通过pH测定仪（雷磁）测定；COD采用重铬酸钾法测定；氨氮采用纳氏试剂法测定，并通过公式（7）计算FA浓度.
式中：ρ为FA浓度（mg/L）；为以N计的氨氮浓度（mg/L）；t为温度（℃）.
2 结果与讨论
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2.1 菌株的筛选与鉴定
从长期运行的处理含高碳、氮浓度废水的反应器中取样，在含高浓度FA（210.4~302.8mg/L）的HNAD固体培养基上初步筛选得到6株菌株，在经过HNAD液体培养基复筛得到1株脱氮除碳效果较好的菌株，命名为WN1.菌株WN1在HNAD固体培养基上菌落具有以下特征：呈黄色，圆形隆起，边缘整齐，表面湿润、光滑.
菌株WN1的16S rDNA基因片段的PCR扩增产物长度约为1400bp，获得序列号为PQ127018.通过NCBI数据库的Blast同源性分析，显示该菌株序列与Corynebacterium glutamicum strain ATCC 13032相似性为100%，结合形态学特征后鉴定菌株WN1为谷氨酸棒状杆菌C. glutamicum，系统发育树如图1（b）所示.
2.2 不同初始氨氮浓度对菌株生长与基质降解的影响
在30℃、pH值为8.4反应条件下，不同初始氨氮浓度（500~3000mg/L）对应的FA浓度分别为97.0，188.9，284.6，412.5，470.3和590.2mg/L，菌株WN1的生长与基质降解情况如图2所示.动力学拟合结果显示（表1），初始氨氮浓度由500mg/L提高到3000mg/L，t0从3.44增加为34.30h，说明随着初始氨氮浓度升高，菌株WN1的适应期不断增加.菌株WN1在初始氨氮为3000mg/L下仍可以较好的生长，48h的OD600为3.264.已有研究表明，大多数HNAD菌如Alcaligenes faecalis TF-1[12]、Pseudomonas L3[17]等能够较好地耐受高浓度氨氮（500~1000mg/L）并进行生长，但氨氮浓度过高时HNAD菌的生长也会受到抑制甚至停止，可能是此时高浓度FA干扰了跨膜质子浓度梯度进而影响了合成代谢的能量生成[18].王秀杰等[19]研究表明Acinetobactor sp. JQ1004能够在0~2000mg/L氨氮条件下生长，但在初始氨氮浓度为2500mg/L时，对应FA为86.7mg/L，菌株生长被完全抑制.菌株WN1在经过一定适应期后，即使在FA浓度高达590.2mg/L时仍能够较好生长，且48h后各组均有较高的生物量（OD600>3.0）.进一步，计算了不同初始氨氮浓度下菌株WN1的最大比生长速率（表2）.菌株WN1最大比生长速率随着氨氮浓度的增加有所下降，初始氨氮浓度1500mg/L时其最大比生长速率仍有0.310h-1，大于已报道的Acinetobacter sp. JQ1004（0.308h-1）[19]、Alcalgens faecalis No.4（0.2h-1）[7]等HNAD菌.上述结果表明菌株WN1可以耐受高浓度氨氮（FA浓度也高）进行生长，对毒物有较好的抗抑制能力，相比其它HNAD菌对高氨氮环境的耐受性更强.
本实验中各组用初始氨氮浓度（500~3000mg/L）均保证了菌株充足底物利用，虽然菌株WN1的表观氨氮转化率随初始氨氮浓度的升高有所降低，但初始氨氮为1000mg/L时其氨氮转化率（48h）达到44.6%，相比其它HNAD菌如Bacillus methylotrophicus strain L7[20]（1121mg/L，36.5%）、Raoultella sp. sari01[21]（1000mg/L，37.6%）等在处理高浓度氨氮（>1000mg/L）时的转化率较高.研究表明，FA浓度过高时，可能会对底物降解相关功能酶产生毒害作用[22-23]，从而抑制菌的代谢活性.然而，从动力学拟合结果可知（表1），随着初始氨氮浓度由500提升至3000mg/L，菌株WN1的最大氨氮转化速率Rm由15.93逐渐增加至43.29mg/（L·h），与Pseudomonas putida YH[13]、Acinetobacter junii NP1[24]等HNAD菌的Rm随氨氮浓度升高而增加的结果类似，说明菌株WN1对较宽的氨氮浓度范围均有良好的响应.计算后得出菌株WN1反应过程的平均氨氮转化速率为7.06~9.36mg/（L·h），均高于处理高浓度氨氮（>1000mg/L）时的Pseudomonas L3[17]（1000mg/L，5.46mg/（L·h））、Alcaligenes faecalis C16[25]（1200mg/L，5.26mg/（L·h））、Pseudomonas. aeruginosa U1[11]（1500mg/L，6.38mg/（L·h））等HNAD菌.以上结果说明菌株WN1不仅能够耐受高浓度氨氮，而且在高氨氮负荷下具有较强的脱氮能力.此外，在氨氮降解过程中，均未检测到硝氮和亚硝氮的浓度变化.分析原因可能是亚硝氮和硝氮作为中间产物在代谢过程中被迅速转化且没有产生积累，以致于检测不到；或者是该菌像个别已报道的HNAD菌一样具有能将氨氮直接转化为气态氮的脱氮路径[26]，并未转化为亚硝氮或硝氮等中间产物.上述特点使得该菌具有较佳的脱氮效能，在应用于高氨氮废水处理时可以减少工艺流程并降低处理成本.
相比于自养硝化菌，HNAD菌能耐受较高氨氮浓度，而多数关于HNAD菌的筛选和降解特性的研究只关注离子态氨氮浓度而忽视了FA浓度.Wang等[27]研究发现，HNAD菌Serratia marcescens W5在低氨氮浓度（100mg/L）时pH值变化（7.0~10.0）对其氨氮降解没有明显影响；而在高氨氮浓度（300~500mg/L）时，pH值为10.0时的氨氮转化速率比pH值为7.0时显著降低，推断是由于FA浓度过高所致，因此FA对HNAD菌的影响应该得到关注.本研究采用了高浓度FA环境（210.4~ 302.8mg/L）筛选出菌株WN1；在菌株培养过程中，虽然随着氨氮的降解FA浓度有所下降，但过程中较高的pH值（>8.0）使得FA仍维持在一定的浓度，48h后各组的FA浓度分别为55.2，82.4，166.8，228.2，278.3，369.4mg/L.上述进一步说明了菌株WN1能够耐受高浓度氨氮（FA浓度也高）并对氨氮进行有效转化，在处理高浓度氨氮（FA浓度也高）废水中具有很大的应用潜力.
同时，菌株WN1的生长代谢过程中，COD浓度变化与氨氮浓度的变化一致（图2）.随着菌株生长体系中的COD浓度不断降低，反应过程的COD平均转化速率为95.63~199.13mg/（L·h）.由此可知，该菌对有机物同样具有较好的降解能力，在处理高浓度含N有机废水时可以实现较好地同步脱氮除碳.
实际工程中，在对高浓度含氮有机废水如畜禽养殖废水、污泥脱水液和垃圾渗沥液等处理时，通常采用减少进水或增加回流的方法降低原水中氨氮及FA浓度，并设置较长的水力停留时间，以满足废水处理要求.这不仅降低了处理效率，而且大幅增加了工艺处理成本.而菌株WN1所具的优良特性，使其在通过生物强化等手段进行工程应用后能降低工艺复杂度，实现对上述含氮有机废水的低耗高效处理并提升处理效果，具有极佳的应用前景.
2.3 不同初始氨氮浓度条件下菌株的生长及基质降解动力学特性分析
本文进一步采用Haldane模型，拟合不同氨氮浓度对菌株生长及基质降解特性的影响，得到动力学参数及相关系数（R2）如表3所示.
由图3可知，在不同的初始氨氮浓度条件下，菌株的比生长速率随氨氮浓度的增加呈现逐渐降低的趋势.上文实验结果也发现，随着氨氮浓度升高，菌株的适应期t0增加且最大比生长速率umax下降，可见高浓度氨氮对菌株的生长产生了一定的抑制.采用Haldane模型很好地拟合了上述生长规律（R2=0.99），最大比生长速率时对应的氨氮浓度可以通过以下公式计算得出：
由此可知，氨氮浓度为474mg/L（FA浓度为93mg/L）时，菌株达到最大比生长速率0.36h-1，当氨氮浓度超过该浓度时会对菌株的生长产生抑制.
由图4可知，随着氨氮浓度的升高，菌株对氨氮的比降解速率呈现先上升后下降的趋势.菌株WN1对氨氮的最大比降解速率为6.45gN/（gDCW·d），此时对应的氨氮浓度可以通过以下公式计算得出：
当氨氮浓度超过1816mg/L时，会对氨氮的降解产生抑制.在Haldane模型中，抑制常数Ki越大，表明菌株的抗抑制能力越强.菌株WN1的Ki值为3749.49mg/L，远大于自养氨氧化菌（Ki为59mg/L）和厌氧氨氧化菌（Ki为67.23mg/L）[28]，且约为已报道的HNAD菌Acinetobactor sp. JQ1004（Ki为1445.31mg/L）[19]的2.6倍.高氨氮浓度时，更多的FA分子通过自由扩散穿过细胞膜，会破坏细胞内外质子和钾离子的浓度平衡从而使细胞失活.上述结果表明菌株WN1对高氨氮具有更强的抗抑制能力，原因可能是该菌为革兰氏阳性菌，细胞壁较厚，FA不易渗入细胞内对细胞活性产生抑制.
随着氨氮浓度的升高，菌株对COD的比降解速率也呈现先上升后下降的趋势.菌株WN1对COD的最大比降解速率为122.85gCOD/（gDCW·d），此时对应的氨氮浓度可以通过以下公式计算得出：
当氨氮浓度超过859mg/L时，会对COD的降解产生抑制.随着氨氮浓度的升高，菌株对COD降解会先于氨氮降解受到抑制，COD降解过程对高浓度氨氮更敏感，说明菌株的碳代谢路径如TCA循环等受到了FA更多的抑制影响.
3 结论
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3.1 从处理含高碳、氮浓度废水的反应器中筛选出一株异养硝化-好氧反硝化菌WN1，鉴定为谷氨酸棒杆菌Corynebacterium glutamicum.
3.2 菌株WN1在氨氮浓度高达3000mg/L，对应FA浓度为590.2mg/L时仍能较好生长.在T为30℃、pH值为8.4，随初始氨氮浓度由500升至3000mg/L，最大氨氮转化速率Rm由15.93增至43.29mg/（L·h）.
3.3 菌株WN1在氨氮降解过程中，没有亚硝氮或硝氮的积累.反应过程的平均氨氮和COD转化速率分别为7.06~9.36mg/（L·h）和95.63~199.13mg/（L·h）.
3.4 Haldane模型较好地拟合了不同初始氨氮浓度下菌株WN1的生长及基质降解特性（R2=0.99），菌株WN1的最大比生长速率、最大氨氮比降解速率和最大COD比降解速率分别为0.36h-1、6.45gN/（gDCW·d）和122.85gCOD/（gDCW·d）.菌株WN1的氨氮抑制常数Ki为3749.49mg/L.
基金
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  • 国家自然科学基金青年科学基金项目(52300075)
  • 国家自然科学基金项目(642201053)
文献
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2025年第45卷第1期
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  • 接收时间：2024-06-26
  • 首发时间：2026-03-18
  • 出版时间：2025-01-20
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  • 收稿日期：2024-06-26
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国家自然科学基金青年科学基金项目(52300075)
国家自然科学基金项目(642201053)
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    北京林业大学环境科学与工程学院，北京市水体污染源控制技术重点实验室，北京 100083

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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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