柱前衍生-固相萃取-高效液相色谱荧光测定生物脱氮反应器中痕量信号分子AI-2
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摘要
为揭示污水生物脱氮工艺中污泥菌群间的群体感应作用,建立了柱前衍生-固相萃取-高效液相色谱荧光检测法(HPLC-FLD)定量检测介导细菌种间群体感应信号分子AI-2的方法。取反应器的泥水混合液,经0.45μm滤膜过滤后,用氨基磺酸掩蔽亚硝酸盐干扰,并与2,3-二氨基萘(DAN)发生衍生化反应,衍生化产物用C18固相萃取柱进行固相萃取,经氮吹浓缩后上机分析。采用C18色谱柱(4.6 mm×250 mm,5μm)进行分离,乙腈与水(含0.1%甲酸)作为流动相进行梯度洗脱,使用荧光检测器(激发和发射波长分别为271 nm和503 nm)进行检测。结果表明,该检测方法在1～200 ng·mL~(-1)范围内呈现出了良好的线性关系,检出限为1 ng·mL~(-1),回收率为55.08%～59.25%,相对标准误差为2.98%～10.41%。该方法适用于杂质干扰多的痕量信号分子AI-2定量分析,可为生物脱氮工艺中信号分子AI-2介导群体感应研究提供有效的分析方法。
To reveal the mechanism of quorum sensing in biological nitrogen removal processes, a method usingpre-column derivatization, solid-phase extraction and high-performance liquid chromatography with fluorescencedetector(HPLC-FLD) was established for quantitation of AI-2 signal molecules. The samples collected from thereactor and filtered through 0.45 μm filter membrane, then were reacted with amino-sulfonic acid to mask nitriteand derived with 2,3-diaminonaphthalene(DAN). The derived products were extracted and concentrated by solidphase extraction(SPE), and then determined by HPLC(C18 column, 4.6 mm×250 mm, 5 μm) with fluorescencedetector(excitation wavelength and emission wavelength were 271 nm and 503 nm, respectively). Gradientelution was carried out by acetonitrile and water(containing 0.1% formic acid) as mobile phases. The resultspresented an excellent linearity with the concentration ranging from 1 to 200 ng·mL~(-1), and the detection limitswere 1 ng · mL~(-1). The recovery ratio of sample ranged from 55.08% to 59.25%, with the relative standarddeviations of 2.98% to 10.41%, respectively. This method provided an effective analytical method forquantitation of trace signal molecule AI-2 in biological nitrogen removal processes.
To reveal the mechanism of quorum sensing in biological nitrogen removal processes, a method usingpre-column derivatization, solid-phase extraction and high-performance liquid chromatography with fluorescencedetector(HPLC-FLD) was established for quantitation of AI-2 signal molecules. The samples collected from thereactor and filtered through 0.45 μm filter membrane, then were reacted with amino-sulfonic acid to mask nitriteand derived with 2,3-diaminonaphthalene(DAN). The derived products were extracted and concentrated by solidphase extraction(SPE), and then determined by HPLC(C18 column, 4.6 mm×250 mm, 5 μm) with fluorescencedetector(excitation wavelength and emission wavelength were 271 nm and 503 nm, respectively). Gradientelution was carried out by acetonitrile and water(containing 0.1% formic acid) as mobile phases. The resultspresented an excellent linearity with the concentration ranging from 1 to 200 ng·mL~(-1), and the detection limitswere 1 ng · mL~(-1). The recovery ratio of sample ranged from 55.08% to 59.25%, with the relative standarddeviations of 2.98% to 10.41%, respectively. This method provided an effective analytical method forquantitation of trace signal molecule AI-2 in biological nitrogen removal processes.
引文
[1]MILLER M B,BASSLER B L.Quorum sensing in bacteria[J].Annual Review of Microbiology,2001,55(1):165-199.
[2]叶姜瑜,谭旋,吕冰,等.细菌群体感应现象及其在控制膜生物污染中的应用[J].环境工程,2013,31(S1):196-199.
[3]李松亚,费学宁,焦秀梅,等.废水处理中群体感应调控行为研究进展[J].应用生态学报,2018,29(3):1015-1022.
[4]温晓芳,黄俊生.细菌的群体感应及其信号分子[J].华南热带农业大学学报,2005,11(1):31-35.
[5]TAN C H,KOH K S,XIE C,et al.The role of quorum sensing signalling in EPS production and the assembly of a sludge community into aerobic granules[J].ISME Journal,2014,8(6):1186-1197.
[6]DE C H,DEFOIRDT T,VANHAECKE L,et al.Long-chain acylhomoserine lactones increase the anoxic ammonium oxidation rate in an OLAND biofilm[J].Applied Microbiology&Biotechnology,2011,90(4):1511-1519.
[7]XIONG Y,LIU Y.Essential roles of eDNA and AI-2 in aerobic granulation in sequencing batch reactors operated at different settling times[J].Applied Microbiology&Biotechnology,2012,93(6):2645-2651.
[8]CHEN X,SCHAUDER S,POTIER N,et al.Structural identification of a bacterial quorum-sensing signal containing boron[J].Nature,2002,415(6871):545-549.
[9]XIONG Y,LIU Y.Essential roles of eDNA and AI-2 in aerobic granulation in sequencing batch reactors operated at different settling times[J].Applied Microbiology&Biotechnology,2012,93(6):2645-2651.
[10]TAN C H,KOH K S,XIE C,et al.The role of quorum sensing signalling in EPS production and the assembly of a sludge community into aerobic granules[J].ISME Journal,2014,8(6):1186-1197.
[11]VILCHEZ R,LEMME A,THIEL V,et al.Analysing traces of autoinducer-2 requires standardization of the Vibrio harveyibioassay[J].Analytical and Bioanalytical Chemistry,2007,387(2):489-496.
[12]燕彩玲,李博,顾悦,等.信号分子AI-2的检测方法研究进展[J].微生物学通报,2016,43(6):1333-1338.
[13]BASSLER B L,GREENBERG E P,STEVENS A M.Cross-species induction of luminescence in the quorum-sensing bacterium Vibrio harveyi[J].Journal of Bacteriology,1997,179(12):4043-4045.
[14]ZHU J G,PEI D H.A LuxP-based fluorescent sensor for bacterial autoinducerII[J].ACS Chemical Biology,2008,3(2):110-119.
[15]CAMPAGNA S R,GOODING J R,MAY A L.Direct quantitation of the quorum sensing signal,autoinducer-2,in clinically relevant samples by liquid chromatography-tandem mass spectrometry[J].Analytical Chemistry,2009,81(15):6374-6381.
[16]THIEL V,VILCHEZ R,SZTAJER H,et al.Identification,quantification,and determination of the absolute configuration of the bacterial quorum-sensing signal autoinducer-2 by gas chromatography-mass spectrometry[J].ChemBioChem,2010,10(3):479-485.
[17]SONG X N,QIU H B,XIAO X,et al.Determination of autoinducer-2 in biological samples by high-performance liquid chromatography with fluorescence detection using pre-column derivatization[J].Journal of Chromatography A,2014,1361:162-168.
[18]单瑶瑶,邱秀文,周桂香,等.活性污泥胞外聚合物提取方法比较研究[J].九江学院学报(自然科学版),2016,33(4):5-8.
[2]叶姜瑜,谭旋,吕冰,等.细菌群体感应现象及其在控制膜生物污染中的应用[J].环境工程,2013,31(S1):196-199.
[3]李松亚,费学宁,焦秀梅,等.废水处理中群体感应调控行为研究进展[J].应用生态学报,2018,29(3):1015-1022.
[4]温晓芳,黄俊生.细菌的群体感应及其信号分子[J].华南热带农业大学学报,2005,11(1):31-35.
[5]TAN C H,KOH K S,XIE C,et al.The role of quorum sensing signalling in EPS production and the assembly of a sludge community into aerobic granules[J].ISME Journal,2014,8(6):1186-1197.
[6]DE C H,DEFOIRDT T,VANHAECKE L,et al.Long-chain acylhomoserine lactones increase the anoxic ammonium oxidation rate in an OLAND biofilm[J].Applied Microbiology&Biotechnology,2011,90(4):1511-1519.
[7]XIONG Y,LIU Y.Essential roles of eDNA and AI-2 in aerobic granulation in sequencing batch reactors operated at different settling times[J].Applied Microbiology&Biotechnology,2012,93(6):2645-2651.
[8]CHEN X,SCHAUDER S,POTIER N,et al.Structural identification of a bacterial quorum-sensing signal containing boron[J].Nature,2002,415(6871):545-549.
[9]XIONG Y,LIU Y.Essential roles of eDNA and AI-2 in aerobic granulation in sequencing batch reactors operated at different settling times[J].Applied Microbiology&Biotechnology,2012,93(6):2645-2651.
[10]TAN C H,KOH K S,XIE C,et al.The role of quorum sensing signalling in EPS production and the assembly of a sludge community into aerobic granules[J].ISME Journal,2014,8(6):1186-1197.
[11]VILCHEZ R,LEMME A,THIEL V,et al.Analysing traces of autoinducer-2 requires standardization of the Vibrio harveyibioassay[J].Analytical and Bioanalytical Chemistry,2007,387(2):489-496.
[12]燕彩玲,李博,顾悦,等.信号分子AI-2的检测方法研究进展[J].微生物学通报,2016,43(6):1333-1338.
[13]BASSLER B L,GREENBERG E P,STEVENS A M.Cross-species induction of luminescence in the quorum-sensing bacterium Vibrio harveyi[J].Journal of Bacteriology,1997,179(12):4043-4045.
[14]ZHU J G,PEI D H.A LuxP-based fluorescent sensor for bacterial autoinducerII[J].ACS Chemical Biology,2008,3(2):110-119.
[15]CAMPAGNA S R,GOODING J R,MAY A L.Direct quantitation of the quorum sensing signal,autoinducer-2,in clinically relevant samples by liquid chromatography-tandem mass spectrometry[J].Analytical Chemistry,2009,81(15):6374-6381.
[16]THIEL V,VILCHEZ R,SZTAJER H,et al.Identification,quantification,and determination of the absolute configuration of the bacterial quorum-sensing signal autoinducer-2 by gas chromatography-mass spectrometry[J].ChemBioChem,2010,10(3):479-485.
[17]SONG X N,QIU H B,XIAO X,et al.Determination of autoinducer-2 in biological samples by high-performance liquid chromatography with fluorescence detection using pre-column derivatization[J].Journal of Chromatography A,2014,1361:162-168.
[18]单瑶瑶,邱秀文,周桂香,等.活性污泥胞外聚合物提取方法比较研究[J].九江学院学报(自然科学版),2016,33(4):5-8.
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