谭炳琰1,
李淳峰1,
陈雨佳1,
韦童1,
荣宏伟1,2,
储昭瑞1,2,
1.广州大学土木工程学院，广州510006
2.广东省水质安全及污染控制工程技术研究中心，广州510006
基金项目: 广州大学大学生创新训练项目(201711078014)
广东省教育厅青年创新人才项目(2016KQNCX121)
Detection of AI-2 signal molecules of quorum sensing in biological nitrogen removal processes by pre-column derivatization-solid phase extraction-high performance liquid chromatography with fluorescence detector
HUANG Xiaoyu1,,TAN Bingyan1,
LI Chunfeng1,
CHEN Yujia1,
WEI Tong1,
RONG Hongwei1,2,
CHU Zhaorui1,2,
1.School of Civil Engineering, Guangzhou University, Guangzhou 510006, China
2.Guangdong Engineering Research Center for Water Quality Safety and Pollution Control, Guangzhou 510006, China
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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0.45 μm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，5 μm)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。
关键词: 生物脱氮/
群体感应/
信号分子AI-2/
高效液相色谱荧光检测法(HPLC-FLD)
Abstract:To reveal the mechanism of quorum sensing in biological nitrogen removal processes, a method using pre-column derivatization, solid-phase extraction and high-performance liquid chromatography with fluorescence detector (HPLC-FLD) was established for quantitation of AI-2 signal molecules. The samples collected from the reactor and filtered through 0.45 μm filter membrane, then were reacted with amino-sulfonic acid to mask nitrite and derived with 2,3-diaminonaphthalene(DAN). The derived products were extracted and concentrated by solid phase extraction (SPE), and then determined by HPLC (C18 column, 4.6 mm×250 mm, 5 μm) with fluorescence detector (excitation wavelength and emission wavelength were 271 nm and 503 nm, respectively). Gradient elution was carried out by acetonitrile and water (containing 0.1% formic acid) as mobile phases. The results presented an excellent linearity with the concentration ranging from 1 to 200 ng·mL-1, and the detection limits were 1 ng·mL-1. The recovery ratio of sample ranged from 55.08% to 59.25%, with the relative standard deviations of 2.98% to 10.41%, respectively. This method provided an effective analytical method for quantitation of trace signal molecule AI-2 in biological nitrogen removal processes.
Key words:biological nitrogen removal/
quorum sensing/
autoinducer-2/
high-performance liquid chromatography with fluorescence detector (HPLC-FLD).
| [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. |
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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
黄晓遇1,,谭炳琰1,
李淳峰1,
陈雨佳1,
韦童1,
荣宏伟1,2,
储昭瑞1,2,
1.广州大学土木工程学院，广州510006
2.广东省水质安全及污染控制工程技术研究中心，广州510006
基金项目: 广州大学大学生创新训练项目(201711078014) 广东省教育厅青年创新人才项目(2016KQNCX121)
关键词: 生物脱氮/
群体感应/
信号分子AI-2/
高效液相色谱荧光检测法(HPLC-FLD)
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0.45 μm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，5 μm)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。
English Abstract
Detection of AI-2 signal molecules of quorum sensing in biological nitrogen removal processes by pre-column derivatization-solid phase extraction-high performance liquid chromatography with fluorescence detector
HUANG Xiaoyu1,,TAN Bingyan1,
LI Chunfeng1,
CHEN Yujia1,
WEI Tong1,
RONG Hongwei1,2,
CHU Zhaorui1,2,
1.School of Civil Engineering, Guangzhou University, Guangzhou 510006, China
2.Guangdong Engineering Research Center for Water Quality Safety and Pollution Control, Guangzhou 510006, China
Keywords: biological nitrogen removal/
quorum sensing/
autoinducer-2/
high-performance liquid chromatography with fluorescence detector (HPLC-FLD)
Abstract:To reveal the mechanism of quorum sensing in biological nitrogen removal processes, a method using pre-column derivatization, solid-phase extraction and high-performance liquid chromatography with fluorescence detector (HPLC-FLD) was established for quantitation of AI-2 signal molecules. The samples collected from the reactor and filtered through 0.45 μm filter membrane, then were reacted with amino-sulfonic acid to mask nitrite and derived with 2,3-diaminonaphthalene(DAN). The derived products were extracted and concentrated by solid phase extraction (SPE), and then determined by HPLC (C18 column, 4.6 mm×250 mm, 5 μm) with fluorescence detector (excitation wavelength and emission wavelength were 271 nm and 503 nm, respectively). Gradient elution was carried out by acetonitrile and water (containing 0.1% formic acid) as mobile phases. The results presented an excellent linearity with the concentration ranging from 1 to 200 ng·mL-1, and the detection limits were 1 ng·mL-1. The recovery ratio of sample ranged from 55.08% to 59.25%, with the relative standard deviations of 2.98% to 10.41%, respectively. This method provided an effective analytical method for quantitation of trace signal molecule AI-2 in biological nitrogen removal processes.
