生物质能源研究中微生物发酵的拉曼光谱法检测
|
摘要
随着化石能源的日益枯竭和环境问题的日趋严重,开发洁净可再生资源已成了紧迫的课题。一些微生物在合适的生长环境下能在细胞内部合成或者代谢转化出乙醇、油脂,或者有重要经济价值的代谢产物,如类胡萝卜素等,其中微生物油脂能够替代化石能源来制取生物柴油,类胡萝卜素可以作为食物色素添加剂及药物添加剂,而乙醇则可以提纯后直接做为燃料,可见这些产物均具有比较高的经济价值。因此,本文在前人研究的基础上,通过结合激光镊子拉曼光谱系统,尝试进一步探索研究生物质能源微生物发酵过程中,探测发酵产物、底物以及细胞内部的物质动态变化过程,为实际发酵生产提供理论依据和重要参数参考,同时结合拉曼光谱和光学操控便利,探索发展分选微生物的新方法。主要开展了以下研究工作：
1.微生物乙醇发酵中,实验室研究和工业生产的必检项目是发酵液乙醇含量的测定。为此应用拉曼光谱在倒置显微镜上建立一种用96孔板高通量快速检测发酵液乙醇含量的新方法,通过最小二乘法拟合标准乙醇溶液与内标物的拉曼信号比值,得到回归方程,根据回归方程计算乙醇溶液和发酵液中的乙醇含量,与气相色谱法的检测结果进行对比。用本方法检测了以木薯淀粉为发酵基质的菌株筛选、三角瓶发酵和500升发酵罐发酵过程的乙醇含量,t-测验表明,与气相色谱法结果有很好的重现性(p=0.05),准确度高。并在此基础上,对比利用一元线性回归法、多元线性回归法、主成分回归法和偏最小二乘法建立定量分析模型,探索同时定量检测乙醇和葡萄糖的快捷方法。结果表明基于拉曼光谱技术定量方法,能满足实验和生产对乙醇和葡萄糖检测精度的要求。
2.采用拉曼光谱在分子水平上对单个活细胞的结构变化,物质转换和生物活性进行研究。基于拉曼光谱,探索实验室500 L中式发酵罐木薯淀粉浓醪发酵乙醇过程中,酿酒酵母细胞内生物大分子的生化信息,以及发酵底物和细胞代谢产物的变化。分别追踪发酵液光谱中乙醇和葡萄糖的特征峰880 cm-1和1127 cm-1谱带的变化,与气相色谱法和DNS比色法检测结果比较发现,其变化曲线基本吻合,表明特征峰面积与物质的含量存在对应关系。此外,采用多种分析方法从拉曼光谱数据中提取细胞在生长发育过程中的演变信息。结果表明,应用激光镊子拉曼光谱技术,探索微生物发酵研究的新方法,为优化发酵参数,指导乙醇发酵生产调控提供借鉴。
3.为了探求建立准确快速分选酵母细胞的方法,尝试利用拉曼光谱与激光镊子技术,从细胞群体中分选出目标酵母。收集各酵母细胞的拉曼光谱,经去背景、17点S-G平滑滤波、多项式拟合基线校正和矢量归一化等数据预处理,结合主成分分析,从酵母细胞的拉曼光谱中提取胞内物质的主要特征信息,根据物质构成的差异来识别目标酵母,进而建立分选模型。激光镊子俘获单个酵母细胞并收集其拉曼光谱,实时判别细胞的归属,并根据判别结果收集细胞。将收集的酵母细胞经菌落培养和苏丹黑B进行染色等方法进行验证,结果显示,分选出的细胞均含有目标产物。这一方法在实现分选所选目标酵母的同时,也为利用激光镊子拉曼光谱分选其它单细胞提供了借鉴方法。
Along with the depletion of fossil fuels and the environmental problem is becoming increasingly serious, the development of clean and renewable resources has become a pressing issue. There are some microorganisms can be used to synthesize or convert some special material if cultured in the right condition, such as the microbial oil which can substitute for fossil fuels to produce biodiesel, the carotenoid can be used as food and drug additives, and ethanol can be used as fuel after purification, This means that these products all have relatively high economic value. This work, combined with the previous research, and the laser tweezers Raman spectroscopy system was used, trying to further explore the dynamic development of the products and the substrates of fermentation liquor and the material of intracellular during the process of fermentation, then we can provide a theoretical and an important reference parameters for the ferment industrial production, At the same time, we trying to carve from a selected group of yeast cells with the production capacity of some special products of the target cells. For that reason, the main research was carried out the following:
1. This work is an attempt to study about fermentation detection of ethanol which is important to the laboratory research and industrial production. A high-throughput method used in our study is based on Raman Spectroscopy in the inverted microscope and 96-well plates. Base on the Raman signal ratio of standard ethanol solution and the internal, I establish a regression equation by the least squares fitting. And then the equation was used to calculate the content of ethanol, it was stem from the strains with cassava starch as a fermentation substrate, flask fermentation and the 500 liters of ethanol fermentation tank separately. At the same time the gas chromatography was used to detect the foregoing item. The t-test indicate that the results of Raman and gas chromatography have good reproducibility (p=0.05), this means that Raman test has a good accuracy. On the basis of Raman spectroscopy and 96-well plate, the linear regression, multiple linear regression, principal component regression and partial least squares (PLS) were employed to measure the concentration of ethanol and glucose aqueous mixtures simultaneously. A group of standard concentration of ethanol and glucose mixtures are was detected and fermentation broth of cassava starch was used to validate. The results show that this method could be applied to the real-time monitoring of ethanol fermentation.
2. The structure, transformation, and bioactivity of single living cells at the molecular level have been studied by Raman spectroscopy. In this study, I explore the biochemical information about their biological macromolecules and the metabolic product from the Raman spectra of Saccharomyces cerevisiae, which was cultured by cassava starch with the 500 L fermentor. At first, in order to measure the ethanol and glucose concentration of the fermentation broth, we track the 880 cm-1 and 1127 cm-1 spectra band, and the results were compared with that of gas chromatography and the DNS colorimetry. In addition, several approaches such as the study of mean spectra, the difference spectra, the usage of histogram of characteristic absorption peak area and the application of principal component analysis were applied. These methods were applied on a data set containing Raman spectra of yeast in three stages of its growth, aiming to extract information about the evolution of cell components during growth. The result show that the laser tweezers Raman spectroscopy approach has the potential to open a new frontier to study microorganisms, which were used to optimize the fermentation parameters, and direct practical production of ethanol.
3. Extensive research has been carried out in an effort to screen the microorganisms. Here, Raman Spectroscopy and laser tweezers were used to sort yeast from mixed yeast cells. The preprocessing of subtracted background,17 points S-G smoothing filter, polynomial fitting baseline correction and vector normalization were performed and the main features information of intracellular substances from the Raman Spectroscopy of yeast cells were extracted by combining principal component analysis. Basing on the distinguished composition of the traget yeast and the other yeast, a sorting model was established. The test yeast cell in optical trapping was distinguished real-time by the model referring to its Raman spectra. The cells distinguished as target yeast was collected by means of optical manipulation. The sorted traget yeast cells were verified by microbial culture and Sudan black B test etc. The result illustrates that Raman spectroscopy combining with optical manipulation is an effective technique for sorting yeast and other economic microorganisms.
1.微生物乙醇发酵中,实验室研究和工业生产的必检项目是发酵液乙醇含量的测定。为此应用拉曼光谱在倒置显微镜上建立一种用96孔板高通量快速检测发酵液乙醇含量的新方法,通过最小二乘法拟合标准乙醇溶液与内标物的拉曼信号比值,得到回归方程,根据回归方程计算乙醇溶液和发酵液中的乙醇含量,与气相色谱法的检测结果进行对比。用本方法检测了以木薯淀粉为发酵基质的菌株筛选、三角瓶发酵和500升发酵罐发酵过程的乙醇含量,t-测验表明,与气相色谱法结果有很好的重现性(p=0.05),准确度高。并在此基础上,对比利用一元线性回归法、多元线性回归法、主成分回归法和偏最小二乘法建立定量分析模型,探索同时定量检测乙醇和葡萄糖的快捷方法。结果表明基于拉曼光谱技术定量方法,能满足实验和生产对乙醇和葡萄糖检测精度的要求。
2.采用拉曼光谱在分子水平上对单个活细胞的结构变化,物质转换和生物活性进行研究。基于拉曼光谱,探索实验室500 L中式发酵罐木薯淀粉浓醪发酵乙醇过程中,酿酒酵母细胞内生物大分子的生化信息,以及发酵底物和细胞代谢产物的变化。分别追踪发酵液光谱中乙醇和葡萄糖的特征峰880 cm-1和1127 cm-1谱带的变化,与气相色谱法和DNS比色法检测结果比较发现,其变化曲线基本吻合,表明特征峰面积与物质的含量存在对应关系。此外,采用多种分析方法从拉曼光谱数据中提取细胞在生长发育过程中的演变信息。结果表明,应用激光镊子拉曼光谱技术,探索微生物发酵研究的新方法,为优化发酵参数,指导乙醇发酵生产调控提供借鉴。
3.为了探求建立准确快速分选酵母细胞的方法,尝试利用拉曼光谱与激光镊子技术,从细胞群体中分选出目标酵母。收集各酵母细胞的拉曼光谱,经去背景、17点S-G平滑滤波、多项式拟合基线校正和矢量归一化等数据预处理,结合主成分分析,从酵母细胞的拉曼光谱中提取胞内物质的主要特征信息,根据物质构成的差异来识别目标酵母,进而建立分选模型。激光镊子俘获单个酵母细胞并收集其拉曼光谱,实时判别细胞的归属,并根据判别结果收集细胞。将收集的酵母细胞经菌落培养和苏丹黑B进行染色等方法进行验证,结果显示,分选出的细胞均含有目标产物。这一方法在实现分选所选目标酵母的同时,也为利用激光镊子拉曼光谱分选其它单细胞提供了借鉴方法。
Along with the depletion of fossil fuels and the environmental problem is becoming increasingly serious, the development of clean and renewable resources has become a pressing issue. There are some microorganisms can be used to synthesize or convert some special material if cultured in the right condition, such as the microbial oil which can substitute for fossil fuels to produce biodiesel, the carotenoid can be used as food and drug additives, and ethanol can be used as fuel after purification, This means that these products all have relatively high economic value. This work, combined with the previous research, and the laser tweezers Raman spectroscopy system was used, trying to further explore the dynamic development of the products and the substrates of fermentation liquor and the material of intracellular during the process of fermentation, then we can provide a theoretical and an important reference parameters for the ferment industrial production, At the same time, we trying to carve from a selected group of yeast cells with the production capacity of some special products of the target cells. For that reason, the main research was carried out the following:
1. This work is an attempt to study about fermentation detection of ethanol which is important to the laboratory research and industrial production. A high-throughput method used in our study is based on Raman Spectroscopy in the inverted microscope and 96-well plates. Base on the Raman signal ratio of standard ethanol solution and the internal, I establish a regression equation by the least squares fitting. And then the equation was used to calculate the content of ethanol, it was stem from the strains with cassava starch as a fermentation substrate, flask fermentation and the 500 liters of ethanol fermentation tank separately. At the same time the gas chromatography was used to detect the foregoing item. The t-test indicate that the results of Raman and gas chromatography have good reproducibility (p=0.05), this means that Raman test has a good accuracy. On the basis of Raman spectroscopy and 96-well plate, the linear regression, multiple linear regression, principal component regression and partial least squares (PLS) were employed to measure the concentration of ethanol and glucose aqueous mixtures simultaneously. A group of standard concentration of ethanol and glucose mixtures are was detected and fermentation broth of cassava starch was used to validate. The results show that this method could be applied to the real-time monitoring of ethanol fermentation.
2. The structure, transformation, and bioactivity of single living cells at the molecular level have been studied by Raman spectroscopy. In this study, I explore the biochemical information about their biological macromolecules and the metabolic product from the Raman spectra of Saccharomyces cerevisiae, which was cultured by cassava starch with the 500 L fermentor. At first, in order to measure the ethanol and glucose concentration of the fermentation broth, we track the 880 cm-1 and 1127 cm-1 spectra band, and the results were compared with that of gas chromatography and the DNS colorimetry. In addition, several approaches such as the study of mean spectra, the difference spectra, the usage of histogram of characteristic absorption peak area and the application of principal component analysis were applied. These methods were applied on a data set containing Raman spectra of yeast in three stages of its growth, aiming to extract information about the evolution of cell components during growth. The result show that the laser tweezers Raman spectroscopy approach has the potential to open a new frontier to study microorganisms, which were used to optimize the fermentation parameters, and direct practical production of ethanol.
3. Extensive research has been carried out in an effort to screen the microorganisms. Here, Raman Spectroscopy and laser tweezers were used to sort yeast from mixed yeast cells. The preprocessing of subtracted background,17 points S-G smoothing filter, polynomial fitting baseline correction and vector normalization were performed and the main features information of intracellular substances from the Raman Spectroscopy of yeast cells were extracted by combining principal component analysis. Basing on the distinguished composition of the traget yeast and the other yeast, a sorting model was established. The test yeast cell in optical trapping was distinguished real-time by the model referring to its Raman spectra. The cells distinguished as target yeast was collected by means of optical manipulation. The sorted traget yeast cells were verified by microbial culture and Sudan black B test etc. The result illustrates that Raman spectroscopy combining with optical manipulation is an effective technique for sorting yeast and other economic microorganisms.
引文
[1]Jose Goldemberg. Ethanol for a sustainable energy future. Science[J],2007,315(5813):808-810.
[2]Arthur J. Ragauskas, Charlotte K. Williams, Brian H. Davison, et al. The path forward for biofuels and biomaterials[J]. Science,2006,311(5760):484-489.
[3]Barry D. Solomon, Justin R. Barnes, Kathleen E. Halvorsen. Grain and cellulosic ethanol:History, economics, and energy policy[J]. Biomass and Bioenergy,2007.31(6):416-425.
[4]石元春.发展生物质产业[J].中国农业科技导报,2006,8(1)：1-5.
[5]章克昌.酒精与蒸馏酒工艺学[M],北京：中国轻工业出版社,1995.
[6]Marcel Blanco, Antonio C. Peinado, Jordi Mas. Analytical monitoring of alcoholic fermentation using NIR spectroscopy[J]. Biotechnology and Bioengineering,2004,88(4):536-542.
[7]Sivakesava S, Irudayaraj J, Demirci A. Monitoring a bioprocess for ethanol production using FT-MIR and FT-Raman spectroscopy[J]. Journal of Industrial Microbiology and Biotechnology.2001,26(4):185-190.
[8]Eliana M Alhadeff, Andrea M Salgado, Nei Pereira, Belkis Valdman. A sequential enzymatic microreactor system for ethanol detection of gasohol mixtures[J]. Applied Biochemistry and Biotechnology,2005, 121(1-3):361-371.
[9]Choengchan N, Mantim T, Wilairat P, Dasgupta P K, Motomizu S, Nacapricha D. A membraneless gas diffusion unit:design and its application to determination of ethanol in liquors by spectrophotometric flow injection[J]. Analytica Chimica Acta,2006,579(1):33-37.
[10]Cai Zhao-Xia, Yang Hong, Zhang Yi, Yan Xiu-Ping. Preparation, characterization and evaluation of water-soluble L-cysteine-capped-CdS nanoparticles as fluorescence probe for detection of Hg(II) in aqueous solution[J]. Analytica Chimica Acta,2006,559(2):234-239.
[11]史月华,陆勇,张荣,徐铸德.傅里叶变换近红外光谱同时定量分析水溶液中的乙醇、果糖和葡萄糖[J].分析化学,2001,29(10)：1213-1215.
[12]Soohwa Cho, Hoeil Chung, Young-Ah Woo, Kim Hyo-Jim, Determination of water content in ethanol by miniaturized Near-Infrared (NIR) system[J]. Bull. K. Chem. Soc,2005,26(1):115-118.
[13]Leornardo S. Mendes, Flavia C. C. Oliveira, Paulo A. Z. Suarez, Joel C. Rubin. Determination of ethanol in fuel ethanol and beverages by fouier transform (FT)-near infrared and FT-Raman spectrometries[J]. Analytica Chimica Acta,2003,493:219-231.
[14]Romina Kaushik, William R. LaCourse, Barry Levine. Determination of ethyl glucuronide in urine using reversed-phase HPLC and pulsed electrochemical detection (Part II) [J]. Analytica Chimica Acta,2006, 556(2):267-274.
[15]A. Ashkin, J.M. Dziedzic, J.E. Bjorkholm, Steven Chu, Observation of a single-beam gradient force optical trap for dielectric articles[J], Optics Letters.1986,11(5):288-290.
[16]A. Ashkin, J. M. Dziedzic. Optical trapping and manipulation of single cells using inferred laser beams[J], Berichte der Bunsengesellschaft fur phsikalische chemie[J],1989,93(3):254-260..
[17]Xie Chang-An, Mumtaz A. Dinno, Li Yong-Qing. Near-infrared Raman spectroscopy of single optically rapped biological cells[J]. Optics Letters.2002,27:249-251.
[18]Xie Chang-An, Li Yong-Qing. Confocal micro-Raman spectroscopy of single biological cell using optical trapping and shifted excitation difference techniques[J]. Journal Applied Physics,2003,93(5):2982-2987.
[19]杨序纲,吴琪琳,拉曼光谱的分析与应用[M],北京,国防工业出版社,2008,11(1)：19-25.
[20]陈秀丽,王桂文,刘军贤,陈萍,黎永青.基于拉曼光谱的地贫红细胞种类识别方法的研究[J],分析测试学报,2009,28(4)：403-408.
[21]冯听韡,朱仲良,沈梦洁,丛培盛.基于多项式拟合的拉曼光谱基线漂移校正方法[J].计算机与应用化学.2009,26(6)：759-762.
[22]Xie Chang-An, Charles Goodman, Mumtaz Dinno, Li Yong-Qing. Real-time Raman spectroscopy of optically trapped living cells and organells[J]. Optics Express,2004,12(25),6208-6214.
[23]Gajendra Pratap Singh, Giovanni Volpe, Caitriona M. Creely, Helga Grotsch, Isabel M. Geli, Dmitri Petrov. The lag phase and G1 phase of a single yeast cell monitored by Raman microspectroscopy[J]. Journal of Raman spectroscopy,2006,37(8),858-864.
[24]王桂文,姚辉璐,彭立新,何碧娟,黎永青.一种酵母细胞生长现象的实时单细胞拉曼光谱观察[J].微生物学通报,2007,34(6),1109-1113.
[25]Li Hai-Long, Chai Xin-Sheng, Deng Yu-Lin, Zhan Huai-Yu, Fu Shi-Yu. Rapid determination of ethanol in fermentation liquor by full evaporation headspace gas chromatography[J]. Journal of Chromatography A,2009,1216(1):169-172.
[26]Marcelo Firmino de Oliveira, Adelir Aparecida Saczk, Leonardo Luiz Okumura, Nelson Ramos Stradiotto. Analytical methods employed at quality control of fuel ethaol[J]. Energy Fuels,2009,23(10):4852-4859.
[27]覃建军,柳畅先,察东梅.酶催化动力学荧光法测定乙醇[J].分析科学学报.2008,24(2)：224-226.
[28]李燕萍,许宏贤,钱莹,段钢.发酵液中乙醇含量的近红外光谱NIRS定量分析与验证[J].食品与发酵工业,2008,34(7)：136-140.
[29]袁文杰,孔亮,孜力汗,白凤武.高效液相色谱法测定克鲁维酵母菊芋发酵液中的乙醇,糖和有机酸类代谢成分[J].分析化学,2009,37(6)：850-854.
[30]Zhao Ren-Yong, Scott R. Bean, Brian P. Ioerger, Wang Dong-Hai, Daniel L. Boyle. Impact of mashing on sorghum proteins and its relationship to ethanol fermentation[J]. Journal of Agricultural and Food Chemistry,2008,56(3):946-953.
[31]Landulfo Silveira Jr., Leonardo Marmo Moreira, Viviane G. B. Conceicao, Heliodora L. Casalechi, Ingrid S. Munoz, Fabiano Fernandes da Silva, Marcos Augusto S. R. Silva, Renato Aparecido de Souza, Marcos Tadeu T. Pacheco. Determination of sucrose concentration in lemon-type soft drinks by dispersive Raman spectroscopy[J]. Spectroscopy,2009,23(3-4):217-226.
[32]Chien-Ju Shih, Emily A. Smith. Determination of glucose and ethanol after enzymatic hydrolysis and fermentation of biomass using Raman spectroscopy[J]. Analytica Chimica Acta,2009,653(2):200-206
[33]王桂文,姚辉璐,彭立新,何碧娟,黎永青.拉曼镊子分析单个苏云金芽孢杆菌伴孢晶体蛋白[J].分析化学,2007,35(9)：1351-1354.
[34]Ye Qing, Xu Qin-Feng, Yu Yong-Gai, Qu Rong-Hui, Fang Zu-Jie. Rapid and quantitative detection of ethanol proportion in ethanol-gasoline mixtures by Raman spectroscopy [J]. Optics Communications,2009, 282(18):3785-3788.
[35]A. Picard, I. Daniel, G. Montagnac, P. Oger. In situ monitoring by quantitative Raman spectroscopy of alcoholic fermentation by Saccbaromyces cerevisiae under high pressure[J]. Extremophiles,2007,11(3): 445-452.
[36]A. A. Abreu, A. S. Danko, J. C. Costa, E. C. Ferreira, M. M. Alves. Inoculum type response to different pHs on biohydrogen production from L-arabinose, a component of hemicellulosic biopolymers[J]. International Journal of Hydrogen Energy,2009,34(4):1744-1751
[37]刘文涵,杨未,吴小琼,林振兴.激光拉曼光谱内标法直接测定乙醇浓度[J].分析化学,2007,3(35)：416-418.
[38]李燕萍,钱莹,段钢.采用近红外光谱测定木薯乙醇发酵液中乙醇、甘油和葡萄糖含量[J].食品与发酵工业,2009,35(8)：117-121.
[39]Marcelo F O, Adelir A S, Leonardo L O,Nelson R S. Analytical Methods Employed at Quality Control of Fuel Ethanol[J]. Brasil Energy Fuels,2009,23(10):4852-4859.
[40]Leornardo S M, Flavia C C O, Paulo A Z S, Determination of ethanol in fuel ethanol and beverages by Fourier transform (FT)-near infrared and FT-Raman spectrometries[J]. Joel C R.Chimica Acta,2003, 493(10):219-231.
[41]吴小琼,郑建珍,刘文涵,林振兴.激光拉曼光谱内标法测定葡萄糖液浓度[J].光谱学与光谱分析,2007,27(7)：1344-1346.
[42]吴正洁,黄耀熊,王成,黎绍发.多种拉曼光谱归一化法对乙醇定量分析的研究[J].光谱学与光谱分析,2010,30(4)：971-974.
[43]潘立登.先进控制与在线优化技术及其应用[M].北京：机械工业出版社,2009：78-118.
[44]杜一平,潘铁英,张玉兰.化学计量学应用[M].北京：化学工业出版社,2008：133-170.
[45]陈敏伯.化学计量学[M].北京：科学出版社,2009：295-316.
[46]严航贞,张安平,林春绵.96孔板法用于除草剂高通量筛选的研究[J].浙江工业大学学报,2009,4,37(2)：119-122.
[47]Sumitra Ramachandran, Pierre Fontanille, Ashok Pandey, Christian Larroche. Stability of Glucose Oxidase Activity of Aspergillus niger Spores Produced by Solid-State Fermentation and Their Role as Biocatalysts in Bioconversion Reaction[J]. Food Technol. Biotechnol.,2008,46 (2):190-194.
[48]王桂文,姚辉璐,何碧娟,彭立新,黎永清.单个血小板的拉曼光谱分析[J].光谱学与光谱分析,2007,22(7)：1347-1350.
[49]Gobinet Cyril, Valeriu Vrabie, Manfait Michel, Piot Olivier. Preprocessing methods of spectra for source extraction on biomedical samples:application on paraffin-embedded skin biopsies[J]. IEEE Transactions on Biomedical Engineering,2009,56(5):1371-1382.
[50]彭帮柱,龙明华,岳田利,袁亚宏,赵志华.用偏最小二乘法及傅立叶变换近红外光谱快速检测白酒酒精度[J].农业工程学报,2007,23(4)：233-237.
[51]刘振,王金鹏,张立峰,曾爱武,袁希钢.木薯干原料同步糖化发酵生产乙醇[J].过程工程学报,2005,5(3)：353-356.
[52]Fabien Joux, and Philippe Lebaron. Use of fluorescent probes to assess physiological functions of bacteria at single-cell level[J]. Microbes and Infection.2000,2(12):1523-1535.
[53]K. Christian Schuster, Ingo Reese, Eva Urlaub, J. Richard Gapes, Bernhard Lendl. Multidimensional information on the chemical composition of single bacterial cells by confocal Raman microspectroscopy [J]. Anal. Chem.,2000,72(22):5529-5534.
[54]S Sivakesava, J Irudayraj, A Demirci. Monitoring a bioproceess for ethanol production using FT-MIR and FT-Raman spectroscopy[J]. Journal of Industrial Microbiology and Biotechnology,2001,26(4):185-190.
[55]Xie Chang-an, Mumtaz A. Dinno, Li Yong-qing. Near-infrared Raman spectroscopy of single optically trapped biological cells[J]. Optics Letters,2002,27(4):249-251.
[56]Xie Chang-an, Charles Goodman, Mumtaz A. Dinno, and Li Yong-Qing. Real-time Raman spectroscopy of optically trapped living cells and organelles[J]. Optics Express,2004,12(25):6208-6214.
[57]Yusuf Chisti. Biodiesel from microalgae beats bioethanol[J]. Trends in Biotechnology,2008,26(3): 126-131.
[58]Meng Xin, Yang Jian-Ming, Xu Xin, Zhang Lei, Nie Qin-Juan, Xian Mo. Biodiesel production from oleaginous microorganisms[J], Renewable Energy,2009,34(1):1-5.
[59]Zhang Wei, Li Zhi-Yong, Miao Xiao-Ling, Zhang Feng-Li. The screening of antimicrobial bacteria with diverse novel nonribosomal peptide synthetase (NRPS) genes from south china sea sponges[J]. Marine Biotechnology,2009,11(3):346-355.
[60]Xie Chang-An, Mumtaz A. Dinno, Li Yong-Qing. Near-infrared Raman spectroscopy of single optically trapped biological cells[J]. Optics Letters,2002,27(4):249-251.
[61]Ai Min, LIU Jun-xian, Huang Shu-Shi, Wang Gui-Wen, Chen Xiu-Li, Chen Zhi-Cheng, YAO Hui-lu. Application and progress of Raman Tweezers in single cells[J]. Chinese Journal of Analytical Chemistry, 2009,37(5):758-763.
[62]Notingher I., Verrier S., Romanska H., Bishop A. E., Polak J. M., Hench L. L.. In situ characterization of living cells by Raman spectroscopy [J]. Spectroscopy,2002,16(2):43-51.
[63]Davey H., Kell D. B. Flow cytometry and cell sorting of heterogeneous microbial populations:the importance of single-cell analyses [J]. Microbiol. Rev.,1996,60(4):641-696.
[64]Wei E. Huang, Robert I. Griffiths, Ian P. Thompson, Mark J. Bailey, Anderw S. Whiteley. Raman microscopic analysis of single microbial Cells [J]. Anal. Chem.,2004,76(15),4452-4458.
[65]Wei E. Huang, Kilian Stoecker, Robert Griffiths, Lyndsay Newbold, Holger Daims, Andrew S. Whiteley, Michael Wagner. Raman-FISH:combining stable-isotope Raman spectroscopy and fluorescence in situ hybridization for the single cell analysis of identity and function [J]. Environmental Microbiology,2007, 9(8),1878-1889.
[66]Wei E. Huang, Mark J. Bailey, Ian P. Thompson, Andrew S. Whiteley, Andrew J. Spiers. Single-Cell Raman Spectral Profiles of Pseudomonas fluorescens SBW25 Reflects in vitro and in planta Metabolic History [J]. Microbial Ecology,2007,53(3),414-425.
[67]John M. Sanderson, Andrew D. Ward. Analysis of liposomal membrane composition using Raman tweezers [J]. Chemical Communications,2004,7,1120-1121.
[68]Xie Chang-an, Chen De, Li Yong-qing. Raman sorting and identification of single living micro-organisms with optical tweezers, Optics Letters,2005,30(14),1800-1802.
[69]杨洲平,黄乾明,任君,裴亮.高产类胡萝卜素红酵母的筛选及发酵条件的优化[J].四川农业大学学报.2009,27(3)：327-332.
[70]Gelder J. D., Gussem K. D., Vandenabeele P. Methods for xtracting biochemical information frombacterial Raman spectra:Focus on a Group of structurally similar biomolecules-Fatty acids [J]. Analytica chimica acta,2007,603(2):167-175.
[71]王微微,李亮,李占龙,孙成林,等.温度对短链多烯生物分子β胡萝卜素拉曼光谱的影响[J].高等学校化学学报,2010,31(9)：1864-1867.
[72]李艳,孟令晶,刘军贤,郑禹,宋春胜,姚辉璐.拉曼光谱法测定单个大米淀粉微粒糊化过程的实时研究[J].光子学报,2010,39(7)：1324-1329.
[73]Ritsuko Fujii, Chen Chun-hai, Tadashi Mizoguchi, Yasushi Koyama. HNMR, electronic-absorption and resonance-Raman spectra of isomeric okenone as compared with those of isomeric β-carotene, canthaxanthin, β-apo-8'-carotenal and spheroidene. Spectrochimica Acta Part A,1998,54(5):727-743.
[74]Huang Y. S., Karashima T., Yamamoto M. Molecular-Level investigation of the structure, transformation, and bioactivity of single living feyeast cells by time- and space-resolved Raman spectroscopy [J]. Biochemistry,2005,44(30):10009-10019.
[75]Wei E. Huang., Ward A. D., Whiteley A. S. Raman tweezers sorting of single microbial cells [J]. Environmental Microbiology Reports,2009,1(1):44-49.
[76]Marovai, Cameka M., Haliebova A. Production of carotenoid-/ergosterol- supplemented biomass by red yeast Rhodotorula glutinis grown under external stress [J]. Food Technol. Biotechnd.2010,48(1):56-61.
[2]Arthur J. Ragauskas, Charlotte K. Williams, Brian H. Davison, et al. The path forward for biofuels and biomaterials[J]. Science,2006,311(5760):484-489.
[3]Barry D. Solomon, Justin R. Barnes, Kathleen E. Halvorsen. Grain and cellulosic ethanol:History, economics, and energy policy[J]. Biomass and Bioenergy,2007.31(6):416-425.
[4]石元春.发展生物质产业[J].中国农业科技导报,2006,8(1)：1-5.
[5]章克昌.酒精与蒸馏酒工艺学[M],北京：中国轻工业出版社,1995.
[6]Marcel Blanco, Antonio C. Peinado, Jordi Mas. Analytical monitoring of alcoholic fermentation using NIR spectroscopy[J]. Biotechnology and Bioengineering,2004,88(4):536-542.
[7]Sivakesava S, Irudayaraj J, Demirci A. Monitoring a bioprocess for ethanol production using FT-MIR and FT-Raman spectroscopy[J]. Journal of Industrial Microbiology and Biotechnology.2001,26(4):185-190.
[8]Eliana M Alhadeff, Andrea M Salgado, Nei Pereira, Belkis Valdman. A sequential enzymatic microreactor system for ethanol detection of gasohol mixtures[J]. Applied Biochemistry and Biotechnology,2005, 121(1-3):361-371.
[9]Choengchan N, Mantim T, Wilairat P, Dasgupta P K, Motomizu S, Nacapricha D. A membraneless gas diffusion unit:design and its application to determination of ethanol in liquors by spectrophotometric flow injection[J]. Analytica Chimica Acta,2006,579(1):33-37.
[10]Cai Zhao-Xia, Yang Hong, Zhang Yi, Yan Xiu-Ping. Preparation, characterization and evaluation of water-soluble L-cysteine-capped-CdS nanoparticles as fluorescence probe for detection of Hg(II) in aqueous solution[J]. Analytica Chimica Acta,2006,559(2):234-239.
[11]史月华,陆勇,张荣,徐铸德.傅里叶变换近红外光谱同时定量分析水溶液中的乙醇、果糖和葡萄糖[J].分析化学,2001,29(10)：1213-1215.
[12]Soohwa Cho, Hoeil Chung, Young-Ah Woo, Kim Hyo-Jim, Determination of water content in ethanol by miniaturized Near-Infrared (NIR) system[J]. Bull. K. Chem. Soc,2005,26(1):115-118.
[13]Leornardo S. Mendes, Flavia C. C. Oliveira, Paulo A. Z. Suarez, Joel C. Rubin. Determination of ethanol in fuel ethanol and beverages by fouier transform (FT)-near infrared and FT-Raman spectrometries[J]. Analytica Chimica Acta,2003,493:219-231.
[14]Romina Kaushik, William R. LaCourse, Barry Levine. Determination of ethyl glucuronide in urine using reversed-phase HPLC and pulsed electrochemical detection (Part II) [J]. Analytica Chimica Acta,2006, 556(2):267-274.
[15]A. Ashkin, J.M. Dziedzic, J.E. Bjorkholm, Steven Chu, Observation of a single-beam gradient force optical trap for dielectric articles[J], Optics Letters.1986,11(5):288-290.
[16]A. Ashkin, J. M. Dziedzic. Optical trapping and manipulation of single cells using inferred laser beams[J], Berichte der Bunsengesellschaft fur phsikalische chemie[J],1989,93(3):254-260..
[17]Xie Chang-An, Mumtaz A. Dinno, Li Yong-Qing. Near-infrared Raman spectroscopy of single optically rapped biological cells[J]. Optics Letters.2002,27:249-251.
[18]Xie Chang-An, Li Yong-Qing. Confocal micro-Raman spectroscopy of single biological cell using optical trapping and shifted excitation difference techniques[J]. Journal Applied Physics,2003,93(5):2982-2987.
[19]杨序纲,吴琪琳,拉曼光谱的分析与应用[M],北京,国防工业出版社,2008,11(1)：19-25.
[20]陈秀丽,王桂文,刘军贤,陈萍,黎永青.基于拉曼光谱的地贫红细胞种类识别方法的研究[J],分析测试学报,2009,28(4)：403-408.
[21]冯听韡,朱仲良,沈梦洁,丛培盛.基于多项式拟合的拉曼光谱基线漂移校正方法[J].计算机与应用化学.2009,26(6)：759-762.
[22]Xie Chang-An, Charles Goodman, Mumtaz Dinno, Li Yong-Qing. Real-time Raman spectroscopy of optically trapped living cells and organells[J]. Optics Express,2004,12(25),6208-6214.
[23]Gajendra Pratap Singh, Giovanni Volpe, Caitriona M. Creely, Helga Grotsch, Isabel M. Geli, Dmitri Petrov. The lag phase and G1 phase of a single yeast cell monitored by Raman microspectroscopy[J]. Journal of Raman spectroscopy,2006,37(8),858-864.
[24]王桂文,姚辉璐,彭立新,何碧娟,黎永青.一种酵母细胞生长现象的实时单细胞拉曼光谱观察[J].微生物学通报,2007,34(6),1109-1113.
[25]Li Hai-Long, Chai Xin-Sheng, Deng Yu-Lin, Zhan Huai-Yu, Fu Shi-Yu. Rapid determination of ethanol in fermentation liquor by full evaporation headspace gas chromatography[J]. Journal of Chromatography A,2009,1216(1):169-172.
[26]Marcelo Firmino de Oliveira, Adelir Aparecida Saczk, Leonardo Luiz Okumura, Nelson Ramos Stradiotto. Analytical methods employed at quality control of fuel ethaol[J]. Energy Fuels,2009,23(10):4852-4859.
[27]覃建军,柳畅先,察东梅.酶催化动力学荧光法测定乙醇[J].分析科学学报.2008,24(2)：224-226.
[28]李燕萍,许宏贤,钱莹,段钢.发酵液中乙醇含量的近红外光谱NIRS定量分析与验证[J].食品与发酵工业,2008,34(7)：136-140.
[29]袁文杰,孔亮,孜力汗,白凤武.高效液相色谱法测定克鲁维酵母菊芋发酵液中的乙醇,糖和有机酸类代谢成分[J].分析化学,2009,37(6)：850-854.
[30]Zhao Ren-Yong, Scott R. Bean, Brian P. Ioerger, Wang Dong-Hai, Daniel L. Boyle. Impact of mashing on sorghum proteins and its relationship to ethanol fermentation[J]. Journal of Agricultural and Food Chemistry,2008,56(3):946-953.
[31]Landulfo Silveira Jr., Leonardo Marmo Moreira, Viviane G. B. Conceicao, Heliodora L. Casalechi, Ingrid S. Munoz, Fabiano Fernandes da Silva, Marcos Augusto S. R. Silva, Renato Aparecido de Souza, Marcos Tadeu T. Pacheco. Determination of sucrose concentration in lemon-type soft drinks by dispersive Raman spectroscopy[J]. Spectroscopy,2009,23(3-4):217-226.
[32]Chien-Ju Shih, Emily A. Smith. Determination of glucose and ethanol after enzymatic hydrolysis and fermentation of biomass using Raman spectroscopy[J]. Analytica Chimica Acta,2009,653(2):200-206
[33]王桂文,姚辉璐,彭立新,何碧娟,黎永青.拉曼镊子分析单个苏云金芽孢杆菌伴孢晶体蛋白[J].分析化学,2007,35(9)：1351-1354.
[34]Ye Qing, Xu Qin-Feng, Yu Yong-Gai, Qu Rong-Hui, Fang Zu-Jie. Rapid and quantitative detection of ethanol proportion in ethanol-gasoline mixtures by Raman spectroscopy [J]. Optics Communications,2009, 282(18):3785-3788.
[35]A. Picard, I. Daniel, G. Montagnac, P. Oger. In situ monitoring by quantitative Raman spectroscopy of alcoholic fermentation by Saccbaromyces cerevisiae under high pressure[J]. Extremophiles,2007,11(3): 445-452.
[36]A. A. Abreu, A. S. Danko, J. C. Costa, E. C. Ferreira, M. M. Alves. Inoculum type response to different pHs on biohydrogen production from L-arabinose, a component of hemicellulosic biopolymers[J]. International Journal of Hydrogen Energy,2009,34(4):1744-1751
[37]刘文涵,杨未,吴小琼,林振兴.激光拉曼光谱内标法直接测定乙醇浓度[J].分析化学,2007,3(35)：416-418.
[38]李燕萍,钱莹,段钢.采用近红外光谱测定木薯乙醇发酵液中乙醇、甘油和葡萄糖含量[J].食品与发酵工业,2009,35(8)：117-121.
[39]Marcelo F O, Adelir A S, Leonardo L O,Nelson R S. Analytical Methods Employed at Quality Control of Fuel Ethanol[J]. Brasil Energy Fuels,2009,23(10):4852-4859.
[40]Leornardo S M, Flavia C C O, Paulo A Z S, Determination of ethanol in fuel ethanol and beverages by Fourier transform (FT)-near infrared and FT-Raman spectrometries[J]. Joel C R.Chimica Acta,2003, 493(10):219-231.
[41]吴小琼,郑建珍,刘文涵,林振兴.激光拉曼光谱内标法测定葡萄糖液浓度[J].光谱学与光谱分析,2007,27(7)：1344-1346.
[42]吴正洁,黄耀熊,王成,黎绍发.多种拉曼光谱归一化法对乙醇定量分析的研究[J].光谱学与光谱分析,2010,30(4)：971-974.
[43]潘立登.先进控制与在线优化技术及其应用[M].北京：机械工业出版社,2009：78-118.
[44]杜一平,潘铁英,张玉兰.化学计量学应用[M].北京：化学工业出版社,2008：133-170.
[45]陈敏伯.化学计量学[M].北京：科学出版社,2009：295-316.
[46]严航贞,张安平,林春绵.96孔板法用于除草剂高通量筛选的研究[J].浙江工业大学学报,2009,4,37(2)：119-122.
[47]Sumitra Ramachandran, Pierre Fontanille, Ashok Pandey, Christian Larroche. Stability of Glucose Oxidase Activity of Aspergillus niger Spores Produced by Solid-State Fermentation and Their Role as Biocatalysts in Bioconversion Reaction[J]. Food Technol. Biotechnol.,2008,46 (2):190-194.
[48]王桂文,姚辉璐,何碧娟,彭立新,黎永清.单个血小板的拉曼光谱分析[J].光谱学与光谱分析,2007,22(7)：1347-1350.
[49]Gobinet Cyril, Valeriu Vrabie, Manfait Michel, Piot Olivier. Preprocessing methods of spectra for source extraction on biomedical samples:application on paraffin-embedded skin biopsies[J]. IEEE Transactions on Biomedical Engineering,2009,56(5):1371-1382.
[50]彭帮柱,龙明华,岳田利,袁亚宏,赵志华.用偏最小二乘法及傅立叶变换近红外光谱快速检测白酒酒精度[J].农业工程学报,2007,23(4)：233-237.
[51]刘振,王金鹏,张立峰,曾爱武,袁希钢.木薯干原料同步糖化发酵生产乙醇[J].过程工程学报,2005,5(3)：353-356.
[52]Fabien Joux, and Philippe Lebaron. Use of fluorescent probes to assess physiological functions of bacteria at single-cell level[J]. Microbes and Infection.2000,2(12):1523-1535.
[53]K. Christian Schuster, Ingo Reese, Eva Urlaub, J. Richard Gapes, Bernhard Lendl. Multidimensional information on the chemical composition of single bacterial cells by confocal Raman microspectroscopy [J]. Anal. Chem.,2000,72(22):5529-5534.
[54]S Sivakesava, J Irudayraj, A Demirci. Monitoring a bioproceess for ethanol production using FT-MIR and FT-Raman spectroscopy[J]. Journal of Industrial Microbiology and Biotechnology,2001,26(4):185-190.
[55]Xie Chang-an, Mumtaz A. Dinno, Li Yong-qing. Near-infrared Raman spectroscopy of single optically trapped biological cells[J]. Optics Letters,2002,27(4):249-251.
[56]Xie Chang-an, Charles Goodman, Mumtaz A. Dinno, and Li Yong-Qing. Real-time Raman spectroscopy of optically trapped living cells and organelles[J]. Optics Express,2004,12(25):6208-6214.
[57]Yusuf Chisti. Biodiesel from microalgae beats bioethanol[J]. Trends in Biotechnology,2008,26(3): 126-131.
[58]Meng Xin, Yang Jian-Ming, Xu Xin, Zhang Lei, Nie Qin-Juan, Xian Mo. Biodiesel production from oleaginous microorganisms[J], Renewable Energy,2009,34(1):1-5.
[59]Zhang Wei, Li Zhi-Yong, Miao Xiao-Ling, Zhang Feng-Li. The screening of antimicrobial bacteria with diverse novel nonribosomal peptide synthetase (NRPS) genes from south china sea sponges[J]. Marine Biotechnology,2009,11(3):346-355.
[60]Xie Chang-An, Mumtaz A. Dinno, Li Yong-Qing. Near-infrared Raman spectroscopy of single optically trapped biological cells[J]. Optics Letters,2002,27(4):249-251.
[61]Ai Min, LIU Jun-xian, Huang Shu-Shi, Wang Gui-Wen, Chen Xiu-Li, Chen Zhi-Cheng, YAO Hui-lu. Application and progress of Raman Tweezers in single cells[J]. Chinese Journal of Analytical Chemistry, 2009,37(5):758-763.
[62]Notingher I., Verrier S., Romanska H., Bishop A. E., Polak J. M., Hench L. L.. In situ characterization of living cells by Raman spectroscopy [J]. Spectroscopy,2002,16(2):43-51.
[63]Davey H., Kell D. B. Flow cytometry and cell sorting of heterogeneous microbial populations:the importance of single-cell analyses [J]. Microbiol. Rev.,1996,60(4):641-696.
[64]Wei E. Huang, Robert I. Griffiths, Ian P. Thompson, Mark J. Bailey, Anderw S. Whiteley. Raman microscopic analysis of single microbial Cells [J]. Anal. Chem.,2004,76(15),4452-4458.
[65]Wei E. Huang, Kilian Stoecker, Robert Griffiths, Lyndsay Newbold, Holger Daims, Andrew S. Whiteley, Michael Wagner. Raman-FISH:combining stable-isotope Raman spectroscopy and fluorescence in situ hybridization for the single cell analysis of identity and function [J]. Environmental Microbiology,2007, 9(8),1878-1889.
[66]Wei E. Huang, Mark J. Bailey, Ian P. Thompson, Andrew S. Whiteley, Andrew J. Spiers. Single-Cell Raman Spectral Profiles of Pseudomonas fluorescens SBW25 Reflects in vitro and in planta Metabolic History [J]. Microbial Ecology,2007,53(3),414-425.
[67]John M. Sanderson, Andrew D. Ward. Analysis of liposomal membrane composition using Raman tweezers [J]. Chemical Communications,2004,7,1120-1121.
[68]Xie Chang-an, Chen De, Li Yong-qing. Raman sorting and identification of single living micro-organisms with optical tweezers, Optics Letters,2005,30(14),1800-1802.
[69]杨洲平,黄乾明,任君,裴亮.高产类胡萝卜素红酵母的筛选及发酵条件的优化[J].四川农业大学学报.2009,27(3)：327-332.
[70]Gelder J. D., Gussem K. D., Vandenabeele P. Methods for xtracting biochemical information frombacterial Raman spectra:Focus on a Group of structurally similar biomolecules-Fatty acids [J]. Analytica chimica acta,2007,603(2):167-175.
[71]王微微,李亮,李占龙,孙成林,等.温度对短链多烯生物分子β胡萝卜素拉曼光谱的影响[J].高等学校化学学报,2010,31(9)：1864-1867.
[72]李艳,孟令晶,刘军贤,郑禹,宋春胜,姚辉璐.拉曼光谱法测定单个大米淀粉微粒糊化过程的实时研究[J].光子学报,2010,39(7)：1324-1329.
[73]Ritsuko Fujii, Chen Chun-hai, Tadashi Mizoguchi, Yasushi Koyama. HNMR, electronic-absorption and resonance-Raman spectra of isomeric okenone as compared with those of isomeric β-carotene, canthaxanthin, β-apo-8'-carotenal and spheroidene. Spectrochimica Acta Part A,1998,54(5):727-743.
[74]Huang Y. S., Karashima T., Yamamoto M. Molecular-Level investigation of the structure, transformation, and bioactivity of single living feyeast cells by time- and space-resolved Raman spectroscopy [J]. Biochemistry,2005,44(30):10009-10019.
[75]Wei E. Huang., Ward A. D., Whiteley A. S. Raman tweezers sorting of single microbial cells [J]. Environmental Microbiology Reports,2009,1(1):44-49.
[76]Marovai, Cameka M., Haliebova A. Production of carotenoid-/ergosterol- supplemented biomass by red yeast Rhodotorula glutinis grown under external stress [J]. Food Technol. Biotechnd.2010,48(1):56-61.