脂溶性荧光染料测定微生物油脂的研究进展
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摘要
微生物油脂因生产不受气候、季节影响,占地少,所需人力较少等优点,被认为是生物柴油的理想原料。不同种类的油脂微生物的脂质含量差别较大,因此,高效筛选油脂含量高的微生物菌株是开发微生物油脂资源的关键问题,而如何分析、测定微生物的油脂含量是筛选高油脂含量菌株不可缺少的步骤。荧光分析法具有速度快、污染少、可原位测定等优势正逐渐取代传统氯仿/甲醇重量分析方法,被广泛应用于定性定量分析微生物油脂。本文综述了脂溶性荧光染料(以尼罗红与BODIPY 505/515为主)测定微生物油脂的研究进展,以及其应用中存在的问题,并提出了未来改进该方法的研究思路。
Microbial lipid was deemed as an ideal raw material to produce biodiesel, possessing characteristics of no limit of season and climate, occupation less land space and manpower, etc. The lipid contents vary with different strains, so the key factor for developing the source of microbial lipid is to screen the microbial species with high lipid content. The essential step for screening the strains with high lipid content is to analyze and measure the lipid content of microorganism.Fluorescent method, which is quick, less pollution, measuring in stiu, have been applied to analyze microbial lipid qualitatively and quantitatively, is now gradually replacing the traditional gravimetric method by chloroform/methyl. This review narrated the development and some problems of microbial lipid analyzed by liposoluble fluorescent probe(mianly Nile red and BODIPY 505/515). Also, some research means for developing this method were put forward.
Microbial lipid was deemed as an ideal raw material to produce biodiesel, possessing characteristics of no limit of season and climate, occupation less land space and manpower, etc. The lipid contents vary with different strains, so the key factor for developing the source of microbial lipid is to screen the microbial species with high lipid content. The essential step for screening the strains with high lipid content is to analyze and measure the lipid content of microorganism.Fluorescent method, which is quick, less pollution, measuring in stiu, have been applied to analyze microbial lipid qualitatively and quantitatively, is now gradually replacing the traditional gravimetric method by chloroform/methyl. This review narrated the development and some problems of microbial lipid analyzed by liposoluble fluorescent probe(mianly Nile red and BODIPY 505/515). Also, some research means for developing this method were put forward.
引文
[1]HU Q,SOMMERFELD M,JARVIS E,et al.Microalgal triacylglycerols as feedstocks for biofuel production:perspectives and advances[J].The plant journal,2008,54(4):621-639.DOI:10.1111/j.1365-313X.2008.03492.x.
[2]GUERFALI M,AYADI I,BELHASSEN A,et al.Single cell oil production by Trichosporon cutaneum and lignocellulosic residues bioconversion for biodiesel synthesis[J].Process safety and environmental protection,2018,113:292-304.DOI:10.1016/j.psep.2017.11.002.
[3]BHARATHIRAJA B,SRIDHARAN S,SOWMYA V,et al.Microbial oil-A plausible alternate resource for food and fuel application[J].Bioresource technology,2017,233:423-432.DOI:10.1016/j.biortech.2017.03.006.
[4]HEREDIA-ARROYO T,WEI W,RUAN R G,et al.Mixotrophic cultivation of Chlorella vulgaris and its potential application for the oil accumulation from nonsugar materials[J].Biomass and bioenergy,2011,35(5):2245-2253.DOI:10.1016/j.biombioe.2011.02.036.
[5]BLIGH E G,DYER W J.A rapid method of total lipid extraction and purification[J].Canadian journal of biochemistry and physiology,1959,37(8):911-917.DOI:10.1139/o59-099.
[6]FOLCH J,LEES M,SLOANE STANLEY G H.A simple method for the isolation and purification of total lipides from animal tissues[J].Journal of biological chemistry,1957,226(1):497-509.
[7]郝翠翠,梁成伟,石蕾.尼罗红和BODIPY荧光染料在微藻油脂含量测定中的应用[J].生物学杂志,2017,34(1):70-74.DOI:10.3969/j.issn.2095-1736.2017.01.070.
[8]RUMIN J,BONNEFOND H,SAINT-JEAN B,et al.The use of fluorescent Nile red and BODIPY for lipid measurement in microalgae[J].Biotechnology for biofuels,2015,8:42.DOI:10.1186/s13068-015-0220-4.
[9]XU D,GAO Z Q,LI F,et al.Detection and quantitation of lipid in the microalga Tetraselmis subcordiformis(Wille)Butcher with BODIPY 505/515 staining[J].Bioresource technology,2013,127:386-390.DOI:10.1016/j.biortech.2012.09.068.
[10]GREENSPAN P,FOWLER S D.Spectrofluorometric studies of the lipid probe,nile red[J].Journal of lipid research,1985,26(7):781-789.
[11]MUTANDA T,RAMESH D,KARTHIKEYAN S,et al.Bioprospecting for hyper-lipid producing microalgal strains for sustainable biofuel production[J].Bioresource technology,2011,102(1):57-70.DOI:10.1016/j.biortech.2010.06.077.
[12]ELSEY D,JAMESON D,RALEIGH B,et al.Fluorescent measurement of microalgal neutral lipids[J].Journal of microbiological methods,2007,68(3):639-642.DOI:10.1016/j.mimet.2006.11.008.
[13]O'ROURKE E J,SOUKAS A A,CARR C E,et al.C.elegans major fats are stored in vesicles distinct from lysosome-related organelles[J].Cell metabolism,2009,10(5):430-435.DOI:10.1016/j.cmet.2009.10.002.
[14]BROWN W J,SULLIVAN T R,GREENSPAN P.Nile red staining of lysosomal phospholipid inclusions[J].Histochemistry,1992,97(4):349-354.DOI:10.1007/BF00270037.
[15]DUTTA A K,KAMADA K,OHTA K.Spectroscopic studies of nile red in organic solvents and polymers[J].Journal of photochemistry and photobiology A:chemistry,1996,93(1):57-64.DOI:10.1016/1010-6030(95)04140-0.
[16]HOUNSLOW E,NOIREL J,GILMOUR D J,et al.Lipid quantification techniques for screening oleaginous species of microalgae for biofuel production[J].European journal of lipid science and technology,2017,119(2):1500469.DOI:10.1002/ejlt.201500469.
[17]FOWLER S D,GREENSPAN P.Application of Nile red,a fluorescent hydrophobic probe,for the detection of neutral lipid deposits in tissue sections:comparison with oil red O[J].Journal of histochemistry&cytochemistry,1985,33(8):833-836 DOI:10.1177/33.8.4020099.
[18]COOKSEY K E,GUCKERT J B,WILLIAMS S A,et al.Fluorometric determination of the neutral lipid content of microalgal cells using Nile Red[J].Journal of microbiological methods,1987,6(6):333-345.DOI:10.1016/0167-7012(87)90019-4.
[19]LEE S J,YOON B D,OH H M.Rapid method for the determination of lipid from the green alga Botryococcus braunii[J].Biotechnology techniques,1998,12(7):553-556.DOI:10.1023/A:1008811716448.
[20]KIMURA K,YAMAOKA M,KAMISAKA Y.Rapid estimation of lipids in oleaginous fungi and yeasts using Nile red fluorescence[J].Journal of microbiological methods,2004,56(3):331-338.DOI:10.1016/j.mimet.2003.10.018.
[21]HUANG G H,CHEN G,CHEN F.Rapid screening method for lipid production in alga based on Nile red fluorescence[J].Biomass and bioenergy,2009,33(10):1386-1392.DOI:10.1016/j.biombioe.2009.05.022.
[22]DE LA JARA A,MENDOZA H,MARTEL A,et al.Flow cytometric determination of lipid content in a marine dinoflagellate,Crypthecodinium cohnii[J].Journal of applied phycology,2003,15(5):433-438.DOI:10.1023/A:1026007902078.
[23]DA SILVA T L,REIS A,MEDEIROS R,et al.Oil production towards biofuel from autotrophic microalgae semicontinuous cultivations monitorized by flow cytometry[J].Applied biochemistry and biotechnology,2009,159(2):568-578.DOI:10.1007/s12010-008-8443-5.
[24]SITEPU I R,IGNATIA L,FRANZ A K,et al.An improved high-throughput Nile red fluorescence assay for estimating intracellular lipids in a variety of yeast species[J].Journal of microbiological methods,2012,91(2):321-328.DOI:10.1016/j.mimet.2012.09.001.
[25]CHEN Z,GONG Y M,FANG X T,et al.Scenedesmus sp.NJ-1 isolated from Antarctica:a suitable renewable lipid source for biodiesel production[J].World journal of microbiology and biotechnology,2012,28(11):3219-3225.DOI:10.1007/s11274-012-1132-0.
[26]DE LA HOZ SIEGLER H,AYIDZOE W,BEN-ZVI A,et al.Improving the reliability of fluorescence-based neutral lipid content measurements in microalgal cultures[J].Algal research,2012,1(2):176-184.DOI:10.1016/j.algal.2012.07.004.
[27]PICK U,RACHUTIN-ZALOGIN T.Kinetic anomalies in the interactions of Nile red with microalgae[J].Journal of microbiological methods,2012,88(2):189-196.DOI:10.1016/j.mimet.2011.10.008.
[28]GOVENDER T,RAMANNA L,RAWAT I,et al.BODIPY staining,an alternative to the Nile Red fluorescence method for the evaluation of intracellular lipids in microalgae[J].Bioresource technology,2012,114:507-511.DOI:10.1016/j.biortech.2012.03.024.
[29]WONG D M,NGUYEN T T N,FRANZ A K.Ethylenediaminetetraacetic acid(EDTA)enhances intracellular lipid staining with Nile red in microalgae Tetraselmis suecica[J].Algal research,2014,5:158-163.DOI:10.1016/j.algal.2014.08.002.
[30]CHEN W,ZHANG C W,SONG L R,et al.A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae[J].Journal of microbiological methods,2009,77(1):41-47.DOI:10.1016/j.mimet.2009.01.001.
[31]YEN DOAN T T,OBBARD J P.Enhanced lipid production in Nannochloropsis sp.using fluorescenceactivated cell sorting[J].Global change biology bioenergy,2011,3(3):264-270.DOI:10.1111/j.1757-1707.2010.01076.x.
[32]CHEN W,SOMMERFELD M,HU Q.Microwave-assisted Nile red method for in vivo quantification of neutral lipids in microalgae[J].Bioresource technology,2011,102(1):135-141.DOI:10.1016/j.biortech.2010.06.076.
[33]SU L C,HSU Y H,WANG H Y.Enhanced labeling of microalgae cellular lipids by application of an electric field generated by alternating current[J].Bioresource technology,2012,111:323-327.DOI:10.1016/j.biortech.2012.01.180.
[34]BRENNAN L,BLANCO FERNáNDEZ A,MOSTAERTA S,et al.Enhancement of BODIPY505/515 lipid fluorescence method for applications in biofuel-directed microalgae production[J].Journal of microbiological methods,2012,90(2):137-143.DOI:10.1016/j.mimet.2012.03.020.
[35]COOPER M S,HARDIN W R,PETERSEN T W,et al.Visualizing“green oil”in live algal cells[J].Journal of bioscience and bioengineering,2010,109(2):198-201.DOI:10.1016/j.jbiosc.2009.08.004.
[36]CIRULIS J T,STRASSER B C,SCOTT J A,et al.Optimization of staining conditions for microalgae with three lipophilic dyes to reduce precipitation and fluorescence variability[J].Cytometry part A,2012,81A(7):618-626.DOI:10.1002/cyto.a.22066.
[37]KURNIASIH I N,LIANG H,MOHR P C,et al.Nile red dye in aqueous surfactant and micellar solution[J].Langmuir,2015,31(9):2639-2648.DOI:10.1021/la504378m.
[38]FELBECK T,BEHNKE T,HOFFMANN K,et al.Nile-Red-nanoclay hybrids:red emissive optical probes for use in aqueous dispersion[J].Langmuir,2013,29(36):11489-11497.DOI:10.1021/la402165q.
[2]GUERFALI M,AYADI I,BELHASSEN A,et al.Single cell oil production by Trichosporon cutaneum and lignocellulosic residues bioconversion for biodiesel synthesis[J].Process safety and environmental protection,2018,113:292-304.DOI:10.1016/j.psep.2017.11.002.
[3]BHARATHIRAJA B,SRIDHARAN S,SOWMYA V,et al.Microbial oil-A plausible alternate resource for food and fuel application[J].Bioresource technology,2017,233:423-432.DOI:10.1016/j.biortech.2017.03.006.
[4]HEREDIA-ARROYO T,WEI W,RUAN R G,et al.Mixotrophic cultivation of Chlorella vulgaris and its potential application for the oil accumulation from nonsugar materials[J].Biomass and bioenergy,2011,35(5):2245-2253.DOI:10.1016/j.biombioe.2011.02.036.
[5]BLIGH E G,DYER W J.A rapid method of total lipid extraction and purification[J].Canadian journal of biochemistry and physiology,1959,37(8):911-917.DOI:10.1139/o59-099.
[6]FOLCH J,LEES M,SLOANE STANLEY G H.A simple method for the isolation and purification of total lipides from animal tissues[J].Journal of biological chemistry,1957,226(1):497-509.
[7]郝翠翠,梁成伟,石蕾.尼罗红和BODIPY荧光染料在微藻油脂含量测定中的应用[J].生物学杂志,2017,34(1):70-74.DOI:10.3969/j.issn.2095-1736.2017.01.070.
[8]RUMIN J,BONNEFOND H,SAINT-JEAN B,et al.The use of fluorescent Nile red and BODIPY for lipid measurement in microalgae[J].Biotechnology for biofuels,2015,8:42.DOI:10.1186/s13068-015-0220-4.
[9]XU D,GAO Z Q,LI F,et al.Detection and quantitation of lipid in the microalga Tetraselmis subcordiformis(Wille)Butcher with BODIPY 505/515 staining[J].Bioresource technology,2013,127:386-390.DOI:10.1016/j.biortech.2012.09.068.
[10]GREENSPAN P,FOWLER S D.Spectrofluorometric studies of the lipid probe,nile red[J].Journal of lipid research,1985,26(7):781-789.
[11]MUTANDA T,RAMESH D,KARTHIKEYAN S,et al.Bioprospecting for hyper-lipid producing microalgal strains for sustainable biofuel production[J].Bioresource technology,2011,102(1):57-70.DOI:10.1016/j.biortech.2010.06.077.
[12]ELSEY D,JAMESON D,RALEIGH B,et al.Fluorescent measurement of microalgal neutral lipids[J].Journal of microbiological methods,2007,68(3):639-642.DOI:10.1016/j.mimet.2006.11.008.
[13]O'ROURKE E J,SOUKAS A A,CARR C E,et al.C.elegans major fats are stored in vesicles distinct from lysosome-related organelles[J].Cell metabolism,2009,10(5):430-435.DOI:10.1016/j.cmet.2009.10.002.
[14]BROWN W J,SULLIVAN T R,GREENSPAN P.Nile red staining of lysosomal phospholipid inclusions[J].Histochemistry,1992,97(4):349-354.DOI:10.1007/BF00270037.
[15]DUTTA A K,KAMADA K,OHTA K.Spectroscopic studies of nile red in organic solvents and polymers[J].Journal of photochemistry and photobiology A:chemistry,1996,93(1):57-64.DOI:10.1016/1010-6030(95)04140-0.
[16]HOUNSLOW E,NOIREL J,GILMOUR D J,et al.Lipid quantification techniques for screening oleaginous species of microalgae for biofuel production[J].European journal of lipid science and technology,2017,119(2):1500469.DOI:10.1002/ejlt.201500469.
[17]FOWLER S D,GREENSPAN P.Application of Nile red,a fluorescent hydrophobic probe,for the detection of neutral lipid deposits in tissue sections:comparison with oil red O[J].Journal of histochemistry&cytochemistry,1985,33(8):833-836 DOI:10.1177/33.8.4020099.
[18]COOKSEY K E,GUCKERT J B,WILLIAMS S A,et al.Fluorometric determination of the neutral lipid content of microalgal cells using Nile Red[J].Journal of microbiological methods,1987,6(6):333-345.DOI:10.1016/0167-7012(87)90019-4.
[19]LEE S J,YOON B D,OH H M.Rapid method for the determination of lipid from the green alga Botryococcus braunii[J].Biotechnology techniques,1998,12(7):553-556.DOI:10.1023/A:1008811716448.
[20]KIMURA K,YAMAOKA M,KAMISAKA Y.Rapid estimation of lipids in oleaginous fungi and yeasts using Nile red fluorescence[J].Journal of microbiological methods,2004,56(3):331-338.DOI:10.1016/j.mimet.2003.10.018.
[21]HUANG G H,CHEN G,CHEN F.Rapid screening method for lipid production in alga based on Nile red fluorescence[J].Biomass and bioenergy,2009,33(10):1386-1392.DOI:10.1016/j.biombioe.2009.05.022.
[22]DE LA JARA A,MENDOZA H,MARTEL A,et al.Flow cytometric determination of lipid content in a marine dinoflagellate,Crypthecodinium cohnii[J].Journal of applied phycology,2003,15(5):433-438.DOI:10.1023/A:1026007902078.
[23]DA SILVA T L,REIS A,MEDEIROS R,et al.Oil production towards biofuel from autotrophic microalgae semicontinuous cultivations monitorized by flow cytometry[J].Applied biochemistry and biotechnology,2009,159(2):568-578.DOI:10.1007/s12010-008-8443-5.
[24]SITEPU I R,IGNATIA L,FRANZ A K,et al.An improved high-throughput Nile red fluorescence assay for estimating intracellular lipids in a variety of yeast species[J].Journal of microbiological methods,2012,91(2):321-328.DOI:10.1016/j.mimet.2012.09.001.
[25]CHEN Z,GONG Y M,FANG X T,et al.Scenedesmus sp.NJ-1 isolated from Antarctica:a suitable renewable lipid source for biodiesel production[J].World journal of microbiology and biotechnology,2012,28(11):3219-3225.DOI:10.1007/s11274-012-1132-0.
[26]DE LA HOZ SIEGLER H,AYIDZOE W,BEN-ZVI A,et al.Improving the reliability of fluorescence-based neutral lipid content measurements in microalgal cultures[J].Algal research,2012,1(2):176-184.DOI:10.1016/j.algal.2012.07.004.
[27]PICK U,RACHUTIN-ZALOGIN T.Kinetic anomalies in the interactions of Nile red with microalgae[J].Journal of microbiological methods,2012,88(2):189-196.DOI:10.1016/j.mimet.2011.10.008.
[28]GOVENDER T,RAMANNA L,RAWAT I,et al.BODIPY staining,an alternative to the Nile Red fluorescence method for the evaluation of intracellular lipids in microalgae[J].Bioresource technology,2012,114:507-511.DOI:10.1016/j.biortech.2012.03.024.
[29]WONG D M,NGUYEN T T N,FRANZ A K.Ethylenediaminetetraacetic acid(EDTA)enhances intracellular lipid staining with Nile red in microalgae Tetraselmis suecica[J].Algal research,2014,5:158-163.DOI:10.1016/j.algal.2014.08.002.
[30]CHEN W,ZHANG C W,SONG L R,et al.A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae[J].Journal of microbiological methods,2009,77(1):41-47.DOI:10.1016/j.mimet.2009.01.001.
[31]YEN DOAN T T,OBBARD J P.Enhanced lipid production in Nannochloropsis sp.using fluorescenceactivated cell sorting[J].Global change biology bioenergy,2011,3(3):264-270.DOI:10.1111/j.1757-1707.2010.01076.x.
[32]CHEN W,SOMMERFELD M,HU Q.Microwave-assisted Nile red method for in vivo quantification of neutral lipids in microalgae[J].Bioresource technology,2011,102(1):135-141.DOI:10.1016/j.biortech.2010.06.076.
[33]SU L C,HSU Y H,WANG H Y.Enhanced labeling of microalgae cellular lipids by application of an electric field generated by alternating current[J].Bioresource technology,2012,111:323-327.DOI:10.1016/j.biortech.2012.01.180.
[34]BRENNAN L,BLANCO FERNáNDEZ A,MOSTAERTA S,et al.Enhancement of BODIPY505/515 lipid fluorescence method for applications in biofuel-directed microalgae production[J].Journal of microbiological methods,2012,90(2):137-143.DOI:10.1016/j.mimet.2012.03.020.
[35]COOPER M S,HARDIN W R,PETERSEN T W,et al.Visualizing“green oil”in live algal cells[J].Journal of bioscience and bioengineering,2010,109(2):198-201.DOI:10.1016/j.jbiosc.2009.08.004.
[36]CIRULIS J T,STRASSER B C,SCOTT J A,et al.Optimization of staining conditions for microalgae with three lipophilic dyes to reduce precipitation and fluorescence variability[J].Cytometry part A,2012,81A(7):618-626.DOI:10.1002/cyto.a.22066.
[37]KURNIASIH I N,LIANG H,MOHR P C,et al.Nile red dye in aqueous surfactant and micellar solution[J].Langmuir,2015,31(9):2639-2648.DOI:10.1021/la504378m.
[38]FELBECK T,BEHNKE T,HOFFMANN K,et al.Nile-Red-nanoclay hybrids:red emissive optical probes for use in aqueous dispersion[J].Langmuir,2013,29(36):11489-11497.DOI:10.1021/la402165q.