脂肽类生物表面活性剂的发酵优化及其性质和应用研究
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摘要
生物表面活性剂主要是由微生物产生的天然表面活性化合物,可被分为六大类:糖脂类、脂肽类、磷脂类、脂多糖类、脂肪酸、中性脂以及多糖蛋白复合物等。和化学合成表面活性剂一样,具有降低表面张力、稳定乳化液等功能。除此之外,由于其具有生物可降解性;低毒或无毒,可用于医药、化妆品和食品领域;在极端温度、pH、盐浓度下的耐受性和选择性;具有更高的起泡性等;结构功能多样性,在特殊领域具有一定的更好的可适用性。由于这些显著特点,生物表面活性剂的研究日益增多,应用也迅速扩展至环保、石油开采、制药、食品、土壤修复等领域。
本研究以中国科学院南海海洋研究所提供的一株生物表面活性剂产生菌为研究对象,分别从菌株鉴定、发酵条件和产物特性等方面进行了研究,主要结论如下:
1.经菌落、菌体形态和16S rDNA序列分析,鉴定为芽孢杆菌属,命名为Bacillus SCUT09。该菌株以葡萄糖为碳源时培养基表面张力可降至46.2 mN/m,对煤油的乳化能力达到34%。
2. Bacillus SCUT09的培养基优化研究结果表明,最利于Bacillus SCUT09生长和生物表面活性剂积累的培养条件为:木薯淀粉20 g/L,牛肉膏7 g/L,NaCl 10 g/L,MgSO_4·7H_2O 0.7 g/L,Na_2HPO_4·12H_2O 3.8 g/L,酵母提取物0.5 g/L,28oC,初始pH值7.0,接种量2%,转速为200 rpm。优化后以木薯淀粉为碳源时,培养基的表面张力达到28 mN/m,对煤油的乳化能力达到57%。发酵40 h后,菌体生长进入稳定期,菌体干重达1.6 g/L,表面活性剂产量约为0.8 g/L。
3.产物TLC和FTIR分析结果表明,该生物表面活性剂为脂肽类物质,其临界胶束浓度为0.2 g/L;对盐浓度的耐受性为21% NaCl。在pH4-11范围内性质变化不大。样品经121 oC高温处理2 h后的表面活性和乳化能力保持稳定。
4.经重力沉降法分析,脂肽表面活性剂可以作为纳米材料的分散剂。同时利用该脂肽物质经水热法合成的纳米ZnO,经扫描电镜和电子显微镜分析,和利用CTAB合成的纳米ZnO相比,具有更小的粒径和较好的分散稳定性。
Biosurfactants synthesized by microorganisms are one of natural surface-active compounds produced by microorganisms, which could be classified into six groups. Basically there are six major classes of biosurfactants:, glycolipids, lipopeptide, phospholipids, lipopolysaccharides, substituted fatty acids and neutral lipids. Like synthetic surfactants, biosurfactants can reduce the surface tensions of aqueous media. However, biosurfacants have several outstanding advantages, such as higher biodegradability, lower toxicity, higher selectivity and specific activity at extreme temperatures, pH, and salinity, and they also have higher foaming, better environmental compatibility and the ability to be synthesized from renewable feed stocks and so on.
A biosurfactant-producing bacterium provided by South China Sea Institute of Oceanology was studied. The detailed conclusions were as follows:
(1). The strain was classified into Bacillus sp. according to its 16S rDNA sequences and designated as Bacillus SCUT09. The surface tension is lowered to 46.2 mN/m and the emulsification in kerosene-water is 34% when glucose was used as carbon source.
(2). The culture conditions of the strain were primarily optimized. The best carbon and nitrogen source are the cassava starch and beef extract, respectively. Under the conditions of 1% NaCl, pH 6.5 and 28 oC, the biomass and emulsifying ability reach the maximum values.
(3). Fourier transform infrared spectroscopy and thin layer chromatography analysis revealed that SCUT09 produced lipopeptide biosurfactant in the process of metabolism. Its critical micelle concentration (CMC) was 0.2 g/L and its salt tolerance was 21% of NaCl. Also, the biosurfactant has higher stability in the pH range from 4 to 11. Also, the surface tension and the emulsifying ability remained steady after 2 h incubation at 121oC.
(4). Gravity sedimentation analysis results showed that lipopeptide prepared in this study was a good kind of nano-materials dispersant. Compared to CTAB, the size of nano-ZnO particles using lipopeptide prepared in this study as dispersant is smaller by scanning electron microscopy and electron microscopy analysis.
本研究以中国科学院南海海洋研究所提供的一株生物表面活性剂产生菌为研究对象,分别从菌株鉴定、发酵条件和产物特性等方面进行了研究,主要结论如下:
1.经菌落、菌体形态和16S rDNA序列分析,鉴定为芽孢杆菌属,命名为Bacillus SCUT09。该菌株以葡萄糖为碳源时培养基表面张力可降至46.2 mN/m,对煤油的乳化能力达到34%。
2. Bacillus SCUT09的培养基优化研究结果表明,最利于Bacillus SCUT09生长和生物表面活性剂积累的培养条件为:木薯淀粉20 g/L,牛肉膏7 g/L,NaCl 10 g/L,MgSO_4·7H_2O 0.7 g/L,Na_2HPO_4·12H_2O 3.8 g/L,酵母提取物0.5 g/L,28oC,初始pH值7.0,接种量2%,转速为200 rpm。优化后以木薯淀粉为碳源时,培养基的表面张力达到28 mN/m,对煤油的乳化能力达到57%。发酵40 h后,菌体生长进入稳定期,菌体干重达1.6 g/L,表面活性剂产量约为0.8 g/L。
3.产物TLC和FTIR分析结果表明,该生物表面活性剂为脂肽类物质,其临界胶束浓度为0.2 g/L;对盐浓度的耐受性为21% NaCl。在pH4-11范围内性质变化不大。样品经121 oC高温处理2 h后的表面活性和乳化能力保持稳定。
4.经重力沉降法分析,脂肽表面活性剂可以作为纳米材料的分散剂。同时利用该脂肽物质经水热法合成的纳米ZnO,经扫描电镜和电子显微镜分析,和利用CTAB合成的纳米ZnO相比,具有更小的粒径和较好的分散稳定性。
Biosurfactants synthesized by microorganisms are one of natural surface-active compounds produced by microorganisms, which could be classified into six groups. Basically there are six major classes of biosurfactants:, glycolipids, lipopeptide, phospholipids, lipopolysaccharides, substituted fatty acids and neutral lipids. Like synthetic surfactants, biosurfactants can reduce the surface tensions of aqueous media. However, biosurfacants have several outstanding advantages, such as higher biodegradability, lower toxicity, higher selectivity and specific activity at extreme temperatures, pH, and salinity, and they also have higher foaming, better environmental compatibility and the ability to be synthesized from renewable feed stocks and so on.
A biosurfactant-producing bacterium provided by South China Sea Institute of Oceanology was studied. The detailed conclusions were as follows:
(1). The strain was classified into Bacillus sp. according to its 16S rDNA sequences and designated as Bacillus SCUT09. The surface tension is lowered to 46.2 mN/m and the emulsification in kerosene-water is 34% when glucose was used as carbon source.
(2). The culture conditions of the strain were primarily optimized. The best carbon and nitrogen source are the cassava starch and beef extract, respectively. Under the conditions of 1% NaCl, pH 6.5 and 28 oC, the biomass and emulsifying ability reach the maximum values.
(3). Fourier transform infrared spectroscopy and thin layer chromatography analysis revealed that SCUT09 produced lipopeptide biosurfactant in the process of metabolism. Its critical micelle concentration (CMC) was 0.2 g/L and its salt tolerance was 21% of NaCl. Also, the biosurfactant has higher stability in the pH range from 4 to 11. Also, the surface tension and the emulsifying ability remained steady after 2 h incubation at 121oC.
(4). Gravity sedimentation analysis results showed that lipopeptide prepared in this study was a good kind of nano-materials dispersant. Compared to CTAB, the size of nano-ZnO particles using lipopeptide prepared in this study as dispersant is smaller by scanning electron microscopy and electron microscopy analysis.
引文
[1] Muthusamy K., et al. Biosurfactants: Properties, comercial production and application [J]. Current Science, 2008, 94(6): 736-747
[2]李祖义,童正新.生物表面活性剂发酵液的组成及表面活性[J].工业微生物, 2002, 32(002): 1-4
[3] Biria D. Purification and characterization of a novel biosurfactant produced by Bacillus licheniformis MS3 [J]. World Journal of Microbiology & Biotechnology, 2010, 26(5): 871-878
[4] Mukherjee S., Das P., Sen R.. Towards commercial production of microbial surfactants [J]. Trends in Biotechnology, 2006, 24(11): 509-515
[5] Rodrigues L., et al. Biosurfactants: potential applications in medicine [J]. Journal of Antimicrobial Chemotherapy, 2006, 57(4): 609-618
[6] Suresh Kumar, Mody A.K., Jha B.. Evaluation of Biosurfactant/Bioemulsifier Production by a Marine Bacterium [J]. Bulletin of Environmental Contamination and Toxicology, 2007, 79(6): 617-621
[7] Arima K., Kakinuma A., Tamura G. Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation [J]. Bioche-mical and Biophysical Research Communications, 1968, 31(3): 488-&
[8] Calvo C., et al. Application of bioemulsifiers in soil oil bioremediation processes. Future prospects [J]. Science of the Total Environment, 2009, 407(12): 3634-3640
[9] Konishi M., et al. Production of New Types of Sophorolipids by Candida batistae [J]. Journal of Oleo Science, 2008, 57(6): 359-369
[10] Mulligan C.N. Environmental applications for biosurfactants [J]. Environmental Pollution, 2005, 133(2): 183-198
[11] Karanth N.G.K., P.G. Deo, Veenanadig N.K.. Microbial production of biosurfactants and their importance[J]. Current Science, 1999, 77(1): 116-126
[12] Neu T.R., Hfirtner T., Poralla K.. Surface active properties of viscosin: a peptid-olipid antibiotic [J]. Appl Microbiol Biotechnol, 1990, 32: 518-520
[13] Kaeppeli O., Finnerty W.. Characteristics of hexadecane partition by the growth medium of Acinetobacter sp [J]. Biotechnology and Bioengineering, 1980, 22(3):495-503
[14] Belsky I., Gutnick D.L., Rosenberg E.. Emulsifier of Arthrobacter RAG-1:determination of emulsifier-bound fatty acids. [J]. Febs Letters, 1979, 101(1): 175-178
[15] Iqbal S., Khalid Z.M., Malik K.A.. Enhanced biodegradation and emulsification of crude oil and hyperproduction of biosurfactants by a gamma ray-induced mutant of Pseudomonas aeruginosa [J]. Lett Appl Microbiol, 1995, 21(3): 176-179
[16] Dogan I., et al. Expression of Vitreoscilla hemoglobin in Gordonia amarae enhances biosurfactant production [J]. Journal of Industrial Microbiology & Biotechnolo-gy, 2006, 33(8): 693-700
[17] Cooper D., et al. Enhanced production of surfactin from Bacillus subtilis by continuous product removal and metal cation additions [J]. Applied and Environmental Microbiology, 1981, 42(3): 408
[18] Clenns O.N., Cooper D.G.. Effect of substrate on sophorolipid properties [J]. Journal of the American Oil Chemists Society, 2006, 83(2): 137-145
[19] Sobrinho H.B.S., et al. Utilization of two agroindustrial byproducts for the production of a surfactant by Candida sphaerica UCP0995 [J]. Process Biochemistry, 2008, 43(9): 912-917
[20] Dehghan-Noude G., Housaindok M.t, Bazzaz B.. Isolation characterization and investigation of surface and hemolytic activities of a lipopeptide biosurfactant produced by Bacillus subtilis ATCC 6633 [J]. J. Microbiol, 2005, 436: 272
[21]陈云瑛,脂肽生物表面活性剂的制备,理化性质和抑菌活性研究[J]. 2008,中国海洋大学
[22] Abouseoud M., et al. Evaluation of different carbon and nitrogen sources in production of biosurfactant by Pseudomonas fluorescens [J]. Desalination, 2008, 223(1-3): 143-151
[23] Thavasi R., et al. Biosurfactant production by Corynebacterium kutscheri from waste motor lubricant oil and peanut oil cake [J]. Letters in applied microbiology, 2007, 45(6): 686-691
[24]陈静,张云瑞,宋欣.槐糖脂的生产及其应用研究进展[J].食品科学, 2007, 28(008): 525-529
[25] Haddadin M., et al. Kinetics of hydrocarbon extraction from oil shale using biosurfactant producing bacteria [J]. Energy Conversion and Management, 2009, 50(4): 983-990
[26] Mutalik S., et al. Use of response surface optimization for the production of biosurfactant from Rhodococcus spp. MTCC 2574 [J]. Bioresource technology, 2008,99(16): 7875-7880
[27] Ilori M., Amobi C., Odocha A.. Factors affecting biosurfactant production by oil degrading Aeromonas spp. isolated from a tropical environment [J]. Chemosphere, 2005, 61(7): 985-992
[28] Abu-Ruwaida A., et al. Nutritional requirements and growth characteristics of a biosurfactant-producing Rhodococcus bacterium [J]. World Journal of Microbiology and Biotechnology, 1991, 7(1): 53-60
[29] Davis D., Lynch H., Varley J.. The production of surfactin in batch culture by Bacillus subtilis ATCC 21332 is strongly influenced by the conditions of nitrogen metabolism [J]. Enzyme and Microbial Technology, 1999, 25(3-5): 322-329
[30] Abushady H., et al. Molecular Characterization of Bacillus subtilis Surfactin Producing Strain and the Factors Affecting its Production
[31] Martinez A., Soberon-Chavez G.. Characterization of the lipA gene encoding the major lipase from Pseudomonas aeruginosa strain IGB83 [J]. Applied microbiology and biotechnology, 2001, 56(5): 731-735
[32] Oliveira F., Vazquez L., Fran F.. Production of biosurfactants by Pseudomonas alcaligenes using palm oil [J]. 2005
[33]Makkar R.S., Cameotra S.S.. An update on the use of unconventional substrates for biosurfactant production and their new applications [J]. Applied Microbiology and Biotechnology, 2002, 58(4): 428-434
[34] Parawira W., et al. Profile of hydrolases and biogas production during two-stage mesophilic anaerobic digestion of solid potato waste [J]. Process Biochemistry, 2005, 40(9): 2945-2952
[35] Thompson D.N., Fox S.L., Bala C.A.. Biosurfactants from potato process effluents [J]. Applied Biochemistry and Biotechnology, 2000, 84-6: 917-930
[36] Thompson D.N., Fox S.L., Bala G.A.. The effect of pretreatments on surfactin production from potato process effluent by Bacillus subtilis [J]. Applied Biochemistry and Biotechnology, 2001, 91-3: 487-501
[37] Noah K.S., Bruhn D.F., Bala G.A.. Surfactin production from potato process effluent by Bacillus subtilis in a chemostat [J]. Applied Biochemistry and Biotechnology, 2005, 121: 465-473
[38] Noah K.S., et al. Development of continuous surfactin production from potato process effluent by Bacillus subtilis in an airlift reactor [J]. Applied Biochemistry and Biotechnology, 2002, 98: 803-813
[39] Fox S.L., Bala G.A.. Production of surfactant from Bacillus subtilis ATCC 21332 using potato substrates [J]. Bioresource Technology, 2000, 75(3): 235-240
[40] Nitschke M., Pastore G.M.. Production and properties of a surfactant obtained from Bacillus subtilis grown on cassava wastewater [J]. Bioresource Technology, 2006, 97(2): 336-341
[41] Haba E., et al. Screening and production of rhamnolipids by Pseudomonas aeruginosa 47T2 NCIB 40044 from waste frying oils [J]. Journal of Applied Microbiology, 2000, 88(3): 379-387
[42] Nitschke M., et al. Oil wastes as unconventional substrates for rhamnolipid biosurfactant production by Pseudomonas aeruginosa LBI [J]. Biotechnology Progress, 2005, 21(5): 1562-1566
[43] Thavasi R., et al. Biosurfactant production by Corynebacterium kutscheri from waste motor lubricant oil and peanut oil cake [J]. Letters in Applied Microbiology, 2007, 45: 686-691
[44] Trummler K., Effenberger F., Syldatk C.. An integrated microbial/enzymatic process for production of rhamnolipids and L-(+)-rhamnose from rapeseed oil with Pseudomonas sp DSM 2874 [J]. European Journal of Lipid Science and Technology, 2003, 105(10): 563-571
[45] Pekin G., F. Vardar-Sukan N., Kosaric. Production of sophorolipids from Candida bombicola ATCC 22214 using Turkish corn oil and honey [J]. Engineering in Life Sciences, 2005, 5(4): 357-362
[46] Kim H.S., et al. Extracellular production of a glycolipid biosurfactant, mannosylerythritol lipid, by Candida sp SY16 using fed-batch fermentation [J]. Applied Microbiology and Biotechnology, 2006, 70(4): 391-396
[47] Thaniyavarn J., et al. Biosurfactant production by Pseudomonas aeruginosa A41 using palm oil as carbon source [J]. Journal of General and Applied Microbiology, 2006, 52(4): 215-222
[48] Bednarski W., et al. Application of oil refinery waste in the biosynthesis of glycolipids by yeast [J]. Bioresource Technology, 2004, 95(1): 15-18
[49] Benincasa M., et al. Chemical structure, surface properties and biological activities of the biosurfactant produced by Pseudomonas aeruginosa LBI from soapstock [J]. Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, 2004, 85(1): 1-8
[50] Lee H., et al. Production of Ganoderma lucidum mycelium using cheese whey as analternative substrate: response surface analysis and biokinetics [J]. Biochemical Engineering Journal, 2003, 15(2): 93-99
[51] Rodrigues L., et al. Kinetic study of fermentative biosurfactant production by Lactobacillus strains [J]. Biochemical Engineering Journal, 2006, 28(2): 109-116
[52] Dubey K.A., Juwarkar. Distillery and curd whey wastes as viable alternative sources for biosurfactant production [J]. World Journal of Microbiology & Biotechnology, 2001, 17(1): 61-69
[53] Makkar R.S., Cameotra S.S. Utilization of molasses for biosurfactant production by two Bacillus strains at thermophilic conditions [J]. Journal of the American Oil Chemists Society, 1997, 74(7): 887-889
[54] Onbaslil D. B., Aslim. Biosurfactant production in sugar beet molasses by some Pseudomonas spp [J]. Journal of Environmental Biology, 2009. 30(1): 161-163
[55] Joshi S., et al. Biosurfactant production using molasses and whey under thermophilic conditions [J]. Bioresource Technology, 2008, 99(1): 195-199
[56] da Silva, G.P., Mack M., Contiero J.. A promising and abundant carbon source for industrial microbiology[J]. Biotechnology Advances, 2009, 27(1): 30-39
[57] Ciapina E.M.P., et al. Biosurfactant production by Rhodococcus erythropolis grown on glycerol as sole carbon source [J]. Applied Biochemistry and Biotechnology, 2006, 131(1-3): 880-886
[58] Das P., Mukherjee S., Sen R.. Substrate dependent production of extracellular biosurfactant by a marine bacterium [J]. Bioresource Technology, 2009, 100(2): 1015-1019
[59] Sarachat T., et al. Purification and concentration of a rhamnolipid biosurfactant produced by Pseudomonas aeruginosa SP4 using foam fractionation [J]. Bioresource technology, 2010, 101(1): 324-330
[60] Qiao N., Shao Z.. Isolation and characterization of a novel biosurfactant produced by hydrocarbon-degrading bacterium Alcanivorax dieselolei B-5 [J]. Journal of applied microbiology, 2010, 108(4): 1207-1216
[61] Mulligan C.N., Wang S.L.. Remediation of a heavy metal-contaminated soil by a rhamnolipid foam[J]. Engineering Geology, 2006, 85(1-2): 75-81
[62] Syldatk C., et al. Chemical and physical characterization of 4 interfacial active rhamnolpids from Pseudomonas spec DSM 2847 grown on normal alkanes [J]. Zeitschrift Fur Naturforschung C-a Journal of Biosciences, 1985, 40(1-2): 51-60
[63] Edwards J.R., Hayashi J.A.. Structure of a rhamnolipid from Pseudomonasaeruginosa [J]. Archives of Biochemistry and Biophysics, 1965, 111(2): 415-418
[64] Ashby R.D., Solaiman D.K.Y., Foglia T.A.. The use of fatty acid esters to enhance free acid sophorolipid synthesis [J]. Biotechnology Letters, 2006, 28(4): 253-260
[65] Hu Y.M.,. Ju L.K. Purification of lactonic sophorolipids by crystallization [J]. Journal of Biotechnology, 2001, 87(3): 263-272
[66] Desai J.D., Banat I.M.. Microbial production of surfactants and their commercial potential [J]. Microbiology and Molecular Biology Reviews, 1997, 61(1): 47-&.
[67] Morita T., et al. Production of a novel glycolipid biosurfactant, mannosylmannitol lipid, by Pseudozyma parantarctica and its interfacial properties [J]. Applied microbiology and biotechnology, 2009, 83(6): 1017-1025
[68]刘飞,田延军.脂肽类生物表面活性剂产生及鉴定[J].山东食品发酵, 2009, 4(155): 3-7
[69]马歌丽.生物表面活性剂及其应用[J].中国生物工程杂志, 2003, 23(005): 42-45
[70] Helmy Q.,. Kardena E, Nurachman Z.. Application of Biosurfactant Produced by Azotobacter vinelandii AV01 for Enhanced Oil Recovery and Biodegradation of Oil Sludge. 2010
[71] Salehizade H., Mohammadizad S.. Microbial enhanced oil recovery using biosurfactant produced by Alcaligenes faecalis [J]. Iranian journal of Biotechnology, 2009, 7(4): 216-223
[72] Zhang H., et al. Biotreatment of oily wastewater by rhamnolipids in aerated active sludge system [J]. Journal of Zhejiang University-Science B, 2009, 10(11): 852-859
[73] Torrens J.L., Herman D.C., Miller-Maier R.M.. Biosurfactant (rhamnolipid) sorption and the impact on rhamnolipid-facilitated removal of cadmium from various soils under saturated flow conditions [J]. Environmental science & technology, 1998, 32(6): 776-781
[74] Mulligan C.N., et al. Metal removal from contaminated soil and sediments by the biosurfactant surfactin [J]. Environmental science & technology, 1999, 33(21): 3812-3820
[75] Mulligan C.N., Yong R.N., Gibbs B.F.. Heavy metal removal from sediments by biosurfactants [J]. Journal of hazardous materials, 2001, 85(1-2): 111-125
[76]候万国,孙德军,张春光.应用胶体化学[M]. 1998,北京:科学出版社
[77] Velraeds M., et al. Inhibition of initial adhesion of uropathogenic Enterococcus faecalis by biosurfactants from Lactobacillus isolates [J].Applied and EnvironmentalMicrobiology, 1996, 62(6): 1958
[78] Shepherd R., et al. Novel bioemulsifiers from microorganisms for use in foods [J]. Journal of Biotechnology, 1995, 40(3): 207-217
[79]凌关庭.天然食品添加剂手册[M]. 2009:化学工业出版社
[80]梅建凤,闵航.生物表面活性剂及其应用[J]. Industrial Microbiology, 2001, 31(1): 41-45
[81]夏咏梅,方云.生物表面活性剂的开发和应用[J].日用化学工业, 1999(001): 27-31
[82]吴清平,姚汝华.表面活性素的发酵生产及应用[J].微生物学通报, 1999, 26 (001): 74-76
[83] Peypoux F., J. Bonmatin, J. Wallach. Recent trends in the biochemistry of surfactin [J]. Applied microbiology and biotechnology, 1999, 51(5): 553-563
[84] Uchida Y., et al. Factors affecting the production of succinoyl trehalose lipids by Rhodococcus erythropolis SD-74 grown on n-alkanes[J]. Agricultural and biological chemistry, 1989, 53(3): 765-769
[85] Kitamoto D., et al. Surface active properties and antimicrobial activities of mannosylerythritol lipids as biosurfactants produced by Candida Antarctica [J]. Journal of Biotechnology, 1993, 29(1-2): 91-96
[86] White R.T., et al. Isolation and characterization of the human pulmonary surfactant apoprotein gene [M]. 1985
[87] Yamane T. Enzyme technology for the lipids industry: an engineering overview [J]. Journal of the American Oil Chemists' Society, 1987, 64(12): 1657-1662
[88] Inoue, S., Y. Kimwa, M. Kinta. German Patent 2905252. Kao Soap Co., Japan, 1979
[89] Thompson J.D., Higgins D.G.,. Gibson T.J. Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice [J]. Nucleic Acids Research, 1994, 22(22): 4673-4680
[90] Tamura K., et al. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0 [J]. Molecular Biology and Evolution, 2007, 24(8): 1596-1599
[91]孙燕,洪青,李顺鹏.一株生物表面活性剂产生菌的分离及其特性研究[J].微生物学通报, 2009(008): 1110-1116
[92] Ghojavand H., et al. Isolation of thermotolerant, halotolerant, facultative biosurfactant-producing bacteria [J]. Applied Microbiology and Biotechnology, 2008,80(6): 1073-1085
[93] Das P., Mukherjee S., Sen R.. Improved bioavailability and biodegradation of model polyaromatic hydrocarbon by a biosurfactant producing bacterium of marine origin [J]. Chemosphere, 2008, 72(9): 1229-1234
[94] Pansiripat S., et al. Biosurfactant production by Pseudomonas aeruginosa SP4 using sequencing batch reactors: Effect of oil-to-glucose ratio [J]. Biochemical Engineering Journal, 2010, 49(2): 185-191
[95] Yin H., et al. Characteristics of biosurfactant produced by Pseudomonas aeruginosa S6 isolated from oil-containing wastewater [J]. Process Biochemistry, 2009, 44(3): 302-308
[96] Wattanaphon H.T., et al. A biosurfactant from Burkholderia cenocepacia BSP3 and its enhancement of pesticide solubilization [J]. Journal of Applied Microbiology, 2008, 105(2): 416-423
[97] Barros F.F.C., Ponezi A.N., Pastore G.M.. Production of biosurfactant by Bacillus subtilis LB5a on a pilot scale using cassava wastewater as substrate [J]. Journal of Industrial Microbiology & Biotechnology, 2008, 35(9): 1071-1078
[98] Abouseoud M., et al. Biosurfactant production by free and alginate entrapped cells of Pseudomonas fluorescens [J]. Journal of Industrial Microbiology and Biotechnology, 2008, 35(11): 1303-1308
[99] Fukuoka T., et al. Structural characterization and surface-active properties of a new glycolipid biosurfactant, mono-acylated mannosylerythritol lipid, produced from glucose by Pseudozyma antarctica [J]. Applied microbiology and biotechnology, 2007, 76(4): 801-810
[100]李国龙,吴勘,谭镜明.纳米在水基体系中分散的研究[J].现代涂料与涂装,2006,11:31-34
[101]孙秀果,张建民.分散剂对纳米二氧化钛粉体分散性的影响[J].半导体技术,2006,31(5):363-366
[102]沈勇,秦伟庭,张慧芳等.改性纳米二氧化钛的性能研究[J].印染,2006,10:8-10
[2]李祖义,童正新.生物表面活性剂发酵液的组成及表面活性[J].工业微生物, 2002, 32(002): 1-4
[3] Biria D. Purification and characterization of a novel biosurfactant produced by Bacillus licheniformis MS3 [J]. World Journal of Microbiology & Biotechnology, 2010, 26(5): 871-878
[4] Mukherjee S., Das P., Sen R.. Towards commercial production of microbial surfactants [J]. Trends in Biotechnology, 2006, 24(11): 509-515
[5] Rodrigues L., et al. Biosurfactants: potential applications in medicine [J]. Journal of Antimicrobial Chemotherapy, 2006, 57(4): 609-618
[6] Suresh Kumar, Mody A.K., Jha B.. Evaluation of Biosurfactant/Bioemulsifier Production by a Marine Bacterium [J]. Bulletin of Environmental Contamination and Toxicology, 2007, 79(6): 617-621
[7] Arima K., Kakinuma A., Tamura G. Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation [J]. Bioche-mical and Biophysical Research Communications, 1968, 31(3): 488-&
[8] Calvo C., et al. Application of bioemulsifiers in soil oil bioremediation processes. Future prospects [J]. Science of the Total Environment, 2009, 407(12): 3634-3640
[9] Konishi M., et al. Production of New Types of Sophorolipids by Candida batistae [J]. Journal of Oleo Science, 2008, 57(6): 359-369
[10] Mulligan C.N. Environmental applications for biosurfactants [J]. Environmental Pollution, 2005, 133(2): 183-198
[11] Karanth N.G.K., P.G. Deo, Veenanadig N.K.. Microbial production of biosurfactants and their importance[J]. Current Science, 1999, 77(1): 116-126
[12] Neu T.R., Hfirtner T., Poralla K.. Surface active properties of viscosin: a peptid-olipid antibiotic [J]. Appl Microbiol Biotechnol, 1990, 32: 518-520
[13] Kaeppeli O., Finnerty W.. Characteristics of hexadecane partition by the growth medium of Acinetobacter sp [J]. Biotechnology and Bioengineering, 1980, 22(3):495-503
[14] Belsky I., Gutnick D.L., Rosenberg E.. Emulsifier of Arthrobacter RAG-1:determination of emulsifier-bound fatty acids. [J]. Febs Letters, 1979, 101(1): 175-178
[15] Iqbal S., Khalid Z.M., Malik K.A.. Enhanced biodegradation and emulsification of crude oil and hyperproduction of biosurfactants by a gamma ray-induced mutant of Pseudomonas aeruginosa [J]. Lett Appl Microbiol, 1995, 21(3): 176-179
[16] Dogan I., et al. Expression of Vitreoscilla hemoglobin in Gordonia amarae enhances biosurfactant production [J]. Journal of Industrial Microbiology & Biotechnolo-gy, 2006, 33(8): 693-700
[17] Cooper D., et al. Enhanced production of surfactin from Bacillus subtilis by continuous product removal and metal cation additions [J]. Applied and Environmental Microbiology, 1981, 42(3): 408
[18] Clenns O.N., Cooper D.G.. Effect of substrate on sophorolipid properties [J]. Journal of the American Oil Chemists Society, 2006, 83(2): 137-145
[19] Sobrinho H.B.S., et al. Utilization of two agroindustrial byproducts for the production of a surfactant by Candida sphaerica UCP0995 [J]. Process Biochemistry, 2008, 43(9): 912-917
[20] Dehghan-Noude G., Housaindok M.t, Bazzaz B.. Isolation characterization and investigation of surface and hemolytic activities of a lipopeptide biosurfactant produced by Bacillus subtilis ATCC 6633 [J]. J. Microbiol, 2005, 436: 272
[21]陈云瑛,脂肽生物表面活性剂的制备,理化性质和抑菌活性研究[J]. 2008,中国海洋大学
[22] Abouseoud M., et al. Evaluation of different carbon and nitrogen sources in production of biosurfactant by Pseudomonas fluorescens [J]. Desalination, 2008, 223(1-3): 143-151
[23] Thavasi R., et al. Biosurfactant production by Corynebacterium kutscheri from waste motor lubricant oil and peanut oil cake [J]. Letters in applied microbiology, 2007, 45(6): 686-691
[24]陈静,张云瑞,宋欣.槐糖脂的生产及其应用研究进展[J].食品科学, 2007, 28(008): 525-529
[25] Haddadin M., et al. Kinetics of hydrocarbon extraction from oil shale using biosurfactant producing bacteria [J]. Energy Conversion and Management, 2009, 50(4): 983-990
[26] Mutalik S., et al. Use of response surface optimization for the production of biosurfactant from Rhodococcus spp. MTCC 2574 [J]. Bioresource technology, 2008,99(16): 7875-7880
[27] Ilori M., Amobi C., Odocha A.. Factors affecting biosurfactant production by oil degrading Aeromonas spp. isolated from a tropical environment [J]. Chemosphere, 2005, 61(7): 985-992
[28] Abu-Ruwaida A., et al. Nutritional requirements and growth characteristics of a biosurfactant-producing Rhodococcus bacterium [J]. World Journal of Microbiology and Biotechnology, 1991, 7(1): 53-60
[29] Davis D., Lynch H., Varley J.. The production of surfactin in batch culture by Bacillus subtilis ATCC 21332 is strongly influenced by the conditions of nitrogen metabolism [J]. Enzyme and Microbial Technology, 1999, 25(3-5): 322-329
[30] Abushady H., et al. Molecular Characterization of Bacillus subtilis Surfactin Producing Strain and the Factors Affecting its Production
[31] Martinez A., Soberon-Chavez G.. Characterization of the lipA gene encoding the major lipase from Pseudomonas aeruginosa strain IGB83 [J]. Applied microbiology and biotechnology, 2001, 56(5): 731-735
[32] Oliveira F., Vazquez L., Fran F.. Production of biosurfactants by Pseudomonas alcaligenes using palm oil [J]. 2005
[33]Makkar R.S., Cameotra S.S.. An update on the use of unconventional substrates for biosurfactant production and their new applications [J]. Applied Microbiology and Biotechnology, 2002, 58(4): 428-434
[34] Parawira W., et al. Profile of hydrolases and biogas production during two-stage mesophilic anaerobic digestion of solid potato waste [J]. Process Biochemistry, 2005, 40(9): 2945-2952
[35] Thompson D.N., Fox S.L., Bala C.A.. Biosurfactants from potato process effluents [J]. Applied Biochemistry and Biotechnology, 2000, 84-6: 917-930
[36] Thompson D.N., Fox S.L., Bala G.A.. The effect of pretreatments on surfactin production from potato process effluent by Bacillus subtilis [J]. Applied Biochemistry and Biotechnology, 2001, 91-3: 487-501
[37] Noah K.S., Bruhn D.F., Bala G.A.. Surfactin production from potato process effluent by Bacillus subtilis in a chemostat [J]. Applied Biochemistry and Biotechnology, 2005, 121: 465-473
[38] Noah K.S., et al. Development of continuous surfactin production from potato process effluent by Bacillus subtilis in an airlift reactor [J]. Applied Biochemistry and Biotechnology, 2002, 98: 803-813
[39] Fox S.L., Bala G.A.. Production of surfactant from Bacillus subtilis ATCC 21332 using potato substrates [J]. Bioresource Technology, 2000, 75(3): 235-240
[40] Nitschke M., Pastore G.M.. Production and properties of a surfactant obtained from Bacillus subtilis grown on cassava wastewater [J]. Bioresource Technology, 2006, 97(2): 336-341
[41] Haba E., et al. Screening and production of rhamnolipids by Pseudomonas aeruginosa 47T2 NCIB 40044 from waste frying oils [J]. Journal of Applied Microbiology, 2000, 88(3): 379-387
[42] Nitschke M., et al. Oil wastes as unconventional substrates for rhamnolipid biosurfactant production by Pseudomonas aeruginosa LBI [J]. Biotechnology Progress, 2005, 21(5): 1562-1566
[43] Thavasi R., et al. Biosurfactant production by Corynebacterium kutscheri from waste motor lubricant oil and peanut oil cake [J]. Letters in Applied Microbiology, 2007, 45: 686-691
[44] Trummler K., Effenberger F., Syldatk C.. An integrated microbial/enzymatic process for production of rhamnolipids and L-(+)-rhamnose from rapeseed oil with Pseudomonas sp DSM 2874 [J]. European Journal of Lipid Science and Technology, 2003, 105(10): 563-571
[45] Pekin G., F. Vardar-Sukan N., Kosaric. Production of sophorolipids from Candida bombicola ATCC 22214 using Turkish corn oil and honey [J]. Engineering in Life Sciences, 2005, 5(4): 357-362
[46] Kim H.S., et al. Extracellular production of a glycolipid biosurfactant, mannosylerythritol lipid, by Candida sp SY16 using fed-batch fermentation [J]. Applied Microbiology and Biotechnology, 2006, 70(4): 391-396
[47] Thaniyavarn J., et al. Biosurfactant production by Pseudomonas aeruginosa A41 using palm oil as carbon source [J]. Journal of General and Applied Microbiology, 2006, 52(4): 215-222
[48] Bednarski W., et al. Application of oil refinery waste in the biosynthesis of glycolipids by yeast [J]. Bioresource Technology, 2004, 95(1): 15-18
[49] Benincasa M., et al. Chemical structure, surface properties and biological activities of the biosurfactant produced by Pseudomonas aeruginosa LBI from soapstock [J]. Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, 2004, 85(1): 1-8
[50] Lee H., et al. Production of Ganoderma lucidum mycelium using cheese whey as analternative substrate: response surface analysis and biokinetics [J]. Biochemical Engineering Journal, 2003, 15(2): 93-99
[51] Rodrigues L., et al. Kinetic study of fermentative biosurfactant production by Lactobacillus strains [J]. Biochemical Engineering Journal, 2006, 28(2): 109-116
[52] Dubey K.A., Juwarkar. Distillery and curd whey wastes as viable alternative sources for biosurfactant production [J]. World Journal of Microbiology & Biotechnology, 2001, 17(1): 61-69
[53] Makkar R.S., Cameotra S.S. Utilization of molasses for biosurfactant production by two Bacillus strains at thermophilic conditions [J]. Journal of the American Oil Chemists Society, 1997, 74(7): 887-889
[54] Onbaslil D. B., Aslim. Biosurfactant production in sugar beet molasses by some Pseudomonas spp [J]. Journal of Environmental Biology, 2009. 30(1): 161-163
[55] Joshi S., et al. Biosurfactant production using molasses and whey under thermophilic conditions [J]. Bioresource Technology, 2008, 99(1): 195-199
[56] da Silva, G.P., Mack M., Contiero J.. A promising and abundant carbon source for industrial microbiology[J]. Biotechnology Advances, 2009, 27(1): 30-39
[57] Ciapina E.M.P., et al. Biosurfactant production by Rhodococcus erythropolis grown on glycerol as sole carbon source [J]. Applied Biochemistry and Biotechnology, 2006, 131(1-3): 880-886
[58] Das P., Mukherjee S., Sen R.. Substrate dependent production of extracellular biosurfactant by a marine bacterium [J]. Bioresource Technology, 2009, 100(2): 1015-1019
[59] Sarachat T., et al. Purification and concentration of a rhamnolipid biosurfactant produced by Pseudomonas aeruginosa SP4 using foam fractionation [J]. Bioresource technology, 2010, 101(1): 324-330
[60] Qiao N., Shao Z.. Isolation and characterization of a novel biosurfactant produced by hydrocarbon-degrading bacterium Alcanivorax dieselolei B-5 [J]. Journal of applied microbiology, 2010, 108(4): 1207-1216
[61] Mulligan C.N., Wang S.L.. Remediation of a heavy metal-contaminated soil by a rhamnolipid foam[J]. Engineering Geology, 2006, 85(1-2): 75-81
[62] Syldatk C., et al. Chemical and physical characterization of 4 interfacial active rhamnolpids from Pseudomonas spec DSM 2847 grown on normal alkanes [J]. Zeitschrift Fur Naturforschung C-a Journal of Biosciences, 1985, 40(1-2): 51-60
[63] Edwards J.R., Hayashi J.A.. Structure of a rhamnolipid from Pseudomonasaeruginosa [J]. Archives of Biochemistry and Biophysics, 1965, 111(2): 415-418
[64] Ashby R.D., Solaiman D.K.Y., Foglia T.A.. The use of fatty acid esters to enhance free acid sophorolipid synthesis [J]. Biotechnology Letters, 2006, 28(4): 253-260
[65] Hu Y.M.,. Ju L.K. Purification of lactonic sophorolipids by crystallization [J]. Journal of Biotechnology, 2001, 87(3): 263-272
[66] Desai J.D., Banat I.M.. Microbial production of surfactants and their commercial potential [J]. Microbiology and Molecular Biology Reviews, 1997, 61(1): 47-&.
[67] Morita T., et al. Production of a novel glycolipid biosurfactant, mannosylmannitol lipid, by Pseudozyma parantarctica and its interfacial properties [J]. Applied microbiology and biotechnology, 2009, 83(6): 1017-1025
[68]刘飞,田延军.脂肽类生物表面活性剂产生及鉴定[J].山东食品发酵, 2009, 4(155): 3-7
[69]马歌丽.生物表面活性剂及其应用[J].中国生物工程杂志, 2003, 23(005): 42-45
[70] Helmy Q.,. Kardena E, Nurachman Z.. Application of Biosurfactant Produced by Azotobacter vinelandii AV01 for Enhanced Oil Recovery and Biodegradation of Oil Sludge. 2010
[71] Salehizade H., Mohammadizad S.. Microbial enhanced oil recovery using biosurfactant produced by Alcaligenes faecalis [J]. Iranian journal of Biotechnology, 2009, 7(4): 216-223
[72] Zhang H., et al. Biotreatment of oily wastewater by rhamnolipids in aerated active sludge system [J]. Journal of Zhejiang University-Science B, 2009, 10(11): 852-859
[73] Torrens J.L., Herman D.C., Miller-Maier R.M.. Biosurfactant (rhamnolipid) sorption and the impact on rhamnolipid-facilitated removal of cadmium from various soils under saturated flow conditions [J]. Environmental science & technology, 1998, 32(6): 776-781
[74] Mulligan C.N., et al. Metal removal from contaminated soil and sediments by the biosurfactant surfactin [J]. Environmental science & technology, 1999, 33(21): 3812-3820
[75] Mulligan C.N., Yong R.N., Gibbs B.F.. Heavy metal removal from sediments by biosurfactants [J]. Journal of hazardous materials, 2001, 85(1-2): 111-125
[76]候万国,孙德军,张春光.应用胶体化学[M]. 1998,北京:科学出版社
[77] Velraeds M., et al. Inhibition of initial adhesion of uropathogenic Enterococcus faecalis by biosurfactants from Lactobacillus isolates [J].Applied and EnvironmentalMicrobiology, 1996, 62(6): 1958
[78] Shepherd R., et al. Novel bioemulsifiers from microorganisms for use in foods [J]. Journal of Biotechnology, 1995, 40(3): 207-217
[79]凌关庭.天然食品添加剂手册[M]. 2009:化学工业出版社
[80]梅建凤,闵航.生物表面活性剂及其应用[J]. Industrial Microbiology, 2001, 31(1): 41-45
[81]夏咏梅,方云.生物表面活性剂的开发和应用[J].日用化学工业, 1999(001): 27-31
[82]吴清平,姚汝华.表面活性素的发酵生产及应用[J].微生物学通报, 1999, 26 (001): 74-76
[83] Peypoux F., J. Bonmatin, J. Wallach. Recent trends in the biochemistry of surfactin [J]. Applied microbiology and biotechnology, 1999, 51(5): 553-563
[84] Uchida Y., et al. Factors affecting the production of succinoyl trehalose lipids by Rhodococcus erythropolis SD-74 grown on n-alkanes[J]. Agricultural and biological chemistry, 1989, 53(3): 765-769
[85] Kitamoto D., et al. Surface active properties and antimicrobial activities of mannosylerythritol lipids as biosurfactants produced by Candida Antarctica [J]. Journal of Biotechnology, 1993, 29(1-2): 91-96
[86] White R.T., et al. Isolation and characterization of the human pulmonary surfactant apoprotein gene [M]. 1985
[87] Yamane T. Enzyme technology for the lipids industry: an engineering overview [J]. Journal of the American Oil Chemists' Society, 1987, 64(12): 1657-1662
[88] Inoue, S., Y. Kimwa, M. Kinta. German Patent 2905252. Kao Soap Co., Japan, 1979
[89] Thompson J.D., Higgins D.G.,. Gibson T.J. Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice [J]. Nucleic Acids Research, 1994, 22(22): 4673-4680
[90] Tamura K., et al. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0 [J]. Molecular Biology and Evolution, 2007, 24(8): 1596-1599
[91]孙燕,洪青,李顺鹏.一株生物表面活性剂产生菌的分离及其特性研究[J].微生物学通报, 2009(008): 1110-1116
[92] Ghojavand H., et al. Isolation of thermotolerant, halotolerant, facultative biosurfactant-producing bacteria [J]. Applied Microbiology and Biotechnology, 2008,80(6): 1073-1085
[93] Das P., Mukherjee S., Sen R.. Improved bioavailability and biodegradation of model polyaromatic hydrocarbon by a biosurfactant producing bacterium of marine origin [J]. Chemosphere, 2008, 72(9): 1229-1234
[94] Pansiripat S., et al. Biosurfactant production by Pseudomonas aeruginosa SP4 using sequencing batch reactors: Effect of oil-to-glucose ratio [J]. Biochemical Engineering Journal, 2010, 49(2): 185-191
[95] Yin H., et al. Characteristics of biosurfactant produced by Pseudomonas aeruginosa S6 isolated from oil-containing wastewater [J]. Process Biochemistry, 2009, 44(3): 302-308
[96] Wattanaphon H.T., et al. A biosurfactant from Burkholderia cenocepacia BSP3 and its enhancement of pesticide solubilization [J]. Journal of Applied Microbiology, 2008, 105(2): 416-423
[97] Barros F.F.C., Ponezi A.N., Pastore G.M.. Production of biosurfactant by Bacillus subtilis LB5a on a pilot scale using cassava wastewater as substrate [J]. Journal of Industrial Microbiology & Biotechnology, 2008, 35(9): 1071-1078
[98] Abouseoud M., et al. Biosurfactant production by free and alginate entrapped cells of Pseudomonas fluorescens [J]. Journal of Industrial Microbiology and Biotechnology, 2008, 35(11): 1303-1308
[99] Fukuoka T., et al. Structural characterization and surface-active properties of a new glycolipid biosurfactant, mono-acylated mannosylerythritol lipid, produced from glucose by Pseudozyma antarctica [J]. Applied microbiology and biotechnology, 2007, 76(4): 801-810
[100]李国龙,吴勘,谭镜明.纳米在水基体系中分散的研究[J].现代涂料与涂装,2006,11:31-34
[101]孙秀果,张建民.分散剂对纳米二氧化钛粉体分散性的影响[J].半导体技术,2006,31(5):363-366
[102]沈勇,秦伟庭,张慧芳等.改性纳米二氧化钛的性能研究[J].印染,2006,10:8-10