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Biosurfactant 姓名：吕梅玲 学号：SY1227111 化学与环境学院 导师：江雷第1页Introduction Biosurfactants(amphiphilic compounds)(synthesized by microorganisms)hydrophobic(nonpolar)hydrophilic(polar)that confer ability to accumulate between fluid phases such as oil/water or air/water,reducing the surface and interfacial tensions and forming emulsions.household、industry and agriculturemono-,oligo-or polysaccharides(多糖）,peptides（缩氨酸）or proteinssaturated,unsaturated and hydroxylated fatty acids or fatty alcohols第2页Pseudomonas aeruginosa（绿脓假单胞菌）（绿脓假单胞菌）Acinetobacter calcoaceticus（醋酸钙不动杆菌）（醋酸钙不动杆菌）emulsan（乳化剂）（乳化剂）Candida bombicola（假丝酵母菌）（假丝酵母菌）sophorolipids（槐糖脂）（槐糖脂）(Originally)hydrocarbons dissolution agentsfood,pharmaceutical and oil industry(recently)applicationenvironmental friendlyeasily biodegradablelow toxicityunique structuresRhamnolipids(鼠李糖脂）鼠李糖脂）第3页.Classification Biosurfactants are categorized by their chemical composition,molecular weight,physico-chemical properties and mode of action and microbial origin.Based on molecular weight they are divided into low-molecular-mass biosurfactants and into high-molecular-mass biosurfactants.第4页 One of the best-studied glycolipids is rhamnolipid,produced by several species of P.seudomonads（假单胞菌）,which consists of two moles of rhamnose and two moles of-hydroxydecanoic acid.The low molecular weight biosurfactants are generally glycolipids in which carbohydrates are attached to a long-chain aliphatic acid(脂肪酸）or lipopeptides（脂肽）.Glycolipid bioemulsifiers,such as rhamnolipids（鼠李糖脂）,trehaloselipids（海藻糖脂）and sophorolipids（槐糖脂）,are disaccharides（二糖）that are acylated with long-chain fatty acids or hydroxy fatty acids.第5页The high molecular weight surfactants are less effective in reducing interfacial tension,but are efficient at coating the oil droplets and preventing their coalescence.These are highly efficient emulsifiers that work at low concentrations(0.01%0.001%),representing emulsifier-to-hydrocarbon ratios of 1:1001:1000.The high molecular weight bacterial surfactants are produced by a large number of bacterial species from different genera and are composed of polysaccharides（多糖）,proteins,lipopolysaccharides（脂多糖）,lipoproteins（脂蛋白）or complex mixtures of these biopolymers.第6页 others can also emulsify pure hydrocarbons but only of a high molecular weight.The best-studied biosurfactants are the bioemulsans produced by different species of Acinetobacter.These high molecular weight bioemulsifiers exhibit considerable substrate specificity.For example,some emulsify efficiently mixtures of aliphatic and aromatic (or cyclic alkane)hydrocarbons,but will not emulsify pure aliphatic,aromatic or cyclic hydrocarbons;第7页Based on the microbial origin,the major class of biosurfactant include:Glycolipids(糖脂）Rhamnolipids（鼠李糖脂）Trehalolipids(海藻糖脂）Sophorolipids（槐糖脂）Lipopeptide and Lipoprotein（脂肽和脂蛋白）Fatty Acids,Phospholipids（磷脂质),and Neutral LipidsPolymeric BiosurfactantsParticulate Biosurfactant第8页PropertiesSurface and interface activitybiodegradability(bioremediation).emulsifying and demulsifying abilityantimicrobial activity第9页Surface and interface activity In general,biosurfactants are more effective and efficient and their CMC(critical micelle concentration)is about 10-40 times lower than chemical surfactants,i.e.,less surfactant is necessary to get a maximal decrease on ST.A good surfactant can lower surface tension(ST)of water from 72 to 35 mN/m and the interfacial tension(IT)water/hexadecane（十六烷）from 40 to 1 mN/m.第10页The biosurfactants accumulate at the interface between two immiscible fluids or between a fluid and a solid.(figure 1)By reducing surface(liquid-air)and interfacial(liquid-liquid)tension they reduce the repulsive forces between two dissimilar phases and allow these two phases to mix and interact more easily第11页Biosurfactant activities depend on the concentration of the surface-active compounds until the critical micelle concentration(CMC)is obtained.At concentrations above the CMC,biosurfactant molecules associate to form micelles,bilayers and vesicles(Figure 2).The CMC is commonly used to measure the efficiency of surfactant.Efficient biosurfactants have a low CMC,which means that less biosurfactant is required to decrease the surface tension.第12页 The biosurfactant effectiveness is determined by measuring its ability to change surface and interfacial tensions,stabilization of emulsions and by studying its hydrophilic-lipophilic balance(HLB).Emulsifiers with low HLB are lipophilic and stabilize water-in-oil emulsification,whereas emulsifiers with high HLB have the opposite effect and confer better water solubility 第13页Low toxicity For example:A biosurfactant from P.aeruginosa（绿脓假单胞菌）（was compared with a synthetic surfactant(Marlon A-350)widely used in industry in terms of toxicity and mutagenic properties.Both assays indicated the higher toxicity and mutagenic effect of the chemical-derived surfactant whereas biosurfactant was considered slightly to non-toxic and non-mutagenic.low or non-toxic products and therefore,appropriate for pharmaceutical,cosmetic and food uses.第14页Emulsion forming and emulsion breaking An emulsion is a heterogeneous system,consisting of at least one immiscible liquid intimately dispersed in another in the form of droplets,whose diameter in general exceeds 0.1 m.Emulsions have an internal or dispersed and an external or continuous phase,so there are generally two types:oil-in-water(o/w)or water-in-oil(w/o)emulsions.Such systems possess a minimal stability,which may be accentuated by additives such as surface-active agents(surfactants).Thus,stable emulsions can be produced with a life span of months and years.Biosurfactants may stabilize(emulsifiers)or destabilize(de-emulsifiers)the emulsion.High-molecular-mass biosurfactants are in general better emulsifiers than low-molecular-mass biosurfactants.第15页Antimicrobial activity A significative reduction on the mycoflora（真菌群）present in stored grains of corn,peanuts and cottonseeds was observed at iturin concentration of 50-100 ppm).Inactivation of enveloped virus such as herpes（疱疹）and retrovirus（逆转录酶病毒）was observed with 80 m M of surfactin（脂肽）.Several biosurfactants have shown antimicrobial action against bacteria,fungi（真菌）,algae（藻类）and viruses.The lipopeptide iturin（脂肽伊枯草菌素）from B.subtilis（枯草芽孢杆菌）showed potent antifungal activity.第16页Production Biosurfactants do not compete economically with synthetic surfactants.To reduce production costs,other carbon sources,such as olive oil（橄榄油），mill effluent（工厂废水）,whey（乳清）from cheese making,and cassava flour water（木薯粉水）,used vegetable oils,molasses（糖浆）(by-product).Biosurfactants have been synthesized by various researchers using different microorganisms and carbon sources.The carbon sources used for biosurfactant production are hydrocarbons,carbohydrates,and vegetable oils.第17页Production methods Natural biological extract method、Microorganism fermentation method、Enzyme catalytic methodPurification methodsPrecipitation method、Extraction method、Superfiltering method、Foam separation method、Adsorption method、Column chromatography method、Thin layer chromatography method、High performance liquid chromatography method、Liquid surface adsorption enrichment method、Liquid membrane separation method.第18页Fig.2.Structure of four different rhamnolipids produced by P.aeruginosa.第19页第20页Factors Affecting Biosurfactant Production Effect of Carbon Source on Biosurfactants ProductionEffect of Carbon Source on Biosurfactants Production carbohydrate hydrocarbonvegetable oils第21页Effect of Support Material and Relationship with Water Support material for immobilized enzyme affects the water content in the proximity of the enzyme and the partitioning of reactants and/or products in the reaction mixture.Since thermal stability is closely related to the amount of water in close vicinity of the enzymes molecule.The ideal carrier should not retain water than necessary to reduce the risk of enzyme denaturation（变性）.Immobilization on hydrophilic supports often leads to a loss of lipase（脂肪酶）activity as the enzyme undergoes a conformational（构象）change to a form of reduced activity.These support materials may also reduce hydrophobic substrate solubility in hydrophilic regions,thereby reducing the accessibility of substrate to the active sites.第22页Effect of Environmental Factors on Biosurfactants Production pHtemperatureagitation（搅拌）speedoxygen availabilityRhamnolipid production in pseudomonas sp（假单胞菌）was its maximum at a pH range from 6 to 6.5 and decreased sharply above pH 7.In addition,surface tension and CMCs of a biosurfactant product remained stable over a wide range of pH values,whereas emulsification had a narrower pH range.第23页 A thermophilic Bacillus sp（芽孢杆菌）grew and produced biosurfactant at temperature above 40 .Heat treatment of some biosurfactants caused no appreciable change in biosurfactant properties,such as the lowering of the surface tension and interfacial tension and the emulsification efficiency,all of that remained stable after autoclaving（高压灭菌）at 120 for 15 min.An increase in agitation speed result in the reduction of biosurfactant yield due to the effect of shear in Nocardia（土壤丝菌属）.On other hand,in yeast,biosurfactant production increases when the agitation and aeration rates are increased.Salt concentration also affected biosurfactant production depending on its effects on cellular activity.Some biosurfactant products,however,were not affected by salt concentrations up to 10%(wt/vol),although slight reduction in the CMCs were detected.第24页 a modified drop-collapse technique for surfactant quantitation and screening of biosurfactant producing microorganismsQualitative drop-collapse testA drop of water applied to a hydrophobic surface in the absence of surfactantswill form a bead,as shown in Fig.1(A).The bead forms because the polar water molecules are repelled from the hydrophobic surface.In contrast,if the water droplet contains surfactant,the force or interfacial tension between the water drop and the hydrophobic surface is reduced,which results in the spreading of the water drop over the hydrophobic surface(Fig.1,B).The amount of surfactant required to cause drop-collapse is dependent on the ability of the surfactant to reduce surface and interfacial tension.The more potent the surfactant,the smaller the quantity that can be detected.(A)Water control(no surfactant),(B)1000 mg/L rhamnolipid.第25页surfactant quantitation by the drop-collapseQuantitative drop-collapse method:(A)Water control,(B)25 mg/L rhamnolipid,(C)50 mg/L rhamnolipid,(D)75 mg/L rhamnolipid and(E)100 mg/L rhamnolipid.In this case,as the surfactant concentration increased,the diameter of the sample drop increased.第26页 Quantitative results for two surfactants,rhamnolipid and SDS,are presented as standard curves in Fig.2.A linear correlation was found between the rhamnolipid concentration and the drop diameter,in the range of 0 to 100 mg/L,with an r2=of 0.997(Fig.2A).For SDS(Fig.2B),concentrations between 0 and 2400 mg/L were linearly correlated with drop diameter(r2 =50.989).Fig.2.The quantitative drop-collapse method.The figure shows the results obtained with two different surfactants:(A)P.aeruginosa IGB83 with a CMC of 27 mg/L and(B)SDS with a CMC of 1845 mg/L.Each point represents the mean and standard deviation of five replicates from experiments that were carried out in triplicate.第27页 Potential Commercial Applications Most surfactants are chemically synthesized.Nevertheless,in recent years,much attention has been directed toward biosurfactants due to their broad-range functional properties and the diverse synthetic capabilities of microbes.Most important is their environmental acceptability,because they are readily biodegradable and have lower toxicity than synthetic surfactants.A number of applications of biosurfactants have been envisaged.MEOR、Food Industry、Cosmetic Industry、Medicinal Use、Soil Bioremediation第28页Biosurfactants in Microbial Enhanced Oil Recovery(MEOR)Fig.Mechanism of enhanced oil recovery by biosurfactants.the oil is trapped in the pores by capillary pressure.Biosurfactants reduce interfacial tension between oil/water and oil/rock.This reduces the capillary forces preventing oil from moving through rock pores.Biosurfactants can also bind tightly to the oil-water interface and form emulsion.This stabilizes the desorbed oil in water and allows removal of oil along with the injection water.第29页The Application of Biosurfactants for Soil BioremediationThe biological remediation process can be performed(i)in situ(ii)in a prepared bed(iii)in a slurry reactor systemIn situ processes are usually accomplished by addition of microbial nutrients to the soil,which allows considerable growth of soil microbial indigenous population.Thus increased microbial biomass in the soil.(fig1)第30页第31页Figure 3 Mechanism of biosurfactant activity in metal-contaminated soildue to the lowering of the interfacial tension.第32页Conclusion Advantage:higher biodegradability,better environmental compatibility,higher foaming,high selectivity and high specific activity at extreme temperature,pH and salinity.There the demand of biosurfactants is increasing worldwide in recent years.However,biosurfactants do not economically compete with chemically synthesized surfactants.Thats why there is a great scope for further research to find a more economical production process and technology.第33页第34页