characterization of biosurfactant produced from some microorganism

seminar on characterization of biosurfactant produced from some organism written by chinedu. CHAPTER ONE INTRODUCTION Biosurfactants are surface active molecules having hydrophilic and hydrophobic moieties as their constituents which allow them to interact at interfaces and reduce the surface tension. They are produce by diverse group of organism belong to bacteria, fungi and actinomycetes etc., mainly on surfaces of microorganisms or may also secreted extracellularly. They are categorized based on their chemical composition as fatty acids, glycolipids, glycolipopeptides, glycoproteins, lipopeptides, phospholipids, polymeric and particulate biosurfactants. The chemicaldiversity of biosurfactants makes them a potential source for green chemicals having applications in industrial, environmental (agricultural and bioremediation), and medical fields. Almost all surfactants being currently produced are derived from petroleum source. However, these synthetic surfactants are usually toxic and hardly degraded by microorganisms. These are potential source of pollution and damage to the environment. Therefore, in the recent years, much interest and attention have been directed towards biosurfactants over chemically synthesized surfactants due to their superiority to the chemical surfactants with respect to their biocompatibility, lower toxicity, higher biodegradability, higher stability, extreme stability in extreme temperature and pH. With the advent of time, this attribute is contributing its higher demand in the field of biotechnology. The agricultural waste such as whey (a by-product of the manufacture of cheese or casein) are well known for containing high levels of carbohydrates and of lipids -both of which are necessary for substrates for the production of biosurfactants and contains all necessary substances (lactose, protein, organic acids and vitamins) that require for growth of surfactant producing microorganism. This study focus on the screening, production, extraction and purification of biosurfactant from bacteria isolated from whey spilled soil and which is easily available in India. CHAPTER TWO 2.0 LITERATURE REVIEW Surfactants are usually organic compounds that are amphiphilic, meaning they contain both hydrophobic groups (their tails) and hydrophilic groups (their heads). The hydrophilic (polar) end part of the biosurfact ant is insoluble in water and may have a long chain of fatty acids, hydroxyl fatty acids or α-alkyl-β -hydroxy fatty acids. The hydrophilic (polar) end can be a carbohydrate, amino acid, cyclic peptide, phosphate, carboxylic acid or alcohol (Jaysree et al., 2011). Surfactant or surface active agents can be classified into two main groups; synthetic surfactant and bio-surfactant. Synthetic surfactant is produced by chemical reactions, while bio-surfactant is produced by biological processes, being excreted extracellularly by microorganisms such as bacteria, fungi and yeast (Jayrees et al., 2011). Chemically-synthesized surfactants have been used in the oil industry to aid clean up of oil spills, as well as to enhance oil recovery from oil reservoirs. These compounds are not biodegradable and can be toxic to environment (Tabatabaee et al., 2005). When compared to synthetic surfactant, bio-surfactant have several advantages including high bio-degradability, low toxicity, low irritancy, ecological acceptability, compatibility with human skin and ability to be produced from renewable and cheaper substrates (Banat et al.,2000) Therefore, it is reasonable to expect diverse properties and physiological functions of bio-surfactants such as increasing the surface area and bio-availability of hydrophobic water-insoluble substrates, metal binding, bacterial pathogenesis, quorum sensing, and bio-film formation (Priya & Usharani, 2009). Unlike synthetic surfactants, microbial-produced compounds are easily degraded and particularly suited for environmental applications such as bioremediation and dispersion of oil spills (Mohan et al., 2006). The aim of this study is to isolate and screen bacterial species from different hydrocarbon polluted sites for bio-surfactants production. 2.1. Oil spreading technique Cultures of ten isolated strains (APCC S1a, 1b; APCCS 2a, 2b; APCCS 3a, 3b; APCCS 4a, 4b; APCCS 5a, 5b) are to centrifuged and added to the oil containing plates. The strain APCCS 1a, APCCS 1b, APCCS 3b and APCCS 5a the clear zone will be displaced, the oil around the colony will indicate biosurfactant production. 2.2. Hemolytic Activity The isolated will be streaked in the blood agar plates. The hemolytic activity is to be observed all the ten isolated strains, showing (alpha) hemolytic activity of strain APCCS 2a, APCCS 3b,APCCS 4a,and APCCS 5b, the (beta) hemolytic activity of strain APCCS 1a, APCCS 1b, APCCS 2b and APCCS 5a and the (gamma) hemolytic activity of strain APCCS 3a and APCC 4b. 2.3. Drop collapsing test The isolated culture supernatant is to be place on an oil coated solid surface, the drops spread or even collapse because the force or interfacial tension between the liquid drop and the hydrophobic surface is reduced indicate the presence of surfactant in the cell supernatants. 2.4. Characterization of biosurfactant producing organisms The screened biosurfactant producing organism is taken to the extraction surfactants. First the organism is to be identified by different biochemical tests. The results of its will be tabulated. Comparing the results with Bergey’sManual, identification of bacteria is to be performed. 2.5. Extraction of biosurfactants The culture to be inoculated in Mckeen broth with whey is to be centrifuged and the supernatant is to be taken mixing with Chloroform: methanol. White sediment is to be retained while after evaporation for overnight. 3.7. Characterization of biosurfactants The crude biosurfactant produced is characterized by using silica thin layer chromatography (TLC) plates. The sediment obtain is to be place in the TLC plate and the plates when sprayed with ninhydrin reagent and anthrone reagent it will show red spot (for APCCS 1b) and yellow spots (for APCCS 1a,APCCS 2b and APCCS 5a) in the plates respectively. This shows the production of lipopeptide (for APCCS 1b) and glycolipid (for APCCS 1a, APCCS 3b and APCCS 5a) biosurfactants in the organisms. 3.8. Emulsification measurement Emulsification activity is to be measure according to the method of Cooper and Goldenberg (1987). The emulsification activity of isolated strain is to be measure after 24 hours indicate the value varies from 45% to 60%. CHAPTER THREE Materials and methods 3.1. Sampling area For isolation biosurfactant producing bacteria soil samples are to be collected from whey spilled surfaces of five different cheese making farm of West Bengal, India (sample 1-5). The samples are to be collected in sterile container under aseptic condition and are to be taken to the laboratory for analysis. The pH of the samples during collection is to be 7.0 and temperature should be 300C. 3.1.1 Sampling: Soil samples (A-D) are to be collected from oil spilled surfaces of different automobile workshops in Owerri Imo-State, Nigeria. The samples are to be collected in sterile polythene bags and are to be taken to the laboratory for analysis. The pHs of the samples during collection are to be 7. 3.1.2 Isolation and enumeration of bacterial isolates from the sample 5g of the oil spilled soil samples are to be inoculated in 50ml of nutrient broth and incubated at 25 ̊C for72 hours. After incubation the medium is serially diluted from 10-1 to10-6 in sterile water. From the dilutions (10-1 to10-6) 1ml are to be transferred to sterile petri-dish and over that 20mls of nutrient agar are to be poure. The plates then inverted and incubated at 25 ̊C for 48 hours. Control and replica plates - maintained. 3.1.3 Bacteriological isolation techniques After incubation, the different discrete colonies formed on the plate that had between 30 and 300 colony forming unit (cfu) are to be streaked on nutrient agar slant and incubated at ambient temperature (37 ̊C) for 24 hours to obtain their pure cultures. These pure cultures, are to be sub-cultured on nutrient agar slant, incubated at 37 ̊C for 24 hours and stored at 4 ̊C for bio-surfactants production screening. 3.1.4 Oil spreading technique Each of the bacterial species is to be screened for bio-surfactants production using the oil spreading techniques (Anandaraj & Thivakaran, 2010; Priya & Usharani, 2009). The procedure is as follows: the bacterial isolates are to be streaked on nutrient agar slant and incubated for 24 hours at 37 ̊C. After 24 hours, two loops of culture were inoculated in 50ml of nutrient broth in a 50ml Erlenmeyer flask and incubated at 37 ̊C for 48 hours. After the inoculum development, 50ml of distilled water should be added to a large petri dish (25cm in diameter) followed by the addition of 20μl of crude oil to the surfaceof the distilled water and 20μl of the supernatant of the cultures isolated from the soil. 2.1.6 Blood haemolysis test The fresh single colonies from the isolated cultures are to be taken and streaked on blood agar plates. The plates are to be incubated for 48-72 hours at 37 ̊C. The bacterial colonies are to be then observe for the presence of clear zone around the colonies. These clear zones indicate the presence of bio-surfactants producing bacteria. 3.1.7 Charaterization of biosurfactants producing isolates The bio-surfactants producing bacteria are characterized by cultural and biochemical tests. They are gram staining, spore staining, motility test, oxidase test, indole test, catalase test, citrate test, coagulase test, methyl red and vogues proskauer test. 3.1.8 Analytical method Thin layer chromatography Preliminary characterization of biosurfactant is done by TCL method. A portion of the crude biosurfactant is separated on a silica gel plate using a developing solvent system with different colour developing reagents. Ninhydrin reagent (0.5g ninhydrin in 100ml anhydrous acetone) is to be use to detect lipopeptide biosurfactant as red spots and anthrone reagent (1g anthrone reagent in 5ml sulfuric acid mix with 95 ml ethanol) to detect glycolipid biosurfactant as yellow spots (Yin et al., 2008). 3.2. Enrichment, Isolation and enumeration of bacterial isolates 5.0g of the whey spilled soil samples should be dissolve in 100 ml of phosphate buffer saline (PBS). After precipitation of solid debris 5ml liquid suspension are inoculated in 50ml of nutrient broth and incubated at 25°C with agitation speed of 200 rpm for 48 hours. After incubation the medium is serially diluted from 10-1 to 10 in sterile water. From the dilutions (10-1 to 10-6) 1ml was transferred to sterile petri-dish containing 20mls of Reasoner ́s 2A agar (R2A) contained (g/L): Proteose peptone, 0.5; Casamino acids, 0.5; Yeast extract, 0.5; Dextrose, 0.5; Soluble starch, 0.5; Dipotassium phosphate, 0.3; Magnesium sulfate 7H2O, 0.05; Sodium pyruvate, 0.3; Agar, 15; Final pH 7.2 ± 0.2 @ 25 °C, by spread plate techniques. The plates is then inverted and incubated at 25°C for 48 hours. Control and replicaplates is maintained. After incubation 30-300 colonies containing plates should be selected and morphologically different colonies are to be streak on LB agar media and obtained pure culture by incubating at 370C for 24 hours. The pure isolates should be stored in R2A agar slants for further identification. These cultures are stored in R2A agar slants and kept under refrigerated condition (40C) for further experimentation. 3.3. Screening of biosurfactant producing organisms The isolated colonies are tested for their biosurfactant production by different methods; CTAB Agar Plate; Oil Spreading Technique; Blood Hemolysis Test and Drop collapsing test. 3.3.1. CTAB Agar Plate The CTAB agar plate method is a semi-quantitative assay for the detection of extra cellular glycolipids or other anionic surfactants. It is develop by Siegmund and Wagner. Blue agar plates containing cetyltrimethylammonium bromide (CTAB) (0.2 mg ml -1) and methylene blue (5 mg ml-1) are to be use to detect extracellular glycolipid production. Biosurfactants are to be observing by the formation of dark blue halos around the colonies. 3.3.2. Oil spreading technique Isolate bacterial strains should be incubate into 100 Ml of culture medium. The Mckeen medium (20 gL-1 glucose, 5.0 gL-1 glutamic acid, 1.0 gL-1K2 HPO4, 1.02 gL-1MgSO4, 0.5 gL-1KCl) supplemented with 1 mL of trace elements solution (0.5gL-1 MnSO4,.7H2O, 0.16 gL-1CuSO4,.5H2O and 0.015 gL-1 FeSO4,.7H2O) adjusting to pH 7.0 is use as cultural medium. The cultures should be incubated on rotary shaker (150 rpm) for 3 days at 25 °C. The culture suspension is screen for biosurfactant production by the oil spreading techniques. The procedure is as follows: 30ml of distilled water is taken in the petri dish (25cm in diameter). 20μl of crude oil is added to the center of the plates containing distilled water. Now add 20μl of the supernatant of the culture suspension to the center. The biosurfactant producing organism can displace the oil and spread in the water. The diameter and area of clear halo visualized under visible light should be measured and calculated after 1 minute. 3.3.3. Hemolytic activity test Hemolytic activity appears to be a good screening criterion for surfactant-producing strains because biosurfactant producing capacity is found to be associated with hemolytic activity. The fresh single colonies from the isolated cultures is taken and streaked on blood agar plates. The plates should be incubate for 48-72 hours at 37°C. Hemolytic activity is detected as the occurrence of a define clear zone around a colony (Carrillo et al., a 1996). These clear zones indicate the presence of bio-surfactants producing bacteria. 3.3.4. Drop collapsing test Jain et al developed the drop collapse assay. This assay relies on the destabilization of liquid droplets by surfactants. Therefore, drops of culture supernatant are placed on an oil coated, solid surface. If the liquid does not contain surfactants, the polar water molecules are repelled from the hydrophobic surface and the drops remain stable. If the liquid contains surfactants, the drops spread or even collapse because the force or interfacial tension between the liquid drop and the hydrophobic surface is reduced. The stability of drops is dependent on surfactant concentration and correlates with surface and interfacial tension. The assay was done in following way: 2μl of mineral oil is added to each well of a 96-well micro-titer plate lid. The lid is equilibrated for 1 hour at room temperature, and then 5 μl of the cultural supernatant is added to the surface of oil. The shape of the drop on the oil surface should be inspected after 1 min. Biosurfactant-producing cultures giving flat drops are to be scored as positive '+'. Those cultures that gave rounded drops is to be scored as negative '-indicative of the lack of biosurfactant production (Youssef et al., 2004). 3.4. Identification of bacteria The isolated biosurfactant producing bacteria is to be characterized by microbiological and biochemical tests such as Gram staining, carbohydrate fermentation test, H2 Sproduction test, indole production test, methylred test, Voges-Proskauer test, citrate utilization test, urease test, catalase test, oxidase test, litmus milk reaction, starch hydrolysistest, gelatin hydrolysis test, lipid hydrolysis test. Results from the biochemical analysis are to be used to find the closest match with known bacterial genus and to assign the bacterial signature according to Bergey’s manual. 3.5. Extraction of biosurfactants Each culture has to be inoculated in 50 ml of Mckeenbrothwith 1 ml of whey. The culture is to be incubated at 250C for 3 days with shakingcondition. After incubation the bacterialcells is to be removed by centrifugation at 8000rpm, 40C for 10 minutes. The supernatant is to be taken and the pH of the supernatant is to be adjusted to 2, using 1MH2SO4. Now add equal volume of chloroform: methanol (2:1). This mixture is to be shaken well for mixing and left overnight for evaporation. White colored sediment is to be obtained as a result i.e., the “Biosurfactants”. 3.6. Recharacterization of biosurfactants Preliminary characterization of the biosurfactant is to be done by Thin Layer chromatography (TLC) method. A spot of crude biosurfactant is to be placed on the silica plate(Merck & Co., Mumbai, India) and the biosurfactant is to be separated on the plate using chloroform: methanol: water (:10:0.5). The plate is to be developed with different color developing reagents. Ninhydrin reagent (0.5g ninhydrin in 100ml anhydrous acetone) are to be used to detect lipopeptide biosurfactant as red spots and anthrone reagent (1g anthrone reagent in 5ml sulfuric acid is to be to mix with 95 ml ethanol) to detect glycolipid biosurfactant as yellow spots (Yin et al., 2008). 3.7. Emulsification index (E24) The emulsifying capacity is to be evaluated by an emulsification index (E24). The E24 of culture samples determined by adding 2 ml of kerosene and 2 ml of the cell-free broth in test tube, vortexing at high speed for 2 min and allowed to stand for 24h. The E index is given as percentage of the height of emulsified layer (cm) divided by the total height of the liquid column (cm).The percentage of emulsification index calculated by using the following equation (Cooper and Goldenberg, 1987). CHAPTER FOUR 4.0. CONCLUSION AND RECOMMENDATION Uses of biosurfactants are increasingly in almost every sectors of the modern industry as an alternative to chemical surfactants. With increasing public awareness in the environment, biosurfactant would most likely replace the usage of chemical surfactants in the near future. As biosurfactants are derived from natural sources, each of these types is an attractive alternative to synthetic compounds. Biosurfactants are surfactants that are produced extracelluarly or as part of the cell membrane by bacteria, yeast and fungi. The main commercial use of biosurfactant is in oil industry, foods, cosmetics, pharmacology and environmental technology because of their ability to stabilize emulsions. The features that make them commercially promising alternatives to chemically synthesized surfactants are their lower toxicity, higher biodegradability and greater environmental compatibility. 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