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Optimization and production of a biosurfactant from the sponge-associated marine fungus Aspergillus ustus MSF3
Colloids and Surfaces B: Biointerfaces 73 (2009) 250–256
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Optimization and production of a biosurfactant from the sponge-associated marine fungus Aspergillus ustus MSF3 G. Seghal Kiran a,b , T.A. Hema a , R. Gandhimathi a , Joseph Selvin a,∗ , T. Anto Thomas a , T. Rajeetha Ravji a , K. Natarajaseenivasan a a b
Department of Microbiology, Bharathidasan University, Tiruchirappalli – 620 024, India Department of Biotechnology, Bharathidasan University, Tiruchirappalli – 620 024, India
a r t i c l e
i n f o
Article history: Received 11 March 2009 Received in revised form 23 April 2009 Accepted 25 May 2009 Available online 6 June 2009 Keywords: Biosurfactant Optimization Marine fungi Sponge Penicillium ustus
a b s t r a c t Marine endosymbiotic fungi Aspergillus ustus (MSF3) which produce high yield of biosurfactant was isolated from the marine sponge Fasciospongia cavernosa collected from the peninsular coast of India. Maximum production of biosurfactant was obtained in Sabouraud dextrose broth. The optimized bioprocess conditions for the maximum production was pH 7.0, temperature 20 ◦ C, salt concentration 3%, glucose and yeast extract as carbon source and nitrogen sources respectively. The response surface methodology based analysis of carbon and nitrogen ratio revealed that the carbon source can increase the biosurfactant yield. The biosurfactant produced by MSF3 was partially characterized as glycolipoprotein based on the estimation of macromolecules and TLC analysis. The partially purified biosurfactant showed broad spectrum of antimicrobial activity. The strain MSF3 can be used for the microbially enhanced oil recovery process. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Microbial surfactants or biosurfactants are surface active amphiphilic molecules produced by a number of microorganisms. They occur in nature as a diverse group of molecules comprising of glycolipids, lipopeptides and lipoproteins, fatty acids, neutral lipids, phospholipids, polymeric and particulate biosurfactants [1]. They are mainly produced by hydrocarbon utilizing microorganisms exhibiting surface activity [2]. These molecules reduce surface tension and interfacial tension in both aqueous solutions and hydrocarbon mixtures. These properties create microemulsions leading to micelle formation in which hydrocarbons can be solubilized in water or hydrocarbon in water. Almost all surfactants being currently produced are derived from petroleum. However these synthetic surfactants are usually toxic themselves and hardly degraded by microorganisms. They are therefore, a potential source of pollution and damage to the environment. These hazards associated with synthetic surfactants have in recent years; draw much attention to the microbial production of surfactants [3]. They are also easy to produce cheaper and renewable feedstock. The striking advantages of biosurfactants over chemically synthesized surface active compounds includes their broad range of novel structural characteristics and physical
∗ Corresponding author. Tel.: +91 431 2407082; fax: +91 431 2407045. E-mail address: [email protected] (J. Selvin). 0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2009.05.025
properties and their capacity to be modified by genetic engineering, as well as by biotechnological or biochemical techniques. Biosurfactants are produced extracellular or cell wall associated compounds by bacteria, yeasts and fungi. In the present study, a sponge-associated marine fungi was isolated and screened as potent biosurfactant producer. The producer strain (MSF3) was optimized for enhanced production and evaluated for prospective applications including antimicrobial activity and microbially enhanced oil recovery (MEOR). 2. Materials and methods 2.1. Sample collection and isolation of sponge-associated fungi Marine sponge F. cavernosa was collected from the Bay of Bengal region of the Indian peninsular coast by SCUBA diving at 10–15 m depth. To avoid cross-contamination, only unbroken samples were used for microbiological analysis. The specimens were kept 2 h in sterilized aged seawater to remove loosely associated microorganisms from inner and outer sponge surfaces. It has been hypothesized that this process may eliminate nonassociated microbes from the host sponge by digestion. Environmental water representing the sponge habitat was taken prior to sponge sampling and filled up in 1 l sterilized glass bottles. Habitat water was used for isolation of fungi on Sabouraud dextrose agar (SDA) and starch casein agar (supplemented with 2% NaCl) respectively. Isolated colonies were purified on Sabouraud dextrose agar slants and stored in refrig-
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erator for further proceedings. Identification of the sponge was carried out by P.A. Thomas, an eminent sponge taxonomist in India. 2.2. Screening methods for potential biosurfactant producers The potential biosurfactant producer was screened by hemolytic assay [4], drop collapsing test [5], oil displacement test [6] and lipase activity. Hemolytic activity in blood agar plate (Peptone—5 g, yeast extract—3 g, NaCl2 —5 g, Sheep blood—5 ml) is a primary screening method to screen biosurfactant producers. Hemolytic activity was detected using blood agar plates with 5% (v/v) human blood. Plates were examined for hemolysis after incubation at 37 ◦ C for 24 h. The plates were inspected for zone of clearance around the colony. The presence of clearing zone served as an indicator of biosurfactant producing microorganism. In the drop-collapse test method, 2 l of mineral oil was added to 96-well microtitre plates. The plate was equilibrated for 1 h at 37 ◦ C and 5 l of the culture supernatant was added to the surface of the oil. The shape of drop on the oil surface was observed after 1 min. The culture supernatant that make the drop collapsed was indicated as positive result and the drops remain beaded were scored as negative, which is examined with distilled water as control. To perform oil displacement test, 15 l of weathered crude oil were placed on the surface of 40 l distilled water in a Petri dish and 10 l of the culture supernatant were gently put on the surface of the oil film. The diameter and area of clear halo visualized under visible light was measured after 30 s. The isolates that produce lipase were screened using tributyrin agar plates. 1% tributyrin was added to actinomycetes isolation agar. The pH of the medium was adjusted to 7.3–7.4 using 0.1 N NaOH. A loopful of inoculum was streaked on to the tributyrin agar plates. The plates were incubated at 26 ◦ C for 7 days. After incubation, the plates were examined for the formation of clear zone around the colonies. 2.3. Emulsification index The emulsification index was measured using the method described by Cooper and Goldenberg [7] in which 2 ml of the kerosene was added to equal volume of cell free supernatant and homogenized in a vortex at high speed for 2 min. The emulsification stability was measured after 24 h and the emulsification index was calculated by dividing the measured height of the emulsion layer by the total height of the liquid layer and multiplying by 100. The emulsification activity of the strain was compared with the standards including SDS and Tween 80. E24 (%) =
total height of the emulsified layer total height of the liquid layer
2.4. Identification of the strain MSF3 Identification and characterization of the strain MSF3 were carried out with the methods described by Gilman [8]. The morphological and colony appearances were observed by naked eye examination of 3 days old culture grown on SDA medium. The micomorphology and sporulation was observed by light microscopy with LCB (Lactophenol Cotton Blue) staining. 2.5. Cultivation condition The fungal strain MSF3 was grown on SDA (peptone—10 g, dextrose—40 g, agar—15 g, distilled water—1000 ml), potato dextrose agar (PDA) (potato (peeled)—200.0 g, dextrose—20.0 g, agar—15.0 g, distilled water—1000 ml), Zobell Marine broth 2216 (ZMB 2216) (glucose—10.0 g, peptone—5.0 g, yeast extract—1 g, ferric citrate—0.1 g, sodium sulphate—3.24 g, calcium chloride—1.50 g,
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Na2 HCO3 —0.16 g, H3 BO3 —0.22 g, SnCl2 —0.034 g, sodium silicate— 0.004 g, sodium fluorate—0.0024 g, NaNO3 —0.0016 g, Na2 PO4 — 0.008 g, NaCl2 —19.45 g, MgCl2 —8.80 g, KCl2 —0.55 g, KBr—0.08 g, pH 7.6 ± 0.2, distilled water—1000 ml) and MSM (NaNO3 — 15 g, KCl—1.1 g, FeSO4 ·7H2 O—0.00028 g, KH2 PO4 —3.4 g, K2 HPO4 — 4.4 g, MgSO4 ·7H2 O—0.5 g, yeast extract—0.5 g). Trace elements— ZnSO4 ·7H2 O—0.29 g, CaCl2 ·4H2 O—0.24 g, CuSO4 ·5H2 O—0.25 g, MnSO4 ·H2 O—0.17 g/100 ml) medium inorder to determine the growth conditions. The strain MSF3 was inoculated in a 250 ml Erlenmeyer flask containing 50 ml of SDA medium and incubated for 120 h at 20 ◦ C. The cell free supernatant (CFS) was filtered through filter paper and cold centrifuged (Eppendorff 5804 R) at 8000 × g at 4 ◦ C for 20 min. Emulsification activity was determined using the method of Cooper and Goldenberg [7]. 2.6. Time course of biosurfactant production To obtain the production curve, the culture was inoculated in 1000 ml Erlenmeyer flasks each containing 300 ml production media. The experiment was designed for 120 h starting from the log phase to stationary phase under submerged culture conditions. The resultant CFS was removed by filtration followed by cold centrifugation at 8000 × g at 4 ◦ C for 20 min. The resultant CFS was analyzed for emulsification activity and the dry weight of the biomass was weighed using the physical balance. 2.7. Optimization of biosurfactant production To optimize the culture conditions for biosurfactant production, this strain was cultured under the conditions presented in Table 1. The culture was inoculated in the production medium and incubated at 120 h. The resultant CFS was removed by filtration followed by cold centrifugation at 8000 × g at 4 ◦ C for 20 min. The resultant CFS was analyzed for emulsification activity and biomass dry weight. 2.8. Response surface methodology (RSM) experimental design Response surface methodology (RSM) [9,10] is an empirical technique employed for multiple regression analysis by using quantitative data obtained from properly designed experiments to solve multivalent equations simultaneously. The graphical representations of these equations are called response surfaces. A 22 full-factorial central composite design for two test variables with corresponding star points and centre points was employed to fit the procedure and analyzed using the Design Expert Software (version 7.0). Based on the ANOVA of the quadratic regression model demonstrates that the model is highly significant, as is evident from the Fischer F test The two factors carbon (glucose) and nitrogen (yeast extract) were analyzed for the biosurfactant production using this software which is the independent variable. 2.9. Extraction of biosurfactant The strain MSF3 was grown on 250 ml Erlenmeyer flask containing 100 ml of SDA medium and incubated at 120 h at 20 ◦ C. Table 1 Culture conditions for the optimization of biosurfactant production by MSF3. Factors
Ranges
pH Temperature Salinity Carbon sources Nitrogen source Metals
4, 5, 6, 7, 8, 9 10, 20, 30, 40, 50 ◦ C 1.0, 1.5, 2.0, 2.5, 3.0, 3.5% Glucose, paddy straw, olive oil, kerosene, vegetable oil Peptone, yeast extract, beef extract, NaNO3 , urea FeSO4 , CuSO4 , MnCl2 , CaCl2 , MgCl2
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Culture samples were centrifuged at 12,000 × g for 20 min at 4 ◦ C to remove the cells as well as debris and the supernatant was filtered through 0.2 m filter. Filtered supernatant and the harvested cells were used for the extraction of surfactive compounds. Extraction was performed by acid precipitation followed by liquid–liquid extraction method, in both the cell free supernatant (CFS) and the harvested cells respectively. The CFS and the pellet was acidified with conc. HCl to attain a pH 2.0 and extracted with an equal volume of solvents such as ethyl acetate, diethyl ether and dichloromethane. The resultant aliquot was concentrated to dryness in a rotary vacuum evaporator (Yamato) and tested for emulsification activity as described above. 2.10. Chemical analysis of biosurfactant 2.10.1. Analytical methods To identify the chemical nature of the compound, the residue was applied in preparative silica gel TLC plates. Chromatograms were developed with 96% ethanol:water (7:3) for amino acids; chloroform:acetic acid:water (60:30:10) for sugars and chloroform:methanol:water (65:25:4) for lipids. Fractions were isolated and eluted with corresponding buffer and subjected to quantification of macromolecules such as protein, carbohydrate and lipid. Protein was estimated using the method of Lowry et al. [11], using bovine serum albumin as standard. Carbohydrate with phenol–sulphuric acid method by Chaplin and Kennedy [12], with glucose as standard and lipid was estimated for free fatty acid using the method of Sadasivam and Manickam [13] with cholesterol as standard. 2.10.2. Stability of biosurfactant Stability of the surface active compound was determined by dissolving the TLC eluted fraction in 1–2% of NaCl and the emulsification index was calculated. The stability of the biosurfactant at different pH values was performed by dissolving the biosurfactant in 0.1 M sodium acetate buffer (pH 4.0–7.0) and 0.1 M sodium phosphate buffer (pH 8.0–9.0). After 1 h of incubation with reciprocal agitation, the emulsification activity was measured as previously described. The stability of the biosurfactant at different temperatures was carried out by incubating the surface active compound at 0.4% (w/v) in water for 30 min from 25 to 120 ◦ C before measuring the emulsification activity. All these factors were compared to that of the corresponding solution in water. 2.10.3. Antimicrobial activity of surfactive compound The extracted compound as well as the culture supernatant was tested for antimicrobial activity using well diffusion method and area of the zone was calculated [14]. Extracted active compounds were tested against human pathogens such as Candida albicans, E. coli, Proteus mirabilis, hemolytic Streptococcus, Pseudomonas aeruginosa, Micrococcus luteus, Staphylococcus epidermidis, Enterococcus faecalis, Klebsiella pneumoniae, Bacillus subtilis, and Staphylococcus aureus. Mueller Hinton agar (beef infusion solids 4.0 g, starch 1.5 g, casein hydrolysate 17.5 g, agar 15.0 g, final pH 7.4 ± 0.2 at 37 ◦ C) plates were prepared and swabbed with appropriate pathogen. Using a sterile cork borer well was made and 50 l of extracted compound was added in wells, incubated at 30 ◦ C for 24 h. After incubation, the clear zone was measured and calculated. 2.10.4. Microbially enhanced oil recovery (MEOR) (sand pack test) The potential application of the biosurfactant in MEOR was evaluated using the ‘sand pack column’ technique described by Abu-Ruwaida et al. [15]. A glass column was packed with 100 g of acid washed dry sand .The column was then saturated with different hydrocarbon (50 ml). The potential of the isolated surfactant
for oil recovery was estimated by pouring 50 ml of aqueous solution of biosurfactant (extraction from 100 ml culture broth) in the column. The amount of oil released was measured. The experiment was carried out at room temperature, 30, 50 and 70 ◦ C to assess the influence of temperature on biosurfactant-induced oil recovery. 3. Results and discussion 3.1. Isolation and screening of biosurfactant producing fungi Based on the colony morphology and stability in subculturing, 8 sponge-associated marine fungus were isolated from the marine sponge F. cavernosa. Among these 4 strains showed positive result for biosurfactant production, particularly CFS of the strain MSF3 exhibited highest emulsification activity (42.8%) followed by MSF5 (17.5%), MSF1 (11.4%) and MSF8 (7.5%). This strain was selected for characterization and optimization of the biosurfactant production. Marine sponges (Porifera) have attracted significant attention from various scientific disciplines. As sponges produce various novel chemical molecules, they have been a goldmine to chemists and also found their way into biotechnological applications. Microbiologists became fascinated by these unique animals with the discovery that sponges contain an abundance of unusual microorganisms having potential for drug discovery and bioremediation [16]. 3.2. Screening of biosurfactant production Hemolytic activity of MSF3 showed a clear zone diameter 9 mm around the colony. In the present study, a significant correlation was established between the hemolytic activity and biosurfactant production. According to Carillo et al. [4], and Banat [17], biosurfactant production of the new isolates was preliminary screened by hemolytic activity. Blood–agar lysis has been used to quantify surfactant [18] and rhamnolipids [19]. Carillo et al. [4] found an association between hemolytic activity and surfactant production and they recommended the use of blood agar lysis as a primary method to screen biosurfactant production. The drop collapsing test, oil displacement method and lipase activity was also performed as a part of screening. In drop collapsing test a flat drop was observed and in oil displacement method, a clear diameter of 5 mm was observed and the area was calculated as 78.50 mm2 (Table 2). From the above two observation, it was confirmed that the strain MSF3 was a biosurfactant producing marine fungi. Both the techniques have several advantages: small volume of samples was required, rapid and easy to carry out and also do not require specialized equipment. 3.3. Identification of the biosurfactant producer Colonies more or less felted, floccose, with fine hyphae, from white through shades of gray, olive-gray, yellow, yellow-brown toward fuscous, with often a greenish cast, but no true green color, in old cultures purplish and vinaceous at times. Reverse through shades of yellow, orange and brown. Stalks when rising from submerged hyphae up to 1000 m, most branches of aerial hyphae up to 500 × 5–10 m, few septate, sinuous, with walls rather thin, Table 2 Results on the screening of biosurfactant production for the strain MSF3 in SDA medium. Screening tests
Results
Hemolytic activity Drop collapsing test Oil displacement test Lipase activity
9 mm + 78.50 mm2 75 U/mg
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tionary phase. The results obtained was compared with other fungal isolates Candida lipolytica [20] and the marine yeast Yarrowia lipolytica [21] which excreted biosurfactant by microbes only during the stationary phase, significant emulsification activity was detected during the exponential phase of growth being an advantage as it enhance productivity. 3.6. Optimization of biosurfactant production Cell growth and the accumulation of metabolic products were strongly influenced by medium composition such as carbon sources, nitrogen sources, salinity and other growth factors, thus the optimization of organisms can give results in the high yield of metabolites [22].
Fig. 1. Biosurfactant production using different medium at 20 ◦ C for 120 h with the corresponding pH of each medium. Biomass was quantified in mg and the biosurfactant production was denoted in terms of E24 .
smooth, usually partly colored some shade of brown; vesicles 10–20 m in diameter; heads hemispherical to almost columnar; phialides colorless, semi-radiate, loosely arranged into two series, primary 5–8 × 3 m, secondary 7–9 × 2–2.5 m. Conidia globose about 3.6 m, spinulose or with fine faint bars of rosy, reddish yellow or vinaceous color, with chains forming fairly compact columns in old cultures. 3.4. Cultivation conditions and biosurfactant production The isolated strain MSF3 produced biosurfactant when grown in various nutrients. However, the amount of biosurfactant secreted varied in the presence of different nutrient sources of the four investigated culture medium, maximum biosurfactant production was observed in SDA (45%) followed by ZMB (15%). There was no production of biosurfactant in MSM and PDA (Fig. 1). So SDA was selected as the biosurfactant production medium for the strain at 20 ◦ C for 120 h. 3.5. Time course of biosurfactant production
3.6.1. pH and temperature The important characteristics of most organisms are their strong dependence on the pH for cell growth and production of secondary metabolites. The strain MSF3 produced the highest yield of biosurfactant (15%) at pH 7.0 (Fig. 3) even though the growth was higher in pH 8 and 9. There was no correlation established between the growth and biosurfactant production. B. subtilis was able to produce biosurfactant in a pH range of 6.0 to 9.0, although the maximal yield of the biosurfactant was obtained at pH 7.0 [23]. A change in temperature caused alterations in the composition of the biosurfactant in the case of A. paraffineus [24] and Pseudomonas sp. [25]. Temperature was one of the critical parameters that have been controlled in bioprocess. The results in the present study revealed that the biosurfactant activity reached the highest when the strain was grown at 20 ◦ C (20%) as it is a marine strain (Fig. 4). 3.6.2. Salt concentration The salinity was found to be one of the critical parameter in the production of biosurfactant, in the absence of the salt production and growth was very low. Since it is a marine isolate it showed optimum activity in the salt supplemented medium (Fig. 5). The emulsification activity was highest in the production medium contained 3% NaCl. Similar observation was made with the protease production by marine Roseobacter MMD40 [26].
Under the optimal conditions, 75% biosurfactant activity was reached in the culture of the fungal isolate at 72 h of the fermentation medium. When the cell growth reached the late log phase (Fig. 2) there was no biosurfactant activity during the early and midexponential phase. However, the biosurfactant production reached a peak by the end of the exponential phase and continued in the sta-
3.6.3. Multivalent cations Limiting multivalent cation concentrations also causes overproduction of biosurfactant [27]. Guerra-Santos et al. [28] demonstrated that limiting the concentrations of salt of magnesium, calcium, potassium and trace elements resulted in a better
Fig. 2. Growth kinetics and biosurfactant production by the marine fungus MSF3 for 6 days at 20 ◦ C in SDA medium. Biomass was quantified in mg and the biosurfactant production was denoted in terms of E24.
Fig. 3. Effect of different pH on biosurfactant production at 20 ◦ C for 120 h in SDA medium with 3% salinity. Biomass was quantified in mg and the biosurfactant production was denoted in terms of E24.
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Fig. 4. Effect of temperature on biosurfactant production at 120 h with pH 7.0 and 3% salinity in SDA medium. Biomass was quantified in mg and the biosurfactant production was denoted in terms of E24.
yield of rhamnolipid in P. aeruginosa DSM 2659. In our study, supplementation of metal ions increased the biosurfactant production. In the present study, biosurfactant production was increased in FeSO4 (75%) followed by MgCl2 (60%) with a concentration of 0.1 mM (Fig. 6). However the increased supplementation drastically inhibits the biosurfactant production. The total yield of biosurfactant higher in the production medium by adding the metal cations altogether
Fig. 7. Effect of carbon source on biosurfactant production at 20 ◦ C for 120 h with pH 7.0 and 3% salinity in SDA medium with 1% carbon source. Biomass was quantified in mg and the biosurfactant production was denoted in terms of E24 .
than the individual addition of cations. Similar results are observed for the biosurfactant production in B. subtilis MTCC 2423 [29]. 3.6.4. Carbon and nitrogen sources The present study envisaged that the supplementation of glucose as carbon source increase the biosurfactant production (35%) followed by the cheapest raw material paddy straw (30%) (Fig. 7). Many studies revealed the presence of glucose in the production medium increased the biosurfactant production in the culture medium [27,2–31]. Nitrogen source also plays an important role in the production of biosurfactant by microorganisms. Desai and Banat [1] reported that the limitation of nitrogen source increased the biosurfactant production. In the present study, the supplementation of yeast extract (25%) as well as NaNO3 (10%) showed little increase in the production medium (Fig. 8). Ouled-Hadder et al. [32] reported that NaNO3 was a good substrate for the growth with good productivity. It was also reported that NaNO3 and yeast extract used for the production of biosurfactant from different Bacillus sp. [33].
Fig. 5. Effect of salt concentration on biosurfactant production with pH 7.0 in SDA medium at 20 ◦ C for 120 h. Biomass was quantified in mg and the biosurfactant production was denoted in terms of E24 .
3.6.5. Effect of carbon–nitrogen ratio on biosurfactant production The carbon–nitrogen ratio is one of the most important factors which induce the secondary metabolites production. Therefore a wide range of the ratios from 5:1 to1:5 between the glucose and yeast extract were studied for achieving an optimal biosurfactant production. The results suggested that the peak yield of biosurfactant appeared at the ratio of 3:2 whereas the ratio at 1:5 yielded no
Fig. 6. Effect of multivalent cations (01 mM) on biosurfactant production at 20 ◦ C for 120 h with pH 7.0 and 3% salinity in SDA medium. Biomass was quantified in mg and the biosurfactant production was denoted in terms of E24 .
Fig. 8. Effect of nitrogen source on biosurfactant production at 20 ◦ C for 120 h with pH 7.0 and 3% salinity in SDA medium with 1% nitrogen source. Biomass was quantified in mg and the biosurfactant production was denoted in terms of E24 .
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Table 3 Effect of carbon–nitrogen ratio on biosurfactant production at 20 ◦ C for 120 h in SDA medium (pH 7). Carbon nitrogen ratio
E24
5:1 3:2 2.5:2.5 2:3 1:5
30% 75% 45% 20% 5%
biosurfactant production (Table 3). The results were supported by Guerra-Santos et al. [27,28], the increased rhamnolipid production under the limitation of nitrogen rather than carbon sources. 3.7. Statistical analysis of the carbon–nitrogen source using RSM The three dimensional response surface plot was represented in Fig. 9. The model proposed for the response emulsification activity (y) was: y = +25.00 + 9.31 × A + 6.66 × B − 3.75 × A × B + 15.00 × A2 + 13.75 × B2 The ANOVA of the regression model demonstrates that the model is highly significant, as is evident from the Fischer’s F test (F model = 40.49) and also indicated the treatment levels are highly significant with a very low probability value (P model > F = 0.0001) with degree of freedom 5. 3.8. Extraction and chemical analysis of biosurfactant Acid precipitation of the CFS followed by solvent extraction revealed that the biosurfactant production was highest in the CFS of the ethyl acetate extract not in the intracellular extract. TLC of the ethyl acetate extract showed a single spot in the chromatogram of lipid, carbohydrate and protein with Rf values 0.84, 0.73 and 0.80 respectively. From the TLC fractions the macromolecules was estimated and it found to be as follows: protein 925 g/ml, carbohydrate 1865.8 g/ml and lipid 382 g/ml. From the estimation and TLC analysis, the compound was partially characterized as glycolipoprotein.
Fig. 10. Comparison of the biosurfactant extractives of the fungi MSF3 with the standards SDS and Tween 80.
3.9. Stability and comparison of biosurfactant production The biosurfactant produced by this strain exhibited greatest activity in the range of 6–8 with an optimum range of pH 7.0 and in temperature the stability was observed from 20 to 40 ◦ C, it was unstable in higher temperature whereas the biosurfactant produced by bacterial isolates were stable at higher temperature (60–120 ◦ C) [29]. The biosurfactant produced by MSF3 was compared with the synthetic surfactant SDS and Tween 80. The results showed a 40% increase over the synthetic surfactants (Fig. 10). Therefore the strain MSF3 can be used for the scaled-up production of biosurfactant. 3.10. Antimicrobial activity of surface active compound The ethyl acetate extract of fungi MSF3 showed a wide activity against the pathogenic culture. In the ethyl acetate extract C. albicans, hemolytic Streptococcus, M. luteus, E. faecalis, P. aeruginosa and S. epidermidis showed a wide range of activity (Fig. 11). This marine fungal diethyl ether extract under this study also showed a highest activity towards the yeast C. albicans and Gram-negative bacterium. According to Tsuge et al. [34], lipopeptide surfactants are potent antibiotics mainly the surfactin, streptofactin and gramicidin produced by the microorganism had the wide antimicrobial activity [35–37] compared to the glycolipid producing strain. A glycolipid surfactant from the C. antartica has demonstrated antimicrobial activity against Gram-positive bacteria. 3.11. Microbially enhanced oil recovery
Fig. 9. Three dimensional plot on the effect of carbon–nitrogen ratio on the emulsification activity.
The MEOR was analyzed by the sand pack method. Hydrocarbon saturated sand pack column with the biosurfactant extract was treated and subsequently incubated at room temperature, 50 and 70 ◦ C caused release of the hydrocarbon from the column. The released hydrocarbon was quantified and the data thus obtained are presented graphically in Fig. 12. The biosurfactant produced by this organism was stable when exposed to higher temperature thus the surface tension of oil molecules was reduced. From this it was envisaged that since this microorganism are present in the marine environment, it helps in the biodegradation of the oil spillages of the marine environment. As a result it may perhaps leads to the reduction of mortality rate of the marine creatures and increased the
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high price of productivity and low yields, the biosurfactants are not competitive with chemical surfactants. In the present study, we demonstrated the sponge-associated marine fungi as potential source of biosurfactant. The strain MSF3 had the ability to degrade the hydrocarbons in a large amount compared to the synthetic surfactants. Acknowledgements GSK is thankful to CSIR, New Delhi for the award of SRF. JS is thankful to Ministry of Earth Sciences (MoES), New Delhi for financial support. References
Fig. 11. Antimicrobial activity of biosurfactant (ethyl acetate extract) produced by marine fungus MSF3.
Fig. 12. Microbially enhanced oil recovery by the marine fungus MSF3 under different temperatures.
dissolved oxygen level as well as the light penetration. In addition, the future use of this biosurfactant as broad spectrum of antibiotics is highly warranted. 4. Conclusion Sponge-associated marine microbes have been a goldmine for microbiologists and many research workers. At present due to
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Optimization and production of a biosurfactant from the sponge-associated marine fungus Aspergillus ustus MSF3 G. Seghal Kiran a,b , T.A. Hema a , R. Gandhimathi a , Joseph Selvin a,∗ , T. Anto Thomas a , T. Rajeetha Ravji a , K. Natarajaseenivasan a a b
Department of Microbiology, Bharathidasan University, Tiruchirappalli – 620 024, India Department of Biotechnology, Bharathidasan University, Tiruchirappalli – 620 024, India
a r t i c l e
i n f o
Article history: Received 11 March 2009 Received in revised form 23 April 2009 Accepted 25 May 2009 Available online 6 June 2009 Keywords: Biosurfactant Optimization Marine fungi Sponge Penicillium ustus
a b s t r a c t Marine endosymbiotic fungi Aspergillus ustus (MSF3) which produce high yield of biosurfactant was isolated from the marine sponge Fasciospongia cavernosa collected from the peninsular coast of India. Maximum production of biosurfactant was obtained in Sabouraud dextrose broth. The optimized bioprocess conditions for the maximum production was pH 7.0, temperature 20 ◦ C, salt concentration 3%, glucose and yeast extract as carbon source and nitrogen sources respectively. The response surface methodology based analysis of carbon and nitrogen ratio revealed that the carbon source can increase the biosurfactant yield. The biosurfactant produced by MSF3 was partially characterized as glycolipoprotein based on the estimation of macromolecules and TLC analysis. The partially purified biosurfactant showed broad spectrum of antimicrobial activity. The strain MSF3 can be used for the microbially enhanced oil recovery process. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Microbial surfactants or biosurfactants are surface active amphiphilic molecules produced by a number of microorganisms. They occur in nature as a diverse group of molecules comprising of glycolipids, lipopeptides and lipoproteins, fatty acids, neutral lipids, phospholipids, polymeric and particulate biosurfactants [1]. They are mainly produced by hydrocarbon utilizing microorganisms exhibiting surface activity [2]. These molecules reduce surface tension and interfacial tension in both aqueous solutions and hydrocarbon mixtures. These properties create microemulsions leading to micelle formation in which hydrocarbons can be solubilized in water or hydrocarbon in water. Almost all surfactants being currently produced are derived from petroleum. However these synthetic surfactants are usually toxic themselves and hardly degraded by microorganisms. They are therefore, a potential source of pollution and damage to the environment. These hazards associated with synthetic surfactants have in recent years; draw much attention to the microbial production of surfactants [3]. They are also easy to produce cheaper and renewable feedstock. The striking advantages of biosurfactants over chemically synthesized surface active compounds includes their broad range of novel structural characteristics and physical
∗ Corresponding author. Tel.: +91 431 2407082; fax: +91 431 2407045. E-mail address: [email protected] (J. Selvin). 0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2009.05.025
properties and their capacity to be modified by genetic engineering, as well as by biotechnological or biochemical techniques. Biosurfactants are produced extracellular or cell wall associated compounds by bacteria, yeasts and fungi. In the present study, a sponge-associated marine fungi was isolated and screened as potent biosurfactant producer. The producer strain (MSF3) was optimized for enhanced production and evaluated for prospective applications including antimicrobial activity and microbially enhanced oil recovery (MEOR). 2. Materials and methods 2.1. Sample collection and isolation of sponge-associated fungi Marine sponge F. cavernosa was collected from the Bay of Bengal region of the Indian peninsular coast by SCUBA diving at 10–15 m depth. To avoid cross-contamination, only unbroken samples were used for microbiological analysis. The specimens were kept 2 h in sterilized aged seawater to remove loosely associated microorganisms from inner and outer sponge surfaces. It has been hypothesized that this process may eliminate nonassociated microbes from the host sponge by digestion. Environmental water representing the sponge habitat was taken prior to sponge sampling and filled up in 1 l sterilized glass bottles. Habitat water was used for isolation of fungi on Sabouraud dextrose agar (SDA) and starch casein agar (supplemented with 2% NaCl) respectively. Isolated colonies were purified on Sabouraud dextrose agar slants and stored in refrig-
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erator for further proceedings. Identification of the sponge was carried out by P.A. Thomas, an eminent sponge taxonomist in India. 2.2. Screening methods for potential biosurfactant producers The potential biosurfactant producer was screened by hemolytic assay [4], drop collapsing test [5], oil displacement test [6] and lipase activity. Hemolytic activity in blood agar plate (Peptone—5 g, yeast extract—3 g, NaCl2 —5 g, Sheep blood—5 ml) is a primary screening method to screen biosurfactant producers. Hemolytic activity was detected using blood agar plates with 5% (v/v) human blood. Plates were examined for hemolysis after incubation at 37 ◦ C for 24 h. The plates were inspected for zone of clearance around the colony. The presence of clearing zone served as an indicator of biosurfactant producing microorganism. In the drop-collapse test method, 2 l of mineral oil was added to 96-well microtitre plates. The plate was equilibrated for 1 h at 37 ◦ C and 5 l of the culture supernatant was added to the surface of the oil. The shape of drop on the oil surface was observed after 1 min. The culture supernatant that make the drop collapsed was indicated as positive result and the drops remain beaded were scored as negative, which is examined with distilled water as control. To perform oil displacement test, 15 l of weathered crude oil were placed on the surface of 40 l distilled water in a Petri dish and 10 l of the culture supernatant were gently put on the surface of the oil film. The diameter and area of clear halo visualized under visible light was measured after 30 s. The isolates that produce lipase were screened using tributyrin agar plates. 1% tributyrin was added to actinomycetes isolation agar. The pH of the medium was adjusted to 7.3–7.4 using 0.1 N NaOH. A loopful of inoculum was streaked on to the tributyrin agar plates. The plates were incubated at 26 ◦ C for 7 days. After incubation, the plates were examined for the formation of clear zone around the colonies. 2.3. Emulsification index The emulsification index was measured using the method described by Cooper and Goldenberg [7] in which 2 ml of the kerosene was added to equal volume of cell free supernatant and homogenized in a vortex at high speed for 2 min. The emulsification stability was measured after 24 h and the emulsification index was calculated by dividing the measured height of the emulsion layer by the total height of the liquid layer and multiplying by 100. The emulsification activity of the strain was compared with the standards including SDS and Tween 80. E24 (%) =
total height of the emulsified layer total height of the liquid layer
2.4. Identification of the strain MSF3 Identification and characterization of the strain MSF3 were carried out with the methods described by Gilman [8]. The morphological and colony appearances were observed by naked eye examination of 3 days old culture grown on SDA medium. The micomorphology and sporulation was observed by light microscopy with LCB (Lactophenol Cotton Blue) staining. 2.5. Cultivation condition The fungal strain MSF3 was grown on SDA (peptone—10 g, dextrose—40 g, agar—15 g, distilled water—1000 ml), potato dextrose agar (PDA) (potato (peeled)—200.0 g, dextrose—20.0 g, agar—15.0 g, distilled water—1000 ml), Zobell Marine broth 2216 (ZMB 2216) (glucose—10.0 g, peptone—5.0 g, yeast extract—1 g, ferric citrate—0.1 g, sodium sulphate—3.24 g, calcium chloride—1.50 g,
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Na2 HCO3 —0.16 g, H3 BO3 —0.22 g, SnCl2 —0.034 g, sodium silicate— 0.004 g, sodium fluorate—0.0024 g, NaNO3 —0.0016 g, Na2 PO4 — 0.008 g, NaCl2 —19.45 g, MgCl2 —8.80 g, KCl2 —0.55 g, KBr—0.08 g, pH 7.6 ± 0.2, distilled water—1000 ml) and MSM (NaNO3 — 15 g, KCl—1.1 g, FeSO4 ·7H2 O—0.00028 g, KH2 PO4 —3.4 g, K2 HPO4 — 4.4 g, MgSO4 ·7H2 O—0.5 g, yeast extract—0.5 g). Trace elements— ZnSO4 ·7H2 O—0.29 g, CaCl2 ·4H2 O—0.24 g, CuSO4 ·5H2 O—0.25 g, MnSO4 ·H2 O—0.17 g/100 ml) medium inorder to determine the growth conditions. The strain MSF3 was inoculated in a 250 ml Erlenmeyer flask containing 50 ml of SDA medium and incubated for 120 h at 20 ◦ C. The cell free supernatant (CFS) was filtered through filter paper and cold centrifuged (Eppendorff 5804 R) at 8000 × g at 4 ◦ C for 20 min. Emulsification activity was determined using the method of Cooper and Goldenberg [7]. 2.6. Time course of biosurfactant production To obtain the production curve, the culture was inoculated in 1000 ml Erlenmeyer flasks each containing 300 ml production media. The experiment was designed for 120 h starting from the log phase to stationary phase under submerged culture conditions. The resultant CFS was removed by filtration followed by cold centrifugation at 8000 × g at 4 ◦ C for 20 min. The resultant CFS was analyzed for emulsification activity and the dry weight of the biomass was weighed using the physical balance. 2.7. Optimization of biosurfactant production To optimize the culture conditions for biosurfactant production, this strain was cultured under the conditions presented in Table 1. The culture was inoculated in the production medium and incubated at 120 h. The resultant CFS was removed by filtration followed by cold centrifugation at 8000 × g at 4 ◦ C for 20 min. The resultant CFS was analyzed for emulsification activity and biomass dry weight. 2.8. Response surface methodology (RSM) experimental design Response surface methodology (RSM) [9,10] is an empirical technique employed for multiple regression analysis by using quantitative data obtained from properly designed experiments to solve multivalent equations simultaneously. The graphical representations of these equations are called response surfaces. A 22 full-factorial central composite design for two test variables with corresponding star points and centre points was employed to fit the procedure and analyzed using the Design Expert Software (version 7.0). Based on the ANOVA of the quadratic regression model demonstrates that the model is highly significant, as is evident from the Fischer F test The two factors carbon (glucose) and nitrogen (yeast extract) were analyzed for the biosurfactant production using this software which is the independent variable. 2.9. Extraction of biosurfactant The strain MSF3 was grown on 250 ml Erlenmeyer flask containing 100 ml of SDA medium and incubated at 120 h at 20 ◦ C. Table 1 Culture conditions for the optimization of biosurfactant production by MSF3. Factors
Ranges
pH Temperature Salinity Carbon sources Nitrogen source Metals
4, 5, 6, 7, 8, 9 10, 20, 30, 40, 50 ◦ C 1.0, 1.5, 2.0, 2.5, 3.0, 3.5% Glucose, paddy straw, olive oil, kerosene, vegetable oil Peptone, yeast extract, beef extract, NaNO3 , urea FeSO4 , CuSO4 , MnCl2 , CaCl2 , MgCl2
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Culture samples were centrifuged at 12,000 × g for 20 min at 4 ◦ C to remove the cells as well as debris and the supernatant was filtered through 0.2 m filter. Filtered supernatant and the harvested cells were used for the extraction of surfactive compounds. Extraction was performed by acid precipitation followed by liquid–liquid extraction method, in both the cell free supernatant (CFS) and the harvested cells respectively. The CFS and the pellet was acidified with conc. HCl to attain a pH 2.0 and extracted with an equal volume of solvents such as ethyl acetate, diethyl ether and dichloromethane. The resultant aliquot was concentrated to dryness in a rotary vacuum evaporator (Yamato) and tested for emulsification activity as described above. 2.10. Chemical analysis of biosurfactant 2.10.1. Analytical methods To identify the chemical nature of the compound, the residue was applied in preparative silica gel TLC plates. Chromatograms were developed with 96% ethanol:water (7:3) for amino acids; chloroform:acetic acid:water (60:30:10) for sugars and chloroform:methanol:water (65:25:4) for lipids. Fractions were isolated and eluted with corresponding buffer and subjected to quantification of macromolecules such as protein, carbohydrate and lipid. Protein was estimated using the method of Lowry et al. [11], using bovine serum albumin as standard. Carbohydrate with phenol–sulphuric acid method by Chaplin and Kennedy [12], with glucose as standard and lipid was estimated for free fatty acid using the method of Sadasivam and Manickam [13] with cholesterol as standard. 2.10.2. Stability of biosurfactant Stability of the surface active compound was determined by dissolving the TLC eluted fraction in 1–2% of NaCl and the emulsification index was calculated. The stability of the biosurfactant at different pH values was performed by dissolving the biosurfactant in 0.1 M sodium acetate buffer (pH 4.0–7.0) and 0.1 M sodium phosphate buffer (pH 8.0–9.0). After 1 h of incubation with reciprocal agitation, the emulsification activity was measured as previously described. The stability of the biosurfactant at different temperatures was carried out by incubating the surface active compound at 0.4% (w/v) in water for 30 min from 25 to 120 ◦ C before measuring the emulsification activity. All these factors were compared to that of the corresponding solution in water. 2.10.3. Antimicrobial activity of surfactive compound The extracted compound as well as the culture supernatant was tested for antimicrobial activity using well diffusion method and area of the zone was calculated [14]. Extracted active compounds were tested against human pathogens such as Candida albicans, E. coli, Proteus mirabilis, hemolytic Streptococcus, Pseudomonas aeruginosa, Micrococcus luteus, Staphylococcus epidermidis, Enterococcus faecalis, Klebsiella pneumoniae, Bacillus subtilis, and Staphylococcus aureus. Mueller Hinton agar (beef infusion solids 4.0 g, starch 1.5 g, casein hydrolysate 17.5 g, agar 15.0 g, final pH 7.4 ± 0.2 at 37 ◦ C) plates were prepared and swabbed with appropriate pathogen. Using a sterile cork borer well was made and 50 l of extracted compound was added in wells, incubated at 30 ◦ C for 24 h. After incubation, the clear zone was measured and calculated. 2.10.4. Microbially enhanced oil recovery (MEOR) (sand pack test) The potential application of the biosurfactant in MEOR was evaluated using the ‘sand pack column’ technique described by Abu-Ruwaida et al. [15]. A glass column was packed with 100 g of acid washed dry sand .The column was then saturated with different hydrocarbon (50 ml). The potential of the isolated surfactant
for oil recovery was estimated by pouring 50 ml of aqueous solution of biosurfactant (extraction from 100 ml culture broth) in the column. The amount of oil released was measured. The experiment was carried out at room temperature, 30, 50 and 70 ◦ C to assess the influence of temperature on biosurfactant-induced oil recovery. 3. Results and discussion 3.1. Isolation and screening of biosurfactant producing fungi Based on the colony morphology and stability in subculturing, 8 sponge-associated marine fungus were isolated from the marine sponge F. cavernosa. Among these 4 strains showed positive result for biosurfactant production, particularly CFS of the strain MSF3 exhibited highest emulsification activity (42.8%) followed by MSF5 (17.5%), MSF1 (11.4%) and MSF8 (7.5%). This strain was selected for characterization and optimization of the biosurfactant production. Marine sponges (Porifera) have attracted significant attention from various scientific disciplines. As sponges produce various novel chemical molecules, they have been a goldmine to chemists and also found their way into biotechnological applications. Microbiologists became fascinated by these unique animals with the discovery that sponges contain an abundance of unusual microorganisms having potential for drug discovery and bioremediation [16]. 3.2. Screening of biosurfactant production Hemolytic activity of MSF3 showed a clear zone diameter 9 mm around the colony. In the present study, a significant correlation was established between the hemolytic activity and biosurfactant production. According to Carillo et al. [4], and Banat [17], biosurfactant production of the new isolates was preliminary screened by hemolytic activity. Blood–agar lysis has been used to quantify surfactant [18] and rhamnolipids [19]. Carillo et al. [4] found an association between hemolytic activity and surfactant production and they recommended the use of blood agar lysis as a primary method to screen biosurfactant production. The drop collapsing test, oil displacement method and lipase activity was also performed as a part of screening. In drop collapsing test a flat drop was observed and in oil displacement method, a clear diameter of 5 mm was observed and the area was calculated as 78.50 mm2 (Table 2). From the above two observation, it was confirmed that the strain MSF3 was a biosurfactant producing marine fungi. Both the techniques have several advantages: small volume of samples was required, rapid and easy to carry out and also do not require specialized equipment. 3.3. Identification of the biosurfactant producer Colonies more or less felted, floccose, with fine hyphae, from white through shades of gray, olive-gray, yellow, yellow-brown toward fuscous, with often a greenish cast, but no true green color, in old cultures purplish and vinaceous at times. Reverse through shades of yellow, orange and brown. Stalks when rising from submerged hyphae up to 1000 m, most branches of aerial hyphae up to 500 × 5–10 m, few septate, sinuous, with walls rather thin, Table 2 Results on the screening of biosurfactant production for the strain MSF3 in SDA medium. Screening tests
Results
Hemolytic activity Drop collapsing test Oil displacement test Lipase activity
9 mm + 78.50 mm2 75 U/mg
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tionary phase. The results obtained was compared with other fungal isolates Candida lipolytica [20] and the marine yeast Yarrowia lipolytica [21] which excreted biosurfactant by microbes only during the stationary phase, significant emulsification activity was detected during the exponential phase of growth being an advantage as it enhance productivity. 3.6. Optimization of biosurfactant production Cell growth and the accumulation of metabolic products were strongly influenced by medium composition such as carbon sources, nitrogen sources, salinity and other growth factors, thus the optimization of organisms can give results in the high yield of metabolites [22].
Fig. 1. Biosurfactant production using different medium at 20 ◦ C for 120 h with the corresponding pH of each medium. Biomass was quantified in mg and the biosurfactant production was denoted in terms of E24 .
smooth, usually partly colored some shade of brown; vesicles 10–20 m in diameter; heads hemispherical to almost columnar; phialides colorless, semi-radiate, loosely arranged into two series, primary 5–8 × 3 m, secondary 7–9 × 2–2.5 m. Conidia globose about 3.6 m, spinulose or with fine faint bars of rosy, reddish yellow or vinaceous color, with chains forming fairly compact columns in old cultures. 3.4. Cultivation conditions and biosurfactant production The isolated strain MSF3 produced biosurfactant when grown in various nutrients. However, the amount of biosurfactant secreted varied in the presence of different nutrient sources of the four investigated culture medium, maximum biosurfactant production was observed in SDA (45%) followed by ZMB (15%). There was no production of biosurfactant in MSM and PDA (Fig. 1). So SDA was selected as the biosurfactant production medium for the strain at 20 ◦ C for 120 h. 3.5. Time course of biosurfactant production
3.6.1. pH and temperature The important characteristics of most organisms are their strong dependence on the pH for cell growth and production of secondary metabolites. The strain MSF3 produced the highest yield of biosurfactant (15%) at pH 7.0 (Fig. 3) even though the growth was higher in pH 8 and 9. There was no correlation established between the growth and biosurfactant production. B. subtilis was able to produce biosurfactant in a pH range of 6.0 to 9.0, although the maximal yield of the biosurfactant was obtained at pH 7.0 [23]. A change in temperature caused alterations in the composition of the biosurfactant in the case of A. paraffineus [24] and Pseudomonas sp. [25]. Temperature was one of the critical parameters that have been controlled in bioprocess. The results in the present study revealed that the biosurfactant activity reached the highest when the strain was grown at 20 ◦ C (20%) as it is a marine strain (Fig. 4). 3.6.2. Salt concentration The salinity was found to be one of the critical parameter in the production of biosurfactant, in the absence of the salt production and growth was very low. Since it is a marine isolate it showed optimum activity in the salt supplemented medium (Fig. 5). The emulsification activity was highest in the production medium contained 3% NaCl. Similar observation was made with the protease production by marine Roseobacter MMD40 [26].
Under the optimal conditions, 75% biosurfactant activity was reached in the culture of the fungal isolate at 72 h of the fermentation medium. When the cell growth reached the late log phase (Fig. 2) there was no biosurfactant activity during the early and midexponential phase. However, the biosurfactant production reached a peak by the end of the exponential phase and continued in the sta-
3.6.3. Multivalent cations Limiting multivalent cation concentrations also causes overproduction of biosurfactant [27]. Guerra-Santos et al. [28] demonstrated that limiting the concentrations of salt of magnesium, calcium, potassium and trace elements resulted in a better
Fig. 2. Growth kinetics and biosurfactant production by the marine fungus MSF3 for 6 days at 20 ◦ C in SDA medium. Biomass was quantified in mg and the biosurfactant production was denoted in terms of E24.
Fig. 3. Effect of different pH on biosurfactant production at 20 ◦ C for 120 h in SDA medium with 3% salinity. Biomass was quantified in mg and the biosurfactant production was denoted in terms of E24.
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Fig. 4. Effect of temperature on biosurfactant production at 120 h with pH 7.0 and 3% salinity in SDA medium. Biomass was quantified in mg and the biosurfactant production was denoted in terms of E24.
yield of rhamnolipid in P. aeruginosa DSM 2659. In our study, supplementation of metal ions increased the biosurfactant production. In the present study, biosurfactant production was increased in FeSO4 (75%) followed by MgCl2 (60%) with a concentration of 0.1 mM (Fig. 6). However the increased supplementation drastically inhibits the biosurfactant production. The total yield of biosurfactant higher in the production medium by adding the metal cations altogether
Fig. 7. Effect of carbon source on biosurfactant production at 20 ◦ C for 120 h with pH 7.0 and 3% salinity in SDA medium with 1% carbon source. Biomass was quantified in mg and the biosurfactant production was denoted in terms of E24 .
than the individual addition of cations. Similar results are observed for the biosurfactant production in B. subtilis MTCC 2423 [29]. 3.6.4. Carbon and nitrogen sources The present study envisaged that the supplementation of glucose as carbon source increase the biosurfactant production (35%) followed by the cheapest raw material paddy straw (30%) (Fig. 7). Many studies revealed the presence of glucose in the production medium increased the biosurfactant production in the culture medium [27,2–31]. Nitrogen source also plays an important role in the production of biosurfactant by microorganisms. Desai and Banat [1] reported that the limitation of nitrogen source increased the biosurfactant production. In the present study, the supplementation of yeast extract (25%) as well as NaNO3 (10%) showed little increase in the production medium (Fig. 8). Ouled-Hadder et al. [32] reported that NaNO3 was a good substrate for the growth with good productivity. It was also reported that NaNO3 and yeast extract used for the production of biosurfactant from different Bacillus sp. [33].
Fig. 5. Effect of salt concentration on biosurfactant production with pH 7.0 in SDA medium at 20 ◦ C for 120 h. Biomass was quantified in mg and the biosurfactant production was denoted in terms of E24 .
3.6.5. Effect of carbon–nitrogen ratio on biosurfactant production The carbon–nitrogen ratio is one of the most important factors which induce the secondary metabolites production. Therefore a wide range of the ratios from 5:1 to1:5 between the glucose and yeast extract were studied for achieving an optimal biosurfactant production. The results suggested that the peak yield of biosurfactant appeared at the ratio of 3:2 whereas the ratio at 1:5 yielded no
Fig. 6. Effect of multivalent cations (01 mM) on biosurfactant production at 20 ◦ C for 120 h with pH 7.0 and 3% salinity in SDA medium. Biomass was quantified in mg and the biosurfactant production was denoted in terms of E24 .
Fig. 8. Effect of nitrogen source on biosurfactant production at 20 ◦ C for 120 h with pH 7.0 and 3% salinity in SDA medium with 1% nitrogen source. Biomass was quantified in mg and the biosurfactant production was denoted in terms of E24 .
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Table 3 Effect of carbon–nitrogen ratio on biosurfactant production at 20 ◦ C for 120 h in SDA medium (pH 7). Carbon nitrogen ratio
E24
5:1 3:2 2.5:2.5 2:3 1:5
30% 75% 45% 20% 5%
biosurfactant production (Table 3). The results were supported by Guerra-Santos et al. [27,28], the increased rhamnolipid production under the limitation of nitrogen rather than carbon sources. 3.7. Statistical analysis of the carbon–nitrogen source using RSM The three dimensional response surface plot was represented in Fig. 9. The model proposed for the response emulsification activity (y) was: y = +25.00 + 9.31 × A + 6.66 × B − 3.75 × A × B + 15.00 × A2 + 13.75 × B2 The ANOVA of the regression model demonstrates that the model is highly significant, as is evident from the Fischer’s F test (F model = 40.49) and also indicated the treatment levels are highly significant with a very low probability value (P model > F = 0.0001) with degree of freedom 5. 3.8. Extraction and chemical analysis of biosurfactant Acid precipitation of the CFS followed by solvent extraction revealed that the biosurfactant production was highest in the CFS of the ethyl acetate extract not in the intracellular extract. TLC of the ethyl acetate extract showed a single spot in the chromatogram of lipid, carbohydrate and protein with Rf values 0.84, 0.73 and 0.80 respectively. From the TLC fractions the macromolecules was estimated and it found to be as follows: protein 925 g/ml, carbohydrate 1865.8 g/ml and lipid 382 g/ml. From the estimation and TLC analysis, the compound was partially characterized as glycolipoprotein.
Fig. 10. Comparison of the biosurfactant extractives of the fungi MSF3 with the standards SDS and Tween 80.
3.9. Stability and comparison of biosurfactant production The biosurfactant produced by this strain exhibited greatest activity in the range of 6–8 with an optimum range of pH 7.0 and in temperature the stability was observed from 20 to 40 ◦ C, it was unstable in higher temperature whereas the biosurfactant produced by bacterial isolates were stable at higher temperature (60–120 ◦ C) [29]. The biosurfactant produced by MSF3 was compared with the synthetic surfactant SDS and Tween 80. The results showed a 40% increase over the synthetic surfactants (Fig. 10). Therefore the strain MSF3 can be used for the scaled-up production of biosurfactant. 3.10. Antimicrobial activity of surface active compound The ethyl acetate extract of fungi MSF3 showed a wide activity against the pathogenic culture. In the ethyl acetate extract C. albicans, hemolytic Streptococcus, M. luteus, E. faecalis, P. aeruginosa and S. epidermidis showed a wide range of activity (Fig. 11). This marine fungal diethyl ether extract under this study also showed a highest activity towards the yeast C. albicans and Gram-negative bacterium. According to Tsuge et al. [34], lipopeptide surfactants are potent antibiotics mainly the surfactin, streptofactin and gramicidin produced by the microorganism had the wide antimicrobial activity [35–37] compared to the glycolipid producing strain. A glycolipid surfactant from the C. antartica has demonstrated antimicrobial activity against Gram-positive bacteria. 3.11. Microbially enhanced oil recovery
Fig. 9. Three dimensional plot on the effect of carbon–nitrogen ratio on the emulsification activity.
The MEOR was analyzed by the sand pack method. Hydrocarbon saturated sand pack column with the biosurfactant extract was treated and subsequently incubated at room temperature, 50 and 70 ◦ C caused release of the hydrocarbon from the column. The released hydrocarbon was quantified and the data thus obtained are presented graphically in Fig. 12. The biosurfactant produced by this organism was stable when exposed to higher temperature thus the surface tension of oil molecules was reduced. From this it was envisaged that since this microorganism are present in the marine environment, it helps in the biodegradation of the oil spillages of the marine environment. As a result it may perhaps leads to the reduction of mortality rate of the marine creatures and increased the
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high price of productivity and low yields, the biosurfactants are not competitive with chemical surfactants. In the present study, we demonstrated the sponge-associated marine fungi as potential source of biosurfactant. The strain MSF3 had the ability to degrade the hydrocarbons in a large amount compared to the synthetic surfactants. Acknowledgements GSK is thankful to CSIR, New Delhi for the award of SRF. JS is thankful to Ministry of Earth Sciences (MoES), New Delhi for financial support. References
Fig. 11. Antimicrobial activity of biosurfactant (ethyl acetate extract) produced by marine fungus MSF3.
Fig. 12. Microbially enhanced oil recovery by the marine fungus MSF3 under different temperatures.
dissolved oxygen level as well as the light penetration. In addition, the future use of this biosurfactant as broad spectrum of antibiotics is highly warranted. 4. Conclusion Sponge-associated marine microbes have been a goldmine for microbiologists and many research workers. At present due to
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