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Optimization of cultural conditions for biosurfactant production by Pleurotus djamor in solid state fermentation
Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e6, 2015 www.elsevier.com/locate/jbiosc
Optimization of cultural conditions for biosurfactant production by Pleurotus djamor in solid state fermentation Zulfiye Velioglu1, * and Raziye Ozturk Urek2 Biotechnology Department, Graduate School of Natural and Applied Sciences, Dokuz Eylül University, 35160 Buca, Izmir, Turkey1 and Chemistry Department, Biochemistry Division, Faculty of Science, Dokuz Eylül University, 35160 Buca, Izmir, Turkey2 Received 15 October 2014; accepted 5 March 2015 Available online xxx
Being eco-friendly, less toxic, more biodegradable and biocompatible, biological surfactants have higher activity and stability compared to synthetic ones. In spite of the fact that there are abundant benefits of biosurfactants over the synthetic congeners, the problem related with the economical and large scale production proceeds. The utilization of several industrial wastes in the production media as substrates reduces the production cost. This current study aims optimization of biosurfactant production conditions by Pleurotus djamor, grown on sunflower seed shell, grape wastes or potato peels as renewable cheap substrates in solid state fermentation. After determination of the best substrate for biosurfactant production, we indicate optimum size and amount of solid substrate, volume of medium, temperature, pH and Fe2D concentrations on biosurfactant production. In optimum conditions, by reducing water surface tension to 28.82 ± 0.3 mN/m and having oil displacement diameter of 3.9 ± 0.3 cm, 10.205 ± 0.5 g/l biosurfactant was produced. Moreover, chemical composition of biosurfactant produced in optimum condition was determined by FTIR. Lastly, laboratory’s large-scale production was carried out in optimum conditions in a tray bioreactor designed by us and 8.9 ± 0.5 g/l biosurfactant was produced with a significant surface activity (37.74 ± 0.3 mN/m). With its economical suggestions and applicability of laboratory’s large-scale production, this work indicates the possibility of using low cost agro-industrial wastes as renewable substrates for biosurfactant production. Therefore, using economically produced biosurfactant will reduce cost in several applications such as bioremediation, oil recovery and biodegradation of toxic chemicals. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Pleurotus djamor; Solid state fermentation; Biosurfactant; Optimization; Sunflower seed shell]
Surfactants establish an important class of industrial chemicals widely used in modern household and industry. Most of these compounds are synthesized chemically and petroleum-originated, toxic to the environment and not easily biodegradable (1). Their usage in such large quantities indicates that their waste and the potential adverse effects for environment and health are significant. However, rapid improvement in biotechnology has provided invention and development of possible alternatives as biological surfactants. Biosurfactants, biological surfactants, are structurally diverse compounds which exhibit particularly high surface activity and emulsifying activity (2). The three main classes of biosurfactants are glycolipids, lipopeptides and polymeric biosurfactants. They are widely used in the pharmaceutical, biomedical, cosmetic, petroleum and food industries as well as environmental and agricultural applications. Due to high ecological acceptability, less toxicity, easily degradability and biocompatibility, biosurfactants have gained importance recently. These molecules also have higher activity and stability than synthetic surface active agents (3).
* Corresponding author. Tel.: þ90 232 301 8689; fax: þ90 232 453 4188. E-mail addresses: zulfi[email protected] (Z. Velioglu), raziye.urek@ deu.edu.tr (R. Ozturk Urek).
Although there are significant benefits of biosurfactants over the synthetic surfactants, their economical and large scale production has difficulties (4). Using various industrial wastes in the formulation of production media as a substrate is one of the approaches to decrease production cost (5). Biodegradation of these wastes into biotechnological products has double benefit as economical production and a decrease in environmental pollution. The properties of used waste as substrate in the medium and fermentation type are significant factors that influence the biosurfactant production and activity (5e7). Especially agro-industrial wastes such as oily wastes from vegetable oil refineries, starchy materials wastes from potato processing industries were also reported as good substrates for biosurfactant production (4). Solid state fermentation (SSF) is a suitable fermentation system using agro-industrial wastes as cheap substrates (8). In addition, the nature, chemical structure, activity and the amount of produced biosurfactant depends on the producer microorganism (5). As fungi grow in nature on solid substrates, SSF is a productive fermentation when fungi are used (9). Pleurotus spp. belongs to the genus Pleurotus (Quel.) Fr., tribe Lentineae Fayod, family Polyporaceae (Fr.) Fr., and they are saprophytic, white rot fungi which are cultivated on substrates containing lignin or cellulose such as agro-industrial wastes. Utilization of these materials is dependent on lignolytic enzymes
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.03.007
Please cite this article in press as: Velioglu, Z., and Ozturk Urek, R., Optimization of cultural conditions for biosurfactant production by Pleurotus djamor in solid state fermentation, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.007
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secreted by Pleurotus spp. (10). Although, there are several studies on lignolytic enzyme production by Pleurotus spp. as far as we know, there is one of the publications which indicate biosurfactant production potential of Pleurotus spp. showing production of an emulsifying agent by Pleurotus ostreatus (11). This current study aims to optimize biosurfactant production condition by P. djamor, in SSF using sunflower seed shell, grape waste or potato peels as sustainable low-cost substrates. In the first part of the study, the best substrate for biosurfactant production in SSF was determined. In the second part, we investigated effects of size and amount of solid substrate, volume of medium, temperature, pH and Fe2þ concentrations to optimize biosurfactant production conditions. The biosurfactant production was screened by oil spreading technique, emulsification index and surface tension assay. Moreover, biosurfactant produced in optimum production conditions was further characterized by Fourier transform infrared spectroscopy (FTIR) and its chemical composition was determined. Lastly, laboratory’s large-scale production was carried out in optimum conditions.
MATERIALS AND METHODS Organism and culture conditions P. djamor (Rumph. Ex Fr.) Boedijn (MCC15) was grown on potato dextrose agar (39 g/l) at 25 C for 7 days (7) and was stored at 4 C. For SSF in control condition, biosurfactant production medium was a Gıda, Izmir, Turkey), grape prepared with 5 g sunflower seed shell (from Dog wastes (dried at 60 C for 24 h) (from Dimes, Izmir, Turkey) or potato peels (dried at 60 C for 24 h) (from Izmir, Turkey) in 250-ml Erlenmeyer flasks. The used substrates were chosen as 1 substrate particle/cm2 surface area. The medium (pH 6.0) contained (g/l) NH4NO3, 0.724; KH2PO4, 1.0; MgSO4$7H2O, 1.0; KCl, 0.5; yeast extract, 0.5; FeSO4$7H2O, 0.001; ZnSO4$7H2O, 0.0028; CaCl2$2H2O, 0.033 and peptone, 10.0 (12). Medium included 10 ml/l sunflower seed oil, as second carbon source. After sterilization (at 121 C for 20 min), 10 ml liquid medium was added into solid substrate (humidity 70%) and agar plugs (1 cm2) were inoculated. Flasks were incubated at 29 C and samples were extracted on 13th day of incubation period. According to our previous study, maximum biosurfactant production was determined on 13th day (13). For extraction, each flask received 25 ml of distilled water and was agitated for 1 h at 150 rpm, 29 C (13). The suspension was centrifuged at 5000 rpm and 4 C for 15 min and supernatant was used for analysis determining below. Chemical analysis of solid substrate Lignin concentrations of solid substrates used in SSF were determined by thioglycolic acid lignin analysis method (14). Analysis of cellulose content was performed by Kürschner-Hanack method (15). Optimization of culture condition After determination of the best solid substrate for biosurfactant production, we used this substrate in optimization steps. First of all the amounts of solid substrate (with 1 cm2 surface area) were changed as 1, 3, 5, 7 and 10 g. Also solid substrate which had 0.25 cm2 surface area was used in amounts of 1, 3, 5, 7 and 10 g. After determination of optimum surface area and amount of solid substrate, medium volume was changed as 10, 25 and 50 ml. Lastly three different fermentation temperatures (25 C, 29 C and 35 C), four different pH levels (pH 5.5, 6, 7 and 8), different concentrations of Fe2þ (0, 3.5, 18 and 35 mM) were analyzed. During optimization steps, levels of protein (16), reducing sugar (17), total carbohydrate (18) and nitrogen (19) were determined in the extracted supernatant. In addition, uronic acid (20) assay was used. As liquid medium containing sunflower seed oil, we investigated lipase activity using p-nitrophenyl palmitate (pNPP) as the substrate (21). One unit (U) was defined as the amount of enzyme that liberated 1 mmol p-nitrophenol per min. Emulsification index measurements Emulsification index was measured according to Pinto et al. by combining 3.5 ml of extracted supernatant and 2 ml of sunflower seed oil (22). The mixture was agitated in a vortex agitator at high speed for 1 min. Nonfermented culture medium was used as blank. The emulsion index was calculated after 24 h (E24) and 48 h (E48) by the height of the emulsion layer divided by the total height, and multiplied by 100 (Eq. 1) and emulsifying activity determined according to Eq. 2. Emulsification index stability was preserved value of emulsification index for 24 h. EðsampleÞ ¼
Hðemulsion layerÞ *100 HðtotalÞ
(1)
where H is height. EI ¼ ðEðsampleÞ EðblankÞÞ*D where D is dilution of sample in water.
(2)
Oil spreading test Sunflower seed oil (200 ml) was added onto the surface of distilled water filled in a 50 ml Petri dish to form a layer on the surface. 20 ml extracted supernatant was gradually added to the centre of oil layer. The diameter of the clear zone on the oil surface was measured in a relation to biosurfactant concentration (23). Determination of surface tension The extracted supernatants obtained during optimization process in SSF were used for the determination of the surface tension using a Sigma 701 digital surface tensiometer (KSV Instruments Ltd., Finland) and working on the principle of the Du Nuoy ring method at room temperature. The surface tension measurement was carried out by immersing the platinum ring into the sample for a while, in order to attain the equilibrium. We use surface tension results as base to determine optimum conditions. Isolation and chemical analysis of biosurfactant To isolate produced biosurfactant in optimum conditions, the concentrated HCl was added with 20 ml extracted supernatant to bring final pH of 2.0 and kept for overnight at 4 C (24). Resulted precipitation was collected by centrifugation at 5000 rpm and 4 C for 30 min. The supernatant was removed and 10 ml of chloroform: methanol (2:1, v/ v) was added to precipitate pellet and incubated in a rotary shaker at 30 C and 200 rpm for 20 min. The content was centrifuged at 5000 rpm and 4 C for 30 min and the supernatant was evaporated by air drying (25). Levels of protein (16), reducing sugar (17), total carbohydrate (18), nitrogen (19) and lipid (26) were determined to demonstrate chemical composition of biosurfactant isolated from optimum production medium. Also uronic acid (20) assay was used. In addition we investigate lipase activity of isolated biosurfactant (21). Fourier transform infrared spectroscopy The FTIR spectra were recorded on the Perkin Elmer Spectrum BX, in the spectral region of 4000e400 cm1. The biosurfactant, produced in optimum condition was analyzed and a KBr pellet was used as a background reference. Laboratory’s large-scale production in SSF According to obtained results, we produced biosurfactant in laboratory’s large-scale in optimum conditions. We used 35 g sunflower seed shell and 70 ml medium (included 10 ml/l sunflower seed oil). A tray, 24 cm diameter and made by heat-resistant glass, was designed as bioreactor. Sample of 13th day was extracted. Surface tension and emulsification index of extract were measured. Statistical analysis The Tukey test was used for statistical significance analyses. The values were the mean of three separate experiments. Comparisons were also made with Pearson’s correlation.
RESULTS This current study focused on optimization of biosurfactant production conditions by P. djamor in SSF with various industrial wastes such as carbon sources, complex substrates. Using several industrial wastes, which are substrate in green biotechnological production, is important for economical production as well as being an alternative solution to environmental problems. In this current study, we determined the most suitable solid substrate for biosurfactant production in the first step and then we optimized production conditions in the second step. Lastly, we produced biosurfactant in laboratory’s large-scale according to obtained optimum conditions. Determination of suitable solid substrate for biosurfactant production In order to determine suitable solid substrate, we analyzed surface tension of biosurfactants produced in control conditions with three different wastes as carbon source. Surface tension values of biosurfactant produced on sunflower seed shell, grape wastes and potato peels were 29.79 0.3, 42.6 0.4 and 34.66 0.3 mN/m, respectively. According to these results sunflower seed shell was established as the most suitable solid substrate and also its lignin and cellulose concentrations were detected as 18.65 1.1 and 45 3.8%, respectively. Determination of optimum amount and surface area of solid substrate for biosurfactant production First of all, we determined optimum amount and surface area of sunflower seed shell (Fig. 1) in optimization steps, after choosing the best solid substrate. As mentioned above, the maximum biosurfactant activity was determined on 13th day of incubation (13), so in this present study samples of 13th day were analyzed. As minimum surface tension value (29.79 0.3 mN/m) was detected in control condition, optimum surface area and amount of solid substrate were determined as 1 cm2 and 5 g, respectively (Fig. 1A and B).
Please cite this article in press as: Velioglu, Z., and Ozturk Urek, R., Optimization of cultural conditions for biosurfactant production by Pleurotus djamor in solid state fermentation, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.007
VOL. xx, 2015
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FIG. 2. Emulsification indexes (E24 and E48) and surface tensions values of produced biosurfactants with different volume of medium and different temperature. The values are the mean SD for experiments of three separate experiments.
pH value of culture medium. The minimum surface tension value (28.82 0.3 mN/m) was detected at pH 5.5 (Fig. 3). In this condition, biosurfactant maintained its emulsification index by 90 4.5% (p < 0.05). Also oil spreading activity was maximum (3.9 0.2 cm) in this condition. Considering the results, we changed pH value of control condition as 5.5.
FIG. 1. Effects of different amount of solid substrate on emulsification indexes (E24 and E48) and surface tensions of produced biosurfactants with 1 cm2 surface area (A) and 0.25 cm2 surface area (B). The values are the mean SD for experiments of three separate experiments.
Also, maximum E24 value (45.71 4.0%) has been determined in control condition and diameter of clear zone according to oil spreading was 3.5 0.2 cm. The emulsification index stability designates the strength of a surfactant and in control condition the produced biosurfactant conserved the emulsification index by 90.07 4.5% for 48 h. In control condition levels of protein, total carbohydrate, reducing sugar, lipid, nitrogen, and uronic acid were 5320 85 ppm, 7151 187 ppm, 183.2 8.9 mM, 1600.2 53 ppm, 18,691.3 252 ppm and 31.833 1.1 M respectively. Also, we determined lipase activity as 1.652 0.01 U/ml.
Determination of optimum Fe2D concentration for biosurfactant production In the last optimization step, effects of different Fe2þ concentrations on biosurfactant production were investigated (Fig. 4). There was no significant effect of Fe2þ concentration on biosurfactant production in investigated culture condition and concentrations (rs ¼ 0.157, p > 0.05). According to surface tension values, the optimum Fe2þ concentration was 3.5 mM (control condition). Furthermore, maximum emulsification index (E24 ¼ 44.44 4.0% and E48 ¼ 40 3.7%) and diameter of clear zone (3.9 0.3 cm) were determined in control condition. According to results we determined optimum conditions as surface area and amount of solid substrate 1 cm2 and 5 g, volume of medium 10 ml, temperature 29 C, pH level 5.5 and Fe2þ concentration 3.5 mM. In these condition, level of protein, total carbohydrate, reducing sugar, lipid, nitrogen, and uronic acid were 3214.29 98 ppm, 8969.69 102 ppm, 205.3 9.2 mM, 1395.5 42 ppm, 12,933.03 162 ppm, and 32,750 471 mM, respectively. Also we determined lipase activity as 1.467 0.01 U/ml.
Determination of optimum volume of medium and process temperature for biosurfactant production In the next optimization step, the volume of medium and process temperature was indicated (Fig. 2). The correlation between surface tension and medium volume was strong (rs ¼ 0.946, p < 0.05). According to surface tension results, optimum volume of medium was 10 ml (control condition). Also the maximum emulsification index (E24 ¼ 45.71 4.0% and E48 ¼ 41.17 4.0%) and oil spreading activity (3.5 0.2 cm) values were determined in control condition while no growth was observed in 50 ml medium. As maximum emulsification index values (E24 ¼ 45.71 4.0% and E48 ¼ 41.17 4.0%) and minimum surface tension value (29.79 0.3 mN/m) were observed in control condition, optimum temperature for biosurfactant production process was 29 C (Fig. 2). A meaningful correlation was determined between temperature and surface tension (rs ¼ 0.775, p < 0.05). Determination of optimum pH value of production medium for biosurfactant production The next step was optimization of
FIG. 3. Emulsification indexes (E24 and E48) and surface tensions values of produced biosurfactants with different pH values. The values are the mean SD for experiments of three separate experiments.
Please cite this article in press as: Velioglu, Z., and Ozturk Urek, R., Optimization of cultural conditions for biosurfactant production by Pleurotus djamor in solid state fermentation, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.007
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J. BIOSCI. BIOENG., E24 ¼ 34.21 3.2% and E48 ¼ 32.43 3.1% and also it conserved its stability by 94.8 4.8% (p < 0.05). The amount of produced biosurfactant was 8.9 0.5 g/l in laboratory’s large-scale. DISCUSSION
FIG. 4. Emulsification indexes (E24 and E48), and surface tensions of produced biosurfactants with different Fe2þ concentrations. The values are the mean SD for experiments of three separate experiments.
Partial chemical characterization of produced biosurfactant After optimization steps produced biosurfactant in optimum conditions was extracted and its partial chemical characterization was analyzed. The amount of extracted biosurfactant was 10.205 0.5 g/l. The chemical composition of 1 mg extracted biosurfactant was formed 92.38 5.2 mg protein, 15.8 0.5 mg total carbohydrate, 13 0.5 mg reducing sugar, 13.27 0.8 mg lipid, 56.2 2.4 mg nitrogen, and 14.97 0.5 mg uronic acid. Moreover lipase activity was determined as 0.695 0.01 U/mg extracted biosurfactant. The molecular composition of biosurfactant produced in optimum conditions was evaluated by FTIR (Fig. 5). The most important bands were located at 3294 cm1 (OeH stretching) and at 2991e2924 cm1 (CeH bands: CH2eCH3 stretching) was associated with the stretching vibration of CeH bond of constituent sugar residues. The peak at 1658 cm1 (C]O stretching) suggested the presence of carbonyl functionality present in carboxylate or amide moieties of protein and peptide amines and also the peaks at 1539 cm1 (NeH bending) were indication of proteins. The peaks at 1200e1400 cm1 were indication of hydrocarbon chain (13). Laboratory’s large-scale production After optimization and partial characterization of produced biosurfactant, we produced biosurfactant in laboratory’s large-scale in optimum conditions. As far as we are concerned, used bioreactor was designed and it was used for the first time in this study. The surface tension value of produced biosurfactant in laboratory’s large-scale was 37.74 0.3 mN/m. Emulsification index activities were
In this current study, we optimized biosurfactant production conditions by P. djamor on renewable substrates. First of all, we decided the most suitable solid substrate. Three different agroindustrial wastes, abundant around Izmir-Turkey, were used as solid substrates, According to surface tension values the best solid substrate was sunflower seed shell as it contains hydrocarbon that activates biosurfactant production due to its insoluble property. Such waste materials are mostly burned in order to dispose and release CO2 that advances to greenhouse effect. This study demonstrated the potential of bioconversion of these waste materials to value-added biotechnological products. In some studies glucose was used as carbon source which was an expensive substrate especially in large-scale production (27e29). In our study, sunflower seed oil was also used as second carbon source. According to Makkar and Cameotra producing biosurfactant from oily substrates is an advisable strategy of waste management and low cost production (1). It is important to note that the selection of suitable waste with the right balance of nutrients that permits cell growth and product accumulation. The lignocellulosic and oily property of sunflower seed shell permitted production of biosurfactant as well as lipase enzyme. In the next step of optimization experiments, we investigated the effects of amount and surface area of solid particles on biosurfactant production. Surface tension values did not show alterations except control condition. To surface tension and emulsification index values, the optimum amount and surface area of solid particles were in control condition. Also, according to oil spreading test, diameter of clear zone (3.5 0.2 cm) supported these results. Biosurfactants exhibit important roles in increasing bioavailability of substrates (30). Increment of surface area expedited getting substrates, thus microorganisms did not need large amounts of biosurfactant production. Similarly production with large amount of solid substrate made easier to reach substrate although microorganisms produced biosurfactant to get more nutrients in control condition. In addition, the effects of medium volume on biosurfactant production were searched. As humidity exceeded 70% no growth was observed in 50 ml medium. The minimum surface tension was determined in control condition besides the maximum emulsification index and oil spreading activity. In this present study, we
FIG. 5. FTIR spectra of biosurfactant produced by P. djamor in optimum conditions.
Please cite this article in press as: Velioglu, Z., and Ozturk Urek, R., Optimization of cultural conditions for biosurfactant production by Pleurotus djamor in solid state fermentation, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.007
VOL. xx, 2015 also aimed economic production, so the volume of used medium is an important factor that effects production cost. The optimum medium volume was determined as 10 ml which was suitable for an economic production. In order to get large amounts of biosurfactant, it is necessary to optimize the environmental conditions (5). Temperature is one of the important factors that affect type and amount of produced biosurfactant. In our study, surface tension values of biosurfactant produced at different temperature showed considerable changes. Hence temperature effects biochemical reactions in microorganism cells, changes in biosurfactant production were expected. The minimum surface tension and maximum emulsification index were determined at control condition temperature (29 C). pH is another environmental factor that affects yield and characteristics of the produced biosurfactant. In reference to Zinjarde and Pant (31), the best production of biosurfactant occurred at pH 8.0 while in another study (32) the production of biosurfactant reached maximum at pH 5.5. The optimum pH value shows changes according to the biosurfactant type, production condition and producer microorganism. We did not determine significant changes between surface tensions of biosurfactant produced at different pH values. The best surface tension value (28.82 0.3 mN/ m) was detected at pH 5.5. Lastly, effects of Fe2þ concentration on biosurfactant production were examined. Metal ions play an important role in biosurfactant production since they form cofactors of some enzymes (5). Fe is an important activator of isocitrate lyase enzyme which is involved in cell growth on hydrophobic substrates (33). In our study, there were not meaningful alterations in surface tension values according to chancing Fe2þ concentrations (rs ¼ 0.157, p > 0.05). Excessive concentration or deficiency of Fe2þ did not considerably effect biosurfactant production. Similarly in the study of Wei et al. (34), biosurfactant production by Bacillus subtilis was optimal with 4 mM Fe2þ. The authors supposed that poor growth was observed at low iron concentration due to the chelating effects of biosurfactant resulting in less iron for cell metabolism (34). At high iron concentration, growth was reduced due to the acidification of media. In this current study, we used sunflower seed oil as second carbon source and likewise in the study of Kalyani et al. (35), olive oil was used as carbon source to produce rhamnolipid by Streptomyces coelicoflavus and they produced 0.475 g/l biosurfactant in optimum conditions. Rufino et al. (36) studied the cultivation of Candida lipolytica grown on ground nut oil and produced 4.5 g/l biosurfactant. Similarly, a low-cost agricultural byproduct palm oil was used as carbon source and achieved biosurfactant amount was 2.3 g/l (37). In another study (38), sunflower and olive oil were used as carbon sources and 2.7 g/l biosurfactant produced with 32e36 mN/m surface tension. Different from studies mentioned above, we used two low cost substrates, as sunflower seed shell and oil, and the amount of produced biosurfactant was 10.205 0.5 g/l with high surface tension reducing activity (28.82 0.3 mN/m). In the economical production strategy over production is important as well as high activity of the product. As far as we are concerned this is the first study that indicated such over production and high activity of biosurfactant in SSF. Also, 8.9 0.5 g/l biosurfactant was produced in laboratory’s large-scale that reduced surface tension to 37.74 0.3 mN/m. When compared to similar studies, the achieved results in large scale production were significantly higher. Thaniyavarn et al. (39) examined biosurfactant production in large scale with olive oil and maximum amount of produced biosurfactant was 6.58 g/l. Optimization of laboratory scale to large scale is problematic and there are not always same results in different scale productions. The amounts and surface activities of produced biosurfactant in small and large scales were not as expected. Nevertheless, the determined results from large scale
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production demonstrated applicability of designed tray bioreactor and possibility of high production in case of optimization. In optimum conditions, levels of protein and lipid reduced while the level of total carbohydrate increased in reference to control condition. The increasing level of carbohydrate was supposed by the increase in level of reducing sugar, and uronic acid. Due to the reduced level of lipid, decrease of lipase activity in optimum conditions was an unexpected result. In a study of Açıkel et al. (40), the activity of lipase produced in optimum conditions was 0.964 U/ml. Although lipase production was not primer aim of this present study, the lipase activity (1.467 0.01 U/ml) was higher than several studies. In order to optimize lipase production conditions, it is also possible to achieve higher lipase activity in a biosurfactant production process. The produced biosurfactant also demonstrated considerable lipase activity (0.695 0.01 U/mg). Insoluble substrates can be resourceful for microbial growth; however, there is a great restriction on the availability of substrate by the cell (41). As microorganisms need to metabolize insoluble compounds, they synthesis lipase and biosurfactant (42). The main effect of the biosurfactants is to reduce the interfacial tension at the surface of substrate and to increase the availability of substrate by microorganism. In this current study, simultaneous production of lipase and biosurfactant facilitated utilization of oily substrates. Simultaneous and economic production of biosurfactant and lipase enzyme is important for economical applications like bioremediation of oil residues, waste management and cosmetic industry. The economically produced biosurfactant in control condition was analyzed by FTIR. In the FTIR spectrum of biosurfactant, since the peaks at 1539 cm1 showed the presence of protein, it could be concluded that the produced biosurfactants were protein containing type such as lipopeptide or polymeric biosurfactants (43,44). Besides, the peaks at 2991e2929 cm1 are found in structure of polysaccharide containing biosurfactants as glycolipids and polymeric ones (25,43,45,46). The obtained results suggested that produced biosurfactant possibly had protein-polysaccharide-lipid complex structure (12). Meanwhile, results of chemical composition analysis supported complex structure of biosurfactant produced. In conclusion, this current study demonstrated an active and economical biosurfactant production by P. djamor in SSF in determined optimum condition. In this condition 10.205 0.5 g/l biosurfactant was produced which reduced water surface tension to 28.82 0.3 mN/m. In laboratory’s large-scale production 8.9 0.5 g/l biosurfactant was produced, which was carried out in optimum condition in a tray bioreactor designed by us. With regard to dual product strategies lipase enzyme was simultaneously produced. Simultaneous and economic production of eco-friendly biosurfactant and lipase enzyme is important for environmental applications like mineralization of environmental contaminants and also in cosmetics and food industry. Furthermore, this study demonstrated the potential of bioconversion of waste materials to value-added biotechnological products. According to Syldatk and Wagner (47), the hydrophilic and hydrophobic moieties of biosurfactant are synthesized de novo by two independent pathways; One of these moieties is synthesized de novo while the synthesis of other moiety is induced by substrate, or both of the moieties have substrate dependent synthesis (47). In this current study, solid substrate has lignocellulosic and oily character, which might induce hydrophilic and hydrophobic moieties of produced biosurfactant. The second carbon source, sunflower seed oil, also might stimulate production of hydrophobic moiety while the carbohydrate groups from lignocellulosic waste induce production hydrophilic part. Large amounts of agroindustrial wastes are generated during the manufacture of agricultural products every year. Utilization of these by-products in biotechnological production processes has importance in
Please cite this article in press as: Velioglu, Z., and Ozturk Urek, R., Optimization of cultural conditions for biosurfactant production by Pleurotus djamor in solid state fermentation, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.007
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economical and ecological fields. For that reason, determination the feasibility of usage of these wastes as substrates to produce valuable biotechnological products has gained importance, recently. Our offer is a new bioconversion of sunflower seed shells to biosurfactant. ACKNOWLEDGMENTS We thank Dr. Ufuk Malayoglu for surface tension analysis and a Gıda and Dimes Meyve Suyu Izmir-Turkey for providing also Dog solid substrates. This work was supported by the Scientific and Technological Research Council of Turkey (grant number 113M801). References 1. Makkar, R. S. and Cameotra, S. S.: An update on the use of unconventional substrates for biosurfactant production and their new applications, Appl. Microbiol. Biotechnol., 58, 428e434 (2002). 2. Banat, I. M., Makkar, R. S., and Cameotra, S. S.: Potential commercial applications of microbial surfactants, Appl. Microbiol. Biotechnol., 53, 495e508 (2000). 3. Bhardwaj, G., Cameotra, S. S., and Chopra, H. K.: Biosurfactants from fungi: a review, Petrol. Environ. Biotechnol., 4, 160 (2013). 4. Makkar, R. S., Cameotra, S. S., and Banat, I. M.: Advances in utilization of renewable substrates for biosurfactant production, AMB Express, 1, 1e19 (2011). 5. Saharan, B. S., Sahu, R. K., and Sharma, D.: A review on biosurfactants: fermentation, current developments and perspectives, Gen. Eng. Biotechnol. J., 29, 1e39 (2011). 6. Massadeh, M. I., Fraij, A., and Fandi, K. G.: Effect of carbon sources on the extracellular lignocellulolytic enzymetic system of Pleurotus sajor-caju, Jordan J. Biol. Sci., 3, 51e54 (2010). 7. Stajic, M., Persky, L., Friesem, D., Hadar, Y., Wasser, S. P., Nevo, E., and Vukojevic, J.: Effect of different carbon and nitrogen sources on laccase and peroxidases production by selected Pleurotus species, Enzyme Microb. Technol., 38, 65e73 (2006). 8. Pandey, A., Selvakumar, P., Soccol, C. R., and Nigam, P.: Solid state fermentation for the production of industrial enzymes, Curr. Sci., 77, 149e162 (1999). 9. Bhargav, S., Panda, B. P., Ali, M., and Javed, S.: Solid-state fermentation: an overview, Chem. Biochem. Eng. Q., 22, 49e70 (2008). 10. Cohen, R., Persky, L., and Hadar, Y.: Biotechnological applications and potential of wood-degrading mushrooms of the genus Pleurotus, Appl. Microbiol. Biotechnol., 58, 582e594 (2002). 11. Nikiforova, S. V., Pozdnyakova, N. N., and Turkovskaya, O. V.: Emulsifying agent production during PAHs degradation by the white rot fungus Pleurotus ostreatus D1, Curr. Microbiol., 58, 554e558 (2009). 12. Bazalel, L., Hadar, Y., and Cerniglia, C.: Enzymatic mechanisms involved in phenanthrene degradation by the white rot fungus Pleurotus ostreatus, Appl. Environ. Microbiol., 63, 2495e2501 (1997). 13. Velioglu, Z. and Ozturk Urek, R.: Concurrent biosurfactant and ligninolytic enzyme production by Pleurotus spp. in solid state fermentation, Appl. Biochem. Biotechnol., 174, 1354e1364 (2014). 14. Campbel, M. M. and Ellis, B. E.: Fungal elicitor-mediated responses in pine cell cultures, Planta, 186, 409e417 (1992). 15. Kuli c, G. J. and Radoji ci c, V. B.: Analysis of cellulose content in stalks and leaves of large leaf tobacco, J. Agric. Sci., 56, 207e215 (2011). 16. Bradford, M. M.: A rapid and sensitive method for the quantitation of microorganisms qualities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72, 248e254 (1976). 17. Miller, G. L.: Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal. Chem., 31, 426e428 (1959). 18. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F.: Colorimetric method for determination of sugars and related substances, Anal. Chem., 28, ,350e356 (1956). 19. Weatherburn, M. W.: Phenol-hypochlorite reaction for determination of ammonia, Anal. Chem., 28, 971e974 (1967). 20. Chandrasekaran, E. V. and Bemiller, J. N.: Constituent analysis of glycosaminoglycans, pp. 89e96, in: Whister, L. and Wolfrom, L. (Eds.), Methods in carbohydrate chemistry. Academic Press, New York (1980). 21. Winkler, U. K. and Stuckmann, M.: Glycogen, hyaluronate and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens, J. Bacteriol., 138, 663e670 (1979). 22. Pinto, M. H., Martins, R. G., and Costa, J. A. V.: Bacteria biosurfactants production kinetic evaluation, Quím. Nova, 32, 2104e2108 (2009).
J. BIOSCI. BIOENG., 23. Youssef, N. H., Duncana, K. E., Naglea, D. P., Savagea, K. N., Knappb, R. M., and McInerney, M. J.: Comparison of methods to detect biosurfactant production by diverse microorganisms, J. Microbiol. Methods, 56, 339e347 (2004). 24. Yin, H., Qiang, J., Jia, Y., Ye, J., Peng, H., Qin, H., Zhang, N., and He, B.: Characteristics of biosurfactant produced by Pseudomonas aeruginosa S6 isolated from oil-containing wastewater, Process Biochem., 44, 302e308 (2009). 25. Chander, C. R., Lohitnath, T., Mukesh Kumar, D. J., and Kalaichelvan, P. T.: Production and characterization of biosurfactant from Bacillus subtilis MTCC441 and its evaluation to use as bioemulsifier for food bioepreservative, Adv. Appl. Sci. Res., 3, 1827e1831 (2012). 26. Mishra, S. K., Suh, W. I., Farooq, W., Moon, M. H., Shrivastav, A., Park, M. S., and Yang, J. W.: Rapid quantification of microalgal lipids in aqueous medium by a simple colorimetric method, Bioresour. Technol., 155, 330e333 (2013). 27. Rau, U., Hammen, S., Heckmann, R., Wray, V., and Lang, S.: Sophorolipids: a source for novel compounds, Ind. Crops Prod., 13, 85e92 (2001). 28. Reis, F. A., Servulo, E. F., and De Franca, F. P.: Lipopeptide surfactant production by Bacillus subtilis grown on low-cost raw materials, Appl. Biochem. Biotechnol., 115, 899e912 (2004). 29. Prieto, L. M., Michelon, M., Burkert, J. F. M., Kalil, S. J., and Burkert, C. A. V.: The production of rhamnolipid by a Pseudomonas aeruginosa strain isolated from a southern coastal zone in Brazil, Chemosphere, 71, 1781e1785 (2008). 30. Van Hamme, J. D., Singh, A., and Ward, O. P.: Physiological aspects. Part 1 in a series of papers devoted to surfactants in microbiology and biotechnology, Biotechnol. Adv., 24, 604e620 (2006). 31. Zinjarde, S. S. and Pant, A.: Emulsifier from tropical marine yeast, Yarrowia lipolytica NCIM 3589, J. Basic Microbiol., 42, 67e73 (2002). 32. Bednarski, W., Adamczak, M., and Tomasik, J.: Application of oil refinery waste in the biosynthesis of glycolipids by yeast, Bioresour. Technol., 95, 15e18 (2004). 33. Hommel, R. K. and Ratledge, C.: Biosynthetic mechanisms of low molecular weight surfactants and their precursor molecules, pp. 3e63, in: Kosaric, N. (Ed.), Biosurfactant: Production, properties and applications. Marcel Dekker Inc., New York (1993). 34. Wei, Y., Wang, L., and Chang, J.: Optimization iron supplement strategies for enhanced surfacting production with Bacillus subtilis, Biotechnol. Prog., 20, 979e983 (2004). 35. Kalyani, A. L. T., Naga Sireesha, G., Aditya, A. K. G., Girija Sankar, G., and Prabhakar, T.: Production optimization of rhamnolipid biosurfactant by Streptomyces coelicoflavus (NBRC 15399T) using Plackett-Burman design, Eur. J. Biotechnol. Biosci., 1, 7e13 (2014). 36. Rufino, R., Sarubbo, L., and Campos-Takaki, G.: Enhancement of stability of biosurfactant produced by Candida lipolytica using industrial residue as substrate, World. J. Microbiol. Biotechnol., 8, 313e316 (2007). 37. Oliveira, F. J. S., Vazquez, L., de Campos, N. P., and de França, F. P.: Production of rhamnolipids by a Pseudomonas alcaligenes strain, Process Biochem., 44, 383e389 (2009). 38. Haba, E., Espuny, M., Busquets, M., and Manresa, A.: Screening and production of rhamnolipids by Pseudomonas aeruginosa 47T2 NCIB 40044 from waste frying oils, J. Appl. Microbiol., 88, 379e387 (2000). 39. Thaniyavarn, J., Chongchin, A., Wanitsuksombut, N., Thaniyavarn, S., Pinphanichakarn, P., Leepipatpiboon, N., Morikawa, M., and Kanaya, S.: Biosurfactant production by Pseudomonas aeruginosa A41 using palm oil as carbon source, J. Gen. Appl. Microbiol., 52, 215e222 (2006). Açıkel, Y.: The effects of the composition of 40. Açıkel, Ü., Ers¸an, M., and Sag growth medium and fermentation conditions on productions on the production of lipase by R. delemar, Turk. J. Biol., 35, 35e44 (2011). 41. Gerson, D. F.: The biophysics of microbial surfactants: growth on insoluble substrates, pp. 269e286, in: Kosaric, N. (Ed.), Biosurfactant: Production, properties and applications, 1st ed. Marcel Dekker, New York (1993). 42. Desai, J. D. and Banat, I. M.: Microbial production of surfactants and their commercial potential, Microbiol. Mol. Biol. Rev., 61, 47e64 (1997). 43. Chamanrokh, P., Mazaheri Assadi, M., Amoabediny, G., and Rashedi, H.: Cleaning oil-contaminated vessel by emulsan producers (Autochthonous bacteria), Iran. J. Environ. Health Sci. Eng., 7, 209e222 (2010). 44. Das, K. and Mukherjee, A. K.: Comparison of lipopeptide biosurfactants production by Bacillus subtilis strains in submerged and solid state fermentation systems using a cheap carbon source: some industrial applications of biosurfactants, Process Biochem., 42, 1191e1199 (2007). 45. Jain, R. M., Mody, K., Mishra, A., and Jha, B.: Isolation and structural characterization of biosurfactant produced by an alkaliphilic bacterium Cronobacter sakazakii isolated from oil contaminated wastewater, Carbohydr. Polym., 87, 2320e2326 (2012). 46. Pulate, V. D., Bhagwat, S., and Prabhune, A.: Microbial oxidation of medium chain fatty alcohol in the synthesis of sophorolipids by Candida bombicola and its physicochemical characterization, J. Surfactants Deterg., 16, 173e181 (2013). 47. Syldatk, C. and Wagner, F.: Production of biosurfactants, pp. 89e120, in: Kosaric, N., Cairns, W. L., and Gray, N. C. C. (Eds.), Biosurfactants and biotechnology. Marcel Dekker, New York (1987).
Please cite this article in press as: Velioglu, Z., and Ozturk Urek, R., Optimization of cultural conditions for biosurfactant production by Pleurotus djamor in solid state fermentation, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.007
Optimization of cultural conditions for biosurfactant production by Pleurotus djamor in solid state fermentation Zulfiye Velioglu1, * and Raziye Ozturk Urek2 Biotechnology Department, Graduate School of Natural and Applied Sciences, Dokuz Eylül University, 35160 Buca, Izmir, Turkey1 and Chemistry Department, Biochemistry Division, Faculty of Science, Dokuz Eylül University, 35160 Buca, Izmir, Turkey2 Received 15 October 2014; accepted 5 March 2015 Available online xxx
Being eco-friendly, less toxic, more biodegradable and biocompatible, biological surfactants have higher activity and stability compared to synthetic ones. In spite of the fact that there are abundant benefits of biosurfactants over the synthetic congeners, the problem related with the economical and large scale production proceeds. The utilization of several industrial wastes in the production media as substrates reduces the production cost. This current study aims optimization of biosurfactant production conditions by Pleurotus djamor, grown on sunflower seed shell, grape wastes or potato peels as renewable cheap substrates in solid state fermentation. After determination of the best substrate for biosurfactant production, we indicate optimum size and amount of solid substrate, volume of medium, temperature, pH and Fe2D concentrations on biosurfactant production. In optimum conditions, by reducing water surface tension to 28.82 ± 0.3 mN/m and having oil displacement diameter of 3.9 ± 0.3 cm, 10.205 ± 0.5 g/l biosurfactant was produced. Moreover, chemical composition of biosurfactant produced in optimum condition was determined by FTIR. Lastly, laboratory’s large-scale production was carried out in optimum conditions in a tray bioreactor designed by us and 8.9 ± 0.5 g/l biosurfactant was produced with a significant surface activity (37.74 ± 0.3 mN/m). With its economical suggestions and applicability of laboratory’s large-scale production, this work indicates the possibility of using low cost agro-industrial wastes as renewable substrates for biosurfactant production. Therefore, using economically produced biosurfactant will reduce cost in several applications such as bioremediation, oil recovery and biodegradation of toxic chemicals. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Pleurotus djamor; Solid state fermentation; Biosurfactant; Optimization; Sunflower seed shell]
Surfactants establish an important class of industrial chemicals widely used in modern household and industry. Most of these compounds are synthesized chemically and petroleum-originated, toxic to the environment and not easily biodegradable (1). Their usage in such large quantities indicates that their waste and the potential adverse effects for environment and health are significant. However, rapid improvement in biotechnology has provided invention and development of possible alternatives as biological surfactants. Biosurfactants, biological surfactants, are structurally diverse compounds which exhibit particularly high surface activity and emulsifying activity (2). The three main classes of biosurfactants are glycolipids, lipopeptides and polymeric biosurfactants. They are widely used in the pharmaceutical, biomedical, cosmetic, petroleum and food industries as well as environmental and agricultural applications. Due to high ecological acceptability, less toxicity, easily degradability and biocompatibility, biosurfactants have gained importance recently. These molecules also have higher activity and stability than synthetic surface active agents (3).
* Corresponding author. Tel.: þ90 232 301 8689; fax: þ90 232 453 4188. E-mail addresses: zulfi[email protected] (Z. Velioglu), raziye.urek@ deu.edu.tr (R. Ozturk Urek).
Although there are significant benefits of biosurfactants over the synthetic surfactants, their economical and large scale production has difficulties (4). Using various industrial wastes in the formulation of production media as a substrate is one of the approaches to decrease production cost (5). Biodegradation of these wastes into biotechnological products has double benefit as economical production and a decrease in environmental pollution. The properties of used waste as substrate in the medium and fermentation type are significant factors that influence the biosurfactant production and activity (5e7). Especially agro-industrial wastes such as oily wastes from vegetable oil refineries, starchy materials wastes from potato processing industries were also reported as good substrates for biosurfactant production (4). Solid state fermentation (SSF) is a suitable fermentation system using agro-industrial wastes as cheap substrates (8). In addition, the nature, chemical structure, activity and the amount of produced biosurfactant depends on the producer microorganism (5). As fungi grow in nature on solid substrates, SSF is a productive fermentation when fungi are used (9). Pleurotus spp. belongs to the genus Pleurotus (Quel.) Fr., tribe Lentineae Fayod, family Polyporaceae (Fr.) Fr., and they are saprophytic, white rot fungi which are cultivated on substrates containing lignin or cellulose such as agro-industrial wastes. Utilization of these materials is dependent on lignolytic enzymes
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.03.007
Please cite this article in press as: Velioglu, Z., and Ozturk Urek, R., Optimization of cultural conditions for biosurfactant production by Pleurotus djamor in solid state fermentation, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.007
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secreted by Pleurotus spp. (10). Although, there are several studies on lignolytic enzyme production by Pleurotus spp. as far as we know, there is one of the publications which indicate biosurfactant production potential of Pleurotus spp. showing production of an emulsifying agent by Pleurotus ostreatus (11). This current study aims to optimize biosurfactant production condition by P. djamor, in SSF using sunflower seed shell, grape waste or potato peels as sustainable low-cost substrates. In the first part of the study, the best substrate for biosurfactant production in SSF was determined. In the second part, we investigated effects of size and amount of solid substrate, volume of medium, temperature, pH and Fe2þ concentrations to optimize biosurfactant production conditions. The biosurfactant production was screened by oil spreading technique, emulsification index and surface tension assay. Moreover, biosurfactant produced in optimum production conditions was further characterized by Fourier transform infrared spectroscopy (FTIR) and its chemical composition was determined. Lastly, laboratory’s large-scale production was carried out in optimum conditions.
MATERIALS AND METHODS Organism and culture conditions P. djamor (Rumph. Ex Fr.) Boedijn (MCC15) was grown on potato dextrose agar (39 g/l) at 25 C for 7 days (7) and was stored at 4 C. For SSF in control condition, biosurfactant production medium was a Gıda, Izmir, Turkey), grape prepared with 5 g sunflower seed shell (from Dog wastes (dried at 60 C for 24 h) (from Dimes, Izmir, Turkey) or potato peels (dried at 60 C for 24 h) (from Izmir, Turkey) in 250-ml Erlenmeyer flasks. The used substrates were chosen as 1 substrate particle/cm2 surface area. The medium (pH 6.0) contained (g/l) NH4NO3, 0.724; KH2PO4, 1.0; MgSO4$7H2O, 1.0; KCl, 0.5; yeast extract, 0.5; FeSO4$7H2O, 0.001; ZnSO4$7H2O, 0.0028; CaCl2$2H2O, 0.033 and peptone, 10.0 (12). Medium included 10 ml/l sunflower seed oil, as second carbon source. After sterilization (at 121 C for 20 min), 10 ml liquid medium was added into solid substrate (humidity 70%) and agar plugs (1 cm2) were inoculated. Flasks were incubated at 29 C and samples were extracted on 13th day of incubation period. According to our previous study, maximum biosurfactant production was determined on 13th day (13). For extraction, each flask received 25 ml of distilled water and was agitated for 1 h at 150 rpm, 29 C (13). The suspension was centrifuged at 5000 rpm and 4 C for 15 min and supernatant was used for analysis determining below. Chemical analysis of solid substrate Lignin concentrations of solid substrates used in SSF were determined by thioglycolic acid lignin analysis method (14). Analysis of cellulose content was performed by Kürschner-Hanack method (15). Optimization of culture condition After determination of the best solid substrate for biosurfactant production, we used this substrate in optimization steps. First of all the amounts of solid substrate (with 1 cm2 surface area) were changed as 1, 3, 5, 7 and 10 g. Also solid substrate which had 0.25 cm2 surface area was used in amounts of 1, 3, 5, 7 and 10 g. After determination of optimum surface area and amount of solid substrate, medium volume was changed as 10, 25 and 50 ml. Lastly three different fermentation temperatures (25 C, 29 C and 35 C), four different pH levels (pH 5.5, 6, 7 and 8), different concentrations of Fe2þ (0, 3.5, 18 and 35 mM) were analyzed. During optimization steps, levels of protein (16), reducing sugar (17), total carbohydrate (18) and nitrogen (19) were determined in the extracted supernatant. In addition, uronic acid (20) assay was used. As liquid medium containing sunflower seed oil, we investigated lipase activity using p-nitrophenyl palmitate (pNPP) as the substrate (21). One unit (U) was defined as the amount of enzyme that liberated 1 mmol p-nitrophenol per min. Emulsification index measurements Emulsification index was measured according to Pinto et al. by combining 3.5 ml of extracted supernatant and 2 ml of sunflower seed oil (22). The mixture was agitated in a vortex agitator at high speed for 1 min. Nonfermented culture medium was used as blank. The emulsion index was calculated after 24 h (E24) and 48 h (E48) by the height of the emulsion layer divided by the total height, and multiplied by 100 (Eq. 1) and emulsifying activity determined according to Eq. 2. Emulsification index stability was preserved value of emulsification index for 24 h. EðsampleÞ ¼
Hðemulsion layerÞ *100 HðtotalÞ
(1)
where H is height. EI ¼ ðEðsampleÞ EðblankÞÞ*D where D is dilution of sample in water.
(2)
Oil spreading test Sunflower seed oil (200 ml) was added onto the surface of distilled water filled in a 50 ml Petri dish to form a layer on the surface. 20 ml extracted supernatant was gradually added to the centre of oil layer. The diameter of the clear zone on the oil surface was measured in a relation to biosurfactant concentration (23). Determination of surface tension The extracted supernatants obtained during optimization process in SSF were used for the determination of the surface tension using a Sigma 701 digital surface tensiometer (KSV Instruments Ltd., Finland) and working on the principle of the Du Nuoy ring method at room temperature. The surface tension measurement was carried out by immersing the platinum ring into the sample for a while, in order to attain the equilibrium. We use surface tension results as base to determine optimum conditions. Isolation and chemical analysis of biosurfactant To isolate produced biosurfactant in optimum conditions, the concentrated HCl was added with 20 ml extracted supernatant to bring final pH of 2.0 and kept for overnight at 4 C (24). Resulted precipitation was collected by centrifugation at 5000 rpm and 4 C for 30 min. The supernatant was removed and 10 ml of chloroform: methanol (2:1, v/ v) was added to precipitate pellet and incubated in a rotary shaker at 30 C and 200 rpm for 20 min. The content was centrifuged at 5000 rpm and 4 C for 30 min and the supernatant was evaporated by air drying (25). Levels of protein (16), reducing sugar (17), total carbohydrate (18), nitrogen (19) and lipid (26) were determined to demonstrate chemical composition of biosurfactant isolated from optimum production medium. Also uronic acid (20) assay was used. In addition we investigate lipase activity of isolated biosurfactant (21). Fourier transform infrared spectroscopy The FTIR spectra were recorded on the Perkin Elmer Spectrum BX, in the spectral region of 4000e400 cm1. The biosurfactant, produced in optimum condition was analyzed and a KBr pellet was used as a background reference. Laboratory’s large-scale production in SSF According to obtained results, we produced biosurfactant in laboratory’s large-scale in optimum conditions. We used 35 g sunflower seed shell and 70 ml medium (included 10 ml/l sunflower seed oil). A tray, 24 cm diameter and made by heat-resistant glass, was designed as bioreactor. Sample of 13th day was extracted. Surface tension and emulsification index of extract were measured. Statistical analysis The Tukey test was used for statistical significance analyses. The values were the mean of three separate experiments. Comparisons were also made with Pearson’s correlation.
RESULTS This current study focused on optimization of biosurfactant production conditions by P. djamor in SSF with various industrial wastes such as carbon sources, complex substrates. Using several industrial wastes, which are substrate in green biotechnological production, is important for economical production as well as being an alternative solution to environmental problems. In this current study, we determined the most suitable solid substrate for biosurfactant production in the first step and then we optimized production conditions in the second step. Lastly, we produced biosurfactant in laboratory’s large-scale according to obtained optimum conditions. Determination of suitable solid substrate for biosurfactant production In order to determine suitable solid substrate, we analyzed surface tension of biosurfactants produced in control conditions with three different wastes as carbon source. Surface tension values of biosurfactant produced on sunflower seed shell, grape wastes and potato peels were 29.79 0.3, 42.6 0.4 and 34.66 0.3 mN/m, respectively. According to these results sunflower seed shell was established as the most suitable solid substrate and also its lignin and cellulose concentrations were detected as 18.65 1.1 and 45 3.8%, respectively. Determination of optimum amount and surface area of solid substrate for biosurfactant production First of all, we determined optimum amount and surface area of sunflower seed shell (Fig. 1) in optimization steps, after choosing the best solid substrate. As mentioned above, the maximum biosurfactant activity was determined on 13th day of incubation (13), so in this present study samples of 13th day were analyzed. As minimum surface tension value (29.79 0.3 mN/m) was detected in control condition, optimum surface area and amount of solid substrate were determined as 1 cm2 and 5 g, respectively (Fig. 1A and B).
Please cite this article in press as: Velioglu, Z., and Ozturk Urek, R., Optimization of cultural conditions for biosurfactant production by Pleurotus djamor in solid state fermentation, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.007
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FIG. 2. Emulsification indexes (E24 and E48) and surface tensions values of produced biosurfactants with different volume of medium and different temperature. The values are the mean SD for experiments of three separate experiments.
pH value of culture medium. The minimum surface tension value (28.82 0.3 mN/m) was detected at pH 5.5 (Fig. 3). In this condition, biosurfactant maintained its emulsification index by 90 4.5% (p < 0.05). Also oil spreading activity was maximum (3.9 0.2 cm) in this condition. Considering the results, we changed pH value of control condition as 5.5.
FIG. 1. Effects of different amount of solid substrate on emulsification indexes (E24 and E48) and surface tensions of produced biosurfactants with 1 cm2 surface area (A) and 0.25 cm2 surface area (B). The values are the mean SD for experiments of three separate experiments.
Also, maximum E24 value (45.71 4.0%) has been determined in control condition and diameter of clear zone according to oil spreading was 3.5 0.2 cm. The emulsification index stability designates the strength of a surfactant and in control condition the produced biosurfactant conserved the emulsification index by 90.07 4.5% for 48 h. In control condition levels of protein, total carbohydrate, reducing sugar, lipid, nitrogen, and uronic acid were 5320 85 ppm, 7151 187 ppm, 183.2 8.9 mM, 1600.2 53 ppm, 18,691.3 252 ppm and 31.833 1.1 M respectively. Also, we determined lipase activity as 1.652 0.01 U/ml.
Determination of optimum Fe2D concentration for biosurfactant production In the last optimization step, effects of different Fe2þ concentrations on biosurfactant production were investigated (Fig. 4). There was no significant effect of Fe2þ concentration on biosurfactant production in investigated culture condition and concentrations (rs ¼ 0.157, p > 0.05). According to surface tension values, the optimum Fe2þ concentration was 3.5 mM (control condition). Furthermore, maximum emulsification index (E24 ¼ 44.44 4.0% and E48 ¼ 40 3.7%) and diameter of clear zone (3.9 0.3 cm) were determined in control condition. According to results we determined optimum conditions as surface area and amount of solid substrate 1 cm2 and 5 g, volume of medium 10 ml, temperature 29 C, pH level 5.5 and Fe2þ concentration 3.5 mM. In these condition, level of protein, total carbohydrate, reducing sugar, lipid, nitrogen, and uronic acid were 3214.29 98 ppm, 8969.69 102 ppm, 205.3 9.2 mM, 1395.5 42 ppm, 12,933.03 162 ppm, and 32,750 471 mM, respectively. Also we determined lipase activity as 1.467 0.01 U/ml.
Determination of optimum volume of medium and process temperature for biosurfactant production In the next optimization step, the volume of medium and process temperature was indicated (Fig. 2). The correlation between surface tension and medium volume was strong (rs ¼ 0.946, p < 0.05). According to surface tension results, optimum volume of medium was 10 ml (control condition). Also the maximum emulsification index (E24 ¼ 45.71 4.0% and E48 ¼ 41.17 4.0%) and oil spreading activity (3.5 0.2 cm) values were determined in control condition while no growth was observed in 50 ml medium. As maximum emulsification index values (E24 ¼ 45.71 4.0% and E48 ¼ 41.17 4.0%) and minimum surface tension value (29.79 0.3 mN/m) were observed in control condition, optimum temperature for biosurfactant production process was 29 C (Fig. 2). A meaningful correlation was determined between temperature and surface tension (rs ¼ 0.775, p < 0.05). Determination of optimum pH value of production medium for biosurfactant production The next step was optimization of
FIG. 3. Emulsification indexes (E24 and E48) and surface tensions values of produced biosurfactants with different pH values. The values are the mean SD for experiments of three separate experiments.
Please cite this article in press as: Velioglu, Z., and Ozturk Urek, R., Optimization of cultural conditions for biosurfactant production by Pleurotus djamor in solid state fermentation, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.007
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J. BIOSCI. BIOENG., E24 ¼ 34.21 3.2% and E48 ¼ 32.43 3.1% and also it conserved its stability by 94.8 4.8% (p < 0.05). The amount of produced biosurfactant was 8.9 0.5 g/l in laboratory’s large-scale. DISCUSSION
FIG. 4. Emulsification indexes (E24 and E48), and surface tensions of produced biosurfactants with different Fe2þ concentrations. The values are the mean SD for experiments of three separate experiments.
Partial chemical characterization of produced biosurfactant After optimization steps produced biosurfactant in optimum conditions was extracted and its partial chemical characterization was analyzed. The amount of extracted biosurfactant was 10.205 0.5 g/l. The chemical composition of 1 mg extracted biosurfactant was formed 92.38 5.2 mg protein, 15.8 0.5 mg total carbohydrate, 13 0.5 mg reducing sugar, 13.27 0.8 mg lipid, 56.2 2.4 mg nitrogen, and 14.97 0.5 mg uronic acid. Moreover lipase activity was determined as 0.695 0.01 U/mg extracted biosurfactant. The molecular composition of biosurfactant produced in optimum conditions was evaluated by FTIR (Fig. 5). The most important bands were located at 3294 cm1 (OeH stretching) and at 2991e2924 cm1 (CeH bands: CH2eCH3 stretching) was associated with the stretching vibration of CeH bond of constituent sugar residues. The peak at 1658 cm1 (C]O stretching) suggested the presence of carbonyl functionality present in carboxylate or amide moieties of protein and peptide amines and also the peaks at 1539 cm1 (NeH bending) were indication of proteins. The peaks at 1200e1400 cm1 were indication of hydrocarbon chain (13). Laboratory’s large-scale production After optimization and partial characterization of produced biosurfactant, we produced biosurfactant in laboratory’s large-scale in optimum conditions. As far as we are concerned, used bioreactor was designed and it was used for the first time in this study. The surface tension value of produced biosurfactant in laboratory’s large-scale was 37.74 0.3 mN/m. Emulsification index activities were
In this current study, we optimized biosurfactant production conditions by P. djamor on renewable substrates. First of all, we decided the most suitable solid substrate. Three different agroindustrial wastes, abundant around Izmir-Turkey, were used as solid substrates, According to surface tension values the best solid substrate was sunflower seed shell as it contains hydrocarbon that activates biosurfactant production due to its insoluble property. Such waste materials are mostly burned in order to dispose and release CO2 that advances to greenhouse effect. This study demonstrated the potential of bioconversion of these waste materials to value-added biotechnological products. In some studies glucose was used as carbon source which was an expensive substrate especially in large-scale production (27e29). In our study, sunflower seed oil was also used as second carbon source. According to Makkar and Cameotra producing biosurfactant from oily substrates is an advisable strategy of waste management and low cost production (1). It is important to note that the selection of suitable waste with the right balance of nutrients that permits cell growth and product accumulation. The lignocellulosic and oily property of sunflower seed shell permitted production of biosurfactant as well as lipase enzyme. In the next step of optimization experiments, we investigated the effects of amount and surface area of solid particles on biosurfactant production. Surface tension values did not show alterations except control condition. To surface tension and emulsification index values, the optimum amount and surface area of solid particles were in control condition. Also, according to oil spreading test, diameter of clear zone (3.5 0.2 cm) supported these results. Biosurfactants exhibit important roles in increasing bioavailability of substrates (30). Increment of surface area expedited getting substrates, thus microorganisms did not need large amounts of biosurfactant production. Similarly production with large amount of solid substrate made easier to reach substrate although microorganisms produced biosurfactant to get more nutrients in control condition. In addition, the effects of medium volume on biosurfactant production were searched. As humidity exceeded 70% no growth was observed in 50 ml medium. The minimum surface tension was determined in control condition besides the maximum emulsification index and oil spreading activity. In this present study, we
FIG. 5. FTIR spectra of biosurfactant produced by P. djamor in optimum conditions.
Please cite this article in press as: Velioglu, Z., and Ozturk Urek, R., Optimization of cultural conditions for biosurfactant production by Pleurotus djamor in solid state fermentation, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.007
VOL. xx, 2015 also aimed economic production, so the volume of used medium is an important factor that effects production cost. The optimum medium volume was determined as 10 ml which was suitable for an economic production. In order to get large amounts of biosurfactant, it is necessary to optimize the environmental conditions (5). Temperature is one of the important factors that affect type and amount of produced biosurfactant. In our study, surface tension values of biosurfactant produced at different temperature showed considerable changes. Hence temperature effects biochemical reactions in microorganism cells, changes in biosurfactant production were expected. The minimum surface tension and maximum emulsification index were determined at control condition temperature (29 C). pH is another environmental factor that affects yield and characteristics of the produced biosurfactant. In reference to Zinjarde and Pant (31), the best production of biosurfactant occurred at pH 8.0 while in another study (32) the production of biosurfactant reached maximum at pH 5.5. The optimum pH value shows changes according to the biosurfactant type, production condition and producer microorganism. We did not determine significant changes between surface tensions of biosurfactant produced at different pH values. The best surface tension value (28.82 0.3 mN/ m) was detected at pH 5.5. Lastly, effects of Fe2þ concentration on biosurfactant production were examined. Metal ions play an important role in biosurfactant production since they form cofactors of some enzymes (5). Fe is an important activator of isocitrate lyase enzyme which is involved in cell growth on hydrophobic substrates (33). In our study, there were not meaningful alterations in surface tension values according to chancing Fe2þ concentrations (rs ¼ 0.157, p > 0.05). Excessive concentration or deficiency of Fe2þ did not considerably effect biosurfactant production. Similarly in the study of Wei et al. (34), biosurfactant production by Bacillus subtilis was optimal with 4 mM Fe2þ. The authors supposed that poor growth was observed at low iron concentration due to the chelating effects of biosurfactant resulting in less iron for cell metabolism (34). At high iron concentration, growth was reduced due to the acidification of media. In this current study, we used sunflower seed oil as second carbon source and likewise in the study of Kalyani et al. (35), olive oil was used as carbon source to produce rhamnolipid by Streptomyces coelicoflavus and they produced 0.475 g/l biosurfactant in optimum conditions. Rufino et al. (36) studied the cultivation of Candida lipolytica grown on ground nut oil and produced 4.5 g/l biosurfactant. Similarly, a low-cost agricultural byproduct palm oil was used as carbon source and achieved biosurfactant amount was 2.3 g/l (37). In another study (38), sunflower and olive oil were used as carbon sources and 2.7 g/l biosurfactant produced with 32e36 mN/m surface tension. Different from studies mentioned above, we used two low cost substrates, as sunflower seed shell and oil, and the amount of produced biosurfactant was 10.205 0.5 g/l with high surface tension reducing activity (28.82 0.3 mN/m). In the economical production strategy over production is important as well as high activity of the product. As far as we are concerned this is the first study that indicated such over production and high activity of biosurfactant in SSF. Also, 8.9 0.5 g/l biosurfactant was produced in laboratory’s large-scale that reduced surface tension to 37.74 0.3 mN/m. When compared to similar studies, the achieved results in large scale production were significantly higher. Thaniyavarn et al. (39) examined biosurfactant production in large scale with olive oil and maximum amount of produced biosurfactant was 6.58 g/l. Optimization of laboratory scale to large scale is problematic and there are not always same results in different scale productions. The amounts and surface activities of produced biosurfactant in small and large scales were not as expected. Nevertheless, the determined results from large scale
BIOSURFACTANT PRODUCTION OPTIMIZATION
5
production demonstrated applicability of designed tray bioreactor and possibility of high production in case of optimization. In optimum conditions, levels of protein and lipid reduced while the level of total carbohydrate increased in reference to control condition. The increasing level of carbohydrate was supposed by the increase in level of reducing sugar, and uronic acid. Due to the reduced level of lipid, decrease of lipase activity in optimum conditions was an unexpected result. In a study of Açıkel et al. (40), the activity of lipase produced in optimum conditions was 0.964 U/ml. Although lipase production was not primer aim of this present study, the lipase activity (1.467 0.01 U/ml) was higher than several studies. In order to optimize lipase production conditions, it is also possible to achieve higher lipase activity in a biosurfactant production process. The produced biosurfactant also demonstrated considerable lipase activity (0.695 0.01 U/mg). Insoluble substrates can be resourceful for microbial growth; however, there is a great restriction on the availability of substrate by the cell (41). As microorganisms need to metabolize insoluble compounds, they synthesis lipase and biosurfactant (42). The main effect of the biosurfactants is to reduce the interfacial tension at the surface of substrate and to increase the availability of substrate by microorganism. In this current study, simultaneous production of lipase and biosurfactant facilitated utilization of oily substrates. Simultaneous and economic production of biosurfactant and lipase enzyme is important for economical applications like bioremediation of oil residues, waste management and cosmetic industry. The economically produced biosurfactant in control condition was analyzed by FTIR. In the FTIR spectrum of biosurfactant, since the peaks at 1539 cm1 showed the presence of protein, it could be concluded that the produced biosurfactants were protein containing type such as lipopeptide or polymeric biosurfactants (43,44). Besides, the peaks at 2991e2929 cm1 are found in structure of polysaccharide containing biosurfactants as glycolipids and polymeric ones (25,43,45,46). The obtained results suggested that produced biosurfactant possibly had protein-polysaccharide-lipid complex structure (12). Meanwhile, results of chemical composition analysis supported complex structure of biosurfactant produced. In conclusion, this current study demonstrated an active and economical biosurfactant production by P. djamor in SSF in determined optimum condition. In this condition 10.205 0.5 g/l biosurfactant was produced which reduced water surface tension to 28.82 0.3 mN/m. In laboratory’s large-scale production 8.9 0.5 g/l biosurfactant was produced, which was carried out in optimum condition in a tray bioreactor designed by us. With regard to dual product strategies lipase enzyme was simultaneously produced. Simultaneous and economic production of eco-friendly biosurfactant and lipase enzyme is important for environmental applications like mineralization of environmental contaminants and also in cosmetics and food industry. Furthermore, this study demonstrated the potential of bioconversion of waste materials to value-added biotechnological products. According to Syldatk and Wagner (47), the hydrophilic and hydrophobic moieties of biosurfactant are synthesized de novo by two independent pathways; One of these moieties is synthesized de novo while the synthesis of other moiety is induced by substrate, or both of the moieties have substrate dependent synthesis (47). In this current study, solid substrate has lignocellulosic and oily character, which might induce hydrophilic and hydrophobic moieties of produced biosurfactant. The second carbon source, sunflower seed oil, also might stimulate production of hydrophobic moiety while the carbohydrate groups from lignocellulosic waste induce production hydrophilic part. Large amounts of agroindustrial wastes are generated during the manufacture of agricultural products every year. Utilization of these by-products in biotechnological production processes has importance in
Please cite this article in press as: Velioglu, Z., and Ozturk Urek, R., Optimization of cultural conditions for biosurfactant production by Pleurotus djamor in solid state fermentation, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.007
6
VELIOGLU AND OZTURK UREK
economical and ecological fields. For that reason, determination the feasibility of usage of these wastes as substrates to produce valuable biotechnological products has gained importance, recently. Our offer is a new bioconversion of sunflower seed shells to biosurfactant. ACKNOWLEDGMENTS We thank Dr. Ufuk Malayoglu for surface tension analysis and a Gıda and Dimes Meyve Suyu Izmir-Turkey for providing also Dog solid substrates. This work was supported by the Scientific and Technological Research Council of Turkey (grant number 113M801). References 1. Makkar, R. S. and Cameotra, S. S.: An update on the use of unconventional substrates for biosurfactant production and their new applications, Appl. Microbiol. Biotechnol., 58, 428e434 (2002). 2. Banat, I. M., Makkar, R. S., and Cameotra, S. S.: Potential commercial applications of microbial surfactants, Appl. Microbiol. Biotechnol., 53, 495e508 (2000). 3. Bhardwaj, G., Cameotra, S. S., and Chopra, H. K.: Biosurfactants from fungi: a review, Petrol. Environ. Biotechnol., 4, 160 (2013). 4. Makkar, R. S., Cameotra, S. S., and Banat, I. M.: Advances in utilization of renewable substrates for biosurfactant production, AMB Express, 1, 1e19 (2011). 5. Saharan, B. S., Sahu, R. K., and Sharma, D.: A review on biosurfactants: fermentation, current developments and perspectives, Gen. Eng. Biotechnol. J., 29, 1e39 (2011). 6. Massadeh, M. I., Fraij, A., and Fandi, K. G.: Effect of carbon sources on the extracellular lignocellulolytic enzymetic system of Pleurotus sajor-caju, Jordan J. Biol. Sci., 3, 51e54 (2010). 7. Stajic, M., Persky, L., Friesem, D., Hadar, Y., Wasser, S. P., Nevo, E., and Vukojevic, J.: Effect of different carbon and nitrogen sources on laccase and peroxidases production by selected Pleurotus species, Enzyme Microb. Technol., 38, 65e73 (2006). 8. Pandey, A., Selvakumar, P., Soccol, C. R., and Nigam, P.: Solid state fermentation for the production of industrial enzymes, Curr. Sci., 77, 149e162 (1999). 9. Bhargav, S., Panda, B. P., Ali, M., and Javed, S.: Solid-state fermentation: an overview, Chem. Biochem. Eng. Q., 22, 49e70 (2008). 10. Cohen, R., Persky, L., and Hadar, Y.: Biotechnological applications and potential of wood-degrading mushrooms of the genus Pleurotus, Appl. Microbiol. Biotechnol., 58, 582e594 (2002). 11. Nikiforova, S. V., Pozdnyakova, N. N., and Turkovskaya, O. V.: Emulsifying agent production during PAHs degradation by the white rot fungus Pleurotus ostreatus D1, Curr. Microbiol., 58, 554e558 (2009). 12. Bazalel, L., Hadar, Y., and Cerniglia, C.: Enzymatic mechanisms involved in phenanthrene degradation by the white rot fungus Pleurotus ostreatus, Appl. Environ. Microbiol., 63, 2495e2501 (1997). 13. Velioglu, Z. and Ozturk Urek, R.: Concurrent biosurfactant and ligninolytic enzyme production by Pleurotus spp. in solid state fermentation, Appl. Biochem. Biotechnol., 174, 1354e1364 (2014). 14. Campbel, M. M. and Ellis, B. E.: Fungal elicitor-mediated responses in pine cell cultures, Planta, 186, 409e417 (1992). 15. Kuli c, G. J. and Radoji ci c, V. B.: Analysis of cellulose content in stalks and leaves of large leaf tobacco, J. Agric. Sci., 56, 207e215 (2011). 16. Bradford, M. M.: A rapid and sensitive method for the quantitation of microorganisms qualities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72, 248e254 (1976). 17. Miller, G. L.: Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal. Chem., 31, 426e428 (1959). 18. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F.: Colorimetric method for determination of sugars and related substances, Anal. Chem., 28, ,350e356 (1956). 19. Weatherburn, M. W.: Phenol-hypochlorite reaction for determination of ammonia, Anal. Chem., 28, 971e974 (1967). 20. Chandrasekaran, E. V. and Bemiller, J. N.: Constituent analysis of glycosaminoglycans, pp. 89e96, in: Whister, L. and Wolfrom, L. (Eds.), Methods in carbohydrate chemistry. Academic Press, New York (1980). 21. Winkler, U. K. and Stuckmann, M.: Glycogen, hyaluronate and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens, J. Bacteriol., 138, 663e670 (1979). 22. Pinto, M. H., Martins, R. G., and Costa, J. A. V.: Bacteria biosurfactants production kinetic evaluation, Quím. Nova, 32, 2104e2108 (2009).
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Please cite this article in press as: Velioglu, Z., and Ozturk Urek, R., Optimization of cultural conditions for biosurfactant production by Pleurotus djamor in solid state fermentation, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.007