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INT. J. BIOL. BIOTECH., 15 (3): 419-429, 2018. CONCURRENT PRODUCTION OF BIOSURFACTANT AND ENZYME PROTEASE BY BACTERIA AND ESTIMATION OF APPLE POMACE WASTE FOR LOW-COST PRODUCTION Faiza A. Ansari*, Bashir Ahmed, Erum Shoeb, Jameela Akhtar, Fouad M. Qureshi and Obaid Y. Khan Department of Genetics, University of Karachi, Karachi-75270, Pakistan *Corresponding Author: faiza.ansari22@gmail.com ABSTRACT The present study is an attempt to optimize production of biosurfactant and protease simultaneously from bacterial isolates of the oil-contaminated region of Karachi coastal area. Bacterially produced biosurfactant and proteases well known for many applications in various industries and due to their environmentally friendly nature. Their production cost, however, remains high because of the high cost of culture media and low yield that is why only a few microbial sources recognized as commercial producers for biosurfactant and protease. For the present study, bacterial isolates selected for rhamnolipid production followed by screening for protease activity. Total 24 isolates selected for biosurfactant production through oil spreading, hemolytic activity, CTAB agar plate, drop collapse test, BATH assay, and emulsification activity (E24). Skim milk agar plate used for screening of protease producing isolates by producing clear zone. As a cheap source for the production of biosurfactants, the apple pomace successfully used in culture media. This study accomplishes that these isolates have the ability to produce commercially important biosurfactants and proteases respectively. It also suggested that apple pomace is a cost-effective substrate for the production of commercially important biomolecules. Keywords: Biosurfactants, proteases, adherence, skim milk agar INTRODUCTION Surfactants are chemicals, which help to reduce surface tension, interfacial tension and solubilize hydrocarbons. Based on production surfactants classified into two categories; chemically derived surfactants and biologically produced surfactants known as biosurfactants. Microorganisms work as chemical factories in producing commercially significant biosurfactants which are preferred over their synthetic counterparts for having lower toxicity, biodegradability and stability at high temperature, pH and salinity (Das et al., 2008; Pandy, 2012). These bio-molecules also have pharmaceutical properties as well as antiviral, antifungal and antibacterial properties (Singh and Cameotra, 2004). Proteases are the distinct class of enzymes, which have important applications in both physiological and commercial fields. Catalytic function of proteases is to hydrolyze peptide bonds of proteins and break them down into polypeptides or free amino acids. Proteases are widely used in leather processing, detergent industry, food industries, bioremediation process, pharmaceutical, textile industry, waste processing companies, and in the film industry (Rao et al., 1998). Microbes serve as a preferred source of production of these enzymes in limited space because of their rapid growth and the ease with which they can be genetically manipulated to generate new enzymes with altered properties that are desirable for various applications (Kocher and Mishra, 2009). Enzyme producing bacteria are widely distributed in soil and water, and certain strains tolerate extreme environmental conditions including highly alkaline conditions. One of the most important characteristics that determine the industrial suitability of proteases is their requirement of high pH for optimum enzyme activity. Screening of proteases producing bacterial species from different ecological environments can result in isolation of new proteases with unique characteristics for the various industrial application (Singh et al., 1999). Due to high production cost and low yield, the commercial production of biosurfactants and proteases is limited. (Nitschke and Pastore, 2002; Pornsunthorntawee et al., 2010). Alternative low-cost substrates like agro-industrial wastes, hydrophobic wastes, used frying oil, sludge from petroleum refineries and peels of different fruits or vegetables utilized for reducing the production cost of biosurfactants (Cameotra and Makkar, 1998). This study mainly focused on the screening of bacterial isolates capable of producing both biosurfactants and proteases and assessment of apple pomace as a cheap source for reducing the production cost of these biomolecules for the future industrial application. 420 F.A. ANSARI ET AL., MATERIALS AND METHODS Bacterial Isolates Previously isolated and preserved bacterial isolates were utilized in the present study (Shoeb et al., 2012). For enrichment Luria Bertani (LB) broth was used (Bertani, 1951). Cultures grown at 37°C and stored at 4°C. The isolates coded as DGEF11-DGEF34. Identification through GSP agar plate method For the detection of Pseudomonas and Aeromonas from the samples Glutamate Starch Phenol Red (GSP) agar (Oxoid) used for preliminary screening (Stanier et al., 1966) . The purified isolates streaked on GSP agar plates and incubated at 37°C for 24–48 hours (Martínez-Martínez et al., 1998). Result analyzed by a change in color from redviolet to yellow. Screening Methods for Bio-surfactant Production Isolates were grown aerobically for the screening of biosurfactant production through oil spreading method, hemolytic activity, CTAB agar plate, drop collapse test, BATH assay, emulsification activity (E24). Oil spreading method Oil spreading technique is a primary screening test of biosurfactant. Oil spreading was performed according to the method described previously by (Youssef et al., 2004). The occurrence of the clear zone on the oil surface was an indication of biosurfactant production. The diameter of a clear zone measured and compared to 10μL of distilled water as negative control. Hemolytic activity Hemolytic assay performed on blood agar plates. O/N culture was spot-streaked on blood agar plates and incubated for 48 h at 37°C. The plates visually inspected for the zone of clearance (hemolysis) around the colony which was used as an indicator of biosurfactant production (Mulligan et al., 1984). CTAB Agar Plate Blue agar plates containing cetyltrimethylammonium bromide (CTAB) and methylene blue used to detect extracellular glycolipid production (Siegmund and Wagner, 1991). Biosurfactant production observed by the formation of dark blue halos around the colonies. Drop-collapse test Screening of biosurfactant production performed using the qualitative drop-collapse test described by Jain et al. (1991) as modified by Bodour and Maier (1998). A result considered positive for biosurfactant production when the drop diameter was at least 1 mm larger than that produced by deionized water (negative control). Bacterial adhesion to hydrocarbons (BATH) Assay BATH assays were performed as previously described by (Rosenberg et al., 1980). Hydrophobicity expressed as the percentage of cell adherence to hydrocarbon calculated using following formula: 1-(OD of the aqueous phase/OD of initial cell suspension) ×100 Emulsification activity (E24) Emulsification activity performed using cell-free supernatant with xylene as hydrocarbon (Freitas et al., 2009). The emulsification activity was determined using the following formula: E24 = (Height of emulsion layer/ Height of liquid column) ×100 Screening for Protease activity by bacterial isolates Proteolytic activity of the bacterial cultures screened on skimmed milk agar plates containing skimmed milk powder 1.0%, peptone 0.5%, and sodium chloride 5% and agar 2.5%. The pH of the medium adjusted to 9.0 with 1N HCl/1N NaOH, before sterilization at 121°C for 15 minutes. The plates then incubated at 37°C for 48 hrs. The formation of the clear zone around the colonies confirmed the production of alkaline protease (Amoozegar et al., 2008). Hydrolysis expressed as the diameter of clear zone in mm. INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 15 (3): 419-429, 2018. PRODUCTION OF BIOSURFACTANT AND ENZYME PROTEASE BY BACTERIA 421 Effect of Sodium chloride on protease activity The isolates with positive protease activity further tested with varying concentration of NaCl on skimmed milk agar plate. The protease producing positive bacterial isolates streaked on skimmed milk agar plates containing 3%, 5% and 10% NaCl separately, incubated at 37°C for 48 hrs. Hydrolysis expressed as the diameter of clear zone in mm. The bacterial isolates with prominent zones of clearance considered as positive. Use of Apple pomace as a cheap source for biosurfactant production Preparation of substrate Apple (Malus pumilla Mill.) fruit pomace used as a cheap nutritional source for biosurfactant production in this study. Apple pomace collected from the canteen at the University of Karachi. Firstly, apple pomace washed with water and then air-dried. After drying apple pomace crushed into powder and autoclave for 15 min. Stored at room temperature for further use. Production media and cultivation conditions A mineral salt medium (MSM) containing (g/L): KH2PO4, 1.4; Na2HPO4, 2.2; (NH4)2SO4, 3; MgSO4.7H2O, 0.6; NaCl, 0.05; yeast extract, 1; FeSO4.7H2O, 0.01 and CaCl2.7H2O, 0.02; was used. The pH of the medium adjusted to 7.0.The mineral medium supplemented with apple pomace 2% (w/v) as the sole carbon source for biosurfactant production. All cultivations carried out in 250 mL flasks containing 50 ml of MSM medium incubated at 37 °C, agitation rate 150 rpm for 5 days. Absorbance at 600nm was taken on every 24 hrs and the cell-free supernatant subjected to emulsification activity (Ilori et al., 2005). RESULTS AND DISCUSSION The present study aimed to focus on the investigation of industrially important biomolecules produced by microorganisms. Biosurfactants and enzymes have great importance in our daily life and beneficent to the ecosystem (Pandy, 2012). Biosurfactant, protease well known for their industrial significance too, and it is very desirable to find bacterial isolates capable of simultaneous production of biosurfactant and protease. In this study, we have successfully isolated twenty-four bacterial isolates, which previously purified from samples of Arabian Sea coast of Karachi (Shoeb et al., 2015). Species identification on GSP agar plates showed that among the 24 isolates, 10 (41%) isolates belong to the genus Pseudomonas (Thavasi and Jayalakshmi, 2003). The existence of biosurfactant producing Pseudomonas species in hydrocarbon polluted environment is reported by many researchers (Das and Mukherjee, 2005). A number of methods are reported for the screening of biosurfactant producing bacteria (Kiran et al., 2009); (Walter et al., 2010). We used six methods, which are, oil spreading method, hemolytic activity, CTAB agar plate, drop collapse, BATH assay and emulsification activity (E24), to screen the biosurfactant producing isolates. Out of 24 bacterial isolates, 16 (66%) isolates significantly displaced oil layer and started to spread in the water, showing clear zone on oil plate. The maximum size of a zone formed by isolates DGEF11 and DGEF31 (35mm and 33mm) respectively as shown in Table 1 and in Fig. 1. Oil spreading results were in support of drop collapse assay results. Isolates, which were positive for oil spreading assay also showed positive results with drop collapse test (Table 2 and Fig. 2). These results confirmed the presence of biosurfactant in cell-free supernatant. It is the most effective tools to prove the biosurfactant production in many bacterial isolates. Youssef et al. (2004) reported similar findings with oil spreading and drop collapse assay. It reported that clear area formed due to the displacement of oil reflects the activity of biosurfactant. Larger the displacement area signifying a high biosurfactant activity (Sidkey and Al Hadry, 2014). Similarly, 95% isolates showed clear zone around the streaks of the colony in blood agar plates, confirming hemolytic activity as shown in Table 3 and Fig. 3. Lysis of red blood cells suggested as a simple and easy method to test for biosurfactant activity (Yonebayashi et al., 2000) and it is widely used to screen biosurfactant production (Shoeb et al., 2015). The CTAB is a semi-quantitative assay for the detection of extracellular glycolipids or other anionic surfactants (Saravanan and Vijayakumar, 2012). All twenty-four isolates subjected to CTAB agar plate method and results revealed that 95% isolates produced the dark blue halos around the colony and considered as positive. Isolates possess highest biosurfactant activity confirmed the presence of anionic biosurfactant as shown in table and figure. The maximum size of zone formation 26mm, 25mm observed in DGEF32 and DGEF31 respectively (Table 4 and Fig. 4). Anitha et al. (2015) used CTAB assay for screening of newly isolated bacterial strain. INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 15 (3): 419-429, 2018. 422 F.A. ANSARI ET AL., Table 1. Result for oil-spreading assay, indicated Table 2. Drop collapse used for biosurfactant oil displacement produced by isolates. activity. Code # Oil displacement (mm) DGEF11 DGEF12 DGEF13 DGEF14 DGEF15 DGEF16 DGEF17 DGEF18 DGEF19 DGEF20 DGEF21 DGEF22 DGEF23 DGEF24 DGEF25 DGEF26 DGEF27 DGEF28 DGEF29 DGEF30 DGEF31 DGEF32 DGEF33 DGEF34 35 24 25 25 19 10 13 10 30 20 15 5 3 5 5 15 10 12 12 2 33 20 5 5 Code # DGEF11 DGEF12 DGEF13 DGEF14 DGEF15 DGEF16 DGEF17 DGEF18 DGEF19 DGEF20 DGEF21 DGEF22 DGEF23 DGEF24 DGEF25 DGEF26 DGEF27 DGEF28 DGEF29 DGEF30 DGEF31 DGEF32 DGEF33 DGEF34 Drop Collapse + + + + + + + + + + + + + + + + + + + + + + + + BS drop Fig. 1. oil spreading assay showing highly active biosurfactant producers, the changes seen in the oil present in the systems, compared to the control (water) without any change. Fig. 2. for drop collapse assay, drop containing biosurfactant may collapse on oil layer as compare to water. INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 15 (3): 419-429, 2018. water drop 423 PRODUCTION OF BIOSURFACTANT AND ENZYME PROTEASE BY BACTERIA Table 3. Results for hemolytic activity. Code # DGEF11 DGEF12 Hemolytic Activity β α DGEF13 DGEF14 DGEF15 DGEF16 DGEF17 α β β β α DGEF18 DGEF19 DGEF20 DGEF21 DGEF22 α α α α α DGEF23 DGEF24 DGEF25 DGEF26 DGEF27 β β β β β DGEF28 DGEF29 DGEF30 DGEF31 DGEF32 β β Ƴ α α DGEF33 DGEF34 α α Table 4. Results for CTAB assay indicated zone formation (mm). Code # CTAB test Zone size (mm) DGEF11 DGEF12 DGEF13 DGEF14 DGEF15 DGEF16 DGEF17 DGEF18 DGEF19 DGEF20 DGEF21 DGEF22 DGEF23 DGEF24 DGEF25 DGEF26 DGEF27 DGEF28 DGEF29 DGEF30 DGEF31 DGEF32 DGEF33 DGEF34 16 15 16 14 21 15 20 19 20 22 16 15 21 20 17 19 20 20 20 19 25 26 22 18 * α’ hemolysis indicates complete lysis cells, β’ hemolysis indicates partial lysis of red cells, Ƴ’ no hemolysis. Fig. 3. Hemolytic activity of bacterial isolates showed lysis of red blood cells. INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 15 (3): 419-429, 2018. 424 F.A. ANSARI ET AL., Table 5. Bath assay of isolates against xylene as hydrocarbon. Code # Table 6. Emulsification index (E24) against generator oil and motor oil. Motor oil (%) Bath Assay (%) Code # DGEF11 Generator oil (%) 33.3 33.3 DGEF11 33.5 DGEF12 33.3 40 DGEF12 29.3 DGEF13 35.7 31 DGEF13 19.2 DGEF14 33.3 43.7 DGEF14 38.9 DGEF15 35.7 26.6 DGEF15 34.7 DGEF16 31.2 41.1 DGEF16 30.6 DGEF17 33 27 DGEF17 37 DGEF18 31.2 26.6 DGEF18 16.5 DGEF19 DGEF19 33.3 35.2 6.9 DGEF20 DGEF20 30.4 26.6 31.4 DGEF21 DGEF21 40 31 38.6 DGEF22 DGEF22 26.3 43.7 35.4 DGEF23 DGEF23 50 41.1 39.6 DGEF24 DGEF24 21.9 35.2 26.6 DGEF25 DGEF25 44.6 41.1 33.3 DGEF26 DGEF26 29.4 35.7 40 DGEF27 DGEF27 16.7 28.5 31 DGEF28 29.4 33.3 DGEF29 29.4 33.3 DGEF30 46.6 26.6 DGEF31 33 53 DGEF32 35.7 43.7 DGEF33 28.5 40 DGEF34 26.6 26.6 DGEF28 37.9 DGEF29 30.3 DGEF30 17.8 DGEF31 25% DGEF32 27.1 DGEF33 16.1 DGEF34 26.5 Fig. 4. In CTAB assay, dark blue halo was formed around bacterial growth indicated the biosurfactant activity. INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 15 (3): 419-429, 2018. 425 PRODUCTION OF BIOSURFACTANT AND ENZYME PROTEASE BY BACTERIA Table 7. Screening of selected protease producing bacteria on skim milk agar plates. Codes # DGEF11 DGEF12 DGEF13 DGEF14 DGEF15 DGEF16 DGEF17 DGEF18 DGEF19 DGEF20 DGEF21 DGEF22 DGEF23 DGEF24 DGEF25 DGEF26 DGEF27 DGEF28 DGEF29 Zone size after 24hr (mm) 30 13 12 18 14 22 30 10 20 22 22 11 29 20 32 25 15 18 31 Zone size after 48hr (mm) 45 16 12 29 32 22 30 10 30 22 22 11 29 29 32 25 15 18 31 Fig. 5. Image of the emulsification assay showed emulsion formed against generator oil. Table 8. To check the effect of sodium chloride (NaCl) on positive protease producing isolates. Code # NaCl Concentration DGEF11 DGEF12 DGEF13 DGEF14 DGEF15 DGEF16 10% 10% 10% 10% 10% 3% DGEF17 DGEF18 DGEF19 DGEF20 DGEF21 DGEF22 DGEF23 DGEF24 DGEF25 DGEF26 DGEF27 DGEF28 DGEF29 5% 5% 5% 5% 5% 5% 10% 5% 5% 3% 5% 5% 5% Fig. 6. Protease production by producing clear zone on skim milk agar plates. Bacterial adhesion to hydrocarbons (BATH assay) performed to estimate the affinity of the cell surface to hydrocarbon. It is a photometric based method, used to measures the degree of adhesion to hydrocarbon. Interaction with hydrophobic compounds considers an indirect method to screen biosurfactant producer. For this purpose, xylene used as the hydrophobic compound. Result for BATH assay indicated that all twenty-four isolates were positive and showed affinity of the bacterial cells with xylene. Cell attachment for positive isolates with xylene was in the range of 16.5-44.6%. Maximum cell attachment with xylene observed in isolate DGEF25 (44.6%) followed by DGEF14, DGEF21, DGEF23 (38.9, 38.9 and 39.6%) respectively as shown in the Table 5. Bacterial strains with high cell hydrophobicity are reported as potential biosurfactant producers (Volchenko et al., 2007). According to (Zhang and Miller, 1994) strains of Pseudomonas genus showed highest cell adherence with crude oil as compared to other bacterial isolates. Many reports mention BATH assay as principle method for screening of biosurfactant producers (Volchenko et al., 2007). INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 15 (3): 419-429, 2018. 426 F.A. ANSARI ET AL., (c) (a) (b) Fig. 7(a-c) Production of Biosurfactant in presence of apple pomace as nutritional source. (a)Showing growth curves at OD600 indicating growth with apple pomace. (b) Emulsification indexes values of produced biosurfactants against xylene.(c) bath assay result showed affinity of the cell with xylene with respect of time. Emulsification activity (E24) is another method used to determine the potential and stability of biosurfactant (Ilori et al., 2005). An emulsion formed when one liquid phase dispersed as microscopic droplets in another liquid phase. Analysis of emulsification activity indicated that isolate DGEF23 and DGEF31 exhibited highest emulsification capacity of 50% and 46.6% in against generator oil. Isolates, which were positive with generator oil, showed emulsification in range of 26 -50 %. While the isolates that positive with motor oil were in the range of 2643% of E24. The maximum emulsion formed by isolate DGEF31 (53%) with motor oil correspondingly (Table 6 and Fig. 5). Similarly, (Khalid, 2011) found an emulsion of 51% from the bacterial strain of Bacillus subtilis DSM 15029. According (Willumsen and Karlson, 1996) an emulsification index of 50.0% represents some good emulsifier properties of a biosurfactant. Although the stability of emulsion formed by bacterial isolates is irrespective of bio emulsifier produced (Cameotra and Makkar, 1998). Emulsification index (E24) is the speedy and consistent measure of produced biosurfactant (Asfora Sarubbo et al., 2006). The proteolytic activities of all the isolates were assayed using skim milk agar plate method. Proteolytic bacteria hydrolyze casein and form soluble nitrogenous compounds exhibited as a clear zone around colonies. Researchers (Vermelho et al., 1996) suggested that the hydrolysis zone produced on the casein agar could be related to the amount of protease produced. Similarly, Gupta (Gupta and Gupta, 2005) performed isolation of bacterial isolates from environmental samples and recommended skim milk agar for the screening of protease producing organisms. Out of 24 isolates streaked on skim milk agar plates, 19 (79%) isolates produced clear zone on skim milk agar plates after 48 hours of incubation. Among all tested isolates, DGEF11 showed highest protease activity by producing the extensive clear zone of 45mm after 48 hours (Table 7 and Fig. 6). Bacterial Isolates with positive Protease activity further treated with different sodium chloride (NaCl) concentration (3%, 5% and 10% w/v). All isolates were able to grow on salt containing skim milk agar plates. Isolates considered positive by producing clear zone on skim milk agar plates by tolerating concentration of NaCl INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 15 (3): 419-429, 2018. PRODUCTION OF BIOSURFACTANT AND ENZYME PROTEASE BY BACTERIA 427 from 3% to 10% after 48 hours of incubation (Table 8). According to (Sanchez-Porro et al., 2003) proteases was more active and stable in a wide range of NaCl concentration (Souza et al., 2012). The use of waste material as carbon sources to produce biosurfactants is an interesting and low-cost alternative (Abouseoud et al., 2008). In present study for biosurfactant production, apple pomace used as a cheap nutritional source in the cultivation media. Biosurfactant may produce during stationary growth phase as a typical secondary metabolite. It also depends upon the type of carbon substrate select for biosurfactant production (Davis et al., 1999). Results suggested that bacterial growth in apple pomace medium gradually increased and best absorbance obtained after the 5th day of incubation by isolate DGEF12 as shown in Fig. 7a. Similar findings were obtained by Rocha (Rocha et al., 2006) using natural cashew apple juice in the mineral complex medium for biosurfactant production. Sobrinho (1999) produced biosurfactant by utilizing 4% corn steep liquor and refinery waste as a substrate. In our study, after completion of incubation, the biosurfactant activity measured through emulsion formation of cell-free supernatant against xylene showed maximum activity i.e. 50% after 96 hours (Fig.7b). Results for cell attachment to hydrophobic compound indicated that the attachment of a cell to xylene was in the range of 13-29% (Fig. 7c). The biosurfactant production and bath assay dependent on the growth of culture in the fermentation medium. Results of the present study indicated positive prospects for use of apple pomace as a sole carbon source for biosurfactant production. Approximately 10–30% accounts the total production cost for biotechnological processes. The use of agro-industrial waste not only reduce cost but also help to clean the environment. Conclusion Biomolecules produced by microorganism have many pharmaceutical, food and industrial applications. In this study, we have screened the twenty-four isolates for biosurfactant production, nineteen of which showed positive protease production. This concurrent production of protease from biosurfactant producing isolates and their activity on different salt concentration is an interesting application for biotechnological processes. For an economical point of view, the use of apple pomace as a promising substrate for biosurfactant production demonstrated. 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