Isolation of biosurfactant producing bacteria from Potwar oil fields: Effect of non-fossil fuel based carbon sources

Biosurfactants are surface-active metabolites produced by microorganisms. Biosurfactants tend to solubilize hydrocarbons in the surrounding environment, by reducing surface tension and increasing carbon uptake. In this study, isolation of biosurfactant producing bacteria and effect of renewable, non-fossil fuel based carbon sources on production were assessed. Oil field produced water was collected from Potwar oil fields and thirteen strains were isolated. Strains were screened for biosurfactant production by hemolysis test, emulsification index test, emulsification assay, oil displacement test, drop collapse test, tilted glass slide test and oil coated agar emulsification test. Strains were further screened for maximum production on cost effective renewable carbon source e.g. glucose, waste glycerol and animal fat. Promising strains were identified as Bacillus subtilis (MH142143), Pseudomonas aeruginosa (MH142144), Bacillus tequilensis (MH142145) and Bacillus safensis (MH142146) by 16S rRNA gene sequencing. Among all isolates, highest biosurfactant production on glucose (37%), glycerol (48%) and animal fat oil (49%), after 24 h cultivation was by Pseudomonas aeruginosa. Biosurfactant showed similarity to rhamnolipids by Thin Layer Chromatography (TLC). Assessment of bioactive propertiaes of rhamnolipid showed strong antimicrobial activity against Bacillus spp. Future investigations can be focused on application of these strains in environmental as well as industrial sectors.


Introduction
Let F denote a eld and let V denote a vector space over F with nite positive d pair A, A * of diagonalizable F-linear maps on V, each of which acts on an eigenba irreducible tridiagonal fashion. Such a pair is called a Leonard pair (see [13, De nit A, A * is said to be self-dual whenever there exists an automorphism of the endom swaps A and A * . In this case such an automorphism is unique, and called the dua The literature contains many examples of self-dual Leonard pairs. For instance ( ated with an irreducible module for the Terwilliger algebra of the hypercube (see [4, Leonard pair of Krawtchouk type (see [10, De nition 6.1]); (iii) the Leonard pair asso module for the Terwilliger algebra of a distance-regular graph that has a spin mode bra (see [1,Theorem], [3,Theorems 4.1,5.5]); (iv) an appropriately normalized tota (see [11,Lemma 14.8]); (v) the Leonard pair consisting of any two of a modular Leo De nition 1.4]); (vi) the Leonard pair consisting of a pair of opposite generators fo bra, acting on an evaluation module (see [5,Proposition 9.2]). The example (i) is a s examples (iii), (iv) are special cases of (v).
Let A, A * denote a Leonard pair on V. We can determine whether A, A * is self-By [13, Lemma 1.3] each eigenspace of A, A * has dimension one. Let {θ i } d i= denote values of A. For ≤ i ≤ d let v i denote a θ i -eigenvector for A. The ordering {θ i } whenever A * acts on the basis {v i } d i= in an irreducible tridiagonal fashion. If the or then the ordering {θ d−i } d i= is also standard, and no further ordering is standard. S A * . Let {θ i } d i= denote a standard ordering of the eigenvalues of A. Then A, A * is sel is a standard ordering of the eigenvalues of A * (see [7,Proposition 8.7]).

Introduction
Nowadays, use of petroleum and petroleum base polymers has increased to an alarming degree. These polymers facilitate us in many different ways but their accumulation, non-biodegradability and contamination has been a concern -voiced by many. Oil fields contain large quantities of produced water, as it is the largest by-product associated with oil and gas exploration [1]. Oil field produced water contain large quantities of hydrocarbons especially petroleum, diesel oils, many aromatic compounds, dispersed oil and radionuclides [2]. Certain indigenous hydrocarbon degrading microorganisms -able to use these toxic metabolic wastes in a bioremediation process to help their own survival -are present in produced water. Hydrocarbon degrading bacteria are found to be most active among these. Biosurfactants (BS) are heterogeneous amphiphilic surface-active compounds that reduce surface and interfacial tension [3]. Biosurfactants help the cell survival and promote growth kinetics by reduction of surface tension and facilitation of uptake of hydrocarbon by hydrocarbon solubilization [4]. Biosurfactant producers are a group of hydrocarbon degraders, which show high surface activity and aid in degradation of harmful metabolites. Surface activity of biosurfactants helps in formation of microemulsions in which hydrocarbon solubilization can occur [5]. Bacteria belonging to genera Pseudomonas, Bacillus, Corynebacterium, Klebsiella, Acinetobacter, Archromobacter, Falvobacterium and Proteobacteria have been reported to produce efficient biosurfactants [6]. Most biosurfactants, known to date, are produced on immiscible hydrocarbons but some are produced on miscible hydrocarbons [5]. Biosurfactants consist of hydrophilic and hydrophobic moieties. Hydrophilic moieties help in reduction of surface tension levels, promotion of hydrocarbon desorption and solubilization. Hydrophobic moieties are concerned with the relationship between interface of surfactant molecules and liquid droplets [7].
Biosurfactants can be classified as low molecular weight biosurfactants; dealing mainly with the reduction in surface and interfacial tension levels, and high molecular weight biosurfactants; dealing mainly with emulsification and stabilization [8,9]. The chemical composition of biosurfactants depends on the biosynthetic pathway and enzymes (mainly transferases and synthases) needed for their production. Based on chemical composition, biosurfactants have been classified as glycolipids, lipopeptides, phospholipids, and neutral lipids. There are also polymeric biosurfactants e.g. emulsions, biodispersan, etc. [10]. Sophorolipids, rhamnolipids, lipomanan, surfactin, emulsan, iturin, liposen, lichenysin, alasan etc. are some of the best studied biosurfactants [11]. Two most common bacterial biosurfactants are rhamnolipids (produced by Pseudomonas aeruginosa), and surfactins (produced by Bacillus subtilis) [10]. Out of all these classes, glycolipids, mainly rhamnolipids are most thoroughly studied. Importance of rhamnolipids is, in part, due to their high bioactivity and antimicrobial properties. Production and synthesis of biosurfactants is susceptible to many external factors including pH, temperature, carbon, and nitrogen sources etc. Maximum growth was observed using glucose at 37 o C and pH 7 [10]. RhlA enzyme is involved in initiating rhamnolipid biosynthesis [12]. rhlC and rhlB genes encodes the enzyme needed for synthesis of rhamnolipids [13]. Gene rmlA, rmlB, rmlC, and rmlD are needed for the synthesis of rhamnose skeleton.
Recent advancements have provided the information that biosurfactants are a vital part of growth and reproduction of most biosurfactant producers e.g. Bacillus needs surfactin (biosurfactant) for formation of fruiting body, needed in order to reproduce [9]. Few biosurfactants adhere to cell membrane but most are released into the extracellular environment where these aid in cell survival through various biophysicochemical routes. Biosurfactants are set apart from their synthetic counterparts because of their biodegradable nature, low toxic levels, low irritancy, specific selectivity for human skin cells, effectiveness as well as ease of synthesis and extraction [5]. Although, most significant use of biosurfactants, to date, has been in petroleum and biodiesel industries pertaining to clearing oil spills, enhancing oil quality and removal of sludge from oil tanks, biosurfactants also have multifarious applications in the field of environmental sciences. These biosurfactants, due to their hydrocarbon degradation capability and biodegradative nature, have high potential for ecofriendly bioremediation of oil-contaminated sites, heavy metal pollution and pesticides, washing and cleaning of oil reservoirs etc. [14]. This property of biosurfactants can also be employed for management of wastewater pollution [10]. Emulsification and surface active properties of biosurfactants have made their use as detergents, foaming and wetting agents, flocculants, etc. possible [15]. Due to their high compatibility with human skin, most cosmetic industries employs biosurfactants in shampoos, tooth pastes and other products [4]. Food industries are also employing biosurfactants, as these tend to raise the quality of fat-based products [9]. Some biosurfactants display therapeutic and antimicrobial properties. Biosurfactants are used as biocontrol, for plant disease protection, due to their microbicidal effect [6]. Rhamnolipids possess strong antifungal properties. Rhamnolipids also serve to reduce metal toxicity [10].
Presence of biosurfactant producers in produced water samples collected from oil fields of Potwar, Pakistan was assessed. Bacterial flora of PW was screened for biosurfactant production by different qualitative and quantitative tests. Isolated strains were screened for biosurfactant production by hemolysis test, for which 31% isolates tested positive. These hemolytic strains were further screened using criteria such as emulsification index test, emulsification assay, oil displacement test, drop collapse test, tilted glass slide test and by using oil coated agar emulsification test. The effect of carbon sources -glucose, glycerol, and animal fat -on biosurfactant production was also evaluated. Biosurfactant samples were analyzed using thin layer chromatography (TLC).

Sample collection and isolation of bacterial strains
Potwar oil fields have tons of waste produced water, which was collected in sterile bottle and stored at 4°C. Sample was characterized by measuring pH, temperature, color, texture, and smell of sample. Dilutions of sample were prepared by serial dilution method. Dilutions 10 0 , 10 -1 and 10 -2 were aseptically transferred to Luria Agar plates by spread plate method. Plates were incubated at 37°C for 24 h. Colonies with distinguishing morphological features were restreaked two to three times to obtain pure culture. Microscopy was used for identification and differentiation

Screening of biosurfactant (BS) producers
Isolated strains were screened for by hemolysis test for direct screening. Ability of strains to produce biosurfactant was further analyzed by emulsification index, emulsification assay, oil displacement, drop collapse, tilted glass slide tests and by using oil coated agar plates [10,[17][18][19][20][21].

Hemolytic activity
Blood agar media containing 5% v/v human blood was streaked with isolated strains and incubated at 37°C for 24 h. After incubation, zone formation around culture was observed. Hemolytic activity is qualitative indication of biosurfactant production [17].

Emulsification assay
Strains were cultivated on MSM at 37°C for 24 h. Culture broth was centrifuged at 6000 rpm for 15 min and cell free supernatant was obtained. 3 mL of supernatant was mixed with 0.5 mL oil, vortexed for 2 min and then left undisturbed for 1 h. Absorption of aqueous phase was measured at 400 nm [17,18]. Emulsification activity per mL (EU/mL) was calculated as product of absorbance and dilution factor.

Emulsification index test
2 mL of volume of supernatant and oil were mixed vigorously for 2 min and then left undisturbed for 24 h. After 24 h, height of emulsified layer and total height of mixture was observed [17,19]. Emulsification index was calculated as follows: = ×

Oil spreading assay
40 mL of distilled water was taken in clean glass petri plates. 20 µL oil and 10 µL cell free supernatant was added. System was monitored carefully for formation of halo around supernatant, indicative of positive test result [10,20].

Drop collapse assay
A single droplet of oil and supernatant was taken on a clean glass slide. Droplet of supernatant was monitored carefully to notice whether it remained beaded or collapsed [20,21]. Collapsed supernatant drop was scored as positive "+," indicative of biosurfactant presence.

Tilted glass slide assay
A single droplet of 0.9% NaCl was taken on a clean glass slide angled at 45 degrees and a single colony of isolated was transferred to it. Colony was not mixed in 0.9% NaCl droplet. Droplet was monitored carefully to observe whether it remained beaded or collapsed [17,18]. Beaded drop was scored as negative "-," indicative of biosurfactant absence.

Oil coated agar plate test
Surface of Nutrient agar media plates were coated with oil. Plates were streaked with isolated strains and incubated at 37°C for 7 days. Plates were observed for presence of emulsification halo around culture growth, indicative of biosurfactant activity [22].

Molecular analysis
Strains were cultivated on Nutrient agar slants and genomic DNA was isolated by rapid DNA isolation method. 16S rRNA sequencing of promising strains was done by Sanger dideoxy sequencing [23]. Reverse primer sequence was converted to complementary sequence and assembled with forward sequences, to obtain consensus sequences, using Chromas Pro 2.6.5 [24] and Cap3 [25], respectively. Sequences were inspected for maximum homology against GenBank using BlastN [26]. Sequences were submitted to GenBank. Evolutionary relationships of promising strains were mapped using MEGA4 to construct phylogenetic trees by neighbor joining method [27].

Optimization of biosurfactant production
Mineral salts media (MSM) used, in this study, a modification of mineral salts solution developed by Knetting and Zajic [5] . Promising strains were cultivated on MSM supplemented separately with glucose, waste glycerol, and animal fat oil [28]. Animal fat oil was extracted by heating animal fat and decanting the residue. Extracted oil was filtered to remove any impurities. Each carbon source was used in 2% v/v concentration in one liter of mineral salt media. Biosurfactant productions were conducted in 500 mL flasks of 300 mL MSM supplemented with 2% carbon source. Media was incubated at 37°C for 96 h. Growth kinetics of promising biosurfactant producers were observed by measuring culture density at 600 nm at regular intervals [29]. Dry weight of biomass and biosurfactant was recorded over a period of 96 h using dry weight method [30]. Biomass and biosurfactant are mean of values recorded during production kinetics. Standard error of each value was calculated.

Thin layer chromatography (TLC)
Biosurfactant samples of strains were extracted and analyzed by TLC. Polysorbate 80 was taken as control.
TLC plates were developed in a solvent system of chloroform: methanol (65:7). Developed plates were visualized under UV at longer and shorter wave lengths and then subjected to iodine fumes for detection of lipids [5,10,31].

Antimicrobial activity
Rhamnolipid sample of Pseudomonas aeruginosa was analyzed for antibacterial activity by preparing rhamnolipid discs and placing discs on culture lawn of Bacillus subtilis and Pseudomonas aeruginosa. Dilution of ampicillin was taken as negative control. Zone formation around rhamnolipid discs is an indication of antimicrobial activity [32].

Sample collection and isolation of bacterial strains
Produced water sample was observed to have an oily texture with a diesel smell and brown color. Temperature of sample was measured as 27°C while pH was recorded as 6.0. Thirteen discrete colonies were selected from mixed culture plates of sample dilutions 10 0 , 10 -1 , 10 -2 and labelled as (produced water strains) PWA to PWM. Microscopic measurements of bacterial cells were recorded after observation under light microscope. Out of thirteen isolates, eleven were gram-positive rods, while one was gram-positive cocci and one was gramnegative rods. Seven strains were spore-formers and capsulated, DNase positive and motile. Nine strains were catalase positive and utilized citrate. Eleven strains were oxidase positive. Three were urease positive. Ten strains hydrolyzed starch. Staining results and biochemical characterization of strains indicated that most isolates belong to genus Pseudomonas, Bacillus and Rhodococcus.

Screening of biosurfactant (BS) producers
On screening for biosurfactant production, only strains that gave positive result for all tests were selected as producers i.e. PWA, PWC, PWF, and PWG. PWC, PWF, and PWG showed β-hemolysis while PWA showed α-hemolysis.
Emulsification index (EI) of PWA was highest (21.72), while that of PWG was lowest (7.81). Emulsification assay (EI/mL) of PWC was highest (125.0) while that of PWG was lowest (87.6). On oil spreading assay, PWA had largest halo diameter (0.42 cm), while PWC had smallest diameter (0.14 cm). On oil coated agar plate assay, these four strains showed oil emulsification around culture growth. On drop collapse assay and tilted glass slide assay, supernatant drops of these strains collapsed while others remained in beaded form (Table 1).

Kinetics of biosurfactant (BS) production
Production kinetics were mapped for all four biosurfactant producers. Over a period of 96 h, all strains gave high biomass and biosurfactant productions on all carbon sources. Biomass of Bacillus subtilis increased exponentially after 24 h. After 24 h, biosurfactant production on animal fat oil was 39%. After 96 h, highest biosurfactant production (36%) was also on animal fat oil. Lowest biosurfactant production (31%) was on glycerol.
In case of Pseudomonas aeruginosa, highest biomass was obtained on glucose but largest quantity of biosurfactant was produced on animal fat oil. Results depicted in Figure 2, show the biomass and biosurfactant production by Pseudomonas aeruginosa on Mineral salt media containing glucose, glycerol and animal fat oil. Maximum biomass production was obtained after 96 h (110.4 g/L) on using glucose as sole carbon source. Biosurfactant obtained after 24 h (49%) was highest using animal fat oil. Shift in biosurfactant concentration, after initial build up, was also noted in case of animal fat oil after 96 h (35%).

Biosurfactant analyzation
Preliminary analysis of biosurfactant sample of Pseudomonas aeruginosa indicated the presence of glycolipids. Preliminary analysis of biosurfactant samples indicated the presence of glycolipids and lipoproteins ( Figure 5). Spots on TLC plate developed in chloroform: methanol (65:7) were labelled as V: visible

Discussion
One major pollution associated problem of oil fields is produced water whose rich hydrocarbon content of produced water accounts for presence of high number of hydrocarbon degrading microorganisms, including biosurfactant-producing bacteria. Sample enumeration conducted to determine bacterial load present in sample indicated that it was significantly higher than in water. Sample enumeration and subsequent pure culture isolation resulted in isolation of thirteen morphologically different colonies from mixed culture of sample. Out of  these thirteen, 84.6% were gram-positive rods whereas gram-positive cocci and gram-negative rods were both 7.69% each. Cultural and biochemical characterization of these isolated strains aided in further classification of these pure cultures. For screening of biosurfactant production, hemolysis test was employed, as hemolytic activity is considered a clear indication of biosurfactant production by many authors [5]. Emulsification assay, emulsification index and hydrocarbon layered assay test only give an indication of biosurfactant presence, as often these do not correlate with surface activity. Collapse and spreading assay rely on destabilization of droplets, due to reduction in interfacial tension, in the presence of surfactants and surfactant activity respectively [18]. Out of thirteen isolates, 31% were biosurfactant producers (Table 1). Present study deals with isolation of biosurfactant producing bacteria and effect of non-fossil fuel based carbon sources on production kinetics. Production kinetics were mapped using glucose, waste glycerol, and animal fat oil [33]. Glucose and related compounds have a large hydrocarbon skeleton and bacteria, which can successfully utilize them, are able to obtain an easily replenishable supply of carbon, as compared to other carbon sources [34]. Waste glycerol is a main byproduct of biodiesel industry. Large quantities of waste glycerol are generated annually. Utilization of glycerol, therefore, imparts an evolutionary and biodegradability advantage to bacteria that can catabolize glycerol into simple components [35]. Many industrial sectors such as meat processing industries, tanneries, oil processing industries, and food industries, etc. produce significant quantities of animal fat, lard, grease, and tallow as waste [36]. This waste animal fat is used considerably less than other industrial wastes and poses a problem for disposal as well as utilization. Using animal fat as substrate for commercial production of biosurfactants etc. resolves this issue [28]. Animal fat was used in this study as an easily replenishable carbon source to obtain a measure of biodegradative activity of isolated strains [37]. Differences in biosurfactant production over the period of 96 h showed that initially biosurfactant was used up after 24 h, to solubilize hydrocarbons. Biosurfactant quantity was insufficient to form micelles at 48 h, but it started to increase again after 48 h. High biosurfactant production was observed, in case of PWA, Bacillus subtilis. In a similar study, Pereira et al. reported 21.58 g/L biosurfactant production by Bacillus subtilis [38]. Strain PWC (Pseudomonas aeruginosa; MH142144) showed highest growth rate on glucose, followed by animal fat oil and lowest growth rate on waste glycerol supplemented media ( Figure 2). Biomass also increased exponentially up to 24 h. Fall of biomass relates to stationary phase of growth indicating a regulatory switch to rhamnolipid (RL) synthesis [13]. Biomass of Pseudomonas increased after 24 h but biosurfactant production decreased after 24 h. Although highest growth was observed when glucose supplemented media was utilized, overall, highest production of biosurfactant was observed when animal fat oil was used. Similar studies were conducted by Nitschke et al. [39] who reported 75% biosurfactant production by Pseudomonas aeruginosa using oil wastes. Also Robert et al. [29] reported 38.2% production using olive oil.
Analysis of TLC spots of biosurfactant samples indicated presence of triglycerides, diglycerides, glycolipid, sterols, free fatty acids, and lipoproteins ( Figure 5). Triglycerides and diglycerides spots were observed in case of biosurfactant produced by Bacillus safensis indicating neutral lipids presence. In case of Bacillus tequilensis, lipoprotein spots were observed after treatment with iodine. These spots indicate the reaction of iodine vapors with lipid and protein parts of biosurfactant sample. Similar results were reported by Al-Wahaibi et al. during screening of minimal salt media for biosurfactant production by Bacillus subtilis and Bacillus licheniformis [42]. Further investigation focusing on identification and characterization of the type of biosurfactants are required. Glycolipid spots of Pseudomonas aeruginosa showed TLC spots of Rf value 0.68, which was assigned to rhamnolipids [31]. Comparable results were reported by Elshikh et al. [32], who studied natural antimicrobial activity of rhamnolipids. According to screening results and production kinetics, strains isolated from Potwar oil fields, Pakistan produced large amounts of biosurfactant, using renewable carbon sources as feedstock. This suggests potential exploitation as a promising resource conserving innovative, for large-scale production and application in environmental sectors.