CRUDE PALM OIL BY BACTERIA PSEUDOMONAS PUTIDA MTCC 2467 AND ITS

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1 International Journal of Civil Engineering and Technology (IJCIET) Volume 9, Issue 11, November 2018, pp , Article ID: IJCIET_09_ Available online at aeme.com/ijciet/issues.asp?jtype=ijciet&vtype= =9&IType=11 ISSN Print: and ISSN Online: IAEME Publication Scopus Indexed PRODUCTION OF BIOSURFACTANTT USING CRUDE PALM OIL BY BACTERIA PSEUDOMONAS PUTIDA MTCC 2467 AND ITS APPLICATION IN IMPROVED OIL RECOVERY Department of Petroleum Engineering, Academy of Maritime Education and Training (AMET) 135 East Coast Road, Kanathur, Chennai ABSTRACT Present work is to produce biosurfactant and evaluate its effectiveness in oil recovery process. Strain Pseudomonas putida MTCC 2467 was grown in mineral salt medium supplemented with NaCl and crude palm oil. The crude and purified form of biosurfactants have been examined for potential oil recovery from a laboratory oil contaminated with sand in shake flask. Moreover, Chemical surfactants (SDS, Tween- the results. It 80) and water (control) were used to evaluate oil recovery and compare is observed that removal of crude oil from the oil contaminated sand by both crude and purified biosurfactants were 64.10±2.10% and 36.91±2.13% respectively. The results obtained in this study indicate that produced biosurfactant has the potential to remove oil from sand. Also the efficiency was 12% higher than the previous reports. Keywords: Biosurfactant, Crude palm oil, SDS, Tween-80, Oil recovery. Cite this Article:, Production of Biosurfactant Using Crude Palm Oil By Bacteria Pseudomonass PUTIDA MTCC 2467 and Its Application In Improved Oil Recovery, International Journal of Civil Engineering and Technology (IJCIET) 9(11), 2018, pp IET/issues.asp?JType=IJCIET&VType=9&ITy ype=11 1. INTRODUCTION Crude oil recovery consists of primary recovery method where, due to natural energy (pressure) oil is recovered. Once there is decline in pressure secondary recovery which includes injection of water to drive oil to the surface. 10% and 25% of crude is recovered using primary and secondary methods. Crude oil which were trapped in wells secondary phase makes up to 67% of the total reserves [4; 26].In heavy crude, oil viscosity is one of the key factors for selection of recovery method, mode of transportation and production [24]. Many chemical and physical methods are applied to improve the recovery of this residual oil. Enhanced Oil Recovery (EOR) is a tertiary recovery process to recover residual oil from editor@iaeme.com

2 Production of Biosurfactant Using Crude Palm Oil By Bacteria Pseudomonas PUTIDA MTCC 2467 and Its Application In Improved Oil Recovery reservoir by injecting chemicals. Using synthetic surfactants in chemical flooding process of EOR, its restructure wettability alteration [2]. Majority of chemical surfactants are considered to be high toxic and found to be hazard that affect the human and water environment [4]. Utilization of microbes and its metabolites is considered to be the most promising methods in oil recovery [12]. Biosurfactants are microbial compounds which are amphiphilic in nature and are considered to be surface active agents produced by variety of bacteria. These biosurfactants are highly potential and used in various industrial applications such as farming, cosmetics, food and petroleum industries [11]. As biosurfactants are surface active compounds, they are considered to be an alternative to replace the synthetic surfactants in EOR technology due to biodegradable and low toxicity [5].Pseudomonas aeruginosa ATCC 9027 was grown on both mesophilic and thermophilic conditions. Biomass, biosurfactant production was analyzed by subjecting to temperature and ph conditions which typically resembles petroleum reservoir. Laboratory scale experiment was carried out analysis such as involving physical and biochemical parameters were studied to optimize the growth and biosurfactant production. Moreover, analysis such as surface and interfacial tension have revealed that Pseudomonas aeruginosa ATCC 9027 is capable to grow and produce maximum biosurfactants and which in-turn helped in interfacial tension reduction to 33 mn/m at ph 8.0 and 30 C [12]. Acinetobacter calcoaceticus RAG-1 is potential to produce emulsan, an extracellular lipopolysaccharide. The produced biosurfactant has been used in flooding process of EOR. Due to the activity of surfactant produced by Acinetobacter calcoaceticus, reduction in interfacial tension between the oil/water and oil/rock was achieved [10]. A kinetic modelling was performed by experimental investigations using Pseudomonas putida which was subjected to different ph, resident time and water saturation with different concentrations of bacteria, nutrients and biosurfactant was studied and the results suggest biosurfactant production are found to be maximum at ph 8 and 7.5 respectively [23]. Candida lipolytica UCP 0988 was employed to produce biosurfactant with cost effective medium formulation along with 2% of waste frying oil, 2% corn steep liquor and 5% molasses at 120 h, 30 C and 180 rpm. Surface and interfacial tension was found to be reduced upto 24 mn/m and 11 mn/m respectively. Further, it is reported that CMC to be 0.32% [13]. Flooding experiment was conducted using biosurfactants produced by Bacillus mojavensis (PTCC 1696) and results showed about 25% of improvement in oil recovery was achieved. Further, biosurfactants reduce the interfacial tension from 65 to 26.7 mn/m under extreme saline environment [9]. Rhodococcussp strain TA6 produced biosurfactant, which is an extracellular lipids and glycolipids could reduce surface tension while grown in hydrocarbon as carbon source medium 68 to 30 mn/m with residual oil recovery around 70% using sand packs [20]. Present work is mainly focused on application of crude and purified biosurfactants produced by Pseudomonas putida MTCC 2467 using palm oil as substrate in laboratory oil contaminated sand, agitated flask. The result was further compared to evaluate their potential in oil recovery with synthetic surfactants (Sodium Dodecyl Sulphate and Tween-80) and water as control. 2. MATERIALS & EXPERIMENTAL PROCEDURES 2.1. Microorganism and Maintenance condition Crude Palm oil was obtained from Tamil Nadu Palm Development Board, Madhavaram, Chennai. Crude oil was obtained from Chennai Petroleum Corporation Limited, Manali, Chennai. Pseudomonas putida MTCC 2467 was procured from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, India for the present investigations. The procured culture was maintained in nutrient agar plates with the following composition (g/l): beef editor@iaeme.com

3 extract, 1.0; peptone, 5.0; yeast extract, 2.0; NaCl, 5.0; agar, 15.0; ph 6.0 ± 0.2, storage temperature 2 C to 8 C Media Cultivation Nutrient broth was used for media cultivating with the following composition (g/l) was used for preparation of inoculum. Beef extract, 1.0; yeast extract, 2.0; peptone, 5.0; NaCl, 5.0. Pseudomonas putida (MTCC 2467) was grown in nutrient broth for 8 12 h at 30 C (A 600nm 0.7) and 2% (v/v) inoculum was used for biosurfactant production using mineral salt medium with the following composition (g/l) KNO 3, 0.3; Na 2 HPO 4, 0.2; KH 2 PO 4, 0.013; NaCl, 0.001; MgSO 4, 0.05; CaCl 2, 0.003; FeSO 4, ml of trace elements are added with following composition (g/l) Na 2 MoO 4.2H 2 O, 0.39; CoCl 2.6H 2 O, 0.42; EDTA, 0.5; NiCl 2.6H 2 O, 0.004; MnSO 4.4H 2 O, 1.78; KI, 0.66; ZnSO 4.7H 2 O, 2.32; H 3 BO 3, 0.56; CuSO 4.5H 2 O, Biosurfactant Production Production of the biosurfactants have been carried following the methods of our previous work [9]. Cultures of bacteria were maintained on nutrient agar. Mineral salt media (10 g/l) was used for production of biosurfactants along with NaCl 5.0 g/l and crude palm oil (10 % v/v). Media was sterilized before inoculation process. All the experiment were carried out in standard 250 ml Erlenmeyer flask with working volume of 100 ml. Media were inoculated with 2% (w/v) overnight pre-culture of the strain was maintained in incubator cum shaker at 30 C, ph 7.0 and 180 rpm. Fermented samples were collected at regular interval of 12 h. Samples were centrifuged at 12,000 rpm for 20 min Purification of Biosurfactant Fermented culture of P. putida was centrifuged. The supernatant was then acidified to ph 2.0 with 3 N HCL for overnight at 4 C. Further it was extracted twice with solvent dodecane for removing of the remaining crude palm oil (CPO). Solution was extracted using dichloromethane. Sodium sulphate anhydrous was then added to the organic layer. The organic layer which was free form water was evaporated to get purified biosurfactant Evaluating Oil Recovery Using Produced Biosurfactant by Batch Method In order to evaluate the potential of biosurfactants in oil recovery using batch method was carried [19]. To replicate typical porous petroleum reservoir, sand samples were screened, cleaned and air dried in hot air oven at 50 C for overnight. Sand samples were subjected to 100 Mesh particle size (20 g) were washed with NaCl 5.0% and mixed with 2 g of crude oil, and transferred to 250 ml Erlenmeyer flasks. Add 20 ml of the biosurfactants were added and incubate with agitation of 180 rpm at room temperature for 24 h in a rotary shaker. The solution was further separated with the sand. Solvent dodecane (10 ml) were added to the sand and extracted. Post evaporation to retrieve residual oil was determined by gravimetric analysis. The percentage of crude oil removal was calculated using the equation (A). Crude oil recovered (%) = (G i G r ) / G i x 100% (A) G i - Initial oil in the sand before washing (g) G r - Oil remaining in the sand after washing (g) editor@iaeme.com

4 Production of Biosurfactant Using Crude Palm Oil By Bacteria Pseudomonas PUTIDA MTCC 2467 and Its Application In Improved Oil Recovery SDS, Tween-80 and distilled water as a control were analysed at the same conditions in order to compare recovered crude oil using biosurfactants. All experiments have been carried out in triplicate to maintain consistency. 3. RESULTS AND DISCUSSION Chemical surfactants are used in oil recovery applications. Alternatively biosurfactants have been used as a replacement for synthetic surfactant due to their toxicity and resistance to degradation [16; 18]. Biosurfactant producing microbes can able to grow on both water immiscible hydrocarbons and carbohydrate containing mineral salt medium [9]. While implementing MEOR process, the produced biosurfactant reduces the IFT between oil and water and helps remove residual oil. Recovery of residual oil during MEOR process is due to decrease in oil saturation and improving consecutively results in the increase of water saturation (Sw + So = 1) [23]. In biosurfactant flooding, contact time is a critical parameter which affects the efficiency of oil removal, as a sufficient contact time is required for effective oil removal. Almost 24 h is sufficient contact time for solubilisation of the hydrocarbon to mobile phase [17]. To evaluate the capacity of the crude biosurfactants, the recovery ability of the cell-free broth was determined (Table 1). Experiments was conducted by batch method in Erlenmeyer flasks for recovering crude oil from oil contaminated sand using both crude and purified form of biosurfactant. As biosurfactant has the potential for oil recovery, we compared the result with two synthetic surfactants (SDS and Tween-80) and water as a control. Experiment were conducted above CMC values of the biosurfactants/surfactants. Removing ability of crude oil from the oil contaminated sand by purified and crude biosurfactants were 79.40±3.10 and 46.84±2.23 %, respectively. On other hand the recoveries obtained with the SDS and Tween-80 as chemical surfactants at the same concentration were 64.10±2.10 and 36.91±2.13 % respectively. Water as a control removed only 83.33±0.47, 65.84±6.31 and 36.42±2.23% respectively. Also, recovery efficiency obtained with the SDS, Tween-80 and water were 83.33±0.47, 65.84±6.31 and 36.42±2.23% respectively. Table 1 Removal of crude oil by Biosurfactants, SDS and Tween-80 using batch method. Sample Oil recovered (%) Water 36.42±2.23% Tween ±6.31 SDS 83.33±0.47 Crude biosurfactant 64.10±2.10 Purified biosurfactant 36.91±2.13% Conventionally water injection is applied before any EOR recovery process is done in order to diminish the crude oil content and consequently the consumed any surfactant quantity. From earlier report, water washing of a diesel-polluted soil could remove 24% of alkanes compounds [14]. Similar report showed that upon water flooding around 51.5 % of oil was recovered [10]. Similarly according to another report, by using water washing removal of oil was 40-85% of hydrocarbons [5]. From the experimental result derived from present investigation, it is in accordance with the reported researches the ability of positive controls of synthetic surfactants (SDS and Tween-80) for crude oil removal [5; 14]. It was observed that % of PAHs were removed in the presence of SDS [5] and Triton X-100 and Tween- 80 injection gave a recovery of 70% and 50.5 % respectively [11]. It is a fact that the purified form of biosurfactants were generally more effective in oil recovery when compared to crude editor@iaeme.com

5 biosurfactants because the purified biosurfactants oil removal ability is slightly higher than that of the SDS, but it showed a lower values of that Tween-80. Present study showed that the ability of crude biosurfactants in removing oil were about 36%. It is a clear indicator that biosurfactants in crude form has lower ability than that of reported results. Biosurfactant produced from Candida tropicalis in the presence of waste frying oil removed 70-95% of the petroleum, motor oil from sand [3]. Whereas the crude biosurfactant from Candida guilliermondii grown in residues from industries could remove about 90% of motor oil from sand [7]. Similarly, biosurfactant produced by Rhodococcus sp. removed approximately 82% of crude oil [1]. Critical micelle concentration is of biosurfactants is an important parameter which begin to form micelle in solution at the CMC value so that solubilization of hydrocarbons in soil-water at the above CMC. The mobilization of oil is attracted towards the aqueous phase resulting in stable emulsification and micelle formation which leads for oil removal [8]. The result from current experiment, showed that the crude biosurfactants are less effective in oil recovery the when compared with purified biosurfactants but very few reports are in contradictory with our results. Reports on earlier studies show that the crude biosurfactants have equal ability in removing oil with the purified biosurfactants [26]. But there are reports that the crude biosurfactants have better ability removing oil than that of the purified biosurfactants. It was reported that biosurfactants produced by yeast C. sphaerica and with bacterium Bacillus sp. for removing motor oil from soil both in crude and purified form. Another report stated that the crude biosurfactants from P. aeruginosa was more effective as the purified biosurfactants resulted in 80% diesel oil removal from sand packing [21]. The crude biosurfactant from C. lipolytica grown in medium containing corn liquor and animal fat found to be more effective in removing motor oil than that of the isolated biosurfactant [19]. Studies using sand packed column had been used to evaluate the exact and accurate result in removing crude oil. Results showed that produced biosurfactant using P. cepacia in mineral medium supplemented with corn steep liquor and soybean waste frying oil achieved for the removal of motor oil packed with sand and clay soil [22]. Our earlier report where experiment on sand packed column was saturated with brine followed by oil saturation. Water injection followed by biosurfactant was done to analyze the amount of oil recovered. Further, the injection of biosurfactant volume was varied and checked how effectively oil recovery can be achieved. A comparative study was also injected by injecting Triton X-100 which is one of the chemical surfactants. Since biosurfactant could reduce interfacial tension oil can be easily recovered from the porous sand packed column. It was clear that recovery was 79.3% with triton by injecting 5 ml. Bio-surfactant flooding achieved 60% of recovery. Water flooding and tween flooding gave higher recovery of 83% and 90% by injecting 5 ml respectively [11]. Hence, biosurfactants produced by Pseudomonas putida MTCC 2467 grown in media containing CPO have good prospect for crude oil removal in the presence of sand. 4. CONCLUSIONS Mineral salt medium used as production media has produced biosurfactants cultivated by Pseudomonas putida containing crude palm oil (CPO) for crude oil recovery has been studied. We had investigated further in the presence of crude and purified biosurfactants for crude oil recovery from a laboratory oil-contaminated sand. Results showed that purified biosurfactants were more effective than crude biosurfactants in removal crude oil from sand. It is also observed that the purified biosurfactants were able to remove crude oil which were equally effective in the oil removal in par with commercial surfactants (SDS and Tween-80) Hence, we conclude the purified biosurfactants produced by Pseudomonas putida may be applied in crude oil recovery applications editor@iaeme.com

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