Journal of Petroleum Exploration and Production Technology https://doi.org/10.1007/s13202-019-0619-8 ORIGINAL PAPER - PRODUCTION ENGINEERING Screening and characterization of biosurfactant produced by Pseudoxanthomonas sp. G3 and its applicability for enhanced oil recovery Dea Indriani Astuti1 · Isty Adhitya Purwasena1 · Ratna Eka Putri1 · Maghfirotul Amaniyah1 · Yuichi Sugai2 Received: 8 September 2018 / Accepted: 7 February 2019 © The Author(s) 2019 Abstract Biosurfactants are one of the microbial bioproducts that are in most demand from microbial-enhanced oil recovery (MEOR). We isolated and screened potential biosurfactant-producing bacteria, followed by biosurfactant production and characteriza- tion, and a simulation of the MEOR application to biosurfactants in a sand-packed column. Isolate screening was conducted based on qualitative (hemolytic blood assay and oil-spreading test) and semi-qualitative (emulsification assay and interfacial tension measurement) parameters. Bacterial identification was performed using 16S rRNA phylogenetic analysis. Sequential isolation yielded 32 bacterial isolates, where Pseudomonas sp. G3 was able to produce the most biosurfactant. Pseudomonas sp. G3 had the highest emulsification activity (Ei = 72.90%) in light crude oil and could reduce the interfacial tension between oil and water from 12.6 to 9.7 dyne/cm with an effective critical-micelle concentration of 0.73 g/L. The Fourier transform infrared spectrum revealed that the biosurfactant produced was a glycolipid compound. A stable emulsion of crude extract and biosurfactant formed at pH 2–12, up to 100 °C, and with a NaCl concentration of up to 10% (w/v) in the response-surface method, based on the Box–Behnken design model. The sand-packed column experiment with biosurfactant resulted in 20% additional oil recovery. Therefore, this bacterium and its biosurfactant show potential and the bacterium is suitable for use in MEOR applications. Keywords Enhanced oil recovery · Biosurfactant · Sand-packed column · Pseudoxanthomonas Introduction reservoirs. This method is considerably more economic and environmentally friendly than other EOR methods. Energy The demand for energy resources continues to increase with that is used in microbial processes to enhance oil recovery time. Developments in renewable energy are expected to does not depend on the price of crude oil. Microbes can provide sustainable energy and environmentally friendly growth independently under many conditions and produce industries. However, many projects that are related to renew- large amounts of useful products rapidly from cheap, renew- able energy face challenges, including technical, social, and able materials that are available in large quantities. As a bio- economic challenges. Existing energy resources should logical agent, microbial bioproducts are often biodegradable, optimize production, while avoiding critical environmen- which results in lower levels of pollution and a low toxicity tal risks. Microbial-enhanced oil recovery (MEOR) is an (Youssef et al. 2009; Khire 2010). MEOR uses microbial alternative approach to optimize oil production from existing activities and various bioproducts to help release residual oil that is trapped inside the rock pores and to stimulate oil flow to the production wells (Safdel et al.2017 ). * Isty Adhitya Purwasena Microorganisms produce biosurfactants, and the latter [email protected] are important in the MEOR mechanism. Biosurfactants act as surface-active molecules that reduce the interfacial ten- 1 School of Life Sciences and Technology, Institut Teknologi Bandung, Jalan Ganesha 10, Bandung 40132, Indonesia sion (IFT) between different fluid components, and enhance pseudosolubilization of oil in water by creating smaller oil 2 Earth Resources Engineering Department, Kyushu University, 744 Motooka, Nishi-Ku, Fukuoka 819-0395, droplets (Khire 2010). Injection of partially purified biosur- Japan factants has increased the amount of recovered oil to 40% Vol.:(0123456789)1 3 Journal of Petroleum Exploration and Production Technology (Mcinerney et al. 2003). Compared with chemically syn- Table 2 Physicochemical characteristics of the formation brine thesized surfactants, biosurfactants are biodegradable, non- Parameter Unit Value toxic, characteristically diverse, and stable under extreme conditions. Biosurfactant production can be significantly Salinity % 7.9 more affordable, as it may be produced using biomass waste Conductivity μS/cm 14,990 (Gautam and Tyagi 2008; Jing et al. 2011; Dhasayan et al. Water hardness mg/L CaCO3 253 2+ 2014). Ca mg/L 21.5 − Limited studies exist on the in situ production of bio- HCO3 mg/L 2083 2− surfactants for MEOR implementation and biosurfactant CO3 mg/L 0 − stability under various physiological conditions. This study Cl mg/L 237 − aimed to isolate, screen, and identify potential biosurfactant- NO3 mg/L 0.09 − producing bacteria from oil reservoir samples. We character- NO2 mg/L 0.034 + ized the biosurfactant stability under several physiological NH4 mg/-NH3-N 0.056 2− conditions using Fourier transform infrared (FT-IR) spec- SO4 mg/L 3.65 3− troscopy and response-surface methodology. MEOR simula- PO4 mg/L 0.044 tions were performed on a sand-packed column to determine Total P mg/L 0.071 the biosurfactant’s applicability to recover residual crude oil. Organic component mg/L 166 S2− mg/L < 0.01 Ni mg/L 0.142 Materials and methods Material 7 days. A plate count was conducted every 48 h using the pour method on Nutrient Agar Difco™. Residual oil from Samples were from a petroleum reservoir in South Sumatra. the first stage was used as a substrate in the second stage of Sample that contained crude oil (heavy oil) and formation sequential isolation using the same procedure. Purification water (brine) were extracted from its wellhead. All the sam- of potential biosurfactant-producing bacteria was conducted ples were kept in a cold box during transportation and they with a four-way streak method on NA medium. The purified were kept at 4 °C in the laboratory until they were analyzed. cultures were used for further screening and analysis. Light crude oil was utilized in these experiments. The char- acteristics of the crude oil and formation brine are listed in Screening of potential biosurfactant‑producing Tables 1 and 2. bacteria Isolation of biosurfactant‑producing bacteria Screening was performed based on two qualitative (hemo- lytic blood assay and oil-spreading test) and two semi- Isolation was carried out in two sequential stages (Halim qualitative (emulsification index and IFT measurement) et al. 2008) to obtain bacteria with varying oil-degrading methods. Preliminary screening used the hemolytic blood potential. Sample was added into Stone Mineral Salt Solu- assay method according to Youssef et al. (2004). Further tion (SMSS) medium with 0.1% (w/v) yeast extract and confirmation of isolate biosurfactant production was done incubated at 50 °C and 70 °C with 120 rpm agitation for using the oil-spreading test, based on a study by Satpute Table 1 Characteristics of crude oil Properties Heavy oil Light oil Value Gravity (API) 19 22–28 Viscosity at 25 °C (cP) 10.11 58.9 Experiment Unit Standard Results Saturates content Mass percent SARA 43 41.5 Aromatic content Mass percent SARA 5 9 Asphaltene Mass percent SARA 30 13 Resin content Mass percent SARA 22 36.5 1 3 Journal of Petroleum Exploration and Production Technology et al. (2010). The emulsification activity of the produced Biosurfactant‑stability test biosurfactant to emulsify crude oil and water was investi- gated from the emulsification index (Ei/E24) (Satpute et al. The Box–Behnken design model was used to study the 2010). IFT measurements were performed using a Surface interactive effects of the biosurfactant emulsification stabil- Tensiomat Model 21 (Fisher Scientific™, USA) with a 6-cm ity at different pH conditions, temperatures, and salinities. ring diameter to investigate the ability of the biosurfactant- The temperature ranged from 40 to 120 °C, the pH from 2 producing bacteria to decrease the IFT between the oil and to 12, and the salinity from 2 to 10% (w/v) NaCl. The pH the water. was adjusted using 1 N NaOH and 1 N HCl. The stability of a formed emulsion was determined by measuring the Ei as a generated response after 24 h of incubation. All the Critical‑micelle‑concentration determination experiments were performed in triplicate. A regression equa- tion was derived from the response analysis, including the The critical-micelle concentration (CMC) was obtained by determinant coefficients (F andR 2), which were evaluated measuring the IFT and the biosurfactant concentration (g/L) to generate contour plots. These plots were used to deter- at a certain time. The effective CMC was determined when mine the interaction between different factors and to predict an increase in the biosurfactant concentration did not result the emulsification stability at certain points (Dhasayan et al. in a significant decrease in IFT (Satpute et al.2010 ). 2014). All the statistical analyses were carried out using Minitab™ 17 Software. Identification of potential biosurfactant‑producing Sand‑packed column assay bacteria Biosurfactant application for enhanced oil recovery was The selected biosurfactant-producing bacteria was identi- evaluated using a modified sand-packed column described fied using a 16S ribosomal DNA analysis and was desig- by Suthar et al. (2008). Glass columns packed with sand (50 nated MEOR_G3. DNA sequencing was performed using