Recently, several investigations have been carried out on the in situ bacteria flooding, but the ex situ biosurfactant production and addition to the sand pack as agents for microbial enhanced oil recovery (MEOR) has little been studied. In order to develop suitable technology for ex situ MEOR processes, it is essential to carry out tests about it. Therefore, this work tries to fill the gap. The intention of this study was to investigate whether the rhamnolipid mix could be produced in high enough quantities for enhanced oil recovery in the laboratory scale and prove its potential use as an effective material for field application. In this work, the ability of Pseudomonas aeruginosa MM1011 to grow and produce rhamnolipid on sunflower as sole carbon source under nitrogen limitation was shown. The production of Rha-C10-C10 and Rha2-C10-C10 was confirmed by thin-layer chromatography and high-performance liquid chromatography analysis. The rhamnolipid mixture obtained was able to reduce the surface and interfacial tension of water to 26 and 2 mN/m, respectively. The critical micelle concentration was 120 mg/L. Maximum rhamnolipid production reached to about 0.7 g/L in a shake flask. The yield of rhamnolipid per biomass (Y RL/x ), rhamnolipid per sunflower oil (Y RL/s ), and the biomass per sunflower oil (Y x/s ) for shake flask were obtained about 0.01, 0.0035, and 0.035 g g(-1), respectively. The stability of the rhamnolipid at different salinities, pH and temperature, and also, its emulsifying activity has been investigated. It is an effective surfactant at very low concentrations over a wide range of temperatures, pHs, and salt concentrations, and it also has the ability to emulsify oil, which is essential for enhanced oil recovery. With 120 mg/L rhamnolipid, 27 % of original oil in place was recovered after water flooding from a sand pack. This result not only suggests rhamnolipids as appropriate model biosurfactants for MEOR, but it even shows the potential as a biosurfactant of choice for actual MEOR applications.

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There is a lack of fundamental knowledge about the scale up of biosurfactant production. In order to develop suitable technology of commercialization, carrying out tests in shake flasks and bioreactors was essential. A reactor with integrated foam collector was designed for biosurfactant production using Bacillus subtilis isolated from agricultural soil. The yield of biosurfactant on biomass (Y(p/x)), biosurfactant on sucrose (Y(p/s)), and the volumetric production rate (Y) for shake flask were obtained about 0.45 g g(-1), 0.18 g g(-1), and 0.03 g l(-1) h(-1), respectively. The best condition for bioreactor was 300 rpm and 1.5 vvm, giving Y(x/s), Y(p/x), Y(p/s), and Y of 0.42 g g(-1), 0.595 g g(-1), 0.25 g g(-1), and 0.057 g l(-1) h(-1), respectively. The biosurfactant maximum production, 2.5 g l(-1), was reached in 44 h of growth, which was 28% better than the shake flask. The obtained volumetric oxygen transfer coefficient (K(L)a) values at optimum conditions in the shake flask and the bioreactor were found to be around 0.01 and 0.0117 s(-1), respectively. Comparison of K(L)a values at optimum conditions shows that biosurfactant production scaling up from shake flask to bioreactor can be done with K(L) a as scale up criterion very accurately. Nearly 8% of original oil in place was recovered using this biosurfactant after water flooding in the sand pack.

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Saponins are the main group of phytogenic biosurfactants extracted from plants. One of the significant applications of these compounds is upgrading and viscosity reduction of heavy crude oil water in oil (W/O) emulsions. In this research, use of saponin extracted from Glycyrrhiza glabra was investigated for viscosity reduction of heavy crude oil and upgrading its API properties. The extracted saponin was characterized using Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopy. Phase behavior analysis demonstrated a reduction in initial viscosity and improved API gravity of the heavy oil from 2350 mPa·s and 19 to 900 mPa·s and 27, respectively. In addition, the emulsification index (E24) was found to be 98 % at a saponin concentration of 8 % w/v. It was observed that the emulsions were stable in the pH range of 5.5–13, temperature from 30 to 80 °C and salinity up to 6 % w/v of NaCl solution. Average diameter of W/O droplets evaluated by dynamic light scattering (DLS) were in the range of 10–15 µm. The results obtained from the present research revealed that the extracted saponin improved the physical characteristics of heavy crude oil. We propose the use of saponin as a potential alternative to conventional emulsifiers for upgrading heavy crude oil in petroleum industry.

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Recently, nanoparticles have become an attractive agent for enhanced oil recovery (EOR). Because much of the work on nanoparticles for enhanced oil recovery is still at the laboratory stage and to gain a better understanding of this technique, it is essential to study the effect of nanoparticles on EOR. In addition, the world is now more environmentally aware, presenting the opportunity to use biosurfactants for EOR. In this paper, the synergistic effect of biosurfactant and nanoparticles on the removal of oil in a glass micromodel was evaluated. In this study, an aqueous solution of emulsan biosurfactant with addition of SiO 2 nanoparticles was used as a nanofluid. The emulsan biosurfactant was produced by Acinetobacter calcoaceticus PTCC1318. The production of emulsan was confirmed by FTIR and 1 H NMR analysis. According to our results, the use of the mixture of biosurfactant and nanoparticle (nanofluid) permitted a 90% reduction of interfacial tension in comparison with biosurfactant solution alone. Micromodel oil displacement experiments with kerosene showed around 10 and 20% recovery of residual oil after water flooding when the emulsan and nanofluid were injected, respectively. These results are useful in extending the application of nanostructures in ex situ microbial enhanced oil recovery.