Sanket J. Joshi

Biosurfactant production by Bacillus subtilis B30 and its application in enhancing oil recovery

The fermentative production of biosurfactants by Bacillus subtilis strain B30 and the evaluation of biosurfactant based enhanced oil recovery using core-flood were investigated. Different carbon sources (glucose, sucrose, starch, date molasses, cane molasses) were tested to determine the optimal biosurfactant production. The isolate B30 produced a biosurfactant that could reduce the surface tension and interfacial tension to 26.63±0.45 mN/m and 3.79±0.27 mN/m, respectively in less than 12h in both glucose or date molasses based media. A crude biosurfactant concentration of 0.3-0.5 g/l and critical micelle dilution (CMD) values of 1:8 were observed. The biosurfactants gave stable emulsions with wide range of hydrocarbons including light and heavy crude oil. The biosurfactants were partially purified and identified as a mixture of lipopeptides similar to surfactin, using high performance thin layer chromatography and Fourier transform infrared spectroscopy. The biosurfactants were stable over wide range of pH, salinity and temperatures. The crude biosurfactant preparation enhanced light oil recovery by 17-26% and heavy oil recovery by 31% in core-flood studies. The results are indicative of the potential of the strain for the development of ex situ microbial enhanced oil recovery processes using glucose or date molasses based minimal media.

Isolation and characterization of nodule-associated Exiguobacterium sp. from the root nodules of fenugreek (Trigonella foenum-graecum) and their possible role in plant growth promotion

One of the ways to increase the competitive survivability of rhizobial biofertilizers and thus achieve better plant growth under such conditions is by modifying the rhizospheric environment or community by addition of nonrhizobial nodule-associated bacteria (NAB) that cause better nodulation and plant growth when coinoculated with rhizobia. A study was performed to investigate the most commonly associated nodule-associated bacteria and the rhizospheric microorganisms associated with the Fenugreek (Trigonella foenum-graecum) plant. Isolation of nonrhizobial isolates from root nodules of Fenugreek was carried out along with the rhizospheric isolates. About 64.7% isolates obtained from Fenugreek nodules were gram-negative coccobacilli, 29.41% were gram-positive bacilli, and all rhizospheric isolates except one were gram-positive bacilli. All the isolates were characterized for their plant growth promoting (PGP) activities. Two of the NAB isolates M2N2c and B1N2b (Exiguobacterium sp.) showed maximum positive PGP features. Those NAB isolates when coinoculated with rhizobial strain—S. meliloti, showed plant growth promotion with respect to increase in plants root and shoot length, chlorophyll content, nodulation efficiency, and nodule dry weight.

Production of bioethanol using agricultural waste: Banana pseudo stem

India is amongst the largest banana (Musa acuminata) producing countries and thus banana pseudo stem is commonly available agricultural waste to be used as lignocellulosic substrate. Present study focuses on exploitation of banana pseudo stem as a source for bioethanol production from the sugars released due to different chemical and biological pretreatments. Two fungal strains Aspergillus ellipticus and Aspergillus fumigatus reported to be producing cellulolytic enzymes on sugarcane bagasse were used under co-culture fermentation on banana pseudo stem to degrade holocellulose and facilitate maximum release of reducing sugars. The hydrolysate obtained after alkali and microbial treatments was fermented by Saccharomyces cerevisiae NCIM 3570 to produce ethanol. Fermentation of cellulosic hydrolysate (4.1 g%) gave maximum ethanol (17.1 g/L) with yield (84%) and productivity (0.024 g%/h) after 72 h. Some critical aspects of fungal pretreatment for saccharification of cellulosic substrate using A. ellipticus and A. fumigatus for ethanol production by S. cerevisiae NCIM 3570 have been explored in this study. It was observed that pretreated banana pseudo stem can be economically utilized as a cheaper substrate for ethanol production.

Microbial enhanced heavy oil recovery by the aid of inhabitant spore-forming bacteria: an insight review.

Crude oil is the major source of energy worldwide being exploited as a source of economy, including Oman. As the price of crude oil increases and crude oil reserves collapse, exploitation of oil resources in mature reservoirs is essential for meeting future energy demands. As conventional recovery methods currently used have become less efficient for the needs, there is a continuous demand of developing a new technology which helps in the upgradation of heavy crude oil. Microbial enhanced oil recovery (MEOR) is an important tertiary oil recovery method which is cost-effective and eco-friendly technology to drive the residual oil trapped in the reservoirs. The potential of microorganisms to degrade heavy crude oil to reduce viscosity is considered to be very effective in MEOR. Earlier studies of MEOR (1950s) were based on three broad areas: injection, dispersion, and propagation of microorganisms in petroleum reservoirs; selective degradation of oil components to improve flow characteristics; and production of metabolites by microorganisms and their effects. Since thermophilic spore-forming bacteria can thrive in very extreme conditions in oil reservoirs, they are the most suitable organisms for the purpose. This paper contains the review of work done with thermophilic spore-forming bacteria by different researchers.

Sophorolipids Production by Candida bombicola ATCC 22214 and its Potential Application in Microbial Enhanced Oil Recovery

Biosurfactant production using Candida bombicola ATCC 22214, its characterization and potential applications in enhancing oil recovery were studied at laboratory scale. The seed media and the production media were standardized for optimal growth and biosurfactant production. The production media were tested with different carbon sources: glucose (2%w/v) and corn oil (10%v/v) added separately or concurrently. The samples were collected at 24 h interval up to 120 h and checked for growth (OD660), and biosurfactant production [surface tension (ST) and interfacial tension (IFT)]. The medium with both glucose and corn oil gave better biosurfactant production and reduced both ST and IFT to 28.56 + 0.42mN/m and 2.13 + 0.09mN/m, respectively within 72 h. The produced biosurfactant was quite stable at 13–15% salinity, pH range of 2–12, and at temperature up to 100°C. It also produced stable emulsions (%E24) with different hydrocarbons (pentane, hexane, heptane, tridecane, tetradecane, hexadecane, 1-methylnaphthalene, 2,2,4,4,6,8-heptamethylnonane, light and heavy crude oil). The produced biosurfactant was extracted using ethyl acetate and characterized as a mixture of sophorolipids (SPLs). The potential of SPLs in enhancing oil recovery was tested using core-flooding experiments under reservoir conditions, where additional 27.27% of residual oil (Sor) was recovered. This confirmed the potential of SPLs for applications in microbial enhanced oil recovery.

Bench-Scale Production of Biosurfactants and their Potential in Ex-Situ MEOR Application

The fermentative production of biosurfactants by five Bacillus strains in a bench-scale bioreactor and evaluation of biosurfactant-based enhanced oil recovery using sand pack columns were investigated. Adjusting the initial dissolved oxygen to 100% saturation, without any further control and with collection of foam and recycling of biomass, gave higher biosurfactant production. The microorganisms were able to produce biosurfactants, thus reducing the surface tension and interfacial tension to 28 mN/m and 5.8–0.5 mN/m, respectively, in less than 10 hours. The crude surfactant concentration of 0.08–1.1 g/L, and critical micelle concentration (CMC) values of 19.4–39 mg/L, corresponding to the biosurfactants produced by the different Bacillus strains, were observed. The efficiency of crude biosurfactant preparation obtained from Bacillus strains for enhanced oil recovery, by sand pack column studies, revealed it to vary from 30.22–34.19% of the water flood residual oil saturation. The results are indicative of the potential of the strains for the development of ex-situ, microbial-enhanced, oil recovery processes.

Occurrence of Biosurfactant Producing Bacillus spp. in Diverse Habitats

Diversity among biosurfactant producing Bacillus spp. from diverse habitats was studied among 77 isolates. Cluster analysis based on phenotypic characteristics using unweighted pair-group method with arithmetic averages (UPGMAs) method was performed. Bacillus isolates possessing high surface tension activity and five reference strains were subjected to amplified 16S rDNA restriction analysis (ARDRA). A correlation between the phenotypic and genotypic characterization of Bacillus spp. is explored. Most of the oil reservoir isolates showing high surface activity clustered with B. licheniformis and B. subtilis, the hot water spring isolates clustered in two ingroups, while the petroleum contaminated soil isolates were randomly distributed in all the three ingroups. Present work revealed that diversity exists in distribution of Bacillus spp. from thermal and hydrocarbon containing habitats where majority of organisms belonged to B. licheniformis and B. subtilis group. Isolate B. licheniformis TT42 produced biosurfactant which reduced the surface tension of water from 72 mNm−1 to 28 mNm−1, and 0.05 mNm−1 interfacial tension against crude oil at 80°C. This isolate clustered with B. subtilis and B. licheniformis group on the basis of ARDRA. These findings increase the possibility of exploiting the Bacillus spp. from different habitats and their possible use in oil recovery.

Lipopeptide production by Bacillus subtilis R1 and its possible applications

The possible application of a bacterial strain – Bacillus subtilis R1, isolated from an oil contaminated desert site in India, as biocontrol agent and its biosurfactant in microbial enhanced oil recovery are discussed. The biosurfactant production in minimal medium was carried out at different temperatures and salt concentrations, where it produced an efficient biosurfactant at 30–45 °C and in presence of up to 7% salt. It significantly reduced the surface tension from 66 ± 1.25 mN/m to 29 ± 0.85 mN/m within 24 h. In order to enhance the biosurfactant production, random mutagenesis of B. subtilis R1 was performed using chemical mutagen – ethyl methanesulfonate. Majority of the isolated 42 mutants showed biosurfactant production, but the difference was statistically insignificant as compared with parent strain R1. Therefore none of the mutants were selected for further study, and only parent strain R1 was studied. The biosurfactant was quite stable under harsh conditions for up to 10 days. The biosurfactant was extracted and characterized as similar to the lipopeptide group – surfactins and fengycin. The crude oil displacement experiments using biosurfactant broth in sand pack glass columns showed 33 ± 1.25% additional oil recovery. The strain also showed inhibition of various plant pathogenic fungi on potato dextrose agar medium.

Biosurfactant's Role in Bioremediation of NAPL and Fermentative Production

Surfactants and biosurfactants are amphipathic molecules with both hydrophilic and hydrophobic moieties that partition preferentially at the interface between fluid phases that have different degrees of polarity and hydrogen bonding which confers excellent detergency, emulsifying, foaming and dispersing traits, making them most versatile process chemicals. One of the major applications of (bio)surfactants is in environmental bioremediation field. Most synthetic organic compounds present in contaminated soils are only weakly soluble or completely insoluble in water, so they exist in the subsurface as separate liquid phase, often referred as a non-aqueous phase liquids (NAPL), which poses as threat to environment. Several studies have revealed the use of surfactants for remediation; however, several factors limit the use of surfactants in environmental remediation, mainly persistence of surfactants or their metabolites and thus potentially pose an environmental concern. Biosurfactants may provide a more cost-effective approach for subsurface remediation when used alone or in combination with synthetic surfactants. There are several advantages of biosurfactants when compared to chemical surfactants, mainly biodegradability, low toxicity, biocompatibility and ability to be synthesized from renewable feedstock. Despite having many commercially attractive properties and clear advantages compared with their synthetic counterparts, biosurfactants have not yet been employed extensively in industry because of their low yields and relatively high production and recovery costs. However, the use of mutants and recombinant hyperproducing microorganisms along with the use of cheaper raw materials and optimal growth and production conditions and more efficient recovery processes, the production of biosurfactant can be made economically feasible. Therefore, future research aiming for high-level production of biosurfactants must be focused towards the development of appropriate combinations of hyperproducing microbial strains, optimized cheaper production media and optimized process conditions, which will lead to economical commercial level biosurfactant production.

Production, Characterization, and Application of Bacillus licheniformis W16 Biosurfactant in Enhancing Oil Recovery

The biosurfactant production by Bacillus licheniformis W16 and evaluation of biosurfactant based enhanced oil recovery (EOR) using core-flood under reservoir conditions were investigated. Previously reported nine different production media were screened for biosurfactant production, and two were further optimized with different carbon sources (glucose, sucrose, starch, cane molasses, or date molasses), as well as the strain was screened for biosurfactant production during the growth in different media. The biosurfactant reduced the surface tension and interfacial tension to 24.33 ± 0.57 mN m−1 and 2.47 ± 0.32 mN m−1 respectively within 72 h, at 40°C, and also altered the wettability of a hydrophobic surface by changing the contact angle from 55.67 ± 1.6 to 19.54°± 0.96°. The critical micelle dilution values of 4X were observed. The biosurfactants were characterized by different analytical techniques and identified as lipopeptide, similar to lichenysin-A. The biosurfactant was stable over wide range of extreme environmental conditions. The core flood experiments showed that the biosurfactant was able to enhance the oil recovery by 24–26% over residual oil saturation (Sor). The results highlight the potential application of lipopeptide biosurfactant in wettability alteration and microbial EOR processes.