Isolation and Characterization of Gram-Positive Biosurfactant-Producing Halothermophilic Bacilli from Iranian Petroleum Reservoirs

Abstract

Background:Petroleum reservoirs have long been known as the hosts of extremophilic microorganisms. Some of these microorganisms are known for their potential biotechnological applications, particularly production of extra and intracellular polymers and enzymes.Objectives:Here, 14 petroleum liquid samples from southern Iranian oil reservoirs were screened for presence of biosurfactant‐producing halothermophiles.Materials and Methods:Mixture of the reservoir fluid samples with a minimal growth medium was incubated under an N2 atmosphere in 40°C; 0.5 mL samples were transferred from the aqueous phase to agar plates after 72 hours of incubation; 100 mL cell cultures were prepared using the MSS-1 (mineral salt solution 1) liquid medium with 5% (w/v) NaCl. The time-course samples were analyzed by recording the absorbance at 600 nm using a spectrophotometer. Incubation was carried out in 40°C with mild shaking in aerobic conditions. Thermotolerance was evaluated by growing the isolates at 40, 50, 60 and 70°C with varying NaCl concentrations of 5% and 10% (w/v). Halotolerance was evaluated using NaCl concentrations of 5%, 10%, 12.5% and 15% (w/v) and incubating them at 40°C under aerobic and anaerobic conditions. Different phenotypic characteristics were evaluated, as outlined in Bergey's manual of determinative bacteriology. Comparing 16S rDNA sequences is one of the most powerful tools for classification of microorganisms.Results:Among 34 isolates, 10 demonstrated biosurfactant production and growth at temperatures between 40°C and 70°C in saline media containing 5%‐15% w/v NaCl. Using partial 16S rDNA sequencing (and amplified ribosomal DNA restriction analysis [ARDRA]) and biochemical tests (API tests 20E and 50 CHB), all the 10 isolates proved to be facultative anaerobic, Gram-positive moderate thermohalophiles of the genus Bacillus (B. thermoglucosidasius, B. thermodenitrificans, B. thermoleovorans, B. stearothermophilus and B. licheniformis), exhibiting surface-active behaviors.Conclusions:General patterns include decreasing the thermotolerance with increasing the salt concentrations and also more halotolerance in the aerobic environment compared with anaerobic conditions. The results demonstrated that Iranian petroleum reservoirs enjoy a source of indigenous extremophilic microorganisms with potential applications in microbial enhanced oil recovery and commercial enzyme production.

Investigating the mechanism of interfacial tension reduction through the combination of low-salinity water and bacteria

“…The samples were collected from various well-sampling lines. The evaluation of the isolated bacterium has been conducted in previous studies, including those by Zargari et al 69 , Sarafzadeh et al 71 , 72 , and Abdi et al 13 . These bacteria exhibit moderate halotolerance, ranging between 5 and 15%, indicating their moderate halophilic nature.…”

“…In previous investigations, BHI was proven to be effective in cultivating G. Stearothermophilus under harsh conditions 13 , 69 , 71 , 72 . According to Zargari et al 69 , the isolation process was carried out. Initially, the cultured bacterial solution was evaluated for IFT.…”

“…Moreover, numerous studies have employed BHI as an effective medium for biosurfactant production by different bacteria types [76][77][78][79] . In previous investigations, BHI was proven to be effective in cultivating G. Stearothermophilus under harsh conditions 13,69,71,72 . According to Zargari et al 69 , the isolation process was carried out.…”

“…Moreover, Geobacillus strains are thermophilic, allowing them to tolerate high-temperature environments like oil fields [66][67][68] . Zargari et al 69 first introduced G. Stearothermophilus (strain bio14) for MEOR purposes, demonstrating acceptable tolerance to temperature and salinity. Subsequently, Abdi et al 13 used G. Stearothermophilus (strain bio14) along with the smart water method to reduce IFT and enhance recovery factors.…”

Commentary: Oil degradation and biosurfactant production by the deep sea bacterium Dietzia maris As-13-3

“…Nevertheless, there is enormous scope for investigating new and extreme environments so as to look for novel strains which can mass produce these biosurfactants using relatively cheap substrates. Microbial consortia ERCPPI-2 isolated from heavy crude oil contaminated site in Iran, Virgibacillus salarius isolated from soil and sea water samples from Saudi Arabia, 10 thermohalophilic isolates of genus Bacillus isolated from Iranian petroleum reservoirs are few examples of extremophilic microorganisms that produce biosurfactants (Darvishi et al, 2011 ; Zargari et al, 2014 ; Elazzazy et al, 2015 ). Although alkanes are considerably not very cheap but interestingly Dietzia maris As-13-3 could utilize three different alkanes such as n-tetradecane, n-hexadecane, and pristane, for biosurfactant production.…”