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

Research into the microbial life inhabiting the subterranean environments was traditionally important in biogeosciences, where it was crucial for understanding and explaining the chemistry and ecology of such systems. More recently, such knowledge has been useful in a number of engineered systems involving deep underground environments. A good example is oil reservoirs, considered for microbial enhanced oil recovery (MEOR) projects (1). Insight into the microbial community nature of a reservoir can be crucial in determining the suitability of the reservoir for an MEOR project, by providing information about the presence or absence of favorable species versus the unfavorable ones (e.g. sulfur-reducing bacteria) (2). Furthermore, such information contributes to understanding the possible mechanisms of enhanced oil recovery (interfacial tension reduction, selective plugging, etc.).

This in turn can be used in generating models that predict ultimate enhanced recovery, and optimizing the chemistry of injection fluids to promote a certain mechanism (e.g. biosurfactant production). Alternatively, lack of a potent microbial community can lead engineers to introduce exogenous species (3). Similar to other extreme environments, petroleum reservoirs typically host extremophilic organisms like haloalkaliphiles and thermohalophiles; a diverse group of microorganisms, characterized by their haloadaptive capabilities, enabling them to survive in hypersaline environments such as salt lakes, saline soils, salt mines, oil reservoirs, etc. (4-6). These microorganisms are generally categorized in two groups of extreme and moderate halophiles.

Extreme halophiles grow optimally in NaCl concentrations of 15%-30% (w/v) and generally belong to archaea, while moderate halophiles tolerate NaCl concentrations of 3%-15% (w/v) and are dominated by Bacteria (7). Moderate halophiles are better known for their potential biotechnological applications, particularly production of extra and intracellular polymers and enzymes (2). Enzymes produced by moderate halophiles have been the subject of extensive research because of their unique ability to perform optimally in high salt concentrations, which potentially eliminates the need for extensive sterilization procedures.

Amylase (8-13), esterase (12, 13), protease (5, 14-16), leucine dehydrogenase (17) and other halotolerant enzymes (18) have been successfully purified from moderate halophiles. In addition to extracellular enzymes, production of extracellular biosurfactants and bioemulsifiers has been reported by some moderate halophiles (1, 19, 20). Producing biosurfactant is favorable in the MEOR processes. In the present study, we reported isolation and characterization of biosurfactant-producing moderate halophiles from southern Iranian petroleum reservoirs. The results of this work demonstrated the potential of these reservoirs for MEOR and provided further information about their indigenous microbial populations.

The aim of this study was isolation of biosurfactant-producing microorganisms from petroleum reservoirs with potential applications in microbially enhanced oil recovery and commercial enzyme production.

Thirty four bacterial isolates were isolated from the liquid samples through the procedure previously explained, out of which, 10 produced extracellular surface active compounds through the oil spreading and E24 tests (Table 1). Biochemical and molecular assays suggested that the isolates most resembled B. thermoglucosidasius, B. thermodenitrificans, B. thermoleovorans, B. stearothermophilus and B. licheniformis, as presented in Table 1. The results were confirmed by API tests 20E and 50 CHB, performed using the commercially available kits (data are not shown).

The isolates are fairly similar in growth kinetics in the studied conditions (Figure 1). As it is given in Tables 2 and 3, all the isolates demonstrated similar behaviors in varying saline and thermal conditions. General patterns included decreasing thermotolerance with increasing salt concentrations and also more halotolerance in the aerobic environment compared with anaerobic conditions.

Figure 1.

All the Isolates had Similar Growth Patterns.

Figure 2 demonstrates that all the isolates of 1114 bp were specific to bacilli. Taq1 digested the PCR product identically and produced two amplicons with approximate sizes of 420 and 690 bp (Figure 3). Similarly, Alu1 digested the PCR product identically and produced five amplicons of 53, 59, 250, 252 and 600 bp (Figure 4) (the precise amplicon sizes were calculated using Vector NTI software, based on the sequencing data). Mse1 and Mbo1 were also tested, but failed to differentiate between the isolates. The similarity search using BLAST revealed over 99% homology with more than 1000 different bacilli.

Figure 2.

Amplified Ribosomal DNA Restriction Analysis Using the Bacilli Group-Specific Primer Developed by Wu et al. (24)

Figure 3.

Amplified Ribosomal DNA Restriction Analysis Using Taq1 (TCGA)

Figure 4.

Amplified Ribosomal DNA Restriction Analysis Using Alu1 (AGCT)

The results of 16S rDNA sequencing identical a 1114-bp sequence for these thermophilic bacilli. Therefore, implemented primers are not recommended for this class of bacilli. Wu et al. (24) successfully used this sequence to differentiate between varieties of bacilli including B. licheniformis, B. subtilis and B. ciculans. Manachini et al. (25) also reported that sequencing of 16s rDNA could not differentiate between Bacillus thermodenitrificans and their closest phylogenetic neighbors, e.g. the type strains of B. stearothermophilus and B. thermoleovorans, but they concluded that amplification of the internal transcribed spacer (ITS) region of rrn operons could significantly separate these strains (25). On the other hand, Vaerewijck et al. (26) reported that ARDRA using universal primers could not distinguish B. licheniformis and B. subtilis from B. pumilus and B. amyloliquefaciens. Kuisiene et al. (27), however, successfully identified the thermophilic strain Geobacillus stearothermophilus from similar strains, using ARDRA with Alu1 as their sequencing enzyme.

As given in Table 3, all the isolates demonstrated similar halotolerance behaviors in aerobic and anaerobic conditions, except for isolate 71, which did not growth under anaerobic conditions with salt concentrations more than 5%. It may be concluded that this isolate can only survive in low salt concentration reservoirs, or had a kind of symbiosis under high salt concentration conditions. The results of the present work contributed to the mounting evidence that Iranian petroleum reservoirs are host to a rich community of extremophiles. Previous studies demonstrated presence of Pseudomonas, Bacillus and Rhodococcus species in these areas and their successful implementation for MEOR in experimental core flooding setups (28-31). All of these studies focused specifically on biosurfactant-producing species. However, the results of isolation experiments suggest that microbial communities of these reservoirs are more diverse and might not be limited to bacteria.

Although, little is known about the obligate anaerobes in these reservoirs of which preliminary experiments suggest their existence (data are not shown). Future work can address various questions related to basic microbiology of the organisms, or focus on optimizing the parameters important to engineering their growth. The methods used in this study have been tailored for selective isolation of biosurfactant-producing extremophiles; however, similar experimental approaches can be used to study the microbial communities in broader microbiological contexts, to encompass nonbacterial organisms, extreme halophiles, and obligate anaerobes. Finally, potential applications of such organisms in enzyme production can be explored.