DESC:BACTERIOPHAGE MEDIATED BIOCONTROL IN OIL RESERVOIRS
FIELD OF INVENTION
The invention generally relates to the field of biotechnology and specifically to a bacteriophage mediated bio-control for controlling bacterial growth in oil reservoirs.
BACKGROUND
The ‘crude’ oil is a mixture of water termed as formation, produced or co-produced water (commonly referred to as produced water), and oil & combustible gases. Resulting ‘water in oil emulsion’ is rich in variety of minerals and other toxic substances, microbial flora, both aerobic and anaerobic. During processing, the crude obtained from different wells is pooled and fed into tanks where gaseous components are first separated followed by separation of most of the oil component. This leaves behind produced water which is actually an ‘oil in water’ emulsion highly conducive to the growth of indigenous microbial population. Bacteria, such as Sulphate Reducing Bacteria, herein referred to as “SRB” converts sulphate found in the wells at the oil processing installations, into hydrogen sulphide causing acidification in the local environment and thus, corrosion of the pipelines in the presence of water.
The anaerobic SRB and associated facultative anaerobes are present in the produced water and multiplies exponentially. Using sulphate as a terminal electron acceptor, and organic acids present in the produced water as substrates, the bacteria causes the production of sulphide. The growth of SRBs leads to sulphate being reduced to sulfide. The ‘production water’ containing formed hydrogen sulfide herein referred to as H2S is highly toxic, posing health risk to the personnel and environment. To dispose, the production water has to be pumped back into the depths of the earth through water disposal wells. The water disposal wells are generally at a depth of about 1500 meters. Since the water stored in the wells is rich in SRBs constantly producing H2S, the enhanced presence of SRBs promotes corrosion of iron and steel storage tanks, precipitates ferrous sulfide and blocks water disposal wells, drastically reducing the injectivity of water disposal wells. Replacement of the iron and steel storage tanks is highly expensive. Frequent blocking of the wells greatly incapacitates the disposal of production water. One method known in the art for removal of the block caused due to corrosion is through mechanical scrapping. Removal of the block by mechanical scrapping is highly expensive & time consuming. Another method known in the art is the method of removal of corrosion through acid wash, which is also an expensive and time consuming process. Thus, appropriate microbial control in oil and gas recovery processes is critical for safe and efficient operations. Since hydrogen sulphide gas is toxic and highly flammable, inhalation of the fumes of the hydrogen sulphide gas increases the risk to the people working in the proximity of the wells. Yet another method known in the art employs the use of biocides for treatment of the blocks in the oil processing installations. Biocide control microbes and thereby reduces microbe mediated corrosion process. Biocides are not only hazardous with the potential risk to the environment, but also highly expensive.
Further during the oil recovery process, the bacteria in the formation water constantly encounter favourable environment for growth that results in their colonization and eventual biofilm formation. These biofilms are impervious to biocides and prevent their action on the bacteria. Increasing amounts of biocides further would result in resistance to these chemicals. The Microbiologically-induced corrosion (MIC) can damage pipelines resulting in spills and thus cause threat to mankind. There is need in the art for an alternate, better and eco-friendly methods for the control of microbes in highly anaerobic environment such as oil processing installations.
BRIEF DESCRIPTION OF DRAWINGS
So that the manner in which the recited features of the invention can be understood in detail, some of the embodiments are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.
Fig.1 shows Transmission electron micrograph of Bacteriophages, according to an embodiment of the invention.
Fig.2 shows pure culture of D. vulgaris on SSA medium following incubation at 37 °C in anaerobic condition for 3 days, according to an example of the invention.
Fig.3 shows isolation and enrichment of bacteriophages against SRB in formation water, according to an example of the invention.
Fig.4 shows two types of plaques following agar overlay of enriched bacteriophages on to SSB culture, according to an example of the invention.
Fig.5 shows the efficacy of bacteriophage against SRB in the presence of facultative anaerobic bacteria isolated from formation water, according to an example of the invention.
Fig.6 shows the layout plan of oil processing installation in Duliajan, Assam and sites of phage application for efficient control of bacteria, according to an example of the invention.
SUMMARY OF THE INVENTION
One aspect of the invention provides a heat resistant cocktail of bacteriophage for bio-control in oil reservoirs. The cocktail of bacteriophage comprises bacteriophages having MTCC accession no. MTCC 25355 and MTCC 25353. The cocktail of bacteriophage is heat resistant and stable in the temperature range of 45°C to 55°C.
DETAILED DESCRIPTION OF THE INVENTION
Various embodiments of the invention provide a heat resistant cocktail of bacteriophage for bio-control in oil reservoirs.
Specific embodiments of the invention provide for bio-control of the bacterial growth by the use of heat resistant cocktail of bacteriophage, particularly heat resistant lytic bacteriophages in highly anaerobic conditions. The cocktail of bacteriophage in the present invention have MTCC accession no. MTCC 25355 and MTCC 25353.
According to an embodiment, the heat resistant cocktail of bacteriophage is stable in the temperature range of 45°C to 55°C.
Another embodiment provides the cocktail of bacteriophage specific to anaerobic bacteria, particularly to SRB, that include but are not limited to Desulfovibrio spp, Desulfosporosinu.ssp, Desulfotomaculum.spp, Desulfomicrobiumspp, Desulfobacter.spp, Thermodesulfobacteium.spp and Desuffanium.spp
Yet another embodiment provides the cocktail of bacteriophage specific to facultative bacteria that include but are not limited to Clostridium, Enterobacter, Bacillus, Kleibsiella, Pseudomonas, Proteus, Thermoproteus.
The tailed bacteriophage with double stranded DNA belonging to order Caudovirale is isolated from water samples collected from various locations using SRB cultures as bait.
Fig. 1 provides Transmission electron micrograph of bacteriophages, TEM images reveals phage MTCC 25353 as well as MTCC 25355 belongs to family Podoviridae.
Genome sequencing of bacteriophage having MTCC accession no. MTCC 25355, using NGS, reveals that it has a double-stranded genome with a length of 37,586 base pairs and G+C content of 55.5%. 57 putative open reading frames are predicted, of these only 12 ORFs encoded proteins has known functions and include proteins matching to DNA polymerase, DNA helicase, tail and capsid.
A prerequisite for isolating bacteriophage and demonstrating efficacy against bacteria is to isolate bacteria as pure cultures. Described herein are methods of reviving and culturing anaerobic bacteria from lyophilised powder in the presence of associated facultative anaerobic bacteria isolated from produced water obtained from highly anaerobic conditions of oil processing installations. In one example, lyophilized culture of D. vulgar is having MTCC accession number 25354, procured from German Collection of Microorganisms and Cell Cultures, DSMZ, is resuspended in SAPI broth and cultured in the presence of Klebsiella sp. in SAPI broth. In another example, the method is further extended to isolate SRBs from produced water samples obtained from oil processing installations of Duliajan, Assam. The method further involves isolating the various bacteria present in the produced water as pure cultures. In one example, the blackened culture indicating the presence of anaerobic bacteria is serially diluted and spread plated on SSA medium and incubated at 37 °C in anaerobic condition for 3 days. Fig.2 shows pure culture of D. vulgar is on SSA medium following incubation at 37°C in anaerobic condition for 3 days, by single colony isolation method known in the art, according to an embodiment of the invention.
Accordingly, in one example, bacteriophages are enriched from sewage water samples collected from Bengaluru and produced water from OIL labs. Specifically the 1 ml of water samples and 0.5 ml SRB culture suspensions of 108 CFU are mixed, inoculated into appropriate medium, such as SAPI broth, sparged with nitrogen, and incubated at 37°C in anaerobic condition till the cell control of SRB pure culture shows blackening at concentration range of 108 to109 CFU. Following incubation, the water sample and cell culture mixture are filtered through 0.2 µm filter for further enrichment of bacteriophages and the same is spotted on SRB cultures grown on SSA plates with and without Ferrous ammonium sulphate, herein after referred to as FAS. The enriched filtrate samples shows plaques on SSA plates with SRB indicating presence of bacteriophages. Fig.3 shows isolation and enrichment of bacteriophages against SRB in formation water, according to an example of the invention. The bactericidal efficiency of isolated bacteriophages is seen by formation on plaques and prevention of growth of SRB as shown in Fig. 4.
The cocktail of bacteriophage shows bactericidal activity that is comparable to that shown by biocides. SRB culture at concentration range of 1×108 CFU suspension is mixed with cocktail of bacteriophage at concentration range of 1×108PFU or with biocide (THPS 50ppm) and incubated in anaerobic conditions till the SRB culture control shows blackening. Phage treatment is able to prevent growth of SRB present in the concentration range of1×108 PFU in the absence of biocide that is comparable to the killing seen in the presence of 50 ppm of biocide. Biocides are added in multiple locations for maximum bactericidal effect; however the activity may be largely compromised in the presence of emulsion treaters. Fig. 5 shows the efficacy of bacteriophage against SRB in the presence of facultative anaerobic bacteria isolated from formation water, according to an example of the invention. The lytic bacteriophages cocktail of the invention are viable at high temperatures ranges from 450C to 550C and continue to kill bacteria.
Cocktail of bacteriophage efficacy studies on pure culture of SRB and consortium of SRB and Klebsiella at 45 0C demonstrates utility of bacteriophages cocktail in effective controlling of SRB.
Groups Combination of cultures of Klebsiella (103CFU/mL)
And Observation(Blackening at)
Cell Control
(Untreated)
D. vulgaris 106 CFU/mL 2 days

D. vulgaris 105 CFU/mL 5 days
D. vulgaris 104 CFU/mL
D. vulgaris 103 CFU/mL 10 days
D. vulgaris 102 CFU/mL
Phage treatment
D. vulgaris 106 CFU/mL 7 days
D. vulgaris 105 CFU/mL 6 days
D. vulgaris 104 CFU/mL 17 days
D. vulgaris 103 CFU/mL No Blackening at the end of 29 days
D. vulgaris 102 CFU/mL 17 days
D. vulgaris 106 CFU/mL+ Klebsiella 103CFU/mL with biocide No Blackening at the end of 29 days
Table 1 : Efficacy of phages at 45 0C on consortium of SRB and Klebsiella

Table 1 shows efficacy of bacteriophage at 450C on SRB and associated bacteria in consortium. Bacteriophages cocktail treatment is effective in controlling blackening by consortium culture of SRB and Klebsiella. Single dose bacteriophages cocktail is able to prevent the bacterial growth. Cell control, untreated with bacteriophages cocktail grows in 48 hour, indicating blackening of broth, while broth treated with bacteriophages cocktail does not yield blackening, indicating bacteriophages cocktail is stable at 450C and continue to kill the bacterial consortium.
One embodiment of the invention additionally provides a carrier vehicle for reasonably retaining the cocktail of bacteriophage. The carrier includes but is not limited to alginate, chitosan based nanoparticles, silver based nanoparticles, selenium nanoparticles.
Fig. 6 shows the layout plan of oil processing installation in Duliajan, Assam and sites of phage application for efficient control of bacteria, according to an example of the invention. Crude oil from oil wells is collected (1) and passed through the Three Phase Separator (2) where initial separation of gas, oil and water occurs. The crude oil stream is then fed into Emulsion Treater (3) to further reduce the water content by breaking the oil and water emulsion by heating up to 60°C. Oil Field Chemicals like Oil Soluble Demuslifier (OSD) are often injected in the Emulsion Treater to further augment the emulsion breaking process. Separation of Gas, Crude oil and produced water is therefore achieved fully in the Emulsion Treaters. The crude oil stream now referred to as ‘dry crude’ is collected and stored in oil tanks(7) and dispatched through crude oil dispatch pumps ( CODP)(10) to Tank Farms(8) from where they are transported to end users i.e. Refineries. The gas collected in three phase separator (TPS) and emulsion treater (ET) is transported to Gas Collecting Stations. The produced water stream from the TPS and ETs is routed / transported to Formation Water Tanks(4,5) from where the produced water or formation water is disposed of into Water Disposal Wells (WDW,9). Bio-Fouling initiates primarily in the ETs where produced water is separated from the oil. As such the cocktail of bacteriophage dosing points are located upstream of ET (6). Additionally Bacteriophage injection points or Bacteriophage delivery points can also be located at the water stream after ET before the formation water tanks (11).
The invention, as described herein advantageously provides a composition for bacteriophage mediated bio-control in oil reservoirs. One significant advantage of the composition is a cost effective approach for controlling bacterial growth in oil processing installations. Additionally, the composition as described herein is non-hazardous in nature.
The foregoing description of the invention has been set merely to illustrate the invention and is not intended to be limiting. Since modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to person skilled in the art, the invention should be construed to include everything within the scope of the appended claims and equivalents thereof.
,CLAIMS:We Claim,
1. A heat resistant cocktail of bacteriophage for bio-control in oil reservoirs, comprising bacteriophages having MTCC accession no. MTCC 25355 and MTCC 25353.
2. The cocktail of bacteriophage as claimed in claim 1, wherein the cocktail of bacteriophage belongs to the order caudovirale.
3. The cocktail of bacteriophage as claimed in claim 1, wherein the cocktail of bacteriophage is specific to sulphur reducing bacteria selected from the group comprising of Desulfovibriospp, Desulfosporosinu.ssp, Desulfotomaculum.spp, Desulfomicrobiumspp, Desulfobacter.spp, Thermodesulfobacteium.spp and Desuffanium.spp.
4. The cocktail of bacteriophage as claimed in claim 1, wherein the cocktail of bacteriophage is specific to facultative anaerobes selected from the group comprising of Clostridium, Enterobacter, Bacillus, Kleibsiella, Pseudomonas, Proteus, Thermoproteus.
5. The cocktail of bacteriophage as claimed in claim 1, wherein the cocktail of bacteriophage is stable in the temperature range of 45°C to 55°C.
6. The cocktail of bacteriophage as claimed in claim 1, wherein additionally the cocktail of bacteriophage contains carrier vehicle for reasonably retaining the cocktail of bacteriophage selected from alginate, chitosan based nanoparticles, silver based nanoparticles, selenium nanoparticles.