Isolation and identification of 101C5
A hyperthermophilic strain, designated as 101C5, was isolated from a high-temperature oil reservoir at 101 °C by enrichment, followed by roll tube isolation technique. Strain 101C5 was selected for further studies based on its ability to grow at 101 °C and produce metabolites desirable for MEOR. Morphological characterization revealed 101C5 as Gram-negative cocci (0.3 µ to 0.5 µ in diameter), which occurred in clusters (Fig. S1). The strain 101C5 shared the highest 16S rRNA gene sequence homology of 100% with phylogenetic affiliate Thermococcus litoralis DSM 5473 strain NS-C. The genome of strain 101C5 was sequenced, and the sequence homology of the extracted housekeeping genes was determined. Relatively low sequence homologies with closest phylogenetic affiliate DSM 5473 (as identified by 16S rRNA gene sequence) for each of the housekeeping genes (90.8% for rpoB, 88.95% for gly A- serine methyltransferase and 83.31% for pfkB- carbohydrate kinase) identified strain 101C5 to be taxonomically different from DSM 5473. Further, the entire 101C5 genome was compared with other publically available genomes of Thermococcus (Thermococcus litoralis DSM 5473 and Thermococcus sp. PK) using Digital DNA-DNA-hybridization (DDH) and Average Nucleotide Identity (ANI) tool. The strain 101C5 shared only 33.6% and 67.9% DDH homology with DSM 5473 and PK, respectively, which was less than the threshold of 70%. Similarly, lower ANI homologies of 87.7% and 96.5% were shared with DSM 5473 and PK, respectively, which was less than the threshold of 95% (Chun et al. 2018). Thus, strain 101C5 was identified as a putative novel member of genus Thermococcus based on lesser threshold homologies in DDH and ANI.
The genus Thermococcus comprises strictly anaerobic and hyperthermophilic archaea belonging to the order Thermococcales within the phylum of euryarchaeota. The euryarchaeal rrder Thermococcales consists of three genera Pyrococcus, Palaeococcus and Thermococcus. Members of the genus Thermococcus include 19 different species and are common inhabitants of extreme niches such as hot springs, hydrothermal vents, deep subsurface oil reservoirs, etc., with growth temperature ranging from 45 °C to 90 °C (Adams 1993; Summit and Baross, 1998). Majority of Thermococcus sp. are organoheterotrophs fermenting organic compounds into CO2 and H2 as end products (Teske et al. 2019), while few are capable of lithotrophic CO-dependent hydrogenogenic growth such as Thermococcus strain AM4 and Thermococcus onnurineus (Svetlitchnyi et al. 2001). Though the presence of Thermococcus sp. has been reported from high-temperature oil reservoirs (Zahner et al. 2010; Orphan et al. 2000), only two reports have documented its role in the MEOR application (Takahata et al. 2001; Lal et al. 2009), thus warranting further study in this area.
General genome features and comparative genomics of 101C5 with related species
The sequencing of the 101C5 genome yielded a genome size of 1,926,104 bp with 119 contigs and GC content of 44%. A circular map illustrating the graphical display of the distribution of genome annotations was generated (Fig. S2) (Wattam et al. 2017), wherein, outermost circle 1 represented the contigs, circle 2 represents CDS on the forward strand, circle 3 represents CDS on the reverse strand, circle 4 represents RNA genes, circle 5 represents CDS with homology to known antimicrobial resistance genes, circle 6 represents GC content, and innermost circle 7 represents GC skew. The colors of the CDS on the forward and reverse strand indicate the subsystem that protein-coding genes belong to. RAST functional annotation predicted 146 subsystems having 2304 total genes, with 2258 protein-coding genes and 46 RNA encoding genes. Majority of the 101C5 proteins showed similarity to those of Thermococcus sp. PK. The phylogenomic tree constructed using PATRIC indicated that 101C5 was placed at the same node with PK, revealing a close taxonomic relationship of 101C5 with Thermococcus sp. PK (Fig. 1). However, 101C5 was placed distantly from DSM5473, indicating the taxonomic difference between both species.
Genome reports of six different species of Thermococcus namely, T. sibricus, T. barophilus, T. kodakaraensis, T. gammotolerans, T. onnurineus and T. litoralis are publically available and have provided insights into their genetic makeup and metabolic machinery. Except for T. sibricus, none of the reported species of Thermococcus originated from oil reservoirs. T. sibricus was isolated from a high-temperature oil reservoir located in Western Siberia, growing anaerobically at 78 ℃. The genome size of T. sibricus was 1.84 Mb with 2061 protein coding genes, providing metabolic insights into substrate utilization and energy generation. The T. sibricus genome established its indigenous origin from oil reservoir based on the presence of enzymes involved in metabolizing organic matter (cellulose, agarose, laminarin, and triglycerides) from oceanic sediments and alkanes from crude oil (Mardanov et al. 2009). However, this study did not provide any information regarding the adaptive starategy employed by T. sibricus to survive in harsh reservoir conditions. T. barophilus MP a hyperthermophilic, piezophilic archaeon capable of growing at 95°C and 40 MPa was isolated from chimney samples. The MP genome consisted of a circular chromosome of size 2.01 Mb and a circular plasmid of size 54,159 bp, harboring the hydrogenase complexes and genes involved in carboxydotrophic pathway (Vannier et al. 2011). T. kodakaraensis KOD1 was isolated from Kodakara Island, Japan and possessed a genome size of 2.08 Mb with 2306 coding sequences. KOD1 genome reported several proteins involved in nucleotide metabolism, stress response, etc (Fukui et al. 2005). T. gammotolerans, a radioresistant archaea isolated from hydrothermal chimneys had a genome size of 2.045 Mb with 2157 coding sequences. Genome analysis of T. gammotolerans provided insights into catabolism, hydrogenase complexes, detoxification systems and DNA repair (Zivanovic et al. 2009). T. onnurineus NA1 isolated from a deep-sea hydrothermal vent possessed a genome size of 1.84 Mb with 1976 coding sequences. Genome analysis of NA1 reported the carboxydotrophic pathway in combination with heterotrophy as well as provided the first evidence of lithotrophic feature of Thermococcus (Lee et al. 2008). T. litoralis NS-C isolated from a hot spring Italy had a genome size of 2.3 Mb with 2724 coding sequences. The NS-C genome provided crucial information regarding the mobile genetic elements inteins (Gardner et al. 2012).
101C5 genome was compared with the publically available Thermococcus genomes of PK, EP1, DSM 5473 and oil based on general genome features using RAST (Table S1). The genome size of Thermococcus sp. used for comparison in the present study ranged from 1.82 – 2.22 Mb, with DSM 5473 having the largest genome size among the genomes used. Interestingly, the genomes sued in the study had similar GC contents (39.3% to ~44%). A noticeable difference in the number of genes between 101C5 and DSM 5473 was observed mainly in carbohydrate, DNA, protein, phosphorus, and amino acids metabolism, membrane transport and stress response. RAST analysis revealed a total of 31, 26, 12 and 31 unique genes respectively, in 101C5 genome compared with other genomes, namely, PK, EP1, DSM 5473 and oil.
BRIG tool (Alikhan et al. 2011) was used for comparing multiple genomes of Thermococcus sp. available in the NCBI database with 101C5 genome. The circular map (Fig. S3) representing the BLASTn results of each query genome (Thermococcus litoralis DSM 5473, Thermococcus sp. PK, Thermococcus sp. EP1, and Thermococcus sp. oil) against the reference strain 101C5 was generated. BRIG image displayed lighter color intensities and pronounced gaps in each of the query genomes, highlighting the difference between strain 101C5 and the other Thermococcus genomes used and further confirmed the novelty of strain 101C5.
Stress resistance mechanisms: Survival under extreme reservoir conditions
An oil reservoir is characterized as an extreme niche with extremes of temperature, pH, salinity, pressure, oxidative stress, etc. These harsh conditions can have a detrimental effect on exogenously injected microbes and can severely impair MEOR efficiency. Thus, adaptation to the environment becomes crucial for microbes to survive, which is controlled by several mechanisms at the molecular level. Genome analysis of strain 101C5 revealed the presence of diverse and abundant genes known to have a role in combating these physiological stresses (Table S2).
Temperature is one of the critical factors affecting microbial growth and metabolism in oil reservoirs. Elevated temperature affects the integrity of the cell membranes and can thus hamper bacterial growth. 101C5 genome harbored genes encoding various heat shock proteins, spermidine synthases, Clp, etc., having a role in thermo-tolerance. Spermidine synthases are physiological polyamines that are reported to be strong candidates responsible for thermophily in bacteria and archaea (Takami et al. 2004). Genes conferring resistance against oxidative stress (which might be transiently introduced during water flushing used for oil production), such as superoxide reductases, rubrerythrin, etc., were also detected in the 101C5 genome. Rubrerythrins are non‐haem iron proteins having a potential role in oxidative stress protection in anaerobes (Sztukowska et al. 2002). 101C5 genome was also found to be well equipped with the genetic arsenal required for adaptation to the saline environment present in a reservoir (osmo-adaptation), such as Trk like transporters, genes involved in compatible solutes (choline and betaine) synthesis. Under suddenly imposed and prolonged osmotic stress, uptake of K+ ions via Trk-like transporter could act as an essential feature in the cellular defense of strain 101C5 (Kraegeloh et al. 2005). Genes encoding betaine synthesis were also detected in the genome, which indicated its osmo-adaptive trait towards hypersaline environments. Plausible genes involved in tolerance of strain 101C5 to high pressure were also assessed. Previous reports have suggested the involvement of specific heat shock proteins in piezo tolerance (Aertsen et al. 2004; Simonato et al. 2006; Karatzas et al. 2007). The presence of GroEL in the 101C5 genome suggested its role as a protectant against high-pressure stress, which may also aid in repairing damages caused by high pressure.
Thus, in silico analysis of 101C5 predicted the excellent reservoir adaptive features of the strain, suggesting its strong adaptability to harsh conditions.
Prediction of functional genes in 101C5 genome to elucidate its potential role in MEOR
Genome mining revealed an arsenal of candidate genes involved in producing various metabolites like hydrogen, exopolysaccharide (EPS), organic acids, bio-emulsifier (Table S3), highlighting the potential of strain 101C5 for MEOR. 101C5 genome contains a wide range of genes encoding various membrane-bound hydrogenases, NiFe hydrogenases, hydrogenase maturation, expression, and formation proteins. These enzymes are actively involved in the generation of H+ ions and the production of bio-hydrogen, which could enhance light oil recovery by the mechanisms of reservoir re-pressurization (Van Hamme et al. 2003). Numerous NiFe hydrogenases have been identified and well characterized from Thermococcus litoralis (Rákhely et al. 1999). NiFe hydrogenases are multisubunit membrane bound enzymes involved in hydrogen production.
Biopolymers or exopolysaccharide selectively plug the high permeability zones and divert the displacement fluid towards the low permeability oil-rich zones in the reservoir thus, providing better sweep efficiency of oil, leading to enhanced oil recovery (Guo et al. 2015). Genes encoding Phosphomannomutase, mannose-1-phosphate guanylyltransferase, mannosyl-3-phosphoglycerate phosphatase, and mannosyl-3-phosphoglycerate synthase suggested mannose to be the significant component of EPS produced by strain 101C5. EPS is also known to act as a bio-emulsifier. Emulsification is one of the most efficient mechanisms in MEOR processes. Bio-emulsifiers work by desorbing the oil from rocks and forming a stable oil/water emulsion, consequently facilitating oil mobilization. 101C5 harbored genes encoding key enzymes involved in the Mannoprotein nature of bio-emulsifier production, indicative of crude oil emulsifying and solubilizing ability of strain 101C5. 101C5 genome harbored enzymes such as Pyruvate: Ferredoxin oxidoreductase (POR), Acetyl-CoA synthetase (ADP-forming) (ACS), along with other auxiliary enzymes involved in acetate production. Organic acids, such as acetic acid, play a vital role in oil recovery by acting on the reservoir properties, particularly by dissolving the carbonate rock, thereby increasing its porosity and permeability.
101C5 genome presented the ability to produce gases, EPS, bio-emulsifiers and organic acids simultaneously, suggesting its strong potential for MEOR, which was further validated with wet-lab evidence. Thus, genome-level analysis aided in identifying candidate genes that can act as biomarkers for selecting potential MEOR strains.
Genomic insights into crude oil degradation ability of 101C5
Genome analysis revealed the presence of a cascade of genes involved in the production of enzymes that play a significant role in the biotransformation/ bio-degradation of crude oil. Genes encoding enzymes such as dioxygenases, 4-oxalocrotonate tautomerase, acylphosphatase, etc.having a role in benzoate degradation, alcohol dehydrogenase and haloacid dehalogenase, rubredoxin involved in alkane degradation were present in 101C5 genome (Table S4). The presence of these genes indicated the biotransformation potential of 101C5, implicating its prospects in improving the flow characteristics of heavier crudes. Also, several oxidoreductases, dehydrogenases, and dioxygenases involved in the degradation of PAHs, cyclic hydrocarbons and other aromatic compounds were detected in the genome. Members of genus Thermococcus have been reported as efficient hydrocarbon degraders, particularly in high-temperature oil reservoirs, making them ideal candidates for MEOR (Nikolova & Gutierrez 2020). Heavy crude oil, because of its high viscosity and density, is challenging to extract, transport, and refine by conventional technologies (Shibulal et al. 2014). However, if strain 101C5 can bio-transform or partially degrade heavy oil into light oil, then this trait can be considered desirable by many.
Thus, genome analysis unraveled the ability of strain 101C5 to produce MEOR desired metabolites, which was further validated in wet-lab experiments.
Metabolite profiling of hyperthermophile 101C5
The highest production of CO2, organic acids and emulsifying activity was obtained with strain 101C5 in molasses medium (20 ml) in 14 days at 101 °C (Table 1). This validates the potential of the hyperthermophilic strain 101C5 towards microbial enhanced oil recovery. For successful implementation of the MEOR process, microbial performance in terms of sustained growth and production of desired metabolites such as CO2, biosurfactants, solvents, acids, etc., is of utmost importance. Microbial production of carbon dioxide is a desired property for MEOR as it enhances oil recovery by increasing the reservoir pressure and reducing the viscosity of heavy oil (Al-Sulaimani et al. 2011). In the present study, 101C5 produced significant quantities of gas, with carbon dioxide (43.8 ml molasses g-1) as the major component. It was interesting to note that strain 101C5 produced substantial quantities of hydrogen (33.1 ml molasses g-1) and lower quantities of CO2 (3.2 ml molasses g-1) at 96 °C, however at 101 °C, a high quantity of CO2 was produced with negligible H2 production (Table 4). Bio-acids such as lactate and acetate, which are produced as the byproducts of anaerobic fermentation of sugars, can be helpful in carbonate reservoirs or sandstone formations cemented by carbonates. Acids are known to dissolve the carbonate rocks, resulting in improved porosity and permeability and also aids in the solubilization of oil (Adkins et al. 1992; Fardeau et al. 2004). Strain 101C5 produced 1477 mg/l of acetate, 466.4mg/l of lactate using molasses as fermentable C source and Yeast extract as N source. Bio-emulsifier production was analyzed in terms of the emulsification index. Arora et al. (2014) have reported EI of 43 % for thermophilic Clostridium sp. growing at 96 °C. The highest EI24% of 60.8% was obtained in the present study by strain 101C5 at 101 °C. This was suggestive of bio-emulsifier activity known to form stable oil-water emulsions. This property is known to mobilize trapped oil in reservoirs.
101C5 mediated oil recovery under simulated reservoir conditions
Reservoir characteristics can drastically influence the final outcome of MEOR as they significantly influence the microbial performance and, thus, the efficiency of MEOR. Such reservoir characteristics include in situ temperature, pressure, the porosity of reservoir rocks, carbonaceous or otherwise nature of the formation rocks, viscosity and pour point of the oil in place, etc. Thus, it becomes imperative to evaluate the oil recovery potential of the microbe under simulated reservoir conditions. The pore size of the rock will determine the microbial penetration and extent of the growth in the pores and crevices. The efficiency of MEOR will be directly affected by lesser pore size. Oil properties can have a significant impact on the oil recovery process. The high viscosity and low flow characteristics of heavy oil make it difficult to extract oil from the reservoir. The viscosity of the oil will determine the concentration of metabolites required for dissolution/ emulsification/ dissipation of oil. Additional oil recoveries as compared to water control were observed at 96 °C and 101 °C (Table 2). With increasing temperature, increased oil recovery was observed. A maximum oil recovery of 56.5% was observed at 101 °C by strain 101C5. To our best knowledge, this is the first report of the Microbial Enhanced Oil Recovery at 101 °C.
Further, >56% recovery of the residual oil by MEOR is one of the highest ever reported efficiency of MEOR for any microorganisms at any temperature in sand pack studies. This ability of strain 101C5 to efficiently recover heavy oil may be attributed to the significant quantities of acetic acid as well as gases (H2 and CO2) generated by the culture, which may have reduced the viscosity of the heavy oil, allowing it to display improved flow features and thereby, facilitating enhanced oil recovery. Organic acids are reported to be the active metabolite involved in the viscosity reduction of heavy crudes, thereby improving their mobility. Laboratory experiments in Daqing Oilfield revealed the viscosity reduction of 36 % with the aid of microbial acids (Han et al. 2001). Studies have also reported the importance of CO2 in the viscosity reduction of crude oil by expanding it when produced by microbes in a supercritical state of high temperature and high pressure (She et al. 2019). Treatment of heavy oils with strain 101C5 can thus improve their flow characteristics, thereby increasing the additional oil recoveries.