Taxonomic distributions
The phylum level distribution of SRB at both the Galo de Campina and Periquito oilfields was dominated by the Proteobacteria, 93.8%, and Firmicutes with 6.2%. This was not a surprise and has been reported previously Belgini et al.41 also identified Proteobacteria, Firmicutes and Bacteroidetes as dominant when analysing samples from the Gabriel Passos Refinery (REGAP) in the city of Betim (MG, Brazil). Proteobacteria (29.2%), Firmicutes (8.3%) and Bacteroidetes (8.3%) were found in produced water of Diyarbakır oil Fields in Turkey42. In Turkey, the Proteobacteria were the predominant phylum in all samples, similar to the GC and PQO samples. In a study from the Mae Soon Luang Field, Fang Basin, Thailand43, Proteobacteria, Firmicutes and Actinobacteria dominated the bacterial communities in most samples. This has also been reported from Texas, USA by Santillan et al.9, Li et al.44 and Lan et al.45 in China. Silva et al.23 studied the microbial communities in petroleum samples from Brazilian oil fields and found that the Proteobacteria was the most abundant phylum, followed by Bacilli and Clostridia which belong to the Firmicutes phylum. Lipus et al.46, analyzed microbial abundance in produced water samples in the Bakken region by qPCR. the analysis was done directly from produced water, without a pre-enrichment medium. The anaerobic, fermentative Firmicutes orders (Bacillales and Halanaerobiales) and the Proteobacteria Order Pseudomonadales were the most abundant taxa across all evaluated samples, representing between 57% and 99% of the total microbial population. Despite the differences in geographical locations of oilfields, their physicochemical properties and methods used for analyses, it is interesting to note that these two phyla dominate and perhaps not surprising if we consider conditions within a reservoir will strongly select for certain groups of bacteria. No differences in bacterial diversity were observed by using three enrichment media at phylum level. The dominant phyla are dominant regardless. Use of enrichment is a deliberate strategy for recovering cultivable bacteria with desired metabolic rates47. The main Orders detected in the produced water from Galo de Campinas e Periquito were Desulfovibrionales, Pseudomonadales, Enterobacteriales, Clostridiales, Alteromonadales, Rhizobiales and Bacillalles and their distributions are shown in (Fig. 3). Li et al.44, in his work on biofilms in water injection systems in the Daqing oil field (China), also identified Pseudomonadales, Enterobacteriales, Clostridiales and Desulfovibrionales orders, some organisms of these orders are already known to cause problems associated with hydrogen sulphide production, biofilm formation and biocorrosion8,9,19. Sequences from the order Desulfovibrionales were present in all samples and it was the dominant order found in both oil fields accounting for 46% of all sequences. The majority of bacteria in this order are SRB and are responsible for biocorrosion and possibly related to problems with hydrogen sulphide at the Periquito oilfield (personal communication). Pseudomonadales was predominant in Galo de Campinas oilfield and the, Enterobacteiriales in both oil fields. In the Galo de Campinas oilfield, no significative difference was observed among the media. The Firmicutes, and Proteobacteria Orders seem to have a worldwide distribution in oilfieds and are abundant in produced water. Sun et al.48 isolated 61 phylogenetic groups that belong to 32 genera in the phyla Actinobacteria, Firmicutes, and Proteobacteria in oil-production water from the Karamay Oilfield, Xinjiang, China. The Enterobacteriales and Alteromonadales orders that were detected in this study are in agreement with other reports in the literature that relate these orders to oil environments49,50. Liu and Liu51 analyzed the bacterial community of oil collected from the sea surface of the northern Gulf of Mexico, as a result they found high proportions of Alteromonas, Marinobacter, Thalassospira, Bartonella, Rhodovulum and Stappia. And they point out that Marinobacter and Alteromonas, Gammaproteobacteria, are common oil-degrading microorganisms. This result is in agreement with Bacosa et al.52 who analyzed the bacterial diversity of the same region and concluded that Alteromonas is an important class of bacteria in the fate of oil, being effective in degrading the alkanes in oil. Wang et al.53 studied the microbial community of oil-polluted soil on agricultural land in Fushun, Liaoning province in China, where they attested that the abundance of Enterobacteriales was greater in areas with oil-contaminated soil.
Figure 4 provides analysis genus level diversity of the produced water and the main genera are summarized in Table 2. Desulfovibrio is the main and dominant genus in produced water from Galo de Campinas (16,08%) and Periquito (29,7%) followed by Pseudomonas in Galo de Campinas (19,88%) and Shewanella in Periquito (4,69%). Some bacterial sequences were too divergent for classification and that was the case for both oilfields. Clostridia were detected in low numbers from both oil fields. Postgate C, in the majority of the samples, favoured Desulfovibrio isolation over the other media used. Pseudomonas was detected only in groups GCc and GCb, being a possible biomarker for these sites. Pleomorphomona were identified only at Periquito. Desulfovibrio, Pseudomonas and Clostridium are routinely found in produced water samples where strains from the Genus Desulfovibrio are noted as of major concern for the oil and gas industry. Comparing Fig. 4 with Fig. 3 we note that the figures look similar. This happens because a limited number of dominant genera are responsible for the Order level diversity. What stands out immediately is the importance of the Desulfovibrio sequences40,54. Desulfovibrio impact negatively causing corrosion, hydrogen production, souring and biofouling caused by biofilm formation within the operational plants. More than 220 species from 60 genera of SRB have been reported; of which, the most commonly isolated mesophilic SRB from produced water are from the Desulfovibrio genus40. Also of note were the Pseudomonads from two of the three Galo de Campinas sites, and, the as yet unidentified bacteria at genus level were from the Enterobacteriales. Pelobacter, Marinobacterium and Geotoga were detected in produced water from Petrobras Ilha Grande Bay Oil Terminal in Brazil, as well as the SRB Desulfoplanes formicivoran55. Bacteria of the genera Desulfovibrio and Clostridium are producers of hydrogen sulphide, it is toxic and accelerates the corrosion of metallic structures20,56. The most frequently described SRB genera from produced water are the Delta Proteobacteria Desulfovibrio and Desulfomicrobium21. Although Desulfovibrio was the dominant SRB in this study other related genera have been reported from petroleum derived samples, including Desulfomicrobium, Desulfobacterium, Desulfosarcina, Desulfococcus, Desulfotignum, Desulfobotulus, Desulfobulbus, Desulfacinum, Thermodesulforhabdus, Desulforhabdus, Desulfatibacillum, Desulfoglaeba, Desulfonauticus, Desulfocurvus (Delta-proteobacteria) Desulfotomaculum (Firmicutes), Thermodesulfobacterium (Thermodesulfobacteria), Thermodesulfovibrio (Nitrospira), Archaeoglobus (Euryarchaeota) and Caldivirga (Crenarchaeota)44,57−59. The genera Pleomorphomonas and Shewanella were more abundant in samples from the Periquito oil field. Pleomorphomonas was described in 2005 by Xie and Yokota60 and is composed of Gram-negative, pleomorphic, nitrogen-fixing, non-spore-forming, non-motile rods61,62. At the time of writing, there is no data correlating this genus to problems in oil industry. Shewanella are facultative anaerobic, gram-negative, motile and rod-shaped bacteria, most of which have been isolated from marine environments, such as seawater, marine sediments or sand, tidal flats or marine invertebrates. Some species have, however, been isolated from clinical samples, oilfield fluids, activated sludge and coal-mine sludge63,64. Shewanella strains were described as a potential hazard to the oil industry causing souring of crude oil65.
Table 2
Top 5 dominant genus found in Galo de Campinas e Periquito oilfield
| Oil well |
Genera | Galo de Campinas | Periquito |
Desulfovibrio | 16,08% | 29,87% |
Pseudomonas | 19,88% | 0,18% |
Clostridium_sensu_stricto | 2,40% | 2,81% |
Shewanella | 0,07% | 4,69% |
Pleomorphomonas | 0,07% | 3,77% |
* unclassified genera from Galo de Campinas is 11,05% and Periquito 7,62 |
In Fig. 5, the heat map provides a clearer representation of the genus level differences between the sites and oilfields. The heatmap illustrates the mutual occurrence of Pseudomonas and Desulfovibrio only at the GCb site. The genera Anaerosalibacter, Pleomorphominas and Shewanella are important and exclusive biomarkers of the Periquito oilfield. Non-metric multidimensional scaling analysis of Galo de Campina and Periquito oil fields (Fig. 2) was used to study the correlation between the three sampling locations with the two oil fields (PQO and GC) and culture media (Postgate B, Postgate C and Baars) and the influence of each on the diversity detected. That analysis indicated that sampling points within each oil field and culture media did not significantly influence the microbial populations detected and the main difference in microbial distributions at the genus level was related to the two oil fields.
The metagenomic data obtained here by enriching produced water and then large scale PCR-DNA sequencing is consistent with the results reported in literature based on isolation methods and on methods where organisms were neither enriched nor isolated21,23,66.
It is only at the genus level that differences between individual wells and oil fields become evident. Bacterial strains of the Genus Pseudomonas are producers of extracellular polymeric substances that form biofilms within oil facilities17. The presence of mesophilic Pseudomonas strains that are sensitive to high temperatures is not believed to originate from pristine oil reservoirs. Their presence in systems with high temperature oil wells is likely to follow flooding with cooler produced water, and contamination with Pseudomonas strains that are very versatile heterotrophs with the competitive capability to survive including formation of biofilms in oil/water mixtures21.
Zdanowski et al.67 analyzed the anaerobic microbiome of subglacial samples. In their work, the authors compared the phylogenetic diversity of native samples with enriched ones. This enrichment was done by incubating native sediments in Postgate C medium for 8 weeks using airtight bottles to emulate subglacial conditions. As a result, they found that the following genera were found more abundantly in the enriched medium: Psychrosinus, Clostridium, Paludibacter, Acetobacterium, Pseudomonas, Carnobacterium, and Desulfosporosinus. Pseudomonas and Carnobacterium were found only in the enriched medium. Thus, the authors suggest that it may be that bacteria of the genus Pseudomonas have proliferated under enrichment conditions, depleting available oxygen in the early stages of the process, and then helping anaerobes to develop. Kliushnikova et al.68 described a microorganism of the genus Pseudomonas with sulphate-reducing activity. According to the authors, this strain when grown under strictly anaerobic conditions was able to reduce sulphate more intensely than under aerobic conditions. In a study by Guo et al.69, the authors demonstrated that Pseudomonas strain sp. C27 has an enzymatic system to perform sulfide removal. The authors cultivated the C27 strain in an anaerobic environment and demonstrated that the sulfide metabolism occurred through the expression of succinate dehydrogenase, iron–sulfur protein, oxidoreductase, serine hydroxymethyltransferase, and iron superoxide dismutase. Brahmacharimayum and Ghosh70 analyzed the removal of sulfate in an anaerobic environment by metagenomics. As a result, they show that the P. aeruginosa strain was predominant in the consortium and that it was involved in reducing Sulfate. Tüccar et al.42, found Pseudomonas as the dominant genus in produced water from Diyarbakir oil fields in Turkey and the authors suggested that these strains may have been inoculated into the oil reservoirs through the injection of fluids, and point out that these strains may adapt to the conditions of the reservoir to survive. Several studies have found the occurrence of the genus Pseudomonas in oil reservoirs71–77, according to Cui et al.78, Pseudomonas and Acinetobacter are genera that can effectively use crude oil as a carbon source, being able to survive and reproduce at the oil-water interface. Species of the genus Pseudomonas are facultative anaerobes capable of performing nitrification and nitrate reduction using various carbon substrates79,80. In a study by Braun and Gibson[11], the authors reported two bacteria of the genus Pseudomonas that were able to degrade, under anaerobic conditions, 2-aminobenzoate (anthranilic acid) to CO2 and NH4+. According to Cai et al.81, in the petroleum area, despite the fact that the oil is considered a hostile and toxic environment for microorganisms, there is lot of evidence that demonstrates the presence of microbes in the crude oil. The authors analyzed the microorganisms found in oil and water samples from four oil wells. According to them, as an unexpected result, they found that the genus Pseudomonas dominated the oil samples, where several groups of functional genes were identified.
According to Arai (2001)82, Pseudomonas aeruginosa has a remarkable ability to grow in the most diverse environmental conditions, such as in soil and water and on and in animals, humans and plants. This versatility is related to its metabolic flexibility through a branched respiratory chain with multiple terminal oxidases and denitrification enzymes. Its set of denitrification enzymes are capable of reducing nitrate to nitrogen via nitrite, nitric oxide (NO), and nitrous oxide. Nitrogen oxides function as electron acceptors, which allows P. aeruginosa to grow in anaerobic conditions. It is interesting to note that in the absence of nitrate, P. aeruginosa is able to metabolize arginine via arginine deiminase and in the absence of both, it can ferment mixed acid pyruvate and survive for long periods in anoxic conditions83.