Bacteria in the produced water and wastewater samples from the oil industry

. Today, studying the diversity of microbial communities associated with samples of highly mineralized oil industry waters is expanding our knowledge of the ecology of polyextremophilic microorganisms. During this work, samples of produced water and wastewater from the oil industry were thoroughly analyzed. The analyzed waters were characterized by very high concentrations of Na + , Ca 2+ , Mg 2+ , and Cl – ions. Furthermore, enriched and pure bacterial cultures from oilfield waters were obtained. Additionally, enriched cultures were analyzed using high-throughput sequencing of the bacterial 16S rRNA gene on the Illumina platform. Among the representatives of the studied bacterial communities, members of the genera Halomonas , Marinobacter , Modicisalibacter , Bacillus , Clostridium , Prauserella , and Rubrobacter were identified. They can be considered for various biotechnological applications.


Introduction
The petroleum industry is a leading industry where a lot of research is carried out related to production, refining, transportation, and corrosion control.Monitoring of microorganisms, both living in the subsurface and introduced during the operation of oil reservoirs, is one of the main tasks of the oil industry, since it is necessary to control and reduce the proportion of microorganisms that synthesize aggressive products for pipelines.Corrosion is a result of chemical or electrochemical reactions between materials and substances in their environment, and this process affects several sectors, including the petroleum industry [1].Corrosion caused by microorganisms (biocorrosion or microbial corrosion) causes millions of dollars in losses every year [2] and is a serious problem in a wide range of industries [3].
Biocorrosion is affected by the presence and activity of various microorganisms on the surface of the corroding material [4,5].Despite the relatively long history of microbial corrosion research, there are still many unresolved problems in this area.The list of microorganisms that researchers have linked to corrosion is certainly growing, but the gaps in biocorrosion are not narrowing, especially in relation to non-sulfate-reducing microorganisms.The effective operation of metal structures and product pipelines (water, gas, and oil pipelines) directly depends on the identification of microbial agents that cause biocorrosion.In this regard, it is necessary to study the mechanisms of both abiotic and biotic damage to metal and develop effective methods to combat them [5].
Bacteria, fungi, and methanogenic archaea can directly or indirectly initiate or accelerate biocorrosion.Microorganisms associated with biocorrosion alter the electrochemical conditions at the metal/solution interface by forming biofilms and subsequent release of aggressive metabolites [6] or removing protective films [7].Aerobic representatives of microbial communities consume molecular oxygen, helping to create conditions for the development of anaerobic microorganisms, which may have mechanisms that trigger biocorrosion [8,9].Particular attention is paid to sulfate-reducing prokaryotes and acid-producing bacteria, which produce highly corrosive hydrogen sulfide and organic acids [6,10].Researchers also study sulfur-reducing, Fe(III)-reducing, and nitrate-reducing bacteria [11,12].Certain microorganisms that are part of the normal microbiota of oil reservoirs [13] have a metabolism that initiates or accelerates the breakdown of metals.
The culture-dependent method is a widely used method for the detection and quantification of microorganisms in the petroleum industry and is commonly used to monitor corrosive microbes [14,15].However, only a small percentage of microbes present in environmental samples are cultivable.Culture-independent molecular methods have made it possible to detect and identify other important microbes.In recent decades, the biodiversity of many oilfields around the world has been studied using the 16S rRNA gene amplicon sequencing approach [2,5], which allowed us to compile a list of the most important microorganisms.Despite the existing scientific developments in the field of biocorrosion inhibition, microbial composition as well as the interaction between various functional groups within complex microbial communities, especially those associated with internal biocorrosion, are not well understood.Today, there remains a noticeable gap between scientific results and effective approaches to recognizing and solving practical problems arising from microbial metabolism.
Understanding microbial processes and interactions is critical to developing new strategies to minimize or prevent biocorrosion and facilitates the development of new biotechnologies.Therefore, the purpose of this work was to assess the composition of bacterial communities at oil production sites using different techniques.

Sample collection
Produced water (oil-water mixture) was sampled from four wells currently under development by the Oil and Gas Production Department "Almetyevneft", № 3 (Republic of Tatarstan, Russia).Additionally, water samples (recycled produced water) from treatment facilities for preparing water for pumping it into the oil reservoir were analyzed.The samples of produced waters were labeled as prod_water (1-4), whereas the samples of recycled produced waters were labeled as rec_water (1-2).Water samples were collected in sterile, airtight plastic bottles in the autumn of 2022.Samples were immediately transported to the laboratory and then used for chemical and microbiological characterization.

Characterization of water samples
After separating the oil and water phases, the composition of the water samples was analyzed.Analyses included the measurement of pH and concentrations of the main cations and anions.pH was measured with a Starter 5000 pH meter and STMICRO8 electrode (OHAUS Corporation, China).The capillary electrophoresis system Capel 105M (Lumex, Russia) was used to analyze the concentrations of sodium, potassium, calcium, and magnesium ions.The ammonium concentration was measured with the Nessler reagent (Sigma-Aldrich, USA).The chloride ion concentration was analyzed using the chloride test kit (Hanna Instruments, Romania).The concentration of nitrite and nitrate ions was analyzed using the nitrite/nitrate test kits (Hack, USA).All analyses were conducted in triplicate, and the mean values are presented with standard deviations.

Cultivation of bacteria
The detection and quantification of planktonic mesophilic bacteria were performed using the plate count (colony-forming units, CFU) and the most probable number (MPN) techniques, while the detection of sulfate-reducing bacteria was performed using the MPN technique.Nutrient agar medium, thioglycolate medium, and Postgate's B medium were used for the cultivation of bacteria.Water samples for the analysis of strict anaerobic bacteria were injected into serum bottles with sterile medium, purged with N2, and clamped with sterile rubbers and aluminum caps.Plates, the experimental bottles, and bottles only with the nutrient media as the negative control were incubated at +32 °C in a thermostat RI 53 Red Line (Binder, Germany).

Sequence analysis of the 16S rRNA gene of bacterial isolates
Bacterial isolates obtained using the plate count technique were identified by sequence analysis of amplified fragments of the bacterial 16S rRNA gene.Amplification of 16S rRNA gene was carried out using the universal primers UniBac27f (5′-AGA GTT TGA TCM TGG CTC AG-3′) and Univ1492r (5′-TAC GGY TAC CTT GTT ACG ACT T-3′).The library of bacterial isolates was analyzed for restriction fragment length polymorphisms using the restriction endonucleases HaeIII and RsaI (Thermo Fisher Scientific, Lithuania) as described previously [16,17].The 16S rRNA genes of representative isolates from each main cluster were partially sequenced.For the purification of amplicons, a QIAquick PCR purification kit (Qiagen, Germany) was used.The sequencing of bacterial 16S rRNA genes was carried out using an Applied Biosystems 3500 Series Genetic Analyzer (Thermo Fisher Scientific, USA), and the information was searched using the NCBI-BLAST search tool for the identification of bacteria.

High-throughput sequencing of bacterial 16S rRNA gene
Considering the coexistence of bacteria in oilfield waters in mixed consortia as well as the low initial content of microbial cells in the samples, to clarify the bacterial composition of the studied waters, the composition of enriched cultures was analyzed using Illumina sequencing.Total DNA was extracted using the FastDNA spin kit for soil (MP Biomedicals, USA).DNA concentrations were quantified using a Qubit 2.0 fluorometer (Invitrogen, USA).PCR amplicons were generated using Bakt_341F (5′-CCT ACG GGN GGC WGC AG-3′) and Bakt_805R (5′-GAC TAC HVG GGT ATC TAA TCC-3′) primers.The results of PCR amplifications in the negative controls were negative.Purified PCR products were used to prepare the DNA library for sequencing on an Illumina MiSeq platform with a MiSeq Reagent Kit v3 (Illumina, USA).The analysis of bioinformatic data was performed using the QIIME software pipeline.The filtered sequences were clustered and assigned to operational taxonomy units (OTUs) using the SILVA database as a reference at a 97% sequence similarity.

Characteristics of samples
Table 1 presents the characteristics of the studied oilfield waters.The salinity of oilfield waters is predominantly due to dissolved sodium and chloride ions and a smaller contribution from calcium and magnesium ions, as reported earlier [18].In general, all the water samples analyzed here contained very high concentrations of sodium, calcium, and magnesium cations, as well as chloride anion.Sodium was the most common dominant cation in the produced waters, with values in the waters tested 2-7 times higher than those typically observed in seawater samples.The concentrations of calcium and magnesium were also higher than usually observed in seawater.The pH value varied from 4.4 to 6.2.Nitrate and nitrite ions were not detected; nitrogen was predominantly presented in the form of ammonium.The chloride level was also high in all samples.The results obtained indicate the aggressiveness of these waters.

Cultivable bacteria
In the present work, individual bacteria and bacterial communities were analyzed based on the 16S rRNA gene sequencing approach.The total number of microorganisms was in low concentrations (maximum 700 CFU mL -1 ).Sulfate-reducing bacteria (using Postgate's B medium) were not detected in the analyzed samples of formation and recycled waters.In a study conducted by Oliveira et al. [14], the content of all detected bacteria in the produced water samples collected from the Brazilian oil production plant was higher than the values observed in this study.Low concentration of extracted DNA from the initial produced waters and low values of the number of cultivable microbes allow us to conclude that the microbial load was low due to the aggressiveness of the samples.The results obtained are consistent with the idea that such waters are a storehouse of extremophilic bacteria with rich functional potential [19].Individual bacterial cultures were additionally obtained.Based on 16S rRNA gene sequencing of thirty isolates, species belonging to five major genera were identified (Halomonas, Marinobacter, Bacillus, Micrococcus, and Kocuria).Among the cultivable bacteria, we note the halophilic genera Halomonas and Marinobacter of the phylum Proteobacteria, which are also found in oil reservoirs [19], petroleum-product-transporting pipelines [20], and fuel ballast systems [21].Halomonas and Marinobacter species have been identified as hydrocarbon-degrading bacteria and are subjects of microbial corrosion research [5,22,23].Interestingly, the dominant species of corrosive bacterial consortia identified in some other oil pipeline samples were bacteria of the genus Bacillus [20].Today there is only a narrow range of research that investigates the biological significance of strains of the genus Micrococcus in the petroleum industry.However, these actinomycetes have oil-oxidizing ability and are isolated from biotopes contaminated with crude oil [24].Thus, bacterial isolates obtained from such samples can have high biotechnological potential.

Biodiversity of enriched bacterial communities based on high-throughput sequencing
Only enriched cultures of three oilfield water samples (obtained using thioglycolate medium) were subjected to high-throughput sequencing; enriched cultures of the remaining samples were not studied due to insufficient amount of extracted DNA.The relative abundance of bacterial phyla and families found in the studied water samples is demonstrated in Figures 1 and 2.  The bacterial communities of the produced water (prod_water 3) were represented by the dominant genus Bacillus within the phylum Firmicutes.OTUs associated with rec_water 1 and rec_water 2 were more diverse and spanned genera mainly belonging to the phyla Proteobacteria and Firmicutes.The genera with high abundance included unclassified Lachnospiraceae UCG-008, Marinobacter, Halomonas, Clostridium sensu stricto 2, Bacillus, and Modicisalibacter in rec_water 1. Bacteria of the genera Modicisalibacter, Moraxella, Alloprevotella, Acinetobacter, Sphingomonas, Finegoldia, Prauserella, and Rubrobacter were mainly detected in rec_water 2. Modicisalibacter is involved in aromatic and aliphatic hydrocarbon biodegradation at high salinity and was isolated from produced water [25].Prauserella species belong to the halophilic and thermotolerant crude-oil-degrading bacteria that were recently isolated from an oilcontaminated mud pit [26].Members of the genus Rubrobacter are known for their polyextremophilic growth conditions: they are halotolerant, thermotolerant, and highly radiation-resistant [27].However, their participation in biocorrosion processes requires further careful study, since their subsequent injection into wells can aggravate the existing problem.

Conclusions
Cultivation of ecologically important bacteria and culture-independent analysis of microbial community structure reinforce each other's importance.Thus, considering the characteristics of the studied objects, we conclude that high salinity and substrate deficiency impose strong selection on the microorganisms of the studied biotypes, which led to extremely uneven microbial communities.Different environmental conditions may also explain differences between water samples.Thus, despite the widespread belief that oilfield is a harsh habitat, here we show that samples from such objects have unexpectedly microbial functional potential.Finally, many of the identified bacterial groups may be useful in a variety of biotechnological applications, and some may indicate the potential for biocorrosion of metal surfaces.

Fig. 1 .
Fig. 1.The relative abundance of bacterial phyla found in produced and recycled waters from the oil industry.

Fig. 2 .
Fig. 2. The relative abundance of bacterial families found in produced and recycled waters from the oil industry.

Table 1 .
Characteristics of investigated oilfield waters.