Characterization of the biofilm structure and microbial diversity of sulfate-reducing bacteria from petroleum produced water supplemented by different carbon sources.

Colonization by sulfate-reducing bacteria (SRB) in environments associated with oil is mainly dependent on the availability of sulfate and carbon sources. The formation of biofilms by SRB increases the corrosion of pipelines and oil storage tanks, representing great occupational and operational risks and respective economic losses for the oil industry. The aim of this study was to evaluate the influence of the addition of acetate, butyrate, lactate, propionate and oil on the structure of biofilm formed in carbon steel coupons, as well as on the diversity of total bacteria and SRB in the planktonic and sessile communities from petroleum produced water. The biofilm morphology, chemical composition, average roughness and the microbial diversity was analyzed. In all carbon sources, formation of dense biofilm without morphological and/or microbial density differences was detected, with the most of cells observed in the form of individual rods. The diversity and richness indices of bacterial species in the planktonic community was greater than in the biofilm. Geotoga was the most abundant genus, and more than 85% of SRB species were common to all treatments. The functional predicted profile shown that the observed genres in planktonic communities were related to the reduction of sulfate, sulfite, elementary sulfur and other sulfur compounds, but the abundance varied between treatments. For the biofilm, the functions predicted profile for the oil treatment was the one that most varied in relation to the control, while for the planktonic community, the addition of all carbon sources interfered in the predicted functional profile. Thus, although it does not cause changes in the structure and morphology biofilm, the supplementation of produced water with different carbon sources is associated with changes in the SRB taxonomic composition and functional profiles of the biofilm and the planktonic bacterial communities.

Physicochemical characterization of Pseudomonas stutzeri UFV5 and analysis of its transcriptome under heterotrophic nitrification/aerobic denitrification pathway induction condition.

Biological ammonium removal via heterotrophic nitrification/aerobic denitrification (HN/AD) presents several advantages in relation to conventional removal processes, but little is known about the microorganisms and metabolic pathways involved in this process. In this study, Pseudomonas stutzeri UFV5 was isolated from an activated sludge sample from oil wastewater treatment station and its ammonium removal via HN/AD was investigated by physicochemical and molecular approaches to better understand this process and optimize the biological ammonium removal in wastewater treatment plants. Results showed that P. stutzeri UFV5 removed all the ammonium in 48-72 hours using pyruvate, acetate, citrate or sodium succinate as carbon sources, C/N ratios 6, 8, 10 and 12, 3-6% salinities, pH 7-9 and temperatures of 20-40 °C. Comparative genomics and PCR revealed that genes encoding the enzymes involved in anaerobic denitrification process are present in P. stutzeri genome, but no gene that encodes enzymes involved in autotrophic nitrification was found. Furthermore, transcriptomics showed that none of the known enzymes of autotrophic nitrification and anaerobic denitrification had their expression differentiated and an upregulation of the biosynthesis machinery and protein translation was observed, besides several genes with unknown function, indicating a non-conventional mechanism involved in HN/AD process.

Desulfovibrio alaskensis prophages and their possible involvement in the horizontal transfer of genes by outer membrane vesicles.

Desulfovibrio alaskensis is a Gram-negative bacterial species that belongs to the group of Sulphate Reducing Bacteria (SRB) and presents prophages in genomes, a common characteristic of the genus Desulfovibrio. Genetic material can be transported by outer membrane vesicles, however, no data regarding the production of these vesicles has been reported for D. alaskensis. To verify the expression of D. alaskensis prophages and their involvement with outer membrane vesicles, the DSM16109 strain was used. The DSM16109 strain had three prophages and presented reduced growth after mitomycin C addition when compared to the control culture. This reduction was accompanied by the presence of virus-like particles (VLPs), indicating mitomycin C dependent prophage induction. The increase in the number of cap gene copies and transcriptions of the three prophages was verified in the control sample, however, without the formation of VLPs. Prophage genes were identified in outer membrane vesicles from cultures treated and not treated with mitomycin C. DSM16109 prophages are expressed spontaneously but only in the presence of mitomycin C was it possible to observe VLP formation. Due to the genetic material detection from the prophages within outer membrane vesicles, this property may be related to the horizontal transfer of viral genes.

Screening and characterization of prophages in Desulfovibrio genomes.

Bacteria of the genus Desulfovibrio belong to the group of Sulphate Reducing Bacteria (SRB). SRB generate significant liabilities in the petroleum industry, mainly due to their ability to microbiologically induce corrosion, biofilm formation and H2S production. Bacteriophages are an alternative control method for SRB, whose information for this group of bacteria however, is scarce. The present study developed a workflow for the identification of complete prophages in Desulfovibrio. Poly-lysogenesis was shown to be common in Desulfovibrio. In the 47 genomes analyzed 53 complete prophages were identified. These were classified within the order Caudovirales, with 69.82% belonging to the Myoviridade family. More than half the prophages identified have genes coding for lysozyme or holin. Four of the analyzed bacterial genomes present prophages with identity above 50% in the same strain, whose comparative analysis demonstrated the existence of colinearity between the sequences. Of the 17 closed bacterial genomes analyzed, 6 have the CRISPR-Cas system classified as inactive. The identification of bacterial poly-lysogeny, the proximity between the complete prophages and the possible inactivity of the CRISPR-Cas in closed bacterial genomes analyzed allowed the choice of poly-lysogenic strains with prophages belonging to the Myoviridae family for the isolation of prophages and testing of related strains for subsequent studies.

Growth Inhibition of Sulfate-Reducing Bacteria in Produced Water from the Petroleum Industry Using Essential Oils.

Strategies for the control of sulfate-reducing bacteria (SRB) in the oil industry involve the use of high concentrations of biocides, but these may induce bacterial resistance and/or be harmful to public health and the environment. Essential oils (EO) produced by plants inhibit the growth of different microorganisms and are a possible alternative for controlling SRB. We aimed to characterize the bacterial community of produced water obtained from a Brazilian petroleum facility using molecular methods, as well as to evaluate the antimicrobial activity of EO from different plants and their major components against Desulfovibrio alaskensis NCIMB 13491 and against SRB growth directly in the produced water. Denaturing gradient gel electrophoresis revealed the presence of the genera Pelobacter and Marinobacterium, Geotoga petraea, and the SRB Desulfoplanes formicivorans in our produced water samples. Sequencing of dsrA insert-containing clones confirmed the presence of sequences related to D. formicivorans. EO obtained from Citrus aurantifolia, Lippia alba LA44 and Cymbopogon citratus, as well as citral, linalool, eugenol and geraniol, greatly inhibited (minimum inhibitory concentration (MIC) = 78 µg/mL) the growth of D. alaskensis in a liquid medium. The same MIC was obtained directly in the produced water with EO from L. alba LA44 (containing 82% citral) and with pure citral. These findings may help to control detrimental bacteria in the oil industry.

Ammonium removal from high-salinity oilfield-produced water: assessing the microbial community dynamics at increasing salt concentrations.

Water generated during oil exploration is chemically complex and contains high concentrations of ammonium and, in some cases, high salinity. The most common way to remove ammonium from effluent is a biological process, which can be performed by different routes and different groups of microorganisms. However, the presence of salts in the effluents could be an inhibiting factor for biological processes, interfering directly with treatment. This study aimed to evaluate changes in the profile of a microbial community involved in the process of ammonium removal when subjected to a gradual increase of salt (NaCl), in which the complete inhibition of the ammonium removal process occurred at 125 g L-1 NaCl. During the sludge acclimatization process, samples were collected and submitted to denaturing gradient gel electrophoresis (DGGE) and massive sequencing of the 16S ribosomal RNA (rRNA) genes. As the salt concentration increased in the reactor, a change in the microbial community was observed by the DGGE band profiles. As a result, there was a reduction in the presence of bacterial populations, and an increase in archaeal populations was found. The sequencing data suggested that ammonium removal in the reactor was carried out by different metabolic routes by autotrophic nitrifying bacteria, such as Nitrosococcus, Nitrosomonas, Nitrosovibrio, Nitrospira, and Nitrococcus; ammonium-oxidizing archaea Candidatus nitrosoarchaeum; ANAMMOX microorganisms, such as Candidatus brocadia, Candidatus kuenenia, and Candidatus scalindua; and microorganisms with the potential to be heterotrophic nitrifying, such as Paracoccus spp., Pseudomonas spp., Bacillus spp., Marinobacter sp., and Alcaligenes spp.