Metagenomic Investigation of a Low Diversity, High Salinity Offshore Oil Reservoir.

Oil reservoirs can represent extreme environments for microbial life due to low water availability, high salinity, high pressure and naturally occurring radionuclides. This study investigated the microbiome of saline formation water samples from a Gulf of Mexico oil reservoir. Metagenomic analysis and associated anaerobic enrichment cultures enabled investigations into metabolic potential for microbial activity and persistence in this environment given its high salinity (4.5%) and low nutrient availability. Preliminary 16S rRNA gene amplicon sequencing revealed very low microbial diversity. Accordingly, deep shotgun sequencing resulted in nine metagenome-assembled genomes (MAGs), including members of novel lineages QPJE01 (genus level) within the Halanaerobiaceae, and BM520 (family level) within the Bacteroidales. Genomes of the nine organisms included respiratory pathways such as nitrate reduction (in Arhodomonas, Flexistipes, Geotoga and Marinobacter MAGs) and thiosulfate reduction (in Arhodomonas, Flexistipes and Geotoga MAGs). Genomic evidence for adaptation to high salinity, withstanding radioactivity, and metal acquisition was also observed in different MAGs, possibly explaining their occurrence in this extreme habitat. Other metabolic features included the potential for quorum sensing and biofilm formation, and genes for forming endospores in some cases. Understanding the microbiomes of deep biosphere environments sheds light on the capabilities of uncultivated subsurface microorganisms and their potential roles in subsurface settings, including during oil recovery operations.

Oil field microorganisms cause highly localized corrosion on chemically inhibited carbon steel.

Carbon steel pipelines, a means for crude oil transportation, occasionally experience highly localized perforation caused by microorganisms. While microorganisms grown in laboratory culture tend to corrode steel specimens unevenly, they rarely inflict a corrosion morphology consistent with that of pipelines, where centimetre-sized corrosion features are randomly distributed within vast stretches of otherwise pristine metal surface. In this study, we observed that corrosion inhibitors (CIs), widely used for the control of acid gas (H2 S, CO2 ) corrosion in oil fields, also affect microbial growth and activity. Inhibited carbon steel resisted biofilm formation and underwent negligible corrosion (< 0.002 mm Fe0 year-1 ), despite 15 months of exposure to oil field waters harbouring a diverse microbiome. In contrast, physical scavenging of CI in these waters led to severe and highly localized corrosion (up to 0.93 mm Fe0 year-1 ) underneath biofilms dominated by methanogenic archaea and sulfate-reducing bacteria. A sharp decline in CI concentration, as well as its active components, quaternary ammonium compounds (QACs), correlated with microbial sulfidogenesis. CIs are ubiquitously present in oil field waters and play an underappreciated role in microbial corrosion mitigation. Physical and biological scavenging of CIs may create local differences in steel inhibition effectiveness and thus result in highly localized corrosion.

Severe Corrosion of Carbon Steel in Oil Field Produced Water Can Be Linked to Methanogenic Archaea Containing a Special Type of [NiFe] Hydrogenase.

Methanogenic archaea have long been implicated in microbially influenced corrosion (MIC) of oil and gas infrastructure, yet a first understanding of the underlying molecular mechanisms has only recently emerged. We surveyed pipeline-associated microbiomes from geographically distinct oil field facilities and found methanogens to account for 0.2 to 9.3% of the 16S rRNA gene sequencing reads. Neither the type nor the abundance of the detected methanogens was correlated with the perceived severity of MIC in these pipelines. Using fluids from one pipeline, MIC was reproduced in the laboratory, both under stagnant conditions and in customized corrosion reactors simulating pipeline flow. High corrosion rates (up to 2.43 mm Fe0 · yr-1) with macroscopic, localized corrosion features were attributed to lithotrophic, mesophilic microbial activity. Other laboratory tests with the same waters yielded negligible corrosion rates (<0.08 mm Fe0 · yr-1). Recently, a novel [NiFe] hydrogenase from Methanococcus maripaludis strain OS7 was demonstrated to accelerate corrosion. We developed a specific quantitative PCR (qPCR) assay and detected the gene encoding the large subunit of this hydrogenase (labeled micH) in corrosive (>0.15 mm Fe0 · yr-1) biofilms. The micH gene, on the other hand, was absent in noncorrosive biofilms, despite an abundance of methanogens. Reconstruction of a nearly complete Methanococcus maripaludis genome from a highly corrosive mixed biofilm revealed micH and associated genes in nearly identical genetic configuration to that in strain OS7, thereby supporting our hypothesis that the encoded molecular mechanism contributed to corrosion. Lastly, the proposed MIC biomarker was detected in multiple oil fields, indicating a geographically widespread involvement of this [NiFe] hydrogenase in MIC.IMPORTANCE Microorganisms can deteriorate built environments, which is particularly problematic in the case of pipelines transporting hydrocarbons to industrial end users. MIC is notoriously difficult to detect and monitor and, as a consequence, is a particularly difficult corrosion mechanism to manage. Despite the advent of molecular tools and improved microbial monitoring strategies for oil and gas operations, specific underlying MIC mechanisms in pipelines remain largely enigmatic. Emerging mechanistic understanding of methanogenic MIC derived from pure culture work allowed us to develop a qPCR assay that distinguishes technically problematic from benign methanogens in a West African oil field. Detection of the same gene in geographically diverse samples from North America hints at the widespread applicability of this assay. The research presented here offers a step toward a mechanistic understanding of biocorrosion in oil fields and introduces a binary marker for (methanogenic) MIC that can find application in corrosion management programs in industrial settings.

Comparison of sulfide-oxidizing Sulfurimonas strains reveals a new mode of thiosulfate formation in subsurface environments.

Sulfur-oxidizing Sulfurimonas spp. are widespread in sediments, hydrothermal vent fields, aquifers and subsurface environments such as oil reservoirs where they play an important role in the sulfur cycle. We determined the genome sequence of the oil field isolate Sulfurimonas sp. strain CVO and compared its gene expression during nitrate-dependent sulfide oxidation to the coastal sediment isolate Sulfurimonas denitrificans. Formation of elemental sulfur (S0 ) and high expression of sulfide quinone oxidoreductase (SQR) genes indicates that sulfide oxidation in both strains is mediated by SQR. Subsequent oxidation of S0 was achieved by the sulfur oxidation enzyme complex (SOX). In the coastal S. denitrificans, the genes are arranged and expressed as two clusters: soxXY1 Z1 AB and soxCDY2 Z2 H, and sulfate was the sole metabolic end product. By contrast, the oil field strain CVO has only the soxCDY2 Z2 H cluster and not soxXY1 Z1 AB. Despite the absence of the soxXY1 Z1 AB cluster, strain CVO oxidized S0 to thiosulfate and sulfate, demonstrating that soxCDY2 Z2 H genes alone are sufficient for S0 oxidation in Sulfurimonas spp. and that thiosulfate is an additional metabolic end product. Screening of publicly available metagenomes revealed that Sulfurimonas spp. with only the soxCDY2 Z2 H cluster are widespread suggesting this mechanism of thiosulfate formation is environmentally significant.

Metabolites of an Oil Field Sulfide-Oxidizing, Nitrate-Reducing Sulfurimonas sp. Cause Severe Corrosion.

Oil reservoir souring and associated material integrity challenges are of great concern to the petroleum industry. The bioengineering strategy of nitrate injection has proven successful for controlling souring in some cases, but recent reports indicate increased corrosion in nitrate-treated produced water reinjection facilities. Sulfide-oxidizing, nitrate-reducing bacteria (soNRB) have been suggested to be the cause of such corrosion. Using the model soNRB Sulfurimonas sp. strain CVO obtained from an oil field, we conducted a detailed analysis of soNRB-induced corrosion at initial nitrate-to-sulfide (N/S) ratios relevant to oil field operations. The activity of strain CVO caused severe corrosion rates of up to 0.27 millimeters per year (mm y-1) and up to 60-µm-deep pitting within only 9 days. The highest corrosion during the growth of strain CVO was associated with the production of zero-valent sulfur during sulfide oxidation and the accumulation of nitrite, when initial N/S ratios were high. Abiotic corrosion tests with individual metabolites confirmed biogenic zero-valent sulfur and nitrite as the main causes of corrosion under the experimental conditions. Mackinawite (FeS) deposited on carbon steel surfaces accelerated abiotic reduction of both sulfur and nitrite, exacerbating corrosion. Based on these results, a conceptual model for nitrate-mediated corrosion by soNRB is proposed.IMPORTANCE Ambiguous reports of corrosion problems associated with the injection of nitrate for souring control necessitate a deeper understanding of this frequently applied bioengineering strategy. Sulfide-oxidizing, nitrate-reducing bacteria have been proposed as key culprits, despite the underlying microbial corrosion mechanisms remaining insufficiently understood. This study provides a comprehensive characterization of how individual metabolic intermediates of the microbial nitrogen and sulfur cycles can impact the integrity of carbon steel infrastructure. The results help explain the dramatic increases seen at times in corrosion rates observed during nitrate injection in field and laboratory trials and point to strategies for reducing adverse integrity-related side effects of nitrate-based souring mitigation.

Corrosion of iron by sulfate-reducing bacteria: new views of an old problem.

About a century ago, researchers first recognized a connection between the activity of environmental microorganisms and cases of anaerobic iron corrosion. Since then, such microbially influenced corrosion (MIC) has gained prominence and its technical and economic implications are now widely recognized. Under anoxic conditions (e.g., in oil and gas pipelines), sulfate-reducing bacteria (SRB) are commonly considered the main culprits of MIC. This perception largely stems from three recurrent observations. First, anoxic sulfate-rich environments (e.g., anoxic seawater) are particularly corrosive. Second, SRB and their characteristic corrosion product iron sulfide are ubiquitously associated with anaerobic corrosion damage, and third, no other physiological group produces comparably severe corrosion damage in laboratory-grown pure cultures. However, there remain many open questions as to the underlying mechanisms and their relative contributions to corrosion. On the one hand, SRB damage iron constructions indirectly through a corrosive chemical agent, hydrogen sulfide, formed by the organisms as a dissimilatory product from sulfate reduction with organic compounds or hydrogen ("chemical microbially influenced corrosion"; CMIC). On the other hand, certain SRB can also attack iron via withdrawal of electrons ("electrical microbially influenced corrosion"; EMIC), viz., directly by metabolic coupling. Corrosion of iron by SRB is typically associated with the formation of iron sulfides (FeS) which, paradoxically, may reduce corrosion in some cases while they increase it in others. This brief review traces the historical twists in the perception of SRB-induced corrosion, considering the presently most plausible explanations as well as possible early misconceptions in the understanding of severe corrosion in anoxic, sulfate-rich environments.

Marine sulfate-reducing bacteria cause serious corrosion of iron under electroconductive biogenic mineral crust.

Iron (Fe(0) ) corrosion in anoxic environments (e.g. inside pipelines), a process entailing considerable economic costs, is largely influenced by microorganisms, in particular sulfate-reducing bacteria (SRB). The process is characterized by formation of black crusts and metal pitting. The mechanism is usually explained by the corrosiveness of formed H(2) S, and scavenge of 'cathodic' H(2) from chemical reaction of Fe(0) with H(2) O. Here we studied peculiar marine SRB that grew lithotrophically with metallic iron as the only electron donor. They degraded up to 72% of iron coupons (10 mm × 10 mm × 1 mm) within five months, which is a technologically highly relevant corrosion rate (0.7 mm Fe(0) year(-1) ), while conventional H(2) -scavenging control strains were not corrosive. The black, hard mineral crust (FeS, FeCO(3) , Mg/CaCO(3) ) deposited on the corroding metal exhibited electrical conductivity (50 S m(-1) ). This was sufficient to explain the corrosion rate by electron flow from the metal (4Fe(0) → 4Fe(2+) + 8e(-) ) through semiconductive sulfides to the crust-colonizing cells reducing sulfate (8e(-) + SO(4) (2-) + 9H(+) → HS(-) + 4H(2) O). Hence, anaerobic microbial iron corrosion obviously bypasses H(2) rather than depends on it. SRB with such corrosive potential were revealed at naturally high numbers at a coastal marine sediment site. Iron coupons buried there were corroded and covered by the characteristic mineral crust. It is speculated that anaerobic biocorrosion is due to the promiscuous use of an ecophysiologically relevant catabolic trait for uptake of external electrons from abiotic or biotic sources in sediments.

The role of nitric-oxide-synthase-derived nitric oxide in multicellular traits of Bacillus subtilis 3610: biofilm formation, swarming, and dispersal.

BACKGROUND: Bacillus subtilis 3610 displays multicellular traits as it forms structurally complex biofilms and swarms on solid surfaces. In addition, B. subtilis encodes and expresses nitric oxide synthase (NOS), an enzyme that is known to enable NO-mediated intercellular signalling in multicellular eukaryotes. In this study, we tested the hypothesis that NOS-derived NO is involved in the coordination of multicellularity in B. subtilis 3610. RESULTS: We show that B. subtilis 3610 produces intracellular NO via NOS activity by combining Confocal Laser Scanning Microscopy with the NO sensitive dye copper fluorescein (CuFL). We further investigated the influence of NOS-derived NO and exogenously supplied NO on the formation of biofilms, swarming motility and biofilm dispersal. These experiments showed that neither the suppression of NO formation with specific NOS inhibitors, NO scavengers or deletion of the nos gene, nor the exogenous addition of NO with NO donors affected (i) biofilm development, (ii) mature biofilm structure, and (iii) swarming motility in a qualitative and quantitative manner. In contrast, the nos knock-out and wild-type cells with inhibited NOS displayed strongly enhanced biofilm dispersal. CONCLUSION: The results suggest that biofilm formation and swarming motility in B. subtilis represent complex multicellular processes that do not employ NO signalling and are remarkably robust against interference of NO. Rather, the function of NOS-derived NO in B. subtilis might be specific for cytoprotection against oxidative stress as has been proposed earlier. The influence of NOS-derived NO on dispersal of B. subtilis from biofilms might be associated to its well-known function in coordinating the transition from oxic to anoxic conditions. Here, NOS-derived NO might be involved in fine-tuning the cellular decision-making between adaptation of the metabolism to (anoxic) conditions in the biofilm or dispersal from the biofilm.