On the benefits of desulfated seawater flooding in mature hydrocarbon fields.

Removal of sulfate from the injection seawater (desulfation) in hydrocarbon reservoirs is a Modified Salinity Water (MSW) flooding method that mitigates microbial reservoir souring, improves oil recovery, and enables produced-water re-injection (PWRI). Aside from the Improved Oil Recovery (IOR) effect, desulfation results in a cleaner production of oil through enabling PWRI and reducing the environmental impacts associated with reservoir souring and nitrate treatment. However, whether desulfation is still beneficial for mature fields, after years of the injection of untreated seawater, is a valid common concern. In such cases, sulfate concentration inside the reservoir has already increased due to years of untreated seawater injection. The high sulfate concentration inside the subsurface reservoir before desulfated water flooding may render desulfation pointless. The present study investigates the potential benefits of desulfation after around 20 years of untreated seawater injection in a sector of an oil field in the Danish North Sea. The results show that depending on the cessation of production point in time and the efficiency of residual oil saturation reduction of MSW flooding, desulfation results in a significant increase in oil production. Even if improving oil recovery is no longer a priority, modification of injected seawater would still help reduce the amount of water required to support a given oil production rate. Moreover, desulfation is considerably more effective than nitrate treatment in mitigating microbial reservoir souring. Furthermore, the possibility of scale formation is decreased considerably due to desulfation, which further encourages PWRI.

Assessing Microbial Corrosion Risk on Offshore Crude Oil Production Topsides under Conditions of Nitrate and Nitrite Treatment for Souring.

Oilfield souring is a detrimental effect caused by sulfate-reducing microorganisms that reduce sulfate to sulfide during their respiration process. Nitrate or nitrite can be used to mitigate souring, but may also impart a corrosion risk. Produced fluids sampled from the topside infrastructure of two floating, production, storage, and offloading (FPSO) vessels (Platform A and Platform B) were assessed for microbial corrosion under nitrate and nitrite breakthrough conditions using microcosm tests incubated at 54 °C. Microbial community compositions on each individual FPSO were similar, while those between the two FPSO vessels differed. Platform B microbial communities responded as expected to nitrate breakthrough conditions, where nitrate-reducing activity was enhanced and sulfate reduction was inhibited. In contrast, nitrate treatments of Platform A microbial communities were not as effective in preventing sulfide production. Nitrite breakthrough conditions had the strongest sulfate reduction inhibition in samples from both platforms, but exhibited the highest pitting density. Live experimental replicates with no nitrate or nitrite additive yielded the highest general corrosion rates in the study (up to 0.48 mm/year), while nitrate- or nitrite-treated fluids revealed general corrosion rates that are considered low or moderate (<0.12 mm/year). Overall, the results of this study provide a description of nitrogen- and sulfur-based microbial activities under thermophilic conditions, and their risk for MIC that can occur along fluid processing lines on FPSO topsides that process fluids during offshore oil production operations.

Bio-augmentation with dissimilatory nitrate reduction to ammonium (DNRA) driven sulfide-oxidizing bacteria enhances the durability of nitrate-mediated souring control.

Biological souring (producing sulfide) is a global challenge facing anaerobic water bodies, especially the oil reservoir fluids. Nitrate injection has demonstrated great potential in souring control, and dissimilatory nitrate reduction to ammonium (DNRA) bacteria was proposed to play crucial roles in the process. How to durably control souring with nitrate amendment, however, remains undiscovered. Herein, Gordonia sp. TD-4, a DNRA-driven sulfide-oxidizing bacterium, was used to elucidate the effects of bio-augmentation with DNRA bacteria on the durability of nitrate-mediated souring control. The results revealed that nitrate amendment combined with bio-augmentation with TD-4 after souring could effectively control souring and enhance the durability of nitrate-mediated souring control, while nitrate amendment before souring failed to persistently control souring. Nitrate amendment before and after souring resulted in different evolution dynamics of nitrate-reducing bacteria. Denitrifying bacteria were enriched in reactors amended with nitrate before souring or in dissolved sulfide exhausted reactors amended with nitrate after souring. The heterotrophic denitrifying activity of denitrifying bacteria, however, decreased the durability of nitrate-mediated souring control. Comparative and functional genomics analysis identified potential niche adaptation mechanisms (autotrophic and heterotrophic nitrate/nitrite reduction, including DNRA and denitrification) of predominant SRB in nitrate-amended environments, which were responsible for the rapid resumption of sulfide accumulation after the depletion of nitrate and nitrite. Pulsed injection of nitrate combined with bio-augmentation with DNRA-driven sulfide-oxidizing bacteria was proposed as a potential method to enhance the durability of nitrate-mediated souring control. The findings were innovatively applied to simultaneous bio-demulsification and souring control of emulsified and sour produced water from the petroleum industry.

Importance of thermodynamics dependent kinetic parameters in nitrate-based souring mitigation studies.

Souring is the unwanted formation of hydrogen sulfide (H2S) by sulfate-reducing microorganisms (SRM) in sewer systems and seawater flooded oil reservoirs. Nitrate treatment (NT) is one of the major methods to alleviate souring: The mechanism of souring remediation by NT is stimulation of nitrate reducing microorganisms (NRM) that depending on the nitrate reduction pathway can outcompete SRM for common electron donors, or oxidize sulfide to sulfate. However, some nitrate reduction pathways may challenge the efficacy of NT. Therefore, a precise understanding of souring rate, nitrate reduction rate and pathways is crucial for efficient souring management. Here, we investigate the necessity of incorporating two thermodynamic dependent kinetic parameters, namely, the growth yield (Y), and FT, a parameter related to the minimum catabolic energy production required by cells to utilize a given catabolic reaction. We first show that depending on physiochemical conditions, Y and FT for SRM change significantly in the range of [0-0.4] mole biomass per mole electron donor and [0.0006-0.5], respectively, suggesting that these parameters should not be considered constant and that it is important to couple souring models with thermodynamic models. Then, we highlight this further by showing an experimental dataset that can be modeled very well by considering variable FT. Next, we show that nitrate based lithotrophic sulfide oxidation to sulfate (lNRM3) is the dominant nitrate reduction pathway. Then, arguing that thermodynamics would suggest that S° consumption should proceed faster than S0 production, we infer that the reason for frequently observed S0 accumulation is its low solubility. Last, we suggest that nitrate based souring treatment will suffer less from S0 accumulation if we (i) act early, (ii) increase temperature and (iii) supplement stoichiometrically sufficient nitrate.

Sulfate reducing microorganisms in high temperature oil reservoirs.

High temperature reservoirs offer a window into the microbial life of the deep biosphere. Sulfate reducing microorganisms have been recovered from high temperature oil reservoirs around the globe and characterized using culture-dependent and culture-independent approaches. The activities of sulfate reducers contribute to reservoir souring and hydrocarbon degradation among other attracting considerable interest from the oil industry for the last 100 years. The extremes of temperature and pressure shape the activities and distribution of sulfate reducing bacteria and archaea in high temperature reservoirs. This chapter will attempt to summarize the key findings on the diversity and activities of sulfate reducing microorganisms in high temperature reservoirs.

Long-Term Biocide Efficacy and Its Effect on a Souring Microbial Community.

Reservoir souring, which is the production of H2S mainly by sulfate-reducing microorganisms (SRM) in oil reservoirs, has been a long-standing issue for the oil industry. While biocides have been frequently applied to control biogenic souring, the effects of biocide treatment are usually temporary, and biocides eventually fail. The reasons for biocide failure and the long-term response of the microbial community remain poorly understood. In this study, one-time biocide treatments with glutaraldehyde (GA) and an aldehyde-releasing biocide (ARB) at low (100 ppm) and high (750 ppm) doses were individually applied to a complex SRM community, followed by 1 year of monitoring of the chemical responses and the microbial community succession. The chemical results showed that souring control failed after 7 days at a dose of 100 ppm regardless of the biocide type and lasting souring control for the entire 1-year period was achieved only with ARB at 750 ppm. Microbial community analyses suggested that the high-dose biocide treatments resulted in 1 order of magnitude lower average total microbial abundance and average SRM abundance, compared to the low-dose treatments. The recurrence of souring was associated with reduction of alpha diversity and with long-term microbial community structure changes; therefore, monitoring changes in microbial community metrics may provide early warnings of the failure of a biocide-based souring control program in the field. Furthermore, spore-forming sulfate reducers (Desulfotomaculum and Desulfurispora) were enriched and became dominant in both GA-treated groups, which could cause challenges for the design of long-lasting remedial souring control strategies. IMPORTANCE Reservoir souring is a problem for the oil and gas industry, because H2S corrodes the steel infrastructure, downgrades oil quality, and poses substantial risks to field personnel and the environment. Biocides have been widely applied to remedy souring, but the long-term performance of biocide treatments is hard to predict or to optimize due to limited understanding of the microbial ecology affected by biocide treatment. This study investigates the long-term biocide performance and associated changes in the abundance, diversity, and structure of the souring microbial community, thus advancing the knowledge toward a deeper understanding of the microbial ecology of biocide-treated systems and contributing to the improvement of current biocide-based souring control practices. The study showcases the potential application of incorporating microbial community analyses to forecast souring, and it highlights the long-term consequences of biocide treatment in the microbial communities, with relevance to both operators and regulators.

Denitrification Biokinetics: Towards Optimization for Industrial Applications.

Denitrification is a microbial process that converts nitrate (NO3 -) to N2 and can play an important role in industrial applications such as souring control and microbially enhanced oil recovery (MEOR). The effectiveness of using NO3 - in souring control depends on the partial reduction of NO3 - to nitrite (NO2 -) and/or N2O while in MEOR complete reduction of NO3 - to N2 is desired. Thauera has been reported as a dominant taxon in such applications, but the impact of NO3 - and NO2 - concentrations, and pH on the kinetics of denitrification by this bacterium is not known. With the goal of better understanding the effects of such parameters on applications such as souring and MEOR, three strains of Thauera (K172, NS1 and TK001) were used to study denitrification kinetics when using acetate as an electron donor. At low initial NO3 - concentrations (∼1 mmol L-1) and at pH 7.5, complete NO3 - reduction by all strains was indicated by non-detectable NO3 - concentrations and near-complete recovery (> 97%) of the initial NO3-N as N2 after 14 days of incubation. The relative rate of denitrification by NS1 was low, 0.071 mmol L-1 d-1, compared to that of K172 (0.431 mmol L-1 d-1) and TK001 (0.429 mmol L-1 d-1). Transient accumulation of up to 0.74 mmol L-1 NO2 - was observed in cultures of NS1 only. Increased initial NO3 - concentrations resulted in the accumulation of elevated concentrations of NO2 - and N2O, particularly in incubations with K172 and NS1. Strain TK001 had the most extensive NO3 - reduction under high initial NO3 - concentrations, but still had only ∼78% of the initial NO3-N recovered as N2 after 90 days of incubation. As denitrification proceeded, increased pH substantially reduced denitrification rates when values exceeded ∼ 9. The rate and extent of NO3 - reduction were also affected by NO2 - accumulation, particularly in incubations with K172, where up to more than a 2-fold rate decrease was observed. The decrease in rate was associated with decreased transcript abundances of denitrification genes (nirS and nosZ) required to produce enzymes for reduction of NO2 - and N2O. Conversely, high pH also contributed to the delayed expression of these gene transcripts rather than their abundances in strains NS1 and TK001. Increased NO2 - concentrations, N2O levels and high pH appeared to cause higher stress on NS1 than on K172 and TK001 for N2 production. Collectively, these results indicate that increased pH can alter the kinetics of denitrification by Thauera strains used in this study, suggesting that liming could be a way to achieve partial denitrification to promote NO2 - and N2O production (e.g., for souring control) while pH buffering would be desirable for achieving complete denitrification to N2 (e.g., for gas-mediated MEOR).

Physicochemical and biological controls of sulfide accumulation in a high temperature oil reservoir.

In order to maintain the reservoir pressure during secondary oil production large volumes of seawater are injected into reservoirs. This practice introduces high concentrations of sulfate into the reservoir promoting the growth of sulfate-reducing microorganisms (SRM) and results in the production of an increasing volume of produced water (PW) that needs to be discharged. SRM reduce sulfate to sulfide causing reservoir souring and as a mitigation strategy nitrate is injecting along with the seawater into the reservoir. We used PW from the Halfdan oil field (North Sea) to set up microcosms to determine the best reinjection strategy in order to inhibit SRM activity and minimize the environmental impact of PW during secondary oil production. We discuss the effect of temperature, electron donor, and sulfate and nitrate availability on sulfide production and microbial community composition. Temperature and the terminal electron acceptor played a key role in shaping the microbial community of the microcosms. PW reinjection at 62 °C inhibited SRM activity due to nitrite toxicity by encouraging nitrate reduction to nitrite by thermophilic nitrate reducers, while at 74 °C we observed complete absence of any microbial activity over the course of 150 days. KEY POINTS: • Temperature and the presence/ absence of nitrate shaped the microbial community structure. • Thermophilic nitrate reducers convert nitrate to ammonia with the accumulation of nitrite that inhibits sulfide production. • Nitrite inhibition is the most effective nitrate-based souring mitigation mechanisms. • The reinjection of hot produced water to oil reservoirs is a promising souring mitigation approach.

Fagos de bactérias redutoras de sulfato em água de produção de um reservatório de petróleo offshore: método tentativo de concentração e purificação / Phages of Sulphate Reducing Bacteria in reactor water from offshore oil wells: tentative method for concentration and purification

RESUMO O controle do crescimento microbiano é um desafio crescente na produção de petróleo e gás, uma vez que a presença de determinadas bactérias traz impactos econômica e ambientalmente negativos. As bactérias redutoras de sulfato (BRS) são particularmente problemáticas, uma vez que são responsáveis pela corrosão biológica ligada à produção de sulfeto de hidrogênio, efeito conhecido como souring. A principal forma de controle das BRS atualmente é a injeção de biocidas, no entanto essa estratégia, além de requerer aplicação contínua, tem se revelado pouco efetiva na eliminação de biofilmes e é associada a um alto risco de contaminação das águas. Portanto, é necessário que se busquem abordagens mais eficientes e específicas em relação ao controle microbiológico. O uso de vírus bacteriófagos vem ao encontro dessas necessidades, pois eles, após se multiplicarem, geralmente provocam a lise celular, liberando novas partículas virais e evitando que a bactéria se prolifere. Diante disso, este estudo propõe estabelecer um método para a concentração e a determinação da eficiência de recuperação de bacteriófagos de BRS presentes em água de reator oriunda de poços de petróleo. As amostras foram coletadas de dois reatores operados em batelada alimentada e que simulam um poço de petróleo. As amostras de água de reator foram primeiramente clarificadas, os vírus eluídos desse sedimento e, em seguida, concentrados por ultracentrifugação. O concentrado viral foi então purificado com Vertrel XF. Ensaios de semeadura experimental de miofago P1 nas amostras de água do reator revelaram taxa de recuperação viral de 27,7%, contra ao 16% obtidos com outros protocolos.

ABSTRACT The control of microbial growth is an increasing challenge in the production of oil and gas, since the presence of certain bacteria has economic and environmental negative impacts. Sulphate reducing bacteria are particularly problematic, since they are responsible for the biological corrosion associated with the production of hydrogen sulfide, an effect known as souring. The main form of control is the use of biocides; however, this strategy, in addition to requiring continuous application, has proven to be ineffective in the elimination of biofilms and is associated with a high risk of water contamination. Therefore, it is necessary to seek more efficient and specific approaches to microbiological control. The use of bacteriophage viruses meets these needs, because after they multiply, they usually cause cell lysis, releasing new viral particles and preventing the bacteria from proliferating. In view of this, this study proposes to establish a method for the concentration and detection of bacteriophages of Sulphate Reducing Bacteria present in reactor water from oil wells. The samples were collected from two reactors, operated in a batch fed to simulate an oil well. The reactor water samples were first clarified, viruses adsorbed to sediment were eluted and then concentrated by ultracentrifugation. The viral concentrate was then purified with Vertrel-XF. Experimental seeding of P1 myophage in water samples from the reactor revealed a viral recovery rate of 27.7%, compared to the 16% obtained by use of other protocols.

Towards sulfide removal and sulfate reducing bacteria inhibition: Function of biosurfactants produced by indigenous isolated nitrate reducing bacteria.

The effectiveness of nitrate-mediated souring control highly depends on the interactions of sulfate reducing bacteria (SRB) and nitrate reducing bacteria (NRB). Biosurfactants produced by natural NRB are promising bio-agents for enhancing NRB competence towards SRB. However, the function of NRB-produced biosurfactants in NRB-SRB interactions remains unexplored due to the rarely successful isolation of natural biosurfactant-producing NRB. Hereby, biosurfactant-aided inhibitory control of SRB strain Desulfomicrobium escambiense ATCC 51164 by biosurfactant-producing NRB strain Pseudomonas stutzeri CX3, reported in our previous work, was investigated. Under non-sour conditions, insufficient nitrate injection resulted in limited SRB inhibition. Phospholipid fatty acid (PLFA) biomarkers traced the overall bacterial responses. Compositional PLFA patterns revealed biosurfactant addition benefitted both SRB and NRB towards stressful conditions. Under sour conditions, nitrite oxidation of sulfide proved to be the primary mechanism for sulfide removal. The subsequent elevation of redox potential and pH inhibited SRB activities. NRB-produced biosurfactants significantly enhanced SRB inhibition by NRB through more efficient sulfide removal and effective duration of nitrate in the microcosms. Biosurfactants specially produced by the NRB strain are for the first time reported to significantly strengthen SRB inhibition by NRB via reduced nitrate usage and prolonged effective duration of nitrate, which has encouraging potential in nitrate-dependent souring control.

A two-compartment upflow pilot scale bioreactor system for microbial sulfate reduction control studies.

Souring in oil fields occurs mainly due to the activity of sulfate reducing bacteria (SRB). Most of the studies on SRB are performed using upflow packed-bed reactors that have a limitation to describe the region close to the injection wells in oil fields, which is characterized by void and saturated porous bed regions. Here, it is described the design and operation of a pilot scale system to investigate the SRB activity, inhibition and control in oil fields. •The bioreactor is composed by two-compartments (empty and packed-bed).•The reactor system has two parallel bioreactors that can be supplied with the same source of nutrients through a single pump or can be supplied separately with different solutions using two pumps.•The hydrodynamics for conventional packed bed bioreactors has a mixing behavior dependent of the flow rate and has a significant by-pass. In contrast, the two-compartment system presented here has a mixing behavior almost independent of the flow rate.

The potential impact of using a surfactant and an alcoholic co-surfactant on SRB activity during EOR.

Surfactants and co-surfactants play an important role in enhanced oil recovery for they improve petroleum solubility and reduce interfacial tensions between oil, water and the rock formation. Ethanol is receiving renewed attention as potential co-surfactant because of the negative results obtained with the use of salts and alkaline substances. Sulphate-reducing bacteria (SRB) can use surfactants and co-surfactants as carbon sources and, consequently, this can increase the biological accumulation of sulphide (souring). The aim of this research is to correlate SRB activity with different concentrations of co-surfactant (ethanol) as an attempt to quantifying in which concentration such compound can potentially increase or inhibit souring. The results show that the combination of surfactant (lauryl glucoside) and co-surfactant (ethanol) can increase SRB activity to about 2.3-fold. The highest sulphate consumption rate of 591 µg l-1 h-1 was observed in experiments with 0.03% and 1.5% (v/v) of surfactant and ethanol, respectively. The experiments indicated that SRB activity is only controlled by ethanol concentrations above 6.5% (v/v). Ethanol can potentially decrease costs with the use of biocides and significantly increase oil recovery ratios. Tests with the model Desulfovibrio vulgaris were not comparable with the results obtained with the SRB consortium.

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.

Comparison of Nitrate and Perchlorate in Controlling Sulfidogenesis in Heavy Oil-Containing Bioreactors.

Control of microbial reduction of sulfate to sulfide in oil reservoirs (a process referred to as souring) with nitrate has been researched extensively. Nitrate is reduced to nitrite, which is a strong inhibitor of sulfate-reducing bacteria (SRB). Perchlorate has been proposed as an alternative souring control agent. It is reduced to chlorate (ClO3 -) and chlorite (ClO2 -), which is dismutated to chloride and O2. These can react with sulfide to form sulfur. Chlorite is also highly biocidal. Here we compared the effectiveness of perchlorate and nitrate in inhibiting SRB activity in medium containing heavy oil from the Medicine Hat Glauconitic C (MHGC) field, which has a low reservoir temperature and is injected with nitrate to control souring. Using acetate, propionate and butyrate as electron donors, perchlorate-reducing bacteria (PRB) were obtained in enrichment culture and perchlorate-reducing Magnetospirillum spp. were isolated from MHGC produced waters. In batch experiments with MHGC oil as the electron donor, nitrate was reduced to nitrite and inhibited sulfate reduction. However, perchlorate was not reduced and did not inhibit sulfate reduction in these incubations. Bioreactor experiments were conducted with sand-packed glass columns, containing MHGC oil and inoculated with an oil-grown mesophilic SRB enrichment. Once active souring (reduction of 2 mM sulfate to sulfide) was observed, these were treated with nitrate and/or perchlorate. As in the batch experiments, 4 mM nitrate completely inhibited sulfide production, while partial inhibition occurred with 1 and 2 mM nitrate, but injection of 4 mM perchlorate did not inhibit sulfate reduction and perchlorate was not reduced. The enriched and isolated PRB were unable to use heavy oil components, like alkylbenzenes, which were readily used by nitrate-reducing bacteria. Hence perchlorate, injected into a low temperature heavy oil reservoir like the MHGC, may not be reduced to toxic intermediates making nitrate a preferable souring control agent.

Mitigating Sulfidogenesis With Simultaneous Perchlorate and Nitrate Treatments.

Sulfide biogenesis (souring) in oil reservoirs is an extensive and costly problem. Nitrate is currently used as a souring inhibitor but often requires high concentrations and yields inconsistent results. Recently, perchlorate has displayed promise as a more potent inhibitor in lab scale studies. However, combining the two treatments to determine synergy and effectiveness in a dynamic system has never been tested. Nitrate inhibits perchlorate consumption by perchlorate reducing bacteria, suggesting that the combined treatment may allow deeper penetration of the perchlorate into the reservoir matrix. Furthermore, the metabolic intermediates of perchlorate and nitrate reduction (nitrite and chlorite, respectively) are synergistic with the primary electron acceptors for inhibition of sulfate reduction. To assess the possible synergies between nitrate and perchlorate treatments, triplicate glass columns packed with pre-soured marine sediment were flushed with media containing sulfate and an inhibitor treatment [(i) perchlorate; (ii) nitrate; (iii) perchlorate and nitrate; or (iv) none]. Internal geochemistry and microbial community changes were monitored along the length of the columns during six phases of increasing treatment concentrations. In a final phase all treatments were removed. Sulfide production decreased in all treated columns in conjunction with increased inhibitor concentrations relative to the untreated control. Interestingly, the potency of the "mixed" treatment was additive relative to the individual treatments suggesting no interaction. Microbial community analyses indicated community shifts and clustering by treatment. The mixed treatment column community's trajectory closely resembled that of the community found in the perchlorate only treatment, suggesting that perchlorate was the dominant control on the "mixed" community structure. In contrast, the nitrate and untreated column communities had unique trajectories. This study indicates that concurrent nitrate and perchlorate treatment is not more effective than perchlorate treatment alone but is more effective than nitrate treatment. As such, treatment decisions may be based on economic factors.

[A Thermotolerant and Halotolerant Sulfate-reducing Bacterium in Produced Water from an Offshore High-temperature Oilfield in Bohai Bay, China: Isolation, Phenotypic Characterization, and Inhibition].

The growth and activity of sulfate-reducing prokaryotes (SRP) in oilfield environments could produce large amounts of H2S, leading to multifaceted problems, including oilfield souring and microbially-influenced corrosion, yet knowledge about the diversity and physiology of SRP therein was quite limited. To further understand the phenotypic characteristics of SRP residing in an offshore high-temperature oilfield at Bohai Bay, China, and to explore the potential methods for control of SRP-mediated problems, we isolated, using Hungate techniques, a thermotolerant, halotolerant SRP strain, designated BQ1, from the produced water of a high-temperature. We also presented the phenotypic features of BQ1, and investigated the efficacy of five biocides, or metabolic inhibitors, in suppressing the sulfidogenic activity of BQ1. Cells of BQ1 were motile, short rod-shaped, 1.2-2.5 µm in length and 0.5-0.8 µm in width. Although BQ1 shared 99% 16S rRNA gene sequence similarity with Desulfovibrio vulgaris Hildenborough, distinct phenotypic traits between them were observed. Isolated BQ1 could grow at 14-70℃(optimum at 30℃) and pH 6.0-9.0 (optimum pH 7.0), and in the presence of 0%-10% NaCl. Isolated BQ1 utilized a wide range of carbon substrates, including sodium formate, sodium lactate, and acetate. Sulfate, sulfite, thiosulfate, and sulfur were utilized as electron acceptors, but not nitrate or nitrite. Sodium hypochlorite (600 mg·L-1), Benzyltrimethylammonium chloride (300 mg·L-1), or nitrate (800 mg·L-1) failed to inhibit H2S production by BQ1. By contrast, glutaraldehyde (50 mg·L-1), bronopol (30 mg·L-1), chlorine dioxide (50 mg·L-1), and nitrite (70 mg·L-1) inhibited H2S production by BQ1 for at least 30 d, indicating that these compounds may be suitable for the mitigation of microbial souring in this specific, high-temperature, offshore oilfield at Bohai Bay, China.

Attenuating Sulfidogenesis in a Soured Continuous Flow Column System With Perchlorate Treatment.

Hydrogen sulfide production by sulfate reducing bacteria (SRB) is the primary cause of oil reservoir souring. Amending environments with chlorate or perchlorate [collectively denoted (per)chlorate] represents an emerging technology to prevent the onset of souring. Recent studies with perchlorate reducing bacteria (PRB) monocultures demonstrated that they have the innate capability to enzymatically oxidize sulfide, thus PRB may offer an effective means of reversing souring. (Per)chlorate may be effective by (i) direct toxicity to SRB; (ii) competitive exclusion of SRB by PRB; or (iii) reversal of souring through re-oxidation of sulfide by PRB. To determine if (per)chlorate could sweeten a soured column system and assign a quantitative value to each of the mechanisms we treated columns flooded with San Francisco bay water with temporally decreasing amounts (50, 25, and 12.5 mM) of (per)chlorate. Geochemistry and the microbial community structure were monitored and a reactive transport model was developed, Results were compared to columns treated with nitrate or untreated. Souring was reversed by all treatments at 50 mM but nitrate-treated columns began to re-sour when treatment concentrations decreased (25 mM). Re-souring was only observed in (per)chlorate-treated columns when concentrations were decreased to 12.5 mM and the extent of re-souring was less than the control columns. Microbial community analyses indicated treatment-specific community shifts. Nitrate treatment resulted in a distinct community enriched in genera known to perform sulfur cycling metabolisms and genera capable of nitrate reduction. (Per)chlorate treatment enriched for (per)chlorate reducing bacteria. (Per)chlorate treatments only enriched for sulfate reducing organisms when treatment levels were decreased. A reactive transport model of perchlorate treatment was developed and a baseline case simulation demonstrated that the model provided a good fit to the effluent geochemical data. Subsequent simulations teased out the relative role that each of the three perchlorate inhibition mechanisms played during different phases of the experiment. These results indicate that perchlorate addition is an effective strategy for both souring prevention and souring reversal. It provides insight into which organisms are involved, and illuminates the interactive effects of the inhibition mechanisms, further highlighting the versatility of perchlorate as a sweetening agent.

Dissimilatory Sulfate Reduction Under High Pressure by Desulfovibrio alaskensis G20.

Biosouring results from production of H2S by sulfate-reducing microorganisms (SRMs) in oil reservoirs. H2S is toxic, corrosive, and explosive, and as such, represents a significant threat to personnel, production facilities, and transportation pipelines. Since typical oil reservoir pressures can range from 10 to 50 MPa, understanding the role that pressure plays in SRM metabolism is important to improving souring containment strategies. To explore the impact of pressure, we grew an oil-field SRM isolate, Desulfovibrio alaskensis G20, under a range of pressures (0.1-14 MPa) at 30°C. The observed microbial growth rate was an inverse function of pressure with an associated slight reduction in sulfate and lactate consumption rate. Competitive fitness experiments with randomly bar-coded transposon mutant library sequencing (RB-TnSeq) identified several genes associated with flagellar biosynthesis and assembly that were important at high pressure. The fitness impact of specific genes was confirmed using individual transposon mutants. Confocal microscopy revealed that enhanced cell aggregation occurs at later stages of growth under pressure. We also assessed the effect of pressure on SRM inhibitor potency. Dose-response experiments showed a twofold decrease in the sensitivity of D. alaskensis to the antibiotic chloramphenicol at 14 MPa. Fortuitously, pressure had no significant influence on the inhibitory potency of the common souring controlling agent nitrate, or the emerging SRM inhibitors perchlorate, monofluorophosphate, or zinc pyrithione. Our findings improve the conceptual model of microbial sulfate reduction in high-pressure environments and the influence of pressure on souring inhibitor efficacy.

Synergy of Sodium Nitroprusside and Nitrate in Inhibiting the Activity of Sulfate Reducing Bacteria in Oil-Containing Bioreactors.

Sodium nitroprusside (SNP) disrupts microbial biofilms through the release of nitric oxide (NO). The actions of SNP on bacteria have been mostly limited to the genera Pseudomonas, Clostridium, and Bacillus. There are no reports of its biocidal action on sulfate-reducing bacteria (SRB), which couple the reduction of sulfate to sulfide with the oxidation of organic electron donors. Here, we report the inhibition and kill of SRB by low SNP concentrations [0.05 mM (15 ppm)] depending on biomass concentration. Chemical reaction of SNP with sulfide did not compromise its efficacy. SNP was more effective than five biocides commonly used to control SRB. Souring, the SRB activity in oil reservoirs, is often controlled by injection of nitrate. Control of SRB-mediated souring in oil-containing bioreactors was inhibited by 4 mM (340 ppm) of sodium nitrate, but required only 0.05 mM (15 ppm) of SNP. Interestingly, nitrate and SNP were found to be highly synergistic with 0.003 mM (1 ppm) of SNP and 1 mM (85 ppm) of sodium nitrate being sufficient in inhibiting souring. Hence, using SNP as an additive may greatly increase the efficacy of nitrate injection in oil reservoirs.

Control of microbial sulfide production by limiting sulfate dispersal in a water-injected oil field.

Oil production by water injection often involves the use of makeup water to replace produced oil. Sulfate in makeup water is reduced by sulfate-reducing bacteria to sulfide, a process referred to as souring. In the MHGC field souring was caused by using makeup water with 4mM (384ppm) sulfate. Mixing with sulfate-free produced water gave injection water with 0.8mM sulfate. This was amended with nitrate to limit souring and was then distributed fieldwide. The start-up of an enhanced-oil-recovery pilot caused all sulfate-containing makeup water to be used for dissolution of polymer, which was then injected into a limited region of the field. Produced water from this pilot contained 10% of the injected sulfate concentration as sulfide, but was free of sulfate. Its use as makeup water in the main water plant of the field caused injection water sulfate to drop to zero. This in turn strongly decreased produced sulfide concentrations throughout the field and allowed a decreased injection of nitrate. The decreased injection of sulfate and nitrate caused major changes in the microbial community of produced waters. Limiting sulfate dispersal into a reservoir, which acts as a sulfate-removing biofilter, is thus a powerful method to decrease souring.