Transformation of iron sulfide to greigite by nitrite produced by oil field bacteria

Nitrate, injected into oil fields, can oxidize sulfide formed by sulfate-reducing bacteria (SRB) through the action of nitrate-reducing sulfide-oxidizing bacteria (NR-SOB). When reservoir rock contains siderite (FeCO₃), the sulfide formed is immobilized as iron sulfide minerals, e.g. mackinawite (FeS). The aim of our study was to determine the extent to which oil field NR-SOB can oxidize or transform FeS. Because no NR-SOB capable of growth with FeS were isolated, the well-characterized oil field isolate Sulfurimonas sp. strain CVO was used. When strain CVO was presented with a mixture of chemically formed FeS and dissolved sulfide (HS⁻), it only oxidized the HS⁻. The FeS remained acid soluble and non-magnetic indicating that it was not transformed. In contrast, when the FeS was formed by adding FeCl₂ to a culture of SRB which gradually produced sulfide, precipitating FeS, and to which strain CVO and nitrate were subsequently added, transformation of the FeS to a magnetic, less acid-soluble form was observed. X-ray diffraction and energy-dispersive spectrometry indicated the transformed mineral to be greigite (Fe₃S₄). Addition of nitrite to cultures of SRB, containing microbially formed FeS, was similarly effective. Nitrite reacts chemically with HS⁻ to form polysulfide and sulfur (S⁰), which then transforms SRB-formed FeS to greigite, possibly via a sulfur addition pathway (3FeS + S⁰ → Fe₃S₄). Further chemical transformation to pyrite (FeS₂) is expected at higher temperatures (>60°C). Hence, nitrate injection into oil fields may lead to NR-SOB-mediated and chemical mineral transformations, increasing the sulfide-binding capacity of reservoir rock. Because of mineral volume decreases, these transformations may also increase reservoir injectivity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Microbial analysis of backflowed injection water from a nitrate-treated North Sea oil reservoir

Reservoir souring in offshore oil fields is caused by hydrogen sulphide (H₂S) produced by sulphate-reducing bacteria (SRB), most often as a consequence of sea water injection. Biocide treatment is commonly used to inhibit SRB, but has now been replaced by nitrate treatment on several North Sea oil fields. At the Statfjord field, injection wells from one nitrate-treated reservoir and one biocide-treated reservoir were reversed (backflowed) and sampled for microbial analysis. The two reservoirs have similar properties and share the same pre-nitrate treatment history. A 16S rRNA gene-based community analysis (PCR-DGGE) combined with enrichment culture studies showed that, after 6 months of nitrate injection (0.25 mM NO₃ ⁻), heterotrophic and chemolithotrophic nitrate-reducing bacteria (NRB) formed major populations in the nitrate-treated reservoir. The NRB community was able to utilize the same substrates as the SRB community. Compared to the biocide-treated reservoir, the microbial community in the nitrate-treated reservoir was more phylogenetically diverse and able to grow on a wider range of substrates. Enrichment culture studies showed that SRB were present in both reservoirs, but the nitrate-treated reservoir had the least diverse SRB community. Isolation and characterisation of one of the dominant populations observed during nitrate treatment (strain STF-07) showed that heterotrophic denitrifying bacteria affiliated to Terasakiella probably contributed significantly to the inhibition of SRB. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effects of nitrate treatment on a mixed species, oil field microbial biofilm

Biofilms of bacteria, indigenous to oil field produced water, were grown in square section, glass capillary flow cells at 45 °C. Initially, in situ image analysis microscopy revealed predominantly coccoid bacteria (length-to-width ratio measurements (l _c:w _c) of bacterial cells gave a mean value of 1.1), while chemical measurements confirmed sulphate reduction and sulphide production. After nitrate ion addition at 100 and 80 mg/l, in the two repeat experiments respectively, the dominance of rod-shaped bacteria (mean l _c:w _c = 2.8) was observed. This coincided with the occurrence of nitrate reduction in the treated flow cells. Beneficially, no significant increase in biofilm cover was observed after the addition of nitrate. The dominant culturable nitrate-reducing bacterium was Marinobacter aquaeolei. The l _c:w _c ratio measured here concurs with previously reported cell dimensions for this organism. Several Marinobacter strains were also isolated from different oil fields in the North Sea where nitrate treatment has been applied to successfully treat reservoir souring, implying that this genus may play an important role in nitrate treatment.     

Dynamics of corrosion rates associated with nitrite or nitrate mediated control of souring under biological conditions simulating an oil reservoir

Representative microbial cultures from an oil reservoir and electrochemical techniques including potentiodynamic scan and linear polarization were used to investigate the time dependent corrosion rate associated with control of biogenic sulphide production through addition of nitrite, nitrate and a combination of nitrate-reducing, sulphide-oxidizing bacteria (NR-SOB) and nitrate. The addition of nitrate alone did not prevent the biogenic production of sulphide but the produced sulphide was eventually oxidized and removed from the system. The addition of nitrate and NR-SOB had a similar effect on oxidation and removal of sulphide present in the system. However, as the addition of nitrate and NR-SOB was performed towards the end of sulphide production phase, the assessment of immediate impact was not possible. The addition of nitrite inhibited the biogenic production of sulphide immediately and led to removal of sulphide through nitrite mediated chemical oxidation of sulphide. The real time corrosion rate measurement revealed that in all three cases an acceleration in the corrosion rate occurred during the oxidation and removal of sulphide. Amendments of nitrate and NR-SOB or nitrate alone both gave rise to localized corrosion in the form of pits, with the maximum observed corrosion rates of 0.72 and 1.4 mm year⁻¹, respectively. The addition of nitrite also accelerated the corrosion rate but the maximum corrosion rate observed following nitrite addition was 0.3 mm year⁻¹. Furthermore, in the presence of nitrite the extent of pitting was not as high as those observed with other control methods.     

Determination of nitrate and nitrite by high-performance liquid chromatography in human plasma

A new, accurate, fast and simple method has been implemented by which nitrite and nitrate ions, as stable forms of nitric oxide production were studied. A study of these two ions was carried out by a sensitive and accurate HPLC method with two detectors. The most important advantages of the reported method are: short time of analysis, minimal sample pre-treatment, long life of the analytical column and stable eluent solution. The photodiode array UV-Vis detector detected nitrite and nitrate ions at an absorbance of 212 nm. Much more sensitive electrochemical detection with a WE (glassy carbon) electrode was used for the detection of nitrite ions. An analytical chromatographic column was formed by a sorbent, containing strong base anion-exchange groups bound in Cl(-) form in the hydrophilic hydroxyethyl methacrylate matrix. The anions were analysed in human plasma without deproteinization using 0.02 M sodium perchlorate monohydrate as eluent solution at pH 3.9. At this pH organic substances do not affect the analysis. The retention times for nitrite and nitrate were 3.62 and 3.72 min (by electrochemical detection) and 4.44 min, respectively. The method was linear (r=0.9992, 0.9998, 0.996) within a 1-100 (nitrate), 1-20 micro mol/l (nitrite) concentration range.     

High-resolution microanalysis of nitrite and nitrate in neuronal tissues by capillary electrophoresis with conductivity detection

Nitrites and nitrates are widely used reporters of endogenous activity of nitric oxide synthases (NOS), an important group of enzymes producing the gaseous signal molecule nitric oxide (NO). However, due to the great chemical heterogeneity of neuronal tissues, standard analytical protocols for evaluation of neuronal nitrite/nitrate concentrations are inefficient. We optimized a high-performance capillary zone electrophoresis (CZE) technique to analyze nitrite/nitrate concentrations in submicroliter samples from mammalian neuronal tissues. The measurements were made using a PrinCE 476 computerized capillary electrophoresis system with a Crystal 1000 contact conductivity detector. Isotachophoretic stacking injection of 1000- to 10000-fold diluted samples, which had been pretreated with a custom-designed solid-phase microextraction (SPME) cartridge, was employed to assay micromolar and nanomolar nitrite and nitrate levels in the presence of the high millimolar chloride concentrations characteristic of many biological samples. In the presented technique, a 10-microl volume of diluted ganglionic sample was used for chloride removal and sample cleanup. The method yields high analytical performance, including good reproducibility, resolution, and accuracy. The limits of detection relative to undiluted sample matrix were 8.9 nM (0.41 ppb) and 3.54 nM (0.22 ppb) for nitrite and nitrate, respectively. In addition, this technique resolves other anions that are present in neuronal tissues at sub-nanomolar concentrations and can be broadly applied for high-throughput anionic profiling. In rat dorsal root ganglia, endogenous levels of nitrate (231+/-29 microM; n=6) and nitrite (24-96 microM) were found. These concentrations exceeded those previously found in neuronal tissue homogenates using different techniques.     

Simple and rapid method for the measurement of nitrite and nitrate in human plasma and cerebrospinal fluid by capillary electrophoresis

Nitrite and nitrate levels in physiological fluids are commonly used as an index of nitric oxide production. We developed simple and rapid method for the determination of these anions by capillary zone electrophoresis employing borate buffer (pH 10, 100 mmol/l) as running electrolyte. The anions were analyzed in plasma and cerebrospinal fluid (CSF) without deproteinization of the samples. Electrophoresis was carried out in a capillary (36.5 cm×75 μm) at a potential of 15 kV, with on-column UV detection at 214 nm. Mean retention times for nitrite and nitrates were 4.631 and 5.152 min, respectively. The method was linear (r=0.999) within a 1–500 μmol/l concentration range. Physiological levels of nitrate in plasma (40.2 μmol/l) and CSF (15.3 μmol/l) could be determined with good precision (coefficients of variation <6%) and accuracy (recoveries of added nitrate to plasma and CSF were 97.4 and 104.5%, respectively). Measurements of the physiological levels of nitrite in plasma (6.1 μmol/l) and CSF (0.9 μmol/l) were less precise and accurate.     

Effect of nitrate injection on the bacterial community in a water-oil tank system analyzed by PCR-DGGE

Sulfide production by sulfate-reducing bacteria (SRB) is a major concern for the petroleum industry since it is toxic and corrosive, and causes plugging due to the formation of insoluble iron sulfides (reservoir souring). In this study, PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE) using two sets of primers based on the 16S rRNA gene and on the aps gene (adenosine-5-phosphosulfate reductase) was used to track changes in the total bacterial and SRB communities, respectively, present in the water-oil tank system on an offshore platform in Brazil in which nitrate treatment was applied for 2 months (15 nitrate injections). PCR-DGGE analysis of the total bacterial community showed the existence of a dominant population in the water-oil tank, and that the appearance and/or the increase of intensity of some bands in the gels were not permanently affected by the introduction of nitrate. On the other hand, the SRB community was stimulated following nitrate treatment. Moreover, sulfide production did not exceed the permissible exposure limit in the water-oil separation tank studied treated with nitrate. Therefore, controlling sulfide production by treating the produced water tank with nitrate could reduce the quantity of chemical biocides required to control microbial activities.     

The bioremoval of nitrate and sulfate alterations on artistic stonework: The case-study of Matera Cathedral after six years from the treatment

The bioremoval of nitrate and sulfate salts from the tuff stone surfaces of 12th century Matera Cathedral, which had been altered by nitrates and sulfates, using nitrate and sulphate reducing bacteria was performed.The bioremoval treatment was based on the direct application onto the altered stone surfaces of a previously-isolated Pseudomonas pseudoalcaligenes KF707 strain and Desulfovibrio vulgaris ATCC 29579 cells.The two strains were entrapped in a Carbogel and applied individually and together to the vertical wall; the protocol adopted included a multilayer biosystem, as a solution to maximise the efficacy of the biocleaning process on artistic stonework.The biological procedure resulted in the efficient, homogeneous removal of surface deposits and salt alterations. In fact, at 24 h the strains had removed 55% of the nitrate and 85% of the sulfate deposits, respectively.After six years from the original application the results show further improvements in microbial technology when applied to “bioremoval” and “biocleaning” of cultural heritage surface alterations.     

Simultaneous determination of nitrite and nitrate anions in plasma, urine and cell culture supernatants by high-performance liquid chromatography with post-column reactions

A high-performance liquid chromatographic method for the determination of nitrite and nitrate anions derived from nitric oxide in biological fluids is presented. After separation on a strong anion-exchange column (Spherisorb SAX, 250×4.6 mm I.D., 5 μm), two on-line post-column reactions occur. The first involves nitrate reduction to nitrite on a copper-plated cadmium-filled column. In the second, the diazotization-coupling reaction between nitrite and the Griess reagent (0.05% naphtylethylendiamine dihydrochloride plus 0.5% sulphanilamide in 5% phosphoric acid) takes place, and the absorbance of the chromophore is read at 540 nm. This methodology was applied to biological fluids. Before injection into the chromatographic system, the samples were diluted and submitted to suitable clean-up procedures (urine and cell culture supernatant samples are passed through C₁₈ cartridges, and serum samples were deproteinized by ultrafiltration through membranes with a molecular mass cut-off of 3000). The method has a sensitivity of 30 pmol for both anions, as little as 0.05–0.1 ml sample volume is required and linearity is observed up to 60 nmol for each anion.     

The effect of long-term nitrate treatment on SRB activity,corrosion rate and bacterial community composition in offshore water injection systems

Biogenic production of hydrogen sulphide (H₂S) is a problem for the oil industry as it leads to corrosion and reservoir souring. Continuous injection of a low nitrate concentration (0.25–0.33 mM) replaced glutaraldehyde as corrosion and souring control at the Veslefrikk and Gullfaks oil field (North Sea) in 1999. The response to nitrate treatment was a rapid reduction in number and activity of sulphate-reducing bacteria (SRB) in the water injection system biofilm at both fields. The present long-term study shows that SRB activity has remained low at ≤0.3 and ≤0.9 μg H₂S/cm²/day at Veslefrikk and Gullfaks respectively, during the 7–8 years with continuous nitrate injection. At Veslefrikk, 16S rRNA gene based community analysis by PCR–DGGE showed that bacteria affiliated to nitrate-reducing sulphide-oxidizing Sulfurimonas (NR–SOB) formed major populations at the injection well head throughout the treatment period. Downstream of deaerator the presence of Sulfurimonas like bacteria was less pronounced, and were no longer observed 40 months into the treatment period. The biofilm community during nitrate treatment was highly diverse and relative stable for long periods of time. At the Gullfaks field, a reduction in corrosion of up to 40% was observed after switch to nitrate treatment. The present study show that nitrate injection may provide a stable long-term inhibition of SRB in sea water injection systems, and that corrosion may be significantly reduced when compared to traditional biocide treatment.     

Diverse effects of formate on the dissimilatory metabolism of nitrate in Pseudomonas denitrificans ATCC 13867: Growth,nitrite accumulation in culture,cellular activities of nitrate and nitrite reductases

The growth of Pseudomonas denitrificans ATCC 13867 under denitrifying conditions was significantly stimulated by adding an appropriate amount of formate (2.5 mM or above) to the growth medium. The accumulation of nitrite in the culture was markedly depressed so long as formate remained in the culture above a certain level. Cellular activities of enzymes participating in denitrification also changed. The cells grown in the presence of formate exhibited a lower nitrate reductase activity and, in contrast, a higher nitrite reductase activity than the cells grown without added formate.     

The increase in plasma nitrite after a dietary nitrate load is markedly attenuated by an antibacterial mouthwash

Recent studies surprisingly show that dietary inorganic nitrate, abundant in vegetables, can be metabolized in vivo to form nitrite and then bioactive nitric oxide. A reduction in blood pressure was recently noted in healthy volunteers after dietary supplementation with nitrate; an effect consistent with formation of vasodilatory nitric oxide. Oral bacteria have been suggested to play a role in bioactivation of nitrate by first reducing it to the more reactive anion nitrite. In a cross-over designed study in seven healthy volunteers we examined the effects of a commercially available chlorhexidine-containing antibacterial mouthwash on salivary and plasma levels of nitrite measured after an oral intake of sodium nitrate (10 mg/kg dissolved in water). In the control situation the salivary and plasma levels of nitrate and nitrite increased greatly after the nitrate load. Rinsing the mouth with the antibacterial mouthwash prior to the nitrate load had no effect on nitrate accumulation in saliva or plasma but abolished its conversion to nitrite in saliva and markedly attenuated the rise in plasma nitrite. We conclude that the acute increase in plasma nitrite seen after a nitrate load is critically dependent on nitrate reduction in the oral cavity by commensal bacteria. The removal of these bacteria with an antibacterial mouthwash will very likely attenuate the NO-dependent biological effects of dietary nitrate.     

The influence of nitrate on microbial processes in oil industry production waters

Sulfide accumulation due to bacterial sulfate reduction is responsible for a number of serious problems in the oil industry. Among the strategies to control the activity of sulfate-reducing bacteria (SRB) is the use of nitrate, which can exhibit a variety of effects. We investigated the relevance of this approach to souring oil fields in Oklahoma and Alberta in which water flooding is used to enhance oil recovery. SRB and nitrate-reducing bacteria (NRB) were enumerated in produced waters from both oil fields. In the Oklahoma field, the rates of sulfate reduction ranged from 0.05 to 0.16 μM S day⁻¹ at the wellheads, and an order of magnitude higher at the oil–water separator. Sulfide production was greatest in the water storage tanks in the Alberta field. Microbial counts alone did not accurately reflect the potential for microbial activities. The majority of the sulfide production appeared to occur after the oil was pumped aboveground, rather than in the reservoir. Laboratory experiments showed that adding 5 and 10 mM nitrate to produced waters from the Oklahoma and Alberta oil fields, respectively, decreased the sulfide content to negligible levels and increased the numbers of NRB. This work suggests that sulfate reduction control measures can be concentrated on aboveground facilities, which will decrease the amount of sulfide reinjected into reservoirs during the disposal of oil field production waters. Journal of Industrial Microbiology & Biotechnology (2001) 27, 80–86. Received 30 January 2001/ Accepted in revised form 30 June 2001     

Interference by S-nitroso compounds with the measurement of nitrate in methods requiring reduction of nitrate to nitrite by cadmium

Various methods suited for the measurement of nitrate require its reduction to nitrite by cadmium under acidic or alkaline conditions. N^G-Nitroarginine analogs have been shown to interfere with the measurement of nitrate by such assays. In the present work we show by gas chromatography−mass spectrometry that under alkaline reduction conditions the S-nitroso compounds S-nitrosoglutathione and S-nitrosohomocysteine but not S-nitroso-N-acetylcysteine and S-nitroso-N-acetylpenicillamine can considerably contribute to nitrate and thus interfere with its measurement. Our results suggest that S-nitroso compounds may interfere with the measurement of nitrate in methods requiring cadmium-catalyzed reduction of nitrate to nitrite.     

Effect of nitrite on a thermophilic, methanogenic consortium from an oil storage tank

Samples from an oil storage tank (resident temperature 40 to 60 °C), which experienced unwanted periodic odorous gas emissions, contained up to 2,400/ml of thermophilic, lactate-utilizing, sulfate-reducing bacteria. Significant methane production was also evident. Enrichments on acetate gave sheathed filaments characteristic of the acetotrophic methanogen Methanosaeta thermophila of which the presence was confirmed by determining the PCR-amplified 16S rDNA sequence. 16S rDNA analysis of enrichments, grown on lactate- and sulfate-containing media, indicated the presence of bacteria related to Garciella nitratireducens, Clostridium sp. and Acinetobacter sp. These sulfidogenic enrichments typically produced sulfide to a maximum concentration of 5–7 mM in media containing excess lactate and 10 mM sulfate or thiosulfate. Both the production of sulfide and the consumption of acetate by the enrichment cultures were inhibited by low concentrations of nitrite (0.5–1.0 mM). Hence, addition of nitrite may be an effective way to prevent odorous gas emissions from the storage tank.     

基质氮素对大球盖菇生长及亚硝酸盐含量的影响

目的探索氮源对大球盖菇生长及亚硝酸盐、硝酸盐含量的影响。方法在培养基中添加不同含氮化合物,培养菌丝,定时测定生长量,并采用重氮偶合分光光度法测定其亚硝酸盐、硝酸盐含量。结果添加亚硝酸盐的基质,菌丝生长速度比对照慢。尿素、硫酸铵、硝酸铵和硝酸钾四处理之间差异无显著性,其生长速度最快。各处理间亚硝酸盐、硝酸盐含量差异均存在非常显著性。亚硝酸钾处理,亚硝酸盐含量高达402.03 mg/kg,不宜食用。硝酸钾处理,亚硝酸盐含量为30.87 mg/kg,食用100 g,就会超出亚硝酸盐日限量;硝酸铵处理,硝酸盐含量远远超过日常蔬菜,也不宜食用。结论在栽培大球盖菇时氮源不宜使用亚硝酸盐、硝酸盐,应使用尿素或硫酸铵,有机氮源也可以。