Use of nitrate to control sulfide generation by sulfate-reducing bacteria associated with oily waste

Received 13 November 1998/ Accepted in revised form 03 March 1999     

Planktonic nitrate-reducing bacteria and sulfate-reducing bacteria in some western Canadian oil field waters

Oil fields that use water flooding to enhance oil recovery may become sour because of the production of H₂S from the reduction of sulfate by sulfate-reducing bacteria (SRB). The addition of nitrate to produced waters can stimulate the activities of nitrate-reducing bacteria (NRB) and control sulfide production. Many previous studies have focused on chemolithotrophic bacteria that can use thiosulfate or sulfide as energy sources while reducing nitrate. Little attention has been given to heterotrophic NRB in oil field waters. Three different media were used in this study to enumerate various types of planktonic NRB present in waters from five oil fields in western Canada. The numbers of planktonic SRB and bacteria capable of growth under aerobic conditions were also determined. In general, microbial numbers in the produced waters were very low (<10 ml⁻¹) in samples taken near or at wellheads. However, the numbers increased in the aboveground facilities. No thiosulfate-oxidizing NRB were detected in the oil field waters, but other types of NRB were detected in 16 of 18 produced water samples. The numbers of heterotrophic NRB were equal to or greater than the number of sulfide-oxidizing, chemolithotrophic NRB in 12 of 15 samples. These results showed that each of the oil fields contained NRB, which might be stimulated by nitrate amendment to control H₂S production by SRB. Journal of Industrial Microbiology & Biotechnology (2002) 29, 83–92 doi:10.1038/sj.jim.7000274 Received 20 February 2002/ Accepted in revised form 14 May 2002     

高温油藏生物膜内硫酸盐还原菌的腐蚀机理及防治策略

硫酸盐还原菌(sulfate-reducing bacteria,SRB)广泛分布于高温、高压及高盐的石油油藏中,在油藏硫循环中起主导作用。SRB能在油藏生物膜内生长,有微量低分子有机酸时利用硫酸盐为电子受体并将其还原成硫化氢。硫化氢会腐蚀管道,导致原油泄露等其他安全问题,每年造成的经济损失超过7 000亿元。本文首先总结了油藏生物膜内微生物菌群多样性,分析了生物膜内SRB及其相关菌群的协同腐蚀机理;然后讨论了高温油藏SRB介导的硫氮氢生物地球化学循环过程、胞外电子传递机制及其腐蚀作用,并通过几个高温油藏SRB生物膜内腐蚀的现场案例进一步阐明了SRB的腐蚀机制。在此基础上,提出了应对高温油藏生物膜内SRB腐蚀的生物纳米防治策略,这为高温油藏管道防腐提供了新思路。     

胜利油田单12高温油藏非培养和富集培养样品的细菌组成特性

[目的]通过比较分析油藏样品的微生物群落结构特点,认识油藏微生物的生态功能.[方法]利用3种油藏微生物研究中常用的富集培养方法,对胜利油田单12区块S12-4油井产出水样品进行了选择性富集培养,运用构建16S rRNA基因文库的方法分析了富集样品和非培养样品的细菌多样性.[结果]通过16S rRNA基因序列比对发现,非培养样品、异养菌富集样品、烃降解菌富集样品和硫酸盐还原菌富集样品中的优势菌分别为Pseudomonas属,Thermotoga属,Thermaerobacter属和Thermotoga属的成员.多样性分析结果表明,非培养样品的微生物多样性最丰富,同时非培养样品和富集样品的微生物群落结构存在很大的差异,富集样品中的微生物包括优势菌在油藏原位环境中含量很低.[结论]细菌组成差异的比较结果,对油藏微生物的生态功能研究和微生物驱油潜力评估具有重要意义.     

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     

三元(碱-表面活性剂-聚合物)复合驱油藏采出水微生物群落特征

与水驱技术相比,向油藏中注入碱、表面活性剂和聚合物(简称三元复合驱,ASP)能大幅提高石油采收率,但这些驱油剂对油藏中微生物多样性与群落结构的影响尚亟待阐明,这对油田水质管理与腐蚀控制均具有的重要意义. 本研究采用高通量测序技术解析了大庆油田ASP油藏4口油井采出水中的微生物多样性与群落结构. 结果表明: ASP油藏采出水的pH高达9.65. 采出水中微生物Shannon多样性指数为2.00～3.56,采出井间菌群多样性存在差异. 在门、纲、属分类水平上,变形菌门(85.5%～98.3%)、γ-变形菌纲(83.7%～97.8%)、栖碱菌属(51.8%～82.5%)是绝对优势菌群. 共检测到12个属的潜在硫化氢产生菌,以硫磺单胞菌属丰度最高(0.4%～7.4%). 与已发表的水驱油藏研究结果相比,三元复合驱油藏采出水微生物群落组成独特,呈嗜/耐碱趋势,其多样性偏低,群落结构更单一.     

Chemical and microbiological changes in laboratory incubations of nitrate amendment "sour" produced waters from three western Canadian oil fields

Nitrate addition to oil field waters stops the biogenic formation of sulfide because the activities of nitrate-reducing bacteria (NRB) suppress the activities of sulfate-reducing bacteria (SRB). In general, there are two types of NRB — the heterotrophic NRB and the chemolithotrophic NRB. Within the latter group are the nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB). To date, no study has specifically addressed the roles of these different NRB in controlling sulfide concentrations in oil field produced waters. This study used different culture media to selectively enumerate heterotrophic NRB and NR-SOB by most probable number (MPN) methods. Produced waters from three sulfide-containing western Canadian oil fields were amended with nitrate as an electron acceptor, but no exogenous electron donor was added to the serum bottle microcosms. Changes in the chemical and microbiological characteristics of the produced waters were monitored during incubation at 21°C. In less than 4 days, the sulfide was removed from the waters from two of the oil fields (designated P and C), whereas nearly 27 days were required for sulfide removal from the water from the third oil field (designated N). Nitrate addition stimulated large increases in the number of the heterotrophic NRB and NR-SOB in the waters from oil fields P and C, but only the NR-SOB were stimulated in the water from oil field N. These data suggest that stimulation of the heterotrophic NRB is required for rapid removal of sulfide from oil field-produced waters. Received 25 March 2002/ Accepted in revised form 10 June 2002     

A quick method for the determination of dissolved and precipitated sulfides in cultures of sulfate-reducing bacteria

Dissolved sulfide was determined spectrophotometrically as a colloidal solution of copper sulfide. Calibration curves were linear. Maximal deviation error was below 5%. Sulfide precipitated as FeS was determined after acidification of the medium.     

山西阳泉老窑水硫酸盐还原菌的分离鉴定及其还原功能驯化

【背景】含硫煤矿开采后,地表水/地下水回流至采空区形成酸性老窑水,含有高浓度重金属离子和硫酸盐,严重危害生态系统健康。利用微生物自身生长处理老窑水具有成本低、环境友好等特点,具有良好的应用前景。目前利用的硫酸盐还原菌大多只在适宜温度和中性pH条件下具有较高活性,在北方低温和酸性条件下难以发挥作用。【目的】本研究旨在从山西阳泉山底河流域的老窑水环境中分离硫酸盐还原菌,并调节温度和pH进行驯化,从而得到高效耐低温耐酸菌株,为北方老窑水微生物治理提供可用菌种资源。【方法】对山底河流域典型老窑水样品中的微生物进行富集培养,并筛选硫酸盐还原菌。通过革兰氏染色、扫描电镜对菌株形貌特性进行表征,利用16SrRNA基因序列比对进行菌种鉴定,探究其生长特性和硫酸盐还原性能。在此基础上降低温度和pH,对高效还原硫酸盐菌株进行驯化,探讨其在北方老窑水污染治理中的应用潜力。【结果】本研究筛选得到2株硫酸盐还原菌,命名为YQ-1和YQ-2,分别属于革兰氏阴性瘤胃解蛋白质菌属(Proteiniclasticum)和脱硫弧菌属(Desulfovibrio)。在30°C、pH 7.5条件下,YQ-1和YQ-2对1 1...     

Synthesis and crystal structure of a highly selective colorimetric and fluorometric sensor for hydrogen sulfide

A novel Cu(II) complex chemosensor for hydrogen sulfide with azo as the colorimetric group has been synthesized. The complex and ligand crystals were obtained and the molecular structures were characterized by X‐ray diffraction and Electrospray ionization High resolution mass spectrometer (ESI‐HRMS). The photophysical and recognition properties were examined. The complex can recognize S^2?, with an obvious color change from yellow to red based on a copper ion complex displacement mechanism. By contrast, no obvious changes were observed in the presence of other anions (AcO^?, H₂PO₄^?, F^?, Cl^?, Br^? and I^?). We present a simple, easily prepared, yet efficient, inorganic reaction‐based sensor for the detection of S^2?. The complex should have many chemical and analytical applications in the sensing of hydrogen sulfide.     

Usefulness of the hydrogen sulfide test for assessment of water quality in Bangladesh

Aim: To evaluate the usefulness of the hydrogen sulfide (H₂S) test for assessing water quality in Bangladesh. Methods and Results: We tested 382 water samples from a variety of sources using locally produced H₂S test kits and laboratory‐based membrane filtration for the detection of Escherichia coli. Compared with membrane filtration, H₂S tests, when incubated for 24 h, had both a sensitivity and positive predictive value (PPV) of <40% when analysis was restricted to water samples with E. coli levels below 100 colony forming units (CFU) per 100 ml. In contrast, for E. coli levels from 1000 to 9999 CFU per 100 ml, sensitivity was 94% and PPV 88%; specificity was 97% and negative predictive value was 99%. Conclusions: The hydrogen sulfide test, when incubated at 24 h, is a promising alternative for assessing water quality where E. coli levels may be high. An improved understanding of the incremental impact of contamination level on health is needed to better determine its usefulness. Significance and Impact of the Study: The hydrogen sulfide test is inexpensive, easy to use and portable. Its use may allow rapid assessment of water quality in situations where cost or logistics prevent use of other testing methods, such as in remote areas or during floods and other natural disasters.     

Benchmarking the Activity,Stability, and Inherent Electrochemistry of Amorphous Molybdenum Sulfide for Hydrogen Production

Anodically electrodeposited amorphous molybdenum sulfide (AE‐MoS_x) has attracted significant attention as a non‐noble metal electrocatalyst for its high activity toward the hydrogen evolution reaction (HER). The [Mo₃S₁₃]^2? polymer‐based structure confers a high density of exposed sulfur moieties, widely regarded as the HER active sites. However, their intrinsic complexity conceals full understanding of their exact role in HER catalysis, hampering their full potential for water splitting applications. In this report, a unifying approach is adopted accounting for modifications in the inherent electrochemistry (EC), HER mechanism, and surface species to maximize the AE‐MoS_x electroactivity over a broad pH region (0–10). Dramatic enhancements in HER performance by selective electrochemical cycling within reductive (overpotential shift, η_HER ≈ ?350 mV) and electro‐oxidative windows (η_HER ≈ ?290 mV) are accompanied by highly stable performance in mildly acidic electrolytes. Joint analysis of X‐ray photoelectron spectroscopy, Raman, and EC experiments corroborate the key role of bridging and terminal S ligands as active site generators at low pH, and reveal molybdenum oxysulfides (Mo⁵⁺O_xS_y) to be the most active HER moiety in AE‐MoS_x in mildly acidic‐to‐neutral environments. These findings will be extremely beneficial for future tailoring of MoS_x materials and their implementation in commercial electrolyzer technologies.     

Waste Rock Biogeochemistry in a Permafrost Environment: Examination of a Cover Design for a Low-Sulfide,Granitic Waste Rock

At the Diavik Waste Rock Project's mine-research site, the microbial colonization and oxidation of waste rock sulfide minerals are attenuated by the extreme freeze-thaw cycle of a permafrost environment. The closure design for the waste rock stockpile consists of a low-sulfide waste rock and low-permeability till, covering a relatively higher sulfide waste rock. This design was examined at the mine site through construction of experimental waste rock piles and active zone lysimeters with and without the till cover. Leachate from these experiments indicates variable pH and SO₄ concentrations that correlate with sulfide content and the thermal moderating influence of the till cover. The till initially provided a moderated environment for the production of acid, growth of acidophilic Fe- and S-oxidizing bacteria, and enhanced weathering until wet up and freezing of the till and underlying waste rock as a permafrost. Greater sulfide oxidation was observed above the till cover because of greater exposure to the annual freeze-thaw cycle. An examination of the bacterial communities at the genus level indicates the prevalence of Pseudomonas, Rhodanobacter, Sideroxydans, and Thiobacillus in the waste rock. Pseudomonas spp. were dominant in the drier and more extreme temperature environment above the till cover, while Thiobacillus spp. were dominant in the more sulfide-rich, wetter/frozen environment below the till. A decreasing trend in Thiobacillus spp. from the exterior to the interior and an opposing trend in Acidithiobacillus spp. suggest greater acid generation deeper in the waste rock further from the extreme temperature variation of the tundra climate. The presence of the till cover moderated temperature variations, enhanced the initial rate of sulfide oxidation, and allowed for greater microbial diversity, but the freezing of the till cover and underlying waste rock drastically reduced sulfide oxidation and the generation of acid rock drainage. These results highlight the importance of temperature on microbially catalyzed acid production and our ability to use the extreme temperatures of the tundra climate to minimize potential environmental effects from mining through formation of waste rock permafrost.     

Development of europium(III) complex fluorescent probe for hydrogen sulfide detection and its application in water samples

Hydrogen sulfide (H₂S) is a crucial endogenous signaling component in organisms that is involved in redox homeostasis and numerous biological processes. Modern medical research has confirmed that hydrogen sulfide plays an important role in the pathogenesis of many diseases. Herein, a fluorescent probe Eu(ttbd)₃abt based on europium(III) complex was designed and synthesized for the detection of H₂S. Eu(ttbd)₃abt exhibited significant quenching for H₂S at long emission wavelength (625 nm), with rapid detection ability (less than 2 min), high sensitivity [limit of detection (LOD) = 0.41 μM], and massive Stokes shift (300 nm). Additionally, this probe showed superior selectivity for H₂S despite the presence of other possible interference species such as biothiols. Furthermore, the probe Eu(ttbd)₃abt was successfully applied to detect H₂S in water samples.     

Isolation of pig colonic crypts for cytotoxic assay of luminal compounds: effects of hydrogen sulfide,ammonia, and deoxycholic acid

Some colonic luminal molecules resulting from bacterial metabolism of alimentary or endogenous compounds are believed to exert various effects on the colonic epithelial cell physiology. We isolated surface epithelial cells and intact colonic crypts in order to test bacterial metabolites in the pig model, which is often considered relevant for extrapolation to the physiopathology of the human gastrointestinal tract. Using colonocytes isolated with EDTA, we found that the initial cell viability, estimated by the membrane integrity and oxidative capacity measurement, fell rapidly despite several experimental attempts to preserve it such as the use of a medium designed to increase the adherence of epithelial cells and of a coated extracellular matrix, the presence in the culture medium of the oxidative substrate butyrate, and the use of an inhibitor of the caspases involved in cell apoptosis. In contrast, using dispase and collagenase as proteolytic agents, we were able to obtain pig colonic crypts that maintain an excellent membrane integrity after 4 h. Using this preparation, we were able to test the presumably cytotoxic luminal compounds hydrogen sulfide, ammonia, and deoxycholic acid on colonic crypt viability. Of these, only deoxycholic acid was found to significantly alter the cellular membrane integrity. It is concluded that pig colonic crypts can be useful for thein vitro appraisal of the cytotoxic properties of luminal compounds. This revised version was published online in July 2006 with corrections to the Cover Date.     

Minimal sulfur requirement for growth and sulfur-dependent metabolism of the hyperthermophilic archaeon Staphylothermus marinus

Staphylothermus marinus is an anaerobic hyperthermophilic archaeon that uses peptides as carbon and energy sources. Elemental sulfur (S(o)) is obligately required for its growth and is reduced to H2S. The metabolic functions and mechanisms of S(o) reduction were explored by examining S(o)-dependent growth and activities of key enzymes present in this organism. All three forms of S(o) tested--sublimed S(o), colloidal S(o) and polysulfide--were used by S. marinus, and no other sulfur-containing compounds could replace S(o). Elemental sulfur did not serve as physical support but appeared to function as an electron acceptor. The minimal S(o) concentration required for optimal growth was 0.05% (w/v). At this concentration, there appeared to be a metabolic transition from H2 production to S reduction. Some enzymatic activities related to S(o)-dependent metabolism, including sulfur reductase, hydrogenase, glutamate dehydrogenase and electron transfer activities, were detected in cell-free extracts of S. marinus. These results indicate that S(o) plays an essential role in the heterotrophic metabolism of S. marinus. Reducing equivalents generated by the oxidation of amino acids from peptidolysis may be transferred to sulfur reductase and hydrogenase, which then catalyze the production of H2S and H2, respectively.     

Application of biological activated carbon as a low pH biofilter medium for gas mixture treatment

Packing material is a crucial component of a bioreactor as it is the microbial population's habitat. This study assessed potential improvements to current biofiltration processes by investigating use of a novel support medium. Biological activated carbon (BAC) with microorganisms growing on granular activated carbon can produce a novel medium in which both adsorption and biodegradation contribute to pollutants removal. Investigation of carbon characteristics demonstrated that BAC was an ideal packing medium for biofiltration. The application of the novel packing medium for gas mixture treatment was evaluated in a low pH biofilter. Results demonstrated that BAC biofilter obtained high removal efficiency for both H(2)S and toluene. The removal mechanisms of BAC were investigated after the biofilter operation and it demonstrated that the performance of the BAC system was mainly controlled by the additive contributions of two removal mechanisms - adsorption and biodegradation. This study also indicated the potential for simultaneous treatment of hydrogen sulfide and toluene at low pH condition.     

青石斑鱼海水循环水养殖水体的细菌群落特征北大核心CSCD

【目的】为揭示工厂化循环水青石斑鱼养殖水体的细菌群落特征,比较患病养殖池与健康养殖池的细菌群落结构差异,探讨细菌群落结构与青石斑鱼病害相关的相关性。【方法】采用Illumina Miseq高通量测序方法,分析比较了患病和健康养殖水体细菌群落结构、α-多样性指数(包括多度、均一度和系统发育多样性);并结合传统方法从患病青石斑鱼病灶部位分离疑似病原菌。【结果】患病和健康养殖水体中细菌群落的α-多样性并无太大差异,但主坐标分析与热图样本聚类分析表明细菌群落结构明显不同。二者的优势细菌门均为Proteobacteria、Verrucomicrobia和Bacteroidetes,但它们的相对丰度差异显著。患病养殖水体中α-Proteobacteria(25.07%)和γ-Protebacteria(22.74%)丰度相当,而健康养殖水体中γ-Protebacteria(40.49%)显著高于α-Proteobacteria(10.87%)。患病水体的Verrucomicrobia丰度(26.4%)远高于健康水体(10.9%);而Bacteroidetes的相对丰度则相反(12.3%vs 20.9%)。主要的差异类群包括α-Proteobacteria的Rhodobacteraceae和Rhodospirillaceae,γ-Proteobacteria的Alteromonadaceae、HTCC2188和Oceanospirillaceae,Verrucomicrobia的Verrucomicrobiaceae和Bacteroidetes的Cryomorphaceae。更表现在核心微生物类群的差异,健康养殖池水体以Glaciecola、HTCC、Sediminicola、Prevotella等对于养殖动物有益或无害的属为核心微生物;而患病养殖池水体则以Vibrio、Rubritalea、Oleibacter等病原菌或对养殖动物不利的属为核心微生物。从患病青石斑鱼的皮肤、肝脏和脾脏共分离得到弧菌20株,Acinetobacter haemolyticus 1株。【结论】患病的青石斑鱼循环水养殖水体中的细菌群落明显不同于健康养殖水体,特别是核心微生物的差异,其以Vibrio等病原菌或对养殖动物不利的属为主。该结果为青石斑鱼循环水养殖系统的管理、病害的诊断和监测提供理论与实验基础。     

Terbium (III) coordination polymer–copper (II) compound as fluorescent probe for time‐resolved fluorescence ‘turn‐on’ detection of hydrogen sulfide

With recognition of the biological importance of hydrogen sulfide (H₂S), we present a simple and effective fluorescent probe for H₂S using a Tb³⁺ coordination polymer–Cu²⁺ compound (DPA/Tb/G–Cu²⁺). Dipicolinic acid (DPA) and guanosine (G) can coordinate with Tb³⁺ to form a macromolecular coordination polymer (DPA/Tb/G). DPA/Tb/G specifically binds to Cu²⁺ in the presence of coexisting cations, and obvious fluorescence quenching is observed. The quenched fluorescence can be exclusively recovered upon the addition of sulfide, which is measured in the mode of time‐resolved fluorescence. The fluorescence intensities of the DPA/Tb/G–Cu²⁺ compound enhance linearly with increasing sulfide concentrations from 1 to 30 μM. The detection limit for sulfide in aqueous solution is estimated to be 0.3 μM (at 3σ). The DPA/Tb/G–Cu²⁺ compound was successfully applied to sense H₂S in human serum samples and exhibited a satisfactory result. It displays some desirable properties, such as fast detection procedure, high selectivity and excellent sensitivity. This method is very promising to be utilized for practical detection of H₂S in biological and environmental samples.