Mechanistic study of microbial control of hydrogen sulfide production in oil reservoirs.

Microbial control of biogenic production of hydrogen sulfide in oil fields was studied in a model system consisting of pure cultures of the nitrate-reducing, sulfide-oxidizing bacterium (NR-SOB) Thiomicrospira sp. strain CVO and the sulfate-reducing bacterium (SRB) Desulfovibrio sp. strain Lac6, as well as in microbial cultures enriched from produced water of a Canadian oil reservoir. The presence of nitrate at concentrations up to 20 mM had little effect on the rate of sulfate reduction by a pure culture of Lac6. Addition of CVO imposed a strong inhibition effect on production of sulfide. In the absence of added nitrate SRB we were able to overcome this effect after an extended lag phase. Simultaneous addition of CVO and nitrate stopped the production of H2S immediately. The concentration of sulfide decreased to a negligible level due to nitrate-dependent sulfide oxidation activity of CVO. This was not prevented by raising the concentration of Na-lactate, the electron donor for sulfate reduction. Similar results were obtained with enrichment cultures. Enrichments of produced water with sulfide and nitrate were dominated by CVO, whereas enrichments with sulfate and Na-lactate were dominated by SRB. Addition of an NR-SOB enrichment to an SRB enrichment inhibited the production of sulfide. Subsequent addition of sufficient nitrate caused the sulfide concentration to drop to zero. A similar response was seen in the presence of nitrate alone, although after a pronounced lag time, it was needed for emergence of a sizable CVO population. The results of the present study show that two mechanisms are involved in microbial control of biogenic sulfide production. First, addition of NR-SOB imposes an inhibition effect, possibly by increasing the environmental redox potential to levels which are inhibitory for SRB. Second, in the presence of sufficient nitrate, NR-SOB oxidize sulfide, leading to its complete removal from the environment. Successful microbial control of H2S in an oil reservoir is crucially dependent on the simultaneous presence of NR-SOB (either indigenous population or injected) and nitrate in the environment.     

Action of glutaraldehyde and nitrite against sulfate-reducing bacterial biofilms

A continuous flow reactor system was developed to evaluate the efficacy of antimicrobial treatments against sulfate-reducing bacterial biofilms. An annular reactor operating at a nominal dilution rate of 0.5 h⁻¹ was fed one-tenth strength Postgate C medium diluted in 1.5% NaCl and was inoculated with a mixed culture enriched from oilfield-produced water on the same medium. Thin biofilms developed in this reactor after 2 days of operation. The activity of these biofilms resulted in approximately 50 mg S l⁻¹ of sulfide at steady state prior to biocide treatment. Biocide efficacy was quantified by recording the time required for sulfide production to recover following an antimicrobial treatment. In a control experiment in which pure water was applied, the time required to reach 10 mg S l⁻¹ sulfide after the treatment was 1.7±1.2 h, whereas the time to reach this level of sulfide after a pulse dose of 500 mg l⁻¹ glutaraldehyde was delayed to 61±11 h. Nitrite treatment suppressed sulfide production as long as the nitrite concentration remained above 15 mg N l⁻¹. Sulfide production recovered more rapidly after nitrite treatment than it did after glutaraldehyde treatment. Received 01 February 2002/ Accepted in revised form 13 June 2002     

Estimating the Abundance of Endospores of Sulfate-Reducing Bacteria in Environmental Samples by Inducing Germination and Exponential Growth

It is a challenge to quantitatively distinguish active from dormant microbial populations in environmental samples. Here we present an approach for estimating the abundance of dormant sulfate-reducing bacteria (SRB), present as viable endospores in environmental samples. This is achieved by inducing endospores to germinate and grow exponentially. We demonstrate this approach for thermophilic SRB in temperate sediment from Aarhus Bay, Denmark. The approach is based on measuring bulk sulfate reduction rates (SRRs) in pasteurized sediment and calculating associated cell-specific SRRs based on average values for SRB growth yield and cell size known from exponentially growing pure cultures. The method presented is a faster bioassay than most probable number enumerations and has the potential to distinguish between slow- and fast-growing SRB populations in the same sample. This bioassay is attractive given the challenges posed by endospores with respect to cell permeabilization and lysis, which are prerequisite in quantitative microscopy- and nucleic acid extraction-based strategies. These molecular approaches additionally rely on designing target-appropriate oligonucleotide probes, whereas the method presented here considers the trait of interest more broadly. This strategy thus presents a useful complement to other methods in ecological investigations of microbial biogeography and for specific industrial applications such as reservoir souring and corrosion risk assessments in the oil and gas sector.     

Microwave-assisted pretreatment of woody biomass with ammonium molybdate activated by H2O2

Pretreatments for enzymatic saccharification are crucial for the establishment of lignocellulosic biorefineries. In this study, we focused on ammonium ions and peroxometal complexes as potential delignifying agents. We first examined the pretreatment of beech wood with nine different ammonium salts in the presence of H₂O₂. Significant pretreatment effects were found only for ammonium molybdate, which is transformed to a peroxometal complex on reacting with H₂O₂. Since microwave sensitizer effects are expected for (peroxo)molybdate, beech wood was pretreated using external heating and microwave irradiation. As a result, a maximum sugar yield of 59.5% was obtained by microwave irradiation at 140 °C for 30 min, while external heating in an autoclave gave a sugar yield of 41.8%. We also found that an ammonium ion is the key counterion accelerating the pretreatment with molybdate. These results highlight the powerful selective delignifying capability of the H₂O₂-activated ammonium molybdate system energised by microwave radiation.     

Changes in soil microbial community composition induced by cometabolism of toluene and trichloroethylene

The effects of trichloroethylene (TCE) on microbial community composition were analyzed by reverse sample genome probing. Soil enrichments were incubated in dessicators containing an organic phase of either 1 or 10% (w/w) toluene in vacuum pump oil, delivering constant equilibrium aqueous concentrations of 16 and 143mg/l, respectively. Increasing the equilibrium aqueous concentration of TCE from 0 to 10mg/l led to shifts in community composition at 16, but not at 143mg/l of toluene. In closed system co-degradation studies, TCE at an aqueous concentration of 1mg/1 was effectively degraded by toluene-degrading soil enrichments once the aqueous toluene concentration dropped below 25mg/l. Little TCE degradation was observed at higher toluene concentrations (50–250mg/l). The results indicate that TCE changes microbial community composition under conditions where it is being actively metabolized.     

Phylogeny of nitrite reductase (nirK) and nitric oxide reductase (norB) genes from Nitrosospira species isolated from soil

Ammonia-oxidizing bacteria are believed to be an important source of the climatically important trace gas nitrous oxide (N(2)O). The genes for nitrite reductase (nirK) and nitric oxide reductase (norB), putatively responsible for nitrous oxide production, have been identified in several ammonia-oxidizing bacteria, but not in Nitrosospira strains that may dominate ammonia-oxidizing communities in soil. In this study, sequences from nirK and norB genes were detected in several cultured Nitrosospira species and the diversity and phylogeny of these genes were compared with those in other ammoniaoxidizing bacteria and in classical denitrifiers. The nirK and norB gene sequences obtained from Nitrosospira spp. were diverse and appeared to be less conserved than 16S rRNA genes and functional ammonia monooxygenase (amoA) genes. The nirK and norB genes from some Nitrosospira spp. were not phylogenetically distinct from those of denitrifiers, and phylogenetic analysis suggests that the nirK and norB genes in ammonia-oxidizing bacteria have been subject to lateral transfer.     

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.     

2-bromoethanesulfonate (BES) Enhances Sulfate-reducing Bacterial Population and Dichlorodiphenyltrichloroethane (DDT) Dechlorination in an Anaerobic Paddy Soil

2-bromoethanesulfonate (BES) is a structural analogue of 2-mercaptoethanesulfonic acid (coenzyme M) and often used to specifically inhibit methanogenesis. The role of BES and sulfate on the reductive dechlorination of dichlorodiphenyltrichloroethane (DDT) was compared in an anaerobic soil slurry reactor of sulfate-reducing system in this study. The population of soil sulfate-reducing bacteria (SRB) was markedly decreased under DDT condition compared to DDT-free reactor, while greatly increased by sulfate and slightly increased by BES. However, the dechlorination rate of DDT was the highest in the DDT+BES treatment, followed in order by DDT+Sulfate and the control condition. In the DDT+BES treatment, more than 60% of DDT-Cl was cleaved within 16 weeks, which was about 124% and 369% greater than that in the DDT+Sulfate treatment and under the control condition, respectively. The results suggested that the inhibition of methanogenesis by BES was another pathway to improve sulfate-reducing activity and the related dechlorination rate of DDT in waterlogged soils.     

Ecology of some Species of Thiobacillus and Sulfate-reducing Bacteria in the Middle Course of the River Guadalquivir (Spain)

During 1985, a study was made of the physical, chemical and biological characteristics of the River Guadalquivir. At the same time, the density of certain microorganisms involved in the transformation of sulfur compounds in the water was studied. The results obtained give the following numerical order of density: T. thioparus >sulfate reducers > T. denitrificans > T. thiooxidans. Urban waste was found to increase density levels of these microorganisms. Their relationship with certain environmental parameters is discussed in this study. Regression equations have been obtained of bacteria numbers for three of the four groups studied, using water temperature and permanganate oxidability as dependent variables.     

Vertical distribution of nitrite reductase genes (nir S) in continental margin sediments of the Gulf of Mexico

Marine sediments account for up to 66% of the loss of nitrogen load to coastal areas. Sedimentary denitrification is the main sink for fixed nitrogen in the global nitrogen budget, and thus it is important to understand the structure and composition of denitrifying communities. To understand the structure and composition of denitrifying communities, the diversity of nitrite reductase (nirS) genes from sediments along the Gulf of Mexico was examined using a PCR-based cloning approach. Sediments were collected at three different depths (0-0.5, 4-5 and 19-21 cm). Geochemical analysis revealed decreasing nitrate and oxygen concentrations with increasing sediment depth. This trend coincided with the decrease in diversity of denitrifying bacteria. LIBSHUFF analysis indicated that the clone library in the shallowest sediment (depth, 0-0.5 cm) was significantly different from that in the deepest sediment (depth, 19-21 cm), and that the deeper sediments (depths of 4-5 and 19-21 cm) were significantly similar. Community structural shifts were evident between the shallowest (oxic zone) and deepest (anoxic zone) sediments. Community changes within the deepest sediments were more subtle, with the presence of different nirS clone sequences gradually becoming dominant or, alternatively, decreasing with depth. The changes in community structure at this depth are possibly driven by nutrient availability, with lower quality sources of carbon and energy leading to the disappearance of nirS sequences common in the top layer. The majority of recovered nirS sequences were phylogenetically divergent relative to known denitrifying bacteria in the database.     

Oscillation of NAD(P)H fluorescence in Escherichia coli culture performing dissimilative nitrate/nitrite reduction

When nitrate was added to anaerobic resting cultures of Escherichia coli, two different profiles of NAD(P)H fluorescence were observed. E. coli is known to reduce nitrate to ammonia via nitrite as an anaerobic respiration mechanism. The profile showing single-stage response corresponded to situations where the nitrite formed from nitrate reduction was immediately converted to ammonia. The other profile showing two-stage response resulted from a much slower reduction of nitrite than nitrate. Nitrite thus accumulated during the first stage and was gradually reduced to ammonia when nitrate was depleted, i.e. in the second stage. An undamped oscillation of NAD(P)H fluorescence was also observed in the cultures showing the two-stage response. The oscillation was always detected during the second stage and seldom during either the first stage or the recovered anaerobic stage (after complete nitrite reduction). It never occurred in the cultures showing the single-stage response. The period of oscillation ranged from 1 to 5min. The possibility of the common glycolytic oscillation being responsible is low, as judged from the current knowledge of the nitrate/nitrite reductases of E. coli and the observations in this study. This is the first report on the occurrence of oscillatory NAD(P)H fluorescence in E. coli.     

The reaction of H(2)S with oxidized thiols: generation of persulfides and implications to H(2)S biology

Hydrogen sulfide is an endogenously generated molecule with many reported physiological functions. Although several biological targets have been proposed, the biochemical mechanisms by which it elicits activity are not established. Thus, in an effort to begin to delineate the fundamental biological chemistry of H₂S, we have examined the reaction of H₂S with oxidized thiols and thiol proteins in order to determine whether persulfide formation occurs, is stable and how this may affect protein function. We have found that persulfides are easily generated, relatively stable and can alter enzyme activity. Moreover, we have begun to develop methodology for in situ generation of persulfides to facilitate further study of this potentially important species.     

Induction of interleukin 8 (IL-8) production by Pseudomonas nitrite reductase in human alveolar macrophages and epithelial cells.

Interleukin-8 (IL-8) participates in the generation of dense neutrophil accumulations in bronchopulmonary infections caused by Pseudomonas aeruginosa (P. aeruginosa). We have recently reported that nitrite reductase, a bifunctional enzyme located in the periplasmic space of P. aeruginosa, induces IL-8 generation in bronchial epithelial cells (K. Oishi et al. Infect. Immun. 65: 2648-2655, 1997). We examined whether or not Pseudomonas nitrite reductase (PNR) could also stimulate human alveolar macrophages (AM) and pulmonary type II epithelial-like cells (A549) to induce IL-8 production and mRNA expression as well as the production of TNF alpha and IL-1beta. We demonstrated a time- and dose-dependent IL-8 protein synthesis and IL-8 mRNA expression, but no TNF alpha or IL-1beta production, by A549 cells in response to PNR. New protein translation was not required for PNR-mediated IL-8 mRNA expression in the same cells. Furthermore, simultaneous stimulation of PNR with serial doses of TNF alpha or IL-1beta resulted in additive IL-8 production in A549 cells. In adherent AM, PNR enhanced IL-8 protein synthesis and IL-8 mRNA expression in a time-dependent fashion. PNR similarly induced a time-dependent production of TNF alpha and IL-1beta by human adherent AM. Neutralization of TNF alpha or IL-1beta did not influence the levels of IL-8 production in adherent AM culture. We also evaluated whether the culture supernatants of the A549 cells or AM stimulated with PNR could similarly mediate neutrophil migration in vitro. When anti-human IL-8 immunoglobulin G was used for neutralizing neutrophil chemotactic factor (NCF) activities in the culture supernatants of these cells stimulated with 5 microg/ml of PNR, the mean percent reduction of NCF activities were 49-59% in A549 cells and 24-34% in AM. Our present data support that PNR directly stimulates AM and pulmonary epithelial cells to produce IL-8. PNR also mediates neutrophil migration, in part, through IL-8 production from AM and pulmonary epithelial cells. These data suggest the contribution of PNR to the pathogenesis of bronchopulmonary infections due to P. aeruginosa.     

两类产NO的亚硝酸盐还原酶的分布,结构与序列比较及宏基因组分析

【目的】为了体现亚硝酸盐还原酶在环境中氮生物循环的重要性,研究了它们的分布情况。【方法】利用现有亚硝酸盐还原酶序列在已经测序的基因组数据库中进行查找,研究该酶的分布情况,通过多序列比对比较了它们的序列相似性,通过构建系统发育树比较其进化关系,并利用宏基因组学的方法研究了它们在海洋宏基因组中的分布。【结果】分析结果显示,两类亚硝酸盐还原酶在已测序的细菌和古生菌基因组中分别有397和812个,分别占总量的8%和15.7%,几乎所有的古生菌都含有Ⅱ类酶;它们自身的序列相似性很高,在Ⅰ类酶和Ⅱ类酶中底物结合位点以及Ⅱ类酶的铜离子结合位点保守性都很高,显示该酶序列保守性与其环境功能相适应的特点;进化分析显示它们可能具有共同的进化来源;在海洋宏基因组中,平均每100000读数中分别有6个Ⅰ类和35个Ⅱ类,且2类酶都在热带南太平洋区域有最大分布。【结论】NIR在氮的生物循环及环境修复中可能起到重要作用。     

Purification and properties of nitrite reductase from roots of pea (Pisum sativum cv. Meteor)

Nitrite reductase (EC 1.6.6.4) prepared from pea roots was found to be immunologically indistinguishable from pea leaf nitrite reductase. Comparisons of the pea root enzyme with nitrite reductase from leaf sources showed a close similarity in inhibition properties, light absorption spectrum, and electron paramagnetic resonance signals. The resemblances indicate that the root nitrite reductase is a sirohaem enzyme and that it functions in the same manner as the leaf enzyme in spite of the difference in reductant supply implicit in its location in a non-photosynthetic tissue.Abbreviations DEAE diethylaminoethyl - EPR electron paramagnetic resonance - NIR nitrite reductase - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Acute toxicity of ammonia and nitrite to different ages of Pacific cod (Gadus macrocephalus) larvae

Abstract

This study was conducted to evaluate the acute toxicity of ammonia and nitrite to three developmental stages of Pacific cod (Gadus macrocephalus) larvae (11, 22, and 35 days after hatching, with mean total lengths of 4.63 ± 0.14, 5.83 ± 0.17, and 7.46 ± 0.23 mm, respectively). The results showed for the first time that the acute toxicity of ammonia and nitrite is closely related to the age of Pacific cod larvae, and the acute toxicity of ammonia or nitrite increased with increased Pacific cod larval growth. Lethal concentrations (LC₅₀) of un-ionized ammonia nitrogen (UIAN) for a 48-h exposure in 11-day post-hatch, 22-day post-hatch, and 35-day post-hatch Pacific cod larvae were 1.72, 0.69, and 0.32 mg L^?1, respectively. The 48-h LC₅₀ of nitrite nitrogen to Pacific cod larvae 11-day post-hatch, 22-day post-hatch, and 35-day post-hatch were 831.76, 269.15, and 223.87 mg L^?1, respectively. The present findings demonstrate that the acute toxicity of ammonia for Pacific cod larvae is much higher than that of nitrite.     

Microbial Reductive Dechlorination of Weathered and Exogenous Co-planar Polychlorinated Biphenyls (PCBs) in an Anaerobic Sediment of Venice Lagoon

The occurrence of reductive dechlorination processes towards pre-existing PCBs and five exogenous coplanar PCBs were investigated in a contaminated sediment of Porto Marghera (Venice Lagoon, Italy) suspended, under strictly anaerobic conditions, in water collected from the same site. PCB dechlorination started after five months of incubation, when sulfate initially occurring in the microcosms was completely depleted and methanogenesis was in progress. It was ascribed to sulfate-reducing bacteria. Several pre-existing hexa-, penta- and tetra-chlorinated biphenyls were slowly bioconverted into tri- and di-, ortho-substituted PCBs from the 5th to the 16th month of experiment. Spiked coplanar PCBs, i.e., 3,3′,4,4′-tetrachlorobiphenyl, 3,3′,4,4′,5- and 2,3′,4,4′,5-pentachlorobiphenyls, 3,3′,4,4′,5,5′- and 2,3,3′,4,4′,5-hexachlorobiphenyls, were extensively transformed (by about 90%) into lower chlorinated congeners, such as 3,3′,5,5′-/2,3′,4,4′-tetrachlorobiphenyl, 3,3′,5-, 2,4,4′-, 2,3′,4- and 2,3′,5-trichlorobiphenyl, 3,4-/3,4′- and 3,3′-dichlorobiphenyl and 2-chlorobiphenyl. The reductive dechlorination of spiked PCBs did not influence significantly the biotransformation rate and extent of pre-existing PCBs.     

硫化氢与细胞的增殖和凋亡

硫化氢是内源性气体分子家族中的一员,是一种气体递质（gasotransmitter）。近年来,内源性硫化氢的产生及生理意义已经被认识,其代谢异常与许多疾病有关。本文综述了最近发现的硫化氢对细胞增殖和凋亡的调节作用,并重点概述硫化氢细胞效应的分子机制,包括丝裂原活化蛋白激酶、细胞周期相关激酶、细胞死亡相关基因以及离子通道等的改变。对硫化氢调节细胞生长或死亡的深入了解将为新药设计及许多疾病的治疗提供新的思路。     

Physiology, phylogenetic relationships, and ecology of filamentous sulfate-reducing bacteria (genus Desulfonema)

Microscopy of organic-rich, sulfidic sediment samples of marine and freshwater origin revealed filamentous, multicellular microorganisms with gliding motility. Many of these neither contained sulfur droplets such as the Beggiatoa species nor exhibited the autofluorescence of the chlorophyll-containing cyanobacteria. A frequently observed morphological type of filamentous microorganism was enriched under anoxic conditions in the dark with isobutyrate plus sulfate. Two strains of filamentous, gliding sulfate-reducing bacteria, Tokyo 01 and Jade 02, were isolated in pure cultures. Both isolates oxidized acetate and other aliphatic acids. Enzyme assays indicated that the terminal oxidation occurs via the anaerobic C₁ pathway (carbon monoxide dehydrogenase pathway). The 16S rRNA genes of the new isolates and of the two formerly described filamentous species of sulfate-reducing bacteria, Desulfonema limicola and Desulfonema magnum, were analyzed. All four strains were closely related to each other and affiliated with the δ-subclass of Proteobacteria. Another close relative was the unicellular Desulfococcus multivorans. Based on phylogenetic relationships and physiological properties, Strains Tokyo 01 and Jade 02 are assigned to a new species, Desulfonema ishimotoi. A new, fluorescently labeled oligonucleotide probe targeted against 16S rRNA was designed so that that it hybridized specifically with whole cells of Desulfonema species. Filamentous bacteria that hybridized with the same probe were detected in sediment samples and in association with the filamentous sulfur-oxidizing bacterium Thioploca in its natural habitat. We conclude that Desulfonema species constitute an ecologically significant fraction of the sulfate-reducing bacteria in organic-rich sediments and microbial mats. Received: 30 December 1998 / Accepted: 19 July 1999     

Role of Organic Carbon Sources and Sulfate in Controlling Net Methylmercury Production in Riverbank Sediments of the South River,VA (USA)

Mercury (Hg) transport and methylmercury (MeHg) production in riverbank sediments are complex processes influenced by site-specific physical and biogeochemical conditions. The South River watershed in VA, USA, contains elevated concentrations of Hg in riverbank and floodplain sediments, which has the potential to methylate. The role of specific organic carbon sources in promoting methylation reactions in natural sediments under dynamic flow conditions is not well understood. Four saturated column experiments were conducted, including a control column, which received South River water as an influent solution, and three columns that received South River water amended with: acetate (5.8 mM); lactate (5.7 mM); and lactate (5.7 mM) with SO₄^2? (10.1 mM). The amendments were selected to promote growth of different microorganisms to gain an understanding of the microbial processes, controlling rates of methylation. The column receiving lactate and SO₄^2? had the highest MeHg concentrations in the effluent and in the pore water near the effluent at 1.8 and 4.9 μg L^?1, respectively. At the cessation of the column experiments, the lactate–sulfate column sediments contained the highest populations of enumerable sulfur-reducing bacteria and the highest solid-phase MeHg at 530 ± 100 ng g^?1 dry wt. from the interval closest to the influent. The results suggest that the form and availability of electron donors and acceptors are primary factors controlling rates of methylation in the South River sediment.