Sulfide-induced dissimilatory nitrate reduction to ammonia in anaerobic freshwater sediments

Abstract: Different reduced sulfur compounds (H₂S, FeS, S₂O₃²⁻) were tested as electron donors for dissimilatory nitrate reduction in nitrate-amended sediment slurries. Only in the free sulfide-enriched slurries was nitrate appreciably reduced to ammonia (     ), with concomitant oxidation of sulfide to S⁰ (     ). The initial concentration of free sulfide appears as a factor determining the type of nitrate reduction. At extremely low concentrations of free S²⁻ (metal sulfides) nitrate was reduced via denitrification whereas at higher S²⁻ concentrations, dissimilatory nitrate reduction to ammonia (DNRA) and incomplete denitrification to gaseous nitrogen oxides took place. Sulfide inhibition of NO- and N₂O- reductases is proposed as being responsible for the driving part of the electron flow from S²⁻ to NH₄⁺.     

Determination of tetrathionate and thiosulfate in natural samples and microbial cultures by a new, fast and sensitive ion chromatographic technique

Abstract A new isocratic ion chromatographic technique is described for the sensitive measurement of tetrathionate, trithionate and thiosulfate. The sulfur oxyanions were separated on a polymer-coated, silica-based anion exchange column and directly detected by UV absorption at 216 nm. Aqueous saline acetonitrile/methanol mixtures were used as eluent. The three anions could be quantified in less than 10 min with detection limits of about 0.6 pmol for tetrathionate and of 40 and 10 pmol for trithionate and thiosulfate, respectively. The retention times of tetrathionate responded to changes of the solvent concentration, whereas the elution of thiosulfate depended predominatly on the ionic strenght of the eluent. Starting at the lowest detectable concentrations, calibration curves for all three compounds were linear over a concentration range of three orders of magnitude. The analysis of freshwater and saline samples worked equally well. Since contact of the eluent with metallic components caused shifts in retention times during operation, the solvent delivery system had to consist of plastic material. Examples of applicaton are given for determination of tetrathionate and thiosulfate in natural samples and for the turnover of these two compounds in sediment slurries and anaerobic enrichment cultures.     

In situ stimulation of bacterial sulfate reduction in sulfate-limited freshwater lake sediments

Abstract By adding sulfate in the form of solid gypsum, it was possible to transform in situ a predominantly methanogenic sediment ecosystem into a sulfate-reducing one. The concentrations of sulfate, sulfide, methane, acetate, propionate, soluble iron, and manganese were determined in the porewater before and after the transition. Although sulfate was no longer limiting, acetate and propionate continued to accumulate and reached much higher concentrations than under sulfate-limited conditions. Metabolic activities of fermenting bacteria and of sulfate reducers, which belong to the group that incompletely oxidizes organic material, might be responsible for the increased production of volatile fatty acids. The elevated concentrations of soluble Fe(II)²⁺ and Mn(II)²⁺ observed in the porewater stem from iron and manganese compounds which may be reduced chemically by hydrogen sulfide and other microbially produced reducing agents or directly through increased activities of the iron and manganese reducing bacteria. In the horizon with high sulfate-reducing activities the methane concentrations in the porewater were lower than in non-stimulated sediment regions. The shape of the concentration depth profile indicates methane consumption through sulfate reducing processes. The in situ experiment demonstrates the response of a natural microbial ecosystem to fluctuations in the environmental conditions.     

Stable sulfur isotopic distributions and sulfate reduction in lake sediments of the Adirondack Mountains,New York

Cores from five lakes of the Adirondack Mountains, New York, were analyzed for sedimentary sulfur concentrations and stable sulfur isotopic compositions. Isotopic values of total sedimentary sulfur were as much as 6 to 8 lower than isotopic values of sulfur sources (soils, tree leaves and lake water sulfate) which showed little isotopic variation in the Adirondacks. The low isotopic values of recent sediments indicate increased sulfate reduction, probably in response to increased sulfate loading and acid deposition. Detailed historical reconstruction of sulfate loading histories from sedimentary sulfur profiles is premature, however, since model calculations indicate that sulfur can be added to deeper older sediments by ongoing, contemporary sulfate reduction.     

Effects of sulfate concentration in the overlying water on sulfate reduction and sulfur storage in lake sediments

We investigated the effects of sulfate concentration on sulfate reduction and net S storage in lake sediments using³⁴S as a tracer. The water overlying intact sediment cores from the hypolimnion of Mares Pond, MA, was replaced with two Na₂ ³⁴SO₄ solutions at either ambient (70 M) or elevated (260 M) sulfate concentrations. The ³⁴S of the added sulfate was 4974 . Over two months, the net sulfate reduction rate in the ambient sulfate treatment was zero, while the net rate for the high sulfate treatment was 140 moles/m²/d. The water overlying the cores was kept under oxic conditions and the sediment received no fresh carbon inputs, thus the net rate reported may underestimate the in situ rate. Gross sulfate reduction rates calculated by isotope dilution were approximately 350 moles/m²/d for both treatments. While the calculation of gross sulfate reduction rates in intact sediment cores can be complicated by differential diffusion of³⁴S and³²S, isotopic fractionation, and the possible formation of ester sulfates, we believe these effects to be small. The results suggest that sulfate reduction is not strongly sulfate-limited in Mares Pond. The difference in net sulfate reduction rates between treatments resulted from a decrease in sulfide oxidation and suggests the importance of reoxidation in controlling net S storage in lake sediments. In both treatments the CRS and organic S fractions were measurably labelled in³⁴S. Below the sediment surface, the CRS fraction was the more heavily labelled storage product for reduced sulfides.     

Demonstration of HOQNO and antimycin A sensitive coupling of NADH oxidation and APS and sulfite reduction in a marine Desulfovibrio strain

Abstract In cell suspensions of the marine sulfate-reducing bacterium Desulfovibrio 20020 (DSM 3099) permeabilized with formaldehyde or Triton X-100, sulfite-dependent NADH oxidation activities of 0.05 μmol · min⁻¹· mg⁻¹ protein were detected. NADH oxidation coupled to APS, thiosulfate and fumarate reduction was also demonstrated. All the activities were subject to inhibition by HOQNO and antimycin A. The rate of NADH oxidation coupled to the reduction of sulfite was extremely low in cell-free extracts. The physiological function and possible mechanism of the NADH oxidation coupled to the reduction of various electron acceptors are discussed.     

Major substrates for microbial sulfate reduction in the sediments of Ise Bay, Japan

To clarify the anaerobic microbial interactions in the process of carbon mineralization in marine eutrophic environments, the microbial sulfate reduction and methane production rates were examined in coastal marine sediments of Ise Bay, Japan, in autumn 1990. Sulfate reduction rates (51–210 nmol ml⁻¹ day⁻¹ at 24°C) were much higher than the methane production ones (<1.78 nmol ml⁻¹ day⁻¹) in the surface sediments (top 2 cm) at the six stations surveyed (water depth: 10.7–23.3 m). Substrates for sulfate-reducing bacteria (SRB) were estimated after the addition of a specific inhibitor for SRB (20 mmol l⁻¹ molybdate) into the sediment slurry, from the substrate accumulation rates. In the presence of the inhibitor, sulfate reduction was completely stopped and volatile fatty acids (mainly acetate) were accumulated, although hydrogen was not. Methane production occurred markedly accompanied by consumption of the accumulated acetate from the third day after the addition of molybdate. The maximum rate of methane production was 1.2–1.9 μmol ml⁻¹ day⁻¹, which was similar to those in highly polluted freshwater sediments such as the Tama River, Tokyo, Japan. These results show that acetate is a common major substrate for sulfate reduction and methane production, and SRB competitively inhibit potential acetoclastic methanogenesis in coastal sediments. Methanogens may potentially inhabit the sediments at low levels of population density and activity.     

Uptake of sulfate,sulfite and thiosulfate by proton-anion symport in Desulfovibrio desulfuricans

pH changes and sulfide production upon addition of sulfate, sulfite or thiosulfate to non-buffered H₂-saturated cell suspensions of Desulfovibrio desulfuricans were studied by means of electrodes. The addition of these electron acceptors resulted in a rapid alkalinization of the suspension which was accompanied by sulfide production. At-2° C, alkalinization without immediate sulfide production could be obtained. After addition of ³⁵S-labelled sulfate at-2° C, the label was found to be concentrated 7,500-fold in the cells, while 2 protons per sulfate molecule had disappeared from the outer bulk phase. Alkalinization and sulfide production from micromolar electron acceptor additions depended on the transmembraneous proton gradient ( pH), and were reversibly inhibited in alkaline solution (pH>8.0) or by the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP). Protonophore-inhibited sulfide production from sulfite or thiosulfate could be restored if the cell membranes were permeabilized by the detergent cetyltrimethylammonium bromide (CTAB), or if downhill transport was made possible by the addition of electron acceptors at millimolar concentrations. Sulfate was not reduced under these conditions, presumably because the cells did not contain ATP for its activation. K⁺-and Na⁺-ionophores such as nigericin, valinomycin or monensin appeared to be of limited efficiency in D. desulfuricans. In most experiments, sulfate reduction was inhibited by the K⁺–H⁺ antiporter nigericin in the presence of K⁺, but not by the thiocyanate anion or the K⁺-transporter valinomycin. The results indicate that sulfate, sulfite and thiosulfate are taken up by proton-anion symport, presumably as undissociated acids with an electroneutral mechanism, driven by the transmembraneous pH gradient ( pH) or by a solute gradient. Kinetics of alkalinization and sulfide production in cells grown with different electron acceptors revealed that D. desulfuricans has different specific uptake systems for sulfate and thiosulfate, and obviously also for sulfite. It is proposed that the electron acceptor transport finally will not consume net energy during growth in buffered medium: The protons taken up during active electron acceptor transport leave the cell with the reduced end-product by simple passive diffusion of H₂S.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - FCCP carbonyl cyanide p-trifluoromethoxy phenylhydrazone - CTAB cethyltrimethylammonium bromide     

Characterization of sulphate-reducing bacterial populations within marine and estuarine sediments with different rates of sulphate reduction

Viable counts of sulphate-reducing bacteria, able to use a range of different growth substrates were determined in sediments from two Sea Lochs (Etive and Eil) and an estuarine site (Tay), in Scotland. The composition of the sulphate-reducing bacterial population, in terms of substrate utilization, broadly corresponded to the in situ substrates for sulphate reduction and concentration of substrates at each site. Addition of acetate, lactate, propionate, butyrate, hydrogen and glutamate/serine (20 mM) to replicate slurries from each site resulted in stimulation of the corresponding population of sulphate-reducing bacteria and the in situ rates of sulphate reduction. The metabolism of the added substrates and changes in bacterial phospholipid fatty acids (PLFA) were quantified. With the exception of acetate and hydrogen, added substrates were incompletely oxidised, producing a mixture of further substrates, which predominantly were sequentially oxidised, and resulted in the stimulation of a mixed population of sulphate-reducing bacteria. There were significant changes in the PLFA of slurries with added substrate compared to controls. Acetate was completely removed at all sites and the small increase in even chain PLFA together with the absence of stimulation of any other biomarker, indicated that acetate was oxidised by sulphate-reducing bacteria distinctly different from those using other substrates. A biomarker for Desulfobacter, 10 Methyl 16:0, was not stimulated in any of the acetate slurries or in slurries where acetate was produced. Biomarkers for the propionate utilizing Desulfobulbus sp (17:1w6, 15:1w6) were always stimulated in propionate slurries and also in lactate slurries, where partial lactate fermentation produced propionate and acetate. In lactate and glutamate / serine slurries from the Tay estuary and lactate and hydrogen slurries from Loch Etive the biomarker for Desulfovibrio sp (i17:1w7) as well as those for Desulfobulbus were stimulated. This provides direct evidence for the significance of Desulfovibrio sp. within sediment slurries and demonstrates the competitive interaction between members of this genus and Desulfobulbus sp. for lactate, hydrogen and amino acid metabolism. At the estuarine site, sulphate reduction was limited at higher sulphate concentrations (about 3.5 mM) than the Sea Loch sites (<2 mM) and this had a significant effect on propionate and butyrate metabolism, as well as on methane production. These results demonstrate that although the sulphate-reducing bacterial population at each site could metabolise identical substrates, the types of sulphate-reducing bacteria involved and their sulphate thresholds were characteristically different.     

Characterizing the distribution and rates of microbial sulfate reduction at Middle Valley hydrothermal vents

Few studies have directly measured sulfate reduction at hydrothermal vents, and relatively little is known about how environmental or ecological factors influence rates of sulfate reduction in vent environments. A better understanding of microbially mediated sulfate reduction in hydrothermal vent ecosystems may be achieved by integrating ecological and geochemical data with metabolic rate measurements. Here we present rates of microbially mediated sulfate reduction from three distinct hydrothermal vents in the Middle Valley vent field along the Juan de Fuca Ridge, as well as assessments of bacterial and archaeal diversity, estimates of total biomass and the abundance of functional genes related to sulfate reduction, and in situ geochemistry. Maximum rates of sulfate reduction occurred at 90 °C in all three deposits. Pyrosequencing and functional gene abundance data revealed differences in both biomass and community composition among sites, including differences in the abundance of known sulfate-reducing bacteria. The abundance of sequences for Thermodesulfovibro-like organisms and higher sulfate reduction rates at elevated temperatures suggests that Thermodesulfovibro-like organisms may have a role in sulfate reduction in warmer environments. The rates of sulfate reduction presented here suggest that—within anaerobic niches of hydrothermal deposits—heterotrophic sulfate reduction may be quite common and might contribute substantially to secondary productivity, underscoring the potential role of this process in both sulfur and carbon cycling at vents.     

Seasonal variation in sulfate reduction and methanogenesis in peaty sediments of eutrophic Lake Loosdrecht,The Netherlands

During one year, concentration profiles of sulfate and methane were measured in sediment cores of eutrophic Lake Loosdrecht. Sulfate concentrations decreased exponentially with depth towards a constant threshold value of 7.6 ± 6.1 μM. Concentration profiles were used to calculate fluxes of sulfate and methane and to estimate the anaerobic mineralization rate. Anaerobic mineralization was highest in autumn which was probably due to an increased sedimentation of easily degradable organic carbon. At high rates (>600 μ mol organic carbon .m⁻².h⁻¹), sulfate reduction appeared to be limited by sulfate and methanogenesis accounted for over 80% of the anaerobic mineralization. At low anaerobic mineralization rates, measured in winter and spring, sulfate reduction was predominant. There was little methanogenesis below 5 cm depth in the sediment which indicated a rapid decrease of degradable organic matter with depth. There was a remarkable difference, especially in winter, between methane fluxes which were measured in batch experiments and those calculated from the concentration profiles in the sediment. These differences may be due to methane diffusing upward from deep layers.     

Rates and regulation of microbial iron reduction in sediments of the Baltic-North Sea transition

The rates and pathways of anaerobic carbon mineralization processes were investigated at seven stations, ranging from 10 to 56 m water depth, in the Kattegat and Belt Sea, Denmark. Organic carbon mineralization coupled to microbial Mn and Fe reduction was quantified using anaerobic sediment incubation at two stations that were widely separated geographically within the study area. Fe reduction accounted for 75% of the anaerobic carbon oxidation at the station in the northern Kattegat, which is the highest percentage so far reported from subtidal marine sediment. By contrast, sulfate reduction was the dominant anaerobic respiration pathway (95%) at the station in the Great Belt. Dominance of Fe reduction was related to a relatively high sediment Fe content in combination with active reworking of the sediment by infauna. The relative contribution of Fe reduction to anaerobic carbon oxidation at both stations correlated with the concentration of poorly crystalline Fe(III), confirming that the concentration of poorly crystalline Fe(III) exerts a strong control on rates of Fe reduction in marine sediments. The dependence of microbial Fe reduction on concentrations of poorly crystalline Fe(III) was used to quantify the importance of Fe reduction at sites where anaerobic incubations were not applied. This study showed that Fe reduction is an important process in anaerobic carbon oxidation in a wider area of the seafloor in the northern and eastern Kattegat (contribution 60 – 75%). By contrast, Fe reduction is of little significance (6 – 25%) in the more coarse-grained sediments of the shallower western and southern Kattegat, where a low Fe content was an important limiting factor, and in fine-grained sediments of the Belt Sea (4 – 28%), where seasonal oxygen depletion limits the intensity of bioturbation and thereby the availability of Fe(III). A large fraction of the total deposition of organic matter in the Kattegat and Belt Sea occurs in the northern Kattegat, and we estimate 33% of benthic carbon oxidation in the whole area is conveyed by Fe reduction.     

The effects of sediment slurrying on microbial processes,and the role of amino acids as substrates for sulfate reduction in anoxic marine sediments

In sediment slurry experiments with anoxic marine sediments collected in Cape Lookout Bight, NC, and a site in mid-Chesapeake Bay, the rates of sulfate reduction and ammonium production decrease with increasing dilution of sediment with oxygen-free sea-water. The effect of sediment dilution on the rates of these processes can be described by a simple mathematical relationship, and when these rates are corrected for sediment dilution they yield values which agree well with direct measurements of these processes.In sediment slurry studies of amino acid utilization in Cape Lookout Bight sediments, the fermentative decarboxylation of glutamic acid (to -aminobutyric acid) or aspartic acid (to alanine or -alanine) did not occur when either of these amino acids were added to Cape Lookout Bight slurries. The addition of glutamic acid did however lead to a small (1) transient build-up of -aminoglutaric acid. Measured rates of glutamic acid uptake in these slurries also decreased with increasing sediment dilution.Molybdate inhibition experiments demonstrated that dissolved free amino acids represent 1–3% of the carbon sources/electron donors used for sulfate reduction in Cape Lookout Bight sediments. The direct oxidation of amino acids by sulfate reducing bacteria also accounts for 13–20% of the total ammonium produced. Glutamic acid, alanine, -aminoglutaric acid, aspartic acid and asparagine are the major amino acids oxidized by sulfate reducing bacteria in Cape Lookout Bight sediments.     

A new sensitive, rapid fluorescence technique for the determination of proteins in gel electrophoresis and in solution

We describe the use of o-phthalaldehyde (OPT) in a simple, rapid, fluorimetric technique for the proteins in the concentration range of 0.1–50 μg/ml. Fluorescence excitation is near 338 nm and emission is measured near 446 nm.The technique is eminently suitable for simple rapid, high-resolution evaluation of protein/peptide separations in polyacrylamide gel electrophoresis. For this purpose the mixture to be fractionated is treated first with β-mercaptoethanol, followed by OPT, prior to electrophoresis. The separation is evaluated after electrophoresis by exciting the fluorescence of protein/peptide-bound OPT with a conventional (365 nm) “black light”. The technique provides exceptional resolution and far greater sensitivity than conventional detection techniques; its detection limits are ca. 10 ng protein. When used without β-mercaptoethanol, the technique could also allow definition of the distribution of SH-containing proteins/peptides.Quantitative evaluation of protein/peptide distribution is best achieved by photometric scanning of photographic negatives obtained under defined exposure and developing times.     

Application of microcolumn ion chromatography using anion exchangers modified with dextran sulfate for the determination of alkali and alkaline-earth metal ions

Microcolumn ion chromatography using anion exchangers modified with dextran sulfate has been applied to the determination of alkali and alkaline-earth metal ions contained in guinea pig serum and bovine serum. These serums contained Na⁺, NH₄⁺, K⁺, Mg²⁺ and Ca²⁺ and they were indirectly detected at 200 nm. The determination was done without any pretreatment procedure other than dilution.     

Impact of sampling methods on sulfate reduction rates and dissolved organic carbon (DOC) concentrations in vegetated salt marsh sediments

The impact of sediment coring on measured rates of sulfate reduction(SRR) by the whole core ³⁵S-injection technique was assessed inmarshsediment vegetated by Spartina anglica. Simultaneously,therole of extraction method (centrifugation vs. sippers) for determination ofporewater DOC in vegetated sediment was evaluated. SRR was measuredinsitu with radiotracer injected directly into the sediment and in atime series from 1 to 24 h after coring. SRR incubations carriedout within 6 h (June) or 12 h (August) of coringyielded up to an order of magnitude higher rates than measured insitu. The enhancement of SRR was instantaneous but temporary andcorrelated with measured porewater DOC concentrations. Cores sampled fromrootedsediments should therefore not be used for sulfate reduction incubations withinthe first 12 h due to the effect of DOC leaching from roots cutduring the coring procedure. The labile fraction of leached DOC appears to beexhausted after a pre-incubation period of at least 12 h.Measurement of porewater DOC is also problematic in vegetated sediment.Porewater extraction by centrifugation of sediment may result in up to oneorderof magnitude higher DOC concentrations than in porewater obtained by anondestructive sipper technique. DOC is probably forced out of roots duringcentrifugation resulting in erroneously high porewater DOC concentrations.     

气相色谱法测定生物制品中苯酚含量

本文采用气相色谱法测定了几种生物制品中苯酚含量。样品无需进一步处理,加入内标液后直接上柱测定,色谱峰形良好,线性范围宽,平均回收率和变异系数分别为１０１．８％ 和１．０８５％ ,最小检测限为１０ μｇ／ｍ ｌ,本法操作简单,样品用量少,可作为测定生物制品中苯酚含量的一种常规方法。     

Gas chromatographic determination of total amino acids and protein in sediments using the ninhydrin-CO2 reaction

A sensitive modification of the ninhydrin-CO₂ method involving the gas chromatographic determination of the total protein and amino acid content of sediment is described. The method gives a linear response over the amino acid concentration range 10^–5 M to 4 × 10^–2 M. It can be used for whole sediment, hydrolysates and interstitial water. The performance of the method is compared with the fluorescamine method for primary amines.     

Factors influencing the potential mobility and bioavailability of exchangeable ammonium ion in dried lake sediments

Abstract

The effect of drying temperature and oxidation on the level of exchangeable ammonium ion found in sediments has been examined using samples collected from along a polluted creek and from shallow lake bays. The sediments were dried at temperatures between 20°C and 100°C (either in air or under a nitrogen atmosphere), and the ammonium ion content was extracted into 0.1 M KCl prior to analysis using an ion selective electrode. Exposure to air during the drying stage usually resulted in lower ammonium values, while increasing the drying temperature altered the amount of displaceable (i.e. available) ammonium ion extracted, generally in an upward direction. The amount detected (5–25 μ g^?1) varied between sites, and surface sediment values differed from the 10–50 cm core material results. The pH of the extracts varied with the drying temperature used, indicating that the heating process promoted some chemical changes in the test samples. The study has demonstrated that in nutrient level surveys, the analytical data produced can depend greatly on the sample preparation procedure selected. It also indicated the type of changes which could occur when dredged sediments are land dumped.