Sulfur-Containing Amino Acids as Precursors of Thiols in Anoxic Coastal Sediments

Sulfur-containing amino acids were examined as precursors for thiols in anoxic coastal sediments. Substrates (10 to 100 μM) were anaerobically incubated with sediment slurries; thiols were assayed as isoindole derivatives by high-performance liquid chromatography; and microbial transformations of thiols, in contrast to their chemical binding by sediment particles, were identified by inhibition with a mixture of chloramphenicol and tetracycline. Methionine and homocysteine were transformed to methanethiol and 3-mercaptopropionate (3-MPA); methionine stimulated mainly methanethiol production, whereas homocysteine generated more 3-MPA than methanethiol. 2-Keto-4-methiolbutyrate yielded results similar to those with methionine, indicating that demethiolation yields methanethiol at the keto-acid level. Glutathione gave rise to cysteine, which was further transformed to 3-mercaptopyruvate and thence to mercaptoacetate and mercaptoethanol. Mercaptoethanol was oxidized to mercaptoacetate, which was biologically consumed. In conclusion, sulfurcontaining amino acids contribute to the range of thiols that occur in anoxic coastal sediments. New metabolic and environmental transformations were identified: the production of 3-MPA as a metabolite of methionine and the transformation of mercaptopyruvate to mercaptoethanol and mercaptoacetate.     

Mercury Methylation and Demethylation in Anoxic Lake Sediments and by Strictly Anaerobic Bacteria

After spiking anoxic sediment slurries of three acidic oligotrophic lakes with either HgCl₂ at 1.0 μg/ml or CH₃HgI at 0.1 μg/ml, both mercury methylation and demethylation rates were measured. High mercury methylation potentials were accompanied by high demethylation potentials in the same sediment. These high potentials correlated positively with the concentrations of organic matter and dissolved sulfate in the sediment and with mercury levels in fish. Adjustment of the acidic sediment pH to neutrality failed to influence either the methylation or the demethylation rate of mercury. The opposing methylation and demethylation processes converged to establish similar Hg²⁺-CH₃Hg⁺ equilibria in all three sediments. Because of their metabolic dominance in anoxic sediments, mercury methylation and demethylation in pure cultures of sulfidogenic, methanogenic, and acetogenic bacteria were also measured. Sulfidogens both methylated and demethylated mercury, but the methanogen tested only catalyzed demethylation and the acetogen neither methylated nor demethylated mercury.     

Production and Consumption of Dimethylsulfoniopropionate in Marine Microbial Mats

The fate of dimethylsulfoniopropionate (DMSP), a major sulfonium compound in marine ecosystems, was examined in Microcoleus chthonoplastes-dominated microbial mats. Chemical decomposition of DMSP was observed under laboratory conditions at pH values higher than 10.0. pH profiles measured in situ showed that these highly alkaline conditions occurred in microbial mats. Axenic cultures of M. chthonoplastes contained 37.3 μmol of DMSP g of protein⁻¹, which was partially liberated when the cells were subjected to an osmotic shock. DMSP-amended mat slurries showed a rapid turnover of this compound. The addition of glutaraldehyde blocked DMSP decrease, indicating biological consumption. Populations of potential dimethyl sulfide consumers were found in the top 10 mm of the mat.     

Ammonium Production in Sediments Inhibited with Molybdate: Implications for the Sources of Ammonium in Anoxic Marine Sediments

Ammonium production in the presence of specific inhibitors of sulfate reduction and methanogenesis was investigated in six marine sediments which differed in bulk properties and organic matter input. In all cases, little effect of the inhibitors on ammonium production was observed, although sulfate reduction was suppressed by molybdate. This gives evidence that the processes of fermentation and hydrolysis are of primary importance in ammonium generation at the sites studied. Although sulfate reduction rates may appear to be coupled to ammonium production rates, sulfate reduction does not necessarily contribute directly to generation of ammonium in marine environments.     

Seasonal Rates of Methane Oxidation in Anoxic Marine Sediments

Methane concentrations and rates of methane oxidation were measured in intact sediment cores from an inshore marine sediment at Jutland, Denmark. The rates of methane oxidation, determined by the appearance of ¹⁴CO₂ from injected ¹⁴CH₄, varied with sediment depth and season. Most methane oxidation was anoxic, but oxygen may have contributed to methane oxidation at the sediment surface. Cumulative rates (0- to 12-cm depth) for methane oxidation at Kysing Fjord were 3.34, 3.48, 8.60, and 17.04 μmol m⁻² day⁻¹ for April (4°C), May (13°C), July (17°C), and August (21°C), respectively. If all of the methane was oxidized by sulfate, it would account for only 0.01 to 0.06% of the sulfate reduction. The data indicate that methane was produced, in addition to being oxidized, in the 0- to 18-cm sediment stratum.     

Comparative Analysis of Methane-Oxidizing Archaea and Sulfate-Reducing Bacteria in Anoxic Marine Sediments

The oxidation of methane in anoxic marine sediments is thought to be mediated by a consortium of methane-consuming archaea and sulfate-reducing bacteria. In this study, we compared results of rRNA gene (rDNA) surveys and lipid analyses of archaea and bacteria associated with methane seep sediments from several different sites on the Californian continental margin. Two distinct archaeal lineages (ANME-1 and ANME-2), peripherally related to the order Methanosarcinales, were consistently associated with methane seep marine sediments. The same sediments contained abundant ¹³C-depleted archaeal lipids, indicating that one or both of these archaeal groups are members of anaerobic methane-oxidizing consortia. ¹³C-depleted lipids and the signature 16S rDNAs for these archaeal groups were absent in nearby control sediments. Concurrent surveys of bacterial rDNAs revealed a predominance of δ-proteobacteria, in particular, close relatives of Desulfosarcina variabilis. Biomarker analyses of the same sediments showed bacterial fatty acids with strong ¹³C depletion that are likely products of these sulfate-reducing bacteria. Consistent with these observations, whole-cell fluorescent in situ hybridization revealed aggregations of ANME-2 archaea and sulfate-reducing Desulfosarcina and Desulfococcus species. Additionally, the presence of abundant ¹³C-depleted ether lipids, presumed to be of bacterial origin but unrelated to ether lipids of members of the order Desulfosarcinales, suggests the participation of additional bacterial groups in the methane-oxidizing process. Although the Desulfosarcinales and ANME-2 consortia appear to participate in the anaerobic oxidation of methane in marine sediments, our data suggest that other bacteria and archaea are also involved in methane oxidation in these environments.     

Non-Growth-Associated Demethylation of Dimethylsulfoniopropionate by (Homo)acetogenic Bacteria

The demethylation of the algal osmolyte dimethylsulfoniopropionate (DMSP) to methylthiopropionate (MTPA) by (homo)acetogenic bacteria was studied. Five Eubacterium limosum strains (including the type strain), Sporomusa ovata DSM 2662^T, Sporomusa sphaeroides DSM 2875^T, and Acetobacterium woodii DSM 1030^T were shown to demethylate DMSP stoichiometrically to MTPA. The (homo)acetogenic fermentation based on this demethylation did not result in any significant increase in biomass. The analogous demethylation of glycine betaine to dimethylglycine does support growth of acetogens. In batch cultures of E. limosum PM31 DMSP and glycine betaine were demethylated simultaneously. In mixed substrates experiments with fructose-DMSP or methanol-DMSP, DMSP was used rapidly but only after exhaustion of the fructose or the methanol. In steady-state fructose-limited chemostat cultures (at a dilution rate of 0.03 h⁻¹) with DMSP as a second reservoir substrate, DMSP was biotransformed to MTPA but this did not result in higher biomass values than in cultures without DMSP; cells from such cultures demethylated DMSP at rates of approximately 50 nmol min⁻¹ mg of protein⁻¹, both after growth in the presence of DMSP and after growth in its absence. In cell extracts of glycine betaine-grown strain PM31, DMSP demethylation activities of 21 to 24 nmol min⁻¹ mg of protein⁻¹ were detected with tetrahydrofolate as a methyl acceptor; the activities seen with glycine betaine were approximately 10-fold lower. A speculative explanation for the demethylation of DMSP without an obvious benefit for the organism is that the DMSP-demethylating activity is catalyzed by the glycine betaine-demethylating enzyme and that a transport-related factor, in particular a higher energy demand for DMSP transport across the cytoplasmic membrane than for glycine betaine transport, may reduce the overall ATP yield of the fermentation to virtually zero.     

Methane Production in Shallow-Water, Tropical Marine Sediments

The in situ production of methane was monitored in several types of tropical benthic communities. A bed of Thalassia testudinum located in Caesar Creek (Florida Keys) exhibited the highest methanogenic activity (initial rates = 1.81 to 1.86 mumol CH4/m2 per h) as compared with another seagrass (Syringodium sp., 0.15 to 0.33 mumol/m2 per h) and two coral reef environments (Hydro-Lab, 0.016 to 0.10 mumol/m2 per h; Curacao, 0.14 to 0.47 mumol/m2 per h). The results suggest that a wide variety of benthic metabolic processes (e.g., photosynthetic oxygen production) influences methane production rates.     

Simultaneous Measurements of Organic Carbon Mineralization and Bacterial Production in Oxic and Anoxic Lake Sediments

Based on work in marine sediments it can be hypothesized that (i) overall OM mineralization depends on the enzymatic capacity and is largely independent from the energy yield, (ii) similar oxic and anoxic rates are expected for fresh OM, while oxic rates should be faster for old OM that is partially degraded or adsorbed to particles, and (iii) that the thermodynamic energy yield does not regulate mineralization, but primarily determines the energy fraction allocated to bacterial production (BP). We addressed these hypotheses by simultaneous measurements of mineralization rates (MR) and BP in sediments from a eutrophic lake, along with MR measurements in sediments of a dystrophic lake. Anoxic MR were 44 and 78% of oxic MR in the eutrophic and dystrophic lake, respectively, which was always higher than expected given the theoretical energy yields. The BP:MR ratio was 0.94 and 0.24 in the oxic and anoxic treatments, respectively, in accordance with the expected energy yields. Thus, the results support all three hypotheses above. We also critically discuss BP measurements in sediments and suggest that bacterial growth efficiency values from simultaneous MR and BP measurements can be used to evaluate the reliability of BP estimates.     

Microbial Formation of Ethane in Anoxic Estuarine Sediments

Estuarine sediment slurries produced methane and traces of ethane when incubated under hydrogen. Formation of methane occurred over a broad temperature range with an optimum above 65°C. Ethane formation had a temperature optimum at 40°C. Formation of these two gases was inhibited by air, autoclaving, incubation at 4 and 80°C, and by the methanogenic inhibitor, 2-bromoethanesulfonic acid. Ethane production was stimulated by addition of ethylthioethanesulfonic acid, and production from ethylthioethanesulfonic acid was blocked by 2-bromoethanesulfonic acid. A highly purified enrichment culture of a methanogenic bacterium obtained from sediments produced traces of ethane from ethylthioethanesulfonic acid. These results indicate that the small quantities of ethane found in anaerobic sediments can be formed by certain methanogenic bacteria.     

Formation of Dimethyl Sulfide and Methanethiol in Anoxic Freshwater Sediments

Concentrations of volatile organic sulfur compounds (VOSC) were measured in water and sediment columns of ditches in a minerotrophic peatland in The Netherlands. VOSC, with methanethiol (4 to 40 nM) as the major compound, appeared to be mainly of sediment origin. Both VOSC and hydrogen sulfide concentrations decreased dramatically towards the water surface. High methanethiol and high dimethyl sulfide concentrations in the sediment and just above the sediment surface coincided with high concentrations of hydrogen sulfide (correlation factors, r = 0.91 and r = 0.81, respectively). Production and degradation of VOSC were studied in 32 sediment slurries collected from various freshwater systems in The Netherlands. Maximal endogenous methanethiol production rates of the sediments tested (up to 1.44 (mu)mol per liter of sediment slurry (middot) day(sup-1)) were determined after inhibition of methanogenic and sulfate-reducing populations in order to stop VOSC degradation. These experiments showed that the production and degradation of VOSC in sediments are well balanced. Statistical analysis revealed multiple relationships of methanethiol production rates with the combination of methane production rates (indicative of total anaerobic mineralization) and hydrogen sulfide concentrations (r = 0.90) or with the combination of methane production rates and the sulfate/iron ratios in the sediment (r = 0.82). These findings and the observed stimulation of methanethiol formation in sediment slurry incubations in which the hydrogen sulfide concentrations were artificially increased provided strong evidence that the anaerobic methylation of hydrogen sulfide is the main mechanism for VOSC formation in most freshwater systems. Methoxylated aromatic compounds are likely a major source of methyl groups for this methylation of hydrogen sulfide, since they are important degradation products of the abundant biopolymer lignin. Increased sulfate concentrations in several freshwater ecosystems caused by the inflow of water from the river Rhine into these systems result in higher hydrogen sulfide concentrations. As a consequence, higher fluxes of VOSC towards the atmosphere are conceivable.     

Dimethylsulfoniopropionate and Methanethiol Are Important Precursors of Methionine and Protein-Sulfur in Marine Bacterioplankton

Organic sulfur compounds are present in all aquatic systems, but their use as sources of sulfur for bacteria is generally not considered important because of the high sulfate concentrations in natural waters. This study investigated whether dimethylsulfoniopropionate (DMSP), an algal osmolyte that is abundant and rapidly cycled in seawater, is used as a source of sulfur by bacterioplankton. Natural populations of bacterioplankton from subtropical and temperate marine waters rapidly incorporated 15 to 40% of the sulfur from tracer-level additions of [³⁵S]DMSP into a macromolecule fraction. Tests with proteinase K and chloramphenicol showed that the sulfur from DMSP was incorporated into proteins, and analysis of protein hydrolysis products by high-pressure liquid chromatography showed that methionine was the major labeled amino acid produced from [³⁵S]DMSP. Bacterial strains isolated from coastal seawater and belonging to the α-subdivision of the division Proteobacteria incorporated DMSP sulfur into protein only if they were capable of degrading DMSP to methanethiol (MeSH), whereas MeSH was rapidly incorporated into macromolecules by all tested strains and by natural bacterioplankton. These findings indicate that the demethylation/demethiolation pathway of DMSP degradation is important for sulfur assimilation and that MeSH is a key intermediate in the pathway leading to protein sulfur. Incorporation of sulfur from DMSP and MeSH by natural populations was inhibited by nanomolar levels of other reduced sulfur compounds including sulfide, methionine, homocysteine, cysteine, and cystathionine. In addition, propargylglycine and vinylglycine were potent inhibitors of incorporation of sulfur from DMSP and MeSH, suggesting involvement of the enzyme cystathionine γ-synthetase in sulfur assimilation by natural populations. Experiments with [methyl-³H]MeSH and [³⁵S]MeSH showed that the entire methiol group of MeSH was efficiently incorporated into methionine, a reaction consistent with activity of cystathionine γ-synthetase. Field data from the Gulf of Mexico indicated that natural turnover of DMSP supplied a major fraction of the sulfur required for bacterial growth in surface waters. Our study highlights a remarkable adaptation by marine bacteria: they exploit nanomolar levels of reduced sulfur in apparent preference to sulfate, which is present at 10⁶- to 10⁷-fold higher concentrations.     

Tube-dwelling Meiofauna in Marine Sediments

Tube-dwelling has been recognized previously as a life-style for several meiobenthic species, but behavioural observation of living specimens has rarely been reported. The extent to which tube-building and tube-dwelling occurs within meiofauna, and how they have influenced evolutionary and ecological processes as well as morphology within these organisms, is relatively unknown but potentially of great significance. In addition to direct observation of tube-building and the occurence of tubes in natural habitats, the internal anatomy associated with tube-building in two nematode species (Ptycholaimellus jacobi, P. ponticus) and one harpacticoid copepod species (Stenhelia palustris) is the focus of this study. Special attention is given to the secretory products, glands, and their association with secretory pores. Also, the role of meiobenthic tube-dwelling activities in relationship to their environment is extensive discussed.     

Sources of Lipids in Anoxic Lacustrine Sediments Using Stable Carbon Isotopes

The origin of organic matter in recent anoxic sediments of the alpine Lake Bled (NW Slovenia) was determined by analyzing the carbon isotope composition of lipid biomarkers, i.e. alkanes, alcohols, sterols and fatty acids, busing compound specific, carbon isotope analysis. The results indicate that, although biomarker analysis indicated mostly plankton and terrestrial sources for lipids, an important part of sedimentary lipids, especially sterols, are autochthonous, of anaerobic microbial (methanotrophic) origin. Marked differences were observed in δ¹³C values of lipid biomarkers in settling particles collected 2 m above the bottom, and in δ¹³C values determined in surface sediment. These results indicate that even some compounds found in both particulate organic matter and sediments are the same in terms of chemical structures, their sources can be different and thus, isotopic composition should be used as a complementary tool for source identification.     

Purification and Characterization of Dimethylsulfoniopropionate Lyase from an Alcaligenes-Like Dimethyl Sulfide-Producing Marine Isolate

Dimethyl sulfide (DMS) is quantitatively the most important biogenic sulfur compound emitted from oceans and salt marshes. It is formed primarily by the action of dimethylsulfoniopropionate (DMSP) lyase which cleaves DMSP, an algal osmolyte, to equimolar amounts of DMS and acrylate. This report is the first to describe the isolation and purification of DMSP lyase. The soluble enzyme was purified to electrophoretic homogeneity from a facultatively anaerobic gram-negative rod-shaped marine bacterium identified as an Alcaligenes species by the Vitek gram-negative identification method. The key to successful purification of the enzyme was its binding to, and hydrophobic chromatography on, a phenyl-Sepharose CL-4B column. DMSP lyase biosynthesis was induced by its substrate, DMSP; its product, acrylate; and also by acrylamide. The relative effectivenesses of the inducers were 100, 90, and 204%, respectively. DMSP lyase is a 48-kDa monomer with a Michaelis-Menten constant (K(infm)) for DMSP of 1.4 mM and a V(infmax) of 408 (mu)mol/min/mg of protein. It converted DMSP to DMS and acrylate stoichiometrically. The similar K(infm) values measured for pure DMSP lyase and the axenic culture, seawater, and surface marsh sediment suggest that the microbes in these ecosystems must have enzymes similar to the one purified from our marine isolate. Anoxic sediment populations, however, have a 40-fold-lower K(infm) for this enzyme (30 (mu)M), possibly giving them the capability to metabolize much lower levels of DMSP than the aerobes.     

Method for Measuring Rates of NH4+ Turnover in Anoxic Marine Sediments, Using a 15N-NH4+ Dilution Technique

A method is described for the determination of the net and total rates of NH₄⁺ production and NH₄⁺ incorporation at different depths in an anoxic marine sediment. ¹⁵N-NH₄⁺ was added to the sediment NH₄⁺ pool, and the ¹⁵N content was assayed after 0, 2, and 5 days of incubation. The pool size changed during incubation; this change in pool size is incorporated into a model which predicts the dynamics of ¹⁵N-NH₄⁺ dilution. A simple microdiffusion of NH₃ was followed by an emission spectrometry analysis of ¹⁵N content. This procedure avoided all problems of cross-contamination. The model was tested and rates were measured in four sediment cores, at seven different depths. The high correlation coefficients (mean, 0.96 for the 0- to 2-, 2- to 4-, 4- to 6-, and 6- to 8-cm sediment fractions) indicated that the model was correct and that the measured rates were valid. The immediate distribution of ¹⁵N-NH₄⁺ between interstitial and exchangeable NH₄⁺ pools indicated that it was the combined pool that was turning over. In the 0- to 2-cm fraction at 17°C the net rate of NH₄⁺ production was 274 (standard deviation, 31) nmol cm⁻³ day⁻¹, and the mean total rate of NH₄⁺ production was 309 (standard deviation, 39) nmol cm⁻³ day⁻¹; both rates decreased to <1% of these values in the 12- to 14-cm fractions.     

Anaerobic versus Aerobic Degradation of Dimethyl Sulfide and Methanethiol in Anoxic Freshwater Sediments

Degradation of dimethyl sulfide and methanethiol in slurries prepared from sediments of minerotrophic peatland ditches were studied under various conditions. Maximal aerobic dimethyl sulfide-degrading capacities (4.95 nmol per ml of sediment slurry · h⁻¹), measured in bottles shaken under an air atmosphere, were 10-fold higher than the maximal anaerobic degrading capacities determined from bottles shaken under N₂ or H₂ atmosphere (0.37 and 0.32 nmol per ml of sediment slurry · h⁻¹, respectively). Incubations under experimental conditions which mimic the in situ conditions (i.e., not shaken and with an air headspace), however, revealed that aerobic degradation of dimethyl sulfide and methanethiol in freshwater sediments is low due to oxygen limitation. Inhibition studies with bromoethanesulfonic acid and sodium tungstate demonstrated that the degradation of dimethyl sulfide and methanethiol in these incubations originated mainly from methanogenic activity. Prolonged incubation under a H₂ atmosphere resulted in lower dimethyl sulfide degradation rates. Kinetic analysis of the data resulted in apparent K_m values (6 to 8 μM) for aerobic dimethyl sulfide degradation which are comparable to those reported for Thiobacillus spp., Hyphomicrobium spp., and other methylotrophs. Apparent K_m values determined for anaerobic degradation of dimethyl sulfide (3 to 8 μM) were of the same order of magnitude. The low apparent K_m values obtained explain the low dimethyl sulfide and methanethiol concentrations in freshwater sediments that we reported previously. Our observations point to methanogenesis as the major mechanism of dimethyl sulfide and methanethiol consumption in freshwater sediments.     

Dimethyl Sulfide Production from Dimethylsulfoniopropionate in Coastal Seawater Samples and Bacterial Cultures

Dimethyl sulfide (DMS) was produced immediately after the addition of 0.1 to 2 μM β-dimethylsulfonio-propionate (DMSP) to coastal seawater samples. Azide had little effect on the initial rate of DMS production from 0.5 μM added DMSP, but decreased the rate of production after 6 h. Filtration of water samples through membrane filters (pore size, 0.2 μm) greatly reduced DMS production for approximately 10 h, after which time DMS production resumed at a high rate. Autoclaving completely eliminated the production of DMS. The antibiotics chloramphenicol, tetracycline, kanamycin, and vancomycin all had little effect on the accumulation of DMS over the first few hours of incubation, but produced significant inhibition thereafter. The effects of individual antibiotics were additive. Chloroform over a range of concentrations (0.25 to 1.25 mM) had no effects on DMS production. Similarly, organic amendments, including acrylate, glucose, protein, and starch, did not affect DMS accumulation from DMSP. Acrylate, a product of the enzymatic cleavage of DMSP, was metabolized in seawater samples, and two strains of bacteria were isolated with this compound as the growth substrate. These bacteria produced DMS from DMSP. The sensitivity to inhibitors with respect to growth and DMSP-lyase activity varied from strain to strain. These results illustrate the significant potential for microbial conversion of dissolved DMSP to DMS in coastal seawater.     

Production of N2 through Anaerobic Ammonium Oxidation Coupled to Nitrate Reduction in Marine Sediments

In the global nitrogen cycle, bacterial denitrification is recognized as the only quantitatively important process that converts fixed nitrogen to atmospheric nitrogen gas, N₂, thereby influencing many aspects of ecosystem function and global biogeochemistry. However, we have found that a process novel to the marine nitrogen cycle, anaerobic oxidation of ammonium coupled to nitrate reduction, contributes substantially to N₂ production in marine sediments. Incubations with ¹⁵N-labeled nitrate or ammonium demonstrated that during this process, N₂ is formed through one-to-one pairing of nitrogen from nitrate and ammonium, which clearly separates the process from denitrification. Nitrite, which accumulated transiently, was likely the oxidant for ammonium, and the process is thus similar to the anammox process known from wastewater bioreactors. Anaerobic ammonium oxidation accounted for 24 and 67% of the total N₂ production at two typical continental shelf sites, whereas it was detectable but insignificant relative to denitrification in a eutrophic coastal bay. However, rates of anaerobic ammonium oxidation were higher in the coastal sediment than at the deepest site and the variability in the relative contribution to N₂ production between sites was related to large differences in rates of denitrification. Thus, the relative importance of anaerobic ammonium oxidation and denitrification in N₂ production appears to be regulated by the availability of their reduced substrates. By shunting nitrogen directly from ammonium to N₂, anaerobic ammonium oxidation promotes the removal of fixed nitrogen in the oceans. The process can explain ammonium deficiencies in anoxic waters and sediments, and it may contribute significantly to oceanic nitrogen budgets.