Demethylation of Dimethylsulfoniopropionate and Production of Thiols in Anoxic Marine Sediments

Dimethylsulfoniopropionate (DMSP) is a natural product of algae and aquatic plants, particularly those from saline environments. We investigated whether DMSP could serve as a precursor of thiols in anoxic coastal marine sediments. The addition of 10 or 60 μM DMSP to anoxic sediment slurries caused the concentrations of 3-mercaptopropionate (3-MPA) and methanethiol (MSH) to increase. Antibiotics prevented the appearance of these thiols, indicating biological formation. Dimethyl sulfide (DMS) and acrylate also accumulated after the addition of DMSP, but these compounds were rapidly metabolized by microbes and did not reach high levels. Acrylate and DMS were probably generated by the enzymatic cleavage of DMSP. MSH arose from the microbial metabolism of DMS, since the direct addition of DMS greatly increased MSH production. Additions of 3-methiolpropionate gave rise to 3-MPA at rates similar to those with DMSP, suggesting that sequential demethylation of DMSP leads to 3-MPA formation. Only small amounts of MSH were liberated from 3-methiolpropionate, indicating that demethiolation was not a major transformation for 3-methiolpropionate. We conclude that DMSP was degraded in anoxic sediments by two different pathways. One involved the well-known enzymatic cleavage to acrylate and DMS, with DMS subsequently serving as a precursor of MSH. In the other pathway, successive demethylations of the sulfur atom proceeded via 3-methiolpropionate to 3-MPA.     

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.     

Factors Controlling Anaerobic Ammonium Oxidation with Nitrite in Marine Sediments

Factors controlling the anaerobic oxidation of ammonium with nitrate and nitrite were explored in a marine sediment from the Skagerrak in the Baltic-North Sea transition. In anoxic incubations with the addition of nitrite, approximately 65% of the nitrogen gas formation was due to anaerobic ammonium oxidation with nitrite, with the remainder being produced by denitrification. Anaerobic ammonium oxidation with nitrite exhibited a biological temperature response, with a rate optimum at 15°C and a maximum temperature of 37°C. The biological nature of the process and a 1:1 stoichiometry for the reaction between nitrite and ammonium indicated that the transformations might be attributed to the anammox process. Attempts to find other anaerobic ammonium-oxidizing processes in this sediment failed. The apparent K_m of nitrite consumption was less than 3 μM, and the relative importance of ammonium oxidation with nitrite and denitrification for the production of nitrogen gas was independent of nitrite concentration. Thus, the quantitative importance of ammonium oxidation with nitrite in the jar incubations at elevated nitrite concentrations probably represents the in situ situation. With the addition of nitrate, the production of nitrite from nitrate was four times faster than its consumption and therefore did not limit the rate of ammonium oxidation. Accordingly, the rate of this process was the same whether nitrate or nitrite was added as electron acceptor. The addition of organic matter did not stimulate denitrification, possibly because it was outcompeted by manganese reduction or because transport limitation was removed due to homogenization of the sediment.     

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.     

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.     

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.     

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.     

Evidence for the Existence of Psychrophilic Methanogenic Communities in Anoxic Sediments of Deep Lakes

In order to obtain evidence for the existence of psychrophilic methanogenic communities in sediments of deep lakes that are low-temperature environments (4 to 5°C), slurries were first incubated at temperatures between 4 and 60°C for several weeks, at which time they were amended, or not, with an additional substrate, such as cellulose, butyrate, propionate, acetate, or hydrogen, and further incubated at 6°C. Initial methane production rates were highest in slurries preincubated at temperatures between 4 and 15°C, with maximal rates in slurries kept at 6°C. Hydrogen-amended cultures were the only exceptions, with the highest methane production rates at 6°C after preincubation at 30°C.     

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.     

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.     

Method for Measuring Rates of NH(4) Turnover in Anoxic Marine Sediments, Using a N-NH(4) Dilution Technique

A method is described for the determination of the net and total rates of NH(4) production and NH(4) incorporation at different depths in an anoxic marine sediment. N-NH(4) was added to the sediment NH(4) pool, and the N 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 N-NH(4) dilution. A simple microdiffusion of NH(3) was followed by an emission spectrometry analysis of N 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 N-NH(4) between interstitial and exchangeable NH(4) pools indicated that it was the combined pool that was turning over. In the 0- to 2-cm fraction at 17 degrees C the net rate of NH(4) production was 274 (standard deviation, 31) nmol cm day, and the mean total rate of NH(4) production was 309 (standard deviation, 39) nmol cm day; both rates decreased to <1% of these values in the 12- to 14-cm fractions.     

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.     

Reduction of Elemental Selenium to Selenide: Experiments with Anoxic Sediments and Bacteria that Respire Se-Oxyanions

A selenite-respiring bacterium, Bacillus selenitireducens, produced significant levels of Se(-II) (as aqueous HSe^?) when supplied with Se(0). B. selenitireducens was also able to reduce selenite [Se(IV)] through Se(0) to Se(-II). Reduction of Se(0) by B. selenitireducens was more rapid in cells grown on colloidal sulfur [S(0)] or Se(IV) as their electron acceptor than for cell lines grown on fumarate. In contrast, three cultures of selenate-respiring bacteria, Sulfurospirillum barnesii, B. arsenicoselenatis, and Selenihalanaerobacter shriftii either were unable to reduce Se(0) to Se(-II) or had only a very limited capacity to achieve this reduction. Biological reduction of Se(0) to Se(-II) was observed during incubation of estuarine sediment slurries, while no such activity was noted in formalin-killed controls. The majority of the Se(-II) produced was found in the sediments as a solid precipitate of FeSe, rather than in solution as HSe^?. These results demonstrate that certain anaerobic bacteria have the capacity to reduce Se(0) to Se(-II), providing a possible biological explanation for the occurrence of the selenide species in some sedimentary rocks.     

Evidence for Production of Sexual Resting Cysts by the Toxic Dinoflagellate Karenia mikimotoi in Clonal Cultures and Marine Sediments

The toxic dinoflagellate Karenia mikimotoi has been well-known for causing large-scale and dense harmful algal blooms (HABs) in coastal waters worldwide and serious economic loss in aquaculture and fisheries and other adverse effects on marine ecosystems. Whether K. mikimotoi forms resting cysts has been a puzzling issue regarding to the mechanisms of bloom initiation and geographic expansion of this species. We provide morphological and molecular confirmation of sexually produced thin-walled resting cysts by K. mikimotoi based on observations of laboratory cultures and their direct detection in marine sediments. Light and scanning electron microscopy evidences for sexual reproduction include attraction and pairing of gametes, gamete fusion, formation of planozygote and thin-walled cyst, and the documentation of the thin-walled cyst germination processes. Evidence for cysts in marine sediments was in three aspects: positive PCR detection of cysts using species-specific primers in the DNA extracted from whole sediments; fluorescence in situ hybridization detection of cysts using FISH probes; and single-cell PCR sequencing for cysts positively labeled with FISH probes. The existence of sexually produced, thin-walled resting cysts by K. mikimotoi provides a possible mechanism accounting for the initiation of annually recurring blooms at certain regions and global expansion of the species during the past decades.     

Organic Carbon Degradation in Anoxic Organic-Rich Shelf Sediments: Biogeochemical Rates and Microbial Abundance

Identifying and explaining bottlenecks in organic carbon mineralization and the persistence of organic matter in marine sediments remain challenging. This study aims to illuminate the process of carbon flow between microorganisms involved in the sedimentary microbial food chain in anoxic, organic-rich sediments of the central Namibian upwelling system, using biogeochemical rate measurements and abundances of Bacteroidetes, Gammaproteobacteria, and sulfate-reducing bacteria at two sampling stations. Sulfate reduction rates decreased by three orders of magnitude in the top 20 cm at one sampling station (280 nmol cm^?3 d^?1 – 0.1 nmol cm^?3 d^?1) and by a factor of 7 at the second station (65 nmol cm^?3 d^?1 – 9.6 nmol cm^?3 d^?1). However, rates of enzymatic hydrolysis decreased by less than a factor of three at both sampling stations for the polysaccharides laminarin (23 nmol cm^?3 d^?1– 8 nmol cm^?3 d^?1 and 22 nmol cm^?3 d^?1– 10 nmol cm^?3 d^?1) and pullulan (11 nmol cm^?3 d^?1– 4 nmol cm^?3 d^?1 and 8 nmol cm^?3 d^?1– 6 nmol cm^?3 d^?1). Increasing imbalance between carbon turnover by hydrolysis and terminal oxidation with depth, the steep decrease in cell specific activity of sulfate reducing bacteria with depth, low concentrations of volatile fatty acids (less than 15 μM), and persistence of dissolved organic carbon, suggest decreasing bioavailability and substrate limitation with depth.     

Association of Bacteria with Marine Invertebrates: Implications for Ballast Water Management

Bacteria associated with plankton are of importance in marine bioinvasions and the implementation of ship’s ballast water treatment technologies. In this study, epibiotic and endobiotic bacteria associated with zooplankton, including barnacle nauplii, veliger larvae, and adults of the copepod Oithona sp., were characterized and quantified. Barnacle nauplius and veliger larva harbored ~4.4 × 10⁵ cells ind^?1 whereas Oithona sp. had 8.8 × 10⁵ cells ind^?1. Computation of bacterial contribution based on biovolume indicated that despite being the smallest zooplankton tested, veliger larvae harbored the highest number of bacteria, while barnacle nauplii, the largest of the zooplankton, tested in terms of volume contributed the least. Pulverization of zooplankton led to an increase in bacterial numbers; for example, Vibrio cholerae, which was initially 3.5 × 10³, increased to 5.4 × 10⁵ CFU g^?1; Escherichia coli increased from 5.0 × 10² to 1.3 × 10⁴ CFU g^?1; and Streptococcus faecalis increased from 2.1 × 10² to 2.5 × 10⁵ CFU g^?1, respectively. Pulverized zooplankton was aged in the dark to assess the contribution of bacteria from decaying debris. Aging of pulverized zooplankton led to emergence of Chromobacterium violaceum, which is an opportunistic pathogen in animals and humans.