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.     

Temperature Dependence of Methane Production in Tropical Rice Soils

Although CH 4 production is sensitive to temperature, it is not clear how temperature controls CH 4 production directly versus the production of organic substrates that methanogens convert into CH 4 . Therefore, this study was done to better understand how CH 4 production in rice paddy soil responded to temperature when the process was not limited by the availability of substrates. In a laboratory-incubation study using three Indian rice soils under flooded conditions, the effect of temperature on CH 4 production was examined. CH 4 production in acid sulphate, laterite, and alluvial soil samples under flooded conditions distinctly increased with increase in temperature from 15°C to 35°C. Laterite and acid sulphate soils produced distinctly less CH 4 than alluvial soils. CO 2 production increased with increase in temperature in all the soils. The readily mineralizable carbon C and Fe 2+ contents in soils were least at 15°C and highest at 35°C, irrespective of soil type. Likewise, a significant correlation existed between microbial population (methanogens and sulphate reducers) and CH 4 production. Comparing the temperature coefficients ( Q 10 ) for methane production within each soil type at low (15°C-25°C) and medium (25°C-35°C) temperature intervals revealed that these values were not uniform for both alluvial and laterite soils. But acid sulphate soil had Q 10 values that were near 2 at both temperature intervals. When these soil samples were amended with substrates (acetate, H 2 -CO 2 , and rice straw), there were stimulatory effects on methane production rates and consequently on the Q 10 values. The pattern of temperature coefficients was characteristic of the soil type and the nature of substrates used for amendment.     

Microbial Communities from Methane Hydrate-Bearing Deep Marine Sediments in a Forearc Basin

Microbial communities in cores obtained from methane hydrate-bearing deep marine sediments (down to more than 300 m below the seafloor) in the forearc basin of the Nankai Trough near Japan were characterized with cultivation-dependent and -independent techniques. Acridine orange direct count data indicated that cell numbers generally decreased with sediment depth. Lipid biomarker analyses indicated the presence of viable biomass at concentrations greater than previously reported for terrestrial subsurface environments at similar depths. Archaeal lipids were more abundant than bacterial lipids. Methane was produced from both acetate and hydrogen in enrichments inoculated with sediment from all depths evaluated, at both 10 and 35°C. Characterization of 16S rRNA genes amplified from the sediments indicated that archaeal clones could be discretely grouped within the Euryarchaeota and Crenarchaeota domains. The bacterial clones exhibited greater overall diversity than the archaeal clones, with sequences related to the Bacteroidetes, Planctomycetes, Actinobacteria, Proteobacteria, and green nonsulfur groups. The majority of the bacterial clones were either members of a novel lineage or most closely related to uncultured clones. The results of these analyses suggest that the microbial community in this environment is distinct from those in previously characterized methane hydrate-bearing sediments.     

Substrates for Sulfate Reduction and Methane Production in Intertidal Sediments

The activity of and potential substrates for methane-producing bacteria and sulfate-reducing bacteria were examined in marsh, estuary, and beach intertidal sediments. Slow rates of methane production were detected in all sediments, although rates of sulfate reduction were 100- to 1,000-fold higher. After sulfate was depleted in sediments, the rates of methane production sharply increased. The addition of methylamine stimulated methanogenesis in the presence of sulfate, and [¹⁴C]methylamine was rapidly converted to ¹⁴CH₄ and ¹⁴CO₂ in freshly collected marsh sediment. Acetate, hydrogen, or methionine additions did not stimulate methanogenesis. [methyl-¹⁴C]methionine and [2-¹⁴C]acetate were converted to ¹⁴CO₂ and not to ¹⁴CH₄ in fresh sediment. No reduction of ¹⁴CO₂ to ¹⁴CH₄ occurred in fresh sediment. Molybdate, an inhibitor of sulfate reduction, inhibited [2-¹⁴C]acetate metabolism by 98.5%. Fluoracetate, an inhibitor of acetate metabolism, inhibited sulfate reduction by 61%. These results suggest that acetate is a major electron donor for sulfate reduction in marine sediments. In the presence of high concentrations of sulfate, methane may be derived from novel substrates such as methylamine.     

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.     

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.     

Temporal Variations of Microbial Activity and Diversity in Marine Tropical Sediments (New Caledonia Lagoon)

Temporal variations of oxygen consumption, sensitivity to metal spiking, and microbial diversity were investigated during a one-year survey at the sediment–water interface in the tropical lagoon of New Caledonia. Sediment oxygen consumption (SOC) exhibited strong variations with time with maximum rates during February (Austral summer) and minimum values during July (cold period). SOC was strongly positively correlated with temperature, with an apparent activation energy (E _a) of 41 kJ mol⁻¹, corresponding to an apparent Q ₁₀(20–30 °C) of 1.75. Strong short-term variations of SOC were also observed with ratios between two consecutive samplings reaching up to twofold of magnitude within one week, whereas the maximum/minimum ratio over the whole year was equal to 2.73. In most cases, metal spiking led to a strong decrease of SOC; however, in a third of sampling dates, spiking did not significantly decrease activity. These periods of apparent metal tolerance were not characterized by a particular bacterial community structure. Bacterial community structure estimated from terminal restriction fragment length polymorphism (T-RFLP) analysis exhibited strong variations over the one-year survey, and no seasonality was observed for bacterial richness. However, on average, the Whittaker similarity index between two consecutive T-RFLP profiles was above 60% suggesting a relative stability of the bacterial community structure on the short timescale with prominent T-RFs representing on average more than 67% of relative abundance occurring over most of the year, whereas other T-RFs only occurred during some periods.     

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.     

Molecular Identification and Comparative Evaluation of Tropical Marine Microalgae for Biodiesel Production

Marine microalgae have emerged as important feedstock for liquid biofuel production. The identification of lipid-rich native microalgal species with high growth rate and optimal fatty acid profile and biodiesel properties is the most challenging step in microalgae-based biodiesel production. In this study, attempts have been made to bio-prospect the biodiesel production potential of marine and brackish water microalgal isolates from the west coast of India. A total of 14 microalgal species were isolated, identified using specific molecular markers and based on the lipid content; seven species with total lipid content above 20% of dry cell weight were selected for assessing biodiesel production potential in terms of lipid and biomass productivities, nile red fluorescence, fatty acid profile and biodiesel properties. On comparative analysis, the diatoms were proven to be promising based on the overall desirable properties for biodiesel production. The most potential strain Navicula phyllepta MACC8 with a total lipid content of 26.54 % of dry weight of biomass, the highest growth rate (0.58 day^?1) and lipid and biomass productivities of 114 and 431 mgL^?1 day^?1, respectively, was rich in fatty acids mainly of C16:0, C16:1 and C18:0 in the neutral lipid fraction, the most favoured fatty acids for ideal biodiesel properties. The biodiesel properties met the requirements of fuel quality standards based on empirical estimation. The marine diatoms hold a great promise as feedstock for large-scale biodiesel production along with valuable by-products in a biorefinery perspective, after augmenting lipid and biomass production through biochemical and genetic engineering approaches.     

Cell Turnover and Detritus Production in Marine Sponges from Tropical and Temperate Benthic Ecosystems

This study describes in vivo cell turnover (the balance between cell proliferation and cell loss) in eight marine sponge species from tropical coral reef, mangrove and temperate Mediterranean reef ecosystems. Cell proliferation was determined through the incorporation of 5-bromo-2′-deoxyuridine (BrdU) and measuring the percentage of BrdU-positive cells after 6 h of continuous labeling (10 h for Chondrosia reniformis). Apoptosis was identified using an antibody against active caspase-3. Cell loss through shedding was studied quantitatively by collecting and weighing sponge-expelled detritus and qualitatively by light microscopy of sponge tissue and detritus. All species investigated displayed substantial cell proliferation, predominantly in the choanoderm, but also in the mesohyl. The majority of coral reef species (five) showed between 16.1±15.9% and 19.0±2.0% choanocyte proliferation (mean±SD) after 6 h and the Mediterranean species, C. reniformis, showed 16.6±3.2% after 10 h BrdU-labeling. Monanchora arbuscula showed lower choanocyte proliferation (8.1±3.7%), whereas the mangrove species Mycale microsigmatosa showed relatively higher levels of choanocyte proliferation (70.5±6.6%). Choanocyte proliferation in Haliclona vansoesti was variable (2.8–73.1%). Apoptosis was negligible and not the primary mechanism of cell loss involved in cell turnover. All species investigated produced significant amounts of detritus (2.5–18% detritus bodyweight⁻¹·d⁻¹) and cell shedding was observed in seven out of eight species. The amount of shed cells observed in histological sections may be related to differences in residence time of detritus within canals. Detritus production could not be directly linked to cell shedding due to the degraded nature of expelled cellular debris. We have demonstrated that under steady-state conditions, cell turnover through cell proliferation and cell shedding are common processes to maintain tissue homeostasis in a variety of sponge species from different ecosystems. Cell turnover is hypothesized to be the main underlying mechanism producing sponge-derived detritus, a major trophic resource transferred through sponges in benthic ecosystems, such as coral reefs.     

Methane, Carbon Dioxide, and Hydrogen Sulfide Production from the Terminal Methiol Group of Methionine by Anaerobic Lake Sediments

A significant portion of the sulfide in lake sediments may be derived from sulfur-containing amino acids. Methionine degradation in Lake Mendota (Wisconsin) sediments was studied with gas chromatographic and radiotracer techniques. Temperature optimum and inhibitor studies showed that this process was biological. Methane thiol and dimethyl sulfide were produced in sediments when 1-μmol/ml unlabeled methionine was added. When chloroform (an inhibitor of one-carbon metabolism) was added to the sediments, methane thiol, carbon disulfide, and n-propane thiol were produced, even when no methionine was added. When ³⁵S-labeled methionine was added to the sediments in tracer quantities (1.75 nmol/ml), labeled hydrogen sulfide was produced, and a roughly equal amount of label was incorporated into insoluble material. Methane and carbon dioxide were produced from [methyl-¹⁴C]methionine. Evidence is given favoring methane thiol as an intermediate in the formation of methane, carbon dioxide, and hydrogen sulfide from the terminal methiol group of methionine. Methionine may be an important source of sulfide in lake sediments.     

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.     

Determination of Virus Abundance in Marine Sediments

In this study, we optimized procedures to enumerate viruses from marine sediments by epifluorescence microscopy using SYBR Green I as a stain. The highest virus yields from the bulk of the sediments were obtained by utilizing pyrophosphate and 3 min of sonication. The efficiency of extraction benthic viruses by pyrophosphate-ultrasound treatment was about 60% of the extractable virus particles. Samples treated with nucleases had increased virus counts, suggesting a masking effect of extracellular DNA. No significant differences were observed between virus counts obtained by epifluorescence microscopy and transmission electron microscopy. Both formaldehyde and glutaraldehyde gave significant reductions of virus counts after only 24 h of sediment storage, but no further loss occurred after 7 days.     

Prokaryotic Diversity in Zostera noltii-Colonized Marine Sediments

The diversity of microorganisms present in a sediment colonized by the phanerogam Zostera noltii has been analyzed. Microbial DNA was extracted and used for constructing two 16S rDNA clone libraries for Bacteria and Archaea. Bacterial diversity was very high in these samples, since 57 different sequences were found among the 60 clones analyzed. Eight major lineages of the Domain Bacteria were represented in the library. The most frequently retrieved bacterial group (36% of the clones) was δ-Proteobacteria related to sulfate-reducing bacteria. The second most abundant group (27%) was γ-Proteobacteria, including five clones closely related to S-oxidizing endosymbionts. The archaeal clone library included members of Crenarchaeota and Euryarchaeota, with nine different sequences among the 15 analyzed clones, indicating less diversity when compared to the Bacteria organisms. None of these sequences was closely related to cultured Archaea organisms.     

Biodegradation of Pyrene in Marine Beach Sediments

The potential of chitosan (0.1% dry weight equivalent) as a bioremediation additive for removal of the recalcitrant polycyclic aromatic hydrocarbon (PAH) pyrene in marine beach sediments was investigated using an open irrigation system over a 63-day period. Osmocote, a slow release fertilizer, was used as the key nutrient supplement at a concentration of 1% in sediment (dry weight equivalent). Osmocote significantly (p < .05) enhanced nutrient levels, and the metabolic activity of the indigenous microbial biomass. Both additives were comparable in stimulating pyrene biodegradation rates; with chitosan (0.062 day^?1) being slightly more effective as an amendment than Osmocote (0.051 day^?1). Loss of pyrene in a control sediment (i.e., pyrene, without additives) was 66.6% over a 63-day period. The concurrent application of additives yielded the greatest biodegradation rates (0.072day^?1), resulting in a 98.2% loss of pyrene over 63 days. The treatment of oil contaminated beach sediments with both osmocote (1%) and chitosan (0.1%) is therefore recommended as an effective treatment for the intrinsic biodegradation of recalcitrant PAHs in oil-contaminated beach sediments.     

Interactions between Methane Oxidation and Nitrification in Coastal Sediments

Galveston Bay sediments exhibit substantial spatial and seasonal variability in rates of nitrification and aerobic methane oxidation. We examined the biogeochemical and microbiological controls on these processes using aerobic enrichment slurries. Potential aerobic methane and ammonia oxidation rates from unamended control slurries were compared to rates in slurries amended with methane, ammonium, or methane + ammonium. Bacterial community composition was monitored using denaturing gradient gel electrophoresis (DGGE) analysis of PCR amplified ribosomal and functional gene DNA. Potential methane and ammonia oxidation rates increased over time in sediments amended with methane and ammonium, respectively. The highest potential methane oxidation rates occurred in treatments receiving both ammonium and methane suggesting that methanotrophs in the enrichment cultures were nitrogen limited. The highest ammonia oxidation rates occurred in treatments amended with ammonium only. Treatments receiving both ammonium and methane exhibited ammonia oxidation rates and porewater ammonium concentrations similar to those measured in the unamended control suggesting that methanotrophs may have inhibited ammonia oxidation by sequestering available ammonia. Sequence analysis revealed a decrease in general bacterial community diversity over time and a shift in ammonia-oxidizing bacterial composition corresponding with methane availability. However, methanotroph community composition similarities between treatments with different relative methane oxidation rates suggest that changes in physiological activity, as well as shifts in community composition, contributed to the observed patterns in potential rates.     

Characterization of beta-Glucosidase Activity in Intertidal Marine Sediments

Glycoside derivatives of 4-methylumbelliferone (MUF) were used to characterize the polysaccharidase enzyme systems present in sediments from an intertidal mud flat. The formation of highly fluorescent MUF on hydrolysis of the various glycosides was determined at low substrate concentrations (<1 muM) and with short incubation periods (>5 min). The hydrolysis of MUF-beta-d-glucose in sediments from depth intervals of 0 to 2 cm was insensitive to the presence of oxygen, dissolved sulfide, and iron; magnesium and calcium were stimulatory, however. A pronounced temperature optimum was observed at 40 degrees C, a salinity optimum at 30 per thousand, and a pH optimum at 8.5. Rates of hydrolysis were completely inhibited by the addition of mercuric chloride and sodium azide, but only partially inhibited by toluene and the specific beta-glucosidase inhibitor delta-1,5-gluconolactone. The response to delta-1,5-gluconolactone suggested that about 50% of the observed hydrolysis of MUF-beta-d-glucoside was due to exo- and endoglucanases. A wide variety of hydrolytic activities was observed, with at least some nonspecificity occurring in the case of MUF-beta-d-fucoside. Depth profiles indicated maximal activity in surface sediments with a rapid decline below 2 cm. MUF-glycosides provided a convenlent tool for initial analyses of the dynamics and controls of polymer hydrolysis in marine sediments.     

Competitive Mechanisms for Inhibition of Sulfate Reduction and Methane Production in the Zone of Ferric Iron Reduction in Sediments

Mechanisms for inhibition of sulfate reduction and methane production in the zone of Fe(III) reduction in sediments were investigated. Addition of amorphic iron(III) oxyhydroxide to sediments in which sulfate reduction was the predominant terminal electron-accepting process inhibited sulfate reduction 86 to 100%. The decrease in electron flow to sulfate reduction was accompanied by a corresponding increase in electron flow to Fe(III) reduction. In a similar manner, Fe(III) additions also inhibited methane production in sulfate-depleted sediments. The inhibition of sulfate reduction and methane production was the result of substrate limitation, because the sediments retained the potential for sulfate reduction and methane production in the presence of excess hydrogen and acetate. Sediments in which Fe(III) reduction was the predominant terminal electron-accepting process had much lower concentrations of hydrogen and acetate than sediments in which sulfate reduction or methane production was the predominant terminal process. The low concentrations of hydrogen and acetate in the Fe(III)-reducing sediments were the result of metabolism by Fe(III)-reducing organisms of hydrogen and acetate at concentrations lower than sulfate reducers or methanogens could metabolize them. The results indicate that when Fe(III) is in a form that Fe(III)-reducing organisms can readily reduce, Fe(III)-reducing organisms can inhibit sulfate reduction and methane production by outcompeting sulfate reducers and methanogens for electron donors.