Methanethiol accumulation exacerbates release of N2 O during denitrification in estuarine sediments and bacterial cultures.

Microbes play critical roles in the biogeochemical cycling of nitrogen and sulfur in aquatic environments. Here we investigated the interaction between the naturally occurring organic sulfur compound methanethiol (MeSH) and the final step of the denitrification pathway, the reduction of nitrous oxide (N2 O) to dinitrogen (N2 ) gas, in sediment slurries from the temperate Douro and Ave estuaries (NW Portugal) and in pure cultures of the marine bacterium Ruegeria pomeroyi. Sediment slurries and cell suspensions were amended with a range of concentrations of either MeSH (0-120 µM) or methionine (0-5 mM), a known precursor of MeSH. MeSH or methionine additions caused N2 O to accumulate and this accumulation was linearly related to MeSH concentrations in both coastal sediments (R(2) = 0.7-0.9, P < 0.05) and R. pomeroyi cell suspensions (R(2) = 0.9, P < 0.01). Our results suggest that MeSH inhibits the final step of denitrification resulting in N2 O accumulation.

Dynamics of nitrous oxide reductase genes (nosZ) in intertidal rocky biofilms and sediments of the Douro River estuary (Portugal), and their relation to N-biogeochemistry.

In this study, temporal variability of nosZ genotypes was evaluated in two intertidal rocky biofilms and two intertidal sediment sites of the Douro River estuary, Portugal. The results were compared to rates of key N-cycle processes and environmental variables to examine possible links between denitrifier community dynamics and N biogeochemistry. Genetic heterogeneity of the nosZ gene was evaluated by terminal restriction fragment length polymorphism analysis (T-RFLP) and by sequencing cloned nosZ gene fragments. Phylogenetic analysis showed that the majority of the nosZ genes detected were most similar to nosZ genes from isolates affiliated with alpha-subclass of the class Proteobacteria. Results revealed low nosZ genotype richness, and hierarchical cluster analysis showed significant differences in the composition of denitrifier communities that inhabit different intertidal environments of the Douro River estuary. Monthly surveys of nosZ genotypes from sandy sediments showed that, while the same T-RFLP peaks were present in all samples, shifts in the relative peak areas of the different nosZ genotypes occurred. Canonical correspondence analysis, based on data from the monthly survey, revealed a strong relationship between the relative peak areas of some T-RFLP operational taxonomic units (OTUs) with denitrification rate and NO3- availability. Results suggest that denitrifiers with specific nosZ genotypes (OTUs) have competitive advantage over others when NO3- fluctuates in the system; these fluctuations reflect, in turn, variability in denitrification rates.

Composition and activity of beta-Proteobacteria ammonia-oxidizing communities associated with intertidal rocky biofilms and sediments of the Douro River estuary, Portugal.

AIMS: To characterize the phylogenetic composition of ammonia-oxidizing bacteria (AOB) of the beta-subclass of the class Proteobacteria in intertidal sediment and rocky biofilms of the Douro estuary, and evaluate relationships with environmental variables and N-biogeochemistry. METHODS AND RESULTS: Cluster analysis of denaturing gradient gel electrophoresis profiles showed differences in beta-Proteobacteria AOB assemblage composition between rocky biofilms and sediments. All sequences obtained from intertidal rocky biofilm sites exhibited phylogenetic affinity to Nitrosomonas sp. lineages, whereas a majority of the sequences from the sediment sites were most similar to marine Nitrosospira cluster 1. Hierarchical cluster analysis based on environmental variables identified two main groups of samples. The first contained samples from rocky biofilm sites characterized by high concentrations of NO2- and NH4+, and high organic matter and chlorophyll a content. The second group contained all of the sediment samples; these sites were characterized by lower values for the variables above. In addition, rocky biofilm sites exhibited higher nitrification rates. CONCLUSIONS: Intersite differences in environmental and/or physical conditions led to the selection of different populations of beta-Proteobacteria AOB, supporting different magnitudes of N-cycling regimes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents an important step in establishing the influence of environmental factors on the distribution of beta-Proteobacteria AOB with possible consequences for N-biogeochemistry.

Prokaryotes: the unseen majority.

The number of prokaryotes and the total amount of their cellular carbon on earth are estimated to be 4-6 x 10(30) cells and 350-550 Pg of C (1 Pg = 10(15) g), respectively. Thus, the total amount of prokaryotic carbon is 60-100% of the estimated total carbon in plants, and inclusion of prokaryotic carbon in global models will almost double estimates of the amount of carbon stored in living organisms. In addition, the earth's prokaryotes contain 85-130 Pg of N and 9-14 Pg of P, or about 10-fold more of these nutrients than do plants, and represent the largest pool of these nutrients in living organisms. Most of the earth's prokaryotes occur in the open ocean, in soil, and in oceanic and terrestrial subsurfaces, where the numbers of cells are 1.2 x 10(29), 2.6 x 10(29), 3.5 x 10(30), and 0. 25-2.5 x 10(30), respectively. The numbers of heterotrophic prokaryotes in the upper 200 m of the open ocean, the ocean below 200 m, and soil are consistent with average turnover times of 6-25 days, 0.8 yr, and 2.5 yr, respectively. Although subject to a great deal of uncertainty, the estimate for the average turnover time of prokaryotes in the subsurface is on the order of 1-2 x 10(3) yr. The cellular production rate for all prokaryotes on earth is estimated at 1.7 x 10(30) cells/yr and is highest in the open ocean. The large population size and rapid growth of prokaryotes provides an enormous capacity for genetic diversity.

Evidence for an enhanced substrate requirement by marine mesophilic bacterial isolates at minimal growth temperatures.

Bacterial isolates from the subtropical southeastern continental shelf were cultured in a matrix of temperature and substrate concentrations encompassing a range of temperature and substrate concentrations equal to and exceeding natural ones. At the annual minimum temperature, marine heterotrophic bacterial isolates required higher concentrations of dissolved substrates for active growth than are usually found in seawater. We show this to result from a nonlinear interaction of the combined effects of temperature and substrate concentration on bacterial growth and respiratory rate. As a result, bacterial and protozoan utilization of phytoplankton production during winter and early spring is low, permitting greater energy flow to zooplankton and benthic animals, while in late spring, summer, and fall, the microbial loop dominates energy flux and organic carbon utilization. Escherichia coli shows a similar nonlinear response to temperature at minimal substrate concentrations, albeit at a higher range of concentrations than were utilized by the marine isolates. Thus, bacteria from subtropical regions are shown to have a differential growth response near the minimum temperature for growth, depending on the concentration of available substrates.

Bacterial growth in the cold: evidence for an enhanced substrate requirement.

Growth responses and biovolume changes for four facultatively psychrophilic bacterial isolates from Conception Bay, Newfoundland, and the Arctic Ocean were examined at temperatures from - 1.5 to 35 degrees C, with substrate concentrations of 0.15, 1.5, and 1,500 mg of proteose peptone-yeast extract per liter. For two cultures, growth in 0.1, 1.0, and 1,000 mg of proline per liter was also examined. At 10 to 15 degrees C and above, growth rates showed no marked effect of substrate concentration, while at - 1.5 and 0 degrees C, there was an increasing requirement for organic nutrients, with generation times in low-nutrient media that were two to three times longer than in high-nutrient media. Biovolume showed a clear dependence on substrate concentration and quality; the largest cells were in the highest-nutrient media. Biovolume was also affected by temperature; the largest cells were found at the lowest temperatures. These data have implications for both food web structure and carbon flow in cold waters and for the effects of global climate change, since the change in growth rate is most dramatic at the lowest temperatures.

Populations of methanogenic bacteria in a georgia salt marsh.

Methanogens represented about 0.5% of the total bacteria in sediments from a Georgia salt marsh in which Spartina alterniflora is the predominant vegetation. The population of methanogens was composed of at least two groups of nearly equal size. One group was represented by cocci which were able to utilize trimethylamine and were unable to use H(2) or acetate. The second group was composed of two subgroups which were able to utilize H(2) but were unable to use trimethylamine or acetate. The more common subgroup included rod- or plate-shaped methanogens which could utilize isopropanol in addition to H(2) and formate. The second subgroup included Methanococcus maripaludis, which utilized only H(2) and formate. Other groups of methanogens were also present, including Methanosarcina sp. which utilized acetate, H(2), and methylamines. In addition to the overall variability in the types of methanogens, the numbers of methanogens in sediments also exhibited significant spatial variability both within and between tall- and short-Spartina zones.

Tracer analysis of methanogenesis in salt marsh soils.

Differences in paths of carbon flow have been found in soils of the tall (TS) and short (SS) Spartina alterniflora marshes of Sapelo Island, Ga. Gaseous end products of [U-C]glucose metabolism were CO(2) and CH(4) in the SS region and primarily CO(2) in the TS region. Sulfate concentration did not demonstrably affect glucose catabolism or the distribution of end products in either zone. [U-C]acetate was converted to CO(2) and CH(4) in the SS soils and almost exclusively to CO(2) in the TS soils. Sulfate concentration did not affect acetate metabolism in the SS soils; however, a noticeable effect of sulfate dilution was seen in TS soils. Sulfate dilution in TS samples resulted in increased methane formation. Total glucose and acetate metabolism were similar in TS and SS soils despite differences in end products. A microbial community characterized by fermentative/sulfate-reducing processes has developed in TS soils as opposed to the fermentative/methanogenic/sulfate-reducing community found in SS soils.

Arylsulfatase activity in salt marsh soils.

The presence of arylsulfatase(s) was confirmed in salt marsh soils. The temperatures of maximum activity and inactivation, the pH range over which the enzyme was active, and the K(m) values were similar to those of soil enzymes. Unlike soil arylsulfatases, however, the salt marsh enzymes do not appear to be repressed by sulfate. It is postulated that these enzymes may be necessary for the initiation of arylsulfate ester metabolism.

Nitrogen-fixing communities in an intertidal ecosystem.

Nitrogen gixation (acetylene reduction) associated with various communities in the intertidal sand and mud flat was measured in situ. Areas which were colonized by algae, plants, and animals generally had significantly higher activities than areas which were visibly uncolonized. The highest activities were measured at sites colonized by a mixed bloom of Oscillatoria-Euglena-photosynthetic bacteria. These areas occupied only about 1% of the surface of the sediments, yet contributed nearly 50% of the biologically fixed nitrogen. Enteromorpha communities also exhibited relatively high activities. Sites were grouped according to the statistical significance of the mean of their activities: group I: uncolonized sand, mud and gravel flats, Eelgrass (Zostera) communities, and snail (amphibola) beds, with an estimated contribution of 1 g N ha-1 day-1; group II: Cockel (Chione) beds, 3 g N ha-1 day-1; group III: Salicornia, Ulva, Juncus, and Enteromorpha, 10 g N ha-1 day-1; and group IV: Oscillatoria-Euglena-photosynthetic bacteria association, 200 g N ha-1 day-1. Enteromorpha and Oscillatoria-Euglena-photosynthetic bacteria communities showed significantly lower rates of nitrogen fixation in the dark than in the light.

(14)C-labeling of the compounds excreted by phytoplankton for employment as a realistic tracer in secondary productivity measurements.

Preparations of the dissolved organic compounds released by photosynthesizing marine phytoplankton have been obtained with(14)carbon activities as high as 1.5 × 10(5) dpm/ml. The radioisotope content of the preparations resides wholly in dissolved organic compounds of low molecular weight (MW<3500), which are uncontaminated by residual(14)C-labeled inorganic carbon. The labeled compunds arise through photosynthetic fixation and do not appear to be products of cell lysis during the incubation or to originate from cell damage during the filtration step employed.

Constant release of photosynthate from marine phytoplankton.

The release rate of dissolved organic carbon (DOC) by unialgal cultures and natural phytoplankton assemblages was constant over a wide range of dissolved inorganic carbon concentrations. DOC release was not proportional to the particulate organic carbon production rate. We postulate that intracellular DOC, fated for release, exists either as a separate pool from that leading to particulate organic carbon production or that there is active metabolic control on one portion of a common pool.

Nitrification on a coral reef.

We report that the algal pavement just behind the reef crest at Enewetak Atoll produces nitrate at measurable rates. In situ and in vitro incubations with N-Serve indicate that the autotrophic pathway involving two separate organisms is effective in this oxidation of ammonia to nitrate. Significant nitrification is indicated throughout the reef environment; Nitrobacter agilis has specifically been identified as at least one of the organisms responsible for the terminal oxidation of nitrite to nitrate.

Use of the adenylate energy charge ratio to measure growth state of natural microbial communities.

Measurement of the adenylate energy charge ratio is proposed as a means of determining the growth state of natural microbial communities and the effect of environmental changes on them. Observations on microbial cultures and on natural microbial populations from the Western North Atlantic Ocean water and from sediments of a costal salt marsh show that energy charge measurements do show the metabolic state of communities as well as species populations.

Nitrogen fixation in a coral reef community.

Algal reef flats at Enewetak Atoll, Marshall Islands, fix atmospheric nitrogen at rates comparable to those in managed agriculture. The dominant nitrogen fixer appears to be the blue-green alga Calothrix crustacea. Since this nutrient enrichment contributes to the high productivity of adjacent coral reefs and undoubtedly to atoll lagoons, it is recommended that the algal reef flats receive increased conservation priority.

Ecological application of antibiotics as respiratory inhibitors of bacterial populations.

Two terregenous and four marine bacterial isolates were treated with six antibiotics and antibiotic combinations. Comparisons made between responses of cells in early and late logarithmic and stationary growth phases indicated variable sensitivity to the agents. Bacteria in stationary and late log-phase cultures exhibited the greatest resistance, whereas the early log-phase cells exhibited greatest antibiotic susceptibility. We conclude that the tested antibiotics cannot be used for ecological purposes to delineate bacterial respiration in mixed microbial communities.

Simple, reliable cold tray for the recovery and examination of thermosensitive organisms.

A simple chamber is described for the isolation and handling of thermosensitive bacteria on board ship and in conventional research or classroom laboratories.

Fine structure of selected marine pseudomonads and achromobacters.

The fine structure of more than 20 marine pseudomonads and more than 15 achromobacters was examined. Under the conditions extant, clear differences between members of these two groups were seen. The pseudomonads displayed the characteristic gram-negative morphology: the cell wall was irregularly undulant and the cytoplasmic membrane more nearly planar, ribonucleoprotein (RNP) particles were loosely packed throughout the periphery of the cytoplasm, and the deoxyribonucleic acid (DNA) was axially disposed. Cell division appeared to be by constriction. Some strains characteristically produced evaginations or blebs of the cell wall. Occasionally, thick, densely stained ring structures were seen which are possibly analogous to mesosomes. In contrast, the achromobacters demonstrated a regularly undulant outer cell wall element and a planar inner wall. The cytoplasmic membrane was thin and not readily observed. RNP particles were densely stained and tightly packed in the cytoplasm; the DNA was most often lobate in disposition. Cellular division was mediated by the formation of a septum which consisted of the cytoplasmic membrane and the inner element of the cell wall. Mesosomes were observed in all of the strains examined. Dense inclusion bodies were also seen in many strains.