Seasonal movements of American horseshoe crabs Limulus polyphemus in the Great Bay Estuary,New Hampshire (USA)

<正> The goal of this study was to determine the year round movement patterns of American horseshoe crabs,Limuluspolyphemus,in the Great Bay Estuary,New Hampshire (USA) by using acoustic telemetry to track the movements of 37 adultLimulus,for periods ranging from 2 to 31 months.During the winter (December-March) horseshoe crabs moved very little.In thespring,when water temperatures exceeded 11℃,horseshoe crabs moved at least 1 km further up into the estuary to shallowersubtidal areas about a month prior to spawning.The mean distance traveled during spring migrations was 2.6 ± 0.5 (n=20) km upthe estuary.Mating occurred in May and June and during these months animals spent most of their time in shallow subtidal areasadjacent to mating beaches.In the summer (July-August),animals moved 1.5 ± 0.5 (n=26) km down the estuary,towards theocean,and ranged widely,using extensive portions of the estuary.In the fall (September-November) movement was more limited(0.5 ± 0.5 km;n = 24) while animals settled into wintering locations,where they remained until spring.The mean annual linearrange for all animals was 4.5 ± 0.3 km (n =35) and the maximum distance traveled by an individual horseshoe crab within oneyear was 9.2 km.There was no evidence that any of the horseshoe crabs tracked during this study left the estuary     

Impacts of Seawalls on Saltmarsh Plant Communities in the Great Bay Estuary,New Hampshire USA

Seawalls are often built along naturally dynamic coastlines, including the upland edge of salt marshes, in order to prevent erosion or to extend properties seaward. The impacts of seawalls on fringing salt marshes were studied at five pairs of walled and natural marshes in the Great Bay Estuary of New Hampshire, USA. Marsh plant species and communities showed no difference in front of walls when compared with similar elevations at paired controls. However, seawalls eliminated the vegetative transition zone at the upper border. Not only did the plant community of the transition zone have high plant diversity relative to the low marsh, but it varied greatly from site to site in the estuary. The effects of seawall presence on other marsh processes, including sediment movement, wrack accumulation, groundwater flow, and vegetation distribution and growth, were examined. Although no statistically significant effects of seawalls were found, variation in the indicators of these processes were largely controlled by wave exposure, site-specific geomorphology and land use, and distance of the sampling station from the upland. Trends indicated there was more sediment movement close to seawalls at high energy sites and less fine grain sediment near seawalls. Both trends are consistent with an increase in energy from wave reflection. The distribution of seawalls bordering salt marshes was mapped for Great and Little Bays and their rivers. Throughout the study area, 3.54% of the marshes were bounded by shoreline armoring (5876 m of seawalls along 165.8 km of marsh shoreline). Localized areas with high population densities had up to 43% of marshes bounded by seawalls. Coastal managers should consider limiting seawall construction to preserve plant diversity at the upper borders of salt marshes and prevent marsh habitat loss due to transgression associated with sea level rise.     

Adhesion of Dissimilatory Fe(III)-Reducing Bacteria to Fe(III) Minerals

The metabolism of dissimilatory iron-reducing bacteria (DIRB) may provide a means of remediating contaminated subsurface soils. The factors controlling the rate and extent of bacterial F(III) mineral reduction are poorly understood. Recent research suggests that molecular-scale interactions between DIRB cells and Fe(III) mineral particles play an important role in this process. One of these interactions, cell adhesion to Fe(III) mineral particles, appears to be a complex process that is, at least in part, mediated by a variety of surface proteins. This study examined the hypothesis that the flagellum serves as an adhesin to different Fe(III) minerals that range in their surface area and degree of crystallinity. Deflagellated cells of the DIRB Shewanella algae BrY showed a reduced ability to adhere to hydrous ferric oxide (HFO) relative to flagellated cells. Flagellated cells were also more hydrophobic than deflagellated cells. This was significant because hydrophobic interactions have been previously shown to dominate S. algae cell adhesion to Fe(III) minerals. Pre-incubating HFO, goethite, or hematite with purified flagella inhibited the adhesion of S. algae BrY cells to these minerals. Transposon mutagenesis was used to generate a flagellum-deficient mutant designated S. algae strain NF. There was a significant difference in the rate and extent of S. algae NF adhesion to HFO, goethite, and hematite relative to that of S. algae BrY. Amiloride, a specific inhibitor of Na + -driven flagellar motors, inhibited S. algae BrY motility but did not affect the adhesion of S. algae BrY to HFO. S.algae NF reduced HFO at the same rate as S. algae BrY. Collectively, the results of this study support the hypothesis that the flagellum of S. algae functions as a specific Fe(III) mineral adhesin. However, these results suggest that flagellum-mediated adhesion is not requisite for Fe(III) mineral reduction.     

Direct and Fe(II)-Mediated Reduction of Technetium by Fe(III)-Reducing Bacteria

The dissimilatory Fe(III)-reducing bacterium Geobacter sulfurreducens reduced and precipitated Tc(VII) by two mechanisms. Washed cell suspensions coupled the oxidation of hydrogen to enzymatic reduction of Tc(VII) to Tc(IV), leading to the precipitation of TcO₂ at the periphery of the cell. An indirect, Fe(II)-mediated mechanism was also identified. Acetate, although not utilized efficiently as an electron donor for direct cell-mediated reduction of technetium, supported the reduction of Fe(III), and the Fe(II) formed was able to transfer electrons abiotically to Tc(VII). Tc(VII) reduction was comparatively inefficient via this indirect mechanism when soluble Fe(III) citrate was supplied to the cultures but was enhanced in the presence of solid Fe(III) oxide. The rate of Tc(VII) reduction was optimal, however, when Fe(III) oxide reduction was stimulated by the addition of the humic analog and electron shuttle anthaquinone-2,6-disulfonate, leading to the rapid formation of the Fe(II)-bearing mineral magnetite. Under these conditions, Tc(VII) was reduced and precipitated abiotically on the nanocrystals of biogenic magnetite as TcO₂ and was removed from solution to concentrations below the limit of detection by scintillation counting. Cultures of Fe(III)-reducing bacteria enriched from radionuclide-contaminated sediment using Fe(III) oxide as an electron acceptor in the presence of 25 μM Tc(VII) contained a single Geobacter sp. detected by 16S ribosomal DNA analysis and were also able to reduce and precipitate the radionuclide via biogenic magnetite. Fe(III) reduction was stimulated in aquifer material, resulting in the formation of Fe(II)-containing minerals that were able to reduce and precipitate Tc(VII). These results suggest that Fe(III)-reducing bacteria may play an important role in immobilizing technetium in sediments via direct and indirect mechanisms.     

Role of Hydrophobicity in Adhesion of the Dissimilatory Fe(III)-Reducing Bacterium Shewanella alga to Amorphous Fe(III) Oxide

The mechanisms by which the dissimilatory Fe(III)-reducing bacterium Shewanella alga adheres to amorphous Fe(III) oxide were examined through comparative analysis of S. alga BrY and an adhesion-deficient strain of this species, S. alga RAD20. Approximately 100% of S. alga BrY cells typically adhered to amorphous Fe(III) oxide, while less than 50% of S. alga RAD20 cells adhered. Bulk chemical analysis, isoelectric point analysis, and cell surface analysis by time-of-flight secondary-ion mass spectrometry and electron spectroscopy for chemical analysis demonstrated that the surfaces of S. alga BrY cells were predominantly protein but that the surfaces of S. alga RAD20 cells were predominantly exopolysaccharide. Physicochemical analyses and hydrophobic interaction assays demonstrated that S. alga BrY cells were more hydrophobic than S. alga RAD20 cells. This study represents the first quantitative analysis of the adhesion of a dissimilatory Fe(III)-reducing bacterium to amorphous Fe(III) oxide, and the results collectively suggest that hydrophobic interactions are a factor in controlling the adhesion of this bacterium to amorphous Fe(III) oxide. Despite having a reduced ability to adhere, S. alga RAD20 reduced Fe(III) oxide at a rate identical to that of S. alga BrY. This result contrasts with results of previous studies by demonstrating that irreversible cell adhesion is not requisite for microbial reduction of amorphous Fe(III) oxide. These results suggest that the interaction between dissimilatory Fe(III)-reducing bacteria and amorphous Fe(III) oxide is more complex than previously believed.     

Reduction of Actinides and Fission Products by Fe(III)-Reducing Bacteria

Microbial metabolism plays a pivotal role in controlling the solubility and mobility of radionuclides in waters contaminated by nuclear waste. The distribution and activity of dissimilatory Fe(III)-reducing bacteria are of particular importance because they can alter the solubility of radionuclides via direct enzymatic reduction or by indirect mechanisms catalyzed via a range of electron shuttling compounds. Using a combination of the techniques of microbiology, biochemistry, and molecular biology, we have characterized the mechanisms of electron transfer to key radionuclides by Fe(III)-reducing bacteria. The mechanisms of enzyme-mediated reduction of problematic actinides, principally U(VI) but including Pu(IV) and Np(V), are described in this review. In addition, the mechanisms by which the fission product Tc(VII) is reduced are also discussed. Direct enzymatic reductions of Tc(VII), catalyzed by microbial hydrogenases, are described along with indirect mechanisms catalyzed by microbially produced Fe(II). Finally, we describe new results that demonstrate the transfer of electrons from biogenic U(IV) to Tc(VII), leading to coprecipitation of Tc(IV) and U(IV), and opening the way for treatment of liquid wastes cocontaminated with both uranium and technetium in one step.     

Using Functional Trajectories to Track Constructed Salt Marsh Development in the Great Bay Estuary, Maine/New Hampshire, U.S.A.

A growing number of studies have assessed the functional equivalency of restored and natural salt marshes. Several of these have explored the use of functional trajectories to track the increase in restored marsh function over time; however, these studies have disagreed as to the usefulness of such models in long‐term predictions of restored marsh development. We compared indicators of four marsh functions (primary production, soil organic matter accumulation, sediment trapping, and maintenance of plant communities) in 6 restored and 11 reference (matched to restored marshes using principal components analysis) salt marshes in the Great Bay Estuary. The restored marshes were all constructed and planted on imported substrate and ranged in age from 1 to 14 years. We used marsh age in a space‐for‐time substitution to track constructed salt marsh development and explore the use of trajectories. A high degree of variability was observed among natural salt marsh sites, displaying the importance of carefully chosen reference sites. As expected, mean values for constructed site (n = 6) and reference site (n = 11) functions were significantly different. Using constructed marsh age as the independent variable and functional indicator values as dependent variables, nonlinear regression analyses produced several ecologically meaningful trajectories (r ²> 0.9), demonstrating that the use of different‐aged marshes can be a viable approach to developing functional trajectories. The trajectories illustrated that although indicators of some functions (primary production, sediment deposition, and plant species richness) may reach natural site values relatively quickly (<10 years), others (soil organic matter content) will take longer.     

Naphthalene and Benzene Degradation under Fe(III)-Reducing Conditions in Petroleum-Contaminated Aquifers

Naphthalene was oxidized anaerobically to CO₂ in sediments collected from a petroleum-contaminated aquifer in Bemidji, Minnesota in which Fe(III) reduction was the terminal electron-accepting process. Naphthalene was not oxidized in sediments from the methanogenic zone at Bemidji or in sediments from the Fe(III)-reducing zone of other petroleum-contaminated aquifers studied. In a profile across the Fe(III)-reducing zone of the Bemidji aquifer, rates of naphthalene oxidation were fastest in sediments with the highest proportion of Fe(III), which was also the zone of the most rapid degradation of benzene, toluene, and acetate. The comparative studies attempted to elucidate factors that might account for the fact that unsubstituted aromatic hydrocarbons such as benzene and naphthalene were degraded under Fe(III)-reducing conditions at Bemidji, but not at the other aquifers examined. These studies indicated that the ability of Fe(III)-reducing microorganisms to degrade benzene and naphthalene at the Bemidji site cannot be attributed to groundwater components that make Fe(III) more available for reduction or other potential factors that were evaluated. However, unlike the other aquifers evaluated, uncontaminated sediments at the Bemidji site could be adapted for anaerobic benzene degradation merely with the addition of benzene. These findings indicate that Bemidji sediments naturally contain Fe(III) reducers capable of degradation of unsubstituted aromatic hydrocarbons.     

Reductive Precipitation of Gold by Dissimilatory Fe(III)-Reducing Bacteria and Archaea

Studies with a diversity of hyperthermophilic and mesophilic dissimilatory Fe(III)-reducing Bacteria and Archaea demonstrated that some of these organisms are capable of precipitating gold by reducing Au(III) to Au(0) with hydrogen as the electron donor. These studies suggest that models for the formation of gold deposits in both hydrothermal and cooler environments should consider the possibility that dissimilatory metal-reducing microorganisms can reductively precipitate gold from solution.     

Deflocculation of Activated Sludge by the Dissimilatory Fe(III)-Reducing Bacterium Shewanella alga BrY

The influence of microbial Fe(III) reduction on the deflocculation of autoclaved activated sludge was investigated. Fe(III) flocculated activated sludge better than Fe(II). Decreasing concentrations of Fe(III) caused an increase in sludge bulk water turbidity, while bulk water turbidity remained relatively constant over a range of Fe(II) concentrations. Cells of the dissimilatory metal-reducing bacterium Shewanella alga BrY coupled the oxidation of H(inf2) to the reduction of Fe(III) bound in sludge flocs. Cell adhesion to the Fe(III)-sludge flocs was a prerequisite for Fe(III) reduction. The reduction of Fe(III) in sludge flocs by strain BrY caused an increase in bulk water turbidity, suggesting that the sludge was deflocculated. The results of this study support previous research suggesting that microbial Fe(III) respiration may have an impact on the floc structure and colloidal chemistry of activated sludge.     

Characterization of Metabolism in the Fe(III)-Reducing Organism Geobacter sulfurreducens by Constraint-Based Modeling

Geobacter sulfurreducens is a well-studied representative of the Geobacteraceae, which play a critical role in organic matter oxidation coupled to Fe(III) reduction, bioremediation of groundwater contaminated with organics or metals, and electricity production from waste organic matter. In order to investigate G. sulfurreducens central metabolism and electron transport, a metabolic model which integrated genome-based predictions with available genetic and physiological data was developed via the constraint-based modeling approach. Evaluation of the rates of proton production and consumption in the extracellular and cytoplasmic compartments revealed that energy conservation with extracellular electron acceptors, such as Fe(III), was limited relative to that associated with intracellular acceptors. This limitation was attributed to lack of cytoplasmic proton consumption during reduction of extracellular electron acceptors. Model-based analysis of the metabolic cost of producing an extracellular electron shuttle to promote electron transfer to insoluble Fe(III) oxides demonstrated why Geobacter species, which do not produce shuttles, have an energetic advantage over shuttle-producing Fe(III) reducers in subsurface environments. In silico analysis also revealed that the metabolic network of G. sulfurreducens could synthesize amino acids more efficiently than that of Escherichia coli due to the presence of a pyruvate-ferredoxin oxidoreductase, which catalyzes synthesis of pyruvate from acetate and carbon dioxide in a single step. In silico phenotypic analysis of deletion mutants demonstrated the capability of the model to explore the flexibility of G. sulfurreducens central metabolism and correctly predict mutant phenotypes. These results demonstrate that iterative modeling coupled with experimentation can accelerate the understanding of the physiology of poorly studied but environmentally relevant organisms and may help optimize their practical applications.     

pH Gradient-Induced Heterogeneity of Fe(III)-Reducing Microorganisms in Coal Mining-Associated Lake Sediments

Lakes formed because of coal mining are characterized by low pH and high concentrations of Fe(II) and sulfate. The anoxic sediment is often separated into an upper acidic zone (pH 3; zone I) with large amounts of reactive iron and a deeper slightly acidic zone (pH 5.5; zone III) with smaller amounts of iron. In this study, the impact of pH on the Fe(III)-reducing activities in both of these sediment zones was investigated, and molecular analyses that elucidated the sediment microbial diversity were performed. Fe(II) was formed in zone I and III sediment microcosms at rates that were approximately 710 and 895 nmol cm⁻³ day⁻¹, respectively. A shift to pH 5.3 conditions increased Fe(II) formation in zone I by a factor of 2. A shift to pH 3 conditions inhibited Fe(II) formation in zone III. Clone libraries revealed that the majority of the clones from both zones (approximately 44%) belonged to the Acidobacteria phylum. Since moderately acidophilic Acidobacteria species have the ability to oxidize Fe(II) and since Acidobacterium capsulatum reduced Fe oxides at pHs ranging from 2 to 5, this group appeared to be involved in the cycling of iron. PCR products specific for species related to Acidiphilium revealed that there were higher numbers of phylotypes related to cultured Acidiphilium or Acidisphaera species in zone III than in zone I. From the PCR products obtained for bioleaching-associated bacteria, only one phylotype with a level of similarity to Acidithiobacillus ferrooxidans of 99% was obtained. Using primer sets specific for Geobacteraceae, PCR products were obtained in higher DNA dilutions from zone III than from zone I. Phylogenetic analysis of clone libraries obtained from Fe(III)-reducing enrichment cultures grown at pH 5.5 revealed that the majority of clones were closely related to members of the Betaproteobacteria, primarily species of Thiomonas. Our results demonstrated that the upper acidic sediment was inhabited by acidophiles or moderate acidophiles which can also reduce Fe(III) under slightly acidic conditions. The majority of Fe(III) reducers inhabiting the slightly acidic sediment had only minor capacities to be active under acidic conditions.     

Dissimilatory Fe(III) Reduction by the Marine Microorganism Desulfuromonas acetoxidans

The ability of the marine microorganism Desulfuromonas acetoxidans to reduce Fe(III) was investigated because of its close phylogenetic relationship with the freshwater dissimilatory Fe(III) reducer Geobacter metallireducens. Washed cell suspensions of the type strain of D. acetoxidans reduced soluble Fe(III)-citrate and Fe(III) complexed with nitriloacetic acid. The c-type cytochrome(s) of D. acetoxidans was oxidized by Fe(III)-citrate and Mn(IV)-oxalate, as well as by two electron acceptors known to support growth, colloidal sulfur and malate. D. acetoxidans grew in defined anoxic, bicarbonate-buffered medium with acetate as the sole electron donor and poorly crystalline Fe(III) or Mn(IV) as the sole electron acceptor. Magnetite (Fe₃O₄) and siderite (FeCO₃) were the major end products of Fe(III) reduction, whereas rhodochrosite (MnCO₃) was the end product of Mn(IV) reduction. Ethanol, propanol, pyruvate, and butanol also served as electron donors for Fe(III) reduction. In contrast to D. acetoxidans, G. metallireducens could only grow in freshwater medium and it did not conserve energy to support growth from colloidal S⁰ reduction. D. acetoxidans is the first marine microorganism shown to conserve energy to support growth by coupling the complete oxidation of organic compounds to the reduction of Fe(III) or Mn(IV). Thus, D. acetoxidans provides a model enzymatic mechanism for Fe(III) or Mn(IV) oxidation of organic compounds in marine and estuarine sediments. These findings demonstrate that 16S rRNA phylogenetic analyses can suggest previously unrecognized metabolic capabilities of microorganisms.     

Comparison of Humic Substance- and Fe(III)-Reducing Microbial Communities in Anoxic Aquifers

Humic substances can mediate electron transfer between microorganisms and Fe(III) minerals. Because it is unknown which microorganisms reduce humics in anoxic aquifers, we analyzed the diversity and physiological flexibility of Fe(III)-, humics-, and AQDS-reducers, which were present at up to 10⁶ cells g^?1. No significant differences in 16S rRNA gene based diversity were found between enrichment cultures reducing ferrihydrite, humics or AQDS. Even after repeated transfers many of the enrichments retained the ability to switch to other electron acceptors. This suggests that humics- and Fe(III)-reducing microorganisms in anoxic aquifers are rather versatile and able to reduce different extracellular electron acceptors.     

Requirement for a Microbial Consortium To Completely Oxidize Glucose in Fe(III)-Reducing Sediments

In various sediments in which Fe(III) reduction was the terminal electron-accepting process, [¹⁴C]glucose was fermented to ¹⁴C-fatty acids in a manner similar to that observed in methanogenic sediments. These results are consistent with the hypothesis that, in Fe(III)-reducing sediments, fermentable substrates are oxidized to carbon dioxide by the combined activity of fermentative bacteria and fatty acid-oxidizing, Fe(III)-reducing bacteria.     

Competition between Fe(III)-Reducing and Methanogenic Bacteria for Acetate in Iron-Rich Freshwater Sediments

The kinetics of acetate uptake and the depth distribution of [2-¹⁴C]acetate metabolism were examined in iron-rich sediments from a beaver impoundment in northcentral Alabama. The half-saturation constant (K_m) determined for acetate uptake in slurries of Fe(III)-reducing sediment (0.8 µM) was more than 10-fold lower than that measured in methanogenic slurries (12 µM) which supported comparable rates of bulk organic carbon metabolism and V_max values for acetate uptake. The endogenous acetate concentration (S _n) was also substantially lower (1.7 µM) in Fe(III)-reducing vs methanogenic (9.0 µM) slurries. The proportion of [2-¹⁴C]acetate converted to ¹⁴CH₄ increased with depth from ca 0.1 in the upper 0.5 cm to ca 0.8 below 2 cm and was inversely correlated (r² = 0.99) to a decline in amorphous Fe(III) oxide concentration. The results of the acetate uptake kinetics experiments suggest that differences in the affinity of Fe(III)-reducing bacteria vs methanogens for acetate can account for the preferential conversion of [2-¹⁴C]acetate to ¹⁴CO₂ in Fe(III) oxide-rich surface sediments, and that the downcore increase in conversion of [2-¹⁴C]acetate to ¹⁴CH₄ can be attributed to progressive liberation of methanogens from competition with Fe(III) reducers as Fe(III) oxides are depleted with depth.     

Protein-Mediated Adhesion of the Dissimilatory Fe(III)-Reducing Bacterium Shewanella alga BrY to Hydrous Ferric Oxide

The rate and extent of bacterial Fe(III) mineral reduction are governed by molecular-scale interactions between the bacterial cell surface and the mineral surface. These interactions are poorly understood. This study examined the role of surface proteins in the adhesion of Shewanella alga BrY to hydrous ferric oxide (HFO). Enzymatic degradation of cell surface polysaccharides had no effect on cell adhesion to HFO. The proteolytic enzymes Streptomyces griseus protease and chymotrypsin inhibited the adhesion of S. alga BrY cells to HFO through catalytic degradation of surface proteins. Trypsin inhibited S. alga BrY adhesion solely through surface-coating effects. Protease and chymotrypsin also mediated desorption of adhered S. alga BrY cells from HFO while trypsin did not mediate cell desorption. Protease removed a single peptide band that represented a protein with an apparent molecular mass of 50 kDa. Chymotrypsin removed two peptide bands that represented proteins with apparent molecular masses of 60 and 31 kDa. These proteins represent putative HFO adhesion molecules. S. alga BrY adhesion was inhibited by up to 46% when cells were cultured at sub-MICs of chloramphenicol, suggesting that protein synthesis is necessary for adhesion. Proteins extracted from the surface of S. alga BrY cells inhibited adhesion to HFO by up to 41%. A number of these proteins bound specifically to HFO, suggesting that a complex system of surface proteins mediates S. alga BrY adhesion to HFO.