Interrelationships between Rates of Microbial Production,Exopolymer Production,Microbial Biomass,and Sediment Stability in Biofilms of Intertidal Sediments

Abstract The upper few millimeters of intertidal sediment supports a varied biomass of microbial consortia and microphytobenthos. Many of these organisms release extracellular polymers into the surrounding sediment matrix that can result in sediment cohesion and the increased stability of the sediment. The relationship between the heterotrophic and autotrophic components of these biofilms is not well understood. A combination of mesocosm and field investigations were used to investigate the relationship between microbial production rate (algae and bacteria), the extracellular carbohydrates, biomass, and stability in conjunction with a variety of environmental factors. An inverse relationship was found between rates of algal production and sediment stability both in the field and in laboratory mesocosms, though the relationship was significant only in the field (P < 0.001). Stability of sediments increased with increasing bacterial production rate (P < 0.001). Positive correlations were found between sediment stability and a range of other variables, including algal biomass (P < 0.001), colloidal-S EPS (P < 0.001), colloidal-S carbohydrate (P < 0.01), colloidal-S EDTA (P < 0.01), and sediment water content (P < 0.001). Using the data acquired, a preliminary model was developed to predict changes in sediment stability. Chlorophyll a, water content, and colloidal-S EPS were found to be the most important predictors of stability in intact cores incubated under laboratory conditions. Differences observed in patterns of the surface (0–2 mm) distribution of colloidal-S carbohydrate and chlorophyll a when expressed on a dry weight or areal basis were attributed to effects of dewatering and concomitant changes in wet bulk density. The polymeric carbohydrate (colloidal-S EPS) component of the biofilms was not found to be a constant fraction of the colloidal-S carbohydrate extract, varying from 16 to 58%, and the percentage of polymer decreased logarithmically as chlorophyll a concentrations increased and the biofilms matured (P < 0.001). Changes in the relationships between these variables over the period of biofilm development and maturation highlight the difficulties in their use to predict sediment stability. Exopolymer concentrations were more closely correlated with algal biomass than with bacterial numbers. Rates of algal carbon fixation were considerably greater than those for bacteria, suggesting that the algae have a much greater potential for exopolymer production. It is suggested that the microphytobenthos secretions make a more important contribution to sediment stability. Received: 12 May 1999; Accepted: 13 October 1999; Online Publication: 24 March 2000     

Denitrification in San Francisco Bay Intertidal Sediments

The acetylene block technique was employed to study denitrification in intertidal estuarine sediments. Addition of nitrate to sediment slurries stimulated denitrification. During the dry season, sediment-slurry denitrification rates displayed Michaelis-Menten kinetics, and ambient NO₃⁻ + NO₂⁻ concentrations (≤26 μM) were below the apparent K_m (50 μM) for nitrate. During the rainy season, when ambient NO₃⁻ + NO₂⁻ concentrations were higher (37 to 89 μM), an accurate estimate of the K_m could not be obtained. Endogenous denitrification activity was confined to the upper 3 cm of the sediment column. However, the addition of nitrate to deeper sediments demonstrated immediate N₂O production, and potential activity existed at all depths sampled (the deepest was 15 cm). Loss of N₂O in the presence of C₂H₂ was sometimes observed during these short-term sediment incubations. Experiments with sediment slurries and washed cell suspensions of a marine pseudomonad confirmed that this N₂O loss was caused by incomplete blockage of N₂O reductase by C₂H₂ at low nitrate concentrations. Areal estimates of denitrification (in the absence of added nitrate) ranged from 0.8 to 1.2 μmol of N₂ m⁻² h⁻¹ (for undisturbed sediments) to 17 to 280 μmol of N₂ m⁻² h⁻¹ (for shaken sediment slurries).     

Structure and Dynamics of Exopolymers in an Intertidal Diatom Biofilm

Diatom biofilms growing at the surface of the intertidal mudflat of Marennes Oléron, France, were incubated for 48 h in the laboratory under simulated conditions of high- and low tide (immersed and emersed in seawater) and day and night (illuminated or dark conditions). The biofilms were subsequently sampled using the cryolander technique, without disturbing the structure. The samples were kept in liquid nitrogen until they were transferred to the cooled stage of a field-emission cryo-scanning electron microscope, which was used to study the structural relationships between the sediment particles, the diatoms and the different types of extracellular polymeric substances (EPS) produced by these organisms. The diatoms were most abundant at the sediment surface when incubated in the light under emersed conditions. In the dark or when immersed, the diatoms migrated into the sediment. In the light, the diatoms were coated with EPS, while this was not the case when incubated in the dark. When immersed, the sediment surface appeared smooth as the result of the deposition of mud. Under emersed conditions, the coarser silt grains were prominently present. These grains were wrapped with organic matter and bound together through threads of EPS. This was the case both in light and in dark incubated sediment. It is proposed that this latter type of EPS contributes to the increased erosion threshold of intertidal mudflats colonized by biofilms of diatoms.     

The Fate of Nitrate in Intertidal Permeable Sediments

Coastal zones act as a sink for riverine and atmospheric nitrogen inputs and thereby buffer the open ocean from the effects of anthropogenic activity. Recently, microbial activity in sandy permeable sediments has been identified as a dominant source of N-loss in coastal zones, namely through denitrification. Some of the highest coastal denitrification rates measured so far occur within the intertidal permeable sediments of the eutrophied Wadden Sea. Still, denitrification alone can often account for only half of the substantial nitrate (NO₃ ⁻) consumption. Therefore, to investigate alternative NO₃ ⁻ sinks such as dissimilatory nitrate reduction to ammonium (DNRA), intracellular nitrate storage by eukaryotes and isotope equilibration effects we carried out ¹⁵NO₃ ⁻ amendment experiments. By considering all of these sinks in combination, we could quantify the fate of the ¹⁵NO₃ ⁻ added to the sediment. Denitrification was the dominant nitrate sink (50–75%), while DNRA, which recycles N to the environment accounted for 10–20% of NO₃ ⁻ consumption. Intriguingly, we also observed that between 20 and 40% of ¹⁵NO₃ ⁻ added to the incubations entered an intracellular pool of NO₃ ⁻ and was subsequently respired when nitrate became limiting. Eukaryotes were responsible for a large proportion of intracellular nitrate storage, and it could be shown through inhibition experiments that at least a third of the stored nitrate was subsequently also respired by eukaryotes. The environmental significance of the intracellular nitrate pool was confirmed by in situ measurements which revealed that intracellular storage can accumulate nitrate at concentrations six fold higher than the surrounding porewater. This intracellular pool is so far not considered when modeling N-loss from intertidal permeable sediments; however it can act as a reservoir for nitrate during low tide. Consequently, nitrate respiration supported by intracellular nitrate storage can add an additional 20% to previous nitrate reduction estimates in intertidal sediments, further increasing their contribution to N-loss.     

Spatial and Temporal Variation of Phenanthrene-Degrading Bacteria in Intertidal Sediments

Phenanthrene-degrading bacteria were isolated from a 1-m² intertidal sediment site in Boston Harbor. Samples were taken six times over 2 years. A total of 432 bacteria were isolated and characterized by biochemical testing. When clustered on the basis of phenotypic characteristics, the isolates could be separated into 68 groups at a similarity level of approximately 70%. Several groups (a total of 200 isolates) corresponded to well-characterized species belonging the genera Vibrio and Pseudomonas. Only 51 of the 437 isolates (<11.7% of the total) hybridized to a DNA probe that encodes the upper pathway of naphthalene and phenanthrene degradation in Pseudomonas putida NCIB 9816. A cluster analysis indicated that the species composition of the phenanthrene-degrading community changed significantly from sampling date to sampling date. At one sampling time, 12 6-mm-diameter core subsamples were taken within the 1-m² site to determine the spatial variability of the degrading communities. An analysis of molecular variance, performed with the phenotypic characteristics, indicated that only 6% of the variation occurred among the 12 subsamples, suggesting that the subsamples were almost identical in composition. We concluded that the communities of phenanthrene-degrading bacteria in the sediments are very diverse, that the community structure undergoes significant change with time but does not vary significantly on a spatial scale of centimeters, and that the predominant genes that encode phenanthrene degradation in the communities are not well-characterized.     

Glucose Uptake and End Product Formation in an Intertidal Marine Sediment

Glucose uptake was monitored on a seasonal basis, using [6-³H]glucose and undisturbed cores collected from an intertidal mud flat. The fate of glucose carbon, including the formation of CO₂ and biomass, was assayed by using undisturbed cores and [U-¹⁴C]glucose; the production of short-chain fatty acids was monitored with [U-¹⁴C]glucose and sediment slurries. Rate constants for glucose uptake varied temporally, with temperature accounting for much of the variability; turnover times ranged from about 2 to 10 min. Rate constants decreased with increasing sediment depth and in the following order for several common monosaccharides: glucose>galactose>mannose~fucose. Time course analyses of ¹⁴CO₂ production provided evidence of significant isotopic dilution; although pore water glucose turnover times were on the order of minutes, ¹⁴CO₂ did not plateau until after approximately 6 h of incubation. At this time a maximum of about 40% of the added radioglucose had been respired. The extent of respiration varied as a function of sediment depth and season, with the highest values below the surface (4 to 7 cm) and in summer and fall. Incorporation of radiolabelled glucose into biomass also varied seasonally, but the greatest extent of incorporation (about 40%) was observed in the fall and for the 0- to 1-cm depth interval. The production of short-chain fatty acid end products was largely limited to acetate, which accounted for only a small percentage of the added radiolabel. Other organic acids, pyruvate in particular, were observed in pore water and were due to artifacts in the heat-kill procedure used to terminate incubations. An accurate assessment of the distribution and importance of short-chain fatty acids as end products required the use of an enzymatic technique coupled with high-pressure liquid chromatography to verify qualitative identities.     

Chemoautotrophic Carbon Fixation Rates and Active Bacterial Communities in Intertidal Marine Sediments

Chemoautotrophy has been little studied in typical coastal marine sediments, but may be an important component of carbon recycling as intense anaerobic mineralization processes in these sediments lead to accumulation of high amounts of reduced compounds, such as sulfides and ammonium. We studied chemoautotrophy by measuring dark-fixation of ¹³C-bicarbonate into phospholipid derived fatty acid (PLFA) biomarkers at two coastal sediment sites with contrasting sulfur chemistry in the Eastern Scheldt estuary, the Netherlands. At one site where free sulfide accumulated in the pore water right to the top of the sediment, PLFA labeling was restricted to compounds typically found in sulfur and ammonium oxidizing bacteria. At the other site, with no detectable free sulfide in the pore water, a very different PLFA labeling pattern was found with high amounts of label in branched i- and a-PLFA besides the typical compounds for sulfur and ammonium oxidizing bacteria. This suggests that other types of chemoautotrophic bacteria were also active, most likely Deltaproteobacteria related to sulfate reducers. Maximum rates of chemoautotrophy were detected in first 1 to 2 centimeters of both sediments and chemosynthetic biomass production was high ranging from 3 to 36 mmol C m⁻² d⁻¹. Average dark carbon fixation to sediment oxygen uptake ratios were 0.22±0.07 mol C (mol O₂)⁻¹, which is in the range of the maximum growth yields reported for sulfur oxidizing bacteria indicating highly efficient growth. Chemoautotrophic biomass production was similar to carbon mineralization rates in the top of the free sulfide site, suggesting that chemoautotrophic bacteria could play a crucial role in the microbial food web and labeling in eukaryotic poly-unsaturated PLFA was indeed detectable. Our study shows that dark carbon fixation by chemoautotrophic bacteria is a major process in the carbon cycle of coastal sediments, and should therefore receive more attention in future studies on sediment biogeochemistry and microbial ecology.     

Hydrocarbon Mineralization in Sediments and Plasmid Incidence in Sediment Bacteria from the Campeche Bank

Rates of degradation of radiolabeled hydrocarbons and incidence of bacterial plasmid DNA were investigated in sediment samples collected from the Campeche Bank, Gulf of Mexico, site of an offshore oil field containing several petroleum platforms. Overall rates of mineralization of [¹⁴C]hexadecane and [¹⁴C]phenanthrene measured for sediments were negligible; <1% of the substrate was converted to CO₂ in all cases. Low mineralization rates are ascribed to nutrient limitations and to lack of adaptation by microbial communities to hydrocarbon contaminants. Plasmid frequency data for sediment bacteria similarly showed no correlation with proximity to the oil field, but, instead, showed correlation with water column depth at each sampling site. Significant differences between sites were observed for proportion of isolates carrying single or multiple plasmids and mean number of plasmids per isolate, each of which increased as a function of depth.     

Seasonal Variations in Chlorophyll a Concentrations in Relation to Potentials of Sediment Phosphate Release by Different Mechanisms in a Large Chinese Shallow Eutrophic Lake (Lake Taihu)

The relationship between chlorophyll a and fractionation of sediment phosphorus, inorganic phosphate-solubilizing bacteria (IPB), and organic phosphate-mineralizing bacteria (OPB) was evaluated in a large Chinese shallow eutrophic lake (Lake Taihu) and its embayment (Wuli Bay). At the three study sites, the increase of chlorophyll a concentrations in April paralleled those of the iron bound phosphate accounting for major portion of sediment inorganic phosphate, and in June significantly higher OPB and IPB numbers (especially OPB) in sediment were main contributors to the peaks of chlorophyll a concentration. Even though IPB peaked from February to June, it should serve as an unimportant P source due to the irrelevancy with chlorophyll a and soluble reactive phosphorus (SRP). By contrast, at the other site in the embayment, the calcium-bound phosphate was predominant and solid, which was difficult to be released, and neither IPB nor OPB were detectable in the sediment, indicating weak potential for phosphorus release from the sediment, which was reflected in the small seasonal variation in SRP concentration in water column. Hence, the extents to which the three general mechanisms behind phosphate release from sediment (desorption of iron bound phosphate, solubilization by IPB and enzymatic hydrolysis by OPB) operated were different depending on seasons and sites in Lake Taihu, they may jointly drive phosphate release and accelerate the eutrophication processes.     

Measurements of Phytoplankton of Sub-Nanomolar Chlorophyll Concentrations by a Modified Double-Modulation Fluorometer

A modification of the double-modulation fluorometer is described that allows measuring very dilute phytoplankton samples. The high sensitivity is achieved by increasing the sample volume and by collecting the fluorescence from the large volume by an integrating sphere. The sensitivity of the instrument increased approximately proportionally to the volume of the sample. A further improvement of the sensitivity was achieved by replacing the PIN photodiode of the earlier versions by a photomultiplier. The instrument was used to measure fluorescence induction, F₀ and F_m parameters, and Q_A ^-reoxidation kinetics at concentrations at and below 100 pM chlorophyll. This revised version was published online in August 2006 with corrections to the Cover Date.     

Competition for Sulfate and Ethanol Among Desulfobacter, Desulfobulbus, and Desulfovibrio Species Isolated from Intertidal Sediments

Competition for sulfate and ethanol among Desulfobacter, Desulfobulbus, and Desulfovibrio species isolated from estuarine sediments was studied in energy-limited chemostats. Desulfovibrio baculatus was the most successful competitor for limiting amounts of sulfate and ethanol, followed by Desulfobulbus propionicus. The success of Desulfovibrio baculatus was dependent on the availability of sufficient iron. Of the three species studied, Desulfobacter postgatei was the least successful competitor for limiting amounts of sulfate. Although stimulating the growth of Desulfobacter postgatei, addition of Ca-saturated illite particles to culture media did not affect the outcome of competition for sulfate. Thus, under sulfate limitation acetate accumulated. This phenomenon was briefly discussed in relation to the flow of electrons during anaerobic mineralization in marine and estuarine sulfate-limited sediments.     

Depth-Related Differences in Organic Substrate Utilization by Major Microbial Groups in Intertidal Marine Sediment

Stable isotope probing of magnetic-bead-captured rRNA (Mag-SIP) indicated clear differences in in situ organic substrate utilization by major microbial groups between the more oxidized (0 to 2 cm) and sulfate-reducing (2 to 5 cm) horizons of marine intertidal sediment. We also showed that cyanobacteria and diatoms may survive by glucose utilization under dark anoxic conditions.     

The Diverse Bacterial Community in Intertidal, Anaerobic Sediments at Sapelo Island, Georgia

The phylogenetic diversity and composition of the bacterial community in anaerobic sediments from Sapelo Island, GA, USA were examined using 16S rRNA gene libraries. The diversity of this community was comparable to that of soil, and 1,186 clones formed 817 OTUs at 99% sequence similarity. Chao1 estimators for the total richness were also high, at 3,290 OTUs at 99% sequence similarity. The program RDPquery was developed to assign clones to taxonomic groups based upon comparisons to the RDP database. While most clones could be assigned to describe phyla, fewer than 30% of the clones could be assigned to a described order. Similarly, nearly 25% of the clones were only distantly related (<90% sequence similarity) to other environmental clones, illustrating the unique composition of this community. One quarter of the clones were related to one or more undescribed orders within the γ-Proteobacteria. Other abundant groups included the δ-Proteobacteria, Bacteroidetes, and Cyanobacteria. While these phyla were abundant in other estuarine sediments, the specific members at Sapelo Island appeared to be different from those previously described in other locations, suggesting that great diversity exists between as well as within estuarine intertidal sediments. In spite of the large differences in pore water chemistry with season and depth, differences in the bacterial community were modest over the temporal and spatial scales examined and generally restricted to only certain taxa. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Migration of Electronic Energy from Chlorophyll b to Chlorophyll a in Solutions

Absorption, emission, and fluorescence excitation spectra of pure solutions of chlorophyll a (Chl a) and chlorophyll b (Chl b) in diethyl ether and of equimolecular mixed solutions of the two pigments, were determined at room temperature as functions of concentration (in the range from 5 × 10^-6 M to 4 × 10^-3 M) and of wavelength of the exciting light (in the regions 380-465 and 550-650 nm). The efficiency of energy transfer from Chl b to Chl a, derived from these data, was found to depend on the wavelength of exciting light. Furthermore, the transfer efficiency calculated from sensitization of Chl a fluorescence by Chl b was substantially smaller than that calculated from quenching of Chl b fluorescence by Chl a. Both these effects are tentatively explained as evidence of superposition of a “fast” energy transfer (taking place before the Boltzmann distribution of vibrational energy had been reached) upon the “delayed” transfer, which takes place after vibrational equilibration. The first-named mechanism is made possible by overlapping of the absorption bands of the two pigments; the second, by overlapping of the emission band of Chl b and the absorption band of Chl a. The first mechanism can lead to repeated transfer of excitation energy between pigment molecules, the second only to a one-time transfer from the donor to the acceptor. Both mechanisms could be of the same, second-order type, with the transfer rate proportional to r^-6. An alternative is for the fast mechanism to be of the first order, with the transfer rate proportional to r^-3, but spectroscopic evidence seems to make this alternative less probable.     

Degradation and Mineralization of the Polycyclic Aromatic Hydrocarbons Anthracene and Naphthalene in Intertidal Marine Sediments

The degradation of the polynuclear aromatic hydrocarbons (PAHs) anthracene and naphthalene by the microbiota of intertidal sediments was investigated in laboratory studies. No mineralization of either PAH was observed in the absence of oxygen. Both rates and total amounts of PAH mineralization were strongly controlled by oxygen content and temperature of the incubations. Inorganic nitrogen and glucose amendments had minimal effects on PAH mineralization. The rates and total amounts of PAH mineralized were directly related to compound concentration, pre-exposure time, and concentration. Maximum mineralization was observed at the higher concentrations (5 to 100 μg/g [ppm]) of both PAHs. Optimal acclimation to anthracene and naphthalene (through pre-exposures to the compounds) occurred at the highest acclimation concentration (1,000 ppm). However, acclimation to a single concentration (100 ppm) resulted in initial relative mineralization rates over a range of re-exposure concentrations (1 to 1,000 ppm) being nearly identical. Maximum mineralization of both PAHs occurred after intermediate periods (1 to 2 weeks) of pre-exposure. The fraction of the total heterotrophic population capable of utilizing anthracene or naphthalene as sole carbon source was also greatest after 2 weeks.     

Interactions between Benthic Copepods,Bacteria and Diatoms Promote Nitrogen Retention in Intertidal Marine Sediments

The present study aims at evaluating the impact of diatoms and copepods on microbial processes mediating nitrate removal in fine-grained intertidal sediments. More specifically, we studied the interactions between copepods, diatoms and bacteria in relation to their effects on nitrate reduction and denitrification. Microcosms containing defaunated marine sediments were subjected to different treatments: an excess of nitrate, copepods, diatoms (Navicula sp.), a combination of copepods and diatoms, and spent medium from copepods. The microcosms were incubated for seven and a half days, after which nutrient concentrations and denitrification potential were measured. Ammonium concentrations were highest in the treatments with copepods or their spent medium, whilst denitrification potential was lowest in these treatments, suggesting that copepods enhance dissimilatory nitrate reduction to ammonium over denitrification. We hypothesize that this is an indirect effect, by providing extra carbon for the bacterial community through the copepods'' excretion products, thus changing the C/N ratio in favour of dissimilatory nitrate reduction. Diatoms alone had no effect on the nitrogen fluxes, but they did enhance the effect of copepods, possibly by influencing the quantity and quality of the copepods'' excretion products. Our results show that small-scale biological interactions between bacteria, copepods and diatoms can have an important impact on denitrification and hence sediment nitrogen fluxes.     

Inorganic Nutrient Concentrations and Chlorophyll in the Euphotic Zone of Lake Tanganyika

The taxonomic value of nematocyst size in sea anemones is still being assessed. We evaluate size distribution of nematocysts of one type in a single individual anemone. Length of unfired nematocysts was measured along the column, tentacles, and actinopharynx of a preserved specimen of Actinodendron arboreum (Quoy & Gaimard, 1833). Mean, range, minimum, and maximum length of nematocysts vary along the column, those in the middle region being least variable. The length of nematocysts in mature (split) acrospheres is less variable than in immature (unsplit) acrospheres. There is significant variability between nematocysts in tentacles of the primary and quaternary cycles, and along a tentacle, the middle being least variable. Size distribution of actinopharynx nematocysts is complex. The results of this study suggest that assembling data on nematocysts from multiple individuals for taxonomic purposes should be used with an awareness that sampling site can be an important variable. Ideally, the position of tissue sampled should be documented, an attempt should be made to be consistent in sampling from the same position in individuals being compared, and the variability of nematocyst length at each sampled site should be assessed. Inferences can also be made on ontogeny from these data; we conclude that an actinodendrid tentacle grows from the base and at the tips of its branches.     

Aerobic Biofilms Grown from Athabasca Watershed Sediments Are Inhibited by Increasing Concentrations of Bituminous Compounds

Sediments from the Athabasca River and its tributaries naturally contain bitumen at various concentrations, but the impacts of this variation on the ecology of the river are unknown. Here, we used controlled rotating biofilm reactors in which we recirculated diluted sediments containing various concentrations of bituminous compounds taken from the Athabasca River and three tributaries. Biofilms exposed to sediments having low and high concentrations of bituminous compounds were compared. The latter were 29% thinner, had a different extracellular polysaccharide composition, 67% less bacterial biomass per μm², 68% less cyanobacterial biomass per μm², 64% less algal biomass per μm², 13% fewer protozoa per cm², were 21% less productive, and had a 33% reduced content in chlorophyll a per mm² and a 20% reduction in the expression of photosynthetic genes, but they had a 23% increase in the expression of aromatic hydrocarbon degradation genes. Within the Bacteria, differences in community composition were also observed, with relatively more Alphaproteobacteria and Betaproteobacteria and less Cyanobacteria, Bacteroidetes, and Firmicutes in biofilms exposed to high concentrations of bituminous compounds. Altogether, our results suggest that biofilms that develop in the presence of higher concentrations of bituminous compounds are less productive and have lower biomass, linked to a decrease in the activities and abundance of photosynthetic organisms likely due to inhibitory effects. However, within this general inhibition, some specific microbial taxa and functional genes are stimulated because they are less sensitive to the inhibitory effects of bituminous compounds or can degrade and utilize some bitumen-associated compounds.