Enumeration of Particle-Bound and Unattached Respiring Bacteria in the Salt Marsh Environment

Proportions of respiring bacteria determined with a 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride dye-epifluorescent technique were significantly elevated in the 300-μm surface layer of a salt marsh estuary. Almost all the detectably respiring bacteria in the particle-laden surface layer and a significant proportion in subsurface waters were attached to particles.     

Ectoenzymatic activity and glucose heterotrophic metabolism in a shallow estuary (Ria de Aveiro,Portugal): influence of bed sediments and salt marshes

Bacterioplankton abundance and activity were studied in the estuarine system of Ria de Aveiro (Portugal) to test if tidal resuspension of sediments and transport of particles from the salt marshes may act as factors of variability of bacterial communities. The total and attached cell abundance, ectoenzymatic activity and the heterotrophic metabolism of glucose, as well as seston, chlorophyll a and particulate organic carbon (POC) were monitored during four 10-h periods along the tidal cycle at four sampling sites across a transect. The variation of particulate materials (seston, POC and chlorophyll a ) along the transect was not significantly correlated with either distance to the margin or distance to the sediment surface. Nevertheless, proximity to the salt marsh or to the bottom sediment surface favoured glucose incorporation and aminopeptidase activity. A multiple stepwise linear regression analysis using temperature, salinity, seston, POC, chlorophyll a , distance to sediment surface and distance to the margin as independent variables explained 66.5% of the variability of the fraction of particle-attached bacteria and only a very small proportion (12–43%) of the observed variability of total bacterial abundance, ectoenzymatic activity and glucose utilization. The spatial patterns of variation of the concentration of particulate material (seston, POC and chlorophyll a) do not clearly indicate the occurrence of sediment resuspension and runoff from the margins. This, together with the poor contribution of these parameters to the transversal and tidal variability of bacterial activity, dismisses the importance of inputs of suspended material across the sediment/water interface and from neighbouring salt marshes in the control of bacterial density and activity.     

Dissolved organic carbon and bacterial populations in the gelatinous surface microlayer of a Norwegian fjord mesocosm

The sea surface microlayer is the interfacial boundary layer between the marine environment and the troposphere. Surface microlayer samples were collected during a fjord mesocosm experiment to study microbial assemblage dynamics within the surface microlayer during a phytoplankton bloom. Transparent exopolymer particles were significantly enriched in the microlayer samples, supporting the concept of a gelatinous surface film. Dissolved organic carbon and bacterial cell numbers (determined by flow cytometry) were weakly enriched in the microlayer samples. However, the numbers of Bacteria 16S rRNA genes (determined by quantitative real-time PCR) were more variable, probably due to variable numbers of bacterial cells attached to particles. The enrichment of transparent exopolymer particles in the microlayer and the subsequent production of a gelatinous biofilm have implications on air–sea gas transfer and the partitioning of organic carbon in surface waters.     

Macrophyte disturbance alters aquatic surface microlayer structure,metabolism, and fate

Macrophytes drive the functioning of many salt marsh ecosystem components. We questioned how temporary clearing of the macrophyte community, during restoration, would impact processes at the scale of the aquatic surface microlayer. Development, deposition, and breakup of the tidal creek surface microlayer were followed over tidal cycles seasonally in a cleared “former” Phragmites marsh and an adjacent restored Spartina marsh. Metabolic and physical processes of the mobile surface microlayers and underlying water were compared, along with distribution of organic and inorganic components onto simulated plant stems. In July and October, chlorophyll-a quantities were less on simulated stems in the cleared site than in the restored site. The aquatic microlayer in the cleared site creek exhibited lower photosynthesis and respiration rates, fewer diatoms and green algae, and less chlorophyll-a. There was a lower concentration (250 times) and reduced diversity of fatty acids in the surface microlayer of the cleared site, reflecting a smaller and less diverse microbial community and reduced food resources. Fiddler crab activity was an order of magnitude higher where macrophytes had been cleared. Their consumption of edaphic algae on the mud surface may account for the reduced algae and other organics in the creek surface microlayer, thus representing a redirection of this food resource from creek consumers. Overall, there were less total particulates in the creek surface microlayer at the cleared site, and they dropped out of the surface microlayer sooner in the tidal cycle, resulting in a lower sediment load available for deposit onto marsh surfaces.     

Numerical dominance and phylotype diversity of marine Rhodobacter species during early colonization of submerged surfaces in coastal marine waters as determined by 16S ribosomal DNA sequence analysis and fluorescence in situ hybridization

Early stages of surface colonization in coastal marine waters appear to be dominated by the marine Rhodobacter group of the alpha subdivision of the division Proteobacteria (alpha-Proteobacteria). However, the quantitative contribution of this group to primary surface colonization has not been determined. In this study, glass microscope slides were incubated in a salt marsh tidal creek for 3 or 6 days. Colonizing bacteria on the slides were examined by fluorescence in situ hybridization by employing DNA probes targeting 16S or 23S rRNA to identify specific phylogenetic groups. Confocal laser scanning microscopy was then used to quantify and track the dynamics of bacterial primary colonists during the early stages of surface colonization and growth. More than 60% of the surface-colonizing bacteria detectable by fluorescence staining (Yo-Pro-1) could also be detected with the Bacteria domain probe EUB338. Archaea were not detected on the surfaces and did not appear to participate in surface colonization. Of the three subdivisions of the Proteobacteria examined, the alpha-Proteobacteria were the most abundant surface-colonizing organisms. More than 28% of the total bacterial cells and more than 40% of the cells detected by EUB338 on the surfaces were affiliated with the marine Rhodobacter group. Bacterial abundance increased significantly on the surfaces during short-term incubation, mainly due to the growth of the marine Rhodobacter group organisms. These results demonstrated the quantitative importance of the marine Rhodobacter group in colonization of surfaces in salt marsh waters and confirmed that at least during the early stages of colonization, this group dominated the surface-colonizing bacterial assemblage.     

The hydrophobicity of bacteria — An important factor in their initial adhesion at the air-water inteface

Bacteria isolated from the surface and the subsurface water at four stations along the Swedish west coast were assessed for their hydrophobicity with hydrophobic interaction chromatography (HIC). The surface bacteria were sampled by the Teflon sheet technique. [³H]-l-leucine metabolically labeled isolates were run on a column packed with Octyl-Sepharose CL-4B gel. The relative hydrophobicity of the bacteria was expressed as the ratio, g/e, between the radioactivity of the gel and the eluate. The results revealed a positive correlation between the degree of enrichment of bacteria at the surface and their hydrophobicity. The subsurface bacteria exhibited a broader spectrum of g/e-values than the surface bacteria. The initial adhesion of bacteria to the surface microlayer depends on several factors of which the hydrophobic interaction may be one of the most important.Abbreviations HIC hydrophobic interaction chromatography - NSS nine salt solution     

Attached and Free-Floating Bacteria in a Diverse Selection of Water Bodies

The contribution of attached and free-floating bacteria to the bacterial numbers and heterotrophic uptake in 44 diverse aquatic environments was studied. A factor analysis reduced the variability of the raw data base to three major factors explaining 53.6% of total variance. These factors were (i) salinity, (ii) heterotrophic uptake, and (iii) particulate load. A cluster analysis categorized the 44 habitats into five distinct environmental types based on these three factors. There was no significant pattern in the distribution of attached versus free-floating bacteria when assessed by epifluorescent microscopy. However the contribution of attached bacteria to the uptake of an amino acids mix was reduced in marine waters. Heavy particulate loads resulted in an increased percentage uptake of amino acids and glucose from the attached bacteria. Uptake response was found to be substrate specific especially in oliogotrophic freshwater. Amino acid uptake was more associated with the attached fraction, whereas glucose uptake was mediated more by the free-floating fraction.     

Exploiting wild population diversity and somaclonal variation in the salt marsh grass Distichlis spicata (Poaceae) for marsh creation and restoration

The salt marsh grass Distichlis spicata was regenerated from tissue culture and propagated in a greenhouse. Selected regenerants, along with selections from six wild populations, were grown for two years in a common garden flood-irrigated thrice weekly with tidal creek water. Selected wild and regenerated plants were also planted in a created salt marsh. Significant differences among regenerant and wild population selections were found in several functionally important salt marsh plant characteristics, including potential detritus production, belowground organic matter production, canopy structure, and decomposition rate. A combination of characteristics not found in the wild populations was evident in a regenerated line that exhibited both a high detritus production potential and a high decomposition rate. The amount of variation that occurred among regenerants from one parental line via somaclonal variation was similar to that which occurred among the wild population selections. Results of this study suggest that tissue culture may provide a means of producing marsh grasses with specific characteristics for directing the functional development of newly created salt marshes.     

A survey on bacteria inhabiting the sea surface microlayer of coastal ecosystems

Bacterial populations inhabiting the sea surface microlayer from two contrasted Mediterranean coastal stations (polluted vs. oligotrophic) were examined by culturing and genetic fingerprinting methods and were compared with those of underlying waters (50 cm depth), for a period of two years. More than 30 samples were examined and 487 strains were isolated and screened. Proteobacteria were consistently more abundant in the collection from the pristine environment whereas Gram-positive bacteria (i.e., Actinobacteria and Firmicutes) were more abundant in the polluted site. Cythophaga-Flavobacter-Bacteroides (CFB) ranged from 8% to 16% of total strains. Overall, 22.5% of the strains showed a 16S rRNA gene sequence similarity only at the genus level with previously reported bacterial species and around 10.5% of the strains showed similarities in 16S rRNA sequence below 93% with reported species. The CFB group contained the highest proportion of unknown species, but these also included Alpha- and Gammaproteobacteria. Such low similarity values showed that we were able to culture new marine genera and possibly new families, indicating that the sea-surface layer is a poorly understood microbial environment and may represent a natural source of new microorganisms. Genetic fingerprinting showed, however, no consistent differences between the predominant bacterial assemblages from surface microlayer and underlying waters, suggesting that the presence of a stable and abundant neustonic bacterial community is not a common trait of coastal marine environments.     

Compositions isotopiques naturelles des bactéries hétérotrophes et détermination de l'origine du carbone organique dissous biodisponible

Several studies of salt marsh systems have attempted to quantify the flow of organic matter between the land and coastal waters. However, the techniques used could not identify sources of dissolved organic carbon (DOC) rapidly assimilated by heterothrophic bacteria. Recently, the assay of carbon isotope ratios has allowed characterization of the different sources of organic matter in salt marshes. In this study, we wanted to find out if the natural isotopic composition assayed in heterotrophic bacteria distinguished the origin of bioavailable DOC. We determined the δ¹³C values for 1) three bacterial strains and their nucleic acids cultured on glucose and tryptose substrates, respectively, and 2) naturally occurring bacteria recovered from seawater in which salt marsh vegetation had been immersed. First, we showed that the isotopic fractionation was the same for the three bacterial strains cultured on the same synthetic substrate, but could vary depending on the nature of DOC. There was no significant difference between the δ¹³ C values of bacteria and their nucleic acids. Second, natural bacteria were grown in a medium enriched in DOC from halophytic plants. The δ¹³C values of this community were close to those of dissolved organic carbon from plant leachates. The comparison between the isotopic ratios of natural bacteria in Vibrio alginolyticus showed that the heterogeneity of the bacterial community averaged the isotopic fractionation from the preferential assimilation of organic compounds in the medium by each bacterial strain. The δ¹³ C values recorded for the bacterial community in the field and their nucleic acids made it possible to identify the source of organic matter readily accessible to microorganisms in a coastal ecosystem.     

Influence of salt marsh on bacterial activity in two estuaries with different hydrodynamic characteristics (Ria de Aveiro and Tagus Estuary)

The influence of salt marsh on estuarine bacterioplankton was investigated in two estuaries with different hydrodynamic characteristics (Ria de Aveiro and Tagus Estuary). In the Ria de Aveiro, bacteria in the flood water overlying the marsh were two times more abundant and five to six times more active than in the main channel. In the Tagus Estuary, bacterial abundance was similar in flooding and channel water, but bacterial activity was up to two times higher in the main channel. The two salt marshes have distinct influences on estuarine bacterioplankton abundance and activity. In the Ria de Aveiro, salt marsh enhanced estuarine bacterial communities, increasing their size and stimulating their activity. By contrast, the salt marsh in the Tagus Estuary does not seem to increase the bacterial abundance and production in the channel water. These distinct influences may be explained by the hydrodynamic characteristics of the salt marshes, which were confirmed by the hydrodynamic model implemented for both systems.     

Numerical Dominance and Phylotype Diversity of Marine Rhodobacter Species during Early Colonization of Submerged Surfaces in Coastal Marine Waters as Determined by 16S Ribosomal DNA Sequence Analysis and Fluorescence In Situ Hybridization

Early stages of surface colonization in coastal marine waters appear to be dominated by the marine Rhodobacter group of the α subdivision of the division Proteobacteria (α-Proteobacteria). However, the quantitative contribution of this group to primary surface colonization has not been determined. In this study, glass microscope slides were incubated in a salt marsh tidal creek for 3 or 6 days. Colonizing bacteria on the slides were examined by fluorescence in situ hybridization by employing DNA probes targeting 16S or 23S rRNA to identify specific phylogenetic groups. Confocal laser scanning microscopy was then used to quantify and track the dynamics of bacterial primary colonists during the early stages of surface colonization and growth. More than 60% of the surface-colonizing bacteria detectable by fluorescence staining (Yo-Pro-1) could also be detected with the Bacteria domain probe EUB338. Archaea were not detected on the surfaces and did not appear to participate in surface colonization. Of the three subdivisions of the Proteobacteria examined, the α-Proteobacteria were the most abundant surface-colonizing organisms. More than 28% of the total bacterial cells and more than 40% of the cells detected by EUB338 on the surfaces were affiliated with the marine Rhodobacter group. Bacterial abundance increased significantly on the surfaces during short-term incubation, mainly due to the growth of the marine Rhodobacter group organisms. These results demonstrated the quantitative importance of the marine Rhodobacter group in colonization of surfaces in salt marsh waters and confirmed that at least during the early stages of colonization, this group dominated the surface-colonizing bacterial assemblage.     

Isolation of Atypical Candida albicans from the North Sea

Isolates of Candida albicans with sparse filamentation and weak fermentation were isolated from the surface microlayer of the North Sea, but not from subsurface waters. Such atypical isolates may be misidentified by using normal taxonomic procedures.     

Influence of the surface microlayer on nutrients,chlorophyll and algal diversity of a small eutrophic bog pond

Nutrients, chlorophyll, phaeophytin and algal abundances were investigated in the surface microlayer and at subsurface depths in a small eutrophic bog pond. Nutrient levels were consistently higher in the microlayer while algal abundance was sometimes higher but sometimes lower in the microlayer than at near subsurface depths. Algal diversity values were strongly influenced by the depth of flagellate blooms, and in contrast to previous studies, diversity in the microlayer was higher than at near subsurface depths. These results are discussed in terms of weather parameters, affinity of algal species for the surface and differences between microlayer ecology in shallow and deep water systems.     

Distribution of urea-decomposing bacteria in waters of lake suwa

The present study deals with the distribution of free and attached urea-decomposing bacteria in waters of Lake Suwa, one of the typical eutrophic lakes in Japan.Urea-decomposing bacteria are proved to be not an exceptional population among the populations of the heterotrophic bacteria studied. The percentage of attached urea-decomposing bacteria versus attached heterotrophic bacteria seems to be higher than that of free urea-decomposing bacteria versus free heterotrophic bacteria. Based on the assumption that most bacterial ureases are inducible ones, it is inferred that their ureases may not be induced under a such degree of urea concentration as is generally encountered in natural waters.     

Stratification of microorganisms and nutrients in the surface microlayer of small freshwater ponds

Using two different surface microlayer sampling devices (the Harvey and Burzell glass plate and the Garrett screen), two microlayer fractions could be distinguished. The first was from 0 to 50 µm and the second was from 51 to 320 µm. Significantly different (p < 0.05) concentrations of dissolved nutrients between the two microlayer fractions strongly suggests stratification within the surface microlayer. This apparent stratification is also examined for phototrophs, bacteria and other material found within the surface microlayer.Based in part on an M.S. thesis, submitted to the Graduate School of University of Wisconsin - Milwaukee.     

Attached and free-liviing dividing bacteria in two aquatic systems

The percentage of dividing biomass was calculated for attached and free-living bacteria, in a coastal marine and a freshwater system. In the marine system with low concentrations of total and dissolved organic carbon (TOC and DOC) the percentage of dividing biomass was higher for attached (41.4 ± 13.9) than for the free-living bacteria (22.0 ± 11.7). However, in the freshwater system, which had a higher concentration of TOC and DOC, the percentage of dividing biomass was similar for both communities-attached (53.4 ± 26.5) and free-living (78.4 ± 21.9). Thus the attachment to particulate material is not necessarily an advantage in waters where dissolved organic nutrients are readily available.     

Attached and Free-Floating Bacterioplankton in Howe Sound, British Columbia, a Coastal Marine Fjord-Embayment

Factors which influence the attachment of bacterioplankton to particles (including phytoplankton) were investigated by using (i) water samples removed from a coastal temperate fjord over an annual cycle and (ii) unialgal cultures of Prorocentrum minimum, Dunaliella tertiolecta, and Skeletonema costatum. Silt and salinity levels in this fjord seawater did not appear to influence bacterial attachment, but the percent attached bacteria was inversely related to both chlorophyll a concentrations and primary productivities. During periods of high primary productivities the percent attached bacteria was low, whereas during periods of low, increasing, and declining primary productivities the percent attached bacteria was high. A similar pattern of bacterial attachment was observed when the three phytoplankton were grown as batch cultures. The percent attached bacterial numbers increased upon the initiation of algal growth and after these cells stopped growing, but not while the algae were growing. We suggest that a major factor influencing the attachment of bacterioplankton is the physiological condition of their major nutrient source, the phytoplankton; mainly free-living bacteria are associated with growing phytoplankton, whereas a much greater proportion of the bacteria are attached among senescent phytoplankton populations.     

Effects of surfactant adsorption and biodegradability on the distribution of bacteria between sediments and water in a freshwater microcosm.

A microcosm containing resuspended river sediment was used to investigate the effect of anionic surfactants on the distribution of bacteria between planktonic and attached populations. Freshwater river sediment containing viable bacteria was preequilibrated in the microcosm, which was subsequently supplemented with biodegradable or recalcitrant surfactants and a non-surface-active carbon and energy source. Population dynamics of both free-living and attached bacteria were measured by epifluorescence microscopy with simultaneous analysis of the residual solution concentration of the xenobiotic carbon source. The addition of the readily biodegradable anionic surfactants sodium decyl sulfate and sodium dodecyl sulfate in separate experiments caused an increase in the number of attached bacteria and a concomitant decrease in the number of free-living bacteria. As biodegradation of the surfactants progressed, these trends reversed and the bacterial populations had returned to their preaddition values by the time when biodegradation was completed. In contrast, sodium tetradecyl sulfate or sodium dodecane sulfonate did not stimulate bacterial association with sediment, nor were they biodegraded in the microcosm. Sodium pyruvate, a non-surface-active carbon and energy source, was readily utilized but caused no bacterial attachment to the sediment. These results indicate that for an anionic surfactant to induce bacterial attachment to river sediment, it must be biodegradable. The bacterial attachment to the sediment appears to be reversible and may be dependent on the accumulation of the surfactant at the surface or as a result of alteration of the surface free energies.     

Effects of surfactant adsorption and biodegradability on the distribution of bacteria between sediments and water in a freshwater microcosm.

A microcosm containing resuspended river sediment was used to investigate the effect of anionic surfactants on the distribution of bacteria between planktonic and attached populations. Freshwater river sediment containing viable bacteria was preequilibrated in the microcosm, which was subsequently supplemented with biodegradable or recalcitrant surfactants and a non-surface-active carbon and energy source. Population dynamics of both free-living and attached bacteria were measured by epifluorescence microscopy with simultaneous analysis of the residual solution concentration of the xenobiotic carbon source. The addition of the readily biodegradable anionic surfactants sodium decyl sulfate and sodium dodecyl sulfate in separate experiments caused an increase in the number of attached bacteria and a concomitant decrease in the number of free-living bacteria. As biodegradation of the surfactants progressed, these trends reversed and the bacterial populations had returned to their preaddition values by the time when biodegradation was completed. In contrast, sodium tetradecyl sulfate or sodium dodecane sulfonate did not stimulate bacterial association with sediment, nor were they biodegraded in the microcosm. Sodium pyruvate, a non-surface-active carbon and energy source, was readily utilized but caused no bacterial attachment to the sediment. These results indicate that for an anionic surfactant to induce bacterial attachment to river sediment, it must be biodegradable. The bacterial attachment to the sediment appears to be reversible and may be dependent on the accumulation of the surfactant at the surface or as a result of alteration of the surface free energies.