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.     

Large differences in the fraction of active bacteria in plankton,sediments, and biofilm

Generally, only a small fraction of free-living pelagic bacteria are metabolically active, while particle-associated bacteria usually exhibit a larger proportion of active bacteria. Most previous studies on the active fraction of bacteria focus on planktonic communities, and there are only a few studies on sediment and epiphytic biofilm bacteria. We compared the active fraction of the total number of bacteria in three different habitats of the littoral zone of Lake Erken, Sweden, including the sediments, the epiphytic biofilm on the submerged macrophyte Ranunculus circinatus, and the water column. Active bacteria were detected as those with an active electron transport system, identified by the capacity to reduce the tetrazolium salt CTC (5-cyano-2,3-ditolyltetrazolium chloride) into its fluorescent, water insoluble state. There were large differences between habitats. The active fraction of the total number of bacteria detected by fluorescence microscopy (annual mean +/- SD) in the sediments was 46 +/- 10%, on R. circinatus 37 +/- 18%, and in the water column 4 +/- 4%. The abundance of CTC-reducing cells was correlated with total bacterial abundance, and the fraction of CTC-reducing bacteria generally increased with total bacterial abundance, for all the habitats. Consequently, the difference in the fraction of CTC-reducing bacteria between the habitats could be attributed to different densities of bacteria, with a larger proportion of active bacteria at higher bacterial densities.     

Comparison of Free-Living and Particle-Associated Bacterial Communities in the Chesapeake Bay by Stable Low-Molecular-Weight RNA Analysis

Free-living and particle-associated bacterial communities in the Chesapeake Bay estuary were analyzed and compared by using acridine orange direct counts and low-molecular-weight (LMW) RNA analysis. Samples were taken from top and bottom waters at upper- and mid-bay sites in December 1992. Free-living bacteria dominated the bacterial numbers at all sampling sites, although particle-associated bacteria increased in areas with greater particle loads. LMW RNAs (5S rRNA and tRNA) obtained directly from free-living, particle-associated, and total bacterioplankton communities were analyzed by high-resolution electrophoresis. There were distinct differences in the migration distances between LMW RNAs of free-living and particle-associated communities taken from the same site, indicating that the two communities differ in composition. In addition, LMW RNA profiles differed minimally with depth for all of the communities examined, presumably because of vertical mixing. 5S rRNAs of free-living communities from the upper- and mid-bay regions differed considerably. Particle-associated RNAs, on the other hand, were very similar, suggesting consistent environmental conditions on particles that select for similar community members. Lastly, several isolated bacteria had 5S rRNAs that were not detected in their respective extracted community 5S rRNAs, indicating that these isolated organisms were not representative of dominant members.     

Relationship between the Intracellular Integrity and the Morphology of the Capsular Envelope in Attached and Free-Living Marine Bacteria

The integrity of the intracellular structures and the presence and dimension of the capsular envelope were investigated in marine snow-associated and marine free-living bacteria by transmission electron microscopy and special fixation techniques. Three categories depending on the presence of internal structures were differentiated. In marine snow, 51% of the marine snow-associated bacterial community was considered intact, 26% had a partly degraded internal structure, and 23% were empty with only the cell wall remaining. For the free-living bacterial community, 34% were intact cells, 42% exhibited damage, and 24% of the cells were lacking any internal structure. We also investigated the morphology and the extent of the bacterial capsular envelope. More than 95% of all intact marine snow-associated bacteria were surrounded by a capsule while (apprx=)55% of empty marine snow-associated bacteria had no capsule. For free-living bacteria, (apprx=)65% of the intact cells had a capsule while (apprx=)80% of the empty free-living bacteria lacked a capsule. Thus there is a clear trend from intact cells which are commonly surrounded by a capsular envelope to empty bacteria for which only the cell wall is remaining. Since bacterioplankton represent the largest living surface in the ocean, it is concluded that the release of intracellular material from bacteria into the environment as well as the release of extracellular capsular material might fuel the dissolved organic matter pool of the ocean.     

Diel and Seasonal Variations in Abundance,Activity, and Community Structure of Particle-Attached and Free-Living Bacteria in NW Mediterranean Sea

Diel and seasonal variations in abundance, activity, and structure of particle-attached vs free-living bacterial communities were investigated in offshore NW Mediterranean Sea (0–1000 m). Attached bacteria were always less abundant and less diverse but generally more active than free-living bacteria. The most important finding of this study was that the activity of attached bacteria showed pronounced diel variations in the upper mixed water column with higher activities at night. Under mesotrophic conditions, the contribution of attached bacteria to total bacterial activity increased from less than 10% at day time to 83% at night time. At high chlorophyll a concentration, the highest cell-specific activities and contribution to total bacterial activity were due to free-living bacteria at day and to attached bacteria at night. Under summer oligotrophic conditions, free-living bacteria dominated and contributed to the most important part of the bacterial activity at both day and night, whereas attached bacteria were much less abundant but presented the highest cell-specific activities. These diel and seasonal variations in activities were concomitant to changes in bacterial community structure, mainly in the upper layer. The number of attached ribotypes was fairly constant suggesting that particles are colonized by a relatively limited number of ubiquitous ribotypes. Most of these ribotypes were also free-living ribotypes suggesting that attached bacteria probably originate from colonization of newly formed particles by free-living bacteria in the upper layer. These results reinforce the biogeochemical role of attached bacteria in the cycling of particulate organic carbon in the NW Mediterranean Sea and the importance of diel variability in these processes.     

Complexity of bacterial communities in a river-floodplain system (Danube, Austria)

Natural floodplains play an essential role in the processing and decomposition of organic matter and in the self-purification ability of rivers, largely due to the activity of bacteria. Knowledge about the composition of bacterial communities and its impact on organic-matter cycling is crucial for the understanding of ecological processes in river-floodplain systems. Particle-associated and free-living bacterial assemblages from the Danube River and various floodplain pools with different hydrological characteristics were investigated using terminal restriction fragment length polymorphism analysis. The particle-associated bacterial community exhibited a higher number of operational taxonomic units (OTUs) and was more heterogeneous in time and space than the free-living community. The temporal dynamics of the community structure were generally higher in isolated floodplain pools. The community structures of the river and the various floodplain pools, as well as those of the particle-associated and free-living bacteria, differed significantly. The compositional dynamics of the planktonic bacterial communities were related to changes in the algal biomass, temperature, and concentrations of organic and inorganic nutrients. The OTU richness of the free-living community was correlated with the concentration and origin of organic matter and the concentration of inorganic nutrients, while no correlation with the OTU richness of the particle-associated assemblage was found. Our results demonstrate the importance of the river-floodplain interactions and the influence of damming and regulation on the bacterial-community composition.     

Complexity of Bacterial Communities in a River-Floodplain System (Danube, Austria)

Natural floodplains play an essential role in the processing and decomposition of organic matter and in the self-purification ability of rivers, largely due to the activity of bacteria. Knowledge about the composition of bacterial communities and its impact on organic-matter cycling is crucial for the understanding of ecological processes in river-floodplain systems. Particle-associated and free-living bacterial assemblages from the Danube River and various floodplain pools with different hydrological characteristics were investigated using terminal restriction fragment length polymorphism analysis. The particle-associated bacterial community exhibited a higher number of operational taxonomic units (OTUs) and was more heterogeneous in time and space than the free-living community. The temporal dynamics of the community structure were generally higher in isolated floodplain pools. The community structures of the river and the various floodplain pools, as well as those of the particle-associated and free-living bacteria, differed significantly. The compositional dynamics of the planktonic bacterial communities were related to changes in the algal biomass, temperature, and concentrations of organic and inorganic nutrients. The OTU richness of the free-living community was correlated with the concentration and origin of organic matter and the concentration of inorganic nutrients, while no correlation with the OTU richness of the particle-associated assemblage was found. Our results demonstrate the importance of the river-floodplain interactions and the influence of damming and regulation on the bacterial-community composition.     

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.     

Community composition and activity of prokaryotes associated to detrital particles in two contrasting lake ecosystems

The composition, distribution and extracellular enzyme activities of bacteria attached to small (2-50 microm in size) transparent exopolymer and Coomassie-stained proteinaceous particles (TEP and CSP) were examined in two lakes of different trophic status located in the Massif Central of France. TEP concentrations (10(4)-10(6) particle per L) were significantly higher in the more productive lake and were significantly related to chlorophyll a concentrations. The majority of TEP and CSP were colonized by bacteria that constituted 2.6% and 7.4% of the total 4',6-diamidino-2-phenylindole-stained bacteria in lakes Pavin and Aydat, respectively. In both lakes, the composition of particle-associated bacteria was different from that of free-living bacteria, the Betaproteobacteria and Bacteroidetes (i.e. former Cytophaga-Flavobacteria group) being the dominant groups on particles. We also found that 2-5 microm TEP were more colonized than 2-5 microm CSP in the two lakes, and that TEP colonization was higher in the less productive lake. Measurements of Leucine aminopeptidase and alpha-glucosidase activities in fractionated lake water (0.2-1.2, 1.2-5 and >5 microm fractions) indicated that proteolytic activity was always higher and that particle-associated bacteria have higher enzymatic activities than free-living bacteria. The glycolytic activities in the 1.2-5 and >5 microm fractions were related to the abundance of TEP. We conclude that small freshwater detrital organic particles constitute microhabitats with high bacterial activities in pelagic environments and, undoubtedly, present significant ecological implications for the prokaryotic community structure and function in aquatic ecosystems.     

Phylogenetic analysis of particle-attached and free-living bacterial communities in the Columbia river, its estuary, and the adjacent coastal ocean.

The Columbia River estuary is a dynamic system in which estuarine turbidity maxima trap and extend the residence time of particles and particle-attached bacteria over those of the water and free-living bacteria. Particle-attached bacteria dominate bacterial activity in the estuary and are an important part of the estuarine food web. PCR-amplified 16S rRNA genes from particle-attached and free-living bacteria in the Columbia River, its estuary, and the adjacent coastal ocean were cloned, and 239 partial sequences were determined. A wide diversity was observed at the species level within at least six different bacterial phyla, including most subphyla of the class Proteobacteria. In the estuary, most particle-attached bacterial clones (75%) were related to members of the genus Cytophaga or of the alpha, gamma, or delta subclass of the class Proteobacteria. These same clones, however, were rare in or absent from either the particle-attached or the free-living bacterial communities of the river and the coastal ocean. In contrast, about half (48%) of the free-living estuarine bacterial clones were similar to clones from the river or the coastal ocean. These free-living bacteria were related to groups of cosmopolitan freshwater bacteria (beta-proteobacteria, gram-positive bacteria, and Verrucomicrobium spp.) and groups of marine organisms (gram-positive bacteria and alpha-proteobacteria [SAR11 and Rhodobacter spp.]). These results suggest that rapidly growing particle-attached bacteria develop into a uniquely adapted estuarine community and that free-living estuarine bacteria are similar to members of the river and the coastal ocean microbial communities. The high degree of diversity in the estuary is the result of the mixing of bacterial communities from the river, estuary, and coastal ocean.     

Bacterial production and growth rate estimation from [h]thymidine incorporation for attached and free-living bacteria in aquatic systems

Production and specific growth rates of attached and free-living bacteria were estimated in an oligotrophic marine system, La Salvaje Beach, Vizcaya, Spain, and in a freshwater system having a higher nutrient concentration, Butron River, Vizcaya, Spain. Production was calculated from [methyl-H]thymidine incorporation by estimating specific conversion factors (cells or micrograms of C produced per mole of thymidine incorporated) for attached and free-living bacteria, respectively, in each system. Conversion factors were not statistically different between attached and free-living bacteria: 6.812 x 10 and 8.678 x 10 mug of C mol for free-living and attached bacteria in the freshwater system, and 1.276 x 10 and 1.354 x 10 mug of C mol for free-living and attached bacteria in the marine system. Therefore, use of a unique conversion factor for the mixed bacterial population is well founded. However, conversion factors were higher in the freshwater system than in the marine system. This could be due to the different trophic conditions of the two systems. Free-living bacteria contributed the most to production in the two systems (85% in the marine system and 67% in the freshwater system) because of their greater contribution to total biomass. Specific growth rates calculated from production data and biomass data were similar for attached and free-living bacteria.     

Long-Term Characterization of Free-Living and Particle-Associated Bacterial Communities in Lake Tiefwaren Reveals Distinct Seasonal Patterns

Seasonal changes in environmental conditions have a strong impact on microbial community structure and dynamics in aquatic habitats. To better elucidate the response of bacterial communities to environmental changes, we have measured a large variety of limnetic variables and investigated bacterial community composition (BCC) and dynamics over seven consecutive years between 2003 and 2009 in mesotrophic Lake Tiefwaren (NE Germany). We separated between free-living (FL, >0.2, <5.0?μm) and particle-associated (PA, >5.0?μm) bacteria to account for different bacterial lifestyles and to obtain a higher resolution of the microbial diversity. Changes in BCC were studied by DGGE based on PCR-amplified 16S rRNA gene fragments. Sequencing of DGGE bands revealed that ca. 70?% of all FL bacteria belonged to the Actinobacteria, whereas PA bacteria were dominated by Cyanobacteria (43?%). FL communities were generally less diverse and rather stable over time compared to their PA counterpart. Annual changes in reoccurring seasonal patterns of dominant freshwater bacteria were supported by statistical analyses, which revealed several significant correlations between DGGE profiles and various environmental variables, e.g. temperature and nutrients. Overall, FL bacteria were generally less affected by environmental changes than members of the PA fraction. Close association of PA bacteria with phytoplankton and zooplankton suggests a tight coupling of PA bacteria to organisms of higher trophic levels. Our results indicate substantial differences in bacterial lifestyle of pelagic freshwater bacteria, which are reflected by contrasting seasonal dynamics and relationships to a number of environmental variables.     

Community Dynamics of Free-living and Particle-associated Bacterial Assemblages during a Freshwater Phytoplankton Bloom

Bacterial community dynamics were followed in a 19-day period during an induced diatom bloom in two freshwater mesocosms. The main goal was to compare diversity and succession among free-living (<10 MM) AND PARTICLE-ASSOCIATED (>10 mm) bacteria. Denaturing gradient gel electrophoresis (DGGE) of PCR amplified 16S rDNA showed the highest number of bands among free-living bacteria, but with a significant phylogenetic overlap in the two size fractions indicating that free-living bacteria were also important members of the particle-associated bacterial assemblage. Whereas the number of bands in the free-living fraction decreased during the course of the bloom, several phylotypes unique to particles appeared towards the end of the experiment. Besides the primer set targeting Bacteria, a primer set targeting most members of the Cytophaga-Flavobacterium (CF)-cluster of the Cytophaga-Flavobacterium-Bacteroides group and a primer set mainly targeting a-Proteobacteria were applied. PCR-DGGE analyses revealed that a number of phylotypes targeted by those primer sets were found solely on particles. Almost all sequenced bands from the bacterial DGGE gel were related to phylogenetic groups commonly found in freshwater: a-Proteobacteria, CF, and Firmicutes. Despite the use of primers intended to be specific mainly for a-Proteobacteria most bands sequenced from the a-proteobacterial DGGE gel formed a cluster within the Verrucomicrobiales subdivision of the Verrucomicrobia division and were not related to a-Proteobacteria. Bands sequenced from the CF DGGE gel were related to members of the CF cluster. From the present study, we suggest that free-living and particle-associated bacterial communities should not be perceived as separate entities, but rather as interacting assemblages, where the extent of phylogenetic overlap is dependent on the nature of the particulate matter.     

Temporal variability of attached and free-living bacteria in coastal waters

The temporal variability of the abundance and the incorporation of ³H-thymidine and ¹⁴C-glucose by attached and free-living bacteria, as well as their relation with environmental factors, were analyzed in a coastal marine ecosystem during a year. Both communities were quantitatively very different. Attached bacteria represented only 6.8% of the total bacterial abundance, whereas free-living bacteria represented 93.2%. The environmental factors most closely linked to the abundance and activity of free-living bacteria were temperature and the concentration of dissolved nutrients. Moreover, the free-living community showed similar temporal variations in abundance and in activity, with lower values in the cold months (from October to May). The attached community did not present the same pattern of variation as the free-living one. The abundance of the attached bacteria was mainly correlated to the concentration of particulate material, whereas their activity was correlated to temperature. We did not find a significant correlation between the abundance and the activity of the attached community. On the other hand, the activity per cell of the two communities did not present a clear temporal variation. Attached bacteria were more active than free-living ones in the incorporation of radiolabeled substrates on a per cell basis (five times more in the case of glucose incorporation and twice as active in thymidine incorporation). However, both communities showed similar specific growth rates. The results suggest that the two aquatic bacterial communities must not be considered as being independent of each other. There appears to be a dynamic equilibrium between the two communities, regulated by the concentrations of particulate matter and nutrients and by other environmental factors.Offprint requests to: M. Unanue.     

Amino Acid Assimilation and Electron Transport System Activity in Attached and Free-Living Marine Bacteria

Amino acid assimilation and electron transport system activity of a marine Pseudomonas sp. was evaluated to determine whether the activity of bacteria attached to solid surfaces differed from that of free-living bacteria or bacteria which had been attached but subsequently desorbed from the substratum (detached bacteria). Bacteria were allowed to attach to glass and to a range of plastic surfaces (Thermanox, polyvinylidene fluoride, polyethylene, polytetrafluoroethylene). Microautoradiography and staining with a tetrazolium salt to demonstrate electron transport system activity were used to compare the activity of these organisms with that of free-living or detached cells. The water-wettability of the surfaces was evaluated by measuring the advancing contact angle (θ_A) of water on each surface, to determine whether there was a relationship between activity and substratum hydrophilicity. There was an increase in the proportion of leucine-assimilating attached bacteria and in the proportion of attached cells demonstrating electron transport system activity with an increase in substratum θ_A, but the relationship between activity of attached and free-living cells depended on the substratum. Activity appeared to promote firm attachment, and detached bacteria assimilated fewer amino acids than did attached cells. There was no general effect of surfaces on attached bacterial activity, and attached cells may be more, or less, active than free-living cells, depending on the amino acid, its concentration, and substratum properties.     

Seasonal variations in the contributions of different bacterial groups to the uptake of low-molecular-weight compounds in northwestern Mediterranean coastal waters

We analyzed the contributions of different heterotrophic bacterial groups to the uptake of several low-molecular weight compounds during a seasonal cycle on the northwestern Mediterranean coast (Blanes Bay Microbial Observatory). The bacterial assemblage structure had been shown to change substantially year-round for this site, but whether changes in the activities of the different bacterial groups also occurred on the seasonal scale was unknown. Microautoradiography combined with catalyzed reporter deposition fluorescence in situ hybridization was used to analyze the patterns of glucose, amino acid, and ATP uptake by different bacterial groups. Gammaproteobacteria and Bacteroidetes were not very active in the uptake of glucose at any time of the year (<10% of cells were active) compared to Alphaproteobacteria (generally >20% of cells were active). Dissolved free amino acids were taken up considerably by Alphaproteobacteria and Gammaproteobacteria but not by Bacteroidetes. Relatively high percentages of cells of the three broad phylogenetic groups actively took up ATP, which could be related to the important phosphorous limitation of bacterial production during most of the year in Blanes Bay. The contribution of SAR11 to the uptake of the monomers was variable year-round, generally with fewer than 30% of the cells being active. By contrast, Roseobacter were highly overrepresented in the uptake of all the substrates throughout all the year, with more than 50% of cells being active in all the samples and for all substrates. Our results suggest that substantial changes in the activity of some phylogenetic groups of bacteria occur throughout the year.     

Bacterial Activity at -2 to -20 degrees C in Arctic wintertime sea ice

Arctic wintertime sea-ice cores, characterized by a temperature gradient of -2 to -20 degrees C, were investigated to better understand constraints on bacterial abundance, activity, and diversity at subzero temperatures. With the fluorescent stains 4',6'-diamidino-2-phenylindole 2HCl (DAPI) (for DNA) and 5-cyano-2,3-ditoyl tetrazolium chloride (CTC) (for O(2)-based respiration), the abundances of total, particle-associated (>3- micro m), free-living, and actively respiring bacteria were determined for ice-core samples melted at their in situ temperatures (-2 to -20 degrees C) and at the corresponding salinities of their brine inclusions (38 to 209 ppt). Fluorescence in situ hybridization was applied to determine the proportions of Bacteria, Cytophaga-Flavobacteria-Bacteroides (CFB), and ARCHAEA: Microtome-prepared ice sections also were examined microscopically under in situ conditions to evaluate bacterial abundance (by DAPI staining) and particle associations within the brine-inclusion network of the ice. For both melted and intact ice sections, more than 50% of cells were found to be associated with particles or surfaces (sediment grains, detritus, and ice-crystal boundaries). CTC-active bacteria (0.5 to 4% of the total) and cells detectable by rRNA probes (18 to 86% of the total) were found in all ice samples, including the coldest (-20 degrees C), where virtually all active cells were particle associated. The percentage of active bacteria associated with particles increased with decreasing temperature, as did the percentages of CFB (16 to 82% of Bacteria) and Archaea (0.0 to 3.4% of total cells). These results, combined with correlation analyses between bacterial variables and measures of particulate matter in the ice as well as the increase in CFB at lower temperatures, confirm the importance of particle or surface association to bacterial activity at subzero temperatures. Measuring activity down to -20 degrees C adds to the concept that liquid inclusions in frozen environments provide an adequate habitat for active microbial populations on Earth and possibly elsewhere.     

Comparison of free-living and particle-associated bacterial communities in a coastal Lagoon

We analyzed, by terminal restriction fragment length polymorphisms (T-RFLPs) of PCR-amplified 16S rDNA, microbial diversity in water collected during the dry and wet seasons in a human-impacted coastal lagoon. Water samples were fractionated by prefiltration to differentiate particle-associated and free-living microbes. From a sample collected during the dry season, prefiltration removed 23 to 44% of bacteria, as assessed by direct counts and MPN, and 99% of phytoplankton, as assessed by chlorophyll a. Restriction with RsaI yielded fewer peaks than restriction with HhaI. Diversity indices calculated from T-RFLPs were higher in the lagoon than adjoining coastal waters and higher in the particle-associated than the free-living fraction. In the dry season, peaks found only in bulk and particle-associated T-RFLPs were consistent with plastid and cyanobacterial ribotypes. These peaks matched those observed in the sequence of a clone generated from the bulk fraction with plastid and cyanobacterial specific primers. This clone appeared related to plastids found in the diatom genus Skeletonema. Principal component analysis of T-RFLPs suggested that the difference between the free-living and particle-associated fractions in the dry season was less than temporal variability in this lagoon and that these fractions varied significantly only in the wet season. This fractionation of microbial populations into particle-associated and free-living guilds during the wet season, when water residence time in the lagoon is relatively low, suggests an external source of particle-associated bacteria such as erosion of upland soils by runoff.     

Microbial community diversity in the phycosphere of natural populations of the toxic alga, Alexandrium fundyense

The dinoflagellate Alexandrium fundyense is the major causative organism of paralytic shellfish poisoning in the Gulf of Maine. While laboratory studies have shown that A. fundyense population dynamics can be affected dramatically by co-occurring bacteria, little is known about these interactions in nature. Because A. fundyense is typically a minor Gulf of Maine phytoplankton community member, analyses of the bulk community cannot be used to address bacterium-A. fundyense associations. Therefore, an immunomagnetic bead method was used to selectively capture A. fundyense cells, and the bacteria attached to them, from complex natural samples. Bulk particle-associated and free-living bacterial communities were collected simultaneously. DNA was extracted from all sample types and subjected to 16S rRNA gene fragment amplification, denaturing gradient gel electrophoresis (DGGE) and sequence analysis. Ordination analysis of DGGE profiles confirmed that A. fundyense-associated bacteria community profiles were distinct from bulk bacterial community profiles, indicating selection of specific phylotypes in the A. fundyense phycosphere. Phylogenetic analyses confirmed that Alexandrium-associates were distinct from bulk particle-associated bacteria and that they included a greater prevalence and broader diversity of Gammaproteobacteria than previously thought to be associated with toxic algae. Phylogenetic groups known to be associated with dinoflagellates were also found, including members of the families Alteromonadaceae, Pseudoalteromonadaceae, Rhodobacteraceae and Flavobacteraceae.     

Ecophysiology of a group of uncultured Gammaproteobacterial glycogen-accumulating organisms in full-scale enhanced biological phosphorus removal wastewater treatment plants

The presence of glycogen-accumulating organisms (GAOs) in enhanced biological phosphorus removal (EBPR) plants can seriously deteriorate the biological P-removal by out-competing the polyphosphate-accumulating organisms (PAOs). In this study, uncultured putative GAOs (the GB group, belonging to the Gammaproteobacteria) were investigated in detail in 12 full-scale EBPR plants. Fluorescence in situ hybridization (FISH) revealed that the biovolume of the GB bacteria constituted 2-6% of total bacterial biovolume. At least six different subgroups of the GB bacteria were found, and the number of dominant subgroups present in each plant varied between one and five. Ecophysiological investigations using microautoradiography in combination with FISH showed that, under aerobic or anaerobic conditions, all subgroups of the GB bacteria could take up acetate, pyruvate, propionate and some amino acids, while some subgroups in addition could take up formate and thymidine. Glucose, ethanol, butyrate and several other organic substrates were not taken up. Glycolysis was essential for the anaerobic uptake of organic substrates. Polyhydroxyalkanoates (PHA) but not polyphosphate (polyP) granules were detected in all GB bacterial cells. Polyhydroxyalkanoate formation after anaerobic uptake of acetate was confirmed by measuring the increase in fluorescence intensity of PHA granules inside GB bacterial cells after Nile blue staining. One GB subgroup was possibly able to denitrify, and several others were able to reduce nitrate to nitrite. PAOs were also enumerated by FISH in the same treatment plants. Rhodocyclus-related PAOs and Actinobacteria-related PAOs constituted up to 7% and 29% of total bacterial biovolume respectively. Rhodocyclus-related PAOs always coexisted with the GB bacteria and showed many physiological similarities. Factors of importance for the competition between the three groups of important bacteria in EBPR plants are discussed.