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.     

Phylogenetic Differences in Attached and Free-Living Bacterial Communities in a Temperate Coastal Lagoon during Summer,Revealed via High-Throughput 16S rRNA Gene Sequencing

Most of what is known about coastal free-living and attached bacterial diversity is based on open coasts, with high particulate and nutrient riverine supply, terrestrial runoffs, and anthropogenic activities. The Magdalen Islands in the Gulf of St. Lawrence (Canada) are dominated by shallow lagoons with small, relatively pristine catchments and no freshwater input apart from rain. Such conditions provided an opportunity to investigate coastal free-living and attached marine bacterial diversity in the absence of confounding effects of steep freshwater gradients. We found significant differences between the two communities and marked temporal patterns in both. Taxonomic richness and diversity were greater in the attached than in the free-living community, increasing over summer, especially within the least abundant bacterial phyla. The highest number of reads fell within the SAR 11 clade (Pelagibacter, Alphaproteobacteria), which dominated free-living communities. The attached communities had deeper phylum-level diversity than the free-living fraction. Distance-based redundancy analysis indicated that the particulate organic matter (POM) concentration was the main variable separating early and late summer samples with salinity and temperature changes also significantly correlated to bacterial community structure. Our approach using high-throughput sequencing detected differences in free-living versus attached bacteria in the absence of riverine input, in keeping with the concept that marine attached communities are distinct from cooccurring free-living taxa. This diversity likely reflects the diverse microhabitats of available particles, implying that the total bacterial diversity in coastal systems is linked to particle supply and variability, with implications for understanding microbial biodiversity in marine systems.     

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.     

Diversity and dynamics of free-living and particle-associated Betaproteobacteria and Actinobacteria in relation to phytoplankton and zooplankton communities

The diversity of attached and free-living Actinobacteria and Betaproteobacteria, based on 16S rRNA gene sequences, was investigated in a mesotrophic lake during two periods of contrasting phytoplankton dominance. Comparison analyses showed a phylogenetic difference between attached and free-living communities for the two bacterial groups. For Betaproteobacteria, the betaI clade was detected at all sampling dates in free-living and attached bacterial communities and was the dominant clade contributing to 57.8% of the total retrieved operational taxonomic units (OTUs). For Actinobacteria, the acIV cluster was found to be dominant, followed by acI contributing to 45% and 25% of the total retrieved OTUs, respectively. This study allows the determination of eight new putative clades among the Betaproteobacteria termed lbI-lbVIII and a new putative clade named acLBI belonging to the Actinobacteria. The seasonal dynamics of phytoplankton and zooplankton communities have been reflected as changes in distinct bacterial phylotypes for both attached and free-living communities. For attached communities, relationships were observed between Actinobacteria and Chrysophyceae, and between Betaproteobacteria and Dinophyceae and Chlorophyceae biomass. On the other hand, within free-living communities, few actinobacterial clades were found to be dependent on either nutrients or phytoplankton communities, whereas Betaproteobacteria were mainly associated with biological parameters (i.e. phytoplankton and copepod communities).     

Bacteria of the gamma-subclass Proteobacteria associated with zooplankton in Chesapeake Bay

The seasonal abundance of gamma-subclass Proteobacteria, Vibrio-Photobacterium, Vibrio cholerae-Vibrio mimicus, Vibrio cincinnatiensis, and Vibrio vulnificus in the Choptank River of Chesapeake Bay associated with zooplankton was monitored from April to December 1996. Large (>202- microm) and small (64- to 202- microm) size classes of zooplankton were collected, and the bacteria associated with each of the zooplankton size classes were enumerated by fluorescent oligonucleotide direct count. Large populations of bacteria were found to be associated with both the large and small size classes of zooplankton. Also, the species of bacteria associated with the zooplankton showed seasonal abundance, with the largest numbers occurring in the early spring and again in the summer, when zooplankton total numbers were correspondingly large. Approximately 0.01 to 40.0% of the total water column bacteria were associated with zooplankton, with the percentage of the total water column bacteria population associated with zooplankton varying by season. A taxonomically diverse group of bacteria was associated with zooplankton, and a larger proportion was found in and on zooplankton during the cooler months of the year, with selected taxa comprising a larger percent of the Bacteria in the summer. V. cholerae-V. mimicus and V. vulnificus comprised the bulk of the large and small zooplankton-associated Vibrio-Photobacterium species. In contrast, V. cincinnatiensis accounted for less than 0.1 to 3%. It is concluded that water column and zooplankton bacterial populations vary independently with respect to species composition since no correlation was observed between taxa occurring with highest frequency in the water column and those in association with zooplankton.     

Bacterial Colonization and Ectoenzymatic Activity in Phytoplankton-Derived Model Particles: Cleavage of Peptides and Uptake of Amino Acids

Abstract Phytoplankton-derived model particles were created in laboratory from a mixture of autoclaved diatom cultures. These particles were colonized by a marine bacterial community and incubated in rolling tanks in order to examine the relationship between aminopeptidase activity and leucine uptake. Bacteria inhabiting particles and ambient water were characterized for abundance, biovolume, aminopeptidase activity, leucine uptake, and growth rate. Particles were a less favorable habitat than ambient water for bacterial growth since growth rates of particle-attached bacteria were similar or even lower than those of free-living bacteria. During the first ∼100 h of the particle decomposition process, there were not statistically significant differences in the aminopeptidase activity:leucine uptake ratio between attached and free-living bacteria. From ∼100 h to ∼200 h, this ratio was higher for attached bacteria than for free-living bacteria. This indicates an uncoupling of aminopeptidase activity and leucine uptake. During this period, attached and free-living bacteria showed similar hydrolytic activities on a cell-specific basis. In the free-living bacterial community, variations in aminopeptidase activity per cell were associated with variations in leucine uptake per cell and growth rates. However, in the attached bacterial community, when leucine uptake and growth rates decreased, aminopeptidase activity remained constant. Thus, after ∼100 h, particle-attached bacteria were not taking advantage of their high aminopeptidase activity; consequently the hydrolysed amino acids were released into the ambient water, supporting the growth of free-living bacteria. These results demonstrate that over the particle decomposition process, the relationship between hydrolysis and uptake of the protein fraction shows different patterns of variation for attached and free-living bacterial communities. However, in our experiments, this uncoupling was not based on a hyperproduction of enzymes by attached bacteria, but on lower uptake rates when compared to the free-living bacteria. Received: 4 February 1997; Accepted: 9 May 1997     

Protist abundance and carbon concentration during a Phaeocystis-dominated bloom at an Antarctic coastal site

Summary Changes in the concentrations of bacteria, phytoplankton, protozoa, dissolved organic carbon (DOC), particulate organic carbon (POC), particulate carbohydrate (PCHO) and particulate organic nitrogen (PON) were followed throughout the summer at an Antarctic coastal site. The colonial prymnesiophyte Phaeocystis pouchetii was the first major phytoplankton species to bloom, reaching concentrations of 6 × 10⁷ cells · 1^–1 and remained numerically dominant for most of the summer. During the P. pouchetii bloom the concentration of most other autotrophs did not increase. Microheterotroph abundance peaked during or immediately after the Phaeocystis bloom. Their peak coincided with very high concentrations of organic carbon, particularly DOC which exceeded 100 mg · 1^–1, and low bacterial abundance. Maximum bacterial abundance was reached after the decline in microheterotroph numbers. Bacterial utilization of carbon substrates and microheterotroph grazing of bacteria and uptake of DOC may form an important link to higher trophic levels during Antarctic Phaeocystis blooms.     

Massive Regime Shifts and High Activity of Heterotrophic Bacteria in an Ice-Covered Lake

In winter 2009/10, a sudden under-ice bloom of heterotrophic bacteria occurred in the seasonally ice-covered, temperate, deep, oligotrophic Lake Stechlin (Germany). Extraordinarily high bacterial abundance and biomass were fueled by the breakdown of a massive bloom of Aphanizomenon flos-aquae after ice formation. A reduction in light resulting from snow coverage exerted a pronounced physiological stress on the cyanobacteria. Consequently, these were rapidly colonized, leading to a sudden proliferation of attached and subsequently of free-living heterotrophic bacteria. Total bacterial protein production reached 201 µg C L⁻¹ d⁻¹, ca. five times higher than spring-peak values that year. Fluorescence in situ hybridization and denaturing gradient gel electrophoresis at high temporal resolution showed pronounced changes in bacterial community structure coinciding with changes in the physiology of the cyanobacteria. Pyrosequencing of 16S rRNA genes revealed that during breakdown of the cyanobacterial population, the diversity of attached and free-living bacterial communities were reduced to a few dominant families. Some of these were not detectable during the early stages of the cyanobacterial bloom indicating that only specific, well adapted bacterial communities can colonize senescent cyanobacteria. Our study suggests that in winter, unlike commonly postulated, carbon rather than temperature is the limiting factor for bacterial growth. Frequent phytoplankton blooms in ice-covered systems highlight the need for year-round studies of aquatic ecosystems including the winter season to correctly understand element and energy cycling through aquatic food webs, particularly the microbial loop. On a global scale, such knowledge is required to determine climate change induced alterations in carbon budgets in polar and temperate aquatic systems.     

Links between Phytoplankton and Bacterial Community Dynamics in a Coastal Marine Environment

Bacteria and phytoplankton dynamics are thought to be closely linked in coastal marine environments, with correlations frequently observed between bacterial and phytoplankton biomass. In contrast, little is known about how these communities interact with each other at the species composition level. The purpose of the current study was to analyze bacterial community dynamics in a productive, coastal ecosystem and to determine whether they were related to phytoplankton community dynamics. Near-surface seawater samples were collected in February, May, July, and September 2000 from several stations in the Bay of Fundy. Savin et al. (M.C. Savin et al., Microb Ecol 48: 51-65) analyzed the phytoplankton community in simultaneously collected samples. The attached and free-living bacterial communities were collected by successive filtration onto 5 m and 0.22 m pore-size filters, respectively. DNA was extracted from filters and bacterial 16S rRNA gene fragments were amplified and analyzed by denaturing gradient gel electrophoresis (DGGE). DGGE revealed that diversity and temporal variability were lower in the free-living than the attached bacterial community. Both attached and free-living communities were dominated by members of the Roseobacter and Cytophaga groups. Correspondence analysis (CA) ordination diagrams showed similar patterns for the phytoplankton and attached bacterial communities, indicating that shifts in the species composition of these communities were linked. Similarly, canonical CA revealed that the diversity, abundance, and percentage of diatoms in the phytoplankton community accounted for a significant amount of the variability in the attached bacterial community composition. In contrast, ordination analyses did not reveal an association between free-living bacteria and phytoplankton. These results suggest that there are specific interactions between phytoplankton and the bacteria attached to them, and that these interactions influence the composition of both communities.     

Host-Specificity and Dynamics in Bacterial Communities Associated with Bloom-Forming Freshwater Phytoplankton

Many freshwater phytoplankton species have the potential to form transient nuisance blooms that affect water quality and other aquatic biota. Heterotrophic bacteria can influence such blooms via nutrient regeneration but also via antagonism and other biotic interactions. We studied the composition of bacterial communities associated with three bloom-forming freshwater phytoplankton species, the diatom Aulacoseira granulata and the cyanobacteria Microcystis aeruginosa and Cylindrospermopsis raciborskii. Experimental cultures incubated with and without lake bacteria were sampled in three different growth phases and bacterial community composition was assessed by 454-Pyrosequencing of 16S rRNA gene amplicons. Betaproteobacteria were dominant in all cultures inoculated with lake bacteria, but decreased during the experiment. In contrast, Alphaproteobacteria, which made up the second most abundant class of bacteria, increased overall during the course of the experiment. Other bacterial classes responded in contrasting ways to the experimental incubations causing significantly different bacterial communities to develop in response to host phytoplankton species, growth phase and between attached and free-living fractions. Differences in bacterial community composition between cyanobacteria and diatom cultures were greater than between the two cyanobacteria. Despite the significance, major differences between phytoplankton cultures were in the proportion of the OTUs rather than in the absence or presence of specific taxa. Different phytoplankton species favoring different bacterial communities may have important consequences for the fate of organic matter in systems where these bloom forming species occur. The dynamics and development of transient blooms may also be affected as bacterial communities seem to influence phytoplankton species growth in contrasting ways.     

Similarities and seasonal variations in bacterial communities from the blood of rodents and from their flea vectors

Vector-borne microbes are subject to the ecological constraints of two distinct microenvironments: that in the arthropod vector and that in the blood of its vertebrate host. Because the structure of bacterial communities in these two microenvironments may substantially affect the abundance of vector-borne microbes, it is important to understand the relationship between bacterial communities in both microenvironments and the determinants that shape them. We used pyrosequencing analyses to compare the structure of bacterial communities in Synosternus cleopatrae fleas and in the blood of their Gerbillus andersoni hosts. We also monitored the interindividual and seasonal variability in these bacterial communities by sampling the same individual wild rodents during the spring and again during the summer. We show that the bacterial communities in each sample type (blood, female flea or male flea) had a similar phylotype composition among host individuals, but exhibited seasonal variability that was not directly associated with host characteristics. The structure of bacterial communities in male fleas and in the blood of their rodent hosts was remarkably similar and was dominated by flea-borne Bartonella and Mycoplasma phylotypes. A lower abundance of flea-borne bacteria and the presence of Wolbachia phylotypes distinguished bacterial communities in female fleas from those in male fleas and in rodent blood. These results suggest that the overall abundance of a certain vector-borne microbe is more likely to be determined by the abundance of endosymbiotic bacteria in the vector, abundance of other vector-borne microbes co-occurring in the vector and in the host blood and by seasonal changes, than by host characteristics.     

Small core communities and high variability in bacteria associated with the introduced ascidian Styela plicata

The solitary ascidian Styela plicata is an introduced species in harbors of temperate and tropical oceans around the world. The invasive potential of this species has been studied through reproductive biology and population genetics but no study has yet examined the microbial diversity associated with this ascidian and its potential role in host ecology and invasiveness. Here, we used 16S rRNA gene tag pyrosequencing and transmission electron microscopy to characterize the abundance, diversity and host-specificity of bacteria associated with 3 Mediterranean individuals of S. plicata. Microscopy revealed low bacterial abundance in the inner tunic and their absence from gonad tissues, while pyrosequencing revealed a high diversity of S. plicata-associated bacteria (284 OTUs from 16 microbial phyla) in the inner tunic. The core symbiont community was small and consisted of 16 OTUs present in all S. plicata hosts. This core community included a recently described ascidian symbiont (Hasllibacter halocynthiae) and several known sponge and coral symbionts, including a strictly anaerobic Chloroflexi lineage. Most recovered bacterial OTUs (79.6 %) were present in single S. plicata individuals and statistical analyses of genetic diversity and community structure confirmed high variability of bacterial communities among host individuals. These results suggest that diverse and variable bacterial communities inhabit the tunic of S. plicata, including environmental and host-associated bacterial lineages that appear to be re-established each host generation. We hypothesize that bacterial communities in S. plicata are dynamic and have the potential to aid host acclimation to new habitats by establishing relationships with beneficial, locally sourced bacteria.     

Experimental study of Microcystis-associated and free-living bacteria

In the experiment with water from the hypereutrophic Lake Frederiksborg Slotso (Denmark) sampled during the autumn peak of Microcystis growth, the quantity and production of free-living and cyanobacteria-associated heterotrophic bacteria were determined, as well as the extracellular enzymatic (aminopeptidase) activity. The functional diversities of associated and free-living bacterial communities were additionally compared using BIOLOG GN microplates to reveal the possible export of Microcystis-attached bacteria into ambient water. It has been shown that the cell size, production values, and growth rates of associated bacteria were less than those of free-living bacteria. At the same time, the potential aminopeptidase activity of associated bacteria was always higher than that of free-living bacteria. The experimental results have shown significant compositional differences in the structure of bacterial communities from different habitats.     

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.     

Effect of Abandonment on Diversity and Abundance of Free-Living Nitrogen-Fixing Bacteria and Total Bacteria in the Cropland Soils of Hulun Buir,Inner Mongolia

In Inner Mongolia, steppe grasslands face desertification or degradation because of human over activity. One of the reasons for this condition is that croplands have been abandoned after inappropriate agricultural management. The soils in these croplands present heterogeneous environments in which conditions affecting microbial growth and diversity fluctuate widely in space and time. In this study, we assessed the molecular ecology of total and free-living nitrogen-fixing bacterial communities in soils from steppe grasslands and croplands that were abandoned for different periods (1, 5, and 25 years) and compared the degree of recovery. The abandoned croplands included in the study were natural restoration areas without human activity. Denaturing gradient gel electrophoresis and quantitative PCR (qPCR) were used to analyze the nifH and 16S rRNA genes to study free-living diazotrophs and the total bacterial community, respectively. The diversities of free-living nitrogen fixers and total bacteria were significantly different between each site (P<0.001). Neither the total bacteria nor nifH gene community structure of a cropland abandoned for 25 years was significantly different from those of steppe grasslands. In contrast, results of qPCR analysis of free-living nitrogen fixers and total bacteria showed significantly high abundance levels in steppe grassland (P<0.01 and P<0.03, respectively). In this study, the microbial communities and their gene abundances were assessed in croplands that had been abandoned for different periods. An understanding of how environmental factors and changes in microbial communities affect abandoned croplands could aid in appropriate soil management to optimize the structures of soil microorganisms.     

Seasonal dynamics of free and attached heterotrophic nanoflagellates in an oligomesotrophic lake

1. The seasonal development of heterotrophic nanoflagellates (HNF), bacteria, rotiferans and crustacean zooplankton was studied in the epilimnion of Lake Pavin, an oligomesotrophic lake in the Massif Central of France.
2. HNF abundance varied from 0.1 to 2.5 × 10³ mL^–1. Free-living HNF reached their highest density in spring when the copepod Acanthodiaptomus denticornis dominated the metazooplankton. They were present in low numbers when rotifers and cladocerans were numerous.
3. Attached HNF, consisting of bicoecids and choanoflagellates, were fixed to large diatoms and to the colonial cyanobacterium Anabaena flos-aquae . The abundance of attached HNF was significantly correlated to bacterial abundance, which fluctuated between 1.1 and 2.7 × 10⁶ mL^–1. Highest abundance of these epiphytic protists was recorded when free-living heterotrophic nanoflagellates declined.
4. The comparison of the dynamics of heterotrophic nanoflagellates, bacteria, and the impact of zooplankton grazing suggested that prey abundance, the presence of suitable attachment sites and limited competition from the free-living forms were the main factors controlling the development of the epiphytic flagellate protists. In contrast, the low abundance of free-living forms during the period of rotiferan and cladoceran development suggests the prevalence of a top-down control by predation of the metazoopankton.     

Production and Turnover of Planktonic Bacteria in Two Southeastern Blackwater Rivers

Production by attached and free-living planktonic bacteria in two blackwater rivers in the Southeastern United States was measured over a period of 14 months by using the rate of incorporation of [methyl-³H]thymidine into DNA. Production rates and biomass dynamics were compared to determine the potential for in situ production to supply planktonic biomass. Bacterial production in these rivers was moderate and varied seasonally. Rates varied from 0.058 to 2.120 mg of C m⁻³ h⁻¹ in the Ogeechee River and from 0.002 to 2.418 mg of C m⁻³ h⁻¹ in Black Creek. Regressions of growth rate on various environmental variables showed that temperature and total dissolved organic carbon concentration were the best predictors of growth. Although attached bacteria were <21% of the total biomass, they accounted for up to 53% of the total production. Turnover times for attached bacteria ranged from <1 day to >3 years depending on season. Turnover times of free-living bacteria varied from 4.4 days to 11.8 years. Comparisons of biomass with production indicated that during most seasons, the majority of bacterial biomass in these rivers was of allochthonous origin. During summer, when water temperatures were high, bacterial growth in the river may have supplied a greater percentage of the standing stock of bacteria than allochthonous inputs.     

Bacterial Production and Growth Rate Estimation from [3H]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-³H]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 × 10¹¹ and 8.678 × 10¹¹ μg of C mol⁻¹ for free-living and attached bacteria in the freshwater system, and 1.276 × 10¹¹ and 1.354 × 10¹¹ μg of C mol⁻¹ 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.     

Occurrence and distribution of plankton-associated and free-living toxigenic Vibrio cholerae in a tropical estuary of a cholera endemic zone

Cholera epidemics are thought to be influenced by changes in populations of estuarine Vibrio cholerae. We investigated the abundance and distribution of this bacterium, as ??free-living?? (<20???m fraction) and associated with microphytoplankton (>20???m) or zooplankton (>60???m), in the Karnaphuli estuary of Bangladesh during pre- and post-monsoon seasons. Cultivable Vibrio populations were ~10²?C10⁴ colony forming units (CFU) ml^?1 in the high saline zone (19?C23 practical salinity unit, PSU) and declined in freshwater (<10¹?CFU?ml^?1). Culture independent detection of toxigenic V. cholerae O1 and O139 serogroups revealed a higher abundance of ??free-living?? (10⁴?C10⁵ cells?l^?1) than those attached to plankton (10¹?C10³ cells?l^?1). However, ??free-living?? O1 and O139 cells were sometimes absent in the medium saline and freshwater areas (0.0?C11 practical salinity unit [PSU]). In contrast, plankton samples always harbored these serogroups despite changes in salinity and other physico-chemical properties. Microphytoplankton and zooplankton were dominated by diatoms and blue-green algae, and copepods and rotifers, respectively. Toxigenic V. cholerae abundance did not correlate with plankton abundance or species but had a positive correlation with chitin in the <20???m fraction, where suspended particulate matter (SPM), V. cholerae and chitin concentrations were highest. C:N ratios indicated that organic matter in SPM originated predominantly from plankton. The differential occurrence of ??free-living?? and attached V. cholerae suggests a pivotal function of plankton in V. cholerae spreading into freshwater areas. The probable association of this pathogen with organisms and particles in the nanoplankton (<20???m) fraction requires validation of the concept of the ??free living?? state of V. cholerae in aquatic habitats.