Elemental Composition (C, N, P) and Cell Volume of Exponentially Growing and Nutrient-Limited Bacterioplankton

Marine bacterioplankton were isolated and grown in batch cultures until their growth became limited by organic carbon (C), nitrogen (N), or phosphorus (P). Samples were taken from the cultures at both the exponential and stationary phases. The elemental composition of individual bacterial cells was analyzed by X-ray microanalysis with an electron microscope. The cell size was also measured. The elemental content was highest in exponentially growing cells (149 ± 8 fg of C cell⁻¹, 35 ± 2 fg of N cell⁻¹, and 12 ± 1 fg of P cell⁻¹; average of all isolates ± standard error). The lowest C content was found in C-limited cells (39 ± 3 fg of C cell⁻¹), the lowest N content in C- and P-limited cells (12 ± 1 and 12 ± 2 fg of N cell⁻¹, respectively), and the lowest P content in P-limited cells (2.3 ± 0.6 fg of P cell⁻¹). The atomic C:N ratios varied among treatments between 3.8 ± 0.1 and 9.5 ± 1.0 (average ± standard error), the C:P ratios between 35 ± 2 and 178 ± 28, and the N:P ratios between 6.7 ± 0.3 and 18 ± 3. The carbon-volume ratios showed large variation among isolates due to different types of nutrient limitation (from 51± 4 to 241 ± 38 fg of C μm⁻¹; average of individual isolates and treatments ± standard error). The results show that different growth conditions and differences in the bacterial community may explain some of the variability of previously reported elemental and carbon-volume ratios.     

Isotopic tracing reveals single-cell assimilation of a macroalgal polysaccharide by a few marine Flavobacteria and Gammaproteobacteria

Algal polysaccharides constitute a diverse and abundant reservoir of organic matter for marine heterotrophic bacteria, central to the oceanic carbon cycle. We investigated the uptake of alginate, a major brown macroalgal polysaccharide, by microbial communities from kelp-dominated coastal habitats. Congruent with cell growth and rapid substrate utilization, alginate amendments induced a decrease in bacterial diversity and a marked compositional shift towards copiotrophic bacteria. We traced ¹³C derived from alginate into specific bacterial incorporators and quantified the uptake activity at the single-cell level, using halogen in situ hybridization coupled to nanoscale secondary ion mass spectrometry (HISH-SIMS) and DNA stable isotope probing (DNA-SIP). Cell-specific alginate uptake was observed for Gammaproteobacteria and Flavobacteriales, with carbon assimilation rates ranging from 0.14 to 27.50 fg C µm⁻³ h⁻¹. DNA-SIP revealed that only a few initially rare Flavobacteriaceae and Alteromonadales taxa incorporated ¹³C from alginate into their biomass, accounting for most of the carbon assimilation based on bulk isotopic measurements. Functional screening of metagenomic libraries gave insights into the genes of alginolytic Alteromonadales active in situ. These results highlight the high degree of niche specialization in heterotrophic communities and help constraining the quantitative role of polysaccharide-degrading bacteria in coastal ecosystems.Subject terms: Water microbiology, Microbial ecology, Marine microbiology, Biogeochemistry, Microbial ecology     

Determination of Total Protein Content of Bacterial Cells by SYPRO Staining and Flow Cytometry

An assay has been developed for measuring protein biomass of marine planktonic bacteria by flow cytometry. The method was calibrated by using five species of Bacteria (an Arcobacter sp., a Cytophaga sp., an Oceanospirillum sp., a Pseudoalteromonas sp., and a Vibrio sp.) recently isolated from seawater samples and grown in culture at different temperatures. The intensity of SYPRO-protein fluorescence of these bacteria strongly correlated with their total protein content, measured by the bicinchoninic acid method to be in the range of 60 to 330 fg of protein cell⁻¹ (r² = 0.93, n = 34). According to the calibration, the mean biomass of planktonic bacteria from the North Sea in August 1998 was 24 fg of protein cell⁻¹.     

Determination of Bacterial Cell Dry Mass by Transmission Electron Microscopy and Densitometric Image Analysis

We applied transmission electron microscopy and densitometric image analysis to measure the cell volume (V) and dry weight (DW) of single bacterial cells. The system was applied to measure the DW of Escherichia coli DSM 613 at different growth phases and of natural bacterial assemblages of two lakes, Piburger See and Gossenköllesee. We found a functional allometric relationship between DW (in femtograms) and V (in cubic micrometers) of bacteria (DW = 435 · V^0.86); i.e., smaller bacteria had a higher ratio of DW to V than larger cells. The measured DW of E. coli cells ranged from 83 to 1,172 fg, and V ranged from 0.1 to 3.5 μm³ (n = 678). Bacterial cells from Piburger See and Gossenköllesee (n = 465) had DWs from 3 fg (V = 0.003 μm³) to 1,177 fg (V = 3.5 μm³). Between 40 and 50% of the cells had a DW of less than 20 fg. By assuming that carbon comprises 50% of the DW, the ratio of carbon content to V of individual cells varied from 466 fg of C μm⁻³ for Vs of 0.001 to 0.01 μm³ to 397 fg of C μm⁻³ (0.01 to 0.1 μm³) and 288 fg of C μm⁻³ (0.1 to 1 μm³). Exponentially growing and stationary cells of E. coli DSM 613 showed conversion factors of 254 fg of C μm⁻³ (0.1 to 1 μm³) and 211 fg of C μm⁻³ (1 to 4 μm³), respectively. Our data suggest that bacterial biomass in aquatic environments is higher and more variable than previously assumed from volume-based measurements.     

Relationships between Biovolume and Biomass of Naturally Derived Marine Bacterioplankton

Microscopic estimation of bacterial biomass requires determination of both biovolume and biovolume-to-biomass conversion. Both steps have uncertainty when applied to the very small bacteria typically found in natural seawater. In the present study, natural bacterioplankton assemblages were freshly collected, passed through 0.6-μm-pore-size Nuclepore filters to remove larger particulate materials, and diluted for growth in 0.22-μm-pore-size Millipore filter-sterilized unenriched seawater. This provided cells comparable in size and morphology to those in natural seawater, but the cultures were free of the interfering particulate detritus naturally present. Cells were collected on glass-fiber GF/F filters, and biovolumes were corrected for cells passing these filters; C and N were measured with a CHN analyzer. Our criteria for size measurement by epifluorescence photomicrography were confirmed with fluorescent microspheres of known diameters. Surprisingly, in six cultures with average per-cell biovolumes ranging from 0.036 to 0.073 μm³, the average per-cell carbon biomass was relatively constant at 20 ± 0.08 fg of C (mean ± standard error of the mean). The biovolume-to-biomass conversion factor averaged 0.38 ± 0.05 g of C cm⁻³, which is about three times higher than the value previously estimated from Escherichia coli, and decreased with increasing cell volume. The C:N ratio was 3.7 ± 0.2. We conclude that natural marine bacterial biomass and production may be higher than was previously thought and that variations in bacterial size may not reflect variations in biomass per cell.     

Frequency of Dividing Cells as an Estimator of Bacterial Productivity

It has recently been proposed that the frequency of dividing bacterial cells (FDC) can be used to predict growth rates of natural aquatic bacterial assemblages. We have examined the relationship between FDC and growth rate in bacteria from southern-temperate, coastal marine waters by using incubation under conditions of manipulated nutrient availability and exclusion of bacterivores. The regression of the natural logarithm of bacterial instantaneous growth rate (μ) on FDC resulted in a better fit than regression of untransformed μ on FDC. The regression equation was ln μ = 0.299FDC − 4.961. The coefficient of variation for predicted ln μ at mean FDC was 7%. The range of FDC-estimated bacterial instantaneous generation times for coastal Georgia waters was 12 to 68 h, and range of calculated bacterial production rates was 0.6 to 17.6 mg of C·m⁻³· h⁻¹. Unresolved problems of and suggested improvements on the FDC method of predicting growth rate are discussed.     

Growth of Vibrio cholerae O1 in Red Tide Waters off California

Vibrio cholerae serotype O1 is autochthonous to estuarine and coastal waters. However, its population dynamics in such environments are not well understood. We tested the proliferation of V. cholerae N16961 during a Lingulodinium polyedrum bloom, as well as other seawater conditions. Microcosms containing 100-kDa-filtered seawater were inoculated with V. cholerae or the 0.6-μm-pore-size filterable fraction of seawater assemblages. These cultures were diluted 10-fold with fresh 100-kDa-filtered seawater every 48 h for four cycles. Growth rates ranged from 0.3 to 14.3 day⁻¹ (4.2 day⁻¹ ± 3.9) for V. cholerae and 0.1 to 9.7 day⁻¹ (2.2 ± 2.8 day⁻¹) for bacterial assemblage. Our results suggest that dissolved organic matter during intense phytoplankton blooms has the potential to support explosive growth of V. cholerae in seawater. Under the conditions tested, free-living V. cholerae was able to reach concentrations per milliliter that were up to 3 orders of magnitude higher than the known minimum infectious dose (10⁴ cell ml⁻¹) and remained viable under many conditions. If applicable to the complex conditions in marine ecosystems, our results suggest an important role of the growth of free-living V. cholerae in disease propagation and prevention during phytoplankton blooms.     

Dimethylsulfoniopropionate Turnover Is Linked to the Composition and Dynamics of the Bacterioplankton Assemblage during a Microcosm Phytoplankton Bloom

Processing of the phytoplankton-derived organic sulfur compound dimethylsulfoniopropionate (DMSP) by bacteria was studied in seawater microcosms in the coastal Gulf of Mexico (Alabama). Modest phytoplankton blooms (peak chlorophyll a [Chl a] concentrations of ~2.5 μg liter⁻¹) were induced in nutrient-enriched microcosms, while phytoplankton biomass remained low in unamended controls (Chl a concentrations of ~0.34 μg liter⁻¹). Particulate DMSP concentrations reached 96 nM in the enriched microcosms but remained approximately 14 nM in the controls. Bacterial biomass production increased in parallel with the increase in particulate DMSP, and nutrient limitation bioassays in the initial water showed that enrichment with DMSP or glucose caused a similar stimulation of bacterial growth. Concomitantly, increased bacterial consumption rate constants of dissolved DMSP (up to 20 day⁻¹) and dimethylsulfide (DMS) (up to 6.5 day⁻¹) were observed. Nevertheless, higher DMSP S assimilation efficiencies and higher contribution of DMSP to bacterial S demand were found in the controls compared to the enriched microcosms. This indicated that marine bacterioplankton may rely more on DMSP as a source of S under oligotrophic conditions than under the senescence phase of phytoplankton blooms. Phylogenetic analysis of the bacterial assemblages in all microcosms showed that the DMSP-rich algal bloom favored the occurrence of various Roseobacter members, flavobacteria (Bacteroidetes phylum), and oligotrophic marine Gammaproteobacteria. Our observations suggest that the composition of the bacterial assemblage and the relative contribution of DMSP to the overall dissolved organic sulfur/organic matter pool control how efficiently bacteria assimilate DMSP S and thereby potentially divert it from DMS production.     

Protozoan Grazing, Bacterial Activity, and Mineralization in Two-Stage Continuous Cultures

In two-stage continuous cultures, at bacterial concentrations, biovolumes, and growth rates similar to values found in Lake Vechten, ingestion rates of heterotrophic nanoflagellates (HNAN) increased from 2.3 bacteria HNAN⁻¹ · h⁻¹ at a growth rate of 0.15 day⁻¹ to 9.2 bacteria · HNAN⁻¹ · h⁻¹ at a growth rate of 0.65 day⁻¹. On a yeast extract medium with a C/N/P ratio of 100:15:1.2 (Redfield ratio), a mixed bacterial population showed a yield of 18% (C/C) and a specific carbon content of 211 fg of C · μm⁻³. The HNAN carbon content and yield were estimated at 127 fg of C · μm⁻³ and 47% (C/C). Although P was not growth limiting, HNAN accelerated the mineralization of PO₄-P from dissolved organic matter by 600%. The major mechanism of P remineralization appeared to be direct consumption of bacteria by HNAN. N mineralization was performed mainly (70%) by bacteria but was increased 30% by HNAN. HNAN did not enhance the decomposition of the relatively mineral-rich dissolved organic matter. An accelerated decomposition of organic carbon by protozoa may be restricted to mineral-poor substrates and may be explained mainly by protozoan nutrient regeneration. Growth and grazing in the cultures were compared with methods for in situ estimates. Thymidine incorporation by actively growing bacteria yielded an empirical conversion factor of 1.1 × 10¹⁸ bacteria per mol of thymidine incorporated into DNA. However, nongrowing bacteria also showed considerable incorporation. Protozoan grazing was found to be accurately measured by uptake of fluorescently labeled bacteria, whereas artificial fluorescent microspheres were not ingested, and selective prokaryotic inhibitors blocked not only bacterial growth but also protozoan grazing.     

Steady-State Effects of 2,5,2′,5′-Tetrachlorobiphenyl on Growth, Photosynthesis, and P Uptake in Selenastrum capricornutum

The steady-state effect of 2,5,2′,5′-tetrachlorobiphenyl (TCBP) on the green alga Selenastrum capricornutum was investigated in a P-limited two-stage chemostat system. The partition coefficient of this polychlorinated biphenyl congener was 5.9 × 10⁴ in steady-state cultures. At a cellular TCBP concentration of 12.2 × 10⁻⁸ ng · cell⁻¹, growth rate was not affected. However, photosynthetic capacity (P_max) was significantly enhanced by TCBP (56 × 10⁻⁹ μmol of C · cell⁻¹ · h⁻¹ versus 34 × 10⁻⁹ μmol of C · cell⁻¹ · h⁻¹ in the control). Photosynthetic efficiency, or the slope of the photosynthesis-irradiance curve, was also significantly higher. There was little difference in the cell chlorophyll a content, and therefore the difference in these photosynthetic characteristics was the same even when they were expressed on a per-chlorophyll a basis. Cell C content was higher in TCBP-containing cells than in TCBP-free cells, but approximately 36% of the C fixed by cells with TCBP was not incorporated as cell C. The maximum P uptake rate was also enhanced by TCBP, but the half-saturation concentration appeared to be unaffected.     

Bacterial Community Affects Toxin Production by Gymnodinium catenatum

The paralytic shellfish toxin (PST)-producing dinoflagellate Gymnodinium catenatum grows in association with a complex marine bacterial community that is both essential for growth and can alter culture growth dynamics. Using a bacterial community replacement approach, we examined the intracellular PST content, production rate, and profile of G. catenatum cultures grown with bacterial communities of differing complexity and composition. Clonal offspring were established from surface-sterilized resting cysts (produced by sexual crosses of strain GCDE06 and strain GCLV01) and grown with: 1) complex bacterial communities derived from each of the two parent cultures; 2) simplified bacterial communities composed of the G. catenatum-associated bacteria Marinobacter sp. strain DG879 or Alcanivorax sp. strain DG881; 3) a complex bacterial community associated with an untreated, unsterilized sexual cross of the parents. Toxin content (STX-equivalent per cell) of clonal offspring (134–197 fmol STX cell⁻¹) was similar to the parent cultures (169–206 fmol STX cell⁻¹), however cultures grown with single bacterial types contained less toxin (134–146 fmol STX cell⁻¹) than offspring or parent cultures grown with more complex mixed bacterial communities (152–176 fmol STX cell⁻¹). Specific toxin production rate (fmol STX day⁻¹) was strongly correlated with culture growth rate. Net toxin production rate (fmol STX cell⁻¹ day⁻¹) did not differ among treatments, however, mean net toxin production rate of offspring was 8-fold lower than the parent cultures, suggesting that completion of the sexual lifecycle in laboratory cultures leads to reduced toxin production. The PST profiles of offspring cultures were most similar to parent GCDE06 with the exception of cultures grown with Marinobacter sp. DG879 which produced higher proportions of dcGTX2+3 and GC1+2, and lower proportions of C1+2 and C3+4. Our data demonstrate that the bacterial community can alter intracellular STX production of dinoflagellates. In G. catenatum the mechanism appears likely to be due to bacterial effects on dinoflagellate physiology rather than bacterial biotransformation of PST toxins.     

Influence of Cyanobacterial Hyperscum on Heterotrophic Activity of Planktonic Bacteria in a Hypertrophic Lake

The response of the planktonic heterotrophic bacterial community to the buildup and breakdown of a semipermanent, crusted, floating cyanobacterial mat, or hyperscum, that covered 1 to 2 ha was studied in a hypertrophic lake (Hartbeespoort Dam, South Africa). The initial response of bacteria in the main basin to the release of dissolved organic carbon (DOC) from the hyperscum 1 km away was an increase in activity per cell from 35 × 10⁻¹² to 153 × 10⁻¹² μg of C cell⁻¹ h⁻¹ for total cell counts, while activity per cell for metabolically active cells increased from 19 × 10⁻¹¹ to 85 × 10⁻¹¹ μg of C cell⁻¹ h⁻¹. No major population growth occurred at this stage. Later, with the continuous supply of DOC from the hyperscum, total bacterial numbers increased from 6.6 × 10⁶ to 20 × 10⁶ cells ml⁻¹, while the activity per cell declined. Metabolically active bacteria followed the same trend. Shorter-term DOC increases caused only increases in bacterial activity per cell. The data from Hartbeespoort Dam demonstrate an interesting and little-documented mechanism by which aquatic bacteria respond to increased DOC concentration and which may be universal for aquatic systems.     

Comparison of Extracellular Enzyme Activities and Community Composition of Attached and Free-Living Bacteria in Porous Medium Columns

Free-living and surface-associated microbial communities in sand-packed columns perfused with groundwater were compared by examination of compositional and functional characteristics. The composition of the microbial communities was assessed by bulk DNA extraction, PCR amplification of 16S ribosomal DNA fragments, separation of these fragments by denaturing gradient gel electrophoresis, and sequence analysis. Community function was assessed by measurement of β-glucosidase and aminopeptidase extracellular enzyme activities. Free-living populations in the aqueous phase exhibited a greater diversity of phylotypes than populations associated with the solid phase. The attached bacterial community displayed significantly greater β-glucosidase and aminopeptidase enzyme activities per volume of porous medium than those of the free-living community. On a per-cell basis, the attached community had a significantly higher cell-specific aminopeptidase enzyme activity (1.07 × 10⁻⁷ nmol cell⁻¹ h⁻¹) than the free-living community (5.02 × 10⁻⁸ nmol cell⁻¹ h⁻¹). Conversely, the free-living community had a significantly higher cell-specific β-glucosidase activity (1.92 × 10⁻⁶ nmol cell⁻¹ h⁻¹) than the surface-associated community (6.08 × 10⁻⁷ nmol cell⁻¹ h⁻¹). The compositional and functional differences observed between these two communities may reflect different roles for these distinct but interacting communities in the decomposition of natural organic matter or biodegradation of xenobiotics in aquifers.     

Pure-Culture Growth of Fermentative Bacteria, Facilitated by H2 Removal: Bioenergetics and H2 Production

We used an H₂-purging culture vessel to replace an H₂-consuming syntrophic partner, allowing the growth of pure cultures of Syntrophothermus lipocalidus on butyrate and Aminobacterium colombiense on alanine. By decoupling the syntrophic association, it was possible to manipulate and monitor the single organism's growth environment and determine the change in Gibbs free energy yield (ΔG) in response to changes in the concentrations of reactants and products, the purging rate, and the temperature. In each of these situations, H₂ production changed such that ΔG remained nearly constant for each organism (−11.1 ± 1.4 kJ mol butyrate⁻¹ for S. lipocalidus and −58.2 ± 1.0 kJ mol alanine⁻¹ for A. colombiense). The cellular maintenance energy, determined from the ΔG value and the hydrogen production rate at the point where the cell number was constant, was 4.6 × 10⁻¹³ kJ cell⁻¹ day⁻¹ for S. lipocalidus at 55°C and 6.2 × 10⁻¹³ kJ cell⁻¹ day⁻¹ for A. colombiense at 37°C. S. lipocalidus, in particular, seems adapted to thrive under conditions of low energy availability.     

Muramic Acid Measurements for Bacterial Investigations in Marine Environments by High-Pressure Liquid Chromatography

Muramic acid, a constituent of procaryotic cell walls, was assayed by high-pressure liquid chromatography in samples from several marine environments (water column, surface microlayer, and sediment) and a bacterial culture. It is used as a microbial biomass indicator. The method gave a good separation of muramic acid from interfering compounds with satisfactory reproducibility. A pseudomonad culture had a muramic acid content of 4.7 × 10⁻¹⁰ to 5.3 × 10⁻¹⁰ μg per cell during growth. In natural water samples, highly significant relationships were found between muramic acid concentrations and bacterial numbers for populations of 10⁸ to 10¹¹ cells per liter. The muramic acid content in natural marine water decreased from 5.3 × 10⁻¹⁰ to 1.6 × 10⁻¹⁰ μg per cell with increasing depth. In coastal sediments exposed to sewage pollution, concentrations of muramic acid, ATP, organic carbon, and total amino acids displayed a parallel decrease with increasing distance from the sewage outlet. Advantages of muramic acid measurement by high-pressure liquid chromatography are its high sensitivity and reduction of preparation steps, allowing a short time analysis.     

Large Fraction of Dead and Inactive Bacteria in Coastal Marine Sediments: Comparison of Protocols for Determination and Ecological Significance

It is now universally recognized that only a portion of aquatic bacteria is actively growing, but quantitative information on the fraction of living versus dormant or dead bacteria in marine sediments is completely lacking. We compared different protocols for the determination of the dead, dormant, and active bacterial fractions in two different marine sediments and at different depths into the sediment core. Bacterial counts ranged between (1.5 ± 0.2) × 10⁸ cells g⁻¹ and (53.1 ± 16.0) × 10⁸ cells g⁻¹ in sandy and muddy sediments, respectively. Bacteria displaying intact membrane (live bacterial cells) accounted for 26 to 30% of total bacterial counts, while dead cells represented the most abundant fraction (70 to 74%). Among living bacterial cells, nucleoid-containing cells represented only 4% of total bacterial counts, indicating that only a very limited fraction of bacterial assemblage was actively growing. Nucleoid-containing cells increased with increasing sediment organic content. The number of bacteria responsive to antibiotic treatment (direct viable count; range, 0.3 to 4.8% of the total bacterial number) was significantly lower than nucleoid-containing cell counts. An experiment of nutrient enrichment to stimulate a response of the dormant bacterial fraction determined a significant increase of nucleoid-containing cells. After nutrient enrichment, a large fraction of dormant bacteria (6 to 11% of the total bacterial number) was “reactivated.” Bacterial turnover rates estimated ranged from 0.01 to 0.1 day⁻¹ but were 50 to 80 times higher when only the fraction of active bacteria was considered (on average 3.2 day⁻¹). Our results suggest that the fraction of active bacteria in marine sediments is controlled by nutrient supply and availability and that their turnover rates are at least 1 order of magnitude higher than previously reported.     

Active Transport and Accumulation of Iodide by Newly Isolated Marine Bacteria

Iodide (I⁻)-accumulating bacteria were isolated from marine sediment by an autoradiographic method with radioactive ¹²⁵I⁻. When they were grown in a liquid medium containing 0.1 μM iodide, 79 to 89% of the iodide was removed from the medium, and a corresponding amount of iodide was detected in the cells. Phylogenetic analysis based on 16S rRNA gene sequences indicated that iodide-accumulating bacteria were closely related to Flexibacter aggregans NBRC15975 and Arenibacter troitsensis, members of the family Flavobacteriaceae. When one of the strains, strain C-21, was cultured with 0.1 μM iodide, the maximum iodide content and the maximum concentration factor for iodide were 220 ± 3.6 (mean ± standard deviation) pmol of iodide per mg of dry cells and 5.5 × 10³, respectively. In the presence of much higher concentrations of iodide (1 μM to 1 mM), increased iodide content but decreased concentration factor for iodide were observed. An iodide transport assay was carried out to monitor the uptake and accumulation of iodide in washed cell suspensions of iodide-accumulating bacteria. The uptake of iodide was observed only in the presence of glucose and showed substrate saturation kinetics, with an apparent affinity constant for transport and a maximum velocity of 0.073 μM and 0.55 pmol min⁻¹ mg of dry cells⁻¹, respectively. The other dominant species of iodine in terrestrial and marine environments, iodate (IO₃⁻), was not transported.     

Bacterial Biomass, Metabolic State, and Activity in Stream Sediments: Relation to Environmental Variables and Multiple Assay Comparisons

Bacterial biomass, metabolic condition, and activity were measured over a 16-month period in the surface sediments of the following four field sites with differing dissolved organic matter regimes: a woodlot spring seep, a meadow spring seep, a second-order stream, and a third-order stream. Total bacterial biomass was measured by lipid phosphate and epifluorescence microscopic counts (EMC), and viable biomass was measured by ¹⁴C most probable number, EMC with 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride reduction, and ATP. Bacterial metabolic condition was determined from the percentage of respiring cells, poly-β-hydroxybutyrate concentrations, and adenylate energy charge. Activity measures included ¹⁴C-lipid synthesis, ³²P-phospholipid synthesis, the rate of uptake of algal lysate dissolved organic carbon, and respiration, from which biosynthesis was calculated (dissolved organic carbon uptake corrected for respiration). Total bacterial biomass (from EMC) ranged from 0.012 to 0.354 μg of C/mg of dry sediment and was usually lowest in the third-order stream. The percentage of cells respiring was less than 25% at all sites, indicating that most bacteria were dormant or dead. Adenylate energy charge was measured only in the third-order stream and was uniformly low. Poly-β-hydroxybutyrate concentrations were greater in the woodlot spring seep than in the second- and third-order streams. Uptake of algal lysate dissolved organic carbon ranged from undetectable levels to 166 mg of C · m⁻² · h⁻¹. Little community respiration could be attributed to algal lysate metabolism. Phospholipid synthesis ranged from 0.006 to 0.354 pmol · mg of dry sediment⁻¹ · h⁻¹. Phospholipid synthesis rates were used to estimate bacterial turnover at the study sites. An estimated 375 bacterial generations per year were produced in the woodlot spring seep, and 67 per year were produced in the third-order stream.     

Seasonal Bacterial Production in a Dimictic Lake as Measured by Increases in Cell Numbers and Thymidine Incorporation

Rates of primary and bacterial production in Little Crooked Lake were calculated from the rates of incorporation of H¹⁴CO₃⁻ and [methyl-³H]thymidine, respectively. Growth rates of bacteria in diluted natural samples were determined for epilimnetic and metalimnetic bacterial populations during the summers of 1982 and 1983. Exponential growth was observed in these diluted samples, with increases in cell numbers of 30 to 250%. No lag was observed in bacterial growth in 14 of 16 experiments. Correlation of bacterial growth rates to corresponding rates of thymidine incorporation by natural samples produced a conversion factor of 2.2 × 10¹⁸ cells produced per mole of thymidine incorporated. The mass of the average bacterial cell in the lake was 1.40 × 10⁻¹⁴ ± 0.05 × 10⁻¹⁴ g of C cell⁻¹. Doubling times of natural bacteria calculated from thymidine incorporation rates and in situ cell numbers ranged from 0.35 to 12.00 days (median, 1.50 days). Bacterial production amounted to 66.7 g of C m⁻² from April through September, accounting for 29.4% of total (primary plus bacterial) production during this period. The vertical and seasonal distribution of bacterial production in Little Crooked Lake was strongly influenced by the distribution of primary production. From April through September 1983, the depth of maximum bacterial production rates in the water column was related to the depth of high rates of primary production. On a seasonal basis, primary production increased steadily from May through September, and bacterial production increased from May through August and then decreased in September.     

Comparison of the Seasonal Variations of Synechococcus Assemblage Structures in Estuarine Waters and Coastal Waters of Hong Kong

Seasonal variation in the phylogenetic composition of Synechococcus assemblages in estuarine and coastal waters of Hong Kong was examined through pyrosequencing of the rpoC1 gene. Sixteen samples were collected in 2009 from two stations representing estuarine and ocean-influenced coastal waters, respectively. Synechococcus abundance in coastal waters gradually increased from 3.6 × 10³ cells ml⁻¹ in March, reaching a peak value of 5.7 × 10⁵ cells ml⁻¹ in July, and then gradually decreased to 9.3 × 10³ cells ml⁻¹ in December. The changes in Synechococcus abundance in estuarine waters followed a pattern similar to that in coastal waters, whereas its composition shifted from being dominated by phycoerythrin-rich (PE-type) strains in winter to phycocyanin-only (PC-type) strains in summer owing to the increase in freshwater discharge from the Pearl River and higher water temperature. The high abundance of PC-type Synechococcus was composed of subcluster 5.2 marine Synechococcus, freshwater Synechococcus (F-PC), and Cyanobium. The Synechococcus assemblage in the coastal waters, on the other hand, was dominated by marine PE-type Synechococcus, with subcluster 5.1 clades II and VI as the major lineages from April to September, when the summer monsoon prevailed. Besides these two clades, clade III cooccurred with clade V at relatively high abundance in summer. During winter, the Synechococcus assemblage compositions at the two sites were similar and were dominated by subcluster 5.1 clades II and IX and an undescribed clade (represented by Synechococcus sp. strain miyav). Clade IX Synechococcus was a relatively ubiquitous PE-type Synechococcus found at both sites, and our study demonstrates that some strains of the clade have the ability to deal with large variation of salinity in subtropical estuarine environments. Our study suggests that changes in seawater temperature and salinity caused by the seasonal variation of monsoonal forcing are two major determinants of the community composition and abundance of Synechococcus assemblages in Hong Kong waters.