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.     

Seasonal variations of metabolically active bacteria in a hypertrophic lake (Hartbeespoort Dam,South Africa)

The number of metabolically active bacteria was measured with nalidixic acid over two annual cycles at three depths in the epilimnion of hypertrophic Hartbeespoort Dam, South Africa. Concurrent measurements were made of water temperature, DOC, phytoplankton production of dissolved (EDOC) and particulate organic carbon, chlorophyll a and the uptake of glucose (V_max). The objective was to determine the dominant factors correlated to the number of metabolically active bacteria and the relationship between active bacterial numbers and heterotrophic activity.The number of active bacteria was usually highest at the surface and ranged between 0.70 and 6.82 x 10⁶ cells ml^–1. The dominant factors correlated to the number of bacteria at the surface were water temperature (r = 0.65, n = 54, p<0.001), primary production (r = 0.53, n = 51, p<0.001) and EDOC (r = 0.37, n = 45, p = 0.005). Surface V_max for glucose ranged between 0.11 and 4.0 µgC 1^–1 h^–1 and was positively correlated to the number of active bacteria (r = 0.61, n = 53, p<0.001). The specific activity index (10^–12 µgC cell^–1 h^–1) varied between 80 and 2290 at the surface and was most strongly correlated to EDOC (r = 0.70, n = 48, p<0.001). Relationships between active bacterial numbers, water temperature, phytoplankton activity and glucose uptake were also found at two additional depths within the epilimnion. These data suggest that bacterial populations in nutrient enriched lakes contain a large number of metabolically active cells with high individual activity as a result of enhanced phytoplankton growth.     

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.     

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.     

Protozoan Bacterivory and Escherichia coli Survival in Drinking Water Distribution Systems

The development of bacterial communities in drinking water distribution systems leads to a food chain which supports the growth of macroorganisms incompatible with water quality requirements and esthetics. Nevertheless, very few studies have examined the microbial communities in drinking water distribution systems and their trophic relationships. This study was done to quantify the microbial communities (especially bacteria and protozoa) and obtain direct and indirect proof of protozoan feeding on bacteria in two distribution networks, one of GAC water (i.e., water filtered on granular activated carbon) and the other of nanofiltered water. The nanofiltered water-supplied network contained no organisms larger than bacteria, either in the water phase (on average, 5 × 10⁷ bacterial cells liter⁻¹) or in the biofilm (on average, 7 × 10⁶ bacterial cells cm⁻²). No protozoa were detected in the whole nanofiltered water-supplied network (water plus biofilm). In contrast, the GAC water-supplied network contained bacteria (on average, 3 × 10⁸ cells liter⁻¹ in water and 4 × 10⁷ cells cm⁻² in biofilm) and protozoa (on average, 10⁵ cells liter⁻¹ in water and 10³ cells cm⁻² in biofilm). The water contained mostly flagellates (93%), ciliates (1.8%), thecamoebae (1.6%), and naked amoebae (1.1%). The biofilm had only ciliates (52%) and thecamoebae (48%). Only the ciliates at the solid-liquid interface of the GAC water-supplied network had a measurable grazing activity in laboratory test (estimated at 2 bacteria per ciliate per h). Protozoan ingestion of bacteria was indirectly shown by adding Escherichia coli to the experimental distribution systems. Unexpectedly, E. coli was lost from the GAC water-supplied network more rapidly than from the nanofiltered water-supplied network, perhaps because of the grazing activity of protozoa in GAC water but not in nanofiltered water. Thus, the GAC water-supplied network contained a functional ecosystem with well-established and structured microbial communities, while the nanofiltered water-supplied system did not. The presence of protozoa in drinking water distribution systems must not be neglected because these populations may regulate the autochthonous and allochthonous bacterial populations.     

Estimating Bacterioplankton Production by Measuring [3H]thymidine Incorporation in a Eutrophic Swedish Lake

Bacterioplankton abundance, [³H]thymidine incorporation, ¹⁴CO₂ uptake in the dark, and fractionated primary production were measured on several occasions between June and August 1982 in eutrophic Lake Norrviken, Sweden. Bacterioplankton abundance and carbon biomass ranged from 0.5 × 10⁹ to 2.4 × 10⁹ cells liter⁻¹ and 7 to 47 μg of C liter⁻¹, respectively. The average bacterial cell volume was 0.185 μm³. [³H]thymidine incorporation into cold-trichloroacetic acid-insoluble material ranged from 12 × 10⁻¹² to 200 × 10⁻¹² mol liter⁻¹ h⁻¹. Bacterial carbon production rates were estimated to be 0.2 to 7.1 μg of C liter⁻¹ h⁻¹. Bacterial production estimates from [³H]thymidine incorporation and ¹⁴CO₂ uptake in the dark agreed when activity was high but diverged when activity was low and when blue-green algae (cyanobacteria) dominated the phytoplankton. Size fractionation indicated negligible uptake of [³H]thymidine in the >3-μm fraction during a chrysophycean bloom in early June. We found that >50% of the ³H activity was in the >3-μm fraction in late August; this phenomenon was most likely due to Microcystis spp., their associated bacteria, or both. Over 60% of the ¹⁴CO₂ uptake in the dark was attributed to algae on each sampling occasion. Algal exudate was an important carbon source for planktonic bacteria. Bacterial production was roughly 50% of primary production.     

Direct Determination of Carbon and Nitrogen Contents of Natural Bacterial Assemblages in Marine Environments

In order to better estimate bacterial biomass in marine environments, we developed a novel technique for direct measurement of carbon and nitrogen contents of natural bacterial assemblages. Bacterial cells were separated from phytoplankton and detritus with glass fiber and membrane filters (pore size, 0.8 μm) and then concentrated by tangential flow filtration. The concentrate was used for the determination of amounts of organic carbon and nitrogen by a high-temperature catalytic oxidation method, and after it was stained with 4′,6-diamidino-2-phenylindole, cell abundance was determined by epifluorescence microscopy. We found that the average contents of carbon and nitrogen for oceanic bacterial assemblages were 12.4 ± 6.3 and 2.1 ± 1.1 fg cell⁻¹ (mean ± standard deviation; n = 6), respectively. Corresponding values for coastal bacterial assemblages were 30.2 ± 12.3 fg of C cell⁻¹ and 5.8 ± 1.5 fg of N cell⁻¹ (n = 5), significantly higher than those for oceanic bacteria (two-tailed Student’s t test; P < 0.03). There was no significant difference (P > 0.2) in the bacterial C:N ratio (atom atom⁻¹) between oceanic (6.8 ± 1.2) and coastal (5.9 ± 1.1) assemblages. Our estimates support the previous proposition that bacteria contribute substantially to total biomass in marine environments, but they also suggest that the use of a single conversion factor for diverse marine environments can lead to large errors in assessing the role of bacteria in food webs and biogeochemical cycles. The use of a factor, 20 fg of C cell⁻¹, which has been widely adopted in recent studies may result in the overestimation (by as much as 330%) of bacterial biomass in open oceans and in the underestimation (by as much as 40%) of bacterial biomass in coastal environments.     

Spatial Heterogeneity of Bacterial Populations along an Environmental Gradient at a Shallow Submarine Hydrothermal Vent near Milos Island (Greece)

The spatial heterogeneity of bacterial populations at a shallow-water hydrothermal vent in the Aegean Sea close to the island of Milos (Greece) was examined at two different times by using acridine orange staining for total cell counts, cultivation-based techniques, and denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA gene fragments. Concurrent with measurements of geochemical parameters, samples were taken along a transect from the center of the vent to the surrounding area. Most-probable-number (MPN) counts of metabolically defined subpopulations generally constituted a minor fraction of the total cell counts; both counting procedures revealed the highest cell numbers in a transition zone from the strongly hydrothermally influenced sediments to normal sedimentary conditions. Total cell counts ranged from 3.2 × 10⁵ cells ml⁻¹ in the water overlying the sediments to 6.4 × 10⁸ cells g (wet weight) of sediment⁻¹. MPN counts of chemolithoautotrophic sulfur-oxidizing bacteria varied between undetectable and 1.4 × 10⁶ cells g⁻¹. MPN counts for sulfate-reducing bacteria and dissimilatory iron-reducing bacteria ranged from 8 to 1.4 × 10⁵ cells g⁻¹ and from undetectable to 1.4 × 10⁶ cells g⁻¹, respectively. DGGE revealed a trend from a diverse range of bacterial populations which were present in approximately equal abundance in the transition zone to a community dominated by few populations close to the center of the vent. Temperature was found to be an important parameter in determining this trend. However, at one sampling time this trend was not discernible, possibly due to storm-induced disturbance of the upper sediment layers.     

Initial Effects of the Mount St. Helens Eruption on Nitrogen Cycle and Related Chemical Processes in Ryan Lake

Ryan Lake, a 1.6-hectare basin lake near the periphery of the tree blowdown area in the blast zone 19 km north of Mount St. Helens, was studied from August to October 1980 to determine the microbial and chemical response of the lake to the eruption. Nutrient enrichment through the addition of fresh volcanic material and the organic debris from the surrounding conifer forest stimulated intense microbial activity. Concentrations of such nutrients as phosphorus, sulfur, manganese, iron, and dissolved organic carbon were markedly elevated. Nitrogen cycle activity was especially important to the lake ecosystem in regulating biogeochemical cycling owing to the limiting abundance of nitrogen compounds. Nitrogen fixation, both aerobic and anaerobic, was active from aerobic benthic and planktonic cyanobacteria with rates up to 210 nmol of N₂ cm⁻¹ h⁻¹ and 667 nmol of N₂ liter⁻¹ h⁻¹, respectively, and from anaerobic bacteria with rates reaching 220 nmol of N₂ liter⁻¹ h⁻¹. Nitrification was limited to the aerobic epilimnion and littoral zones where rates were 43 and 261 nmol of NO₂ liter⁻¹ day⁻¹, respectively. Potential denitrification rates were as high as 30 μmol of N₂O liter⁻¹ day⁻¹ in the anaerobic hypolimnion. Total bacterial numbers ranged from 1 × 10⁶ to 3 × 10⁸ ml⁻¹ with the number of viable sulfur-metal-oxidizing bacteria reaching 2 × 10⁶ ml⁻¹ in the hypolimnion. A general scenario for the microbial cycling of nitrogen, carbon, sulfur, and metals is presented for volcanically impacted lakes. The important role of nitrogen as these lakes recover from the cataclysmic eruption and proceed back towards their prior status as oligotrophic alpine lakes is emphasized.     

Dynamics of Microbial Communities on Marine Snow Aggregates: Colonization, Growth, Detachment, and Grazing Mortality of Attached Bacteria

We studied the dynamics of microbial communities attached to model aggregates (4-mm-diameter agar spheres) and the component processes of colonization, detachment, growth, and grazing mortality. Agar spheres incubated in raw seawater were rapidly colonized by bacteria, followed by flagellates and ciliates. Colonization can be described as a diffusion process, and encounter volume rates were estimated at about 0.01 and 0.1 cm³ h⁻¹ for bacteria and flagellates, respectively. After initial colonization, the abundances of flagellates and ciliates remained approximately constant at 10³ to 10⁴ and ~10² cells sphere⁻¹, respectively, whereas bacterial populations increased at a declining rate to >10⁷ cells sphere⁻¹. Attached microorganisms initially detached at high specific rates of ~10⁻² min⁻¹, but the bacteria gradually became irreversibly attached to the spheres. Bacterial growth (0 to 2 day⁻¹) was density dependent and declined hyperbolically when cell density exceeded a threshold. Bacterivorous flagellates grazed on the sphere surface at an average saturated rate of 15 bacteria flagellate⁻¹ h⁻¹. At low bacterial densities, the flagellate surface clearance rate was ~5 × 10⁻⁷ cm² min⁻¹, but it declined hyperbolically with increasing bacterial density. Using the experimentally estimated process rates and integrating the component processes in a simple model reproduces the main features of the observed microbial population dynamics. Differences between observed and predicted population dynamics suggest, however, that other factors, e.g., antagonistic interactions between bacteria, are of importance in shaping marine snow microbial communities.     

Production of NO2- and N2O by Nitrifying Bacteria at Reduced Concentrations of Oxygen

Pure cultures of the marine ammonium-oxidizing bacterium Nitrosomonas sp. were grown in the laboratory at oxygen partial pressures between 0.005 and 0.2 atm (0.18 to 7 mg/liter). Low oxygen conditions induced a marked decrease in the rate for production of NO₂^-, from 3.6 × 10⁻¹⁰ to 0.5 × 10⁻¹⁰ mmol of NO₂^- per cell per day. In contrast, evolution of N₂O increased from 1 × 10⁻¹² to 4.3 × 10⁻¹² mmol of N per cell per day. The yield of N₂O relative to NO₂^- increased from 0.3% to nearly 10% (moles of N in N₂O per mole of NO₂^-) as the oxygen level was reduced, although bacterial growth rates changed by less than 30%. Nitrifying bacteria from the genera Nitrosomonas, Nitrosolobus, Nitrosospira, and Nitrosococcus exhibited similar yields of N₂O at atmospheric oxygen levels. Nitrite-oxidizing bacteria (Nitrobacter sp.) and the dinoflagellate Exuviaella sp. did not produce detectable quantities of N₂O during growth. The results support the view that nitrification is an important source of N₂O in the environment.     

Improved Most-Probable-Number Method To Detect Sulfate-Reducing Bacteria with Natural Media and a Radiotracer

A greatly improved most-probable-number (MPN) method for selective enumeration of sulfate-reducing bacteria (SRB) is described. The method is based on the use of natural media and radiolabeled sulfate (³⁵SO₄²⁻). The natural media used consisted of anaerobically prepared sterilized sludge or sediment slurries obtained from sampling sites. The densities of SRB in sediment samples from Kysing Fjord (Denmark) and activated sludge were determined by using a normal MPN (N-MPN) method with synthetic cultivation media and a tracer MPN (T-MPN) method with natural media. The T-MPN method with natural media always yielded significantly higher (100- to 1,000-fold-higher) MPN values than the N-MPN method with synthetic media. The recovery of SRB from environmental samples was investigated by simultaneously measuring sulfate reduction rates (by a ³⁵S-radiotracer method) and bacterial counts by using the T-MPN and N-MPN methods, respectively. When bacterial numbers estimated by the T-MPN method with natural media were used, specific sulfate reduction rates (qSO₄²⁻) of 10⁻¹⁴ to 10⁻¹³ mol of SO₄²⁻ cell⁻¹ day⁻¹ were calculated, which is within the range of qSO₄²⁻ values previously reported for pure cultures of SRB (10⁻¹⁵ to 10⁻¹⁴ mol of SO₄²⁻ cell⁻¹ day⁻¹). qSO₄²⁻ values calculated from N-MPN values obtained with synthetic media were several orders of magnitude higher (2 × 10⁻¹⁰ to 7 × 10⁻¹⁰ mol of SO₄²⁻ cell⁻¹ day⁻¹), showing that viable counts of SRB were seriously underestimated when standard enumeration media were used. Our results demonstrate that the use of natural media results in significant improvements in estimates of the true numbers of SRB in environmental samples.     

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.     

Chlorine Demand and Inactivation of Fungal Propagules

Conidia of filamentous fungi, vegetative yeast cells, and coliform bacteria were tested to determine their chlorine demand and their sensitivity to chlorine inactivation. Levels of chlorine demand for the various conidia, yeast, and coliforms were, respectively, 3.6 × 10⁻⁹ to 3.2 × 10⁻⁸, 1.2 × 10⁻⁹ to 8.0 × 10⁻⁹, and 2.5 × 10⁻¹¹ to 6.3 × 10⁻¹⁰ mg of chlorine per propagule. Preliminary evidence suggests that the chlorine demand per propagule increases as the number of propagules per milliliter decreases. In general, conidia showed greatest resistance to chlorine inactiviation, followed by the yeast and coliforms. Inactivation by chlorine was influenced by pH, with inactivation (chlorine activity) falling in the order pH 5 > 7 > 8.     

Distribution of Sulfate-Reducing and Methanogenic Bacteria in Anaerobic Aggregates Determined by Microsensor and Molecular Analyses

Using molecular techniques and microsensors for H₂S and CH₄, we studied the population structure of and the activity distribution in anaerobic aggregates. The aggregates originated from three different types of reactors: a methanogenic reactor, a methanogenic-sulfidogenic reactor, and a sulfidogenic reactor. Microsensor measurements in methanogenic-sulfidogenic aggregates revealed that the activity of sulfate-reducing bacteria (2 to 3 mmol of S²⁻ m⁻³ s⁻¹ or 2 × 10⁻⁹ mmol s⁻¹ per aggregate) was located in a surface layer of 50 to 100 μm thick. The sulfidogenic aggregates contained a wider sulfate-reducing zone (the first 200 to 300 μm from the aggregate surface) with a higher activity (1 to 6 mmol of S²⁻ m⁻³ s⁻¹ or 7 × 10⁻⁹ mol s⁻¹ per aggregate). The methanogenic aggregates did not show significant sulfate-reducing activity. Methanogenic activity in the methanogenic-sulfidogenic aggregates (1 to 2 mmol of CH₄ m⁻³ s⁻¹ or 10⁻⁹ mmol s⁻¹ per aggregate) and the methanogenic aggregates (2 to 4 mmol of CH₄ m⁻³ s⁻¹ or 5 × 10⁻⁹ mmol s⁻¹ per aggregate) was located more inward, starting at ca. 100 μm from the aggregate surface. The methanogenic activity was not affected by 10 mM sulfate during a 1-day incubation. The sulfidogenic and methanogenic activities were independent of the type of electron donor (acetate, propionate, ethanol, or H₂), but the substrates were metabolized in different zones. The localization of the populations corresponded to the microsensor data. A distinct layered structure was found in the methanogenic-sulfidogenic aggregates, with sulfate-reducing bacteria in the outer 50 to 100 μm, methanogens in the inner part, and Eubacteria spp. (partly syntrophic bacteria) filling the gap between sulfate-reducing and methanogenic bacteria. In methanogenic aggregates, few sulfate-reducing bacteria were detected, while methanogens were found in the core. In the sulfidogenic aggregates, sulfate-reducing bacteria were present in the outer 300 μm, and methanogens were distributed over the inner part in clusters with syntrophic bacteria.     

Protozoan Grazing and Bacterial Production in Stratified Lake Vechten Estimated with Fluorescently Labeled Bacteria and by Thymidine Incorporation

In stratified Lake Vechten, The Netherlands, protozoan grazing was estimated on the basis of uptake of fluorescently labeled bacteria and compared with bacterial production estimated on the basis of thymidine incorporation. By using a grazer-free mixed bacterial population from the lake in continuous culture, an empirical relationship between cell production and thymidine incorporation was established. Thymidine incorporation into total cold-trichloroacetic-acid-insoluble macromolecules yielded a relatively constant empirical conversion factor of ca. 10¹⁸ (range, 0.38 × 10¹⁸ to 1.42 × 10¹⁸) bacteria mol of thymidine⁻¹ at specific growth rates (μ) ranging from 0.007 to 0.116 h⁻¹. Although thymidine incorporation has been assumed to measure DNA synthesis thymidine incorporation appeared to underestimate the independently measured bacterial DNA synthesis by at least 1.5- to 13-fold, even if all incorporated label was assumed to be in DNA. However, incorporation into DNA was found to be insignificant as measured by conventional acid-base hydrolysis. Methodological problems of the thymidine technique are discussed. Like the cultures, Lake Vechten bacteria showed considerable thymidine incorporation into total macromolecules, but no significant incorporation into DNA was found by acid-base hydrolysis. This applied not only to the low-oxygen hypo- and metalimnion but also to the aerobic epilimnion. Thus, the established empirical conversion factor for thymidine incorporation into total macromolecules was used to estimate bacterial production. Maximum production rates (141 × 10⁶ bacteria liter⁻¹ h⁻¹; μ, 0.012 h⁻¹) were found in the metalimnion and were 1 order of magnitude higher than in the epi- and hypolimnion. In all three strata, the estimated bacterial production was roughly balanced by the estimated protozoan grazing. Heterotrophic nanoflagellates were the major consumers of the bacterial production and showed maximum numbers (up to 40 × 10⁶ heterotrophic nanoflagellates liter⁻¹) in the microaerobic metalimnion.     

Experimental Evaluation of Conversion Factors for the [3H]Thymidine Incorporation Assay of Bacterial Secondary Productivity

The relationship between bacterial growth and incorporation of [methyl-³H]thymidine in oligotrophic lake water cultures was investigated. Prescreening, dilution, and addition of organic and inorganic nutrients were treatments used to prevent bacterivory and stimulate bacterial growth. Growth in unmanipulated samples was estimated through separate measurements of grazing losses. Both bacterial number and biovolume growth responses were measured, and incorporation of [³H]thymidine in both total macromolecules and nucleic acids was assayed. The treatments had significant effects on conversion factors used to relate thymidine incorporation to bacterial growth. Cell number-based factors ranged from 1.1 × 10¹⁸ to 38 × 10¹⁸ cells mol of total thymidine incorporation⁻¹ and varied with treatment up to 10-fold for the same initial bacterial assemblage. In contrast, cell biovolume-based conversion factors were similar for two treatment groups across a 16-fold range of [³H]thymidine incorporation rates: 5.54 × 10¹⁷ μm³ mol of total thymidine incorporation⁻¹ and 15.2 × 10¹⁷ μm³ mol of nucleic acid incorporation⁻¹. Much of the variation in cell number-based conversion factors was related to changes in apparent mean cell volume of produced bacteria. Phosphorus addition stimulated [³H]thymidine incorporation more than it increased bacterial growth, which resulted in low conversion factors.     

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.     

High-Frequency Phage-Mediated Gene Transfer among Escherichia coli Cells, Determined at the Single-Cell Level

Recent whole-genome analysis suggests that lateral gene transfer by bacteriophages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency of phage-mediated gene transfer, we employed cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) and investigated the movement of the ampicillin resistance gene among Escherichia coli cells mediated by phage at the single-cell level. Phages P1 and T4 and the newly isolated E. coli phage EC10 were used as vectors. The transduction frequencies determined by conventional plating were 3 × 10⁻⁸ to 2 × 10⁻⁶, 1 × 10⁻⁸ to 4 × 10⁻⁸, and <4 × 10⁻⁹ to 4 × 10⁻⁸ per PFU for phages P1, T4, and EC10, respectively. The frequencies of DNA transfer determined by CPRINS-FISH were 7 × 10⁻⁴ to 1 × 10⁻³, 9 × 10⁻⁴ to 3 × 10⁻³, and 5 × 10⁻⁴ to 4 × 10⁻³ for phages P1, T4, and EC10, respectively. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viabilities. These results revealed that the difference in the number of viable cells carrying the transferred gene and the number of cells capable of growth on the selective medium was 3 to 4 orders of magnitude, indicating that phage-mediated exchange of DNA sequences among bacteria occurs with unexpectedly high frequency.     

Gene Transfer by Transduction in the Marine Environment

To determine the potential for bacteriophage-mediated gene transfer in the marine environment, we established transduction systems by using marine phage host isolates. Plasmid pQSR50, which contains transposon Tn5 and encodes kanamycin and streptomycin resistance, was used in plasmid transduction assays. Both marine bacterial isolates and concentrated natural bacterial communities were used as recipients in transduction studies. Transductants were detected by a gene probe complementary to the neomycin phosphotransferase (nptII) gene in Tn5. The transduction frequencies ranged from 1.33 × 10⁻⁷ to 5.13 × 10⁻⁹ transductants/PFU in studies performed with the bacterial isolates. With the mixed bacterial communities, putative transductants were detected in two of the six experiments performed. These putative transductants were confirmed and separated from indigenous antibiotic-resistant bacteria by colony hybridization probed with the nptII probe and by PCR amplification performed with two sets of primers specific for pQSR50. The frequencies of plasmid transduction in the mixed bacterial communities ranged from 1.58 × 10⁻⁸ to 3.7 × 10⁻⁸ transductants/PFU. Estimates of the transduction rate obtained by using a numerical model suggested that up to 1.3 × 10¹⁴ transduction events per year could occur in the Tampa Bay Estuary. The results of this study suggest that transduction could be an important mechanism for horizontal gene transfer in the marine environment.