Estimates of bacterial productivity in marine sediments and water from a temperate saltmarsh lagoon

Tritiated thymidine incorporation (TTI) into DNA was used to estimate bacterial productivity in sediment and water samples from two sites in Langebaan Lagoon, South Africa. Routine analysis of isotope dilution showed seasonal variations of approximately threefold in the thymidine precursor pool sizes for bacterial assemblages from each site. Dual label incorporation of [³H]-thymidine and ¹⁴C-leucine into DNA and protein, respectively, showed that pelagic but not sediment assemblages were in a balanced state of growth during TTI. This is the first report of dual label measurements of bacterial production in sediments. Sediments supported bacterial productivities that exceeded those in the water column by factors from five- to 950-fold, whereas bacterial abundance supported by sediments exceeded that in the water column by more than 3 orders of magnitude. Estimates of bacterial productivities in sediments were coincident with levels of organic content in sediments, but not with bacterial abundance. Measurements of TTI activity for 5 different benthic microhabitats at one lagoon site showed highest activity associated with seagrass beds (2.11 ± 0.84 nmol thymidine hours^–1 g-1 dry weight), whereas activities decreased with depth (0.46 ± 0.21 nmol thymidine hours^–1 g^–I dry weight) below sediment surface.Offprint requests to: B. J. Tibbles.     

Assessment of [3H]Thymidine Incorporation into DNA as a Method To Determine Bacterial Productivity in Stream Bed Sediments

We performed several checks on the underlying assumptions and procedures of the thymidine technique applied to stream bed sediments. Bacterial production rates were not altered when sediments were mixed to form a slurry. Incubation temperature did affect production rates. Controls fixed and washed with formaldehyde had lower backgrounds than trichloroacetic acid controls. DNA extraction by base hydrolysis was incomplete and variable at 25°C, but hydrolysis at 120°C extracted 100% of the DNA, of which 84% was recovered upon precipitation. Production rates increased as thymidine concentrations were increased over 3 orders of magnitude (30 nM to 53 μM thymidine). However, over narrower concentration ranges, thymidine incorporation into DNA was independent of thymidine concentration. Elevated exogenous thymidine concentrations did not eliminate de novo synthesis. Transport of thymidine into bacterial cells occurred at least 5 to 20 times faster than incorporation of label into DNA. We found good agreement between production rates of bacterial cultures based upon increases in cell numbers and estimates based upon thymidine incorporation and amount of DNA per cell. Those comparisons emphasized the importance of isotopic dilution measurements and validated the use of the reciprocal plot technique for estimating isotopic dilution. Nevertheless, the thymidine technique cannot be considered a routine assay and the inability to measure the cellular DNA content in benthic communities restricts the accuracy of the method in those habitats.     

Benthic bacterial biomass and production in the Hudson River estuary

Bacterial biomass, production, and turnover were determined for two freshwater marsh sites and a site in the main river channel along the tidally influenced Hudson River. The incorporation of [methyl-³H]thymidine into DNA was used to estimate the growth rate of surface and anaerobic bacteria. Bacterial production at marsh sites was similar to, and in some cases considerably higher than, production estimates reported for other aquatic wetland and marine sediment habitats. Production averaged 1.8–2.8 mg C·m^–2·hour^–1 in marsh sediments. Anaerobic bacteria in marsh sediment incorporated significant amounts of [methyl-³H]thymidine into DNA. Despite differences in dominant vegetation and tidal regime, bacterial biomass was similar (1×10³±0.08 mg C·m^–2) inTrapa, Typha, andNuphar aquatic macrophyte communities. Bacterial abundance and productivity were lower in sandy sediments associated withScirpus communities along the Hudson River (0.2×10³±0.05 mg C·m^–2 and 0.3±0.23 mg C·m^–2·hour^–1, respectively).     

Assessment of [h]thymidine incorporation into DNA as a method to determine bacterial productivity in stream bed sediments

We performed several checks on the underlying assumptions and procedures of the thymidine technique applied to stream bed sediments. Bacterial production rates were not altered when sediments were mixed to form a slurry. Incubation temperature did affect production rates. Controls fixed and washed with formaldehyde had lower backgrounds than trichloroacetic acid controls. DNA extraction by base hydrolysis was incomplete and variable at 25 degrees C, but hydrolysis at 120 degrees C extracted 100% of the DNA, of which 84% was recovered upon precipitation. Production rates increased as thymidine concentrations were increased over 3 orders of magnitude (30 nM to 53 muM thymidine). However, over narrower concentration ranges, thymidine incorporation into DNA was independent of thymidine concentration. Elevated exogenous thymidine concentrations did not eliminate de novo synthesis. Transport of thymidine into bacterial cells occurred at least 5 to 20 times faster than incorporation of label into DNA. We found good agreement between production rates of bacterial cultures based upon increases in cell numbers and estimates based upon thymidine incorporation and amount of DNA per cell. Those comparisons emphasized the importance of isotopic dilution measurements and validated the use of the reciprocal plot technique for estimating isotopic dilution. Nevertheless, the thymidine technique cannot be considered a routine assay and the inability to measure the cellular DNA content in benthic communities restricts the accuracy of the method in those habitats.     

Heterotrophic microbial activity in shallow aquifer sediments of Long Island,New York

Bacterial numbers and activities (as estimated by glucose uptake and total thymidine incorporation) were investigated at two sites in Long Island, New York aquifer sediments. In general, bacterial activities were higher in shallow (1.5–4.5 m below the water table or BWT), oxic sediments than in deep (10–18 m BWT), anoxic sediments. The average total glucose uptake rates were 0.18 ± 0.10 ng gdw^–1 h^–1 in shallow sediments and 0.09 ± 0.11 ng gdw^–1 h^–1 in deep sediments; total thymidine incorporation rates were 0.10 ± 0.13 pmol gdw^–1 h^–1 and 0.03 ± 0.03 pmol gdw^–1 h^–1 in shallow and deep sediments, respectively. Incorporation of glucose was highly efficient, as only about 10% of added label was recovered as CO₂. Bacterial abundance (estimated from acridine orange direct counts) was 2.5 ± 2.0 × 10⁷ cells gdw^–1 and 2.0 ± 1.3 × 10⁷ cells gdw^–1 in shallow and deep sediments, respectively. These bacterial activity and abundance estimates are similar to values found in other aquifer environments, but are 10- to 1000-fold lower than values in soil or surface sediment of marine and estuarine systems. In general, cell specific microbial activities were lower in sites from Connetquot Park, a relatively pristine site, when compared to activities found in sites from Jamesport, which has had a history of aldicarb (a pesticide) contamination. To our knowledge, this is the first report of bacterial activity measurements in the shallow, sandy aquifers of Long Island, New York.Correspondence to: D.G. Capone     

Measurement of bacterial growth rates on the rhizoplane using 3H-thymidine incorporation into DNA

Bacterial growth rates on the rhizoplane of rape seedlings grown in sand were determined using ³H-thymidine incorporation into DNA. Axenic roots incorporated thymidine into DNA, which had to be subtracted from values for roots with associated bacteria. Thymidine incorporation into rhizoplane bacterial DNA ranged between 0.6 and 1.4 pmol thymidine h^–1 root^–1 for 6 to 26-day-old plants. Using a conversion factor, the turnover time of bacteria was calculated to decrease from 9.2 h for 6-day-old plants to 160h for 26-day-old plants. A similar value was found for rhizosphere bacteria of plants grown for 26 days in natural soil.     

Microbial biomass and activities associated with subsurface environments contaminated with chlorinated hydrocarbons

Soil microcosms and enrichment cultures from subsurface sediments and groundwaters contaminated with trichloroethylene (TCE) were examined. Total lipids, [I‐‘⁴C]acetate incorporation into lipids, and [Me‐³H]thymidine incorporation into DNA were determined in these subsurface environments. In heavily TCE‐contam‐inated zones (greater than 500 mg/L) radioisotopes were not incorporated into lipids or DNA. Radioisotope incorporation occurred in sediments both above and below the TCE plume. Phospholipid fatty acids (PLFA) were not detected, i.e., less than 0.5 pmol/L in heavily contaminated groundwater samples. In less contaminated waters, extracted PLFA concentrations were greater than 100 pmollL and microbial isolates were readily obtained. Degradation of 30–100 mg/L TCE was observed when sediments were amended with a variety of energy sources. Microorganisms in these subsurface sediments have adapted to degrade TCE at concentrations greater than 50 mg/L.     

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.     

Microbial activities in deep subsurface environments

Activities of microorganisms residing in terrestrial deep subsurface sediments were examined in 46 sediment samples from three boreholes. Radiolabeled time course experiments assessing in situ microbial activities were initiated within 30 min of core recovery. [1‐C⁴] Acetate incorporation into lipids, [ methyl‐³H] thymidine incorporation into DNA, [2‐¹⁴C]acetate, and [U‐¹⁴C]glucose mineralization in addition to microbial enrichment and enumeration studies were examined in surface and subsurface sediments. Surface soils contained the greatest biomass and activities, followed by the shallow aquifer zones. Water‐saturated subsurface sands exhibited three to four orders of magnitude greater activity and culturable microorganisms than the dense clay zones, which had low permeability. Regardless of depth, sediments that contained more than 20% clays exhibited the lowest activities and culturable microorganisms.     

Adaptation of thymidine utilization to changing rates of DNA synthesis in the cell cycle

Summary In synchronous cultures of P-815 murine mastocytoma and of Chinese hamster ovary (CHO) cells, the relative contribution of exogenous thymidine to DNA synthesis was studied by comparing rates of (³H)thymidine incorporation with the rate of DNA synthesis as derived from incorporation of (³H)thymidine (10^–5 m) in the presence of amethopterin. In synchronous P-815 cultures, time-dependent variations of DNA synthesis rates were in close agreement with those of (³H)thymidine incorporation rates at concentrations of the precursor ranging from 5 × 10^–8 to 10^–5 m. Similarly, in synchronous CHO cell cultures prepared by two different methods, time-dependent changes in DNA synthesis rate were almost identical with those of the rate of incorporation of (³H)thymidine supplied at 5 × 10^–8 m. Thus, at a given thymidine concentration in the medium, the proportion of thymine residues in DNA that were derived from exogenous thymidine remained nearly constant, even though rates of cellular DNA synthesis underwent pronounced changes. This indicates that in the synchronous culture systems used, utilization of exogenous thymidine is efficiently adapted to changing rates of DNA synthesis.In partial fulfillment of the requirements for the degree of Ph.D. by G.G.M.     

Spatial and Temporal Variations in Bacterial Macromolecule Labeling with [methyl-3H]Thymidine in a Hypertrophic Lake

The incorporation of [methyl-³H]thymidine into three macromolecular fractions, designated as DNA, RNA, and protein, by bacteria from Hartbeespoort Dam, South Africa, was measured over 1 year by acid-base hydrolysis procedures. Samples were collected at 10 m, which was at least 5 m beneath the euphotic zone. On four occasions, samples were concurrently collected at the surface. Approximately 80% of the label was incorporated into bacterial DNA in surface samples. At 10 m, total incorporation of label into bacterial macromolecules was correlated to bacterial utilization of glucose (r = 0.913, n = 13, P < 0.001). The labeling of DNA, which ranged between 0 and 78% of total macromolecule incorporation, was inversely related to glucose uptake (r = -0.823), total thymidine incorporation (r = -0.737), and euphotic zone algal production (r = -0.732, n = 13, P < 0.005). With decreased DNA labeling, increasing proportions of label were found in the RNA fraction and proteins. Enzymatic digestion followed by chromatographic separation of macromolecule fragments indicated that DNA and proteins were labeled while RNA was not. The RNA fraction may represent labeled lipids or other macromolecules or both. The data demonstrated a close coupling between phytoplankton production and heterotrophic bacterial activity in this hypertrophic lake but also confirmed the need for the routine extraction and purification of DNA during [methyl-³H]thymidine studies of aquatic bacterial production.     

Bacterial production in freshwater sediments: Cell specific versus system measures

Estimates of bacterial production based on total trichloroacetic acid (TCA)-precipitable [methyl-³H]thymidine incorporation and frequency of dividing cell (FDC) techniques were compared to sediment respiration rates in Lake George, New York. Bacterial growth rates based on thymidine incorporation ranged from 0.024 to 0.41 day^–1, while rates based on FDC ranged from 1.78 to 2.48 day^–1. Respiration rates ranged from 0.11 to 1.8mol O₂·hour^–1·g dry weight sediment^–1. Thymidine incorporation yielded production estimates which were in reasonable agreement with respiration rates. Production estimates based on FDC were 4- to 190-fold higher than those predicted from respiration rates.     

Measurement of the incorporation rates of four amino acids into proteins for estimating bacterial production

In aquatic ecosystems, [³H]thymidine incorporation into bacterial DNA and [³H]leucine incorporation into proteins are usually used to estimate bacterial production. The incorporation rates of four amino acids (leucine, tyrosine, lysine, alanine) into proteins of bacteria were measured in parallel on natural freshwater samples from the basin of the river Meuse (Belgium). Comparison of the incorporation into proteins and into the total macromolecular fraction showed that these different amino acids were incorporated at more than 90% into proteins. From incorporation measurements at four subsaturated concentrations (range, 2–77 nm), the maximum incorporation rates were determined. Strong correlations (r > 0.91 for all the calculated correlations) were found between the maximum incorporation rates of the different tested amino acids over a range of two orders of magnitude of bacterial activity. Bacterial production estimates were calculated using theoretical and experimental conversion factors. The productions calculated from the incorporation rates of the four amino acids were in good concordance, especially when the experimental conversion factors were used (slope range, 0.91–1.11, and r > 0.91). This study suggests that the incorporation of various amino acids into proteins can be used to estimate bacterial production.     

DNA extractions from deep subseafloor sediments: novel cryogenic-mill-based procedure and comparison to existing protocols

Extracting DNA from deep subsurface sediments is challenging given the complexity of sediments types, low biomasses, resting structures (spores, cysts) frequently encountered in deep sediments, and the potential presence of enzymatic inhibitors. Promising results for cell lysis efficiency were recently obtained by use of a cryogenic mill (Lipp et al., 2008). These findings encouraged us to devise a DNA extraction protocol using this tool. Thirteen procedures involving a combination of grinding in liquid nitrogen (for various durations and beating rates) with different chemical solutions (phenol, chloroform, SDS, sarkosyl, proteinase, GTC), or with use of DNA recovery kits (MagExtractor®) were compared. Effective DNA extraction was evaluated in terms of cell lysis efficiency, DNA extraction efficiency, DNA yield and determination of prokaryotic diversity. Results were compared to those obtained by standard protocols: the FastDNA®SPIN kit for soil and the Zhou protocol. For most sediment types grinding in a cryogenic mill at a low beating rate in combination with direct phenol-chloroform extraction resulted in much higher DNA yields than those obtained using classical procedures. In general (except for clay-rich sediments), this procedure provided high-quality crude extracts for direct downstream nested-PCR, from cell numbers as low as 1.1 × 10⁶ cells/cm³. This procedure is simple, rapid, low-cost, and could be used with minor modifications for large-scale DNA extractions for a variety of experimental goals.     

Assessment of Preparation Methods for Organic Phosphorus Analysis in Phosphorus-Polluted Fe/Al-Rich Haihe River Sediments Using Solution 31P-NMR

Fe/Al-rich river sediments that were highly polluted with phosphorus (P) were used in tests to determine the optimum preparation techniques for measuring organic P (Po) using solution ³¹P nuclear magnetic resonance spectroscopy (³¹P-NMR). The optimum pre-treatment, extraction time, sediment to solution ratio and sodium hydroxide-ethylenediaminetetraacetic acid (NaOH-EDTA) extractant solution composition were determined. The total P and Po recovery rates were higher from freeze- and air-dried samples than from fresh samples. An extraction time of 16 h was adequate for extracting Po, and a shorter or longer extraction time led to lower recoveries of total P and Po, or led to the degradation of Po. An ideal P recovery rate and good-quality NMR spectra were obtained at a sediment:solution ratio of 1∶10, showing that this ratio is ideal for extracting Po. An extractant solution of 0.25 M NaOH and 50 mM EDTA was found to be more appropriate than either NaOH on its own, or a more concentrated NaOH-EDTA mixture for ³¹P-NMR analysis, as this combination minimized interference from paramagnetic ions and was appropriate for the detected range of Po concentrations. The most appropriate preparation method for Po analysis, therefore, was to extract the freeze-dried and ground sediment sample with a 0.25 M NaOH and 50 mM EDTA solution at a sediment:solution ratio of 1∶10, for 16 h, by shaking. As lyophilization of the NaOH-EDTA extracts proved to be an optimal pre-concentration method for Po analysis in the river sediment, the extract was lyophilized as soon as possible, and analyzed by ³¹P-NMR.     

Significance of bacterial biomass in the nutrition of a freshwater isopod (Lirceus sp.)

The quantitative significance of bacterial biomass in the nutrition of detritivores remains equivocal. We have used tritiated thymidine to specifically label stable macromolecules in natural assemblages of sediment-associated and detritus-associated bacteria. This material was presented to the isopod (Lirceus sp.) and incorporation of bacterial biomass measured. The isopod incorporated roughly 1 ng bacterial carbon (mg wet wt.)^-1 h^-1 from leaf discs and about 6 ng mg^-1 h^-1 from sediment. Calculation of grazing rate from changes in cell counts yields grazing rates from 2.3–17.9 ng C mg^-1 h^-1. Even the maximum grazing rate, which is an overestimate of C assimilated, represents only 14.7% of C respired by the isopod.     

Contribution of heterotrophic bacterial production to the carbon budget of the river Seine (France)

Bacterial activity was measured in the river Seine by two methods, ³H-thymidine incorporation into DNA and ³H-leucine incorporation into proteins. Both incorporation rates are characterized by low values upstream of Paris, a large increase just downstream of the outfall of the Achères treatment plant effluents, and then decreasing values further downstream. The covariation of both activities is demonstrated by the constancy of the molar ratio (leucine to thymidine incorporation rate) in the range of 6 to 8 for all the samples, except in the perturbed area where it is higher (15 to 35). These high values of molar ratio are linked to the introduction into the river of large sized bacteria (1 µm) with higher incorporation rates per cell or biomass unit than the small autochthonous bacteria (< 1 µm). Growth rates of large bacteria were on average 3.7 times higher than those of small bacteria. Bacterial production was calculated with experimentally determined conversion factors (0.5 × 10¹⁸ cells per mole of thymidine incorporated and 900 gC per mole of leucine incorporated) and by taking into account the activity of both size classes of bacteria measured through fractionation experiments (post-incubation filtration). Production estimated in the perturbed area downstream of Ach6res was very high, up to 60 µgC liter^–1h^–1 in the summer. Carbon consumption by bacteria in the area perturbed by the Ach6res effluents was calculated assuming a growth yield of 0.2 and compared to the load of biodegradable organic matter discharged by the treatment plant. In summer, an additional supply of organic matter is required to account for the intense bacterial activity, suggesting the importance of phytoplankton production in the carbon budget. Offprint requests to: Pierre Servais     

DETERMINATION OF RATES OF DNA SYNTHESIS IN CULTURED MAMMALIAN CELL POPULATIONS

In cultures of a murine mastocytoma, endogenous synthesis of thymidine phosphates, as determined by the incorporation of [³H]deoxyuridine into DNA, was reduced within 15 min to less than 3% of control values by the addition of amethopterin (10 µM) in combination with hypoxanthine and glycine. If [³H]thymidine and unlabeled thymidine were added simultaneously with amethopterin, the increase with time of radioactivity in cellular DNA was linear at least between 30 and 90 min, while radioactivity in the acid-soluble nucleotide fraction remained constant during this time interval, indicating that intracellular thymidine nucleotides had the same specific activity as exogenously supplied [³H]thymidine. This permitted calculation of the amount of thymidine incorporated per hour into DNA of 10⁶ cells. In conjunction with the base composition of mouse DNA, these results were used to calculate rates of DNA synthesis. Cell proliferation rate, cell cycle time, and the duration of the S period were not affected to any appreciable extent by the addition of amethopterin and thymidine. Rates of DNA synthesis, as derived from thymidine incorporation rates, were in good agreement with those derived from the measured mean DNA content of exponentially multiplying cells and rates of cell proliferation.     

Bacterial productivity in ponds used for culture of penaeid prawns

The quantitative role of bacteria in the carbon cycle of ponds used for culture of penaeid prawns has been studied. Bacterial biomass was measured using epifluorescence microscopy and muramic acid determinations. Bacterial growth rates were estimated from the rate of tritiated thymidine incorporation into DNA. In the water column, bacterial numbers ranged from 8.3×10⁹ 1^–1 to 2.57×10¹⁰ 1^–1 and production ranged from 0.43 to 2.10 mg Cl^–1 d^–1. In the 0–10 mm zone in sediments, bacterial biomass was 1.4 to 5.8 g C m^–2 and production was 250 to 500 mg C m^–2 d^–1. The results suggested that most organic matter being supplied to the ponds as feed for the prawns was actually being utilized by the bacteria. When the density of meiofauna increased after chicken manure was added, bacterial biomass decreased and growth rates increased.     

DNA SYNTHESIS IN THE OOPLASM OF DROSOPHILA MELANOGASTER

Tritiated thymidine was injected into 2-day-old Drosophila melanogaster females, and tissue sections were prepared from the ovary for radioautography with both the light and electron microscopes. Besides the expected incorporation of H³-thymidine into nuclei of nurse cells and follicle cells, there was a relatively high level of incorporation of label into ooplasmic DNA. The highest level of incorporation occurred at stage 12. At the same time, the 15 nurse cell nuclei also incorporate thymidine in spite of the fact that they are breaking down and degenerating. The label in the ooplasm is not removed by extraction with DNase (although this removes nuclear label) unless extraction is preceded by a treatment with protease. Electron microscopic radioautography revealed that 36% of the silver grains resulting from decay of H³-thymidine are found over mitochondria, with a further 28% being located within 0.25 µ of these organelles. The remaining 36% of the silver grains was not found to be associated with any organelles, and it probably represents synthesis in the cytoplasm by the "storage DNA" characteristic of many eggs. It is suggested that one mechanism acting throughout the egg chamber is responsible for the synchronous synthesis of DNA in the degenerating nurse cells, in the mitochondria of the egg, and in the "storage DNA" of the ooplasm.