Bacterial activity and production in near-surface estuarine and freshwater sediments

Abstract: Two indices of bacterial production, thymidine incorporation and the frequency of divided and dividing cells were measured, along with a suite of measurements of aerobic and anaerobic bacterial activity, to investigate the relationship between bacterial cell production and organic carbon mineralisation at three different sediment sites: a sheltered intertidal estuarine mudflat (Kingoodie Bay), a riverside mudbank (Ashleworth Quay) and an intertidal mudflat in a hydraulically dynamic estuary (Aust Warth). Organic carbon mineralisation was dominated by anaerobic processes at all three sites: sulfate reduction at the two estuarine sites (equivalent to 76% and 61% of oxygen uptake) and methanogenesis at the freshwater site (56%). Although all three sites had similar bacterial population sizes, activities in Kingoodie Bay were 2–3 times higher than at Aust Warth or Ashleworth Quay. Thymidine incorporation rates and Numbers of Dividing and Divided Cells correlated strongly at all three sites. Thymidine incorporation rates were spatially uncoupled from zones of principal anaerobic activity, providing in situ evidence that sulfate-reducing bacteria and methanogens do not incorporate radiolabelled thymidine into DNA during growth. Cell yield was lower in the anaerobic zone, as subsurface peaks in anaerobic mineralisation were not matched by increases in bacterial productivity. However, as anaerobic degradation processes were so dominant, anaerobic productivity still accounted for the majority of cell production.     

Bacterial secondary production on vascular plant detritus: relationships to detritus composition and degradation rate.

Bacterial production at the expense of vascular plant detritus was measured for three emergent plant species (Juncus effusus, Panicum hemitomon, and Typha latifolia) degrading in the littoral zone of a thermally impacted lake. Bacterial secondary production, measured as tritiated thymidine incorporation into DNA, ranged from 0.01 to 0.81 microgram of bacterial C mg of detritus-1 day-1. The three plant species differed with respect to the amount of bacterial productivity they supported per milligram of detritus, in accordance with the predicted biodegradability of the plant material based on initial nitrogen content, lignin content, and C/N ratio. Bacterial production also varied throughout the 22 weeks of in situ decomposition and was positively related to the nitrogen content and lignin content of the remaining detritus, as well as to the temperature of the lake water. Over time, production was negatively related to the C/N ratio and cellulose content of the degrading plant material. Bacterial production on degrading plant material was also calculated on the basis of plant surface area and ranged from 0.17 to 1.98 micrograms of bacterial C cm-2 day-1. Surface area-based calculations did not correlate well with either initial plant composition or changing composition of the remaining detritus during decomposition. The rate of bacterial detritus degradation, calculated from measured production of surface-attached bacteria, was much lower than the actual rate of weight loss of plant material. This discrepancy may be attributable to the importance of nonbacterial organisms in the degradation and loss of plant material from litterbags or to the microbially mediated solubilization of particulate material prior to bacterial utilization, or both.     

Fate of heterotrophic bacteria in Lake Tanganyika (East Africa)

Bacterial mortality was studied using two complementary methods between 2002 and 2004 in the two main basins (north and south) of Lake Tanganyika. The disappearance of radioactivity from the DNA of natural assemblages of bacteria previously labeled with tritiated thymidine was used to estimate the mortality due to grazing by predators (72%) and due to the cell lysis (28%). Measurements of ingestion rate of bacteria by protozoa using fluorescent micro-particles yielded protozoan grazing rates similar to those provided by the thymidine method, and showed that heterotrophic nano-flagellates were responsible for most of the grazing pressure on the bacterial community of the pelagic zone (92-99%). Bacterial cell lysis was the second process involved in bacterial mortality, ranking before ciliate grazing. Overall, bacterial mortality was balanced with bacterial production. With regard to the assessment of the trophic role of bacteria, it was estimated that c. 5-8% of the organic carbon taken up by bacteria was converted into protozoan biomass and was thus available for metazoans.     

Incorporation of [methyl-3H]thymidine by obligate and facultative anaerobic bacteria when grown under defined culture conditions

Abstract Incorporation of [ methyl -³H]thymidine into bacterial DNA was determined for a range of axenic anaerobic bacterial cultures: fermentative heterotrophs, sulphate-reducing bacteria, purple sulphur bacteria, acetogens and methanogens. Anaerobically growing Bacillus sp. and the obligate aerobe Thiobacillus ferrooxidans were also investigated. Actively growing cultures of sulphate-reducing bacteria belonging to the genera Desulfovibrio, Desulfotomaculum, Desulfobacter, Desulfobotulus and Desulfobulbus , purple sulphur bacteria ( Chromatium vinosum OP2 and Thiocapsa roseopersicina OP1), methanogens ( Methanococcus GS16 and Methanosarcina barkeri ) and an acetogen ( Acetobacterium woodii ) did not incorporate [ methyl -³H]thymidine into DNA. The only obligate anaerobes in which thymidine incorporation into DNA could be unequivocally demonstrated were members of the genus Clostridium . Anaerobically growing Bacillus sp. also incorporated thymidine. These data demonstrate that pure culture representatives of major groups of anaerobic bacteria involved in the terminal oxidation of organic carbon and anoxygenic phototrophs within sediments are unable to incorporate [ methyl -³H]thymidine into DNA, although some obligate and facultative anaerobes can. Variability in thymidine incorporation amongst pure culture isolates indicates that unless existing techniques can be calibrated to take this into consideration then productivity estimates in both aerobic and anaerobic environments may be greatly underestimated using the [ methyl -³H]thymidine technique.     

Measuring Bacterial Production in Deep-Sea Sediments using 3H-Thymidine Incorporation: Ecological Significance

Bacteria play a major role in the decomposition of organic matter arriving at the deep-sea floor, and hence there is a need to determine accurate rates of bacterial production associated with sediment particles. However, sediment-based procedures are not well defined and sampling deep-sea sediments is technically difficult, time consuming, and expensive, often only producing relatively small amounts of undisturbed sediment for analysis. We describe and test a small-scale method (requiring 0.25 ml sediment) for the examination of bacterial production in deep-sea calcium carbonate rich sediments. Time course experiments showed variation in the period of linear [3H]thymidine uptake between 1 and 3 hr depending on station depth. The average concentration of natural thymidine in deep-sea sediments was 0.61 nmol per 0.5 ml slurry sample. Isotope dilution was significant, ranging between 26 and 51%. There was substantial small-scale (0.2-1.0 m) variation in deep-sea benthic bacterial [3H]thymidine incorporation rates (39%). Deep-sea surficial sediment bacterial production (assuming zero isotope dilution due to its potential high variability) in surficial sediments of the deep NE Atlantic varied between 0.014 and 0.48 mg C g-1 d-1 (mean = 0.23 mg C g-1 d-1) over 3 locations of depths between 1,092 and 3,572 m and at 3 times. Bacterial biomass varied between 1.1 and 12 mg C g-1 (mean = 6.1 mg C g-1). Bacterial growth rate estimates in these deep-sea sediments varied between 0.003 and 0.13 d-1 (mean = 0.050 d-1) giving doubling times of 5.3-216 d (mean = 44.5 d); which are similar to those of bacteria inhabiting waters in the upper mixed layer (2-<40 m) of the water column (2.6-57.8 d). comparison with shallow and coastal sea sediments (0.13-116 d) indicates that deep-sea sediment bacteria in the NE Atlantic are able to grow at rates similar to those in shallow sediment systems given sufficient food. However, the range is broader for deep-sea sediment bacteria, which may indicate a more "feast" and "fast" life than their counterparts in shallower environments. waters >2,000 m cover 60% of the Earth's surface; thus bacterial production in deep-sea sediments must contribute an important fraction of oceanic and global bacterial production. It is therefore important to establish an accurate method of measuring bacterial production so that the full roles and controls of bacteria from this environment can be determined.     

Long-term, high-rate anaerobic biological treatment of whey wastewaters at psychrophilic temperatures

Two laboratory-scale anaerobic hybrid reactors, R1 and R2, treated low- (1 kg COD m-3) and high-strength (10 kg COD m-3) whey-based wastewaters, respectively, in a 500-day trial. The chemical oxygen demand (COD) removal efficiencies of R1 averaged 70-80%, at organic loading rates of 0.5-1.3 kg COD m-3 day-1, between 20 and 12 degrees C. The COD removal efficiencies of R2 exceeded 90%, at organic loading rates up to 13.3 kg COD m-3 day-1, between 20 and 14 degrees C. Lowering the operating temperature of R2 to 12 degrees C resulted in a decrease in COD removal efficiency, to between 50% and 60%, and a disintegration of granular sludge. The decline in performance, and granule disintegration, was reversed by decreasing the organic loading rate of R2 to 6.6 kg m-3 day-1. Specific methanogenic activity profiles revealed mesophilic (37 degrees C) temperature optima for biomass in both reactors, even after 500 days of psychrophilic operation, although the development of psychrotolerance in the biomass was noted.     

Comparative evaluation of anaerobic bacterial communities associated with roots of submerged macrophytes growing in marine or brackish water sediments

Sediment microbial communities are important for seagrass growth and carbon cycling, however relatively few studies have addressed the composition of prokaryotic communities in seagrass bed sediments. Selective media were used enumerate culturable anaerobic bacteria associated with the roots of the seagrass, Halodule wrightii, the fresh to brackish water plant, Vallisneria americana, and the respective vegetated and unvegetated sediments. H. wrightii roots and sediments had high numbers of sulfate-reducing bacteria whereas iron-reducing bacteria appeared to have a more significant role in V. americana roots and sediments. Numbers of glucose-utilizing but not acetate-utilizing iron reducers were higher on the roots of both plants relative to the vegetated sediments indicating a difference within the iron reducing bacterial community. H. wrightii roots had lower glucose-utilizing iron reducers, and higher acetogenic bacteria than did V. americana roots suggesting different aquatic plants support different anaerobic microbial communities. Sulfur-disproportionating and sulfide-oxidizing bacteria were also cultured from the roots and sediments. These results provide evidence of the potential importance of sulfur cycle bacteria, in addition to sulfate-reducing bacteria, in seagrass bed sediments.     

Degradation and total mineralization of monohalogenated biphenyls in natural sediment and mixed bacterial culture

Mixed bacterial cultures obtained from polychlorinated biphenyl-contaminated river sediments are capable of degrading monohalogenated biphenyls under simulated natural conditions. Culture conditions include river water as supportive medium and mixed bacterial cultures obtained from river sediments. Degradation occurs when the substrates are supplied as the sole carbon source or when added together with glucose. The degradation rates of 2-, 3-, and 4-chlorobiphenyl, at 30 micrograms ml-1, were 1.1, 1.6, and 2.0 micrograms ml-1 day-1, respectively. Monobrominated biphenyls, including 2-, 3-, and 4-bromobiphenyl, were degraded at rates of 2.3, 4.2, and 1.4 micrograms ml-1 day-1, respectively. Metabolites, including halogenated benzoates, were detected by high-performance liquid chromatography and mass spectrometry. By using chlorophenyl ring-labeled monochlorobiphenyls as substrates, total mineralization (defined as CO2 production from the chlorophenyl ring) was observed for 4-chlorobiphenyl but not for 2-chlorobiphenyl. Rates of total mineralization of 4-chlorobiphenyl (at 39 to 385 micrograms ml-1 levels) were dependent on substrate concentration, whereas variation of cell number in the range of 10(5) to 10(7) cells ml-1 had no significant effects. Simulated sunlight enhanced the rate of mineralization by ca. 400%.     

Degradation and total mineralization of monohalogenated biphenyls in natural sediment and mixed bacterial culture.

Mixed bacterial cultures obtained from polychlorinated biphenyl-contaminated river sediments are capable of degrading monohalogenated biphenyls under simulated natural conditions. Culture conditions include river water as supportive medium and mixed bacterial cultures obtained from river sediments. Degradation occurs when the substrates are supplied as the sole carbon source or when added together with glucose. The degradation rates of 2-, 3-, and 4-chlorobiphenyl, at 30 micrograms ml-1, were 1.1, 1.6, and 2.0 micrograms ml-1 day-1, respectively. Monobrominated biphenyls, including 2-, 3-, and 4-bromobiphenyl, were degraded at rates of 2.3, 4.2, and 1.4 micrograms ml-1 day-1, respectively. Metabolites, including halogenated benzoates, were detected by high-performance liquid chromatography and mass spectrometry. By using chlorophenyl ring-labeled monochlorobiphenyls as substrates, total mineralization (defined as CO2 production from the chlorophenyl ring) was observed for 4-chlorobiphenyl but not for 2-chlorobiphenyl. Rates of total mineralization of 4-chlorobiphenyl (at 39 to 385 micrograms ml-1 levels) were dependent on substrate concentration, whereas variation of cell number in the range of 10(5) to 10(7) cells ml-1 had no significant effects. Simulated sunlight enhanced the rate of mineralization by ca. 400%.     

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.     

The effect of nutrient additions on bacterial activity in seagrass (Posidonia oceanica) sediments

The influence of nutrient additions on benthic bacterial activity under seagrass meadows was tested by enriching five seagrass (Posidonia oceanica) meadows with nutrients over one year. We found a highly significant response of benthic bacterial activity to nutrient additions, which was reflected in greater (about two-fold) ammonification rates and, to a smaller extent, a significant tendency for a greater exoenzymatic activity. Nutrient additions significantly raised bacterial activity, without altering the seasonal changes in bacterial activity. As a result of the increased bacterial activity, the organic content of the sediments declined significantly, by about 33%, after one year of nutrient addition. Hence, nutrient additions to the seagrass meadows enhance seagrass production but also accelerate bacterial decomposition of seagrass carbon, thereby reducing the capacity of the sediments to store organic carbon. These results demonstrate that sediment nutrient availability limits bacterial activity in these Posidonia oceanica meadows, and identify bacteria as important nutrient consumers in these systems.     

Bacterial [methyl-3H]thymidine incorporation in substrate-amended estuarine sediment slurries

Abstract Five different bacterial communities were enriched in substrate-amended slurries of sediment from the Tay Estuary, Scotland. During incubation of the slurries, concentrations of volatile fatty acids, sulphate, sulphide and methane were monitored to clearly define the activity of the stimulated populations. An aerobic population, a ‘microaerophilic’ population and three anaerobic populations (fermentative heterotrophs, sulphate-reducing bacteria and methanogens plus acetogens) were established to reflect community growth and metabolism both in surface oxic and deeper anoxic layers. Similar numbers of cells involved in division were observed in all five slurries, demonstrating the potential for bacterial production. Thymidine incorporation rates in glucose-stimulated slurries under both aerobic and fully anaerobic conditions were similar, confirming the ability of fermentative anaerobic heterotrophs to incorporate [ methyl -³H]thymidine into DNA during growth. Although anaerobic communities of sulphate-reducing, acetogenic plus methanogenic bacteria were stimulated and actively growing, they did not incorporate [ methyl -³H]thymidine into DNA. Since the thymidine technique does not measure the growth of these important groups, calculated productivity values based upon thymidine incorporation within anoxic sediment systems will be substantially underestimated, even if growth substrates are not limiting.     

Estimates of bacterial growth from changes in uptake rates and biomass.

Rates of nucleic acid synthesis have been used to examine microbiol growth in natural waters. These rates are calculated from the incorporation of [3H]adenine and [3H]thymidine for RNA and DNA syntheses, respectively. Several additional biochemical parameters must be measured or taken from the literature to estimate growth rates from the incorporation of the tritiated compounds. We propose a simple method of estimating a conversion factor which obviates measuring these biochemical parameters. The change in bacterial abundance and incorporation rates of [3H]thymidine was measured in samples from three environments. The incorporation of exogenous [3H]thymidine was closely coupled with growth and cell division as estimated from the increase in bacterial biomass. Analysis of the changes in incorporation rates and initial bacterial abundance yielded a conversion factor for calculating bacterial production rates from incorporation rates. Furthermore, the growth rate of only those bacteria incorporating the compound can be estimated. The data analysis and experimental design can be used to estimate the proportion of nondividing cells and to examine changes in cell volumes.     

Comparison of Rates of Flagellate Bacterivory and Bacterial Production in a Marine Coastal System

Protozoan predation on bacteria and bacterioplankton secondary production were simultaneously determined in La Salvaje Beach water during 1990. Protozoan grazing on bacterioplankton was measured from fluorescently labeled bacterium uptake rates; estimates of bacterial secondary production were obtained from [³H]thymidine incorporation rates. Two different conversion factors were used to transform thymidine incorporation rates into bacterial production rates; both of them were specific for La Salvaje Beach and were calculated by using empirical and semitheoretical approaches. The average flagellate predation rate was 14.0 bacteria flagellate^-1 h^-1; the average population predation rate was 7.35 x 10⁶ bacteria liter^-1 h^-1. The estimates of bacterial production differed greatly depending on the conversion factor used, and so did the percentages of bacterial production consumed by flagellated protozoa (4.6% when the empirical conversion factor for La Salvaje Beach was used and 113% when the semitheoretical conversion factor specific for this system was used). The ecological implications of each of these values are discussed.     

Validity of the tritiated thymidine method for estimating bacterial growth rates: measurement of isotope dilution during DNA synthesis.

The rate of tritiated thymidine incorporation into DNA was used to estimate bacterial growth rates in aquatic environments. To be accurate, the calculation of growth rates has to include a factor for the dilution of isotope before incorporation. The validity of an isotope dilution analysis to determine this factor was verified in experiments reported here with cultures of a marine bacterium growing in a chemostat. Growth rates calculated from data on chemostat dilution rates and cell density agreed well with rates calculated by tritiated thymidine incorporation into DNA and isotope dilution analysis. With sufficiently high concentrations of exogenous thymidine, de novo synthesis of deoxythymidine monophosphate was inhibited, thereby preventing the endogenous dilution of isotope. The thymidine technique was also shown to be useful for measuring growth rates of mixed suspensions of bacteria growing anaerobically. Thymidine was incorporated into the DNA of a range of marine pseudomonads that were investigated. Three species did not take up thymidine. The common marine cyanobacterium Synechococcus species did not incorporate thymidine into DNA.     

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.     

Structure and activity of lacustrine sediment bacteria involved in nutrient and iron cycles

Knowledge of the bacterial community structure in sediments is essential to better design restoration strategies for eutrophied lakes. In this regard, the aim of this study was to quantify the abundance and activity of bacteria involved in nutrient and iron cycling in sediments from four Azorean lakes with distinct trophic states (Verde, Azul, Furnas and Fogo). Inferred from quantitative PCR, bacteria performing anaerobic ammonia oxidation were the most abundant in the eutrophic lakes Verde, Azul and Furnas (4.5-16.6%), followed by nitrifying bacteria (0.8-13.0%), denitrifying bacteria (DNB) (0.5-6.8%), iron-reducing bacteria (0.2-1.4%) and phosphorus-accumulating organisms (<0.3%). In contrast, DNB dominated sediments from the oligo-mesotrophic lake Fogo (8.8%). Activity assays suggested that bacteria performing ammonia oxidation (aerobic and anaerobic), nitrite oxidation, heterothrophic nitrate reduction, iron reduction and biological phosphorus storage/release were present and active in all Azorean lake sediments. The present work also suggested that the activity of DNB might contribute to the release of phosphorus from sediments.     

Growth and population dynamics of anaerobic methane-oxidizing archaea and sulfate-reducing bacteria in a continuous-flow bioreactor

The consumption of methane in anoxic marine sediments is a biogeochemical phenomenon mediated by two archaeal groups (ANME-1 and ANME-2) that exist syntrophically with sulfate-reducing bacteria. These anaerobic methanotrophs have yet to be recovered in pure culture, and key aspects of their ecology and physiology remain poorly understood. To characterize the growth and physiology of these anaerobic methanotrophs and the syntrophic sulfate-reducing bacteria, we incubated marine sediments using an anoxic, continuous-flow bioreactor during two experiments at different advective porewater flow rates. We examined the growth kinetics of anaerobic methanotrophs and Desulfosarcina-like sulfate-reducing bacteria using quantitative PCR as a proxy for cell counts, and measured methane oxidation rates using membrane-inlet mass spectrometry. Our data show that the specific growth rates of ANME-1 and ANME-2 archaea differed in response to porewater flow rates. ANME-2 methanotrophs had the highest rates in lower-flow regimes (mu(ANME-2) = 0.167 . week(-1)), whereas ANME-1 methanotrophs had the highest rates in higher-flow regimes (mu(ANME-1) = 0.218 . week(-1)). In both incubations, Desulfosarcina-like sulfate-reducing bacterial growth rates were approximately 0.3 . week(-1), and their growth dynamics suggested that sulfate-reducing bacterial growth might be facilitated by, but not dependent upon, an established anaerobic methanotrophic population. ANME-1 growth rates corroborate field observations that ANME-1 archaea flourish in higher-flow regimes. Our growth and methane oxidation rates jointly demonstrate that anaerobic methanotrophs are capable of attaining substantial growth over a range of environmental conditions used in these experiments, including relatively low methane partial pressures.     

Magnetite accelerates syntrophic acetate oxidation in methanogenic systems with high ammonia concentrations

Ammonia accumulation is a major inhibitory substance causing anaerobic digestion upset and failure in CH₄ production. At high ammonia levels, CH₄ production through syntrophic acetate oxidization (SAO) pathways is more tolerant to ammonia toxicity than the acetoclastic methanogenesis pathway, but the low CH₄ production rate through SAO constitutes the main reason for the low efficiency of energy recovery in anaerobic digesters treating ammonia‐rich substrates. In this study, we showed that acetate fermentation to CH₄ and CO₂ occurred through SAO pathway in the anaerobic reactors containing a high ammonia concentration (5.0 g l^?1 NH₄⁺–N), and the magnetite nanoparticles supplementation increased the CH₄ production rates from acetate by 36–58%, compared with the anaerobic reactors without magnetite under the same ammonia level. The mechanism of facilitated methanogenesis was proposed to be the establishment of direct interspecies electron transfer (DIET) for SAO, in which magnetite facilitated DIET between syntrophic acetate oxidizing bacteria and methanogens. High‐throughput 16S rRNA gene sequencing analysis revealed that the bacterial Geobacteraceae and the archaeal Methanosarcinaceae and Methanobacteriaceae might be involved in magnetite‐mediated DIET for SAO and CH₄ production. This study demonstrated that magnetite supplementation might provide an effective approach to accelerate CH₄ production rates in the anaerobic reactors treating wastewater containing high ammonia.     

Depth Distribution of Bacterial Production in a Stratified Lake with an Anoxic Hypolimnion

The purpose of this study was to determine the depth distribution of bacterial biomass and production in a stratified lake and to test techniques to measure bacterial production in anaerobic waters. Bacterial abundance and incorporation of both [³H]thymidine and [³H]leucine into protein were highest in the metalimnion, at the depth at which oxygen first became unmeasurable. In contrast, [³H]thymidine incorporation into DNA was highest in the epilimnion. The ratios of incorporation into DNA/protein averaged 2.2, 0.49, and 0.95 for the epilimnion, metalimnion, and hypolimnion, respectively. Low incorporation into DNA was not due to artifacts associated with the DNA isolation procedure. Recovery of added [³H]DNA was about 90% in waters in which the portion of [³H]thymidine incorporation into DNA was about 40%. At least some obligate anaerobic bacteria were capable of assimilating thymidine since aeration of anaerobic hypolimnion waters substantially inhibited thymidine incorporation. The depth profile of bacterial production estimated from total thymidine and leucine incorporation and the frequency of dividing cells were all similar, with maximal rates in the metalimnion. However, estimates of bacterial production based on frequency of dividing cells and leucine incorporation were usually significantly higher than estimates based on thymidine incorporation (using conversion factors from the literature), especially in anaerobic hypolimnion waters. These data indicate that the thymidine approach must be examined carefully if it is to be applied to aquatic systems with low oxygen concentrations. Our results also indicate that the interface between the aerobic epilimnion and anaerobic hypolimnion is the site of intense bacterial mineralization and biomass production which deserves further study.