Effects of four aromatic organic pollutants on microbial glucose metabolism and thymidine incorporation in marine sediments.

The metabolism of D-[U-14C]glucose and the incorporation of [methyl-3H]thymidine by aerobic and anaerobic marine sediment microbes exposed to 1 to 1,000 ppm anthracene, naphthalene, p,p'-dichlorodiphenyltrichloroethane, and pentachlorophenol were examined. Cell-specific rates of [14C]glucose metabolism averaged 1.7 X 10(-21) and 0.5 X 10(-21) mol/min per cell for aerobic and anaerobic sediment slurries, respectively; [3H]thymidine incorporation rates averaged 43 X 10(-24) and 9 X 10(-24) mol/min per cell for aerobic and anaerobic slurries, respectively. Aerobic sediments exposed to three of the organic pollutants for 2 to 7 days showed recovery of both activities. Anaerobic sediments showed little recovery after 2 days of pre-exposure to the pollutants. We conclude that (i) anaerobic sediments are more sensitive than aerobic sediments to pollutant additions; (ii) [3H]thymidine incorporation is more sensitive to pollutant additions than is [14C]glucose metabolism; and (iii) the toxicity of the pollutants increased in the following order: anthracene, p,p'-dichlorodiphenyltrichloroethane, naphthalene, and pentachlorophenol.     

Seasonal and spatial distribution of extracellular enzymatic activities and microbial incorporation of dissolved organic substrates in marine sediments

Seasonal and spatial distributions of extracellular enzymatic activities and microbial incorporations of dissolved organic substrates were followed in sediments of the brackish water Kiel Bight (Baltic Sea, Federal Republic of Germany). Enzymatic hydrolysis of polymeric organic compounds was determined by means of fluorogenic substrates (4-methylumbelliferyl-beta-d-glucoside, l-leucine-4-methylcoumarinyl-7-amide hydrochloride); incorporation of dissolved organic substrates into microbial biomass was measured by using tritiated substances (acetate, leucine, and thymidine). Based on a recently developed core injection technique, substrates were injected in microliter portions into undisturbed sediment cores. Enzymatic and incorporation activities underwent strong seasonal variations related to the enrichment of organic material in the sediment surface following sedimentation events. The input of the phytoplankton bloom during autumn caused stimulation of both enzymatic hydrolysis of polymeric organic compounds and microbial incorporation of dissolved organic substrates. Following input by spring phytoplankton bloom, mainly incorporation activities were stimulated. In late spring the development of the benthic fauna obviously greatly influenced microbial activities. During summer individual periods of high microbial activities were observed which might be traced back to short-term sedimentation events. The high microbial incorporation of leucine and thymidine during winter demonstrated that the nutrient supply rather than temperature is the dominating factor determining microbial production. Stimulation of microbial activities arose from the sediment surface and spread out relatively quickly into deeper horizons. Generally, the sediments were characterized by distinct patterns of interrelationships between the individual parameters of microbial activities measured.     

Chromatographic separation of marine organic pollutants

A review and discussion of the chromatographic separation of marine organic pollutants is given, including sampling and clean-up procedures, fractionation and enrichment of marine pollutants, capillary gas chromatography (cGC) and high-performance liquid chromatography applying both classical and chiral stationary phases. The potential of multi-dimensional cGC for the analysis of marine organic trace pollutants is discussed for polychlorinated biphenyls (PCBs). The chromatographic separation of coplanar PCBs and of the enantiomers of chiral pollutants provides a further insight into the toxic potential of these marine organic pollutants.     

Thymidine uptake, thymidine incorporation, and thymidine kinase activity in marine bacterium isolates

One assumption made in bacterial production estimates from [3H]thymidine incorporation is that all heterotrophic bacteria can incorporate exogenous thymidine into DNA. Heterotrophic marine bacterium isolates from Tampa Bay, Fla., Chesapeake Bay, Md., and a coral surface microlayer were examined for thymidine uptake (transport), thymidine incorporation, the presence of thymidine kinase genes, and thymidine kinase enzyme activity. Of the 41 isolates tested, 37 were capable of thymidine incorporation into DNA. The four organisms that could not incorporate thymidine also transported thymidine poorly and lacked thymidine kinase activity. Attempts to detect thymidine kinase genes in the marine isolates by molecular probing with gene probes made from Escherichia coli and herpes simplex virus thymidine kinase genes proved unsuccessful. To determine if the inability to incorporate thymidine was due to the lack of thymidine kinase, one organism, Vibrio sp. strain D19, was transformed with a plasmid (pGQ3) that contained an E. coli thymidine kinase gene. Although enzyme assays indicated high levels of thymidine kinase activity in transformants, these cells still failed to incorporate exogenous thymidine into DNA or to transport thymidine into the cells. These results indicate that the inability of certain marine bacteria to incorporate thymidine may not be solely due to the lack of thymidine kinase activity but may also be due to the absence of thymidine transport systems.     

Thymidine uptake, thymidine incorporation, and thymidine kinase activity in marine bacterium isolates.

One assumption made in bacterial production estimates from [3H]thymidine incorporation is that all heterotrophic bacteria can incorporate exogenous thymidine into DNA. Heterotrophic marine bacterium isolates from Tampa Bay, Fla., Chesapeake Bay, Md., and a coral surface microlayer were examined for thymidine uptake (transport), thymidine incorporation, the presence of thymidine kinase genes, and thymidine kinase enzyme activity. Of the 41 isolates tested, 37 were capable of thymidine incorporation into DNA. The four organisms that could not incorporate thymidine also transported thymidine poorly and lacked thymidine kinase activity. Attempts to detect thymidine kinase genes in the marine isolates by molecular probing with gene probes made from Escherichia coli and herpes simplex virus thymidine kinase genes proved unsuccessful. To determine if the inability to incorporate thymidine was due to the lack of thymidine kinase, one organism, Vibrio sp. strain D19, was transformed with a plasmid (pGQ3) that contained an E. coli thymidine kinase gene. Although enzyme assays indicated high levels of thymidine kinase activity in transformants, these cells still failed to incorporate exogenous thymidine into DNA or to transport thymidine into the cells. These results indicate that the inability of certain marine bacteria to incorporate thymidine may not be solely due to the lack of thymidine kinase activity but may also be due to the absence of thymidine transport systems.     

Effects of beta-lactam antibotics on thymidine incorporation of bacteria

Five strains each ofEscherichia coli, Proteus mirabilis, andStaphylococcus aureus were grown in Trypticase soy broth (TSB) with or without penicillin, oxacillin, ampicillin, amoxicillin, or cefamandole. [³H]Thymidine incorporation and the optical density of the cultures were determined at an hourly interval for the duration of incubation. All strains ofP. mirabilis showed after 2 and 3 h of incubation of 5-to 16-fold increase in the specific activity of [³H]thymidine incorporated in the presence of MIC of the antibiotics as compared to controls grown without the drugs.E. coli andS. aureus showed smaller increases in thymidine incorporation than didP. mirabilis. After 4–5 h the antibiotics produced an inhibition of [³H]thymidine incorporation. At the MAC, the responses were of a smaller magnitude. Regardless of whether these changes are the result of specific interference or just lack of [³H]thymidine incorporation, they are directly related to the antibiotic activity of agents known not to affect DNA synthesis. The monitoring of [³H]thymidine incorporation detects early antibiotic activity probably earlier than other current systems which are used for this purpose.     

Enantioselective and nonenantioselective degradation of organic pollutants in the marine ecosystem

Enantiomeric ratios of 11 chiral environmental pollutants determined in different compartments of the marine ecosystem by chiral capillary gas chromatography and chiral high-performance liquid chromatography allow discrimination between the following processes: enantioselective decomposition of both enantiomers with different velocities by marine microorganisms (α-HCH, β-PCCH, γ-PCCH); enantioselective decomposition of one enantiomer only by marine microorganisms (DCPP); enantioselective decomposition by enzymatic processes in marine biota (α-HCH, β-PCCH, trans-chlordane, cis-chlordane, octachlordane MC4, octachlordane MC5, octachlordane MC7, oxychlordane, heptachlor epoxide); enantioselective active transport through the “blood–brain barrier” (α-HCH); nonenantioselective photochemical degradation (α-HCH, β-PCCH). © 1993 Wiley-Liss, Inc.     

Effects of augmentation of coarse particulate organic matter on metabolism and nutrient retention in hyporheic sediments

SUMMARY 1. Metabolic and biogeochemical processes in hyporheic zones may depend on inputs of coarse particulate organic matter. Our research focused on how differing quantity and quality of organic matter affects metabolism and nutrient retention in the hyporheic zone of a first-order Appalachian stream.
2. Sixteen plots were established on a tributary of Hugh White Creek, NC, U.S.A. Sediment was extracted and treated with leaves, wood, plastic strips or remained unamended. Following treatment, sediment was returned to the stream and, approximately 3 months later, samples were removed from each plot.
3. Aerobic and anaerobic metabolism were measured as the change in O₂ and CO₂ in recirculating microcosms. At the same time, we monitored other possible terminal electron accepting processes and changes in nutrient concentrations. Aerobic metabolism was low in all treatments and respiratory quotients calculated for all treatments indicated that metabolism was dominated by anaerobic processes.
4. Rates of anaerobic respiration and total (combined aerobic and anaerobic) respiration were significantly greater ( P  < 0.05) in plots treated with leaf organic matter compared to controls.
5. Addition of leaves, which had a low C:N ratio, stimulated respiration in hyporheic sediments. Anaerobic processes dominated metabolism in both control and amended sediments. Enhanced metabolic rates increased retention of many solutes, indicating that energy flow and nutrient dynamics in the subsurface of streams may depend upon the quantity and quality of imported carbon.     

Observations on microbial percent respiration values in arctic and subarctic marine waters and sediments

Percent respiration was measured in over 1,100 arctic and subarctic marine water and sediment samples using¹⁴C-labeled glucose and glutamate. These measurements were made at different times of the year in 4 regions. Percent respiration values were typically lower in regions where the waters of large rivers mixed with seawater. They were also lower in sediments and in waters collected near the bottom than in surface waters. They were higher in winter arctic waters than water samples collected in the summer; however, a similar seasonal trend was not observed in subarctic waters. There were a number of studies in which there were significant positive rank correlations between percent respiration and salinity and between percent respiration and temperature. From what is known about the range of temperature and salinity encountered in samples collected during these studies and the results of temperature and salinity effects experiments, it was concluded that changes in these 2 variables did not explain the variation observed in percent respiration. Correlations between percent respiration and the inorganic nutrients PO₄ ^–3, NH₄ ⁺ and NO₃ ^– showed that of the 3 variables, only NO₃ ^– showed relatively high correlations with all the same sign. From this it was concluded that there may be situations in which NO₃ ^– levels may influence percent respiration in nearshore marine waters. It is also likely that qualitative characteristics of the available organic nutrients may also influence percent respiration levels. Although no organic nutrient data is available for statistical analysis, the patterns of percent respiration near river plumes and the relatively strong negative correlation often observed between uptake rates (heterotrophic activity) and percent respiration suggests that organic nutrients may be a factor in controlling percent respiration. It is suggested that there are situations in which percent respiration measurements may be used to document stress in natural microbial populations due to nutrient deficiencies.     

Effects of specific inhibitors on anammox and denitrification in marine sediments

The effects of three metabolic inhibitors (acetylene, methanol, and allylthiourea [ATU]) on the pathways of N2 production were investigated by using short anoxic incubations of marine sediment with a 15N isotope technique. Acetylene inhibited ammonium oxidation through the anammox pathway as the oxidation rate decreased exponentially with increasing acetylene concentration; the rate decay constant was 0.10+/-0.02 microM-1, and there was 95% inhibition at approximately 30 microM. Nitrous oxide reduction, the final step of denitrification, was not sensitive to acetylene concentrations below 10 microM. However, nitrous oxide reduction was inhibited by higher concentrations, and the sensitivity was approximately one-half the sensitivity of anammox (decay constant, 0.049+/-0.004 microM-1; 95% inhibition at approximately 70 microM). Methanol specifically inhibited anammox with a decay constant of 0.79+/-0.12 mM-1, and thus 3 to 4 mM methanol was required for nearly complete inhibition. This level of methanol stimulated denitrification by approximately 50%. ATU did not have marked effects on the rates of anammox and denitrification. The profile of inhibitor effects on anammox agreed with the results of studies of the process in wastewater bioreactors, which confirmed the similarity between the anammox bacteria in bioreactors and natural environments. Acetylene and methanol can be used to separate anammox and denitrification, but the effects of these compounds on nitrification limits their use in studies of these processes in systems where nitrification is an important source of nitrate. The observed differential effects of acetylene and methanol on anammox and denitrification support our current understanding of the two main pathways of N2 production in marine sediments and the use of 15N isotope methods for their quantification.     

Effects of erythropoietin on glucose metabolism

We purposed to determine the impact of erythropoietin on altering glucose metabolism in the settings of in vitro and in vivo experiments. The acute effect of erythropoietin on lowering blood glucose levels was studied in animal experiments. In [3H]-deoxy-D-glucose isotope studies we measured glucose uptake with insulin and erythropoietin using 3T3-L1 cells cultured under normal or high glucose conditions. Altered activation of Akt and ERK pathways was evaluated in immunoblot analyses. Immunocytochemistry was conducted to determine the glucose transporter 4 translocation to the plasma membrane. Addition of erythropoietin significantly lowered blood glucose levels in vivo in rats. The glucose uptake was markedly increased by erythropoietin treatment (at concentrations 0.15, 0.3, and 0.625 ng/ml) in adipocytes grown in high glucose medium (p<0.05), but it remained unaltered in cells under normal glucose conditions. Significant increase of phosphorylation of ERK and Akt was detected due to erythropoietin (p<0.05). Co-administration of erythropoietin and insulin resulted in higher phosphorylation of Akt and [3H]-deoxy-D-glucose uptake in adipocytes than insulin treatment alone. We found that erythropoietin induced the trafficking of glucose transporter 4 to the plasma membrane. Our data showed that erythropoietin significantly decreased blood glucose levels both in vivo and in vitro, in part, by increasing glucose uptake via the activation of Akt pathway. Preliminary data revealed that adipocytes most likely exhibit a specific receptor for erythropoietin.     

Measurement of bacterial growth rates in subsurface sediments using the incorporation of tritiated thymidine into DNA

Microbial growth rates in subsurface sediment from three sites were measured using incorporation of tritiated thymidine into DNA. Sampling sites included Lula, Oklahoma, Traverse City, Michigan, and Summit Lake, Wisconsin. Application of the thymidine method to subsurface sediments required (1) thymidine concentrations greater than 125 nM, (2) incubation periods of less than 4 hours, (3) addition of SDS and EDTA for optimum macromolecular extraction, and (4) DNA purification, in order to accurately measure the rate of thymidine incorporation into DNA. Macromolecule extraction recoveries, as well as the percentage of tritium label incorporated into the DNA fraction, were variable and largely dependent upon sediment composition. In general, sandy sediments yielded higher extraction recoveries and demonstrated a larger percentage of label incorporated into DNA than sediments that contained a high silt-clay component. Reported results also indicate that the acid-base hydrolysis procedure routinely used for macromolecular fractionation in water samples may not be routinely applicable to the modified sediment procedure where addition of SDS and EDTA are required for macromolecule extraction. Growth rates exhibited by subsurface communities are relatively slow, ranging from 5.1 to 10.2×10⁵ cells g^–1 day^–1. These rates are 2–1,000-fold lower than growth rates measured in surface sediments. These data lend support to the supposition that subsurface microbial communities are nutritionally stressed.     

Thermodynamics of microbial growth coupled to metabolism of glucose, ethanol, short-chain organic acids, and hydrogen

A literature compilation demonstrated a linear relationship between microbial growth yield and the free energy of aerobic and anaerobic (respiratory and/or fermentative) metabolism of glucose, ethanol, formate, acetate, lactate, propionate, butyrate, and H(2). This relationship provides a means to estimate growth yields for modeling microbial redox metabolism in soil and sedimentary environments.