Competition between Fe(III)-reducing and methanogenic bacteria for acetate in iron-rich freshwater sediments.

The kinetics of acetate uptake and the depth distribution of [2-14C]acetate metabolism were examined in iron-rich sediments from a beaver impoundment in northcentral Alabama. The half-saturation constant (Km) determined for acetate uptake in slurries of Fe(III)-reducing sediment (0.8 mM) was more than 10-fold lower than that measured in methanogenic slurries (12 mM) which supported comparable rates of bulk organic carbon metabolism and Vmax values for acetate uptake. The endogenous acetate concentration (Sn) was also substantially lower (1.7 mM) in Fe(III)-reducing vs methanogenic (9.0 mM) slurries. The proportion of [2-14C]acetate converted to 14CH4 increased with depth from ca 0.1 in the upper 0.5 cm to ca 0.8 below 2 cm and was inversely correlated (r2 = 0.99) to a decline in amorphous Fe(III) oxide concentration. The results of the acetate uptake kinetics experiments suggest that differences in the affinity of Fe(III)-reducing bacteria vs methanogens for acetate can account for the preferential conversion of [2-14C]acetate to 14CO2 in Fe(III) oxide-rich surface sediments, and that the downcore increase in conversion of [2-14C]acetate to 14CH4 can be attributed to progressive liberation of methanogens from competition with Fe(III) reducers as Fe(III) oxides are depleted with depth.

New spatially explicit method for detecting extracellular protease activity in biofilms.

A novel method of detecting extracellular protease activity at biofilm-substratum interfaces was developed. This method utilizes fluorescent molecules bound to cellulose substrata with a lectin. Extracellular proteases degrade the lectin and release the fluorochrome into solution. This new technique and a standard dissolved-substrate assay detected similar responses of biofilm extracellular protease activity to experimental manipulation of N supply. Combination of this technique with confocal scanning laser microscopy allowed direct visualization of microspatial patterns of bacterial distribution and extracellular protease activity at the biofilm-substratum interface.

Influence of Algal Photosynthesis on Biofilm Bacterial Production and Associated Glucosidase and Xylosidase Activities.

Natural photosynthetic biofilms were incubated under light (100 mmol m-2 s-1) and dark conditions to elucidate the impact of photosynthesis on bacterial production, abundance, biovolume, biomass, and enzyme activities over 24 h. Use of organic carbon-free media limited carbon sources to algal photosynthesis and possibly the polysaccharides of the biofilm matrix. Bacterial production of biofilm communities was significantly higher in light incubations (p <0.001). The greatest differences in production rates between light and dark incubations occurred between 8 and 24 h. Biomass-specific a- and b-glucosidase and b-xylosidase activities were stimulated by photosynthesis, with significantly greater activities occurring at hours 16 and 24 in the light treatment (p <0.01). The results indicate that algal photosynthesis can have a significant impact on bacterial productivity, biomass, biovolume, and enzyme production over longer time periods at low photon flux densities (?100 mmol m-2 s-1).

Complexation, Stabilization, and UV Photolysis of Extracellular and Surface-Bound Glucosidase and Alkaline Phosphatase: Implications for Biofilm Microbiota.

Biofilm-produced and commercially-purified a- and b-glucosidase and alkaline phosphatase were subjected to different spectral portions of natural and artificial light and exposed to various humic substances to elucidate their impact on enzyme activities. Photochemical degradation of all enzymes occurred under different portions of the light spectrum. UVB irradiance produced the greatest overall photochemical degradation of enzymes, with significant rates occurring with UVA and PAR irradiance. The complexation of enzymes with humic substances resulted in inhibition, stabilization, and photochemical protection of the enzyme. Inhibition of enzyme activity occurred via reductions in overall enzyme activity in the presence of humic substances. However, humic-enzyme complexation also resulted in stabilization by restricting enzyme degradation while retaining high activities. Enzymes exposed to natural and artificial light sources had significantly lower reductions in enzyme activities in the presence of humic substances, which indicates that humic-enzyme complexes may protect enzymes from light-induced photochemical degradation. Bacterial surface-bound a- and b-glucosidase activities were significantly reduced in the presence of humic substances. Photosynthetically induced pH changes within biofilm communities can cause large reductions in a- and b-glucosidase activities while enhancing the hydrolytic activity of alkaline phosphatase.

Effects of Photosynthesis on Bacterial Phosphatase Production in Biofilms.

The fraction of bacteria displaying phosphatase activity within natural photosynthetic biofilms was examined in relation to phosphorus limitation and algal photosynthesis. An artificial substrate that forms a fluorescent precipitate was used in conjunction with the nucleic acid stain DAPI to enumerate extracellular phosphatase expression by biofilm bacteria exposed to different photosynthetic activities and phosphorus supplies. The proportion of bacteria displaying phosphatase activity changed in response to the presence or absence of algal photosynthesis. In general, phosphate-deprived biofilms had positive linear trends in bacterial phosphatase activity (p <0.001), with greater proportions of bacteria displaying phosphatase under photosynthetic inhibition compared to active photosynthesis. Under sufficient phosphate supplies, biofilms had negative linear trends (p <0.05) or were lower in the proportion of bacteria displaying phosphatase activity in the presence of algal photosynthesis, whereas bacterial phosphatase activity was generally maintained when photosynthesis was inhibited. it is suggested that the amount of extracellular organic carbon released within the biofilm matrix during photosynthesis indirectly affected bacterial phosphatase synthesis.

Fundamental processes within natural and constructed wetland ecosystems: short-term versus long-term objectives.

Use of wetland ecosystems for water pollution control consists essentially of sustained induced disturbances as pollutants are loaded to complex biological communities. Objectives are to maximize pollutant loading, incorporation, and retention while maintaining highest levels of community metabolism and minimal alteration of community structure. Several basic processes are emphasized: (a) macrophyte productivity in relation to shoot:root ratios, and nutrient availability; (b) macrophyte life history strategies, succession, and biodiversity under constant pollutant stress; (c) importance of standing dead and particulate detritus; (d) functions and controlling mechanisms of heterotrophic and autotrophic periphyton in pollutant retention and recycling; (e) coupling of microbial metabolism to macrophyte retention of pollutants; (f) gaseous losses to the atmosphere; (g) losses of dissolved organic matter and its utilization; and (h) water losses by evapotranspiration and effects on wetland efficacy. Short-term wetland removal efficiencies are confounded by massive variations in retentive capacities diurnally, seasonally, and spatially, in exceeding physiological tolerance levels, and in species succession. Problems of channelization, altered microhydrology, and assimilation/retention are major in natural and non-engineered ecosystems. Wetlands are highly ephemeral and variable in their capabilities for sequestering and retention of nutrients and other pollutants.

Allelochemical autotoxicity in the emergent wetland macrophyte Juncus effusus (Juncaceae).

Bioassays for allelochemical toxicity of aboveground Juncus effusus tissues were conducted with seeds and seedlings of Eleocharis obtusa and Scirpus cyperinus, two emergent sedge species (Cyperaceae) found sympatric with J. effusus, and with seeds and seedlings of J. effusus itself to evaluate potential autotoxicity. Bioassays were performed under controlled, axenic conditions with aqueous shoot extract treatments simulating in situ dissolved organic carbon concentrations. With respect to the two sedge species, neither shoot development nor seedling biomass accrual was significantly suppressed by lyophilized whole extracts from J. effusus. Although the extracts induced no significant reduction in growth of E. obtusa or S. cyperinus, biomass-specific chlorophyll a concentration was significantly reduced in E. obtusa seedlings. In contrast, seedlings of J. effusus exhibited significant reductions of biomass and chlorophyll a concentrations, and seedling shoot development was retarded in response to leachate exposure. Results of the present study suggest that J. effusus seedlings possess autotoxic sensitivity to extracts of dead, aboveground tissues of adult plants.

Effect of substrate and cell surface hydrophobicity on phosphate utilization in bacteria.

We measured the rates of utilization of hydrophobic and hydrophilic phosphate compounds in gram-negative bacteria with different surface hydrophobicities, isolated from wetland habitats. Three hydrophobic and two hydrophilic bacterial species were selected for study by measuring cell adherence to hydrocarbons. The bacteria were grown under phosphorus-limited conditions with P(infi), hydrophilic (beta)-glycerophosphate, or hydrophobic phosphatidic acid as the phosphate source. Hydrophilic bacteria grew most rapidly on P(infi), followed by (beta)-glycerophosphate. Phosphatidic acid did not support growth or did so at a much later time (40 h) than did the other phosphate treatments. Although all hydrophobic species grew well on these substrates, the rate of growth of two Acinetobacter baumannii isolates on phosphatidic acid exceeded the rate of growth on phosphate or (beta)-glycerophosphate. A membrane phospholipid and lipopolysaccharide were used as a source of phosphorus by hydrophobic species, whereas hydrophilic species could not use the membrane phospholipids and used lipopolysaccharide to a lesser extent. Besides hydrophobic interaction between cells and substrate, phosphatase activity, which was cell bound in hydrophilic species but 30 to 50% unbound in hydrophobic species, affected cell growth. Dialyzed culture supernatant containing phosphatase from hydrophobic species increased the phosphate availability to hydrophilic species. Additionally, cellular extracts from a hydrophilic species, when added to hydrophilic cells, permitted growth on hydrophobic phosphate sources. Naturally occurring amphiphilic humic acids affected the utilization of P(infi) and (beta)-glycerophosphate in bacteria with hydrophilic surfaces but did not affect hydrophobic bacteria. Our results indicate that hydrophobic phosphate sources can be used by bacteria isolated from aquatic environments as the sole phosphorus source for growth. This utilization, in part, appears to be related to cell surface hydrophobicity and extracellular enzyme production.

Simultaneous use of (14)C and (3)H to determine autotrophic production and bacterial protein production in periphyton.

A method of simultaneously quantifying photoautotrophic (algae and cyanobacteria) and bacterial production in periphyton communities by (14)C-bicarbonate and (3)H-leucine incorporation was investigated and applied to communities subjected to specific intensities of photosynthetically active radiation (400-700 nm). Maximum photosynthetic output (2.23 ± 0.29 (SE) µg C cm(-2) h(-1)) and bacterial production (0.07 ± 0.006 µg C cm(-2) h(-1)) occurred at the highest photon flux density (400 µmol m(-2) s(-1)). Over a photon flux density range of 20-400 µmol m(-2) s(-1), bacterial and autotroph productivity were significantly and positively correlated (r = 0.89). Furthermore, application of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, a photosystem 11 inhibitor, to periphyton films reduced bacterial production by 46%, but it had no such effect on bacteria-only cultures. Therefore, the magnitude of bacterial production in periphyton was coupled to the photosynthesis/metabolism of algae and/or cyanobacteria.

[(3)H]Leucine incorporation methodology to estimate epiphytic bacterial biomass production.

[(3)H]leucine incorporation into protein, as a method of measuring bacterial biomass production (BBP), was adapted to epiphytic bacteria. Incorporation of the isotope was saturated at concentrations higher than 400 nM. Disruption of thicker biofilms by sonication resulted in higher values of BBP and ratios of BBP/biomass when compared to those of intact biofilm. Thin biofilms formed early in the decomposition process did not show this phenomenon. These results support to evidence that more internally located cells of the matrices either have greatly reduced access to the leucine from the overlying medium or that fast recycling of leucine occurs in the biofilm.

Acclimation of Photosynthesis and Dark Respiration of a Submersed Angiosperm beneath Ice in a Temperate Lake.

Ceratophyllum demersum L. remained physiologically active beneath ice of a southeastern Michigan lake. The effect of seasonally low photosynthetic photon flux density (PPFD) and cold but nonfreezing temperature on whole-plant physiology was studied. Net photosynthesis was measured at six temperatures and 12 PPFDs. Net photosynthesis, soluble protein concentration, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) protein concentration, and Rubisco activity of winter plants were 32, 31, 33, and 70% lower, respectively, than those of plants collected in the summer. Optimum temperatures for net photosynthesis of winter and summer plants were 5 and 30[deg]C, respectively. Dark respiration of winter plants was up to 313% greater than that of summer plants. Reduced Rubisco activity and increased dark respiration interacted to reduce net photosynthesis. Interaction of reduced net photosynthesis and increased dark respiration increased CO2 and light compensation points and the light saturation point of winter plants. Growth of C. demersum was limited by the ambient phosphorus concentration of lake water during summer. Apical stem segments of winter-collected plants had 54 and 35% more phosphorus and nitrogen, respectively, than summer-collected plants. Physiologically active perennation beneath ice enabled C. demersum to accumulate phosphorus during the winter when it was most abundant. Partial uncoupling of phosphorus acquisition from utilization may reduce phosphorus limitation upon growth during the summer when phosphorus concentration is seasonally the lowest.

Effects of nutrients on specific growth rate of bacterioplankton in oligotrophic lake water cultures.

The effects of organic and inorganic nutrient additions on the specific growth rates of bacterioplankton in oligotrophic lake water cultures were investigated. Lake water was first passed through 0.8-mum-pore-size filters (prescreening) to remove bacterivores and to minimize confounding effects of algae. Specific growth rates were calculated from changes in both bacterial cell numbers and biovolumes over 36 h. Gross specific growth rates in unmanipulated control samples were estimated through separate measurements of grazing losses by use of penicillin. The addition of mixed organic substrates alone to prescreened water did not significantly increase bacterioplankton specific growth rates. The addition of inorganic phosphorus alone significantly increased one or both specific growth rates in three of four experiments, and one experiment showed a secondary stimulation by organic substrates. The stimulatory effects of phosphorus addition were greatest concurrently with the highest alkaline phosphatase activity in the lake water. Because bacteria have been shown to dominate inorganic phosphorus uptake in other P-deficient systems, the demonstration that phosphorus, rather than organic carbon, can limit bacterioplankton growth suggests direct competition between phytoplankton and bacterioplankton for inorganic phosphorus.

5'-nucleotidase activity in a eutrophic lake and an oligotrophic lake.

Differences in enzymatic hydrolysis of dissolved organic phosphorus and subsequent phosphorus uptake were compared by using dual-labeled (gamma-P and 2-H) ATP in oligotrophic Lake Michigan and a moderately eutrophic lake in southeastern Michigan. More than 50% of the phosphate that was hydrolyzed was immediately taken up into bacterium-sized particles in the eutrophic lake and at a near-shore site in Lake Michigan. Less than 50% of the hydrolyzed phosphate was taken up into bacterium-sized particles at an offshore site in Lake Michigan. It is hypothesized that differences in size-fractionated uptake were the result of greater phosphorus utilization capacity in bacteria in habitats where loading of organic carbon is greater. Substantial isotope dilution of labeled phosphate uptake by unlabeled phosphate occurred, which implied that the phosphate was hydrolyzed extracellularly in both systems. Comparable nucleotidase activities were measured in the eutrophic lake and Lake Michigan, but the significance of the phosphate regenerated relative to particulate phosphorus pools was an order of magnitude greater in Lake Michigan. Seventy percent of the nucleotidase activity was inhibited by 100 muM phosphate in the eutrophic lake, which suggests that most hydrolysis was by phosphatase. Therefore, nucleotidase activity may be more important to phosphorus regeneration in oligotrophic habitats than phosphatase activity.

Direct Comparison of Phosphate Uptake by Adnate and Loosely Attached Microalgae within an Intact Biofilm Matrix.

We report a direct comparison of phosphate uptake by adnate and loosely attached microalgae in an intact biofilm matrix, with resolution at the level of individual cells. Track scanning electron microscope autoradiography enabled assay of [P]phosphate uptake from the overlying water by adnate algae left undisturbed on mature leaves of the macrophyte Potamogeton illinoensis or on artificial plant mimics. The epiphyte communities developed in either phosphate-poor or moderately phosphate-enriched water, and they were assayed on both natural and artificial plants. All adnate taxa examined from both natural and artificial plants in both habitats took up significantly less radiolabel when assayed beneath the overlying matrix than when they were exposed to the water upon removal of the overstory material. Track scanning electron microscope autoradiography and track light microscope autoradiography were intercalibrated to enable comparison of [P]phosphate uptake by adnate and loosely attached components of the epiphyte matrix. Loosely attached cells on substrata from both habitats took up significantly more radiolabel than did underlying adnate cells, indicating that access to phosphate supplies from the water depended on the position of microbial cells in the matrix. In this short-term assay, the adnate microalgae were relatively isolated from the water column nutrient source.

Experimental evaluation of conversion factors for the [h]thymidine incorporation assay of bacterial secondary productivity.

The relationship between bacterial growth and incorporation of [methyl-H]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 [H]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 x 10 to 38 x 10 cells mol of total thymidine incorporation 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 [H]thymidine incorporation rates: 5.54 x 10 mum mol of total thymidine incorporation and 15.2 x 10 mum mol of nucleic acid incorporation. 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 [H]thymidine incorporation more than it increased bacterial growth, which resulted in low conversion factors.

Improved double-vial radiorespirometric technique for mineralization of C-labeled substrates.

New procedures which simplify sample preparation and improve counting efficiency were developed for double-vial radiorespirometry. Under certain conditions, efficiency of fluor-NaOH-impregnated wicks was not stable, but was adversely affected by water vapor, NaOH concentration, and CO(2) loading. Glass fiber wick material treated with a methanolic fluor-NaOH solution showed improved C counting efficiency (58%) compared with previous methods.

Photosynthesis in submersed macrophytes of a temperate lake.

The photosynthetic carbon fixation pathways and levels of carbon-fixing enzymes of four dominant submersed macrophytes of Lawrence Lake, southern Michigan, were investigated during the main growth season (May to November). All four species (Scirpus subterminalis Torr., Najas flexilis (Willd.) Rostk. and Schmidt, Potamogeton praelongus Wulf., and Myriophyllum heterophyllum Michx.) were C(3) plants based on their patterns of (14)C pulse-chase incorporation. High levels of phosphoenolpyruvate carboxylase were also found in these species. These levels, as well as the ribulose 1,5-biphosphate carboxylase/phosphoenolpyruvate carboxylase ratio of the leaves, varied throughout the growing season and exhibited highest values in July. No shift in carbon fixation pathways, however, could be detected from July to October. The possible functions of phosphoenolypyruvate carboxylase in these plants, as well as the significance of C(3) metabolism in submersed plants of temperate lakes, are delineated.

Measuring chlorophyll a and C-labeled photosynthate in aquatic angiosperms by the use of a tissue solubilizer.

A compound that quantitatively correlated with chlorophyll a could be measured fluorometrically in the extracts of leaves of three aquatic angiosperms (Myriophyllum heterophyllum Michx., Potamogeton crispus L., Elodea canadensis Michx.) treated with the tissue solubilizer BTS-450. Fluorescent characteristics of the solubilized plant tissues were stable for several weeks in the dark at temperatures up to 60 degrees C but rapidly degraded in sunlight or when acidified. (14)C-Labeled photosynthate, which had been fixed by leaf discs during 1- to 10-hour exposure to H(14)CO(3), was also readily extracted by the tissue solubilizer. Solubilizer extraction can, therefore, be used to determine both chlorophyll a content and (14)C incorporation rates in the same leaf sample. The method is practical, because no grinding is required, the fluorescent characteristics of the extracts are stable, and analyses can be performed with very little plant material (about 3 milligrams).

Photolithotrophy, photoheterotrophy, and chemoheterotrophy: Patterns of resource utilization on an annual and a diurnal basis within a pelagic microbial community.

An annual investigation of rates of photolithotrophy, photoheterotrophy, and chemoheterotrophy utilizing glucose and bicarbonate was made within the pelagic zone of a small, hardwater, southwestern Michigan lake. Sampling proceeded on a monthly, diurnal, and depth-wise basis. Annual mean photoheterotrophic uptake was estimated at 2.6µg C m(-3)h(-1). Two periods of relatively high activity were observed: one during spring overturn and the second during the late summer period. In general, greatest contributions to overall carbon cycling occurred during morning to midday incubation periods and at intermediate depths within the water column. Rates of chemoheterotrophy averaged 6.9µg C m(-3)h(-1) and were relatively uniform throughout the annual period. Greatest overall chemoheterotrophic activity was associated with periods of overturn. In general, this activity increased throughout the day and with increasing depth within the water column. The annual mean for photolithotrophic fixation was 1.33 mg C m(-3)h(-1). Greatest contributions to rates of photosynthesis were associated with epilimnetic waters during early morning and midday incubations. Relatively minor contributions to inorganic fixation were made by waters below the 6-meter contour. Spring overturn and late summer represented periods of particularly great photolithotrophic activity. Quantitative comparisons among carbon pathways indicate that rates of pelagic heterotrophy, both photo- and chemoheterotrophy combined, contribute small quantities of carbon to overall carbon metabolism in this oligotrophic system. Qualitative comparisons among pathways indicate strong spatial and temporal separation. The late summer period showed greatest seasonal separation of the three pathways. Spring values represented a period of relatively high activity for all three pathways. On a depth-wise basis, photolithotrophic activity was greatest near the surface and chemolithotrophic activity greatest near the bottom. Photoheterotrophy took an intermediate position between the two. Diurnally, photoheterotrophy and photolithotrophy showed greatest activity during midday and early morning periods, whereas chemoheterotrophy increased throughout the daylight period and reached maximal values in sunset incubations.