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.     

Protozoan Grazing Increases Mineralization of Naphthalene in Marine Sediment

Bacterial decomposition of organic matter is frequently enhanced when protozoa are present. Various mechanisms have been proposed to account for this phenomenon, including effects associated with grazing by protozoa (such as increased recycling of limiting nutrients, removal of senescent cells, or reduction of competition among bacteria) and indirect effects of grazers (such as excretion of bacterial growth factors). Few studies have examined the role of protozoa in bacterial degradation of xenobiotic compounds in sediment containing a natural community of microbes. The effect of protozoa on mineralization of naphthalene was investigated in this study. Laboratory experiments were conducted using field-contaminated estuarine sediment, with the indigenous microbial populations. Mineralization of naphthalene was up to four times greater in treatments with actively grazing protozoa than in treatments containing the grazing inhibitor cytochalasin B. Control experiments confirmed that the grazing inhibitor was not toxic to ciliates but did prevent them from grazing. The grazing inhibitor did not affect growth rates of a mixed culture of sediment bacteria or a pure polycyclic-aromatic-hydrocarbon-degrading strain. Once grazing had been inhibited, supplementing treatments with inorganic N and P, glucose, or additional protozoa failed to stimulate naphthalene mineralization. Naphthalene-degrading bacteria were four to nine times less abundant when protozoan grazing was suppressed. We suggest that protozoa enhance naphthalene mineralization by selectively grazing on those sediment bacteria that ordinarily would outcompete naphthalene-degrading bacteria.     

Use of Monodispersed, Fluorescently Labeled Bacteria to Estimate In Situ Protozoan Bacterivory

We have developed a procedure for preparing monodispersed, fluorescently labeled bacteria (FLB), which may be used to measure virtually instantaneous rates of protozoan bacterivory in natural waters. FLB can be prepared both from natural bacterioplankton assemblages and from clonal isolates and can be stored in frozen suspension or freeze-dried without apparent loss of fluorescence intensity. They are not toxic to protozoa and can be metabolized to support bacterivorous protozoan growth rates equal to those on the same strain of unstained, viable bacteria. In experiments comparing uptake of FLB with uptake of fluorescent latex microspheres by protozoan assemblages in a salt marsh tidal creek, we found that both pelagic oligotrichous ciliates and phagotrophic flagellates ingested FLB with a frequency 4- to 10-fold greater than they ingested the microspheres. Consequently, it appears that the use of latex microspheres leads to underestimation of protozoan bacterivory and that the FLB technique is superior for estimating instantaneous rates of in situ protozoan grazing on bacterioplankton.     

Attached and Free Bacteria in an Estuary with Abundant Suspended Solids

Concentrations and glucose mineralization potentials ( V _max) of attached and free bacteria in waters from the Humber estuary were determined. The waters had high concentrations of suspended solids. Most bacteria were attached to suspended solids and V _max of attached bacteria was greater than that of free bacteria. These observations suggest that breakdown of organic matter is carried out mainly by attached bacteria. Examination of samples from other estuaries which contained less suspended solids than the Humber suggested that in these estuaries free bacteria might be more abundant than attached bacteria.     

Inadequacy of the Eucaryote Inhibitor Cycloheximide in Studies of Protozoan Grazing on Bacteria at the Freshwater-Sediment Interface

Four guilds from a lake sediment-water interface microbial community were isolated and tested for sensitivity to cycloheximide (0.1 to 200 mg liter⁻¹). Field experiments were conducted to compare the inhibition, dilution, and filtration methods for determining grazing rates. Cycloheximide inhibited anaerobic bacteria at 50 mg liter⁻¹, and inhibition of bacterial growth was observed in the grazing experiments. The results show that the assumption of selective inhibition of heterotrophic eucaryotes was violated and preclude the use of cycloheximide in grazing experiments.     

The Effect of Grazing by a Ciliated Protozoan on Phosphorus Limitation of Heterotrophic Bacteria in Batch Culture1

ABSTRACT. The yield of the bacterium Enterobacter aerogenes and the ciliate Colpidium colpoda was dependent on initial phosphorus concentrations in batch cultures containing 125 or 250 mg/liter glutamate and 50–1000 μg/liter phosphorus. For both, yield per unit phosphorus declined at higher phosphorus concentrations. A marked decline in growth rate in bacterial cultures was coincident with the depletion of dissolved phosphorus and the development of rapid orthophosphate turnover times. Colpidium introduced to these cultures consumed about 16,000 bacteria/h/ciliate while multiplying exponentially and relieved phosphorus limitation, as indicated by a longer turnover-time for phosphate. The longer turnover-time was due to the reduction of bacterial numbers; in cultures with ciliates, bacteria appear to be more active in taking up phosphate, and much of the total phosphorus accumulates in ciliates. Ciliates released both inorganic and organic phosphorus, but the organic phosphorus did not accumulate to excess in the cultures to an extent that would indicate that it is less used by bacteria. Although ciliates release enough phosphorus to account for ca. 20% of the bacterial uptake, ciliates appear to behave as phosphorus sinks as much as phosphorus recyclers in these closed systems.     

Effects of Deposit-Feeding Macrofauna on Benthic Bacteria,Viruses, and Protozoa in a Silty Freshwater Sediment

In microcosm experiments, we simultaneously tested the effects of increased numbers of deposit-feeding macrofauna (chironomids, oligochaetes and cladocerans) on the standing stock, activities and interactions of heterotrophic bacteria, viruses, and bacterivorous protozoa (heterotrophic nanoflagellates and ciliates) in the aerobic layer of a silty littoral freshwater sediment. On average, bacterial secondary production was stimulated between 11 and 29% by all macrofaunal groups compared to control experiments without macrofauna addition. Bacterial standing stock increased significantly by 8 and 13% in case of chironomids and cladocerans, respectively. Oligochaetes and chironomids produced significant negative effects on viral abundance while the results with cladocerans were inconsistent. The addition of oligochaetes and chironomids resulted in a significant decrease by on average 68 and 32% of viral decay rates, respectively, used as a measure of viral production. The calculated contribution of virus-induced lysis to benthic bacterial mortality was low, with 2.8 to 11.8% of bacterial secondary production, and decreased by 39 to 81% after the addition of macrofauna compared to the control. The abundances of heterotrophic nanoflagellates were significantly reduced by 20% by all tested macrofauna groups, while ciliates showed inconsistent results. The importance of heterotrophic nanoflagellate grazing on benthic bacteria was very low (<1% of bacterial secondary production) and was further reduced by elevated numbers of macrofauna. Thus, the selected deposit feeding macrofauna groups seem to have several direct and indirect and partly antagonistic effects on the benthic bacterial compartment through the enhancement of bacterial production and the reduction of virus-induced cell lysis and protozoan grazing.     

Grazing, Growth, and Ammonium Excretion Rates of a Heterotrophic Microflagellate Fed with Four Species of Bacteria

We studied aspects of the population growth of a microflagellate, Monas sp., isolated from Lake Kinneret, Israel. The protozoan growth rates, rates of ingestion of bacteria, and final population yields generally increased with increasing bacterial concentrations, although the exact relationship varied depending on the species of bacteria used as food. Grazing rates decreased hyperbolically with increasing food density. Gross growth efficiencies and ammonia excretion rates were similar over a range of food densities among the four species of bacteria. Population doubling times and ammonia excretion rates were lowest, and growth efficiencies were highest, at temperatures between 18 and 24°C. Under optimum conditions, the microflagellates had average population doubling times of 5.0 to 7.8 h, average growth efficiencies of 23.7 to 48.7%, and average ammonia excretion rates of 0.76 to 1.23 μmol of NH₄⁺ per mg (dry wt) per h.     

Rapid Virus Production and Removal as Measured with Fluorescently Labeled Viruses as Tracers

Pelagic marine viruses have been shown to cause significant mortality of heterotrophic bacteria, cyanobacteria, and phytoplankton. It was previously demonstrated, in nearshore California waters, that viruses contributed to up to 50% of bacterial mortality, comparable to protists. However, in less productive waters, rates of virus production and removal and estimates of virus-mediated bacterial mortality have been difficult to determine. We have measured rates of virus production and removal, in nearshore and offshore California waters, by using fluorescently labeled viruses (FLV) as tracers. Our approach is mathematically similar to the isotope dilution technique, employed in the past to simultaneously measure the release and uptake of ammonia and amino acids. The results indicated overall virus removal rates in the dark ranging from 1.8 to 6.2% h⁻¹ and production rates in the dark ranging from 1.9 to 6.1% h⁻¹, corresponding to turnover times of virus populations of 1 to 2 days, even in oligotrophic offshore waters. Virus removal rates determined by the FLV tracer method were compared to rates of virus degradation, determined at the same locations by radiolabeling methods, and were similar even though the current FLV method is suitable for only dark incubations. Our results support previous findings that virus impacts on bacterial populations may be more important in some environments and less so in others. This new method can be used to determine rates of virus degradation, production, and turnover in eutrophic, mesotrophic, and oligotrophic waters and will provide important inputs for future investigations of microbial food webs.     

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

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

Rates of Sediment Resuspension and Erosion Following Green Turtle Grazing in a Shallow Caribbean Thalassia testudinum Meadow

Ecosystems - Seagrass meadows buffer sediments against resuspension and erosion by reducing water velocity and attenuating wave energy, thereby promoting accumulation of sediment and associated...     

Fluid and Sediment Dynamic Effects on Marine Benthic Community Structure

Fluid and sediment dynamics affect benthic community dynamicsand structure in manifold ways. We single out three community-structuringprocesses that are both strongly affected and amenable to controlledmanipulation: microbial population growth, faunal recruitment,and particle feeding. Attachment and colony growth rates ofmicrobes depend on the details of near-bed fluid exchange. Theiremigration and colony growth rates are affected by erosion ofmicrobial filmsand by abrasion during sediment transport. Recruitment and successional patterns of metazoans, especiallythose resulting from the settlement of weakly swimming, smalllarvae and juveniles, also are very sensitive to local variationsin boundary layer flow pattern and strength. While the importanceof particle fluxes to suspension feeders has long been apparent,the foraging patterns of a growing number of surface depositfeeders are being found to reflect a dependence upon sedimenttransport. Although these three processes have spatial and temporalscales amenable to both laboratory and field experimentation,proper dynamic scaling of laboratory model flows may not alwaysbe easy. Even the simplest two-phase (particles plus liquids,where particles can be bacteria, floes, larvae, or sediments)flows must match appropriate laboratory and field Reynolds number,Froude number, particle-fluid density (weight per unit volume)ratio, and the ratio of boundary layer thickness to particlesize, if the laboratory flow model is to provide accurate results.     

Influence of Physical Disruption on Growth of Attached Bacteria

Attached bacterial populations cultured without an exogenous carbon source or grown in conjunction with attached diatoms incorporated [³H]thymidine at a rate between four and five times lower than that of replicate bacterial populations which were dispersed before being assayed.     

Protozoan Colonization Rates and Trophic Status of Some Freshwater Wetland Lakes

Primary productivity, chlorophyll a, phosphorus and nitrogen nutrients, and other chemical and physical parameters were measured in 13 wetland lakes in northern lower Michigan. These lakes included several examples located in each of the four major wetland types—bogs, fens, marshes, and swamps. Of the four types, the brown-colored waters of the acid bog lakes generally had the highest levels of primary productivity, chlorophyll a, phosphorus, and nitrogen. Primary productivity correlated positively with water color, total-N, and NH₃-N (α≤ 0.05). By these measures, waters of the bogs were the most eutrophic of the four types of wetland lakes. These findings would seem to contradict the generally-held concept that “dystrophic” bog lakes are extremely oligotrophic. Protozoan colonization of artificial substrate islands was monitored at each wetland site. The correlation between protozoan colonization rates (G values in the MacArthur-Wilson noninteractive model) and primary productivity, measured by 24-h light and dark bottle incubations, was significant at the 95% confidence level (r= 0.850, P= 0.001) and with water color at the 90% confidence level (r= 0.599, P= 0.084). It was concluded that protozoan colonization rate was an excellent indicator of the trophic status of wetland lakes.     

Production and Vertical Flux of Attached Bacteria in the Hudson River Plume of the New York Bight as Studied with Floating Sediment Traps

We investigated the growth and vertical flux of attached bacteria with floating sediment traps in the Hudson River Plume of the New York Bight during the spring diatom blooms. Traps were floated at the base of the mixed layer (ca. 10 m) for 1-day periods. After recovery, we measured bacterial abundance and rates of [methyl-³H]thymidine incorporation in the trap samples. The vertical flux of attached bacteria was estimated with a model formulated to distinguish between bacterial accumulation in traps due to in situ growth and that due to vertical flux. Attached bacterial flux ranged from 0.6 × 10¹¹ to 2.0 × 10¹¹ cells m⁻² day⁻¹, and attached bacterial settling rates of 0.1 to 1.0 m day⁻¹ were observed during periods of vertical particulate organic carbon flux ranging from 254 to 1,267 mg of C m⁻² day⁻¹. In situ growth of bacteria in sediment traps was unimportant as a source of bacterial increase when compared with vertical flux during our study. The vertical flux of attached bacteria removed 3 to 67% of the total daily bacterial production from the water column. Particulate organic carbon is not significantly mineralized by attached bacteria during its descent to the sea floor in the plume area during this period, when water temperature and grazing rates are at their annual minima.     

Permanent Stained Agar Preparation of Mycoplasma and of L Forms of Bacteria

A procedure is described that permits the preparation of permanent stained mounts of Mycoplasma and of bacterial L forms grown on the surface of and within agar media. These preparations are especially useful for making representative photographs. The cultures are fixed with Formalin vapor. Thin slices of agar are stained at elevated temperature between 50 and 60 C and at a low pH, are dried rapidly, and are mounted in Canada balsam. The results with this staining procedure are illustrated by photographs of various strains of Mycoplasma and of bacterial L forms.     

Interaction of Nutrient Limitation and Protozoan Grazing Determines the Phenotypic Structure of a Bacterial Community

We examined the impact of nutrient conditions (carbon and phosphorus limitation) and grazing by protozoans on the phenotypic community structure of freshwater bacteria in continuous culture systems. Lakewater bacteria were grown on mineral medium, which was supplemented with glucose and amino acids and adjusted by different phosphorus concentrations to achieve either carbon or phosphorus limitation. Each nutrient treatment was inoculated with the same bacterial community and consisted of a nongrazing and a grazing treatment, to which the heterotrophic nanoflagellates Spumella sp. and Ochromonas sp. were added. We found that nutrient conditions alone resulted in differences in the phenotypic structure of the bacterial community: small and motile bacteria dominated under C limitation while large, elongated, and capsulated bacteria were characteristic for P limitation. The genotypic community composition as measured by T-RFLP (terminal restriction fragment length polymorphism) was not severely influenced by the two nutrient treatments. In the presence of flagellate predators, grazing-resistant bacteria developed under both nutrient conditions, but with different survival mechanisms: highly motile bacteria prevailed under C limitation, whereas the P-limited grazing treatment was dominated by filamentous forms. T-RFLP analysis revealed only moderate changes in bacterial community composition due to grazing, which were most pronounced under P limitation. Analysis by video microscopy revealed that high swimming speed is an efficient nonmorphological survival mechanism for bacteria to reduce the capture success of the flagellate predator. The rejection of optimal-sized, nonmotile bacteria under P limitation suggests the importance of other nonmorphological, surface-located cell properties. Our results illustrate that the realized mechanisms of grazing resistance are linked to the actual limitation conditions, and that the combined effects of nutrient limitation and grazing are major determinants of bacterial community structure.     

Protozoan Response to the Addition of Bacterial Predators and Other Bacteria to Soil

Representatives of several categories of bacteria were added to soil to determine which of them might elicit responses from the soil protozoa. The various categories were nonobligate bacterial predators of bacteria, prey bacteria for these predators, indigenous bacteria that are normally present in high numbers in soil, and non-native bacteria that often find their way in large numbers into soil. The soil was incubated and the responses of the indigenous protozoa were determined by most-probable-number estimations of total numbers of protozoa. Although each soil was incubated with only one species of added bacteria, the protozoan response for the soil was evaluated by using most-probable-number estimations of several species of bacteria. The protozoa did not respond to incubation of the soil with either Cupriavidus necator, a potent bacterial predator, or one of its prey species, Micrococcus luteus. C. necator also had no effect on the protozoa. Therefore, in this case, bacterial and protozoan predators did not interact, except for possible competition for bacterial prey cells. The soil protozoa did not respond to the addition of Arthrobacter globiformis or Bacillus thuringiensis. Therefore, the autochthonous state of Arthrobacter species in soil and the survival of B. thuringiensis were possibly enhanced by the resistance of these species to protozoa. The addition of Bacillus mycoides and Escherichia coli cells caused specific responses by soil protozoa. The protozoa that responded to E. coli did not respond to B. mycoides or any other bacteria, and vice versa. Therefore, addition to soil of a nonsoil bacterium, such as E. coli, did not cause a general increase in numbers of protozoa or in protozoan control of the activities of other bacteria in the soil.     

Prediction of Substrate Removal Rates of Attached Microorganisms and of Relative Contributions of Attached and Suspended Communities at Field Sites

A mathematical model composed of a direct proportionality relationship between bulk water velocities and field-determined second-order microbial transformation rate coefficients, and the relative rate coefficient of a benchmark chemical, was developed for estimating the substrate removal rates of rapidly degraded chemicals by attached organisms in shallow (<1 m deep) aquatic ecosystems. Data from 31 field experiments involving the addition of 2,4-dichlorophenoxyacetic acid methyl ester (2,4-DME) in nine field areas were used to determine a field-derived second-order rate coefficient for microbial transformation of the ester. By using 2,4-DME as a benchmark chemical, the model was used to predict microbial transformation rates of the butoxyethyl ester of 2,4-dichlorophenoxyacetic acid (2,4-DBE) at five other field sites. The predicted half-lives of 2,4-DBE varied 1,500-fold and were within about a threefold range or less of the measured half-lives. Under conditions of mass transport limitation, the contributions of attached microorganisms relative to total microbial activities at various field sites were related to the ratio of water velocity, U, and depth, D, showing that historical definitions of ecosystems according to flow and depth characteristics are also valid for describing the process-related structure of ecosystems. An equation was developed for predicting the relative contributions of attached and suspended communities with values of U and D for lotic and lentic ecosystems. On the basis of this equation, attached microorganisms were expected to be insignificant in deep lentic ecosystems and suspended microorganisms were expected to be insignificant in shallow lotic systems for the same process carried out by both populations. Neglecting epiphytic microorganisms, both suspended and attached organisms were expected to be significant in wetlands.     

Intertidal Benthic Invertebrates and Sediment Stability at the Mouth of Delaware Bay

From December 1970 to December 1972, the monthly species composition, density, species richness, and biomass of marine macroscopic invertebrates were quantitatively studied from a wave-protected, intertidal sand flat at the mouth of Delaware Bay. Considerable erosion, transportation, and deposition of sand occurred throughout the study period. The fauna was extremely flexible and resilient in its response to a gradient of considerable sediment mobility. This response was similar in scope to one recognized along the coast of California. Comparison of the local biota with other sand flats and beaches from Maine to South Carolina showed that (1). haustoriids were among the dominant species, (2). densities were roughly comparable, and (3). secondary species were similar. Based on the biological index value, three species contained 87.8% of the fauna from the Cape Henlopen tide flat. These species were the polychaete Scoloplos fragilis and the amphipod Neohaustorius biarticulatus, which were characteristic of sand bars, and the polychaete Heteromastus filiformis, which was more characteristic of troughs or depressions. Mean density varied considerably both monthly and annually with almost no cyclic repetition for any of the three species. There was evidence to indicate that specific habitat requirements of a few species can strongly affect the structure which emerges for a total fauna. Morphology, behavior, physiology, and reproductive strategy are aspects of biology which link dominant benthic species of intertidal sand beaches together.