Alternative model and approach for determining microbial heterotrophic activities in aquatic systems.

Increasing amounts of high-specific-activity tritiated organic compounds were added to samples of several natural waters such that in situ substrate concentrations might be approximated. The uptake responses by the native heterotrophic microflora suggested that (i) heterotrophic populations metabolize the added nutrients, but (ii) these responses are not necessarily a reflection of Michaelis-Menten enzyme kinetics. The uptake kinetics appeared to be due to dilution of the naturally occurring metabolite by added radioactive substrate and physiological responses of the microflora to organic enrichment.     

Acclimation of stream-bed heterotrophic microflora: metabolic responses to dissolved organic matter

SUMMARY.

• 1 Studies were performed to assess the acclimation of the stream-bed heterotrophic microflora to sources of dissolved organic matter (DOM) typical of its environment and microfloral responses to pulses of DOM.
• 2 Microcosm measurements of dissolved organic carbon (DOC) uptake, dissolved oxygen uptake. ATP concentration and epitluorescence microscopic counts (EMC) were performed using stream-bed sediments and heterogeneous dissolved organic matter (DOM) sources.
• 3 Three study sites included an undisturbed woodlot spring seep, a small stream traversing a cattle pasture and a larger stream draining a catchment used for silage crops, pastures and woodlands.
• 4 The DOM sources were cold water extracts of forest floor leaf litter, bovine manure, the green alga Ulothrix and jewel weed (Impaliens capensis L.) leaves.
• 5 DOC uptake occurring in 2.5–5.0 h incubations indicated an acclimation of the microflora at each site to DOM sources generated by surrounding land use.
• 6 The sediment microflora from the larger stream did not readily metabolize bovine manure DOM and the latter was used in an acclimation experiment.
• 7 A minimum of 48 h of cumulative exposure to bovine manure DOM at 15–20°C were required to yield measurable changes in sediment microbial activity of sediment microbial biomass.
• 8 The same microflora retained an ability to readily metabolize the added DOM source after 72 h of exposure to unamended stream water.
• 9 The time frame of microfloral responses during acclimation indicated that changes leading to the metabolism of a DOM source were initially enzymatic and eventually involved growth and selection for specific decomposers within the microbial community.
• 10 We conclude that in order to utilize naturally occurring pulses of carbon and energy, stream-bed heterotrophs must be already enzymatically prepared, induced, when the pulse occurs.

     

ALGAE AS COMPETITORS FOR GLUCOSE WITH HETEROTROPHIC BACTERIA1

Dissolved organic carbon (DOC) constitutes the bulk of organic carbon in aquatic environments. The importance of DOC utilization by mixotrophic algae is unclear since heterotrophic bacteria are regarded as more efficient users. We tested the hypothesis that algae decrease the DOC concentration in the light to lower levels than in darkness resulting in competitive exclusion of heterotrophic bacteria according to the mechanistic competition theory. We investigated (a) the uptake kinetics of glucose as a model substrate by two cultured algae and mixed bacteria populations, (b) the competition for glucose between algae and bacteria in chemostats, (c) the effect of discontinuous glucose supply in chemostats, and (d) the minimum glucose concentrations achieved in cultures of algae and bacteria. Bacteria showed higher specific‐glucose‐uptake rates than algae. In chemostats, algae became extinct in the dark and coexisted in the light where they decreased bacteria to lower densities. Discontinuous glucose supply promoted the algae compared to continuous substrate addition. Several algae consumed glucose to lower concentrations in the dark than in the light and showed lower or equal residual glucose concentrations than bacteria. Residual concentrations were not related to allometric traits (cell volume) and photosynthetic potential (chl content). Overall, the hypothesis was not supported, and mechanisms of competition for DOC obviously differed from those for particulate prey. However, since some algae showed lower or equal residual glucose concentrations than bacteria, algal dark uptake of DOC may be important in deep layers of many waters.     

Measurement of aquatic biodegradation rates by determining heterotrophic uptake of radiolabeled pollutants.

The heterotrophic uptake technique was modified to provide a rapid and simple technique for estimating the rates of biodegradation of organic pollutants under environmental conditions. The methodology is based on an evaluation of uptake into cells and subsequent respiration of radiolabeled organic substrates in short-term experiments. The resulting data can be used to calculate either turnover times or, if multiple concentrations of substrate are used, kinetic parameters. The procedure was applied to assess the biodegradation rates of m-cresol, chlorobenzene, nitrilotriacetic acid, and 1,2,4-trichlorobenzene in fresh, brackish, and marine water samples from the coastal areas of North Carolina. Saturation kinetics for uptake were obtained with each of the compounds tested. Rates of metabolism were shown to be dependent on sample location and time of year.     

Heterotrophic Potential for Amino Acid Uptake in a Naturally Eutrophic Lake

The uptake of sixteen (14)C-labeled amino acids by the indigenous heterotrophic microflora of Upper Klamath Lake, Oregon, was measured using the kinetic approach. The year-long study showed a seasonal variation in the maximum uptake velocity, V(max), of all the amino acids which was proportional to temperature. The maximum total flux of amino acids by the heterotrophic microflora ranged from 1.2 to 11.9 mumol of C per liter per day (spring to summer). Glutamate, asparagine, aspartate, and serine had the highest V(max) values and were respired to the greatest extent. The percentages of the gross (net + respired) uptake of the amino acids which were respired to CO(2) ranged from 2% for leucine to 63% for glutamate. Serine, lysine, and glycine were the most abundant amino acids found in Upper Klamath Lake surface water; at intermediate concentrations were alanine, aspartate, and threonine; and the remaining amino acids were always below 7.5 x 10(-8) M (10 mug/liter). The amino acid concentrations determined chemically appear to be the sum of free and adsorbed amino acids, since the values obtained were usually greater than the (K(t) + S(n)) values obtained by the heterotrophic uptake experiments.     

The stoichiometry of nitrogen and phosphorus spiralling in heterotrophic and autotrophic streams

1. Nutrient spiralling provides a conceptual framework and a whole‐system approach to investigate ecosystem responses to environmental changes. We use spiralling metrics to examine how the coupling of nitrogen and phosphorus uptake varies between streams dominated by either heterotrophic (i.e. bacteria‐dominated) or autotrophic (algal‐dominated) microbial communities. 2. Algae generally exhibit greater capacity to store nutrients than bacteria because of differences in cellular structures. These differences led us to hypothesise that the uptake of N and P in heterotrophic ecosystems should have reduced stoichiometric variation in response to changes in supply N : P compared to autotrophic ecosystems when assimilation dominates nutrient uptake. 3. To test this hypothesis, we used an array of serial nutrient additions in several streams in the South Fork Eel River watershed in Northern California. In one set of experiments, N and P were added alone and simultaneously in separate experiments to two small, heterotrophic streams to assess uptake rates and interactions between nutrient cycles. In a second set of experiments, N and P were added simultaneously at a range of N : P in one heterotrophic and one autotrophic stream to assess differences in uptake responses to changes in supply N : P. 4. Results of these experiments suggest two important conclusions. First, increased N supply significantly shortened P uptake lengths, while P addition had little impact on N uptake in both streams, indicating that uptake of non‐limiting nutrients is tightly coupled to the availability of the limiting element. Second, changes in P uptake and uptake ratios (U_N : U_P) with increased supply N : P supported our hypothesis that heterotrophic streams are more homeostatic in their responses to changes in nutrient supply than autotrophic streams, suggesting that physiological controls on nutrient use scale up to influence ecosystem‐scale patterns in nutrient cycling.     

Polymerized coumaric acid as a model substrate for terrestrial-derived dissolved organic carbon utilized by aquatic microorganisms

It is now widely accepted that many surface waters receive more terrestrial carbon than assumed in the past, and that aquatic food webs are largely based on the supply of external dissolved organic carbon. However, very little information is available on how efficiently external carbon is utilized by microorganisms and transported to consumers of higher trophic levels. To address this issue, we prepared and tested polymers of ¹⁴C-p-coumaric acid (PCA) as a model substrate for terrestrial organic carbon. Photodegradation products that can be considered potential substrates for microorganisms were identified using hyphenated techniques, including gas chromatography-mass spectrometry (GC/MS) and ion chromatography-electrospray ionization mass spectrometry (IC/MS). Photolysis of PCA released monomeric phenol derivatives, e.g. 4-hydroxybenzaldehyde. The photolysis products observed were similar to those characteristic for natural organic carbon. Both a heterotrophic bacteria assemblage and a cultured algae strain exhibiting heterotrophic capabilities proved capable of utilizing the model substrate. Irradiation of PCA increased the uptake rate approximately eight times for the bacteria, but no significant increase was observed for the algae. Potential sources of interferences, e.g. the uptake of ¹⁴CO₂ released by photolysis, were addressed. It was concluded that PCA is a suitable substrate to study the metabolism of terrestrial DOC within aquatic communities.     

Microbial Activity at the Sediment-Water Interface in Halifax Harbor, Canada

The sediment-water interface in Halifax Harbor supports a microbial population of 6.95 × 10⁹ cells per g (dry weight). As determined by the standard technique of suspending subsamples in filtered seawater, the uptake of added glutamic acid by this population is 113.5 ng g (dry weight)⁻¹ h⁻¹. An alternate technique was developed to measure the heterotrophic activity of the interface over longer periods of time, using undisturbed cores with the sediment-water interface intact. Under these conditions, the microbes in the water column and the interface increased exponentially in number, with mean doubling times of 9.6 and 4.5 days, respectively. The uptake of glutamic acid by the microbial population of the interface was determined to be 12.7 ng g (dry weight)⁻¹ h⁻¹, almost an order of magnitude less than the uptake determined by the previous method. This indicates that substrate diffusion and competition for substrate by the microbes in the water column are important factors when considering the heterotrophic activity of the sediment microbial population. After 48 h of incubation, uptake and respiration ceased, probably due to the exhaustion of labeled substrate. Additional substrate added after 48 h of incubation was taken up at a rate similar to that measured after the first addition. It appears that the microbial population of the interface is able to respond quickly and repeatedly to relatively large nutrient additions. After 10 days of incubation, the number of “viable” cells as determined by autoradiography was much smaller than the increase in numbers as determined by direct counts. Apparently a large part of the viable population is unaffected by nutrient addition.     

Shifting N and P limitation along a north-south gradient of mangrove estuaries in South Florida

The heterotrophic utilization of organic substrates by diatoms is likely an important survival strategy when light levels are too low for photosynthesis. The objectives of this study were: (1) to determine if heterotrophic utilization of a large array of organic compounds by eight common freshwater benthic diatom taxa was light-dependent, and (2) to determine if organic substrate utilization patterns differed between dark-grown diatoms and bacteria as a possible means of reducing competition by niche separation. Eight light- and dark-grown diatom taxa and five bacterial species were incubated in 96-well Biolog^® Microtiter plates with each well containing 1 of 95 different organic substrates. Oxidation rates of each organic substrate were measured through time. There was a substantial increase in the number of organic substrates oxidized by diatoms grown in the dark compared to their light-grown counterparts, indicating that the transport systems for these molecules may be light activated. Therefore, diatoms likely only utilize these metabolically expensive uptake mechanisms when they are necessary for survival, or when substrates are plentiful. A principal components analysis indicated discernible differences in the types of organic-C substrates utilized by dark-grown diatoms and bacteria. Although bacteria were able to oxidize a more diverse array of organic substrates including carboxylic acids and large polymers, diatoms appeared to more readily utilize the complex carbohydrates. By oxidizing different organic substrates than bacteria, heterotrophically metabolizing diatoms may be reducing direct competition and enhancing coexistence with bacteria.     

Bacterial responses to natural organic inputs in a marine subAntarctic area

Quantitative investigations of bacteriological and organic matter parameters were conducted regularly at least once a week for a year at three subAntarctic benthic stations. The different bacteriological methods used (direct counts, viable counts and heterotrophic potentials) gave complementary results. Despite substantial differences in organic matter content of the different sediments, the bacterial communities were of similar density. Seasonal variations observed in the studied area were regular enough to allow a detailed analysis. A general scheme describing the reactions of the bacterial microflora to the different periods of organic input is discussed. This model, which includes three parts (lag phase, growing phase and return to the initial state), allows the interpretation of the bacterial responses observed during the benthic surveys.     

Short-term changes in polysaccharide utilization mechanisms of marine bacterioplankton during a spring phytoplankton bloom

Spring phytoplankton blooms in temperate environments contribute disproportionately to global marine productivity. Bloom-derived organic matter, much of it occurring as polysaccharides, fuels biogeochemical cycles driven by interacting autotrophic and heterotrophic communities. We tracked changes in the mode of polysaccharide utilization by heterotrophic bacteria during the course of a diatom-dominated bloom in the German Bight, North Sea. Polysaccharides can be taken up in a ‘selfish’ mode, where initial hydrolysis is coupled to transport into the periplasm, such that little to no low-molecular weight (LMW) products are externally released to the environment. Alternatively, polysaccharides hydrolyzed by cell-surface attached or free extracellular enzymes (external hydrolysis) yield LMW products available to the wider bacterioplankton community. In the early bloom phase, selfish activity was accompanied by low extracellular hydrolysis rates of a few polysaccharides. As the bloom progressed, selfish uptake increased markedly, and external hydrolysis rates increased, but only for a limited range of substrates. The late bloom phase was characterized by high external hydrolysis rates of a broad range of polysaccharides and reduced selfish uptake of polysaccharides, except for laminarin. Substrate utilization mode is related both to substrate structural complexity and to the bloom-stage dependent composition of the heterotrophic bacterial community.     

Biodegradation of di-n-butyl phthalate (DnBP) in bioaugmented bioslurry phase reactor

Bioremediation of di-n-butyl phthalate (DnBP) in soil was studied with various concentrations in a bioslurry phase batch reactor operated in sequenting batch mode (bioaugmented with effluent treatment plant (ETP) microflora) for a total cycle period of 96h. Process performance during the reactor operation was assessed by monitoring DnBP concentration and biochemical process parameters viz., pH, dissolved oxygen (DO), colony forming units (CFU) and oxygen uptake rate (OUR), during the sequence phase operation. The degradation rate was observed to be rapid at lower substrate concentrations and found to be slow as the substrate concentration increased. The potent bacterial strain was also isolated from the slurry phase reactor. Metabolites formed during the degradation of DnBP in the slurry phase reactor were identified. Studies on the kinetics and half-life of the reaction revealed that the degradation process followed zero-order kinetic model.     

Autotrophic and Heterotrophic Metabolism of Hydrogenomonas I. Growth Yields and Patterns Under Dual Substrate Conditions

Auxotrophic mutants of Hydrogenomonas eutropha and H. facilis requiring utilizable amino acids were employed to demonstrate the simultaneous utilization of H(2) and an organic substrate for growth. The ratio of the cell yields under dual substrate conditions compared to heterotrophic conditions indicated the relative contributions of the autotrophic and heterotrophic systems to the growth of the organism. Wildtype H. eutropha grown under simultaneous conditions exhibited a dicyclic growth pattern, the first cycle representing either heterotrophic or simultaneous growth and the second cycle representing autotrophic growth. The duration of the changeover period was either very short with no plateau or long with a plateau up to 8 hr, depending upon the organic substrate. The growth rate under simultaneous conditions with some organic substrates was faster than either the autotrophic or heterotrophic rate, but was not the sum of the two rates. The data suggest that, in the presence of both organic and inorganic substrates, heterotrophic metabolism functions normally but autotrophic metabolism is partially repressed.     

The aerobic mineralization of organic compounds in the saline Lake Grevelingen (The Netherlands) (VI)

Summary During a one year period the uptake of aspartic acid and of a mixture of amino acids was determined using¹⁴C-labeled substrates as described by WRIGHT and HOBBIE (1966). By this technique the activity is analyzed of that part of the bacterial population which is able to utilize the added substrate. For comparison purposes the activity of the total heterotrophic bacterial population was determined by measurement of the oxygen consumption rate. From the oxygen consumption rate (mg O₂.l^–1.h^–1) the carbon mineralization rate (mg C.l^–1.h^–1) was calculated by applying a conversion factor of 0.29.Aspartic acid was respired for 80% and the amino acid mixture for 43%. From the maximum uptake rates, the potential yearly uptake of the substrate in question can be calculated. These data indicate the relative importance of the several subpopulations in the carbon mineralization process as a whole. The highest value of the potential yearly uptake was obtained for the amino acid mixture; the comparable value for the uptake of aspartic acid was slightly lower.The carbon mineralization rate as calculated from the oxygen uptake experiments was about 150–200 g C.m^–2.year^–1. The potential yearly uptake as determined with the¹⁴C-labeled amino acid mixture was only 2.8% of the amount of mineralized carbon, as calculated from the oxygen uptake experiments. This percentage is very low in view of the fact that 35–55% of the organic carbon of living phytoplankton and zooplankton consists of protein (HAGMEIER, 1961) and that the aerobic mineralization of amino acids is a very common property among the heterotrophic bacterial population (SEPERS, 1979). The value of the applied activity measurements was investigated in order to obtain information about the relation between the uptake process as measured with¹⁴C-labeled substrates and the activity of the bacterial population in situ. The results of this study have been published bij SEPERS and VAN ES (1979).     

HETEROTROPHIC GROWTH OF ULVA LACTUCA (CHLOROPHYCEAE)1

The effect of external glucose (51 mM) and acetate (13 mM) on growth and photosynthetic capacity of Ulva lactuca L. was tested in laboratory cultures over 41 days in the dark and in dim light (0.9 μmol photons·m^?2·s^?1) at 7–8° C. Glucose and acetate had a significant positive effect on growth rate, chlorophyll content, and quantum yield for discs grown in the dark and in dim light. The carbon gain from heterotrophic uptake was low and only allowed U. lactuca to maintain a specific uptake was low and only allowed U. lactuca to maintain a specific growth rate of 0.005 day^?1 compared to 0.06–0.1 day^?1 at higher light intensities. However, plants with added organic substrate maintained a normal chlorophyll content and were able to photosynthesize whereas control plants lost pigmentation and photosynthetic capability after 41 days in both dim light and darkness, probably because of disorganization of the photosynthetic apparatus. This suggest that the ecological significance of heterotrophic uptake is to allow U. lactuca to survive during prolonged low light conditions with an intact photosynthetic apparatus.     

How interactions between microbial resource demands,soil organic matter stoichiometry,and substrate reactivity determine the direction and magnitude of soil respiratory responses to warming

Recent empirical and theoretical advances inform us about multiple drivers of soil organic matter (SOM) decomposition and microbial responses to warming. Absent from our conceptual framework of how soil respiration will respond to warming are adequate links between microbial resource demands, kinetic theory, and substrate stoichiometry. Here, we describe two important concepts either insufficiently explored in current investigations of SOM responses to temperature, or not yet addressed. First, we describe the complete range of responses for how warming may change microbial resource demands, physiology, community structure, and total biomass. Second, we describe how any relationship between SOM activation energy of decay and carbon (C) and nitrogen (N) stoichiometry can alter the relative availability of C and N as temperature changes. Changing availabilities of C and N liberated from their organic precursors can feedback to microbial resource demands, which in turn influence the aggregated respiratory response to temperature we observe. An unsuspecting biogeochemist focused primarily on temperature sensitivity of substrate decay thus cannot make accurate projections of heterotrophic CO₂ losses from diverse organic matter reservoirs in a warming world. We establish the linkages between enzyme kinetics, SOM characteristics, and potential for microbial adaptation critical for making such projections. By examining how changing microbial needs interact with inherent SOM structure and composition, and thus reactivity, we demonstrate the means by which increasing temperature could result in increasing, unchanging, or even decreasing respiration rates observed in soils. We use this exercise to highlight ideas for future research that will develop our abilities to predict SOM feedbacks to climate.     

The uptake of inorganic nutrients by heterotrophic bacteria

It is now well known that heterotrophic bacteria account for a large portion of total uptake of both phosphate (60% median) and ammonium (30% median) in freshwaters and marine environments. Less clear are the factors controlling relative uptake by bacteria, and the consequences of this uptake on the plankton community and biogeochemical processes, e.g., new production. Some of the variation in reported inorganic nutrient uptake by bacteria is undoubtedly due to methodological problems, but even so, uptake would be expected to vary because of variation in several parameters, perhaps the most interesting being dissolved organic matter. Uptake of ammonium by bacteria is very low whereas uptake of dissolved free amino acids (DFAA) is high in eutrophic estuaries (the Delaware Bay and Chesapeake Bay). The concentrations and turnover of DFAA are insufficient, however, in oligotrophic oceans where bacteria turn to ammonium and nitrate, although the latter only as a last resort. I argue here that high uptake of dissolved organic carbon, which has been questioned, is necessary to balance the measured uptake of dissolved inorganic nitrogen (DIN) in seawater culture experiments. What is problematic is that this DIN uptake exceeds bacterial biomass production. One possibility is that bacteria excrete dissolved organic nitrogen (DON). A recent study offers some support for this hypothesis. Lysis by viruses would also release DON.While ammonium uptake by heterotrophic bacteria has been hypothesized to affect phytoplankton community structure, other impacts on the phytoplankton and biomass production (both total and new) are less clear and need further work. Also, even though bacteria account for a very large fraction of phosphate uptake, how this helps to structure the plankton community has not been examined. What is clear is that the interactions between bacterial and phytoplankton uptake of inorganic nutrients are more complicated than simple competition.     

Mycorrhizal functioning of Phialocephala fortinii with Pinus contorta on glacier forefront soil: interactions with soil nitrogen and organic matter

 Plants growing on an environmentally stressed glacier forefront on soil low in N and organic matter have abundant root colonizations by dark-septate fungi. As the plants appeared fit for this severe habitat, it was hypothesized that the dark-septate endophytes were neutral or beneficial rather than detrimental to the plants. To test this hypothesis, we designed a growth-room experiment with Pinus contorta grown on forefront soil inoculated with the dark-septate fungus Phialocephala fortinii in the absence of climatic stress. N and organic matter treatments were included to explore their interaction with the fungal inoculation. P. fortinii colonized roots inter- and intracellularly and occasionally formed microsclerotia. Inoculated plants absorbed significantly more P than noninoculated plants in all combinations of N and organic matter. Without added N, neither inoculation nor organic matter addition improved plant growth or N uptake, showing that N indeed limits plant growth in this substrate. With added N, however, both organic matter addition and inoculation significantly increased total pine biomass and N uptake. The enhanced P uptake by the P. fortinii-inoculated pine as well as the increased pine growth and N uptake in the treatment combining P. fortinii and N appear as typical mycorrhizal responses. Accepted: 20 October 1997     

Transport of organic sulfur and nitrogen in the roots of young mycorrhizal pedunculate oak trees (Quercus robur L.)

Uptake and xylem loading of organic sulfur and nitrogen were analyzed in detached mycorrhizal (Laccaria laccata L.) roots of pedunculate oak (Quercus robur L.) seedlings using radiolabeled reduced glutathione (GSH) and glutamine (Gln) for transport analyses. The experiments showed for the first time that GSH is taken up by plant roots from the nutrient solution and is partially allocated to the shoot. Apparently, GSH produced during mineralization processes in the soil can be used by plant roots as a sulfur source. GSH uptake into the roots showed biphasic kinetics within the concentration range studied (0–500 M) with maximum transport velocities (v _max) and substrate affinities (K _m) that were similar to the kinetics of Gln uptake. GSH uptake kinetics were also in the same range as previously reported for sulfate uptake by mycorrhizal roots of pedunculate oak. It may therefore be assumed that GSH and sulfate uptake can be of comparable significance for sulfur nutrition, provided both sulfur sources are available at similar concentrations at the sites of uptake. Xylem loading of GSH and Gln showed monophasic transport kinetics with v _max significantly lower than observed for the two respective uptake systems and, as indicated by the K _m-values, a substrate affinity between the high and the low affinity uptake systems. The possible nature of the transport systems for GSH and Gln is discussed.     

Number of bacteria and features of their activity in hypersaline reservoirs of the Crimea

The incidence of bacteria, their biomass production, and heterotrophic assimilation of CO2 by bacterioplankton were studied in the Crimean hypersaline water reservoirs from May to October of 1974. The total incidence of bacteria in the natural brine of these reservoirs varied from 20 to 70 x 10(6) cells per 1 ml. Such a high bacterial number may be caused by the combined action of water evaporation which increased the concentration of bacterial cells and active growth of microflora. Low values of bacterial production and heterotrophic CO2 assimilation should be attributed to weak activity of microflora in the reservoirs.