Are dissolved free amino acids free?

Microbial assimilation of 3 amino acids (glutamic acid, alanine, and ornithine) was characterized in 3 lakes and 2 marine stations using the Michaelis-Menten kinetic approach. The calculated K_t + S_n concentrations were related to chemical concentration measurements of dissolved free amino acids (DFAA) to evaluate the biological and the chemical determinations of the DFAA pools. Concentrations of K_t + S_n always were larger than chemical measurements of the S_n concentrations. K_t + S_n and S_n varied from 11.5 and 9.5 nM (alanine, oligotrophic lake) to 288.7 and 89.9 nM (ornithine, marine harbor station), respectively. Subtracting S_n from the K_t + S_n concentrations, K_t was found to range from 12–897% of the chemically measured S_n concentrations. To test whether the DFAA actually were free, dissolved molecules, dissolved material in the water samples was separated into various molecular size classes by means of gel permeation chromatography. From 47–116% of the DFAA in the untreated water samples was recovered in the low molecular fraction (<700 Daltons). Variation in recoveries mainly appeared to be due to an incomplete chromatographic separation and difficulties in obtaining proper blank levels. The present observations suggest that labeled tracers can be used in the study of DFAA assimilation and that the DFAA are free, dissolved molecules. This partly conflicts with previously published reports.     

Net uptake of dissolved free amino acids by four scleractinian corals

High pressure liquid chromatography was employed to provide the first definitive proof of the net uptake of dissolved free amino acids (DFFA) at nanomolar levels by four scleractinian corals (Montastrea annularis, Madracis mirabilis, Agaricia fragilìs, and Favia fragum). During 2 h incubations all species exhibited simultaneous net uptake of eight amino acids. For M. annularis and F. fragum uptake of some dissolved amino acids occurred at concentrations lower than those found in reef waters. Microbial activity or adsorption of DFAA to exposed coral skeletons during these experiments did not appear to be important. Although it seems unlikely that DFAA uptake can provide a significant energy source for corals under ambient condìtions, it may be important in the acquisition and retention of nitrogen by these animals.     

Gradient of dissolved free amino acids and phytoplankton in a shallow bay

A 3 h survey of the concentrations of individual free amino acids, chlorophyll a and phytoplankton species biomass was conducted in the surface waters of a shallow bay. Significant coefficients of correlation were found between chlorophyll a, nanoflagellates, and DFAA concentrations. Although phytoplankton biomass variations sometimes relate to DFAA concentration patterns, consideration of the physiological activity of both phytoplankton and microheterotrophs would undoubtedly explain a more significant fraction of the DFAA variation.This work is a contribution of GREPMA (Groupe Régional d'Etudes Pélagiques en Manche-Atlantique)     

Fates of dissolved free amino acids in groundwater discharged through stream bed sediments

Laboratory simulations were used to investigate the immobilization of dissolved free amino acids (DFAAs) from groundwater discharged up through cores of stream-bed sediments from a first order stream. At natural concentrations, 99% of DFAAs supplied in groundwater were immobilized, with 14–25% of this material respired and the remainder retained in the lower layers of the cores (depth = 7.5 cm). Immobilization efficiencies increased with increasing groundwater DFAA concentrations and discharge rates. Moderate enrichments (up to 1 mg l^–1) appeared to stimulate biotic immobilization of DFAAs, while abiotic processes accounted for much of the increased immobilization at higher enrichments (tested up to 100 mg l^–1). Variability in groundwater discharge rates induced no changes in the proportional contribution of biotic and abiotic immobilization processes. Thus relative contributions of DFAA concentration and groundwater discharge rate to a given DFAA load (concentration × discharge rate) influenced the degree to which immobilized DFAAs were retained (as microbial biomass or adsorbed to the biofilm) or respired. Results showed that DFAAs in groundwater discharged through the stream-bed are in a highly dynamic state of flux, suggesting that these compounds may be more significant to the transfer of organic matter to the benthic trophic structure than their normally low concentrations in groundwater would imply.     

Zooplankton induced changes in dissolved free amino acids and in production rates of freshwater bacteria

This study examined the importance of zooplankton in the flux of dissolved free amino acids (DFAA) in the water and into bacteria. DFAA release rates were followed in laboratory grazing experiments usingDaphnia galeata andEudiaptomus graciloides as grazers, andScenedesmus acutus andSynechococcus elongatus as food sources. Except for minor initial peaks, DFAAs were released continuously during the first 2 hours and made up 6–12% (in one experiment 50%) of the calculated ingestion rates. During three diel studies in lakes, effects of removal and increase of the density of zooplankton (>200m) on the pools of DFAA as well as on the bacterial production were followed. During two of the diel studies, higher DFAA pools were measured when 3–4 times the natural zooplankton density was present, and in one study a minor increase also occurred in the bacterial production, compared with results from experiments without zooplankton and with a natural zooplankton density. The increase in bacterial growth coincided with a decline in DFAA. During the third study, neither DFAA nor the bacterial production changed significantly when the zooplankton density was increased 3 times. Removal of zooplankton, however, caused a decline in both DFAA and bacterial production. Our data suggest a close relationship between occurrence of zooplankton and release of DFAA, but the factors regulating the amount of DFAA released and its effect on bacterial growth are not yet understood.     

Volatile fatty and dissolved free amino acids in Organic Lake,Vestfold Hills,East Antarctica

The distributions of volatile fatty acids (C1–C5) (VFAs) and dissolved free amino acids (DFAAs) in Organic Lake, a shallow hypersaline meromictic lake in the Vestfold Hills, East Antarctica, were determined during the winter and spring of 1987. The concentrations of the two VFAs detected, formic and acetic acid, were low (<10 M) in the oxic upper waters of the lake, but increased dramatically (up to 250M) beneath the oxycline. The distribution of DFAAs was similar, with a total concentration of 280M in the anoxic water early in the study. The amino acids occurring at the highest concentrations were alanine, glutamic acid, leucine and valine. Total VFA concentrations in the anoxic zone increased during the study period, but the total amino acid concentration dropped significantly to 64 M by the end of the study. The high concentrations beneath the oxycline were probably the result of bacterial utilisation of these substrates that was slow due to the high salinity and low temperature of the lake water.     

Kinetics of amino acids mineralization by a pond sediment bacterial community

Bacterial degradation of a high concentration of amino acids (up to 150 mg l^–1), released by quail manure pellets, was studied in a fertilized fish pond. Aspartic acid, Glutamic acid and Alanine were major components of the free amino acid pool (respectively 14.6%, 16.4% and 11.1%). Laboratory experiments showed a fast decomposition of these three amino acids. The degradation was a zero order reaction. Activation energy, Ea, was of the same order for the three amino acids (from 64.2 to 70.3 kg-mol^–1). There was no significant difference between the behaviour of aspartic acid in the presence or not of other carbon and nitrogen supply, but stimulation of degradation of glutamic acid and of Alanine by NH₄ ⁺ and C, was marked. This process may be important in the initial mineralisation of the fertilizer in fish ponds.     

Effect of dissolved free amino acids (DFAA) on the biomass and production of microphytobenthic communities

Mixed microphytobenthos communities, manipulated in two different ways in the laboratory (semi-natural and sediment-stripped), were examined for their response to dissolved free amino acids (DFAA) and nitrate (NO₃⁻). The semi-natural manipulation involved only the removal of macrofauna; and the sediment-stripped community used microfauna and flora that were separated from natural sediment and re-established on clean sediment, that is, certain indigenous nutrient sources were removed. Using sediment collected on two different occasions, two sets of experiments were made (3 and 4 week), under different light conditions. The response by the communities to the added nitrogen was measured as biomass (Chl a) and primary production. The stimulus from the DFAA addition on microphytobenthos biomass and primary production was similar to or higher than that from NO₃⁻. A conclusively positive effect from the nitrogen additions could be measured only in the sediment-stripped community. Compared to the semi-natural community, the sediment-stripped community responded faster to the added DFAA. When light was limiting, biomass and production by the sediment-stripped community was enhanced by the N additions, in particular by the DFAA addition. Results suggest that an efficient recycling of nitrogen allows generally nitrogen-poor sandy sediments to sustain high microphytobenthos productivity, thereby retaining nitrogen within the system.     

Adsorption and release of amino acids from epilithic biofilms in streams

SUMMARY. I. Stream sediment with a natural biofilm generally adsorbed more glycine, aspartic acid, and lysine than sterile sediment. Lysine was adsorbed to a greater extent than the other two amino acids.
2. In the presence of Ca²⁺, adsorption of aspartic acid increased and adsorption of lysine decreased. Glycine was not affected.
3. Stream sediment preferentially removed amino acids with a positive charge or a polar R group from maple leaf leachate.
4. It is estimated that stream invertebrates may have access to a maximum of 7.3% of the amino acids present in epilithic biofilms. More amino acids were available from biofilms exposed to leaf leachate.     

Thiophosphorylation of free amino acids and enzyme protein by thiophosphoramidate ions

In search of an activity-preserving protein thiophosphorylation method, with thymidylate synthase recombinant protein used as a substrate, potassium thiophosphoramidate and diammonium thiophosphoramidate salts in Tris- and ammonium carbonate based buffer solutions were employed, proving to serve as a non-destructive environment. Using potassium phosphoramidate or diammonium thiophosphoramidate, a series of phosphorylated and thiophosphorylated amino acid derivatives was prepared, helping, together with computational (using density functional theory, DFT) estimation of ³¹P NMR chemical shifts, to assign thiophosphorylated protein NMR resonances and prove the presence of thiophosphorylated lysine, serine and histidine moieties. Methods useful for prediction of ³¹P NMR chemical shifts of thiophosphorylated amino acid moieties, and thiophosphates in general, are also presented. The preliminary results obtained from trypsin digestion of enzyme shows peak at m/z 1825.805 which is in perfect agreement with the simulated isotopic pattern distributions for monothiophosphate of TVQQQVHLNQDEYK where thiophosphate moiety is attached to histidine (His²⁶) or lysine (Lys³³) side-chain.     

The role of dissolved organic matter, particularly free amino acids and humic substances, in freshwater ecosystems

1. Although the mass of dissolved organic matter (DOM) often exceeds that of living organisms in freshwaters, little is known about the roles of its constituent molecules as sources of energy and information for aquatic organisms. In the present review attention is focused on free amino acids (FAA) and humic substances (HS) as examples of labile and refractory components within DOM. 2. The following questions are addressed. (i) What are spatiotemporal patterns in the distribution of DOM, HS and FAA? (ii) What are the origins of the components of DOM and how are their concentrations regulated? (iii) What is the significance of the spatial and temporal distributional patterns of DOM, HS and FAA to detritivorous invertebrates and other organisms associated with them? (iv) What is the relevance of DOM to the food web concept and to the biochemical ecology of freshwater ecosystems? 3. Concentrations of DOM, FAA and HS within lentic ecosystems are ranked as follows: Sediment pore water > Air–water interface > Midwater column. Comparisons between water bodies show that the concentrations of labile constituents of DOM, such as FAA, are usually positively correlated with base cations, nutrients and biological activity. In contrast, HS concentrations are negatively correlated with base cations or nutrients but positively correlated with the rate of biological degradation (the maximum values occurring in the autumn). The FAA : HS ratios might serve therefore as an indicator of the potential productivity of a water body. 4. External sources of DOM in general, and FAA and HS in particular, include rainwater, windborne material, surface flow and groundwater. The relative importance of these allochthonous sources of DOM decreases along the length of lotic ecosystems and also with increase in size of lentic ecosystems. Internal sources of FAA and HS include synthesis or polymerization from existing organic matter, degradation of organic matter and release from both living and dead organisms. The net accumulation of DOM released by living bacteria, phytoplankton, epilithon, macrophytes and invertebrates is much reduced due to heterotrophic uptake. Hence, most of the allochthonous DOM in freshwater originates from dead organic matter deposited on the sediment. Phytoplankton-dominated ecosystems may, however, differ, as most of their DOM may be recycled within the water column. 5. The factors that determine the external concentrations of DOM, FAA and HS are discussed. Evidence is cited in support of the following testable hypotheses. (i) The rates of production of DOM components will be favoured by increasing base cation and nutrient concentrations. (ii) Colloidal clay, base cations, biopolymers and living organisms, particularly bacteria, facilitate the removal of HS. Consequently, base-rich eutrophic waters tend to have lower HS concentrations than oligotrophic, base-deficient waters. (iii) As a result of higher productivity and selective removal of FAA, eutrophic waters tend to have higher FAA concentrations than those that are oligotrophic. 6. Labile DOM components, such as FAA, act as sources of information for aquatic organisms. More research is needed in this field. There is a consensus that DOM acts as an important source of energy for aquatic bacteria, thus forming the microbial loop. However, higher eukaryotic organisms also utilize DOM, including components released by bacteria and plants as metabolic end-products and photoassimilates, respectively. As a result, these DOM components may be more important as food for macrodecomposers than the microdecomposers themselves. HS may also benefit aquatic organisms by promoting their growth and protecting them from inimical forces. Conversely, the removal of photons and the release of toxins by HS may be detrimental to aquatic organisms. 7. It is concluded that the central dogma of the foodweb, and its implicit assumption that the energy flow in aquatic ecosystems can be quantified solely by measuring rates of photosynthesis, ingestion of solid food and its digestion by higher organisms, is invalid. To extend our understanding of the role of DOM as a source of nutrition and information to aquatic organisms it is suggested that the subject should be studied within the context of ‘modules’ which have the following properties: (i) the components have co-evolved; (ii) the more vulnerable components will have protective mechanisms; (iii) the components will derive mutual benefits from co-existence; (iv) sedentary components will release kairomonal attractants or developmental primers; (v) living components will exchange energy and information; (vi) the module will collapse following the removal of strongly interactive keystone species. An example of a three-component, three-subset module, is provided by tubificid worms, epilithic bacteria and algae. A more complex module consisting of pulmonate snails, associated macrophytes, their epiphytic bacteria and algae has four components and six subsets. The elucidation of the interactive mechanisms within such modules demands an interdisciplinary approach, involving microbiology, biochemistry and behavioural biology.     

Fluxes of dissolved amino acids between sea water and Echinaster

• 1.1. Measurement of free amino acid (primary amine) influx and efflux into the starfish, Echinaster, were accomplished utilizing improved methods of sea water purification and analysis.
• 2.2. Specimens placed in amino acid depleted sea water (5 × 10⁻⁸ M) demonstrated net release as measured with the fluorescamine method. Similarly, specimens placed in the same water to which amino acid mixtures had been reintroduced to normal levels demonstrated net uptake.
• 3.3. A mathematical model indicated an equilibrium amino acid concentration (when influx equals efflux) of 5.26 × 10⁻⁷ M, or about one fourth the level of natural sea water.
• 4.4. Since at normal environmental levels (20.65 × 10⁻⁷ M) net flux is inward by a ratio of nearly 4-1, it is concluded that the previous suggestions of some workers that such would not be the case for marine invertebrates are no longer valid.
• 5.5. The net uptake of amino acid from environmental levels would account for 5.67% of the measured total respiration if all were being metabolized.
• 6.6. This figure appears to be in line with the previously developed hypothesis that the epidermis largely obtains its nutrition directly from the environment. However, the real benefit of the uptake mechanism may be to prevent loss of the body amino acid pools.

     

Net uptake of dissolved free amino acids by the giant clam, Tridacna maxima: alternative sources of energy and nitrogen?

 The role of dissolved free amino acids (DFAA) in nitrogen and energy budgets was investigated for the giant clam, Tridacna maxima, growing under field conditions at One Tree Island, at the southern end of the Great Barrier Reef, Australia. Giant clams (121.5–143.7 mm in shell length) took up neutral, acidic and basic amino acids. The rates of net uptake of DFAA did not differ between light and dark, nor for clams growing under normal or slightly enriched ammonium concentrations. Calculations based on the net uptake concentrations typical of the maximum concentrations of DFAA found in coral reef waters (∼0.1 μM ) revealed that DFAA could only contribute 0.1% and 1% of the energy and nitrogen demands of giant clams, respectively. These results suggest that DFAA does not supply significant amounts of energy or nitrogen for giant clams or their symbionts. Accepted: 7 October 1998     

Production of extracellular free amino acids by cyanobacteriumAnabaena siamensis Antarikanonda

The cyanobacteriumAnabaena siamensis Antarikanonda, isolated from rice paddies of Bangkok, Thailand, liberates substantial quantities of free amino acids into the external medium irrespective of whether it is growing on N₂, NH₄ ⁺, NO₃ ^– or under nitrogen-starved conditions. Addition of such combined nitrogen causes changes in both intracellular and extracellular free amino acid pool patterns. No overall relationship exists between the amino acid efflux and the intracellular pools. The most abundant free amino acids found in the external media of N₂, NO₃ ^–, NH₄ ⁺-grown and N-starved cultures were phenylalanine, threonine, glutamate, and glycine, respectively. These investigations suggest that amino acid liberation by the cyanobacterium is a selective diffusional process that is sensitive to environmental changes.     

Impact of UV radiation on the production patterns and composition of dissolved free and combined amino acids in marine phytoplankton

Using the ¹³C tracer technique in conjunction with gas chromatographic-massspectro-metric (GC-MS) techniques, we examined the patternsof synthesis and the composition of dissolved free and combinedamino acids within phytoplankton photosynthesizing in the presenceand absence of natural solar ultraviolet radiation (UVR). Atlevels that still permitted the uptake of carbon assimilationinto the cells, UVR caused a marked decline in the overall rateof carbon incorporated into amino acids and a reduction in thepool size of total cellular amino acids (TCAA). In contrast,absolute concentrations of amino acids within the intracellulardissolved free amino acid (INDFAA) pool (measured using an aminoacid analyzer) were higher in the presence of UVR. An examinationof the production patterns and composition of amino acids constitutingthe INDFAA and TCAA pools revealed a marked diminution in thesynthesis and accumulation of alanine and valine in the presenceof UVR. On the other hand, the rates of synthesis and concentrationsof glutamic acid (glutamic acid + glutamine) in the INDFAA andTCAA pools of phytoplankton were higher in samples exposed toUVR. These changes are discussed with reference to the knowneffects of UVR on nitrogen and carbon assimilation within phytoplankton.     

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.

     

Uptake of free amino acids by the diatom,Melosira mediocris

Individual ¹⁴C-labelled amino acids are rapidly removed from dilute solution in artificial sea water (0.2 mol 1^–1) by suspensions of Meliosira medocris. The rate of disappearance of radioactivity corresponds closely to removal of primary amines as determined by measurement of the rate of decrease of fluorescamine-positive material. Net removal of naturally occurring free amino acids from the sea water habitat from which the alga was isolated is demonstrated using high performance liquid chromatography. Removal of amino acids from natural sources makes a significant contribution to the carbon requirements of the alga as well as supplying significant amounts of amino nitrogen.