Asparagine and glutamine metabolism in Rhodopseudomonas acidophila

Rhodopseudomonas acidophila strain 7050 achieved balanced growth when provided with either asparagine or glutamine as nitrogen source. Under these growth conditions R. acidophila synthesized a mixed amidase which exhibited similar activity (223–422 nmol/min·mg protein) against either nitrogen source. Determination of the free intracellular amino acid pools show that deamidation of asparagine and glutamine resulted in elevated levels of both aspartate and glutamate. Cell-free extracts of R. acidophila showed significant aminotransferase activity, particulary glutamine-oxaloacetate aminotransferase (89.7–209.3 nmol/min·mg protein), glycine oxaloacetate aminotransferase (135–227 nmol/min ·mg protein), alanine glyoxylate aminotransferase (66.3–163.2 nmol/min·mg protein) and serineglyoxylate aminotransferase (57.1–68.4 nmol/min ·mg protein). Short term labelling experiments using ¹⁴C-glyoxylate show that glycine plays an important role in amino nitrogen transfer in R. acidophila and that the enzymes for the metabolism of glyoxylate via glycine, serine and hydroxypyruvate were present in cell-free extracts. These data confirm that R. acidophila can satisfy all its' nitrogen requirements by transamination.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase - MSO methionine sulfoximine - GOT glutamate—oxaloacetate aminotransferase - GPT glutamate-pyruvate aminotransferase - AGAT alanineglyoxylate aminotransferase - GOAT glycine-oxaloacetate aminotransferase - GOGAT glycine-2-oxoglutarate aminotransferase - AOAT alanine-oxaloacetate aminotransferase - SGAT serineglyoxylate aminotransferase - INH isonicotinylhydrazide     

Indigo formation by aromatic hydrocarbon-degrading bacteria

A variety of aromatic hydrocarbon-degrading bacteria expressing different oxygenases were tested for their ability to produce indigo from indole. Styrene-grown cells of Pseudomonas putida S12 and CA-3 expressing styrene mono-oxygenase produced indigo at rates of 4–8 nmol min–1 mg dry wt–1. Toluene-grown cells of P. putida F1 and naphthalene-grown cells of P. putida PpG7 expressing dioxygenases formed indigo at rates of 1.5 and 2.5 nmol min–1 mg dry wt–1, respectively. © Rapid Science Ltd. 1998     

Chitinolytic Activity of Filamentous Fungi

The chitinolytic activity of nine species of filamentous fungi, classified with seven genera (specifically, Aspergillus, Penicillium, Trichoderma, Paecilomyces, Sporotrichum, Beaueria, and Mucor), was studied. When cultured in liquid medium containing 1% crystalline chitin, all fungi produced extracellular chitosans with activity varying from 0.2 U/mg protein (Sporotrichum olivaceum, Mucorsp., etc.) to 4.0–4.2 U/mg protein (Trichoderma lignorum, Aspergillus niger).     

Catalysis of an isotopic exchange between CO2 and the carboxyl group of acetate by Methanosarcina barkeri grown on acetate

Cell suspensions of Methanosarcina barkeri (strain Fusaro) grown on acetate were found to catalyze the formation of methane and CO₂ from acetate (30–40 nmol/min·mg protein) and an isotopic exchange between the carboxyl group of acetate and ¹⁴CO₂ (30–40 nmol/min·mg protein). An isotopic exchange between [¹⁴C]-formate and acetate was not observed. Cells grown on methanol mediated neither methane formation from acetate nor the exchange reactions. The data indicate that the isotopic exchange between CO₂ and the carboxyl group of acetate is a partial reaction of methanogenesis from acetate. Both reactions were completely inhibited by low concentrations of cyanide (20 M) or of hydrogen (0.5% in the gas phase). Methane formation from acetate was also completely inhibited by low concentrations of carbon monoxide (0.2% in the gas phase) whereas only significantly higher concentrations of CO had an effect on the exchange reaction. In the concentration range tested KCN, H₂ and CO had no effect on methane formation from methanol or from H₂ and CO₂; however, cyanide (20 M) also affected methane formation from CO. The results are discussed with respect to proposed mechanisms of methane and CO₂ formation from acetate.     

Methyl mercury uptake by free and immobilized cyanobacterium

Methyl mercury uptake in free cells and different immobilizates of the cyanobacteriumNostoc calcicola has been examined. The general growth of the immobilized cyanobacterial cells could be negatively correlated with methyl mercury uptake. Alginate spheres proved most efficient in terms of uptake rate (0.48 nmol mg protein^–1 min^–1, 10 min) and total bioaccumulation (10.71 nmol mg protein^–1, 1 h) with a bioconcentration factor of 3.3×10³. Alginate biofilms showed a faster methyl mercury accumulation rate (0.83 nmol mg protein^–1 min^–1, 10 min) with a saturation of 10.28 nmol mg protein^–1 reached within only 30 min (bioconcentration factor, 3.1×10³). Foam preparations with a slow initial uptake approximated biofilms but were characterized by a lower bioconcentration factor (2.8×10³). Free cells, in comparison, maintained the initial slow rate of uptake (0.62 nmol mg protein^–1 min^–1, 10 min), saturating at 30 min (8.81 nmol mg protein^–1), and the resultant lowest bioconcentration factor (2.7×10³). Cell ageing (30 days) brought a drastic reduction (3-fold) in organomercury uptake by free cells while alginate spheres maintained the same potential. Foam preparations of the same age showed a significant improvement in methyl mercury uptake followed by only a marginal decline in alginate biofilms. Data are discussed in the light of the physiological efficiency and longevity of immobilized cells.     

Oxidation of Benzene to Phenol, Catechol, and 1,2,3-Trihydroxybenzene by Toluene 4-Monooxygenase of Pseudomonas mendocina KR1 and Toluene 3-Monooxygenase of Ralstonia pickettii PKO1

Aromatic hydroxylations are important bacterial metabolic processes but are difficult to perform using traditional chemical synthesis, so to use a biological catalyst to convert the priority pollutant benzene into industrially relevant intermediates, benzene oxidation was investigated. It was discovered that toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1, toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1, and toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 convert benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by successive hydroxylations. At a concentration of 165 μM and under the control of a constitutive lac promoter, Escherichia coli TG1/pBS(Kan)T4MO expressing T4MO formed phenol from benzene at 19 ± 1.6 nmol/min/mg of protein, catechol from phenol at 13.6 ± 0.3 nmol/min/mg of protein, and 1,2,3-trihydroxybenzene from catechol at 2.5 ± 0.5nmol/min/mg of protein. The catechol and 1,2,3-trihydroxybenzene products were identified by both high-pressure liquid chromatography and mass spectrometry. When analogous plasmid constructs were used, E. coli TG1/pBS(Kan)T3MO expressing T3MO formed phenol, catechol, and 1,2,3-trihydroxybenzene at rates of 3 ± 1, 3.1 ± 0.3, and 0.26 ± 0.09 nmol/min/mg of protein, respectively, and E. coli TG1/pBS(Kan)TOM expressing TOM formed 1,2,3-trihydroxybenzene at a rate of 1.7 ± 0.3 nmol/min/mg of protein (phenol and catechol formation rates were 0.89 ± 0.07 and 1.5 ± 0.3 nmol/min/mg of protein, respectively). Hence, the rates of synthesis of catechol by both T3MO and T4MO and the 1,2,3-trihydroxybenzene formation rate by TOM were found to be comparable to the rates of oxidation of the natural substrate toluene for these enzymes (10.0 ± 0.8, 4.0 ± 0.6, and 2.4 ± 0.3 nmol/min/mg of protein for T4MO, T3MO, and TOM, respectively, at a toluene concentration of 165 μM).     

Identification of a Tsetse Fly Salivary Protein with Dual Inhibitory Action on Human Platelet Aggregation

Background

Tsetse flies (Glossina sp.), the African trypanosome vectors, rely on anti-hemostatic compounds for efficient blood feeding. Despite their medical importance, very few salivary proteins have been characterized and functionally annotated.

Methodology/Principal Findings

Here we report on the functional characterisation of a 5′nucleotidase-related (5′Nuc) saliva protein of the tsetse fly Glossina morsitans morsitans. This protein is encoded by a 1668 bp cDNA corresponding at the genomic level with a single-copy 4 kb gene that is exclusively transcribed in the tsetse salivary gland tissue. The encoded 5′Nuc protein is a soluble 65 kDa glycosylated compound of tsetse saliva with a dual anti-hemostatic action that relies on its combined apyrase activity and fibrinogen receptor (GPIIb/IIIa) antagonistic properties. Experimental evidence is based on the biochemical and functional characterization of recombinant protein and on the successful silencing of the 5′nuc translation in the salivary gland by RNA interference (RNAi). Refolding of a 5′Nuc/SUMO-fusion protein yielded an active apyrase enzyme with K_m and V_max values of 43±4 µM and 684±49 nmol Pi/min×mg for ATPase and 49±11 µM and 177±37 nmol Pi/min×mg for the ADPase activity. In addition, recombinant 5′Nuc was found to bind to GPIIb/IIIa with an apparent K_D of 92±25 nM. Consistent with these features, 5′Nuc potently inhibited ADP-induced thrombocyte aggregation and even caused disaggregation of ADP-triggered human platelets. The importance of 5′Nuc for the tsetse fly hematophagy was further illustrated by specific RNAi that reduced the anti-thrombotic activities in saliva by approximately 50% resulting in a disturbed blood feeding process.

Conclusions/Significance

These data show that this 5′nucleotidase-related apyrase exhibits GPIIb/IIIa antagonistic properties and represents a key thromboregulatory compound of tsetse fly saliva.     

The removal of nickel from mine waters using bacterial sulfate reduction

Summary Experiments were done to determine if a compost-based sulfate-reduction system could be used to treat nickel-contaminated mine waters. Sulfate-reduction systems were established in columns containing acid-washed mushroom compost. Simulated mine waters containing 2000 mg sulfate 1^–1 and 50–1000 mg nickel 1^–1 were adjusted to pH 4.5 and pumped through the columns at flow rates between 15 and 25 ml h^–1. Initially, almost all of the influent nickel was removed in the columns by sorptive and ion exchange mechanisms. The nickel removal rate then dropped to 18–30 mg nickel day (7.8 to 12.8 nmol g ^–1 compost day), where it remained relatively constant. The mechanisms responsible for the low and sustained rates of nickel removal on unamended compost are unclear. When sodium lactate was added to the inflow, sulfate reduction rates between 250 and 650 nmol day cm^–3 compost were obtained and a sevenfold increase in the nickel removal rate was observed. The maximum nickel removal rate observed was 540 mg Ni kg^–1 compost day (92 nmol Ni g^–1 compost day) for columns receiving 1000 mg Ni 1^–1. Correspondence to: H. M. Edenborn     

An isolate of Rhizopus nigricans capable of tolerating and removing pentachlorophenol

Rhizopus nigricans, isolated from an industrial effluent (paper mill), was resistant to pentachlorophenol (PCP) in Petri dishes and in submerged cultures (100 and 25 mg l^–1 respectively). It was shown that this strain of R. nigricans can remove PCP in submerged culture. When 12.5 mg of PCPl^–1 were added at 48 h, this compound had been completely removed by 144h. Results indicated that the fungus did not produce extracellular lignin peroxidase (LiP) and laccase, but extracellular phenoloxidase production was observed. The synthesis of the latter enzyme was stimulated by the presence of PCP and/or tyrosine. These results indicate that this fungus, and probably other filamentous fungi, have an interesting potential to be used in processes for chlorophenol biodegradation.     

Nocistatin and nociceptin given centrally induce opioid-mediated gastric mucosal protection

Nociceptin (N/OFQ) and nocistatin (NST) are two endogenous neuropeptides derived from the same precursor protein, preproN/OFQ. The aim of the present work was to study the effect of NST on the ethanol-induced mucosal damage compared with that of N/OFQ following intracerebroventricular (i.c.v.) administration in the rat and to analyze the mechanism of the gastroprotective action. It was found that both NST and N/OFQ reduced the mucosal lesions in the same dose range (0.2–1 nmol i.c.v.), but in higher doses (2–5 nmol i.c.v.) the gastroprotective effect of both peptides was highly diminished. The gastroprotective effect of N/OFQ (1 nmol), but not that of NST (1 nmol), was reduced by the selective nociceptin receptor antagonist J-113397 (69 nmol i.c.v.). Similarly, decrease of the gastroprotective effect was observed after the combination of NST (1 nmol) with N/OFQ (0.6 or 1 nmol). However, addition of the gastroprotective effects was observed, when lower dose (0.2 nmol) of NST was given prior to N/OFQ (0.6 nmol). The gastroprotective effect of both N/OFQ and NST was antagonized by naloxone (27 nmol), β-funaltrexamine (20 nmol), naltrindole (5 nmol) and norbinaltorphimine (14 nmol), the μ-, δ- and κ-opioid receptor antagonists, respectively, given i.c.v. The mucosal protection was significantly decreased after bilateral cervical vagotomy. The present findings suggest that NST similar to N/OFQ, may also induce gastric mucosal protective action initiated centrally in a vagal-dependent mechanism. Opioid component is likely to be involved in the gastroprotective effect of both NST and N/OFQ.     

H+/ATP stoichiometry for the gastric (K++H+)-ATPase

Summary The initial rate of ATP-dependent proton uptake by hog gastric vesicles was measured at pH's between 6.1 and 6.9 by measuring the loss of protons from the external space with a glass electrode. The apparent rates of proton loss were corrected for scalar proton production due to ATP hydrolysis. For vesicles in 150mm KCl and pH 6.1, corrected rates of proton uptake and ATP hydrolysis were 639±84 and 619±65 nmol/min×mg protein, respectively, giving an H⁺/ATP ratio of 1.03±0.7. Furthermore, at all pH's tested the ratio of the rate of proton uptake to the rate of ATP hydrolysis was not significantly different than 1.0. No proton uptake (<10 nmol/min×mg protein) was exhibited by vesicles in 150mm NaCl at pH 6.1 despite ATP hydrolysis of 187±46 nmol/min×mg (nonproductive hydrolysis). Comparison of the rates of proton transport and ATP hydrolysis in various mixture of KCl and NaCl showed that the H⁺/ATP stoichiometries were not significantly different than 1.0 at all concentrations of K⁺ greater than 10mm. This fact suggests that the nonproductive rate is vanishingly small at these concentrations, implying that the measured H⁺/ATP stoichiometry is equal to the enzymatic stoichiometry. This result shows that the isolated gastric (K⁺+H⁺)-ATPase is thermodynamically capable of forming the observed proton gradient of the stomach.     

Leaf developmental stage and tissue location affect the detection of β-glucuronidase in transgenic tobacco plants

Expression in Nicotiana tabaccum L. plants containing the -glucuronidase (GUS) gene under the control of the 35S (CaMV promoter) was affected by tissue type and ontogenic development of the leaves. GUS activity in ontogenetically younger leaves was 1003–1022 nmol 7-hydroxy-4-methylcoumarin (MU) formed mg^–1 (protein) min^–1 and in ontogenetically older leaves was only 140–198 nmol (MU) mg^–1 (protein) min^–1.     

A comparison of dinitrogen fixation rates in wood litter decayed by white-rot and brown-rot fungi

Nitrogen fixation rates, as estimated by the acetylene reduction technique, were determined in conifer wood litter being decayed by brown- and white-rot fungi. Average ethylene production rates were significantly higher in white-rotted wood (15.1 nmol g^–1 day^–1) than in brown-rotted wood (2.3 nmol g^–1 day^–1). This difference may be related to a higher soluble sugar content in white-versus brown-rotted wood. The nitrogen-fixing bacteriumAzospirillum was not detected in any of the decaying wood samples examined. Greater nitrogen additions from nitrogen-fixing bacteria may be a factor in the more rapid white-rot decay of hardwood litter, as compared to the slower brown-rot decay of conifer wood.     

The role of muscle development in the transition to endothermy in nestling bank swallows,Riparia riparia

Summary We examined the transition from ectothermy to endothermy in nestling bank swallows (Riparia riparia) by measuring the peak metabolic response to cold (PMR) in groups of nestlings. Additionally aerobic capacity, as assessed by citrate synthase activity (CS), and contractile function, as assessed by myofibrillar ATPase activity (mATPase) were measured in the pectoralis and mixed leg muscles during development. During the first 65% of their growth (from 2–12 g) bank swallows do not increase their metabolic rate in response to cold (Fig. 1). Between 12 and 16 g the PMR increased from 4 to more than 10 ml O₂ (g·h)^–1. Citrate synthase activity increased throughout development, starting at 20 moles (min·g fresh mass)^–1 in both tissues and increasing to 150 and 50 moles (min·g)^–1 in the pectoralis and leg muscles, respectively (Fig. 5). The augmented aerobic capacity combined with large increases in muscle mass undoubtedly contributes to the improved thermoregulatory abilities of older nestlings. However, muscle mass and aerobic capacity increase continuously and do not show the sharp transition noted in PMR. In the leg muscle mATPase activity is constant throughout growth, but in the pectoralis muscle it undergoes an abrupt increase from 0.5 moles (min·mg myofibrillar protein)^–1 in animals weighing less than 12 g to 0.9 moles (min·mg)^–1 in nestlings weighing more than 15 g (Fig. 6). The similar pattern of development of PMR and mATPase suggests a critical role for muscle development in the transition to endothermy in this species.Abbreviations CS citrate, synthase - mATPase myofibrillar adenosine triphosphatase - PMR peak metabolic rate during cold stress - rate of oxygen consumption     

Aerobic nitrate and nitrite reduction in continuous cultures of Escherichia coli E4

Nitrate and nitrite was reduced by Escherichia coli E4 in a l-lactate (5 mM) limited culture in a chemostat operated at dissolved oxygen concentrations corresponding to 90–100% air saturation. Nitrate reductase and nitrite reductase activity was regulated by the growth rate, and oxygen and nitrate concentrations. At a low growth rate (0.11 h^–1) nitrate and nitrite reductase activities of 200 nmol · mg^–1 protein · min^–1 and 250 nmol · mg^–1 protein · min^–1 were measured, respectively. At a high growth rate (0.55 h^–1) both enzyme activities were considerably lower (25 and 12 nmol mg^–1 · protein · min^–1). The steady state nitrite concentration in the chemostat was controlled by the combined action of the nitrate and nitrite reductase. Both nitrate and nitrite reductase activity were inversely proportional to the growth rate. The nitrite reductase activity decreased faster with growth rate than the nitrate reductase. The chemostat biomass concentration of E. coli E4, with ammonium either solely or combined with nitrate as a source of nitrogen, remained constant throughout all growth rates and was not affected by nitrite concentrations. Contrary to batch, E. coli E4 was able to grow in continuous cultures on nitrate as the sole source of nitrogen. When cultivated with nitrate as the sole source of nitrogen the chemostat biomass concentration is related to the activity of nitrate and nitrite reductase and hence, inversely proportional to growth rate.     

A comparison of proline, thiol levels and GAPDH activity in cyanobacteria of different origins facing temperature-stress

Three cyanobacterial strains originating from different habitats were subjected to temperature shift exposures and monitored for levels of proline, thiol and activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Thermophile Mastigocladus laminosus (growth optimum, 40 °C), raised the proline level 4.2-fold at low temperature (20 °C), for the psychrophile Nostoc 593 (growth optimum, 20 °C), it was raised 8-fold at 40 °C while in the mesophile Nostoc muscorum (growth optimum, 30 °C), the imino acid level increased 2.3-fold during temperature shiftdown to 20 °C or 3.5-fold in sets facing shiftup (40 °C). Alterations in thiol levels in the above strains were in line with proline. It is suggested that such fluctuations reflect metabolic shifts as a response to stress. Interestingly, GAPDH activity was maximum at the respective growth temperature optimum of M. laminosus (122 nmol NADPH oxidized min ^–1 mg ^–1 protein) and Nostoc 593 (141 nmol NADPH oxidized min ^–1 mg ^–1 protein) while in N. muscorum, it increased at 40 °C (101 nmol NADPH oxidized min ^–1 mg ^–1 protein) and to 93.3 nmol NADPH oxidized min ^–1 mg ^–1 protein (20 °C) relative to 86 nmol NADPH oxidized min ^–1 mg ^–1 protein at 30 °C. It seems that extremophiles maintain the GAPDH activity/level during growth at their respective temperatures optimal while the mesophile increases it in order to cope up with temperature-stress.     

Decolorization of synthetic dyes and production of manganese-dependent peroxidase by new fungal isolates

Two yeasts, Debaryomyces polymorphus, Candida tropicalis, and two filamentous fungi, Umbelopsis isabellina, Penicillium geastrivorus, could completely decolorize 100 mg Reactive Black 5 (RB 5) l^–1 within 16–48 h. Manganese-dependent peroxidase (MnP) activities between 60 and 424 U l^–1 were detected in culture supernatants of three of these organisms indicating the color removal by enzymatic biodegradation but with P. geastrivorus there was no ligninolytic enzyme activity in its culture and the decolorization was mainly due to biosorption to mycelium. Extensive decolorization by D. polymorphus (69–94%) and C. tropicalis (30–97%) was obtained with five other azo dyes and one anthraquinone dye. Except for Reactive Brilliant Blue KNR and Reactive Yellow M-3R, the four azo dyes, Reactive Red M-3BE, Procion Scharlach H-E3G, Procion Marine H-EXL and Reactive Brilliant Red K-2BP, induced D. polymorphus to produce MnP (105–587 U l^–1). However, MnP activities of 198–329 U l^–1 were only detected in the culture of C. tropicalis containing Reactive Red M-3BE and Reactive Brilliant Red K-2BP, respectively.     

Microbial introductions to apple leaves: Influences of altered immigration on fungal community dynamics

Fungal immigration to apple leaves in the field was altered by the introduction of populations ofChaetomium globosum orAureobasidium pullulans to surface-disinfested leaves either immediately following, or 6 days after, disinfestation. Total numbers of fungal individuals and numbers of filamentous fungal and yeast individuals were estimated and compared over time for 4–7 weeks on control leaves (leaves disinfested but no populations applied), onAureobasidium-treated, and onChaetomium-treated leaves. Fungal communities developing on leaves during three experiments in two different time frames (experiment 1: July 9–August 27; experiments 2 and 3: July 29–August 27), and thus under different immigration regimes, were also compared. Survival of introduced populations was not related to the presence of prior fungal immigrants. Rates of increase in total numbers of fungi and numbers of filamentous fungi and yeasts per leaf varied among experiments, apparently in relation to differences in immigration and environmental history. Differences among leaves in immigration had a short-term (days) influence on community size. However, no long-term effects of altered immigration on phylloplane fungal community size were evident.     

Stimulation of potato tuber respiration by cold stress is associated with an increased capacity of both plant uncoupling mitochondrial protein (PUMP) and alternative oxidase

The CO₂ evolution of intact potato tubers (Solanum tuberosum, L., var. Bintje) was analyzed during a 10-day period of their warm (25 ± 2°C) or cold (5 ± 1°C) storage, to evaluate cold-stress effects on expression and activities of plant uncoupling mitochondrial protein (PUMP) and alternative oxidase (AOX). CO₂ evolution rates were analyzed at 20°C, to reflect their possible capacities. The 20°C CO₂ production declined from 13 to 8 mg kg^–1 h^–1 after 2 days of warm storage and then (after 3 to 7 days) decreased from 8 to 6.5 mg kg^–1 h^–1. In contrast, 20°C CO₂ evolution did not change after the first day of cold storage, increased up to 14.5 mg kg^–1 h^–1 after 2 days, and decreased to about 12 mg kg^–1 h^–1 after 3 to 7 days of cold storage. Cold storage increased PUMP expression as detected by Western blots and led to elevated capacities of both PUMP (44%) and CN-resistant AOX (10 times), but not the cytochrome pathway. Since we found that cold storage led to about the same mitochondrial respiration of 40 nmol O₂ min^–1 mg^–1 attributable to each of the respective proteins, we conclude that both AOX and PUMP equally contribute to adaptation of potato tubers to cold.     

Release of sedimentary nitrogen and phosphorus in polder ditches of a low-moor peat area

The release of N and P from the sediment of two ditches, one (A) dominated by filamentous algae and the other (B) by water-lilies, was estimated by core and enclosure experiments. The release rates for ditch A tended to be higher than those for ditch B. Sediment cores covered by a filamentous algae layer released about 1.5 times more N and P than those from which the layer had been removed. During the incubation of the cores in the dark at 20°C for 2–3 weeks, about 10% of the N in the filamentous algae layer was mineralized. The mineralization could be described as a first-order reaction with a rate constant of about 0.2 d^–1. On average the cores of ditches A and B released about 40 mg mineral N and 3 mg.m^–2.d^–1 soluble reactive phosphorus. Defining the release from the sediment in the enclosures as the net increase of N and P in the water phase and in the vegetation minus the input, a negative net release,i.e. net accumulation of N and P in the sediment, was found over the summer half of the year. The negative values were due to the significant N and P input, resulting from pumping ditch water into the enclosures in order to compensate for downward seepage. From the enclosure experiments a downward seepage rate of 14 mm.d^–1 and an external load of about 6 g.m^–2 total N and 0.6 g.m^–2 total P during the summer half of the year —i.e. 33 mg.m^–2.d^–1 N and 3 mg.m^–2.d^–1 P. respectively — was calculated for the ditches. Tentative gross release rates — based on the sum of the positive net release of N and P into the water phase over 1–2 weeks intervals and the net increase of N and P in the vegetation — converted to 20°C and allowing for underestimation of the primary production by a factor of 5, amounted to 58 mg mineral N and 7 mg.m^–2.d^–1 soluble reactive phosphorus during the summer half of the year. Combining the rates estimated by cores and enclosures and converting them to rates at the mean water temperature during the summer half of the year, the release of mineral N and soluble reactive phosphorus roughly amounted to 40 and 4 mg.m^–2.d^–1, respectively. The release rates as well as the external load indicated a relatively low eutrophication of the ditches.