Survival of nonculturable Aeromonas salmonicida in lake water.

The survival of Aeromonas salmonicida subsp. salmonicida was investigated in sterile and untreated lake water. In sterile lake water (filtered and autoclaved), it was found that cells of A. salmonicida entered a nonculturable but viable condition. Viability was determined by flow cytometry with the dye rhodamine 123, which is taken up and maintained within cells with a membrane potential. For survival studies in untreated lake water, A. salmonicida was marked with the xylE gene by using the plasmid pLV1013. Marked cells were detected by growth on tryptone soy agar and tryptone soy agar supplemented with kanamycin. Cells were also detected by polymerase chain reaction DNA amplification of the xylE gene and a chromosomal DNA fragment specific for A. salmonicida (pLV1013). The results indicated that A. salmonicida entered a nonculturable condition in untreated lake water over a 21-day study. The viability of nonculturable cells could not be determined in mixed samples; however, the presence of nonculturable cells containing both chromosomal and plasmid DNA was confirmed.     

Validation of microbial community structure and ecological functional parameters in an aquatic microcosm designed for testing genetically engineered microorganisms

Microcosms were designed to facilitate studies of the fate, functioning, and ecological effects of microorganisms released into the aquatic environment. The microcosms were three-phase systems (sediment/water/air) with three compartments (a primary producer component, a herbivore grazer component, and intact sediment cores). The microcosms were validated by comparing gross ecological parameters and microbial community structure between the microcosms and the eutrophic Lake Bagsværd, which was simulated in the model. The photosynthetic potential and chlorophyll a concentrations were significantly lower in the microcosms than in the lake, which apparently was due to inorganic nutrient limitation. In the microcosms, total bacterial numbers and metabolic activity by [³H]thymidine incorporation were unaffected by the reduced algal biomass and primary production, simulating field conditions closely, with a strong dependence on temperature. Two days after filling the microcosms, the percentage of similarity of the microbial communities in the microcosm and Lake Bagsværd was 40%, measured by hybridizations of total microbial DNA. The similarity increased during the 10-day experimental period to 63–76%. In two experiments, Alcaligenes eutrophus AEO106(pRO101) was released into the microcosms. The release reduced the similarity between microcosms and lake to 2% and 27%, depending on the number of introduced cells. Concomitant to a decline in the A. eutrophus AEO106(pRO101) population, the similarity gradually recovered. It is concluded that the microcosms can simulate a freshwater lake ecosystem, but care has to be taken when extrapolating microcosm results to the source ecosystem because of the possible different selective conditions in the microcosm.     

Transport of a genetically engineered Pseudomonas fluorescens strain through a soil microcosm.

Vertical soil microcosms flushed with groundwater were used to study the influence of water movement on survival and transport of a genetically engineered Pseudomonas fluorescens C5t strain through a loamy sand and a loam soil. Transport of cells introduced into the top 1 cm of the vertical soil microcosms was dependent on the flow rate of water and the number of times microcosms were flushed with groundwater. The presence of wheat roots growing downward in the microcosms contributed only slightly to the movement of P. fluorescens C5t cells to lower soil regions of the loamy sand microcosms, but enhanced downward transport in the loam microcosms. Furthermore, the introduced P. fluorescens C5t cells were detected in the effluent water samples even after three flushes of groundwater and 10 days of incubation. As evidenced by a comparison of counts from immunofluorescence and selective plating, nonculturable C5t cells occurred in day 10 soil and percolated water samples, primarily of the loamy sand microcosms. Vertical soil microcosms that use water movement may be useful in studying the survival and transport of genetically engineered bacteria in soil under a variety of conditions prior to field testing.     

Transport of a genetically engineered Pseudomonas fluorescens strain through a soil microcosm

Vertical soil microcosms flushed with groundwater were used to study the influence of water movement on survival and transport of a genetically engineered Pseudomonas fluorescens C5t strain through a loamy sand and a loam soil. Transport of cells introduced into the top 1 cm of the vertical soil microcosms was dependent on the flow rate of water and the number of times microcosms were flushed with groundwater. The presence of wheat roots growing downward in the microcosms contributed only slightly to the movement of P. fluorescens C5t cells to lower soil regions of the loamy sand microcosms, but enhanced downward transport in the loam microcosms. Furthermore, the introduced P. fluorescens C5t cells were detected in the effluent water samples even after three flushes of groundwater and 10 days of incubation. As evidenced by a comparison of counts from immunofluorescence and selective plating, nonculturable C5t cells occurred in day 10 soil and percolated water samples, primarily of the loamy sand microcosms. Vertical soil microcosms that use water movement may be useful in studying the survival and transport of genetically engineered bacteria in soil under a variety of conditions prior to field testing.     

Survival of Aeromonas salmonicida in lake water

The survival of Aeromonas salmonicida subsp. salmonicida in lake water was investigated by using a variety of techniques. They included acridine orange epifluorescence, respiration, cell culture, cell revival, flow cytometry, plasmid maintenance, and membrane fatty acid analysis. During a 21-day study, A. salmonicida became nonculturable in sterile lake water samples. Flow cytometry and direct microscopy indicated that cells were present. Although the nonculturable cells could not be revived, the recovery method did indicate that the presence of low numbers of culturable cells within samples could produce misleading results. Plasmid DNA, genomic DNA, and RNA were maintained in the nonculturable cells; in addition, changes in the fatty acid profiles were also detected. Although viability could not be proven, it was shown that the morphological integrity of nonculturable cells was maintained.     

Survival of Aeromonas salmonicida in lake water.

The survival of Aeromonas salmonicida subsp. salmonicida in lake water was investigated by using a variety of techniques. They included acridine orange epifluorescence, respiration, cell culture, cell revival, flow cytometry, plasmid maintenance, and membrane fatty acid analysis. During a 21-day study, A. salmonicida became nonculturable in sterile lake water samples. Flow cytometry and direct microscopy indicated that cells were present. Although the nonculturable cells could not be revived, the recovery method did indicate that the presence of low numbers of culturable cells within samples could produce misleading results. Plasmid DNA, genomic DNA, and RNA were maintained in the nonculturable cells; in addition, changes in the fatty acid profiles were also detected. Although viability could not be proven, it was shown that the morphological integrity of nonculturable cells was maintained.     

Influence of ecosystematic factors on survival of Escherichia coli after large-scale release into lake water mesocosms.

Mass cultures of an Escherichia coli K-12 strain were released into exposed mesocosms in a eutrophic lake. The release was performed with and without additional input of the E. coli culture medium to stimulate the scenario of leakage of a production fermenter on one hand and to compare the influence of the added organic nutrients with that of the added strain on the other hand. The survival of the introduced strain and the influence on ecological processes in the mesocosms were monitored for 10 weeks after release. For comparison, survival of the strain in microcosms with sterile lake water was also monitored. Survival of the strain was determined by means of immunofluorescence and growth on selective agar medium. In lake mesocosms, E. coli showed a rapid and constant dieback during the first week. After 4 days, cells were mostly restricted to particles, which seemed to provide niches for survival. From the second week onward, survival was improved in mesocosms with culture medium added. In microcosms with sterile lake water, plate counts of E. coli showed a strong decrease within 2 weeks, while total cell numbers remained approximately the same. The rapid elimination of E. coli from the free-water phase of the mesocosms was probably due to the combined effect of the inability to grow in lake water and grazing. The better survival of E. coli (mainly on particles) in mesocosms with added medium was attributed to the medium-induced enhancement of primary production, which was the source of a large quantity of particles. These particles, in turn, may have functioned as niches for prolonged survival as well as transport vehicles for sedimentation of the E. coli cells.     

Influence of ecosystematic factors on survival of Escherichia coli after large-scale release into lake water mesocosms.

Mass cultures of an Escherichia coli K-12 strain were released into exposed mesocosms in a eutrophic lake. The release was performed with and without additional input of the E. coli culture medium to stimulate the scenario of leakage of a production fermenter on one hand and to compare the influence of the added organic nutrients with that of the added strain on the other hand. The survival of the introduced strain and the influence on ecological processes in the mesocosms were monitored for 10 weeks after release. For comparison, survival of the strain in microcosms with sterile lake water was also monitored. Survival of the strain was determined by means of immunofluorescence and growth on selective agar medium. In lake mesocosms, E. coli showed a rapid and constant dieback during the first week. After 4 days, cells were mostly restricted to particles, which seemed to provide niches for survival. From the second week onward, survival was improved in mesocosms with culture medium added. In microcosms with sterile lake water, plate counts of E. coli showed a strong decrease within 2 weeks, while total cell numbers remained approximately the same. The rapid elimination of E. coli from the free-water phase of the mesocosms was probably due to the combined effect of the inability to grow in lake water and grazing. The better survival of E. coli (mainly on particles) in mesocosms with added medium was attributed to the medium-induced enhancement of primary production, which was the source of a large quantity of particles. These particles, in turn, may have functioned as niches for prolonged survival as well as transport vehicles for sedimentation of the E. coli cells.     

Survival of Vibrio anguillarum and Vibrio salmonicida at different salinities

The fish pathogenic bacteria Vibrio anguillarum and V. salmonicida showed the capacity to survive for more than 50 and 14 months, respectively, in seawater microcosms. A salinity of 5% proved lethal to V. anguillarum harvested in the late-exponential growth phase, whereas a salinity of 9% was lethal to the bacterium after it had been starved at a salinity of 30% for 67 days. The lethal salinity for V. salmonicida harvested in the late-exponential growth phase was probably in the vicinity of 10%. V. anguillarum and V. salmonicida were very sensitive to nalidixic acid. Direct determination of viable cells after incubation with nalidixic acid was not possible, since the cells did not elongate. Samples of V. salmonicida were double stained with fluorescein isothiocyanate-labeled antibodies and 4',6-diamidino-2-phenylindole. After 3 or 4 days of starvation, there was a discrepancy between the total numbers of cells as determined by immunofluorescence versus by staining with 4',6-diamidino-2-phenylindole. The immunofluorescence counts remained high, which indicated the presence of intact cell envelopes but leakage of DNA and other cytoplasm components. After 2 weeks of starvation, for some of the cells, the region stained with 4',6-diamidino-2-phenylindole (i.e., DNA) was markedly smaller than the cell envelope. I attributed this to a shrinkage of the cytoplasm or a confined nucleoid or both. V. anguillarum lost its exoproteolytic activity before 11 days of starvation.     

Fate of Pseudomonas putida after release into lake water mesocosms: Different survival mechanisms in response to environmental conditions

To study the fate of Pseudomonas putida DSM 3931 in an aquatic environment, cultures of the strain were released into lake water mesocosms. P. putida, bearing the TOL-plasmid, was released as a representative xenobiotic-degrading microorganism. The release was carried out in mesocosms with unamended lake water and in lake water with added culture medium to compare the survival of the strain due to the influence of different organic load. As a comparison, the survival of P. putida was followed in microcosms with sterile lake water. Survival and fate of the strain were determined by means of immunofluorescence with highly specific monoclonal antibodies and growth on selective agar medium for up to ten weeks after release. Addition of medium had a pronounced influence on survival in mesocosms. In mesocosms without added medium, the number of P. putida cells decreased within ten days by over 2 orders of magnitude. In mesocosms with medium, cell numbers increased in the first two days by an order of magnitude and were, after ten days, in the same range as at the time of introduction. Over time, cell numbers decreased but remained detectable in both types of mesocosms for up to ten weeks after release. In mesocosms with unamended lake water, the major fraction of the cells was attached to particles after two days. In mesocosms with medium, large aggregates of P. putida cells formed which included algae. The observed decrease in cell numbers in mesocosms was attributed mainly to grazing. Sedimentation was an additional factor contributing to loss of cells out of the water column, which especially affected aggregate-forming cells in mesocosms with medium in the long run (beyond two weeks). These studies demonstrate that experimental tools on a mesoscale are crucial in order to understand the complex processes microorganisms are subjected to after release into a natural environment, and that single cell detection, such as immunofluorescence, is essential to understand mechanisms of survival and elimination.Correspondence to: M.G. Höfle     

Survival of Vibrio anguillarum and Vibrio salmonicida at different salinities.

The fish pathogenic bacteria Vibrio anguillarum and V. salmonicida showed the capacity to survive for more than 50 and 14 months, respectively, in seawater microcosms. A salinity of 5% proved lethal to V. anguillarum harvested in the late-exponential growth phase, whereas a salinity of 9% was lethal to the bacterium after it had been starved at a salinity of 30% for 67 days. The lethal salinity for V. salmonicida harvested in the late-exponential growth phase was probably in the vicinity of 10%. V. anguillarum and V. salmonicida were very sensitive to nalidixic acid. Direct determination of viable cells after incubation with nalidixic acid was not possible, since the cells did not elongate. Samples of V. salmonicida were double stained with fluorescein isothiocyanate-labeled antibodies and 4',6-diamidino-2-phenylindole. After 3 or 4 days of starvation, there was a discrepancy between the total numbers of cells as determined by immunofluorescence versus by staining with 4',6-diamidino-2-phenylindole. The immunofluorescence counts remained high, which indicated the presence of intact cell envelopes but leakage of DNA and other cytoplasm components. After 2 weeks of starvation, for some of the cells, the region stained with 4',6-diamidino-2-phenylindole (i.e., DNA) was markedly smaller than the cell envelope. I attributed this to a shrinkage of the cytoplasm or a confined nucleoid or both. V. anguillarum lost its exoproteolytic activity before 11 days of starvation.     

Evaluation of aquatic sediment microcosms and their use in assessing possible effects of introduced microorganisms on ecosystem parameters.

In this paper we describe a sediment microcosm system consisting of 20 undisturbed, layered sediment cores with overlying site water which are incubated under identical conditions of temperature, light, stirring rate of overlying water, and water exchange rate. Ecosystem parameters (nutrient level, photosynthetic potential, community structure of heterotrophic bacteria, thymidine incorporation rate, and oxygen microgradients) of the laboratory microcosms and the source ecosystem were compared and shown to be indistinguishable for the first 2 weeks. In weeks 3 and 4, small differences were detectable in the nutrient level, community structure of heterotrophic bacteria, and thymidine incorporation rate. However, the photosynthetic potential, depth profiles of heterotrophic bacterial community structure, and oxygen microgradients were maintained throughout the incubation period and did not differ between laboratory microcosms and the source ecosystem. The microcosm system described here would thus appear to be a valid model of aquatic sediments for up to 4 weeks; the actual period would depend on the sediment source and incubation temperature. The validated systems were used with Rhine river sediment to assess possible effects on ecosystem parameters of Pseudomonas sp. strain B13 FR1(pFRC20P), a genetically engineered microorganism (GEM) that had been constructed to degrade mixtures of halo- and alkylbenzoates and -phenols. The GEM survived in the surface sediment at densities of 5 x 10(4) to 5 x 10(5)/g (dry weight) for 4 weeks and degraded added chloro- and methylaromatics. The GEM did not measurably influence ecosystem parameters such as photosynthesis, densities of selected heterotrophic bacteria, thymidine incorporation rate, and oxygen microgradients. Thus, the microcosm system described here would seem to be useful for the study of the ecology of biodegradation and the fate and effect of microorganisms introduced into the environment.     

Evaluation of aquatic sediment microcosms and their use in assessing possible effects of introduced microorganisms on ecosystem parameters.

In this paper we describe a sediment microcosm system consisting of 20 undisturbed, layered sediment cores with overlying site water which are incubated under identical conditions of temperature, light, stirring rate of overlying water, and water exchange rate. Ecosystem parameters (nutrient level, photosynthetic potential, community structure of heterotrophic bacteria, thymidine incorporation rate, and oxygen microgradients) of the laboratory microcosms and the source ecosystem were compared and shown to be indistinguishable for the first 2 weeks. In weeks 3 and 4, small differences were detectable in the nutrient level, community structure of heterotrophic bacteria, and thymidine incorporation rate. However, the photosynthetic potential, depth profiles of heterotrophic bacterial community structure, and oxygen microgradients were maintained throughout the incubation period and did not differ between laboratory microcosms and the source ecosystem. The microcosm system described here would thus appear to be a valid model of aquatic sediments for up to 4 weeks; the actual period would depend on the sediment source and incubation temperature. The validated systems were used with Rhine river sediment to assess possible effects on ecosystem parameters of Pseudomonas sp. strain B13 FR1(pFRC20P), a genetically engineered microorganism (GEM) that had been constructed to degrade mixtures of halo- and alkylbenzoates and -phenols. The GEM survived in the surface sediment at densities of 5 x 10(4) to 5 x 10(5)/g (dry weight) for 4 weeks and degraded added chloro- and methylaromatics. The GEM did not measurably influence ecosystem parameters such as photosynthesis, densities of selected heterotrophic bacteria, thymidine incorporation rate, and oxygen microgradients. Thus, the microcosm system described here would seem to be useful for the study of the ecology of biodegradation and the fate and effect of microorganisms introduced into the environment.     

Biotransformations of Aroclor 1242 in Hudson River test tube microcosms.

A microcosm system to physically model the fate of Aroclor 1242 in Hudson River sediment was developed. In the dark at 22 to 25 degrees C with no amendments (nutrients, organisms, or mixing) and with overlying water being the only source of oxygen, the microcosms developed visibly distinct aerobic and anaerobic compartments in 2 to 4 weeks. Extensive polychlorinated biphenyl (PCB) biodegradation was observed in 140 days. Autoclaved controls were unchanged throughout the experiments. In the surface sediments of these microcosms, the PCBs were biologically altered by both aerobic biodegrading and reductive dechlorinating microorganisms, decreasing the total concentration from 64.8 to 18.0 micromol/kg of sediment in 1140 days. This is the first laboratory demonstration of meta dechlorination plus aerobic biodegradation in stationary sediments. In contrast, the primary mechanism of microbiological attack on PCBs in aerobic subsurface sediments was reductive dechlorination. The concentration of PCBs remained constant at 64.8 micromol/kg of sediment, but the average number of chlorines per biphenyl decreased from 3.11 to 1.84 in 140 days. The selectivities of microorganisms in these sediments were characterized by meta and para dechlorination. Our results provide persuasive evidence that naturally occurring microorganisms in the Hudson River have the potential to attack the PCBs from Aroclor 1242 releases both aerobically and anaerobically at rapid rates. These unamended microcosms represent a unique method for determining the fate of released PCBs in river sediments.     

Survival of and wheat-root colonization by alginate encapsulated Herbaspirillum spp

The survival ofHerbaspirillum spp. cells added directly or encapsulated in alginate beads and colonization of wheat roots was evaluated in soil microcosms. Cells entrapped in alginate in the presence of JNFb-broth and introduced into unplanted non-sterile clay loamy and sandy soils survived better than cells added directly to the same soils after 50 d incubation. On amendment by JNFb broth and/or skim milk the entrapped cells survived better than those prepared in water. Encapsulated cells survived better in a heavier textured soil (clay-loamy) than in a lighter (sandy) soil. Wheat plants growing in microcosms inoculated with various bead types from day 0 to day 30 exhibited high levels of histosphere colonization, nitrogenase activity (in situ) measured by acetylene reduction assay, plant dry mass and total N content but no symptoms of mottled stripe disease were observed. Comparable results of growth criteria and nitrogenase activity, but relatively lower bacterial populations, were obtained with wheat grown for 45 d after the inoculant had been introduced into the soil with different bead types.     

Luminometry and PCR-based monitoring of gene-tagged cyanobacteria in Baltic Sea microcosms

Abstract The cyanobacterium Synechocystis 6803 was tagged by chromosomal integration of the firefly luciferase gene, lue , resulting in the modified strain Synechocystis 6803- luc . The tagged cells were monitored in Baltic Sea microcosms both by detection of the luc gene by PCR amplification and by measurement of luc gene expression (bioluminescence) in total protein extracted from sediment and water. A new method was developed for isolation and concentration of total protein from sediment for optimization of luciferase quantitation. The detection limit for Synechocystis with a chromosomal luc insertion by bioluminescence was in the order of 4 × 10³ cells per g sediment, a considerable improvement in sensitivity over previous methods. Another improvement was to use an internal luciferase standard to correct for quenching of light output by impurities in the samples. Baltic sea microcosms were inoculated with Synechocystis 6803- luc , and the luc DNA and luciferase protein specific to the tagged cells were monitored over time. A decrease in luminescence in the microcosm water was observed, simultaneously with an increase in luminescence in the sediment, suggesting settling of the luc -tagged cells in the sediment layer.     

Survival and activity of Pseudomonas sp. strain B13(FR1) in a marine microcosm determined by quantitative PCR and an rRNA-targeting probe and its effect on the indigenous bacterioplankton.

Genetically engineered Pseudomonas sp. strain B13(FR1) was released into laboratory-scale marine ecosystem models (microcosms). Survival of the introduced population in the water column and the sediment was determined by plating on a selective medium and by quantitative competitive PCR. The activity of the released bacteria was determined by in situ hybridization of single cells with a specific rRNA-targeting oligonucleotide probe. Two microcosms were inoculated with 10(6) cells ml-1, while an uninoculated microcosm served as a control. The number of Pseudomonas sp. strain B13(FR1) cells decreased rapidly to ca. 10(2) cells ml-1 within 2 days after the release, which is indicative of grazing by protozoa. Three days after the introduction into seawater, cells were unculturable, but PCR continued to detect cells in low numbers. Immediately after the release, the ribosomal content of Pseudomonas sp. strain B13(FR1) corresponded to a generation time of 2 h. The growth rate decreased to less than 0.04 h-1 in 5 days and remained low, probably because of carbon limitation of the cells. Specific amendment of the microcosms with 10 mM 4-chlorobenzoate resulted in a rapid increase of the growth rate and an exponentially increasing number of cells detected by PCR, but not in resuscitation of the cells to a culturable state. The release of Pseudomonas sp. strain B13(FR1) into the microcosms seemed to affect only the indigenous bacterioplankton community transiently. Effects on the community were also apparent from the handling of water during filling of the microcosms and the amendment with 4-chlorobenzoate.     

Methane formation and release in a small Wisconsin lake

Abstract

Tharae rate of methane released from the sediment‐water interface and from the surface of the water of Lake Wingra, Madison, Wisconsin, was measured during the summer months for 2 years. The amount of methane escaping the lake is estimated to be an important factor in the carbon budget of the lake. Most rapid methanogenesis was in shallow water (less than 1 m deep) and in the uppermost 5 cm of sediment. The numbers of methanogenic bacteria were estimated by a most probable number technique to vary from approximately 10² to 3 × 10⁴ methanogens per gram of dry weight sediment during winter and summer, respectively.     

Phosphorus budgetary analysis of sediment–water interface in a short-term anoxic condition in shallow Lake Kasumigaura,Japan

To investigate the effective depth from the surface sediment, and phosphorus fractions related to phosphorus release under short-term anoxic conditions, varying lengths of sediment cores taken from Lake Kasumigaura, a large shallow polymictic lake in Japan, were incubated for a few weeks and then analyzed. Results showed few differences in total phosphorus (TP) amount per unit area in overlying waters irrespective of the core thickness, and sums of TP in both overlying water and 0- to 2-cm sediment layers were nearly equal before and after the experiment, indicating that phosphorus was released mainly from the 0- to 2-cm layer by dissolution. In contrast, phosphorus was decreased in pore water below a 2-cm depth, probably through sorption to sediment solids. The citrate-dithionite-bicarbonate total phosphorus (CBD-TP) and non-reactive phosphorus extracted by NaOH (NaOH-NRP) in sediment solids in the 0- to 2-cm layer decreased during the experiment. The decreases of CBD-TP were 10 times higher than those of NaOH-NRP, suggesting that the released phosphorus came mainly from the fraction bound to iron in Lake Kasumigaura.     

The impact of Chironomus plumosus larvae on organic matter decay and nutrient (N, P) exchange in a shallow eutrophic lake sediment following a phytoplankton sedimentation

The importance of Chironomus plumosus larvae onbenthic metabolism and nutrient exchange across thesediment–water interface was evaluated in a shalloweutrophic lake (Lake Arreskov, Denmark) following aphytoplankton sedimentation. Chironomus plumosuslarvae were added to laboratory sediment microcosms,corresponding to a density of 2825 larvae m⁻².Non-inhabited microcosms served as controls. Asedimentation pulse of organic matter was simulated byadding fresh algal material (Chlamydomonasreinhardii) to sediment cores (36 g dryweight m⁻²). The mineralization was followed bymeasuring fluxes of O₂, CO₂, dissolvedinorganic nitrogen and phosphate. A rapid clearance ofalgae from the water column in faunated microcosmssuggested that chironomids may be of major importancein controlling phytoplankton concentrations in shalloweutrophic lakes. Chironomids increased the sedimentO₂ uptake ≈ 3 times more than what wouldbe expected from their own respiration, indicating astimulation of microbial activity and decomposition oforganic matter in the sediment. Addition of algaeenhanced the release of CO₂, NH⁺ ₄ ando-P. The excess inorganic C, N and P released inamended non-inhabited sediment after 36 dayscorresponded to 65, 31 and 58% of the C, N and P inthe added algae. In sediment inhabited by Chironomus plumosus the corresponding numbers were147, 45 and 73%, indicating that mineralization oforganic matter also from the indigenous sediment poolwas stimulated by chironomids. This revised version was published online in July 2006 with corrections to the Cover Date.