Aerobic stepwise hydrocarbon degradation and formation of biosurfactants by an original soil population in a stirred reactor

Summary Using a model system containing 10% soil and a 1.35% hydrocarbon mixture of tetradecane, pentadecane, hexadecene, pristane (2,6,10,14-tetramethylpentadecane), trimethylcyclohexane, phenyldecane and naphthalene suspended in a mineral salts medium, the hydrocarbon degradation rate by a soil population was 25.7 g model oil per kg soil dry weight per day under non-limited conditions within two degradation phases. During the first degradation phase only the most water-soluble naphthalene was degraded, while the other components could only be metabolized when the interfacial tension was lowered by the production of surfactants at the beginning of the second degradation phase. This second degradation phase ended when 89% of the hydrocarbons were metabolized.Dedicated to Professor F. Wagner on the occasion of his 60th birthday     

Distribution of Hydrocarbon-Utilizing Microorganisms and Hydrocarbon Biodegradation Potentials in Alaskan Continental Shelf Areas

Hydrocarbon-utilizing microorganisms were enumerated from Alaskan continental shelf areas by using plate counts and a new most-probable-number procedure based on mineralization of ¹⁴C-labeled hydrocarbons. Hydrocarbon utilizers were ubiquitously distributed, with no significant overall concentration differences between sampling regions or between surface water and sediment samples. There were, however, significant seasonal differences in numbers of hydrocarbon utilizers. Distribution of hydrocarbon utilizers within Cook Inlet was positively correlated with occurrence of hydrocarbons in the environment. Hydrocarbon biodegradation potentials were measured by using ¹⁴C-radiolabeled hydrocarbon-spiked crude oil. There was no significant correlation between numbers of hydrocarbon utilizers and hydrocarbon biodegradation potentials. The biodegradation potentials showed large seasonal variations in the Beaufort Sea, probably due to seasonal depletion of available nutrients. Non-nutrient-limited biodegradation potentials followed the order hexadecane > naphthalene pristane > benzanthracene. In Cook Inlet, biodegradation potentials for hexadecane and naphthalene were dependent on availability of inorganic nutrients. Biodegradation potentials for pristane and benzanthracene were restricted, probably by resistance to attack by available enzymes in the indigenous population.     

Biosurfactant-enhanced degradation of residual hydrocarbons from ship bilge wastes

The use of Bacillus subtilis O9 biosurfactant (surfactin) and of bioaugmentation to improve the treatment of residual hydrocarbons from ship bilge wastes was studied. A biodegradation experiment was conducted in aquaria placed outdoors under non-aseptic conditions. Three treatments were examined: culture medium plus bilge wastes, bioaugmentation with microorganisms from bilge wastes, and bioaugmentation plus biosurfactant. Samples were analyzed for viable counts, aliphatic and aromatic hydrocarbon concentrations, n-C17/pristane and n-C18/phytane ratios. While the addition of biosurfactant stimulated hydrocarbon degradation, bioaugmentation did not produce any remarkable effect. At day 10, the remaining percentages of aliphatic and aromatic hydrocarbons in aquaria, which received biosurfactant, were 6.8 and 7.2, respectively, while it took 20 days to reach comparable results with the other treatments. The biosurfactant did not affect the preferential biodegradation of n-C17/pristane and n-C18/phytane. This biosurfactant, which can be produced in a relatively simple and inexpensive process, is a promising alternative in the optimization of hydrocarbon waste treatment. Journal of Industrial Microbiology & Biotechnology (2000) 25, 70–73. Received 26 January 2000/ Accepted in revised form 09 June 2000     

Changes in iso- and n-alkane distribution during biodegradation of crude oil under nitrate and sulphate reducing conditions

Crude oil consists of a large number of hydrocarbons with different susceptibility to microbial degradation. The influence of hydrocarbon structure and molecular weight on hydrocarbon biodegradation under anaerobic conditions is not fully explored. In this study oxygen, nitrate and sulphate served as terminal electron acceptors (TEAs) for the microbial degradation of a paraffin-rich crude oil in a freshly contaminated soil. During 185 days of incubation, alkanes from n-C11 to n-C39, three n- to iso-alkane ratios commonly used as weathering indicators and the unresolved complex mixture (UCM) were quantified and statistically analyzed. The use of different TEAs for hydrocarbon degradation resulted in dissimilar degradative patterns for n- and iso-alkanes. While n-alkane biodegradation followed well-established patterns under aerobic conditions, lower molecular weight alkanes were found to be more recalcitrant than mid- to high-molecular weight alkanes under nitrate-reducing conditions. Biodegradation with sulphate as the TEA was most pronounced for long-chain (n-C32 to n-C39) alkanes. The observation of increasing ratios of n-C17 to pristane and of n-C18 to phytane provides first evidence of the preferential degradation of branched over normal alkanes under sulphate reducing conditions. The formation of distinctly different n- and iso-alkane biodegradation fingerprints under different electron accepting conditions may be used to assess the occurrence of specific degradation processes at a contaminated site. The use of n- to iso-alkane ratios for this purpose may require adjustment if applied for anaerobic sites.     

Effect of addition ofPseudomonas aeruginosa UG2 inocula or biosurfactants on biodegradation of selected hydrocarbons in soil

Summary A laboratory study was undertaken to assess the effect of adding eitherPseudomonas aeruginosa UG2 cells or the biosurfactants produced by this m microorganism on the biodegradation of a hydrocarbon mixture in soil at 20°C over a 2-month incubation period. The addition of 100 g of UG2 biosurfactants per g soil significantly enhanced the degradation of tetradecane, hexadecene and pristane but not 2-methylnaphthalene, the most water-soluble of the hydrocarbons. Addition of UG2 cells at densities of 10⁶, 10⁷, and 10⁸ per g soil did not have a significant effect on biodegradation of the hydrocarbon mixture.     

Treatment of Jet Fuel-Contaminated Runoff Water by Subsurface Infiltration

Subsurface infiltration of jet fuel-contaminated surface runoff from airport activities has been shown to be a feasible treatment method. Complete removal of monoaromatic hydrocarbons and naphthalene was observed in 1-m-long (0.2-m-diameter) soil columns. Mineral soil mixed with 5% peat (MinP) showed the best hydrocarbon removal at variable hydraulic loads (50 to 375 mm/d). Natural mineral soil from the C-horizon (Min) showed good hydrocarbon removal after an initial phase, during which hydrocarbons were found in the leachate. This is believed to be a result of adaptation of the soil microflora to the applied hydrocarbons. When hydrocarbon application was stopped and resumed, a new adaptation phase was observed. Cleaned soil (335 mg/kg total petroleum hydrocarbons [TPH]) from a former diesel-polluted site (MinO) showed good hydrocarbon removal but poor hydraulic conductivity. Removal rates of 4.7 and 3.0 mg/kg/d total hydrocarbons were found for Min and MinP, respectively, over the total column length. The highest removal rates (20 and 18 mg/kg/d total hydrocarbons) are observed in the top 20 cm for Min and MinP. Respectively, Potassium acetate (KAc), used as a deicing chemical on airport runways, caused breakthrough of hydrocarbons in the soil columns. The observed breakthrough was caused by oxygen depletion in MinP and MinO. No oxygen depletion was observed in Min, but substrate competition might have limited the hydrocarbon degradation. Based on the results from this study, a full-scale experimental infiltration plant is being constructed and will be tested during 1997. The full-scale tests will focus on the observed relation between oxygen availability and hydrocarbon removal.     

Anaerobic degradation of polycyclic aromatic hydrocarbons and alkanes in petroleum-contaminated marine harbor sediments.

Although polycyclic aromatic hydrocarbons (PAHs) have usually been found to persist under strict anaerobic conditions, in a previous study an unusual site was found in San Diego Bay in which two PAHs, naphthalene and phenanthrene, were oxidized to carbon dioxide under sulfate-reducing conditions. Further investigations with these sediments revealed that methylnaphthalene, fluorene, and fluoranthene were also anaerobically oxidized to carbon dioxide in these sediments, while pyrene and benzo[a]pyrene were not. Studies with naphthalene indicated that PAH oxidation was sulfate dependent. Incubating the sediments with additional naphthalene for 1 month resulted in a significant increase in the oxidation of [14C]naphthalene. In sediments from a less heavily contaminated site in San diego Bay where PAHs were not readily degraded, naphthalene degradation could be stimulated through inoculation with active PAH-degrading sediments from the most heavily contaminated site. Sediments from the less heavily contaminated site that had been adapted for rapid anaerobic degradation of high concentrations of benzene did not oxidize naphthalene, suggesting that the benzene- and naphthalene-degrading populations were different. When fuels containing complex mixtures of alkanes were added to sediments from the two sites, there was significant degradation in the alkanes. [14C]hexadecane was also anaerobically oxidized to 14CO2 in these sediments. Molybdate, a specific inhibitor of sulfate reduction, inhibited hexadecane oxidation. These results demonstrate that a wide variety of hydrocarbon contaminants can be degraded under sulfate-reducing conditions in hydrocarbon-contaminated sediments, and they suggest that it may be possible to use sulfate reduction rather than aerobic respiration as a treatment strategy for hydrocarbon-contaminated dredged sediments.     

Isolation of Eikenella corrodens in a General Hospital

The carbon source markedly influenced the qualitative and quantitative composition of cellular hydrocarbons in Cladosporium resinae. Total lipid and hydrocarbon content was greater in cells grown on n-alkanes than in cells grown on glucose or glutamic acid. Glucose-grown cells contained a spectrum of aliphatic hydrocarbons from C(7) to C(36); pristane and n-hexadecane comprised 98% of the total. Cells grown on glutamic acid contained C(7) to C(23) hydrocarbons; n-tridecane, n-tetradecane, n-hexadecane, and pristane made up 74% of the total. n-Decane-grown cells yielded C(8) to C(32) compounds, and n-hexadecane (96%) was the major hydrocarbon. Cells grown on individual n-alkanes from C(11) to C(15) all contained C(11) to C(28) hydrocarbons, and cells grown on n-hexadecane contained C(11) to C(32) hydrocarbons. In n-undecane-grown cells, n-hexadecane and pristane made up 92% of the total, but in cells grown on C(12) to C(16)n-alkanes the major cellular hydrocarbon was the one on which the cells were grown. This suggests that cells cultured on n-alkanes of C(12) or longer accumulate n-alkanes prior to oxidizing them.     

Microbial Utilization of Naturally Occurring Hydrocarbons at the Guaymas Basin Hydrothermal Vent Site

The Guaymas Basin (Gulf of California; depth, 2,000 m) is a site of hydrothermal activity in which petroliferous material is formed by thermal alteration of deposited planktonic and terrestrial organic matter. We investigated certain components of these naturally occurring hydrocarbons as potential carbon sources for a specific microflora at these deep-sea vent sites. Respiratory conversion of [1-¹⁴C]hexadecane and [1(4,5,8)-¹⁴C]naphthalene to ¹⁴CO₂ was observed at 4°C and 25°C, and some was observed at 55°C, but none was observed at 80°C. Bacterial isolates were capable of growing on both substrates as the sole carbon source. All isolates were aerobic and mesophilic with respect to growth on hydrocarbons but also grew at low temperatures (4 to 5°C). These results correlate well with previous geochemical analyses, indicating microbial hydrocarbon degradation, and show that at least some of the thermally produced hydrocarbons at Guaymas Basin are significant carbon sources to vent microbiota.     

Effect of the addition of microbial surfactants on hydrocarbon degradation in a soil population in a stirred reactor

Summary The hydrocarbon degradation rate could be doubled by the addition of sophorose lipids as biosurfactants in a model system containing 10% soil and a 1.35% hydrocarbon mixture of tetradecane, pentadecane, hexadecene, 1,2,4-trimethylcyclohexane, pristane (2,6,10,14-tetramethylpentadecane) phenyldecane and naphthalene suspended in mineral salts medium. The adaptation phases for two degradation phases were shortened, and the extent of degradation and final biomass were increased. The added biosurfactants were degraded after they had facilitated degradation of all hydrocarbon components.     

Adding handles to unhandy substrates: anaerobic hydrocarbon activation mechanisms

In spite of their chemical inertness, hydrocarbons are degraded by microorganisms in the complete absence of oxygen. As all known aerobic hydrocarbon degradation pathways start with oxygen-dependent reactions, hydrocarbon catabolism in anaerobes must be initiated by novel biochemical reactions. In recent years, the enzymes catalyzing oxygen-independent activation of several hydrocarbons have been identified. Surprisingly, a variety of reactions seems to be employed to overcome the activation barrier of different hydrocarbons. This review presents the current understanding on some of these reactions and the associated degradation pathways: oxygen-independent hydroxylation as employed in ethylbenzene metabolism, fumarate addition to methyl or methylene carbons in toluene or alkane degradation, and only recently discovered reactions such as methylation of naphthalene or anaerobic methane oxidation via reverse methanogenesis.     

Enhanced Biodegradation of Anthracene by Bacillus Cereus Strain JMG-01 Isolated from Hydrocarbon Contaminated Soils

The present study displays the biodegradation capacities of native bacteria toward polycyclic aromatic hydrocarbons with particular emphasis to anthracene. A total of 23 bacterial strains were isolated from hydrocarbon-contaminated sites of Guwahati city, using anthracene as the carbon source. Among all these isolates, one Gram-positive strain (JMG-01) was selected as an efficient anthracene degrader, based on its maximum growth ability in anthracene enriched medium (100 ppm–700 ppm). At 500 ppm concentration, strain JMG-01 showed the maximum growth rate with 98% of anthracene degradation within 21 days of observation. The strain also demonstrated its potentiality by utilizing naphthalene and higher molecular hydrocarbons like pyrene, and benzo(a)pyrene at 500 ppm. The morphological, biochemical and molecular characterization identified the strain as Bacillus cereus. Surface morphology of the biomass, captured by Atomic Force Microscope, showed a distinctive modification, during the process of degradation. Study revealed that the effect of hydrocarbon exhibited the alteration, which concurrently enhanced the metabolic activity. Further, Gas chromatography-mass spectrometer analysis elucidates the possible metabolic pathway of anthracene degradation, depending on the intermediate metabolites produced. The finding thus suggests the essence of Bacillus cereus strain JMG-01 in enhanced anthracene degradation along the utilization of other hydrocarbons.     

Emergence of Chironomidae (Diptera) from a delta-swamp receiving thermal effluent

Chironomid pupal exuviae were collected over an eight-week period from a delta-swamp which receives thermal effluent from a nuclear reactor on the Savannah River Plant, South Carolina, USA. Two sites were sampled using box-type emergence traps. Site 1 was directly in a major thermal plume channel, and site 2 was outside the plume in a stand of stressed bald cypress trees. Sixteen chironomid taxa were collected, of these, species of Chironomus (42%) and Tanytarsus (35%) dominated the warmer site (site 1, maximum temperature 46 °C), whereas Tanypus neopunctipennis comprised > 84 % at site 2 (maximum temperature 43 °C). Emergence substantially increased at both sites after the reactor was shut down and water temperatures returned to ambient (27–28 °C). Tanytarsus sp. 1 and T. neopunctipennis were capable of successfully emerging during water temperature periods of 40–46 °C. The deep organic sediment, characteristic of the delta-swamp apparently served as a refugium for these and other species of midges during high temperature periods. It is suggested that the ability of some taxa to tolerate these elevated temperatures may be a combination of several factors: behavioral and ecological adaptations to utilize available refugia; and physiological adaptations to withstand higher temperatures and low dissolved oxygen concentrations.     

Phenanthrene Biodegradation in Freshwater Environments

Phenanthrene, a low-molecular-weight polycyclic aromatic hydrocarbon, was incubated with water samples from various reservoir systems in Tennessee to evaluate the potential for significant polycyclic aromatic hydrocarbon degradation by the indigenous microbial populations. Biodegradation was assessed by comparison of total polycyclic aromatic hydrocarbon substrate recovery in degradation flasks relative to sterile control flasks. During 1977 field studies, the mean phenanthrene biodegradation was approximately 80% after a 4-week incubation. Within a given habitat, 45% of the total variability in phenanthrene biodegradation was attributable to the physical, chemical, and microbiological site characteristics examined. Polycyclic aromatic hydrocarbon degradation was directly related to the historical environmental pollution of the sampling sites examined, the length of biodegradation assessment, temperature, and the molecular size of the polycyclic aromatic hydrocarbon substrate.     

Metabolic and phylogenetic analysis of microbial communities during phytoremediation of soil contaminated with weathered hydrocarbons and heavy metals

In the current study, the microbial ecology of weathered hydrocarbon and heavy metal contaminated soil undergoing phytoremediation was studied. The relationship of functional diversity, measured as carbon source utilisation in Biolog plates and extracellular enzymatic activities, and genetic diversity of bacteria was evaluated. Denaturing gradient gel electrophoresis was used for community analyses at the species level. Bulk soil and rhizosphere soil from pine and poplar plantations were analysed separately to determine if the plant rhizosphere impacted hydrocarbon degradation. Prevailing microbial communities in the field site were both genetically and metabolically diverse. Furthermore, both tree rhizosphere and fertilisation affected the compositions of these communities and increased activities of extracellular aminopeptidases. In addition, the abundance of alkane hydroxylase and naphthalene dioxygenase genes in the communities was low, but the prevalence of these genes was increased by the addition of bioavailable hydrocarbons. Tree rhizosphere communities had greater hydrocarbon degradation potential than those of bulk soil. Hydrocarbon utilising communities were dominated generally by the species Ralstonia eutropha and bacteria belonging to the genus Burkholderia. Despite the presence of viable hydrocarbon-degrading microbiota, decomposition of hydrocarbons from weathered hydrocarbon contaminated soil over four years, regardless of the presence of vegetation, was low in unfertilised soil. Compost addition enhanced the removal of hydrocarbons.     

Degradation of crude oil by an arctic microbial consortium

The ability of a psychrotolerant microbial consortium to degrade crude oil at low temperatures was investigated. The enriched arctic microbial community was also tested for its ability to utilize various hydrocarbons, such as long-chain alkanes (n-C₂₄ to n-C₃₄), pristane, (methyl-)naphthalenes, and xylenes, as sole carbon and energy sources. Except for o-xylene and methylnaphthalenes, all tested compounds were metabolized under conditions that are typical for contaminated marine liquid sites, namely at pH 6–9 and at 4–27°C. By applying molecular biological techniques (16S rDNA sequencing, DGGE) nine strains could be identified in the consortium. Five of these strains could be isolated in pure cultures. The involved strains were closely related to the following genera: Pseudoalteromonas (two species), Pseudomonas (two species), Shewanella (two species), Marinobacter (one species), Psychrobacter (one species), and Agreia (one species). Interestingly, the five isolated strains in different combinations were unable to degrade crude oil or its components significantly, indicating the importance of the four unculturable microorganisms in the degradation of single or of complex mixtures of hydrocarbons. The obtained mixed culture showed obvious advantages including stability of the consortium, wide range adaptability for crude oil degradation, and strong degradation ability of crude oil.     

Microbial degradation of model petroleum at low temperatures

Two areas of Chesapeake Bay, Colgate Creek in Baltimore Harbor and Eastern Bay, are presently under study, with routine sampling of water and sediment for petroleum-degrading microorganisms (bacteria, yeasts, and fungi) by direct plating and enrichment culture. Selected physical and chemical parameters are recorded for each sampling site, and water and sediment samples are extracted for hydrocarbons. Numbers of petroleum-degrading microorganisms enumerated by direct plating were found to correlate with the concentration of benzene-extractable material and were higher for the Colgate Creek than for the Eastern Bay site. Petroleum-degrading microorganisms were isolated from water and sediment samples at environmental temperatures of 0°, 5°, and 10°C. A salts medium supplemented with nitrate and phosphate was used to provide optimum conditions for petroleum degradation, whereas Chesapeake Bay water was used to simulate natural environmental conditions. Use of a model petroleum permitted quantitative measurement of utilization of individual hydrocarbons ranging in complexity from simple alkanes to polynuclear aromatic hydrocarbons. Higher growth yields and maximum hydrocarbon degradation was observed for microorganisms in the salts medium at 0°, 5°, and 10°C, although significant quantities of hydrocarbons were utilized in some samples grown in a medium for which Chesapeake Bay water was the diluent. Bacterial hydrocarbon degradation accounted for most of the model petroleum utilization at 0° and 5°C. However, oscillations of bacterial populations, with significant growth of yeasts, was observed at 10°C. Photomicroscopy and scanning electron microscopy revealed aggregates of bacteria, yeasts, and fungi associated with oil globules. From preliminary identification and classification of the hydrocarbon-utilizing bacteria, members of the generaVibrio, Aeromonas, Pseudomonas, andAcinetobacter were present in the enrichment cultures. From results of this study, it is concluded that utilization of model petroleum at low temperatures is a function of the types and numbers of microorganisms present in an original inoculum taken from the natural environment.     

Methane in maritime Antarctic freshwater lakes

Summary Methane was found to occur in all freshwater lakes, irrespective of trophic status, sampled during this preliminary investigation at Signy Island, South Orkney Islands, Antarctica. Methane accumulated in the water column of these lakes during the winter period when ice cover prevented wind-induced mixing. Maritime Antarctic lakes are usually subject to wind-induced complete mixing during the summer open-water period but two major exceptions to the rule were found during this study. Methanogenesis occurred in both littoral and profundal regions of oligotrophic Sombre Lake. The presence of a substantial algal mat stabilized the Eh status of underlying sediments at the littoral site. Methane production was confined to the sediments in both littoral and profundal sediments during the study period (December–March) but in winter probably migrated to the sediment surface at the profundal site. All Signy Island lakes sampled were sulphate-poor and addition of sulphate markedly inhibited methanogenesis. Radio-isotope studies indicated that the H₂/CO₂ pathway was probably the predominant route for methanogenesis in these sediments through the acetate pathway appeared equally important at the sediment surface. In the absence of sulphate, sulphate reducers probably acted as net hydrogen donors to the methanogens. The process rate was permanently limited by the consistent low temperature (annual range 1–3°C). Rates increased with increasing temperature over the range 4–32°C, but no evidence was found to suggest cold sensitivity or psychrophily. The optimum temperature for methanogenesis was in excess of 30°C, temperatures never experienced at Signy Island. Rates of methanogenesis during the study period (Dec–Mar) ranged from 0.29 to 0.45 mg of carbon m^-2 and on an annual basis methanogenesis was calculated equivalent to 13% of the organic carbon deposition rate.     

Stimulating the anaerobic degradation of aromatic hydrocarbons in contaminated sediments by providing an electrode as the electron acceptor

The possibility that electrodes might serve as an electron acceptor to simulate the degradation of aromatic hydrocarbons in anaerobic contaminated sediments was investigated. Initial studies with Geobacter metallireducens demonstrated that although toluene was rapidly adsorbed onto the graphite electrodes it was rapidly oxidized to carbon dioxide with the electrode serving as the sole electron acceptor. Providing graphite electrodes as an electron acceptor in hydrocarbon‐contaminated sediments significantly stimulated the removal of added toluene and benzene. Rates of toluene and benzene removal accelerated with continued additions of toluene and benzene. [¹⁴C]‐Toluene and [¹⁴C]‐benzene were quantitatively recovered as [¹⁴C]‐CO₂, demonstrating that even though the graphite adsorbed toluene and benzene they were degraded. Introducing an electrode as an electron acceptor also accelerated the loss of added naphthalene and [¹⁴C]‐naphthalene was converted to [¹⁴C]‐CO₂. The results suggest that graphite electrodes can serve as an electron acceptor for the degradation of aromatic hydrocarbon contaminants in sediments, co‐localizing the contaminants, the degradative organisms and the electron acceptor. Once in position, they provide a permanent, low‐maintenance source of electron acceptor. Thus, graphite electrodes may offer an attractive alternative for enhancing contaminant degradation in anoxic environments.     

Mechanisms regulating abundance of submerged vegetation in shallow eutrophic lakes

Shallow eutrophic lakes tend to be either in a turbid state dominated by phytoplankton or in a clear-water state dominated by submerged macrovegetation. Recent studies suggest that the low water turbidity in the clear-water state is maintained through direct and indirect effects of the submerged vegetation. This study examined what mechanisms may cause a recession of the submerged vegetation in the clear-water state, and thereby a switch to the turbid state. The spatial distribution of submerged vegetation biomass was investigated in two shallow eutrophic lakes in the clear-water state in southern Sweden. Biomass of submerged vegetation was positively correlated with water depth and wave exposure, which also were mutually correlated, suggesting that mechanisms hampering submerged vegetation were strongest at shallow and/or sheltered locations. The growth of Myriophyllum spicatum, planted in the same substrate and at the same water depth, was compared between sheltered and wave exposed sites in two lakes. After 6 weeks the plants were significantly smaller at the sheltered sites, where periphyton production was about 5 times higher than at the exposed sites. Exclosure experiments were conducted to evaluate the effects of waterfowl grazing on macrophyte biomass. Potamogeton pectinatus growth was decreased by grazing, whereas M. spicatum was not affected. The effects were greater at a sheltered than at a wave-exposed site, and also negatively related to distance from the reed belt. These results suggest that competition from epiphytes and waterfowl grazing hamper the development of submerged vegetation at sheltered and/or shallow locations. An increased strength of these mechanisms may cause a recession of submerged vegetation in shallow eutrophic lakes in the clear-water state and thereby a switch to the turbid state. Received: 24 June 1996 / Accepted: 8 September 1996