Epilithic Biofilms Research Articles

Effect of nutrients on the sporulation and diversity of aquatic hyphomycetes on submerged substrata in a Mediterranean stream

Eutrophication in streams often results in enhanced decomposition rates and chemical conditions that greatly affect the suitability of the habitat for many species. We performed an enrichment experiment in a 50-m reach of a Mediterranean stream by the continuous addition of N and P (enriched reach) while another consecutive reach upstream served as the reference (unenriched reach). After 1.5 years of enrichment, conidia production and species composition of aquatic hyphomycetes were measured in communities colonizing four streambed substrata ( Platanus acerifolia and Populus nigra leaves, epipsammic and epilithic biofilms). Samples collected from the two reaches were subjected to two nutrient treatments in the laboratory [low nutrient (−NP) and high nutrient (+NP) concentrations] to test for short-term (48 h) nutrient effects. Long-term nutrient additions did not affect potential sporulation rates while short-term nutrient enrichment produced insignificant increase in these rates on most substrata collected in the unenriched reach. Major differences in potential sporulation rates were observed between substrata, the highest rate being for leaves (especially Platanus acerifolia). However, enrichment produced significant changes in fungal community composition. Clavariopsis aquatica, Alatospora acuminata and Lemonniera sp. were dominant in the enriched reach while Heliscella stellata, Trisceloporus acuminatus and Clavatospora longibrachiata were characteristic of the unenriched reach. Our results suggest that fungal N demands can be fulfilled at relatively low levels of dissolved nitrate and further increases in the nutrients availability may not result in enhanced fungal activity.

Detailing biogeochemical N budgets in riverine epilithic biofilms

Dissolved inorganic N fluxes, including nitrification and denitrification rates, were measured in situ under dark and light conditions using epilithic biofilms from a 6th to 8th Strahler-order reach of the River Garonne, France. A broad range of epilithic biofilm biomasses (6–51 g ash-free dry mass [AFDM]/m2) was assessed in 39 benthic chamber experiments. Nitrification was detected in 31 cases (0.1–7.7 mg N m−2 h−1) and caused a net release of NO3– in spite of algal assimilation in light conditions in 5 cases. Denitrification (Dw) (0–8.1 mg N m−2 h−1), which accounted for 83% of gross NO3– removal in the dark, was a significant, permanent N sink. Calculation of mean (±1 SE) N budgets indicated epilithon release of inorganic N in the dark (1.5 ± 0.3 mg N m−2 h−1) that was significantly related to biofilm mass. Calculation of mean N budgets under light indicated net uptake (1.8 ± 0.4 mg N m−2 h−1). However, a net release was observed when biomass was >40 g AFDM/m2. A calculated daily budget showed evidence for a threshold between photoautotrophy (N assimilation) and heterotrophy (N mineralization) of 23 g AFDM/m2. This threshold approximated mean biomass at low water and may correspond to an equilibrium value for epilithic biomass in the river. A conceptual scheme of the epilithic biofilm function and the consequences on the water-column N content in rivers is proposed.

Localization of EPS components secreted by freshwater diatoms using differential staining with fluorophore-conjugated lectins and other fluorochromes

Diatoms produce extracellular polymeric substances (EPS), which mainly consist of carbohydrates and may form different morphological structures. We studied the localization and structure of EPS secreted by 17 diatom species that were isolated from epilithic biofilms from the littoral zone of Lake Constance (Germany). We used six different lectins labelled with FITC (fluorescein isothiocynate) and DAPI (diamidino phenylindole) to localize and visualize the structure of secreted EPS and/or cell-wall-associated EPS, while DTAF (dichlorotriazinyl aminofluorescein) was used to label cell-wall-associated EPS only. The diatoms were categorized according to the structure of the secreted EPS i.e. pads, capsules, tubes or stalks. Among eight pad-producing diatoms, three Fragilaria species showed variable lectin binding indicating the presence of different carbohydrate components. Other pad-forming diatoms like Synedra, Diatoma, Asterionella, and Melosira generally showed binding to at least two different lectins. On the other hand, we did not observe any lectin binding to the capsules of Staurosira and one Achnanthes isolate. We further detected differences in the carbohydrate composition of tube-like EPS structures in two Cymbella species and were able to demonstrate developmental stages of tube formation. The stalk secreting species Gomphonema and Cymbella showed labelling by only one lectin, while EPS of Caloneis alpestris were stained by all lectins and fluorochromes used indicating a highly complex composition.

Nitrogen dynamics in grassland streams along a gradient of agricultural development

We examined nitrogen (N) uptake in seven grassland streams lying in catchments along a gradient (0–84%) of agricultural land use in New Zealand using a stable isotope (15N) addition of NH4+ to quantify whole‐stream uptake rates and uptake by specific compartments within the streams. Whole‐stream uptake of NH4+ ranged from 1.2 mmol N m−2 min−1 to 7.9 mmol N m−2 min−1 and showed no evidence of strong saturation of uptake with increasing NH4+ availability along the land use gradient. The relatively simple pattern of increasing N uptake along the gradient occurred despite substantial shifts in uptake among community members. Uptake of NH4+ by epilithic biofilms followed a subsidy‐stress relationship, with highest uptake rates at intermediate levels of pastoral development and lowest uptake rates at high pastoral development. At high (>60%) pastoral development, reduced uptake by epilithon was compensated for by enhanced uptake by macrophytes. Compensatory uptake can play an important role in streams when community composition is reorganized by human activity.

Production of biogenic manganese oxides by repeated-batch cultures of laboratory microcosms

We investigated the production of manganese (Mn) oxides using repeated-batch bioreactors maintained over long periods under laboratory conditions. Freshwater epilithic biofilms were used as the initial inocula. The bioreactors yielded suspended solids that could remove 0.1 mM dissolved Mn(II) within a few days. Chemical titration, X-ray absorption near-edge structure spectroscopy, and X-ray diffraction analysis revealed that the Mn(II) had been converted to poorly crystallized layer-type Mn(IV) oxides, which were similar to known biogenic Mn oxides from pure bacterial cultures. Spherical or rod-shaped Mn microconcretions occurred in the suspended solids; transmission electron microscopy showed that these structures likely resulted from the microbial activity but not represent living cells. Instead, the presence of encapsulated, sheathed, and hyphal budding cells in the suspended solids indicated that a range of Mn-depositing bacteria contributed to the Mn oxide formation. To our knowledge, our data represent the first observation of production of such Mn oxides in a laboratory microcosm wherein a range of Mn-depositing bacteria coexist. The fact that sorption of trace Zn(II) and Ni(II) ions onto the suspended solids co-occurred with the removal of dissolved Mn(II) emphasizes the important role of Mn-oxidizing microorganisms in the fates of trace or contaminant metals in the aquatic environment.

EFFECTS OF THE DISCHARGE INCREASE ON ATTACHED ALGAE IN THE OHYAMA RIVER (UPPER CHIKUGO RIVER)

The maintenance flow discharge of the Ohyama River (upper Chikugo River) had been restricted to 1.5m3/s by a hydroelectric dam. To improve the river environment, the river administrator has changed it to 4.5m3/s in summer and to 1.8m3/s in winter. In this study, we aim at investigating the effects of discharge increase on epilithic biofilms composed of algae. We conducted sampling of attached algae every two weeks from March to July, 2006, at an impact reach increasing the disharge, at a control reach maintained a constant discharge, and at two reference reaches in upstream of the hydroelectric dam. Results of the field surveys show that chlorophyll-a at the impact reach is higher than one at the control reach. However, in case of controling flood disturbance by a dam, there is no difference in chlorophyll-a between the impact reach and the control reach, because attached algae is regulated by heterotrophic process.

Landscape variation in C sources of scraping primary consumers in streams

AbstractWe studied variation in C and energy flow in stream food webs by examining primary consumer diets and potential food sources at 8 sites of different drainage areas in the South Fork Eel River drainage. Both heptageniid mayfly nymphs and Glossosoma caddisfly larvae are considered scrapers in traditional functional feeding group classification, but past studies suggested that they differed in their relative use of terrestrial and algal C in some streams. In our study, microscopic examination and stable C isotope ratios (δ13C) suggested an increasing contribution of algae to both epilithic biofilms and fine particulate organic matter as stream drainage area and productivity increased. The proportion of algal cells in biofilms of small, unproductive streams was low, and biofilm δ13C values were similar to those of terrestrial detritus, suggesting that biofilms were composed primarily of heterotrophic microorganisms. Glossosoma larvae fed selectively on algae where it was scarce within the biofilms of ...

Virulence traits associated with verocytotoxigenic Escherichia coli O157 recovered from freshwater biofilms

To investigate whether epilithic biofilms in freshwater streams in a mixed UK agricultural river catchment harbour Escherichia coli O157, and if so, whether they demonstrate an association with those excreted by grazing farm animals. Flint shingle, native to the study site, was used as a surface for biofilm development within cages of metal lath set into a stream bed at four locations on a chalkland farm. Shingle was collected from all sites once a month, as were pooled faecal samples from five farm animal populations. Subpopulations of E. coli, including E. coli O157 that demonstrated significant phenotypic and genotypic similarity with animal faecal isolates (t-test, P = 0.05) were isolated. Of 1002 E. coli isolates from biofilms and animal faeces, 48 were confirmed as the O157 strain by latex agglutination. The presence of five virulence traits associated with incidence of human disease was tested using PCR. Stx(2) was the most frequently isolated single gene (30 isolates), while stx(1) was the least frequently recovered (four isolates). Escherichia coli O157, expressing up to four virulence factors associated with human disease, reside within freshwater biofilms in this agricultural environment. Aquatic biofilms may potentially act as a reservoir for these pathogens, and the implications of the findings for the protection of drinking water resources should be further investigated.

Genetic Characterization of Epilithic Cyanobacteria and Their Associated Bacteria

Epilithic phototrophic biofilms develop inside Roman Necropolis and Catacombs on rock surfaces exposed to artificial light sources and are composed by a microbial consortium dominated by cyanobacteria. In this work, six non-axenic cultures of Leptolyngbya sp. strains isolated from biofilms from different Roman hypogea and maintained in cultures from 11 to 20 years were analysed along with their associated bacteria isolated in culture. The employment of PCR-fingerprinting techniques, using HIP1 and ERIC derived primers, allowed the clustering in three groups of the six Leptolyngbya strains and the typing of their isolated bacteria. The bacterial fingerprinting patterns were in agreement with the 16S rRNA gene sequencing and showed the presence in Leptolyngbya isolates of Pseudomonas, Stenotrophomonas, Agrobacterium and Bacillus representatives that were detected also in biofilms sampled from catacombs.

Response of nutrients, biofilm, and benthic insects to salmon carcass addition

Salmon carcass addition to streams is expected to increase stream productivity at multiple trophic levels. This study examined stream nutrient (nitrogen, phosphorus, and carbon), epilithic biofilm (ash-free dry mass and chlorophyll a), leaf-litter decomposition, and macroinvertebrate (density and biomass) responses to carcass addition in three headwater streams of southwestern Washington State, USA. We used stable isotopes (δ13C and δ15N) to trace incorporation of salmon-derived (SD) nutrients into stream food webs. SD nutrients were assimilated by biofilm, benthic insects (Perlidae and Limnephilidae spp.), and age-1 steelhead (Oncorhynchus mykiss gairdneri). SD nutrients peaked ~2 weeks after carcass addition for insects and fish feeding on carcasses, but indirect uptake of SD nutrients by biofilm and insects was delayed by ~2 months. A strong stable isotope signal did not always correspond with measurable biological change. At reaches 10–50 m downstream from carcasses, ammonium concentration, leaf-litter decomposition, and benthic insect density all increased relative to upstream control sites. The strongest responses and greatest SD-nutrient uptake were observed 10 m from decomposing carcasses, with effects generally decreasing to undetectable levels 250 m downstream. Carcass addition to headwater streams can have a transient effect on primary and secondary trophic levels, but responses may be limited to specific taxa near carcass locations.

Benthic Algae in High Altitude Streams of the Alps – a Neglected Component of the Aquatic Biota

This is a review on benthic algae from streams situated above the tree line in the Alps. It integrates published and unpublished data from alpine streams in Austria and in the Trento Province (Northern Italy). The main focus is on the structural and taxonomic composition of benthic algae including macro- and micro-algae and their contribution to the epilithic biofilm and the stream food-web. The environmental conditions relevant to algae within the two major stream types, the glacial (glacier-fed) and non-glacial krenal (spring-fed) stream are discussed. The paper considers both the maximum possible structural complexity of transverse algae zonation in cascading alpine/subalpine stream segments, and the effects of glacial water on species richness in the Central Alps in Austria. Autecological data are given for 46 common diatoms from 42 sites in the Central Eastern and Southern Central Alps and for 30 algae in addition to diatoms for 22 streams in the Central Eastern Alps. Since data on responses of benthic algae to the harsh conditions in high altitude Alpine streams are very scarce, results from literature and the author’s experiences from these and other mountain stream types are synthesized to formulate major objectives for future research in benthic high altitude Alpine stream ecology.

Assessing the importance of a self‐generated detachment process in river biofilm models

Summary1. Epilithic biofilm biomass was measured for 14 months in two sites, located up‐ and downstream of the city of Toulouse in the Garonne River (south‐west France). Periodical sampling provided a biomass data set to compare with simulations from the model of Uehlinger, Bürher and Reichert (1996: Freshwater Biology, 36, 249–263.), in order to evaluate the impact of hydraulic disturbance.2. Despite differences in application conditions (e.g. river size, discharge, frequency of disturbance), the base equation satisfactorily predicted biomass between low and high water periods of the year, suggesting that the flood disturbance regime may be considered a universal mechanism controlling periphyton biomass.3. However modelling gave no agreement with biomass dynamics during the 7‐month long low water period that the river experienced. The influence of other biomass‐regulating factors (temperature, light and soluble reactive phosphorus) on temporal biomass dynamics was weak.4. Implementing a supplementary mechanism corresponding to a temperature‐dependent self‐generated loss because of heterotrophic processes allowed us to accurately reproduce the observed pattern: a succession of two peaks. This case study suggests that during typical summer low water periods (flow stability and favourable temperature) river biofilm modelling requires self‐generated detachment to be considered.

Impact of Salmon Spawning on Microbial Communities in a Northern California River

ABSTRACT We studied microbial communities in the water column during the fall 2002 Chinook salmon (Oncorhynchus tshawytscha) run on the Feather River in northern California. We quantified culturable heterotrophic bacteria, live/dead bacteria, and Aeromonas spp. bacteria in the water column. Concentrations of each group peaked two weeks following the peak of new salmon carcasses in the river then declined. During the fall 2003 Chinook salmon spawning season we used fluorescent in situ hybridization with domain-specific DNA probes to characterize changes in epilithic biofilm communities. Eukarya counts were lower than Bacteria counts earlier in the salmon run; however, by the end of the salmon migration season, cell concentrations of Bacteria and Eukarya were approximately equal. Chlorophyll a concentrations from epilithic biofilm began to increase in parallel with the increase in Eukarya. Since the biofilms persisted through the spawning season, it is possible that these communities support a food chain that leads to insect populations which become nutrients for the newly hatched salmon fry in the winter and spring.

Mercury Methylation in the Epilithon of Boreal Shield Aquatic Ecosystems

Methylation rates by periphyton growing on the rocky shore of a remote boreal shield lake were measured over diurnal cycles at temperatures representative of summer and fall conditions. The measurements were carried out in vitro with natural communities grown on artificial Teflon substrates submerged along the lake's shore for 1-2 years. At temperatures above 20 degrees C, epilithon Hg methylation rates were fast and reached a steady state within 12 h upon exposure to 2 ng L(-1) of inorganic mercury. A variety of inhibitors were used to identify which microorganisms in the epilithic biofilm are responsible for the methylation. The addition of molybdate, which is believed to suppress the activity of sulfate-reducing bacteria, decreased methylmercury production rates by 60% in both light and dark experiments. The prokaryote inhibitor chloramphenicol reduced the methylation rate by 40% only during dark periods whereas an algal inhibitor (DCMU), which suppresses photosynthesis, decreased the methylation rate by 60% during light periods. Results of this study reveal that epilithon communities may be a significant source of MeHg to higher aquatic organisms in lakes and that the integrity of the epilithic biofilm is important for its ability to methylate Hg.

Limitations on the effects of ultraviolet radiation on benthic algae in a clear boreal forest lake

We examined the effects of ultraviolet radiation (UVR) (280–400 nm) on epilithic algal metabolism, biomass, and taxonomy from early June to mid August 1999 (66 d) in Lake 224, an oligotrophic lake in the Experimental Lakes Area, northwestern Ontario, Canada. Epilithon colonized bare clay tiles placed under UVR-transparent (UVR+) and UVR-shielding (UVR−) acrylic filters, at depths of 0.5, 0.8, and 1.4 m. Filamentous chlorophytes and cyanophytes were the dominant algal taxa at 0.5 and 1.4 m depths, respectively, on day 28. Biovolumes did not differ between UVR treatments for any major algal class. We measured epilithic metabolism on days 28, 45, and 66 after the tiles were deployed. Photosynthesis was not affected by UVR on any date. On day 28, dark respiration was 33 to 49% greater in UVR+ than in UVR− treatments at all depths. On day 45, respiration was 11% greater in UVR+ than in UVR− treatments only at 0.5 m, and by day 66, respiration did not differ between treatments at any depth. Thus, the effects of UVR on respiration diminished with depth and over time and were strongest when epilithic biofilms were least developed. UVR probably has little impact on naturally colonized, fully developed epilithon deeper than 0.5 m in Lake 224, i.e., UVR affects <6% of the littoral zone. Comparison of our findings with those of an earlier study in Lake 224 suggests that the intensity and specific nature of UVR effects depend on the taxonomic composition of the benthic algal assemblage.

New strategies for the monitoring and control of cyanobacterial films on valuable lithic faces

A survey of studies on the structure and functioning of sub-aerial epilithic phototrophic biofilms, which develop inside hypogea sites, was conducted in the framework of projects whose aim is to investigate cyanobacteria and associated microorganisms causing deterioration of stone artefacts. Using different methodological approaches, it was possible to identify and characterize cyanobacterial taxa, and to deduce their relationships with other microorganisms and the mineral substrata. Part of studies also aimed at identifying biotransformation and biomineralization mechanisms affecting stone, and the biofilm response to various light conditions. The calcium distribution and the structure of the cyanobacterial sheath gave an indication on the most deteriogenic species. Monochromatic light experiments allowed us to develop non-destructive methodological approaches for the monitoring and control/prevention of cyanobacterial biofilm growth on stone.

Bacterial Community Succession in Natural River Biofilm Assemblages

Temporal bacterial community changes in river biofilms were studied using 16S rRNA gene-based polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) followed by sequence analysis. Naturally occurring biofilms were sampled in 2001 during an undisturbed 7-month low-water period in the River Garonne (SW France). During the sampling period epilithic biomass exhibited a particular pattern: two 3-month periods of accumulation that resulted in two peaks in summer and fall, each at about 25 g ash-free dry mass per square meter. Bacterial community DGGE profiles differed between the summer and fall biomass peaks and shared only 30% common operational taxonomic units (OTUs), suggesting the influence of seasonal factors on these communities. During the second biomass accrual phase, bacterial richness and the appearance of new OTUs fitted a conceptual model of bacterial biofilm succession. During succession, five OTUs (corresponding to Dechloromonas sp., Nitrospira sp., and three different Spirosoma spp.) exhibited particular patterns and were present only during clearly defined successional stages, suggesting differences in life-history strategies for epilithic bacteria. Co-inertia analysis of DGGE banding patterns and physical-chemical data showed a significant relationship between community structure and environmental conditions suggesting that bacterial communities were mainly influenced by seasonal changes (temperature, light) and hydrodynamic stability. Within the periods of stability, analysis of environmental variables and community patterns showed the dominant influence of time and maturation on bacterial community structure. Thus, succession in these naturally occurring epilithic biofilm assemblages appears to occur through a combination of allogenic (seasonal) and autogenic changes.

Spatial patchiness of epilithic biofilm caused by refuge-inhabiting high shore gastropods

Aggregations of Melarhaphe neritoides and Littorina saxatilis are common on the high shore in north-east England. These aggregations are frequently found in crevices which act as a refuge from desiccation. The aggregations are surrounded by patches of epilithic biofilm which are visibly lighter in colour than the biofilm present on the remainder of the high shore. Chlorophyll a levels were lower in regions of close proximity to aggregated littorinids (mean = 4.9 μg cm−2) as compared to areas >‰5 cm away from the edge of the visible colour change (mean = 15.9 μg cm−2). Manipulations of littorinid density showed that areas of high density had significantly lower levels of chlorophyll a than those where littorinids were excluded. This difference was mainly due to the presence of higher numbers of small grazed areas or an increase in size of grazed areas rather than a homogenous change in chlorophyll a levels across the entire manipulated area. These results are supported by observations showing that only 7% of the total foraging time of littorinids was spent outside visibly lighter patches of biofilm suggesting littorinids only control local biofilm levels through grazing.

Molecular diversity and biomass of epilithic biofilms from intertidal rocky shores in the Gulf of Mexico

Epilithic biofilms play key roles in rocky shore ecosystems. The diversity and biomass of epilithic biofilms were determined along a subtropical intertidal rocky shore at Xpicob, Southern Gulf of Mexico (Campeche, Mexico). Biofilm diversity was assessed by polymerase chain reaction (PCR) amplification of partial 16 S rRNA genes using single-strand conformation polymorphism (SSCP). Triplicate 2 cm×2 cm biofilm samples per site were taken randomly from 0.6 m×0.6 m quadrats located in two sampling sites, established 20 m apart within a homogeneous calcareous intertidal platform. Twenty-two partial rRNA sequences, belonging to four bacterial divisions (Cyanobacteria, Bacteroidetes, Actinobacteria and Proteobacteria), were recovered from these biofilms; of these, cyanobacteria were the most abundant (41%). The occurrence of cyanobacterial sequences in most samples, along with the detection of high levels of chlorophyll a and phycobiliprotein, indicates that these organisms are dominant within the biofilms. Consistent with previous reports, thick-sheathed cyanobacteria such as those found in this study (Xenococcus, Myxosarcina and Chroococcidiopsis) are typical in habitats in intertidal zones. In addition, most of the detected organisms from other bacterial lineages had closest relatives displaying biofilm phenotypes that suggested stressful conditions (i.e. desiccation) prevailed in the intertidal shores; this was selecting for biofilm-forming or thick-sheathed organisms as an ecological adaptation to withstand the conditions. Unweighted pair-group method with arithmetic mean (UPGMA) analysis of SSCP profiles indicated that there was significant spatial heterogeneity in biofilm community composition across different scales of analysis and that this heterogeneity was related to the distance at which samples were taken. This analysis showed that certain organisms appeared to be station specific and that the biofilm community structure varied even on a small scale (1 cm). Since no significant differences in bulk sea water or substratum physicochemistry were observed, these results suggest that small-scale variability of environmental conditions, surface properties and/or biological interactions (i.e. allelopathy) may be important factors structuring these biofilm communities.

Structuring of epilithic biofilms by the caddisfly Tinodes rostocki: photosynthetic activity and photopigment distribution in and beside larval retreats

Tinodes rostocki larvae (Trichoptera: Psychomyiidae) cover large proportions of stream hard substrata with retreats constructed of mineral particles and larval silk. We consider these retreats as 3-dimensional extensions of the epilithic biofilm that may possess a distinct microenviron- ment, community metabolism and composition. Therefore, we compared the photosynthetic/respira- tory activities (O2 and pH microsensors) and the photopigment composition (HPLC) of larval retreats and the surrounding epilithic biofilms. In retreats, pigment contents and photosynthetic/respiratory activities were highest in sections with a visible microphytobenthic biofilm that were mostly the older parts of the retreats. In contrast, newly constructed sections of the retreats and the surrounding epilithic biofilm had approximately 5-fold lower values. The fucoxanthin-to-chlorophyll ratio of the retreat biofilm was high (fuco/chl a = 1.27) and indicated diatom dominance, which was not evident in the surrounding epilithic biofilm (fuco/chl a = 0.15). Experimental transplantation of larval retreats to microscope slides allowed microsensor measurements through the 500 to 700 µm-thick wall and inside the lumen. In the light, O2 concentration and pH values increased significantly across the wall and remained high in the lumen of the retreat, whereas in darkness O2 and pH depressions in both wall and lumen were moderate or even absent. Our data suggest that T. rostocki larvae construct and maintain retreats with a particular physico-chemical microenvironment that favours a distinct micro- bial community. Thereby, abundant T. rostocki larvae might significantly influence benthic primary production and heterotrophic metabolism in small streams.