Epilithic Biofilms Research Articles

Deciphering biodiversity and interactions between bacteria and microeukaryotes within epilithic biofilms from the Loue River, France

Epilithic river biofilms are complex matrix-enclosed communities harboring a great diversity of prokaryotic and eukaryotic microorganisms. Interactions between these communities and the relative impacts of environmental factors on their compositions are poorly understood. In this study, we assessed the spatio-temporal variation in the diversity and composition of bacterial and microeukaryotic communities within biofilms in a French river. Significant changes were found in the composition of these microbial communities over the sampling period and between the upstream and downstream stations. In addition, the beta diversity of the bacterial community tended to decrease along the river, mostly as a result of turnover. These changes could be caused by the different water temperatures and geological and hydrological river contexts at the sampling sites (from karst landscape to river plain). Finally, our network analysis showed multiple correlations among dominant OTUs. Among them, negative correlations between Rhodobacteraceae and two other dominant groups of photosynthetic microorganisms (cyanobacteria and diatoms) were particularly interesting, which raises the question of what environmental factors trigger the changes occurring in benthic microbial photosynthetic communities.

Bacterial weathering of fossil organic matter and organic carbon mobilization from subterrestrial Kupferschiefer black shale: long-term laboratory studies.

A large part of the organic carbon present in the lithosphere is trapped in fossil organic matter deposited in sedimentary rocks. Only specialized microorganisms are able to degrade it contributing to the return of the carbon to the global cycle. The role of bacteria in this process is not yet completely understood. In the present laboratory studies, subterrestrial organic-rich ∼256-million-year-old Kupferschiefer black shale was exposed to the activity of an indigenous consortium of lithobiontic bacteria for 365 days under aerobic conditions. An interdisciplinary research approach was applied, consisting of a detailed comparison of the chemical composition of extractable bitumens as well as resistant to extraction kerogen of the unweathered black shale to that of the bioweathered and chemically weathered, identification of mobilized organic compounds and spectrometry-based determination of proteomic composition of the bacterial biofilm. The oxidative bioweathering of bitumens and kerogen was confirmed. The mobilization of organic carbon in the form of oxidized organic compounds, such as monohydroxy and dihydroxy alcohols, aldehydes, monocarboxylic and dicarboxylic acids and esters due to the microbial activity, was documented. The enzymes crucial for the aerobic metabolism of aliphatic and aromatic hydrocarbons such as monooxygenases and dehydrogenases were identified in the epilithic biofilm inhabiting the black shale.

Bottom-up effects of streambed drying on consumer performance through changes in resource quality

Stream flow intermittency and subsequent streambed drying, which already occurs in most biomes worldwide, is expected to increase in many regions due to both climate change and increased water demand. We studied the effects of streambed drying on leaves and epilithic biofilm and their effects on potential consumers. In the field, resources were conditioned according to the following treatments: (i) continuously submerged (PERM), (ii) submerged, exposed to the dry streambed and then submerged again (INT), or (iii) conditioned in the dry streambed and only allowed instream conditioning for 1 week (DRY, only for leaves). The results showed that drying affects resource quality, and the effects on biofilm were more severe than those on leaves. Both DRY leaves and INT biofilm showed lower microbial colonization and nitrogen accrual, whereas INT leaves had similar characteristics to PERM leaves. Drying resulted in decreased shredder and herbivore consumption rates and detritivore growth. Our results suggest that bottom-up effects of drying through changes in resource quality can constrain detritivore growth in temporary streams, potentially affecting stream secondary production and invertebrate-mediated organic matter cycling under a drier climate scenario.

Seasonal Variations of Epilithic Biofilm Biomass and Community Structure at Byeonsan Peninsula, Korea

Seasonal Variations of Epilithic Biofilm Biomass and Community Structure at Byeonsan Peninsula, Korea

Photoacoustic monitoring of water transport process in calcareous stone coated with biopolymers

Moisture is a critical control of chemical and physical processes leading to stone deterioration. These processes can be enhanced by microbial biofilms and associated exopolymers (EPS). There is limited current understanding of the water transport process across rocks covered by EPS. In the present work, we employed the photoacoustic technique to study the influence of three biopolymers (xanthan, microbactan and arabic gum) in the water transport process of two types of limestone rock of similar mineralogy but contrasting porosity. Both controls of RL (low porosity) and RP (high porosity) presented the higher values of water diffusion coefficient (D) than biopolymer-coated samples, indicating that biopolymer layers slowed down the transport of water. This trend was steeper for RP samples as water was transported seven times faster than in the more porous rock. Important differences of D values were observed among samples coated by different biopolymers. Scanning electron microscopy and optical microscopy showed that surface topography was different between both types of rocks; adherence of coatings was seen predominantly in the less porous rocks samples. FTIR and NMR analysis showed the presence of pyruvate and acetate in microbactan and xanthan gum, suggesting their participation on adherence to the calcareous surfaces, sealing surface pores. These results indicate that water transport at rock interfaces is dependent on the chemistry of biopolymer and surface porosity. The implications for reduced water transport in stone conservation under the influence of biopolymers include both enhanced and lower deterioration rates along with altered efficiency of biocide treatment of epilithic biofilms.

Impact of wastewater treatment plant discharge on the contamination of river biofilms by pharmaceuticals and antibiotic resistance.

Wastewater treatment plants (WWTPs) are one of the main sources of pharmaceutical residue in surface water. Epilithic biofilms were collected downstream from 12 WWTPs of various types and capacities to study the impacts of their discharge through the changes in biofilm composition (compared to a corresponding upstream biofilm) in terms of pharmaceutical concentrations and bacterial community modifications (microbial diversity and resistance integrons). The biofilm is a promising indicator to evaluate the impacts of WWTPs on the surrounding aquatic environment. Indeed, the use of biofilms reveals contamination hot spots. All of the downstream biofilms present significant concentrations (up to 965ng/g) of five to 11 pharmaceuticals (among the 12 analysed). Moreover, the exposition to the discharge point increases the presence of resistance integrons (three to 31 fold for Class 1) and modifies the diversity of the bacterial communities (for example cyanobacteria). The present study confirms that the discharge from WWTPs has an impact on the aquatic environment.

Multiple‐stressor effects on river biofilms under different hydrological conditions

Summary We studied the relative importance of environmental factors (river flow, nutrients, dissolved organic matter) and organic micro‐pollutants [herbicides, insecticides, industrial organic compounds, personal care products, antibiotics and pharmaceuticals, (PhCs)] to the structure and function of epilithic biofilms under various hydrological conditions and land uses. Biofilms from four river basins in the Iberian Peninsula (Llobregat, Ebro, Júcar and Guadalquivir) were analysed in summer–autumn during a wet period and base‐flow conditions. The sites experienced different levels of pollution. We used variance partitioning (Redundancy Detrended Analysis, RDA) to link environmental stressors to the structure and function of biofilms, which were assessed as diatom community composition, algal biomass, photosynthetic activity, bacterial density and extracellular phosphatase activity. RDAs showed that industrial organic compounds, herbicides and PhC products were the pollutants most strongly associated with measures of biofilm structure and function, whereas dissolved inorganic nitrogen, dissolved organic carbon and hydrological variability were the environmental factors most strongly associated with biofilm responses. Thicker biofilms developing during base flow were most affected by pollutants and nutrients. In contrast, thinner biofilms developing during periods of high river flow were less affected by chemicals.

Succession and seasonal variation in epilithic biofilms on artificial reefs in culture waters of the sea cucumber Apostichopus japonicus

Periphytic biofilms in aquaculture waters are thought to improve water quality, provide an additional food source, and improve the survival and growth of some reared animals. In the Asia- Pacific region, particularly in China, artificial reefs are commonly used in the commercial farming of sea cucumbers. However, few studies have examined the epilithic biofilms on the artificial reefs. To gain a better understanding of the succession of epilithic biofilms and their ecological processes in sea cucumber culture waters, two experiments were conducted in culture waters of the sea cucumber Apostichopus japonicus in Rongcheng, China, using artificial test panels. On the test panels of succession experiment, more than 67 species were identified in the biofilms. On the test panels of seasonal variation experiment, more than 46 species were recorded in the biofilms. In both experiments, communities of epilithic biofilms were dominated by diatoms, green algae and the annelid Spirorbis sp. In the initial colonization, the dominant diatoms were Cocconeis sp., Amphora spp. and Nitzschia closterium in June, which were succeeded by species of Navicula, Cocconeis and Nitzschia (July to September), and then by Licmophora abbreviata, Nitzschia closterium and Synedra spp. in the following months. A diatom bloom in the autumn and filamentous green algae burst in the summer were also observed. Ecological indices well annotated the succession and seasonal changes in epilithic communities. Multidimensional scaling (MDS) analysis found significant differences in diatom community composition among months and seasons. Fast growth of biofilms was observed in the summer and autumn, whereas the biomass of summer biofilms was largely made up of filamentous green algae. Present results show that the components of epilithic biofilms are mostly optimal foods of A. japonicus, suggesting that biofilms on artificial reefs may contribute important nutritional sources for sea cucumbers during their growth seasons. Future works should include quantitative determination of the contribution of epilithic biofilms to the diet of A. japonicus, potential roles of epilithic biofilms in regulating the water quality of sea cucumber ponds, and the regulation of epilithic biofilms in sea cucumber culture ponds.

Bacterial production and their role in the removal of dissolved organic matter from tributaries of drinking water reservoirs

Enhanced concentrations of dissolved organic matter (DOM) in freshwaters are an increasing problem in drinking water reservoirs. In this study we investigated bacterial DOM degradation rates in the tributaries of the reservoirs and tested the hypotheses that (1) DOM degradation is high enough to decrease DOM loads to reservoirs considerably, (2) DOM degradation is affected by stream hydrology, and (3) phosphorus addition may stimulate bacterial DOM degradation. Bacterial biomass production, which was used as a measure of DOM degradation, was highest in summer, and was usually lower at upstream than at downstream sites. An important proportion of bacterial production was realized in epilithic biofilms. Production of planktonic and biofilm bacteria was related to water temperature. Planktonic production weakly correlated to DOM quality and to total phosphorus concentration. Addition of soluble reactive phosphorus did not stimulate bacterial DOM degradation. Overall, DOM was considerably degraded in summer at low discharge levels, whereas degradation was negligible during flood events (when DOM load in reservoirs was high). The ratio of DOM degradation to total DOM release was negatively related to discharge. On annual average, only 0.6–12% of total DOM released by the catchments was degraded within the tributaries.

Phylogenetically Diverse Aerobic Anoxygenic Phototrophic Bacteria Isolated from Epilithic Biofilms in Tama River, Japan.

The diversity of aerobic anoxygenic phototrophic (AAP) bacteria in freshwater environments, particularly in rivers, has not been examined in as much detail as in ocean environments. In the present study, we investigated the phylogenetic and physiological diversities of AAP bacteria in biofilms that developed on submerged stones in a freshwater river using culture methods. The biofilms collected were homogenized and inoculated on solid media and incubated aerobically in the dark. Sixty-eight red-, pink-, yellow-, orange-, or brown-colored colonies were isolated, and, of these, 28 isolates contained the photosynthetic pigment, bacteriochlorophyll (BChl) a. Phylogenetic analyses based on 16S rRNA gene sequences showed that the isolates were classified into 14 groups in 8 operational taxonomic units (OTUs) and distributed in the orders Rhodospirillales, Rhodobacterales, and Sphingomonadales of Alphaproteobacteria and in Betaproteobacteria. Physiological analyses confirmed that none of the representative isolates from any of the groups grew under anaerobic phototrophic conditions. Seven isolates in 4 OTUs showed a 16S rRNA gene sequence identity of 98.0% or less with any established species, suggesting the presence of previously undescribed species of AAP bacteria. Six isolates in 2 other OTUs had the closest relatives, which have not been reported to be AAP bacteria. Physiological comparisons among the isolates revealed differences in preferences for nutrient concentrations, BChl contents, and light-harvesting proteins. These results suggest that diverse and previously unknown AAP bacteria inhabit river biofilms.

La bioprecipitación de carbonato de calcio por la biota nativa como un método de restauración.

Partiendo de 30 aislados bacterianos de diferentes tipos (fundamentalmente Bacillus), previamente aislados e identificados por métodos microbiológicos y moleculares, provenientes de biopelículas epilíticas traídas del Castillo de Chapultepec de la ciudad de México, DF. Se realizaron diferentes metodologías como comprobación y determinación de formación biopelículas y su cuantificación, prueba de ureasa y la producción de cristales de carbonato de calcio en placa Petry con LB enriquecido con carbonato de calcio al 0.2 %. Los cristales fueron enviados a difracción de rayos X y Microscopia Electrónica de Barrido.Los resultados muestran que en alguna medida todos los microorganismos probados participan en el proceso de formación de biopelículas y un total de 12 aislados destacaron como los mejores formadores de biopelículas, sobresaliendo las cepas IS16 y IIIS10 (Bacillus subtilis), IIS15a (Bacillus cereus) por ser las que mayores UFC/mL presentaron en la biopelícula. Todos los buenos formadores de biopelículas resultan también adecuados en cuanto a bioprecipitación de carbonatos, destacando las cepas IIIS4 (Bacillus megaterium), IS5 (Bacillus subtilis) e IIIS9b (Bacillus subtilis), 21 (Pantoea agglomerans) y la IIIS5 (Bacillus simplex). Poco o nada se sabe hasta el momento de la participación de Pantoea agglomerans en procesos de biorrestauración mediante bioprecipitación de cristales de carbonato de calcio y debido a que se obtuvo buena respuesta a la prueba de ureasa y los cristales obtenidos fueron mayormente Calcita (99 %), este trabajo muestra la factibilidad de usar la biota nativa para su activación y así lograr la restauración de los monumentos biodeteriorados.

Spatial Patterns in Biofilm Diversity across Hierarchical Levels of River-Floodplain Landscapes.

River-floodplain systems are among the most diverse and productive ecosystems, but the effects of biophysical complexity at multiple scales on microbial biodiversity have not been studied. Here, we investigated how the hierarchical organization of river systems (i.e., region, floodplain, zone, habitats, and microhabitats) influences epilithic biofilm community assemblage patterns by characterizing microbial communities using 16S rRNA gene sequence data and analyzing bacterial species distribution across local and regional scales. Results indicate that regional and local environmental filters concurrently sort bacterial species, suggesting that spatial configuration of epilithic biofilms resembles patterns of larger organisms in floodplain ecosystems. Along the hierarchical organization of fluvial systems, floodplains constitute a vector of maximum environmental heterogeneity and consequently act as a major landscape filter for biofilm species. Thus, river basins and associated floodplains may simply reflect very large scale ‘patches’ within which environmental conditions select for community composition of epilithic biofilms.

Coastal Upwelling Drives Intertidal Assemblage Structure and Trophic Ecology.

Similar environmental driving forces can produce similarity among geographically distant ecosystems. Coastal oceanic upwelling, for example, has been associated with elevated biomass and abundance patterns of certain functional groups, e.g., corticated macroalgae. In the upwelling system of Northern Chile, we examined measures of intertidal macrobenthic composition, structure and trophic ecology across eighteen shores varying in their proximity to two coastal upwelling centres, in a hierarchical sampling design (spatial scales of >1 and >10 km). The influence of coastal upwelling on intertidal communities was confirmed by the stable isotope values (δ13C and δ15N) of consumers, including a dominant suspension feeder, grazers, and their putative resources of POM, epilithic biofilm, and macroalgae. We highlight the utility of muscle δ15N from the suspension feeding mussel, Perumytilus purpuratus, as a proxy for upwelling, supported by satellite data and previous studies. Where possible, we used corrections for broader-scale trends, spatial autocorrelation, ontogenetic dietary shifts and spatial baseline isotopic variation prior to analysis. Our results showed macroalgal assemblage composition, and benthic consumer assemblage structure, varied significantly with the intertidal influence of coastal upwelling, especially contrasting bays and coastal headlands. Coastal topography also separated differences in consumer resource use. This suggested that coastal upwelling, itself driven by coastline topography, influences intertidal communities by advecting nearshore phytoplankton populations offshore and cooling coastal water temperatures. We recommend the isotopic values of benthic organisms, specifically long-lived suspension feeders, as in situ alternatives to offshore measurements of upwelling influence.

Do the threats of predation and competition alter larval behaviour and selectivity at settlement under field conditions?

The temporary ability of larvae to select optimal substrata during settlement can strongly influence the success of sedentary benthic invertebrates. Larval responses to cues from algae, epilithic microbial assemblages and conspecific species are well recognised. Comparatively, little is known about how the threat of predation or of competition influences settlement. We used a manipulative field experiment to test if the presence of a predator or spatial competitor affects settlement patterns of two intertidal mussel species, Perna perna and Mytilus galloprovincialis. By deploying treated artificial units of settlement habitat (larval collectors) in ‘no choice’ and ‘choice’ combinations, we determined whether the observed settlement patterns resulted from active larval choices (preference/avoidance). Among treatments where no choice was offered, settlement was generally greater on the predator treatment, lower on treatments with epilithic biofilm or competitor presence, and least on collectors without biofilm. In all but one case, the observed proportions of settlement in the ‘choice’ tests were significantly different from that expected by random association based on the ‘no choice’ tests. Choices related primarily to the absence of biofilm and to the threat of predation, with little influence of competitor presence. Although active preferential behaviour was confirmed, patterns of selectivity differed between the two study sites. Settlers of both species preferred the predator treatment at one site, possibly suggesting a predator swamping strategy. In contrast, settlers at the second site avoided the presence of predators. This suggests that settlement behaviour may be contingent on site-specific factors and demonstrates a greater degree of flexibility in settlement behaviour than expected.

Draft genome of Elstera litoralis, a freshwater epilithic biofilm associated bacterium from littoral zone of Lake Constance

Elstera litoralis, is a Rhodospirillaceae member which was isolated from the littoral zone of Lake Constance from a stone biofilm using diatom extracellular polymeric substances (EPS) as C source. We present here the draft genome of E. litoralis which has a genome size of 3.83Mb and 61.2% G+C content. Genome analysis indicated utilization of multiple C substrates explaining its heterotrophic lifestyle as a bacterium present in natural biofilms. Further comparative genome analysis of Elstera with other members of Rhodospirillaceae would be helpful to understand the evolutionary relationships and divergence of hydrobacteria from terrabacteria.

Abiotic autumnal organic matter deposition and grazing disturbance effects on epilithic biofilm succession.

Stream epilithic biofilm community assembly is influenced in part by environmental factors. Autumn leaf deposition is an annual resource subsidy to streams, but the physical effects of leaves settling on epilithic biofilms has not been investigated.We hypothesized that bacterial and microeukaryotic community assembly would follow a successional sequence that was mediated by abiotic effects that were simulating leaf deposition (reduced light and flow) and by biotic (snail grazing)disturbance. This hypothesis was tested using an in situ experimental manipulation. Ambient biofilms had greater algal biomass and distinct ARISA community profiles compared to biofilms developed under manipulated conditions. There were no significant differences in biofilm characteristics associated with grazing, suggesting that results were driven by reduced light/flow rather than invertebrate disturbance; however, grazing appeared to increase bacterial taxon richness.Interestingly at day 38, all treatments grouped together in ordination space and had similar algal/total biomass ratios. We suggest that algal priming promoted a shift in ambient biofilms but that this effect is dependent upon successional timing of algal establishment. These data demonstrate that abiotic effects were more influential than local grazing disturbance and imply that leaf litter deposition may have bottom-up effects on the stream ecosystem through altered epilithic biofilms.

Microscale aspects in the diet of the limpet Patella vulgata L.

The limpet Patella vulgata is a key species of northern Atlantic rocky shore-associated communities, and is commonly considered to be important in regulating populations of canopy-forming Ascophyllum nodosum, through consumption of propagules and young recruits. Although P. vulgata is usually regarded as a non-selective epilithic biofilm grazer, a role in the collapse of established A. nodosum through grazing of adult plants has been repeatedly suggested. Factors controlling the preference of P. vulgata for epilithic biofilm or adult algae are still not clearly established. Here, we test the hypothesis that the diet of P. vulgata is mainly driven by the local availability of food sources. Limpets were sampled along the first 6 metres of an A. nodosum bed–bare rock gradient. Stable isotope ratios of their muscle tissue and digestive glands were measured. The contribution of A. nodosum to the diet of limpets was the highest in the immediate vicinity of macroalgae beds, which confirmed our initial hypothesis. However, the contribution of epilithic biofilm did not match our hypothesis, being the lowest for limpets colonizing bare rock. Instead, these limpets relied on a wide array of sources, including ephemeral green algae, biofilm and drifting A. nodosum fragments. Overall, our results indicate that A. nodosum can be readily grazed by limpets, which challenges the hypothesis that these macroalgae dominate rocky shores due to the absence of strong top-down control exerted by herbivores. Our results also highlight the need to consider the small spatial scale to understand the dynamic of herbivore–algae interactions in natural environments.

Evidence for the assimilation of ancient glacier organic carbon in a proglacial stream food web

AbstractWe used natural abundance δ13C, δ15N, and Δ14C to compare trophic linkages between potential carbon sources (leaf litter, epilithic biofilm, and particulate organic matter) and consumers (aquatic macroinvertebrates and fish) in a nonglacial stream and two reaches of the heavily glaciated Herbert River. We tested the hypothesis that proglacial stream food webs are sustained by organic carbon released from glacial ecosystems. Carbon sources and consumers in the nonglacial stream had carbon isotope values that ranged from −30‰ to −25‰ for δ13C and from −14‰ to 53‰ for Δ14C reflecting a food web sustained mainly on contemporary primary production. In contrast, biofilm in the two glacial stream sites was highly Δ14C‐depleted (−215‰ to 175‰) relative to the nonglacial stream consistent with the assimilation of ancient glacier organic carbon. IsoSource modeling showed that in upper Herbert River, macroinvertebrates (Δ14C = −171‰ to 22‰) and juvenile salmonids (Δ14C = −102‰ to 17‰) reflected a feeding history of both biofilm (∼ 56%) and leaf litter (∼ 40%). We estimate that in upper Herbert River on average 36% of the carbon incorporated into consumer biomass is derived from the glacier ecosystem. Thus, 14C‐depleted glacial organic carbon was likely transferred to higher trophic levels through a feeding history of bacterial uptake of dissolved organic carbon and subsequent consumption of 14C‐depleted biofilm by invertebrates and ultimately fish. Our findings show that the metazoan food web is sustained in part by glacial organic carbon such that future changes in glacial runoff could influence the stability and trophic structure of proglacial aquatic ecosystems.

Effects of organic pollution on biological communities of marine biofilm on hard substrata

We examined the effect of organic enrichment on diatom and bacterial assemblages of marine epilithic biofilms on two locations in the Mediterranean, one situated in Spain and the other in Greece. Total organic carbon, total organic nitrogen, stable isotopes (δ13C and δ15N) and chlorophyll a indicated significant incorporation of organic wastes, increased primary production and trophic niche modifications on the biofilms close to the organic enrichment source. In Spain, where the organic load was higher than in Greece, diatom and, to some extent, bacterial assemblages varied following the organic enrichment gradient. The taxonomic richness of diatom and bacterial communities was not influenced by organic enrichment. Classical community parameters showed consistent patterns to organic pollution in both locations, whereas community assemblages were only influenced when organic pollution was greatest. The successional patterns of these communities were similar to other epilithic communities. The modification of community assemblages induced by organic pollution may affect ecological functions.

The spatial organization and microbial community structure of an epilithic biofilm.

Microbial biofilms are common on lithic surfaces, including stone buildings. However, the ecology of these communities is poorly understood. Few studies have focused on the spatial characteristics of lithobiontic biofilms, despite the fact that spatial structure has been demonstrated to influence ecosystem function (and hence biodegradation) and community diversity. Furthermore, relatively few studies have utilized molecular techniques to characterize these communities, even though molecular methods have revealed unexpected microbial diversity in other habitats. This study investigated (1) the spatial structure and (2) the taxonomic composition of an epilithic biofilm using molecular techniques, namely amplicon pyrosequencing and terminal restriction fragment length polymorphism. Dispersion indices and Mantel correlograms were used to test for the presence of spatial structure in the biofilm. Diversity metrics and rank-abundance distributions (RADs) were also generated. The study revealed spatial structure on a centimetre scale in eukaryotic microbes (fungi and algae), but not the bacteria. Fungal and bacterial communities were highly diverse; algal communities much less so. The RADs were characterized by a distinctive 'hollow' (concave up) profile and long tails of rare taxa. These findings have implications for understanding the ecology of epilithic biofilms and the spatial heterogeneity of stone biodeterioration.