Molecular diversity and tools for deciphering the methanogen community structure and diversity in freshwater sediments

Methanogenic archaeal communities existing in freshwater sediments are responsible for approximately 50 % of the total global emission of methane. This process contributes significantly to global warming and, hence, necessitates interventional control measures to limit its emission. Unfortunately, the diversity and functional interactions of methanogenic populations occurring in these habitats are yet to be fully characterized. Considering several disadvantages of conventional culture-based methodologies, in recent years, impetus is given to molecular biology approaches to determine the community structure of freshwater sedimentary methanogenic archaea. 16S rRNA and methyl coenzyme M reductase (mcrA) gene-based cloning techniques are the first choice for this purpose. In addition, electrophoresis-based (denaturing gradient gel electrophoresis, temperature gradient gel electrophoresis, and terminal restriction fragment length polymorphism) and quantitative real-time polymerase chain reaction techniques have also found extensive applications. These techniques are highly sensitive, rapid, and reliable as compared to traditional culture-dependent approaches. Molecular diversity studies revealed the dominance of the orders Methanomicrobiales and Methanosarcinales of methanogens in freshwater sediments. The present review discusses in detail the status of the diversity of methanogens and the molecular approaches applied in this area of research.     

Multifunctionality and diversity in bacterial biofilms

Bacteria are highly diverse and drive a bulk of ecosystem processes. Analysis of relationships between diversity and single specific ecosystem processes neglects the possibility that different species perform multiple functions at the same time. The degradation of dissolved organic carbon (DOC) followed by respiration is a key bacterial function that is modulated by the availability of DOC and the capability to produce extracellular enzymes. In freshwater ecosystems, biofilms are metabolic hotspots and major sites of DOC degradation. We manipulated the diversity of biofilm forming communities which were fed with DOC differing in availability. We characterized community composition using molecular fingerprinting (T-RFLP) and measured functioning as oxygen consumption rates, the conversion of DOC in the medium, bacterial abundance and the activities of five specific enzymes. Based on assays of the extracellular enzyme activity, we calculated how the likelihood of sustaining multiple functions was affected by reduced diversity. Carbon source and biofilm age were strong drivers of community functioning, and we demonstrate how the likelihood of sustaining multifunctionality decreases with decreasing diversity.     

CARD-FISH and confocal laser scanner microscopy to assess successional changes of the bacterial community in freshwater biofilms

Bacterial community composition was assessed during riverine biofilm development by the Catalyzed Reporter Deposition Fluorescence In Situ Hybridization (CARD-FISH) in combination with Confocal Laser Scanning Microscopy. Using artificial substrates, it was possible to follow the dynamics of specific bacterial clusters, while maintaining the unaltered structure and architecture of the biofilm.     

Combined eukaryotic and bacterial community fingerprinting of natural freshwater biofilms using automated ribosomal intergenic spacer analysis

Biofilms are complex communities playing an important role in aquatic ecosystems. Automated ribosomal intergenic spacer analysis (ARISA) has been used successfully to explore biofilm bacterial diversity. However, a gap remains to be filled as regards its application to biofilm eukaryotic populations. The aim of this study is to use ARISA to detect eukaryotic population shifts in biofilm. We designed a new set of primers to focus specifically on the ITS1-5.8S-ITS2 region of diatoms and tested it on natural biofilms. Additionally, we tested universal primers, used previously to perform ARISA on fungal communities. Cloning and sequencing showed that the universal primer set amplified various eukaryotes, whereas the new set was diatom specific. The new set amplified a wider variety of diatoms. Therefore, the universal set is appropriate to study the general eukaryotic population shifts in biofilms, whereas the new set is more appropriate to study diatoms specifically. We used both primer sets, along with a bacterial set, to study the population shifts in natural river biofilms. Principal component analysis of the ARISA fingerprints revealed seasonal shifts that did not coincide for bacterial and eukaryotic communities. Therefore, the use of both eukaryotic and bacterial primers provides a useful insight to assess microbial succession in biofilms.     

Microbial diversity and community respiration in freshwater sediments influenced by artificial light at night

An increasing proportion of the Earth''s surface is illuminated at night. In aquatic ecosystems, artificial light at night (ALAN) may influence microbial communities living in the sediments. These communities are highly diverse and play an important role in the global carbon cycle. We combined field and laboratory experiments using sediments from an agricultural drainage system to examine how ALAN affects communities and alters carbon mineralization. Two identical light infrastructures were installed parallel to a drainage ditch before the start of the experiment. DNA metabarcoding indicated that both sediment communities were similar. After one was lit for five months (July–December 2012) we observed an increase in photoautotroph abundance (diatoms, Cyanobacteria) in ALAN-exposed sediments. In laboratory incubations mimicking summer and winter (six weeks each), communities in sediments that were exposed to ALAN for 1 year (July 2012–June 2013) showed less overall seasonal change compared with ALAN-naive sediments. Nocturnal community respiration was reduced in ALAN-exposed sediments. In long-term exposed summer-sediments, we observed a shift from negative to positive net ecosystem production. Our results indicate ALAN may alter sediment microbial communities over time, with implications for ecosystem-level functions. It may thus have the potential to transform inland waters to nocturnal carbon sinks.     

The molecular diversity of the methanogenic community in a hypereutrophic freshwater lake determined by PCR-RFLP

AIMS: To combine database-held sequence information with a programme of experimental molecular ecology to define the methanogenic community of a hypereutrophic lake by a PCR-restriction fragment length polymorphism (RFLP) analysis. METHODS AND RESULTS: Methanogen diversity in a hypereutrophic freshwater lake was analysed using 16S rDNA PCR-RFLP. Database-held 16S rRNA gene sequences for 76 diverse methanogens were analysed for specific restriction sites that permitted unequivocal differentiation of methanogens. Restriction digestion and agarose gel electrophoresis of the 16S rDNA from selected methanogen pure cultures generated observed restriction profiles that corroborated the expected patterns. This method was then tested by analysing methanogen diversity in samples obtained over 1 year from sediment and water samples taken from the same sampling site. CONCLUSIONS: Restriction analysis of the 16S rRNA gene sequences from 157 methanogen clones generated from lakewater and sediment samples showed that over 50% were similar to Methanoculleus spp. Furthermore, a total of 16 RFLP types (1-16) were identified, eight of which contained no cultured representative archaeal 16S rRNA gene sequences. SIGNIFICANCE AND IMPACT OF THE STUDY: This RFLP strategy provides a robust and reliable means to rapidly identify methanogens in the environment.     

Microbial diversity in biofilms from corroding heating systems

Culture-independent investigations of the bacterial diversity and activity in district heating systems with and without corrosion did not make it possible to relate one group of microorganisms with the observed corrosion. Fluorescence in situ hybridization by oligonucleotide probes revealed the dominance of beta-proteobacteria, sulphate reducing prokaryotes and alpha-proteobacteria. Analysis of a clone library from one Danish heating (DH) system showed that the most sequences formed two clusters within the alpha-proteobacteria affiliated to the families Rhizobiaceae and Acetobacteraceae and two clusters within the beta-proteobacteria belonging to the family Comamonadaceae. Functional groups were determined by microautoradiography showing aerobic and anaerobic bacteria (sulphate reducing and methanogenic bacteria). The corrosion study showed that pitting corrosion rates were five to ten times higher than the general corrosion rates, suggesting the presence of biocorrosion. The results indicate that several bacterial groups could be involved in corrosion of DH system piping including sulphate reducing prokaryotes, Acidovorax (within the beta-proteobacteria), methanogenic bacteria and others.     

Habitat patch size alters the importance of dispersal for species diversity in an experimental freshwater community

Increased dispersal of individuals among discrete habitat patches should increase the average number of species present in each local habitat patch. However, experimental studies have found variable effects of dispersal on local species richness. Priority effects, predators, and habitat heterogeneity have been proposed as mechanisms that limit the effect of dispersal on species richness. However, the size of a habitat patch could affect how dispersal regulates the number of species able to persist. We investigated whether habitat size interacted with dispersal rate to affect the number of species present in local habitats. We hypothesized that increased dispersal rates would positively affect local species richness more in small habitats than in large habitats, because rare species would be protected from demographic extinction. To test the interaction between dispersal rate and habitat size, we factorially manipulated the size of experimental ponds and dispersal rates, using a model community of freshwater zooplankton. We found that high‐dispersal rates enhanced local species richness in small experimental ponds, but had no effect in large experimental ponds. Our results suggest that there is a trade‐off between patch connectivity (a mediator of dispersal rates) and patch size, providing context for understanding the variability observed in dispersal effects among natural communities, as well as for developing conservation and management plans in an increasingly fragmented world.     

Biophysical controls on community succession in stream biofilms

Biofilm formation is controlled by an array of coupled physical, chemical, and biotic processes. Despite the ecological relevance of microbial biofilms, their community formation and succession remain poorly understood. We investigated the effect of flow velocity, as the major physical force in stream ecosystems, on biofilm community succession (as continuous shifts in community composition) in microcosms under laminar, intermediate, and turbulent flow. Flow clearly shaped the development of biofilm architecture and community composition, as revealed by microscopic investigation, denaturing gradient gel electrophoresis (DGGE) analysis, and sequencing. While biofilm growth patterns were undirected under laminar flow, they were clearly directed into ridges and conspicuous streamers under turbulent flow. A total of 51 biofilm DGGE bands were detected; the average number ranged from 13 to 16. Successional trajectories diverged from an initial community that was common in all flow treatments and increasingly converged as biofilms matured. We suggest that this developmental pattern was primarily driven by algae, which, as "ecosystem engineers," modulate their microenvironment to create similar architectures and flow conditions in all treatments and thereby reduce the physical effect of flow on biofilms. Our results thus suggest a shift from a predominantly physical control to coupled biophysical controls on bacterial community succession in stream biofilms.     

Microbial diversity of biofilms in dental unit water systems

We investigated the microbial diversity of biofilms found in dental unit water systems (DUWS) by three methods. The first was microscopic examination by scanning electron microscopy (SEM), acridine orange staining, and fluorescent in situ hybridization (FISH). Most bacteria present in the biofilm were viable. FISH detected the beta and gamma, but not the alpha, subclasses of Proteobacteria: In the second method, 55 cultivated biofilm isolates were identified with the Biolog system, fatty acid analysis, and 16S ribosomal DNA (rDNA) sequencing. Only 16S identified all 55 isolates, which represented 13 genera. The most common organisms, as shown by analyses of 16S rDNA, belonged to the genera Afipia (28%) and Sphingomonas (16%). The third method was a culture-independent direct amplification and sequencing of 165 subclones from community biofilm 16S rDNA. This method revealed 40 genera: the most common ones included Leptospira (20%), Sphingomonas (14%), Bacillus (7%), Escherichia (6%), Geobacter (5%), and Pseudomonas (5%). Some of these organisms may be opportunistic pathogens. Our results have demonstrated that a biofilm in a health care setting may harbor a vast diversity of organisms. The results also reflect the limitations of culture-based techniques to detect and identify bacteria. Although this is the greatest diversity reported in DUWS biofilms, other genera may have been missed. Using a technique based on jackknife subsampling, we projected that a 25-fold increase in the number of subclones sequenced would approximately double the number of genera observed, reflecting the richness and high diversity of microbial communities in these biofilms.     

Global diversity of aquatic macrophytes in freshwater

Aquatic macrophytes are aquatic photosynthetic organisms, large enough to see with the naked eye, that actively grow permanently or periodically submerged below, floating on, or growing up through the water surface. Aquatic macrophytes are represented in seven plant divisions: Cyanobacteria, Chlorophyta, Rhodophyta, Xanthophyta, Bryophyta, Pteridophyta and Spermatophyta. Species composition and distribution of aquatic macrophytes in the more primitive divisions are less well known than for the vascular macrophytes (Pteridophyta and Spermatophyta), which are represented by 33 orders and 88 families with about 2,614 species in c. 412 genera. These c. 2,614 aquatic species of Pteridophyta and Spermatophyta evolved from land plants and represent only a small fraction (∼1%) of the total number of vascular plants. Our analysis of the numbers and distribution of vascular macrophytes showed that whilst many species have broad ranges, species diversity is highest in the Neotropics, intermediate in the Oriental, Nearctic and Afrotropics, lower in the Palearctic and Australasia, lower again in the Pacific Oceanic Islands, and lowest in the Antarctic region. About 39% of the c. 412 genera containing aquatic vascular macrophytes are endemic to a single biogeographic region, with 61–64% of all aquatic vascular plant species found in the Afrotropics and Neotropics being endemic to those regions. Aquatic macrophytes play an important role in the structure and function of aquatic ecosystems and certain macrophyte species (e.g., rice) are cultivated for human consumption, yet several of the worst invasive weeds in the world are aquatic plants. Many of the threats to fresh waters (e.g., climate change, eutrophication) will result in reduced macrophyte diversity and will, in turn, threaten the faunal diversity of aquatic ecosystems and favour the establishment of exotic species, at the expense of native species. Guest editors: E. V. Balian, C. Lévêque, H. Segers & K. Martens Freshwater Animal Diversity Assessment     

Metagenomic analysis of viral community in the Yangtze River expands known eukaryotic and prokaryotic virus diversity in freshwater

Viruses in aquatic ecosystems are characterized by extraordinary abundance and diversity. Thus far, there have been limited studies focused on viral communities in river water systems. Here, we investigated the virome of the Yangtze River Delta using viral metagenomic analysis. The compositions of viral communities from six sampling sites were analyzed and compared. By using library construction and next generation sequencing, contigs and singlet reads similar to viral sequences were classified into 17 viral families, including nine dsDNA viral families, four ssDNA viral families and four RNA viral families. Statistical analysis using Friedman test suggested that there was no significant difference among the six sampling sites (P > 0.05). The viromes in this study were all dominated by the order Caudovirales, and a group of Freshwater phage uvFW species were particularly prevalent among all the samples. The virome from Nanjing presented a unique pattern of viral community composition with a relatively high abundance of family Parvoviridae. Phylogenetic analyses based on virus hallmark genes showed that the Caudovirales order and CRESS-DNA viruses presented high genetic diversity, while viruses in the Microviridae and Parvoviridae families and the Riboviria realm were relatively conservative. Our study provides the first insight into viral community composition in large river ecosystem, revealing the diversity and stability of river water virome, contributing to the proper utilization of freshwater resource.     

A flow-lane incubator for studying freshwater and marine phototrophic biofilms

Phototrophic biofilms are defined as interfacial microbial communities mainly driven by light as energy source and are studied for both ecological and technological reasons. Field investigations of biofilms usually do not offer the opportunity to study the effects of a large number of external parameters. In order to investigate the temporal development of phototrophic communities a laboratory flow-lane incubator for cultivation of freshwater and marine biofilms was developed. The incubator has four lanes which accommodate microscope slides used as substratum and for sampling. The slides can be of different material and may be employed for characterisation of phototrophic biofilms by means of gravimetry, microscopy, taxonomy, molecular biology and chemical analysis. The design allows control of irradiance, temperature and flow velocity. Furthermore, on-line control of biomass accumulation via specially adapted light sensors was proved to be a suitable indicator of temporal developmental stages (initial adhesion, active growth and mature stage). Spatial heterogeneity of the cultivated phototrophic biofilms along the flow direction within each flow-lane was low. Biofilm growth characteristics (e. g. lag time, net accrual rate, peak biomass) recorded in dependency from external conditions may be used as input data for training of artificial neural networks (ANN) and mechanistic modelling. The material and devices used in combination with low maintenance costs and ease of handling suggests the flow-lane incubator as a useful tool for studying the influence of abiotic and biotic factors on the development of freshwater and marine phototrophic biofilms.     

Global diversity of freshwater mussels (Mollusca, Bivalvia) in freshwater

The term freshwater bivalve is very inclusive and not very informative. There are representatives of at least 19 families that have at least one representative living in freshwater. This suggests at least 14 different invasions of freshwater. At least nine families have small to large radiations in the freshwater environment: Corbiculidae, Sphaeriidae, Dreissenidae, and the unioniforme families: Hyriidae, Margaritiferidae, Unionidae, Etheriidae, Iridinidae, and Mycetopodidae. The unioniforme families contain at least 180 genera and about 800 species. This order is characterized by the unique parasitic larval stage on the gills, fins or the body of a particular host fish. This order of freshwater bivalves is suffering a very high rate of extinction, with about 37 species considered presumed extinct in North America alone. The level of endangerment and extinction facing these animals is primarily the result of habitat destruction or modification. Guest editors: E. V. Balian, C. Lévêque, H. Segers & K. Martens Freshwater Animal Diversity Assessment     

Characterizing the roughness of freshwater biofilms using a photogrammetric methodology

The physical roughness of a surface changes when freshwater biofilms colonize and grow on it and this has significant implications for surfaces enclosing water conveying systems such as pipelines and canals. Plates with surfaces initially artificially roughened with varying grit size were deployed in an open channel system and biofilms were allowed to grow on the exposed surface. The plates were retrieved at intervals in time and their surfaces mapped using close range photogrammetry. For a fine grit surface (0.5–4 mm particles), diatom-dominated biofilms initially grew between the roughness elements; they subsequently developed as a mat to create a physically smoother outer surface than the underlying rough surface. For a coarse grit surface (2–4 mm), biofilms colonized faster; in one instance, larger clumps of biofilm were observed as transverse ripples across the plate.     

Cell-cell influences on bacterial community development in aquatic biofilms

Dialysis tubing containing spent culture media, when placed in a lake, was colonized by a low diversity of bacteria, whereas abiotic controls had considerable diversity. Changes were seen in the presence and absence of acylated homoserine lactones, suggesting that these molecules and other factors may influence adherent-population composition.     

Microbial diversity and function in Antarctic freshwater ecosystems

Freshwater lakes occur through much of Antarctica and are characterized by short food chains dominated by microbes. Comparatively few studies have been made of continental freshwater lakes until recently, with the main emphasis being on the less extreme maritime Antarctic lakes. The wide range of trophic status seen at the northern extremes of the maritime Antarctic reduces markedly further south, but a wide range of micro-organisms occur throughout the latitudinal range. Information on seasonal and spatial patterns of microbial activity for freshwater lakes demonstrate rapid changes in community composition at certain times of year despite constant low temperatures. Benthic communities of cyanobacteria and bacteria are a feature of most lakes and are involved in a wide range of geochemical cycling. There is a need for more detailed taxonomic information on most groups and considerable potential for molecular studies.     

Acidity and species diversity in freshwater crustacean faunas

SUMMARY. On the basis of material from over seventy acidic water bodies in three different areas a relationship is revealed between pH and the species diversity of their crustacean faunas. With increasing acidity species diversity decreases. No such relationship is shown between calcium concentration and species diversity. The very low calcium levels of such waters may, however, play a part in the exclusion of certain species.     

Genetic diversity in populations of a freshwater ciliate

RAPD fingerprinting with nine different primers revealed that all of 18 E. aediculatus isolates from nine ponds and streams in western Germany, France and the U.S.A. were genetically different. The extent of genetic similarity between genotypes from different waters did not show a significant relationship with the geographical distance among habitats, although genotypes isolated from the same habitat showed a higher genetic similarity than genotypes isolated from different habitats. Phylogenetic analyses of RAPD patterns indicate a separation of E. aediculatus strains into subgroups within one species, but all strains were genetically more similar to one another than to strains from two other Euplotes species. Crossings of the different E. aediculatus strains revealed they belonged to seven mating types of one gene pool. The high genetic diversity observed is explained by a frequent occurrence of conjugation in the studied populations.     

Microbial community structure and biomass in developing drinking water biofilms

Traditional techniques to study microbes, such as culturable counts, microbial biomass, or microbial activity, do not give information on the microbial ecology of drinking water systems. The aim of this study was to analyze whether the microbial community structure and biomass differed in biofilms collected from two Finnish drinking water distribution systems (A and B) receiving conventionally treated (coagulation, filtration, disinfection) surface water. Phospholipid fatty acid methyl esters (PLFAs) and lipopolysaccharide 3-hydroxy fatty acid methyl esters (LPS 3-OH-FAs) were analyzed from biofilms as a function of water residence time and development time. The microbial communities were rather stabile through the distribution systems, as water residence time had minor effects on PLFA profiles. In distribution system A, the microbial community structure in biofilms, which had developed in 6 weeks, was more complex than those grown for 23 or 40 weeks. The microbial communities between the studied distribution systems differed, possibly reflecting the differences in raw water, water purification processes, and distribution systems. The viable microbial biomass, estimated on the basis of PLFAs, increased with increasing water residence time in both distribution systems. The quantitative amount of LPS 3-OH-FAs increased with increasing development time of biofilms of distribution system B. In distribution system A, LPS 3-OH-FAs were below the detection limit.