Processing deep-sea particle-rich water samples for fluorescence in situ hybridization: consideration of storage effects, preservation, and sonication

Particles are often regarded as microniches of enhanced microbial production and activities in the pelagic ocean and are vehicles of vertical material transport from the euphotic zone to the deep sea. Fluorescence in situ hybridization (FISH) can be a useful tool to study the microbial community structures associated with these particles, and thus their ecological significance, yet an appropriate protocol for processing deep-sea particle-rich water samples is lacking. Some sample processing considerations are discussed in the present study, and different combinations of existing procedures for preservation, size fractionation sequential filtration, and sonication were tested in conjunction with FISH. Results from this study show that water samples should be filtered and processed within no more than 10 to 12 h after collection, or else preservation is necessary. The commonly used prefiltration formaldehyde fixation was shown to be inadequate for the rRNA targeted by FISH. However, prefiltration formaldehyde fixation followed by immediate freezing and postfiltration paraformaldehyde fixation yielded highly consistent cell abundance estimates even after 96 days or potentially longer storage. Size fractionation sequential filtration and sonication together enhanced cell abundance estimates by severalfold. Size fractionation sequential filtration effectively separated particle-associated microbial communities from their free-living counterparts, while sonication detached cells from particles or aggregates for more-accurate cell counting using epifluorescence microscopy. Optimization in sonication time is recommended for different specific types of samples. These tested and optimized procedures can be incorporated into a FISH protocol for sampling in deep-sea particle-rich waters.     

Processing Deep-Sea Particle-Rich Water Samples for Fluorescence In Situ Hybridization: Consideration of Storage Effects, Preservation, and Sonication

Particles are often regarded as microniches of enhanced microbial production and activities in the pelagic ocean and are vehicles of vertical material transport from the euphotic zone to the deep sea. Fluorescence in situ hybridization (FISH) can be a useful tool to study the microbial community structures associated with these particles, and thus their ecological significance, yet an appropriate protocol for processing deep-sea particle-rich water samples is lacking. Some sample processing considerations are discussed in the present study, and different combinations of existing procedures for preservation, size fractionation sequential filtration, and sonication were tested in conjunction with FISH. Results from this study show that water samples should be filtered and processed within no more than 10 to 12 h after collection, or else preservation is necessary. The commonly used prefiltration formaldehyde fixation was shown to be inadequate for the rRNA targeted by FISH. However, prefiltration formaldehyde fixation followed by immediate freezing and postfiltration paraformaldehyde fixation yielded highly consistent cell abundance estimates even after 96 days or potentially longer storage. Size fractionation sequential filtration and sonication together enhanced cell abundance estimates by severalfold. Size fractionation sequential filtration effectively separated particle-associated microbial communities from their free-living counterparts, while sonication detached cells from particles or aggregates for more-accurate cell counting using epifluorescence microscopy. Optimization in sonication time is recommended for different specific types of samples. These tested and optimized procedures can be incorporated into a FISH protocol for sampling in deep-sea particle-rich waters.     

Metagenomics and biodiversity of sphagnum bogs

Biodiversity of sphagnum bogs is one of the richest and less studied, while these ecosystems are among the top ones in ecological, conservation, and economic value. Recent studies focused on the prokaryotic consortia associated with sphagnum mosses, and revealed the factors that maintain sustainability and productivity of bog ecosystems. High-throughput sequencing technologies provided insight into functional diversity of moss microbial communities (microbiomes), and helped to identify the biochemical pathways and gene families that facilitate the spectrum of adaptive strategies and largely foster the very successful colonization of the Northern hemisphere by sphagnum mosses. Rich and valuable information obtained on microbiomes of peat bogs sets off the paucity of evidence on their eukaryotic diversity. Prospects and expectations of reliable assessment of taxonomic profiles, relative abundance of taxa, and hidden biodiversity of microscopic eukaryotes in sphagnum bog ecosystems are briefly outlined in the context of today’s metagenomics.     

Alteration and resilience of the soil microbial community following compost amendment: effects of compost level and compost-borne microbial community

Compost amendment has been reported to impact soil microbial activities or community composition. However, little information is available on (i) to what extent compost amendment concurrently affects the activity, size and composition of soil microbial community, (ii) the relative effect of the addition of a material rich in organic matter versus addition of compost-borne microorganisms in explaining the effects of amendment and (iii) the resilience of community characteristics. We compared five treatments in microcosms: (i) control soil (S), (ii) soil + low level of compost (Sc), (iii) soil + high level of compost (SC), (iv) sterilized soil + high level of compost [(S)C] and (v) soil + high level of sterilized compost [S(C)]. The actual C mineralization rate, substrate-induced respiration, size of microbial community (biomass and heterotrophic cells number), and structure of total microbial (phospholipid fatty acids) and bacterial (automated ribosomal intergenic spacer analysis, A-RISA) communities were surveyed during 6 months after amendment. Our results show that (i) compost amendment affected the activity, size and composition of the soil microbial community, (ii) the effect of compost amendment was mainly due to the physicochemical characteristics of compost matrix rather than to compost-borne microorganisms and (iii) no resilience of microbial characteristics was observed 6-12 months after amendment with a high amount of compost.     

Growth enhancement of Citrus reshni after inoculation with Glomus intraradices and Trichoderma aureoviride and associated effects on microbial populations and enzyme activity in potting mixes

There have been some scientific reports suggesting that dual inoculations with arbuscular mycorrhizal (AM) and saprophytic soil fungi may cause an additive or synergistic growth enhancement of the inoculated host plant. Some Trichoderma spp. have shown antagonistic potential against pathogenic fungi and a beneficial effect on plant growth. Joint inoculations of the mycorrhizal fungus Glomus intraradices Schenck and Smith, isolated from a citrus nursery (Tarragona, Spain) and a strain of Trichoderma aureoviride Rifai, isolated from an organic compost, were tested on a citrus rootstock, Citrus reshni Hort. ex Tan. The interactions between both microorganisms and their influence on mycorrhizal root colonization and plant growth enhancement, the changes produced in the soil microbial activity, like esterase, trehalase, phosphatase and chitinase activities, and on microbial populations were evaluated in three organic substrates: (1) sphagnum peat and autoclaved sandy soil (1/1, v/v), (2) sphagnum peat, quartz sand and perlite (1/1/1, v/v) and (3) pine bark compost (BVU, Prodeasa Product). Substrate characteristics were more important than the AM inoculation treatment in the determination of enzyme activity. In bark compost, the number of bacterial colonies obtained on soil-dilution plates was significantly higher than in peat and sand mixtures. Inoculation with T. aureoviride alone produced no significant effect on growth enhancement of C. reshni. However, dual inoculation with both, T. aureoviride and G. intraradices significantly increased plant growth in two of the substrates used and was the best treatment in pine bark amended compost. The inoculation with T. aureoviride did not affect the development of mycorrhizal root colonization. These results show a synergistic effect of G. intraradices and T. aureoviride on the growth of C. reshni in organic substrates and indicate the potential benefits of using combined inoculations.     

Bacteria of the genus Burkholderia as a typical component of the microbial community of sphagnum peat bogs

Bacteria of the genus Burkholderia are a typical component of the microbial complex of sphagnum peat bogs and constitute a substantial portion of the aerobic chemoorganotrophic isolates which are routinely obtained from these environments on acidic nutrient media. The ecophysiological characteristics of the 27 strains of such organisms, which were isolated from the peat of acidic sphagnum bogs of the boreal and tundra zones of Russia, Canada, and Estonia, were investigated in the present paper. The overwhelming majority of the Burkholderia strains isolated from these bogs were phylogenetically close to the species B. glathei, B. phenazinium, B. fungorum, and B. caryophylli, the typical inhabitants of soil and plant rhizosphere. The bog isolates utilized a broad range of substrates as carbon and energy sources, including organic acids, sugars, polyalcohols, and certain aromatic compounds. All the strains studied were capable of growth on nitrogen-free media. They developed in the pH ranges of 3.5 to 7.4 and from 3 to 37 degrees C, with the optima at pH 5-7 and 11-23 degrees C, respectively. They were therefore moderately acidophilic, psychroactive, dinitrogen-fixing microorganisms well adapted to the conditions of acidic northern sphagnum bogs.     

Responses of Pinus halepensis growth, soil microbial catabolic functions and phosphate-solubilizing bacteria after rock phosphate amendment and ectomycorrhizal inoculation

We examined the effects of an ectomycorrhizal (EM) fungus, Pisolithus sp., on of the growth of Pinus halepensis (Allepo pine) seedlings, soil microbial functions and rock phosphate solubilization in a un-disinfected soil amended or not with a Moroccan rock phosphate. Allepo pine seedlings were inoculated with an EM fungus (Pisolithus sp. strain PH4) isolated from a P. halepensis plantation and selected for its high ability to mobilize P from an inorganic form of phosphate. After 4 month’s culture in a disinfected substrate, plants were transferred in 10 l-containers filled with a natural forest soil and amended or not with rock phosphate powder. After 12 month’s culturing, the growth, needle nutrient concentrations of P. halepensis plants were measured. Soil microbial catabolic diversity was assessed by measuring CO₂ production of substrate induced respiration responses. Fluorescent pseudomonads were isolated from each soil treatment and tested in axenic conditions for their ability to solubilize a source of inorganic phosphate. The results clearly showed that (i) P. halepensis growth was greatly promoted by the presence of the ectomycorrhizal fungus Pisolithus strain PH4 in a disinfected soil/vermiculite mixture and in a non disinfected soil, (ii) ectomycorrhizal inoculation induced significant changes in the functions of soil microbial communities and selected microorganisms potentially beneficial to the plant growth (i.e. phosphate-solubilizing fluorescent pseudomonad) and (iii) rock phosphate solubilisation was mainly dependent on EM inoculation and mycorrhizosphere microorganisms. These results were in accordance with previous studies where it was demonstrated that EM symbiosis has a beneficial effect on plant growth through a direct effect on the host plant but also an indirect effect via a selective pressure on soil microbiota that favours microorganisms potentially beneficial to plant growth.     

Use of flow cytometric methods for single-cell analysis in environmental microbiology

Flow cytometry (FCM) is emerging as an important tool in environmental microbiology. Although flow cytometry applications have to date largely been restricted to certain specialized fields of microbiology, such as the bacterial cell cycle and marine phytoplankton communities, technical advances in instrumentation and methodology are leading to its increased popularity and extending its range of applications. Here we will focus on a number of recent flow cytometry developments important for addressing questions in environmental microbiology. These include (i) the study of microbial physiology under environmentally relevant conditions, (ii) new methods to identify active microbial populations and to isolate previously uncultured microorganisms, and (iii) the development of high-throughput autofluorescence bioreporter assays.     

Methanotrophic bacteria of acid sphagnum bogs

Acid sphagnum bogs cover a considerable part of the territory of Russia and are an important natural source of biogenic methane, which is formed in their anaerobic layers. A considerable portion of this methane is consumed in the aerobic part of the bog profile by acidophilic methanotrophic bacteria, which comprise the methane filter of sphagnum bogs and decrease CH4 emission to the atmosphere. For a long time, these bacteria escaped isolation, which became possible only after the elucidation of the optimal conditions of their functioning in situ: pH 4.5 to 5.5; temperature, from 15 to 20 degrees C; and low salt concentration in the solution. Reproduction of these conditions and rejection of earlier used media with a high content of biogenic elements allowed methanotrophic bacteria of two new genera and species--Methylocella palustris and Methylocapsa acidophila--to be isolated from the peat of sphagnum bogs of the northern part of European Russia and West Siberia. These bacteria are well adapted to the conditions in cold, acid, oligotrophic sphagnum bogs. They grow in a pH range of 4.2-7.5 with an optimum at 5.0-5.5, prefer moderate temperatures (15-25 degrees C) and media with a low content of mineral salts (200-500 mg/l), and are capable of active nitrogen fixation. Design of fluorescently labeled 16S rRNA-targeted oligonucleotide probes for the detection of Methylocella palustris and Methylocapsa acidophila and their application to the analysis of sphagnum peat samples showed that these bacteria represent dominant populations of methanotrophs with a density of 10(5)-10(6) cells/g peat. In addition to Methylocella and Methylocapsa populations, one more abundant population of methanotrophs was revealed (10(6) cells/g peat), which were phylogenetically close to the genus Methylocystis.     

Natural Microbial Community Compositions Compared by a Back-Propagating Neural Network and Cluster Analysis of 5S rRNA

The community compositions of free-living and particle-associated bacteria in the Chesapeake Bay estuary were analyzed by comparing banding patterns of stable low-molecular-weight RNA (SLMW RNA) which include 5S rRNA and tRNA molecules. By analyzing images of autoradiographs of SLMW RNAs on polyacrylamide gels, band intensities of 5S rRNA were converted to binary format for transmission to a back-propagating neural network (NN). The NN was trained to relate binary input to sample stations, collection times, positions in the water column, and sample types (e.g., particle-associated versus free-living communities). Dendrograms produced by using Euclidean distance and average and Ward's linkage methods on data of three independently trained NNs yielded the following results. (i) Community compositions of Chesapeake Bay water samples varied both seasonally and spatially. (ii) Although there was no difference in the compositions of free-living and particle-associated bacteria in the summer, these community types differed significantly in the winter. (iii) In the summer, most bay samples had a common 121-nucleotide 5S rRNA molecule. Although this band occurred in the top water of midbay samples, it did not occur in particle-associated communities of bottom-water samples. (iv) Regardless of the season, midbay samples had the greatest variety of 5S rRNA sizes. The utility of NNs for interpreting complex banding patterns in electrophoresis gels was demonstrated.     

Geographical isolation in hot spring cyanobacteria

It has been proposed that free-living microorganisms exhibit ubiquitous dispersal, do not form geographically isolated populations and rarely (if ever) speciate via allopatry. We studied island-like hot spring cyanobacterial communities in which geographical isolation should be prominent and detectable if it influences the evolution of bacteria. The genetic diversity of cyanobacteria indigenous to North American, Japanese, New Zealand and Italian springs was surveyed by (i) amplification and cloning of 16S rRNA and 16S-23S internal transcribed spacer regions; (ii) lineage-specific oligonucleotide probing (used to verify the predominance of cloned sequences); and (iii) lineage-specific polymerase chain reaction (PCR) (used to search for possible rare genotypes). Phylogenetic and distribution patterns were found to be consistent with the occurrence of geographical isolation at both global and local spatial scales, although different cyanobacterial lineages were found to vary in their distribution. A lack of correspondence between biological patterning and the chemical character of springs sampled suggested that the geographical distribution of thermophilic cyanobacteria cannot be explained by the 20 potential niche-determining chemical parameters that we assayed. Thus, geographical isolation (i.e. genetic drift) must in part be responsible for driving the observed evolutionary divergences. Geographical isolation may be an important underestimated aspect of microbial evolution.     

Intertwined interspecies relationships: approaches to untangle the microbial network

In nature, microorganisms live by interacting with each other. Microbiological studies that only consider pure cultures are not sufficient to adequately describe the natural behaviour of microbes. Several microbial interactions have been recognized to affect the growth or metabolism of others; e.g. syntrophic cometabolism, competition, production of inhibitors or activators, and predation. It is believed that third‐party organisms easily affect the two‐species relationships and these relationships form the basis of interspecies networks within microbial communities. A microbial network contributes to ‘functional redundancy’ or ‘structural diversity’ and the microbial communities effectively act as a multicellular organism. It is necessary to understand not only the physiological activity of members within microbial communities but also their roles to regulate the activity or population of others. To access the microbial network, we require (i) comprehensive determination of all possible interspecies relationships among microbes, (ii) knock‐out experiments by which certain members can be removed or suppressed, and (iii) supplemental addition of microbes or activation of certain members. Microbial network studies have started using defined microbial communities, i.e. a mixed culture that is composed of three or four species. In order to expand these studies to microflora in nature, microbial ecology requires the help of mathematical biology.     

A rapid and quantitative method for the isolation of gangliosides and neutral glycosphingolipids by DEAE-silica gel chromatography

DEAE-silica gel has been shown to be an improvement over DEAE-Sephadex for the quantitative isolation of gangliosides and neutral glycosphingolipids from animal tissues or cells. Preliminary results indicated that it can also be used for protein separation. Direct comparative studies of DEAE-silica gel with DEAE-Sephadex showed preferences for the former for the following reasons: i) faster flow rate; ii) more rapid equilibration with the starting buffer; iii) easier regeneration; iv) more economical; and v) a lesser susceptibility to microbial attack.     

Marine microorganisms associated with the food of young salmon

The biomass of microorganisms in the digestive tracts of young salmon in the sea of Saanich Inlet was examined by (i) the direct microscopic method, (ii) the plate count method and (iii) the physiological method (microbial biomass assumed from the release of carbon dioxide). By all methods, most microbial biomass in foods was shown to decrease during its stay in the digestive organ. This observation was supported by the detection of many bacteria at different stages of digestion. The microbial biomass was quantitatively influenced by the composition of food ingested and by the sampling time.     

Fungal importance extends beyond litter decomposition in experimental early‐successional streams

Fungi are important decomposers of leaf litter in streams and may have knock‐on effects on other microbes and carbon cycling. To elucidate such potential effects, we designed an experiment in outdoor experimental channels simulating sand‐bottom streams in an early‐successional state. We hypothesized that the presence of fungi would enhance overall microbial activity, accompanied by shifts in the microbial communities associated not only with leaf litter but also with sediments. Fifteen experimental channels received sterile sandy sediment, minimal amounts of leaf litter, and one of four inocula containing either (i) fungi and bacteria, or (ii) bacteria only, or (iii) no microorganisms, or (iv) killed microorganisms. Subsequently, we let water from an early‐successional catchment circulate through the channels for 5 weeks. Whole‐stream metabolism and microbial respiration associated with leaf litter were higher in the channels inoculated with fungi, reflecting higher fungal activity on leaves. Bacterial communities on leaves were also significantly affected. Similarly, increases in net primary production, sediment microbial respiration and chlorophyll a content on the sediment surface were greatest in the channels receiving a fungal inoculum. These results point to a major role of fungal communities in stream ecosystems beyond the well‐established direct involvement in leaf litter decomposition.     

High-throughput molecular analyses of microbiomes as a tool to monitor the wellbeing of aquatic environments

Aquatic environments are the recipients of many sources of environmental stress that trigger both local and global changes. To evaluate the associated risks to organisms and ecosystems more sensitive and accurate strategies are required. The analysis of the microbiome is one of the most promising candidates for environmental diagnosis of aquatic systems. Culture-independent interconnected meta-omic approaches are being increasing used to fill the gaps that classical microbial approaches cannot resolve. Here, we provide a prospective view of the increasing application of these high-throughput molecular technologies to evaluate the structure and functional activity of microbial communities in response to changes and disturbances in the environment, mostly of anthropogenic origin. Some relevant topics are reviewed, such as: (i) the use of microorganisms for water quality assessment, highlighting the incidence of antimicrobial resistance as an increasingly serious threat to global public health; (ii) the crucial role of microorganisms and their complex relationships with the ongoing climate change, and other stress threats; (iii) the responses of the environmental microbiome to extreme pollution conditions, such as acid mine drainage or oil spills. Moreover, protists and viruses, due to their huge impacts on the structure of microbial communities, are emerging candidates for the assessment of aquatic environmental health.     

Evaluating the use of dominant microbial consumers (testate amoebae) as indicators of blanket peatland restoration

Peatlands represent globally-important ecosystems and carbon stores. However, large areas of peatland have been drained for agriculture, or peat has been harvested for use as fuel or in horticulture. Increasingly, these landscapes are being restored through ditch blocking and rewetting primarily to improve biodiversity and promote peat accumulation. To date we have little knowledge of how these interventions influence the microbial communities in peatlands. We compared the responses of dominant microbial consumers (testate amoebae) to drainage ditch restoration relative to unblocked ditches in a UK upland blanket peatland (Migneint, North Wales). Two techniques were used for restoration: (i) dammed ditches with re-profiling; and (ii) dammed ditches with pools of open water behind each dam. Testate communities in the inter-ditch areas changed markedly over time and between treatments illustrating the potential of this group of organisms as indicators of blanket peatland restoration status. However, the responses of testate amoebae to peat rewetting associated with restoration were partially obscured by inter-annual variability in weather conditions through the course of the experiment. Although there was considerable variability in the response of testate amoebae communities to peatland drain blocking, there were clearly more pronounced changes in samples from the dammed and reprofiled treatments including an increase in diversity, and the appearance of unambiguous wet-indicator species in relatively high abundances (including Amphitrema stenostoma, Archerella flavum, Arcella discoides type, Difflugia bacillifera and Difflugia bacillarium). This reflects a shift towards overall wetter conditions across the site and the creation of new habitats. However, water-table was not a significant control on testate amoebae in this case, suggesting a poor relationship between water table and surface moisture in this sloping blanket peatland. Our findings highlight the potential of testate amoebae as bioindicators of peatland restoration success; however, there is a need for caution as mechanisms driving change in the microbial communities may be more complex than first assumed. Several factors need to be taken into account when implementing biomonitoring studies in peatlands including: (i) the natural variability of the peatland ecosystem under changing weather conditions; (ii) any disturbance connected with the restoration procedures; and (iii) the timescales over which the ecosystem responds to the management intervention. Our results also suggest an indicator species approach based on population dynamics may be more appropriate for biomonitoring peatland restoration than examining changes at the community level.     

Genomics, environmental genomics and the issue of microbial species

A microbial species concept is crucial for interpreting the variation detected by genomics and environmental genomics among cultivated microorganisms and within natural microbial populations. Comparative genomic analyses of prokaryotic species as they are presently described and named have led to the provocative idea that prokaryotes may not form species as we think about them for plants and animals. There are good reasons to doubt whether presently recognized prokaryotic species are truly species. To achieve a better understanding of microbial species, we believe it is necessary to (i) re-evaluate traditional approaches in light of evolutionary and ecological theory, (ii) consider that different microbial species may have evolved in different ways and (iii) integrate genomic, metagenomic and genome-wide expression approaches with ecological and evolutionary theory. Here, we outline how we are using genomic methods to (i) identify ecologically distinct populations (ecotypes) predicted by theory to be species-like fundamental units of microbial communities, and (ii) test their species-like character through in situ distribution and gene expression studies. By comparing metagenomic sequences obtained from well-studied hot spring cyanobacterial mats with genomic sequences of two cultivated cyanobacterial ecotypes, closely related to predominant native populations, we can conduct in situ population genetics studies that identify putative ecotypes and functional genes that determine the ecotypes' ecological distinctness. If individuals within microbial communities are found to be grouped into ecologically distinct, species-like populations, knowing about such populations should guide us to a better understanding of how genomic variation is linked to community function.     

Microarray-based analysis of subnanogram quantities of microbial community DNAs by using whole-community genome amplification

Microarray technology provides the opportunity to identify thousands of microbial genes or populations simultaneously, but low microbial biomass often prevents application of this technology to many natural microbial communities. We developed a whole-community genome amplification-assisted microarray detection approach based on multiple displacement amplification. The representativeness of amplification was evaluated using several types of microarrays and quantitative indexes. Representative detection of individual genes or genomes was obtained with 1 to 100 ng DNA from individual or mixed genomes, in equal or unequal abundance, and with 1 to 500 ng community DNAs from groundwater. Lower concentrations of DNA (as low as 10 fg) could be detected, but the lower template concentrations affected the representativeness of amplification. Robust quantitative detection was also observed by significant linear relationships between signal intensities and initial DNA concentrations ranging from (i) 0.04 to 125 ng (r2 = 0.65 to 0.99) for DNA from pure cultures as detected by whole-genome open reading frame arrays, (ii) 0.1 to 1,000 ng (r2 = 0.91) for genomic DNA using community genome arrays, and (iii) 0.01 to 250 ng (r2 = 0.96 to 0.98) for community DNAs from ethanol-amended groundwater using 50-mer functional gene arrays. This method allowed us to investigate the oligotrophic microbial communities in groundwater contaminated with uranium and other metals. The results indicated that microorganisms containing genes involved in contaminant degradation and immobilization are present in these communities, that their spatial distribution is heterogeneous, and that microbial diversity is greatly reduced in the highly contaminated environment.