Automated image analysis for quantitative fluorescence in situ hybridization with environmental samples

When fluorescence in situ hybridization (FISH) analyses are performed with complex environmental samples, difficulties related to the presence of microbial cell aggregates and nonuniform background fluorescence are often encountered. The objective of this study was to develop a robust and automated quantitative FISH method for complex environmental samples, such as manure and soil. The method and duration of sample dispersion were optimized to reduce the interference of cell aggregates. An automated image analysis program that detects cells from 4',6'-diamidino-2-phenylindole (DAPI) micrographs and extracts the maximum and mean fluorescence intensities for each cell from corresponding FISH images was developed with the software Visilog. Intensity thresholds were not consistent even for duplicate analyses, so alternative ways of classifying signals were investigated. In the resulting method, the intensity data were divided into clusters using fuzzy c-means clustering, and the resulting clusters were classified as target (positive) or nontarget (negative). A manual quality control confirmed this classification. With this method, 50.4, 72.1, and 64.9% of the cells in two swine manure samples and one soil sample, respectively, were positive as determined with a 16S rRNA-targeted bacterial probe (S-D-Bact-0338-a-A-18). Manual counting resulted in corresponding values of 52.3, 70.6, and 61.5%, respectively. In two swine manure samples and one soil sample 21.6, 12.3, and 2.5% of the cells were positive with an archaeal probe (S-D-Arch-0915-a-A-20), respectively. Manual counting resulted in corresponding values of 22.4, 14.0, and 2.9%, respectively. This automated method should facilitate quantitative analysis of FISH images for a variety of complex environmental samples.     

Removal of Nonvolatile Hydrophobic Compounds from Artificially and Naturally Contaminated Soils by Column Flotation

Removal of a nonvolatile paraffin oil from spiked soils using column flotation with countercurrent bubbles was explored at both ambient and elevated temperatures. Up to 80% of the contaminant was separated from the coarse fraction (250 to 800?µm) by flotation at 45°C using aqueous solutions of anionic and nonionic surfactants or alkali salt as collectors. With the 75 to 800?µm fraction, removal efficiencies of up to 65% was achieved. Sodium dodecyl-sulfate and Triton 100X at 50?ppm concentrations as well as sodium carbonate at pH 10 were found to yield similar removal efficiencies. Same surfactants were tested in soil washing experiments at similar and higher dosages. Removal efficiency by flotation was higher than those obtained by soil washing in all cases. In addition, as high surfactant dosage are not used in flotation, unlike in the case of soil washing, the problem of formation of stable emulsions was absent. Experiments with soil polluted by hydrocarbons from a contaminated site demonstrated the feasibility of the flotation process for decontamination of coarse (250 to 830?µm) fractions. A 70% reduction of petroleum hydrocarbon in soil was achieved as a result of flotation at 45°C using the above surfactants.     

Alpha- and beta-Proteobacteria control the consumption and release of amino acids on lake snow aggregates

We analyzed the composition of aggregate (lake snow)-associated bacterial communities in Lake Constance from 1994 until 1996 between a depth of 25 m and the sediment surface at 110 m by fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes of various specificity. In addition, we experimentally examined the turnover of dissolved amino acids and carbohydrates together with the microbial colonization of aggregates formed in rolling tanks in the lab. Generally, between 40 and more than 80% of the microbes enumerated by DAPI staining (4',6'-diamidino-2-phenylindole) were detected as Bacteria by the probe EUB338. At a depth of 25 m, 10.5% +/- 7.9% and 14.2% +/- 10.2% of the DAPI cell counts were detected by probes specific for alpha- and beta-Proteobacteria. These proportions increased to 12.0% +/- 3.3% and 54.0% +/- 5.9% at a depth of 50 m but decreased again at the sediment surface at 110 m to 2.7% +/- 1.4% and 41.1% +/- 8.4%, indicating a clear dominance of beta-Proteobacteria at depths of 50 and 110 m, where aggregates have an age of 3 to 5 and 8 to 11 days, respectively. From 50 m to the sediment surface, cells detected by a Cytophaga/Flavobacteria-specific probe (CF319a) comprised increasing proportions up to 18% of the DAPI cell counts. gamma-Proteobacteria always comprised minor proportions of the aggregate-associated bacterial community. Using only two probes highly specific for clusters of bacteria closely related to Sphingomonas species and Brevundimonas diminuta, we identified between 16 and 60% of the alpha-Proteobacteria. In addition, with three probes highly specific for close relatives of the beta-Proteobacteria Duganella zoogloeoides (formerly Zoogloea ramigera), Acidovorax facilis, and Hydrogenophaga palleroni, bacteria common in activated sludge, 42 to 70% of the beta-Proteobacteria were identified. In the early phase (<20 h) of 11 of the 15 experimental incubations of aggregates, dissolved amino acids were consumed by the aggregate-associated bacteria from the surrounding water. This stage was followed by a period of 1 to 3 days during which dissolved amino acids were released into the surrounding water, paralleled by an increasing dominance of beta-Proteobacteria. Hence, our results show that lake snow aggregates are inhabited by a community dominated by a limited number of alpha- and beta-Proteobacteria, which undergo a distinct succession. They successively decompose the amino acids bound in the aggregates and release substantial amounts into the surrounding water during aging and sinking.     

Spectral imaging detection and counting of microbial cells in marine sediment

Semiautomated detection and counting techniques for microbial cells in soil and marine sediment using microscopic-spectral-imaging analysis were developed. Microbial cells in microscopic fields were selectively detected from other fluorescent particles by their fluorescent spectrum, based on the spectral shift between the conjunction and nonconjunction of DNA fluorochrome (SYBR Green II) with nucleic acids. Using this technique, microbial cells could be easily detected in soil and 30-cm deep sediment samples from Tokyo Bay, both of which contain particles other than microbial cells. Total cell density was semiautomatically estimated at 1-6 x 10(9) cells cm(-3) of sediment sampled at different depths in Tokyo Bay, which corresponded to 65-106% (mean 88%) of visual direct counting. This technique may be useful for detecting microbial cells in soil and sediment samples from the deeper subsurface environment.     

The prokaryotic community of subglacial bottom sediments of Antarctic Lake Untersee: Detection by cultural and direct microscopic techniques

The heterotrophic mesophilic microbial component was studied in microbial communities of the samples of frozen regolith collected from the glacier near Lake Untersee collected in 2011 during the joint Russian-American expedition to central Dronning Maud Land (Eastern Antarctica). Cultural techniques revealed high bacterial numbers in the samples. For enumeration of viable cells, the most probable numbers (MPN) method proved more efficient than plating on agar media. Fluorescent in situ hybridization with the relevant oligonucleotide probes revealed members of the groups Eubacteria (Actinobacteria, Firmicutes) and Archaea. The application of the methods of cell resuscitation, such as the use of diluted media and prevention of oxidative stress, did not result in a significant increase in the numbers of viable cells retrieved from subglacial sediment samples. Our previous investigations demonstrated the necessity for special procedures for efficient reactivation of the cells from microbial communities of replace with buried soil and permafrost samples collected in the Arctic zone. The differing responses to the special resuscitation procedures may reflect the differences in the physiological and morphological state of bacterial cells in microbial communities subject to continuous or periodic low temperatures and dehydration.     

rRNA sequence-based scanning electron microscopic detection of bacteria

A new scanning electron microscopic method was developed for gaining both phylogenetic and morphological information about target microbes using in situ hybridization with rRNA-targeted oligonucleotide probes (SEM-ISH). Target cells were hybridized with oligonucleotide probes after gold labeling. Gold enhancement was used for amplification of probe signals from hybridized cells. The hybridized cells released a strong backscatter electron signal due to accumulation of gold atoms inside cells. SEM-ISH was applied to analyze bacterial community composition in freshwater samples, and bacterial cell counts determined by SEM-ISH with rRNA-targeted probes for major phyla within the domain Bacteria were highly correlated to those by fluorescent in situ hybridization (FISH). The bacterial composition on surface of river sediment particles before and after cell dispersion treatment by sonication was successfully revealed by SEM-ISH. Direct enumeration of bacterial cells on the surface of sonicated sediment particles by SEM-ISH demonstrated that members of Cytophaga-Flavobacterium existed tightly on the surface of particles. SEM-ISH allows defining the number and distribution of phylogenetically defined cells adherent to material surfaces, which is difficult in FISH, and it gives new insight into electron microscopic studies of microorganisms in their natural environment.     

Desulfurization of coal by microbial column flotation

Twenty-three strains capable of oxidizing iron were isolated from coal and ore storage sites as well as coal and ore mines, volcanic areas, and hot spring. Four strains were found to have high iron-oxidizing activity. One strain (T-4) was selected for this experiment since the strain showed the fastest leaching rate of iron and sulfate from pyrite among the four strains. The T-4 strain was assigned for Thiobacillus ferrooxidans from its cultural and morphological characteristics.Bacterial treatment was applied to column flotation. An increase of cell density in the microbial column flotation resulted in the increase of pyrite removal from a coal-pyrite mixture (high sulfur imitated coal) with corresponding decrease of coal recovery. The addition of kerosene into the microbial column flotation increased the recovery of the imitated coal from 55% (without kerosene) to 81% (with 50 muL/L kerosene) with the reduction of pyrite sulfur content from 11% (feed coal) to 3.9% (product coal). The kerosene addition could reduce the pyritic sulfur content by collecting the coal in the recovery. However, the addition could not enhance separation of pyrite from the coal-pyrite mixture, since pyrite rejection was not affected by the increase of the kerosene addition. An excellent separation was obtained by the microbial flotation using a long column which had a length-diameter (L/D) ratio of 12.7. The long column flotation reduced the pyritic sulfur content from 11% (feed coal) to 1.8% (product coal) when 80% of the feed coal was recovered without the kerosene addition. The long column flotation not only attained an excellent separation but also reduced the amount of cells for desulfurization to as little as one-tenth of the reported amount. (c) 1994 John Wiley & Sons, Inc.     

Electron microscopic detection and in situ characterization of bacterial nanoforms in extreme biotopes

The morphology, ultrastructure, and quantity of bacterial nanoforms were studied in extreme biotopes: East Siberia permafrost soil (1–3 Ma old), petroleum-containing slimes (35 years old), and biofilms from subsurface oil pipelines. The morphology and ultrastructure of microbial cells in natural biotopes in situ were investigated by high-resolution transmission electron microscopy and various methods of sample preparation: ultrathin sectioning, cell replicas, and cryofractography. It was shown that the biotopes under study contained high numbers of bacterial nanoforms (29–43% of the total number of microorganisms) that could be assigned to ultramicrobacteria due to their size (diameter of ≤ 0.3 μm and volume of ≤ 0.014 μm³) and structural characteristics (the presence of the outer and cytoplasmic membranes, nucleoid, and cell wall, as well as their division patterns). Seven different morphostructural types of nanoforms of vegetative cells, as well as nanospores and cyst-like cells were described, potentially representing new species of ultramicrobacteria. In petroleum-containing slimes, a peculiar type of nanocells was discovered, gram-negative cells mostly 0.18–0.20 × 0.20–0.30 μm in size, forming in situ spherical aggregates (microcolonies) of dividing cells. The data obtained promoted the isolation of pure cultures of ultramicrobacteria from petroleum-containing slimes; they resembled the ultramicrobacterium observed in situ in their morphology and ultrastructure.     

Electron microscopic detection and in situ characterization of bacterial nanoforms in extreme biotopes

The morphology, ultrastructure, and quantity of bacterial nanoforms were studied in extreme biotopes: East Siberia permafrost soil (1-3 Ma old), petroleum-containing slimes (35 years old), and biofilms from subsurface oil pipelines. The morphology and ultrastructure of microbial cells in natural biotopes in situ were investigated by high-resolution transmission electron microscopy and various methods of sample preparation: ultrathin sectioning, cell replicas, and cryofractography. It was shown that the biotopes under study contained high numbers of bacterial nanoforms (29-43% of the total number of microorganisms) that could be assigned to ultramicrobacteria due to their size (diameter of < or =0.3 microm and volume of < or =0.014 microm3) and structural characteristics (the presence of the outer and cytoplasmic membranes, nucleoid, and cell wall, as well as their division patterns). Seven different morphostructural types of nanoforms of vegetative cells, as well as nanospores and cyst-like cells were described, potentially representing new species of ultramicrobacteria. In petroleum-containing slimes, a peculiar type of nanocells was discovered, gram-negative cells mostly 0.18-0.20 x 0.20-0.30 microm in size, forming spherical aggregates (microcolonies) of dividing cells in situ. The data obtained promoted the isolation of pure cultures of ultramicrobacteria from petroleum-containing slimes; they resembled the ultramicrobacterium observed in situ in their morphology and ultrastructure.     

Investigation of lotic microbial aggregates by a combined technique of fluorescent in situ hybridization and lectin-binding-analysis.

A technique combining fluorescent in situ hybridization and lectin-binding-analysis (FISH-LBA) was developed and applied for the simultaneous detection of cellular components and glycoconjugates in lotic microbial aggregates (river snow). River snow aggregates were directly collected from the bulk water phase into coverslip chambers, in which the complete procedure including fixation, fluorescent in situ hybridization, lectin-binding and optical analysis by confocal laser scanning microscopy was performed. Neither autofluorescence originating from phyotosynthetic organisms nor inorganic particles did negatively interfere with the FISH-LBA technique. In river snow samples obtained from the river Elbe, Germany, distinct compartments of the river snow structure could be visualized with FITC-labelled lectins from Triticum vulgaris, Limulus polyphemus, Arachis hypogaea, Phaseolus vulgaris and Pseudomonas aeruginosa, binding to frequently occurring saccharide residues in the river snow matrix. The analysis could be performed on different levels of complexity. The combined technique visualized bacteria of different phylogenetic groups in the entire river snow structure as well as glycoconjugate components linked with various microcolonies. Different lectins stained slime layers and cell-envelopes of individual eukaryotic and prokaryotic cells. Consequently, application of the FISH-LBA technique allows the linkage between cellular and glycoconjugate identity in complex microbial communities.     

rRNA Sequence-Based Scanning Electron Microscopic Detection of Bacteria

A new scanning electron microscopic method was developed for gaining both phylogenetic and morphological information about target microbes using in situ hybridization with rRNA-targeted oligonucleotide probes (SEM-ISH). Target cells were hybridized with oligonucleotide probes after gold labeling. Gold enhancement was used for amplification of probe signals from hybridized cells. The hybridized cells released a strong backscatter electron signal due to accumulation of gold atoms inside cells. SEM-ISH was applied to analyze bacterial community composition in freshwater samples, and bacterial cell counts determined by SEM-ISH with rRNA-targeted probes for major phyla within the domain Bacteria were highly correlated to those by fluorescent in situ hybridization (FISH). The bacterial composition on surface of river sediment particles before and after cell dispersion treatment by sonication was successfully revealed by SEM-ISH. Direct enumeration of bacterial cells on the surface of sonicated sediment particles by SEM-ISH demonstrated that members of Cytophaga-Flavobacterium existed tightly on the surface of particles. SEM-ISH allows defining the number and distribution of phylogenetically defined cells adherent to material surfaces, which is difficult in FISH, and it gives new insight into electron microscopic studies of microorganisms in their natural environment.     

Microorganisms in Unamended Soil as Observed by Various Forms of Microscopy and Staining

A light-diffraction microscope was modified to allow sequential viewing of the microorganisms in a soil smear by transmitted, reflected, and reflected-polarized incandescent light and by reflected ultraviolet light. Observations were also made by conventional incandescent and ultraviolet transmitted-light microscopy. All results for the various forms of bright-field microscopy with stained and unstained soils were in agreement, but they differed from the results obtained for two types of ultraviolet-fluorescence microscopy. The latter proved to be nonspecific for in situ soil microorganisms. Capsule-like areas were noted surrounding many of the resident microbial cells of soil when viewed by the various forms of bright-field microscopy. These areas could not be stained or removed by a variety of treatments, but they apparently often did take up stain after in situ soil growth had been initiated. It was concluded that these areas are not capsules but may represent a structural component of nonmultiplying microbial cells in soil.     

Role of proteins in soil carbon and nitrogen storage: controls on persistence

Mechanisms of soil organic carbon (C) and nitrogen (N) stabilization are of great interest, due to the potential for increased CO₂ release from soil organic matter (SOM) to the atmosphere as a result of global warming, and because of the critical role of soil organic N in controlling plant productivity. Soil proteins are recognized increasingly as playing major roles in stabilization and destabilization of soil organic C and N. Two categories of proteins are proposed: detrital proteins that are released upon cell death and functional proteins that are actively released into the soil to fulfill specific functions. The latter include microbial surface-active proteins (e.g., hydrophobins, chaplins, SC15, glomalin), many of which have structures that promote their persistence in the soil, and extracellular enzymes, responsible for many decomposition and nutrient cycling transformations. Here we review information on the nature of soil proteins, particularly those of microbial origin, and on the factors that control protein persistence and turnover in the soil. We discuss first the intrinsic properties of the protein molecule that affect its stability, next possible extrinsic stabilizing influences that arise as the proteins interact with other soil constituents, and lastly controls on accessibility of proteins at coarser spatial scales involving microbial cells, clay particles, and soil aggregates. We conclude that research at the interface between soil science and microbial physiology will yield rapid advances in our understanding of soil proteins. We suggest as research priorities determining the relative abundance and turnover time (age) of microbial versus plant proteins and of functional microbial proteins, including surface-active compounds.     

Influence of maize root mucilage on soil aggregate stability

This study was undertaken to determine the effects of root exudates on soil aggregate stability. Root mucilage was collected from two-month old maize plants (Zea mays L.) Mucilage and glucose solutions were added at a rate of 2.45 g C kg⁻¹ dry soil to silty clay and silt loam soils. Amended soils, placed in serum flasks, were incubated for 42 d with a drying-wetting cycle after 21 d. Evolved CO₂ was measured periodically as well as the water-stable aggregates and soluble sugar and polysaccharide content of the soil. In mucilage-amended soils CO₂ evolution started with a lag phase of 2–3 days, which was not observed in glucose-amended soils. There was then a sharp increase in evolved CO₂ up to day 7. During the second incubation period there were only small differences in evolved C between treatments. Incorporation of mucilage in both soils resulted in a spectacular and immediate increase in soil aggregate stability. Thereafter, the percent of water-stable aggregates quickly decreased parallel to microbial degradation. On completion of the incubation, aggregate stability in the silty clay soil was still significantly higher in the presence of mucilage than in the control. This work supports the assumption that freshly released mucilage is able to stick very rapidly to soil particles and may protect the newly formed aggregates against water destruction. On the silty clay, microbial activity contributes to a stabilization of these established organo-mineral bounds.     

四种土壤微生物总DNA的纯化方法的比较

比较了4种从土壤中直接抽提的微生物总DNA的纯化方法,实验结果表明1 %的琼脂糖凝胶电泳纯化方法及葡聚糖凝胶G 2 0 0离心层析纯化方法均不能完全纯化从土壤中抽提的微生物总DNA。若将直接抽提的总DNA先经葡聚糖凝胶G 2 0 0离心层析纯化,再用1 %的琼脂糖凝胶电泳纯化,则能取得较好的纯化效果。含2 %PVP的1 %琼脂糖凝胶电泳纯化,用DNA凝胶回收试剂盒回收后没有得到纯化后的土壤微生物总DNA。     

Microbial properties of soil aggregates created by earthworms and other factors: spherical and prismatic soil aggregates from unreclaimed post-mining sites

Soil aggregates between 2 and 5 mm from 35- and 45-year-old unreclaimed post-mining sites near Sokolov (Czech Republic) were divided into two groups: spherical and prismatic. X-ray tomography indicated that prismatic aggregates consisted of fragments of claystone bonded together by amorphous clay and roots while spherical aggregates consisted of a clay matrix and organic fragments of various sizes. Prismatic aggregates were presumed to be formed by plant roots and physical processes during weathering of Tertiary mudstone, while earthworms were presumed to contribute to the formation of spherical aggregates. The effects of drying and rewetting and glucose addition on microbial respiration, microbial biomass, and counts of bacteria in these aggregates were determined. Spherical aggregates contained a greater percentage of C and N and a higher C-to-N ratio than prismatic ones. The C content of the particulate organic matter was also higher in the spherical than in the prismatic aggregates. Although spherical aggregates had a higher microbial respiration and biomass, the growth of microbial biomass in spherical aggregates was negatively correlated with initial microbial biomass, indicating competition between bacteria. Specific respiration was negatively correlated with microbial biomass. Direct counts of bacteria were higher in spherical than in prismatic aggregates. Bacterial numbers were more stable in the center than in the surface layers of the aggregates. Transmission electron microscopy indicated that bacteria often occurred as individual cells in prismatic aggregates but as small clusters of cells in spherical aggregates. Ratios of colony forming units (cultivatable bacteria) to direct counts were higher in spherical than in prismatic aggregates. Spherical aggregates also contained faster growing bacteria.     

Survival and colonisation potential of photoautotrophic microorganisms within a glacierised catchment on Svalbard, High Arctic

The survival and colonisation potential of photoautotrophic microbes (cyanobacteria and microalgae) were investigated in three terrestrial environments within a glacierised catchment on Svalbard: old vegetation-covered soil, recently deglaciated barren soil and subglacial sediments. One-year reciprocal transplant incubations of photoautotrophic microbial communities from the three soil/sediment environments were conducted in order to reveal the autochthonous or allochthonous origin of the present photoautotrophs. The abundance and taxonomic composition of photoautotrophic microbes and their changes over time and between soil/sediment types and physico-chemical characteristics of the soils/sediments were determined. The recovery time of a photoautotrophic community by import of cells was between several months in subglacial and vegetated soils and up to 27 years in proglacial soils. No active growth was recorded in subglacial sediments, whilst positive growth, and so the potential for autochthonous recovery, was found in proglacial and vegetated soils. The most suitable environment for the survival of transplanted microbes was provided in proglacial soil. We show here that the new proglacial substrata can be successfully colonised by photoautotrophic microbes, and that input of allochthonous cells may, in some cases, exceed in situ microbial growth. Whilst the subglacial environment is rather a conduit for photoautotrophic microbes than a place of growth and production, the supply of viable photoautotrophs in it is relatively high and may serve as a significant resource of nutrients for subglacial microbial communities.     

Release of microorganisms from soil with respect to transmission electron microscopy viewing and plate counts

Microorganisms were detached and washed from soil by various procedures involving blending and sonication of the soil in water or pyrophosphate solution, followed by successive low-speed, centrifugal-washing separations of the suspended cells from the soil debris. Some of these procedures were previously used for separating and concentrating cells from soil for transmission electron microscopy viewing. Exhaustive applications of these procedures separated up to 27% of the platable cells from the soil. Based on filterability, these cells either were no longer attached to soil particles, or were attached to very small particles. The cells fractionating with the soil debris, however, seemed to be strongly attached to it or to other cells so that they were not filterable. Laboratory-grown cultures added to sterile and non-sterile soil did not attach to the soil materials and were easily recovered from the soil even though low-speed centrifugations were being used. Electron microscopy evidence for cells released and concentrated from non-inoculated natural soil, and for cells remaining with the soil debris, suggests that the very small, probably non-platable, cells tend to release more easily than do cells in the size range of 0.3 to 0.5 μm in diameter, and that cells larger than this, including bacterial and fungal spores, are more difficult to separate from soil. Plating data for heated preparations are in agreement with this for bacterial spores. The results are considered in relation to the validity of plate counts and direct soil transmission electron microscopy for evaluating the microbial flora of soil. This research was authorized for publication as paper no. 5131 in the journal series of the Pennsylvania Agricultural Experiment Station on 7/20/76.     

Microbial community structure and ecology of subglacial sediments in two polythermal Svalbard glaciers characterized by epifluorescence microscopy and PLFA

Biological and physico-chemical characteristics of subglacial sediments were studied in Svalbard. Sediment from close proglacial and supraglacial environments was used for a comparison. Viable bacteria, cyanobacteria and microalgae were detected in subglacial sediments from two polythermal glaciers using epifluorescence microscopy and phospholipid fatty acid (PLFA) analyses. The subglacial samples were generally of higher pH values, coarser texture and lower water content, organic matter, organic carbon, and nitrogen compared to proglacial and supraglacial sediments). Bacterial counts of 1.6 × 10⁷ cells mg^{− 1} OM (organic matter) were found. Cyanobacteria and algae were also of low abundance [4.2 cells mg^{− 1} DW (dry weight)]. Cyanobacteria comprised the major proportion of the autophotothrophic assemblages of subglacial soils. Deglaciated soils were similar to subglacial sediment in physico-chemical properties and microbial structure and numbers, unlike soil from vegetated sites or cryoconite sediment. In subglacial and deglaciated soil, relatively low diversity of microorganisms and low substrate availability was detected by PLFA analyses. Good accordance in microbial community structure assessments between epifluorescence microscopy and PLFA analyses was found. Our results suggest that the subglacial microbial populations can be divided into two groups: autochthonous microorganims (chemoheterotrophic bacteria) and allochthonous that retain the ability to proliferate and give rise to active population when conditions become favorable. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Influence of co-substrates on structure of microbial aggregates in long-chain fatty acid-fed anaerobic digesters

AIMS: The purpose of this study was to investigate the influence of co-substrates, such as glucose and cysteine, on the structure of microbial aggregates in anaerobic digesters treating oleate, a long-chain fatty acid (LCFA). METHODS AND RESULTS: Transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) were used to examine the structure of microbial aggregates. Fluorescence in situ hybridization (FISH) techniques were also used to characterize and localize the different trophic groups present in the aggregates. Oleate was found to inhibit the methanogenic activity and formation of granular biomass in digesters. The addition of co-substrates, such as glucose and cysteine either singly or in combination, increased the methanogenic activity and formation of granular biomass. Glucose was more effective than cysteine in reducing the inhibition by oleate on the methanogenic bacteria and in enhancing the formation of granules. CONCLUSIONS: The addition of nutrient substrate, such as glucose and cysteine could decrease the toxicity of LCFA on anaerobic granulation. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that the addition of other substrates might decrease the toxicity of LCFA on the granulation of biomass in anaerobic digesters and enhance methanogenic activity. A combination of TEM, CLSM and FISH techniques provides a better tool for visualizing microbial aggregates and for differentiating and localizing different microbial groups within these aggregates.