FEMS Microbiology Ecology

Although the existence of 0.2 µm filterable bacteria has been known since the early 80's, they are not taken into consideration when modeling microbial food webs, due to an overall lack of information concerning this specific size class. According to physiological studies on starvation forms and investigations on small bacterial cells in marine ecosystems, a 0.2 µm filtrate may consist of different phenotypes: starvation forms of typical marine bacteria, ultramicrobacteria or bacterial cells, even larger than 0.2 µm, but flexible enough to pass the nominal filter pore-size. In this pilot study we examined three filtered seawater fractions from the Western Mediterranean Sea (Bay of Calvi, Corsica/France) - the total bacterial population, the bacterial fraction above 0.2 µm and the 0.2 µm filtrate - to investigate the bacterial community structure of each of those fractions by the molecular approach of denaturing gradient gel electrophoresis (DGGE) of 16S rDNA fragments. The analysis of the resulting DGGE profiles revealed different patterns of dominant bands for the 0.2 µm filterable and the total bacterial populations within the samples. Additionally the 0.2 µm filterable bacterial compartment exhibited obvious differences in band patterns for winter and summer samples, which were not observed for the total bacterial fraction. According to the current knowledge concerning the status of 0.2 µm filterable bacteria, DGGE patterns indicate that most of the fragments representing 0.2 µm filterable bacteria were rather starvation forms of marine bacteria than ultramicrobacteria. The sequencing of excised and cloned DNA bands of the DGGE profiles characterized the phylogenetic affiliation of the corresponding 0.2 µm filterable bacteria, clustering mainly with known, typical marine isolates of both alpha-subclass and gamma-subclass of the Proteobacteria and the Cytophaga-Flavobacterium-Bacteroides branch.

The origins of the biological complexity and the factors that regulate the development of community composition, diversity and richness in soil remain largely unknown. To gain a better understanding of how bacterial communities change during soil ecosystem development, their composition and diversity in soils that developed over c. 77 000 years of intermittent aeolian deposition were studied. 16S rRNA gene clone libraries and fatty acid methyl ester (FAME) analyses were used to assess the diversity and composition of the communities. The bacterial community composition changed with soil age, and the overall diversity, richness and evenness of the communities increased as the soil habitat matured. When analysed using a multivariate Bray-Curtis ordination technique, the distribution of ribotypes showed an orderly pattern of bacterial community development that was clearly associated with soil and ecosystem development. Similarly, changes in the composition of the FAMEs across the chronosequence were associated with biomarkers for fungi, actinomycetes and Gram-positive bacteria. The development of the soil ecosystem promoted the development of distinctive microbial communities that were reminiscent of successional processes often evoked to describe change during the development of plant communities in terrestrial ecosystems.

The fermentability of rice bran (RB), alone or in combination with one of two probiotics, by canine faecal microbiota was evaluated in stirred, pH-controlled, anaerobic batch cultures. RB enhanced the levels of bacteria detected by probes Bif164 (bifidobacteria) and Lab158 (lactic acid bacteria); however, addition of the probiotics did not have a significant effect on the predominant microbial counts compared with RB alone. RB sustained levels of Bifidobacterium longum 05 throughout the fermentation; in contrast, Lactobacillus acidophilus 14 150B levels decreased significantly after 5-h fermentation. RB fermentation induced changes in the short-chain fatty acid (SCFA) profile. However, RB combined with probiotics did not alter the SCFA levels compared with RB alone. Denaturing gradient gel electrophoresis analysis of samples obtained at 24 h showed a treatment effect with RB, which was not observed in the RB plus probiotic systems. Overall, the negative controls displayed lower species richness than the treatment systems and their banding profiles were distinct. This study illustrates the ability of a common ingredient found in pet food to modulate the canine faecal microbiota and highlights that RB may be an economical alternative to prebiotics for use in dog food.

The effect of probiotic bacteria Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07 on the composition of the Lactobacillus group, Bifidobacterium and the total bacterial population in feces from young children with atopic dermatitis was investigated. The study included 50 children randomized to intake of one of the probiotic strain or placebo. Microbial composition was characterized by denaturing gradient gel electrophoresis, quantitative PCR and, in a subset of subjects, by pyrosequencing of the 16S rRNA gene. The core population of the Lactobacillus group was identified as Lactobacillus gasseri, Lactobacillus fermentum, Lactobacillus oris, Leuconostoc mesenteroides, while the bifidobacterial community included Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum and Bifidobacterium catenulatum. The fecal numbers of L. acidophilus and B. lactis increased significantly after intervention, indicating survival of the ingested bacteria. The levels of Bifidobacterium correlated positively (P=0.03), while the levels of the Lactobacillus group negatively (P=0.01) with improvement of atopic eczema evaluated by the Severity Scoring of Atopic Dermatitis index. This correlation was observed across the whole study cohort and not attributed to the probiotic intake. The main conclusion of the study is that administration of L. acidophilus NCFM and B. lactis Bi-07 does not affect the composition and diversity of the main bacterial populations in feces.

Micromanipulated filamentous bacteria from bulking and foaming activated sludge morphologically identified as Eikelboom type 0803 were shown to be affiliated to the genus Caldilinea within the phylum Chloroflexi. Specific FISH probes were designed for their in situ detection and quantification in seven Danish wastewater treatment plants with biological nutrient removal. The survey applied all species-specific probes for Chloroflexi of relevance in activated sludge treatment plants as well as the phylum-specific probes. Type 0803 filaments constituted around 20% of the total Chloroflexi population. In four of the treatment plants, type 0803 and type 0092 co-occurred and were the dominating fraction of the Chloroflexi population. In the other plants, most Chloroflexi could not be identified beyond the phylum level, suggesting a yet far larger diversity. On average, for all plants, the total Chloroflexi population constituted 12% of the entire microbial population and seems to play an important structural role in the sludge floc formation. Ecophysiological characterization of type 0803 showed their potential role in macromolecule conversion as evident by high levels of exoenzyme expression. Acetate was not consumed. Glucose was consumed with oxygen, nitrite and nitrite as electron acceptors, suggesting that type 0803 may be a denitrifier. Their surfaces were hydrophobic, explaining their occasional occurrence in foaming incidents.

Evidence is presented demonstrating the ability of Ralstonia eutropha A5 to degrade 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (DDD) aerobically. Strain A5 was able to effect significant transformation of [(14)C]DDD: the hexane extractable radioactivity decreased to approximately 50% of the controls while more than 25% of the total radioactivity became associated with the acidified culture supernatant. There was also an increase in the amount of radioactivity associated with the cell pellet when compared to the biotic control. A meta-fission pathway for the degradation of DDD is proposed based on the recovery of seven chlorinated metabolites identified by gas chromatography-mass spectrometry analysis.

The bacterial diversity of an anaerobic 1,2-dichloropropane (DCP) dechlorinating bioreactor consortium derived from river sediment has been investigated by a combined molecular approach. By using rDNA clone libraries, denaturing gradient gel electrophoresis and quantitative real-time PCR, both Dehalococcoides ethenogenes- and Dehalobacter restrictus-like 16S rDNA sequences were found within the community. Both species are known for reductive dechlorination of tetrachloroethene. Furthermore, numerous yet-uncultured members of the Green non-sulfur bacteria occurred within the consortium. The community analyses over a period of 14 months revealed a clear population shift. D. restrictus 16S rDNA was enriched significantly and became the most abundant rDNA sequence type, suggesting that Dehalobacter spp. play a key role within the reductive dechlorination of DCP in this consortium. We propose the use of this species as an indicator to monitor the transformation process within the bioreactor.

Thermodynamic calculations were coupled with time-series measurements of chemical species (parent and daughter chlorinated solvents, H(2), sulfite, sulfate and methane) to predict the anaerobic transformation of cis-1,2-dichloroethene (cis-1,2-DCE) and 1,2-dichloroethane (1,2-DCA) in constructed wetland soil microcosms inoculated with a dehalorespiring culture. For cis-1,2-DCE, dechlorination occurred simultaneously with sulfite and sulfate reduction but competitive exclusion of methanogenesis was observed due to the rapid H(2) drawdown by the dehalorespiring bacteria. Rates of cis-1,2-DCE dechlorination decreased proportionally to the free energy yield of the competing electron acceptor and proportionally to the rate of H(2) drawdown, suggesting that H(2) competition between dehalorespirers and other populations was occurring, affecting the dechlorination rate. For 1,2-DCA, dechlorination occurred simultaneously with methanogenesis and sulfate reduction but occurred only after sulfite was completely depleted. Rates of 1,2-DCA dechlorination were unaffected by the presence of competing electron-accepting processes. The absence of a low H(2) threshold suggests that 1,2-DCA dechlorination is a cometabolic transformation, occurring at a higher H(2) threshold, despite the high free energy yields available for dehalorespiration of 1,2-DCA. We demonstrate the utility of kinetic and thermodynamic calculations to understand the complex, H(2)-utilizing reactions occurring in the wetland bed and their effect on rates of dechlorination of priority pollutants.

Many organisms have been found to readily oxidize the prevalent contaminant 1,2-dichloroethane (1,2-DCA) to CO2 under aerobic conditions. Some organisms have also been isolated that can reduce 1,2-DCA to ethene via dihaloelimination under anaerobic, fermentative conditions. However, none have been described that can metabolize 1,2-DCA under anoxic, nitrate-reducing conditions. In microcosms prepared from aquifer material and groundwater samples from a contaminated site in eastern Louisiana, USA, 1,2-DCA was observed to degrade with nitrate as the terminal electron acceptor. Nitrate-dependent enrichment cultures were developed from these microcosms that sustained rapid 1,2-DCA degradation rates of up to 500 microM day(-1). This degradation was tightly coupled to complete reduction of nitrate via nitrite to nitrogen gas. A novel 1,2-DCA-degrading organism belonging to the Betaproteobacteria (affiliated with the genus Thauera) was isolated from this enrichment culture. However, degradation rates were much slower in cultures of the isolate than observed in the parent mixed culture. Complete mineralization of 1,2-DCA to CO2 was linked to cell growth and to nitrate reduction in both enrichment and isolated cultures. Monochloroacetate, a putative metabolite of 1,2-DCA degradation, could also be mineralized by these cultures.

The dynamics and composition of microbial communities in the aqueous phase of a model wetland supplied with cis- and trans-1,2-dichloroethenes (DCE)-contaminated groundwater was characterized. PCR-denaturing gradient gel electrophoresis analysis of water samples obtained from different parts of the wetland revealed that changes of the bacterial community structure coincided with a succession of the hydrochemical conditions in the wetland, from oxic towards anoxic conditions. During this transition phase, the appearance of vinyl chloride and ethene correlated with the presence of putative dechlorinating bacteria (Dehalococcoides spp., Geobacter spp. and Dehalobacter spp.). Additionally, a shift of the DCE isotopic composition indicated the progressive prevalence of reductive dechlorination in the wetland. Although the DCE degradation processes varied over time, biodegradation activity was maintained in the wetland system. 16S rRNA gene libraries revealed that Proteobacteria accounted for >50% of 16S rRNA genes clone libraries, whereas approximately 17% of the sequences from the wetland were related to sulphate reducers. Based on a multiple-method approach, this study illustrates the linkage between microbial community dynamics and composition, changes of hydrochemical conditions and processes of DCE degradation in a wetland system.

Gene sequence alignments of the reductive dehalogenases PceA (Dehalospirillum multivorans) and CprA (Desulfitobacterium dehalogenans) were used to develop specific PCR primers binding to conserved regions of these sequences. These primers enabled us to amplify and subsequently sequence cprA-like gene fragments from the chlororespiring species Dehalobacter restrictus, Desulfitobacterium sp. strain PCE1, and D. hafniense. No specific amplicons were obtained from the chlororespiring species D. frappieri, D. chlororespirans, and Desulfomonile tiedjei. Furthermore, we were able to amplify and sequence cprA/pceA-like gene fragments from both trichlorobenzene (TCB)- and 1,2-dichloropropane (DCP)-dechlorinating microbial consortia using the novel primers. Subsequent sequence analysis of the fragments obtained from the microbial consortia revealed a group of four clusters (I-IV). Of these, clusters I and II showed the highest similarities to the cprA-like gene of Dehalobacter restrictus (79.0 and 96.2%, respectively). Cluster III comprised cprA-like sequences found in both the TCB- and the DCP-dechlorinating consortia, whereas sequences of cluster IV were most similar to the pceA gene of Dehalospirillum multivorans (97.8%). Our detection of genes encoding reductive dehalogenases, the key enzymes of chlororespiration, supports the hypothesis that reductive dechlorination of TCB and DCP occurs via a respiratory pathway.

A suite of experiments were conducted to ascertain whether dehalogenation of a model dioxin compound could be stimulated in marine sediments by supplementation with halogenated analogues to enrich for dehalogenating bacteria and if growth by members of the Chloroflexi-like group was associated with dioxin removal. Five halogenated compounds (tetrachlorobenzene, tetrachloroanisole, tetrachlorophenol, tetrachlorobenzoic acid and trichloroacetophenone) were added with 1,2,3,4-tetrachlorodibenzo-p-dioxin (TeCDD) to estuarine sediments from four sites in San Diego Bay and the coast of southern New Jersey to test for dioxin dehalogenation. Most of the halogenated additives were found to stimulate dechlorination of the model dioxin. Molecular analysis of the bacterial population using 16S rRNA and reductive dehalogenase genes indicated that distinct microbial populations were enriched with each halogenated co-amendment. Additionally, Chloroflexi-like ribosomal genes associated with dehalogenation were detected. For example, quantitative real-time PCR analysis of 16S rRNA and reductive dehalogenase gene copy number in the microcosms showed a positive correlation with 1,2,3,4-TeCDD reductive dechlorination in coastal sediments amended with different halogenated additives. These results suggest that specific Chloroflexi-like microorganisms related to Dehalococcoides are involved in 1,2,3,4-TeCDD reductive dechlorination.

Five obligate anaerobes that were most closely related to Clostridium bifermentans, Clostridium celerecrescens, Clostridium saccharolyticum, Clostridium butyricum and Desulfovibrio desulfuricans by their 16S rRNA genes sequences were isolated from enrichment cultures using hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) as a nitrogen source. The above isolates transformed RDX at rates of 24.0, 5.4, 6.2, 2.5, 5.5 mumol h(-1) g (dry weight) of cells(-1), respectively, to nitrite, formaldehyde, methanol, and nitrous oxide. The present results indicate that clostridia are major strains responsible for RDX removal, and all isolates seemed to mainly transform RDX via its initial reduction to MNX and subsequent denitration. Since clostridia are commonly present in soil, we suggest that they may contribute to the removal of RDX in the subsurface (anoxic) soil.

Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) is a toxic explosive known to be resistant to biodegradation. In this study, we found that sediment collected from two unexploded ordnance (UXO) disposal sites (UXO-3, UXO-5) and one nearby reference site (midref) in Hawaii contained anaerobic bacteria capable of removing HMX. Two groups of HMX-removing bacteria were found in UXO-5: group I contained aerotolerant anaerobes and microaerophiles, and group II contained facultative anaerobes. In UXO-3 and midref sediments, HMX-metabolizing bacteria were strictly anaerobic (group III and group IV). Using 16S rRNA sequencing, group I was assigned to a novel phylogenetic cluster of Clostridiales, and groups II and III were related to Paenibacillus and Tepidibacter of Firmicutes, respectively. Group IV bacteria were identified as Desulfovibrio of Deltaproteobacteria. Using [UL-(14)C]-HMX, group IV isolates were found to mineralize HMX (26.8% in 308 d) as determined by liberated (14)CO(2), but negligible mineralization was observed in groups I-III. Resting cells of isolates metabolized HMX to N(2)O and HCHO via the intermediary formation of 1-nitroso-octahydro-3,5,7-trinitro-1,3,5,7-tetrazocine together with methylenedinitramine. These experimental findings suggest that HMX biotransformation occurred either via initial denitration followed by ring cleavage or via reduction of one or more of the N-NO(2) group(s) to the corresponding N-NO bond(s) prior to ring cleavage.

The distributions of bacterial form IA and form IC ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) were investigated using Lowes Cove intertidal mudflat and Damariscotta Lake littoral sediments by PCR amplification of 492-495 bp fragments of the large subunit RuBisCO gene, cbbL. Genomic extracts for amplification were obtained from lake surface (upper 2 mm), mudflat surface (upper 2 mm), subsurface (5-7 cm), and soft-shell clam (Mya arenaria) burrow-wall sediments, as well as from a sulfide-oxidizing mat. Phylogenetic analyses of cbbL clone libraries revealed that Lowes Cove sediments were dominated by form IA cbbL-containing sequences most closely related to cbbL genes of sulfur-oxidizing bacteria or sulfide-oxidizing mats. In contrast, Damariscotta Lake cbbL clones contained primarily form IC cbbL sequences, which typify aerobic CO- and hydrogen-oxidizing facultative chemolithotrophs. Statistical analyses supported clear differentiation of intertidal and lake chemolithotroph communities, and provided evidence for some differentiation among intertidal communities. amova and libshuff analyses of Lowes Cove libraries suggested that M. arenaria burrow-wall sediments did not harbour distinct communities compared with surface and subsurface sediments, but that surface and subsurface libraries displayed moderate differences. The results collectively support a conceptual model in which the relative distribution of form IA- and IC-containing bacterial chemolithotrophs depends on sulfide availability, which could reflect the role of sulfate reduction in sediment organic matter metabolism, or the presence of geothermal sulfide sources.

The Calvin cycle is known to be the major pathway for CO2 fixation, but our current understanding of its occurrence and importance in paddy soils is poor. In this study, the diversity of three ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit genes (cbbLG, cbbLR, cbbM) was investigated by clone library, T-RFLP, qPCR, and enzyme assay in five paddy soils in China. The cbbLG sequences revealed a relatively low level of diversity and were mostly related to the sequences of species from Thiobacillus. In contrast, highly diverse cbbLR and cbbM sequences were dispersed on the phylogenetic trees, and most of them were distantly related to known sequences, even forming separate clusters. Abundances of three cbbL genes ranged from 10(6) to 10(9) copies g(-1) soil, and cbbLR outnumbered cbbM and cbbLG in all soil samples, indicating that cbbLR may play a more important role than other two cbbL genes. Soil properties significantly influenced cbbL diversity in five paddy soils, of which clay content, C/N ratio, CEC, pH, and SOC correlated well with variations in microbial composition and abundance. In summary, this study provided a comparison of three cbbL genes, advancing our understanding of their role in carbon sequestration and nutrient turnover in the paddy soil.

Archaeal communities in many acidic forest soil systems are dominated by a distinct crenarchaeal lineage Group 1.1c. In addition, they are found consistently in other acidic soils including grassland pasture, moorland and alpine soils. To determine whether soil pH is a major factor in determining their presence and abundance, Group 1.1c community size and composition were investigated across a pH gradient from 4.5 to 7.5 that has been maintained for > 40 years. The abundances of Group 1.1c Crenarchaeota, total Crenarchaeota and total bacteria were assessed by quantitative PCR (qPCR) targeting 16S rRNA genes and the diversity of Group 1.1c crenarchaeal community was investigated by denaturing gradient gel electrophoresis (DGGE) and phylogenetic analysis. The abundance of Group 1.1c Crenarchaeota declined as the pH increased, whereas total Crenarchaeota and Bacteria showed no clear trend. Community diversity of Group 1.1c Crenarchaeota was also influenced with different DGGE bands dominating at different pH. Group 1.1c Crenarchaeota were also quantified in 13 other soils representing a range of habitats, soil types and pH. These results exhibited the same trend as that shown across the pH gradient with Group 1.1c Crenarchaeota representing a greater proportion of total Crenarchaeota in the most acidic soils.

The aim of this study was to investigate the diversity and variability of bacterial communities associated with the marine sponge Halichondria panicea with respect to tissue compartmentalization as well as seasonal and small-scale geographic variation. Diversity of microorganisms in sponges was investigated recently, but work on the variability and succession of associated bacterial communities is rare. Despite some information on Pacific and Mediterranean sponges, it is still uncertain whether bacteria and sponges are specifically associated. In this study, H. panicea specimens were sampled throughout the year at different stations around the island of Helgoland (North Sea) and investigated using molecular tools. The bacterial community associated with H. panicea was diverse, consisting of one denaturing gradient gel electrophoresis (DGGE) band occurring in most 'tissue' samples and additional variable bands. Variability was observed between different sponge fractions (i.e. the aquiferous system and the 'tissue'), sampling locations, and sampling dates. A PCR-DGGE specific for the Roseobacter group of marine Alphaproteobacteria displayed low diversity and a marked similarity between all samples. Phylogenetic analysis also pointed to specific Alphaproteobacteria of the Roseobacter group, which was predominant in most sponge 'tissue' samples. We conclude that H. panicea harbour a specific Roseobacter population with varying bacterial co-populations occurring seasonally or on a small-scale geographically, sometimes even dominating the bacterial community.

The thermal springs of Zerka Ma'in, with waters emerging at temperatures up to 63 degrees C, have been of interest to biologists already from the beginning of the 19th century. These waters, springing out from below ground and flowing into the hypersaline Dead Sea, form an isolated environment from a biogeographic point of view. We have investigated the molecular diversity of the cyanobacteria in the springs. The diversity discovered was large, defining operational taxonomic units (OTUs) by a cutoff of 97% similarity; 10 major OTUs were found, including an as yet unidentified cluster of cyanobacteria. The various patterns of similarities of our sequences to others obtained from different thermal environments worldwide led us to rethink the common theories in biogeography. Based on the data obtained, we suggest that there is no constant geographical separation of microorganisms; however, local speciation does occur at a rate dictated mainly by local community dynamics and the rate of entrance of new organisms into the ecosystem.

Bacterial populations and pathways involved in acetate and propionate consumption were studied in anoxic brackish sediment from the Grosser Jasmunder Bodden, German Baltic Sea. Uptake of acetate and propionate from the porewater was studied using stable carbon isotope-labeled compounds. Labeled acetate was not produced as an intermediate during propionate uptake experiments, and propionate consumption was not affected by the addition of acetate. In parallel, incorporation of labeled acetate and propionate into phospholipid-derived fatty acids (PLFA) was studied to indicate bacterial populations involved in the consumption of these substrates. The (13)C-acetate label was mainly recovered in even-numbered PLFA (16:1omega7c, 16:0 and 18:1omega7c). In contrast, primarily odd-numbered PLFA (a15:0, 15:0, 17:1omega6 and 17:0) and the even-numbered i16:0 were labeled after incubation with (13)C-propionate. Although single PLFA labeled with propionate are commonly found in sulfate reducers, the complete PLFA-labeling pattern does not resemble any of the know strains. However, the acetate-labeling pattern is similar to Desulfotomaculum acetoxidans and Desulfofrigus spp., two acetate-consuming, sulfate reducers. In conclusion, our data suggest that acetate and propionate were predominantly consumed by different, specialized groups of sulfate-reducing bacteria.

Vibrio cholerae, the causative agent of cholera, is a natural inhabitant of the aquatic ecosystem. Chironomid (nonbiting midges) egg masses were recently found to harbour V. cholerae non-O1 and non-O139, providing a natural reservoir for the cholera bacterium. Chironomid populations and the presence of V. cholerae in chironomid egg masses were monitored. All V. cholerae isolates were able to degrade chironomid egg masses. The following virulence associated genes were detected in the bacterial isolates: hapA (100%), toxR (100%), hlyA (72%) and ompU (28%). The chironomid populations and the V. cholerae in their egg masses followed the phenological succession and interaction of host-pathogen population dynamics. A peak in the chironomid population was followed by a peak in the V. cholerae population. If such a connection is further substantiated for the pathogenic serogroups of V. cholerae in endemic areas of the disease, it may lead to a better understanding of the role of chironomids as a host for the cholera bacterium.

Abstract Alterations in soil microfungal community structure across a transect between a semi-natural upland grassland and an agriculturally improved enclosure were assessed using an indirect measurement of active fungal biomass (ergosterol), together with a nucleic acid approach, terminal restriction fragment length polymorphism (TRFLP), which was compared to a commonly used but less sensitive community fingerprinting technique, denaturing gradient gel electrophoresis (DGGE). These techniques indicated that there was no reduction in numbers of fungal ribotypes across the floristic transect, despite decreased floristic diversity and a reduction of more than two-fold in ergosterol concentration. Although there were no differences in ribotype number, there was a decrease in diversity and an increase in dominance in only one of the transitional areas. The highest degree of variability within fungal communities was also found in this transitional area, with 84% of ribotypes only being detected in one of three replicates. Comparison of the two fungal community fingerprinting approaches indicated that TRFLP (26-33 ribotypes) was more sensitive for monitoring alterations in fungal community structure than DGGE (13-18 ribotypes). Using a measurement of the relative percentage of each ribotype within communities, a decrease in abundance of prominent ribotypes of the natural grassland soil fungal community was indicated together with an emergence of previously undetected ribotypes towards the improved area. This may have important implications for ecosystem stability or productivity, particularly if agricultural inputs to managed grasslands are suspended.

In order to develop effective bioremediation strategies for radionuclide contaminants, the composition and metabolic potential of microbial communities need to be better understood, especially in highly contaminated subsurface sediments for which little cultivation-independent information is available. In this study, we characterized metabolically active and total microbial communities associated with uranium-contaminated subsurface sediments along geochemical gradients. DNA and RNA were extracted and amplified from four sediment-depth intervals representing moderately acidic (pH 3.7) to near-neutral (pH 6.7) conditions. Phylotypes related to Proteobacteria (Alpha-, Beta-, Delta- and Gammaproteobacteria), Bacteroidetes, Actinobacteria, Firmicutes and Planctomycetes were detected in DNA- and RNA-derived clone libraries. Diversity and numerical dominance of phylotypes were observed to correspond to changes in sediment geochemistry and rates of microbial activity, suggesting that geochemical conditions have selected for well-adapted taxa. Sequences closely related to nitrate-reducing bacteria represented 28% and 43% of clones from the total and metabolically active fractions of the microbial community, respectively. This study provides the first detailed analysis of total and metabolically active microbial communities in radionuclide-contaminated subsurface sediments. Our microbial community analysis, in conjunction with rates of microbial activity, points to several groups of nitrate-reducers that appear to be well adapted to environmental conditions common to radionuclide-contaminated sites.

Autotrophic ammonia-oxidizing bacteria were considered to be responsible for the majority of ammonia oxidation in soil until the recent discovery of the autotrophic ammonia-oxidizing archaea. To assess the relative contributions of bacterial and archaeal ammonia oxidizers to soil ammonia oxidation, their growth was analysed during active nitrification in soil microcosms incubated for 30 days at 30 degrees C, and the effect of an inhibitor of ammonia oxidation (acetylene) on their growth and soil nitrification kinetics was determined. Denaturing gradient gel electrophoresis (DGGE) analysis of bacterial ammonia oxidizer 16S rRNA genes did not detect any change in their community composition during incubation, and quantitative PCR (qPCR) analysis of bacterial amoA genes indicated a small decrease in abundance in control and acetylene-containing microcosms. DGGE fingerprints of archaeal amoA and 16S rRNA genes demonstrated changes in the relative abundance of specific crenarchaeal phylotypes during active nitrification. Growth was also indicated by increases in crenarchaeal amoA gene copy number, determined by qPCR. In microcosms containing acetylene, nitrification and growth of the crenarchaeal phylotypes were suppressed, suggesting that these crenarchaea are ammonia oxidizers. Growth of only archaeal but not bacterial ammonia oxidizers occurred in microcosms with active nitrification, indicating that ammonia oxidation was mostly due to archaea in the conditions of the present study.

Crop plants genetically modified for the expression of Bacillus thuringiensis (Bt) insecticidal toxins have broad appeal for reducing insect damage in agricultural systems, yet questions remain about the impact of Bt plants on symbiotic soil organisms. Here, arbuscular mycorrhizal fungal (AMF) colonization of transgenic maize isoline Bt 11 (expressing Cry1Ab) and its non-Bt parental line (Providence) was evaluated under different fertilizer level and spore density scenarios. In a three-way factorial design, Bt 11 and non-Bt maize were inoculated with 0, 40, or 80 spores of Glomus mosseae and treated weekly with 'No' (0 g L(-1) ), 'Low' (0.23 g L(-1) ), or 'High' (1.87 g L(-1) ) levels of a complete fertilizer and grown for 60 days in a greenhouse. While no difference in AMF colonization was detected between the Bt 11 and Providence maize cultivars in the lower spore/higher fertilizer treatments, microcosm experiments demonstrated a significant reduction in AMF colonization in Bt 11 maize roots in the 80 spore treatments when fertilizer was limited. These results confirm previous work indicating an altered relationship between this Bt 11 maize isoline and AMF and demonstrate that the magnitude of this response is strongly dependent on both nutrient supply and AMF spore inoculation level.