Parallel Microbial Ecology of Pasteuria and Nematode Species in Scottish Soils.

Pasteuria spp. are endospore forming bacteria which act as natural antagonists to many of the most economically significant plant parasitic nematodes (PPNs). Highly species-specific nematode suppression may be observed in soils containing a sufficiently high density of Pasteuria spp. spores. This suppression is enacted by the bacteria via inhibition of root invasion and sterilization of the nematode host. Molecular methods for the detection of Pasteuria spp. from environmental DNA (eDNA) have been described; however, these methods are limited in both scale and in depth. We report the use of small subunit rRNA gene metabarcoding to profile Pasteuria spp. and nematode communities in parallel. We have investigated Pasteuria spp. population structure in Scottish soils using eDNA from two sources: soil extracted DNA from the second National Soil Inventory of Scotland (NSIS2); and nematode extracted DNA collected from farms in the East Scotland Farm Network (ESFN). We compared the Pasteuria spp. community culture to both nematode community structure and the physiochemical properties of soils. Our results indicate that Pasteuria spp. populations in Scottish soils are broadly dominated by two sequence variants. The first of these aligns with high identity to Pasteuria hartismeri, a species first described parasitizing Meloidogyne ardenensis, a nematode parasite of woody and perennial plants in northern Europe. The second aligns with a Pasteuria-like sequence which was first recovered from a farm near Edinburgh which was found to contain bacterial feeding nematodes and Pratylenchus spp. encumbered by Pasteuria spp. endospores. Further, soil carbon, moisture, bulk density, and pH showed a strong correlation with the Pasteuria spp. community composition. These results indicate that metabarcoding is appropriate for the sensitive, specific, and semi-quantitative profiling of Pasteuria species from eDNA.

Fate of tetracycline and sulfonamide resistance genes in a grassland soil amended with different organic fertilizers.

This study provided an assessment of the environmental fate of antibiotic resistance genes (ARGs) in a Scottish grassland field repeatedly treated with different organic fertilizers. The impacts of manure, biosolids and municipal food-derived compost on the relative abundances of tetracycline ARGs (tetA, tetB, tetC, tetG and tetW), sulfonamide ARGs (sul1 and sul2) and class 1 integron-integrase gene (IntI1) in soils were investigated, with inorganic fertilizer (NPK) as a comparison. The background soil with a history of low intensity farming showed a higher total relative abundance of tet ARGs over sul ARGs, with tetracycline efflux genes occurring in a higher frequency. In all treatments, the relative abundances of most ARGs detected in soils decreased over time, especially IntI1 and tet ARGs. This general attenuation of soil ARGs is a reflection of changes in the soil microbial community, which is supported by the result that almost all the soils at the end of the experiment had different bacterial communities from the untreated soil at the beginning of the experiment. Multiple applications of organic fertilizers to some extent counteracted the decreasing trend of soil ARGs relative abundances, which resulted in higher ARGs relative abundances in comparison to NPK, either by a lesser decrease of IntI1 and tet ARGs or an increase of sul ARGs. The enhancement of existing soil ARG prevalence by organic fertilizers was strongly dependent on the organic fertilizer type and the particular ARG. Compost contained the lowest relative abundance of inherent ARGs and had the least effect on the soil ARG decrease after application. The relative increase of tet ARGs caused by biosolids was larger than that of sul ARGs, while manure caused the opposite effect. Fertilization practices did not exert effective impacts on the soil bacterial community, although it caused significant changes in the profile of the ARG pool. Organic fertilization may thus accelerate the dissemination of ARGs in soil mainly through horizontal gene transfer (HGT), consistent with the enrichment of IntI1 in organic fertilized soils.

It is elemental: soil nutrient stoichiometry drives bacterial diversity.

It is well established that resource quantity and elemental stoichiometry play major roles in shaping below and aboveground plant biodiversity, but their importance for shaping microbial diversity in soil remains unclear. Here, we used statistical modeling on a regional database covering 179 locations and six ecosystem types across Scotland to evaluate the roles of total carbon (C), nitrogen (N) and phosphorus (P) availabilities and ratios, together with land use, climate and biotic and abiotic factors, in determining regional scale patterns of soil bacterial diversity. We found that bacterial diversity and composition were primarily driven by variation in soil resource stoichiometry (total C:N:P ratios), itself linked to different land uses, and secondarily driven by other important biodiversity drivers such as climate, soil spatial heterogeneity, soil pH, root influence (plant-soil microbe interactions) and microbial biomass (soil microbe-microbe interactions). In aggregate, these findings provide evidence that nutrient stoichiometry is a strong predictor of bacterial diversity and composition at a regional scale.

The Role of Microbial Community Composition in Controlling Soil Respiration Responses to Temperature.

Rising global temperatures may increase the rates of soil organic matter decomposition by heterotrophic microorganisms, potentially accelerating climate change further by releasing additional carbon dioxide (CO2) to the atmosphere. However, the possibility that microbial community responses to prolonged warming may modify the temperature sensitivity of soil respiration creates large uncertainty in the strength of this positive feedback. Both compensatory responses (decreasing temperature sensitivity of soil respiration in the long-term) and enhancing responses (increasing temperature sensitivity) have been reported, but the mechanisms underlying these responses are poorly understood. In this study, microbial biomass, community structure and the activities of dehydrogenase and ß-glucosidase enzymes were determined for 18 soils that had previously demonstrated either no response or varying magnitude of enhancing or compensatory responses of temperature sensitivity of heterotrophic microbial respiration to prolonged cooling. The soil cooling approach, in contrast to warming experiments, discriminates between microbial community responses and the consequences of substrate depletion, by minimising changes in substrate availability. The initial microbial community composition, determined by molecular analysis of soils showing contrasting respiration responses to cooling, provided evidence that the magnitude of enhancing responses was partly related to microbial community composition. There was also evidence that higher relative abundance of saprophytic Basidiomycota may explain the compensatory response observed in one soil, but neither microbial biomass nor enzymatic capacity were significantly affected by cooling. Our findings emphasise the key importance of soil microbial community responses for feedbacks to global change, but also highlight important areas where our understanding remains limited.

A compositional shift in the soil microbiome induced by tetracycline, sulfamonomethoxine and ciprofloxacin entering a plant-soil system.

Antibiotics entering the soil likely disturb the complex regulatory network of the soil microbiome, which is closely associated with soil quality and ecological function. This study investigated the effects of tetracycline (TC), sulfamonomethoxine (SMM), ciprofloxacin (CIP) and their combination (AM) on the bacterial community in a soil-microbe-plant system and identified the main bacterial responders. Antibiotic effects on the soil microbiome depended on antibiotic type and exposure time. TC resulted in an acute but more rapidly declining effect on soil microbiome while CIP and SMM led to a delayed antibiotic effect. The soil exposed to AM presented a highly similar bacterial structure to that exposed to TC rather than to SMM and CIP. TC, SMM and CIP had their own predominantly impacted taxonomic groups that include both resistance and sensitive bacteria. The antibiotic sensitive responders predominantly distributed within the phylum Proteobacteria. The potential bacteria resistant to each antibiotic exhibited phyla preference to some extent, particularly those resistant to TC. CIP and SMM resistance in soil was increased with exposure time while TC resistance gave the opposite result. Overall, the work extended the understanding of antibiotic effects on soil microbiome after introduced into the soil during greenhouse vegetable cultivation.

Effects of manure and mineral fertilization strategies on soil antibiotic resistance gene levels and microbial community in a paddy-upland rotation system.

This work investigated the responses of antibiotic resistance genes (ARGs) and the soil microbial community in a paddy-upland rotation system to mineral fertilizer (NPK) and different application dosages of manure combined with NPK. The occurrence of five tetracycline ARGs (tetA, tetB, tetC, tetG and tetW), two sulfonamide ARGs (sul1 and sul2) and one genetic element (IntI1) was quantified. NPK application showed only slight or no impact on soil ARGs abundances compared with the control without fertilizer. Soil ARGs abundances could be increased by manure-NPK application but was related to manure dosage (2250-9000 kg ha(-1)). Principal component analysis (PCA) showed that the soil ARG profile of the treatment with 9000 kg ha(-1) manure separated clearly from the other treatments; the ARGs that contributed most to the discrimination of this treatment were tetA, tetG, tetW, sul1, sul2 and IntI1. Community level physiological profile (CLPP) analysis showed that increasing manure dosage from 4500 kg ha(-1) to 9000 kg ha(-1) induced a sharp increase in almost all of the detected ARGs but would not change the microbial community at large. However, 9000 kg ha(-1) manure application produced a decline in soil microbial activity. Determination of antibiotics and heavy metals in soils suggested that the observed bloom of soil ARGs might associate closely with the accumulation of copper and zinc in soil.

Distinct circular single-stranded DNA viruses exist in different soil types.

The potential dependence of virus populations on soil types was examined by electron microscopy, and the total abundance of virus particles in four soil types was similar to that previously observed in soil samples. The four soil types examined differed in the relative abundances of four morphological groups of viruses. Machair, a unique type of coastal soil in western Scotland and Ireland, differed from the others tested in having a higher proportion of tailed bacteriophages. The other soils examined contained predominantly spherical and thin filamentous virus particles, but the Machair soil had a more even distribution of the virus types. As the first step in looking at differences in populations in detail, virus sequences from Machair and brown earth (agricultural pasture) soils were examined by metagenomic sequencing after enriching for circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) virus genomes. Sequences from the family Microviridae (icosahedral viruses mainly infecting bacteria) of CRESS-DNA viruses were predominant in both soils. Phylogenetic analysis of Microviridae major coat protein sequences from the Machair viruses showed that they spanned most of the diversity of the subfamily Gokushovirinae, whose members mainly infect obligate intracellular parasites. The brown earth soil had a higher proportion of sequences that matched the morphologically similar family Circoviridae in BLAST searches. However, analysis of putative replicase proteins that were similar to those of viruses in the Circoviridae showed that they are a novel clade of Circoviridae-related CRESS-DNA viruses distinct from known Circoviridae genera. Different soils have substantially different taxonomic biodiversities even within ssDNA viruses, which may be driven by physicochemical factors.

Multi-factorial drivers of ammonia oxidizer communities: evidence from a national soil survey.

The factors driving the abundance and community composition of soil microbial communities provide fundamental knowledge on the maintenance of biodiversity and the ecosystem services they underpin. Several studies have suggested that microbial communities are spatially organized, including functional groups and much of the observed variation is explained by geographical location or soil pH. Soil ammonia-oxidizing archaea (AOA) and bacteria (AOB) are excellent models for such study due to their functional, agronomic and environmental importance and their relative ease of characterization. To identify the dominant drivers of different ammonia oxidizers, we used samples (n = 713) from the National Soil Inventory of Scotland (NSIS). Our results indicate that 40-45% of the variance in community compositions can be explained by 71 environmental variables. Soil pH and substrate, which have been regarded as the two main drivers, only explained 13-16% of the total variance. We provide strong evidence of multi-factorial drivers (land use, soil type, climate and N deposition) of ammonia-oxidizing communities, all of which play a significant role in the creation of specific niches that are occupied by unique phylotypes. For example, one AOA phylotype was strongly linked to woodland/semi-natural grassland, rainfall and N deposition. Some soil typologies, namely regosols, have a novel AOA community composition indicating typology as one of the factors which defines this ecological niche. AOA abundance was high and strongly linked the rate of potential nitrification in the highly acidic soils supporting the argument that AOA are main ammonia oxidizers in acidic soils. However, for AOB, soil pH and substrate (ammonia) were the main drivers for abundance and community composition. These results highlight the importance of multiple drivers of microbial niche formation and their impact on microbial biogeography that have significant consequences for ecosystem functioning.

Links between methane flux and transcriptional activities of methanogens and methane oxidizers in a blanket peat bog.

The relationship between biogeochemical process rates and microbial functional activity was investigated by analysis of the transcriptional dynamics of the key functional genes for methanogenesis (methyl coenzyme M reductase; mcrA) and methane oxidation (particulate methane monooxygenase; pmoA) and in situ methane flux at two peat soil field sites with contrasting net methane-emitting and -oxidizing characteristics. qPCR was used to quantify the abundances of mcrA and pmoA genes and transcripts at two soil depths. Total methanogen and methanotroph transcriptional dynamics, calculated from mcrA and pmoA gene : transcript abundance ratios, were similar at both sites and depths. However, a linear relationship was demonstrated between surface mcrA and pmoA transcript dynamics and surface flux rates at the methane-emitting and methane-oxidizing sites, respectively. Results indicate that methanotroph activity was at least partially substrate-limited at the methane-emitting site and by other factors at the methane-oxidizing site. Soil depth also contributed to the control of surface methane fluxes, but to a lesser extent. Small differences in the soil water content may have contributed to differences in methanogen and methanotroph activities. This study therefore provides a first insight into the regulation of in situ, field-level surface CH(4) flux at the molecular level by an accurate reflection of gene : transcript abundance ratios for the key genes in methane generation and consumption.

Stable isotope probing analysis of interactions between ammonia oxidizers.

The response of natural microbial communities to environmental change can be assessed by determining DNA- or RNA-targeted changes in relative abundance of 16S rRNA gene sequences by using fingerprinting techniques such as denaturing gradient gel electrophoresis (DNA-DGGE and RNA-DGGE, respectively) or by stable isotope probing (SIP) of 16S rRNA genes following incubation with a (13)C-labeled substrate (DNA-SIP-DGGE). The sensitivities of these three approaches were compared during batch growth of communities containing two or three Nitrosospira pure or enriched cultures with different tolerances to a high ammonia concentration. Cultures were supplied with low, intermediate, or high initial ammonia concentrations and with (13)C-labeled carbon dioxide. DNA-SIP-DGGE provided the most direct evidence for growth and was the most sensitive, with changes in DGGE profiles evident before changes in DNA- and RNA-DGGE profiles and before detectable increases in nitrite and nitrate production. RNA-DGGE provided intermediate sensitivity. In addition, the three molecular methods were used to follow growth of individual strains within communities. In general, changes in relative activities of individual strains within communities could be predicted from monoculture growth characteristics. Ammonia-tolerant Nitrosospira cluster 3b strains dominated mixed communities at all ammonia concentrations, and ammonia-sensitive strains were outcompeted at an intermediate ammonia concentration. However, coexistence of ammonia-tolerant and ammonia-sensitive strains occurred at the lowest ammonia concentration, and, under some conditions, strains inhibited at high ammonia in monoculture were active at high ammonia in mixed cultures, where they coexisted with ammonia-tolerant strains. The results therefore demonstrate the sensitivity of SIP for detection of activity of organisms with relatively low yield and low activity and its ability to follow changes in the structure of interacting microbial communities.

Correlation of methane production and functional gene transcriptional activity in a peat soil.

The transcription dynamics of subunit A of the key gene in methanogenesis (methyl coenzyme M reductase; mcrA) was studied to evaluate the relationship between process rate (methanogenesis) and gene transcription dynamics in a peat soil ecosystem. Soil methanogen process rates were determined during incubation of peat slurries at temperatures from 4 to 37 degrees C, and real-time quantitative PCR was applied to quantify the abundances of mcrA genes and transcripts; corresponding transcriptional dynamics were calculated from mcrA transcript/gene ratios. Internal standards suggested unbiased recovery of mRNA abundances in comparison to DNA levels. In comparison to those in pure-culture studies, mcrA transcript/gene ratios indicated underestimation by 1 order of magnitude, possibly due to high proportions of inactive or dead methanogens. Methane production rates were temperature dependent, with maxima at 25 degrees C, but changes in abundance and transcription of the mcrA gene showed no correlation with temperature. However, mcrA transcript/gene ratios correlated weakly (regression coefficient = 0.76) with rates of methanogenesis. Methanogen process rates increased over 3 orders of magnitude, while the corresponding maximum transcript/gene ratio increase was only 18-fold. mcrA transcript dynamics suggested steady-state expression in peat soil after incubation for 24 and 48 h, similar to that in stationary-phase cultures. mcrA transcript/gene ratios are therefore potential in situ indicators of methanogen process rate changes in complex soil systems.

Growth, activity and temperature responses of ammonia-oxidizing archaea and bacteria in soil microcosms.

Ammonia oxidation, as the first step in the nitrification process, plays a central role in the global cycling of nitrogen. Although bacteria are traditionally considered to be responsible for ammonia oxidation, a role for archaea has been suggested by data from metagenomic studies and by the isolation of a marine, autotrophic, ammonia-oxidizing, non-thermophilic crenarchaeon. Evidence for ammonia oxidation by non-thermophilic crenarchaea in marine and terrestrial environments is largely based on abundance of bacterial and archaeal ammonia monooxygenase (amo) genes, rather than activity. In this study, we have determined the influence of temperature on the response of ammonia-oxidizing bacteria and archaea in nitrifying soil microcosms using two approaches, involving analysis of transcriptional activity of 16S rRNA genes and of a key functional gene, amoA, which encodes ammonia monooxygenase subunit A. There was little evidence of changes in relative abundance or transcriptional activity of ammonia-oxidizing bacteria during nitrification. In contrast, denaturing gradient gel electrophoresis analysis of crenarchaeal 16S rRNA and crenarchaeal amoA genes provided strong evidence of changes in community structure of active archaeal ammonia oxidizers. Community structure changes were similar during incubation at different temperatures and much of the activity was due to a group of non-thermophilic crenarchaea associated with subsurface and marine environments, rather than soil. The findings suggest a role for crenarchaea in soil nitrification and that further information is required on their biogeography.

Comparison of PCR primer-based strategies for characterization of ammonia oxidizer communities in environmental samples.

PCR-based techniques are commonly used to characterize microbial communities, but are subject to bias that is difficult to assess. This study aimed to evaluate bias of several PCR primer-based strategies used to study diversity of autotrophic ammonia oxidizers. 16S rRNA genes from soil- or sediment-DNA were amplified using primers considered either selective or specific for betaproteobacterial ammonia oxidizers. Five approaches were assessed: (a) amplification with primers betaAMO143f-betaAMO1315r; (b) amplification with primers CTO189f-CTO654r; (c) nested amplification with betaAMO143f-betaAMO1315r followed by CTO189f-CTO654r primers; (d) nested amplification with betaAMO143f-betaAMO1315r and CTO189f-Pf1053r primers; (e) nested amplification with 27f-1492r and CTO189f-CTO654r primers. Amplification products were characterized by denaturing gradient gel electrophoresis (DGGE) analysis after further amplification with 357f-GC-518r primers. DGGE profiles of soil communities were heterogeneous and depended on the approach followed. Ammonia oxidizer diversity was higher using approaches (b), (c) and (e) than using (a) and (d), where sequences of the most prominent bands showed similarities to nonammonia oxidizers. Profiles from marine sediments were more consistent, regardless of the approach adopted, and sequence analysis of excised bands indicated that these consisted of ammonia oxidizers only. The study demonstrates the importance of choice of primer, of screening for sequences of nontarget organisms and use of several approaches when characterizing microbial communities in natural environments.

Changes in the community structure and activity of betaproteobacterial ammonia-oxidizing sediment bacteria along a freshwater-marine gradient.

To determine whether the distribution of estuarine ammonia-oxidizing bacteria (AOB) was influenced by salinity, the community structure of betaproteobacterial ammonia oxidizers (AOB) was characterized along a salinity gradient in sediments of the Ythan estuary, on the east coast of Scotland, UK, by denaturant gradient gel electrophoresis (DGGE), cloning and sequencing of 16S rRNA gene fragments. Ammonia-oxidizing bacteria communities at sampling sites with strongest marine influence were dominated by Nitrosospira cluster 1-like sequences and those with strongest freshwater influence were dominated by Nitrosomonas oligotropha-like sequences. Nitrosomonas sp. Nm143 was the prevailing sequence type in communities at intermediate brackish sites. Diversity indices of AOB communities were similar at marine- and freshwater-influenced sites and did not indicate lower species diversity at intermediate brackish sites. The presence of sequences highly similar to the halophilic Nitrosomonas marina and the freshwater strain Nitrosomonas oligotropha at identical sampling sites indicates that AOB communities in the estuary are adapted to a range of salinities, while individual strains may be active at different salinities. Ammonia-oxidizing bacteria communities that were dominated by Nitrosospira cluster 1 sequence types, for which no cultured representative exists, were subjected to stable isotope probing (SIP) with 13C-HCO3-, to label the nucleic acids of active autotrophic nitrifiers. Analysis of 13C-associated 16S rRNA gene fragments, following CsCl density centrifugation, by cloning and DGGE indicated sequences highly similar to the AOB Nitrosomonas sp. Nm143 and Nitrosomonas cryotolerans and to the nitrite oxidizer Nitrospira marina. No sequence with similarity to the Nitrosospira cluster 1 clade was recovered during SIP analysis. The potential role of Nitrosospira cluster 1 in autotrophic ammonia oxidation therefore remains uncertain.

Influence of inorganic nitrogen management regime on the diversity of nitrite-oxidizing bacteria in agricultural grassland soils.

To assess links between the diversity of nitrite-oxidizing bacteria (NOB) in agricultural grassland soils and inorganic N fertilizer management, NOB communities in fertilized and unfertilized soils were characterized by analysis of clone libraries and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Previously uncharacterized Nitrospira-like sequences were isolated from both long-term-fertilized and unfertilized soils, but DGGE migration patterns indicated the presence of additional sequence types in the fertilized soils. Detailed phylogenetic analysis of Nitrospira-like sequences suggests the existence of one newly described evolutionary group and of subclusters within previously described sublineages, potentially representing different ecotypes; the new group may represent a lineage of noncharacterized Nitrospira species. Clone libraries of Nitrobacter-like sequences generated from soils under different long-term N management regimes were dominated by sequences with high similarity to the rhizoplane isolate Nitrobacter sp. strain PJN1. However, the diversity of Nitrobacter communities did not differ significantly between the two soil types. This is the first cultivation-independent study of nitrite-oxidizing bacteria in soil demonstrating that nitrogen management practices influence the diversity of this bacterial functional group.

Links between ammonia oxidizer species composition, functional diversity and nitrification kinetics in grassland soils.

Molecular approaches have revealed considerable diversity and uncultured novelty in natural prokaryotic populations, but not direct links between the new genotypes detected and ecosystem processes. Here we describe the influence of the structure of communities of ammonia-oxidizing bacteria on nitrogen cycling in microcosms containing natural and managed grasslands and amended with artificial sheep urine, a major factor determining local ammonia concentrations in these environments. Nitrification kinetics were assessed by analysis of changes in urea, ammonia, nitrite and nitrate concentrations and ammonia oxidizer communities were characterized by analysis of 16S rRNA genes amplified from extracted DNA using ammonia oxidizer-specific primers. In natural soils, ammonia oxidizer community structure determined the delay preceding nitrification, which depended on the relative abundance of two Nitrosospira clusters, termed 3a and 3b. In batch cultures, pure culture and enrichment culture representatives of Nitrosospira 3a were sensitive to high ammonia concentration, while Nitrosospira cluster 3b representatives and Nitrosomonas europaea were tolerant. Delays in nitrification occurred in natural soils dominated by Nitrosospira cluster 3a and resulted from the time required for growth of low concentrations of Nitrosospira cluster 3b. In microcosms dominated by Nitrosospira cluster 3b and Nitrosomonas, no substantial delays were observed. In managed soils, no delays in nitrification were detected, regardless of initial ammonia oxidizer community structure, most probably resulting from higher ammonia oxidizer cell concentrations. The data therefore demonstrate a direct link between bacterial community structure, physiological diversity and ecosystem function.

Differences between betaproteobacterial ammonia-oxidizing communities in marine sediments and those in overlying water.

To assess links between betaproteobacterial ammonia-oxidizing bacteria (AOB) in marine sediment and in overlying water, communities in Loch Duich, Scotland, were characterized by analysis of clone libraries and denaturant gradient gel electrophoresis of 16S rRNA gene fragments. Nitrosospira cluster 1-like sequences were isolated from both environments, but different sequence types dominated water and sediment samples. Detailed phylogenetic analysis of marine Nitrosospira cluster 1-like sequences in Loch Duich and surrounding regions suggests the existence of at least two different phylogenetic subgroups, potentially indicative of new lineages within the betaproteobacterial AOB, representing different marine ecotypes.

Community structure of ammonia-oxidizing bacteria within anoxic marine sediments.

The potential for oxidation of ammonia in anoxic marine sediments exists through anaerobic oxidation by Nitrosomonas-like organisms, utilizing nitrogen dioxide, coupling of nitrification, manganese reduction, and anaerobic oxidation of ammonium by planctomycetes (the Anammox process). Here we describe the presence of microbial communities with the potential to carry out these processes in a natural marine sediment system (Loch Duich, Scotland). Natural microbial communities of Planctomycetales-Verrucomicrobia and beta- and gamma-proteobacterial ammonia-oxidizing bacteria were characterized by analysis of 16S rRNA genes amplified using group-specific primers by PCR- and reverse transcription-PCR amplification of 16S rDNA and RNA, respectively. Amplification products were analyzed by sequencing of clones and by denaturant gradient gel electrophoresis (DGGE). Amplification of primers specific for Planctomycetales-Verrucomicrobia and beta-proteobacterial ammonia-oxidizing bacteria generated products at all sampling sites and depths, but no product was generated using primers specific for gamma-proteobacterial ammonia-oxidizing bacteria. 16S rDNA DGGE banding patterns indicated complex communities of beta-proteobacterial ammonia-oxidizing bacteria in anoxic marine sediments. Phylogenetic analysis of sequences from clones and those excised from DGGE gels suggests dominance of Nitrosospira cluster 1-like organisms and of strains belonging to a novel cluster represented in dominant bands in 16S rRNA DGGE banding patterns. Their presence indicates a group of organisms closely related to recognized beta-proteobacterial ammonia-oxidizing bacteria that may be selected in anoxic environments and may be capable of anoxic ammonia oxidation. Sequence analysis of planctomycete clone libraries and sequences excised from DGGE gels also demonstrated a diverse microbial community and suggested the presence of new subdivisions, but no sequence related to recognized Anammox organisms was detected.

Effect of anoxia and high sulphide concentrations on heterotrophic microbial communities in reduced surface sediments (Black Spots) in sandy intertidal flats of the German Wadden Sea.

Abstract Black reduced sediment surfaces (Black Spots) in sandy intertidal flats of the German Wadden Sea (southern North Sea) are characterised by elevated sulphide concentrations (up to 20 mM) and low redox potentials. It is assumed that the appearance of Black Spots is linked to elevated levels of organic matter content within the sediments. In order to establish the effect of high substrate and sulphide concentrations on the heterotrophic microbial communities in Black Spot sediments, bacterial abundances and the potential C-source utilisation patterns of microbial communities were compared in natural and artificially induced Black Spots and unaffected control sites. Bacterial numbers were estimated by direct counts and the most probable number technique for different physiological groups, while patterns of C-substrate utilisation of entire aerobic microbial communities were assessed using the Biolog sole-carbon-source-catabolism assay. Bacterial abundances at Black Spot sites were increased, with increases in mean cell numbers, more disperse data distributions and more extreme values. Substrate utilisation patterns of aerobic microbial communities were significantly different in Black Spot sediment slurries, showing diminished richness (number of C-sources catabolised) and substrate diversity (Shannon diversity index) in comparison to unaffected sites. Principal component analysis clearly discriminated Black Spot utilisation patterns from controls and indicated that microbial communities in individual Black Spot sites are functionally diverse and differ from communities in oxidised surface sediments and reduced subsurface sediments at control sites. This work suggests that potentially negative effects on microbial communities in Black Spot sediments, through anoxia and high sulphide concentrations, are balanced by the stimulating influence of substrate availability, leading to comparable or higher bacterial numbers, but lower functional microbial diversity of aerobic microbial communities.