The Stochastic Assembly of Nitrobacter winogradskyi-Selected Microbiomes with Heterotrophs from Sewage Sludge or Grassland Soil.

Chemolitho-autotrophic microorganisms like the nitrite-oxidizing Nitrobacter winogradskyi create an environment for heterotrophic microorganisms that profit from the production of organic compounds. It was hypothesized that the assembly of a community of heterotrophic microorganisms around N. winogradskyi depends on the ecosystem from which the heterotrophs are picked. To test this hypothesis, pure cultures of N. winogradskyi were grown in continuously nitrite-fed bioreactors in a mineral medium free of added organic carbon that had been inoculated with diluted sewage sludge or with a suspension from a grassland soil. Samples for chemical and 16S rRNA gene amplicon analyses were taken after each volume change in the bioreactor. At the end of the enrichment runs, samples for shotgun metagenomics were also collected. Already after two volume changes, the transformations in community structure became less dynamic. The enrichment of heterotrophs from both sewage and soil was highly stochastic and yielded different dominant genera in most of the enrichment runs that were independent of the origin of the inoculum. Hence, the hypothesis had to be refuted. Notwithstanding the large variation in taxonomic community structure among the enrichments, the functional compositions of the communities were statistically not different between soil- and sludge-based enrichments. IMPORTANCE In the process of aerobic nitrification, nitrite-oxidizing bacteria together with ammonia-oxidizing microorganisms convert mineral nitrogen from its most reduced appearance, i.e., ammonium, into its most oxidized form, i.e., nitrate. Because the form of mineral nitrogen has large environmental implications, nitrite-oxidizing bacteria such as Nitrobacter winogradskyi play a central role in the global biogeochemical nitrogen cycle. In addition to this central role, the autotrophic nitrite-oxidizing bacteria also play a fundamental role in the global carbon cycle. They form the basis of heterotrophic food webs, in which the assimilated carbon is recycled. Little is known about the heterotrophic microorganisms that participate in these food webs, let alone their assembly in different ecosystems. This study showed that the assembly of microbial food webs by N. winogradskyi was a highly stochastic process and independent of the origin of the heterotrophic microorganisms, but the functional characteristics of the different food webs were similar.

A Physiological and Genomic Comparison of Nitrosomonas Cluster 6a and 7 Ammonia-Oxidizing Bacteria.

Ammonia-oxidizing bacteria (AOB) within the genus Nitrosomonas perform the first step in nitrification, ammonia oxidation, and are found in diverse aquatic and terrestrial environments. Nitrosomonas AOB were grouped into six defined clusters, which correlate with physiological characteristics that contribute to adaptations to a variety of abiotic environmental factors. A fundamental physiological trait differentiating Nitrosomonas AOB is the adaptation to either low (cluster 6a) or high (cluster 7) ammonium concentrations. Here, we present physiological growth studies and genome analysis of Nitrosomonas cluster 6a and 7 AOB. Cluster 6a AOB displayed maximum growth rates at ≤ 1 mM ammonium, while cluster 7 AOB had maximum growth rates at ≥ 5 mM ammonium. In addition, cluster 7 AOB were more tolerant of high initial ammonium and nitrite concentrations than cluster 6a AOB. Cluster 6a AOB were completely inhibited by an initial nitrite concentration of 5 mM. Genomic comparisons were used to link genomic traits to observed physiological adaptations. Cluster 7 AOB encode a suite of genes related to nitrogen oxide detoxification and multiple terminal oxidases, which are absent in cluster 6a AOB. Cluster 6a AOB possess two distinct forms of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and select species encode genes for hydrogen or urea utilization. Several, but not all, cluster 6a AOB can utilize urea as a source of ammonium. Hence, although Nitrosomonas cluster 6a and 7 AOB have the capacity to fulfill the same functional role in microbial communities, i.e., ammonia oxidation, differentiating species-specific and cluster-conserved adaptations is crucial in understanding how AOB community succession can affect overall ecosystem function.

Tide as Steering Factor in Structuring Archaeal and Bacterial Ammonia-Oxidizing Communities in Mangrove Forest Soils Dominated by Avicennia germinans and Rhizophora mangle.

Mangrove species are adapted to grow at specific zones in a tidal gradient. Here we tested the hypothesis that the archaeal and bacterial ammonia-oxidizing microbial communities differ in soils dominated by the mangrove species Avicennia germinans and Rhizophora mangle. Two of the sampling locations were tidal locations, while the other location was impounded. Differences in the community compositions of ammonia-oxidizing archaea (AOA) and bacteria (AOB) were analyzed by denaturing gradient gel electrophoresis (DGGE) of amoA genes and by MiSeq 16S rRNA gene-sequencing. The abundances of AOA and AOB were established by quantitative PCR of amoA genes. In addition, we analyzed the total microbial community composition based on 16S rRNA genes and explored the influence of soil physicochemical properties underneath Avicennia germinans and Rhizophora mangle on microbial communities. AOA were always more abundant than AOB, but the effect of mangrove species on total numbers of ammonia oxidizers was location-specific. The microbial communities including the ammonia oxidizers in soils associated with A. germinans and R. mangle differed only at the tidal locations. In conclusion, potential site-specific effects of mangrove species on soil microbial communities including those of the AOA and AOB are apparently overruled by the absence or presence of tide.

Numerical Relationships Between Archaeal and Bacterial amoA Genes Vary by Icelandic Andosol Classes.

Bacterial amoA genes had not been detectable by qPCR in freshly sampled Icelandic Andosols thus far. Hence, a new primer set yielding shorter gene fragments has been designed to verify the absence of ammonia-oxidizing bacteria in different Icelandic Andosol classes. At the same time, a new primer set was also constructed for archaeal amoA genes that should improve the quality of PCR products. Although a large part of the soil samples were found to be amoA-negative, bacterial amoA genes were detectable with new as well as old primer sets. The same results were obtained for the archaeal amoA genes. The relative distribution of archaeal and bacterial amoA genes varied between Andosol classes. Archaeal amoA genes were significantly more abundant in Brown than in Histic Andosols, while the opposite was observed for bacterial amoA genes. The numbers of archaeal and bacterial amoA genes in Gleyic Andosols were not significantly different from those in Histic and Brown Andosols. The numbers of bacterial amoA genes, but not the numbers of archaeal amoA genes, correlated significantly and positively with potential ammonia oxidation activities. The presence of the bacterial nitrification inhibitor allylthiourea inhibited the potential ammonia oxidation activities during the first 12 h of incubation. Hence, it was concluded that ammonia-oxidizing bacteria profited most from the conditions during the measurements of potential ammonia oxidation activities.

Effects of Bacterial Community Members on the Proteome of the Ammonia-Oxidizing Bacterium Nitrosomonas sp. Strain Is79.

UNLABELLED: Microorganisms in the environment do not exist as the often-studied pure cultures but as members of complex microbial communities. Characterizing the interactions within microbial communities is essential to understand their function in both natural and engineered environments. In this study, we investigated how the presence of a nitrite-oxidizing bacterium (NOB) and heterotrophic bacteria affect the growth and proteome of the chemolithoautotrophic ammonia-oxidizing bacterium (AOB) Nitrosomonas sp. strain Is79. We investigated Nitrosomonas sp. Is79 in co-culture with Nitrobacter winogradskyi, in co-cultures with selected heterotrophic bacteria, and as a member of the nitrifying enrichment culture G5-7. In batch culture, N. winogradskyi and heterotrophic bacteria had positive effects on the growth of Nitrosomonas sp. Is79. An isobaric tag for relative and absolute quantification (iTRAQ) liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics approach was used to investigate the effect of N. winogradskyi and the co-cultured heterotrophic bacteria from G5-7 on the proteome of Nitrosomonas sp. Is79. In co-culture with N. winogradskyi, several Nitrosomonas sp. Is79 oxidative stress response proteins changed in abundance, with periplasmic proteins increasing and cytoplasmic proteins decreasing in abundance. In the presence of heterotrophic bacteria, the abundance of proteins directly related to the ammonia oxidation pathway increased, while the abundance of proteins related to amino acid synthesis and metabolism decreased. In summary, the proteome of Nitrosomonas sp. Is79 was differentially influenced by the presence of either N. winogradskyi or heterotrophic bacteria. Together, N. winogradskyi and heterotrophic bacteria reduced the oxidative stress for Nitrosomonas sp. Is79, which resulted in more efficient metabolism. IMPORTANCE: Aerobic ammonia-oxidizing microorganisms play an important role in the global nitrogen cycle, converting ammonia to nitrite. In their natural environment, they coexist and interact with nitrite oxidizers, which convert nitrite to nitrate, and with heterotrophic microorganisms. The presence of nitrite oxidizers and heterotrophic bacteria has a positive influence on the growth of the ammonia oxidizers. Here, we present a study investigating the effect of nitrite oxidizers and heterotrophic bacteria on the proteome of a selected ammonia oxidizer in a defined culture to elucidate how these two groups improve the performance of the ammonia oxidizer. The results show that the presence of a nitrite oxidizer and heterotrophic bacteria reduced the stress for the ammonia oxidizer and resulted in more efficient energy generation. This study contributes to our understanding of microbe-microbe interactions, in particular between ammonia oxidizers and their neighboring microbial community.

Effects of increased summer flooding on nitrogen dynamics in impounded mangroves.

Mangroves are important for coastal protection, carbon sequestration and habitat provision for plants and animals in the tropics and subtropics. Mangroves are threatened by habitat destruction and sea level rise, but management activities such as impounding for mosquito control can also have negative effects. We studied the effects of Rotational Impoundment Management (RIM) on nitrogen dynamics in impoundments dominated by three types of Black mangrove (Avicennia germinans) stands along the Indian River Lagoon (Florida). RIM, designed for noxious insect control, involves pumping estuarine water into impoundments in this area during spring and summer to raise water levels by 30 cm. We compared aspects of the nitrogen cycle before and after the start of the RIM and measured the same variables in an impoundment without RIM management. RIM led to the accumulation of ammonium in the substrate which coincided with a lowering of nitrification rates and decreased denitrification rates. Salt pan habitats dominated by dwarf mangroves became less saline following RIM initiation. Shoot growth of mangroves increased in response to higher nitrogen availability and lower pore water salinity. Mangrove responses were greatest in areas with dwarf and sparse mangrove cover. Overall, RIM resulted in lower nitrification and denitrification leading to lower nitrogen losses and increased Black mangrove growth, all benefits of RIM beyond those associated with noxious insect control.

Temporal and spatial coexistence of archaeal and bacterial amoA genes and gene transcripts in Lake Lucerne.

Despite their crucial role in the nitrogen cycle, freshwater ecosystems are relatively rarely studied for active ammonia oxidizers (AO). This study of Lake Lucerne determined the abundance of both amoA genes and gene transcripts of ammonia-oxidizing archaea (AOA) and bacteria (AOB) over a period of 16 months, shedding more light on the role of both AO in a deep, alpine lake environment. At the surface, at 42 m water depth, and in the water layer immediately above the sediment, AOA generally outnumbered AOB. However, in the surface water during summer stratification, when both AO were low in abundance, AOB were more numerous than AOA. Temporal distribution patterns of AOA and AOB were comparable. Higher abundances of amoA gene transcripts were observed at the onset and end of summer stratification. In summer, archaeal amoA genes and transcripts correlated negatively with temperature and conductivity. Concentrations of ammonium and oxygen did not vary enough to explain the amoA gene and transcript dynamics. The observed herbivorous zooplankton may have caused a hidden flux of mineralized ammonium and a change in abundance of genes and transcripts. At the surface, AO might have been repressed during summer stratification due to nutrient limitation caused by active phytoplankton.

Pro- and eukaryotic keystone taxa as potential bio-indicators for the water quality of subtropical Lake Dongqian.

The microbial community plays an important role in the biogeochemical cycles in water aquatic ecosystems, and it is regulated by environmental variables. However, the relationships between microbial keystone taxa and water variables, which play a pivotal role in aquatic ecosystems, has not been clarified in detail. We analyzed the seasonal variation in microbial communities and co-occurrence network in the representative areas taking Lake Dongqian as an example. Both pro- and eukaryotic community compositions were more affected by seasons than by sites, and the prokaryotes were more strongly impacted by seasons than the eukaryotes. Total nitrogen, pH, temperature, chemical oxygen demand, dissolved oxygen and chlorophyll a significantly affected the prokaryotic community, while the eukaryotic community was significantly influenced by total nitrogen, ammonia, pH, temperature and dissolved oxygen. The eukaryotic network was more complex than that of prokaryotes, whereas the number of eukaryotic keystone taxa was less than that of prokaryotes. The prokaryotic keystone taxa belonged mainly to Alphaproteobacteria, Betaproteobacteria, Actinobacteria and Bacteroidetes. It is noteworthy that some of the keystone taxa involved in nitrogen cycling are significantly related to total nitrogen, ammonia, temperature and chlorophyll a, including Polaromonas, Albidiferax, SM1A02 and Leptolyngbya so on. And the eukaryotic keystone taxa were found in Ascomycota, Choanoflagellida and Heterophryidae. The mutualistic pattern between pro- and eukaryotes was more evident than the competitive pattern. Therefore, it suggests that keystone taxa could be as bio-indicators of aquatic ecosystems.

Genome sequence of Nitrosomonas sp. strain AL212, an ammonia-oxidizing bacterium sensitive to high levels of ammonia.

Nitrosomonas sp. strain AL212 is an obligate chemolithotrophic ammonia-oxidizing bacterium (AOB) that was originally isolated in 1997 by Yuichi Suwa and colleagues. This organism belongs to Nitrosomonas cluster 6A, which is characterized by sensitivity to high ammonia concentrations, higher substrate affinity (lower K(m)), and lower maximum growth rates than strains in Nitrosomonas cluster 7, which includes Nitrosomonas europaea and Nitrosomonas eutropha. Genome-informed studies of this ammonia-sensitive cohort of AOB are needed, as these bacteria are found in freshwater environments, drinking water supplies, wastewater treatment systems, and soils worldwide.

Distribution and diversity of Gallionella-like neutrophilic iron oxidizers in a tidal freshwater marsh.

Microbial iron oxidation is an integral part of the iron redox cycle in wetlands. Nonetheless, relatively little is known about the composition and ecology of iron-oxidizing communities in the soils and sediments of wetlands. In this study, sediment cores were collected across a freshwater tidal marsh in order to characterize the iron-oxidizing bacteria (FeOB) and to link their distributions to the geochemical properties of the sediments. We applied recently designed 16S rRNA primers targeting Gallionella-related FeOB by using a nested PCR-denaturing gradient gel electrophoresis (DGGE) approach combined with a novel quantitative PCR (qPCR) assay. Gallionella-related FeOB were detected in most of the samples. The diversity and abundance of the putative FeOB were generally higher in the upper 5 to 12 cm of sediment than in deeper sediment and higher in samples collected in April than in those collected in July and October. Oxygen supply by macrofauna appears to be a major force in controlling the spatial and temporal variations in FeOB communities. The higher abundance of Gallionella-related FeOB in April coincided with elevated concentrations of extractable Fe(III) in the sediments. Despite this coincidence, the distributions of FeOB did not exhibit a simple relationship to the redox zonation inferred from the geochemical depth profiles.

Methane emission from natural wetlands: interplay between emergent macrophytes and soil microbial processes. A mini-review.

BACKGROUND: According to the Intergovernmental Panel on Climate Change (IPCC) 2007, natural wetlands contribute 20-39 % to the global emission of methane. The range in the estimated percentage of the contribution of these systems to the total release of this greenhouse gas is large due to differences in the nature of the emitting vegetation including the soil microbiota that interfere with the production and consumption of methane. SCOPE: Methane is a dominant end-product of anaerobic mineralization processes. When all electron acceptors except carbon dioxide are used by the microbial community, methanogenesis is the ultimate pathway to mineralize organic carbon compounds. Emergent wetland plants play an important role in the emission of methane to the atmosphere. They produce the carbon necessary for the production of methane, but also facilitate the release of methane by the possession of a system of interconnected internal gas lacunas. Aquatic macrophytes are commonly adapted to oxygen-limited conditions as they prevail in flooded or waterlogged soils. By this system, oxygen is transported to the underground parts of the plants. Part of the oxygen transported downwards is released in the root zone, where it sustains a number of beneficial oxidation processes. Through the pores from which oxygen escapes from the plant into the root zone, methane can enter the plant aerenchyma system and subsequently be emitted into the atmosphere. Part of the oxygen released into the root zone can be used to oxidize methane before it enters the atmosphere. However, the oxygen can also be used to regenerate alternative electron acceptors. The continuous supply of alternative electron acceptors will diminish the role of methanogenesis in the anaerobic mineralization processes in the root zone and therefore repress the production and emission of methane. The role of alternative element cycles in the inhibition of methanogenesis is discussed. CONCLUSIONS: The role of the nitrogen cycle in repression of methane production is probably low. In contrast to wetlands particularly created for the purification of nitrogen-rich waste waters, concentrations of inorganic nitrogen compounds are low in the root zones in the growing season due to the nitrogen-consuming behaviour of the plant. Therefore, nitrate hardly competes with other electron acceptors for reduced organic compounds, and repression of methane oxidation by the presence of higher levels of ammonium will not be the case. The role of the iron cycle is likely to be important with respect to the repression of methane production and oxidation. Iron-reducing and iron-oxidizing bacteria are ubiquitous in the rhizosphere of wetland plants. The cycling of iron will be largely dependent on the size of the oxygen release in the root zone, which is likely to be different between different wetland plant species. The role of the sulfur cycle in repression of methane production is important in marine, sulfate-rich ecosystems, but might also play a role in freshwater systems where sufficient sulfate is available. Sulfate-reducing bacteria are omnipresent in freshwater ecosystems, but do not always react immediately to the supply of fresh sulfate. Hence, their role in the repression of methanogenesis is still to be proven in freshwater marshes.

Response of the sulfate-reducing community to the re-establishment of estuarine conditions in two contrasting soils: a mesocosm approach.

We studied the response of the sulfate-reducing prokaryote (SRP) communities to the experimental variation of salinity and tide in an outdoor mesocosm setup. Intact soil monoliths were collected at two areas of the Haringvliet lagoon (The Netherlands): one sampling location consisted of agricultural grassland, drained and fertilized for at least the last century; the other of a freshwater marshland with more recent sea influence. Two factors, i.e., "salinity" (freshwater/oligohaline) and "tide" (nontidal/tidal), were tested in a full-factorial design. Soil samples were collected after 5 months (June-October). Dissimilatory (bi)sulfite reductase beta subunit-based denaturing gradient gel electrophoresis (dsrB-DGGE) analysis revealed that the SRP community composition in the agricultural grassland and in the freshwater marshland was represented mainly by microorganisms related to the Desulfobulbaceae and the Desulfobacteraceae, respectively. Desulfovibrio-related dsrB were detected only in the tidal treatments; Desulfomonile-related dsrB occurrence was related to the presence of oligohaline conditions. Treatments did have an effect on the overall SRP community composition of both soils, but not on the sulfate depletion rates in sulfate-amended anoxic slurry incubations. However, initiation of sulfate reduction upon sulfate addition was clearly different between the two soils.

Tannins from senescent Rhizophora mangle mangrove leaves have a distinctive effect on prokaryotic and eukaryotic communities in a Distichlis spicata salt marsh soil.

Due to climate warming, tannin-rich Rhizophora mangle migrates into tannin-poor salt marshes, where the tannins interfere with the biogeochemistry in the soil. Changes in biogeochemistry are likely associated with changes in microbial communities. This was studied in microcosms filled with salt marsh soil and amended with leaf powder, crude condensed tannins, purified condensed tannins (PCT), all from senescent R. mangle leaves, or with tannic acid. Size and composition of the microbial communities were determined by denaturing gradient gel electrophoresis, high-throughput sequencing and real-time PCR based on the 16S and 18S rRNA genes. Compared with the control, the 16S rRNA gene abundance was lowered by PCT, while the 18S rRNA gene abundance was enhanced by all treatments. The treatments also affected the composition of the 16S rRNA and 18S rRNA gene assemblies, but the effects on the 18S rRNA gene were greater. The composition of the 18S rRNA gene, but not of the 16S rRNA gene, was significantly correlated with the mineralization of carbon, nitrogen and phosphorus. Distinctive microbial groups emerged during the different treatments. This study revealed that migration of mangroves may affect both the prokaryotic and the eukaryotic communities in salt marsh soils, but that the effects on the eukaryotes will likely be greater.

Biological nitrification inhibition in the rhizosphere: determining interactions and impact on microbially mediated processes and potential applications.

Nitrification is the microbial conversion of reduced forms of nitrogen (N) to nitrate (NO3-), and in fertilized soils it can lead to substantial N losses via NO3- leaching or nitrous oxide (N2O) production. To limit such problems, synthetic nitrification inhibitors have been applied but their performance differs between soils. In recent years, there has been an increasing interest in the occurrence of biological nitrification inhibition (BNI), a natural phenomenon according to which certain plants can inhibit nitrification through the release of active compounds in root exudates. Here, we synthesize the current state of research but also unravel knowledge gaps in the field. The nitrification process is discussed considering recent discoveries in genomics, biochemistry and ecology of nitrifiers. Secondly, we focus on the 'where' and 'how' of BNI. The N transformations and their interconnections as they occur in, and are affected by, the rhizosphere, are also discussed. The NH4+ and NO3- retention pathways alternative to BNI are reviewed as well. We also provide hypotheses on how plant compounds with putative BNI ability can reach their targets inside the cell and inhibit ammonia oxidation. Finally, we discuss a set of techniques that can be successfully applied to solve unresearched questions in BNI studies.

Niche separation of ammonia-oxidizing bacteria across a tidal freshwater marsh.

Like many functional groups or guilds of microorganisms, the group of ammonia-oxidizing bacteria (AOB) consists of a number of physiologically different species or lineages. These physiological differences suggest niche differentiation among these bacteria depending on the environmental conditions. Species of AOB might be adapted to different zones in the flooding gradient of a tidal marsh. This issue has been studied by sampling sediments from different sites and depths within a tidal freshwater marsh along the river Scheldt near the village of Appels in Belgium. Samples were taken in February, April, July and October 1998. Communities of AOB in the sediment were analysed on the basis of the 16S rRNA gene by application of polymerase chain reaction in combination with denaturing gradient gel electrophoresis (DGGE). In addition, moisture content and concentrations of ammonium and nitrate were determined as well as the potential ammonia-oxidizing activities. Six different DGGE bands belonging to the beta-subclass of the Proteobacteria were observed across the marsh. The community composition of AOB was determined by the elevation in the flooding gradient as well as by the sampling depth. The presence of plants was less important for the community composition of AOB. DGGE bands affiliated with the Nitrosospira lineage were mostly found in the upper part of the marsh and in the deeper layers of the sediment. Two of the three DGGE bands related to the Nitrosomonas oligotropha lineage were more broadly distributed over the marsh, but were predominantly found in the upper layers of the sediment. Members of the environmental Nitrosomonas lineage 5 were predominantly detected in the deeper layers in the lower parts of the marsh. Potential driving factors for niche differentiation are discussed.

Biogeography of sulfate-reducing prokaryotes in river floodplains.

In this study, a large-scale field survey was conducted to describe the biogeography of sulfate-reducing prokaryotes (SRPs) in river floodplains. Fingerprints obtained with three methods, i.e. 16S rRNA gene-based oligonucleotide microarray, dsrB-based denaturing gradient gel electrophoresis (DGGE) and polar lipid-derived fatty acid (PLFA) analyses, were used as a proxy to describe the SRPs community diversity. Each set of profiles was subjected to a combined multivariate/correlation analysis in order to compare SRP community profiles and to highlight the environmental variables influencing the SRPs distribution along environmental gradients. Floodplain soils harbored distinct SRP communities displaying biogeographic patterns. Nearly all profiles from the tidal sites consistently separated from the nontidal sites, independently from the screening method and the multivariate statistics used. The distribution of the microarray/DGGE/PLFA-based fingerprints in the principal component plots could be correlated to eight soil variables, i.e. soil organic matter, total nitrogen, total phosphorous and total potassium, and extractable ammonium, nitrate, phosphate and sulfate, as well as seven pore water variables, i.e. phosphate, sulfate, sulfide, chloride, sodium, potassium and magnesium ions. Indication of a salinity- and plant nutrient-dependent distribution of SRPs related to Desulfosarcina, Desulfomonile and Desulfobacter was suggested by microarray, DGGE and PLFA analyses.

Limitations of the use of group-specific primers in real-time PCR as appear from quantitative analyses of closely related ammonia-oxidising species.

To study the ecology of ammonia-oxidising bacteria (AOB), quantitative techniques are essential. Real-time PCR assays based on the 16S rRNA or on the structural amoA gene are routinely used. The CTO primer set rooted on the 16S rRNA gene has a number of mismatches with some of the cultures of AOB. To examine if these mismatches have an effect on the outcome of real-time PCR assays, the assay was tested with DNA from a number of closely related isolates of AOB. Standard curves of known amounts of initial DNA were similar among most of the tested cultures of AOB, except for the standard curves of Nitrosomonas strain AL212 and Nitrosospira strain NpAV. Nitrosomonas strain AL212 had 3 mismatches with the CTO primer set. Adaptation of the CTO primer set in order to perfectly match the Nitrosomonas strain AL212 gave a standard curve similar to the majority of the AOB tested. As Nitrosospira strain NpAV has no mismatches with the original CTO primer set, there must be another reason for the less efficient amplification than the sequence itself. Application of an existing sigmoidal mathematical model gave no other results with respect to the standard curves of Nitrosomonas europaea and Nitrosomonas strain AL212, but also demonstrated that primer mismatches can seriously underestimate the initial target concentration. It was concluded that in general correct interpretation of real-time PCR results requires knowledge of the target community composition, in particular of the target sequences of the dominant community members.

Improved PCR-DGGE for high resolution diversity screening of complex sulfate-reducing prokaryotic communities in soils and sediments.

In this study we evaluated a high resolution PCR-DGGE strategy for the characterization of complex sulfate-reducing microbial communities inhabiting natural environments. dsrB fragments were amplified with a two-step nested PCR protocol using combinations of primers targeting the dissimilatory (bi)sulfite reductase genes. The PCR-DGGE conditions were initially optimized using a dsrAB clone library obtained from a vegetated intertidal riparian soil along the river Rhine (Rozenburg, the Netherlands). Partial dsrB were successfully amplified from the same environmental DNA extracts used to construct the library, DGGE-separated and directly sequenced. The two approaches were in good agreement: the phylogenetic distribution of clones and DGGE-separated dsrB was comparable, suggesting the presence of sulfate-reducing prokaryotes (SRP) belonging to the families 'Desulfobacteraceae,' 'Desulfobulbaceae' and 'Syntrophobacteraceae,' and to the Desulfomonile tiedjei- and Desulfobacterium anilini-groups. The nested PCR-DGGE was also used to analyze sediment samples (Appels, Belgium) from a series of microcosms subjected to a tidal flooding regime with water of different salinity, and proved to be a valid tool also to monitor the SRP community variation over time and space as a consequence of environmental changes.

Water management strategies against toxic Microcystis blooms in the Dutch delta.

To prevent flooding of the Dutch delta, former estuaries have been impounded by the building of dams and sluices. Some of these water bodies, however, experience major ecological problems. One of the problem areas is the former Volkerak estuary that was turned into a freshwater lake in 1987. From the early 1990s onward, toxic Microcystis blooms dominate the phytoplankton of the lake every summer. Two management strategies have been suggested to suppress these harmful algal blooms: flushing the lake with fresh water or reintroducing saline water into the lake. This study aims at an advance assessment of these strategies through the development of a mechanistic model of the population dynamics of Microcystis. To calibrate the model, we monitored the benthic and pelagic Microcystis populations in the lake during two years. Field samples of Microcystis were incubated in the laboratory to estimate growth and mortality rates as functions of light, temperature, and salinity. Recruitment and sedimentation rates were measured in the lake, using traps, to quantify benthic-pelagic coupling of the Microcystis populations. The model predicts that flushing with fresh water will suppress Microcystis blooms when the current flushing rate is sufficiently increased. Furthermore, the inlet of saline water will suppress Microcystis blooms for salinities exceeding 14 g/L. Both management options are technically feasible. Our study illustrates that quantitative ecological knowledge can be a helpful tool guiding large-scale water management.

Potential for Sulfate Reduction in Mangrove Forest Soils: Comparison between Two Dominant Species of the Americas.

Avicennia and Rhizophora are globally occurring mangrove genera with different traits that place them in different parts of the intertidal zone. It is generally accepted that the oxidizing capacity of Avicennia roots is larger than that of Rhizophora roots, which initiates more reduced conditions in the soil below the latter genus. We hypothesize that the more reduced conditions beneath Rhizophora stands lead to more active sulfate-reducing microbial communities compared to Avicennia stands. To test this hypothesis, we measured sulfate reduction traits in soil samples collected from neighboring Avicennia germinans and Rhizophora mangle stands at three different locations in southern Florida. The traits measured were sulfate reduction rates (SRR) in flow-through reactors containing undisturbed soil layers in the absence and presence of easily degradable carbon compounds, copy numbers of the dsrB gene, which is specific for sulfate-reducing microorganisms, and numbers of sulfate-reducing cells that are able to grow in liquid medium on a mixture of acetate, propionate and lactate as electron donors. At the tidal locations Port of the Islands and South Hutchinson Islands, steady state SRR, dsrB gene copy numbers and numbers of culturable cells were higher at the A. germinans than at the R. mangle stands, although not significantly for the numbers at Port of the Islands. At the non-tidal location North Hutchinson Island, results are mixed with respect to these sulfate reduction traits. At all locations, the fraction of culturable cells were significantly higher at the R. mangle than at the A. germinans stands. The dynamics of the initial SRR implied a more in situ active sulfate-reducing community at the intertidal R. mangle stands. It was concluded that in agreement with our hypothesis R. mangle stands accommodate a more active sulfate-reducing community than A. germinans stands, but only at the tidal locations. The differences between R. mangle and A. germinans stands were absent at the non-tidal, impounded location.