Project description:The community composition (in terms of abundance, distribution and contribution of diverse clades) of bacteria involved in nitrogen transformations in the oxygen minimum zones may be related to the rates of fixed N loss in these systems. The abundance of both denirifying and anammox bacteria, and the assemblage composition of denitrifying bacteria were investigated in the Eastern Tropical South Pacific and the Arabian Sea using assays based on molecular markers for the two groups of bacteria. The abundance and distribution of bacteria associated with the fixed N removal processes denitrification and anammox were investigated using quantitative PCR for genes encoding nitrite reductase (nirK and nirS) in denitrifying bacteria and hydrazine oxidase(hzo) and 16S rRNA genesin anammox bacteria. All of these genes had depth distributions with maxima associated with the secondary nitrite maximum in low oxygen waters. NirS was mch more abundant than nirK, and much more abundant than the 16S rRNA gene from anammox bacteria. The ratio of hzo:16S rRNA for anammox was low and variable implying greater unexplored diversity in the the hzo gene. Assemblage composition of the abundant nirS-type denitrifiers was evaluated using a funcitonal gene microarray. Of the nirS archetypes represented on the microarray, very few occurred speficically in one region or depth interval, but the assemblages varied significantly. Community composition of denitrifiers based on microarray analysis of the nirS gene was most different between geographical regions. Within each region, the surface layer and OMZ assemblages clustered distinctly. Thus, in addition to spatial and temporal variation in denitrificaiton and anammox rates, both microbial abundance and community composition also vary between OMZ regions and depths.
Project description:Pristine groundwater is a highly stable environment with microbes adapted to dark, oligotrophic conditions. Input events like heavy rainfalls can introduce excess particulate organic matter including surface-derived microbes into the groundwater, hereby creating a disturbance to the groundwater microbiome. Some of the translocated bacteria are not able to thrive in groundwater and will form necromass. Here, we investigated the effects of necromass addition to the microbial community in fractured bedrock groundwater, using groundwater mesocosms as model systems. We followed the uptake of 13C-labeled necromass by the bacterial and eukaryotic groundwater community quantitatively and over time by employing a combined protein and DNA stable isotope probing approach. Necromass was rapidly depleted in the mesocosms within four days, accompanied by a strong decrease of Shannon diversity and an increase of bacterial 16S rRNA gene copy numbers by one order of magnitude. Species of Flavobacterium, Massilia, Rheinheimera, Rhodoferax and Undibacterium dominated the microbial community within two days and were identified as key players in necromass degradation, based on a 13C incorporation of > 90% in their peptides. Their proteomes showed various uptake and transport related proteins, and many proteins involved in metabolizing amino acids. After four and eight days of incubation, autotrophic and mixotrophic groundwater species of Nitrosomonas, Limnohabitans, Paucibacter and Acidovorax increased in abundance, with a 13C incorporation between 0.5 and 23%. Our data point towards a very fast and exclusive uptake of labeled necromass by a few specialists followed by a concerted action of groundwater microorganisms, including autotrophs presumably fueled by released, reduced nitrogen and sulfur compounds generated during necromass degradation.
Project description:Gene expression microarrays were performed to investigate the molecular effects of exposure to environmental polluted groundwater. Zebrafish was treated with polluted waters collected from dumps located upstream and downstream a sanitary landfills. Gene expression profiling of zebrafish liver was analyzed after acute exposure to sampled waters.
Project description:Gene expression microarrays were performed to investigate the molecular effects of exposure to environmental polluted groundwater. Mice were treated with polluted waters collected from dumps located upstream and downstream a sanitary landfills. Gene expression profiling of mouse liver was analyzed after acute and chronic exposure to sampled waters.
Project description:The community composition (in terms of abundance, distribution and contribution of diverse clades) of bacteria involved in nitrogen transformations in the oxygen minimum zones may be related to the rates of fixed N loss in these systems. The abundance of both denirifying and anammox bacteria, and the assemblage composition of denitrifying bacteria were investigated in the Eastern Tropical South Pacific and the Arabian Sea using assays based on molecular markers for the two groups of bacteria. The abundance and distribution of bacteria associated with the fixed N removal processes denitrification and anammox were investigated using quantitative PCR for genes encoding nitrite reductase (nirK and nirS) in denitrifying bacteria and hydrazine oxidase(hzo) and 16S rRNA genesin anammox bacteria. All of these genes had depth distributions with maxima associated with the secondary nitrite maximum in low oxygen waters. NirS was mch more abundant than nirK, and much more abundant than the 16S rRNA gene from anammox bacteria. The ratio of hzo:16S rRNA for anammox was low and variable implying greater unexplored diversity in the the hzo gene. Assemblage composition of the abundant nirS-type denitrifiers was evaluated using a funcitonal gene microarray. Of the nirS archetypes represented on the microarray, very few occurred speficically in one region or depth interval, but the assemblages varied significantly. Community composition of denitrifiers based on microarray analysis of the nirS gene was most different between geographical regions. Within each region, the surface layer and OMZ assemblages clustered distinctly. Thus, in addition to spatial and temporal variation in denitrificaiton and anammox rates, both microbial abundance and community composition also vary between OMZ regions and depths. Two color array (Cy3 and Cy5): the universal standard 20-mer oligo is printed to the slide with a 70-mer oligo (an archetype). Environmental DNA sequences (fluoresced with Cy3) within 15% of the 70-mer conjugated to a 20-mer oligo (fluoresced with Cy5) complementary to the universal standard will bind to the oligo probes on the array. Signal is the ratio of Cy3 to Cy5. Three replicate probes were printed for each archetype. Two replicate arrays were run on duplicate targets.
Project description:Metagenome-assembled genomes (MAGs) have revealed the existence of novel bacterial and archaeal groups and provided insight into their genetic potential. However, metagenomics and even metatranscriptomics cannot resolve how the genetic potential translates into metabolic functions and physiological activity. Here, we present a novel approach for the quantitative and organism-specific assessment of the carbon flux through microbial communities with stable isotope probing-metaproteomics and integration of temporal dynamics in 13C incorporation by Stable Isotope Cluster Analysis (SIsCA). We used groundwater microcosms labeled with 13CO2 and D2O as model systems and stimulated them with reduced sulfur compounds to determine the ecosystem role of chemolithoautotrophic primary production. Raman microspectroscopy detected rapid deuterium incorporation in microbial cells from 12 days onwards, indicating activity of the groundwater organisms. SIsCA revealed that groundwater microorganisms fell into five distinct carbon assimilation strategies. Only one of these strategies, comprising less than 3.5% of the community, consisted of obligate autotrophs (Thiobacillus), with a 13C incorporation of approximately 95%. Instead, mixotrophic growth was the most successful strategy, and was represented by 12 of the 15 MAGs expressing pathways for autotrophic CO2 fixation, including Hydrogenophaga, Polaromonas and Dechloromonas, with varying 13C incorporation between 5% and 90%. Within 21 days, 43% of carbon in the community was replaced by 13C, increasing to 80% after 70 days. Of the 31 most abundant MAGs, 16 expressed pathways for sulfur oxidation, including strict heterotrophs. We concluded that chemolithoautotrophy drives the recycling of organic carbon and serves as a fill-up function in the groundwater. Mixotrophs preferred the uptake of organic carbon over the fixation of CO2, and heterotrophs oxidize inorganic compounds to preserve organic carbon. Our study showcases how next-generation physiology approach like SIsCA can move beyond metagenomics studies by providing information about expression of metabolic pathways and elucidating the role of MAGs in ecosystem functioning.
Project description:The ecophysiology of complete ammonia oxidizing Nitrospira (CMX) and their widespread occurrence in groundwater suggests that CMX bacteria have a competitive advantage over ammonia-oxidizing bacteria (AOB) and archaea (AOA) in these environments. However, the relevance of their activity from the ecosystem-level process perspective has remained unclear. We investigated oligotrophic carbonate rock aquifers as a model system to assess the contribution of CMX, AOA and AOB to nitrification and to identify the environmental drivers of their niche differentiation at different levels of ammonium and oxygen. CMX accounted for up to 95% of the ammonia oxidizer communities. Nitrification rates were positively correlated to CMX clade A-associated phylotypes and AOB affiliated with Nitrosomonas ureae. Surprisingly, short-term incubations amended with the nitrification inhibitors allylthiourea and chlorate suggested that AOB contributed more than 90% to overall ammonia oxidation, while metaproteomics analysis confirmed an active role of CMX in both ammonia and nitrite oxidation. Ecophysiological niche differentiation of CMX clades A and B, AOA and AOB was linked to their requirements for ammonium, oxygen tolerance, and metabolic versatility. Our results demonstrate that despite numerical predominance of CMX, the first step of nitrification in oligotrophic groundwater is primarily governed by AOB. Higher growth yields at lower NH4+ turnover rates and energy derived from nitrite oxidation most likely enable CMX to maintain consistently high populations. Activity measurements combined with differential inhibition allowed a refined understanding of ammonia oxidizer coexistence, competition and cooperation beyond the insights from molecular data alone.
Project description:The use of aqueous film-forming foams (AFFF) at fire-training areas (FTAs) has introduced into ground- and surface waters a complex mixture of per- and poly-fluorinated alkyl substances (PFAS). The toxicity of environmental PFAS mixtures to wildlife is not well understood and presents a knowledge gap that limits accurate risk assessment. To evaluate reproductive biomarker responses to complex environmental PFAS mixtures, we conducted a series of on-site experiments using flow-through mobile laboratories exposing fish to groundwater impacted by a legacy FTA and an adjacent reference site A 60K fathead minnow microarray was used to quantify gene expression patterns in the testis and liver of fish exposed to water from Fire Training Area 1 and 2 relative to a reference site.
Project description:The use of aqueous film-forming foams (AFFF) at fire-training areas (FTAs) has introduced into ground- and surface waters a complex mixture of per- and poly-fluorinated alkyl substances (PFAS). The toxicity of environmental PFAS mixtures to wildlife is not well understood and presents a knowledge gap that limits accurate risk assessment. To evaluate reproductive biomarker responses to complex environmental PFAS mixtures, we conducted a series of on-site experiments using flow-through mobile laboratories exposing fish to groundwater impacted by a legacy FTA and an adjacent reference site A 60K fathead minnow microarray was used to quantify gene expression patterns in the testis and liver of fish exposed to water from Fire Training Area 1 and 2 relative to a reference site.
Project description:Anaerobic ammonium-oxidising (anammox) bacteria, members of the ‘Candidatus Brocadiaceae’ family, play an important role in the nitrogen cycle and are estimated to be responsible for about half of the oceanic nitrogen loss to the atmosphere. Anammox bacteria combine ammonium with nitrite and produce dinitrogen gas via the intermediates nitric oxide and hydrazine (anammox reaction) while nitrate is formed as a by-product. These reactions take place in a specialized, membrane-bound compartment called the anammoxosome. Therefore, the substrates ammonium, nitrite and product nitrate have to cross the outer-, cytoplasmic- and anammoxosome membranes to enter or exit the anammoxosome. The genomes of all anammox species harbour multiple copies of ammonium-, nitrite- and nitrate transporter genes. Here we investigated how the distinct genes for ammonium-, nitrite- and nitrate- transport were expressed during substrate limitation in membrane bioreactors. Transcriptome analysis of Kuenenia stuttgartiensis planktonic cells under ammonium-limitation showed that three of the seven ammonium transporter genes and one of the six nitrite transporter genes were significantly upregulated, while another ammonium and nitrite transporter gene were downregulated in nitrite limited growth conditions. The two nitrate transporters were expressed to similar levels in both conditions. In addition, genes encoding enzymes involved in the anammox reaction were differentially expressed, with those using nitrite as a substrate being upregulated under nitrite limited growth and those using ammonium as a substrate being upregulated during ammonium limitation. Taken together, these results give a first insight in the potential role of the multiple nutrient transporters in regulating transport of substrates and products in and out of the compartmentalized anammox cell.