Project description:This experiment aimed to understand stress responses of microbial communities differing in chronic exposure to the photosynthesis inhibitor diuron, combining untargeted metatranscriptomics (RNA-seq) and dose-response design. First, river microbial communities were incubated for 5-weeks in microcosms 1/ under constant exposure to 4µg/L of diuron (stressed community) or 2/ without contamination (non-stressed community). Then, both communities were exposed for 1 hour to a gradient of diuron concentrations to investigate differences in stress responses after chronic exposure. This experimental design enabled the determination of contig response trends as well as sensitivity thresholds.
Project description:Identify and characterize two distinct communities, the aerobic community and the anaerobic community in the partial nitritation/anammox reactors using metaproteomics approach
Project description:Widespread organic pollutants such as BTEX (benzene, toluene, ethylbenzene, and xylene) are traditionally considered to enhance soil carbon loss through mineralization and ecotoxicity. Challenging this paradigm, we reveal that BTEX can stimulate microbial carbon chain elongation (CE)—a previously overlooked carbon fixation pathway—thereby reshaping soil carbon dynamics. Through phased amplicon sequencing, metagenomics, and metaproteomics, we demonstrate that BTEX exerts bidirectional regulation on CE at both taxonomic and molecular levels. Specifically, BTEX selectively enriches Clostridium_sensu_stricto_12 and Rummelibacillus, while suppressing Acinetobacter, a key CE contributor in natural soils. BTEX also inhibits Petrimonas, a syntrophic degrader of medium-chain fatty acids (MCFAs), promoting MCFAs accumulation. Moreover, BTEX-degrading bacteria establish cooperative interactions with CE bacteria, facilitating the sequestration of carbon as MCFAs rather than complete mineralization to CO₂, with Bacillus bridging both metabolic roles. At the molecular level, BTEX enhances CE by accelerating substrate uptake and acetyl-CoA flux into the reverse β-oxidation (RBO) pathway. Multi-omics analysis revealed that BTEX downregulates fatty acid biosynthesis (FAB), another pathway of CE, through fabR, acrR, and fadR while maintaining NADH availability to relieve Rex-mediated inhibition of the key RBO enzyme gene bcd. However, excessive BTEX disrupts metabolic homeostasis and suppresses CE activity. Collectively, our findings redefine the ecological implications of aromatic hydrocarbon contamination by uncovering its capacity to modulate anaerobic carbon fixation and retention in soil microbial communities. This work highlights a previously unrecognized link between pollutant degradation and biogenic carbon sequestration, with broader implications for understanding soil biogeochemical resilience under anthropogenic pressure.
Project description:Two parallel anaerobic digestion lines were designed to match a "bovid-like" digestive structure. Each of the lines consisted of two Continuous Stirred Tank Reactors placed in series and separated by an acidic treatment step. The first line was inoculated with industrial inocula whereas the second was seeded with cow digestive tract contents. After three month of continuous sewage sludge feeding, samples were recovered for shotgun metaproteomic and DNA-based analysis. Strikingly, protein inferred and 16S rDNA tags based taxonomic community profiles were not fully consistent. Principal Component analysis however revealed a similar clustering pattern of the samples, suggesting that reproducible methodological and/or biological factors underlie this observation. The performances of the two digestion lines did not differ significantly and the cow derived inocula did not establish in the reactors. A low throughput metagenomic dataset (3.4x106 reads, 1.1 Gb) was also generated for one of the samples. It allowed a substantial increase of the analysis depth (increase of the spectral identification rate). For the first time, a high level of proteins expressed by members of the "Candidatus Competibacter" group is reported in an anaerobic digester, a key microbial player in environmental bioprocess communities.
Project description:Industrial anaerobic digestion (AD) represents a relevant energy source beyond today’s fossil fuels, wherein organic matter is recycled to methane gas via an intricate and complex microbial food web. Despite its potential, anaerobic reactors often undergo process instability over time, mainly caused by substrate composition perturbations, making the system unreliable for stable energy production. To ensure the reliability of AD technologies, it is crucial to identify microbial- and system responses to better understand the effect of such perturbations and ultimately detect signatures indicative of process failure . Here, we investigate the effect of microalgal organic loading rate (OLR) on the fermentation products profile, microbiome dynamics, and disruption/recovery of major microbial metabolisms. Reactors subjected to low- and high-OLR disturbances were operated and monitored for fermentation products and biogas production over time, while microbial responses were investigated via 16S rRNA gene amplicon data, shotgun metagenomics and metagenome-centric metaproteomics.
Project description:Through chemical contamination of natural environments, microbial communities are exposed to many different types of chemical stressors; however, research on whole genome responses to this contaminant stress is limited. This study examined the transcriptome response of a common soil bacterium, Pseudomonas aeruginosa, to the common environmental contaminant pentachlorophenol (PCP). Cells were grown in chemostats at a low growth rate to obtain substrate-limited, steady-state, balanced-growth conditions. The PCP stress was administered as a continuous increase in concentration, and samples taken over time were examined for physiological function changes with whole cell acetate uptake rates (WAUR) and cell viability, and for gene expression changes using Affymetrix GeneChip technology and RT-PCR. Cell viability, measured by heterotrophic plate counts, showed a moderately steady decrease after exposure to the stressor, but WAURs did not change in response to PCP. In contrast to the physiological data, the microarray data showed significant changes in the expression of several genes. In particular, genes coding for multi-drug efflux pumps, including MexAB-OprM, were strongly upregulated. The upregulation of these efflux pumps protected the cells from the potentially toxic effects of PCP, allowing the physiological whole-cell function to remain constant. Experiment Overall Design: Cells of P. aeruginosa were grown in steady-state nutrient-limiting conditions using a minimal medium with acetate as the sole carbon source. PCP was added as a continuous input, and the samples were taken at timepoints corresponding to approximately 0.5, 1, and 2 generation times. Reactors were run at least in triplicate, and samples from duplicate WT reactors and a single RpoS- reactor were used for the microarrays. Gene expression is reported as the average fold-change associated with gene expression of PCP-shocked cells compared with the pre-PCP timepoint (0 hours) for the WT.
Project description:By using metagenome resolved protein stable isotope probing (protein-SIP) through incubations of identical reactors with 13C labelled bicarbonate over a period of 48 hours, the study aims to map differences in the metabolic behaviour of the microbial community during anaerobic digestion.
Project description:Low concentrations of pharmaceutical compounds were shown to induce transcriptional responses in isolated microorganisms, which could have consequences on ecosystem dynamics. In order to test if these transcriptional responses could also be observed in complex river microbial communities, biofilm reactors were inoculated with water from two distinct rivers and supplemented with environmentally relevant doses of four pharmaceutical products (erythromycin-ER, gemfibrozil-GM, sulfamethazine-SN and sulfamethoxazole-SL). To follow the expression of functional genes, we constructed a 9,600 features anonymous DNA microarray platform onto which cDNA from the various biofilms was hybridized. The reactor design for biofilm development has been previously described (Lawrence et al., 2004; Lawrence et al., 2000). Two duplicate experiments were carried out, with reactors being inoculated with either water from the WC (nutrient rich) or the SSR (nutrient poor). Treatments consisted in the addition of various pharmaceutical compounds: 1 µg l-1 erythromycin (ER), 1 µg l-1 gemfibrozil (GM), 0.5 µg l-1 sulfamethazine (SN), 0.5 µg l-1 sulfamethoxazole (SL). Nothing was added to control reactors (CO). All treatments were replicated independently three times. A reference sample (composite sample from Wascana Creek reactors used to construct the microarray) was hybridized (Cy5) on each slide.
Project description:Biofilms are ubiquitous in nature, forming diverse adherent microbial communities that perform a plethora of functions. Here, we operated two laboratory-scale sequence batch reactors enriched with Candidatus Accumulibacter phosphatis (Accumulibacter) performing enhanced biological phosphorus removal (EBPR). Reactors formed two distinct biofilms, a floccular biofilm, consisting of small, loose, microbial aggregates, and a granular biofilm, forming larger, dense, spherical aggregates. Using metaproteomic methods we investigated the proteomic differences between these two biofilm communities, identifying a total of 2022 unique proteins. Both biofilms contained proteins that were indicative of core EBPR metabolisms and cellular function. To understand the proteomic differences between floccular and granular biofilm communities, we compared protein abundances that were statistically enriched in both biofilm states (alpha level = 0.05). Floccular biofilms were enriched with pathogenic secretion systems suggesting a previously unrecognized, highly competitive, mixed microbial community. Comparatively, granular biofilms revealed a high stress environment with evidence of nutrient starvation, phage predation pressure, extracellular polymeric substance (EPS) synthesis, and increased cell lysis. Granular biofilms enriched outermembrane transport proteins to scavenge the extracellular milieu for amino acids and other metabolites, likely released through cell lysis, to supplement core EBPR metabolic pathways. This study provides the first detailed proteomic comparison between Accumulibacter–enriched floccular and granular biofilm communities, proposes a conceptual model for the granule biofilm, and offers novel insights into granule biofilm formation and stability.