Project description:We developed a laboratory-scale model to improve our understanding and capacity to assess the biological risks of genetically engineered bacteria and their genetic elements in the natural environment. Our hypothetical scenario concerns an industrial bioreactor failure resulting in the introduction of genetically engineered bacteria to a downstream municipal wastewater treatment plant (MWWTP). As the first step towards developing a model for this scenario, we sampled microbial communities from the aeration basin of a MWWTP at three seasonal time points. Having established a baseline for community composition, we investigated how the community changed when propagated in the laboratory, including cell culture media conditions that could provide selective pressure in future studies. Specifically, using PhyloChip 16S rRNA gene-targeting microarrays, we compared the compositions of sampled communities to those of inoculates propagated in the laboratory in simulated wastewater conditionally amended with various carbon sources (glucose, chloroacetate, D-threonine) or the ionic liquid 1-ethyl-3-methylimidazolium chloride ([C2mim]Cl). Proteobacteria, Bacteroidetes, and Actinobacteria were predominant in aeration basin and laboratory-cultured populations. Laboratory-cultured populations were enriched in Gammaproteobacteria. Enterobacteriaceae and Aeromonadaceae were enriched by glucose, Pseudomonadaceae by chloroacetate and D-threonine, and Burkholderiaceae by high (50 mM) concentrations of chloroacetate. Microbial populations cultured with chloroacetate and D-threonine were more similar to sampled populations than thoes cultured with glucose or [C2mim]Cl. Although observed relative richness in operational taxonomic units was lower for laboratory cultures than for sampled populations, both flask and reactor systems cultured phylogenetically diverse communities. These results importantly provide a foundation for laboratory models of industrial bioreactor failure scenarios.

Project description:We developed a laboratory-scale model to improve our understanding and capacity to assess the biological risks of genetically engineered bacteria and their genetic elements in the natural environment. Our hypothetical scenario concerns an industrial bioreactor failure resulting in the introduction of genetically engineered bacteria to a downstream municipal wastewater treatment plant (MWWTP). As the first step towards developing a model for this scenario, we sampled microbial communities from the aeration basin of a MWWTP at three seasonal time points. Having established a baseline for community composition, we investigated how the community changed when propagated in the laboratory, including cell culture media conditions that could provide selective pressure in future studies. Specifically, using PhyloChip 16S rRNA gene-targeting microarrays, we compared the compositions of sampled communities to those of inoculates propagated in the laboratory in simulated wastewater conditionally amended with various carbon sources (glucose, chloroacetate, D-threonine) or the ionic liquid 1-ethyl-3-methylimidazolium chloride ([C2mim]Cl). Proteobacteria, Bacteroidetes, and Actinobacteria were predominant in aeration basin and laboratory-cultured populations. Laboratory-cultured populations were enriched in Gammaproteobacteria. Enterobacteriaceae and Aeromonadaceae were enriched by glucose, Pseudomonadaceae by chloroacetate and D-threonine, and Burkholderiaceae by high (50 mM) concentrations of chloroacetate. Microbial populations cultured with chloroacetate and D-threonine were more similar to sampled populations than thoes cultured with glucose or [C2mim]Cl. Although observed relative richness in operational taxonomic units was lower for laboratory cultures than for sampled populations, both flask and reactor systems cultured phylogenetically diverse communities. These results importantly provide a foundation for laboratory models of industrial bioreactor failure scenarios. 46 samples, flask and reactor experiments were conducted in triplicate with two exceptions: [C2mim]Cl_flask and No-Carbon_flask treatments had only one sample (no replicates).

Project description:Anthropogenic nutrient inputs alter soil biodiversity; however, it remains largely unknown whether changes in soil microeukaryotes (fungi and protists) are primarily driven by direct effects, such as modifications in soil properties, or by indirect effects, such as plant diversity loss. To disentangle these mechanisms, we investigated the long-term effects (11 years) of fertilization and manipulated plant diversity (1, 2, or 4 plant species) on soil microeukaryote communities in a temperate grassland experiment using long-amplicon rRNA sequencing. Our results indicate that fertilization generally had a stronger influence on microeukaryote communities than plant species richness. Fertilization altered the community composition of fungi and protists, increased OTU richness by 20.8% and 52.7%, respectively, and shifted community dominance from fungi to protists. Regarding plant diversity, we observed an effect exclusively on the protist community. Changes were primarily explained by increased plant biomass (driven by both fertilization and plant diversity) and by higher soil phosphorus and lower soil pH levels (driven exclusively by fertilization). Regarding life strategies, we observed synergistic treatment effects: fertilization primarily enhanced fungal saprophytes (only richness), fungal animal pathogens, and protist consumers, whereas plant diversity affected phototrophic protists (reduction) and protist animal pathogens (enhancement). Notably, fertilization and plant diversity decline together led to a cumulative increase in fungal plant pathogens. In conclusion, we highlight that fertilisation alone has a significant effect on soil microeukaryotes, while the additional decline in plant diversity affects different soil groups that are not directly affected by fertilisation. This synergistic pattern indicates that fertilization can influence the entire microeukaryote community through direct and indirect mechanisms, with a cumulative enhancement on certain groups, such as plant pathogens.

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