Grantee Research Project Results
2024 Progress Report: Microbial Community Models for Measuring Survival and Persistence of SynBio Microbes in Soil
EPA Grant Number: R840206Title: Microbial Community Models for Measuring Survival and Persistence of SynBio Microbes in Soil
Investigators: Farny, Natalie G
Institution: Worcester Polytechnic Institute
EPA Project Officer: Callan, Richard
Project Period: July 1, 2021 through May 10, 2025
Project Period Covered by this Report: July 1, 2023 through June 30,2024
Project Amount: $449,213
RFA: Assessment Tools for Biotechnology Products (2020) RFA Text | Recipients Lists
Research Category: Safer Chemicals , Chemical Safety for Sustainability
Objective:
The overarching goal of the work supported by this grant is to understand, predict, and control the relationships between soil microbial communities (SMCs) and genetically engineered microbes (GEMs). In order to better understand and predict the behavior of GEMs within a soil environment, we aim to develop scalable, high-throughput laboratory models to measure survival and persistence of GEMs in soils. These models will enable us to understand how a GEM released into an environment – for example to remediate contaminated soils – will behave. The models also permit us to test how best to control the GEM after release. We propose that GEM control could be achieved by altering the balance of members within an existing SMC.
Progress Summary:
During the current project report period, we made three new advancements to the project:
i. Liquid SESOM model is an accurate and stable representation of the native soil microbiome in environmental samples. In the previous annual report, we confirmed that the liquid SESOM (solubilized extract of soil organic material) extracted soil microbial community (SMC) is stable in liquid culture at room temperature over a period of 28 days, and confirmed the SESOM model maintains species that are not culturable in isolation, in relatively similar proportions to the solid soil. Thus, we achieved our goals for development of a facile soil model for GEM risk assessment. However, its translatability to environmental samples remained to be determined. We have now confirmed these results using genuine environmental samples from a wooded and a roadside area in suburban Massachusetts. Using 16S rDNA sequencing and beta diversity (UniFrac) analysis, we show that even after 28 days, the SESOM samples are more akin to the starting SESOM extracts than they are to extracts from other soils cultured over the 28-day period. We conclude that each SESOM-extracted SMC therefore retains the unique SMC signature of its starting soil, regardless of its origin.
ii. The SESOM model is scalable to high throughput. We have established that the SESOM model can be scaled to high-throughput analysis. The experiments shown in Figure 2 (below) were completed using samples grown in 3mL cultures in 24-well deep well assay plates. These plates unable us to use multichannel pipettors and to move samples into 96-well plates for flow cytometry analysis using an autosampler. Analytical laboratories using this technique could, therefore, use technologies such as robotic liquid handling to perform this assay in the future. Thus, our system has met our goal of generating a soil model that is capable of adaptation to high-throughput analysis.
iii. Engineered microbes display reproducible, distinct, soil-specific patterns of survival and persistence. A major goal of the program was to apply our soil survival assay (SESOM) to identify soil type-specific differences in the survival and persistence of a GEM. Using our flow cytometry-based SESOM assay, we compared the survival profiles of our model GEM (P. putida expressing tdTomato) in two soil types, a commercial potting soil and a sample taken from the roadside of a suburban neighborhood in eastern Massachusetts. As hypothesized, we observe these survival and persistence dynamics to be specific to different soil types.
Future Activities:
The No-Cost Extension period (Year 4) will be used to complete the experiments proposed in Objective 2, related to the co-culture of our GEM with native species as a potential biocontrol mechanism. We will perform the continuous co-culture assay in our liquid soil model with additional species, and then add selected co-cultured species to liquid extracted soil communities to measure the impact of the species on GEM survival and persistence. We will further determine the impact of lead contamination on GEM survival and persistence. Finally, we will use the additional program time in Year 4 to write and publish the results of our studies.
Journal Articles:
No journal articles submitted with this report: View all 2 publications for this projectSupplemental Keywords:
Risk assessment, bioremediation, biocontainment, soil ecology, TNT, soil, bacteria, soil microbial community (SMC), genetically engineered microbe (GEM).Relevant Websites:
Natalie Farny’s faculty website Exit
Natalie Farny’s Google Scholar profile Exit
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.