Grantee Research Project Results

2022 Progress Report: Microbial Community Models for Measuring Survival and Persistence of SynBio Microbes in Soil

EPA Grant Number: R840206
Title: 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, 2021 through June 30,2022
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 soil contaminated with TNT – 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:

We have established that we can measure survival and persistence of our sample GEM within a natural SMC in solid soil, using both a traditional colony forming unit assay (CFU) and a streamlined protocol using flow cytometry which we call the solid soil survival assay (solid SSA). We determined, in working on the solid SSA, that adapting the solid SSA to a high-throughput platform would be technically problematic. In the process of testing alternative approaches, we developed the liquid soil survival assay (liquid SSA). This process extracts soluble organic matter and a subset of the microbial community into an easy-to-manipulate liquid medium. We find that GEM survival and persistence in the liquid SSA recapitulates the behavior of the GEM as observed in the solid SSA. The composition and stability of SMCs within the solid SSA and liquid SSA are being assessed using deep sequencing (Illumina) of the bacterial 16S rDNA region. The liquid SSA represents a significant advance in our ability to model GEM behavior in the soil within a high-throughput laboratory environment. As part of our work to identify interspecies relationships, we have isolated culturable bacteria from various soil samples. Applying our liquid SSA, and using two competitor strains, we established a protocol to measure interspecies relationships by flow cytometry. We further demonstrated that we can differentiate live and dead cells, and wild strains versus GEMs, by flow cytometry. We are currently initiating competition assays between our GEM strain and culturable strains isolated from our soil samples.

Future Activities:

We plan to adhere to our original goals and milestones. With SOPs now in place, we project we will make rapid progress in three key areas: 1. Utilizing the solid and liquid SSAs to determine the effects of TNT contamination on GEM survival and persistence. We will add low (100 mg/kg) and high (400 mg/kg) TNT to both the solid and liquid SSAs to measure GEM survival and persistence in the presence of TNT. 2. Building a stable engineered SMC (E-SMC) in the liquid SSA model using culturable species. Applying our 16S sequencing data as a model, we will assemble E-SMCs and track the stability of these communities in both the solid and liquid SSA. Once a stable community is achieved, we will introduce the GEM and measure survival and persistence. 3. Completing individual competition assays between our model GEM and culturable species. We will continue to assess interspecies relationships between our GEM and culturable strains. We will further apply live/dead staining analysis to better narrow down the nature of interspecies relationships.

Journal Articles:

No journal articles submitted with this report: View all 2 publications for this project

Supplemental Keywords:

Risk assessment, bioremediation, biocontainment, soil ecology, TNT, soil, bacteria, soil microbial community (SMC), genetically engineered microbe (GEM).

Relevant Websites:

Farny Lab website Exit
Natalie Farny’s faculty website Exit
Natalie Farny’s Google Scholar profile Exit

Progress and Final Reports:

Original Abstract

The 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.