HR001123S0012.pdf

Broad Agency Announcement Ceres

BIOLOGICAL TECHNOLOGIES OFFICE

HR001123S0012

October 13, 2022

HR001123S0012, Ceres

TABLE OF CONTENTS

PART I: OVERVIEW INFORMATION

PART II: FULL TEXT OF ANNOUNCEMENT

1. Funding Opportunity Description

1.1. Program Overview

1.2. Technical Approach

1.3. Program Structure

1.4. Application Tracks

1.5. Technical Areas

1.6. Independent Verification and Validation (IV&V)

1.7. Program Milestones, Metrics, and Deliverables

1.8. General Requirements

2. Award Information

2.1. General Award Information

2.2. Fundamental Research

3. Eligibility Information

3.1. Eligible Applicants

3.2. Organizational Conflicts of Interest

3.3. Cost Sharing/Matching

4. Application and Submission Information

4.1. Address to Request Application Package

4.2. Contact and Form of Application Submission

4.2.4. Disclosure of Information and Compliance with Safeguarding Covered Defense Information Controls

4.3. Funding Restrictions

4.4. Other Submission Information

5. Application Review Information

5.1. Evaluation Criteria

5.2. Review of Proposals

5.2.1. Countering Foreign Influence Program (CFIP)

6. Award Administration Information

6.1. Submission Status Notifications

6.2. Administrative and National Policy Requirements

6.3. Reporting

6.4. Electronic Systems

7. Agency Contacts

8. Other Information

8.1. Proposers Day

9. APPENDIX 1 – Volume II checklist

PART I: OVERVIEW INFORMATION

Federal Agency Name – Defense Advanced Research Projects Agency (DARPA), Biological Technologies Office (BTO) Funding Opportunity Title – Ceres Announcement Type – Initial Announcement Funding Opportunity Number – HR001123S0012 North American Industry Classification System (NAICS) – 541714 Catalog of Federal Domestic Assistance Numbers (CFDA) – 12.910 Research and

Technology Development Dates o Posting Date: October 13, 2022 o Proposal Abstract Due Date and Time: 4:00 PM ET, November 8, 2022 o Full Proposal Due Date and Time: 4:00 PM ET, January 4, 2023 o BAA Closing Date: January 4, 2023 o Proposers’ Day: October 14, 2022 https://sam.gov/opp/96e4de3319e342238a25eadaee40cbca/view

Concise description of the funding opportunity – Conventional soil bioremediation is laborious, inefficient, and expensive, requiring soil preparation, nutrient delivery to remediating organisms, and continuous site monitoring. Plants and their rhizosphere microbiomes have innate potential to inexpensively perform the costliest aspects of bioremediation—soil preparation, nutrient augmentation, and site monitoring—without routine human intervention. Leveraging the inherent plant-rhizosphere community, Ceres will enhance community performance by engineering synthetic communities to achieve autonomous bioremediation of a fuel, jet propellant 8 (JP-8), and an explosive, 2,4,6-trinitrotoluene (TNT), from soil while overtly reporting the status of this remediation.

Advances in both synthetic biology and synthetic ecology could contribute to the creation of these sophisticated synthetic communities.

Types of instruments that may be awarded – Procurement contract, cooperative agreement, or Other Transaction.

Agency contact The BAA Coordinator for this effort may be reached at:

Ceres@darpa.mil

DARPA/BTO

ATTN: HR001123S0012

675 North Randolph Street Arlington, VA 22203-2114 https://sam.gov/opp/96e4de3319e342238a25eadaee40cbca/view mailto:Ceres@darpa.mil

PART II: FULL TEXT OF ANNOUNCEMENT

1. Funding Opportunity Description

This publication constitutes a Broad Agency Announcement (BAA) as contemplated in Federal Acquisition Regulation (FAR) 6.102(d)(2) and 35.016 and 2 C.F.R. § 200.203. Any resultant award negotiations will follow all pertinent laws and regulations, and any negotiations and/or awards for procurement contracts will use procedures under FAR 15.4, Contract Pricing, as specified in the BAA.

The Biological Technologies Office (BTO) is soliciting proposals to develop innovative technologies and approaches to build synthetic plant-microbe communities that can either (1) cleanse soil contaminants from military installations and battlefields; or (2) overtly report soil status when cleanup is finished. Proposed research should combine innovative approaches to enable revolutionary advances in synthetic ecology, synthetic biology, and high throughput testing of terrestrial systems. Research that generates incremental improvements to the existing state of these scientific fields is specifically excluded.

1.1. PROGRAM OVERVIEW

Residual contaminants can remain in the environment following defense operations, including training. Spilled fuel and explosives such as JP-8 and TNT can significantly degrade local habitats and negatively impact the surrounding communities. Soil remediation often relies on conventional ex situ methods that laboriously and destructively excavate soil for off-site decontamination followed by extensive monitoring to track progress. Ceres aims to greatly reduce the cost of remediation by generating a platform that requires minimal energy and human intervention, leveraging the natural behaviors and processes performed by plant-microbe communities.

Specifically, Ceres seeks to develop modular platforms that demonstrate proof-of-concept capabilities for two specific applications – one focused on cleaning TNT or JP-8 and the other on indicating the presence of TNT and JP-8. The Cleaner Track will develop synthetic plant-rhizosphere microbial communities that would degrade the targeted contaminant down to Environmental Protection Agency (EPA)-approved levels without dangerous byproducts. The Beacon Track will develop a complementary plant-microbe synthetic community that would provide an overt signal that the target contamination has been reduced to EPA-approved levels.

Proposers may chose a Combined Track to address both Cleaner and Beacon for TNT or JP-8.

1.2. TECHNICAL APPROACH

Plants communicate with and control the surrounding soil community in myriad ways. For instance, root exudates enhance rhizosphere organisms, controlling both free-living and root-symbiont microbial community dynamics and metabolism. Reciprocally, microorganisms in the rhizosphere communicate with the plant via metabolites that enable, for example, the plant to increase nutrient uptake. Ceres will leverage these multi-kingdom plant-rhizosphere communities, engineering them into cohesive synthetic communities with optimized track-specific functions.

Proposers are expected to (1) isolate or assemble functional plant-microbe communities; (2) optimize the communities through biological and ecological engineering into synthetic communities; (3) develop controls so that the synthetic communities can be easily removed post remediation; and (4) generate realistic testbeds that mimic field conditions for synthetic community optimizing and testing. Proposers should describe how they will biocontrol the genetically modified organisms (GMOs) using current biocontainment strategies, such as those established through synthetic biology, while also developing ecological containment approaches.

1.3. PROGRAM STRUCTURE

Ceres will be divided into two 24-month phases, and one 6-month phase comprised of a final demonstration performed by an independent verification and validation (IV&V) team.

HR001123S0012 is not soliciting proposals for IV&V, and the Ceres IV&V team will be selected separately by the Government. Thehe Cleaner Track, Beacon Track, and Combined Track all require proposers to develop a functionally resilient synthetic community via four technical areas (TAs) across the first two phases.

In TA1 (Engineer Root Communities), proposers must identify or engineer rhizosphere microorganisms (bacteria, fungi, other microbial eukaryotes, archaea, etc.) that persist in contaminated soil with the support of the plant host and subsequently refine these microorganisms into track-specific functional communities.

In TA2 (Engineer Plants), for either the Cleaner and Beacon tracks, the plant engineered by the proposer must support TA1 microbial community growth. Moreover, for the Cleaner Track, the plant should enhance the Cleaner community’s metabolic activity to increase contaminant degradation rates. Likewise, for the Beacon Track, the plant should also detect signals from the rhizosphere community indicating contamination and then relay those signals to the surface for unaided human observation. Additionally, because rhizosphere community establishment, maintenance, and movement are tightly connected to the plant, the engineered rhizosphere community (TA1) and engineered plant host (TA2) for each track must be functionally integrated by the end of Phase I.

In TA3 (Engineer Ecological Containment), proposers must demonstrate robust biocontainment strategies for controlling the GMOs in the synthetic communities to enable future regulatory approval for soil-controlled greenhouse-testing. Such strategies may include synthetic biology approaches, such as evolutionarily stable ‘off switches’, but must include ecological containment, such as the removal of keystone species or auxotrophy, that leads to the removal of engineered organisms within the rhizosphere. The intent is that the rhizosphere community collapses in the absence of the supporting plant host. The plant component of the synthetic community may be removed through conventional techniques or may be engineered to drive itself out of its own niche.

Finally, in TA4 (Develop Testbeds), proposers will develop high-throughput testbeds that emulate realistic environmental conditions in the laboratory to test synthetic community functionality and biocontainment. Teams must detail in the proposals the key abiotic parameters they will target that characterize the environmental conditions of the target deployment biomes.

In Phase I, the developed testbeds will be parallelizable microcosms that provide rapid screening feedback for synthetic community development. In Phase II, terrestrial testbeds will increase in size and complexity to better emulate the field. Phase III will consist of a single 6-month test at greenhouse scale, performed by the IV&V partner(s). Proposals must address all three phases of their chosen application track.

1.4. APPLICATION TRACKS

To focus technology development, proposals must concentrate on one of four possible application tracks for Department of Defense (DoD) relevant environmental contaminants: a TNT Cleaner, a JP-8 Cleaner, a Beacon for both TNT and JP-8, or a Combined Track to address both Cleaner and Beacon for one contaminant. Because of its greater complexity, Combined Track proposals are allowed an additional five pages in Volume 1 to describe their approach and to outline mitigation for the substantially increased risk associated with the more challenging problem. Combined Track proposals and budgets should be modular so they can be separated into single-track components (Cleaner and Beacon). Synthetic community organisms, including the rhizosphere microorganisms and the plant host, should be chosen and subsequently optimized to function in the three major U.S. biomes: Eastern Woodlands, Great Plains, or Mediterranean (West Coast), regardless of track.

Proposers must identify a soil from one of these U.S. biomes for synthetic community optimization from the start of the project.

Description of Tracks:

► Cleaner Track:

TNT and JP-8 jet fuel contaminate a number of DoD sites in every U.S. state,1 potentially causing acute and long-term harm to humans through the inhalation of soil and dust particles and through consumption of food produced on contaminated land.2,3 TNT often enters soil via low-order detonations, or duds.4 Residue remains in the top 10–300 cm and persists for decades, primarily binding to organic matter or readily photodegrading to DNT at the surface.5

The DoD purchases more than 50 million barrels of jet fuel annually, which comprises ≈ 45% of all military operational energy, being used for transport, tactical power systems, generators, etc.6,7 Such widespread fuel use is associated with soil contamination on military bases: fuel handling leads to small but ubiquitous surface spills while large punctuated fuel spills primarily stem from storage tank leakage. While the lower molecular weight components of JP-8 rapidly volatilize, the higher molecular weight components tend to adsorb to organic matter to persist within the top several meters of soil.8,9 This soil contamination not only poses a risk to those directly exposed to the soil but also hinders the land transitioning into residential or other productive uses.

TNT and JP-8 fundamentally differ in their chemistry and their expected degradation products.

For instance, mineralization is more readily achieved in an aerobic environment for fuel, while an anaerobic environment is more favorable for explosive degradation. Consequently, proposers

1 Lustgarten. (2018) Bombs in our backyard. (propublica.org/series/bombs-in-our-backyard).

2 Kalderis et al (2011) Pure Appl. Chem. doi.org/10.1351/PAC-REP-10-01-05.

3 Gorecki et al (2017) Sci Total Environ. doi.org/10.1016/j.scitotenv.2017.04.213.

4 DoD Technical Report (2014): Operational Range Sustainability through Management of Munitions Constituents.

5 Sharma et al (2013) Chemosphere. doi.org/j.chemosphere.2013.03.028.

6 Defense Working Capital Fund; Defense-Wide FY2021 Budget Estimates, Operating and Capital Budgets Congressional data.

7 OSD Fiscal Year 2020 Operational Energy Annual Report.

8 Risher et al (2017) TP 121 Toxguide, Chapter 6.

9 Arthur et al (1992) NCEL Contract Report.

choosing the Cleaner Track must select ONE contaminant to degrade. Additionally, because jet fuels are compound mixtures and explosives chemically react in the soil, proposers must outline and justify the "marker” compound(s) that will reflect overall contaminant degradation. For example, the marker compound may be some combination of a primary component of the contaminant mixture itself, the most recalcitrant in soil, or a major intermediate degradation product. The concentration of the marker should be a surrogate for the contaminant as a whole. The marker compounds must be remediated to concentrations indicating that the target contaminants are below the environmental thresholds determined by the EPA: industrial screening level (ISL, Phase I) and residential screening level (RSL, Phase II). The EPA’s ISLs and RSLs may be found at comptox.epa.gov/dashboard/. Target contaminants, potential default marker compounds, and corresponding marker ISL/RSL are shown in Table 1. Synthetic communities must function in soils with high and low levels of contamination. Locating contaminants in soil is easier if concentrations are high and more challenging as soil concentrations decrease. As such, synthetic communities optimized for highly contaminated soil may degrade the contaminant readily. In contrast, soil with lower concentrations, but which are still above EPA thresholds, may require more engineering of microbes and/or plant support to enhance microbial growth towards the soil contaminant.

Table 1: Default target contaminants Target Contaminant

Starting Levels (mg/kg soil)

Potential Marker Compound

Marker ISL (mg/kg soil)

Marker RSL (mg/kg soil)

TNT High: 3000 Low: 30

• DNT (Dinitrotoluene)

• ADNT (2-Amino-4,6-DNT/

4-Amino-2,6-DNT)

• p-Cresol (4-Hydroxytoluene)

1.2 0.77

JP-8 Total petroleum hydrocarbons:

High: 20,000 Low: 2000

• Butylbenzene

• Naphthalene

8.6

► Beacon Track:

Conventional bioremediation requires significant time and effort to track the remediation process and thus identify when the process is complete. Contaminated soil in situ can appear ‘normal’ with verdant forests obscuring otherwise uninhabitable soil. Note the continued persistence of the Zone Rouge in France from World War I. The presence of contamination may not be apparent to either the warfighter or the local population living on the contaminated land. To this end, some plants can naturally report soil contamination. The European Blue Zinc Violet (Viola lutea var.

westfalica) grows selectively on soil contaminated with heavy metals and, thus, the flower’s presence denotes soil contamination. Recreating such a natural and overt beacon but for a broader range of contaminants is the goal of the Beacon Track, which will develop plants that present an overt signal indicating the state of soil contamination thus informing decisions about land management. The signal should be binary based on the set threshold and can indicate either that the soil remains contaminated or that the soil has been cleaned. Proposers must detail how this overt signal produced by the plant will be unambiguous, outline how false signals will be mitigated, and should propose metrics for establishing the accuracy and sensitivity of signaling.

Beacon Track proposals will outline and develop modular plant-microbe synthetic communities where rhizosphere communities identify the soil’s contamination state denoting whether JP-8 and/or TNT contaminated soil is at or below the EPA-approved screening levels (see Table 1). It will amplify and relay that signal to the plant, which will in turn produce an overt, above-ground phenotypic signal. The system will ultimately have a limit of detection matching the EPA’s RSL for the corresponding marker for the selected contaminant. Since TNT rapidly degrades to other compounds and JP-8 is a complex mixture of compounds, proposals must identify and justify their choices for contaminant-specific marker compound(s). Marker compounds may be some combination of a major component of the contaminant mixture, a recalcitrant component in the soil, or a major intermediate degradation product of the contaminant.

Due to the long development timelines and high cost of engineering plants relative to microbes, the Beacon approach should be modular, enabling the system to be directed towards a new target without completely re-engineering the plant. For example, while the plant component scans for a particular relay signal molecule produced by the rhizosphere microbiome, that microbiome can be engineered to detect different contaminants but relay the same signal molecule to the plants for above-ground reporting. Thus, by swapping out engineered microbiomes, a wide range of contaminants could, in principle, be detected without having to re-engineer the plant. Given the central role of the relay signal molecule(s), proposers should identify and clearly defend their choice of signaling molecule(s) and suggest alternates for risk mitigation. Proposers addressing just the Beacon Track must develop plant-microbiome synthetic communities that detect both JP- 8 and TNT individually (see Table 1). Phase I performance will focus on a single contaminant, while Phase II will both improve the limit of detection for the initial contaminant and develop a second community contaminant signal. To be successful within the Beacon Track, the microbial community must relay a message to the plant regarding contaminant concentration, and the plant must relay that message to a human via an overt signal detectable by an unaided human. Signals could include, but are not limited to, unique and significant changes in leaf or flower color, distinct changes in smell, etc. Proposals should address the potential for false positive/negative signals due to circuit failure, community breakdown, seasonality, or other confounding factors and describe appropriate risk mitigation strategies. Ultimately, the Beacon Track synthetic communities must overtly signal when TNT or JP-8 is below RSL. This may be done directly or through the use of a marker molecule associated with the primary degradation pathway. This choice should be defended.

► Combined Track:

The Cleaner and Beacon Tracks fundamentally center on the detection of contaminants in soil with engineered plant-microbe systems. As such, proposers may submit proposals that address developing one Cleaner and one Beacon that collectively focus on a single contaminant (JP-8 or TNT). Otherwise, they must address all the requirements for the Cleaner and the Beacon Tracks.

Proposers must justify their choice for a marker compound(s). Combined Track proposals are allowed five additional pages in the technical volume to address the significant risks of optimizing two distinct plant-microbe synthetic communities and to outline corresponding mitigation strategies.

1.5. TECHNICAL AREAS

Ceres will comprise two 24-month phases focused on technology development and biological system integration. Integrated biological components will be tested by an IV&V team during a third and final phase (6 months); the final test will be conducted in large-scale greenhouse studies.

Both the testbeds and the plant–microbe synthetic communities will be developed through four technical areas (TAs): TA1 – Engineer Root Communities, TA2 – Engineer Plants, TA3 – Engineer Ecological Containment, and TA4 – Develop Testbeds. Cleaner and Beacon Tracks must address all TAs.

1.5.1. Technical Area 1 (TA1): Engineer Root Communities

Plants and their rhizosphere communities consist of many intricate biotic relationships. During Phase I, proposers will obtain or generate rhizosphere microbial communities specific to the chosen plant species. Rhizosphere communities can be assembled or supplemented with organisms from existing collections or identified through bioprospecting at the beginning of the program.

This community will be optimized and integrated into the target plant’s rhizosphere. Optimization for the target function, Beacon or Cleaner, can leverage techniques from both synthetic ecology (e.g., iterative selective enrichment) and synthetic biology (e.g., CRISPR). For the Beacon Track, proposers should implement a clear signaling pathway that avoids crosstalk with compounds common in the soil. During Phase II, the function and stability of the rhizosphere microbial communities will be advanced. Track-specific functionality will be further enhanced, including level of detection (Beacon) or degradation rate (Cleaner). Proposals should justify the choice of obtaining the microbial community and the relative merits of the community engineering strategy.

Moreover, the proposal should include risk mitigation for their strategies, addressing both potential deleterious processes, such as enhancement of pathogenicity, and the known challenges such as those associated with eventual field-deployment.

1.5.2. Technical Area 2 (TA2): Engineer Plants

TA2 will focus on generating plant scaffolds that successfully support the engineered microbial communities (TA1) in their track-specific function. The many, rich pathways where plants support the rhizosphere and the diverse communication pathways that control that support are finally being revealed. Proposers will modify plants to specifically enhance the target rhizosphere community developed in in TA1. Example strategies include the manipulation of root exudates, the release of signaling molecules, and the creation of a niche. Given the challenges associated with engineering plants, proposals should identify and justify plant selection, briefly delineate the extent to which that plant has been engineered to date, and describe the proposer’s specific engineering strategy with clear risk mitigation. Additionally, proposals should outline which tools (e.g., transcriptomics, metabolomics, etc.) will be used to identify key metabolites to communicate directly with and enhance the rhizosphere community. For the Cleaner Track, the plant supports the TA1 generated community’s metabolic activity to increase contaminant degradation rates. For the Beacon Track, the plant should support and stabilize the TA1 generated community with specific emphasis on receiving engineered orthogonal molecular signals from the rhizosphere and converting the relayed signals into an overt change in aboveground plant phenotype. By the end of Phase I, the plant–microbe synthetic communities should be fully integrated. By the end of Phase II, synthetic community s will either detect and signal contaminant presence at the EPA approved low RSL concentration (Beacon) or fully degrade the contaminant to EPA approved concentrations (Cleaner).

1.5.3. Technical Area 3 (TA3): Engineer Ecological Containment Biosafety, biosecurity, and preventing the uncontained release of GMOs into the native landscape is a core goal of Ceres. The plant component of the synthetic community can be removed ecologically, working themselves out of a niche in the absence of the contaminant, and/or in a straightforward fashion, via uprooting or similar strategy, but should not be an invasive species.

Removing engineered rhizosphere community members from the soil is the greater challenge and consequently the primary effort and target for TA3. To this end, proposers must develop synthetic community biocontainment strategies that will collapse the rhizosphere community in the absence of the plant host. For TA3, proposers must detail what approach(es) they will employ for synthetic community biocontainment, and how risks associated with such approaches will be mitigated.

Biocontainment strategies can include, but are not limited to, targeted ‘off switches’ for community collapse10, engineering ecological wrappers using autotoxicity,11,12 or auxotrophy.13 Biocontainment stringency standards are developed and set by the U.S. National Institutes of Health (NIH) for biosafety and recombinant DNA, including GMO escape.14 NIH defines acceptable escape frequency at less than 1 in 108 cells, where combining containment strategies is strongly advised in order to meet this biosafety standard.15 The Ceres program will build towards this goal over the initial two phases of the program. The ultimate goal for these biocontainment levels is to restrict invasion or persistence of the GMO microorganisms once their purpose is served. Contribution to containment from synthetic biology control and ecological containment should each be at least 103 towards the final combined goal of a reduction by a factor of 108.

1.5.4. Technical Area 4 (TA4): Develop Testbeds

To realistically and quickly test the technology developed in the other technical areas, proposals must develop high-throughput testbeds that recapitulate terrestrial systems and specifically enable plant-soil interactions. Liquid-based microcosms are not of interest. Proposals must describe technological advances to increase ecological realism at the bench and to close the gap between laboratory testing and terrestrial field deployment. Proposals should describe the type and relative frequency of measurements (pH, omics, metabolites, etc.) to track technical progress and how these measurements will be taken from the testbed. Proposals should outline how platforms will increase in complexity, such that Phase I synthetic community optimization will occur in more simplified microcosms for easy parallel testing.

Phase I testbeds should scale to ≥ 50 samples in tandem and fit within controlled chambers. The testbeds must contain and support the soil, root microbial community and plants, and allow monitoring of community interactions. Performers must indicate which parameters are critical for their system and will be tracked.

10 Ke et al. (2021) Trends in Biotech. doi.org/10.1016/j.tibtech.2020.07.008.

11 Čelešnik et al (2016) Biol Open. doi/10.1242/bio.017129.

12 Sebesta et al (2022) Front Plant Sci. doi.org/10.3389/fpls.2022.839446.

13 Kato et al (2015) PeerJ. doi/10.7717/peerj.1247.eCollection2015.

14 NIH Guidelines – Biosafety & Recombinant DNA Policy (2019).

15 Haskett (2021) ISME J. doi.org/10.1038/s41396-020-00835-4.

Phase II will develop larger testbeds for increased synthetic community complexity. Specifically, these testbeds must capture soil depth and spatial heterogeneity and must operate in contained greenhouses per biosafety standards. Performers must indicate which parameters are critical for their system and how they will be tracked in this larger format. Proposers should consider soils disposal and provide effective containment strategies, for instance those set by the Animal and Plant Health Inspection Service (APHIS) for soil biosafety, to prevent environmental release of GMOs or residual contaminates.

1.6. INDEPENDENT VERIFICATION AND VALIDATION (IV&V)

Proposals must outline how synthetic communities will be shared with IV&V for the final 6-month Phase III testing. During Phase II, the IV&V partner(s) will work with teams to begin testing the synthetic communities in one DoD soil from a distinct biome and chosen by the performer from the following: Eastern Woodlands, Great Plains, and Mediterranean (West Coast).

At the end of the program, IV&V will evaluate synthetic community generalizability by testing optimized synthetic communities in 1x1x1 m3 greenhouse plots using soil from contaminated DoD sites from all three biomes. (54 months) If needed, samples of these soils can be provided during Phase I.

To avoid potential conflicts of interest, proposers for this solicitation (HR001123S0012) will not be allowed to compete for the IV&V contract. HR001123S0012 is not soliciting proposals for

IV&V.

Government-furnished Property/Equipment/Information:

If needed, DoD soil samples from the DoD target biomes can be provided in Phase I of the program by IV&V partner(s). The Government will select and fund IV&V partner(s) for the Ceres program at a later date. Proposals submitted in response to HR001123S0012 should not include Phase III development tasks or costs associated with conducting IV&V.

Schedule As shown in Table 2, Ceres will span 4.5 years and consist of a 24-month Phase I, a 24-month Optional Phase II, and a final IV&V 6-month demonstration (Phase III). Proposals should outline work with appropriate metrics/milestones for Phases I and II with final milestones to be completed at 48-months. Proposers should also budget for minimal support to aide IV&V testing at the end of Phase II and at 54-months for Phase III. Progress towards the stated goals will be assessed throughout the program with Phase II funding dependent on both funding availability and performance towards the Phase I-specific milestones.

Table 2: Overall Project Timeline

Technical Area Phase I (0-24 mo) Phase II (25-48 mo) Phase III (49-54 mo)

TA1 –

Engineer Root Communities

Rhizosphere community can be engineered to track-specific function

Integrated synthetic community

Demonstrate track-specific synthetic communities robustly function across three

Technical Area Phase I (0-24 mo) Phase II (25-48 mo) Phase III (49-54 mo)

TA2 –

Engineer Plants

Plant supports the engineered community’s track-specific function

Integrated synthetic community

Integrated

TA1/TA2

Plant-microbe synthetic community survives in non-sterile soil for ≥ 30 days synthetic community functions to track-specific EPA thresholds for ≥ 60 days in greenhouse.

TA3 –

Engineer Ecological Containment

Engineer biocontainment into GMO members of the synthetic community

Optimize GMO biocontainment in the synthetic community to NIH standards

TA2 –

Develop Testbeds

Develop high-throughput soil microcosms for laboratory testing.

Develop larger, more complex high-throughput testbeds greenhouse distinct U.S. biomes in DoD contaminated soils.

1.7. PROGRAM MILESTONES, METRICS, AND DELIVERABLES

Progress toward the program goal will be determined through the use of regular milestones, metrics, and deliverables. The Government specifies the following minimally-required milestones, metrics, and deliverables to bound the effort while still affording the maximum flexibility, creativity, and innovation in proposing solutions to the stated problems. Proposers must propose additional quantitative and qualitative success criteria as needed. Proposers must clearly and uniquely itemize tasks needed to accomplish planned milestones and deliverables.

Proposals must be written to address milestones in all TAs: TA1 – Engineer Root Communities;

TA2 – Engineer Plants; TA3 – Engineer Ecological Containment; and TA4 – Develop Testbeds —with common metrics and application-track specific metrics. Proposals that do not address all TAs will be considered non-conforming and may be removed from consideration (rejected without review).

As noted above, proposers may address the Cleaner Track, the Beacon Track, or a Combined Track. TAs 1 and 2 differ by track and, for clarity, the minimum milestones and metrics for these two TAs are separately outlined below by track and by phase. TAs 3 and 4 are common across all the tracks and are also listed below. Again, all four TAs must be addressed. Proposers must explain quantitative success criteria for each milestone and provide detailed information on how these will be achieved in their Statement of Work (SOW). Proposers submitting proposals to the Combined Track have five (5) additional pages in Volume 1 to detail the substantial added risk of addressing both tracks and how they plan to mitigate this risk. In particular, as contaminant concentration is likely to impact synthetic communities’ fitness and function, proposers should identify and justify intermediate starting concentrations for demonstrations.

TA1 and TA2 METRICS and MILESTONES BY TRACK AND BY PHASE

►CLEANER TRACK

Phase I (Months 1 through 24) Goal: Phase I, 24 months, will require proposers to obtain and engineer intact rhizosphere communities (TA1) and engineer the plant host (TA2) as integrated contaminant-specific Cleaner synthetic communities. Synthetic communities will be optimized to sense and respond to the soil contaminant(s) with engineered plants supporting and enhancing rhizosphere community contaminant degradation. By the end of Phase I, proposers must demonstrate integrated TA1/TA2 synthetic community retains degradation function for ≥ 15 days in the simplified testbeds.

Proposals must concisely describe known metabolic pathways pertinent to the proposed synthetic community function, by-products produced, and potential environmental or health impacts of biodegradation of the contaminant and how the proposers plan to mitigate these risks.

A principal goal of Ceres is achieving rates of bioremediation comparable to conventional bioremediation, such as biopiling or windrowing, but with significantly less effort or energy.

Consequently, proposals should outline strategies for referencing the rate of synthetic community degradation to that achieved via biopiling to serve as the final target degradation rate for the synthetic community. Biopiling, where excavated contaminated soil is supplemented with specific microbes, nutrients, and environmental conditions in order to optimize the targeted microbial degradation activity, can accelerate soil bioremediation ≈10-fold versus native rates. The rate of biopiling varies substantially by specific rate limitation(s), e.g., temperature in cold biomes, water availability in dry climates, or nutrient availability in oligotrophic soil. The goal for the Cleaner synthetic community is to match the decontamination rate achievable locally with biopiling but without the added human effort and energetic inputs associated with biopiling.

TA1

Demonstrate the rhizosphere community can be engineered either through exogenous gene circuit expression or by a ≥5-fold increase in a Track-specific metabolic pathway. (12 months)

Demonstrate rhizosphere community doubles the degradation rate of the target contaminant compared to matched native soils in simplified testbeds. At the end of the remediation period, no side products of the bioremediation should exceed the Industrial Screening Level as defined by the EPA. (12 months)

Demonstrate rhizosphere community degrades the soil contaminant from the high (Table

1) concentration 2x faster than matched native soil, i.e., matched soil without any biopiling.

(15 months)

TA2

Demonstrate the plant supports the engineered microbiome community and its track-specific function in non-sterile soil. By either increasing the functional population or enhancing the target metabolism, the overall targeted metabolic activity should increase by ≥2x. (15 months)

The plant support, such as via root exudates, must be engineered to selectively enhance the targeted rhizosphere community’s metabolic activity in vitro, doubling contaminant-specific degradation from high concentration compared to matched native soil. (12 months)

TA1/TA2 synthetic community Demonstrate the synthetic community can survive together in non-sterile soil with native microbes for at least 30 days. (12 months)

Demonstrate the plant-microbe synthetic community degrades the soil contaminant from high starting concentration (Table 1) in non-sterile soil 2x faster than matched native soil.

(18 months)

Demonstrate the synthetic community degrades the soil contaminant from high concentration (Table 1) to ISL for the marker compound 4x faster than matched native soil.

The Cleaner synthetic community is functional ≥15 days in the simplified testbeds (24 months)

Phase II Option (Months 25 through 48) Goal: The Phase II Option, 24 months, will focus on further synthetic community refinement under increasingly stringent conditions. Proposers will optimize the integrated synthetic community (TA1/TA2) function for the following Cleaner-specific metrics. By the end of Phase II, the Cleaner synthetic community must be functionally operational for ≥60 days in greenhouse testbeds.

TA1/TA2 synthetic community

Delivery of the Cleaner synthetic community to the IV&V partner(s) for testing in one DoD target biome soil capable of degrading to ISL. (26 months)

Demonstrate the plant host supports the rhizosphere community function, for instance via modulated and orthogonal root exudation under the track-specific EPA thresholds. (36 months)

Demonstrate synthetic community degrades target soil contaminant from both high and low concentrations (synthetic community 1) down to RSL for the marker compound.

Remediation must occur 4x faster than matched native soil. (36 months)

Demonstrate that the synthetic community functions to the track-specific EPA threshold for at least 60 days in a greenhouse testbed. (48 months)

Demonstrate synthetic community degrades target soil contaminant down to RSL for the marker compound(s) 10x faster than matched native soil, is functionally operational for at least 60 days, and scales to a 1 m3 greenhouse plot. (48 months)

Delivery of the Cleaner synthetic community to the IV&V partner(s) for final Phase III demonstrations. (48 months)

Phase III Demonstration (Months 49 through 54)

IV&V teams will demonstrate that the Cleaner synthetic community can biodegrade the chosen contaminant to RSL 10x faster than matched native soil in the three distinct U.S.

biomes—Eastern Woodlands, Great Plains, and Mediterranean Climate. The biome is defined as the soil, the associated microbiota, and the local abiotic conditions. The synthetic community must be functionally operational to biodegrade the target contaminant for at least 60 days and scale to a 1 m3 greenhouse plot. (54 months)

BEACON TRACK

Phase I (Months 1 through 24) Goal: Phase I, 24 months, will require proposers to obtain and engineer intact rhizosphere communities (TA1) and engineer the plant host (TA2) as integrated Beacon synthetic communities. Synthetic communities will be optimized to sense soil contaminants with the engineered plant supporting the rhizosphere community and relaying its detection of the contaminant above ground into overt plant changes in phenotype. By the end of Phase I, proposers must demonstrate integrated TA1/TA2 synthetic communities are stable and can detect contamination for ≥15 days in the simplified testbeds. The EPA’s industrial screening level (ISL) and residential screening level (RSL) for each compound may be found at comptox.epa.gov/dashboard/. Default target contaminants, potential marker compounds, and corresponding marker ISL/RSL are shown in Table 1. The ultimate goal for the Beacon synthetic community is engineer plant-microbe propagate soil contamination status without sampling soil to monitor and test.

TA1

Demonstrate the rhizosphere community can generate a signal in response to the target contaminant at the marker-specific ISL in vitro. (12 months)

Demonstrate the target rhizosphere community can generate the signal molecule(s) for orthogonal microbe-to-plant communication to the target contaminant in non-sterile soil.

(18 months)

TA2

Demonstrate the plant can detect and respond to a target signal from the rhizosphere community for microbe-to-plant communication. (12 months) Demonstrate the plant supports the engineered microbiome community and its track-specific function in non-sterile soil. By either increasing the functional population or enhancing the target metabolism, the overall targeted metabolic activity should increase by ≥2x. (15 months)

Demonstrate the plant phenotypically responds aboveground to the rhizosphere community’s metabolic signal(s). (18 months)

TA1/TA2

Demonstrate the synthetic community can survive together in non-sterile soil with native microbes for at least 30 days. (12 months) Demonstrate the synthetic community can communicate with microbe-to-plant metabolic signals in non-sterile soil. (18 months) Demonstrate synthetic community(ies) can detect and report on a contaminant status at or below ISL for the marker after at least 15 days of living in simplified testbeds. (24 months)

Demonstrate the Beacon synthetic community has a positive predictive value (PPV) of 0.7 for contaminant concentrations above and below the ISL in small scale tests.

Phase II Option (Months 25 through 48) Goal: Phase II Option, 24 months, will focus on further synthetic community refinement under increasingly stringent conditions. Proposers will optimize the integrated synthetic community (TA1/TA2) function for the specific Beacon Track metrics outlined below. By the end of Phase II, track-specific synthetic communities must be functionally operational for ≥60 days in greenhouse testbeds.

TA1/TA2

Demonstrate the plant host supports the rhizosphere community function, for instance via modulated and orthogonal root exudation under the track-specific EPA thresholds. (36 months)

Demonstrate that the synthetic community om functions to the track-specific EPA threshold for at least 60 days in a greenhouse testbed. (48 months)

Demonstrate the plant host supports track-specific rhizosphere community function EPA screening levels, relative to the rhizosphere community function without the plant host, for at least 60 days in a greenhouse testbed. (48 months)

Demonstrate the synthetic community(ies) limit of detection occurs at or below RSL for one contaminant marker and at or below ISL for the second contaminant marker. (36 months)

Demonstrate the synthetic community(ies) limit of detection and signal generation occurs at or below RSL for both TNT and JP-8. The signal must be overt (detectable by an unaided human at 2m), and the synthetic community must detect and report soil contaminant status and be functionally operational for at least 60 days in the simplified testbeds with native soils. (48 months)

Demonstrate the synthetic community(ies) have a positive predictive value (PPV) of 0.85 for contaminant concentrations above and below the RSL as demonstrated in small scale tests.

Phase III Demonstration (Months 49 through 54) IV&V teams will demonstrate the Beacon synthetic community can generate overt signals (as defined above) of contamination status with a limit of detection at or below RSL and is generalizable to functioning in the three U.S. distinct biomes—Eastern Woodlands, Great Plains, and Mediterranean Climate. The biome is defined as the soil, the associated microbiota, and the local abiotic conditions. (54 months)

►COMBINED TRACK

Phase I (Months 1 through 24) Goal: Phase I, 24 months, will require proposers to obtain and engineer intact rhizosphere communities (TA1) and engineer the plant host (TA2) as integrated track-specific synthetic communities outlined above. synthetic communities will be optimized to sense and respond to the soil contaminant(s) with engineered host plants that support and enhance the track-specific function of the rhizosphere community. By the end of Phase I, proposers must demonstrate integrated TA1/TA2 synthetic communities retain track-specific function for ≥ 15 days in the Phase I testbeds.

TA1

Demonstrate the rhizosphere community can be engineered either through exogenous gene circuit expression or by a ≥ 5-fold increase in a Track-specific metabolic pathway. (12 months)

Demonstrate the Cleaner rhizosphere community doubles the degradation of the target contaminant compared to matched native soils in Phase I testbeds. Degradation products must not be contaminants. (12 months)

Demonstrate the Beacon rhizosphere community can generate a signal in response to the target contaminant at the marker-specific ISL in vitro. (12 months)

Demonstrate the Cleaner rhizosphere community degrades the soil contaminant from the high (and from low) concentration 2x faster than matched native soil. (18 months)

Demonstrate the Beacon rhizosphere community can generate the signal molecule(s) for orthogonal microbe-to-plant communication. (18 months)

TA2

Demonstrate the plant supports the engineered microbiome community and its track-specific function in non-sterile soil. By either increasing the functional population or enhancing the target metabolism, the overall targeted metabolic activity should increase by ≥2x. (15 months)

The Cleaner plant support, such as via root exudates, must be engineered to selectively enhance the targeted rhizosphere community’s metabolic activity in vitro, doubling contaminant-specific degradation from high concentration compared to matched native soil. (12 months)

Demonstrate Beacon plant can detect and respond to the target signal molecule(s) from the rhizosphere community for microbe-to-plant communication. (12 months)

Demonstrate Beacon plant phenotypically responds aboveground to the rhizosphere community’s metabolic signal(s). (18 months)

TA1/TA2

Demonstrate each synthetic community can survive together in non-sterile soil with native microbes for at least 30 days. (12 months) Demonstrate functionality of the synthetic community to the track-specific EPA threshold.

(24 months) Demonstrate Cleaner synthetic community degrades the soil contaminant from the high

(and separately from low) concentration in non-sterile soil 2x faster than matched native soil. (18 months)

Demonstrate Cleaner synthetic community degrades the soil contaminant from the high (and separately from low) concentration down to ISL for the marker compound 4x faster than matched native soil. The Cleaner synthetic community is functional ≥15 days in the simplified testbeds (24 months)

Demonstrate Beacon synthetic community can communicate with microbe-to-plant metabolic signals in non-sterile soil. (18 months)

Demonstrate Beacon synthetic community can detect and report on a contaminant status at or below ISL for the marker after at least 15 days of living in Phase I testbeds. (24 months)

Phase II Option (Months 25 through 48) In Phase II, teams will focus on further synthetic community refinement under increasingly stringent conditions. Proposers will optimize both integrated synthetic communities (TA1/TA2) function for the specific application-track metrics outlined above. By the end of Phase II, both synthetic communities must be functionally operational for ≥ 60 days in greenhouse testbeds.

TA1/TA2

Demonstrate the plant host supports the rhizosphere community function, for instance via modulated and orthogonal root exudation under the track-specific EPA thresholds. (36 months)

Demonstrate that the synthetic community functions to the track-specific EPA threshold for at least 60 days in a greenhouse testbed. (48 months)

Demonstrate the plant host supports track-specific rhizosphere community function EPA screening levels, relative to the rhizosphere community function without the plant host, for at least 60 days in a greenhouse testbed. (48 months)

Demonstrate Cleaner synthetic community degrades target soil contaminant from high (and separately from low) concentration down to RSL for the marker compound 4x faster than matched native soil. (36 months)

Demonstrate Cleaner synthetic community degrades target soil contaminant down to RSL for the marker compound(s) 10x faster than matched native soil and is functionally operational to biodegrade the target contaminant for at least 60 days and scale to a 1 m3 greenhouse plot. (48 months)

Demonstrate Beacon synthetic community limit of detection occurs at or below RSL for one contaminant marker and at or below ISL for the second contaminant marker. (36 months)

Demonstrate Beacon synthetic community limit of detection and signal generation occurs at or below RSL for both TNT and JP-8. The signal must be overt (detectable by an unaided human at 2m), and the synthetic community must detect and report soil contaminant status and be functionally operational for at least 60 days in the simplified testbeds with native soils. (48 months)

By the end of Phase II, teams will have worked with IV&V partner(s) to test both synthetic communities each in one biome soil. (48 months)

Phase III Demonstration (Months 49 through 54) IV&V teams will demonstrate that the Cleaner and Beacon synthetic communities can function in the three distinct U.S. biomes—Eastern Woodlands, Great Plains, and Mediterranean Climate. The biome is defined as the soil, the associated microbiota, and the local abiotic conditions. The Cleaner biodegrades the target contaminant to RSL 10x faster than matched native soil. The Beacon synthetic community generates an overt signal of target contamination status with a limit of detection at or below RSL. Both synthetic communities must be functionally operational for at least 60 days and scale to a 1 m3 greenhouse plot. (54 months)

TA3, TA4, and IV&V METRICS COMMON TO ALL TRACKS

Phase I (Months 0-24) All proposals, regardless of track, must address TA3 and TA4. For TA3, proposals must outline biocontainment strategies for GMOs in the synthetic communities that collapse the…