HR001121S0035.pdf

Broad Agency Announcement Environmental Microbes as a Bioengineering Resource

(EMBER)

BIOLOGICAL TECHNOLOGIES OFFICE

HR001121S0035

July 13, 2021

HR001121S0035, EMBER

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 Tasks, Milestones, and Deliverables

1.4. Permits, Licenses, and Compliance

1.5. 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. Content and Form of Application Submission

4.3. Funding Restrictions

4.4. Other Submission Information

5. Application Review Information

5.1. Evaluation Criteria

5.2. Review of Proposals

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

9. APPENDIX 1 – List of Attachments

10. APPENDIX 2 – Volume II checklist

PART I: OVERVIEW INFORMATION

Federal Agency Name – Defense Advanced Research Projects Agency (DARPA), Biological Technologies Office (BTO)

Funding Opportunity Title – Environmental Microbes as a Bioengineering Resource (EMBER)

Announcement Type – Initial Announcement Funding Opportunity Number – HR001121S0035 North American Industry Classification System (NAICS) – 541714 Catalog of Federal Domestic Assistance Numbers (CFDA) – 12.910 Research and

Technology Development Dates o Posting Date: July 13, 2021 o Proposal Abstract Due Date and Time: 4:00 PM ET, August 16, 2021 o Full Proposal Due Date and Time: 4:00 PM ET, September 27, 2021 o BAA Closing Date: September 27, 2021 o Proposers Day: July 27, 2021 https://sam.gov/opp/6f7b9d46a7cb4e12b17440165b6f5dc8/view

Concise description of the funding opportunity – The Environmental Microbes as a BioEngineering Resource (EMBER) program aims to develop novel, bio-based technologies to overcome key challenges facing domestic supply of Rare Earth Elements (REEs) critical to the U.S. and Department of Defense (DoD). The EMBER program will leverage the diversity, specificity, and customizability of environmental microbiology to enable new biomining methods for the separation, purification, and conversion of REEs into manufacturing-ready forms. Microbes (and/or biomolecules), including those from extreme or metal-rich environments, can be biologically engineered or adapted to bind, assimilate, and manipulate individual REEs. These biological components, once developed, may be assembled into an in-line separation, purification, and recovery workflow resulting in individual, purified REEs. Scalability of EMBER’s approach will be demonstrated with proof-of-concept, pilot scale studies aligned with existing mining/waste treatment infrastructure.

Anticipated individual awards – Multiple awards are anticipated.

Types of instruments that may be awarded – Award instruments may include procurement contract, grant, cooperative agreement, or Other Transaction; however, only procurement contracts and Other Transactions may be used for proposers whose proposed solution includes Controlled Unclassified Information (CUI).

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

EMBER@darpa.mil

DARPA/BTO

ATTN: HR001121S0035

675 North Randolph Street Arlington, VA 22203-2114 https://sam.gov/opp/6f7b9d46a7cb4e12b17440165b6f5dc8/view mailto:EMBER@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 CFR § 200.203. Any resultant award negotiations will follow all pertinent law and regulation, and any negotiations and/or awards for procurement contracts will use procedures under FAR 15.4, Contract Pricing, as specified in the BAA.

The Defense Advanced Research Projects Agency (DARPA) is soliciting innovative proposals to develop a cohesive, domestic Rare Earth Element (REE) separation and purification technology that uses biologically engineered microbes and/or biomolecules. Proposed research and development should investigate and exploit environmental microbial strategies for recovering and separating individual REEs from complex mixtures. To accomplish this goal, new design-build-test-learn synthetic biology paradigms that capitalize on specific metal binding, transport, and mineralization mechanisms employed by microbial and biomolecular systems are needed.

Resultant bioengineering approaches will be coupled to process engineering workflows to extract and purify REEs from domestic source materials. Demonstration of viable schemes will be supported by compelling techno-economic analyses and culminate in an aggressive, proof-of-concept pilot-scale REE biomining operation.

1.1. PROGRAM OVERVIEW

The Environmental Microbes as a BioEngineering Resource (EMBER) program will develop a biotechnology-based separation and purification strategy for REEs from under-utilized domestic sources such as phosphate mine waste, acid mine drainage, and electronics recycling processes.

The program aims to deliver multiple capabilities such as the separation of REE mixtures into individual elements using aqueous processes; inter-conversion of REE salts/oxides to facilitate production of manufacturing-ready forms (e.g., halides, phosphates, nitrates); and new assays for high-throughput analysis of REE-containing cells and biomolecules.

REEs, which include the 15-element lanthanide group plus yttrium and scandium, are critical ingredients in many DoD systems: e.g., in permanent magnets for electric motors, high-temperature ceramics, and lasers. REE purification is challenging – similarities between the 17 REEs require many physical and chemical extraction steps that are energy-intensive, hazardous to the environment and personnel, and often inefficient. As a group, these elements exhibit only modest differences between their Lewis acidities, molecular weights, and atomic radii;

subsequently, separation of mixtures of these species into distinct concentrates of isolated elements remains technically challenging.

Biomining is an alternative approach that utilizes microbes to recover metals (e.g., copper, gold) from source materials, often using redox processes to liberate the target metal from a mineral source. Biosorbent and biofiltration approaches show promise in the extraction or removal of metals from contaminated milieu (e.g., for bioremediation) but need to be able to function with complex REE source materials and enable efficient recovery of the bound metals. Using microbes or biomolecules to separate REEs from mixtures is under study, but this approach currently lacks the required specificity to separate all individual REEs, is slow, and is not yet viable at scale.

Advances in microbial and biomolecular engineering could help address these limitations. While synthetic biology tools are well-developed for conventional lab-adapted chassis organisms, and may be suitable for production of organic molecules at circumneutral pH and moderate temperatures (T°), these approaches are underdeveloped for environmental and extremophile microbes, including those known to tolerate and utilize heavy metals. Engineering of organisms that thrive at acidic or alkaline pH and elevated T°, and those that bind, uptake, or store metals, has been impeded by cultivation and isolation challenges. Pathway design and engineering to produce organic molecules are informed by vast libraries of enzymes and regulatory parts, but a lack of annotated genomes, regulatory components, and genome integration tools has impeded advances to develop organisms that specifically utilize inorganic elements, including REEs.

Lastly, current assays for precise measurement of organism-associated REEs are low-throughput, destructive, and, thus, incompatible with typical synthetic biology pipelines. Overcoming these deficiencies could enable selective, specific, and high-capacity biomining of individual REEs.

1.2. TECHNICAL APPROACH

Performers for the EMBER program will develop bioengineered organism/biomolecular approaches for REE separation and purification, then translate these to practical platforms for biomining (e.g., biosorbent, biofiltration, bioleaching) modules that will be used to extract REEs from domestic REE sources. Some of the key technical challenges to be addressed by EMBER include:

Design and engineering of chassis organisms tolerant of pH and temperature extremes, and high metal concentrations.

Selective and specific biologically-driven extraction of individual REEs from complex mixtures.

Development of high-throughput, sensitive, non-destructive assays for REEs associated with cells/biomolecules.

Optimization of REE accumulation, rates, and regenerability/reuse of the bio-extraction process.

Process engineering to integrate the chosen REE source material (e.g., mine waste, acid mine drainage, recycled electronics) with bio-based REE extraction modules to form a complete REE purification system. Demonstration of the developed technology at the pilot scale will likely require co-location at the facility that provides the REE source material.

Techno-economic analysis of the developed bio-based approach that supports its scalability and commercial viability.

Developing these REE separation and purification solutions will require two Technical Areas (TAs) – Bioengineering for REE Utilization (TA1) and REE Biomining (TA2), which are described in Section 1.2.1 below. Proposing teams are required to address both TA1 and TA2, and must provide an integrated, multidisciplinary approach addressing each element of TA1 and TA2. Strategies for optimizing, integrating, and expanding capabilities should be elaborated throughout. Proposals should also discuss mitigation of technical challenges that may arise within each Phase and TA. Proposals that fail to address both technical areas will be considered non-conforming and will not be evaluated.

Proposers are strongly encouraged to team with domestic industry partners focused on activities such as mining or reclamation, mine waste treatment, and/or electronics waste/recycling.

Partners with this experience can identify procedural, regulatory, economic, and technical challenges that will need to be overcome; provide real-world REE source material for development of biological chassis for REE extraction; and maximize compatibility of the end-of-program pilot scale biomining demonstration with an industrial operation. Performer teams must describe their intended domestic REE source materials/processing sites and work within the specific parameters of those sites (e.g., pH, metal content/concentrations, halide content, REE composition, radioactive contaminants, redox potential, sulfur content) to guide bioengineering decisions. Performers may elect to pursue different strategies to extract each individual REE (e.g., multi-stage approaches that first remove other competing ions/metals/radioactive elements from their selected REE source material, separate light from heavy REEs, or “one-pot” approaches). Since diverse strategies are possible, performers are encouraged to explore multiple biomolecules/chassis. Ultimately, the selected bio-based strategies must converge to demonstrate the program goal to specifically bind and purify at least 8 different REEs. Because demonstrating the feasibility of industrial scale up and the economic viability of developed processes are critical aims of EMBER, proposers should describe how they will develop and substantiate techno-economic analyses of their approach.

Specifically excluded from this opportunity is research that: (1) results in incremental improvements to the existing state of practice or lacks a bioengineering approach; (2) focuses on coal/coal by-products as a domestic source of REEs; or, (3) utilizes intact, living terrestrial plants or macroalgae to extract the REEs. (NOTE: employment of genetic parts, pathways, biomolecules, or biopolymers obtained from these sources is permitted). Proposals falling into these categories may be considered non-conforming and may not be evaluated.

1.2.1. TA1: Bioengineering for REE Utilization

The overall goal of TA1 is to create the tools, both organismal and biomolecular, that will enable the TA2 effort to develop efficient REE biomining processes. Teams will need to establish a design-build-test-learn (DBTL) platform for engineering organisms and/or biomolecules that manipulate REEs and enable their separation and purification from complex mixtures.

“Organisms” envisioned for this TA include microbes, fungi, or bacteriophage; use of photosynthetic organisms (cyanobacteria, microalgae) as chassis must include justification of the additional energy demands that these organisms require.

Design: Performers will explore and develop pathway and design tools that incorporate genomes, genes, biomolecules, and regulatory parts required to build organisms/biomolecules capable of selective, specific, and regenerable REE extraction and manipulation under harsh reaction conditions. This program component should focus, at a minimum, on developing the capability to design chassis organisms possessing the following phenotypes:

Survival in harsh conditions: Chassis will need to function in hot, acidic (or basic) milieu, and overcome toxicity associated with high concentrations of REEs and other metals.

Ability to specifically bind/extract REEs: Performers should consider mechanisms of cellular metal transport, binding and uptake, and possible synergistic approaches.

Bioengineered strategies for removal or mitigation of radioactive contaminants may also be required if present in the selected source material.

Bio-conversion of REE compounds for manufacturing: Bio-focused methods that interconvert between different REE chemical forms (e.g., oxides into halides or nitrates, halides into phosphates, etc.) are also sought. Design strategies that explore biological routes to convert REE compounds into fully reduced chemical states, with individual REE metallic elements as end products, are of great interest.

Build: While conventional DNA synthesis, amplification, and sequencing verification steps are expected and will be acceptable over the course of the program, codon usage and genetic pathway assembly may require adaptation to facilitate insertion into non-traditional chassis.

Performers will also develop the tools necessary to cultivate, isolate (if necessary), and genetically transform selected chassis organisms, to enable expression of biological components identified in the Design component.

Test: An effective DBTL platform requires assays to measure improvements in strain performance. To this end, development of high-throughput, non-destructive assays is required:

Assessment of survival of low and high pH, elevated temperatures, and high REE/metal concentration relative to growth in minimal media.

Specific REE association with individual cells with sensitivity at the femtogram scale.

Performers will need to advance beyond existing limits of assay capabilities in order to detect specific REE association and kinetics with biomolecules, biomass, and/or cells.

Learn: Identify the relationships between observed REE specificity/selectivity/accumulation levels and design factors through the use of statistical methods and machine learning, with appropriate experimental validation. Experimental and computational analysis of results with 1st generation strains will be re-iterated as needed to meet program goals for REE specificity/purity and growth in TA2-driven biomining conditions. The minimum performer objectives for TA1 are as follows:

Selective and specific binding of ≥ 8 target REEs.

Chassis organisms that function at extremes of pH and T°.

High-throughput assays compatible with genetic screening protocols.

Methods for REE bioconversion to aid in downstream manufacturing of REE-containing products.

1.2.2. TA2: REE Biomining

The overall goal for TA2 is to develop technology and processes to utilize organisms, biomolecules, or biopolymers as the key means to separate, purify, and recover individual REEs from domestic REE source materials. Recovered REE materials may be in the form of salts (e.g., halides or nitrates), phosphates, oxides/hydroxides, or reduced metals, and should be devoid of biomass and impurities. Teams will develop and test a biomining workflow to purify individual REEs from complex source mixtures, likely using a combination of geological, chemical, and/or process engineering steps. Studies will progress from the laboratory bench scale to a pilot scale demonstration capable of generating at least 700 grams total REEs (tREE) per week. It is anticipated that the pilot scale demo will need to be co-located with the REE source material site.

REE source material: Many domestic sources can enable the U.S. to meet current and growing demand (e.g., phosphate deposits, acid mine drainage, recycled electronics). Due to extensive concurrent research investment from other Government agencies, coal/coal by-product sources are specifically excluded from the EMBER funding opportunity. While it is anticipated that the processes developed under this program can be generalizable to many varied REE sources, proposals must identify an intended domestic (fully contained on land of U.S. and territories and/or within the economic exclusion zone of the U.S.) REE source. To limit the focus on the separation and purification of REEs, rather than the mining process as a whole, the source material for the program should consist of downstream concentrates, which contain at least 300 parts per million (ppm) total REEs and at least 8 different REEs. Proposals should also describe researcher’s access to sufficient quantities of the source material to complete the work outlined in the proposal as well as the current and projected annual production amounts.

REE biomining workflow: REE source material characteristics are anticipated to guide the development of modular components of biomining platforms that incorporate REE-specific organisms/biomolecules, optimize their safe and effective deployment, and integration with existing infrastructure to maximize accumulation rates and throughput. Performers should describe their strategy for incorporating organisms/biomolecules, identify the initial REE-binding organisms, biomolecules, and/or biopolymers to be used, and the performance requirements (e.g., binding profile kinetics, structural stability, scalability of production) required of the improved strains/biomolecules developed in TA1. Workflow may remove/mitigate radioactive or other toxic/hazardous contaminants (if present in source material), concentrate REEs, separate light from heavy REEs, and purify individual REEs. It is anticipated that novel processes enabled by sophisticated bioengineering accomplished in TA1 that can facilitate specific REE separation and purification may include the following:

Expression of chelators and proteins (e.g., lanthanophores, siderophores) to sequester and/or solubilize target REEs.

Secretion of biolixiviants, such as organic acids to solubilize metals.

Hyperaccumulation/biomineralization by cells.

Biosorbents/biofiltration media fabricated from living or dead/dried biomass.

Bioengineering to facilitate recovery of REEs from biomass.

Process engineering: Proposers should also discuss the anticipated physical infrastructure required for their biomining workflow, from the preparation of biomining organisms/biomolecules to intake of REE source material through output of purified REEs and waste by-products. Performers are likely to consider a combination of processing steps that facilitate the extraction and separation of REEs depending on their selected REE source material and specific biomining approach. Physical infrastructure may include, but is not limited to, the following:

Bioreactors for organism growth or biomolecule preparation, and for processing biomass to recover REEs.

Energy requirements and sources.

Liquid/solid handling pumps, piping, and valving.

Aeration and/or stirring equipment.

Columns/tanks/bioreactors for REE accumulation/separation/extraction.

Effluent and waste discharge and disposal.

Physical safeguards to contain living genetically engineered organisms within the workflow cycle.

Demonstrate feasibility and economics of scale-up: TA2 will emphasize development of regenerable and/or reusable bio-based components; will require multiple technoeconomic analyses; and culminate with a pilot scale demonstration of the fully integrated system. Proposals should describe how the developed REE bioextraction steps will integrate within a complete processing pipeline and outline the ways in which the new process mitigates the central challenges to domestic purification of REEs (e.g., waste and hazard reduction, energy efficiency, cost reduction). Over the course of the program, three techno-economic analyses of the developed process, including material and energy balances, will be required (toward the end of each program Phase) in order to help inform the feasibility and viability of the performer’s approach.

Performer requirements for TA2 are to demonstrate pilot scale extraction with the following minimal objectives:

Process should be regenerable or reusable.

Strategy to recover REE from biomass should be practical (e.g., feasible at industrial scale up).

Alignment/integration at pilot scale on site with existing mining/waste treatment infrastructure is required.

Safeguards against accidental release if living genetically engineered organisms are used.

Process occurs in-line and extraction of each REE only adds one additional step.

Techno-economic analysis supports feasibility at industrial scale.

1.2.3. Integration of TA1 and TA2

Proposing teams must address both TAs to ensure a complete, integrated system tailored to specific REE source and site infrastructure by the end of Phase 3. TA1 and TA2, as described above, must converge to deliver a pilot scale REE extraction operation. Each of the TAs will remain active for all three phases as it is anticipated that iterative improvement and optimization will be required throughout the project.

Performers must plan their approach for TA1 to integrate with their strategy for TA2 to address the following program objectives:

Developed REE-utilizing organisms and/or biomolecules need to function and grow under conditions relevant to the chosen source material (e.g., REE- and mineral-rich mixtures, extremes of pH, elevated temperatures).

Selective, specific, and efficient binding/accumulation of multiple, individual REEs from these complex source materials.

Recovery of the REEs from the biomass should be efficient: remove 95% REEs from source media, recover 95% REE bound to biomass; with ≥ 95% final purity for each

REE.

Purity of extracted REEs, selectivity and efficiency of separation steps must be analytically confirmed using validated materials characterization methods (e.g., Inductively coupled plasma mass spectroscopy (ICP-MS), X-ray diffraction, other spectroscopic methods, elemental analysis, electron microscopy, titration analysis).

Removal or mitigation of radioactive or other hazardous contaminants, should the selected REE source material contain them.

Multiple safeguards (genetic, physical) against inadvertent environmental release, should the TA2 Biomining process depend upon genetically engineered living organisms used in a manner where such accidental release is possible.

1.2.4. Independent Validation and Verification

Throughout the program, the performers will work with an Independent Verification and Validation (IV&V) team established by the Government. The IV&V team will consist of subject matter experts from the Government, Federally Funded Research and Development Centers (FFRDCs), academia and/or other relevant domains. The IV&V team will test and validate the ability of the EMBER bio-based technologies to separate and purify REEs from similar (or different) source materials. IV&V partners with expertise in chemical and materials analysis will confirm the identity, purity, and chemical composition of extracted and separated REEs. IV&V partners with expertise in process engineering and technoeconomic analysis of mining, biomining, or waste bio-treatment systems will review and comment on performer’s initial and final designs for REE biomining workflows and performer-generated technoeconomic analyses.

The milestone and metrics section below describes the schedule for delivery of performer-developed strains, biomolecules, integrated systems, and protocols to the IV&V team for testing and evaluation.

To avoid potential conflicts of interest, performers for HR001121S0035 will not be allowed to compete for the IV&V contract. HR001121S0035 is not soliciting proposals for IV&V.

1.2.5. Schedule

The EMBER program spans four (4) years and consists of an 18-month Phase 1, 18-month Phase 2 Option, and 12-month Phase 3 Option. Progress towards the stated goals will be assessed throughout the program. Participation in Phase 1 does not guarantee funding in the Option Phases; the Government’s unilateral determination to exercise an option will be contingent on performance in the previous phase and availability of funds.

During Phase 1, performers will focus on developing micro-organisms and biomolecules suitable for use in REE separation, assays for non-destructive monitoring of REE accumulation in or on microorganisms and biomolecules, and initial system designs. An initial techno-economic analysis utilizing acquired data will be used to inform feasibility/viability of the performer’s bio-based REE extraction and purification process.

During Phase 2, performer teams will focus on improving the efficiency and scale of REE separation from actual source materials and providing a second techno-economic analysis incorporating information gained from this initial scale up phase.

In Phase 3, performer teams will integrate components from Phase 2, to execute a pilot-scale demonstration of the REE-separation technology, co-located at the facility where the selected REE source material is generated. A final techno-economic analysis, including data obtained from pilot-scale studies, will be executed.

1.3. PROGRAM TASKS, MILESTONES, AND DELIVERABLES

In order for the Government to evaluate the effectiveness of a proposed solution in achieving the stated program objectives, proposers should note that the Government hereby promulgates the following program milestones that may serve as the basis for determining whether satisfactory progress is being made to warrant continued funding of the program. Continued funding for each subsequent phase is contingent upon meeting or exceeding the milestones prescribed for the current phase.

Although the following program milestones are specified, proposers should note that the Government has identified these goals with the intention of bounding the scope of effort, while affording the maximum flexibility, creativity, and innovation in proposing solutions to the stated problem, to include variations in performance. The milestones identified for this program were designed to encourage and drive innovative solutions that significantly increase the understanding of the biological, geochemical, and physical processes that will enable optimized REE Biomining systems. Only satisfaction of the milestones for both TAs will be considered as successful completion of a given phase.

1.3.1. Program Tasks, Specifications, and Milestones

The milestones are listed in tables below, separated Phase and TA, on pages 12-17.

Phase TA Task Task Specifications Milestone Task 1: Develop engineerable chassis organisms from different genera which function and grow under the extreme conditions relevant to REE biomining.

• Chassis strains reliably express exogenous (reporter) genes.

• Growth (rate or # of cells) ≥ 25% of that seen with minimal media (after transformants selected; @ extreme conditions).

• Extreme conditions defined as: T ≥ 42°C; pH ≤ 2, pH ≥ 9.

• Total REE (tREE) comprised of equimolar concentrations of 10 different REE.

• Composition/concentrations of other metals/ions/sulfides should simulate the selected TA2 REE source material.

(Month 6) Assays under extreme conditions help reduce DBTL cycle time by 20% (relative to Month 2) (Month 6) ≥ 2 engineered chassis strains able to survive and grow in 500 µM tREE.

(Month 12) Assays under extreme conditions that reduce DBTL cycle time by 40% (relative to Month 2).

(Month 18) ≥ 5 engineered chassis strains, with some functional at pH ≤ 2 and some at pH ≥ 9; 500 µM tREEs + other metal/ions/ sulfides.

Task 2: Develop a platform to assay REE association with organisms.

Assays should be non-destructive in nature or allow recovery of sequenceable nucleic acids post-analysis, or otherwise allow for pairing of sequence data to specific organisms.

(Month 15) Detect ≥ 3 different REEs at concentration of femtograms/cell at a rate of 103 cell samples per day. Ph as e

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Task 3:

Demonstrate organisms (and/or identify/express biomolecules) that bind and/or modify REEs.

• Specific binding and conversion of REEs using cells, biomolecules, or cell-free systems. Approach should be adaptable to TA2 workflow.

• Specificity is defined as analytically verified ratio of target bound REEs to non-targeted REEs.

• Media should simulate the components of the planned TA2 source material.

• Specificity, purity, and yields of REEs must be demonstrated analytically.

(Month 12) Biologically convert 1 REE from one chemical form into another (e.g., oxide to salt, salt to elemental metal) at ≥ 75% yield.

(Month 15) Specific binding of ≥ 3 individual REE with 4:1 specificity.

Phase TA Task Task Specifications Milestone Task 4: Continue to advance engineerable chassis strains which function and grow under the extreme conditions relevant to REE biomining by increasing the extremes of conditions.

• Actual REE source material selected for TA2 will be employed.

• Chassis strains reliably express pathways for interactions with REE.

• Growth (rate or # of cells) ≥ 25% of that seen with minimal media (after transformants selected; @ extreme conditions).

• REEs added if needed to reach tREE concentrations indicated; equimolar concentrations of 10 different REEs.

(Month 24) ≥ 2 engineered chassis strains able to survive and grow at pH ≤

2.0 or T° ≥ 42°C, in actual source material with ≥ 500 µM tREE.

(Month 27) ≥ 2 engineered chassis strains that survive and grow in source material with 10 mM tREE.

(Month 36) ≥ 5 engineered chassis strains that survive and grow in source material with 10 mM tREE at T° ≥ 80°C, with multiple strains functional at pH ≤ 0.5, and pH ≥10.

Task 5: Build on the progress made in Phase 1 to expand the numbers of organisms and or biomolecules which specifically bind and/or modify REEs.

• Specific binding and conversion of REEs using cells, biomolecules, or cell-free systems.

• Specific binding approaches must be adaptable to TA2 workflow.

• Actual REE source material selected for TA2 must be employed for specific binding studies.

• Specificity is defined as analytically verified ratio of target bound REEs to non-targeted REEs.

• Purity of converted REE chemical forms (e.g., oxide to salt, salt to elemental metal) must be verified analytically.

(Month 30) Specific binding of ≥ 8 REE (≥ 4 heavy REE) with specificity ≥ 10:1. (Month 30) Biologically convert 1 light, and 1 heavy REE from one chemical form into another at ≥ 90% yield.

(Month 36) 8 or more distinct organisms or biomolecules, each capable of specifically binding a different REE (≥ 4 ‘heavy’ REE), with specificity ≥ 20:1.

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Task 6: Develop a platform to assay REE association with organisms.

Assays should be non-destructive in nature or allow recovery of sequenceable nucleic acids post-analysis, or otherwise allow for pairing of sequence data to specific organisms.

(Month 36) Detect ≥ 8 REEs at concentration of femtograms/cell at a rate of 105 cell samples per day.

Phase TA Task Task Specifications Milestone Task 7: Expand the capability to biologically interconvert and purify chemical forms of multiple, biologically extracted REEs.

• Specific binding or conversion of REEs using cells, biomolecules, or cell-free systems.

• Actual TA2 REE source material will be employed for specific binding studies.

• Specificity is defined as analytically verified ratio of target bound REEs to non-targeted REEs.

• Purity of converted REE chemical forms (e.g., oxide to salt, salt to elemental metal) must be verified analytically.

(Month 42) Biologically convert ≥ 5 REE (≥ 2 heavy REE) from one chemical form into another ≥ 90% yield.

(Month 48) Selective binding ≥ 1 REE with specificity of 1000:1 using a biological mechanism.

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Task 8: Develop a platform to assay REE association with single cells.

Assays should be non-destructive in nature or allow recovery of sequenceable nucleic acids post-analysis, or otherwise allow for pairing of sequence data to specific organisms.

(Month 48) Ability to identify association of different REEs with single cells at a rate of 106 cell samples per day.

Phase TA Task Task Specifications Milestone

Task 1: Design, develop, and justify a modular REE-biomining workflow capable of purifying individual REEs from complex domestic source materials.

• Clear outline of purification pipeline compatible with current mining operations and detailed cost, energy, and scalability analyses.

• REE source material must have a minimum tREE concentration of 300 ppm and ≥ 8 different REEs.

• Techno-economic analysis (TEA, with Life Cycle Analysis, LCA) should project financial realities and other benefits/ bottlenecks such as energy efficiency and state-of-the-art costs (SOA) for REE.

(Month 3) Characterize components of REE source material to inform TA1 and TA2 activities.

(Month 6) Provide descriptions/ schematics of initial biomining concept pipeline along with preliminary format/matrix for individual steps.

(Month 15) Conceptual

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Task 2: Develop and demonstrate biomining modules for REE separation and recovery.

• REE separation and recovery modules capable of isolating individual REEs from simulated source material (modeled on the selected TA2 REE source material in terms of REE composition/ concentrations, pH, and other components).

• Processes should be robust and scalable.

• Purity of recovered REEs must be analytically confirmed.

(Month 12) Bind/remove ≥ 50% of a target REE at milligram scale from simulated source material.

(Month 15) Separate and recover ≥ 3 individual REEs, each at mg scale, ≥ 75% final purity from simulated source material.

(Month 18) Demonstrate a 2nd cycle of REE binding and recovery with 90% efficiency relative to 1st cycle.

Phase TA Task Task Specifications Milestone

Task 3: Design, develop, and justify a modular REE-biomining workflow capable of purifying individual REEs from complex domestic source materials.

• Refined outline of purification pipeline compatible with current mining operations.

• Physical/genetic containment strategies for any living genetically engineered organisms must be included.

• TEA (with LCA) should project financial realities and other benefits/ bottlenecks such as energy efficiency and state-of-the-art costs (SOA) for

REE.

• TEA should be based on data acquired during the project.

(Month 19) Established process for transfer of REE source material to, and disposal of waste from, the processing site at quantities reflective of increased scale.

(Month 24) Updated descriptions/drawings of biomining concept pipeline along with format/matrix for individual steps.

(Month 33) Updated TEA.

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Task 4: Develop and demonstrate biomining modules for REE separation and recovery.

• Demonstrate pipeline of REE separation and recovery modules capable of isolating individual REEs from actual source material.

• Processes should be robust, scalable, and operate as a unified pipeline where purification of each REE adds one (or less) additional step(s).

• Actual source material processed through the upstream stage used as input media.

• Purity of recovered REEs must be analytically confirmed.

(Month 24) Bind/remove ≥ 80% of a target REE at milligram scale.

(Month 30) Bind/remove ≥ 80% of target REE at the gram scale at a rate of ≥ 2 g total REE/day.

(Month 36) As a full pipeline, separate and recover ≥ 8 individual REEs, ≥ 14 g/week total REE, recover ≥ 80% of bound REE from biomass; ≥ 90% final purity for each REE.

(Month 36) For an individual ‘module’ demonstrate 10 cycles of REE binding and recovery with 90% efficiency (at 10th cycle) relative to 1st cycle.

1.3.2. Deliverables

The following will be delivered by the performer teams to the IV&V team(s):

Phase 1 (15, 16 months) Biological - Performance of strains, proteins, and materials will be tested using performer provided protocols (16 mo).

Elemental - REE materials produced with TA2 bioengineering approaches will be tested for composition and purity (16 mo).

Analytical - Performers will provide a techno-economic analysis of their approach at bench scale (15 mo).

Phase 2 (33, 36 months) Biological - Performance of strains, proteins, materials, will be tested using performer-provided protocols (36 mo).

Elemental - REE materials produced with TA2 bioengineering approaches will be tested for composition and purity (36 mo).

Phase TA Task Task Specifications Milestone

Ph as e

II

I ( M on th s 3 7-

T ec hn ic al A re a

2:

R

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B io m in in g

Task 5:

Demonstrate pilot scale operations.

• Pilot scale pipeline of REE separation and recovery modules must be capable of isolating individual REEs from actual source material.

• Processes should be robust, scalable, and capable of operating in-line with operation at source site.

• Controls for regeneration, biofouling mitigation, contaminants (e.g., radioactive elements, sulfides);

biological and chemical waste disposal must be included.

• Efficacy of physical/genetic containment strategies for any living genetically engineered organisms to prevent accidental release must meet local, state and/or Federal compliance thresholds.

• TEA (LCA) should project financial realities and other benefits/ bottlenecks such as energy efficiency and state-of-the-art costs (SOA) for REE. TEA should be refined from prior versions using data acquired from the pilot study.

(Month 48) Bind/remove > 95% of ≥ 8 individual REEs from actual source material and recover > 95% of ≥ 8 individual REEs at a scale of ≥ 700 g/week total REE, with a final purity of ≥ 95%.

(Month 48) Final TEA.

Analytical - Performers will provide an updated techno-economic analysis of their approach reflecting scaled up experiments, and IV&V will review (33 mo).

Phase 3 (48 months) Biological - Performance of strains, proteins, and materials will be tested using performer provided protocols.

Elemental - REE materials produced with TA2 bioengineering approaches will be tested for composition and purity.

Analytical - Performers will provide a final techno-economic analysis of their approach as they approach the end of their pilot scale demo.

1.4. PERMITS, LICENSES, AND COMPLIANCE

Performers will obtain any necessary permits, licenses or certifications required for acquisition, transportation, storage, and/or disposal of REE source materials, radioactive elements, biomass, waste by-products, and other system components. Performers will describe anticipated regulatory guidance (if any) and safety practices applicable to their planned research activities using their selected REE source material. This may include the Environmental Protection Agency (EPA), Department of the Interior, Nuclear Regulatory Commission, State or local regulators for certification, licensing, packaging, handling, transportation, research uses, and disposal of REEs, heavy metals, radioactive elements, other hazardous materials, and genetically modified living organisms (if used in the biomining workflow).

1.5. GENERAL REQUIREMENTS

1.5.1. Proposing Teams

Proposer teams must address both TA1 and TA2 described above, which should run in parallel.

Consequently, it is expected that the teams will include experts from the multiple disciplines related to the program challenges and goal (e.g., synthetic biology, environmental microbiology, geology, chemical engineering). Because several different technologies must ultimately work together, teams must identify one or more members as project integrators who will ensure those team members focused on a specific TA are also appropriately working towards the overall program goal. The project integrator should also address all risks specifically associated with integration.

Specific content, communications, networking, and team formation are the sole responsibility of the proposer teams. Proposer teams must submit a single, integrated proposal led by a single Principal Investigator or Prime Contractor.

1.5.2. Controlled Unclassified Information (CUI)

Because REEs are considered to be critical materials for the DoD, and their natural mineral deposits sometimes also contain radioactive elements, it is expected that certain aspects of the proposed research may be considered CUI if they reveal proprietary or other sensitive technical information, and may require safeguarding or dissemination controls pursuant to and consistent with applicable law, regulations, and government-wide policies. Proposals that anticipate the production of any such information must deliver a detailed CUI risk mitigation plan to DARPA (see Section 4.2.2.A. Volume I, Technical and Management Proposal, Section III; Attachment 1

- CUI Guide; Attachment 2 - CUI Management Plan Template). Performers must partition potentially sensitive tasks from non-sensitive research efforts, and are asked to identify sensitive tasks within their Statement of Work (Attachment 3 – Statement of Work Template). All performers (prime contractor and subcontractors) desiring public release of project information will be required to submit a request for public release from DARPA in accordance with their contractual requirements. As such, organizations that can comply with DoD CUI requirements as described in Attachment 1 must be part of the proposed team.

1.5.3. Other Requirements

Performers are expected to attend semi-annual program reviews to provide updates to the DARPA program management team, IV&V partners, government stakeholders and other EMBER performers on progress towards their milestones and scientific goals on the EMBER program. Performers will also summarize outstanding challenges and limitations that must still be overcome to achieve the overarching goals of the program.

2. Award Information

2.1. GENERAL AWARD INFORMATION

Multiple awards are possible. The amount of resources made available under this BAA will depend on the quality of the proposals received and the availability of funds.

The Government reserves the right to select for negotiation all, some, one, or none of the proposals received in response to this solicitation and to make awards without discussions with proposers. The Government also reserves the right to conduct discussions if it is later determined to be necessary. If warranted, portions of resulting awards may be segregated into pre-priced options. Additionally, DARPA reserves the right to accept proposals in their entirety or to select only portions of proposals for award. In the event that DARPA desires to award only portions of a proposal, negotiations may be opened with that proposer. The Government reserves the right to fund proposals in phases with options for continued work, as applicable.

The Government reserves the right to request any additional, necessary documentation once it makes the award instrument determination. Such additional information may include but is not limited to Representations and Certifications (see Section VI.B.2., “Representations and Certifications”). The Government reserves the right to remove proposers from award consideration should the parties fail to reach an agreement on award terms, conditions, and/or cost/price within a reasonable time, and the proposer fails to timely provide requested additional information. Proposals identified for negotiation may result in a procurement contract, cooperative agreement, or Other Transaction, depending upon the nature of the work proposed, the required degree of interaction between parties, whether or not the research is classified as Fundamental Research, and other factors.

Proposers looking for innovative, commercial-like contractual arrangements are encouraged to consider requesting Other Transactions. To understand the flexibility and options associated with Other Transactions, consult http://www.darpa.mil/work-with-us/contract-management#OtherTransactions.

In accordance with 10 U.S.C. § 2371b(f), the Government may award a follow-on production contract or Other Transaction (OT) for any OT awarded under this solicitation if: (1) that participant in the OT, or a recognized successor in interest to the OT, successfully completed the http://www.darpa.mil/work-with-us/contract-management#OtherTransactions http://www.darpa.mil/work-with-us/contract-management#OtherTransactions entire prototype project provided for in the OT, as modified; and (2) the OT provides for the award of a follow-on production contract or OT to the participant, or a recognized successor in interest to the OT.

In all cases, the Government contracting officer shall have sole discretion to select award instrument type, regardless of instrument type proposed, and to negotiate all instrument terms and conditions with selectees. DARPA will apply publication or other restrictions, as necessary, if it determines that the research resulting from the proposed effort will present a high likelihood of disclosing performance characteristics of military systems or manufacturing technologies that are unique and critical to defense. Any award resulting from such a determination will include a requirement for DARPA permission before publishing any information or results on the program. For more information on publication restrictions, see the section below on Fundamental Research.

2.2. FUNDAMENTAL RESEARCH

It is DoD policy that the publication of products of fundamental research will remain unrestricted to the maximum extent possible. National Security Decision Directive (NSDD) 189 defines fundamental research as follows:

‘Fundamental research’ means basic and applied research in science and engineering, the results of which ordinarily are published and shared broadly within the scientific community, as distinguished from proprietary research and from industrial development, design, production, and product utilization, the results of which ordinarily are restricted for proprietary or national security reasons.

As of the date of publication of this solicitation, the Government expects that program goals as described herein may be met by proposed efforts for fundamental research and non-fundamental research. Some proposed research may present a high likelihood of disclosing performance characteristics of military systems or manufacturing technologies that are unique and critical to defense. Based on the anticipated type of proposer (e.g., university or industry) and the nature of the solicited work, the Government expects that some awards will include restrictions on the resultant research that will require the awardee to seek DARPA permission before publishing any information or results relative to the program.

More specifically, research conducted for TA1 Bioengineering for REE Utilization is expected to constitute fundamental research, and research conducted on REE Biomining (TA2) is expected to produce Controlled Unclassified Information (CUI). However, the overall program will require protection as CUI, thus the Government expects that all awards, to include subawardees, will include restrictions on the resultant research that will require the awardee to seek DARPA permission before publishing any information or results relative to the program.

Proposers should indicate in their proposal whether they believe the scope of the research included in their proposal is fundamental or not. While proposers should clearly explain the intended results of their research, the Government shall have sole discretion to determine whether the proposed research shall be considered fundamental and to select the award instrument type. Appropriate language will be included in resultant awards for non-fundamental research to prescribe publication requirements and other restrictions, as appropriate. This language can be found at http://www.darpa.mil/work-with-us/additional-baa.

http://www.darpa.mil/work-with-us/additional-baa

For certain research projects, it may be possible that although the research to be performed by a potential awardee is non-fundamental research, its proposed subawardee’s effort may be fundamental research. It is also possible that the research performed by a potential awardee is fundamental research while its proposed subawardee’s effort may be non-fundamental research.

In all cases, it is the potential awardee’s responsibility to explain in its proposal which proposed efforts are fundamental research and why the proposed efforts should be considered fundamental research.

3. Eligibility Information

3.1. ELIGIBLE APPLICANTS

All responsible sources capable of satisfying the Government’s needs may submit a proposal that shall be considered by DARPA.

3.1.1. Federally…