DOE Bioengery Technologies Office (BETO) 2019 Project Peer Review Success through Synergy: Increasing Cultivation Yield and Stability with Rationally Designed Consortia

March 6, 2019 Advanced Algal Systems Shawn Starkenburg, Los Alamos National Laboratory (LANL) Alina Corcoran, New Mexico Consortium (New Mexico Consortium)

This presentation does not contain any proprietary, confidential, or otherwise restricted information. 2 of 30 Goal Statement Algae Bacteria-algae consortia consortia Project Goal: to increase productivity of open, outdoor Nannochloropsis cultures from 7 g/m2/d to HIGH-THROUGHPUT 2 >14 g/m /d (doubling of fall State of Technology SCREENING value) via development of (1) rationally-designed intrageneric Nannochloropsis consortia & (2) Nannochloropsis-bacteria consortia Complex Outputs: molecular toolkits, pipelines, consortia consortia

Outcomes: increased productivity & stability BIOREACTOR TRIALS

Industry Relevance: increased yield, fewer crop losses, & enhanced economic feasibility FIELD TRIALS 3 of 30 Quad Chart

Timeline Barriers Addressed  start date -- February 1, 2018 ➢ Biomass Genetics & Development (Aft-C); to improve the  end date -- January 31, 2021 productivity and robustness of  30% complete (with verification) algae strains against perturbations

FY FY 18 Total Objective 17 Costs Planned  to rationally design intrageneric Costs Funding Nannochloropsis consortia & (FY 19-21) Nannochloropsis-bacteria DOE $0 LANL: LANL: consortia to increase productivity, Funded $351 K $1.19 M stability and yield of open, NMC: NMC: outdoor cultures $173 K $1.29 M Project $0 LANL: LANL: End of Project Goal Cost $0 $0  to reach a productivity target Share >14 g/m2/d with consistent NMC: NMC: biomass composition $41 K $276 K 4 of 30 1. Project Overview -- Context and Project History

• industrial economics of production systems limited by low crop productivity and stability • consortia of microbial assemblages may address this limitation  combining algal strains with complementary traits increases yield and stability photo of “dead” and (Shurin 2014, Corcoran & Boeing live ponds, photo 2012, Stockenreiter et al. 2011) credit: Sapphire  addition of bacteria enhances Energy, Inc. productivity and reduces crashes (Sapp et al. 2007, Croft et al. 2005, LLNL work) learning from nature: microbial consortia photo credit: A. Dohnalkova/ PNNL 5 of 30 1. Project Overview -- Context and Project History

• despite strong theoretical and Types of consortia empirical underpinnings, Intrageneric algal – group of algae containing consortia are not commonly strains from the same genus cultivated at scale Intergeneric algal – group of algae containing strains from different genera • we aim to develop intrageneric algal consortia Algal-bacteria – group of algae & bacteria as well as algal-bacteria consortia (important for consistent biomass composition, downstream processing) conceptual • novel approaches model of taxa  rational design represented by different  high-throughput screening colored shapes filling up niche space 6 of 30 2. Approach -- Management

• WBS, SOPO, and Gantt chart with Milestones and Go/No-Go Points serve as guiding documents for the team

• PIs enable communication Task Key Team Members and collaboration  weekly PI meetings 1. Verification All  weekly team meetings to plan 2. Design of Nannochloropsis Garcia (Postdoc), Hanschen work, review Consortia & Tracking Tools (Postdoc) progress towards 3. Screening & Identification of Hovde (Research Scientist), milestones, and Growth-Promoting Bacteria Ohan (Post-MS Student) discuss technical 4. Verification & Optimization Garcia (Postdoc), Hanschen of Consortia Performance (Postdoc) barriers • team members drive 5. PEAK Challenge Holguin (Assistant Professor), Boeing (Professor) Rodriguz- progress on specific tasks Uribe (Postdoc) (see chart) 7 of 30 2. Approach -- Technical

Task #2: Design of Candidate Nannochloropsis Algae Bacteria-algae consortia consortia Consortia & Tracking Tools • rational design HIGH-THROUGHPUT SCREENING  match strains to outdoor regimes by combining taxa with different phenotypes  use Sapphire Energy, Inc.* data and strains *original award to Starkenburg at LANL and Corcoran at Sapphire Complex consortia • molecular tracking of strains BIOREACTOR TRIALS  needed to distinguish consortia members FIELD TRIALS  cox1 amplicon sequencing light micrograph of Nannochloropsis strain 8 of 30 2. Approach -- Technical

Task #3: Screening & Identification of Growth- Algae Bacteria-algae consortia consortia Promoting Bacteria Liquid Microdroplet (MD) ~150 µm • High-throughput Screening of HIGH-THROUGHPUT Cell-to-cell Interactions (HiSCI) Agarose Gel SCREENING system Microdroplet (GMD) ~20 – 70 µm • sorting, scale-up, and ID of Algal Cell ~5 µm beneficial bacteria Bacterial Cells ~1 – 3 µm Complex • sourcing of environmental consortia samples 1 algal BIOREACTOR TRIALS cell

3-5 FIELD TRIALS bacterial cells 9 of 30 2. Approach -- Technical

Task #4: Verification & Optimization of Consortia Algae Bacteria-algae consortia consortia Performance • individual/complex consortia HIGH-THROUGHPUT tested at increasing scales SCREENING  high-throughput screens to confirm co-existence (no stressors)  high-throughput screens Complex consortia with perturbations (temperature fluctuations, BIOREACTOR TRIALS invasions)  bioreactor trials to better simulate field conditions

FIELD TRIALS  β-field trials in micro- (100 L) or mini- (300 L) ponds 10 of 30 2. Approach -- Technical

Task #5: PEAK Field Challenge Algae Bacteria-algae consortia consortia • best performing consortia HIGH-THROUGHPUT SCREENING validated through outdoor field trials • 300-L miniponds located at the Complex Fabian Garcia consortia Center in Las

BIOREACTOR TRIALS Cruces, NM • metrics of productivity (e.g.,

FIELD TRIALS AFDW) and stability (e.g., Aerial view (top left) of miniponds (top right, temporal CV) bottom) at the Fabian Garcia Center at NMSU 11 of 30 2. Approach -- Technical Potential Challenges Critical Success Factors • insufficient  access to data/analysis methods phenotypic  creative design approaches variability across algal strains  access to bioreactors, ponds, infrastructure • data gaps  trained pond operators/technicians • challenges in  coordination across team members/sites scaling • variability in The success of this project hinges on consortia media due to performance exceeding the baseline -- this is the sourcing critical success factor for commercial viability. • environmental variability difficult to simulate in lab • control failures 3. Accomplishments/Progress/Results 12 of 30 Task 2 (Design of Consortia & Tracking Tools)

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Task 2 (Design of ConsortiaRotifer &trial 1Tracking Tools)

1) strain data compiled 2) screenings to enhance database (added assays to overcome data gaps)  salinity  pests

Pest challenges showed differential tolerances (i.e., crash rates). 3. Accomplishments/Progress/Results 16 of 30 Task 2 (Design of Consortia & Tracking Tools)

1) strain data compiled # Rationale 2) screenings to 1 top performing strains from each salinity enhance database top performing strains from each salinity plus 2 (added assays to baseline strain top performing strains from temperature overcome data gaps) 3 screenings  salinity top performing strains from temperature and 4 & 5  pests salinity screenings (two species compositions) 3) 9 consortia designed 6 consortia with new field isolates kitchen sink (non-rational design for 7 comparison) 8 strains with different intrinsic growth rates 9 strains with known genetic diversity 3. Accomplishments/Progress/Results 17 of 30 Task 2 (Design of Consortia & Tracking Tools)

1) strain data compiled Gene/ Amplicon Species Strain SNPs 2) screenings to loci size SNPs enhance database cox1 322 5-40 0 (added assays to rbcL 990 4-76 0 overcome data gaps) ITS 324 6-50 0-6  salinity N. salina ccsA 415 6-50  pests N. oceanica 40 Nannochloropsis strains including the following 3) 9 consortia designed species: australis, gaditana, salina, oculata, 4) molecular tracking granulata, oceanica, limnetica tool developed and Evaluation of conserved genes to differentiate species and strains revealed chloroplast ccsA gene validated (changed a good candidate, compared to cox1 with multiple target gene to copies in Nannochloropsis. overcome technical barrier) 3. Accomplishments/Progress/Results 18 of 30 Task 2 (Design of Consortia & Tracking Tools)

1) strain data compiled Custom pipeline 2) screenings to with amplicon sequence data enhance database determined species (added assays to and relative overcome data gaps) abundance with Nannochloropsis  salinity DNA and cells at  pests equal and unequal proportions. 3) 9 consortia designed 4) molecular tracking tool developed and validated (changed target gene to overcome technical barrier) 3. Accomplishments/Progress/Results 19 of 30 Task 3 (Screening & ID of Growth-Promoting Bacteria)

1) HiSCI system optimized Droplet of Cultivation for Nannochloropsis Medium

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Gell Novel platform Microdroplet HiSCI (High- (GMD) throughput screening of cell- day 0 to-cell interactions),

previously used Nannochloropsis for larger cells, Microcolony optimized for Nannochloropsis cells and used with field day 4 medium. 3. Accomplishments/Progress/Results 20 of 30 Task 3 (Screening & ID of Growth-Promoting Bacteria)

1) HiSCI system optimized for Nannochloropsis 2) environmental samples collected

Environmental samples collected from 30+ locations across NM, including Sapphire and Fabian Garcia ponds. 3. Accomplishments/Progress/Results 21 of 30 Task 3 (Screening & ID of Growth-Promoting Bacteria)

1) HiSCI system optimized algae for Nannochloropsis and 2) environmental samples bacteria collected 3) screenings conducted  3 iterations with algae environmentally-sourced only bacteria  3 isolates being scaled for further testing

Initial data show increased algal productivity, verified by cell sorting and microscopy during each round of selection when compared to controls. 3. Accomplishments/Progress/Results 22 of 30 Task 4 (Verification & Optimization of Consortia Performance)

• intrageneric Nannochloropsis (algae only) consortia testing in progress 1) high-throughput bioassays

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• intrageneric Nannochloropsis (algae only) consortia testing in progress 1) high-throughput bioassays 2) flask-scale bioassays

Flask-scale assay (50 mL) in field medium with 12/12 LD cycle and diel temperature changes (20 to 30 °C) showed higher productivity by consortia than baseline field strain (which crashed after final dilution). Task 4 (Verification &Optimization Consortia of Performance) • intrageneric intrageneric 3) 2) 1) bioreactor bioreactor trials flask high inoculation compared to 4 to compared inoculation strain consortium (n = 3). (n consortium strain showed lag in growth by growth in lag showed field - 3. Accomplishments/Progress/Results Bioreactor trial under under trial Bioreactor - throughput bioassays throughput bioassays scale scale bioassays baseline strain upon upon strain baseline temperature profiles profiles temperature - relevant light and and light relevant Nannochloropsis -

OD740 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 (algae only) progress testing in consortia only) (algae 0 1 2 d a ys 3 co b a 4 n se so l i n rt e i u m 5 24 of of 30 co b a n se so

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u m 3. Accomplishments/Progress/Results 25 of 30 Summary of Milestones 26 of 30 4. Relevance Project Goal:  double productivity of open, outdoor Nannochloropsis cultures via through intrageneric consortia & algae-bacteria consortia  important in its utilization of ecological and high throughput approaches

Industry Relevance:  project has potential to advance the state of technology and enhance the economic viability of algal biofuels by increasing yield & reducing losses in cultivation systems  project supports BETO goals by advancing toolkits and technologies to meet 2020 DOE PEAK targets of 18 g/m2/d and 80 GGE ton-1 biomass

Technology Transfer:  consortia will be available for use in industry  information will be available to broader community via publications 27 of 30 5. Future Work Task 3 (Screening & ID of Growth-Promoting Bacteria)

• Main Tasks  scale-up to larger volumes in flasks to verify phenotype(s)  new HiSCI runs with field isolate and known growth-promoting taxa • Stretch Goal  assays with taxa described in literature or acquired from collaborators (Sandia) • Milestones  3.3.ML – At least one bacterium identified that improves biomass production rates >30% in fall cultivation simulations for one strain/species of Nannochloropsis over the monoculture baseline  GNG.2 – Bacteria-augmented Nannochloropsis cultures demonstrate a 30% productivity improvement over the monoculture baseline

mitigation: low-throughput screening with known beneficial taxa 28 of 30 5. Future Work Task 4 (Verification/Optimization of Consortia Performance)

• Main Tasks  scale-up to larger volumes to verify phenotype(s)  continue perturbations • Stretch Goal  testing of intergeneric consortia • Milestones  4.1.ML – Intrageneric consortia demonstrating a 30% productivity improvement over a Nannochloropsis monoculture grown in triplicate under fall Las Cruces environmental conditions in commercial cultivation media  GNG.3 – Simulations or fall field trials demonstrating productivity improvements over the monoculture baseline to reach target of 14 g/m2/d

mitigation: UV mutagenesis, intergeneric consortia 29 of 30 5. Future Work Task 5 (PEAK Field Challenge)

• Main Tasks (at NMSU)  scale consortia members for field trials  grow consortia and baseline control in miniponds (25 cm depth, 300-L) semi- continuously for >2 growth cycles and sample for metrics of biomass • Stretch Goal  repeat with pest challenges or intergeneric consortia • Milestones  5.1 ML – Algae and bacteria inocula scaled to >200L for 300-L PEAK field challenge  5.2.ML – Field trials demonstrating >30% productivity and stability improvements over monoculture baseline for a target of at least 14 g/m2/d in the fall growth season (Sept-October) 30 of 30 Summary • Overview: project aimed to improve productivity and robustness of strains, resulting in a doubling of fall productivity • Approach: development of intrageneric algal consortia via rational design and algal-bacteria consortia via high throughput screening • Accomplishments/Progress/Results – Task 2: strain data compiled, additional screenings conducted, 9 consortia designed, molecular tracking tool developed and validated – Task 3: HiSCI system optimized, >30 environmental samples collected, 3 iterations conducted, additional screenings in progress – Task 4: high-throughput and flask-scale bioassays completed, additional trials in progress • Future Work: continuation of Tasks 3 and 4 (consortia development and testing) and PEAK Field Challenge • Relevance: direct relevance to the bioenergy industry because project addresses challenges associated with open ponds