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Algal-Bacteria Consortia 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) 1) strain data compiled temperature 16 25 32 0.012 0.008 f i Growth rate bioassay e l data from Sapphire d e t 0.004 a Energy, Inc. r h m for ~30 t w 0.000 e o Nannochloropsis d r i g a strains (along x-axis) 0.012 e v i in different media types t a l 0.008 and temperatures were e l a r compiled. Strains grew b better in lab media at 0.004 low or intermediate temperatures. 0.000 strain 3. Accomplishments/Progress/Results 13 of 30 Task 2 (Design of Consortia & Tracking Tools) 1) strain data compiled 1.2 1 6 0.8 C 0.4 0.0 temperature 1.2 T 0 2 e 5 5 m 7 Within the bioassay 0.8 C p D data, a single top O 0.4 performer (black line) was revealed. This 0.0 strain was a production 1.2 3 strain at Sapphire 2 0.8 C Energy and is our baseline strain for 0.4 consortia comparisons. 0.0 0 1 2 3 4 5 6 7 day 3. Accomplishments/Progress/Results 14 of 30 Task 2 (Design of Consortia & Tracking Tools) 1) strain data compiled 0.6 1 2 2) screenings to 0.3 enhance database 0.0 0.6 1 (added assays to 4 e t 0.3 a r overcome data gaps) h 0.0 t s w a o l 1 r salinity 0.6 i n 6 g i t y m 0.3 u m 0.0 i x a 0.6 1 m Salinity screenings 8 showed some differential 0.3 tolerances by some 0.0 strains. Baseline strain 0.6 2 0 (indicated by box) grew 0.3 well under all salinities. 0.0 strain 3. Accomplishments/Progress/Results 15 of 30 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
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