Biochemical shifts in Chlorella lipid metabolism for two-stage bioprocessing Julian Rosenberg, PhD Candidate Department of Chemical & Biomolecular Engineering Johns Hopkins University, Baltimore, MD September 30, 2014 Algae Biomass Summit, San Diego, CA
Mature and emerging markets for algae Chlamydomonas reinhardtii Schizochytrium sp. Haematococcus pluvialis Dunaliella salina Chlorella vulgaris Chlorella spp. Chlorella and Scenedesmus sp. $ 2
Sensitivity analysis for cost of biocrude production Lipid Content (35% : 25% : 15%) Oil Extraction Efficiency (100% : 80% : 60%) Areal Productivity, g m -2 d -1 (20 : 15 : 10) Pond Liner (none : liner : replace once) Pond Cover (none : cover : replace once) Paddlewheel Energy per Pond (1.74 kw : 5.96 kw) Biomass Harvesting Efficiency (100% : 85%) Biomass Harvesting Cost (50% : base : 150%) Flocculant Cost, ton -1 algae ($50 : $100 : $150) CO 2 Cost, ton -1 ($20 : $40 : $80) Nutrient Recycle (100% : none) Oil Extraction Cost (50% : base : dry algae) Evaporation Rate, cm day -1 (0.05 : 0.5) Pumping Capacity, ft head (20 : 40) DOE TABB 2014: Reduce modeled mature price below $5 per gal with value-added coproducts. Baseline cost of production: $15 gal -1 crude algal oil Rogers, Rosenberg et al. (2014) A critical analysis of raceway-driven ponds for algal biofuel production. Algal Research 4:76-88. 3
Chlorella genus: long history as model organisms The Boeing Company (1961) Large algal systems; Benson & Calvin (1950) Annual Review of Plant Physiology 1: 25-42. 4
Physiological response to light and sugar: green fuels and golden opportunities Stage 1: Photoautotrophy ATP NADPH Photosynthesis ATP NADPH Photosynthesis Organic Compounds accd Biosynthesis TCA Glycolysis Biomass acc1 1 Biomass Biosynthesis TCA 2 Glycolysis Organic Compounds Stage 2: Heterotrophy Mixotrophic Two-Stage 5
Strain development pipeline for Chlorella spp. Chlorella & Scenedesmus spp. C. vulgaris UTEX 265 C. sorokiniana UTEX 1230 Ribosomal RNA Fingerprint 18S ITS1 5.8S ITS2 28S C. protothecoides UTEX 411 NCBI PopSet: 631798695 Wan, Rosenberg, et al. (2011) An improved colony PCR procedure for genetic of Chlorella algae. Biotech Lett, 33:1615-1619. 6
Lead candidate lipid and biomass productivity C. sorokiniana UTEX 1230 1.5 L Autotrophy (100 µe m -2 s -1 ) Growth similar to other strains 10-15% total lipids Hetrotrophy (10 g L -1 glucose) 5-fold growth vs. autotrophy 25-40% total lipids Heterotrophy C. vulgaris, C. protothecoides strains did not exceed 20 million cells ml -1 Autotrophy Rosenberg et al. (2014) Comparative analyses of Chlorella species in response to light and sugar. PLoS ONE. 9 (4), e92460 7
Fatty Acid Content (% of dry weight) Classification of auto- and heterotrophic lipid types 90% * + UTEX 265 C. vulgaris UTEX 411 C. protothecoides UTEX 1230 C. sorokiniana AUTO HETERO Rosenberg et al. (2014) Influence of light and sugar. PLoS ONE. 9 (4), e92460 Chain Length : Unsaturated Bonds 8
Scale-up of cultivation, harvesting, & lipid analysis 3 L 25 L Dionex Solvent Extraction System Slurry Paste Evodos Model T-10 Continuous Centrifuge 300 L 9
UTEX 1230 mixotrophic growth & lipid accumulation HETEROTROPHIC 38.7±0.9% Total Lipids 21.1±0.7% Total MIXOTROPHIC 31.8±0.7% Total Lipids 11.8±0.8% Total Day 3 Day 6 Day 10 Day 13 Fatty Acid Content (% of dry weight) Rosenberg et al. (2014) Comparative analyses of Chlorella species in response to light and sugar. PLoS ONE. 9 (4), e92460 10
Two-stage bioprocess demonstration at 100+ L 140 L 36 L 1: Photoautotrophic Scale-Up 2: Heterotrophic Stage 11
Harmonization of Chlorella genome projects Species and Strain Number Genome Size Status Auxenochlorella protothecoides UTEX 25 21.4 Mbps Sequenced Auxenochlorella protothecoides 0710 (Tsinghua University, China) 22.9 Mbps Sequenced Chlorella sorokiniana DOE 1412 (aka NAABB 2412) 55 Mbps Sequenced C. sorokiniana Phycal 1228 55 Mbps Sequenced C. sorokiniana UTEX 1230 (University of Nebraska-Lincoln) 55 Mbps Sequenced C. variabilis NC64a (University of Nebraska) 46 Mpbs Sequenced C. vulgaris UTEX 259 (UC-San Diego) 54 Mbps Sequenced C. vulgaris UTEX 395 (NREL) 55 Mbps Sequenced C. emersonii (University of Mumbai) TBA, NCBI: PRJNA243839 In Progress C. minutissima UTEX 2341 (State Oceanic Admin, China) TBA, NCBI: PRJNA245012 In Progress C. zofingiensis UTEX 32 (NREL) TBA In Progress Transcriptomic data sets with UTEX 1230 and UTEX 395 Significant Changes during Heterotrophy Function PFam ID Sugar, citrate, ABC, and other transporters Carbon metabolism PF00083 Phospholipid methyltransferase Lipid remodeling PF04191 Lipase (class 3) Lipid remodeling PF01764 Multicopper oxidase, ferrooxidase (hephaestin) Metal micronutrient uptake PF07731 12
Concluding Remarks Influence of light and sugar on C. sorokiniana UTEX 1230 Genome has been recently sequenced, transcriptomes available (UNL) UTEX 1230: unique fatty acid profile for biofuels & nutritional lipids Uncouple carbon metabolism with two-stage bioprocess Beneficial physiological changes occur during this trophic transition Sugars Fatty Acids Chlorosis: turnover of photosynthetic biomolecules 13
Acknowledgements WHITING SCHOOL OF ENGINEERING JOHNS HOPKINS UNIVERSITY Dr. Michael Betenbaugh Jon Rogers Dr. Marc Donohue Pavlo Bohutskyi Dr. Scott Williams Dr. Minxi Wan Dr. George Oyler Dr. Donald Weeks Dr. Naoko Kobayashi Eric Noel Austin Barnes Dr. George Oyler Gunjan Andlay Adithya Balasubramanian Dr. Ben Hobbs Dr. Cindy Parker ENVIRONMENT, ENERGY, SUSTAINABILITY, & HEALTH INSTITUTE Katie Igrec Lima L. Gordon Croft Drs. Michael Guarnieri, Phil Pieknos October 2014
Thank you! algae@jhu.edu October 2014