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Ethanol/Biomass Concentrations

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Ethanol/Biomass Concentrations A Cyanobacteria-Based Photosynthetic Process for Bioethanol Production: Modeling of Productivities in Laboratory and Outdoor Photobioreactors Yanhui Yuan, Ryan Adams, Laura Belicka, Josee Bouchard, Kofi Dalrymple, Harlan L Miller III, William Porubsky, Ed Malkiel, Karl Ziegler, and Ron Chance Algenol Biofuels, Fort Myers, Florida ABO Summit October 1, 2014 1 Algenol Overview 2 Algenol Overview Advanced Industrial Research and Development Commercializing Direct To Biotechnology Company Facilities Ethanol ® Technology . 60,000 ft2 of Research . $200M equity capital . Started up in 2006 and Development lab . $25M Department of . Headquartered in Fort space in Fort Myers and Energy Integrated Myers, Florida Berlin, Germany Biorefinery grant . 200 employees including . 4 acre Process . $10M economic 70 with advanced Development Unit (PDU) development grant from degrees . 36 acre Integrated Bio- Lee County, FL Refinery (IBR) Fort Myers Research Labs Process Development Unit Integrated Biorefinery [Paul Woods & Ed Legere] 3 Technology Overview Algenol's Direct to Ethanol® process has three key components: A Very Productive Algal Specialized VIPER™ Energy Efficient Platform Photobioreactors Downstream Processing Proprietary cyanobacteria Cyanobacteria are grown in Energy efficiency is critical make ethanol and biomass saltwater contained in for economics and for low directly from CO2, water, proprietary PBRs that are carbon footprint and sunlight. exposed to the sun and are . Water-ethanol mixture is sent to patented downstream processing . 2013 ethanol productivity > 8000 fed CO2 and nutrients. gal/acre-yr (gepay) equipment that provides a 10-fold increase in concentration, then on . 2014 target 7,000 gepay to fuel grade “annualized” . Spent algae are processed into a bio-crude that can be refined into diesel, gasoline, and jet fuel 4 Process Scale-up in 2013 at the IBR 10 x Scale Up 4000 Module 40 Block 400 Block 4000 Block First Inoculation February 6 First Inoculation March 15 First Inoculation July 19 Still in operation 5 Biology and Productivity Modeling 6 Metabolic Pathway for Ethanol Production Metabolically enhanced cyanobacteria: key proprietary component of the Algenol technology 2 CO2 + 3 H2O C2H5OH + 3 O2 Enhanced ethanol production via over-expression of fermentation pathway enzymes • Pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) are found widely in nature • PDC catalyzes the non-oxidative decarboxylation of pyruvate to produce acetaldehyde • ADH converts acetaldehyde to ethanol • Ethanol diffuses from the cell into the culture medium and accumulates for eventual harvesting 77 Photosynthetic Efficiency and Productivity Targets Ethanol Production Target • Algenol target is >7000 gal ethanol/acre-yr • Corn is about 400 gal/acre-yr; sugarcane about 1000 • 2013 actual outdoor production exceeded 8000 gal/acre-yr at Algenol’s Florida facility • Target and recent results corresponds to 2-3% solar energy conversion efficiency (all % referenced to average US solar radiation) • Efficiency similar to commercial biomass conversion for Chlorella (food supplements) as well as more Algenol Vertical Photobioreactors conventional crops • Absolute theoretical limit (8 photons per C fixed) is about 30,000 gal/acre-yr of ethanol Potential Yield Limitations Ethanol branching ratio Light (photosaturation, photoinhibition) Contaminants CO2 and/or Nutrient supply Photosaturation Illustration (Melis, Plant Science (2009)) 8 Algenol Productivity Model Ethanol E0 Cyano- Fixed Biomass bacteria Carbon Maintenance Respiration (scales with cell count or chlorophyll concentration, C0) φ = ethanol branching ratio fixed carbon rate serving ethanol production φ = total fixed carbon rate Obtainable from short time behavior (dilute limit) “Static” Range = 0 % – 85 % All light 2 Areal Ethanol Productivity (mol EtC/m -s): Pe = αφEk ln(1 + E0/Ek) absorbed (kD>>1) 2 Areal Net Biomass Productivity (mol BmC/m -s): Pb = α(1-φ)Ek ln(1 + E0/Ek) – R0C0D α = limiting quantum yield (Cfix/photon) 2 Ek = photosaturation parameter (μmol photons/m -sec) R0 = maintenance respiration rate (µmol C/mg Chl.a-min) D = culture depth or thickness (m) 9 A Recent Culture Management Experiment Objective: Compare productivity of two ethanologenic modifications of Strain 2 Compare photobiological responses of the ethanologenic strains with wild type Treatment Strain # of PBRs Controls Strain 2a 2 New Strain Strain 2b 3 Wild Type Strain 2-WT 2 Operating Conditions: . Outdoors (Florida) . pH-controlled CO2 delivery . Vertical PBRs constructed in-house . Inoculation Day: 14 May 2013 . Collect samples periodically for . Ethanol/biomass concentrations . Productivity-Irradiance (PE) curves . Optical absorption spectra . Enzymatic activities 10 Modeling an Outdoor Experiment in Florida (Strain 2b) Model Fit: α, 흋0, and R0 Ek taken from PE curves α = 0.09 (11 photons/fixed C) 흋0= 72 % R0 = 0.05 µmol C/mg Chl.a-min (consistent with dark O2 consumption) Optical Absorption Spectra (integrating sphere) 60 50 day - 2 40 Experimental Data in Triplicate 30 photons/m 20 mol 10 PAR, PAR, 0 0 10 20 30 40 50 60 Time-days Net Fixed C (TC) = all non-ethanol organic C plus 3/2 C in ethanol Average Daily PAR irradiance Net Ethanol C (EtC) = 3/2 C in ethanol May 14 - July 4, 2013 Experimental data from Algenol aquaculture group (Dr. Laura Belicka, Dr. Lanny Miller) 11 Modeling an Earlier Strain with Lower Ethanol Branching (Strain 1) Model Fit: α, 흋0, and R0 Ek taken from PE curves α = 0.09 (11 photons/fixed C) 흋0= 40 % R0 = 0.05 µmol C/mg Chl.a-min (consistent with dark O2 consumption) Optical Absorption Spectra (integrating sphere) 50 40 day - 2 Experimental Data in Duplicate 30 photons/m 20 mol 10 PAR, PAR, 0 0 10 20 30 40 50 60 70 Net Fixed C (TC) = all non-ethanol organic C plus 3/2 C in ethanol Time-days Net Ethanol C (EtC) = 3/2 C in ethanol Average Daily PAR irradiance Sept 12, 2012 - Nov 25, 2012 Experimental data from Algenol aquaculture group (Dr. Laura Belicka, Dr. Lanny Miller) 12 Modeling a Laboratory Experiment Strain 2c, 12-12 constant light-dark cycle • Same modeling approach as outdoors • Experiment designed to be highly predictive of Model Fit: α, 흋0, and R0 outdoor (vertical) configuration α = 0.09 (12 photons/fixed C) = 75%, Q =0.5, R =0.08 • Same model parameters derived for range of 흋0 e 0 R0 consistent with dark O2 consumption 2 irradiance levels (E0 = 90 – 350 µE/m -sec) Ek derived from PE measurements on • Ek acclimation derived from PE curves samples extracted at various time points Experimental Data in Triplicate 2 E0 = 350 µE/m -sec 13 Biomass Production Model (φ = 0) Productivity Model α = 0.10 molC/molphotons, R0 = 0.05 µmolC/mgChl.a-min α = 0.075 molC/molphotons, R0 = 0.10 µmolC/mgChl.a-min Cell density [OD750nm] densityCell Laboratory Experiment (n=6) 2 Strain 2 Wild Type, E0=230 µE/m -s Note: Biomass concentration (g/L) = 0.4 OD750 14 Conclusions . A relatively simple model based on Michaelis-Menten kinetics is remarkably successful in describing biomass and ethanol production in long term indoor and outdoor cultures . Application of the model to Algenol’s ethanologenic strains yields: . Very high limiting quantum yields (α ~ 0.08-0.10) corresponding to quantum demands of 10-12 photons/fixed carbon . High branching ratios, with about 80% of the fixed carbon diverted to the ethanol pathway . Reasonable estimates of maintenance respiration that are quite consistent with inferences from measured rates of oxygen consumption in the dark . Reasonable estimates of the impact of light-related acclimation on culture productivity . High confidence in the translation of laboratory experiments to outdoor conditions 15 Acknowledgements Fort Myers Research Lab Berlin Research Lab 16.
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