Switchgrass for ethanol and lipid production Field based ethanol yields from established SG plots Yeast discovery for single cell oil production from SG hydrolysates Bruce S. Dien* Patricia. J. Slininger Josh Quarterman Patricia J. O Bryan *Contact information; bruce.dien@ars.usda.gov Michael Casler Robert Mitchell Ken Vogel Gene Lester (NPS ARS National Program 213 Biorefineries Agriculture and Food Research Initiative Competitive Grant No. 2011-68005-30411 from the USDA National Institute of Food and Agriculture (CenUSA; field operations and phenotypic measurements) AICHE, Boston December 13, 2016
Commodity Credit Corporation BioPreferred Program USDA Certified Biobased Label Quantification of product s new carbon measurement FP on label indicates Federal Procurement Preference 2,500 products certified/labeled (2015)
ARS Biological Refining Technology Centers Eastern Regional Research Center National Center for Agricultural Wyndmoor, PA Utilization Research, Peoria, IL Southern Regional Research Center, New Orleans Western Regional Research Center, Albany, CA
Switchgrass for production on marginal farmland. Mike Casler Ken Vogel Rob Mitchell CENUSA: Agriculture and Food Research Initiative Competitive Grant No. 2011-68005-30411
Why Switchgrass? Good Biomass Productivity Low Inputs Native grass Winter Adapted Established Management Genetic Diversity Molecular Knowledge La and Footprint (x10 000 acres) Co-Feeding Switchgrass & Corn Stover Land Footprint for Cellulosic Ethanol Plant with Annual Production of 60 million gal/yr 4000 3500 3000 2500 2000 1500 1000 500 0 20 40 60 80 100 Corn Stover (% used) Estimates harvests of 10 ton/acre for SG & 2 ton/acre for corn stover with ethanol yields of 80 gal/ton. Switchgrass + corn stover allows for better scaling.
Vogel KP, Mitchell RB, Casler MD, Sarath G. Registration of Liberty s switchgrass. Journal of Plant Registrations. 2014;8(3):242-7.
Crop management and land-based ethanol yields Objectives Determine yield response of Summer & Kanlow versus Liberty Determine fertilizer application for 50 vs. 100 lb N/acre Determine harvest time for anthesis versus postfrost Responses: biomass, chemical composition, & enzymatic yield of sugars (e.g. ethanol)
Bioma ass (Mg g/ha) Switchgrass Biomass Production 25 20 15 10 5 Liberty Summer 2014 2015 Significanceifi Variety Year Fertilizer Harvest 0 H1 H2 H1 H2 H1 H2 H1 H2 Fertilizer (N lb/acre): 50 100 50 100 Treatment
SG Theoretical Ethanol Yield per Mg 600 Max Eth hanol (L L/Mg) 500 400 300 200 100 Liberty Summer Significance Harvest Year 2014 2015 0 H1 H2 H1 H2 H1 H2 H1 H2 Fertilizer (N lb/acre): 50 100 50 100 Treatment
% Glucose Extra acted Cellulase Treatment Efficiency 90 Liberty Summer 80 70 60 50 40 30 20 10 0 H1 H2 H1 H2 H1 H2 H1 H2 Fertilizer (N lb/acre): 50 100 50 100 Treatment Significance Cultivar & Harvest Year 2014 2015 Note: Ammonia Pretreatment
Cellulase efficiency negatively correlated with lignin content 90 % Glucose Extracted d80 70 60 50 40 160 170 180 190 200 210 220 Lignin Content (g/kg)
Estimated Ethanol Land Productivity Es st. Etha anol (L/ /ha) 7000 Liberty Summer 6000 5000 4000 2000 Significance Cultivar Year Nitrogen 2014 3000 2015 1000 0 H1 H2 H1 H2 H1 H2 H1 H2 Fertilizer (N lb/acre): 50 100 50 100 Treatment Yield of corn ethanol = 200 bu/acre x 2.8 gal/bu = 560 gal/acre 5300 L/ha
Beyond Ethanol with Lipid Production Jin et al., 2015
Lipid Producing Yeast Plan: To screen Lipomyces and Yarrowia yeasts on dilute-acid pretreated switchgrass sugar hydrolysates Lipomyces: better lipid producer & ARS culture collection has diverse collection. Yarrowia: widely accepted commercial yeast, GRAS, & developed transformation system. Advantage of oleaginous yeast Accumulate 20-70+% of their biomass as lipids In general, oleaginous yeast have faster growth, higher density growth, and allow lower ph conditions than microalgae Similar in fatty acid composition to seed oils
Lipomyces In C:N 60:1 dilute acid pretreated switch grass hydrolyzate (1000 x) 24 h 120 h 144h
Lipomyces clade screening Screening Lipid Production on 100 g/l Lipid Glucose Production Medium 25 80 Titer (g/l) Lipid 20 15 10 5 Lipids % Lipids 60 40 20 Lipid Con ntent (%w/w w) 0 0 ABCDE FGH I J KLMNOPQ RSTUVWXY Yeast Isolates Winners: L. tetrasporous, L. spencer-martin, L. lipofer, & Rhodosporidium toruloides
Biomass hydrolysates are challenging to ferment because they contain inhibitors. 10% sugars Hydrolysis sugars Inhibi itors H 3 C O OH Acetic O HO O O O HMF Furfural Ferulic O O HO O O OH CH 3 H 3 C OH Levulinic O Phenolics OH Formic
Dilute-acid pretreated switchgrass - Superior Strains Utilize Undetoxified Hydrolyzate - Two Stage Process Optimizes High Lipid Titers Lipomyces tetrasporus Y-11562 70 35 70 35 Bio omass (A 620 ) 100 10 1 Transfer Cells to Stage 2 Biomass (A620) Glucose (g/l) Xylose (g/l) Arabinose (g/l) Acetic Acid Oil 60 50 40 30 20 10 Sugars an nd Acetic Acid (g/l) 30 25 20 15 10 5 Oil (g/l) mass (A 620 ) Bio 100 10 1 60 50 40 30 20 10 Sugars an nd Acetic Acid (g/l) 30 25 20 15 10 Oil (g/l) 0.1 0 0 50 100 150 200 250 Time (h) 0 0.1 0 0 50 100 150 Time (h) 5 Stage 1 - Growth Stage 2 - Lipid Amplification 75% Switchgrass Hydrolyzate 100% Switchgrass Hydrolyzate 62:1 C:N 600:1 C:N Pat Slininger
35 30 25 A 2- to 5-Fold Lipid Titer Improvement by Strain and Process Optimization Lipid Titer (g/l) Yield x 100 (g/g) 20 15 10 5 0 Prior Art Lipomyces spencer-martin Lipomyces tetrasporus Rhodosporidium toruloides Lipomyces tetrasporus Pat Slininger 2-Stage process Dilute acid pretreated Switchgrass 2 or Multi-Stage process SPORL pretreated Douglas Fir
Summary Switchgrass cultivated on marginal land can compete with corn grain for ethanol production. Biomass productivity controlled overall landbased ethanol yield. Demonstrated lipid titers from switchgrass hydrolysate that are 2-5x higher than maximum previously reported for lignocellulose. Identified a lesser-known Yarrowia species that was superior to the control in terms of lipid content (49.7% of DW) and lipid titer (2.9-fold improvement). Demonstrated successful transformation of top performing strains (not shown).