Switchgrass for ethanol and lipid production

Transcription

1 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 from the USDA National Institute of Food and Agriculture (CenUSA; field operations and phenotypic measurements) AICHE, Boston December 13, 2016

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3 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)

4 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

5 Switchgrass for production on marginal farmland. Mike Casler Ken Vogel Rob Mitchell CENUSA: Agriculture and Food Research Initiative Competitive Grant No

6 Why Switchgrass? Good Biomass Productivity Low Inputs Native grass Winter Adapted Established Management Genetic Diversity Molecular Knowledge La and Footprint (x acres) Co-Feeding Switchgrass & Corn Stover Land Footprint for Cellulosic Ethanol Plant with Annual Production of 60 million gal/yr 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.

7 Vogel KP, Mitchell RB, Casler MD, Sarath G. Registration of Liberty s switchgrass. Journal of Plant Registrations. 2014;8(3):242-7.

8 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)

9 Bioma ass (Mg g/ha) Switchgrass Biomass Production Liberty Summer Significanceifi Variety Year Fertilizer Harvest 0 H1 H2 H1 H2 H1 H2 H1 H2 Fertilizer (N lb/acre): Treatment

10 SG Theoretical Ethanol Yield per Mg 600 Max Eth hanol (L L/Mg) Liberty Summer Significance Harvest Year H1 H2 H1 H2 H1 H2 H1 H2 Fertilizer (N lb/acre): Treatment

11 % Glucose Extra acted Cellulase Treatment Efficiency 90 Liberty Summer H1 H2 H1 H2 H1 H2 H1 H2 Fertilizer (N lb/acre): Treatment Significance Cultivar & Harvest Year Note: Ammonia Pretreatment

12 Cellulase efficiency negatively correlated with lignin content 90 % Glucose Extracted d Lignin Content (g/kg)

13 Estimated Ethanol Land Productivity Es st. Etha anol (L/ /ha) 7000 Liberty Summer Significance Cultivar Year Nitrogen H1 H2 H1 H2 H1 H2 H1 H2 Fertilizer (N lb/acre): Treatment Yield of corn ethanol = 200 bu/acre x 2.8 gal/bu = 560 gal/acre 5300 L/ha

14 Beyond Ethanol with Lipid Production Jin et al., 2015

15 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 % 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

16 Lipomyces In C:N 60:1 dilute acid pretreated switch grass hydrolyzate (1000 x) 24 h 120 h 144h

17 Lipomyces clade screening Screening Lipid Production on 100 g/l Lipid Glucose Production Medium Titer (g/l) Lipid Lipids % Lipids 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

18 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

19 Dilute-acid pretreated switchgrass - Superior Strains Utilize Undetoxified Hydrolyzate - Two Stage Process Optimizes High Lipid Titers Lipomyces tetrasporus Y Bio omass (A 620 ) Transfer Cells to Stage 2 Biomass (A620) Glucose (g/l) Xylose (g/l) Arabinose (g/l) Acetic Acid Oil Sugars an nd Acetic Acid (g/l) Oil (g/l) mass (A 620 ) Bio Sugars an nd Acetic Acid (g/l) Oil (g/l) Time (h) 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

20 A 2- to 5-Fold Lipid Titer Improvement by Strain and Process Optimization Lipid Titer (g/l) Yield x 100 (g/g) 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

21 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).