2015 DOE BioEnergy Technologies Office Principal Investigators: (BETO) Project Peer Review Gregg Beckham (NREL) Date: March 25th, 2015 John Gladden (SNL) Technology Review Area: Biochemical Conversion Organizations: National Biological Lignin Depolymerization (WBS Renewable Energy 2.3.2.100) Laboratory and Sandia National Laboratory 1 | Bioenergy Technologies Office eere.energy.gov Project Goal Goal and Outcome: develop a biological approach to depolymerize solid lignin for upgrading of low MW aromatic compounds to co-products Relevance to BC Platform, BETO, industry: lignin valorization is key for meeting HC fuel cost and sustainability targets in integrated biorefineries Residual Ligninolytic Aromatic- Biorefinery Lignin Enzymes catabolizing bacteria 2 | Bioenergy Technologies Office Quad Chart Timeline Barriers • Start date: October 2014 • Bt-F Hydrolytic Enzyme Production • End date: September 2017 • Bt-I Catalyst Efficiency • Percent complete: ~30% • Bt-J Biochemical Conversion Process Integration Budget Partners and Collaborators • BETO Projects: Pretreatment and Process Total Planned Hydrolysis, Lignin Utilization, Biochemical Funding FY14 Costs Platform Analysis, Pilot Scale Integration, (FY15-Project Enzyme Engineering and Optimization End Date) • BETO-funded National Lab Projects: Oak Ridge National Laboratory (A. Guss) DOE $222,410 $1,752,590 • Office of Science funded efforts: Joint funded BioEnergy Institute, Environmental Molecular Sciences Laboratory, Pacific Northwest National Funds are split 50/50 between NREL Laboratory (R. Robinson, E. Zink) and SNL • Academic collaborators: Swedish University of Agricultural Sciences, University of Portsmouth 3 | Bioenergy Technologies Office Project Overview Project History: • Began as a BETO seed project in FY14 between NREL and SNL • Achieved major milestone at end of FY14 • Complementary effort to other BETO-funded lignin projects Project Context: • Lignin valorization is a primary challenge in biochemical conversion processes • Leverage studies in biological lignin depolymerization with new synthetic biology techniques and new process concepts in biological funneling High-Level Objective: • Employ biology for depolymerization and aromatic catabolism of residual lignin Lignin Depolymerization Lignin Upgrading • Focus on residual solids from • Develop aromatic metabolic map Deacetylation-Mechanical Refining Prt. • Elucidate aromatic transport mechanisms • Examine fungal and bacterial ligninolytic • Understand rate limitations in aromatic enzymes for solid lignin depolymerization catabolism • Develop effective enzyme secretion • Develop optimized modules for production systems of value-added products 4 | Bioenergy Technologies Office Technical Approach DMR Pretreatment Biomass Degraded lignin Sugars for fuels DDR-EH lignin Lignin-degrading Enzymes: Laccases & Peroxidases Enzymatic Hydrolysis Substrate: residual lignin from deacetylation and mechnical-refining pretreatment and enzymatic hydrolysis (DMR-EH) Approach: • Characterize enzymatic degradation of DMR-EH lignin • Identify parameters for optimal biological lignin degradation • Engineer organisms convert depolymerized lignin into valuable bioproducts Primary challenges: • Effective analytical tools to quantify lignin depolymerization • Minimizing repolymerization of high MW lignin during depolymerization • Identifying and overcoming aromatic transport and catabolic limitations Use microbial cell factories to upgrade lignin Success factors: • Achieving high yields of low MW lignin species • Generating aromatics that can be metabolized by microbes • Producing products with high market value from lignin 5 | Bioenergy Technologies Office Management Approach Critical Success Factors: Management Approach: • Employ tools of synthetic biology and • Use quarterly milestones to track lignin analytical chemistry progress and down-select options • Leverage experience from two • Milestone priority dictated by biomass conversion centers depolymerization and overcoming key risks in yields • Strategic hire in biological lignin depolymerization • Phone calls every 3 weeks, site visits every 6 months • Work with Biochemical Platform Analysis Project to identify optimal co- • Contributions from both partners on product targets lignin analytical chemistry leveraging respective expertise • Leverage input and collaborations from other BETO-funded projects in lignin • Divide research in a manner that leverages each partners strengths 6 | Bioenergy Technologies Office Technical Results – Outline • Examined purified enzymes and fungal- derived secretomes on several lignin substrates for ability to depolymerize lignin Lignin • Determined that reaction conditions and enzyme composition are key factors in Repolymerization ENZYMES Depolymerization promoting lignin depolymerization • Found evidence that lignin repolymerization is an issue that must be addressed to Co- achieve maximum lignin depolymerization. products Solution: microbial “sink” Aromatic Catabolism • For FY15, we have assembled a list of Strain Consumes Growth on Lignin Aromatics microbial “sinks” and initiated a detailed Amycolatopsis sp. Y Y Pseudomonas fluorescens Y Y characterization of DMR-EH lignin Pseudomonas putida KT2440 Y Y Etc… Y Y 7 | Bioenergy Technologies Office FY15 Milestone: Characterization of solid DMR-EH Compositional analysis Molecular weight distribution Sample ID Content (%) Ash 2.18 Mn Mw PD Lignin 66.0 2,000 10,000 5.0 Glucan 9.24 Xylan 9.36 Galactan 1.04 Arabinan 1.62 Fructan 0.00 Acetate 0.72 Total sugar 21.26 Total 90.2 2D-NMR for lignin chemical composition (HSQC) Moiety Abundance 1 1 6 2 6 H 2 5 S 3 5 3 H3 C O 4 OC H 3 4 S 17% O O R R O O G 45% b a 1 O O H 2% 6 FA 2 b 5 3 1 a 4 PCA 27% 6 G 2 1 O 5 3 6 2 R 4 OC H PCA 3 FA 9% O 5 R 4 O C H 3 O R 8 | Bioenergy Technologies Office Microbial Lignin Depolymerization Enzymatic cocktail Laccases Peroxidases Manganese (MnP) Lignin (LiP) Dye-decolorizing (DyP) Versatile (VP) Enzymatic assays O2 H2O2 Aryl-alcohol (AAO) H O 2 Free radicals LIGNIN DEPOLYMERIZATION FY14 Research: Start with Fungi • Use purified fungal enzymes to optimize conditions for lignin depolymerization • Examine ligninolytic enzyme cocktails produced by basidiomycete fungi to optimize conditions and identify the enzymes present in natural secretomes 9 | Bioenergy Technologies Office Optimizing Enzymatic Lignin Depolymerization Ligninolytic enzyme activity is a strong function of temperature and pH Top Enzymes • Screened 9 commercial purified fungal laccases Enzyme Function Source Organism Name and peroxidases for ability to solubilize lignin Laccase Trametes versicolor Tv • Used active enzymes to optimize the reaction Laccase Agaricus bisporus Ab Laccase Rhus vernicifera Rv conditions and create cocktail Laccase Pleurotus ostreatus Po • pH and Temp are critical factors for Lignin depolymerization Peroxidase Unknown LiP Green enzymes used in ligninolytic cocktails • Optimal enzyme loads depend on pH, T Ligninolytic Enzymes Depolymerize or Solubilization of DMR-EH Lignin by Repolymerize Lignin Depending on Reaction Ligninolytic Cocktail 60 Conditions 0.8 50 0.6 40 30 0.4 20 Solubility(%) 0.2 Depolymerize Depolymerize 10 0 Repolymerize Repolymerize 0 ugE/mgL 0.4 4 0.4 4 inLignin 1x 0.5x 1x 0.5x -0.2 -10 Temp 30°C 30°C Tv Lac -20 20°C 30°C -0.4 pH Po Lac Change Change -30 DDR-EH Lignin Solubilization (ABS 280nm) Solubilization Lignin -0.6 LiP Enzyme Loadings are also very important! 10 | Bioenergy Technologies Office Screening of natural fungal secretomes Screen with 12 white-rot fungi Ligninolytic Enzyme induction on DDR-EH lignin • Pleurotus ostreatus • Irpex lacteus • Panus tigrinus • Bjerkandera adusta • Bjerkandera sp. • Cerioporopsis subvermispora • Pleurotus eryngii • Phellinus robustus • Polyporus alveolaris • Stereum hirsutum Production of fungal secretomes in the presence of • Trametes versicolor lignocellulose can induce and/or accelerate the • Phanerochaete chrysosporium production of ligninolytic and cellulolytic enzymes Profile ligninolytic enzyme activities Production of ligninolytic cocktails ABTS assay Reactive Blue 19 assay 11 | Bioenergy Technologies Office Characterization of ligninolytic cocktails Pleurotus eryngii Phellinus robustus Bjerkandera sp. ABTSLaccase (MnPs) ( laccase, AAO, VP, MnP ) ( laccase, MnP, DyP, AAO) 2000 ABT 2000 ABTS + H2O2 2000S 2000 Peroxidase 1500 ABTS + H2O2+ MnP I 1500 1500 Mn 1500 9 days 9 days MnSO4 ABT 7 days mU/mL 1000 MnP II S + 1000 1000H2O 1000 mU/mL mU/mL Laccase x 10 2 mU/mL VeratrylAAO alcohol 500 500 500 500 VeratrylLiP alcohol+ ABT H2O2 S + 0 H2O 0 RBDyP 19 0 2+0 0 5 10 15 0 5 10 15 Mn 0 5 10 15 0 5 10 15 Days Days DaysDays Control Secretomes from: • Identified organisms that Pleurotus eryngii Bjerkandera sp. Phellinus robustus 9000 produce the most activity of 8000 pH4.5 7000 specific ligninolytic enyzmes pH7 6000 60% decrease P. eryngii 50% decrease MW • shows highest 5000 MW 4000 lignin depolymerization, 3000 lowest MW pH 7 +2 +2 2000 H2O2 + H2O2 + Mn H2O2 + Mn Mn+2 • Surpassed FY14 Go/No-Go 1000 Molecular weight average (kDa) weight Molecular average 0 • pH plays a major role in lignin 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 12 | Bioenergy Technologies Office depolymerization extents P. eryngii secretome depolymerizes DMR-EH Lignin Number-averaged Molecular Weight Distribution upon ligninolytic enzyme treatment at pH 7 2500 3 U laccase/g DDR Lignin 11 U laccase/g DDR 2000 30 U laccase/g DDR Repolymerization Depolymerization 1500 (kDa) n 1000 M 500 Need to create