Ensilage as a Microbial Platform for Biomass Pretreatment

Tom L. Richard Agricultural and Biological Engineering, Penn State University

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Feedstock storage: the missing box

Harvest

Pretreatment/Conditioning

Bioconversion

Sugars, ethanol, chemicals, materials

Biomass

Storage

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Biorefinery Regional Pretreatment

Transport Transport

Anaerobic Storage

Standing Crop

Harvest

After – Searcy – AETC – 2009

Field Drying

Mobile Pyrolysis

Transport Oil

Aerobic Storage

Potential Biomass Logistic Schemes

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Harvest Frequency and Yield Dry matter .. ton DM / ac

Switchgrass PSU1 UW2

Late summer 3.7 4.9

Late fall 3.1 4.5

Spring 2.0 3.5

1 – Adler et al., 2006

2 – Shinners et al., 2008

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Impacts of storage on feedstock value ! Dry matter losses !  Biomass compositional changes ! Conversion efficiencies

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Dry storage

!  Biomass moisture content < 20% "  Field drying may be

necessary !  Potential for high dry

matter losses !  High risk of fire

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Cost of dry matter losses

(Adapted from Hess et al., 2009)

Dry storage strategy

Dry matter loss, DML (%)

Dry climate

Wet climate

Stack on ground

1-9 7-39

Covered stack on ground

3-13 6-25

Enclosed buildling

1-4 2-8

Cost of DML ($/DM ton)

Dry climate

Wet climate

1.20 4.30

1.50 3.30

0.40 0.90

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US DOE Projections – Near Term

Perlack and Hess 2006 9

The Ensilage Process

0

1

2

3

4

5

6

7

8

0 5 10 15 20

water soluble carbohydrateslactic acidacetic acid

% D

M

ensilage duration (d)

pH = 6.62

pH = 4.05

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Ensiled Storage Advantages: !  High moisture content !  Low pH for long term storage !  Synergies may allow less intensive pretreatment

through hydrolysis and saccharification Disadvantages: !  Hydrolysis products (sugar) may degrade and be lost !  Additional cost if cell wall degrading enzymes,

microbes, and/or chemicals are applied

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Process area contributions to Minimum Ethanol Selling Price, 2012 projection

(Aden and Foust, 2009) 12

Bioreactor Schematics

Silo Bunker Bags 13

Bioreactors on the Landscape

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Scalable Technology

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Feedstock storage opportunities

Harvest

Storage Pretreatment/Conditioning

Bioconversion

Sugars, ethanol, chemicals, materials

Biomass

Cell wall degrading enzymes

Microbes

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Switchgrass ensiled storage experiments

Wrapped bales in field

FoodSaver bags in lab

August harvest; initial moisture = 33% 17

Stover storage experiments !  Moisture content:

adjusted to 6 different levels (15% - 65% w.b.)

!  Dry bulk density: 159 kg/m3 (~10 lb/ft3)

!  Temperature: 22oC and 37oC

!  Oxygen status: anaerobic and aerobic

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Dry matter losses

0

10

20

30

40

10 20 30 40 50 60 70

21 d, aerobic90 d, aerobic

21 d, anaerobic210 d, anaerobic

dry

mat

ter

loss

(%)

initial moisture content (% w.b.) 19

Ensilage stabilizes biomass composition

Component (%DM)

Storage duration

Day 0 Day 180

Lignin 21.2 21.9

Glucan 28.7 28.5

Xylan 17.4 17.6

Extractable sugars

3.31 1.73

October harvest; initial moisture = 47% 20

Ethanol yields from ensiled switchgrass with or without reduced severity pretreatment

0

20

40

60

80

100

0 217

nativepretreated

etha

nol (

mg/

g D

M)

days of ensilage

Chen, 2009 21

Why add enzymes during ensilage? !  Achieve biological pretreatment !  Initiate lactic acid fermentation in feedstocks with

low water soluble carbohydrate (WSC) levels

(Jones et al., 2004)

Feedstock Initial WSC (% DM)

Switchgrass, November

2

Switchgrass, August

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For maximum silage fermentation in grasses:

range = 2-19%

typical = 10-20%

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Screening Commercial Enzyme Products Microorganism source Brand name Hemicellulase Cellulase C:H

Aspergillus niger Enzecoxylanase A Concertrate

24805. U/g 1904 U/g 0.08 AN0.08

Deerland Hemicellulase

1075 U/g 384 U/g 0.36 AN0.36

Trichoderma reesei Multifect xylanase 5712 U/ml 109 U/ml 0.02 TR0.02

DeerlandCellulase TR 1219. U/g 2607 U/g 2.14 TR2.14

Multifect CL 116 U/ml 278 U/ml 2.38 TR2.38

Trichoderma longibrachiatum

Dyadic Xylanase 2XP 18624 U/g 5144 U/g 0.28 TL0.28

Safizym fl300 390 U/ml 543 U/ml 1.39 TL 1.39

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Impact of enzyme characteristics

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Long-term Storage Effects

Enzyme Control TR 2.37 Control

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Enzyme stability during ensilage

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Lignin-degrading enzymes and wet storage !  Ensiled corn stover treated with laccase,

mediator, and oxygen

(Vicuna, 2000) 28

Effect of laccase on sugar release during enzymatic hydrolysis

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28

30

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0 1000 2000 3000 4000 5000

% c

ellu

lose

con

vers

ion

laccase loading rate (U/g)

Chen, 2009 29

Work in progress

Ensiled Storage

Synthetic Lactobacillus

Controlled environments

Biomass characterization

Downstream processes:

Hot water pretreatment (Dionex ASE 350)

Enzymatic hydrolysis

Fermentation

Stover, cobs, switchgrass

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Silage Inoculants

Reasons farmers use Inoculants

• Rapidly reduce pH • Reduce dry matter losses • Increase bunk life • Maximize forage nutrient value • Improve NDF digestibility

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Lactobacillus plantarum !  Established methods for laboratory use

Sequenced genome Promoter libraries Proven heterologous enzyme producer Amylase Cellulase Esterase

!  Robust in silage !  Common silage inoculant

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Conditions in Silage !  Low pH !  Low Oxygen Using promoters that are induced by these

conditions can offer many benefits #  Reduced metabolic load during growth phase #  Higher cell densities #  Greater long-term enzyme delivery #  More stable storage conditions

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Natural System

Activates

P170 Conditional Promoter

Keto-steroid Isomerase

At low pH

Stress Promoter

rcfB Regulatory Protein

yxbE Stress Protein

yxbD Stress Protein

Constitutive Promoter

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rcfB

At low pH

Activates

Constitutive Promoter Library

Regulatory Protein

P170 Conditional Promoter

mRFP Functional Gene B14

Secretion Signal Library

Synthetic System

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RFP Test of Construct

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Hydrolysis Enzymes to Express

Functional Gene Sources Information

Enzyme Type Function Organism Reference

laccase Depolymerize lignin

Streptomyces lavendulae

Suzuki et al. (2003) Expressed successfully in E. coli

Ferulic-acid

Esterase

Ferulic acid esterase

Lactobacillus johnsonii

Lai et al. (2009)

Ferulic acid crosslinks lignin and xylan and using inoculants producing a ferulic acid esterase has been shown to increase fiber digestability in ruminants by over 30%

Xylanase Depolymerize Xylan

Clostridium Stercorarium

Adelsberger et al. (2004)

Used by Keasling Lab in Steen et al. (2010)

Xylanase Depolymerize Xylan

Bacteroides ovatus

Whitehead et al. (1990)

Used by Keasling Lab in Steen et al. (2010)

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Acknowledgements !  Co-investigators: Megan Marshall, Mike Speer,

Haiyu Ren, Qin Chen, Irene Darku, Farzaneh Rezaei, Deepti Tanjore, Allison Ray, Richard Hess, Ken Moore, Kevin Shinners, and Richard Muck

!  Lab and field assistance: Matt Myers, Jessica Schwartz, Rosa Jarvis, Jimmy Truong

!  Idaho National Lab - OBP !  Morgan Family Foundation !  USDA-DOE Biomass Research and Development

Initiative 38