The Lignocellulosic Biomass Digestibility

Challenge

Simon McQueen-Mason, Centre for Novel Agricultural products, University of York, UK

Mary Rose after 500 years of AD

Lignocellulosic plant biomass

• Strong fibre

composite materials

• Durable

• Rich in

polysaccharides

• Hard to digest

Cellulose

Hemicellulose

Lignin

BIOLOGY TO BENEFIT SOCIETY

Hemicellulose

Cellulose

Lignin

Courtesy,

Watanabe

and Tokuda

Cellulose hydrolysis is very slow

because of its crystalline nature

Lytic Polysaccharide Monooxygenases

potentiate cellulose hydrolysis by oxidative

attack of surface glucans

Courtesy

Greg

Beckham,

NREL

Requires

Oxygen!

Lignin is typically comprised of three monolignols;

ρ-coumaryl, coniferyl and sinapyl alcohols

Dicot lignin contains

mostly coniferyl and

sinapyl units

Monocots have a

lot more coumaryl

units- roughly

equal quantities

of all 3

monolignols

Conifers have

coniferyl and

coumaryl units

only

• Sinapyl

referred to as

S-lignin

• Coniferyl as

G-lignin

• Coumaryl as

H- lignin

Lignin

Monolignols polymerised

in wall by free radical

chemistry to form

complex polymers with

no repeat structure

Thus, no specific

enzymes have evolved to

digest this material

Lignin attack

accomplished using

peroxidases and laccases

that NEED OXYGEN!

The biomass digestibility

challenge

Two approaches to improve digestibility:

• Make the cell walls more digestible without

compromising field performance

• Produce more effective enzymes and

pretreatments for conversion

Fuel

Second Generation,

Sustainable, Bacterial BiofuelsNigel Minton (Nottingham)

• Newcastle University

• TMO Renewables Ltd

ENVIRONMENTAL, SOCIAL, ECONOMIC SUSTAINABILITY

Perennial

Bioenergy

Crops

(BSBEC-

BioMASS)Angela Karp

(Rothamsted)• IBERS

• Imperial College

• University of

Cambridge

• Ceres Inc

Cell Wall SugarsPaul Dupree

(Cambridge)

• Newcastle University

• Novozymes A/G

Cell Wall LigninClaire Halpin

(Dundee)

• University of York

• SCRI

• RERAD

• Limagrain UK Ltd

• Syngenta

• AgroParisTec – INRA

Marine

Wood Borer

Enzyme DiscoverySimon McQueen-

Mason

(York)

• University of Portsmouth

• Novozymes

Lignocellulosic Conversion to

BioethanolKatherine Smart (Nottingham)

• University of Bath • University of Surrey

• BP • Bioethanol Ltd

• Briggs of Burton • British Sugar Ltd

• Coors Brewers Ltd • DSM

• Ethanol Technology Ltd • HGCA

• Pursuit Dynamics • SABMiller

• Scottish Whisky Research Institute

Tackling major barriers of sustainable biofuel production

Six integrated programmes; 20 million investment

Automated to

measure cell

wall

digestibility

On the left,

automated

sample

grinding and

weighing, on

the right,

automated

digestion

assay

Screening for mutations that improve

digestibility

Screen plants for

digestibility

Identify plants

with high

digestibility

Use linkage

disequilibrium

in segregating

generations to

map the

mutation

Large population of

randomly mutagenised

plants

Brachypodium as a model grass

• Small genome (300 Mbp)

• Fully sequenced

• Genetic tool available

• Diploid

• Small and easy to grow

• Self fertile

• Short life cycle

Reducing sugars (nmoles hour-1

mg DW-1

)

40 60 80 100 120 140 160

Num

ber

of

pla

nts

0

100

200

300

400

Highest saccharification

Lowest saccharification

+72 % -51 %

37 high

digestibility

mutants selected

Distribution of saccharification potential

Heritable increases in

digestibility

100

110

120

130

140

150

160

170

sac1 sac2 sac3 sac4 sac5 sac6 sac7 sac8 sac9 sac10 sac11 sac12

% s

acch

ari

ficati

on

o

f W

T

12 mutant lines

out of

3000 had a

heritable

increase in

digestibility

0

50

100

150

200

250

300

350

WT sac1 sac2 sac3 sac4 sac5 sac6 sac7 sac8 sac9 sac10 sac11 sac12

Lig

nin

co

nte

nt

(mg

g-1

)

**

** *

*

Lignin

Acetyl bromide method

0 arrows: 0-5% increase/decrease

1 arrow: 5-10% increase/decrease

2 arrows: 10-20% increase/decrease

3 arrows: 20-30% increase/decrease

4 arrows: 30-40% increase/decrease

PNAS 2014 111(40):14601-6

Association genetics in barley- with Robbie Waugh

and Claire Halpin, University of Dundee

• High density single nucleotide polymorphism (SNP)

markers defined in 850 elite barley cultivars from

around the world

• Phenotypic screening of these cultivars can then be

used with SNP association mapping to define

quantitative trait loci QTL at high resolution

• Quantitative traits (such as digestibility) are

determined by many genes, QTL analysis allows the

identification of major genetic contributors to a trait

Ranked barley genotypes – Model

Su

ga

r re

lea

sed

nm

ol/

mg

DM

Ranked barley genotypes – Raw data

Su

ga

r re

lea

sed

nm

ol/

mg

DM

A B

Ranked barley genotypes – Model

Su

ga

r re

lea

sed

nm

ol/

mg

DM

Ranked barley genotypes – Raw data

Su

ga

r re

lea

sed

nm

ol/

mg

DM

A B

640 genotypes of elite spring barley ranked by yield of

sugar released in a simple saccharification assay.

Datapoints are means of 5 replicate plants per genotype

(incorporating 3 technical assay replicates per plant)

2H 3H 7H5H4H3H 6H 7H5H2H 3H 7H5H4H3H 6H 7H5H

GWAS identifies several QTL for digestibility in

barley straw

So, what happened to the other half?

Wood borer mid term

Enzymes and mechanisms of wood digestion

in Limnoria quadripunctata

Simon McQueen-MasonNeil BruceSimon Cragg

Marine Wood Borer Enzyme Programme

Singletons

(not annotated)

18.3%

Proteases

2.7%

GH Family

proteins

27.0%

Other sequences

29.7%

Leucine-rich

repeat proteins

2.7%

Hemocyanins

17.3%

Ferritins

1.1%

Fatty acid binding

protein

1.3% GH7

53.3%

GH9

37.0%

GH30

1.5%

GH5

3.9%

Others

1.6%GH35

2.7%

A

B C

D ESingletons

(not annotated)

18.3%

Proteases

2.7%

GH Family

proteins

27.0%

Other sequences

29.7%

Leucine-rich

repeat proteins

2.7%

Hemocyanins

17.3%

Ferritins

1.1%

Fatty acid binding

protein

1.3% GH7

53.3%

GH9

37.0%

GH30

1.5%

GH5

3.9%

Others

1.6%GH35

2.7%Singletons

(not annotated)

18.3%

Singletons

(not annotated)

18.3%

Proteases

2.7%

Proteases

2.7%

GH Family

proteins

27.0%

GH Family

proteins

27.0%

Other sequences

29.7%

Other sequences

29.7%

Leucine-rich

repeat proteins

2.7%

Leucine-rich

repeat proteins

2.7%

Hemocyanins

17.3%

Hemocyanins

17.3%

Ferritins

1.1%

Ferritins

1.1%

Fatty acid binding

protein

1.3%

Fatty acid binding

protein

1.3% GH7

53.3%

GH7

53.3%

GH9

37.0%

GH9

37.0%

GH30

1.5%

GH30

1.5%

GH5

3.9%

GH5

3.9%

Others

1.6%

Others

1.6%GH35

2.7%

GH35

2.7%

A

B C

D E

Limnoria

quadripunctata is a

marine isopod that

lives on a diet of

wood, and has a

digestive tract devoid

of microbial life.

The Limnoriid gut is

effectively an enzyme

reactor for

lignocellulose

mobilisation

GH20

0.1%

GH31

0.2%

GH13

0.5%

GH2

0.1%GH38

0.0%

GH30

1.5%GH16

0.4%GH18

0.3%

GH35

2.7%

GH5

3.9%

GH9

37.0%

GH7

53.3%

King et al. 2010 Proc. Natl. Acad. Sci. U S A.

107: 5345-5350

Singletons

18.3%

GH Family

27.0%

Other

30.1%

Ferretin

1.1%

Fatty acid

binding

protein

1.3%

O.

gracilorostris

oxygenase

homologue

2.1%

Haemocyanin

17.3%Protease

2.7%Lipase

0.2%

Marine Wood Borer Enzyme Programme : Discovery

We used 454 DNA sequencing to analyse ~360,000 ESTs from a

cDNA library made from the hepatopancreas of L. quadripuctata

LqCel7B, a salt-stable cellobiohydrolase

haemocyanin

cellulase

Cellulose microfibril

Cellobiose orCellusose oligosaccharides

Matrix oligosaccharides

lignin

Reactive oxygen species

A two-chamber bioreactor for wood digestion:1. Wood digestion potentiated by free radical attack that opens up substrates for cellulases, and is contained in a cuticle-lined reactor2. Sensitive living processes carried out in hepatopancreas away from free radical generation

LBNet is an active community of industrial practitioners and leading

academics dedicated to creating economic value by developing novel

chemical, material and fuel processes using lignocellulosic biomass

instead of petroleum-derived inputs

LBNet provides both access to a range of funding to kick-start

innovation in the sector and organises events

Acknowledgements

York:Leonardo GomezKatrin BesserLuisa EliasMarcelo KernPoppy MarriottCaragh WhiteheadRachel SimisterLaura FaasClare steele-KingWill EborallShaza Mohamad

Neil BrucePaul Walton

Portsmouth:Simon CraggGraham MalyonSimon StreeterAmaia ExtabeJohn McGeehan

NREL:Gregg BeckhamKirsty PayneMike Himmel

Dundee:Claire HalpinRobbie WaughReza ShafeiHelena Oakey

VIB:Wout BoerjanRueben

NovozymesKirk Schnorr

USPIgor PolikarpovFrancisco Gumeraes