Cellulosic ethanol: facing the challenges

October 2014

British Society of Sugar Technologists

Leonardo Gomez

Center for Novel Agricultural Products

University of York

Why cellulosic biofuels?

• Environmental concerns about the impact

of human greenhouse gases on climate

change

• Dwindling petroleum reserves

• Security of oil supply

DRIVERS FOR THE EXPANSION OF BIOFUELS

(2004)

Shale gas

• Environmental concerns about the impact

of human greenhouse gases on climate

change

DRIVERS FOR THE EXPANSION OF BIOFUELS

(present)

• Biofuels should avoid competing with

food production

• Biofuels should be sustainable

Liquid biofuels

First generation = produced from food crops such as oils and starch

(corn ethanol, biodiesel)

Second generation = made from lignocellulosic

plant biomass

Third generation biofuels = designer fuels made from engineered microorganisms

Advanced

Biofuels

Different procedures to produce liquid biofuels from biomass

EU Renewable Energy Directive (RED, 2009) - 10% of renewable transport

fuels by 2020

Food crop derived biofuels limited to 7% (July 2014)

Lignocellulosic Biomass = Cell wall

CELLULOSE

40%

MATRIX

20%

LIGNIN

25%

ASHES

5%

Cell walls have evolved to develop recalcitrance

Cellulose

molecules

Cellulose

microfibrils

Hemicelluloses

Plasma

membrane

Lignins

Glucose

Cell walls have evolved to develop recalcitrance

- Cellulose is a B-1-4 linked glucan that is grouped

in a crystalline status in microfilbrils which are very

difficult to hydrolise to release glucose

- Matrix polysaccharides are complex in

composition and structure. They are largely

composed by pentoses.

- Lignins are polyphenolics of quasi-random structure

that creates a hydrophobic coating for the

polysaccharides

Size

reduction Pretreatment

Feedstock

Quality

Hydrolysis

Fermentation

Enzyme production

Co

nso

lida

ted

bio

pro

ce

ssin

g

SS

F

Bio

log

ica

l s

tep

s

Product

recovery

Residue

processing

Heat and

electricity

General biomass processing for cellulosic biofuels

The success of cellulosic biofuels depends on their cost-competitiveness and sustainability

Cellu

losic

Eth

an

ol price (

cents

/gal)

Enzyme Feedstock Conversion

0

100

200

300

400

500

600

700

2001

2002

2003

2004

2005

2006

2007

2008

2009

2010

20

11

2012

Commercial Cellulosic Ethanol Plants

- GranBio (Alagoas, Brazil) Operational since

October 2014. 82 million litres per year.

Investment $208M. Sugar cane bagasse.

- POET-DSM (Iowa, US) Operational since September

2014. 95 million litres per year.

Investment $250M. Corn stover.

- Beta Renewables (Crescentino, Italy)

Operational since July 2013. 76 million

litres per year. Investment $190M.

Develop novel pretreatments

Identify the molecular basis of recalcitrance to

saccharification

Discover new enzymes for more efficient hydrolysis

and tailored fractionation

Improving biomass for industrial applications

HTBP

Species involved: Arabidopsis, Brachypodium, Poplar, Barley, Maize, Tobacco, Sugar cane, Sorghum, Willow,

Rice, Wheat, etc.

Biomass Analytical Facility:

Developing tools for tailoring

biomass for industrial processes

Developing novel chemical

pretreatments

Biomass pretreatment can be

considered biomass fractionation to

add value into different fractions

LIGNOCELLULOSIC MATERIAL

Supercritical extraction

Microwave assisted Thermochemical

pretreatment

Fermentation

Anaerobic Digestion

Pyrolysis

-400

-300

-200

-100

0

100

200

300

400

-400 -300 -200 -100 0 100 200 300 400 500

t[2

]

t[1]

Scores Comp[1] vs. Comp[2] colored by File Text

123

1_180

1_180

1_180

1_20

1_201_20

2_180A2_180A2_180A2_180B

2_180B

2_180B

2_180C

2_180C2_180C

2_20A2_20A

2_20A

2_20B2_20B2_20B

2_20C

2_20C2_20C

3_180A3_180A

3_180A

3_180B3_180B3_180B

3_180C

3_180C

3_180C

3_20A3_20A3_20A3_20B3_20B3_20B3_20C3_20C3_20C

EZinf o 2 - bagasse3 (M2: PCA-X) - 2012-08-30 18:17:00 (UTC+1)

H2SO4

NaOH

WATER

Sugar cane bagasse

Biomass derivatives in the pretreatment liquor

Gomez et al. Bioenergy Research 2014

Developing novel chemical pretreatments

Control

Maize

Antifoam Test at Ecover

Supercritical CO2 Extraction and

Fractionation of Maize wax

Andy Hunt-Tom Attard-

BDC

Identify the molecular basis of recalcitrance to saccharification

Fermentation

Chemical

catalysis

Synthetic biology

Consolidated

bioprocessing

Alc

oh

ols

/Hyd

rocarb

on

s

The process of breaking a complex carbohydrate

into its monosaccharide components

High Throughput Saccharification Analysis:

“Hypothesis free” approaches to understand the main determinants

of the saccharification potential

- Screening of mutagenised populations Brachypodium

Mutagen M1

M2 M3

Saccharification analysis of M2 plants

Characterisation and mapping of mutants with

altered saccharification

Forward genetic screening of Brachypodium for variability in saccharification

0

20

40

60

80

100

120

140

160

180

200

40

45

50

55

60

65

70

75

80

85

90

95

10

0

10

5

11

0

11

5

12

0

12

5

13

0

13

5

14

0

14

5

15

0

15

5

16

0

16

5

17

0

Mo

re

No

. of

mu

tan

t p

lan

ts

Saccharification Potential (nmoles reducing sugars hour-1 mg DW-1)

WT mean overall mean

Distribution of saccharification potential

Highest saccharification

Lowest saccharification

INRA

+72 %

-51 %

USDA

+74 %

-46 %

John Innes Centre

+65 %

-33 %

% = % higher/lower than WT for plate

Characterising the sac1 mutant

Allele frequency plot for the five chromosomes of sac1

Association genetics of saccharification in barley populations

BSBEC Cell Wall Lignin Programme

Association Genetics experiment

• 640 elite 2-row spring barley genotypes

• Grown in polytunnel; 5 reps, watered, collared

• Various phenotypes measured (including straw biomass)

• Samples collected and SNP genotyped

• 3240 samples of 2nd internode base collected;

powdered

• Evaluated for saccharification at York

0

1

2

3

4

5

6

-lo

g10 (

p-v

alu

e)

Saccharification

1H 2H 3H 4H 5H 6H 7H

GWAS Saccharification

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

Improving Enzymes

3 mm

Enzyme discovery in

Limnoria

Teredinid bivalves (shipworm)

Isopod crustacean Limnoria (gribble)

Lignocellulosic biomass is an important food source

for marine organisms

Termite guts are microbial bioreactors

Image: John Breznak, Michigan State University

Image: Genome Management Information System, Oak Ridge National

Laboratory

Limnoria guts are sterile

DNA pyrosequencing of

hepatopancreas cDNA libraries

• Total of 106 million bp of sequence

• 418,749 DNA sequences

• Average length of 247 base pairs

• 12,306 contiguous sequences

• 4,336 had close sequence similarity to known

genes

Exploring lignocellulose degradation in

Limnoria

Glycosyl hydrolases dominate EST representation

Singletons

18%

Proteases

2%

Others

31%

Fatty acid

binding

protein

1% Lipase

0.2%

Glycosyl Hydrolases

27%

Haemocyanin

17%

Ferritin

1%

Oxygenase

2%

GH7

53.2%

GH9

37.0%

GH5

3.9%

GH38

0.1%

GH2

0.1%

GH16

0.4%

GH13

0.5%

GH35

2.7%

GH31

0.2%

GH20

0.1%

GH30

1.5%

GH18

0.3%

GH7 family - cellobiohydrolases

• GH7 family enzymes found only

in fungi and protists

• Limnoria GH7 has 41% identity

to the closest relative

• Three distinct genes that

account for 14.4 % of all transcripts

0.1

USP - Md

P. grassii

USP - Cp 2

USP - Hs 2

USP - Rs 6

L. quadripunctata GH7C

L. edodes

C. rolfsii

Pleurotus sp.

V. volvacea I - I

P. arcularius

S. commune

H. jecorina

A. nidulans A. terreus

A. clavatus

A. fumigatus N. fischeri

T. aurantiacus A. niger

C. thermophilum

G. zeae

L. quadripunctata GH7A

L. quadripunctata GH7B

USP - Rs 5 USP - Rs 2 USP - Rs 3

USP - Rs 4 USP - Rs 1

USP - Hs 1

USP - Hs 3

PROTISTS

FUNGI

V. volvacea I - II

USP - Cp 1 USP - Cp 3

100

87

97

64 43

0.1

USP - Md

P. grassii

USP - Cp 2

USP - Hs 2

USP - Rs 6

L. quadripunctata GH7C

L. edodes

C. rolfsii

Pleurotus sp.

V. volvacea I - I

P. arcularius

S. commune

H. jecorina

A. nidulans A. terreus

A. clavatus

A. fumigatus N. fischeri

T. aurantiacus A. niger

C. thermophilum

G. zeae

L. quadripunctata GH7A

L. quadripunctata GH7B

USP - Rs 5 USP - Rs 2 USP - Rs 3

USP - Rs 4 USP - Rs 1

USP - Hs 1

USP - Hs 3

PROTISTS

FUNGI

V. volvacea I - II

USP - Cp 1 USP - Cp 3

100

87

97

64 43

0.1

USP - Md

P. grassii

USP - Cp 2

USP - Hs 2

USP - Rs 6

L. quadripunctata GH7C

L. edodes

C. rolfsii

Pleurotus sp.

V. volvacea I - I

P. arcularius

S. commune

H. jecorina

A. nidulans A. terreus

A. clavatus

A. fumigatus N. fischeri

T. aurantiacus A. niger

C. thermophilum

G. zeae

L. quadripunctata GH7A

L. quadripunctata GH7B

USP - Rs 5 USP - Rs 2 USP - Rs 3

USP - Rs 4 USP - Rs 1

USP - Hs 1

USP - Hs 3

PROTISTS

FUNGI

V. volvacea I - II

USP - Cp 1 USP - Cp 3

100

87

97

64 43

0.1

USP - Md

P. grassii

USP - Cp 2

USP - Hs 2

USP - Rs 6

L. quadripunctata GH7C

L. edodes

C. rolfsii

Pleurotus sp.

V. volvacea I - I

P. arcularius

S. commune

H. jecorina

A. nidulans A. terreus

A. clavatus

A. fumigatus N. fischeri

T. aurantiacus A. niger

C. thermophilum

G. zeae

L. quadripunctata GH7A

L. quadripunctata GH7B

USP - Rs 5 USP - Rs 2 USP - Rs 3

USP - Rs 4 USP - Rs 1

USP - Hs 1

USP - Hs 3

PROTISTS

FUNGI

V. volvacea I - II

USP - Cp 1 USP - Cp 3

100

87

97

64 43

Acknowledgements

University of York

Simon McQueen Mason

Caragh Whitehead

Poppy Marriott

Rachael Simister

Susannah Bird

Marcelo Kern

Katrin Besser

Andrew Hunt

Tom Attard

SCRI-Dundee

Claire Halpin

Robby Waugh

Reza Shafiei

VIB-Gent

Wout Boerjan

Ruben Vanholme

INRA-Versailles

Richard Sibout

Herman Hofte