Biological approach to breakdown of complex

polysaccharides

Igor Polikarpov

IFSC/USP

_______________________________________________ Igor Polikarpov , (e): ipolikarpov@ifsc.usp.br Instituto de Física de São Carlos

UNIVERSIDADE

DE SÃO PAULO

Workshop conjunto do Centro Paulista

de Pesquisa em Bioenergia e

universidades de Nottingham e

Birmingham (FAPESP, 14 de maio 2012)

UK

France

Belgium Holland

Norway Sweden

Cellulose and glucose Hemicellulose and Pentose

sugars

Lignins

Marcos Buckeridge & Wanderley dos Santos

Exo-

glucanases

Endo-

glucanases

Beta-

glucosidases

Expansins GH61

Xylanases Xylosidases

Capillary isoelectric focusing (CIEF)

Hui, J.P.M., Lanthier, P., White, T.C., et al., Journal of Chromatography B, 752 (2001) 349–368

Biomass recalcitrance: Lignin and crystallinity.

Biomass is recalcitrant, but can be transformed into hexoses and pentoses in technological process that evolves pre-treatment, enzymatic hydrolysis, fermentation and distillation.

It is difficult, if not impossible, to develop customized enzymatic cocktails without profound comprehension of structure-functional relationships that govern glycoside hydrolysis and the enzymes interaction with the substrates (i.e. pre-processed biomass) and to engineer microorganisms capable of expressing the enzymes, inexpensively and in bulk quantities.

All these major technological steps should be developed in an integrated manner, optimizing the process as a whole.

KEY POINTS

• Pretreatment of Biomass

Efficiency of enzymatic hydrolysis of alkaline pretreated cellulignin increases with severity of pre-treatment

-3 0 3 6 9 12 15 18 21 24

-5

0

5

10

15

20

25

30

35

40

45

Glu

cose

(g

/L)

Time (h)

0.10

0.25

0.50

1.00

2.00

4.00

Bagasse

Cellulignin

Maeda, Serpa, et al. (2011) Proc. Biochem. 46:1196 - 1201

NaOH

concentration,

%

EF

FIC

IEN

CY

OF

PR

ET

RE

AT

ED

SU

GA

R

CA

NE

BA

GA

SS

E H

YD

RO

LY

SIS

Composition of bagasse samples after pretreatment steps

Rezende, et al., Biotechnology for Biofuels (2011) 4:54

CPMAS-TOSS NMR spectra of

sugarcane bagasse: (a) untreated; (b)

bagasse treated with H2SO4 1.0% and

(c) bagasse treated with acid and NaOH

4.0%. The spectra were normalized by

the intensity of line 10 (C1 carbon of

cellulose).

ssNMR

H L

Solid vs Hydrolisate fractions

The solid fraction spectra (a) exhibit a

progressive decrease of the lignin lines

with pretreatments using increasing NaOH

concentrations (note particularly the

methoxy carbon at 56.2 ppm on the

highlighted region).

The cellulose signals at 62.5, 64.8, 72.5,

83.5 and 105 ppm (indicated by arrows in b)

are not observed in samples pretreated with

NaOH concentrations below 0.5%, but

these lines clearly show up for higher NaOH

concentrations.

C C

C C

Line Number Chemical Group 13C Chemical Shift

(ppm)

1 CH3 in acetyl groups of hemicelluloses 21.5

2 Aryl methoxyl carbons of lignin 56.2

3 C6 carbon of non-crystalline cellulose, C6 carbon of

hemicelluloses, OCH2 carbons of lignin

62.5

4 C6 carbon of crystalline cellulose 64.8

5 C2,3,5 of cellulose, OCH2 carbons of lignin 72.5

6 C2,3,5 of cellulose and hemicelluloses 74.4

7 C4 carbon of non-crystalline cellulose and hemicelluloses,

OCH2 carbons of lignin

83.5

8 C4 carbon of crystalline cellulose 87.9

9 Shoulder of C1 carbon of hemicelluloses 101.8

10 C1 carbon of cellulose 105.0

11 C2 and C6 aromatic carbons of Syringyl and C5 and C6

aromatic carbons of Guaiacyl in lignin

110-115

12 C2 of aromatic carbons Guaiacyl in lignin 126.6

13 C1 and C4 aromatic carbons of Syringyl (e) 134.5

13 C1 and C4 aromatic carbons of Syringyl (ne) 136.9

14 C3 and C5 aromatic carbons of Syringyl (ne) and C1 and C4

aromatic carbons of Guaiacyl in lignin

148.0

15 C3 and C5 aromatic carbons of Syringyl (e) in lignin 153.5

16 Carboxyl groups of lignin 163.0-180.0

17 Carboxyl groups of hemicelluloses 173.6

ssNMR

Morphology of untreated and acid pre-treated bagasse (SEM)

Untreated

Acid pre-treated

Morphology of acid+alkaline pre-treated bagasse

SEM surface images of the sugarcane bagasse sample treated with alkaline

solutions: (a) NaOH 0.5% with bundles starting to come apart; (b) and (c) NaOH 2%,

(unstructured and unattached bundles); and (d) NaOH 4%, (individual fibers).

0,5%

2%

2%

4%

Rezende, et al., Biotechnology for Biofuels (2011) 4:54

Crystalinity

Combined pre-treatment and hydrolysis yields of sugar cane bagasse

Enzymatic hydrolysis yields of eucalyptus bark

• Enzymatic Hydrolysis

Efficiency of biomass saccharification by commercial and home-made enzymatic cocktails.

M=Multifect

Maeda, Serpa, et al. (2011) Proc. Biochem. 46:1196 - 1201

M+P+T M+T

M+P

M=Multifect

Cellobiose (or lack of beta-glucosidases) might be one of problems

Others

A-gal (T. reesei) Exo-Inul (A. awamori)

TrAsP (T. reesei) Lamin (Rhodothermus

marinus)

Peroxidase

(Roystonea regia)

B-man

(T. reesei)

B-gal (Penicilium sp)

Endo-Inul

(Arthrobacter sp.)

0

10

20

30

40

50

60

70

80

90

100

0

500

1000

1500

2000

2500

3000

0 50 100 150 200 C

on

cen

tração

B (

%)

Ab

s (

mA

u)

Volume (mL)

Native gel electrophoresis of CBHI (6, 3, 1.5 e 1 mg/mL)

CBHI

66 kDa

Topt=50ºC, pHopt=5

Colussi, F., Textor, L.C., et al. J. Microbiol. Biotech. (2011) 21: 808–817

CBHI from Trichoderma harzianum

A

Loop 6

Loop 5

Loop 6

B

Loop 6

Loop 5

Loop 5

C

A384

V216

Y371

I203

A386

T216

Tr_CBHI

Th_CBHI

Ph. crys_CBHI

Catalytic side loops movements

are strongly anticorrelated!

DYNAMIC CROSS-CORRELATION MATRIX & ESSENTIAL DYNAMICS

3D structure of EGIII (Cel12, T. harzianum): A cellulase without CBM

X-ray Structure of Palm Tree Peroxidase

Watanabe, et al. & Polikarpov, I. J. Struct. Biol. (2010) 169: 226-242

Oxidative enzymes

Statistical Coupling Analysis of Peroxidase Superfamily

Bleicher, L., et al., & Polikarpov, I. J. Phys. Chem. B (2011) 115: 7940–7949

Hyperthermostable Rhodothermus marinus β-1,3-glucanase

2 C L 2

H 2 Y K

L a m R

2 5 o C 9 0 o C

2 C L 22 C L 2

H 2 Y KH 2 Y K

L a m RL a m R

2 5 o C 9 0 o C2 5 o C 9 0 o C

Rodothermus

Nocardiopsis

P. chrysosporium Salt bridges within the hydrophobic

environment facilitate water

penetration

(not every salt bridge favors

thermal stability)

Water penetration into the

hydrophobic layer of LamR

is reduced relative to less

thermostable proteins.

New and novel enzymes

Targeted analysis of microbial lignocellulolytic secretomes -

a new approach to enzyme discovery

São Paulo State (Brazil):

- Prof. Igor Polikarpov (PI,

IFSC/USP),

- Dr. Sandro José de Souza

(Ludwig Institute),

- Prof. Eduardo Ribeiro de

Azevedo (IFSC/USP) &

- Prof. Wanius José Garcia da

Silva (UFABC)

UK, University of York

- Prof. Neil Bruce (PI),

- Profs. Simon McQueen-Mason &

- Peter Young (Co-PIs).

Results

The addition of a steam dryer, doubling of the enzyme dosage in enzymatic hydrolysis,

including leaves as raw material in the 2G process, heat integration and the use of more

energy-efficient equipment led to a 37 % reduction in MESP-2G compared to the Base

case. Modelling showed that the MESP for 2G ethanol was 0.97 US$/L, while in the

future it could be reduced to 0.78 US$/L. In this case the overall production cost of 1G +

2G ethanol would be about 0.40 US$/L with an output of 102 L/ton dry sugar cane

including 50 % leaves. Sensitivity analysis of the future scenario showed that a 50 %

decrease in the cost of enzymes, electricity or leaves would lower the MESP-2G by about

20%, 10% and 4.5%, respectively.

Conclusions

According to the simulations, the production of 2G bioethanol from sugar cane bagasse

and leaves in Brazil is already competitive (without subsidies) with 1G starch-based

bioethanol production in Europe. Moreover 2G bioethanol could be produced at a lower

cost if subsidies were used to compensate for the opportunity cost from the sale of

excess electricity and if the cost of enzymes continues to fall.

Techno-economic evaluation of 2nd generation bioethanol production from sugar

cane bagasse and leaves integrated with the sugar-based ethanol process

Stefano Macrelli1*, Johan Mogensen2 and Guido Zacchi1

Biotechnology for Biofuels 2012, 5:22 doi:10.1186/1754-6834-5-22

Brazilian E2G is already cost-effective

Acknowledgements

Thematic project & CeProBIO

Prof. Munir Skaf (UNICAMP)

CeProBIO team

Prof. Marcos Buckeridge (CTBE& IB/USP)

Prof. Paulo Seleghim Jr. (EESC/USP)

Profa. Anete P. de Souza (CBMEG/UNICAMP)

Prof. A. Augusto F. Garcia (ESALQ/USP)

Profa. Glaucia M. de Souza (IQ/USP)

Prof. Carlos Labate (ESALQ/USP)

Prof. Marcelo E. Loureiro (UFV)

Dr. Itamar Soares de Melo (EMBRAPA)

Dr. Jose Geraldo Pradella (CTBE)

Prof. Luiz Antonio Martinelli (CENA/USP)

Prof. Armando Augusto H. Vieira (UFSCar)

&

all the SUNLIBB collaborators

FAPESP-BBSRC project team

Dr. Sandro J. Souza (Ludwig Institute),

Prof. Eduardo R. Azevedo (IFSC/USP)

Prof. Wanius J. Garcia Silva (UFABC)

&

Our collaborators from the U. of York

Thank you !

Chemical and morphological characterization of sugarcane bagasse submitted to delignification process for enhanced enzymatic digestibility

Bagasse samples

Bagasse composition/%

Cellulose Hemicellulose Lignin Ash Total

Untreated 35.2 ± 0.9 24.5 ± 0.6 22.2 ± 0.1 20.9 ± 4.3 102.8 ± 2.6

H2SO4 1% 51.2 ± 0.2 7.8 ± 0.7 29.5 ± 0.6 12.2 ± 1.5 100.7 ± 1.5

NaOH 0.25% 66.0 ± 0.5 5.2 ± 0.1 25.2 ± 0.3 3.3 ± 0.1 99.7 ± 0.9

NaOH 0.5% 68.0 ± 1.3 3.3 ± 0.1 23.1 ± 6.7 4.3 ± 5.9 98.8 ± 0.5

NaOH 1% 81.6 ± 0.6 3.1 ± 0.1 11.0 ± 0.9 1.9 ± 0.3 97.7 ± 1.1

NaOH 2% 84.7 ± 0.3 3.3 ± 0.1 9.5 ± 0.5 1.1 ± 0.4 98.8 ± 1.1

NaOH 3% 85.3 ± 0.1 3.2 ± 0.1 9.5 ± 0.5 2.3 ± 0.2 100.1 ± 0.4

NaOH 4% 83.4 ± 3.8 3.2 ± 0.1 9.3 ± 0.4 1.8 ± 0.4 97.7 ± 4.6

Chemical composition of the untreated bagasse sample and samples submitted to

acid and alkali pretreatments, as determined by HPLC measurements.

A384

Y260

Y371

V216

I203

Y247

A B

Loop 5

Loop 6

COLAPSE OF THE ACTIVE SITE FOR P. CHRYSOSPORIUM LAMINARINASE,

WHILE IT IS PRESERVED IN LAMINARINASE RH. MARINUS SIMULATIONS AT

HIGHER TEMPERATURE

Collapsed active site

Solvent-accessible active site

Rhodothermus

P. chrysosporium

Topology of the salt bridges

Bleicher, L., et al., & Polikarpov, I. J. Phys. Chem. B (2011) 115: 7940–7949

Some of our glycosyl hydrolases structural studies

Aparicio, I. et al. & Polikarpov, I. (2002) Biochemistry 41: 9370-9375.; Rojas, A.L., Nagem, R.A.P. et al., & Polikarpov, I. (2004) J. Mol. Biol. 343: 1281-1292; Golubev, A.M. et al., and Polikarpov, I. (2004) J. Mol. Biol. 339: 413-422; Nagem, R.A.P. et al. & Polikarpov, I. (2004) J. Mol. Biol., 344: 471-480; Rojas, A.L. et al. & Polikarpov, I. (2005) Biochemistry 44: 15578-15584; Watanabe, L., et al, & Polikarpov, I. (2007) Acta Cryst. F63: 780-783; Kim, K.-Y., Nascimento, A.S. et al. & Polikarpov, I. (2008) BBRC 371: 600-605; Golubev A.M., et al., & Polikarpov, I. (2008) Prot. Pept. Lett. 15:1142-1144; Nascimento, A.S., et al. & Polikarpov, I. (2008) J. Mol. Biol. 382:763-778; Zamorano, L.S., et. al., Polikarpov, I. and Shnyrov, V.L. (2008) Biochimie 90: 1737-1749; Watanabe, et al. & Polikarpov, I. J. Struct. Biol. (2010) 169: 226-242.

Pre-treatment and hydrolysis yields

Bagasse samples

Enzymatic hydrolysis (48 h)

Total released

glucose/

g/g substrate

Partial hydrolysis

yield/ %

Pretreatment

yield/ %

Total hydrolysis

yield/ %

Untreated 0.085 ± 0.001 22.0 ± 0.3 100.0 ± 5.1 22.0 ± 1.4

H2SO4 1% 0.170 ± 0.001 30.3 ± 0.3 93.5 ± 2.7 28.3 ± 8.4

NaOH 0.25% 0.342 ± 0.006 47.1 ± 0.9 90.5 ± 3.2 42.6 ± 2.3

NaOH 0.5% 0.591 ± 0.019 79.0 ± 2.5 84.5 ± 3.7 66.2 ± 5.2

NaOH 1% 0.896 ± 0.066 99.8 ± 7.4 72.5 ± 2.8 72.3 ± 8.1

NaOH 2% 0.903 ± 0.018 96.9 ± 1.9 68.3 ± 2.0 66.2 ± 3.3

NaOH 3% 0.896 ± 0.098 95.5 ± 10.5 70.7 ± 1.8 67.5 ± 9.1

NaOH 4% 0.940 ± 0.182 97.4 ± 19.9 65.9 ± 4.7 67.5 ± 10.6