New opportunities to develop bio-based products related to

2nd generation ethanol production

Workshop sustainable production of biopolymers and other bio-based products 26/07/2012

(email: jpradella@bioetanol.org.br) 1

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Some history: the sugarcane

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1532: Martim Afonso de Souza introduced sugarcane and built the country's first mill, in the coastal town of São Vicente, in São Paulo

State

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5

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1931: The Federal Government issued Law No. 19717 – mandatory purchase of ethanol by gasoline importers; mix

up to 5% in gasoline

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0

20

40

60

80

100

120

0

3000

6000

9000

12000

15000

18000

21000

24000

27000

Oil

Pri

ce

/Ba

rre

l W

TIU

S$

Eth

an

ol P

rod

uc

tio

n1

06

Lit

res

Ethanol Production_Brazil Oil Price/Barrel

Source: Produced based on P/EIA & UNICA (2009)

The international oil price crisis and ethanol production in Brazil

1st oil

crisis

2nd oil

crisis

ProAlcool

establishment

“Instability”

crisis

Flexfuel

cars

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Sugarcane main characteristics

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Sugar and Ethanol Mills location

N-NE C-S

Sugarcane 15% 85%

Ethanol 10% 90%

Source: IBGE and Conab (2006)

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Mechanized sugarcane harvesting (100% up to 2014)

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The sugarcane plant

BASE: 01 year (medium values)

Fresh biomass : 80 t/ha Bagasse + straw: 20 t/ha 1 ton of fresh sugarcane Straw: 140 kg Fiber: 140 kg Sucrose: 150 kg H2O+salts: 570 kg

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Catalytic conversion of biomass to biofuels - D.M. Alonso, J. Q. Bond and J. A. Dumesic (GREEN CHEMISTRY, 2010)

Biomass composition

Componentes Composição (%)

Celulose 43.4

Hemiceluloses 25.6

Lignin 23.2

Ash 2.9

Extratives 4.8 Rocha, G. J. M. et al, 2010 *Average from more

than 50 analysis

Sugarcane bagasse

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Fiber (60% w/w) Pith (40% w/w)

(Olivares, 2009) 15

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Bagasse MEV Fiber Pith

(Borges, Squina, Pradella, 2009) 16

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AFM images of fiber fraction

(Courtesy of Lucia Vieira Santos, UNIVAP, 2012)

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Catalytic conversion of biomass to biofuels - D.M. Alonso, J. Q. Bond and J. A. Dumesic (GREEN CHEMISTRY, 2010)

Biomass composition

Componentes Composição (%)

Celulose 43.4

Hemiceluloses 25.6

Lignin 23.2

Ash 2.9

Extratives 4.8 Rocha, G. J. M. et al, 2010 *Average from more

than 50 analysis

Sugarcane bagasse

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Hemicellulose fraction

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Block flow diagram - Integrated 1st and 2nd generation bioethanol production from sugarcane

Pentose liquour

• Xylo-oligomers

• Xylose

• Glucose

• Furfural

• Hydroxymethylfurfural

• Acetic acid

• Phenols

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The C5 Biorefinery

• High amount of C5 carbohydrates

• (>10.000.000 t/year)

• C5 stream valorization

• Use of low emission technology

•

•

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Biological platforms • Escherichia coli

• Corynebacterium

• Clostridium

• Lactobacillus

• Aspergillus

• rSaccharomyces cerevisiae

• Candida, Rhodotorula, Yarrovia, Pichia

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• Biofuels

• Ethanol

• Butanol

• Lipids

• Isoprenoids

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Potential products from C5 Biorefinery

Potential products from C5 Biorefinery

• Organic acids

• Acetic acid

• Propionic acid

• Lactic acid

• Butyric acid

• 1-4 diacids: Succinic; Fumaric; Malic acid

• Adipic acid

• Terephtalic acid ??

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• Alcohol, ketone and aldehyde

• Isopropanol

• Acetone

• Propanol

• 1-3 propanediol, 1-4 butanediol

• Butanol

• Acetic aldehyde

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Potential products from C5 Biorefinery

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AS: ácido succínico

Some ideas

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Xylose consumption

29 (Matsushika, 2009)

Chemical catalysis

Enzimatic catalysis

“ex cellula”

30 E coli (semi) synthetic anaerobic pathway (Clomburg, 2010)

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Fig. 6. Conversion of glucose/glycerol to 1,3-propanediol by the expression of glycerol-3-phosphatase in organisms already

transformed with dhaB and dhaT. DHAP=DihydroxyAcetone Phosphate, G-3-P=Glycerol 3-phosphate, GA-3-Pglyceral=dehyde

3-phosphate, 3HPA=hydroxypropionaldehyde, 1,3-propanediol,

GPP1/2=glycerol-3-phosphatease

dha B=glycerol dehydratase,

dhaT=1,3-propanediol oxidoreductase. ( adapted from Saxena, 2009)

32 E coli (semi) synthetic aerobic pathway (Clomburg, 2010)

33 (Pradella, 1980)

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E coli synthetic pathway to adipic acid (cited in Pradella, 2008)

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Some economics

A case study: ethanol from xylose

Xylose fermentation kinetics

Tao et al., (2001) M. Sedlak, et al. (2003) Silva et al. (2009)

rE coli P. stipitis YNRRL Y-7124 rS. cerevisiae

Prod = 1 g/Lh

μp = 0.30 h-1

Yp/s = 0.5 g/g

Prod = 0.3 g/Lh

μp = 0.02 h-1

Yp/s = 0.32 g/g

Prod = 1.5 g/Lh

μp = 0.15 h-1

Yp/s = 0.45 g/g

maxμμ = ISK

S

S max

- 1P

Pn

P: inhibition by product of interest I: inhibition by substance present culture media S: carbon source

Inhibition kinetics

P-2 / PM-101

Fluid Flow

P-3 / HX-101

Heating

P-5 / HX-102

CoolingS-102

S-103

P-8 / PM-102

Fluid Flow

P-9 / DS-101

Centrifugation

S-109

S-110

P-4 / FR-101

Stoich. Fermentation

P-6 / FR-102

Stoich. Fermentation

P-10 / FR-104

Stoich. Fermentation

S-104

S-105

S-106

S-107

S-108

P-1 / MX-101

Mixing

S-101

S-112 S-113

S-114

S-115 S-116

P-7 / PM-103

Fluid Flow

S-111

S-117

Simulation of Ethanol production from xylose using E. coli KO11

Yp/s = 0,5 g/g; Pmax = 50 g/L; n = 1,0;

μmax = 0,8 h-1;V1=2 *V2=2 *V3; Θ = 14 h;

Sin = 95 g/L

P out= 47,5 g/L

Pp = 14,2 g/L h

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Some final comments

• Seek for xylose consumers

• Reinforcement /redirection/implantation of metabolic pathways to final products

• Pressure, adaptation and selection:

overcoming either C5 liquor and product inhibition

• Bioreactor intensification: increase yield and productivity

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R&D route