BIOPROCESS SYSTEMS ENGINEERING (BSE) APPLIED TO THE PRODUCTION OF BIOETHANOL FROM SUGARCANE

BAGASSE

Coord.: Roberto C. Giordano

roberto@ufscar.br (DEQ/UFSCar)

Proc. 2008/56246-0

WORKSHOP BIOEN – November 2012

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Software Engineering:

Biorefinery simulation, optimization

Enzyme Engineering +

Bioreactor Engineering (“industrial” yeast strains)

Validation: Process

Non-conventional

bioreactors

Where we contributing (for the best of our knowledge) Biorefinery multiobjective optimization in EO simulator Application of immobilized enzymes Production of cellulases (in situ) in air lift reactors Simultaneous isomerization and fermentation (SIF) of C5 3

Subproject 1: Development, implementation and validation of a user-friendly computational environment

Senior researchers (UFSCar):

Antonio J. G. Cruz Caliane B. B. Costa Jose A. S. Gonçalves Luiz F. Moura Roberto C. Giordano Ruy Sousa Jr

Graduate students:

Cassia M. Oliveira (MSc) Fabio H. P. B. Pinto (PhD) Felipe F. Furlan (PhD) Gabriel C. Fonseca (MSc) Herbert A. S. P. M. Jardim (MSc) José I. S. Silva (MSc) Karina Matugi (MSc) Renato Tonon Filho (MSc)

Undergraduate students:

Anderson R. A. Lino Fernando Foramiglio Jorge A. R. Fanton Leonardo C. G. Gonçalves Lucas Passos

Collaboration (EMSO developers):

Argimiro R. Secchi (PEQ-COPPE/UFRJ) Rafael P. Soares (DEQ/UFRGS)

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Simulation platform: EMSO (www.enq.ufrgs.br/alsoc)

Novelty: global simulator, open access models; efficient solver for static & dynamic models; our group has access to the source code.

Research horizon: Optimization: static dynamic (natural for this process) Super-structural optimization, coupling economics,

sustainability, energetic integration, water balance, etc (tool: Pareto + PSO, Particle Swarm Optimization)

Dynamic real time optimization

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Biorefinery flowsheet: an example (autonomous distillery, integrated 1G + 2G, organosolv/ethanol pretreatment; other pretreatments are implemented)

Dimensionless cash flow: burning sugarcane trash (50%); C6 + C5 fermentation

Infeasible region

Dimensionless cash flow: burning sugarcane trash (50%); only C6 fermentation

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Furlan et al. Assessing the production of first and second generation bioethanol from sugarcane through the integration of global optimization and process detailed modeling. Computers & Chemical Engineering, 2012.

Bagasse partition in the biorefinery.

Pareto + PSO in EMSO – an example: Cellulignin hydrolysis (hydrotherm pretreatment) + Vapor demand in evaporator

Pareto + PSO in EMSO – an example: Cellulignin hydrolysis (hydrotherm pretreatment) + Vapor demand in evaporator

Ethanol mol fraction in outflow side stream

Mass balances: (a) ethanol; (b) global.

Distillation: Transients due to variations in the feed tank

Subproject 2: Cultivation of microorganisms for the production of cellulases and xylanases in non-conventional triphase reactors

Senior researchers: UFSCar:

Alberto C. Badino Jr. Teresa C. Zangirolami Paulo W. Tardioli Rosineide G. Silva

EMBRAPA: Cristiane S. Farinas

Graduate students: Fernanda M. Cunha (PhD) Camila Florencio (PhD) Gabriel D. Torresam (MSc) Mateus N. Esperança (MSc)

Undergraduate students: Daniel Botta RafaelRodrigues Esteves

Project Goals: Cellulase production by Aspergillus sp. and Streptomyces sp. in conventional and pneumatic bioreactors Enzymes purification and characterization Methodology: Inoculum development (with/without bagasse) Effect of the mass transfer and shear conditions Chromatographic purification

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Collaboration: Michael Ladisch and Eduardo Ximenes Purdue University, USA

SolSF

SubF 12

13 Cunha et al. Biotechnology and Bioprocess Engineering 17:100-108, 2012.

24h under SSF

Methodology for inoculum cultivation

in a triphase medium established.

Hydrodymanics and

transport parameters in

pneumatic bioreactor in the

presence of sugarcane

bagasse determined.

14 Cunha et al. Bioresource Technology 112:270-274, 2012.

BIN <0,5

BIN 0,5-1

,0

BIN 1,0-2

,0

BEX <0,5

BEX 0,5-1,0

BEX 1,0-2,0

0

200

400

600

800

1000

1200

1400

1600

1800

En

do

glu

ca

nas

e a

cti

vit

y (

IU.L

-1)

Submerged Fermentation

Sequencial Fermentation

Effects of sugar

cane bagasse

particle size and

pretreatment

Almost 2-fold

improvement of

cellulase

biosynthesis

Sub-Project 3: Physical-chemical pretreatment of SCB bagasse: removal of hemicellulose, delignification. Biomass characterization.

Senior Researchers: Antonio J. G. Cruz (UFSCar) Cristiane S. Farinas (EMBRAPA) Raquel L. C. Giordano (UFSCar)

Pos-Doc Fellow: Úrsula F. Rodrigues-Zuñiga

Graduate Students: Gislene M. Silva (PhD) Luciano J. Corrêa (PhD) Renata B. A. Souza (PhD)

Specially acknowledged collaboration: Adilson R. Gonçalves (EEL/USP)

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Collaboration: Celina L. Duarte (IPEN, Brazil)

Claus Felby (University of Copenhagen, Denmark)

Doris Schieder and Martin Faulstich (TUM, Germany)

George J. M. Rocha (CTBE, Brazil)

Mark R. Wilkins (Oklahoma State University, USA)

Renato L. Carneiro (Chemistry Department, UFSCar, Brazil)

Biomass characterization (Methodology)

Biomass pretreatment: bagasse deconstruction

Cellulose loss;

Hemicellulose: oligos or monomers?

Lignin removal: cost-effectiveness

Enhancement of enzymatic hydrolysis

Data for simulations of the biorefinary

(Sub-project 1)

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4000 4500 5000 5500 6000 6500 7000 7500 0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

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Setting the conventional methodology for an accurate compositional analysis

Based in: Laboratory Analytical Procedure: provided by the National Renewable Energy Laboratory (NREL) (Sluiter et al., 2006) with modifications proposed by Gouveia et al. (2009).

Bagasse components In-natura (w/w %) Steam exploded (w/w %)

Cellulose 39,0% 41,8%

Hemicellulose 29,7% 20,7%

Soluble lignin 2,5% 4,3%

Insoluble lignin 19,2% 30,0%

Extractives 5,0% -

Ashes 4,9% 2,2%

Mass balance (total) 100,3% 99,0%

Rapid alternative methodology based in Near Infrared Spectroscopy (NIR) Database generated in the subproject 3 + Chemiometrics management

4000 4500 5000 5500 6000 6500 7000 7500-0.06

-0.04

-0.02

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

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a) BC in natura b) Ammonia

c) Alkaline d) Organosolv

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Biomass pretreatment

Organosolv-ethanol 1, 2, 3

Experimental conditions:

Temperature: 150, 170 and 190 oC.

Reaction time: 10, 30, 60 and 90 minutes.

Degree of severity: 3.2 to 4.7

Agitation speed: 300 rpm.

Ethanol solution: 30, 50 and 70% (w/w)

Solid liquid ratio: 1:10 (w/v)

1 – Autohydrolysis only;

2 – Autohydrolysis in presence of 1% w/w of sulfuric acid;

3 – Autohydrolysis followed by alkaline delignification (1% w/v of sodium hydroxide).

Parr reactor

Best condition: 190 oC, 10 min., 50% ethanol

• Loss of cellulose (%): < 2.0

• Hemicellulose removal (%): 86.7

• Lignin removal (%): 78.3

• Enzymatic Cellulose Conversion *, ECC (%): 61.2

• Specific produtivity (mgglucoseFPU-1h-1): 0.303

* Enzyme load: 20 FPU/gcellulignin (Accellerase 1500,

Genencor)

Solid load: 10% w/w

100x(%)(g)m

0.9(g)mECC(%)

initial

glucose

ECC: Enzymatic cellulose

conversion

x: cellulose content in the

lignocellulosic material

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Subproject 4: C6 processing

Senior researchers: Antonio J. G. Cruz Paulo W. Tardioli Ruy Sousa Jr Teresa C. Zangirolami Raquel L. C. Giordano Roberto C. Giordano

Graduate students: Carlos E. Galeano-Suárez (PhD) Inti D. Cavalcante-Montaño (PhD) Gislene M. Silva (PhD) Luciano J. Corrêa (PhD) Pedro L. M. Aquino (PhD) Renata B. A. Souza (PhD) William Kopp (PhD)

Pos-doc fellow:

Ursula F. Rodríguez-Zúñiga

Collaboration

Jose M. Guisan (ICP/CSIC, Spain) Claus Felby (Univ. of Copenhagen, Denmark)

Challenge: Immobilized cellullolitic enzymes •Which in the pool? •Mechanism of action? •Half-life vs immobilization cost? •Biorreactor operation?

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Sequential batches with immobilized pool

0 10 20 30 40

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40

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50T

ota

l R

ed

ucin

g S

ug

ar

Co

nce

ntr

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n (

g.L

-1)

Time (hours)

1st batch

0 10 20 30 40

Time (hours)

2nd

batch

0 10 20 30 40

Time (hours)

3rd batch

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Silica Magnetic Micro-Particles SMMP

Superparamagnetic Enzyme Aggregates,

SEAs)

Subproject 5: C5 processing

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Senior researchers:

Raquel L. C. Giordano Roberto C. Giordano Ruy Sousa Jr. Teresa C. Zangirolami

Graduate students:

Anny Manrich (PhD) Carlos E. Galeano-Suarez (PhD) Claudia R. Silva (PhD) Inti D. Cavalcante-Montano (PhD)

Guilherme S. Moraes (MSc) Patrícia M. Aquino (MSc)

Challenges: Xylulose metabolism in S. cerevisiae Directed evolution of strains Stabilizing ex-vivo immobilized enzyme (XI) Controlled hydrolysis of hemicellulose for production of xylooligosaccharides (XOS)

Collaboration: Eugénio. Ferreira, Isabel Rocha and Sónia Carneiro (Pos Doc) UMinho, Portugal

Andreas K Gombert EPUSP, Brazil

GI comercial livre

IGI-Ch

IGI- comercial

30 oC; pH 5,0;

60 g/L de xilose ISOMERIZATION UNDER FERMENTATION

CONDITIONS MADE FEASIBLE BY

IMMOBILIZATION

Silva CR, Zangirolami TC, Rodrigues JP, Matugi K, Giordano RC, Giordano RLC. . An innovative biocatalyst for production of ethanol from xylose in a continuous bioreactor. Enzyme and Microbial Technology 50:35-42, 2012.

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SCREENING FOR SUITABLE S. cerevisiae STRAINS

Experimental conditions: batches conducted at T= 35 oC; biocatalyst containing 20 % enzyme, 10 % yeast and 1 % CaCO3;

microaerophile. Fleischmann and Itaiquara: bakery yeasts; BG-1, CAT-1 and PE-2: industrial strains; CEN.PK117-7D: lab strain.

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ASSESSING PROCESS PERFORMANCE

EtOH – Ethanol; XOH – Xylitol; X – Conversion; PrEtOH – Productivity in ethanol; Se – Selectivity, YEtOH/S0 – Overall yield

acel/ml; b recombinant yeast; *hemicellulose hydrolysate (FOGEL et al., 2005)

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OBRIGADO