Bioenergy Research: BIOEN  Program

Bioenergy Research: BIOEN Program

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Description: Improvement of Biomass (Agronomy, Breeding, Biotechnology)Identify new paths to genetically manipulate the energy metabolism of cultivated plants, creating new bio-fuel alternatives.

 
Author: GlauciaMendes Souza (Fellow) | Visits: 2749 | Page Views: 2758
Domain:  Green Tech Category: Biomass Subcategory: Government Policy 
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Contents:
Glaucia Mendes Souza Professor Institute of Chemistry University of S�o Paulo BIOEN Program Coordinator

The Brazilian Bioenergy Research Program BIOEN

http://bioenfapesp.org

BIOEN DIVISIONS
BIOMASS Contribute with knowledge and technologies for Sugarcane Improvement Enable a Systems Biology approach for Biofuel Crops PROCESSING AND ETHANOL TECHNOLOGIES Increasing productivity (amount of ethanol by sugarcane ton), energy saving, water saving and minimizing environmental impacts
ENGINES Flexfuel engines with the same performance, consumption, pollutant emissions and durability as the engines would run on a particular fuel blend

BIOREFINERIES AND ALCOHOL CHEMISTRY Complete substitution of fossil fuel derived compounds Sugarchemistry for intermediate chemical production and alcoholchemistry as a petrochemistry substitute IMPACTS Studies to consolidate sugarcane ethanol as the leading technology path to ethanol and derivatives production Horizontal themes: Social and Economic Impacts, Environmental studies and Land Use

ENGINES DIVISION

Research to consolidate ethanol as the renewable substitute for gasoline on a short to medium term (10 to 20 years), with the evolution of internal combustion engines, and on a long term with fuel cells. Flex-fuel engines with the same performance, consumption, pollutant emissions and durability as the engines would run on a particular fuel blend

Adjust the engine compression ratio Solve the cold-starting problem when using only ethanol Understand the role of ethanol in new engine configurations (direct fuel injection together with port injection and turbo-chargers to decrease fuel consumption and CO2 emission) Study compatibility with after-treatment devices used for emissions control Develop ethanol or sugarcane products with physical-chemical properties adequate for compression ignition engines

BIOREFINERIES DIVISION

Industrial units near sugar and alcohol industrial facilities that take advantage of renewable raw materials available (ethanol, sugar, CO2, bagasse, trash, yeasts) to produce high added value products Sugarchemistry for intermediate chemical production Alcoholchemistry as a petrochemistry substitute � Consorted ethanol-biodiesel production with oleaginous in the sugarcane reform area � Develop products from ethanol via acetaldehyde and ethylene route � Perform Chemical synthesis of intermediate oxygenated chemicals (alcohols, acid ketones), polymers (PHA, lactic acid), nutraceuticals directly from sucrose � Develop biocatalysis for transformations of carbohydrates in valuable chemicals Complete substitution of fossil fuel derived compounds

IMPACTS DIVISION

Horizontal themes: Social and Economic Impacts, Environmental studies and Land Use Ethanol as a global strategic fuel
� Certification Methodology for ethanol produced in a sustainable environmentally friendly manner � Research on new agronomical practices (precision agriculture, mecanization, no-till farming, low input practices, new crop protection systems) and their impact on soil loss, management and efficiency in different production environments � Improve recycling plant nutrients of crop and industry residues in the sugarcane farm and industry system � Define changes in carbon sequestration, greenhouse gases emission gains, carbon and energy balances impacted through the use of Bioenergy � Evaluate the environmental impact of GM sugarcane and biosafety � Risk assessment of effects on environment, on social relations and other economic activities (competition with food supply, energy supply and local materials)

Studies to consolidate sugarcane ethanol as the leading technology path to ethanol and derivatives production

Participating Institutions
UNIVERSIDADE DE SAO PAULO UNIVERSIDADE ESTADUAL DE CAMPINAS SECR EST AGRICULTURA E ABASTECIMENTO DE SAO PAULO MINISTERIO DA CIENCIA E TECNOLOGIA UNIVERSIDADE FEDERAL DE SAO CARLOS SECR EST DESENVOLVIMENTO DE SAO PAULO UNIVERSIDADE FEDERAL DO ABC FUNDACAO GETULIO VARGAS SAO PAULO INST DE ESTUDOS DO COMERCIO E NEGOCIACOES INTERNACIONAIS UNIVERSIDADE ESTADUAL PAULISTA JULIO DE MESQUITA FILHO

# Projects
27 8 6 4 3 2 2 1 1 1

One of the most productive cultivated plants - a large biomass
Commercial sugarcane is vegetatively propagated through stem cuttings In 12 months the plant will reach 4-5 meters with the extractable culm measuring 2-3 meters After harvest, underground buds will sprout giving rise to a new crop (6 harvests) C4 carbohydrate metabolism - large amount of carbon partitioned into sucrose (up to 42% of the stalk dry weight, around 0.7 M in mature internodes)

Fonte: International Energy Agencia (2005). *ISPA

Biomass and Ethanol Production in Brazil

70 65 60 55 50 45 40 35 30 25 20 15 10 5 0
at� 1990 1992 1993 1995 1996 1998 1999 2000 2001 2002 2003 2005 2006

Biomass/ha

Sugar

70's

Hoje

82 %

Sugarcane production
Brazil has great tradition in sugarcane cultivation (since 1532) � � � 8.0 million ha World leader Several public and private institutions dedicated to P&D � Very competitive costs and production

technology � Breeding Programs (since 1910) IAC (1934) Campos (1946-1972) CTC (ex Copersucar, 1968) Ridesa (1971) Canavialis (2003)

Fertilizer experiments. IAC, 1938

BIOMASS DIVISION

Improvement of Biomass (Agronomy, Breeding, Biotechnology) Identify new paths to genetically manipulate the energy metabolism of cultivated plants, creating new biofuel alternatives
� Uncover metabolic networks related to the production of carbohydrates and sucrose through the use of "omics" technologies � Integrate the results in a single platform and develop bioinformatic tools to assess the information � Discovery of genes associated with agronomic characteristics of interest � Development of new sugar cane cultivars � Signaling, regulation of gene expression and regulatory networks � Genetic transformation of sugarcane and other grasses � Molecular markers, statisticalgenetics, mapping and breeding � Sequencing, physical, genetic and molecular mapping of genomes � Understand cell wall structure, architecture and biological function � Discover new cellulolytic fungi species capable of degrading biomass � Refine field practices for enhancing crop production including soil management, fertilization and precision agriculture � Improve control of weed, pests, and diseases though chemical or biological control, resistant varieties and field practices

Contribute with knowledge and technologies for Sugarcane Improvement Enable a Systems Biology approach for Biofuel Crops

Domestication and early evolution of sugarcane
Saccharum officinarum

Chewing

Sugar extraction

Manufacturing

Cottage industries

Modern Breeding

0 00 8

BC

SE Asia Pacific Islands

00 15 000 1 BC Intertropics

50

0

AD

-8 ry 6 ntu Ce
th

th

th y 5 tur 1 n Ce

th y 6 tur 1 n Ce

th y 9 tur 1 n Ce

Persia

Mediterranean Spain

Canary Madeira West Africa

Dominican Republic Brasil

Java India

Modern sugarcane cultivars

Saccharum sinence Saccharum barberi (crosses to wild relatives natural hybrids)

Saccharum officinarum clones World trade plantation factories

S. officinarum X S. spontaneum (interspecific hybrids)

Interspecific breeding: a major breakthrough in modern sugarcane breeding
Solved some of the disease problems but also provided increased yields, improved ratooning ability and adaptability for growth under various stress conditions
Contributing genera: Saccharum, Erianthus, Miscanthus, Sclerostachya and Narenga Saccharum genus (six polyploid taxonomic groups): Wild species Early cultivars Marginal species S. spontaneum (2n=40 to 128) S. officinarum (2n= 80) S. edule (2n = 60 to 122)) S. robustum (2n= 60, 80 and up to 200) S. barberi (2n=81-124) S. sinense (2n=116-120)
Genome organization of a modern cultivar Each bar represents a chromosome Chromosomes in the same column are homologues
S. officinarum S. spontaneum

Giant Genome (n 750-930 Mpb)

Polyploid (2n = 70-120 cromossomos)

~10 Gb

Breeding

Bi-parental Crossings Bi-parental Crossings Area Crossings Area Crossings

Seedlings Seedlings

Multiple Crossings Multiple Crossings

Selection

New Varieties
2006
H ar rc ga Su on
13 clones in final phase

ve s

168 clones in Experimental Trials 619 clones in T3

ta bi lit
g r in lle Ti

d el Yi

y

D ea is se re si st an ce

te nt

6.792 clones in T2 (1.349 Clones Brix > RB855156) 398.477 Seedlings in T1

12 years

Total: 406,069 genotypes

The SUCEST EST Sequencing Project

50 labs 200 researchers 238000 ESTs 43000 Transcripts

26 libraries - 13 cultivars - Over 90% of sugarcane genes tagged

SUCEST: A Defining Initiative for Sugarcane Genomics and Biotechnology in Brazil

Sugarcane Genome Research Network in Brazil

50 artigos 20 artigos 01 artigo

Empresas e institui��es paulistas Empresas e institui��es de outros estados

Setting up the priorities

Biotechnological Roadmap for Sugarcane Improvement
1 � Gene Discovery Genes associated to agronomic traits of interest Gene function evaluation C4 model plant system 2 � Physiology Sucrose metabolism, carbon partitioning, photosynthesis, stress responses 3 Genome Sequence Full length transcripts Surveys of several genomes Gene enrichment methods for regions to be sequenced BAC library with 10x coverage BAC screening mthods 4 International Bioinformatics International Consortium 5 � Transgenics Expression vectors Complete ORFeome Promoters Screening methods for phenotyping (metabolomics, qPCR, biochemical assays, cell wall) 6 Marker identification Integrated databases and translation of transcriptome data to marker assays

How far can we go?

Waclawovsky, A. J., Sato, P. M., Lembke, G. M., Moore, P. H. and Souza, G. M. Sugarcane for Bioenergy Production: an assessment of yield and regulation of sucrose content (Plant Biotech. J. 2010)

High yield variety: 260 ton/ha in 13 months (commercial at Agrovale, Bahia) and 299 ton/ha (experimental at Fazenda Busato, Bahia)

Waclawovsky, A. J., Sato, P. M., Lembke, G. M., Moore, P. H. and Souza, G. M. Sugarcane for Bioenergy Production: an assessment of yield and regulation of sucrose content (Plant Biotech. J. 2010)

Potential yield of sugarcane

Agilent Technologies : 2 color gene expression 14,000 genes represented
Array 1 (Experiment 1) 4x44k Chip image

LogRatio vs Log Processed Signal

Sucrose Biomass Lignin Fiber CO2 Sugars

Drought ABA
Herbivory Phosphate

MeJA
Endophytes

7000 genes expression profiled

Genetical Genomics of Traits of interest

Progeny 1 Genotypes CTC98-241 CTC98-242 CTC98-243 CTC98-244 CTC98-246 CTC98-252 CTC98-253 CTC98-258 CTC98-261 CTC98-262 CTC98-265 CTC98-268 CTC98-271 CTC98-272 CTC98-277 CTC98-279

Brix 18.00 18.60 19.20 14.60 18.80 18.00 19.60 18.00 7.00 7.40 6.40 4.80 6.00 6.80 7.40 7.80

Sucrose % m/m 7.311 9.183 10.956 11.161 10.974 6.370 11.120 6.739 1.14 1.37 0.49 0.70 0.92 1.07 1.58 1.74

Glucose % m/m 1.322 1.430 0.649 0.633 0.709 0.840 0.660 1.116 0.878 0.968 1.200 0.342 1.098 0.725 0.774 1.318

Fructose % m/m 0.988 1.014 0.602 0.646 0.545 0.579 0.643 0.865 0.755 0.823 1.090 0.326 0.992 0.632 0.716 1.066

Progeny 2 Genotypes C158 C121 C171 C496 C11 C6 C113 C436 C292 C231 C38 C250 C405 C144

Brix 18.3 18.8 16.8 17 19.2 18.2 21 13.9 15 13.9 12.9 11.5 15.2 13.2

SnRK1
Phosphorylates sucrose phosphate synthase (SPS) and nitrate reductase (NR), which together with binding of 1433 proteins inhibits their activity
category adapter adapter adapter adapter protein kinase sub category 1 14-3-3 protein 14-3-3 protein 14-3-3 protein 14-3-3 protein SNF-like kinase sub category 2 GF14 GF14 GF14 GF14 caneSnRK1-2 HB vs LB MIn vs IIn 1 4 2 1 2 Drought ABA Sucrose 3 Glucose

1

3

A

B R R

C

D
Storage parenchyma (R), Phloem (f), Fiber and bundle parenchymal cells (arrows), Bundle distal cells (ce), Bundle proximal cells (ci)

f

Physiology of sucrose and biomass accumulation

Waclawovsky, A. J., Sato, P. M., Lembke, C. G., Moore, P. H and Souza, G. M. Sugarcane for Bioenergy Production: an assessment of yield and regulation of sucrose content. Plant Biotechnology Journal (2010)

Regulation of sucrose accumulation and biomass

Hemicellulose and pectin synthesis

Cellulose synthesis

Sucrose synthesis

Sucrose synthesis

Waclawovsky, A. J., Sato, P. M., Lembke, C. G., Moore, P. H and Souza, G. M. Sugarcane for Bioenergy Production: an assessment of yield and Drought regulation of sucrose responses content. Plant Biotechnology Journal (accepted)

Drought responses

Climate Change: +60% biomass +35% photosyntesis

Transport Transcription factors Stress response Secondary metabolism Functional categories Receptor Protein metabolism Photosynthesis Acid nucleic metabolism No match Lipid metabolism Development Cell cycle Carbon metabolism 0 1 2 3 4

Repressed Induced
5

370 ppm CO2 Ambient

720 ppm CO2 Elevated CO2

Number of genes

60% more Biomass

Cultivated land expansion

High Sucrose Content High Biomass Drought Tolerance High Fiber Enhanced saccharification .. .. .. .. .

65% of expansion land is pasture

CONAB, 2008; J. Goldemberg (2008) Biotechnology for Biofuels 1:6.

Water Deficit SE and CW Regions

Drought Field Experiments with 6 cultivars: S�o Paulo, Pernambuco, Alagoas (SE, NE)

irrigated

Nonirrigated

USP, UNICAMP, UFV, UFAL, UFPE, UFRPE

Drought Field Experiments with 10 genotypes: Goian�sia, Goi�s (CW)

10 genotypes (preselected based on performance over 2009 from a group of 100 clones)

Sensors (30, 60, 90 cm) Irrigation

IAC

Production of transgenic sugarcane plants

Explants: Immature Leaves

Callus Induction

Regeneration Selective Medium

Rooting

PCR Shoot Growth Greenhouse

CEBTEC - Esalq/Usp

Sugarcane transgenic plants with increased sucrose content: gene silencing using RNAi

PapiniTerzi, F. S., Rocha, F. R., V�ncio, R. Z. N., Felix, J. M., Branco, D., Waclawovsky, A. J., DelBem, L. E. V., Lembke, C. G., Costa, M. DB. L., NishiyamaJr, M. Y., Vicentini, R., Vincentz, M., Ulian, E. C., Menossi, M., Souza, G. M. (2009). Genes associated with sucrose content. BMC Genomics 10, 120. doi:10.1186/1471 216410120 Genes associated to sucrose content, sugarcane with increased sucrose levels USPTO Patent US 11/716,262. PCT/BR2007/000282. Gl�ucia Mendes Souza, Fl�via Stal PapiniTerzi, Fl�via Riso Rocha, Alessandro Jaquiel Waclawovsky, Ricardo Zorzetto Nicollielo V�ncio, Jos�lia Oliveira Marques, Juliana de Maria Felix, Marcelo Menossi Teixeira, Marcos Buckeridge, Amanda Pereira de Souza, Eug�nio C�sar Ulian. Universidade de S�o Paulo, Unicamp, Centralcool, CTC and FAPESP

Lignin Biosynthesis is associated to sucrose content

Genes associated to sucrose content, sugarcane with increased sucrose levels USPTO Patent US 11/716,262. PCT/BR2007/000282. Gl�ucia Mendes Souza, Fl�via Stal PapiniTerzi, Fl�via Riso Rocha, Alessandro Jaquiel Waclawovsky, Ricardo Zorzetto Nicollielo V�ncio, Jos�lia Oliveira Marques, Juliana de Maria Felix, Marcelo Menossi Teixeira, Marcos Buckeridge, Amanda Pereira de Souza, Eug�nio C�sar Ulian. Universidade de S�o Paulo, Unicamp, Centralcool, CTC and FAPESP

Energy Cane

Cogeneration in 2008 = 1.400 MW In 2020 = 14.000 MW (equals 1 Itaipu)

Sugarcane Cane Energy Mill

Fazenda Busato � Bom Jesus da Lapa BA

320

65 60 55 50 45 40 35 30
RB855113 RB863129 RB931003 RB961552 RB98710

300

RB92579

280

TCH

260

240 RB951541

RB93509

220

RB9629 RB931566 RB72454 RB867515 VAT90-212

SP79-1011

200 15,0

15,5

16,0

16,5

17,0

17,5

18,0

18,5

19,0

19,5

20,0

20,5

BRIX

Breeding and Genetics Workgroup Challenge
To understand the genetic architecture of quantitative traits in sugarcane, in order to implement marker assisted selection

Why is this a challenge
Marker systems that are informative in other scenarios (e. g. Microssatelites) provide less information in polyploids, having a dominant action Commonly, only markers that have a single copy (dosage) on the genome have been used Single Nucleotide Polymorphisms (SNP) are usefull (codominant), but the data provided by current approaches and technologies cannot be readily used Good genetic maps and QTL (quantitative trait loci) results are not available to date

State of the Art
Up to 400 SNPs were developed and used to genotype a biparental brazilian population Methods to interpret this data were developed and are ready to use Statistical methods to build genetic maps and to map QTL using markers with higher doses have been developed

Group Leaders: Anete P Souza (UNICAMP) and Augusto Garcia (USP)

Sugarcane Map incorporating double and triple dose markers (SSR, ESTSSR, RFLP)
SP80180 x SP804966, 200 individuals 934 markers Final map with 347 linked markers 329 single dose (239 1:1 and 90 3:1) 16 double dose 2 triple dose Assembled in 102 linkage groups The total map length 2,880.3 cM (4,361.3) with a density of 7.6 cM (11.6)

Mollinari, M; Silva, RR; Margarido, GRA; Marconi, TG; Souza, AP; Garcia, AAF

Sequencing Workgroup 1 � BAC Sequencing Strategies R570 BAC selection (3D pools, PCR, membrane hybridization) Construction of new BAC libraries (SP803280) Sequence 1000 BACs BAC assembly from pyrosequencing and Sanger (454 and ABI) Anchoring to sorghum BACend sequencing and BAC homeologues sequencing 2 � Whole Genome Sequencing WGS pilot experiments: generich enrichment, methylation filtration chip hybridization, preliminary surveys, ChIPSeq 3 � SSR, SNP discovery Sequenon 4 Bioinfo and database development http://sucestfun.org 5 � Sugarcane Gene Nomenclature Transcription factor recommendation

The SUGESI Project

A draft of the sugarcane monoploid genome (1 Gb)

Genome organization of a modern cultivar Each bar represents a chromosome Chromosomes in the same column are homologues
S. officinarum S. spontaneum

Giant Genome (n 750-930 Mpb)

Polyploid (2n = 70-120 cromossomos)

~10 Gb

SUGESI Partners

UGA
International Consortium for Sugarcane Biotechnology South African Sugarcane Research Institute University of KwaZuluNatal Sugar Milling Research Institute

UIUC

Which Genotype should we sequence?

Which Genotype should we sequece?
Sugarcane is a collection of alleles: ideally one needs to sequence a hybrid cultivar and ancestor genotypes (relatively pure autopolyploids)

Initial Candidates: SP803280 (most ESTs) R570 (BAC library and dense genetic map) Q165 (genetic map) LA Purple (S. officinarum) SES208 (S. spontaneum) Mol6081 (S. robustum)

LA Purple S. officinarum

There are no homozigous diploid genotypes SES208 S. spontaneum
Angelique D'Hont, Glaucia Mendes Souza,Marcelo Menossi, Michel Vincentz, MarieAnne VanSluys, Jean Christophe Glaszmann and Eug nio Ulian

Syntheny with Sorghum

Saccharum and Sorghum diverged between 5 and 9 million years ago Some genotypes can still be crossed to one another

Grivet et al, 1996; Dufour et al, 1997 D'Hont et Paulet (Personnal Comm.)

Prediction of Ortholog Groups

Sugarcane and Sorghum promoter alignments

Fragment of 2169bp

Segment 724bp (81% identity)
Fragment of 936bp

5 Segments Length Identity 22bp 97% 52bp 94% 126bp 89% 240bp 94% 751bp 80%

Genome Walking: A putative promoter that does not align to sorghum
Fragment 5 (SASGMS09692) Length: 1298bp Total aligned: 110bp in 3 segments

Alignment of the SASGMS09692 (walking: Fragment 5)

Alignment of the SASGMS09692 (walking: Fragment 5)

ChIPSeq Experiments: RNApolII ChIP to construct a promoter database

Phatnani and Greenleaf (2006)

We are using RNA polymerase II repeat YSPTSPS antibody to generate a promoter and active genes database. This antibody reacts with the nonphosphorylated heptapeptide repeat of the largest subunit of eukaryotic RNA polymerase II.

ChIPSeq: 7 Abs raised against TFs associated to drought and sucrose

BAC mapping on sorghum generich regions
Overgo hybridization against sugarcane BACs

http://bacman.sourceforge.net

UGA

6.021 overgo sequences (40 nt) BLASTed against the sorghum and rice genomes and 438 sugarcane SAS of interest (genes associated to sucrose content and drought)

Select only stringent hits (38 nt aligned with no gaps)

Select only if presented 10 copies or less against the BAC library

Select only if correspond to unique locus in both sorghum and rice

Genome walking

generich BACs
Collaboration: Glaucia Souza, Roberta Campos Andrew Paterson and Changsoo Kim University of Georgia USA

The Sugarcane Transcriptome Project & CaneRegNet

Bioinformatics Workgroup
Challenge
Create and maintain a Database, tools and resources for the community for a grass with a giant genome, hundreds of cultivars and not enough hands

Why is this a challenge
� Sequencing of the sugarcane genome is one of the most challenging projects in genomics nowadays �We want to develop an evolving database that can grow and host heterogenous data as projects progress �We want to advance data collection and storage to a Systems Biology approach integrating genomics, functional genomics, molecular markers, statisticalgenetics tools, physiological and agronomical data �Large multigene families and polyplody makes allele identification difficult

State of the Art

�The SUCESTFUN Database has been created �It currently hosts data on ESTs, BACs, shotgun sequencing (caneGenome) �ESTs and SAS can be related to results of over 300 hybridizations (caneGeneExpress) �We started developing a sorghum vs. sugarcane ortholog dataset �We started identifying gene promoters �We are creating a database dictionary

Group Leader: Glaucia Souza (Instituto de Qu�mica � USP)

SUCESTFUN DB (http://sucestfun.org)
1 � Genome (CaneGenomeDB) WGS Promoters ChIPSeq BACs Methylation 2 � Transcriptome (CaneGeneExpressDB) genes/ESTs/proteins probes Microarray experiments RTPCR 3 � Proteome proteins/domains Ontology/Nomenclature Structure PDB 4 � Metabolome Metabolic Pathways Mass Spectrometry Ontology 5 � Protein Catalogue (CaneCatalogueDB) Transcription Factors(CaneTFDB) Kinases(CanePKDB) Phosphatases (CanePPaseDB) Cell Wall (CaneCellWallDB) 6 � Genotypes and Transgenics (CaneTransgenicDB) 7 � Regulatory Networks (CaneRegNetDB) 8 � Comparative (CaneSimDB)

DB Architecture designed to facilitate autonomous and heterogenous DB correlations Data Dictionary (descriptions, attributes and domains) Import/export scheme (XML) Software JAVA Jo�o Eduardo Ferreira (IMEUSP) DB MySQL, Postgree Hibernate Framework

http://sucestfun.org

PROCESSING DIVISION

Engineering, processing and equipment design aspects of bioethanol production Cellulosic Ethanol Increasing productivity (amount of ethanol by sugarcane ton), energy saving, water saving and minimizing environmental impacts

� Identify and improve bottlenecks in ethanol production chain at the mill (reception, juice extraction, hydrolysis process, among others) � Improve fermentation process yield � Optimization of water recycling and energy efficiency � Improve separation processes especially to dehydrated ethanol � Develop cellulosic ethanol technology focusing the following objectives: Characterization and development of physical-chemical pretreatment of bagasse for ligninocellulose hydrolysis Development of acid catalyzed and biocatalyzed saccharification Development of high performance cellulases and hydrolases Reduction of the impact of fermentation inhibitors Development of microorganism strains capable to efficient fermentation of pentoses and hexoses Byproducts recovery

Industrial aspects: research in all aspects of the production

Total sugarcane production is estimated to be 664,33 ton/ha for 2010/2011 Total bioethanol production for 2010/11 is projected at 28.5 billion L 54,6% (362,8 million tons) for ethanol (20,14 billlion L hydrated and 8,4 billion L anyhidride) 45,4% (301,6 million tons) for sugar (38,7 million tons) 405 plants of which 157 are exclusive to ethanol Brazilian Bioethanol production costs are the cheapest in the world Industry estimates the cost of producing ethanol from sugarcane at approximately US$ 0.29/L (1 gallon = US$ 1.00).

Cellulosic Ethanol

In 10 years? Today

70's

82 %

40-60 %

12.000/h a

Engineering Workgroup
Challenge: Process Development for High Performance Operation Why is this a challenge? Existing first generation processes needed to be improved in several topics as juice production and treatment, fermentation and separation process Sugar cane bagasse and straw conditioning Lignocellulosic pretreatment process development Hydrolysis Process Development and Enzymes Production Process Development C5 fermentation Process Development Alternative routes for bioethanol production from lignocellulosic feedstock Process Development for cheaper anhydride bioethanol production State of the art? Industrial fermentation well established but with high water content Very few works dealing with lignocellulosic materials from sugar cane feedstock Pretreatment methods are still to defined, in spite of many works using steam explosion for other feedstock C5 fermentation is not suitable yet Hydrolysis is still expensive and not efficient and third generation has to be deloped Anhydride bioethanol processes need to be improved to become cheaper Group Leader: Prof. Rubens Maciel Filho (School of Chem. Eng- UNICAMP)

National Institute of Science and Technology of Bioethanol CNPq, FAPESP

BIOREFINERIES DIVISION

Industrial units near sugar and alcohol industrial facilities that take advantage of renewable raw materials available (ethanol, sugar, CO2, bagasse, trash, yeasts) to produce high added value products Sugarchemistry for intermediate chemical production Alcoholchemistry as a petrochemistry substitute � Consorted ethanol-biodiesel production with oleaginous in the sugarcane reform area � Develop products from ethanol via acetaldehyde and ethylene route � Perform Chemical synthesis of intermediate oxygenated chemicals (alcohols, acid ketones), polymers (PHA, lactic acid), nutraceuticals directly from sucrose � Develop biocatalysis for transformations of carbohydrates in valuable chemicals Complete substitution of fossil fuel derived compounds

Green Acrylic Acid
A��CAR metionina metilcitrato DMSP Piruvato -alanina Lactato metilmal-CoA propanoil-CoA Acetil-CoA glicerol oxaloacetato aspartato

Mathematic modeling and metabolic route selection (structured model) Microorganism Selection (bioprospection and collection analysis)

Sugarcane sucrose

-alanina malonil-CoA -alanina-CoA mal.semiald 3-HP-CoA 3-HP 3-hidroxipropanol

lactoil-CoA Acrilil-CoA

propanoato �cido Acr�lico

Culture Medium Optimization

Fermentation Lactic Acid L- and DSeparation/Purification Acrylic Acid Dehydration

Production Kinetics
reduction Propionic Acid

Processes

Kinetics

Modeling

Process Optimization

Process Control

Products obtained from Lactic Acid

SUCROSE descarboxilation
Fermentation

O H

+

CO + H2O CO2+ H2

Acetaldehyde dehydration + H2O

Lactic Acid
reduction

OH O Acrylic Acid
H2

O Propionic Acid O condensation

O H

+ 1/2 O2

+ CO2 + 2H2O O 2,3 pentanodione

Conceptual Plant design for Green Ethyl Acrylate

ETHANOL

R EC 3

R EC 2 D IS TIL 1

STRIPPER AC ID TOPO C OOLER FE ED R EAC TOR C OOL

EXTR ACT

R AF AC R YLATE

D IS TIL 2 R EC 1 EXT

WAS TE WATER

IMPACTS DIVISION

Horizontal themes: Social and Economic Impacts, Environmental studies and Land Use Ethanol as a global strategic fuel
� Certification Methodology for ethanol produced in a sustainable environmentally friendly manner � Research on new agronomical practices (precision agriculture, mecanization, notill farming, low input practices, new crop protection systems) and their impact on soil loss, management and efficiency in different production environments � Improve recycling plant nutrients of crop and industry residues in the sugarcane farm and industry system � Define changes in carbon sequestration, greenhouse gases emission gains, carbon and energy balances impacted through the use of Bioenergy � Evaluate the environmental impact of GM sugarcane and biosafety � Risk assessment of effects on environment, on social relations and other economic activities (competition with food supply, energy supply and local materials)

Studies to consolidate sugarcane ethanol as the leading technology path to ethanol and derivatives production

Energy Balance

GHG Reduction

Expansion of sugarcane to new land areas 8,1 million hectares 2010/11 = 9,2% increase over the last cycle
In Brasil: Total arable land 355 mi ha Total cropland 76.7 mi ha Total pasture land 172 mi ha Total available land 105 mi ha Sugarcane 23%

CONAB, 2008; J. Goldemberg (2008) Biotechnology for Biofuels 1:6. � USDA 2007

65% of expansion land is pasture

S�o Paulo 4,4 million ha; Minas Gerais 648 thousand ha; Parana 608 thousand ha; Goi�s 601 thousand ha; Alagoas, 464 thousand ha. Total = 0,95% national territory

GHG Mitigation

Conceptual framework for measuring LUC (using an economic model)

Sugarcane Agroecological Zoning in S�o Paulo

Burned X Green Cane: Burning phasing out in 2014/2017 in S�o Paulo
Green cane: Thick mulch of plant residues (8 14 t/ha DM) better soil protection and nutrient cycling (C, N) Increased soil Carbon Challenges: Problems with some insects Difficult to incorporate fertilizers (lower efficiency, nutrient losses) Some varieties have reduced sprouting Research needs: Assess environmental gains due to cycling, soil protection, C accumulation in soil Create varieties adapted to green cane Adequate management practices to green cane Biological Control (viable for several pests in sugarcane) Chemical control and crop management: combination of best management practices to minimize use of pesticides

Environmental challenges
� � � � Pollution of soil and water with chemicals and residues Fossil fuel use to produce ethanol and GHG (CO2, N2O, CH4) must be low to justify production of biofuels N fertilizer : 25% of fossil energy used to produce and transport cane (Production of N fertilizer requires lots of energy: 53.8 MJ/kg N or 1400 m3 natural gas per ton N) N2O release after N fertilization (1-4%, N2O has a GWP 300 greater than that of CO2)

Research: � Good management, precision agriculture, efficient tool to monitor pests and diseases � Management practices to reduce GHG in agricultural processes � Optimize nutrient use. Biological fixation of N � Improve recycling of residues (vinasse, filter cake, ashes, plant residues etc )

Vinasse chanel
Sugarcane industry is in a privileged position: only C, O and H are exported (all mineral nutrients can be recycled in the farmindustry system) Leaching losses that may affect deep water quality has not been a problem associated with sugarcane cultivation For each ton of ethanol used as fuel 2.3t of CO2 are not emitted to the atmosphere with a simultaneous reduction in SO2 emission

Expansion of sugarcane to new land areas
Southwest: dry winter Marginal land, pastureland, and poor soils Research: � Drought resistance � Crop breeding to new environments � Soil/chemical management for deep rooting (addition of calcium) � Chemical/fertilizer supply to compensate for deficiencies in new land areas � Revise nutritional needs: (inorganic nutrients are 5% of plant dry matter) � Optimize the use of fertilizers and chemicals (Sugarcane: 13% of fertilizer used in Brazil) � Recycle nutrients of crop and industry residues

Recycling of residues

Nutrient recycling

Fertilization for biofuel crops 2007/2008

Million t N, P2O5, K2O Biological Nitrogen Fixation in Sugarcane: Strong Research Effort

FAPESP BIOENERGY PROGRAM BIOEN http://bioenfapesp.org

Committee
Program Coordinator Glaucia Mendes Souza Departamento de Bioqu�mica Instituto de Qu�mica Universidade de S�o Paulo
MarieAnne VanSluys Instituto de Bioci�ncias Universidade de S�o Paulo Anete de Souza Centro de Biologia Molecular e Engenharia Gen�tica Universidade Estadual de Campinas Marcos Buckeridge Instituto de Bioci�ncias Universidade de S�o Paulo Rubens Maciel Faculdade de Engenharia Qu�mica Universidade Estadual de Campinas Heitor Cantarella Instituto Agron�mico Secretaria de Agricultura e Abastecimento do Estado de S�o Paulo

Thank You!

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