Technological Trajectories in Enzymatic Hydrolisis

José Maria Ferreira Jardim da Silveira & Thays MurakamiCenter of Agriculture and Environmental Research (NEA); Center of Interdisciplinary Research on Energy (NIPE/BE-BASIC & Institute of Economics (IE), University of Campinas (UNICAMP/), Brazil/

Paper prepared for presentation at the 19th ICABR ConferenceIMPACT OF THE BIOECONOMY ON AGRICULTURAL SUSTAINABILITY, THE ENVIRONMENT AND HUMAN HEALTH Ravello, June 17-19, 2015Copyright 2015 by author(s). All rights reserved. Readers may make verbatim copies of this document for non-commercial purposes by any means, provided that this copyright notice appears on all such copies.

Motivation: description of the technologyEnzimatic Hydrolisis in the context of Second Generation

Motivation of the paper: what is the role of Enzymatic Hydrolisis (EH) ?

Scenarios of Brazil:

link between products, technological routes and biomass;

First generation still has an expected technological evolution;

The concept of modularity/specialization shows up;

The possibilities of co-evolution of different trajectories: first generation and second generation;

Simultaneous fermentation 5C with 6C as a focusing device, but separate 5C fermentation (Xylose and others) is still another option for tech evolution: microorganisms, enzymes and biomass.

Short term Medium term Long term

2G Perspectives

1 Medium and long term scenarios include a small 1G plant processing energy cane juice

2 Including straw

Increase on 2G yields and recoveries

2G1 All year: sugarcane bagasse+ straw

All year: energy cane bagasse2

All year: energy cane bagasse2

1G2G Season: sugarcane bagasse+ straw

Season: sugarcane bagasse+ straw

Off-season: energy cane bagasse2

Season: sugarcane bagasse+ straw + energy

cane bagasse2

Off-season: energy cane bagasse2

Source: Junqueira, 2015 CTBE/CNPEM

2G Flowsheet

5

Short term and “A” scenarios– Separated C5 fermentation

C5 fermentation - Partial cell recycling is considered.

2G Flowsheet

6

C5/C6 co-fermentation

C5/C6 co-fermentation - No cell recycling due to solids presence.

Biomass

¹ Does not consider mineral impurities;² Composition for baled straw. Integral harvested straw moisture varies according to recovery fraction: 33.6 % for 50% recovery; 31.3% for 60% recovery; and 29.6% for 70% recovery;3 Energy cane composition includes its straw (100%). Source: Junqueira, 2015 CTBE/CNPEM

Biomass composition (wt %) ¹

Sugarcane stalks Sugarcane straw ² Energy cane3

Water 70.3% 15.0% 66.8%

Sucrose 14.0% 4.3% 8.1%

Reducing sugars 0.6% 0.2% 2.5%

Fibers 12.7% 77.9% 21.3%

Cellulose 6.0% 32.4% 10.0%

Hemicellulose 3.5% 24.8% 5.9%

Lignin 3.2% 20.6% 5.4%

Others 2.4% 2.6% 1.3%

Steam explosion pretreatmentShort term Medium term Long term

Temperature (°C) 190 200 210

Residence time (min) 15 10 5

Solids content (%) defined by the steam required totemperature

achieve reactor

Cellulose conversion to glucose (%) 0.5% 1.0% 1.0%

Cellulose conversion to glucose oligomers (%) 3.0% 3.0% 3.0%

Cellulose degradation to HMF (%) 1.5% 1.5% 1.5%

Xylan conversion to xylose (%) 30% 45% 60%

Xylan conversion to xylose oligomers (%) 30% 25% 20%

Xylan degradation to furfural (%) 10% 10% 10%

Lignin solubilization (%) 10% 10% 10%

Acetyl group conversion to acetic acid (%) 70% 80% 90%

Source: Junqueira, 2015 CTBE/CNPEM

9

Enzymatic HydrolysisShort term Medium term Long term

Temperature (°C) 50 50 65

Pressure (bar) 1.0 1.0 1.0

Residence time (h) 48 36 36

Solids content (%) 15 20 25

Cellulose conversion to glucose (%) 60% 70% 80%

Xylan conversion to xylose (%) 60% 70% 80%

Acetyl group conversion to acetic acid (%) 60% 70% 80%

Xylose oligomers to xylose (%) 60% 70% 80%

Source: Junqueira, 2015 CTBE/CNPEM

Fermentation

C6/C12 fermentation Short term Medium term Long term

Operational conditions same as 1G same as 1G same as 1G

C6/C12 conversion to ethanol (%)

88 90 90

Maximum alcoholic content (g/L)

70 85 85

Short term and “A” scenarios – Separated C5 fermentationDeoligomerization and C5 fermentation Short term Medium term Long term

Temperature (°C) 33 33 33

Residence time (h) 48 36 24

Xylose oligomers conversion to xylose (%) 80% 90% 90%

Glucose oligomers conversion to glucose (%) 80% 90% 90%

C6 conversion to ethanol (%) 90% 90% 90%

C5 conversion to ethanol (%) 80% 80% 85%

Maximum alcoholic content (g/L) 70 70 70

Cell Recycling (%) * 80% 90% 95%

Source: Junqueira, 2015 CTBE/CNPEM

Methodology of Identification of PatentsRelated only to EH

Methodology

Searching for a clear delimitation of the subject Using IPC Search: EH

Step 1

  Key-words combination (Ten Groups- take Group 1 for instance

Group 1

biochemical + conversion + biomassbiochemical + conversion + biomass + lignocellulosicbiochemical + conversion + biomass + cellulosisbiochemical + conversion + biomass + hemicellulosisbiochemical + conversion + biomass + lignocellulosic + ethanolbiochemical + conversion + biomass + lignocellulosic + ethanol + fuel

biochemical + conversion + biomass

SubclassImportance(

%)Subgroups

C12P 25 C12P7/10 C12P7/06 C12P19/14 C12P19/00  

C12N 18 C12N1/20 C12N15/29 C12N1/12 C12N1/14 C12N1/22

C10G 13 C10G3/00 C10G1/00 C10G1/08 C10G11/18  

C10L 10 C10L5/44 C10L3/08      

B01D 8 B01D1/14 B01D21/01 B01D25/00 B01D53/62 B01D53/78

C08H 7 C08H8/00        

C10B 7 C10B49/22 C10B53/02      

C13K 7 C13K1/02 C13K13/00      

D21C 7 D21C1/02 D21C3/04      

B01J 6 B01J8/18 B01J8/24 B01J29/40 B01J8/00 B01J8/32

Strongly concentrated on

those sub-classes

Selection of 44 IPC codes

MethodologySearching for EH (rigorous processes of convergence to the goal)

STEP 2 Selecting Patents in the Data Base Derwent Innovations Index (1970-2014)

“IP=(C12P-007/06 OR C12P-007/08 OR C12P-007/10 OR C12P-007/14 OR C12P-019/00 OR C12P-019/02 OR C12P-019/04 OR C12P-019/12 OR C12P-019/14 OR C12P-039/00 OR C12N-001/00 OR C12N-001/12 OR C12N-001/13 OR C12N-001/14 OR C12N-001/15 OR C12N-001/16 OR C12N-001/18 OR C12N-001/19 OR C12N-001/20 OR C12N-001/21 OR C12N-001/22 OR C12N-009/00 OR C12N-009/02 OR C12N-009/04 OR C12N-009/14 OR C12N-009/24 OR C12N-009/42 OR C12N-015/01 OR C12N-015/02 OR C12N-015/03 OR C12N-015/04 OR C12N-015/05 OR C12N-015/09 OR C12N-015/10 OR C12N-015/11 OR C12N-015/52 OR C12N-015/53 OR C12N-015/54 OR C12N-015/55 OR C12N-015/56 OR C12N-015/63 OR C12N-015/80 OR C12N-015/81 OR C12N-015/82)”

“biochemical conversion OR bioconversion OR hydrolysis OR saccharification OR biomass OR lignocellulose OR lignocellulosic OR cellulose OR cellulosic OR hemicellulose OR enzyme OR enzymes OR enzymatic OR ethanol OR bioethanol OR fuel OR biofuel”

“Acetivibrio OR Bacillus OR Bacteroides OR Cellulomonas OR Clostridium OR Erwinia OR Ruminococcus OR Streptomyces OR Microbispora OR Thermomonospora OR (genus AND bacterium) OR (genus AND bacteria) OR (genus AND bacteria*) OR (genera AND bacterium) OR (genera AND bacteria) OR (genera AND bacteria*) OR (specie AND bacterium) OR (specie AND bacteria) OR (specie AND bacteria*) OR (species AND bacterium) OR (species AND bacteria) OR (species AND bacteria*) OR (bacterium OR bacteria) OR Aspergillus OR Penicillium OR Phanerochaete OR Schizophyllum OR Sclerotinia OR Trichoderma OR (genus AND fungus) OR (genus AND fungi) OR (genus AND fung*) OR (genera AND fungus) OR (genera AND fungi) OR (genera AND fung*) OR (specie AND fungus) OR (specie AND fungi) OR (specie AND fung*) OR (species AND fungus) OR (species AND fungi) OR (species AND fung*) OR (fungus OR fungi)”

“swichgrass OR miscanthus OR prairie grass OR eucalyptus OR softwood OR hardwood OR willow OR poplar OR spruce OR loblolly pine OR pinus taeda OR cane bagasse OR bagasse OR corn stover OR stover OR husk OR stalk OR treetop OR treetops OR branch OR branches OR perennial grass OR perennial grasses OR residue OR residues OR waste OR forest”

IPC

Adventious terms

Microorg.

Raw materials

Methodology: EH

Step 2: Patents from Derwent Innovations Index (1970-2014)

Period Filterby

IPCs

Filter byAdventious

terms

By micro-organisms

By Raw materials

Crossing: micro versus raw material

1970-1979 673 268 89 44 9

1980-1989 10.566 3.852 1.368 499 168

1990-1999 35.553 8.831 2.634 1.360 454

2000-2009 98.852 29.832 9.364 6.758 2.311

2010-2014 48.012 15.827 5.631 3.266 1.176

Total 193.656 58.610 19.086 11.927 4.118

CHART1 – Number of Patetns by filter

Source: base Derwent Innovations Index, in <http://www.periodicos.capes.gov.br/>. Downloads in: 14, 15, 16, 19 e 20 May 2014.

Methodology: Identifying EH

STEP 3: Consulting a expert group: 304 patents were selected

1975-79 1980-84 1985-89 1990-94 1995-99 2000-04 2005-09 2010-140

20

40

60

80

100

120

140

2

13

512

24

45

79

124

By year of filling

Nu

mb

er o

f p

aten

ts

Preliminary Results

Coutry Filling Agent N. Pat.

1 USA

Genencor International Inc. 21

Danisco A/S 18

DuPont Nutrition Biosciences* 2

2 Denmark

Novozymes 19Novo Nordisk 11

Allopartis Biotechnologies Inc. 1

3 USA Codexis Inc. 11

4 GermanyVerenium Corp. 5Diversa Corp. 4Basf AG* 2

5 France IFP Energies Nouvelles 106 Canada Iogen Energy Corp. 97 USA Mascoma Corp. 88 GB BP Corp North America Inc. 8

9Netherlands DSM 6Netherlands Gist-brocades 2

10 USA Qteros Inc. 711 USA Dyadic International Inc. 7

12

USA MRIGlobal - Midwest Research Institute 5

USA Alliance for Sustainable Energy, LLC 1

USA Battelle Memorial Institute 1

CHART 2– Main Companies Filling Patents in EH field (1970-2010)

Applying the NK modelWhy to choose NK modelling ?

FRENKEN, K. A complexity approach to innovation networks. The case of the aircraft industry (1909 - 1997). Research Policy, v. 29, n. 2, p.257-272, Feb. 2000.

FRENKEN, K. Technological Innovation and Complexity Theory. Economics of Innovation and New Technology, v. 15, n. 2, p.137-155, 2006Koen Frenken (2006), Murakamy (2015)

Description of the Model

Methodology to apply NK model: mutual information indicators

First Category of information: Research Fields

  Contents

1 Culture of Microorganisms – definition of parameters

2 OGM

3 Gene Expression in yeast/plants for expression of lignocellolitic Enzymes

4 Discovery of new microorganisms

5 Isolation, cloning and modification of genes that codifies enzymes

6 Changing aminoacids sequences

7 Preparing mix of Enzymes

Mutual Information Indicators(2) Microorganisms

(90 different genus of microorg).

  Genus N. de pat Classification Species

1 Trichoderma 97 FungiT. reesei; T. viride; T. harzianum; T. longibrachiatum; T. asperellum; T. atroviride; T. cremeum; T. inhamatum; T. konilangbra; T. koningii; T. pseudokoningii; T. virens

2 Aspergillus 52 FungiA. niger; A. aculeatus; A. oryzae; A. fumigatus; A. tubingensis; A. terreus; A. usamii; A. awamori; A. japonicus; A. cellulolyticus; A. foetidus; A. saccharolyticus

3 Bacillus 37 BacteriaB. subtilis; B. licheniformis; B. halodurans; B. agaradhaerens; B. amyloliquefaciens; B. circulans; B. acidocaldarius; B. cereus; B. pumilus; B. stearothermophillus; B. velezensis

4 Penicillium 24 FungiP. funiculosum; P. brasilianum; P. verruculosum; P. janthinellum; P. decumbens; P. oxalicum; P. pinophilum; P. canescens; P. chrysogenum; P. citrinum; P. emersonii; P. funiculosum; P. griseofulvum; P. swollenin

5 Clostridium 20 Bacteria

C. phytofermentans; C. thermocellum; C. cellulolyticum; C. stercorarium; C. thermosulfurogenes; C. aldrichii; C. celerecrescens; C. cellobioparum; C. cellulosi; C. cellulovorans; C. chartatabidum; C. herbivorans; C. hungatei; C. josui;C. lentocellum; C. papyrosolvens; C. polysaccharolyticum; C. populeti; C. termitidis; C. thermocopriae

Mutual information Indicators

33 kinds of Lignocellulosic Enzymes

  Enzymes N. de pat Group

1 endo-beta-1,4-glucanase 127 Celulase

2 endo-beta-1,4-xilanase 96 Hemicelulase

3 exo-beta-1,4-glucanase 92 Celulase

4 beta-glucosidase 79 Celulase

5 celulase 60 Celulase

Mutual information indicators

Formulas

Tri-dimensional

Bi-dimensionals

𝑇 ( 𝑋 ,𝑌 ,𝑍 )=∑𝑥=1

7

∑𝑦=1

83

∑𝑧=1

34

𝑝𝑥𝑦𝑧 log2(𝑝 𝑥𝑦𝑧

𝑝𝑥 .𝑝 𝑦 .𝑝𝑧

)

 

 

Results: values of T(X,Y,Z) of patentes, 1975-2013

Modelo NK Two dimensions T(X,Y) co-occurence

Modelo NK T(Y,Z) co-ocurrence

Dimension:content

definition of the parameters of the culture mean

application of the biotechnological techniques

Dimensionenzyme

cellulases (endo-beta-1,4-glucanase, exo-beta-1,4-glucanase and beta-glucosidase) and endo-beta-1,4-xylanase

TrichodermaAspergillus

Others

Bacillus

Penicillium

others

others

Chrysosporium

Humicola

Dimension:

micro-organism

Clostridium

Myceliophthora

StreptomycesTalaromyces

Technology trajectories in 2G

Conclusions

A substantial growth in the number of patents, even before having a convergence of Technologies,

Few corporations fiiled a considerable part of patentes, showing the importance of cumulativeness and technological tradition (DUPONT e NOVOZYMES)

Merger and acquisitions

Barriers to new entrants

Brazil: only one patent: Petrobrás (2009)????????

Questions

NK model allow us to detect the main combinations of the three: reinforced trajectories

Pre-paradigmatic stage: Windows of opportunities?

There is some convinience to guide research and patenting to a certain microorganisms, Enzymes and even methods?

Direction of TT: accelerating convergence of Technologies to a standart packges?

Supply from Global Corporations

If it is so, Why researcher in Brazil is focused on EH?