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ReNEW Technology Foresight Workshop23‐24th April 2013
Enzyme Production & Enzymatic conversion of lignocellulosic feedstocks & wastestreams
Dr. Maria TuohyTCBB & NUI Galway
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Technology Centre for Biorefining & Bioenergy (TCBB;
www.tcbb.ie)Technology Leader: Bart Bonsall
Industry‐led Technology Centre, supported by Enterprise Ireland & the Industrial Development Authority – National network
SME, ME & Large Enterprise partners
5 Principal Investigators: NUI Galway, University College Dublin, University of Limerick and Trinity College Dublin
Objectives: • Engage with Industry to develop a Bio‐Economy in Biorefining & Bioenergy• Best‐practice Centre of Excellence – strong focus on Sustainable, Innovative & Costeffective processes – maximize use of wastes
• International partnerships & Networks• Develop demonstration test facilities – test‐bed for industry & technology transfer• Demonstration to commercial reality
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• Enzymes - Natures own (bio)catalysts
• Found in all living creatures – simplest life-forms (single cells) to most complex (multicellular, e.g. humans)
Material/Compound(Substrate)
Product
Enyzmes are not changed during catalysisEnzymes are highly specific (‘lock & key’)
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• Produced in Nature by microorganisms – Fungi & Bacteria
Plant biomass/wastes Nutrients
Application of the Enzymes can be selective (singleenzyme/selected combinations –specific end products) orextensive (cocktail of enzymes – broad spectrum ofactivities)
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Lignocellulose
Cellulose (25-40%) Hemicelluloses (18-40%); Pectins(2-18%); Lignin (8-25%)Proteins (2-12%); Starch [Lipid, phenolics/extractives, Ash]
Polymer‐fragmenting Enzymes (Endo‐acting)
Oligomers
Simple Building ‘blocks’Monomers
Exo‐acting Enzymes
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Cellulose‘Cellulases’
Glucose (C6)
Hemicellulose C5 & C6; Sugar acids; acetyl, phenolic acids
‘Hemicellulases’
Pectins C5 & C6; Sugar acids; methyl, acetyl, phenolic acids
‘Pectinases’
‘Amylases’Starch Glucose (C6)
Non‐cellulosic β‐Glucans
Glucose (C6)‘Non‐cellulolytic glucanases’
‘Peptidases’Proteins Amino acids; small
peptides
Lignin‘Lignin‐modifying Enzymes’ Phenolic monomers;
oligomers
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• Complex task: Accessory + ‘Main‐chain’ polymer‐converting enzymes– e.g. xylans, pectins
• ‘Cocktails’ of these enzymes are essential for biomass/waste conversion
• Design & optimization of cocktails essential
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The need for Accessory & Depolymerizing enzymes, e.g. Xylan decomposition
Deconstruction of Hemicellulose: Cereal Xylans
Acetyl(xylan)esterase
-Glucuronidase
‘Accessory’ and main-chain enzymes required
Arabinofuranosidase
Ferulic (phenolic) acid esterases
Endoxylanase, Exoxylanase –act on polymer backbone -Xylosidsae – acts on oligosaccharides
+
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Cross‐links exist between biopolymers in Plant biomass: additional non‐carbohydrase
enzymes requiredOxidative enzymes, Esterases & peptidases essential
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Schematic of the polysaccharide structures in Pectins: A multitude of different enzymes required
RG IIRG I
Homogalacturonan
Arabino-3,4-galactan
Arabinan
Galactan
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Relevance of Accessory enzymes
Cellulose structure
Inter- & Intra-chain H-bonds between cellulose chains
In planta localization
Example 1: Cellulose Hydrolysis
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Classic Model of Hydrolysis: 3 essential types of ‘Cellulases’
ReducingEnd
Non-reducing end
CBH or Cellobiohydrolase; EG or Endoglucanase and BG or -Glucosidase.-Glucosidase hydrolyzes cellooligosaccharides and cellobiose to Glucose
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Current working model: Discovery of additional, very important Accessory enzymes
Horn et al (2012) Biotech Biofuels 5: 45
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Impact on Cellulose Hydrolysis
Langston et al (2011) AEM 77: 7007-7015
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Similar observations with effect of Accessory enzymes on Hemicellulose hydrolysis
Tuohy et al Biochem. J.
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Fungal Enzyme Systems or ‘Cocktails’– Focus on Thermophilic fungal sources– More recent work on Psychrophilic, Mesophilic, Highly thermophilic & fungi from isoteric ecological niches
– Detailed biochemical & molecular understandingAdvantages of Fungal ‘cell factories’
– Very adaptive, respond to nutrients/growth conditions– Enzymes produced extracellularly (secreted)– Eukaryotic processing can yield more stable enzymes– Multicomponent enzyme systems– Good biomass yields – scalable – existing commercial processes
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TCBB programme: • Biomass/Wastes to Energy & Biorefinery feedstocks• Collaboration with Industry partners – developing
solutions to waste problems & creating added value• Four ‘pillars’ or sub‐programmes: Agrigas, Wastegas,
CELLACTIC; CELLPHAWastes: • Agri‐residues – Straws/Stovers, Spent Mushroom
Compost, AD digestates, Manures, Dairy whey wastes• OFMSW ‐ Food Wastes, recovered paper & cardboard• Forestry – toppings, wood chip, sawdust, etc• Marine – Seaweed processing residues & liquors
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Wastes/Residuals Enzymes
FEEDSTOCKS
BIOFUELS
Fermentation
BiogasBioethanol
Hydrolysis**
ORGANIC ACIDS
Lactic acidSuccinic acid
BIOPOLYMERS
PHAPHB
Nutraceuticals; Bioactives
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OFMSW: Bioenergy
+ Fungal Enzymes
Starch & Cellulose-rich wastes/plant materials
Simple Sugars (glucose)
Biogas Bioethanol
Motor Fuel blendsHeat & Electricity
Example 1
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NUI, Galway Thermozyme cocktail: Bioconversion of Cellulose-rich PAPER wastes
0
1
2
3
4
5
6
7
8
9
10
0 3 12 24 48 72
Time (h
0
10
20
30
40
50
60
% E
than
ol y
ield
Ethanol (g/l)% Ethanol yiel
NUI, GalwayThermozymes
Paper productsbefore treatment
After 6 h
Copyright to National University of Ireland, Galway, 2008
Ethanol production
Example 2
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OP Control OP – E1 (40oC)
OP – E1 (70oC)
Physical effects of Enzyme action: Waste Office paper (SEM)
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Example 3: Similar study
with Newsprint
NP-control 40oC NP-control 70oC
NP-E1 40oC NP-E1 70oC
NP-E2 40oC NP-E2 70oC
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Cereal/ cereal residue
Mixing tank
Water
Thermozymes
Saccharification
Yeast
Fermentation Distillation & Dehydration
Storage tank
By-products for animal feed, Biogas or Thermochemical Energy/products
Biomass preparation & Pretreatment
Distillation column
CO2
16.6-27.9 L EtOH
~255 kg CO2
~ 17-20 kg residual biomass
16.6-27.9 L EtOHDepending on yeast used
100 kg dry Oat grains
~54.4 Kg C6 sugar+ ~20.5 KgC5 sugar
Steaming 121oC/30 min
67-70oC
24 h/67-70oC
Cooling to ~70oC
25-79 h/30oC
EtOH
~75.9% conversion
(~77.3% complex Carbohydrates)
Example 5: Bioconversion of Cereals: Small Pilot‐scale
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GRASS/ENSILED GRASS
FOOD WASTE
Direct & Indirect Anaerobic Digestion (AD)
STEP 1
Hydrolysis
STEP 4
MethanogenesisBIOGAS
Heat, Power, Biofuel
15‐30 Days
Enzymatic pretreament
6‐24 h3‐5 Days
Tuohy Laboratory Team & Prof. V. O’Flaherty’s Team
Acidogenesis AcetogenesisSTEP 2 STEP 3
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60oC C E1 E2 70oC C E1 E2 80oC C E1 E2
Food waste: 6 h post enzyme treatment
60oC C E1 E2 70oC C E1 E2 80oC C E1 E2
Food waste: 24 h post enzyme treatment
Example 4: Food Waste to Biogas
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GRASS/ENSILED GRASS
FOOD WASTEHydrolysis Methanogenesis
Volatile Fatty acids (VFAs)
Separation & Concentration (Dr. R. Babu, TCD)
VFA concentrate
BIOPLASTICS/BIOPOLYMERS
(Dr. K. O’Connor, UCD)
BIOPOLYMER Characterization/Composites
(Dr. R. Babu, TCD)
BIOGAS
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Marine waste streams
Marine Algal Ingredients
Textural, Gelling & Bulking agentsNutrition
Health & Pharma
Crop production
Food Security
Dietary Fibre Sensory & Organoleptic
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Work to‐date on Marine value‐added products Some examples from funded projects
• Novel Functional Beverages of Algal origin • Novel prebiotic bioactive oligosaccharides (clinical trial)• BiaSlan: Novel Carbohydrate bioactives for control of
Campylobacter infection in Poultry • Novel enzymes & functional ingredients with anti‐microbial
activities• Applications at Small pilot to Pilot scale• Close collaboration with Industry• New value‐added processes/value streams ‐ new partner
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Current Technology/Process development/IP
Mixed wastes(MSW)
Agri‐wastes
Food wastes
Mesophilic or Thermophilicenzymes
Mesophilic or Psychrophlilicenzymes
Thermophilicenzymes
AD Reactors
CSTR
Leachbed
UASB
Bioenergy
BiorefineryBioplastics
Waste Management
Specific Food waste streams
Thermozymes Nutraceuticals; Bioplastics
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Current Scale‐up & Challenges: focus on low‐cost, sustainable approach
Enzymes – ‘holy grail’ – reduce cost of enzymes in bioconversion applications by:
– Low‐cost production– Increased enzyme performance in conversion at lower dosages– Increasing enzyme half‐life and ‘recyclability
Breakthroughs to‐date:– Novel non‐GM approach to increase enzyme yield – 3‐50 fold
increase in enzyme output (associated IP) – Novel natural ingredient to increase bioconversion – lower
dosages possible; shorter reaction times (associated IP)– New non‐GM Fungal cell factories (associated IP)
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Small pilot‐scale Facility in NUI Galway
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Scale‐up & Development Targets
Demonstration Scale
Enzyme production
Target Waste to Bioenergy applications
Commercial Scale
2nd Generation & 3rd Generation Feedstocks
‘On‐site’ Integrated modules
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Interest in Thermophilic Fungi
• Ecological niche, e.g. compost• Grow after ‘mesophilic phase’• Complex polysaccharides in plant biomass = only substrates
• Produce complex, multicomponent enzyme systems to convert polysaccharides to simple sugars
• Very little known or understood to‐date –opportunities
• Regulation of enzyme production?• Higher stability of proteins – factors that enhance stability?
• Applications – advantages (pasteurization)
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Filamentous fungi – very important role in carbon recycling