ReNEW Technology Foresight Workshop23‐24th April 2013
Enzyme Production & Enzymatic conversion of lignocellulosic feedstocks & wastestreams
Dr. Maria TuohyTCBB & NUI Galway
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
• 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’)
• 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)
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
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
• 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
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
+
Cross‐links exist between biopolymers in Plant biomass: additional non‐carbohydrase
enzymes requiredOxidative enzymes, Esterases & peptidases essential
Schematic of the polysaccharide structures in Pectins: A multitude of different enzymes required
RG IIRG I
Homogalacturonan
Arabino-3,4-galactan
Arabinan
Galactan
Relevance of Accessory enzymes
Cellulose structure
Inter- & Intra-chain H-bonds between cellulose chains
In planta localization
Example 1: Cellulose Hydrolysis
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
Current working model: Discovery of additional, very important Accessory enzymes
Horn et al (2012) Biotech Biofuels 5: 45
Impact on Cellulose Hydrolysis
Langston et al (2011) AEM 77: 7007-7015
Similar observations with effect of Accessory enzymes on Hemicellulose hydrolysis
Tuohy et al Biochem. J.
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
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
Wastes/Residuals Enzymes
FEEDSTOCKS
BIOFUELS
Fermentation
BiogasBioethanol
Hydrolysis**
ORGANIC ACIDS
Lactic acidSuccinic acid
BIOPOLYMERS
PHAPHB
Nutraceuticals; Bioactives
OFMSW: Bioenergy
+ Fungal Enzymes
Starch & Cellulose-rich wastes/plant materials
Simple Sugars (glucose)
Biogas Bioethanol
Motor Fuel blendsHeat & Electricity
Example 1
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
OP Control OP – E1 (40oC)
OP – E1 (70oC)
Physical effects of Enzyme action: Waste Office paper (SEM)
Example 3: Similar study
with Newsprint
NP-control 40oC NP-control 70oC
NP-E1 40oC NP-E1 70oC
NP-E2 40oC NP-E2 70oC
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
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
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
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
Marine waste streams
Marine Algal Ingredients
Textural, Gelling & Bulking agentsNutrition
Health & Pharma
Crop production
Food Security
Dietary Fibre Sensory & Organoleptic
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
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
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)
Small pilot‐scale Facility in NUI Galway
Scale‐up & Development Targets
Demonstration Scale
Enzyme production
Target Waste to Bioenergy applications
Commercial Scale
2nd Generation & 3rd Generation Feedstocks
‘On‐site’ Integrated modules
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)
Filamentous fungi – very important role in carbon recycling