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