Applied Biochemistry: Eirzyme's Molecular Enzymology for ...

Applied Biochemistry: Eirzyme’s MolecularEnzymology for Bioenergy production

Copyright to National University of Ireland, Galway, 2008

ABIC 2008University College Cork

Dr. Maria Tuohy

Molecular Glycobiotechnology GroupDepartment of Biochemistry, NUI, Galway, & Eirzyme, Ireland

Our Location: NUI, Galway!


Biomass to Bioenergy: Key Goals, Challenges &Barriers

Global Energy targetsEnvironmental benefits

SustainabilityBiofuel crops

Technological challenges?Barriers?

CO2


Market Size and Targets: Bioethanol

Global Bioethanol Market:2002: 17 mTon (€10,249 billion)2020: Predicted 5-fold market increase

European Bioethanol market:2004: 0.77 mTon (€367 million)Annual growth rate of 72%

Irish Bioethanol market:2005: 0 mTon2010: 0.07 mTon

221 mL BioETOH (€34.1million)

1 hectare ≈8-10 tonnes wheat≈2.4-3.0 ton bioethanol

Bio-Energy from MSW (Irl)2004: 6,438,085 tonnes to landfill

3,476,565 tons = paper/organics (54% total waste)

Cost: €906 million - €1,416 million

At 60% conversion to Biofuel: 160,952 MegaW Power Heat 70,000 homes /annum Cost saving to landfill:€560 million - €850 million


1st Generation Biofuel technology:Corn to Ethanol


Food vs Biofuel!


Bioenergy Process targets

Turner et al. Microbial Cell Factories 2007 6:9

Non-food crops& wastes

Novel crops, incl.transgenic crops

Better pretreatmentsNovel low-cost Enyzmes/

Enzyme systemsHeat stable &

improved specificity

Improved fermentation yieldsOvercoming the C5 problem

Novel microorganismsAlternative biofuels

Recovery & Exploitationof Co-products

The ‘Biorefinery’ Concept


2nd Generation Biofuels:Technologies & Goals

• Lignocellulosic biomass – woody residues, cereals,miscanthus, sugar beet, novel crops – marine & terrestrial

• Transgenic crops & novel crop species/strains• Agri- & Municipal wastes• More cost effective, less toxic pretreatments• Novel cellulases & hemicellulases

– heat stable; better specificity/performance• Novel accessory enzymes (oxidases, esterases)• Pentose-fermenting, EtOH-tolerant yeast strains• Novel fermentation microorganisms/next generation

biofuels• Co-product recovery & By-product recycling


Eirzyme’s Core Objectives & Goals

• Scale-up and commercial development of Optimizedthermostable enzyme technology to unlock the fullpotential of biomass to produce feedstocks enriched insimple sugars & to enhance downstream bioenergyproduction

• Target biomass substrates: 2nd generationfeedstocks/crops; Wastes, including Municipal SolidWaste, key source-segregated wastes and 1st generationbiofuel wastes (DDGS & Corn stover)


Molecular GlycobiotechnologyGroup, NUI, Galway

Spin-out Company: Eirzyme

Research Team BioEnzTech Team (Pilot plant)Senior scientist, Post-doc & Project ManagerPhD Students Research AssistantsErasmus students, national Fermentation scientist& international summer students M.Sc. student


Molecular Glycobiotechnology Core Facilities:• Molecular Biochemistry, Cell Biology & Genetics

Laboratory – enzyme biodiscovery, functionalcharacterization & co-product analysis

• BioEnzTech Fermentation Technology Unit (100 LEnzyme production capacity) – enzyme scale-up

• Eirzyme Product & Process Development laboratory;Development of Large-scale enzyme productionfacility


Technology development: Eirzyme

NUI,Galway Enzyme TechnologySource Microorganism

Enzymeproduction Metabolism &

Cell Biology

Genetics &gene regulation

Optimized Enzyme Cocktails


Proteomics Genomics

Each Cocktail – biochemical ‘fingerprint’

T. emersonii - a treasure trove ofBiotechnological ‘goodies’!!

Cellulases

Pectin-modifyingenzymes

Hemicellulases

Peptidases

Oxidases/PeroxidasesOxidoreductases

Starch-modifyingenzymes

Lignin-modifyingenzymes

Novel carbohydrate-activeenzymes

Various esterases

Key Advantages of NUI,Galway EnzymeTechnology over competitors

• Derived from a ‘Generally regarded as Safe’(GRAS) Source’

• Unique & large portfolio of enzyme preparations• Multiple applications, different industrial sectors• Readily optimized for target applications

• Characterized by Thermostability– higher reactivity and efficiency,– shorter reaction times,– pasteurization of feedstock,– greater product yield


Target substrates: A wide range ofCarbohydrate-rich raw materials

Primary sources

Terrestrial & Marine plants,Fruits to woody tissues

Secondary sources

Processing by-productsVarious Wastestreams

Value-added products

NUI, Galway PlatformTechnology


Overview of Applied Biochemistry

Patents Patents

Biofuel production– lab-scale studies


Key steps in Cocktail development

• Detailed understanding of profile of enzyme activitiespresent

• ‘Fingerprint’ for each cocktail – chromatographic andproteomic analyses (‘de novo’ sequence data)

• In-depth biochemical characterization – functionalitytests, mode of action/products of hydrolysis with modeland target susbtrates

• Effect of reaction parameters on enzyme function• Novel activities characterized/cloned – detailed

characterization studies• Thermostability and stability on storage• QC & validation

BioEnzTech FermentationFacility


MGBG: Technology Development to date

Development of a unique portfolio of proprietary,intelligent/designer enzyme preparations of native andrecombinant origin

Novel approaches for the optimized production of targetenzyme cocktails – low-cost strategies

Application of novel enzymes/enzyme cocktails to thebioconversion of existing and new biofuel feedstocks

Increased enzyme yields ~3-5 fold at pilot-scale andproduction time by decreased 36-72 h (COSTSAVINGS)

Pre-pilot and pilot scale validation of enzyme production& catalytic properties.


Biomass conversion, Bioenergy &Biorefinery applications

Applied Biochemistry: Selected Examples


Biofuel/Bioenergy Strategies

+ Thermozymes

Substrate

Residue (lignin)

Simple sugars

+

Biogas Bioethanol Flavour & AromaprecursorsAntioxidantsBiopharmaceuticals

Value-added products


Complexity of the plant materials: Challenging!

Somerville et al., (2004) Science, 306: 2206-2211

Enzymes that fragment : Attacks internal linkages in a random manner(Endo Hydrolases) [ ]n

Enzymes that convertsmaller sugar chains to

simple sugars(Endo & Exo Hydrolases)

Generate Monomers (monosaccharides) for Anaerobic digestion(biogas) & Yeast fermentation (bioethanol)

Enzymes – tools to convert polymericcarbohydrates in biomass to simple sugars


Low-cost Enzyme cocktail for conversion of Woodyresidues

Best, optimized Commercial enzymepreparation (40oC)

T. emersonii enzyme cocktail (40oC)

Spruce fibres – no enzyme

Complete lossof fibre structureat highertemperatures

Enzyme stable

Complete loss ofenzyme activityat highertemperatures

Extensive cellulosedegradation


Other Terrestrial Biomass Substrates• Project title: ‘Thermozyme technology for

production of high-value products from Sugarbeet’

• Project title: ‘A sustainable strategy forBioethanol production in Ireland’ - A wide range ofCereals, Cereal Residues & Grasses

• Others: Sugarcane, various Agri-residues, Foodprocessing residues.

• Results: Enzyme cocktails – highly efficientrelease of simple, fermentable sugars (68-89%conversion)


Enzyme technology for the bioconversion ofCereals & Sugar beet

0.0 5.0 10.0 15.0 20.0 25.0 30.00

20

40

60

80

100 nC

min

0.0 5.0 10.0 15.0 20.0 25.0 30.00

20

40

60

80

100 nC

min

Arabinose

Arabinose

Xylose

Xylose

Glucose/Galactose

Glucose/Galactose

Sucrose

Sucrose

Galacturonic acid

Galacturonic acid

nCn

C

Min

Min

(B)

(A)

Simple sugars released from sugar beetby cocktails 1 and 5

Sugar Beet - no chemical pretreatmentCocktail 1: Glc (47.6%), Gal (8.8%), Xylose (20.2%),Ara (19.3%) & GalUA (4.1%)Cocktail 5: Glc (49.3%), Gal (5.6%), Xyl (26.0%),Ara (9.3%) & GalUA (9.9%)

Cereals & cereal Grains: mechanical discruptionoat straw, oat grain, wheat bran and mixedretail flour: 70-92% conversion of polymericcarbohydrates to simple sugars~65-72% are monosaccharides, with Glc(42-51%),Gal/Man (6-8%), Xyl (17-23%), Ara (6.5-21%)

In progress: Distillers grains (with Teagasc)


Waste Valorization & Management

RESOURCE RECOVERYEnergy & Value-added

By-productsWASTE MANAGEMENT

Volume reduction

Environmentally friendly Bio-Technology


A wide variety of Wastes: Cellulose-rich Clinical Wastes, MSW,Food wastes, Print & card wastes

NUI, Galway Thermozyme cocktail: Bioconversion ofCellulose-rich PAPER wastes

0

1

2

3

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5

6

7

8

9

10

0 3 12 24 48 72

Time (h)

0

10

20

30

40

50

60

%E

tha

nol

yiel

d

Ethanol (g/l)% Ethanol yield

NUI, GalwayThermozymes

Paper productsbefore treatment

After 6 h


Ethanol production

Visible changes in paper products

No Enzyme treatment Enzyme treatment for 24 h

Reduced reaction time at 65-80oCNo chemical pre-treatment required – shredding or roughmaceration enhances sugar yield

XyloseGlucose


0.0 5.0 10.0 15.0 20.0 25.0 30.00.0

10.0

20.0

30.0

40.0

50.0 nC

min12

3 4 5 6

7

8

9 10 11

Az

Range of Paper products screened

• Newsprint• Office Paper• Brown paper & Card• Coloured print & Card• Glossy print• Catering products• Tissue products• Packaging

Process parameters:PretreatmentTemperaturepHDuration of reactionMoisture content

Steam pretreatment > chemical pretreatment


Feedstocks for Biogas production

Target Biomass Substrate

Sugar-rich hydrolyzate

Anaerobic Digestion

Thermozyme treatment

Biogas

Other gases

~45-65% Methane

Shorter Retention times –potential for higher loadingrates and reduced reactor size/costs

Upflow Anaerobic Hybrid Reactors


Sources of OFMSW (Food Waste – Nonanimal)

Catering &Restaurants

DomesticUniversity Caterers

Local Coffee shops

Fruit & VegetableSuppliers

Fruit processors

Alcoholic beverageproducers

Bakery & Confectionery

Florists

Grain processors


0.0 5.0 10.0 15.0 20.0 25.0 30.00.0

5.0

10.0

15.0

20.0

25.0

30.0 T24 A1 Dil 1&20 12Oct07 #1 T24 A1 dil20 12Oct07 ED_1nC

min

12

34

5

6

7

89

10 11

12

13

Glc

Gal

/Man Dis Suc

Maltose

Food Waste – 24 h E1

FW#2-Enzyme 1 (Replicate batches)

0

100

200

300

400

500

600

700

800

900

0 h 6 h 17 h 24 h 48 h 72 h

Timepoint (h)

mg

/gR

ed

uci

ng

Su

gar

Sub Control A

Sub Control B

Sub Control C

Test A

Test B

Test C

Mixed Food Waste

% Conversion of Food waste polysaccharides:65.4-94% on a DW basis. In addition, 72.0-92.5%of the available carbohydrate was converted toreducing sugars at 24 h and 72.8-96.2% at 72 h.~18-30% = monosaccharides; remainder = di &trisaccharides


Mixed Food Waste

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

%C

onv

ers

ion

of

Av

aila

ble

Ca

rbo

hyd

rate

Control Test 1 Test 2

Reactor

% Conversion of Available Carbohydrate in Mixed Food Waste

% Conversion

Fermentable sugars(leachate)

3.5d/~40oC

Biogas: 317 kWh/tonne(Heat for 10.8-17.8 houses p.a.)

Food Waste

Thermozyme40oC

71-83% conversion

+ E


Unsorted MSW%Conversion of Carbohydrate in Biodegradable fraction of Initial

waste

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

0 h 3 h 6 h 21 h 24 h 30 h 48 h 72 h 96 h 120 h 144 h

Incubation time

%C

onve

rsio

n

100 E (Initial)

No E(Initial)

~40% of the Biodegradablefraction

Fermentable sugars(leachate)

6 h/~40oCThermozyme

0 h 6 h

Yield can be increased ~2-fold at 70oC>80-85% is monosaccharide;remainder = di- and higher oligo-saccharides.Glc ~ 50-55%; Fru ~29-33%.

Methane: 40-60% of biogas


Steam pretreatment > chemical pretreatment

AcknowledgementsEnterprise Ireland (Basic, Strategic, Applied Research, RIF & C-Plus

awards; Innovation Partnership; IP protection; Start-up and IP protection);DAF/FIRM and RSF NDP HEA; European Union; EPA – ERTDI

MGBG Team, NUI, GalwayBioEnzTech team & Eirzyme, NUI, Galway; Prof. E. Colleran and Prof.V. O’Flaherty, Dept. of Microbiology, NUI, GalwayTTO, NUI, Galway

Teagasc Oakpark Research Centre, CarlowInternational Collaborators:VTT, Helsinki, Finland; KTH, Stockholm; RUGhent, Belgium


The Future: Power to the Plants!!


Applied Biochemistry: Eirzyme's Molecular Enzymology for ...

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