CSIRO Enzyme Based Biorem

Technology

Cameron Begley, Commercial General MCSIROJune 2010
CSIRO.

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Recipient should conduct and rely upon its own investigation and analysis of the informat

that may be relevant to it in considering whether to acquire the Shares. Each Recipient co

make, and will be taken to have made, its own independent investigation and analysis of tIndependent expert advice (including from a Recipient's accountant, stockbroker, lawyer osought before making a decision to invest in the Shares.Not a document requiring lodgement with ASICThis Business Plan is not a prospectus or other disclosure document required to be lodgeCorporations Act as the Offer is only made to persons who, or in circumstances that, fall wdisclosure requirement contained in section 708 of the Corporations Act. Nor is a producrequired under Chapter 7 of the Corporations Act. Accordingly, this Business Plan does n

contained in a prospectus or other disclosure document prepared under the Corporations 

information that may be necessary or desirable to enable a potential investor to properly eShares.No responsibility for contents of Business PlanTo the maximum extent permitted by law, no representation, warranty or undertaking, expextent permitted by law, no responsibility or liability is accepted by CSIRO and its adviser

or advisers or any other person as to the adequacy, accuracy, completeness or reasonablextent permitted by law, no responsibility for any errors or omissions from this Business 

otherwise is accepted.This Business Plan contains various opinions, estimates and forecasts which are based u

correct or appropriate. Except to the extent implied by law, no representation or warranty of any of the assumptions or the accuracy of the information, opinions, estimates or forecby any of CSIRO and its advisers or any of their officers, employers, agents or advisers.

IMPORTANT NOTICE
CSIRO.

About CSIRO

• Australia’s major strategic and applied R&D • ~ Au$1Billion p.a. (~70% Parliamentary ap

external)

• Ranks in top 1% of world science institutio• Over 4000 science papers p.a.• ~4000 granted or pending patents• >100 successful spin-off companies
CSIRO.

LandGuardTM - Background (1) LANDGUARD TM• Unique suite of enzyme technologies• Tailored bio-remediation solutions to target 

specific compound classes

• Rapid acting, superior potency• Technology platform that addresses 

pesticide contamination in water extending to POS / POU horticultural produce

• Equity raising now commencing to 

accelerate production, product portfolio and market acceleration
CSIRO.

LandGuardTM - Background (2) 

• Widespread use of pesticide products has give

which trace residues of the applied products arpublic health and/or to the environment. Examp

• where pesticides are subsequently washed into d

enters public water-ways and ultimately into sour

• where pesticide residues remain on treated prod

restrictions to trade and consumption

• situations involving disposal of ‘waste’ after treat

livestock.

• ‘Landguard’ proprietary enzyme products have 

achieve low cost, rapid degradation of undesiraresidues without the need for high energy rememitigation or dilution through flushing high volu
CSIRO.

LandGuardTM - Market Opportunities 

Environmental Water

• UK vet pesticides remediation legislation passed in 

4Q10

• http://www.environment-agency.gov.uk/research/library/position/116250• http://www.environment-agency.gov.uk/research/library/position/116246• Incentive to use the product has been created for per

penalties for breach of permit is also anticipated 

• California broad acre pesticide remediation in draft

end 2011

• http://www.cdpr.ca.gov/docs/emon/surfwtr/regulatory.

Regulations

• http://www.waterboards.ca.gov/centralcoast/water_iss

order.shtml

• regulatory changes in California are expected to resu

15 parts per trillion (ppt) applicable to both OP and SP 

• There are also reports emerging in the media from 

increasing concern over environmental water qualipesticide use

• In Australia, there are drivers emerging for remedia

forestry operations and on sugarcane crops, with repotential damage to the Barrier Reef
CSIRO.

LandGuardTM - Market Opportunities (1 Municipal & Potable water

• Rising interest in public health and water quality a

consumer level

• Municipal and point of use / entry opportunities 

emerging in US, China and India

• Most significant segment identified to date is 

atrazine residues in mid west USA

• segmented by municipal water treatment and poin

of use (POU) / point of entry (POE) segments. 

• certain municipal opportunities will lend 

themselves to the use of enzyme based technologies, most likely where membrane systems require concentrate or waste disposal

• POU / POE opportunities will likely emerge where 

carbon (rather than membrane) media is the favoured and enzymes can be immobilized on a media to remove pesticide residues.
CSIRO.

LandGuardTM - Market Opportunities 

Industrial water and Horticulture & Sp

1. Industrial water

• Waste water treatment for specific organic 

residues

2. Horticulture & Specialty

• Commodity wash applications at point of 

packing / point of sale

• Commodity pesticide removal applications at 

point of sale / point of use

• Remediation of golf course irrigation run off
CSIRO.

Technical performance criteria

• We discover & improve enzymes with mod• The prerequisites are demanding:

• >2 logs detoxification• Preferably single step detoxification• No expensive co-factors• low Km (μM or ppm)

• respectable kcat (>100 turnovers/minute)

• broad substrate range within a pesticide c• bacterial expression (≥ 5% yield as enzym

• broad temperature range• broad pH range• shelf life in months• preferably proprietary platform IP

• But are generally achievable with modern b
CSIRO.

OpdA meets all the technical pe

Single step detoxif

• One step hyd

nontoxic prod

High catalytic effici

• Acts upon its 

diffusion- lim

Cofactor-less 

• Non-diffusible 

cost to enzy

Expression and sta

• Good expres

Patent position

• CSIRO propr

Suitable substrate 

• Targets most 

except for N-aliphatic non-

dimethoate)
CSIRO.

0

20406080

100120140160180200

0 5 10 15 20 

Time post enzyme addition (min)

Diazinon Concentration (ppm)

20,000 Units/100L 40,000 Units/100L 60

*: One Unit of enzyme activity is defined as the amount of OpdA that hparathion per minute at 25oC, in 50 mM Tris.HCl pH 8.0, 100 μM ethyl 

Approachidegradatio

LandguardTMOP-A: typical pesti
CSIRO.

Fate of Diazinon following La

By-products are always significantly less tox

LC50 Bluegill 96 hr 

EC50 Daphnid 48 hr 

LD50 Trout 

N

N

H3C

(H3C)2HC

O P

S

OCH2CH3

OCH2CH3

N

N

H3C

(H3C)2HC

OH P

S

OCH2CH3

OCH2CH3

HO
CSIRO.

Product safety

• Metabolite impacts of degraded OP using Lan

• Ecotoxicity issues for CSIRO’s ‘Landguard’ 

degrading enzyme, Dr John Oakeshott, CSIRO 2008

• Chlorpyrifos Residues in Corn Runoff Water

Management Specialist, UC Davis.

• Toxicity Assessment of Country Diazinon tre

A, Orica Australia Pty Ltd, Test Report, Novemb

• Project Report: The Use of Landguard OP-A to 

Chlorpyrifos in Alfalfa tail water – January 20

• Environmental Toxicity (from NICNAS, REACH 

• Acute toxicity (zebra fish) – not observed effect • Acute toxicity (daphnia) – not observed immobil

• Acute toxicity (algae) – not observed effect at 10

• Biodegradation rate after 28 days is 95%• Considered readily biodegradable and highly wa

term adverse effects in the aquatic environment 

1. Full N
CSIRO.

Current Product range and statu

• Landguard OP

• Degrades a broad range of organophosphate 

chemistries

• Freeze dried powder added manually (batch 

treatment) or via dosing system (continuous treatment)

• Distribution partners identified in California and 

• Landguard ZIM-A

• Degrades fungicidal carbamates

• Landguard SP

• Degrades a broad range of synthetic pyrethroid 

chemistries

• Landguard TRZ

• Trials completed in Queensland with 1 log 

reduction in 4 hours in a 1 ML pond starting with 160 ppb atrazine
CSIRO.

LandGuardTM - Technology pipe• Advanced research is underway to d

enzymatic products for degradation other pesticides of interest:

• Phenyl-ureas (diuron)

• Neonicotinoids (specifically imida

• Insecticidal Carbamates

• Benzimidazoles• Strobilurin • Paraquat• Glyphosate• Chlorinated hydrocarbons
Thank you

For further information:Cameron BegleyGeneral ManagerCameron.Begley@csiro.au

Acknowledgements:CSIRO: John Oakeshott, Robyn Russell, Colin ScHartley, Colin Jackson, Matt TaylorOrica: Steve Kotsonis, Craig Clarke, Kate DawsonHorticulture Australia Limited, Rural Industries ReCorporation, CRC Sugar Industry Innovation throuCentral Science Laboratory (UK), CEMAS (UK), RAgentase (USA), Hydrophilix corporation (USA), U

(Davis), Stewart Agricultural Research Services (UUniversity of The PhilippinesChung-Hsin University, Taiwan

Australian Centre for International Agricultural ReServices, Agrifood Technology, Entox (Queenslan

Ag and Virbac Pty Limited 
CSIRO Enzyme Based Biorem

Technology – Technical Inform

Cameron Begley, Commercial General ManagerColin Scott, Research ScientistCSIROFebruary 2010
CSIRO.

3⁄4 17 Hectares of cotton was sprayed with methyl3⁄4 Field flood irrigated 20 hrs later3⁄4 Irrigation return channel (80,000L) treated with 

 Untreated irrigation run-off residue levels var

the first 40 min Treated irrigation run-off residue levels were reto the water source

3⁄4 Repeat performance on 4000ha

Irrigation Run-Off Treatmen
CSIRO.

0

20406080

100120140160180

0 

[Atrazine] μg/L

AB

An atrazine contaminated holding damVolume = 1.5 megalitres

Application of Enzyme

Ti

All very good but – can we use T

bioremediant?

>90
CSIRO.

Use Landguard OP in pesticide evaporation ponds 

Addition of Landguard at low rates (200g/ML), achi>50% reduction in profenofos>90% reduction in m-parathion and chlorpyrifos 

in 2 weeks
CSIRO.

Time (min) 0 2 5 10 

OpdA treated 31 21(32%) 6.2(80%) 3.4(89%) 0

Methyl parathion concentrati

achieved by OpdA enzyme 

Treatment of contaminated sprrinsate using Landguard OPA
CSIRO.

Diazinon in sludge (soil and organic matter) reduwithin 3 hours following the application of Land

20g/100L

Treatment of sheep dips using L
CSIRO.

Applications of Landguard OPA to spesticide applications

Landguard

Dose rate

g/ha

0 

100 250 500 

500 + 1-mm rain 

0 

100 250 500 

500 + 1-mm rain 

LandguardTM OP-A sprayed onto the ground directly afte

to prevent pesticide run off into surface water following ra
CSIRO.

Using Landguard OPA for on-commdetoxification

• Tested cucumbers,

tomatoes, egg plant, bitter gourd, mangoes... (& wool)

• Tested ~10 OPs• Low dose (2g/100L) 

treatment for ~ 5 minutes

• Up to 80% OP removal• But not yet consistent*
CSIRO.

9Single step detoxification

9 One step hydrolysis yielding short-lived, nont

9High catalytic efficiency 

8 Wild-type has low activity9 Activity improved by enzyme engineering

9Cofactor-less 

9 No cofactor

9Expression and stability

9 Good expression and extremely stable

9Patent position

9 CSIRO proprietary

9Suitable substrate range

9 Targets most major SPs)

•

SP performance: E3 did not initiathe technical performance criteri

05

101520253035
CSIRO.

9Single step detoxification

9 One step hydrolysis yielding, nonherbicial pro

9High catalytic efficiency 

9 Acts upon its best substrates at high rates (> 1

9Cofactor-less 

9 Non-diffusible metal cofactor, no added cost t

use

9Expression and stability

8 Wild-type yields little protein9 Improved yield by enzyme engineering

9Patent position

9 FTO 9 Improved enzyme CSIRO owned

9Suitable substrate range

9 Targets all major triazine herbicides

TrzN did not initially meet all the performance criteria

kDa 

5037.52575