1

Environmental Risk Assessment of Genetically Modified algae and cyanobacteria:

potential concerns and possible solutions

Santanu Dasgupta

Reliance Industries Limited, Navi Mumbai, India

2

DISCLAIMER

Any statement, opinion, prediction, comment, or observation made in thispresentation/publication are those of the presenter/author only and in no condition should beconstrued necessarily representing the policy and intent of Reliance Industries Ltd. (RIL).

The information presented herein are of the presenter/author’s own and in no way RILattracts any liability for any inconsistency or irregularity in terms of the accuracycompleteness, veracity, or truth of the content of the presentation/publication. In addition,RIL shall not be liable for any copyright infringement and misrepresentation for thepresented content as the content is presumed in good faith to be a creation ofpresenter’s/author’s own mind.

The scope of this presentation/publication is strictly for knowledge sharing purposes andnot necessarily to provide any advice or recommendation to the audience/readers. Anyendorsement, recommendation, suggestion, or advice made by the presenter/author shall bein his personal capacity and not in professional capacity as an employee of RIL. Any personacting on such endorsement, recommendation, suggestion, or advice will himself/herself beresponsible for any injury/damage.

3

DhirubhaiH.AmbaniFounderChairmanRelianceGroup

• 1966- Establishes Textile mill in Naroda

• 1977- Public share offering Raised money from public

offerings oversubscribed 7X

• 1982- Builds fiber/filament plant in Patalganga

• 1995- Builds Polyolefin plants at Hazira

• 1997- Builds multi-feed cracker at Hazira

• 1999- Builds Jamnagar refinery

• 2002- Acquired IPCL a state run company

• 2008- Builds JERP refinery at Jamnagar

• 2009- Begins KG D6 gas production

• 2013- Builds PBR/SBR plants at Hazira

• 2014- Builds PTA plant at Dahej

• 2014- Builds PET plant at Dahej

Reliance Industries Limited - brief introduction

© Reliance Industries Ltd., 2016

4RIL: A significant commitment to renewables R&D, major focus on advanced biotechnology§ Large number of scientists and engineers working in India, trained in the best

institutions globally§ International collaborations with top notch institutions, leveraging talent of

additional Two hundred scientists and engineers§ Solutions for the entire spectrum of landmass

Ø Barren land: Algae (use sea-water and desert land at coastline)Ø Marginal land: Jatropha (develop high yield varieties in low rainfall areas)Ø Fertile land: Surplus agri-residue (at least 100 MM MTpa biomass resulting in 25 MM

MTpa biofuel)§ Adopt PM’s triple S mantra: Skill, Scale and Speed for these challenging problems

Algae oil

RIL committed to largest cutting-edge R&D program by any private enterprise to help India leap-frog

Agri-residue to kerosene Jatropa to bio-diesel Algae to bio-crude

© Reliance Industries Ltd., 2015

5

Stacking traits

Genetic Engineering

Mutation

Cultivation Optimization

Outdoor stabilization

Growth Optimization

Lab domestication

Native Strain from Nature <1 gm/m2/day

10 gm/m2/day

20 gm/m2/day

15 gm/m2/day

25 gm/m2/day

45 gm/m2/day

35 gm/m2/day

35 gm/m2/day

Incremental productivity improvement through genetic engineering could be the key for successful algae business

{

FTO with Genetic Engineered algae is a must

6

USE OF BIOLOGICAL MATERIAL FOR RESEARCH AND INDUSTRY IS CONTROLLED BY REGULATIONS LAID DOWN BY THE GOVERNMENT OF INDIA

Regulatory implications are much more complex in an R&D with living organisms

7

One of the Must-Win Battles in Any Algal Biotechnology Centres Around Regulatory Matters

Challenges Are:

§ Collecting organism§ Import and export of live algae involve complex governmental procedures embroiled

in bureaucracy§ Algae modified through genetic manipulation need to go through complex regulations

for environmental release§ A regulatory environment enabling complete FTO for algae research and

commercialization is a must

Regulatory Challenges for Algal Biotechnology

8Collection of Bioresources and Exchange with

Overseas Collaborators

§ India – A Party to the Convention on Biological Diversity (CBD) (1992).

§ Recognizing the sovereign rights of States to use their own biological resources,CBD expects the Parties to facilitate access to genetic resources by other Partiessubject to national legislation and on mutually agreed upon terms (Article 3and 15 of CBD).

§ The Biological Diversity Act, 2002 (Biological Diversity Rules, 2004) is as perCBD Guidelines

§ Collection of germplasm from Indian coastline, export of strains to overseascollaborators and filing of IP using the germplasm need clearances requiredfrom the National Biodiversity Authority (NBA), Ministry of Environment &Forests

9

Regulatory Challenges for Algal Biotechnology

In India, import of strains needs a clearance from the Directorate of Plant Protection, Quarantine & Storage (PPQS), Ministry of Agriculture

Classifies algae as an agricultural pest/pathogen!

Regulated by Plant Quarantine (Regulation of Import into India) Order, 2003 (revised guidelines 2015)issued under the Destructive Insects & Pests Act, 1914

q Lack of a defined regulatory framework for algal biotechnology is an impedimentq Lack of adequate understanding of algal biology among regulators leaves requirements to

individual interpretation; elaborate studies are arbitrarily asked forq These elaborate and arbitrary studies result in increasing the project costs and timeq Consequently, there are inordinate delays in receiving strains from overseas collaborators and start

of projects

10India Regulatory Framework for GMO:

Key players- Current

n Ministry of Environment and Forests (MoEF)– Responsible for the manufacture, import, use, research, and commercialization of

GMO’s under ‘Rules 1989’ (EPA Act, 1986)– GEAC: Genetic Engineering Appraisal Committee (31+ members)

Large Scale trial approvals and Environmental release recommendation

n Ministry of Science Technology- Department of Biotechnology (DBT)– Responsible for guidelines for the safety evaluation of GMO– RCGM: Review Committee on Genetic Manipulation (28+ members)

• Transgenic import for research, Research trial approvals, safety studies

n Ministry of Agriculture- Indian Council of Agricultural Research (ICAR)

10

11

RCGM(DBT)

Small-scaleFT Permit

Monitoring & Evaluation

Committee (MEC)

ImportInstituitional

Biosafety Committee (IBSC)

Large Scale FT Permit

State BiosafetyControl

Committee (SBCC)

GEAC(MoEF)

Department of

Biotechnology (DBT)

State BiosafetyControl

Committee (SBCC

Testing by ICARCommittee

for the Purpose of Control

&Supervision of

Experiments on Animals (CPCSEA)

Regulatory Process for field testing of GM crops

Research Commercial

12

Conceptual Flow Chart for Environmental Risk Problem Formulation for Escaping GM Algae

13

Algae modified through genetic manipulation need to go through complex regulations for environmental release

Recombinant DNA Safety Guidelines

§ Greenhouse and PBR facility design forcompliance with the biosafety guidelines

§ Proof-of-concept experiments using wildtype strains to test their ability to spreadand persist in environment

§ Data from these experiments will helpformulating guidelines for biosafetyexperiments using recombinant algae

GEAC (MoEF) Approval

14

Proof of Concept

• Conceptualization and execution of a “limit-of-spread/dispersal” experiment

PCR

• Prior to and post cultivation monitoring• Detection in soils and water bodies in a defined radius

Water bodies

• Drainage channels, streams, rivers, wells, ponds, lakes and sea

Molecular Monitoring Methods (for mechanical and natural means of spread)

Regulatory Science: GM Strains

15

Avianfauna

• Controlled experiments with chicken fed with culture

Water animals

• Controlled experiments with catfish and tadpoles grown in tanks with algal strain as a monoculture

Toxicology• As defined in Codex Alimentarius

Additional Data Required(for mechanical and natural means of spread)

Regulatory Science: GM Strains

16

PCR • Recombinant gene’s sequences to be used for validation

PCR

• Site of integration to be determined to assist in investigating:• (a) Disruption of any important functional gene

Water bodies

• (b) Identification of wild type versus GM strains in relevant water bodies

Additional Molecular Data for GM Strains

Regulatory Science GM Strains

17

Regulatory Challenges for Algal Biotechnology

Environmental Release of GM Algaeq Environmental Risk Assessment procedures for GM algae not formulatedq Guidelines yet to be preparedq Department of Biotechnology, GOI is preparing a set of regulations for environmental release of

GM algaeq GM algae is the only way-forward for trait development; active engagement between stakeholders

and government regulators required

Precedence of Open Pond Trial of GM Algae

Only one report so far – In 2013, EPA approved an open pond trial of a genetically modifiedScenedesmus conducted by Sapphire Energy within the UCSD campus

18

§ Algal research and associated Regulatory framework – a comparatively new field

§ Major learnings from plant research can be taken to formulate one

§ Bottlenecks anticipated due to the worldwide absence of regulatory framework for algae

§ More regulatory science based experiments to be carried out for GM algae to anticipate and mitigateenvironmental hazard associated with it

§ Genetically modified algae currently can be grown in closed bioreactors or green houses till thetime adequate information available regarding their spread or effect on the native ecosystem ifcultivated outdoors.

§ National Biodiversity Authority’s provisions were highly restrictive and their processing time andapprovals took an extraordinarily long time

§ Algae and Cyanobacteria either need to be classified as a “crop” (as done with legislations in Ohio,Arizona and Iowa) OR as an economically useful microbe (e.g., yeast for brewing)

Summarized Recommendations of Different Panels

Opinion Building Among Key Stakeholders

19

§ A framework for biosafety guidelines for algal cultivation needs to be created.Governments and international bodies could invite relevant stakeholders from industryand academia to brainstorm with the objective of defining the protocols for both wild-type and recombinant algae and cyanobacteria

§ The International Society for Biosafety Research (ISBR) is organizing the 14th

International Symposium on GMO at Guadalajara, Mexico in June 2017

§ A session on Environmental Risk Assessment (ERA) of GM Algae

§ This will be a unique opportunity to brainstorm on the subject and discuss the possibleguidelines

A UNIQUE REGULATORY BODY FACILITATING SINGLE-WINDOW CLEARANCE OF R&D PROJECTS INVOLVING BOTH WILD-TYPE AND GM ALGAE IS DESIRABLE FOR SUCCESS

OF THE ANY ALGAL RESEARCH PROGRAM