David Glass Presentation at Alternative Fuels & Chemicals Coalition Biobased Economy Conference, November 15, 2021. Discusses the potential for uses of modified algae and cyanobacteria for production of chemicals and fuels, and how such uses would be regulated in the US and elsewhere in the world.

1. Applications and Regulation of
the Use of Photosynthetic
Microorganisms in Biobased
Manufacturing
David J. Glass, Ph.D.
D. Glass Associates, Inc.
November 15, 2021

2. Photosynthetic Microorganisms
• Algae (microalgae)
– Eukaryotic organisms, often aquatic
– Microalgae can be cultivated for industrial
purposes (open ponds or photobioreactors)
– Common industrial species include Chlorella,
Chlamydomonas, Nannochloropsis
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• Cyanobacteria (aka blue-green algae)
– Prokaryotic microorganisms
– Can be grown in photobioreactors for
industrial purposes
– Common industrial species include
Prochlorococcus, Synechocystis, Synechococcus

3. POTENTIAL USES OF PHOTOSYNTHETIC
MICROORGANISMS TO PRODUCE VALUABLE
BIOBASED PRODUCTS
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4. Industrial Uses of Algae or Cyanobacteria
• Foods, feed, dietary supplements, fragrances, etc.
• Production of Chemicals
– Butanol
– Ethylene
– Farnesenes
• Production of Fuels
– Ethanol
– Gasoline, Jet Fuel, Diesel
• Carbon sequestration
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5. Strategies to Improve Algae or Cyanobacteria
for Industrial Use
• Enhance or alter lipid biosynthesis for improved diesel, jet fuel
production.
• Enable or improve secretion of lipids.
• Enhance photosynthesis; improve carbon fixation.
• Metabolic engineering to enhance existing pathways.
– Maximize carbon flow to desired product(s)
– Eliminate competing pathways
– Remove toxic, harmful compounds
• Introduce new pathways for desired products.
• Impart tolerance to desired chemical products.
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6. Companies Modifying Algae or Cyanobacteria
for Chemical Production
• CemVita Factory: photosynthetic bacteria for CO2 capture and the
production of bioethylene.
• Photanol: cyanobacteria to manufacture organic acids and other
chemicals.
• Phytonix: modified cyanobacteria to produce biobutanol,
biooctanol.
• Solaga: phototrophic bacteria to produce biogas (also algae for air
purification).
• Viridos (formerly Synthetic Genomics): algal genomics to produce
low-carbon intensity biofuels.
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7. REGULATION OF INDUSTRIAL BIOTECHNOLOGY
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8. Overview of U.S. Biotechnology Regulation
Environmental Protection Agency
• Microbial pesticides, plant pesticides. (FIFRA)
• Engineered microorganisms used for other industrial purposes.
(TSCA)
U.S. Department of Agriculture
• Transgenic plants, potential plant pests.
• Plant-produced industrial products.
Food and Drug Administration
• Foods, food additives, pharmaceuticals, cosmetics.
• Animal drugs, foods and food additives
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Uses of algae, cyanobacteria for foods, feed, food
additives, cosmetics.
Uses of algae, cyanobacteria for fuels
and chemicals.

9. History and Scope of TSCA Biotechnology
Regulation
• Toxic Substances Control Act of 1976: “Gap-filling” statute to
cover chemicals not regulated elsewhere in the government.
• In the 1986 Biotechnology “Coordinated Framework”, it was
decided to use TSCA to regulate uses of microorganisms not
regulated by other federal agencies.
• Interim policy took effect in 1986, but final regulations (40 CFR
Part 725) not issued until 1997.
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10. Industrial Applications Subject to TSCA Jurisdiction
TSCA Biotechnology Rule covers uses of “new microorganisms”
used for the following purposes.
• Nonpesticidal agricultural microorganisms.
– Rhizobia for nitrogen fixation
– Biostimulants
• Production of pesticide intermediates.
• Bioremediation, biotreatment.
• Manufacture of industrial enzymes.
• Biofuel, bio-based chemical manufacture.
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11. Definition of “New Organism” as “Intergeneric”
Organism
• TSCA covers only “new chemicals” used in commerce, so the
Biotech Rule covers only “new microorganisms”.
• “New microorganisms” defined as “intergeneric”: i.e., containing
deliberate combinations of coding nucleic acids from more than
one taxonomic genus.
• Organisms modified solely by classical mutagenesis, gene
deletions, directed evolution or gene editing, with no intergeneric
sequences, would be outside of TSCA scope.
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12. Overview: TSCA Biotechnology Regulation
R&D Use Commercial Use
Contained Use Exempt: self-certifying, no
approval needed; largely
independent of scale (may
include pilot-plant uses)
Microbial Commercial Activity
Notice (MCAN):
File 90 days before commercial use
or importation
Open-Pond or Outdoor Use TSCA Experimental Release
Application (TERA):
File 60 days before beginning
outdoor experimentation
Microbial Commercial Activity
Notice (MCAN):
File 90 days before commercial use
or importation
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13. Microbial Commercial Activity Notifications
(MCANs)
• Commercial use or importation of “new microorganisms”
requires MCAN reporting at least 90 days before commencing
commercialization or importing microorganism.
• Applies to contained manufacturing and commercial uses in
the environment.
• MCAN requires submission of data package and other
information to EPA.
• Note: in some cases, the chemical product may itself require
Premanufacture Notification as a “new chemical” under TSCA.
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14. TSCA Environmental Release Application
(TERA)
• Outdoor R&D (”deliberate releases”) requires prior EPA approval
under TSCA Environmental Release Applications (TERAs).
• TERA must be submitted at least 60 days before planned use,
requires EPA approval.
• If EPA determines that the proposed R&D does not present an
unreasonable risk to health or the environment, EPA will approve
the TERA.
• EPA may include requirements and conditions (beyond those
specified in the application) in its approval of the TERA.
• Experience from field research under TERAs will provide the basis
to proceed to commercial approval under an MCAN.
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15. European Regulation of Industrial Uses of
Algae, Cyanobacteria
• European Union countries regulate biotechnology under national
laws based on two EU Directives:
• Directive 2009/41/EC, on the contained use of genetically modified
microorganisms (GMMs).
• Directive 2001/18/EC, on the deliberate release into the
environment of GMOs (plants and microorganisms).
• Definition of Genetically modified microorganism:
“genetically modified micro-organism” (GMM) means a micro-organism in which
the genetic material has been altered in a way that does not occur naturally by
mating and/or natural recombination (includes CRISPR-mediated deletions).
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16. EXAMPLES OF SUCCESSFUL REGULATORY
APPLICATIONS
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17. Approved MCANs: Algae and Cyanobacteria
• MCANs approved with consent
orders for use of cyanobacteria
in photobioreactors.
– Joule MCAN J-12-0006, signed
July 2013.
– Algenol, MCANs J-14-0007 to -
0009, December 2014.
• Solazyme, Inc., Four MCANs,
2013-14, heterotrophic growth
of microalgae.
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18. Approved TERAs for Algae
• R-13-0003 thru 0007. Sapphire/UCSD,
August 2013. Five genetically modified
strains of Scenedesmus dimorphus.
• R-17-0002. Arizona State University, May
2016. Two modified strains of Chlorella
sorokiniana.
• R-18-0001. Arizona State University, August
2017. Modified strain of Chlorella
sorokiniana, expressing SNF related kinase
from Picochlorum soloecismus.
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Source: Szyjka, et al. (2017) Algal Research,
Volume 24, Part A, pages 378-386.

19. Approved TERAs for Algae
• R-19-0001. Synthetic
Genomics, Inc.
(Viridos), April 2019,
small-scale open-pond
testing of a proprietary
Parachlorella sp.
strain, engineered with
a marker gene.
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Image courtesy of Viridos, Inc.: L. Brown “The path to scale at the California
Advanced Algae Facility” 2021 Algal Biomass Summit (virtual).

20. Approved TERAs for Algae
• R-20-0001. Synthetic
Genomics, Inc. (Viridos), May
2020, open-pond testing of
multiple green microalgal
strains engineered for
enhanced lipid productivity.
• R-21-0002. Synthetic
Genomics, Inc. (Viridos),
March 2021, open-pond
testing of three green
microalgal strains engineered
for enhanced lipid
productivity.
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Image courtesy of Viridos, Inc.: L. Brown “The path to scale at the California
Advanced Algae Facility” 2021 Algal Biomass Summit (virtual).

21. Brazilian Approval for Contained Use of Algae
Solazyme Renewable Oils and Bioproducts Brazil Ltda.
Subject: Request for Opinion on the commercial release of genetically
modified Class I biohazard microorganism.
October 17, 2013. Decision: Granted
The microalgae species Prototheca moriformis is a single-celled non-
chlorophyll-containing obligatory heterotroph, which reproduces
asexually and does not produce spores. The Brazilian biotechnology
regulatory authority CTNBio approved the commercial use of the
genetically modified Prototheca moriformis, strain S2014, for the
production of triglyceride oils and other bioproducts.
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22. Regulatory Approvals in Europe
• In June 2020, AlgaePARC at Wageningen
University received permits to grow
modified microorganisms in pilot scale
photobioreactors, in trials from 20 to
1500 liters.
• In 2019 and 2021, Photanol received
Dutch government approval for use of
modified cyanobacteria in pilot-scale
photobioreactors for the production of
an organic acid.
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23. STRATEGIC CONSIDERATIONS TO ACHIEVE
REGULATORY APPROVAL
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24. Regulatory Strategies
• Plan ahead: begin internal planning at least 9-12 months before
intended start date.
• Request early presubmission consultation with regulatory agency.
• U.S. EPA: Guidance document available for submissions involving
modified algae.
• Where applicable: gather data from outdoor or commercial use of
nonmodified strain, to support similar use of a modified strain.
• In U.S., studies such as toxicology are generally not necessary, but
data on environmental fate would be important for proposed
outdoor activities.
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Planning for TERA would follow similar
timeline, but with shorter EPA review time.
Planning for
Regulatory Approval

26. David J. Glass, Ph.D.
D. Glass Associates, Inc.
124 Bird Street
Needham, MA 02492
Phone 617-653-9945
dglass@dglassassociates.com
www.dglassassociates.com
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27. ADDITIONAL SLIDES
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28. Detailed Contents of TERAs: §725.255(e)
(1) Detailed description of the proposed research and development
activity.
i. The objectives and significance of the activity; rationale for the test.
ii. Number of microorganisms released; method(s) of application or
release.
iii. Characteristics of the test site(s), including location, geographical,
biological and other features of the site.
iv. Target organisms (e.g., prey) of the modified microorganism (if any)
v. Planned start date and duration of each activity.
vi. Whether State and/or local authorities have been notified of the
activity, evidence of notification.
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29. Detailed Contents of TERAs : §725.255(e)
(2) Information on monitoring, confinement, mitigation, and emergency
termination procedures.
i. Confinement procedures, access and security measures, procedures for
routine termination.
ii. Mitigation and emergency procedures.
iii. Measures to detect and control potential adverse effects.
iv. Name of principal investigator and chief of site personnel responsible for
emergency procedures.
v. Personal protective equipment, engineering controls, procedures to be
followed to minimize dispersion.
vi. Procedures for disposal of articles, waste, or equipment involved in the
release, methods for inactivation of the microorganism(s), and for
containment, disinfection, and disposal of contaminated items.
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30. Additional Contents of TERAs: §725.260
Each TERA must contain all available data concerning actual or
potential effects on health or the environment of the new
microorganism that are in the possession or control of the
submitter and a description of other data known to or
reasonably ascertainable by the submitter that will permit a
reasoned evaluation of the planned test in the environment.
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31. Approved TERAs
• 1998-2015: 30 approved, 3 withdrawn
– Rhizobia for nitrogen fixation: 5
– Pesticide research: 3
– Hazwaste detection (bioindicators): 13
– Bioremediation: 2
– Biofuel research (algae): 5
– Enzyme production: 2 (using B. thuringiensis Israelensis).
• 2016 to date: 8 approved (5 for algae), 1 withdrawn
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