Growing Products in Microalgae: From Nutraceuticals to Human Therapeutic Proteins and Remediation

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Growing Products in Microalgae From
Nutraceuticals to Human Therapeutic Proteins and
Remediation

• BIOMAN 2006
• New Hampshire Community Technical College
• Portsmouth, NH
• July 24 2006

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Outline

• Introduction
• Microalgae what they are, do and need
• History of microalgal cultivation
• Scaling up processes
• High value products (I)
• Natural products astaxanthin
• High value products (II)
• Therapeutics (monoclonal antibodies)
• Environmental remediation
• CO2 sequestration to fuel stocks
• Examples from my own experiences at Mera
  Pharmaceuticals and GreenFuel

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Microalgae are

• Very diverse 30,000-50,000 species
• Ubiquitous wherever there is water
• Fast growing
• Largely unexplored!
• Source of new and valuable substances

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What makes an organism a microalga
CO2
LIGHT

• Small (usually microscopic)
• Eukaryotic or Prokaryotic
• Unicellular (colonial)
• Colorful (photosynthetic and other pigments)
• Aquatic (but not necessarily)
• Likely photoautrotophic (but not necessarily all
  the time)

WATER
NUTRIENTS
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3 um cocci 8 um Chlorophyte
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Scenedesmus Botryococcus Lyngbya
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Characteristics of microalgae of interest in
biotechnology

• Very diverse
• but
• Largely unexplored
• ( of species)

• Why not go synthetic route?
• Natural products are a consistent source of new
  drugs
• Natural products posses advantages over synthetic
  products (unique chemistries, solubility,
  permeability, bioavailability)

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Scale up in microalgal biotechnology

• Low abundance of source material
• Material needed for testing beyond initial
  discovery
• Industrial applications
• Need to scale up cultures

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History of microalgae domestication
Ancient times
Accidental consumption by coastal communities
Ancient times
Consumption of Nostoc and others in China Japan
lt1500s
Spirulina by Aztecs in Mexico
Pre-domestication
lt1900s
Spirulina in Lake Rombou, Chad
Awareness Anton van Leeuwenhoek
1673
Studies in plant nutrition
XVIII/XIX c.
1871
Famintzin microalgal nutrition
Beijerink bacteria-free Chlorella, pure cultures
1890
Chick nutrition, growth and metabolism
1903
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Production Technology History
1905
First marine cultures (Plymouth, UK - 60 ml)
5-gal reactor 1st chemical measurements (WHOI)
1938
Automated photobioreactor - 10 L Yield
optimization dilution/harvesting strategies
1940s
1953
Burlew, 000s of L, enclosed reactor, economics
Open pond technology - many 000s of
L paddle wheel raceways (Dortmund) round
ponds (Japan)
1950s
Many species attempted
1970s
Only 3 species are commercial
1990s
MGM 1st new commercial species
Issues of cost, low value but very large markets
2000
GreenFuel
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Laboratory scale Collections, maintenance,
experiments
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Laboratory scale Experiments (3 liter pH-stat)

• Limited capacity for production in lab cultures
  (but very well controlled)
• Need to scale up

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Lab Scale-Up inefficient
Need to scale up to outdoor PBRs
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Scale-up photobioreactor requirements

• Control light (sun)
• Control temperature
• Control pH
• Control nutrients
• Control turbulence
• Control pests/weeds
• at INDUSTRIAL SCALE

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Photobioreactor characteristics

• High area productivity (g/m2/d)
• High volumetric productivity (g/l/d)
• Large volume (l/PBR)
• Inexpensive (/PBR)
• Easy to control culture parameters
• Reliable

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The technology

• open ponds
• easy to operate
• easy to contaminate
• no control
• limited to special case species and processes

• enclosed photobioreactors
• easy to operate with the right technology
• keep contaminants out
• control growth parameters
• grow many different species

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Open ponds
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Sun Chlorella ponds
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Dunaliella in Australia
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Enclosed photobioreactors
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Enclosed photobioreactors
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Enclosed PBRs
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Enclosed photobioreactors
Cyanotechs Phytodome
MicroGaias Biodome
Meras Growth Module
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Enclosed PhotobioreactorsNatural gas power
plant, Red Hawk (AZ)
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Enclosed Photobioreactors Coal power plant, NRG,
Dunkirk (NY)
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How is it supposed to work
Goal of biotechnology to make by developing
marketable products

• Identify a desirable metabolite (or process) and
  the microalga that produces it
• Establish a large scale production process for
  the desired metabolite
• Market and sell the metabolite or process
• THREE EXAMPLES
• Astaxanthin (Haematococcus pluvialis)
• Monoclonal antibodies (Chlamydomonas)
• Bioremediation (Flue gas to biofuels)

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Haematococcus astaxanthin
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Haematococcus astaxanthin

• Carotenoid
• Algal response to environment
• Excellent antioxidant
• Applications in human health
• Photoprotectant
• Eye health
• Skin health
• Anti-inflamatory
• Heart health
• Anticancer
• Neurodegenerative diseases
• Immunomodulator
• Safe for human consumption
• High retail value (10s thousands /kg)

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Production of Haematococcus astaxanthin

• Scale up
• Photobioreactors enclose and open
• Harvest
• Separation and recovery
• End products and formulation

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Scale up of Haematococcus
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The Mera Growth Module

• The MGM can be programmed to control
• Nutrient concentrations
• Turbulence
• Gas exchange
• Temperature
• pH
• The production MGMs are one of the largest
  enclosed, computer controlled, photobioreactors
  in commercial production in the world
• Produce green biomass

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Pond process

• Inoculate on day 0 with H. pluvialis biomass from
  MGMs
• Stress the cells into carotenogenesis
• Harvest on day 5
• Inoculate again on day 6
• Produce algal meal at 2.5-3.5 astax DW

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Harvest of Haematococcus biomass
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Separation and recovery

• Break cell walls high pressure homogenizer
  running at gt10,000 psi
• Dry cell broken material to lt5 moisture
• Package dried material and prepare for
• incorporating into pills (nutraceutical market)
• incorporating into foods/feeds (ingredient)
• extract material/purify astaxanthin

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End products and formulation
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Monoclonalantibodies
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Monoclonal antibodies GM algae

• Collaboration with Rincon Pharmaceuticals
• Scott Franklin, chief scientist
• Production of therapeutic proteins (monoclonal
  antibodies) in Chlamydomonas
• Scale up capabilities

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Antibody therapeutics

• Antibodies are naturally occurring proteins, made
  by all vertebrates, used by the body to fight
  disease and confer immunity
• Antibody therapeutics are made today using
  mammalian cells, purified, and administered
  through injection
• Antibody therapeutics are the fastest growing
  segment of the pharmaceutical industry because
  these proteins are
• inherently safe (minimal side effects)
• highly effective in treating major diseases
• gt 250 therapeutic proteins in clinical trials
  150 monoclonal antibodies
• Tremendous industry shortfall in production
  capability
• Current production methods are very slow and
  expensive - hence the enormously high cost of
  these therapies ( 20,000 per treatment)

Representative antibody products and disease
treated Product Company Indication
Remicade J J Rheumatoid
Arthritis/Crohns Rituxan BiogenIdec/Genen. Non-
Hodgkins Lymphoma Enbrel Amgen Rheumatoid
Arthritis Herceptin Genentech Metastatic
Breast Cancer Synagis MedImmune RSV Infection
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Tremendous Industry Need for Alternative Protein
Expression Systems

• Existing technologies (mammalian, bacteria,
  yeast) have
• long development timelines 24 months
• long manufacturing scale-up 4-5 years
• extraordinary capital requirements gt 200 million
  
• inherently high cost of goods gt 500/gram
• Massive barrier to entry / restricts product
  development
• Federal insurance/HMO pressure to significantly
  reduce the cost of increasingly expensive
  biologics

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Transgenic algae (Chlamydomonas)

• Same fundamental science that has been applied
  for over 40 years to mammalian cells, bacteria,
  and yeast - for example to make human insulin
• Site specific gene modification
• Target protein can be used as therapeutic - once
  purified from the cell and injected

C. reinhardtii
antibody protein
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Why Algae as an Expression Platform?

• Speed
• Cost
• Safety
• G.R.A.S

• RESULTS
• Expression of two MABs
• Expression levels higher than lab cultures
• Field demonstration was accomplished within 2
  months

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Environmentalbioremediation(flue gas to biofuel)
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Concept overview
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Advantages

• Wide temperature and pH optima
• High purity CO2 not required for microalgae
  culture
• simplify gas separation
• Noxious gas tolerant some combustion products
  can be used as nutrients by microalgae
• simplify flue gas scrubbing
• Microalgae culturing yields high value commercial
  products, even fuel feed stocks, and can be
  scaled up
• offset capital and operation cost
• Process is based on renewable photosynthetic
  carbon fixation
• minimal negative impacts on environment

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Selection of microalgae

• Characterization of Physiology and Metabolism of
  Microalgae
• Temperature tolerance
• pH tolerance
• Noxious gas tolerance
• High value chemical content
• Achievable CO2 Capture Rates
• Effects of pH
• Effects of gas concentrations
• Non-biomass carbon capture

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Temperature tolerance
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CO2 capture rates at different pH
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Effects of pH on CO2 capture efficiency
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Typical flue gas compositions for different fuels
and combustion systems

• Concentrations of trace acid gas species such as
  NOx and SOx depend on the composition of the fuel
  and on the air pollution control system
• Natural gas-fired combustors have virtually no
  SOx in the flue gas, while coal-fired systems
  have hundred of parts per millions

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CO2 capture rates from simulated combustion gases
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Effects of gas concentrations on CO2 capture
efficiency
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Red Hawk data Water source
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NRG data 3 species, CO2 vs coal FG
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Growing Products in Microalgae From
Nutraceuticals to Human Therapeutic Proteins and
Remediation

• Thanks to Dr. Wallman and her crew!