Micro‐Algal
as
a
Pla>orm
for
the



ProducAon
of
Biofuels
and
BioProducts

Stephen
Mayfield

The
San
Diego
Center
for
Algae
Biotechnology

University
of
California
San
Diego

 Alga: Latin “Seaweed”

•  Any of a the group of aquatic eukaryotic organisms

that contain chlorophyll and other pigments and can
carry on photosynthesis
–  Range from microscopic single cells to very large
multicellular structures resembling roots, stems and leaves
–  Further categorized as brown algae, red algae, green algae,
also organisms such as dinoflagellates

•  Photosynthetic prokaryotes are known as

cyanobacteria or blue-green algae
BioFuel:
Conversion
of
solar
energy
into

chemical
energy:
photosynthesis



Fermenta2on

Food
 Into
Fuel


Photosynthesis

In
crops

Burn
for


Fiber

Electricity


Photosynthesis

In
Algae
 Natural
Oils
 Liquid
Fuels

 RelaAonship
of
Energy
and
Power

Oil
is
energy
 Electricity
is
power


Stored
energy
for
use
any
.me
 Delivered
by
transmission
for
immediate

use

 Energy
is
the
world’s
largest
market,
and
is
growing
rapidly


1900
 Today

$0.4
billion
 $5.8
trillion

2020

$10.4
trillion


Source:

World
Trade
OrganizaAon;
CIA
World
Factbook;
OICA.net;
IMS
Health;
just‐food.com;
Gartner;
EIA

 Primary
sources
of
energy
today
Hydroelectricity

Uranium
 Other
renewable
sources


#" !" 1%

0%


Coal
 23%


36%
 Crude
oil


•  Demand
is
growing

rapidly
to
~600

quadrillion
Btu
by
2020

•  Without
significant

$%" subsAtutes
for
crude
oil

Natural
gas


Sources:
EIA
Annual
Energy
Review,
June
2009;
EIA
InternaAonal
Energy
Outlook,
May
2009

 Something
has
to
fill
the
gap
le[
by
shrinking
oil
reserves
Current
fossil
reserve
by
type
 2030
reserves
by
oil
type


Non‐conven2onal

petroleum

1st
genera2on

biofuels

Extra
heavy
oil
 Oil
sands

bitumen


Heavy
oil
 Advanced

biofuels


Conven2onal
oil


Conven2onal


petroleum



Source:
 CIA
World
Factbook,
2008;
HighlighAng
Heavy
Oil,
Oilfield
review,
2006

 World’s
proven
reserves
of
oil
total
1.4
trillion
barrels
today,
but

unconvenAonal
sources
of
oil
already
outnumber
convenAonal
sources
•  Proven
reserves
of
oil,
by
region*
 •  Current
fossil
reserves,
by
oil

•  Billions
of
barrels
 type

ConvenAonal
oil

•  Percent

Middle
East
 ()" Oil
sands
bitumen

Heavy
oil

Extra
heavy
oil

North
America
 222


Europe
&
 !"#
Eurasia
 25%

30%

Africa
 126


Central
&
 !#'
South
America

15%

Asia
Pacific
 42

!"#
Total
 !$"%&


 *
 Including
Canadian
oil
sands

Source:

 BP
Review
of
World
Energy
2009;
HighlighAng
Heavy
Oil,
Oilfield
review,
2006

 The End of the Hydrocarbon Era
Total
World
Energy
Reserves*

Coal
 
 
 1013
TW‐yr


Oil
 
 
 582
TW‐yr

Natural
Gas
 
 539
TW‐yr


Nuclear
 
 60‐300
TW‐yr


Total
Reserves





~2194‐2434
TW‐yr


Assuming
conAnued
2
‐3
%
growth
in
consumpAon*


We
will
run
out
of
all
energy
reserves
~2099

If
world
was
at
US
consumpAon
rates
now:
we
would
run
out
in
2048

*
InternaAonal
Energy
Agency,
World
Energy
Outlook
2001:Assessing
Today‘s
Supplies
to
Fuel
Tomorrow‘s
Growth

Can
that
really
be
true?
 Energy Return on Investment

The
EROI
for
the
Alberta
Tar
Sands
is
5:1,
in
Saudi
Arabia
it
is
100:1

How
will
we
replace
this
energy
source?


*Energy
can
neither
be
created

nor
destroyed,
it
can
only
be

transformed
from
one
state
to

another




*First
law
of
thermodynamics:
ConservaAon
of
Energy
 Every
form
of
fuel
has
a
limiAng
factor


Mode
of
energy
 Most
significant
limi2ng
factor


Petroleum
 Finite
reserves,
GHG


1st
generaAon
ethanol
 Food
supply


2nd
gen
Advanced
biofuels
 Easily
recoverable
plant
waste


Electric
baheries
 Lithium
and
half
life


Hydrogen
 Electricity
and
natural
gas


Algae
 Scale
and
Cost


Why
algae
as
a
energy
source


Scalability


Cost


Sustainability


Oil
Source

BioFuels
are
the
Biological
conversion
of

sunlight
energy
into
chemical
energy

Carbohydrate
 Hydrocarbons

Protein

The World Consumes 15 Terawatts of
Energy every Year - 85% Fossil Fuel

The
Good
News

The
Sun
Provide
86,000
Terawahs
of
Energy
Every
Year
 Processing
Green
Crude
to
Fuel


Grow
 Harvest
 Extract
 Concentrate


Fungible
fuels
have
been
made
from
algae
oil


Algaeus,
the
world's
first
hybrid
vehicle


to
cross
the
country
on
fuel
containing
a
blend


of
algae‐based
renewable
gasoline.

27
August
2009


A
90‐minute
flight
by
a
ConAnental
Boeing

737.

One
engines
powered
by
a
50‐50
blend

of
biofuel
and
normal
aircra[
fuel.

8
January
2009

Algal
Biofuels
ProducAon
Chain

CO2

BioprospecAng
 Water
 Pond
Design

GeneAcs
 Nutrients
 Water
Management

Breeding
 Sunlight
 Crop
ProtecAon
 3
Strain
 Dewatering

Development

Input
 ProducAon
 Harvest


1 2 4
ProducAon
 Co‐products

ExtracAon


Refining
 Pre‐Refining


Fuel

Gasoline

ExtracAon

Diesel

jet

Residual

5 Biomass


Animal
feed

Anaerobic
DigesAon

Nutrient
recycling

Improving
the
Economic
Viability
of
Algal
Biofuels


Cost
Per
Gallon
(in
2009
dollars)


21


A. Bio‐prospecAng


18

 Engineering


15
 Breeding‐selecAon


12

co‐products

B. D.
9

C.
6


3

petroleum
ppg


0

2009
 2010
 2011
 2012
 2013
 2014
 2015
 2016
 2017
 2018

Year

DomesAcaAon
of
crops
and
livestock
has

taken
7,000
of
years
of
breeding


•  No
commercial
system
uses
wild
type
organisms

•  All
large
scale
producAon
relies
on
species
that
are

geneAcally
modified
(breeding
and
engineering)

 The
agricultural
revoluAon
was
accomplished
first
by

breeding
and
now
by
molecular
science

•  Crop
technology

"(!
revolu2on

"'! •  Corn
yield
in
the
U.S.
 Molecular
science

"&!
•  Bushels
per
year

"%! •  Agricultural

"$! commercializa2on

"#!
""! Industrial
prac2ces

"!!
*!
)!
(!
'!
&!
%!
$!
#!
"!
!
")'! ")(! "))! ")*! "*!! "*"! "*#! "*$! "*%! "*&! "*'! "*(! "*)! "**! #!!! #!"!


 Source:
 NaAonal
Agricultural
StaAsAcs
Service,
2009

DomesAcaAon
will
require
source
genes,

engineering
and
hosts
strains


IdenAficaAon

AnnotaAon

Cloning


OpAmizaAon

Engineering

TransformaAon


Engineering/

Source
genes
 Gene2cs
 Produc2on
strains

Algae
are
the
most
diverse


organisms
in
nature


Cyanobacteria


Brown
Algae


Green
Algae


Red
Algae

 Algae
are
capable
of
rapid
geneAc
improvement

Scales
from
liters
to
1000s
liters
producAon
in
weeks


Research 1 Initial Transformation:

Scale day 1

2 Selection of primary
transformants: day 10
3 Homoplasmic lines
with high expression
levels: week 6

4 Scale-up to multi-liter
volumes: week 8

5 Scale-up to 100s
liters: week 10

6 Scale to 1000s
liters: week 14 Commercial
Scale
Patented large-scale photobioreactors - 25,000 liters*

*Algal facility as currently used to manufacture nutraceuticals

TransformaAon
of
algae
is
relaAvely
easy

Need
selectable
markers
for
each
genome


nucleus

Chloroplast
 Chloroplast
transformaAon
proceeds


by
homologous
recombinaAon


psbA
promoter





5’
UTR
 rbcL
3’
UTR


Codon
opAmized
coding
region



• 
need
promoter
and
UTRs
flanking
region
of
homology

• 
fewer
transformaAon
events

• 
recombinant
proteins
can
accumulate
to
very
high
levels

• 
can
make
proteins
there
that
you
can’t
make
elsewhere

 Nuclear
transformaAon
proceeds


by
random
integraAon

Promoters larger
More complex 5’ UTR 3’ UTR

Codon optimized coding region

• 
need
more
transformaAon
events
to
get
good
expression

• 
gene
expression
more
complex,
regulaAon
potenAal
greater

• 
can
export
proteins
from
the
cell

 Engineering Algal Chloroplast

  Complete set of genetic material

  Simple promoters / expression elements

  Consistent transformation by homologous recombination

 Proteins have disulfide bonds but are non-glycosylated

 Recombinant proteins can accumulate to high levels

 Metabolic engineering by plastid transformation possible

C. reinhardtii
Chloroplast genome
203,395 bp
 psbA
replacement
vector
for
improved


Serum
amyloid
A3
expression


EcoR1 D1 gene Xho I

D1 5’ genomic flanking D1 3’ genomic flanking

EcoR1 Xho I
D1 promoter SAA coding region
D1 3’UTR
and UTR
Expression
of
Bovine
SAA3
in
chloroplast


SAA-22 mem
Total Sol Mem

Saa-22 Sol
SAA-22

SAA-22

SAA-22

Wt total
SAA-1

Wt Sol
Wt

Wt

Wt

Coomassie stain gel Western anti-SAA3

 Analysis
of
expression
of
seven
human


recombinant
genes
in
C.
reinhard<i
chloroplast


psbA 1xFLAG
promoter/ psbA
5’ UTR 3’ UTR
recombinant gene AtpA KanR rbcL

psbA
1xFLAG
promoter/ psbA
5’ UTR 3’ UTR
SAA recombinant gene AtpA KanR rbcL

protease
cleavage
site

AtpA
promoter/ rbcL
5’ UTR 3’ UTR

recombinant gene
AccumulaAon
of
human
therapeuAc


proteins
in
transgenic
algae


Interferon β
Proinsulin

HMGB1
10FN3
14FN3

VEGF
EPO

WT
39

25
psbA 20 µg
18

13

39

atpA 25 40 µg
18

13
74

48
psbA::SAA 20 µg
39

25

α-FLAG AP
 VEGF
receptor‐binding
assay


Elution
0.3
FT

0.25

0.2

0.15
A450 R6

0.1
Commercial R&D

50
0.05 algal
37
0
0.00001 0.0001 0.001 0.01 0.1 1
25 -0.05

20 Relative concentration
Expression
of
a
full
length
human
monoclonal

anAbody
in
algal
chloroplasts


AccumulaAon
of
human
IgG1
heavy
chain
and


light
chain
in
C.
reinhardAi
chloroplasts


C.r 83K7C
C.r 83K7C
CHO 83K7C
CHO 83K7C

CHO 83K7C
C.r 83K7C

C.r 83K7C

CHO 83K7C
MW

MW
MW

MW 170
170
110 110
79 110 110
79 Dimmer
60 60 79 79
47 47 Monomer 60 60

35 35
47 47
25 25
35 35
18 18

α-kappa LC α-gamma HC α-kappa LC α-gamma HC

Chloroplast
expressed
a‐Anthrax
anAbody
bind
PA83


at
same
affinity
as
mammalian
expressed
anAbody


Algal 83K7C Mammalian 83K7C

anti-gamma HC anti-gamma HC
1.4 0.7

1.2 0.6
Absorbance @ 450nm

Absorbance @ 450nm
1 0.5

0.8 0.4

0.6 0.3

0.4 0.2

0.2 0.1

0 0
0 2 4 6 8 10 12 0 2 4 6 8 10 12
Concentration (ng) Concentration (ng)
 Hydrocarbon
Biosynthesis

Isopentyl‐PP

Monoterpenes

Geranyl‐PP
synthase


Geranyl‐PP

Geranylgeranyl‐PP
synthase


Geranylgeranyl‐PP

Phytoene
synthase


Phytoene

Carotenoid
isomerase


Lycopene


Xanthophyll

 Beta‐Carotene

Cycle

DetecAon
of
Limonene
(C‐10
hydrocarbon)


in
C.
reinhard<i
chloroplasts

Western
anA‐LS
transgenic
algae


Kd
 WT
 13
 20
 59
 68
 79
 +


LS

70

55


41


30


GC‐MS
idenAficaAon
of
limonene
 *

 AccumulaAon
of
a
Novel
C30
Isoprenoid


in
Engineered
Chloroplasts

Clone
27

Clone
16

Clone
51

Clone
66

Wt


FPPS

Syn


C.
reinhardAi

Western
blot


Isoprenoid
accumulaAon
in
transgenic
algae

Technology
C
–
Co‐Products
Fuel
ProducAon
in
Algae

 

Economic
viability
will
likely
require
many
posiAve
factors


protein

biofuel


Co‐products


Municipal
wastewater
 Waste
carbon
dioxide

Bio‐Products
made
in
Algae

Product
 Price
USD/kg
 Market
Size
 Year
Available

Biomass
for
Humans
 $50
 $3.5
Billion
 Presently
Sold

Omega‐3
Fahy
Acids
 $5,000
 $1.5
Billion
 Presently
Sold

Aquaculture
Feed
 $160
 $700
Million
 Presently
Sold

B‐Carotene
 $2,000
 $280
Million
 Presently
Sold

Astaxanthin
 $3,000
 $200
Million
 Presently
Sold

Specialty
Chemicals
 ~$10,000
 $
50
Million
 Presently
Sold


TherapeuAc
Proteins
 $100,000
 $20
Billion
 2015


Human
NutraceuAcals
 $1,000
 $20
Billion
 2012

Industrial
enzymes
 $50
 $10
Billion
 2012

Bio‐Polymers
 $4
 $100
Billion
 2015


Biofuels
 $1
 $1.5
Trillion
 2020


 Efficient
Growth
and
Oil
producAon
Growth Efficiency (MT/acre/yr) Oil
 Biodiesel

content

 yield
gal./
50 Crop
 (%)
 acre

Canola
 40‐45
 113

40
Mustard
 25‐27
 70

30 Safflower
 42‐48
 146


20 Soy
 20‐22
 55


10
Jatropha
 32‐35
 202

Palm
 48‐52
 635

0
algae switchgrass sugarcane corn Algae
 20‐40
 ~
5,000


5,000
gal/acre
X
60
million
acres
=
300
billion
gal

How much food do we grow
 Can
We
Really
Replace

Petroleum
with
Algae
Oil

“It
is
not
the
criAc
who
counts
...
The
credit
belongs
to
the
man
who
is
actually
in

the
arena
…
who
at
the
best
knows
in
the
end
the
triumph
of
high
achievement

and
who
at
the
worst,
if
he
fails,
at
least
he
fails
while
daring
greatly.
So
that
his

place
shall
never
be
with
those
cold
and
<mid
souls
who
know
neither
victory
nor

defeat.”


Theodore
Roosevelt
1901
 The
San
Diego
Center
for
Algae
Biotechnology

SD‐CAB

hhp://algae.ucsd.edu/

Mission:
 The
 San
 Diego
 Center
 for
 Algae
 Biotechnology
 (SD‐CAB)
 was
 established
 to
 support
 the

development
of
innovaAve,
sustainable
and
commercially
viable
algae‐based
biotechnology
soluAons
for

renewable
energy,
green
chemistry,
bio‐products,
water
conservaAon
and
CO2
abatement.