A presentation to UCSD on the role of algae in addressing the food and fuel needs in the 21st century.

1. Big Red is The New Green

2. The Agro/BioEnergy Systems: Addressing the
Integrated Vision of UNL Algae Program Triad.
BioEnergy
ENERGY
Agriculture
Products
FOOD
Environmental
Enhancement
WATER
Base of pyramid: Basic Life Science and Agricultural Strengths

4. Population is increasing, calories per capita are
increasing, meat consumption is increasing.

6. Heart Land Problem
•
•
Hypoxic Zone
Midwest corn production
for biofuels has lead to
record size of Dead Zone
this summer.

7. Hypoxic zones are a world problem

8. NREL Harmonized Algae Biofuels Models

9. UNL Superloop Biorefinery
Example of Integrated AD, Algae, Animal and Aquaculture
Feed System
Biofuels
Biofuels
Aquaculture
Aquaculture
Sun light
Grain
Grain

10. Algae can be combined with animal agriculture, AD, and
Aquaculture - an integrated industry
Algae
productio
n
Aquaculture
production

11. Fish Meal Market
6.0 million tons/yr with decreasing catch and increasing demand
Result skyrocketing prices and need for replacement

14. Poultry Farms
Cattle Farms
Dairy Farms
Hog Farms

15. Selection & Phylogenetic Pedigree of Chlorella spp.
UCSD
NREL
Autoflocculation, heterotrophic capacity
UNL
JHU, CSU - Isolate from Inner Mongolia, China
Isolate from Texas, thermophilic ≤ 40°C
Lutein, oil, 10,000-L cultures
NAABB
Austin Barnes
Wan et al. Biotech Lett (2011); Kim et al. J Appl Phycol (2009); Shi et al. Enz Microb Tech (2000); Sorokin, Sci (1953)
September 27, 2012

16. A.
2.0 micron
C.
B.
UTEX395
Time (Hours)
% FAME (dcw)
wt % Total FAME
D.
Cell Density (OD750)
Chlorella vulgaris: NREL Model System
Nitrogen
Replete
Nitrogen
Deplete
Nitrogen Nitrogen
Replete Deplete

17. Cattle AD effluent for Algae and compositions of AD effluent
Organic Matter
AD Effluent
Composition of AD Effluent
% of DM
WDGS
Manure
WDGS
Effluent
Total N
3.79
6.02
Organic N
2.66
4.35
Ammonium
Nitrate
P2O5
1.13
1.67
0.001
0.001
1.46
2.94
0.51
0.75
Ca
2.13
4.47
Mg
0.65
1.19
Na
Algae biomass
4.82
S
Algae culture
in hanging bags
2.64
K2O
0.23
0.58
Zn
0.04
0.06
Fe
0.09
0.61
pH
5.7
7.7
DM = dry matter
WDGS = wet distillers grains plus soluble cattle
diet
Andrea Watson, Dr. Galen Erikson (Department of Animal Science, UNL)

18. Algal Strain Selection on Wastewater and Anaerobic Digester Effluent
April 2013

19. Cultivation of Chlorella in hanging bag in comparison of
10% ADE and BBM
Day 10
Day 0
ADE
ADE BBM
Day 1
ADE
BBM
Day 21
BBM
ADE
BBM
Day 6
ADE
BBM

20. Dynamics of phosphorus, ammonia and nitrate/nitrite levels in
Chlorella under 10% ADE and BBM conditions
BBM components
NaNO3
CaCl2· 2H2O
MgSO4· 7H2O
K2HPO4
KH2PO4
NaCl
KOH
Na2EDTA
FeSO4· 7H2O
H3BO3
ZnSO4· 7H2O
MnCl2· 4H2O
CuSO4· 5H2O
(NH4)6Mo7O24· 4H2O
CoCl2· 6H2O
ADE components
Days
Days
Total N
Organic N
Ammonium
NaNO3
P2O5
K2O
S
Ca
Mg
Na
Zn
Fe
mg/L
250
25
75
75
175
25
31
50
4.98
11.42
17.64
2.88
3.14
1.74
0.8
% of DM
6.02
4.35
1.67
0.001
4.82
2.94
0.75
4.47
1.19
0.58
0.06
0.61

21. Growth curves of CS-01, UTEX 1230 and UTEX 2714 under
10% ADE and BBM conditions
10%ADE
BBM

22. % DW
Levels of protein, lipid and starch in CS-01, UTEX 1230 and UTEX 2714
in comparison of 10% ADE and BBM conditions
Days

25. Installation of Lining within the Raceway Pond
Wendell Leimbach
April 2013

26. Greenhouse Algal Growth Facility
George Oyler

27. Harvesting Algal Biomass with Continuous Centrifuge
April 2013

28. Two-Stage Process Demonstration at 100+ L Scale
0.5 L
40 L
60 L
150 L
600 L
1000 L
UNL
Eric Noel, Austin Barnes
Phase 1: Photoautotrophic Scale-Up
SR and CGC
Doug Morton, Gunjan Andlay

29. Chlorella: Mixotrophy vs. Two-Stage Growth
CO2
ATP
NADPH
accD
H2O
ATP
NADPH
Photosynthesis
TCA
Photosynthesis
TCA
1
Biomass
2
Glycolysis
TAG
Biosynthesis
Biomass
acc1
Organic
Compounds
Lipids Types:
TAG
Biosynthesis
Glycolipds,
Phospholipids,
TAGs, PUFAs,
Carotenoids,
Tocopherols
Mixotrophic: No Synergy,
Risk of Contamination
Biodiesel
Crude Algae Oil
Glycolysis
Organic
Compounds
Two-Stage: Uncouple Biomass
and Lipid Contributions
Bio-Jet Fuel
Wan et al. Appl Microbiol Biotechnol (2011); Rosenberg et al. Curr Opin Biotechnol (2008)
Green Gasoline
September 27, 2012

30. UTEX 1230
Total lipids by weighing
Total lipids by GC/MS
35
30
35
20
% of DW
% of DW
30
25
15
10
5
25
20
15
10
5
0
0
Auto 8/1-28-1
Auto 8/219/24-1
Hetero 8/68/28-1
Auto 8/1-28-1
Hetero 7/30-1
TAG levels by HPLC-ELSD
30
20
15
% of DW
% of DW
25
10
5
0
Auto 8/219/24-1
Hetero 8/68/28-1
Hetero 8/68/28-1
Hetero 7/30-1
TAG levels by GC/MS
35
Auto 8/1-28-1
Auto 8/219/24-1
Hetero 7/30-1
35
30
25
20
15
10
5
0
Auto 8/1-28-1
Auto 8/219/24-1
Hetero 8/68/28-1
Hetero 7/30-1

31. Effect of Glucose on Biomass, Lipid Composition
Naoko Kobayashi
Dionex Automated Solvent Extraction
Auto
Hetero
Wan et al. Biotechnology & Bioengineering
September 27, 2012

32. Biomass Productivity & Lipid Storage Classes
Phase 2: High Density Heterotrophic Phase
September 27, 2012

33. Comparison of Lipid Profiles:
Chlorella sorokiniana vs Nannochloropsis oceanica
•
Chlorella may complement lipid deficiencies of Nannochloropsis
•
PUFA accumulation in Chlorella induced by heterotrophy
September 27, 2012

34. Conclusions
• Goal: target effective use of sugar for producing desirable lipid profiles
• Higher TAG and PUFA content compared to autotrophic growth
• Additional advantage of two-stage process:
Chlorosis: degradation of chlorophyll & thylakoid membranes
• Two-fold contribution to TAG accumulation:
Fatty Acid
Biosynthesis
1) Conversion of sugars
2) Turnover of photosynthetic biomolecules
• Cellular biorefinery concept: TAGs are ideal for biodiesel
• Convenient, but not sufficient for biofuels
• TAGs require input of sugars or extreme stress
Aim for total lipid recovery for maximum hydrocarbon yield

35. Use the pressure chamber for mixing, H2O&algae + solvent(s)
Compare to hand mixing with test tubes

36. Butanol then
heptane
Heptane
alone
Mix
by
hand
Mix
in
chambe
r

37. Nile Tilapia Aquaculture
April 2013

38. Tilapia Donated to High School for Aquaponics
April 2013

40. Fig. 1 viral promoter function test in mammalian cell

41. Fig. 2 GUS gene expression in Arabidopsis thaliana controlled by viral promoters

42. fig. 3. Viral promoters function in Saccharomyces cerevisiae

43. 5x10 $
7
5x10 $
7
5x10 $
7
5x10 $
pGOrbcS2%
pGOaUQ%
pGOatu%
pGOpsaD%
0.5$
0.5$
0.5$
0.5$
6$
4$
6$
6$
11$
5$
4$
5$
8$
6$
6$
10$
0$
6$
11$
10$
$
$
$
$
Appendix C.2.5 (updated 4/5/2013)
Chlorella and Chlorella Virus Promoters Used for the transformation of Unicellular Green Algae and
Other Eukaryotic Systems
Promoter
Resources
Promoter
Size(bp)
Chlamy
Transformation Recipient System
Chlorella
Yeast
Arabidopsis
Mammalian
Chlorella variabilis Promoters*
rbcS1
600
rbcS2
600
psaD
600
αtubulin
600
ubiquitin
600
tested
no function
tested
no function
tested
no function
tested
no function
tested
no function
on going
on going
on going
on going
on going
Chlorella Virus Promoters
Previously Studied Promoters
tested
no function**
NVP1
361
tested
VP54
636
no function
NVP5
213
Promoters Chosen from Transcriptomic Profile
NVP8
253
tested
NVP14
384
A158L
500
no function**
NVP19
226
tested
A312L
500
NVP20
150
no function**
NVP25
104
A348R
500
NVP28
251
tested
A404R
500
NVP30
>5,000
to function**
NVP35
196
Promoters Selected from Shotgun Library***
AMT
851
on going
functional
on going
functional
to be tested
tested
need repeat
tested
need repeat
to be tested
to be tested
to be tested
to be tested
tested
no function
tested
no function
tested
no function
to be tested
to be tested
functional
NVP37
329
to be tested
to be tested
NVP40
667
to be tested
to be tested
NVP63
79
NVP80
176
tested
need repeat
tested
need repeat
tested
need repeat
tested
no function
to be tested
functional

44. Acknowledgements
Dr. George Oyler
Dr. Naoko Kobayashi
Eric Noel
Austin Barnes
Galen Erickson
Maya Khasin
WHITING
SCHOOL OF
ENGINEERING
JOHNS HOPKINS UNIVERSITY
Dr. Michael Betenbaugh
Dr. Marc Donohue
Dr. Scott Williams
Dr. Minxi Wan
Jon Rogers
Gunjan Andlay
Adithya Balasubramanian
Scott Johnson