Algae food and fuel 21st century 04

Big Red is The New Green

The Vision of UNL Algae Program

AgricultureProductsFOOD

EnvironmentalEnhancement

WATER

BioEnergyENERGY

Base of pyramid: Basic Life Science and Agricultural Strengths Base of pyramid: Basic Life Science and Agricultural Strengths of UNL of UNL

Integrated Agro/BioEnergy Systems: Addressing the Triad.

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

Heart Land ProblemHeart Land Problem

•

• Midwest corn production for biofuels has lead to record size of Dead Zone this summer.

Hypoxic Zone

Hypoxic zones are a world problem

NREL Harmonized Algae Biofuels Models

Biofuels Biofuels Biofuels Biofuels

GrainGrainGrainGrain

UNL Superloop BiorefineryUNL Superloop BiorefineryExample of Integrated AD, Algae, Animal and Aquaculture Example of Integrated AD, Algae, Animal and Aquaculture

Feed SystemFeed System

Sun lightSun light

AquacultureAquacultureAquacultureAquaculture

Algae productio

nAquaculture production

Algae can be combined with animal agriculture, AD, and Aquaculture - an integrated industry

Fish Meal Market

6.0 million tons/yr with decreasing catch and increasing demand

Result skyrocketing prices and need for replacement

Poultry Farms Cattle Farms

Hog FarmsDairy Farms

Selection & Phylogenetic Pedigree of Chlorella spp.

Austin Barnes

UCSD

NREL

UNL

NAABB

JHU, CSU

Autoflocculation, heterotrophic capacity

Isolate from Texas, thermophilic ≤ 40°C

Lutein, oil, 10,000-L cultures

- Isolate from Inner Mongolia, China

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

UTEX395

Time (Hours)

Ce

ll D

en

sit

y (O

D7

50)

Chlorella vulgaris: NREL Model System%

FA

ME

(d

cw

)

NitrogenReplete

NitrogenDeplete

wt

% T

ota

l FA

ME

NitrogenReplete

NitrogenDeplete

A. B.

C. D.

2.0 micron

Organic Matter AD Effluent

Algae biomass Algae culture in hanging bags

% of DMWDGS Manure

WDGS Effluent

Total N 3.79 6.02

Organic N 2.66 4.35

Ammonium 1.13 1.67

Nitrate 0.001 0.001P2O5 2.64 4.82K2O 1.46 2.94

S 0.51 0.75

Ca 2.13 4.47

Mg 0.65 1.19

Na 0.23 0.58

Zn 0.04 0.06

Fe 0.09 0.61

pH 5.7 7.7

DM = dry matterWDGS = wet distillers grains plus soluble cattle diet

Composition of AD Effluent

Cattle AD effluent for Algae and compositions of AD effluent

Andrea Watson, Dr. Galen Erikson (Department of Animal Science, UNL)

Algal Strain Selection on Wastewater and Anaerobic Digester Effluent

April 2013

ADE BBM

ADE

ADE

ADE

ADE

BBM

BBM

BBM

BBM

Day 0

Day 1

Day 6

Day 10

Day 21

Cultivation of Chlorella in hanging bag in comparison of 10% ADE and BBM

Dynamics of phosphorus, ammonia and nitrate/nitrite levels in Chlorella under 10% ADE and BBM conditions

BBM components mg/L

NaNO3 250

CaCl2· 2H2O 25

MgSO4· 7H2O 75

K2HPO4 75

KH2PO4 175

NaCl 25

KOH 31

Na2EDTA 50

FeSO4· 7H2O 4.98

H3BO3 11.42

ZnSO4· 7H2O 17.64

MnCl2· 4H2O 2.88

CuSO4· 5H2O 3.14

(NH4)6Mo7O24· 4H2O 1.74

CoCl2· 6H2O 0.8

ADE components % of DM

Total N 6.02

Organic N 4.35

Ammonium 1.67

NaNO3 0.001

P2O5 4.82

K2O 2.94

S 0.75

Ca 4.47

Mg 1.19

Na 0.58

Zn 0.06

Fe 0.61

Days Days

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

10%ADE

BBM

% D

W

Days

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

Installation of Lining within the Raceway Pond

Wendell LeimbachApril 2013

Greenhouse Algal Growth FacilityGeorge Oyler

Harvesting Algal Biomass with Continuous Centrifuge

April 2013

Two-Stage Process Demonstration at 100+ L Scale

Phase 1: Photoautotrophic Scale-UpUNLEric Noel, Austin Barnes

0.5 L 40 L

60 L 150 L

1000 L600 LSR and CGCDoug Morton, Gunjan Andlay

Chlorella: Mixotrophy vs. Two-Stage Growth

Biodiesel Green Gasoline

Lipids Types:Glycolipds, Phospholipids,TAGs, PUFAs, Carotenoids, Tocopherols

OrganicCompounds

TCA

ATP NADPH

Photosynthesis

H2O CO2

Bio-Jet Fuel

Glycolysis

TCA

ATP NADPH

Photosynthesis

TAGBiosynthesis

Glycolysis

Mixotrophic: No Synergy,Risk of Contamination

acc1

accD

Wan et al. Appl Microbiol Biotechnol (2011); Rosenberg et al. Curr Opin Biotechnol (2008)

Two-Stage: Uncouple Biomassand Lipid Contributions

Biomass

Crude Algae Oil

OrganicCompounds

1

2Biomass

TAGBiosynthesis

September 27, 2012

Total lipids by weighing

0

5

10

15

20

25

30

35

Auto 8/1-28-1 Auto 8/21-9/24-1

Hetero 8/6-8/28-1

Hetero 7/30-1

% o

f DW

Total lipids by GC/MS

0

5

10

15

20

25

30

35

Auto 8/1-28-1 Auto 8/21-9/24-1

Hetero 8/6-8/28-1

Hetero 7/30-1

% o

f DW

TAG levels by HPLC-ELSD

0

5

10

15

20

25

30

35

Auto 8/1-28-1 Auto 8/21-9/24-1

Hetero 8/6-8/28-1

Hetero 7/30-1

% o

f DW

TAG levels by GC/MS

0

5

10

15

20

25

30

35

Auto 8/1-28-1 Auto 8/21-9/24-1

Hetero 8/6-8/28-1

Hetero 7/30-1

% o

f DW

UTEX 1230

Effect of Glucose on Biomass, Lipid Composition

Wan et al. Biotechnology & Bioengineering (2012)

Auto Hetero

Dionex Automated Solvent Extraction

Naoko Kobayashi

September 27, 2012

Biomass Productivity & Lipid Storage Classes

Phase 2: High Density Heterotrophic PhaseSeptember 27, 2012

Comparison of Lipid Profiles:Chlorella sorokiniana vs Nannochloropsis oceanica

September 27, 2012

• Chlorella may complement lipid deficiencies of Nannochloropsis

• PUFA accumulation in Chlorella induced by heterotrophy

Conclusions

• 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

• 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:

1) Conversion of sugars

2) Turnover of photosynthetic biomoleculesFatty Acid

Biosynthesis

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

Mixby

hand

Mixin

chamber

Heptane alone

Butanol then heptane

Nile Tilapia Aquaculture

April 2013

Tilapia Donated to High School for Aquaponics

April 2013

Fig. 1 viral promoter function test in mammalian cell

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

fig. 3. Viral promoters function in Saccharomyces cerevisiae

Table 1, Chlorella Promoter Function Test in Chlamydomonas reinhardtii

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

Transformation Recipient System Promoter Resources

Promoter Size(bp) Chlamy Chlorella Yeast Arabidopsis Mammalian

Chlorella variabilis Promoters*

rbcS1 600 tested

no function on going

rbcS2 600 tested

no function on going

psaD 600 tested

no function on going

αtubulin 600 tested

no function on going

ubiquitin 600 tested

no function on going

Chlorella Virus Promoters Previously Studied Promoters

AMT 851 tested

no function** on going functional

tested need repeat

functional

VP54 636 tested

no function on going functional

tested need repeat

tested no function

Promoters Chosen from Transcriptomic Profile

A158L 500 tested

no function** to be tested to be tested

tested no function

A312L 500 tested

no function** to be tested

tested no function

A348R 500 to be tested to be tested

A404R 500 tested

to function** to be tested to be tested

tested no function

Promoters Selected from Shotgun Library***

Ble r%Colonies%Constructs%

C.reinhardtii%CC503%Cells%

Plasmid%(µg)% Set$I$ Set$II$ Set$III$ Set$IV$

GFP%Detection%

No%DNA%%(negative%control)%

5x107$ 0.5$ 0$ 0$ 0$ 0$$

pGO25%%(negative%control)%

5x107$ 0.5$ 12$ 8$ 6$ 5$$

pGO24%(positive%control)%

5x107$ 0.5$ 660$ 607$ 496$ 563$$

pGOrbcS1% 5x107$ 0.5$ 6$ 9$ 5$ 5$ $pGOrbcS2% 5x107$ 0.5$ 6$ 11$ 8$ 0$ $pGOaUQ% 5x107$ 0.5$ 4$ 5$ 6$ 6$ $pGOatu% 5x107$ 0.5$ 6$ 4$ 6$ 11$ $pGOpsaD% 5x107$ 0.5$ 6$ 5$ 10$ 10$ $

NVP1 361 NVP5 213 NVP8 253 NVP14 384 NVP19 226 NVP20 150 NVP25 104 NVP28 251 NVP30 >5,000 NVP35 196

NVP37 329 to be tested to be tested tested

need repeat to be tested

NVP40 667 to be tested to be tested tested

need repeat functional

NVP63 79 tested

need repeat

NVP80 176 NVP83 264 NVP96 520 NVP99 269

NVP100 >4,000

PVP4 >4,000

PVP7 122

PVP13 385 * Maya Khasin and Ming are working on these projects together **Mayfield lab tested these promoters in Chlamy ***NVP Chlorella variabilis NC64A virus promoter, abbreviated as "N"; PVP Micractinium conductrix Pbi virus promoters, abbreviated as "P"

Acknowledgements

WHITINGSCHOOL OFENGINEERINGJOHNS HOPKINS UNIVERSITY

Dr. George OylerDr. Naoko KobayashiEric NoelAustin BarnesGalen EricksonMaya Khasin

Dr. Michael BetenbaughDr. Marc DonohueDr. Scott WilliamsDr. Minxi WanJon Rogers

Gunjan AndlayAdithya BalasubramanianScott Johnson