Comparison of Chlorella sorokiniana (DOE 1412) grown in outdoor

Comparison of Chlorella sorokiniana (DOE 1412)
grown in outdoor raceway ponds, indoor LEDlighted and temperature-controlled raceway
ponds, and PhenometricsTM photobioreactors
AARON CHAVIS, BRADEN CROWE, DON VALENTINE, AND MICHAEL HUESEMANN
Pacific Northwest National Laboratory, Sequim, WA
September 30, 2014
1
The Problem:
How to determine what laboratory strains should be selected for
outdoor cultivation
High biomass productivity
Resistant to invasive species
Tolerant to wide temperature ranges
The Solution:
A multi-step approach to predict the industrial potential for promising
strains
Strain
Characterization
September 30, 2014
Biomass Growth
Modeling
Productivity
Mapping
Climate-Simulated
Pond Culturing
2
The Problem:
How to determine what laboratory strains should be selected for
outdoor cultivation
High biomass productivity
Resistant to invasive species
Tolerant to wide temperature ranges
The Solution:
A multi-step approach to predict the industrial potential for promising
strains
Strain
Characterization
September 30, 2014
Biomass Growth
Modeling
Productivity
Mapping
Climate-Simulated
Pond Culturing
3
Overview
Cultivation conditions for growing Chlorella sorokiniana (Chlorella
1412) in outdoor raceway ponds at the University of Arizona
Agricultural Research Station in Rimrock, AZ
Capabilities of the PhenometricsTM environmental photobioreactors
(ePBR)
Capabilities of PNNL’s indoor climate simulation raceway ponds
Comparison of Chlorella 1412 growth in Phenometrics ePBRs,
PNNL’s indoor ponds, and outdoor Arizona ponds
September 30, 2014
4
Cultivation of Chlorella 1412 in Rimrock, AZ
from June 6th to August 10th 2012
30 days of batch culture
28 days of semi-continuous
culture
45% and 21% dilution rate
BG-11 Medium
17.6 mM nitrate
0.7 mM phosphate
25 cm culture depth
pH maintained at 7 via CO2
injection
Paddle wheel mixing at ~30 cm/s
September 30, 2014
5
Pond Culture Temperature Measurements
taken in Rimrock, AZ during Batch Phase
30
Pond Temperature [oC]
28
26
24
22
20
18
16
14
12
10
Date
September 30, 2014
6
Light Intensity at Pond Surface [µmol m-2 s-1]
Light Intensity Measurements taken in
Rimrock, AZ during Batch Phase
2500
2000
1500
1000
500
0
Date
September 30, 2014
7
PhenometricsTM Environmental
Photobioreactors (ePBRs)
Simulates pond with a water
column 6 cm in diameter
Temperature control via
thermoelectric heater/cooler
Illuminated with sunlight intensity
white LED
Intermittent CO2 sparging
regulates pH via feedback control
Stir bar mixing up to 800 rpm
Environmental conditions
programmed and maintained via
Algal Command software
September 30, 2014
8
Phenometric’s ePBR Replicate Temperature
Set Points
30
Measured ePBR Temperature
28
Temperature Set Point
ePBR Temperature [oC]
26
24
22
20
18
16
14
12
10
6
September 30, 2014
7
8
9
10
11
Day of Experiment
12
13
14
9
PNNL Climate Simulation Ponds
• 800 L volume
• 4500 LEDs
• Temperature control
• pH control (CO2)
• Paddle wheel mixing
• LabVIEW software
10
PNNL’s Climate Simulation Ponds Closely
Replicate Temperature Set Points
30
Measured Pond Temperature
Temperature Set Point
28
Pond Temperature [oC]
26
24
22
20
18
16
14
12
10
6
7
8
9
10
11
12
13
14
Day of Experiment
11
PNNL’s Climate Simulation Ponds Closely
Replicate Light Set Points
Light Intensity at Pond Surface [µmol m-2 s-1]
2500
Measured Light Intensity
Light Set Point
2000
1500
1000
500
0
6
7
8
9
10
11
12
13
14
Day of Experiment
12
Is the Productivity of Chlorella 1412 the
Same in Outdoor Arizona Pond Cultures,
ePBRs, and PNNL’s LED-Lighted Ponds?
?
=
September 30, 2014
?
=
13
Experimental Methods
3 cultivation phases
Batch phase (30 days)
45% dilution rate semi-continuous phase (14 days)
21% dilution rate semi-continuous phase (14 days)
Algal biomass measured by two metrics
Optical Density at 750 nm (OD750)
Ash-Free Dry Weight (AFDW)
Experiment repeated twice in climate simulation ponds due to rotifer
crash
September 30, 2014
14
Batch Culture Comparison (AFDW)
Rotifer Crash
Ash-Free Dry Weight [mg/L]
1200
AZ Ponds
LED Ponds R1
1000
LED Ponds R2
800
113%
600
108%
400
200
0
0
5
10
15
20
25
30
Day of Experiment
September 30, 2014
*Graph shows average AFDW values for duplicate LED ponds and triplicate AZ ponds
15
Batch Culture Comparison (OD750)
7
Optical Density (OD750)
AZ Ponds
6
LED Ponds R1
5
LED Ponds R2
Rotifer Crash
ePBRs
4
136%
3
107%
2
1
61%
0
0
5
10
15
20
25
30
Day of Experiment
September 30, 2014
*Graph shows average OD750 values for duplicate LED ponds and ePBRs and triplicate AZ ponds
Semi-Continuous Culture Comparison (AFDW)
Ash-Free Dry Weight [mg L-1]
300
143%
AZ Ponds
LED Ponds
ePBRs
250
200
150
104%
100
71%
56%
50
Dilution Rate = 0.21/day
Dilution Rate = 0.45/day
0
36
41
46
51
56
Day of Experiment
September 30, 2014
*Graph shows average AFDW values for duplicate LED ponds and ePBRs and triplicate AZ ponds
17
Conclusions
PNNL climate simulation ponds and Phenometrics ePBRs replicate culture
temperature and light intensity set points extremely accurately
PNNL climate simulation ponds over-predict biomass productivity (AFDW) of
Chlorella 1412 outdoor AZ pond cultures by 4-13%
May be caused by lack of UV radiation and minimal exposure to elements and
invasives compared to outdoor ponds?
Phenometrics ePBRs under-predict biomass productivity of Chlorella 1412
outdoor AZ pond cultures by 39-44%
May be caused by LED spectrum, lack of mixing, salt/algae build up on lens?
Phenometrics ePBR Chlorella 1412 cultures exhibit ~50% of the biomass
productivity observed in PNNL climate simulation ponds
Independent study comparing Picochlorum sp. productivity in ePBRs at LANL and
in PNNL’s LED ponds showed a similar trend
September 30, 2014
18
Acknowledgements
Support provided by the Department of Energy’s BioEnergy
Technology Office under the National Alliance for Advanced Biofuels
and Bioproducts
September 30, 2014
19
Questions?
Aaron Chavis: aaron.chavis@pnnl.gov
Michael Huesemann: michael.huesemann@pnnl.gov
September 30, 2014
20
Supplementary Slides
September 30, 2014
21
Culturing of LANL’s Picochlorum sp. in
ePBRs and PNNL’s LED-lighted Ponds
ePBR Productivity (initial 15 day slope) is
about Half (50%) of Pond Productivity
September 30, 2014
22
Semi-Continuous Culture Comparison (OD750)
160%
AZ Ponds
1.4
Optical Density (OD750)
LED Ponds
1.2
ePBRs
1
0.8
119%
0.6
0.4
75%
52%
0.2
Dilution Rate = 0.45/day
0
35
40
Dilution Rate = 0.21/day
45
50
55
60
Day of Experiment
September 30, 2014
*Graph shows average OD750 values for duplicate LED Ponds and ePBRs and triplicate AZ ponds
23
Rotifer Infestation in the First Batch
Cultivation in the LED Ponds
September 30, 2014
24
ePBR White LED Light Spectrum
Blue Dot-Dashed Line = Fluorescent Light
Red Dashed Line = White LED
Black = Solar
September 30, 2014
25
PNNL Pond LED Light Spectrum
9.00E+04
Metal Halide
8.00E+04
Arbitrary Unit
7.00E+04
6.00E+04
Sunlight
LEDs
5.00E+04
4.00E+04
3.00E+04
2.00E+04
1.00E+04
0.00E+00
400
September 30, 2014
450
500
550
Wavelength, nm
600
650
700
26