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Development of a pH drift assay for rapid carbon uptake measurements for mass algal culture
Niu Du Scripps Institution of Oceanography | J. Craig Venter Institute
Algae Biomass Summit 2016Phoenix
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ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
Biomass/m2/day
Evolutionintheprospectiveofpursuinghigherproductivity
Aquaticspeciesprogram1978
Duetooilcrisis
Aimedtosavehumanity
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
Biomass/m2/day
Evolutionintheprospectiveofpursuinghigherproductivity
Fromnomoredrivethroughdonuts
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
Biomass/m2/day
Photosyntheticefficiency
Evolutionintheprospectiveofpursuinghigherproductivity
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
How to determine the efficiency of photosynthesis?
Lightabsorption
Oxygenevolution
&Electrontransfer
Carbonuptake
&fixation
Productofinterest
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
Lightabsorption
Oxygenevolution
&Electrontransfer
Carbonuptake
&fixation
Productofinterest
How to determine the efficiency of photosynthesis?
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
A good method for determine carbon uptake rate
- Reliable
- Representative
- Fastandeasytooperate
- Safe
- Compatible
þ Goodforquantitativeresearch
þ Variablesarecontrolled
þ Highfrequencyforkineticsstudy
þ Nodangerouschemicalsinvolved
þ Coupledwithotheranalyses
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
Why old methods do not work very well?
http://ocean.stanford.edu/
14Cincubation
Pros:
Cons:
SensitiveArbitraryRadioactive
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
Why old methods do not work very well?
http://ocean.stanford.edu/
14Cincubation In-linegasanalysis
Pros:
Cons:
SensitiveArbitraryRadioactive
NoninvasiveImplementationLaggingCO2 signal
Oakleyetal.,2012
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
Why old methods do not work very well?
http://ocean.stanford.edu/
14Cincubation In-linegasanalysis
Pros:
Cons:
SensitiveArbitraryRadioactive
NoninvasiveImplementationLaggingCO2 signal
Oakleyetal.,2012
OldschoolpHdrift
InvivoPoorcalculation,Non– steadystate
Allenetal.,1981
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
The principles of pH-drift assay
O CO
OC
O OH
O CO
HCO3− +H + H2O+CO2
CO32− +H + HCO3
−
OC
O OH
H + pH
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
O CO
O CO
OC
O OH
HCO3− +H + H2O+CO2
H + pH
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
0
500
1000
1500
2000
2500
7 8 9 10 11 12
Cspeciesc
oncentratio
n(uM)
pH
CO2
HCO3
CO3
Dicksonetal.,2007SOP
Constantcarbonatealkalinity
CT [H+] CO2 removal
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
A good method for mass culture (Lab and Industry)
- Reliable
- Representative
- Fastandeasytooperate
- Safe
- Compatible
pHbuffers+Tris buffer
ReferenceseawaterSIOCRM
pHcalibration
MediumAlkalinity
f (x)
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
A good method for mass culture (Lab and Industry)
- Reliable
- Representative
- Fastandeasytooperate
- Safe
- CompatibleWaterbath
CreeLED
Algaesample
ALGinstrumentsTM
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
Hille etal2014
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
A good method for mass culture (Lab and Industry)
- Reliable
- Representative
- Fastandeasytooperate
- Safe
- Compatible
Sample
Wash
MeasurePvs Icurvein30minorless
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
A good method for mass culture (Lab and Industry)
- Reliable
- Representative
- Fastandeasytooperate
- Safe
- Compatible
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
A good method for mass culture (Lab and Industry)
- Reliable
- Representative
- Fastandeasytooperate
- Safe
- Compatible
pHacquisitionsystem
Additionaldata
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
Currentsetup
Integratedwithamembraneinletmass-spectrometry(MIMS)system
Simultaneousmeasurementoftotalinorganiccarbonandothersolublegases
Fabricatedand3-Dprintedpartsforconnection
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
Case study VerifyingachloroplastHCO3- transporter
Transcriptome +Westernblot Up-regulationunderlowCO2condition
Signalsequence Chloroplastlocalized
LowergrowthrateinknockdownmutantGrowthexperiment
Yakajima etal.,inprep
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
Case study VerifyingachloroplastHCO3- transporter
Transcriptome +Westernblot Up-regulationunderlowCO2condition
Signalsequence Chloroplastlocalized
LowergrowthrateinknockdownmutantGrowthexperiment
Indicate
Chloroplastlocalizedbicarbonatetransporter
Yakajima etal.,inprep
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
Case study VerifyingachloroplastHCO3- transporter
Transcriptome +Westernblot Up-regulationunderlowCO2condition
Signalsequence Chloroplastlocalized
LowergrowthrateinknockdownmutantGrowthexperiment
Indicate
ChloroplastlocalizedbicarbonatetransporterMoredirectevidence?
Yakajima etal.,inprep
Chloroplast
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
Case study
Bicarbonatetransporter
VerifyingachloroplastHCO3- transporter
-5E-09
0
5E-09
1E-08
1.5E-08
2E-08
2.5E-08
0 500 1000 1500 2000 2500
Carboninflux
Suppressed
μmol/cell/s
Normal
Lightintensity
CO2
HCO3-
Ci Pool
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
-2E-09
-1E-09
0
1E-09
2E-09
3E-09
4E-09
5E-09
6E-09
7E-09
0 500 1000 1500 2000 2500
Carboneflux
μmol/cell/s
LightintensityChloroplast
Bicarbonatetransporter
CO2 Ci PoolSuppressedNormal
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
Summary
• DevelopedapHdriftsystemforrapidcarbonuptakemeasurements
• Moreefficientmeasurementformassculturethanclassicmethods
• Variableswellcontrolledduringthemeasurement
• Applicabletoscientificandpotentialindustrialapplications
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
Acknowledgements
Dr.GregMitchellDr.DominickMendolaMitchellLab
Dr.AndrewAllenAllenLabDr.ChrisDuPont
Dr.JonathanMeuserALGinstrumentsBrianCreggerSafeHarborAssociates
Dr.AndrewDickson
RobertBuzzelli EndowedFellowshipFund EdnaBaileySussman Foundation
DepartmentofEnergy,Biodesign AwardDE-SC0008593GordonandBettyMooreFoundationInvestigatorAwardGMBF3828
ABS2016, Phoenix Niu Du: ndu@ucsd.edu
Scripps Institution of Oceanography | J. Craig Venter Institute
Thankyou!
UCSDGreenhouse/BiologyFieldStation
Photo:Niu Du