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

1 2 3 4 5 6 7 8 9 10 11 12 13 14

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