Parameters Affecting the Growth
and Hydrogen Production of the
Green Algae Chlamydomonas
Reinhardtii
Bojan Tamburic
Prof. Geoff Maitland
Dr. Klaus Hellgardt
Chemical Engineering
Bojan Tamburic
Solar Hydrogen Project
Introduction
1) Hydrogen production and
utilisation
– PEM fuel cells
– Clean and green H2 production
2) Green algal routes to solar
hydrogen
– Photosynthetic H2 production
– Two stage growth and
hydrogen production
4) Preliminary H2 measurements
– Procedure
– Results
– Improvements
5) Future plans
– Cultivation reactor
– 96-well plate detector
– Masters targets
3) Growth and sulphur deprivation
of C.reinhardtii
– Growth of C.reinhardtii
– Sulphur deprivation
– Measuring Chlorophyll content
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Content
• Hydrogen production and utilisation
• Green algal routes to solar hydrogen
• Growth and sulphur deprivation of
C.reinhardtii
• Preliminary H2 measurements
• Future plans
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PEM fuel cells
• Environmental concerns over:
– CO2 emissions
– Vehicle exhaust gasses (SOx, NOx)
• Sustainability concerns:
– Peak oil
– Global warming
• Proton exchange membrane (PEM)
fuel cells use H2 to drive an
electrochemical engine
• Only product is water
• Barriers that must be overcome:
– Compression of H2
– Hydrogen infrastructure required
– Sustainable H2 production
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Clean and green H2 production
• Bulk Hydrogen is typically produced by
the steam reforming of Methane,
followed by the gas-shift reaction:
– CH4 + H2O → CO + 3H2
– CO + H2O → CO2 + H2
• Negates many of the benefits of PEM
fuel cells
• Renewable and sustainable H2
production required
• Can be achieved by renewable
electricity generation, followed by water
electrolysis, but:
– Low efficiency
– High costs
– Makes more sense to just use electricity
directly
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“Photosynthetic H2
production by green algae
may hold the promise of
generating renewable fuel
from nature’s most plentiful
resources – sunlight and
water” – Melis et al. (2007)
Content
• Hydrogen production and utilisation
• Green algal routes to solar hydrogen
• Growth and sulphur deprivation of
C.reinhardtii
• Preliminary H2 measurements
• Future plans
Bojan Tamburic
Solar Hydrogen Project
Photosynthetic H2 production
2 H 2O  2 H 2  O2
Ferredoxin
Hallenbeck & Benemann (2002)
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• Discovered by Gaffron in 1942
• Direct H2 photoproduction
– 2H2O → 2H2 + O2
• Solar energy absorbed by Photosystem II
and used to split water
• Electrons transported by Ferredoxin
• H2 production governed by the
Hydrogenase enzyme – a natural
catalyst
• Anaerobic photosynthesis required
• Process provides ATP – energy source
• No toxic or polluting bi-products
• Potential for value-added products
derived from algal biomass
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Two-stage growth and hydrogen
production
• Hydrogenase enzyme deactivated in
the presence of Oxygen – limit on
volume and duration of H2 production
• Two-stage process developed by
Melis et al. (2000)
– Grow algae in oxygen-rich conditions
– Deprive algae of sulphur
– Photosystem II protons cannot
regenerate their genetic structure
– Algae use up remaining oxygen by
respiration and enter anaerobic state
– Algae produce H2 and ATP
– H2 production slows after about 5
days as algae begin to die
Melis et al. (2002)
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• Use the model green algae
C.reinhardtii
Solar Hydrogen Project
Content
• Hydrogen production and utilisation
• Green algal routes to solar hydrogen
• Growth and sulphur deprivation of
C.reinhardtii
• Preliminary H2 measurements
• Future plans
Bojan Tamburic
Solar Hydrogen Project
Growth of C.reinhardtii
• Prepare Tris-Acetate Phosphate (TAP) growth
media
• Algae stocks are initially grown on a Petri dish
• Cultivation:
– Scrape algae off Petri dish and transfer into 25ml
flask filled with TAP medium
– Flask is kept under “ideal” algal growth conditions:
•
•
•
•
room temperature
pH 7.0
cool white light
constantly shaken to provide aeration
– Regularly scale up culture
– Algae should be adequately grown after two
weeks
• Can check the stage of algal growth by
measuring Chlorophyll content
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Sulphur deprivation
• Centrifugation
• Dilution
– Prepare sulphur-deprived
media (TAP-S)
– Spin down culture in a
centrifuge (4000rpm for
15min)
– Pellet of algal cells forms
– Pour out liquid – algal cells
are lost in this process
– Wash cells in TAP-S
– Re-suspend washed algae
in about 3l of TAP-S
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1) TAP medium prepared with different
(small) sulphur concentrations



TAP medium pre-set with various
sulphur concentrations (20-250μM)
Inoculated with 1.4% v/v of growing
culture
Algae grow until all sulphur is used
up, then produce H2
2) TAP-S medium inoculated with
different concentrations of algal cells


2-50% v/v of culture used
Procedure using 10% cell inoculum
identified as the best in literature
(Laurinavichene, 2002)
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Measuring Chlorophyll content
• Take small sample (20ml) of
culture and dilute with Acetone
• Vortex for 1min to release all
Chlorophyll
• Micro-centrifuge (13000rpm,
5min)
• Use spectrophotometer to
measure absorbance at 645nm
and 663nm
• Calculate Chlorophyll
concentration
• About 20% of algal cells lost
through the centrifugation
procedure!
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Chlorophyll
content
(μg/ml)
Our data
Tsygankov
et al.
(2004)
Prior to
sulphur
deprivation
8.42
18-22
Following
sulphur
deprivation
6.94
14-16
Solar Hydrogen Project
Content
• Hydrogen production and utilisation
• Green algal routes to solar hydrogen
• Growth and sulphur deprivation of
C.reinhardtii
• Preliminary H2 measurements
• Future plans
Bojan Tamburic
Solar Hydrogen Project
Procedure
• Use Helium as the inert carrier gas
• Use gas-tight, sterile syringe to
inject 0.5ml gas sample from the
sulphur-deprived algal culture into
the He stream
• Measure the amount of H2 in the
stream with a mass spectrometer
• Take 5+ injections
• Repeat measurements at 24h
intervals – experiment maintained
for only 2 days
• Calibrate with an injection of pure
H2 to obtain the total volume of
hydrogen produced
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Results
Hydrogen Production During 2 Days of Mass Spectrometer
Measurements
6.00E-09
30
5.00E-09
25
00h after Sulphur Deprivation
4.00E-09
24h
48h
3.00E-09
2.00E-09
Volume of Hydrogen produced (ml)
Ion Current (A) at Atomic Mass = 2
Mass Spectrometer Measurement of Hydrogen Production
following Sulphur Deprivation of C.reinhardtii
20
15
10
5
1.00E-09
0
0.00E+00
0
0
200
400
600
800
1000
1200
Time (s)
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12
24
Time After Sulphur Deprivation (h)
36
48
Improvements
• Run experiment again:
Author
H2 production after
48h of sulphur
deprivation (ml/l)
Ghirardi et al.
(2000)
25
Melis et al. (2002)
45
Kosourov et al.
(2007)
70
Our results
11
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– More care with preparation and
sterilisation to avoid fungal
contamination
– Use heated mass spectrometer
capillary system to prevent water
blockage
– Use bioreactor (improved stirring,
lighting, control)
• Use water/oil bath system as
alternative method of H2 detection
• Develop a continuous method of
H2 measurement (membrane
mass spectrometry)
• Develop a means of measuring
dissolved Hydrogen
Solar Hydrogen Project
Content
• Hydrogen production and utilisation
• Green algal routes to solar hydrogen
• Growth and sulphur deprivation of
C.reinhardtii
• Preliminary H2 measurements
• Future plans
Bojan Tamburic
Solar Hydrogen Project
Cultivation reactor
• Micro-algal cultivation unit from Aqua Medic
• We have 3 units in lab
• Store algal cultures after they are grown in
Biology
– Wild type
– DUM24 mutant
• Can also be used for short-term storage of
sulphur-deprived algal cultures
• Improvements to the system:
– Need a closed bioreactor system – vessel
requires a silicon seal
– Circulation pump needed to provide a source
of CO2, which is needed for algal nutrition
• Expect to test reactor with water/algae in the
coming weeks
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96-Well plate detector
•
•
•
•
Use Tungsten Oxide (WO3) as the
hydrogen detector
It turns blue in the presence of
hydrogen – transient change
Catalyse with Palladium (Pd) for
better responsiveness
Detector targets:
– Identify the wells where hydrogen
production is taking place
– Quantify the hydrogen production
•
Best way to proceed:
– Coat microscope slides with
PdWO3
– Test responsiveness with hydrogen
stream
– Test with de-sulphurised algal
culture
– Build detector
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Masters targets
• Conduct Hydrogen measurements
using the Sartorius photobioreactor
• Attempt the dilution method of sulphur
deprivation
• Complete 96-well plate detector
• Develop a continuous method of
monitoring H2 production (membrane
mass spectrometry)
• Compare H2 production of wild type
and DUM24 strains of C.reinhardtii
• Begin to optimise parameters affecting
growth and H2 production of
C.reinhardtii
• Attempt to prolong algal lifetime using
sulphur re-insertion techniques
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References
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•
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•
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Gaffron, H. 1942, "Fermentative and photochemical production of hydrogen from algae", Journal of
General Physiology, pp. 219-240.
Ghirardi, M.L., Zhang, L., Lee, J.W., Flynn, T., Seibert, M., Greenbaum, E. & Melis, A. 2000,
"Microalgae: a green source of renewable H2", Trends in Biotechnology, vol. 18, no. 12, pp.
506-511.
Hallenbeck, P.C. & Benemann, J.R. 2002, "Biological hydrogen production; fundamentals and
limiting processes", International Journal of Hydrogen Energy, vol. 27, no. 11-12, pp. 11851193.
Kosourov, S., Patrusheva, E., Ghirardi, M.L., Seibert, M. & Tsygankov, A. 2007, "A comparison of
hydrogen photoproduction by sulfur-deprived Chlamydomonas reinhardtii under different
growth conditions", Journal of Biotechnology, vol.
128, no. 4, pp. 776-787.
Laurinavichene, T., Tolstygina, I. & Tsygankov, A. 2004, “Dilution methods to deprive
Chlamydomonas reinhardtii cultures of sulfur for subsequent hydrogen photoproduction",
International Journal of Hydrogen Energy, vol. 27, pp. 1245-1249.
Melis, A. 2000, "Sustained photobiological hydrogen gas production upon reversible inactivation of
oxygen evolution in the green algae Chlamydomonas reinhardtii", Plant Physiology, vol. 122,
pp. 127-135.
Melis, A. 2002, "Green alga hydrogen production: progress, challenges and prospects",
International Journal of Hydrogen Energy, vol. 27, no. 11-12, pp. 1217-1228.
Melis, A. 2007, "Photosynthetic H2 metabolism in Chlamydomonas reinhardtii (unicellular green
algae)", Planta, vol. 226, no. 5, pp. 1075-1086.
Tsygankov, A.A., Kosourov, S.N., Tolstygina, I.V., Ghirardi, M.L. & Seibert, M. 2006, "Hydrogen
production by sulfur-deprived Chlamydomonas reinhardtii under photoautotrophic
conditions", International Journal of Hydrogen Energy, vol. 31, no. 11, pp. 1574-1584.
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