Bojan Tamburic Prof. Geoff Maitland Dr. Klaus Hellgardt Chemical Engineering

1. Parameters Affecting the Growth and Hydrogen Production of the Green Algae Chlamydomonas Reinhardtii Bojan Tamburic Prof. Geoff Maitland Dr. Klaus Hellgardt Chemical Engineering

2. 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 3) Growth and sulphur deprivation of C.reinhardtii Growth of C.reinhardtii Sulphur deprivation Measuring Chlorophyll content 4) Preliminary H2 measurements Procedure Results Improvements 5) Future plans Cultivation reactor 96-well plate detector Masters targets Introduction

3. Content • Hydrogen production and utilisation • Green algal routes to solar hydrogen • Growth and sulphur deprivation of C.reinhardtii • Preliminary H2 measurements • Future plans

4. 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

5. 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 “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)

6. Content • Hydrogen production and utilisation • Green algal routes to solar hydrogen • Growth and sulphur deprivation of C.reinhardtii • Preliminary H2 measurements • Future plans

7. Photosynthetic H2 production • 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 Hallenbeck & Benemann (2002)

8. 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 • Use the model green algae C.reinhardtii Melis et al. (2002)

9. Content • Hydrogen production and utilisation • Green algal routes to solar hydrogen • Growth and sulphur deprivation of C.reinhardtii • Preliminary H2 measurements • Future plans

10. 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

11. Centrifugation 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 Dilution 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 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) Sulphur deprivation

12. 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!

13. Content • Hydrogen production and utilisation • Green algal routes to solar hydrogen • Growth and sulphur deprivation of C.reinhardtii • Preliminary H2 measurements • Future plans

14. 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

15. Results

16. Improvements • Run experiment again: • 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

17. Content • Hydrogen production and utilisation • Green algal routes to solar hydrogen • Growth and sulphur deprivation of C.reinhardtii • Preliminary H2 measurements • Future plans

18. 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

19. 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 96-Well plate detector

20. 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

21. References • 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. 1185- 1193. • 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.

22. Thank you for listening! Any questions?