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

2. Bojan TamburicSolar Hydrogen Project Introduction 1)Hydrogen production and utilisation –PEM fuel cells –Clean and green H 2 production 2)Green algal routes to solar hydrogen –Photosynthetic H 2 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 H 2 measurements –Procedure –Results –Improvements 5)Future plans –Cultivation reactor –96-well plate detector –Masters targets

3. Bojan TamburicSolar Hydrogen Project Content Hydrogen production and utilisation Green algal routes to solar hydrogen Growth and sulphur deprivation of C.reinhardtii Preliminary H 2 measurements Future plans

4. Bojan TamburicSolar Hydrogen Project PEM fuel cells Environmental concerns over: –CO 2 emissions –Vehicle exhaust gasses (SO x, NO x ) Sustainability concerns: –Peak oil –Global warming Proton exchange membrane (PEM) fuel cells use H 2 to drive an electrochemical engine Only product is water Barriers that must be overcome: –Compression of H 2 –Hydrogen infrastructure required –Sustainable H 2 production

5. Bojan TamburicSolar Hydrogen Project Clean and green H 2 production Bulk Hydrogen is typically produced by the steam reforming of Methane, followed by the gas-shift reaction: –CH 4 + H 2 O → CO + 3H 2 –CO + H 2 O → CO 2 + H 2 Negates many of the benefits of PEM fuel cells Renewable and sustainable H 2 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 H 2 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. Bojan TamburicSolar Hydrogen Project Content Hydrogen production and utilisation Green algal routes to solar hydrogen Growth and sulphur deprivation of C.reinhardtii Preliminary H 2 measurements Future plans

7. Bojan TamburicSolar Hydrogen Project Photosynthetic H 2 production Discovered by Gaffron in 1942 Direct H 2 photoproduction –2H 2 O → 2H 2 + O 2 Solar energy absorbed by Photosystem II and used to split water Electrons transported by Ferredoxin H 2 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. Bojan TamburicSolar Hydrogen Project Two-stage growth and hydrogen production Hydrogenase enzyme deactivated in the presence of Oxygen – limit on volume and duration of H 2 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 H 2 and ATP –H 2 production slows after about 5 days as algae begin to die Use the model green algae C.reinhardtii Melis et al. (2002)

9. Bojan TamburicSolar Hydrogen Project Content Hydrogen production and utilisation Green algal routes to solar hydrogen Growth and sulphur deprivation of C.reinhardtii Preliminary H 2 measurements Future plans

10. Bojan TamburicSolar 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

11. Bojan TamburicSolar Hydrogen Project Sulphur deprivation 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 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 H 2 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)

12. Bojan TamburicSolar Hydrogen Project 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! Chlorophyll content (μg/ml) Our data Tsygankov et al. (2004) Prior to sulphur deprivation 8.4218-22 Following sulphur deprivation 6.9414-16

13. Bojan TamburicSolar Hydrogen Project Content Hydrogen production and utilisation Green algal routes to solar hydrogen Growth and sulphur deprivation of C.reinhardtii Preliminary H 2 measurements Future plans

14. Bojan TamburicSolar 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 H 2 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 H 2 to obtain the total volume of hydrogen produced

15. Bojan TamburicSolar Hydrogen Project Results

16. Bojan TamburicSolar Hydrogen Project 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 H 2 detection Develop a continuous method of H 2 measurement (membrane mass spectrometry) Develop a means of measuring dissolved Hydrogen Author H 2 production after 48h of sulphur deprivation (ml/l) Ghirardi et al. (2000) 25 Melis et al. (2002)45 Kosourov et al. (2007) 70 Our results11

17. Bojan TamburicSolar Hydrogen Project Content Hydrogen production and utilisation Green algal routes to solar hydrogen Growth and sulphur deprivation of C.reinhardtii Preliminary H 2 measurements Future plans

18. Bojan TamburicSolar 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 CO 2, which is needed for algal nutrition Expect to test reactor with water/algae in the coming weeks

19. Bojan TamburicSolar Hydrogen Project 96-Well plate detector Use Tungsten Oxide (WO 3 ) 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 PdWO 3 –Test responsiveness with hydrogen stream –Test with de-sulphurised algal culture –Build detector

20. Bojan TamburicSolar Hydrogen Project 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 H 2 production (membrane mass spectrometry) Compare H 2 production of wild type and DUM24 strains of C.reinhardtii Begin to optimise parameters affecting growth and H 2 production of C.reinhardtii Attempt to prolong algal lifetime using sulphur re-insertion techniques

21. Bojan TamburicSolar Hydrogen Project 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. Bojan TamburicSolar Hydrogen Project Thank you for listening! Any questions?