1. Group 2: CHUAY JIE QI OW CHOON LIN FITRI HIDAYAH AKMAL Production of Bio-hydrogen by Microorganism

2. Fact of Fossil Fuel Non-renewable energy Excessive global climate change Emission of greenhouse pollutant Negative impact on environment Source: http://harmonscience6.wikispaces.com/; fossilfuelsenergy.weebly.com; www.theguardian.com

3. Introduction to Hydrogen Fuel Act as energy carrier High energy content Almost 3x greater than H-C fuels It can be operate at ambient temperature and pressure with minimum energy consumption Clean renewable energy Sustainably: contribution to the world economic growth Environmentally friendly Most abundant element

4. Raw Materials for H ₂ Production Source:www.tullowoil.com; reachingutopia.com; globalresourceadvisorsllc.com; www.biologyexams4u.com

5. Microorganisms Produce H ₂ 1. Plant base: Green Microalgae Chlamydomonas sp. Produce x2 efficient compared to wild type strain for PS I 2. Microbe base: Cyanobacteria Anabaena variabilis ATCC 29413 Source: www.fytoplankton.cz Source: microbewiki.kenyon.edu

6. Other Information: Registration Product Strain used for Hydrogen production 1 Ideal microbes for hydrogen production 2 Unique prokaryotes with diverse range properties 3 Production can undergo i) Direct photolysis ii) Indirect photolysis 5 Species divided into 3 groups: i) Heterocystous ii) Non- heterocystous iii) Marine 4 Use two distinct enzymes to generate H2: i) Nitrogenase ii) Hydrogenase Cyanobacteria Source: http://waynesword.palomar.edu/

7. Factors affecting Hydrogen production in Cyanobacteria

8. Role of environmental factors light temperature Nitrogen source Molecular nitrogen Carbon source oxygen sulphur Methanesalinity micronutrients

9. Condition of production Growth of algae in photobioreactors (PBR) Light source and hydrodynamics – conditions affect algae growth Must be in closed system – to prevent hydrogen escape and facilitate collection of gas Design must allow for easy sterilization Provide high surface area to volume ratio

10. Types of PBR Tubular PBR 1) Consists of long transparent tube (d = 3-6 cm, L = 10- 100m) 2) Used to pumped culture liquid by mechanical or airlift pumps 3) Flexibility in volume to surface area ratio 4) Flexibility in shifting the place receiving light

11. Flat panel PBR 1) Consists of stainless steel frame and 3 polycarbonate panels 2) Comprises of 2 compartments – placed side by side 3) Front compartment – bacterial culture 4) Hind compartment – to circulate the water by temperature controlled water bath 5) To maintain the desired temperature of the culture

12. Vertical PBR 1) Consists of transparent column 2) Made up of high glass and surrounded by a water jacket 3) Allows maintenance of the temperature with circulating waters 4) Circulating water is to allows entry of light

13. Method to Produce H ₂ 1. Biophotolysis 2. Photo-fermentation Photosynthesis process 3. Dark-fermentation Anaerobic process: Organic  H ₂ Better than photo-fermentation: not depend on sunlight as energy source, can perform continuous process of hydrogen production 4. Hybrid Biological Hydrogen Production Electrolysis : continuous produce H ₂ assisted electron exchange by bacteria Adv: ↓ Energy needed to produce H ₂, electrochemical process is not limited only to carbohydrates Bacteria

14. Biophotolysis Definition: Plant-type photosynthesis process by microorganism to split waster to H ₂ with light in anaerobic condition. H₂H₂ O₂O₂ H₂OH₂O Enzyme: Hydrogenase Bacteria: Green algae/cyanobacteria Sunlight CO ₂

15. Advantages of Biophotolysis 1. No requirement of adding substrate as nutrients 2. Hydrogen produced: Does not emit greenhouse gas Abundant for avaibility Renewable energy 3. H ₂ used in fuel cell to generate electricity

16. Overview of Production & Utilization of H ₂

17. Production of H ₂ Hydrogenase H₂H₂ Direct Biophotolysis

18. Type of Hydrogenase 1. FeFe-hydrogenase Catalyses the reversible oxidation of molecular hydrogen.

19. Contd.. 2. NiFe-bidirectional hyrogenase 3. Metal-free hydrogenase

20. Contd Problem: Hydrogenase, Ferredoxin and Nitrogenase is very sensitive to O ₂ Solution: [O ₂ ] < 0.1% Yield = Max H ₂

21. Direct Biophotolysis

22. Indirect Biophotolysis Definition: Process to produce H ₂ from water using microorganism (microalgae and cyanobacteria) by conversion of sunlight energy into chemical energy (H ₂ ) though 1. Light dependent process 2. Light independent process

23. Reaction Equation 1. Biomass production by photosynthesis 2. Biomass concentration 3. Dark aerobic fermentation 4. Conversion of 2 mol of acetate into H ₂

24. Role of Nitrogenase This enzyme is found in the heterocysts of filamentous cyanobacteria Responsible for nitrogen fixation It reduce N ₂ to NH ₃ to produce hydrogen with consumption of reducing power and ATP 16ATP+16H ₂ O+N ₂ +10H ⁺ +8e ⁻ 16ADP+16Pi+2NH ₄⁺ + H ₂

25. In N ₂ fixing strains, the net H ₂ production is the result of H ₂ evolution by nitrogenase H ₂ consumption mainly catalyzed by the uptake hydrogenase.

26. Structure of Nitrogenase Nitrogenase consists of two part : 1. Dinitrogenase (MoFe Protein, encoded by the genes nifD and nifK, a and b respectively) breaks apart the atoms of nitrogen 2. Dinitrogenase reductase (Fe Protein, encoded by nifH ) transfer of electrons from the external electron donor (a ferredoxin or a flavodoxin) to the dinitrogenase

27. Role of Nitrogenase & Hydrogenase In Cyanobacterial Hydrogen Production

28. Limitation of Hydrogen

29. System for producing, storing, and delivering the Hydrogen gas 1 Complex storage capability 2 Highly Flammable 3 Less information exchange between scientist & engineers 4

30. Cost hundreds of billions of dollars to build. More expensive than a system based on liquid fuels such as gasoline or methanol System for producing, storing, and delivering the Hydrogen gas 1

31. Hydrogen itself is hard to move around Required to be stored as compressed gas Stored in gas cylinders or spherical containers Complex storage capability 2

32. Explosive range is a 13- to 79-percent concentration in air Colorless and odorless and burns with a nearly invisible flame Hydrogen mixes with air faster than does gasoline vapor (diffusion coefficient for hydrogen is 0.61 cm 3 /sec) Highly Flammable 3

33. Rates of hydrogen produced by biological systems are unknown to fuel cell engineers Amounts of H 2 required for practical applications, such as fuel cells, are unknown to biohydrogen researchers Rates of hydrogen produced by the various biohydrogen systems are expressed in different units. Difficult to assess and compare the rates and amounts of hydrogen synthesized by different biohydrogen technologies Less information exchange between scientist & engineers 4