A LOS ANGELES Y A LOS SANTOS

Dark fermentation is carried out most efficiently by purple non-sulphur bacteria, such as

Rhodospirillum sp. or Rhodobacter sp., or by green sulphur bacteria such as Chlorobium vibrioforme (Rupprecht et al., 2006). These bacteria are grown on carbohydrate-rich organic

substrates, typically glucose or sucrose, in the dark. The organic substrates are degraded by oxidation to provide the building blocks and energy for growth; the use of an organic carbon source for growth is known as heterotrophic growth. This process also produces electrons, which would normally be used to reduce O2 to water. However, under anaerobic conditions, the electrons are used to drive the process of organic acid, alcohol and H2 formation by dark fermentation (Hallenbeck & Ghosh, 2009). In the case of glucose fermentation, glucose catabolism initially produces pyruvate. Pyruvate can be oxidised to produce formic acid and acetyl-coenzyme A (acetyl CoA), which can be converted to acetyl phosphate, resulting in the formation of the energy carrier adenosine triphosphate (ATP) and the excretion of acetic acid (Manish & Banerjee, 2008). Pyruvate oxidation requires reduction of the electron carrier ferredoxin (Fd). Reduced Fd is re-oxidised by the nitrogenase or hydrogenase enzymes, which release electrons as H2 (Nath & Das, 2004).

Dark fermentation redox pathways:

pyruvate + CoA → acetyl-CoA + formate

pyruvate + CoA + 2 Fdoxidised → acetyl-CoA + CO2 + 2 Fdreduced 2 Fdreduced → 2 Fdoxidised + H2

Dark fermentation reaction:

C6H12O6 (glucose) + 2 H2O → 2 CH3COOH (acetate) + 4 H2 + 2 CO2

The maximum theoretical yield of dark-fermentative H2 production is 4 molH2·mol-1 of glucose if all the glucose is completely metabolised to acetate (Das, 2009). In a practical experiment, the substrate conversion is approximately 53% efficient and the H2 production rate is 1,300-3,000 ml of H2 per litre of culture per hour (mlH2·l-1·h-1) (Levin, 2004). Glucose is not the ideal choice of

substrate because it needs to be derived from an energy crop such as sugarcane. Practical and economic dark fermentation would use organic substrates from the food and forestry industry or from sewage sludge (Das, 2009). The main barrier facing the commercialisation of waste-to-H2 by dark fermentation is the relatively low H2 yield and the formation of large quantities of side- products, such as acetic, propionic, butyric acids and ethanol (Hallenbeck & Ghosh, 2009). The main concern is therefore one of selection and separation efficiency.

2.6.2. Photofermentation

As well as carrying out dark fermentation, purple non-sulphur bacteria are also capable of carrying out photofermentation in the light, which is sometimes also referred to as anaerobic photosynthesis (Das & Veziroglu, 2001). In this process, the bacteria use a reduced carbon substrate, typically an organic acid such as acetate, together with the light energy captured by their photosystem to produce ATP and high-energy electrons that reduce Fd. Reduced Fd drives H2 formation via the nitrogenase enzyme (Akkerman et al., 2002). This reaction requires an energy input of 75.2 kJ·mol-1_{, as defined by its Gibbs free energy (ΔG}0_).

Photofermentation reaction (purple non-sulphur bacteria): CH3COOH (acetate) + 2 H2O → 2 CO2 + 4 H2 ΔG0 _{= +75.2 kJ·mol}-1 _{(light energy requirement)}

The nitrogenase enzyme is highly sensitive to O2 and inhibited in the presence of ammonium ions. Photofermentation by purple non-sulphur bacteria must therefore be carried out in closed PBRs, free of N2 (Akkerman et al., 2002). The process also suffers from low light conversion efficiencies and a high energy demand by the nitrogenase enzyme. Nevertheless, anaerobic photosynthesis has its merits because it can completely convert organic wastes to H2 and CO2 (Hallenbeck & Ghosh, 2009). H2 production rates of 50-180 mlH2·l-1·h-1 have been documented (Levin, 2004).

An alternative route to photofermentative H2 production involves the use of nitrogen-fixing cyanobacteria (blue-green algae), such as Anabaena sp. PCC 7120 and Cyanothece sp. ATCC 51142. Anabaena contains a structure called a heterocyst, a walled-off metabolic block within the

cell, which allows it to spatially separate photosynthetic and photofermentative processes (Lopes et

al., 2002). The main part of the cell fixes CO2 to sucrose, which is used to power the nitrogenase enzyme in the heterocyst (Ghirardi et al., 2009). Cyanothece does not contain a heterocyst; instead, it separates photosynthesis and photofermentation temporally, fixing CO2 to glycogen during the day and using this glycogen as an electron donor to the nitrogenase enzyme during the night (Bandyopadhyay et al., 2010). In the absence of molecular nitrogen (N2), photofermentative cyanobacteria divert all of the fermentative electrons to H2 production, introducing the stoichiometric H2 yield by a factor of 4 (Ghirardi et al., 2009). These photofermentative electrons involve the breakup of ATP into adenosine diphosphate (ADP) and an inorganic phosphate ion (Pi), a process that releases energy (ΔG0 _{= -30.5 kJ·mol}-1_).

Photofermentation reactions (cyanobacteria): In the presence of N2:

N2 + [8 H+ + 8 e- + 16 ATP] (sucrose/glycogen) → 2 NH3 (ammonia) + H2 + 16 ADP + 16 Pi In the absence of N2:

[8 H+ _{+ 8 e}- _{+ 16 ATP] (sucrose/glycogen) → 4 H2 + 16 ADP + 16 Pi}

H2 production rates depend on the quantity of sucrose/glycogen that has been biosynthesised. Photofermentative H2 production by cyanobacteria could be further increased by feeding the bacteria with an organic substrate such as glucose or glycerol (Bandyopadhyay et al., 2010).

2.6.3. Biophotolysis

Green algae such as Chlamydomonas reinhardtii and cyanobacteria such as Synechocystis sp. PCC6803 have the ability to produce H2 from two of nature’s most plentiful resources, sunlight and water, by biophotolysis (Benemann, 1997). The PSII protein complex captures sunlight and uses this energy to split water into O2, protons and electrons. The photosynthetic electrons are transported by Fd and other intermediates to the hydrogenase enzyme, which catalyses the process of proton-electron recombination to produce H2 (Esper et al., 2006).

Biophotolysis reactions: Water-splitting:

2 H2O → O2 + 4 H+ + 4 e- Proton-electron recombination: 4 H+ _{+ 4 e}- _{→ 2 H2}

Biophotolysis is a completely sustainable H2 production process. H2 is produced from water, and it also returns to water once it has been used up as a fuel. Renewable solar power is collected and chemically stored as H2 by algae. The problem with direct biophotolysis is that the hydrogenase enzyme is inactivated in the presence of O2 so that the process only works under anoxic or anaerobic conditions (Ghirardi et al., 2009). Since photosynthetic water-splitting constantly evolves O2, this O2 needs to be removed as it is being produced. A more detailed description of biophotolytic H2 production by Chlamydomonas reinhardtii, as well as an overview of the techniques used to overcome the O2 sensitivity of the hydrogenase enzyme, will be discussed in Chapter 3.