Purple non-sulfur (PNS) photosynthetic bacteria are a physiological group of different kinds of gram negative aquatic bacteria. They are considered to be as old as the first photototrophic organisms on our planet. The common characteristics of this group are their ability to perform an anaerobic type of photosynthesis without the production of oxygen. Depending on the degree of anaerobiosis, and avail­ability of carbon and light source they can grow as photoheterotrophs, photoau — totrophos or chemoheterotrophs. Because they do not use hydrogen sulfide as electron donor while growing photoautotrophically they are called purple non­sulfur bacteria. While growing facultative anaerobically they give purple to deep red pigments. Under photoheterotrophic conditions (light, anaerobiosis, organic electron donor) they can produce hydrogen. The mostly studied species are the members of Rhodobacter, Rhodopseudomonas and Rhodospirillum [93]. The enzyme systems, the carbon flow (specially TCA cycle) and photosynthetic membrane apparatus make up the overall production of hydrogen by intercon­necting the exchange of electrons, protons and ATP [94]. Because of the reasons like; high substrate/product conversion yields, lack of oxygen evolving activity, a wide range of light can be used and different kinds of organic substrates can be used; photofermentative hydrogen production has been an interest of many researchers [93]. Because of not producing hydrogen during photosynthesis in anaerobic conditions both nitrogenase and hydrogenase in bacterial membrane are active. Hydrogen production is mainly associated with nitrogenase action (Fig. 10.3).

These nitrogen-fixing bacteria can utilize the enzyme nitrogenase to catalyze the reduction of molecular nitrogen (N2) to ammonia (NH3) while producing hydrogen. Mo-nitrogenase is the most common and the most efficient nitrogenase for converting N2 to NH3 (Eqs. 10.1-10.3). It is also found in all nitrogen-fixing bacteria and is thus the most studied [95]. Three different kinds of nitrogenase and the reactions are:

Mo — nitrogenase: N2 + 8H+ + 8e~ + 16ATP! 2NH3 + H2 + 16ADP + 16Pi

(10.10)

V — nitrogenase: N2 + 12H+ + 12e“ + 24ATP! 2NH3 + 3H2 + 24ADP + 24Pi

(10.11)

Fe — nitrogenase: N2 + 24H+ + 24e~ + 48ATP! 2NH3 + 9H2 + 48ADP + 48Pi

(10.12)

In the absence of molecular nitrogen this enzyme catalyzes the hydrogen production with the reaction below (Eq. 3.4) [94]:

2H+ + 2e~ + 4ATP! H2 + 4ADP + 4Pi (10.13)

For efficient operation of nitrogenase large amounts of ATP and reducing power are needed. Oxygen is a potent inhibitor of nitrogenase that can destroy the enzyme irreversibly. Ammonium is a second important inhibitor because it can repress the synthesis of nitrogenase and can inhibit nitrogenase activity. The inhibition is reversible because nitrogenase can recover activity after consuming or removing ammonium [96].

Hydrogenase enzyme is a common feature of photosynthetic bacteria and it can be responsible for both hydrogen production and consumption. Because hydrogen production is by nitrogenase hydrogen producing activity by hydrogenase can be ignored. Studies have shown that hydrogen producing activity of hydrogenase is less than hydrogen consuming activity [97, 98]. Hydrogenase is generally accepted as a metabolic antagonist of nitrogenase. Since it is very critical to eliminate the hydrogen uptake property of hydrogenase studies on mutations on organisms to eliminate hydrogenase synthesis have been reported. Hup-mutants have been found to have more hydrogen production capacity [99-101]. Another way is to make inhibitions chemically, using carbon monoxide or oxygen, limiting the amount of nickel since hydrogenases are nickel enzymes [102] and finally the presence of ethylendiamintetraacetic acid (EDTA) is known to inhibit hydrogenase activity [101, 102]. For reducing 2H+ to H2 nitrogenase re-oxidizes electron carriers and also another reductive process can compete with hydrogen production. A good and common example is the formation of the carbon storage polymer poly — b-hydroxybutyrate (PHB) from acetate [103].

9nCH3COOH ! 4(CH3CH2CHOO~)n+2nCO2 + 6nH2O (10.14)