Biophotolysis of water, fermentation and photofermentation of organic substrates are considered to be the best biological methods of hydrogen generation. Reversibility, lack of toxic substances generated in these processes, mild conditions for microbiological reactions, as well as operation at low pressure of these processes are the conditions required for

modern microbiological systems. Moreover, the possibility of application of different waste waters (containing organic carbon) in these processes is an additional benefit.

Fermentation is the process generating basically two gaseous metabolites: hydrogen and carbon dioxide. The volatile fatty acids (VFA) and alcohols represent liquid metabolites of dark fermentation. The low yield of generated hydrogen and high concentration of CO2 (almost 50%) in gaseous products are the main disadvantages of microbiological hydrogen generation. In contrary, high reaction rate and possibility of biodegradation of many organic substances can be assigned to the benefits of this process.

In photofermentation, the photosynthetic heterotrophoic bacteria under anaerobic conditions and in the absence of nitrogen generate hydrogen in presence of organic compounds. Nitrogenase is the enzyme catalyzing hydrogen generation reaction. Presence of molecular nitrogen or nitrogen compounds directs the reaction route towards ammonia formation. The possibility of application of wide spectrum of light (400-950 nm), lack of methabolism generating molecular oxygen, as well as possibility of use of organic substances originating from wastes are the main advantages of photobiological method of hydrogen generation.

Both fermentation and photofermentation require presence of anaerobic microorganisms and the light in case of photofermentation. Photosynthesis, and in consequence also photofermentation is the series of complex reactions transforming energy of light into chemical energy.

The photosynthetic apparatus is localized in invaginations of the cytoplasmic membrane. The photosystem is built of three multimeric (transmembrane) proteins: antennas making the light-harvesting complex (LHC), the reaction centre (RC) and the complex of cytochromes bci (Fig.1) (Vermeglio, 1999). The LHC antennas contain molecules of bacteriochlorophyll and carotenoides. The carotenoides play a double role in LHC systems;

they absorb light from the visible part of the light spectrum in which bacteriochlorophyll is not active and protect the antenna system against damage by singlet oxygen (Isaacs, 1995, Jones, 1997). The majority of the purple bacteria have two different antenna complexes known as LH1 and LH2. The number of LH2 complexes depends on such parameters like light intensity and partial pressure of oxygen, while the number of LH1 complexes is directly correlated with that of the reaction center (RC) to form RC-LH1 center. High ratio of pigment molecules to RC (e. g. 100 molecules of chlorophyll to one RC) increases the area capable of light absorption. Upon absorption of photon by LHC, the reaction centre becomes excited with simultaneous charge separation in a time shorter than 100 picoseconds (ps). The high reaction rate of this process is a consequence of the mutual arrangement of LH1 and RC: one RC is surrounded by a ring of 15-17 LH1 subunits. The closed structure of LH1 complexes in combination with the dense packing of bacteriochlorophyll molecules ensures fast delocalization of the excited state and possibility of energy transfer towards the reaction centre from every point of the ring (Vermeglio, 1999). The reaction centre is an integral part of protein membrane composed of three polypeptides (subunits L, M and H), containing four molecules of bacteriochlorophyll a (PA, PB, BA, BB), two molecules of bacteriofeophityne a (HA, HB), two molecules of ubichinone(QA, QB), one molecule of carotenoid (Crt) and one atom of non-heme iron (Fig.2).

All pigments are linked to the heterodimeric protein skeleton of L and M subunits forming five transmembrane protein helixes (Paschenkoa, 2003). The main source of electrons is the "special pair" of the excited bacteriochlorophylls a located close to side of the cytoplasmic membrane. The excitation is realized by direct absorption of light by the "special pair" of bacteriochlorophylls absorbing at 870 nm and by energy transfer from other pigment molecules located at RC or LHC. The transfer of electrons from the special pair to bacteriopheophytin, located in the middle of the dielectric cytoplasmic membrane occurs in 3-4 ps. This reaction is probably intermediated by a transient product of monomeric bacteriochlorophyll BA. In the next 200 ps the electron is transferred to ubiquinone Qa (connected with RC) and subsequently to ubiquinone QB. The transfer of electron to ubiquinone QB is accompanied by its protonation. The full reduction of ubiquinone QB requires two subsequent cycles in RC after which electrons finally leave RC with electrostatically neutral doubly reduced ubiquinol QH2 (Jones, 1997). The two protons required for protonation originate from cytoplasmic space. In the next step ubiquinol is oxidized by the bc2 cytochrome complex. This complex caused reduction of the [Fe2S2] unit which is a part of cytochrome (part of Rieske unit) and releases two protons to periplasmic space. Then the cycle of electron transfer is closed by recombination of cytochrome c2 by reduction of the special pair of bacteriochlorophylls. The cyclic transfer of electrons is accompanied by transfer of protons from cytoplasm to periplasm leading to the proton gradient between the two sides of cytoplasmic membrane, which is the most important effect of photosynthesis because it stimulates ATP synthesis and reduction of NAD+ (Vermeglio, 1999). Protons accumulated on the periplasmic space of the membrane return to the cytoplasmic space through the ATP synthase channel, which closes the transfer of protons (Paschenkoa, 2003).