Genetic engineering is one of the methods for improvement of activity in hydrogen generation by microorganisms. Although the yields of generated hydrogen can be performed by optimization of the reaction conditions, genetic modifications seems to be the appropriate solution at the moment. The main idea of modification rely on implantation of other genes into the bacteria strains containing hydrogenase.

decomposition of formic acid in presence of formate-hydrogen liaze (FHL) representing the set of enzymes localized in the inner cell membrane. Hydrogenase 3 coded as hycA and formate dehydrogenase known as fdhF are the main components of the FHL. The presence of hycA gene limits the synthesis of fhlA, responsible for better activity of FHL towards hydrogen. Therefore the removal of hycA increases the fhlA gene expression and in consequence hydrogen production by 5-10%. (Hallenback, 2009). The research of the FHL genes expression were performed by Bisaillon et al. and other authors (Bisaillon, 2006, Turcot, 2008, Penfold, 2003) and they found almost two times higher rate of hydrogen generation for modified strain of E. coli HD701. Genes responsible for nickel-iron hydrogenases (hydrogenase I and II) coded by hya and hyb operons were found in the E. coli genom as well. It was found that elimination of these enzymes by genetic modification can result with almost 35% higher production of hydrogen (Hallenback, 2009, Bisaillon, 2006, Turcot, 2008). Other profits originating from genetic engineering are related to deactivation of enzymes responsible for transformations of glucose into lactic, succinic and fumaric acids. The removal of ldhA (lactic acid) and frdBC (succinic and fumaric acids) genes results in increase of hydrogen formation. The 1.4 fold higher amount of hydrogen were found by Yoshida et al. (Yoshida, 2006) in this situation. The new mutant strain of SR 15 can produce 1.82 mol H2/ mol glucose what is close to the theoretical value (2 mol H2/mol glucose). Studies performed by Maeda et al. (Maeda, 2007) showed that bacteria BW2513 with seven modified genes (hyaB, hybC, hycA, fdoG, frdC, ldhA and aceE) generate 4.6 fold more hydrogen than wild-type strain.

The nitrogenase and uptake hydrogenase play an important role in the photofermentation process of hydrogen generation by PNS bacteria. The engineering of the mutants free of uptake hydrogenase is the basic task of gene modifications. Genes coding hydrogenase (hup) can be modified by resistance gene insertion into the hup genes or by deletion of hup genes (Kars, 2009, Kars, 2008, Kim, 2006). Appropriately modified Rhodobacter spheroids can generate hydrogen also in the absence of light (Kim, 2008).

Production of polyhydroxybutyrate (PHB) accompany hydrogen generation by PNS bacteria what applies the excess of reducing equivalents in other metabolic pathway. The PHB is the storage material stored in cytoplasm. This compound is formed in the environment rich in carbon compounds but lean in nitrogen (Kemavongse, 2007). The PHB is unwanted competition product accompanying hydrogen generation. The removal of genes responsible for formation of PHB syntase effectively stops generation of the polymer (Kim, 2006). Low activity in PHB formation not always results in an increase of hydrogen yield. Whereas in presence of lactate, malate or malate the amount of photogenerated hydrogen is not influenced by PHB (Hustede, 1993) the presence of acetate can increase photofermentation towards hydrogen. However, the importance of PHB as biodegradable polymer significantly increased in recent years. Therefore, simultaneous photogeneration of hydrogen and PHB gained economic dimension (Yigit, 1999).

There are genetic modifications influencing changes in the amount of LHC (light harvesting complexes). The reduction of pigment present in LHC diminish the self-shadow effect and therefore better access of light into deeper located cells. The decrease of amount of LH1 (Vasilyeva, 1999) complexes with maximum of absorption at 875 nm or those with absorption maximum at 800 and 850 nm (LH2) (Kim, 2006) can increase the amount of photo generated hydrogen. Genetic manipulations cannot lead to total elimination of the pigments (Kim, 2006).

The negative influence of ammonium ions on nitrogenase is well recognized. Therefore, genetic modifications of nonsensitive to NH+ ions should be the subject for considerations. Among many methods reducing the role of ammonium ions in photofermentation is blockage of Calvin cycle via mutation of genes coding the RuBisCO enzyme. This way the excess electron stream is directed to nitrogenase even in the presence of NH4+ ions. Another modification can be achieved by disruption of proteins transporting NH4+ ions through cytoplasmic membrane. Strains of this type ( e. g. Rhodobacter capsulatus) loose their ability to regulate nitrogenase in presence of ammonium ions. (Qian, 1996). Such modifications allow to perform photofermentation even in the presence of molecular nitrogen. Although the amount of generated hydrogen is lower than in nitrogen free atmosphere but economically much more favorable (Yakunin, 2002).

Genetic modifications can be very effective but also troublesome and very expensive. Therefore other methods of process improvement are under investigations. Optimum value of pH equals 7. Photofermentation with Rhodobacter sphaeroides starting at pH=6.8 and ending at pH=7.5 results in significant drop of activity ( 7 times) but PHB concentration is tripled (Jamil, 2009).

Photofermentative bacteria belongs to mesophilic microorganisms and operate between 30 and 35 oC. Therefore, any critical temperatures act against high yield of hydrogen. For example Rhodobacter capsulatus operating at temperatures varying from 15-40 oC produce 50% less hydrogen than the same bacteria kept at constant temperature of 30 oC (Ozgura, 2010).

The access of photobacteria to the light with appropriate length and intensity play a crucial role m hydrogen photogeneration. Better access of light induce better phosphorylation and in consequence more effective synthesis of ATP and better yield of photofermentation (Kars, 2010).

Although the PNS bacteria absorb light in wide spectrum 400-950 nm the range of 750-950 nm is the most important (Eroglu, 2009, Ko, 2002). The light intensity is as well important as their wavelength. For Rhodobacter sphaeroides the amount of generated hydrogen grows linearly from 270 W/m2 (4klx) to 600 W/m2 (~ 10 klx). Below 270 W/m2 no activity of bacteria is observed (Miyake, 1999, Uyar, 2007).

Application of illumination with wavelength longer than 900 nm results in overheating of the system. This require additional cooling systems because of decrease the amount of generated hydrogen. An application of appropriate filters cutting the unwanted range of spectrum seems to be the only solution in this situation (Ko, 2002). Considering natural irradiation one should remember about day-night periodicity. It was found, however, that amount of generated hydrogen is even higher under periodic irradiation than under the continuous one (Eroglu, 2010, Koku, 2003). The day-night illumination induces better activity of nitrogenase what results from better adjustment of PNS bacteria to live in natural conditions (Meyer, 1978).

The presence of organic compounds, also those containing nitrogen (except NH4+ ions) is the key issue for the photofermentation. However, presence of macro and microelements at appropriate concentration can influence the hydrogen productivity. Iron belongs to the most important ones. This element exists mainly as the cofactor of proteins engaged in metabolism. Process of photofermentation, strictly related to the transport of electrons.

There are many electrons carriers such as cytochromes (proteins containing Fe) or ferredoxin. Moreover, the main enzyme in photofermentation — nitrogenase contains 24 atoms of iron in each molecule. The presence of iron ions in medium containing PNS bacteria is one of the very important factors influencing hydrogen productivity. At concentrations of Fe2+ ions lower than 2.4 mg/l there is no hydrogen in products. At concentrations higher than 3.2 mg/l the gradual decrease of evolved hydrogen is observed. It was assumed that non physiological coagulation of the cells can occurs (Zhu, 2007). Molybdenum is the second microelement playing an important role in photofermentaive hydrogeneration. The optimal concentration of molybdenum is 16.5 pmol/l (Kars, 2006).

The substrate yield in hydrogen production can be significantly improved by adding other strains of bacteria into the liquid medium. Improvement in photofermentation was achieved by adding halofilic archeons of Halobacterium salinarum type. The integral membrane protein — bacteriorhodopsin as the pump for the light excited electrons. The H+ ions are pumped out from cytoplasm outside the cell. The proton gradient is then engaged in ATP synthesis by Rhodobacter sphaeroides and this way increasing hydrogen generation. In this case, it is advised to use strains of PNS bacteria tolerating high concentrations of salts (Zabut, 2006) because of the high activity of bacteriorodopsyne in aqueous solution with high ionic strength.