Photoheterotrophic or photo-fermentative hydrogen production refers to the microbial process, during which organic substrates are oxidized under anaerobic conditions in the presence of light, generating hydrogen and carbon dioxide. Photo-fermentative hydrogen production is generally carried out by prokaryotic microorganisms called purple non-sulfur bacteria (PNS) (Basak and Das, 2007), although lately the process has also been reported to be carried out by eukaryotic microorganisms, that is, green algae (Hemschemeier and Happe, 2005). Photosynthetic microorganisms convert light energy into chemical energy in the form of chemical bonds, via the pathway of photosynthesis.

Contrary to dark fermentation (see Section 13.3), in which the enzyme hydrogenase catalyses hydrogen production, nitrogenase is the key enzyme for the photo-fermentative process of PNS. Under nitrogen-deficient conditions, nitrogenases can also catalyse the generation of molecular hydrogen using light energy and reduced compounds (such as organic acids) as the electron donors, where ferredoxin acts as the electron carrier (Das and Veziroglu, 2001). Light as an energy source is necessary for such reactions to take place, since their Gibbs energy is positive and thus they are not thermodynamically favored:

CH3COOH + 2H2O + “hv” ^2CO2 + 4H2 DGo= +75.2 kJ/mol [13.6]

As shown in Table 13.3, in most photo-fermentative biohydrogen studies pure cultures of the genera Rhodopseudomonas, Rhodobacter and Rhodospirillum have been used, whereas studies with other genera such as Rubrivivax (Li and Fang, 2008) and Rhodobium (Kawaguchi et al., 2001), as well as with mixed cultures (Zhang et al., 2002; Fang et al, 2005) have also been reported. Malate and glutamate were commonly selected as carbon and nitrogen sources, respectively. However, the use of other carbon sources such as the acids lactic, succinic, acetic, propionic and butyric, or their salts, has also been investigated for their potential to be converted into hydrogen either in the form of synthetic substrates or as parts of actual waste streams.

In order to evaluate the performance of a photo-fermentative hydrogen production system, the efficiency with which light energy is converted to energy in the form of hydrogen, the so-called photochemical efficiency (PE) or light conversion efficiency, has to be taken into consideration (Akkerman et al., 2002). It thus becomes obvious that the efficient utilization of light energy, provided either by a physical (sunlight) or an artificial source, is of extreme importance for the construction of a feasible energy production system (Miyake and Kawamura, 1987). Factors affecting PE include wavelength and intensity of light, cell concentration in the culture, surface to volume ratio of the culture (reactor geometry) and light penetration in the reactor. It is widely accepted that optimal light utilization is indispensable for maximal hydrogen production.

As shown in Table 13.3, in most studies one or more artificial light sources have been selected among florescent lamps, halogen lamps, optical fibers, neon tubes, light-emitting diodes and photosynthetically active radiations (PARs), which however could become a hindrance to the overall economic viability of a full-scale application. Sunlight on the other hand, is a free and abundant light source, which

Microorganism Substrate Reactor operation/ configuration Nitrogen source Condition of microorganisms Light source/lenergy Reference Rhodopseudomonas palustris Malic acid, acetic acid Fed-batch/ cylindrical glass Glutamic acid Suspended Lamps light source from one and two sides/460 |iE m-2 s-1 Carlozzi and Lambardi, 2007 Palm oil mill effluent (POME) Batch/serum bottles No addition, TKN ~60 mg I-1 Suspended Tungsten light bulbs/2-5 klux Jamil ef a/., 2009 Glycerol Batch/serum bottles Glutamate, 2-6 mM Ammonium, 0-4 mM Suspended Panel of 50 W halogen spotlights/- Sabourin- Provost and Hallenbeck, 2009 Glucose Continuous/flat, polymethyl methacrylate Ammonium Immobilized in (PVA)-boric acid gel LED mounted on the topside/3-11 klux Tian ef a/., 2009 Rhodopseudomonas faecalis Sodium Acetate Fed-batch/serum bottles Sodium glutamate, 10 mM Suspended 60 W incandescent lamps/4 klux Ren ef a/., 2009a Glucose Batch/serum bottles Sodium glutamate, 1 g I-1 Immobilized in agar gel 60 W incandescent lamps/4 klux Ding ef a/., 2009 Sodium acetate Batch/serum bottles Glutamate, 10 mM Suspended 60 W incandescent lamps/4 klux Ren ef a/., 2009b Rhodobacter capsulatus Acidified Miscanthus hydrolysate, acetate, lactate, fructose Batch, sealed glass bottles Sodium glutamate, 2 mM Suspended 150 W halogen lamp/1,370 [imol photons/m2/s Uyar ef a/., 2009 Lactate Batch/flat glass Sodium glutamate, 7 mM Suspended Sodium-vapour lamp 600 W/ Obeid ef a/., 2009 Malate Batch Sodium glutamate, 2 mM Suspended lamps/4 klux Ozturk ef a/., 2006 IContinued)

Microorganism Substrate Reactor operation/ configuration Nitrogen source Condition of microorganisms Light source/lenergy Reference Rhodobacter sphaeroides DL — malate Batch/triple jacketed vertical, cylindrical, glass Glutamate, 2 mM Suspended Tungsten filament lamp placed in central axis of reactor/15± 1.1 W m-2 Basak and Das, 2009 Mixed substrate of acetate, butyrate, ethanol Batch/water jacket glass column Sodium glutamate, 10 mM Suspended 100 W lamps 5.5±0.5 klux Nath ef a/., 2008 Raw, acidified or clay pretreated olive mill wastewater Batch/glass vessels No addition Suspended 150 W tungsten lamp/4 klux Eroglu efa/., 2006 Succinate Batch Ammonium chloride 0.04 w/v Suspended Lamps/2.4 klux Chalam ef a/., 1996 Rhodospirillum rubrum Sodium succinate Batch Glutamate 3 mM Suspended Fluorescent and incandescent light bulbs/60 W m-2 Melnicki efa/., 2008 Acidified cassava wastewater Batch/serum bottles Glutamic acid, ammonium nitrate Suspended Fluorescence lamp/6000 candela/m2 Reungsang ef a/., 2007 Mixed substrate of acetate, malate Batch/cylindrical, glass L-glutamate 23 mM Immobilized in agar gel Lamps/20 klux Planchard ef a/., 1989 Lactate, cheese whey Continuous, HRT 74 h rectangular L-glutamate 15 mM Suspended 100-W spot-light tungsten Zurrer and Bachofen, 1979

can be used for direct irradiation of the bioreactor or amplified by the use of solar — energy-excited optical fibers (Chen et al., 2008a). A drawback of using sunlight could be the periodicity of the light source; an obstacle that could be surpassed by the addition of solar-energy-excited optical fibers, accompanied by light-dependent resistors, which can ensure the stability of light energy (Chen et al., 2008a).

In order to develop commercially viable processes, the influence of many other factors has to be taken into consideration. The nitrogen source is one of the most critical parameters for effective photo-fermentative hydrogen production. An organic nitrogen source, such as glutamic acid or inorganic salts or more complex organic nitrogen sources such as yeast extract, seems to be necessary for efficient hydrogen production regardless of the species of microorganism used. The effect of the type and concentration of the carbon source used as substrate (Carlozzi and Lambardi, 2009), the C/N and C/N/P ratios (Reungsang et al., 2007) as well as the physicochemical conditions of growth such as pH (Tian et al., 2009) and temperature (He et al., 2006) have been widely studied and optimized, since they seem to have a severe effect on both the final hydrogen yield and the hydrogen production rate. Regarding pH, the optimum value is reported to be 7 in most cases, whereas the optimum temperature is reported to be 30°C. A general conclusion from all these studies is that the photo-fermentation processes seem to be favored by a high ratio of C/N, irradiation with light of saturating intensity, under anaerobic conditions with optimal temperature and pH, depending on the specific microorganism used.

There are three major types of photo-bioreactors developed for hydrogen production that is tubular, flat panel and bubble column reactors. The features of these photo-bioreactors have been reviewed by Akkerman et al. (2002) and the importance of PE in hydrogen production was strongly emphasized. The main advantage of tubular and column photo-bioreactors is that their geometry allows for quite efficient mixing of the culture, and thus the exposure of the microbial cell to light is more equally distributed. The way to scale-up is to connect a number of tubes via manifolds. Flat panel reactors consist of a rectangular transparent box with a depth of only 1-5 cm. The height and width can be varied to some extent, but in practice only panels with a height and width both smaller than 1 m have been studied. The advantage of these systems is the large surface that can be illuminated either using sunlight or artificial means. The main disadvantage of such type of reactors is the high consumption of energy used for maintaining efficient air supply and mixing of the liquid. Many scaled-up versions of photo-bioreactors consist of repeating many of the smaller photo-bioreactor units, with its practical implications.

Finally, the quantitative description of photo-fermentative hydrogen production seems to be quite complex, due to the large number of parameters that have to be taken into account. Simple models such as the Luedeking-Piret model (Basak and Das, 2009), the logistic model (He et al, 2009), the Monod equations (Obeid et al., 2009) and the Gompertz equation (Nath et al., 2008) have been used in order to fit experimental results regarding biomass growth and cumulative hydrogen generation, but so far very few studies have dealt with the development of complex structured kinetic models, properly incorporating specialized for photo-fermentative hydrogen production parameters such as light intensity and wavelength influence. A simple kinetic model for photo-fermentative biohydrogen production has been developed by Gadhamshetty et al. (2008) for batch bioreactors, where it was assumed that sufficient light intensity and optimal C/N ratio were available under stressful nitrogen concentrations. The proposed model used Rhodobacter sphaeroides as the model biomass and contained 17 parameters to describe cell growth, substrate consumption, and hydrogen evolution as well as inhibition of the process by biomass, light intensity, and substrate. Based on sensitivity analysis performed with the validated model, only 6 of the 17 parameters were found to be significant and it was indicated that the range of optimal light intensity for maximum hydrogen yield from malate by R. sphaeroides was 150-250 W/m2.