5. Factors affecting photo-fermentative hydrogen production

#### 5.1 Carbon sources

Various kinds of substrates can be used as carbon source by PNSB. Short-chain organic acids such as malic, lactic, succinic, acetic, propionic, and butyric acids

Bio-hydrogen and Methane Production from Lignocellulosic Materials DOI: http://dx.doi.org/10.5772/intechopen.85138

[137–141] are the most generally used substrates for photo-hydrogen production. VFAs in the hydrogenic effluent can also be used to produce hydrogen by PNSB [142–145]. Additionally, other carbohydrate substrates [37, 146, 147] and organic acids from industrial wastewaters can be utilized as carbon source by PNSB [148–151]. Carbon affects the metabolism of cell growth and photo-hydrogen fermentation system [152, 153]. Cell formation utilizes large fraction of carbon, while hydrogen production utilizes a smaller fraction. The efficiency of photo-hydrogen production is different according to the types of carbon substrates. This is due to the variations in the electron transfer capabilities in the different metabolic pathways of photosynthetic microbes [154]. Substrate concentration can also affect the photohydrogen production. The optimum concentrations of VFAs for photo-hydrogen production were reported in the range of 1800–2500 mg/L [155, 156]. The maximum theoretical HY from different carbon substrates are as follows:

$$\text{Lactate}: \text{C}\_3\text{H}\_6\text{O}\_3 + \text{3H}\_2\text{O} \rightarrow \text{6H}\_2 + \text{3CO}\_2 \tag{3}$$

$$\text{Malate}: \text{C}\_4\text{H}\_6\text{O}\_5 + 3\text{H}\_2\text{O} \to \text{6H}\_2 + 4\text{CO}\_2\tag{4}$$

$$\text{Butyrate} : \text{C}\_4\text{H}\_8\text{O}\_2 + \text{6H}\_2\text{O} \to \text{10H}\_2 + 4\text{CO}\_2\tag{5}$$

$$\text{Acetate} : \text{C}\_2\text{H}\_4\text{O}\_2 + 2\text{H}\_2\text{O} \to 4\text{H}\_2 + 2\text{CO}\_2 \tag{6}$$

$$\text{Propionate} : \text{C}\_3\text{H}\_6\text{O}\_2 + 4\text{H}\_2\text{O} \to 7\text{H}\_2 + \text{3CO}\_2 \tag{7}$$

$$\text{Formula}: \text{CH}\_2\text{O}\_2 \rightarrow \text{H}\_2 + \text{CO}\_2 \tag{8}$$

#### 5.2 Nitrogen sources

Nitrogen is an essential nutrient for cell synthesis and hydrogen production. The activity of nitrogenase, an enzyme involved in the hydrogen production by photosynthetic bacteria, is greatly affected by nitrogen. Glutamate is a preferred nitrogen source for PNSB. It was rapidly consumed and could also improve hydrogen production of photo-hydrogen-producing bacteria [157–159]. Ammonia has an adverse effect on hydrogen production. High concentration of ammonium ions powerfully inhibited the synthesis and activity of nitrogenase. However, a low ammonium concentration less than a non-inhibitory level can support the growth of cells and is able to enhance the photo-hydrogen production.

#### 5.3 pH

attributed to the protonation of undissociated acids in medium which can penetrate the microbial cell membrane and inhibit the growth and activities of microorganism [127]. Acidic pH of 4.5–6.0 favors the acetic and butyric acid production pathway. High initial pH leads to the production of ethanol and propionate rather than hydrogen production [128]. The propionate production pathways consume reduc-

Fe, Ni, and Mg are required for bio-hydrogen production process. These metals are cofactors for enzymes facilitating transport processes in the microorganisms [122, 129, 130]. Fe2+ is an important element to form hydrogenase and other enzymes. Fe-S affects protein functions by acting as an electron carrier and involving in oxidation of pyruvate to acetyl-CoA, CO2, and H2 [122]. Additionally, Fe2+ induces metabolic alteration and is involved in Fe-S and non-Fe-S protein operation

Hydraulic retention time (HRT) is defined as the time that fermentation broth remains in a reactor. It is related to the working volume of the reactor and the influent flow rate. HRT affects a continuous hydrogen production. Hydrogenproducing bacteria are fast-growing bacteria, so they prefer short HRT, while the methanogens are slow-growing microorganisms, so they prefer long HRT [134]. Therefore, HRT can be used as controlling parameters to suppress the community of methanogens [102]. Jung et al. [134] reported that the HRT for treating liquidtype substrate is shorter than that of solid-type substrate because the times to

Hydrogen partial pressure affects hydrogenase activity because it is involved in reversibly oxidizing and reducing ferredoxin [102]. High accumulation of hydrogen partial pressure in the fermentation broth decreases the hydrogen production because the reaction tends to be reducing ferredoxin rather than oxidizing ferredoxin [135]. Hydrogen partial pressure can be reduced by biogas sparging [136], agitation, and reduction of headspace pressure using vacuum pump or enlarging the

Various kinds of substrates can be used as carbon source by PNSB. Short-chain organic acids such as malic, lactic, succinic, acetic, propionic, and butyric acids

5. Factors affecting photo-fermentative hydrogen production

hydrolyze substrate containing high solid are much longer.

ing powers that are potentially used for hydrogen synthesis [108].

Biomass for Bioenergy - Recent Trends and Future Challenges

in hydrogenase [122, 131]. Nickel is a fundamental component of [NiFe] hydrogenase. It has the influences on the activity of [NiFe]-hydrogenase. High concentration of nickle inhibits the activity of [NiFe]-hydrogenase, promoting fermentative hydrogen production [122, 132]. Mg2+ is an element that is found abundantly in microbial cells. It stabilizes ribosomes, cell membranes, and nucleic acids and plays a crucial role as an activator of many kinases and synthetases [133]. Cu, Cr, and Zn also have influences on hydrogen fermentation process [122]. The relative toxicity of these heavy metals are Zn (most toxic) > Cu > Cr (least toxic).

4.6 Metal ion

4.7 Hydraulic retention time

4.8 Hydrogen partial pressure

headspace volume.

5.1 Carbon sources

116

pH affects the ionic concentration in the medium. These ionic forms influence the active site of nitrogenase and affect the biochemical characteristic in microbial cells during metabolism process [154, 160]. Optimal pH for photo-hydrogen production of PNSB was 7.0 [140, 161–164].

#### 5.4 Temperature

An increase in the environmental temperature until the optimal temperature can improve the activities of the nitrogenase and proteins associated with the cell growth or hydrogen production. An imbalance of incubation temperature on cells growth inhibits the physiological activity, intracellular enzyme activity, and metabolism of cells. Unstable temperature may cause bacteria to spend their energy for adaptation to low/high temperatures in order to be able to survive [165] which results in a reduction in the hydrogen production, HPR, HY, and substrate conversion efficiency [139, 154, 166].

### 5.5 Light energy

Light energy is a necessary resource for the reaction, electron transport, ATP synthesis, and hydrogen production [165, 167]. Light intensity influences the HPR and cell synthesis [160, 163, 168]. At the optimal light intensity, large amounts of ATP and reductive power are sufficient for nitrogenase activity to produce hydrogen and generate the cells. However, a further increase in light intensity greater than the saturation condition became an inhibitory for hydrogen production by PNSB. Photo inhibition occurs when the photosynthetic system supplies excess ATP and Fdred in comparison to the capacity of nitrogenase enzyme [169]. Consequently, the cell is damaged by the bleaching bacteriochlorophyll pigment during the extra-light cultivation [170].

support the cell synthesis and metabolism process [140]. At a high S0/X0 ratio, i.e., low seed inoculation, microorganisms require more adaptation to utilize the high substrate concentration leading to a delay of the lag period for photo-hydrogen fermentation [140, 162, 163]. A further increase in cell concentration to greater than the optimal level resulted in a decreased hydrogen production [140, 162, 163]. A low S0/X0 ratio, i.e., high seed inoculation, resulted in an insufficiency of the substrate to supply the growth of cells [140]. In addition, excess biomass prevents penetration of light into the cultivation system due to a self-shading effect. This leads to a decrease in light intensity that causes a reduction of ATP creation resulting in the decrease of hydrogen production by photosynthetic bacteria. Moreover, extracellular concentrations might promote the formation of bacterial flocs or biofilm creation which can limit substrate distribution inside the bioreactor

Bio-hydrogen and Methane Production from Lignocellulosic Materials

DOI: http://dx.doi.org/10.5772/intechopen.85138

pH influences the growth of microorganisms in various stages of the anaerobic

digestion (AD) process [184, 185]. Optimum pH for methanogens to produce methane ranges from 7.0 to 7.2 [186]. pH outside the range of 6.0–8.5 is toxic to methanogens. pH values below 6.6 starts to adversely affect the activities of the methanogens, and the values below 6.2 are significantly toxic to the methanogens. During the acidogenesis stage of AD process, the pH in an anaerobic digester decreases to below 6.0 due to VFAs accumulation and carbon dioxide production. After this, the pH rises to 7.0–8.0 or above. Yu and Fang [186] and Kim et al. [187]

found that the optimal hydrolysis and acidogenesis stage were achieved at

inhibited. Thus, it is recommended that the hydrolysis, acidification, acetogenesis/methanogenesis stage in AD process should be carried out

from mesophilic to thermophilic temperature [191].

depending on bacterial stains, operation condition, and so on.

pH 5.5–6.5, and the acidogenic bacteria continue to produce the acids until the pH drops to 4.5–5.0 [186, 188, 189]. As a consequence, the activity of methanogens is

Most of the methanogens are mesophile which are active in the temperature ranges of 30–35°C, while only a few are thermophile which are active in the temperature ranges of 50–60°C [186]. Deublein and Steinhauser [190] reported that the methanogenic activity is inhibited at the temperatures between 40 and 50°C especially at the values near 42°C. This is believed to be a transition temperature

HRT affects the rate and extent of methane production. A long HRT results in higher total VS mass reduction, which in turn leads to higher cumulative biogas production as well as to allow the microorganisms to acclimate to toxic compounds [191]. Methanogens have a long generation time. Thus, the HRT is usually set at 10–15 days to avoid the washout from the reactor [186]. The length of HRT can vary

system [140, 157, 167].

6.1 pH

separately [190].

6.2 Temperature

119

6.3 Hydraulic retention time

6. Factors affecting methane production

Halogen [141, 152, 171], tungsten [155, 161], fluorescent [172], infrared [172], and light-emitting diode (LED) lamps [173, 174] have been used as the light source for photo-hydrogen fermentation. Among these lamps, LED has the high operational stability and can improve the performance of photo-hydrogen fermentation [154]. Other advantages of LED include specific wavelengths (770–920 nm), lower electricity consumption, lower heat generation, and longer life expectancy [154, 174].

### 5.6 Iron concentration

Iron is the major cofactor at the active site of FeMo-nitrogenase [157, 175]. There are 24 atoms of Fe as the composition in each molecule of nitrogenase [176]. It is also an essential component in ferredoxin and cytochrome b-c complex, which are electron carriers of the photosynthetic electron transport system. Ferredoxin also contains Fe4S4 in a cluster of nitrogenase [177]. Photo-hydrogen production is functioned by nitrogenase, which receives electron carriers from ferredoxin and reduces protons to molecular hydrogen. The optimal Fe2+ concentration for photo-hydrogen fermentation are in the range of 1.68–35 mg/L [164, 177–179]. Concentration of iron greater than the requirement of regular physiological metabolisms can disrupt the cell surface of microorganisms. As a consequence, the production of hydrogen is reduced [177].

#### 5.7 Vitamin solution

Vitamins are essential for carbohydrate, protein, lipid, and cell metabolism [180, 181]. Vitamin B1 (thiamine) is a precursor of thiamine pyrophosphate (TPP), a coenzyme of the pyruvate dehydrogenase complex, essential for catabolism of carbohydrates, organic acids, and amino acids. This is important in the conversion of pyruvic acid and provides acetyl-CoA in the TCA cycle which supports cell synthesis. Biotin is a part of an enzymatic carboxylation and is a cofactor for carbon dioxide fixing enzymes such as pyruvate carboxylase. Oxaloacetate is supplied by pyruvate carboxylase. This is important in the citric acid cycle and in the production of biochemical energy. Vitamin B6 (pyridoxamine) is necessary for the metabolism of amino acid and in glycogen hydrolysis [181–183]. Nicotinic acid is a precursor of NAD<sup>+</sup> /NADP, which are electron carrier and play an important role in electron transfer during the photo-fermentation process [180].

#### 5.8 Inoculum concentration

The ratio of initial cell concentration (X0) to initial substrate concentration (S0) affects the initial energy level of microorganisms. This energy is necessary to

Bio-hydrogen and Methane Production from Lignocellulosic Materials DOI: http://dx.doi.org/10.5772/intechopen.85138

support the cell synthesis and metabolism process [140]. At a high S0/X0 ratio, i.e., low seed inoculation, microorganisms require more adaptation to utilize the high substrate concentration leading to a delay of the lag period for photo-hydrogen fermentation [140, 162, 163]. A further increase in cell concentration to greater than the optimal level resulted in a decreased hydrogen production [140, 162, 163]. A low S0/X0 ratio, i.e., high seed inoculation, resulted in an insufficiency of the substrate to supply the growth of cells [140]. In addition, excess biomass prevents penetration of light into the cultivation system due to a self-shading effect. This leads to a decrease in light intensity that causes a reduction of ATP creation resulting in the decrease of hydrogen production by photosynthetic bacteria. Moreover, extracellular concentrations might promote the formation of bacterial flocs or biofilm creation which can limit substrate distribution inside the bioreactor system [140, 157, 167].
