6. Factors affecting methane production

#### 6.1 pH

5.5 Light energy

[154, 174].

5.6 Iron concentration

5.7 Vitamin solution

5.8 Inoculum concentration

NAD<sup>+</sup>

118

during the extra-light cultivation [170].

Biomass for Bioenergy - Recent Trends and Future Challenges

production of hydrogen is reduced [177].

transfer during the photo-fermentation process [180].

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

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

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

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

/NADP, which are electron carrier and play an important role in electron

The ratio of initial cell concentration (X0) to initial substrate concentration (S0)

affects the initial energy level of microorganisms. This energy is necessary to

electricity consumption, lower heat generation, and longer life expectancy

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 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 inhibited. Thus, it is recommended that the hydrolysis, acidification, acetogenesis/methanogenesis stage in AD process should be carried out separately [190].

#### 6.2 Temperature

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 from mesophilic to thermophilic temperature [191].

#### 6.3 Hydraulic retention time

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