4. Factors affecting dark fermentative hydrogen production

#### 4.1 Types of inoculum

Pure and mixed cultures are two types of inoculum used to produce hydrogen by dark fermentation. Clostridium sp. and Enterobacter sp. are the pure culture widely used to produce hydrogen. Pure cultures give the high HPR and HY [100]. The major disadvantage of using pure culture is the sterile conditions which are required during the start-up and operations resulting in high operation costs from an energy use. This problem can be mitigated by using mixed cultures. Using mixed cultures as an inoculum in bio-hydrogen fermentation process is more practical than those using pure culture because it is simpler to operate, the process is easier to be controlled [101], and its feasibility to use complex organic wastes [100]. Inoculum sources for mixed cultures are animal dung, anaerobic sludge, municipal solid waste, soil, and compost [102]. The presence of hydrogen consumers such as methanogens and homoacetogens is the drawbacks of using mixed cultures. In order to inhibit these hydrogen consumers while harvesting the hydrogen producers, the pretreatment methods including heat treatment; acid treatment; alkali treatment; sonication; aeration; freezing and thawing; addition of specific chemical compounds, e.g., 2-bromoethanesulfonic acid; and addition of long-chain fatty acids are needed [103, 104].

#### 4.2 Feedstocks

Various kinds of feedstock have been used to produce hydrogen by dark fermentation. They can be classified into three generations. First-generation feedstocks are food crops such as sugarcane, sugar beet, corn, and cassava which can be Bio-hydrogen and Methane Production from Lignocellulosic Materials DOI: http://dx.doi.org/10.5772/intechopen.85138

easily digested by fermentative microorganisms. However, there is the concern on food competition and arable land when food crops are used to produce biofuels [105]. Thus, lignocellulosic biomass is developed as the second-generation feedstocks. Due to its compositions, lignocellulosic biomass is difficult to be digested by microorganisms. Therefore, the pretreatment and hydrolysis of the lignocellulosic biomass are needed in order to obtain its underlying monosugars prior the fermentation. Recently, the third-generation feedstock, i.e., microalgae has received high attention to produce hydrogen. Microalgae have rapid growth rate with a high capturing ability for CO2 and other greenhouse gases. They can be cultivated without soil and have a very short harvesting cycle (1–10 days) [106, 107]. Microalgae biomass consists of high carbohydrates (cellulose and starch) and lipid contents that can be converted to hydrogen by hydrogen producers. HY, HPR, and the overall economy of the process [102] are affected by the differences in carbohydrate content, bioavailability, and biodegradation rate of the first-, second-, and thirdgeneration feedstocks. In addition, the concentrations of feedstocks must be considered because a feedstock or product inhibition can occur in the fermentation process [108].

#### 4.3 Nitrogen and phosphate

Herbaceous biomasses are common lignocellulosic feedstocks for methane production. Corn stover yielded 320–335 mL-CH4/g-VS [79, 84]. Co-digestion of corn stover with goose manure increased the methane yield (MY) to 393 mL-CH4/g-VS [85]. The straws of wheat, rice, and corn gave 240–329 mL-CH4/g-VS [86–91].

301 mL-CH4/g-VS) [36, 83, 92, 93] and woody biomass (136–205 mL-CH4/g-TS)

Two-stage hydrogen and methane production was reported as a successful process to produce hydrogen together with methane and enhance energy recovery from lignocellulosic biomass. Energy yield from OPT hydrolysate increased from 0.8 to 10.6 kJ/g-COD by applying two-stage thermophilic hydrogen and mesophilic methane production in comparison to one-stage thermophilic hydrogen production [57]. The HY of 53.8 mL-H2/g-VS together with HY of 133.9 mL-CH4/g-VS was achieved by two-stage fermentation of maize silage [97]. Sequential hydrogen and methane fermentation of sugarcane bagasse hydrolysate obtained by steam explosion yielded a total energy of 304.11 kJ/L-substrate [78]. The gaseous (hydrogen and methane) recovery from mixed sugarcane bagasse hydrolysate and water hyacinth was maximized by continuous two-stage hydrogen and methane production at a hydraulic retention time of 8 h and 10 days, respectively, providing energy yield of 8.97 KJ/g-COD [98]. Continuous two-stage hydrogen and methane production from agave bagasse enzymatic hydrolysate was optimized at an organic loading rate of 44 g-COD/L-d (for hydrogen) and 20 g-COD/L-d (for methane), in which 9.22

Relatively low values HY were observed from biomass of grasses (142–

Biomass for Bioenergy - Recent Trends and Future Challenges

[79, 94], while bagasse feedstocks yielded relatively high values of 330–

4. Factors affecting dark fermentative hydrogen production

Pure and mixed cultures are two types of inoculum used to produce hydrogen by dark fermentation. Clostridium sp. and Enterobacter sp. are the pure culture widely used to produce hydrogen. Pure cultures give the high HPR and HY [100]. The major disadvantage of using pure culture is the sterile conditions which are required during the start-up and operations resulting in high operation costs from an energy use. This problem can be mitigated by using mixed cultures. Using mixed cultures as an inoculum in bio-hydrogen fermentation process is more practical than those using pure culture because it is simpler to operate, the process is easier to be controlled [101], and its feasibility to use complex organic wastes [100]. Inoculum sources for mixed cultures are animal dung, anaerobic sludge, municipal solid waste, soil, and compost [102]. The presence of hydrogen consumers such as methanogens and homoacetogens is the drawbacks of using mixed cultures. In order to inhibit these hydrogen consumers while harvesting the hydrogen producers, the pretreatment methods including heat treatment; acid treatment; alkali treatment; sonication; aeration; freezing and thawing; addition of specific chemical compounds, e.g., 2-bromoethanesulfonic acid; and addition of long-chain fatty

Various kinds of feedstock have been used to produce hydrogen by dark fermentation. They can be classified into three generations. First-generation feedstocks are food crops such as sugarcane, sugar beet, corn, and cassava which can be

420 mL-CH4/g-VS [95, 96].

kJ/g-bagasse was recovered [99].

4.1 Types of inoculum

acids are needed [103, 104].

4.2 Feedstocks

114

Nitrogen is required for growth of hydrogen-producing bacteria. Nitrogen source for fermentative hydrogen production is classified into inorganic and organic. Examples of inorganic nitrogen are ammonia nitrogen [109], ammonium bicarbonate [110], and ammonium chloride [111, 112]. Ammonia nitrogen is the most widely used inorganic nitrogen with its optimal concentration in the range 0.1 to 7.0 g/L [113, 114]. Peptone, yeast extract, and corn steep liquor are the examples of organic nitrogen. Ferchichi et al. [115] and Ueno et al. [116] reported that a higher HY was obtained when organic nitrogen is supplied to the fermentation medium.

In fermentative hydrogen production, phosphate is needed due to its nutritious value as well as buffering capacity. An increase in phosphate concentration results in increase of the capability of the bacteria to produce hydrogen. However, too high concentrations of phosphate could cause the substrate inhibition [113, 117]. The optimum C/N and C/P ratios are 74:200 and 599:1000, respectively [118, 119].

#### 4.4 Temperature

Temperature affects the maximum specific growth rate, substrate utilization rate, hydrolysis of the substrate, mass transfer rate, hydrogen partial pressure, hydrogenase activity, and the metabolic pathway of the bacteria resulting in a shift of byproduct compositions [101, 120, 121]. Fermentative hydrogen production can be operated under a wide range of temperature, i.e., mesophilic (25–40°C), thermophilic (40–65°C), or hyperthermophilic (>80°C) ranges [122]. Thermophilic condition gave a higher hydrogen production than the mesophilic condition. Sotelo-Navarro et al. [123] reported that the bio-hydrogen production from disposable diapers at 55°C was greater at 35°C. This could be due to the increased pace of microbial metabolism in the thermophilic condition. The optimal temperature for fermentative hydrogen production varies depending on the inoculum and substrate types.

#### 4.5 pH

pH affects the activity of hydrogenase as well as the metabolism pathway of the microorganisms [109]. Low pH inhibited hydrogenase activity [124, 125] resulting in longer lag time [126] and the inhibition of dark fermentation process. This can be 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 reducing powers that are potentially used for hydrogen synthesis [108].

[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 maxi-

Bio-hydrogen and Methane Production from Lignocellulosic Materials

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

mum theoretical HY from different carbon substrates are as follows:

5.2 Nitrogen sources

5.3 pH

117

5.4 Temperature

sion efficiency [139, 154, 166].

able to enhance the photo-hydrogen production.

duction of PNSB was 7.0 [140, 161–164].

Lactate : C3H6O3 þ 3H2O ! 6H2 þ 3CO2 (3) Malate : C4H6O5 þ 3H2O ! 6H2 þ 4CO2 (4) Butyrate : C4H8O2 þ 6H2O ! 10H2 þ 4CO2 (5) Acetate : C2H4O2 þ 2H2O ! 4H2 þ 2CO2 (6) Propionate : C3H6O2 þ 4H2O ! 7H2 þ 3CO2 (7)

Formate : CH2O2 ! H2 þ CO2 (8)

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

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 pro-

An increase in the environmental temperature until the optimal temperature can

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 conver-

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
