**4.4. Hydraulic retention time (HRT)**

important in dark fermentation. Phosphate helps in maintaining buffered condition during fermentation and provides the building blocks of nucleic acid and ATPs. In dark fermenta-

**Table 3.** Main factors affecting the two-stage anaerobic fermentation for biohythane production from organic wastes.

**Factors Effects on biohythane process References**

Inoculum • Fermentation metabolism and microbial community [69]

Feedstocks • Fermentation metabolism, microbial activity, and microbial

pH and Alkalinity • Fermentation metabolism, microbial activity, and microbial

• Metabolic shift to solvent production

Temperature • Fermentation metabolism, microbial activity, and microbial

HRT • Fermentation metabolism, microbial activity, and microbial

• Activity of acetogens and methanogens

community

92 Advances in Biofuels and Bioenergy

community • Cell membrane charge

community

community • Microbial growth rate

Partial Pressure • Fermentation metabolism and activity

• Enzyme activity

Trace element • Essential for cell growth,

consumers. Different selective procedures such as heat, acid, ultra-




production [47].



in first stage fermentation.

[68]

[70]

[71]

[72]

[70]

[73]


widely exist in natural


tion, an increase in phosphate concentration leads to enhancement of the H2

In the enrichment process, selection procedure was applied to selectively promote H2

sonic, ultraviolet, organic and alkali treatment were commonly used [58]. Most of H<sup>2</sup>

ing, which get eliminated with selection methods. The selection methods are promoting endo-

environment in the form of mixed cultures such as anaerobic sludge, municipal sewage sludge, hot spring sediment, compost and soil have been widely used as inoculum for fermentative H2 production [82–84]. Using mixed cultures is more practical than using pure cultures due to the easy operating and control under the non-sterile condition. Mixed cultures also have a broader

 reactor may be regarded as inoculum preparation. It should consider the revival of bacteria from the stock, successive of subculturing to active bacteria, short lag phase and high active

Developing an enriched inoculum is very important for obtaining H2

spores formation in a certain group of bacteria that also include H2

and *Clostridium* sp. Furthermore, the bacteria capable of producing H<sup>2</sup>

under favorable conditions, the endospores germinate and the H2

**4.2. Inoculums**

H2

bacteria and eliminate H2

in the system. The H2

H2

bacteria are spore forming, while H2

source of feedstock [85]. The selection of H2

The total time that cells and soluble nutrients reside in the reactor is called the HRT. H<sup>2</sup> production occurring at low HRT is dependent on the volume of the reactor and the flow rate of feed. It is generally well known that the H2 -producing bacteria are fast growing [70]. By applying this principle, Liu et al. [48] produced H2 free of CH4 in continuously CSTR feeding with household solid waste at acidic pH range of 5.0–5.5 and a short HRT of 3 days without any pretreatment to inhibit methanogens contained in the initial digested manure. HRT is the main optimization parameters of continuous H2 dark fermentation bioprocesses. In the CSTRs, short HRTs or high dilution (D) rates can be used to eliminate methanogens, which have significant low growth rate [70, 89]. However, HRT is needed to be maintained in a proper level that still gives a D value less than specific growth rate of H<sup>2</sup> -producing bacteria. Generally, short HRT is considered to favor the H<sup>2</sup> fermentation metabolism [3]. On the other hand, too high loading rates may result in substrate inhibition effects, improper food to microorganism (F/M) ratios of H<sup>2</sup> producers or washout of microorganisms [90]. These shock loads could reduce the H2 production metabolism through decreasing of pH and metabolite inhibition (accumulation of intermediates). The HRT could also help in the enrichment of microbial consortium, since it directly affects the specific growth rate of bacteria. By manipulating the HRT, slow-growing microbes like methanogens and H<sup>2</sup> -consuming microbes can be expelled out of the reactor, thus leading to selective enrichment of H<sup>2</sup> -producing bacteria [91]. This approach of using short HRT for suppressing methanogens led to improvement in H<sup>2</sup> production [92]. In second stage, the HRT is a measure to describe the average time that a certain substrate resides in a digester. If the HRT is shorter, the system will fail due to washout of microorganisms. HRT for anaerobic digestion process are typically in the range of 15–30 days at mesophilic conditions and 10–20 days at thermophilic conditions [13]. Long retention times also benefit hydrolysis of the particulate matter of complex structure such as lignocellulose biomass [93]. On the other hand, organic loading rate (OLR) or amount of organic matter in the system is relative with HRT. The shorter HRT will achieve high OLR that leads to the accumulation of VFA which consequently leads to a pH drop and inhibition of methanogenic activity. This causes a system failure. During methanogenesis, the HRT should be kept twofold greater than the generation time of the slow-growing microbes [94]. The HRT should be held for a suitable duration so that the dead zones get eliminated, and it would also help in promoting an efficient syntrophy among the microorganisms present in the mixed culture.

their cells. Thermophilic temperature makes the H2

mesophilic temperatures. Thermophilic H2

tages such as low solubility of H2

yield of ∼2.1 mol H2

/mol glucose [101]. Although the H<sup>2</sup>

ture was slightly higher than that for mesophilic temperatures, the specific H<sup>2</sup>

and CO2

reactions and give higher degradation efficiency as well as higher CH<sup>4</sup>


the stable temperature is important for biohythane production.

favorable with the H2

(mmol H<sup>2</sup>

CH4

CH4

**4.7. Trace elements**

ions such as Fe2+, Zn2+, Ni2+, Na+

tion (0–4000 mg/L) on H<sup>2</sup>

with increasing H2

the yield of ∼1.7 mol H2

production process thermodynamically

http://dx.doi.org/10.5772/intechopen.74392


yield from thermophilic tempera-

production gave

95

production rate

partial pressure, better

production rates com-

formation [104].

production increased with iron

/g sucrose. Ferchichi et al. [106] sug-

, and destruction of pathogens

/mol glucose, while mesophilic H2

Biohythane Production from Organic Wastes by Two-Stage Anaerobic Fermentation Technology

/h·gVSS) for thermophilic temperatures was 5–10 times higher than that from the

solubility of the substrate, improved hydrolysis reaction as well as thermodynamic efficiency. Temperature is also a very important operation factor in the second stage for anaerobic digestion process. It determines the rate of anaerobic digestion process, particularly the rate of hydrolysis and methanogenesis. The thermophilic process could accelerate the biochemical

pared to mesophilic condition [102]. As temperature increases, the rate of retention time process is much faster and this results in more efficient operation and lowers the retention time requirement [97]. Thermophilic condition also increases in thermodynamic favorability of

in the reactor effluent. Methanogens are extremely subtle to change in temperature and even a small temperature variation (2–3°C) can lead to VFA accumulation [103]. This decreases the

Biohydrogen and biomethane production required various types of metal ions as micronutrients. These metal ions play a critical role in the metabolism of microorganisms. Metal

biomethane process. Metals are essential to supplement in media for dark fermentation. These micronutrients might be required in trace amounts but they have an influential role as cofactors, transport processes facilitators, and structural skeletons of many enzymes (Fe-Fe

Therefore, several researchers have studied the effect of supplementation of Fe ion on biohydrogen production. For example, Lee et al. [105] studied the effect of Fe ion concentra-

concentration of 200 mg/L. The addition of Fe ion 200 mg/L influences the system positively

gested that the supplementation with Fe2+ ions (12 mg/l) led to a shift in their metabolic profile, for example, supplementation with Fe2+ ion concentration of 12 mg/l caused a metabolic shift from lactic acid fermentation to butyric acid fermentation. Magnesium ions function as a cofactor of many enzymes such as kinases and synthetases. In glycolysis, many enzymes require magnesium ions as a cofactor. The activation of hexokinase, phosphofructokinases, glutaraldehyde-3-phosphate dehydrogenases, and enolases helps bacteria to metabolize substrate and produce energy component ATP [107]. Fe ion also plays a critical role in biomethane stage. The Fe ion is required by methanogenic archaea like *Methanosarcina barkeri* to synthesize protocheme via precorrin-2, which is formed from uroporphyrinogen III in two consecutive methylation reaction utilizing S-adenosyl-L-methionine [108]. Nickel is also an

fermentation and found that the H2

hydrogenase and Ni-Fe hydrogenase) involved in the biochemistry of H<sup>2</sup>

production from 131 to 196 mL H<sup>2</sup>

production rate for methanogens, especially at the thermophilic conditions. Maintaining

, less influenced by the H<sup>2</sup>

and CO2

, Mg2+, and Co2+ play a pivotal role in both biohydrogen and

#### **4.5. pH and alkalinity**

Among all the chemical factors influencing dark fermentation, pH is considered the most influential. It influences the stability of the acid-producing fermentative bacteria and acetoclastic CH4 -producing archaea. It plays a major role in the oxidation-reduction potential of the anaerobic process. Thus, it directly impacts the metabolic pathway. In most of literature reports, a pH of 5.5 has been considered to be the optimum pH for H2 production [3, 47, 70, 95]. The optimal initial pH range for the maximum H<sup>2</sup> yield or specific H<sup>2</sup> production rate is between pH 5.5 and 6.5 [95]. The optimal pH is highly dependent on the microorganism. The control of pH and alkalinity of a substrate is essential for first stage dark fermentation since organic acids produced tend to decrease the pH. The pH lower than 4.5 trends to inhibit the activity of hydrogenases. Low pH also causes in shift of metabolic pathways of dark fermentation microorganisms away from H2 production. H2 -producing bacteria like *Clostridium acetobutylicum* can change metabolism from H2 (acetate and butyrate pathway) to the production of solvents (acetone and butanol pathway) when the pH is decreased to less than 5.0. Alternatively, depending on the organism, low pH can shift the metabolism toward ethanol production [72]. Carbohydrate-based substrates provide good carbon and energy sources for H2 -producing bacteria. The fermentation process needs buffering of the growth medium, and to be supplemented with nutrients to enhance the growth of microorganisms and resist the pH change caused by organic acids produced [9, 55, 96]. CH4 production is favored at alkaline pH exhibiting maximum activity at pH of 7.8–8.2 [97]. The rate of CH4 production may decrease if the pH is lower than this optimal range. The pH is also an important factor for the stability of CH4 production. The H2 effluent which is rich in VFA, may cause a drop in pH if fed with high OLR. The pH adjustment can be achieved by an addition of alkali chemical, typically calcium carbonate or sodium hydroxide. A cheap material like ash was used to adjust the pH in an anaerobic reactor [98]. A stable CH<sup>4</sup> production process is characterized by the bicarbonate alkalinity in the range of 1000–5000 mg/L as CaCO3 . The ratio between VFA and alkalinity should be in the range of 0.1–0.25.

#### **4.6. Temperature**

Temperature is one of the most important factors affecting the growth of microorganisms. The operating temperature influences the growth rate of bacteria by influencing the biochemical reactions responsible for the maintenance of homeostasis and their metabolism. H2 -producing dark fermentation reactors can be operated in various temperature ranges from mesophilic (35–45°C), thermophilic (55–60°C) to extreme thermophilic (70–80°) conditions. Most of the H2 dark fermentation studies have been conducted at temperature range of 35–45°C. Many mesophilic bacteria such as *Clostridium* sp. and *Enterobacter* sp. showed optimal H2 production in the temperature range of 35–45°C [99]. A thermophilic H<sup>2</sup> -producing bacterium gave higher H2 yield compared to mesophilic bacteria [100]. When temperature rises, microbial growth rates increase due to the increase in the rates of chemical and enzymatic reactions in their cells. Thermophilic temperature makes the H2 production process thermodynamically favorable with the H2 yield of ∼2.1 mol H2 /mol glucose, while mesophilic H2 production gave the yield of ∼1.7 mol H2 /mol glucose [101]. Although the H<sup>2</sup> yield from thermophilic temperature was slightly higher than that for mesophilic temperatures, the specific H<sup>2</sup> production rate (mmol H<sup>2</sup> /h·gVSS) for thermophilic temperatures was 5–10 times higher than that from the mesophilic temperatures. Thermophilic H2 -producing bacteria has certain operation advantages such as low solubility of H2 and CO2 , less influenced by the H<sup>2</sup> partial pressure, better solubility of the substrate, improved hydrolysis reaction as well as thermodynamic efficiency. Temperature is also a very important operation factor in the second stage for anaerobic digestion process. It determines the rate of anaerobic digestion process, particularly the rate of hydrolysis and methanogenesis. The thermophilic process could accelerate the biochemical reactions and give higher degradation efficiency as well as higher CH<sup>4</sup> production rates compared to mesophilic condition [102]. As temperature increases, the rate of retention time process is much faster and this results in more efficient operation and lowers the retention time requirement [97]. Thermophilic condition also increases in thermodynamic favorability of CH4 -producing reactions, decreases solubility of CH4 and CO2 , and destruction of pathogens in the reactor effluent. Methanogens are extremely subtle to change in temperature and even a small temperature variation (2–3°C) can lead to VFA accumulation [103]. This decreases the CH4 production rate for methanogens, especially at the thermophilic conditions. Maintaining the stable temperature is important for biohythane production.

#### **4.7. Trace elements**

activity. This causes a system failure. During methanogenesis, the HRT should be kept twofold greater than the generation time of the slow-growing microbes [94]. The HRT should be held for a suitable duration so that the dead zones get eliminated, and it would also help in promoting an efficient syntrophy among the microorganisms present in the mixed culture.

Among all the chemical factors influencing dark fermentation, pH is considered the most influential. It influences the stability of the acid-producing fermentative bacteria and acetoclastic

yield or specific H<sup>2</sup>

6.5 [95]. The optimal pH is highly dependent on the microorganism. The control of pH and alkalinity of a substrate is essential for first stage dark fermentation since organic acids produced tend to decrease the pH. The pH lower than 4.5 trends to inhibit the activity of hydrogenases. Low pH also causes in shift of metabolic pathways of dark fermentation microorganisms away

pathway) when the pH is decreased to less than 5.0. Alternatively, depending on the organism, low pH can shift the metabolism toward ethanol production [72]. Carbohydrate-based

tion process needs buffering of the growth medium, and to be supplemented with nutrients to enhance the growth of microorganisms and resist the pH change caused by organic acids

which is rich in VFA, may cause a drop in pH if fed with high OLR. The pH adjustment can be achieved by an addition of alkali chemical, typically calcium carbonate or sodium hydroxide. A cheap material like ash was used to adjust the pH in an anaerobic reactor [98]. A stable CH<sup>4</sup>

duction process is characterized by the bicarbonate alkalinity in the range of 1000–5000 mg/L as

Temperature is one of the most important factors affecting the growth of microorganisms. The operating temperature influences the growth rate of bacteria by influencing the biochemical

dark fermentation reactors can be operated in various temperature ranges from mesophilic (35–45°C), thermophilic (55–60°C) to extreme thermophilic (70–80°) conditions. Most of the

growth rates increase due to the increase in the rates of chemical and enzymatic reactions in

dark fermentation studies have been conducted at temperature range of 35–45°C. Many

yield compared to mesophilic bacteria [100]. When temperature rises, microbial

. The ratio between VFA and alkalinity should be in the range of 0.1–0.25.

reactions responsible for the maintenance of homeostasis and their metabolism. H2

mesophilic bacteria such as *Clostridium* sp. and *Enterobacter* sp. showed optimal H2

tion in the temperature range of 35–45°C [99]. A thermophilic H<sup>2</sup>


production is favored at alkaline pH exhibiting maximum activity at

production may decrease if the pH is lower than this optimal

(acetate and butyrate pathway) to the production of solvents (acetone and butanol


production [3, 47, 70, 95]. The optimal

production rate is between pH 5.5 and


production. The H2

effluent

pro-


produc-


**4.5. pH and alkalinity**

94 Advances in Biofuels and Bioenergy

of 5.5 has been considered to be the optimum pH for H2

substrates provide good carbon and energy sources for H2

range. The pH is also an important factor for the stability of CH4

initial pH range for the maximum H<sup>2</sup>

production. H2

produced [9, 55, 96]. CH4

pH of 7.8–8.2 [97]. The rate of CH4

CH4

from H2

CaCO3

H2

higher H2

**4.6. Temperature**

lism from H2

Biohydrogen and biomethane production required various types of metal ions as micronutrients. These metal ions play a critical role in the metabolism of microorganisms. Metal ions such as Fe2+, Zn2+, Ni2+, Na+ , Mg2+, and Co2+ play a pivotal role in both biohydrogen and biomethane process. Metals are essential to supplement in media for dark fermentation. These micronutrients might be required in trace amounts but they have an influential role as cofactors, transport processes facilitators, and structural skeletons of many enzymes (Fe-Fe hydrogenase and Ni-Fe hydrogenase) involved in the biochemistry of H<sup>2</sup> formation [104]. Therefore, several researchers have studied the effect of supplementation of Fe ion on biohydrogen production. For example, Lee et al. [105] studied the effect of Fe ion concentration (0–4000 mg/L) on H<sup>2</sup> fermentation and found that the H2 production increased with iron concentration of 200 mg/L. The addition of Fe ion 200 mg/L influences the system positively with increasing H2 production from 131 to 196 mL H<sup>2</sup> /g sucrose. Ferchichi et al. [106] suggested that the supplementation with Fe2+ ions (12 mg/l) led to a shift in their metabolic profile, for example, supplementation with Fe2+ ion concentration of 12 mg/l caused a metabolic shift from lactic acid fermentation to butyric acid fermentation. Magnesium ions function as a cofactor of many enzymes such as kinases and synthetases. In glycolysis, many enzymes require magnesium ions as a cofactor. The activation of hexokinase, phosphofructokinases, glutaraldehyde-3-phosphate dehydrogenases, and enolases helps bacteria to metabolize substrate and produce energy component ATP [107]. Fe ion also plays a critical role in biomethane stage. The Fe ion is required by methanogenic archaea like *Methanosarcina barkeri* to synthesize protocheme via precorrin-2, which is formed from uroporphyrinogen III in two consecutive methylation reaction utilizing S-adenosyl-L-methionine [108]. Nickel is also an essential metal which plays a critical role in functioning of many enzymes that are responsible for CH4 production such as monoxide dehydrogenase, hydrogenase, and methyl coenzyme M reductases.

#### **5. Reactors configuration for biohythane production**

The bioreactors in which the microorganisms are grown also play a crucial role. The design and the configuration of the fermenter help in the improvement of mixing characteristics and manipulation of overhead gas partial pressure. Parameters such as HRT and recycle ratio are influenced by the bioreactors configuration. The progress on two-stage system was presented based on the type of feeding substrates, classified as sugar-rich biomass, food/municipal waste, cellulose-based biomass, and palm oil mill effluent (POME). Over 20% of the publications reported so far focused on a system using sugar-rich synthetic wastewater. The most commonly used sugars were glucose and sucrose [10]. The maximum biohythane production was 3.21 mol H2 /mol hexose and 3.63 mol CH<sup>4</sup> /mol hexose from glucose and acetic acid (synthetic wastewater) in CSTR reactor [109]. The summarized H2 and CH4 yield from various two-stage reactors configuration used for biohythane production is shown in **Table 4**. The schematic flow diagrams of each two-stage anaerobic fermentation systems for biohythane production are shown in **Figure 2**. The two-stage anaerobic fermentation is suitable for individual optimization of the H2 and CH4 production processes. For example, temperaturedependent process will be favored by the two-stage process, where high yield of H2 could be achieved under thermophilic conditions, and stable maintaining of CH4 production might be achieved under mesophilic conditions [13, 15, 21, 110]. Solubilization and saccharification of organic wastes with high solid content can be realized simultaneously during the first stage H2 production [17, 74]. The two-stage anaerobic fermentation systems by integrated continuous stirred-tank reactor (CSTR) with anaerobic baffled reactor (ABR), CSTR with UASB, CSTR with CSTR, UASB with UASB, ASBR with UASB and stepped anaerobic baffled (SAB) were used for biohythane production (**Figure 2**.). The system with a CSTR and an upflow biofilter reactor for H2 and CH4 production from sucrose was established [89]. This system inoculated with heat-treated sludge as inoculum achieved a maximum H<sup>2</sup> yield of 1.62 mol H2 /mol hexose. The second stage reactor inoculated with raw anaerobic sludge achieved a maximum CH4 yield of 323 L CH4 /kg COD. The analysis of COD balance showed that 13.5% of the influent COD was transformed to H<sup>2</sup> and 70% of the influent COD was transformed to CH<sup>4</sup> . A CSTR H<sup>2</sup> and CSTR CH<sup>4</sup> system fed with synthetic glucose medium using the same anaerobic sludge as inoculums was reported [18]. By optimizing the inoculums-to-substrate ratio (2:1) in this CSTR-CSTR system, the H<sup>2</sup> yield and the methane yield increased to 2.75 and 2.13 mol/ mol hexose, respectively, with 10 g/L glucose as a substrate, which corresponded to a total energy recovery of 82%. A similar reactor configuration was also used by Lee et al. [25] and Hafez et al. [109]. A synthesis wastewater containing glucose and acetic acid produced 2.6 mol H2 /mol hexose and 426 mL CH<sup>4</sup> /kg COD via continuous fermentation in CSTR [109]. The stable H2 production in the CSTR was possibly due to the introduction of a gravity settler after the H2 CSTR for H<sup>2</sup> -producer retention. A complete CSTR system for H<sup>2</sup> and CH4 production from cassava stillage was developed [12]. The gas yields under thermophilic conditions with high

organic loading (13 g COD/L·d) were 56.6 L H<sup>2</sup>

and CH4

**Reactors (H2 and CH4 )**

CSTR and CSTR

UASB and UASB

CSTR and UASB

ASBR and UASB

CSTR and UASB

CSTR and CSTR

CSTR and UASB with gas upgrade systems

CSTR and ABR

**Feedstock and conditions H2**

Olive pulp, temperature of 35 and 35°C, pH of 5 and 7

Sugarcane syrup, temperature of 37 and 30 °C, pH of 5.5 and

POME, temperature of 55 and 35°C, pH of 5.5 and 7.5

POME, temperature of 55 and 35 °C, pH of 5.5 and 7.5

Biowaste, temperature of 55 and 35 °C, pH of 5.5 and 8

Wheat straw, temperature of 70 and 37°C, pH of 6.9 and 7.5

Food waste, temperature of 55 and 35°C, pH of 5.5 and 7.5

temperature of 21 and 21°C, pH

Desugared molasses, temperature of 70 and 55°C,

pH of 5 and 7

7.5

 **production yield (L-H2**

**VS)**

**/kg** 

**CH4**

Biohythane Production from Organic Wastes by Two-Stage Anaerobic Fermentation Technology

**kg VS)**

190 160 1.6% H2

89 307 16.5% H2

88 271 19.6% H<sup>2</sup>

210 315 14% H2

135 414 13.3% H2

41 102 6.7% H2

270 179 46–57% H2

205 464 15% H2

88 318 16% H2

 **production yield (L-CH4**

**/**

**Biogas composition**

http://dx.doi.org/10.5772/intechopen.74392

38.3% CO2 60% CH4

38.7% CO2 44.8% CH4

62.6% CO2 10.9% CH<sup>4</sup>

32% CO2 51% CH4

32.2% CO2 54.4% CH4

40.1% CO2 52.3% CH4

0.4% CO2 43–54% CH4

54.5% CO2 30.5% CH4

27% CO2 52% CH4 **References**

97

[110]

[11]

[111]

[13]

[15]

[112]

[113]

[21]

[114]

tion of H2

and CH4

SAB Petrochemical wastewater,

of 5.5 and 7.5

/kg TS, and 249 L CH<sup>4</sup>

respectively. Chu et al. [21] developed a two-stage thermophilic CSTR reactor and a mesophilic ABR reactor with the heat-treated digested sludge to recirculation to first reactor for H<sup>2</sup>

**Table 4.** Hydrogen and methane yield from various reactor configurations used for two-stage biohythane production.

of 1.3 days, and pH of 5.5. Kongjan et al. [11] established a biohythane process from wheat straw hydrolysate by two-stage extreme thermophilic UASB and thermophilic UASB. Specific

production was successful by operating the H2

production from organic fraction of municipal solid wastes (OFMSW). The separa-

/kg volatile solid (VS),

reactor at a controlled HRT


essential metal which plays a critical role in functioning of many enzymes that are responsible

The bioreactors in which the microorganisms are grown also play a crucial role. The design and the configuration of the fermenter help in the improvement of mixing characteristics and manipulation of overhead gas partial pressure. Parameters such as HRT and recycle ratio are influenced by the bioreactors configuration. The progress on two-stage system was presented based on the type of feeding substrates, classified as sugar-rich biomass, food/municipal waste, cellulose-based biomass, and palm oil mill effluent (POME). Over 20% of the publications reported so far focused on a system using sugar-rich synthetic wastewater. The most commonly used sugars were glucose and sucrose [10]. The maximum biohythane produc-

two-stage reactors configuration used for biohythane production is shown in **Table 4**. The schematic flow diagrams of each two-stage anaerobic fermentation systems for biohythane production are shown in **Figure 2**. The two-stage anaerobic fermentation is suitable for indi-

achieved under mesophilic conditions [13, 15, 21, 110]. Solubilization and saccharification of organic wastes with high solid content can be realized simultaneously during the first stage

 production [17, 74]. The two-stage anaerobic fermentation systems by integrated continuous stirred-tank reactor (CSTR) with anaerobic baffled reactor (ABR), CSTR with UASB, CSTR with CSTR, UASB with UASB, ASBR with UASB and stepped anaerobic baffled (SAB) were used for biohythane production (**Figure 2**.). The system with a CSTR and an upflow biofilter

ose. The second stage reactor inoculated with raw anaerobic sludge achieved a maximum

sludge as inoculums was reported [18]. By optimizing the inoculums-to-substrate ratio (2:1)

mol hexose, respectively, with 10 g/L glucose as a substrate, which corresponded to a total energy recovery of 82%. A similar reactor configuration was also used by Lee et al. [25] and Hafez et al. [109]. A synthesis wastewater containing glucose and acetic acid produced 2.6 mol

production in the CSTR was possibly due to the introduction of a gravity settler after the

cassava stillage was developed [12]. The gas yields under thermophilic conditions with high


production from sucrose was established [89]. This system inoculated

/kg COD. The analysis of COD balance showed that 13.5% of the

system fed with synthetic glucose medium using the same anaerobic

and 70% of the influent COD was transformed to CH<sup>4</sup>

yield and the methane yield increased to 2.75 and 2.13 mol/

/kg COD via continuous fermentation in CSTR [109]. The stable

/mol hexose from glucose and acetic acid

yield of 1.62 mol H2

and CH4

yield from various

production might be

could be

/mol hex-

production from

. A

and CH4

production processes. For example, temperature-

**5. Reactors configuration for biohythane production**

/mol hexose and 3.63 mol CH<sup>4</sup>

and CH4

achieved under thermophilic conditions, and stable maintaining of CH4

with heat-treated sludge as inoculum achieved a maximum H<sup>2</sup>

dependent process will be favored by the two-stage process, where high yield of H2

(synthetic wastewater) in CSTR reactor [109]. The summarized H2

production such as monoxide dehydrogenase, hydrogenase, and methyl coenzyme

for CH4

M reductases.

96 Advances in Biofuels and Bioenergy

tion was 3.21 mol H2

H2

CH4

H2

H2

H2

CSTR for H<sup>2</sup>

CSTR H<sup>2</sup>

reactor for H2

vidual optimization of the H2

and CH4

influent COD was transformed to H<sup>2</sup>

and CSTR CH<sup>4</sup>

in this CSTR-CSTR system, the H<sup>2</sup>

/mol hexose and 426 mL CH<sup>4</sup>

yield of 323 L CH4

**Table 4.** Hydrogen and methane yield from various reactor configurations used for two-stage biohythane production.

organic loading (13 g COD/L·d) were 56.6 L H<sup>2</sup> /kg TS, and 249 L CH<sup>4</sup> /kg volatile solid (VS), respectively. Chu et al. [21] developed a two-stage thermophilic CSTR reactor and a mesophilic ABR reactor with the heat-treated digested sludge to recirculation to first reactor for H<sup>2</sup> and CH4 production from organic fraction of municipal solid wastes (OFMSW). The separation of H2 and CH4 production was successful by operating the H2 reactor at a controlled HRT of 1.3 days, and pH of 5.5. Kongjan et al. [11] established a biohythane process from wheat straw hydrolysate by two-stage extreme thermophilic UASB and thermophilic UASB. Specific

H2

and CH4

38.7% CO2

the H2

H2

CH4

135mL H2

during H2

yields of 89 mL-H<sup>2</sup>


4.4 L/L·d with biogas composition of 14% H2

/gVS and 414 mL CH<sup>4</sup>

*Methanoculleus* sp. were dominated in the CH4

, 54.4% CH4

effluent recirculation to H<sup>2</sup>

/gVS, respectively.

tation process for H2

degradation and H2

posed with 13.3% H2

/g-VS (190 mL H<sup>2</sup>

with total HRT of 4 days. A biohythane gas with the composition of 16.5% H<sup>2</sup>

*Caldanaerobacter subteraneus*, and *Caloramator fervidus* were responsible for H2

production when compared with non-recirculation systems. The H2

genic effluent recirculation flavored *Thermoanaerobacterium* sp. in the H2

retention time (HRT) of 72 h with hydrogen and methane yield of 88 mL H<sup>2</sup>

Reactors are considered to be practical and economical for industrial H<sup>2</sup>

substrate conversion efficiency [101, 115]. Most studies on H<sup>2</sup>

, and 32.2% CO2

were achieved simultaneously with the overall VS removal efficiency of 81% by operating

of *Methanosarcina mazei* and *Methanothermobacter defluvii*. Successful biohythane production from palm oil mill effluent (POME) by two-stage thermophilic ASBR followed by mesophilic UASB was achieved by Mamimin et al. [13]. The continuous biohythane production rate of

et al. [15] established two-stage thermophilic CSTR and mesophilic UASB with methanogenic

tion rate of methanogenic effluent could keep pH at optimal pH with two times increase in

for energy recovery from POME. Elreedy et al. [114] established biohythane production from petrochemical wastewater containing mono-ethylene glycol by a novel stepped anaerobic baffled (SAB) reactor. The reactor was continuously operated for 5 months at constant hydraulic

ticularly via mixed culture fermentation [70, 100]. The two main bioreactor configurations: suspended and attached, or immobilized, growth types have been applied to optimize fermen-

rich substrates have been conducted in suspended CSTRs, which are simple to construct, easy to regulate both acidity and temperature, and give complete homogeneous mixing for direct contact between the substrate and active biomass [1, 70, 72]. Furthermore, the CSTR is very suitable for substrates with a high-suspended solid (SS) content, typically with a volatile solid (VS) content of 2–12% [48]. However, in CSTR reactor, HRTs must be greater than the specific growth rate of the microorganisms in order to control the proper concentration of microbial biomass, but faster dilution rates risk active biomass washout [1, 67] leading to process failure. In addition, cell density retained in CSTR is limited, since the active biomass has the same retention time as HRT, resulting in process instability caused by the fluctuation of environmental parameters, including acidity and then having the consequence of limiting substrate

tion of a continuous flow reactor is required to decouple the cell mass retention from HRT and subsequently retain higher cell densities in the reactor, such as UASB and ASBR, which can be achieved through granules and biofilm [47, 91, 115, 116]. Cells immobilization can be

production through advancements in active biomass concentration and

production. To overcome the above mention problem, a new configura-

, 51% CH4

production from POME, whereas archaea belonging to *Methanosarcina* sp. and

could be produced at high production rates (3.5 L/L·d). *Thermoanaerobacter wiegelii*,

Biohythane Production from Organic Wastes by Two-Stage Anaerobic Fermentation Technology

/g sugars) and 307 mL CH<sup>4</sup>

and 35% CO2

/gVS, respectively. Biohythane gas composition was com-

reactor for biohythane production from POME. The 30% recircula-


/gVS, respectively

production in

yields were

, and

99

, 44.8% CH4

http://dx.doi.org/10.5772/intechopen.74392

was achieved. O-Thong

reactor and efficiently

/gVS and 318 mL

production, par-

production from carbohydrate

and CH4

. *Thermoanaerobacterium* sp. was dominated

reactor. A two-stage process with methano-

**Figure 2.** Schematic flow diagrams of two-stage anaerobic fermentation systems for biohythane production by integrated CSTR with ABR (A), CSTR with UASB (B), CSTR with CSTR (C), UASB with UASB (D), ASBR with UASB (E) and SAB (F).

H2 and CH4 yields of 89 mL-H<sup>2</sup> /g-VS (190 mL H<sup>2</sup> /g sugars) and 307 mL CH<sup>4</sup> /gVS, respectively were achieved simultaneously with the overall VS removal efficiency of 81% by operating with total HRT of 4 days. A biohythane gas with the composition of 16.5% H<sup>2</sup> , 44.8% CH4 , and 38.7% CO2 could be produced at high production rates (3.5 L/L·d). *Thermoanaerobacter wiegelii*, *Caldanaerobacter subteraneus*, and *Caloramator fervidus* were responsible for H2 production in the H2 -UASB reactor. Meanwhile, the CH<sup>4</sup> -UASB reactor was dominated with methanogens of *Methanosarcina mazei* and *Methanothermobacter defluvii*. Successful biohythane production from palm oil mill effluent (POME) by two-stage thermophilic ASBR followed by mesophilic UASB was achieved by Mamimin et al. [13]. The continuous biohythane production rate of 4.4 L/L·d with biogas composition of 14% H2 , 51% CH4 and 35% CO2 was achieved. O-Thong et al. [15] established two-stage thermophilic CSTR and mesophilic UASB with methanogenic effluent recirculation to H<sup>2</sup> reactor for biohythane production from POME. The 30% recirculation rate of methanogenic effluent could keep pH at optimal pH with two times increase in H2 production when compared with non-recirculation systems. The H2 and CH4 yields were 135mL H2 /gVS and 414 mL CH<sup>4</sup> /gVS, respectively. Biohythane gas composition was composed with 13.3% H2 , 54.4% CH4 , and 32.2% CO2 . *Thermoanaerobacterium* sp. was dominated during H2 production from POME, whereas archaea belonging to *Methanosarcina* sp. and *Methanoculleus* sp. were dominated in the CH4 reactor. A two-stage process with methanogenic effluent recirculation flavored *Thermoanaerobacterium* sp. in the H2 reactor and efficiently for energy recovery from POME. Elreedy et al. [114] established biohythane production from petrochemical wastewater containing mono-ethylene glycol by a novel stepped anaerobic baffled (SAB) reactor. The reactor was continuously operated for 5 months at constant hydraulic retention time (HRT) of 72 h with hydrogen and methane yield of 88 mL H<sup>2</sup> /gVS and 318 mL CH4 /gVS, respectively.

Reactors are considered to be practical and economical for industrial H<sup>2</sup> production, particularly via mixed culture fermentation [70, 100]. The two main bioreactor configurations: suspended and attached, or immobilized, growth types have been applied to optimize fermentation process for H2 production through advancements in active biomass concentration and substrate conversion efficiency [101, 115]. Most studies on H<sup>2</sup> production from carbohydrate rich substrates have been conducted in suspended CSTRs, which are simple to construct, easy to regulate both acidity and temperature, and give complete homogeneous mixing for direct contact between the substrate and active biomass [1, 70, 72]. Furthermore, the CSTR is very suitable for substrates with a high-suspended solid (SS) content, typically with a volatile solid (VS) content of 2–12% [48]. However, in CSTR reactor, HRTs must be greater than the specific growth rate of the microorganisms in order to control the proper concentration of microbial biomass, but faster dilution rates risk active biomass washout [1, 67] leading to process failure. In addition, cell density retained in CSTR is limited, since the active biomass has the same retention time as HRT, resulting in process instability caused by the fluctuation of environmental parameters, including acidity and then having the consequence of limiting substrate degradation and H2 production. To overcome the above mention problem, a new configuration of a continuous flow reactor is required to decouple the cell mass retention from HRT and subsequently retain higher cell densities in the reactor, such as UASB and ASBR, which can be achieved through granules and biofilm [47, 91, 115, 116]. Cells immobilization can be

**Figure 2.** Schematic flow diagrams of two-stage anaerobic fermentation systems for biohythane production by integrated CSTR with ABR (A), CSTR with UASB (B), CSTR with CSTR (C), UASB with UASB (D), ASBR with UASB

(E) and SAB (F).

98 Advances in Biofuels and Bioenergy

employed successfully by using a diluted waste stream with relatively small reactor volumes in ASBR, SAB, and UASB reactors. However, such a reactor configuration has a poor mass transfer system, which is mainly caused by a lack of mixing; this can lead to gases accumulating in the biofilm or granular sludge that risk losing H<sup>2</sup> by H2 -consuming bacteria [92, 101]. Mass transfer can be improved by mechanical stirring or liquid recirculation, depending on the reactor type and configuration. Also, applying proper bioreactor shapes and optimizing reactor dimensions such as the height to diameter ratio can help to improve mass transfer efficiency [91, 98, 117–119].

due to the large surface area of granular sludge, which provides fast biofilm development and improves methanogenesis. Also clogging and channeling occur less in the UASB reactor than

Biohythane Production from Organic Wastes by Two-Stage Anaerobic Fermentation Technology

Methane is being commonly used, not only in the chemical industry but also in transport as compressed natural gas (CNG), which has been regarded as the clean energy carrier in

considered one of the important fuels involved in achieving the transition of technical mod-

has weak points on its narrow range of flammability, slow burning speed, poor combustion efficiency as well as requirement for high ignition temperature of CNG-powered vehicles.

thus, improves the fuel efficiency and can extend the narrow range of flammability of CH<sup>4</sup>

process technique, combining acidogenesis and methanogesis appears to give more efficient waste treatment and energy recovery than a single methanogenic process [13]. As the results

could be achieved. This specification was found to be most suitable for burning directly in the internal combustion engines [131] and could be biohythane. In addition to economical concern, the two-stage thermophilic anaerobic process has been previously evaluated that the payback time is around 2–6 years, depending on the disposal costs of organic wastes/

Various types of organic wastes can be used as substrate for biohythane production such as starch wastewater, palm oil mill effluent (POME), biowaste, sugarcane syrup, olive pulp,

/gCOD, respectively [13], which were higher than those of starch wastewater

/gCOD, respectively) [18], sugarcane syrup (88mL H<sup>2</sup>

two-stage biohythane production of palm oil mill effluent (POME) was 201 mL H<sup>2</sup>

and CH4

/gCOD, respectively) [111], and biowaste (21 mL H<sup>2</sup>

was achieved with biohythane production rate of 4.4 L/L·d with biogas composition of 51%

of high biogas production volume. Energy analysis of two-stage anaerobic fermentation

[13]. POME is a suitable substrate for H<sup>2</sup>

can be greatly increased by adding H2

perfectly complements the weak points of CH4

tion duration and improving heat efficiency. The quenching distance of CH<sup>4</sup>

ric content of 44.8, 38.7, and 16.5%, respectively, containing approx. 10% H<sup>2</sup>

desugared molasses, food waste, and organic solid waste [13, 18, 19]. H2

/gVS and 160 mL CH<sup>4</sup>

production from POME by two-stage thermophilic H<sup>2</sup>

/gVS and 464 mL CH<sup>4</sup>

and CH4

http://dx.doi.org/10.5772/intechopen.74392

, which reduces greenhouse gas emissions. Adding H<sup>2</sup>

, CO2

, and H2

yield from two-stage biohythane production of

/gVS, respectively) [110] was lower than that of

/gVS, respectively) [21]. Successful biohythane

reactor and mesophilic CH4

, making the engine easy to ignite with less input energy. A two-stage

, biohythane is

can be reduced

with the volumet-

on energy basis

yield from

/gCOD and 55 mL

production in terms

/gCOD

/gCOD

reactor

,

101

.

used as a fuel for vehicle

such as the hydrogen/carbon

, eventually reducing combus-

and CH4

comparison to gasoline or diesel. By combining the advantages of H2

els from a fossil fuel-based society to renewable-based society. CH4

reported by Kongjan and Angelidaki [129], mixed gas of CH<sup>4</sup>

other biofilm systems [121].

Interestingly, H2

The flame speed of CH<sup>4</sup>

by the addition of H2

residues [28].

and 315 mL CH4

and 271 mL CH4

, 14% H2

olive pulp (190 mL H<sup>2</sup>

food waste (205 mL H<sup>2</sup>

/gCOD and 230mL CH4

/gCOD, respectively) [112]. H2

, and 35% CO2

(130mL H<sup>2</sup>

CH4

CH4

**6. Application of biohythane process**

ratio which is increased by adding H2

The anaerobic conversion of VFA to CH<sup>4</sup> is mainly associated with sequential stages of acetogenesis and methanogesis. When optimizing a methanogenic process using VFA rich, soluble organic matters, the goal is to maximize both CH<sup>4</sup> production and VFA degradation, while keeping the reactor stable [37]. The acetogenesis is limited mainly by VFA degradation, especially propionate that is the rate-limiting factor in the second stage anaerobic process. The investigation into optimizing the methanogenic reactor is mostly carried out by varying OLRs via increasing the substrate concentration or decreasing the HRTs to obtain satisfactory performance [25, 120]. The main signs of methanogenic reactor instability or overloading are decrease in pH [121]. As a drop of pH actually corresponds to VFA accumulation, pH below 6.3 has an impact on enzyme activity in the microorganisms involved in the second stage anaerobic digestion. Methanogenic archaea can function properly in a pH range between 6.5 and 7.8 [122]. Thus, a buffering solution is needed in order to resist a pH drop from VFA accumulation in the methanogenic process and maintain stability. The main buffer in the anaerobic digester is bicarbonate (HCO<sup>3</sup> ), which is usually added to carbohydrate rich substrates before feeding them to the first stage of H<sup>2</sup> fermentation because the first stage needs to be controlled with pH within the favorable range of 5–6 for H2 -producing bacteria [123, 124]. Lee et al. [25] found that the pH drop below 6.4 caused by the accumulation of 122 mM VFA in the attached growth reactor operated at 55°C and fed with 11.0 gVS/L·d (5.13 d HRT) of the food waste fermentation. The pH could inhibit the bioactivity of methanogenesis. Meanwhile, the maximum CH<sup>4</sup> production rate of 2100 mL CH4 /L·d with a CH4 content of 65% was obtained at pH around 7.5, where the reactor was operated at a 7.7 day HRT (7.9 gVS/L·d OLR) and almost VFA degradation was achieved. For the high rate anaerobic reactor, UASB reactor was operated at double OLR comparing to CSTR at thermophilic temperature (55°C) which providing better VFAs degradation than mesophilic temperature (35°C) [125]. This is mainly attributed to the increase of chemical and biological reaction rates for operating temperature of thermophilic condition and the organic acid oxidation reactions become more energetic at higher temperature [126, 127]. Because the H2 reactor effluents are in soluble form of organic matters as the consequence of hydrolysis and acidogenesis in the first stage, the reactor type used to convert these soluble organic matters to CH<sup>4</sup> in the second stage are based on high rate biofilm systems as reviewed by Demirel et al. [27]. Cell mass is retained well in the biofilm/ granular aggregates in biofilm systems, leading to have much higher sludge retention time (SRT) compared to HRT, which provides the advantage that the reactor can run at a higher flow rate and can tolerate higher toxic concentrations [128]. Various types of high rate biofilm systems such as UASB, ABR, and SAB can be operated by continuous feeding with the H<sup>2</sup> reactor effluent, with HRTs of less than 5 days [114, 125, 129, 130]. Among the high rate reactor types, the UASB is the most popular for anaerobic treatment of soluble organic matters due to the large surface area of granular sludge, which provides fast biofilm development and improves methanogenesis. Also clogging and channeling occur less in the UASB reactor than other biofilm systems [121].
