**2. Principles of biohythane process**

Most of wastewater and organic wastes were usually treated in an anaerobic process for CH<sup>4</sup> recovery as energy. Regarding clean energy of H<sup>2</sup> , anaerobic process was modified for H<sup>2</sup> production by suppression of methanogenic activity. To harvest H2 from the first stage, the H<sup>2</sup> consuming pathway has to be inhibited [23]. Most H<sup>2</sup> -producing bacteria can form endospores in stress environment. Various selection methods can be used to enrich H<sup>2</sup> -producing bacteria [24]. The most common selection methods are heat treatment and pH control. However, some researchers reported the invalidity of such selection methods [25], because not all H2 -producing bacteria are associated with the ability to form endospores. In addition, there are many H2 consuming bacteria that can form endospores, such as acetogens and sulfate-reducing bacteria [26]. The pH control is an important method for maintaining H2 -producing bacteria in continuous systems of first stage. The pH varies depending on the microbial species, microbial activities, reactor configuration, feedstock characteristics, organic loading rate, buffer capacity, and temperature. The change of pH is due to acetic acid and butyric acid production accompanies with H2 production, whereas the low pH influences on the shift of metabolic products from acidogenesis to solventogenesis [27]. Low pH is also critical strategies to inhibit the activity of methanogenesis. The suggestion for optimal pH of H2 production could range from 5.0 to 6.5. From the perspective of thermodynamics, changes of Gibbs free energy during H<sup>2</sup> production were much larger than those of methanogenesis. This means faster rates for microbial growth in biohydrogen fermentation. On the basis of this characteristic, the manipulation of hydraulic retention time (HRT), temperature, and oxidation-reduction potential (ORP) can achieve microbial H2 process feasible in continuous operation.

H2

CO2

to CH4

into H2

13–18 days [10].

, CO2

, VFA, lactic acid, and alcohols. High H2

the acetic acid in the H2

fermentation process.

production was achieved by fermentative bacteria

and CO2

and CO2

and CO2

are consequently converted

via the

and

by acetoclastic

via acidogenesis process under pH range of 5-6 and operating at short HRT of 1-3 days. Under

**Figure 1.** Modification of anaerobic digestion for biohythane production from organic wastes via two-stage anaerobic

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

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

87

acetate and butyrate pathways and competition to other microorganisms. In the second stage,

effluent is anaerobically converted to CH<sup>4</sup>

The two-stage anaerobic fermentation process is based on two physiologically different groups of microorganisms. One group of acidogenic bacteria that converts organic matter

environment of pH 5–6, and is less sensitive to environmental changes. A large number of microbial species, including strict and facultative anaerobic bacteria such as *Clostridium* sp.,

 by hydrogenotrophic methanogens [29]. These reactions occur under an optimal pH range of 7–8 and HRT of 10–15 days [30]. The two-stage anaerobic fermentation process is also characterized by a significantly reduced fermentation time with overall fermentation time of

, soluble VFA, lactic acid, and alcohols, is fast growing, prefers a slightly acidic

methanogens. The acetogenic bacteria could produce acetic acid along with additional H2

the optimum condition, acidogenic bacteria could convert carbohydrate to H2

from butyric acid, propionic acid, and lactic acid. H2

Continuous biohythane production by integrating biohydrogen with biomethane process worth for commercialization could get the biogas that has composition like hythane gas. In the first stage, substrate is fermented to H<sup>2</sup> , CO2 , VFA, lactic acid, and alcohols whereby the non-gas metabolites are converted to CH4 and CO2 in the second stage [10]. The fermentation products from H2 production process are very important for the whole biohythane system performance because they can affect the loading, degradation efficiency, and operating stability of the methanogenesis stage [28]. The conversion rate from VFA to acetic acid will affect the methanogenic archaea quantity, and subsequently affect the degradation rate of acetic acid and CH<sup>4</sup> yield. The basic principle of a two-stage process is shown in **Figure 1**. The first stage includes hydrolysis and acidogenesis where hydrolytic and fermentative bacteria excrete enzymes to break down complex organic compounds of carbohydrate, protein, and lipid into single molecules of mono sugar, amino acid, and long chain fatty acids and/or glycerol respectively. The acidogenesis, fermentative, and acidogenic bacteria convert the hydrolysis products into CO2 , Biohythane Production from Organic Wastes by Two-Stage Anaerobic Fermentation Technology http://dx.doi.org/10.5772/intechopen.74392 87

and CH4

with H2

microbial H2

products from H2

ganisms involved in H2

86 Advances in Biofuels and Bioenergy

and CH4

**2. Principles of biohythane process**

recovery as energy. Regarding clean energy of H<sup>2</sup>

consuming pathway has to be inhibited [23]. Most H<sup>2</sup>

production and technical challenges toward the scale-up process.

duction by suppression of methanogenic activity. To harvest H2

[26]. The pH control is an important method for maintaining H2

process feasible in continuous operation.

methanogenesis. The suggestion for optimal pH of H2

the first stage, substrate is fermented to H<sup>2</sup>

non-gas metabolites are converted to CH4

in stress environment. Various selection methods can be used to enrich H<sup>2</sup>

researchers reported the invalidity of such selection methods [25], because not all H2

From the perspective of thermodynamics, changes of Gibbs free energy during H<sup>2</sup>

fermentation process. This chapter provides the information on general approach of

production, reactor configuration for biohythane produc-

, anaerobic process was modified for H<sup>2</sup>


from the first stage, the H<sup>2</sup>


production could range from 5.0 to 6.5.

, VFA, lactic acid, and alcohols whereby the

in the second stage [10]. The fermentation



production

,

pro-



biohythane via two-stage anaerobic fermentation, principles of biohythane process, microor-

tion, methods for improve biohythane production, process parameters affecting biohythane

Most of wastewater and organic wastes were usually treated in an anaerobic process for CH<sup>4</sup>

[24]. The most common selection methods are heat treatment and pH control. However, some

bacteria are associated with the ability to form endospores. In addition, there are many H2

consuming bacteria that can form endospores, such as acetogens and sulfate-reducing bacteria

ous systems of first stage. The pH varies depending on the microbial species, microbial activities, reactor configuration, feedstock characteristics, organic loading rate, buffer capacity, and temperature. The change of pH is due to acetic acid and butyric acid production accompanies

acidogenesis to solventogenesis [27]. Low pH is also critical strategies to inhibit the activity of

were much larger than those of methanogenesis. This means faster rates for microbial growth in biohydrogen fermentation. On the basis of this characteristic, the manipulation of hydraulic retention time (HRT), temperature, and oxidation-reduction potential (ORP) can achieve

Continuous biohythane production by integrating biohydrogen with biomethane process worth for commercialization could get the biogas that has composition like hythane gas. In

, CO2

and CO2

formance because they can affect the loading, degradation efficiency, and operating stability of the methanogenesis stage [28]. The conversion rate from VFA to acetic acid will affect the methanogenic archaea quantity, and subsequently affect the degradation rate of acetic acid and CH<sup>4</sup> yield. The basic principle of a two-stage process is shown in **Figure 1**. The first stage includes hydrolysis and acidogenesis where hydrolytic and fermentative bacteria excrete enzymes to break down complex organic compounds of carbohydrate, protein, and lipid into single molecules of mono sugar, amino acid, and long chain fatty acids and/or glycerol respectively. The acidogenesis, fermentative, and acidogenic bacteria convert the hydrolysis products into CO2

production process are very important for the whole biohythane system per-

production, whereas the low pH influences on the shift of metabolic products from

**Figure 1.** Modification of anaerobic digestion for biohythane production from organic wastes via two-stage anaerobic fermentation process.

H2 , VFA, lactic acid, and alcohols. High H2 production was achieved by fermentative bacteria via acidogenesis process under pH range of 5-6 and operating at short HRT of 1-3 days. Under the optimum condition, acidogenic bacteria could convert carbohydrate to H2 and CO2 via the acetate and butyrate pathways and competition to other microorganisms. In the second stage, the acetic acid in the H2 effluent is anaerobically converted to CH<sup>4</sup> and CO2 by acetoclastic methanogens. The acetogenic bacteria could produce acetic acid along with additional H2 and CO2 from butyric acid, propionic acid, and lactic acid. H2 and CO2 are consequently converted to CH4 by hydrogenotrophic methanogens [29]. These reactions occur under an optimal pH range of 7–8 and HRT of 10–15 days [30]. The two-stage anaerobic fermentation process is also characterized by a significantly reduced fermentation time with overall fermentation time of 13–18 days [10].

The two-stage anaerobic fermentation process is based on two physiologically different groups of microorganisms. One group of acidogenic bacteria that converts organic matter into H2 , CO2 , soluble VFA, lactic acid, and alcohols, is fast growing, prefers a slightly acidic environment of pH 5–6, and is less sensitive to environmental changes. A large number of microbial species, including strict and facultative anaerobic bacteria such as *Clostridium* sp., *Enterobacter* sp., *Caldicellulosiruptor* sp., *Thermotoga* sp., and *Thermoanaerobacterium* sp., are efficient H<sup>2</sup> producers, while degrading various types of carbohydrates [31]. The other group in second stage is methanogenic archaea, which converts VFA, lactic acid, and alcohols into CH4 and CO2 , is slow growing, prefers neutral to slightly alkaline environments, and is very sensitive to environmental changes. *Methanosarcina* sp. and *Methanoculleus* sp. were dominant and played an important role in second stage [14, 15]. *Methanosarcina* species were reported to be dominant at high acetate concentration (>1.2 mM), and the results were consistent with the high acetate concentrations in H2 effluent that feed to CH<sup>4</sup> reactors. *Methanoculleus* species were responsible for hydrogenotrophic methanogenesis that convert H2 and CO2 to CH4 [11]. Obtaining the optimum environmental conditions for each group of organisms by the two-stage anaerobic fermentation process provides several advantages over the conventional single stage [32–34], e.g., high net energy efficiencies, more stable operation, allowing higher organic loading rate operation, smaller-size reactor (40–60% smaller), thus better economics for construction cost and higher CH4 content in the biogas (65–75%) [15, 35]. High CH4 content and production was found in the second stage due to CO2 in the second stage is mainly generated by aceticlastic methanogen and then consumed partly by hydrogenotrophic methanogen also existed in the second stage. The higher CH4 content is definitely a better fuel value for on-site use and higher digestion efficiency, thus more CH<sup>4</sup> is recovered [36].

and less affected by the partial pressure of H<sup>2</sup>

**Table 2.** Microorganisms involved in the first stage H<sup>2</sup>

anaerobic fermentation process.

**Stages Mesophilic condition (30–35°C)**

> *Clostridium* sp. *Enterobacter* sp. *Citrobacter* sp. *Bacillus* sp.

*Clostridium* sp. *Bacillus* sp.

*Desulfobacterium* sp.

*Methanobacterium* sp. *Methanoculleus* sp. *Methanospirillum* sp. *Methanococcus* sp. *Methanobacter* sp.

1st hydrogen production (Bacteria)

2nd methane production (Bacteria)

2nd methane production (Archaea)

ture (60°C) and can convert carbohydrate to H<sup>2</sup>

with the yield of 1.6 mol H2

but cannot degrade cellulose. These species produce H2

H2

H2

H2

in the liquid phase. Dark fermentation under

via ethanol- and acetate-type fermentation,

/mol hexose [53]. Dark fermentation at extreme thermophilic

, ethanol, lactate, acetate, and CO2

via butyr-

production via two-stage

produc-

production


**Extreme thermophilic condition** 

89

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

**(70–90°C)**

*Caldanaerobacter* sp. *Caloramator* sp. *Thermotoga* sp.

*Caloramator* sp.

*Methanothermus* sp. *Methanothermococcus* sp.

thermophilic condition was involved with *Thermoanaerobacterium* sp., *Thermoanaerobacter* sp., and *Clostridium* sp. [15]. *Thermoanaerobacterium thermosaccharolyticum* has an optimal growth

production, and the second stage CH4

**Thermophilic condition** 

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

T*hermoanaerobacterium* sp.

*Thermoanaerobacterium* sp. *Desulfomicrobium* sp.

*Methanothermobacter* sp. *Methanosarcina* sp.

**(55–60°C)**

*Clostridium* sp. *Thermoanaerobacter* sp.

*Clostridium* sp.

ate- and acetate-type fermentation [46]. *Thermoanaerobacterium* species are well known as good

tion from various types of substrate under the thermophilic conditions. Various *Tbm. thermosaccharolyticum* strains have been isolated such as strain PSU2 [46], strain GD17 [48], strain W16 [49], strain KKU19 [50], and strain IIT BT-ST1 [51]. In addition, *Tbm. thermosaccharolyticum* can grow on various organic wastes including hemicellulosic waste and lignocellulosic waste [48, 52]. *Thermoanaerobacter* sp. has optimal growth at moderate thermophilic tempera-

as the major products, but no butyrate production. Thermophilic *Clostridium* sp. was found to degrade cellulose using cellulase enzymes and can ferment the lignocellulosic biomass to

compared to the thermophilic and mesophilic systems. Dark fermentation under extreme thermophilic condition was involved with *Thermotoga* sp. and *Caldicellulosiruptor* sp. [54]. The

 production ability of *Caldicellulosiruptor* sp. was explored at extreme temperatures. These microbes are known to have various kinds of hydrolytic enzymes that can utilize a wide range of substrate such as cellulose, cellubiose, and xylan. *Caldicellulosiruptor* sp. has high poten-

temperatures (70–90°C) showed more favorable kinetics and stoichiometry of H<sup>2</sup>


at moderate thermophilic temperature (60°C) and can convert carbohydrate to H<sup>2</sup>

Genus *Thermoanaerobacterium*, especially *Tbm. thermosaccharolyticum*, is capable of H2

thermophilic microorganisms previously found in thermophilic H2

#### **3. Microorganisms in biohythane process**

The two-stage anaerobic fermentation process is based on the differences between acidogens and methanogens in physiology, nutrition needs, growth kinetics, and sensitivity to environmental conditions. The acidogens and methanogens are enriched separately in two tanks enabling optimized growth by maintaining proper environmental conditions in each reactor [37]. Microorganisms involved in the first stage H<sup>2</sup> production and in the second stage CH4 production via two-stage anaerobic fermentation process are shown in **Table 2**. First stage (H<sup>2</sup> reactor) involved with the several bacterial strains is capable to produce H<sup>2</sup> through dark fermentation of various carbohydrates. Obligate anaerobic *Clostridia* are potential H2 producers and are well known for high H2 yield [38]. *C. butyricum*, *C. welchii*, *C. pasteurianum*, and *C. beijerinckii* were used for H2 production [39]. *Clostridium* sp. is capable of utilizing a wide range of carbohydrates such as xylose, arabinose, galactose, glucose, cellobiose, sucrose and fructose with a H2 yield of 2.1–2.2 mol H2 /mol sugars [40]. Facultative anaerobes *Enterobacteriaceae* are H2 producers that are resistant to trace amount of dissolved oxygen. *Enterobacter* sp. has lower yield (1.0 mol H<sup>2</sup> /mol sugars) when compared to *Clostridium* sp. [41]. *Citrobacter* sp. also belongs to family *Enterobacteriaceae* known to produce H2 from CO and H2 O by water-gas shift reaction under anaerobic condition [42]. *Escherichia coli* is capable of producing H2 and CO2 from formate in the absence of oxygen. The H<sup>2</sup> yields of *E. coli* were 0.6–1.3 mol H2 /mol glucose [43]. *Bacillus* sp. also has been identified as H<sup>2</sup> producers such as *B. licheniformis* [44] and *B. coagulans* [45]. Its H2 yield was 0.5 mol H2 /mol glucose with lactic acid as main soluble metabolites. Dark fermentation at thermophilic temperatures (55–60°C) showed favorable kinetics and stoichiometry of H2 production compared to the mesophilic systems. Metabolism at higher temperatures becomes thermodynamically more favorable


*Enterobacter* sp., *Caldicellulosiruptor* sp., *Thermotoga* sp., and *Thermoanaerobacterium* sp., are

in second stage is methanogenic archaea, which converts VFA, lactic acid, and alcohols into

sensitive to environmental changes. *Methanosarcina* sp. and *Methanoculleus* sp. were dominant and played an important role in second stage [14, 15]. *Methanosarcina* species were reported to be dominant at high acetate concentration (>1.2 mM), and the results were consistent with

[11]. Obtaining the optimum environmental conditions for each group of organisms by the two-stage anaerobic fermentation process provides several advantages over the conventional single stage [32–34], e.g., high net energy efficiencies, more stable operation, allowing higher organic loading rate operation, smaller-size reactor (40–60% smaller), thus better economics

generated by aceticlastic methanogen and then consumed partly by hydrogenotrophic meth-

The two-stage anaerobic fermentation process is based on the differences between acidogens and methanogens in physiology, nutrition needs, growth kinetics, and sensitivity to environmental conditions. The acidogens and methanogens are enriched separately in two tanks enabling optimized growth by maintaining proper environmental conditions in each reac-

production via two-stage anaerobic fermentation process are shown in **Table 2**. First

reactor) involved with the several bacterial strains is capable to produce H<sup>2</sup>

dark fermentation of various carbohydrates. Obligate anaerobic *Clostridia* are potential H2

ing a wide range of carbohydrates such as xylose, arabinose, galactose, glucose, cellobiose,

O by water-gas shift reaction under anaerobic condition [42]. *Escherichia coli* is capable

from formate in the absence of oxygen. The H<sup>2</sup>

yield of 2.1–2.2 mol H2

[41]. *Citrobacter* sp. also belongs to family *Enterobacteriaceae* known to produce H2

/mol glucose [43]. *Bacillus* sp. also has been identified as H<sup>2</sup>

acid as main soluble metabolites. Dark fermentation at thermophilic temperatures (55–60°C)

systems. Metabolism at higher temperatures becomes thermodynamically more favorable

cies were responsible for hydrogenotrophic methanogenesis that convert H2

tent and production was found in the second stage due to CO2

value for on-site use and higher digestion efficiency, thus more CH<sup>4</sup>

anogen also existed in the second stage. The higher CH4

**3. Microorganisms in biohythane process**

tor [37]. Microorganisms involved in the first stage H<sup>2</sup>

producers and are well known for high H2

*Enterobacter* sp. has lower yield (1.0 mol H<sup>2</sup>

and CO2

*B. licheniformis* [44] and *B. coagulans* [45]. Its H2

showed favorable kinetics and stoichiometry of H2

*num*, and *C. beijerinckii* were used for H2

sucrose and fructose with a H2

*Enterobacteriaceae* are H2

producers, while degrading various types of carbohydrates [31]. The other group

, is slow growing, prefers neutral to slightly alkaline environments, and is very

content in the biogas (65–75%) [15, 35]. High CH4

reactors. *Methanoculleus* spe-

in the second stage is mainly

content is definitely a better fuel

production and in the second stage

/mol sugars [40]. Facultative anaerobes

production compared to the mesophilic

yield [38]. *C. butyricum*, *C. welchii*, *C. pasteuria-*

/mol sugars) when compared to *Clostridium* sp.

production [39]. *Clostridium* sp. is capable of utiliz-

producers that are resistant to trace amount of dissolved oxygen.

yield was 0.5 mol H2

is recovered [36].

and CO2

to CH4

con-

through

from CO

yields of *E. coli* were

/mol glucose with lactic

producers such as

effluent that feed to CH<sup>4</sup>

efficient H<sup>2</sup>

and CO2

88 Advances in Biofuels and Bioenergy

the high acetate concentrations in H2

for construction cost and higher CH4

CH4

CH4

stage (H<sup>2</sup>

and H2

of producing H2

0.6–1.3 mol H2

**Table 2.** Microorganisms involved in the first stage H<sup>2</sup> production, and the second stage CH4 production via two-stage anaerobic fermentation process.

and less affected by the partial pressure of H<sup>2</sup> in the liquid phase. Dark fermentation under thermophilic condition was involved with *Thermoanaerobacterium* sp., *Thermoanaerobacter* sp., and *Clostridium* sp. [15]. *Thermoanaerobacterium thermosaccharolyticum* has an optimal growth at moderate thermophilic temperature (60°C) and can convert carbohydrate to H<sup>2</sup> via butyrate- and acetate-type fermentation [46]. *Thermoanaerobacterium* species are well known as good H2 -producing bacteria [8, 47]. *Thermoanaerobacterium* sp. represents anaerobic spore forming thermophilic microorganisms previously found in thermophilic H2 -producing reactors [8, 9]. Genus *Thermoanaerobacterium*, especially *Tbm. thermosaccharolyticum*, is capable of H2 production from various types of substrate under the thermophilic conditions. Various *Tbm. thermosaccharolyticum* strains have been isolated such as strain PSU2 [46], strain GD17 [48], strain W16 [49], strain KKU19 [50], and strain IIT BT-ST1 [51]. In addition, *Tbm. thermosaccharolyticum* can grow on various organic wastes including hemicellulosic waste and lignocellulosic waste [48, 52]. *Thermoanaerobacter* sp. has optimal growth at moderate thermophilic temperature (60°C) and can convert carbohydrate to H<sup>2</sup> via ethanol- and acetate-type fermentation, but cannot degrade cellulose. These species produce H2 , ethanol, lactate, acetate, and CO2 as the major products, but no butyrate production. Thermophilic *Clostridium* sp. was found to degrade cellulose using cellulase enzymes and can ferment the lignocellulosic biomass to H2 with the yield of 1.6 mol H2 /mol hexose [53]. Dark fermentation at extreme thermophilic temperatures (70–90°C) showed more favorable kinetics and stoichiometry of H<sup>2</sup> production compared to the thermophilic and mesophilic systems. Dark fermentation under extreme thermophilic condition was involved with *Thermotoga* sp. and *Caldicellulosiruptor* sp. [54]. The H2 production ability of *Caldicellulosiruptor* sp. was explored at extreme temperatures. These microbes are known to have various kinds of hydrolytic enzymes that can utilize a wide range of substrate such as cellulose, cellubiose, and xylan. *Caldicellulosiruptor* sp. has high potential to use lignocellulosic waste for H2 production with the yield of 3.3 mol H2 /mol hexose. The predominant metabolites formed by these organisms are acetic acid and lactic acid [55]. *Thermotoga* sp. was isolated from geothermal spring and capable to grow and produce H2 at temperatures of 90°C. *Thermotoga* sp. can use elemental sulfur as electron source with H2 yield of 3.5 mol H2 /mol hexose [56]. The soluble metabolites of these strains are mostly acetic acid, H2 , CO2 , and trace amount of ethanol [57].

sp., *Citrobacter* sp., *Thermoanaerobacterium* sp., and *Caldicellulosiruptor* sp. After H<sup>2</sup>

*Thermoanaerobacterium* sp.*, Clostridium roseum*, and *Clostridium isatidis,* which are H2

and *Methanothermobacter thermautotrophicus* could possibly consume H2

detected when the methanogenic stage reached stable conditions [67].

**4. Process parameters affecting biohythane production**

stage, confirming that some H<sup>2</sup>

However, the presence of the hydrogenotrophic methanogens of *Methanothermobacter defluvii*

Biohythane production processes are greatly influenced by complex biochemical and physical parameters. The process parameters such as inoculum properties, complexity of substrate,

have influence on biohythane process (**Table 3**). Inoculums and feedstocks compositions

stocks [1, 70, 74]. Environmental and physical factors greatly affect the second stage CH<sup>4</sup>

metabolic pathway toward acetic acid and/or butyric acid and also to maintain the right H2

The main factors affecting two-stage anaerobic fermentation are described as follows.

tides, while fat is considered very limited [77]. Most of dark fermentation for H<sup>2</sup>

fermentative microorganisms showed improvement in H2

fermentation [79, 80]. The H2

producing bacteria during first stage operation. The performance of microorganisms in the

Biohythane can be produced from various substrates mainly carbohydrate. In terms of H2 rate and yields, carbohydrates are the most suitable feedstock followed by protein and pep-

has been conducted with glucose or sucrose. Glucose is the monomeric unit of cellulose and starch which is a major component in organic wastes [78]. Carbohydrate-rich organic waste is

ing waste was significantly low compared to carbohydrate-rich substrates [80]. For stable H<sup>2</sup> fermentation, a carbon/nitrogen (C/N) ratio of feedstock greater than 20 is recommended [81].

grown in a fermentation media having a C/N ratio greater than 20. The C/N ratio of 20–30

concentration, hydraulic retention time (HRT), and toxic compounds

fermentation when using mixed cultures and non-sterile feed-

is also dependent on the efficiency of its enzymatic machinery.

production stage. Phosphate concentration in feedstock is also

*Clostridium*, *Bacillus,* and *Desulfobacterium* in CH4

are able to degrade lactic acid to acetate and/or H2

production [75, 76]. To stabilize and maximize H<sup>2</sup>

significant removal of lactic acid in the H<sup>2</sup>

[64–66] were also detected in CH4

nutrient, alkalinity, H2

greatly affect first stage H<sup>2</sup>

conversion of substrate to H2

a favorable substrate for H2

also has positive effect on CH<sup>4</sup>

**4.1. Feedstocks**

The H2

effluents rich in VFA such as acetic acid, butyric acid, lactic acid, and ethanol would be consumed by methanogenic archaea at neutral pH. High acetic acid concentration promotes the growth of *Methanosarcina* sp. On the contrary, lower acetic acid concentration is preferred by *Methanosaeta* sp. For acetoclastic methanogens such as *Methanosarcina* sp., the minimum thresholds for acetate utilization are typically in the range of 0.5 mM and higher. The minimum thresholds for acetic acid utilization of *Methanoseata* sp. are in the micromole range. The presence of

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

production,

91

producers

could be


production

were also produced.

; thus, no H2

production stage is in accordance with the

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

[63]. Meanwhile, some acidogenic bacteria,

production, it is necessary to direct the

yield from bean curd manufactur-

production when they were

effluent since *Clostridium* and *Desulfobacterium* spp.

and CO2

Microbial consortium or mixed cultures are providing more enzymes for the utilization of complex substrate than pure cultures. Mixed microbial consortium can be developed from various sources such as anaerobic digested sludge, soil samples, and wastewater by heat treatment and load-shock treatment [58]. These two treatments could eliminate unwanted microorganisms such as methanogens and H2 -consuming bacteria while enriching an H2 -producing bacterium. Heat treatment inhibits the activity of the methanogens and H2 consumers, while the spore forming H2 -producing bacteria was survived. Additionally, continuous operation at a low hydraulic retention time (1–2 days) helps in washing out slow-growing methanogens from H<sup>2</sup> reactor. Industrially, the use of mixed cultures for H<sup>2</sup> production from organic wastes in the first stage could be more advantage than pure cultures. Enriched H<sup>2</sup> -producing bacteria from anaerobic sludge could utilize cellulose as a substrate for H2 production with the yield of 2.4 mol H2 /mol hexose [59]. The fermentation of various organic wastes by mixed cultures gave the H<sup>2</sup> yields in the range of 57–128 mL H2 /gCOD, depending on type of waste [6–9]. This indicates the practical potential to commercialize H2 production from organic wastes by mixed microbial consortium.

The second stage CH4 reactor involved with several archaea strains is capable to produce CH4 through anaerobic fermentation of VFA, lactic acid, and alcohols. The order *Methanobacteriales* comprises of two families (*Methanobacteriaceae* and *Methanothermaceae*) is CO2 , H2 , and methanol consuming methanogens. The family *Methanobacteriaceae* including *Methanobacterium* sp., *Methanothermobacter* sp., *Methanobrevibacter* sp., *Methanothermus* sp., and *Methanospaera* sp. are commonly found in CH4 -producing reactor. *Methanothermobacter* sp. is a thermophilic *Methanobacteriaceae* that is commonly found in thermophilic CH4 producing reactor. *Methanothermus* sp. is an extreme thermophilic *Methanobacteriaceae* that is commonly found in extreme thermophilic CH<sup>4</sup> -producing reactor. *Methanothermus* sp. grows at a temperature of 83–85°C and assimilates CO<sup>2</sup> and H2 [60]. The order *Methanococcales* consists of *Methanocaldococcus* sp., *Methanothermococcus* sp., and *Methanococcus* sp. These archaea produces CH4 from CO2 and H2 or formate as the energy source. [61]. The order *Methanomicrobiales* consists of *Methanomicrobium* sp., *Methanocorpusculum* sp., *Methnanoplanus* sp., *Methanospirillum* sp., and *Methanoculleus* sp. These archaea produce CH4 from acetic acid and exception of *Methanocorpusculum* sp. and *Methanoculleus* sp. using CO2 and H2 for CH4 production [62]. The order *Methanosarcinales* consists of *Methanosarcina* sp., *Methanohalobium* sp., *Methanohalophilus* sp., *Methanolobus* sp., and *Methanosaeta* sp. *Methanosarcina* sp. are hydrogenotrophic or acetoclastic and thus can reduce CO2 to CH4 or can utilize acetic acid to CH4 and CO2 . *Methanosarcina* sp. also can convert methyl-group-containing compounds such as methanol, methylamines, and methyl sulfides to CH<sup>4</sup> and CO2 . *Methanosaeta* sp. utilizes acetic acid as the energy source through acetoclastic reaction.

Acidogenic H<sup>2</sup> producers grow faster than methanogens and eventually produce VFA in effluent. Major genuses related to acidogenic H<sup>2</sup> production are *Enterobacter* sp., *Clostridium*

sp., *Citrobacter* sp., *Thermoanaerobacterium* sp., and *Caldicellulosiruptor* sp. After H<sup>2</sup> production, effluents rich in VFA such as acetic acid, butyric acid, lactic acid, and ethanol would be consumed by methanogenic archaea at neutral pH. High acetic acid concentration promotes the growth of *Methanosarcina* sp. On the contrary, lower acetic acid concentration is preferred by *Methanosaeta* sp. For acetoclastic methanogens such as *Methanosarcina* sp., the minimum thresholds for acetate utilization are typically in the range of 0.5 mM and higher. The minimum thresholds for acetic acid utilization of *Methanoseata* sp. are in the micromole range. The presence of *Clostridium*, *Bacillus,* and *Desulfobacterium* in CH4 production stage is in accordance with the significant removal of lactic acid in the H<sup>2</sup> effluent since *Clostridium* and *Desulfobacterium* spp. are able to degrade lactic acid to acetate and/or H2 [63]. Meanwhile, some acidogenic bacteria, *Thermoanaerobacterium* sp.*, Clostridium roseum*, and *Clostridium isatidis,* which are H2 producers [64–66] were also detected in CH4 stage, confirming that some H<sup>2</sup> and CO2 were also produced. However, the presence of the hydrogenotrophic methanogens of *Methanothermobacter defluvii* and *Methanothermobacter thermautotrophicus* could possibly consume H2 ; thus, no H2 could be detected when the methanogenic stage reached stable conditions [67].
