**1. Introduction**

Currently, development of biofuels to replace fossil fuels by the biological process has been attracting attention as an environmentally friendly process. Among the various processes, biohydrogen and biohythane are the promising future energy carriers due to their potentially higher conversion efficiency and low pollutants generation [1]. Dark fermentation shows high H2 production rate under realistic conditions, which is approaching practical levels [2]. In addition, the major advantages are rapid bacterial growth rates, relatively high H2 production capacities, operation without light sources, no oxygen limitation problems, and low capital cost of at least at small-scale production facilities [3, 4]. The dark fermentation process can utilize organic materials for H2 gas production, such as cellulose and starch-containing agricultural and food industry wastes, and some food industry wastewaters, such as cheese whey, olive mill, palm oil mill, and baker's yeast industry wastewaters [5]. H2 yields from dark fermentation of organic wastes such as food waste, apple processing wastewater, starch wastewater, palm oil mill effluent, and potato processing wastewater were 57, 92, 92, 115, and 128 mL H2 /gCOD, respectively [6–9]. However, dark fermentation has low substrate conversion efficiency as only 7.5–15% of the energy contained in organic wastes are converted to H<sup>2</sup> and the rest of the energy still remains in the liquid (H<sup>2</sup> effluent) as VFA (mainly butyric acid and acetic acid), lactic acid, and alcohols [1]. The disadvantage of dark fermentation must be overcome before biohydrogen can become economically feasible. The conversion of VFA, lactic acid, and alcohols to CH4 through anaerobic digestion (AD) [10] is faster and simpler than the conversion of these components to H2 by photo-fermentation and microbial-electrolysis process [1]. In addition, it has been shown to be an energy efficiency strategy for the production of a mixture of H<sup>2</sup> and CH4 , known as biohythane, via two-stage anaerobic fermentation [11, 12].

The two-stage anaerobic fermentation process could increase energy recovery, degradation

improving negative impacts of inhibitive compounds in feedstocks (such as wheat hydrolysate, molasses, and skim latex serum), operated at high organic loading rates and reduced fermentation time with total HRT of 10–18 days for overall processes. Advantages of biohythane over traditional biogas are improved energy recovery, shortened fermentation time, flexible H<sup>2</sup>

ratio, and more environmentally benign and process robustness for handling the organic wastes [10, 16]. Integrated biohydrogen with biomethane process worth for commercialization could

10–15% by volume. Biohythane is considered to be a clean fuel for vehicles compared to gaso-

Biohythane via two-stage anaerobic fermentation using organic wastes could be a promising technology for higher energy recovery and cleaner transport biofuel than biogas. Various types of organic wastes can be used as substrate for biohythane production such as starch wastewater, wheat straw hydrolysate, palm oil mill effluent, food waste, and organic solid waste [13, 18–20]. Wheat straw hydrolysate was used for biohythane production by *Caldicellulosiruptor* 

production rate of 5.2 L H2

and CH4

straw with energy recovery of 57% of energy contained in the wheat straw [20]. Biohythane

/gCOD, respectively [18]. Biohythane production of food waste achieved H2

/L·d. The maximum energy output of the process was 10.9 kJ/g of

and CH4

. Energy analysis suggested that the two-stage fermentation process

/gCOD, respectively [13]. Nathao et al. [22] obtained two-stage process for bio-

and CH4

production from wheat straw hydrolysate via a two-stage anaerobic fermentation process.

Successful continuous biohythane production from POME by two-stage thermophilic fermentation and mesophilic anaerobic digestion was reported by Mamimin et al. [13]. The continuous biohythane production rate of 4.4 L/L·d was achieved with biogas containing 51% CH4

/gVS and 307 mL CH<sup>4</sup>

yields of 130 mL H2

/gVS, respectively [21]. Biohythane production of

yields of 201 mL H2

yields of 55 and 94 mL/gVS at F/M of

/gVS, respectively, were achieved.

line or diesel due to low greenhouse gas emission from the combustion process [17].

get the biogas in the form of biohythane. Typically, the suggested H2

**Technology Processes Substrates Products** Hythane Thermo-chemical Natural gas 5–7% H2

Biomethane Anaerobic digestion (AD) Organic wastes 50–60% CH4

Biohydrogen Fermentation Organic wastes 40–60% H2

Biohythane Two-stage fermentation/AD Organic wastes 5–10% H2

**Table 1.** Biohythane technology development from two-stage anaerobic fermentation technology.

production rates, and purity of gas products when compared

, 90% CH<sup>4</sup>

, 60% CH4

and 5% CO2

and 30% CO2

and 40–50% CO2

and 40–60% CO2

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

/CH4

85

pro-

and CH4

/gCOD and

fermentation

,

/gCOD and 230 mL

and thermophilic CH4

content in biohythane is

/L·d and maximum CH<sup>4</sup>

fermentation [15]. In addition, the two-stage process has advantages of

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

efficiency, reactor stability, CH<sup>4</sup>

or CH4

*saccharolyticus* with maximum H<sup>2</sup>

production of starch wastewater achieved H2

hythane production from food waste with H2

palm oil mill effluent (POME) was achieved with H<sup>2</sup>

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

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

7.5. Kongjan et al. [11] used UASB reactors for extreme thermophilic H<sup>2</sup>

for biohythane production had greater net energy recovery than the single H2

duction rate of 2.6 L CH4

and CH4

, and 35% CO2

yields of 205 mL H2

315 mL CH4

Specific H<sup>2</sup>

14% H2

CH4

to one-stage H2

Biohythane has attracted growing attention worldwide due to its potential use as vehicle fuel, high potential to produce from conversion of organic wastes and probably an alternative to the fossil-based hythane [10]. Normally, hythane gas was produced from a thermo-chemical process using natural gas as a starting material. This process is a high-energy consumption and still depends on fossil fuel. Biohydrogen and biomethane production from organic wastes by fermentation process and anaerobic digestion process, respectively, are already established. The combination of these two processes via two-stage anaerobic fermentation processes could yield a H2 and CH4 gas with a composition like hythane (10–15% H<sup>2</sup> , 50–55% CH4 , and 30–40% CO2 ) called biohythane [13], which could be upgraded to biobased hythane by removing of CO2 . The two-stage anaerobic fermentation for biohythane production is involved with the fermentation of organic wastes to H2 , CO2 , VFA, lactic acid, and alcohols in the first stage and conversion of these substances in H2 effluent to CH<sup>4</sup> and CO2 via anaerobic digestion process in the second stage (**Table 1**). The optimum condition for the first stage is a pH range between 5 and 6 and a hydraulic retention time (HRT) range of 1–3 days that are suitable for acidogens for the conversion of organic wastes to H2 via the acetate and butyrate pathways. In the second stage, the acetic acid in the H2 effluent is converted to CH<sup>4</sup> and CO2 by acetoclastic methanogens under an anaerobic condition with optimal pH range of 7–8 and optimal HRT of 10–15 days [11]. Others VFA, lactic acid, and alcohols in the H<sup>2</sup> effluent are anaerobically converted by acetogens to H<sup>2</sup> and CO2 , which are consequently converted to CH4 by hydrogenotrophic methanogens [14].


**Table 1.** Biohythane technology development from two-stage anaerobic fermentation technology.

**1. Introduction**

84 Advances in Biofuels and Bioenergy

can utilize organic materials for H2

and the rest of the energy still remains in the liquid (H<sup>2</sup>

high H2

128 mL H2

acid, and alcohols to CH4

a mixture of H<sup>2</sup>

and CH4

of organic wastes to H2

these substances in H2

acetic acid in the H2

and CO2

version of organic wastes to H2

VFA, lactic acid, and alcohols in the H<sup>2</sup>

a H2

conversion of these components to H2

and CH4

, CO2

effluent to CH<sup>4</sup>

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

, which are consequently converted to CH4

Currently, development of biofuels to replace fossil fuels by the biological process has been attracting attention as an environmentally friendly process. Among the various processes, biohydrogen and biohythane are the promising future energy carriers due to their potentially higher conversion efficiency and low pollutants generation [1]. Dark fermentation shows

In addition, the major advantages are rapid bacterial growth rates, relatively high H2

whey, olive mill, palm oil mill, and baker's yeast industry wastewaters [5]. H2

duction capacities, operation without light sources, no oxygen limitation problems, and low capital cost of at least at small-scale production facilities [3, 4]. The dark fermentation process

agricultural and food industry wastes, and some food industry wastewaters, such as cheese

dark fermentation of organic wastes such as food waste, apple processing wastewater, starch wastewater, palm oil mill effluent, and potato processing wastewater were 57, 92, 92, 115, and

sion efficiency as only 7.5–15% of the energy contained in organic wastes are converted to H<sup>2</sup>

and acetic acid), lactic acid, and alcohols [1]. The disadvantage of dark fermentation must be overcome before biohydrogen can become economically feasible. The conversion of VFA, lactic

cess [1]. In addition, it has been shown to be an energy efficiency strategy for the production of

Biohythane has attracted growing attention worldwide due to its potential use as vehicle fuel, high potential to produce from conversion of organic wastes and probably an alternative to the fossil-based hythane [10]. Normally, hythane gas was produced from a thermo-chemical process using natural gas as a starting material. This process is a high-energy consumption and still depends on fossil fuel. Biohydrogen and biomethane production from organic wastes by fermentation process and anaerobic digestion process, respectively, are already established. The combination of these two processes via two-stage anaerobic fermentation processes could yield

gas with a composition like hythane (10–15% H<sup>2</sup>

called biohythane [13], which could be upgraded to biobased hythane by removing of CO2

and CO2

two-stage anaerobic fermentation for biohythane production is involved with the fermentation

stage (**Table 1**). The optimum condition for the first stage is a pH range between 5 and 6 and a hydraulic retention time (HRT) range of 1–3 days that are suitable for acidogens for the con-

anaerobic condition with optimal pH range of 7–8 and optimal HRT of 10–15 days [11]. Others

and CO2

/gCOD, respectively [6–9]. However, dark fermentation has low substrate conver-

through anaerobic digestion (AD) [10] is faster and simpler than the

, known as biohythane, via two-stage anaerobic fermentation [11, 12].

by photo-fermentation and microbial-electrolysis pro-

, 50–55% CH4

via anaerobic digestion process in the second

by acetoclastic methanogens under an

by hydrogenotrophic methanogens [14].

, VFA, lactic acid, and alcohols in the first stage and conversion of

via the acetate and butyrate pathways. In the second stage, the

effluent are anaerobically converted by acetogens to H<sup>2</sup>

, and 30–40% CO2

)

. The

production rate under realistic conditions, which is approaching practical levels [2].

gas production, such as cellulose and starch-containing

effluent) as VFA (mainly butyric acid

pro-

yields from

The two-stage anaerobic fermentation process could increase energy recovery, degradation efficiency, reactor stability, CH<sup>4</sup> production rates, and purity of gas products when compared to one-stage H2 or CH4 fermentation [15]. In addition, the two-stage process has advantages of improving negative impacts of inhibitive compounds in feedstocks (such as wheat hydrolysate, molasses, and skim latex serum), operated at high organic loading rates and reduced fermentation time with total HRT of 10–18 days for overall processes. Advantages of biohythane over traditional biogas are improved energy recovery, shortened fermentation time, flexible H<sup>2</sup> /CH4 ratio, and more environmentally benign and process robustness for handling the organic wastes [10, 16]. Integrated biohydrogen with biomethane process worth for commercialization could get the biogas in the form of biohythane. Typically, the suggested H2 content in biohythane is 10–15% by volume. Biohythane is considered to be a clean fuel for vehicles compared to gasoline or diesel due to low greenhouse gas emission from the combustion process [17].

Biohythane via two-stage anaerobic fermentation using organic wastes could be a promising technology for higher energy recovery and cleaner transport biofuel than biogas. Various types of organic wastes can be used as substrate for biohythane production such as starch wastewater, wheat straw hydrolysate, palm oil mill effluent, food waste, and organic solid waste [13, 18–20]. Wheat straw hydrolysate was used for biohythane production by *Caldicellulosiruptor saccharolyticus* with maximum H<sup>2</sup> production rate of 5.2 L H2 /L·d and maximum CH<sup>4</sup> production rate of 2.6 L CH4 /L·d. The maximum energy output of the process was 10.9 kJ/g of straw with energy recovery of 57% of energy contained in the wheat straw [20]. Biohythane production of starch wastewater achieved H2 and CH4 yields of 130 mL H2 /gCOD and 230 mL CH4 /gCOD, respectively [18]. Biohythane production of food waste achieved H2 and CH4 yields of 205 mL H2 /gVS and 464 mL CH<sup>4</sup> /gVS, respectively [21]. Biohythane production of palm oil mill effluent (POME) was achieved with H<sup>2</sup> and CH4 yields of 201 mL H2 /gCOD and 315 mL CH4 /gCOD, respectively [13]. Nathao et al. [22] obtained two-stage process for biohythane production from food waste with H2 and CH4 yields of 55 and 94 mL/gVS at F/M of 7.5. Kongjan et al. [11] used UASB reactors for extreme thermophilic H<sup>2</sup> and thermophilic CH4 production from wheat straw hydrolysate via a two-stage anaerobic fermentation process. Specific H<sup>2</sup> and CH4 yields of 89 mL H<sup>2</sup> /gVS and 307 mL CH<sup>4</sup> /gVS, respectively, were achieved. Successful continuous biohythane production from POME by two-stage thermophilic fermentation and mesophilic anaerobic digestion was reported by Mamimin et al. [13]. The continuous biohythane production rate of 4.4 L/L·d was achieved with biogas containing 51% CH4 , 14% H2 , and 35% CO2 . Energy analysis suggested that the two-stage fermentation process for biohythane production had greater net energy recovery than the single H2 fermentation and CH4 fermentation process. This chapter provides the information on general approach of biohythane via two-stage anaerobic fermentation, principles of biohythane process, microorganisms involved in H2 and CH4 production, reactor configuration for biohythane production, methods for improve biohythane production, process parameters affecting biohythane production and technical challenges toward the scale-up process.
