**4. Process parameters affecting biohythane production**

Biohythane production processes are greatly influenced by complex biochemical and physical parameters. The process parameters such as inoculum properties, complexity of substrate, nutrient, alkalinity, H2 concentration, hydraulic retention time (HRT), and toxic compounds have influence on biohythane process (**Table 3**). Inoculums and feedstocks compositions greatly affect first stage H<sup>2</sup> fermentation when using mixed cultures and non-sterile feedstocks [1, 70, 74]. Environmental and physical factors greatly affect the second stage CH<sup>4</sup> production [75, 76]. To stabilize and maximize H<sup>2</sup> production, it is necessary to direct the metabolic pathway toward acetic acid and/or butyric acid and also to maintain the right H2 producing bacteria during first stage operation. The performance of microorganisms in the conversion of substrate to H2 is also dependent on the efficiency of its enzymatic machinery. The main factors affecting two-stage anaerobic fermentation are described as follows.

#### **4.1. Feedstocks**

tial to use lignocellulosic waste for H2

90 Advances in Biofuels and Bioenergy

such as methanogens and H2

the range of 57–128 mL H2

The second stage CH4

archaea produces CH4

duce CH4

CO2 , H2

CH4

and CO2

Acidogenic H<sup>2</sup>

potential to commercialize H2

, and trace amount of ethanol [57].

Industrially, the use of mixed cultures for H<sup>2</sup>

sludge could utilize cellulose as a substrate for H2

and *Methanospaera* sp. are commonly found in CH4

is commonly found in extreme thermophilic CH<sup>4</sup>

grows at a temperature of 83–85°C and assimilates CO<sup>2</sup>

from CO2

hydrogenotrophic or acetoclastic and thus can reduce CO2

as methanol, methylamines, and methyl sulfides to CH<sup>4</sup>

effluent. Major genuses related to acidogenic H<sup>2</sup>

acetic acid as the energy source through acetoclastic reaction.

and H2

sp., *Methanospirillum* sp., and *Methanoculleus* sp. These archaea produce CH4

and exception of *Methanocorpusculum* sp. and *Methanoculleus* sp. using CO2

Heat treatment inhibits the activity of the methanogens and H2

could be more advantage than pure cultures. Enriched H<sup>2</sup>

of 3.5 mol H2

H2 , CO2

ing H2

production with the yield of 3.3 mol H2

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

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



/mol hexose [56]. The soluble metabolites of these strains are mostly acetic acid,

temperatures of 90°C. *Thermotoga* sp. can use elemental sulfur as electron source with H2

retention time (1–2 days) helps in washing out slow-growing methanogens from H<sup>2</sup>

hexose [59]. The fermentation of various organic wastes by mixed cultures gave the H<sup>2</sup>

/mol hexose.


consumers, while the spore form-




or formate as the energy source. [61]. The order

production are *Enterobacter* sp., *Clostridium*

[60]. The order *Methanococcales*

from acetic acid

for CH4

and H2

or can utilize acetic acid to

. *Methanosaeta* sp. utilizes

production from organic wastes in the first stage

production with the yield of 2.4 mol H2

/gCOD, depending on type of waste [6–9]. This indicates the practical

reactor involved with several archaea strains is capable to pro-

and H2

to CH4

and CO2

through anaerobic fermentation of VFA, lactic acid, and alcohols. The order

, and methanol consuming methanogens. The family *Methanobacteriaceae* including

*Methanobacteriales* comprises of two families (*Methanobacteriaceae* and *Methanothermaceae*) is

*Methanobacterium* sp., *Methanothermobacter* sp., *Methanobrevibacter* sp., *Methanothermus* sp.,

sp. is a thermophilic *Methanobacteriaceae* that is commonly found in thermophilic CH4

producing reactor. *Methanothermus* sp. is an extreme thermophilic *Methanobacteriaceae* that

consists of *Methanocaldococcus* sp., *Methanothermococcus* sp., and *Methanococcus* sp. These

*Methanomicrobiales* consists of *Methanomicrobium* sp., *Methanocorpusculum* sp., *Methnanoplanus*

production [62]. The order *Methanosarcinales* consists of *Methanosarcina* sp., *Methanohalobium* sp., *Methanohalophilus* sp., *Methanolobus* sp., and *Methanosaeta* sp. *Methanosarcina* sp. are

. *Methanosarcina* sp. also can convert methyl-group-containing compounds such

producers grow faster than methanogens and eventually produce VFA in

production from organic wastes by mixed microbial consortium.

at

yield

reactor.

/mol


yields in

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 peptides, while fat is considered very limited [77]. Most of dark fermentation for H<sup>2</sup> production 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 a favorable substrate for H2 fermentation [79, 80]. The H2 yield from bean curd manufacturing 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]. The H2 fermentative microorganisms showed improvement in H2 production when they were grown in a fermentation media having a C/N ratio greater than 20. The C/N ratio of 20–30 also has positive effect on CH<sup>4</sup> production stage. Phosphate concentration in feedstock is also


cells [86]. Inoculum size for dark H2

**4.3. Hydrogen partial pressure**

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

microorganism (F/M) ratios of H<sup>2</sup>

loads could reduce the H2

of feed. It is generally well known that the H2

applying this principle, Liu et al. [48] produced H2

the main optimization parameters of continuous H2

ria. Generally, short HRT is considered to favor the H<sup>2</sup>

lating the HRT, slow-growing microbes like methanogens and H<sup>2</sup>

expelled out of the reactor, thus leading to selective enrichment of H<sup>2</sup>

The H2

fermentation was varied in the range of 10–20% (v/v). This

and CH4

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

partial pressure, the NADH, which is

partial pressure.


dark fermentation bioprocesses. In the

fermentation metabolism [3]. On the



producers or washout of microorganisms [90]. These shock

production metabolism through decreasing of pH and metabolite

in continuously CSTR feeding


production.

production, as high H2

partial pressure by

production during

pro-

93

depends on the characteristics of the species and medium used. Obligate anaerobes produce very less amount of biomass; thus, larger inoculum volume and concentration are required. The inoculum age also matters during the fermentation. Cells growing at the exponential phase

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

an electron carrier in the cell, will be oxidized mainly to lactate during extreme thermophilic fermentation with *Caldicellulosiruptor saccharolyticus* [88]. The formation of lactate during the

The total time that cells and soluble nutrients reside in the reactor is called the HRT. H<sup>2</sup>

duction occurring at low HRT is dependent on the volume of the reactor and the flow rate

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

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

other hand, too high loading rates may result in substrate inhibition effects, improper food to

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 manipu-

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

proper level that still gives a D value less than specific growth rate of H<sup>2</sup>

free of CH4

have the entire enzymatic machinery active which is required for H2

thermophilic fermentation [87]. In addition to a high H2

overloading or unstable conditions might be caused by a high H2

partial pressure in the liquid phase is the major factor affecting H<sup>2</sup>

partial pressure causes deactivation of hydrogenase enzyme. Decreasing H2

intermittent nitrogen sparging of batch reactor headspace could enhance H<sup>2</sup>

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

important in dark fermentation. Phosphate helps in maintaining buffered condition during fermentation and provides the building blocks of nucleic acid and ATPs. In dark fermentation, an increase in phosphate concentration leads to enhancement of the H2 production [47].

#### **4.2. Inoculums**

Developing an enriched inoculum is very important for obtaining H2 in first stage fermentation. In the enrichment process, selection procedure was applied to selectively promote H2 -producing bacteria and eliminate H2 consumers. Different selective procedures such as heat, acid, ultrasonic, ultraviolet, organic and alkali treatment were commonly used [58]. Most of H<sup>2</sup> -producing bacteria are spore forming, while H2 -consuming bacteria and methanogens are non-spore forming, which get eliminated with selection methods. The selection methods are promoting endospores formation in a certain group of bacteria that also include H2 -producing bacteria. Thus, under favorable conditions, the endospores germinate and the H2 -producing bacteria dominate in the system. The H2 -producing inoculum might consist of sporulating bacteria like *Bacillus* sp. and *Clostridium* sp. Furthermore, the bacteria capable of producing H<sup>2</sup> widely exist in natural 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 source of feedstock [85]. The selection of H2 -producing bacteria suitable for introduction into H2 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 cells [86]. Inoculum size for dark H2 fermentation was varied in the range of 10–20% (v/v). This depends on the characteristics of the species and medium used. Obligate anaerobes produce very less amount of biomass; thus, larger inoculum volume and concentration are required. The inoculum age also matters during the fermentation. Cells growing at the exponential phase have the entire enzymatic machinery active which is required for H2 and CH4 production.

#### **4.3. Hydrogen partial pressure**

The H2 partial pressure in the liquid phase is the major factor affecting H<sup>2</sup> production, as high H2 partial pressure causes deactivation of hydrogenase enzyme. Decreasing H2 partial pressure by intermittent nitrogen sparging of batch reactor headspace could enhance H<sup>2</sup> production during thermophilic fermentation [87]. In addition to a high H2 partial pressure, the NADH, which is an electron carrier in the cell, will be oxidized mainly to lactate during extreme thermophilic fermentation with *Caldicellulosiruptor saccharolyticus* [88]. The formation of lactate during the overloading or unstable conditions might be caused by a high H2 partial pressure.
