18.4 Factors Affecting Biohydrogen

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18.4.3

Inoculum

Inoculum refers to the microbial catalyst used for fermentative hydrogen pro-

duction. Size and type of inoculum are the two factors affecting biological H2

productivity. H2 production by dark fermentation is driven by both pure and mixed

cultures of strict and facultative anaerobic bacteria belonging to family Enterobac-

teriaceae or Clostridiaceae. Obligate anaerobes are highly sensitive to atmospheric

oxygen and mostly belong to Clostridium sps. such as Clostridium pasteurianum,

Clostridium acetobutylicum, Clostridium butyricum, Clostridium thermocellum,

Clostridium tyrobutyricum, Clostridium paraputrificum. Obligate anaerobes are

highly efficient for H2 production compared to facultative anaerobes, which

includes Enterobacter sps., Escherichia coli, Bacillus sps. etc. This is mainly due

to the existence of broad diversity in enzymes catalyzing the hydrogen-producing

pathway in both types of anaerobes. Several authors have investigated the capacity

of numerous pure bacterial strains to produce H2 by using various substrates.

Table 18.2 represents various studies on H2 production by pure cultures with the

strain Clostridium bifermentans demonstrating highly efficient H2 yields [15–31].

The yield of 3.29 mol-H2/mol-glucose has been reported, which is near to the equiv-

alent of the theoretical yield of 4 mol-H2/mol-glucose [15]. The strains were isolated

from landfill leachate sludge and have been reported to favor butyrate-associated H2

production pathway. On the other hand, another genus of Clostridium species C. ace-

tobutylicum produces H2 with a maximum yield of 2 mol-H2/mol-glucose [20]. Thus,

it could be corroborated from these observations that the maximum yield of H2 varies

from strain to strain and widely depends on the sources of isolation, type of substrate

utilized, and operating conditions favorable for a particular microbial strain.

Mixed culture comprises naturally developed consortia of microbes, which feeds

on various organic wastes or pure carbon sources to produce H2 as the major

metabolic product. Table 18.2 summarizes the various sources of mixed culture

which are enriched to produce H2. Anaerobic sludge, cow dung slurry, sewage

sludge, dairy sludge, municipal sewage sludge, and compost are commonly used

natural sources of mixed culture for fermentative H2 production. Mixed culture as

inoculum for H2 production is advantageous over pure culture as it can feed on

complex carbon sources like cellulose, food waste hydrolysate, dairy wastewater,

corn starch, and other organic wastes. Moreover, H2 generation from organic

wastes using natural consortia is more cost-effective and feasible for industri-

alization, thereby resulting in energy production along with waste reduction.

Depending on the source, mixed cultures may contain both H2-producing and

H2-consuming bacteria along with methanogens. Therefore, various pretreatment

methods are recommended for deactivating the H2-consuming bacteria and enrich

the H2-producing spore-forming bacteria. Heat shock, acid/base treatment, and

freeze–thaw are the most prevalent pretreatment methods applied to enrich

H2-producing bacteria in mixed culture. The range of temperature, duration of heat

shock or freeze–thaw cycles, and concentration of acid/base varies and requires

optimization to produce maximum fermentative hydrogen.