Dark fermentative H2 production can be carried out at mesophilic (25–40 °C), thermophilic (40–
65 °C) or hyperthermophilic (>80 °C) conditions depending on the microbial inoculum used (Sinha and Pandey, 2011). H2-producers are quite diverse and widespread in nature. They have been found in environments such as animal manure (Yokoyama et al., 2007), sewage sludge (Kotay and Das, 2006) and hot springs (Koskinen et al., 2008). Fermentative microorganisms are either
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facultative anaerobes or obligate anaerobes (Cabrol et al., 2017; Ghimire et al., 2015; Rittmann and Herwig, 2012).
Facultative anaerobic H2 producers are attractive for their lower oxygen sensitivity and their presence in dark fermentative process is useful for rapidly depleting O2 possibly present in the culture (Khanna et al., 2011). However, their presence is often limited by harsh inoculum pretreatment methods (described in more detail in section 2.4) such as heat treatment and acid treatment used to inactivate H2 consuming organisms, which are typically found from the same environments as H2 producing microorganisms (Cabrol et al., 2017). Expect for Bacillales (phylum Firmicutes, class Bacilli) all other known facultative H2-producers belong to the class gammaproteobacteria (phylum proteobacteria) and are able to thrive at mesophilic conditions.
These include for example, Citrobacter spp., Klebsiellaspp., Enterobacter spp. (Patel et al., 2014), Shewanella oneidensis (Meshulam-Simon et al., 2007) and Pseudomonas stutzeri (Goud et al., 2014). An interesting characteristic of the H2 producing Bacillales is that some of the members of this group possess the ability to resist shock conditions (such as heat-shock) via sporulation (Kumar et al., 2013).
Obligate anaerobes constitute a diverse array of microorganisms which are either spore formers or non-spore formers (For review, see e.g. Cabrol et al., 2017). One of the most studied obligately anaerobic microorganisms able to produce H2 at mesophilic conditions is Clostridium butyricum, which belongs to the class Clostridia. Clostridia have been found to dominate mesophilic fermentative microbial communities irrespective of the source of inoculum or pre-treatment method (Cabrol et al., 2015; Das and Veziroglu, 2008; Ueno et al., 2001).
H2 producing microorganisms that can produce H2 at thermophilic conditions include both bacteria and archaea (Pawar and van Niel, 2013). Some thermophilic H2 producers, such as Caldicellulosiruptor saccharolyticus have the ability to effectively hydrolyze complex carbohydrates for their metabolism (Carver et al., 2011; Nissilä et al., 2011). Their ability to hydrolyze complex carbohydrates makes them valuable for H2 production as well as other industrial processes for enhancing the rate of hydrolysis (De Vrije et al., 2009; Islam et al., 2009).
Among the thermophilic H2 producers that have been reported in literature, Thermoanaerobacterium spp. have been the most often found from mixed cultures (Karadag and Puhakka, 2010; Koskinen et al., 2008; O-Thong et al., 2011). Other genera of thermophilic H2
producing microorganisms include for example Caldicellulosiruptor (Willquist et al., 2010), Thermotoga (Nguyen et al., 2008), and Thermoanaerobacter (Koskinen et al., 2008).
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Thermophilic microorganisms typically produce higher H2 yields than H2 producers thriving at mesophilic conditions, because H2 yielding reactions are thermodynamically more favorable at higher temperatures (Foglia et al., 2011; Qiu et al., 2016; Verhaart et al., 2010). For example, H2
yield of approximately 96% of the theoretical value (4 mol H2 mol-1 glucose) has been reported with Thermotoga neapolitana at 80 °C (Ippolito et al., 2010), while about 90% of the theoretical yield has been obtained with Caldicellulosiruptor saccharolyticus at 72.5 °C (De Vrije et al., 2007).
Dark fermentative H2 production at both mesophilic and thermophilic conditions can be carried out using either mixed or pure cultures (Hung et al., 2011; Li and Fang, 2007; Zhang et al., 2007).
Pure microbial cultures have mainly been utilized at laboratory-scale for studying H2 production mechanisms, the effect of environmental conditions on dark fermentation and the ability of different species to utilize different carbon sources (Table 2.1). However, Van Groenestijn et al.
(2009) demonstrated pilot-scale H2 production (400 L cylindrical stainless steel trickle bed reactor of 1.2 m height filled with 190 L polyurethane foam) using sucrose for studying the performance of pure cultures of C. saccharolyticus at a temperature of 73 °C under non-aseptic conditions (Van Groenestijn et al., 2009). They obtained a yield of 2.8 mol H2 mol-1 hexose and a volumetric productivity of 22 mmol H2 L-1 filter bed h-1. Even though pure cultures often generate H2 with very high efficiency, they are typically quite sensitive to contamination and can require aseptic conditions throughout the fermentation, which is not feasible for large scale production (Taherdanak et al., 2015). Conversely, mixed cultures do not require an aseptic environment for H2 production (for a review, see e.g. Hallenbeck, 2009). Additionally, single microorganisms have less hydrolytic capacity when compared to a consortium of microorganism which offers the possibility to work on a wide spectrum of low-cost, easily available substrates. Therefore, mixed microbial cultures are preferred for H2 production especially when complex organic materials such as lignocellulosic biomass or wastewaters are used as substrates (Anh et al., 2011; Cappelletti et al., 2012; Kim et al., 2008; Kumar et al., 2015; Hiroshi Yokoyama et al., 2007).
The microbial diversity often observed in mixed cultures can sometimes be disadvantageous for dark fermentative H2 production due to the activity of H2 consuming microorganisms and/or microorganisms that compete with H2 producers for substrates without producing H2 (Cabrol et al., 2017). Microorganisms that contribute negatively towards H2 production in mixed cultures are phylogenetically diverse. Hydrogenotrophic methanogens and homoacetogens are the most common H2 consuming microorganisms (Cabrol et al., 2017). Examples of hydrogenotrophic methanogens, which use H2 as the major electron donor to reduce CO2 and produce methane, include methanoarchaea in the order Methanobacteriales and Methanomicrobiales (Chaganti et
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al., 2012). Homoacetogens are strict anaerobes, which can consume H2 and CO2 to form acetate and include e.g. certain Clostridium spp. such as C. aceticum, C. thermoautotrophicum, C.
thermoaceticum and C. stercorarium (Guo et al., 2010; Ueno et al., 2006). Their growth is typically favored at acidic pH and long hydraulic retention time ( Guo et al., 2010; Ren et al., 2007).
Microorganisms that compete with the organic substrate and are not able to produce H2 include for example lactic acid bacteria and nitrate-reducing bacteria (Bundhoo and Mohee, 2016; Cabrol et al., 2017; Ghimire et al., 2015; Mizuno et al., 1998; Wang and Wan, 2009). The presence of such microorganisms in the mixed cultures can lead to low H2 yields and increase in the formation of soluble metabolites.
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Table 2.1 Dark fermentative H2 yields obtained with selected pure cultures under different growth conditions.
Microbial species Mode Substrate Temperature (°C) pH Substrate concentration (g L-1)
H2 yield (mol H2
mol-1 glucose) Reference
Mesophiles
Enterobacter aerogenes Batch Glucose 38 6.5 10 1.0 Yokoi et al., 1995
Enterobacter cloacae Batch Glucose 36 6 10 2.2 Kumar and Das,
2000
Clostridium beijerinckii Batch Glucose 35 7.2 3 2.8 Lin et al., 2007
Clostridium butyricum Batch Glucose 36 7.2 3 2.3 Lin et al., 2007
Clostridium butyricum Batch Palm oil mill
effluent 37 5.5 15─100 0.22 Chong et al., 2009a
Thermophiles
Caldicellulosiruptor saccharolyticus
Batch Glucose 70 7 10 3.4 Budde et al., 2010
Batch Miscanthus
hydrolysate 72 7 28 2.4 De Vrije et al., 2009
Clostridium thermocellum Batch Cellulose 60 6.8 1 1.9 Islam et al., 2009
Thermoanaerobacterium
thermosaccharolyticum Batch
Glucose 60 6.5 10 2.4 O-Thong et al., 2008
Xylose 60 6.5 10 2.6 Ren et al., 2008
Thermotoga neapolitana Batch
Xylose 75 7 5 3.4 Ngo et al., 2012
Glucose 75 7 10 3.5 De Vrije et al., 2010
Carrot pulp
hydrolysate 75 7 10 2.7 De Vrije et al., 2010