Fermentation reactions can be carried out at mesophilic (25-40°C), thermophilic (40-65°C), extreme thermophilic (65-80°C), or hyperthermophilic (>80°C)

Table 13.4 Actual wastes/wastewaters used for fermentative hydrogen production Type of waste/ wastewater Microorganisms Operation mode H2 production rate Maximum H2 yield References Rice winery wastewater Mixed culture Continuous 9.33 L/gVSS/d 3.81 L/L/d 2.14 mol/mol hexose Yu etal., 2002 Sugar factory wastewater Mixed thermophilic culture Continuous 4.4 L/L/d 2.6 mol/mol hexose Ueno ef a/., 1996 Potato processing wastewater Mixed mesophilic culture Batch 2.8 L/L wastewater Van Ginkel etal., 2005 Olive pulp Mixed mesophilic culture Continuous 0.26 L/L/d 0.19 mole/kg TS Koutrouli etal.,2009 Cheese whey Mixed mesophilic indigenous microbial culture Continuous 2.51 L/L/d 0.9 mol/mol hexose Antonopoulou etal., 2008a Dairy wastewater Mixed mesophilic culture Continuous 1.59 mmol H2/L/d Venkata Mohan etal., 2007 Molasses Mixed mesophilic culture Continuous 4.8 L/L/d Ren ef at., 2007 Food waste — sewage sludge Mixed mesophilic culture Batch 2.67 L/gVSS/d 122.9 mL/g COD carbohydrate Kim ef at., 2004 Food waste Mixed thermophilic culture Batch 0.28 8 L/gVSS/d 1.8 mol/mol hexose Shin ef al., 2004 OFMSW Mixed mesophilic culture Batch 0.4 L/g VSS/d 0.15 L/g OFMSW Lay ef al., 1999 OFMSW: organic fraction of municipal solid wastes

Crops/residues Microorganisms Operation mode H2 production rate Maximum H2 yield References Corn starch Mixed mesophilic cultures Continuous 2.57 L/L/d 0.51 mol/mol hexose added Arooj et at., 2008 Wheat starch Mixed mesophilic cultures Continuous 1.26 mol/mol hexose Hussy et ai, 2003 Sweet sorghum extract Indigenous microbial mesophilic culture Continuous 8.52 L/L/d 0.86 mol/mol hexose Antonopoulou et ai, 2008b Sweet sorghum extract Rumicococcus albus Batch 2.61 mol/mol hexose Ntaikou et al., 2008 Sweet sorghum residues Rumicococcus albus Batch 2.59 mol/mol hexose Ntaikou et ai, 2008 Wheat straw Caldicellulosiruptor saccharolyticus Batch 3.8 mol/mol glucose (44.7 L/kg dry biomass) Ivanova et ai, 2009 Maize leaves Pretreatment: enzymatic hydrolysis Caldicellulosiruptor saccharolyticus Batch 3.6 mol/mol glucose (81.5 L/kg dry biomass Ivanova et ai, 2009 Corn stalks Pretreatment: acid hydrolysis Mixed mesophilic cultures Batch 0.1824 L/d 0.150 L/kg TVS Zhang et ai, 2007a Corn stover Pretreatment: acid hydrolysis Thermoanaerobacterium thermosaccharolyticum W16 Batch 3.305 L/d 2.24 mol/mol hexose Cao et ai, 2009 Sugarcane bagasse hydrolysate Pretreatment: acid hydrolysis Clostridium butyricum Batch 1.611 L/L/d 1.73 mol/mol total sugar Pattra et ai, 2008

temperatures. Hydrogen production could be achieved either by using pure cultures of hydrogen producing bacteria grown in the dark on carbohydrate-rich substrates or by mixed acidogenic microbial cultures, selected by natural environments such as soil, wastewater sludge, and compost. At a full-scale application, a mixed culture system would be cheaper to operate, easier to control, and would have a broader choice of feedstocks (Valdez-Vazquez et al., 2005). In Tables 13.4 and 13.5 the different feedstocks used by pure or mixed microbial cultures in lab — scale experiments are presented, since data from full scale applications are not available so far.

Hydrogen production is a specific mechanism to dispose of excess electrons through the activity of the enzyme hydrogenase in bacteria. Bacteria that possess such capability include strict anaerobes (Clostridia, methylotrophs, rumen bacteria, methanogenic bacteria, archaea), facultative anaerobes (Escherichia coli, Enterobacter, Citrobacter), and even aerobes (Alcaligenes, Bacillus). Figure 13.4 presents the morphology of fermentative bacteria selected from a hydrogen producing reactor, at pH 5.5. Among the hydrogen-producing bacteria, Clostridium sp. and Enterobacter, are the most widely studied. Species of genus Clostridium such as C. butyricum (Chong et al., 2009), C. acetobutyricum (Lin et al., 2007), C. beijerinckii (Lin et al., 2007), C. thermolacticum (Collet et al.,

2004) , C. tyrobutyricum (Jo et al., 2008), C. thermocellum (Levin et al., 2006) and C. paraputrificum (Evvyernie et al., 2000) are examples of strict anaerobic and spore-forming microorganisms, generating hydrogen gas during the exponential

growth phase. In parallel, facultative anaerobes such as the species of genus E. coli and its modified strains (Manish et al., 2007) and the species of genus Enterobacter, such as E. aerogenes (Tanisho and Ishiwata, 1994; Yokoi et al., 2001) and E. cloacae (Kumar and Das, 2001) have also been used for hydrogen production. In recent years, extensive research has also been carried out in hydrogen production at high temperature, using thermophilic or hyperthermophilic bacteria, since the increase of temperature favours the reaction kinetics. The thermophiles include Caldicellulosiruptor saccharolyticus (van Niel et al., 2002), Thermoanaerobacterium sp. such as T. thermosaccharolyticum (O-Thong et al., 2008) and Thermotoga sp. such as Thermotoga maritima (Schroder et al., 1994) and Thermotoga elfii (de Vrije et al., 2002).

Degradation of glucose (or its isomer hexoses or its polymers, starch, glycogen and cellulose) by mixed microbial culture, under anaerobic conditions is accompanied by the production of hydrogen and various metabolic products, mainly volatile fatty acids ((VFAs) acetic, propionic, and butyric acid), lactic acid, and alcohols (butanol and ethanol), depending on the microbial species present and the prevailing conditions. The hydrogen yield can be correlated stoichiometrically with the final metabolic products, through the reactions describing the individual processes of acidogenesis:

For complex substrates, the hydrogen production could also be expressed in terms of hydrogen productivity (HP) which is defined as the percentage of influent substrate electrons which are distributed to hydrogen gas (gaseous and dissolved phases) (Kraemer and Bagley, 2005). It is obvious that the production of acetic and butyric acids favors the simultaneous production of hydrogen, with the fermentation of glucose to acetic acid giving the highest theoretical yield of 4 mol of H2/mol of glucose (HP = 33%) (reaction [13.7]) and the conversion to butyric acid resulting in 2 mol of H2/mol of glucose (HP = 17%) (reaction [13.8]), while the production of propionic acid consumes hydrogen (reaction [13.9]).

From the reactions [13.7], [13.8] and [13.9], it is obvious that the metabolism should be shifted towards acetate and/or butyrate production in order to achieve a high hydrogen yield. Clostridia sp. produce a mixture of acids, with butyrate in excess of acetate, upon biological degradation of glucose (Mizuno et al., 2000; Fang and Liu, 2002). In practice, the production of more metabolic products (lactate or ethanol), accompanied by a negative or zero hydrogen yield, results in lower overall yields of hydrogen (HP: 10-20%). Moreover, the metabolism towards acetate may occur via different, non-hydrogen-yielding pathways. In mixed fermentation processes, the microorganisms may select different pathways while converting sugars, as a response to changes in their environment (pH, sugar concentration, etc.). The absence or presence of hydrogen-consuming microorganisms in the microbial consortium also affects the microbial metabolic balance and consequently, the fermentation end products.

In order to harness hydrogen from a fermentative hydrogen production process, the mixed cultures need to be pre-treated in order to suppress as much hydrogen­consuming bacterial activity as possible, while still preserving the activity of the hydrogen-producing bacteria. The pre-treatment method is achieved mostly by relying on the spore-forming characteristics of the hydrogen-producing Clostridium, which is ubiquitous in anaerobic sludge and sediment (Brock et al, 1994). Treating an anaerobic sludge under harsh conditions, Clostridium would have a better chance to survive than the non-spore-forming bacteria, many of which are hydrogen consumers (Lay, 2001). Effective pre-treatment processes include heating (100°C, 15 minutes), acidic (pH = 3, adjusted with ortho-phosphoric acid, 24 hours) or basic treatment, aeration, chemicals addition (chloroform, acetylene), and application of an electric current (3-4.5 V). Another approach involves the use of the indigenous mixed microbial culture already contained in a wastewater through its activation for one day at mesophilic temperatures, a practice that has been applied and proposed by Antonopoulou et al. (2008a; 2008b). The most widely used pre-treatment method for enriching hydrogen — producing bacteria from

mixed microbial inocula is heat — pre-treatment, which combines the simplicity with the effectiveness, securing that Clostridium sp. will survive.