Análisis detallado de inundaciones

Dark fermentation is the conversion of organic substrates to bio-H2 through a series of biochemical reactions by anaerobic bacteria in the absence of light (Figure 2.4). In comparison to other bio-H2 production methods, dark fermentation is a promising technology (Levin et al., 2004). Dark fermentation is an intermediate step in the anaerobic digestion process and involves multiple series of oxidation and reduction reactions (Pavlostathis and Giraldo, 1991). Anaerobic digestion involves 4 major steps: hydrolysis; acidogenesis; acetogenesis; and methanogenesis (Figure 2.4).

Complex Organic Matter (Carbohydrates, Proteins and Fats)

Soluble organic molecules (sugars, amino-acids, fatty acids)

Volatile Fatty Acids

Acetic Acid Methane+CO2 Hydrogen+ CO2 1 2 3 4 4

Figure 2.4 Schematic representation of anaerobic digestion pathway (Erickson and Fung, 1988)

2.4.1.1 Hydrolysis

During this step, all of the macro-molecules of lignocellulosic or carbohydrate polymers are broken down into monomeric or fermentable sugars. The rate of hydrolysis is mainly dependent on the particle size, the composition of the biomass material and the conditions under which hydrolysis take place (Sanders, 2001). Hydrolysis is carried out by obligate or facultative anaerobes, which convert the biomass into a soluble form that can be assimilated by the fermenting organisms (Gerardi, 2003).

28 2.4.1.2 Acidogenesis

Fermentable sugars are degraded into liquid byproducts such as volatile fatty acids (VFAs), alcohols and gaseous products including H2 and carbon-dioxide (CO2). The VFAs produced at this stage are diverse and typically include succinic acid, lactic acid (HLa), HAc, HPr, and HBu. There are a variety of factors that may affect acidogenesis, such as: pH, temperature, substrate composition, inoculum source and type, and HRT in the case of continuous operating systems (Banerjee et al., 1998; Zhang et al., 2005). The acidogenesis reaction is carried out strictly by anaerobes that are not tolerant to oxygen; however, some facultative anaerobes can utilize trace amounts of oxygen. The most common genera that include acidogens are Clostridium, Pseudomonas, Bacillus, Micrococcus, Flavobacterium and Enterobacterium (Ziemiński and Frac, 2012).

2.4.1.3 Acetogenesis

During acetogenesis, organic compounds having more than two carbons are degraded to HAc. Acetic acid is not only produced from compounds with multiple carbon atoms, but also from a molecule with a single carbon atom, in which CO2 and H2 produced during acidogenesis are used to form HAc. Acetogenesis by obligate proton-reducing bacteria is thermodynamically favorable under low partial pressure for H2 (pH2) (Khanal, 2011). Formation of H2, CO2 and HAc (although formate is found in a few cases) from the degradation of VFAs containing longer carbon chains lowers the pH levels and enhances the H2 production (Denac et al., 1988). The syntrophic relationship between H2- consuming methanogens, H2-consuming acetogens and H2 producers, assist in maintaining the low pH2 and a balance in the system making thermodynamically favorable conditions for the fermentation reactions to proceed (Schink, 1997). The HAc produced by homoacetogens includes two types: one type grows autotrophically using H2 and CO/CO2 and the other heterotrophically by producing HAc from organic compounds (Ryan et al., 2008). Thus, acetogenesis and acidogenesis are the two steps in anaerobic digestion during which H2 is produced.

2.4.1.4 Methanogenesis

Methanogenesis is the final stage of anaerobic digestion where methane is the end product. Methane is primarily produced from H2 and CO2 (hydrogenotrophic

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methanogens) or from HAc (aceteclastic methanogens). The methane-producing bacteria belong to the Archaea class of microorganisms that are sensitive to oxygen and live in a syntrophic relationship with acetogens. Methanogens are affected by pH, high levels of VFAs produced during acidogenesis and acetogenesis, and the amount of H2 produced (Zeikus, 1977).

2.4.2 Electron flow in the metabolic pathway for hydrogen production through dark fermentation

The metabolic mechanisms for dark fermentation are derived from anaerobic digestion. Hydrogen production arising from dark fermentation takes place if organic carbon is available as an energy source for the microflora. Dark fermentative H2 production is preferred for bio-fuel production because of its high HPR (Levin et al., 2004). The stoichiometric reaction Equation 2.1 explains how H2 is produced from glucose metabolism when HAc is the end product. The maximum possible H2 yield per mole of glucose is 4 mol corresponding to only 33% of the substrate conversion. However, in practice, attaining this theoretical maximum yield is not possible.

C6H12O6 + 2H2O → 2CH3COOH + 4H2 + 2CO2 (2.1) When the end product is HBu (Equation 2.2), 2 mol H2 is produced: C6H12O6 + 2H2O → CH3CH2CH2COOH + 2H2 + 2CO2 (2.2)

These stochiometric equations reveal that the HAc/HBu ratio controls the maximum H2 yield possible, and that acetogenic fermentation is preferred over HBu fermentation. Furthermore, low yields are characteristic of HPr, HLa or EtOH fermentation (Azbar and Levin, 2012; Levin et al., 2004). A description of glucose metabolism in the following sections describes the different stochiometric reactions.

The metabolic pathway for glucose degradation via anaerobic fermentation is described in Figure 2.5. The pathway integrates the formation of an intermediate, pyruvate, by glycolysis during the breakdown of complex sugars. The pathway shows that H2 can be produced from pyruvate decarboxylation where electrons are transferred to ferrodoxin (Fd). In subsequent a reaction, the reduction of protons (hydrogen ions, H+) takes place resulting in the release of H2 gas (Jungermann et al., 1973; Saint-Amans et

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al., 2001). In this pathway, the NADH formed from glycolysis is used in the formation of H2, releasing the oxidized form of NAD+. Equation 2.3 and 2.4 represent the formation of pyruvate and NADH through glycolysis, following which evolution of H2 via oxidation occurs.

C6H12O6 + 2NAD+ → 2CH3COCOOH + 2NADH + 2H+ (2.3)

NADH + H+ → H2 + NAD+ (2.4) Glucose 2-Pyruvate 2 Acetyl-CoA Acetoacetyl-CoA Butyryl-CoA Acetone Propanol or iso-propnaol Butanol Butyrate Ethanol 2 Acetate Lactate Propionate 2 Formate + 2 Acetyl-CoA 2 NAD+ 2 NADH 2 NAD+ 2 NADH 2 NAD+ 4 Fd2+ 4 Fd+ 2 H2 4 NADH 4 NAD+ 2 NADH 2 NAD+ 2 NADH 2 NAD+ 1 ATP 2 ATP 2 ATP 2 NAD+ 2 NADH NAD+ _NADH N H B S Q R C A F G E D P O I J M K L

Figure 2.5 Simplified metabolic pathway for glucose degradation by Clostridium sp.* (Adapted from Jones and Woods (1986) and Chaganti et al. (2011))

Notes: *Enzymes are indicated as follows: (A) hydrogenase; (B) pyruvate-ferredoxin oxidoreductase; (C) NADH-ferredoxin oxidoreductase; (D) phosphate acetyltransferase; (E) acetate kinase; (F) acetaldehyde dehydrogenase; (G) ethanol dehydrogenase; (H) thiolase; (I) acetoacetate decarboxylase; (J) isopropanol dehydrogenase; (K) 3- hydroxybutyryl-CoA dehydrogenase; (L) butyryl-CoA dehydrogenase; (M) phosphate butyryltransferase; (N) butyrate kinase; (O) butyaldehyde dehydrogenase; (P) butanol dehydrogenase; (Q) lactic dehydrogenase; (R) Propionate dehydrogenase; and (S) Pyruvate formate lyase.

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The metabolic pathway is based on the intermediate, pyruvate, from which the pathway branches to different intermediates in anaerobic fermentation. The major fermentation products in the gaseous phase includes H2, CO2 and CH4 while soluble metabolites in the liquid includes HAc, HBu, HLa, EtOH and butanol (Hawkes et al., 2002; Zhou et al., 2007). Figure 2.5 shows the metabolites formed and the enzymes involved at each step of the metabolic pathway. Electron/carbon flow from glucose to other metabolites (e.g. H2, HAc, HBu, HPr, etc.) occurs and the NADH2 produced is balanced with the NADH2 consumed in this pathway.

From pyruvate, the metabolic pathway proceeds into two different branches which are distinguished by the nature of the associated bacterial system (i.e., enteric or Clostridial). In the enteric bacterial system, pyruvate is broken down to acetyl-CoA and formate by pyruvate formate lyase (S in Figure 2.5). The latter metabolite (formate) is then converted to H2 and CO2 by formate hydrogenase. The former metabolite (acetyl-CoA) is used for acetic acid production via substrate level phosporylation (D and E in Figure 2.5) and regeneration of NAD+ to maintain glycolysis. However, the NAD+ regeneration directly from pyruvate which is also possible under acidic conditions by lactate dehydrogenase results in low H2 yields. The regeneration of NAD+ via non-H2 producing reactions (such as HLa, EtOH, and butanol formation as shown in Figure 2.5 and described by equations 2.5, 2.6 and 2.7) in enteric bacterial systems results in H2 yield less than 2 mol H2 mol-1 glucose, which is only 50% of the theoretical maximum (Hallenbeck, 2005).

CH3COCOOH+ NADH+ H+ → CH3CHOHCOOH+ NAD+ (2.5) CH3COCOOH+ NADH+ H+ → CH3CH2OH+ CO2+ NAD+ (2.6) 2CH3COCOOH+ 2NADH+ 2H+ → CH3(CH2)2CH2OH+2CO2+ H2O+ 2NAD+ (2.7) In Clostridial bacterial systems, pyruvate is broken down into acetyl-CoA and reduced ferredoxin (Fd+) by a pyruvate ferrodoxin oxidoreductase (B in Figure 2.5) (Hallenbeck, 2005; Zajic et al., 1978). The Fd+ is then oxidized to ferrodoxin (Fd2+) and the associated electron transfer through hydrogenase activity (A in Figure 2.5) results in evolution of H2 from the electron acceptance of a proton (hydrogen ion, H+). Acetyl-CoA

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produced by the Clostridial system is further degraded to HAc and HBu via ATP generation. The maximum H2 production that is accompanied by HAc and HBu formation is described by the stochiometric reactions (Equations 2.1 and 2.2). The production of other reduced metabolites (such as HLa, HPr, butanol and EtOH) is also important in this pathway for maintaining the balance through which NAD+ regeneration occurs (Nandi and Sengupta, 1998).

The NADH consumption is marked by HPr and HLa formation. The formation of these by-products is essential for balancing the NADH produced during glycolysis, since the acceptance of electrons by protons (H+ ions) is affected by the corresponding levels of acetyl-CoA and NADH (Lee et al., 2011). In glycolysis, 2 mol of NADH are produced for every mole of glucose consumed and 2 mol of Fd+ is produced during pyruvate decarboxylation. Maximum H2 production is determined by the mechanism in which NADH is recycled through the conversion of pyruvate to fermentation products (Manish et al., 2007). In theory, a maximum of 4 mol of H2 can be produced if HAc is the end product, but in actuality such an ideal state cannot be achieved because of the fact that the accumulation of H2 affects the activity of the hydrogenase enzyme and the types of electron carriers present in the metabolic pathway.

As shown in Figure 2.5, the electron flow from acetyl co-A is diverted to HAc and EtOH, and then to HBu and butanol through butyryl Co-A. Note, the electron source is NADH in the case of EtOH and butanol. The depiction of the metabolic pathway shows that the H2 is produced via the Fd:hydrogenase system, implying that the reduction of ferrodoxin (Fd2+ to Fd+) is the sole electron source for proton reduction and the release of H2. The presence of reduced ferrodoxin (Fd+) is based on electron flow from the pyruvate node. The electron equivalent (e- eq) of H2 measured and its e- eq relative to the e- eq of reduced ferrodoxin determines the direction of electron flow between NAD+/NADH and the Fd2+(oxidized)/Fd+(reduced) pools (Lee et al., 2009a).