Glycerol, as carbon and energy source has been used for production of H2 and different biochemical
products. Glycerol metabolisms are a dismutation process occurring through coupled oxidative and reductive pathways [100]. Both oxidative and reductive of glycerol are known for different species [101,102]. As shown in Figures 2.8 and 2.9 in the oxidative pathway, glycerol is first converted to dihydroxyacetone with the formation of NADH2 [103]. This intermediate is then phosphorylated by
the glycolytic enzyme dihydroxyacetone kinase. Finally, the phosphorylated product is metabolized through glycolysis to pyruvate which then may be oxidized to different end-products [101]. Ethanol, butanol, 2,3-butanediol, acetate, butyrate and lactate are some of the possible metabolites of the oxidative metabolism of glycerol (Figure 2.8 and 2.9) [15,101,104-106]. The glycerol bioconversion pathway to H2 is based on a simple redox reaction: 2H+ + 2e− ↔ H2 [107]. Enzymes that emanate
from hydrogen producing organisms catalyze this reaction. Three of the main such enzymes are nitrogenases, [NiFe]-hydrogenases, and [FeFe]-hydrogenases [107,108]. As it is shown in Figure 2.8, the oxidative metabolism of glycerol, first pyruvate is produced and then converted to different metabolites and H2 via different pathways. Pyruvate is broken down to acetyl-CoA via reduction of a
ferredoxin (Fd) catalyzed by pyruvate ferredoxin oxidoreductase. Reduced ferredoxin (Fd) is then oxidized by a hydrogenase that reproduces oxidized Fd and hydrogen gas [107,109].
In the reducing pathway, glycerol is finally converted to 1,3-PDO via production of the intermediate product 3-hydroxypropionaldehyde. Conversion of glycerol to 3-hydroxypropionaldehyde is catalyzed by B12-dependent glycerol dehydratase and related diol dehydratases, which is then
reduced to 1,3-PDO by 1,3-propanediol dehydrogenase [101,110,111]. For species unable to synthesize 1,3-PDO, such as E. coli, the reductive pathway takes place through a respiratory pathway that requires an external electron acceptor. Alternatively, Gonzalez et al. [112] and Ko et al [109] reported that 1,2-PDO can be synthesized from the glycolytic intermediate dihydroxyacetone- phosphate (DHAP) in E. coli.
C H O + H O3 8 3 2 CH COOH (Acetic acid ) + CO + 3H3 2 2 C H O 3 8 3 C H OH (Ethanol) + CO + H2 5 2 2
2C H O 3 8 3 C H O (Butyric acid ) + 2CO + 4H4 8 2 2 2 2C H O3 8 3 C H4 10O (Butanol) + 2CO + H O + 2H2 2 2
Equation (2.2)[84]
Equation (2.2) Stoichiometric equations showing hydrogen yield during glycerol bioconversion. From Equation (2.2), a theoretical maximum of 3 mol H2 can be produced per mole of glycerol when
acetate is the fermentation end product. However, only 2 or 1 mol H2 per mol glycerol can be
generated during butyrate and ethanol production respectively. For reduced end-products such as diols and lactic acid H2 generation can be even lower [107, 113].
Figure 2.8. Biochemical pathways of glycerol fermentation of representative microorganism (from
[89,100].
2.2.7.2 Enzymes and genes involved in metabolic path way for glycerol uptake by bacteria
A number of microorganisms can grow anaerobically on glycerol as the sole carbon and energy source. Klebsiella spp., Citrobacter spp., Clostridium spp., and Enterobacter spp. metabolize glycerol both oxidative and reductive [114]. Constructing and identifying the genes and enzymes evolved in metabolitic pathways is a very important step for the metabolic engineering and to understand its biochemistry.
Figure 2.9 shows general biochemical pathways for glycerol fermentation. During this process, glycerol is dehydrogenated to dihydroxyacetone which then can be converted (after phosphorylation) to pyruvate. This then enters to the glycolysis catabolism pathway. This process is regulated by GldA dehydrogenase and DHAK dihydroxyacetone kinase for obtaining ethanol, succinate, acetate, and formate (Figure 2.9) [67].
In general terms, the enzymes involved in the pathways for glycerol conversion to glycolytic intermediates (i.e., GlpK-GlpD and GldA- DHAK) and the enzyme involved in the pathway for D-lactic acid synthesis from pyruvic acid are i.e., D-lactate dehydrogenase [67].
In Klebsiella, Citrobacter, Clostridium, Enterobacter, and E. coli glycerol is metabolized both oxidatively and reductively [114]. In the oxidative pathway, the NAD+-dependent enzyme glycerol dehydrogenase (EC 1.1.1.6) catalyzes the conversion of glycerol to dihydroxyacetone and the glycolytic enzyme dihydroxyacetone kinase (EC 2.7.1.29) phosphorylates the latter product [104- 106], which is then funneled, to glycolysis. The reducing pathway is catalyzed by coenzyme B12- dependent glycerol dehydratase (EC 4.2.1.30) and related diol dehydratases (EC 4.2.1.28) [115-117], converting glycerol to 3- hydroxypropionaldehyde [118-120], and by the NADH+ H+-dependent enzyme 1,3-propanediol dehydrogenase (1,3-propanediol-oxydoreductase, EC 1.1.1.202), reducing 3-hydroxypropionaldehyde to 1,3-propanediol and regenerating NAD+ [106, 110,111, 121, 122] (Figure. 2.9). The final 1,3-propanediol (1,3-PDO) product is highly specific for glycerol fermentation and cannot be obtained from any other anaerobic conversion [123,124].
In K. pneumoniae (Forage and Lin, 1982) and C. freundii, the genes encoding the functionally linked activities of glycerol dehydratase (dhaB), 1,3-PDO dehydrogenase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK) are encompassed by the dha regulon [114] (Fig. 2.9). The 1,3-PDO operon of C. butyricum is composed of three genes, a different type of glycerol dehydratase (dhaB1), its activator protein (dhaB2) and dhaT [125]. In this bacterium, glycerol dehydratase is extremely oxygen sensitive, strongly associated with the cell membrane and vitamin- B12 independent [43, 69, 87, 125-127]. Fermentative production of 1,3-propanediol (PD) under
anaerobiosis takes place in two parallel ways. First, a fraction of glycerol is oxidezed by glycerol- dehydrogenase (Glyc-DH) to dihydroxy-acetone (DHA), and then phosphorrylated by DHA kinase to enter glycollysis. The remaining glycerol is then dehydrated to 3-hydroxypropionaldehyde (3HPA) by glyceroldehydratase, where reduction continues by propanedioldehydrogenase (PPD-DH) and by a dependent NAD oxidorreductase to 1,3- propanediol [128,129].
Fermentation from glycerol to ethanol or butanol by C. pasteurianum does not depend on the formation of by-products [130], since hydrogen carriers are completely regenerated in the pathway [89]. Another example of a redox-balanced process is the conversion of glycerol into succinic acid. Although the pathways for ethanol and succinate are equivalent regarding the overall redox balance, the energetic contribution of the ethanologenic pathway is much higher, as 1 ATP is produced per each molecule of glycerol converted into ethanol, while production of energy in the succinate
pathway is limited to the potential generation of a proton motive force by fumarate reductase [57] (Figure. 2.8). Such a complication can be effectively overcome by the use of microaerobic conditions. ATP will be gained through oxidative phosphorylation resulting from the reducing equivalents generated during the utilization of glycerol, including those generated by the incorporation of glycerol into cell mass (i.e. cell mass is less reduced on average than glycerol) [131] (See Figure 2.8). However, inducing microaerobic can ultimately reduce the H2 production.
Figure 2.9. Glycerol conversion overview Figure 1 b) Metabolic pathways to 1.2- Propanediol(1,2-PD) and 1, 3-propanediol (1,3-PD) from dihydroxyacetone (DHAP), a common intermediate of sugar metabolism [15,70]. Glycerol Glycerol sn-Glycerol 3-P sn-Glycerol 3-P Dihydroxyacetone 1.1.1.6 Pyruvate 3-hydroxypropionalde hyde 1,3 propanediol ATP ADP + Pi 2.7.1.30 H2O 4.2.3.3 1.1.1.7 1.1.1.202 4.2.1.30 ADP ATP NAD+ NADH + H+ 1.1.1.94 NAD+ NADH + H+ 1.1.5.3 Dihydroxyacetone-P 2.7.1.29 ADP ATP Gluconeogenes is 2.7.1.121 PEP PYR quinone quinol Acetyl-CoA Acetol Lactaldehyde Methylglyoxal ADP 1,2 propanediol 2[H] 2[H] NADH + H+ NAD+ Lactate 1.2.1.- 1.1.1.283 1.1.1.- 1.1.1.78 -.-.-.- NAD+ NADH + H+ 2Fdox 2Fdre d NADH + H+ NAD+ Lactate 1.1.1.27 NADH NAD+ 2Fdre d 2Fdox H+ 2H+ H2 H2 NADH + H+ NAD+ CO2+ H2 2.3.1.54 1.2.7.1 2NADH + H+ 2NAD+ Ethanol Acetate 1.2.1.10 1.1.1.1