absolute value of activation energy in the temperature of range of 34o–40oC corresponds to the activation energy of thermal deactivation or denaturation of the intracellular enzymes.
On the basis of these results, we have defined two regimes for thermodynamic analysis, viz. fermentation regime (in the range of 23°–34°C) and deactivation regime (in the range of 34°–40°C). Figs. 2.7C and D show the Eyring plot for pure and crude glycerol in the two regimes. Ea for crude glycerol for fermentation regime is significantly lower than pure glycerol, which indicates easier and faster reactivity requiring lesser energy input. The values of thermodynamic parameters also corroborate this conclusion. Crude glycerol has lesser ∆H and ∆G values than pure glycerol indicating smaller energy input required for bioconversion. The positive values of ∆S (entropy change) for biohydrogen production from both pure and crude glycerol is indicative of marginal rise in the randomness of the tertiary structure of enzyme during formation of enzyme–substrate complex (or formation of the activated or transition state) during the reaction. Ea and ∆G values for crude glycerol in the thermal deactivation regime are higher than pure glycerol. These values indicate greater resistance and stability of the intracellular enzymes towards deactivation with crude glycerol as substrate. ∆H and ∆S in the thermal deactivation regime are negative for both pure and crude glycerol, which indicates disruption of the secondary structure of the enzyme and the concurrent reduction in the tendency for enzyme-substrate complex formation.
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study is higher yield of hydrogen using crude glycerol as compared to pure glycerol. This result has also been reported in previous studies by (Lo et al., 2013; Mangayil et al., 2015).
This result is quite a typical, as one would expect the impurities in crude glycerol to inhibit the fermentation leading to lower yields of biohydrogen. Indeed, lowering of the yield of other fermentation products such as 1,3– propanediol and butanol using Clostridium pasteurianum due to impurities present in crude glycerol has been reported in previous studies (Venkataramanan et al., 2012; Khanna et al., 2012; Khanna et al., 2013). The main impurities in crude glycerol are short chain alcohols (usually methanol or ethanol), alkali (NaOH/KOH), inorganic salts (NaCl/Na2SO4/KCl/K2SO4 etc.) and unsaturated fatty acids.
Each of these impurities has effect on growth and metabolism of Clostridia. Previous studies have investigated the effect of impurities in glycerol on fermentation (in terms of yield/selectivity of products) using different microbial cultures (Venkataramanan et al., 2012; Xiaolong et al., 2006; Cao et al., 2009; Khanna et al., 2011; Chatzifragkou et al., 2012; ). In the context of present study, the influence of various impurities on hydrogen production vis–à–vis their concentrations in crude glycerol is discussed below:
Alcohols and unsaturated fatty acids: Alcohols are known to enhance the fluidity of the membrane. Ingram et al. (1976) has reported that bacterial membrane fluidity is significantly affected by alcohol only for concentrations ≥ 1% w/v. In the present study, the concentration of alcohol in glycerol is 2.6 g/L (or 0.26% w/v), which is far lower than the limit reported (Ingram et al., 1976). Hence, influence of alcohol impurity on glycerol metabolism is expected to be minimal. Venkataramanan et al. (2012), have given an account of the effect of fatty acids on growth/ metabolism of Clostridia. As a result, the diffusive mass transfer is affected. More unsaturated fatty acids containing two or more double bonds create hindrance to mass transfer of nutrients and metabolites through the
adverse effect of fatty acids is expected to be minimal, as the substrate used for transesterification was refined soybean oil, with very little content of fatty acids.
Alkali metals, salts and ions: Monovalent salts can cause swelling of the cell membrane due to weakening of Vander Waal’s forces between lipid tails of the membrane. This affects the energy barrier within lipid bilayer of the cells, which results in obstruction of substrate transport across the membrane. In the present study, the probability of formation of metal salts is rather miniscule due to pure version of the chemicals used.
The alkali contamination in glycerol is a source of sodium ions, which have marked effect on biohydrogen production, as reported by previous studies. Cao and Zhao (2009) have reported enhancement in hydrogen concentration and yield in anaerobic fermentation of food waste in presence of sodium ions. Typically, sodium ion concentrations below 14 g/L promoted biohydrogen synthesis. Cao and Zhao (2009) have proposed that sodium can build a Na–K–ATP enzyme pump to transfer nutrients and substrates such as glucose or glycerol to the intracellular region to improve the bioreactions leading to biohydrogen production. This is manifested in terms of reduction in activation energy, increase in Vmax and 1st order kinetic constant. Impurities in crude glycerol, however, may affect the intracellular enzymes in glycerol metabolism making them more susceptible to inhibition, which is reflected in terms of reduction in KI. The inhibition substrate concentration (24.93 g/L) predicted by the Haldane kinetic model for crude glycerol is significantly higher than the optimum concentration (7.4 g/L) predicted by the CCD experimental design, and hence, substrate inhibition effect is expected to be minimal for crude glycerol fermentation at optimum conditions.
Xiaolong et al. (2006) have studied effect of sodium ion concentration on hydrogen production from sucrose. The optimum sodium ion concentration reported by Xiaolong et
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reduction in hydrogen production for Na+ concentrations above 4 g/L. This reduction is attributed to adverse effect of high Na+ concentration on microbial enzyme activities such as dehydrogenase activity, alkaline phosphatase and adenosine tri phosphate. Khanna et al.
(2011) have assessed the effect of sodium ions on hydrogen production. Sodium ion concentration up to 250 mM has been found to be beneficial for metabolism and growth of hydrogen producing bacteria. The maximum Na+ concentration in fermentation mixture in present study is 35.2 mM, which is below the limit of 250 mM given by Cao and Zhao (2009) and Khanna et al. (2011). The Na+ ions contributed by alkali in crude glycerol can help enhance biohydrogen production, as compared to pure glycerol.