Prior to main experiments for biohydrogen production, initial or preliminary experiments have been conducted for determination of following parameters: (1) incubation time, (2) inoculum size, (3) incubation temperature, (4) initial pH of fermentation, (5) initial glycerol concentration. The approach used in these experiments is
“one variable at a time (OVAT)”. The results of these experiments have been used to design the main CCD experiments. Procedure and results of these preliminary experiments are described as follows.
2.2.5.1 Incubation period
Optimum incubation period for maximum H2 production was determined by conducting batch experiments with varying periods with crude glycerol (10 g/L) as the sole carbon source. H2 content in the gas phase increased from 8.6 ± 0.80% v/v at 4 h to 33.36 ± 0.98% v/v at 15 h (data not shown). Beyond incubation period of 15 h, the H2
content in the gas phase remained practically constant. On the basis of this result, the optimum incubation period was determined as 15 h.
2.2.5.2 Inoculum size
Batch experiments were conducted with varying inoculum sizes from an overnight–grown culture of Clostridium pasteurianum. Inoculum size of 1, 5, 10, 15, 20%
(v/v) was used to inoculate freshly prepared anaerobic BSH medium containing 10 g/L crude glycerol as substrate. Incubation was carried out for 15 h at 37°C and 150 rpm, and the gaseous phase was analyzed for H2 content after completion of incubation. The H2
content of gas phase increased from 22.5 ± 0.7% v/v for inoculum size of 1% to 32.76 ± 0.45% v/v for inoculum size of 10% (v/v). Thereafter, the H content showed slight
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reduction till inoculum size of 20% v/v. Fig. 2.1A depicts the variations in H2 content of product gas with inoculum size. A probable cause leading to reduction in H2 content for higher inoculum size is over consumption of the substrate (glycerol) that results in overgrowth of the cells with synthesis of other metabolites like acetate and butyrate.
Formation of the acidic side products in the medium results in rapid reduction of the pH (Zhao et al., 2011). Fig. 2.1B depicts the time profiles of pH of the reaction mixture for 5, 10 and 15% v/v inoculum. It could be inferred from Fig. 2.1B that the rate of reduction in pH varies proportionately with the inoculum size. The final pH attended at the end of 24 h fermentation for 5, 10 and 15% v/v inoculum is 5.9, 5.7 and 5.3, respectively.
Comparative analysis of biohydrogen yield and pH profiles reveals that hydrogen production is a strong function of the pH in the medium and shows an optimum with the pH. The optimum value of the inoculum was determined as 10% v/v as presented in Figs.
2.1A and 2.1B.
2.2.5.3 pH of fermentation medium
To determine the optimum value of this parameter, experiments have been conducted in serum bottles containing BSH media with initial pH in the range of 5 to 10.
Initial pH of the medium was adjusted by addition of 1 M NaOH or 1 N ortho–phosphoric acid. H2 content in the gas phase increased from 14.3 ± 0.33% v/v at initial pH of 5 to 31.65 ± 0.62% v/v at initial pH of 7. Gradual reduction in H2 content was observed on further increase in initial pH. These trends have been shown in Fig. 2.1C. Maximum H2
content of gas phase was obtained at initial pH 7, which is consistent with the previous literature (Zhao et al., 2011; Pan et al., 2008). Lower or higher pH of medium (or cell environment) results in a lower level of ATP in the cell, which in turn inhibits bacterial growth and enzyme activity resulting in reduction in H2 production (Bowles et al., 1985;
2.2.5.4 Incubation temperature
To evaluate effect of temperature on H2 production, batch tests containing BSH media were conducted at various temperatures (30°, 35°, 37°, 40° and 42°C) with other parameters at their optimum values: initial pH 7, crude glycerol concentration 10 g/L and shaking at 150 rpm. Maximum H2 content of 34.56 ± 0.44% v/v in the gas phase was observed at 37°C, which gradually decreased to 25.0 ± 0.71% v/v at 42°C, as depicted in Fig. 2.1D. The optimum temperature for H2 production was thus determined as 37°C, which is consistent with results of Zhao et al. (2011).
2.2.5.5 Initial glycerol concentration
Crude glycerol resulting from transesterification reaction for biodiesel synthesis was used as the sole carbon source (or substrate) for hydrogen production. The substrate concentration was varied in the range of 0 to 20 g/L (0.4, 0.8, 1, 2, 4, 6, 10, 15, 17, 20 g/L). The experiments were conducted in serum bottles at pH 7, 37°C and 150 rpm. H2
content of product gas was found to increase with crude glycerol concentration. The maximum H2 content of 37.76 ± 0.92% v/v in the gaseous phase was obtained for crude glycerol concentration of 6 g/L, which gradually reduced to 28.92 ± 1.1% v/v (reduction of 23%) at crude glycerol concentration of 20 g/L. The profile of H2 production with varying initial crude glycerol concentration is shown in Fig. 2.2A, which is consistent with results of Lo et al. (2013). Experiments have also been conducted with varying initial concentrations of pure glycerol as substrate, and the trends in H2 production are depicted in Fig. 2.2A. For pure glycerol, the highest H2 content of 31.01 ± 1% v/v in the gas phase was obtained for initial glycerol concentration of 10 g/L.
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(A) (B)
(C) (D)
Figure 2.1. Results of preliminary experiments. Effect of various parameters on hydrogen production in batch fermentation for initial crude glycerol concentration of 10 g/L. (A) inoculum size, (B) pH profiles for different inoculums size, (C) initial pH, (D) temperature.
(A)
(B)
Figure 2.2. (A) Effect of initial concentration (g/L) of crude and pure glycerol on hydrogen content of product gas. (B) Results of fitting of Haldane substrate–inhibition model to data of initial reaction velocities at different initial concentrations of pure and crude glycerol as substrate.
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