Chapter VI Simultaneous Ethanol and Hydrogen
6.2 Materials and Method
6.2.1 Preparation of Substrate for Fermentation
The biomass material (Bonbogori) was collected from the local forest near IIT Guwahati of Assam. All the biomass samples were chopped; air-dried, milled and prepared samples were used for ultrasound assisted lime pretreatment process. The ultrasound-assisted lime pretreatment was conducted according to the recommended design (Lime loading ratio 0.75; Biomass loading ratio 20 and duration of ultrasound 180 min). The residual pretreated biomass samples were then washed and followed by saccharification process as described in Chapter 4 (Temperature –50 °C; RPM 150, pH 4.5; Enzyme loading 20 FPU g-1 of cellulose). The saccharified sugar solution was then used as the substrate of fermentation process.
6.2.2 Bioreactors
Fig 6.2 (a) and (b) shows the schematic diagrams of both fluidized bed bioreactor and stirred tank bioreactor respectively. Fluidized bed bioreactor (FBBR) was constructed of glass with the following dimensions: 55 cm height, an internal diameter of 5 cm, and a total volume of 1500 mL. The substrate was recycled through a recycling pump. The stirred tank bioreactor (STBR) was made of glass with the following dimensions: 27 cm height, an internal diameter of 11 cm, and a total working volume of 1500 mL. The temperature of both types of reactors was maintained by recirculating heated water from a thermostatic bath through the column water jackets. A gas liquid separator was used at the outlet to collect gaseous and soluble products separately. The hydrogen gas was collected by means of a water displacement method. The liquid flow rate velocity maintained throughout the process for each experiment was 95 to 110 L h-1
to fluidize the static bed materials. The biogas (CO2 and H2) was measured by means of liquid dispersing method and the gas components were analyzed by gas chromatography.
6.2.3 Immobilization of Sludge and Media Preparation
The wastewater sludge was collected from the wastewater treatment plant of IIT Guwahati. Hydrogen productivity of the sludge was enhanced by pretreating with HCl (0.1 N) at pH 3.0 for 24 h to eliminate the methanogenic activity [47].
50 mL of pretreated sludge was mixed with 100 g polyethylene–octene elastomer (POE). The resulting mixture was cell-entrapped colloid beads (1.5 mm diameter). After being rinsed with deionized water, the colloid bead was immersed into a solution containing 0.5 g sodium alginate, 0.5 g zirconium oxide and 5 g of pretreated sludge and was then transferred to 0.1M CaCl2 for solidification. The resulting immobilized-cell beads showed an average density of 1.4 g cm-3.
The medium used for this study was hydrolysate of pretreated biomass (bon bogori) along with the wastewater sludge. The other inorganic supplements to support the growth of microorganisms for both ethanol and hydrogen production was: CaCl2·2H2O, 100 mg; MnSO4·6H2O, 1.5 mg; NH4Cl, 2600 mg; K2HPO4, 250 mg; MgCl2, 6H2O, 125 mg; FeSO4,7H2O, 5 mg; CoCl2, 6H2O, 2.5 mg; MnCl2, 4H2O, 2.5 mg; Na2MoO4 , 2H2O, 0.5 mg; H3BO4, 0.5 mg; NiCl2, 6H2O, 0.5 mg; ZnCl2, 0.5 mg antifoaming agent. The chemical oxygen demand (COD) value of the prepared substrate solution was found to be in the range of 30-50 kg COD/L.
Fig. 6.2. (a) Schematic diagram of fluidized bed bioreactor
1. acid tank, 2. base tank, 3. controller 4. fluidized reactor, 5. media storage tank, 6. circulation pump, 7.
hot water inlet at jacket of fluidized bed reactor, 8. hot water outlet from jacket of fluidized bed reactor, 9.
media outlet, 10. water displacement tank, 11. provision of gas inlet (nitrogen for making the environment anaerobic), 12. beads of waste water sludge, 13. medium inlet to fluidized bed, 14. provision to collect the gas.
Fig. 6.2. (b) Schematic diagram of Stirred Tank Bioreactor
1. acid tank, 2. base tank, 3. Controller, 4. Motor, 5. Reactor, 6. hot water inlet at jacket of fluidized bed reactor, 7. hot water outlet from jacket of fluidized bed reactor, 8. Baffles, 9. media/sampling port, 10.
water displacement tank anaerobic, 11. provision to collect the gas, 12. beads of wastewater sludge, 13.
impeller.
6.2.4 Fermentation
The simultaneous ethanol and bio-hydrogen optimization experiments were conducted according to the Taguchi method of design experiment. The experiments were designed with four factors in combination of two and three different levels viz. type of reactor, temperature, pH and organic loading rate and their different levels that will be optimized were tabulated in Table 6.2. Selection of this parameter was based on the through literature survey of various researchers. “1”, “2” or “3” represent the three levels of each factor in the matrix. For analysis of the results and optimization of conditions for setting the control factors, MINITAB software was used.
50 g (approximately) of immobilized acid treated wastewater sludge was poured into the reactor to make a static bed. The height of this static bed was about 5 cm. The broth was fed from the bottom of the reactor along with the nitrogen gas thoroughly to create the anaerobic condition. The same amount of (50 g) of immobilized acid treated wastewater sludge was poured into the STBR. The broth was fed from the top of the reactor along with the nitrogen gas thoroughly to create the anaerobic condition. The speed of stirrer was maintained at 150 RPM. The pH of the medium was adjusted by using pH controller as described in diagrams (Fig. 6.2 (a) and (b)). The detail design matrix of experimental design and results for simultaneous ethanol and hydrogen production was described in Table 6.3.
Table 6.2 Different levels of the parameters used for experimental design
Factor Parameters
Level
1 Level 2
Leve l 3
A Reactor FBBR
*
STBR*
* -
B Temperature (°C) 30 40 50
C pH 4.5 5.5 6.5
D Organic Loading Rate 30 40 50
* FBBR = Fluidized Bed Bioreactor
** STBR = Stirred Tank Bioreactor
Table 6.3. The Design matrix for simultaneous ethanol and hydrogen production
Run A B C D
1 1 1 1 1
2 1 1 2 2
3 1 1 3 3
4 1 2 1 1
5 1 2 2 2
6 1 2 3 3
7 1 3 1 2
8 1 3 2 3
9 1 3 3 1
10 2 1 1 3
11 2 1 2 1
12 2 1 3 2
13 2 2 1 2
14 2 2 2 3
15 2 2 3 1
16 2 3 1 3
17 2 3 2 1
18 2 3 3 2
6.2.5 Analytical Methods
The analysis of gas samples was carried out by gas chromatography (GC) using a thermal conductivity (TCD) detector and molecular sieve column with argon as a carrier gas. The injector, detector and column temperatures were 30 °C, 200 °C and 230 °C respectively. The liquid samples were analyzed by HPLC (Repromer H+, 9 µm, 300 × 7.8 mm; Temperature 25 °C; Eluant: 9 mM sulfuric acid; Flow 1 mL min-1; Pressure: 90 bar). Standard method was used to determine the COD, VSS, TS of the wastewater sludge [48].