were used. The main culture bottles with a volume of 500 mL each for the three
different sources of soil were also maintained. These bottles were also used for
acclimatization purposes. In acclimatization period of 2 years, the substrate’s NaCl
concentration was kept at 26%. Gas production was periodically measured, and
substrate was changed after no gas production was detected. Anaerobic condition
was maintained under nitrogen atmosphere.
Biohydrogen production at a NaCl concentration of 26% after 2-year period of
acclimatization experiments was also studied under the same conditions, except that
the initial pH was not adjusted, the serum bottles had a volume of 75 mL, and
shaking incubator temperature was at 37
C.
2.3 Analytical Method
The initial pH was adjusted by using a pH meter (D-13; Horiba Co. Ltd., Japan). The
volume of the produced biogas was measured with a glass syringe. The composition
of H 2 , N 2 , CH 4 and CO 2 was analyzed via gas chromatography (GC-8APT;
Shimadzu Corp., Japan) with a 60/80 activated charcoal mesh column
(1.5 m  3.0-mm internal diameter) and argon as a carrier gas, with operational
temperatures of the injector, column, and TCD detector of 50
C, 60
C, and 50
C,
respectively. The compositions of volatile fatty acids (VFAs) were determined by
gas chromatography (GC-8APF; Shimadzu Corp., Japan) with a flame ion detector
(FID) and a Unisole F-200 30/60 glass mesh column (3 m  3.2-mm internal
diameter). The operational temperatures for the injection port, FID detector, and
oven were 250
C, 140
C, and 140
C, respectively.
The water content and total organic matter of the soil was determined via the JIS
A 1203 test method for water content of soils and JIS A 1226–2000 test method for
ignition loss of soils [17, 18]. Sample masses of 20.5506–44.1789 g were used to
determine the soil’s water content and ignition loss. Volatile suspended solid (VSS)
was determined according to method 2540 E of Standard Methods [19].
Soil salinity was determined by mixing soil and distilled water in 1:2.5 dry soil to
water ratio, shaken for 3 hours at 180 rpm (Eyela Multishaker MMS; Tokyo
Rikakikai Co. Ltd., Japan). After 25 minutes of settling time, the supernatant is
measured by a thermosalinity meter (TS-391; As One Corp., Japan). The measurement results were then multiplied by the dilution factor of water content of soil.
2.4 Theoretical Hydrogen Production and Yield
The theoretical hydrogen production was determined using Eqs. (1) and (2) in
Table 11.1. Based on Eq. (1), 1 mol of glucose will produce 4 mol of hydrogen.
Thus, 1 g of glucose at standard temperature and pressure (STP) conditions will
produce 498 mL of H 2 via the acetic acid (HAc) pathway. Based on Eq. (2), 1 mol of
416
D. A. H. Taroepratjeka et al.
different sources of soil were also maintained. These bottles were also used for
acclimatization purposes. In acclimatization period of 2 years, the substrate’s NaCl
concentration was kept at 26%. Gas production was periodically measured, and
substrate was changed after no gas production was detected. Anaerobic condition
was maintained under nitrogen atmosphere.
Biohydrogen production at a NaCl concentration of 26% after 2-year period of
acclimatization experiments was also studied under the same conditions, except that
the initial pH was not adjusted, the serum bottles had a volume of 75 mL, and
shaking incubator temperature was at 37
C.
2.3 Analytical Method
The initial pH was adjusted by using a pH meter (D-13; Horiba Co. Ltd., Japan). The
volume of the produced biogas was measured with a glass syringe. The composition
of H 2 , N 2 , CH 4 and CO 2 was analyzed via gas chromatography (GC-8APT;
Shimadzu Corp., Japan) with a 60/80 activated charcoal mesh column
(1.5 m  3.0-mm internal diameter) and argon as a carrier gas, with operational
temperatures of the injector, column, and TCD detector of 50
C, 60
C, and 50
C,
respectively. The compositions of volatile fatty acids (VFAs) were determined by
gas chromatography (GC-8APF; Shimadzu Corp., Japan) with a flame ion detector
(FID) and a Unisole F-200 30/60 glass mesh column (3 m  3.2-mm internal
diameter). The operational temperatures for the injection port, FID detector, and
oven were 250
C, 140
C, and 140
C, respectively.
The water content and total organic matter of the soil was determined via the JIS
A 1203 test method for water content of soils and JIS A 1226–2000 test method for
ignition loss of soils [17, 18]. Sample masses of 20.5506–44.1789 g were used to
determine the soil’s water content and ignition loss. Volatile suspended solid (VSS)
was determined according to method 2540 E of Standard Methods [19].
Soil salinity was determined by mixing soil and distilled water in 1:2.5 dry soil to
water ratio, shaken for 3 hours at 180 rpm (Eyela Multishaker MMS; Tokyo
Rikakikai Co. Ltd., Japan). After 25 minutes of settling time, the supernatant is
measured by a thermosalinity meter (TS-391; As One Corp., Japan). The measurement results were then multiplied by the dilution factor of water content of soil.
2.4 Theoretical Hydrogen Production and Yield
The theoretical hydrogen production was determined using Eqs. (1) and (2) in
Table 11.1. Based on Eq. (1), 1 mol of glucose will produce 4 mol of hydrogen.
Thus, 1 g of glucose at standard temperature and pressure (STP) conditions will
produce 498 mL of H 2 via the acetic acid (HAc) pathway. Based on Eq. (2), 1 mol of
416
D. A. H. Taroepratjeka et al.