2 Materials and Methods
2.1 Seed Microorganisms and Medium
The soil samples were obtained from salt-damaged soil in Khon Kaen, Thailand, and
a commercial salt pan field near Bangkok (Samut Sakhon), Thailand. The soil
samples were mixed with a substrate for cultivation in anaerobic conditions. The
composition of the substrate used for biohydrogen production experiments at different salinity concentrations, i.e., between 3–10% and 15–26% NaCl, before the
acclimatization experiments is as follows: 2 g/L NaHCO 3 , 2 g/L K 2 HPO 4 , 1 g/L
yeast extract, 0.7 g/L (NH 4 ) 2 HPO 4 , 0.75 g/L KCl, 0.85 g/L NH 4 Cl, 0.42 g/L
FeCl 3 Á6H 2 O, 0.82 g/L MgCl 2 Á6H 2 O, 0.25 g/L MgSO 4 Á7H 2 O, 0.018 g/L
CoCl 2 Á6H 2 O, 0.15 g/L CaCl 2 Á2H 2 O, and 0.018 g/L NiCl 2 Á6H 2 O. Glucose concentration was adjusted according to each experiment. All chemicals were purchased
from FUJIFILM Wako Pure Chemical Corp., Japan. The composition of the substrate for the experiments on the bacteria’s requirement for chloride ions in high
salinity conditions, acclimatization period, and biohydrogen production at 26%
NaCl after 2 years of acclimatization was the same as that above, except that
NiCl 2 Á6H 2 O was omitted.
2.2 Culture Conditions and Experimental Procedures
The first step of the experiment was to evaluate the hydrogen production in conditions of 3–10% salinity before acclimatization. The experiments were done under six
NaCl concentrations of 3%, 3.5%, 5%, 7%, 7.5%, and 10%. The second step of
experiment was to evaluate the hydrogen production in conditions of 15–26%
salinity before acclimatization and at 26% salinity after an acclimatization period
of 2 years. The experiment was done at NaCl concentrations of 15%, 20%, and 26%.
The third step of the experiment was the evaluation of the bacteria’s requirement
for chloride ions in high salinity conditions. The experiment was done by comparing
the culture under Na 2 SO 4 /NaCl ratios of 1:1 and 4:1. The ratio was prepared by
weight to reach 26% (351.35 g/L) concentration of the mix. The third step was done
after 1 year of acclimatization.
The experiments to determine biohydrogen production under salinity concentrations of 3–10% and 15–26% NaCl before acclimatization and various F/M ratio at
15% NaCl were done under the following conditions: 100 mL sealed serum bottles
in a nitrogen atmosphere with an initial pH of 6.8 adjusted with 1 M HCl and 1 M
NaOH and incubated in a shaking incubator (BT100 and BT300; Yamato Scientific
Co., Ltd. Japan) at 35
C and a 100-rpm shaking speed. Biogas samples were
periodically collected, and the compositions were analyzed via gas chromatography.
The experiment to determine the bacteria’s requirement for chloride ions in high
salinity conditions followed the same conditions, except that 125-mL serum bottles
11 Biohydrogen Production from Lignocellulosic Biomass by Extremely Halotolerant. . . 415
2.1 Seed Microorganisms and Medium
The soil samples were obtained from salt-damaged soil in Khon Kaen, Thailand, and
a commercial salt pan field near Bangkok (Samut Sakhon), Thailand. The soil
samples were mixed with a substrate for cultivation in anaerobic conditions. The
composition of the substrate used for biohydrogen production experiments at different salinity concentrations, i.e., between 3–10% and 15–26% NaCl, before the
acclimatization experiments is as follows: 2 g/L NaHCO 3 , 2 g/L K 2 HPO 4 , 1 g/L
yeast extract, 0.7 g/L (NH 4 ) 2 HPO 4 , 0.75 g/L KCl, 0.85 g/L NH 4 Cl, 0.42 g/L
FeCl 3 Á6H 2 O, 0.82 g/L MgCl 2 Á6H 2 O, 0.25 g/L MgSO 4 Á7H 2 O, 0.018 g/L
CoCl 2 Á6H 2 O, 0.15 g/L CaCl 2 Á2H 2 O, and 0.018 g/L NiCl 2 Á6H 2 O. Glucose concentration was adjusted according to each experiment. All chemicals were purchased
from FUJIFILM Wako Pure Chemical Corp., Japan. The composition of the substrate for the experiments on the bacteria’s requirement for chloride ions in high
salinity conditions, acclimatization period, and biohydrogen production at 26%
NaCl after 2 years of acclimatization was the same as that above, except that
NiCl 2 Á6H 2 O was omitted.
2.2 Culture Conditions and Experimental Procedures
The first step of the experiment was to evaluate the hydrogen production in conditions of 3–10% salinity before acclimatization. The experiments were done under six
NaCl concentrations of 3%, 3.5%, 5%, 7%, 7.5%, and 10%. The second step of
experiment was to evaluate the hydrogen production in conditions of 15–26%
salinity before acclimatization and at 26% salinity after an acclimatization period
of 2 years. The experiment was done at NaCl concentrations of 15%, 20%, and 26%.
The third step of the experiment was the evaluation of the bacteria’s requirement
for chloride ions in high salinity conditions. The experiment was done by comparing
the culture under Na 2 SO 4 /NaCl ratios of 1:1 and 4:1. The ratio was prepared by
weight to reach 26% (351.35 g/L) concentration of the mix. The third step was done
after 1 year of acclimatization.
The experiments to determine biohydrogen production under salinity concentrations of 3–10% and 15–26% NaCl before acclimatization and various F/M ratio at
15% NaCl were done under the following conditions: 100 mL sealed serum bottles
in a nitrogen atmosphere with an initial pH of 6.8 adjusted with 1 M HCl and 1 M
NaOH and incubated in a shaking incubator (BT100 and BT300; Yamato Scientific
Co., Ltd. Japan) at 35
C and a 100-rpm shaking speed. Biogas samples were
periodically collected, and the compositions were analyzed via gas chromatography.
The experiment to determine the bacteria’s requirement for chloride ions in high
salinity conditions followed the same conditions, except that 125-mL serum bottles
11 Biohydrogen Production from Lignocellulosic Biomass by Extremely Halotolerant. . . 415