Hydrogen is an attractive alternative energy carrier due to the high energy density, and the cleaner by-products generated when used in automobiles (Khan et al. 2017). Biological hydrogen production is a technique of producing hydrogen through biological processes, using microorganisms as the biocatalysts. Among all the biological processes, bacterial dark fermentation is the most promising one, due to the high biohydrogen yield, and the ability to ferment different substrates to produce biohydrogen (Khan et al. 2017; Miandad et al. 2017).
Psychrophiles and psychrotolerant bacteria are abundant in the colder environment, e.g. Antarctica. Psychrophiles grow optimally at 20°C, and their fermentative processes have been considered beneficial due to the unique enzymes they possess (Corr and Murphy 2011). However, the reduced metabolic rate in psychrophiles is one key factor that affects substrate uptake and synthesis, which invariably affect the rate of substrate degradation and fermentative yield (Lettinga et al. 2001; Thauer et al. 2010; Lu et al. 2011). Psychrotolerant bacteria on the other hand, can grow above 20°C (Morita 1975; Pesciaroli et al. 2012). Thus, they are expected to be more useful for biohydrogen production at ambient temperature. Some psychrotolerant strains can thrive between 0–40°C (Pikuta et al. 2016), giving them an advantage over psychrophiles. Temperature contributes significantly to fermentative biohydrogen yield due to its influence on the rate of metabolism and enzyme activity (Hallenbeck et al. 2012). Antarctic seawater is subjected to a wide variation in environmental conditions which permits the survival of a wide range of bacteria, including the fermentative strains (Delille 1992). This study therefore focused on the isolation and characterization of psychrotolerant biohydrogen producing bacteria from Antarctic seawater.
More recently, a growing number of research has focused on biohydrogen production using cold-active bacteria isolated from Antarctica. For instance, Alvarez-Guzmán et al. (2016) investigated biohydrogen productivity of psychrophilic bacteria at 25°C under anaerobic condition. They reported a prolonged biohydrogen production lag phase and carbohydrate uptake after the start of fermentation. We hypothesized that utilizing facultative psychrotolerant bacteria with tolerance to mesophilic temperature may be a good option for improving substrate uptake and biohydrogen productivity of cold-adapted bacteria. Therefore, the objective of this study was to investigate the biohydrogen production ability of Antarctic facultative psychrotolerant bacteria under mesophilic temperature conditions. In addition, the influence of oxygen on the substrate uptake and biohydrogen productivity was also examined.
While growth yield (YX/S) was defined as the fraction of the dry cell weight (g/ml) and the substrate consumed (mg/ml).
Out of the five isolates screened for biohydrogen production, only two isolates (Fig. 1) is able to produce biohydrogen, namely ABZ11 (0.49 mol H2/mol glucose) and ABZ4 (0.02 mol H2/mol glucose). Due to higher biohydrogen production, ABZ11 was selected for further characterizations.
ABZ11 has a short growth lag phase (1 h) (Fig. 2) but a longer stationary phase between 2 and 18 h of incubation. The cells of ABZ11 are rod-shaped, with an approximate length of 1.7 µm and diameter of 0.4 µm (Fig. 3). Capsule staining revealed no capsule around the cells as a result of the absence of visible halo zones after staining (Fig. 4).
Analysis of the partial 16S rRNA sequence showed that ABZ11 is closely related to
Temperature tolerance of the
Oxygen tolerance of ABZ11 was then investigated by measuring the level of dissolved oxygen in the culture medium. An oxygen uptake of 4.17 ± 0.03 mg/l was observed after 2 h, representing 95% of the oxygen in the medium that was scavenged by
Biohydrogen production and kinetic parameters of substrate utilization by
Carbohydrate concentrations (g/l) | 5 | 7.5 | 10 | 12.5 | ||
Biohydrogen production at initial incubation time and at maximum production (mol/l) | Glucose | Initial | 0.21 ± 0.00 | 0.21 ± 0.02 | 2.35 ± 0.03 | 0.77 ± 0.01 |
Maximum | 26.23 ± 2.18 | 23.80 ± 3.29* | 38.55 ± 2.19* | 21.11 ± 0.14* | ||
Fructose | Initial | 0.21 ± 0.00 | 0.47 ± 0.05 | 0.65 ± 0.46 | 0.81 ± 0.09 | |
Maximum | 25.47 ± 2.02 | 22.77 ± 2.01 | 19.97 ± 1.60 | 16.97 ± 0.12 | ||
Sucrose | Initial | 0.21 ± 0.00 | 0.23 ± 0.04 | 0.23 ± 0.01 | 4.52 ± 0.36 | |
Maximum | 28.24 ± 2.96* | 22.37 ± 2.19 | 18.21 ± 0.77 | 15.91 ± 1.49 | ||
Ketic analysis | YP/S (mol/mg) | 23.53 | 17.00 | 24.09 | 11.11 | |
YX/S (g/mg) | 0.170 | 0.424 | 0.532 | 0.205 |
Maximum biohydrogen production in the group and the productivity considered for the kinetic
Biohydrogen yield in relation to the biomass during fermentation is 24.09 mol/mg while cell growth in relation to substrate utilization is 0.532 g/mg, at 10 g/l of glucose (Fig. 9). The maximum biohydrogen production and kinetic data for all the parameters investigated are summarized in Table I.
Generally, there is a prolonged period between the fermentation start time and the beginning of biohydrogen production in psychrophilic bacteria, providing a challenge for application in lower temperature biohydrogen reactors. This study was thus initiated to overcome this challenge by using Antarctic psychrotolerant bacteria, with higher metabolic activity in order to improve its substrate uptake and biohydrogen production. We have successfully isolated a psychrotolerant, biohydrogen-producing
The carbon sources tested in the fermentation experiment were glucose, fructose and sucrose. The highest biohydrogen production of 38.55 ± 2.19 mol/l was observed at 10 g/l glucose concentration, at pH 5.6. A decrease in biohydrogen production with the increase in substrate concentration was also observed in almost all carbon sources tested, as reported previously (Ginkel et al. 2001; Kamalaskar et al. 2010) except for glucose. The optimum glucose concentration of 10 g/l observed in this study is in agreement with mesophilic
Biohydrogen production was detected within the exponential and stationary phase with maximum production found in the stationary phase of
A psychrotolerant, facultative, oxygen-insensitive biohydrogen-producing bacterial strain has successfully been isolated from Antarctic seawater. The strain, identified as