Biohydrogen Production by Antarctic Psychrotolerant Klebsiella sp. ABZ11

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.

Isolation and characterization of biohydrogen psychrotolerant bacteria. Five pure colonies were obtained from the Antarctic seawater sample, with milky mucoid color. All isolates grew on Marine Agar plates after three days incubation. These isolates were designated ABZ4, ABZ7, ABZ10, ABZ11 and ABZ12.

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.

Fig. 1.

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).

Fig. 2.

Fig. 3.

Fig. 4.

Analysis of the partial 16S rRNA sequence showed that ABZ11 is closely related to Klebsiella pneumoniae (99% identity). The isolate was thus designated Klebsiella sp. ABZ11, with a Genbank accession number KX266892. Based on the phylogenetic relationship identified using neighbor-joining method, ABZ11 cluster with Klebsiella pneumoniae subsp. rhinoscleromatis R-70 strain on the same taxon among the 13 closely related strains that were used (Fig. 5).

Fig. 5.

Temperature tolerance of the Klebsiella sp. ABZ11 was tested by observing the viable cells after incubation at several different temperatures. ABZ11 is able to grow at 20, 30, 37, and 40°C, but no growth was observed at 45°C (Fig. 6). The optimum growth was observed at 37°C after three days of incubation.

Fig. 6.

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 Klebsiella sp. ABZ11 (Fig. 7). Meanwhile, oxygen content was slightly reduced by 0.5% in 6 h and 0.02% in 24 h leading to a total of 96.8% of oxygen uptake in 24 h.

Fig. 7.

Biohydrogen production using different carbon sources. Biohydrogen production was observed to start after 2 hours of fermentation for all the substrate concentration except 12.5 g/l sucrose concentration that begins production at the 4^th hour. Table I shows the summary of the biohydrogen production of bacterial with different concentrations of the carbohydrate sources. Meanwhile, the biohydrogen production was observed to increase with fermentation time for all the carbohydrate concentrations with maximum productivity observed at different time. Productivity was also observed to decrease with increased substrate concentration. A maximum biohydrogen of 38.55 mol/l observed at 10 g/l was significantly (p > 0.05) different from the productivity of other substrate concentrations. Further, carbohydrate consumption started immediately with 74% of the total carbohydrate consumed after 2 h (Fig. 8). It was also observed that the carbohydrate in the medium was totally exhausted before 48 h in all tested parameters.

Fig. 8.

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.

Fig. 9.

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 Klebsiella sp. (closely related to Klebsiella pneumoniae) from Antarctic seawater. This Klebsiella sp. strain ABZ11 has the ability to withstand a wide growth temperature, with optimum at 37°C, suggesting that this strain is psychrotolerant (Carrión et al. 2011; Lavin et al. 2016).

Klebsiella spp. has previously been isolated from Antarctic sea animals, samples obtained from islands, as well as other environments such as sewage sludge and hot springs (Minnan et al. 2005; Kargel et al. 2008; Brat et al. 2016). The diverse environmental conditions from which this genus was isolated suggest the unique physiological characteristics that allows their survival. Klebsiella sp. ABZ11 that we isolated from Antarctic seawater is devoid of capsules and does not show the ability to lyse blood cells, suggesting the non-pathogenic nature of this strain.

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 Bacillus sp. FS2011 and Clostridium beijerinckii Fanp3 strains (Pan et al. 2008; Song et al. 2013) but differs with other work on hydrogen producing Klebsiella strains (Niu et al. 2010; Chookaew et al. 2012). Strain ABZ11 also shows high carbohydrate uptake, with 74% of glucose consumed after 2 h of fermentation due to high substrate conversion efficiency attributed to the incubation temperature and oxygen, before complete utilization around 48 h.

Biohydrogen production was detected within the exponential and stationary phase with maximum production found in the stationary phase of Klebsiella sp. ABZ11 growth, indicating that hydrogen production is more of a secondary metabolite in its fermentative process. This is similar to the report on biohydrogen production by mesophilic Clostridium sp. DMHC-10 (Kamalaskar et al. 2010). More importantly, strain ABZ11 shows a very short production lag phase, rapid carbohydrate uptake, and is able to substantially decrease culture oxygen level in a short period of time. Our findings thus suggest an improved metabolic biosynthesis for biohydrogen production compared to the psychrophiles and psychrotolerant strains (Alvarez-Guzmán et al. 2016). There is a possibility that Klebsiella sp. ABZ11 possesses oxygen tolerant hydrogenases, as previously reported in K. oxytoca HP1 (Minnan et al. 2005). The improvement in metabolic activity yielding early productivity may be attributed to the temperature condition at which the fermentation was performed. The rapid decrease in oxygen concentration in the fermentation medium could be another factor that influences the rate of the subsequent carbohydrate hydrolysis due to release of large extracellular hydrolytic enzymes and increase in their activity, which in turn increases hydrogen biosynthesis. A small amount of oxygen in the fermentation medium has been shown before to enhance the rate of substrate hydrolysis (Johansen and Bakke 2006; Ramos and Fdz-Polanco 2013).

Klebsiella sp. ABZ11 biohydrogen production kinetics show that the strain is inclined towards biohydrogen production rather than growth at the stationary phase, under the experimental conditions described here. Experiments performed under batch fermentation revealed the potential of this strain to be able to utilize different carbon sources for biohydrogen production. In the future, optimization of experimental parameters may further improve the biohydrogen-producing ability of this facultative psychrotolerant bacteria.

A psychrotolerant, facultative, oxygen-insensitive biohydrogen-producing bacterial strain has successfully been isolated from Antarctic seawater. The strain, identified as Klebsiella sp. ABZ11, has been investigated in terms of its biohydrogen productivity, temperature tolerance, and dissolved oxygen uptake. This strain shows up to 95% oxygen uptake, and can tolerate growth temperature of 20–40°C, with optimum growth observed at 37°C. Maximum biohydrogen productivity of 38.55 mol/l was observed at 30°C. This study demonstrates the feasibility of using a psychrotolerant bacterial strain for biohydrogen production at lower temperature, with high oxygen tolerance.