Batch fermentation system for biohydrogen production by klebsiella sp. abz11 isolated from antarctica

Abstract

The study of cold-adapted bacteria for biohydrogen production has attracted much interest in the last few decades due to the lower energy input required during the fermentation process. However, an extended lag phase of growth and slow metabolic rate of the bacteria remain the obstacles for the process to be feasible, particularly for obligate psychrophilic and psychrotolerant bacteria. Bacteria with the oxygen-tolerant ability are also favourable for large-scale fermentation. Thus, there is a need to find oxygen-tolerant bacteria capable of producing biohydrogen at mesophilic temperature. In this study, Antarctic soil and seawater samples were used for bacterial isolation, before being screened for biohydrogen production ability. Twelve bacteria were successfully isolated and six were found capable of producing biohydrogen. The bacterium with the highest biohydrogen production was characterised. The optimum physicochemical parameters, such as temperature, pH and carbohydrate concentration were determined using one-factor-at-a-time (OFAT) approach. Appropriate nitrogen source, temperature tolerance and the effects of dissolved oxygen on the growth and biohydrogen productivity were also investigated. Precise optimal factors for biohydrogen productivity were then examined using the three-level factorial design of Response Surface Methodology (RSM). Identification of bacterium with the highest biohydrogen production showed that it was closely related to Klebsiella pneumoniae with 99% similarity based on the 16S rRNA analysis. The bacterium was therefore designated as Klebsiella sp. ABZ11. It was a Gram-negative bacillus, with no capsule detected and grew at a temperature range of 20-40°C, and exhibited 95% uptake of dissolved oxygen in two hours. Screening using OFAT suggested that the optimum conditions for biohydrogen production were 30°C, an initial pH of 6.5, and with glucose supplemented with concentration of 10 g/L. The bacterium utilised various types of carbon and nitrogen sources for biohydrogen production but preferred glucose as the carbon source and beef extract as the nitrogen source. Further optimisation using RSM revealed that the highest biohydrogen productivity (110.15 mol/L) was obtained at 33.5°C, with an initial pH of 6.75 and glucose concentration of 9.15 g/L. For each gram of glucose supplied, the yield for biohydrogen and cell-biomass was 122 mol/L/g and 0.87 g, respectively. Kinetics showed that the bacterium used more of the glucose for biohydrogen production than for biomass formation in the fermentation process. A scale-up culture using the optimised conditions recorded a biohydrogen production of 137.56 mol/L in 36 h with a cumulative yield of 533.51 mol/L. In conclusion, batch fermentation using Klebsiella sp. ABZ11 under mesophilic temperature was found to have decreased lag phase of growth and increased metabolic rate, thereby influencing faster and higher biohydrogen production.