Analysis Of Biohydrogen Production With Klebsiella Pneumoniae ECU-15 And Experiment Studies On Its Fermentation Using Lignocellulosic Hydrolysate

Serious global environment pollution and energy crisis have been caused by excessive use of fossil fuels. Thus, there is a pressing need to develop nonpolluting and renewable energy resource. Hydrogen is widely thought to be an ideal and efficient energy carrier in the future due to its high conversion efficiency, recyclability and nonpolluting nature. It could be produced by thermochemical process, electrochemical process and bioconversion process. Among them, bioconversion process has attracted more and more attentions for two reasons: utilization of renewable resource, and usually operated at ambient temperature and atmospheric pressure. It can be conducted by photosynthetic fermentation, dark fermentation and two-stage fermentation. Dark fermentation is a promising method for its high hydrogen-producing rate, simple fermentative equipment, and bioconversion feasibility from the recycle resources. However, the low yield of hydrogen production and high cost of the substrate are the main constraints for industrialization of biohydrogen production. Based on these problems, the researches of the effect of hydrogen producing strains, culture conditions arid the fermentative substrates on biohydrogen process were carried out in this paper. Firstly, high-producing strains were isolated and identified with the optimal culture conditions obtained. The effect of culture conditions on metabolic pathway was investigated to provide the theoretical foundation for the microorganism recombination. Secondly, the characteristics of biohydrogen production from hydrolysate were investigated, and the key factors and its action rules that influenced the hydrogen production in hydrolysate were analyzed. These results offered the fundamental data for the development of hydrogen production from lignocellulosic hydrolysate.In this paper,54 hydrogen producing strains were isolated from the anaerobic sewage sludge. Among them, only three strains with the highest hydrogen producing efficiency were identified by morphological observation, physiological and biochemical experiments and 16S rRNA analysis. The results indicated that the all the three strains were belonging to the Klebsiella pneumoniae. A strain named Klebsiella pneumoniae ECU-15 with the highest hydrogen-producing capability was chosen as the target strain for the following investigations.In order to optimize the culture conditions and medium compositions of the biohydrogen fermentation, the hydrogen production characteristics of K. pneumoniae ECU-15 were firstly investigated. It was found that the anaerobic condition was in favor of hydrogen production, and the hydrogen production was improved by 17.97% at the anaerobic condition than that under the micro-aerobic condition. The optimum culture temperature and pH were 37℃and 6.0 for the hydrogen fermentation, but the optimal pH for cell growth was observed at pH7.0. The strain of ECU-15 could grow in several kinds of monosaccharide and disaccharide but starch and cellulose. Fermentation process through glucose exhibited the maximum hydrogen productivity and production among these carbon sources. The hydrogen yield was decreased with the increasing of initial glucose concentration. The maximum yield of 2.07mol/mol glucose was obtained at the initial glucose concentration of 5g/L. The hydrogen production rate was increased with the increasing of initial glucose concentration, and the maximum production rate of 482ml/l/h was obtained at the initial glucose concentration of 30g/L. However, the cell growth and hydrogen production would be inhibited at the initial glucose concentration of 40g/L. In addition, the concentration of phosphate buffer was optimized to be 200mM. The hydrogen yield was improved by 31.03%,18.89% and 24.83% by adding concentration of 300mg/L Fe2+,120mg/L Mg2+ and 35mg/L Ni2+, respectively. The processes of cell growth, hydrogen production and glucose consumption in batch fermentation were simulated by Logistic model and Gompertz model. It was indicated that the experimental data of these three processes could fit well with these models. From the above results, it could be seen that K. pneumoniae ECU-15 exhibited the highest hydrogen production rate compared with the other strains.In order to find out the influence of culture conditions on the metabolic pathway of K. pneumoniae ECU-15, the network of glucose metabolism under anaerobic conditions was calculated and analyzed by metabolic flux analysis (MFA). The effects of temperature, pH and initial glucose concentration on the intracellular fluxes, as well as the fluxes of hydrogen producing and uptake were caculated and analyzed. The results indicated that the uptake flux of hydrogen was affected by temperature, pH and initial glucose concentration significantly. It was decreased with the increasing temperature from 26℃to 37℃. Compared with the weak acidity pH, the neutral or weak basicity pH was in favor of the uptake of hydrogen. Under the limited glucose concentration conditions, the uptake flux of hydrogen would also decrease. The hydrogen producing flux and the flux distribution of some key nodes in the whole metabolic networks could also be affected by these factors. The flux of the ethanol producing pathway was decreased with the temperature. Thus NADH would be remained for hydrogen production. And the fluxes of pyruvate dehydrogenase pathway and the pyruvate formate lyase metabolic pathway were higher at pH>7.0 than that of other pH values. Moreover, the flux of pyruvate formate lyase metabolic pathway was increased under the limited glucose concentration condition, which would in favor of the hydrogen production.As for the biohydrogen fermentation, both the types and the cost of fermentative substrates were important factors that restricted the practicality and industrialization of the process. Therefore, the lignocellulosic hydrolysate which had rich resource and lower cost was used for biohydrogen fermentation in this paper. In order to decrease the biological resistance and increase the enzymatic hydrolysis of lignocellulose, the pretreatment technology of rice straw through NaOH solution assisted with photocatalysis was investigated through experiments. The results showed that the degradation rates of the lignin and hemicellulose were 67.84% and 53.03%, respectively under the condition of 2g/L nano-TiO2 and 1h of photocatalysis time in 1.5% NaOH solution. The enzymatic hydrolysis rate of the pretreated straw was 73.96%, which was higher than that with the alkali procedure. The biohydrogen production with this hydrolysate was 0.78 V/V, which was higher than that of the corn stalk hydrolysate pretreated by steam-explosion process (0.65V/V). These results indicated that the hydrogen production of the hydrolysate was lower than that of glucose. In order to determine the main compositions in hydrolysate that influenced the biohydrogen production, the fermentation with various substrates of this bacterium was investigated. It was found that glucose, xylose and cellobiose were the main compositions in hydrolysate. The lower hydrogen production of the hydrolysate might be due to the existence of xylose. In addition, the fermentation results of K. pneumoniae ECU-15 with various proportions of glucose and xylose indicated that the consumption rate of xylose was lower than that of glucose. The biohydrogen metabolic pathway of glucose was seriouslly influenced by xylose, which resulted in the decrease the hydrogen efficiency of ECU-15 from hydrolysate.