Improving the microbial production of biofuels through metabolic engineering

The joint challenges of anthropogenic climate change and dwindling fossil fuel reserves are driving intense research into alternative energy sources. One attractive avenue is to use a biological process to produce a biofuel. Among the various biofuel options, biohydrogen gas is an attractive future energy carrier due to its potentially higher efficiency of conversion to usable power, low to non-existent generation of pollutants and high energy density. However, low yields and production rates have been major barriers to the practical application of biohydrogen technologies. Intensive research on biohydrogen is underway, and in the last few years several novel approaches have been proposed and studied to surpass these drawbacks. To this end the main aim of this thesis was to improve the molar hydrogen yield with special emphasis of metabolic engineering using the interactive effect with bioprocess independent variable.  One investigated hypothesis was that H2 production could be improved and made more economically viable by metabolic engineering on the facultative hydrogen producer Escherichia coli from glucose as well as various other carbon sources, including pentoses. The effects of pH, temperature and carbon source were investigated in batch experiments. Maximal hydrogen production from glucose was obtained at an initial pH of 6.5 and temperature of 35°C. Kinetic growth studies showed that the μmax was 0.0495 h−1 with a Ks of 0.0274 g L−1 when glucose was the sole carbon source in M9 (1X) minimal medium. Among the many sugar and sugar derivatives tested, hydrogen yields were highest with fructose, sorbitol and d-glucose; 1.27, 1.46 and 1.51 mol H2 mol−1 substrate respectively.  In addition, to obtain the interactions between the variables important for achieving maximum hydrogen production, a 3K full factorial Box–Behnken design and response surface methodology (RSM) were employed for experimental design and analysis on a metabolically engineered Escherichia coli strain, DJT135. A maximum molar hydrogen yield of 1.69 mol H2 mol−1 glucose was obtained under the optimal conditions of 75 mM glucose, 35°C and pH 6.5. Thus, RSM with Box–Behnken design was a useful statistical tool for achieving higher molar hydrogen yields by metabolically engineered organisms.  Furthermore, the heterologous expression of the soluble [Ni-Fe] hydrogenase from Ralstonia eutropha H16 (the SH hydrogenase) was attempted to demonstrate the introduction of a pathway capable of deriving hydrogen from NADH to surpass the stoichiometric molar hydrogen yield. Successful expression was demonstrated by in vitro assay of enzyme activity.  Moreover, expression of SH restored anaerobic growth on glucose to adhE strains, normally blocked for growth due to the inability to re-oxidize NADH.  Measurement of in vivo hydrogen production showed that several metabolically engineered strains were capable of using the SH hydrogenase to derive 2 mol H2 per mol of glucose consumed, close to the theoretical maximum.   Using another strategy, it was shown that crude glycerol could be converted to hydrogen, a possible future clean energy carrier, by photofermentation using Rhodopseudomonas palustris through photofermentation. Here, the effects of nitrogen source and different concentrations of crude glycerol on this process were assessed. At 20 mM glycerol, 4 mM glutamate, 6.1 mol hydrogen/mole of crude glycerol were obtained under optimal conditions, a yield of 87% of the theoretical, and significantly higher than what was achieved previously. As a continuation of this study, multiprocess parameter optimization was also involved. Under optimal conditions, a light intensity of 175 W/m2, 30 mM glycerol, and 4.5 mM glutamate, 6.69 mol hydrogen/mole of crude glycerol were obtained, a yield 96% of theoretical. Determination of nitrogenase activity and expression levels showed that there was relatively little variation in levels of nitrogenase protein with changes in process variables whereas nitrogenase activity varied considerably, with maximal nitrogenase activity (228 nmol of C2H4/ml/min) at the optimal central point. In the final section, hydrogen production from glucose via single-stage photofermentation was examined with the photosynthetic bacterium Rhodobacter capsulatus JP91 (hup-). Response surface methodology with Box–Behnken design was used to optimize the independent experimental variables of glucose concentration, glutamate concentration and light intensity, as well as examining their interactive effects for maximization of molar hydrogen yield. Under optimal condition with a light intensity of 175 W/m2, 35 mM glucose, and 4.5 mM glutamate, a maximum hydrogen yield of 5.5 (±0.15) mol H2/mol glucose, and a maximum nitrogenase activity of 246 (±3.5) nmol C2H4/ml/min were obtained. Densitometric analysis of nitrogenase Fe-protein expression under different conditions showed significant variation in Fe-protein expression with a maximum at the optimized central point. Even under optimum conditions for hydrogen production, a significant fraction of the Fe-protein was found in the ADP-ribosylated state, suggesting that further improvement in yields might be possible. To this end an AmtB- derivative of Rhodobacter capsulatus JP91 (hup-) was created by conjugating in amtB::Km using the suicide vector pSUP202. Preliminary experimental results showed that the newly metabolically engineered strain, R. capsulatus DG9, produced 8.2 (±0.06) mol hydrogen/mole of glucose under optimal conditions in batch cultures (light intensity, 175 W/m2; 35 mM glucose, and 4.5 mM glutamate).  Western blot analyses of the ADP-ribosylation status of the nitrogenase Fe-protein were investigated on metabolically engineered strain R. capsulatus DG9.  In brief, the progress on hydrogen production technology has been limited due to the metabolic barrier. The major metabolic barrier is due to lacking of potential consistent molecular tools to reach or surpass the stochiometric biohydrogen yield since last decades using microbes. To this end a novel approach “metabolic engineering” seems very promising to overcome this constraint towards industrialization to ensure the feasibility of hydrogen production technology. In this present study it has been shown that metabolically engineered facultative (Escherichia coli) anaerobe and photosynthetic bacteria (Rhodobacter capsulatus and Rhodopseudomonas palustris) can produce hydrogen as a major product through fermentative mode by metabolic redirection toward potential energy generation. On the other hand, response surface methodology has depicted in this study as another potential tool to statistically optimize the hydrogen production by generating suitable information concerning interactive correlation between process variables and metabolically engineered biohydrogen producers. Thus, multi process parameter optimization tool has been creating a novel avenue to make a crosslink between lab scale and pilot scale hydrogen production by providing potential mathematical model for scaling up biohydrogen production using metabolically engineered biohydrogen producers. Therefore, it has been clearly revealed in this project that combined effort of metabolic engineering and response surface methodology can make biohydrogen production technology potentially feasible towards its commercialization in near future.

Keyphrases: Photofermentation, Fermentation, Biofuel, Hydrogen production, Photofermentation, Dark fermentation, Metabolic engineering