Biohydrogen production by nitrogen fixing cyanobacterium anabaena siamensis TISTR 8012

Abstract

In this thesis,we focused to study biohydrogen production by using the cyanobacterium Anabaena siamensis TISTR 8012, a strain isolated from rice paddy field in Thailand. The phylogenetic analysis using 16S rDNA gene sequence revealed that A. siamensis TISTR 8012 is classified into a single group which possesses akinete and heterocyst differentiations when grown under N-deprivation. Optimum physical parameters to enhance growth rate of A. siamensis TISTR 8012 under N₂ fixing condition were temperature at 30 ℃, light intensity between 40-50 µE m⁻² s⁻¹ and pH 7.5. The efficiency of H₂ production depends on several factors, nitrogen compound available in medium and high salt stress could immediately decrease H₂ production in A. siamensis TISTR 8012. Optimizing key factors affecting H₂ production such as cell age, light intensity, time of light incubation and source of carbon resulted in enhanced H₂ production. The maximum H₂ production was about 32 µmolH₂ mg chl a⁻¹h⁻¹ when cells at log phase were adapted in BG11₀ medium without N-source supplemented with 0.5% fructose and incubated under continuous illumination of 200µE m⁻² s⁻¹ for 12 h under anaerobic condition. this rate was higher than that observed in the model organisms Anabaena PCC 7120, Nostoc punctiforme ATCC 29133 and Synechocystis PCC 6803. This higher production was likely caused by higher nitrogenase and bidirectional hydrogenase activities. Light and fructose were found to induce activity of nitrogenase. A study of sugar uptake by A. siamensis TISTR 8012 showed that fructose was taken up by cells more than other sugars tested. However, The production of H₂ did not increase after 12 h of light incubation which was probably due to an increased uptake hydrogenase activity, indicating that a proper adjustment of light conditions such as intensity and duration is important to minimize both the photodamage of the cells and the uptake hydrogenase activity. Nevertheless, the main obstacle for high H₂ production by A. siamensis is H₂ consumption by uptake hydrogenase under light condition. To overcome this, we created the hydrogen uptake deficient mutant by interrupting the hypS gene with antibiotic resistance cassette. We could demonstrate that the mutant strain produced about 3-folds more H₂ than wild type strain. Moreover, The ∆hupS mutant could sustain H₂ production under light exposure for long period with about 2-folds higher activity of nitrogenase when compared to wild type. Gene expression analysis by RT-PCR showed that more electrons and ATP molecules required for H₂ production in ∆hupS mutant may be obtained from the electron transport chain associated with the photosynthetic oxidation of water in PSII in vegetative cells. The results obtained from this study indicated that A. siamensis TISTR 8012 has a high potential for H₂ production leading to further development for the production of biohydrogen in an expanded scale, thus serving as a renewable and clean energy source for the future.