Screening of hydrogen-producing algae and optimization for enhanced hydrogen production

Abstract

Water samples collected from fresh water sources and rice paddy fields located in central part of Thailand were used in screening for hydrogen-producing green microalgae. One out of 210 isolates was identified as belonging to the genus Tetraspora under light microscope for morphology identification. Confirmation by phylogenetic analysis of 18S rDNA sequence revealed that the green alga, identified as Tetraspora sp. CU2551, is closely related to other unicellular green algal species. Tetraspora sp. CU2551 had the shortest doubling time when grown in Tris-Acetate-Phosphate (TAP) medium under a light intensity of 37 - 92 µE/m2/s and a temperature of 36 oC. A 24 h culture under light intensity of 37 µE/m2/s showed the optimal hydrogen production rate when cells were incubated at 35 oC. The production under light incubation increased with increasing pH from 5.75 to 9.30; however, almost no production was observed at a pH of 5.25. Addition of 0.5 mM β-mercaptoethanol to the TAP medium stimulated hydrogen production rate about 2-fold. During the hydrogen production phase, the use of TAP medium lacking both nitrogen and sulfur resulted in about 50% increase in the hydrogen production. This was in contrast to only a small increase in the production when either nitrogen or sulfur was omitted in TAP medium. The stimulation of hydrogen production by 0.5 mM β-mercaptoethanol under nitrogen- and sulfur-deprived conditions occurred only when the cells were grown at a light intensity lower than 5 µE/m2/s with no effects at higher intensities. Incubating the culture in TAP-N-S medium under a light intensity of 29 µE/m2/s gave a maximal calculated hydrogen production of 17.3 - 61.7 µmol/mg Chl a/h. This is a very high production rate compared to other green algae and makes Tetraspora sp. CU2551 an interesting model strain for photobiological hydrogen production. The newly identified chloroplast envelope-localized sulfate permease gene (sulP) and the hydrogenase gene (hydA) from the green alga Tetraspora sp. CU2551 are reported in this study. The sulP showed an open reading frame of 1,014 bp with the 5’- and 3’ UTR being 285 and 225 bp, respectively. Tetraspora sulP also contained four introns, whereas other known photosynthetic organisms, except Chlamydomonas reinhardtii, show no intron at the DNA level. The deduced amino acid sequence of SulP revealed an extended N-terminus where the putative chloroplast transit peptide was identified. This suggests a close relationship between Tetraspora and Chlamydomonas reinhardtii SulPs, as confirmed by phylogenetic tree analysis. In addition, the Tetraspora hydA was identified. The cDNA sequence showed an 878 bp encoding 292 amino acid residues. Two introns of 261 and 282 bp, respectively were also found in hydA structural gene. The deduced amino acid sequence of Tetraspora HydA is closely related to HydA of Chlorella fusca. The transcript levels of both sulP and hydA of Tetraspora showed an up-regulation of about 2.3 times after sulfur deprivation, whereas upon sulfur repletion the expression of both genes decreased. The production of H2 and PSII activity decreased in cells grown under sulfur-deprived condition. These two activities could be restored upon transferring the cells to sulfur-replete medium. Our results highlight the importance of sulfur for the regulation of hydrogen metabolism in Tetraspora sp. CU 2551.