Effects of Extracellular pH on the Metabolic Pathways in Sulfur-Deprived H2-Producing Chlamydomonas Reinhardtii Cultures

Sustained photoproduction of H₂ by the green alga, Chlamydomonas reinhardtii, can be obtained by incubating cells in sulfur-deprived medium [Ghirardi et al. (2000b) Trends Biotechnol. 18: 506; Melis et al. (2000) Plant Physiol. 122: 127]. The current work focuses on (a) the effects of different initial extracellular pHs on the inactivation of photosystem II (PSII) and O₂-sensitive H₂-production activity in sulfur-deprived algal cells and (b) the relationships among H₂-production, photosynthetic, aerobic and anaerobic metabolisms under different pH regimens. The maximum rate and yield of H₂ production occur when the pH at the start of the sulfur deprivation period is 7.7 and decrease when the initial pH is lowered to 6.5 or increased to 8.2. The pH profile of hydrogen photoproduction correlates with that of the residual PSII activity (optimum pH 7.3-7.9), but not with the pH profiles of photosynthetic electron transport through photosystem I or of starch and protein degradation. In vitro hydrogenase activity over this pH range is much higher than the actual in situ rates of H₂ production, indicating that hydrogenase activity per se is not limiting. Starch and protein catabolisms generate formate, acetate and ethanol; contribute some reductant for H₂ photoproduction, as indicated by 3-(3,4-dichiorophenyl)-1,1-dimethylurea and 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone inhibition results; and are the primary sources of reductant for respiratory processes that remove photosynthetically generated O₂. Carbon balances demonstrate that alternative metabolic pathways predominate at different pHs, and these depend on whether residual photosynthetic activity is present or not.