Optimization of metabolic capacity and flux through environmental cues to maximize hydrogen production by the Cyanobacterium "Arthrospira (Spirulina) maxima"

Environmental and nutritional conditions that optimize the yield of hydrogen (H₂) from water using a two-step photosynthesis/fermentation (P/F) process are reported for the hypercarbonate-requiring cyanobacterium "Arthrospira maxima." Our observations lead to four main conclusions broadly applicable to fermentative H₂ production by bacteria: (i) anaerobic H₂ production in the dark from whole cells catalyzed by a bidirectional [NiFe] hydrogenase is demonstrated to occur in two temporal phases involving two distinct metabolic processes that are linked to prior light-dependent production of NADPH (photosynthetic) and dark/anaerobic production of NADH (fermentative), respectively; (ii) H₂ evolution from these reductants represents a major pathway for energy production (ATP) during fermentation by regenerating NAD⁺ essential for glycolysis of glycogen and catabolism of other substrates; (iii) nitrate removal during fermentative H₂ evolution is shown to produce an immediate and large stimulation of H₂, as nitrate is a competing substrate for consumption of NAD(P)H, which is distinct from its slower effect of stimulating glycogen accumulation; (iv) environmental and nutritional conditions that increase anaerobic ATP production, prior glycogen accumulation (in the light), and the intracellular reduction potential (NADH/NAD⁺ ratio) are shown to be the key variables for elevating H₂ evolution. Optimization of these conditions and culture age increases the H₂ yield from a single P/F cycle using concentrated cells to 36 ml of H₂/g (dry weight) and a maximum 18% H₂ in the headspace. H₂ yield was found to be limited by the hydrogenase-mediated H₂ uptake reaction.