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Environmental and nutritional conditions that optimize the yield of hydrogen (H 2 ) 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 H 2 production by bacteria: (i) anaerobic H 2 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) H 2 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 H 2 evolution is shown to produce an immediate and large stimulation of H 2 , 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 H 2 evolution. Optimization of these conditions and culture age increases the H 2 yield from a single P/F cycle using concentrated cells to 36 ml of H 2 /g (dry weight) and a maximum 18% H 2 in the headspace. H 2 yield was found to be limited by the hydrogenase-mediated H 2 uptake reaction.

Hydrolysis of plant biomass generates a mixture of simple sugars that is particularly rich in glucose and xylose. Fermentation of the released sugars emits CO2 as byproduct due to metabolic inefficiencies. Therefore, the ability of a microbe to simultaneously convert biomass sugars and photosynthetically fix CO2 into target products is very desirable. In this work, the cyanobacterium, Synechocystis 6803, was engineered to grow on xylose in addition to glucose. Both the xylA (xylose isomerase) and xylB (xylulokinase) genes from Escherichia coli were required to confer xylose utilization, but a xylose-specific transporter was not required. Introduction of xylAB into an ethylene-producing strain increased the rate of ethylene production in the presence of xylose. Additionally, introduction of xylAB into a glycogen-synthesis mutant enhanced production of keto acids. Isotopic tracer studies found that nearly half of the carbon in the excreted keto acids was derived from the engineered xylose metabolism, while the remainder was derived from CO2 fixation.