Enhancing the metabolic capacity of cyanobacteria for biological hydrogen production: Biofuel applications of Cyanothece and Arthrospira spp

The dissertation presented herein focuses on understanding the biological pathways leading to hydrogen production in cyanobacteria and optimizing their capacity to funnel intracellular reductant to hydrogen.;In Chapter 1, we characterize hydrogen production from the nitrogen-fixing (diazotrophic) cyanobacterium Cyanothece sp. Miami BG 043511. This organism is capable of producing H2 via the bidirectional hydrogenase as well as the nitrogenase complex. We illustrate that dark, autofermentative H2 production is catalyzed by the hydrogenase, whereas photo-fermentative H2 production (photo-H2) is catalyzed by nitrogenase and is dependent upon excitation of Photosystem I. Internal carbohydrate reserves supply the reductant for both pathways; this reductant is therefore shared between the two H2 producing modes. We illustrate that by allowing increased time for autofermentation, the available reductant (generated from carbohydrate catabolism) is increased and an increased photo-H2 production rate is observed.;In Chapter 2, we advance our understanding of Cyanothece sp. Miami BG 043511 and demonstrate methods to increase its catabolic rate. Internal carbohydrate catabolism serves as the source of reductant for biological H2 production from this strain, and by increasing the rate of catabolism in the strain we are able to supply increased reductant to hydrogenase and nitrogenase and subsequently observe increased H2 yield. We demonstrate that the addition of "ATP sinks" to the cell under fermentative conditions (e.g. cyanophycin synthesis and/or amino acid import) causes an increased redox poise of the cell, due to increased glycolytic flux to generate ATP by substrate level phosphorylation. While increased H2 production is observed under conditions showing a moderate increase in redox poise, further increased redox stress causes the cell to increase production of ethanol and formate, fermentative pathways that recycle NADH to NAD+.;In Chapter 3, we present a two channel instrument that allows the simultaneous detection of dissolved H2 (measured electrochemically) and intracellular NAD(P)H concentration (measured by NAD(P)H fluorescence). This tool is applied to the cyanobacterium Arthrospira maxima and utilized to study its metabolic response under fermentative conditions to the availability of the macronutrient nitrate. Nitrate is shown to compete with protons for intracellular reductant; a clock-like delay is observed where all extracellular nitrate must be consumed prior to the onset of fermentative H2 production. Moreover, nitrate is shown to induce a metabolic switch from the glycolytic to the oxidative pentose phosphate (OPP) pathway. This pathway generates more reductant per glucose equivalent catabolized, but provides this reductant in the form of NADPH rather than NADH, the latter being the obligate substrate for hydrogenase.;In Chapter 4, we continue our studies with Arthrospira maxima and illustrate methods to increase the yield of autofermentative H2 production by eliminating H2 backpressure. We define the continuous removal of H2 as "milking" and illustrate that by selective "milking" of H2 by its electrochemical consumption produces an increase in H2 yield (11-fold) and rate (3.4-fold). Smaller increases are observed when H2 is "milked" non-selectively by dilution of the biomass in the incubation media (3.7-fold yield increase, 3.1-fold rate increase). The addition of a mixture of excreted carbon fermentative products (lactate, acetate, and ethanol) that form in competition with other NADH sinks is shown to increase H2 yield by 1.4-fold.;Lastly, in Chapter 5, we illustrate degenerate primers to amplify a fragment of the large subunit of the bidirectional hydrogenase ([NiFe] hydrogenase) from cyanobacteria. Two established limitations of [NiFe] hydrogenases are their oxygen sensitivity and relatively slow turnover rates. These primers will allow the screening of large collections of cyanobacterial culture collections for novel hydrogenases that may not suffer from these limitations. (Abstract shortened by UMI.)...