ENHANCING THE METABOLIC CAPACITY OF CYANOBACTERIA FOR BIOLOGICAL HYDROGEN PRODUCTION: BIOFUEL APPLICATIONS OF CYANOTHECE AND ARTHROSPIRA SPP.

Abstract

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. With increasing global population and the continued development of previously unindustrialized nations, it is now more important than ever to meet the worldwide demand for energy to allow economies to continue to develop and grow. Coupling this demand to the decreasing availability of our finite reserves of fossil fuels and the increasing global climate, the development of clean and renewable energy sources is clearly a critical societal need. The biological production of hydrogen from cyanobacteria, photosynthetic bacteria which solely require minimal nutrients and sunlight to grow, is one promising approach to meeting this need. The energy-dense fuel hydrogen is completely carbon-free and can be used in combustion engines or for electricity generation through fuel cells. The production of hydrogen from nitrogen-fixing cyanobacteria (e.g. Cyanothece spp.) and non-nitrogen-fixing cyanobacteria (e.g. Arthrospira spp.) is explored within this dissertation.

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. For photo-H2, this reductant is supplied to Photosystem I via non-photochemical reduction of the plastoquinone pool by either of two NADH dehydrogenases. By inhibiting the class of NADH dehydrogenase that does not pump protons, we observe a 2-fold increase in photo-H2 production rate, due to the production of additional ATP (via the proton gradient).

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+. The growth of Cyanothece in medium with increased osmotic strength is shown to cause hyper-accumulation of osmolytes during photosynthetic growth and the subsequent hypotonic salt stress of these cells by transfer to low salt media correlates with increased photo-H2 production (maximal increase 9-fold). Thirdly, co-fermentation of Cyanothece with the lactic acid producing cyanobacterium Synechococcus sp. PCC 7002 (WT and lactate dehydrogenase overexpression mutant) causes increased H2 production from the mixed culture. A maximal increase of 2.5-fold is observed from the mixed culture of Cyanothece and the overexpression mutant; this increase is attributed to (a) providing Cyanothece with increased substrate (lactate) for photo-H2 production and (b) removing extracellular lactate, leading to an increase in the catabolic rate of the Synechococcus strain.

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. A mechanism for the utilization of this NADPH by a membrane associated nitrate reductase is presented. This pathway would not only utilize NADPH for nitrate reduction, but would also pump protons and lead to PMF dependent ATP generation (so called nitrate respiration). This is the first observation of nitrate respiration in cyanobacteria and supports the intense competition between nitrate and proton reduction in Arthrospira maxima.

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. We attribute this to shifting the reversible reaction equilibria toward substrate (NADH) and thereby increasing the availability of NADH to hydrogenase. Photosynthetic activity of the cultures after an exhaustive 7 day autofermentation is demonstrated to be completely recoverable by low intensity light incubation, allowing regeneration of carbohydrate reserves by subsequent growth/fermentation cycles.

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. The method described herein was applied to screen a collection of 24 axenic strains of cyanobacteria (from the University of Hawaii Culture Collection) and 10 strains were identified as containing hydrogenase. Two such strains (which not only exhibited presence of hydrogenase but also grew well in liquid culture) were shown to possess both in vitro and in vivo hydrogenase activity.

Throughout the thesis we illustrate methods to increase the biological production of H2 from the biotechnologically relevant strains of the genera Cyanothece and Arthrospira presented herein. We demonstrate physiological approaches to redirect metabolism for increased H2 yield and identify future targets (physiological and genetic) to be explored to further increase biofuel production from these strains and bring them closer to the realization of their biotechnological potential.