Oxygenic phototrophs have the ability to oxidize water via a well characterized photosystem complex yielding protons, oxygen, and electrons. Electrons and protons are used to fix carbon dioxide to produce reduced carbon substrates to be used for growth and energy storage. The use of aquatic microbial oxygenic phototrophs such as algae, cyanobacteria, or diatoms rather than land-based crops to produce fuels has been described as an advantageous potential strategy for carbon-neutral solar energy to fuel conversion.1 Cyanobacteria of the genus Arthrospira have particular advantages for mass-cultivation 2, and produce fermentative products such as ethanol and hydrogen that could be used as fuels. Here photosynthesis and fermentation were studied in the organism Arthrospira maxima (formerly known as Spirulina maxima) strain CS-328. This dissertation presents several strategies for control over photosynthesis and fermentation in A. maxima that elucidate the role of bicarbonate, nickel, sodium gradients, and osmostic stress.
Chapter 1 demonstrates A. maxima’s strong requirement for inorganic carbon for efficient Photosystem II turnover in vivo . The largest reversible bicarbonate effect on PSII activity ever observed is reported, which is due to the requirement for bicarbonate at the water oxidizing complex. This work also demonstrates a requirement for sodium-ion gradients in order to efficiently uptake inorganic carbon in the form of bicarbonate.
Chapter 2 demonstrates A. maxima’s requirement for low (1.5 μM) concentrations of nickel in growth medium for increased in vitro hydrogenase activity and in vivo hydrogen evolution rates and yields. This nickel requirement is shown to inhibit growth at high light levels, indicating a trade-off between high-light growth and high hydrogenase activities in this organism.
Chapter 3 reports a new method for studying fermentative product formation from microbial suspensions. All excreted fermentative products from A. maxima can be identified and quantified within 5.3% error using cryoprobe-assisted proton nuclear magnetic resonance spectroscopy in concentrations ranging from 50 μM – 3 mM with sampling times under 10 minutes. This method is used in chapters 4 and 5 to measure total fermentative end-product formation from cultures of A. maxima under conditions designed to increase fermentative flux.
In Chapter 4, excreted carbon products are detected during anaerobic metabolism under conditions that remove a sodium ion gradient. The results show that A. maxima utilizes on a sodium ion gradient under anaerobic conditions to perform an energy requiring process. Removal of this ion gradient causes cells to increase their cellular demand for ATP and thus increases carbohydrate consumption and total fermentative product formation by approximately 67%.
In Chapter 5, osmotic stress created by growing cells photoautotrophically in growth medium with added sodium (up to 1M additional NaCl) and fermenting in a hypotonic buffer is shown to increase sugar catabolism and fermentative product formation of some products. While hydrogen production is not increased by this strategy, ethanol formation increases by 121-fold. Hypotonic stress is thus a strategy for increasing mobilization of stored carbohydrates through fermentation.
1G. Charles Dismukes, Damian Carrieri, Nicholas Bennette, Gennady M. Ananyev, and Matthew C Posewitz. “Aquatic phototrophs: efficient alternatives to land-based crops for biofuels” Current Opinion in Biotechnology. 2008, 19, 235-240. 2Gennady Ananyev, Damian Carrieri, and G. Charles Dismukes. “Optimization of Metabolic Capacity and Flux through Environmental Cues To Maximize Hydrogen Production by the Cyanobacterium Arthrospira (Spirulina ) maxima” Applied and Environmental Microbiology. 2008, 74, (19), 6102-6113.