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Evaluation of wastewater as a nutrient source for the cultivation of the model cyanobacterium Synechocystis sp. PCC6803




Hughes, Alexander, author
Peers, Graham, advisor
Sloan, Daniel, committee member
Reardon, Kenneth, committee member

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The rising demand for more sustainable and renewable energy sources has led to the development of using photosynthetic microalgae and cyanobacteria for biofuel feedstocks. Microalgae and cyanobacteria offer an attractive solution over the cellulosic and lignocellulosic feedstocks of first and second generation biofuels that compete for arable land, nutrients and water necessary for sufficient food crop production. It has been proposed for several decades that wastewater could be a sustainable and affordable source of water and nutrients. Phycoremediation of wastewater by microalgae as biofuel feedstocks could provide beneficial environmental health impacts by preventing eutrophication of fresh water supplies. Many eukaryotic microalgae have been grown in diluted and/or modified wastewaters. The growth of cyanobacteria on wastewaters has not been nearly as well characterized. Cyanobacteria grown on wastewaters could be an ideal feedstock for advanced biofuels, since cyanobacteria have a more extensively established molecular toolbox for genetic engineering. The first aim of this thesis was to evaluate wastewater centrate as a growth medium for the cultivation of the cyanobacterium Synechocystis sp. strain PCC6803 (Chapter 1). Centrate was collected from the Drake Water Reclamation Facility (Fort Collins, CO) and filter sterilized to allow axenic culturing of PCC6803 under controlled laboratory conditions. PCC6803 was grown in up to 21% filtered centrate diluted in sterile water; while higher concentrations completely inhibited growth. Nitrogen drawdown from centrate by PCC6803 was then analyzed. Surprisingly, the drawdown of nitrogen from the centrate media correlated poorly with the amount of cyanobacterial biomass. The cell densities of cultures grown in centrate were all significantly lower than that of PCC6803 grown in BG-11 indicating that diluted centrate does not provide adequate nutrients to support optimal growth. Abiotic precipitation of nitrogen was then determined to dominate the removal of nitrogen from the cultivation media. Furthermore, it suggested that centrate lacks a critical nutrient to support robust growth of PCC6803. The second aim of this thesis was to augment the nutrient composition of wastewater in order to optimize PCC6803 growth and nutrient removal (Chapter 2). A series of bioassays were performed to elucidate the limiting macronutrient in centrate. Adding 304 μM Na2SO4 – equivalent to the concentration of SO42in BG-11 media – yielded final cell densities that were only 4% lower than those observed in cultures grown in the synthetic, standard media (BG-11). Exogenous supplementation of Na2SO4 also improved total dissolved nitrogen (TN) drawdown for centrate grown PCC6803 cultures. In Na2SO4 amended centrate, PCC6803 was able to grow to significantly higher cell densities, permitting the removal of 69% of the TN in diluted centrate. Transcript abundance of the sulfate transporters encoded by the spbA-cysTWA operon were found to be upregulated when grown in centrate, confirming that PCC6803 experienced S-limitation during growth on this media. Hydrogen sulfide gas (H2S) is an undesirable product of the biological nutrient removal process due to its pungent odor. Currently, H2S produced at the DWRF is vented to biofilters consisting of wood chips and compost where sulfide oxidizing microbes convert sulfide into elemental sulfur. Therefore, endogenously sourced sulfur supplementation from H2S into centrate could provide a viable sulfur source to support the cultivation of PCC6803. We have shown that sulfur supplementation improves the phycoremediation of nutrients in centrate. Cultivation of PCC6803 on centrate supplemented with endogenously sourced sulfur provides an industrially feasible method for combining wastewater treatment with advanced biofuel production.


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