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Item Influence of calcium and other cations on surface adhesion of bacteria and diatoms: a review(2000-05) Geesey, Gill G.; Wigglesworth-Cooksey, Barbara; Cooksey, Keith E.Association with a surface is an important aspect of survival for microorganisms in natural and manmade environments. Both bacteria and diatoms are involved in such associations. In many cases, this leads to surface fouling, which often results in surface deterioration and mechanical failure in industrial systems. We now know that microorganisms exploit many strategies to establish associations with surfaces. As in the case of other cellular processes, calcium ions seem to play an important role in adhesion of cells to surfaces. Calcium is involved in non-specific interactions such as neutralization of the electrical double layer between cell and substratum surface as well as specific adhesive interactions that cannot be replaced by other cations. The unique properties of calcium ions promote both specific and non-specific interactions with protein and polysaccharide adhesin molecules at the cell surface. As important, but less well understood, calcium ions also influence the way microbial cells interact with different substrata.Item Biofilms and microbial fouling(1983) Characklis, William G.; Cooksey, Keith E.Microbial cells attach firmly to almost any surface submerged in an aquatic environment. The immobilized cells grow, reproduce, and produce extracellular polymer substances (EPS) that frequently extend from the cell, forming a tangled mass of fibers lending structure to the entire assemblage which shall be termed a biofilm. The term biofilm does not necessarily imply a surface accumulation that is uniform in time and/or space.Item Influence of a calcium-specific chelant on biofilm removal(1983-11) Turakhia, Mukesh Harilal; Cooksey, Keith E.; Characklis, William G.This paper describes the influence of ethylene glycol-bis(β-aminoethyl ether)-N,N-tetraacetic acid (EGTA) on biofilm removal. The addition of EGTA resulted in the immediate detachment of biofilm which suggests that the chelant removed essential calcium from the biofilm, causing it to detach.Item Medium pH and nitrate concentration effects on accumulation of triacylglycerol in two members of the chlorophyta(2010-12) Gardner, Robert D.; Peters, P.; Peyton, Brent M.; Cooksey, Keith E.Algal-derived biodiesel is of particular interest because of several factors including: the potential for a near-carbon-neutral life cycle, the prospective ability for algae to capture carbon dioxide generated from coal, and algae’s high per acre yield potential. Our group and others have shown that in nitrogen limitation, and for a single species of Chlorella, a rise in culture medium pH yields triacylglycerol (TAG) accumulation. To solidify and expand on these triggers, the influence and interaction of pH and nitrogen concentration on lipid production was further investigated on Chlorophyceae Scenedesmus sp. and Coelastrella sp. Growth was monitored optically and TAG accumulation was monitored by Nile red fluorescence and confirmed by gas chromatography. Both organisms grew in all treatments and TAG accumulation was observed by two distinct conditions: high pH and nitrogen limitation. The Scenedesmus sp. was shown to grow and produce lipids to a larger degree in alkaliphilic conditions (pH >9) and was used to further investigate the interplay between TAG accumulation from high pH and/or nitrate depletion. Results given here indicate that TAG accumulation per cell, monitored by Nile red fluorescence, correlates with pH at the time of nitrate depletion.Item Use of sodium bicarbonate to stimulate triacylglycerol accumulation in the chlorophyte Scenedesmus sp. and the diatom Phaeodactylum tricornutum(2012-10) Gardner, Robert D.; Cooksey, Keith E.; Mus, Florence; Macur, Richard E.; Moll, Karen M.; Eustance, E. O.; Carlson, Ross P.; Gerlach, Robin; Fields, Matthew W.; Peyton, Brent M.There is potential for algal-derived biofuel to help alleviate part of the world’s dependency on petroleum based fuels. However, research must still be done on strain selection, induction of triacylglycerol (TAG) accumulation, and fundamental algal metabolic studies, along with large-scale culturing techniques, harvesting, and biofuel/biomass processing. Here, we have advanced the knowledge on Scenedesmus sp. strain WC-1 by monitoring growth, pH, and TAG accumulation on a 14:10 light–dark cycle with atmospheric air or 5% CO2 in air (v/v) aeration. Under ambient aeration, there was a loss of pH-induced TAG accumulation, presumably due to TAG consumption during the lower culture pH observed during dark hours (pH 9.4). Under 5% CO2 aeration, the growth rate nearly doubled from 0.78 to 1.53 d−1, but the pH was circumneutral (pH 6.9) and TAG accumulation was minimal. Experiments were also performed with 5% CO2 during the exponential growth phase, which was then switched to aeration with atmospheric air when nitrate was close to depletion. These tests were run with and without the addition of 50 mM sodium bicarbonate. Cultures without added bicarbonate showed decreased growth rates with the aeration change, but there was no immediate TAG accumulation. The cultures with bicarbonate added immediately ceased cellular replication and rapid TAG accumulation was observed, as monitored by Nile Red fluorescence which has previously been correlated by gas chromatography to cellular TAG levels. Sodium bicarbonate addition (25 mM final concentration) was also tested with the marine diatom Phaeodactylum tricornutum strain Pt-1 and this organism also accumulated TAG.Item Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum(2012-06) Valenzuela, Jacob J.; Mazurie, Aurélien J.; Carlson, Ross P.; Gerlach, Robin; Cooksey, Keith E.; Peyton, Brent M.; Fields, Matthew W.BACKGROUND: Phaeodactylum tricornutum is a unicellular diatom in the class Bacillariophyceae. The full genome hasbeen sequenced (<30 Mb), and approximately 20 to 30% triacylglyceride (TAG) accumulation on a dry cell basis hasbeen reported under different growth conditions. To elucidate P. tricornutum gene expression profiles duringnutrient-deprivation and lipid-accumulation, cell cultures were grown with a nitrate to phosphate ratio of 20:1 (N:P)and whole-genome transcripts were monitored over time via RNA-sequence determination.RESULTS: The specific Nile Red (NR) fluorescence (NR fluorescence per cell) increased over time; however, theincrease in NR fluorescence was initiated before external nitrate was completely exhausted. Exogenous phosphatewas depleted before nitrate, and these results indicated that the depletion of exogenous phosphate might be anearly trigger for lipid accumulation that is magnified upon nitrate depletion. As expected, many of the genesassociated with nitrate and phosphate utilization were up-expressed. The diatom-specific cyclins cyc7 and cyc10were down-expressed during the nutrient-deplete state, and cyclin B1 was up-expressed during lipid-accumulationafter growth cessation. While many of the genes associated with the C3 pathway for photosynthetic carbonreduction were not significantly altered, genes involved in a putative C4 pathway for photosynthetic carbonassimilation were up-expressed as the cells depleted nitrate, phosphate, and exogenous dissolved inorganic carbon(DIC) levels. P. tricornutum has multiple, putative carbonic anhydrases, but only two were significantly up-expressed(2-fold and 4-fold) at the last time point when exogenous DIC levels had increased after the cessation of growth.Alternative pathways that could utilize HCO-3 were also suggested by the gene expression profiles (e.g., putativepropionyl-CoA and methylmalonyl-CoA decarboxylases).CONCLUSION: The results indicate that P. tricornutum continued carbon dioxide reduction when population growthwas arrested and different carbon-concentrating mechanisms were used dependent upon exogenous DIC levels.Based upon overall low gene expression levels for fatty acid synthesis, the results also suggest that the build-up ofprecursors to the acetyl-CoA carboxylases may play a more significant role in TAG synthesis rather than the actualenzyme levels of acetyl-CoA carboxylases per se. The presented insights into the types and timing of cellularresponses to inorganic carbon will help maximize photoautotrophic carbon flow to lipid accumulation.Item Comparison of CO2 and bicarbonate as inorganic carbon sources for triacylglycerol and starch accumulation in Chlamydomonas reinhardtii(2013-01) Gardner, Robert D.; Lohman, Egan J.; Gerlach, Robin; Cooksey, Keith E.; Peyton, Brent M.Microalgae are capable of accumulating high levels of lipids and starch as carbon storage compounds. Investigation into the metabolic activities involved in the synthesis of these compounds has escalated because these compounds can be used as precursors for food and fuel. Here, we detail the results of a comprehensive analysis of Chlamydomonas reinhardtii using high or low inorganic carbon concentrations and speciation between carbon dioxide and bicarbonate, and the effects these have on inducing lipid and starch accumulation during nitrogen depletion. High concentrations of CO2 (5%;v/v) produced the highest amount of biofuel precursors, transesterified to fatty acid methyl esters, but exhibited rapid accumulation and degradation characteristics. Low CO2 (0.04%;v/v) caused carbon limitation and minimized triacylglycerol (TAG) and starch accumulation. High bicarbonate caused a cessation of cell cycling and accumulation of both TAG and starch that was more stable than the other experimental conditions. Starch accumulated prior to TAG and then degraded as maximum TAG was reached. This suggests carbon reallocation from starch-based to TAG-based carbon storage.Item Physiological and molecular analysis of carbon source supplementation and pH stress-induced lipid accumulation in the marine diatom Phaeodactylum tricornutum(2013-03) Mus, Florence; Toussaint, Jean-Paul; Cooksey, Keith E.; Fields, Matthew W.; Gerlach, Robin; Peyton, Brent M.; Carlson, Ross P.A detailed physiological and molecular analysis of lipid accumulation under a suite of conditions including nitrogen limitation, alkaline pH stress, bicarbonate supplementation, and organic acid supplementation was performed on the marine diatom Phaeodactylum tricornutum. For all tested conditions, nitrogen limitation was a prerequisite for lipid accumulation and the other culturing strategies only enhanced accumulation highlighting the importance of compounded stresses on lipid metabolism. Volumetric lipid levels varied depending on condition; the observed rankings from highest to lowest were for inorganic carbon addition (15 mM bicarbonate), organic acid addition (15 carbon mM acetate), and alkaline pH stress (pH9.0). For all lipidaccumulating cultures except acetate supplementation, a common series of physiological steps were observed. Upon extracellular nitrogen exhaustion, culture growth continued for approximately 1.5 cell doublings with decreases in specific protein and photosynthetic pigment content. As nitrogen limitation arrested cell growth, carbohydrate content decreased with a corresponding increase in lipid content. Addition of the organic carbon source acetate appeared to activate alternative metabolic pathways for lipid accumulation. Molecular level data on more than 50 central metabolism transcripts were measured using real-time PCR. Analysis of transcripts suggested the central metabolism pathways associated with bicarbonate transport, carbonic anhydrases, and C4 carbon fixations were important for lipid accumulation. Transcriptomic data also suggested that repurposing of phospholipids may play a role in lipid accumulation. This study provides a detailed physiological and molecular-level foundation for improved understanding of diatom nutrient cycling and contributes to a metabolic blueprint for controlling lipid accumulation in diatoms.Item Cellular cycling, carbon utilization, and photosynthetic oxygen production during bicarbonate-induced triacylglycerol accumulation in a Scenedesmus sp.(2013-11) Gardner, Robert D.; Lohman, Egan J.; Cooksey, Keith E.; Gerlach, Robin; Peyton, Brent M.Microalgae are capable of synthesizing high levels of triacylglycerol (TAG) which can be used as precursor compounds for fuels and specialty chemicals. Algal TAG accumulation typically occurs when cellular cycling is delayed or arrested due to nutrient limitation, an environmental challenge (e.g., pH, light, temperature stress), or by chemical addition. This work is a continuation of previous studies detailing sodium bicarbonate-induced TAG accumulation in the alkaline chlorophyte Scenedesmus sp. WC-1. It was found that upon sodium bicarbonate amendment, bicarbonate is the ion responsible for TAG accumulation; a culture amendment of approximately 15 mM bicarbonate was sufficient to arrest the cellular cycle and switch the algal metabolism from high growth to a TAG accumulating state. However, the cultures were limited in dissolved inorganic carbon one day after the amendment, suggesting additional carbon supplementation was necessary. Therefore, additional abiotic and biotic experimentation was performed to evaluate in- and out-gassing of CO2. Cultures to which 40–50 mM of sodium bicarbonate were added consumed DIC faster than CO2 could ingas during the light hours and total photosynthetic oxygen production was elevated as compared to cultures that did not receive supplemental inorganic carbon.Item Direct measurement and characterization of active photosynthesis zones inside wastewater remediating and biofuel producing microalgal biofilms(2014-03) Bernstein, Hans C.; Kessano, M.; Moll, Karen M.; Smith, Terrence; Gerlach, Robin; Carlson, Ross P.; Miller, Charles D.; Peyton, Brent M.; Cooksey, Keith E.; Gardner, Robert D.; Sims, R. C.Microalgal biofilm based technologies are of keen interest due to their high biomass concentrations and ability to utilize light and CO2. While photoautotrophic biofilms have long been used for wastewater remediation, biofuel production represents a relatively new and under-represented focus area. However, the direct measurement and characterization of fundamental parameters required for industrial control are challenging due to biofilm heterogeneity. This study evaluated oxygenic photosynthesis and respiration on two distinct microalgal biofilms cultured using a novel rotating algal biofilm reactor operated at field- and laboratory-scales. Clear differences in oxygenic photosynthesis and respiration were observed based on different culturing conditions, microalgal composition, light intensity and nitrogen availability. The cultures were also evaluated as potential biofuel synthesis strategies. Nitrogen depletion was not found to have the same effect on lipid accumulation compared to traditional planktonic microalgal studies. Physiological characterizations of these microalgal biofilms identify fundamental parameters needed to understand and control process optimization.