Browsing by Author "Gardner, Robert D."

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Assessment of Nannochloropsis gaditana growth and lipid accumulation with increased inorganic carbon delivery
(2018-04) Pedersen, Todd C.; Gardner, Robert D.; Peyton, Brent M.
Algal biomass refineries for sustainable transportation fuels, in particular biodiesel, will benefit from algal strain enhancements to improve biomass and lipid productivity. Specifically, the supply of inorganic carbon to microalgal cultures represents an area of great interest due to the potential for improved growth of microalgae and the possibility for incorporation with CO2 mitigation processes. Combinations of bicarbonate (HCO3−) salt addition and application of CO2 to control pH have shown compelling increases in growth rate and lipid productivity of fresh water algae. Here, focus was placed on the marine organism, Nannochloropsis gaditana, to investigate growth and lipid accumulation under various strategies of enhanced inorganic carbon supply. Three gas application strategies were investigated: continuous sparging of atmospheric air, continuous sparging of 5% CO2 during light hours until nitrogen depletion, and continuous sparging of atmospheric air supplemented with 5% CO2 for pH control between 8.0 and 8.3. These gas sparging schemes were combined with addition of low concentrations (5 mM) of sodium bicarbonate at inoculation and high concentration (50 mM) of sodium bicarbonate amendments just prior to nitrogen depletion. The optimum scenario observed for growth of N. gaditana under these inorganic carbon conditions was controlling pH with 5% CO2 on demand, which increased both growth rate and lipid accumulation. Fatty acid methyl esters were primarily comprised of C16:0 (palmitic) and C16:1 (palmitoleic) aliphatic chains. Additionally, the use of high concentration (50 mM) of bicarbonate amendments further improved lipid content (up to 48.6%) under nitrogen deplete conditions when paired with pH-controlled strategies.
Carbon partitioning in lipids synthesized by Chlamydomonas reinhardtii when cultured under three unique inorganic carbon regimes
(2014-07) Lohman, Egan J.; Gardner, Robert D.; Halverson, L.; Peyton, Brent M.; Gerlach, Robin
Inorganic carbon is a fundamental component for microalgal lipid biosynthesis. Understanding how the concentration and speciation of dissolved inorganic carbon (DIC) influences lipid metabolism in microalgae may help researchers optimize the production of these high value metabolites. Using relatively straight forward methods for quantifying free fatty acids (FFAs), mono- (MAG), di- (DAG), tri-acylglycerides (TAG), and total cellular fatty acids (FAME), lipid profiles over time were established for Chlamydomonas reinhardtii when grown under three unique inorganic carbon regimes. Specifically, cultures sparged with atmospheric air were compared to cultures which were sparged with 5% CO2 (v/v) and cultures supplemented with 50 mM NaHCO3 just prior to medium nitrogen depletion. All three conditions exhibited similar lipid profiles prior to nitrogen depletion in the medium, with FFA and MAG being the predominant lipid metabolites. However, these precursors were quickly reallocated into DAG and subsequently TAG after nitrogen depletion. C16 DAG did not accumulate significantly in any of the treatments, whereas the C18 DAG content increased throughout both exponential growth and stationary phase. C16 and C18 TAG began to accumulate after nitrogen depletion, with C16 TAG contributing the most to overall TAG content. C16 fatty acids exhibited a shift towards saturated C16 fatty acids after nitrogen depletion. Results provide insight into inorganic carbon partitioning into lipid compounds and how the organism's lipid metabolism changes due to N-deplete culturing and inorganic carbon source availability. The methodologies and findings presented here may be adapted to other organisms with high industrial relevance.
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 40â€“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.
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
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 chlorophyteScenedesmus 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.
Combining multiple nutrient stresses and bicarbonate addition to promote lipid accumulation in the diatom RGd-1
(2014-07) Moll, Karen M.; Gardner, Robert D.; Eustance, E. O.; Gerlach, Robin; Peyton, Brent M.
Algal biofuels represent a renewable, potentially viable, solution to mitigate transportation fuel demands. A novel diatom strain, RGd-1, isolated from Yellowstone National Park, produces high concentrations of lipids that can be converted to biodiesel. To increase the cell concentration and determine optimal conditions for growth, RGd-1 was grown without added Si, in the presence of four Si concentrations within the soluble range (0.5â€“2 mM), and one above the soluble range (2.5 mM). Medium Si concentrations and intracellular triacylglycerol (TAG) content were monitored daily by inductively coupled plasma mass spectrometry and Nile Red fluorescence, respectively (end-point TAG values were measured using gas chromatography). Si depletion with or without combined nitrate (NO3âˆ’) limitation was shown to induce TAG accumulation. Additionally, the effects of sodium bicarbonate (NaHCO3) supplementation were examined on cultures grown using two NO3âˆ’ concentrations (2.94 and 1 mM NO3âˆ’), which also resulted in increased TAG accumulation. It was determined that utilizing a combination of two independent physiological stresses and HCO3âˆ’ supplementation resulted in the highest total and per cell TAG accumulation.
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.
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.
Dissolved inorganic carbon enhanced growth, nutrient uptake, and lipid accumulation in wastewater grown microalgal biofilms
(2015-03) Kesaano, M.; Gardner, Robert D.; Moll, Karen M.; Lauchnor, Ellen G.; Gerlach, Robin; Peyton, Brent M.; Sims, R. C.
Microalgal biofilms grown to evaluate potential nutrient removal options for wastewaters and feedstock for biofuels production were studied to determine the influence of bicarbonate amendment on their growth, nutrient uptake capacity, and lipid accumulation after nitrogen starvation. No significant differences in growth rates, nutrient removal, or lipid accumulation were observed in the algal biofilms with or without bicarbonate amendment. The biofilms possibly did not experience carbon-limited conditions because of the large reservoir of dissolved inorganic carbon in the medium. However, an increase in photosynthetic rates was observed in algal biofilms amended with bicarbonate. The influence of bicarbonate on photosynthetic and respiration rates was especially noticeable in biofilms that experienced nitrogen stress. Medium nitrogen depletion was not a suitable stimulant for lipid production in the algal biofilms and as such, focus should be directed toward optimizing growth and biomass productivities to compensate for the low lipid yields and increase nutrient uptake.
An efficient and scalable extraction and quantification method for algal derived biofuel
(2013-09) Lohman, Egan J.; Gardner, Robert D.; Halverson, L.; Macur, Richard E.; Peyton, Brent M.; Gerlach, Robin
Microalgae are capable of synthesizing a multitude of compounds including biofuel precursors and other high value products such as omega-3-fatty acids. However, accurate analysis of the specific compounds produced by microalgae is important since slight variations in saturation and carbon chain length can affect the quality, and thus the value, of the end product. We present a method that allows for fast and reliable extraction of lipids and similar compounds from a range of algae, followed by their characterization using gas chromatographic analysis with a focus on biodiesel-relevant compounds. This method determines which range of biologically synthesized compounds is likely responsible for each fatty acid methyl ester (FAME) produced; information that is fundamental for identifying preferred microalgae candidates as a biodiesel source. Traditional methods of analyzing these precursor molecules are time intensive and prone to high degrees of variation between species and experimental conditions. Here we detail a new method which uses microwave energy as a reliable, single-step cell disruption technique to extract lipids fromlive cultures of microalgae. After extractable lipid characterization (including lipid type (free fatty acids, mono-, di- or tri-acylglycerides) and carbon chain length determination) by GCâ€“FID, the same lipid extracts are transesterified into FAMEs and directly compared to total biodiesel potential by GCâ€“MS. This approach provides insight into the fraction of total FAMEs derived from extractable lipids compared to FAMEs derived fromthe residual fraction (i.e. membrane bound phospholipids, sterols, etc.). This approach can also indicate which extractable lipid compound, based on chain length and relative abundance, is responsible for each FAME. This method was tested on three species of microalgae: the marine diatom Phaeodactylum tricornutum, the model Chlorophyte Chlamydomonas reinhardtii, and the freshwater green alga Chlorella vulgaris. The method is shown to be robust, highly reproducible, and fast, allowing for multiple samples to be analyzed throughout the time course of culturing, thus providing time-resolved information regarding lipid quantity and quality. Total time from harvesting to obtaining analytical results is less than 2 h.
Evaluating the relative impacts of operational and financial factors on the competitiveness of an algal biofuel production facility
(2016-11) Hise, Adam M.; Characklis, William G.; Kern, L.; Gerlach, Robin; Viamajala, Sridhar; Gardner, Robert D.; Vadlamani, A.
Algal biofuels are becoming more economically competitive due to technological advances and government subsidies offering tax benefits and lower cost financing. These factors are linked, however, as the value of technical advances is affected by modeling assumptions regarding the growth conditions, process design, and financing of the production facility into which novel techniques are incorporated. Two such techniques, related to algal growth and dewatering, are evaluated in representative operating and financing scenarios using an integrated techno-economic model. Results suggest that these techniques can be valuable under specified conditions, but also that investment subsidies influence cost competitive facility design by incentivizing development of more capital intensive facilities (e.g., favoring hydrothermal liquefaction over transesterification-based facilities). Evaluating novel techniques under a variety of operational and financial scenarios highlights the set of site-specific conditions in which technical advances are most valuable, while also demonstrating the influence of subsidies linked to capital intensity.
Growth, nitrogen utilization and biodiesel potential for two chlorophytes grown on ammonium, nitrate or urea
(2013-03) Eustance, E. O.; Gardner, Robert D.; Moll, Karen M.; Menicucci, Joseph A. Jr.; Gerlach, Robin; Peyton, Brent M.
Nitrogen removal from wastewater by algae provides the potential benefit of producing lipids for biodiesel and biomass for anaerobic digestion. Further, ammonium is the renewable form of nitrogen produced during anaerobic digestion and one of the main nitrogen sources associated with wastewater. The wastewater isolates Scenedesmus sp. 131 and Monoraphidium sp. 92 were grown with ammonium, nitrate, or urea in the presence of 5 % CO2, and ammonium and nitrate in the presence of air to optimize the growth and biofuel production of these chlorophytes. Results showed that growth on ammonium, in both 5 % CO2 and air, caused a significant decrease in pH during the exponential phase, causing growth inhibition due to the low buffering capacity of the medium. Therefore, biological buffers and pH controllers were utilized to prevent a decrease in pH. Growth on ammonium with pH control (synthetic buffers or KOH dosing) demonstrated that growth (rate and yield), biodiesel production, and ammonium utilization, similar to nitrate- and urea-amended treatments, can be achieved if sufficient CO2 is available. Since the use of buffers is economically limited to laboratory-scale experiments, chemical pH control could bridge the gap encountered in the scale-up to industrial processes.
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.
Optimized Inorganic Carbon Regime for Enhanced Growth and Lipid Accumulation in Chlorella Vulgaris
(2015-06) Lohman, Egan J.; Gardner, Robert D.; Pedersen, Todd C.; Peyton, Brent M.; Cooksey, Keith E.; Gerlach, Robin
Background Large-scale algal biofuel production has been limited, among other factors, by the availability of inorganic carbon in the culture medium at concentrations higher than achievable with atmospheric CO 2 . Life cycle analyses have concluded that costs associated with supplying CO 2 to algal cultures are significant contributors to the overall energy consumption. Results A two-phase optimal growth and lipid accumulation scenario is presented, which (1) enhances the growth rate and (2) the triacylglyceride (TAG) accumulation rate in the oleaginous Chlorophyte Chlorella vulgaris strain UTEX 395, by growing the organism in the presence of low concentrations of NaHCO 3 (5 mM) and controlling the pH of the system with a periodic gas sparge of 5 % CO 2 (v/v). Once cultures reached the desired cell densities, which can be “fine-tuned” based on initial nutrient concentrations, cultures were switched to a lipid accumulation metabolism through the addition of 50 mM NaHCO 3 . This two-phase approach increased the specific growth rate of C. vulgaris by 69 % compared to cultures sparged continuously with 5 % CO 2 (v/v); further, biomass productivity (g L −1 day −1 ) was increased by 27 %. Total biodiesel potential [assessed as total fatty acid methyl ester (FAME) produced] was increased from 53.3 to 61 % (FAME biomass −1 ) under the optimized conditions; biodiesel productivity (g FAME L −1 day −1 ) was increased by 7.7 %. A bicarbonate salt screen revealed that American Chemical Society (ACS) and industrial grade NaHCO 3 induced the highest TAG accumulation (% w/w), whereas Na 2 CO 3 did not induce significant TAG accumulation. NH 4 HCO 3 had a negative effect on cell health presumably due to ammonia toxicity. The raw, unrefined form of trona, NaHCO 3 ∙Na 2 CO 3 (sodium sesquicarbonate) induced TAG accumulation, albeit to a slightly lower extent than the more refined forms of sodium bicarbonate. Conclusions The strategic addition of sodium bicarbonate was found to enhance growth and lipid accumulation rates in cultures of C. vulgaris, when compared to traditional culturing strategies, which rely on continuously sparging algal cultures with elevated concentrations of CO 2(g) . This work presents a two-phased, improved photoautotrophic growth and lipid accumulation approach, which may result in an overall increase in algal biofuel productivity.
Sources and Resources: Importance of nutrients, resource allocation, and ecology in microalgal cultivation for lipid accumulation
(2014-04) Fields, Matthew W.; Hise, Adam M.; Lohman, Egan J.; Bell, Tisza A. S.; Gardner, Robert D.; Corredor, Luisa; Moll, Karen M.; Peyton, Brent M.; Characklis, Greg W.; Gerlach, Robin
Regardless of current market conditions and availability of conventional petroleum sources, alternatives are needed to circumvent future economic and environmental impacts from continued exploration and harvesting of conventional hydrocarbons. Diatoms and green algae (microalgae) are eukaryotic photoautotrophs that can utilize inorganic carbon (e.g., CO2) as a carbon source and sunlight as an energy source, and many microalgae can store carbon and energy in the form of neutral lipids. In addition to accumulating useful precursors for biofuels and chemical feed stocks, the use of autotrophic microorganisms can further contribute to reduced CO2 emissions through utilization of atmospheric CO2. Because of the inherent connection between carbon, nitrogen, and phosphorus in biological systems, macronutrient deprivation has been proven to significantly enhance lipid accumulation in different diatom and algae species. However, much work is needed to understand the link between carbon, nitrogen, and phosphorus in controlling resource allocation at different levels of biological resolution (cellular versus ecological). An improved understanding of the relationship between the effects of N, P, and micronutrient availability on carbon resource allocation (cell growth versus lipid storage) in microalgae is needed in conjunction with life cycle analysis. This mini-review will briefly discuss the current literature on the use of nutrient deprivation and other conditions to control and optimize microalgal growth in the context of cell and lipid accumulation for scale-up processes.
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 worldâ€™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 lightâ€“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 dâˆ’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.
Using life cycle assessment and techno-economic analysis in a real options framework to inform the design of algal biofuel production facilities
(2017-02) Kern, Jordan D.; Hise, Adam M.; Characklis, William G.; Gerlach, Robin; Viamajala, Sridhar; Gardner, Robert D.
This study investigates the use of “real options analysis” (ROA) to quantify the value of greater product flexibility at algal biofuel production facilities. A deterministic optimization framework is integrated with a combined life cycle assessment/techno-economic analysis model and subjected to an ensemble of 30-year commodity price trajectories. Profits are maximized for two competing plant configurations: 1) one that sells lipid-extracted algae as animal feed only; and 2) one that can sell lipid-extracted algae as feed or use it to recover nutrients and energy, due to an up-front investment in anaerobic digestion/combined heat and power. Results show that added investment in plant flexibility does not result in an improvement in net present value, because current feed meal prices discourage use of lipid-extracted algae for nutrient and energy recovery. However, this study demonstrates that ROA provides many useful insights regarding plant design that cannot be captured via traditional techno-economic modeling.