Browsing by Author "Lohman, Egan J."

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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.
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.
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.
Evaluation of portability and cost of a fluorescent PCR ribotyping protocol for Clostridium difficile epidemiology
(2015-01) Martinson, Jonathan N.V.; Broadaway, Susan C.; Lohman, Egan J.; Johnson, Christina; Alam, M. Jahangir; Khaleduzzaman, Mohammed; Garey, Kevin W.; Schlackman, Jessica; Young, Vincent B.; Santhosh, Kavitha; Rao, Krishna; Lyons, Robert H. Jr; Walk, Seth T.
Clostridium difficile is the most commonly identified pathogen among health care-associated infections in the United States. There is a need for accurate and low-cost typing tools that produce comparable data across studies (i.e., portable data) to help characterize isolates during epidemiologic investigations of C. difficile outbreaks and sporadic cases of disease. The most popular C. difficile-typing technique is PCR ribotyping, and we previously developed methods using fluorescent PCR primers and amplicon sizing on a Sanger-style sequencer to generate fluorescent PCR ribotyping data. This technique has been used to characterize tens of thousands of C. difficile isolates from cases of disease. Here, we present validation of a protocol for the cost-effective generation of fluorescent PCR ribotyping data. A key component of this protocol is the ability to accurately identify PCR ribotypes against an online database (http://walklab.rcg.montana.edu) at no cost. We present results from a blinded multicenter study to address data portability across four different laboratories and three different sequencing centers. Our standardized protocol and centralized database for typing of C. difficile pathogens will increase comparability between studies so that important epidemiologic linkages between cases of disease and patterns of emergence can be rapidly identified.
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.