Grade 12 | Colorado
Grade 12 | Colorado
Microalgae hold great potential as a renewable source of oil for biofuel production, but there are many inherent difficulties involved in making their use commercially viable (Singh and Gu, 2010). Algae offer numerous advantages over other, more conventional oil sources: they can grow more rapidly and densely than land crops, they can be cultivated on non-arable land, and they have high rates of atmospheric carbon dioxide sequestration( Hu et al, 2008). However, algae oil yields must increase to give algae biofuels the economic feasibility necessary to impact our current dependence on fossil fuels (Raehtz, 2009).
Studies show that nitrate deprivation can decrease growth rates and increase lipid accumulation in algae by changing lipid metabolism (Wang et al, 2009). My previous work confirmed that environmental stress can inhibit growth while increasing lipid productivity. I also conducted experiments to characterize the biochemical processes underlying this shift in lipid metabolism, including a transcriptional study of the enzyme acetyl-CoA carboxylase (ACCase), which plays a crucial part in lipid synthesis (Ke et al, 2000). More work is necessary to thoroughly characterize multiple strains of algae at varying levels of nitrogen stress, along with a systematic refinement of lipid analytical techniques to ensure accurate results. Most critically, additional research must move past characterization steps to a scheme for the manipulation of the lipid metabolism to induce higher lipid synthesis (Griffiths and Harrison, 2009).
An essential part of the lipid biosynthetic pathway, the enzyme ACCase catalyzes the first committed step in the process of fatty acid synthesis: the conversion of acetyl-CoA to malonyl-CoA (Figure 1). Because of its rate-limiting role in lipid production, ACCase has been studied for its potential to effect higher algae oil yields, including some efforts to overexpress the native enzyme (Dunahay et al, 1995). However, due to the complex nature of lipid metabolism, it is likely that other factors involved in the ACCase mechanism also have critical roles to play in observed changes in lipid synthesis (Ohlrogge and Browse, 1995) (Nikolau et al, 2003). Direct genetic change requires a sophisticated understanding of the precise alterations in gene expression desired, along with knowledge of exactly how those changes will contribute to the sought-after phenotype. As such, attempts at targeted genetic manipulation, such as a study that introduced an extra copy of the ACCase gene into Cyclotella cryptica with the hope of improving lipid synthesis, have met with only limited success (Sheenan et al, 1998) (Raadakovits et al, 2010). My approach is distinct from those tactics. Inspired by a study of how these adaptations came about in the first place, I sought a more natural solution. Instead of struggling with a particular genetic manipulation, I sought to pressure the lipid synthetic pathway through phenotypic selection, forcing the organism to independently alter its own metabolism to increase lipid production.
As an alternative to direct genetic transformation, my work uses applied natural selection (artificial selection) to establish algae cell lines with higher lipid yields. The herbicide sethoxydim inhibits ACCase by competing for its binding site with acetyl-CoA (Figure 1). (Delye, 2005). In order to survive this stress, cells must increase their activity of ACCase to overcome obstructed lipid production. Hypothetically, treating algae populations with incrementally increasing levels of sethoxydim would select for cells with higher-than-normal ACCase activity and lipid productivity. Developing cell lines with sethoxydim tolerance through artificial selection can then isolate populations with heightened activity or expression of ACCase, with concomitant increases in lipid synthesis (Figure 2) (Parker et al, 1990). A similar selection strategy applied to foxtail millet cell cultures resulted in cell lines not only with increased ACCase expression, but with heightened lipid content as well (Dong et al, 2011).
This use of artificial selection to induce lipid accumulation in microalgae represents a novel approach to improving algae oil yields. It is my hope that my work could positively impact the feasibility and production of algae biofuel in the not-too-distant future.
The project goal was to use the ACCase-inhibitor sethoxydim to select for tolerant algae cultures with increased ACCase activity and lipid production. It was first necessary for me to establish protocols for measurement of (1) lipid content and (2) ACCase abundance and/or activity, so I could determine if heightened ACCase activity and lipid content were the results of sethoxydim selection. To accomplish this, I used nitrogen limitation as a control condition that has been shown to increase lipid synthetic activity and oil content of algae cells (Hu and Gao, 2006). I also characterized the cellular effects of sethoxydim to determine effective selection treatments. Finally, I employed my artificial selection technique, using sethoxydim to isolate cell lines with unusually high lipid synthesis. These populations required characterization to confirm the basis for their successful selection.
Materials and Methods
Culture Maintenance and Monitoring: Nannochloropsis salina, chosen for its high oil production, was used for all sethoxydim experimentation (the cultures were obtained from my own established cell lines; the seed culture was initially obtained in the spring of 2011 from AlgaGen LLC). Tetraselmis suecica and Scenedesmus obliquus, obtained from and used at the U.S. Air Force Academy (USAFA), were two supplemental strains used for nitrogen limitation experimentation, as additional controls for protocol development. N. salina and T. suecica were grown in f/2-Si Medium, while S. obliquus was grown in Bold Basal Medium (Andersen, 2005). The cultures were grown and maintained with light between 1000 and 2000 lux, on a 16:8 hour light/dark cycle. At USAFA, cultures were grown in a growth chamber on shake platforms; at my home laboratory (partially pictured in Figure 3), air pumps connected to Pasteur pipettes bubble cultures for cellular suspension and exposure to light and CO2. Microscopy was used to view cellular morphology during sethoxydim experimentation, as well as to estimate the degree of cellular disruption for cell lysis and permeabilization during the lipid and ACCase analyses. Hemacytometry was used to count cells for growth measurements, sampling in triplicate and using volumes of 10 µl.
Nitrogen Limitation: At USAFA, cultures of Nannochloropsis salina,Tetraselmis suecica and Scenedesmus obliquus were acclimatized for one week (to ameliorate the effects of shock) to one of four distinct levels of sodium nitrate abundance: 100%, 67%, 33% and 1%, relative to the amounts of nitrate added to the standard growth media. Cultures of 400 ml were inoculated in triplicate at 15 cells/µL with acclimatized cells, grown for 14 days, and then analyzed for growth and lipid content.
Lipid Analytical Development: At USAFA, the sample biomass was harvested by centrifugation. As methods to lyse cells and release the lipids into solution, grinding in liquid nitrogen and sonication were compared, separately and in conjunction, by evaluating the disruption in microscopic wet cell mounts. Two extraction steps, using a 2:1 ratio (v/v) of chloroform/methanol, were used to solubilize the lipids and separate them from the disrupted cell masses (Bligh and Dwyer, 1959). Hydrochloric acid catalyzed the esterification of cellular lipids in methanol and toluene to form fatty acid methyl esters (FAMEs) (Ichihara and Fukubayashi, 2010) (Xu and Mi, 2011). An internal standard of methyl tridecanoate was added to samples for FAME quantification. Esterifying known quantities of tridecanoic acid and using NMR spectroscopy to analyze the relative reactant and product concentrations at particular time points allowed for the kinetic analysis of the esterification efficiency. Gas chromatography-mass spectrometry was used to analyze the lipid profiles of samples (Christie, 1993).
Determination of ACCase Abundance: At Colorado State University, samples were harvested by microcentrifugation. Pellets were boiled in Laemmli buffer to lyse the cells and extract the proteins, which were separated based on size by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Gels were stained with Coomasie Blue to visualize the proteins. Proteins from replicate gels were transferred to nitrocellulose membranes. The membranes were blocked, exposed to antibodies and washed using standard molecular techniques for immunoblotting (Gallagher, 2006). Two sets of anti-human-ACCase antibodies (a polyclonal rabbit antibody raised against a synthetic peptide, and a monoclonal mouse antibody raised against a bacterially-synthesized protein) were used, with appropriate secondary antibodies conjugated to horseradish peroxidase (HRP). Additionally, immunoblotting with anti-biotin antibodies directly conjugated to HRP was used to compare total biotin-containing protein expression between cultures, as an alternative metric of ACCase abundance (Rohringer and Holden, 1985). Following antibody incubation, a chemiluminescence reaction involving HRP and subsequent exposure to photographic film allowed the antibody-derived signal strength to be observed.
Adaption of a colorimetric enzymatic assay was undertaken for measurement of ACCase activity (Willis at el, 2008). The assay indirectly quantifies ACCase activity by spectrophotometrically measuring the depletion of ACCase-dependent acetyl-CoA carboxylation reactants. Three strategies were tested for permeabilizing N. salina’s strong cell walls to allow enzyme interactions with reagents: the addition of CTAB (hexadecyltrimethyl-ammonium bromide), 16-hour and 4-hour cellulase digestion, and French pressing (Salerno, 1985: 259-264).
Sethoxydim testing was independently conducted in my home laboratory. The behavior of sethoxydim in liquid media and its effects on Nannochloropsis salina were characterized through a series of experiments on replicate cultures. Dose response and time course experiments included concentrations of sethoxydim from 0.2 µM to 400 mM and were monitored using hemacytometry and microscopy.
Sethoxydim selection was independently conducted in my home laboratory. Artificial selection was implemented by the addition of sethoxydim at levels of 1 mM and 2 mM in healthy cultures of N. salina in f/2-Si medium. Six control and eight sethoxydim-treated cultures were grown in 250 Erlenmeyer flasks. After two weeks, the initial amount of sethoxydim was added a second time. After a total of three weeks, aliquots of selected cells were pelleted, removed from the sethoxydim-treated media and subcultured in media lacking sethoxydim.
Nitrogen Limitation: Nitrogen-limited cultures provided a well-characterized experimental condition as the basis for testing lipid and ACCase analytical techniques for subsequent use on sethoxydim selection experiments. For development of the ACCase assay and immunoblotting measurements of ACCase protein abundance, nitrogen-limited cultures grown under home laboratory conditions were used as representative cells with high lipid content and ACCase activity. As shown in Figures 4 and 5, nitrogen limitation causes decreased cell growth and increased lipid content: a balance of these factors is key to the increases in lipid productivity in Figure 6.
Lipid Analytical Development: Drawing upon results and observations from a range of sources, I aimed to establish an effective methodology for determining lipid profiles for small amounts of cells. I tested a colorimetric procedure unsuccessfully (Wawrik and Harriman, 2012). Instead, I chose a modified lipid extraction/lipid esterification/GC-MS analysis.
Sonication, grinding in liquid nitrogen and lyophilization were tested as techniques for cell lysis (Zheng et al, 2011). After observing wet mounts and analyzing the lipid profiles of cultures that had been lysed with these methods, thoroughly grinding in liquid nitrogen without lyophilization was deemed reliable for cellular disruption. Esterification kinetic analysis shows that in a span of 60 minutes, the reaction converted more than 99% of the initial tridecanoic acid into methyl tridecanoate (Figure 7). Following esterification, FAMEs were extracted into a volatile hexane phase suitable for GC-MS analysis. Several different GC-MS programs were tested to decide upon an effective one to show the full range of extracted lipids with distinct peaks (O’Fallon et al, 2007). Methyl tridecanoate was added to blank samples before esterification for reference. Running a Supelco® Fatty Acid Methyl Ester Standard Mix (C8-C24) confirmed the FAME identities. Multiple identical samples were analyzed using this protocol to establish repeatability.
Determination of ACCase Abundance: Immunoblotting was used to identify an effective method for detecting the differential expression of ACCase. Stained protein abundance demonstrated the success of protein extraction and purification procedures with N. salina cultures (not pictured). The use of anti-human-ACCase antibodies to measure ACCase production in algae cells was not successful—understandably so, given the vast genomic differences between H. sapiens and N. salina. Application of anti-biotin antibodies showed distinct banding patterns representing all of the biotin-conjugated proteins extracted. Comparison of the biotin bands of nitrogen-limited and untreated cultures evidenced a large (130–250 kDa) protein with greater expression in stressed than in unstressed cultures—the predicted size of ACCase is approximately 220 kDa (Roessler et al, 1994). This band is a likely candidate for ACCase, given its size, biotinylation and differential expression in cells with different amounts of lipid production (Figure 8) (Alban et al, 1994).
Development of an enzymatic assay for ACCase based upon a published technique used in Corynebacterium glutamicum was undertaken to measure ACCase activity (Willis et al, 2008). Various aspects of the assay were altered for use with algae, most notably the step of cellular permeabilization to allow cellular ACCase interaction with assay reagents. French pressing successfully formed protoplasts, confirmed by microscopic observation of the treated cells. In the assay, the activity of ACCase depletes the initial acetyl-CoA present in the reagent volumes, which corresponds downstream to a decrease in the colorimetrically observable formation of 2-nitro, 5-thiobenzoic acid (absorbance at 412 nm, measureable by spectrophotometry). However, as indicated by equal absorbance between cell-containing samples and pure acetyl-CoA standards, the ACCase-catalyzed reaction (acetyl-CoA’s carboxylation into malonyl-CoA) did not take place even in successfully permeabilized cells. Standard curves of acetyl-CoA tested with the latter steps of the assay show a direct linear relationship between spectrophotometrically measured absorption and acetyl-CoA concentrations (data not shown), indicating that the assay sensitivity threshold is in an effective range to make quantifiable differences in ACCase activity perceptible as soon as its execution can be perfected.
Sethoxydim Testing: Dose response experiments established sethoxydim treatments to reliably exert a suitably strong selection pressure on algae cultures. Most cells had to be adversely affected by sethoxydim to indicate that it was potently acting to inhibit ACCase, but some cells had to remain alive so that the population could adapt to the new environment. Figure 9 displays the dosage response curve of N. salina to treatments of sethoxydim: after nine days, treatments of 2 mM sethoxydim inhibited growth by approximately 40%–60%. The time course experiment depicted in Figure 10 shows growth inhibition in 2 mM-sethoxydim-treated cultures over the course of 25 days. Visually, cultures treated with 2 mM sethoxydim became cloudy and more yellow; clumps of cells, cellular detritus and bacteria aggregated; and dead and abnormally sized cells were apparent using microscopy (Figure 11).
Sethoxydim Selection: Cells were treated with sethoxydim to gauge if artificial selection has the potential to establish cell lines with increased lipid metabolism by establishing algae populations resistant to a strong inhibitor of the lipid synthesis pathway. As shown in Figure 9, the preliminary dose response experiments determined that dosages near a 2 mM treatment constitute a suitable selection pressure. Cultures were grown in initial concentrations of 1 mM and 2 mM sethoxydim, which were increased over the course of the selection process. Resultant populations were analyzed for their lipid content, with the data presented in Figure 12. Declines in growth (Figure 10) and significant increases in lipid content (Figure 12) were observed in the selected cultures, with more drastic differences in the cultures consistent with higher treatments of sethoxydim. Groups with 2 mM treatment show an approximately sevenfold increase in overall lipid productivity, with 1 mM-treated groups showing a fivefold increase. Aliquots of the cells were pelleted and transferred to untreated media to determine if the sethoxydim-induced change in lipid metabolism is dependent on continued selection exposure. Figure 12 shows that the populations recultured in sethoxydim-free media for multiple generations produce less cellular lipids than cells remaining in the selection environment. Nonetheless, these cultures still maintain a lipid content that is approximately threefold greater than that of untreated algae.
The overall aim of my work was to use artificial selection to establish algae populations with increased ACCase activity and lipid production. To this end, sethoxydim treatments had to be determined empirically, and lipid content and ACCase abundance had to be made measurable by using nitrogen-limited cultures for protocol development.
Multiple experiments established effective treatments of the ACCase inhibitor sethoxydim for artificial selection of cell lines with sethoxydim tolerance. Sethoxydim treatment testing identified treatments of 1 mM and 2 mM as suitable dosages that significantly decrease cell populations and act as a potent selective pressure while maintaining a stable culture.
Gradual nitrogen stress causes decreased growth and generally increased lipid content in multiple strains of algae, an effect that has been reported previously. This work adds to such research by characterizing the lipid profiles of three algae strains under nitrogen deprivation. Maximum lipid productivities were observed at 33% N for N. salina, 67% N for T. suecica and 100% N for S. obliquus.
Nitrogen-limited cultures were used in the development of a technique for lipid extraction, esterification and analysis. The GC-MS lipid profiles obtained were repeatable and reliable. I confirmed high esterification efficiency with kinetic reaction analysis using NMR spectroscopy.
Protein purification and staining success indicates that current techniques are efficiently extracting protein for downstream analyses. The colorimetric assay for ACCase activity currently in development would be a more accurate representation of change in ACCase’s role in the lipid metabolism than methods that represent only protein abundance, as it could be used to distinguish increases in enzyme activity not necessarily correlated with translational change; however, preliminary tests with anti-biotin immunoblotting are more promising. The detectable presence of a large (close to 250 kDa) biotin-containing protein with differential expression between stressed and unstressed cultures using this technique indicates its probable identity as ACCase. Following confirmatory experimentation, anti-biotin immunoblotting could prove to be an effective means of ACCase expression comparison between experimental cultures. Further troubleshooting to minimize the background signaling and provide for band clarity of large proteins will help to establish this procedure.
Finally, the increased lipid content in the sethoxydim-treated cultures may indicate the successful selection of algae populations with abnormally high lipid production. Confirmatory analyses are required to positively identify the selected cells as the desired algae strain, as selection acted upon a realistic community rather than on an axenic population. It is also necessary for me to show that the predicted mechanism of ACCase overexpression and/or overactivity is occurring consistent with sethoxydim’s mode of action. Selected populations recultured in normal media decrease in lipid content relative to their counterparts remaining in sethoxydim; this is most likely a consequence of unadapted, low-oil-producing cells still alive in the selected line repopulating the culture following removal from selection pressure. Nevertheless, removal from the selection environment does not eliminate the increase in lipid content that sethoxydim selection confers. Cells removed from sethoxydim maintain a lipid content significantly higher than untreated cells even after multiple weeks, indicating the potential for sustaining elevated lipid yields.
My work is especially pertinent in today’s petroleum-dependent world. Finding a viable energy source that is not reliant upon dwindling fossil fuel supplies is critical for reasons ranging from national security to environmental preservation and economic stability.
By profiling growth and lipid content as well as overall oil productivity, I provide important information on the impacts of nutrient conditions on particular strains of algae. My described method for algae lipid analysis utilizing GC-MS, optimized protocols at multiple points and demonstrated repeatable results. Plus, I have characterized sethoxydim’s effects on microalgae to establish its use for artificial selection.
Moreover, I’ve demonstrated ACCase induction in nitrogen-stressed cells by studying biotin-conjugated protein expression, and went through considerable troubleshooting to adapt a spectrophotometric assay to measure algae ACCase activity.
Finally, my technique, sethoxydim selection, shows promise as a method to increase the oil yields of microalgae. This is a novel application of artificial selection to increase microalgae lipid production, and requires more research. Nonetheless, these initial results suggest that sethoxydim selection holds great promise as a method to isolate cells with improved rates of lipid synthesis. With more development, artificial selection could constitute a viable way to make algae biofuels more feasible.
These findings constitute important steps toward a tomorrow not dependent upon fossil fuels: a world powered by sustainable, “green” algae biofuel.
Ongoing and Future Work
My refinement of sethoxydim characterization experiments is ongoing, including more narrowly defined dose response tests. I am confirming lipid results for sethoxydim-selected cultures by repeating lipid analysis on current cultures, replicating experiments with greater numbers of samples, and selecting individual sethoxydim-resistant clonal colonies from plated cultures. I am also treating larger cultures (up to four liters) to more efficiently confer resistance to larger numbers of cells. Selected cell lines are being maintained in both sethoxydim-treated and untreated media to demonstrate successful artificial selection, by continuing to show that resistance and high lipid yields are maintained in the absence of sethoxydim over long-term growth. To confirm the microalgal identity of selected populations, the culture DNA will be extracted, PCR amplified, and the 18S ribosomal RNA gene sequenced and checked against a genetic database.
I am continuing to develop ACCase analyses using nitrogen-limited cultures in order to establish that, consistent with the mechanism of selection, increased ACCase expression and activity are concomitant with observed increases in lipid synthesis. I am refining immunoblotting techniques with anti-biotin by troubleshooting the antibody concentrations to minimize background signaling and sharpen band focus. I’ll also adjust protein separation and blotting procedures to optimize the transfer efficiency and visualization of large proteins. Moreover, I am still attempting to effectively expose assay reagents to ACCase to measure enzymatic activity.
I will also use cell sorting to expedite artificial selection. Combining cellular size and complexity measurements with fluorimetric indications of photosynthetic viability, using flow cytometry, could identify healthy N. salina cells in sethoxydim-selected cultures (Carrol, 2004). Using stringent gating criteria based on multiple types of control cultures, possibly also incorporating a fluorimetric measurement of stained lipid content, could be used to select only the most viable cells, with the most robust growth and lipid production, during cell line development. Using this cell sorting in conjunction with sethoxydim treatment would eliminate the unadapted cells in cell lines acquiring resistance, facilitating population evolution and increasing the likelihood of maintaining increased lipid content when removed from the selection environment.
Finally, following my confirmation of sethoxydim-resistant cell lines, future avenues of investigation should characterize the genetic mechanism(s) of conferred resistance. Study of the different resistant cell lines could reveal multiple different metabolic strategies used by the cells to overcome sethoxydim’s lipid production inhibition—for instance, by increasing ACCase’s abundance, by altering the enzyme target site, or by improving its activity through regulation or other molecular changes.
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