by Paul Held, Xavier Amouretti and Peter Banks
Increasing global energy demands, oil pricing, and the hotly debated environmental impact of fossil fuels are driving research for alternative, economic and sustainable energy sources including solar, wind, and organic options. Bioethanol as an energy source focuses on fermentation of sugars from corn, sugar cane, grasses, wood byproducts, soybean, and other plant crops. Although each yields promising data , potential direct or indirect interference with food crops and resources needed for production could hinder full-scale implementation. Yeasts, bacteria, fungi and algae are also investigated as biofuel potentials, each requiring less arable land for production than the aforementioned crops, but each have their own unique challenges. In particular, algae can potentially produce significantly more lipids, which are then converted to biofuels, and more harvests per area than traditional crops with fewer CO2 emissions.
Algae-based biofuel research is typically done using two growth methods: closed loop, where the cultures are grown in controlled, closely monitored bioreactors and open pond, where the algae is exposed to the outside environment including weather fluctuations and possible contaminating organisms. Regardless of the growth method, optimizing biofuel production requires screening and experimentation to select the best algal strain for lipid production, determining optimal growth conditions and assay development for lipid and other metabolite byproduct analysis.
For example, the single-celled green algae Chlorella vulgaris has chloroplasts containing chlorophyll-a and chlorophyll-b. Under favourable and unlimited growth conditions, this microalgae produces polar lipids to enrich chloroplasts and cellular membranes, reaching approximately 20% by weight of the overall dried culture mass . However, during unfavourable or restricted growth conditions, the algae increases neutral lipid production in the cytoplasm to be used for energy, accounting for up to 80% or more of the dry biomass [3,4]. It is these neutral lipids, composed primarily of triacylglycerol esters (TAG), that are harvested for biofuels including biodiesel.
Nile Red is a red lipid soluble phenoxazone dye that is relatively photostable and highly fluorescent in non-polar hydrophobic environments, yet very poorly fluorescent in aqueous solutions . The dye is amenable for use when determining neutral lipids present in an algae sample as it rapidly passes through many algae cell walls when dissolved in aqueous DMSO solutions. Inside the algal cells, the dye partitions to cytoplasmic lipid droplets produced by the cell in response to nutrient deprivation stress and becomes measurable via fluorescence methods . The extent of fluorescent intensity is indicative of the amount of neutral lipid present.
Here, we detail a method to quantify C. vulgaris under various growth media conditions and using absorbance and fluorescence microplate-based measurements. Algae-based screening in a microplate-based format allows rapid and simultaneous measurement of many samples and multiple experimental conditions, thus reducing time and consumable materials needed while increasing repeatability and precision.
Chlorella vulgaris (2714) cultures were obtained from the UTEX Culture Collection of Algae at The University of Texas at Austin. BG-11 media was made according to UTEX Culture Collection of Algae, while complete tris-acetate-phosphate (TAP) and tris-phosphate (TP) media, as well as nitrogen and sulphur-deficient variants, were made following the process described by Deng et al . Nile Red dye (catalog no. ENZ-52551) was obtained from Enzo Life Sciences (Farmingdale, New York, USA). Sterile, TC-treated black polystyrene 96-well microplates (Catalog no. 3603) with clear bottoms were purchased from Corning, Inc. (Corning, New York, USA)
Absorbance and fluorescence measurements were made using the Synergy™ H4 Hybrid Multi-Mode Microplate Reader from BioTek Instruments (Winooski, Vermont, USA). This patented reader combines filter- and monochromator-based detection systems in the same unit, with the monochromators used in this application to detect absorbance at 600 nm and fluorescence from the top of the microplate using 530 nm excitation and 570 nm emission wavelengths. All reader parameters and data management were performed via integrated Gen5™ Data Analysis Software.
C. vulgaris cultures were grown in BG-11, TAP and TP media, and their nitrogen and sulphur-deficient variants in sterile 250 mL Erlenmeyer flasks on a platform shaker rotating at 100 rpm. Cultures were maintained at room temperature with constant light illumination. 100 μL aliquots were removed and pipetted into microplates for light scatter readings at 600 nm in the Synergy H4 using Gen5’s discontinuous kinetics feature.
Nile Red powder was dissolved in DMSO at 1 mg/mL and stored at -20°C. Working 2X (1 μg/mL) solutions were freshly prepared with final dye and DMSO concentrations of 0.5 μg/mL and 25% respectively. Immediately following the aforementioned absorbance readings, 100 μL 2X working Nile Red solution was added to the culture samples in the microplates, and the plates were incubated in the microplate reader’s reading chamber for 10 minutes. After incubation, fluorescence measurements were made from the top of the microplate using 530 nm excitation and 570 nm emission wavelengths. Algal stress response was accomplished by media exchange. Log phase stock cultures grown in complete media were centrifuged at 4000 x g, after which the supernatant was discarded and the cell pellets resuspended in nitrogen- or sulphur-deficient TP or TAP media for further growth.
Different media formulations showed a marked influence in the C. vulgaris growth rate and final cell density as measured by absorbance at 600 nm. Per Figure 1a, cultures grown in TAP media, with high carbon levels from acetate, grew to the highest absorbance density; approximately 10-fold greater than cultures grown in BG-11 media, which is carbon poor. Cultures grown in TP media, with less carbon than TAP, have an intermediate final density. It is also seen that while maximal absorbance is lower with TP media, the culture’s log growth phase begins sooner than in TAP media.
Additionally, per Figure 1b, C. vulgaris cultures grown in TAP media deficient in either nitrogen or sulphur displayed significantly slower growth rates and lower final densities than the complete media formulation. Sulphur deficiency reduced growth to a greater extent than did nitrogen.
Neutral Lipid Production
When comparing C. vulgaris neutral lipid production in complete and nitrogen- or sulpfur-deficient TP and TAP media [Figure 2a], cultures grown in nitrogen-deficient media were found to have over twice the lipid per culture volume as those using complete media. This is in spite of the complete media cultures having 5-8 times greater total cells. Sulphur-deficient cultures also produced significant amounts of lipid compared to complete media cultures and despite having substantially fewer cells. When the data is normalized for cell number using 600 nm optical densities [Figure 2b], the amount of lipid per cell is consistent between the different media formulations. Nitrogen-deficient cell growth results in a nearly 15-fold increase in lipid amount per cell, irrespective of the presence or absence of acetate as the carbon source. Sulphur-deficient cultures show a 5-fold increase in cellular lipid content, while complete media does not show any lipid increase on a per cell basis.
When algal cultures grown in complete media were switched to deficient media, the stress results in rapid lipid production. Regardless of media type tested, nitrogen deficiency induced a 10-fold increase in lipid production relative to complete media lipid production [Figure 3a]. The increase is rapid, with significant lipid increase within a few hours. The stress-induced lipid production is reversed when the C. vulgaris cultures are returned back to complete media, with lipid levels returning to normal levels within 20 hours [Figure 3b].
Algae are promising alternative energy sources, yet much research still remains before commercially viable options are available. Selection of the optimal algal strain, nutrients and environmental conditions are critical to maximize neutral lipid yields. Towards this selection, light scatter absorbance is a simple, non-destructive method to assess cell density and growth state, while Nile Red dye is proven to be a suitable method to monitor lipid production in microplate-based algae samples such as Chlorella vulgaris. Microplate-based absorbance and fluorescence measurements provide increased sample throughput and parameter testing compared to traditional tube- and flask-based formats. The Synergy H4 Multi-Mode Microplate Reader combines absorbance and fluorescence, thus allowing cell number and lipid content on the same sample and at the same time for increased efficiency. It also features luminescence for enhanced assay flexibility. The flexible array of microplate well density allows researchers to choose the optimal format for their needs.
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Paul Held, Xavier Amouretti and
BioTek Instruments, Inc.
Winooski, Vermont, USA