Response of growth and fatty acid compositions of Nannochloropsis sp. to environmental factors under elevated CO2 concentration

Nannochloropsis sp. was grown with different levels of nitrate, phosphate, salinity and temperature with CO₂ at 2,800 μl l⁻¹. Increased levels of NaNO₃ and KH₂PO₄ raised protein and polyunsaturated fatty acids (PUFAs) contents but decreased carbohydrate, total lipid and total fatty acids (TFA) contents. Nannochloropsis sp. grew well at salinities from 22 to 49 g l⁻¹, and lowering salinity enhanced TFA and PUFAs contents. TFA contents increased with the increasing temperature but PUFAs contents decreased. The highest eicosapentaenoic acid (EPA, 20:5ω3) content based on the dry mass was above 3% under low N (150 μM NaNO₃) or high N (3000 μM NaNO₃) condition. Excessive nitrate, low salinity and temperature are thus favorable factors for improving EPA yields in Nannochloropsis sp.     

Stress-Induced Changes in Optical Properties,Pigment and Fatty Acid Content of <Emphasis Type="Italic">Nannochloropsis</Emphasis> sp.: Implications for Non-destructive Assay of Total Fatty Acids

In order to develop a practical approach for fast and non-destructive assay of total fatty acid (TFA) and pigments in the biomass of the marine microalga Nannochloropsis sp. changes in TFA, chlorophyll, and carotenoid contents were monitored in parallel with the cell suspension absorbance. The experiments were conducted with the cultures grown under normal (complete nutrient f/2 medium at 75 μmol PAR photons/(m² s)) or stressful (nitrogen-lacking media at 350 μmol PAR photons/(m² s)) conditions. The reliable measurement of the cell suspension absorbance using a spectrophotometer without integrating sphere was achieved by deposition of cells on glass–fiber filters in the chlorophyll content range of 3–13 mg/L. Under stressful conditions, a 30–50% decline in biomass and chlorophyll, retention of carotenoids and a build-up of TFA (15–45 % of dry weight) were recorded. Spectral regions sensitive to widely ranging changes in carotenoid-to-chlorophyll ratio and correlated changes of TFA content were revealed. Employing the tight inter-correlation of stress-induced changes in lipid metabolism and rearrangement of the pigment apparatus, the spectral indices were constructed for non-destructive assessment of carotenoid-to-chlorophyll ratio (range 0.3–0.6; root mean square error (RMSE) = 0.03; r ² = 0.93) as well as TFA content of Nannochloropsis sp. biomass (range 5.0–45%; RMSE = 3.23 %; r ² = 0.89) in the broad band 400–550 nm normalized to that in chlorophyll absorption band (centered at 678 nm). The findings are discussed in the context of real-time monitoring of the TFA accumulation by Nannochloropsis cultures under stressful conditions.     

Investigation of continuous-batch mode of two-stage culture of Nannochloropsis sp. for lipid production

Microalgal lipid induction through nitrogen stress often suffers from a contradiction between biomass productivity and lipid content, i.e., either high biomass productivity with low lipid content or vice versa. A two-stage nitrogen-replete and nitrogen-deplete (NR–ND) culture was suggested to be an option to attain high lipid productivity. In this study, the effects of culture conditions and modes on biomass productivity and lipid productivity of Nannochloropsis sp. in the two stages were comprehensively investigated. The optimal culture conditions for the two stages, aiming to high biomass productivity and lipid productivity respectively, were consistent, i.e., CO₂ content in aeration (1 %), phosphorus concentration in medium (181 μmol/L), incident light intensity (150 μE/(m²s)), temperature (25 °C). Different culture modes of the two stages were compared. The overall lipid productivity of the two-stage continuous-batch mode achieved 0.123 g/(L day), which was 60.3, 48.2, 34.9 and 13.5 % higher than that of single nitrogen-replete batch, single nitrogen-limited batch, continuous nitrogen-replete culture and two-stage batch–batch culture, respectively, and also higher than most reported values. This contribution provides fundamental data for the two-stage NR–ND cultivation process design of Nannochloropsis sp.     

Optimization of growth and fatty acid composition of a unicellular marine picoplankton,Nannochloropsis sp., with enriched carbon sources

A unicellular marine picoplankton, Nannochloropsis sp., was grown under CO₂-enriched photoautotrophic or/and acetate-added mixotrophic conditions. Photoautotrophic conditions with enriched CO₂ of 2800 l CO₂ l^–1 and aeration gave the highest biomass yield (634 mg dry wt l^–1), the highest total lipid content (9% of dry wt), total fatty acids (64 mg g^–1 dry wt), polyunsaturated fatty acids (35% total fatty acids) and eicosapentaenoic acid (EPA, 20:53) (16 mg g^–1 dry wt or 25% of total fatty acids). Mixotrophic cultures gave a greater protein content but less carbohydrates. Adding sodium acetate (2 mM) decreased the amounts of the total fatty acids and EPA. Elevation of CO₂ in photoautotrophic culture thus enhances growth and raises the production of EPA in Nannochloropsis sp.     

Effects of initial population density (IPD) on growth and lipid composition of Nannochloropsis sp.

The microalga Nannochloropsis sp. was cultured under different initial population densities (IPDs) ranging from 0.11 to 9.09 g L^?1. The IPD affected the biomass and lipid accumulation significantly. The algal cultured with higher IPD resulted higher biomass concentration (up to 13.07 g L^?1) in 10 days growth. The biomass productivity with 0.98 g L^?1 IPD was 0.75 g L^?1 d^?1 which was higher than that of other IPDs. For IPDs ranging from 0.11 to 0.98 g L^?1, with the increase of IPD, the biomass productivity increased, while for IPD over 0.98 g L^?1, the biomass productivity decreased. Lipid content of the algal culture started with 0.11 g L^?1 IPD reached to 42 % of dry weight. But with the increase of IPD, the lipid content decreased. Lipid composition was analyzed using thin layer chromatography coupled with flame ionization detection (TLC/FID). Seven lipid classes were identified and quantified. The main reserve lipid, triacylglyceride (TAG), accumulated under all different IPD conditions. However, with the increasing IPD values, TAG content decreased from 59.1 to 23.5 % of the total lipids. Based on these results, to obtain the maximal biomass productivity and lipid productivity of Nannochloropsis sp. in mass cultivation systems, it is necessary to select an appropriate IPD.     

Factors affecting lipid accumulation by <Emphasis Type="Italic">Nannochloropsis oculata</Emphasis> in a two-stage cultivation process

Reserve lipids of microalgae are promising for biodiesel production. However, optimization of cultivation conditions for both biomass yield and lipid production of microalgae is a contradictory problem because required conditions for both targets are different. In this study, a two-stage cultivation strategy is proposed to enhance lipid production of the microalga Nannochloropsis oculata. Biomass growth and lipid production were carried out in two separate and non-interacting stages. In first-stage cultivation, microalgae were cultivated in optimal conditions for cell growth. Then, microalgae were harvested and transferred into a growth-limited environment, thus enhancing lipid production of microalgae. Here, optimization of the lipid production stage (second stage) with respect to different levels of inoculum concentration, salinity of culture broth, and intensity of irradiance was performed. The results show that irradiance exhibits a significant influence on lipid production. The highest lipid productivity of 0.324 g L⁻¹ day⁻¹ was obtained with an inoculum concentration of 2.3 g L⁻¹, a salinity of 35 g L⁻¹, and an irradiance of 500 μmol photons m⁻² s⁻¹. The final yield of lipid obtained from the two-stage process was 2.82-times higher than that from traditional single-stage batch cultivation systems.     

Growth,lipid content,productivity, and fatty acid composition of tropical microalgae for scale‐up production

Biomass and lipid productivity, lipid content, and quantitative and qualitative lipid composition are critical parameters in selecting microalgal species for commercial scale‐up production. This study compares lipid content and composition, and lipid and biomass productivity during logarithmic, late logarithmic, and stationary phase of Nannochloropsis sp., Isochrysis sp., Tetraselmis sp., and Rhodomonas sp. grown in L1‐, f/2‐, and K‐medium. Of the tested species, Tetraselmis sp. exhibited a lipid productivity of 3.9–4.8 g m^?2 day^?1 in any media type, with comparable lipid productivity by Nannochloropsis sp. and Isochrysis sp. when grown in L1‐medium. The dry biomass productivity of Tetraselmis sp. (33.1–45.0 g m^?2 day^?1) exceeded that of the other species by a factor 2–10. Of the organisms studied, Tetraselmis sp. had the best dry biomass and/or lipid production profile in large‐scale cultures. The present study provides a practical benchmark, which allows comparison of microalgal production systems with different footprints, as well as terrestrial systems. Biotechnol. Bioeng. 2010;107: 245–257. © 2010 Wiley Periodicals, Inc.     

Effect of cell density and irradiance on growth,proximate composition and eicosapentaenoic acid production ofPhaeodactylum tricornutum grown in a tubular photobioreactor

Growth and eicosapentaenoic acid (EPA) productivity of the diatomPhaeodactylum tricornutum grown semicontinuously in a helical tubular photobioreactor were examined under a range of irradiances (approximately 56 to 1712 µmol photons m^-2 s^-1) and cell densities (3 × 10⁶ to 18 × 10⁶ cells mL^-1). Self shading sets the upper limit of operational maximum cell density. Higher irradiance increases this upper limit and also increase the growth rate. Biomass productivity and EPA productivity were enhanced at those cell densities which support the fastest growth rate irrespective of irradiance. The cell protein content increased with increasing irradiance and the carbohydrate and lipid content increased with increasing cell density. EPA productivity was greatest at the highest irradiance. This study shows that biomass productivity and EPA productivity can be maximised by optimising cell density and irradiance, as well as by addition of CO₂.Author for correspondence     

Effect of 24-epibrassinolide on biomass, growth and free proline concentration in Spirulina platensis (Cyanophyta) under NaCl stress

In this study, biomass, growth and free proline concentration were investigated in Spirulina platensis treated with different concentrations of NaCl (50, 100, 150, and 200 mM) and 24-epibrassinolide (24-epiBL) hormone (0.5, 1.0, and 3.0 μM) over 5 days. As a result of analysing the cultures under salinity stress, it was determined that biomass and growth rate decreased significantly, while proline concentration increased considerably under salinity stress. The increase in the concentration of proline suggests a role in response to NaCl stress. Among the cultures treated with different concentrations of 24-epiBL, maximum growth was determined at the cultures at 1.0 μM 24-epiBL. Algal growth was also greater at the 0.5 and 3.0 μM concentrations of 24-epiBL with respect to control cultures. With respect to control, 24-epiBL affected growth rate and biomass positively, but proline concentration did not change. Among the cultures supplied with different combinations of NaCl and 24-epiBL, growth rate increased in 150/0.5 and 150/3.0 mM/μM concentrations, but was maximal for the culture containing 150/1.0 mM/μM combination. The increase in algal growth suggests a role for 24-epiBL in partially alleviated to NaCl stress. These results suggest that 24-epiBL may have a protective role for S. platensis reducing the inhibitor effects of salinity stress.     

Isolation and characterization of polyunsaturated fatty acid producing Thraustochytrium species: screening of strains and optimization of omega-3 production

An isolation program targeting Thraustochytrids (marine fungoid protists) from 19 different Atlantic Canadian locations was performed. Sixty-eight isolates were screened for biomass, total fatty acid (TFA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) content. Analysis of fatty acid methyl ester results discerned four distinctive clusters based on fatty acid profiles, with biomass ranging from 0.1 to 2.3 g L⁻¹, and lipid, EPA, and DHA contents ranging from 27.1 to 321.14, 2.97 to 21.25, and 5.18 to 83.63 mg g⁻¹ biomass, respectively. ONC-T18, was subsequently chosen for further manipulations. Identified using 18S rRNA gene sequencing techniques as a Thraustochytrium sp., most closely related to Thraustochytrium striatum T91-6, ONC-T18 produced up to 28.0 g L⁻¹ biomass, 81.7% TFA, 31.4% (w/w biomass) DHA, and 4.6 g L⁻¹ DHA under optimal fermentation conditions. Furthermore, this strain was found to produce the carotenoids and xanthophylls astaxanthin, zeaxanthin, canthaxanthin, echinenone, and β-carotene. Given this strain’s impressive productivity when compared to commercial strains, such as Schizochytrium sp. SR21 (which has only 50% TFA), coupled with its ability to grow at economical nitrogen and very low salt concentrations (2 g L⁻¹), ONC-T18 is seen as an ideal candidate for both scale-up and commercialization.     

Effects of light intensity and nitrogen starvation on growth, total fatty acids and arachidonic acid in the green microalga Parietochloris incisa

The effects of light and nitrogen deficiency on biomass, fatty acid content and composition were studied in Parietochloris incisa, the unicellular freshwater chlorophyte accumulating very high amounts of arachidonic-acid-rich triacylglycerols. P. incisa cultures grown on complete nutrient medium and under high light (400 μmol photons m^{− 2} s⁻¹) showed the highest rate of growth in comparison to medium (200 μmol photons m⁻² s⁻¹) and low (35 μmol photons m⁻² s⁻¹) light intensity. Cultures grown under high light (on complete BG-11 medium) attained higher volumetric contents of total fatty acids and arachidonic acid due to greater increase in biomass. Nitrogen starvation brought about a strong increase in the arachidonic acid proportion of total fatty acids. Thus, adjustments to cultivation conditions could serve as an efficient tool for manipulation of yield and relative content of arachidonic acid in P. incisa. The significance of the changes in lipid metabolism for adaptation of P. incisa to high-light stress and nitrogen deficiency is also discussed.     

Effect of salinity on growth,biochemical composition,and lipid productivity of Nannochloropsis oculata CS 179

Effect of salinity (15, 25, 35, 45, and 55‰) on growth, biochemical composition, and lipid productivity of Nannochloropsis oculata CS 179 was investigated under controlled cultivation in a 19‐day study. The results demonstrate that the dry biomass of N. oculata was the highest at a salinity of 25‰ among the treatments in the first 10‐day cultivation (P<0.05). During days 14–19 (stage III), the dry biomass productivity was the highest at a salinity of 35‰ (P<0.05). The algae had the highest chlorophyll a content (26.47 mg g^?1) at 25‰ in stage I, and it decreased continuously at stage III. Protein content (as% of dry biomass) of algae reached the highest value of 42.25 ± 2.10% at 15‰, and the lipid content was the highest of 32.11 ± 1.30% of dry biomass at 25‰. However, the lipid productivity of these algae was the highest at 35‰ (64.71 mg L^?1 d^?1; P<0.001). C16 series content was the highest among the total fatty acid methyl esters (FAME), and eicosapentaenoic acid C20:5n‐3 (EPA) content was high at the low salinity. Fatty acid profiles of N. oculata varied significantly under different salinities.     

Effects of nitrogen sources on cell growth and lipid accumulation of green alga <Emphasis Type="Italic">Neochloris oleoabundans</Emphasis>

Microalgal lipids are the oils of future for sustainable biodiesel production. However, relatively high production costs due to low lipid productivity have been one of the major obstacles impeding their commercial production. We studied the effects of nitrogen sources and their concentrations on cell growth and lipid accumulation of Neochloris oleoabundans, one of the most promising oil-rich microalgal species. While the highest lipid cell content of 0.40 g/g was obtained at the lowest sodium nitrate concentration (3 mM), a remarkable lipid productivity of 0.133 g l⁻¹ day⁻¹ was achieved at 5 mM with a lipid cell content of 0.34 g/g and a biomass productivity of 0.40 g l⁻¹ day⁻¹. The highest biomass productivity was obtained at 10 mM sodium nitrate, with a biomass concentration of 3.2 g/l and a biomass productivity of 0.63 g l⁻¹ day⁻¹. It was observed that cell growth continued after the exhaustion of external nitrogen pool, hypothetically supported by the consumption of intracellular nitrogen pools such as chlorophyll molecules. The relationship among nitrate depletion, cell growth, lipid cell content, and cell chlorophyll content are discussed.     

The cell composition of Nannochloropsis sp. changes under different irradiances in semicontinuous culture

Nannochloropsis sp. was grown semicontinuously with a rate of daily renewal of the culture media of 40% of the volume of the culture under different irradiances (40, 60, 80, 220 and 480 mol quanta m^–2 s^–1). Under the conditions tested, light saturation was achieved at 220 mol quanta m^–2 s^–1 with no significant increase in steady-state cell density or of dry weight productivity with higher irradiance, reaching values of 115 × 10⁶ cells ml^–1 and 375 mg l^–1 day^–1 respectively. C/N ratios clearly indicated the point of light saturation, decreasing with increasing irradiance for light-limited conditions and increasing for light-saturated conditions. Under light-limited conditions, an increase in the irradiance produced an increase in the protein percentage of the organic fraction to the detriment of lipids and carbohydrates, while small changes were recorded under light-saturated conditions. The degree of unsaturation of fatty acids was lower with increasing irradiance, with a three-fold decrease of the percentage of total n–3 fatty acids, from 29 to 8% of total fatty acids, caused mainly by a decrease of eicosapentaenoic acid (EPA) (20:5n–3). The microalga reached its maximal value of dry weight productivity (375 mg l^–1 day^–1), EPA productivity (3.2 mg l^–1 day^–1) and maximal protein content (36% of the organic content) at the point at which light saturation was achieved. Results demonstrate the efficiency of the use of the irradiance for the modification of the biochemical composition of Nannochloropsis sp.     

Effect of nitrate feeding strategies on lipid and biomass productivities in fed-batch cultures of Nannochloropsis gaditana

Controlled nitrate feeding strategies for fed-batch cultures of microalgae were applied for the enhancement of lipid production and microalgal growth rates. In particular, in this study, the effect of nitrate feeding rates on lipid and biomass productivities in fed-batch cultures of Nannochloropsis gaditana were investigated using three feeding modes (i.e., pulse, continuous, and staged) and under two light variations on both lipid productivity and fatty acid compositions. Higher nitrate levels negatively affected lipid production in the study. Increasing the light intensity increased the lipid contents of the microalgae in all three fed-batch feeding modes. A maximum of 58.3% lipid- to dry weight ratio was achieved when using pulse-fed cultures at an illumination of 200 μmol photons m⁻² s⁻¹ and 10 mg/day of nitrate feeding. This condition also resulted in the maximum lipid productivity of 44.6 mg L⁻¹ day⁻¹. The fatty acid compositions of the lipids consisted predominantly of long-chain fatty acids (C:16 and C:18) and accounted for 70% of the overall fatty acid methyl esters. Pulse feeding mode was found to significantly enhance the biomass and lipid production. The other two feeding modes (continuous and staged) were not ideal for lipid and biomass production. This study demonstrates the applicability of pulse feeding strategies in fed-batch cultures as an appropriate cultivation strategy that can increase both lipid accumulation and biomass production.     

Mixotrophic growth of Phaeodactylum tricornutum on glycerol: growth rate and fatty acid profile

Mixotrophic growth of the eicosapentaenoic acid (EPA)producing diatom Phaeodactylum tricornutum UTEX640 was carried out in 1-L batch cultures under anexternal irradiance of 165 mol photons m^-2s^-1 by supplementing the inorganic culture mediumwith glycerol. The effect on the growth and the fattyacid profile was studied for different initialglycerol concentrations (0–0.1 M). The optimalglycerol concentration was 0.1 M.A lag phase was observed at high glycerolconcentrations. The present study also shows thatsuccessive additions of glycerol at 0.1M concentrationand using ammonium chloride as a nitrogen sourceremarkably increased the maximum biomass concentration(16.2 g L^-1) and maximum biomass productivity(61.5 mg L^-1 h^-1). These values wererespectively 9 and 8-fold higher than in thephotoautotrophically grown control. The level ofsaponifiable lipids in mixotrophically cultured cellswas significantly higher than in photoautotrophicallycultured cells and increased with the glycerolconcentration in the medium. The concentration ofstorage lipids, saturated and monounsaturated fattyacids, were enhanced but the EPA content did notchange significantly. The EPA content was around 2.2%of biomass dry weight. The maximum EPA yield was33.5 mg L^-1 d^-1 and was obtained in aculture containing 0.1 M glycerol, supplementedperiodically by ammonium chloride. This productivitywas 10-fold higher than the EPA productivity obtainedunder mixotrophic conditions.     

The effect of degree and timing of nitrogen limitation on lipid productivity in Chlorella vulgaris

Improvements in lipid productivity would enhance the economic feasibility of microalgal biodiesel. In order to optimise lipid productivity, both the growth rate and lipid content of algal cells must be maximised. The lipid content of many microalgae can be enhanced through nitrogen limitation, but at the expense of biomass productivity. This suggests that a two-stage nitrogen supply strategy might improve lipid productivity. Two different nitrogen supply strategies were investigated for their effect on lipid productivity in Chlorella vulgaris. The first was an initial nitrogen-replete stage, designed to optimise biomass productivity, followed by nitrogen limitation to enhance lipid content (two-stage batch) and the second was an initial nitrogen-limited stage, designed to maximise lipid content, followed by addition of nitrogen to enhance biomass concentration (fed-batch). Volumetric lipid yield in nitrogen-limited two-stage batch and fed-batch was compared with that achieved in nitrogen-replete and nitrogen-limited batch culture. In a previous work, maximum lipid productivity in batch culture was found at an intermediate level of nitrogen limitation (starting nitrate concentration of 170 mg L^?1). Overall lipid productivity was not improved by using fed-batch or two-stage culture strategies, although these strategies showed higher volumetric lipid concentrations than nitrogen-replete batch culture. The dilution of cultures prior to nitrogen deprivation led to increased lipid accumulation, indicating that the availability of light influenced the rate of lipid accumulation. However, dilution did not lead to increased lipid productivity due to the resulting lower biomass concentration.     

Salicylic Acid Confers Salt Tolerance in Giant Juncao Through Modulation of Redox Homeostasis,Ionic Flux,and Bioactive Compounds: An Ionomics and Metabolomic Perspective of Induced Tolerance Responses

We investigated the stimulatory and/or inhibitory role of exogenous SA in alleviating the salt stress (250, 500 mM NaCl) in Pennisetum giganteum (Giant Juncao) through coordinated induction of redox homeostasis, ionic flux, and bioactive compounds. Salt stress radically impaired root and shoot (growth, fresh, and dry biomass as well as tolerance indices), leaf relative water content, and leaf chlorophyll a/b ratio of Juncao due to higher Na⁺ and Cl^? accumulation followed by H₂O₂ generation, lipid peroxidation (MDA contents), and electrolyte leakage. However, the innate defense response of Juncao counteracted salt-induced damages by osmolytes accumulation combined with orchestrating antioxidants and ionic homeostasis mechanisms. Furthermore, the application of SA had an incremental impact on the development and productivity of high-salinity-exposed Juncao plants by increasing root length, plant biomass, tolerance indices, chlorophyll a/b ratio, and protein contents. Furthermore, SA treatment considerably decreased Na⁺ and Cl^? toxicity by orchestrating antioxidant enzymes, ion transport, and secondary metabolism. Notably, the application of SA substantially mitigated the adverse effects of high salinity concentration (500 mM NaCl), owing to the simultaneous upregulation in enzymatic and non-enzymatic antioxidants, nutrient ion flux, alongside chlorogenic acid production. Thus, we concluded that SA enhanced the tolerance capability of Juncao plants in a NaCl concentration-dependent manner. The findings of this study will enable environmentalists and pharmacologists to gain dual farm-level benefits, including animal therapeutics and restoration of salinized soils for arable purposes.

     

Improved Nile Red staining of <Emphasis Type="Italic">Nannochloropsis</Emphasis> sp.

High-throughput screening of microalgae for use as a potential feedstock for biodiesel requires a reliable method for the rapid detection of intracellular neutral lipid content. In this study, we report a modified and improved Nile Red (NR) fluorescence staining procedure for use as a rapid and sensitive screening tool to estimate levels of intracellular neutral lipid in the picopleustonic microalgae, Nannochloropsis sp. Addition of either glycerol or dimethyl sulfoxide (DMSO) into microalgae cultures greatly enhances lipid staining efficiency and increases the fluorescence intensity of stained cells. The optimized procedure requires glycerol and DMSO at the concentration of 0.1 and 0.165 g mL⁻¹, respectively, for peak fluorescence in a live culture of Nannochloropsis sp. Incubation for 5 min for glycerol-NR staining and 10 min for DMSO-NR staining at room temperature, in darkness, is used for the NR concentration of 0.3 and 0.7 μg mL⁻¹ for glycerol and DMSO, respectively. For the selection of lipid-rich cells of Nannochloropsis sp. using flow cytometric cell sorting, the glycerol-NR procedure is recommended as glycerol, unlike DMSO, does not inhibit subsequent growth of sorted cells.     

Triacylglycerol,total fatty acid,and biomass accumulation of metabolically engineered energycane grown under field conditions confirms its potential as feedstock for drop-in fuel production

Metabolic engineering for hyperaccumulation of lipids in vegetative tissues of high biomass crops promises a step change in oil yields for the production of advanced biofuels. Energycane is the ideal feedstock for this approach due to its exceptional biomass production and persistence under marginal conditions. Here, we evaluated metabolically engineered energycane with constitutive expression of the lipogenic factors WRINKLED1 (WRI1), DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1), and OLEOSIN1 (OLE1) for the accumulation of triacylglycerol (TAG), total fatty acid (TFA), and biomass under field conditions at the University of Florida-IFAS experiment station near Citra, Florida. TAG and TFA accumulation were highest in leaves (up to 9.9% and 12.9% of DW, respectively), followed by juice from crushed stems, stems, and roots. TAG and TFA accumulation increased up to harvest time and correlated highest with OLE1 and DGAT1 expression. Biomass dry weight, TAG, and TFA content differed greatly depending on DGAT1 and OLE1 expression in transgenic lines with similar WRI1 expression. Biomass did not significantly differ between WT and line L2 with DAGT1 and OLE1 expressed at low levels and TAG and TFA accumulating to 12- and 1.6-fold that of WT leaves, respectively. In contrast, line L13, with intron-mediated enhancement of DGAT1 expression, displayed a 245- to 330-fold increase in TAG and a 4.75- to 6.45-fold increase in TFA content compared with WT leaves and a biomass reduction of 52%. These results provide the basis for developing novel feedstocks for expanding plant lipid production and point to new prospects for advanced biofuels.