Biomass, lipid accumulation kinetics and the transcriptome of heterotrophic oleaginous microalga Tetradesmus bernardii under different carbon and nitrogen regime

Background: Heterotrophic cultivation of microalgae has been proposed as a viable alternative method for novel high-value biomolecules, enriched biomass and biofuel production because of their allowance of high cell density levels, as well as simple production technology. Tetradesmus bernardii, a newly isolated high-yielding oleaginous microalga under photoautotrophic conditions, is able to grow heterotrophically, meaning that it can consume organic carbon sources in dark condition. We investigated the effect of different carbon/nitrogen (C/N) ratios on the growth and lipid accumulation of T. bernardii in heterotrophic batch culture under two nitrogen sources (NaNO3, CO(NH2)2). In addition, we conducted time-resolved transcriptome analysis to reveal the metabolic mechanism of T. bernardii in heterotrophic culture. Results: T. bernardii can accumulated high biomass concentrations in heterotrophic batch culture which the highest biomass of 46.09 g/L was achieved at 100 g/L glucose concentration. The rate of glucose to biomass was exceed 55% when the glucose concentration was less than 80 g/L, and the C/N ratio was 44 at urea treatment. The culture was beneficial to lipid accumulation at a C/N ratio between 110 and 130. NaNO3 used as a nitrogen source enhanced the lipid content more than urea, and the highest lipid content was 45% of dry weight. We performed RNA-seq to analyze the time-resolved transcriptome of T. bernardii. As the nitrogen was consumed in the medium, nitrogen metabolism related genes were significantly up-regulated to speed up the N metabolic cycle. As chloroplasts were destroyed in the dark, the metabolism of cells was transferred from chloroplasts to cytoplasm. However, storage of carbohydrate in chloroplast remained active, mainly the synthesis of starch, and the precursor of starch synthesis in heterotrophic culture may largely came from the absorption of organic carbon source (glucose). With regard to lipid metabolism, the related genes of fatty acid synthesis in low nitrogen concentration increased gradually with the extension of cultivation time.Conclusion: T. bernardii exhibited rapid growth and high lipid accumulation in heterotrophic culture. It may be a potential candidate for biomass and biofuel production. Transcriptome analysis showed that multilevel regulation ensured the conversion from carbon to the synthesis of carbohydrate and lipid.

Biomass, lipid accumulation kinetics, and the transcriptome of heterotrophic oleaginous microalga Tetradesmus bernardii under different carbon and nitrogen sources

Background Heterotrophic cultivation of microalgae has been proposed as a viable alternative method for novel high-value biomolecules, enriched biomass, and biofuel production because of their allowance of high cell density levels, as well as simple production technology. Tetradesmus bernardii, a newly isolated high-yielding oleaginous microalga under photoautotrophic conditions, is able to grow heterotrophically, meaning that it can consume organic carbon sources in dark condition. We investigated the effect of different carbon/nitrogen (C/N) ratios on the growth and lipid accumulation of T. bernardii in heterotrophic batch culture under two nitrogen sources (NaNO3 and CO(NH2)2). In addition, we conducted time-resolved transcriptome analysis to reveal the metabolic mechanism of T. bernardii in heterotrophic culture. Results T. bernardii can accumulate high biomass concentrations in heterotrophic batch culture where the highest biomass of 46.09 g/L was achieved at 100 g/L glucose concentration. The rate of glucose to biomass exceeded 55% when the glucose concentration was less than 80 g/L, and the C/N ratio was 44 at urea treatment. The culture was beneficial to lipid accumulation at a C/N ratio between 110 and 130. NaNO3 used as a nitrogen source enhanced the lipid content more than urea, and the highest lipid content was 45% of dry weight. We performed RNA-seq to analyze the time-resolved transcriptome of T. bernardii. As the nitrogen was consumed in the medium, nitrogen metabolism-related genes were significantly up-regulated to speed up the N metabolic cycle. As chloroplasts were destroyed in the dark, the metabolism of cells was transferred from chloroplasts to cytoplasm. However, storage of carbohydrate in chloroplast remained active, mainly the synthesis of starch, and the precursor of starch synthesis in heterotrophic culture may largely come from the absorption of organic carbon source (glucose). With regard to lipid metabolism, the related genes of fatty acid synthesis in low nitrogen concentration increased gradually with the extension of cultivation time. Conclusion T. bernardii exhibited rapid growth and high lipid accumulation in heterotrophic culture. It may be a potential candidate for biomass and biofuel production. Transcriptome analysis showed that multilevel regulation ensured the conversion from carbon to the synthesis of carbohydrate and lipid.

Antioxidant and Cytotoxic Effects on Tumor Cells of Exopolysaccharides from Tetraselmis suecica (Kylin) Butcher Grown Under Autotrophic and Heterotrophic Conditions

Marine microalgae produce extracellular metabolites such as exopolysaccharides (EPS) with potentially beneficial biological applications to human health, especially antioxidant and antitumor properties, which can be increased with changes in crop trophic conditions. This study aimed to develop the autotrophic and heterotrophic culture of Tetraselmis suecica (Kylin) Butcher in order to increase EPS production and to characterize its antioxidant activity and cytotoxic effects on tumor cells. The adaptation of autotrophic to heterotrophic culture was carried out by progressively reducing the photoperiod and adding glucose. EPS extraction and purification were performed. EPS were characterized by Fourier-transform infrared spectroscopy and gas chromatography-mass spectrometry. The antioxidant capacity of EPS was analyzed by the 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) method, and the antitumor capacity was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, showing high activity on human leukemia, breast and lung cancer cell lines. Although total EPS showed no cytotoxicity, acidic EPS showed cytotoxicity over the gingival fibroblasts cell line. Heterotrophic culture has advantages over autotrophic, such as increasing EPS yield, higher antioxidant capacity of the EPS and, to the best of our knowledge, this is the first probe that T. suecica EPS have cytotoxic effects on tumor cells; therefore, they could offer greater advantages as possible natural nutraceuticals for the pharmaceutical industry.

Biomass and Lipid Accumulation Kinetics and the Transcriptome of Oleaginous Microalga Tetradesmus Bernardii (Chlorophyceae) Under Heterotrophic Culture With Different Nitrogen Source Supply and Carbon/Nitrogen Ratio

Background: Heterotrophic cultivation of microalgae has been proposed as a viable alternative method for novel high-value biomolecules, enriched biomass and biofuel production because of their allowance of high cell density levels, as well as simple production technology. Tetradesmus bernardii, a newly isolated high-yielding oleaginous microalga under photoautotrophic conditions, is able to grow heterotrophically, meaning that it can consume organic carbon sources in dark condition. We investigated the effect of different carbon/nitrogen (C/N) ratios on the growth and lipid accumulation of T. bernardii in heterotrophic batch culture under two nitrogen sources (NaNO3, CO(NH2)2). In addition, we conducted time-resolved transcriptome analysis to reveal the metabolic mechanism of T. bernardii in heterotrophic culture. Results: T. bernardii can accumulated high biomass concentrations in heterotrophic batch culture which the highest biomass of 46.09 g/L was achieved at 100 g/L glucose concentration. The rate of glucose to biomass was exceed 55% when the glucose concentration was less than 80 g/L, and the C/N ratio was 44 at urea treatment. The culture was beneficial to lipid accumulation at a C/N ratio between 110 and 130. NaNO3 used as a nitrogen source enhanced the lipid content more than urea, and the highest lipid content was 45% of dry weight. We performed RNA-seq to analyze the time-resolved transcriptome of T. bernardii. As the nitrogen was consumed in the medium, nitrogen metabolism related genes were significantly up-regulated to speed up the N metabolic cycle. As chloroplasts were destroyed in the dark, the metabolism of cells was transferred from chloroplasts to cytoplasm. However, storage of carbohydrate in chloroplast remained active, mainly the synthesis of starch, and the precursor of starch synthesis in heterotrophic culture may largely came from the absorption of organic carbon source (glucose). With regard to lipid metabolism, the related genes of fatty acid synthesis in low nitrogen concentration increased gradually with the extension of cultivation time.Conclusion: T. bernardii exhibited rapid growth and high lipid accumulation in heterotrophic culture. It may be a potential candidate for biomass and biofuel production. Transcriptome analysis showed that multilevel regulation ensured the conversion from carbon to the synthesis of carbohydrate and lipid.

Denitrification Kinetics of Nitrate by a Heterotrophic Culture in Batch and Fixed-Biofilm Reactors

Herein, the progress of nitrate removal by a heterotrophic culture in a batch reactor and continuous-flow fixed-biofilm reactor was examined. Two batch experiments for nitrate reduction with acetate degradation using 250 mL batch reactors with acclimated denitrifying biomass were conducted. The experimental results indicated that the nitrate was completely reduced; however, the acetate remained at a concentration of 280 mg/L from initial nitrate concentration of 100 mg/L. However, the acetate was fully biodegraded by the denitrifying biomass at an initial nitrate concentration of 300 mg/L. To evaluate the biokinetic parameters, the concentration data of nitrate, nitrite, acetate, and denitrifying biomass from the batch kinetic experiments were compared with those of the batch kinetic model system. A continuous-flow fixed-biofilm reactor was used to verify the kinetic biofilm model. The removal efficiency of nitrate in the fixed-biofilm reactor at the steady state was 98.4% accompanied with 90.5% acetate consumption. The experimental results agreed satisfactorily with the model predictions. The modeling and experimental approaches used in this study could be applied in the design of a pilot-scale, or full-scale, fixed-biofilm reactor for nitrate removal in water and wastewater treatment plants.

Maximizing Biomass and Lipid Production in Heterotrophic Culture of Chlorella vulgaris: Techno-Economic Assessment

Background: Nowadays, chlorophycean microalgae have attained a broad-spectrum attention as a potential candidate for biomass and bioenergy production. Despite their appreciated benefits, one of major problems is their low biomass and lipid productivity. Here we investigated the heterotrophic culture in shake flasks and stirred tank bioreactor to improve the lipid and biomass production in a naturally isolated strain of Chlorella vulgaris. Methods: A naturally isolated C. vulgaris strain was cultivated in BG-11 medium in shake flask and bioreactor. Its biochemical composition and growth kinetic parameters were investigated. Results: The biomass productivity was improved (3.68 fold) under heterotrophic culture compared to basal autotrophic culture condition in shake flask experiment. The total lipid content increased to 44% of total Dry Cell Weight (DCW) during heterotrophic growth after 21 days. Moreover, a great Fatty Acid Methyl Esters (FAME) yield was observed under heterotrophic cultivation. Total biomass and lipid content of microalgae in bioreactor experiment increased to 4.95 and 2.18 g L-1 respectively, during 5 days of the experiment compared to its basic autotrophic culture. Conclusion: The techno-economic aspects of exploiting C. vulgaris as a biodiesel feedstock werealso evaluated. The results imply that heterotrophic cultivation could compensate the low biomass productivity in microalgae for green energy production. Ever growing rates of established patents on application of various genetic and bioengineering-based methods have made it possible to achieve higher lipid contents with reduced total costs for microalgal biodiesel production as well.