Nitrogen and phosphorus stress as a tool to induce lipid production in microalgae.

Microalgae, capable of accumulating large amounts of lipids, are of great value for biodiesel production. The high cost of such production stimulates the search for cultivation conditions that ensure their highest productivity. Reducing the content of nitrogen and phosphorus in the culture medium is widely used to change the content and productivity of lipids in microalgae. Achieving the right balance between maximum growth and maximum lipid content and productivity is the primary goal of many experimental works to ensure cost-effective biodiesel production from microalgae. The content of nitrogen and phosphorus in nutrient media for algal cultivation after converted to nitrogen (-N) and phosphorus (-P) lies in an extensive range: from 0.007 g L- 1 to 0.417 g L- 1 and from 0.0003 g L- 1 to 0.227 g L- 1 and N:P ratio from 0.12:1 to 823.33:1. When studying nutritional stress in microalgae, no single approach is used to determine the experimental concentrations of nitrogen and phosphorus. This precludes the possibility of correct interpretation of the data and may lead to erroneous conclusions. This work results from the systematisation of information on using nitrogen and phosphorus restriction to increase the lipid productivity of microalgae of different taxonomic and ecological groups to identify future research directions. The results of 301 experiments were included in the analysis using the principal components method. The investigation considered various divisions and classes: Cyanobacteria, Rhodophyta, Dinophyta, Haptophyta, Cryptophyta, Heterokontophyta/Ochrophyta (Bacillariophyceae, Eustigmatophyceae, Xanthophyceae), Chlorophyta, and also the ratio N:P, the time of the experiment, the light intensity during cultivation. Based on the concentrations of nitrogen and phosphorus existing in various nutrient media, a general scheme for designating the supply of nutrient media for nitrogen (as NO3- or NH4+, N g L- 1) and phosphorus (as РO4-, P g L- 1) has been proposed: replete -N (˃0.4 g L- 1), moderate -N (0.4-0.2), moderate N-limitation (0.19-0.1), strong N-limitation (˂0.1), without nitrogen (0), replete -Р (˃0.2), moderate -P (0.2-0.02), moderate P-limitation (0.019-0.01), strong P-limitation (˂0.01), without phosphorus (0).

Optimization of Biomass Accumulation and Production of Phenolic Compounds in Callus Cultures of Rhodiola rosea L. Using Design of Experiments.

Rhodiola rosea L. is a valuable medicinal plant with adaptogenic, neuroprotective, antitumor, cardioprotective, and antidepressant effects. In this study, design of experiments methodology was employed to analyze and optimize the interacting effects of mineral compounds (concentration of NO3- and the ratio of NH4+ to K+) and two plant growth regulators [total 6-benzylaminopurine (BAP) and α-naphthylacetic acid (NAA) concentration and the ratio of BAP to NAA] on the growth and the production of total phenolic compounds (TPCs) in R. rosea calluses. The overall effect of the model was highly significant (p < 0.0001), indicating that NH4+, K+, NO3-, BAP, and NAA significantly affected growth. The best callus growth (703%) and the highest production of TPCs (75.17 mg/g) were achieved at an NH4+/K+ ratio of 0.33 and BAP/NAA of 0.33, provided that the concentration of plant growth regulators was 30 µM and that of NO3- was ≤40 mM. According to high-performance liquid chromatography analyses of aerial parts (leaves and stems), in vitro seedlings and callus cultures of R. rosea contain no detectable rosarin, rosavin, rosin, and cinnamyl alcohol. This is the first report on the creation of an experiment for the significant improvement of biomass accumulation and TPC production in callus cultures of R. rosea.

Lipid accumulation by Coelastrella multistriata (Scenedesmaceae, Sphaeropleales) during nitrogen and phosphorus starvation.

A novel freshwater strain of Coelastrella multistriata MZ-Ch23 was discovered in Tula region, Russia. The identification is based on morphological features, phylogenetic analysis of SSU rDNA gene and ITS1-5.8S rDNA-ITS2 region and predicted secondary structure of the ITS2. Phylogenetic analysis places the novel strain in the "core" Coelastrella clade within the Chlorophyceae. This is the first record of Coelastrella multistriata in the algal flora of Russia. Cultivation experiments were carried out to evaluate growth dynamics of the newly identified strain and the impact of nitrogen and/or phosphorus depletion on the fatty acid profiles and lipid productivity. On the fully supplemented Bold's basal medium and under phosphorus-depleted conditions as well, the fatty acid profiles were dominated by α-linolenic acid (29.4-38.1% of total fatty acids). Depletion of either nitrogen or both nitrogen and phosphorus was associated with increased content of oleic acid (32.9-33.7%) and linoleic acid (11.9%). Prolongation of the growth to two months (instead of 25 days) resulted in increased content and diversity of very long-chain fatty acids including saturated species. The total very long-chain fatty acid content of 9.99% achieved in these experiments was 1.9-12.3-fold higher than in stress experiments. The highest variation was observed for oleic acid (3.4-33.7%). The novel strain showed the ability to accumulate lipids in amounts up to 639.8 mg L-1 under nitrogen and phosphorus starvation, which exceeds the previously obtained values for most Coelastrella strains. Thus, the newly identified MZ-Ch23 strain can be considered as a potential producer of omega-3 fatty acids on fully supplemented Bold's basal medium or as a source of biomass with high content of saturated and monounsaturated fatty acids after nitrogen and phosphorus starvation.

A New Species of Freshwater Algae Nephrochlamys yushanlensis sp. nov. (Selenastraceae, Sphaeropleales) and Its Lipid Accumulation during Nitrogen and Phosphorus Starvation.

The new species Nephrochlamys yushanlensis sp. nov. is described from a freshwater plankton sample. A comparison of morphology, 18S rDNA gene and ITS2 sequences, and fatty acid profiles showed that the novel strain represents a new lineage within the genus Nephrochlamys. For the first time with a member of the Selenastraceae, experiments with phosphate and nitrate deprivation were conducted to evaluate changes in biomass, lipid and triacylglycerol (TAGs) accumulation, and composition of fatty acids. Biomass dry weight under simultaneous nitrogen and phosphorus depletion was 1.73 g · L-1 , which is significantly lower than the 2.41 g · L-1 observed in the control. All conditions of nutrient restriction significantly increased the lipid content in comparison with the control. The largest increase in the total lipid content, reaching 58.64% DW per cell at the end of cultivation, occurred with nitrogen deficiency. Significant increases in TAGs content (to 23.69% and 21.74%, respectively) occurred in phosphorus- and nitrogen-depleted conditions in comparison to the control (16.90%). Oleic (49.8-64.1%), palmitic (21.1-22.7%), and linoleic (8.6-10.3%) acids were the dominant fatty acids when cultured on standard BBM medium, as well as with the shortage of nutrients. Phosphorus deprivation as well as absence of both nitrogen and phosphorus led to the appearance of FAMEs α-linolenic (1.5-4.1%) and stearidonic (1.0-1.8%) acids. In general, FAME profiles revealed that the relative percentage of saturated and monounsaturated fatty acids increased (88.9% of total fatty acids) in nitrogen-depletion conditions, suggesting this strain may be suitable for biodiesel production.

Fatty Acid Content and Profile of the Novel Strain of Coccomyxa elongata (Trebouxiophyceae, Chlorophyta) Cultivated at Reduced Nitrogen and Phosphorus Concentrations.

A novel freshwater strain of Coccomyxa elongata (MZ-Ch64) was isolated from the Zaporizhia region, Ukraine. The identification was based on the phylogenetic analysis of SSU rDNA gene and ITS1-5.8S rDNA-ITS2 region and predicted secondary structure of the ITS2. Phylogenetic analysis placed this strain in the Coccomyxa group, within the class Trebouxiophyceae. The novel strain MZ-Ch64 formed a strongly supported lineage closest with C. elongata. The MZ-Ch64 strain differed from the morphological description of the species by the size of vegetative cells and absence of small mucilaginous caps at one end of the cell. A number of experiments with different concentrations of phosphate and nitrate were conducted to evaluate changes in the resulting fatty acid profiles and biomass productivity. The fatty acid profile and total fatty acids varied significantly under different nutrient deficiencies. The dominant fatty acid during cultivation on standard BBM medium, as well as in phosphorus-depleted conditions, was oleic acid (to 48.0%-54.6% of total fatty acids). Absence of nitrogen alone, and absence of both nitrogen and phosphorus, led to an increase of palmitic acid (to 24.7%-25.6%), cis-7-hexadecenoic acid (to 14.8%) and α-linolenic acid (to 9.1%-10.1%) in comparison with the control sample. The greatest variation was found for oleic acid (31.9%-54.6%). Thus, this strain can be considered as a potential producer of oleic acid or cis-7-hexadecenoic and α-linolenic acids for biotechnological applications.