Random Mutagenesis as a Promising Tool for Microalgal Strain Improvement towards Industrial Production.

Microalgae have become a promising novel and sustainable feedstock for meeting the rising demand for food and feed. However, microalgae-based products are currently hindered by high production costs. One major reason for this is that commonly cultivated wildtype strains do not possess the robustness and productivity required for successful industrial production. Several strain improvement technologies have been developed towards creating more stress tolerant and productive strains. While classical methods of forward genetics have been extensively used to determine gene function of randomly generated mutants, reverse genetics has been explored to generate specific mutations and target phenotypes. Site-directed mutagenesis can be accomplished by employing different gene editing tools, which enable the generation of tailor-made genotypes. Nevertheless, strategies promoting the selection of randomly generated mutants avoid the introduction of foreign genetic material. In this paper, we review different microalgal strain improvement approaches and their applications, with a primary focus on random mutagenesis. Current challenges hampering strain improvement, selection, and commercialization will be discussed. The combination of these approaches with high-throughput technologies, such as fluorescence-activated cell sorting, as tools to select the most promising mutants, will also be discussed.

Drying Microalgae Using an Industrial Solar Dryer: A Biomass Quality Assessment.

Microalgae are considered a promising resource of proteins, lipids, carbohydrates, and other functional biomolecules for food and feed markets. Competitive drying solutions are required to meet future demands for high-quality algal biomass while ensuring proper preservation at reduced costs. Since often used drying methods, such as freeze or spray drying, are energy and time consuming, more sustainable processes remain to be developed. This study tested an indirect and hybrid solar dryer as an alternative to conventional freeze drying of industrially produced Tetraselmis chui and Nannochloropsis oceanica wet paste. The effects of the drying method on biomass quality parameters, including biochemical profiles, functional properties, and microbial safety, were assessed. No significant differences were found between the applied drying technologies for total proteins, carbohydrates, lipids, and fatty acid profiles. On the other hand, some pigments showed significant differences, displaying up to 44.5% higher contents in freeze-dried samples. Minor differences were also registered in the mineral profiles (<10%). Analyses of microbial safety and functional properties of the solar-dried biomass appear adequate for food and feed products. In conclusion, industrial solar drying is a sustainable technology with a high potential to preserve high-quality microalgal biomass for various markets at expected lower costs.

Nutritional and Functional Evaluation of Inula crithmoides and Mesembryanthemum nodiflorum Grown in Different Salinities for Human Consumption.

The nutritional composition and productivity of halophytes is strongly related to the biotic/abiotic stress to which these extremophile salt tolerant plants are subjected during their cultivation cycle. In this study, two commercial halophyte species (Inula crithmoides and Mesembryanthemum nodiflorum) were cultivated at six levels of salinity using a soilless cultivation system. In this way, it was possible to understand the response mechanisms of these halophytes to salt stress. The relative productivity decreased from the salinities of 110 and 200 mmol L-1 upwards for I. crithmoides and M. nodiflorum, respectively. Nonetheless, the nutritional profile for human consumption remained balanced. In general, I. crithmoides vitamin (B1 and B6) contents were significantly higher than those of M. nodiflorum. For both species, ß-carotene and lutein were induced by salinity, possibly as a response to oxidative stress. Phenolic compounds were more abundant in plants cultivated at lower salinities, while the antioxidant activity increased as a response to salt stress. Sensory characteristics were evaluated by a panel of culinary chefs showing a preference for plants grown at the salt concentration of 350 mmol L-1. In summary, salinity stress was effective in boosting important nutritional components in these species, and the soilless system promotes the sustainable and safe production of halophyte plants for human consumption.

Carotenoid biosynthetic gene expression, pigment and n-3 fatty acid contents in carotenoid-rich Tetraselmis striata CTP4 strains under heat stress combined with high light.

In this study, two carotenoid-rich strains of the euryhaline microalga Tetraselmis striata CTP4 were isolated by random mutagenesis combined with selection via fluorescence activated cell sorting and growth on norflurazon. Both strains, ED5 and B11, showed an up to 1.5-fold increase in carotenoid contents as compared with the wildtype, independent of the growth conditions. More specifically, violaxanthin, ß-carotene and lutein contents reached as high as 1.63, 4.20 and 3.81 mg g-1 DW, respectively. Genes coding for phytoene synthase, phytoene desaturase, lycopene-ß-cyclase and ε-ring hydroxylase involved in carotenoid biosynthesis were found to be upregulated in ED5 and B11 cells as compared to the wildtype. Both strains showed higher contents of eicosapentaenoic acid as compared with those of the wildtype, reaching up to 4.41 and 2.88 mg g-1 DW, respectively. Overall, these results highlight the complexity of changes in carotenoid biosynthesis regulation that are required to improve pigment contents in microalgae.

Flashing light emitting diodes (LEDs) induce proteins, polyunsaturated fatty acids and pigments in three microalgae.

As the periodic emission of light pulses by light emitting diodes (LEDs) is known to stimulate growth or induce high value biocompounds in microalgae, this flashing light regime was tested on growth and biochemical composition of the microalgae Nannochloropsis gaditana, Koliella antarctica and Tetraselmis chui. At low flashing light frequencies (e.g., 5 and 50 Hz, Duty cycle = 0.05), a strain-dependent growth inhibition and an accumulation of protein, polyunsaturated fatty acids, chlorophyll or carotenoids (lutein, ß-carotene, violaxanthin and neoxanthin) was observed. In addition, a 4-day application of low-frequency flashing light to concentrated cultures increased productivities of eicosapentaenoic acid (EPA) and specific carotenoids up to three-fold compared to continuous or high frequency flashing light (500 Hz, Duty cycle = 0.05). Therefore, applying low-frequency flashing light as finishing step in industrial production can increase protein, polyunsaturated fatty acids or pigment contents in biomass, leading to high-value algal products.

Improvement of carotenoid extraction from a recently isolated, robust microalga, Tetraselmis sp. CTP4 (chlorophyta).

In recent years, there has been increasing consumer interest in carotenoids, particularly of marine sustainable origin with applications in the food, cosmeceutical, nutritional supplement and pharmaceutical industries. For instance, microalgae belonging to the genus Tetraselmis are known for their biotechnologically relevant carotenoid profile. The recently isolated marine microalgal strain Tetraselmis sp. CTP4 is a fast-growing, robust industrial strain, which has successfully been produced in 100-m3 photobioreactors. However, there are no reports on total carotenoid contents from this strain belonging to T. striata/convolutae clade. Although there are several reports on extraction methods targeting chlorophytes, extraction depends on the strength of cell coverings, solvent polarity and the nature of the targeted carotenoids. Therefore, this article evaluates different extraction methods targeting Tetraselmis sp. CTP4, a strain known to contain a mechanically resistant theca. Here, we propose a factorial experimental design to compare extraction of total carotenoids from wet and freeze-dried microalgal biomass using four different solvents (acetone, ethanol, methanol or tetrahydrofuran) in combination with two types of mechanical cell disruption (glass beads or dispersion). The extraction efficiency of the methods was assessed by pigment contents and profiles present in the extracts. Extraction of wet biomass by means of glass bead-assisted cell disruption using tetrahydrofuran yielded the highest amounts of lutein and ß-carotene (622 ± 40 and 618 ± 32 µg g-1 DW, respectively). Although acetone was slightly less efficient than tetrahydrofuran, it is preferable due to its lower costs and toxicity.

Flashing LEDs for Microalgal Production.

Flashing lights are next-generation tools to mitigate light attenuation and increase the photosynthetic efficiency of microalgal cultivation systems illuminated by light-emitting diodes (LEDs). Optimal flashing light conditions depend on the reaction kinetics and properties of the linear electron transfer chain, energy dissipation, and storage mechanisms of a phototroph. In particular, extremely short and intense light flashes potentially mitigate light attenuation in photobioreactors without impairing photosynthesis. Intelligently controlling flashing light units and selecting electronic components can maximize light emission and energy efficiency. We discuss the biological, physical, and technical properties of flashing lights for algal production. We combine recent findings about photosynthetic pathways, self-shading in photobioreactors, and developments in solid-state technology towards the biotechnological application of LEDs to microalgal production.

Urban wastewater treatment by Tetraselmis sp. CTP4 (Chlorophyta).

The ability of a recent isolate, Tetraselmis sp. CTP4, for nutrient removal from sewage effluents before and after the nitrification process under batch and continuous cultivation was studied. Biomass productivities in both wastewaters were similar under continuous conditions (0.343±0.053gL-1d-1) and nutrient uptake rates were maximal 31.4±0.4mgNL-1d-1 and 6.66±1.57mgP-PO43-L-1d-1 in WW before nitrification when cultivated in batch. Among batch treatments, cellular protein, carbohydrate and lipid levels shifted with aging cultures from 71.7±6.3 to 29.2±1.2%, 17.4±7.2 to 57.2±3.9% and 10.9±1.7 to 13.7±4.7%, respectively. In contrast, CTP4 cultivated continuously in Algal medium (control) showed lower biomass productivities (0.282gVSSL-1d-1) although improved lipid content (up to 20% lipids) in batch cultivation. Overall, Tetraselmis sp. CTP4 is promising for WW treatment as a replacement of the costly nitrification process, fixating more nutrients and providing a protein and carbohydrate-rich biomass as by-product.