Growth performance, biochemical composition and sedimentation velocity of Tetraselmis sp. CTP4 under different salinities using low-cost lab- and pilot-scale systems

Biomass harvesting is one of the most expensive steps of the whole microalgal production pipeline. Therefore, the present work aimed to understand the effect of salinity on the growth performance, biochemical composition and sedimentation velocity of Tetraselmis sp. CTP4, in order to establish an effective low-cost pilot-scale harvesting system for this strain. At lab scale, similar growth performance was obtained in cultures grown at salinities of 5, 10 and 20 g L-1 NaCl. In addition, identical settling velocities (2.4-3.6 cm h-1) were observed on all salinities under study, regardless of the growth stage. However, higher salinities (20 g L-1) promoted a significant increase in lipid contents in this strain compared to when this microalga was cultivated at 5 or 10 g L-1 NaCl. At pilot-scale, cultures were cultivated semi-continuously in 2.5-m3 tubular photobioreactors, fed every four days, and stored in a 1-m3 harvesting tank. Upon a 24-hour settling step, natural sedimentation of the microalgal cells resulted in the removal of 93% of the culture medium in the form of a clear liquid containing only vestigial amounts of biomass (0.07 ± 0.02 g L-1 dry weight; DW). The remaining culture was recovered as a highly concentrated culture (19.53 ± 4.83 g L-1 DW) and wet microalgal paste (272.7 ± 18.5 g L-1 DW). Overall, this method provided an effective recovery of 97% of the total biomass, decreasing significantly the harvesting costs.Agência financiadora Portuguese national budget P2020 Foundation for Science and Technology (FCT) CMAR/Multi/04326/2013 ALGARED+ 1398 EP - INTERREG V-A Espana Portugal project ALGACO2 project 023310 COST Action 1408 - European Network for Bio-products FCT SFRH/BD/105541/2014info:eu-repo/semantics/publishedVersio

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.info:eu-repo/semantics/publishedVersio

Development of an organic culture medium for autotrophic production of chlorella vulgaris biomass

Microalgal biomass has gained increasing attention in the last decade for various biotechnological applications, including human nutrition. Certified organic products are currently a growing niche market in which the food industry has shown great interest. In this context, this work aimed at developing a certified organic culture medium for the production of autotrophic Chlorella vulgaris biomass. A preliminary assay in 2 L bubble column photobioreactors was performed in order to screen di erent commercial organic substrates (OS) at a normalized concentration of N (2 mmol L1). The highest growth performance was obtained using EcoMix4 and Bioscape which showed similar biomass concentrations compared to the synthetic culture medium (control). In order to meet the nutrient needs of Chlorella, both OS underwent elemental analyses to assess their nutrient composition. The laboratory findings allowed the development of a final organic culture medium using a proportion of Bioscape/EcoMix4 (1:1.2, m/m). This organic culture medium was later validated outdoors in 125 L flat panel and 10 m3 tubular flow through photobioreactors. The results obtained revealed that the developed organic medium led to similar microalgal growth performance and biochemical composition of produced biomass, as compared to the traditional synthetic medium. Overall, the formulated organic medium was e ective for the autotrophic production of organic C. vulgaris biomass.Portugal 2020 program (grant agreement nº POCI-01-0247-FEDER-035234; LISBOA-01-0247-FEDER-035234; ALG-01-0247-FEDER-035234); FCT: UIDB/04326/2020info:eu-repo/semantics/publishedVersio

Heterotrophic and photoautotrophic media optimization using response surface methodology for the Novel Microalga Chlorococcum amblystomatis

The nutritional requirements of novel microalgal strains are key for their effective cultivation and metabolite content. Therefore, the optimization of heterotrophic and photoautotrophic culture media is crucial for novel Chlorococcum amblystomatis growth. Heterotrophic and photoautotrophic biomass samples were characterized to identify the differences between their heterotrophic and photoautotrophic biomass composition and their biotechnological potential. Media optimization through surface response methodology led to 44.9 and 51.2% increments in C. amblystomatis-specific growth rates under heterotrophic and photoautotrophic growth, respectively. This microalga registered high protein content (61.49–73.45% dry weight), with the highest value being observed in the optimized photoautotrophic growth medium. The lipid fraction mainly constituted polyunsaturated fatty acids, ranging from 44.47 to 51.41% for total fatty acids (TFA) in cells under heterotrophy. However, these contents became significantly higher (70.46–72.82% TFA) in cultures cultivated under photoautotrophy. An interesting carotenoids content was achieved in the cultures grown in optimized photoautotrophic medium: 5.84 mg·g−1 β-carotene, 5.27 mg·g−1 lutein, 3.66 mg·g−1 neoxanthin, and 0.75 mg·g−1 violaxanthin. Therefore, C. amblystomatis demonstrated an interesting growth performance and nutritional profile for food supplements and feed products that might contribute to meeting the world’s nutritional demand.info:eu-repo/semantics/publishedVersio

Scenedesmus rubescens Heterotrophic Production Strategies for Added Value Biomass

Microalgae attract interest worldwide due to their potential for several applications. Scenedesmus is one of the first in vitro cultured algae due to their rapid growth and handling easiness. Within this genus, cells exhibit a highly resistant wall and propagate both auto- and heterotrophically. The main goal of the present work is to find scalable ways to produce a highly concentrated biomass of Scenedesmus rubescens in heterotrophic conditions. Scenedesmus rubescens growth was improved at the lab-scale by 3.2-fold (from 4.1 to 13 g/L of dry weight) through medium optimization by response surface methodology. Afterwards, scale-up was evaluated in 7 L stirred-tank reactor under fed-batch operation. Then, the optimized medium resulted in an overall productivity of 8.63 g/L/day and a maximum biomass concentration of 69.5 g/L. S. rubescens protein content achieved approximately 31% of dry weight, similar to the protein content of Chlorella vulgaris in heterotrophy.publishedVersio

Isolation and characterization of novel chlorella vulgaris mutants with low chlorophyll and improved protein contents for food applications

Microalgae are widely used as food supplements due to their high protein content, essential fatty acids and amino acids as well as carotenoids. The addition of microalgal biomass to food products (e.g., baked confectioneries) is a common strategy to attract novel consumers. However, organoleptic factors such as color, taste and smell can be decisive for the acceptability of foods supplemented with microalgae. The aim of this work was to develop chlorophyll-deficient mutants of Chlorella vulgaris by chemically induced random mutagenesis to obtain biomass with different pigmentations for nutritional applications. Using this strategy, two C. vulgaris mutants with yellow (MT01) and white (MT02) color were successfully isolated, scaled up and characterized. The changes in color of MT01 and MT02 mutant strains were due to an 80 and 99% decrease in their chlorophyll contents, respectively, as compared to the original wild type (WT) strain. Under heterotrophic growth, MT01 showed a growth performance similar to that of the WT, reaching a concentration of 5.84 and 6.06 g L-1, respectively, whereas MT02 displayed slightly lower growth (4.59 g L-1). When grown under a light intensity of 100 μmol m-2 s-1, the pigment content in MT01 increased without compromising growth, while MT02 was not able to grow under this light intensity, a strong indication that it became light-sensitive. The yellow color of MT01 in the dark was mainly due to the presence of the xanthophyll lutein. On the other hand, phytoene was the only carotenoid detected in MT02, which is known to be colorless. Concomitantly, MT02 contained the highest protein content, reaching 48.7% of DW, a 60% increase as compared to the WT. MT01 exhibited a 30% increase when compared to that of the WT, reaching a protein content of 39.5% of DW. Taken together, the results strongly suggest that the partial abrogation of pigment biosynthesis is a factor that might promote higher protein contents in this species. Moreover, because of their higher protein and lower chlorophyll contents, the MT01 and MT02 strains are likely candidates to be feedstocks for the development of novel, innovative food supplements and foods.FCT: UIDB/04085/2020info:eu-repo/semantics/publishedVersio

Lab-Scale Optimization of Aurantiochytrium sp. Culture Medium for Improved Growth and DHA Production

Thraustochytrids have gained increasing relevance over the last decades, due to their fast growth and outstanding capacity to accumulate polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA). In this context, the present work aimed to optimize the growth performance and DHA yields by improving the culture medium of Aurantiochytrium sp. AF0043. Accordingly, two distinct culture media were optimized: (i) an inorganic optimized medium (IOM), containing only monosodium glutamate and glucose as nitrogen and carbon sources, respectively; and (ii) an organic and sustainable waste-based optimized medium (WOM), containing corn steep powder and glycerol, added in fed-batch mode, as nitrogen and carbon sources, respectively. Overall, the lab-scale optimization allowed to increase the biomass yield 1.5-fold and enhance DHA content 1.7-fold using IOM. Moreover, WOM enabled a 2-fold increase in biomass yield and a significant improvement in lipid contents, from 22.78% to 31.14%. However, DHA content was enhanced almost 3-fold, from an initial content of 10.12% to 29.66% of total fatty acids contained in the biomass. Therefore, these results strongly suggest, not only that the production pipeline was significantly improved but also confirmed the potential use of Aurantiochytrium sp. AF0043 as a source of DHA

Heterotrophic growth of thraustochytrids strains: screening, optimization and scale-up

Thraustochytrids are well-reported microorganisms with an outstanding ability to produce and accumulate polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA). In the present study, a lab-scale optimization on medium and fermentation parameters, was carried out with Aurantiochytrium sp. 0043 AA aiming at high cell biomass, lipids and DHA yields. Medium optimization was performed based on the literature, focused on the screening of different C and N sources, on the concentration of each source as well as on the ratio and feeding strategies. In addition, other fermentation parameters were optimized, such as initial inoculum conditions (volume, age and stage), different oxygen transfer rates and pH. Later on, the growth performances of Aurantiochytrium sp. 0050 AA and 0051 AA, were compared to strain 0043 AA. The respective cryopreservation processes weres completed and the banks validated, with the exception of the master cell bank (MCB1) of strain 0051 AA. Overall, lab-scale optimization of strain 0043 AA allowed to increase biomass yield by 2fold and improve lipid and DHA contents from 22.78% and 1.25% to 31.14% and 29.66%, respectively. Furthermore, with strain 0051 AA, a maximum dry weight (DW) of 80.15 g/L was obtained, with concomitant lipid and DHA contents of 60.34% and 38.81%, respectively. Although several conditions were tested in the 5 L bench-top reactor, cultivation was not successful due to the shear-sensitive cells of this species and further trials on fermentation parameters and reactor design are required to enable the scale-up