Effects of Temperature, pH, and NaCl Concentration on Biomass and Bioactive Compound Production by Synechocystis salina.

Synechocystis salina is a cyanobacterium that has biotechnological potential thanks to its ability to synthesize several bioactive compounds of interest. Therefore, this study aimed to find optimal conditions, in terms of temperature (15-25 °C), pH (6.5-9.5), and NaCl concentration (10-40 g·L-1), using as objective functions the productivities of biomass, total carotenoids, total PBPs, phycocyanin (PC), allophycocyanin (APC), phycoerythrin (PE), and antioxidants (AOXs) capacity of Synechocystis salina (S. salina) strain LEGE 06155, based in factorial design resorting to Box-Behnken. The model predicted higher biomass productivities under a temperature of 25 °C, a pH of 7.5, and low NaCl concentrations (10 g·L-1). Maximum productivities in terms of bioactive compounds were attained at lower NaCl concentrations (10 g·L-1) (except for PE), with the best temperature and pH in terms of carotenoids and total and individual PBPs ranging from 23-25 °C to 7.5-9.5, respectively. PE was the only pigment for which the best productivity was reached at a lower temperature (15 °C) and pH (6.5) and a higher concentration of NaCl (≈25 g·L-1). AOX productivities, determined in both ethanolic and aqueous extracts, were positively influenced by lower temperatures (15-19 °C) and higher salinities (≈15-25 g·L-1). However, ethanolic AOXs were better recovered at a higher pH (pH ≈ 9.5), while aqueous AOXs were favored by a pH of 8. The model showed that biomass production can be enhanced by 175% (compared to non-optimized conditions), total carotenoids by 91%, PC by 13%, APC by 50%, PE by 130%, and total PBPs by 39%; for AOX productivities, only water extracts exhibited a (marginal) improvement of 1.4%. This study provided insightful information for the eventual upgrading of Synechocystis salina biomass in the biotechnological market.

Potential of Microalgae Extracts for Food and Feed Supplementation-A Promising Source of Antioxidant and Anti-Inflammatory Compounds.

Microalgae are known producers of antioxidant and anti-inflammatory compounds, making them natural alternatives to be used as food and feed functional ingredients. This study aimed to valorise biomass and exploit new applications and commercial value for four commercially available microalgae: Isochrysis galbana, Nannochloropsis sp., Tetraselmis sp., and Phaeodactylum tricornutum. For that, five extracts were obtained: acetone (A), ethanol (E), water (W), ethanol:water (EW). The antioxidant capacity (ABTS•+/DPPH•/•NO/O2•-/ORAC-FL) and anti-inflammatory capacity (HBRC/COX-2) of the extracts were screened. The general biochemical composition (carbohydrates, soluble proteins, and lipids) and the main groups of bioactive compounds (carotenoids, phenolic compounds, and peptides) of extracts were quantified. The results of antioxidant assays revealed the potential of some microalgae extracts: in ABTS•+, Nannochloropsis sp. E and Tetraselmis sp. A, E, and P; in DPPH•, Tetraselmis sp. A and E; in •NO, P. tricornutum E and EW; in O2•-, Tetraselmis sp. W; and in ORAC-FL, I. galbana EW and P. tricornutum EW. Concerning anti-inflammatory capacity, P. tricornutum EW and Tetraselmis sp. W showed a promising HBRC protective effect and COX-2 inhibition. Hence, Tetraselmis sp. and P. tricornutum extracts seem to have potential to be incorporated as feed and food functional ingredients and preservatives.

Cosmetic Potential of Pigments Extracts from the Marine Cyanobacterium Cyanobium sp.

The current mindset in the cosmetics market about sustainable ingredients had increased the search for new sources of natural active ingredients. Cyanobacteria are a great source of functional ingredients for cosmetics, as a producer of pigments with described bioactive potential (carotenoids and phycobiliproteins). This work aimed to evaluate the cosmetic potential of marine cyanobacterium Cyanobium sp. pigment-targeted extracts (carotenoids and phycobiliproteins), evaluating their in vitro safety through cytotoxicity assays, cosmetic-related enzyme inhibition, ingredient stability, and putative product (serum formulation). Results showed no cytotoxicity from the extracts in skin-related cell lines. Carotenoid extract showed anti-hyaluronidase capacity (IC50 = 108.74 ± 5.74 mg mL-1) and phycobiliprotein extract showed anti-hyaluronidase and anti-collagenase capacity (IC50 = 67.25 ± 1.18 and 582.82 ± 56.99 mg mL-1, respectively). Regarding ingredient and serum stability, both ingredients showed higher stability at low-temperature conditions, and it was possible to maintain the pigment content and bioactive capacity stable during the tested period, although in higher temperatures the product was degraded in a week. As a major conclusion, both extracts can be potential natural and sustainable ingredients for cosmetic uses, with relatively simple formulation and storage, and can be promising natural anti-aging ingredients due to their bioactive capacity.

Light Modulation for Bioactive Pigment Production in Synechocystis salina.

Cyanobacteria are microorganisms that are well-adapted to sudden changes in their environment, namely to light conditions. This has allowed them to develop mechanisms for photoprotection, which encompass alteration in pigment composition. Therefore, light modulation appears to be a suitable strategy to enhance the synthesis of specific pigments (e.g., phycocyanin) with commercial interest, in addition to conveying a more fundamental perspective on the mechanisms of acclimatization of cyanobacterium species. In this study, Synechocystis salina was accordingly cultivated in two light phase stages: (i) white LED, and (ii) shift to distinct light treatments, including white, green, and red LEDs. The type of LED lighting was combined with two intensities (50 and 150 µmolphotons·m-2·s-1). The effects on biomass production, photosynthetic efficiency, chlorophyll a (chl a) content, total carotenoids (and profile thereof), and phycobiliproteins (including phycocyanin, allophycocyanin, and phycoerythrin) were assessed. White light (under high intensity) led to higher biomass production, growth, and productivity; this is consistent with higher photosynthetic efficiency. However, chl a underwent a deeper impact under green light (high intensity); total carotenoids were influenced by white light (high intensity); whilst red treatment had a higher effect upon total and individual phycobiliproteins. Enhanced PC productivities were found under modulation with red light (low intensities), and could be achieved 7 days earlier than in white LED (over 22 days); this finding is quite interesting from a sustainability and economic point of view. Light modulation accordingly appears to be a useful tool for supplementary studies pertaining to optimization of pigment production with biotechnological interest.

Karlodinium veneficum: Growth optimization, metabolite characterization and biotechnological potential survey.

AIM OF THIS STUDY: The major aim of this work was to consistently optimize the production of biomass of the dinoflagellate Karlodinium veneficum and evaluate its extracts biotechnological potential application towards food, nutraceutical or/and pharmaceutical industries. METHODS AND RESULTS: A successful approach of biomass production of K. veneficum CCMP 2936 was optimized along with the chemical characterization of its metabolite profile. Several temperatures (12, 16, 20, 25, 30°C), L1 nutrient concentrations (0.5×, 2×, 2.5×, 3×) and NaCl concentrations (20, 25, 30, 40 g L-1 ) were tested. The growth rate was maximum at 16°C, 2× nutrient concentration and 40 g L-1 of NaCl; hence, these conditions were chosen for bulk production of biomass. Methanolic extracts were prepared, and pigments, lipids and phenolic compounds were assessed; complemented by antioxidant and anti-inflammatory capacities, and cytotoxicity. Fucoxanthin and derivatives accounted for 0.06% of dry weight, and up to 60% (w/w) of all quantified metabolites were lipids. Said extracts displayed high antioxidant capacity, as towards assessed via the NO•- and ABTS•+ assays (IC50  = 109.09 ± 6.73 and 266.46 ± 2.25 µgE  ml-1 , respectively), unlike observed via the O2 •- assay (IC25 reaching 56.06 ± 5.56 µgE  ml-1 ). No signs of cytotoxicity were observed. CONCLUSIONS: Karlodinium veneficum biomass production was consistently optimized in terms of temperature, L1 nutrient concentrations and NaCl concentration. In addition, this strain appears promising for eventual biotechnological exploitation. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides fundamental insights about the growth and potential of value-added compounds of dinoflagellate K. veneficum. Dinoflagellates, as K. veneficum are poorly studied regarding its biomass production and added-value compounds for potential biotechnological exploitation. These organisms are difficult to maintain and grow in the laboratory. Thus, any fundamental contribution is relevant to share with the scientific community.

Continuous pressurized extraction versus electric fields-assisted extraction of cyanobacterial pigments.

Cyanobacteria pigments, in special carotenoids and phycobiliproteins, are usually used in industry as raw extracts, although there is still no standard methodology for their extraction. For the co-extraction of carotenoids and phycobiliproteins from the marine cyanobacterium Cyanobium sp., a continuous pressurized solvent extraction (CPSE) system and an electric fields-assisted extraction system based in ohmic heating were optimized using Central Composite Designs, with three factors each: time (t), temperature (T) and, flow (f) for CPSE; and time, temperature and frequency (F) for ohmic heating. The content of pigments and the antioxidant capacity of extracts were evaluated. All tested factors seem to influence the extraction of pigments in different ways: a high temperature (70 °C) had a positive impact on the extraction rate in both methods, while the influence of time depended on the extraction principle. Flow and frequency affected directly the extraction efficiency and these methods are indeed suitable for cyanobacterial pigments extraction, achieving good extraction results. Optimal conditions for co-extraction of carotenoids and phycobiliproteins in CPSE were T = 70 °C, t = 20 min and f = 1.5 mL min-1, and for ohmic heating they were T = 70 °C, t = 5 min and F = 20 kHz. Both, CPSE and ohmic heating systems allowed obtaining better extraction yields when compared with a previously optimized extraction method (homogenization), used here as a reference. However, ohmic heating was the best methodology for pigments co-extraction from Cyanobium sp.

White and red LEDs as two-phase batch for cyanobacterial pigments production.

Carotenoids and phycobiliproteins have a high economic value, due to their wide range of biological and industrial applications. The implementation of strategies to increase their production, such as the application of two-phase light cultivation systems, can stimulate pigments production, increasing economic turnover. In this sense, Cyanobium sp. was grown in seven different two-phase white/red cultivation arrangements, varying the time of each light from 0 to 21 days. Biomass, photosynthetic activity, pigments profile and antioxidant capacity were measured along time. Red light increased photosynthetic activity and pigments content (ca. 1.8-fold), and the use of a two-phase cultivation system generally raised bioactivity and production of phytochemicals. Among the studied, the optimal cultivation condition was found with 10 days of white followed by 4 days of red light. The optimized growth led to a productivity of 137.4 ± 0.8 mg L-1 d-1 of biomass, 17.0 ± 0.2 mg L-1 d-1 of total phycobiliproteins and 4.5 ± 0.2 mg L-1 d-1 of carotenoids.

Phycobiliproteins from cyanobacteria: Chemistry and biotechnological applications.

Phycobiliproteins are a group of water soluble proteins with an associated chromophore, responsible for the light-harvesting in cyanobacteria. They are divided in four main types: phycoerythrin, phycocyanin, phycoerythrocyanin and allophycocyanin, and they are characterized according to their structure and light quality absorption. Phycobiliproteins from cyanobacteria have been described as potential bioactive compounds, and recognized as high-valued natural products for biotechnological applications. Moreover, phycobiliproteins have been associated to antioxidant, anticancer and anti-inflammatory capacities among others. Thus, in order to produce phycobiliproteins from cyanobacteria for industrial application, it is necessary to optimize the whole bioprocess, including the processing parameters (such as light, nitrogen and carbon source, pH, temperature and salinity) that affects the growth and phycobiliprotein accumulation, as well as the optimization of phycobiliproteins extraction and purification. The aim of this review is to give an overview of phycobiliproteins not only in terms of their chemistry, but also in terms of their biotechnological applicability and the advances and challenges in the production of such compounds.

Gloeothece sp. as a Nutraceutical Source-An Improved Method of Extraction of Carotenoids and Fatty Acids.

The nutraceutical potential of microalgae boomed with the exploitation of new species and sustainable extraction systems of bioactive compounds. Thus, a laboratory-made continuous pressurized solvent extraction system (CPSE) was built to optimize the extraction of antioxidant compounds, such as carotenoids and PUFA, from a scarcely studied prokaryotic microalga, Gloeothece sp. Following "green chemical principles" and using a GRAS solvent (ethanol), biomass amount, solvent flow-rate/pressure, temperature and solvent volume-including solvent recirculation-were sequentially optimized, with the carotenoids and PUFA content and antioxidant capacity being the objective functions. Gloeothece sp. bioactive compounds were best extracted at 60 °C and 180 bar. Recirculation of solvent in several cycles (C) led to an 11-fold extraction increase of ß-carotene (3C) and 7.4-fold extraction of C18:2 n6 t (5C) when compared to operation in open systems. To fully validate results CPSE, this system was compared to a conventional extraction method, ultrasound assisted extraction (UAE). CPSE proved superior in extraction yield, increasing total carotenoids extraction up 3-fold and total PUFA extraction by ca. 1.5-fold, with particular extraction increase of 18:3 n3 by 9.6-fold. Thus, CPSE proved to be an efficient and greener extraction method to obtain bioactive extract from Gloeothece sp. for nutraceutical purposes-with low levels of resources spent, while lowering costs of production and environmental impacts.

Viability of dietary substitution of live microalgae with dry Ulva rigida in broodstock conditioning of the Pacific oyster (Crassostrea gigas).

The current study evaluated the microalgae replacement by dry macroalgae (Ulva rigida) in the reproductive success and biochemical composition of the Pacific oyster (Crassostrea gigas) during broodstock conditioning. Five nutritional regimes were tested: 100% macroalgae (diet 1), 50% macroalgae+50% microalgae (diet 2), 25% macroalgae+75% microalgae (diet 3) and 100% microalgae (diet 4). An unfed group was used as a negative control. The microalgae blend was composed of 33% Isochrysis galbana and 67% diatoms (75% Skeletonema costatum+25% Chaetoceros calcitrans). Gonadal maturation was reflected in the physiological condition of the individuals. All treatments, except diet 1, showed an increase in condition index and were fully matured at the end of the trial, with the best physiological condition observed in oysters fed diet 3 and diet 4. Protein and total lipid content increased during the conditioning period, whereas glycogen content decreased. Oysters conditioned with diet 3 had higher protein and total lipid content and lower glycogen content than the other treatments. In addition, diet 3 showed the highest percentage of viable veliger larvae. The current study demonstrated that it is possible to replace 25% of microalgae with macroalgae in the broodstock conditioning, minimizing the operative cost in bivalve hatcheries.This article has an associated First Person interview with the first author of the paper.

Biotechnological and Pharmacological Applications of Biotoxins and Other Bioactive Molecules from Dinoflagellates.

The long-lasting interest in bioactive molecules (namely toxins) produced by (microalga) dinoflagellates has risen in recent years. Exhibiting wide diversity and complexity, said compounds are well-recognized for their biological features, with great potential for use as pharmaceutical therapies and biological research probes. Unfortunately, provision of those compounds is still far from sufficient, especially in view of an increasing demand for preclinical testing. Despite the difficulties to establish dinoflagellate cultures and obtain reasonable productivities of such compounds, intensive research has permitted a number of advances in the field. This paper accordingly reviews the characteristics of some of the most important biotoxins (and other bioactive substances) produced by dinoflagellates. It also presents and discusses (to some length) the main advances pertaining to dinoflagellate production, from bench to large scale-with an emphasis on material published since the latest review available on the subject. Such advances encompass improvements in nutrient formulation and light supply as major operational conditions; they have permitted adaptation of classical designs, and aided the development of novel configurations for dinoflagellate growth-even though shearing-related issues remain a major challenge.

Effect of Solvent System on Extractability of Lipidic Components of Scenedesmus obliquus (M2-1) and Gloeothece sp. on Antioxidant Scavenging Capacity Thereof.

Microalgae are well known for their biotechnological potential, namely with regard to bioactive lipidic components-especially carotenoids and polyunsaturated fatty acids (PUFA), well-known for therapeutic applications based on their antioxidant capacity. The aim of this work was to evaluate the influence of four distinct food-grade solvents upon extractability of specific lipidic components, and on the antioxidant capacity exhibited against both synthetic (2,2-diphenyl-1-picrylhydrazyl (DPPH(•)) and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS(+•))) and biological reactive species (O2(•)⁻ and (•)NO⁻). A eukaryotic microalga (Scenedesmus obliquus (M2-1)) and a prokaryotic one (Gloeothece sp.) were used as case studies. Concerning total antioxidant capacity, the hexane:isopropanol (3:2) and acetone extracts of Sc. obliquus (M2-1) were the most effective against DPPH(•) and ABTS(+•), respectively. Gloeothece sp. ethanol extracts were the most interesting scavengers of O2(•)⁻, probably due the high content of linolenic acid. On the other hand, acetone and hexane:isopropanol (3:2) extracts were the most interesting ones in (•)NO⁻ assay. Acetone extract exhibited the best results for the ABTS assay, likely associated to its content of carotenoids, in both microalgae. Otherwise, ethanol stood out in PUFA extraction. Therefore, profiles of lipidic components extracted are critical for evaluating the antioxidant performance-which appears to hinge, in particular, on the balance between carotenoids and PUFAs.

Evaluation of the antioxidant activity of cell extracts from microalgae.

A growing market for novel antioxidants obtained from non-expensive sources justifies educated screening of microalgae for their potential antioxidant features. Characterization of the antioxidant profile of 18 species of cyanobacteria (prokaryotic microalgae) and 23 species of (eukaryotic) microalgae is accordingly reported in this paper. The total antioxidant capacity, accounted for by both water- and lipid-soluble antioxidants, was evaluated by the (radical cation) ABTS method. For complementary characterization of cell extracts, a deoxyribose assay was carried out, as well as a bacteriophage P22/Salmonella-mediated approach. The microalga Scenedesmus obliquus strain M2-1 exhibited the highest (p > 0.05) total antioxidant capacity (149 ± 47 AAU) of intracellular extracts. Its scavenger activity correlated well with its protective effects against DNA oxidative damage induced by copper(II)-ascorbic acid; and against decay in bacteriophage infection capacity induced by H2O2. Finally, performance of an Ames test revealed no mutagenic effects of the said extract.

Microalgal compounds modulate carcinogenesis in the gastrointestinal tract.

Gastrointestinal cancers rank second in overall cancer-related deaths. Carotenoids, sulfated polysaccharides, and polyunsaturated fatty acids (PUFAs) from microalgae exhibit cancer chemopreventive features at different stages of carcinogenesis. For instance, sulfated polysaccharides bear a prophylactic potential via blocking adhesion of pathogens to the gastric surface, whereas carotenoids are effective against Helicobacter pylori infection. This effect is notable because H. pylori has been targeted as the primary cause of gastric cancer. Recent results on antitumor and antibacterial compounds synthesized by microalgae are reviewed here, with an emphasis on their impact upon H. pylori infection and derived pathologies accompanying the progression of gastric carcinogenesis.

Optimization of ABTS radical cation assay specifically for determination of antioxidant capacity of intracellular extracts of microalgae and cyanobacteria.

A renewed interest in antioxidants has arisen in recent years; microalgae and cyanobacteria are potential sources thereof for use as food/feed ingredients. However, improved methods for comprehensive screening of antioxidant capacity specifically in intracellular extracts of marine microorganisms are required - encompassing lipophilic and hydrophilic compounds simultaneously. The original ABTS method was thus improved, and in particular the procedures of cell disruption and storage were optimized. The best solvent found was ethanol/water (1:1, v/v). The reaction to form ABTS(+) in said solvent was essentially complete by eight hours, and this radical cation was stable for at least 6 days; at room temperature, the ABTS(+) solution remained within an allowable analytical range for up to 13 h. Ultra Turrax was the best cell disruption method, and refrigeration was the best preservation method. This improved methodology was validated with four representative strains that respond poorly to cell disruption.

Effects of temperature and pH on growth and antioxidant content of the microalga Scenedesmus obliquus.

Reactive forms of oxygen can damage DNA (among other molecules), thus triggering, e.g., atherogenesis and carcinogenesis. However, such dietary antioxidants as lutein and ß-carotene can effectively inactivate them; these compounds were found to high levels in a novel strain (M2-1) of the microalga Scenedesmus obliquus. The independent and combined effects of pH and temperature on its rates of growth and production of antioxidants were experimentally assessed, via a full factorial experimental design; the effects of each parameter independently, and of their interactions were accordingly quantified by ANOVA. Our results indicated that temperature plays a more important role on the maximum specific growth rate than pH; in terms of antioxidant content, pH and, to a lesser extent, temperature also have relevant effects. Consequently, the highest rate of biomass specific growth (0.294 ± 0.013 day(-1)) and biomass productivity (0.837 ± 0.054 mg L(-1) day(-1)) were associated with relatively low pH (6) and relatively high temperature (30°C). Conversely, the antioxidant production rate increased with pH; hence, the highest productivity (0.638 mg L(-1) day(-1)) was attained at pH 8 and 30°C. At the best operating conditions for antioxidant content, the levels of lutein and ß-carotene were 203.57 ± 1.41 and 18.20 ± 0.33 mg mL(-1), respectively; the maximum production of either one occurred at the early exponential phase.

Microalgae as sources of high added-value compounds--a brief review of recent work.

Microalgae have found commercial applications as natural sources of valuable macromolecules, including carotenoids, long-chain polyunsaturated fatty acids, and phycocolloids. As photoautotrophs, their simple growth requirements make them attractive for bioprocesses aimed at producing high added-value compounds that are in large demand by the pharmaceutical market. A few compounds synthesized by microalgae have indeed proven to possess anti-inflammatory, antiviral, antimicrobial, and antitumoral features; astaxanthin, a known antioxidant produced by Haematococcus pluvialis, is an illustrative example with important anti-inflammatory and antitumoral roles. From a chemical standpoint, several such compounds are polysaccharides or long chain fatty acids, where the latter can be either saturated or unsaturated. Additionally, their chemical structures are often atypical, whereas their concentrations can exceed those found in many other natural sources. The productivity and biochemical composition of microalgae depend strongly on the mode of cultivation, medium composition, and nutrient profile. Consequently, numerous efforts aimed at elucidating the practical impacts of the aforementioned parameters have been developed. This review accordingly covers the knowledge produced in the last two decades on the uses of microalgae to obtain physiologically active compounds, and on the optimization of the underlying production and purification processes. It also identifies major gaps and opportunities in this field that should be addressed or exploited in the near future.

Lipid class composition of the microalga Pavlova lutheri: eicosapentaenoic and docosahexaenoic acids.

The lipid classes of Pavlova lutheri, cultivated in semicontinuous mode, were studied by thin-layer chromatography and gas chromatography in attempts to describe the distribution of fatty acid residues within its lipid pool, with special emphasis on eicosapentaenoic (C20:5n-3, EPA) and docosahexaenoic (C22:6n-3, DHA) acids. Neutral lipids and glycolipids were the major constituents and accounted for approximately 57 and 24% of the total fatty acid residues (TFA), respectively. Phospholipids accounted for approximately 10% of TFA. Two lipid classes, acylated steryl glycosides (SG) and diphosphatidylglycerols (DPG), were eventually identified in P. lutheri for the first time. The nonpolar fraction was mainly composed of triacylglycerol (TAG), whereas the polar fraction was mainly composed of monogalactosylacylglycerols (MGDG). The distribution of total EPA and DHA within the lipid pool was calculated in attempts to ascertain the quality of said microalgae as a feed source, as well as the possibility of enhancement of individual fatty acid production and extraction thereafter. EPA was especially concentrated in MGDG (approximately 45%) and TAG (approximately 33%); conversely, DHA was dispersed through various classes, especially within TAG (approximately 27%), DPG (approximately 22%), and betaine lipids (21%).

Increase of the yields of eicosapentaenoic and docosahexaenoic acids by the microalga Pavlova lutheri following random mutagenesis.

The high commercial values of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids have driven a strain-improvement program, aimed at increasing the content of those fatty acids in the microalga Pavlova lutheri (SMBA 60) as parent strain. After a round of mutation using UV-light as mutagenic agent, an isolate strain (tentatively called II#2) was obtained, the EPA and DHA contents of which (in % dry biomass) were 32.8% and 32.9% higher than those of the control, native strain. The final EPA yields, when the cultures were maintained under appropriate conditions, were 17.4 and 23.1 mg. g(-1) dry biomass, for the wild-type and the II#2 strain, respectively, whereas the final DHA yields were 8.0 and 10.6 mg. g(-1) dry biomass, respectively. These results suggest that random mutagenesis can successfully be applied to increase the yield of n-3 fatty acids by microalgae.