Development of a high-performance thin-layer chromatography-based method for targeted glycerolipidome profiling of microalgae.

The conditionally essential very-long-chain polyunsaturated fatty acids (VLC-PUFAs), such as eicosapentaenoic acid (EPA, C20:5 n-3), play a vital role in human nutrition. Their biological activity is thereby greatly influenced by the distinct glycerolipid molecule that they are esterified to. Here, microalgae differ from the conventional source, fish oil, both in quantity and distribution of VLC-PUFAs among the glycerolipidome. Therefore, the aim of this study was to develop a fast and reliable one-dimensional high-performance thin-layer chromatography (HPTLC)-based method that allows the separation and quantification of the main microalgal glycerolipid classes (e.g., monogalactosyldiacylglycerol (MGDG), sulfoquinovosyl diacylglycerol (SQDG), phosphatidylglycerol (PG)), as well as the subsequent analysis of their respective fatty acid distribution via gas chromatography (GC) coupled to mass spectrometry (MS). Following optimization, method validation was carried out for 13 different lipid classes, based on the International Conference on Harmonization (ICH) guidelines. In HPTLC, linearity was effective between 100 and 2100 ng, with a limit of quantification between 62.99 and 90.09 ng depending on the glycerolipid class, with strong correlation coefficients (R2 > 0.995). The recovery varied between 93.17 and 108.12%, while the inter-day precision measurements showed coefficients of variation of less than 8.85%, close to the limit of detection. Applying this method to crude lipid extracts of four EPA producing microalgae of commercial interest, the content of different glycerolipid classes was assessed together with the respective FA distribution subsequent to band elution. The results showed that the described precise and accurate HPTLC method offers the possibility to be used routinely to follow variations in the glycerolipid class levels throughout strain screening, cultivation, or bioprocessing. Thus, additional quantitative analytical information on the complex lipidome of microalgae can be obtained, especially for n-3 and n-6 enriched lipid fractions.

Meilensteine in der Algenbiotechnologie.

Recent progress in algal biotechnology has identified new products based on their broad evolutionary origin. Novel metabolites were found for pharmacy, food industry, medicine e.g. tumor suppression and antibiotics. However, sustainable and economical algal production for crude oil replacement is limited by extremely low space time yields in photobioreactors. The consequences are a high energy burden for mass flow dependent processes and the need of space being in conflict with sustainable landscape management. New concepts using algae not as biomass producers but as living catalysts may open new options.

Variability in Macro- and Micronutrients of 15 Rarely Researched Microalgae.

Microalgae have enormous potential for human nutrition, yet the European Commission has authorized the consumption of only eleven species. Strains of fifteen rarely researched microalgae from two kingdoms were screened regarding their nutritional profile and value for human health in two cultivation phases. Contents of protein, fiber, lipids, fatty acids, minerals, trace elements and heavy metals were determined. In the growth phase, microalgae accumulated more arginine, histidine, ornithine, pure and crude protein, Mg, Mn, Fe and Zn and less Ni, Mo and I2 compared to the stationary phase. Higher contents of total fat, C14:0, C14:1n5, C16:1n7, C20:4n6, C20:5n3 and also As were observed in microalgae from the chromista kingdom in comparison to microalgae from the plantae kingdom (p < 0.05). Conversely, the latter had higher contents of C20:0, C20:1n9 and C18:3n3 as well as Ca and Pb (p < 0.05). More precisely, Chrysotila carterae appeared to have great potential for human nutrition because of its high nutrient contents such as fibers, carotenoids, C20:6n3, Mg, Ca, Mn, Fe, Se, Zn, Ni, Mo and I2. In summary, microalgae may contribute to a large variety of nutrients, yet the contents differ between kingdoms, cultivation phases and also species.

Impact of Regular Intake of Microalgae on Nutrient Supply and Cardiovascular Risk Factors: Results from the NovAL Intervention Study.

A 14-day randomized controlled study with a parallel design was conducted with 80 healthy participants. Intervention groups I (IG1) and II (IG2) received a defined background diet and consumed a smoothie enriched with either 15 g of Chlorella dry weight (d.w.) or 15 g of Microchloropsis d.w. daily. Control group II (CG2) received a defined background diet without the smoothie. Control group I (CG1) received neither. Blood samples and 24-h urine were collected at the beginning and the end of the study. Serum concentrations of 25-hydroxyvitamin D3, vitamin D3, selenium, iron, ferritin, transferrin saturation, total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, non-HDL cholesterol and the LDL-cholesterol/HDL cholesterol ratio decreased in IG1 (p < 0.05), while 25-hydroxyvitamin D2 increased (p < 0.05). In IG2, vitamin D3, 25-hydroxyvitamins D2 and D3 decreased (p < 0.05), while concentrations of fatty acids C20:5n3 and C22:5n3 increased. Serum and urine uric acid increased in IG1 and IG2 (p < 0.05). Microchloropsis is a valuable source of n3 fatty acids, as is Chlorella of vitamin D2. Regular consumption of Chlorella may affect the iron and selenium status negatively but may impact blood lipids positively. An elevated uric acid concentration in blood and urine following the regular consumption of microalgae poses potential risks for human health.

Testing for terrestrial and freshwater microalgae productivity under elevated CO2 conditions and nutrient limitation.

BACKGROUND: Microalgae CO2 fixation results in the production of biomass rich in high-valuable products, such as fatty acids and carotenoids. Enhanced productivity of valuable compounds can be achieved through the microalgae's ability to capture CO2 efficiently from sources of high CO2 contents, but it depends on the species. Culture collections of microalgae offer a wide variety of defined strains. However, an inadequate understanding of which groups of microalgae and from which habitats they originate offer high productivity under increased CO2 concentrations hampers exploiting microalgae as a sustainable source in the bioeconomy. RESULTS: A large variety of 81 defined algal strains, including new green algal isolates from various terrestrial environments, were studied for their growth under atmospheres with CO2 levels of 5-25% in air. They were from a pool of 200 strains that had been pre-selected for phylogenetic diversity and high productivity under ambient CO2. Green algae from terrestrial environments exhibited enhanced growth up to 25% CO2. In contrast, in unicellular red algae and stramenopile algae, which originated through the endosymbiotic uptake of a red algal cell, growth at CO2 concentrations above 5% was suppressed. While terrestrial stramenopile algae generally tolerated such CO2 concentrations, their counterparts from marine phytoplankton did not. The tests of four new strains in liquid culture revealed enhanced biomass and chlorophyll production under elevated CO2 levels. The 15% CO2 aeration increased their total carotenoid and fatty acid contents, which were further stimulated when combined with the starvation of macronutrients, i.e., less with phosphate and more with nitrogen-depleted culture media. CONCLUSION: Green algae originating from terrestrial environments, Chlorophyceae and Trebouxiophyceae, exhibit enhanced productivity of carotenoids and fatty acids under elevated CO2 concentrations. This ability supports the economic and sustainable production of valuable compounds from these microalgae using inexpensive sources of high CO2 concentrations, such as industrial exhaust fumes.

Microalgae-derived sterols do not reduce the bioavailability of oral vitamin D3 in mice.

Microalgae have drawn increasing attention as sustainable food sources, also because of their lipid-lowering phytosterols. As phytosterols are also discussed critically regarding their effect on the availability of fat-soluble vitamins, this study aimed to investigate microalgae-derived phytosterols and their effect on vitamin D status. GC-MS analysis showed large variations in the phytosterol profiles of microalgal species. The most frequent sterols were ß-sitosterol and stigmasterol. To investigate their effects on vitamin D status, 40 mice were randomized to four groups and fed a vitamin D3-adequate (25 µg/kg) Western-style diet with 0% phytosterols (control) or 1% ergosterol (a fungal sterol not typical for microalgae), ß-sitosterol or stigmasterol for four weeks. Contrary to the hypothesis that phytosterols adversely affect vitamin D uptake, mice fed ß-sitosterol had significantly higher concentrations of vitamin D3 in plasma (3.15-fold, p<0.01), liver (3.15-fold, p<0.05), and skin (4.12-fold, p<0.005) than the control group. Small increases in vitamin D3 in plasma and skin were also observed in mice fed stigmasterol. In contrast, vitamin D3 levels in the ergosterol and control groups did not differ. The increased tissue levels of vitamin D3 in mice fed ß-sitosterol and stigmasterol were not attributable to the observed reduction in liver triglycerides in these groups. The data rather suggest that changes in bile acid profiles were responsible for the beneficial effect of microalgae sterols on the bioavailability of vitamin D3. In conclusion, consumption of microalgae might not adversely affect vitamin D status.

Associated bacterial microbiome responds opportunistic once algal host Scenedesmus vacuolatus is attacked by endoparasite Amoeboaphelidium protococcarum.

The interactions of microalgae and their associated microbiomes have come to the fore of applied phycological research in recent years. However, the functional mechanisms of microalgal interactions remain largely unknown. Here, we examine functional protein patterns of the microalgae Scenedesmus vacuolatus and its associated bacterial community during algal infection by the endoparasite Amoeboaphelidium protococcarum. We performed metaproteomics analyses of non-infected (NI) and aphelid-infected (AI) S. vacuolatus cultures to investigate underlying functional and physiological changes under infectious conditions. We observed an increase in bacterial protein abundance as well as a severe shift of bacterial functional patterns throughout aphelid-infection in comparison to NI treatment. Most of the bacterial proteins (about 55%) upregulated in AI were linked to metabolism and transport of amino acids, lipids, coenzymes, nucleotides and carbohydrates and to energy production. Several proteins associated with pathogenic bacterial-plant interactions showed higher protein abundance levels in AI treatment. These functional shifts indicate that associated bacteria involved in commensalistic or mutualistic interactions in NI switch to opportunistic lifestyles and facilitate pathogenic or saprotrophic traits in AI treatment. In summary, the native bacterial microbiome adapted its metabolism to algal host die off and is able to metabolize nutrients from injured cells or decompose dead cellular material.

Natural Products from Plants and Algae for Treatment of Alzheimer's Disease: A Review.

Neurodegenerative disorders including Parkinson's disease (PD), Huntington's disease (HD) and the most frequent, Alzheimer's disease (AD), represent one of the most urgent medical needs worldwide. Despite a significantly developed understanding of disease development and pathology, treatments that stop AD progression are not yet available. The recent approval of sodium oligomannate (GV-971) for AD treatment in China emphasized the potential value of natural products for the treatment of neurodegenerative disorders. Many current clinical studies include the administration of a natural compound as a single and combination treatment. The most prominent mechanisms of action are anti-inflammatory and anti-oxidative activities, thus preserving cellular survival. Here, we review current natural products that are either approved or are in testing for a treatment of neurodegeneration in AD. In addition to the most important compounds of plant origin, we also put special emphasis on compounds from algae, given their neuroprotective activity and their underlying mechanisms of neuroprotection.

Leptolyngbya sp. NIVA-CYA 255, a Promising Candidate for Poly(3-hydroxybutyrate) Production under Mixotrophic Deficiency Conditions.

Cyanobacteria are a promising source for the sustainable production of biodegradable bioplastics such as poly(3-hydroxybutyrate) (PHB). The auto-phototrophic biomass formation is based on light and CO2, which is an advantage compared to heterotrophic PHB-producing systems. So far, only a handful of cyanobacterial species suitable for the high-yield synthesis of PHB have been reported. In the present study, the PHB formation, biomass, and elemental composition of Leptolyngbya sp. NIVA-CYA 255 were investigated. Therefore, a three-stage cultivation process was applied, consisting of a growth stage; an N-, P-, and NP-depleted phototrophic stage; and a subsequent mixotrophic deficiency stage, initiated by sodium acetate supplementation. The extracted cyanobacterial PHB was confirmed by FTIR- and GC-MS analyses. Furthermore, the fluorescent dyes LipidGreen2 and Nile red were used for fluorescence-based monitoring and the visualization of PHB. LipidGreen2 was well suited for PHB quantification, while the application of Nile red was limited by fluorescence emission crosstalk with phycocyanin. The highest PHB yields were detected in NP- (325 mg g-1) and N-deficiency (213 mg g-1). The glycogen pool was reduced in all cultures during mixotrophy, while lipid composition was not affected. The highest glycogen yield was formed under N-deficiency (217 mg g-1). Due to the high carbon storage capacity and PHB formation, Leptolyngbya sp. NIVA-CYA 255 is a promising candidate for PHB production. Further work will focus on upscaling to a technical scale and monitoring the formation by LipidGreen2-based fluorometry.

In situ quantification of poly(3-hydroxybutyrate) and biomass in Cupriavidus necator by a fluorescence spectroscopic assay.

Fluorescence spectroscopy offers a cheap, simple, and fast approach to monitor poly(3-hydroxybutyrate) (PHB) formation, a biodegradable polymer belonging to the biodegradable polyester class polyhydroxyalkanoates. In the present study, a fluorescence and side scatter-based spectroscopic setup was developed to monitor in situ biomass, and PHB formation of biotechnological applied Cupriavidus necator strain. To establish PHB quantification of C. necator, the dyes 2,2-difluoro-4,6,8,10,12-pentamethyl-3-aza-1-azonia-2-boranuidatricyclo[7.3.0.03,7]dodeca-1(12),4,6,8,10-pentaene (BODIPY493/503), ethyl 5-methoxy-1,2-bis(3-methylbut-2-enyl)-3-oxoindole-2-carboxylate (LipidGreen2), and 9-(diethylamino)benzo[a]phenoxazin-5-one (Nile red) were compared with each other. Fluorescence staining efficacy was obtained through 3D-excitation-emission matrix and design of experiments. The coefficients of determination were ≥ 0.98 for all three dyes and linear to the high-pressure liquid chromatography obtained PHB content, and the side scatter to the biomass concentration. The fluorescence correlation models were further improved by the incorporation of the biomass-related side scatter. Afterward, the resulting regression fluorescence models were successfully applied to nitrogen-deficit, phosphor-deficit, and NaCl-stressed C. necator cultures. The highest transferability of the regression models was shown by using LipidGreen2. The novel approach opens a tailor-made way for a fast and simultaneous detection of the crucial biotechnological parameters biomass and PHB content during fermentation. KEY POINTS: • Intracellular quantification of PHB and biomass using fluorescence spectroscopy. • Optimizing fluorescence staining conditions and 3D-excitation-emission matrix. • PHB was best obtained by LipidGreen2, followed by BODIPDY493/503 and Nile red.

Variability in Macro- and Micronutrients of 15 Commercially Available Microalgae Powders.

The nutrient composition of 15 commercially available microalgae powders of Arthrospira platensis, Chlorella pyrenoidosa and vulgaris, Dunaliella salina, Haematococcus pluvialis, Tetraselmis chuii, and Aphanizomenon flos-aquae was analyzed. The Dunaliella salina powders were characterized by a high content of carbohydrates, saturated fatty acids (SFAs), omega-6-polyunsaturated fatty acids (n6-PUFAs), heavy metals, and α-tocopherol, whereas the protein amounts, essential amino acids (EAAs), omega-3-PUFAs (n3-PUFAs), vitamins, and minerals were low. In the powder of Haematococcus pluvialis, ten times higher amounts of carotenoids compared to all other analyzed powders were determined, yet it was low in vitamins D and E, protein, and EAAs, and the n6/n3-PUFAs ratio was comparably high. Vitamin B12, quantified as cobalamin, was below 0.02 mg/100 g dry weight (d.w.) in all studied powders. Based on our analysis, microalgae such as Aphanizomenon and Chlorella may contribute to an adequate intake of critical nutrients such as protein with a high content of EAAs, dietary fibers, n3-PUFAs, Ca, Fe, Mg, and Zn, as well as vitamin D and E. Yet, the nutritional value of Aphanizomenon flos-aquae was slightly decreased by high contents of SFAs. The present data show that microalgae are rich in valuable nutrients, but the macro- and micronutrient profiles differ strongly between and within species.

Fast, inexpensive, and reliable HPLC method to determine monomer fractions in poly(3-hydroxybutyrate-co-3-hydroxyvalerate).

The determination of the monomer fractions in polyhydroxyalkanoates is of great importance for research on microbial-produced plastic material. The development of new process designs, the validation of mathematical models, and intelligent control strategies for production depend enormously on the correctness of the analyzed monomer fractions. Most of the available detection methods focus on the determination of the monomer fractions of the homopolymer poly(3-hydroxybutyrate). Only a few can analyze the monomer content in copolymers such as poly(3-hydroxybutyrate-co-3-hydroxyvalerate), which usually require expensive measuring devices, a high preparation time or the use of environmentally harmful halogenated solvents such as chloroform or dichloromethane. This work presents a fast, simple, and inexpensive method for the analysis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with high-performance liquid chromatography. Samples from a bioreactor experiment for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with Cupriavidus necator H16 were examined regarding their monomer content using the new method and gas chromatography analysis, one of the most frequently used methods in literature. The results from our new method were validated using gas chromatography measurements and show excellent agreement.Key points∙ The presented HPLC method is an inexpensive, fast and environmentally friendly alternative to existing methods for quantification of monomeric composition of PHBV.∙ Validation with state of the art GC measurement exhibits excellent agreement over a broad range of PHBV monomer fractions.

The use of LipidGreen2 for visualization and quantification of intracellular Poly(3-hydroxybutyrate) in Cupriavidus necator.

Numerous studies have been conducted to develop a rapid protocol for the quantification of poly(3-hydroxybutyrate) during bacterial fermentation as an alternative to time-consuming gravimetric or analytical methods. Fluorescence spectroscopy is one of the most promising approaches. In this study, it could be demonstrated that the novel fluorescent probe LipidGreen2 is able to stain selectively poly(3-hydroxybutyrate) in Cupriavidus necator. Optimal excitation and emission wavelengths were evaluated using 3D-Excitation-Emission-Matrix, displaying the best intensities between 440-460 nm and 490-520 nm for excitation and emission, respectively. The lipophilic fluorophore LipidGreen2 showed a high long-term stability even when incubated under ambient lighting. Due to a strong linear relationship between side scatter and biomass concentration, the influence of the inner filter effects could be incorporated, and adjusting the sample to a specific OD is thus superfluous. The developed method allows a very accurate quantification of poly(3-hydroxybutyrate) in just 15 min, following a comprehensible and simple protocol. It is also excellently suited for bioimaging of intracellular poly(3-hydroxybutyrate) granules.

Natural Products from Microalgae with Potential against Alzheimer's Disease: Sulfolipids Are Potent Glutaminyl Cyclase Inhibitors.

In recent years, many new enzymes, like glutaminyl cyclase (QC), could be associated with pathophysiological processes and represent targets for many diseases, so that enzyme-inhibiting properties of natural substances are becoming increasingly important. In different studies, the pathophysiology connection of QC to various diseases including Alzheimer's disease (AD) was described. Algae are known for the ability to synthesize complex and highly-diverse compounds with specific enzyme inhibition properties. Therefore, we screened different algae species for the presence of QC inhibiting metabolites using a new "Reverse Metabolomics" technique including an Activity-correlation Analysis (AcorA), which is based on the correlation of bioactivities to mass spectral data with the aid of mathematic informatics deconvolution. Thus, three QC inhibiting compounds from microalgae belonging to the family of sulfolipids were identified. The compounds showed a QC inhibition of 81% and 76% at concentrations of 0.25 mg/mL and 0.025 mg/mL, respectively. Thus, for the first time, sulfolipids are identified as QC inhibiting compounds and possess substructures with the required pharmacophore qualities. They represent a new lead structure for QC inhibitors.

Time- and media-dependent secondary carotenoid accumulation in Haematococcus pluvialis.

The green microalgae Haematococcus pluvialis synthesizes secondary carotenoids after exposure to environmental stress, a process that is used for the biotechnological production of astaxanthin (Ax). This study reports, for the first time, the medium-dependent changes in the carotenoid pattern throughout the cultivation process as well as the exact composition of carotenoids and their fatty acid mono- and diesters using LC-MS. Secondary carotenoid formation started immediately upon exposure to nutrient depletion and high light conditions. Ax and its corresponding mono- and diesters were detected simultaneously. After 15 days of cultivation, no significant changes were detected in carotenoid composition; however, the ratio between carotenoid mono- and diesters still varied. Main carotenoids were identified as Ax linolenate and Ax oleate, but also five adonirubin and one lutein monoester were detected. The influence of three different autotroph media was studied on carotenoid content, which reached a maximum 16.1 mg/g dry weight. The results indicate that media composition has an influence on the ratio of Ax mono- to diester but not on the qualitative composition of secondary carotenoids in H. pluvialis. Beside the pathway via echinenone, canthaxanthin and adonirubin the results indicate that Ax biosynthesis takes place via another route: from beta-carotene via beta-cryptoxanthin, zeaxanthin and adonixanthin.

Enantioselective separation of all-E-astaxanthin and its determination in microbial sources.

A method for the enantioselective separation of all-E-astaxanthin (3,3'-dihydroxy-beta,beta-carotene-4,4'-dione), an important colorant in the feed industry, was developed. Different chiral stationary phases (CSPs) such as Pirkle phases (R,R Ulmo and l-leucine), modified polysaccharides and a beta-cyclodextrin have been investigated on their separation performance of astaxanthin enantiomers. Direct resolution was only achieved employing the Chiralcel OD-RH (cellulose-tris-3,5-dimethylphenyl-carbamate) under reversed phase conditions. The chiral separation of the enantiomeric forms of astaxanthin produced in microalgae and yeasts was reported. The yeast Xanthophyllomyces sp. produces astaxanthin predominantly in the R,R configuration, whereas in the green microalgae Scenedesmus sp. astaxanthin is built primarily in the S,S form. The separation method for the identification of astaxanthin enantiomers is of great interest since astaxanthin is used as functional food additive in human nutrition. Moreover the method may be used as a food chain indicator in farmed salmon.