Isolation, Characterization and Immunomodulatory Activity Evaluation of Chrysolaminarin from the Filamentous Microalga Tribonema aequale.

The aim of this study is to investigate the differences in the accumulation capacity of chrysolaminarin among six Tribonema species and to isolate this polysaccharide for immunomodulatory activity evaluation. The results showed that T. aequale was the most productive strain with the highest content and productivity of chrysolaminarin, which were 17.20% (% of dry weight) and 50.91 mg/L/d, respectively. Chrysolaminarin was then extracted and isolated from this alga, and its monosaccharide composition was mainly composed of a glucose (61.39%), linked by ß-D-(1→3) (main chain) and ß-D-(1→6) (branch chain) glycosidic bonds, with a molecular weight of less than 6 kDa. In vitro immunomodulatory assays showed that it could activate RAW264.7 cells at a certain concentration (1000 µg/mL), as evidenced by the increased phagocytic activity and upregulated mRNA expression levels of IL-1ß, IL6, TNF-α and Nos2. Moreover, Western blot revealed that this polysaccharide stimulated the phosphorylation of p-65, p-38 and JNK in NF-κB and MAPK signaling pathways. Overall, these findings provide a reference for the further development and utilization of algae-based chrysolaminarin, while also offering an in-depth understanding of the immunoregulatory mechanism.

Nutrient Deprivation Coupled with High Light Exposure for Bioactive Chrysolaminarin Production in the Marine Microalga Isochrysis zhangjiangensis.

Chrysolaminarin, a kind of water-soluble bioactive ß-glucan produced by certain microalgae, is a potential candidate for food/pharmaceutical applications. This study identified a marine microalga Isochrysis zhangjiangensis, in which chrysolaminarin production was investigated via nutrient (nitrogen, phosphorus, or sulfur) deprivations (-N, -P, or -S conditions) along with an increase in light intensity. A characterization of the antioxidant activities of the chrysolaminarin produced under each condition was also conducted. The results showed that nutrient deprivation caused a significant increase in chrysolaminarin accumulation, though this was accompanied by diminished biomass production and photosynthetic activity. -S was the best strategy to induce chrysolaminarin accumulation. An increase in light intensity from 80 (LL) to 150 (HL) µE·m-2·s-1 further enhanced chrysolaminarin production. Compared with -N, -S caused more suitable stress and reduced carbon allocation toward neutral lipid production, which enabled a higher chrysolaminarin accumulation capacity. The highest chrysolaminarin content and concentration reached 41.7% of dry weight (%DW) and 632.2 mg/L, respectively, under HL-S, with a corresponding productivity of 155.1 mg/L/day achieved, which exceeds most of the photoautotrophic microalgae previously reported. The chrysolaminarin produced under HL-N (Iz-N) had a relatively competitive hydroxyl radical scavenging activity at low concentrations, while the chrysolaminarin produced under HL-S (Iz-S) exhibited an overall better activity, comparable to the commercial yeast ß-glucan, demonstrating I. zhangjiangensis as a promising bioactive chrysolaminarin producer from CO2.

Potentially Beneficial Effects on Healthy Aging by Supplementation of the EPA-Rich Microalgae Phaeodactylum tricornutum or Its Supernatant-A Randomized Controlled Pilot Trial in Elderly Individuals.

Dietary supplements that promote healthy aging are mostly warranted in an aging society. Because of age-related risks, anti-inflammatory and anti-oxidative agents such as microalgae are potential candidates for intervention. In a randomized controlled trial, we tested Phaeodactylum tricornutum (PT), a microalgae rich in eicosapentaenoic acid (EPA), carotenoids, vitamins, and ß-glucans, cultured in bioreactors. In this pilot trial, 19 healthy elderly received supplements for two weeks based on either the whole PT (A), the ß-1,3-glucan-rich PT supernatant (SupB), the combination thereof (A+SupB), or a Comparator product (Comp). The primary outcome variable plasma interleukin-6 was reduced after treatment with A+SupB compared to the Comp group (p = 0.04). The mobility parameters 5 s sit-to-stand test (p = 0.04 in the A group) and by trend gait speed (p = 0.08 in the A+SupB diet) were improved compared to Comp. No treatment effects were observed for fatty acids, compared to Comp but omega-6 to -3 fatty acid ratio (p = 0.006) and arachidonic acid/EPA ratio (p = 0.006) were reduced within group A+SupB. Further, the SupB study product reduced faecal zonulin (p = 0.03) compared to the Comp. The data revealed an anti-inflammatory and potentially anti-oxidative effect of particular PT preparations, suggesting that they might be suitable for effects in healthy elderly.

Reduced vacuolar ß-1,3-glucan synthesis affects carbohydrate metabolism as well as plastid homeostasis and structure in Phaeodactylum tricornutum.

The ß-1,3-glucan chrysolaminarin is the main storage polysaccharide of diatoms. In contrast to plants and green algae, diatoms and most other algal groups do not accumulate storage polysaccharides in their plastids. The diatom Phaeodactylum tricornutum possesses only a single gene encoding a putative ß-1,3-glucan synthase (PtBGS). Here, we characterize this enzyme by expressing GFP fusion proteins in P. tricornutum and by creating and investigating corresponding gene silencing mutants. We demonstrate that PtBGS is a vacuolar protein located in the tonoplast. Metabolite analyses of two mutant strains with reduced amounts of PtBGS reveal a reduction in their chrysolaminarin content and an increase of soluble sugars and lipids. This indicates that carbohydrates are shunted into alternative pathways when chrysolaminarin production is impaired. The mutant strains show reduced growth and lower photosynthetic capacities, while possessing higher photoprotective abilities than WT cells. Interestingly, a strong reduction in PtBGS expression also results in aberrations of the usually very regular thylakoid membrane patterns, including increased thylakoid thickness, reduced numbers of thylakoids per plastid, and increased numbers of lamellae per thylakoid stack. Our data demonstrate the complex intertwinement of carbohydrate storage in the vacuoles with carbohydrate metabolism, photosynthetic homeostasis, and plastid morphology.

Diatoms as cell factories for high-value products: chrysolaminarin, eicosapentaenoic acid, and fucoxanthin.

Diatoms are unicellular photosynthetic microalgae existing ubiquitously in marine and freshwater environments. This review focuses on high-value compounds produced from diatoms, including chrysolaminarin (Chrl), eicosapentaenoic acid (EPA), and fucoxanthin (Fx), which can be applied in aquaculture, human health foods, pharmaceuticals, and cosmetics. In addition, this review provides an overview of their biosynthesis in diatoms and technologies for production. EPA and Fx typically accumulate synergistically in diatoms, while Chrl competes with EPA and Fx for carbon precursors. Several diatom strains have been employed that simultaneously accumulate these three compounds, but limitations and challenges still exist during commercialization. To address the bottleneck in biomass and high-value compound production, the optimization of cultivation parameters, the trophic mode, elicitor- or bacteria-assisted stimulations, and genetic modifications via mutant breeding, adaptive evolution engineering, and metabolic engineering have been developed in diatoms to establish improved technologies. Currently, large-scale cultivation of diatoms occurs mostly in open ponds and photobioreactors in autotrophic mode. Mixotrophic cultivation and coextraction approaches for multiple products represent novel strategies for economically enhancing the future production of biomass and high-value compounds on an industrial scale.

Diatom Vacuolar 1,6-ß-Transglycosylases can Functionally Complement the Respective Yeast Mutants.

Diatoms are unicellular photoautotrophic algae, which can be found in any aquatic habitat. The main storage carbohydrate of diatoms is chrysolaminarin, a nonlinear ß-glucan, consisting of a linear 1,3-ß-chain with 1,6-ß-branches, which is stored in cytoplasmic vacuoles. The metabolic pathways of chrysolaminarin synthesis in diatoms are poorly investigated, therefore we studied two potential 1,6-ß-transglycosylases (TGS) of the diatom Phaeodactylum tricornutum which are similar to yeast Kre6 proteins and which potentially are involved in the branching of 1,3-ß-glucan chains by adding d-glucose as 1,6-side chains. We genetically fused the full-length diatom TGS proteins to GFP and expressed these constructs in P. tricornutum, demonstrating that the enzymes are apparently located in the vacuoles, which indicates that branching of chrysolaminarin may occur in these organelles. Furthermore, we demonstrated the functionality of the diatom enzymes by expressing TGS1 and 2 proteins in yeast, which resulted in a partial complementation of growth deficiencies of a transglycosylase-deficient ∆kre6 yeast strain.

Resolving the dynamics of chrysolaminarin regulation in a marine diatom: A physiological and transcriptomic study.

Diatom containing different active biological macromolecules are thought to be an excellent microbial cell factory. Phaeodactylum tricornutum, a model diatom, is a superb chassis organism accumulating chrysolaminarin with important bioactivities. However, the characteristic of chrysolaminarin accumulation and molecular mechanism of the fluctuated chrysolaminarin in diatom are still unknown. In this study, physiological data and transcriptomic analysis were carried out to clarify the mechanism involved in chrysolaminarin fluctuation. The results showed that chrysolaminarin content fluctuated, from 7.41 % dry weight (DW) to 40.01 % DW during one light/dark cycle, increase by day and decrease by night. The similar fluctuated characteristic was also observed in neutral lipid content. Genes related to the biosynthesis of chrysolaminarin and neutral lipid were up-regulated at the beginning of light-phase, explaining the accumulation of these biological macromolecules. Furthermore, genes involved in carbohydrate degradation, cell cycle, DNA replication and mitochondria-localized ß-oxidation were up-regulated at the end of light phase and at the beginning of dark phase hinting an energy transition of carbohydrate to cell division during the dark period. Totally, our findings provide important information for the regulatory mechanism in the diurnal fluctuation of chrysolaminarin. It would also be of great help for the mass production of economical chrysolaminarin in marine diatom.

Influence of light conditions on the production of chrysolaminarin using Phaeodactylum tricornutum in artificially illuminated photobioreactors.

The light conditions are of utmost importance in any microalgae production process especially involving artificial illumination. This also applies to a chrysolaminarin (soluble 1,3-ß-glucan) production process using the diatom Phaeodactylum tricornutum. Here we examine the influence of the amount of light per gram biomass (specific light availability) and the influence of two different biomass densities (at the same amount of light per gram biomass) on the accumulation of the storage product chrysolaminarin during nitrogen depletion in artificially illuminated flat-panel airlift photobioreactors. Besides chrysolaminarin, other compounds (fucoxanthin, fatty acids used for energy storage [C16 fatty acids], and eicosapentaenoic acid) are regarded as well. Our results show that the time course of C-allocation between chrysolaminarin and fatty acids, serving as storage compounds, is influenced by specific light availability and cell concentration. Furthermore, our findings demonstrate that with increasing specific light availability, the maximal chrysolaminarin content increases. However, this effect is limited. Beyond a certain specific light availability (here: 5 µmolphotons gDW -1 s-1 ) the maximal chrysolaminarin content no longer increases, but the rate of increase becomes faster. Furthermore, the conversion of light to chrysolaminarin is best at the beginning of nitrogen depletion. Additionally, our results show that a high biomass concentration has a negative effect on the maximal chrysolaminarin content, most likely due to the occurring self-shading effects.

Production of lipids and proteins from marine diatoms under changing pH and silica.

Diatom algae are increasingly explored as an alternative sustainable source for functional biomolecules likes fucoxanthin, and eicosapentaenoic acid. But biomolecule quantity and quantity are influenced by growth conditions. So, effect of differential silica concentration (0-120 mg L-1) and medium pH (5.5-9.5) on growth and cellular biochemical composition of commercially important marine diatom species were studied. Growth rate of Thalassiosira sp., Skeletonema sp., and Chaetoceros sp., was higher with 30 mg L-1 Si at a pH of 7.5-8.5. Highest carbohydrate (153.71 mg g-1) and protein (17.34 mg g-1) content was found in Skeletonema sp. Silica concentration positively influenced chlorophyll and carotenoid content in a dose dependent manner. A medium pH of 8.5 and Si concentration between 60 and 120 mg L-1 was ideal for lipid production. The optimum concentration of Si and pH for maximum biomolecule production have been reported with further scope of utilizing these conditions in commercial scale systems.

Mass cultivation of marine diatoms using local salts and its impact on growth and productivity.

Diatoms are of great interest for many biotechnological applications. The present study highlights the comparative analysis for mass cultivation under the effect of seawater made from table salt (TS), rock salt (RS), and synthetic seawater in the presence of normal silica and induction coupled plasma (ICP) nanosilica (Nano Si) for inducing diatom growth. Out of all the test formulations, RS-f/2 Nano Si showed the best results with maximum cell density (3.16x107±0.04 and 3.24x107±0.05 cells mL-1), carbohydrate (403.0±3.4 and 398.0±8.1 mg g-1), and chrysolaminarin yield (66.2±5.5 and 49.3±5.1 mg g-1) in both Chaetoceros gracilis and Thalassiosira weissflogii respectively. The presence of a rich pigment profile and lipids further highlights the importance of TS and RS for cost-effective mass culturing. Results reveal that mass cultivation of marine diatoms with TS and RS in the presence of nanosilica not only reduces costs but also enhances metabolite production.

The Microalgae Phaeodactylum tricornutum Is Well Suited as a Food with Positive Effects on the Intestinal Microbiota and the Generation of SCFA: Results from a Pre-Clinical Study.

Microalgae such as Phaeodactylum tricornutum (PT) are a sustainable source of nutrients, especially eicosapentaenoic acid (EPA), fucoxanthin (Fx), and chrysolaminarin (Chrl), the concentrations of which can vary depending on the culture conditions. We generated three types of diets containing either an EPA- and Fx-rich (EPA/Fx) or Chrl-rich microalgae (with 5, 15, or 25% added to the diet) or an isocaloric control diet (CD). These diets were evaluated over 14 days in young C57BL/6J mice for safety and bioavailability, short-chain fatty acid (SCFA) production, and microbiome analysis. Both microalgae diets increased body weight gain dose-dependently compared to the CD. Microalgae-derived EPA was well absorbed, resulting in increased liver and fat tissue levels and a decrease in the n-6:n-3 ratio in liver tissue. Both microalgae diets increased the production of selected SCFA and decreased the Firmicutes/Bacteriodota ratio, whereas the Chrl-rich diet led to an increase in Akkermansia. Doses of up to 4621 mg Chrl, 920 mg EPA, and 231 mg Fx per kg body weight daily were tolerated without adverse effects. This pre-clinical study shows that PT is suitable for mouse feed, with positive effects on microbiota composition and SCFA production, suggesting beneficial effects on gut health.

Nutrient acclimation in benthic diatoms with adaptive laboratory evolution.

The growth of marine diatom algaeChaetoceros gracilisandThalassiosira weissflogiiin agricultural fertilizers and additional carbon sources were evaluated. The main objective behind the study was to increase the growth and productivity of the diatom acclimatized under adaptive laboratory culture conditions. In optimized conditions,C.gracilisshowed the highest cell density in NPK (202.5 ± 2.6 × 105 cells mL-1), maximum carbohydrate (212.8 ± 4.0 mg g-1) and protein (133.9 ± 1.5 mg g-1) in urea. In contrast,T.weissflogiishowed the highest cell density in glycerol (148.2 ± 2.5x105 cells mL-1), maximum carbohydrate in glycerol (273.7 ± 3.3 mg g-1), and protein in sucrose (126.2 ± 0.7 mg g-1). Lipid content was maximum in glycerol (73.4 ± 0.6%) and glucose (39.7 ± 0.2%) in C. gracilisand T. weissflogii respectively. Increased pigment production and chrysolaminarin concentration were obtained in both marine species. The study highlights the importance of adaptive laboratory evolution as an promising tool in enhancing productivity in diatom algae.

Storage Compound Accumulation in Diatoms as Response to Elevated CO2 Concentration.

Accumulation of reserve compounds (i.e., lipids and chrysolaminarin) in diatoms depends on the environmental conditions, and is often triggered by stress conditions, such as nutrient limitation. Manipulation of CO2 supply can also be used to improve both lipids and carbohydrates accumulation. Given the high diversity among diatoms, we studied the two marine model diatoms-Thalassiosira pseudonana and Phaeodactylum tricornutum, a freshwater diatom, Asterionella formosa, and Navicula pelliculosa-found in fresh- and sea-water environments. We measured the accumulation of reserve compounds and the activity of enzymes involved in carbon metabolism in these diatoms grown at high and atmospheric CO2. We observed that biomass and lipid accumulation in cells grown at high CO2 differ among the diatoms. Lipid accumulation increased only in P. tricornutum and N. pelliculosa grown in seawater in response to elevated CO2. Moreover, accumulation of lipids was also accompanied by an increased activity of the enzymes tested. However, lipid accumulation and enzyme activity decreased in N. pelliculosa cultured in fresh water. Chrysolaminarin accumulation was also affected by CO2 concentration; however, there was no clear relation with lipids accumulation. Our results are relevant to understand better the ecological role of the environment in the diatom adaptation to CO2 and the mechanisms underpinning the production of storage compounds considering diatom diversity.

Integrated biorefinery strategy for tofu wastewater biotransformation and biomass valorization with the filamentous microalga Tribonema minus.

This study focused on the feasibility of using different concentrations of tofu wastewater (TW) as alternative media for Tribonema minus cultures to produce valuable biorefinery feedstock. T. minus grew mixotrophically in 100% TW with larger carbohydrate (30.99% of dry weight (DW)), protein (15.56% of DW) and chrysolaminarin (6.93% of DW) accumulations than that of in mBG-11 medium. The highest biomass concentration, 7.77 g/L, was achieved in 100% TW, and nutrient removal efficiencies of T. minus at this concentration ranged from 60.49% to 93.60%. Although smaller neutral lipid and palmitoleic acid amounts were detected in 100% TW, their productivities reached 133.77 and 67.19 mg/L/d, respectively, due to the largest biomass yield contribution, which were comparable to those in mBG-11 medium. These findings demonstrated that TW is a promising alternative medium, and an integrated TW biotransformation and biomass valorization process is proposed to achieve better economic performance and environmental sustainability.

Overproduction of Bioactive Algal Chrysolaminarin by the Critical Carbon Flux Regulator Phosphoglucomutase.

Chrysolaminarin, the primary polysaccharide reservoir in some marine algae, has attracted much attention due to its broad health properties. However, its biosynthetic pathway and regulation mechanisms have rarely been reported which hinders the improvement of production efficiency. Therefore, this study aims to identify key metabolic nodes in the chrysolaminarin biosynthetic pathway. A phosphoglucomutase (PGM) in the model microalga Phaeodactylum tricornutum, revealing its critical role in chrysolaminarin biosynthesis is identified. PGM overexpression significantly elevates chrysolaminarin content by 2.54-fold and reaches 25.6% of cell dry weight; while algal growth and photosynthesis are not impaired. Besides, PGM overexpression up- and down-regulates the expression of chrysolaminarin and lipid biosynthetic genes, respectively. Microscopic analysis of aniline blue stained cells reveals that overproduced chrysolaminarin localized predominantly in vacuoles. Lipidomic analyses reveal that PGM overexpression significantly reduces the lipid content. The findings reveal the critical role of PGM in regulating the carbon flux between carbohydrate and lipid biosynthesis in microalgae, and provide a promising candidate for high efficiency production of chrysolaminarin.

Production of fucoxanthin, chrysolaminarin, and eicosapentaenoic acid by Odontella aurita under different nitrogen supply regimes.

Microalgae are recognized as promising producers of many bioactive products, but their utility is limited due to high production costs. We subjected the marine diatom Odontella aurita to three nitrogen supply regimes [initial low nitrogen (ILN), initial high nitrogen (IHN), and initial high nitrogen plus supplementary nitrogen (SN)] to investigate the accumulation of three high-value bioactive components: fucoxanthin, chrysolaminarin and eicosapentaenoic acid (EPA). We found that SN conditions maximized fucoxanthin accumulation: a maximum productivity of 6.01 mg L-1 d-1 was obtained, a 4.32-fold and 1.42-fold increase over production in the ILN and IHN groups, respectively. After nitrogen was depleted in the growth medium, chrysolaminarin became the dominant energy storage compound. Chrysolaminarin content rose to 60.33% of dry weight (DW) in the ILN group, and 46.27% of DW in the IHN group. Variations in fatty acid composition across the different nitrogen supply regimes indicated that EPA primarily accumulated in the glycolipids, especially when nitrogen supply was sufficient. The maximum productivity of chrysolaminarin (161.55 mg L-1 d-1) and EPA (9.37 mg L-1 d-1) was observed in the IHN group. However, IHN conditions did not maximize overall content of either compound. Our results demonstrated that O. aurita is potentially useful as a producer of a variety of bioactive products; the compounds produced by this species can be controlled by altering the nitrogen supply.

Laminarin Quantification in Microalgae with Enzymes from Marine Microbes.

The marine beta-glucan laminarin is an abundant storage polysaccharide in microalgae. High production rates and rapid digestion by heterotrophic bacteria turn laminarin into an ideal carbon and energy source, and it is therefore a key player in the marine carbon cycle. As a main storage glucan laminarin also plays a central role in the energy metabolism of the microalgae (Percival and Ross, 1951; Myklestad, 1974; Painter, 1983). We take advantage of enzymes that digest laminarin selectively and can thereby quantify only this polysaccharide in environmental samples. These enzymes hydrolyze laminarin into glucose and oligosaccharides, which are measured with a standard reducing sugar assay to obtain the laminarin concentration. Prior to this assay, the three enzymes need to be produced via heterologous expression and purification. The assay can be used to monitor laminarin concentrations in environmental microalgae, which were concentrated from seawater by filtering, or in samples derived from algal lab cultures.

A novel Vibrio beta-glucosidase (LamN) that hydrolyzes the algal storage polysaccharide laminarin.

The metabolic versatility, tractability and rapid growth potential of the Vibrio spp. have made them increasingly attractive systems for investigating carbon cycling in the marine environment. In this study, an in silico subtractive proteomic strategy was used to identify a novel 101 kDa GH3 family ß-glucosidase (LamN) that was found in bioluminescent Vibrio campbellii strains capable of utilizing the algal storage glucan laminarin. A heterologous overexpression system verified the sequence-predicted function of LamN as it enabled the growth of Escherichia coli on laminarin as a sole carbon source. Quantitative reverse transcription PCR analyses revealed that V. campbellii grown on laminarin demonstrated a 4- to 314-fold induction of lamN gene expression when compared to the same strains grown on glucose or glycerol. Corresponding tandem mass spectrometric analyses detected LamN protein expression only in cells grown on laminarin. Heterologous expression, purification and biochemical characterization identified LamN as a heat stable laminarinase with ß-1,3, ß-1,4 and ß-1,6 glucosidase activity. Collectively, these data identify an enzyme that may allow V. campbellii to exploit some of the most abundant polysaccharides associated with deteriorating phytoplankton blooms and provide support for the potential involvement of V. campbellii in the formation of bioluminescent milky seas.