Effective isolation of cannabidiol and cannabidiolic acid free of psychotropic phytocannabinoids from hemp extract by fast centrifugal partition chromatography.

Cannabidiol (CBD), together with its precursor cannabidiolic acid (CBDA), is the major phytocannabinoid occurring in most hemp cultivars. To ensure the safe use of these compounds, their effective isolation from hemp extract is required, with special emphasis on the elimination of ∆9-tetrahydrocannabinol (∆9-THC) and ∆9-tetrahydrocannabinolic acid (∆9-THCA-A). In this study, we demonstrate the applicability of fast centrifugal partition chromatography (FCPC) as a challenging format of counter-current preparative chromatography for the isolation of CBD and CBDA free of psychotropic compounds that may occur in Cannabis sativa L. plant extracts. Thirty-eight solvent mixtures were tested to identify a suitable two-phase system for this purpose. Based on the measured partition coefficients (KD) and separation factors (α), the two-phase system consisting of n-heptane:ethyl acetate:ethanol:water (1.5:0.5:1.5:0.5; v:v:v:v) was selected as an optimal solvent mixture. Employing UHPLC-HRMS/MS for target analysis of collected fractions, the elution profiles of 17 most common phytocannabinoids were determined. Under experimental conditions, the purity of isolated CBD and CBDA was 98.9 and 95.1% (w/w), respectively. Neither of ∆9-THC nor of ∆9-THCA-A were present; only trace amounts of other biologically active compounds contained in hemp extract were detected by screening against in-house spectral library using UHPLC-HRMS.

Use of saline waste water from demineralization of cheese whey for cultivation of Schizochytrium limacinum PA-968 and Japonochytrium marinum AN-4.

Saline waste water from demineralization of cheese whey was used as the main component of waste saline medium (WSM) for cultivation of thraustochytrids. The suitability of WSM for cultivation of Schizochytrium limacinum PA-968 and Japonochytrium marinum AN-4 was evaluated by comparison with cultivation on nutrient medium (NM) in shake flask and fermenter cultures. Biomass productivities achieved in WSM for the thraustochytrids were comparable with those in NM for both shake flask and fermenter cultures. The maximum total lipid content (56.71% dry cell weight) and docosahexaenoic acid productivity (0.86 g/L/day) were achieved by J. marinum AN-4 grown on WSM in shake flask and fermenter cultures, respectively. A cost estimate of WSM suggests that this medium could result in lower production costs for thraustochytrid biomass and lipids and contribute to the effective reduction in saline diary process waste water.

Microalgae for bioenergy: key technology nodes.

Microalgae have increasingly gained research interest as a source of lipids for biodiesel production. The wet way processing of harvested microalgae was suggested and evaluated with respect to the possible environmental impacts and production costs. This study is focused on the three key steps of the suggested process: flocculation, water recycling, and extraction of lipids. Microalgae strains with high content of lipids were chosen for cultivation and subsequent treatment process. Ammonium hydroxide was tested as the flocculation agent and its efficiency was compared with chitosan. Determined optimal flocculation conditions for ammonium hydroxide enable the water recycling for the recurring microalgae growth, which was verified for the use of 30, 50, and 80% recycled water. For extraction of the wet microalgae hexane, hexane/ethanol and comparative chloroform/methanol systems were applied. The efficiency of hexane/ethanol extraction system was found as comparable with chloroform/methanol system and it seems to be promising owing to its low volatility and toxicity and mainly the low cost.

Marine microalgae Schizochytrium demonstrates strong production of essential fatty acids in various cultivation conditions, advancing dietary self-sufficiency.

Introduction: Polyunsaturated fatty acids (PUFAs) are essential nutrients that humans obtain from their diet, primarily through fish oil consumption. However, fish oil production is no longer sustainable. An alternative approach is to produce PUFAs through marine microalgae. Despite the potential of algae strains to accumulate high concentrations of PUFAs, including essential fatty acids (EFAs), many aspects of PUFA production by microalgae remain unexplored and their current production outputs are frequently suboptimal. Methods: In this study, we optimized biomass and selected ω-3 PUFAs production in two strains of algae, Schizochytrium marinum AN-4 and Schizochytrium limacinum CO3H. We examined a broad range of cultivation conditions, including pH, temperature, stirring intensity, nutrient concentrations, and their combinations. Results: We found that both strains grew well at low pH levels (4.5), which could reduce bacterial contamination and facilitate the use of industrial waste products as substrate supplements. Intensive stirring was necessary for rapid biomass accumulation but caused cell disruption during lipid accumulation. Docosahexaenoic acid (DHA) yield was independent of cultivation temperature within a range of 28-34°C. We also achieved high cell densities (up to 9 g/L) and stable DHA production (average around 0.1 g/L/d) under diverse conditions and nutrient concentrations, with minimal nutrients required for stable production including standard sea salt, glucose or glycerol, and yeast extract. Discussion: Our findings demonstrate the potential of Schizochytrium strains to boost industrial-scale PUFA production and make it more economically viable. Additionally, these results may pave the way for smaller-scale production of essential fatty acids in a domestic setting. The development of a new minimal culturing medium with reduced ionic strength and antibacterial pH could further enhance the feasibility of this approach.

Whole genome sequencing and characterization of Pantoea agglomerans DBM 3797, endophyte, isolated from fresh hop (Humulus lupulus L.).

Background: This paper brings new information about the genome and phenotypic characteristics of Pantoea agglomerans strain DBM 3797, isolated from fresh Czech hop (Humulus lupulus) in the Saaz hop-growing region. Although P. agglomerans strains are frequently isolated from different materials, there are not usually thoroughly characterized even if they have versatile metabolism and those isolated from plants may have a considerable potential for application in agriculture as a support culture for plant growth. Methods: P. agglomerans DBM 3797 was cultured under aerobic and anaerobic conditions, its metabolites were analyzed by HPLC and it was tested for plant growth promotion abilities, such as phosphate solubilization, siderophore and indol-3-acetic acid productions. In addition, genomic DNA was extracted, sequenced and de novo assembly was performed. Further, genome annotation, pan-genome analysis and selected genome analyses, such as CRISPR arrays detection, antibiotic resistance and secondary metabolite genes identification were carried out. Results and discussion: The typical appearance characteristics of the strain include the formation of symplasmata in submerged liquid culture and the formation of pale yellow colonies on agar. The genetic information of the strain (in total 4.8 Mb) is divided between a chromosome and two plasmids. The strain lacks any CRISPR-Cas system but is equipped with four restriction-modification systems. The phenotypic analysis focused on growth under both aerobic and anaerobic conditions, as well as traits associated with plant growth promotion. At both levels (genomic and phenotypic), the production of siderophores, indoleacetic acid-derived growth promoters, gluconic acid, and enzyme activities related to the degradation of complex organic compounds were found. Extracellular gluconic acid production under aerobic conditions (up to 8 g/l) is probably the result of glucose oxidation by the membrane-bound pyrroloquinoline quinone-dependent enzyme glucose dehydrogenase. The strain has a number of properties potentially beneficial to the hop plant and its closest relatives include the strains also isolated from the aerial parts of plants, yet its safety profile needs to be addressed in follow-up research.

Modulation of the bacterial virulence and resistance by well-known European medicinal herbs.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia officinalis L., Sambucus nigra L., Matricaria chamomilla L., Agrimonia eupatoria L., Fragaria vesca L. and Malva sylvestris L. are plants that have a long tradition in European folk medicine. To this day, they are part of medicinal teas or creams that help with the healing of skin wounds and the treatment of respiratory or intestinal infections. However, so far these plants have not been investigated more deeply than in their direct antibacterial effect. AIM OF THE STUDY: Our research is focused on adjuvants that inhibit the mechanism of antibiotic resistance or modulate bacterial virulence. Based on a preliminary screening of 52 European herbs, which commonly appear as part of tea blends or poultice. Six of them were selected for their ability to revert the resistant phenotype of nosocomial bacterial strains. METHODS: Herbs selected for this study were obtained from commercially available sources. For the extraction of active compounds ethanol was used. Modulation of virulence was observed as an ability to inhibit bacterial cell-to-cell communication using two mutant sensor strains of Vibrio campbellii. Biofilm formation, and planktonic cell adhesion was measured using a static antibiofilm test. Ethidium bromide assay was used to checked the potential of inhibition bacterial efflux pumps. The antibacterial activities of the herbs were evaluated against resistant bacterial strains using macro dilution methods. RESULTS: Alcohol extracts had antibacterial properties mainly against Gram-positive bacteria. Of all of them, the highest antimicrobial activity demonstrated Malva sylvestris, killing both antibiotic resistant bacteria; Staphylococcus aureus with MIC of 0.8 g/L and Pseudomonas aeruginosa 0.7 g/L, respectively. Fragaria vesca extract (0.08 g/L) demonstrated strong synergism with colistin (4 mg/L) in modulating the resistant phenotype to colistin of Pseudomonas aeruginosa. Similarly, the extract of S. officinalis (0.21 g/L) reverted resistance to gentamicin (1 mg/L) in S. aureus. However, Sambucus nigra and Matricaria chamomilla seem to be a very promising source of bacterial efflux pump inhibitors. CONCLUSION: The extract of F. vesca was the most active. It was able to reduce biofilm formation probably due to the ability to decrease bacterial quorum sensing. On the other hand, the activity of S. nigra or M. chamomilla in reducing bacterial virulence may be explained by the ability to inhibit bacterial efflux systems. All these plants have potential as an adjuvant for the antibiotic treatment.

Cannabidiol nanoemulsion for eye treatment - Anti-inflammatory, wound healing activity and its bioavailability using in vitro human corneal substitute.

Cannabidiol (CBD) is the non-psychoactive component of the plant Cannabis sativa (L.) that has great anti-inflammatory benefits and wound healing effects. However, its high lipophilicity, chemical instability, and extensive metabolism impair its bioavailability and clinical use. Here, we report on the preparation of a human cornea substitute in vitro and validate this substitute for the evaluation of drug penetration. CBD nanoemulsion was developed and evaluated for stability and biological activity. The physicochemical properties of CBD nanoemulsion were maintained during storage for 90 days under room conditions. In the scratch assay, nanoformulation showed significantly ameliorated wound closure rates compared to the control and pure CBD. Due to the lower cytotoxicity of nanoformulated CBD, a higher anti-inflammatory activity was demonstrated. Neither nanoemulsion nor pure CBD can penetrate the cornea after the four-hour apical treatment. For nanoemulsion, 94 % of the initial amount of CBD remained in the apical compartment while only 54 % of the original amount of pure CBD was detected in the apical medium, and 7 % in the cornea, the rest was most likely metabolized. In summary, the nanoemulsion developed in this study enhanced the stability and biological activity of CBD.

Factors Influencing the Production of Extracellular Polysaccharides by the Green Algae Dictyosphaerium chlorelloides and Their Isolation, Purification, and Composition.

The freshwater green microalgae, Dictyosphaerium chlorelloides (CCALA 330), has the ability to produce extracellular polysaccharides (EPS). Conditions for optimum growth and EPS overproduction were determined in laboratory-scale tubular photobioreactors (PBR) with a working volume of 300 mL. Multiple limitations in nutrient supply were proven to be an effective method for EPS overproduction. Salinity stress was also applied to the culture, but no significant increase in EPS production was observed. The effects of different nitrogen sources were examined and the microalgae exhibited the fastest growth and EPS production in medium containing ammonium nitrate. Under determined optimal conditions, EPS concentration reached 10 g/L (71% of the total biomass) and a total biomass of 14 g/L at the end of 17 days cultivation. Pilot-scale cultivation was also carried out in a column type airlift photobioreactor (PBR) with a working volume of 60 L. A new and efficient methodology was developed for separating cells from the EPS-containing culture broth. Due to the strong attachment between cells and EPS, high-pressure homogenization was carried out before a centrifugation process. The EPS in the supernatant was subsequently purified using ultrafiltration. The green microalgae Dictyosphaerium chlorelloides may therefore be appropriate for the commercial production of EPS.

Fractionation of the water insoluble part of the heterotrophic mutant green microalga Parachlorella kessleri HY1 (Chlorellaceae) biomass: Identification and structure of polysaccharides.

The water-insoluble part of Parachlorella kessleri HY1 biomass was subjected to the extraction of cell-wall polysaccharides using polar aprotic solvents (DMSO, LiCl/DMSO) and aqueous alkaline solutions (0.1, 1 and 4 mol·l-1 of NaOH). Proteins predominated in all the crude extracts and in the insoluble residues were partially removed by treatment with proteolytic enzymes (pepsin and pronase), and in some cases with the HCl/H2O2 reagent, yielding purified polysaccharide-enriched fractions. These treatments led to the solubilisation of some products in water. The composition and structure of isolated polysaccharides were characterised based on monosaccharide composition, glycosidic linkage and spectroscopic analyses. The DMSO extract contained mainly proteins, and polysaccharides were not detected. The water-soluble parts isolated from the LiCl/DMSO extract contained α-l-rhamnan, α-d-glucan and ß-d-glucogalactan; the water-insoluble part contained (1 â†’ 4)-ß-d-xylan, first isolated from the biomass of green microalgae. The alkali extracts contained polysaccharides of similar structure, and also water-insoluble (1 â†’ 4)-ß-d-mannan. The insoluble part after all extractions contained α-chitin as the main polysaccharide, which was confirmed by spectroscopic methods. All these polysaccharides can play a certain role in the cell wall structure of this microalga.

In-field experimental verification of cultivation of microalgae Chlorella sp. using the flue gas from a cogeneration unit as a source of carbon dioxide.

A complex treatment of agricultural waste including the following major steps: anaerobic fermentation of suitable waste, cogeneration of the obtained biogas and growth of microalgae consuming the CO(2) from biogas and flue gas was verified under field conditions in a pilot-scale photobioreactor. The growth kinetics of microalgae Chlorella sp. consuming mixture of air and carbon dioxide (2% (v/v) of CO(2)), or flue gas (8-10% (v/v) of CO(2)) was investigated. The results obtained in the pilot photobioreactor were compared with results previously measured in laboratory photobioreactors. The field tests were performed in a pilot-scale outdoor solar-bubbled photobioreactor located at a biogas station. The pilot-scale photobioreactor was in the shape of a flat and narrow vertical prism with a volume of 300 L. The microalgae growth rates were correlated with empirical formulas. Laboratory analyses of the produced microalgae confirmed that it meets the strict EU criteria for relevant contaminants level in foodstuffs. Utilization of flue gases from cogeneration therefore was not found to be detrimental to the quality of microalgal biomass, and may be used in these types of bioreactors.

Immunoactive polysaccharides produced by heterotrophic mutant of green microalga Parachlorella kessleri HY1 (Chlorellaceae).

Hot water extract from biomass of heterotrophic mutant green alga Parachlorella kessleri HY1 (Chlorellaceae) was deproteinised, and three polysaccharidic fractions were obtained by preparative chromatography. The low-molecular fraction (1.5 × 104g mol-1) was defined mainly as branched O-2-ß-xylo-(1→3)-ß-galactofuranan where xylose is partially methylated at O-4. Two high-molecular fractions (3.05 × 105 and 9.84 × 104g mol-1) were complex polysaccharides containing α-l-rhamnan and xylogalactofuranan parts in different ratios. The polysaccharides were well soluble in hot water and, upon cooling, tended to self-segregate. Immunomodulatory activities of the obtained fractions were preliminary tested using ELISA, FACS and ImmunoSpot kits. The polysaccharides increased the TNF-α production in melanoma bearing mice with much higher intensity than in healthy mice. This was in agreement with the FACS results on T and B cells indicating their possibly secondary activation by innate immunity cells.

Phytochemical Composition and In Vitro Biological Activity of Iris spp. (Iridaceae): A New Source of Bioactive Constituents for the Inhibition of Oral Bacterial Biofilms.

The inhibition and eradication of oral biofilms is increasingly focused on the use of plant extracts as mouthwashes and toothpastes adjuvants. Here, we report on the chemical composition and the antibiofilm activity of 15 methanolic extracts of Iris species against both mono-(Pseudomonas aeruginosa, Staphylococcus aureus) and multi-species oral biofilms (Streptococcus gordonii, Veillonella parvula, Fusobacterium nucleatum subsp. nucleatum, and Actinomyces naeslundii). The phytochemical profiles of Iris pallida s.l., Iris versicolor L., Iris lactea Pall., Iris carthaliniae Fomin, and Iris germanica were determined by ultra-high performance liquid chromatography-high-resolution tandem mass spectroscopy (UHPLC-HRMS/MS) analysis, and a total of 180 compounds were identified among Iris species with (iso)flavonoid dominancy. I. pallida, I. versicolor, and I. germanica inhibited both the quorum sensing and adhesion during biofilm formation in a concentration-dependent manner. However, the extracts were less active against maturated biofilms. Of the five tested species, Iris pallida s.l. was the most effective at both inhibiting biofilm formation and disrupting existing biofilms, and the leaf extract exhibited the strongest inhibitory effect compared to the root and rhizome extracts. The cytotoxicity of the extracts was excluded in human fibroblasts. The inhibition of bacterial adhesion significantly correlated with myristic acid content, and quorum sensing inhibition correlated with the 7-ß-hydroxystigmast-4-en-3-one content. These findings could be useful for establishing an effective tool for the control of oral biofilms and thus dental diseases.

Estimation of growth and exopolysaccharide production by two soil cyanobacteria, Scytonema tolypothrichoides and Tolypothrix bouteillei as determined by cultivation in irradiance and temperature crossed gradients.

Two filamentous cyanobacteria of the genera Scytonema and Tolypothrix were reported to be effective for stabilizing soil in arid areas due to the production of significant amounts of extracellular polysaccharides (EPS). These EPS may also have applications in the biotechnology industry. Therefore, two cyanobacterial species, Scytonema tolypothrichoides and Tolypothrix bouteillei were examined using crossed gradients of temperature (8-40°C) and irradiance (3-21 W m-2) to identify their temperature and irradiance optima for maximum biomass and EPS production. According to their reported temperature requirements, both strains were considered mesophilic. The optimum growth range of temperature in S. tolypothrichoides (27 to 34°C) was higher than T. bouteillei (22-32°C). The optimum irradiance range for growth of S. tolypothrichoides (9-13 W m-2) was slightly lower than T. bouteillei (7-18 W m-2). Maximum EPS production by S. tolypothrichoides occurred at similar temperatures (28-34°C) as T. bouteillei (27-34°C), both slightly higher than for maximum growth. The optimum irradiance range for EPS production was comparable to that for growth in S. tolypotrichoides (8-13 W m-2), and slightly lower in T. bouteillei (7-17 W m-2). The Redundancy Analysis confirmed that temperature was the most important controlling factor and protocols for field applications or for mass cultivation can now be developed.

The green alga Dictyosphaerium chlorelloides biomass and polysaccharides production determined using cultivation in crossed gradients of temperature and light.

The green microalga Dictyosphaerium chlorelloides was identified as promising microorganism for biotechnological production of exopolysaccharides (EPS). In stationary phase the culture suspension solidifies to thick gel, with very high viscosity and high content of EPS which may be interesting for many biotechnological applications. To develop cultivation protocol for maximum biomass/polysaccharide production, the optimum conditions for growth and polysaccharides production were determined in this study using the crossed gradient cultivation method. Temperature and irradiance requirements of Dictyosphaerium chlorelloides were evaluated by statistical analyses for growth rate/biomass, extracellular (EPS) and intracellular (IPS) polysaccharides contents in crossed gradients of temperature (4-45°C) and irradiance (2-18 W/m2, 9.1 - 82.3 µmol/(m2 s)). The maximum relative growth rate was observed at temperatures around 19.2°C and relatively low irradiances in range 2.6-11 W/m2 (11.9-50.3 µmol/(m2 s)). The maximum IPS production was observed at temperatures around 19.2°C and irradiance around 11 W/m2 (50.3 µmol/(m2 s)). The maximum production of EPS was observed at temperatures around 25.7°C and similar irradiances as IPS production. Due to temperature separation of growth and EPS production, development of cultivation protocol based controlled temperature manipulation is possible.

Characterizing Biochar as Alternative Sorbent for Oil Spill Remediation.

Biochar (BC) was characterized as a new carbonaceous material for the adsorption of toluene from water. The tested BC was produced from pine wood gasification, and its sorption ability was compared with that of more common carbonaceous materials such as activated carbon (AC). Both materials were characterized in terms of textural features and sorption abilities by kinetic and equilibrium tests. AC and BC showed high toluene removal from water. Kinetic tests demonstrated that BC is characterized by faster toluene removal than AC is. Textural features demonstrated that the porosity of AC is double that of BC. Nevertheless, equilibrium tests demonstrated that the sorption ability of BC is comparable with that of AC, so the materials' porosity is not the only parameter that drives toluene adsorption. The specific adsorption ability (mg sorbed m-2 of surface) of the BC is higher than that of AC: toluene is more highly sorbed onto the biochar surface. Biochar is furthermore obtained from biomaterial thermally treated for making energy; this also makes the use of BC economically and environmentally convenient compared with AC, which, as a manufactured material, must be obtained in selected conditions for this type of application.

Bioprospecting of microalgae: Proper extraction followed by high performance liquid chromatographic-high resolution mass spectrometric fingerprinting as key tools for successful metabolom characterization.

Currently, the interest in microalgae as a source of biologically active components exploitable as supplementary ingredients to food/feed or in cosmetics continues to increase. Existing research mainly aims to focus on revealing and recovering the rare, cost competitive components of the algae metabolom. Because these components could be of very different physicochemical character, a universal approach for their isolation and characterization should be developed. This study demonstrates the systematic development of the extraction strategy that represents one of the key challenges in effective algae bioprospecting, which predefines their further industrial application. By using of Trachydiscus minutus as a model microalgae biomass, following procedures were tested and critically evaluated in order to develop the generic procedure for microalgae bioprospecting: (i) various ways of mechanical disintegration of algae cells enabling maximum extraction efficiency, (ii) the use of a wide range of extraction solvents/solvent mixtures suitable for optimal extraction yields of polar, medium-polar, and non-polar compounds, (iii) the use of consecutive extractions as a fractionation approach. Within the study, targeted screening of selected compounds representing broad range of polarities was realized by ultra-high performance liquid chromatography coupled with high resolution tandem mass spectrometric detection (UHPLC-HRMS/MS), to assess the effectiveness of undertaken isolation steps. As a result, simple and high-throughput extraction-fractionation strategy based on consecutive extraction with water-aqueous methanol-hexane/isopropanol was developed. Moreover, to demonstrate the potential of the UHPLC-HRMS/MS for the retrospective non-target screening and compounds identification, the collected mass spectra have been evaluated to characterize the pattern of extracted metabolites. Attention was focused on medium-/non-polar extracts and characterization of lipid species present in the T. minutus algae. Such detailed information on the composition of native (non-hydrolyzed) lipids of this microalga has not been published yet.

Combined decontamination processes for wastes containing PCBs.

This project has focused on the development of a complex assembly of mutually corresponding technological units: a low temperature thermal process for the desorption of PCBs and other organics from soils and other contaminated solid wastes; the extraction of PCBs from soils by an ecological friendly aqueous solution of selected surfactants; the chemical decontamination of PCBs in oils and in-oil-in-water emulsions by metallic sodium and potassium in polyethylene glycols in the presence of aluminum powder; the modified alkaline catalyzed chemical decontamination of PCBs in oil-in-water dispersions in a solid-state reactor (in a film of reacting emulsion on solid carriers); and the breakdown of PCBs in aqueous emulsions with activated hydroxyl radicals enhanced by UV radiation. The processes operate in a closed loop configuration with effluents circulating among the process unit. These technologies have been verified at laboratory and pilot-plant scales.

Harvesting freshwater Chlorella vulgaris with flocculant derived from spent brewer's yeast.

One of the key bottlenecks of the economically viable production of low added value microalgal products (food supplements, feed, biofuels) is the harvesting of cells from diluted culture medium. The main goals of this work were to prepare a novel flocculation agent based on spent brewer's yeast, a brewery by-product, and to test its harvesting efficiency on freshwater Chlorella vulgaris in different environments. The yeast was first autolyzed/hydrolyzed and subsequently chemically modified with 2-chloro-N,N-diethylethylamine hydrochloride (DEAE). Second, optimal dosage of modified spent yeast (MSY) flocculant for harvesting C. vulgaris was determined in culture media of various compositions. It was found that the absence of phosphorus ions decreased (0.4 mg MSY/g biomass), while the presence of algogenic organic matter (AOM) increased (51 mg MSY/g biomass) the required dosage of flocculant as compared to complete mineral medium with phosphorus and without AOM (12 mg MSY/g biomass).

ZOOSPOROGENESIS, MORPHOLOGY, ULTRASTRUCTURE, PIGMENT COMPOSITION, AND PHYLOGENETIC POSITION OF TRACHYDISCUS MINUTUS (EUSTIGMATOPHYCEAE, HETEROKONTOPHYTA)(1).

The traditional order Mischococcales (Xanthophyceae) is polyphyletic with some original members now classified in a separate class, Eustigmatophyceae. However, most mischococcalean species have not yet been studied in detail, raising the possibility that many of them still remain misplaced. We established an algal culture (strain CCALA 838) determined as one such species, Trachydiscus minutus (Bourr.) H. Ettl, and studied the morphology, ultrastructure, life cycle, pigment composition, and phylogeny using the 18S rRNA gene. We discovered a zoosporic part of the life cycle of this alga. Zoospore production was induced by darkness, suppressed by light, and was temperature dependent. The zoospores possessed one flagellum covered with mastigonemes and exhibited a basal swelling, but a stigma was missing. Ultrastructural investigations of vegetative cells revealed plastids lacking both a connection to the nuclear envelope and a girdle lamella. Moreover, we described biogenesis of oil bodies on the ultrastructural level. Photosynthetic pigments of T. minutus included as the major carotenoids violaxanthin and vaucheriaxanthin (ester); we detected no chl c. An 18S rRNA gene-based phylogenetic analysis placed T. minutus in a clade with species of the genus Pseudostaurastrum and with Goniochloris sculpta Geitler, which form a sister branch to initially studied Eustigmatophyceae. In summary, our results are inconsistent with classifying T. minutus as a xanthophycean and indicate that it is a member of a novel deep lineage of the class Eustigmatophyceae.