Inhibition of growth rate and cylindrospermopsin synthesis by Raphidiopsis raciborskii upon exposure to macrophyte Lemna trisulca (L).

Impact of macrophyte Lemna trisulca on the growth rate and synthesis of cylindrospermopsin (CYN) by cyanobacterium Raphidiopsis raciborskii was determined. The presence of L. trisulca inhibited the biomass accumulation of the cyanobacterium by 25% compared to the control during co-cultivation. The simultaneous cultivation of these organisms slightly affected the inhibition of macrophyte growth rate by 5.5% compared to the control. However, no morphological changes of L. trisulca after incubation with cyanobacteria were observed. It was also shown that the long-term (35 days) co-cultivation of R. raciborskii and L. trisulca led to a decrease in CYN concentration in media and cyanobacterial cells by 32 and 38%, respectively, compared to the values obtained for independent cultivation of cyanobacterium. Excessive absorption of phosphate ions by L. trisulca from the medium compared to nitrate ions led to a significant increase in the nitrate:phosphate ratio in the media, which inhibits the development of cyanobacterium. The obtained results indicate that L. trisulca in the natural environment may affect the physiology of cyanobacteria. The presented study is the first assessment of the allelopathic interaction of macrophyte and R. raciborskii.

Analytical Technique Optimization on the Detection of ß-cyclocitral in Microcystis Species.

ß-Cyclocitral, specifically produced by Microcystis, is one of the volatile organic compounds (VOCs) derived from cyanobacteria and has a lytic activity. It is postulated that ß-cyclocitral is a key compound for regulating the occurrence of cyanobacteria and related microorganisms in an aquatic environment. ß-Cyclocitral is sensitively detected when a high density of the cells is achieved from late summer to autumn. Moreover, it is expected to be involved in changes in the species composition of cyanobacteria in a lake. Although several analysis methods for ß-cyclocitral have already been reported, ß-cyclocitral could be detected using only solid phase micro-extraction (SPME), whereas it could not be found at all using the solvent extraction method in a previous study. In this study, we investigated why ß-cyclocitral was detected using only SPME GC/MS. Particularly, three operations in SPME, i.e., extraction temperature, sample stirring rate, and the effect of salt, were examined for the production of ß-cyclocitral. Among these, heating (60 °C) was critical for the ß-cyclocitral formation. Furthermore, acidification with a 1-h storage was more effective than heating when comparing the obtained amounts. The present results indicated that ß-cyclocitral did not exist as the intact form in cells, because it was formed by heating or acidification of the resulting intermediates during the analysis by SPME. The obtained results would be helpful to understand the formation and role of ß-cyclocitral in an aquatic environment.

Mycosporine-Like Amino Acids: Potential Health and Beauty Ingredients.

Human skin is constantly exposed to damaging ultraviolet radiation (UVR), which induces a number of acute and chronic disorders. To reduce the risk of UV-induced skin injury, people apply an additional external protection in the form of cosmetic products containing sunscreens. Nowadays, because of the use of some chemical filters raises a lot of controversies, research focuses on exploring novel, fully safe and highly efficient natural UV-absorbing compounds that could be used as active ingredients in sun care products. A promising alternative is the application of multifunctional mycosporine-like amino acids (MAAs), which can effectively compete with commercially available filters. Here, we outline a complete characterization of these compounds and discuss their enormous biotechnological potential with special emphasis on their use as sunscreens, activators of cells proliferation, anti-cancer agents, anti-photoaging molecules, stimulators of skin renewal, and functional ingredients of UV-protective biomaterials.

Cyanobacterial blue color formation during lysis under natural conditions.

Cyanobacteria produce numerous volatile organic compounds (VOCs), such as ß-cyclocitral, geosmin, and 2-methylisoborneol, which show lytic activity against cyanobacteria. Among these compounds, only ß-cyclocitral causes a characteristic color change from green to blue (blue color formation) in the culture broth during the lysis process. In August 2008 and September 2010, the lysis of cyanobacteria involving blue color formation was observed at Lake Tsukui in northern Kanagawa Prefecture, Japan. We collected lake water containing the cyanobacteria and investigated the VOCs, such as ß-cyclocitral, ß-ionone, 1-propanol, 3-methyl-1-butanol, and 2-phenylethanol, as well as the number of cyanobacterial cells and their damage and pH changes. As a result, the following results were confirmed: the detection of several VOCs, including ß-cyclocitral and its oxidation product, 2,2,6-trimethylcyclohexene-1-carboxylic acid; the identification of phycocyanin based on its visible spectrum; the lower pH (6.7 and 5.4) of the lysed samples; and characteristic morphological change in the damaged cyanobacterial cells. We also encountered the same phenomenon on 6 September 2013 in Lake Sagami in northern Kanagawa Prefecture and obtained almost the same results, such as blue color formation, decreasing pH, damaged cells, and detection of VOCs, including the oxidation products of ß-cyclocitral. ß-Cyclocitral derived from Microcystis has lytic activity against Microcystis itself but has stronger inhibitory activity against other cyanobacteria and algae, suggesting that the VOCs play an important role in the ecology of aquatic environments.

Metabolism of Mycosporine-Glutamicol in the Lichen Cladonia arbuscula subsp. squarrosa under Seasonal Changes and Elevated Exposure to UV-B or PAR Irradiation.

Cladonia arbuscula in its environmental niches is regularly affected by daily and annual variations in solar radiation. Mycosporine-glutamicol, Myc-Glu(OH), which it synthesizes, may act as a significant cellular UV-protector. Therefore, we studied this compound concentration in lichen thalli concerning seasonal changes and increased exposure to UV-B and photosynthetically active radiation (PAR) with/without simultaneous CO2 deprivation. Myc-Glu(OH) occurred year-round and exhibited a strong seasonality. The most crucial role in the control of its synthesis played UV-B radiation, although its high concentration was also found after PAR irradiation at 1000 µmol m-2 s-1. As PAR intensity increased to 2000 µmol m-2 s-1, the rate of Myc-Glu(OH) synthesis slowed down. In turn, under dark/PAR irradiation with simultaneous deprivation of CO2 in the atmosphere surrounding C. arbuscula and during darkness with continuous access to atmospheric CO2, its production was insignificant. Obtained data confirmed that Myc-Glu(OH) plays an important role in protecting C. arbuscula from UV damage and favours its adaptation to environmental stress in its natural habitat. They also suggest that its synthesis is a synergism of multiple factors. Consequently, further studies should focus on their evaluation and the identification of a lichen partner actively involved in Myc-Glu(OH) biogenesis.

Differences in susceptibility of cyanobacteria species to lytic volatile organic compounds and influence on seasonal succession.

Cyanobacteria produce numerous volatile organic compounds (VOCs) that show a lytic activity against other cyanobacteria. We found the lytic phenomenon under natural conditions and during densification experiments, and also observed the species change of the cyanobacteria during the lysis processes, in which Microcystis finally became dominant. The species change of the cyanobacteria was strongly suggested to depend on the susceptibility of the cyanobacteria toward the VOCs. To verify this suggestion, the susceptibility of the species was evaluated by the minimal inhibitory concentration (MIC) using axenic cyanobacterial strains against ß-cyclocitral, its oxidation products and ß-ionone with the aid of log D. It was found that the difference depended on the susceptibility of the cyanobacteria toward the VOCs, in which ß-cyclocitral played a crucial role and Microcystis had a significantly protective ability compared to the other cyanobacteria. In addition, the species change of cyanobacteria was consistent with the cyanobacterial seasonal succession in Lakes Sagami and Tsukui, based on data that had been accumulated for 10 years. Conventionally, although this phenomenon could be explained by nutrient availability or the physical structure of the environment, the results of this study revealed that it was controlled by the VOCs, particularly ß-cyclocitral produced by the cyanobacteria.

Determination of the toxicity of the freshwater cyanobacterium Woronichinia naegeliana (Unger) Elenkin.

Cyanobacterial blooms are undesirable for ecological and health reasons. While Woronichinia naegeliana is a cyanobacterial species that appears frequently in freshwater, information about it is limited. An evaluation of its toxicity was conducted via tests based on the crustaceans Thamnocephalus platyurus and Daphnia pulex. The greatest effect of the aqueous extract obtained from W. naegeliana cells was observed for T. platyurus. The denoted semi-lethal concentration (LC50) after 24 h of exposure was 0.99 mg of dry weight (d.w.) mL-1. A lower toxicity was displayed for D. pulex, although it grew with time. Among the 18 fractions separated from cyanobacterial extract, only one containing the microginin FR3 (MG-FR3) displayed biological activeness against T. platyurus. The remaining products synthesized by W. naegeliana displayed an absence or a low level of toxicity making it impossible to determine the LC50 value. Detailed research revealed that MG-FR3 did not affect the activity of enzymes such as trypsin, chymotrypsin, elastase and thrombin, which indicates another mode of action. The results demonstrated that blooms of W. naegeliana showed toxic activity towards invertebrate zooplankton.

Characteristic oxidation behavior of ß-cyclocitral from the cyanobacterium Microcystis.

The cyanobacterium Microcystis produces volatile organic compounds such as ß-cyclocitral and 3-methyl-1-butanol. The lysis of cyanobacteria involving the blue color formation has been occasionally observed in a natural environment. In this study, we focused on the oxidation behavior of ß-cyclocitral that contributed to the blue color formation in a natural environment and compared ß-cyclocitral with a structurally related compound concerning its oxidation, acidification, and lytic behavior. The oxidation products of ß-cyclocitral were identified by the addition of ß-cyclocitral in water, in which 2,2,6-trimethylcyclohex-1-ene-1-yl formate and 2,2,6-trimethylcyclohexanone were structurally characterized. That is, ß-cyclocitral was easily oxidized to produce the corresponding carboxylic acid and the enol ester in water without an oxidizing reagent, suggesting that this oxidation proceeded according to the Baeyer-Villiger oxidation. The oxidation behavior of ß-cyclocitral in a laboratory was different from that in the natural environment, in which 2,2,6- trimethylcyclohexanone was detected at the highest amount in the natural environment, whereas the highest amount in the laboratory was ß-cyclocitric acid. A comparison of ß-cyclocitral with structurally similar aldehydes concerning the lytic behavior of a Microcystis strain and the acidification process indicated that only ß-cyclocitral was easily oxidized. Furthermore, it was found that a blue color formation occurred between pH 5.5 and 6.5, suggesting that chlorophyll a and ß-carotene are unstable and decomposed, whereas phycocyanin was stable to some extent in this range. The obtained results of the characteristic oxidation behavior of ß-cyclocitral would contribute to a better understanding of the cyanobacterial life cycle.

Secondary metabolites of the lichen Hypogymnia physodes (L.) Nyl. and their presence in spruce (Picea abies (L.) H. Karst.) bark.

Lichen species typically have a characteristic profile of secondary metabolites. Dense populations of Hypogymnia physodes growing frequently as epiphytes on tree branches have harmful effects on the host, likely due to their secondary compounds, which were undetected in tree tissues until now. The aim of the present study was to re-characterise the suite of secondary metabolites of H. physodes thalli and to estimate their translocation into spruce (Picea abies) bark. Thallus and bark extracts were compared using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The compounds were identified based on their UV, MS and MS/MS spectra as well as retention factors of their TLC analysis. In addition to the previously described secondary metabolites (protocetraric, physodalic, 3-hydroxyphysodic, physodic, and 2'-O-methylphysodic acids, atranorin and chloroatranorin) of H. physodes, further three were identified in its thalli: conphysodalic, 4-O-methylphysodic and α-alectoronic acids. Fragmentation patterns from the negative ionisation of each compound were proposed, some of which were described for the first time. Among all of the detected lichen substances, a few, e.g., physodalic, 3-hydroxyphysodic, physodic acids and atranorin, were present in the bark of spruce branches that were abundantly colonised by lichen. The newly identified compounds of H. physodes thalli may belong to its constant or accessory secondary metabolites. These compounds may be useful in the chemotaxonomic classification of this species. The presence of some lichen substances in spruce bark confirmed their ability to penetrate host tissues. These data suggest that H. physodes compounds may cause long-term effects on spruces in nature.

Sensitised decomposition of microcystin-LR using UV radiation.

UV radiation was applied to degrade cyanobacterial hepatotoxin, microcystin-LR in the presence of phycocyanin as a model natural sensitiser. The concentrations of both the toxin and the pigment used in the experiments were higher by several orders of magnitude than found in the environment. The photoreaction parameters were optimised. The process was found to be of limited use for water treatment due to its low efficacy. Additionally, pronounced UV-induced bleaching of the pigment significantly reducing the photoreaction rates of the toxin was observed for the highest UV radiation intensities applied.

Phytoremediation of anatoxin-a by aquatic macrophyte Lemna trisulca L.

The neurotoxin anatoxin-a (ANTX-a), one of the most common cyanotoxin, poses a health risk to people and can be lethal to aquatic organisms. This paper presents results on its bioremediation by the aquatic macrophyte Lemna trisulca. We show that the plant is resistant to the harmful impact of toxin and is capable of removing ANTX-a from water. Some of the ANTX-a concentrations which were used in our experiments were much higher than those found in natural conditions. The exposition of L. trisulca to 2.5 µg ANTX-a/mL did not affect its biomass accumulation within 24 d. Significant decreases in biomass content by 21% and 30% were demonstrated in samples cultivated in media containing 12.5 µg ANTX-a/mL after 18 and 24 day of experiment, respectively. One gram of fresh weight (f.w.) of L. trisulca cultured for 14 d in the media containing 50 µg ANTX-a removed 95% of the initial toxin concentration; for media with 250 µg ANTX-a, 86% was removed. In tests of ANTX-a binding stability and degradation we transferred the macrophyte to fresh media without added toxin; within 14 d the content of accumulated ANTX-a in the macrophyte decreased by 76% (from initial 19.3 µg ANTX-a/gf.w.), 71% (from 37.3 µg ANTX-a/g f.w.) and 47% (from 63.7 µg ANTX-a/g f.w.). The quantity of ANTX-a released to media was minimal: from 3.5% to 5.1% of the initial bioaccumulated value. The data show that part of the ANTX-a was degraded. Mass spectra analyses did not indicate transformation of ANTX-a to already known forms. These findings suggest that L. trisulca has much potential as a phytoremediation agent for stabilization of aquatic environments.

Degradation of microcystin-LR by ozone in the presence of Fenton reagent.

In this study the effectiveness and feasibility of the degradation of microcystin-LR (MC-LR) using a combined method ozone/Fenton reagent was investigated. The decomposition of the toxin was determined by the chromatographic technique. The effect of the Fenton reaction on the ozonation was observed at various concentrations of ozone at pH 3.0 and 6.8. A low concentration of ozone (0.01 mg/L) given simultaneously with Fenton reagent was more effective in MC-LR degradation than ozone or Fenton reagent treatment individually regardless of the pH. At higher concentrations of ozone the use together with Fenton reagent at pH 6.8 was less efficient in MC-LR degradation than treatment with ozone alone, whereas at pH 3.0 Fenton process turned out to be more effective. The mixture of ozone and hydrogen peroxide was more striking than using ozone with Fenton reagent regardless of the pH. The complete degradation of MC-LR was achieved using ozone alone at a concentration of 0.10 mg/L, whereas the same result using ozonation with Fenton reagent required a dose of ozone of 0.20 mg/L at pH 6.8. In acidic pH the total removal of MC-LR was achieved using ozone alone at a concentration of 0.20 mg/L and the same result was obtained for the combined method of ozone/Fenton reagent.

Macroporous polyacrylamide monolithic gels with immobilized metal affinity ligands: the effect of porous structure and ligand coupling chemistry on protein binding.

Macroporous polyacrylamide gels (MPAAG) with iminodiacetic acid (IDA) functionality were prepared by (i) chemical modification of polyacrylamide gel, (ii) co-polymerization of acrylamide with allyl glycidyl ether (AGE) and N,N'metylene-bis(acrylamide) (MBAAm) followed by coupling IDA ligand or (iii) by copolymerization of acrylamide and MBAAm with functional monomer carrying IDA-functionality (1-(N,N-bis(carboxymethyl)amino-3-allylglycerol). Screening for optimized conditions for the production of the MPAAG with required porous properties was performed in a 96-well chromatographic format that allowed parallel production and analysis of the MPAAG prepared from reaction mixtures with different compositions. Scanning electron microscopy of the fabricated MPAAG revealed two different types of the porous structures: monomodal macroporous structure with large interconnected pores separated by dense non-porous pore walls in case of plain gels or gels produced via copolymerization with AGE. The other type of the MPAAG (gel produced via co-polymerization with functional monomer carrying IDA-functionality) had bimodal pore structure with large interconnected pores separated by the pore walls pierced through with micropores. The effect of different modifications of MPAAG monoliths and of porous structure of the MPAAG (monomodal and bimodal porous structure) on protein binding has been evaluated.