Chemical Composition of Salvia fruticosa Mill. Essential Oil and Its Protective Effects on Both Photosynthetic Damage and Oxidative Stress in Conocephalum conicum L. Induced by Environmental Heavy Metal Concentrations.

The genus Salvia L., belonging to the Lamiaceae family, contains more than 900 species distributed in various parts of the world. It is a genus containing aromatic plants used both in the culinary field and above all in the cosmetic area to produce several perfumes. Salvia fruticosa Mill., notoriously known as Greek Salvia, is a plant used since ancient times in traditional medicine, but today cultivated and used in various parts of Europe and Africa. Polar and apolar extracts of this plant confirmed the presence of several metabolites such as abietane and labdane diterpenoids, triterpenoids, steroids, and some flavonoids, causing interesting properties such as sedative, carminative, and antiseptic, while its essential oils (EOs) are mainly characterized by compounds such as 1,8-cineole and camphor. The aim of this work concerns the chemical analysis by GC and GC-MS, and the investigation of the biological properties, of the EO of S. fruticosa plants collected in eastern Sicily. The gas-chromatographic analysis confirmed the presence of 1,8-cineole (17.38%) and camphor (12.81%), but at the same time, also moderate amounts of α-terpineol (6.74%), ß-myrcene (9.07%), camphene (8.66%), ß-pinene (6.55%), and α-pinene (6.45%). To study the protective effect of EOs from S. fruticosa (both the total mixture and the individual compounds) on possible damage induced by heavy metals, an in vitro system was used in which a model organism, the liverwort Conocephalum conicum, was subjected to the effect of a mix of heavy metals (HM) prepared using values of concentrations actually measured in one of the most polluted watercourses of the Campania region, the Regi Lagni. Finally, the antioxidant response and the photosynthetic damage were examined. The exogenous application of the EO yields a resumption of the oxidative stress induced by HM, as demonstrated by the reduction in the Reactive Oxygen Species (ROS) content and by the increased activity of antioxidant enzyme catalase (CAT) and glutathione-S-transferase (GST). Furthermore, plants treated with HMs and EO showed a higher Fv/Fm (maximal quantum efficiency of PSII in the dark) with respect to HMs-only treated ones. These results clearly indicate the protective capacity of the EO of S. fruticosa against oxidative stress, which is achieved at least in part by modulating the redox state through the antioxidant pathway and on photosynthetic damage.

Non-Protein Thiol Compounds and Antioxidant Responses Involved in Bryophyte Heavy-Metal Tolerance.

Heavy-metal pollution represents a problem which has been widely discussed in recent years. The biological effects of heavy metals have been studied in both animals and plants, ranging from oxidative stress to genotoxicity. Plants, above all metal-tolerant species, have evolved a wide spectrum of strategies to counteract exposure to toxic metal concentrations. Among these strategies, the chelation and vacuolar sequestration of heavy metals are, after cell-wall immobilization, the first line of defence that prevent heavy metals from interacting with cell components. Furthermore, bryophytes activate a series of antioxidant non-enzymatic and enzymatic responses to counteract the effects of heavy metal in the cellular compartments. In this review, the role of non-protein thiol compounds and antioxidant molecules in bryophytes will be discussed.

Biological and metabolic effects of the association between the microalga Galdieria sulphuraria and the fungus Penicillium citrinum.

Contamination of microalgae cultures can reduce their productivity and affect the quality of biomass and valuable bioproducts. In this article, after having isolated and identified for the first time the filamentous fungus Penicillium citrinum from heterotrophic cultures of the red polyextremophilic microalga Galdieria sulphuraria, we investigated the biological and metabolic significance of this alga-fungus association. In the same medium, both organisms grow better in each other's presence than separately. Both cell density and cell size of G. sulphuraria increase in co-cultures compared to pure alga cultures. In co-cultures, despite very severe growth conditions, the load of P. citrinum increases compared to pure fungus cultures. Optical microscope images have shown physical contact between cells of P. citrinum hyphae and G. sulphuraria which, however, retain their morphology and cell wall intact. GC-MS-based metabolomics analysis of metabolites excreted in the culture medium shows that pure cultures of the fungus and alga and co-cultures of alga plus fungus can be easily differentiated based on their metabolic products. Indeed, a richer assortment of extracellular metabolites (comprising both products of primary and secondary metabolism) is a distinct feature of co-cultures compared to both pure alga and pure fungus cultures.

Fungal Contamination in Microalgal Cultivation: Biological and Biotechnological Aspects of Fungi-Microalgae Interaction.

In the last few decades, the increasing interest in microalgae as sources of new biomolecules and environmental remediators stimulated scientists' investigations and industrial applications. Nowadays, microalgae are exploited in different fields such as cosmeceuticals, nutraceuticals and as human and animal food supplements. Microalgae can be grown using various cultivation systems depending on their final application. One of the main problems in microalgae cultivations is the possible presence of biological contaminants. Fungi, among the main contaminants in microalgal cultures, are able to influence the production and quality of biomass significantly. Here, we describe fungal contamination considering both shortcomings and benefits of fungi-microalgae interactions, highlighting the biological aspects of this interaction and the possible biotechnological applications.

Mycorrhized Wheat Plants and Nitrogen Assimilation in Coexistence and Antagonism with Spontaneous Colonization of Pathogenic and Saprophytic Fungi in a Soil of Low Fertility.

The aim of the work was to study the biological interference of the spontaneous colonization of pathogenic and saprophytic endophytes on the nitrogen assimilation of mycorrhized wheat plants cultivated in soils deficient in N and P. The nitrogen assimilation efficiency of mycorrhized plants was determined by measuring the activities of nitrate reductase assimilatory and glutamine synthetase enzymes and free amino acid patterns. Mycorrhizal plants at two different sites showed an assimilative activity of nitrate and ammonium approximately 30% greater than control plants. This activity was associated with significant increases in the amino acids Arg, Glu Gln and Orn in the roots where those amino acids are part of the inorganic nitrogen assimilation of mycorrhizal fungi. The nutrient supply of mycorrhizal fungi at the root guaranteed the increased growth of the plant that was about 40% greater in fresh weight and 25% greater in productive yield than the controls. To better understand the biological interaction between plant and fungus, microbiological screening was carried out to identify colonies of radicular endophytic fungi. Fourteen fungal strains belonging to nine different species were classified. Among pathogenic fungi, the genus Fusarium was present in all the examined roots with different frequencies, depending on the site and the fungal population present in the roots, providing useful clues regarding the principle of spatial conflict and fungal spread within the root system.

Antioxidant response to heavy metal pollution of Regi Lagni freshwater in Conocephalum conicum L. (Dum.).

Conocephalum conicum L. is a cosmopolitan liverwort species able to respond to local environmental pollution by changing its biological features. In the present study, we assessed the different biological responses in C. conicum to heavy metal contamination of Regi Lagni channels, a highly polluted freshwater body. As for the in field experiment, we set up moss bags containing collected samples of the local wild growing C. conicum, from the upstream site (non-polluted area), and we exposed them in the three selected sites characterized by different and extreme conditions of heavy metal pollution. In addition, to better understand the contribution of heavy metals to the alterations and response of the liverwort, we performed in vitro tests, using the same concentration of heavy metals measured in the sites at the moment of the exposition. In both experimental settings, bioaccumulation, ultrastructural damage, reactive oxygen species production and localization, antioxidant enzymes activity (superoxide dismutase, catalase and glutathione S-transferases), glutathione (reduced and oxidized) levels, localization of compounds presenting thiol groups and phenolic content were investigated. The results showed that the samples from different sites and conditions (for in vitro tests) showed significant differences. In particular, the ultrastructural alterations show a trend correlated to the different exposure situations; ROS contents, glutathione, antioxidant enzyme activities, and phenolic contents were increased showing an enhancement of the antioxidant defense both by the enzymatic way and by using the synthesis of antioxidant phenolic compounds. This study confirms the ability of C. conicum to respond to heavy metal pollution and the responses studied are, at least partially, correlated to the presence of heavy metals. All the responses considered respond consistently with the pollution trend and they can be proposed as pollution biomarkers. Therefore, we suggest the use of C. conicum to identify local hot spots of pollution in further investigation.

Sulfur Starvation in Extremophilic Microalga Galdieria sulphuraria: Can Glutathione Contribute to Stress Tolerance?

This study reports the effects of sulfur (S) deprivation in cultures of Galdieria sulphuraria (Cyanidiophyceae). Galdieria is a unicellular red alga that usually grows, forming biomats on rocks, in S-rich environments. These are volcanic areas, where S is widespread since H2S is the prevalent form of gas. The glutathione content in Galdieria sulphuraria is much higher than that found in the green algae and even under conditions of S deprivation for 7 days, it remains high. On the other hand, the S deprivation causes a decrease in the total protein content and a significant increase in soluble protein fraction. This suggests that in the conditions of S starvation, the synthesis of enzymatic proteins, that metabolically support the cell in the condition of nutritional stress, could be up regulated. Among these enzymatic proteins, those involved in cell detoxification, due to the accumulation of ROS species, have been counted.

Long-term multi-endpoint exposure of the microalga Raphidocelis subcapitata to lanthanum and cerium.

Significant release of rare earth elements (REEs) into the environment is mainly due to active or abandoned mining sites, but their presence is globally increasing due to their use in several industrial sectors. The effects on primary producers as Raphidocelis subcapitata are still limited. This research focused on La and Ce as the two most widespread REEs that can be currently found up to hundreds of µg/L in water and wastewater. Microalgae were exposed to La and Ce for 3 days (pH = 7.8) (short-term exposure) to derive the effective concentrations inhibiting the growth on 10% (EC10) of the exposed population. EC10 values (0.5 mg/L of La and 0.4 mg/L of Ce) were used for the 28 days long-term exposure (renewal test) to observe after 7, 14, 21, and 28 days on a multi-endpoint basis microalgae growth inhibition (GI), biomarkers of stress (reactive oxygen species (ROS), superoxide dismutase (SOD), and catalase (CAT)), and bioconcentration. Results evidenced that La and Ce EC10 increased GI (day 28) up to 38% and 28%, respectively. ROS, CAT, and SOD activities showed differential responses from day 7 to day 14, 21, and 28, suggesting, in most of the cases, that La and Ce effects were counteracted (i.e., being the values at day 28 not significantly different, p > 0.05, from the relative negative controls), except for La-related ROS activities. La and Ce significantly bioconcentrated in microalgae populations up to 2- and 5-fold (i.e., at day 28 compared to day 7), in that order. Bioconcentrated La and Ce were up to 3157 and 1232 µg/g dry weight (day 28), respectively. These results suggested that low La and Ce concentrations can be slightly toxic to R. subcapitata having the potential to be bioaccumulated and potentially transferred along the food web.

Simultaneous Biochemical and Physiological Responses of the Roots and Leaves of Pancratium maritimum (Amaryllidaceae) to Mild Salt Stress.

Pancratium maritimum (Amaryllidaceae) is a bulbous geophyte growing on coastal sands. In this study, we investigated changes in concentrations of metabolites in the root and leaf tissue of P. maritimum in response to mild salt stress. Changes in concentrations of osmolytes, glutathione, sodium, mineral nutrients, enzymes, and other compounds in the leaves and roots were measured at 0, 3, and 10 days during a 10-day exposure to two levels of mild salt stress, 50 mM NaCl or 100 mM NaCl in sandy soil from where the plants were collected in dunes near Cuma, Italy. Sodium accumulated in the roots, and relatively little was translocated to the leaves. At both concentrations of NaCl, higher values of the concentrations of oxidized glutathione disulfide (GSSG), compared to reduced glutathione (GSH), in roots and leaves were associated with salt tolerance. The concentration of proline increased more in the leaves than in the roots, and glycine betaine increased in both roots and leaves. Differences in the accumulation of organic osmolytes and electron donors synthesized in both leaves and roots demonstrate that osmoregulatory and electrical responses occur in these organs of P. maritimum under mild salt stress.

UV-C Irradiation as a Tool to Reduce Biofilm Growth on Pompeii Wall Paintings.

This study focuses on the experimentation of a method based on the use of UV-C irradiation to eliminate the biofilms present in a tomb located in the necropolis of Porta Nocera, in Pompeii. For this study, the autotrophic component of the biofilm was isolated in the laboratory, while, contemporarily, the characterization of the composition of the pigments of the frescoes took place on original fragments, which had already detached from the tomb and were examined in situ. These preliminary analyses were necessary for the recreation of test samples in the laboratory, which closely matched the original surfaces. Artificial biofilms were used for experimental exposure to UV-C radiation. The exposure to UV-C radiation was carried out at different distances for a fixed time interval. The effectiveness of the biocidal action was assessed by employing optical microscopy techniques, through a careful visual assessment of the area occupied by the biofilm on the different test samples, using a photographic survey, as well as by means of colorimetric measurements using spectrometric techniques. In order to obtain an additional parameter to evaluate the death rate of microorganism cultures exposed to the UV-C radiation, the concentrations of the photosynthetic pigments were also measured by spectrophotometry. Results showed that biofilms were completely eradicated by radiation, and no change in pigment color was observed.

Use of an immobilised thermostable α-CA (SspCA) for enhancing the metabolic efficiency of the freshwater green microalga Chlorella sorokiniana.

There is significant interest in increasing the microalgal efficiency for producing high-quality products that are commonly used as food additives in nutraceuticals. Some natural substances that can be extracted from algae include lipids, carbohydrates, proteins, carotenoids, long-chain polyunsaturated fatty acids, and vitamins. Generally, microalgal photoautotrophic growth can be maximised by optimising CO2 biofixation, and by adding sodium bicarbonate and specific bacteria to the microalgal culture. Recently, to enhance CO2 biofixation, a thermostable carbonic anhydrase (SspCA) encoded by the genome of the bacterium Sulfurihydrogenibium yellowstonense has been heterologously expressed and immobilised on the surfaces of bacteria. Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes, which catalyse the physiologically reversible reaction of carbon dioxide hydration to bicarbonate and protons: CO2 + H2O ⇄ HCO3- + H+. Herein, we demonstrate for the first time that the fragments of bacterial membranes containing immobilised SspCA (M-SspCA) on their surfaces can be doped into the microalgal culture of the green unicellular alga, Chlorella sorokiniana, to significantly enhance the biomass, photosynthetic activity, carotenoids production, and CA activity by this alga. These results are of biotechnological interest because C. sorokiniana is widely used in many different areas, including photosynthesis research, human pharmaceutical production, aquaculture-based food production, and wastewater treatment.

Antioxidant and anti-proliferative properties of extracts from heterotrophic cultures of Galdieria sulphuraria.

This study explores the possibility to use the extremophilic microalga Galdieria sulphuraria (strain 064) as a source of natural biomolecules with beneficial and protective effects on human health. Galdieria was cultivated in heterotrophy conditions and cells extracts for their antioxidant and anti-proliferative properties were tested. Galdieria extracts showed high antioxidant power tested through ABTS assay and revealed high glutathione and phycocyanin contents. Based on Annexin-V FITC/propidium iodide and MTT analysis, algae extracts inhibited the proliferation of human adenocarcinoma A549 cells (51.2% inhibition) through the induction of apoptosis without cell cycle arrest. Besides, cytotoxicity and cytometry assays showed a positive pro-apoptotic mechanism. On these bases, we suggest that G. sulphuraria from heterotrophic culture, for its therapeutic potential, could be considered a good candidate for further studies with the aim to isolate bioactive anti-cancer molecules.

Comparative analysis of photosynthetic and respiratory parameters in the psychrophilic unicellular green alga Koliella antarctica, cultured in indoor and outdoor photo-bioreactors.

Effect of temperatures and illumination of temperate winter on photosynthesis and respiration was studied in the psychrophilic microalgae, Koliella antarctica (Trebouxiophyceae). Outdoor and indoor algal cultures were compared. Photosynthetic as well as respiration rates increased as light and temperature increased, until 35 °C, more in outdoor than in indoor cells, in agreement with the calculated Q 10 values. K. antarctica showed important strategy mechanisms of adaption to the several temperature and light conditions. These significant photo-acclimation and thermo-acclimation abilities make it possible to cultivate Koliella for different uses, under less expensive outdoor conditions. Therefore, K. antarctica shows important strategy mechanisms of adaption to various temperature and light conditions; moreover, by varying the culture conditions, it is possible to modulate and optimize the growth and accordingly the biomass production. This is a very interesting point since it has been proved that this microalga is a promising potential source of functional ingredients, such as polyunsaturated fatty acids and carotenoids, suitable for industrial purposes.

O-acetylserine(thio)lyase (OAS-TL) molecular expression in Pancratium maritimum L. (Amaryllidaceae) under salt stress.

MAIN CONCLUSION: Different levels of salt stress affected the OAS-TL expression levels in Pancratium maritimum organs (bulb, leaf and root). A detailed method has been described for the identification of the conserved domain of the OAS-TL cDNA in sea daffodil given the scarce data available for the Amaryllidaceae family. Pancratium maritimum or sea daffodil (Amaryllidaceae) is a bulbous geophyte growing on coastal sands. In this study, we investigated the involvement of cysteine synthesis for salt tolerance through the expression of the enzyme O-acetylserine(thio)lyase (OAS-TL) during the stress response to NaCl treatments in P. maritimum. Quantitative real-time PCR was used in different organs (bulb, leaf and root).

Extraction and Activity of O-acetylserine(thiol)lyase (OASTL) from Microalga Chlorella sorokiniana.

O-acetylserine(thiol)lyase (OASTL) is an enzyme catalysing the reaction of inorganic sulphide with O-acetylserine to form the S-containing amino acid L-cysteine. Here we describe an improved protocol to evaluate the activity of this enzyme from the microalga Chlorella sorokiniana. It is a colorimetric assay based on the reaction between cysteine, the product of OASTL activity, and ninhydrin reagent, which forms a thiazolidine (Thz).

Determination of Reduced and Total Glutathione Content in Extremophilic Microalga Galdieria phlegrea.

Glutathione is an important molecule involved in the primary and secondary metabolism of all organisms. The Glutathione redox status is an indicator of the cellular redox state. Therefore, it is important to have precise methods on hand to determine the glutathione redox status in the cell. In this protocol, we describe an improved spectrophotometric method to estimate the content of reduced (GSH) and oxidized (GSSG) forms of glutathione in the extremophilic microalga Galdieria phlegrea.

Impact of Sulfur Starvation in Autotrophic and Heterotrophic Cultures of the Extremophilic Microalga Galdieria phlegrea (Cyanidiophyceae).

In plants and algae, sulfate assimilation and cysteine synthesis are regulated by sulfur (S) accessibility from the environment. This study reports the effects of S deprivation in autotrophic and heterotrophic cultures of Galdieria phlegrea (Cyanidiophyceae), a unicellular red alga isolated in the Solfatara crater located in Campi Flegrei (Naples, Italy), where H2S is the prevalent form of gaseous S in the fumarolic fluids and S is widespread in the soils near the fumaroles. This is the first report on the effects of S deprivation on a sulfurous microalga that is also able to grow heterotrophically in the dark. The removal of S from the culture medium of illuminated cells caused a decrease in the soluble protein content and a significant decrease in the intracellular levels of glutathione. Cells from heterotrophic cultures of G. phlegrea exhibited high levels of internal proteins and high glutathione content, which did not diminish during S starvation, but rather glutathione significantly increased. The activity of O-acetylserine(thiol)lyase (OASTL), the enzyme synthesizing cysteine, was enhanced under S deprivation in a time-dependent manner in autotrophic but not in heterotrophic cells. Analysis of the transcript abundance of the OASTL gene supports the OASTL activity increase in autotrophic cultures under S deprivation.

Dietary supplementation with the microalga Galdieria sulphuraria (Rhodophyta) reduces prolonged exercise-induced oxidative stress in rat tissues.

We studied the effects of ten-day 1% Galdieria sulphuraria dietary supplementation on oxidative damage and metabolic changes elicited by acute exercise (6-hour swimming) determining oxygen consumption, lipid hydroperoxides, protein bound carbonyls in rat tissue (liver, heart, and muscle) homogenates and mitochondria, tissue glutathione peroxidase and glutathione reductase activities, glutathione content, and rates of H2O2 mitochondrial release. Exercise increased oxidative damage in tissues and mitochondria and decreased tissue content of reduced glutathione. Moreover, it increased State 4 and decreased State 3 respiration in tissues and mitochondria. G. sulphuraria supplementation reduced the above exercise-induced variations. Conversely, alga supplementation was not able to modify the exercise-induced increase in mitochondrial release rate of hydrogen peroxide and in liver and heart antioxidant enzyme activities. The alga capacity to reduce lipid oxidative damage without reducing mitochondrial H2O2 release can be due to its high content of C-phycocyanin and glutathione, which are able to scavenge peroxyl radicals and contribute to phospholipid hydroperoxide metabolism, respectively. In conclusion, G. sulphuraria ability to reduce exercise-linked oxidative damage and mitochondrial dysfunction makes it potentially useful even in other conditions leading to oxidative stress, including hyperthyroidism, chronic inflammation, and ischemia/reperfusion.

Sulfur Deprivation Results in Oxidative Perturbation in Chlorella sorokiniana (211/8k).

Sulfur deficiency in plant cells has not been considered as a potential abiotic factor that can induce oxidative stress. We studied the antioxidant defense system of Chlorella sorokiniana cultured under sulfur (S) deficiency, imposed for a maximum period of 24 h, to evaluate the effect of an S shortage on oxidative stress. S deprivation induced an immediate (30 min) but transient increase in the intracellular H2O2 content, which suggests that S limitation can lead to a temporary redox disturbance. After 24 h, S deficiency in Chlorella cells decreased the glutathione content to <10% of the value measured in cells that were not subjected to S deprivation. Consequently, we assumed that the cellular antioxidative mechanisms could be altered by a decrease in the total glutathione content. The total ascorbate pool increased within 2 h after the initiation of S depletion, and remained high until 6 h; however, ascorbate regeneration was inhibited under limited S conditions, indicated by a significant decrease in the ascorbate/dehydroascorbate (AsA/DHA) ratios. Furthermore, ascorbate peroxidase (APX) and superoxide dismutase (SOD) were activated under S deficiency, but we assumed that these enzymes were involved in maintaining the cellular H2O2 balance for at least 4 h after the initiation of S starvation. We concluded that S deprivation triggers redox changes and induces antioxidant enzyme activities in Chlorella cells. The accumulation of total ascorbate, changes in the reduced glutathione/oxidized glutathione (GSH/GSSG) ratios and an increase in the activity of SOD and APX enzymes indicate that oxidative perturbation occurs during S deprivation.

Affinity Purification of O-Acetylserine(thiol)lyase from Chlorella sorokiniana by Recombinant Proteins from Arabidopsis thaliana.

In the unicellular green alga Chlorella sorokiniana (211/8 k), the protein O-acetylserine(thiol)lyase (OASTL), representing the key-enzyme in the biosynthetic cysteine pathway, was isolated and purified to apparent homogeneity. The purification was carried out in cells grown in the presence of all nutrients or in sulphate (S) deprived cells. After 24 h of S-starvation, a 17-fold increase in the specific activity of OASTL was measured. In order to enable the identification of OASTL proteins from non-model organisms such as C. sorokiniana, the recombinant his-tagged SAT5 protein from Arabidopsis thaliana was immobilized by metal chelate chromatography. OASTL proteins from C. sorokiniana were affinity purified in one step and activities were enhanced 29- and 41-fold, from S-sufficient and S-starved (24 h) cells, respectively. The successful application of SAT/OASTL interaction for purification confirms for the first time the existence of the cysteine synthase complexes in microalgae. The purified proteins have apparent molecular masses between 32-34 kDa and are thus slightly larger compared to those found in Arabidopsis thaliana and other vascular plants. The enhanced OASTL activity in S-starved cells can be attributed to increased amounts of plastidic and the emergence of cytosolic OASTL isoforms. The results provide proof-of-concept for the biochemical analysis of the cysteine synthase complex in diverse microalgal species.