Functional Feeds to Tackle Meagre (Argyrosomus regius) Stress: Physiological Responses under Acute Stressful Handling Conditions

Marine algae are recognised sources of bioactive compounds that have attracted great interest as nutritional supplements for aquaculture fish. Intensive rearing conditions often expose fish to husbandry-related stressors, rendering fish more susceptible to disease and reducing production yields. The present work evaluated the potential of two marine algae extracts (Fucus vesiculosus and Nannochloropsis gaditana) as nutritional supplements to mitigate stress effects in meagre (Argyrosomus regius) exposed to an acute handling stress (AS). A plant-based diet was used as a control, and three other diets were prepared, which were similar to the control diet but supplemented with 1% of each algal extract or a combination of the two extracts (0.5% each). The effects of supplemented diets on stress biomarkers, antioxidant enzyme activities, and immune response were analysed in fish exposed to AS after 4 weeks of feeding. Supplemented diets did not affect growth performance but the inclusion of F. vesiculosus promoted higher feed efficiency, as compared to the control group. Dietary algal extracts supplementation reduced plasma glucose levels, increased white blood cell counts, and reduced the expression of pro-inflammatory genes when compared with the control. N. gaditana supplementation led to a reduction in hepatic antioxidant enzyme activity and glutathione levels, while F. vesiculosus supplementation increased muscle glutathione reductase activity and reduced lipid peroxidation. These findings support the potential of algal extracts as nutraceuticals in aquafeeds to enhance the ability of fish to cope with husbandry-related stressful conditions and ultimately improve fish health and welfare.     

Bioactive Compounds,Antioxidant and Binding Activities and Spear Yield of Asparagus officinalis L.

The aim of this investigation was to find a proper harvesting period and establishing fern number, which effects the spear yield, bioactive compounds and antioxidant activities of Asparagus officinalis L. Spears were harvested at 2, 4, and 6 weeks after sprouting. Control for comparison was used without harvest. Spears and total yield increased with prolonged spear harvest period. In harvest of 6 weeks long optimum spear yield was the highest and fern numbers were 5?~?8. Bioactive compounds (polyphenols, flavonoids, flavanols, tannins and ascorbic acid) and the levels of antioxidant activities by ferric-reducing/antioxidant power (FRAP) and cupric reducing antioxidant capacity (CUPRAC) assays in asparagus ethanol extracts significantly differed in the investigated samples and were the highest at 6 weeks harvest period (P?<?0.05). The first and the second segments from the tip significantly increased with the increase of catalase (CAT). It was interesting to investigate in vitro how human serum albumin (HSA) interacts with polyphenols extracted from investigated vegetables. Therefore the functional properties of asparagus were studied by the interaction of polyphenol ethanol extracts with HSA, using 3D- FL. In conclusion, antioxidant status (bioactive compounds, binding and antioxidant activities) improved with the harvesting period and the first segment from spear tip. Appropriate harvesting is effective for higher asparagus yield and its bioactivity.     

Chemical Characterization and Release of Polyphenols from Pecan Nut Shell [<Emphasis Type="Italic">Carya illinoinensis</Emphasis> (Wangenh) C. Koch] in Zein Microparticles for Bioactive Applications

The pecan nut [Carya illinoinensis (Wangenh) C. Koch] is a natural source of polyphenols with antioxidant properties. In this study, the encapsulation of aqueous and hydroalcoholic extracts of pecan nut shell were evaluated for the release of bioactive compounds and antioxidant potential in order to explore food applications using zein as encapsulating agent. The extracts showed high contents of total phenolics, condensed tannins and high antioxidant activity. Concentrations of proanthocyanidins were 9-fold higher in hydroalcoholic extracts. The LC-DAD analysis showed that catechins were the major phenolic compounds in samples, with epigallocatechin levels up to 138.62 mg mL^?1. Zein microparticles loaded with aqueous extract released 2.3 times more phenolic compounds than the hydroalcoholic extracts and the DSC thermograms showed that extracts of pecan nut shell remained thermally stable up to 240 °C. The zein microcapsules obtained in this study were efficiently encapsulated and represent an interesting additive due its high antioxidant capacity, physicochemical characteristics and morphology. The use of zein microparticles combined with natural extracts constitute a step forward in the improvement of current technology for delivering phenolic compounds with applications in functional foods and nutraceuticals.     

Bacterial Bioeffectors Modify Bioactive Profile and Increase Isoflavone Content in Soybean Sprouts (Glycine max var Osumi)

The effect of two bacterial strains to enhance bioactive contents (total phenolic compounds, total flavonoid compounds and isoflavones) and antioxidant activity on 3-day-old soybean sprouts were investigated. To identify bacterial determinants responsible for these effects, viable and UV-treated strains were delivered to wounded seeds at different concentration. Multivariate analysis performed with all the evaluated parameters indicated the different effectiveness of Stenotrophomonas maltophilia N5.18 and Pseudomonas fluorescens N21.4 based on different structural and metabolic determinants for each. N21.4 increased total phenolics and isoflavones from the genistein family, while N5.18 triggered biosynthesis of daidzein and genistein families coupled to a decrease in total phenolics, suggesting different molecular targets in the phenilpropanoid pathway. Only extracts from N5.18 treated seeds showed an improved antioxidant activity according to the β-carotene bleaching prevention method. In summary, bioeffectors from both bacterial strains are effective tools to improve soybean sprouts quality; structural elicitors from N5.18 also enhanced antioxidant activity, being the best alternative for further development of a biotechnological procedure.     

Effect of Solvent System on Extractability of Lipidic Components of Scenedesmus obliquus (M2-1) and Gloeothece sp. on Antioxidant Scavenging Capacity Thereof

Microalgae are well known for their biotechnological potential, namely with regard to bioactive lipidic components—especially carotenoids and polyunsaturated fatty acids (PUFA), well-known for therapeutic applications based on their antioxidant capacity. The aim of this work was to evaluate the influence of four distinct food-grade solvents upon extractability of specific lipidic components, and on the antioxidant capacity exhibited against both synthetic (2,2-diphenyl-1-picrylhydrazyl (DPPH^•) and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS^+•)) and biological reactive species (O₂^•− and ^•NO⁻). A eukaryotic microalga (Scenedesmus obliquus (M2-1)) and a prokaryotic one (Gloeothece sp.) were used as case studies. Concerning total antioxidant capacity, the hexane:isopropanol (3:2) and acetone extracts of Sc. obliquus (M2-1) were the most effective against DPPH^• and ABTS^+•, respectively. Gloeothece sp. ethanol extracts were the most interesting scavengers of O₂^•−, probably due the high content of linolenic acid. On the other hand, acetone and hexane:isopropanol (3:2) extracts were the most interesting ones in ^•NO⁻ assay. Acetone extract exhibited the best results for the ABTS assay, likely associated to its content of carotenoids, in both microalgae. Otherwise, ethanol stood out in PUFA extraction. Therefore, profiles of lipidic components extracted are critical for evaluating the antioxidant performance—which appears to hinge, in particular, on the balance between carotenoids and PUFAs.     

Physicochemical assessment and bioactive properties of condensed distillers solubles,a by-product from the sorghum bio-fuel industry

Large volumes of condensed distillers solubles (CDS) are generated as by-products, from the sorghum bioethanol industry. The objective was to assess the physico-chemical and bioactive properties of CDS. The unfractionated CDS showed the highest content of phenolic compounds (16 mg GAE/g), antioxidant (522 μM Trolox/g) and antimicrobial activity (MIC 1%(w/v) against Campylobacter spp.) compared to its extracts. The water and methanol extracts also showed high levels of phenolic compounds and antioxidant activity (11.6 and 9.2 mg GAE/g and 349 and 409 μM Trolox/g respectively), followed by ethanol and acetone extractions (7.5 and 6.6 mg GAE/g; 337 and 346 μM Trolox/g respectively). A positive correlation was revealed between total phenol and antioxidant activity. The main phenolic compounds found in the extracts were protocatechuic acid, 4-hydroxybenzoic acid, taxifolin, ferulic acid, cinnamic acid and p-coumaric acid. This study indicates the potential of using CDS as a functional ingredient for other food and feed applications.     

Topical anti-inflammatory plant species: Bioactivity of Bryonia dioica, Tamus communis and Lonicera periclymenum fruits

The practice of rubbing different plant material juices or extracts into the skin to relieve pain and rheumatic symptoms is deeply rooted in folk medicine and has been used for a long time. Several common species, usually available in agroecosystems of the Iberian Peninsula, were/are used for topical medicinal preparations as reported in recent ethnobotanical surveys. Based on these studies, the fruits of three relevant species (Bryonia dioica or white-bryony, Lonicera periclymenum or common honeysuckle and Tamus communis or black-bryony) were gathered and different analyses and assays were performed in order to characterize their phytochemical composition and to find biologically active compounds for pharmaceutical application. Black-bryony ripened fruits revealed the highest antioxidant properties which are in agreement to its highest concentration in phenolics, flavonoids, ascorbic acid, tocopherols and lycopene. The studied fruits revealed interesting antioxidant properties and bioactive phytochemicals that could provide scientific evidence for their folk uses as anti-inflammatory species.     

Evaluation of the Antioxidant Activity of Cell Extracts from Microalgae

A growing market for novel antioxidants obtained from non-expensive sources justifies educated screening of microalgae for their potential antioxidant features. Characterization of the antioxidant profile of 18 species of cyanobacteria (prokaryotic microalgae) and 23 species of (eukaryotic) microalgae is accordingly reported in this paper. The total antioxidant capacity, accounted for by both water- and lipid-soluble antioxidants, was evaluated by the (radical cation) ABTS method. For complementary characterization of cell extracts, a deoxyribose assay was carried out, as well as a bacteriophage P22/Salmonella-mediated approach. The microalga Scenedesmus obliquus strain M2-1 exhibited the highest (p > 0.05) total antioxidant capacity (149 ± 47 AAU) of intracellular extracts. Its scavenger activity correlated well with its protective effects against DNA oxidative damage induced by copper(II)-ascorbic acid; and against decay in bacteriophage infection capacity induced by H₂O₂. Finally, performance of an Ames test revealed no mutagenic effects of the said extract.     

Marine Benthic Diatoms Contain Compounds Able to Induce Leukemia Cell Death and Modulate Blood Platelet Activity

In spite of the high abundance and species diversity of diatoms, only a few bioactive compounds from them have been described. The present study reveals a high number of mammalian cell death inducing substances in biofilm-associated diatoms sampled from the intertidal zone. Extracts from the genera Melosira, Amphora, Phaeodactylum and Nitzschia were all found to induce leukemia cell death, with either classical apoptotic or autophagic features. Several extracts also contained inhibitors of thrombin-induced blood platelet activation. Some of this activity was caused by a high content of adenosine in the diatoms, ranging from 0.07 to 0.31 μg/mg dry weight. However, most of the bioactivity was adenosine deaminase-resistant. An adenosine deaminase-resistant active fraction from one of the extracts was partially purified and shown to induce apoptosis with a distinct phenotype. The results show that benthic diatoms typically found in the intertidal zone may represent a richer source of interesting bioactive compounds than hitherto recognized.     

Bioactivity of Different Enriched Phenolic Extracts of Wild Fruits from Northeastern Portugal: A Comparative Study

Arbutus unedo, Prunus spinosa, Rosa micrantha and Rosa canina are good sources of phenolic compounds, including anthocyanins. These compounds have potent antioxidant properties, which have been related to anticancer activity. Herein, the in vitro antioxidant and antitumor properties of enriched phenolic extracts (non-anthocyanin phenolic compounds enriched extract- PE and anthocyanins enriched extract- AE) of the mentioned wild fruits were evaluated and compared. PE gave higher bioactive properties than the corresponding AE. It was observed a high capacity of A. unedo phenolic extract to inhibit lipid peroxidation in animal brain homogenates (EC₅₀?=?7.21 μg/mL), as also a high antitumor potential against NCI-H460 human cell line (non-small lung cancer; GI₅₀?=?37.68 μg/mL), which could be related to the presence of galloyl derivatives (exclusively found in this species). The bioactivity of the studied wild fruits proved to be more related to the phenolic compounds profile than to the amounts present in each extract, and could be considered in the design of new formulations of dietary supplements or functional foods.     

Nutritional and Pharmaceutical Properties of Bioactive Compounds in Organic and Conventional Growing Kiwifruit

The bioactivity of two kiwifruit’s cultivars growing under organic and conventional conditions were studied and compared. The bioactive compounds were extracted with water and ethanol using similar conditions which are applied in pharmaceutical applications and for daily fruit consumption such as tea drink. Antioxidant radical scavenging assays [ferric-reducing/antioxidant power (FRAP); cupric reducing antioxidant capacity (CUPRAC); 2, 2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS)], fourier transform infrared (FT-IR) and ultraviolet spectroscopy, two (2D-FL) and three-dimensional (3D-FL) fluorometry were used for the detection of biologically active metabolites derived from kiwifruits (total phenols, flavonoids, chlorophylls, carotenoids and ascorbic acid). The correlation between the total phenol content (TPC) and other bioactive compounds, and their total antioxidant capacities (TAC) was calculated for studied kiwifruit’s extracts. The interaction between drugs and human serum albumin (HSA) plays an important role in the distribution and metabolism of drugs. The properties of kiwifruit’s phenol extracts showed their ability to quench HSA, forming the complexes similar to the ones between the proteins and pure flavonoids such as quercetin. The cultivar ‘Bidan’ exhibited significantly higher TAC than the classic ‘Hayward’. In conclusion, for the first time ‘Bidan’ organic kiwifruit was analyzed and compared with widely consumed ‘Hayward’, using its bioactive and fluorescence properties. The influence of physiologically active kiwifruit’s compounds on human health, through our investigations in vitro and scientifically proven information, was explained. Relatively high content of bioactive compounds, high antioxidant and fluorescence properties of kiwifruit justify its use as a source of valuable antioxidants.     

Biobased Solvents for Pressurized Liquid Extraction of Nannochloropsis gaditana Omega-3 Lipids

To develop greener extraction alternatives for microalgae biomass, ultrasound assisted extraction (UAE) and pressurized liquid extraction (PLE) with different biobased solvents were investigated, demonstrating that both techniques are useful alternatives for algal lipid extraction. Specifically, Nannochloropsis gaditana lipids were extracted by UAE and PLE at different temperatures and extraction times with sustainable solvents like 2-Methyltetrahydrofuran (2-MeTHF) and its mixtures with ethanol and other alcohols. The best oil yields for both PLE and UAE of N. gaditana were achieved with the mixture of 2-MeTHF:ethanol (1:3), reaching yields of up to 16.3%, for UAE at 50 °C and up to 46.1% for PLE at 120 °C. Lipid composition of the extracts was analyzed by HPLC-ELSD and by GC-MS to determine lipid species and fatty acid profile, respectively. Different fractionation of lipid species was achieved with PLE and solvent mixtures of different polarity. Thus, for the extraction of glycolipids, ethanolic extracts contained higher amounts of glycolipids and EPA, probably due to the higher polarity of the solvent. The optimized method was applied to microalgae Isochrysis galbana and Tetraselmis chuii showing the potential of mixtures of biobased solvents like 2-methyl-THF and ethanol in different proportions to efficiently extract and fractionate lipids from microalgal biomass.     

Marine Demospongiae: A Challenging Treasure of Bioactive Compounds

In the last decades, it has been demonstrated that marine organisms are a substantial source of bioactive compounds with possible biotechnological applications. Marine sponges, in particular those belonging to the class of Demospongiae, have been considered among the most interesting invertebrates for their biotechnological potential. In this review, particular attention is devoted to natural compounds/extracts isolated from Demospongiae and their associated microorganisms with important biological activities for pharmacological applications such as antiviral, anticancer, antifouling, antimicrobial, antiplasmodial, antifungal and antioxidant. The data here presented show that this class of sponges is an exciting source of compounds, which are worth developing into new drugs, such as avarol, a hydroquinone isolated from the marine sponge Disidea avara, which is used as an antitumor, antimicrobial and antiviral drug.     

Development of the “Applied Proteomics” Concept for Biotechnology Applications in Microalgae: Example of the Proteome Data in Nannochloropsis gaditana

Most of the marine ecosystems on our planet are still unknown. Among these ecosystems, microalgae act as a baseline due to their role as primary producers. The estimated millions of species of these microorganisms represent an almost infinite source of potentially active biocomponents offering unlimited biotechnology applications. This review considers current research in microalgae using the “omics” approach, which today is probably the most important biotechnology tool. These techniques enable us to obtain a large volume of data from a single experiment. The specific focus of this review is proteomics as a technique capable of generating a large volume of interesting information in a single proteomics assay, and particularly the concept of applied proteomics. As an example, this concept has been applied to the study of Nannochloropsis gaditana, in which proteomics data generated are transformed into information of high commercial value by identifying proteins with direct applications in the biomedical and agri-food fields, such as the protein designated UCA01 which presents antitumor activity, obtained from N. gaditana.     

Valorization of the Red Algae Gelidium sesquipedale by Extracting a Broad Spectrum of Minor Compounds Using Green Approaches

Until now, the red algae Gelidium sesquipedale has been primarily exploited for agar production, leaving an undervalued biomass. In this work, the use of eco-friendly approaches employing ultrasound-assisted extraction (UAE) and green solvents was investigated to valorize the algal minor compounds. The green methods used herein showed an attractive alternative to efficiently extract a broad spectrum of bioactive compounds in short extraction times (15 to 30 min vs. 8 h of the conventional method). Using the best UAE conditions, red seaweed extracts were characterized in terms of total phenolics (189.3 ± 11.7 mg GAE/100 g dw), flavonoids (310.7 ± 9.7 mg QE/100 g dw), mycosporine-like amino acids (MAAs) (Σ MAAs = 1271 mg/100 g dw), and phycobiliproteins (72.4 ± 0.5 mg/100 g dw). Additionally, produced algal extracts exhibited interesting antioxidant and anti-enzymatic activities for potential applications in medical and/or cosmetic products. Thus, this study provides the basis to reach a superior valorization of algal biomass by using alternative methods to extract biologically active compounds following eco-friendly approaches. Moreover, the strategies developed not only open new possibilities for the commercial use of Gelidium sesquipedale, but also for the valorization of different algae species since the techniques established can be easily adapted.     

Black currant polyphenols: Their storage stability and microencapsulation

Interest in the consumption of fresh fruits is, to a large extent, due to its content of bioactive nutrients and their importance as dietary antioxidants. There is a growing demand for delivery of antioxidants through functional foods with the related challenge of protecting their bioactivity during food processing and subsequent passage through the gastrointestinal tract. This study focuses on the evaluation of concentration of bioactive compounds in black currant berries (Ribes nigrum L.) and retention of black currant polyphenol compounds and their antioxidant activity after microencapsulation by spray-drying. The polyphenol contents and antioxidant activities were assessed for 5 black currant cultivars grown in Canada in fresh and stored fruits at −20 °C for 9 months. The Ben Alder cultivar was the richest in anthocyanins and flavonols content with high antioxidant potential measured with ABTS and DPPH radicals (4.5 and 2.4 mM/100 g). Black currant polyphenols were stable during 9 month of storage at −20 °C. The results of this study indicated that in black currants storage did not affect negatively the antioxidant capacity. The good stability of bioactive compounds of black currants during freezer storage allows prolonging the storage time of these berries. Maltodextrins with different DE (dextrose equivalent): DE11, DE18, DE21 and inulin were selected as wall material. The stability of encapsulated polyphenols during 12 months storage at 8 °C and 25 °C was evaluated. Compared to maltodextrins DE18 and DE21, maltodextrin DE11 had not only higher drying yield but also offered better protection for phenolics during storage. Radical scavenging activity studies demonstrated significant antioxidant activity of microencapsulated powders before and after storage. The black currant polyphenol microcapsules described in this study represent a promising food additive for incorporation into functional foods due to antioxidant content.     

Cytotoxicity of the ascidian Cystodytes dellechiajei against tumor cells and study of the involvement of associated microbiota in the production of cytotoxic compounds

Many cytotoxic compounds of therapeutic interest have been isolated from marine invertebrates, and some of them have been reported to be of microbial origin. Pyridoacridine alkaloids are the main compounds extracted from the ascidian Cystodytes dellechiajei. Here we describe the in vitro antiproliferative activity against different tumor cell lines of the ascidian extracts and provide some insights on the role of the microbial community associated with the tunicate in the production of these compounds. C. dellechiajei extracts showed remarkably high antiproliferative activity (IC₅₀ ≤5 μg/mL) in human lung carcinoma A-549, colon adenocarcinoma H-116, pancreatic adenocarcinoma PSN-1 and breast carcinoma SKBR3 cell lines. Moreover, we found that the maximum activity was located in the tunic tissue of the colony, which harbours a microbial community. In order to ascertain the involvement of this community in the synthesis of the bioactive compounds different approachs that included culture and culture independent methods were carried out. We undertook a screening for antiproliferative activities of the bacterial isolates from the ascidian, as well as a comprative analysis of the cytotoxic activities and the microbial communities from two color morphs of the ascidian, green and blue. In addition, the changes of the antiproliferative activities and the composition of the microbial communities were studied from ascidians kept in aquaria and treated with antibiotics for one month. Our data obtained from the different experiments did not point out to bacteria as the source of the cytotoxic compounds, suggesting thus an ascidian origin.     

Exploring Bioactive Properties of Marine Cyanobacteria Isolated from the Portuguese Coast: High Potential as a Source of Anticancer Compounds

The oceans remain a major source of natural compounds with potential in pharmacology. In particular, during the last few decades, marine cyanobacteria have been in focus as producers of interesting bioactive compounds, especially for the treatment of cancer. In this study, the anticancer potential of extracts from twenty eight marine cyanobacteria strains, belonging to the underexplored picoplanktonic genera, Cyanobium, Synechocystis and Synechococcus, and the filamentous genera, Nodosilinea, Leptolyngbya, Pseudanabaena and Romeria, were assessed in eight human tumor cell lines. First, a crude extract was obtained by dichloromethane:methanol extraction, and from it, three fractions were separated in a Si column chromatography. The crude extract and fractions were tested in eight human cancer cell lines for cell viability/toxicity, accessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactic dehydrogenase release (LDH) assays. Eight point nine percent of the strains revealed strong cytotoxicity; 17.8% showed moderate cytotoxicity, and 14.3% assays showed low toxicity. The results obtained revealed that the studied genera of marine cyanobacteria are a promising source of novel compounds with potential anticancer activity and highlight the interest in also exploring the smaller filamentous and picoplanktonic genera of cyanobacteria.     

European pennyroyal (Mentha pulegium) from Portugal: Chemical composition of essential oil and antioxidant and antimicrobial properties of extracts and essential oil

There is a growing interest of industry to replace synthetic chemicals by natural products with bioactive properties from plant origin. The aim of this study was to determine the chemical composition of European pennyroyal (Mentha pulegium) essential oil and to characterize the in vitro antioxidant and antimicrobial activities of its water (hot and cold) and ethanolic extracts and of the essential oil. The essential oil revealed menthone, pulegone and neo-menthol as the main constituents, comprising 35.9, 23.2 and 9.2% of the essential oil, respectively. The hot water extract exhibited the highest antioxidant activity and phenol content. In contrast, the extracts were not very effective to inhibit the growth of the seven foodborne spoilage and pathogenic bacteria tested, but the essential oil showed antibacterial activity against all bacterial strains. In conclusion, extracts and essential oil of M. pulegium from Mediterranean origin have huge potential as an alternative to chemical additives for the food industry.     

New approaches concerning the utilization of natural amendments in cadmium phytoremediation

Different vegetal biomass resources such as Picea abies bark, Castanea sativa chestnuts shell and Asclepias syriaca plant were considered to provide a source of natural bioactive compounds, which can be properly used in several directions such as: plant growth regulators and amendments in bioremediation. Having in mind a complex processing biomass technology, these raw materials are used in a first step to separate bioactive compounds by an aqueous extraction. The obtained extracts containing polyphenols were tested in germination tests to evaluate their influence on cadmium bioaccumulation in oat plant (Avena sativa). It was observed that the mentioned extracts modulated cadmium bioaccumulation, photosynthesizing pigments biosynthesis, plant growth and its development depending on metal ion concentration (5, 12.5, 25 mg/L), and extracts composition determined by vegetal raw material and their total polyphenols content (130, 122, 114 mg/L related to gallic acid). FTIR spectra and histo-anatomical cross section of the roots certify that P. abies bark and A. syriaca plant extracts amendments stimulated cadmium bioaccumulation, promoting the translocation of heavy metals to the aerial part of the plant, meanwhile chestnuts shell extract reduced the mobility of cadmium determining in situ inactivation of heavy metal ions.