Features of the microalga Raphidocelis subcapitata: physiology and applications.

The microalga Raphidocelis subcapitata was isolated from the Nitelva River (Norway) and subsequently deposited in the collection of the Norwegian Institute of Water Research as "Selenastrum capricornutum Printz". This freshwater microalga, also known as Pseudokirchneriella subcapitata, acquired much of its notoriety due to its high sensitivity to different chemical species, which makes it recommended by different international organizations for the assessment of ecotoxicity. However, outside this scope, R. subcapitata continues to be little explored. This review aims to shed light on a microalga that, despite its popularity, continues to be an "illustrious" unknown in many ways. Therefore, R. subcapitata taxonomy, phylogeny, shape, size/biovolume, cell ultra-structure, and reproduction are reviewed. The nutritional and cultural conditions, chronological aging, and maintenance and preservation of the alga are summarized and critically discussed. Applications of R. subcapitata, such as its use in aquatic toxicology (ecotoxicity assessment and elucidation of adverse toxic outcome pathways) are presented. Furthermore, the latest advances in the use of this alga in biotechnology, namely in the bioremediation of effluents and the production of value-added biomolecules and biofuels, are highlighted. To end, a perspective regarding the future exploitation of R. subcapitata potentialities, in a modern concept of biorefinery, is outlined. KEY POINTS: • An overview of alga phylogeny and physiology is critically reviewed. • Advances in alga nutrition, cultural conditions, and chronological aging are presented. • Its use in aquatic toxicology and biotechnology is highlighted.

Palmelloid-like phenotype in the alga Raphidocelis subcapitata exposed to pollutants: A generalized adaptive strategy to stress or a specific cellular response?

This work focuses on the formation of palmelloid-like phenotype in the freshwater alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata and Selenastrum capricornutum), when exposed to adverse conditions generated by the presence of organic [the antibiotic erythromycin (ERY) and the herbicide metolachlor (MET)] or inorganic [the heavy metals, cadmium (Cd) and zinc (Zn)] pollutants, at environmentally relevant concentrations. This alga in absence of stress or when exposed to ERY or Zn, up to 200 µg/L, essentially showed a single-nucleus state, although algal growth was reduced or stopped. R. subcapitata "switched" to a multinucleated state (palmelloid-like morphology) and accumulated energy-reserve compounds (neutral lipids) when stressed by 100-200 µg/L MET or 200 µg/L Cd; at these concentrations of pollutants, growth was arrested, however, the majority of the algal population (≥83 %) was alive. The formation of palmelloid-like phenotype, at sub-lethal concentrations of pollutants, was dependent on the pollutant, its concentration and exposure time. The multinucleated structure is a transitory phenotype since R. subcapitata population was able to revert to a single-nucleus state, with normal cell size, within 24-96 h (depending on the impact of the toxic in the alga), after being transferred to fresh OECD medium, without pollutants. The obtained results indicate that the formation of a palmelloid-like phenotype in R. subcapitata is dependent on the mode of action of toxics and their concentration, not constituting a generalized defense mechanism against stress. The observations here shown contribute to understanding the different strategies used by the unicellular alga R. subcapitata to cope with severe stress imposed by organic and inorganic pollutants.

Life and death of Pseudokirchneriella subcapitata: physiological changes during chronological aging.

The green alga Pseudokirchneriella subcapitata is widely used in ecotoxicity assays and has great biotechnological potential as feedstock. This work aims to characterize the physiology of this alga associated with the aging resulting from the incubation of cells for 21 days, in the OECD medium, with continuous agitation and light exposure, in a batch mode. After inoculation, cells grow exponentially during 3 days, and the culture presents a typical green color. In this phase, "young" algal cells present, predominantly, a lunate morphology with the chloroplast occupying a large part of the cell, maximum photosynthetic activity and pigments concentration, and produce starch as a reserve material. Between the 5th and the 12th days of incubation, cells are in the stationary phase. The culture becomes less green, and the cells stop dividing (≥ 99% have one nucleus) and start to age. "Old" algal cells present chloroplast shrinkage, an abrupt decline of chlorophylls content, and photosynthetic capacity (Fv/Fm and ɸPSII), accompanied by a degradation of starch and an increase of neutral lipids content. The onset of the death phase occurs after the 12th day and is characterized by the loss of cell membrane integrity of some algae (cell death). The culture stays, progressively, yellow, and the majority of the population (~93%) is composed of live cells, chronologically "old," with a significant drop in photosynthetic activity (decay > 75% of Fv/Fm and ɸPSII) and starch content. The information here achieved can be helpful when exploring the potential of this alga in toxicity studies or in biotechnological applications. KEY POINTS: • Physiological changes of P. subcapitata with chronological aging are shown • "Young" algae exhibit a semilunar shape, high photosynthetic activity, and accumulated starch • "Old"-live algae show reduced photosynthetic capacity and accumulated lipids.

Exposure of the alga Pseudokirchneriella subcapitata to environmentally relevant concentrations of the herbicide metolachlor: Impact on the redox homeostasis.

This study investigated the effect of the herbicide metolachlor (MET) on the redox homeostasis of the freshwater green alga Pseudokirchneriella subcapitata. At low MET concentrations (≤40 µg L-1), no effects on algal cells were detected. The exposure of P. subcapitata to 45-235 µg L-1 MET induced a significant increase of reactive oxygen species (ROS). The intracellular levels of ROS were particularly increased at high (115 and 235 µg L-1) but environmentally relevant MET concentrations. The exposure of algal cells to 115 and 235 µg L-1 MET originated a decrease in the levels of antioxidants molecules (reduced glutathione and carotenoids) as well as a reduction of the activity of scavenging enzymes (superoxide dismutase and catalase). These results suggest that antioxidant (non-enzymatic and enzymatic) defenses were affected by the excess of MET. As consequence of this imbalance (ROS overproduction and decline of the antioxidant system), ROS inflicted oxidative injury with lipid peroxidation and damage of cell membrane integrity. The results provide further insights about the toxic modes of action of MET on a non-target organism and emphasize the relevance of toxicological studies in the assessment of the impact of herbicides in freshwater environments.

Toxicological effects induced by the biocide triclosan on Pseudokirchneriella subcapitata.

Triclosan, a widely used biocide broadly found in aquatic environments, is cause of concern due to its unknown effects on non-targets organisms. In this study, a multi biomarker approach was used in order to evaluate the 72 h-effect of triclosan on the freshwater alga Pseudokirchneriella subcapitata (Raphidocelis subcapitata). Triclosan, at environmental relevant concentrations (27 and 37 µg L-1), caused a decrease of proliferative capacity, which was accompanied by an increase of cell size and a profound alteration of algae shape. It was found that triclosan promoted the intracellular accumulation of reactive oxygen species, the depletion of non-enzymatic antioxidant defenses (reduced glutathione and carotenoids) and a decrease of cell metabolic activity. A reduction of photosynthetic pigments (chlorophyll a and b) was also observed. For the highest concentration tested (37 µg L-1), a decrease of photosynthetic efficiency was detected along with a diminution of the relative transport rate of electrons on the photosynthetic chain. In conclusion, triclosan presents a deep impact on the microalga P. subcapitata morphology and physiology translated by multiple target sites instead of a specific point (cellular membrane) observed in the target organism (bacteria). Additionally, this study contributes to clarify the toxicity mechanisms of triclosan, in green algae, showing the existence of distinct modes of action of the biocide depending on the microalga.

Reproductive cycle progression arrest and modification of cell morphology (shape and biovolume) in the alga Pseudokirchneriella subcapitata exposed to metolachlor.

Metolachlor (MET) is an herbicide widely used and frequently found (at µg L-1) in aquatic systems. This work aimed to study the modes of action of MET on the green microalga Pseudokirchneriella subcapitata. Algae exposed to 115 or 235 µg L-1 MET, for 48 or 72 h, presented a reduction of metabolic activity, chlorophyll a and b content and photosynthetic efficiency. The exposure to 115 or 235 µg L-1 MET also induced growth yield reduction, mean cell biovolume increase and alteration of the typical algae shape (cells lunate or helically twisted) to "French croissant"-type; at these MET concentrations, algal population was mainly composed by multinucleated cells (≥ 4 nuclei), which suggest that MET impairs the normal progression of the reproductive cycle but did not hinder nuclear division. The accumulation of multinucleated cells seems to be the consequence of the incapacity of the parent cell to release the autospores. In conclusion, MET disrupts the physiology of P. subcapitata cells; the disturbance of the progression of the reproductive cycle should be in the origin of growth slowdown (or even its arrest), increase of mean cell biovolume and modification of algal shape. This work contributed to elucidate, in a systematically and integrated way, the toxic mechanism of MET on the non-target organism, the alga P. subcapitata.

Impact of erythromycin on a non-target organism: Cellular effects on the freshwater microalga Pseudokirchneriella subcapitata.

The increasing and indiscriminate use of antibiotics is the origin of their introduction in aquatic systems through domestic and livestock effluents. The occurrence of erythromycin (ERY), a macrolide antibiotic, in water bodies raises serious concerns about its potential toxic effect in aquatic biota (non-target organisms), particularly in microalgae, the first organisms in contact with aquatic contaminants. This study aimed to evaluate the possible toxic effects of ERY on relevant cell targets of the freshwater microalga Pseudokirchneriella subcapitata. Algal cells incubated with significant environmental ERY concentrations presented disturbance of the photosynthetic apparatus (increased algal autofluorescence and reduction of chlorophyll a content) and mitochondrial function (hyperpolarization of mitochondrial membrane). These perturbations can apparently be attributed to the similarity of the translational machinery of these organelles (chloroplasts and mitochondria) with the prokaryotic cells. P. subcapitata cells treated with ERY showed a modification of metabolic activity (increased esterase activity) and redox state (alteration of intracellular levels of reactive oxygen species and reduced glutathione content) and an increased biovolume. ERY induced an algistatic effect: reduction of growth rate without loss of cell viability (plasma membrane integrity). The present study shows that chronic exposure (72 h), at low (µg L-1) ERY concentrations (within the range of concentrations detected in surface and ground waters), induce disturbances in the physiological state of the alga P. subcapitata. Additionally, this work alerts to the possible negative impact of the uncontrolled use of ERY on the aquatic systems.

Influence of the metabolic state on the tolerance of Pichia kudriavzevii to heavy metals.

This work aims to examine the influence of the metabolic state of the yeast Pichia kudriavzevii on the susceptibility to a metals mixture (5 mg L-1 Cd, 10 mg L-1 Pb, and 5 mg L-1 Zn). Cells exposed to the metals mixture in the presence of 25 mmol L-1 glucose displayed a higher loss of membrane integrity and proliferation capacity, compared to cells incubated in the absence of glucose. The analysis of the effect of individual metals revealed that glucose increased the toxic effect of Cd marginally, and of Pb significantly. The increased susceptibility to heavy metals due to glucose was attenuated in the simultaneous presence of a mitochondrial respiration inhibitor such as sodium azide (NaN3 ). ATP-depleted yeast cells, resulting from treatment with the non-metabolizable glucose analogue 2-deoxy-d-glucose, showed an increased susceptibility to heavy metals mixture. Pre-incubation of yeast cells with 1 or 1.5 mmol L-1 Ca2+ reduced significantly (P < 0.05) the loss of membrane integrity induced by the metals mixture. These findings contribute to the understanding of metals mechanisms of toxicity in the non-conventional yeast P. kudriavzevii.

Responses of the alga Pseudokirchneriella subcapitata to long-term exposure to metal stress.

The green alga Pseudokirchneriella subcapitata has been widely used in ecological risk assessment, usually based on the impact of the toxicants in the alga growth. However, the physiological causes that lead algal growth inhibition are not completely understood. This work aimed to evaluate the biochemical and structural modifications in P. subcapitata after exposure, for 72 h, to three nominal concentrations of Cd(II), Cr(VI), Cu(II) and Zn(II), corresponding approximately to 72 h-EC10 and 72 h-EC50 values and a high concentration (above 72 h-EC90 values). The incubation of algal cells with the highest concentration of Cd(II), Cr(VI) or Cu(II) resulted in a loss of membrane integrity of ~16, 38 and 55%, respectively. For all metals tested, an inhibition of esterase activity, in a dose-dependent manner, was observed. Reduction of chlorophyll a content, decrease of maximum quantum yield of photosystem II and modification of mitochondrial membrane potential was also verified. In conclusion, the exposure of P. subcapitata to metals resulted in a perturbation of the cell physiological status. Principal component analysis revealed that the impairment of esterase activity combined with the reduction of chlorophyll a content were related with the inhibition of growth caused by a prolonged exposure to the heavy metals.

Impact of multi-metals (Cd, Pb and Zn) exposure on the physiology of the yeast Pichia kudriavzevii.

Metal contamination of the environment is frequently associated to the presence of two or more metals. This work aimed to study the impact of a mixture of metals (Cd, Pb and Zn) on the physiology of the non-conventional yeast Pichia kudriavzevii. The incubation of yeast cells with 5 mg/l Cd, 10 mg/l Pb and 5 mg/l Zn, for 6 h, induced a loss of metabolic activity (assessed by FUN-1 staining) and proliferation capacity (evaluated by a clonogenic assay), with a small loss of membrane integrity (measured by trypan blue exclusion assay). The staining of yeast cells with calcofluor white revealed that no modification of chitin deposition pattern occurred during the exposure to metal mixture. Extending for 24 h, the exposure of yeast cells to metal mixture provoked a loss of membrane integrity, which was accompanied by the leakage of intracellular components. A marked loss of the metabolic activity and the loss of proliferation capacity were also observed. The analysis of the impact of a single metal has shown that, under the conditions studied, Pb was the metal responsible for the toxic effect observed in the metal mixture. Intracellular accumulation of Pb seems to be correlated with the metals' toxic effects observed.

Siderophore production by Bacillus megaterium: effect of growth phase and cultural conditions.

Siderophore production by Bacillus megaterium was detected, in an iron-deficient culture medium, during the exponential growth phase, prior to the sporulation, in the presence of glucose; these results suggested that the onset of siderophore production did not require glucose depletion and was not related with the sporulation. The siderophore production by B. megaterium was affected by the carbon source used. The growth on glycerol promoted the very high siderophore production (1,182 µmol g(-1) dry weight biomass); the opposite effect was observed in the presence of mannose (251 µmol g(-1) dry weight biomass). The growth in the presence of fructose, galactose, glucose, lactose, maltose or sucrose, originated similar concentrations of siderophore (546-842 µmol g(-1) dry weight biomass). Aeration had a positive effect on the production of siderophore. Incubation of B. megaterium under static conditions delayed and reduced the growth and the production of siderophore, compared with the incubation in stirred conditions.

Modification of cell volume and proliferative capacity of Pseudokirchneriella subcapitata cells exposed to metal stress.

The impact of metals (Cd, Cr, Cu and Zn) on growth, cell volume and cell division of the freshwater alga Pseudokirchneriella subcapitata exposed over a period of 72 h was investigated. The algal cells were exposed to three nominal concentrations of each metal: low (closed to 72 h-EC10 values), intermediate (closed to 72 h-EC50 values) and high (upper than 72 h-EC90 values). The exposure to low metal concentrations resulted in a decrease of cell volume. On the contrary, for the highest metal concentrations an increase of cell volume was observed; this effect was particularly notorious for Cd and less pronounced for Zn. Two behaviours were found when algal cells were exposed to intermediate concentrations of metals: Cu(II) and Cr(VI) induced a reduction of cell volume, while Cd(II) and Zn(II) provoked an opposite effect. The simultaneous nucleus staining and cell image analysis, allowed distinguishing three phases in P. subcapitata cell cycle: growth of mother cell; cell division, which includes two divisions of the nucleus; and, release of four autospores. The exposure of P. subcapitata cells to the highest metal concentrations resulted in the arrest of cell growth before the first nucleus division [for Cr(VI) and Cu(II)] or after the second nucleus division but before the cytokinesis (release of autospores) when exposed to Cd(II). The different impact of metals on algal cell volume and cell-cycle progression, suggests that different toxicity mechanisms underlie the action of different metals studied. The simultaneous nucleus staining and cell image analysis, used in the present work, can be a useful tool in the analysis of the toxicity of the pollutants, in P. subcapitata, and help in the elucidation of their different modes of action.

Evaluation of the role of glutathione in the lead-induced toxicity in Saccharomyces cerevisiae.

The effect of intracellular reduced glutathione (GSH) in the lead stress response of Saccharomyces cerevisiae was investigated. Yeast cells exposed to Pb, for 3 h, lost the cell proliferation capacity (viability) and decreased intracellular GSH level. The Pb-induced loss of cell viability was compared among yeast cells deficient in GSH1 (∆gsh1) or GSH2 (∆gsh2) genes and wild-type (WT) cells. When exposed to Pb, ∆gsh1 and ∆gsh2 cells did not display an increased loss of viability, compared with WT cells. However, the depletion of cellular thiols, including GSH, by treatment of WT cells with iodoacetamide (an alkylating agent, which binds covalently to thiol group), increased the loss of viability in Pb-treated cells. In contrast, GSH enrichment, due to the incubation of WT cells with amino acids mixture constituting GSH (L-glutamic acid, L-cysteine and glycine), reduced the Pb-induced loss of proliferation capacity. The obtained results suggest that intracellular GSH is involved in the defence against the Pb-induced toxicity; however, at physiological concentration, GSH seems not to be sufficient to prevent the Pb-induced loss of cell viability.

Development of a short-term assay based on the evaluation of the plasma membrane integrity of the alga Pseudokirchneriella subcapitata.

Membrane integrity has been used as a criterion for the definition of cell viability. In the present work, staining conditions (time and dye concentration) for the evaluation of membrane integrity in a fluorescence microplate reader, using the membrane-impermeant nucleic-acid dye SYTOX Green, were optimized. Incubating Pseudokirchneriella subcapitata algal cells with 0.5 µmol/l SYTOX Green for 40 min allowed a clear discrimination between live (intact plasma membrane) and dead cells (with compromised plasma membrane). Algal cell suspensions, labelled with SYTOX Green, exhibited a green fluorescence proportional to the fraction of the cells with a permeabilized plasma membrane. The optimized staining conditions were used to assess the toxicity of 1-pentanol on P. subcapitata in a short-term exposure (6 h) assay. The loss of membrane integrity in the cell population increased with the concentration of 1-pentanol. The 6-h EC(10) and EC(50) values were 7,617 mg/l 1-pentanol (95 % confidence limits 4,670-9,327) and 12,818 mg/l 1-pentanol (95 % confidence limits 10,929-15,183), respectively. The developed microplate-based short-term assay can be useful in the high-throughput screening of toxics or environmental samples using the alga P. subcapitata.

Flocculation in ale brewing strains of Saccharomyces cerevisiae: re-evaluation of the role of cell surface charge and hydrophobicity.

Flocculation is an eco-friendly process of cell separation, which has been traditionally exploited by the brewing industry. Cell surface charge (CSC), cell surface hydrophobicity (CSH) and the presence of active flocculins, during the growth of two (NCYC 1195 and NCYC 1214) ale brewing flocculent strains, belonging to the NewFlo phenotype, were examined. Ale strains, in exponential phase of growth, were not flocculent and did not present active flocculent lectins on the cell surface; in contrast, the same strains, in stationary phase of growth, were highly flocculent (>98%) and presented a hydrophobicity of approximately three to seven times higher than in exponential phase. No relationship between growth phase, flocculation and CSC was observed. For comparative purposes, a constitutively flocculent strain (S646-1B) and its isogenic non-flocculent strain (S646-8D) were also used. The treatment of ale brewing and S646-1B strains with pronase E originated a loss of flocculation and a strong reduction of CSH; S646-1B pronase E-treated cells displayed a similar CSH as the non-treated S646-8D cells. The treatment of the S646-8D strain with protease did not reduce CSH. In conclusion, the increase of CSH observed at the onset of flocculation of ale strains is a consequence of the presence of flocculins on the yeast cell surface and not the cause of yeast flocculation. CSH and CSC play a minor role in the auto-aggregation of the ale strains since the degree of flocculation is defined, primarily, by the presence of active flocculins on the yeast cell wall.

Selective recovery of chromium, copper, nickel, and zinc from an acid solution using an environmentally friendly process.

PURPOSE: Real electroplating effluents contain multiple metals. An important point related with the feasibility of the bioremediation process is linked with the strategy to recover selectively metals. In this work, a multimetal solution, obtained after microwave acid digestion of the ashes resulted from the incineration of Saccharomyces cerevisiae contaminated biomass, was used to recover selectively chromium, copper, nickel, and zinc. RESULTS: The acid solution contained 3.8, 0.4, 2.8, and 0.2 g/L of chromium(III), copper, nickel, and zinc, respectively. The strategy developed consisted of recovering copper (97.6%), as a metal, by electrolyzing the solution at a controlled potential. Then, the simultaneous alkalinization of the solution (pH 14), addition of H(2)O(2), and heating of the solution led to a complete oxidation of chromium and nickel recovery (87.9% as a precipitate of nickel hydroxide). After adjusting the pH of the remaining solution at pH 10, selective recovery of zinc (82.7% as zinc hydroxide) and chromium (95.4% as a solution of cromate) was achieved. CONCLUSION: The approach, used in the present work, allowed a selective and efficient recovery of chromium, copper, nickel, and zinc from an acid solution using a combined electrochemical and chemical process. The strategy proposed can be used for the selective recovery of metals present in an acid digestion solution, which resulted from the incineration of ashes of biomass used in the treatment of heavy metals rich industrial effluents.

Selective recovery of copper, nickel and zinc from ashes produced from Saccharomyces cerevisiae contaminated biomass used in the treatment of real electroplating effluents.

The aim of this work was to seek an environmentally friendly process for recycling metals from biomass-sludges generated in the treatment of industrial wastewaters. This work proposes a hybrid process for selective recovery of copper, nickel and zinc from contaminated biomass of Saccharomyces cerevisiae, used in the bioremediation of electroplating effluents. The developed separation scheme comprised five consecutive steps: (1) incineration of the contaminated biomass; (2) microwave acid (HCl) digestion of the ashes; (3) recovery of copper from the acid solution by electrolysis at controlled potential; (4) recycle of nickel, as nickel hydroxide, by alcalinization of the previous solution at pH 14; (5) recovery of zinc, as zinc hydroxide, by adjusting the pH of the previous solution at 10. This integrated approach allowed recovering each metal with high yielder (>99% for all metals) and purity (99.9%, 92% and 99.4% for copper, nickel and zinc, respectively). The purity of the metals recovered allows selling them in the market or being recycled in the electroplating process without waste generation.

Removal of heavy metals using a brewer's yeast strain of Saccharomyces cerevisiae: chemical speciation as a tool in the prediction and improving of treatment efficiency of real electroplating effluents.

In the present work, the influence of the competitive effect of inorganic ligands (carbonates, chlorides, fluorides, phosphates, nitrates and sulphates), which can be present in real multi-metal electroplating effluents, on the biosorption of chromium, copper, nickel and zinc ions by yeast cells of Saccharomyces cerevisiae was rationally examined. Additionally, chemical speciation studies allowed optimizing the amount of yeast biomass to be used in the treatment of effluents contaminated with nickel. The applicability of chemical simulation studies was tested using two simulated effluents and validated using one real electroplating effluent, all containing high concentrations of nickel (about 303 micro mol l(-1)). For nickel removal, heat-killed biomass of a brewing flocculent strain of S. cerevisiae was used, in a batch mode. After the implementation of the bioremediation process (12 g dry weight l(-1) of yeast cells), the concentration of nickel in the real effluent (34 micro mol l(-1)) reached the quality criteria for industrial effluents discharge, after the second or third batch according to the U.S.-Environmental Protection Agency and Portuguese law, respectively. This corresponded to a removal of nickel of 89%.

Removal of heavy metals using a brewer's yeast strain of Saccharomyces cerevisiae: the flocculation as a separation process.

In this work, a brewer's yeast strain was used to remove heavy metals from a synthetic effluent. The solid-liquid separation process was carried out using the flocculation ability of the strain. The yeast strain was able to sediment in the presence of Cu2+, Ni2+, Zn2+, Cd2+ and Cr3+, which evidences that the flocculation can be used as a cheap and natural separation process for an enlarged range of industrial effluents. For a biomass concentration higher than 0.5 g/l, more than 95% of the cells were settled after 5 min; this fact shows that the auto-aggregation of yeast biomass is a rapid and efficient separation process. Cells inactivated at 45 degrees C maintain the sedimentation characteristics, while cells inactivated at 80 degrees C lose partially (40%) the flocculation. The passage of metal-loaded effluent through a series of sequential batches allowed, after the second batch, the reduction of the Ni2+ concentration in solution for values below the legal limit of discharge of wastewater in natural waters (2mg/l); this procedure corresponds to a removal of 91%. A subsequent batch had a marginal effect on Ni2+ removal (96%). Together, the results obtained suggest that the use of brewing flocculent biomass looks a promising alternative in the bioremediation of metal-loaded industrial effluents since the removal of the heavy metals and cell separation are simultaneously achieved.