Biosorption of Heavy Metals from Acid Mine Drainage ... - Springer Link

Biotechnology and Bioprocess Engineering 16: 1262-1272 (2011) DOI 10.1007/s12257-010-0465-5

RESEARCH PAPER

Biosorption of Heavy Metals from Acid Mine Drainage onto Biopolymers (Chitin and α (1,3) β-D-glucan) from Industrial Biowaste Exhausted Brewer’s Yeasts (Saccharomyces cerevisiae L.) Fernando I. Ramírez-Paredes, Teresa Manzano-Muñoz, Juan C. Garcia-Prieto, Galina G. Zhadan, Valery L. Shnyrov, John F. Kennedy, and Manuel G. Roig

Received: 19 December 2010 / Revised: 16 June 2011 / Accepted: 3 July 2011 © The Korean Society for Biotechnology and Bioengineering and Springer 2011

Abstract A biosorption process has been developed for the bioremediation of heavy metal-contaminated acid drainages from Merladet and Faith open-cast mines, located in western Spain. The process is based on the physico-chemical properties for the adsorption, ion exchange, and complexation of metal ions by biopolymers (chitin and α (1,3) β-D-glucan) from industrial biowaste exhausted brewer's yeast (Saccharomyces cerevisiae L.). Firstly, the chemical composition (U, Mn, Al, Fe, Cu, Zn, and Ni) and the physico-chemical and ecological states of these acid mine drainages were characterised. Furthermore, the selectivity for Zn, Cu, Mn, Ni, and Al the first order kinetics and the performance of the metals biosorption process by exhausted brewer's yeast were evaluated with polluted acid synthetic waters and mine drainages. The biosorption equilibria were reached in 10 ~ 15 min following Langmuir type isotherms with higher affinity constants for metal-biosorbent binding for synthetic waters than for acid mine drainages. The efficiency of the process Fernando I. Ramírez-Paredes, Manuel G. Roig* Departamento de Química Física, Facultad de Química, Universidad de Salamanca, 37008 Salamanca, Spain Tel: +34-923-294-487; Fax: +34-923-294-579 E-mail: [email protected] Teresa Manzano-Muñoz, Juan C. Garcia-Prieto, Manuel G. Roig Centro de Investigación y Desarrollo Tecnológico del Agua (CIDTA), Universidad de Salamanca, 37007 Salamanca, Spain Galina G. Zhadan, Valery L. Shnyrov Departamento de Bioquímica y Biología Molecular, Universidad de Salamanca, 37007 Salamanca, Spain John F. Kennedy Chembiotech Laboratories, Advanced Science and Technology Institute, Kyrewood House, Tenbury Wells, Worcestershire, WR15 8SG, UK

with real water samples was markedly lower for the case of Mn, and zero for Zn and Al. An antagonistic interference on the biosorption of a metal due to the presence of other metals is proposed. Finally, the ecotoxicity of the acid mine drainage was removed when it was incubated with brewer’s yeast trapped in polyurethane foam. Keywords: biopolymers, biosorption, chitin, β-glucans, isotherms, kinetics, heavy metals, mine drainage, toxicity, industrial wastes, brewer’s yeasts

1. Introduction Although mining is a local activity and the greatest impact on terrestrial and aquatic ecosystems is essentially also local in nature, this is not always the case and the dispersion of heavy metals may become regional and even global. Mines generate considerable amounts of rock and slag that must be dumped on the land, generating bioleachates that eventually flow into aquatic ecosystems [1-7]. In terms of heavy metal pollution, the main result of this is to render useless lands for agriculture that were previously fertile; metal-rich waters arise from residues and slag dumps on the land, with the consequent pollution of land, rivers, lakes and coastal zones. A very distressing example of this in Spain was the dispersal of the pollution by heavy metals, and their noxious effects, along the basin of the River Guadalquivir and over the Doñana Nature Reserve, brought about by the collapse of the acid mine water reservoir that occurred at Aznalcóllar (Seville) on 28th April 1998. The first aim of the present work was to investigate the

Biosorption of Heavy Metals from Acid Mine Drainage onto Biopolymers (Chitin and α (1,3) β-D-glucan) from Industrial…

capacity of an abundantly available biopolymer from industrial biowastes for the bioremediation of acid mine drainages from metallogenic (W, U, Sn, Bi, etc) areas of the province of Salamanca (Spain) by means of biosorption processes. 1.1. Biosorption of metals There are natural dead and inactive biomasses that have the capacity to accumulate metals through processes of biosorption because they contain biopolymers with functional groups that are able to bind metal ions present in water. In view of the complexity of most biopolymers, the binding of metal ions and biopolymers may simultaneously involve different types of physico-chemical processes: ion exchange, the formation of coordination complexes, physical adsorption, microprecipitation processes and redox reactions. Regarding the exchange of metal cations in aqueous solution, the main ionisable groups for cationic binding in biopolymers are organic carboxyl, phosphate, and sulphate groups. An example is the carboxyl group, which is widely distributed in biopolymers and is nearly always present as a constituent of the side chains of proteins and in the uronic, neuraminic and muramic acids, and related substituted monosaccharides that constitute certain polysaccharides. Regarding the exchange of metal anions, this may occur through a broad range of nitrogen-based organic compounds. In proteins, the amino, the imidazole and the guanidine groups are common sites of positive charge. All these groups are susceptible to possible post-translational changes by in vivo methylation and this will affect all their charge characteristics. Also, in nucleic acids there are positive charge sites on the amino groups of the protonated rings of purine or pyrimidine or on the heterocyclic nitrogen, which is also protonated. Considered as a group, polysaccharides are acid or neutral macromolecules with few basic functional groups and they appear as non-acetylated aminosugars. Chitin is a noteworthy example; in chitin, some of the glucosamine residues are not acetylated, which facilitates the formation of the deacetylated derivative, chitosan, with a high proportion of protonatable positive-charge sites. Some of the representative thermodynamic ionisation constants for some of these groups are shown in Table 1. The formation of complexes between the functional organic groups of adsorbent biopolymers and metal atoms involves the presence within the organic moieties of one or several atoms that have a pair of lone electrons available for donation. Among them, the most likely candidates are trivalent neutral atoms of nitrogen and neutral and divalent atoms of oxygen and sulphur, which exist in a broad range of amino, imino and heterocyclic groups of proteins and nucleic acids, polysaccharides, polyphenols and poly-

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Table 1. Ionisable groups in biopolymers susceptible to participating in metal binding [40] Group

Location

pKa

Carbohydrate as an ester. Tyrosine Sulphonic acid Cysteic acid Carboxyl Sialic acid Carboxyl Protein C-terminal Carboxyl Beta Aspartic Carboxyl Gamma Glutamic Carboxyl Uronic acid Carboxyl Lactate (Muramic acid) Phosphate Diester-Nucleic acid Phosphate Sugar or polyol monoester Phosphate Serine as an ester Sulfhydril Cysteine Hydroxyl Phenolic tyrosine Hydroxyl Serine. Threonine-alcohol Hydroxyl Saccharide-alcohol Amino Guanosine (Purine) Amino Adenosine (Purine) Amino Cytidine (Pyramidine) Hexosamine-ManNH2, Amino GalNH, GlcNH2 Amino Protein N-terminal Amino ε-Lisine Imidazole-NH Guanosine (Purine) Imidazole Histidine Lactam-NH Adenosine (Purine) Lactam-NH Uridine (Pyramidine) Lactam-NH Guanosine (Purine) Lactam-NH Thymidine (Pyramidine) Guanidine-NH2 Arginine Imino Peptide

< −0.5

Sulphate

1.3 2.6 3.5 ~ 4.0 4.0 ~ 5.0 4.0 ~ 5.0 3.0 ~ 4.4 3.8 1.5 0.9 ~ 2.1; 6.1 ~ 6.8 6.8 10.0 12.3 ~ 13.0 > 13 > 13 1.6 3.4 4.1 7.3; 7.7; 7.8 7.5 ~ 8.0 10.0 2.3 6.0 ~ 7.0 3.6 9.2 9.2 9.8 12.0 13.0

heterocyclic compounds and, finally, in the sulphur of the thiol and thioether groups of the cysteine and methionine amino acids, respectively. In 2007, B. Volesky [8] presented a personal overview of biosorption, focusing on R&D reasoning and know-how that is not normally published in the scientific literature. In 2009, G. M. Gadd [9] critically reviewed aspects of biosorption research regarding the benefits, disadvantages, and future potential of biosorption as an industrial process, the rationale, scope and scientific value of biosorption research, and the significance of biosorption in other waste treatment processes and in the environment. 1.2. Biopolymers from industrial wastes: active agents in the biosorption of metals Human productive activity generates residues and, through a growing social demand, these residues are now increasing

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Biotechnology and Bioprocess Engineering 16: 1262-1272 (2011)

in volume to such an extent that they have become a severe risk to the sustainable balance of the environment, with unforeseeable consequences for human life. It is thus necessary to promote the re-use of residues as secondary raw materials for new technological processes and, within the bounds of the technology available, to foster clean production. Over the past few years, the number of studies devoted to examining the metal-decontaminating properties of a large variety of industrial wastes has significantly increased. In 1995 and 1998, Volesky and co-authors reviewed the findings reported in the eighties and the beginning of the nineties within this field [10,11]. In these studies, the authors mentioned certain natural biomaterials, such as the algae of the genera Ascophyllum or Sargassum, or the mycelia of certain industrial fungi, such as Rhizopus and Absidia, comparing them in terms of their ability to remove metals (such as lead, cadmium, copper, zinc, and uranium) from water. Only in the past decade has it become possible to glimpse the potential of metal biosorption by different types of biomass. From the economic point of view, owing to their lower cost, biomasses (biopolymers) that are abundant and that are generated as residual sub-products of large-scale industrial processes (fermentations, the meat, sugar, and paper industries, etc) are of particular interest. These biomasses may serve as a basis for new processes for metal biosorption, considered to be highly competitive in the decontamination of metal-polluted industrial effluents. Adsorption isotherms, obtained from experiments on batch adsorption equilibria, should be used to assess the capacity of different bioadsorbents to eliminate metals. In this sense, elucidation of the mechanisms that are active in the biosorption of metals is crucial if the phenomenon is to be exploited to its full extent, and also for the regeneration of bioadsorbent materials in multiple cycles of re-use. The complex nature of these bioadsorbents makes investigation in this field extremely promising, although very difficult to implement. Recent reviews have provided a selective overview of past achievements and of the present scenario of biosorption studies carried out on certain promising natural biosorbents (algae, fungi, bacteria, and yeast) and some waste materials that could serve as an economical means of treating effluents charged with toxic metallic ions [12,13]. There are undoubtedly clear differences between the biosorption yields obtained with synthetic waters and with residual waters, since the latter not only contain a significantly greater content and variety of metal ions but also a considerable diversity of products in solution, such as calcium, magnesium or other divalent cations, responsible for the complexation phenomena with the bioadsorbent. In

some cases, a simple increase in the capacity of the bioreactor (batch, packed-bed) will suffice to solve the problem, although this is not always the case because there will be a strong dependence on the type of water being treated. Moreover, it should be remembered that waste waters are often not completely characterised before being subjected to the action of the biomass, such that operating conditions will vary considerably and will always be a function of the physico-chemical characteristics of the waste water. Among the natural bioadsorbents most studied (owing to their abundance, economic aspects and efficiency), are the different biopolymers, such as alginates, cellulose and derivatives, pectins and the components of fermentation yeasts. 1.3. Alginates Several studies have addressed the adsorption capacity of alginates and of other components of algae [14]. Alginates function as a mixture of ion exchange resins, and they exhibit processes of anion-cation exchange and of adsorption. In many cases, binding to certain metals is a direct function of the pH, or the presence of competing ligands or of ions [15]. Aderhold et al. (1996) [16] studied the capacity of Pheophyte algae such as Ecklonia maxima, Lessonia flavicans and Durvillea potatorum to eliminate copper, nickel, zinc, and cadmium. In these experiments, the authors found it essential to determine a relationship between the percentages of adsorption in the case of synthetic waters (with a single metal), and in waste water that was strongly polluted with several metals and hence susceptible to undergoing competition phenomena. In most cases studied, pH values of around 4 have been established as appropriate for the adsorption of most metal cations. However, it has also been found that the removal of certain metals, such as Au (III), Ag (I) and Hg (II), is not significantly affected by the pH [17]. In comparative studies performed on the adsorption of Cu, Cd, and Zn by cells of Ascophyllum nodosum, De Carvalho et al. (1994) [18] have shown that, in algae, alginates are not the final agents responsible for adsorption processes. 1.4. Cellulose and derivatives Shukla and Sakhardande (1991) [19] conducted studies on the adsorption of different metal cations (Fe2+, Fe3+, Pb2+, and Hg2+) on cellulosic materials such as cotton fibres, blanched bamboo pulp, jute fibres and sawdust, in some cases observing biosorption capacities of up to 96%. Other abundant and inexpensive agricultural cellulosic materials are able to remove fairly large amounts of metal ions from aqueous solutions. Thus, Eromosele and


Otitolaye (1994) [20] used the husks of seeds from the wild plant Butyrospermun parkii, available in large amounts as a sub-product that is normally discarded. They found that this material was able to bioadsorb iron, zinc and lead ions from aqueous solutions. Also, owing to their high cellulose content, almond husks (a sub-product of the almond industry), could find application in the removal of toxic metals from industrial wastewaters. Cellulose is insoluble in most solvents but can be dissolved if the hydroxyl groups are blocked as adducts of carbon sulphide. The remaining functional group is called xanthate; xanthates have also been studied as products readily obtainable from industrial wastes such as sawdust, maize pulp, straw, and cellulose filter cartridges. Considering this, Holland (1975) [21] observed that the capacity to eliminate metals varied as a function of the number of hydroxyl groups blocked. The same author also reported that complications arose, such as the strong smell produced by the decomposition of xanthates, the large amount of washing water required, etc., and concluded that it was not practically viable to develop these processes on a large scale. 1.5. Pectins Pectins are heteropolysaccharides that are found as cementing substances in cell walls and in the intercellular layers of all terrestrial plants, among them sugar beet. Chemically, they are formed by polymers of galacturonic acid, partially or wholly esterified with methanol. The completely demethylated polymer is called pectic acid and its salts are called pectates. Sugar beet pulp is an abundant and inexpensive residue from the sugar industry and (owing to its content in pectins and due to the processes of ion exchange, adsorption and chelation), is able to trap heavy metals from water (Cu2+ efficiency is similar to that for Pb2+ and both are more efficient than for Cd2+, which is similar to Zn2+, Ni2+, and Ca2+) [22]. Likewise, after a process of acylation by simple alkaline hydrolysis, the pectin extracted from sugar beet pulp accumulates Cd2+ as efficiently as alginate, and it can be eluted with 0.3 M calcium chloride [23]. 1.6. Yeasts As sub-products of the fermentation industry (brewing, antibiotic production, etc), yeast residues show considerable affinity for metal ions in solution (Zn, Cd, Ni, Pb, Cr, and Ag). This affinity is pH-dependent and such residues can thus be used in biosorption processes for the decontamination of waters polluted with metals [24]. The use of fungal biomass for bioremediation studies is based on the capacity of the cell walls of these microorganisms (of which the major components are the poly-

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saccharides chitin and α (1, 3) β-D-glucan) to biosorb heavy metals regardless of the internal processes occurring inside the cell. That is, it is possible to commence with a dead biomass without altering the properties of the process, and hence take advantage of its greater ease of use. Common processes involve ion exchange and the adsorption capacity can be manipulated via a series of physical and chemical mechanisms that take place at the cell surface. This biomass can be used in packed-bed or continuousflow bioreactors [25]. In studies carried out using IR spectroscopy, Guibal et al. [26] have reported the binding of the uranyl cation to the amino groups of the chitosan of the cell wall of several biomasses (Aspergillus niger, Penicillium chrysogenum, and Mucor miehei) typical of certain industrial fermentation processes. This binding of uranyl to the amino sites confers them a structure similar to that of amide groups, such that the binding of the metal elicits changes in the relative intensities of the amine or amide spectral bands. The complexing of uranyl by amino ligands is governed by pH. Thus, Tsezos and Volesky [27] described some of the properties of the binding of uranium to Rhizopus arrhizus, reporting that the processes of uranyl adsorption to the chitin walls of the fungus occur very rapidly, while the precipitation of uranyl hydroxide inside the structure of the cell wall only occurs minimally. They also stated that processes of interference by other ions such as iron and zinc occurred. In 1995, Simmons et al. [28] conducted a study addressing the metal-binding capacity of brewer’s yeast (Saccharomyces cerevisiae) (detailed below) and of Candida albicans, both obtained as industrial wastes. Regarding the elimination of metals (copper, silver) from water, these authors found noteworthy differences among different strains of S. cerevisiae (with different structures in their cell walls) and between the latter microorganism and Candida albicans. According to Huang et al. [29], the adsorption of Cu (II) by S. cerevisiae is strongly affected by pH. When these authors used selective ion electrodes, the release of large amounts of copper-complexing agents (possibly proteins and carbohydrates) was observed. In contrast, these agents did not show the same type of behaviour with silver. Brady and Duncan [30] studied the biosorption properties of Saccharomyces in greater depth, concluding that Saccharomyces is a high-quality bioadsorbent agent at pH between 5 and 9. Simmons and Singleton [31] studied the capacity of Saccharomyces to biosorb silver as a function of the age of the biomass. They concluded that older strains had a lower metal elimination capacity than younger strains and that this was mainly due to a decrease in the number of silver complexes inside the cell. It is

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therefore clear that in these processes it is not only the outer cell wall of the fungus that is involved but also its interior, where there are agents that affect the efficiency of the elimination process. This is also why a stronger elimination has been observed when using biomass formed by intact cells than when only the cell wall is used. The most recent findings in the field of biosorption of heavy metals by S. cerevisiae have been reviewed by Wang and Chen [32].

sound device, Centrolit “Selecta P” and Centromix centrifuges, Biohit Proline automatic pipettes, Millex-Gs Sterile 0.22 µm Millipore filters, a Moulinex blender, Mettler Toledo AB204 and Precisa 600C balances, a WTB Binder drying oven, tank and glass and methacrylate column reactors, GILSON Minipuls 2 and Ismatec peristaltic pumps, a conductimeter, a turbidometer, and a thermometer (WTW Profilab multiparametric probe), a GEM Biochemical Inc. Bacterial Systems “BG-1” ecotoxicity device, a UV-Visible Hitachi 150-20 spectrophotometer, an ICP (Emission Spectroscopy by Induction-Coupled Plasma) spectrophotometer for quantitative analysis of metals and other chemical elements (Central Analysis Services of the University of Salamanca), and an atomic absorption spectrophotometer (facilitated by the Department of Analytical Chemistry, Nutrition and Food Sciences of the University of Salamanca). In the biosorption experiments, this biomass was used (a) in free form, with no modification (i.e., suspending it directly in the waters contaminated with metals and then separating these by centrifugation, and (b) after it had been physically immobilised in synthetic reticulated polyurethane foam polymer. The immobilisation of S. cerevisiae cells was accomplished by physically trapping them in polyurethane reticulated foam, the microorganisms becoming physically embedded in the matrix of the strongly cross-linked polymer [33,34]. This matrix was synthesised from a hydrophilic polyisocyanate Hypol, 2002, supplied by Grace Service Chemicals (Heidelberg, Germany). The experimental procedure used to immobilise the cells consisted of 1) dissolving 1.00 g of lyophilised yeast in 40.0 cm3 of NaCl solution (8.5 g/dm3), determining the protein content (mg protein/cm3) by the Lowry method; (2) slowly mixing, with continuous stirring, the 40.0 cm3 of S. cerevisiae with 20.0 g of the prepolymer Hypol 2002; and (3) leaving the mixture to stand for 10 min; (4) cutting the polyurethane reticulated foam with cells trapped inside into small strips or cubes, conserving these in the NaCl solution (8.5 g/dm3) at 4ºC. The synthesis of the polyurethane reticulated foam was achieved under the same conditions and following the same procedure as described above, except that in this case the mixture was made up with 40.9 cm3 of NaCl (8.5 g/ dm3) plus 20.0 g of Hypol 2002. This plastic matrix, devoid of the microorganism, served as a control in all the experiments with a view to assessing the possible adsorption of metals onto the polyurethane polymer matrix. The physico-chemical parameters determined (some in situ and others at the laboratory) in the mine drainage were temperature, pH, specific conductivity, dissolved oxygen, turbidity, chemical oxygen demand (COD), and biochemical oxygen demand (BOD), following the usual methods

2. Materials and Methods Several wet samples of Saccharomyces cerevisiae were donated by the Cruz Campo brewery industry (Seville, Spain). These exhausted yeasts were collected from the industrial fermentors of the company and were kept under refrigeration at 4ºC. After samples of the water from the mine cuts had been taken, they were transported to the lab and stored at room temperature in PVC recipients, previously washed with 2 N hydrochloric acid. The samples were sourced from the following mines: (1) the Feli or Alabancos mine in the mining district of Barruecopardo-La Fregeneda (La Fregeneda, Salamanca), with the minerals casiterite (SnO2), wolframite [(Fe,Mn)WO4], arsenopyrite (FeAsS), pyrite (FeS2), and lepidolite [K2Li3Al4Si7O21(OH,F)3]; (2) the Merladet mine in the mining district of Barruecopardo-La Fregeneda (Barruecopardo, Salamanca), with the minerals scheelite (CaWO4), wolframite, arsenopyrite, and pyrite; (3) the Terrubias mine in the mining district of MorilleMartinamor (San Pedro de Rozados, Salamanca), with the minerals scheelite, wolframite, and casiterite; (4) the Alegría mine in the mining district of Morille-Martinamor (Morille, Salamanca), with the minerals scheelite, wolframite and casiterite; (5) the Dorinda mine in the mining district of Granito de Ricobayo (Villalcampo, Zamora), with the mineral casiterite; (6) the ENUSA Faith mine in the mining district of Ciudad Rodrigo (Saelices El Chico, Salamanca), with the minerals pitchblende [Complex], pyrite and marcasite (FeS2). The reagents of sulphuric acid, 35% hydrochloric acid (Panreac, Barcelona, Spain), sodium hydroxide, hexahydrated uranyl nitrate (UO2(NO3)2·6H2O) (Merck, Darmstadt, Germany), arsenazo III, powdered lead (Fluka Chimie Gmbh, Buchs, Switzerland), Triton X-100 (Sigma Chem Co), Hypl 2002 (Grace Service Chemicals Gmbh, Heidelberg, Germany) and Milli-Q ultra-pure water (Millipore Spain, Madrid) were obtained from the sources indicated. The instruments and apparatus used in the present work were as follows: a BUNSEN MC-8 magnetic stirrers, a CRISON micropH 2001 pH-meter, a Branson 2200 ultra-


(Standard Methods) for the analysis of waters and waste waters (APHA, AWWA, WPCF) [35]. Qualitative chemical analysis of the waters was accomplished by obtaining dry residues from the samples (dried in an oven at 98ºC) or by progressively increasing the pH of the waters to produce a selective chemical precipitation. The dry residues and precipitates obtained were subjected to chemical analysis by X-ray dispersive energy (EDAX). Quantitative chemical analyses of the water samples were carried out using ICP (Emission Spectroscopy by Induction-Coupled Plasma) spectroscopy at the Central Analysis Services of the University of Salamanca. With a view to checking the possible radioactivity of some water samples, replicate determinations were made at the Environmental Radioactivity Laboratory of the University of Salamanca of the α and β radioactivities of acid water samples from cut 1 of the ENUSA Faith uranium mine. Standard methods (APHA, AWWA, WPCF) [35], improved by Moron et al. [36] and Sill [37], were used. According to the bioluminescence test employing Photobacterium phosphoreum (Ames test), a liquid sample is considered to be toxic when an inhibition of the luminescence of the bacteria greater than 20% occurs (toxicity threshold) in an analysis carried out at 15ºC for 15 min. Quantification of the toxicity of the samples is carried out according to the EC50 (maximum effective concentration: the dilution value of the sample for which 50% inhibition of luminescence occurs) and according to the ANFORT T-90-320 directive. A dual-channel merge-point flow injection (FIA) system was used. The carrier was a stream of 3.6 M HCl in the presence of 1% Triton X-100. The flow rate of the carrier and reagent streams was 2.0 cm3/min. Analysis of the uranyl cation in the acid mine drainage was accomplished using a rapid and sensitive method [38] that avoids manipulation in order to achieve the reduction of U(VI) to U(IV) upon inserting a lead- reducing minicolumn in the system [39]. Standard solutions and samples were introduced in the FIA system by means of an injection valve (100 µL), after which the uranium (VI) solution was passed through the lead-reducing column inserted in the sample entry channel (CR, 2 mm inner diameter and 5.5 cm length), eliciting the reduction of uranium (VI) and hence avoiding contact with atmospheric oxygen. The sample bolus was then merged with the reagent stream and during its passage through the helicoidal reactor (1 m), the U(IV)- Arsenazo (III) complex was formed, its absorbance being recorded at a wavelength of 650 nm. The peak height obtained for each of the solutions injected is directly related to the amount of sample injected and to the concentration of uranium in the sample.

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The kinetic experiments on biosorption were performed at 20ºC by incubation with the biomass (25 g) in a stirredtank reactor (120 rpm) of 5 aqueous solutions of 10 dm3 volume, each with different concentrations of a single metal (between 114 and 0.6 mg/dm3). Water samples (aliquots of 10 cm3) were taken at preset times between 0 and 24 h, separating the yeast by centrifugation and stabilising each supernatant metal solution by adding a further 10 cm3 of 0.01 M H2SO4. The equilibrium experiments (biosorption isotherms) were carried out in batch mode in the presence of a constant concentration of biomass. Equilibrium was considered to have been reached at 20ºC after an incubation period of 1 h, after which a sample of the aqueous solution was removed to measure the concentration of the metal. Analysis of the metals in these acid supernatant solutions was accomplished by flame atomic absorption spectrophotometry, with the exception of Al, which was analysed by UV-Vis spectrophotometry, following the eriochromocyanine-R method and the analysis was performed in continuous mode by flow injection analysis (FIA). The fitting of the kinetic and equilibrium data on biosorption to different equations and models was carried out using the SIMFIT statistical package, authored by Prof. W. G. Bardsley of the University of Manchester (UK).

3. Results and Discussion 3.1. Physico-chemical, ecotoxicological, and chemical characterizations of the AMDs Since a primary goal of this study was to identify and characterise acid mine waters in the Duero basin, and after carrying out a screening of the water samples from six mines in the provinces of Salamanca and Zamora, we observed that the waters from the cuts of the Merladet and Faith mines were the only acid and ecotoxic waters with which to perform later studies on bioremediation (Table 2). In order to determine the chemical composition of these acid waters, we carried out quantitative elemental chemical analyses by ICP emission spectroscopy; the concentrations (mg/dm3) were as follows: Drainage Faith: 1502 (S), 782 (Mg), 268 (Ca), 35.0 (Na), 6.36 (K), 0.63 (Li), 12.1 (Si), 114 (Mn), 89.4 (Al), 6.26 (Fe), 7.12 (Zn), 0.54 (Cu), 4.68 (Ni), 42 (UO22+) Drainage Merladet: 177 (S), 38.6 (Mg), 119 (Ca), 25.3 (Na), 3.97 (K), 0.19 (Li), 28.6 (Si), 3.10 (Mn), 8.76 (Al), 2.39 (Fe), 3.6 (Zn), 0.50 (Cu), 0.50 (Ni) One of the main observations of the present study was a tight correlation between acid pH and the concentration of

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Table 2. Physico-chemical parameters of the different mine drainages evaluated O2(ac) (mg/L)

Mine

T (oC)

pH

Merladet Faith Feli Terrubias Alegria Dorinda

12.9 9.0 20.6 11.6 11.8 11.2

3.16 3.1 6.8 6.45 6.45 6.9

, 12.2 1,001 , 12.5 ,

8.8

k (mS/cm) 0.91 6.4

Turbidity (nephelu)

Acidity (mg CaCO3/L)

Ecotoxicity (equitos/m3)

Radioactivity (Bq/L)

0.91 15

96.0 100.0

14.6 69.2

0 978 ± 11 0 0 0 0

349 60.2

11.3

DQO and DB05 were zero for all waters; i.e., no organic pollution.

metals in the waters analysed. Thus, the acid pH waters from the cuts (the Merladet and Faith mines) were those showing the highest levels of metals and sulphates in solution (data not shown) and were the only mine waters analysed that gave positive results in the Ames test. The metals mainly present in these waters were aluminium, manganese, iron, nickel, zinc, and copper, the acid water from the Faith mine being significantly more polluted (1,870 mg/dm3 of inorganic chemical load) than that of the Merladet mine (411 mg/dm3 of chemical load) and the former also having a differentiating character: namely, the presence of the uranyl UO22+ cation (42 mg/dm3). Moreover, we found that the waters analysed showed a certain degree of mineralisation (a fairly low specific conductivity), low turbidity, and a high degree of oxygenation.

Fig. 1. Biosorption kinetics of Cu from synthetic aqueous acid solutions in contact with Saccharomyces sp. at different initial concentrations of the metal.

3.2. Biosorption of metals from synthetic acid waters In view of the abundance and low cost of exhausted brewer’s yeasts, a biomass of S. cerevisiae was chosen as the main candidate to assess the possible bioremediation of the acid mine waters studied. Since Al, Cu, Mn, Ni, and Zn were the major metals, in the first experiments we attempted to study the selectivity, kinetics, and biosorption equilibrium of each of these five metals dissolved in water at acid pH (3.3 ~ 4.5) by exhausted Brewer’s yeast. The results of this part of the study are shown in Figs. 1

(for the case of Cu as an example) and 2 and in Table 3. The following conclusions may be drawn: (1) Regarding the selectivity of the biomass for the different metals and their capacity to be biosorbed (Table 3), the highest percentages of elimination were obtained for Zn (76%), followed by Cu (70%), Mn (62%), Ni (48%), and Al (30%). (2) The removal kinetics of each metal dissolved in synthetic water by biosorption onto Saccharomyces sp.

Table 3. Maximum percentages of metal biosorbed from acid aqueous solutions of a single metal in batch incubation with Saccharomyces sp. at different initial concentrations of the metal Cu Co

Mn %

(mg/dm3)

84 17 7 2 0.8

53 45 47 70 42

Co

Ni %

(mg/dm3)

11 19 5 2 0.6

*Samples contaminated with Cladosporium sp.

43 61 48 62 61

Co

Zn %

(mg/dm3)

114 15 6 1.5 0.6

40 44 48 43 36

Co

Al %

(mg/dm3)

107 24 6 2 0.8

76 71 66 64 58

Co

% (mg/dm3)

84 17 7 1.3 0.7

100* 100* 30 100* 26


Fig. 2. Biosorption isotherms of the metals from synthetic aqueous acid solutions by the action of Saccharomyces sp. in free suspension.

were of first order with respect to the concentration of the metal ([Metal]solution= Ae−kt + C), equilibrium being reached in the first 15 ~ 20 min. This indicates a rapid process of surface adsorption of the metal onto the biomass. In the case of the biosorption of Cu, k = 0.139/min; that is, 50% of the biosorption of the metal onto the yeast occurred in the first five minutes of the process (t1/2). (3) The isotherms of the biosorption of the metals onto the biomass (Fig. 2) seemed to follow Langmuir type behaviour [Metaladsorbed/adsorbent mass = Kb[Metal]equilibrium/ (1 + K[Metal]equilibrium)]. The affinity constants for metalbiosorbent binding were: K = 7.29 (Zn); 3.65 (Cu); 3.31 (Mn); and 3.20 (Ni) dm3/mmol and the binding capacity constants were: b = 0.076 (Zn); 0.069 (Cu); 0.060 (Mn), and 0.059 (Ni) mmol/g. We propose that mainly specific physico-chemical interactions (ion-exchange, coordination, metal complexing) would be involved in the biosorption process, although more physical and labile and less specific interactions (hydrophobic effect, van der Waals forces) between the metal ion and the different functional groups on the surface of the microorganisms may also participate, albeit to a lesser extent. (4) During the experiments on the biosorption of the dissolved metals by the yeasts, the pH of the waters increased spontaneously from its initial values (pH 3.3 ~ 4.5) up to values around 5.5 ~ 6.0 (data not shown). That is, the process of biosorption of the metal onto the yeast partially neutralized the acid aqueous solutions. From the titration curve (pH versus NaOH added) of the biomass dissolved in water (data not shown) the existence in the biomass of at least four acid-base chemical groups can be deduced, with apparent pK values of 3.7, 4.5, 6.8, and 9.5. Interestingly, in the experiments involving the bio-

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sorption of aluminium onto the yeast, we observed that the supernatants of some aliquot samples collected from the bioreactor, after filtering and acidifying with 0.01 M H2SO4, were contaminated with a microorganism that later proved to be the filamentous fungus Cladosporium sp. Accordingly, the results corresponding to the elimination of Al by S. cerevisiae would have been artefacts. However, this could be considered a positive finding because the sample of yeast contaminated with Cladosporium sp. eliminated percentages of around 70% of the Al present in the waters. This novel observation (hitherto unreported in the literature) could serve as a basis for future studies on the bioremediation of acid waters with heavy metals by Cladosporium, which additionally grew spontaneously and at high rates in waters with a pH of 2.0 ~ 3.5. 3.3. Biosorption of metals from acid mine drainages Having determined the selectivity, kinetics and biosorption capacity of each metal dissolved in acid synthetic water through the action of S. cerevisiae, in the next part of the study our aim was to check these parameters for the biosorption processes of metals contaminating the real acid waters from the Merladet mine (Barruecopardo, Salamanca) through the action of brewer’s yeast. The experiments were the same as those conducted in the previous study, maintaining the same conditions and using the same type of sample analysis. In view of the low metallogenic contamination of the acid waters from the main cut of the Merladet mine (3.6 mg/dm3 Zn; 3.1 mg/dm3 Mn; 0.5 mg/dm3 Cu; and 0.6 mg/ dm3 Ni), the water samples were spiked, each one separately, with each metal to be studied in terms of its biosorption capacity until seven different concentrations between 118 and 0.5 mg/dm3 were obtained. Although the analysis and follow-up were performed for only one metal at a time, the

Fig. 3. Langmuir-type adsorption isotherm of Cu from the acid drainage of the Merladet mine on Saccharomyces cerevisiae in free suspension.

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Table 4. Maximum percentages of metal biosorbed from the acid drainage of the Merladet mine by free Saccharomyces sp. at different initial concentrations of the metal Cu Co

Mn %

Co

(mg/dm3) 94 55 30 7 1.8 0.9 0.5

Ni %

Co

(mg/dm3) 12 5 16 39 47 77 > 80

110 67 33 11 5 4 3

Zn %

Co

(mg/dm3) 2.4 38 37 3.5 0.2 2.7 3.2

remaining metals, together with other chemical species that might interfere or compete in the actual biosorption process by the yeast, were also present in the waters from the cut. The results of this part of the study are shown in Fig. 3 (for the case of Cu, as an example) and in Table 4. The following conclusions can be drawn: (1) Regarding the selectivity and biosorption capacity of the different metals by the biomass, the highest percentages of elimination corresponded to Cu (80% elimination; that is, 0.19 mg Cu/g biosorbent), followed by Ni (25%; 0.24 mg Ni/g biosorbent) and Mn (3%; 0.026 mg Mn/g biosorbent). (2) The biosorption kinetics of the dissolved metals in acid mine water by S. cerevisiae was rapid and was of first order with respect to the concentration of the metal ([Metal]solution= Ae−kt + C), and equilibrium was reached in the first 10 ~ 15 min (data not shown), suggesting a rapid phase of surface adsorption of the metal onto the biomass. (3) The adsorption isotherms of Cu (Fig. 3) and of the other metals (data not shown) onto the biomass seemed to follow Langmuir type behaviour [Metaladsorbed/adsorbent mass = Kb[Metal]equilibrium/(1 + K[Metal]equilibrium)]. The affinity constant for Cu-biosorbent binding was K = 2.46 dm3/mmol and the binding capacity constant was b = 0.1 mmol/g. As with synthetic acid waters, we propose that the biosorption process mainly involves specific physico-chemical interactions (ion-exchange, coordination, metal complexing), although more physical and labile and less specific interactions (hydrophobic effect, polar, van der Waals) between the metal cation and the different functional groups on the surface of the microorganism could also participate, albeit to a lesser extent. (4) During the experiments involving the incubation of the acid mine cut water with the yeast, the pH of the waters increased spontaneously from its initial values (pH 3.3) up to values of around 4.0 ~ 4.6 (data not shown). That is, the

87 61 36 9 2.6 1 0.5

% (mg/dm3)

42 16 44 9 11 16 40

118 70 45 12 5 4 3

12 7 0 0 0 0 0

process of the biosorption of the metal onto the yeast partially neutralised the acid mine waters. (5) On comparing the process of the biosorption of metals by S. cerevisiae in synthetic waters and in waters from the Merladet mine, very similar values were seen regarding the elimination of Cu and Ni, although the efficiency of the process with real water samples was markedly lower for the case of Mn, and zero for the other metals. We propose an antagonistic interference on the biosorption of a metal due to the presence of other metals. 3.4. Removal of ecotoxicity from acid mine drainage by immobilised yeast The experiments were carried out in discontinuous mode in a stirred tank reactor at 120 rpm and at a temperature of 20ºC with acid water from the Merladet mine (Barruecopardo, Salamanca) (0.06 mg/dm3 Al; 0.06 mg/dm3 Ni; 0.56 mg/ dm3 Cu, 1.91 mg/dm3 Zn, and 2.91 mg/dm3 Mn), treated using the official standard Spanish method (B.O.E. 10/11/ 1989) for ecotoxicity analyses. The results of the study of the evolution of the ecotoxicity of two different bioleachate samples (200 cm3 each) from the Merladet mine after their incubation with 11.4 g of polyurethane support, empty or loaded with 2.3 g of immobilized S. cerevisiae cells, were as follows: the EC50 ecotoxicity values decreased from 14.6 and 8.20 to undetectable levels. The conclusion from this study is that the ecotoxicity of the acid mine drainage was removed when it was incubated with brewer’s yeast trapped in polyurethane foam.

4. Conclusion According to their chemical composition and the physicochemical and ecological states, only the acid waters from the Faith (polluted with U, Al, Cu, Mn, etc) and Merladet (with Al, Mn, Zn, and Cu) mines are contaminated with toxic heavy metals.


Following the kinetics and the equilibrium of elimination of metals from the Merladet acid drainage by biosorption processes on brewer’s yeast, it may be proposed that these abundant industrial residues could serve as efficient agents for the bioremediation of this type of metal-polluted water. Furthermore, these biowastes proved to be efficient in abolishing the levels of ecotoxicity of such acid mine drainage.

8. 9. 10. 11. 12.

Acknowledgements The authors wish to thank the Iberdrola Instituto Tecnológico and the Centro de Investigación y Desarrollo Tecnológico del Agua (CIDTA) of the University of Salamanca for funding. They also acknowledge the excellent collaboration of the Director and staff at the CIDTA for allowing the use of the equipment and installations to develop some parts of this work; the Department of Analytical Chemistry, Nutrition and Food Science of the University of Salamanca for allowing the use of atomic absorption spectrophotometers; the collaboration of the companies that provided the acid waters and biowastes used in this work - Empresa Nacional del Uranio S. A. (ENUSA) (Saelices el Chico, Salamanca), Merladet S.A. (Barruecopardo, Salamanca), Grupo Cruz Campo S. A. Finally, the authors thank Prof. William G. Bardsley from the University of Manchester (UK) for free use of the last version of SIMFIT.

13. 14. 15.

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