Compost as a source of microbial isolates for the bioremediation of heavy metals: In vitro selection

Science of the Total Environment 431 (2012) 62–67

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Compost as a source of microbial isolates for the bioremediation of heavy metals: In vitro selection María del Carmen Vargas-García ⁎, María José López, Francisca Suárez-Estrella, Joaquín Moreno Department of Applied Biology, Engineering Higher School, University of Almería-International Excellence Campus in Agri-Food, CeiA3, 04120 Almería, Spain

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Article history: Received 26 January 2012 Received in revised form 3 May 2012 Accepted 3 May 2012 Available online 3 June 2012 Keywords: Heavy metal Bioremediation Biosorption Intracellular accumulation

a b s t r a c t Heavy metal pollution has become a major environmental concern nowadays and the bioremediation of polluted habitats is an increasingly popular strategy due to both its efficiency and safety. A screening and selection protocol based on different composting processes was designed in order to isolate heavy metal-resistant microorganisms. A collection of 51 microorganisms was obtained and most of them showed the capability to tolerate heavy metals in multi-polluted aqueous systems (Cd(II), Cr(VI), Ni, Pb, Zn(II)), as well as to remove them. The highest detoxification ratios were observed for Pb. Some of the isolates detoxifying more than a 90% of this metal, while the other metals were removed in a range between 20% and 60%. The best isolates (Graphium putredinis, Fusarium solani, Fusarium sp. and Penicillium chrysogenum) were further assayed in order to determine the predominant removal mechanism and the potential use of their dead biomass as a biosorbent. Intracellular accumulation was the prevalent mechanism for most isolates and metals, with the exception of Ni. In this case, the proportion removed by extracellular adsorption was similar or even higher than that removed by intracellular accumulation. Thus, the efficiency of living cells was higher than that of dead biomass except in the case of Ni. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Heavy metal (HM) pollution is nowadays one of the most important environmental concerns. Despite the essential role that some of these elements play in biological functions (Nagajyoti et al., 2010), all of them have the potential to cause toxic effects on living organisms if they are taken in a higher proportion than demanded. Damages on human beings, fauna, flora and microbiota have been described (Berny et al., 2010; Beyersmann and Hartwig, 2008; Hinojosa et al., 2005; Nagajyoti et al., 2010; Soderland et al., 2010). Metallic elements can be naturally released from the Earth´s crust through weathering processes, volcanic eruptions, wood fires or wind-blown dusts (Balabanova et al., 2011; MacKenzie and Canil, 2008; Shcherbov et al., 2008). Nevertheless, anthropogenic activities such as agriculture, industry, combustion processes or domestic effluents are nowadays major contributors to heavy metal accumulation in the environment (Nagajyoti et al., 2010; Singh et al., 2010; Sinicropi et al., 2010). Since HMs cannot be chemically or biologically degraded (Pérez de Mora et al., 2006), the different alternatives for their control proposed over the last decades rely on their removal or containment. Ex situ and in situ strategies can be applied in both cases. Ex situ methods are usually quite expensive and cause structural and functional disturbances ⁎ Corresponding author at: Department of Applied Biology, CITE III-B, University of Almeria, 04120 Almería, Spain. Tel.: + 34 950 015892; fax: + 34 950 015476. E-mail addresses: [email protected] (M..C. Vargas-García), [email protected] (M.J. López), [email protected] (F. Suárez-Estrella), [email protected] (J. Moreno). 0048-9697/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2012.05.026

(Helmisaari et al., 2007), while in situ techniques have been described as time consuming and showing some uncertainty (Farrell et al., 2010). Among these in situ strategies, physical, chemical and biological methodologies can be applied. Although both physical and chemical processes are extensively applied, they have proved to be not completely satisfactory. On one hand, they are not completely effective in reducing HM toxicity, especially when the concentration of pollutants in materials is below 100 mg L − 1 (Wang and Chen, 2009) and, on the other hand, they show a high polluting potential on account of the use of some toxic reagents (Neagoe et al., 2009; Shi et al., 2009). On the contrary, biological reclamation of heavy metal-polluted environments is increasingly gaining attention since it is considered as a clean and non-environmentally disruptive method and an efficient strategy for the remediation of a wide range of HMs (Wei et al., 2004; Wu et al., 2010). Phyto and micro-remediation, the use of plants and microorganisms respectively, are the two major biological techniques for the removal or containment of HMs. Microbial actions on HMs availability include biosorption (metal sorption to cell surface by physico-chemical mechanisms), bioleaching (HM mobilization through the excretion of organic acids or methylation reactions), biomineralization (HM immobilization through the formation of insoluble sulfides or polymeric complexes), intracellular accumulation and enzyme-catalyzed transformation by redox processes (Lloyd, 2002). Biosorption seems to be the most common mechanism (Haferburg and Kothe, 2007) as it does not have any energetic requirements. In fact, when dead cells are used as bioremediation agents it is the only feasible process for them. On the contrary, living cells can act through different strategies, which positively affect the efficiency of

M..C. Vargas-García et al. / Science of the Total Environment 431 (2012) 62–67

the process, as well as the self-replenishment capacity of the cells (Malik, 2004). According to the above mentioned, the isolation of HM‐resistant microorganisms can be a good strategy for the treatment of the deleterious effect these pollutants exert. Different habitats have been proposed for the screening and isolation of microbial strains able to biotransform and/or bioaccumulate heavy metals. Among them: soil, tannery effluents from different industrial activities or compost have been considered (Anjana et al., 2007; Morales-Barrera and Cristiani-Urbina, 2008; Shukla et al., 2009). Composting is the aerobic transformation of organic residues through the action of a diverse microbial community. Most of the microorganisms involved show metabolic capabilities of industrial and environmental interest. With regard to HMs, different works have reported the positive effect that the composting process has on the bioavailability of these pollutant elements (Yu et al., 2009; Rihani et al., 2010). This decrease in the exchangeable fraction of HMs is due to both abiotic and biotic factors, since it is a consequence of the capability of some organic compounds, mainly humic substances, to bind metals and the ability of microbial cells to adsorb or uptake them (Barker and Bryson, 2002; Lister and Line, 2004). Thus, composting comes up not only as a suitable strategy for the treatment of HM-polluted materials, but also as the means to create an environment with a relatively high population of microorganisms which might be useful in HM bioremediation. According to what is mentioned above, the goal of this work was to verify the potential of different composting processes to create the adequate conditions for the growth of HM-resistant microorganisms. Therefore, material at different evolutionary stages might be considered as a source from which microbial strains can be isolated on the basis of their capability to remove HM from a multi-polluted solution. The predominant mechanisms for HM removal were established for the most efficient isolates. 2. Materials and methods 2.1. Composting processes Three composting processes were carried out using different raw materials: horticultural waste (HW), sewage sludge (SS) and municipal solid waste (MSW). Horticultural waste, composed of tomato plant residues lacking fruits, was collected from a municipal composting facility located in El Ejido (Southeast Spain) and sewage sludge was provided by the Almería Municipal Sewage Treatment Plant. Piles of 1.5 m 3 (1.5 m length × 1 m width × 1 m height) were disposed on a cement pad and prepared by mixing the wastes with milled almond shell in order to get an appropriate C:N ratio (25) in the following proportions (volume basis): Pile HW : HWð75% Þ þ Milled almond shell ð25% Þ Pile SS : SSð37:5% Þ þ HWð37:5% Þ þ Milled almond shell ð25% Þ Previous experiences of this research group showed the impossibility of composting sewage sludge on its own, making it necessary to add some structuring material. Proper quantities of the different wastes were weighed in an industrial weighing platform (FHI-TC/W 600 ± 0.2 kg, BACSA, Barcelona, Spain) and distributed in big bags. Piles were formed by the sequential adding of thin layers of the materials, just to guarantee the correct mixing and a homogeneous wetting. To ensure aeration, piles were treated with forced aeration (7.5–9.0 L kg− 1 every 4 h) and turned fortnightly. The moisture content was initially set between 40 and 50% and it was maintained by watering on turning days. Samples of MSW composting process were obtained from the Almería Municipal Composting Plant. Piles at this facility were constituted forming a 2.5 m high line and turnings were applied every week.

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Samples of each pile were obtained by mixing nine sub-samples extracted from the HW, SS and MSW piles. The sub-samples were taken from different sections resulting from the division of a pile in three vertical and three horizontal sections. Thus, materials representative from diverse microenvironments in each pile were considered. This fresh material was used for the screening of HM-resistant microorganisms. Sampling was done at different stages of the composting process, according to thermal values prevailing in the piles: -

Phase 1: at the start of the composting process (0 days). Phase 2: at the thermophilic stage (2 days; 7 days for MSW piles). Phase 3: at the cooling stage (60 days; 30 days for MSW piles) Phase 4: at the middle of the curing stage (106 days; 85 days for MSW piles). - Phase 5: at the end of the process (185 days; 125 days for MSW piles).

2.2. HM-resistant microorganisms screening and selection Samples from the composting piles were processed in order to isolate HM-resistant microorganisms. A three-step selective pressure design was developed for this purpose using the fresh solid material, as well as two different aqueous extracts. These extracts were included in the assay to check the viability and survival capability of microorganisms in the specific conditions under which the composted material is treated so as to produce different commercial formats. Two processes were applied to obtain the extracts: a short-time treatment, in which the composted solid material was mixed with water in 1:2 ratio, and shaken for 24 h at 40 °C and 100 rpm, and a longtime treatment, in which the suspension of the composted material (fresh material:water in a ratio 1:2), was maintained at 100 rpm and 25 °C for 48 h. For the isolation of HM-resistant microorganisms, the following selective procedure was carried out: 95 mL of Nutrient Broth media (peptone 5 g L − 1, meat extract 1 g L − 1, yeast extract 2 g L − 1, NaCl 5 g L − 1) were put in 250 mL Erlenmeyer flasks. The medium was supplemented with the necessary concentration of compound containing the metal up to 50 mg L − 1 of each one: Cd (II), Cr(VI), Ni, Pb and Zn(II), using analytical grade Cd(NO3)·4H2O, Na2CrO4, Ni(NO3)2·4H2O, Pb(NO3)2 and Zn(NO3)2·6H2O, respectively. 5 g of composted material (5 mL in the case of the extracts) were added to the sterilized media of and incubated for 7 days at 30 °C and 80 rpm. Subsequent transfers to fresh media were carried out to ensure the selective pressure needed for the isolation of HM-resistant microorganisms. Media and incubation conditions were the same as those previously described. After three transfers, the HM-tolerant microorganisms in the liquid media were isolated in the same HM-modified solid media (Nutrient Agar supplemented with the selected heavy metals). Plates were incubated for 3 days at 30 °C. Morphotypes grown in plates were isolated and maintained in HM-modified Nutrient Agar slants at 4 °C, as well as kept by freeze-drying. All the isolates were assayed in triplicate in relation to their capability to remove HMs in multi-polluted aqueous solutions. Three replicates 250 mL Erlenmeyer flasks containing 50 mL of HM-supplemented Nutrient Agar were inoculated with 1 mL aliquots from a microbial suspension. The suspension was obtained by resuspending the biomass of an HM-supplemented Nutrient Agar slant, grown at 30 °C for 72 h, in 3 mL 0.9% sterile saline solution. Erlenmeyers were incubated for 5 days at 30 °C and 100 rpm, and then the remaining HM concentration in the biomass-free supernatant was measured. The microbial biomass was removed by centrifugation (5 min, 10000 rpm) and the supernatant was digested with 67% HNO3 (5 mL) and 30% H2O2 (1 mL) firstly at 90 °C for 30 min and then at 200 °C until no turbidity (Bloc Digest Unit 12, Selecta, Barcelona, Spain). Non-inoculated controls were processed in the same way. The remaining HM concentration was determined by atomic absorption spectrometry (ThermoElectron Corporation S Series

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AASpectrometer, MA, USA). The most efficient isolates were selected for further assays. 2.3. HM removal studies: accumulation/adsorption mechanisms Previously selected isolates were subjected to further studies to elucidate the dominant mechanisms by which microorganisms remove these inorganic pollutants. The conditions for media preparation and incubation were as described in Section 2.2, and the HM concentration was determined in the supernatant and the biomass using atomic absorption spectrometry. Both fractions were digested as previously described, although the biomass was washed with 1 N NaOH before the digestion. Since this treatment leads to the regeneration of the binding sites at the microbial surface, the estimation of the HM concentration in the biomass provides information regarding intracellular accumulation, while adsorption was calculated according to the equation:   ½HMad ¼ ½HMc – ½HMacc þ ½HMsp ; where [HM]ad is HM concentration removed by adsorption, [HM]c is the HM concentration at the control, [HM]acc is the intracellular accumulation and [HM]sp is the remaining HM concentration at the supernatant. A second assay was carried out in order to establish the potential of dead biomass to act as a biosorbent. Biomass from cultures grown in the previously described conditions was harvested by centrifugation and washed with distilled water three times. The pellet was dried and milled to a final particle size of 1 mm. Aliquots of 0.025 g of dried biomass were added to Erlenmeyer flasks containing 25 mL of multi-polluted HM solution (50 mg L − 1 of each Cd (II), Cr(VI), Ni, Pb and Zn(II)). Erlenmeyers were shaken (120 rpm) for 90 min at 30 °C and then the suspensions were centrifuged and filtered for biomass removal. HM concentration in the digested supernatant was measured as previously done. All the assays were performed in triplicate. 2.4. Molecular identification of selected isolates DNA from axenic cultures of the selected isolates was extracted using the Wizard Genomic DNA Purification kit (Promega Corporation, Madison, WI, USA). For the PCR amplifications, NS1-GC Forward and NS2 + 10 Reverse primers were used. Reactions were performed in 50 μL volumes using the ReadyMix Taq PCR Reaction Mix (SigmaAldrich, St. Louis, MO, USA) in a MyCycler thermal cycler (BioRad, Hercules, CA, USA) and following a protocol with a denaturation step at 94 °C for 5 min, followed by 35 amplification cycles (95 °C, 60 s; 52 °C, 60 s; 68 °C, 90 s) and a final extension step at 68 °C for 10 min. The amplification products were cleaned using the GenElute PCR Clean-Up kit (Sigma-Aldrich, St. Louis, MO, USA), sequenced and compared to the nucleotide database at GenBank by using BLAST (NCBI). The isolates were identified as Graphium putredinis (accession number AB007683), Penicillium chrysogenum (accesion number EU409812), Fusarium solani (accession numbers FJ613599 and EF397944) and Fusarium sp. (accession number EF397944).

horticultural waste process. Treatment of the samples promoted more differences. Thus, while 20 microorganisms were isolated from the extracts obtained applying the short treatment (24 h at 40 °C), only 14 were isolated from the long-treatment hydrolysates (48 h at room temperature). Regarding sample time, the number of isolates fluctuated between 8 and 10 microorganisms in most of the cases, with the exception of the initial time; in that case the count of HM-tolerant microorganisms was 14. All the isolates were tested regarding their capability to remove HMs in multi-polluted growth media (Figs. 1–3). In comparison to control media, most of the cell-free supernatants in the inoculated media showed a final HM concentration lower than the initial one, although differences on account of isolate, origin and HM were detected. Thus, generally speaking, fungi were more efficient than bacteria in removing HMs, while the number of isolates from the MSW process showing a high removal capability was lower than in the case of SS and HW processes. In most cases Pb was the HM removed in a higher proportion. Thus, the remaining Pb concentration in media at the end of the assay did not exceed 10% at 45% of the isolates. However, for those isolates showing medium-low removal capability, this metal was present in higher levels than the others at the end of the assay. No big differences were observed for the other HMs, since similar results were detected for every isolate in most cases. Isolates slh48t1f (Graphium putredinis), slh48t4f (Fusarium solani), hwh48t3f (Fusarium sp.), hwh48t4f (Penicillium chrysogenum) and hwt3f (Fusarium solani) were selected on account of their high detoxifying capability and culturable properties. In all cases, removal levels were above 60%, with the maximum percentages for Pb, always above 95%. Zn(II) removal, the second highest, ranged between 70% and 80%, while levels decreased to 60–70% for Cd(II), Cr(VI) and Ni.

3.2. HM removal studies: bioaccumulation/bioadsorption mechanisms Heavy metals were efficiently removed by the growing cells of the five fungi assayed, although the results from Graphium putredinis and Penicillium chrysogenum were slightly better than those from Fusarium spp. Ni was the metal removed in the lowest proportion and Pb the most affected by the microbial action (Fig. 4). Pb removal efficiency was above 90%, whereas fungal action on Cd(II), Cr(VI) and Zn(II) ranged between 85% and 60%. In most of the cases, intracellular accumulation was the predominant removal mechanism. A major exception to this behaviour was Ni, which was removed primarily through adsorption.

3. Results 3.1. HM-resistant microorganisms screening and selection The implementation of the screening and selection protocol led to the isolation of 51 HM-resistant microorganisms, 28 bacteria and 23 fungi. The distribution within the three different processes was quite similar, with 16 isolates coming from the sewage sludge process, 17 from the municipal solid waste process and 18 from the

Fig. 1. HM removal capability of microorganisms isolated from sewage sludge composting process, expressed as relative percentage in comparison to control. Key for the isolates name: original material →ss: sewage sludge; state of the sample (samples with no specific reference comes from solid material)→ h24: hydrolysate from 24 h treatment; h48: hydrolysate from 48 h treatment; time of sampling→t1: phase 1; t2: phase 2; t3: phase 3; t4: phase 4; t5: phase 5; type of microorganism → b: bacteria; f: fungi. Vertical bars represent the standard error.

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Fig. 2. HM removal capability of microorganisms isolated from sewage sludge composting process, expressed as relative percentage in comparison to control. Key for the isolates name: original material → msw: municipal solid waste; state of the sample (samples with no specific reference comes from solid material) → h24: hydrolysate from 24 h treatment; h48: hydrolysate from 48 h treatment; time of sampling → t1: phase 1; t2: phase 2; t3: phase 3; t4: phase 4; t5: phase 5; type of microorganism → b: bacteria; f: fungi. Vertical bars represent the standard error.

Fungi dead biomass showed to be less efficient in removing HMs than growing cells (Fig. 5). The biosorptive capability ranged between 6% and 34%, with the exception of Pb. This element was removed in a higher proportion than the rest, going over 65% in most cases, analogously to the results with growing cells. On the other hand, differences between the removed percentage of Ni and the other elements were not so steep as in the case of growing cells. Contrarily to growing cells, the best results were observed for Fusarium sp., and the Penicillium chrysogenum biomass showed the least biosorption capability. The most notable feature was the poor Pb removal capability, which was very similar to the other HMs. 4. Discussion 4.1. HM-resistant microorganisms screening and selection Microorganisms are recognized bioremediation agents, both regarding organic and inorganic pollutants. Screening and selection programs are usually designed in order to isolate effective species. The election of the adequate environment for the screening process is a key point for success. In the screening of heavy metal detoxifying microorganisms, the most usual habitats are related to mining and tannery activities. Compost has been applied for the bioremediation of polluted environments, although its use as a source of HM-removing microbial isolates is not so common. In this study, different composting processes have

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been screened with this purpose. The characteristics of the raw material barely affected the number of HM-tolerant microorganisms, although it remarkably affected the removal efficiency. Specifically, isolates from MSW process showed lower capability in comparison to those from SS and HW processes. The predominance of bacterial isolates in the former might explain such difference, as bacteria were less capable of removing than fungi. In a high percentage, the tolerance capability of HMresistant microorganisms depends on their action as biosorbent and, therefore, on the presence of proper functional groups, with a high affinity for binding metals, at the cell coatings. In this sense, both bacteria and fungi have surface compounds useful for HMs sorption (Gadd, 2004; Joo et al., 2010). Other authors have reported the specific properties of fungi for HM bioremediation processes, regarding both bioaccumulation and biosorption applications (Kumar et al., 2012; Taştan et al., 2010). According to Fomina et al. (2007), fungi are usually more tolerant to metallic elements and have a higher surface to volume ratio than bacteria. Among the actions that fungi can exert on metals, some of them ascribable exclusively or mostly to fungal species, are the complexation with cell wall chitin and other polysaccharides, as well as glycoproteins (Skorik et al., 2010), the synthesis of metallothioenins and other functional groups containing thiol (Guimaraes-Soares et al., 2006), the production of organic acids (Arwidsson et al., 2010), or the compartmentalization in vacuoles (Sun and Shao, 2007), which results in three main mechanisms: biosorption on the surface, intracellular uptake and chemical transformation (Leitao, 2009). There were also differences in the removal of the five metals used in the assay, revealing Pb as the most sensitive one to microbial action. Efficiency levels close to 100% have been previously reported for lead detoxification (Kim et al., 2007). As in this case, these results are mostly associated to multi-metal studies, since the presence of other inorganic pollutants seems to have a positive influence on the Pb removal capability of microorganisms, as well as helping to minimize the toxicity of this metal (Pan et al., 2009). Moreover, the covalent index value of Pb points out a greater capability to form covalent bonds with biological ligands in comparison to other metallic ions such as Cd(II) or Zn(II) (Pan et al., 2010). On the contrary, the affinity for other elements, such as Cd(II), Cr(VI) or Ni has been reported to decrease when found in multi-metal solutions (Pakshirajan and Swaminathan, 2009). The competition among different metals for the available binding sites is responsible for this effect, although other factors might be involved since the effect depends on the accompanying HMs and the metal class in which they are categorized (Kiran and Kaushik, 2012). 4.2. HM removal studies: bioaccumulation/bioadsorption mechanisms

Fig. 3. HM removal capability of microorganisms isolated from sewage sludge composting process, expressed as relative percentage in comparison to control. Key for the isolates name: original material → hw: horticultural waste; state of the sample (samples with no specific reference comes from solid material)→ h24: hydrolysate from 24 h treatment; h48: hydrolysate from 48 h treatment; time of sampling →t1: phase 1; t2: phase 2; t3: phase 3; t4: phase 4; t5: phase 5; type of microorganism → b: bacteria; f: fungi. Vertical bars represent the standard error.

Microorganisms act on heavy metals through two main mechanisms: bioadsorption, which is a metabolism-independent binding, and bioaccumulation, which is an energy-dependent flux into the cell. Results from this study point at bioaccumulation as the predominant mechanism, since Cd(II), Cr(VI), Pb and Zn(II) were mostly removed by bioaccumulation. However, in the case of Ni, with significantly lower rates of removal, bioadsorption was more efficient than bioaccumulation. Nickel has been previously described as one of the most persistent HM (Malik, 2004), both regarding extracellular adsorption and intracellular accumulation. This poor biosorptive capability has been mostly attributed to steric hindrances caused by their cationic properties (Gomathi Priya et al., 2009), while the lack of some specific carrier proteins, which are inducible under high Ni-adsorption conditions (Pal and Pail, 2010), might be responsible for the low intracellular accumulation (Pervez et al., 2009). Besides, Ni has been referred to as an inhibitor of some metabolic processes in fungi and an element with capability for altering the cell permeability, thus decreasing the uptake of important nutrients (Ge et al., 2011), resulting in a lesser potential of microorganisms for intracellular accumulation.

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Fig. 4. HM partition after microbiological treatment: bioaccumulation → intracellular concentration; biosorption → surface sorption; supernatant → not removed. Vertical bars represent the standard error.

Several detoxification studies have reported the role of Pb as the most easily removable heavy metal in multi-polluted systems (De et al., 2008; Guo et al., 2010). Some lead properties, such as atomic weight or ionic radius, which make it different from the other tested HMs, probably justify this preferential detoxification action (Pakshirajan and Swaminathan, 2009). Fusarium and Penicillium are well-recognized fungi in terms of their capability in the bioremediation of heavy metals (Pan et al., 2009; Sen et al., 2007; Sun and Shao, 2007). On the contrary, Graphium has been referenced in the bioremediation of organic pollutants and xenobiotics (Santos and Linardi, 2004; Skinner et al., 2008), and to our knowledge its practical applications as an HM detoxifying agent are non-existent so far. In our study, however, Graphium not only acts as a bioremediation agent in HM multi-polluted systems but also matches the results of Fusarium and Penicillium. This result agrees with that described by Pan et al. (2009) using Fusarium and Penicillium as bioremediation agents. Ting and Choong (2009) reported a higher capability of a Trichoderma isolate in removing HMs through intracellular accumulation in comparison to adsorption and a similar result was referenced by Chojnacka (2007) regarding the ability of Spirulina to remove Cr(III). Sun and Shao (2007) too hypothesize on the predominance of intracellular accumulation in detoxifying processes using fungi. According to Chojnacka (2010) and Zucconi et al. (2003) intracellular accumulation firstly proceeds through adsorption, which gives way to uptake when equilibrium is reached. Thus, intracellular accumulation becomes predominant as contact time between HMs and growing

cells increases and metabolic processes and intracellular transport systems are activated. Together with living cells, dead microbial cells can be used in HM bioremediation processes as biosorption agents. Actually, biomass shows some advantages such as easy storage or no need to use expensive enriched media (Velásquez and Dussan, 2009). However, dead biomass showed less efficiency than living cells, even for Pb. although this element was still removed to a greater extent than Cd(II), Cr(VI), Ni and Zn(II). Similar results were described by Amini and Younesi (2009), who found out that Pb. was the most efficiently removed heavy metal in multi-polluted systems. On the other hand, differences between the percentage of Ni removed by dead biomass and of other elements removed by the same cells were not as steep as in the case of growing cells, probably as a consequence of the prevalence of the intracellular accumulation mechanism in these elements when growing cells were used. Results regarding comparison between growing cells and dead biomass removal efficiency are controversial. Thus, Al-Garni et al. (2009) reported a lower capability, ranging between 15% and 44%, when they substituted growing cells of Aspergillus fumigatus and Penicillium chrysogenum for dead biomass, and Zucconi et al. (2003) referred a decrease from 30% to 23% using Paecilomyces lilacinus. On the contrary, Gabr et al. (2008) and Kaushik et al. (2008) described a higher removal capability for dead biomass. A factor which potentially explains these differences might be the methodology used to prepare the dead biomass, as it affects the efficiency of the biosorbent (Baik et al., 2002).

5. Conclusions

Fig. 5. HM biosorption capability of selected composting isolates dead biomass, expressed as relative percentage in comparison to control. Vertical bars represent the standard error.

Composting has proven to produce the proper environmental and nutritional conditions for the existence of HM-resistant microorganisms, which makes the composting pile a significant reservoir of such microorganisms. Some of these isolates showed suitable properties for their use in bioremediation processes of multi-polluted systems. All of the selected isolates in our tests were fungi, which showed a higher efficiency against most of the tested pollutants, especially Pb, and the predominant removal mechanism was intracellular accumulation, which made growing cells more efficient than dead biomass as detoxifying agents. The isolation of a Graphium putredinis strain was especially significant, since no references on this fungus regarding HMs bioremediation had previously been reported.

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Acknowledgments This research has been funded by the Spanish “Ministerio de Ciencia y Tecnología” (CICYT project no. CTM2006-02149/TECNO). References Al-Garni S, Ghanem KM, Bahobail AS. Biosorption characteristics of Aspergillus fumigatus in removal of cadmium from an aqueous solution. Afr J Biotechnol 2009;8:4163–72. Amini M, Younesi H. Biosorption of Cd(II), Ni(II) and Pb(II) from aqueous solution by dried biomass of Aspergillus niger: application of response surface methodology to the optimization of process parameters. Clean-Soil Air Water 2009;37:776–86. Anjana K, Kaushik A, Kiram B, Nisha R. Biosorption of Cr(VI) by immobilized biomass of two indigenous strains of cyanobacteria isolated from metal contaminated soil. J Hazard Mater 2007;148:383–6. Arwidsson Z, Johansson E, von Kronhelm T, Allard B, van Hees P. Remediation of metal contaminated soil by organic metabolites from fungi I-Production of organic acids. Water Air Soil Pollut 2010;205:215–26. Baik WY, Bae JH, Cho KM, Hartmeier W. Biosorption of heavy metals using whole mold mycelia and parts thereof. Bioresour Technol 2002;81:167–70. Balabanova B, Stafilov T, Šjan R, Bačeva K. Distribution of chemical elements in Attic dust as refelction on their geogenic and anthropogenic sources in the vicinity of the copper mine and flotation plant. Arch Environ Contam Toxicol 2011;61: 173–84. Barker AV, Bryson GM. Bioremediation of heavy metals and organic toxicants by composting. ScientificWorldJournal 2002;2:407–20. Berny P, Caloni F, Croubels S, Sachan M, Vandenbroucke V, Davanzo F, et al. Animal poisoning in Europe. Part 2: Companion animals. Vet J 2010;183:255–9. Beyersmann D, Hartwig A. Carcinogenic metal compound: recent insight into molecular and cellular mechanisms. Arch Toxicol 2008;82:493–512. Chojnacka K. Bioaccumulation of Cr(III) ions by blue-green alga Spirulina sp. Part I. A comparison with biosorption. Am J Agric Biol Sci 2007;2:218–23. Chojnacka K. Biosorption and bioaccumulation - the prospects for practical applications. Environ Int 2010;36:299–307. De J, Ramaiah N, Vardanyan L. Detoxification of toxic heavy metals by marine bacteria highly resistant to mercury. Mar Biotechnol 2008;10:471–7. Farrell M, Perkins WT, Hobbs PJ, Griffith GW, Jones D. Migration of heavy metals in soil as influenced by compost amendments. Environ Pollut 2010;158:55–64. Fomina M, Charnock J, Bowen AD, Gadd GM. X-ray absorption spectroscopy (XAS) oftoxic metal mineral transformations by fungi. Environ Microbiol 2007;9:308–21. Gabr RM, Hassan SHA, Shoreit AAM. Biosorption of lead and nickel by living and nonliving cells of Pseudomonas aeruginosa ASU 6a. Int Biodeterior Biodegrad 2008;62: 195–203. Gadd GM. Geomicology: biogeochemical transformation of rocks, minerals, metals, and radionuclides by fungi, bioweathering and bioremediation. Mycol Res 2004;111: 3-49. Ge W, Zamri D, Mineyama H, Valix M. Bioaccumulation of heavy metals on adapted Aspergillus foetidus. Adsorpt -J Int Adsorpt Soc 2011;17:901–10. Gomathi Priya P, Ahmed Basha C, Ramamurthi V, Nathira Begum S. Recovery and reuse of Ni(II) from rinsewater of electroplating industries. J Hazard Mater 2009;163: 899–909. Guimaraes-Soares L, Felicia H, Bebianno MJ, Cassio F. Metal-binding proteins and peptides in the aquatic fungi Fontanospora fusiramosa and Flagellopora curta exposed to severe metal stress. Sci Total Environ 2006;372:148–56. Guo H, Luo S, Chen L, Xiao X, Xi Q, Wei W, et al. Bioremediation of heavy metals by growing hyperaccumulator endophytic bacterium Bacillus sp. L14. Bioresour Technol 2010;101:8599–605. Haferburg G, Kothe E. Microbes and metals: interactions in the environment. J Basic Microbiol 2007;47:453–67. Helmisaari HS, Salemaa M, Derome J, Kiikkilä O, Uhligh C, Nieminem T. Remediation of heavy metal-contaminated forest soil using recycled organic matter and native woody plants. J Environ Qual 2007;36:1145–53. Hinojosa MB, Carreira JA, García-Ruiz R, Dick RP. Microbial response to heavy metalpolluted soils: community analysis from phospholipid-linked fatty acids and ester-linked fatty acid extracts. J Environ Qual 2005;34:1789–800. Joo JH, Hassan SHA, Oh SE. Comparative study of biosorption of Zn2 + by Pseudomonas aeruginosa and Bacillus cereus. Int Biodeterior Biodegrad 2010;64:734–41. Kaushik S, Juwarkar A, Malik A, Satya S. Biological removal of Cr(VI) by bacterial isolates obtained from metal contaminated sites. J Environ Sci Health A Tox Hazard Subst Environ Eng 2008;43:419–23. Kim SU, Cheong YH, Seo DC, Hur JS, Cho JS. Characterisation of heavy metal tolerance and biosorption capacity of bacterium strain CPB4 (Bacillus spp.). Water Sci Technol 2007;55:105–11. Kiran B, Kaushik A. Equilibrium sorption study of Cr(VI) from multimetal systems in aqueous solutions by Lyngbya putealis. Ecol Eng 2012;38:93–6. Kumar R, Bhatia D, Singh R, Bishnoi NR. Metal tolerance and sequestration of Ni(II), Zn(II) and Cr(VI) ions from simulated and electroplating wastewater in batch process: kinetics and equilibrium study. Int Biodeterior Biodegrad 2012;66:82–90. Leitao AL. Potential of Penicillium species in the bioremediation field. Int J Environ Res Public Health 2009;6:1393–417.

67

Lister SK, Line MA. Enhacement of metal sorption by composted paper mill waste following microbial manipulation. Compost Sci Util 2004;2:31–4. Lloyd JR. Bioremediation of metals: the application of microorganisms that make and break minerals. Microbiol Today 2002;29:67–9. MacKenzie JM, Canil D. Volatile heavy metal mobility in silicate liquids: implication for volcanic degassing and eruption prediction. Earth Planet Sci Lett 2008;269:487–95. Malik A. Metal bioremediation through growing cells. Environ Int 2004;30:261–78. Morales-Barrera L, Cristiani-Urbina E. Hexavalent chromium removal by a Trichoderma inhumatum fungal strain isolated from tannery effluent. Water Air Soil Pollut 2008;187:327–36. Nagajyoti PC, Lee KD, Sreekanth TVM. Heavy metals, occurrence and toxicity for plants: a review. Environ Chem Lett 2010;8:199–216. Neagoe A, Merten D, Iordache V, Büchel G. The effect of bioremediation methods involving different degrees of soil disturbance on the export of metals by leaching and by plant uptake. Chem Erde-Geochem 2009;69(S2):57–73. Pakshirajan K, Swaminathan T. Biosorption of lead, copper, and cadmium by Phanerochaete chrysosporium in ternary metal mixtures: statistical analysis of individual and interaction effects. Appl Biochem Biotechnol 2009;158:457–69. Pal A, Pail AK. Nickel uptake and intracellular localization in Cupriavidus pauculus KPS 201, native to ultramafic ecosystem. Adv Biosci Biotechnol 2010;1:276–80. Pan R, Cao L, Zhang R. Combined effects of Cu, Cd, Pb and Zn on the growth and uptake of consortium of Cu-resistant Penicillium sp. A1 and Cd-resistant Fusarium sp. A19. J Hazard Mater 2009;171:761–6. Pan R, Cao L, Huang H, Zhang R, Mo Y. Biosorption of Cd, Cu, Pb, and Zn from aqueous solutions by the fruiting bodies of jelly fungi (Tremella fuciformis and Auricularia polytricha). Appl Microbiol Biotechnol 2010;88:997-1005. Pérez de Mora A, Burgos P, Madejón E, Cabrera F, Jaeckel P, Schloter M. Microbial community structure and function in soil contaminated by heavy metals: effects on plant growth and different amendments. Soil Biol Biochem 2006;38:327–41. Pervez A, Shahbaz S, Shah MM, Mahmood Q, Mirza N. Assesing bioaccumulation of heavy metals in sporocarp of Pleurotus ostreatus. World Appl Sci J 2009;7:1498–503. Rihani M, Malamis D, Bihaoui B, Etahiri S, Loizidou M, Assobhei O. In-vessel treatment of urban primary sludge by aerobic composting. Bioresour Technol 2010;101:5988–95. Santos VL, Linardi VR. Biodegradation of phenol by a filamentous fungi isolated from industrial effluents-identification and degradation potential. Process Biochem 2004;39:1001–6. Sen M, Dastidar MG, Roychoudhury PK. Biological removal of Cr(VI) using Fusarium solani in batch and continuous modes of operation. Enzyme Microb Technol 2007;41:51–6. Shcherbov BL, Strakhovenko VD, Sukhorukov FV. The ecogeochemical role of forest fires in the Baikal region. Geogr Nat Resour 2008;9:150–5. Shi W, Shao H, Li H, Shao M, Su S. Progress in the remediation of hazardous heavy metal-polluted soils by natural zeolite. J Hazard Mater 2009;170:1–6. Shukla OP, Rai UN, Dubey S. Involvement and interaction of microbial communities in the transformation and stabilization of chromium during the composting of tannery effluent treated biomass of Vallisneria spiralis L. Bioresour Technol 2009;100:2198–203. Singh A, Agrawal M, Marshall FM. The role of organic fertilizers vs. inorganic fertilizers in reducing phytoavailability of heavy metals in a waste-water irrigated area. Ecol Eng 2010;36:1733–40. Sinicropi MS, Amantea D, Caruso A, Saturnino C. Chemical and biological properties of toxic metals and use of chelating agents for the pharmacological treatment of metal poisoning. Arch Toxicol 2010;84:501–20. Skinner KM, Martinez-Prado A, Hyman MR, Williamson KJ, Ciuffetti LM. Pathway, inhibition and regulation of methyl tertiary butyl ether oxidation in a filamentous fungi, Graphium sp. Appl Microbiol Biotechnol 2008;77:1359–65. Skorik YA, Pestov AV, Yatluk YG. Evaluation of various chitin-glucan derivatives from Aspergillus niger as transition metal adsorbents. Bioresour Technol 2010;101:1769–75. Soderland P, Lovekar S, Weiner DE, Brooks DR, Kaufman JS. Chronic kidney disease associated with environmental toxins and exposure. Adv Chronic Kidney Dis 2010;17:254–64. Sun F, Shao Z. Biosorption and bioaccumulation of lead by Penicillium sp. Psf-2 isolated from the deep sea sediment of the Pacific Ocean. Extremophiles 2007;11:853–8. Taştan BE, Ertuğrul S, Dönmez G. Effective bioremoval of reactive dye and heavy metals by Aspergillus versicolor. Bioresour Technol 2010;101:870–6. Ting ASY, Choong CC. Bioaccumulation and biosorption efficacy of Trichoderma isolate SP2F1 in removing copper (Cu(II)) from aqueous solutions. World J Microbiol Biotechnol 2009;25:1431–7. Velásquez L, Dussan J. Biosorption and bioaccumulation of heavy metals on dead and living biomass of Bacillus sphaericus. J Hazard Mater 2009;167:713–6. Wang J, Chen C. Biosorbents for heavy metals removal and their future. Biotechnol Adv 2009;27:196–226. Wei SH, Zhou QX, Wang X, Cao W, Ren LP, Song YF. Potential of weed species applied to remediation of soils contaminated with heavy metals. J Environ Sci 2004;16:868–73. Wu G, Kang H, Zhang X, Shao H, Chu L, Ruan C. A critical review on the bio-removal of hazardous heavy metals from contaminated soils: issues, progress, eco-environmental concerns and opportunities. J Hazard Mater 2010;174:1–8. Yu G, Lei H, Bai T, Li Z, Yu Q, Song X. In-situ stabilization followed by ex-situ composting for treatment and disposal of heavy metals polluted sediments. J Environ Sci 2009;21:877–83. Zucconi L, Ripa C, Alianiello F, Benedetii A, Onofri S. Lead resistance, sorption and accumulation in a Paecilomyces lilacinus strain. Biol Fertil Soils 2003;37:17–22.