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Mechanisms of interaction of chromium with Aspergillus niger var tubingensis strain Ed8
Bioresource Technology 158 (2014) 188–192
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Mechanisms of interaction of chromium with Aspergillus niger var tubingensis strain Ed8 A. Coreño-Alonso a, A. Solé c, E. Diestra c, I. Esteve c, J.F. Gutiérrez-Corona b, G.E. Reyna López b, F.J. Fernández a, A. Tomasini a,⇑ a Departamento de Biotecnología, Universidad Autónoma Metropolitana, Unidad Iztapalapa, Av. San Rafael Atlixco no. 186, Col Vicentina México, Del. Iztapalapa, A.P. 55-535, C.P. 09340 México D.F., Mexico b Departamento de Biología, DCNyE, Universidad de Guanajuato, Apartado Postal 187, Guanajuato, Gto 36000, Mexico c Genetics and Microbiology Department, Biosciences Faculty, Universitat Autónoma de Barcelona, Bellaterra, 08193 Barcelona, Spain
h i g h l i g h t s A. niger Ed8 uses reduction as the principle mechanism of interaction with Cr(VI). A. niger Ed8 also shows the ability to capture chromium intracellularly. The biotransformation of Cr(VI) was carried out in a bubble column bioreactor. A. niger Ed8 reduces 2.62 mg Cr(VI) g
a r t i c l e
1
dry biomass h
i n f o
Article history: Received 18 December 2013 Received in revised form 7 February 2014 Accepted 10 February 2014 Available online 17 February 2014 Keywords: A. niger var tubingensis Cr(VI) reduction Cr accumulation Bubble column bioreactor
1
in bubble column bioreactor.
a b s t r a c t Experiments were conducted to determine the mechanisms of interaction with chromium of Aspergillus niger var tubingensis strain Ed8 in batch culture and in bioreactor experiments. Results obtained in this work showed that the interaction of A. niger var tubingensis Ed8 with Cr(VI) is based mainly in a reduction process and also, secondly, in a sorption process. Using electron microscopy techniques the ultrathin sections obtained from the mycelium biomass produced by the fungus in batch cultures showed the ability to incorporate Cr intracellulary, into low electron-dense inclusions, but not extracellularly. On the other hand, cultures without Cr(VI) of A. niger var tubingensis Ed8, grown in a bubble column bioreactor, reduced Cr(VI) immediately after repeated addition of this oxyanion; after six loads, 460 mg Cr(VI) was reduced to Cr(III) in 60 h, corresponding to a reduction rate of 2.62 mg Cr(VI) g 1 dry biomass h 1. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Chromium compounds are widely used in different anthropogenic activities, which include industries such as mining, leather tanning, stainless-steel production, use and waste of chromium (Calder, 1988). Their extensive use has caused that chromium is present in soil and sediments in high concentration causing toxicity to life forms. In nature, Cr is present as trivalent Cr(III) and hexavalent Cr(VI) species. The higher oxidation state, in the form of chromates and (or) dichromates, is highly toxic to most organisms, whereas Cr(III) is relatively innocuous; in this sense, it has been reported that Cr(III) is 100-times less toxic than Cr(VI) (De Flora et al., 1990). Chromium is cataloged within group 1 of
⇑ Corresponding author. Tel.: +52 55 5804 4600x2874; fax: +52 55 5558 4712. E-mail address: [email protected] (A. Tomasini). http://dx.doi.org/10.1016/j.biortech.2014.02.036 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
priority of toxic substances reported by Toxic Substance and Disease Agency. EPA (Environmental Protection Agency, USA) has established the discharge limit of Cr(VI) to surface water below 0.05 mg L 1 while the total Cr including Cr(III), Cr(VI) and its other forms to below 2 mg L 1 (Baral and Engelken, 2002). The microbial mechanisms of interaction with chromium are of basic and technological relevance, due to the fact that the physicochemical methods currently considered for the removal of chromium at large scale are expensive and cause the generation of a large amount of secondary waste products (Bai and Abraham, 2001). Bioremediation is an alternative actually considered to remove Cr from the environment (Mulligan et al., 2001); this process exploits the genetic and metabolic potential of microorganisms to remove metals by biotransformation and/or biosorption (Chen and Hao, 1998; Cervantes et al., 2001). Filamentous fungi belonging to the genera Aspergillus, Penicillium, Trichoderma and Mucor have been described as having the
A. Coreño-Alonso et al. / Bioresource Technology 158 (2014) 188–192
ability to reduce Cr(VI) to Cr(III) in cultures of actively growing cells (Acevedo-Aguilar et al., 2006; Fukuda et al., 2008; MoralesBarrera and Cristiani-Urbina, 2006; Liu et al., 2007). Other works have described fungal strains with the ability to cause the uptake of chromium to the biomass by bioaccumulation/biosorption processes, using growing or inactivated cells; the strains described included isolates of Aspergillus (Nasseri et al., 2002; Prasenjit and Sumathi, 2005; Srivastava and Thakur, 2006a,b; Congeevaram et al., 2007; Mungasavalli et al., 2007; Khambhaty et al., 2009), Hirsutella sp (Srivastava and Thakur, 2006a) and Rhizopus (Bai and Abraham, 2001). An additional process, called ‘‘adsorptioncoupled reduction’’, which occurs mainly at acidic pH, has been described with inactivated cells of Aspergillus and Rhyzopus (Park et al., 2005, 2007). The bioadsorption process takes place at the cell surface mainly at extracellular polymeric substances (EPS), followed by transport into the cytoplasm compartments, through two processes. The first is rapid, nonspecific, and usually based on the chemiosmotic gradient (passive transport), and since it is used by a wide variety of substrates, it is expressed constitutively. The second transport system has a high specificity for the substrate, is slower and often uses ATP hydrolysis as the energy source (active transport), but can also function by osmotic gradient. The captured metal can form complexes with compounds such as proteins rich in thiol groups physiologically present in cells (enzymes of metabolic pathways) or synthesized in the presence of metals (metallothioneins and phytochelatins) (Wang and Chen, 2009). In previous works, the Ed8 strain of A. niger var tubingensis, included in the A. niger species complex, demonstrated high efficiency to transform Cr(VI) to Cr(III) in the extracellular medium, with a restricted chromium sorption to biomass (Acevedo-Aguilar et al., 2006). In Ed8 strain Cr(VI) reduction occurs by a mechanism stimulated by carboxylic acids and metal-chelating agents (Coreño-Alonso et al., 2009). The aim of this paper was to study the ability of A. niger var tubingensis Ed8 to capture chromium extra and intracellularly and its efficiencies to reduce Cr(VI), by means of chemical analysis and electron microscopy techniques in cultures developed in a bubble column bioreactor. 2. Methods 2.1. Microorganism and culture conditions A. niger var tubingensis Ed8 described and characterized previously (Acevedo-Aguilar et al., 2006; Coreño-Alonso et al., 2009) was used in this work. This fungus strain was grown and propagated in PDA medium. Cr(VI) reduction and chromium sorption experiments using this microorganism were realized in batch cultures or in a bubble column bioreactor, using modified Lee minimal medium (LMM) (Lee et al., 1975) containing 0.25% KH2PO4, 0.20% MgSO4, 0.50% (NH4)2SO4, 0.5% NaCl and 0.25% glucose; which were dissolved in citrate buffer 45 mM and the pH of the medium was adjusted to 5.3 with 45 mM citric acid and NaOH (Coreño-Alonso et al., 2009). Cr(VI) reduction experiments in LMM medium containing 3 mM sodium salicylate (LMMS) supplemented with the proper amount of K2CrO4 to reach the indicated concentration of Cr(VI) in the medium were conducted in batch cultures as previously described (Coreño-Alonso et al., 2009). Reduction medium used in bioreactor experiments was based in LMMS, but twofold concentrated (2X) and with saccharose instead of glucose as carbon source; the components of the medium were technique grade reactives, which were dissolved in citrate buffer 45 mM, and pH was 5.3. Medium was supplemented with the proper amount of K2CrO4, to reach the initial Cr(VI) concentration.
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2.2. Batch culture experiments Biomass production in modified LMM medium and Cr(VI) reduction experiments in LMMS medium supplemented with the proper amount of K2CrO4 to reach the indicated concentration of Cr(VI) in the medium, were conducted in batch cultures as previously described (Coreño-Alonso et al., 2009). Then fungal biomass was transferred to LMM containing 50 and 100 mg L 1 of Cr(VI), and samples were analyzed at 0, 12 and 24 h. Cultures were filtered and broth cultures free of biomass were used to determine Cr(VI) reduction and, in another hand, the biomasses were used to analyze the extra or intracellular accumulation of Cr in individual cells. Unpolluted culture was used as a control experiment. 2.3. Scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) microanalysis For SEM analysis, fungal biomass samples from batch cultures were fixed in 4% glutaraldehyde diluted in phosphate buffer (0.1 M and pH7) for 2 h and washed four times in the same buffer, dehydrated in increasing concentrations of ethanol (30%, 50%, 70%, 90%, and 100%) and dried by critical-point drying. Finally, all samples were mounted on metal stubs and coated with gold for better image contrast. A Jeol JSM-6300 scanning electron microscope (Jeol, Tokyo, Japan) was used to view the images. For energy dispersive X-ray microanalysis, cells were homogenously distributed and filtered with polycarbonate membrane filters. These filters were dehydrated and dried by critical-point drying and then coated with gold. An energy dispersive X-ray spectrophotometer (EDX) Link Isis-200 (Oxford Instruments, Bucks, England) coupled to a Zeiss EVOÒ MA 10 scanning electron microscope (Carl Zeiss NTS GmbH, Oberkochen, Germany) operated at 20 kV was used. Finally, EDX–SEM spectra from individual cells were obtained. 2.4. Transmission electron microscopy (TEM) and energy dispersive Xray (EDX) microanalysis For TEM analysis, fungal biomass samples from batch cultures were fixed in glutaraldehyde as mentioned above. Later, samples were post-fixed in 1% OsO4 at 4 °C for 2 h, and washed four times in the same buffer. They were then dehydrated in a graded series of acetone (50%, 70%, 90%, 95% and 100%) and embedded in Spurr’s resin. Ultrathin sections (70 nm), realized with a Leica UCG Ultramicrotome, were mounted on carbon coated copper grids and stained with uranyl acetate and lead citrate. Samples were viewed in a Hitachi H-7000 transmission electron microscope (Hitachi LTD, Tokyo, Japan). For energy dispersive X-ray microanalysis, sections of 200 nm thick were also stained with uranyl acetate and mounted on carbon-coated grids. Samples were analyzed with an EDX Link Isis-200 (Oxford Instruments, Bucks, England) coupled to a Jeol Jem-2011 (Jeol Ltd., Tokyo, Japan) operating at 20 kV. Finally, EDX–TEM spectra were obtained. In all EDX spectra (SEM and TEM) the main Cr peak was detected at 5.4 keV of energy. 2.5. Bubble column bioreactor experiment A laboratory scale bubble column bioreactor was fabricated from glass column of 7 cm diameter by 40 cm high with a total capacity of 1 L. The reactor was worked in two stages: growth and reduction stages. The column containing 800 mL of LMM was inoculated with 5 105 spores of A. niger var tubingensis Ed8 mL 1 with an air flux of 8.5 L min 1 and incubated at room temperature during 60 h (growth stage). After growth, in the
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reduction stage, a concentration of 100 mg Cr(VI) L 1 from a synthetic stock solution (55,000 mg L 1) was added to the culture containing fungal biomass, and incubated during 12 h. After this time, 50% of initial volume of the culture was removed and replaced by the same volume of fresh 2X Lee modified medium and the mixture was incubated for 12 h. In further 12 h-incubation periods, 12.5% of initial volume was removed and replaced by 2X fresh medium containing 100 mg Cr(VI) L 1; these incubations were continued until a total of 60 h from the start of the experiment. Immediately after the addition of Cr(VI) (T = 0) and then every 3 min, 5 mL samples were taken, which were used for the determination of Cr(VI) and total chromium. The biomass obtained at the end of incubation (60 h) was recovered by filtration, washed repeatedly with distilled water and lyophilized to obtain its dry weight, and then the biomass was processed for determining the chromium incorporated therein. 2.6. Analytical determinations Cr(VI) was quantified by a spectrophotometric method employing diphenylcarbazide (DPC) (Greenberg et al., 1981). Total chromium content in the supernatant of the cultures and in the biomass was determined by ICP-MS as previously described (Acevedo-Aguilar et al., 2008). 3. Results and discussion 3.1. Cr(VI) reduction in batch cultures After 24 h of incubation of strain A. niger var tubingensis Ed8 in LMM medium containing an initial concentration of 50 mg Cr(VI) L 1, a decrease of about 94% in Cr(VI) levels was observed; in the same period, in medium with 75 and 100 mg Cr(VI) L 1, the biomass of Ed8 strain caused a decrease of about 60% and 56% in Cr(VI) levels (Table 1). The pH of the cultures changed only from 5.3 to 5.0 because the buffer citrate in the medium. It has been described that in Ed8 strain cultures incubated with 50 mg Cr(VI) L 1 under conditions identical to those employed in this study, most (>98%) total residual chromium was found in the solution, providing evidence indicative of soluble Cr(III)-complex formation, with the consequent increase in Cr(VI) reduction (Acevedo-Aguilar et al., 2008). The observation of a less marked decrease in Cr(VI) levels in Ed8 strain cultures incubated in the presence 100 mg Cr(VI) L 1, probably is due to toxicity effects of Cr(VI). 3.2. Cr(VI) bioaccumulation/sorption in batch cultures Although it is known that in actively growing fungal cells Cr(VI) is incorporated by means of the sulfate permease proteins (Cervantes et al., 2001) or by type CHR-1 transporters (Flores-Alvarez et al., 2012), less is known about the specific molecules that allow ion binding to the cell surface; in part, this is due to the complex nature of the microbial biomass, which is not readily amenable to instrumental analysis (Kapoor and Viraraghavan, 1995). In this work, different techniques of high resolution electron microscopy, SEM and TEM, and both coupled to EDX were
optimized and applied to determine the capacity of A. niger var tubingensis Ed8 to capture Cr(VI) extra or intracellularly. The results obtained from unpolluted samples, as it was expected, Cr was not detected in cells either at extracellular level (SEM–EDX) or at intracellular level (TEM–EDX). Samples of A. niger var tubingensis Ed8 polluted by Cr(VI), and analyzed by SEM, showed morphological changes on fungus hyphae, at both Cr concentrations 50 mg L 1 and 100 mg L 1 and at different exposure time. The most dramatic effect of Cr(VI) on the morphology of the fungus is observed at the highest concentration of the ion since the produced hyphae were short, curved and constricted along its length. Nevertheless, Cr was not detected in their EDX spectra. From these results it can be concluded that A. niger var tubingensis Ed8 cells cannot adsorb this metal externally, either in the surface and/or the EPS. TEM analysis show ultra-structural changes in the hyphae produced by the fungus when this was grown in the presence of Cr(VI). Two types of inclusions with different electron density were observed. The first type was high electron-dense and increased in number at higher Cr concentrations. These inclusions were distributed through the cell wall into the periplasmic space of the cells. Burnat et al. (2010) identified this type of inclusions, by TEM– EDX, as inclusions of polyphosphate in Microcoleus consortium polluted by lead. Although different authors have demonstrated the capacity of the high electron-dense inclusions to capture metals, chromium was not detected in EDX spectra from these inclusions (Burnat et al., 2010; Maldonado et al., 2010, 2011; Puyen et al., 2012; Seder-Colomina et al., 2013). The second type of inclusions were low electron-dense, and were observed in samples exposed to Cr(VI). The analysis of these inclusions by TEM–EDX allowed the detection of the principal peak of Cr at 5.4 keV of energy in the EDX spectra. The results showed the ability of A. niger var tubingensis Ed8 to capture this metal intracellularly. 3.3. Cr(VI) reduction in bioreactor column Previous studies showed that the strain A. niger var tubingensis Ed8 has the capability to reduce Cr(VI) in the extracellular environment, with low amounts of chromium bound to the biomass (Acevedo-Aguilar et al., 2006) and that this capacity for chromium reduction is stimulated by organic acids (Coreño-Alonso et al., 2009). Taking these observations into consideration, we envisaged a rapid and efficient process for Cr(VI) reduction by culturing A. niger var tubingensis Ed8 in a bubble column reactor in the absence of Cr(VI), which was followed by the addition to the culture of this ion and further incubation periods with subsequent medium and Cr(VI) loads. Thus, a culture of A. niger var tubingensis Ed8 was incubated for 60 h in LMM and Cr(VI) was then added. The pH of the medium did not change appreciably; it was between 5.3 and 5.0 because the reduction medium contains citrate buffer. An abrupt decrease in Cr(VI) content was observed when this ion was added and no Cr(VI) was detected 3 min later (time 0 in Fig. 1a). In the first load of 100 Cr(VI) mg L 1 a reduction of 93% of Cr(VI) content was observed immediately after Cr(VI) addition,
Table 1 Cr(VI) reduction by A. niger var tubingensis Ed8 in LMM broth cultures. Incubation period (h)
Cr(VI) remaining (mg L 50 Cr(VI) mg L
0 12 24
1
57 ± 3.0 (0%) 34.5 ± 2.5 (31%) 2.9 ± 0.3.0 (94.2%)
1
) and rate of Cr(VI) reduction (%) in cultures containing: 75 Cr(VI) mg L
1
78 ± 4.1 (0%) 56.16 ± (28%) 46.8 ± 2.1 (60%)
100 Cr(VI) mg L
1
111 ± 4.5 (0%) 73.4 ± 5.02 (26.6%) 44 ± 3.4 (56%)
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a
70
Cr(VI) (mg L-1)
60 50 40 30 20 10 0
Total Cr (mg/L)
b
0
12
24
Time (h)
36
48
60
in the bubble column bioreactor of 2.54 mg Cr(VI) reduced g 1 dry biomass h 1 (biotransformation) and 0.082 mg Cr(VI) accumulated g 1 dry biomass h 1 (bioaccumulation). Few studies have been reported to reduce Cr(VI) employing bioreactors, by example Srivastava et al. (2007) and Tziotzios et al. (2008) reported a sequential bioreactor and a biofilm packed reactor to reduce Cr(VI) and remove chlorophenols simultaneously. In these studies significant amounts of Cr(VI) were removed, some of them by reduction, although this occurred in periods of several hours or days. Moreover, regarding the chromium incorporated into the biomass of the strain A. niger var tubingensis Ed8, the results showed an incorporation of 4.9 Cr g 1 dry weight, which coincides with the range of chromium incorporation in biomass (1.8–8.9 mg Cr g 1 dry weight) described for A. niger strains (Srivastava and Thakur, 2006b). 4. Conclusions
500
The results of this work indicated that the interaction of A. niger var tubingensis Ed8 with Cr(VI) is based mainly in a reduction process, and low ability of the fungus to incorporate Cr. This Cr is incorporated into low electron-dense inclusions from nature not confirmed, that means, accumulates chromium intracellulary. Due to the loads of fresh medium and the fast reduction of Cr(VI) in the cultures, the fungus is kept viable. The bubble column reactor employed showed advantages as low cost of construction and maintenance, lower stress and fragmentation of mycelia, a high efficiency of Cr(VI) reduction.
400 300 200 100 0
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Acknowledgements
0
12
24
36
48
60
Time (h) Fig. 1. Cr(VI) reduction by A. tubingensis Ed8 in a bubble column bioreactor. White bars indicate Cr(VI) at the moment of Cr(VI) addition and black bars indicate Cr(VI) after 3 min of addition (a). Total Cr detected after each time of addition of Cr(VI) (b).
being such reduction of 100% after 3 min of metal addition. As it was previously mentioned, the loads of Cr(VI) to the bubble column reactor were made each 12 h. In the loads at 24 and 36 h of incubation, Cr(VI) reduction in the supernatant was almost complete (95–100%) immediately after the metal addition. These results indicate that the maximum efficiency to reduce Cr(VI) by the fungal biomass was at these times (24 and 36 h) (Fig. 1a). The total Cr content in the supernatant of the bioreactor increased according to the Cr(VI) added in the successive loads, reaching a final concentration of 460 mg L 1 at 60 h (Fig. 1b). After this time no more loads of chromium were made, due to the high viscosity acquired by the culture as a consequence of mycelium growth. The observation that the total chromium content in the culture supernatant increases as Cr(VI) is added, without noticeable differences with respect to the theoretical amount of the ion added, and that the content of Cr(VI) in the culture decreases quickly and dramatically, indicated that under the conditions of the bioreactor the transformation of Cr(VI) to Cr(III) occurred in a highly efficient form. The biomass obtained after 60 h of incubation was 3.02 g (dry weight) and the total Cr(VI) incorporated into this biomass and reduced to Cr(III) in the same period of time were 14.8 mg and 460 mg of Cr, respectively. Thus, a total concentration of 474.8 mg Cr(VI) was added to the bioreactor during the whole experiment (60 h). From these results, the total Cr(VI) removed in the bioreactor was 2.62 mg Cr(VI) g 1 dry biomass h 1 being the efficiencies of Cr(VI) reduction and accumulation by A. niger var tubingensis Ed8
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Mechanisms of interaction of chromium with Aspergillus niger var tubingensis strain Ed8 A. Coreño-Alonso a, A. Solé c, E. Diestra c, I. Esteve c, J.F. Gutiérrez-Corona b, G.E. Reyna López b, F.J. Fernández a, A. Tomasini a,⇑ a Departamento de Biotecnología, Universidad Autónoma Metropolitana, Unidad Iztapalapa, Av. San Rafael Atlixco no. 186, Col Vicentina México, Del. Iztapalapa, A.P. 55-535, C.P. 09340 México D.F., Mexico b Departamento de Biología, DCNyE, Universidad de Guanajuato, Apartado Postal 187, Guanajuato, Gto 36000, Mexico c Genetics and Microbiology Department, Biosciences Faculty, Universitat Autónoma de Barcelona, Bellaterra, 08193 Barcelona, Spain
h i g h l i g h t s A. niger Ed8 uses reduction as the principle mechanism of interaction with Cr(VI). A. niger Ed8 also shows the ability to capture chromium intracellularly. The biotransformation of Cr(VI) was carried out in a bubble column bioreactor. A. niger Ed8 reduces 2.62 mg Cr(VI) g
a r t i c l e
1
dry biomass h
i n f o
Article history: Received 18 December 2013 Received in revised form 7 February 2014 Accepted 10 February 2014 Available online 17 February 2014 Keywords: A. niger var tubingensis Cr(VI) reduction Cr accumulation Bubble column bioreactor
1
in bubble column bioreactor.
a b s t r a c t Experiments were conducted to determine the mechanisms of interaction with chromium of Aspergillus niger var tubingensis strain Ed8 in batch culture and in bioreactor experiments. Results obtained in this work showed that the interaction of A. niger var tubingensis Ed8 with Cr(VI) is based mainly in a reduction process and also, secondly, in a sorption process. Using electron microscopy techniques the ultrathin sections obtained from the mycelium biomass produced by the fungus in batch cultures showed the ability to incorporate Cr intracellulary, into low electron-dense inclusions, but not extracellularly. On the other hand, cultures without Cr(VI) of A. niger var tubingensis Ed8, grown in a bubble column bioreactor, reduced Cr(VI) immediately after repeated addition of this oxyanion; after six loads, 460 mg Cr(VI) was reduced to Cr(III) in 60 h, corresponding to a reduction rate of 2.62 mg Cr(VI) g 1 dry biomass h 1. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Chromium compounds are widely used in different anthropogenic activities, which include industries such as mining, leather tanning, stainless-steel production, use and waste of chromium (Calder, 1988). Their extensive use has caused that chromium is present in soil and sediments in high concentration causing toxicity to life forms. In nature, Cr is present as trivalent Cr(III) and hexavalent Cr(VI) species. The higher oxidation state, in the form of chromates and (or) dichromates, is highly toxic to most organisms, whereas Cr(III) is relatively innocuous; in this sense, it has been reported that Cr(III) is 100-times less toxic than Cr(VI) (De Flora et al., 1990). Chromium is cataloged within group 1 of
⇑ Corresponding author. Tel.: +52 55 5804 4600x2874; fax: +52 55 5558 4712. E-mail address: [email protected] (A. Tomasini). http://dx.doi.org/10.1016/j.biortech.2014.02.036 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
priority of toxic substances reported by Toxic Substance and Disease Agency. EPA (Environmental Protection Agency, USA) has established the discharge limit of Cr(VI) to surface water below 0.05 mg L 1 while the total Cr including Cr(III), Cr(VI) and its other forms to below 2 mg L 1 (Baral and Engelken, 2002). The microbial mechanisms of interaction with chromium are of basic and technological relevance, due to the fact that the physicochemical methods currently considered for the removal of chromium at large scale are expensive and cause the generation of a large amount of secondary waste products (Bai and Abraham, 2001). Bioremediation is an alternative actually considered to remove Cr from the environment (Mulligan et al., 2001); this process exploits the genetic and metabolic potential of microorganisms to remove metals by biotransformation and/or biosorption (Chen and Hao, 1998; Cervantes et al., 2001). Filamentous fungi belonging to the genera Aspergillus, Penicillium, Trichoderma and Mucor have been described as having the
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ability to reduce Cr(VI) to Cr(III) in cultures of actively growing cells (Acevedo-Aguilar et al., 2006; Fukuda et al., 2008; MoralesBarrera and Cristiani-Urbina, 2006; Liu et al., 2007). Other works have described fungal strains with the ability to cause the uptake of chromium to the biomass by bioaccumulation/biosorption processes, using growing or inactivated cells; the strains described included isolates of Aspergillus (Nasseri et al., 2002; Prasenjit and Sumathi, 2005; Srivastava and Thakur, 2006a,b; Congeevaram et al., 2007; Mungasavalli et al., 2007; Khambhaty et al., 2009), Hirsutella sp (Srivastava and Thakur, 2006a) and Rhizopus (Bai and Abraham, 2001). An additional process, called ‘‘adsorptioncoupled reduction’’, which occurs mainly at acidic pH, has been described with inactivated cells of Aspergillus and Rhyzopus (Park et al., 2005, 2007). The bioadsorption process takes place at the cell surface mainly at extracellular polymeric substances (EPS), followed by transport into the cytoplasm compartments, through two processes. The first is rapid, nonspecific, and usually based on the chemiosmotic gradient (passive transport), and since it is used by a wide variety of substrates, it is expressed constitutively. The second transport system has a high specificity for the substrate, is slower and often uses ATP hydrolysis as the energy source (active transport), but can also function by osmotic gradient. The captured metal can form complexes with compounds such as proteins rich in thiol groups physiologically present in cells (enzymes of metabolic pathways) or synthesized in the presence of metals (metallothioneins and phytochelatins) (Wang and Chen, 2009). In previous works, the Ed8 strain of A. niger var tubingensis, included in the A. niger species complex, demonstrated high efficiency to transform Cr(VI) to Cr(III) in the extracellular medium, with a restricted chromium sorption to biomass (Acevedo-Aguilar et al., 2006). In Ed8 strain Cr(VI) reduction occurs by a mechanism stimulated by carboxylic acids and metal-chelating agents (Coreño-Alonso et al., 2009). The aim of this paper was to study the ability of A. niger var tubingensis Ed8 to capture chromium extra and intracellularly and its efficiencies to reduce Cr(VI), by means of chemical analysis and electron microscopy techniques in cultures developed in a bubble column bioreactor. 2. Methods 2.1. Microorganism and culture conditions A. niger var tubingensis Ed8 described and characterized previously (Acevedo-Aguilar et al., 2006; Coreño-Alonso et al., 2009) was used in this work. This fungus strain was grown and propagated in PDA medium. Cr(VI) reduction and chromium sorption experiments using this microorganism were realized in batch cultures or in a bubble column bioreactor, using modified Lee minimal medium (LMM) (Lee et al., 1975) containing 0.25% KH2PO4, 0.20% MgSO4, 0.50% (NH4)2SO4, 0.5% NaCl and 0.25% glucose; which were dissolved in citrate buffer 45 mM and the pH of the medium was adjusted to 5.3 with 45 mM citric acid and NaOH (Coreño-Alonso et al., 2009). Cr(VI) reduction experiments in LMM medium containing 3 mM sodium salicylate (LMMS) supplemented with the proper amount of K2CrO4 to reach the indicated concentration of Cr(VI) in the medium were conducted in batch cultures as previously described (Coreño-Alonso et al., 2009). Reduction medium used in bioreactor experiments was based in LMMS, but twofold concentrated (2X) and with saccharose instead of glucose as carbon source; the components of the medium were technique grade reactives, which were dissolved in citrate buffer 45 mM, and pH was 5.3. Medium was supplemented with the proper amount of K2CrO4, to reach the initial Cr(VI) concentration.
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2.2. Batch culture experiments Biomass production in modified LMM medium and Cr(VI) reduction experiments in LMMS medium supplemented with the proper amount of K2CrO4 to reach the indicated concentration of Cr(VI) in the medium, were conducted in batch cultures as previously described (Coreño-Alonso et al., 2009). Then fungal biomass was transferred to LMM containing 50 and 100 mg L 1 of Cr(VI), and samples were analyzed at 0, 12 and 24 h. Cultures were filtered and broth cultures free of biomass were used to determine Cr(VI) reduction and, in another hand, the biomasses were used to analyze the extra or intracellular accumulation of Cr in individual cells. Unpolluted culture was used as a control experiment. 2.3. Scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) microanalysis For SEM analysis, fungal biomass samples from batch cultures were fixed in 4% glutaraldehyde diluted in phosphate buffer (0.1 M and pH7) for 2 h and washed four times in the same buffer, dehydrated in increasing concentrations of ethanol (30%, 50%, 70%, 90%, and 100%) and dried by critical-point drying. Finally, all samples were mounted on metal stubs and coated with gold for better image contrast. A Jeol JSM-6300 scanning electron microscope (Jeol, Tokyo, Japan) was used to view the images. For energy dispersive X-ray microanalysis, cells were homogenously distributed and filtered with polycarbonate membrane filters. These filters were dehydrated and dried by critical-point drying and then coated with gold. An energy dispersive X-ray spectrophotometer (EDX) Link Isis-200 (Oxford Instruments, Bucks, England) coupled to a Zeiss EVOÒ MA 10 scanning electron microscope (Carl Zeiss NTS GmbH, Oberkochen, Germany) operated at 20 kV was used. Finally, EDX–SEM spectra from individual cells were obtained. 2.4. Transmission electron microscopy (TEM) and energy dispersive Xray (EDX) microanalysis For TEM analysis, fungal biomass samples from batch cultures were fixed in glutaraldehyde as mentioned above. Later, samples were post-fixed in 1% OsO4 at 4 °C for 2 h, and washed four times in the same buffer. They were then dehydrated in a graded series of acetone (50%, 70%, 90%, 95% and 100%) and embedded in Spurr’s resin. Ultrathin sections (70 nm), realized with a Leica UCG Ultramicrotome, were mounted on carbon coated copper grids and stained with uranyl acetate and lead citrate. Samples were viewed in a Hitachi H-7000 transmission electron microscope (Hitachi LTD, Tokyo, Japan). For energy dispersive X-ray microanalysis, sections of 200 nm thick were also stained with uranyl acetate and mounted on carbon-coated grids. Samples were analyzed with an EDX Link Isis-200 (Oxford Instruments, Bucks, England) coupled to a Jeol Jem-2011 (Jeol Ltd., Tokyo, Japan) operating at 20 kV. Finally, EDX–TEM spectra were obtained. In all EDX spectra (SEM and TEM) the main Cr peak was detected at 5.4 keV of energy. 2.5. Bubble column bioreactor experiment A laboratory scale bubble column bioreactor was fabricated from glass column of 7 cm diameter by 40 cm high with a total capacity of 1 L. The reactor was worked in two stages: growth and reduction stages. The column containing 800 mL of LMM was inoculated with 5 105 spores of A. niger var tubingensis Ed8 mL 1 with an air flux of 8.5 L min 1 and incubated at room temperature during 60 h (growth stage). After growth, in the
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reduction stage, a concentration of 100 mg Cr(VI) L 1 from a synthetic stock solution (55,000 mg L 1) was added to the culture containing fungal biomass, and incubated during 12 h. After this time, 50% of initial volume of the culture was removed and replaced by the same volume of fresh 2X Lee modified medium and the mixture was incubated for 12 h. In further 12 h-incubation periods, 12.5% of initial volume was removed and replaced by 2X fresh medium containing 100 mg Cr(VI) L 1; these incubations were continued until a total of 60 h from the start of the experiment. Immediately after the addition of Cr(VI) (T = 0) and then every 3 min, 5 mL samples were taken, which were used for the determination of Cr(VI) and total chromium. The biomass obtained at the end of incubation (60 h) was recovered by filtration, washed repeatedly with distilled water and lyophilized to obtain its dry weight, and then the biomass was processed for determining the chromium incorporated therein. 2.6. Analytical determinations Cr(VI) was quantified by a spectrophotometric method employing diphenylcarbazide (DPC) (Greenberg et al., 1981). Total chromium content in the supernatant of the cultures and in the biomass was determined by ICP-MS as previously described (Acevedo-Aguilar et al., 2008). 3. Results and discussion 3.1. Cr(VI) reduction in batch cultures After 24 h of incubation of strain A. niger var tubingensis Ed8 in LMM medium containing an initial concentration of 50 mg Cr(VI) L 1, a decrease of about 94% in Cr(VI) levels was observed; in the same period, in medium with 75 and 100 mg Cr(VI) L 1, the biomass of Ed8 strain caused a decrease of about 60% and 56% in Cr(VI) levels (Table 1). The pH of the cultures changed only from 5.3 to 5.0 because the buffer citrate in the medium. It has been described that in Ed8 strain cultures incubated with 50 mg Cr(VI) L 1 under conditions identical to those employed in this study, most (>98%) total residual chromium was found in the solution, providing evidence indicative of soluble Cr(III)-complex formation, with the consequent increase in Cr(VI) reduction (Acevedo-Aguilar et al., 2008). The observation of a less marked decrease in Cr(VI) levels in Ed8 strain cultures incubated in the presence 100 mg Cr(VI) L 1, probably is due to toxicity effects of Cr(VI). 3.2. Cr(VI) bioaccumulation/sorption in batch cultures Although it is known that in actively growing fungal cells Cr(VI) is incorporated by means of the sulfate permease proteins (Cervantes et al., 2001) or by type CHR-1 transporters (Flores-Alvarez et al., 2012), less is known about the specific molecules that allow ion binding to the cell surface; in part, this is due to the complex nature of the microbial biomass, which is not readily amenable to instrumental analysis (Kapoor and Viraraghavan, 1995). In this work, different techniques of high resolution electron microscopy, SEM and TEM, and both coupled to EDX were
optimized and applied to determine the capacity of A. niger var tubingensis Ed8 to capture Cr(VI) extra or intracellularly. The results obtained from unpolluted samples, as it was expected, Cr was not detected in cells either at extracellular level (SEM–EDX) or at intracellular level (TEM–EDX). Samples of A. niger var tubingensis Ed8 polluted by Cr(VI), and analyzed by SEM, showed morphological changes on fungus hyphae, at both Cr concentrations 50 mg L 1 and 100 mg L 1 and at different exposure time. The most dramatic effect of Cr(VI) on the morphology of the fungus is observed at the highest concentration of the ion since the produced hyphae were short, curved and constricted along its length. Nevertheless, Cr was not detected in their EDX spectra. From these results it can be concluded that A. niger var tubingensis Ed8 cells cannot adsorb this metal externally, either in the surface and/or the EPS. TEM analysis show ultra-structural changes in the hyphae produced by the fungus when this was grown in the presence of Cr(VI). Two types of inclusions with different electron density were observed. The first type was high electron-dense and increased in number at higher Cr concentrations. These inclusions were distributed through the cell wall into the periplasmic space of the cells. Burnat et al. (2010) identified this type of inclusions, by TEM– EDX, as inclusions of polyphosphate in Microcoleus consortium polluted by lead. Although different authors have demonstrated the capacity of the high electron-dense inclusions to capture metals, chromium was not detected in EDX spectra from these inclusions (Burnat et al., 2010; Maldonado et al., 2010, 2011; Puyen et al., 2012; Seder-Colomina et al., 2013). The second type of inclusions were low electron-dense, and were observed in samples exposed to Cr(VI). The analysis of these inclusions by TEM–EDX allowed the detection of the principal peak of Cr at 5.4 keV of energy in the EDX spectra. The results showed the ability of A. niger var tubingensis Ed8 to capture this metal intracellularly. 3.3. Cr(VI) reduction in bioreactor column Previous studies showed that the strain A. niger var tubingensis Ed8 has the capability to reduce Cr(VI) in the extracellular environment, with low amounts of chromium bound to the biomass (Acevedo-Aguilar et al., 2006) and that this capacity for chromium reduction is stimulated by organic acids (Coreño-Alonso et al., 2009). Taking these observations into consideration, we envisaged a rapid and efficient process for Cr(VI) reduction by culturing A. niger var tubingensis Ed8 in a bubble column reactor in the absence of Cr(VI), which was followed by the addition to the culture of this ion and further incubation periods with subsequent medium and Cr(VI) loads. Thus, a culture of A. niger var tubingensis Ed8 was incubated for 60 h in LMM and Cr(VI) was then added. The pH of the medium did not change appreciably; it was between 5.3 and 5.0 because the reduction medium contains citrate buffer. An abrupt decrease in Cr(VI) content was observed when this ion was added and no Cr(VI) was detected 3 min later (time 0 in Fig. 1a). In the first load of 100 Cr(VI) mg L 1 a reduction of 93% of Cr(VI) content was observed immediately after Cr(VI) addition,
Table 1 Cr(VI) reduction by A. niger var tubingensis Ed8 in LMM broth cultures. Incubation period (h)
Cr(VI) remaining (mg L 50 Cr(VI) mg L
0 12 24
1
57 ± 3.0 (0%) 34.5 ± 2.5 (31%) 2.9 ± 0.3.0 (94.2%)
1
) and rate of Cr(VI) reduction (%) in cultures containing: 75 Cr(VI) mg L
1
78 ± 4.1 (0%) 56.16 ± (28%) 46.8 ± 2.1 (60%)
100 Cr(VI) mg L
1
111 ± 4.5 (0%) 73.4 ± 5.02 (26.6%) 44 ± 3.4 (56%)
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a
70
Cr(VI) (mg L-1)
60 50 40 30 20 10 0
Total Cr (mg/L)
b
0
12
24
Time (h)
36
48
60
in the bubble column bioreactor of 2.54 mg Cr(VI) reduced g 1 dry biomass h 1 (biotransformation) and 0.082 mg Cr(VI) accumulated g 1 dry biomass h 1 (bioaccumulation). Few studies have been reported to reduce Cr(VI) employing bioreactors, by example Srivastava et al. (2007) and Tziotzios et al. (2008) reported a sequential bioreactor and a biofilm packed reactor to reduce Cr(VI) and remove chlorophenols simultaneously. In these studies significant amounts of Cr(VI) were removed, some of them by reduction, although this occurred in periods of several hours or days. Moreover, regarding the chromium incorporated into the biomass of the strain A. niger var tubingensis Ed8, the results showed an incorporation of 4.9 Cr g 1 dry weight, which coincides with the range of chromium incorporation in biomass (1.8–8.9 mg Cr g 1 dry weight) described for A. niger strains (Srivastava and Thakur, 2006b). 4. Conclusions
500
The results of this work indicated that the interaction of A. niger var tubingensis Ed8 with Cr(VI) is based mainly in a reduction process, and low ability of the fungus to incorporate Cr. This Cr is incorporated into low electron-dense inclusions from nature not confirmed, that means, accumulates chromium intracellulary. Due to the loads of fresh medium and the fast reduction of Cr(VI) in the cultures, the fungus is kept viable. The bubble column reactor employed showed advantages as low cost of construction and maintenance, lower stress and fragmentation of mycelia, a high efficiency of Cr(VI) reduction.
400 300 200 100 0
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Acknowledgements
0
12
24
36
48
60
Time (h) Fig. 1. Cr(VI) reduction by A. tubingensis Ed8 in a bubble column bioreactor. White bars indicate Cr(VI) at the moment of Cr(VI) addition and black bars indicate Cr(VI) after 3 min of addition (a). Total Cr detected after each time of addition of Cr(VI) (b).
being such reduction of 100% after 3 min of metal addition. As it was previously mentioned, the loads of Cr(VI) to the bubble column reactor were made each 12 h. In the loads at 24 and 36 h of incubation, Cr(VI) reduction in the supernatant was almost complete (95–100%) immediately after the metal addition. These results indicate that the maximum efficiency to reduce Cr(VI) by the fungal biomass was at these times (24 and 36 h) (Fig. 1a). The total Cr content in the supernatant of the bioreactor increased according to the Cr(VI) added in the successive loads, reaching a final concentration of 460 mg L 1 at 60 h (Fig. 1b). After this time no more loads of chromium were made, due to the high viscosity acquired by the culture as a consequence of mycelium growth. The observation that the total chromium content in the culture supernatant increases as Cr(VI) is added, without noticeable differences with respect to the theoretical amount of the ion added, and that the content of Cr(VI) in the culture decreases quickly and dramatically, indicated that under the conditions of the bioreactor the transformation of Cr(VI) to Cr(III) occurred in a highly efficient form. The biomass obtained after 60 h of incubation was 3.02 g (dry weight) and the total Cr(VI) incorporated into this biomass and reduced to Cr(III) in the same period of time were 14.8 mg and 460 mg of Cr, respectively. Thus, a total concentration of 474.8 mg Cr(VI) was added to the bioreactor during the whole experiment (60 h). From these results, the total Cr(VI) removed in the bioreactor was 2.62 mg Cr(VI) g 1 dry biomass h 1 being the efficiencies of Cr(VI) reduction and accumulation by A. niger var tubingensis Ed8
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