The objective of this work was to study the removal of chromium (VI) in aqueous solution by the fungus Penicillium sp. IA-01, isolated from polluted air with industrial vapors. To obtain the fungal biomass, pre-inoculums were performed in thioglycolate broth from a strain isolated from vapours contaminated with Cr (VI). The fungus was incubated for four weeks at ambient temperature, filtered, and washed three times with trideionized water. In preparing cellullar fractions, it was necessary to break the fungal cells with glass beads using a homogenizer being careful to keep the samples in frosty cold ice. To obtain the fungal biomass, the fungus was filtered and stored in an oven at 80°C, allowing it to dry for 48 h. Removal of Cr (VI) in vitro was evaluated using different cellular fractions and dead fungal biomass. We determine the optimal characteristics for metal removal in the reaction mixture. Concluding that the ideal conditions for the removal of Cr (VI) in the cell extracts were 37°C and pH 7.0, also we observ that the highest enzyme activity was in the mixed membrane fraction. In dead fungal biomass, the ideal conditions for removal of metal are 60°C and pH 1.0.
- Fugal biomass
- Cellular fractions
- Chromium (VI)
Environmental pollution with heavy metals is caused by anthropogenic and natural actions. Discharges of wastewater from various industrial activities such as electroplating, mining, paint factory, plastics, coating metal cables, and automotive radiators, and certain industries producing energy, metal engineering industry and producers of welding materials contain high concentrations of metals. Several heavy metals are highly toxic and ingestion of these metals by drinking contaminated water or breathing polluted air can cause serious health problems in human beings. Several metals are considered toxic at certain levels of concentrations in wastewater, such as arsenic, cadmium, cobalt, copper, chromium, nickel, lead and mercury. Unlike the organic compounds, heavy metals cannot be biodegraded or destroyd, therefore they must be removed. There are several methods for removal of heavy metals: ion exchange, membrane separation, and separation and electrochemical adsorption on various adsorbents. .
Chromium (Cr) is one of the major environmental pollutants coming from industrial effluents and tannery. It is considered the major pollutant cataloged by the United States Environmental Protection Agency (EPA: www.epa.gov), since it is stable in aqueous solution and hence high in mobility in different environments. Chromium is a metal element in the periodic table. It is odorless and tasteless; is found in rocks, plants, soil, and volcanic dust, humans and animals; and exists in the environment most commonly as the trivalent [(chromium (III))], hexavalent [(chromium (VI))] and metallic [(chromium (0))]. Chromium (III) is generally contained in many vegetables, fruits, meats, grains and yeast. Industrial processes generally produce chromium (VI) and chromium (0). The main sources of chromium (VI) in drinking water are discharges from steel and pulp, and erosion of natural deposits of chromium (III). In many places, chromium compounds have been scattered to the environment through leaks, poor storage or improper disposal practices. The chromium compounds are very persistent in water and sediment .
Chromium is regarded as an environmental pollutant due to its wide use in various industrial activities, such as electrolytic plating, leather tanning, explosives manufacturing etc. The stable forms of chromium in the environment are trivalent (Cr (III)) and hexavalent chromium (Cr (VI)). Further, Cr (VI) is highly soluble, making it mobile in soil and aquatic environments, with consequent toxicity ecosystems. Chromium in their different forms can be use in the production of steel alloys and other metals chromed, for dyes and pigments, and the preservation of leather and wood. It can also be find naturally in the soil. The primary forms of chromium found in nature are chromium (III) and chromium (VI) and these forms are converted to each other depending on environmental conditions . Cr (VI) is consider the most toxic form of chromium, and is usually associated with oxygen as chromates (CrO4–2) and dichromates (Cr2O7–2) , which due to its high solubility are highly mobile in soil environments and water . Moreover, Cr (III) is in the form of oxides, hydroxides or poorly soluble sulfates, by which it is much less mobile, and there joined organic matter in the soil and aquatic environments [5, 6]. Cr (VI) is a strong oxidizing agent, and in the presence of organic matter is reduced to Cr (III); this transformation is faster in acidic environments . However, high levels of Cr (VI) may exceed the reducing capacity of the environment and thus can persist as a contaminant. It has been established now that various chromium compounds as oxides, chromates and dichromates, are environmental contaminants in water, soil, and industrial effluents, because this metal is widely used in various manufacturing, such as electrolytic plating, explosives manufacturing, leather tanning, metal alloy, dyes and pigments manufacturing, etc. [1, 5].
There are studies of many methods for removal of chromium ion present in water industrial waste, for example: ion exchange on resins, coagulation-flocculation, adsorption on activated carbon, reduction, chemical precipitation, sedimentation, etc., , which in most cases are expensive or inefficient, especially when the concentration of these ions is very low . Therefore arise emerging technologies such as biosorption, the process of attracting various chemical species by biomass (live or dead), by physicochemical mechanisms as adsorption or ion exchange .
Fungal cells interact with chromium at different levels from the cell wall and, from the periplasm to the cytoplasm and cell organelles. These microorganisms require detecting and regulating intracellular levels of chromium through homeostasis systems that maintain a balance between the incorporation, expulsion, and arrest of metal . It is common for native microorganisms of sites contaminated with chromate ion, show resistance because they have asset or liability mechanisms that allow them to remove from detoxification. In certain species these mechanisms are know in detail, some of which are of basic interest and biotechnological importance, the latter in the context of developing new technologies for the treatment of industrial effluents and for bioremediation of contaminated sites. These mechanisms generally include biotransformation of Cr (VI) reduced species (chemical reduction), which may be direct (enzymatic) or indirect (enzyme); incorporation and bioaccumulation; biosorption of Cr (III), and Cr (VI); and immobilization [1, 9]. Some filamentous fungi reduce Cr (VI) to Cr (III), by different mechanisms of Cr (VI) detoxification, like reducing power generated by carbon metabolism [10, 11, and 12].
2. Materials and methods
2.1. Screening of the microorganism showing the resistant to Chromium (VI) and chromate resistance test
We isolate a chromate resistant mycelial fungus from polluted air near the Faculty of Chemical Science, UASLP (San Luis Potosí, México), and this was used for the screening. The chromate resistant filamentous fungus contained in the air was grown on the Petri dish containing modified Lee’s minimal medium (LMM) (with 0.25% KH2PO4, 0.20% MgSO4, 0.50% (NH4)2SO4, 0.50% NaCl, 0.25% glucose, and 2% agar) supplemented with 500 mg/L K2CrO4; the pH of the medium was adjusted and maintained at 5.3 with 100 mmol/L citrate-phosphate buffer. The plates were incubated at 28∘C for seven days. The strain was identified based on characteristic macroscopic and microscopic observation . Fungal cultures grown in thioglycolate broth were used as primary inoculums. Chromate-resistant tests of the isolated strain, filamentous fungus
2.2. Biosorption tests by using dry cells
The fungal cells was grown at 28°C in an stirred and aerated liquid media containing thioglycolate broth at a concentration of 8g/L (p/v). After five days of incubation, the cells were recovered by centrifugation (3000 rpm/10 min), and washed twice in the same conditions with deionized wáter, and subsequently it was dry (80°C/24 h) in an oven. Solutions of Cr (VI) for analysis, were prepared by diluting 71.86mg/L of stock metal solution. The concentration range of chromium (VI) solutions was 50-1000mg/L. The pH of each solution was adjusted to the required value by adding 1M H2SO4 solution before mixing with the microorganism. The biosorption of the metal by fungal dry cells was determined at different concentrations (50–1,000mg/L) of 100 mL Cr (VI) solution, with 1g of fungal biomass, at 120 rpm, and the sample was filtered. The filtrate containing the residual concentration of Cr (VI) was determined spectrophotometrically. For the determination of rate of metal biosorption, 200, 400, 600, 800, and 1,000mg/L of Cr (VI) solution was used. The supernatant was analyzed for residual Cr (VI) after the contact period at different times. For determination of the effects of pH and temperature, four solutions (pH 1, 2, 3, and 4) and temperatures (28, 40, 50, and 60℃) were respectively used.
Moreover, biosorption to the contaminated soil and water was examined. Four Erlenmeyer glass flasks containing 5g of fungal biomass and 20g of contaminated soil and 20 mL of water (297mg Cr (VI)/g soil or 155mg Cr(VI)/L water), of tannery (Celaya, Guanajuato, México), was completed to 100 mL with trideionized water, were incubated during seven days at 120 rpm, and filtered in Whatman filter paper No. 1, and the concentration of Cr (VI) of the filtrate analyzed with 1, 5 diphenylcarbazide .
2.3. Reduction of Cr (VI) by living cells
Reduction efficiency of Cr (VI) by living, resting, and permeabilized cells was examined. To examine the living cells, cultures in 100 mL of LMM were inoculated with 5
Reduction efficiency of Cr (VI) was examined by the resting cells. 5
Reduction efficiency of Cr (VI) was also examined by the permeable fungal cells. Culture of
2.4. Activity of chromate reductase
Cell-free extracts (CFE) of
Enzymatic chromate reduction was estimated as described previously using a standard curve of Cr (VI) 0–30 mM. The assay was as follows: The reaction system (1.0 mL) was made up of varying Cr (VI) final concentrations (5–30 mM) in 700 µL of 100 mM potassium phosphate buffer (pH 7.0) added with 250 µL aliquots of CFE for chromate reduction and 50 µL of NADH. The system volume of 1.0 mL was kept constant for all experiments. Chromate reductase activity was measured at 37∘C at different pH values using several buffers (100 mM phosphate citrate, pH 5.0; 50 mM phosphate, pH 6.0–8.0, and 50 mM Tris-HCl, pH 8-9). The effect of temperature was studied by measuring chromate reductase activity at different incubation temperatures between 20 and 60∘C, at optimum pH. The CFE samples were also treated with several metal ions to a final concentration of 1mM at optimal pH and temperature; Na+, Ca2+, Cu2+, Hg2+, Mg2+, Cd2+, and Fe3+ were tested by using 10 mM solutions of Na2SO4, CaCl2, CuCl2, HgCl2, MgCl2, CdCl2, and FeCl3. The electron donors tested were NADH, glucose, sodium acetate, formic acid, citrate, cystin, lactic acid, and ascorbic acid in a final concentration of 1mM, and the inhibitors were EDTA, KCN, NaN3, and β-mercaptoethanol at the same concentration. For chromate reductase activity, one unit was defined as enzyme that reduces 1mmol of Cr (VI)/min/37∘C, and the specific activity was defined as unit chromate reductase activity/min/mg protein in the CFE. Protein concentrations were determined by the Lowry method .
2.5. Determination of hexavalent, trivalent, and total amount of chromium
Hexavalent and trivalent chromium were quantified employing diphenylcarbazide  and chromazurol S , respectively, the total amount of Chromium was determined by electrothermal atomic absorption spectroscopy . Tree dependent experiments were carried out and the mean value was shown
3. Results and discussion
3.1. Isolation and identification of a fungal strain tolerant to Cr (VI)
Microorganism was grown on the LMM agar plates containing 500 mg/L of K2CrO4, and the largest colony of fungi was isolated. Colonies isolated grew rapidly within three days. Colonies are usually fast growing, in shades of green, sometimes white, mostly consisting a dense conidiophores. Microscopically, chains of single-celled conidia (ameroconidia) are produced in basipetal succession from a specialized conidiogenous cell called phialide. In
The cells of the isolated strain grew on LMM supplemented with 2 g/L of Cr (VI) about 50% of growth relative to control (85mg of dry weight without metal) was obtained (Figure 2) and, therefore probably is resistant to the metal. Different microorganisms that are Cr (VI) resistant have been isolated from different contaminated sites [1, 16, 26, and 27], and Chromate tolerance has been described in the mutants of stocked culture, and in native isolates of contaminated sites, as in this work; in several cases, both yeast and filamentous fungi showed that tolerance to Cr (VI) is due to transport of sulfate disturbance that leads to reduced incorporation of chromate  in other cases, phenotypes of hypersensitivity to Cr (VI) are produced as a result of alteration of the vacuolar ATPase and vacuolar structures  or by alteration of proteins that protect the oxidative effect of Cr (VI) as the alkyl hydroperoxide reductase  or Cu-Zn-superoxide dismutase and methionine sulfoxide peptide reductase . However, the mechanism of tolerance in
3.2. Absorption of Cr (VI) by the dry cells of
First, the ability of absorption was examined by using
With respect to the influence of initial pH on removal efficiency, it was found that the highest activity was evident at pH 1.0, at 150 min the metal is removed, while at pHs 2, 3, and 4, the authors did not observe significant differences (20% of removal), and at neutral or alkaline pH´s, there was no removal (Figure 4). A pH optimum has been reported of 1.0 to removal Cr (VI) by fruiting bodies of the jelly fungus
Temperature is found to be a critical parameter in the bioadsorption of Cr (VI) (Figure 5). The highest removal was observed at 50oC and 60°C. At this point the total removal of the metal is carried out at 100%, at 40 min. These results are likely for
At different metal concentrations (200, 400, 600, 800, and 1000mg/L), biomass studied, shows the same results for removal, adsorbing 100% between 210 and 240 min while 1000mg/L of metal is removed 100% up to 90 min of incubation (Figure 6) with respect to other fungal biomasses, some reports argue that the amount of metal increases in direct proportion with the increase in concentration of the metal ion in solution [35, 37], and others author report lower removal efficiencies of metal, for example 25 and 250mg/L of chitin and chitosan , and 1mg/L for cellulose acetate . This was due probably to the increase in the number of ions found competing for the available functions groups on the surface of biomass .
The influence of the biomass on the removal capacity of Cr (VI) was depicted in Figure 7. If we increase the amount of biomass, we also increase the removal of Cr (VI) in the solution (although there is a 100% of remotion, with 3, 4, and 5g of biomass, 60 min), perhaps due to increased of biosorption sites of the same, because the amount of added biosorbent determines the number of binding sites available for metal biosorption . Similar results have been reported for
3.3. Removal of Cr (VI) in industrial wastes with fungal biomass
For the removal of the metal from industrial wastes, we incubate the fungal biomass (5g) with non-sterile oil and contaminated water (297mg Cr (VI)/g, and 155mg Cr (VI)/L), suspended in trideionized water. It was observ that after seven days of incubation with the biomass, the Cr (VI) concentration from soil and water samples decreased to 63.24% and 43%, respectively (Figure 8), without significant change in total chromium (not shown). In the absence of the biomass, the metal concentration of the soil samples decreased slightly (18%, not shown), maybe caused by indigenous microflora and (or) reducing components present in the soil [10, 11, and 18]. The capacity of
3.4. Removal of Cr (VI) by living cells of
Next, the reduction of Cr (VI) by
In Figure 10, the effect of the biomass concentration (72, 141, and 169 mg of dry weight) on Cr (VI) removal, with percentages of removal of 35%, 49%, and 60%, respectively, is shown. Similarly, most of the reports in the literature observe at higher biomass dose resulting in an increase in the percentage removal [3, 7, 8, 13, 16, 47, and 52]. With higher biomass dose, there are more binding sites for complex of Cr (VI) (e.g., HCrO4- and Cr2O7
Figure 11, shows the effect of Cr (VI) concentration (50 to 200mg/L) on the removal of the same. If we increase the concentration of the metal, the removal of metal decreases (60%, 50%, 28%, and 11%, respectively. This is probably because, if we increase initial metal concentration, we increase the number of ions competing for the available functions group on the surface of biomass. Our observations are like most of the reports in the literature [3, 7, 8, 37, 47, 48, 53].
With different carbon sources, like fermentable: glucose, sucrose, and citrate, and oxidable (glycerol). With glucose, sucrose, and citrate, the decrease in Cr (VI) levels occurred at a different rate, at six days of incubation (52%, 47%, and 27%, respectively), and the other carbon sugars were less effective (glycerol 7% of removal). With another inexpensive commercial carbon sources like unrefined sugar and brown sugar, the decrease in Cr (VI) levels occurred at a similar rate (96% and 100%, respectively) (Figures 12(a), (b)). If we incubate the fungal biomass without a carbon source, there are no changes in the initial Cr (VI) concentration during the experiment (data not shown), suggesting that a carbon source is required to decrease Cr (VI) concentration in the growth medium. Our results are similar to some reports: how in chromate-resistant strains of filamentous fungi indigenous to contaminated wastes, with
3.5. Adsorption and reduction by resting and permeable cells
We also estimated the ratio of absorption and/or reduction to adsorption, as we found that the fungi
The cell permeabilization increased the Cr (VI) reduction by the resting cells, as the permeabilized cells with Triton X-100 which could reduce 57%, toluene 52%, SDS 47.4%, and Tween 80 40.4% (Figure 14) of 30 mM Cr (VI) within 6 h, suggesting an efficient intracellular mechanism of chromate reduction. The Cr (VI) reductase activity in CFE of cells grown in the absence of Cr (VI) was 94.07 μmoles/min/mg protein.These results indicate that the Cr (VI) reductase was associated with the CFE. Fungal, yeast, and bacteria chromate reductases have been localized either in CFE of
3.6. Chromate reductase activity
The result of permeable cells (Figure 14) suggests that
The optimal temperature for the Cr (VI) reductase activity was 37°C, but the reductase activity was altered significantly at 20°C (39% of inhibition); but when the assays were performed at 50°C the reductase activity showed 14.2% of inhibition (Figure 16). For
The effect of different metal cations on the chromate reductase activity of
The reductase activity increased on supplementation in the reaction mixtures with electron donors. All the electron donors analyzed increased the activity, and the most efficient were ascorbic acid, NADH, glucose, and citrate by 4.4, 4.0, 2.9, and 2.87 times, respectively (Figure 18), and these results are like those reported for the yeasts
Respiratory inhibitors like azide (1mM), EDTA (1mM), and cyanide (1mM) caused inhibitions of 48%, 47%, and 32%, respectively (Figure 19), in the Cr (VI) reductase activity; these results agree with those obtained in previous studies , and it has been observed that cyanide and azide partially inhibited purified chromate reductase of
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