**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 *Penicillium,* the phialides may be produced singly, in groups or from branched metulae, giving a brushlike appearance known as a penicillus (Figure 1) [25].

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 [28] in other cases, phenotypes of hypersensitivity to Cr (VI) are produced as a result of alteration of the vacuolar ATPase and vacuolar structures [29] or by alteration of proteins that protect the oxidative effect of Cr (VI) as the alkyl hydroperoxide reductase [30] or Cu-Znsuperoxide dismutase and methionine sulfoxide peptide reductase [31]. However, the mechanism of tolerance in *Penicillium* sp IA-01fungus are not investigated. Thus, we precisely examined the characteristics of the *Penicillium* strain to estimate the mechanism in the follow‐ ing experiments.

**Figure 1.** Macroscopic and microscopic morphology of the fungus *Penicillium* sp IA-01

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

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

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

activity/min/mg protein in the CFE. Protein concentrations were determined by the Lowry

Hexavalent and trivalent chromium were quantified employing diphenylcarbazide [22] and chromazurol S [24], respectively, the total amount of Chromium was determined by electro‐ thermal atomic absorption spectroscopy [22]. Tree dependent experiments were carried out

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 *Penicillium,* the phialides may be produced singly, in groups or from branched metulae, giving a brush-

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

**2.5. Determination of hexavalent, trivalent, and total amount of chromium**

**3.1. Isolation and identification of a fungal strain tolerant to Cr (VI)**

like appearance known as a penicillus (Figure 1) [25].

several metal ions to a final concentration of 1mM at optimal pH and temperature; Na+

C at different pH values using several buffers (100 mM phosphate

C, and the specific activity was defined as unit chromate reductase

C, at optimum pH. The CFE samples were also treated with

, Ca2+,

activity was measured at 37<sup>∘</sup>

1mmol of Cr (VI)/min/37<sup>∘</sup>

and the mean value was shown

**3. Results and discussion**

method [23].

temperatures between 20 and 60<sup>∘</sup>

170 Advances in Bioremediation of Wastewater and Polluted Soil

#### **3.2. Absorption of Cr (VI) by the dry cells of** *Penicillium* **sp. IA-01**

**3.2 Absorption of Cr (VI) by the Dry Cells of** *Penicillium* **sp. IA-01** 

First, the ability of absorption was examined by using *Penicillum* sp. IA-01 cells to clarify the mechanism of Cr (VI) tolerance. Figure 3 shows the effect of the incubation time on Cr (VI) removal by *Penicillium* sp biomass. The optimum time for Cr (VI) removal was 150 min at constant values of pH (1.0), biosorbent dosage (1g 100/mL), initial metal concentration (50mg/ L) and temperature (28°C). Some studies [32], report an incubation time of 48 h at pH 1.0 by fruiting bodies of the jelly fungus *Auricularia polytricha,* 24 h for *C. fabianii, W. anomalus* and *C. tropicalis*, at pH range between 2 and 4 for the three species [16], to pH of 2.0 to five days for *Aspergillus niger* [20], the latter with 10 g/L of biomass, and at the same pH of 2.0. Changes in the permeability of the fungal cell wall, of unknown origin, could partly explain the differences found in the incubation time, providing greater or lesser exposure of the functional groups of the cell wall of the biomass tested [33].

**Figure 1.** Macroscopic and microscopic morphology of the fungus *Penicillium* sp IA-01

**Figure 2.** Growth in dry weight of *Penicillium* sp., IA-01with different concentrations of Cr (VI), 1 × 105 spores/mL, 28°C, seven days of incubation, 100 rpm. 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)

First, the ability of absorption was examined by using *Penicillum* sp. IA-01 cells to clarify the mechanism of Cr (VI) tolerance. Figure 3 shows the effect of the incubation time on Cr (VI) removal by *Penicillium* sp biomass. The optimum time for Cr (VI) removal was 150 min at constant values of pH (1.0), biosorbent dosage (1g 100/mL), initial metal concentration (50mg/L) and temperature (28°C). Some studies [32], report an incubation time of 48 h at pH 1.0 by fruiting bodies of the jelly fungus *Auricularia polytricha,* 24 h for *C. fabianii, W. anomalus* and *C. tropicalis*, at pH range between 2 and 4 for the three species [16], to pH of 2.0 to five days for *Aspergillus niger* [20], the latter with 10 g/L of biomass, and at the same pH of 2.0. Changes in the permeability of the fungal

Figure 1. Macroscopic and microscopic morphology

morphology of the fungus Penicillium sp IA-01

permeability of the fungal cell wall, of unknown origin, could

mechanism of Cr (VI) biomass. The optimum 100/mL), initial metal at pH 1.0 by fruiting pH range between 2 g/L of biomass, and at could partly explain the groups of the cell wall of

1.0 g of fungal biomass.

activity was evident at pH significant differences (20% been reported of 1.0 to

on removal efficiency, it was found that the highest activity

**Figure 2.** Growth in dry weight of *Penicillium* sp., IA-01with different concentrations of Cr (VI), 1 × 105 spores/mL, 28°C, seven days of incubation, 100 rpm. differences found in the incubation time, providing the biomass tested [33]. providing greater or lesser exposure of the functional groups

the same pH of 2.0. Changes in the permeability

Figure 3. Effect of incubation time on Cr (VI) removal removal by Penicillium sp. 50mg/L Cr (VI), 100 rpm, 28°C, pH, and 1.0 **Figure 3.** Effect of incubation time on Cr (VI) removal by *Penicillium* sp. 50mg/L Cr (VI), 100 rpm, 28°C, pH 1.0, and 1.0 g of fungal biomass.

With respect to the influence of initial pH on

by fruiting bodies of the jelly fungus *A. polytricha* [32] and for the yeast *Saccharomyces cerevi‐ siae* and the fungi *Rhizopus arrhizus* a pH range of 1.5-2.5, at 4 h [34], although most indicate a pH optimum range of 2.0 to 4.0 with the yeasts *C. fabianii, W. anomalus* and *C. tropicalis,* isolated from sediments in Morocco [16], both *Mucor hiemalis* [35] and *R. arrhizus* [36], at 24 h, *Rhizopus nigricans* in 8 h [37]. The Cr (VI) is found as HCrO4 - , Cr2O7 2-, CrO4 2-, Cr4O132-, and Cr3O102 - [12]. A decrease in pH causes protonation of the surface of the adsorbent, which induces a strong attraction for the Cr (VI) ions from solution negatively charged, so that the biosorption increases with increasing acidity of the solution. However, as the pH increases, the concen‐ 1.0, for a 150 min the metal is removed, while of removal), and at neutral or alkaline pH´s, while at pHs 2, 3, and 4, the authors did not observe significant pH´s, there was no removal (Figure 4). A pH optimum has been

tration of OH- ions increases, causing changes in the surface of the adsorbent and preventing the biosorption negatively chargen of Cr (VI) ions, thereby decreasing the adsorption of metal to high pH values. It has been reported [11] that while Cr (VI) is obtain by eliminating indigenous strains of filamentous fungi, most of the cations may be reduced to Cr (III).

**Figure 4.** Effect of pH on Cr (VI) removal by *Penicillium* sp. 50mg/L Cr (VI), 100 rpm, 28°C, and 1g of fungal biomass.

Incubacion time (min) 28°C, seven days of Temperature is found to be a critical parameter in the bioadsorption of Cr (VI) (Figure 5). The highest removal was observed at 50o C 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 *Paecilomyces* sp [10] and *A. niger* [20] at 45o C and 24 h, but are different for *R. arrhizus* [36]. The increase in temperature increases the rate of removal of Cr (VI) and decreases the contact time required for complete removal of the metal, to increase the redox reaction rate [38].

1.0 g of fungal biomass. activity was evident at pH significant differences (20% been reported of 1.0 to 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 [36], and 1mg/L for cellulose acetate [39]. This was due probably to the increase in the number of ions found competing for the available functions groups on the surface of biomass [38].

by fruiting bodies of the jelly fungus *A. polytricha* [32] and for the yeast *Saccharomyces cerevi‐ siae* and the fungi *Rhizopus arrhizus* a pH range of 1.5-2.5, at 4 h [34], although most indicate a pH optimum range of 2.0 to 4.0 with the yeasts *C. fabianii, W. anomalus* and *C. tropicalis,* isolated from sediments in Morocco [16], both *Mucor hiemalis* [35] and *R. arrhizus* [36], at 24 h, *Rhizopus*

**Figure 3.** Effect of incubation time on Cr (VI) removal by *Penicillium* sp. 50mg/L Cr (VI), 100 rpm, 28°C, pH 1.0, and 1.0

Figure 3. Effect of incubation time on Cr (VI) removal

0 50 100

With respect to the influence of initial pH on 1.0, for a 150 min the metal is removed, while of removal), and at neutral or alkaline pH´s,

**Figure 2.** Growth in dry weight of *Penicillium* sp., IA-01with different concentrations of Cr (VI), 1 × 105

Figure 1. Macroscopic and microscopic morphology

morphology of the fungus Penicillium sp IA-01

Figure 2. Growth in dry weight of Penicillium sp.,

3.2. Absorption of Cr (VI) by the dry

First, the ability of absorption was examined tolerance. Figure 3 shows the effect of the time for Cr (VI) removal was 150 min at concentration (50mg/L) and temperature (28°C). bodies of the jelly fungus Auricularia polytricha, and 4 for the three species [16], to pH of 2.0 the same pH of 2.0. Changes in the permeability differences found in the incubation time, providing

incubation, 100 rpm.

172 Advances in Bioremediation of Wastewater and Polluted Soil

the biomass tested [33].

A decrease in pH causes protonation of the surface of the adsorbent, which induces a strong attraction for the Cr (VI) ions from solution negatively charged, so that the biosorption increases with increasing acidity of the solution. However, as the pH increases, the concen‐


Incubacion time (min)

2-, CrO4

150

2-, Cr4O132-, and Cr3O102 -

removal by Penicillium sp. 50mg/L Cr (VI), 100 rpm, 28°C, pH, and 1.0

on removal efficiency, it was found that the highest activity while at pHs 2, 3, and 4, the authors did not observe significant pH´s, there was no removal (Figure 4). A pH optimum has been

[12].

spores/mL,

, IA-01with different concentrations of Cr (VI), 1 × 10<sup>5</sup> spores/mL,

examined by using Penicillum sp. IA-01 cells to clarify the mechanism incubation time on Cr (VI) removal by Penicillium sp biomass. at constant values of pH (1.0), biosorbent dosage (1g 100/mL), (28°C). Some studies [32], report an incubation time of 48 h polytricha, 24 h for C. fabianii, W. anomalus and C. tropicalis, at pH 2.0 to five days for Aspergillus niger [20], the latter with 10 g/L permeability of the fungal cell wall, of unknown origin, could providing greater or lesser exposure of the functional groups

dry cells of Penicillium sp. IA-01

*nigricans* in 8 h [37]. The Cr (VI) is found as HCrO4

0

20

40

Cr (VI) in solution (%)

g of fungal biomass.

60

80

100

28°C, seven days of incubation, 100 rpm.

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 [30]. Similar results have been

**Figure 5.** Effect of temperature on Cr (VI) removal by *Penicillium* sp. IA-01. 50mg/L Cr (VI), 100 rpm, pH 1.0, and 1g of fungal biomass.

**Figure 6.** Effect of initial metal concentration on chromium (VI) removal by *Penicillium* sp. IA-01. 28o C. pH 1.0, 100 rpm, and 1g of fungal biomass.

reported for *M. hiemalis* and *R. nigricans*, although the latter has 10g of biomass [35, 37], but different from those reported for biomass wastes from the mandarin (gabassa), whit an optimal concentration of biomass of 100mg/L [40]. Consequently, we found out the following results: *Penicillium* sp. IA-01 has the ability of absorption to Cr (VI) and the value of adsorption is as high as in the fruiting bodies of the jelly fungus *A. polytricha* [32], *C. fabianii, W. anomalus* and *C. tropicalis* [16], *A. niger* [20], *M. hiemalis* and *R. nigricans* [35, 37], and *Paecilomyces* sp. [10]. The adsorption rate was affected by pH, temperature, initial concentration of Cr (VI) and dry cells. In the case of polluted soil and water, around 40% of Cr (VI) could not be removed. Therefore, absorption to the organic compounds contained in polluted soil and water may occurr. The results suggest that the reduction of Cr (VI) is necessary to the bioremediation of soil and air.

**Figure 7.** Effect of biomass concentration on chromium (VI) removal by *Penicillium* sp. IA-01. 50mg/L Cr (VI). 28o C, and pH 1.0, 100 rpm.

#### **3.3. Removal of Cr (VI) in industrial wastes with fungal biomass**

reported for *M. hiemalis* and *R. nigricans*, although the latter has 10g of biomass [35, 37], but different from those reported for biomass wastes from the mandarin (gabassa), whit an optimal concentration of biomass of 100mg/L [40]. Consequently, we found out the following results: *Penicillium* sp. IA-01 has the ability of absorption to Cr (VI) and the value of adsorption is as high as in the fruiting bodies of the jelly fungus *A. polytricha* [32], *C. fabianii, W. anomalus* and *C. tropicalis* [16], *A. niger* [20], *M. hiemalis* and *R. nigricans* [35, 37], and *Paecilomyces* sp. [10]. The

C. pH 1.0, 100

**Figure 6.** Effect of initial metal concentration on chromium (VI) removal by *Penicillium* sp. IA-01. 28o

**Figure 5.** Effect of temperature on Cr (VI) removal by *Penicillium* sp. IA-01. 50mg/L Cr (VI), 100 rpm, pH 1.0, and 1g of

fungal biomass.

174 Advances in Bioremediation of Wastewater and Polluted Soil

rpm, and 1g of 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 *Penicillium* sp., biomass to remove the metal are lower to those reported for other biomasses, like *Litchi chinensis,* [41] tamarind shell [42], *Mammea americana* [43], and *C. reticulata* [44], and equal or better than that of *C. maltose* RR1 [17]. The *Penicillium* biomass was more efficient for the chromium removal under acidic conditions. Some studies were carried out at neutral pH [45]. A*. niger* mycelium removal 8.9mg/g dry weight at seven days of incubation (500ppm of Cr (VI)). Otherwise, *Paecilomyces* sp. biomass was incubate with non-sterilized contaminated soil containing 50mg Cr (VI)/g, suspended in LMM, pH 4.0, and it was observed that after eight days of incubation with the biomass, the Cr (VI) concentration of soil sample decreased fully [46]. We found out the following results: *Penicillium* sp, IA-01, has the ability to absorb Cr (VI) and the value of adsorption is as high as the fruiting bodies of the jelly fungus *A. polytricha* [32]; *C. fabianii, W. anomalus* and *C. tropicalis* [16]; *A. niger* [20], *M.* *hiemalis* and *R. nigricans* [35, 37] and *Paecilomyces* sp. [10]. The adsorption rate was affected by pH, temperature, initial concentration of Cr (VI) and dry cells. In the case of the polluted soil and water, around 40% of Cr (VI) could not be removed. Therefore, absorption to the organic compounds contained in polluted soil and water may occurr. The results suggest that the reduction of Cr (VI) is necessary to the bioremediation of soil and air.

**Figure 8.** Removal of Cr (VI) in industrial wastes incubated with 5g of fungal biomass. 100 rpm, 28°C, 20g and 100 mL of contaminated soil and water, respectively (297mg Cr (VI)/g soil and 155mg Cr (VI)/L).

#### **3.4. Removal of Cr (VI) by living cells of** *Penicillum* **sp. IA-01**

Next, the reduction of Cr (VI) by *Penicillium* sp. IA-0 were examined by using living cells. The fungal cells, which were cultured in 100 mL LMM containing 50mg/L Cr (VI), under various pH, inoculated amount, Cr (VI) concentration and carbon sources. The amount of Cr (VI) was determined and the percentage of decreased amount to total amount was calculated. The effect of different pHs (4.0, 5.3, and 6.2), show a pH optimum of 5.5 (77% at four days, 28o C, and 100 rpm), while at pH of 4.0 and 6.2 were of 43% and 65%, respectively (Figure 9). About, Coreño-Alonso et. al. [13], reported a 95% of removal at pH of 5.3 and 24 h with *A. niger* var *tubingen‐ sis* strain Ed8, and also, at pH 5.0 for Cr (VI) removal with *A. niger* MTCC 2594 [14] and at pH 3.0–5.0 for Pb+2, Cd+2 and Cr+3 with the yeast *Saccharomyces cerevisiae* [47]. In contrast to our observations, the máximum adsorption capacities by both living yeasts were found at pH 4.0 for *C. fabianii* HE650139 and *W. anomalus* HE648168, and 3.0 for *C. tropicalis* HE650140, with a percentage of removal of 100%, by all living microorganisms [16], also, a maximum uptake of Cr (VI) at pH 7.0 with *Aspergillus foetidus* [48]. On the other hand, using a *Citrobacter* strain, it has been reported that an enhanced uptake of different heavy metals, is increased if pH is from 2.0 to 7.0 and also a decrease in the removal at higher pH values [49]. Al-Asheh and Duvnjak

Figure 11, shows the effect of Cr (VI) concentration (50 to 200mg/L) on the removal of the same. If we increase the

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 Aspergillus foetidus, A. niger and A. parasiticus [11, 48, and 54] with glucose like carbon source, and other carbon sources like sucrose and citrate by a Paecilomyces sp fungal strain isolated from environment. [10], but are different from the observations of Srivasta and Thakur [55] with Aspergillus sp and Acinetobacter sp, who observed that the main carbon source is the sodium acetate. Consequently, we found out the following results in this section: As shown in Figure 4, the difference between dry cells and living cells were clear. Penicillium sp. IA-01 cells could remove Cr (VI) at pH 4.0-6.2, although combining by absorption did not occurr (Figure 4). The result suggests that Penicillium sp. cells can absorb and/or reduce Cr (VI) as well as adsorption. Additionally, as shown in Figure 12, the removal of Cr (VI) by adding glucose was higher than that of water (without glucose), and some carbon sources such as brown sugar and piloncillo enhanced the removal. There results suggest that the carbon sources induce the absorption rate of Cr (VI) or increase the amount of chromate

[50] also reported most removal increasing pH in the range 4.0–7.0 on Cr (III) uptake using *Aspergillus carbonarius*. At low values of pH, the low efficiency of removal of the metal, may be due to the competition between hydrogen (H+) and metal ions [36], and at higher pH values (7.0), the efficient metal removal may be due to the ionization of functional groups and the increase in the negative charge density on the cell surface. At alkaline pH values (8.0 or higher), a reduction in the solubility of metals may contribute to lower uptake rates [1].

*hiemalis* and *R. nigricans* [35, 37] and *Paecilomyces* sp. [10]. The adsorption rate was affected by pH, temperature, initial concentration of Cr (VI) and dry cells. In the case of the polluted soil and water, around 40% of Cr (VI) could not be removed. Therefore, absorption to the organic compounds contained in polluted soil and water may occurr. The results suggest that the

**Figure 8.** Removal of Cr (VI) in industrial wastes incubated with 5g of fungal biomass. 100 rpm, 28°C, 20g and 100 mL

Next, the reduction of Cr (VI) by *Penicillium* sp. IA-0 were examined by using living cells. The fungal cells, which were cultured in 100 mL LMM containing 50mg/L Cr (VI), under various pH, inoculated amount, Cr (VI) concentration and carbon sources. The amount of Cr (VI) was determined and the percentage of decreased amount to total amount was calculated. The effect

rpm), while at pH of 4.0 and 6.2 were of 43% and 65%, respectively (Figure 9). About, Coreño-Alonso et. al. [13], reported a 95% of removal at pH of 5.3 and 24 h with *A. niger* var *tubingen‐ sis* strain Ed8, and also, at pH 5.0 for Cr (VI) removal with *A. niger* MTCC 2594 [14] and at pH 3.0–5.0 for Pb+2, Cd+2 and Cr+3 with the yeast *Saccharomyces cerevisiae* [47]. In contrast to our observations, the máximum adsorption capacities by both living yeasts were found at pH 4.0 for *C. fabianii* HE650139 and *W. anomalus* HE648168, and 3.0 for *C. tropicalis* HE650140, with a percentage of removal of 100%, by all living microorganisms [16], also, a maximum uptake of Cr (VI) at pH 7.0 with *Aspergillus foetidus* [48]. On the other hand, using a *Citrobacter* strain, it has been reported that an enhanced uptake of different heavy metals, is increased if pH is from 2.0 to 7.0 and also a decrease in the removal at higher pH values [49]. Al-Asheh and Duvnjak

C, and 100

of different pHs (4.0, 5.3, and 6.2), show a pH optimum of 5.5 (77% at four days, 28o

of contaminated soil and water, respectively (297mg Cr (VI)/g soil and 155mg Cr (VI)/L).

**3.4. Removal of Cr (VI) by living cells of** *Penicillum* **sp. IA-01**

reduction of Cr (VI) is necessary to the bioremediation of soil and air.

176 Advances in Bioremediation of Wastewater and Polluted Soil

Figure 9. Effect of pH on chromium (VI) removal by biomass of Penicillium sp IA-01 50mg/L Cr (VI), 100 rpm, 28∘C **Figure 9.** Effect of pH on chromium (VI) removal by biomass of *Penicillium* sp IA-01 50mg/L Cr (VI), 100 rpm, 28°C

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]. The higher biomass dose, the more binding sites for complex of Cr (VI) (e.g., HCrO4 and Cr2O7−<sup>2</sup>ions) [3, 28]. 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 *−*2 ions) [3, 28].

Figure 10. The effect of cell concentration on the removal of Cr (VI). 50mg/L Cr (VI), 100 rpm, 28∘C, and pH 5.3 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].

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]. Figure 11. The effect of the concentration of Cr (VI) in the solution on the removal. 100 rpm, 28∘C, pH 5.3 With different carbon sources, like fermentable: glucose, sucrose, and citrate, and oxidable (glycerol). With glucose, 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,

reductase activity.

Cr (VI) in solution (%)

biomass dose, the more binding sites for complex of Cr (VI) (e.g., HCrO4-

0246 Time (days)

Figure 9. Effect of pH on chromium (VI) removal by biomass of Penicillium sp IA-01 50mg/L Cr (VI), 100 rpm, 28∘C

In Figure 10, the effect of the biomass concentration (72, 141, and 169 mg of dry weight) on Cr (VI) removal, with

pH 4.0 pH 5.3 pH 6.2

Figure 11, shows the effect of Cr (VI) concentration (50 to 200mg/L) on the removal of the same. If we increase the

(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 Aspergillus foetidus, A. niger and A. parasiticus [11, 48, and 54] with glucose like carbon source, and other carbon sources like sucrose and citrate by a Paecilomyces sp fungal strain isolated from environment. [10], but are different from the observations of Srivasta and Thakur [55] with Aspergillus sp and Acinetobacter sp, who observed that the main carbon source is the sodium acetate. Consequently, we found out the following results in this section: As shown in Figure 4, the difference between dry cells and living cells were clear. Penicillium sp. IA-01 cells could remove Cr (VI) at pH 4.0-6.2, although combining by absorption did not occurr (Figure 4). The result suggests that Penicillium sp. cells can absorb and/or reduce Cr (VI) as well as adsorption. Additionally, as shown in Figure 12, the removal of Cr (VI) by adding glucose was higher than that of water (without glucose), and some carbon sources such as brown sugar and piloncillo enhanced the removal. There results suggest that the carbon sources induce the absorption rate of Cr (VI) or increase the amount of chromate

Figure 12. Influence of carbon source on the capability of Penicillium sp., IA-01 to decrease Cr (VI) levels in the growth medium. 100

We also estimated the ratio of absorption and/or reduction to adsorption, as we found that the fungi Penicillium sp. IA-01, has these abilities as well as adsorption from the results in Section in 3.3. The resting cells and permeabilized cells were used for the examination, and heat-killed cells were used to examine the amount of adsorption. The removal was calculated as value of Cr (VI) in resting cells to the value from total value minus the value of Cr (VI) in heat-killed cells (0% of removal). First, the removal of the metal by resting cells was examined. The cell pellets of Penicillium sp, which were cultured in 100 mL thioglycolate broth, were incubated in 100 mM potassium phosphate buffer (pH 7.0) for 6h at 30℃. The resting cells of the fungus were expedient in reducing 0–10mg/100 mL Cr (VI) concentrations in 8 h as shown

and Cr2O7−<sup>2</sup>ions) [3, 28].

Figure 10. The effect of cell concentration on the removal of Cr (VI). 50mg/L Cr (VI), 100 rpm, 28∘C, and pH 5.3 **Figure 10.** The effect of cell concentration on the removal of Cr (VI). 50mg/L Cr (VI), 100 rpm, 28°C, and pH 5.3

Figure 11. The effect of the concentration of Cr (VI) in the solution on the removal. 100 rpm, 28∘C, pH 5.3 **Figure 11.** The effect of the concentration of Cr (VI) in the solution on the removal. 100 rpm, 28°C, pH 5.3

3.5. Adsorption and reduction by resting and permeable cells

reductase activity.

rpm, 28∘C, pH 5.3

with *A. foetidus, A*. *niger* and *A*. *parasiticus* [11, 48, and 54] with glucose like carbon source, and other carbon sources like sucrose and citrate by a *Paecilomyces* sp fungal strain isolated from environment. [10], but are different from the observations of Srivasta and Thakur [55] with *Aspergillus* sp and *Acinetobacter* sp, who observed that the main carbon source is the sodium 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 acetate. Consequently, we found out the following results in this section: As shown in Figure 4, the difference between dry cells and living cells were clear. *Penicillium* sp. IA-01 cells could remove Cr (VI) at pH 4.0-6.2, although combining by absorption did not occurr (Figure 4). The result suggests that *Penicillium* sp. cells can absorb and/or reduce Cr (VI) as well as adsorption. Additionally, as shown in Figure 12, the removal of Cr (VI) by adding glucose was higher than that of water (without glucose), and some carbon sources such as brown sugar and piloncillo enhanced the removal. There results suggest that the carbon sources induce the absorption rate of Cr (VI) or increase the amount of chromate reductase activity.

**A**

**B**

with *A. foetidus, A*. *niger* and *A*. *parasiticus* [11, 48, and 54] with glucose like carbon source, and other carbon sources like sucrose and citrate by a *Paecilomyces* sp fungal strain isolated from environment. [10], but are different from the observations of Srivasta and Thakur [55] with *Aspergillus* sp and *Acinetobacter* sp, who observed that the main carbon source is the sodium

02468

Time (days)

**Figure 11.** The effect of the concentration of Cr (VI) in the solution on the removal. 100 rpm, 28°C, pH 5.3

Figure 11. The effect of the concentration of Cr (VI) in the solution on the removal. 100 rpm, 28∘C, pH 5.3

Figure 9. Effect of pH on chromium (VI) removal by biomass of Penicillium sp IA-01 50mg/L Cr (VI), 100 rpm, 28∘C

Figure 10. The effect of cell concentration on the removal of Cr (VI). 50mg/L Cr (VI), 100 rpm, 28∘C, and pH 5.3

02468

Time (days)

**Figure 10.** The effect of cell concentration on the removal of Cr (VI). 50mg/L Cr (VI), 100 rpm, 28°C, and pH 5.3

Figure 11. The effect of the concentration of Cr (VI) in the solution on the removal. 100 rpm, 28∘C, pH 5.3

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

With different carbon sources, like fermentable: glucose, sucrose, and citrate, and oxidable (glycerol). With glucose,

(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 Aspergillus foetidus, A. niger and A. parasiticus [11, 48, and 54] with glucose like carbon source, and other carbon sources like sucrose and citrate by a Paecilomyces sp fungal strain isolated from environment. [10], but are different from the observations of Srivasta and Thakur [55] with Aspergillus sp and Acinetobacter sp, who observed that the main carbon source is the sodium acetate. Consequently, we found out the following results in this section: As shown in Figure 4, the difference between dry cells and living cells were clear. Penicillium sp. IA-01 cells could remove Cr (VI) at pH 4.0-6.2, although combining by absorption did not occurr (Figure 4). The result suggests that Penicillium sp. cells can absorb and/or reduce Cr (VI) as well as adsorption. Additionally, as shown in Figure 12, the removal of Cr (VI) by adding glucose was higher than that of water (without glucose), and some carbon sources such as brown sugar and piloncillo enhanced the removal. There results suggest that the carbon sources induce the absorption rate of Cr (VI) or increase the amount of chromate

Figure 12. Influence of carbon source on the capability of Penicillium sp., IA-01 to decrease Cr (VI) levels in the growth medium. 100

We also estimated the ratio of absorption and/or reduction to adsorption, as we found that the fungi Penicillium sp. IA-01, has these abilities as well as adsorption from the results in Section in 3.3. The resting cells and permeabilized cells were used for the examination, and heat-killed cells were used to examine the amount of adsorption. The removal was calculated as value of Cr (VI) in resting cells to the value from total value minus the value of Cr (VI) in heat-killed cells (0% of removal). First, the removal of the metal by resting cells was examined. The cell pellets of Penicillium sp, which were cultured in 100 mL thioglycolate broth, were incubated in 100 mM potassium phosphate buffer (pH 7.0) for 6h at 30℃. The resting cells of the fungus were expedient in reducing 0–10mg/100 mL Cr (VI) concentrations in 8 h as shown

3.5. Adsorption and reduction by resting and permeable cells

biomass dose, the more binding sites for complex of Cr (VI) (e.g., HCrO4-

0246 Time (days)

178 Advances in Bioremediation of Wastewater and Polluted Soil

Cr (VI) in solution (%)

reductase activity.

rpm, 28∘C, pH 5.3

% Cr (VI) removal

Cr (VI) in solution (%)

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]. The higher

pH 4.0 pH 5.3 pH 6.2

and Cr2O7−<sup>2</sup>ions) [3, 28].

1 inoculum 2 inoculum 3 inoculum

> 50 ppm 100 ppm 150 ppm 200 ppm

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 **Figure 12.** Influence of carbon source on the capability of *Penicillium* sp., IA-01 **Figure 12.** Influence of carbon source on the capability of *Penicillium* sp., IA-01 to decrease Cr (VI) levels in the growth medium. 100 rpm, 28°C, pH 5.3

**3.5 Adsorption and Reduction by Resting and Permeable Cells** 

to decrease Cr (VI) levels in the growth medium. 100 rpm, 28*◦*C, pH 5.3

We also estimated the ratio of absorption and/or reduction to adsorption, as we found that the fungi *Penicillium* sp. IA-01, has these abilities as well as adsorption from the results in Section in 3.3. The resting cells and permeabilized cells were used for the examination, and heat-killed cells were used to examine the amount of adsorption. The removal was calculated as value of Cr (VI) in resting cells to the value from total value minus the value of Cr (VI) in heat-killed cells (0% of removal). First, the removal of the metal by resting cells was examined. The cell pellets of *Penicillium* sp, which were cultured in 100 mL thioglycolate broth, were incubated in 100 mM potassium phosphate buffer (pH

#### **3.5. Adsorption and reduction by resting and permeable cells**

other several factors are known to affect the biosorption rate [59].

We also estimated the ratio of absorption and/or reduction to adsorption, as we found that the fungi *Penicillium* sp. IA-01, has these abilities as well as adsorption from the results in Section in 3.3. The resting cells and permeabilized cells were used for the examination, and heat-killed cells were used to examine the amount of adsorption. The removal was calculated as value of Cr (VI) in resting cells to the value from total value minus the value of Cr (VI) in heat-killed cells (0% of removal). First, the removal of the metal by resting cells was examined. The cell pellets of *Penicillium* sp, which were cultured in 100 mL thioglycolate broth, were incubated in 100 mM potassium phosphate buffer (pH 7.0) for 6h at 30℃. The resting cells of the fungus were expedient in reducing 0–10mg/100 mL Cr (VI) concentrations in 8 h as shown in Figure 13. The fungus removal was between 53% and 70% (2–10 mg/100/mL) of the metal, and these results resemble those reported by *A. niger* and *A. parasiticus* [54], *Fusarium solani* [56], *Paecilomyces lilacinus* [57], and the bacteria *Pseudomonas* sp. [58] and *Paecilomyces* sp. [46]. Structural properties of the biosorbent including the cellular support and other several factors are known to affect the biosorption rate [59]. in Figure 13. The fungus removal was between 53% and 70% (2–10 mg/100/mL) of the metal, and these results resemble those reported by A. niger and A. parasiticus [54], Fusarium solani [56], Paecilomyces lilacinus [57], and the bacteria Pseudomonas sp. [58] and Paecilomyces sp. [46]. Structural properties of the biosorbent including the cellular support and

Figure 13. Resting cell assays for Cr (VI) reduction by Penicillium sp. IA-01 performed at initial concentrations of 0–10mg/100 mL of Cr (VI), pH 7.0, and 37<sup>∘</sup>C in 8 h **Figure 13.** Resting cell assays for Cr (VI) reduction by *Penicillium* sp. IA-01 performed at initial concentrations of 0– 10mg/100 mL of Cr (VI), pH 7.0, and 37°C in 8 h

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 moles/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 A. niger and A. parasiticus [54], Pichia jadinii M9, Pichia anómala M10 [60], Pichia sp. [61], and Bacillus sp. [62], cytosolic fraction of C.maltosa [17], Pichia sp. [62], and Pannonibacter phragmitetus [63] of membrane fraction of Pseudomonas sp. G1DM21 [58], The cell permeabilization increased the Cr (VI) reduction by the resting cells, as the permea‐ bilized 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 mecha‐ nism 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 *A. niger* and *A. parasiticus* [54], *Pichia jadinii* M9, *Pichia anómala* M10

transport through cell membrane is the rate-limiting steps.

pH 7.0, and 37<sup>∘</sup>C

3.6. Chromate reductase activity

Bacillus megaterium [64], and Enterobacter cloacae [65]. The results by resting and permeable cells suggest as follows: As shown in Figure 13, 70-80% of Cr (VI) could be removed by resting cells, and the result suggests that absorption of Cr (VI) occurrs without energy of carbon sources or ATP. Additionally, as shown in Figure 14, the ratios of the removal of Cr (VI) in case of the pretreatment by Triton X-100, toluene and SDS of glucose were 2-2.5 times higher. Therefore, the

Figure 14. Permeabilized cell assays for Cr (VI) reduction by Penicillium sp. IA-01 performed at initial concentrations of 28mM of Cr (VI),

The result of permeable cells (Figure 14) suggests that Penicillium sp. IA-01 has the enzymatic ability of reduction to Cr (VI). Thus, we investigated the reduction of Cr (VI) by Penicillium sp. IA-01. The activity of chromate reductase is examined in the cell-free extract. The function of the chromate reductase of Penicillium sp., was characterized in different in vitro conditions. In determining the optimal pH for the chromate reductase activity, we used the following buffers at different pH ranges: potassium phosphate, citrate phosphate, and Tris-HCl; and we found the máximum enzymatic activity to be at an optimum pH of 7.0, with potassium phosphate buffer, as depicted in Figure 15, and these results resemble those reported by the fungi A. niger and A. parasiticus [54], the yeast P. jadini M9 [60], and cell-free extract of [60], *Pichia* sp. [61], *and Bacillus* sp. [62], cytosolic fraction of *C.maltosa* [17], *Pichia* sp. [62], and *Pannonibacter phragmitetus* [63] of membrane fraction of *Pseudomonas* sp. G1DM21 [58], *Bacillus megaterium* [64], and *Enterobacter cloacae*[65]. The results by resting and permeable cells suggest as follows: As shown in Figure 13, 70-80% of Cr (VI) could be removed by resting cells, and the result suggests that absorption of Cr (VI) occurrs without energy of carbon sources or ATP. Additionally, as shown in Figure 14, the ratios of the removal of Cr (VI) in case of the pre‐ treatment by Triton X-100, toluene and SDS of glucose were 2-2.5 times higher. Therefore, the transport through cell membrane is the rate-limiting steps.

Figure 14. Permeabilized cell assays for Cr (VI) reduction by Penicillium sp. IA-01 performed at initial concentrations of 28mM of Cr (VI), **Figure 14.** Permeabilized cell assays for Cr (VI) reduction by *Penicillium* sp. IA-01 performed at initial concentrations of 28mM of Cr (VI), pH 7.0, and 37<sup>∘</sup> C

#### 3.6. Chromate reductase activity **3.6. Chromate reductase activity**

28mM Cr (VI) at pH 7.0

pH 7.0, and 37<sup>∘</sup>C

**3.5. Adsorption and reduction by resting and permeable cells**

other several factors are known to affect the biosorption rate [59].

transport through cell membrane is the rate-limiting steps.

are known to affect the biosorption rate [59].

180 Advances in Bioremediation of Wastewater and Polluted Soil

(VI), pH 7.0, and 37<sup>∘</sup>C in 8 h

10mg/100 mL of Cr (VI), pH 7.0, and 37°C in 8 h

Cr (VI) removal (%)

pH 7.0, and 37<sup>∘</sup>C

3.6. Chromate reductase activity

We also estimated the ratio of absorption and/or reduction to adsorption, as we found that the fungi *Penicillium* sp. IA-01, has these abilities as well as adsorption from the results in Section in 3.3. The resting cells and permeabilized cells were used for the examination, and heat-killed cells were used to examine the amount of adsorption. The removal was calculated as value of Cr (VI) in resting cells to the value from total value minus the value of Cr (VI) in heat-killed cells (0% of removal). First, the removal of the metal by resting cells was examined. The cell pellets of *Penicillium* sp, which were cultured in 100 mL thioglycolate broth, were incubated in 100 mM potassium phosphate buffer (pH 7.0) for 6h at 30℃. The resting cells of the fungus were expedient in reducing 0–10mg/100 mL Cr (VI) concentrations in 8 h as shown in Figure 13. The fungus removal was between 53% and 70% (2–10 mg/100/mL) of the metal, and these results resemble those reported by *A. niger* and *A. parasiticus* [54], *Fusarium solani* [56], *Paecilomyces lilacinus* [57], and the bacteria *Pseudomonas* sp. [58] and *Paecilomyces* sp. [46]. Structural properties of the biosorbent including the cellular support and other several factors

in Figure 13. The fungus removal was between 53% and 70% (2–10 mg/100/mL) of the metal, and these results resemble those reported by A. niger and A. parasiticus [54], Fusarium solani [56], Paecilomyces lilacinus [57], and the bacteria Pseudomonas sp. [58] and Paecilomyces sp. [46]. Structural properties of the biosorbent including the cellular support and

Figure 13. Resting cell assays for Cr (VI) reduction by Penicillium sp. IA-01 performed at initial concentrations of 0–10mg/100 mL of Cr

0 1 2 3 4 5 6 7 8 9 10 11

Cr (VI) concentration (mg/100 mL)

**Figure 13.** Resting cell assays for Cr (VI) reduction by *Penicillium* sp. IA-01 performed at initial concentrations of 0–

The cell permeabilization increased the Cr (VI) reduction by the resting cells, as the permea‐ bilized 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 mecha‐ nism 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 *A. niger* and *A. parasiticus* [54], *Pichia jadinii* M9, *Pichia anómala* M10

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 moles/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 A. niger and A. parasiticus [54], Pichia jadinii M9, Pichia anómala M10 [60], Pichia sp. [61], and Bacillus sp. [62], cytosolic fraction of C.maltosa [17], Pichia sp. [62], and Pannonibacter phragmitetus [63] of membrane fraction of Pseudomonas sp. G1DM21 [58], Bacillus megaterium [64], and Enterobacter cloacae [65]. The results by resting and permeable cells suggest as follows: As shown in Figure 13, 70-80% of Cr (VI) could be removed by resting cells, and the result suggests that absorption of Cr (VI) occurrs without energy of carbon sources or ATP. Additionally, as shown in Figure 14, the ratios of the removal of Cr (VI) in case of the pretreatment by Triton X-100, toluene and SDS of glucose were 2-2.5 times higher. Therefore, the

Figure 14. Permeabilized cell assays for Cr (VI) reduction by Penicillium sp. IA-01 performed at initial concentrations of 28mM of Cr (VI),

The result of permeable cells (Figure 14) suggests that Penicillium sp. IA-01 has the enzymatic ability of reduction to Cr (VI). Thus, we investigated the reduction of Cr (VI) by Penicillium sp. IA-01. The activity of chromate reductase is examined in the cell-free extract. The function of the chromate reductase of Penicillium sp., was characterized in different in vitro conditions. In determining the optimal pH for the chromate reductase activity, we used the following buffers at different pH ranges: potassium phosphate, citrate phosphate, and Tris-HCl; and we found the máximum enzymatic activity to be at an optimum pH of 7.0, with potassium phosphate buffer, as depicted in Figure 15, and these results resemble those reported by the fungi A. niger and A. parasiticus [54], the yeast P. jadini M9 [60], and cell-free extract of

The result of permeable cells (Figure 14) suggests that Penicillium sp. IA-01 has the enzymatic ability of reduction to Cr (VI). Thus, we investigated the reduction of Cr (VI) by Penicillium sp. IA-01. The activity of chromate reductase is examined in the cell-free extract. The function of the chromate reductase of Penicillium sp., was characterized in different in vitro conditions. In determining the optimal pH for the chromate reductase activity, we used the following buffers at different pH ranges: potassium phosphate, citrate phosphate, and Tris-HCl; and we found the máximum enzymatic activity to be at an optimum pH of 7.0, with potassium phosphate buffer, as depicted in Figure 15, and these results resemble those reported by the fungi A. niger and A. parasiticus [54], the yeast P. jadini M9 [60], and cell-free extract of Arthrobacter sp. SUK 1201 [66]. Other authors reported stability between 7.0 and 7.4 for the bacteria Pseudomonas sp. G1DM21 [58], 6.5 and 7.5 for E. coli CFE [67], and in the range of 5.0 to 8.0 for Bacillus sp. [68]. The result of permeable cells (Figure 14) suggests that *Penicillium* sp. IA-01 has the enzymatic ability of reduction to Cr (VI). Thus, we investigated the reduction of Cr (VI) by *Penicillium* sp. IA-01. The activity of chromate reductase is examined in the cell-free extract. The function of the chromate reductase of *Penicillium* sp., was characterized in different *in vitro* conditions. In determining the optimal pH for the chromate reductase activity, we used the following buffers at different pH ranges: potassium phosphate, citrate phosphate, and Tris-HCl; and we found the máximum enzymatic activity to be at an optimum pH of 7.0, with potassium phosphate buffer, as depicted in Figure 15, and these results resemble those reported by the fungi *A. niger* and *A. parasiticus* [54], the yeast *P. jadini* M9 [60], and cell-free extract of *Arthrobacter* sp. SUK 1201 [66]. Other authors reported stability between 7.0 and 7.4 for the bacteria *Pseudomo‐ nas* sp. G1DM21 [58], 6.5 and 7.5 for *E. coli* CFE [67], and in the range of 5.0 to 8.0 for *Bacillus* sp. [68].

different buffers (pH 6.5–9.0) with an initial concentration of 5.6mM Cr (VI), at 37<sup>∘</sup>C

Figure 15. Effect of pH on Cr (VI) reductase activity in cell-free-extracts of Penicillium sp. IA-01 determined in

The optimal temperature for the Cr (VI) reductase activity was 37<sup>∘</sup>C, but the reductase activity was altered significantly at 20<sup>∘</sup>C (39% of inhibition); but when the assays were performed at 50<sup>∘</sup>C temperatura, the reductase activity showed 14.2% of inhibition (Figure 16). For P. jadinii M9, incubation at 55<sup>∘</sup>C produced a reduction in activity of 55% [60]. In P. anómala M10, when incubated at 8<sup>∘</sup>C, a decrease in activity of 25% was observed, and at 50<sup>∘</sup>C the activity was at 50%. [69]. For A. niger and A. parasiticus [54], Pseudomonas sp. G1DM21 [58], E. coli a [66], and Bacillus sp. CFEs [67], the thermal stability was of 30<sup>∘</sup>C [66, 67], and for the activity in cell-free extract of Arthrobacter sp. SUK 1201, it was 32<sup>o</sup>C [66].

Figure 16. Effect of temperature on Cr (VI) reductase activity in cell-free extracts of Penicillium sp. IA-01 with initial concentrations of

On the contrary, Pseudomonas putida CFE probed to be more resistant, keeping its stability up to 50<sup>∘</sup>C [69].

3.6. Chromate reductase activity

pH 7.0, and 37<sup>∘</sup>C

Cr (VI) removal (%)

G1DM21 [58], 6.5 and 7.5 for E. coli CFE [67], and in the range of 5.0 to 8.0 for Bacillus sp. [68].

Control Triton X-100 Tween 80 Toluene SDS

Detergents

Figure 14. Permeabilized cell assays for Cr (VI) reduction by Penicillium sp. IA-01 performed at initial concentrations of 28mM of Cr (VI),

The result of permeable cells (Figure 14) suggests that Penicillium sp. IA-01 has the enzymatic ability of reduction to Cr (VI). Thus, we investigated the reduction of Cr (VI) by Penicillium sp. IA-01. The activity of chromate reductase is examined in the cell-free extract. The function of the chromate reductase of Penicillium sp., was characterized in different in vitro conditions. In determining the optimal pH for the chromate reductase activity, we used the following buffers at different pH ranges: potassium phosphate, citrate phosphate, and Tris-HCl; and we found the máximum enzymatic activity to be at an optimum pH of 7.0, with potassium phosphate buffer, as depicted in Figure 15, and these results

Figure 15. Effect of pH on Cr (VI) reductase activity in cell-free-extracts of Penicillium sp. IA-01 determined in different buffers (pH 6.5– 9.0) with an initial concentration of 5.6mM Cr (VI), at 37<sup>∘</sup>C **Figure 15.** Effect of pH on Cr (VI) reductase activity in cell-free-extracts of *Penicillium* sp. IA-01 determined in different buffers (pH 6.5–9.0) with an initial concentration of 5.6mM Cr (VI), at 37<sup>∘</sup> C

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 *P. jadinii* M9, incubation at 55°C produced a reduction in activity of 55% [60]. In *P. anómala* M10, when incubated at 8°C, a decrease in activity of 25% was observed, and at 50°C the activity was at 50%. [69]. For *A. niger* and *A. parasiticus* [54], *Pseudomonas* sp. G1DM21 [58], *E. coli* [66], and *Bacillus* sp. CFEs [67], the thermal stability was of 30°C [66, 67], and for the activity in cell-free extract of *Arthrobacter* sp. SUK 1201, it was 32°C [66]. On the contrary, *Pseudomonas putida* CFE probed to be more resistant, keeping its stability up to 50°C [69].

The effect of different metal cations on the chromate reductase activity of *Penicillium* sp. was determined in Figure 17. All the metal ions tested inhibit the Cr (VI) reductase activity of the CFE of 12% with Cu2+ and 40.2% with Na+ , and these results are different than those reported for the yeast *P. jadinii* M9 chromate reductase because only Cu2+ and Na+ produced an augmentation in the activity of 63 and 30%, respectively [60]; in *Arthrobacter* sp. SUK 1201, Cu2+ also increases the activity [66], and all other ions tested had an inhibitory effect but in different levels. A decrease of 56.5% was observed with Hg2+, while addition of Mg2+, Fe3+, Ca2+, and Cd2+ resulted in a decrease of activity between 40% and 51%. In *P. anomala* M10 chromate reductase, only Cu2+ produced a rise in activity of 31% [69]. Inhibition by Hg2+ was higher in *P. anomala* and *Pseudomonas* sp., than in *Penicillium* sp., with a decrease in activity of 85% and 90%, respectively [58, 60]. Inhibition by Ca2+ and Mg2+ was approximately 50%, while Fe3+ reduced the activity to 32%. These results agree with those reported for *Arthrobacter crystallopoietes* [44] and *Bacillus* sp. [67]. On the other hand, inhibition by Hg2+ can be related The optimal temperature for the Cr (VI) reductase activity was 37<sup>∘</sup>C, but the reductase activity was altered significantly at 20<sup>∘</sup>C (39% of inhibition); but when the assays were performed at 50<sup>∘</sup>C temperatura, the reductase activity showed 14.2% of inhibition (Figure 16). For P. jadinii M9, incubation at 55<sup>∘</sup>C produced a reduction in activity of 55% [60]. In P. anómala M10, when incubated at 8<sup>∘</sup>C, a decrease in activity of 25% was observed, and at 50<sup>∘</sup>C the activity was at 50%.

Figure 14. Permeabilized cell assays for Cr (VI) reduction by Penicillium sp. IA-01 performed at initial concentrations of 28mM of Cr (VI),

The result of permeable cells (Figure 14) suggests that Penicillium sp. IA-01 has the enzymatic ability of reduction to Cr (VI). Thus, we investigated the reduction of Cr (VI) by Penicillium sp. IA-01. The activity of chromate reductase is examined in the cell-free extract. The function of the chromate reductase of Penicillium sp., was characterized in different in vitro conditions. In determining the optimal pH for the chromate reductase activity, we used the following buffers at different pH ranges: potassium phosphate, citrate phosphate, and Tris-HCl; and we found the máximum enzymatic activity to be at an optimum pH of 7.0, with potassium phosphate buffer, as depicted in Figure 15, and these results resemble those reported by the fungi A. niger and A. parasiticus [54], the yeast P. jadini M9 [60], and cell-free extract of Arthrobacter sp. SUK 1201 [66]. Other authors reported stability between 7.0 and 7.4 for the bacteria Pseudomonas sp.

Figure 15. Effect of pH on Cr (VI) reductase activity in cell-free-extracts of Penicillium sp. IA-01 determined in different buffers (pH 6.5–

, and these results are different than those reported

produced an

C

G1DM21 [58], 6.5 and 7.5 for E. coli CFE [67], and in the range of 5.0 to 8.0 for Bacillus sp. [68].

6.5 7 7.5 8 9

pH

**Figure 15.** Effect of pH on Cr (VI) reductase activity in cell-free-extracts of *Penicillium* sp. IA-01 determined in different

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 *P. jadinii* M9, incubation at 55°C produced a reduction in activity of 55% [60]. In *P. anómala* M10, when incubated at 8°C, a decrease in activity of 25% was observed, and at 50°C the activity was at 50%. [69]. For *A. niger* and *A. parasiticus* [54], *Pseudomonas* sp. G1DM21 [58], *E. coli* [66], and *Bacillus* sp. CFEs [67], the thermal stability was of 30°C [66, 67], and for the activity in cell-free extract of *Arthrobacter* sp. SUK 1201, it was 32°C [66]. On the contrary, *Pseudomonas putida* CFE probed

The effect of different metal cations on the chromate reductase activity of *Penicillium* sp. was determined in Figure 17. All the metal ions tested inhibit the Cr (VI) reductase activity of the

augmentation in the activity of 63 and 30%, respectively [60]; in *Arthrobacter* sp. SUK 1201, Cu2+ also increases the activity [66], and all other ions tested had an inhibitory effect but in different levels. A decrease of 56.5% was observed with Hg2+, while addition of Mg2+, Fe3+, Ca2+, and Cd2+ resulted in a decrease of activity between 40% and 51%. In *P. anomala* M10 chromate reductase, only Cu2+ produced a rise in activity of 31% [69]. Inhibition by Hg2+ was higher in *P. anomala* and *Pseudomonas* sp., than in *Penicillium* sp., with a decrease in activity of 85% and 90%, respectively [58, 60]. Inhibition by Ca2+ and Mg2+ was approximately 50%, while Fe3+ reduced the activity to 32%. These results agree with those reported for *Arthrobacter crystallopoietes* [44] and *Bacillus* sp. [67]. On the other hand, inhibition by Hg2+ can be related

for the yeast *P. jadinii* M9 chromate reductase because only Cu2+ and Na+

Control Triton X-100 Tween 80 Toluene SDS

Detergents

pH 7.0, and 37<sup>∘</sup>C

Specific activity (mmol/min/mg protein)

Cr (VI) removal (%)

3.6. Chromate reductase activity

182 Advances in Bioremediation of Wastewater and Polluted Soil

9.0) with an initial concentration of 5.6mM Cr (VI), at 37<sup>∘</sup>C

buffers (pH 6.5–9.0) with an initial concentration of 5.6mM Cr (VI), at 37<sup>∘</sup>

to be more resistant, keeping its stability up to 50°C [69].

CFE of 12% with Cu2+ and 40.2% with Na+

Figure 16. Effect of temperature on Cr (VI) reductase activity in cell-free extracts of Penicillium sp. IA-01 with initial concentrations of 28mM Cr (VI) at pH 7.0 **Figure 16.** Effect of temperature on Cr (VI) reductase activity in cell-free extracts of *Penicillium* sp. IA-01 with initial concentrations of 28mM Cr (VI) at pH 7.0

to its affinity to –SH ligands, then suspecting the presence of this chemical group in the active site of the enzyme related to chromate reductase activity [70]. The effect of different metal cations on the chromate reductase activity of Penicillium sp. was determined in Figure 17. All the metal ions tested inhibit the Cr (VI) reductase activity of the CFE of 12% with Cu2+ and 40.2% with Na<sup>+</sup>, and these results are different than those reported for the yeast P. jadinii M9 chromate reductase because only Cu2+ and Na<sup>+</sup>

Figure 17. Effect of different metal cations on Cr (VI) reductase activity in cell-free extracts of Penicillium sp. IA-01 at pH 7.0 and 37<sup>∘</sup>C **Figure 17.** Effect of different metal cations on Cr (VI) reductase activity in cell-free extracts of *Penicillium* sp. IA-01 at pH 7.0 and 37°C anomala chromate reductase with NADH [60] and Pseudomonas sp. with citrate, acetate, glucose, and formate [58]. In previous reports of Bacillus sp., glucose has been reported to act as an electron donor and has been demonstrated to

compounds may be inhibitor of the enzyme.

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 P. jadinii M9 and P. anomala chromate reductase with NADH [60] and Pseudomonas sp. with citrate, acetate, glucose, and formate [58]. In previous reports of Bacillus sp., glucose has been reported to act as an electron donor and has been demonstrated to increase Cr (VI) reduction [72, 73], and also formate-dependent Cr (VI) reductases have been reported in Shewanella putrefaciens MR-1 [74]. Our work supports other studies reporting NADH or NADPH-dependent enzymatic reduction of Cr (VI) under aerobic conditions [58, 60, 67, 69, and 70]. Ramirez-Díaz et al. [75], report the oxidation of NADH donates an electron to the chromate reductase enzyme, and then the electron is transferred to Cr (VI) reducing it to the

increase Cr (VI) reduction [72, 73], and also formate-dependent Cr (VI) reductases have been reported in Shewanella putrefaciens MR-1 [74]. Our work supports other studies reporting NADH or NADPH-dependent enzymatic reduction of Cr (VI) under aerobic conditions [58, 60, 67, 69, and 70]. Ramirez-Díaz et al. [75], report the oxidation of NADH donates

2.9, and 2.87 times, respectively (Figure 18), and these results are like those reported for the yeasts P. jadinii M9 and P.

produced an augmentation in the activity of 63 and 30%, respectively [60]; in Arthrobacter sp. SUK 1201, Cu2+ also

intermediate form Cr (V) which finally accepts two electrons from other organic substances to produce Cr (III).

Figure 18. Effect of different electron donors on Cr (VI) reductase activity in cell-free extracts of Penicillium sp. IA-01 at pH 7.0 and 37<sup>∘</sup>C

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 [66], and it has been observed that cyanide and azide partially inhibited purified chromate reductase of E. coli ATCC 33456 19 [67] and aerobic chromate reduction by Bacillus subtilis [71] and inhibited more than 50% of membrane-associated chromate reductase activity of S. putrefaciens MR-1 [74], while no inhibition was observed in CFE of Bacillus sp. ES29 [70]. Respiratory inhibitors act on de novo protein synthesis or affect the respiratory chain intermediates responsible for Cr (VI) reduction, wherein Cr (VI) serves as a terminal electron acceptor [69]. As shown in Figures 15 and 16, the optimal pH and temperature of chromate reductase in Penicillium sp. IA-01 were pH 7 and 37℃, and the results were supported by the results of living cells. Therefore, the reduction is mainly occurred to remove Cr (VI) and to show resistance to high concentration of Cr (VI). Whereas, addition of electron donors caused the decrease of the activity, and therefore, these

Figure 19. Effect of different inhibitors on Cr (VI) reductase activity in cell-free extracts of Penicillium sp. IA-01 at pH 7.0 and 37<sup>∘</sup>C

0

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 *P. jadinii* M9 and *P. anomala* chromate reductase with NADH [60] and *Pseudomonas* sp. with citrate, acetate, glucose, and formate [58]. In previous reports of *Bacillus* sp., glucose has been reported to act as an electron donor and has been demonstrated to increase Cr (VI) reduction [72, 73], and also formate-dependent Cr (VI) reductases have been reported in *Shewanella putrefaciens* MR-1 [74]. Our work supports other studies reporting NADH or NADPH-dependent enzymatic reduction of Cr (VI) under aerobic conditions [58, 60, 67, 69, and 70]. Ramirez-Díaz et al. [75], report the oxidation of NADH donates an electron to the chromate reductase enzyme, and then the electron is transferred to Cr (VI) reducing it to the intermediate form Cr (V) which finally accepts two electrons from other organic substances to produce Cr (III). Figure 17. Effect of different metal cations on Cr (VI) reductase activity in cell-free extracts of Penicillium sp. IA-01 at pH 7.0 and 37<sup>∘</sup>C 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 P. jadinii M9 and P. anomala chromate reductase with NADH [60] and Pseudomonas sp. with citrate, acetate, glucose, and formate [58]. In previous reports of Bacillus sp., glucose has been reported to act as an electron donor and has been demonstrated to increase Cr (VI) reduction [72, 73], and also formate-dependent Cr (VI) reductases have been reported in Shewanella putrefaciens MR-1 [74]. Our work supports other studies reporting NADH or NADPH-dependent enzymatic reduction of Cr (VI) under aerobic conditions [58, 60, 67, 69, and 70]. Ramirez-Díaz et al. [75], report the oxidation of NADH donates an electron to the chromate reductase enzyme, and then the electron is transferred to Cr (VI) reducing it to the intermediate form Cr (V) which finally accepts two electrons from other organic substances to produce Cr (III). Metal cations

Na Mg Fe Ca Cd Cu Hg

Figure 18. Effect of different electron donors on Cr (VI) reductase activity in cell-free extracts of Penicillium sp. IA-01 at pH 7.0 and 37<sup>∘</sup>C **Figure 18.** Effect of different electron donors on Cr (VI) reductase activity in cell-free extracts of *Penicillium* sp. IA-01 at pH 7.0 and 37°C

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 [66], and it has been observed that cyanide and azide partially inhibited purified chromate reductase of E. coli ATCC 33456 19 [67] and aerobic chromate reduction by Bacillus subtilis [71] and inhibited more than 50% of membrane-associated 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 [66], and it has been observed that cyanide and azide partially inhibited purified chromate reductase of *E. coli* ATCC 33456 19 [67] and aerobic chromate reduction by *Bacillus subtilis* [71] and inhibited more than 50% of membraneassociated chromate reductase activity of *S. putrefaciens* MR-1 [74], while no inhibition was observed in CFE of *Bacillus* sp. ES29 [70]. Respiratory inhibitors act on de novo protein synthesis or affect the respiratory chain intermediates responsible for Cr (VI) reduction,

wherein Cr (VI) serves as a terminal electron acceptor [69]. As shown in Figures 15 and 16, the optimal pH and temperature of chromate reductase in *Penicillium* sp. IA-01 were pH 7 and 37℃, and the results were supported by the results of living cells. Therefore, the reduction is mainly occurred to remove Cr (VI) and to show resistance to high concentration of Cr (VI). Whereas, addition of electron donors caused the decrease of the activity, and therefore, these compounds may be inhibitor of the enzyme. chromate reductase activity of S. putrefaciens MR-1 [74], while no inhibition was observed in CFE of Bacillus sp. ES29 [70]. Respiratory inhibitors act on de novo protein synthesis or affect the respiratory chain intermediates responsible for Cr (VI) reduction, wherein Cr (VI) serves as a terminal electron acceptor [69]. As shown in Figures 15 and 16, the optimal pH and temperature of chromate reductase in Penicillium sp. IA-01 were pH 7 and 37℃, and the results were supported by the results of living cells. Therefore, the reduction is mainly occurred to remove Cr (VI) and to show resistance to high concentration of Cr (VI). Whereas, addition of electron donors caused the decrease of the activity, and therefore, these

compounds may be inhibitor of the enzyme.

Figure 19. Effect of different inhibitors on Cr (VI) reductase activity in cell-free extracts of Penicillium sp. IA-01 at pH 7.0 and 37<sup>∘</sup>C **Figure 19.** Effect of different inhibitors on Cr (VI) reductase activity in cell-free extracts of *Penicillium* sp. IA-01 at pH 7.0 and 37°C

#### We isolated a Penicillium sp. IA-01 strain, which grow about 50% relative to control (85 mg of dry weight without metal) **4. Conclusion**

4. Conclusion

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 *P. jadinii* M9 and *P. anomala* chromate reductase with NADH [60] and *Pseudomonas* sp. with citrate, acetate, glucose, and formate [58]. In previous reports of *Bacillus* sp., glucose has been reported to act as an electron donor and has been demonstrated to increase Cr (VI) reduction [72, 73], and also formate-dependent Cr (VI) reductases have been reported in *Shewanella putrefaciens* MR-1 [74]. Our work supports other studies reporting NADH or NADPH-dependent enzymatic reduction of Cr (VI) under aerobic conditions [58, 60, 67, 69, and 70]. Ramirez-Díaz et al. [75], report the oxidation of NADH donates an electron to the chromate reductase enzyme, and then the electron is transferred to Cr (VI) reducing it to the intermediate form Cr (V) which finally accepts two

Na Mg Fe Ca Cd Cu Hg

Metal cations

Figure 17. Effect of different metal cations on Cr (VI) reductase activity in cell-free extracts of Penicillium sp. IA-01 at pH 7.0 and 37<sup>∘</sup>C

intermediate form Cr (V) which finally accepts two electrons from other organic substances to produce Cr (III).

Figure 18. Effect of different electron donors on Cr (VI) reductase activity in cell-free extracts of Penicillium sp. IA-01 at pH 7.0 and 37<sup>∘</sup>C

Cystine Lactic

acid

Ascorbic acid

Citric acid

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 [66], and it has been observed that cyanide and azide partially inhibited purified chromate reductase of E. coli ATCC 33456 19 [67] and aerobic chromate reduction by Bacillus subtilis [71] and inhibited more than 50% of membrane-associated

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 P. jadinii M9 and P. anomala chromate reductase with NADH [60] and Pseudomonas sp. with citrate, acetate, glucose, and formate [58]. In previous reports of Bacillus sp., glucose has been reported to act as an electron donor and has been demonstrated to increase Cr (VI) reduction [72, 73], and also formate-dependent Cr (VI) reductases have been reported in Shewanella putrefaciens MR-1 [74]. Our work supports other studies reporting NADH or NADPH-dependent enzymatic reduction of Cr (VI) under aerobic conditions [58, 60, 67, 69, and 70]. Ramirez-Díaz et al. [75], report the oxidation of NADH donates an electron to the chromate reductase enzyme, and then the electron is transferred to Cr (VI) reducing it to the

electrons from other organic substances to produce Cr (III).

NADH Glucose Acetate Formic

acid

**Figure 18.** Effect of different electron donors on Cr (VI) reductase activity in cell-free extracts of *Penicillium* sp. IA-01 at

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 [66], and it has been observed that cyanide and azide partially inhibited purified chromate reductase of *E. coli* ATCC 33456 19 [67] and aerobic chromate reduction by *Bacillus subtilis* [71] and inhibited more than 50% of membraneassociated chromate reductase activity of *S. putrefaciens* MR-1 [74], while no inhibition was observed in CFE of *Bacillus* sp. ES29 [70]. Respiratory inhibitors act on de novo protein synthesis or affect the respiratory chain intermediates responsible for Cr (VI) reduction,

Electron donors

184 Advances in Bioremediation of Wastewater and Polluted Soil

Specific activity (mmol/min/mg protein)

pH 7.0 and 37°C

Specific activity (mmol/min/mg protein)

in LMM, probably is resistant to the metal, and also removes efficiently 1g/100 mL of Cr (VI) after 90 min of incubation, and removes 63.2% and 43% from soil and water samples contaminated, respectively. This strain showed an efficient capacity of reduction (91%) of 50mg/L Cr (VI) in the growth medium after seven days of incubation, at 28°C, pH 5.3, 100 rpm and with an inoculum of 169mg of dry weight. The Cr (VI) reduction potential of the resting cells was increased by cell permeabilization. The optimum temperature and pH of chromate reductase activity of the CFE, were found to be 37<sup>∘</sup>C and 7.0, respectively, and activity was enhanced in the presence of 0.1mM NADH and other electron donors. 1mMol of metal ions like Cu2+, Na<sup>+</sup>, Hg2+, Mg2+, Fe3+, Ca2+, and Cd2+ and respiratory inhibitors resulted in a decrease of the activity. Finally, these results suggest the potential applicability of Penicillium sp for the remediation of Cr (VI) from polluted soils and waters. References [1]. Ahemad, M. Bacterial mechanisms for Cr(VI) resistance and reduction: an overview and recent advances. Folia Microbiologica 2014; 59, 321–332. We isolated a *Penicillium* sp. IA-01 strain, which grow about 50% relative to control (85 mg of dry weight without metal) in LMM, probably is resistant to the metal, and also removes efficiently 1g/100 mL of Cr (VI) after 90 min of incubation, and removes 63.2% and 43% from soil and water samples contaminated, respectively. This strain showed an efficient capacity of reduction (91%) of 50mg/L Cr (VI) in the growth medium after seven days of incubation, at 28°C, pH 5.3, 100 rpm and with an inoculum of 169mg of dry weight. The Cr (VI) reduction potential of the resting cells was increased by cell permeabilization. The optimum temperature and pH of chromate reductase activity of the CFE, were found to be 37°C and 7.0, respectively, and activity was enhanced in the presence of 0.1mM NADH and other electron donors. 1mMol of metal ions like Cu2+, Na+ , Hg2+, Mg2+, Fe3+, Ca2+, and Cd2+ and respiratory inhibitors resulted in a decrease of the activity. Finally, these results suggest the potential applicability of *Penicillium* sp for the remediation of Cr (VI) from polluted soils and waters.

sediments. Applied and Environmental Microbiology 2001; 67, 1517-1521.

[3]. Seng, H. and Wang, Y.T. Biological reduction of chromium by E. coli. Journal of Environmental Engineering

[4]. Marsh, T.L. and McInerney, M.J. Relationship of hydrogen bioavailability to chromate reduction in aquifer

[6]. Lofroth, G. and Ames, B.N. Mutagenicity of inorganic compounds in Salmonella typhimurium: Arsenic,

[5]. Nriagu, J.O. and Nieboer, E. Chromium in the natural and human environments. Wiley-Interscience, New York;

[2]. United States Environmental Protection Agency. www.epa.gov.

chromium and selenium. Mutation Research 1978; 53(2), 65-66.

1994; 120(4), 560-572.

1988.
