**2.3. Reduction of Cr (VI) by living cells**

Recently, the reduction of Cr (VI) to Cr (III) through an enzymatic mechanism has been observed in *Pichia*. Both in intact cells and in cell-free extracts of *P. jadinii* M9 and *P. anomala* M10 strains, chromate was reduced, suggesting the presence of a chromate reductase activity possibly associated with the cytosolic or membrane proteins [18]. In the bacteria *Pseudomonas putida* F1, challenged with Cr (VI) in minimal médium (instead of in the complex LB medium), an ATPase involved in DNA repair-like protein (Pput 2963) was overexpressed compared with untreated cultures, suggesting that DNA damage occurs [19], and a non-enzymatic mechanism of Cr (VI) reduction has been described for *A. niger* [20]. The purpose of this chapter is to elucidate the characteristics of removal of chromium (VI) by *Penicillium* sp. IA-01cells.

**2.1. Screening of the microorganism showing the resistant to Chromium (VI) and chromate**

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

characteristic macroscopic and microscopic observation [21]. Fungal cultures grown in thioglycolate broth were used as primary inoculums. Chromate-resistant tests of the isolated strain, filamentous fungus *Penicillium* sp IA-01, were perform on liquid LMM containing the appropriate nutritional requirements and different concentrations of Cr (VI) (as potassium

The fungal cells was grown at 28°C in an stirred and aerated liquid media containing thiogly‐ colate 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

C for seven days. The strain was identified based on

**2. Materials and methods**

168 Advances in Bioremediation of Wastewater and Polluted Soil

buffer. The plates were incubated at 28<sup>∘</sup>

**2.2. Biosorption tests by using dry cells**

chromate), and the dry weight was determined.

**resistance test**

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 *×* 105 spores/mL (28*◦*C, and 48 h), the cells were centrifugated (2000 rpm, at 4*◦*C/10 min), and washed twice with sterile trideionized water and the pellet was resuspended in 3 mL of the same solution, and was transferred at a fresh LMM (100 mL with 50mg/L Cr (VI)). At different times, 1 mL aliquots were removed and centrifuged (5000 rpm/10 min), and we determine the concentration of Cr (VI) or total Cr in the supernatant.

Reduction efficiency of Cr (VI) was examined by the resting cells. 5 *×* 105 spores/mL of *Penicillium* sp. was inoculated and incubated in 100 mL thioglycolate broth (pH 7.0) for five days, and was harvested (3000 ×g at 4∘ C); cell pellets obtained were washed by centrifugation twice with 100 mM potassium phosphate buffer (pH 7.0) and resuspended in the same buffer. The suspended cell pellets were added in 2-10mg/100 mL of Cr (VI) solution, mixed for 30 min, and incubated at 30<sup>∘</sup> C for 6 h. Heat-killed culture pellets (2 mL), which were treated at 100<sup>o</sup> C for 10 min were used as control. After the incubation, the tubes were centrifuged, and 100 μL aliquots were withdrawn from each sample to estimate the remaining Cr (VI).

Reduction efficiency of Cr (VI) was also examined by the permeable fungal cells. Culture of *Penicillium* sp. IA-01 was grown for five days, harvested, and washed with potassium phos‐ phate buffer (pH 7.0) as described above. The suspended culture pellets were treated with 0.2% (w/v) sodium dodecyl sulphate, 0.2% Tween 80, (v/v), 0.2% Triton X-100 (v/v), and 0.2% toluene (v/v), by vortexing for 30 min to achieve cell permeabilization. Permeabilized cell suspensions (0.5 mL) were then added with 2–10mg/100 mL of Cr (VI) as final concentrations and incubated for 6 h at 30<sup>∘</sup> C.

#### **2.4. Activity of chromate reductase**

Cell-free extracts (CFE) of *Penicillium* sp IA-01 were prepared by modifying the previously published protocols. The pellets were resuspended in 5% (v/v) of the original culture volume in 100 mM potassium phosphate buffer (pH 7.0). These cell suspensions were placed to an ice bath and disrupted using an Ultrasonic Mini Bead Beater (Densply) with 15 cycles of 60 sec for each one. The sonicate thus obtained was then centrifuged at 3000 x g for 10 min at 4∘ C. The pellet was resuspended in 100 mM potassium phosphate buffer (pH 7.0), and this is the CFE.

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<sup>∘</sup> 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<sup>∘</sup> 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<sup>∘</sup> 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 [23].

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

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 and the mean value was shown
