Uptake and Reduction of Hexavalent Chromium by Aspergillus niger and Aspergillus parasiticus

The uptake and reduction of Cr(VI) by Aspergillus niger and A. parasiticus was studied in this journal. After 96 hours of growth, the culture solutions spiked with an initial dichromate concentration of 20 mg/l, were completely decolorized and had residual Cr(VI) concentrations of only 0.74 ± 0.55 and 1.69 ± 0.29 mg/l in A. niger and A. parasiticus cultures representing Cr(VI) removal of 96.3% and 91.6%, respectively. In the A. niger culture, significantly (P < 0.01) lower Cr(VI) concentrations were observed within 72 hours of growth compared to those of A. parasiticus, but in both cultures complete removal was almost achieved by 144 hours of growth. The rate of Cr(VI) removal was 0.21 ± 0.09 mgl-1hr-1 and 0.20 ± 0.07 mgl-1hr-1 for A. niger and A. parasiticus, respectively. Cellular concentrations of Cr(VI) in the two fungi increased significantly (P < 0.05 – 0.001) with increasing concentrations of the dichromate treatments. Although tannic acid as sole source of carbon and energy gave significantly lower Cr(VI) removal than glucose (P < 0.001) and acetate (P < 0.01), it supported the removal of about 85.0% and 68.8% of the metal ion by A. niger and A. parasiticus, respectively. The active mycelia of both fungi showed significantly (P < 0.001) higher Cr(VI) removal than inactivated mycelia after incubation at 30°C for 72 hours. Incubation of cell – free extracts of both fungi with NADH at 30°C for 2 hours showed Cr(VI) reduction of 68.0% and 55.5% for A. niger and A. parasiticus, respectively. These findings suggest that uptake and metabolic reduction may be the process by which the two fungi are able to tolerate the toxic effects of hexavalent chromium. However, Cr removal via uptake by the two fungal biomass was observed to be in the range of 0.5 – 1.78% only, for all the concentrations applied, which is insignificant when compared with the initial Cr concentration in the culture medium. The results obtained through this investigation indicate the possibility of treating waste effluents containing hexavalent chromium using Aspergillus niger and A. parasiticus. Uptake and Reduction of Hexavalent Chromium by Aspergillus niger and Aspergillus parasiticus


Introduction
Chromium pollution is one of the most important environmental problems in many regions of the world. Improper disposal of this heavy metal at facilities in arid and semiarid regions has led to contaminations of underlying vadose zones and aquifers [1]. Hexavalent chromium, which is highly toxic to most biological systems is generated and released to the environment through a large number of industrial operations, including tanning, metal electroplating, iron and steel and inorganic chemical industries [2] and is largely responsible for the pollution of underground water due to its high mobility in soils.
Various solutions have been proposed for bioremediation of chromium contaminated soil and water with selected microbial species. Many species of bacteria [1,[3][4][5][6][7][8], yeasts [9][10][11] and fungi [12][13][14][15][16] have been investigated from this aspect, and the biosorption and bioaccumulation of chromium for bioremediation purposes have been demonstrated. Park et al. [13] had demonstrated that Pseudomonas putida culture was able to carry out enzymatic reduction of chromates. Cr(VI) was also shown to be quantitatively transformed to Cr(III) largely by soluble reductase activities in Bacillus species isolated from chromium-contaminated soil samples [5] Chromium reduction by the Bacillus species was not affected by sulphate and/or nitrate. The removal of chromium ions by the cyanobacterium, Anacystis nidulans immobilised on agar has been documented [3] Reports on chromate reduction by resting cells of sulphate-reducing bacteria particularly, Desulfovibrio vulgaris [17] and Cellulomonas sp. [4] are also available.
The biosorption processes for the removal of Cr(VI) from aqueous solutions and industrial effluents by dead fungal biomass [18] immobilized biomass of Rhizopus arrhizus [11,12] and fungal biomass of Rhizopus nigricans [14,15] have been reported. Biosorption of chromium from aqueous solutions by fungal pellets of Aspergillus niger has also been reported [13]; The bioremediation of chromium by the yeast Pichia guilliermondii [10] and Saccharomyces cerevisiae [9,19] has also been studied. Increased tolerance to chromium and enhanced Cr accumulation capabilities were observed among Cr-resistant mutants of Pichia guilliermondii obtained by means of environmental mutagenesis [10].
The mechanisms of chromium tolerance or resistance of selected microbes are of particular importance in both bioremediation and waste water treatment technologies. Studies of the various microbial uptake mechanisms for heavy metals could result in the identification of specific microbes with promising potential for application in metal bioremediation. Prasenjit and Sumathi [16] had demonstrated the ability of Aspergillus foetidus to take up chromium during the stationary phase of growth and under non-supportive growth conditions. In this work we report the uptake and metabolic transformation of hexavalent chromium by cultures of Aspergillus niger and Aspergillus parasiticus.

Fungal isolates
The fungi, Aspergillus niger and Aspergillus parasiticus, used in this study were isolated from landfill and sludge samples obtained from the Neital Shoe Factory and Tannery, Maiduguri, Nigeria. The isolation and identification of the fungi was carried out in the Mycology laboratory of the Plant Pathology Unit of the Department of Crop Science, University of Maiduguri. The fungi were identified by their colony characteristics as well as their vegetative and reproductive structures as observed under the electron microscope. Some macroscopic characteristics used for the identification include, colour of the colony, patterns of growth of colony and the byproducts released by the organisms. Some of the microscopic characteristic as viewed under the microscope include, the shape of the conidia head, pattern of arrangement of spores on the conidia, shape of the spores and shape of the conidiophores. The isolates were further confirmed in the Department of Microbiology, Ahmadu Bello University, Zaria, Nigeria. The organisms were isolated and maintained on Sabouraud Dextrose Agar, SDA (Lab M, Biotech, England) at pH 5. Inoculated slants were incubated for 7 days at 30°C and then stored at 4°C until used.

Growth medium and culture conditions
For the growth of the fungi in suspension culture, a stock solution containing 0 to 20 mg/l Cr(VI) was prepared by dissolving analytical grade potassium dichromate (K 2 Cr 2 O 7 ) in 1 litre of sterile modified Vogel's mineral salts medium [20][21][22][23]. The solution was adjusted to a final pH of 4.5 ± 0.5 and 100 ml was used as culture medium for the growth of fungi. Spores of the two fungi were harvested from the 7day-old culture slants by washing with 0.2% Tween-80. After vigorous shaking, inocula containing an average of 5.00 × 10 3 spores per ml were obtained for each of the two fungal strains.
A batch system of cultivation in batch reactors using 150-ml Erlenmeyer flasks on a rotary shaker (200 to 300 rpm) at pH 4 to 5 and temperature of 30°C for 96 hours was used for growing the fungi. The culture media in the bioreactor were inoculated with an average biomass of 50 mg per liter of inoculum of the fungal strains. Culture media without chromium treatment, 0 mg/l Cr(VI), served as control for the growth experiment.

Determination of Cr(VI) removal in the fungal culture
The removal of chromate from the culture solutions was determined by estimating the residual Cr(VI) in the culture media after the 96 hour growth period as previously described by Shugaba et al. [21][22][23] Cr(VI) uptake and reduction during growth was followed by loss of the yellow to orange colour of the dichromate solution and by the quantitative decrease in Cr(VI) concentration in culture [6,24,25] also, as part of the Cr(VI) reduction studies, the concentrations of trivalent chromium, [Cr(III)], and total chromium, Cr T were also determined in the culture medium [26] Samples of the spent culture solutions were filtered using a membrane filter before measuring the Cr(VI), Cr(III) and Cr T concentrations.
The role of metabolic activity in Cr(VI) removal by the two fungi was evaluated as described by Desjardin et al. [27]. Active and inactivated mycelia of A. niger and A. parasiticus were incubated in culture medium spiked with 50 mg/l Cr(VI) under the same conditions for 72 hours. Inactivation of the mycelia was achieved by autoclaving at 121 0 C for 20 minutes. In order to determine the effects of carbon sources (electron donors) on chromium removal by the two fungi, glucose, acetate and tannic acid were applied separately as sole carbon and energy sources in fresh culture media containing 20 mg/l Cr(VI).

Determination of Cr(VI), Cr(III) and Cr T concentrations in culture medium
The concentration of hexavalent chromium, Cr(VI), in the culture media was determined spectrophotometrically using 1, 5-diphenylcarbazide as complexing agent [24,[28][29][30]. One milliliter (1 ml) of 0.2 % w/v of 1, 5-diphenylcarbazide solution (prepared in 95% ethanol and 1 ml of 1 / 5 H 2 SO 4 ) was added to 1 ml of the sample solution. The solution was allowed to stand for 10 minutes after which the absorbance of the purple-coloured solution was read at 540 nm. Cr(VI) concentration was extrapolated from a standard curve prepared from standard solutions of potassium dichromate.
Cr(III) and Cr T were determined using atomic absorption spectrophotometer with Zeeman graphite furnacve as described by Xu et al. [31]. For Cr(III) determination, about 20 ml of the filtered medium was passed through anion exchange resin at a flow-rate of 1.0 ml/min, and then it was collected for the determination of trivalent chromium, Cr(III), concentration by Atomic Absorption Spectrophotometer (AAS).

Determination of total chromium uptake by fungal biomass
For the determination of total chromium uptake by the fungi, the mycelia were acid digested [10,32]. The cells were separated from the culture media by filtration, washed with water and dried. The dry cells were weighed and then suspended in a known volume of concentrated nitric acid, kept for 30 min at room temperature, and heated gently on a heating mantle (at 60°C for 30 min). After cooling to room temperature, hydrogen peroxide (H 2 O 2 ) solution was added (in a ratio of 7:3 v/v nitric acid/H 2 O 2 ) and the samples were heated again for 15 minutes and then centrifuged at 1500 x g. The supernatant was then used to determine the total chromium Cr T concentration using Atomic Absorption Spectrophotometer, AAS (Perkin Elmer, Japan Co, Ltd.) with Zeeman graphite furnace.

Determination of Cr(VI) reducing activity of cell-free extracts of fungal mycelia
The ability of the two fungi to reduce hexavalent chromium in solution was studied using cell -free extracts of exponentially grown mycelia. The cell-free extracts were prepared by homogenizing the mycelia in 10 ml of 50 mM Tris-HCl buffer (pH 7.0) and then centrifuged at 3000 x g. The supernatant was then tested for ability to reduce Cr (VI). The Cr(VI) reducing activity was determined by measuring the decrease in Cr(VI) concentration [24] after incubation for 2 hours. The activity was assayed at 30°C in a reaction mixture containing 0.1 mM NADH (40 µl), 50 mM Tris -HCl buffer of pH 7.0 (1.8 ml) 0.05 mM K 2 Cr 2 O 7 (100 µl) and 600 μl of 0.2% 1, 5 -diphenylcarbazide reagent and the absorbance was read at 540 nm against a reference sample incubated without the extract.

Measurement of fungal biomass concentration
The biomass concentration in the culture media was measured by collecting the biomass on pre-dried Whatman No. 1 filter paper, washing the biomass with distilled water and drying to constant weight at 70°C [20]. The biomass concentration was calculated as follows: Where A = weight of the dry cells + filter paper B = weight of the filter paper, and V = Volume of culture media (l)

Measurement of specific growth rate
Culture turbidity was used to determine the specific growth rates (µ) of fungal cultures growing in shake -flask batch cultures [33]. Erlenmeyer flasks containing 100 ml of the growth medium and Cr(VI) at concentrations of 5 -20 mg/l were inoculated with 2 ml of exponential -phase culture grown in identical medium (initial cultures absorbance of 0.1 -0.4 nm). The fungal cultures were then incubated at 30°C on a rotary shaker at 200 rpm and changes in turbidity were determined at 24 hours interval for five days. Culture turbidity was measured spectrophotometrically by taking the absorbance of the growing cultures at 560 nm. The specific growth rate, µ (hr -1 ) was determined by dividing the change in turbidity by the time interval within which growth was assessed.

Statistics
The data obtained in the study were analyzed by one-way analysis of variance, (ANOVA) and the Student t-test (using GraphPad Instat statistical program). Differences between means were considered significant at values of P<0.05.

Results and Discussion
Changes in Cr (VI) concentration during fungal growth Figure 1 shows the changes in hexavalent chromium concentrations during growth of A. niger and A. parasiticus. Cr(VI) was significantly (P<0.001) reduced in both cultures every 24 hours with decolouration of the characteristic pale yellow colour of the dichromate. At 96 hours, the solution became completely colourless, and exhibited a Cr(VI) concentration of only 0.74 ± 0.55 and 1.69 ± 0.29 mg/l in A. niger and A. parasiticus cultures, respectively. Thus, with the initial dichromate concentration of 20 mg/l up to 96.3 % and 91.6 % Cr(VI) removal was achieved within 96 hours by A. niger and A. parasiticus, respectively. The significant reduction in media Cr(VI) concentration following fungal growth in both cultures suggest that A. niger and A. parasiticus are both capable of removing this toxic metal in solutions. A. niger culture showed significantly (P<0.01) lower Cr(VI) concentration compared to that of A. parasiticus within 72 hours of growth, suggesting that it is more efficient than A. parasiticus. However, both organisms showed complete removal of Cr(VI) from their media by 144 hours of growth. The rate of Cr(VI) removal was 0.21 ± 0.09 mgl -1 hr -1 and 0.20 ± 0.07 mgl -1 hr -1 for A. niger and A. parasiticus, respectively.
Chromium removal using bacterial chromate reductase has been reported by several workers [5,6,25,29,]. The complete decolouration and clearance of up to 20 mg/l Cr(VI) from the culture media of A. niger and A. parasiticus suggests that these organisms may have the enzymic capabilities for converting Cr(VI) to Cr(III), thus indicating their ability for adaptation. The observed decrease in Cr(VI) concentration in culture media of A. niger and A. parasiticus may be related to a metabolic reduction of Cr(VI) ions, a metabolic uptake of Cr(VI) with no reduction, or a passive uptake of Cr(VI) with no reduction (i.e. biosorption). Metabolic uptake with reduction [27,25,29,34] as well as active uptake without reduction (bioaccumulation) and passive uptake (biosorption) of metal ions, including hexavalent chromium [27,35,36] are known processes of bioremediation by microorganisms.
The cellular concentration of Cr(VI) in the two fungi ( Figure 2) increased significantly (P<0.05-0.001) with increasing concentration of Cr(VI) treatment; suggesting that the two organisms take up chromium ions from the media solution. Biosorption and bioaccumulation are processes by which microorganisms remove metal ions from solutions and industrial effluents [11,[35][36][37]. Aspergillus mycelia have particularly been found to be efficient for removing zinc and thorium from solutions and polluted water [38,39]. In this study A. parasiticus had significantly (P<0.001) higher cellular Cr(VI) concentration (587.56 ± 47.17 µg/g) compared to A. niger (82.12 ± 3.45 µg/g) at the highest Cr(VI) treatment (20 mg/l). Thus A. parasiticus accumulates more chromium than A. niger.
Accumulation of high concentrations of Cr(VI) up to 34 mg/g dry weight by some microorganisms has been reported [40]. The mechanism of Cr(VI) -resistance and reduction may differ in microbial community from group to group or from strain to strain within a group [41] In some organisms Cr(VI) reduction takes place intracellularly because the enzyme is membrane-bound, and in such cases the substrate is taken up from the medium into the cells [34]. The rate of Cr(VI) uptake and its subsequent conversion to Cr(III) differ from organism to organism and reduced substrate uptake is said to be one of the mechanisms of chromium resistance in many microorganisms [42,43].
The significant difference in intracellular Cr(VI) concentration between A. niger and A. parasiticus may therefore be due to differences in the rate of chromium uptake between the two organisms and/or rate of conversion to Cr(III). It is likely that A. parasiticus may have taken up so much Cr(VI) that it could not convert to Cr(III) since intracellular accumulation of Cr(VI) may also disturb the normal functioning of microbial cells [34]. In studies of chromium toxicity and accumulation, Kshminska et al. [10] reported diverse, individual and complex mechanism of chromium bioremediation in the cells of different strains of the yeast Pichia guilliermondii. They observed that all the investigated strains responded to Cr(VI) treatment with substantial changes in protein profile patterns, which included both induction and suppression of certain polypeptides. In particular they

Influence of carbon source on Cr(VI) reduction
The effect of carbon source on chromium reduction in cultures of A. niger and A. parasiticus is shown in Figure 3. Throughout the growth period, Cr(VI) concentrations were lower in cultures containing glucose compared to those with acetate and tannin as carbon sources; the difference being significant (P<0.001) between 72 and 120 hours. Cultures containing acetate also showed significantly (P<0.01) lower Cr(VI) compared to those with tannic acid as carbon and energy source (electron donor). This finding suggests that glucose and acetate as carbon and energy source support Cr(VI) reduction better than the tannin Figure 4. Glucose and acetate are known electron donors for Cr(VI) reduction by several organisms; indeed, about 2700 mg/l glucose was utilized to reduce Cr(VI) from 20 mg/l to 12 mg/l in 24 hours, and that Cr(VI) reduction ceased when glucose was nearly depleted within the 24 hours growth period [44]. According to Prasenjit and Sumathi [16], the initial rate of chromium uptake is enhanced by higher biomass concentrations and the presence of glucose. In an earlier study, it was shown that Cr(VI) reduction by pure culture of S. thermocarboxydus NH50 was more rapid with glycerol as sole carbon source than with glucose [27].
However, the fact that Cr(VI) concentration was significantly (P<0.001) reduced by about 85.0 % (A. niger) and 68.8 % (A. parasiticus) after 96 hours of growth in media with tannic acid as sole carbon and energy source shows that these fungi are capable of utilizing tannins as carbon source or electron donors for Cr(VI) removal in solutions. The tannic acid, being a complex polyphenol, may not be readily available for use by the organisms; it may therefore have to be degraded to simpler, less toxic products, before it can be used to fuel Cr(VI) reduction. Indeed, it was reported that microorganisms breakdown complex tannins to simpler and less toxic products, which they could use as nutrients for growth [45,46] The growth of Fusarium flocciferum [47] the Penicillium strain Bi 7/2 [46], Lactobacillus hilgardii and Lactobacillus plantarum [46,48] on tannins such as protocatechuic, gallic, ferullic and syringic acids has been described. These organisms were able to grow on the tannins after degrading them into smaller less toxic products, which they used as source of energy and carbon for growth and metabolism.

Cr(VI) removal by active and inactive fungal biomass
The results presented in Table 1 show that in the media with active mycelia, A. niger culture had significantly (P< 0.05) lower Cr(VI) concentrations (16.72 ± 1.25 mg/l) compared to the active A. parasiticus culture (22.83 ± 1.03 mg/l); representing 66.6% and 54.3% Cr(VI) removal, respectively. However, there were no significant (P>0.05) differences in residual Cr(VI) concentrations in the media containing the inactivated mycelia of these fungi. For both organisms, the media with active cells had significantly (P<0.001) lower Cr(VI) concentrations after the 72 hours of incubation compared to those with inactivated mycelia. Also, for both fungi, the residual Cr(VI) concentrations were significantly lower in media with the active mycelia (P<0.001) and inactivated mycelia (P<0.05) compared to media incubated without the mycelia. In the media incubated with the inactivated mycelia, the Cr(VI) removal was 19.1% and 15.7% for A.      These observations suggest that Cr(VI) removal by A. niger and A. parasiticus is mainly a metabolic process involving either a metabolic uptake and reduction or metabolic uptake with no reduction (i.e. bioaccumulation). In addition, the significant decrease in Cr(VI) concentration in the presence of inactivated mycelia compared to values obtained in the absence of mycelia suggests that a passive uptake (biosorption) may also be involved in the removal process for Cr(VI). Bioaccumulation is a growth dependent process by which microbial cells sequester metals ions intracellularly by an active metabolic process; it is therefore mediated only by the living biomass [27,35]. In biosorption, the microbial biomass binds substantial amounts of metal ions by a passive process mediated by living and non -living biomass [27,[35][36][37].
The mechanisms of biosorption mainly involve cell surface complexation, ion-exchange, chelation, and microprecipitation [49]. A large number of microorganisms are known to bind a variety of heavy metals with varying degrees of affinity. However, fungal biomasses exhibit a high percentage of cell wall material, with excellent metalbinding properties [37,49]. Among the fungi that have shown excellent potential of metal biosorption, Aspergillus and Rhizopus species have been well studied [13][14][15]37,50].

Cr(VI) -reducing activity of fungal extracts
The residual Cr(VI) concentrations of the reaction mixture incubated with the cell-free extracts of both organisms decreased significantly (P<0.001) within 2 hours, from an initial concentration of 7.35 ± 0.05 mg/l to 2.35 ± 0.17 mg/l and 3.27 ± 0.07 mg/l for A. niger and A. parasiticus, respectively. This shows Cr(VI) reduction of 68.0 and 55.5% for A. niger and A. Parasiticus, respectively. The observation of Cr(VI) reduction by the cell-free extracts of the two fungi suggests that metabolic uptake and reduction to Cr(III) may be the process by which these organisms carry out bioremediation of Cr(VI) in solutions. The conversion of Cr(VI) to Cr(III) has been recognized as one of the several mechanisms by which Cr(VI)-resistant organisms tolerate the toxic effects of chromium [25,29,34]. Cr(VI) -resistant organisms are known to produce chromium reductase, which mediates the transfer of electrons from NAD(P)H to Cr(VI), converting it to Cr(III) [25,29,51]. The abilities of Aspergillus and Penicillium species to massively reduce Cr(VI) to Cr(III) have earlier been reported [26,[52][53][54][55].

Chromium removal via uptake by the fungal biomass
In Table 2 are results showing Cr uptake determined by multiplying the concentration of Cr in the fungal cells grown for 96 hours at 30°C by the fungal biomass concentration. Cr accumulation by the two organisms ranged from 0.5 -1.78%, for all the concentrations applied; the percentage Cr uptake decreases with increasing concentrations of Cr(VI) treatment in the growth medium. These chromium uptake values are insignificant when compared with the initial Cr concentration in the culture medium. Similar observations on Cr uptake by fungal biomass of Aspergillus (strain Ed8) and Penicillium (strain H13) were reported by Acevedo-Aguilar et al. [26] leading to suggestion that Cr uptake by these fungal strains was almost negligible, as compared to the total Cr present in the growth medium. They reported a total Cr uptake of only about 0.06% and 0.04% by the fungal biomass of the Aspergillus sp. (strain Ed8) and the Penicillium sp. (strain H13), respectively after a growth period of 96 hours in a medium containing initial Cr content of 2500 µg. Thus, very little of the Cr in the culture medium was incorporated into the biomass, indicating that the decrease of Cr(VI) in the culture media of strains Ed8 and H13 resulted from its quantitative reduction to Cr(III) rather than from its uptake by the biomass.
In an earlier study, however, Park et al. [52] reported that when synthetic wastewater containing Cr(VI) was placed in contact with   dead fungal biomass of Aspergillus niger, the Cr(VI) was completely removed from aqueous solution, whereas Cr(III), which was not initially present, appeared in aqueous solution Table 3. By using desorption and X-Ray Photoelectron Spectroscopy (XPS) studies they showed that most of the Cr bound onto the biomass was in the trivalent form and postulated that the main mechanism of Cr(VI) removal was a redox reaction between Cr(VI) and the dead fungal biomass, which is quite different from previously reported mechanisms. It was also reported that Trichoderma inhamatum, grown in a batch culture system, exhibited a remarkable capacity to tolerate and completely reduce Cr(VI) to Cr(III) from initial concentrations of 0.83 up to 2.43 mM [56] and that the specific and volumetric rates of Cr(VI) reduction by this fungus decreased as the initial Cr(VI) concentration increased.
It has been reported that in some chromate-resistant yeasts, the general mechanism of Cr(VI) resistance is related to limited ion uptake rather than to chemical reduction of toxic species of the metal ion . However, other yeasts such as Candida maltosa [57] showed some ability to reduce Cr(VI) and also the capability to accumulate chromium in the biomass. Prasenjit and Sumathi [16] had reported 97% decrease in hexavalent chromium (initial concentration, 5 µg/g) at the end of 92 hours of growth in Aspergillus foetidus culture, possibly due to its reduction to Cr(III) and/or complexation with organic compounds released due the metabolic activity of the fungus. Thus, variations in metal tolerance may be due to the presence of different types of tolerance processes or resistance mechanisms exhibited by different isolates. Sun and Shao [58] had demonstrated that both intracellular bioaccumulation and extracellular biosorption contributed to the high resistance of Penicillium sp. Psf-2 to lead. Sintuprapa et al. [59] also suggested that ion exchange and intracellular accumulation in the form of polyphosphate precipitation are the mechanisms of Zn 2+ uptake by living cells of Penicillium sp. The resistance of the bacterium Ralstonia mettalidurans CH34 to Pb is mediated by a P-type ATPase, which can transport lead out of the cell. Copper resistance in Aspergillus niger is due to an active process involving copper metallothionein synthesis [60].
Among microorganisms, fungal biomass offers the advantage of having a high percentage of cell wall material with excellent metalbinding properties [35][36][37]. Many fungi and yeast, particularly the genera Rhizopus, Aspergillus, Streptoverticillum and Saccharomyces have shown excellent potential for metal biosorption and bioaccumulation. The entry of all metal ions into the plasma membrane and cytoplasm takes place via the cell wall, which consists of a variety of polysaccharides and proteins that offer a number of active sites for metal binding. Differences in the cell wall composition among different species of microorganisms and the intra species differences can thus cause significant differences in the type and amount of metal binding to them. Fungi generally show greater resistance to heavy metals than many bacterial species [61] because mycelial growth gives the organism a larger surface area, which provides greater protection to sensitive organelles of the fungi. In addition, fungi are eukaryotic cells, which contain more genes that provide other means of dealing with inhibitory compounds such as toxic metals. Tables 4 and 5 show the specific growth rates and biomass concentrations, respectively, for both fungi after sub-culturing from the Cr(VI) treated cultures into non-chromium media. The specific growth rates for both organisms sub-cultured from all the treatments were not significantly (P>0.05) different. Similarly, the biomass concentrations of both organisms were not significantly (P>0.05) different, except for those of A. parasiticus sub-cultured from the 20 mg Cr(VI)/l treatment which showed significantly (P<0.05) lower biomass compared to the rest.

Regeneration of fungal cells after initial exposure to Cr(VI)
Biotechnological exploitation of microbial biosorption and bioaccumulation technology for removal of heavy metals from wastewater and industrial effluents depends on the efficiency of the   regeneration of the microbial biomass for reuse in multiple cycles [35]. In bioremediation of metal ions, the performance of living microbial biomass depends much on cell age and toxic effects of heavy metals may result in cell death [36]. Hexavalent chromium is highly toxic to a lot of microorganisms, and like other heavy metal ions such as Cu(II), Pb(II), Zn(II), etc. it may cause structural damage in cells and consequently a permanent effect on cell growth [34,62].
The results obtained in this study show that although A.niger and A. parasiticus were exposed to concentrations of Cr (VI), which severely inhibited their growth; these organisms were capable of regenerating with specific growth rates (µ) and biomass comparable to those of the chromium-free control organisms. This observation suggests a remarkable potential for application of these fungi in chromium biotreatment. Sani et al. [62] had reported similar observation with Desulfovibrio desulfuricans where toxic concentration of the heavy metal ions, Cu(II), Pb(II) and Zn(II) utterly inhibited growth, but did not kill the cells of the organism. Although Ezzouhri et al. [63] reported 10 -15 mM of Cr(VI) as the Minimum Inhibitory Concentration (MIC) for Aspergillus species, the results obtained however revealed that the response of the isolates to heavy metals depended on the metal tested, its concentration in the medium and on the isolate considered. They observed that various genera and also isolates of the same genus did not necessarily have the same heavy metal tolerance. Thus, variation in metal tolerance among fungal species may be due to the presence of one or more strategies of tolerance or resistance mechanisms exhibited by fungi.