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Studies on the growth of some filamentous fungi in culture solutions containing hexavalent chromium.

Introduction

The use of microbial biomass of fungi, bacteria and algae for the removal of toxic pollutants and heavy metals from aqueous solutions is gaining increasing attention (Wainwright, 1992; Filipovic-Kovacevic et al., 2000; Ksheminska et al., 2003; Rockne and Reddy, 2003). This technology employs the use of microorganisms to metabolize contaminants either through oxidative or reductive processes. Several species of microorganisms are capable of accumulating metal ions up to concentrations several orders of magnitude higher than the background concentration (Krauter et al., 1996; Khattar et-al., 1999).

Among microorganisms, filamentous fungi are well recognized for their superior capacities to produce a wide variety of extracellular enzymes, organic acids and other metabolites, and for their capabilities to adapt to severe environmental constraints (Wainwright, 1992). For example, members of the Deuteromycetes such as Aspergillus, Penicillium and Trichoderma species are known to produce numerous extracellular enzymes, which are put to good use in biotechnology (Elander, 1989; Wainwright, 1992; Smith, 1996). Similarly, the Basidiomycetes white rot fungi such as Phanerochaete chrysosporium are noteworthy for their abilities to produce nonspecific ligninases and peroxidases (Bumpus et al, 1985; Aust, 1990; Bonnarme and Jeffries, 1990; Rodriguez et al., 1999) which can be used to degrade pollutants both in liquid effluents and in soils. The capabilities of fungal biomass for treating metal-contaminated effluents with efficiencies several orders of magnitude superior to activated carbon (F-400) or the industrial resin Dowex-50 have been highlighted (Coulibally et al., 2003). In this work we studied the effect of hexavalent chromium on the growth of some filamentous fungi with a view to identifying chromium tolerant species for possible application in the bioremediation of this toxic heavy metal.

Materials and Methods

Organisms

Four species of fungi: Aspergillus, Penicillium, Rhizopus and Scopulariopsis were isolated and tested for hexavalent chromium tolerance. Aspergillus niger, Aspergillus flavus, Aspergillus parasiticus and Penicillium roquefortii were isolated from landfill and sludge samples obtained from the Neital Shoe Factory and Tannery while Rhizopus stolinifer and Scopulariopsis fusca were isolated from the stomach contents of ruminant animals obtained after slaughter at the Central Abattoir in Maiduguri, Nigeria. The organisms were isolated and maintained on Sabouraud Dextrose Agar, SDA (Lab M, Biotech, England). 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 isolates were further confirmed in the Department of Microbiology, Ahmadu Bello University, Zaria, Nigeria.

Screening of the isolated fungi for Cr (VI) tolerance

The tolerance of the isolated fungi to chromium was studied by growing the organisms on culture plates containing varying concentrations of hexavalent chromium. Potassium dichromate solution was diluted with the growth media (SDA) to obtain varying concentrations from 0.5% up to 3.0 % and 0 % served as control. Uniform portions of the test fungi were removed from a 7- day old culture using a 10 mm steel borer and aseptically placed on the assay media as described by Yongabi et al. (2000). Diagonal lines were initially ruled at the back of the agar plates using a bold marker to ease measurements of mycelia length. The set-up was carefully sealed all round with a masking tape to prevent aerial contamination and then incubated at 30oC for 7 days. Fungal growth was used as a measure of viability and it was determined by measuring the change in mycelial length with a vernier caliper at 24 hours interval from the 3rd to the 7th day post inoculation.

Cultivation of fungi in submerged culture

For the growth of the fungi in suspension culture, the modified Vogel's mineral salts medium containing 10 g glucose; 1.65 g [(N[H.sub.4]).sub.2]S[O.sub.4]; 0.67 g N[H.sub.4]Cl; 0.1 g MgS[O.sub.4].7[H.sub.2]O; 2.5 g K[H.sub.2]P[O.sub.4]; 1.5 g Na[H.sub.2]P[O.sub.4].2[H.sub.2]O; 0.03 g [Na.sub.2]S[O.sub.4]; 0.08 g [K.sub.2]S[O.sub.4]; and 0.1 g Mg[Cl.sub.2]per litre (Withers et-al, 1998) was used. Varying quantities of sterile potassium dichromate were added directly to the cultivation media. Spores of the fungi were harvested from 7 days old culture slants by washing with 0.2 % Tween-80 and inoculated into a sterile flask (autoclaved at 121[degrees]C for 15 minutes) containing 100ml fresh medium. Fungal growth was evaluated in media with dichromate concentrations of 5 - 25 mg/l. The fungi were grown in batch reactors using 100 ml Erlenmeyer flasks on a rotary shaker (200-300 rpm) at pH 5.0 and temperature of 30[degrees]C for 96 - 168 hours.

Measurement of specific growth rate

Culture turbidity was used to determine the specific growth rates ([micro]) of fungal cultures growing in shake - flask batch cultures (Trinci, 1972). Erlenmeyer flasks containing 100 ml of the modified Vogel's mineral salt medium and Cr (VI) at concentrations of 5 - 25 mg/l were inoculated with 2 ml of exponential - phase culture grown in identical medium (initial culture absorbance of 0.1 - 0.4 nm). The fungal cultures were then incubated at 30[degrees]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, [micro] ([hr.sup.-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 Effect of Cr (VI) on fungal growth

The effect of Cr (VI) on the growth of the six fungi on agar plates is presented in Figure 1. The growth rates of A. niger, A. parasiticus, A. flavus and P. roquefortii were slightly higher at 0.5 % chromium treatment compared to the values obtained for chromium free control. At 1.0 % treatment, A. niger showed significantly (P<0.05) higher growth rate compared to the control value. As the treatment concentrations increased from 0.5 - 2.0 %, the growth rates for A. flavus and P. roquefortii became significantly (P<0.05) lower than those for the control, with no growth at 2.5 %. A. niger and A. parasiticus also showed significantly (P<0.01) lower growth rates as the treatment concentration increased from 1.0- 2.5 %. This observation is further supported by the progressive decrease in the growth of the two fungi on culture plates treated with varying concentrations of potassium dichromate as presented in Plates 1 and 2. Rhizopus stolinifer and S. fusca both exhibited lower growth rates at 0.5 % and 1.0 % compared to their control values, the differences being significant (P<0.05) at 1.0 % chromium treatment. At treatments above 1.0 % these organisms did not show any sign of growth.

The observed decrease in growth rates with increasing concentrations of chromium treatment indicates that chromate is inhibitory to the growth of the organisms under investigation. However, it appears that A. niger and A. parasiticus were relatively more tolerant to the toxic effect of Cr (VI) since they exhibited minimum inhibitory concentration above 1.0 %. Chromates are known to inhibit the growth of most organisms, although resistance to the toxic metal may be developed by the selection of resistant variants to cope with such toxicities (Olukoya et al., 1997).

The inability of A. flavus to grow at 2.5 % chromium treatment shows that strains of the same species may differ in their response to metal toxicity. Francisco et al. (2002) have studied the diversity of Cr (VI) - resistant and Cr (VI)--reducing bacteria in bacterial population from chromium contaminated activated sludge. They reported that the mechanism of chromium resistance and reduction might differ in microbial community from group to group or from strain to strain within the same species. Ksheminska et al. (2003) have reported differential and varied responses to Cr (VI) in cells of different strains of the yeast, Pichia guilliermondii. Also, in studies of chromium removal efficiency by microorganisms in tannery effluent and landfills, Okonkwo et al. (2000) reported that fungi showed better acclimatization to various concentrations of chromium than bacteria. They observed that the bacterial species decreased in count with increasing chrome concentration and died completely at 0.5 % and 1.0 % concentration. On the other hand, the fungi, Aspergillus fumigatus, A. niger, A. flavus and Rhizopus nigricans tolerated the chromium concentration available. A. niger was reported to be the most chromium tolerant, followed by R. nigricans, A. flavus and A. fumigatus.

Effect of Cr (VI) on fungal specific growth rate

Figure 2 shows the effect of hexavalent chromium on the specific growth rate of the six fungi evaluated for Cr (VI) tolerance in submerged cultures at treatments of 5 - 20 mg/l. A. niger and A. parasiticus showed slightly higher specific growth rates at 5 and 10 mg Cr (VI)/l and slightly lower specific growth rates (at 15 and 20 mg/l) compared to their control values. The differences were however not significant (P>0.05). While the specific growth rates for A. flavus and P. roquefortii at 5 and 10 mg Cr (VI)/l were not significantly (P>0.05) different from the values obtained for the controls, these organisms showed significantly (P<0.05) lower specific growth rates at 15 and 20 mg/l Cr (VI) treatments compared to values obtained for the control and at lower treatments. Rhizopus stolinifer and S. fusca showed significantly (P<0.05) lower specific growth rate values compared to those obtained for untreated controls, with no growth observed beyond Cr (VI) treatment of 10 mg/l.

The result shows that the specific growth rates of the untreated control organisms were not significantly different, but significant (P<0.05 - 0.001) differences were observed between the organisms as the concentrations of chromium treatment increases from 10-20 mg/l. The toxicity and growth inhibitory effect of Cr (VI) compounds may be due to their high solubility in aqueous solutions, rapid transport through biological membranes and subsequent interaction with intracellular proteins and nucleic acids (Upreti et al., 2004). Middleton et al. (2003) reported that the growth of Shewanella oneidensis MR-1 was inhibited in aerobic cultures exposed to 150, 200, and 400, [micro]M Cr (VI). Sixty micromoles (60 [micro]M) of Cr (VI) had little or no effect. They also observed a lag period of about 2 hours before inhibition occurred.

Heavy metals generally, interfere with important microbial processes including aerobic and anaerobic degradation of organic matter (Said and Lewis, 1991; Avery et al., 1996; Kuo and Genther, 1996). Toxic effects include ion displacement and/or substitution of essential ions from cellular sites and blocking of functional groups of important molecules like enzymes, polynucleotide and essential nutrient transport systems (Nies, 1999). These result in denaturation and inactivation of enzymes as well as disruption of cell organelle membrane integrity (Ochiai, 1987; Gadd, 1993). For instance, in yeast particularly, Saccharomyces cerevisiae, high concentrations of heavy metals like Cu (II), Zn (II), Co (II) and Ni (II) have been reported to cause a rapid decline in membrane integrity, which is generally manifested by leakage of mobile cellular solutes (e.g. K+) and cell death (Ohsumi et al., 1988; Cerventes and Gutierrez-Corana, 1994; Stohs and Bagchi, 1995). Similarly, it was reported that the growth of the sulphate-reducing bacterium, Desulfovibrio desulfuricans G-20, in the presence of only 6 [micro]M of Cu (II) was characterized by significantly lower maximum specific growth rate and biomass yield, as well as longer lag period (Sani et-al., 2001). The effects of Cu (II) toxicity increased with increasing concentration of the metal ion in the culture medium.

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(1) * A. Shugabat[dagger], (1) A. J. Nok, (1) D. A. Ameh and (2) J. A. Lori (1) Department of Biochemistry and (2) Department of Chemistry, Faculty of Science, Ahmadu Bello University, Zaria, Nigeria. * Present Address: Department of Biochemistry, Faculty of Science, University of Maiduguri, Maiduguri, Nigeria. [dagger] CorrespondingAuthor: aliyushugaba@yahoo.co.uk, shugaba@hotmail.com
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Author:Shugaba, A.; Nok, A.J.; Ameh, D.A.; Lori, J.A.
Publication:International Journal of Biotechnology & Biochemistry
Date:Nov 1, 2010
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