Journal of Applied Sciences & Environmental Management, Vol. 9, No. 1, 2005, pp. 123-126

Heavy Metal Biosorption potential of Aspergillus and Rhizopus sp. isolated from Wastewater treated soil

IQBAL AHMAD*, SHAHEEN ZAFAR; FARAH AHMAD

Department of Agricultural Microbiology, Faculty of Agricultural Sciences, AligarhMuslimUniversity, Aligarh-202002 E.mail: Iqbalahmad8@yahoo.co.in

 Code Number: ja05022

ABSTRACT: Two isolates belonging to the predominant genera Aspergillus and Rhizopus isolated from agricultural field treated with sewage/ industrial effluents were selected for the biosorption potential evaluation of Cr and Cd. Pretreated, dead biomass of above fungi was used for bioadsorption experiment at pH value 4.5 with the biomass, 1-5 mg in a 100 ml metal solution of different concentration (2, 4, 6 and 8 mM) with a contact time of 18 hrs and agitation, 120 rpm. Bioadsorption of Cr ranged from 6.20- 9.5 mg/g of dry mass at one or other initial metal concentrations by Aspergillus and Rhizopus sp. The bioadsorption of Cd was ranged from 2.3-8.21 mg/g. On the comparative basis Rhizopus sp. could bioadsorbed higher concentration of both metals as compared to Aspergillus sp. Bioadsorption of Cd and Cr was influenced by initial metal concentration and nature of organism. The findings revealed that fungi of metal polluted sites showed higher metal tolerance and bioadsorption capacity of chromium and cadmium. @JASEM

Increased industrialization and human activities have impacted on the environment through disposal waste containing heavy metals. Heavy metals pollution of agricultural soil has been mainly due to the disposal of industrial wastewater, sewage and sewage sludge to agricultural land. These wastewaters contain some plant nutrients, organic matter and varying level of heavy metals (McGrath et al., 1988; Hayat et al., 2002; Korboute et al., 2002). Microorganisms like bacteria, fungi, algae and yeast are known to tolerate and accumulate heavy metals. Bioadsorption mechanism involved in the process may include ion exchange, co-ordination, complexation, chelation, adsorption, micro-precipitation, diffusion through cell walls and membrane which differ depending on the species used, the origin and processing of the biomass and solution chemistry (Guibal et al., 1992; Churchill et al., 1995). Preference of living biomass of metal binding ion depends on nutrient, environmental condition and cell age (Brierley et al., 1986; Kapoor and Viraghavan., 1995). In addition, living biomass may subject to toxic effect of heavy metals at elevated concentration. To overcome the disadvantages; non-viable or dead biomass is preferred (Butter et al., 1998). Potential of filamentous fungi in bioremediation of heavy metal containing industrial effluents and wastewaters has been increasingly reported from different parts of the world (Gadd., 1993). However, filamentous fungi of heavy metals polluted habitat in India are not largely screened and exploited for their bioremediation potential.  It is expected that soil receiving long-term application of wastewater/industrial effluents containing toxic metals may result in the development of selection pressure on soil fungi which may cause increase level of metal tolerance as well as metal adsorption capacity. To test this hypothesis predominant two fungal isolates of agricultural fields receiving long-term application of wastewater were evaluated for bioadsoption capacity of cadmium and chromium in laboratory experiments.

MATERIALS AND METHODS

Agricultural field soil of Aligarh city (Bridge Dham, Mathura Road, Aligarh, India) receiving long-term application sewage and industrial effluent as irrigant was used to isolate metal resistant fungi by spread plate method. Briefly, Sabouraud dextrose agar (Hi-media) medium supplemented with CdCl₂ / K₂Cr₂O₇ at 50μg/ml conc. at 45-50 °C before pouring the medium into petriplates. 0.1gm   of aseptically collected composite soil was serially diluted in sterile NSS. 0.1ml of various dilutions were spread on SDA plates in duplicate and incubated at 29°C for 7 days. The predominant form of fungal growth was tentatively identified and given a laboratory isolated number after purification. Aspergillus sp. (SH-1) and Rhizopus sp. (SH-9) were selected on the basis of their predominant occurrence and resistance to multi-metals. Metal tolerance level was determined in terms of MIC against different metals by spot inoculation of fungal inoculum (10⁶ CFU/ml) on heavy metal supplemented agar plates and incubated at 29 °C for 3-4 days. Presence or absence of growth was observed on the spotted area. Minimum inhibitory concentration (MIC) is defined as the lowest concentration of heavy metal/ml of medium inhibited the visible growth of the test fungi.

 Soil samples collected from wastewater treated and untreated fields were analysed for heavy metal content by atomic absorption spectrophotometer (Model- GBC-932 plus) using standard protocols as described earlier (Hayat et al., 2002).

To determine the heavy metals bioadsorption by filamentous fungi, standard procedure as described by Yan and Viraraghavan (2001) was adapted. Briefly, spores of 6-7 days old culture incubated on Sabouraud dextrose agar slant at 29°C were used for inoculation (conc. of spores suspension 1x10⁵ CFU/ml) in a liquid medium (YPG) which contained (in g/l): yeast, 3; peptone, 10; glucose, 20.The culture was grown at 23°C in the above medium in a conical flask kept on a rotary shaker agitated at 125 rpm. The fungi grew in a filamentous (mold like) form under air and forms spherical bodies/ pellets. The growth of the fungi was harvested after 3-4 days of growth by filtration using 150μm sieve .The harvested biomass was washed with generous amount of deionized water and stored at -20 °C or used immediately.

For the treatment of biomass, live harvested biomass (50gm) was treated with 0.5N NaOH for 30 min followed by washing with generous amount of distilled water until the pH of the solution reached to neutral range (pH 6.8-7.2) and autoclaved at 15 lb/inch²   for 20 min. The pretreated biomass was dried at 60 °C for 24hrs in hot air oven and powdered into in mortar and pestle. 0.1 gm of powdered dead biomass was inoculated into 100ml metal solution containing 2 to 8 mM of CdCl₂.H₂O/ K₂Cr₂0₇ in double distilled water. The flasks were kept on rotary shaker for 18hrs at 30°C and 125 rpm. Then the solution along with dead biomass is centrifuged at 10000 rpm for 15 min. The content of the supernatant was analyzed after proper digestion and dilution by atomic absorption spectrophotometer (GBC 932 Plus) available in our lab. Metal solution without biomass addition served as control.

Bioadsorption experiments were carried out in duplicate and average values were used in the analysis. Bioadsorption capacity i.e. amount of metal ions (mg) bioadsorbed per gm (dry mass) of biomass was calculated using the following equation:

Q   =     ( C_i—C_f/ m ) V

Where Q =mg of metal ion bioadsorbed per gm of biomass, C_i=initial metal ion concentration mg/l, C_f =final metal ion concentration mg/l, m=mass of biomass in the reaction mixture gm, V=volume of the reaction mixture (l).

RESULTS AND DISCUSSION

Heavy metals content of agricultural field soils analysed by AAS showed the presence of higher level of metals in treated soil as compared to untreated soil (Table-1).