Isolation and Screening of Some Filamentous Fungi with Various Trace Metals

Trace metal (TM) pollution of soil is a worldwide problem threatening the quality of human life and a proper environment. We investigated fungal diversity of trace metal polluted site contaminated with paper mill effluent in India. Fourteen fungal strains were identified using ITS of rDNA belonged to Aspergillus, Penicillium, Fusarium, Cunninghamella, Simplicilillium, Trichoderma, Rhizomucor, Cladosporium and Hypocrea and subsequent screening was carried out to assess their TM tolerance in in vitro cultures. The results revealed that the majority of the isolates were tolerant to Ni, Cu, Zn whereas only Penicillium sp. and Aspergillus sp. were able to grow in Cd amended medium. The level of tolerance depended on the fungal isolate and the site of its origin. Minimum inhibitory concentrations (MIC) for Ni, Cu, Zn and Cd were also determined among the all tested fungal isolates. Overall, Aspergillus, Penicillium, Rhizomucor, Trichoderma and Fusarium isolates showed a strong growth in different concentrations of TM. Their MIC ranged between 20 and 25 mM for Zn, 15 and 20 mM for Ni and Cd. Some of these fungal isolates showed a high potential for further investigations regarding the mechanisms of TM tolerance and their use in mycoremediation to clean up TM polluted soil. Introduction Pulp paper industries are the sixth largest effluent generating industries of the world as these generates as low as 1.5 m3 of effluent per ton to as high as 60 M3 tone of paper produced (Gavrilesca, 2004). Trace metals are included among the priority pollutants because of their high toxicity require suitable treatment prior to discharge into the environment. Trace metals cannot be degraded and even at low concentration cause toxicity in humans (Rao et al. 2005). Conventional methods used for removal of metals include chemical precipitation, ion exchange, membrane filtration and activated carbon adsorption. Biological methods provide an attractive alternative to conventional methods because of low operating cost, minimization of chemicals, biological sludge easily disposed and high efficiency in detoxifying very dilute effluent. According to World Health Organization (2006), the metals of most immediate concern are cadmium, cobalt, Zinc, nickel, copper, mercury and lead . Many microorganisms including fungi have been identified as superior candidate for metal bioremediation. Major advantages of fungi are its significant metal uptake ability at low anticipated price. Microbial population present in metal polluted environment have adapted and become tolerant to toxic trace metals (Melgar et al. 2007). Trace metals like Cu, Ni, Zn, Cd, Mn present in paper mill effluent can be removed by indigenous microbes isolated from effluent itself . Fungi and yeast biomasses are known to tolerate trace metals (Baldrian, 2003). The tolerance of some fungi to a variety of trace metals is well documented (Burgstaller and Schinner 1993). In some cases it produces intracellular/ extracellular enzymes to resist the metal concentration or they possess the processes of valence transformation, active uptake, complexation, crystallization and biosorption to cell walls (Wang and Chen, 2006; Zafar et al. 2007). Species like Penicillium, Aspergillus, Pseudomonas, Sporophyticus, Bacillus, Phanerochaete, etc are found to be very useful for the removal of trace metals such as chromium and nickel (Ezzourhi et al. 2009). Many Fungi develop their tolerance with specific metals and ability to influence the mobility of target metals with metal containing ores in a various ways ( Fomina et al.2005). They are a versatile group, as they can adapt and grow under various extreme conditions of pH, temperature and nutrient availability, as well as high metal concentrations (Atlas,1984). They offer the advantage of having cell wall material which shows excellent metal-binding properties( Sabat and Gupta 2009). El-Morsy (2004) isolated 32 fungal species isolated from polluted water in Egypt and studied their resistance to metals and found that Cunninghamella echinulata biomass could be employed as a biosorbent of metal ions in waste water. The present work reports the isolation of filamentous fungi indigenous to paper mill environments contaminated with trace metals and their tolerance capacity with the different concentrations of trace metals. The trace metals MICs (minimal inhibitory concentrations) for each microorganism were determined for the selection of most tolerant strains, which can be used in bioremediation of trace metals in the contaminated site. Materials and Methods Isolation of microorganisms Soil samples were taken from the polluted solid effluent area of the Hindustan paper Mill Corporation, Panchgram, India. The paper mill is surrounded by a vast spread of agriculture land used for paddy cultivation on its eastern side while the northern side has hill areas covered with dense forest. Once this area was full of different kinds of plants but now the dumping of solid wastes near the adjoining area resulted in the depletion of various important plant species. The study area was located between 24° 52’ N and 92° 36’ E longitudes. The altitude of the study area was 16 MSL.The serial dilution technique was carried out with 1gm of soil sample with standard dilution 10-4. The aliquots of 100 μl of different dilutions were plated both onto Potato Dextrose Agar (PDA) plates. After at least 7 days of incubation at 25°C, developed colonies were picked and isolated. Purified strains were obtained by streaking repeatedly colonies in PDA medium and observation under compound microscopy. The cultures were characterized to the genus level on the basis of macroscopic characteristics (colonial morphology, colour and appearance of colony, shape) and microscopic characteristics (septation of mycelium, shape, diameter and texture of conidia).