Bioresource Technology 97 (2006) 1167–1173 Isolation and process parameter optimization of Aspergillus sp. for removal of chromium from tannery effluent Shaili Srivastava a, Indu Shekhar Thakur a b,* Environmental Biotechnology Laboratory, Department of Environmental Sciences, College of Basic Sciences and Humanities, G.B. Pant University of Agriculture and Technology, Pantnagar, Uttaranchal 263 145, India b School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110 067, India Received 25 February 2005; received in revised form 17 May 2005; accepted 26 May 2005 Available online 14 July 2005 Abstract Five morphologically different fungi were isolated from leather tanning effluent in which Aspergillus sp. and Hirsutella sp. had higher potential to remove chromium. The potential of Aspergillus sp. for removal of chromium was evaluated in shake flask culture in different pH, temperature, inoculums size, carbon and nitrogen source. The maximum chromium was removed at pH 6, temperature 30 °C, sodium acetate (0.2%) and yeast extract (0.1%). Aspergillus sp. was applied in 2 l bioreactor for removal of chromium, and it was observed that 70% chromium was removed after 3 days. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Aspergillus sp.; Bioreactor; Chromium; Hirsutella sp.; Biosorption; Fungi 1. Introduction Chromium is considerable environmental concern as it is widely used in leather tanning, electroplating, metal finishing and chromate preparation. Chromium occurs in aqueous system in trivalent and hexavalent forms (Horitsu et al., 1987). Chromium(III) is used in tannery as chromium sulphate, which may be converted into chromium(VI) in the effluent. Hexavalent chromium may be converted to Cr(III) under reduced environment, which is much less toxic and less soluble by several microorganism possess chromate reductase, and thus reduction by these enzymes affords a means of chromate bioremediation. Chromium(III) proved to be biologically essential to mammals as it maintains effective glucose, lipid and protein metabolism. Chromium(VI) is * Corresponding author. Tel.: +91 11 2670 4321; fax: +91 11 2671 7586. E-mail addresses: isthakur@hotmail.com, isthakur@mail.jnu.ac.in (I.S. Thakur). 0960-8524/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2005.05.012 taken up via sulphate or thiosulphate transporter, and oxidize biological molecules resulting in toxicity (Krishna et al., 2004). The hexavalent chromium has higher toxicity, and leads to liver damage, pulmonary congestion, skin irritation resulting in ulcer formation, and carcinogenic (Park et al., 2000). The maximum permissible levels of Cr(VI) in potable and industrial wastewater are 0.05 and 0.1 mg/l, respectively (Goyal et al., 2003). Physicochemical methods employed for removal of heavy metals from the effluent such as precipitation with hydroxide, carbonates and sulphides, adsorption on the activated carbon, use of ion exchange resins and membrane separation processes are responsible for generation of pollution, and the processes related to removal of chromium at large scale are expensive (Kratochvil et al., 1998; Brown, 1991; Volesky and Holzen, 1995). Biotransformation and biosorption are emerging technologies, which utilize the potential of microorganisms to transform or to adsorb metal (Chen and Hao, 1998). Intact microbial cell, live or dead and their products can be highly efficient bioaccumulator of both 1168 S. Srivastava, I.S. Thakur / Bioresource Technology 97 (2006) 1167–1173 soluble and particulate forms of metals (Kratochvil et al., 1998). The cell surfaces of microorganisms are negatively charged owing to the presence of various anionic structures. This gives microorganisms an ability to bind metal cation (Chen and Hao, 1998). Microbial viability is essential for biotransformation as these reactions are enzyme mediated. Generally metal ions are converted into insoluble form by specific enzyme mediated reactions and are removed form the aqueous phase (Brierly et al., 1986). There are reports of live microbial systems for the purpose of remediation of contaminated soils and waters (Kratochvil et al., 1998). Higher fungi (mushrooms), seaweed and plant bark materials are abundantly available in nature and can be a source of low cost biosorbents (Pagnanelli et al., 2000; Brady et al., 1994; Nada et al., 1995). There are potent biosorbents easily available in algae, fungi and bacteria. A source of low cost biomass produced in great quantities, are marine macroalgae. The use of microbial cells as biosorbents of heavy metals is a potential alternative to conventional methods used to decontaminate liquid wastes. Tanneries are mainly responsible for the release of huge amount of chromium in the environment. Pentachlorophenol and related biocides used in the leather tanning processes are refractive for the growth of microorganism and it also reduces removal of chromium in tannery effluent. Physicochemical methods have been practiced for several decades for the removal of toxic heavy metals from the effluent have not been successful (Brierley, 1991; Nada et al., 1995). Bacterial strain, Acinetobacter sp., has been used for removal of chromium from tannery effluent in sequential bioreactor indicated 80% reduction in chromium after 15 days (Micera and Dessi, 1988; Shrivastava and Thakur, 2003). But such a long time for removal of chromium may leads to generation and accumulation of metal and toxic compounds in the environment. Effective and efficient removal of chromium in short time is basic necessity of present time. Therefore, in the present investigation fungal strains have been isolated from tannery effluent and process parameters are optimized in presence of toxic form of chromium [Cr(VI)] with biotechnological methods for removal of chromium from tannery effluent and soil. 2. Methods 2.1. Isolation of fungal strains and fungal inoculums The soil sample was collected from the sediment core of main channel of tannery effluent located at Jazmau, Kanpur, UP. The soil was serially diluted in 10-fold, and diluted sample (0.1 ml) was spread on the potato dextrose agar (PDA) plate. The plates were incubated at 30 °C for 4 days. The microbial colonies (fungi) ap- peared on the PDA plates were isolated, purified and characterized based on their morphological structures as colour, texture, and diameter of the mycelia and microscopic observation of spore formation. Fungal inoculum was prepared in the form of pellets. Erlenmeyer flasks (250 ml) containing potato dextrose broth and streptopenicillin (100 ppm) was taken and inoculated by mycelial discs. These flasks were incubated at 30 °C for 4 days with shaking in orbital shaker. The mycelium was filtered by cheesecloth and placed on petriplates. Water was evaporated and fungal disc was prepared by cutting in approximately 1.5–2.0 mm size. 2.2. Screening of potential strain The fungal isolates were screened for their chromium removal potentiality under minimal salt medium containing (gm/l): Na2HPO4 Æ 2H2O, 7.8; KH2PO4, 6.8; MgSO4, 0.2; Fe(CH3COO)3NH4, 0.01; Ca(NO3)2 Æ 4H2O, 0.05, and pH adjusted to 5.5 as described by Thakur (1995). The salt of potassium chromate (500 ppm) was used as source of hexavalent chromium. The effluent was inoculated in an Erlenmeyer flask with individual fungal isolates and incubated at 30 °C in a rotary shaker for 7 days. Chromium was measured at an interval of 0, 1, 3, 5 and 7 days. On the basis of commiserative analysis percentage reduction in parameter was studied by the individual isolates along with the control and the most potential strains were selected for further analysis. 2.3. Optimization of process parameters Sucrose, dextrose, sodium acetate and sodium citrate were used for optimization of carbon source. Batch study was conducted in the Erlenmeyer flasks containing potassium chromate (500 mg/l) supplemented with MSM, different carbon sources i.e. sodium acetate, dextrose, sodium citrate and sucrose (0.2%) and pH adjusted to 5.5. It was inoculated with 10% (w/v) of the fungal isolate for 7 days at 30 °C with shaking in rotary shaker (150 rpm). Sample was removed on 0, 1, 3, 5 and 7 days and chromium removal was analysed by flame atomic absorption spectroscopy. Sodium nitrate, urea and yeast extract powder were used for the optimization of nitrogen source. The rate of nitrogen source (0.1%), inoculum size, incubation period and temperatures, pH and sampling interval was similar. For the optimization of pH, the MSM-Potassium chromate solution was adjusted at pH 2, 3, 4, 5, 6, 7 and 8, respectively, potassium chromate (500 ppm) and C:N (0.2:0.1%, sodium acetate:sodium nitrate). It was inoculated with 10% (w/v) fungal (FK1) inoculum and incubated as described above. Erlenmeyer flask containing potassium chromate (500 mg/l) supplemented with MSM, carbon and nitrogen sources (0.2:0.1%, sodium acetate:yeast extract) S. Srivastava, I.S. Thakur / Bioresource Technology 97 (2006) 1167–1173 pH 5 was adjusted, inoculated with 5% (w/v),10% (w/v), 20% (w/v) and 30% (w/v) of fungal strain inoculum and incubated for 7 days period at 10, 20, 30 and 40 °C. Samples were removed and chromium was determined. 2.4. Analysis of chromium from supernatant Samples from each experimental flask were withdrawn at 0, 1, 3, 5, and 7 days. Samples were centrifuged at 10,000 rpm for 10 min at 4 °C. Effluent was filtered (Whatman No. 1 filter paper). Sample (50 ml) was taken in conical flask and conc. HNO3 (5 ml) and boiling chips was added. Sample was boiled, evaporated to 16–20 ml on hot plate. Concentrate HCl (12 N, 5 ml) was added in the sample and again boiled till sample become clear, and brownish fumes were evident. Sample was cooled and diluted to 50 ml with distilled water, and finally concentration of chromium was determined with the help of flame atomic absorption spectrophotometer (GBC, Avanta–Sigma; USA) (Greenberg et al., 1995). Chromate reduction was determined by presence of chromium in culture supernatant after treatment by fungus. 1169 2.6. Bioreactor and chromium removal in tannery effluent A laboratory scale bioreactor was fabricated from 2 l glass column as described earlier (Shah and Thakur, 2002). The column was equipped with stirring (250 rev/min) and aeration (500 ml/min) fixed at the top of the bioreactor. The fungal strain enriched in potato dextrose broth was applied for the removal of chromium from potassium chromate solution and chromium of tannery effluent in immobilized bioreactor consisting 3 cm layer of gravel placed at bottom of the columns as solid support for immobilization of microbial cells. The retention time of the reactor was maintained for 3 days in both treatment and control condition. The flow inlet–outlet rate, 20.8 ml/h, was maintained in bioreactor. The pH was maintained between 5.0 and 5.5 throughout the course of treatment. The chromium removal in the bioreactor was determined as described by Greenberg et al. (1995). 3. Results and discussion 2.5. Analysis of chromium from the fungal biomass 3.1. Isolation and characterization of fungal isolates Uptake of chromium was determined by digestion and measuring chromium content in mycelium by flame AAS. The fungal mycelium from the sample of batch culture and bioreactor was centrifuged at 10,000 rpm for 10 min at 4 °C. Supernatant and pellets were separated and stored at 4 °C for further analysis of chromium concentration (ppm). Supernatant of the sample was analysed as described above. Pellets were transferred in the known weight sterile crucible, and then pellets were dried overnight at 60 °C in the oven. Weight of dried pellets was calculated which indicated dry ash form of fungal mycelium. One gram of dry ash of fungal mycelium was taken, and crushed in pestle and mortar. One gram of ground material was placed in conical flask (50 ml) and 20 ml of acid mixture (Tri acid mixture HNO3:H2SO4:HClO3 10:1:4), and the content of flask is mixed properly. The flask was placed on slow heating hot plate in a digestion chamber. Then the flask was heated at higher temperature until the production of red NO2 fumes was ceased. The contents were further evaporated until the volume was reduced to about 3 to 5 ml but not to dryness. The completion of digestion was confirmed when the liquid become colourless (Tondon, 1995). After cooling the flask, concentrated HCl (12 N, 5 ml) was added. Volume was making up to 50 ml with distilled water, and the solution was filtered through Whatman No. 1 filter paper. Aliquot of this solution was used for the determination of chromium in the sample by the flame atomic absorption spectrophotometer (GBC, Avanta–Sigma, USA). Five different types of colonies of the fungus were isolated and further purified on potato dextrose agar plates. The purified fungal isolates were analysed for their morphological characterization. The fungal isolates were identified based on morphological characteristics and microscopic observations, which are presented in Tables 1 and 2. 3.2. Characterization of fungal strains Five fungal isolates (FK1 to FK5) were isolated and tested for their ability to remove chromium in comparison to control to see the comparative performance among the different isolates. The growth of the fungus was determined by measuring dry weight of fungal biomass at different time interval. It was observed that the weight of fungus was increased with the uptake of chromium in the mycelium, which increased the bioaccumulation of chromium in fungus, which is determined by flame atomic absorption spectrophotometer. The result indicated decreased in chromium in culture supernatant was dependent with the time, further substantiated the bioaccumulation of chromium by fungus mycelium. The percentage reduction of chromium by the fungus in supernatant was determined up to day 7 (Fig. 1). The residual chromium after day 7 was not determined in this study. It was observed that chromium removal efficiency in FK1 fungus was higher then that of other fungal isolates. Fungal strain (FK1) has removed 80% chromium while FK2 and FK5 show 58% and 52%, respectively, at 7th day. 1170 S. Srivastava, I.S. Thakur / Bioresource Technology 97 (2006) 1167–1173 Table 1 Morphological characterization of the fungal isolates Isolates Characteristics Surface Reverse FK1 FK2 FK3 FK4 FK5 Black, cottony surface Black, viscous surface White Black with growth ring Parrot green White to yellow Black White Black White to grey Growth Identified Fast growth (+++) Fast growth (+++) Slow growth (+) Slow growth (++) Fast growth (+++) Aspergillus sp. Hirsutella sp. Not identified Not identified Aspergillus sp. Note: +++: growth in whole petriplate, ++: growth 2/3 part in petriplate, +: growth in centre of petriplate. Table 2 Microscopic study of fungal isolates Fungal isolates Characteristics FK1 Septate hyphae biseriate short phialides round radiate vesicle Long smooth colourless conidiophore FK2 Septate hyphae with hairy surface short hairy conidiophore Uniseriate phialides round vesicles FK3 Septate hyphae with branches FK4 Slender hyphae Spores attached directly on hyphae FK5 Septate hyphae short smooth conidiophore uniseriate phialides Round radiate vesicles 3.3. Optimization of process parameters Optimization of process parameters for bioaccumulation of heavy metal ions in fungal biomass is affected by specific surface properties of the fungal cell wall and the physicochemical properties of the adsorption medium such as metal ion concentration, temperature, pH and the amount of biosorbents (Dönmez and Aksu, 2002). It is well known that metal ion adsorption on both non-specific and specific sorbents is pH dependent (Ting and Sun, 2000). The fungal cell wall is mainly composed of polysaccharides, some of which may have associated protein, with other components including lipids and melanins (Gadd, 1993). These biomolecules on the fungal cell wall components have several functional groups (such as, amino, carboxyl, thiol, sulfydryl and phosphate groups). Reduction of chromium with the increase in fungal biomass of the most efficient strain (FK1) was measured after supplementation of minimal salt medium (MSM) with potassium chromate (500 ppm). MSM supports the growth of the fungi and thereby increased reduction percentage of pollution load of chromate. The relationship between pH and reduction of chromium by FK1 isolate was evaluated. In this study it was observed that maximum mycelium growth was obtained at pH 6 after 3 days of incubation, and thereafter the increase become somewhat static. The pH of the solution strongly affected the degree of biosorption of chromium on biomass. The effect of pH on metal biosorption has been studied by many researchers, and the results demonstrated the increasing cation uptake with increasing pH values, by fungal biomass (Tsezos and Volesky, 1981; Guibal et al., 1992). In potassium chromate solution FK1 strain showed maximum reduction of chromate at pH 6.0 (88%) followed by pH 5 (86%) and pH 4 (68%), respectively, and uptake of chromium was 12.9 mg/gm, 12.3 mg/gm and 6.3 mg/gm dry wt of mycelium, respectively (Fig. 2). Adsorption of Cr(VI) at pH 5 and pH 6 suggests that the negatively charged chromium species (chromate in the medium) bind through electrostatic attraction to positively charged functional groups on the surface of fungi (Gupta et al., 2001; Dönmez and Aksu, 2002). Among the various carbon sources, sodium acetate was the best, which increase chromium removal efficiency of FK1 strain at 7th day. Percent chromium Fig. 1. Percentage reduction of chromium by fungi (FK1, FK2, FK3, FK4 and FK5) isolated from tannery soil and effluent. S. Srivastava, I.S. Thakur / Bioresource Technology 97 (2006) 1167–1173 1171 Fig. 2. Optimization of pH for the reduction of chromium in potassium chromate by FK1 strain. reduction in sodium acetate carbon source was 72.8% and uptake of chromate in mycelium was 6.95 mg/gm dry wt of mycelium. While percent chromium reduction in presence of dextrose, sodium citrate and sucrose was 68%, 62% and 48%, respectively, and uptake of chromate in mycelium was 6.52 mg/gm, 5.48 mg/gm and 3.52 mg/gm dry wt. of mycelium (Fig. 3). To determine the influence of nitrogen for the removal of chromium by fungal strain, FK1, was incubated for 7 days with different sources of nitrogen viz. sodium nitrate, urea and glutamate. It was observed that yeast extract was the best for the chromium reduction efficiency of FK1 strain. Percent chromium reduction in yeast extract was 76% and uptake of chromate was 6.85 mg/gm, while percent chromium reduction and uptake of chromate in urea and sodium nitrate was 62% and 44% and 5.75 mg/gm and 3.18 mg/gm dry weight of mycelium, respectively (Fig. 4). The size of biomass (i.e. 5, 10, 20 and 30 gm/100 ml) of FK1 were used for the reduction of chromate in potassium chromate was observed (Fig. 5). In which 20% (20 gm/100 ml) of biomass shown maximum percent chromium reduction i.e. 78% and uptake was 8.25 mg/gm dry wt. of mycelium. The chromium removal efficiency was increased up to an optimum dosage Fig. 3. Optimization of carbon source for the reduction of chromium in potassium chromate by FK1 strain. Fig. 4. Optimization of nitrogen source for the reduction of chromium in potassium chromate by FK1 strain. Fig. 5. Optimization of inoculum size for the reduction of chromium in potassium chromate by fungal strain (FK1). beyond which the removal efficiency did not change. Several researchers reported that the increase in the percentage removal with increase in the adsorbents dosage is due to the increase in the number of adsorption sites (Sharma and Forester, 1993). The temperature of the adsorption medium could be important for energy-dependent mechanisms in metal biosorption by microbial cells. Mostly adsorption is an exothermic process whereas some examples of endothermic adsorption have also been reported (Sharma, 2003). The bioaccumulation of Cr(VI) by the fungus appears to be temperature dependent over the temperature range tested (10–40 °C) (Fig. 6). Similar observations have also reported by other researchers (Goyal et al., 2003; Sharma, 2003; Asheh and Duvanjak, 1995; Marques et al., 1991). To determine the influence of temperature for the removal of chromium by fungal strain, FK1 was incubated for 7 days with different temperature i.e. 10, 20, 30 and 40 °C in shake flask condition. It was observed that 30 °C was the suitable condition for the bioaccumulation of chromium by fungal strain (FK1). Percent chromium reduction at temperature 30 °C was 1172 S. Srivastava, I.S. Thakur / Bioresource Technology 97 (2006) 1167–1173 Fig. 6. Optimization of temperature for the reduction of chromium in potassium chromate by fungal strain (FK1). 78% and uptake of chromate was 8.2 mg/gm dry weight of mycelium, while percent chromium reduction and uptake of chromate at 10, 20 and 40 °C were 32%, 35% and 65%, and uptake of chromium were 2.9, 3.8 and 5.6 mg/gm dry weight of mycelium, respectively (Fig. 6). Although temperature has not been studied as relevant variable in biosorption experiments. However, a slight increase in cation uptake by seaweed in the range of 4–55 °C has been reported (Tsezos and Volesky, 1981; Aksu and Kutsal, 1991). 3.4. Evaluation of chromium reduction in bioreactor The bioreactor was designed for studying the biotreatability of chromium by FK1 fungi in tannery effluent, which contains chromium sulphate [Cr(III)]. Trivalent chromium present in tannery effluent readily oxidized in aqueous environment and converted in to hexavalent chromium. Hexavalent chromium is toxic then trivalent chromium. Therefore, process parameters were optimized with the hexavalent chromium for evaluation of reduction of [Cr(III)] in tannery effluent. Several reports are available on principle and design of bioreactor (Shah and Thakur, 2002). To provide conditions resembling the nature, the bioreactor consisted of gravel in order to immobilize the biomass. Stirrer was placed at the top to provide aeration and also mixing of fungus inoculum to grow properly, which may enhance bioaccumulation of chromium by fungus mycelium In tannery effluent. Cr(III) and chlorinated hydrocarbons like pentachlorophenol and its substitute are major contaminants in tannery effluent. The removal and degradation of these chemical compounds are possible by biotechnological methods of treating the effluent in sequential way. Most of the fungi have not potential to degrade chlorinated phenolic compounds, but they absorb chromium. However, bacteria degrade chlorinated compounds, but they Fig. 7. Percent chromium reduction by FK1 strain in potassium chromate and tannery effluent in bioreactor. cannot absorb higher concentration of chromium. The concentration of chromium in tannery effluent some times reaches up to 7000 ppm, and it will be difficult to remove chromium from tannery effluent. Therefore, in the present study bioreactor is used for removal of chromium by FK1, which will be applied with bacteria in sequence in sequential bioreactor in future studies. In the present study the growth of fungal strain in bioreactor was determined by measuring biomass. In bioreactor, fungus strain achieved natural conditions and proper aeration and regeneration due to presence of sodium acetate as additional carbon source. It is well known that the microorganisms thrive in extreme condition; therefore, sequential bioreactor may be used to sustain the shock load in the reactor. The proper aeration made the reactor aerobic, suitable for the fungal growth. The total concentration of chromium initially in tannery effluent was 557 ppm. The efficiency of chromium removal by FK1 fungus was reached to 85% on 7th day in MSM-potassium chromate culture, while in case of tannery effluent concentration of chromium was 65% (195 ppm) on 7th day. Result of the study indicated that percent reduction of chromium and growth of fungi was reduced significantly in bioreactor containing tannery effluent (Fig. 7). 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