Bioremediation measure to minimize heavy metals accumulation in soils and crops irrigated with city effluent

WFL Publisher Science and Technology Food, Agriculture & Environment Vol.2 (1) : 266-270. 2004 www.world-food.net Bioremediation measure to minimize heavy metals accumulation in soils and crops irrigated with city effluent Tapan Adhikari*, M. C. Manna, M. V. Singh and R. H. Wanjari Indian Institute of Soil Science, Nabibagh, Berasia Road, Bhopal-462038, M.P., India. *e-mail: tapan@iiss.mp.nic.in Received 15 September 2003, accepted 19 January 2004. Abstract A survey was conducted to monitor the influence of city sewage irrigation on the heavy metal build up in soils around Bhopal. Concentration of the heavy metals in effluent was within safe limits. Diethylene triamine penta acetic acid extractable Pb, Cd, Ni, Co, Cr, Fe, Mn, Zn and Cu in the sewage irrigated surface soil ranged 3.5-6.8, 0.15-0.40, 2.56-5.58, 1.59-3.89, 0.45-0.55, 3.5-15.8, 3.6-8.5, 1.2-3.6, 1.53-6.59 mg/kg respectively. Continuous sewage irrigation resulted in accumulation of Pb, Cd, Ni, Co, Cr, Fe, Mn, Zn and Cu in surface 0-15 cm layer by 5, 6, 0.4, 8, 0.6, 3.16, 5, 2, 3 times more compared to adjoining tube well irrigated soils. Studies revealed that accumulation of heavy metals such as Pb, Cd, Ni, Co, Cr, Fe, Mn, Zn and Cu was higher mostly in the roots of various crops irrigated with city sewage effluent compared to that of tube well water irrigated crops. Amongst the crops, carrot and spinach showed the tendency of higher metal accumulation. Bioremediation measure is now a day adopted to reduce the heavy metal load in agricultural field. Keeping in view, a laboratory experiment was also conducted to know the biosorption capacity of heavy metals (cadmium and chromium) by different fungi viz. Pleurotus florida, Fusarium oxysporum, Penicillium sp and Aspergillus awamorii to mitigate the problem of heavy metals accumulation in soils and crops irrigated with sewage water. Growth of those fungi was studied with the graded doses of Cd (2.5, 5, 10, 15 and 20 mg/l) and Cr (2.5, 5, 10, 15 and 20 mg/l). Pleurotus florida which sorbed highest amount of heavy metals and can survive at higher levels of heavy metals, can be used as a bio-filter to remove the heavy metals from sewage water. Key words: Survey, soil, crop, bioremediation, heavy metal. Introduction In recent years, contamination of large areas of land by heavy metals has become a major concern. It originates mainly from municipal waste incinerators, car exhausts, residues from metalliferous mining and the smelting industry, and the use of urban compost, pesticides, fertilizers or sludge and sewage. The sewage is characterized by a complex heterogeneous multifaceted matrix depending upon its origin and production. Sewage generated in rural areas and small cities does not cause serious concern and fits for recycling in agriculture due to its high contents of organic matter and major and micro nutrients. Contrary, urban sewage represents an admixture of sewage and industrial waste effluents, industrial effluents which get contaminated with heavy metal pollutants. With increased industrialization in residential areas, different materials are discharged into sewage which lead to environmental pollution 1. This concern is of special importance where untreated sewage is applied for longer periods to grow crops on urban lands 2. Urban sewage containing industrial effluent was found to carry relatively high amounts of heavy metals such as Ni, Cr, Pb, Cd, Co and salt load causing salinity and alkalinity hazards. Heavy metal pollution deteriorates the quality of soil and crops produced. Extent of soil pollution with heavy metals from various anthropogenic sources and subsequent uptake by crops depend upon several factors such as source, soil type, frequency of application, organic matter content, seasonal variations, major and minor nutrients and load of chemical pollutants. Continuous irrigation with untreated sewage, particularly in arid and semiarid regions of the developing countries, is often a potential 266 source of heavy metal pollution to soils and plants and health risk to animals and humans. Excessive metal concentrations in contaminated soils can result in decreased soil microbial activity and soil fertility, and yield losses 3. Remediation of heavy metal contamination on soils is difficult. The conventional technologies for removing heavy metals from soils, often employ stringent physicochemical agents 4, which can dramatically inhibit soil fertility and leaves subsequent negative impacts on the ecosystem. Numerous methods have been proposed to remove heavy metals from sewage sludge, including chlorination, use of chelating agents and acid treatments at high temperatures. However, those methods are generally ineffective in practical applications due to high cost, operational difficulties and low metal leaching efficiency 5. An alternative way to replace chemical methods in removing heavy metals is bioremediation through microbial leaching 6. Bioremediation is the process of cleaning up hazardous wastes with microorganisms or plants and is the safest method of clearing soil of pollutants 7. The chief concern of bioremediation research on soil contaminated with toxic chemicals is the removal of toxic substances so that neither soil nor water is harmful to animals, humans, plants or beneficial microbes 8. Bioremediation has been proposed as a cost effective, environmental friendly alternative modern emerging technology which can be applied to a number of contaminants and site conditions. The current gap between understanding of bioremediation process and its application in field provides incentive to examine the use of microbes to reduce the heavy

metal load from sewage water. The objectives of this study were to survey the heavy metal accumulation in soils and crops around Bhopal and to find out the probable bioremediation measure to mitigate the problem of heavy metal pollution through city sewage. Materials and Methods In a survey of urban agricultural soils, receiving untreated sewage different sites were selected in urban areas around Bhopal, M.P., India. Sampling sites were selected randomly. This experimental site is located between 23 o 18 / N latitude and 77 o 24 / E longitudes, at 485 m height above mean sea level (altitude). The region is characterized as hot-subhumid ecoregion. Under average climatic condition over the years, the area received 1200 mm rainfall annually. The average monthly maximum and minimum atmospheric temperatures were 41.0 and 7.0 o C, respectively. The soil of the experimental field is described as Typic Haplustert. The soil is clay loam in texture with montmorillonite as dominant clay mineral. The area under each sampling site was about one acre where different crops had been grown with the untreated sewage irrigation for about 10 years. Untreated sewage samples were collected from specific sites. After filtration ph and EC 9, Ca 2+ and Mg 2+ (titration with standard Versenate method), CO 3 2- and HCO 3 - (titration with standard H 2 SO 4 ) and Cl - (titration with standard silver nitrate) were determined and concentrations of heavy metals viz. Pb, Cd, Ni, Co, Cr, Fe, Mn, Zn and Cu were analyzed by atomic absorption spectrophotometry. Soil samples were also collected from the same fields. Soils were extracted using DTPA method 10 and concentrations of Pb, Cd, Ni, Co, Cr, Fe, Mn, Zn and Cu in extracts were analyzed by atomic absorption spectrophotometry. Soil particle size analysis was accomplished by hydrometer method 11. Plant samples were collected from the selected locations. Shoot and root samples were collected separately. The plant samples were washed with 1% HCl to remove foreign materials. Thereafter samples were air-dried, oven-dried at 70 o C and digested in di-acid (HNO 3 : HClO 4 ; 3:1) mixture. Heavy metal concentrations in the prepared samples were determined by atomic absorption spectrophotometry. Bio-sorption study: A laboratory experiment was conducted to study the bio sorption capacity of heavy metals by different fungi viz. Pleurotus florida, Fusarium oxysporum, Penicillium digitatum and Aspergillus awamorii. Pleurotus florida was collected from the division of Biotechnology, Barkatullah University, Bhopal and other species were isolated from sewage water. The individual strains were inoculated in the sterilized Rose Bengal growth media with graded levels of Cd and Cr (0 to 20 mg L -1 ) and allowed to incubate for 60 days at 30 o C. After 60 days of incubation, the fungal biomass was collected. For that whole solution was shaken with 125 rpm for 6 h and filtered with 0.22 µm millipore filter with generous amount of deionized water. After that the mycelial mat was oven dried at 50 o C for 24 h. The dry weight was recorded. The content of heavy metals in the mycelial mat was estimated after digestion of the whole mat with di-acid mixture by atomic absorption spectrophotometer. The amount of heavy metals absorbed by the living mat was calculated after deducting the amount of heavy metals present in final solution from initial solution 12. Results and Discussion Composition of untreated city effluent: Mean values of chemical composition of the city effluent collected from different places around Bhopal are presented in Table 1. There was a considerable variation with respect to location. The mean values of different heavy metals indicated that use of these effluents as a source of irrigation for a longer period may cause accumulation of heavy metals in soils. City waste irrigated soils: The soils in the study area were swell-shrink clay soil, alkaline in reaction, non-saline (EC < 4.0 ds m -1 ), CEC 43 cmol (p+) kg -1, and contained 0.4 0.55% organic matter. Average concentrations of DTPA-extractable metals in the upper layer of soil (0-15 cm) are presented in Table 2. Accumulation of these metals was mostly confined to plough layer and markedly decreased up to 30 cm depth and Table 2. DTPA-extractable metals (mg/kg) in sewage irrigated soils under different crops. Crops Metal Soil depth (cm) 0-15 15-30 30-45 Wheat Zn 1.20-3.60 0.70-2.10 0.14-0.30 Cu 1.53-6.59 0.62-3.00 0.34-0.89 Fe 3.5-15.80 2.00-10.10 1.10-3.80 Mn 3.60-8.50 2.20-6.90 0.90-3.20 Pb 3.70-5.90 1.10-1.90 0.20-0.60 Cd 0.14-0.39 0.00-0.10 Tr Ni 2.60-5.50 0.13-2.60 0.00-1.40 Co 1.59-3.80 0.10-1.20 0-00.90 Cr 0.45-0.550 0.00-0.22 Tr- Carrot Zn 1.50-3.20 0.90-2.20 0.15-0.40 Cu 1.62-5.83 0.72-2.90 0.30-0.84 Fe 3.80-14.90 2.10-11.40 1.00-3.50 Mn 3.90-7.90 2.40-5.80 0.80-2.50 Pb 4.20-6.20 1.30-2.20 0.30-0.80 Cd 0.15-0.40 0.00-0.12 Tr- Ni 2.70-5.20 0.12-2.30 0.00-0.12 Co 1.63-3.50 0.12-1.10 0.00-0.80 Cr 0.48-0.50 0.00-0.24 Tr Onion Zn 1.50-3.60 0.80-2.40 0.20-0.50 Cu 1.72-6.20 0.94-2.60 0.38-0.78 Fe 3.90-13.90 2.30-9.20 1.20-2.60 Mn 3.80-8.20 2.30-6.30 1.00-2.90 Pb 3.50-6.80 0.90-1.50 0.40-0.80 Cd 0.18-0.37 0.00-0.12 Tr- Ni 2.56-5.58 0.12-2.80 0.00-1.20 Co 1.67-3.20 0.15-1.00 1.00-0.70 Cr 0.46-0.52 0.00-2.50 Tr Radish Zn 1.40-3.40 0.60-2.80 0.30-0.50 Cu 1.68-6.40 0.81-2.50 0.39-0.68 Fe 4.50-12.80 2.80-9.10 1.10-2.60 Mn 4.10-7.90 2.50-5.90 0.90-3.00 Pb 3.90-6.30 0.80-1.60 0.30-0.70 Cd 0.19-0.38 0.00-0.13 Tr- Ni 2.60-5.50 0.15-2.20 0.00-1.50 Co 1.70-3.50 0.18-1.20 0.00-0.90 Cr 0.47-0.50 0.00-0.22 Tr Spinach Zn 1.60-3.40 0.50-2.70 0.40-0.50 Cu 1.55-5.80 0.67-2.20 0.33-0.70 Fe 3.50-12.50 2.20-7.90 1.00-2.50 Mn 3.90-8.10 2.20-6.10 1.00-2.60 Pb 4.00-6.70 1.20-1.80 1.00-0.90 Cd 0.20-0.40 0.00-0.15 Tr Ni 2.70-5.40 0.16-2.50 0.00-1.00 Co 1.75-3.42 0.13-1.10 0.00-0.80 Cr 0.49-0.52 0.00-0.25 Tr 267

Table 1. Chemical characteristics of untreated city waste samples collected from different sites around Bhopal, M.P. Location ph EC Ca +2 Mg +2 CO - - 3 HCO 3 Cl - Fe Mn Zn Cu Co Cr Pb Ni (1:2.5 H 2 O) dsm -4 meq/l meq/l meq/l meq/l meq/l (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) Tap water 7.2 0.29 3.9 2.30-1.6 1.00 4.99 0.16 0.05 0.08 0.02 Tr Tr Tr Jahangir abad 8.1 1.19 4.2 4.2-2.4 3.81 10.10 0.30 0.04 0.10 0.04 0.01 0.11 0.00 By-Passroad-I 7.9 0.98 3.87 5.13-3.2 3.31 7.80 0.60 0.06 0.05 0.03 0.02 0.15 0.00 By-Passroad-II 7.9 0.59 4.30 4.1-2.4 1.80 3.28 0.80 0.07 0.09 0.05 0.01 0.09 0.00 BHEL 7.8 0.45 3.50 0.9-1.6 1.15 8.09 0.39 0.15 0.18 0.08 0.03 0.22 0.15 Ashok Nagar 7.6 1.00 3.20 8.6 0.8 2.4 1.94 5.85 0.40 0.22 0.22 0.04 0.01 0.52 0.20 Super Bazar 8.1 1.89 4.10 8.7 0.8 2.4 3.16 9.39 0.65 0.13 0.13 0.02 Tr 0.44 0.24 Table 3. Heavy metal contents (mg/kg) in plant samples grown in normal soil. Plants Cd Pb Ni Cr Co Fe Mn Zn Cu Wheat Shoot - 0.20 - - 0.25 65 48.40 12.50 6.05 Wheat Root - 0.30 - - 0.30 85 60.50 26.50 7.25 Carrot Shoot - 0.50 - - 0.45 58 55.30 8.90 4.39 Carrot Root - 0.70 - - 0.55 80 68.00 18.50 6.06 Onion Shoot - 0.12 - - 0.15 56 29.00 9.20 6.50 Onion Root - 0.30 - - 0.10 70 40.00 15.6 7.31 Raddish Shoot - 0.55 - - 0.22 79 39.00 11.5 2.01 Raddish Root - 0.65 - - 0.30 Spinach Shoot - 0.35 - - 0.40 Spinach Root - 0.40 - - 0.50 Table 4. Heavy metal contents (mg/kg) in plant samples grown in sewage treated soil. Plants Cd Pb Ni Cr Cu Fe Mn Zn Co Wheat Shoot 1.75 6.25 5.05 2.95 9.30 98 48.40 31.20 0.25 Wheat Root 2.50 7.75 6.35 4.20 10.25 120 60.50 37.50 0.30 Carrot Shoot 2.89 8.80 7.75 4.39 8.50 205 55.30 34.50 0.45 Carrot Root 3.56 9.39 8.59 7.17 9.25 285 68.00 42.50 0.55 Onion Shoot 1.47 6.40 6.30 2.97 9.30 150 29.00 19.50 0.15 Onion Root 2.19 9.30 8.43 5.32 10.20 180 40.00 23.50 0.10 Raddish Shoot 2.01 5.55 9.05 4.04 7.35 160 39.00 29.80 0.22 Raddish Root 3.03 8.45 10.62 6.95 8.25 190 47.00 36.50 0.30 Spinach Shoot 3.11 7.39 10.32 4.75 8.85 250 50.20 33.05 0.40 Spinach Root 4.46 10.30 11.47 6.80 10.32 300 56.80 40.50 0.50 268

Figure 1. Bio-sorption capacity of cadmium by different fungi. Figure 2. Bio-sorption capacity of chromium by different fungi. Figure 3. Effect of Cd and Cr on the growth of different fungi. 269

there was hardly any variation in their concentration beyond 30 cm depth of the soil profile. Therefore, continuous disposal of sewage treated water will increase the concentration of metals in the feeding zone of plant roots, which may create toxicity to plants and also clinical problems in animals and human beings consuming the food grown on such soils. The continuous use of sewage effluents for irrigation of light textured soils can increase metal concentrations also in deeper soil layers. E.g in Punjab soils of India concentrations of Fe, Mn, Zn, Al and Ni were significantly increased up to 60 cm depth 13. Sewage irrigated crops: A survey was conducted to determine the heavy metal contamination in crops grown with urban sewage water irrigation in soils around Bhopal districts. The heavy metal and micronutrient contents were determined in root and shoot samples and results are presented in Tables 3 and 4. The plants grown on the soil polluted with sewage effluents were found to record higher uptake of heavy metals when compared to plants grown on normal soils. In general, accumulation of heavy metal contents was in the order of root >shoot. A perusal of the data revealed that Pb content in different parts of the crops like wheat, carrot, onion, radish, and spinach ranged from 5.63 to 10.30 mg kg -1 dry matter. Next to Pb, Ni content was higher in different parts of these crops. The contents of Co, Cd and Cr were lesser as compared to the Pb and Ni content. But as far as the distribution within plant parts and severity in the area is concerned, it followed the same trend like above Pb and Ni. Bio-sorption: Two separate laboratory experiments were conducted consecutively to study the bio sorption capacity of different fungi. In the first experiment, different fungi viz. Pleurotus florida, Fusarium oxysporum, Penicillium digitatum and Aspergillus awamorii were subjected to graded doses of Cd ( 0-250 mg L -1 ) and it has been observed that amongst the fungi Pleurotus florida sorbed 1.2 to 2.5 times more Cd than the others. Higher sorption of Cd by Pleurotus florida could be explained by its (i) surface properties, such as higher charge and orientation of the functional groups on the cell surface (ii) congenial chemical micro-environments on the cell surface absorb more metals (iii) release of metal-complexing exudates etc. 12. Cd sorption was recorded higher with increasing doses, but after 50 mg L -1 Cd, growth of microbes was badly affected. Pleurotus florida could survive up to higher level of Cd but growth was reduced above 75 mg L -1 Cd level. The growth of other fungi was severely affected after 25 mg L -1 Cd level and virtually there was no growth after 25 mg L -1 Cd level for Aspergillus awamorii and Fusarium oxysporum. Based on this experiment, second experiment was formulated by selecting lower levels of Cd and Cr with the same fungi (Figure 1 and 2). Results revealed that the order of sorption for Cd and Cr by different fungi was as follows: Pleurotus florida > Penicillium sp > Aspergillus awamorii > Fusarium oxysporum. It has also been found that growth of fungi was more affected by Cr than Cd (Figure 3). Experimental results revealed that Pleurotus florida can be used as a bio-filter to reduce heavy metal load from sewage water as well as from soils. Conclusions Heavy metal contents of sewage samples were recorded below the toxic level, but continuous heavy use of sewage may cause toxicity in soils in future. Soils and plant samples collected from sewage irrigated field showed the accumulation of heavy metals in soils, and vegetable crops recorded higher amounts than that of cereal crop. Roots contained higher amounts of heavy metals than other parts of the crops. Findings of this study suggest that for reducing the entry of heavy metals into human food chain there should be strict restriction on untreated sewage irrigation to food crops. The industries may be allowed to throw the effluents into sewage drains only after some pretreatment. Heavy metal accumulation in the surveyed field (Vertisol) occurred in the root zone of plants and it may create health hazards of humans, animals and plants. In this connection, bioremediation measure may be helpful to mitigate the toxicity problem. Amongst the fungi, Pleurotus florida has been found effective and may be used as a bio-filter to remove heavy metals from untreated sewage which can be used for irrigation purpose in agricultural fields. References 1 Reiman, C. and Caritat, P. 1988. Chemical Elements in the Environment. Heidelberg, Germany, Springer-Verlag. 2 Nriagu, J.O. and Pacyna, J.M. 1988. Quantitative assessment of worldwide contamination of air, water and soils by trace metals. Nature 333:134-139. 3 McGrath, S.P., Shen, Z.G. and Zhao, F.J. 1997. Heavy metal uptake and chemical changes in the rhizosphere of Thlaspi caerulescens and Thlaspi ochroleucum grown in contaminated soil. Plant Soil 188: 153-159. 4 Neilson, J.W., Artiola, J.F. and Maier, M. 2003. Characterization of lead removal from contaminated soils by non-toxic soil washing agents. J. Environ. Qual. 32: 899-908. 5 SreeKrishnan, T.R. and Tyagi, R.D. 1996. A comparative study of the cost of leaching out heavy metals from sewage sludges. Process Biochem (Oxford) 31: 31-41. 6 Ryu, H.W., Moon, H.S., Lee, E.Y., Cho, K.S. and Choi, H. 2003. Leaching characteristics of heavy metals from sewage sludge by Acidithiobacillus thiooxidans Met. J. Environ. Qual. 32: 751-759. 7 Alleman, B.C. and Leeson, A. (Eds) 1997. In situ and on site bioremediation. Battelle Press, Columbus, OH. 8 Dutta, S.K., Hollowell, G.P., Fawzy, M., Hashen, L. and KyKendall, D. 2003. Enhanced bioremediation of soil containing 2, 4- dinitrotoluene by a genetically modified Sinorhizobium meliloti. Soil Biol. Biochem. 35: 667-675. 9 Page, A.L. 1982. Methods of soil analysis, Part 2.(ed) Chemical and Microbiological Properties, Soil Science Society of America Journal, Inc. Madison, Wisconsin, U.S.A. 10 Lindsay, W.L. and Norvell, W.A. 1978. Development of a DTPA soil test for zinc, iron, manganese, and copper. Soil Sci. Soc. Am. J. 42 : 421-428. 11 Bouyoucos, G.J. 1962. Hydrometer method improved for making particle-size analysis of soils. Agron. J. 54: 464-465. 12 Kapoor, A. and Viraraghavan, T. 1997. Heavy metal biosorption sites in Aspergillus niger. Biores. Technol. 61: 24-227. 13 Brar, M.S., Malhi, S.S., Singh, A.P., Arora, C.L. and Gill, K.S. 2000. Effect of sewage effluent on heavy metal content in light textured soils of Punjab. Canadian J. Soil Sci. 80: 461-465. 270