(VI) through biosorption by the Pseudomonas ... - Wiley Online Library

Journal of Basic Microbiology 2008, 48, 135 – 139

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Short Communication Removal of chromium (VI) through biosorption by the Pseudomonas spp. isolated from tannery effluent Jatin Srivastava1, Harish Chandra2, Kirti Tripathi3, Ram Naraian3 and Ranjeev K Sahu1 1

2 3

Department of Environmental Sciences, Chatrapati Shahu Ji Maharaj University, Kalyanpur – Kanpur – 208024 UP, India Department of Microbiology, Gayatri College of Biomedical Sciences, Dehradun (UK), India Department of Microbiology, Chatrapati Shahu Ji Maharaj University, Kalyanpur – Kanpur – 208024 UP, India

Heavy metal contamination of the rivers is a world wide environmental problem and its removal is a great challenge. Kanpur and Unnao two closely located districts of Uttar Pradesh India are known for their leather industries. The tanneries release their treated effluent in the near by water ways containing Cr metal that eventually merges with the river Ganges. Untreated tannery effluent contains 2.673 ± 0.32 to 3.268 ± 0.73 mg l–1 Cr. Microbes were isolated, keeping the natural selection in the view, from the tannery effluent since microbes present in the effluent exposed to the various types of stresses and metal stress is one of them. Investigations include the exposure of higher concentrations of Cr(VI) 1.0 to 4.0 mg l–1 to the bacteria (presumably the Pseudomonas spp.) predominant on the agar plate. The short termed study (72 h) of biosorption showed significant reduction of metal in the media especially in the higher concentrations with a value from 1.0 ± 0.02, 2.0 ± 0.01, 3.0 ± 0, and 4.0 ± 0.09 at zero h to 0.873 ± 0.55, 1.840 ± 1.31, 2.780 ± 0.03 and 3.502 ± 0.68 at 72 h respectively. The biosorption of metal show in the present study that the naturally occurring microbes have enough potential to mitigate the excessive contamination of their surroundings and can be used to reduce the metal concentrations in aqueous solutions in a specific time frame. Keywords: Natural selection / Tannery effluent / Pseudomonas spp. / Cr (VI) / Biosorption Received: October 16, 2007: accepted December 07, 2007 DOI 10.1002/jobm.200700291

Introduction* Heavy metal contamination of the rivers is a world wide environmental problem and its removal is a great challenge. The di-chromate compounds are used as oxidizing agents in quantitative analysis of various water quality parameters such as chemical oxygen demand (COD) and in tanning processes. Chromium compounds are used in the textile and aircraft industry as mordents and anodizing agents respectively. The fate of chromium in the environment is strongly dependent on its valence state [1]. However, chromium levels in air, water, and food are generally very low; the major Correspondence: Dr. Jatin K. Srivastava, Department of Environmental Sciences, Chatrapati Shahu Ji Maharaj University, Kanpur – 208024 UP, India E-mail: [email protected] © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

human exposure is occupational [2]. Cr(VI) is reactive and a potent carcinogenic species [3]. Wastewater containing chromium must be treated before being discharged into the environment. The most commonly used method to remove Chromium from liquid effluents is alkaline precipitation, but the method is expensive, therefore cheaper and effective bioremediation techniques using bacteria [4, 5], soils [6], algae [7] and plants [8] are being studied all over the world. Microorganisms can physically remove heavy metals from solution through either bioaccumulation or biosorption. In bioaccumulation, metals are transported from the outside of the microbial cell, through the cellular membrane, and into the cell cytoplasm, where the metal is sequestered. Earlier reports of Wong and So [9] suggested the accumulation of Cu(II) ions by the isolated Pseudomonas pudida II-11, from electroplating effluent. www.jbm-journal.com

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immediately after the collection. The bottles, containing diluted effluent were then incubated at 37 °C overnight. For the isolation of bacterial cells 100 µl of effluent was spread over a pre-solidified plates of nutrient agar media (Hi-Media Inc.). After getting the bacterial colonies the cells were sub cultured to get the purest colony of the desired bacteria.

Figure 1. Kanpur and Unnao District of Uttar Pradesh India.

Bioremediation of industrial wastes containing heavy metals has been demonstrated by several researchers [9, 10]. Few studies [11, 12] showed the ability of metaltolerant bacterium to liberate metals from metal compounds with no mass loss. The capabilities of several bacteria in removing uranium, Cd, Pb and toxic metals from polluted effluents have already been demonstrated [10, 13 – 16]. Kanpur and Unnao two closely located districts (Fig. 1) of Uttar Pradesh India are known for their leather industries. The tanneries release their treated effluent in the near by water ways containing Cr metal that eventually merges with the Ganges. The present investigation was carried out the concept of natural selection whereby naturally growing microbes can be isolated from the stressful environment. Investigations were carried with these isolated microbes to monitor the accumulation of Cr. In the present study isolated microbes from diluted tannery effluent was identified as a genera Pseudomona (unidentified species) were used to monitor for the accumulation of Cr.

Material and methods Bacterial isolation The untreated tannery effluent was collected from the Common Effluent Treatment Plant (CETP) Unnao, a district of Uttar Pradesh India and was analyzed for the metal contamination. The effluent contains 2.673 ± 0.32 to 3.268 ± 0.73 mg l–1 Cr. 100 ml of effluent was mixed with autoclaved luke-warm distilled water (1 : 5) in pre-sterilized media bottles under a laminar air flow © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Identification of bacterium The bacterial isolates were grown on various specific media viz., Pseudomonas isolation agar, McConkey agar, Pseudomonas fluorescence isolation agar and King’s medium prior to identify the genera of the bacteria by biochemical assays. For identification of the cells grown on (profuse colonies) Pseudomonas isolation agar were assayed as per Bergey’s manual for Gram positive test, Pigmentation, Catalase and test of carbohydrate (poly-βhydroxybutyrate) present in the cell wall of bacterium. The strain was also presumably detected, belonging to the genera Pseudomonas spp. with the help of enterobacter identifying kit KB-100 (Hi-Media Inc.). Sub-culturing was followed for the maintenance of the bacterial strain. Experimentation Solutions of various concentrations of Cr metals such as 1.0, 2.0, 3.0 and 4.0 mg l–1 were prepared in the nutrient broth by K2Cr2O7. These preparations were taken in sterilized glass vials (Borosil) in 6 – 7 replicates and were inoculated with the bacterium isolated from the tannery effluent with an initial count around 24 × 104 CFUs ml–1. Metal concentration was determined with the help of atomic absorption spectrophotometer (Perkin Elmer Model 5100 PC). The bacterial cell mass was recovered every after 24 h with the help of membrane filter (Millipore). The filtrate was analyzed for the metal concentration. The cell mass was digested in acid (di-acidic) and were analyzed for the accumulated metal traces. Cysteine assay Cysteine a thiol (SH) containing amino-acid was assayed as per the method of Gaitonde [17]. Acid ninhydrin reagent reacts specifically to form a pink product (Emax = 560 nm) with cysteine. The reaction product was stable for at least 3 – 4 h at room temperature and its extinction was proportional to the concentration in the range 0.05 – 0.5 millimole of cysteine. The method was applied for the determination of cysteine in perchloric acid (5%) extracts of bacterial mass (10 mg) recovered from the growth medium on a membrane filter. www.jbm-journal.com


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Table 1. The metal concentration in the nutrient broth after exposure of the isolated bacterium of tannery effluent. Concentration of solution –1

1.0 mg l 2.0 mg l–1 3.0 mg l–1 4.0 mg l–1

Metal concentration in broth (mg l–1) Zero h

24 h

48 h

72 h

1.00 ± 0.02 2.00 ± 0.01 3.00 ± 0 4.00 ± 0.09

0.997 ± 0.04 1.973 ± 0.17 2.916 ± 0.21 3.990 ± 0.90

0.905 ± 0.18 1.891 ± 1.01 2.898 ± 0.92 3.690 ± 1.72

0.873 ± 0.55 1.840 ± 1.31 2.780 ± 0.03 3.502 ± 0.68

Table 2. The metal concentration accumulated by the bacterial cell mass. Concentration of solution –1

1.0 mg l 2.0 mg l–1 3.0 mg l–1 4.0 mg l–1

Metal concentration in the bacterial cell mass (µg g–1) 24 h

48 h

72 h*

0.018 ± 0.10 0.020 ± 0.09 0.081± 0.13 0.131± 0.19

0.097 ± 0.01 0.116 ± 0.102 0.415 ± 0.21 0.323 ± 0.05

0.102 ± 0.31 0.137 ± 0.29 0.509 ± 0.50 0.482 ± 0.52

48 h

72 h*

* Bacterial cell mass could not survive for long.

Table 3. The bacterial cell count in the metal solution. Concentration of solution –1

1.0 mg l 2.0 mg l–1 3.0 mg l–1 4.0 mg l–1

Colony forming units ml–1 24 h 4

24 × 10 ± 12.0 21 × 104 ± 8.0 22 × 104 ± 6.0 21 × 104 ± 14.0

4

9 × 103 ± 1.0 2 × 103 ± 0.2 5 × 102 ± 3.0 7 × 102 ± 5.0

17 × 10 ± 6.0 14 × 104 ± 3.0 19 × 104 ± 9.0 11 × 104 ± 9.0

* Bacterial cell mass could not survive for long.

Results Higher concentrations of Cr(VI) were applied on the isolated bacterial mass identified presumably as Pseudomonas spp. from the tannery effluent. Cr metal in the broth was found reduced in which bacterial cell mass were grown except in the blank sets. The reduction in the metal content was found more in the sets of lower metal content i.e., 1.0 mg l–1 in 24 to 72 h with an extent 0.997 ± 0.04 to 0.873 ± 0.55 mg l–1, respectively. However; maximum reduction was observed in the sets of fourth set having metal content 4.0 mg l–1 in 24 to 72 h with a reduced value 3.990 ± 0.90 to 3.502 ± 0.68 mg l–1, respectively (Table 1). The results of metal accumulation (Table 2) by the bacterial cell mass recovered from the broth was found maximum in the third sets with an initial concentration of 3.0 mg l–1 with a value 0.509 ± 0.50 at 72 h followed by the fourth set with a value at 72 h 0.482 ± 0.52 mg l–1. The bacterial count in the broth (Table 3) which was kept 24 × 104 © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

CFUs ml–1 was found significantly reduced in all the sets i.e., 1.0 to 4.0 mg Cr (VI) per liter at 72 h with a reduced value 9 × 103 ± 1.0, 2 × 103 ± 0.2, 5 × 102 ± 3.0, 7 × 102 ± 5.0. Fig. 2 represents the total cysteine residues in the bacterial cell mass which was found to in0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0

Cyst (72) Cyst (48) Cyst (24)

1

2

3

4

5 –1

Figure 2. The cysteine residues (µg mg ) in the bacterial cell mass at different time intervals. www.jbm-journal.com

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crease in 24 h followed by 48 h however; almost no change was observed in the mass collected after 72 h of exposure.


study least cysteine residues were found in the microbes (Fig. 2).

Conclusion Discussion Metals play an integral role in the life processes of micro-organisms [18]. Some metals such as Ca, CO, Cr, Cu, K, Mg, Zn and Na are required nutrients and are essential for the growth of microbes however; the higher concentrations of these metals are toxic to every living cell. Micro-organisms are highly effective in sequestering heavy metals. In the present study bacterial mass was isolated from tannery effluent which is a source pollutant of Cr(VI) in the local area of district Unnao. The presence of microbial population in the effluent can be considered as the tolerant strains. Out of 9 different types of microbes the bacterium (in the present study) was chosen on the basis of growth performance in laboratory condition and convenient in handling. The bacterial colony was exposed to different concentration of Cr metal which showed significant reduction along with accumulation in the cells. The study showed that bacterial biomass in broth could adsorb the metal on the outer surface initially and died as soon as it gets inside the cell of bacterium. The biosorption of metals does not consume cellular energy. Positively charged metal ions are sequestered primarily through the adsorption of metals to the negative ionic groups on cell surfaces, the polysaccharide coating found on most forms of bacteria, or other extra-cellular structures such as capsules or slime layers. Binding sites on microbial cell surfaces usually are carboxyl residues, phosphate residues, SH groups, or hydroxyl groups. Non-essential metals bind with greater affinity to SH group [19]. Bacterial cells which are capable of forming an extra-cellular polysaccharide coating e.g., Pseudomonas sp. bio-adsorbs (biosorp) metal ions and can prevents them from interacting with vital cellular components [20]. The amount of metal biosorbed to the exterior of bacterial cells often exceeds the amount predicted using information about the charge density of the cell surface. In the present study, longer survival of the bacterial cells were found in lower concentration of Cr ions and less in higher concentration. It indicated that Cr metal might have entered into the bacterial cell either by ligand interaction or by active membrane uptake. Rouch et al. [21] demonstrated the presence of the cysteine rich proteins in bacterial cells such as Pseudomonas sp. and Synechococcus spp. which provide resistance to the bacterial cells. However; in the present © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Significant reduction in the metal concentration was observed in all the sets with a significant accumulation in the cells. However; the bacterial cells could not survive for longer period in the broth exhibiting the toxic response of the metal on the bacterial mass. The findings support the well established fact that living organisms naturally selected and can survive the harsh conditions is naturally selected and can be used for the mitigation of pollution from the water and soil.

References [1] Jeremy, F., Joshua, C., David, F., Ken, K., Bruce, B. and Maxim, B., 2000. Non-metabolic reduction of Cr(VI) by bacterial surfaces under nutrient absent conditions. Journal of Conference & Abstracts, 5, 396. [2] Chou, I. N., 1989. Distinct cytoskeleton injuries induced by As, Cd, Co, Cr, and Ni compounds. Biomedical Environmental Sciences, 2, 358 – 365. [3] Gibbs, H.J., Lees, P.S., Pinsky, P.F. and Rooney, B.C., 2000. Lung Cancer among workers in chromium chemical production. Amer. J. Indust. Medicines, 38, 115 – 126. [4] Ohtake, H, Fujii, E. and Toda, K., 1990. Reduction of toxic chromate reducing strain of Enterobacter cloacae. Environm. Technol., 11, 663 – 668. [5] Coleman, R.N. and Paran, J.H., 1991. Biofilm concentration of chromium. Environm. Technol., 12, 1079 – 1093. [6] Losi, M.E., Amrhein, C. and Frankenberger, W.T., Jr., 1994. Bioremediation of chromate contaminated groundwater by reduction and precipitation in surface soils. J. Environm. Quality, 23, 1141 – 1150. [7] Brady, D., Letebele, B., Duncan, J.R. and, Rose, P.D., 1994. Bioaccumulation of metals by Scenedesmus, Selenastrum and Chlorella algae. Water SA, 20, 213 – 218. [8] Wolverton, B.C. and Mc Donald, R.C., 1979. The water hyacinth: from prolific pest to potential provider. Ambio, 8, 2 – 9. [9] Wong, P.K. and So, C.M., 1993. Copper accumulation by a strain of Pseudomonas putida. Microbios, 73, 113 – 121. [10] Silver, S., 1991. Bacterial heavy metal resistance systems and possibility of bioremediation. In: Biotechnology, Bridging Research and Applications, pp. 265 – 287. Kluwer Academic Publishers, London. [11] Cole, F.A. and Clausen, C.A., 1997. Bacterial biodegradation of CCA treated waste wood. In: Proceedings, Forest Products Society Conference on Use of Recycled Wood and Paper in Building Applications, pp. 201 – 204 (September 9 1996, Madison, WI, USA). Forest Products Society. www.jbm-journal.com


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[12] Clausen, C.A. and Smith, R.L., 1998. Removal of CCA from treated wood by oxalic acid extraction, steam explosion, and bacterial fermentation. J. Indust. Microbiol. Biotechnol., 20, 251 – 257.

[17] Gaitonde, M.K., 1967. A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids. Biochem. J., 104, 627 – 633.

[13] Higham, D.P., Sadler, P.J. and Scawen, M.D., 1985. Cadmium resistance in Pseudomonas putida: growth and uptake of cadmium. J. Gen. Microbiol., 131, 2539 – 2544.

[18] Bruins, M.R., Kapil, S. and Oehma, F.K., 2000. Microbial resistance to metals in the environment. Ecotoxicol. Environm. Safety, 45, 198 – 207.

[14] Lovely, D.R., Widman, P.K., Woodward, J.C. and Phillips, E.J.P., 1993. Reduction of uranium by cytochrome C3 of Desulfovibrio vulgarism J. Appl. Environ. Microbiol., 59, 3572 – 3576.

[19] Hughes, M.N. and Poole, R.K., 1989. Metals and Microorganisms. Chapman and Hall London.

[15] Mullen, M.D., Wolf, D.C., Ferris, F.G., Beveridge, T.J., Fleming, C.A. and Bailey, G.W., 1989. Bacterial sorption of heavy metals. Appl. Environ. Microbiol., 55, 3143 – 3149. [16] Phillips, E.J.P., Landa, E.R. and Lovely, D.R., 1995. Remediation of uranium contaminated soils with bicarbonate extraction and microbial uranium (VI) reduction. J. Indust. Microbiol., 14, 203 – 207.

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[20] Scot, J.A. and Palmer, S.J., 1988. Cadmium bio-sorption by bacterial exo-polysaccharide. Biotechnol. Literature, 10, 21 – 24. [21] Rouch, D.A., Lee, B.T.D. and Morby, A.P., 1995. Understanding cellular responses to toxic agents: A model for mechanism choice in bacterial metal resistance. J. Indust. Microbiol., 14, 132 – 141.

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