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Biodegradation potential of bacterial isolates from tannery effluent with special reference to hexavalent chromium
Sankar Narayan Sinha, Mrinal Biswas, Dipak Paul, Saidur Rahaman
Department of Botany, University of Kalyani, Kalyani, West Bengal, 741235, India, E-mail: sinhasn62@yahoo.co.in

ABSTRACT
The application of isolated bacterial culture as biological tool for hexavalent chromium removal from tannery industry waste water was investigated. A number of bacteria tolerant to high levels of chromate (>400gml-1) were isolated from tannery effluents. These isolates showed varying degree of Cr (VI) reduction under oxic and anoxic conditions at room temperature. Ten isolates were found to be efficient in reducing 70% Cr (VI) under anoxic condition. This included 5 isolates of Pseudomonas, three isolates of Micrococcus and two isolates of Aeromonas. These isolates were subjected to further characterization for possible use in Cr (VI) detoxification of industrial wastes. These bacterial isolates were further examined for tolerance to a variety of other heavy metals. The study indicates the possible use of these bacterial isolates in environmental clean-up. Keywords: biodegradation, tannery effluent, chromium, heavy metals

INTRODUCTION
Extensive application of chromium in industries particularly leather tanning industries leads to the formation of chromium-contaminated soil and ground water which pose a serious threat to living biota particularly to human health. Chromium is a potent pollutant which is mutagenic, carcinogenic and teratogenic in humans [1-3]. It is found to be toxic in plants also [4]. Among different forms of chromium, chromium VI is known to cause serious health hazard effects. It can cause allergic reactions, nose irritation and nose bleeds. It creates respiratory problems and weaken-immune system. It causes renal tubular necrosis and produce cytotoxic and genotoxic effects [5,6]. Hexavalent chromium toxicity appears to be due to its rapid permeability through biological membranes and subsequent interaction of chromium with intracellular proteins and nucleic acids. Usually the tanning industry uses chrome liquor in the tanning process. A large number of tanning industries discharge their effluents into the environment containing chrome salts exceeding the maximum permissible limit. As a result, they provide a natural environment for enrichment of chromium-resistant bacteria and consequently these bacteria may be employed for removal of hexavalent chromium from tannery waste. Cr (VI)-containing tannery effluents are commonly treated by chemical means. These methods may be a source of potential heavy metal pollution from the resultant metal-containing chemical sludge. The commonly employed methods include excavation, to pump and treat, in situ vitrification and chemical treatment with a reductant [7]. The conventional method to detoxify and remove Cr (VI) from the environment involve chemical reduction followed by precipitation, ion exchange and absorption on coal, activated carbon, alum, kaolinite and flyash [8,9]. Several reports have indicated biological reduction of Cr (VI) by aerobic and anaerobic bacteria as biological reduction of Cr (VI) usually occurs at a neutral pH range. It generates an insignificant quantity of chemical sludge. It offers potentially cost-effective remediation strategy [10]. It does not require high energy input or toxic chemical reagents and finally it offers an economical as well as
Research Article, Biotechnol. Bioinf. Bioeng. 2011, 1(3):381-386 2011 Society for Applied Biotechnology. Printed in India; ISSN 2249-9075

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eco-friendly option of metal detoxification and bioremediation. The processes by which microorganisms interact with toxic metals enabling their removal/and recovery are bioaccumulation, biosorption and enzymatic reduction [11]. Recent studies have shown that certain species of bacteria are capable of transforming Cr (VI) into the much less toxic and less mobile Cr (III) [12,13]. The objective of the present investigation was to screen and enumerate chromium-resistant bacterial population from tannery waste, determination of MIC of Cr (VI) for chromium-resistant bacteria, to test the ability of bacterial isolates for tolerance to other heavy metals such as lead, zinc and cadmium, to characterize bacterial isolates and utilization of Cr (VI) and other metal-resistant bacteria isolated from tannery waste to remove toxic Cr (VI) from contaminated environment.

MATERIALS AND METHODS Sampling

Effluent samples were collected from a tannery industry at Kolkata, West Bengal. Samples were collected in sterilized glass bottles aseptically and transported to the laboratory in an ice bucket. Samples were analyzed within 6h of collection.

Isolation of chromium-resistant bacteria

Chromium-resistant bacteria were isolated from untreated tannery effluent on agar plates under anoxic conditions. The medium contains (per litre): casein 20g, dextrose 10g, sodium thioglycollate 2g, sodium chloride 5g, sodium formaldehyde sulphoxylate 1g, methylene blue 2mg, agar agar 20g, pH 7.20.2. These agar plates were amended with 200 g ml-1 and 400 g ml-1 Cr (VI) by standard plate method [14,15]. The inoculated agar plates were incubated in BBL Gas Pak anaerobic Jars at 25C for 5days. After incubation clones representing different colony types were purified on agar plates under anoxic condition. All the bacterial cultures were kept at -20C in agar stabs.

Determination of metal tolerance activity

The anoxic bacteria were examined for their resistance to chromium by agar dilution method [16]. In this method, freshly prepared agar plates amended with Cr (VI) as dichromate at various concentrations (50-800 g ml-1) were inoculated with overnight grown cultures under anoxic condition. All the plates were incubated at room temperature (25C) for 48h. The minimal concentration of metal in the Petri plate showing complete inhibition of growth was considered as minimal inhibitory concentration (MIC). Tolerance to Cr (VI) was confirmed by broth-dilution method [17]. Tolerance to Zn, Cd and Pb was determined by agar dilution method. The salts used were zinc chloride, cadmium chloride and lead nitrate.

Evaluation of Cr (VI) reduction

The test bacteria were inoculated in diluted peptone water amended with Cr (VI) at the rate of 20g ml-1 and incubated at 252C for up to 72h. The decrease in Cr (VI) concentration in the experimental flasks was measured after appropriate time intervals by 1,5-diphenyl carbazide (50g ml-1) method [14]. The test samples were acidified (pH 1-2) and 50g ml-1 1,5-diphenyl carbazide was added and Cr (VI) concentration was detected by spectrophotometer (CECIL CE 7200) at 540 nm. To estimate the total chromium, samples were digested with hydrochloric acid and perchloric acid mixture (6:1). Metal concentration was determined by atomic absorption spectrophotometer.

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Characterization of bacterial isolates

Bacterial isolates from tannery effluents were characterized according to Bergeys Manual of Systematic Bacteriology [18].

RESULTS AND DISCUSSION

The bacterial count in the tannery effluent under anoxic condition is given in figure 1. The bacterial counts of 4x106 cfu were obtained at a hexavalent chromium concentration of 50 g ml-1. At 400 g ml-1 and 800 g ml-1 chromium concentrations, the bacterial counts were 2x106 cfu and 1.4x106 cfu respectively. MIC of hexavalent chromium in isolates is represented in table 1. Nearly 38% of the isolates were found to be tolerant to the hexavalent chromium concentration of more than 400 g ml-1. Around 33.3% isolates recorded tolerant to hexavalent chromium level greater than 800 g ml1 . Reduction of Cr (VI) under anoxic condition and their tolerance to chromium is depicted in table 2. Out of the 42 isolates screened, 30 (nearly 71%) isolates exhibited the ability to reduce hexavalent chromium over 50%. Two isolates were able to reduce over 90% hexavalent chromium and one isolate was found to tolerate to concentration of chromium greater than 400g ml-1.

Figure 1. Cr (VI) tolerant bacteria isolated under anoxic condition from tannery effluents.

Table 1. MIC of hexavalent chromium of 42 chromium resistant bacteria. Cr (VI) concentration (g/ml) <400 >400 >800 Number of isolates 12 16 14

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Isolates capable of hexavalent chromium reduction under anoxic condition greater than 70% were also examined for oxic reduction of hexavalent chromium and interestingly all the isolates were found to reduce the hexavalent chromium under oxic condition (Table 3). The maximum level of hexavalent chromium reduction under oxic condition was 65.2% by an isolate KT2 (Table 4).
Table 2. Reduction of Cr (VI) under anoxic condition. MIC of Cr (VI) (g/ml) <400 >400 >800 Total Number of isolates 12 16 14 42 % reduction of chromium (VI) 51-70 71-90 2 7 10 3 8 3 20 13

<50 2 2 3 7

>90 1 1 0 2

Table 3. Reduction Cr (VI) under oxic condition. Total number of isolates 7 Number of isolates 3 3 1 2 6 4 5 3 1 5 4 3 2 % Cr (VI) oxidation <10 <10 11-20 <10 11-20 21-30 31-40 41-50 11-20 21-30 31-40 41-50 41-50

20

13

Table 4. Reduction of Cr (VI) by some selected bacterial isolates. Isolate KT3 KT4 KT13 KT16 KT18 KT2 KT6 KT8 KT23 KT41 MIC of Cr (VI) (g/ml) 400 >400 400 400 >400 400 400 400 400 400 Percent of anoxic reduction of Cr (VI) 92.5 90.2 85.5 85.0 82.0 81.2 80.2 78.5 82.2 81.1 Percent of oxic reduction of Cr (VI) 25.2 42.5 40.5 42.0 40.0 65.2 60.5 60.4 62.4 60.5

Selected isolates of anoxic bacteria capable of chromium reduction over 70% were characterized and their tolerance to other heavy metal such as Pb, Zn, Cd were also determined and it was observed that except for 3 Gram positive cocci (KT2, KT6 and KT8), all isolates were found

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to be Gram negative rods, demonstrating physiological characters primarily indicative of the genera Pseudomonas (KT3, KT4, KT13, KT16 and KT18) and Aeromonas (KT23 and KT41) (Table 5, 6). The Cr (VI) reduction by Aeromonas was reported earlier [19]. Besides their tolerance to chromium all the selected 10 isolates showed tolerance to different concentration of lead, zinc and cadmium (Table 5).
Table 5. Tolerance to other heavy metals by selected bacterial isolates. Isolates KT3 KT4 KT13 KT16 KT18 KT2 KT6 KT8 KT23 KT41 Metal tolerance (g/ml) Zn 100 100 100 100 200 100 100 200 200 200

Pb 100 50 100 100 200 100 50 50 100 100

Cd 50 100 100 100 100 100 100 100 100 200

Table 6. Morphological, physiological and biochemical tests of the isolates. Tests Cell shape Cell size (m) Gram reaction Motility Endospore formation Aerobic growth Anaerobic growth Indole test VP MR Nitrate reduction Catalase Urease Oxidase Utilization of sugar Glucose Fructose Galactose Maltose Isolates KT18 KT2 rod coccus 0.51.5-3.0 0.8 + + + + -

KT3 rod 0.50.8 + + + -

KT4 rod 0.40.7 + + + -

KT13 rod 0.40.8 + + + -

KT16 rod 0.40.1 + + + -

KT6 coccus 1.5-2.9 +

KT8 coccus 1.5-2.8 +

KT23 rod 1.83.0 + + + + + + +

KT41 rod 1.53.0 + + + + + + +

+ + + + +

+ + + + +

+ + + + +

+ + + +

+ + + +

+ + + +

+ + + +

+ + + +

+ -

+ -

+ -

+ + + +

+ + + +

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Lactose + + + + + Sucrose + + + + + Xylose + + + + + Rhamnose Gelatin + + + + + + indicates presence or positive reactions; - indicates absence or negative reactions.

+ +

+ +

Considerable stress about the toxicity of chromium compounds needs recovery and reuse of chromium from tanneries and other industrial wastes or rendering it to a less toxic form [20]. In the present study all the ten bacterial isolates were found to be very much efficient in the detoxification of Cr (VI), capable of reducing 70-90% of Cr (VI) under anoxic condition within 72h. They were also found to reduce Cr (VI) under oxic condition with some lower efficiency. Overall, all the bacterial strains screened from tannery wastes may be exploited in the detoxification of hexavalent chromium. As these bacterial strains are able to tolerate and reduce Cr (VI), they are potentially used in the bioremediation of heavy metals.

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