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Comparative Analysis of Tannery-Effluent Contaminated Soil and Mixed Culture Bacterial Inoculation on Helianthus annuus L. Growth.

Byline: MUHAMMAD YASIN AND MUHAMMAD FAISAL

Summary: Here we reported the effect of four strains Bacillus pumilus-CrK08, Cellulosimicrobium cellulans-CrK16, Exiguobacterium-CrK19 and Bacillus cereus-CrK20 and tannery contaminated soil on Helianthus annuus L. var Hysun-33 growth parameters. Plants growing in tannery effluent contaminated soil have shown slowed leaf growth, reduced shoot length, burning of leaf margins and tips compared to plants growing in normal garden soil. The inoculated plants had shown overall increase in root length (15%), shoot length (33%) and fresh weight shoot (135%) compared to un-inoculated plants growing in stress conditions. Plants growing in tannery contaminated soil have shown increase in soluble proteins contents (9%), acid phosphatase activity (200%), peroxidase activity (203%) and decrease in chlorophyll a (39%), chlorophyll b (23%) and carotenoids contents (28%) compare to plants growing in normal control soil.

Inoculated plants grown in contaminated soil have shown an increased in peroxidase activity, soluble proteins contents, acid phosphatase activity, chlorophyll a, b and carotenoid contents compare to respective un-inoculated plants.

Introduction

Heavy metal pollution is a world wide problem and human activities have become a major process for mobilization of metals in the biosphere [1]. Use of low quality fertilizers, discharge of effluents from industries, metal smelting, mining practices, pesticides and sewage has increased the level of heavy metals in agriculture land above levels consider harmless for food production [2]. A group of trace elements consist of heavy metals such as iron, nickel, copper, chromium, selenium that are essential for cell in trace amounts for different biochemical reactions, however, when present at higher concentrations, they have toxic effects [3].

Chromium is an essential trace element for organisms and it exists naturally in different oxidation states, Cr+3 and Cr+6 are more stable forms but Cr+6 is more toxic as a slight increase in the level of Cr+6 can cause several environmental and human health related problems because of its mutagenicity, carcinogenicity and toxicity [4]. The allowed level of chromium in drinking water is 50-100 (mu)g L[?]1 [5]. According to Central Pollution Control Board, India, the permissible limit for Cr+6 for discharge of industrial effluents in different water bodies i.e., public severs, land surface water and marine costal areas is 2.0, 0.1and 1.0 mg L-1, respectively [6]. The traditional physicochemical methods (chemical precipitation, lime coagulation, ion exchange and solvent extraction) can be used for clean up of metal- contaminated soils but they are very costly and might be destructive to the normal properties of the soil [7].

Crop plants growing on heavy metal contaminated soil can accumulate high concentrations of trace elements to cause severe health threat to consumers [8]. Sulfate utilizing organisms can take up Cr+6 through membrane transport channels [9]. Under normal physiological conditions the Cr+6 ions react spontaneously with intracellular reductants (glutathione) to give unstable Cr+4 and Cr+5 ions [10] (Xu et al. 2005) which gets oxidized into Cr+6 which further reacts with DNA and proteins causing kidney cancer [11]. Rhizosphere as an important interface of soil and plant which can play an important role in the phytoremediation of metal-contaminated soils. Microbes have potential to enhance the phytoremediation processes [12]. Through the soil acidification, exudation of organic compounds, and redox changes microbial populations are known to modify the chemical properties of the rhizospheric soil with subsequent effects on the environmental mobility and availability of metal contaminants [13].

Here in the present study we used a consortium of bacterial strains (Bacillus pumilus- CrK08, Cellulosimicrobium cellulans-CrK16, Exiguobacterium-CrK19 and Bacillus cereus-CrK20) to establish their role in detoxification of chromium and other heavy metal contaminated soil and plant growth promotion by using Helianthus annuus plants.

Results and discussion

Bacterial Strains

Microbes can enhance the process of phytoremediation and play an important role in bioremediation of heavy metals [12]. Chromium exists in different valance states but Cr+6 is highly toxic and it can inhibit the growth of plants [14]. The present work explains the phytotoxic effects of metal contaminated soil especially chromium on plant growth and role of chromium resistant bacteria in improvement of sunflower growth as a tool for the reclamation of the contaminated land. The bacterial strains isolated from water/soil contaminated from tannery effluents have ability to tolerate very high concentration of K2CrO4 (25-40 mg/ml) and these strains could reduce Cr (VI) efficiently. Bacillus cereus-CrK20 was isolated from tannery waste water and Bacillus pumilus-CrK08, Cellulosimicrobium cellulans-CrK16 and Exiguobacterium-CrK19 were isolated from soil contaminated by tannery effluents. Some of their morphological characteristics are given Table-1.

Faisal and Hasnain [15] have also studied strains which could resist very high level of chromate. Bacterial strains isolated by Basu et al. [16] from tannery waste can also tolerate Cr (VI) up to 250 (mu)g ml-1.

Sunflower Growth Parameters

Seed germination was better (100%) in tannery wastewater contaminated soil compared to normal control soil (78%) (Table-2). Apparently bacterial inoculation had negligible effect on seed germination. Both inoculated and un-inoculated seeds have shown 100% germination under metal stress (contaminated soil) compared to normal control soil (Table-2). Plants growing in tannery waste contaminated soil have shown overall reduced growth of leaves, roots and shoots compared to normal control soil (Fig. 1). Fresh biomass of shoot was severely affected in non-inoculated plants and Auto.P2 has shown 80% reduction in fresh biomass of shoot compared to normal control soil. Inoculated plants (Auto.P2B) have shown 33, 89, 125 and 85% increases in shoot length, fresh weight of shoot, fresh weight of root and dry weight of roots, respectively, compared to respective contaminated soil (Auto.P2) (Table-2). Sunflower seeds growing in tannery waste contaminated soil have shown 100% germination compared to normal control soil.

Peralta et al. [17] also observed seed germination in chromium polluted soil even under very high level of metal. Overall fresh weight of root, shoot in sunflower plants growing in tannery waste contaminated soil was lower compared to normal control soil (Table-2). Limited conductive and absorptive function of roots results in limited growth of top parts of the plants because roots are in direct contact with toxic substances in the soil. Inoculated plants (Auto. P2B) have shown overall increase in fresh weight of root and shoots compared to un-inoculated plants (Auto.P2) (Table-2). In the stress environment, microorganisms present in the rhizosphere zone caused increase biomass production and heavy metal tolerance [18]. The biodegradative bacteria may colonize inside (endophytic) or attach efficiently to plant roots and have ability to enhance plant growth [19].

Biochemical Parameters

Compared to normal control soil, plants growing in tannery waste contaminated soil have shown 9% and 199% increases in soluble proteins and acid phosphatase contents, respectively. In another study, Saleh and Belisle [20] had reported that bacterial strains may secrete acid phosphatase enzyme and stimulate acid phosphatase activity. Acid phosphatase is involved in increase uptake of phosphorous (P) form soil and under heavy metal stress its activity in plants increases [21, 22]. A reduction of 37%, 23% and 27% in chlorophyll a, chlorophyll b and total carotenoids, respectively, was also observed in plants growing in contaminated soil as compared to control soil (Table-3). Many investigators also studied that the toxicity of chromium present in the tannery waste water, chlorophyll contents will decrease in Triticum aestivum and Vigna radiata [23, 24].

The effect of 1-2 mg L[?]1 Cr(VI) on Salvinia minima reduced carotenoid and chlorophylls a and b concentrations significantly [25] Inoculated plants have shown an increase in chlorophyll a (52%), chlorophyll b (31%) and carotenoid contents (23%) compare to un- inoculated plants. Inoculated plants (Auto.P2B) have shown an over all increase in acid phosphatase, soluble proteins, chlorophyll a, chlorophyll b and total carotenoids compared to contaminated control soil (Auto.P2) and a decrease in peroxidase units (Table-3).

Table-1: Cell morphology of chromium resistant strains of bacteria.

Strains code###Closest species###Accession Number###Cr-resistance mg ml-1###Locality###Source###Gram's staining

CrK16###Cellulosimicrobium cellulans###GQ503328###25###Kasur###soil###Gram negative Cocci

CrK19###Exiguobacterium sp###GQ503330###25###Kasur###soil###Gram positive Rods

CrK08###Bacillus pumilus###GQ503326###25###Kasur###soil###Gram positive Rods

CrK20###Bacillus cereus###GQ503329###25###Kasur###water###Gram positive Rods

Table-2 Effect of mixed culture inoculation of Cr reducing bacteria on germination, plant height, fresh biomass of sunflower plants.

Plant sample###Germination (%)###Shoot length cm###Root length cm###Fresh wt. of shoot g/plant###Fresh wt. of root g/plant

Control###78+-1.15###68.5+-5.48###9.5+-0.38###10.82+-0.54###1.54+-0.06

Auto.P2###100+-00###42.96+-2.58###6.34+-0.44###2.17+-0.10###0.36+-0.02

Auto.P2B###100+-00###55.75+-5.04###7.25+-0.65###5.11+-0.15###0.81+-0.04

Auto.P2, sunflower growing in sterilized tannery waste contaminated soil.

Auto.P2B, sunflower growing in sterilized tannery waste contaminated soil having mixed culture bacterial inoculum.

Table-3: Estimation of soluble proteins, peroxidase, acid phosphatase and pigment analysis in sunflower grown under heavy metals stress in tannery waste contaminated soil in the presence of mixed culture bacterial inoculation.

###Soluble###Peroxidase###Amount

###protein###contents###Acid phosphatase###Chlorophyll "a"###Chlorophyll b###Total###of total

Sample###content g1###Unit g-1 of###(mu)g/gm of fresh###(mu)g /gm fresh wt.###(mu)g/gm fresh###Carotenoids###Remediation

###of fresh###fresh wt.###wt. of leaf###of leaf###wt. of leaf###(mu)/gm fresh###Cr mg/kg###factor (RF)

###wt. of leaf###plant dry

###wt.of leaf###of leaf###wt.

Control###4.01+-0.24###0.054+-0.02###5.477+-0.11###29.737+-2.67###26.01+-2.60###17.589+-0.52###-###-

Auto.P2###4.25+-0.12###0.164+-0.01###16.0+-1.12###18.45+-0.73###19.84+-0.99###12.74+-0.51###123###0.496

Auto.P2B###4.36+-0.34###0.038+-0.03###16.416+-0.82###28.11+-2.81###26.02+-2.34###15.718+-1.41###16.96###0.068

Auto.P2, sunflower growing in sterilized tannery waste contaminated soil.

Auto.P2B, sunflower growing in sterilized tannery waste contaminated soil having mixed culture bacterial inoculum.

Table-4: Physico-chemical properties of tannery-effluent contaminated and control garden soil samples.###Grain Size

Soil Type###Electric###Organic###Total###Cr

###conductivity###K###Na###Mg###Ca###CO3###HCO3###SO4###PO4###matter###Cr###(VI)###63(mu)m -###500(mu)m###500(mu)m-###0-63(mu)m

Contaminated###15.1 ms/cm###0.09 gm/kg###1.43 gm/kg###0.36 gm/kg###1.0 gm/kg###Nil###0.61 gm/kg###3.18 gm/kg###20.3 mg/kg###7.92 %###24.8 gm/kg###4.21 mg/kg###23.1 %###24.8 %###26.3 %###25.69 %

soil

Natural###28.7 ms/cm###0.13 gm/kg###5.15 gm/kg###0.61 gm/kg###2.32 gm/kg###Nil###1.76 gm/kg###1.88 gm/kg###35.5 mg/kg###5.35 %###Nil###Nil###41.3 %###33.6 %###23.2 %###1.23 %

Garden Soil

Plant and Soil Analysis

The plant stem samples were analyzed for heavy metals (total chromium) by inductively coupled plasma-spectrometry (ICP-OES). Inoculated plants stem samples have shown 123 mg/kg to 17 mg/kg decrease in Cr uptake compared to contaminated soil control (Table-3). In a previous study it was reported that the microbial inoculated plants have less chromium contents compare to non inoculated ones [15]. The chemical and physical characteristics as well as the concentrations of total metals and available metals of the soil sample are given in Table-4. The soil sample was found to have organic matter content of 7.92 % and electric conductivity value of 15.14 ms/cm and pH was 7.6. Hexavalent chromium and total chromium were 4.21 mg/kg and 24.78 mg kg[?]1, respectively, (Table-4). Compared to control soil the total levels of the studied heavy metals in tannery waste contaminated soils were higher.

Experimental

Bacterial Strains

Bacillus pumilus-CrK08, Cellulosimicrobium cellulans-CrK16 and Exiguobacterium -CrK19 were isolated form tannery effluent contaminated soil and Bacillus cereus-CrK20 from tannery polluted water from Kasur, Pakistan. Identified by 16S rRNA gene sequence analysis. These strains have been characterized and their interaction with sunflower plants was also studied in order to determine their potential role in different bioremediation strategies.

Soil Sampling

The soil contaminated by tanneries effluent was collected from Din Gharr, Kasur, Pakistan and transported to field lab in plastic bags. The location of soil sampling site was as follow: longitude (E) 7427'34.17'' and latitude (N) 3106'18.53''. Four soil samples were taken from four different areas from the sampling site (almost up to 9 inches deep), and mixed thoroughly before filling the pots.

Field Experiments with Sunflower

In a field experiment plants were grown in tannery contaminated soil to study the effect of metal contaminated soil and mixed culture bacterial inoculation on plants growth. Pots were filled with 3 kg of tannery effluent contaminated autoclaved soil (AS) and natural control soil (NCS). Nine seeds were sown in each pot and then were kept almost square feet away from each other. The soil had a pH of 7.6, E.C. 15.14 ms/cm, organic matter 7.92% (Table-4). For bacterial cultures preparation, bacteria were grown in L-Broth overnight (150 rpm at 37@C). Optical density of the cultures was adjusted equally using 2D spectrophotometer at 600 nm with sterilizes glass distilled. For the preparation of mixed cultures of bacteria equal quantities of bacterial cultures were mixed to get a uniform number of cells per ml. Seeds were inoculated for 15 minutes prior to sowing. Un- inoculated seeds were sown as control in natural control soil and tannery waste contaminated soil.

Pots were watered regularly (to field capacity by weighing) when required and allowed to grow till maturity. Plant growth and development were monitored throughout the growing season. At maturity all plants were harvested and various growth and yield parameters were investigated.

Biochemical Parameters

David and Murray [26] method was used for the quantitative estimation of peroxidases. Crush one gram frozen plant material in phosphate buffer (0.1M, pH 7.0) in a cold pestle and mortar in the ratio of 1:4. Centrifuge at 10,000 rpm for 10 minutes at 4degC. The Two sets of test tubes were labeled, one for test and the other for control reactions. To all the test tubes (both sets) 0.2ml of enzyme extract, 2.5ml of phosphate buffer (0.1M, pH 7.0) was added. For test reactions, 0.2 ml of 1% Guaiacol was also added and mixed. Put both sets at room temperature for 15-20 minutes. After that, 0.1ml of 0.3% H2O2 solution was added in both the sets. For blank, 0.2ml glass distilled water; 2.5 ml of phosphate buffer and 0.1ml of 0.3% H2O2 solution were mixed. Optical densityof was taken against blank at 470 nm.

Lowry at el [27] method was used for soluble protein estimation. One gram frozen plant material was crushed with phosphate buffer (0.1 M pH 7.0) in a cold pestle and mortar. Volume of buffer was in the ratio of 1:4 (w/v). Centrifuged for 10 minutes at 4degC at 10,000 rpm. Take 0.4ml of extract 2ml of Folin's mixture was added and test tubes were placed at room temperature for 15 minutes. Then 0.2ml of Folin and Ciocalteu's phenol reagent was added, mixed and placed for 45 minutes at room temperature for the development of color. The absorbance was taken at 750 nm.

The acid phosphatase enzyme was extracted following the method of Iqbal and Rafique [28]. Weighed and frozen plant material (shoots) was crushed in a cold pestle and mortar with cold 0.1M Tris HCl buffer (pH 6.5). The ratio of buffer to plant material was 4:1 (v/w). The crushed samples were centrifuged at 14,000 rpm for 10 minutes. The supernatant thus obtained was used for the estimation of enzyme acid phosphatase. For the activity of acid phosphatase enzyme, the time duration was one hour, temperature was 37degC and pH was 4.9. For the quantitative estimation of enzyme, series of reactions i.e., test, control, standard and blank were carried out.

The Pigment chlorophyll A, chlorophyll B, carotenoids were measured by using the method of Lichtenthaler and Wellburn [29]. 1g of fresh plant material was crushed in 8 ml of acetone, and then each sample was centrifuged at 13000rpm for 5 minutes. The supernatant was separated and the absorbance was taken at 470nm, 645nm and 662nmon 2-D spectrophotometer.

Estimation of Metal Content in Plant and Soil

Plants were harvested after five and half months of sowing. Shoots and roots were separated and then oven-dried at 70degC. The plant stem samples and soil sample were analyzed for heavy metals by inductively coupled plasma-spectrometer (ICP- OES) by Pakistan Council of Scientific and Industrial Research (PCSIR) Laboratories Complex, Lahore.

Methods used and other specifications were consulted from "official method of analysis of AOAC international (2005) 18th Ed., AOAC international Gaithersburg MD, USA, official method 985.01. At the time of plant samples testing the temperature of laboratory was 30-38oC and humidity level was 26-42%.

Conclusion

This study establishes the role of chromium resistant bacterial strains Bacillus cereus-CrK20, Cellulosimicrobium cellulans-CrK16, Bacillus pumilus-CrK08 and Exiguobacterium-CrK19 in detoxification and bioremediation of chromium and other heavy metal contaminated soil in association of sunflower plants. These bacteria are helpful in plant growth promotion and can be utilized for the bioremediation of chromium and tanneries waste water contaminated sites in Pakistan.

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Department of Microbiology and Molecular Genetics, University of the Punjab, Quaid-e-Azam Campus, Lahore-54590, Pakistan., yasin_mmg@yahoo.com*
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Author:Yasin, Muhammad; Faisal, Muhammad
Publication:Journal of the Chemical Society of Pakistan
Article Type:Report
Geographic Code:9PAKI
Date:Dec 31, 2012
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