Microbial removal of Hexavalent chromium from chromite waste dump

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Microbial removal of Hexavalent chromium from chromite waste dump. Sudarshan Singh Rathore1, 2, R. K. Tiwary1, Kumar Birendra1 and P. Padma2*. 1Central ...
Malaya Journal of Biosciences 2014, 1(2):109–116 ISSN 2348 6236 print / 2348 3075 online

Malaya Journal of Biosciences

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Microbial removal of Hexavalent chromium from chromite waste dump Sudarshan Singh Rathore 1, 2 , R. K. Tiwary1 , Kumar Birendra1 and P. Padma2 * 1

Central Institute of Mining and Fuel Research (CSIR), Dhanbad, Jharkhand, India School of Bioscience and technology, Vellore Institute of Technology, Vellore, Tamil Nadu, India . * For correspondence: [email protected] 2

Article Info: Received 19 June 2014; Revised: 24 July 2014; Accepted 19 Aug 2014

ABSTRACT Sukinda Chromite Valley, of Orissa, contains 95% of India’s Chromite ore. The Over Burden (OB) material contain approx. 30-40 thousand ppm of Cr (VI) which, after leaching, enters into the ground water regime and contaminate ground water with respect to Cr (VI). Therefore OB dump is the major source of Cr (VI) which by may be removed by microbial reduction which is most effective and economical. Our objective involves isolating and enriching Cr (VI) reducing microbes for the biological treatment of Cr (VI) from Chromite waste dump and evolving optimal strategy for cost-effective remediation of large scale Cr (VI) contaminated OB dump sites. 25 types of bacterial cultures have been isolated from the Chromite OB waste dump soil. Among them, seven bacterial strains were capable to tolerate 100 mg/l and 50 mg/l of Cr (III) and Cr (VI) respectively are considered as chromium resistant. Nutrient agar is capable of reducing Cr (VI) to Cr (III). At about 50 mg/l initial Cr (VI) concentration, 99% reduction has been achieved by nutrient agar within 12 hours of incubation without any bacterial culture. Due to this reason peptone agar was used to check chromium (VI) resistant capability of strains. The effects of different operating parameters such as pH (9-4 pH), temperature (15, 30 and 45°C) and Cr (VI) concentrations on bio-reduction of Cr (VI) by enriched cultures were also studied in a batch system. All these experiments have been performed on artificial soil (10 ppm Cr (VI), Chromite OB waste dump soil and aqua-phase (standard chromium (VI) solution All these experiments have been performed on artificial soil (10 ppm Cr (VI), Chromite OB waste dump soil and aqua-phase (standard chromium (VI) solution). Keywords: Chromite ore, Bioremediation, Bioreduction.

1. INTRODUCTION India continues to rank first in the world in chromite production. Around 95% of country chromium ore is produced from Sukinda chromite mining area. Chromite is very much vital and strategic material for India. It is indispensable for the development of the nation. Total recoverable reserves of chromite ore in India as on 2005 are estimated at 213 million tones,

comprising of 66 million tones reserves (31%) and 147 million tones remaining resources (69%). More than 95% resources of chromite were located in Orissa, mostly in Sukinda valley in Jajpur districts and Dhenkanal as well as Keonjhar district. Grade wise, charge-chrome grade accounts for 26% resources followed by ferro-chrome grade (20% 109

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each) and refractory grade 2 percent. Low grade, unclassified and some unknown grades together account for 32 percent [1]. Management of waste dump in Sukinda valley is the major environmental concern. These overburden dumps modify the land topography affecting the drainage system; prevent natural succession of plant growth resulting in acute problems of soil erosion and environmental pollution. Normally, waste dumps are maintained up to the height of 20-30 m with 30 m terrace width and slope angle of dump 25 to 350. Mine water discharged from open cast mines generally contain hexavalent chromium and contaminate the surface and ground water. Runoff water from OB dump and seepage through OB dump generally contain high level of Cr (VI), which flows into the nearby surface water and also may seep into ground water. OB material generally contains 5 to 9 % chromite, which may get dissolve during rainy season and contaminate the ground water through leaching and also contaminate the surface water through surface runoff. Due to friable nature of soil large gully erosion are observed on the dumps and Sukinda area also faces high rainfall (1200 mm) with high intensity. Large amount of OB dumps are washed away with rainwater, get sedimented at the bottom of the river, and ultimately increases the level of hexavalent chromium in surface water [2]. Chromium enters the environment, and persist in its most stable oxidation states, Cr (III) and Cr (VI). Trivalent form of chromium is relatively innocuous and immobile, while Cr (VI) moves readily through soils and Aquatic environment. Hexavalent compounds are acute toxic, mutagenic [3, 4], teratogenic [5] and carcinogenic [6, 7, 8]. Chromium (Cr+6) is most toxic water pollutant. The value above 0.05 in drinking water may pose detrimental to human health. In higher concentration of Cr+6 may produce diseases like a corrosive action on the skin and mucous membrane. It is a strong oxidizing agent capable of being absorbed through the skin [9]; it is also an irritant to the plant and animal. In contrast, trivalent compounds have relatively low toxicity and are immobile under moderately alkaline to slightly acidic conditions. Chromium and its compounds are known to cause cancer of the lungs, nasal cavity, and paranasals sinus and suspected of causing cancer of the stomach and larynx. The characteristic lesion is a deep penetrating ulcer, which for the most part, does not lead to suppurate and slow in healing. Skin decoloration and peptic ulcer is a common disease in the inhabitants of the area.

Heavy metals exhibit toxic effects on soil biota, and they can affect key microbial processes and decrease the number and activity of soil microorganisms. Microbial population has often been proposed to be an easy and sensitive indicator of anthropogenic effects on soil ecology. Cr (VI) causes detrimental effects on microbial cell metabolism at high concentrations and it has been to cause mutation in the soil microbial populations. The search for Cr6+ reducing microorganisms (both aerobic and anaerobic) has been enthusiastically pursued, with numerous strains being isolated. Reductases enzyme produce from aerobic bacteria and the enzyme involved in Cr (VI) reduction occurring under anaerobic conditions. Biological processes for treating chromium-contaminated sites are becoming very promising. Some of the emerging technologies for the remediation of Cr (VI) include microbial strategies for in situ and on-site bioremediation strategies. Isolation of microorganisms capable of reducing Cr (VI) to Cr (III) has significant potential in the development of in situ or on-site bioremediation strategies. Several other CrO4- reducing strains have been reported by Russian scientists Romanenko and Korenkov, including other strains such as Bacillus cereus, Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas ambigua, Pseudomonas fluorescens, Escherichia coli, Achromobacter eurydice, Micrococcus roseus, Enterobacter cloacae, Desulfovibrio desulfuricans and Desulfovibrio vulgaris. There has been an increasing demand for chromite ores in recent years. This has resulted into increase in chromite ore production. In the opencast mining process the chromite ore as well as the waste rock material are dumped in the open ground without considering the environmental aspects. The ore OB ratio generally vary in this area is 1:3-8.0 and therefore produces 5 to 8 times waste material to the ore produced. The OB around the mine damaging the topography of the area and it may also allow leaching of hexavalent chromium and other impurities to the groundwater as well as surface water sources.

2.

MATERIALS AND METHOD

2.1. Sample Collection Sites In the Sukinda mining area OB dump cause Cr (VI) contamination. In rainy season, Cr (VI) has been leaches out from soil and mixed with ground water and other water bodies. This soil may contain different types of Cr (VI) reducing microbes. Due to all these reasons, soil and samples have been

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collected from TISCO, OB-dump and OMC, OBdump during January month. 2.2. Hexavalent Chromium Estimation Methods 80 ml of chromium solution was taken in 100 ml volumetric flask. 0.25 ml or 5 drops of H3 PO4 was added to the above solution followed by 2 ml of 0.2N H2 SO4 , and pH was adjusted to 2.2 ml of Diphenylcarbazide solution was added and mixed well. The volume was made up to 100 ml by adding double distilled water and the solution was left for 510 min for full colour development and measures its absorbance at 540 nm [10, 11]. 2.3. Leaching Study of Soil and Determination of Soil Texture Dry soil sample was collected from Chromite waste OB dump area. 50 g of soil sample was weighed. Soil slurry was prepared by mixing soil and water in the ratio of 1:5 in 500 ml beaker. The contents of slurry were mixed properly and soil slurry was shaken for 5 hours at 120-180 rpm. After 5 hours shaking soil slurry was filtered with 0.45 µm Whatsman filter paper. Filtrate was used for estimation of Cr (VI) by using Diphenylcarbazide method. Dry 100 gm OB dump soil sample was taken. Then the soil particles are passed through sieve with 2mm sieve that removes gravel then the remaining soil is sieved with appropriate sieves to separate silt and clay. Then the each separated fraction was weighed. Soil texture was reported on percentage basis. [10, 11] 2.4. Estimation of Total Organic Carbon (Toc) In Soil 500 mg of soil was taken in 500 ml conical flask and 20 ml of concentrated H2 SO4 was added to it followed by 10 ml of 1N K2 Cr2 O7 solution. The solution was left for 30 minutes to stand. The volume was made up to 200 ml by adding 170 ml of double distilled water. 10 ml of 85% ortho-phosphoric acid and 250 mg sodium fluorite was added to the above solution and was mixed properly. 1.5 ml of Diphenyl-carbazide indicator was added to the solution. The contents of the flas k were changed to bluish brown colour. A control was taken without the soil sample (with only reagents). The bluish brown colour solution was titrated with 0.5 N FAS until brilliant green colour was appeared [10, 11]. 2.5. Isolation of Cr (VI) Resistant and Reduction Potential Microorganism

By serial dilution and spread plate method has been used for isolation of microorganisms. Samples were serially diluted up to 10-7 dilution. Peptone agar medium, spiked with 1 ppm (mg/L) of Cr (VI) was prepared and sterilized carefully. All the tubes were incubated at 30-37°C for 24-48 hours. After incubation developed colonies were isolated and pure cultures were prepared to s creen most potential organisms [11, 12]. 2.6. Different pH stress The pure cultures obtained from the contaminated site were evaluated for their pH level. Further studies were conducted using the most promising bacterial strain emerging from the screening test. For the screening test, loop full of the pre-grown bacterial pure culture isolated from different locations was inoculated in nutrient agar slant with different pH condition and incubated for 24-48 hours at 30-37° C. A control (all conditions were same except that the bacterial cells were not added) was employed to quantify the contamination in all the experiments. A loop full of culture from the above slant was streaked on nutrient agar slants, incubated for 24 h and stored in a freezer at 4° C for further use [11, 12]. 2.7. Screening of the pure cultures which are able to resist high concentration of Cr (III) and Cr (VI) For the screening test, loop full of the pre-grown bacterial pure culture isolated from different locations was inoculate in peptone agar with different Cr (III) and (VI) concentration and incubated for 24-48 hours at 30-37° C. A control (all conditions were same except that the bacterial cells were not added) was employed to quantify the resistance in organisms. [11, 12]. 2.8. Characterization of Microorganisms Grams staining, motility test, IMViC test, Urea hydrolysis, TSI, catalase test, oxidase test, nitrate reduction test performed to identify microorganism [11, 12]. 2.9. Stress 2.9.1. Stress in Different Cr (Iii) and Cr (Vi) Concentration (In Aerobic Condition) For the stress condition, loop full of the pre-grown bacterial pure culture isolated by screening test inoculated in peptone agar with different Cr (III) and Cr (VI) concentration at pH 6, culture was incubated for 24-48 hours at 30-37° C, and the bacterial growth was checked on agar slant after 24-48 hours. Loop

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full of culture from pure culture inoculated to peptone agar containing 1mg/L of Cr (III) and Cr (VI). This procedure repeated by progressively increasing Cr (III) and Cr (VI) in the peptone agar up to 100 mg/L and 50 mg/L respectively. 2.9.2. Stress in Different Temperature (15° to 45° C) Loop full of culture from pure culture was inoculated to peptone agar and incubates it at 15° C, 35° C and 45° C. A control (all conditions were same except that the bacterial cells were not added) was employed to quantify the contamination in all the experiments. A loop full of culture from the above slant was streaked on nutrient agar slants, incubated for 24 h and stored in a freezer at 4° C for further use. 2.9.3. Stress in Different pH (4 to 9 pH) Loop full of culture from pure culture inoculated to peptone agar containing pH 9. This procedure repeated by progressively decrease pH up to pH 4. A control (all conditions were same except that the bacterial cells were not added) was employed to quantify the contamination in all the experiments. A loop full of culture from the above slant was streaked on nutrient agar slants, incubated for 24 h and stored in a freezer at 4° C for further use. 2.9.4. Stress in Concentration

different

pH And

Cr (Vi)

Loop full of culture from pure culture inoculated to peptone agar containing 1 mg/L of Cr (VI) and pH 7. Progressively increasing Cr (VI) concentration and decrease pH in the peptone agar up to 50 mg/L and pH 4 respectively repeated this procedure. 2.10. Microbial Reduction of Cr (Vi) For screening of electron donors, the most promising microorganism (those which gave max specific Cr (VI) resistant during screening test) from the enriched cultures was employed. The studies carried out in aerobic conditions. Culture incubated for five days at 30°C with 12 hours shaking and 12 hours stationary condition. Incubated cultures were harvested by centrifuging at 10000×g for 10 min and then transferred to 80 mL of centrifuged peptone broth in 100 mL measuring flasks. Now Cr (VI) has estimated by using Di-phenyl-carbazide method [13].

3. RESULTS AND DISCUSSION 3.1. Culture Media Selection

Peptone agar has been used in place of Nutrient agar because at the time of experiment it has been found that those components, which are present in nutrient agar like peptone, beef extract, and NaCl, have the capability to reduce Cr (VI) to Cr (III). The following test were performed to find out which types of reaction are undergoing in the nutrient broth shown in Table 1, Cr (VI) has been estimated by Di-phenylcarbazide method. Components of nutrient agar like peptone, beef extract and NaCl they have the capability to reduce Cr (VI) to Cr (III). Experiment no 1, 2, 3 has been used to observe the individual effect of peptone, beef extract and NaCl on Cr (VI). In these experiments reduction was not observed, means initial and final Cr (VI) concentration was same. However, in experiment no 4 and 5 peptone and beef extract was mixed with NaCl individually. In these experiments, reduction process was observed and up to 25ppm of Cr (VI) was totally reduced. In the last experiment no 6 peptone, beef extract and NaCl were mixed. In this experiment reduction process was observed and up to 50ppm of Cr (VI) was totally reduced. Krishna and Philip, (2005) reported that the effective Cr(VI) reduction increase with the electron donor concentration increased, afterwards it remained as a constant. 3.2. Leaching Study of Soil and Determination of Soil pH The pH of the soil samples collected from Tata mines OB dump Soil and OMC mines OB dump Soil was 7.77 and 3.61 respectively (Table 3). Soil pH is an important consideration for reduction of Cr (VI) to Cr (III). Bioremediation strategy for remediation of Cr (VI) contaminated soil/aquifer involves detoxification of Cr (VI) by reducing it to Cr (III). Cr (III) forms insoluble Cr (OH)3 in the pH range of 6– 9, severely restricting its ability to migrate through groundwater. pH can affect to availability of nutrient in soil. Nutrient availability also affects the growth of microbes and plant growth. If nutrients available for microbes or plants so they easily reduced or absorbed Cr (VI) from soil. Reduction of Cr (VI) is depend on pH, acidic pH increase the Cr (VI) solubility whereas basic pH decrease. Soluble Cr (VI) bio-reduction at low pH is high, because Cr (VI) freely available for microbes and increase in pH up to10 decreases the extraction of Cr (VI) up to 2.5% [14]. 3.3. Determination of Soil Texture The texture of a soil depends on the percentage of sand, silt, and clay in it. Based on this yardstick soils are designation as clay, sand clay, clay loam, silty clay, silty clay loam, sandy loam, silty loam, loamy

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sand, sand, silt. Soil particle occupy roughly more than half the space in soil. The remaining space between the particles called the pore size is occupied by water and air. Which helps to microorganisms grow in soil. The soil texture determined as follow in Table 2. 3.4. Estimation of Total Organic Carbon (TOC) in Soil A soil organic carbon compound refers to the amount of carbon stored in the soil. Soil organic includes polysaccharides and other organic complex compounds. 20-50% less organic carbon in the Chromite waste OB dump soil samples compare to normal forest soil. Due to open cast mining, all forest has been cut out and for management of OB dumps other forest also has been cut out. Due to absence of forest, there are no other sources for organic carbon in soil. Less amount of organic carbon in soil did not support to growth of microorganisms and plants. By the experiment soil pH value of soil s amples were shown in Table 3. 3.5. Enumeration of the Cr (Vi) Reducing Bacterial Strains Bacteria present in Cr (VI) contaminated soil sample has been counted. Samples were collected from Chromite waste OB dump area. Number of colonies present in soil was counted as follow in Table 4. Chromite waste OB dump soil is the region in the vicinity of Cr (VI). It can be distinguish many microorganism habitats. Chromite waste is characterized by grater amount of Cr (VI) and less amount of microbiological activity than the soil have small amount of Cr (VI). Less amount of micro flora is present in Chromite waste due to less nutrient content. Because of the above reasons, lesser microbial load observed in the Chromite waste OB dump soil sample [15]. 3.6. Isolation and Screening of Cr (VI) Resistant and Reduction Potential Microorganism After 24-48 hours peptone agar plates that contain pH 5 and 1ppm, concentration of Cr (VI) was observed. Total 25 types of colonies were able to grow in peptone agar plates. Out of 25 types of colonies, some colonies were aerobic and some are micro-aerophilic in nature. Some colonies were transparent. Some colonies were yellowish, whitish and some were pure white in colour. Some were so tuff and some were gummy types of colonies. Some colonies were small or medium rounded and some were irregular in shape. These 25 colonies were isolated. A loop full of culture from the above slant

was streaked on nutrient agar slants, incubated for 24 h and stored in a freezer at 4° C for further use. All the bacterial colonies were able to grow in peptone agar plates contains 1ppm concentration of Cr (VI). For normal cells up to 0.05 ppm of Cr (VI) is maximum tolerant limit above this Cr (VI) act as a toxic substance. That is why all the bacteria which are able to resist high level of Cr (VI) they can only able to grow in 1ppm of Cr (VI). These bacteria may have special mechanism to resist towards Cr (VI) [16]. They may have able to reduce Cr (VI) to Cr (III), or their cell wall and capsule was able to resist Cr (VI). Due to all reasons, bacteria were able to grow in high concentration of Cr (VI). These colonies were also able to resist low pH level. 3.7. Stress In the Cr (III) and Cr (VI), stress condition at 1 ppm all 25 types of organisms were show good growth. However, when progressively increasing Cr (III) and Cr (VI) in the peptone agar up to 100 mg/L and 50 mg/L respectively that time out of 25 only 12 and 10 organisms were show good growth respectively. In the pH stress condition at pH 7 all 25 types of organisms were show good growth. However, when progressively decrease pH up to pH 4 out of 25 only 6 organisms were show good growth. In the Cr (III) and Cr (VI), stress condition at 1ppm and pH 6 and pH 5 that time all 25 types of organisms were show good growth. However, when progressively increasing Cr (III) and Cr (VI) in the peptone agar up to 100 mg/L and 50mg/L respectively at pH 6 that time out of 25 only 12 and 10 organisms were show good growth respectively. However, when progressively increasing Cr (III) and Cr (VI) in the peptone agar up to 100 mg/L and 50 mg/L respectively at pH 5 that time out of 25 only 5 and 8 organisms were show good growth respectively. All the bacterial colonies were able to grow in nutrient agar and peptone agar slant contains 1ppm-100 ppm concentration of Cr (III) and 1ppm50ppm concentration of Cr (VI) respectively. For normal cells up to 0.05 ppm of Cr (VI) is maximum tolerant limit above this Cr (VI) act as a toxic substance. Hence they are able to resist high level of Cr (III) and Cr (VI). These bacteria may have special mechanism to resist towards Cr (III) and Cr (VI). They may have able to reduce Cr (VI) to Cr (III), or their cell wall was able to resist Cr (VI) [17, 18]. Due to all reasons, bacteria were able to grow in high concentration of Cr (VI). These colonies were also able to resist low pH level. These cultures were able to grow in pH 9 to pH 4. Few species are having Cr (VI) reducing ability and able to resist Cr (VI) from 20 to 50 ppm.

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Table 1. Nutrient agar effect on Chromium (VI)

Experiment No.

Peptone (in gm)

Beef extract (in gm)

NaCl (in gm)

Amount of 500 ppm K 2Cr2O 7 (in ml)

Initial concentration of Cr (VI) (in ppm)

Double Distilled water (in ml)

Total volume of broth (in ml)

Autoclave

Colour Development

With autocalve With autocalve With autocalve With autocalve With autocalve With or without autoclave

Colour developed Colour developed Colour developed No Colour developed No Colour developed No Colour developed

1.

0.5g

0.0g

0.0g

1ml

5ppm

99ml

100ml

2.

0.0g

0.3g

0.0g

1ml

5ppm

99ml

100ml

3.

0.0g

0.0g

0.5g

1ml

5ppm

99ml

100ml

4.

0.5g

0.0g

0.5g

0.0g

0.3g

0.5g

6.

0.5g

0.3g

0.5g

5ppm25ppm 5ppm25ppm 5ppm50ppm

99ml 95ml 99ml 95ml 99ml 90 ml

100ml

5.

1ml5ml 1ml5ml 1ml10ml

100ml 100ml

Final Cr (VI) concentration in broth estimate by Diphenylcarbazide method 5ppm broth 5ppm broth 5ppm broth 0ppm broth 0ppm broth 0ppm broth

in 100ml in 100ml in 100ml in 100ml in 100ml in 100ml

Table 2. Soil Texture Sampling site

Total soil (in gm)

Stones (in gm)

Sand (in gm)

Silt (in gm)

Clay (in gm)

TISCO

100g

29.17g

35.74

16.23

18.94

OMC

100g

44.48g

24.14

12.96

19.1

Table 3. Soil Total Organic Compound (TOC) Sampling site

TISCO

OM C

TOC (in %)

0.58%

0.72%

pH

7.7

3.61

Table 4. Enumeration of microbes S. No.

1.

2.

Cfu/gm

Soil samples

TISCO, OB-dump

OM C, OB-dump

1. 2. 3.

10-1 Dilution: - 0.6×10-1 Cfu/gm 10-2 Dilution: - 0.3×10-2 Cfu/gm 10-3 Dilution: - 0.1×10-3 Cfu/gm

1. 2. 3.

10-1 Dilution: - 0.6×10-1 Cfu/gm 10-2 Dilution: - 0.4×10-2 Cfu/gm 10-3 Dilution: - 0.4×10-3 Cfu/gm 114

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3.8. Microbial Reduction of Cr (Vi) From Aqueous Phase Condition Five days incubated peptone broth contains 5ppm of Cr (VI) at pH 6 with microbes have ability to reduce Cr (VI) to Cr (III). They can able to reduced Cr (VI) from 5ppm to 0.1 ppm. Five days incubated dextrose broth contains 5 ppm of Cr (VI) at pH 6 with microbes have ability to reduce Cr (VI) to Cr (III). They can able to reduced Cr (VI) from 5ppm to 2.0ppm. So in the comparative study peptone was better than dextrose. Results were show in Figure 1 and 2. at 0th hour

at 72th hour

at 120th hour

pH

10

reduction

5

8

4. CONCLUSION

6

Hexavalent chromium is a highly toxic pollutant introduced into natural water sources due to discharge of mine waste water. Cr (VI) contamination is in their water bodies that has affected their health drastically. The present investigation attempted to identify organism which has high resistance to Cr (VI) and has significant capability to completely reduce Cr (VI) in the water bodies of Sukinda area. Soil sample collected from Sukinda mining area shows the presence of 0.7-2.1 mg/Kg Cr (VI) after 5 hour shaking with distilled water. Water analysis also indicates high level of Cr (VI) in the range 0.247 to 0.990 mg/l in mine water which is many folds higher than its permissible limit (0.05 mg/l). Out of 25 bacterial cultures, seven Cr (VI) resistant species have been isolated from the soil samples which can reduce Cr (VI) to Cr (III). High productivity of bacterial culture reduces Cr (VI) in presence of peptone as nitrogen source. Similarly in presence of Dextrose as sole Carbon source maximum biomass growth could be obtained. But it is less effective than peptone.

4

pH

Cr (VI) concentration (mg/l)

Figure 2. Dextrose used as an electron donor for Cr (VI) 6

3 4

2

2

1 0

0 MO 6

MO 19

MO 25

MO 26

Control

Micro-organism

Figure 1. Peptone used as an electron donor for Cr (VI) reduction

3.9. Microbial Reduction of Cr (VI) From Soil Five days incubated Cr (VI) contaminated artificial soil and OB dump soils with peptone broth at pH 6 with microbes have ability to reduce Cr (VI) to Cr (III). These microbes can able to reduced Cr (VI) from 2 ppm to 0.01 ppm in artificial soil but it was less effective in OB dump soil to reduce Cr (VI). These microbes did not able to reduce Cr (VI) directly from soil. Microbes only reduce that Cr (VI) which can leach out from soil. In the case of artificial soil Cr (VI) was only adsorbed by soil surface only, so it can release Cr (VI) very easily from soil to water. However, in the OB dump soil Cr (VI) was not adsorbed on soil, it is present in form of clumps or bounded form so it can release Cr (VI) very slowly. That is why it shows less reduction in OB dump soil. Jeyasingh et al., (2010) and Mistry et al., (2010) also reported compare to chemical reducing methods microbes can reduce up to 92% of Cr (VI) [19, 20].

Conflict of Interest The authors declare that they have no conflicts of interest

Acknowledgement The valuable support of Director and Department of Water Environment, Central Institute of Mining and Fuel Research (CSIR), Dhanbad, Jharkhand, India and Department of Microbiology, VIT University, Vellore, Tamil Nadu, India are greatly acknowledged.

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