Hexavalent Chromium Bioremoval through Adaptation and Consortia

Article pubs.acs.org/IECR

Hexavalent Chromium Bioremoval through Adaptation and Consortia Development from Sukinda Chromite Mine Isolates Jastin Samuel, Madona L Paul, Mrudula Pulimi, M Joyce Nirmala, Natarajan Chandrasekaran, and Amitava Mukherjee* Centre for Nanobiotechnology, VIT University, Vellore, Tamil nadu, India ABSTRACT: Indigenous isolates from the waters of chromite mining sites at Sukinda Valley, Orissa, India, showed a considerable enhancement in Cr (VI) bioreduction rate through adaptation and consortia development. On the basis of 16S-rRNA sequencing, these isolates were identified as Bacillus subtilis VITSUKMW1, Acinetobacter junii VITSUKMW2, and Escherichia coli VITSUKMW3. The native isolates showed a high tolerance at 500−1000 mg L−1 of Cr (VI). An increase in the reduction rate from 0.199−0.477 mg L−1 h−1 to 0.5−1.16 mg L−1 h−1 at 5−20 mg L−1 of initial Cr (VI) concentration was achieved by the adapted isolates. An increase in the growth rate and Cr (VI) reduction rate [0.86−2.6813 mg L−1 h−1 at 5−100 mg L−1 of initial Cr (VI) concentration] was observed in the ternary consortium of adapted isolates. The FT-IR spectra revealed the active participation of the bacterial surface groups in the reduction. The development of sequential processes (native → adapted → consortia) employing Cr (VI) tolerant isolates, proves to be a potential bioremediation strategy for specific chromite mine sites. reducing it to a less toxic Cr (III).10−14 The extent of hexavalent chromium reduction and the mechanisms involved are variable and depend on several factors including the source from which the species are isolated.15 The existence of chromiumresistant bacteria capable of reducing chromate had been reported from chromium polluted environments in and around Sukinda.16,17 An isolate from the Sukinda mine soil identified as Brevibacterium casie was reported to be Cr (VI) tolerant.5 The isolation of resistant microbial species from the contaminated site has become a prerequisite for in situ or on-site bioremediation processes of Cr (VI) detoxification. There is an overall lack of information on the Cr (VI) reduction studies using site specific microbial isolates from the mine waters of Sukinda, India. Moreover, to the best of our knowledge, the possible strategy for enhancing bioremediation potential through adaptation and consortia development of chromium tolerant indigenous bacterial population present in the mine water was not explored in detail so far. The current study is an initiative to develop a site specific bioremediation process for the polluted mine in Sukinda, India, which may well be contextually applied to other hexavalent chromium contaminated sites.

1. INTRODUCTION Metal mining imposes a major threat to water bodies and related ecosystems through the discharge of heavy metals and the drainage of water from underground and open pit mines.1 In the last few decades, the amount of chromium in both aquatic and terrestrial ecosystems has increased as a consequence of anthropogenic activities.2,3 The oxidation state of chromium determines its level of toxicity. Cr (VI) is highly soluble, thus mobile and biologically available in the ecosystem. In contrast, Cr (III) displays a high affinity for organics resulting in the formation of complexes that precipitate as amorphous hydroxides. Because of its persistence in the environment, anthropogenic release of Cr (VI) is a matter of environmental concern. The United States Environmental Protection Agency (US EPA) had declared chromium as one of the greatest threats to humans.4 The permissible limit of hexavalent chromium in drinking water is 0.05 mg L−1. The Sukinda Valley of Jajpur district, Orissa, India, comprises about 98% of India’s chromite ore reserve. The mining activities in this region generate around 7.6 million tonnes of solid wastes in the form of rejected minerals, overburden materials/waste rock, and subgrade ore.5 Because of the leaching of these solid wastes, the water bodies in and around the mines are contaminated with chromium. The open cast mining of chromite ore from the deposits of Sukinda is one of the most polluting activities mainly because of the natural oxidation of chromite from the overburden dumps, which affects the nearby areas with soluble Cr (VI) species. The conventional environmental remediation of Cr (VI) involves an extensive use of chemical processes that are expensive and often generate other harmful byproducts requiring further treatment.6−8 However, applying plants and microbes for remediation is preferred because of their cost-effectiveness, environmental friendliness, and fewer side-effects.9 In recent years, a number of studies had been carried out using aerobic and anaerobic bacteria for the detoxification of Cr (VI) by © 2012 American Chemical Society

2. MATERIALS AND METHODS 2.1. Sample Collection and Characterization. The mine water from a chromite mine in Sukinda Valley of Jajpur district, Orissa, India, was collected in sterile polypropylene bottles and stored at 4 °C until used for microbiological analysis. The water samples were analyzed for pH, total dissolved solids, total suspended solids, conductivity, and dissolved oxygen. The chemical analyses were conducted to quantify phosphate, chloride, nitrate, Received: Revised: Accepted: Published: 3740

August 16, 2011 February 10, 2012 February 15, 2012 February 15, 2012 dx.doi.org/10.1021/ie201796s | Ind. Eng. Chem. Res. 2012, 51, 3740−3749

Industrial & Engineering Chemistry Research

Article

measuring the dry weight of biomass with a known viable cell concentration (CFU/mL) during the exponential growth phase. 2.7. Adaptation. To enhance the Cr (VI) reduction rate and the growth rate of bacteria, each isolate was adapted serially at 10, 20, 30, 50, 80, and 100 mg L−1 of Cr (VI). The period for sequential adaptation varied with increasing concentration of Cr (VI). The adaptation was carried out until the indigenous isolates showed a maximum growth similar to the growth observed in the control medium (nutrient broth without Cr (VI)) at each Cr (VI) concentration. The adapted isolates were maintained at 100 mg L−1 of Cr (VI). 2.8. Antagonistic/Synergistic Assay and Development of Consortia. To achieve a higher reduction rate of Cr (VI), consortia of the adapted isolates were developed based on the antagonistic/synergistic assay. Two isolates were taken and grown in nutrient broth supplemented with 100 mg L−1 of Cr (VI) separately. After 4 h of incubation, 100 μL of one culture broth was poured onto the surface of nutrient agar supplemented with 100 mg L−1 of Cr (VI), and a loop full culture of the other isolate was streaked in the middle of the plate. It was incubated at 37 °C for 24 h, and then the plate was observed. The zone of inhibition between the two isolates was absent for all adapted isolates, which showed that they lacked competitive inhibition. This showed that they have synergistic effect which is very important for the development of consortia. Three binary consortia and one ternary consortium were developed. The growth and Cr (VI) reduction studies were carried out at 5, 10, 20, 50, and 100 mg L−1 initial concentrations of Cr (VI). 2.9. Reduction Studies. The Cr (VI) stock solution was prepared in sterilized distilled water and then the filter was sterilized with 0.45 μm Whatman syringe filter. The medium was supplemented with different initial Cr (VI) concentrations of 5, 10, and 20 mg L−1 for unadapted isolates, and 5, 10, 20, 50, and 100 mg L−1 for adapted isolates and consortia. The experiments were carried out in separate flasks for each interval, and all the tests were done in duplicates. All the experiments were carried out at a pH of 7.5. The initial inoculum containing 1.5 × 108 per milliliter of bacterial cells was added to each flask. The nutrient broth without the bacterial culture was maintained as a control.

and sulfate by the titrimetry method. The soluble metal content in the mine water was analyzed by inductively coupled plasma− optical emission spectroscopy (PerkinElmer Life and Analytical Sciences, Shelton, CT, USA) following the standard methods of the American Public Health Association.18 2.2. Isolation and Culture Conditions. To isolate the hexavalent chromium resistant bacteria, 1 mL of the mine water sample was serially diluted in sterile distilled water and plated on M9 minimal medium agar plates containing 10 mg L−1 of Cr (VI) supplemented as K2Cr2O7. The M9 minimal medium is composed of M9 salts (Na2HPO4.7H2O, KH2PO4, NaCl, NH4Cl) MgSO4, CaCl2, and glucose.11,19 The bacterial colonies were observed after 24 h of incubation at 37 °C. The isolated colonies were plated onto nutrient agar medium and the pure culture of each isolate was obtained. 2.3. Identification of Isolate. The morphological (shape, size, gram reaction, motility), cultural (nutrient agar), and biochemical characterization of the microbial isolates were undertaken for identification. The biochemical test was carried out using the Himedia kit (KB003). The taxonomic identity of the strains was confirmed by 16S-rRNA gene sequencing. The total genomic DNA of the bacterial strain grown in nutrient broth were extracted by the phenol−chloroform method.20 The 16S-rRNA nucleotide sequences were identified by the fluorescent dye terminator method (ABI Prism Big dye terminator cycle sequencing ready reaction kit v.3.1). The data obtained were compared to the sequences in the NCBI GenBank database using BLAST (basic local alignment search tool) at NCBI server (http://www.ncbi.nlm.nih.gov/). The bacterial nucleotide sequences were aligned by using the CLUSTALX program and the phylogenetic tree was constructed by the neighborjoining method. 2.4. Microbial Tolerance to Hexavalent Chromium. The maximum tolerable concentration (MTC) of the indigenous isolates were determined by the well diffusion method in PYG (peptone yeast glucose) medium with Cr (VI) concentrations ranging from 10 to 1000 mg L−1. The maximum concentration of Cr (VI) in the medium which supported the growth of organisms was taken as the MTC (maximum tolerable concentration).21,22 2.5. Microbial Growth. The growth of the bacterial isolates was studied in nutrient broth with and without Cr (VI) in the medium. A volume of 100 mL of nutrient broth in 250 mL Erlenmeyer flask was sterilized and adjusted to an initial pH 7.5. The growth study was carried out at 30 °C. The growth was measured in terms of optical density at 600 nm. The specific growth rates were calculated from the microbial growth data obtained from various batch experiments. 2.6. Viable Cell Count and Dry Weight of Biomass. The pour plate and the colony counting methods were used to estimate the number of viable cells with the colonies grown on nutrient broth and plate count agar. The samples were withdrawn at regular intervals from the experimental batches and were added to the agar plate. The bacterial count measured after 24 h incubation period was reported as colony forming units (CFU) per milliliter of the sample. The dry weight of biomass per liter of media was obtained to calculate the reduction capacity of the bacterial cells. The cells grown in the nutrient broth were withdrawn by sterile pipet and centrifuged at 12000g for 10 min at 2 °C. The pellet was separated and collected in a dry watch glass. It was dried in an oven at 105 °C. To correlate the CFU count to the biomass concentration, a direct conversion factor was determined by

Table 1. Physico-chemical Analyses of Mine Water Samples of Chromite Mine, Sukinda, Orissa

a

3741

parameters

values

pH TDS TSS Ec D.O phosphate (PO4) nitrate (NO3) chloride (Cl2) sulfate (SO4) copper (Cu) ferrous (Fe) nickel (Ni) lead (Pb) sulfide (S) Cr (VI)

6.7 ± 0.150 130 ± 1.700 mg L−1 19 ± 0.450 mg L−1 0.439 ± 0.007 mS 1.80 ± 0.060 mg L−1 2.43 ± 0.050 mg L−1 3.92 ± 0.070 mg L−1 17.14 ± 0.300 mg L−1 22.3 ± 0.420 mg L−1 0.2 ± 0.050 mg L−1 6.655 ± 0.100 mg L−1 0.145 ± 0.003 mg L−1 BDLa BDLa 0.23 ± 0.002 mg L−1

Below detection limit. dx.doi.org/10.1021/ie201796s | Ind. Eng. Chem. Res. 2012, 51, 3740−3749


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Figure 1. 16S-rRNA gene sequencing: phylogenetic tree and SEM image of B. subtilis VITSUKMW1, A. junii VITSUKMW2, and E. coli VITSUKMW3 at 40 000× magnification.

reduction kinetics for chromium reduction was conducted. Each set of experiments were carried out in triplicate to ensure reproducibility, and the standard error was calculated. 2.10. Estimation of Chromium. A colorimetric method was employed to analyze the concentration of Cr (VI) in the supernatant. The samples were acidified with 0.2 N H2SO4 and reacted with 1,5-diphenylcarbazide to produce a purple color. The measurement was carried out at a wavelength of 540 nm.23 Total Cr was measured at a wavelength of 359.9 nm using a flame atomic adsorption spectrophotometer (AAnalyst400/ HGA 900, PerkinElmer Life and Analytical Sciences, Shelton, CT, USA) equipped with 35 mA chromium hollow cathode lamp with a detection limit of 0.03 mg L−1 and a sensitivity of 0.1 mg L−1. Before analysis using AAS, the samples were acidified by 1 N HNO3 to dissolve the chromium hydroxide precipitates and to extract the adsorbed Cr (VI). The Cr (III) concentration was determined as the difference between the total Cr and Cr (VI) concentration. 2.11. Reduction Capacity. The Cr (VI) reduction capacity of the bacterial cells was determined as the concentration of Cr (VI) reduced per amount of viable bacterial cells inactivated during incubation, which is given by the following equation.24

Table 2. Specific Growth Rate, Reduction Rate, And Time Taken to Reduce 80% of the Initial Cr (VI) Concentration (5, 10, 20 mg L−1) by Un-adapted Isolates at pH 7.5 and Maintained at 30 °Ca

Maximum specific growth rate was observed at 20 mg L−1 of initial Cr (VI) concentration by B. subtilis VITSUKMW1. The highest reduction rate was at 5 mg L−1 initial Cr (VI) concentration by A. junii VITSUKMW2. a

Rc =

Co − C Xo − X

(1)

where Rc = Cr (VI) reduction capacity (mg Cr (VI) removed/ mg cells), Co = initial Cr (VI) concentration (mg L−1), C = Cr (VI) concentration at the time of incubation t, Xo = initial viable cell concentration (mg L−1) and X = cell concentration at the time of incubation t. A viable cell conversion factor was

To measure the reduction of Cr (VI) by bacterial cells, the culture from each flask was harvested and centrifuged (12000g for 10 min at 2 °C). The supernatant was separated and analyzed for Cr (VI) concentration by 1,5-diphenylcarbazide (DPC). The 3742

dx.doi.org/10.1021/ie201796s | Ind. Eng. Chem. Res. 2012, 51, 3740−3749


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with 20 mg L−1 of Cr (VI) and adapted interacted with 100 mg L−1 of Cr (VI)) was mixed with 100 mg KBr and the fine powdered mixture was then pressed in a mechanical die press to form a pellet by applying a pressure of 1200 psi for about 5 min. The transparent tablets were inserted in the instrument and the spectra were recorded from 4000 to 400 cm−1. 2.13. Scanning Electron Microscopy. The glutaraldehyde fixed and ethanol dehydrated indigenous bacteria were attached to 10 mm metal mounts using carbon tape and sputter-coated with gold under vacuum in argon atmosphere. The surface morphology of the coated sample was observed by a scanning electron microscope (S-400, Hitachi, Tokyo, Japan).

Table 3. Specific Growth Rate, Reduction Rate, And Time Taken to Reduce 40% of Initial Cr (VI) Concentration (5, 10, 20, 50, 100 mg L−1) by Adapted Isolates at pH 7.5 and Maintained at 30 °Ca

3. RESULTS AND DISCUSSION 3.1. Physico-chemical Analysis of Mine Water. The results from physicochemical analyses of the mine water sample are shown in (Table 1). The hexavalent chromium concentration in the mine water was found to be 0.23 mg L−1, which was considerably high compared to the permissible concentration of 0.05 mg L−1. The concentration ranges were corroborated by an earlier report.25 The amount of sulfate and chloride present in the mine water sample was well below the permissible limit, 250 mg L−1. A slightly elevated level of phosphate (2.43 mg L−1) and nitrate (3.92 mg L−1) were detected. The pH of the mine water sample was close to neutral. 3.2. Isolation and Characterization of Bacterial Species. The isolation of bacteria from the polluted environments would be an appropriate practice to select the metal resistant strains that could be applied for bioremediation purposes.21 The biochemical and molecular characterization were done for all the isolates and they were identified as Bacillus subtilis VITSUKMW1, Acinetobacter junii VITSUKMW2, and Escherichia coli VITSUKMW3. The isolated mine water native bacteria showed a 99% similarity in BLAST search to the available corresponding sequences. The 16S-rRNA sequences of the strains, B. subtilis VITSUKMW1, A. junii VITSUKMW2, and E. coli VITSUKMW3 were submitted to GenBank and the accession numbers (GenBank: JF309279, JF346549 and JN393206, respectively) were obtained. The phylogenetic trees of all three isolates were obtained (Figure 1).

a Maximum specific growth rate was observed at 100 mg L−1 of initial Cr (VI) concentration by E. coli VITSUKMW3. Highest reduction rate was at 100 mg L−1 initial Cr (VI) concentration by B. subtilis VITSUKMW1.

followed for every isolate to convert colony forming units (CFU) to the mass concentration. 2.12. FT-IR Study. The surface chemical characteristics of the isolated bacteria were characterized by Fourier transform-infrared spectrometer (Nicolet 6700, Thermo Scientific Instruments Group, Madison, WI, USA). One milligram of each lyophilized bacterial sample (unadapted uninteracted, unadapted interacted

Figure 2. Reduction rate of Cr (VI) at increasing concentration by adapted isolates and ternary consortium. (MW1, B. subtilis VITSUKMW1; MW2, A. junii VITSUKMW2; MW3, E. coli VITSUKMW3; Ad MW1, adapted B. subtilis VITSUKMW1; Ad MW2, adapted A. junii VITSUKMW2; Ad MW3; adapted E. coli VITSUKMW3). 3743



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Figure 3. Cr (VI) reduction capacity by adapted isolates and consortia at different initial Cr (VI) concentrations.

Figure 4. SEM images of adapted interacted (a) B. subtilis VITSUKMW1, (b) A. junii VITSUKMW2, (c) E. coli VITSUKMW3.

VITSUKMW2 and E. coli VITSUKMW3 (Table 2). The reduction rate of the unadapted isolates were similar to the earlier reports for Cr (VI) reduction by Bacillus sp.33,34 3.4.2. Adapted Isolates. In comparison with the unadapted isolates, an improved microbial growth was observed after adaptation which was evident from the increased specific growth rate (Table 3). The adapted isolates exhibited an increase in the Cr (VI) reduction rate (Figure 2) and the highest was 2.364 mg L−1 h−1 by B. subtilis VITSUKMW1 at 100 mg L−1 initial Cr (VI) concentration, which was considerably more in comparison to the earlier reports.35,36 A. junii VITSUKMW2 at 100 mg L−1 of initial Cr (VI) concentration had a high reduction rate of 1.5 mg L−1 h−1 which is higher compared to that of the earlier reports.37,38 The current study follows a trend in the increase of reduction rate with increase in Cr (VI) concentration which is similar to the earlier report.39 E. coli VITSUKMW3 had a high reduction rate of 1.85 mg L−1 h−1 at 100 mg L−1 of initial Cr (VI) concentration, which is significantly higher compared to the reduction rate by E. coli of Cr (VI) in earlier reports.29−31 The reduction capacity for the three isolates increased with an increase in the initial Cr (VI) concentration (Figure 3). The highest reduction capacity (10 mg Cr (VI)/mg of viable cell) among the adapted isolates was observed in B. subtilis VITSUKMW1 at a 50 mg L−1 of initial Cr (VI) concentration (Table 3). Both eukaryotic as well as prokaryotic species tend to respond physiologically and metabolically to varied stress conditions

The different genera of Bacillus have been reported to exhibit in vitro Cr (VI) reduction.24,26 The Acinetobacter sp has also been reported earlier to be efficient in both Cr (VI) reduction and oil degradation.27,28 E. coli, which is a model organism in microbiological studies, has also been reported to be vital in Cr (VI) remediation in the prior studies.29−31 The SEM images (Figure 1) show that the isolates had a well-defined morphology. B. subtilis VITSUKMW1 had a welldefined rod like structure. A. junii VITSUKMW2 was found to have an oval shaped structure with a groove in the middle which is commonly found in the Acinetobacter genus. E. coli VITSUKMW3 with a rod shaped structure was also observed. 3.3. MTC (Maximum Tolerable Concentration) of Bacterial Isolate. The resistance to Cr (VI) by the isolate was tested with various concentrations of Cr (VI), ranging from 10 to 1000 mg L−1. All isolates showed a MTC value of 500 to 1000 mg L−1 of Cr (VI), since, they were isolated from chromium rich mine water site. As the organisms showed a high tolerance to Cr (VI), further studies were carried out with these isolates to explore their potential in the Cr (VI) reduction. Similarly, a high tolerance to Cr (VI) were reported for mine isolates earlier.32 3.4.1. Reduction and Growth Studies. Unadapted Isolates. The specific growth rates of unadapted isolates measured were in the range of 0.032−0.104 h−1 at an initial Cr (VI) range of 5−20 mg L−1 at 30 °C, with a pH of 7.5. At 20 mg L−1 initial Cr (VI) concentration, B. subtilis VITSUKMW1 had a higher reduction rate of 0.463 mg L−1 h−1 compared to A. junii 3744



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like draft and heavy metal contaminations in the environment.40−42 The aerobic and anaerobic bacteria adapt to the environmental stress by acquiring distinct morphological changes in the environment. Heavy metal activating responses in the form of surface roughness or cell elongation were reported in the case of Bacillus sp., Acinetobacter hemolyticus and E.coli K12.38,43,44 Reports suggested that the bacteria that interacted with a heavy metal was less stressed than the unadapted uninteracted bacteria.44 In the current study, scanning electron microscopy was carried out to identify the distinct morphological changes occurring to the adapted interacted isolates in comparison with the unadapted uninteracted isolates. The SEM image of the adapted isolates B. subtilis VITSUKMW1 showed an increase in size and rough surfaces compared to the unadapted uninteracted isolates (Figure 4a). Additionally, pores were observed on its surface. A 2-fold increase in the average length of B.subtilis VITSUKMW1 cells was measured in the adapted interacted cells (2.25 μm) compared to the unadapted uninteracted (1.1 μm), which may be due to the Cr (VI) stress. This observation is supported by the earlier reports on stress response to Cr (VI) by a gram positive bacterium, Arthrobacter k-4, which can tolerate a higher level of chromate exhibiting an increase in length.45 The rough surface observed might have resulted from chromium interaction with the bacterial cell wall. A similar observation was reported in the case of Bacillus pumilus subjected to heavy metals like arsenic, cobalt, lead, mercury, and selenium.43 A. junii VITSUKMW2 was found to be swollen in shape and the cells appeared to be in chains compared to the unadapted uninteracted cells (Figure 4b). A slight increase in length from 1.5 to 1.8 μm and breadth from 0.6 to 0.7 μm was observed. The chain-like appearance was earlier reported in a gram negative bacterium, Acidocella sp. GS19h, interacted with Cd or Ni.46 In adapted interacted E. coli VITSUKMW3, rough surfaces were observed in the SEM image (Figure 4c) and also the cells were found to be swollen in shape. A significant increase in the length of E.coli VITSUKMW3 cells was observed as a response to chromium stress. The average bacterial cell length increased from 2.55 μm to 3.36−6.50 μm after the interaction with hexavalent chromium. This corresponds to the previous observations reported in E. coli K-12 exhibiting extreme filamentous morphology due to an increase in length from 4.0 to 6.0 μm on Cr (VI) challenge.44 In gram negative bacteria, Ochrobactrum tritici 5bvl1, similar changes corresponding to increase in length of bacteria was due to stress by Cr (VI).47 The surface roughness observed in E. coli VITSUKMW3 after Cr (VI) interaction may be an adaptive mechanism which is similar to the reports in the case of Vibrio f isheri, a gram negative bacteria subjected to Cr (VI) stress. 3.4.3. Consortia (Binary, Ternary). The binary consortia showed an increase in both specific growth rate as well as reduction rate with increasing initial chromium concentrations, which may be due to the adapted nature of isolates and their synergistic ability (Table. 4). Among the three binary consortia, B. subtilis VITSUKMW1 and E. coli VITSUKMW3 showed the highest reduction rate of 2.984 mg L−1 h−1 at 100 mg L−1 of initial Cr (VI) concentration with 80% of Cr (VI) reduced within 34 h (Table 4). The rate of Cr (VI) reduction by the ternary consortium at 100 mg L−1 of initial Cr (VI) concentration was six times more than the highest reduction rate achieved by the unadapted isolates at 20 mg L−1 of initial Cr (VI) concentration (Table 2, 5). Also, 99% reduction of 100 mg L−1 of initial Cr (VI) con-

Table 4. Specific Growth Rate, Reduction Rate, And Time Taken to Reduce 40% of Initial Cr (VI) Concentration (5, 10, 20, 50, 100 mg L−1) by Binary Consortia at pH 7.5 and Maintained at 30 °Ca

Maximum specific growth rate was observed at 100 mg L−1 of initial Cr (VI) concentration by E. coli VITSUKMW3 and A. junii VITSUKMW2 consortia. The highest reduction rate was at 100 mg L−1 initial Cr (VI) concentration by B. subtilis VITSUKMW1 and E. coli VITSUKMW3 consortia. a

Table 5. Specific Growth Rate, Reduction Rate, And Time Taken to Reduce 80% of Initial Cr (VI) Concentration (5, 10, 20, 50, 100 mg L−1) by Ternary Consortium at pH 7.5 and Maintained at 30 °Ca

Maximum specific growth rate of 0.213 h−1 was observed at 100 mg L−1 of initial Cr (VI) concentration. The highest reduction rate of x3.1233 mg L−1 h−1 was observed at 100 mg L−1 initial Cr (VI) concentration. a

centration was achieved in 64 h with a reduction rate of 3.123 mg L−1h−1, which is the highest reduction rate achieved in this process development (Table 5). The reduction rate achieved was similar to that reported for BRITS consortium, which was isolated from four times higher Cr (VI) concentrated source compared to the present study.24 The consortia 3745



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Table 6. Comparative Cr (VI) Reduction by Bacteria Isolated from Different Sites. The Current Study Shows Highest Reduction Rate Achieved source/site landfill leachates, Australia16 tannery effluent, Australia11 Cr landfill, China13 serpentine soil, India12 chromite mine overburden soil, Orissa, India16 chromite mine seepage water, Orissa, India17 (current study)b chromite mine water, Sukinda, India a

source Cr (VI) (mg L−1)

optimal pH

initial Cr (VI) (mg L−1)

incubation period (h)

Cr (VI) reduced (mg L−1)

Reduction rate (mg L−1 h−1)

0.3

7.4

1656.4−3495.4a 1860a

9.0 6.0 7.0

25−250 5−20 10−80 20 10−500

408 30−70 12−72 24 up to 144

25−100 5−12 10−70 10−12 10−300

0.11−0.21 0.16−0.17 0.83−0.97 0.41−0.5 0.032−0.006

3.12

168

2.93

0.017

10−100

64

5−99.04

0.199−3.12

0.19−3.12 0.23

7.5

Units in mg kg−1. bCurrent study showed a high reduction rate compared to the earlier reports.

Figure 5. Cr (VI) reduction percentage by adapted isolates and consortia at 100 mg L−1 initial Cr (VI) concentration.

showed synergistic phenotypic characteristics by their ability to reduce Cr (VI) completely at a rapid rate compared to the unadapted and adapted individual isolates (Table 2, 3, 4). The comparative reduction rates of the bacterial strains isolated from different chromium contaminated sites are displayed in Table 6, in which, the reduction rate of current study is three times higher than the highest reduction rate reported. In the current study, indigenous bacterial consortia (ternary) exhibited Cr (VI) reduction rate (3.21 mg L−1) many fold times higher compared to the earlier reports of other isolates obtained from Cr (VI) contaminated sites (0.006−0.97 mg L−1).13,16,48 The adapted isolates, Ad MW1, Ad MW2, Ad MW3, and the consortia followed 0th order (R2 value above 0.90 in all the three cases), suggesting that the Cr (VI) reduction was independent of initial Cr (VI) concentration studied. As the initial Cr (VI) concentration increased, the reduction capacity increased to 17.01 mg Cr (VI) removed/mg of viable cell (Figure 3). At a 100 mg L−1 of initial Cr (VI) concentration, the reduction percentage of Cr (VI) increased from adapted isolates to binary consortia and finally to ternary consortium (Figure 5). In SEM images, the consortium of adapted isolates appeared to be arranged in a network (Figure 6a,b). All the isolates were adhering to each other firmly. The ternary consortium was found to be efficient compared to all others (unadapted and adapted isolates and binary consortia) in terms of their specific growth rate and reduction rate (Table 5). The ability of ternary consortium to reduce Cr (VI) in the absence of any external electron donor at a comparatively higher rate suggests that, biostimulation (in situ) of these isolates may lead to effective and long-term Cr (VI)

Figure 6. SEM images of consortia, B. subtilis VITSUKMW1 + A. junii VITSUKMW2 + E. coli VITSUKMW3 at (a) 4000× and (b) 13000×, respectively.

removal. However, lab scale reactors like packed bed column and continuous stirred tank reactor can be modeled to simulate the factors affecting scale-up. The bioreduction approach using ternary consortium can be taken up further for developing in situ (pump and treat, biofilter) or ex-situ (biostimulation or bioaugmantation) remediation processes in the Sukinda mine site. 3746



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Figure 7. FT-IR spectra of Cr (VI) unadapted uninteracted (control), unadapted interacted (20 mg L−1), 100 mg L−1 Cr (VI) adapted interacted cells of (a) B. subtilis VITSUKMW1, (b) A. junii VITSUKMW2, (c) E. coli VITSUKMW3.

3.5. FT-IR Studies. The FT-IR spectra (Figure 7) of Cr (VI) unadapted uninteracted (control), unadapted interacted and adapted interacted cells of B. subtilis VITSUKMW1, A. junii

VITSUKMW2, and E. coli VITSUKMW3 were analyzed to understand the possible cell−metal ion interactions. The N−H and O−H stretching vibrations for polysaccharides and proteins 3747



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were observed at 3307, 3312, 3308 cm−1 in the unadapted uninteracted cells, which, on interaction with Cr (VI) shifted to 3420, 3432, 3303 cm−1. The peak corresponding to N−H stretching, due to the presence of amines (2431, 2420, 2449 cm−1) in B. subtilis VITSUKMW1, A. junii VITSUKMW2, and E. coli VITSUKMW3 had shown a decrease in the intensity with Cr (VI) interaction. The changes in C−O and N−H stretching corresponding to amide I and amide II vibrations were observed at the range of 1668−1541 cm−1 for adapted interacted Bacillus subtilis VITSUKMW1, Acinetobacter junii VITSUKMW2, and Escherichia coli VITSUKMW3, when interacted with Cr (VI). The C−O bond of polysaccharide at 1088, 1090, 1083 cm−1 for adapted interacted Bacillus subtilis VITSUKMW1, Acinetobacter junii VITSUKMW2, and Escherichia coli VITSUKMW3 were found to decrease in peak intensity, with a peak shift. A broad and moderately intense peak was visible at the range of 840−725 cm−1 representing Cr−O vibrations which were found in all the isolates that interacted with Cr (VI). Similar observations are reported for Bacillus sp. interactions with Cr (VI). The peaks corresponding to N−H stretching were observed at 3430−3270 cm−1. The C−O and N−H stretching corresponding to amide I and amide II vibrations is observed at the range of 1660−1550 cm−1. The C−O bond of polysaccharide was observed at 1090 cm−1 and Cr−O vibrations at the range of 840−725 cm−1.16 The presence of Cr (VI) on the cell wall of adapted interacted Bacillus subtilis VITSUKMW1 is confirmed by the FT-IR peak observed at 782−725 cm−1. The rough surface observed on the cell wall (Figure 4a) of the adapted interacted Bacillus subtilis VITSUKMW1 may have resulted due to the interaction of Cr (VI) on the cell wall as confirmed by the FT-IR signals. The FT-IR peak corresponding to the Cr−O vibrations observed at 785−725 cm−1 in the case of adapted interacted Acinetobacter junii VITSUKMW2 suggests the interaction of Cr (VI) with the cell wall. This may have an influence in the morphological changes like swelling and chain-like cell arrangement observed (Figure 4b) after adaptation. The elongation of cells evident in the adapted interacted Escherichia coli VITSUKMW3 (Figure 4c) might have resulted due to the Cr (VI) interaction evident from the peak observed at 796−725 cm−1 and other elemental changes observed in the FT-IR spectra. The spectral data indicates the involvement of surface functional groups present on the cell wall in the Cr (VI) interaction. The decrease in polysaccharide and protein peaks in the Cr (VI) interacted bacterial cells may be due to the participation of these compounds in the Cr (VI) reduction.

Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +91 416 220 2620. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Authors thank the Department of Science and Technology (DST), Govt. of India for funding this project and also thank the management of VIT University for their support in research. The authors also extend their thanks to SAIF, IITMadras for SEM and FT-IR analyses.

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4. CONCLUSIONS The heavy metal tolerance and the reduction capacity of indigenous isolates, B. subtilis VITSUKMW1, A. junii VITSUKMW2, and E. coli VITSUKMW3 from the Sukinda mine water were directly proportional to the initial Cr (VI) concentration, suggesting that, they had an inherent capacity to reduce Cr (VI). This native bioreduction capability of the three indigenous isolates was considerably enhanced by adaptation and by developing consortia from the adapted isolates. The salient changes in the FT-IR spectral data of Cr (VI) interacted biomass indicated the possible involvement of surface chemical groups from the cell wall in the bioreduction. The potential application prospects lie in the scale-up of this batch process, to analyze the efficiency of the process for large scale remediation of the contaminated sites. 3748



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Hexavalent Chromium Bioremoval through Adaptation and Consortia

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