- Email: [email protected]
Hexavalent chromium reduction potential of Cellulosimicrobium sp. isolated from common effluent treatment plant of tannery industries
Ecotoxicology and Environmental Safety 147 (2018) 102–109
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Hexavalent chromium reduction potential of Cellulosimicrobium sp. isolated from common effluent treatment plant of tannery industries
MARK
⁎
Ram Naresh Bharagava , Sandhya Mishra Laboratory for Bioremediation and Metagenomics Research (LBMR), Department of Environmental Microbiology, Babasaheb Bhimrao Ambedkar University (A Central University), Vidya Vihar, Raebareli Road, Lucknow 226025, U.P., India
A R T I C L E I N F O
A B S T R A C T
Keywords: Tannery effluent Chromium reduction Cellulosimicrobium sp. 16S rRNA gene SEM analysis EDX analysis
Present study deals with the isolation and characterization of a bacterium capable for the effective reduction of Cr(VI) from tannery wastewater. Based on the 16S rRNA gene sequence analysis, this bacterium was identified as Cellulosimicrobium sp. (KX710177). During the Cr(VI) reduction experiment performed at 50, 100, 200,and 300 mg/L of Cr(VI) concentrations, the bacterium showed 99.33% and 96.98% reduction at 50 and 100 mg/L at 24 and 96 h, respectively. However, at 200 and 300 mg/L concentration of Cr(VI), only 84.62% and 62.28% reduction was achieved after 96 h, respectively. The SEM analysis revealed that bacterial cells exposed to Cr(VI) showed increased cell size in comparison to unexposed cells, which might be due to either the precipitation or adsorption of reduced Cr(III) on bacterial cells. Further, the Energy Dispersive X-ray (EDX) analysis showed some chromium peaks for cells exposed to Cr(VI), which might be either due to the presence of precipitated reduced Cr (III) on cells or complexation of Cr(III) with cell surface molecules. The bacterium also showed resistance and sensitivity against the tested antibiotics with a wide range of MIC values ranging from 250 to 800 mg/L for different heavy metals. Thus, this multi-drug and multi-metal resistant bacterium can be used as a potential agent for the effective bioremediation of metal contaminated sites.
1. Introduction
Andhra Pradesh, Bihar, Gujarat, and Maharashtra, generating total ~ 1,75,000 m3 wastewater per day (Kaul et al., 2005). In Uttar Pradesh, ~ 444 tanneries are in operation mainly in Kanpur and Unnao region generating 22.1 MLD of wastewater per day (CPCB, 2013) and this wastewater is reported to contain 0.01–4.24 mg/L of Cr(VI) (MOWR, 2013). However, most of the tanneries (nearly 80%) are engaged in chrome tanning process that releases ~ 2000–3200 t of Cr into the environment annually (Belay, 2010). The Cr concentration in tannery wastewater ranges between 2000 and 5000 mg/L, which is much higher than the permissible limit of 2 mg/L for wastewater discharge (Belay, 2010). Like organic pollutants, metals are not degraded and tend to accumulate into the environment, may enter the food chain and cause toxic, genotoxic, mutagenic and carcinogenic effects (Chandra et al., 2011). Chromium compounds are well known to have toxic, genotoxic, mutagenic, and carcinogenic effects on humans, animals, plants, and as well as in microbes (Cheung and Gu, 2007; Mishra and Bharagava, 2016). In nature, chromium exists in several oxidation states ranging from − 2 to + 6, but only trivalent (III) and hexavalent (VI) forms of chromium is most prevalent and stable. Out of these two forms, hexavalent chromium [Cr(VI)] is highly toxic, mutagenic, teratogenic,
The contamination of environments (soil and water) with various toxic metals is a serious threat for ecosystem and human health, and requires the implementation of appropriate remedial measures. Heavy metals, such as chromium, cadmium, mercury, arsenic, lead etc. are considered as major environmental pollutants due to their toxic effects on environment as well as on human health (Ray and Ray, 2009). In developing countries, different types of industrial wastes (solid and liquid) containing a number of toxic metals in high concentration are directly or indirectly discharged into the environment without adequate treatment (Dixit et al., 2015; Chandra et al., 2009). Industries such as metallurgical, chemical, refractory brick, leather, wood preservation, pigments and dyes are the major sources of toxic metals contamination in environment (USEPA, 1998; Ryan et al., 2002). However, tannery industries are the major source of chromium contamination into the environment. Tannery industries consume a huge volume of water in tanning of hides and skin, as it is wholly a wet process and generate ~ 30–35 L of wastewater per kg skin/hides processed (Nandy et al., 1999). There are ~ 3000 tanneries in India, mainly located in the states of Tamil Nadu, West Bengal, Uttar Pradesh,
⁎
Corresponding author. E-mail addresses: [email protected], [email protected] (R.N. Bharagava).
http://dx.doi.org/10.1016/j.ecoenv.2017.08.040 Received 20 May 2017; Received in revised form 1 August 2017; Accepted 17 August 2017 0147-6513/ © 2017 Elsevier Inc. All rights reserved.
Ecotoxicology and Environmental Safety 147 (2018) 102–109
R.N. Bharagava, S. Mishra
method and Atomic Absorption Spectrophotometer (AAS) (VARIAN AS240FS, Australia), following the standard methods for the examination of water and wastewater (APHA, 2012). The digestion of wastewater sample was performed by taking 100 mL filter sterilized wastewater sample in a conical flask containing 6 mL of conc. Nitric acid and 1 mL of perchloric acid (6:1). This mixture was swirled gently covered with watch glass and heated on hot plate at room temperature. The sample was digested on hot plate until yellow fumes were released and the solution become clear. After cooling, the acid solution was filtered by Whattman's filter paper No. 44 and the volume of samples was make up 10 mL by using deionized water and used for metal analysis with their respective standard metal solutions.
carcinogenic to human and animals and has been designated as priority pollutant by US Environmental Protection Agency (USEPA) (1998). If Cr(VI) concentration into the environment exceeds > 0.05 mg/L, then it may affect the human physiology and if enter the food chain, it may cause severe health hazards such as skin irritation, nasal irritation, ulceration, eardrum perforation, and lung carcinoma etc. (WHO, 2011; Srinath et al., 2002). Cr(VI) also acts as a strong oxidizing agent and exists only in oxygenated forms as hydro-chromate (HCrO4-), chromate (CrO4-) and dichromate (Cr2O7−2) ionic species in aqueous systems. Cr(VI) compounds are comparatively more toxic than Cr(III) compounds due to their higher solubility in water, rapid permeability through biological membranes and subsequent interaction with intracellular proteins and nucleic acids (Thacker et al., 2006; Cheung and Gu, 2007). Although a number of conventional/traditional methods are reported either for removal or detoxification of Cr(VI) from industrial wastes such as chemical precipitation, reverse osmosis, ion-exchange, filtration, membrane technologies, evaporation recovery, absorption on coal, activated carbon, alum, kaolinite, and fly ash etc. (Saxena et al., 2016; Ahluwalia and Goyal, 2007). These methods are very costly, less effective and also generate a metal rich sludge as secondary pollutants. Therefore, it becomes very essential to develop an eco-friendly, costcompetitive and effective method for removal/detoxification of Cr(VI) for the safety of environment and human health protection. However, microbial reduction of toxic Cr(VI) to non-toxic Cr(III) by chromium resistant bacteria (CRB) is the most pragmatic approach that offers an economical as well as eco-friendly option for chromate detoxification and bioremediation. Microbes have diverse resistance mechanisms to cope with chromate toxicity that enable them to survive in such harsh environmental conditions (Cervantes and Campos-Gracia, 2007). These detoxification strategies include biosorption, bioaccumulation and biotransformation by enzymatic reduction, diminished intracellular accumulation through either direct obstruction of ion uptake system or active chromate efflux, precipitation, and reduction of Cr(VI) to less toxic and less mobile Cr(III) (Cheung and Gu, 2003; RamirezDiaz et al., 2008). Hence, the objectives of this study were to isolate and characterize chromium resistant bacteria, which should be capable to reduce/detoxify the toxic Cr(VI) into less toxic and less mobile Cr(III) for environmental cleanup and human health safety.
2.3. Isolation of chromium resistant bacterial strain and growth conditions To isolate chromium resistant bacteria, the collected tannery wastewater was serially diluted and spreaded on Luria-Bertani (LB) agar plates amended with potassium dichromate (100 mg/L) and incubated at 37 °C for 24–48 h (Farag and Zaki, 2010). The morphologically distinct colonies appeared on potassium dichromate amended LB agar plates were screened for maximum chromium tolerance potential by subsequent transferring/sub-culturing on LB agar plates amended with increasing concentration (200–1000 mg/L) of potassium dichromate. Out of ten bacterial isolates, only one bacterium (SCRB10) was found capable to tolerate 800 mg/L concentration of Cr(VI) and selected for biochemical characterization, identification by 16S rRNA gene sequencing analysis and other studies.
2.4. Characterization and identification of bacterial isolate 2.4.1. Morphological and biochemical characterization The isolated bacterium was characterized morphologically and biochemically following the standard protocols of Cowan and Steel's manual for the identification of medical bacteria (Barrow and Feltham, 1993) and identified based on 16S rRNA gene sequencing analysis.
2.4.2. 16S rRNA gene sequencing analysis and gene-bank accession number The genomic DNA was prepared from overnight grown bacterial culture following the alkaline lysis method described by Kapley et al. (2001). About 5 µL DNA was used to amplify 16S rDNA gene using universal eubacterial primers (27F) 50-AGAGTTTGATCMTGGCTCAG30 and (1492R) 50-TACGGYTACCTTGTTACGACTT-30 (Narde et al., 2004) and a 1500 bp product was amplified. The reaction mixture contained 5 µL template, 1X PCR buffer, 200 µM of each dNTP, 3.0 mM MgCl2, 25 pmol of primer, and 2.5 units of Amplitaq DNA polymerase (Perkin Elmer) in a final reaction volume of 50 µL (Bharagava et al., 2009). The thermocycling reactions were carried out by using Veriti® 96well Thermal Cycler (Applied Biosystems, USA). The 16S rDNA fragment was amplified by 35-cycles, PCR initial denaturation at 95°C for 5min, subsequent denaturation at 94°C for 30s, annealing temperature at 50°C for 30s, extension temperature 72°C for 1.30min and final extension at 72°C for 7min). The PCR product was analyzed on 1% agarose gel and purified by using gel extraction kit (Merk Biosciences, Bangalore). The gel purified PCR products were made sequenced by Chromous Biotech, Pvt Ltd. (Bangalore, India) on ABI 3500 Genetic Analyzer, using Big Dye Terminator Version 3.1″. The partial sequences obtained were subjected to BLAST analysis using the online option available at www.ncbi.nlm.nih.gov/BLAST (Altschul et al., 1997) suggesting the identity of isolated bacterium. The phylogenetic tree was constructed by neighbour-joining method using NCBI database online phylogenetic tree builder (http://www.ncbi.nlm.nih.gov). Further, the sequences were also deposited to Gene-Bank under the accession no. KX710177.
2. Materials and methods 2.1. Collection of tannery wastewater The tannery wastewater was collected from Common Effluent Treatment Plant (CETP) of Jajmau Unit, Kanpur (26°26'59.7228"N and 80°19'54.7335"E), Uttar Pradesh, India in a pre-sterilized conical flask (Cap. 2 L), brought to laboratory, maintained at 4 °C and used in analysis of physico-chemical parameters as well as for the isolation of bacterial strains capable for the reduction of hexavalent chromium. 2.2. Physico-chemical analysis of tannery wastewater The physico-chemical analysis of tannery wastewater was made in triplicate as per the standard methods for the examination of water and wastewaters (APHA, 2012). The collected tannery wastewater was analyzed for pH, conductivity, BOD (5 days method), COD (open reflux method), total solids (TS), total dissolve solids (TDS) and total suspended solids (TSS) (drying method), Total nitrogen (TN) (Kjeldhal method) and Chloride (AgNO3 titration method). Phosphate and sulphate was measured (Vanadomolybdo-phosphoric acid) colourimetric and (BaCl2 precipitation) methods, respectively (APHA, 2012). 2.2.1. Analysis of heavy metals in tannery wastewater The concentration of heavy metals (Cr, Zn, Mn Ni, Cd, and Fe) in collected tannery wastewater was determined by the acid digestion 103
Ecotoxicology and Environmental Safety 147 (2018) 102–109
R.N. Bharagava, S. Mishra
units), Lomefloxacin (10 mcg), Gatifloxacin (5 mcg), Levofloxacin (5 mcg) (Himedia, India). The plates were swabbed with a faintly opalescent culture, and then antibiotic disks were applied followed by incubation at 30 °C for 24 h (Yadav et al., 2016; Bharagava et al., 2014). The inhibition zones were measured after 24 h of growth period and the bacterium was classified as resistant, intermediate, or susceptible based on the zone size following the standard antibiotic disc sensitivity testing method (DIFCO, 1984).
2.5. Evaluation of growth and Cr (VI) reduction potential of isolated bacterium The growth and Cr(VI) reduction potential of bacterial isolate was evaluated by growing the bacterium in Erlenmeyer flasks (250 mL) containing 100 mL of autoclaved LB broth supplemented with different concentrations (50, 100, 200 & 300 mg/L) of Cr(VI) followed by incubation at 35 °C under shaking condition at 120 rpm (Innova 4230, USA). The broth containing Cr(VI), but without bacterial culture was taken as control. The bacterial growth was monitored spectrophotometrically (Evolution 201, Australia) in terms of increase in absorbance at 600 nm and Cr(VI) reduction potential was evaluated by a colorimetric method in terms of decrease in Cr(VI) concentration by using a Cr(VI) specific colorimetric S-diphenylcarbazide (DPC) method at 540 nm (APHA, 2012; Thacker et al., 2007). The Cr(VI) reduction was calculated by using the following formula:
Cr(VI)% reduction =
2.9. Evaluation of metal resistant property for chromium and other toxic metals The metal resistant property of isolated bacterium for Cu, Cr, Cd, Co, Zn, Fe, Ni, Pb, Mo and As was evaluated in terms of minimum inhibitory concentration (MIC). The stock solutions of the analytical grade salts of CuSO4, K2Cr2O7, CdCl2, CoCl2, ZnSO4, FeCl3, NiCl2, PbNO3, Na2MoO4 and NaAsO2 forCu2+,Cr6+, Cd2+, Co2+, Zn2+, Fe3+, Ni2+, Pb3+, Mo6+ and As3+ ions, respectively were prepared in Millipore water and autoclaved. The nutrient agar plates amended with the increasing concentration (50–1000 mg/L) of selected metals were prepared, streaked with the log phase of isolated bacterium followed by incubation at 35 °C for 48 h. The MIC of different heavy metals for bacterial isolates was designated as the minimum concentration of metal ions at which no visible growth of test organisms occurred. The nutrient agar plates without metal solutions were taken as control (Yadav et al., 2016; Bharagava et al., 2014; Sagar et al., 2012).
Ci − Cf × 100 Ci
[where, Ci = initial Cr(VI) conc. (mg/L) and Cf = final Cr(VI) conc. (mg/L)]. 2.6. Scanning Electron Microscopic analysis (SEM) The SEM analysis was performed to observe the effects of Cr(VI) on cell morphology of isolated bacterium. The bacterium was grown in LB broth containing 100 mg/L of Cr(VI) at 35 °C under shaking flask conditions (120 rpm) for 24 h whereas bacterial cells grown in LB broth without Cr(VI) was taken as control. After 24 h, the bacterial cells were harvested by centrifugation at 8000 rpm for 10 min at 4 °C and the pellets obtained were washed thrice with phosphate buffer saline (PBS) and pre-fixed with 2.5% glutaraldehyde for 4–6 h at 4 °C. These prefixed cells were washed twice with PBS (7.2 pH) and post-fixed with 1% osmium tetraoxide for 1 h followed by washing with PBS and thrice dehydration with 30%, 50%, 70%, 90%, 95% and 100% (v/v) of acetone. The fixed cells were dried with critical point dryer (CPD) and platinum-coated ion sputter coater (JEOL, Japan JFC 1600 Auto Fine Coater), and examined under SEM (JEOL JSM-6490LV) to observe the changes in cell morphology.
2.10. Statistical analysis All observations were carried out in triplicates (n = 3) to improve the analytical precision of the experiment and the data was recorded. To confirm the variability of data obtained and validity of results, the data were subjected for the statistical analysis using one way Analysis of Variance (ANOVA) and the means were compared by (Post Hoc) Tukey's test (p < 0.05) using SPSS software (IBM SPSS Statistics version 20). 3. Results and discussion 3.1. Physico-chemical characteristics of tannery wastewater
2.7. Fourier transform infrared spectrophotometric (FT-IR) analysis Thephysico-chemical analysis of tannery wastewater reveals that it was alkaline in nature (pH 8.49 ± 0.2), light yellowish in color and deficient in dissolved oxygen. In addition, the BOD, COD, total solids, TDS, TSS, phenol, sulphate, and total chromium content was 160 ± 15.8, 322 ± 28.6, 11,028 ± 805.2, 3491.3 ± 239.4, 194 ± 23.5, 12.7 ± 1.2, 1445 ± 67.9 and 5.7 ± 0.2 mg/L, respectively as shown in Table 1. The alkaline pH and high EC of collected tannery wastewater could affect the biological properties of receiving water bodies (Vijayanand and Hemapriya, 2014). The high EC (4.16 ± 0.07 mS/cm) and TDS (3491.3 ± 239.4 mg/L) values indicate the presence of high salt content and inorganic substances in tannery wastewater (Sultan and Hasnain, 2007). The EC depends on the chelating properties of water bodies and reported to create an imbalance in free metal availability for aquatic fauna and flora (Akan et al., 2007), whereas high TDS value causes osmotic stress and disrupts the osmoregulatory functions (Thacker et al., 2006). On the other hand, the settled particles may cause damage to soil fauna and flora, various detrimental changes in soil properties and reduction in water holding capacity (Chowdhury et al., 2013).
The FTIR analysis was carried out to identify the functional groups involved in the adsorption of chromium ions on bacterial cell surface. The active culture of isolated bacterium was inoculated into LB broth containing Cr(VI) (100 mg/L) and control without Cr(VI). After 24 h of incubation, the bacterial cells were pelleted by centrifugation at 8000 rpm for 10 min at 4 °C. The bacterial pellets were washed thrice with 0.85% sodium chloride solution followed by DDI water and dried in air at 50 °C (Kamnev et al., 1997). About one milligram of finely crushed air-dried bacterial biomass was mixed with 400 mg of potassium bromide. The mixture was ground to fine powder and translucent sample disks were obtained by using a manual hydraulic press at a pressure of 100 kg cm−2 for 10 min. The disks were fixed in a FTIR Spectrophotometer (Nicolet™ 6700, Thermo Scientific, USA) for analysis. The FTIR spectrum was recorded at 400–4000 cm−1 (François et al., 2012). 2.8. Evaluation of antibiotic resistance property of isolated bacterium The antibiotic resistance property of isolated bacterium was studied by disk diffusion method using Muller-Hinton agar medium against the following antibiotics: Vancomycin (30 mcg), Tetracycline (30 mcg), Chloramphenicol (30 mcg), Kanamycin (30 mcg), Erythromycin (15 mcg), Azithromycin (15 mcg), Streptomycin (10 mcg), Norfloxacin (10 mcg), Ampicillin (10 mcg), Gentamycin (10 mcg), Penicillin G (10
3.2. Isolated chromium resistant bacterium and its characteristics Initially, ten morphologically different bacterial strains (SCRB1–SCRB10) were isolated from the collected tannery wastewater samples and out of these ten bacterial strains, only one bacterium 104
Ecotoxicology and Environmental Safety 147 (2018) 102–109
R.N. Bharagava, S. Mishra
Table 1 Physicochemical characteristics of collected tannery wastewater sample. S. no.
Parameters
Collected effluent
Permissible limit (CPCB, 2013)
1. 2. 3. 4. 5.
Color Odour pH Temperature (°C) Conductivity (mS/cm) Alkalinity (mg/L) BOD (mg/L) COD (mg/L) TS (mg/L) TDS (mg/L) TSS (mg/L) Chloride (mg/L) Sulphate (mg/L) Phosphate (mg/L) Total nitrogen (mg/L) Phenol (mg/L) PCP (mg/L) Nitrate (mg/L)
Light coloured Pungent 8.49 ± 0.2 22 °C 4.16 ± 0.07
Colorless No odour 5.5–9.0 40 °C NM
680 ± 160 ± 15.8 322 ± 28.6 11,028 ± 805.2 3491.3 ± 239.4 194 ± 23.5 44.32 ± 4.3 1445 ± 67.9 3.7 ± 0.2 10.64 ± 0.8
500 30 250 – 2100 100 1000 1000 5.0 100
12.7 ± 1.2 14.6 ± 1.3 9.8 ± 0.4
0.1 0.1 10
5.7 ± 0.2 0.039 ± 0.003 0.025 ± 0.003 3.93 ± 0.02 1.10 ± 0.02 ND
2.0 1.0 – 3.0 3.0 2.0
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Heavy metal concentration i. Cr (mg/L) ii. Zn (mg/L) iii. Mn (mg/L) iv. Fe (mg/L) v. Ni (mg/L) vi. Cd (mg/L)
Fig. 2. a: Growth curve pattern of Cellulosimicrobium sp. (SCRB10) at different Cr(VI) concentrations b: Chromium reduction by Cellulosimicrobium sp. (SCRB10) at 50–300 mg/ L of Cr(VI) concentration. The differences between treatments are statistically different and significant determined by using one-way ANOVA. Different letters (i.e. a–f) above the bars represent the significant differences according to ANOVA followed by the (Post-hoc) Tukey's test (p < 0.05).
(SCRB10) was found capable to tolerate 800 mg/L of Cr(VI) concentration in screening tests. Whereas, bacterial strains SCRB 1, SCRB 2, SCRB 3, SCRB 4, SCRB 5, SCRB 6, SCRB 7, SCRB 8, and SCRB 9 were found capable to tolerate 200, 300, 250, 500, 300, 150, 500, 250, and 550 mg/L of Cr(VI), respectively. This SCRB10 bacterium appeared as golden white colonies on LB agar plates and was gram positive, rod shaped, non-spore forming, and motile bacterium. This bacterium also showed positive reactions for catalase, oxidase, gelatinase, amylase, cellulase and nitrate reductase activities, but negative reactions for starch hydrolysis, indole test, lipase, urease, citrate utilization and voges-proskauer tests. Further, the 16S rRNA gene sequence analysis showed that isolated bacterium has closest relatedness (99%) with that of genus Cellulosimicrobium (Fig. 1) and thus, based on the sequence similarity and blast analysis, the isolated bacterium (SCRB10) was identified as Cellulosimicrobium sp. with accession number KX710177.
3.3. Growth pattern and chromium reduction by isolated bacterium SCRB10 In present study, it was observed that bacterial growth was stimulated upto 100 mg/L of Cr(VI) concentration and further increase in Cr (VI) concentration had inhibitory effects on bacterial growth (Fig. 2a). The bacterium also exhibited concentration dependent reduction pattern of Cr(VI) that is the reduction potential of bacterium decreases as the concentration of Cr(VI) increases (Fig. 2b). Fig. 2a shows bacterial growth and Fig. 2b shows reduction potential at 50, 100, 200 and 300 mg/L of Cr(VI) concentration. The maximum reduction of Cr(VI)
Fig. 1. Phylogenetic tree showing the relationship of isolated bacterium with their neighbouring sp. The numbers in bracket represent gene accession numbers.
105
Ecotoxicology and Environmental Safety 147 (2018) 102–109
R.N. Bharagava, S. Mishra
Fig. 3. Proposed mechanism of Cr(VI) uptake and reduction in a bacterial cell (Modified from Vincent, 1994; Cheung and Gu, 2007).
Fig. 4. SEM analysis of untreated (a) and chromium treated (b) cells of Cellulosimicrobium sp. (SCRB10).
Table 2 Elemental content in bacterial isolate SCRB10 grown on LB agar amended with 100 mg/L Cr(VI). Element
Weight %
Atomic %
CK OK Na K Mg K PK Cl K KK Ca K Cr K
71.62 12.20 4.65 0.66 6.48 1.96 0.91 0.82 0.71
81.95 10.48 2.78 0.37 2.87 0.76 0.32 0.28 0.19
Total
100.00
Fig. 5. EDX analysis of Cellulosimicrobium sp. (SCRB10) grown on LB agar amended with 100 mg/L of Cr(VI)for elemental analysis.
concentration that might be attributed to the toxicity of Cr(VI). The bacterial growth curve after a certain period of time appears saturated suggesting the adaptive mechanisms allowing isolate to develop resistance for toxic Cr(VI) and grow in its presence (Cervantes and Campos-Garcia, 2007). The decrease in chromium reduction potential of bacterium at higher concentrations might be associated to the mutagenic and toxic effects of Cr(VI) on bacterial cell metabolism (Thacker et al., 2006). Various researchers have reported that only few Cellulosimicrobium sp. were found capable to reduce Cr(VI) at higher
i.e. 99.33% and 96.98% by isolated bacterium was achieved at 50 and 100 mg/L of Cr(VI) concentration at 24 and 96 h, respectively, whereas only 84.62% and 62.28% reduction of Cr(VI) was achieved at 200 and 300 mg/L concentration of Cr(VI) after 96 h, respectively. The difference in percentage reduction between different treatments are statistically different and significant (p < 0.05) as determine by the one-way Analysis of Variance (ANOVA) followed by (Post Hoc) Tukey's Test. The bacterial growth varies inversely with increasing Cr(VI) 106
Ecotoxicology and Environmental Safety 147 (2018) 102–109
R.N. Bharagava, S. Mishra
Fig. 6. FTIR analysis of untreated (a) and chromium treated (b) cells of Cellulosimicrobium sp. (SCRB10).
Table 3 Antibiotic resistance profile of isolated bacterium Cellulosimicrobium sp. (SCRB10). S. no.
Antibiotic disc (conc.)
Observation
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Azithromycin (15 mcg) Chloramphenicol (30 mcg) Erythromycin (15 mcg) Gatifloxacin (5 mcg) Kanamycin (30 mcg) Gentamycin (10 mcg) Lomefloxacin (10 mcg) Tetracycline (30 mcg) Norfloxacin (10 mcg) Ampicillin (10 mcg) Levofloxacin (5 mcg) Penicillin G (10 units) Streptomycin (10 mcg)
R R R S R S R R R R S R R
Table 4 Minimum inhibitory concentration (MIC) of different metal ions for the isolated bacterium (SCRB10).
R = Resistant; S = Sensitive.
Metal ions used in study
Minimum inhibitory concentration (MIC) (µg mL−1) of metal ions for the isolated bacterium (SCRB10)
Cu Cr Cd Co Zn Fe Ni Pb Mo As
250 500 160 190 600 700 500 380 600 350
Cellulosimicrobium cellulans CrK16 and Exiguobacterium CrK19 at 200 and 400 μg/mL of Cr(VI) concentration and found that Cellulosimicrobium cellulans CrK16 was more effective resulting 34% and 27% reduction as compared to Exiguobacterium CrK19 resulting only 34% and
concentrations (Naeem et al., 2013; Chatterjee et al., 2011). Rehman and Faisal (2015), studied Cr(VI) reduction potential of 107
Ecotoxicology and Environmental Safety 147 (2018) 102–109
R.N. Bharagava, S. Mishra
In addition, the spectrum also showed presence of some prominent absorption peaks at 1406, 1240, 1548 and 1648 cm–1 indicating the presence of carboxyl and amide groups on bacterial cell surface. However, a slight shifting in absorption peak from lower (1647 cm–1) to higher (1648 cm–1) was also observed indicating the presence of -C=O stretching mainly conjugated with a –NH deformation mode (Doshi et al., 2007). The chromium exposed bacterial biomass also showed two prominent peaks at 974 and 879 cm−1 (not observed in unexposed bacterial samples) representing -CH=CH of trans-di-substituted alkenes and -CH out of plane deformation showing the strong interaction of chromium with bacterial cell wall. The peak appeared at 1063 cm–1 (showing -C-O stretching) becomes more prominent and significant in Cr(VI) exposed cell biomass indicating the shifting of frequency 1066 cm–1 and suggesting the involvement of -SO3H due to the sym stretching of sulphonic moiety in interaction with chromium.
18% reduction of Cr(VI) within 24 h at 37 °C. In addition, Zahoor and Rehman (2009) also checked the chromium reduction potential of Bacillus sp. and S. capitis at 100 μg/mL of Cr(VI) and found that Bacillus sp. JDM-2-1 could reduce 40%, 66%, 77%, and 85%, whereas S. capitis reduced 29%, 53%, 65%, and 81% Cr(VI) from medium after 24, 48, 72, and 96 h. The uptake and reduction of Cr(VI) by bacteria is thought to be mediated by ‘‘acid adsorption’’ mechanism in which liquid should have enough protons to cause anion exchange. After accumulation, the Cr (VI) may act as terminal electron acceptor getting reduced into Cr(III) and binds to cell wall (Cheung and Gu, 2007; Cabrera et al., 2007; Srinath et al., 2002). The reduction of chromium occurs mainly by two processes i.e. bioaccumulation of metal inside cells or biosorption on microbial cell surface. Bioaccumulation involves two phases: an initial rapid phase of physical adsorption or ion exchange at cell surface followed by slow phase of active metabolism-dependent transport of Cr (VI) into the bacterial cell (Chandra et al., 2011; Cabrera et al., 2007). However, the biosorption of heavy metals is a metabolism dependent process that depends on different mechanism of ion exchange, co-ordination, adsorption, chelation, complexation (Srinath et al., 2002). It has been reported that chromate ions actively cross the biological membranes through sulphate pathway, which reflects the chemical analogy between two oxyanions (Cervantes and Campos-Gracia, 2007). Inside the cell, Cr(VI) is readily reduced into Cr(III) by enzymatic or non-enzymatic activities, resulting in diverse toxic effects in the cytoplasm (Cervantes et al., 2001). The reduction of Cr(VI) into Cr(III) by bacteria may occur under aerobic and anaerobic conditions through electron transport system containing cytochromes. Under aerobic conditions, the reduction of Cr(VI) into the stable end product Cr(III) occurs in two to three steps with generation of Cr(V) and Cr(IV) short lived intermediates (Fig. 3) (Vincent, 1994; Cheung and Gu, 2007). However, the reduction of Cr(V) to Cr(IV) and Cr(IV) to Cr(III) is not known to be either spontaneous or enzyme mediated but, NADH, NADPH and electron from the endogenous reserve are supposed to act electron donor in Cr(VI) reduction process (Cheung and Gu, 2007).
3.6. Antibiotic and metal resistant property of isolated bacterium In this study, results revealed that the isolated bacterium was resistant for azithromycin, chloramphenicol, erythromycin, kanamycin, lomefloxacin, tetracycline, norfloxacin, ampicillin, penicillin G, and streptomycin whereas sensitive for gatifloxacin, gentamycin and levofloxacin as shown in Table 3. The antibiotic and metal resistance property in microbes is a potential health hazard because these properties are generally associated with transmissible plasmids. These properties might be also appearing because of exposure to antibiotics/ metal contaminated environments, which may cause a coincidental coselection for resistance factors for antibiotics and metal ions (Verma et al., 2009; Jain et al., 2009). The bacterial heavy metals and antibiotic resistance is generally confined to plasmid DNA, but sometime it can also be coupled to the chromosomal DNA. Further, the isolated bacterium also showed a wide range of MIC values for tested metals ranging from 250, 500, 160, 190, 600, 700, 500, 380, 600 and 350 µg mL−1 for Cu, Cr, Cd, Co, Zn, Fe, Ni, Pb,Mo and As, respectively (Table 4). The resistance for toxic metals in bacteria probably reflects the degree of environmental contamination with toxic metals and might be directly related to the exposure of bacterial cells with toxic metals (Bharagava et al., 2014). However, the unpolluted environments may also harbour metal resistant organisms or organisms that readily adapted to high concentrations of toxic metals. Malik and Jaiswal (2000) have suggested that the incidence of a high metal resistant population resulted from increasing environmental pollution. However, the bacterial resistance to heavy metals is an important consideration when bacteria are to be introduced into soils for enhancing the bioremediation of metal contaminated sites. Although some heavy metals are required in low concentrations for normal metabolic activities, but at elevated levels, these may act as carcinogenic, mutagenic or teratogenic agents (Feuerpfeil et al., 1999).
3.4. SEM analysis The SEM analysis revealed, that the bacterial cells exposed to Cr(VI) become rough along with surface depression and showed increase in cell size in comparison to unexposed cells having smooth cell surface (Fig. 4a and b). This might be due to either the precipitation or adsorption of reduced Cr(III) on bacterial cell surface (Liu et al., 2006; Dhal et al., 2010; Kumari et al., 2016). Further, the Energy Dispersive X-ray (EDX) analysis showed some chromium peaks (with 0.19 wt% analysis) for cells exposed to Cr(VI) as shown in (Fig. 5), which might be associated either with the presence of precipitated reduced Cr(III) species on cells or complexation of Cr(III) species with cell surface molecules. However, carbon, oxygen, phosphorus, sodium, magnesium, and potassium as shown in Table 2 are well reported to participate in surface localization of chromium on bacterial cells (Niftrik et al., 2008).
4. Conclusion
3.5. FTIR analysis
Metals such as chromium, cadmium, mercury, arsenic, lead etc. are considered as a major environmental pollutant due to their toxic effects on environment as well as on human health. Based on the results of this study, it can be concluded that the isolated bacterium can be a good agent for the reduction/detoxification of hexavalent chromium from contaminated environments. During the chromium reduction experiment, the bacterium was found capable to reduce 99.33% and 96.98% Cr(VI) at 50 and 100 mg/L at 24 and 96 h, respectively and 84.62% and 62.28% at 200 and 300 mg/L concentration of Cr(VI) after 96 h, respectively. It indicates that the isolated bacterium is capable for the effective reduction/detoxification of hexavalent chromium from contaminated environments and hence, can be used as a potential agent for the effective bioremediation of metal contaminated sites for environmental safety and human/animal health protection.
The FTIR analysis of bacterial biomass exposed to chromium has shown a number of absorption peaks indicating the presence of different functional groups such as alkene (C=C), carbonyl (C=O), nitro (-NO2), carboxyl (-COOH), amines (-NH2) and sulphonic (-SO3) at different wavelengths (Fig. 6a and b). The peak observed at 3425 and 3245 cm–1 resulted from -NH2 asymmetric stretching mode of amines indicated the overlapping of amines and hydroxyl stretching on bacterial cell surface (Mungasavalli et al., 2007). However, the absorption peak appeared at 2967 cm–1 remains unchanged in case of both Cr(VI) exposed and unexposed cells indicating the asymmetric stretching of lipids, proteins, polysaccharides and nucleic acid in cell wall (Das and Guha, 2007). 108
Ecotoxicology and Environmental Safety 147 (2018) 102–109
R.N. Bharagava, S. Mishra
Rebuffat, S., 2012. Isolation and characterization of environmental bacteria capable of extracellular biosorption of mercury. Appl. Environ. Microbiol. 1097–1106. Jain, P.K., Ramchandran, S., Shukla, V., Bhakuni, D., Verma, S.K., 2009. Characterization of metal and antibiotic resistance in a bacterial population isolated from a copper mining industry. Int. J. Integr. Biol. 6, 57–61. Kamnev, A.A., Ristic, M., Antonyuka, L.P., Chernyshev, A.V., Ignatov, V.V., 1997. Fourier transform infrared spectroscopic study of intact cells of the nitrogen fixing bacterium Azospirillum brasdense. J. Mol. Struct. 408/409, 201–205. Kapley, A., Lampel, K., Purohit, H.J., 2001. Rapid detection of Salmonella in water samples by multiplex PCR. Water Environ. Res. 73, 461–465. http://dx.doi.org/10. 2175/106143001X139515. Kaul, S.N., Szpyrokowicz, L., Gautam, A., Khanna, D.R., 2005. Wastewater Management with Special Reference to Tanneries. Discovery publishing house, New Delhi, pp. 480. Kumari, V., Yadav, A., Haq, I., Kumar, S., Bharagava, R.N., Singh, S.K., Raj, A., 2016. Genotoxicity evaluation of tannery effluent treated with newly isolated hexavalent chromium reducing Bacillus cereus. J. Environ. Manag. 183, 204–211. Liu, Y., Xu, W., Zeng, G., Li, X., Goa, H., 2006. Cr(Cl) reduction by Bacillus sp. isolated from chromium landfill. Process. Biochem. 41, 1981–1986. Malik, A., Jaiswal, R., 2000. Metal resistance in Pseudomonas strains isolated from soil treated with industrial wastewater. World J. Microbiol. Biotechnol. 16, 177–182. Mishra, S., Bharagava, R.N., 2016. Toxic and genotoxic effects of hexavalent chromium in environment and its bioremediation strategies. J. Environ. Sci. Health Part C. http:// dx.doi.org/10.1080/10590501.2015.1096883. MOWR (Ministry of Water Resources), 2013. Report of the Committee: Pollution Caused by Leather Tanning Industries to the Water Bodies/Ground Water in Unnao District of Uttar Pradesh. pp. 90. Mungasavalli, D.P., Viraraghavan, T., Jin, Y.C., 2007. Biosorption of chromium from aqueous solutions by pre-treated Aspergillus niger batch and column studies. Colloids Surf. A: Physicochem. Eng. Asp. 301, 214–223. Naeem, A., Batool, R., Jamil, N., 2013. Cr(VI) reduction by Cellulosimicrobium sp. isolated from tannery effluent. Turk. J. Biol. 37, 315–322. http://dx.doi.org/10.3906/biy1209-18. Nandy, T., Kaul, S.N., Shastry, S., Manivel, U., Deshpande, C.V., 1999. Wastewater management in cluster of tanneries in Tamil Nadu through implementation of common effluent treatment plants. J. Sci. Ind. Res. 58, 475–516. Niftrik, L.V., Geerts, W.J.C., Donselaar, E.G.V., Humbel, B.M., Yakushevska, A., Verkleij, A.J., Jetten, M.S.M., Strous, M., et al., 2008. Combined structural and chemical analysis of the anammoxosome: a membrane bounded intra-cytoplasmic compartment in anammox bacteria. J. Struct. Biol. 161, 401–410. http://dx.doi.org/10.1016/ j.jsb.2007.05.005. Narde, G., Kapley, A., Purohit, H.J., 2004. Isolation and characterization of Citrobacter strain HPC 255 for broad range substrate specificity for chlorophenol. Curr. Microbiol. 48, 419–423. Ramirez-Diaz, M., Diaz-Perez, Vergas, E., Riveros-Rosas, H., Campos-Garcia, Cervantes, C., 2008. Mechanism of bacterial resistance to chromium compounds. Biometals 21, 321–332. Ray, S.A., Ray, M.K., 2009. Bioremediation of heavy metal toxicity with special reference to chromium. Al Ameen J. Med. Sci. 2 (2), 57–63. Rehman, F., Faisal, M., 2015. Toxic hexavalent chromium reduction by Bacillus pumilis, Cellulosimicrobium cellulans and Exiguobacterium. Chin. J. Oceanol. Limnol. 33 (3), 585–589. http://dx.doi.org/10.1007/s00343-015-4155-1. Ryan, M.P., Williams, D.E., Chater, R.J., Hutton, B.M., McPhail, D.S., 2002. Why stainless steel corrodes? Nature 415, 770–774. Sagar, S., Dwevedi, A., Yadav, S., Tripathi, M., Kaistha, S.D., 2012. Hexavalent chromium reduction and plant growth promotion by Staphylococcus arlettae Strain Cr11. Chemosphere 86, 847–852. Saxena, G., Chandra, R., Bharagava, R.N., 2016. Environmental pollution, toxicity profile and treatment approaches for tannery wastewater and its chemical pollutants. Rev. Environ. Contam. Toxicol. http://dx.doi.org/10.1007/398_2015_5009. Srinath, T., Verma, T., Ramteke, P.W., Garg, S., 2002. Chromium (VI) biosorption and bioaccumulation by chromate resistant bacteria. Chemosphere 48 (4), 427–435. Sultan, S., Hasnain, S., 2007. Reduction of toxic hexavalent chromium by Ochrobactrum intermedium strain SDCr-5 stimulated by heavy metals. Bioresour. Technol. 98 (2), 340–344. Thacker, U., Parikh, R., Shouche, Y., Madamwar, D., 2006. Hexavalent chromium reduction by Providencia sp. Process. Biochem. 41 (6), 1332–1337. Thacker, U., Parikh, R., Shouche, Y., Madamwar, D., 2007. Reduction of chromate by cellfree extract of Brucella sp. isolated from Cr(VI) contaminated sites. Bioresour. Technol. 98, 1541–1547. USEPA (US Environmental Protection Agency), 1998. Toxicological Review on Hexavalent Chromium (CAS No. 18540-29-9). Washington, DC. Verma, T., Garg, S.K., Ramteke, P.W., 2009. Genetic correlation between chromium resistance and reduction in Bacillus brevis isolated from tannery effluent. J. Appl. Microbiol. 107, 1425–1432. Vijayanand, S., Hemapriya, J., 2014. Biosorption and detoxification of Cr(VI) by tannery effluent acclimatized halotolerant bacterial strainpv26. Int. J. Curr. Microbiol. Appl. Sci. 3 (9) (971-pv982). Vincent, J.B., 1994. Chromium: biological relevance. In: King, R.B. (Ed.), Second ed. Encyclopaedia of Inorganic Chemistry 2 Wiley, New York. WHO (World Health Organization), 2011. Guidelines for Drinking Water Recommendations. WHO, Geneva. Yadav, A., Raj, A., Bharagava, R.N., 2016. Detection and characterization of a multidrug and multi-metal resistant Enterobacterium Pantoea sp. from tannery wastewater after secondary treatment process. Int. J. Plant Environ. 1 (2), 37–41. Zahoor, A., Rehman, A., 2009. Isolation of Cr(VI) reducing bacteria from industrial effluents and their potential use in bioremediation of chromium containing wastewater. J. Environ. Sci. 21, 814–820.
Acknowledgements Authors are highly grateful to the University Grant Commission (UGC), Government of India (GOI), New Delhi for UGC Fellowship. Authors also acknowledge the support from USIC B. B. Ambedkar University for SEM and FTIR analysis. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2017.08.040. References Ahluwalia, S., Goyal, D., 2007. Microbial and plant derived biomass for removal of heavy metals from wastewater. Bioresour. Technol. 98, 2243–2257. Akan, J.C., Moses, E.A., Ogugbuaja, V.O., 2007. Assessment of tannery industrial effluent from Kano metropolis, Nigeria Asian network for Scientific information. J. Appl. Sci. 7 (19), 2788–2793. Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. http://dx.doi.org/10.1093/nar/25.17. 3389. APHA, 2012. Standard Methods for the Examination of Water and Wastewater, 22nd edition. American Public Health Association, American Water Works Association, Water Environmental Federation, Washington, D.C. (981 pp). Barrow, G.I., Feltham, R.K.A., 1993. Cowan and Steel's Manual for the Identification of Medical Bacteria, 3rd edn. Cambridge University Press, Cambridge. Belay, A.A., 2010. Impacts of chromium from tannery effluent and evaluation of alternative treatment options. J. Environ. Prot. 1, 53–58. Bharagava, R.N., Chandra, R., Rai, V., 2009. Isolation and characterization of aerobic bacteria capable of the degradation of synthetic and natural melanoidins from distillery wastewater. World J. Microbiol. Biotechnol. 25, 737–744. Bharagava, R.N., Yadav, S., Chandra, R., 2014. Antibiotic and heavy metal resistance properties of bacteria isolated from the aeration lagoons of common effluent treatment plant (CETP) of tannery industries (Unnao, India). Ind. J. Biotechnol. 13, 514–519. Cabrera, G., Viera, M., Gomez, J.M., Cantero, D., Donati, E., 2007. Bacterial removal of chromium (VI) and (III) in a continuous system. Biodegradation 18 (4), 505–513. Central pollution control board (CPCB), 2013. Pollution Assessment: River Ganga. Status of Grossly Polluting Industries (GPI). . 〈www.cpcb.nic.in〉. Cervantes, C., Campos-Garcia, J., 2007. Reduction and efflux of chromate by bacteria. Mol. Microbiol. Heavy Met. 6, 407–419. Cervantes, C., Campos-Gracia, J., Devras, S., Gutierrez-Corona, F., Loza-Tavera, H., Torres-Guzman, J.C., Moreno-Sanchez, R., 2001. Interactions of chromium with microorganisms and plants. FEMS Microbiol. Rev. 25, 335–347. Chandra, R., Bharagava, R.N., Kapley, A., Purohit, J.H., 2011. Bacterial diversity, organic pollutants and their metabolites in two aeration lagoons of common effluent treatment plant during the degradation and detoxification of tannery wastewater. Bioresour. Technol. 102, 2333–2341. Chandra, R., Bharagava, R.N., Yadav, S., Mohan, D., 2009. Accumulation and distribution of toxic metals in wheat (Triticum aestivum L.) and Indian mustard (Brassica campestris L.) irrigated with distillery and tannery effluents. J. Hazard. Matter 162, 1514–1521. Chatterjee, S., Sau, G.B., Mukherjee, S.K., 2011. Bioremediation of Cr(VI) from Chromium-Contaminated Wastewater by Free and Immobilized Cells of Cellulosimicrobium cellulans KUCr3. Bioremediat. J. 15 (3), 173–180. http://dx.doi. org/10.1080/10889868.2011.598488. Cheung, K.H., Gu, J.D., 2003. Reduction of chromate (CrO42-) by an enrichment consortium and an isolate of marine sulphate reducing bacteria. Chemosphere 52, 1523–1529. Cheung, K.H., Gu, J.D., 2007. Mechanism of hexavalent chromium detoxification by microorganisms and bioremediation application potential: a review. Int. Biodeterior. Biodegrad. 59 (1), 8–15. Chowdhury, M., Mostafa, M.G., Biswas, T.K., Saha, A.K., 2013. Treatment of leather industrial effluents by filtration coagulation processes. Water Res. Ind. 3, 11–22. Das, S.K., Guha, A.K., 2007. Biosorption of chromium by Termitomyces clypeatus. Colloid Surf. B: Biointerfaces 60, 46–54. Dhal, B., Thatoi, H.N., Das, N.N., Pandey, B.D., 2010. Reduction of hexavalent chromium by Bacillus sp. isolated from chromite mine soils and characterization of reduced product. J. Chem. Technol. Biotechnol. 85, 1471–1479. DIFCO, 1984. Difco Manual, 10th edn. DIFCO Laboratories Inc., Detroit, Michigan. Dixit, S., Yadav, A., Dwivedi, P.D., Das, M., 2015. Toxic hazards of leather industry and technologies to combat threat: a review. J. Clean. Prod. 87, 39–49. Doshi, H., Ray, A., Kothari, I.L., 2007. Biosorption of cadmium by live and dead spirulina: IR spectroscopic, kinetics, and SEM studies. Curr. Microbiol. 54, 213–218. Farag, S., Zaki, S., 2010. Identification of bacterial strains from tannery effluent and reduction of hexavalent chromium. J. Environ. Biol. 31 (5), 877–882. Feuerpfeil, I., Lopez-Pila, J., Schmidt, R., Schneider, E., Szewzyk, R., 1999. Antibiotic resistant bacteria and antibiotics in the environment. Bundesgesundheitsbl. -Gesundh.-Gesundh. 42, 37–50. François, F., Lombard, C., Guigner, J.M., Soreau, P., Brian-Jaisson, F., Martino, G., Vandervennet, M., Garci, D., Molinier, A.L., Pignol, D., Peduzzi, J., Zirah, S.,
109
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Hexavalent chromium reduction potential of Cellulosimicrobium sp. isolated from common effluent treatment plant of tannery industries
MARK
⁎
Ram Naresh Bharagava , Sandhya Mishra Laboratory for Bioremediation and Metagenomics Research (LBMR), Department of Environmental Microbiology, Babasaheb Bhimrao Ambedkar University (A Central University), Vidya Vihar, Raebareli Road, Lucknow 226025, U.P., India
A R T I C L E I N F O
A B S T R A C T
Keywords: Tannery effluent Chromium reduction Cellulosimicrobium sp. 16S rRNA gene SEM analysis EDX analysis
Present study deals with the isolation and characterization of a bacterium capable for the effective reduction of Cr(VI) from tannery wastewater. Based on the 16S rRNA gene sequence analysis, this bacterium was identified as Cellulosimicrobium sp. (KX710177). During the Cr(VI) reduction experiment performed at 50, 100, 200,and 300 mg/L of Cr(VI) concentrations, the bacterium showed 99.33% and 96.98% reduction at 50 and 100 mg/L at 24 and 96 h, respectively. However, at 200 and 300 mg/L concentration of Cr(VI), only 84.62% and 62.28% reduction was achieved after 96 h, respectively. The SEM analysis revealed that bacterial cells exposed to Cr(VI) showed increased cell size in comparison to unexposed cells, which might be due to either the precipitation or adsorption of reduced Cr(III) on bacterial cells. Further, the Energy Dispersive X-ray (EDX) analysis showed some chromium peaks for cells exposed to Cr(VI), which might be either due to the presence of precipitated reduced Cr (III) on cells or complexation of Cr(III) with cell surface molecules. The bacterium also showed resistance and sensitivity against the tested antibiotics with a wide range of MIC values ranging from 250 to 800 mg/L for different heavy metals. Thus, this multi-drug and multi-metal resistant bacterium can be used as a potential agent for the effective bioremediation of metal contaminated sites.
1. Introduction
Andhra Pradesh, Bihar, Gujarat, and Maharashtra, generating total ~ 1,75,000 m3 wastewater per day (Kaul et al., 2005). In Uttar Pradesh, ~ 444 tanneries are in operation mainly in Kanpur and Unnao region generating 22.1 MLD of wastewater per day (CPCB, 2013) and this wastewater is reported to contain 0.01–4.24 mg/L of Cr(VI) (MOWR, 2013). However, most of the tanneries (nearly 80%) are engaged in chrome tanning process that releases ~ 2000–3200 t of Cr into the environment annually (Belay, 2010). The Cr concentration in tannery wastewater ranges between 2000 and 5000 mg/L, which is much higher than the permissible limit of 2 mg/L for wastewater discharge (Belay, 2010). Like organic pollutants, metals are not degraded and tend to accumulate into the environment, may enter the food chain and cause toxic, genotoxic, mutagenic and carcinogenic effects (Chandra et al., 2011). Chromium compounds are well known to have toxic, genotoxic, mutagenic, and carcinogenic effects on humans, animals, plants, and as well as in microbes (Cheung and Gu, 2007; Mishra and Bharagava, 2016). In nature, chromium exists in several oxidation states ranging from − 2 to + 6, but only trivalent (III) and hexavalent (VI) forms of chromium is most prevalent and stable. Out of these two forms, hexavalent chromium [Cr(VI)] is highly toxic, mutagenic, teratogenic,
The contamination of environments (soil and water) with various toxic metals is a serious threat for ecosystem and human health, and requires the implementation of appropriate remedial measures. Heavy metals, such as chromium, cadmium, mercury, arsenic, lead etc. are considered as major environmental pollutants due to their toxic effects on environment as well as on human health (Ray and Ray, 2009). In developing countries, different types of industrial wastes (solid and liquid) containing a number of toxic metals in high concentration are directly or indirectly discharged into the environment without adequate treatment (Dixit et al., 2015; Chandra et al., 2009). Industries such as metallurgical, chemical, refractory brick, leather, wood preservation, pigments and dyes are the major sources of toxic metals contamination in environment (USEPA, 1998; Ryan et al., 2002). However, tannery industries are the major source of chromium contamination into the environment. Tannery industries consume a huge volume of water in tanning of hides and skin, as it is wholly a wet process and generate ~ 30–35 L of wastewater per kg skin/hides processed (Nandy et al., 1999). There are ~ 3000 tanneries in India, mainly located in the states of Tamil Nadu, West Bengal, Uttar Pradesh,
⁎
Corresponding author. E-mail addresses: [email protected], [email protected] (R.N. Bharagava).
http://dx.doi.org/10.1016/j.ecoenv.2017.08.040 Received 20 May 2017; Received in revised form 1 August 2017; Accepted 17 August 2017 0147-6513/ © 2017 Elsevier Inc. All rights reserved.
Ecotoxicology and Environmental Safety 147 (2018) 102–109
R.N. Bharagava, S. Mishra
method and Atomic Absorption Spectrophotometer (AAS) (VARIAN AS240FS, Australia), following the standard methods for the examination of water and wastewater (APHA, 2012). The digestion of wastewater sample was performed by taking 100 mL filter sterilized wastewater sample in a conical flask containing 6 mL of conc. Nitric acid and 1 mL of perchloric acid (6:1). This mixture was swirled gently covered with watch glass and heated on hot plate at room temperature. The sample was digested on hot plate until yellow fumes were released and the solution become clear. After cooling, the acid solution was filtered by Whattman's filter paper No. 44 and the volume of samples was make up 10 mL by using deionized water and used for metal analysis with their respective standard metal solutions.
carcinogenic to human and animals and has been designated as priority pollutant by US Environmental Protection Agency (USEPA) (1998). If Cr(VI) concentration into the environment exceeds > 0.05 mg/L, then it may affect the human physiology and if enter the food chain, it may cause severe health hazards such as skin irritation, nasal irritation, ulceration, eardrum perforation, and lung carcinoma etc. (WHO, 2011; Srinath et al., 2002). Cr(VI) also acts as a strong oxidizing agent and exists only in oxygenated forms as hydro-chromate (HCrO4-), chromate (CrO4-) and dichromate (Cr2O7−2) ionic species in aqueous systems. Cr(VI) compounds are comparatively more toxic than Cr(III) compounds due to their higher solubility in water, rapid permeability through biological membranes and subsequent interaction with intracellular proteins and nucleic acids (Thacker et al., 2006; Cheung and Gu, 2007). Although a number of conventional/traditional methods are reported either for removal or detoxification of Cr(VI) from industrial wastes such as chemical precipitation, reverse osmosis, ion-exchange, filtration, membrane technologies, evaporation recovery, absorption on coal, activated carbon, alum, kaolinite, and fly ash etc. (Saxena et al., 2016; Ahluwalia and Goyal, 2007). These methods are very costly, less effective and also generate a metal rich sludge as secondary pollutants. Therefore, it becomes very essential to develop an eco-friendly, costcompetitive and effective method for removal/detoxification of Cr(VI) for the safety of environment and human health protection. However, microbial reduction of toxic Cr(VI) to non-toxic Cr(III) by chromium resistant bacteria (CRB) is the most pragmatic approach that offers an economical as well as eco-friendly option for chromate detoxification and bioremediation. Microbes have diverse resistance mechanisms to cope with chromate toxicity that enable them to survive in such harsh environmental conditions (Cervantes and Campos-Gracia, 2007). These detoxification strategies include biosorption, bioaccumulation and biotransformation by enzymatic reduction, diminished intracellular accumulation through either direct obstruction of ion uptake system or active chromate efflux, precipitation, and reduction of Cr(VI) to less toxic and less mobile Cr(III) (Cheung and Gu, 2003; RamirezDiaz et al., 2008). Hence, the objectives of this study were to isolate and characterize chromium resistant bacteria, which should be capable to reduce/detoxify the toxic Cr(VI) into less toxic and less mobile Cr(III) for environmental cleanup and human health safety.
2.3. Isolation of chromium resistant bacterial strain and growth conditions To isolate chromium resistant bacteria, the collected tannery wastewater was serially diluted and spreaded on Luria-Bertani (LB) agar plates amended with potassium dichromate (100 mg/L) and incubated at 37 °C for 24–48 h (Farag and Zaki, 2010). The morphologically distinct colonies appeared on potassium dichromate amended LB agar plates were screened for maximum chromium tolerance potential by subsequent transferring/sub-culturing on LB agar plates amended with increasing concentration (200–1000 mg/L) of potassium dichromate. Out of ten bacterial isolates, only one bacterium (SCRB10) was found capable to tolerate 800 mg/L concentration of Cr(VI) and selected for biochemical characterization, identification by 16S rRNA gene sequencing analysis and other studies.
2.4. Characterization and identification of bacterial isolate 2.4.1. Morphological and biochemical characterization The isolated bacterium was characterized morphologically and biochemically following the standard protocols of Cowan and Steel's manual for the identification of medical bacteria (Barrow and Feltham, 1993) and identified based on 16S rRNA gene sequencing analysis.
2.4.2. 16S rRNA gene sequencing analysis and gene-bank accession number The genomic DNA was prepared from overnight grown bacterial culture following the alkaline lysis method described by Kapley et al. (2001). About 5 µL DNA was used to amplify 16S rDNA gene using universal eubacterial primers (27F) 50-AGAGTTTGATCMTGGCTCAG30 and (1492R) 50-TACGGYTACCTTGTTACGACTT-30 (Narde et al., 2004) and a 1500 bp product was amplified. The reaction mixture contained 5 µL template, 1X PCR buffer, 200 µM of each dNTP, 3.0 mM MgCl2, 25 pmol of primer, and 2.5 units of Amplitaq DNA polymerase (Perkin Elmer) in a final reaction volume of 50 µL (Bharagava et al., 2009). The thermocycling reactions were carried out by using Veriti® 96well Thermal Cycler (Applied Biosystems, USA). The 16S rDNA fragment was amplified by 35-cycles, PCR initial denaturation at 95°C for 5min, subsequent denaturation at 94°C for 30s, annealing temperature at 50°C for 30s, extension temperature 72°C for 1.30min and final extension at 72°C for 7min). The PCR product was analyzed on 1% agarose gel and purified by using gel extraction kit (Merk Biosciences, Bangalore). The gel purified PCR products were made sequenced by Chromous Biotech, Pvt Ltd. (Bangalore, India) on ABI 3500 Genetic Analyzer, using Big Dye Terminator Version 3.1″. The partial sequences obtained were subjected to BLAST analysis using the online option available at www.ncbi.nlm.nih.gov/BLAST (Altschul et al., 1997) suggesting the identity of isolated bacterium. The phylogenetic tree was constructed by neighbour-joining method using NCBI database online phylogenetic tree builder (http://www.ncbi.nlm.nih.gov). Further, the sequences were also deposited to Gene-Bank under the accession no. KX710177.
2. Materials and methods 2.1. Collection of tannery wastewater The tannery wastewater was collected from Common Effluent Treatment Plant (CETP) of Jajmau Unit, Kanpur (26°26'59.7228"N and 80°19'54.7335"E), Uttar Pradesh, India in a pre-sterilized conical flask (Cap. 2 L), brought to laboratory, maintained at 4 °C and used in analysis of physico-chemical parameters as well as for the isolation of bacterial strains capable for the reduction of hexavalent chromium. 2.2. Physico-chemical analysis of tannery wastewater The physico-chemical analysis of tannery wastewater was made in triplicate as per the standard methods for the examination of water and wastewaters (APHA, 2012). The collected tannery wastewater was analyzed for pH, conductivity, BOD (5 days method), COD (open reflux method), total solids (TS), total dissolve solids (TDS) and total suspended solids (TSS) (drying method), Total nitrogen (TN) (Kjeldhal method) and Chloride (AgNO3 titration method). Phosphate and sulphate was measured (Vanadomolybdo-phosphoric acid) colourimetric and (BaCl2 precipitation) methods, respectively (APHA, 2012). 2.2.1. Analysis of heavy metals in tannery wastewater The concentration of heavy metals (Cr, Zn, Mn Ni, Cd, and Fe) in collected tannery wastewater was determined by the acid digestion 103
Ecotoxicology and Environmental Safety 147 (2018) 102–109
R.N. Bharagava, S. Mishra
units), Lomefloxacin (10 mcg), Gatifloxacin (5 mcg), Levofloxacin (5 mcg) (Himedia, India). The plates were swabbed with a faintly opalescent culture, and then antibiotic disks were applied followed by incubation at 30 °C for 24 h (Yadav et al., 2016; Bharagava et al., 2014). The inhibition zones were measured after 24 h of growth period and the bacterium was classified as resistant, intermediate, or susceptible based on the zone size following the standard antibiotic disc sensitivity testing method (DIFCO, 1984).
2.5. Evaluation of growth and Cr (VI) reduction potential of isolated bacterium The growth and Cr(VI) reduction potential of bacterial isolate was evaluated by growing the bacterium in Erlenmeyer flasks (250 mL) containing 100 mL of autoclaved LB broth supplemented with different concentrations (50, 100, 200 & 300 mg/L) of Cr(VI) followed by incubation at 35 °C under shaking condition at 120 rpm (Innova 4230, USA). The broth containing Cr(VI), but without bacterial culture was taken as control. The bacterial growth was monitored spectrophotometrically (Evolution 201, Australia) in terms of increase in absorbance at 600 nm and Cr(VI) reduction potential was evaluated by a colorimetric method in terms of decrease in Cr(VI) concentration by using a Cr(VI) specific colorimetric S-diphenylcarbazide (DPC) method at 540 nm (APHA, 2012; Thacker et al., 2007). The Cr(VI) reduction was calculated by using the following formula:
Cr(VI)% reduction =
2.9. Evaluation of metal resistant property for chromium and other toxic metals The metal resistant property of isolated bacterium for Cu, Cr, Cd, Co, Zn, Fe, Ni, Pb, Mo and As was evaluated in terms of minimum inhibitory concentration (MIC). The stock solutions of the analytical grade salts of CuSO4, K2Cr2O7, CdCl2, CoCl2, ZnSO4, FeCl3, NiCl2, PbNO3, Na2MoO4 and NaAsO2 forCu2+,Cr6+, Cd2+, Co2+, Zn2+, Fe3+, Ni2+, Pb3+, Mo6+ and As3+ ions, respectively were prepared in Millipore water and autoclaved. The nutrient agar plates amended with the increasing concentration (50–1000 mg/L) of selected metals were prepared, streaked with the log phase of isolated bacterium followed by incubation at 35 °C for 48 h. The MIC of different heavy metals for bacterial isolates was designated as the minimum concentration of metal ions at which no visible growth of test organisms occurred. The nutrient agar plates without metal solutions were taken as control (Yadav et al., 2016; Bharagava et al., 2014; Sagar et al., 2012).
Ci − Cf × 100 Ci
[where, Ci = initial Cr(VI) conc. (mg/L) and Cf = final Cr(VI) conc. (mg/L)]. 2.6. Scanning Electron Microscopic analysis (SEM) The SEM analysis was performed to observe the effects of Cr(VI) on cell morphology of isolated bacterium. The bacterium was grown in LB broth containing 100 mg/L of Cr(VI) at 35 °C under shaking flask conditions (120 rpm) for 24 h whereas bacterial cells grown in LB broth without Cr(VI) was taken as control. After 24 h, the bacterial cells were harvested by centrifugation at 8000 rpm for 10 min at 4 °C and the pellets obtained were washed thrice with phosphate buffer saline (PBS) and pre-fixed with 2.5% glutaraldehyde for 4–6 h at 4 °C. These prefixed cells were washed twice with PBS (7.2 pH) and post-fixed with 1% osmium tetraoxide for 1 h followed by washing with PBS and thrice dehydration with 30%, 50%, 70%, 90%, 95% and 100% (v/v) of acetone. The fixed cells were dried with critical point dryer (CPD) and platinum-coated ion sputter coater (JEOL, Japan JFC 1600 Auto Fine Coater), and examined under SEM (JEOL JSM-6490LV) to observe the changes in cell morphology.
2.10. Statistical analysis All observations were carried out in triplicates (n = 3) to improve the analytical precision of the experiment and the data was recorded. To confirm the variability of data obtained and validity of results, the data were subjected for the statistical analysis using one way Analysis of Variance (ANOVA) and the means were compared by (Post Hoc) Tukey's test (p < 0.05) using SPSS software (IBM SPSS Statistics version 20). 3. Results and discussion 3.1. Physico-chemical characteristics of tannery wastewater
2.7. Fourier transform infrared spectrophotometric (FT-IR) analysis Thephysico-chemical analysis of tannery wastewater reveals that it was alkaline in nature (pH 8.49 ± 0.2), light yellowish in color and deficient in dissolved oxygen. In addition, the BOD, COD, total solids, TDS, TSS, phenol, sulphate, and total chromium content was 160 ± 15.8, 322 ± 28.6, 11,028 ± 805.2, 3491.3 ± 239.4, 194 ± 23.5, 12.7 ± 1.2, 1445 ± 67.9 and 5.7 ± 0.2 mg/L, respectively as shown in Table 1. The alkaline pH and high EC of collected tannery wastewater could affect the biological properties of receiving water bodies (Vijayanand and Hemapriya, 2014). The high EC (4.16 ± 0.07 mS/cm) and TDS (3491.3 ± 239.4 mg/L) values indicate the presence of high salt content and inorganic substances in tannery wastewater (Sultan and Hasnain, 2007). The EC depends on the chelating properties of water bodies and reported to create an imbalance in free metal availability for aquatic fauna and flora (Akan et al., 2007), whereas high TDS value causes osmotic stress and disrupts the osmoregulatory functions (Thacker et al., 2006). On the other hand, the settled particles may cause damage to soil fauna and flora, various detrimental changes in soil properties and reduction in water holding capacity (Chowdhury et al., 2013).
The FTIR analysis was carried out to identify the functional groups involved in the adsorption of chromium ions on bacterial cell surface. The active culture of isolated bacterium was inoculated into LB broth containing Cr(VI) (100 mg/L) and control without Cr(VI). After 24 h of incubation, the bacterial cells were pelleted by centrifugation at 8000 rpm for 10 min at 4 °C. The bacterial pellets were washed thrice with 0.85% sodium chloride solution followed by DDI water and dried in air at 50 °C (Kamnev et al., 1997). About one milligram of finely crushed air-dried bacterial biomass was mixed with 400 mg of potassium bromide. The mixture was ground to fine powder and translucent sample disks were obtained by using a manual hydraulic press at a pressure of 100 kg cm−2 for 10 min. The disks were fixed in a FTIR Spectrophotometer (Nicolet™ 6700, Thermo Scientific, USA) for analysis. The FTIR spectrum was recorded at 400–4000 cm−1 (François et al., 2012). 2.8. Evaluation of antibiotic resistance property of isolated bacterium The antibiotic resistance property of isolated bacterium was studied by disk diffusion method using Muller-Hinton agar medium against the following antibiotics: Vancomycin (30 mcg), Tetracycline (30 mcg), Chloramphenicol (30 mcg), Kanamycin (30 mcg), Erythromycin (15 mcg), Azithromycin (15 mcg), Streptomycin (10 mcg), Norfloxacin (10 mcg), Ampicillin (10 mcg), Gentamycin (10 mcg), Penicillin G (10
3.2. Isolated chromium resistant bacterium and its characteristics Initially, ten morphologically different bacterial strains (SCRB1–SCRB10) were isolated from the collected tannery wastewater samples and out of these ten bacterial strains, only one bacterium 104
Ecotoxicology and Environmental Safety 147 (2018) 102–109
R.N. Bharagava, S. Mishra
Table 1 Physicochemical characteristics of collected tannery wastewater sample. S. no.
Parameters
Collected effluent
Permissible limit (CPCB, 2013)
1. 2. 3. 4. 5.
Color Odour pH Temperature (°C) Conductivity (mS/cm) Alkalinity (mg/L) BOD (mg/L) COD (mg/L) TS (mg/L) TDS (mg/L) TSS (mg/L) Chloride (mg/L) Sulphate (mg/L) Phosphate (mg/L) Total nitrogen (mg/L) Phenol (mg/L) PCP (mg/L) Nitrate (mg/L)
Light coloured Pungent 8.49 ± 0.2 22 °C 4.16 ± 0.07
Colorless No odour 5.5–9.0 40 °C NM
680 ± 160 ± 15.8 322 ± 28.6 11,028 ± 805.2 3491.3 ± 239.4 194 ± 23.5 44.32 ± 4.3 1445 ± 67.9 3.7 ± 0.2 10.64 ± 0.8
500 30 250 – 2100 100 1000 1000 5.0 100
12.7 ± 1.2 14.6 ± 1.3 9.8 ± 0.4
0.1 0.1 10
5.7 ± 0.2 0.039 ± 0.003 0.025 ± 0.003 3.93 ± 0.02 1.10 ± 0.02 ND
2.0 1.0 – 3.0 3.0 2.0
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Heavy metal concentration i. Cr (mg/L) ii. Zn (mg/L) iii. Mn (mg/L) iv. Fe (mg/L) v. Ni (mg/L) vi. Cd (mg/L)
Fig. 2. a: Growth curve pattern of Cellulosimicrobium sp. (SCRB10) at different Cr(VI) concentrations b: Chromium reduction by Cellulosimicrobium sp. (SCRB10) at 50–300 mg/ L of Cr(VI) concentration. The differences between treatments are statistically different and significant determined by using one-way ANOVA. Different letters (i.e. a–f) above the bars represent the significant differences according to ANOVA followed by the (Post-hoc) Tukey's test (p < 0.05).
(SCRB10) was found capable to tolerate 800 mg/L of Cr(VI) concentration in screening tests. Whereas, bacterial strains SCRB 1, SCRB 2, SCRB 3, SCRB 4, SCRB 5, SCRB 6, SCRB 7, SCRB 8, and SCRB 9 were found capable to tolerate 200, 300, 250, 500, 300, 150, 500, 250, and 550 mg/L of Cr(VI), respectively. This SCRB10 bacterium appeared as golden white colonies on LB agar plates and was gram positive, rod shaped, non-spore forming, and motile bacterium. This bacterium also showed positive reactions for catalase, oxidase, gelatinase, amylase, cellulase and nitrate reductase activities, but negative reactions for starch hydrolysis, indole test, lipase, urease, citrate utilization and voges-proskauer tests. Further, the 16S rRNA gene sequence analysis showed that isolated bacterium has closest relatedness (99%) with that of genus Cellulosimicrobium (Fig. 1) and thus, based on the sequence similarity and blast analysis, the isolated bacterium (SCRB10) was identified as Cellulosimicrobium sp. with accession number KX710177.
3.3. Growth pattern and chromium reduction by isolated bacterium SCRB10 In present study, it was observed that bacterial growth was stimulated upto 100 mg/L of Cr(VI) concentration and further increase in Cr (VI) concentration had inhibitory effects on bacterial growth (Fig. 2a). The bacterium also exhibited concentration dependent reduction pattern of Cr(VI) that is the reduction potential of bacterium decreases as the concentration of Cr(VI) increases (Fig. 2b). Fig. 2a shows bacterial growth and Fig. 2b shows reduction potential at 50, 100, 200 and 300 mg/L of Cr(VI) concentration. The maximum reduction of Cr(VI)
Fig. 1. Phylogenetic tree showing the relationship of isolated bacterium with their neighbouring sp. The numbers in bracket represent gene accession numbers.
105
Ecotoxicology and Environmental Safety 147 (2018) 102–109
R.N. Bharagava, S. Mishra
Fig. 3. Proposed mechanism of Cr(VI) uptake and reduction in a bacterial cell (Modified from Vincent, 1994; Cheung and Gu, 2007).
Fig. 4. SEM analysis of untreated (a) and chromium treated (b) cells of Cellulosimicrobium sp. (SCRB10).
Table 2 Elemental content in bacterial isolate SCRB10 grown on LB agar amended with 100 mg/L Cr(VI). Element
Weight %
Atomic %
CK OK Na K Mg K PK Cl K KK Ca K Cr K
71.62 12.20 4.65 0.66 6.48 1.96 0.91 0.82 0.71
81.95 10.48 2.78 0.37 2.87 0.76 0.32 0.28 0.19
Total
100.00
Fig. 5. EDX analysis of Cellulosimicrobium sp. (SCRB10) grown on LB agar amended with 100 mg/L of Cr(VI)for elemental analysis.
concentration that might be attributed to the toxicity of Cr(VI). The bacterial growth curve after a certain period of time appears saturated suggesting the adaptive mechanisms allowing isolate to develop resistance for toxic Cr(VI) and grow in its presence (Cervantes and Campos-Garcia, 2007). The decrease in chromium reduction potential of bacterium at higher concentrations might be associated to the mutagenic and toxic effects of Cr(VI) on bacterial cell metabolism (Thacker et al., 2006). Various researchers have reported that only few Cellulosimicrobium sp. were found capable to reduce Cr(VI) at higher
i.e. 99.33% and 96.98% by isolated bacterium was achieved at 50 and 100 mg/L of Cr(VI) concentration at 24 and 96 h, respectively, whereas only 84.62% and 62.28% reduction of Cr(VI) was achieved at 200 and 300 mg/L concentration of Cr(VI) after 96 h, respectively. The difference in percentage reduction between different treatments are statistically different and significant (p < 0.05) as determine by the one-way Analysis of Variance (ANOVA) followed by (Post Hoc) Tukey's Test. The bacterial growth varies inversely with increasing Cr(VI) 106
Ecotoxicology and Environmental Safety 147 (2018) 102–109
R.N. Bharagava, S. Mishra
Fig. 6. FTIR analysis of untreated (a) and chromium treated (b) cells of Cellulosimicrobium sp. (SCRB10).
Table 3 Antibiotic resistance profile of isolated bacterium Cellulosimicrobium sp. (SCRB10). S. no.
Antibiotic disc (conc.)
Observation
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Azithromycin (15 mcg) Chloramphenicol (30 mcg) Erythromycin (15 mcg) Gatifloxacin (5 mcg) Kanamycin (30 mcg) Gentamycin (10 mcg) Lomefloxacin (10 mcg) Tetracycline (30 mcg) Norfloxacin (10 mcg) Ampicillin (10 mcg) Levofloxacin (5 mcg) Penicillin G (10 units) Streptomycin (10 mcg)
R R R S R S R R R R S R R
Table 4 Minimum inhibitory concentration (MIC) of different metal ions for the isolated bacterium (SCRB10).
R = Resistant; S = Sensitive.
Metal ions used in study
Minimum inhibitory concentration (MIC) (µg mL−1) of metal ions for the isolated bacterium (SCRB10)
Cu Cr Cd Co Zn Fe Ni Pb Mo As
250 500 160 190 600 700 500 380 600 350
Cellulosimicrobium cellulans CrK16 and Exiguobacterium CrK19 at 200 and 400 μg/mL of Cr(VI) concentration and found that Cellulosimicrobium cellulans CrK16 was more effective resulting 34% and 27% reduction as compared to Exiguobacterium CrK19 resulting only 34% and
concentrations (Naeem et al., 2013; Chatterjee et al., 2011). Rehman and Faisal (2015), studied Cr(VI) reduction potential of 107
Ecotoxicology and Environmental Safety 147 (2018) 102–109
R.N. Bharagava, S. Mishra
In addition, the spectrum also showed presence of some prominent absorption peaks at 1406, 1240, 1548 and 1648 cm–1 indicating the presence of carboxyl and amide groups on bacterial cell surface. However, a slight shifting in absorption peak from lower (1647 cm–1) to higher (1648 cm–1) was also observed indicating the presence of -C=O stretching mainly conjugated with a –NH deformation mode (Doshi et al., 2007). The chromium exposed bacterial biomass also showed two prominent peaks at 974 and 879 cm−1 (not observed in unexposed bacterial samples) representing -CH=CH of trans-di-substituted alkenes and -CH out of plane deformation showing the strong interaction of chromium with bacterial cell wall. The peak appeared at 1063 cm–1 (showing -C-O stretching) becomes more prominent and significant in Cr(VI) exposed cell biomass indicating the shifting of frequency 1066 cm–1 and suggesting the involvement of -SO3H due to the sym stretching of sulphonic moiety in interaction with chromium.
18% reduction of Cr(VI) within 24 h at 37 °C. In addition, Zahoor and Rehman (2009) also checked the chromium reduction potential of Bacillus sp. and S. capitis at 100 μg/mL of Cr(VI) and found that Bacillus sp. JDM-2-1 could reduce 40%, 66%, 77%, and 85%, whereas S. capitis reduced 29%, 53%, 65%, and 81% Cr(VI) from medium after 24, 48, 72, and 96 h. The uptake and reduction of Cr(VI) by bacteria is thought to be mediated by ‘‘acid adsorption’’ mechanism in which liquid should have enough protons to cause anion exchange. After accumulation, the Cr (VI) may act as terminal electron acceptor getting reduced into Cr(III) and binds to cell wall (Cheung and Gu, 2007; Cabrera et al., 2007; Srinath et al., 2002). The reduction of chromium occurs mainly by two processes i.e. bioaccumulation of metal inside cells or biosorption on microbial cell surface. Bioaccumulation involves two phases: an initial rapid phase of physical adsorption or ion exchange at cell surface followed by slow phase of active metabolism-dependent transport of Cr (VI) into the bacterial cell (Chandra et al., 2011; Cabrera et al., 2007). However, the biosorption of heavy metals is a metabolism dependent process that depends on different mechanism of ion exchange, co-ordination, adsorption, chelation, complexation (Srinath et al., 2002). It has been reported that chromate ions actively cross the biological membranes through sulphate pathway, which reflects the chemical analogy between two oxyanions (Cervantes and Campos-Gracia, 2007). Inside the cell, Cr(VI) is readily reduced into Cr(III) by enzymatic or non-enzymatic activities, resulting in diverse toxic effects in the cytoplasm (Cervantes et al., 2001). The reduction of Cr(VI) into Cr(III) by bacteria may occur under aerobic and anaerobic conditions through electron transport system containing cytochromes. Under aerobic conditions, the reduction of Cr(VI) into the stable end product Cr(III) occurs in two to three steps with generation of Cr(V) and Cr(IV) short lived intermediates (Fig. 3) (Vincent, 1994; Cheung and Gu, 2007). However, the reduction of Cr(V) to Cr(IV) and Cr(IV) to Cr(III) is not known to be either spontaneous or enzyme mediated but, NADH, NADPH and electron from the endogenous reserve are supposed to act electron donor in Cr(VI) reduction process (Cheung and Gu, 2007).
3.6. Antibiotic and metal resistant property of isolated bacterium In this study, results revealed that the isolated bacterium was resistant for azithromycin, chloramphenicol, erythromycin, kanamycin, lomefloxacin, tetracycline, norfloxacin, ampicillin, penicillin G, and streptomycin whereas sensitive for gatifloxacin, gentamycin and levofloxacin as shown in Table 3. The antibiotic and metal resistance property in microbes is a potential health hazard because these properties are generally associated with transmissible plasmids. These properties might be also appearing because of exposure to antibiotics/ metal contaminated environments, which may cause a coincidental coselection for resistance factors for antibiotics and metal ions (Verma et al., 2009; Jain et al., 2009). The bacterial heavy metals and antibiotic resistance is generally confined to plasmid DNA, but sometime it can also be coupled to the chromosomal DNA. Further, the isolated bacterium also showed a wide range of MIC values for tested metals ranging from 250, 500, 160, 190, 600, 700, 500, 380, 600 and 350 µg mL−1 for Cu, Cr, Cd, Co, Zn, Fe, Ni, Pb,Mo and As, respectively (Table 4). The resistance for toxic metals in bacteria probably reflects the degree of environmental contamination with toxic metals and might be directly related to the exposure of bacterial cells with toxic metals (Bharagava et al., 2014). However, the unpolluted environments may also harbour metal resistant organisms or organisms that readily adapted to high concentrations of toxic metals. Malik and Jaiswal (2000) have suggested that the incidence of a high metal resistant population resulted from increasing environmental pollution. However, the bacterial resistance to heavy metals is an important consideration when bacteria are to be introduced into soils for enhancing the bioremediation of metal contaminated sites. Although some heavy metals are required in low concentrations for normal metabolic activities, but at elevated levels, these may act as carcinogenic, mutagenic or teratogenic agents (Feuerpfeil et al., 1999).
3.4. SEM analysis The SEM analysis revealed, that the bacterial cells exposed to Cr(VI) become rough along with surface depression and showed increase in cell size in comparison to unexposed cells having smooth cell surface (Fig. 4a and b). This might be due to either the precipitation or adsorption of reduced Cr(III) on bacterial cell surface (Liu et al., 2006; Dhal et al., 2010; Kumari et al., 2016). Further, the Energy Dispersive X-ray (EDX) analysis showed some chromium peaks (with 0.19 wt% analysis) for cells exposed to Cr(VI) as shown in (Fig. 5), which might be associated either with the presence of precipitated reduced Cr(III) species on cells or complexation of Cr(III) species with cell surface molecules. However, carbon, oxygen, phosphorus, sodium, magnesium, and potassium as shown in Table 2 are well reported to participate in surface localization of chromium on bacterial cells (Niftrik et al., 2008).
4. Conclusion
3.5. FTIR analysis
Metals such as chromium, cadmium, mercury, arsenic, lead etc. are considered as a major environmental pollutant due to their toxic effects on environment as well as on human health. Based on the results of this study, it can be concluded that the isolated bacterium can be a good agent for the reduction/detoxification of hexavalent chromium from contaminated environments. During the chromium reduction experiment, the bacterium was found capable to reduce 99.33% and 96.98% Cr(VI) at 50 and 100 mg/L at 24 and 96 h, respectively and 84.62% and 62.28% at 200 and 300 mg/L concentration of Cr(VI) after 96 h, respectively. It indicates that the isolated bacterium is capable for the effective reduction/detoxification of hexavalent chromium from contaminated environments and hence, can be used as a potential agent for the effective bioremediation of metal contaminated sites for environmental safety and human/animal health protection.
The FTIR analysis of bacterial biomass exposed to chromium has shown a number of absorption peaks indicating the presence of different functional groups such as alkene (C=C), carbonyl (C=O), nitro (-NO2), carboxyl (-COOH), amines (-NH2) and sulphonic (-SO3) at different wavelengths (Fig. 6a and b). The peak observed at 3425 and 3245 cm–1 resulted from -NH2 asymmetric stretching mode of amines indicated the overlapping of amines and hydroxyl stretching on bacterial cell surface (Mungasavalli et al., 2007). However, the absorption peak appeared at 2967 cm–1 remains unchanged in case of both Cr(VI) exposed and unexposed cells indicating the asymmetric stretching of lipids, proteins, polysaccharides and nucleic acid in cell wall (Das and Guha, 2007). 108
Ecotoxicology and Environmental Safety 147 (2018) 102–109
R.N. Bharagava, S. Mishra
Rebuffat, S., 2012. Isolation and characterization of environmental bacteria capable of extracellular biosorption of mercury. Appl. Environ. Microbiol. 1097–1106. Jain, P.K., Ramchandran, S., Shukla, V., Bhakuni, D., Verma, S.K., 2009. Characterization of metal and antibiotic resistance in a bacterial population isolated from a copper mining industry. Int. J. Integr. Biol. 6, 57–61. Kamnev, A.A., Ristic, M., Antonyuka, L.P., Chernyshev, A.V., Ignatov, V.V., 1997. Fourier transform infrared spectroscopic study of intact cells of the nitrogen fixing bacterium Azospirillum brasdense. J. Mol. Struct. 408/409, 201–205. Kapley, A., Lampel, K., Purohit, H.J., 2001. Rapid detection of Salmonella in water samples by multiplex PCR. Water Environ. Res. 73, 461–465. http://dx.doi.org/10. 2175/106143001X139515. Kaul, S.N., Szpyrokowicz, L., Gautam, A., Khanna, D.R., 2005. Wastewater Management with Special Reference to Tanneries. Discovery publishing house, New Delhi, pp. 480. Kumari, V., Yadav, A., Haq, I., Kumar, S., Bharagava, R.N., Singh, S.K., Raj, A., 2016. Genotoxicity evaluation of tannery effluent treated with newly isolated hexavalent chromium reducing Bacillus cereus. J. Environ. Manag. 183, 204–211. Liu, Y., Xu, W., Zeng, G., Li, X., Goa, H., 2006. Cr(Cl) reduction by Bacillus sp. isolated from chromium landfill. Process. Biochem. 41, 1981–1986. Malik, A., Jaiswal, R., 2000. Metal resistance in Pseudomonas strains isolated from soil treated with industrial wastewater. World J. Microbiol. Biotechnol. 16, 177–182. Mishra, S., Bharagava, R.N., 2016. Toxic and genotoxic effects of hexavalent chromium in environment and its bioremediation strategies. J. Environ. Sci. Health Part C. http:// dx.doi.org/10.1080/10590501.2015.1096883. MOWR (Ministry of Water Resources), 2013. Report of the Committee: Pollution Caused by Leather Tanning Industries to the Water Bodies/Ground Water in Unnao District of Uttar Pradesh. pp. 90. Mungasavalli, D.P., Viraraghavan, T., Jin, Y.C., 2007. Biosorption of chromium from aqueous solutions by pre-treated Aspergillus niger batch and column studies. Colloids Surf. A: Physicochem. Eng. Asp. 301, 214–223. Naeem, A., Batool, R., Jamil, N., 2013. Cr(VI) reduction by Cellulosimicrobium sp. isolated from tannery effluent. Turk. J. Biol. 37, 315–322. http://dx.doi.org/10.3906/biy1209-18. Nandy, T., Kaul, S.N., Shastry, S., Manivel, U., Deshpande, C.V., 1999. Wastewater management in cluster of tanneries in Tamil Nadu through implementation of common effluent treatment plants. J. Sci. Ind. Res. 58, 475–516. Niftrik, L.V., Geerts, W.J.C., Donselaar, E.G.V., Humbel, B.M., Yakushevska, A., Verkleij, A.J., Jetten, M.S.M., Strous, M., et al., 2008. Combined structural and chemical analysis of the anammoxosome: a membrane bounded intra-cytoplasmic compartment in anammox bacteria. J. Struct. Biol. 161, 401–410. http://dx.doi.org/10.1016/ j.jsb.2007.05.005. Narde, G., Kapley, A., Purohit, H.J., 2004. Isolation and characterization of Citrobacter strain HPC 255 for broad range substrate specificity for chlorophenol. Curr. Microbiol. 48, 419–423. Ramirez-Diaz, M., Diaz-Perez, Vergas, E., Riveros-Rosas, H., Campos-Garcia, Cervantes, C., 2008. Mechanism of bacterial resistance to chromium compounds. Biometals 21, 321–332. Ray, S.A., Ray, M.K., 2009. Bioremediation of heavy metal toxicity with special reference to chromium. Al Ameen J. Med. Sci. 2 (2), 57–63. Rehman, F., Faisal, M., 2015. Toxic hexavalent chromium reduction by Bacillus pumilis, Cellulosimicrobium cellulans and Exiguobacterium. Chin. J. Oceanol. Limnol. 33 (3), 585–589. http://dx.doi.org/10.1007/s00343-015-4155-1. Ryan, M.P., Williams, D.E., Chater, R.J., Hutton, B.M., McPhail, D.S., 2002. Why stainless steel corrodes? Nature 415, 770–774. Sagar, S., Dwevedi, A., Yadav, S., Tripathi, M., Kaistha, S.D., 2012. Hexavalent chromium reduction and plant growth promotion by Staphylococcus arlettae Strain Cr11. Chemosphere 86, 847–852. Saxena, G., Chandra, R., Bharagava, R.N., 2016. Environmental pollution, toxicity profile and treatment approaches for tannery wastewater and its chemical pollutants. Rev. Environ. Contam. Toxicol. http://dx.doi.org/10.1007/398_2015_5009. Srinath, T., Verma, T., Ramteke, P.W., Garg, S., 2002. Chromium (VI) biosorption and bioaccumulation by chromate resistant bacteria. Chemosphere 48 (4), 427–435. Sultan, S., Hasnain, S., 2007. Reduction of toxic hexavalent chromium by Ochrobactrum intermedium strain SDCr-5 stimulated by heavy metals. Bioresour. Technol. 98 (2), 340–344. Thacker, U., Parikh, R., Shouche, Y., Madamwar, D., 2006. Hexavalent chromium reduction by Providencia sp. Process. Biochem. 41 (6), 1332–1337. Thacker, U., Parikh, R., Shouche, Y., Madamwar, D., 2007. Reduction of chromate by cellfree extract of Brucella sp. isolated from Cr(VI) contaminated sites. Bioresour. Technol. 98, 1541–1547. USEPA (US Environmental Protection Agency), 1998. Toxicological Review on Hexavalent Chromium (CAS No. 18540-29-9). Washington, DC. Verma, T., Garg, S.K., Ramteke, P.W., 2009. Genetic correlation between chromium resistance and reduction in Bacillus brevis isolated from tannery effluent. J. Appl. Microbiol. 107, 1425–1432. Vijayanand, S., Hemapriya, J., 2014. Biosorption and detoxification of Cr(VI) by tannery effluent acclimatized halotolerant bacterial strainpv26. Int. J. Curr. Microbiol. Appl. Sci. 3 (9) (971-pv982). Vincent, J.B., 1994. Chromium: biological relevance. In: King, R.B. (Ed.), Second ed. Encyclopaedia of Inorganic Chemistry 2 Wiley, New York. WHO (World Health Organization), 2011. Guidelines for Drinking Water Recommendations. WHO, Geneva. Yadav, A., Raj, A., Bharagava, R.N., 2016. Detection and characterization of a multidrug and multi-metal resistant Enterobacterium Pantoea sp. from tannery wastewater after secondary treatment process. Int. J. Plant Environ. 1 (2), 37–41. Zahoor, A., Rehman, A., 2009. Isolation of Cr(VI) reducing bacteria from industrial effluents and their potential use in bioremediation of chromium containing wastewater. J. Environ. Sci. 21, 814–820.
Acknowledgements Authors are highly grateful to the University Grant Commission (UGC), Government of India (GOI), New Delhi for UGC Fellowship. Authors also acknowledge the support from USIC B. B. Ambedkar University for SEM and FTIR analysis. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2017.08.040. References Ahluwalia, S., Goyal, D., 2007. Microbial and plant derived biomass for removal of heavy metals from wastewater. Bioresour. Technol. 98, 2243–2257. Akan, J.C., Moses, E.A., Ogugbuaja, V.O., 2007. Assessment of tannery industrial effluent from Kano metropolis, Nigeria Asian network for Scientific information. J. Appl. Sci. 7 (19), 2788–2793. Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. http://dx.doi.org/10.1093/nar/25.17. 3389. APHA, 2012. Standard Methods for the Examination of Water and Wastewater, 22nd edition. American Public Health Association, American Water Works Association, Water Environmental Federation, Washington, D.C. (981 pp). Barrow, G.I., Feltham, R.K.A., 1993. Cowan and Steel's Manual for the Identification of Medical Bacteria, 3rd edn. Cambridge University Press, Cambridge. Belay, A.A., 2010. Impacts of chromium from tannery effluent and evaluation of alternative treatment options. J. Environ. Prot. 1, 53–58. Bharagava, R.N., Chandra, R., Rai, V., 2009. Isolation and characterization of aerobic bacteria capable of the degradation of synthetic and natural melanoidins from distillery wastewater. World J. Microbiol. Biotechnol. 25, 737–744. Bharagava, R.N., Yadav, S., Chandra, R., 2014. Antibiotic and heavy metal resistance properties of bacteria isolated from the aeration lagoons of common effluent treatment plant (CETP) of tannery industries (Unnao, India). Ind. J. Biotechnol. 13, 514–519. Cabrera, G., Viera, M., Gomez, J.M., Cantero, D., Donati, E., 2007. Bacterial removal of chromium (VI) and (III) in a continuous system. Biodegradation 18 (4), 505–513. Central pollution control board (CPCB), 2013. Pollution Assessment: River Ganga. Status of Grossly Polluting Industries (GPI). . 〈www.cpcb.nic.in〉. Cervantes, C., Campos-Garcia, J., 2007. Reduction and efflux of chromate by bacteria. Mol. Microbiol. Heavy Met. 6, 407–419. Cervantes, C., Campos-Gracia, J., Devras, S., Gutierrez-Corona, F., Loza-Tavera, H., Torres-Guzman, J.C., Moreno-Sanchez, R., 2001. Interactions of chromium with microorganisms and plants. FEMS Microbiol. Rev. 25, 335–347. Chandra, R., Bharagava, R.N., Kapley, A., Purohit, J.H., 2011. Bacterial diversity, organic pollutants and their metabolites in two aeration lagoons of common effluent treatment plant during the degradation and detoxification of tannery wastewater. Bioresour. Technol. 102, 2333–2341. Chandra, R., Bharagava, R.N., Yadav, S., Mohan, D., 2009. Accumulation and distribution of toxic metals in wheat (Triticum aestivum L.) and Indian mustard (Brassica campestris L.) irrigated with distillery and tannery effluents. J. Hazard. Matter 162, 1514–1521. Chatterjee, S., Sau, G.B., Mukherjee, S.K., 2011. Bioremediation of Cr(VI) from Chromium-Contaminated Wastewater by Free and Immobilized Cells of Cellulosimicrobium cellulans KUCr3. Bioremediat. J. 15 (3), 173–180. http://dx.doi. org/10.1080/10889868.2011.598488. Cheung, K.H., Gu, J.D., 2003. Reduction of chromate (CrO42-) by an enrichment consortium and an isolate of marine sulphate reducing bacteria. Chemosphere 52, 1523–1529. Cheung, K.H., Gu, J.D., 2007. Mechanism of hexavalent chromium detoxification by microorganisms and bioremediation application potential: a review. Int. Biodeterior. Biodegrad. 59 (1), 8–15. Chowdhury, M., Mostafa, M.G., Biswas, T.K., Saha, A.K., 2013. Treatment of leather industrial effluents by filtration coagulation processes. Water Res. Ind. 3, 11–22. Das, S.K., Guha, A.K., 2007. Biosorption of chromium by Termitomyces clypeatus. Colloid Surf. B: Biointerfaces 60, 46–54. Dhal, B., Thatoi, H.N., Das, N.N., Pandey, B.D., 2010. Reduction of hexavalent chromium by Bacillus sp. isolated from chromite mine soils and characterization of reduced product. J. Chem. Technol. Biotechnol. 85, 1471–1479. DIFCO, 1984. Difco Manual, 10th edn. DIFCO Laboratories Inc., Detroit, Michigan. Dixit, S., Yadav, A., Dwivedi, P.D., Das, M., 2015. Toxic hazards of leather industry and technologies to combat threat: a review. J. Clean. Prod. 87, 39–49. Doshi, H., Ray, A., Kothari, I.L., 2007. Biosorption of cadmium by live and dead spirulina: IR spectroscopic, kinetics, and SEM studies. Curr. Microbiol. 54, 213–218. Farag, S., Zaki, S., 2010. Identification of bacterial strains from tannery effluent and reduction of hexavalent chromium. J. Environ. Biol. 31 (5), 877–882. Feuerpfeil, I., Lopez-Pila, J., Schmidt, R., Schneider, E., Szewzyk, R., 1999. Antibiotic resistant bacteria and antibiotics in the environment. Bundesgesundheitsbl. -Gesundh.-Gesundh. 42, 37–50. François, F., Lombard, C., Guigner, J.M., Soreau, P., Brian-Jaisson, F., Martino, G., Vandervennet, M., Garci, D., Molinier, A.L., Pignol, D., Peduzzi, J., Zirah, S.,
109