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Reduction of hexavalent chromium by a moderately halophilic bacterium, Halomonas smyrnensis KS802 under saline environment

  • Jhuma Biswas
  • Pritha Bose
  • Sukhendu Mandal
  • A. K. Paul
Original Article
  • 32 Downloads

Abstract

In the present study a moderately halophilic bacterium, Halomonas smyrnensis KS802 (GenBank Accession No. KU982965) was isolated from multi-pond solar salterns of Gujarat, India and evaluated for its potential to reduce hexavalent chromium during growth. Complete reduction of 2 mM Cr(VI) was achieved in 12 h when the strain was grown in medium for halophiles (MH medium) supplemented with 4% galactose and 5% NaCl at pH 7 and 32 °C. Presence of Mn, Cu and Pb in the culture media were non-toxic for growth and Cr(VI) reduction, while carbonyl cyanide-m-chlorophenyl hydrazone (CCC) inhibited both growth and Cr(VI) reduction. Hexavalent chromium of the untreated tannery effluent (100 µM) was completely reduced by the isolate in 6 h. The rate kinetics of reduction revealed a well fitted linearized exponential equation at a reduction rate of 5.9–1.8 × 10−2 h−1 in the range of 100–500 µM of Cr(VI) in the tannery effluent. Fourier transformed infrared (FTIR) spectroscopic analysis of chromate reducing cells showed characteristic shifting of functional group specific peaks possibly due to binding of reduced Cr-species. Scanning electron microscopy-energy dispersive X-ray (SEM–EDX) studies also supported deposition of Cr(III) on bacterial cells along with distinct changes in cellular morphology. Moreover, the powder X-ray diffraction (p XRD) pattern indicated the possible formation and complexation of chromium hydrogen phosphate and chromium hydroxide with cells of H. smyrnensis KS802.

Keywords

Halophilic bacteria Halomonas smyrnensis Hexavalent chromium Chromate reduction Tannery effluent 

Introduction

Chromium, one of the highly toxic metals is released into the environment as a consequence of variety of anthropogenic activities and has become a serious human health concern. Of the two stable forms of chromium, the water soluble hexavalent chromium [Cr(VI)] is highly toxic to almost every living organisms including human beings (Losi et al. 1994). Compounds of Cr(VI) are mutagenic, carcinogenic and inhibitory to enzyme activity and synthesis of nucleic acid (Gunaratnam and Grant 2008). The trivalent chromium [Cr(III)] species on the contrary is insoluble in neutral pH, less toxic, immobile and forms stable complexes with organic ligands (Ehrlich 2002).

Bio-transformation of toxic Cr(VI) to relatively non-toxic Cr(III) has been established as an advantageous green technology for remediation of toxic chromium as against the conventional physico-chemical processes, which often generate large volume of sludge, toxic gases and are not environment-friendly. Microbial communities inhabiting chromium contaminated environments have been found to tolerate high concentrations of chromium and play important role in the detoxification as well as removal of Cr(VI) from the polluted effluents following reduction under aerobic as well as anaerobic conditions (Losi et al. 1994; Joutey et al. 2016). Such bio-reduction processes are advantageous as they do not require high energy input nor they require toxic chemical reagents but utilize renewable resources. As a consequence, the chromium reducing bacteria (CRB) and fungi, either as pure culture or consortium have been utilized for Cr(VI) treatment in contaminated sites and industrial effluents by several researchers (Camargo et al. 2003; Meghraj et al. 2004; Upadhyay et al. 2017; Lotlikar et al. 2018).

Halophilic bacteria belonging to the genus Halomonas isolated from different naturally polluted saline environments have also been recently reported to be efficient in reducing hexavalent chromium during growth (Amoozegar et al. 2007; Shapovalova et al. 2009; Murugavelh and Mohanty 2012, 2013; Mabrouk et al. 2014). Reduction of chromate by whole cells, immobilized cells and cell-free extracts of Halomonas are not uncommon (Amoozegar et al. 2007; Murugavelh and Mohanty 2012, 2013). Moreover, efficiency of these halophilic strains in reducing hexavalent chromium is no less than those of the non-halophilic chromium resistant strains, so far recorded from chromium contaminated environment (Meghraj et al. 2004; Camargo et al. 2003; Dey and Paul 2012; Karthik et al. 2017).

Tannery effluents, the major source of aquatic pollution in India are characterized with high salinity, chemical oxygen demand (COD), biological oxygen demand (BOD) and chromium content. The high salinity of the effluent often reduces the rate of growth and bioreduction of Cr(VI) by metal resistant indigenous bacteria. Chromate reducing halophilic bacteria have the dual advantages of being able to tolerate and grow under hypersaline environment and also to detoxify and remove toxic Cr(VI) from the tannery effluents in an effective and environment-friendly manner (Abubacker and Ramanathan 2003; Bhargava and Mishra 2018).

The present study is concerned in screening moderately halophilic bacterial strains for the reduction of hexavalent chromium and selection of a potent Cr(VI) resistant and reducing bacterium Halomonas smyrnensis KS802 (GenBank Accession No. KU982965) from multi-pond solar salterns of Gujarat, India. Hexavalent chromium reducing efficiency of the strain has been assessed under varying cultural conditions and in untreated tannery effluents. Morphological and physico-chemical changes of the organism during the course of Cr(VI) reduction have also been analyzed by FTIR, SEM–EDX, and XRD.

Materials and methods

Cultivation and maintenance of bacterial isolates

A total of 29 moderately halophilic bacteria isolated from saline soil and water samples of multi-pond solar salterns of Gujarat and Odisha, India were used in this study. The isolates were cultivated and maintained on medium for halophiles (MH) agar medium (Ventosa et al. 1982) containing (g L−1): yeast extract 10; protease peptone, 5; glucose, 10; NaCl, 25; MgCl2, 6H2O, 7; MgSO4, 7H2O, 9.6; CaCl2, 2H2O, 0.36; KCl, 2; NaHCO3, 0.06‚ and NaBr 0.026 (pH 7.2) according to Gerhardt et al. (1994)

Screening for Cr(VI) tolerance and reduction

Tolerance to hexavalent chromium by the isolates was evaluated on basal MH agar plates supplemented with 0.1–20 mM of Cr(VI). The media were inoculated in form of streaks with overnight grown culture and incubated at 32 °C for 24–48 h. Growth was recorded visually and minimum inhibitory concentration (MIC) of Cr(VI) was determined as the lowest concentration responsible for complete inhibition of growth.

The isolates were also screened for hexavalent chromium reduction during growth in Cr(VI) supplemented MH medium under continuous shaking (120 rpm) at 32 °C. Since all the isolates showed MIC values > 2 mM Cr(VI), reduction studies were carried out uniformly at a sub-lethal concentration of 2 mM Cr(VI) in the medium. Reduction of Cr(VI) was evaluated following the di-phenyl carbazide (DPC) method as described by Park et al. (2000). The most potent isolate, KS802 showing highest Cr(VI) tolerance and reduction was selected for further study.

Characterization and identification of the selected isolate

Morphological, physiological and biochemical characteristics of the selected chromate reducing isolate was determined following standard methods of Smibert and Krieg (1981). Antibiotic sensitivity pattern of the isolate was determined by disc-diffusion method using antibiotic impregnated discs (Himedia, 6 mm dia.). Fermentation of sugars was tested on phenol red medium supplemented with 1% carbon source. Tolerance of the isolate against the heavy metals Pb, Co, Mn, Ni, Ag, Cd, Hg, Zn, and Cu was determined on MH agar plate supplemented with each of the metal at different concentrations. Characteristics of the bacterial isolates were compared with those described in Bergey’s Manual of Systematics of Archaea and Bacteria (Vreeland 2015) for determination of taxonomic identity.

The molecular identification of the isolate was carried out by 16S rRNA gene sequence based method. For this, chromosomal DNA of KS802 was isolated and purified according to a modified method originally described by Marmur (1961) and used as template for polymerase chain reaction (PCR) to amplify a specific region of DNA of interest using eubacterial universal primers (Forward Primer 8F: 5′-AGAGTTTGATCCTGGCTCAG-3; Reverse Primer 1492R: 5′-GGTTACCTTGTTACGACTT-3′ (Turner et al. 1999). The PCR amplified product was purified and sequenced in ABI 377 automated DNA sequencer. Sequenced data were analyzed by using Genetics Computer Group (GCG, Version 9-UNIX) 26 and retrieved by their accession numbers and key words from the EMBL database by using the BLAST search programs (BLASTN) (Altschul et al. 1997). Multiple sequence alignment as well as pair wise alignment of the sequences retrieved during BLAST search programs were carried out using the program package ClustalW (Thompson et al. 1994). The aligned sequences were then used for phylogenetic analysis by neighbor joining method using MEGA 6 (Tamura et al. 2011). Bootstrap values based on 1000 replications were indicated at nodes of the phylogenetic tree.

Chromium reduction by the selected isolate

Time course of chromate reduction during growth by the selected isolate KS802 was determined under batch culture in MH medium containing 2 mM Cr(VI). The medium was inoculated with 250 µl of a stock culture (108 cells/ml) of the isolate to maintain an initial cell concentration of 105 cells/ml and incubated at 32 °C under continuous shaking (120 rpm) for 6 days. A control set of uninoculated MH medium supplemented with 2 mM Cr(VI) was also maintained. Samples were withdrawn at regular interval of 12 h and the cell mass was harvested by centrifugation (10,000×g, 15 min). Growth in terms of dry weight was determined from the harvested cell mass and the hexavalent chromium in the supernatant was determined by the di-phenyl carbazide method (Park et al. 2000).

To one ml of cell-free culture filtrate, 3 ml of 0.166 M H2SO4 and 1 ml of 0.05% di-phenyl carbazide (DPC) solution was added. The reaction mixture was vortexed and optical density was read at 540 nm. The concentration of the hexavalent chromium was determined from the calibration curve prepared in the same way. The reduction of Cr(VI) was determined from the difference of final and initial concentrations and expressed as % Cr(VI) reduced.

Chromate reduction capacity of the isolate KS802 during growth was routinely evaluated with different initial concentrations (0.5–6 mM) of Cr(VI), carbon source (1%), initial pH (5.0–11.0) and temperatures (25–45 °C) in MH medium. The effects of NaCl (0-20%), different heavy metals (1 mM) like Mn, Ni, Cd, Ag, Hg, Cu, Fe, Co, Zn and inhibitors (2 mM) such as carbonyl cyanide-m-chlorophenyl hydrazone (CCC), N,N,-Di-cyclohexyl carboiimide (DCC), sodium azide (NaN3), and 2, 4-dinitrophenol (DNP) on chromate reduction were measured in MH medium supplemented with 2 mM Cr(VI). All the experiments were done in triplicates and the average ± standard deviation (SD) of data was determined and reported.

Reduction of Cr(VI) in tannery effluent

Untreated tannery effluent containing 100 µM of Cr(VI) and 10% (w/v) NaCl collected from Kolkata based tanneries in Bantala, Kolkata, West Bengal, India was sterilized and used for the reduction studies. Both the raw effluent [100 µM, Cr(VI)] and effluent supplemented with additional hexavalent chromium (100 and 400 µM) were inoculated with freshly grown cultures of H. smyrnensis KS802 and incubated under continuous shaking (120 rpm) at 32 °C. The hexavalent chromium removal was expressed as % Cr(VI) reduced using diphenyl carbazide method.

The kinetics of Cr(VI) reduction in the tannery effluent was evaluated at different concentrations of Cr(VI) and expressed according the following equation (Camargo et al. 2003):
$${\text{y}} = {\text{ a}}.{\text{e}}^{{{-}{\text{kt}}}}$$
$${\text{C}}/{\text{Co }} = {\text{ a}}.{\text{e}}^{{{-}{\text{kt}}}}$$
Linearized form becomes: ln C/Co = lna − kt

where a is constant, C/Co is the fraction of Cr(VI) reduction at time t, C is the concentration of Cr(VI) at time t, Co is the original Cr(VI) concentration, and k is the rate constant.

All experiments were performed in triplicates and the average ± SD of data were reported.

Reduction associated changes of the cell mass

Fourier transformed infrared (FTIR) analysis

FTIR of bacterial cell mass was done following the method of Chandra and Singh (2014). The cell mass harvested at different phases of Cr(VI) reduction during growth was washed thrice with distilled water and lyophilized at − 56 °C. The dried cell mass and potassium bromide (1:24) were mixed, grinded and pressed to form pellet by using a hydraulic press. The FTIR spectrum (400–4000 cm−1) of the pellet was recorded in a Jasco-6300 (USA) IR spectrometer.

Scanning electron microscopy-energy dispersive X-ray (SEM–EDX) analysis

The surface properties of the chromate reducing bacterial cells were analyzed in a SEM, ZEISS EVO-MA 10 microscope and the existence of metal ion on the surface of the bacterial biomass was determined using EDX in a JEOL JSM-7600F; INCA Energy 250 and HKL Advanced EBSD system as per the method of Mishra et al. (2012).

Powder X-ray diffraction (p XRD)

Powder XRD was performed according to the method of Dhal et al. (2010). In brief, p XRD patterns of the dried cell mass was recorded in a Rigaku Ultima Diffractometer 1840 using Ni-filtered CuKα (λ = 1.315Å) radiation generated at 20–60 kV and 2–60 mA and the instrument was calibrated with standard silicon using a scintillation scan detector at slow scan in 2θ = 3–80°. The interplaner d-spacing at different θ values were calculated.

Results

Screening for Cr(VI) tolerance and reduction

Systematic screening of all 29 moderately halophilic bacterial isolates for hexavalent chromate tolerance and reduction during growth in MH medium led to the selection of the isolate, KS802 showing maximum Cr(VI) tolerance (10 mM) as well as complete reduction of 2 mM Cr(VI) during shortest incubation time (4 days).

Characterization and identification of the selected isolate

The selected Cr(VI) tolerant and reducing isolate, KS802 formed characteristic cream colored round colonies on MH agar medium. The isolate was Gram-negative, non-sporulating, motile rods and could tolerate 20% NaCl for growth. The isolate was capable of producing catalase and asparaginase, but was unable to produce amylase, lipase, glutaminase, cellulase, caseinase, xylanase, pectinase, inulinase, urease, phenylalanine deaminase, tryptophan deaminase and nitrate reductase (Table 1). It utilized glucose, fructose, galactose, sucrose, trehalose, ribose, propionate, acetate and fumarate as sole source of carbon and was resistant to antibiotics like bacitracin, erythromycin, vancomycin, neomycin, novobiosin, penicillin, methicillin, streptomycin and tetracycline. In addition to Cr (10 mM), the isolate was capable of tolerating Mn and Co (10 mM) followed by Pb (4 mM), Ni and Cu (2 mM). The morphological and physio-biochemical characteristics of the isolate KS802 as outlined in Table 1 were compared with those of halophilic genera described in Bergey’s Manual of Systematics of Archaea and Bacteria (Vreeland 2015) and the isolate was tentatively identified as a member of the genus Halomonas.
Table 1

Morphological, physiological and biochemical characters of the bacterial isolate KS802

Character

Response

Character

Response

Morphological

Production of

Colony character

Cream, round

Catalase

+

Gram nature

Gram negative (−)

Amylase

Cell morphology

Rods

Asparaginase

+

Arrangement

Single, chains

Glutaminase

Endospore formation

Cellulase

Motility

+

Caseinase

Pigment production

None

Xylanase

Physio-biochemical

 

Pectinase

pH range for growth

4–10

Inulinase

pH optimum for growth

7

Urease

Temperature range (°C)

22–45

Nitrate reductase

Temperature optimum (°C)

32

Phenylalanine deaminase

NaCl tolerance (%)

0.5–20

Tryptophan deaminase

NaCl optimum for growth (%)

7.5

Production of EPS

+

Growth on McConcy agar

Sensitivity to antibiotica

 

Utilization of

 

Ampicillin (Am30)

20 (S)

Glucose

+

Bacitracin (B10)

− (R)

Fructose

+

Ciprofloxacin (Cf30)

40 (S)

Galactose

+

Chloramphenicol (C30)

30 (S)

Ribose

+

Chlortetracycline (Ct30)

18 (S)

Sucrose

+

Erythromycin (E15)

13 (R)

Trehalose

+

Kanamycin (K30)

14 (S)

Glycerol

Methicillin (M5)

− (R)

Mannitol

Neomycin (N30)

9 (R)

Sorbitol

Norfloxacin (Nx10)

20 (S)

Acetate

+

Streptomycin (S10)

11 (R)

Fumarate

+

Tetracycline (TE30)

18 (R)

Propionate

+

Vancomycin (Va30)

− (R)

Tolerance to heavy metal (mM)

Cr(VI)

10

Pb(III)

4.0

Co(II)

10

Mn(II)

10.0

Ni(II)

2.0

Ag(I)

1.5

Cd(II)

1.5

Hg(II)

1.5

Zn(II)

1.5

Cu(II)

2.0

+ Positive response, − negative response, R resistant, S sensitive, values indicate diameter (mm) of inhibition zone

aSensitivity to antibiotic

The molecular identification of the isolate was confirmed by 16S rRNA gene sequence analysis. The amplified 16S rRNA gene from the isolate was subjected to sequencing and search carried out with this sequence using BLASTN revealed that the isolate KS802 (GenBank Accession No. KU982965) was most closely related (99% similarity) to Halomonas smyrnensis (Poli et al. 2013) and designated as H. smyrnensis KS802 (Fig. 1).
Fig. 1

Phylogenetic tree showing relationship of Halomonas KS802 with other closely related halophilic species based on 16S rRNA gene sequence analysis

Chromium reduction by the selected isolate

Time course of growth and chromate reduction

Time course of growth and chromate reduction by H. smyrnensis KS802 in MH medium under batch culture clearly indicated that consumption of glucose has led to the increase in biomass formation and associated reduction of Cr(VI) till the isolate attained the stationary phase (72 h) of growth with complete reduction of 2 mM hexavalent chromium (Fig. 2).
Fig. 2

Time course of growth [dry weight (■)], utilization of glucose (▲) and reduction of hexavalent chromium (∆) by H. smyrnensis KS802 in MH medium

Effect of Cr(VI) concentration, NaCl, carbon source, pH and temperature

Effect of initial concentration of Cr(VI) revealed complete reduction of 0.5–2 mM Cr(VI) in 72 h with minimum negative influence on the growth of the strain. Growth as well as reduction were significantly affected at higher concentrations of Cr(VI) (Table 2).
Table 2

Effect of Cr(VI), NaCl, galactose, pH and temperature on growth and reduction of Cr(VI) by H. smyrnensis KS802

Cultural conditions

Growth, dry weight (g L−1)

% Cr(VI) reduction

Incubation, h

48

72

48

72

Cr(VI) (mM)

0.5

6.68 ± 0.29

8.62 ± 0.12

100 ± 0.00

100 ± 0.00

1

6.28 ± 0.23

8.45 ± 0.11

95 ± 0.50

100 ± 0.00

2

6.18 ± 0.25

7.85 ± 0.11

88 ± 1.25

100 ± 0.00

4

5.59 ± 0.13

7.15 ± 0.15

68.5 ± 2.25

92.12 ± 1.25

6

2.23 ± 0.15

3.12 ± 0.23

32.5 ± 2.5

50 ± 2.5

NaCl (%, w/v)

0

0.01 ± 0.02

0.15 ± 0.29

2.25 ± 1.12

2.81 ± 0.25

5

6.58 ± 0.25

7.78 ± 0.21

88.7 ± 2.5

100 ± 0.00

10

5.42 ± 0.12

6.15 ± 0.25

81.25 ± 5.0

100 ± 0.00

15

2.87 ± 0.15

4.10 ± 0.39

69.75 ± 5.25

90.5 ± 2.5

20

0.56 ± 0.15

1.60 ± 0.25

12.62 ± 2.25

51.7 ± 1.12

Galactose (%, w/v)

1

6.06 ± 0.19

6.98 ± 0.41

95.00 ± 0.0

100 ± 0.00

2

6.22 ± 0.11

8.60 ± 0.14

97.25 ± 0.0

100 ± 0.00

4

8.02 ± 0.15

9.65 ± 0.25

100 ± 0.00

100 ± 0.00

6

6.22 ± 0.51

8.80 ± 0.21

78.50 ± 2.5

100 ± 0.00

Control

4.98 ± 0.35

7.75 ± 0.45

82.50 ± 1.1

100 ± 0.00

pH

5

5.24 ± 0.16

6.42 ± 0.13

78.5 ± 1.12

97.75 ± 2.25

6

5.55 ± 0.19

6.25 ± 0.18

93.25 ± 5.0

100 ± 0.00

7

6.21 ± 0.38

7.52 ± 0.12

100 ± 0.00

100 ± 0.00

8

5.45 ± 0.18

6.85 ± 0.12

88.5 ± 2.5

100 ± 0.00

9

1.78 ± 0.51

2.05 ± 0.25

67.62 ± 1.12

92.35 ± 1.12

Temperature (°C)

27

5.24 ± 0.26

7.25 ± 0.35

39.37 ± 1.12

75.75 ± 1.25

32

6.12 ± 0.23

8.41 ± 0.25

86.00 ± 1.5

100.0 ± 0.00

37

5.75 ± 0.25

8.82 ± 0.22

59.50 ± 1.0

89.50 ± 2.2

42

0.98 ± 0.25

1.15 ± 0.62

29.50 ± 1.5

50.50 ± 1.25

As against the optimum NaCl concentration (7.5%) for growth (Table 1), chromate reduction by H. smyrnensis KS802 was obtained at 5% NaCl (Table 2), while the inability of the isolate to reduce Cr(VI) in medium without NaCl indicated the salt dependency of KS802 for Cr(VI) reduction (Table 2).

Effect of different carbon sources on growth and chromate reduction by H. smyrnensis KS802 indicated potential growth and maximum reduction (93.25%) of 2 mM Cr(VI) with galactose. Significant reduction of chromate was also observed with sucrose (89.75%) followed by glucose (82.50%) (Fig. 3). However, growth as well as reduction of Cr(VI) were not supported by organic acids. Variation of galactose in the reduction medium showed complete chromate reduction in 72 and 48 h with 1 and 4% galactose respectively (Table 2).
Fig. 3

Effect of carbon source on growth () and reduction of Cr(VI) () by H. smyrnensis KS802 after 48 and 72 h of incubation

The optimum pH for maximum reduction by the present strain was pH 7 (100% reduction) and was followed by pH 6 (93.25%) and pH 8 (88.5%). However, at higher acidic and alkaline pH the reduction was not affected severely.

Cr(VI) reduction under optimized cultural conditions

Under optimized cultural conditions (4% galactose, 5% NaCl, pH 7 and 32 °C) biomass production was found to increase till 72 h of growth due to the presence of sufficient galactose in the growth medium but the complete reduction of 2 mM Cr(VI) was achieved only after 12 h.

Effect of metal ions

Presence of Mn, Cu and Pb as co-metal ions were non-toxic to H. smyrnensis KS802 and reduction of Cr(VI) was more or less identical to control. Rest of the metal ions were inhibitory to growth as well as Cr(VI) reduction and silver appeared to be most toxic to the isolate (Fig. 4).
Fig. 4

Effect of heavy metal on growth () and reduction of Cr(VI) () by H. smyrnensis KS802 after 48 and 72 h of incubation

Effect of inhibitors

Among the different inhibitors tested, carbonyl cyanide-m-chlorophenyl hydrazone (CCC) was most inhibitory to growth as well as chromate reduction by KS802. On the contrary, NaN3 and N,N,-Di-cyclohexyl carboiimide (DCC) exerted minor inhibition (12–15%) of the chromate reductase activity (Table 3)‚ while the uncoupler DNP was not inhibitory to Cr(VI) reduction by H. smyrnensis KS802.
Table 3

Effect of inhibitors on growth and reduction of Cr(VI) by H. smyrnensis KS802 after 48 and 72 h of incubation

Inhibitor (2 mM)

Growth, dry weight, g L−1)

% Cr(VI) reduction

Incubation, h

48

72

48

72

DNP

3.85 ± 0.23

7.85 ± 0.35

93.75 ± 0.11

100 ± 0.00

DCC

2.72 ± 0.29

6.7 ± 0.41

77.25 ± 0.75

85.0 ± 0.75

CCC

0.01 ± 0.12

0.09 ± 0.18

2.25 ± 2.5

0.25 ± 4.50

NaN3

2.58 ± 0.15

6.05 ± 0.15

88.00 ± 1.12

97.38 ± 1.12

Control

2.71 ± 0.45

6.05 ± 0.35

83.50 ± 1.50

100 ± 0.00

DNP 2, 4-Dinitrophenol, DCC N,N,-Di-cyclohexyl carboiimide, CCC carbonyl cyanide-m-chlorophenyl hydrazone, NaN3 sodium azide

Reduction of Cr(VI) in tannery effluent

The ability of H. smyrnensis KS802 to sustain the cumulative toxic effect of high NaCl and Cr(VI) concentrations has directed us to evaluate the efficiency of the bacterium in reducing Cr(VI) in tannery effluent. Time course studies of chromium reduction by H. smyrnensis KS802 in raw tannery effluent and tannery effluent amended with additional Cr(VI) [100 and 400 µM] were performed. The isolate completely reduced the Cr(VI) of the untreated tannery effluent (100 µM) in 6 h of incubation, while 95 and 79% of Cr(VI) from the amended samples [final concentrations 200 and 500 µM Cr(VI) respectively] were reduced during the same period of incubation (Fig. 5a).
Fig. 5

Hexavalent chromate reduction of tannery effluent containing 100 µM (■), 200 µM (●) and 500 µM (▲) of Cr(VI) (a) and rates of reduction at different levels of Cr(VI) (b) by H. smyrnensis KS802 in tannery effluent. Error bars indicate mean activities of triplicate assays ± SD

The kinetics of Cr(VI) reduction at different levels of Cr(VI) concentrations in the effluent was evaluated and the data fitted well (R2 ≥ 0.98) to the linearized form of exponential rate of equation. The highest (5.9 × 102 h−1) and lowest (1.8 × 10−2 h−1) rates of reduction were found at 100 and 500 µM respectively (Fig. 5b).

Reduction associated changes of the cell mass

FTIR analysis

Comparison of FTIR spectra of lyophilized Cr(VI) reducing cell mass of H. smyrnensis KS802 with the non-reducing cell mass (Fig. 6) revealed no change of peaks in the range of 2925–2336 cm−1. Appearance of new peaks (1725.99, 1727, 1723 cm−1) and shifting of peaks at 1398, 1311, 1235 and 1087 cm−1 were recorded in cell mass carrying chromate reduction at 2-6 mM Cr(VI). The additional peaks at 912 and 827 cm−1 could possibly be due to the Cr=O vibration of reduced Cr-species.
Fig. 6

Comparative FTIR spectra of lyophilized Cr(VI) reducing cell mass [2 mM (blue), 4 mM (green) and 6 mM (brown)] of H. smyrnensis KS802 with non-reducing one (black)

SEM–EDX

Scanning electron microscopic (SEM) analysis revealed distinct changes in cellular morphology of the isolate during growth and reduction of chromate [2–6 mM Cr(VI)] under shake conditions after 72 h of incubation (Fig. 7). The SEM–EDX of the lyophilized cell mass also showed an increase in the content of Cr with increasing concentrations of Cr(VI) in the reducing medium (Table 4).
Fig. 7

Changes in cellular morphology of H. smyrnensis KS802 during growth associated reduction of hexavalent chromium after 72 h of incubation [a Control (scale, 100 nm; magnification, ×10,000); b 2 mM (scale, 100 nm; magnification, ×5000); c 4 mM (scale, 100 nm; magnification, ×5000) and d 6 mM (scale, 100 nm; magnification, ×5000) Cr(VI)]

Table 4

Elemental analysis of lyophilized cell mass of H. smyrnensis KS802 under SEM–EDX following reduction of Cr(VI) after 72 h of incubation

Element

Weight %

Untreated

Treated

2 mM

4 mM

6 mM

C

43.21

12.12

54.99

55.60

O

23.81

30.77

18.47

36.20

Na

3.80

6.81

2.70

2.94

K

3.52

6.61

1.29

3.84

Cr

0.49

0.70

1.42

Powder XRD

Powder XRD patterns of bacterial cells grown with or without Cr(VI) in MH medium are shown in Fig. 8, which give further insight into the nature of reduced product. It was evident that the peaks (2θ values at 42.96 and 76.36) of lyophilized cell mass of bacteria grown in absence of Cr(VI) have not only disappeared in the XRD patterns of cells associated with chromate reduction but also resulted in the appearance of distinct major peaks of 2θ values at 31.81, 45.54, 56.56 and 75.35, the intensity of which has increased significantly with increase in Cr(VI) concentration in the reducing medium.
Fig. 8

Powder XRD patterns of bacterial cell mass grown without (black) or with 2 mM (brown), 4 mM (green) and 6 mM (blue) Cr(VI) in MH medium

Discussion

The biotransformation of highly toxic and mutagenic Cr(VI) to relatively non-toxic Cr(III) by chromate-reducing bacteria offers an economical as well as eco-friendly option for Cr bioremediation. The present study reports a detailed account of growth-associated reduction of Cr(VI) under different environmental conditions by a halophilic bacterial isolate KS802 which was identified as H. smyrnensis KS802 following morphological, physio-biochemical and 16S rRNA analysis. Chromium resistance of KS802 was similar to other halophilic strains isolated from Soap Lake or metal contaminated soil samples (Van Engelen et al. 2008; Shapovalova et al. 2009; Pinon-Castillo et al. 2010; Subramanian et al. 2012; Voica et al. 2016) and was more efficient in reducing Cr(VI) compared to H. chromatireducens, Halomonas sp. TA-04 and Halomonas sp. CSB5 (Shapovalova et al. 2009; Focardi et al. 2012; Chandra and Singh 2014). Besides Halomonas spp., the present strain also exhibited superior reducing activity over those of Nesterenkonia sp. strain MF2 (Amoozegar et al. 2007), Virgibacillus sp. (Mishra et al. 2012) and Planococcus maritimus (Subramanian et al. 2012).

Chromate reduction efficiency of the isolate KS802 was found to decrease with increasing concentration of Cr(VI) similar to those reported for Halomonas sp. TA-04 (Focardi et al. 2012) and Providencia sp. (Thacker et al. 2006). Chromium reduction in presence of NaCl by halophilic as well by non-halophilic organisms has been reported by several authors (Amoozegar et al. 2007; Shapovalova et al. 2009; Das et al. 2014). The Cr(VI) removal efficiency of KS802 decreased with increase in NaCl concentration above 5% and supported the interdependency of Cr(VI) reduction and NaCl concentration (Okeke et al. 2008). Like most chromate reducing bacteria, KS802 also utilized variety of carbon sources with galactose as the best one for reduction of chromate during growth (Thacker et al. 2006; Dhal et al. 2010; Mabrouk et al. 2014).

As an enzyme-mediated bio-process, reduction of Cr(VI) was influenced by changes in pH and temperature of the medium and revealed its neutrophilic and mesophilic status. However, a higher alkaline (8-10) optima for reduction of Cr(VI) in H. chromatireducens (Shapovalova et al. 2009) and Halomonas sp. M-Cr (Mabrouk et al. 2014) as well as lower temperature (28 °C) in Halomonas sp. strain TA-04 (Focardi et al. 2012) are not uncommon.

Chromate reduction by H. smyrnensis KS802 was highly active in presence of Mn, Cu and Pb similar to Bacillus sp. ES29 (Camargo et al. 2003), which could be effective for Cr(VI) removal from industrial wastewater co-contaminated with heavy metals and soluble salts. Moreover, stimulatory effect of Cu2+ on chromate reduction may be due to participation of Cu2+ as a prosthetic group of the enzymes which acts as an electron redox centre, and help in the shuttle of electron between different subunits (Meghraj et al. 2004; Dey and Paul 2012) of the enzyme. The inhibitors of cytochrome oxidase, enolase and ATPase (Opperman and van Heerden 2007; Dey and Paul 2012) like NaN3 and DCC and the uncoupler DNP (Wani et al. 2007) were not severely inhibitory to chromate reductase of H. smyrnensis KS802.

Reduction of Cr(VI) contained in the untreated tannery effluent with high NaCl was accomplished by H. smyrnensis KS802 without the supplementation of any additional carbon or nitrogen sources and appeared to be much more efficient than the strains reported earlier (Ganguli and Tripathi 1999; Srinath et al. 2002; Sharma and Adholeya 2012). The reduction kinetics of Cr(VI) in tannery effluents as observed with KS802 corroborated well with those of Virgibacillus sp. (Mishra et al. 2012) and Bacillus sp. (Camargo et al. 2003; Dhal et al. 2010).

It is apparent that the product of reduction of Cr(VI) formed during growth could be precipitated or coordinated to active functional sites of bacterial cell surface depending on the type of the bacterial strain and process of reduction. FTIR spectrum of the isolate revealed binding and stabilizing of reduced Cr-species to functional groups like –COO–, –OH and –C=O on the cell surface (Ramesh et al. 2012; Chandra and Singh 2014). The additional peaks at 912 and 827 cm−1 could possibly be due to the Cr=O vibration of reduced Cr-species. The SEM–EDX of the cells showed changes in cellular morphology during Cr(VI) reduction and a sequential increase in Cr in the cell mass with increasing Cr(VI) concentrations in the growth medium. Such transformation of rod-shaped cells of KS802 to filamentous form received support from the work of Ackerley et al. (2004) and the increased Cr in the cell mass was supported by earlier studies of Mishra et al. (2012). Powder XRD patterns of bacterial cells grown with or without Cr(VI) showed peaks that closely match with those of chromium hydrogen phosphate (CrH2P3O10. 2H2O) (Dhal et al. 2010). Moreover, the minor peaks at 28.45, 40.68 and 66.42 could possibly be assigned to crystalline Cr(III) compounds formed during biological reduction of Cr(VI) as has been established by several authors (Meghraj et al. 2004; Mishra et al. 2012; Das et al. 2014).

Conclusions

The present study evaluates the efficiency of Cr(VI) bioreduction by H. smyrnensis KS802 during growth under saline environment over a broad range of pH and toxic metal ions. The isolate was also capable of reducing Cr(VI) in tannery effluent containing high concentration of NaCl. FTIR analysis of cells harvested after reduction process indicated the involvement of functional groups on the cell surface in the complexation of reduced Cr species which was further justified by SEM–EDX studies. Moreover, it may also be concluded from the XRD pattern that Cr(VI) reduction by H. smyrnensis KS802 is more complex than the direct production of insoluble Cr(OH)3 and the formation of organo-Cr(III) complexes could be an integral part of the biogeochemical cycling of chromium.

Notes

Acknowledgements

This study was financially supported by grants from University Grants Commission, India under the scheme of Rajiv Gandhi National Fellowship (Sanction No. F.14-2(SC)/2008 (SA-III), 31 March, 2009).

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest.

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Copyright information

© Society for Environmental Sustainability 2018

Authors and Affiliations

  • Jhuma Biswas
    • 1
  • Pritha Bose
    • 1
  • Sukhendu Mandal
    • 2
  • A. K. Paul
    • 1
  1. 1.Microbiology Laboratory, Department of BotanyUniversity of CalcuttaKolkataIndia
  2. 2.Department of MicrobiologyUniversity of CalcuttaKolkataIndia

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