Isolation, characterization, and multiple heavy metal-resistant and hexavalent chromium-reducing Microbacterium testaceum B-HS2 from tannery effluent

2.1 Isolation and characterization of Cr⁶⁺ resistant bacterial isolate

Wastewater samples from tannery industries were collected aseptically from the various locations in Sheikhupura, and Qasoor, Lahore, Pakistan. Effluent samples were plated onto the modified Luria-Bertani (LB) agar amended with 1 mM Cr⁶⁺ in the form of K₂Cr₂O₇ and incubated for 24–72 h at 37 °C. Screening of the bacteria was done on the basis of their ability to resist and reduce higher Cr⁶⁺ concentrations present in the medium (Fig. S1).

2.2 Bacterial identification

The isolate B-HS2 was characterized biochemically. For molecular characterization, genomic DNA of the selected strain was isolated according to Masneuf-Pomarède et al. (2007) and 16S rRNA gene fragment analysis was carried out by PCR amplification using universal bacterial primers RS1 (5-AAACTCAAATGAATTGACGG-3) and RS3 (5-ACGGGCGGTGTGTA-3) (Rehman et al., 2007). Fermentas purification kit (#K0513) was used to clean PCR product, and sequenced with Genetic analysis system model CEQ-800 (Beckman) Coulter Inc. Fullerton, CA, USA. The data obtained after sequencing was subjected to BLAST analysis.

2.3 Growth conditions of the bacterial isolate

The optimum growth conditions (temperature and pH) were determined according to Elahi and Rehman (2019). Effect of presence of Cr⁶⁺ on growth patterns of B-HS2 was scrutinized by growing bacterial strain with and without Cr⁶⁺ stress. Bacterial isolate B-HS2 was cultivated in LB broth, in mineral salt medium (MSM) broth [g/L: FeSO₄·7H₂O 0.015 g, KH₂PO₄ 4.7 g, MgSO₄·7H₂O 1 g, CaCl₂·2H₂O 0.01 g, Na₂HPO₄ 0.12 g, NH₄NO₃ 4 g, MnSO₄·4H₂O 0.01 g, glucose 10 g and yeast extract 5 g (pH 7–7.2)] without Cr and MSM broth supplemented with 2 mM K₂Cr₂O₇ (experimental). The cell mass was determined by taking O.D at 600 nm with the interval of 4 h until 24 h of growth period.

2.4 Cross metal resistance

Minimum inhibitory concentration (MIC) value is regarded as the minimum concentration of metal able to inhibit bacterial growth. To determine MIC of metal ions, various concentrations of Cr⁶⁺ (K₂Cr₂O₇) and other metal heavy metal ion salts such as CuSO₄·5H₂O, CdCl₂, NiCl₂·6H₂O, PbNO₃, NaAsO₂ and ZnSO₄·7H₂O were separately added to 100 ml of MSM broth and were supplemented with 24 h old culture. These flasks were incubated under shaking conditions (150 rpm) at 37 °C in rotary shaker for 7 days. Growth was determined by taking absorbance at 600 nm.

2.5 Chromate reductase assay

2.6 Effect of pH, temperature, and metals on enzyme activity

Optimal pH for the activity of chromate reductase enzyme was assayed in buffers of different pH values (4.0–9.0). Each reaction contained 50 mM particular pH buffer with NADH (0.1 mM), Cr⁶⁺ (20 µM), and crude enzyme (100 µl), and incubated for 30 min. Chromate reductase activity was checked at different temperatures (30 °C–90 °C). Metals influence on the enzyme activity was determined by supplementing the reaction mixture with 0.1 mM concentration of metal ions (Na⁺, Cu²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Hg²⁺, and Zn²⁺), in the reaction system. Enzyme activities were assayed according to Sarangi and Krishnan (2008).

2.7 Measurement of antioxidatants and glutathione contents

Antioxidants profiling of B-HS2 under Cr⁶⁺ stress was evaluated by growing strain in 100 ml MSM medium and incubated at 37 °C. Then broth was amended with Cr⁶⁺ stress (2 mM) after 24 h, and incubated again for a day. Bacterial cells were harvested by centrifugation at 14,000 rpm for 10 min, and weight of pellet was noted, and re-suspended in PBS before subjected to sonication. The sonicated pellet was centrifuged and the supernatant was used as antioxidant enzymes. Glutathione transferase (GST) was evaluated as mentioned by Habig et al. (1974). Peroxidase (POX) was assayed according to Reuveni et al. (1992) with minor modifications. Beers and Sizer (1952) method was used to determine catalase activity. Ascorbate peroxidase (APOX) activity was evaluated according to Israr et al. (2006) and Nakano and Asada (1987). The superoxide dismutase (SOD) activity was measured according to Ewing and Janero (1995).

Under chromium stress, the alteration in the production of glutathione (GSH) and non-protein thiols (NPSHs) was estimated according to Khan et al. (2015). Briefly, three flasks of culture were prepared and 2 mM K₂Cr₂O₇ stress was given in two flasks after 24 h, and third flask acted as control because no metal was added in this flask. All the flasks were again incubated for another 48 h. Culture was harvested, washed thrice with 1 mM PBS, weighed and re-suspended in 5% sulfosalicyclic acid (1 ml). Pellet was broken down by sonication, followed by centrifugation at 14,000 rpm for 10 min at 4 °C and aliquot was divided into two equal parts. One part was used to assess GSH level and other part was used to estimate NPSHs level. The amount of GSH, oxidized glutathione (GSSG) and NPSHs was quantified by Khan et al. (2015).

2.8 Metal processing ability of the bacterial isolate

The ability of metal processing of the bacterium was evaluated by measuring changes in the quantity of Cr⁶⁺ in the culture medium by atomic absorption spectrophotometer (Zahoor and Rehman, 2009; Elahi and Rehman, 2019).

2.9 Chromate reduction in tannery effluent

The Cr⁶⁺ reduction potential of B-HS2 was determined in tannery effluent. Three plastic containers were used; the first container carried the control 1 (10 L original tannery wastewater) while the second container carried control 2 (10 L distilled water, inoculated with 1.5 L culture), and the third container was filled with tannery wastewater (10 L) with 1.5 L culture. All containers were given 2 mM Cr⁶⁺ stress and incubated at room temperature (25 ± 2 °C). Samples (10 ml) were withdrawn after regular intervals (2, 4, 6, and 8 days of incubation). Cells were harvested at 4000 rpm for 10 min and supernatants were used for the estimation of Cr⁶⁺ by Diphenylcarbazide method. The calibration curve, established under the same experimental conditions using standards of Cr⁶⁺ solution, was used to calculate any change in the Cr amount before and after bacterial treatment.

2.10 Effect of microbial treated wastewater on Vigna radiata

Effectiveness of microbially treated tannery effluent was ascertained for the plant cultivation. In this, autoclaved soil was used to prepare small pots and Vigna radiata (mung beans) seeds were sown into each pot. The experimental pot was watered with the treated effluent while watering of control plants were done with either tap water or untreated (original) wastewater. Seeds were allowed to grow under 1:1 light and dark period for 10 days. To determine the effects of toxic Cr⁶⁺ metal on plant growth, changes in plants growth with bacterially purified wastewater and untreated wastewater, was observed.

2.11 Bacterial protein profiling though SDS PAGE

Bacterial isolate was cultures in MSM liquid medium in the presence and absence of 2 mM Cr⁶⁺. The samples were prepared and SDS-PAGE was done according to Laemmli (1970).

2.12 FTIR, SEM and EDX analysis

FTIR spectroscopy was employed to obtain Infrared spectra for B-HS2 under Cr⁶⁺ stress. Specimens were treated as mentioned by Deokar et al. (2013). In order to investigate the mechanism of metal-microbe interaction, it is vital to confirm the presence of chromium species (Cr⁺³, Cr⁶⁺) within the microbial cell and for this purpose EDX analysis was done.

For scanning electron microscopy, bacterial samples were treated as mentioned by Khan et al. (2016). In short, bacterial culture with and without 2 mM Cr was prepared and a drop of suspension was mounted onto the aluminum stub and treated as described by Khan et al. (2016). With sputter coater, samples were covered with gold film (Denton, Desk V HP) and examined through scanning electron microscope (Nova NanoSEM 450) equipped with Oxford energy dispersive X-ray (EDX) microanalysis system.

2.13 Statistical analysis

Each experiment run in triplicate and observations were made. Each experiment was done in at least three separate flasks. Each time three readings were taken, their mean, and standard error of the mean were calculated.

3.1 Physiochemical characteristics

Temperature of the effluent samples ranged from 22 to 34 °C, and pH range was 6.0–10.0. The colonies appeared on the LB agar plates containing 2 mM Cr⁶⁺ were further streaked on higher concentrations of Cr⁶⁺. The isolate that resisted highest Cr⁶⁺ concentration (48 mM) was selected and named as B-HS2.

3.2 Biochemical and molecular characterization of bacterial isolate

B-HS2 is a gram positive strain and its biochemical characteristics are mentioned in Table S1. Molecular identification through 16S ribotyping showed that it shares 91% homology with Microbacterium testaceum (Accession number JQ916902). The phylogenetic tree of M. testaceum was constructed to determine its similarity with other Microbacterium strains using MEGA7 program taking bootstrap value 500 (Fig. S2).

3.3 Optimization of growth conditions

M. testaceum B-HS2 optimum growth was determined as 37 °C and pH 7. M. testaceum B-HS2 growth was substantially declined in presence of Cr⁶⁺ as compared to the culture containing no Cr⁶⁺ (Fig. 1).

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Fig. 1

Growth curves of M. testaceum B-HS2 in LB medium (control), mineral salt medium (MSM), and MSM supplemented with 2 mM K₂Cr₂O₇ (treated) incubated at 37 °C. Optical density was taken at 600 nm after regular time interval.

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3.4 Cross metal resistance

M. testaceum B-HS2 could resist Cr⁶⁺ upto 48 mM and also capable of tolerating other heavy metals, viz., 7 mM (Pb²⁺), 2 mM (Zn²⁺), 2 mM (Cu²⁺), 2 mM (As³⁺), 1 mM (Cd²⁺), and 1 mM (Ni²⁺). Resistance order according to metal ions concentration was Cr⁶⁺ > Pb²⁺ > As³⁺/Zn²⁺/Cu²⁺ > Cd²⁺/Ni²⁺.

3.5 Characteristics of chromate reductase

Chromate reductase of M. testaceum B-HS2 showed optimum activity at pH 7.0 (Fig. 2a) and at 40 °C (Fig. 2b). All the heavy metals used inhibit the enzyme activity (Fig. 2c).

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Fig. 2

Effect of pH (a), temperature (b), metal ions (c) on the chromate reductase activity and changes in antioxidant enzymes activity profile (d), exhibited by M. testaceum B-HS2 upon exposure of 2 mM Cr⁶⁺.

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3.6 Quantification of antioxidants enzymes, glutathione and Non-protein thiols

Cr⁶⁺ presence stimulates activities of all the antioxidative enzymes. Significant increase in POX (86%) and CAT (42%) activities was estimated, however, a relatively less increase in activities of APOX (19%), SOD (28%), and GST (14%) was also determined (Fig. 2d). Cr⁶⁺ stress also stimulates GSH and NPSHs levels in M. testaceum B-HS2 (Table S2). In the presence of 2 mM Cr⁶⁺, 326% increase in GSH was determined as compared to the control. An increase in non-protein thiols was also determined (122.25%) in metal stressed culture when compared with the non-metal stressed culture.

3.7 Cr⁶⁺bioprocessing potential of B-HS2

3.7.1

3.7.1 Biosorption of Cr⁶⁺

Biosorption potential of M. testaceum B-HS2 was assessed by cultivating it in LB broth supplemented with 2 mM Cr⁶⁺ (Fig. 3a). Biosorption proficiency (q) of M. testaceum B-HS2 was determined after 2, 4, 6 and 8 days which was 31, 38, 66, and 47 mM/g, respectively. Cr⁶⁺ was also found within bacterial cells i.e., 13, 17, 27 and 12 mM/g, after 2, 4, 6, and 8 days respectively. While 9, 14, 29 and 26 mM/g of Cr⁶⁺ got adsorbed on the cell surface after 2, 4, 6, and 8 days, respectively.

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Fig. 3

(a) Biosorption of Cr⁶⁺ by M. testaceum B-HS2 at lab scale incubated for 8 days. (b) FTIR spectra of M. testaceum B-HS2 in the presence and absence (control) of hexavalent chromium stress (2 mM). (c) Images of scanning electron microscopy of M. testaceum B-HS2 in the absence (C-i) and presence (C-ii) of Cr⁶⁺ (2 mM).

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3.8 Phytotoxicity study

The phytotoxicity experiment was conducted in order to determine any change in toxicity of the original wastewater post bacterial treatment. The setup consist of control 1 (water containing 2 mM Cr⁶⁺), control 2 (wastewater containing 2 mM Cr⁶⁺), and treated wastewater (wastewater containing 2 mM Cr⁶⁺ also supplemented with the bacterial culture M. testaceum-B-HS2), and all the three containers were incubated for 6 days at room temperature. The results clearly showed that the Cr⁶⁺ concentration remained unchanged in control 1 and 2, while in treated container, M. testaceum- B-HS2 has potentially removed 99% of Cr⁶⁺, clearly demonstrated by change in color and also confirmed by estimation through diphenylcarbazide method. The treated and control samples were then utilized for the cultivation of seeds of Vigna radiata. Thus phytotoxicity study results revealed that the treated tannery effluent can be safely used for the growth of plants as demonstrated by the decent germination of Vigna radiata when watered with the bacterially processed tannery wastewater as compared to the control (original wastewater). The toxicity of original wastewater get highly reduced after bacterial treatment accompanied by the reduction of noxious toxic Cr⁶⁺ (Fig. S5).

3.9 Protein analysis

Presence of Cr⁶⁺ in the medium imposes changes in cell physiology and proteomics. In this regard protein profiling of M. testaceum B-HS2 was ascertained in absence (control) and presence of 2 mM Cr⁶⁺ (treated) by SDS-PAGE. Cr⁶⁺ presence trigger over expression of some of the proteins and some proteins get diminished. For instance an approximately 65 kDa protein is present in control culture sample while it disappeared under stress (2 mM Cr⁶⁺). Low molecular weight protein bands (20 and 16 kDa) indicate the induction of metallothioneins under Cr⁶⁺ stress (Fig. S6).

3.10 FTIR, SEM and EDX analysis

FTIR analysis of M. testaceum B-HS2 with and without chromium is shown in Fig. 3b. Infrared spectra of M. testaceum B-HS2, when grown normally, showed characteristic absorption peaks of amino, hydroxyl, carboxyl, and sulphonate groups ascertained their existence on cell surface. However, when bacterium was challenged with 2 mM Cr⁶⁺, alteration in peaks were detected in the region of 3275 cm⁻¹ and 1800–1000 cm⁻¹ (Bennett et al., 2010). FTIR results revealed that when chromium first interacts with the cell surface, it gets sequestered on the surface via adsorption. This could be a result of surface complexation of chromium with the functional groups present on the cell wall. As a result, changes in the absorption peaks of the spectrum were witnessed. SEM (Fig. 3ci–ii) and EDX analysis (Fig. S7) also support the evidence of intracellular accumulation of Cr⁶⁺.