Efficient removal potential of Microbacterium sp. strain 1S1 against arsenite isolated from polluted environment

2.1 Sample collection and isolation of arsenic resistant bacteria

For isolation of arsenic resistant bacteria, wastewater samples were collected from drainage of chemical industries situated in the area of District Sheikhupura, Pakistan. This is a hub of industries. The screw capped autoclaved bottles were used for samples collection. The physicochemical features like temperature, pH, total dissolved solids (TDS), and arsenic concentration were measured in wastewater samples (Butt and Rehman, 2011).

For the isolation of arsenic resistant bacteria 100 μl of wastewater sample was spread on Luria-Bertani (LB) agar plates containing 5 mM arsenic stress. LB agar plates were prepared by dissolving 5 g NaCl, 10 g tryptone, 5 g yeast extract in 1 L of distilled water, pH was adjusted at 7 and then 15 g agar was added in 1 L flask. LB medium autoclaved at 121 °C for 15 min and growth of bacterial colonies was observed after overnight incubation at 37 °C.

2.2 Arsenite oxidation determination

Isolated strain was streaked on the LB-agar plate supplemented with 5 mM arsenite stress. After 3 days of incubation silver nitrate solution (0.1%) was used to flood the bacterial colonies. After that, results were noted according to Simeonova et al. (2004).

2.3 Cross metal resistance

The minimum inhibitory concentration (MIC) of arsenite against the bacterium was determined by streaking the strain on LB agar plates with different arsenic stress, 5, 10, 20, 30 mM up to 100 mM and cultured at 37 °C for 24 h. The isolated bacterial strain was also processed for its capability to resist other toxic metal ions like arsenate, lead, cadmium, chromium, nickel, mercury, zinc, selenium and cobalt. For this purpose various concentrations of different heavy metals were used by supplementing them into LB-agar plates and incubating the streaked plates overnight at 37 °C for 24 h (Naureen and Rehman, 2016). MIC is the minimum amount of chemical that inhibits the growth of microorganisms by adding the chemical into the media.

2.4 Morphological and biochemical characterization

After the isolation of arsenic resistant bacteria, various biochemical tests were performed. These are gram staining, spore staining, catalase test, oxidase test, growth on blood, and growth on chocolate and MacConkey agar. Other tests like citrate utilization test, SIM agar test, indole test, nitrate reduction test, MR and VP test were also performed for bacterial identification. The bacterium was also characterized at molecular level. Briefly, genomic DNA of Microbacterium sp. 1S1 was isolated, which was then subjected to 16S rRNA gene amplification through primers (Elahi and Rehman, 2019). The amplified products were mailed for sequencing to Macrogen, Korea and the sequence obtained was then aligned by utilizing BLAST analysis. Moreover, a dendrogram was created by using the MEGA7 program on the basis of homology.

2.5 Growth conditions and effect of metal on bacterial strain

The culturing environment (i.e. optimum pH and temperature) of isolated strain was accessed by growing at different temperature (25, 30, 37, and 42) and pH (5, 7, 9, and 11) in LB broth (Naureen and Rehman, 2016). Effect of arsenite on the isolated bacterial strain was determined by culturing the bacterium in LB broth supplementing with 1.33 mM (100 μg/ml) arsenite into the medium. The growth of bacterium was monitored by taking the optical density at 600 nm after every 4 h for up to 36 h.

2.6 Bacterial oxidizing potential

Strain 1S1 was accessed for its potential to oxidize arsenic through culturing in 250 ml flasks containing 100 ml LB broth. For this, 3 flasks supplemented with arsenite 15 mM were maintained. Out of 3 flasks, one acted as control, having the same amount of metal but without bacterial cells. Three parameters i.e. temperature, pH, and arsenite concentration (mM) were considered to optimize the oxidation potential of bacterium. Temperature (20 °C, 25 °C, 30 °C, 37 °C, and 42 °C), pH (3, 5, 7, 9 and 11) and arsenic concentration (15, 30, 45, 60, and 75 mM ml⁻¹) were maintained during the experiment. The bacterium arsenic oxidizing ability was determined with a suitable time period of 24 h upto 96 h. To harvest bacterial cells from each and every flask 5 ml sample was taken out and centrifuged at 3000 rpm for 5 min. Amount of arsenic was assessed through the P S Analytical Millennium Excalibur Method (Sher et al. 2020a).

2.7 Arsenite removal from industrial wastewater by Microbacterium sp. strain 1S1

The experiment to ascertain the potential of bacterium to eradicate arsenic from the real industrial wastewater at room temperature (25 °C ± 2) along with arsenite concentration (15 mM) was performed according to Elahi and Rehman (2019).

2.8 Measurement of antioxidant enzymes and glutathione contents

Behavior of antioxidant enzymes of bacterial strain 1S1 was evaluated in arsenite presence. Briefly, the strain was grown in 100 ml minimal salt medium and then was placed in a shaking incubator at 37 °C containing 10 mM As³⁺ stress. After 48 h of incubation, cells were harvested through centrifugation at 14,000 rpm for a time period of 10 min. The obtained pellets were weighed, dissolved in phosphate buffer, and finally were sonicated. The sonicated pellets were centrifuged at 14,000 rpm for 10 min and aliquots obtained were used for antioxidant enzymes assays. Peroxidase (POX) enzyme reaction was performed according to Reddy et al. (1995) while the catalase, ascorbate peroxidase (APOX) and superoxide dismutase (SOD) reactions were performed according to protocols of Luck (1974), Ratkevicius et al. (2003), and Marklund and Marklund (1974), respectively.

The level of GSH, GSSG, and NPSHs was estimated with and without arsenite for strain 1S1 according to protocol mentioned in Shamim and Rehman (2015).

2.9 SEM, EDX and FTIR analysis

To perform scanning electron microscopy, bacterial samples were treated and prepared as described in Khan et al. (2016). Briefly, microbial culture in the absence and presence of 15 mM arsenite was prepared and a drop of suspension was mounted onto the aluminum stub and treated as described by Khan et al. (2016). The gold film was used to wrap samples with a sputter coater (Denton, Desk V HP) and examined via Scanning Electron Microscope (Nova NanoSEM 450) attached with Oxford Energy Dispersive X-ray (EDX) analysis system. The Fourier-transform infrared spectroscopy (FTIR) was used to get Infrared spectra for strain 1S1 in arsenite challenge and samples were prepared according to protocols mentioned in Deokar et al. (2013). To understand the underlying mechanism of metal-microbe interaction, it is important to validate the metal presence inside the cells of bacteria. EDX analysis was performed to achieve this objective.

2.10 Statistical analysis

For each experiment at least three separate flasks were maintained. Each time three readings were taken, their mean, and standard error of the mean were calculated.

3.1 Wastewater characteristics and arsenite resistant bacterial isolation

Physicochemical characteristics like temperature, pH, TDS, color and arsenic amount were measured for the environmental sample. The sample characteristics were i.e. temperature 25 °C, pH 6.5, TDS 15500 mg/l and color was muddy. The arsenic concentration, determined from the sample, was 85 µg/l. A lot of bacterial colonies appeared on LB agar supplemented with 5 mM arsenite stress. By increasing the arsenite concentration in agar the number of colonies was decreasing and at the end just one bacterial strain appeared on 75 mM arsenite stress, was selected for further research work. The isolated strain was named as 1SI with MIC of 75 mM corresponding with arsenite.

3.2 Strain identification

The strain isolated in this study was gram positive, rod-shaped and yellow pigmented. Morphological and biochemical characteristics of the isolate are given in Table S1. The growth of bacterial strain on the LB agar plate can be seen in Fig. S1. The 16S rRNA gene sequence of bacterium showed 99% homology with 16S rRNA gene sequence of Microbacterium sp. and was deposited to GenBank under accession number of CP043430. Microbacterium sp. strain 1S1 has also been submitted at First Fungal Culture Bank of Pakistan under accession number FCBP-B-731.

3.3 Arsenite oxidizing test

To check the oxidizing ability of isolated strain, brown precipitation appeared on agar plates having bacterial colonies by applying the AgNO₃ (Fig. S2).

3.4 Cross metal resistance

The MIC of isolated strain against other commonly found metal ions is in following concentration Pb (5.5 mM/ml), Cd (4 mM/ml), Cr (5 mM/ml), Ni (4 mM/ml), Hg (6 mM/ml), Se (3 mM/ml), and Co (8 mM/ml).

3.5 Growth conditions and arsenite impact on bacterium

Isolated strain 1S1 showed maximum cell density at 37 °C and pH of 7. The maximum cell density of the strain was measured after 28 h of incubation. The cell density of strain 1S1 culture was higher in control (without arsenite) as compared to the strain 1S1 culturing in arsenite stress (Fig. S3).

3.6 Metal decontamination potential

The bacterial strain 1S1 was grown for 96 h at its optimum culturing conditions (pH 7; 37 °C) and arsenite oxidizing ability was measured at suitable time period of 24 h up to 96 h. Bacterial efficiency of arsenite oxidation was measured after 24, 48, 72, and 96 h was 40, 65, 85, and 98%, respectively (Fig. 1a).

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Fig. 1

Strain 1S1 was grown at its optimum growth conditions in 250 ml flasks containing 100 ml LB broth with arsenite @15 mM. Bacterium oxidizing potential was checked at time period i.e. 24, 48, 72 and 96 h (a), temperature i.e. 20 °C, 25 °C, 30 °C, 37 °C, and 42 °C (b), pH i.e. 3, 5, 7, 9, and 11 (c), and arsenic concentration i.e.15, 30, 45, 60, and 75 mM (d) after 48 and 96 h of time period. Each treatment was repeated thrice (n = 3).

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3.7 Factors versus arsenite oxidizing potential

The metal oxidizing potential of bacterium at various factors including initial arsenite concentration, pH, and temperature was worked out for a time period of 48 and 96 h of incubation.

3.8 Arsenic biosorption through bacterium heat inactivated biomass

After heat treatment, the bioremediation efficiency (E) was ascertained for 10 h with a time period of 2 h. Microbacterium sp. showed arsenite removal efficiency (E) of 60, 75, 89, and 95% after 2, 4, 6, and 8 h of regular time period. Bacterium inactivated cells eradicated 99% arsenite from the medium after 10 h of incubation contact (Fig. 2a).

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Fig. 2

(a) The bacterial culture was grown, centrifuged to obtain pellet, and pellet was incubated at 70 °C (2–3 times) to inactivate the cells. Initially, 1 g/L bacterial biomass was mixed in 1 L of arsenic solution of 1500 ml flasks containing 15 mM As⁺³ stress. The flasks were incubated at 37 °C on the shaker for 2 to 10 h. Arsenic concentration was estimated before and after incubation through atomic absorption spectrophotometer (b) Arsenite removal potential of strain ISI from industrial wastewater was determined. For this, a set of two plastic containers was used; the first container contained wastewater (10 L) with 1.5 L culture, while the second container contained 10 L distilled water with 1.5 L culture. Each container was supplemented with 15 mM As⁺³ and incubated at room temperature (25 ± 2 °C). Ten milliliter samples were taken after 2, 4, 6, and 8 days of incubation, centrifuged for 10 min at 4000 rpm, and the samples supernatants were analyzed for arsenite estimation through atomic absorption spectrophotometer.

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3.9 Arsenic amelioration from real wastewater

It was ascertained that bacterium has the potential to decrease 15 and 24% arsenite from real and distilled water after 48 h of incubation. After 4 days, bacterium was able to eradicate arsenite 24 and 42% from the real wastewater and distilled water. At day 6, bacterium was able to remove 48 and 72% arsenite from wastewater and distilled water and after incubation of 8 days, the arsenite eradication potential of Microbacterium sp. was 72 and 95% from real wastewater and distilled water (Fig. 2b).

3.10 Antioxidant enzymes

The activities of antioxidant enzymes i.e. catalase, ascorbate peroxidase, peroxidase and superoxide dismutase were determined in strain 1S1 when exposed to 15 mM arsenite. A mixed response of such enzymes was ascertained in metal stressed bacterial cells. The POX and SOD activities were decreased while CAT (240%) and APX (11%) activities were enhanced in metal stressed culture (Table 1).

3.11 SEM, EDX, and FTIR analysis

No significant change in bacterial size was measured when the organism was treated with 15 mM arsenite as revealed by SEM analysis (Fig. 3a). The EDX results confirmed the surface absorption of As⁺³ into the microbial cells when challenged with 15 mM As⁺³ but no arsenite surface absorption was measured in bacterial culture having no arsenite stress (Fig. 3b).

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Fig. 3

(a) SEM analysis; (a) Strain 1S1 cells in exponential growth phase without As⁺³ showing cocci shape morphology. (b) Strain 1S1 cells in exponential growth phase challenged with 15 mM As⁺³ showing no change in bacterial cells shape morphology, (b) (b1) Energy dispersive X-ray spectrum of Microbacterium sp. in exponential phase without 15 mM As⁺³, (b2) Energy dispersive X-ray spectrum of Microbacterium sp. in exponential phase when challenged with 15 mM As⁺³. (c) FTIR analysis of Microbacterium sp. strain 1S1, red line represents cells challenged with 15 mM As⁺³ and black line indicates control (no As⁺³).

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The shifting of various peaks in FTIR analysis in Microbacterium sp. challenged with arsenite (15 mM) could be allocated to various functional groups which might be involved in arsenite adsorption or uptake into the microbial cells. FTIR analysis revealed that amide and hydroxyl moieties play an active role in arsenite adsorption by showing a displacement in the area of 3278–2851 cm⁻¹. The shifting of peaks from 1741 to 1220 cm⁻¹ in the bacterial cell wall is due to amide linkages of peptides and proteins. The possible cause behind the peaks shift from 1228 to 1038 cm⁻¹ is stretching of C–O from an alcohol, carboxylic acid, and ester and stretching of C–N from an aliphatic amine indicating the interaction of arsenite ions with bacterial cell chemical moieties (Fig. 3c).