Multiple Metal Resistant Bacillus cereus 3.1S Isolated from Industrial Effluent has Promising Arsenite Oxidizing Potential

Huge quantity of untreated industrial waste water is being discharged from industries such as metal processing, mining, textile, tanneries, pigment production, pharmaceuticals, pesticides, smelting, organic chemicals, alloy industries and storage batteries, rubber and plastics manufacturing industries, severely pollute the environment (Ghosh et al., 2018; Elahi and Rehman, 2019). Heavy metals containing lumber and wood products aggravate water pollution even more day by day when they get entered into the fresh water bodies (Mohammadi et al., 2005). Arsenic due to its non-degradability and persistent nature accumulates in the water bodies thus decreasing the water potential to sustain life.

Arsenic entry into the water system is the first key step of arsenicosis that seriously affects the health of human beings (Oremland and Stolz, 2005). Arsenic exists mainly in two forms; arsenite (As3+) and arsenate (As5+), the former is more toxic and is present in high concentration in the wastewater as compared to the later one which is less toxic and less available in the environment. Arsenic and its compounds are mutagenic, teratogenic, and carcinogenic in nature (Mead, 2005). It is a major cause of skin, liver, lung, and lymphatic cancer, and also highly toxic for the kidneys (Tseng et al., 2002). Arsenic exacerbates human health by negatively affecting mental capabilities, decreasing the levels of red and white blood cells’ production, and also causes abdominal cramps, weakness, diarrhea, headaches, anemia (Kohnhorst et al., 2002; USEPA, 2004). Moreover, it has also been linked to be a cause of type II diabetes in human beings (Walton et al., 2004). The continuous exposure of high concentration of arsenic compounds causes infertility and miscarriages in women as well as weak immune system in neonates. Only 0.05 mg l-1 arsenic quantity is permissible in drinking water, as recommended by the World Health Organization (WHO).

Arsenic is being removed from the contaminated sites through commonly used techniques such as isolation, physical separation, immobilization, extraction of metal, and toxicity reduction (Lim et al., 2014). These methods are not eco-friendly; they are also expensive due to use of chemicals in order to detoxify arsenic impurities. Microorganisms have the ability to oxidize, reduce, and adsorb arsenic. They are capable of detoxifying arsenic by changing arsenite into arsenate (oxidation), and by adding methyl group to the arsenic (methylation). Thus, arsenite oxidizing bacteria could play a vital role in the bioremediation process (Simeonova et al., 2005; Dey et al., 2016) utilizing their ability to detoxify more toxic and mobile arsenite into less toxic and slowly mobile arsenate. Therefore, they are suggested to be used in environmental clean-up operations (Satyapal et al., 2016, 2018; Mu et al., 2019).

In the present study, a multiple metal resistant bacterium isolated from industrial effluents has been found to have arsenite oxidizing potential. Arsenite oxidase activities, an enzyme responsible to convert arsenite into arsenate, were also evaluated at different pH, temperature, and metal ions. Impact of As3+ on the growth pattern of bacterial strain and changes in proteomics and cell physiology were also determined.

Samples of industrial wastewater were collected in the sterile screw capped bottles, from different industrial sites of Lahore, Pakistan. Then, 100 µl of wastewater specimen was spread on acetate minimal salt medium (MSM) agar plates augmented with 100 µg/ml of arsenite for bacterial isolation. The minimum inhibitory concentration (MIC) was determined by preparing MSM-agar plates (Pattanapipitpaisal et al., 2001) supplemented with increasing concentrations of sodium dihydrogen arsenite (NaH2AsO3) up to 3000 µg/ml. The 13 isolated bacteria were evaluated for their tolerance to As3+ at different concentrations and the bacterium 3.1S showing highest resistance (40 mM) was selected for further study.

Arsenite resistant bacterial isolate was characterized morphologically and biochemically by using standard methods (Cappuccino and Sherman, 2001). The molecular characterization of the bacterium was done by isolating DNA (Supplementary Fig. S1a) according to Sambrook et al. (2001), and 16S rRNA gene amplification (Supplementary Fig. S1b) was done using universal primer pair RS1 and RS3. Fermentas purification kit (# K0513) was used to clean PCR products, and sequenced with Genetic analysis system model CEQ-800 (Beckman) Coulter Inc. Fullerton, CA, USA. The data obtained after sequencing was submitted to GenBank to get the accession number.

Resistance pattern of the bacterial isolate against other heavy metals e.g. as lead (PbNO3), cadmium (CdCl2), copper (CuSO4.5H2O), nickel (NiCl2.6H2O), chromium (K2Cr2O7), and arsenite (NaH2AsO3) were also checked. MSM broth medium with different concentrations of heavy metals was prepared i.e. 100, 300, 500, 1000, 1500, 2000, and 2500 µg/ml (Pattanapipitpaisal et al., 2001), and inoculated with log phase culture of bacterium. Culture was allowed to incubate at 37 oC for 24 h, and then growth (O.D600) was determined.

The optimum cultivation parameters of the bacterium were determined i.e., temperature and pH, by cultivating it at various temperatures (25oC, 30oC, 37oC, and 42oC) and pH (5, 6, 7, 8, and 9) in 100 ml Luria broth, inoculated (100 μl) with log phase culture (inoculum O.D600nm was maintained at 0.5). Cell density was determined by taking absorbance at O.D600nm after 24 h of incubation.

Growth curves of bacterial strain were determined by growing the bacterial isolate in LB broth augmented with 100 μg/ml NaH2AsO3 (treated) and 1% glucose (control), inoculated with log phase bacterial culture (1 ml). Growth was measured by taking optical densities at O.D600nm at 0 h (immediately after inoculation), and after a regular interval of four hours up to 36 h.

Acetate minimal agar plate augmented with NaH2AsO3 (100 µg/ml) was streaked with the loop full of bacterial culture, incubated at 30°C for 48 h and was flooded with 0.1 M silver nitrate solution. Arsenate produces brownish precipitates when combined with AgNO3 and these observations were made as mentioned by Simeonova et al. (2004).

Intracellular enzyme: Acetate minimal medium containing NaH2AsO3 (100 µg/ml) was inoculated with bacterial isolate and without inoculation (control), and incubated for up to four days. Culture was centrifuged, and the pellet was washed with a 50 mM phosphate buffer (pH 7) twice before sonication. Cells were sonicated at 4ºC (on ice) for 15 sec with an interval of 1 minute, centrifuged at 14,000 xg for 10 min and supernatant thus obtained was shifted to new eppendorf. This supernatant was used to assay the intracellular enzyme activity.

Extracellular enzyme: MSM, augmented with 100 µg/ml NaH2AsO3, was prepared and inoculated with the bacterial culture and without inoculation (Control). Flasks were incubated at 28 oC for 24, 48, 72, and 96 h, and after completion of incubation, cells were harvested by centrifugation at 14000 xg for 10 min, and supernatant was separated. This supernatant was used for the estimation of extracellular enzyme activity.

Enzyme activity reaction mixture contained each sample supplemented with As3+ (100 μg/ml) along with 1 ml of 2% potassium iodate, and 1 ml of 1 M hydrochloric acid. The mixture was mixed gently until the color turned bright yellow. Then, 0.02% safranin O (0.5 ml) was added, and volume was made with distilled water up to 100 ml, and mixed gently for 2 -3 min. The pH of the solution was adjusted at 4 with the help of 2 ml acetate buffer, and the flask was shaken well. OD532nm of the reactions was measured against reagent blank (Pasha and Narayana, 2008).

Arsenate was estimated as reported by Lenoble et al. (2003). In this, the reaction mixture contained 4 ml enzyme (intracellular or extracellular), 2 ml of reagent A (dissolving 1390.5 g of (NH4)6-Mo7O24_4H2O in 9 M H2SO4), 1 ml of reagent B (dissolving 0.5 g of ascorbic acid in 100 ml sterilized distilled water) and 50 ml of deionized water. OD870nm was determined after regular time intervals (0, 5, 10, 15, 20, 25 and 30 min). Control was also treated in the same manner and observations were made.

For the determination of arsenite oxidation potential, 4 ml of bacterial enzyme (extracellular or intracellular) was mixed with ascorbic acid solution (100 µl) and reagent A (200 µl), and deionized water was added to make the volume up to 5 ml. Reaction mixture was incubated at room temperature for 30 min and OD870 nm was measured. Arsenite oxidation potential of the bacterial strain was calculated from arsenate standard curve (Lenoble et al., 2003).

The bacterium was cultivated in MSM with and without 100µg/ml NaH2AsO3 for three days. Culture was harvested by centrifugation for 10 min at 6,000 rpm and the cells obtained were washed with 50mM phosphate buffer (pH 7) twice. The cell pellet was sonicated three times, for 15 sec at 4ºC with a time interval of 1 minute between each sonication cycle. The sonicated pellet was centrifuged for 10 min at 14,000 xg, and the supernatant obtained was shifted to a fresh Eppendorf, regarded as the soluble fraction.

Two (50 ml) sterilized flasks, labeled as “treated” and “control”, were taken. The reaction system consist of 0.1 ml bacterial sample (supernatant or resuspended pellet), along with 0.9 ml of reaction mixture containing 20 μl of 1 M phenazine methosulfate (PMS), 2 µl of 1 M 2,4-dichlorophenolindophenol (DCPIP), 2 μl of 1 M NaH2AsO3, and volume of the reaction mixture was made up to 10 ml in properly labeled flask, and incubated for 30 min at 37oC. Similar procedure was performed for the treated as well as control samples. Finally O.D600nm was determined (Noreen and Rehman, 2016).

Optimal conditions for the maximum activity of arsenite oxidase of the B. cereus 3.1S were determined. For the optimal temperature, crude extract (100 µl) was incubated at various temperatures (30, 37, 42, 55, 70 and 90 oC) for 30 min then crude extract (100 µl) was mixed with 0.9 ml reaction mixture [20 μl of 1 M phenazine methosulfate (PMS), 2 µl of 1 M 2, 4-dichlorophenolindophenol (DCPIP), 2 μl of 1 M NaH2AsO3, and 9.76 ml of H2O]. To check the enzyme thermostability, the reaction was carried out by incubating assay mixture at 37°C for 30 min.

For pH optimization, the enzyme extract was incubated at pH values 4, 5, 6, 7, 8, and 9 in different buffers. Buffers used were acetate buffer (pH 4), sodium acetate (pH 5-6), Na2HPO4 (pH 7-8), and Tris HCl (pH 9). Then 200 μl from each buffer and 200 μl of the crude extract mixed and incubated for 60 min at 37oC. After that 200 μl from the enzyme-buffer mixture was mixed with 0.8 ml of reaction mixture [20 μl of 1 M PMS, 2 µl of 1 M DCPIP, 2 μl of 1 M NaH2AsO3, and 9.76 ml of H2O] and incubated at 37oC for 30 min.

The metal ions effect on the enzyme activity was determined. For this, 100 μl crude enzyme was mixed with metal ion solution i.e., Zn2+, Ca2+, Cu2+, Mg2+ and was incubated at 37oC for 30 min. Then 0.9 ml of reaction mixture [20 μl of 1 M PMS, 2 µl of 1 M DCPIP, 2 μl of 1 M NaH2AsO3, and 9.76 ml of H2O] was added and incubated again for 30 min at 37oC. All the enzyme activities were determined by measuring absorbance at 600 nm.

Under arsenite stress, the altered levels of glutathione (GSH) and other non-protein thiols (NPSHs) were measured.

The bacterium was cultivated in MSM broth with and without 100 µgAs3+/ml and SDS- PAGE was done according to Laemmli (1970).

Amplification of arsenite oxidase gene was done by using degenerate primers as reported by Que´me´neur et al. (2008). Briefly, the reaction mixture consisted of 50 µL of distilled water containing 6 µl of genomic DNA and 5 µl of each primer (10 pmol), (Amersham Pharmacia, Piscataway, NJ, USA). The polymerase chain reaction (PCR) was performed according to Jinbo et al. (2007). Plasmid from bacterial isolate was isolated by using mini-preparation protocol according to Sambrook et al. (2001). Extracted plasmid DNA (550bp) was analyzed by gel electrophoresis and E. coli DH5α was transformed with the isolated plasnied DNA (Sambrook et al., 2001). Arsenite-oxidizing ability of transformants was checked by using the method of Simeonova et al. (2004).

The bacterial strain potential to oxidize As3+ was determined by inoculating 300 ml bacterial culture (24 h old) in 1 liter of industrial wastewater. Also as control samples, autoclaved and un-autoclaved wastewater was placed under the same experimental conditions. In the wastewater, As3+ initial concentration was determined according to Noreen and Rehman (2016) and final As3+ concentration (100 µg/ml) was adjusted with NaH2AsO3. Changes in As3+ concentration were calculated with the help of a calibration curve prepared under the same experimental conditions.

To check the effect of treated wastewater on the plant growth system, small pots were filled with autoclaved soil and seeds of Vigna radiata (mung beans) were cultivated in them. At least three experimental pots were employed; each pot differs from one another in watering scenario. Two pots served as controls; one was watered with original wastewater and the other with tap water, while the third one was watered with bacterially treated wastewater (experimental). The plants were cultivated under 1:1 light and dark for 10 days at room temperature. Finally, the growth in control, tap and untreated wastewater was compared.

Observations were made and all the experiments run in triplicate. At least three separate flasks were usually maintained for one treatment. Each time three readings were taken, their mean, and standard error of the mean were calculated.

Wastewater samples were taken from leather, pesticide and steel industry near Lahore, Pakistan. The temperature of wastewater samples ranged between 23-35°C, pH ranged between 5.5 to 8, and the sample color was muddy. Thirteen different colonies, appeared on MS-agar plates (100 µg As3+/ml), were selected and evaluated for their ability to tolerate maximum As3+ concentration. Only one (3.1S) showed growth on MS-agar plates containing up to 40 mM As3+ (3000 µg/ml) which also showed resistance against Pb2+ (8 mM), Cd2+ (6 mM), Cr6+ 6 (mM) and Cu2+ (10 mM). This bacterial isolate 3.1S showed maximum homology with the genus of Bacillus (Table I). The data obtained from 16S rRNA gene sequencing for Bacillus cereus (3.1S) was submitted to GenBank database under accession number of KF003020. The dendrogram on the basis of homology was also created (Fig. 1).

Figure 2 shows effect of As3+ on the bacterial growth at 37oC and pH of 7. The growth of bacterium slowed down and the maximum growth was obtained after 24 h in the presence of 100 µg As3+/ml as against 10 h in the control.

Figure 3A shows production of brownish precipitates after treatment AgNo3 in culture plates where bacteria were grown in the preserve the arsenaite. On the other hand, control plates/ bright showed yellow precipitates in which no bacteria were present. B. cereus 3.1S assay results clearly demonstrated the presence of arsenite oxidase activity that converts As3+ (arsenite) into less toxic form As5+ (arsenate).

Moreover, Safranin O spectrophotometric method was used to determine any change in the concentration of As3+ present in the bacterial culture medium after a specific incubation time period. Assay results revealed that extra cellular and intracellular arsenite oxidase of 3.1S oxidized 63. 8% and 71%, respectively As3+ to As5+ after 96 h of growth. Figure 3B shows that the predominant form of the enzyme was intracellular as compared to the extracellular (OD 0.846nm).

Arsenite oxidase assay was performed after 24, 48, and 72 h of bacterial growth for soluble fraction to determine enzyme activity in µM/min/μg in different time intervals. Arsenite oxidase revealed the maximum activity after 30 min of incubation of reaction mixture of 72 h grown bacterial culture. This assay was performed to confirm that enzyme activity was increased with the increase in time of growth and reaction mixture (Fig. 3C).

Bacterial enzyme showed maximum activity at 37oC i.e. 66.5% (Fig. 4A) and at pH 7 i.e. 124.5% (Fig. 4B). All the tested heavy metal ions enhanced the arsenite oxidase activity but the maximum enzyme activity was determined in the presence of Zn2+ (29.6%) when compared with the control containing no metal ions (Fig. 4C).

It was found that As3+ stress stimulates GSH and NPSHs levels in B. cereus 3.1S (Table II). In the presence of 100 μg As3+/ml, 1597% and 86% increase in GSH and NPSHs was determined as compared to the control. This increase in antioxidants molecules indicates that bacterial cells try to maintain homeostasis by neutralizing the reactive oxygen species which are produced during metal stress.

Table II. Effect of As3+ (100 μg/ml) on the glutathione level (GSH, GSSG) and non-protein thiols (NPT) of B. cereus 3.1S.

A significant increase in intracellular protein content was shown by the bacterium in the presence of As3+, presumptively arsenite oxidase (Fig. 5A). The enzyme has a large subunit of 60kDa protein and smaller subunit with low molecular weight 14kDa protein in the bacterial protein sample under As3+ stress. The enzyme concentration was very low in a control sample containing no As3+ stress.

A 550 bp product was obtained, after amplifying the arsenite oxidase gene, by using degenerate primers which presumably indicates the presence of enzyme protein in the bacterial culture under metal stress (Fig. 5B). The plasmid DNA containing arsenite oxidase gene isolated from B. cereus 3.1S (Supplementary Fig. S2) was transformed into E. coli DH5α (Supplementary Fig. S3A). E. coli strain containing plasmid DNA showed the colonies on minimal acetate medium with arsenite stress (Supplementary Fig. S3B).

Arsenite oxidizing ability of the transformants was observed by brownish precipitation appearing on agar plates when AgNO3 reacts with arsenate. Otherwise bright yellow precipitation takes place when AgNO3 reacts with arsenite. The assay results reveal arsenite oxidase activity that converts arsenite into less toxic arsenate (Supplementary Fig. S4).

The As3+ oxidation in industrial wastewater was clearly observed by observing change in color of the medium supplemented with bacterial culture as compared to the control containing no bacterial cells. As3+ was oxidized 76 and 86.5% by the bacterial strain after 3 and 6 days of incubation at room temperature (Fig. 6) showing the promising potential of the bacterial strain to detoxify the wastewater containing arsenic.

Vigna radiata (mung beans) seeds irrigated with B. cereus 3.1S treated wastewater germinated normally and attained good growth size within 10 days as compared to the seeds irrigated with untreated wastewater. Seeds watered with untreated industrial wastewater showed delayed germination and poor growth (Fig. 7). From this experiment it can be deduced easily that microbially-treated industrial wastewater can be used safely for irrigation purposes.

Microorganisms have evolved survival strategies to thrive in the metal increasing environment. In the current investigation, wastewater samples were collected from pesticide industry near Lahore, Pakistan and the bacterium isolated has the potential to resist As3+ contamination up to 3000 µg/ml (40 mM). This research lab is embarked upon isolating the heavy metals resistant microorganisms including arsenic resistant bacteria from the tannery and industrial effluents (Butt and Rehman, 2011; Noreen and Rehman, 2016; Ilyas and Rehman, 2018).

Besides As3+, B. cereus 3.1S also developed resistance against other heavy metals e.g. Cu2+, Cd2+, Cr6+, and Pb2+ at varying concentration. The arsenite oxidizing bacteria possess the property to resist other toxic metal ions (Rehman et al., 2010). Similarly, Muller et al. (2003), Drewniak et al. (2008), Bachate et al. (2013), and Das and Barooah (2018) also reported that arsenite tolerating bacteria were able to resist other heavy metals including Se, Mn, Cr, Sb, Cd, Ni, Zn, Pb and Cu.

Silver and Phung (2005) reported that arsenite oxidase is located both on membrane and within the periplasmic space. Microorganisms produce arsenite oxidase both intra and extracellularly, which is involved in converting more toxic As3+ to less toxic As5+. The oxidation ability of B. cereus 3.1S was visualized by AgNO3 assay by producing brown precipitates. Our findings are in good agreement with other researchers (Krumova et al., 2008; Heinrich-Salmeron et al., 2011; Raja and Omine, 2012). Liao et al. (2011) found that arsenite oxidase transformed 65% As3+ to As5+ within 30 min of incubation and complete As3+ oxidation was achieved within 50 min. In this study, arsenite oxidase of soluble fraction from B. cereus, 72 h grown culture, was able to transform 43% As3+ to As5+ after 30 min of incubation.

The temperature effect on the enzyme activity was evaluated by pre-incubation of enzyme at a temperature range of 30-90oC. B. cereus arsenite oxidase showed maximum activity at 37 oC as reported by Bachate et al. (2013). Many studies have been reported that the optimum temperature for bacterial arsenite oxidase activity is 30oC (Rehman et al., 2010; Butt and Rehman, 2011; Raja and Omine, 2012). However, the arsenite oxidase isolated from bacteria showed the substantially decreased activity at 90 oC and in case of B. cereus 3.1S it was 9%. Enzyme activity is markedly affected by pH. The optimum pH for arsenite oxidase activity (124.5%) from B. cereus 3.1S was 7 and it was decreased markedly over both sides of optimum pH being 119% at pH 6 and 84% at pH 8 for B. cereus. Our findings are similar with other studies that reported the same results from other bacterial species (Rehman et al., 2010; Butt and Rehman, 2011; Raja and Omine, 2012; Marzan et al., 2017). Generally, pH optimum for the enzyme is in the range of 6 to 7. Enzyme activity was increased in the presence of each metal; however, the maximum activity was obtained in the presence of Zn2+.

In the current study, contents of GSH and NPSHs were increased when grown under metal stress which is indicative of the fact that this bacterium is able to tolerate arsenic induced ROS stress. The level of GSH and NPSHs was increased up to 1597 and 86% in B. cereus 3.1S as compared to the cells grown without metal stress. It has been reported that GSH has reducing potential when arsenate enters into the bacterial cell (E. coli), As5+ reacts with ATP and GSH and converts into As3+ (Tsai et al., 2009). As3+ reduction in plants through GSH has also been documented by Singh et al. (2006). The oxidative stress resulting from the formation of ROS is combated by antioxidant molecules including GSH and NPSHs. Metallothioneins (MTs) are involved in the metal scavenging process (Fig. 7).

The enzyme presence was also confirmed by SDS-PAGE and results showed that approximately 60 and 14kDa protein bands were present in the As3+ treated sample (Fig. 5A). Exactly the similar findings were reported by other researchers (Conrads et al., 2002; Stolz et al., 2006). It has been documented that presence of As3+ induces the enzyme synthesis which is composed of two polypeptides; large catalytic subunit has molecular weight of 98 and smaller has 14kDa (Santini and Hoven, 2004). The same results were reported by Ellis et al. (2001) and Yang and Rosen (2016). Koechler et al. (2010) reported that Herminiimonas arsenicoxydans contains aoxB protein with molecular mass of 92kDa.

The impact of microbial purified wastewater on the growth of V. radiata was also determined. Mung beans plants showed efficient growth in B. cereus 3.1S purified wastewater as compared to the growth in untreated wastewater which indicates the efficiency of bacterial arsenite oxidase. Noreen and Rehman (2016) reported that B. cereus and A. junii treated industrial wastewater was safe for the growth of mung beans plants.

In conclusion, B. cereus showed maximum growth at 37ºC and pH of 7. The multiple metal resistant bacterium tolerated As3+ up to 40 mM due to employing various mechanisms including antioxidant molecules and metallothioneins. The arsenite oxidase, which transforms toxic As3+ into less toxic As5+, showed maximum activity at 37ºC, pH 7 and in the presence of Zn2+. This inevitable role of arsenite oxidase was also confirmed by transformation. The bacterial strain oxidized 76 and 86.5% As3+ after 3 and 6 days of incubation, respectively from the real industrial wastewater. This microbially treated wastewater is safe for the growth of plants. This metal resistant bacterium with good oxidizing ability, may find potential applications in environmental biotechnology.

This is to acknowledgement the support of University of the Punjab (Grant no. Env-67), Lahore, Pakistan to accomplish this research work.

There is supplementary material associated with this article. Access the material online at: https://dx.doi.org/10.17582/journal.pjz/20190620050601

The authors have declared no conflict of interest.