Co-existence of Metal Transporter Genes and Heavy Metal Bioremediation Potential of Bacterial Strains from the Effluent Water

Abstract

Heavy metal contamination and accumulation pose a major threat to all life forms in the environment due to its toxic nature, non-biodegradable properties and accumulative behavior. The present study aims to investigate the metals resistance mechanisms, removal capacity and bioremediation potential of the high metal-resistant indigenous bacterial species Bacillus thuringiensis strain BHR1, Bacillus cereus strain BHR2, Enterobacter cloacae strain BHR4, Bacillus pumilus strain BHR5, Bacillus altitudinis strain BHR6 and Klebsiella pneumonia strain BHR8 isolated from the effluent water. Moreover, the presence of specific metal transporter genes such as chrA, chrB, pbrA and nccA in these isolates indicated their role in metal resistance and removal. Based on minimum inhibitory concentration, bacterial isolates namely, BHR1 and BHR5 exhibited high metal tolerance against Cr (510 and 550 ppm), Pb (250 and 300 ppm), Ni (300 and 150 ppm) and As (200 and 120 ppm), respectively. The bioremediation potential of these bacterial isolates (BHR1 and BHR5) was evaluated under batch-scale conditions. Our results revealed that the addition of nutrients, as well as modification of pH to 6.0, enhances the bioremediation potential of BHR1 enabling the reduction of Pb, Cr, Ni and Zn by 60.9%, 31%, 22.7% and 22%, respectively. These findings suggested the potential application of these metal resistant bacterial isolates for the bioremediation of heavy metal contaminated effluents.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12088-024-01362-y.

Introduction

Heavy metals are among the most problematic contaminants, for their hazardous impact on the environment at even very low concentrations [1]. Toxic effects of heavy metals have been observed in microbes, plants, animals and human beings. Some heavy metals like cobalt, copper, iron, manganese, chromium, selenium and zinc are crucial micronutrients required for the growth of plants, animals and humans in trace amounts, while other metals such as arsenic, cadmium, lead and mercury are not much known for their physiological significance [2]. However, the existence of metals above permissible limits in water resources causes toxic, mutagenic and carcinogenic effects. Heavy metals are non-biodegradable and can enter the ecosystem, leading to biomagnification, bio-accumulation and geo-accumulation processes that have intense public health implications.

Increased levels of these metals in the environment have caused global concern for their mitigation because of the toxicity demonstrated by most of them. The occurrence of toxic heavy metals in the environment is natural and anthropogenic. Natural processes such as weathering of rocks, volcanic eruption, forest fires and emissions from vegetation play an important role in metal pollution. Some studies have revealed that wind-blown particles and atmospheric precipitation are also responsible for the metal contamination of water [3, 4]. Apart from it, the rapid increase in urbanization and industrialization for anthropogenic purposes has increased the indiscriminate use of heavy metals. Anthropogenic activities, including mining, smelting, metallurgical and chemical industries are the major contributors to heavy metal pollution. Other sources such as industrial wastewater, agricultural runoffs and combustion of fossil fuels carry a significant amount of heavy metals [5]. Particularly, the discharge of huge volumes of untreated wastewater and domestic sewage are responsible for catastrophic and devastating heavy metal pollution in the water resources [6, 7]. Recent studies have also shown that the long-term use of untreated wastewater can adversely affect the water quality and make it unsuitable for human consumption [8]. Elevated levels of heavy metals in water resources disrupt the microbial community structure and distribution, hinder the cellular processes and limit their growth. According to the World Health Organization [1], the common toxic heavy metals that may cause public concern include arsenic (As), cadmium (Cd), chromium (Cr), lead (Pb), nickel (Ni) and selenium (Se).

Various conventional physicochemical techniques have been used to remediate heavy metals such as chemical precipitation, reverse osmosis, ion exchange resins, filtration, adsorption, oxidation and reduction [9]. However, these techniques are very expensive and don’t ensure efficient removal when metal concentrations are low, in addition to producing a significant amount of secondary noxious products. Effective removal of heavy metals from the polluted environment demands a cost-effective and eco-friendly holistic approach. Bioremediation is an innovative and promising alternative technology used to decontaminate metal pollution from the environment [10]. Bacteria are known to thrive in all kinds of environmental conditions, ranging from cold poles to hot springs, the wet swamps to dry deserts. Their unique features like small size, high multiplication rate, high surface area to volume ratio and efficient transfer of genetic traits make them potential bioremediation agents.

Microorganisms are regarded as the most abundant and diverse living organisms on earth that contribute to many biogeochemical cycles such as carbon, nitrogen and phosphorous cycles. In the aquatic system, mostly bacterial communities play critical roles in ecological functions. Heavy metals are naturally present in the environment, but many anthropogenic activities have led to the accumulation of high concentrations of metal ions in the environment [11]. To survive in a heavy metal-contaminated environment, several microbes develop resistance mechanisms to metabolize, reduce and transform metals into a less toxic form. These processes give rise to different species of heavy metal-resistant microbes in a polluted environment [12]. Various microorganisms such as bacteria, fungi, yeast and algae have been reported to tolerate and remove heavy metals from the environment.

Several studies carried out on the effluent water sites have reported the presence of heavy metal-resistant bacteria with bioremediation potential [13–15] but there is scanty information available on the enhancement of bioremediation efficiency of the metal resistant bacterial isolates or to trace their bioremediation potential at molecular level. Therefore, the purpose of this study was (1) to isolate and screen the metal-resistant bacterial isolates from effluent water, (2) to observe the co-existence of antibiotic and metal resistance in isolates, (3) identification of genes encoding metal resistance to understand the mechanisms involved in the process and (4) to exploit the selected isolates for bioremediation of heavy metals.

Materials and Methods

Materials

All chemicals and reagents used in this study were of analytical grade (AR). The culture media; Luria–Bertani agar or broth, Brain Heart infusion broth, Mueller Hinton agar, Antibiotic Susceptibility discs, SOC broth, Proteinase K and RNase were purchased from HiMedia (India). The K₂Cr₂O₇, NiCl₂.6H₂O, Pb(CH₃COO)₂, Cd(NO₃)₂, HgCl₂, NaAsO₂, Tris-base, sodium dodecyl sulfate, were obtained from CDH Ltd. company. All the stock solutions and culture media were prepared in deionized water and were sterilized at 121 °C with 15 psi for 20 min. The stock solutions were stored in amber glass bottles in a dark environment at 4 °C.

Water Sampling

The study area (75° 87′ 70.45″ E, 30˚91ˊ72.29″ N) was located in the southern part of highly populated Ludhiana City, Punjab, India, which flows as a drain (Buddha Nallah) through the city and merges with the Sutlej River. The major industries located near the study area include small to large units of different types such as electroplating, paper manufacturing, tanneries, dairies, textiles, batteries and bicycle production industries. The depth of water level in the study area varies from 9 to 26 feet below ground level [7]. During the sampling period, the climate is pre-monsoon with an average 34 °C temperature. Based on pollution sources, seven different sites S1, S2, S3, S4, S5, S6 and S7 were selected along the course of the stream of the study area.

Sampling was carried out in April 2021, just before the start of the monsoon season. The water samples were collected aseptically in pre-sterilized bottles at the depth of about 25 cm from the surface water [1]. During the transit, the samples were stored at 4 °C in the ice box and processed on the same day. One portion of the samples was acidified as per standard protocols [16] for heavy metal analysis while the other was used for physicochemical analysis and isolation of bacteria. The physicochemical analysis of different parameters i.e. pH, electrical conductivity (EC), biological oxygen demand (BOD), chemical oxygen demand (COD), sodium (Na⁺) and potassium (K⁺) was carried out following the methods given by USEPA [17] and APHA [16]. The heavy metal content (Cr, Pb, Ni, As and Cd) of the samples was determined by using the Inductively Coupled Argon Plasma-Atomic Emission Spectroscopy (Agilent iCAP-MS 7700) using the standard protocols described by APHA [16].

Isolation and Screening of Heavy Metal Resistant Bacteria

For the isolation of indigenous bacteria, the water samples were spread onto the nutrient agar (NA) by following the dilution method, incubated at 37 °C for 24–72 h and monitored regularly for colony formation on agar plates. The morphological distinct colonies were picked and screened for heavy metal resistant capacity on the Luria Bertani (LB) agar plates (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 15 g/L with pH-7.5) supplemented with test metals including Cr, Pb, Ni, Cd and As in the form of metal salts such as potassium dichromate (K₂Cr₂O₇), lead acetate [Pb(CH3COO)₂], nickel chloride [NiCl₂.6H₂O], cadmium nitrate [Cd(NO₃)₂] and sodium arsenate [NaAsO₂], respectively with concentration ranging from 10 to 600 ppm. The respective plates were incubated at 37 °C for 72 h and observed for bacterial growth. The bacterial isolates that can grow in the presence of metals were selected, purified and preserved in 30% lycerol stock at  − 20 °C for further analysis.

Statistical Analysis

All the experiments and data analysis were carried out in triplicate and results were presented as mean ± standard deviation (SD). One Way Analysis of Variance (ANOVA) was conducted by comparing the mean through Turkey’s HSD posthoc analysis at 5% significant level using SPSS software (version 20.0, SPSS Inc.).

Characterization of Selected Heavy Metal-Resistant Bacterial Isolates

Morphological and Biochemical Characterization

Selected potential isolates were characterized based on cultural appearance i.e. colony color, shape, elevation, texture and by Gram’s staining reaction according to Bergey’s Manual of Systematic Bacteriology [18]. Isolates were biochemically characterized by different tests such as catalase, IMViC (Indole, Methyl-red, Voges-Proskauer and citrate utilization), triple sugar agar (TSI), motility and carbohydrate utilization (glucose, sucrose, galactose, arabinose and cellobiose).

Pathogenicity Assay

In-vitro pathogenicity assay of selected heavy metal-resistant bacterial isolates was done by streaking onto the Blood Agar base supplemented with 5% defibrinated sheep blood and incubated at 37 °C for 24 h [19]. The pathogenicity of isolates was confirmed by observing the formation of a clear yellow zone around the colonies on the blood-supplemented plates due to the lysis of red blood cells.

Molecular Identification

Molecular characterization of isolated pure strains was done by using 16S rRNA sequencing. Genomic DNA was isolated using the modified method of Cubero et al. [20]. The presumptive isolates were grown in 5 ml of SOC broth medium at 37 °C until 0.5 O.D at 600 nm is achieved. One ml of bacterial culture was centrifuged at 10,000×g for 2 min. In pellet, 567 µl of Tris–HCl (10 mM) buffer, 30 µl of sodium dodecyl sulfate (10% v/v) and 3 µl of proteinase K (20 mg/ml) were added and incubated at 37 °C for overnight. To this mixture 100 µl of NaCl (5 M) and 80 µl of CTAB were added, vortexed and incubated for 2 h at 65 °C. An equal volume of isoamyl alcohol and chloroform was added, swirling mixed and centrifuged at 10,000×g for 5 min. To the upper aqueous layer, 0.6 volume of chilled isopropanol was added, mixed and again centrifuged at 10,000×g for 5 min. The pellet was washed for a few seconds with 70% ethanol and air dried. The DNA pellet was suspended in 100 µl of Tris–HCl (10 mM) buffer and 3 µl of RNase for 3 h at room temperature. The quantification of DNA was done by using a NanoDrop spectrophotometer and the quality was determined using 0.8% agarose gel electrophoresis. PCR amplification of the 16S rRNA of selected bacterial isolates was carried out using universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′). The PCR amplified products were observed on agarose gel and sequenced using a BDT v3.1 Cycle sequencing kit on ABI 3730xl Genetic Analyzer (Medauxin Ltd.). The FASTA sequence obtained after sequencing was used to carry out NCBI- Basic Local Alignment Search Tool database and the phylogenetic trees were constructed by the neighbor-joining method using MEGA 7.0 version software [21].

PCR Amplification of Heavy Metal-Resistant Genes of Selected Bacterial Isolates

The molecular characterization of the metal resistance ability of selected bacterial isolates was performed by the PCR amplification of the chrAB, pbrA, nccA and czcABD genes that encode chromium, lead, nickel, copper, cadmium and zinc resistance, using the specific primers. PCR amplification of the target DNA was carried out in a thermal cycle (Thermo Fischer Inc.) using 200 µl PCR tubes with a 25 µl reaction mixture containing 12.5 µl of 2 X PCR master mix (50 units/ml Taq DNA polymerase, 3 mM MgCl_2, 400 µM of each dNTP and reaction buffer; Promega Inc.), 7.5 µl of nuclease-free water, 1 µl of each primer (100 ng/µl) and 3 µl of template DNA. The thermal cycling conditions for metal-resistant genes consisted of 95 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 40 s, annealing of DNA template for 1 min at specific temperatures and an extension time of 2 min at 72 °C for the primers. The final extension was kept at 72 °C for 5 min to complete the synthesis of all the strands and electrophoresed to determine the amplicon size of the PCR product.

Effect of Heavy Metals on the Growth of Selected Bacterial Isolates

Growth kinetics of selected bacterial isolates in response to different heavy metals i.e. Cr, Pb, Ni, Cd and As was carried out in LB broth (100 ml) flasks supplemented with 100 ppm of each metal separately. The broth with bacterial inoculation of respective isolate without any metal supplementation is considered as a control. All the flasks were then incubated in a rotary shaker incubator with 120 rpm at 37 °C. An aliquot of the sample was taken out every 6 h at regular time intervals for 72 h and absorbance was measured at 600 nm using a UV–Vis spectrophotometer (Shimadzu UV-1800).

Determination of Minimum Inhibitory Concentration (MIC)

For the determination of MIC [22], Luria–Bertani (LB) agar plates were separately supplemented with different concentrations of metal (Cr, Pb, Ni, As and Cd) as per Andrews [22]. All the plates were streaked with 24 h old culture and incubated at 37 °C for 24–72 h. The heavy metal concentration was gradually increased (50 ppm each time) until the isolates failed to grow onto the agar plates. The results obtained in solid media were confirmed using the well diffusion agar method. The isolates were spread plated onto the LB agar plates separately and different concentrations of heavy metal solution (100 µl) were added to the respective wells made on these plates followed by incubation at 37 °C for 48 h. The minimum concentration of metal required to completely inhibit the microbial growth was considered to be the minimum inhibitory concentration (MIC).

Antibiotic Susceptibility Test of Selected Isolates

The antibiotic susceptibility test of selected isolates was done by using the Kirby-Bauer disc diffusion method [23]. Twelve antibiotics belonging to different classes including amoxicillin (30 mcg), gentamicin (10 mcg), co-trimoxazole (30 mcg), nitrofurantoin (300 mcg), vancomycin (30 mcg), ciprofloxacin (5 mcg), chloramphenicol (30 mcg), erythromycin (15 mcg), nalidixic acid (30 mcg), ampicillin (10 mcg), cefoxitin (30 mcg) and cefotaxime (30 mcg) were used for susceptibility test against selected heavy metal resistant bacterial isolates using standard antibiotic discs (HiMedia, Media). An aliquot of 24 h old culture was inoculated into Brain Heart Infusion broth and incubated at 37 °C until OD₆₀₀ of 0.5 was obtained. Then, the 100 µl volume of inoculum was taken and aseptically spread onto the sterilized Mueller–Hinton agar plates and the antibiotic discs were placed on agar plates so that a clear zone of inhibition could be observed. Plates were incubated at 37 °C for 24 h and the zone of inhibition was measured using HiAntibioticZoneScale™-C (HiMedia, Media).

Bioremediation Potential of Selected Bacterial Isolates in Effluent Water

To determine the bioremediation ability, the metal uptake capacity of selected bacterial isolates was estimated in the effluent water. Effluent water (250 ml) was inoculated with the 200 µl of freshly prepared bacterial isolates suspension separately in each flask and incubated in a rotatory shaker with 120 rpm at 37 °C. Samples from the flasks were taken out after 48 h and 96 h of incubation to observe the heavy metal profile by using the Inductively Coupled Argon Plasma-Atomic Emission Spectroscopy (Agilent iCAP-MS 7700). A set of the un-inoculated effluent sample was kept as control. The selected bacterial isolates were inoculated into the following treated effluent water separately:- Treatment 0: Effluent samples without any modification, Treatment 1: Effluent samples supplemented with minimal media MM (2.5 g D-glucose, 0.5 g MgSO₄.7H₂O and 0.18 g KNO₃) broth, Treatment 2: Effluent samples were modified to adjust pH at 6.0 because, at this pH, a metal ion exits in its free state, Treatment 3: Effluent samples were supplemented with minimal media (MM) as well as their pH was adjusted to 6.0.

The metal uptake capacity of the bacterial isolates in effluent water was calculated by the following formula:-

$$\text{Metal uptake capacity}(\text{\%})=\frac{Ci-Cf}{\mathit{Ci}}\times 100$$

where C_i = initial metal concentration; C_f = final metal concentration.

Results and Discussion

Physicochemical Properties and Heavy Metal Content of Effluent Water

The physicochemical analysis of water samples revealed that the pH of samples collected from different sites significantly varies from 6.26 to 7.25, showing a slightly acidic to neutral nature. Electrical conductivity (EC) values were found significantly higher than the permissible limits [24] at all the sites except at the S7 site (Table 1). Both, BOD and COD values of the water samples were found above the permissible limits at all the sites. The high amount of BOD and COD values might be correlated with the discharge of industrial effluent, domestic disposal and agricultural runoffs which contained highly toxic organic and inorganic pollutants [25]. The concentration of all the heavy metals such as Cr, Pb, Ni, Cd and As significantly varies from one site to another (Table 1). The concentration of all the heavy metals was recorded as highest at the S2 site followed by the S1 site. However, the heavy metal content at the S1, S2 and S3 sites was found higher than at the other four sites i.e. S4, S5, S6 and S7 (Table 1), while the S6 site showed the lowest heavy metal content of all the sampling sites except lead. The presence of industries, dyeing units, dairy farms and agricultural lands in the proximity of water streams might be the reason for heavy metal contamination in water. A study reported that the discharge of industrial effluent and sewage wastewater, and runoffs from agricultural fields loaded with pesticides and fertilizers increase the heavy metal content in water sources [26].

Table 1.

Water physicochemical properties and heavy metal at each sampling site of effluent water

Sampling sites#	S1	S2	S3	S4	S5	S6	S7	Permissible limits*
Physicochemical parameters
pH	6.26 ± 0.02d	7.25 ± 0.03a	6.53 ± 0.07c	7.18 ± 0.02ab	6.59 ± 0.02c	7.20 ± 0.01ab	7.15 ± 0.01b	6.5–8.5
EC (ds m−1)	2.92 ± 0.55b	3.29 ± 0.41a	2.74 ± 0.44c	2.37 ± 0.30e	2.48 ± 0.31d	2.17 ± 0.42f	1.47 ± 0.10 g	< 2
BOD (mg L−1)	267 ± 1.95b	323 ± 1.80a	191 ± 2.80d	155 ± 1.10e	226 ± 0.55c	76.0 ± 0.70 g	94.0 ± 1.20f	< 3
COD (mg L−1)	431 ± 1.30b	523 ± 2.10a	350 ± 1.10c	323 ± 2.05d	354 ± 1.45c	294 ± 7.05f	313 ± 0.95e	< 250
Heavy metal content (µg L−1)
Cr	21 ± 1.04b	69 ± 0.85a	22 ± 2.25b	11 ± 1.50c	4 ± 0.50d	ND	5 ± 0.25d	50
Pb	120 ± 1.50b	133 ± 1.25a	80 ± 0.55c	55 ± 0.85d	41 ± 1.00e	40 ± 2.25e	20 ± 1.85f	10
Ni	128 ± 2.00b	180 ± 1.00a	110 ± 3.00c	68 ± 1.20d	61 ± 3.75d	32 ± 1.50e	35 ± 2.25e	20
Cd	2 ± 0.07a	6 ± 0.10a	2 ± 0.08a	ND	ND	ND	ND	3
As	22 ± 0.45c	44 ± 1.15a	35 ± 1.50b	17 ± 2.50d	ND	ND	ND	10

^*Permissible limits as prescribed by BIS [24]. EC-electrical conductivity, BOD-biological oxygen demand; COD-chemical oxygen demand; Cr-chromium; Pb-lead; Ni-nickel; Cd-cadmium; As-arsenic; ND- not detectable. Values in the same row with different alphabets indicate the significant difference among the sampling sites according to post- hoc Turkey’s HSD (p < 0.05) analysis. Each value represents the Mean ± SD (standard deviation)

^#Different sites of Buddha nallah S1—Tajpur road, near Haibowal Kalan; S2—GT Road Ladhowal, New Kitchulu Nagar; S3—Balloke STP; S4—Village Talwara; S5—Near Malakpur-Nurpur Bet Road; S6—Near Hambran Laddowal Road; S7—Near Village Walipur Kalan

Isolation, Screening, Morphological, Biochemical and Pathogenicity assay of Heavy Metal Resistant Bacteria

A total of 43 morphologically distinct bacterial isolates were obtained from water samples of Buddha Nallah, Punjab, India. These isolates were purified, screened for metal resistance, and labeled as BHR1 to BHR43. Out of 43, six bacterial isolates i.e. BHR1, BHR2, BHR4, BHR5, BHR6, and BHR8 were found to be resistant up to 100 ppm concentrations of Cr, Pb, Ni, As, and Cd in comparison to other isolates. The selected bacterial isolates were maintained on Nutrient Agar (NA) slants and also in 20% (v/v) glycerol stock, and stored at 4 °C for further use. These six selected heavy metal-resistant bacterial isolates were subjected to cultural, morphological, and biochemical characterization (Table 2).

Table 2.

Morphological, biochemical and virulence characterization of selected heavy metal resistant bacterial isolates

Characteristics	BHR1	BHR2	BHR4	BHR5	BHR6	BHR8
Morphological
Staining	Gram positive	Gram positive	Gram negative	Gram positive	Gram positive	Gram negative
Shape	Rod	Rod	Rod	Rod	Rod	Rod
Elevation	Raised	Flat	Raised	Flat	Flat	Raised
Texture	Smooth	Smooth	Smooth	Rough	Smooth	Smooth
Appearance	Dull	Glistening	Glistening	Dull	Glistening	Glistening
Pigmentation	White	White	Cream	White	White	White
Biochemical and virulence
Catalase	+	+	+	+	+	+
Indole production	−	+	+	+	+	−
Methyl Red	+	+	−	−	+	−
VogesProskauer	−	+	−	+	−	−
Citrate utilization	+	+	+	+	+	+
Motility	−	+	+	+	+	−
Biofilm formation	+	+	+	−	−	+
Congo Red binding	−	+	+	−	−	+
DNase	−	−	−	−	−	+
Hemolysis	α	α	α	α	α	β
Protease	−	+	+	+	+	−
Lipase	−	+	−	+	−	−

(+) and (−) signs denotes positive and negative reactions, respectively

Effect of Metals on the Growth Profile of Selected Bacterial Isolates

Growth kinetics of bacterial isolates such as BHR1, BHR2, BHR4, BHR5, BHR6, and BHR8 were monitored in the Luria Bertani (LB) broth supplemented with 100 ppm each of Cr, Pb, Ni, Cd, and As metals separately. The growth of these isolates was not affected at 100 ppm concentration of Cr, Pb, and Ni; however, As and Cd at the same concentration, affected the growth of the isolates as was evident from the long lag phase (Fig. 1). The reason of having higher value of OD₆₀₀ at 48 h is the extended stationary phase of isolates because of the presence of heavy metals supplemented in the medium. It is clear from the graph that in control (shown by black line) the value of OD₆₀₀ was not zero after 6 h whereas for all the treatments (As, Pb, Cd, Ni, Cu), the OD was near to zero till 6 h and started to rise exponentially after 12 h of acclimatization and reached almost stationary phase after 48–72 h. Heavy metals such as Pb, Cr, and Ni have significantly affected the growth of BHR2, BHR6, and BHR8 isolates as it took 48 h to reach OD₆₀₀ of 2.0 while failed to grow in the presence of As and Cd, indicating their higher toxicity than other metals. However, BHR1 and BHR5 isolates not only showed the ability to tolerate metal stress but also had a significantly higher growth rate OD₆₀₀ up to 2.0 within 24 h. BHR1, BHR5 and BHR6 showed greater growth efficiency in the presence of Cr, Pb and Ni as compared to the control. The highest metal tolerance capacity was observed with the BHR1 isolate that reach the optimal growth in the presence of Pb, Cr and Ni, though displaying a longer incubation with Pb, as compared to the control; it might be an indication of the activation of the metal adapting mechanism in BHR1 for the survival under metal stress conditions.

Fig. 1.

Growth kinetics of selected metal resistant bacterial isolates in the presence of different heavy metals at 100 ppm concentration. Values are represented in graphs as mean value ± SD (standard deviation)

Minimum Inhibitory Concentration (MIC) of Selected Bacterial Isolates Against Heavy Metals

The six metal-resistant bacterial isolates were selected for assessment of minimum inhibitory concentration (MIC) against different heavy metals using the streak plate method and further confirmed by the well diffusion method. In the present study, all the isolates were found highly resistant to Cr, Pb and Ni in comparison to Cd and As. The highest MIC against Cr was observed in BHR5 (550 ppm) followed by BHR1 (510 ppm), BHR4 (300 ppm), BHR6 (250 ppm), BHR2 (220 ppm) and BHR8 (160 ppm) isolates (Fig. 2). In the case of Pb, the BHR5 tolerated 300 ppm of metal concentration, while BHR1, BHR4 and BHR2 showed tolerance up to 250, 230 and 200 ppm, respectively. The Lowest Pb tolerance (150 ppm) was observed in BHR6 and BHR8 isolates. Maximum Ni tolerance was identified in BHRI (300 ppm) followed by BHR8 (250 ppm) and BHR5 (150 ppm), while the lowest was found in BHR2, BHR4 and BHR6 with tolerance capacity up to 100 ppm. The two bacterial isolates i.e. BHR1 and BHR5 showed maximum tolerance to As (200 and 120 ppm, respectively) and Cd (110 and 60 ppm, respectively), while BHR4, BHR5 and BHR8 were found sensitive toward these metals. The BHR2 isolate showed resistance to As (50 ppm) but was unable to grow in the presence of Cd. Overall the metal resistance in bacterial isolates was increased in order Cr > Pb > Ni > As > Cd (Fig. 2). A study conducted by Lenart and Wolny on indigenous bacteria isolated from the samples of soil and water along the roadside, indicated maximum tolerance towards Cd, Pb, Zn and Cr in bacterial populations belonging to the Bacillus genera [27]. Overall, the isolates BHR1 and BHR5 showed comparatively higher MIC against all the tested heavy metals. The continuous exposure to metal pollution in the environment could have conferred resistance in the native thriving bacterial population. The variation in the metal tolerance capacity among the bacterial isolates could be attributed to differences in their cell wall composition and other metabolic mechanisms [28].

Fig. 2.

Multiple heavy metal resistance capacity of selected bacterial isolates

Co-existence of Antibiotic and Heavy Metal Resistance in Selected Bacterial Isolates

The resistance pattern to 12 antibiotics belonging to different classes in selected five heavy metal-resistant bacteria was determined. The results depicted in Table 3 revealed that all six isolates i.e. BHR1, BHR2, BHR4, BHR5, BHR6 and BHR8 showed a high frequency of multiple antibiotic resistance, most frequently towards amoxicillin (100%), co-trimoxazole (100%), vancomycin (100%), chloramphenicol (100%), nitrofurantoin (83.4%), erythromycin (83.4%), nalidixic acid (83.4%), cefoxitin (83.4%), gentamycin (66.7%), ampicillin (66.7%), ciprofloxacin (50%), and cefotaxime (50%). The high metal resistance among these isolates may suggest that they contain some multi-antibiotic resistance efflux pumps that are known to be involved in bacterial resistance to a wide variety of toxic compounds. Several reports suggested that heavy metal pollution not only triggers metal resistance in bacteria but also increases the level of tolerance to the antibiotic by co-regulating the genes that are responsible for the resistance mechanism and decreasing antibiotic susceptibility [29, 30]. Similarly, another study reported the co-existence of antibiotic resistance with heavy metal resistance in all the metal-resistant bacterial strains isolated from the wastewater of wadi El Harrach in the East of Algiers [31]. Out of 13 metal-resistant bacterial isolates, 15% were resistant to one antibiotic and 85% were resistant to multiple antibiotics. In most of the studies, metal resistance has been reported in association with antibiotic resistance [32, 33]. It has been reported that there is an interrelationship between the antibiotic and heavy metal resistance capacities of all microbes. Under the metal stress conditions, heavy metal and antibiotic resistance in microorganisms possibly help them to adapt faster by the spread of resistant factors than by mutation and natural selection [34].

Table 3.

Antibiotic susceptibility pattern of the selected heavy metal resistant bacterial isolates

Bacterial Isolates	Zone of inhibition (in cm)
	Amoxicillin (AMC 30mcg)	Gentamicin (GEN 10mcg)	Co-trimoxazole (COT 30mcg)	Nitrofurantoin (NIT 300mcg)	Vancomycin (VA 30mcg)	Ciprofloxacin (CIP 5mcg)	Chloramphenico (C 30mcg)	Erythromycin (E 15mcg)	Nalidixic acid (NA 30mcg)	Ampicillin (AMP 10mcg)	Cefoxitin (FOX 30mcg)	Cefotaxine (CTX 30mcg)
BHR1	R	R	R	R	R	S(0.8)	R	R	R	R	R	S(0.6)
BHR2	R	S(0.5)	R	R	R	R	R	S(0.7)	S(0.3)	R	R	S(0.2)
BHR4	R	R	R	R	R	R	R	R	R	S(0.4)	S(0.6)	S(0.5)
BHR5	R	R	R	R	R	S(0.6)	R	R	R	R	R	R
BHR6	R	R	R	R	R	R	R	R	R	S(0.9)	R	R
BHR8	R	S(0.4)	R	S(0.6)	R	S(0.8)	R	R	R	R	R	R

R: resistance; S: sensitive

Molecular Identification, Characterization and Detection of Metal-Resistant Genes in Selected Bacterial Isolates

Molecular identification of the presumptive heavy metal-resistant bacterial isolates was done by using 16S rRNA gene sequencing. Blastn analysis of amplified 1.5 Kb gene fragment of 16S rRNA gene of BHR1, BHR2, BHR4, BHR5, BHR6 and BHR8 isolates showed a close association with Bacillus thuringiensis, Bacillus cereus, Enterobacter cloacae, Bacillus pumilus, Bacillus altitudinis and Klebsiella pneumonia, respectively with ≤ 99% consensus sequence homology. Based on sequence homology the bacterial isolates was assigned as Bacillus thuringiensis strain BHR1, Bacillus cereus strain BHR2, Enterobacter cloacae strain BHR4, Bacillus pumilusstrain BHR5, Bacillus altitudinis strain BHR6 and Klebsiella pneumonia BHR8. The partial 16S rRNA gene sequence of these bacterial isolates was deposited to the GenBank at the NCBI site under the accession numbers MZ882154, MZ902928, MZ902932, MZ902992, MZ902994 and OK258312, respectively (Supplementary Table S1). The phylogenetic tree of isolates was constructed by using the MEGA 7.0 version software revealed that all the isolated bacterial strains have a significant association with other heavy metal-resistant bacterial strains.

In the present work, molecular studies were undertaken to investigate the resistance ability of selected metal isolates i.e. BHR1, BHR2, BHR4, BHR5, BHR6 and BHR8 on a genetic level using the polymerase chain reaction (PCR) technique to check whether the heavy-metal removal capacity of isolates is linked to specific metal transporter genes such as chrA and chrB (chromium resistance), pbrA (lead resistance), nccA (nickel–cobalt-cadmium resistance), czcA, czcB and czcD (cobalt-zinc-cadmium resistance). PCR amplified products showed that all the isolates (BHR1, BHR2, BHR4, BHR5, BHR6 and BHR8) produced a single discrete PCR product band on agarose gel by electrophoresis at the expected chrA, chrB, pbrA and nccA amplicon gene size of about 1290 bp, 450 bp, 2400 bp and 1141 bp, respectively with specific primers (Fig. 3).The presence of transporter gene chrA, which is regulated by chrB confers the chromium resistance in all the potential heavy metal-resistant bacterial isolates and suggested that these isolates also use the efflux system for chromium tolerance. PCR amplification of czcA and czcB genes with bacterial genome yielded an amplified product of the expected band size of 1885 and 1500 bp, respectively.

Fig. 3.

Agarose gel electrophoresis showing the PCR products of potential heavy metal resistant bacterial isolates amplified with A chrA, B chrB, C pbrA, D nccA, E czcA and F czcB gene specific primer. Lane M: 1 kb molecular weight marker, Lane 1: Bacillus thuringensis strain BHR1, Lane 2: Bacillus cereus strain BHR2, Lane 3: Enterobacter cloacae strain BHR4, Lane 4: Bacillus pumilus strain BHR5, Lane 5 Bacillus altitudinis strain BHR6, Lane 6: Klebsiella pneumonia BHR8

Amplification of the czcA gene was detected in four bacterial isolates (BHR1, BHR2, BHR5 and BHR6), while the amplified czcB gene product was reported only in the BHR1 bacterial isolate (Fig. 3E, F). However, no amplification was observed for czcD in the bacterial isolates. This might be the result of the lack of this mechanism responsible for metal resistance in the genome of the organisms but can be dependent on other mechanisms responsible for resistance. The CzcA and CzcB proteins constitute an energy-dependent antiporter efflux system that actively transports the metal ions from the bacterial cells and their activity in cells regulates via the czcD gene [35]. Sensitivity to Co, Zn, and Cd rises with the loss of CzcA and CzcB [36]. Proteomic assessment of the Pseudomonas putida strain Cd-001, which was obtained from a location contaminated with lead (Pb), cadmium (Cd) and zinc (Zn), revealed that Cd treatment altered the expression of many proteins, including those involved in the CzcCBA efflux system [37]. Expression of pbrA (a P1B-type ATPase) individually or in combination with other pbr genes, improved resistance to Zn²⁺ 16-fold and Cd²⁺ 128-fold in C. metallidurans DN440 [38]. Amplified PCR-products of 581 bp were produced using degenerative primer sets designed using a fragment from Alcaligenes xylosoxidans 31A [L31363] for nccA [39]. Mechanism of various heavy metal resistance genes in bacterial cell is exhibited in Fig. 4.

Fig. 4.

Various heavy metal resistance genes exhibiting resistance through efflux of heavy metals in a bacterial cell

Heavy Metal Bioremediation Potential of Selected Bacterial Isolates in Effluent Water

Based on minimal inhibitory concentrations, only two bacterial isolates i.e. Bacillus thuringiensis strain BHR1 and Bacillus pumilus strain BHR5 were found to be highly effective in tolerating the high concentration of heavy metal concentrations and were selected for bioremediation studies. Ndeddy Aka and Babalola also indicated in their study that most heavy metal-resistant bacteria were gram-positive, especially the Bacillus strain [44]. The use of native microorganisms alone in bioremediation limits their potential as a result of competition and elevated heavy metal concentration. Several approaches like biostimulation, involve enhancing microbial growth under stress conditions via nutrient addition and modification of environmental parameters [45]. Thus, to enhance the efficiency rate of the bioremediation process the four sets of treatments were carried out in the present study (a) effluent sample without any treatment (Control), (b) effluent sample supplemented with minimal media (MM) (Treatment 1), (c) effluent sample with altered pH (Treatment 2) and (d) effluent sample simultaneously supplemented with MM and altered pH (Treatment 3).

Control: Untreated effluent sample was inoculated with bacterial isolates i.e. BHR1 and BHR5 and was observed for the residual heavy metal concentration after 48 h and 96 h incubation w.r.t un-inoculated effluent sample (Control). The results presented in Table 4 revealed that there was a significant decrease in Pb and Cr concentration with bacterial inoculation after the 48 h incubation, indicating that both the isolates can uptake and thereby reduce the residual metals as compared to the un-inoculated samples. In the present study, the maximum decrease in Pb and Cr concentrations was observed with BHR1 (57.2% and 30.6%, respectively) inoculation, followed by BHR5 (43.7% and 20.2%, respectively), while no significant reduction was observed in un-inoculated samples even after 96 h incubation. On the other hand, isolates showed very low reduction capacity in the case of As, Cd, Fe, Zn and Ni.

Table 4.

Effect of bacterial inoculation on heavy metal concentration in untreated effluent water (control)

Heavy metals	48 h incubation			96 h incubation
	BHR1	BHR5	Control	BHR1	BHR5	Control
	Concentration in ppm
As	0.631 ± 0.02a (7.34↓)	0.665 ± 0.04a (2.34↓)	0.678 ± 0.04a (0.44↓)	0.622 ± 0.03a (8.66↓)	0.643 ± 0.04a (5.28↓)	0.661 ± 0.02a (2.93↓)
Cd	1.875 ± 0.04a (23.4↓)	2.090 ± 0.45a (14.7↓)	2.440 ± 0.20a (0.40↓)	1.853 ± 0.10a (24.3↓)	1.992 ± 0.04ab (18.6↓)	2.150 ± 0.08b (12.2↓)
Cr	312.5 ± 0.75a (25.7↓)	342.2 ± 2.95b (18.7↓)	430.1 ± 1.05c (2.16↑)	292.1 ± 1.75a (30.6↓)	335.6 ± 1.70b (20.2↓)	436.2 ± 2.05c (3.60↑)
Ni	128.2 ± 1.05a (14.2↓)	138.6 ± 2.25b (7.24↓)	148.8 ± 0.90c (0.41↓)	107.7 ± 1.35a (27.8↓)	130.0 ± 1.45b (12.9↓)	148.2 ± 1.05c (0.81↓)
Pb	46.71 ± 3.30a (38.7↓)	53.24 ± 1.55b (30.2↓)	74.23 ± 2.06c (2.72↓)	32.60 ± 1.30a (57.2↓)	42.93 ± 1.40b (43.7↓)	65.40 ± 1.35c (14.3↓)
Fe	1942 ± 3.50a (5.31↓)	2023 ± 1.50b (1.35↓)	2036 ± 2.50c (0.72↓)	1957 ± 3.50a (4.57↓)	1969 ± 2.00c (3.99↓)	1956 ± 1.50b (4.63↓)
Zn	594.3 ± 2.10a (13.7↓)	612.5 ± 5.65b (11.0↓)	680.2 ± 0.40c (1.27↓)	567.7 ± 2.30a (17.6↓)	581.4 ± 1.60b (15.6↓)	645.2 ± 1.95c (6.34↓)

^#Control-Uninoculated effluent sample; incubation conditions: temperature 37 ± 2 °C with 120 rpm shaking. Values are mean ± SD (standard deviation) of three replicates; (↓): decrease; (↑): increase. Values with different alphabets indicate the significant difference among the bacterial inoculation in respective heavy metals according to post- hoc Tukey’s HSD (p < 0.05)

Treatment 1: The results depicted in Table 5 revealed that after supplementation an increase in the metal uptake of bacterial isolates has been observed with increasing the incubation time. Maximum reduction in residual Pb and Cr concentration was observed with BHR1 (60.5% and 29.8%, respectively) inoculation, followed by BHR5 (49.1% and 25.7%, respectively) after 96 h incubation. However, in the un-inoculated effluent sample (control) the Cr and Ni concentration was found to be increased by 1.61% and 0.58%, respectively in 48 h of incubation, while Ni started to decrease by 0.21% after the 96 h incubation. The slight increase in these two metals' concentrations can be because of the increased solubility of pre-existing metals. Overall, the inoculation of BHR1 isolates significantly reduced the residual concentration of Pb (60.5%), Cr (29.8%), Cd (25.2%), Ni (23.4%), Zn (21.1%), As (17.4%) and Fe (5.17%) from the effluent sample within 96 h of incubation. Similar results were reported in a study in which Bacillus safensis-BS16L had the highest removal rates of Pb, Cr, Ni, Fe, and Co (55.4%, 53.1%, 53.7%, 47.7%, and 61.4%, respectively) from a medium that had been supplemented with the heavy metal mixture [46].

Table 5.

Effect of bacterial inoculation on the heavy metal concentration of effluent water supplemented with minimal media (MM)

Heavy metals	48 h incubation			96 h incubation
	BHR1	BHR5	Control	BHR1	BHR5	Control
	Concentration in ppm
As	0.594 ± 0.03a (12.8↓)	0.623 ± 0.01ab (8.51↓)	0.640 ± 0.02b (6.02↓)	0.562 ± 0.05a (17.4↓)	0.618 ± 0.01b (9.25↓)	0.628 ± 0.03b (7.78↓)
Cd	1.958 ± 0.03a (20.0↓)	1.962 ± 0.30a (19.9↓)	2.360 ± 0.06a (3.67↓)	1.831 ± 0.21a (25.2↓)	1.900 ± 0.02ab (22.4↓)	2.240 ± 0.15b (8.57↓)
Cr	310.2 ± 2.40a (26.3↓)	325.2 ± 1.45b (22.7↓)	427.8 ± 1.10c (1.61↑)	295.4 ± 2.20a (29.8↓)	312.7 ± 2.75b (25.7↓)	423.6 ± 0.25c (0.61↑)
Ni	120.1 ± 1.50a (19.5↓)	131.1 ± 1.50b (12.2↓)	150.3 ± 0.65c (0.58↑)	114.4 ± 0.80a (23.4↓)	125.8 ± 0.65b (15.8↓)	149.1 ± 0.50c (0.21↓)
Pb	43.20 ± 1.54a (43.3↓)	46.50 ± 1.82b (39.0↓)	70.23 ± 1.44c (7.96↓)	30.14 ± 3.47a (60.5↓)	38.78 ± 1.67b (49.1↓)	68.12 ± 1.01c (10.7↓)
Fe	1982 ± 2.00a (3.35↓)	1998 ± 1.00b (2.57↓)	1982 ± 3.00a (3.33↓)	1944 ± 2.00a (5.17↓)	1957 ± 1.00b (4.57↓)	1961 ± 1.50b (4.36↓)
Zn	561.2 ± 3.45a (18.5↓)	578.2 ± 1.50b (16.0↓)	676.2 ± 0.50c (1.85↓)	543.0 ± 1.35a (21.1↓)	555.6 ± 1.30b (19.3↓)	672.3 ± 3.00c (2.41↓)

^#Control-Uninoculated effluent sample; incubation conditions: temperature 37 ± 2 °C with 120 rpm shaking. Values are mean ± SD (standard deviation) of three replicates; (↓): decrease; (↑): increase. Values with different alphabets indicate the significant difference among the bacterial inoculation in respective heavy metals according to post- hoc Tukey’s HSD (p < 0.05)

Treatment 2: The pH is a key factor that affects the bioremediation efficiency and bioavailability of metals ion. It has been reported that at pH 6.0 heavy metals have maximum solubility present which increases the bioremediation rate and availability to microorganisms [47]. Therefore, the pH of the effluent sample was altered to 6.0 and inoculated with the selected bacterial isolates (BHR1 and BHR5). The residual concentration of heavy metals was determined after 48 h and 96 h incubation. The sample with altered pH and without any bacterial inoculation was taken as a control. The results depicted in Table 6 revealed that with an increase in incubation time, the residual concentration of heavy metals reduced with the inoculation of bacterial isolates. Maximum reduction of Pb was observed in the presence of BHR1 (49.4%), followed by BHR5 (44.2%) after 96 h incubation. While, the maximum reduction in residual Cr concentration was observed with inoculation of BHR5 (33.8%), followed by BHR1 (26%) within 96 h of incubation. Similar results were reported by Ali et al. with a maximum removal efficiency of Cd, Fe and Zn by Cyanobacterial mats at pH-6.0 [48].

Table 6.

Effect of bacterial inoculation on the heavy metal concentration of effluent water with altered pH (6.0)

Heavy metals	48 h incubation			96 h incubation
	BHR1	BHR5	Control	BHR1	BHR5	Control
	Concentration in ppm
As	0.683 ± 0.03a (0.29↑)	0.680 ± 0.03a (0.14↓)	0.678 ± 0.04a (0.44↓)	0.642 ± 0.05a (5.72↓)	0.674 ± 0.05a (1.02↓)	0.675 ± 0.01a (0.88↓)
Cd	1.936 ± 0.01a (20.9↓)	1.952 ± 0.02a (20.3↓)	2.380 ± 0.17a (2.85↓)	1.876 ± 0.13a (23.4↓)	1.945 ± 0.03ab (20.6↓)	2.040 ± 0.23b (16.7↓)
Cr	328.6 ± 2.30a (21.9↓)	342.1 ± 1.00b (18.7↓)	412.4 ± 1.30c (2.04↓)	311.2 ± 1.60b (26.0↓)	278.3 ± 2.05a (33.8↓)	404.3 ± 0.90c (3.96↓)
Ni	128.3 ± 1.60a (14.1↓)	138.5 ± 1.70b (7.30↓)	141.5 ± 1.75c (5.30↓)	121.8 ± 2.90a (18.4↓)	127.6 ± 0.25b (14.5↓)	138.6 ± 3.75c (7.24↓)
Pb	46.0 ± 1.44a (39.7↓)	54.22 ± 1.54b (28.9↓)	75.20 ± 2.04c (1.45↓)	38.55 ± 1.67a (49.4↓)	42.58 ± 1.23b (44.2↓)	70.62 ± 2.26c (7.45↓)
Fe	1994 ± 3.00a (2.75↓)	2012 ± 2.00b (1.89↓)	2025 ± 1.00c (1.26↓)	1957 ± 3.50a (4.56↓)	1955 ± 5.00b (4.65↓)	1991 ± 1.00c (2.91↓)
Zn	587.3 ± 3.60a (14.7↓)	596.3 ± 4.55a (13.4↓)	620.5 ± 0.35b (9.93↓)	521.2 ± 2.55a (24.3↓)	574.7 ± 3.25b (16.5↓)	614.5 ± 0.40c (10.8↓)

^#Control-Uninoculated effluent sample; incubation conditions: temperature 37 ± 2 °C with 120 rpm shaking. Values are mean ± SD (standard deviation) of three replicates; (↓): decrease; (↑): increase. Values with different alphabets indicate the significant difference among the bacterial inoculation in respective heavy metals according to post- hoc Tukey’s HSD (p < 0.05)

Treatment 3: Effluent samples supplemented with minimal media as well as altered pH (6.0) were inoculated with selected bacterial isolates. The un-inoculated effluent sample supplemented with MM and modified pH was considered as the control. The maximum reduction in residual Pb and Cr concentration was observed with BHR1 (60.9% and 31%, respectively) inoculation followed by BHR5 (51.1% and 27.5%, respectively) after 96 h incubation (Table 7). While uptake of other metals such as Cd, Ni, As, Fe and Zn were very less as compared to Pb and Cr with the inoculation of bacterial isolates.

Table 7.

Effect of bacterial inoculation on the heavy metal concentration of effluent water simultaneously supplemented with MM and altered pH (6.0)

Heavy metals	48 h incubation			96 h incubation
	BHR1	BHR5	Control	BHR1	BHR5	Control
	Concentration in ppm
As	0.662 ± 0.04a (2.79↓)	0.673 ± 0.03a (1.17↓)	0.678 ± 0.04a (0.44↓)	0.628 ± 0.01a (7.78↓)	0.659 ± 0.01ab (3.23↓)	0.670 ± 0.02b (1.61↓)
Cd	1.911 ± 0.02a (22.0↓)	1.942 ± 0.02a (20.7↓)	2.210 ± 0.16b (9.79↓)	1.845 ± 0.03a (24.6↓)	1.950 ± 0.04a (20.4↓)	2.180 ± 0.05b (11.0↓)
Cr	304.3 ± 1.90a (27.7↓)	321.5 ± 1.30b (23.6↓)	418.6 ± 0.80c (0.57↓)	290.1 ± 1.50a (31.0↓)	305.2 ± 1.00b (27.5↓)	411.7 ± 1.30c (2.21↓)
Ni	121.3 ± 0.40a (18.7↓)	129.3 ± 2.10b (13.4↓)	136.4 ± 2.70c (8.71↓)	115.4 ± 0.30a (22.7↓)	121.1 ± 2.00b (18.8↓)	131.8 ± 0.65c (11.7↓)
Pb	38.62 ± 2.25a (49.4↓)	46.40 ± 2.63b (39.1↓)	72.80 ± 0.84c (4.59↓)	29.83 ± 1.18a (60.9↓)	37.31 ± 1.40b (51.1↓)	70.11 ± 1.00c (8.12↓)
Fe	1962 ± 1.50a (4.33↓)	1988 ± 2.50b (3.06↓)	2031 ± 1.00c (0.97↓)	1923 ± 1.50a (6.22↓)	1969 ± 2.00b (3.95↓)	2022 ± 1.50c (1.36↓)
Zn	577.2 ± 1.55a (16.2↓)	582.2 ± 0.50b (15.4↓)	662.3 ± 1.45c (3.87↓)	536.8 ± 1.25a (22.0↓)	563.5 ± 2.35b (18.2↓)	640.4 ± 0.90c (7.04↓)

^#Control-Uninoculated effluent sample; incubation conditions: temperature 37 ± 2 °C with 120 rpm shaking. Values are mean ± SD (standard deviation) of three replicates; (↓): decrease; (↑): increase. Values with different alphabets indicate the significant difference among the bacterial inoculation in respective heavy metals according to post- hoc Tukey’s HSD (p < 0.05)

The results obtained from the above four treatments indicated a significant (p < 0.05) reduction in heavy metals concentration in effluent sample supplemented with minimal media as well as altered pH with inoculation of bacterial isolates BHR1 and BHR5 after the 96 h of incubation, indicating that both the isolates can have an efficient capacity to uptake/reduce the residual metals as compared to the un-inoculated samples. A study also reported that the bioremediation potential of Klebsiella edwardsii significantly increased with the addition of a carbon source and altered pH to 6.0 by 79.8% and 92.4%, respectively [49]. While the maximum reduction in residual Cr concentration with BHR5 (33.8%) inoculation was observed in the samples of altered pH. The decrease in the concentration of heavy metals in the control samples in all the treatments might be due to the precipitation of metal ions. Overall, the maximum reduction in heavy metal concentration was observed with inoculation of BHR1 bacterial isolate at pH 6.0 in the presence nutrient source. The present study revealed that the effectiveness of bacteria in the remediation process can be enhanced through the addition of nutrients which acts as an energy source for microbial growth and modification in pH that increases the bioavailability of metal ions.

Conclusion

Heavy metals are well-known environmental pollutants due to their toxicity, non-biodegradability and accumulative nature. In view of this, metal-resistant bacteria has been isolated from contaminated water, characterized and assessed for their capability of metal removal from the environment. Out of 43 distinct bacterial isolates, six potential isolates i.e. BHR1, BHR2, BHR4, BHR5, BHR6 and BHR8 were selected for further studies and identified as Bacillus thuringiensis, Bacillus cereus, Enterobacter cloacae, Bacillus pumilus, Bacillus altitudinis and Klebsiella pneumoniae, respectively using 16S rRNA gene sequencing. Assessment of minimum inhibitory concentration (MIC) and growth of isolates in the presence of metal ions revealed that among these isolates, BHR1 and BHR5 served as efficient bioremediation agents with high multi-metal resistance. However, the BHR1 isolate showed the maximum heavy metal reduction in effluent water supplemented with minimal media as well as modified pH compared to the BHR5 isolate. The bacterial isolates described in this work showed a promising ability to remove heavy metals from the contaminated environment and can be suitable candidate in the bioremediation process.

Supplementary Information

Below is the link to the electronic supplementary material.

Funding

Authors of the manuscript are thankful to the Punjab Pollution Control Board (PPCB), Government of Punjab for funding a part of the present project work.

Data Availability

The datasets of the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of interest

All the authors listed above have read and approved the submission. They also declare no competing financial and non-financial competing interests.