The environmental release of mercury is continuously increasing with high degree of mobility, transformation and amplified toxicity. Improving remediation strategies is becoming increasingly important to achieve more stringent environmental safety standards. This study develops a laboratory-scale reactor for bioremediation of aqueous mercury using a biofilm-producing bacterial strain, KBH10, isolated from mercury-polluted soil. The strain was found resistant to 80 mg/L of HgCl2 and identified as Bacillus nealsonii via 16S rRNA gene sequence analysis. The strain KBH10 was characterized for optimum growth parameters and its mercury biotransformation potential was validated through mercuric reductase assay. A packed-bed column bioreactor was designed for biofilm-mediated mercury removal from artificially contaminated water and residual mercury was estimated. Strain KBH10 could grow at a range of temperature (20–50 °C) and pH (6.0–9.0) with optimum temperature established at 30 °C and pH 7.0. The optimum mercuric reductase activity (77.8 ± 1.7 U/mg) was reported at 30 °C and was stable at a temperature range of 20–50 °C. The residual mercury analysis of artificially contaminated water indicated 60.6 ± 1.5% reduction in mercury content within 5 h of exposure. This regenerative process of biofilm-mediated mercury removal in a packed-bed column bioreactor can provide new insight into its potential use in mercury bioremediation.

  • Bioaccumulation of aqueous mercury in the food web is a matter of grave concern.

  • Enzymatic reduction of mercury by resistant bacteria reduces its potential toxicity.

  • Bacillus nealsonii possess the inbuilt mechanisms of necessary adaptation.

  • Mercury content was reduced up to 60.6 ± 1.5% within 5 h of exposure in packed-bed bioreactor.

  • Biofilm-mediated mercury remediation is efficient, regenerative and cost-effective.

Mercury (Hg) is a potentially toxic metal that exists naturally in the Earth's crust. Despite its toxic nature, mercury is widely used in several industrial sectors and released to the environment in finishing waste. The anthropogenic release of mercury accounts for its release in air, water and soil. Mercury released in aquatic bodies is alarming due to its transformation into methyl mercury (MeHg) resulting in biomagnification through the ecosystem. The overall high mobility of mercury facilitates its environmental cycling and bioaccumulation at various levels of food chain that eventually poisons human beings (Tchounwou et al. 2012). The increasing release of recalcitrant mercury continues to be a matter of grave concern to the public health and environment. Before discharge, the industrial waste is treated for undesirable concentrations of mercury to minimize its release to the environment. However, the physicochemical means of mercury remediation are costly, and produce a large volume of biomass and mercury-rich residues, which in turn creates disposal problems. The contemporary use of biological tools for remediation has simultaneous advantages of eco-friendliness and inexpensive approach along with greater flexibility to environmental fluctuations (Fulke et al. 2020; Chang et al. 2020).

Biotransformation of mercury to its least toxic form is an effective and alternative method to traditional remediation techniques. The ability of bacteria to reduce mercury is among the most widely found microbe-mediated enzymatic transformation of toxicants to non/less-toxic forms. Mercury resistant bacteria harbor mer operon, a gene cluster that detoxifies ionic mercury (Hg2+) to elemental mercury (Hg0) through enzymatic reduction (Naguib et al. 2018) and attains resistance to potentially toxic mercury. Such mercury-resistant bacteria can be applied beneficially in the detoxification and bioremediation of mercury. In addition to molecular mechanism of mercury resistance, biofilm formation also provides considerable resistance against unfavorable conditions. Bacterial biofilm established on inert support matrix in a bioreactor brings enzymatic transformation of mercury to minimize its toxicity (Gluszcz et al. 2008). The biofilm formation can substantially enhance the remediation potential of mercury-resistant strains.

In the current study, we isolated mercury-resistant bacteria followed by determination of their tolerance limit to mercury, screening for biofilm production, mercury volatilization and characterization of the most putative strain KBH10 for optimum growth and mercury biotransformation potential. Furthermore, a packed-bed reactor was set up employing Bacillus nealsonii strain KBH10 for bioremediation of mercury through biofilm-mediated enzymatic reduction.

Sampling site

Soil samples were collected from Korangi Industrial Area (KIA), Karachi, Pakistan (24°48′37.5″N, 67°06′52.6″E). The KIA is comprised of 8,500 acres and approximately 4,500 industries, trading and commercial units such as engineering, steel, textile, automobile, pharmaceutical, and chemicals are located in this area. The temperature of this area fluctuates throughout the year from 25 °C to 47 °C with a pH range of 7.5 to 8.5. The soil samples were collected from approximately 10 inches depth after removing the upper layer with a sterile spatula in Nalgene sample bottles. Samples were transported at 4 °C to Applied Environmental and Geomicrobiology (AEG) Research Laboratory, Department of Microbiology, Quaid-i-Azam University, Islamabad, following standard microbiological protocols and analyzed within 24 h after collection.

Sample analysis and isolation of mercury-resistant bacteria

Physicochemical properties of the soil samples including pH, temperature and moisture content were noted. Samples were prepared for elemental analysis of copper (Cu), lead (Pb), nickel (Ni), cadmium (Cd), cobalt (Co) and mercury (Hg) by Aqua regia leach method as previously described (Din et al. 2020) and analyzed through Inductively Coupled Plasma Mass Spectrometry (ICP-MS) (Agilent – 7800, USA). Mercury-resistant bacteria were isolated through selective enrichment in mercury-amended nutrient broth followed by serial dilution according to the method as previously described (Din et al. 2019). The colonies, which differed in shape, color and margins, were sub-cultured on nutrient agar plates supplemented with 20 mg/L concentration of mercury chloride (HgCl2) as an inducer for the expression of mercury-resistant genes.

Mercury tolerance assay

Bacterial isolates were streaked on nutrient agar plates amended with increasing concentration of HgCl2 in multiples of ten up to 100 mg/L to assess their maximum tolerance to HgCl2. A nutrient agar plate with no mercury was streaked for each strain as control. Plates were kept at 30 °C for 72 h and growth was observed after every 24 h. The highest concentration of mercury allowing an apparent growth was considered as the mercury tolerance limit. The tolerance limit was further confirmed by challenging the bacterial strain with the same concentration of HgCl2 in nutrient broth incubated for 24 h in a shaking incubator at 120 rpm and 30 °C. Growth was measured in terms of optical density at 600 nm (OD600) via spectrophotometer (Agilent – 8453, USA).

Quantitative assay for biofilm formation

The biofilm formation was quantified to identify the most putative biofilm-producing strain. The mercury tolerant bacterial isolates were grown overnight in Tryptone Soya Broth (TSB) at 30 °C and 120 rpm in a shaking incubator. Twenty microliter broth culture was added to a flat-bottom 96-well microtiter plate containing 180 μL of freshly prepared TSB and incubated at 30 °C for 24 h. The wells with TSB only and no bacterial culture were considered as control. After incubation, the plates were gently washed three times with phosphate buffer saline (pH 6.7) to eliminate planktonic cells. Then 300 μL methanol was added to each well for 15 minutes, followed by staining with safranin (0.1% w/v) for 20 minutes. The extra stain was discarded, and wells were gently washed twice with PBS. The stained cells adhering to wells were resolubilized by adding glacial acetic acid (33% v/v) and optical density was measured at 490 nm.

Mercury volatilization assay

Mercury resistant isolates producing biofilm were tested for HgCl2 volatilization according to the method as previously described (Nakamura et al. 1999). The bacterial isolates were grown for 24 h in nutrient broth supplemented with 50 mg/L of HgCl2. After incubation, 5 mL of culture broth was centrifuged for 20 minutes at 12,000 × g. The pellet was washed twice with PBS and re-suspended in 50 μL of freshly prepared 0.07 M phosphate buffer (pH 7.0) containing EDTA (0.5 mM), sodium thioglycolate (5 mM), magnesium acetate (0.2 mM) and HgCl2 (50 μg/mL) in a microtiter plate. The plate was covered with X-ray film and incubated at 30 °C for 60 min in dark. Subsequently, the X-ray film was developed and observed for white patches produced due to the reaction of volatile mercury with the silver coating on X-ray film.

Based on mercury tolerance, biofilm formation and mercury volatilization, the most putative strain (KBH10) among all isolates was selected for further study.

Characterization of strain KBH10

Identification and phylogenetic analysis

Strain KBH10 was grown on nutrient agar plates at 30 °C for 24 h and observed for its colony morphology. Gram's reaction was determined using a Gram staining kit (Carlo Erba, France) and cellular morphology was examined under a light microscope (Nikon YS100, Japan). The genomic DNA was extracted using a genomic DNA extraction kit (Norgen Biotek Corp, Canada) and analyzed through a NanoDrop 2000 (Fisher Scientific, USA). The 16S rRNA sequence analysis was carried out at Alpha Genomics, Islamabad, Pakistan, by amplifying the V4 region with universal primer 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) (Caporas et al. 2011) using standard polymerase chain reaction conditions (Din et al. 2019). The obtained sequence, together with closely related sequences from the GenBank BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/), was aligned together in MEGA-X software and a phylogenetic tree was constructed by adopting the Neighbor-Joining method. The partial 16S rRNA sequence of KBH10 was submitted to NCBI and can be obtained under sequence number MT180167.

Optimization of temperature and pH

The effect of temperature and pH on growth was determined by incubating 1% inoculum (OD600 0.08–0.1) of strain KBH10 in 50 mL nutrient broth at a range of temperatures (20, 25, 30, 35, 40, 45 and 50 °C) and pH (6, 7, 8 and 9) for 48 h in a shaking incubator at 120 rpm. Growth was checked every 24 h by measuring its OD at 600 nm through a spectrophotometer.

Mercuric reductase assay

Mercuric reductase (MR) assay was performed according to the method as previously described with slight modification (Freedman et al. 2012). The cells of strain KBH10 in the mid-log phase were harvested through centrifugation at 12,000 × g for 20 min at 4 °C and stored at −20 °C. Frozen cells (200 mg/mL) were re-suspended in a buffer of 20 mM sodium phosphate (pH 7.5), 0.5 mM EDTA, and 1 mM β-mercaptoethanol; then lysed by sonication for 6 × 30 s cycles using an ultrasonic bath (Spectralab, Italy). The cell lysate was centrifuged at 12,000 × g for 10 min and supernatant was collected in clean Eppendorf tubes. Mercuric reductase assay of the obtained crude cell extract was performed in 80 mM sodium phosphate buffer (pH 7.4) containing 1 mM β-mercaptoethanol, 200 μM NAD(P)H and 50 μM HgCl2 in a final volume of 800 μL. Assay was performed on range of temperatures (20, 30, 40 and 50 °C) at which the strain was able to grow. Assay temperature was controlled using a water-jacketed cuvette holder connected to a water bath. A 4 μL of crude extract was added to the 800 μL assay buffer, incubated for 1 min, and mercury-dependent NAD(P)H oxidation was monitored as a decrease in absorbance at 340 nm using a UV-visible spectrophotometer (UV-3600 Shimadzu, Japan). Total protein concentration in crude cell extract was determined by Lowry's method (Lowry et al. 1951). Mercury-dependent NADH oxidation rate was calculated from the absorbance curve and expressed as mU (1U = μmol of NADH oxidized/min/mg of total protein).

Packed-bed column bioreactor for mercury remediation

Strain KBH10 was evaluated for its mercury remediation potential by adopting the methods as previously described with slight modification (Gluszcz et al. 2008). Biofilm of strain KBH10 was developed on sterilized white pebbles (6–10 mm) by growing for 72 h in 500 mL nutrient broth in a static incubator at 30 °C. A control was run in parallel with sterilized white pebbles in culture-free nutrient broth. The biofilm formation was analyzed by sonicating 4–5 pebbles in PBS and CFU/mL was calculated by serial dilution method on a mercury-amended nutrient agar plate. A packed-bed glass column reactor was designed for mercury bioremediation with 53.5 cm length and an internal diameter of 4.5 cm (Figure 1). The total capacity of the column was calculated. The column was loaded with biofilm-coated white pebbles and 500 mL of 10 mg/L HgCl2 solution prepared in deionized water. A similar column setup was used as a control containing sterilized pebbles with no biofilm. At 0 h, a 10 mL sample was collected from the control and test columns followed by successive samples collected at 1 h intervals up to 24 h and analyzed for mercury concentration through ICP-MS.

Figure 1

Packed-bed reactor using a glass column of length 53.5 and internal diameter 4.5 cm. The total capacity (V) of the column was 850 mL. The average particle size was 6–10 mm.

Figure 1

Packed-bed reactor using a glass column of length 53.5 and internal diameter 4.5 cm. The total capacity (V) of the column was 850 mL. The average particle size was 6–10 mm.

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Statistical analysis

Three individual replicates were performed for each experiment and values were presented as mean ± S.D. One-way ANOVA followed by post-Tukey's multiple comparisons were used for the significant results (p < 0.05) during mercury removal experiments. GraphPad Prism version 7 was used for statistical analyses.

Sampling site and isolation

Mercury being recognized as a potent and widely distributed toxicant in the global environment is of major concern in the present-day world. It is actively applied in various industries and released in finishing waste, which in turn pollutes the industrial sites and atmosphere. The metal analysis of our study sample indicated high concentrations of Cu, Pb, Ni, Cd, Co and Hg in comparison to the control sample. Table 1 shows the physicochemical properties and elemental analysis of soil samples collected from Korangi industrial area (Karachi), Pakistan. The high mercury concentration in groundwater and vegetables grown on-site signify the mercury contamination of the sampling site (Haq et al. 2005). A total of 18 bacterial strains, designated as KBH1 to KBH18, were isolated by selective enrichment with 20 mg/L of HgCl2 and recognized as separate strains with distinct cultural characteristics on nutrient agar medium. The high degree of mercury resistant bacteria indicates that a rich microbial community is inhabiting the sampling site of the industrial zone. Strain KBH10 was selected as the most putative strain on the basis of mercury tolerance, biofilm production, and mercury volatilization (Table 2).

Table 1

Elemental analysis of Korangi mud sample

SampleMetal concentration
μg/Kgmg/Kg
MercuryCopperLeadNickelCobaltCadmium
Korangi 712 ± 15 798 ± 63 240 ± 8 983.0 ± 44 29 ± 2 13 ± 2 
Controla 103 ± 7 3 ± 0.09 0.4 ± 0.06 2 ± 0.1 1 ± 0.1 0.2 ± 0.1 
SampleMetal concentration
μg/Kgmg/Kg
MercuryCopperLeadNickelCobaltCadmium
Korangi 712 ± 15 798 ± 63 240 ± 8 983.0 ± 44 29 ± 2 13 ± 2 
Controla 103 ± 7 3 ± 0.09 0.4 ± 0.06 2 ± 0.1 1 ± 0.1 0.2 ± 0.1 

aGarden soil.

Table 2

Results of screening experiments for comparative analysis in order to choose the most putative strain

No.StrainMercury tolerance (mg/L)Biofilm ODHg volatilization
KBH1 40 0.4 ± 0.06 ND 
KBH2 60 0.5 ± 0.06 Weak positive 
KBH3 20 0.5 ± 0.11 Negative 
KBH4 20 0.4 ± 0.08 ND 
KBH5 40 0.5 ± 0.1 Negative 
KBH6 40 0.3 ± 0.1 ND 
KBH7 20 0.5 ± 0.08 Negative 
KBH8 40 0.5 ± 0.11 Weak positive 
KBH9 40 0.7 ± 0.13 Weak positive 
10 KBH10 80 1.0 ± 0.05 Strong positive 
11 KBH11 40 0.3 ± 0.07 ND 
12 KBH12 20 0.3 ± 0.08 ND 
13 KBH13 60 0.4 ± 0.03 ND 
14 KBH14 40 0.5 ± 0.06 Weak positive 
15 KBH15 40 0.4 ± 0.04 ND 
16 KBH16 40 0.3 ± 0.05 ND 
17 KBH17 20 0.4 ± 0.07 ND 
18 KBH18 40 0.6 ± 0.04 Weak positive 
No.StrainMercury tolerance (mg/L)Biofilm ODHg volatilization
KBH1 40 0.4 ± 0.06 ND 
KBH2 60 0.5 ± 0.06 Weak positive 
KBH3 20 0.5 ± 0.11 Negative 
KBH4 20 0.4 ± 0.08 ND 
KBH5 40 0.5 ± 0.1 Negative 
KBH6 40 0.3 ± 0.1 ND 
KBH7 20 0.5 ± 0.08 Negative 
KBH8 40 0.5 ± 0.11 Weak positive 
KBH9 40 0.7 ± 0.13 Weak positive 
10 KBH10 80 1.0 ± 0.05 Strong positive 
11 KBH11 40 0.3 ± 0.07 ND 
12 KBH12 20 0.3 ± 0.08 ND 
13 KBH13 60 0.4 ± 0.03 ND 
14 KBH14 40 0.5 ± 0.06 Weak positive 
15 KBH15 40 0.4 ± 0.04 ND 
16 KBH16 40 0.3 ± 0.05 ND 
17 KBH17 20 0.4 ± 0.07 ND 
18 KBH18 40 0.6 ± 0.04 Weak positive 

ND, Not Determined.

Mercury tolerance assay

All the 18 bacterial isolates were screened for their tolerance against increasing concentration of mercury. A gradual decrease in growth was observed with increase in HgCl2 concentration on nutrient agar. Strain KBH10 was the only strain that could grow efficiently in the presence of 80 mg/L of HgCl2 as shown in Table 3, whereas no growth was observed above this concentration. A number of researchers have reported mercury-resistant bacteria from polluted sites with Bacillus and Pseudomonas as the most representative genera (Mindlin et al. 2005; Dash et al. 2014).

Table 3

Bacterial isolates with their maximum tolerance to mercury on nutrient agar plates

Bacterial
Isolates
HgCl2 concentration (mg/L)
020406080
KBH1 ++ + + ++ + + ++ + ++ – 
KBH2 ++ + + ++ + + ++ + + ++ + – 
KBH3 ++ + + ++ + + ++ – 
KBH4 ++ + + ++ + + ++ – 
KBH5 ++ + + ++ + + ++ + ++ 
KBH6 ++ + + ++ + + ++ + + ++ 
KBH7 ++ + + ++ + + ++ – 
KBH8 ++ + + ++ + + ++ + – 
KBH9 ++ + + ++ + + ++ + + ++ – 
KBH10 ++ + + ++ + + ++ + + ++ + + ++ + 
KBH11 ++ + + ++ + + ++ + ++ – 
KBH12 ++ + + ++ + + – 
KBH13 ++ + + ++ + + ++ + + ++ + – 
KBH14 ++ + + ++ + + ++ + ++ 
KBH15 ++ + + ++ + + ++ + ++ – 
KBH16 ++ + + ++ + + ++ + + – 
KBH17 ++ + + ++ + + – – 
KBH18 ++ + + ++ + + ++ + ++ 
Bacterial
Isolates
HgCl2 concentration (mg/L)
020406080
KBH1 ++ + + ++ + + ++ + ++ – 
KBH2 ++ + + ++ + + ++ + + ++ + – 
KBH3 ++ + + ++ + + ++ – 
KBH4 ++ + + ++ + + ++ – 
KBH5 ++ + + ++ + + ++ + ++ 
KBH6 ++ + + ++ + + ++ + + ++ 
KBH7 ++ + + ++ + + ++ – 
KBH8 ++ + + ++ + + ++ + – 
KBH9 ++ + + ++ + + ++ + + ++ – 
KBH10 ++ + + ++ + + ++ + + ++ + + ++ + 
KBH11 ++ + + ++ + + ++ + ++ – 
KBH12 ++ + + ++ + + – 
KBH13 ++ + + ++ + + ++ + + ++ + – 
KBH14 ++ + + ++ + + ++ + ++ 
KBH15 ++ + + ++ + + ++ + ++ – 
KBH16 ++ + + ++ + + ++ + + – 
KBH17 ++ + + ++ + + – – 
KBH18 ++ + + ++ + + ++ + ++ 

Growth: Excellent (++ + +), Good (+++), Fair (++ ), Poor (+), No Growth (–).

Quantitative assay for biofilm production

All the isolates were screened for biofilm formation in 96-well microtiter plate. Among all the isolates, strain KBH10 was observed as an efficient biofilm producer in terms of optical density (Figure 2). Bacteria adapt to harsh conditions of metal stress by presenting various resistance mechanisms. The production of exopolysaccharides (EPS) and biofilm is one of the resistance mechanisms that contributes to increase the survival chance of bacteria in the metal-polluted site by adoring metal elements (Yin et al. 2019). In quantitative biofilm assay, Bacillus nealsonii strain KBH10 was found as a strong biofilm producer among the rest of the isolates. The role of biofilm in resistance against heavy metal has been addressed by several researchers in their reports (Nocelli et al. 2016).

Figure 2

Quantitative analysis of biofilm production by bacterial strains isolated from mercury polluted soil samples.

Figure 2

Quantitative analysis of biofilm production by bacterial strains isolated from mercury polluted soil samples.

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Mercury volatilization assay

Mercury volatilization potential of selected biofilm-producing strains was determined using the simplified X-ray film method. Strain KBH10, with maximum mercury tolerance, was observed to produce determined foggy formation on X-ray film among all tested isolates (Figure 3). The distinguished foggy spots on X-ray film indicates mercury transformation from Hg2+ into Hg0 with subsequent volatilization (Nakamura et al. 1999). The reduction of mercury from its oxidized to volatile form is the premium resistance mechanism that mercury-resistant bacteria showcase and facilitated by the merA gene (Naguib et al. 2019). Our results coincide with the study (Chatziefthimiou et al. 2007) reporting three strains indicative of mercury volatilization that were among the most mercury-resistant isolates in their culture collection. Several bacterial genera resistant to mercury have also been reported for their mercury volatilization potential (Moller et al. 2011).

Figure 3

Mercury volatilization by biofilm-producing mercury-resistant bacteria KBH2 to KBH18 from left to right on X-ray film in the presence of 50 μg/mL of HgCl2. The intensity of foggy spots indicates the production of Hg0.

Figure 3

Mercury volatilization by biofilm-producing mercury-resistant bacteria KBH2 to KBH18 from left to right on X-ray film in the presence of 50 μg/mL of HgCl2. The intensity of foggy spots indicates the production of Hg0.

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Characterization of strain KBH10

Strain KBH10 was observed as Gram-positive rods under the microscope whereas the colony appeared dull gray, opaque with a rough matted surface, larger in size and with irregular perimeters. Figure 4 shows the phylogenetic tree presenting the evolutionary distance of strain KBH10 with closely related strains obtained from NCBI. The 16S rRNA gene sequencing indicated a close genetic relationship of strain KBH10 to Bacillus nealsonii with a boot strap value of 90. Strain KBH10 exhibited growth at a range of temperature (20–50 °C) and pH (6.0–9.0) with optimum at 30 °C and pH 7.0 (Supplementary Figures 1 and 2). The optimized growth condition of strain KBH10 could be helpful in determining optimum conditions during the application of the strain. The optimum growth conditions usually reflect the physicochemical properties of the sampling site (Kotwal et al. 2018). The temperature of our site was 45 °C at the time of sample collection, which may be a reason for the adaptation of microbiota up to 50 °C. Mercury-resistant bacteria adapted to high temperatures are also suggestive of the enzymatic transformation of mercury and its mobilization in geothermal environments (Chatziefthimiou et al. 2007).

Figure 4

Phylogenetic tree of bacterial strain KBH10 constructed by the Neighbor-Joining method. Number at nodes shows the level of bootstrap support based on data for 1,000 replications. Bar = 0.005 substitutions per nucleotide position and numbers in bracket represent GenBank accession numbers.

Figure 4

Phylogenetic tree of bacterial strain KBH10 constructed by the Neighbor-Joining method. Number at nodes shows the level of bootstrap support based on data for 1,000 replications. Bar = 0.005 substitutions per nucleotide position and numbers in bracket represent GenBank accession numbers.

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Mercuric reductase assay

The activity of mercuric reductase (MR) from B. nealsonii strain KBH10 was calculated in order to determine its role in conversion of Hg2+ to Hg0 under standard assay conditions. MR activity of the crude extract of strain KBH10 was observed at a temperature range of 20–50 °C with no statistically significant difference in MR activity (p-value = 0.1424). Figure 5 shows the Hg-dependent NAD(P)H oxidation at temperatures suitable for growth of strain KBH10 with maximum MR activity 77.8 ± 1.7 U/mg at 30 °C. No substantial MR activity was observed in control. The total protein concentration in crude extract from strain KBH10 was estimated as 0.334 mg/mL using a BSA standard curve (R2 = 0.996). Mercury resistant bacteria carry merA gene a component of mer operon, coded for mercuric reductase enzyme which reduces mercury to its least toxic form i.e. H0 and thus confer resistance to bacteria (Sotero-Martins et al. 2008). In some of the previous reports, the scientists have reported maximum MR activity under both mesophilic as well as thermophilic conditions. The thermal stability of their mercuric reductase enzyme was established at a temperature range of 30–50 °C with gradual inhibition of enzyme activity up to 80 °C (Nakamura et al. 1999; Sayed et al. 2014). The mercury resistant B. nealsonii KHB10 expressed resistance to considerable concentration that could be the possible reason for its high MR activity. The reducing ability of mercury resistant Bacillus sp. is accredited for their excellent bioremediation potential (Zulaika & Sembiring 2013). The mercury volatilization and high MR enzyme activity suggests that B. nealsonii strain KBH10 can be recruited as an efficient tool for bioremediation of aqueous mercury.

Figure 5

Effect of temperature on MR activities. Specific activities of crude cell extracts were determined for strain KBH10 at different temperatures (20–50 °C).

Figure 5

Effect of temperature on MR activities. Specific activities of crude cell extracts were determined for strain KBH10 at different temperatures (20–50 °C).

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Packed-bed column bioreactor for mercury removal

Biofilm-mediated mercury removal was carried out by treating artificially contaminated water samples containing 10 mg/L HgCl2 in a column bioreactor packed with pebbles coated by biofilm of B. nealsonii strain KBH10 (4.4 × 102 CFU/mL). ICP-MS analysis of samples indicated that 60.6 ± 1.5% mercury was removed within 5 h with no further decrease in subsequent samples.

Although the biotic enzymatic reduction of Hg2+ to elemental Hg0 is an important pathway for detoxification of mercury in natural ecosystems (Chang et al. 2021), it is restricted to the bioavailable mercury only and no further reduction is possible, due to the unavailability of mercury to the bacteria. Only 2 ± 0.06% of mercury content was decreased in samples from the control setup (Figure 6). Decrease in the mercury content of the treated water indicates mercury removal by immobilized cells of B. nealsonii KBH10. Our results coincide with the findings of Pepi et al. (2011) and Mahbub et al. (2016) identifying mercury-resistant bacterial strains with substantial mercury volatilization within a few hours to several days. Biofilm forming bacteria possess a dual role in mercury bioremediation by utilizing its EPS for mercury entrapment with subsequent volatilization through expression of mer operon (Dash et al. 2017). Immobilization of biofilm-producing bacteria in a packed-bed bioreactor can efficiently reduce mercury content in wastewater (Dzairi et al. 2004; Leonhauser et al. 2006). The immobilized cells are advantageous over a free cells for bioremediation of various types of wastes (Mitra & Mukhopadhyay 2016). The biofilm-mediated regenerative method adapted in this study could used for efficient removal of mercury in wastewater over a long period of time that will not only lower the operational cost but also boost the remediation strategy.

Figure 6

Mercury removal efficiency of Bacillus nealsonii KBH10 treating water supplemented with 10 mg/L of HgCl2. Values were calculated as mean and bar represents standard deviation (n = 3).

Figure 6

Mercury removal efficiency of Bacillus nealsonii KBH10 treating water supplemented with 10 mg/L of HgCl2. Values were calculated as mean and bar represents standard deviation (n = 3).

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Identifying a suitable mercury-resistant strain is a prerequisite to the technological development of mercury bioremediation strategies. We demonstrate biofilm-mediated mercury treatment in a packed-bed column using mercury-resistant strain Bacillus nealsonii strain KBH10. It is a simple, rapid and regenerative approach that could prove a valuable addition to the previously available bioremediation approaches and could help in minimizing mercury in industrial wastewater.

We acknowledge Higher Education Commission of Pakistan for provision of funds and research facility to conduct this study.

The authors declare no conflict of interest.

All relevant data are included in the paper or its Supplementary Information.

Caporaso
J. G.
Lauber
C. L.
Walters
W. A.
Berg-Lyons
D.
Lozupone
C. A.
Turnbaugh
P. J.
Fierer
N.
Knight
R.
2011
Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample
.
Proceedings of the National Academy of Sciences
108
(
Supplement 1
),
4516
4522
.
Chang
J.
Si
G.
Dong
J.
Yang
Q.
Shi
Y.
Chen
Y.
Zhou
K.
Chen
J.
2021
Transcriptomic analyses reveal the pathways associated with the volatilization and resistance of mercury (II) in the fungus Lecythophora sp. DC-F1
.
Science of The Total Environment
752
,
142172
.
Chatziefthimiou
A. D.
Crespo-Medina
M.
Wang
Y.
Vetriani
C.
Barkay
T.
2007
The isolation and initial characterization of mercury resistant chemolithotrophic thermophilic bacteria from mercury rich geothermal springs
.
Extremophiles
11
(
3
),
469
479
.
Dash
H. R.
Mangwani
N.
Das
S.
2014
Characterization and potential application in mercury bioremediation of highly mercury-resistant marine bacterium Bacillus thuringiensis PW-05
.
Environmental Science and Pollution Research
21
(
4
),
2642
2653
.
Din
G.
Hasan
F.
Conway
M.
Denney
B.
Ripp
S.
Shah
A. A.
2019
Engineering a bioluminescent bioreporter from an environmentally sourced mercury-resistant Enterobacter cloacae strain for the detection of bioavailable mercury
.
Journal of Applied Microbiology
127
(
4
),
1125
1134
.
Din
G.
Hassan
A.
Rafiq
M.
Hasan
F.
Badshah
M.
Khan
S.
Che
G.
Ripp
S.
Shah
A. A.
2020
Characterization of organic acid producing Aspergillus tubingensis FMS1 and its role in metals leaching from soil
.
Geomicrobiol Journal
37
(
4
),
336
344
.
Dzairi
F. Z.
Zeroual
Y.
Moutaouakkil
A.
Taoufik
J.
Talbi
M.
Loutfi
M.
Lee
K.
Blaghen
M.
2004
Bacterial volatilization of mercury by immobilized bacteria in fixed and fluidized bed bioreactors
.
Annals of Microbiology
54
(
4
),
353
364
.
Freedman
Z.
Zhu
C.
Barkay
T.
2012
Mercury resistance and mercuric reductase activities and expression among chemotrophic thermophilic Aquificae
.
Applied and Environmental Microbiology
78
(
18
),
6568
6575
.
Fulke
A. B.
Kotian
A.
Giripunje
M. D.
2020
Marine microbial response to heavy metals: mechanism, implications and future prospect
.
Bulletin of Environmental Contamination and Toxicology
105
,
182
197
.
Gluszcz
P.
Zakrzewska
K.
Wagner-Doebler
I.
Ledakowicz
S.
2008
Bioreduction of ionic mercury from wastewater in a fixed-bed bioreactor with activated carbon
.
Chemical Papers
62
(
3
),
232
238
.
Haq
M. U.
Khattak
R. A.
Puno
H. K.
Saif
M. S.
Memon
K. S.
Sial
N. B.
2005
Toxicity of trace elements in different vegetables grown on potentially contaminated sites of the Korangi industrial area, Karachi Pakistan
.
Asian Journal of Plant Sciences
4
(
2
),
132
135
.
Kotwal
D. R.
Shewale
N. B.
Tambat
U. S.
Thakare
M. J.
Bholay
A. D.
2018
Bioremediation of mercury using mercury resistant bacteria
.
Research Journal of Life Sciences, Bioinformatics, Pharmaceutical and Chemical Sciences
4
,
145
156
.
Leonhauser
J.
Rohricht
M.
Wagner-Dobler
I.
Deckwer
W. D.
2006
Reaction engineering aspects of microbial mercury removal
.
Engineering in Life Sciences
6
(
2
),
139
148
.
Lowry
O. H.
Rosebrough
N. J.
Farr
A. L.
Randall
R. J.
1951
Protein measurement with the Folin phenol reagent
.
Journal of Biological Chemistry
193
,
265
275
.
Mindlin
S.
Minakhin
L.
Petrova
M.
Kholodii
G.
Minakhina
S.
Gorlenko
Z.
Nikiforov
V.
2005
Present-day mercury resistance transposons are common in bacteria preserved in permafrost grounds since the Upper Pleistocene
.
Research in Microbiology
156
(
10
),
994
1004
.
Mitra
A.
Mukhopadhyay
S.
2016
Biofilm mediated decontamination of pollutants from the environment
.
AIMS Bioengineering
3
(
1
),
44
.
Moller
A. K.
Barkay
T.
Al-Soud
W. A.
Sørensen
S. J.
Skov
H.
Kroer
N.
2011
Diversity and characterization of mercury-resistant bacteria in snow, freshwater and sea-ice brine from the High Arctic
.
FEMS Microbiol Ecol
75
(
3
),
390
401
.
Naguib
M. M.
El-Gendy
A. O.
Khairalla
A. S.
2018
Microbial diversity of mer operon genes and their potential rules in mercury bioremediation and resistance
.
Open Biotechnology Journal
12
(
1
),
56
77
.
Naguib
M. M.
Khairalla
A. S.
El-Gendy
A. O.
Elkhatib
W. F.
2019
Isolation and characterization of mercury-resistant bacteria from wastewater sources in Egypt
.
Canadian Journal of Microbiology
65
(
4
),
308
321
.
Nakamura
K.
Naruse
I.
Takizawa
Y.
1999
A new mass screening method for methylmercury poisoning using mercury-volatilizing bacteria from Minamata Bay
.
Ecotoxicology and Environmental Safety
44
(
1
),
100
104
.
Nocelli
N.
Bogino
P. C.
Banchio
E.
Giordano
W.
2016
Roles of extracellular polysaccharides and biofilm formation in heavy metal resistance of rhizobia
.
Materials
9
(
6
),
418
.
Pepi
M.
Gaggi
C.
Bernardini
E.
Focardi
S.
Lobianco
A.
Ruta
M.
Nicolardi
V.
Volterrani
M.
Gasperini
S.
Trinchera
G.
Renzi
P.
Gabellin
M.
Focardi
S. E.
2011
Mercury-resistant bacterial strains Pseudomonas and Psychrobacter spp. isolated from sediments of Orbetello Lagoon (Italy) and their possible use in bioremediation processes
.
International Biodeterioration & Biodegradation
65
(
1
),
85
91
.
Sayed
A.
Ghazy
M. A.
Ferreira
A. J.
Setubal
J. C.
Chambergo
F. S.
Ouf
A.
Adel
M.
Dawe
A. S.
Archer
J. A. C.
Bajic
V. B.
Rania Siam
R.
Hamza El-Dorry
H.
2014
A novel mercuric reductase from the unique deep brine environment of Atlantis II in the Red Sea
.
Journal of Biological Chemistry
289
(
3
),
1675
1687
.
Sotero-Martins
A.
Jesus
M. S. D.
Lacerda
M.
Moreira
J. C.
Filgueiras
A. L. L.
Barrocas
P. R. G.
2008
A conservative region of the mercuric reductase gene (merA) as a molecular marker of bacterial mercury resistance
.
Brazilian Journal of Microbiology
39
(
2
),
307
310
.
Tchounwou
P. B.
Yedjou
C. G.
Patlolla
A. K.
Sutton
D. J.
2012
Heavy metal toxicity and the environment
. In:
Molecular, Clinical and Environmental Toxicology
(A. Luch, ed.).
Springer
,
Basel
, pp.
133
164
.
Yin
W.
Wang
Y.
Liu
L.
He
J.
2019
Biofilms: the microbial ‘Protective clothing’ in extreme environments
.
International Journal of Molecular Sciences
20
(
14
),
3423
.
Zulaika
E.
Sembiring
L.
2013
Mercury Reductase Activity of an Indigenous Mercury Resistant Bacterial Isolate (Bacillus sp. S1) from Kalimas-Surabaya As a Potential Reducing Agent for Mercurial Ion (Hg 2+)
. In:
The International Conference on Biological Science (ICBS)
,
September
, Vol.
20
, p.
21
.

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