Cadmium resistance and uptake by bacterium, Salmonella enterica 43C, isolated from industrial effluent

Abstract

Cadmium resistant bacterium, isolated from industrial wastewater, was characterized as Salmonella enterica 43C on the basis of biochemical and 16S rRNA ribotyping. It is first ever reported S. enterica 43C bared extreme resistance against heavy metal consortia in order of Pb²⁺>Cd²⁺>As³⁺>Zn²⁺>Cr⁶⁺>Cu²⁺>Hg²⁺. Cd²⁺ stress altered growth pattern of the bacterium in time dependent manner. It could remove nearly 57 % Cd²⁺ from the medium over a period of 8 days. Kinetic and thermodynamic studies based on various adsorption isotherm models (Langmuir and Freundlich) depicted the Cd²⁺ biosorption as spontaneous, feasible and endothermic in nature. Interestingly, the bacterium followed pseudo first order kinetics, making it a good biosorbent for heavy metal ions. The S. enterica 43C Cd²⁺ processivity was significantly influenced by temperature, pH, initial Cd²⁺ concentration, biomass dosage and co-metal ions. FTIR analysis of the bacterium revealed the active participation of amide and carbonyl moieties in Cd²⁺ adsorption confirmed by EDX analysis. Electron micrographs beckoned further surface adsorption and increased bacterial size due to intracellular Cd²⁺ accumulation. An overwhelming increase in glutathione and other non-protein thiols levels played a significant role in thriving oxidative stress generated by metal cations. Presence of metallothionein clearly depicted the role of such proteins in bacterial metal resistance mechanism. The present study results clearly declare S. enterica 43C a suitable candidate for green chemistry to bioremediate environmental Cd²⁺.

Introduction

Heavy metal pollution has become a major environmental problem worldwide (Ali et al. 2013). Some heavy metals including arsenic (As), cadmium (Cd), mercury (Hg) and chromium (Cr) are called non-essential metals as these are not needed for the survival of organisms and are toxic in low concentrations. Cd is highly toxic, non-essential and non-biodegradable heavy metal with half-life of 20 years (Martelli et al. 2006; Aksoy et al. 2014; Xu et al. 2014; Vinodini et al. 2015). It is soft, silver-white, electropositive metal having atomic number 48, atomic mass 112.41, density 8.64 g/cm³ and melting point 321 °C. It is divalent in all of its stable compounds and found abundantly in nature in the form of CdS. It may also combine with ammonia and cyanide to form Cd(NH₃) ⁻⁴₆ and Cd(CN) ⁻²₄ , respectively. Various industrial processes such as mining, electroplating, stabilizing plastics, manufacturing batteries, alloy, pigment, cement, fossil fuel combustion, municipal and sewage sludge incineration and high phosphate fertilizers are responsible for the release of huge amount of Cd²⁺ in the environment (Klaassen et al. 2009; Yazdankhah et al. 2010; Liu et al. 2013). According to an estimation 30,000 tons Cd²⁺ is released in our environment annually (Nriagu and Pacyna 1988).

Cd and almost all of its compounds are water soluble and hence easily gain entry in human food chain. No physiological role of cadmium in human cellular metabolism has been reported so far and it is extremely toxic in very minute quantity (Aksoy et al. 2014; Xu et al. 2014). It has also been reported to cause osteoporosis and fractures, anemia, eosinophilia, anosmia, apoptosis, diabetes mellitus, oncogenes activation, Itai–Itai disease and chronic pulmonary problems (Waisberg et al. 2003; Edwards and Prozialeck 2009; Yazdankhah et al. 2010).

No treatment for cadmium toxicity has been approved so far (Zhai et al. 2015). Several chemical and physical methods are used to remove cadmium from industrial effluent prior to release the effluent into the environment but all these methods are expensive and less effective (Kurniawan et al. 2006). So the use of living and dead microbial biomass is gaining attraction to remove heavy metals from environment due to less expensive, effective, time efficient and environmental friendly strategy (Feng and Aldrich 2004; He et al. 2011; Huang et al. 2014). Bacteria remove heavy metal ions including Cd²⁺ from the environment either by metabolism independent adsorption on their cell walls or metabolism dependent intracellular accumulation (Gadd 1990; Srinath et al. 2002; Vargas-García et al. 2012). Hyperaccumulation of Cd²⁺ has been reported to disturb the cell physiology by reactive oxygen species (ROS) production and disruption of bacterial respiratory proteins (Gibbons et al. 2011; Zeng et al. 2012). Bacteria have evolved several resistance mechanisms including efflux transport, precipitation, transformation and intracellular sequestration by metallothionein, glutathione and other thiol containing compounds to combat the negative effects of heavy metal ions intracellular accumulation (Nies 2003; Intorne et al. 2012; Maynaud et al. 2014).

The present study is aimed at the isolation and characterization of Cd²⁺ resistant bacterium from industrial wastewater and its potential use to remove Cd²⁺ from aqueous environment. The effect of culture conditions with reference to pH, temperature, initial Cd²⁺ concentration, biomass dosage and co-metal ions on the Cd²⁺ processing capability of the bacterium was also measured. Adsorption data was evaluated by applying adsorption isotherm models, thermodynamic and kinetic studies. Adsorption and intracellular accumulation of Cd²⁺ were confirmed by FTIR, SEM and EDX analysis. The effect of Cd²⁺ stress on bacterial growth and cell physiology (change in glutathione and non-protein thiol levels) was also determined.

Materials and methods

Sample collection

Industrial wastewater samples were collected in sterilized screw capped bottles from different industrial areas of Sheikhupura and Kotlakhpat, Lahore, Pakistan. Some physiochemical parameters of wastewater samples viz., temperature (°C), pH and cadmium ion concentration (mM) were measured.

Isolation and purification of cadmium resistant bacteria

To isolate cadmium resistant bacteria, 100 µL of wastewater sample was spread on Luria-Bertani (LB) agar (1 % NaCl, 1 % tryptone, 0.5 % yeast extract and 1.5 % agar) plates supplemented with 1 mM Cd²⁺. Bacterial growth was observed after incubation at 37 °C for 24 h. Single colony was picked with sterilized wire loop and re-streaked on Cd²⁺ supplemented LB agar plates and again incubated at 37 °C for 24 h. The process was repeated until the purified single colony obtained.

Determination of minimum inhibitory concentration

The bacterial isolate was grown in minimal salt (MS) broth (1 % (NH₄)₂SO₄, 0.086 % CaCl₂, 0.15 % K₂HPO₄, 0.1 % KH₂PO₄, 0.1 % MgSO₄, 0.02 % FeSO₄ and 1 % glucose) supplemented with 1 mM Cd²⁺ at 37 °C for 24 h. Growth was observed by taking absorbance at 600 nm. The process was repeated with high concentrations of Cd²⁺ until the growth of the isolate was inhibited. The minimum Cd²⁺ concentration at which bacterial isolate did not show growth was considered as its MIC.

Determination of optimum growth conditions

Optimum growth conditions of bacterial isolate were determined with respect to temperature and pH. To determine optimum temperature, isolate was grown in LB broth at different incubating temperatures viz., 20, 25, 30, 37 and 42 °C. After 24 h incubation, their absorbance was measured at 600 nm using spectrophotometer. To determine optimum pH, 250 mL flasks having 100 mL LB broth were prepared in six sets, each set containing three flasks, their pH were adjusted at 5, 6, 7, 8, 9 and 10 and autoclaved. These flasks were inoculated with 1 % freshly prepared culture of the isolate. After 24 h incubation at optimum temperature, absorbance was taken at 600 nm.

Effect of cadmium on bacterial growth

Effect of Cd²⁺ on the growth of bacterial strain was determined by growing in the presence (1 mM Cd²⁺) as well in the absence (control) of Cd²⁺. MS broth (100 mL) was taken in a set of three 250 mL flasks, autoclaved, supplemented with 1 mM Cd²⁺, inoculated with 1 % of freshly prepared inoculum and incubated at 37 °C in shaking incubator at 100 rpm. In one flask no Cd²⁺ was added which worked as a positive control. Growth in each culture was determined every 4 h by taking absorbance of an aliquot (1 mL) at 600 nm. Growth curves were plotted between time and absorbance.

Resistance to heavy metal ions

Resistance of bacterial isolate against heavy metal ions (zinc, lead, copper, chromium, arsenic and mercury) was determined by growing it in MS broth supplemented with respective metal ions. Stock solutions of 1 M concentration of heavy metal ions salts (zinc sulfate, lead nitrate, copper sulfate, potassium dichromate, sodium arsenate and mercuric chloride) were used. The concentration of metal ions was increased, 1 mM every time, in a stepwise manner until the growth of the isolate was inhibited. The bacterial growth was determined by taking optical density (OD) at 600 nm after 24 h of incubation at 37 °C.

Physical, biochemical and molecular characterization

Bacterial isolate was identified on the basis of colony morphology and different biochemical tests such as Gram staining, catalase test, oxidase test, citrate utilization, fermentation of carbohydrates, H₂S production, nitrate reduction, indole and urease test (Benson 1994). For molecular characterization, 16S rRNA gene was amplified through polymerase chain reaction (PCR) by using general primers (RS-1; 5′-AAACTCAAATGAATTGACGG-3′, RS-3; 5′-ACGGGCGGTGTGTAC-3′) (Rehman et al. 2007). Amplification of 16S rRNA gene was carried out by 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 2 min followed by final extension at 72 °C for 5 min. The PCR product (approximately 0.5 kb) was purified through Fermentas GeneJet Gel Extraction kit (#K0691) and sequenced from Macrogen, Korea. The sequence obtained was compared with known sequences in NCBI database using NCBI BLAST tool (http://www.ncbi.nlm.nih.gov/BLAST) to identify the bacterial isolate. The accession number was obtained by submitting the sequence in the GenBank.

Cadmium processing ability of bacterial isolate

To ascertain cadmium processing ability, the bacterial isolate was grown in 100 mL LB broth in 250 mL flask on shaking (100 rpm) at optimum temperature and pH. After 24 h of incubation the medium was supplemented with 1 mM Cd²⁺. In one flask Cd²⁺ was added in the same concentration (1 mM) but not inoculated with the organism, which worked as a control. The experiment was carried out in triplicate. Then 5 mL samples were taken out from each flask after 2, 4, 6 and 8 days. Bacterial cells were harvested through centrifugation at 3000 rpm for 5 min. The pellets [acid digested, 0.2 N HNO₃ (1:1)] and supernatants were used for the estimation of Cd²⁺ by atomic absorption spectrophotometer at a wavelength of 228.8 nm. Each pellet was weighed and divided into two portions, one portion was washed thrice with 0.5 M EDTA to collect Cd²⁺ adsorbed on the cell surface and second portion was acid digested to release absorbed Cd²⁺. Total uptake values were calculated by subtracting the metal concentration in treated flasks from the metal concentration in control flask. Standard curve was prepared to calculate amount of metal ions in pellets and supernatants. Percentage increase and decrease in the amount of metal ions were also calculated.

Removal of Cd²⁺ from industrial effluent

A lab experiment was set up to determine the efficacy of bacterial isolate to remove Cd²⁺ from industrial wastewater. Our set up (experiment design) consisted of three plastic containers; one container carrying 10 L distilled water along with 1.5 L bacterial culture grown to log phase, second container carrying 10 L industrial effluent along with 1.5 L bacterial culture while third container was carrying only 10 L industrial effluent. Experiment was carried out at room temperature (25 ± 2 °C) and 1 mM cadmium stress was maintained in each container. Samples (10 mL) were taken from each container after 2, 4, 6 and 8 days of incubation and cells were harvested by centrifugation at 3000 rpm for 5 min. Cell pellets were weighed and separated whereas supernatants were used to estimate the amount of Cd²⁺ in the wastewater and further removed by bacterial isolate.

Cd²⁺ removal by heat-inactivated bacterial cells

Biosorption of Cd²⁺ by heat inactivated bacterial cells was studied by growing the isolate in 250 mL Enlermayer flask containing 100 mL LB broth medium at 37 °C in shaking incubator (150 rpm). After 24 h of incubation bacterial cells were inactivated by autoclaving at 121 °C and 15 LB pressure for 15 min. Dead bacterial cells were harvested by centrifugation at 10,000 rpm for 10 min. Cells were washed with autoclaved deionized distilled water thrice and dried at 80 °C in oven. Dead bacterial biomass (0.5 g) was added in flask containing 100 mL of 1 mM Cd²⁺ solution and incubated at 37 °C on shaking (150 rpm). After regular time intervals samples were collected and centrifuged at 3000 rpm for 5 min. Pellets and supernatants were used to estimate the quantity of Cd²⁺ by atomic absorption spectrophotometer. The experiment was performed in triplicate.

Effect of temperature, pH, initial Cd²⁺ concentration, biomass dosage and co-metal ions on Cd²⁺ processing ability of bacterial biomass

To ascertain the effect of temperature and pH on the Cd²⁺ processing ability of the bacterial isolate, it was grown in LB broth to log phase in flasks on optimum conditions, then 1 mM Cd²⁺ stress was given in each flask and incubated at different incubating temperatures (25, 30, 37 and 42 °C) and pH (5, 6, 7, 8 and 9). The effect of initial Cd²⁺ concentration on the Cd²⁺ processing ability was checked by growing the isolate in different initial concentration of Cd²⁺ ranging from 0.5 to 5 mM. Presence of competing metal ions is known to have effect on the biosorption ability. Their effect was studied by growing the isolate in the presence of 1 mM Cd²⁺ along with 1 mM of one of the competing ions (Cr⁶⁺, Pb²⁺ and Cu²⁺). A mixture of four metal ions (Cd²⁺, Cr⁶⁺, Pb²⁺ and Cu²⁺) was also used to observe co-metal ions effect. To study the effect of biomass concentration, bacterial isolate was grown in the presence of different initial biomass concentration ranging from 5 to 50 g/L. A blank solution containing only Cd²⁺ was also run as a control in each experiment. After 6 days of incubation, samples (5 mL) were collected and centrifuged at 3000 rpm for 5 min. Pellets were separated, dried and weighed whereas supernatants were used to estimate Cd²⁺ by atomic absorption spectrophotometer. All the experiments were performed in triplicates.

Biosorption isotherms, thermodynamics and kinetics

The amount of Cd²⁺ removed by the living and dead biomass of the bacterial isolate at time t was calculated by the following equation;

$$q = \frac{{V(C_{i} - C_{f} )}}{m}$$

where q is Cd²⁺ uptake (mM of Cd²⁺ removed/g of biomass) at time t, V is sample volume taken (mL), C _i is the Cd²⁺ concentration initially added in the medium (mM), C _f is the final Cd²⁺ concentration (mM) remained in the medium after time t and m is the weight of bacterial biomass (g).

The adsorption capacity at equilibrium (q_e) was calculated by using following equation.

$$q_{e} = \frac{V(Ci - Ce)}{m}$$

where Ce is the Cd²⁺ concentration at equilibrium.

The Langmuir and Freundlich isotherm equations were applied to Cd²⁺ uptake data by the bacterial isolate.

Langmuir equation is represented as

$$q_{e} = \frac{{q_{max} bC_{e} }}{{1 + bC_{e} }}$$

The linearized form of the equation is

$$\frac{{C_{e} }}{{q_{e} }} = \frac{{C_{e} }}{{q_{max} }} + \frac{1}{{bq_{max} }}$$

Values of q_max and b were calculated by drawing a plot of C_e/q_e vs C_e.

where q_e refers to the amount of metal ions sorbed at equilibrium (mM/g), C_e is the metal ions concentration in solution at equilibrium (mM), q_max is the maximum sorption capacity of biosorbent (mM/g) and b is Langmuir constant.

A dimensionless constant separation factor (R_L) was calculated to express the Langmuir isotherm feasibility by using following equation;

$$R_{L} = \frac{1}{{1 + bC_{i} }}$$

where b is Langmuir constant and C_i is the initial concentration of metal ion (Cd²⁺).

Freundlich isotherm model reveals the sorption of metal ions on heterogenous surfaces forming multilayers. Freundlich equation is written as

$$q_{e} = k_{f} C_{e}^{{\frac{1}{n}}}$$

Its linearized form is written as

$$\ln \,\,q_{e} = \ln K_{f} + \frac{1}{n} {\ln}C_{e}$$

The values of K_f and 1/n can be calculated by drawing plot of ln q_e vs ln C_e.

Standard thermodynamic parameters i.e. Gibbs free energy change (∆G°), enthalpy change (∆H°) and entropy change (∆S°) were calculated by using following equations;

$$\Delta {\text{G}}^\circ = - {\text{RTln}}K_{D}$$

$${\ln}\, K_{D} = \frac{{(\Delta {\text{S}}^\circ ) }}{R} + \frac{{(\Delta {\text{H}}^\circ ) }}{RT}$$

where R is universal gas constant (8.314 J/mol K), T refers to temperature (K) and K_D (q_e/C_e) is distribution coefficient. The values of ∆S° and ∆H° were calculated by drawing plot of ln K_D vs 1/T.

The dynamics of Cd²⁺ adsorption by bacterial cells were evaluated by applying pseudo first order and pseudo second order models. The linearized form of pseudo first order model is

$$\ln \left( {q_{e} - q_{t} } \right) = {\ln}q_{e} - k_{1} t$$

where q_e and q_t are the adsorption capacity of Cd²⁺ at equilibrium and given time, t is temperature and k₁ is rate constant. The value of k₁ was calculated by drawing plot of ln(q_e−q_t) vs t.

The linearized form of pseudo second order model is;

$$\frac{t}{{q_{t} }} = \frac{t}{{k_{2} (q_{e}^{2} )}} + \frac{1}{{q_{e} }}$$

k₂ (second order rate constant) was calculated by plot of t/q_t vs t.

Estimation of glutathione (GSH) and other non-protein thiol contents

Reduced and oxidized GSH and total thiol contents were estimated according to Shamim and Rehman (2015).

Protein extraction and SDS–PAGE

Bacterial total protein profiling was done according to Laemmli (1970).

Amplification of smtAB

An internal fragment of 480 bp of known smtAB gene was amplified from both plasmid and genomic DNA through PCR using known primers (smtAB-F; 5′-GAT CGACGTTGCAGA GACAG-3′, smtAB-R; 5′-GATCGAGGGCGTTTTGATAA-3′) reported by Naz et al. (2005). The reaction mixture (20 µL) contained 0.2 mM dNTPs, 20 pM each primer, 10 ng DNA, 0.25 U taq polymerase, 50 mM KCl buffer and 1.5 mM MgCl₂. Amplification was carried out by 35 cycles of denaturation at 94 °C for 1 min, annealing at 56 °C for 1 min and extension at 72 °C for 2 min followed by final extension at 72 °C for 5 min.

Fourier transform infrared spectroscopy (FTIR)

For FTIR analysis bacterial samples were prepared according to Deokar et al. (2013). Briefly, the bacterium was grown in the presence and absence of 1 mM Cd²⁺ for 24 h at 37 °C. The bacterial cells were harvested by centrifugation at 3000 rpm for 10 min and washed several times with saline solution (0.9 % NaCl, pH 6.5). Then bacterial pellets were freeze-dried overnight and their infrared spectra were recorded on a FTIR spectrometer (Bruker, alpha) in the region 4000–500/cm.

Scanning electron microscopy (SEM)

To prepare samples for scanning electron microscopy a drop of bacterial suspension (both untreated and treated with 1 mM Cd²⁺) was mounted on aluminum stub and air dried. Bacterial cells were fixed with 2.5 % glutaraldehyde in phosphate buffer saline (pH 7.2), incubated at room temperature for 30 min and washed thrice with Na-phosphate buffer. Samples were dehydrated with graded acetone series (30, 50, 70, 80, 90 and 100 %) for 10 min for each step. For better contrast, samples were coated with gold film by sputter coater (Denton, Desk V HP) operating at 40 mA for 30 s under vacuum and analyzed by scanning electron microscope (Nova NanoSEM 450) equipped with Oxford energy dispersive X-ray (EDX) microanalysis system.

Statistical analysis

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.

The sequences of the 16S rRNA gene was submitted to Genbank under the accession number of KJ880038.

Results

Physiochemical characteristics of wastewater

Some physicochemical parameters of wastewater were measured at the time of sampling. Temperature of different samples ranged between 25 and 40 °C, pH was between 6.2 and 9.0 and Cd²⁺ ranged between 0.0042 ± 0.01 and 0.0139 ± 0.01 mM.

Screening of Cd²⁺ resistant bacteria

The wastewater samples were spread on LB agar plates supplemented with 1 mM Cd²⁺. The Cd²⁺ concentration was increased gradually and a bacterium which showed highest resistance against Cd²⁺ (13.3 mM) was selected for further studies and dubbed as 43C.

Identification of bacterial isolate

Bacterial isolate was identified on the basis of morphological, biochemical characteristics and 16S rRNA ribotyping. Morphological and biochemical characteristics of the isolate are given in Table 1. The partial sequence of 16S rRNA gene showed 99 % homology with 16S rRNA sequence of Salmonella enterica (Accession number N890524.1) already submitted to NCBI database. Interestingly its identity with S. enterica subsp. enterica serovar typhimurium (ATCC 13311) is nearly 93 %, therefore, it can be regarded as non-pathogenic. The sequence of the 16S rRNA gene was submitted to Genbank under the accession number of KJ880038. The dendrogram on the basis of homology was also created (Additional file 1: Figure S1). The organism has been deposited at First Fungal Culture Bank of Pakistan with FCBP no. 590.

Table 1 Morphological and biochemical characteristics of S. enterica 43C

Optimum growth conditions

The most suitable temperature for S. enterica 43C was found to be 37 °C and it revealed maximum growth at pH 7. The effect of Cd²⁺ on the growth of S. enterica 43C was monitored by growing it in the presence (1 mM Cd²⁺) and absence of Cd²⁺. The presence of Cd²⁺ significantly decreased the growth of the bacterium (Fig. 1).

Fig. 1

Growth pattern of S. enterica 43C in the absence (control) and presence (treated) of 1 mM Cd²⁺

Cross metal resistance

The bacterium resisted Cd²⁺ up to 13.3 mM. It was also capable to resist other heavy metal ions, viz., Zn²⁺ (5.3 mM), Pb²⁺ (16 mM), Cu²⁺ (2.6 mM), Cr⁶⁺ (5.3 mM), As³⁺ (8.8 mM) and Hg²⁺ (0.53 mM). The order of resistance regarding metal ions concentration was Pb²⁺>Cd²⁺>As³⁺>Zn²⁺>Cr⁶⁺>Cu²⁺>Hg²⁺.

Biosorption of Cd²⁺

Biosorption capability of S. enterica 43C was assessed by growing it in culture medium supplemented with 1 mM Cd²⁺ (Fig. 2a). Cd²⁺ biosorption efficiency (q) of S. enterica 43C was as 7, 11.5, 18 and 22 mM/g after 2, 4, 6 and 8 days, respectively. The amount of Cd²⁺ found accumulated within bacterial cells after 2, 4, 6 and 8 days was 4.5, 9, 14 and 15.7 mM/g, respectively. While 2.18, 2.2, 3.6 and 6 mM/g of Cd²⁺ was found to be adsorbed on the bacterial surface after 2, 4, 6 and 8 days, respectively (Fig. 2a).

Fig. 2

a Biosorption capability of S. enterica 43C growing in culture medium supplemented with 1 mM Cd²⁺ and b large scale biosorption of Cd²⁺ by S. enterica 43C

At large scale S. enterica 43C was able to remove 41, 43, 45.8 and 49 mM/g 83.4 % Cd²⁺ from industrial effluent and 43.8, 45, 50 and 51.5 mM/g from distilled water after 2, 4, 6 and 8 days, respectively. Cd²⁺ concentration was maintained at 1 mM in both water and industrial effluent. The microorganisms originally present in industrial effluent could remove 4, 9, 12 and 16 mM/g after 2, 4, 6 and 8 days, respectively from original industrial effluent in which no Cd²⁺ was added but originally contained 0.18 mM Cd²⁺ (Fig. 2b).

Cd²⁺ removal by heat-inactivated bacterial cells

Bacterial cells were inactivated by autoclaving to use as biosorbant material for Cd²⁺ bioremediation. Dead cells of S. enterica 43C were observed removing 8.8, 15, 21 and 23.8 mM/g Cd²⁺ from aqueous medium, out of which 4.27, 7.2, 10.84 and 11.06 mM/g Cd²⁺ was detected adsorbed on outer surface whereas 4.91, 7.92, 10.6 and 12.79 mM/g Cd²⁺ found accumulated within dead yeast cells after 2, 4, 6 and 8 days, respectively (Additional file 1: Figure S2).

Biosorption isotherms, thermodynamics and kinetics

Straight lines were obtained when we evaluated our data by applying Langmuir and Freundlich isotherm equations (Figs. 3, 4a–e) which suggested that our data is correlated with both the isotherm models. The values of Langmuir constants (q_max and b) and Freundlich constant (K_f and n) are given in Additional file 1: Table S1. The regression coefficient (R²) values for Langmuir isotherm ranged 0.933–0.999 whereas these values were found in the range of 0.79–0.995 for Freundlich isotherm (Additional file 1: Table S1) which is an indication that our data is more correlated with Langmuir isotherm model than Freundlich isotherm model. The values of R_L (dimensionless constant separation factor which expresses the Langmuir isotherm feasibility) were calculated to determine the suitability of Langmuir model and found in the range of 0.11–0.66 (Additional file 1: Table S1).

Fig. 3

Langmuir isotherm for a living cells b dead cells c effluent plus S. enterica 43C plus Cd²⁺ (1 mM) d distilled water plus S. enterica 43C plus Cd²⁺ (1 mM) e only effluent

Fig. 4

Freundlich isotherm for a living cells b dead cells c effluent plus S. enterica 43C plus Cd²⁺ (1 mM) d distilled water plus S. enterica 43C plus Cd²⁺ (1 mM) e only effluent

The standard Gibbs free energy change (∆G°) was calculated as −4.96, −5.4, −6.9, −7.5, −7.7 and −8.3 kJ/mol at 20, 25, 30, 33, 37 and 42 °C, respectively. Whereas, standard enthalpy (∆H°) and entropy (∆S°) changes were 48.9 kJ/mol and 216.4 J/mol K, respectively (Additional file 1: Table S2) from plot of ln K_D vs 1/T (Additional file 1: Figure S3).

Adsorption data was evaluated by applying pseudo first and pseudo second order kinetic models. The values of constants (K ₁ and K ₂) and coefficient determinant (R²) are given in Additional file 1: Table S3. Pseudo first order R² values are higher for lab scale experiments (0.9) than pseudo second order R² values. Figure 5a, b showing plots of q_t/t vs time(t). Furthermore, experimental q values (q _exp ) 21 and 9 mM/g were found in good agreement with calculated q values (q _cal ) 31 and 10 mM/g for living and dead cells, respectively by using pseudo first order equation. Whereas, at large scale experiments pseudo second order R² values were higher (0.99) than pseudo first order R² values (0.97). Moreover, q_exp values and q_cal values (calculated by using pseudo second order equation) were exactly similar for industrial effluent and distilled water. In both of them bacterial biomass and Cd²⁺ (1 mM) were added. In general, these results suggest that lab scale experiments follow pseudo first order whereas large scale experiments follow pseudo second order model. But the original effluent in which no bacterial biomass and Cd²⁺ were added was found following pseudo first order kinetic model just like small scale experiments.

Fig. 5

Pseudo first order (a) and pseudo second order (b) reaction for Cd²⁺ adsorption onto S. enterica 43C

Effect of temperature, pH, initial Cd²⁺ concentration, biomass dosage and co-metal ions on Cd²⁺ processing ability of bacterial isolate

Several parameters have been proved to have influence on the Cd²⁺ processing ability of S. enterica 43C. It showed optimum biosorption capability at moderate temperature (30 to 37 °C) where 15 mM/g removal of Cd²⁺ was observed. This removal was reduced to 0.89 mM/g when temperature was increased up to 42 °C. Similarly at low temperature (25 °C), it could remove only 13 mM/g Cd²⁺ from the medium (Fig. 6a). The biosorption ability of S. enterica 43C was also found changing with varying pH. Neutral (pH 7) or nearly neutral pH (pH 6) was found most suitable for S. enterica 43C and it removed 18 and 17.3 mM/g Cd²⁺, respectively. Basic (pH 8) and strongly basic pH (pH 9) decreased Cd²⁺ removal to 2.9 and 0.53 mM/g, respectively. At slightly acidic pH (pH 5), it exhibited 8.3 mM/g removal (Fig. 6b). The sorption of Cd²⁺ by S. enterica 43C was studied by varying initial Cd²⁺ concentration ranging from 0.5 to 5 mM. A direct relationship was observed between initial Cd²⁺ concentration and Cd²⁺ processing ability of S. enterica 43C 12.3, 25.8, 45, 66 and 71 mM/g sorption of Cd²⁺ by S. enterica 43C was recorded at 0.5, 1, 1.5, 3 and 5 mM of initial Cd²⁺ concentration, respectively (Fig. 6c). Figure 6d is indicating the effect of bacterial biomass dosage on the potential of S. enterica 43C to biosorp Cd²⁺. An increase in Cd²⁺ removal was evaluated with the increase in biomass concentration but up to a certain limit. Salmonella enterica 43C showed 46, 58, 66, 68 %, removal at 5, 10, 15 and 30 mg/mL of initial biomass concentrations, respectively. But biosorption efficiency (q) was decreased from 15 to 2 mM/g. Further increase (40 and 50 mg/mL) in biomass concentration did not cause any significant change in Cd²⁺ removal efficiency of S. enterica 43C. When S. enterica 43C was allowed to grow in the presence of Cd²⁺ in combinations with different heavy metal ions as competing ions, a change in bioremediation potential of S. enterica 43C was noticed. S. enterica 43C could remove 22.8, 26.6 and 12.2 mM/g Cd²⁺ when Cr⁶⁺, Pb²⁺ and Cu²⁺ were used as competing ions, respectively. This removal capacity was decreased to 11.7 mM/g when all these heavy metal ions i.e. Cd²⁺, Cr⁶⁺, Pb²⁺ and Cu²⁺ were added simultaneously. It is kept in mind that S. enterica 43C showed 21.8 mM/g removal of Cd²⁺ in the absence of any competing ion (Fig. 6e).

Fig. 6

Effect of temperature (a), pH (b), initial Cd²⁺ concentration (c), biomass dosage (d) and co-metal ions (e) on Cd²⁺ processing ability of S. enterica 43C

Measurement of glutathione and non-protein thiols

The levels of GSH and GSSG were altered in the presence of Cd²⁺ (1 mM). There was 53.96 and 154 % increase in GSH level and GSH/GSSG ratio, respectively. Whereas 87 % increase in non-protein thiols level was recorded in bacterial cells in the presence of Cd²⁺ (Additional file 1: Table S4).

SDS–PAGE analysis

Total protein profiles of S. enterica 43C in the absence and presence of Cd²⁺ were compared by polyacrylamide gel electrophoresis. This comparison clearly indicated the induction of a protein due to Cd²⁺ stress. A band of about 40 kDa was observed intensifying with the increasing concentration of Cd²⁺ (Fig. 7a).

Fig. 7

a Total protein profile of S. enterica 43C in the absence and presence of 1, 5 and 10 mM Cd²⁺. M protein marker; C control (without Cd²⁺) and b amplification of smtAB gene through PCR. M represents DNA ladder

Amplification of smtAB

PCR was carried out to amplify internal fragment of smtAB gene from both plasmid and genomic DNA of S. enterica 43C by using known primers. The primer set showed amplification of 480 bp product. But no amplification was detected when plasmid DNA was used as a template (Fig. 7b).

SEM, EDX and FTIR

The size of S. enterica 43C was observed to increase many folds, when treated with Cd²⁺. This indicated the accumulation of Cd²⁺ and it was confirmed from the difference in size, when SEM images of treated S. enterica 43C were compared with untreated bacterium (Fig. 8a). This biosorption of Cd²⁺ was verified by EDX (point and ID scan) analysis, while doing their SEM (Additional file 1: Figure S4). The FTIR analysis confirmed the presence of carboxyl, amino and phosphate moieties in bacterium and this analysis confirmed our assumption regarding binding of Cd²⁺ with bacterium. It is clear that the peaks attributed to amide linkage, appearing at 1635 and 1538/cm are shifted to 1647 and 1552/cm respectively, in the presence of Cd²⁺ (Fig. 8b).

Fig. 8

Images of scanning electron microscopy of S. enterica 43C in the absence (a) and presence (b) of Cd²⁺ (1 mM) and c FTIR of S. enterica 43C in the absence and presence of Cd²⁺

Discussion

In the present investigation we have demonstrated the potential use of S. enterica 43C to remove Cd²⁺ from aqueous medium at lab scale. S. enterica 43C could remove maximally 22 mM/g Cd²⁺ from the aqueous medium and 2.18, 2.2, 3.6 and 6 mM/g Cd²⁺ were found to be adsorbed on bacterial cell surface after 2, 4, 6 and 8 days, respectively. Whereas Pseudomonas stutzeri and Enterobacter sp. have been reported to remove 43.5 and 46.2 mg/g Cd²⁺ by adsorption (Lu et al. 2006). At large scale, S. enterica 43C maximally removed 24 mM/g (83 %) and 25 mM/g (77 %) after 8 days from effluent and water supplemented with 1 mM Cd²⁺. Effluent naturally contains some amount of heavy metal ions as well as number of microorganisms including bacteria, fungi, and protozoans. Experiment was also performed to check the efficacy of these naturally existing microbes to remove Cd²⁺. Results showed that these organisms could maximally remove only 27 % Cd²⁺. But our bacterium, S. enterica 43C removed 83 % Cd²⁺ suggesting its preferential use over other microbes for Cd²⁺ bioremediation.

There are several adsorption models but Langmuir and Freundlich models are commonly used (Bayramoğlu et al. 2006; Ansari and Malik 2007; Kang et al. 2007) which are based on monolayer and multilayer adsorption, respectively. In Langmuir model we make some assumptions that biosorbent surface is uniform and metal ions do not have mutual interaction and adsorb on the biosorbent surface in single layer. Additional file 1: Table S1 is showing the values of different parameters for Langmuir and Freundlich isotherm models, higher values of regression coefficient for Langmuir isotherm model indicated that our data was more consistent with Langmuir model than Freundlich model. Langmuir model is considered unfavorable for a given data if R_L >1, while it is favorable when 0 < R_L < 1 (Hall et al. 1966). In our experiments, R_L values were found less than 1 and greater than 0 (0.11–0.66), indicating the favorability of Langmuir isotherm model. High biosorption capacity of S. enterica 43C (either living or dead form) for Cd²⁺ as compare to other biosorbent reported in literature (Say et al. 2001; Martins et al. 2004; Sari et al. 2008; Chatterjee et al. 2010) to bioremediate Cd²⁺ makes it highly potential and favorable resource to remove environmental Cd²⁺.

SEM revealed the increase in the size of bacterial cells in the presence of Cd²⁺ which clearly indicated the biosorption of Cd²⁺. It was further confirmed by EDX and FTIR. The characteristic peaks of carboxyl, amino and phosphate groups appearing in FTIR analysis confirmed the presence of these moieties in bacterium and the shift in these peaks was used to address the binding of Cd²⁺ with bacterium. In short, the peaks at 1635 and 1538/cm recognized the presence of amide linkage, and the shift in these peaks to 1647 and 1552/cm respectively was due to chemical interaction of Cd²⁺ with amide group of bacterium. It is presumed that cadmium mainly adsorbed on bacterium by interacting with nitrogen atoms of amide groups. The peaks appearing in range of 1000–1320/cm represented the presence of carbon and phosphorous containing oxygen atoms, and suppression in intensity of these peaks indicated their interaction with Cd²⁺ (Parikh and Chorover 2006). The peak at 1047 and 1157/cm recognized the presence of carbonyl group and the shift in these peaks and transmittance level was due to chemical interaction of Cd²⁺ with carbonyl moieties. Addition to this, the percentage transmittance of peaks in FTIR spectra of bacterium treated with cadmium, was considerably less significant than those of control bacterium. This showed that stretching of bonds, occurred to lesser degree (due to presence of Cd²⁺), resulted to reduce their percentage transmittance as reported by Chakravarty and Banerjee (2012a).

Biosorption is really a complex process and dependent on lot of factors such as functional groups, temperature, pH, metal ions concentration, weight of biomass and presence of competing metal ions (Lesmana et al. 2009). Among all the factors, pH and temperature have been reported as the most critical parameters in the biosorption of Cd²⁺ (Chakravarty and Banerjee 2012). Present investigation clearly revealed the effect of acidic, basic and neutral pH on the Cd²⁺ processing ability of the bacterium. Strong acidic and strong basic pH significantly decreased the biosorption of S. enterica 43C. Maximum biosorption of Cd²⁺ (57.14 %) has been observed at neutral pH. These results are parallel to the findings of Chakravarty and Banerjee (2012). Change in temperature also greatly influences the adsorption and overall biosorption of Cd²⁺. Increase in temperature not only enhances the kinetic energy of reacting molecules but also vibrational energy of constituent elements of bacterial envelop. At high temperature (above 37 °C) the vibrational energy becomes too violent resulting in the breakage of bonds between Cd²⁺ and functional groups on bacterial surface thus decreasing adsorption and intracellular accumulation of Cd²⁺ (Özdemir et al. 2009). Whereas at low temperature chances for reacting molecules to collide with each others are low, thus Cd²⁺ processing ability of the bacterium is declined. S. enterica 43C showed maximum Cd²⁺ removal (15 mM/g) at 37 °C but when the temperature was increased up to 42 °C the Cd²⁺ removal capability of S. enterica 43C was significantly reduced.

Initial biomass dosage showed significant effect on Cd²⁺ biosorption ability of S. enterica 43C. Cd²⁺ biosorption found increasing with the increasing biomass concentration. Highest Cd²⁺ biosorption (68 %) was observed when biomass dosage was increased up to 30 g/L. On contrary, biosorption efficiency (q) decreased up to 3.7 mM/g. Further increase in biomass concentration did not cause any significant effect on percentage removal and biosorption efficiency (q) of S. enterica 43C. Greater concentration of biomass provides greater surface area and binding sites for Cd²⁺ which increases the binding of Cd²⁺ with the biomass and hence increasing the percentage removal of Cd²⁺. However, increase in biomass concentration decreases the ratio of biosorbent to metal ions (as metal ion concentration is fixed i.e. 1 mM) thus a decline in biosorption efficiency (q) was observed (Al-Garni 2007).

Industrial wastewater contains multiple metal ions which may influence the removal of Cd²⁺. Present study clearly depicted the effect of other heavy metal ions on Cd²⁺ biosorption in binary and quaternary systems. In multimetal system the binding of metal ions to biomaterial depends on ionic properties, among them electronegativity and ionic radius are the most critical (Naja et al. 2010). Ion with larger radius has greater ability to adsorb and high electronegativity also helps the ion to bind strongly to the surface functional groups (Sulaymon et al. 2011). In the present study, Cd²⁺ biosorption was significantly reduced from 22 to 15 mM/g in the presence of Cu²⁺. Whereas Pb²⁺ and Cr⁶⁺ showed very slight decrease in Cd²⁺ biosorption in binary system. Electronegativity of Cu²⁺ is 1.9 while Cd²⁺ has 1.7 (Additional file 1: Table S2). This high electronegativity of Cu²⁺ makes it more preferred metal ion over Cd²⁺ to get adsorbed on the surface. Zhou et al. (2009) also reported this preference of Cu²⁺ over Cd²⁺.

Salmonella enterica 43C showed tolerance against Cd²⁺ (13.3 mM) as well as against Zn²⁺ (5.3 mM), Pb²⁺ (16 mM), Cu²⁺ (2.6 mM), Cr⁶⁺ (5.3 mM), As³⁺ (8.8 mM) and Hg²⁺(0.53 mM). The order of resistance regarding metal ions concentration was Pb²⁺>Cd²⁺>As³⁺>Zn²⁺>Cr⁶⁺>Cu²⁺>Hg²⁺. Salmonella enterica 43C was found to be more tolerant against Pb²⁺ as compared to the Cd²⁺. This is due to the fact that Pb²⁺ is more ubiquitous heavy metal. On the other hand Cd²⁺ is highly toxic and its no physiological role has been reported so far (Martelli et al. 2006; Vinodini et al. 2015). Salmonella enterica 43C is notorious for its pathogenic behavior. Anyhow, pathogenicity cannot be considered as a limiting factor to use microorganisms for bioremediation purposes. Number of pathogenic bacteria including Enterobacter sp., P. aeruginosa, Shewanella and Alcaligenes faecalis have been used for bioremediation purposes (Lu et al. 2006; Srivastava and Majumder 2008; Kang et al. 2015).

Bacteria have developed different resistance mechanisms to cope with the negative effects of these metal ions (Sinha and Mukherjee 2008; Hassan et al. 2009; Khan et al. 2015) including alteration in the levels of GSH and GSSG and induction of metal binding proteins i.e. metallothioneins (Fig. 9). GSH, as a strong reducing agent, reduces the H₂O₂ to H₂O and gets itself oxidized to GSSG (Penninckx 2002; Galano and Alvarez-Idaboy 2011). GSH/GSSG is the most important parameter to study oxidative stress. Present study clearly revealed the alteration of GSH level and GSH/GSSG. An increase of 53.96 and 154 % in GSH level and GSH/GSSG ratio was determined in the presence of Cd²⁺, respectively. An increase of 87 % in non-protein thiols level was also determined.

Fig. 9

Proposed Cd²⁺ biosorption and resistance mechanism in gram negative bacterium, S. enterica 43C. (1) Peptidoglycan is the primary site on bacterial surface that binds Cd²⁺ in monolayer. (2) Cd²⁺ enter the cells via metal ions transport channel proteins and are known to cause oxidative stress which is combated by GSH dependent antioxidant system. (3) Metallothioneins, induced by Cd²⁺ stress, are primarily involved in Cd²⁺ sequestration through their thiols groups thus mitigating Cd²⁺ interference with cellular metabolism

MTs are low molecular weight (6–7 kDa), thiol-containing, cysteine rich (20–30 %), metal binding proteins, induced by cadmium and other heavy metal ions and are involved in the sequestration of metal ions (Klaassen et al. 2009; Chaturvedi et al. 2012). MTs are widely associated with eukaryotes (Palmiter 1998) and among prokaryotes, these have been reported in P. putida and Synechococcus spp. (Higham et al. 1984; Huckle et al. 1993). The amplification of smtAB gene from the chromosomal DNA of S. enterica 43C revealed the presence of genetic determinants of bacterial metallothioneins. These metallothioneins encoding genes have been found involve in conferring resistance against heavy metal ions including Cd²⁺ (Turner et al. 1995).

In the present study, S. enterica 43C showed high resistance against heavy metal ions in order of Pb²⁺>Cd²⁺>As³⁺>Zn²⁺>Cr⁶⁺>Cu²⁺>Hg²⁺. The bacterium could remove nearly 57 % Cd²⁺ from the medium over a period of 8 days. Kinetic and thermodynamic studies depicted the Cd²⁺ biosorption as spontaneous, feasible and endothermic in nature. The bacterium followed pseudo first order kinetics, making it a good biosorbent for heavy metal ions. The Cd²⁺ processing was significantly influenced by temperature, pH, initial Cd²⁺ concentration, biomass dosage and co-metal ions. FTIR analysis revealed the active participation of amide and carbonyl moieties in Cd²⁺ adsorption which was confirmed by EDX analysis. Electron micrographs beckoned further surface adsorption and increased bacterial size due to intracellular Cd²⁺ accumulation. An increase in GSH and other non-protein thiols levels played a significant role in thriving oxidative stress generated by metal cations. MTs presence clearly indicated the role of such proteins in bacterial metal resistance mechanism. S. enterica 43C, a non-pathogenic bacterium having strong cytoplasmic redox balance system to overcome metal induced cytotoxicity. Moreover, its pseudo first order kinetic trends make it an effective biosorbent to remove toxic heavy metals from the aqueous environment. In future, after exploring its molecular biology it can become an attractive environmental tool for green chemistry.