1. Introduction
Due to the increasing concern about the public health and environmental problems caused by Pb(II) contamination, developing highly efficient and stable treatment methods is necessary. Although Pb(II) is one of the most widely used nonferrous metals that is necessary for manufacturing daily necessities, it has profound negative effects on living organisms [
1]. Many toxicological studies have reported abnormal symptoms and diseases caused by the accumulation of Pb(II) in human tissues. For examples, hematopoiesis (anemia) and malfunctions of the nervous, digestive, and cardiovascular systems have been triggered by exposure to Pb(II), even at low concentrations. Pb(II) can enter the human body through ingestion, inhalation, and adsorption, which can cause other severe effects, such as permanent damage to cognition and the reproductive system, and neurodegenerative diseases [
2,
3]. Thus, the Agency for Toxic Substance and Disease Registry (ATSDR) and the World Health Organization (WHO) have both designated Pb(II) as an extremely hazardous substance, and have withdrawn its provisional value.
To date, much effort has been dedicated to developing an efficient treatment method for removing Pb(II), such as ion exchange, solvent extraction, adsorption, membrane filtration, precipitation, and reverse osmosis [
4,
5,
6,
7,
8,
9]. However, several hindrances, such as (1) difficulties in adsorbent preparation, (2) the risk of secondary contamination, and (3) the requirement for additional steps, such as resin regeneration or replacement, should be overcome prior to practical application. Therefore, collective approaches that combine chemistry and biology to overcome the drawbacks to the conventional process currently in use must be implemented to develop an efficient Pb(II) treatment method. Of the various potential methods, biosorption, which uses inherent biological features that interact with chemicals, is one of the most promising methods for treating toxic heavy metals as it is ecologically sustainable, inexpensive, and the operation parameters can be easily controlled.
Bacterial biofilms are composed of polysaccharides, extracellular DNA, lipids, and other biopolymers [
10], and therefore play an important role in the cellular adhesion, attachment, and diffusion of a material. In most cases, biofilm-producing microorganisms can quickly and firmly attach to a target surface via electrostatic attraction (such as van der Waals forces) between the negatively charged biofilms [
11] and a positively charged target surface. Most of the heavy metals that are likely to pollute drinking water are positively charged. Thus, the positively charged toxic heavy metals can adhere to the negatively charged biofilms through electrostatic attraction. Therefore, biofilms extracted from various microorganisms in vitro have been examined as potential biosorbents [
12,
13,
14]. However, the adsorption of Pb(II) by pellicle-like biofilm-producing microorganisms has not yet been reported.
As a biosorbent, biofilm-producing microorganisms have a considerable advantage over biofilms extracted in vitro for application in biosorption as they have substantial quantities of anionic functional groups, such as carboxyl-, hydroxyl-, phosphoryl, and amino groups, on their cellular membranes. The metal-binding forces of biofilm-producing microorganisms were found to be 20-times higher, and the interactions with and immobilization of cationic metal ions are more efficient [
15,
16]. A floating biofilm (pellicle) could be more effective for adsorbing metal ions given their higher bio-volume, roughness, and thickness than submerged biofilms [
10,
17].
Together, these observations led us to investigate the potential of pellicle-like biofilm-producing microorganism for the removal Pb(II) by electrostatic attraction. This is the first proof-of-concept study to propose the potential of a pellicle-like biofilm-producing microorganism for the treatment of heavy metals from aqueous media.
2. Materials and Methods
2.1. Soil Sampling and Identification of Methylobacterium hispanicum EM2 Strain
A soil sample was collected from the mine tailings in Cheonan, Republic of Korea (36°54′10.19′′ N, 127°15′17.72′′ E). One gram of the soil sample was suspended in sterile saline (0.85%, w/v NaCl) and serially diluted. The dilutions were spread onto tryptone, glucose, and yeast (TGY) agar plates (0.5% tryptone, 0.1% glucose, and 0.3% yeast extract). The isolated bacterial strains were routinely cultured in the TGY medium at 30 °C, with or without agar.
The full length of the 16S rRNA gene was determined using SeqBuilder software (DNASTAR Inc., Madison, WI, USA) and compared with GenBank entries using the Basic Local Alignment Tool (BLAST), a program developed by the National Center for Biotechnology Information (NCBI,
http://www.ncbi.nlm.nih.gov). Phylogenetic analysis was conducted following previously described methods [
18]. Multiple alignments and phylogenetic trees were reconstructed using the Mega 7.0 program [
19] with the neighbor-joining method [
20] and Kimura two-parameter model [
21] with bootstrap values based on 1000 replications [
22].
The isolates were Gram-stained following previously described methods [
23]. The phenotypic characteristics of the isolates were determined using an API20NE kit (BioMérieux
TM, Marcy-l'Etoile, France) following the manufacturer’s instructions. The growth of the isolates at different temperatures, NaCl concentrations, and pH values were evaluated on TGY agar at 30 °C for three days. A motility test was conducted on a semi-solid medium.
2.2. Effect of Pb(II) on Bacterial Growth
Cobalt(II) chloride hexahydrate (CoCl2·6H2O), zinc sulfate heptahydrate (ZnSO4·7H2O), nickel(II) chloride hexahydrate (NiCl2·6H2O), copper sulfate pentahydrate (CuSO4·5H2O), lead(II) chloride (PbCl2), and cadmium chloride hydrate (CdCl2·H2O) (Sigma Aldrich, St. Louis, MO, USA) were used to determine the minimal inhibitory concentrations (MICs) of heavy metals following the solid agar method. To prepare the samples, a fresh colony was inoculated at 30 °C in 3 mL of TGY broth. After cultivation, the cells were serially diluted with 0.85% saline. Ten microliters of the dilutes were dropped onto TGY agar plates supplemented with 1–10 mM of the metal solutions and incubated at 30 °C until colonies were visible. To investigate the effect of microbial activity on Pb(II) ions, overnight cell cultures were diluted 1:100 in fresh TGY broth supplemented with different concentrations of PbCl2 (0, 50, 100, 250, 500, and 750 mg/L) and then incubated at 30 °C for 72 h with agitation at 200 rpm. Cell growth was monitored by measuring the optical density at 600 nm (OD600) using an Epoch microplate spectrophotometer (Biotek, Winooski, VT, USA).
2.3. Biosorption Profile
The overnight culture was subcultured (1:100, v/v) in 500 mL of the same liquid medium until the stationary phase was reached. The cultured cells were harvested then rinsed twice with deionized water to remove any residual media components. The sample was freeze-dried and stored at −80 °C before use in the biosorption experiments.
The effect of several parameters, such as pH, contact time, biomass dosage, and initial Pb(II) concentration, on the biosorption efficiency of the biomass was evaluated. To determine the optimum pH value, biosorption experiments were conducted in an Erlenmeyer flask containing 10 mL of a Pb(II) solution (initial concentration of 10 mg/L) for 60 min at 30 °C with agitation at 150 rpm. The initial pH of the metal solution was adjusted using 0.1 M HCl or NaOH solutions. The residual Pb(II) in the supernatant was measured by inductively coupled plasma-optical emission spectroscopy (ICP-OES, iCAPTM 7000 series, Thermo Scientific™, Waltham, MA, USA). All biosorption assays were conducted in triplicate, and the mean values were calculated.
The biosorption capacity and biosorption rate of metal ions were determined using Equations (1) and (2), respectively [
24]:
where
qe (mg/g) is the equilibrium metal ion concentration on the biosorbent;
C0 and
Ce (mg/L) are the initial and final concentrations of metal ions in the aqueous solution, respectively; and
X is the biomass concentration (g dry cell/L).
2.4. Study of Biosorption and Kinetic Isotherms
The equilibrium isotherms were determined using the Langmuir and Freundlich isotherm models, which are widely used to describe the adsorption mechanism between an adsorbent and absorbate. The Langmuir adsorption isotherm assumes that the adsorbent has a monolayer and a homogeneous surface. However, the Freundlich adsorption isotherm assumes that the adsorbent has a heterogeneous surface with non-uniform adsorption heat and affinity distributions [
25]. The Langmuir isotherm model is represented by [
26]:
where
Ceq (mg/L) is the concentration of metal ions in the aqueous solution at equilibrium, and
Qmax (mg/g) and
KL (L/mg) are the maximum adsorption capacity and Langmuir adsorption constant related to the affinity of the binding sites, respectively.
The Freundlich model is expressed as [
27]:
where
KF (L/g) and
n are the Freundlich constants correlated with the binding capacity and the affinity of the adsorbent, respectively.
Adsorption kinetics are used to predict the adsorption capacity and reaction rate of the adsorbent [
28]. Both pseudo-first-order and pseudo-second-order equations are used to study the kinetics of metal biosorption, as shown in Equations (5) and (6), respectively:
where
qt is the adsorbed metal ion concentration (mg/g) at time
t (min),
qe is the concentration of adsorbed metal ions (mg/g) at equilibrium, and
K1 and
K2 are the pseudo-first-order (L/min) and pseudo-second-order (g/mg/min) adsorption rate constants, respectively.
2.5. Scanning Electron Microscope Energy Dispersive X-ray Spectroscopy Analysis
The cell morphology and presence of lead on the surfaces were observed using a field emission scanning electron microscope (Inspect F50, FEI, Hillsboro, OR, USA). The elemental composition was analyzed using a scanning electron microscope-energy dispersive X-ray (SEM-EDX) spectrophotometer (EDAX Apollo XL, AMETEK, Mahwah, NJ, USA) with accelerating voltages up to 20 kV. The EDX spectra were recorded in the area scan mode by focusing the electron beam onto a region of the surface of the sample.
2.6. Fourier-Transform Infrared Spectroscopy Analysis
The functional groups that interacted with metal ions on the surfaces of the cells were analyzed using Fourier-transform infrared (FTIR) spectroscopy. The FTIR spectra of the cells before and after treatment with a 10 mg/L Pb(II) solution were recorded using a Nicolet iS10 (ThermoScientific, Waltham, MA, USA) spectrophotometer. The infrared spectra of the samples were captured at a wavenumber range of 500 to 4000 cm−1 and resolution of 1 cm−1.
2.7. Pb(II) Removal from Sewage Water
Sewage water was collected from the sedimentation basin area of the Jungnang sewage treatment plant in Seoul, Republic of Korea. The collected sewage water was filtered through a 1.2 μm Whatman glass microfiber filter (Sigma-Aldrich, St. Louis, MO, USA) and autoclaved at 121 °C for 30 min. The Pb(II) stock solution was added to the sewage water to achieve a final concentration of 10 mg/L. To determine the Pb(II) removal efficiency, 1 g/L of biomass was added to 50 mL of the Pb(II)-contaminated sewage water and incubated at 30 °C for 1 h at 200 rpm.
4. Conclusions
In this study, we investigated the potential of pellicle-like biofilm-producing microorganism for the treatment of Pb(II) using the newly isolated Methylobacterium hispanicum EM2 strain. To achieve this goal, the physiological and biochemical properties of the newly isolated EM2 strain were first investigated by analyzing the 16S rRNA sequence, followed by API and minimum inhibitory tests. Second, the maximum adsorption capacity was evaluated considering various parameters, such as the pH, amount of biomass, initial Pb(II) concentration, and contact time. Equilibrium kinetic and isotherm models were defined to understand the biosorption process. Third, SEM-EDX and FTIR analyses were conducted to identify the mechanisms involved in Pb(II) biosorption. An adsorption rate of 96% and a maximum Pb adsorption capacity of 79.84 mg/g were achieved under optimal conditions, and the removal efficiency remained stable for 24 h. The EM2 strain exhibited remarkable Pb(II) removal capacity for the sewage water sample contaminated by various heavy metals. In conclusion, the EM2 strain has considerable potential as a biosorbent for removing Pb(II), and could be used for the on-site remediation of Pb(II)-contaminated environments.