1. Introduction
The extensive use of chromium (Cr) in many industries (e.g., electroplating, steel production, and leather tanning) [
1] and the disposal of Cr-containing wastes over large areas of land have resulted in the release of chromium-containing effluents into the environment [
2,
3]. Chromium, a metal, is present in the environment in two major stable oxidation states—Cr(VI) and Cr(III) [
1]. Cr(VI) compounds exist mainly as chromates and dichromates [
4]. They are highly soluble and mobile and are considered as acutely toxic because they can readily cross cell membranes via the sulfate anion transport system when the ambient pH exceeds 6 [
3,
5]. Ramirez-Diaz et al. (2008) suggested that Cr(VI) compounds are associated with several diseases, such as allergies, contact dermatitis, and lung cancer [
6]. Hence, the detection of toxic Cr(VI) compounds is of great importance in order to maintain the quality and safety of our environment.
Several chemical analysis methods, such as ion chromatography, atomic absorption spectrometry, inductively coupled plasma mass spectroscopy, and colorimetric methods based on diphenylcarbazide, are currently being used for the detection of chromium in water samples [
7,
8]. These methodologies exhibit a high sensitivity and selectivity but involve expensive equipment, require additional chemical compounds, specialized training, and long measurement times [
2,
4]. Apart from these classical methods, several biosensors have also been developed for Cr(VI) measurement. Examples include amperometric enzyme-based sensors using cytochrome c3 or urease and cell-based sensors using bacteria or V79 cells [
1,
9,
10,
11]. They are attractive alternatives to the classical methods due to their less complex instrumentation and shorter measurement period times [
4]; however, these methods are often limited to measuring low Cr(VI) concentrations (μg/L); thus, their dynamic ranges restrict their use to diluted samples only [
7]. These biosensors need external power sources and/or additional probes (e.g., pH, dissolved oxygen, and electrical conductivity) to measure the changes in the concentration of Cr(VI), which is another unfavorable factor [
12]. Moreover, most of the analytical methods/techniques for Cr(VI) concentration monitoring are conducted offsite and thus do not reflect real-time values.
A microbial fuel cell (MFC) is a self-sustaining device without the need for any power supply [
13]. MFCs have been employed to convert wastewater to electricity by electrogenic bacteria [
14]. Previously, the development of MFC was mainly targeted towards power generation; however, in recent times, the potential of MFCs as biosensors for the measurement of parameters such as biological oxygen demand (BOD), chemical oxygen demand (COD), dissolved oxygen (DO), volatile fatty acids (VFA), and toxins in wastewater, has been demonstrated [
12,
13,
15,
16,
17,
18]. As a biosensor, a MFC possesses several unique characteristics [
19]. First, the power required for the MFC to function is obtained from the wastewater itself without the need for an external supply. Secondly, because MFCs are based on the microorganisms and not enzymes or animal cells, it simplifies the setup of the biosensor and prolongs its lifetime. Third, the voltage output of an MFC is directly dependent on the metabolic activities of the anaerobic electrogenic bacteria, indicating that MFC is considered to be a promising device to monitor the Cr(VI) concentrations in wastewater, if the appropriate bacteria are used.
Many microorganisms capable of reducing Cr(VI) under anaerobic conditions have been reported. These strains include
Pseudomonas dechromaticans,
P. chromatophilia,
Aeromonas dechromatica,
Desulfovibrio desulfuricans,
D. vulgaris,
Geobacter metallireducens,
Shewanella putrefaciens,
S. oneidensis,
Pantoea agglomerans,
Agrobacterium radiobacter,
Thermoanaerobacter ethanolicus,
Pyrobaculum islandicum, and
Exiguobacterium aurantiacum [
20,
21]. These bacteria exhibit potentials for in situ or ex situ measurements Cr(VI) when used in an MFC.
The use of Cr(VI) as an electron acceptor in the cathode of an MFC has been demonstrated in several studies [
22]. In these MFCs, Cr(VI) reduction at the cathode and electricity production were accomplished simultaneously; thus, the MFC is a power generation device [
22]. Xu et al. (2015) developed a flat membrane-based MFC sensor to monitor the toxicity of wastewater containing Cr
6+ but the relationship between the Cr
6+ concentration and voltage output was not fully established [
12]. Liu et al. (2014) used Cr(VI) as an electron acceptor in the anode of an MFC to develop a cube MFC sensor for monitoring Cr
6+ shock (<10 mg/L) [
19]. Similarity, Chung et al. (2016) inoculated
Ochrobactrum anthropi YC152 in the anode of an MFC as an early warning device for excess Cr(VI) concentrations (<5 mg/L) [
7]. These results suggest that the inoculums in the MFCs just tolerated Cr(VI) toxicity but did not remove Cr(VI). Most regulatory agencies prescribe that the maximum allowable level of Cr(VI) in an effluent is 0.5 mg/L [
21]; however, the concentrations of Cr(VI) emitted during the processes should be strictly controlled or monitored for sustainable, clean, and green production. Thus, an MFC-based biosensor should be developed for the in situ measurement of a wide range of Cr(VI) concentrations. Because the wastewater produced during electroplating contains large amounts of chromium as well as electrolytes, isolation of the appropriate bacterial strain is required for application in an MFC. In this study,
Exiguobacterium aestuarii YC211, a facultatively anaerobic, Cr(VI)-reducing, salt-tolerant, and exoelectrogenic bacterium, was isolated from the electroplating wastewater. It was inoculated in the anode of an MFC to evaluate its feasibility as a biosensor for in situ Cr(VI) measurement. Crucial operating parameters were established to optimize the performance of the MFC. The relationship between the voltage output and Cr(VI) concentration was investigated. Cr(VI) concentrations in artificial and actual wastewater were measured using the developed MFC-based biosensor and standard colorimetric methods to illustrate the accuracy of the self-sustaining device.
2. Materials and Methods
2.1. Bacterial Strains, Cultivation, and Identification
Soil and sludge were collected from the vicinity of an electroplating wastewater treatment plant in Sanchong, New Taipei City, Taiwan. The electroplating wastewater treatment plant experienced repeated Cr(VI)-pollution crises in 2016. The collected samples were centrifuged at 9000× g for 30 min and the obtained precipitates were inoculated in a 3 L working volume in a chemostat. Tryptic soy broth (TSB), supplemented with Na2Cr2O7, called TSBCr medium, was continuously fed to the chemostat. The TSBCr medium containing 20–150 mg/L of Cr(VI) was progressively added into the chemostat to acclimate the Cr(VI)-resistant or -reducing bacteria under the anaerobic conditions at a liquid retention time (LRT) of 16 h. After a 45-day acclimation period, several dominant strains were isolated from the chemostat solution containing 150 mg/L Cr(VI) by the spread plate method. Among these dominant strains, YC211 bacterium with the biggest colony size on the agar was selected for subsequent experiments.
To identify the isolated YC211 bacterium, the YC211 cells were lysed and subjected to DNA extraction. 16S rRNA gene amplification and sequencing were performed, as described previously [
23]. The 16S rRNA gene sequence of the YC211 bacterium was compared using BLASTN programs to search for the nucleotide sequences in the NCBI website. The phylogenetic tree was constructed using a bootstrap neighbor-joining program, Clustal X (Ver. 2.0).
2.2. Bacterial Growth and Cr(VI) Removal
To obtain the growth curve of the YC211 bacterium, it was cultured in 300 mL of the TSBCr medium with 60 mg/L of Cr(VI) and incubated at 30 °C at pH 7.0 under aerobic and anaerobic conditions, respectively. Bacterial growth and Cr(VI) removal were monitored during the culture period. Simultaneously, the bacterial growth was measured at a wavelength of 600 nm using a UV-Vis spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) and by the spread plate method. Cr(VI) concentration was determined using a colorimetric method in this study.
2.3. Factors Affecting Cr(VI) Removal by the YC211 Strain
To determine the effect of different culture conditions on Cr(VI) removal by the YC211 bacterium, different kinds and concentrations (original to 1/10,000 dilution) of the broth (LB or TSB), different pH values (4–12), culture temperatures (15–45 °C), and NaCl concentrations (0–20%) were used in anaerobic conditions. In this study, the YC211 bacterium was first grown in a TSBCr medium containing 60 mg/L Cr(VI) at 30 °C for 22 h under anaerobic conditions and then inoculated in fresh broth to conduct subsequent experiments. The culture conditions were at 30 °C, pH 7, 1000X TSBCr medium (60 mg/L Cr(VI)), and 1.6 × 107 cfu/mL of the YC211 bacterium, unless otherwise stated. The Cr(VI) removal efficiency and cell numbers were analyzed after 24 h cultivation periods. All of the experiments were conducted at least in triplicate in order to evaluate the accuracy and reproducibility of the obtained results.
2.4. Construction of the MFC-Based Biosensor and Its Operation
A dual-chambered MFC was constructed, as described in a previous report [
7], with a little modification to work as the Cr(VI) biosensor. The MFC was comprised of an 8 cm × 8 cm × 8 cm acrylic cube. The anode and cathode compartments had working volumes of 170 mL (7 cm × 7 cm × 3.47 cm) each and the 2 compartments were physically separated using a proton exchange membrane (Nafion 117, DuPont Co., Fayetteville, NC, USA) with a surface area of 49 cm
2. Graphite felt (18 cm
2 surface area) was used for the electrodes and an OK line connected the electrodes through a variable resistor. TSBCr medium (500 mL, 60 mg/L Cr(VI)) containing 1.6 × 10
7 cfu/mL of the YC211 bacterium was placed in a glass bottle and continuously recycled in the anode compartment of the MFC with a 500 Ω resistor using a submersible pump for cell enrichment or immobilization under anaerobic conditions at a 10-day LRT. The TSBCr medium and the anode compartment were kept anoxic by purging with nitrogen gas. The catholyte consisted of 100 mM phosphate-buffered saline (PBS) and 100 mM NaCl solution [
13]. When the potential of the inoculated MFC reached a steady state, the biofilm in the anode was considered stable.
To understand the operating characteristics of the MFC biosensor, 1/1000 TSB (water sample I) and three other kinds of water samples were used as the anolytes. The circuit was adjusted using variable resistance (100–10,000 Ω) to evaluate the relationship between current density and power density. Water sample II contained 1/1000 TSB, 30 mg/L Cu2+, 25 mg/L Zn2+, 25 mg/L Ni2+, 10 mg/L Ca2+, and 10 mg/L Mg2+. Water sample III contained 1/1000 TSB and 20 mg/L SO42−. Water sample IV contained 1/1000 TSB, 30 mg/L Cu2+, 25 mg/L Zn2+, 25 mg/L Ni2+, 10 mg/L Ca2+, 10 mg/L Mg2+, and 20 mg/L SO42−. In these batch experiments, the voltage and power density of the MFC were analyzed after 60 min operating periods when the original anolyte in the MFC was completely replaced with the tested water samples. All of the experiments were conducted at least in triplicate in order to verify the accuracy and reproducibility of the obtained results.
After determining the operating external resistance on the basis of the relationship between the current density and the power density, 1 mL Cr(VI) with a final concentration in the range of 0.01–100 mg/L was added to 1/1000 TSB to analyze the relationship between voltage variation and reaction time. Based on these results, the relationship between the Cr(VI) concentration and voltage output of the MFC or the calibration curve (voltage vs. Cr(VI) concentration) was established.
2.5. Cr(VI) Measurement in Artificial and Actual Electroplating Wastewater
Cr(VI) concentrations in artificial and actual electroplating wastewater were measured using the developed MFC biosensor and a standard colorimetric method. As the measurable Cr(VI) concentration ranges are different for the MFC biosensor (2.5–60 mg/L) and the colorimetric method (0.1–1 mg/L), appropriate dilution of the water samples may be required. In this study, artificial wastewater contained the prepared Cr(VI) concentration (7.5–55 mg/L) in 1/1000 TSB solution. To evaluate the feasibility of the MFC biosensor, actual electroplating wastewater samples were collected. Wastewater samples A–D were obtained from the effluents of different electroplating units. To measure the Cr(VI) concentration in the wastewater, the original anolyte in the MFC was completely replaced with the artificial wastewater; however, in the case of the electroplating wastewater, 169 mL of actual wastewater was supplemented with 1 mL of 17/100 TSB to maintain the TSB concentration in the anolyte of the MFC. Then, Cr(VI) concentration in wastewater samples was directly determined by the MFC biosensor in batch mode. Based on the established calibration curve (described in
Section 2.4), the Cr(VI) concentrations in these wastewater samples could be easily analyzed. All of the experiments were conducted using five separate MFCs and each analysis was conducted in triplicate.
2.6. Analysis
The standard colorimetric method for Cr(VI) measurement was performed as described previously [
24]. Briefly, 200 μL of the water sample was initially made up to 1 mL with distilled water. Later, this solution was made up to 10 mL with 400 μL of 0.25% S-diphenyl carbazide, 330 μL of 6 M H
2SO
4, and distilled water. Subsequently, the Cr(VI) concentration in the solution was determined at 540 nm using a UV-Vis spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The Cr(VI) removal efficiency of the YC211 bacterium was then calculated as follows:
where C
i and C
f are the initial and final Cr(VI) concentrations, respectively.
The potential difference between the anode and the cathode was measured using a Model 2700 multimeter (Keithley Instruments Inc., Solon, OH, USA). The data were continuously recorded on a computer using a Model PCI-488 interface card (Keithley Instruments Inc.). The current (I, amp) was calculated at a resistance (R, ohm) from the voltage (V, volt) as I = V/R. The power (P, watt) was calculated by P = I × V. The power density (mW/m2) and current density (mA/m2) were calculated by relating the power and current with the surface area (m2) of the anode, respectively. All of the analyses were conducted in triplicate and the mean values were calculated.
To understand the changes in the bacterial community of the MFC, the biofilm at the anode (i.e., the graphite felt) was collected for bacterial community analysis using NGS before and after determining the Cr(VI) concentration in the electroplating wastewater. Cell lysis, DNA extraction, polymerase chain reaction (PCR) amplification, and 454 pyrosequencing were conducted per the processes described by Naz et al. (2016) [
25]. DNA was extracted using a Fast DNA SPIN Kit (MP Biomedicals). PCR primers GAGTTTGATCNTGGCTCAG (forward) and GTNTTACNGCGGCKGCTG (reverse) were used to amplify the eubacterial 16S ribosomal RNA fragment. PCR profiling was conducted as follows: 95 °C for 10 min, 35 cycles at 94 °C for 45 s, 55 °C for 1 min, 72 °C for 1 min, and a final extension at 72 °C for 10 min. All of the partial 16S rRNA gene sequences were preprocessed per the methods described by Naz et al. (2016) [
25]. Sequence analysis was performed using the Quantitative Insights Into Microbial Ecology software package. These processed sequences were clustered into Operational Taxonomic Units (OTUs) based on 0.97 sequence similarity with the UCLUST algorithm. Representative OTUs were selected based on the most abundant sequences and taxonomic assignment was conducted using the ribosomal database project classifier.
4. Conclusions
In this study, we developed a Cr(VI)-MFC biosensor that was inoculated with E. aestuarii YC211 for the in situ determination of Cr(VI) concentration in electroplating wastewater. E. aestuarii YC211 is facultatively anaerobic, Cr(VI)-reducing, and exoelectrogenic; further, it has high adaptability to pH, temperature, salinity, and water quality. Our results illustrate that the MFC biosensor is a simple but reliable device for measuring a wide range of Cr(VI) concentrations with high accuracy in a short period of time. By NSG analysis, the predominant strain in the biofilm was found to be E. aestuarii, which accounted for 95.3% of the total bacterial sequences in the MFC even after treating actual electroplating wastewater. This gives us to understand that the reliable performance of the MFC may be attributed to the stable bacterial community present in the MFC during the treatment period. Although the limit of detection of the developed MFC biosensor was slightly larger than the maximum allowable level of Cr(VI) in an effluent, the biosensor was not designed to evaluate whether the Cr(VI) concentration met with wastewater discharge standards or not, instead of being early warming function. Thus, the application of MFC biosensors as in situ devices for Cr(VI) determination in the effluents produced from different operational units in an electroplating plant is promising.