Photo-Assisted Removal of Tetracycline Using Bio-Nanocomposite-Immobilized Alginate Beads.

In the present study, we report an efficient method for tetracycline (TC) removal from contaminated wastewater using alginate beads, immobilized with bio nanocomposite (BNC) consisting of Fe3O4 (iron oxide) and TiO2 (titanium dioxide) nanoparticles along with dead biomass of TC-resistant bacteria Acinetobacter sp. Chemically synthesized Fe3O4 nanoparticles and commercially available TiO2 (P25) nanoparticles were combined to form nanocomposite followed by encapsulation within alginate beads along with heat-killed biomass of Acinetobactersp. for the efficient degradation and adsorption of the target pollutant. The primary characterization of chemically synthesized nanoparticles was carried out with Fourier transform infrared, scanning electron microscopy-energy-dispersive X-ray spectrometry, transmission electron microscopy, and X-ray diffraction techniques. The batch studies for TC removal were performed by varying the reaction parameters such as bead weight, initial TC concentration, and pH in a photoreactor with UV-C irradiation. TC concentration of 10 mg/L, bead weight 10 g, and pH 6 were fixed as the optimum condition where 98 ± 0.5% of TC was removed from the solution. The possible removal mechanism was investigated with the help of UV-visible, total organic carbon, oxidation-reduction potential, Brunauer-Emmett-Teller, and liquid chromatography-mass spectroscopy analyses. The applicability of the process was successfully tested with the natural water systems spiked with TC at 10 mg/L. To assess the ecotoxic effects of the treated effluents, the cell viability assay was performed with the algal strains, Chlorella, and Scenedesmussp. and the bacterial strains, Pseudomonas aeruginosaand Escherichia coli. Finally, the reusability of the BNC bead was successfully established up to the 4th cycle.

Introduction

Widespread usage of antibiotics in human and veterinary applications has made natural water system rife with organic pollutants.[1] The very first reported water contamination due to antibiotic pollutants was reported in England by Watts et al.[2] They identified a mixture of antibiotics like tetracycline (TC), macrolides, and sulphonamides in natural water system up to the concentration of 1 mg/L way higher than the permissible limit.[2] Minimal cost and ease of large-scale production favors the wide use of TC in agricultural, human, and veterinary applications.[3] TC administration in humans and animals, in turn, results in about 90% of its release into the environment, especially the natural water systems in the active form.[4] The presence of TC in the natural water system brings in toxic effects to aquatic and human life and may lead to antibiotic resistance in bacteria.[5]

Conventional methods of wastewater treatment make use of membrane filtration, chemical reactors (photochemical, electrochemical, advanced oxidation, ozonation, and photocatalytic process), and adsorbents for the removal of various organic pollutants. Among these, the adsorption-based antibiotic removal is profitable as it makes use of a simple separation technique, cost-effective, easy to perform, and less toxic to the environment.[6,7]

Adsorption of contaminants only involves the transformation of antibiotics from one phase to another without any degradation. Hence, a suitable method is required for degrading the contaminants into less harmful byproducts.[9] A recent method, the advanced oxidation process (AOP) involves the formation of hydroxyl radicals which converts such antibiotics into harmless products under UV irradiation with various nanoparticles like ZnO, TiO2, and H2O2.[10]

Nanoparticles are suitable for the oxidation process because of their high photocatalytic activity, relatively stable in aqueous solution, nontoxic nature, and low cost.[11] It was reported that TiO2/UV-A-based photocatalytic degradation followed by adsorption of pharmaceutical compounds is more advantageous over the conventional antibiotic removal techniques,[12] as it involves fast mineralization and detoxification of the antibiotic by photocatalytic ozonation.[13] However, in spite of its advantages, TiO2-based antibiotic removal has reduced photocatalytic effect because of the formation of electron–hole recombination; hence, to prevent this effect, Ti can be fortified with other metals especially with Fe3+. TiO2 was effectively doped with Fe3+ because of its half-full electron configuration and relatively equal ionic radius with Ti nanoparticles.[14]

Fe3+–TiO2/UV-C based photocatalytic degradation removed 88.92% of amoxicillin from wastewater but it required pretreatment methods for the enhanced removal in natural water samples[11] and the nanophotocatalyst, Fe3+–TiO2 with UV-C radiation was used to remove up to 97% of metronidazole from the aqueous system.[15] Also, the occurrence of penicillin-G was reduced to more than 90% with Fe3+–TiO2/UV-A photocatalytic treatment and this technique was reported as the most efficient and cost-effective method for the removal of the penicillin.[14]

Wastewater treatment technique based on adsorption also relies on biosorbents like bacteria, fungi, algae, yeasts, and agricultural waste. The main attributes of biosorption include rapid adsorption rate, high selectivity and efficiency, and cost-effectiveness. Hence, this method can be used as an efficient alternative to the existing carbon-based adsorption techniques.[16] β-Lactam antibiotics like penicillin-G were effectively adsorbed by dried Rhizopus arrhizus and activated sludge. This material can be used as an alternative for carbon nanomaterials because of its low cost and increased selectivity toward antibiotics.[17]

In the current study, we used the dead biomass of Acinetobactersp. isolated from the TC-contaminated site. Even though, there are several studies regarding the removal of antibiotics by AOP, we did not find any specific report on coupling distinct advantages of antibiotic-resistant bacterial biomass with photocatalytic potential of nanomaterials to degrade the antibiotics. Herein, we report a novel nanobiomaterial, that is, the fortified titanium dioxide (TiO2) (P25) catalyst with iron oxide NP and dead biomass immobilized over biodegradable polymer sodium alginate with UV-C irradiation for TC removal from aqueous solution. Bionanocomposite (BNC) preparation involves (a) the synthesis of iron oxide by chemical precipitation, (b) followed by the preparation of nanocomposite with synthetic TiO2 (P25), and finally combining with the dead biomass of Acinetobacter sp. which has high selectivity toward TC antibiotic. The prepared BNC was characterized and validated for the removal of TC from the natural water system.

Results and Discussion

Characterization Studies

Figure A shows the scanning electron microscopy (SEM) image of the synthesized iron oxide nanoparticle, where the average size of the iron oxide nanoparticle was found in the range of 75–85 nm with a spherical structure. The surface morphology of the BNC beads was found to be smooth and uniform before interaction with TC and the average size of the bead was 500–600 μm (Figure B). Energy-dispersive X-ray spectrometry (EDX) analysis of the BNC bead showed the presence of the Fe, Ti content with 1.60 and 1.35 wt %, respectively (Table S2 Supporting Information). The transmission electron microscopy (TEM) image of the procured TiO2 nanoparticle shows the particle diameter ranging from 50 to 200 nm with irregular shape[19] (Figure C).

Figure 1

(A) SEM image of iron oxide nanoparticle, (B) SEM image of the BNC bead, (C) TEM image of procured TiO2 (P25) nanoparticle, (D) FT-IR spectrum of the Fe3O4 nanoparticle.

The Fourier transform infrared (FT-IR) spectrum (Figure D) of Fe3O4 shows two intense peaks at 539 and 665 cm–1 corresponding to Fe–O stretching vibration[20] and the peak at 3609 cm–1 is assigned to OH bending and 1520 cm–1 corresponds to C=C aromatic vibration.[21]

Batch Removal of TC

Effect of Bead Weight

The BNC beads of different weight percentages (1, 5, and 10 g) were tested with 10 mg/L of TC solution. The anticipated increase in the amount of bead weight enhances the removal efficiency. Therefore, the TC removal increased to 98.61% by the application of 10 g of BNC beads at 30 min. The reduced dosage of the beads to that of the TC produced a lower removal efficiency of 33.72 and 66.46% for 1 and 5 g, respectively (Figure A). This indicates that the increase of the bead weight is proportionate with an increase in the efficiency of the TC removal.

Figure 2

Determination of TC removal percentage by (A) effect of bead weight 1, 5 and 10 g loaded with BNC, (B) effect of pH 4, 6 and 9, (C) effect of initial TC concentration 10, 20, and 40 mg/L.

Effect of pH

The tests were performed by varying the pH at 4, 6, and 9 with 10 g of the BNC bead and 10 mg/L of TC. The data showed that at pH 9 the percentage of removal was 40.2 ± 2.19 and at pH 4 51.34 ± 1.97, whereas at pH 6 optimal TC removal of 98.61 ± 1.07 was noted (Figure B).

Effect of Initial TC Concentration

Further tests were performed with increasing concentrations of TC from 10 to 40 mg/L. This condition leads to a decrease in the removal efficiency of the TC (Figure C). The highest removal efficiency of 98.61 ± 1.07% was obtained at 10 mg/L, whereas for 20 and 40 mg/L the removal percentage declined to 88.92 ± 1.04 and 48.93 ± 1.09, respectively, showing a reduction in removal efficiency by increasing TC concentration.

TC removal efficiency in the current study is compared with similar studies from literature in Table . The control study to determine the effect of individual nanoparticle and the dead biomass was performed under the optimum experimental conditions. The experimental data shows 12.74, 15.9, 16.63, and 19.12% of TC removal by alginate beads without loading, beads loaded with dead biomass, beads with Fe3O4 alone, and beads with TiO2 nanoparticle alone, respectively (Figure S1 Supporting Information). To find out the effect of nonphoto-assisted removal, the control experiments were performed without any irradiation keeping the remaining conditions same and only 16.16% of TC removal was obtained (Figure S2 Supporting Information).

Table 1

Comparison of TC Degradation Efficiency with Different Photocatalyst

photocatalyst	reaction condition	% TC removal	ref
UV/TiO2/H2O2	catalyst conc. 1.0 g/L of TiO2, 100 mg/L of H2O2, TC conc. 55 mg/L, pH 5	100	Safari et al.[10]
UV/MIL-101(Fe)/TiO2	catalyst conc. 0.6 g/L, TC conc. 20 mg/L, pH 7	95.95	He et al.[27]
UV/TiO2	catalyst conc. 0.5 g/L, TC conc. 40 mg/L, pH 6.0	100	Reyes et al.[28]
UV/TiO-rGO-AC	catalyst conc. 0.8 g/L, TC conc. 40 mg/L	70	Qu et al.[29]
Cu2O–TiO2-Pal	catalyst conc. 1 g/L, TC conc. 30 mg/L, pH 8.7	88.81	Shi et al.[30]
UV/BNC bead	catalyst conc. 2:2:1 (TiO2/Fe3O4/dead biomass) bead weight: 10 g, pH 6.0, TC conc. 10 mg/L	98.61	present work

Kinetics of TC Removal

The results from the kinetic study clearly showed that the mechanism followed the pseudo first-order kinetics with the regression value of approximately around 0.976 (Figure ). The study suggests that the adsorption of the TC involves initial degradation followed by adsorption on alginate beads.[22]

Figure 3

Kinetic study of TC removal by BNC with UV-C radiation (A) pseudo-first-order kinetic model with k1 = 0.044 and R1 = 0.976 (B) pseudo second-order kinetic model with k2 = 0.762 and R2 = 0.909.

TC Removal Mechanism

The SEM analysis (Figure A) of the BNC bead after the interaction showed rough surface morphology. It can be deduced that because of the adsorption of TC onto the surface of beads there is a change in surface morphology from smooth to rough.[23]

Figure 4

(A) SEM image of the alginate bead loaded with BNC before and after interaction (B) XRD analysis of the BNC bead before and after interaction with TC (C) FTIR spectrum of before and BNC bead before and after interaction with TC.

The X-ray diffraction (XRD) analysis of the BNC bead before interaction showed intense peaks at (101) and (200) corresponding to the anatase TiO2 nanoparticle,[24] and the peak at (300) denoting the highly pure magnetite Fe formation[21] (Figure B). After the interaction with TC, the intensity of the crystalline phase of both TiO2 and Fe–O reduced, which may be attributed to the degradation of antibiotics by the nanoparticle.

The FT-IR data, after interaction, showed slight shift of peaks observed at 2310.72, 651, and, 551 cm–1. This can be due to adsorption of photocatalytically degraded TC through binding with the nanoparticles in the composite, through the formation of C–C, C–OH, and C–H rings[23] (Figure C). The possible surface adsorption of the antibiotic onto the BNC beads was further confirmed by surface area and porosity analysis of the beads before and after the interaction. From the analysis, it was noted that before interaction the surface area was 1.51 m2/g and after interaction, it reduced to 0.27 m2/g which confirmed that because of surface adsorption the porosity of the BNC beads decreased.

The TC removal mechanism with respect to total organic carbon (TOC) content in the sample is displayed in Figure A, which shows steady decrease in TOC content along with decline in TC concentration. Absence of any degradation and adsorption of TC throughout the control test was confirmed because the TOC remained constant.[25] However, after the interaction the TOC content showed significant reduction proving notable role played by adsorption during the reaction. The maximum drop of TOC data was noted at 30 min corresponding to 98.61% removal.

Figure 5

(A) % TC removal analysis by the TOC study (B) ORP measurement (C) HR-LC–MS analysis of the BNC bead-treated TC sample.

To understand the role of a reducing agent in the degradation of antibiotic oxidation–reduction potential (ORP) analysis was performed and the data are shown in Figure B. The ORP value at time t0 was 380 mV and at time t30 had ORP value reduced to 170 mV indicating the presence of strong reducing environment during the interaction.

The liquid chromatography–mass spectroscopy (LC–MS) analysis of the reacted liquid sample in Figure C shows that the intensity of the initial TC peak at 445 was reduced after 30 min of interaction with the BNC bead. The formation of intermediate byproducts was confirmed by the presence of peak at 172 and the peak at 130, which involves various structural and functional group modifications such as epimerization, dehydration, and dimethylation. The degraded byproducts can be easily adsorbed over the beads after interaction.[26]

Reusability of BNC

The reusability test in Figure A showed that BNC beads used in the experiment were viable until 4 cycles for the better removal of TC. Further, the stability of BNC beads and control beads under UV irradiation was analyzed for the deformities and it established stability of beads after continuous cycle of TC removal. The removal efficiency of control beads decreased after the 4th cycle with 4.8 ± 0.5% and for BNC beads 59 ± 0.5% of TC removal. This decrease in the adsorption capacity is probably due to binding of the TC-degraded products onto the nanoparticles present within the matrix of the BNC beads or because of the escape of the nanoparticles from the agile surface of the BNC beads. No deformation of the beads could be noted even after four cycles of interactions.

Figure 6

(A) Reusability assessment of BNC bead (B) effect of the BNC bead on natural water system (C) residual toxicity study of TC and BNC-treated TC sample against algal and bacterial strains.

BNC for Real Water Sample Treatment

The effect of BNC bead on the natural water system adulterated with TC shows was 85.16 ± 0.4% of TC removal in tap water followed by groundwater with 79.62 ± 1.6% removal and lake water of 71.90 ± 0.2% removal Figure B. The percentage TC removal was reduced for the natural water system because of the presence of colloidal particles that are present in the water sources. Hence, it requires that the natural water system be pretreated before the application of the BNC bead for the most effective removal of TC.

Residual Toxicity of BNC

The toxicity assessment in Figure C showed that the percent cell viability of the two strains of algal species in the range of 98.075 ± 0.1 and 93.644 ± 0.2 for Chlorella and Scenedesmus sp. which states that the Scenedesmus obliquus is more sensitive to the BNC-treated TC sample compared to the Chlorella and which shows the percent cell viability of 69.79 ± 0.2 and 62.74 ± 0.3 against TC control. As for the bacterial strains of Pseudomonas aeruginosa and E. coli, the toxicity assessment was performed, which shows the percent cell viability of 91.83 ± 0.2 and 90.67 ± 0.1 for treated TC sample and 61.53 ± 0.2 and 60.75 ± 0.2 for TC control.

Conclusions

Application of the BNC-loaded alginate bead for the TC removal from the natural water system was proven with the removal percentage of 71.09 ± 0.2 which further increased by pretreatment of the real water sample to remove colloidal impurities. It was found that BNC-loaded alginate bead-based photocatalytic removal of TC will release considerably less toxic byproducts with increased reusability range and it can be easily separated out after the process. Hence, this method can be further modified for the removal of mixture of antibiotics with less cost and higher efficiency.

Materials and Methods

Materials

TC (C22H24N2O8), ferric chloride (FeCl3), and titanium dioxide (TiO2) (P25) were procured from Sigma-Aldrich, India and sodium borohydride (NaBH4) was procured from HiMedia. The glassware used was of high-quality borosilicate glass and all other chemicals were of analytical grade. The bacterial strain Acinetobacter sp. was isolated from the contaminated site and confirmed by 16s rRNA sequencing.

Synthesis of Iron Oxide Nanoparticles

Synthesis of iron oxide nanoparticles by co-precipitation techniques involves the reduction of iron(III) chloride (0.1 M FeCl3·6H2O dissolved in 40 mL of 99% ethanol) by 2.5 M of sodium borohydride (NaBH4) solution. The synthesis steps involve the dropwise addition of NaBH4 to FeCl3 ethanol solution with continuous stirring at 200 rpm. The nanoparticles formed were separated by centrifugation and washed with ethanol twice followed by overnight drying in a hot air oven.[18]

Dead Biomass Preparation

Initial isolation and purification of Acinetobacter sp. was performed followed by identification of strain with 16s rRNA sequencing. The regeneration of the Acinetobacter sp. was performed by adding 100 μL in 250 mL of nutrient broth (NB) medium (beef extract 0.5 g, yeast extract 1 g, peptone 2.5 g, sodium chloride 2.5 g) followed by resuspension of abovementioned culture of 400 μL in 1000 mL NB medium which was performed to achieve a desired quantity of biomass. Dead biomass was prepared by sterilizing the culture at 121 °C/15 psi and drying it in a vacuum oven overnight at 59 °C.

Preparation of BNC Bead

Nanocomposite material was prepared by dissolving equal ratio of synthesized Fe3O4 and commercial TiO2 (1:1 ratio) nanoparticles, followed by probe sonication of the abovementioned solution. Preparation of BNC involves the addition of dead biomass (1:2 ratios) into the abovementioned solution. The sample was subjected to sonication in a water bath to get equally dispersed BNC. Prepared BNC was mixed with 25 mL sodium alginate (7%) solution and added dropwise into 100 mL of 2% calcium chloride solution to obtain BNC-immobilized alginate beads.

Characterization Techniques

The chemically synthesized iron oxide nanoparticles and alginate-encapsulated BNC were characterized by high-resolution scanning electron microscope (FEI Quanta FEG 200) for the analysis of particle size, shape, and morphology with a magnification of 12× to 100 000×. EDX provided elemental composition via backscattered electron detection system and FT-IR spectroscopy (IR Affinity-1, Shimadzu, Japan) was employed for analyzing the functional groups. The crystalline nature of the BNC bead (before and after interaction) was analyzed by XRD (Bruker Advanced D8, Germany). TOC in the TC-treated samples was measured by the TOC analyzer (Shimadzu) and digital potentiometer (model DP003 PICO, Chennai) was used to analyze the reducing agents present in the TC–BNC interacted solution. Brunauer–Emmett–Teller was used to study the surface porosity of the BNC bead before and after interaction. High-resolution-LC–MS (HR-LC–MS) (1290 infinity UHPLC system, 6550 iFunnel Q-TOFs, Agilent Technologies, USA) was used to detect the degraded products of TC after interaction with nanoparticles.

Batch Study for TC Removal

TC stock solution (100 mg/L) was prepared and stored in dark at −4 °C well in advance to the work. From the stock solution 40, 20, and 10 mg/L concentration of TC was prepared, and 1, 5, and 10 g of BNC beads was filtered out and interacted with TC for 30 min interval in the UV photoreactor (Heber compact multiwavelength multilamp photoreactor SW-MW-LW888) under UV-C conditions in borosilicate tubes. The degradation of TC with respect to the control was noted by analyzing the absorbance of the sample at 360 nm. The experiment was carried out in triplicates for statistical purpose. The amount of TC that remained in the test sample can be calculated using the regression equation deduced from the calibration graph, and the percentage of TC removal can be calculated using the equationwhere Ai and Af are the initial and final absorbance, m (g) is the mass of the sorbent, and V (L) is the sample volume.

Kinetic Study

To understand the kinetics of TC removal by the UV-assisted BNC bead, pseudo-first and second-order kinetic models were used. The formula is as followswhere q and qe are used to denote TC adsorbed at time t and at equilibrium te; the rate constant of pseudo-first-order model (min–1) was denoted by k1, which was obtained by plotting log(qe – q) versus t. However, the formula for the pseudo-second-order kinetics is as followswhere k2 is the pseudo-second-order equilibrium rate constant and which was obtained by plotting t/q versus t.

Residual Toxicity Analysis

The bacterial strain and algal strain were used to study the toxicity of TC and BNC bead-treated TC sample. The bacterial strains were cultured in NB and the algal strains in BG-11 medium. Standard plate count assay was used to analyze cell viability of TC and BNC bead-treated TC interacted cell culture.[8]