Adsorption of azithromycin antibiotic from water onto biochar derived from Terminalia chebula and sugarcane bagasse

AMR

antimicrobial resistance

BBC

bagasse-derived biochar

CBC

T. chebula-derived biochar

CCD

central composite design

EDS

energy dispersive X-ray spectroscopy

FTIR

Fourier transform infrared spectroscopy

RSM

response surface methodology

SEM

scanning electron microscopy

XRD

X-ray diffraction

Antimicrobial resistance (AMR) is a serious concern of the 21st century that threatens the public health care system. AMR has effectively harmed the treatment and prevention of emerging microbial infections. Microbes are no longer susceptible to the common medicines used for their treatment (Peng et al. 2022). The World Health Organization declared the coronavirus outbreak in 2019 brought on by the SARS-CoV-2 virus as a pandemic on March 11, 2020. This outbreak reshaped the global public health concern about AMR as there is rampant use of antibiotics. It is well known that a major portion of the antibiotics consumed by humans (and animals) are not completely metabolized by their bodies. Unmetabolized antibiotics are let into the soil and water by various means, such as municipal wastewater, animal manure, sewage sludge, and biosolids. These increased concentrations of antibiotics in the environment have led to the emergence of much higher levels of AMR. AMR occurs when microorganisms, including bacteria, fungi, parasites, and viruses, develop resistance against the medicinal drug that once affected them due to excess exposure (Dadgostar 2019). It is estimated that in the USA, AMR organisms cause about 2 million infections, which lead to approximately 23,000 deaths per year (Marston et al. 2016).

Azithromycin, a broad-spectrum antibiotic, has been licensed for the treatment of respiratory infections, otitis media, skin infections, and sexually transmitted diseases (Koch et al. 2005). With the COVID-19 pandemic, the use of azithromycin as a treatment increased. The human body does not completely metabolize azithromycin, and a sizeable amount is added to wastewater treatment facilities. Furthermore, azithromycin has a slow metabolism, indicating poor degradation in aqueous medium and resulting in higher levels in the environment (Koch et al. 2005).

Antibiotics have been removed using a variety of biological and physicochemical approaches (Xu et al. 2020). Several advanced technologies such as photocatalytic degradation chemical oxidation via ozone or ozone/hydrogen peroxide, electrochemical treatments, membrane filtration techniques (such as nanofiltration and reverse osmosis), surface adsorption onto the carbonaceous adsorbents or nanomagnetic adsorbents have been investigated in recent years to remove these pollutants. However, due to the advantages of adsorbent such as simplicity, low toxicity, cheap energy cost, and high removal effectiveness, adsorption is accepted widely (Xu et al. 2020). Many absorbents, such as resin, bentonite, activated carbon (Shao et al. 2021), zeolite, chitosan, carbon nanotubes (Xu et al. 2020), and biochar are used for the removal of contaminants. Biochar, produced by pyrolysis under oxygen-deficient conditions, is a carbon-rich material (Pan 2020). It is generally produced from organic wastes such as agriculture waste, municipal sewage waste, forest residue, and animal manure. Biochar possesses enhanced properties such as a large surface area, higher cation and anion exchange capacities, and a stable structure. Biochar is preferable to biomass because it increases carbon sequestration, reduces waste, and emits less greenhouse emissions. Lignocellulosic biomasses such as agro-industrial wastes and forest wastes are the preferred raw materials for the preparation of biochar due to their low-cost and abundance (Pandey et al. 2020). Biochar derived from various sources, such as oak wood, paper mill sludge (Masrura et al. 2022), rice husk (Herrera et al. 2022), wood chips (Muter et al. 2019), etc., have been used for the adsorptive removal of azithromycin from the water. Herrera et al. (2022), utilized rice husk biochar to remove azithromycin up to 200 mg/L, with a removal efficiency of 95%. Chen et al. (2019), have found to have maximum removal efficiency of 85.1% for azithromycin with biochar derived from spent mushroom substrate.

Terminalia chebula is a species of Terminalia that is also known as Black Myrobalan or Harrad. It is widely distributed throughout India, Nepal, China, Sri Lanka, Malaysia, and Vietnam (Nallathambi et al. 2022). T. chebula is a flowering and medicinal plant widely used in ayurvedic medicine to treat various diseases. While sugarcane bagasse is one of the most abundantly available agro-industrial wastes and has been widely explored for the production of biochar (Iwuozor et al. 2022). Sugarcane bagasse biochar has been used for the removal of antibiotics like sulfonamide, tetracyclines, and other contaminants like carbofuran (Jacob et al. 2020). However, the removal of azithromycin by adsorption using sugarcane bagasse biochar and T. chebula biochar has not yet been investigated.

The aim of the study was to utilize T. chebula and sugarcane bagasse for the development of biosorbent for the adsorptive removal of azithromycin from wastewater. Biochar was characterized by SEM, FTIR, and XRD. Along with adsorbent dosage, temperature and pH were also investigated. Furthermore, isotherms, kinetics, and thermodynamic studies were also executed to understand the underlying mechanism of adsorptive removal of azithromycin from wastewater through both biochars.

The fruits of T. chebula were collected from the garden of the NIT Rourkela Campus, while sugarcane bagasse was collected from the cane juice vendor on the NIT Rourkela Campus. Azithromycin for injection, USP 500 mg – Ozitop (Oscar Remedies Pvt. Ltd) was purchased from the Community Welfare Society Hospital (CWS Hospital) in Rourkela. A concentration of 25,000 mg/mL was achieved by dissolving 500 mg of azithromycin in 20 mL of 0.01 M phosphate buffer with a pH of 7.5. This solution was prepared due to the compound's low solubility in water and the isotonic conditions maintained by phosphate buffer (Mangal et al., 2018). A homogenized solution was generated due to its high solubility in phosphate buffer. Subsequently, a homogenized solution was diluted using distilled water. All other chemicals were purchased from Himedia.

A stock solution (250,000 mg/L) of azithromycin was prepared from which a working solution of 1,000 mg/L was obtained by diluting with distilled water. The working solution of azithromycin (1,000 mg/L) was further diluted with distilled water to get final concentrations of 100, 150, 200, 250, 300, and 400 mg/L for standard curve preparation. For the estimation of azithromycin, water samples were treated with 9 mL of 13.5 M sulfuric acid and heated in a water bath at 50 °C for 30 min. Through this reaction, the antibiotic undergoes hydrolytic cleavage of the glycosidic linkage and generates an erythronolide aglycone moiety. This reaction results in the formation of erythronolide, a yellow compound that exhibits intense adsorption. The samples were cooled down at room temperature and then absorbance was taken at 482 nm by a UV-Vis spectrophotometer (Double beam spectrophotometer 2203 Systronic) as reported earlier (Kumar et al. 2013).

Various analytical methods were utilized to characterize the biochar. For pH analysis, 1 g of biochar was added to 50 mL of distilled water. The pH was taken using a pH meter after 30 min (Pandey et al., 2022a). The amounts of moisture, volatile matter, ash, and fixed carbon were all calculated using proximate analysis. The morphology and structure of the biochar were analyzed by SEM (Scanning Electron Microscopy) (JEOL JSM-6480 model, Jeol USA Inc., Peabody, MA, USA), with EDS (Energy Dispersive X-ray Spectroscopy). The nitrogen adsorption/desorption isotherms of the biochar were characterized at –196.15 °C by using a surface area and porosity analyzer to obtain the BET-specific surface area and pore size. The functional groups of biochar were determined using Fourier Transform Infrared Spectroscopy (FTIR) (Alpha ATR-FTIR, Bruker, USA) in the 500–4,000 per cm range. X-ray diffraction (XRD) was used to identify the crystalline phase and its ratio with the amorphous phase of the biochar. T. chebula and sugarcane bagasse biochar were analyzed using the following operating parameters: with a step size of 7°, the 2θ angle measurement range was adjusted from 0° to 80° (Kwoczynski & Čmelík 2021).

Kinetics were performed using 50 mL initial concentrations of 100, 150, 200, 250, 300, 350, and 400 mg/L of azithromycin solution. The optimized conditions for CBC were 0.35 g of biochar, pH of 8.47 and 37.5 °C temperature, whereas for BBC were 4.7 gm of biochar, pH 10.19, and 38.7 °C temperature. The flasks were incubated in a shaking incubator at 160 rpm for 180 min. Similarly, a thermodynamic study was performed at three different temperatures 29.9, 37.3, and 43.9 °C and at a constant initial concentration of azithromycin (Lagergren 1898; William Kajumba et al. 2019).

The reusability study of CBC and BBC was carried out by the following method. For adsorption, 0.3 g of biochar sample (CBC or BBC) was mixed with 100 mg/L azithromycin and the solution was incubated for 24 h at 25 °C in an orbital shaker. After 24 h of incubation, the samples were collected and filtered using a vacuum filtering device. Desorption was conducted for a duration of 24 h using 25 mL of desorption solution (0.05 M NaOH for CBC and 0.1 M HCl for BBC) on the filtered biochar. After desorption, the samples were collected and subsequently filtered through a vacuum filtration apparatus. The collected supernatants were used for the estimation of azithromycin. The filtered biochar was then air-dried at room temperature and used for adsorption again. The cycle was repeated three times (Mdlalose 2018).

The textural characteristics of CBC and BBC are present in Table 1. The pH of both the biochar was slightly acidic to neutral, with CBC having a pH value of 6.84 and BBC having a pH value of 6.94. It has been observed that biochar produced with low ash content generally has a lower pH as compared to biochar produced with high ash content (Singh et al. 2017). Also, the pH is higher at pyrolysis temperatures above 400 °C than at temperatures below 400 °C, as of this study (Sharma et al. 2012). After adsorption pH of BBC changed to 7.85, while for CBC, it was 6.80. The point of zero charge, which is the pH at which the net surface charge on the adsorbent is zero, was found to be 5.25 for CBC and 7.73 for BBC (Supplementary material, Figure S1). At this pH, the biochar was in an equilibrium state. The proximate analysis and elemental contents of the two biochars (CBC and BBC) are compared in Table 1. The yield and fixed carbon of CBC were higher than those of BBC.

From the BET analysis (Supplementary material, Figure S2), the surface area of BBC (73.95 m²/g) was higher than that of CBC (6.32 m²/g). Here, pore formation may be due to the release of volatile components (Shikuku & Mishra 2021). The number of small pores present in CBC is more than in BBC, but the surface area was decreased due to the overlapping of pores in CBC (R and Maitra, 2020). Table 1 also illustrates the data from the EDS analysis. From the table, it is evident that carbon is the main component of the biochar. The oxygen in the biochar is from the functional groups containing oxygen, such as –COOH, and –OH (Zeng et al. 2018).

The XRD peak profile was observed for CBC and BBC, shown in Supplementary material, Figure S3. Both samples show a broad diffraction peak around 24°, which suggests both are amorphous in nature. Also, the hkl values for both the biochars are similar, i.e., (002) plane of the fabricated material, which diffuses a peak as graphite. Pandey et al. (2022b) studied the XRD pattern of pine needle for adsorption of Congo Red, and the pattern was similar to the current study i.e., 2Ɵ 24° and hkl as 002 (Pandey et al., 2022b).

The three-dimensional response surface diagrams illustrate the interactive effect of two parameters on % adsorption while the other parameter is held at the center point (Figure 4). The interaction between pH and temperature was significant in the case of BBC (p < 0.05) and in the case of CBC it was not significant (p > 0.05) (Supplementary material, Table S1). From Figure 4(a), it was observed that the change in temperature did not show any significant change in adsorption %. Whereas from Figure 4(d), it could be observed that a decrease in pH and an increase in temperature significantly improve the % adsorption. The interactive effect of dosage and temperature was found to be significant for BBC and insignificant for BBC (Table S1). From Figure 4(b) and 4(e), it could be observed that an increase in dosage initially favored the adsorption process, however after the optimal condition, the increase in dosage negatively affected the adsorption process at low temperatures. An increase in temperature showed a positive impact on both the biochars. The interactive effect of pH and dosage was found to be significant for adsorption by CBC and insignificant in the case of BBC (Supplementary material, Table S1). The minimum adsorption was experienced at the highest and lowest levels of dosage for both the CBC and BBC (Figure 4(c) and 4(f)). Whereas the initial increase in pH improved the adsorption and reached the maximum. Further increase in pH lowered the % adsorption a little for both CBC and BBC.

The optimal conditions for BBC were a biochar dosage of 0.47 g, pH of 10.19 and temperature of 38.72 °C. Supplementary material, Table S2 gives the model summary for the CBC and BBC. Following optimization, the percentage of azithromycin adsorbed by CBC increased from 32.21 to 57.17%, whereas it increased from 28.82 to 60.03% for BBC. After optimization, adsorption increased by 1.77-fold in BBC and 2.08-fold in CBC (Table 3).

Thermodynamic studies were performed to analyze the change in energy of the experiment, as shown in Table 4 and Supplementary material, Figures S4 and S5. In both biochars, the free energy ΔG value is negative and decreases with increasing temperature, which implies that the adsorption process involved is spontaneous and feasible. The azithromycin adsorption on both the biochar is an exothermic reaction and is evident by the negative value of ΔH. Bagasse ΔH is −20.06 kJ/mol, while for T. chebula ΔH is −14.89 kJ/mol, suggesting that bagasse adsorbs more energy than T. chebula for azithromycin adsorption. The stability and unpredictability of the adsorption process are indicated by the positive values of (Pandey et al., 2022a; Yao et al. 2020). Even though the drug has three degrees of freedom in the fluid phase and after adsorption, it loses one degree of freedom, therefore, the system is ordered. However, the entropy change of the entire process remains positive. The total entropy of the system is comprised of the total entropy of azithromycin and its surroundings. Theoretically, the entropy of azithromycin decreases because of a reduction in the degree of freedom however the entropy change was found positive. This rise in the entropy of the system must have been due to a rise in the entropy of the surroundings. was plotted against 1/T, ΔH, and ΔS were calculated from the slope and intercept, respectively (see Supplementary material, Figures S4 and S5). Similar results have also been observed by (Ebisike et al., 2023), where adsorption was spontaneous and exothermic.

Azithromycin comprises functional groups, including hydroxyl and carbonyl groups, which are capable of forming hydrogen bonds with the biochar. The surface interactions enhance the adsorption and retention of azithromycin on biochar. FTIR data further confirm the presence of hydroxyl, carboxylic acid, alkyl, and carbonyl groups which facilitate the formation of H-bond on the surface of BBC and CBC (Guo et al. 2023).

The adsorption process of azithromycin and biochar can be aided by the electrostatic attraction between their charged groups. In the case of both the biochar (CBC and BBC) depicts the electrostatic interaction by FTIR analysis. The bond intensity was higher in CBC than in BBC. The adsorption of azithromycin by biochar was also done by electrostatic induction mechanism because the charge present on the surface of azithromycin was neutral (Upoma et al. 2022) and negative in the biochar surface. The negative charge of the biochar was confirmed by the Pzc analysis, desorption and optimum pH obtained during the optimization study (Dong et al. 2024).

Biochar usually has a porous structure, as confirmed by the data obtained by SEM and BET analysis. More surface area contacts and a longer period of antibiotic release result from azithromycin molecules entering and filling the pores.

The presence of nonpolar groups confirmed by the FTIR analysis of both the biochar (C = O, O–H and C–H) interact to form anhydrous domain in aqueous solution that facilitates adsorption of azithromycin on the biochar (Ambaye et al. 2021; Mansee et al. 2023).

The biochar was successfully prepared from the T. chebula fruits and sugarcane bagasse. After optimizing biochar dosage, pH, and temperature, both CBC and BBC could absorb (about 60%) of azithromycin from water. The meta-analysis (Table 5) of azithromycin adsorption also confirms the maximum 200 mg/L of concentration was carried out using biochar. The Langmuir model was best fitted among the tested isotherm models for both types of biochar. The thermodynamic study suggests that the process was spontaneous and feasible, ΔH was exothermic and ΔS has shown stability and unpredictability for azithromycin adsorption. The yield of CBC was found to be 1.6-fold greater than the yield of BBC. The reproducibility of the BBC was more than CBC suggesting its high reusability. The study concluded that the pristine CBC and BBC both showed better adsorption for azithromycin from water. After optimizing biochar dosage, pH, and temperature, for both CBC and BBC the removal efficiencies of ∼60% may be too low to be economically feasible. Further, the adsorption of azithromycin on the CBC and BBC can be improved by modifying the biochar and their potential with real wastewater need to be tested in future.

The authors would like to thank NIT Rourkela for institute funding and Institute's Central Research Facility (CRF) access.

M.P. conducted experiments and collected data, analyzed samples and data, wrote the draft, reviewed and edited the article. S.S. analyzed data, wrote the draft, reviewed and edited the article. D.K. analyzed data, wrote the draft, reviewed and edited the article. A.D. conceptualized the study, wrote the draft, reviewed and edited the article. K.D. conceptualized, supervised, wrote the draft, reviewed and edited the article, collected funds.

The authors would like to thank NIT Rourkela for providing institute funding to carry out this work.

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

The authors declare there is no conflict.