DEGRADATION OF MALACHITE GREEN USING GREEN SYNTHESIZED IRON NANOPARTICLES BY Coffea arabica LEAF EXTRACTS AND ITS ANTIBACTERIAL ACTIVITY

The synthesis of biocompatible nanomaterials using bio-renewable plant extract has emerged as a promising alternative to traditional methods in recent years due to its cost-effectiveness, eco-friendliness, non-toxicity, and biocompatibility. In this research paper, we demonstrate using Coffea arabica leaf extract to synthesize zero-valent iron nanoparticles (GC-FeNPs). This study suggests that this method is efficient and environmentally friendly, making it a promising approach for developing biocompatible nanomaterials. The synthesized GC-FeNPs were characterized using Fourier-Transform Infrared (FT-IR), X-ray diffraction (XRD), scanning electron microscope (SEM), and UV–Vis spectroscopy techniques. According to the analysis of data, polyphenolic/caffeine compounds in the coffee leaf extract act as reducing/capping agents by converting the Fe 2+ to Fe 0 , and minimizing the aggregation. The FT-IR and XRD data confirm the encapsulation of the GC-FeNPs by the polyphenolic/caffeine compounds in the coffee leaf extract. The GC-FeNPs have a quasi-spherical shape morphology with a particle size of about 80 – 100 nm. Further, dye degrading ability, and the antibacterial activity of the GC-FeNPs were investigated using malachite green (MG) and gram-negative and gram-positive pathogens. Experimental data revealed that GC-FeNPs (~ 20 ± 1 mg) showed degradation activity against MG up to 55 % upon the 120-minute incubation. Furthermore, the kinetic analysis of GC-FeNPs on MG degradation was in accordance with the pseudo-second-order kinetic model (R 2 = 0.9922). In addition, GC-FeNPs showed an antibacterial activity against gram-negative (E. coli, and S. enterica), and gram-positive (S. aureus) pathogens. The E. coli growth was highly inhibited by the GC-FeNPs compared to other strains. Overall, GC-FeNPs facilitate an alternative approach to degrade MG from textile effluents and industrial wastewater and treat the pathogen-contaminated water.


INTRODUCTION
Water is an essential element on the earth, and numerous living organisms rely on it.However, water quality is degrading consistently due to various contaminations.Dyes play a significant role in water contamination all over the world.Annually, 700,000 tons of synthetic dyes are used worldwide for the dyeing operation process; around 15 -20 % end up in wastewater and find their way to the environment (Basturk and Karatas, 2015).Dye is a very harmful compound to the environment and living organisms due to its reactivity (Leme et al., 2015;Xiao et al., 2020).Aquatic plants are highly affected due to dye contamination in water since dye colour promotes turbidity (Khandare and Govindwar, 2015;Verma and Samanta, 2018).Turbidity in water bodies reduces the penetration of the sun's rays into the water body and affects photosynthesis.
Further, the biotransformation of the dyes into different products poses various threats to living organisms and the environment.However, due to the photo and heat stability, most dyes do not degrade quickly in the environment (Basturk and Karatas, 2015).Therefore, for the past few decades, novel techniques have been developed to treat wastewater contaminated by dyes.Also, degradation by techniques such as physicochemical adsorption (Gao, Si and He, 2015), electrocoagulation (Kobya et al., 2014), membrane filtration (Chidambaram, Oren and Noel, 2015), nano-filtration (Paździor et al., 2009), ozonation (Panda and Mathews, 2014), Fenton oxidation, and oxidation processes were identified as good strategies (Soares et al., 2015).However, the formation of toxic compounds may reduce the effectiveness of these methods.Therefore, biodegradation of the dye has been employed using aerobic and anaerobic bacteria (Popli and Patel, 2015), Sequencing Batch Reactor (SBR) (Santos and Boaventura, 2015), etc.These biological processes were further found to be effective to a certain extent but challenging to sustain in dye-contaminated wastewater.

Malachite
green (MG) is a cationic triphenylmethane (Figure 1) dye used in the dyeing process in different industries such as fabrics, paint, inks, leather, etc. (Huang et al., 2014a;Xiao et al., 2020).It is vital to remove MG from wastewater before discharging into the environment due to its toxicity.Therefore, different methods such as the adsorption (Gao, Si and He, 2015), biological degradation (Tsai, and Chen, 2010;Yatome et al., 1993), and photocatalytic degradation (Wu et al., 1999;Hachem et al., 2001;Elango and Roopan, 2015), are employed to remove MG content in the wastewater.However, due to the drawbacks such as high cost and low efficiency associated with these techniques, the applicability in the degradation process of MG is limited to a certain extent.Therefore, novel remediation techniques as an alternative are required to remove MG in wastewater effectively.
Recently, nano zero-valent iron (nZVI) has attracted research interest in groundwater treatment due to the higher intrinsic reactivity of its surface sites (Iravani, 2011;Dutta et al., 2016).Numerous chemical and physical approaches have been deployed for the synthesis of nZVI.However, these physical methods are associated with drawbacks such as energy consumption, expensiveness, the requirement of sophisticated laboratories, etc. (Sun et al., 2006;Nadagouda et al., 2010;Huang et al., 2014b).Sodium borohydride (NaBH4) is commonly used as a reducing agent in the chemical synthesis of nZVI (Devatha, Thalla and Katte, 2016).However, reducing agents such as NaBH4, stabilizing agents, capping agents, and organic solvents in chemical synthesis are limited due to the expense and toxic effect on the environment (Sun et al., 2006;Nadagouda et al., 2010;Huang et al., 2014b).The green synthesis of nZVI has been identified as an alternative approach due to its cost-effectiveness, eco-friendliness, biodegradability, and nontoxicity.Green synthesis received much attention due to the usage of bio-renewable natural sources (Tandon, Shukla and Singh, 2013;Huang et al., 2014a;Wang, Fang and Megharaj, 2014;Das and Eun, 2018).Recently, various nanomaterials have been synthesized using different green extracts (Njagi et al., 2011;Mohan Kumar et al., 2013;Zhu et al., 2018).Generally, components such as polyphenols available in tea and coffee extract have emerged as an alternative for the chemical synthesis of nZVI since they can act as both a reducing and capping agent (Mohan Kumar et al., 2013;Ouyang et al., 2019).This feature of the green extract reduces the aggregation and the oxidation of the nZVI particles (Chen et al., 2011).The green synthesis of nZVI using green extract polyphenols has been investigated for biocompatibility, degrading bromothymol blue by the Fenton oxidation process (Shahwan et al., 2011), and degradation of the trichloroethylene (TCE) by developing Fe/Pd membrane using a green tea extract (Smuleac et al., 2011).Fentonlike catalytic properties of the green synthesized iron nanoparticles (FeNPs) have been investigated for the degradation of dyes.The reactivity of the green synthesized FeNPs is significantly dependent on the reducing and capping agents of the green extract.Various green extracts promote different reactivities of the green synthesized FeNPs, (Raveendran, Fu and Wallen, 2003;Makarov et al., 2014).The FeNPs synthesized using green extracts have been employed for dye adsorption and eutrophication remediation (Lin et al., 2017).
Researchers have recently used tea extracts to synthesize iron nanoparticles for the selective removal of MG from wastewater.(Huang et al., 2014a(Huang et al., , 2014b;;Xiao et al., 2020).In 2014, Huang's group (Huang et al., 2014a) conducted a similar study utilizing FeNPs synthesized using tea extracts.The results indicated that the removal efficiency of MG was 81.6%, with an equilibrium at around 60 minutes.The rate kinetics were better fitted to the pseudo-first-order model (R 2 = 0.925).The experiment was conducted using 0.01 g/L of nanomaterial, which was incubated with an 8 mL 50-ppm solution of MG.A recent study by Xiao's group (Xiao et al., 2020) reported that FeNPs synthesized using green methods could effectively remove MG from wastewater.The researchers reported a degradation efficiency of 95.14% for MG, with an equilibrium achieved at approximately 100 minutes.The rate kinetics of the process were found to be better described by the pseudo-first-order model, with an R 2 value of 0.95.The study was conducted by utilizing 0.01 g of nanomaterial incubated in a 50-ppm solution of MG.The observed deviations in batch experiment analysis may be attributed to the variations in experimental conditions and the quantity and composition of polyphenolic compounds used for encapsulating iron nanomaterials.The specific experimental conditions, such as temperature, pressure, and pH level, as well as the type and concentration of polyphenolic compounds in the plant extracts used for the synthesis process, could impact the physicochemical properties of the iron nanoparticles.These physicochemical properties, in turn, may influence the behaviour and performance of the nanoparticles in the batch experiment analysis, resulting in deviations.This study aims to synthesize FeNPs from Coffea arabica leaf extract and characterize the FeNPs using SEM, FT-IR, UV-Vis, and XRD analysis.The reactivity of FeNPs in malachite green degradation and antibacterial activity against gram-positive and gram-negative bacteria were also evaluated.To the best of our knowledge, this is the first study that reports on the antibacterial and degradation effect of malachite green in the presence of green synthesized FeNPs using Coffea arabica leaf extract.

METHODOLOGY A. Materials
Ferrous sulfate heptahydrate (99%, FeSO4•7H2O) was purchased from Sigma Aldrich, and malachite green (MG) was purchased from DYECHEM (Colombo, Sri Lanka).Coffea arabica (coffee) leaves were collected from local farms.Muller Hinton Agar from Hi-Media Laboratories and deionized (DI) water was used in all experiments.

B. Preparation of leaf extract
Fresh coffee leaves were collected from a local farm and washed thoroughly with DI water to clean the leaf's surface.Leaves were dried at room temperature.Twenty-five grams of dry leaves of C. arabica were cut into small pieces and mixed with 150.0 mL of deionized water (Figure 2).The temperature of the solution was maintained at 60 °C and kept on stirring for 1 hour.The resulting solution was allowed to cool to room temperature and filtered using gravity filtration.The green extract was stored at -4 °C for further use.

C. Green synthesis of iron particles
Coffee FeNPs (GC-FeNPs) were synthesized, as shown in Figure 3.The coffee leaf extract was added to 0.10 M FeSO4 solution with a volume ratio of 1:1 at room temperature and continuously stirred at 75 °C for 1 hr.The immediate colour change to black colour indicated the formation of GC-FeNPs.As synthesized GC-FeNPs were separated using gravity filtration.The product was washed three times with ethanol to remove the remaining residues of the coffee leaf extract and dried using a vacuum oven at 25 °C for 24 hrs.

D. Characterization
The synthesized nanomaterials were characterized using UV-Vis, FT-IR, SEM, and XRD techniques.

Fourier transform infrared spectroscopy (FT-IR)
Fourier Transform Infrared Spectroscopy (FT-IR) analysis of GC-FeNPs was done over the range of 4000 -400 cm -1 to investigate the fabrication of GC-FeNPs by the coffee leaf extract.The measurements were performed on a FT-IR spectrometer (Bruker Vertex80 FT-IR spectrometer, Germany).

Scanning electron microscopy (SEM)
The microstructure and size of the GC-FeNPs were characterized using scanning electron microscopy (Carl Zeiss Evo 18 Research, Germany).SEM images of the sample were obtained at different magnifications using an operating voltage of 20 kV.

X-ray diffraction (XRD)
The crystallinity state of GC-FeNPs was analyzed by X-ray diffraction (Rigaku SmartLab X-Ray Powder Diffractometer, Japan) using with Cu-Kβ radiation source at room temperature.It was operated at 40Kv/30mA over 2θ range of 5 to 80⁰.The scanning speed was maintained at 10 min -1 .

E. Degradation of malachite green -Batch experiment
The removal efficiency of MG was evaluated using GC-FeNPs; 100 μL suspension of GC-FeNPs was (~20 ± 1 mg) added to a solution containing 100 ppm MG (25.0 mL).Then, the conical flasks were placed in a rotary shaker at 298 K and 150 r/min.After a decided specific time, the degraded solutions were taken out and filtered through Whatman No. one filter paper to remove the GC-FeNPs.The resulting solution was analyzed to determine the remaining concentration of MG.A calibration plot of absorbance vs concentration for the MG was prepared using a standard series to evaluate the MG concentration of the degraded solution.According to the UV-Vis spectroscopy the maximum wavelength (ℷmax) for the MG was identified as 617 nm.Hence, the absorbance of the degraded MG solution was measured using a UV-Spectrophotometer at 617 nm.The removal efficiency ( ) and the amount of absorbed dye per unit mass of sorbent at a given time (qt, mg/g) and equilibrium (qe, mg/g) using GC-FeNPs were calculated by using the following equations (Wang and Li, 2013;Katata-Seru et al., 2018;Gao et al., 2019): (1) (2) (3) Where = the MG removal efficiency, = the initial MG concentration in the solution (ppm), = the MG concentration at a time (ppm), and = the MG concentration at the equilibrium (ppm).
All experiments were undertaken in triplicate, and the error values are not very significant.

F. Antibacterial activity studies
The antibacterial activities of green synthesized GC-FeNPs and antibiotic compound streptomycin were evaluated against gram-negative Escherichia coli (E. coli ATCC 25922), Salmonella enterica ATCC 14028, and gram-positive Staphylococcus aureus ATCC 25923 by disk diffusion method using Muller Hinton agar (MHA) medium (Prakash et al., 2013;Elango and Roopan, 2015).Briefly, microbes were grown on nutrient agar at 37 °C for 24 hrs.Afterward, fresh nutrient agar was inoculated with the overnight culture and incubated until the optical density at 600 nm (OD600) reached 0.5.Then, the bacterial suspensions were spread on the MHA plates using a sterile spreader.Sterile Whatman No. one paper discs at 6 mm dimension were impregnated with coffee leaf extract and GC-FeNPs.The disc with streptomycin antibiotic compound was used as the positive control reference.These discs were gently pressed in MHA plates and incubated in an inverted position at 37 °C for 24 hrs to determine the Zone of inhibition.All experiments were undertaken in duplicate.

A. FT-IR analysis Figure 4: FT-IR spectra of the GC-FeNPs
The FT-IR analysis identified different functional groups in the synthesized GC-FeNPs (Figure 4).FT-IR spectra showed eight significant peaks in the 400-1700 cm −1 range.Several studies have reported that different functional groups were responsible for green GC-FeNPs synthesis (Shahwan et al., 2011;Akhbari et al., 2019).
According to the FT-IR spectra, stretching vibrational bands at 2981 cm -1 , 1068 cm -1 , and 3129.54cm -1 , correspond to stretching vibrations of C-H, C-O-C, and OH bonds (Lopez et al., 2010;Wang et al., 2014;T. et al., 2020;Xiao et al., 2020;Parthipan et al., 2021).The peaks at 1616 cm -1 and 1387 cm -1 attribute for C-N of aromatic amines (Lopez et al., 2010;Wang et al., 2014;T. et al., 2020;Xiao et al., 2020;Parthipan et al., 2021).The absorption band at 1563 cm -1 is related to the conjugated system of benzene (Lopez et al., 2010;Wang et al., 2014;T. et al., 2020;Xiao et al., 2020;Parthipan et al., 2021), which confirms the functionalization of the GC-FeNPs by the polyphenols/caffeine compounds in the coffee leaf extract.Furthermore, peaks at 550.10 cm -1 and 500.83cm -1 correspond to the Fe-O vibrational stretches, which confirms the formation FeNPs using coffee extract as a reducing and capping agent (Lopez et al., 2010;Wang et al., 2014;T. et al., 2020;Xiao et al., 2020;Parthipan et al., 2021).The morphologies and size of GC-FeNPs were determined by SEM, as shown in Figure 5, which indicated the successful synthesis of iron nanoparticles.SEM images of GC-FeNPs revealed that the morphology of the iron particles is in quasi-spherical shaped nanoparticles with a diameter ranging from 80 -100 nm (Smuleac et al., 2011).Further, many GC-FeNPs form irregular clusters consistent with the previously reported green synthesized iron-based nanoparticles (Njagi et al., 2011;Shahwan et al., 2011).The size distribution of the GC-FeNPs mainly occurs due to the reducing properties associated with natural compounds in the coffee green extract.Also, these compounds act as a capping agent and a stabilizer to avoid the oxidation of the zero-valent iron particles (α-Fe⁰) when exposed to the air.(Wang et al., 2014)

C. XRD analysis Figure 6: XRD pattern of the GC-FeNPs
The X-ray diffractometer analysis was explored to identify the crystalline nature of the synthesized GC-FeNPs (Figure 6).The XRD pattern shows that the GC-FeNPs are an amorphous structure.The α-Fe 0 surface has been encapsulated by coffee leaf extract polyphenols/caffeine, which function as a dispersive and capping agent in the synthesis process, as confirmed by the FT-IR analysis of the GC-FeNPs.The broad shoulder peak at 21.43 0 is determined to be the absorption peak of polyphenols/caffeine in the coffee extract (Njagi et al., 2011;Cao et al., 2016).The broad peak around 45⁰ on the GnZVI corresponds to the α-Fe 0 (Huang et al., 2014a;Wang et al., 2014;Dutta et al., 2016;Katata-Seru et al., 2018;Xiao et al., 2020).

Degradation of Malachite Green (MG)
Figure 7(a) illustrates the degradation of the MG using the GC-FeNPs at different periods.The characteristic absorption peak of MG, which is attributed to the -C=C-functional group, is located at 617 nm.(Wang et al., 2017;Xiao et al., 2020) As decolorization occurs, the peak at 617 nm decreases with time due to the reactivity of GC-FeNPs.This signifies the remarkable potential of GC-FeNPs in degrading MG from the aqueous system by cleaving the -C=C-bond of the MG.(Wang et al., 2017;Xiao et al., 2020) According to Figure 7 (a), the initial concentration of the MG 100 ppm was reduced to 64.9 ppm after 20 min of incubation with the GC-FeNPs.Then, the MG concentration gradually decreased, and the MG concentration at equilibrium was 45 ppm.According to Figure 7

Kinetic Studies
The adsorption kinetics of the MG onto adsorbent (GC-FeNPs) were investigated using pseudo-firstorder and pseudo-second-order equations.These models are highly dependent on adsorbent material's physical and chemical characteristics.The pseudo-first-order is more suitable for lower concentrations of the solution, and the pseudofirst-order rate equation can be expressed as follows (Wang et al., 2014;Gao et al., 2019): Where and (mg/g) are the amounts of MG molecules adsorbed on the GC-FeNPs at equilibrium and at different times t (min -1 ) and is the rate constant of the pseudo-first-order model for the adsorption process (min -1 ).The linear plot of ln against time, as shown in Figure 8(a), was used to calculate the rate constant k1.The slope of the linear plot gives the for the k1.
The pseudo-second-order kinetic model equation is expressed as follows (Bhattacharyya and Gupta, 2008;Gao et al., 2019): (5) Integrating Eq. ( 5) by applying the boundary conditions t = 0 to t and = 0 to t, gives: (6) When Eq. ( 6) linearized, it expressed as follows: (7) Where, and (mg/g) are the amounts of MG molecules adsorbed on the GC-FeNPs at equilibrium and at different times t (min -1 ) and (g mg -1 min -1 ) is the rate constant of the pseudosecond-order model for the adsorption process.
Values of the and can be determined from the plot of t/qt against t, as shown in Figure 8(b).The correlation coefficients (R 2 ) show that MG adsorption onto the GC-FeNPs was better fitted for the pseudo-second-order (R 2 = 0.9922) compared to the pseudo-first-order model, according to Table 1.Therefore, the adsorption of MG onto GC-FeNPs did not follow the pseudo-first-order model but well-fitted the pseudo-second-order model.
However, the coffee leaf extract shows no antibacterial activity against the bacterial strains selected in this study.

CONCLUSION
In this study, the green synthesis of FeNPs was carried out with Coffea arabica leaf extract, and FT-IR, XRD, and SEM analysis confirmed the formation of GC-FeNPs.The polyphenols/caffeine in the coffee leaf extract acted as a reducing and capping agent that reduced the aggregation of the GC-FeNPs.The FT-IR analysis confirms the functionalization of GC-FeNPs by the polyphenolic/caffeine compounds in the coffee leaf extract.Also, XRD data confirm the availability of α-Fe⁰ and encapsulation of GC-FeNPs by the polyphenolic/caffeine.At the same time, the SEM analysis shows that the synthesized GC-FeNPs have a quasi-spherical shape morphology with a particle size of about 80 -100 nm.The green synthesized GC-FeNPs can be utilized in environmental applications such as the toxic dye MG degradation process.The experimental data confirmed that the GC-FeNPs could effectively degrade the 100 ppm MG in an aqueous solution by using a 100 μL suspension (~ 20 ± 1 mg) of GC-FeNPs compared to the previously reported MG degradation using nanomaterials synthesized using different plant extracts.The MG degradation efficiency by GC-FeNPs reached 45 ppm (55 % degradation efficiency) with an equilibrium around 120 minutes.The kinetic study of the MG degradation revealed that the degradation of MG by GC-FeNPs is well-fitted with the pseudo-second-order adsorption model (R 2 = 0.9922).In addition, the GC-FeNPs showed potential antibacterial activity against E. coli and S. aureus compared to Salmonella.The antibacterial activity of the GC-FeNPs increased with higher concentration, and the growth of E. coli strain was highly inhibited by the GC-FeNPs.Therefore, GC-FeNPs can be explored as a promising nanomaterial for treating contaminated water sources, particularly for removing toxic dyes.

Figure 1 :
Figure 1: Chemical structure of the malachite green

Figure 2 :
Figure 2: Preparation of the coffee extract

Figure 5 :
Figure 5: SEM images of the GC-FeNPs (a) 15 KX and (b) 25 KX magnification Figure7(a) illustrates the degradation of the MG using the GC-FeNPs at different periods.The characteristic absorption peak of MG, which is attributed to the -C=C-functional group, is located at 617 nm.(Wang et al., 2017;Xiao et al., 2020) As decolorization occurs, the peak at 617 nm decreases with time due to the reactivity of GC-FeNPs.This signifies the remarkable potential of GC-FeNPs in degrading MG from the aqueous system by cleaving the -C=C-bond of the MG.(Wang et al., 2017;Xiao et al., 2020) According to Figure7(a), the initial concentration of the MG 100 ppm was reduced to 64.9 ppm after 20 min of incubation with the GC-FeNPs.Then, the MG concentration gradually decreased, and the MG concentration at equilibrium was 45 ppm.According to Figure7(b), degradation efficiency reached 51 % at 40 min, and degradation efficiency reached 55 % at 120 min.These data indicate valuable insights for the development of more effective and sustainable methods for the removal of MG from aqueous systems.

Figure 7 :
Figure 7: (a) Variation of the MG concentration with time upon the GC-FeNPs incubation, (b) MG degradation efficiency with time upon the GC-FeNPs incubation

Figure 9 :
Figure 9: Zone of inhibition (a) E. coli, (b) S. aureus and (c) Salmonella The inhibitory activity of the synthesized GC-FeNPs (10 μL and 20 μL) with varying concentrations against the tested isolates was tested.The Pathogens' Zone of inhibition (DIZ)values ranged from 0.6 to 1.1 cm, as indicated in Figure9.This study used various control groups such as coffee leaf extract, positive control (streptomycin), and distilled water, to inhibit pathogens.