Comparative antibacterial activities of Garcinia cowa and Piper sarmentosum extracts against Staphylococcus aureus and Escherichia coli with studying on disc diffusion assay, material characterizations, and batch experiments

Abstract

Using extracted plants is an attractive option because they consist of good chemical properties against different types of bacteria, and the idea of a form modification in beads may conveniently apply to the disinfection of wastewater systems. Garcinia cowa and Piper sarmentosum leaves were extracted for the synthesis of bead materials and studied material characterizations. A disc diffusion assay, batch experiments, adsorption isotherms, adsorption kinetics, and desorption experiments were studied for investigating the bacteria removal efficiencies of materials. G. cowa and P. sarmentosum leaves were prepared in powder (GCP and PSP), ethanol extracted (EGC and EPS), and synthesized bead materials (GCB and PSB). GCB had a higher surface area than PSB whereas the particle size and pore size were smaller than PSB. GCP and PSP had heterogeneous cracking surfaces whereas GCB and PSB had sphere-shaped and rough surfaces. Carbon (C), oxygen (O), calcium (Ca), and functional groups of O–H, C–H, N–H, C–O, and C–Cl were found in GCP, PSP, GCB, and PSB. Both extracted and beaded materials demonstrated high antibacterial activities on Staphylococcus aureus and Escherichia coli by a disc diffusion assay, and GCB demonstrated high bacteria removals on both bacteria types by almost 100% by batch experiments. Freundlich isotherm and a pseudo-second-order kinetic model are good fit models for explaining the adsorption pattern and mechanism of GCB related to a physiochemical adsorption and chemisorption mechanism. Moreover, GCB could reuse more than 3 cycles, so it is possible to use GCB for disinfection in a wastewater treatment system.

Graphical abstract

Highlights

• •G. cowa and P. sarmentosum leaves were used for material synthesis.
• •Disc diffusion assay confirmed all materials could inhibit S. aureus and E. coli.
• •Batch tests confirmed the high efficiency of G. cowa beads.
• •G. cowa beads corresponded to Freundlich and pseudo-second-order kinetic models.
• •G. cowa beads are potential materials to disinfect in a wastewater treatment system.

Plant extraction; Gram-positive bacteria; Gram-negative bacteria; Disinfection; Wastewater treatment.

1. Introduction

The release of wastewater with bacteria contaminations from industrial, agricultural, and community activities into water resources may cause many human diseases such as typhoid fever, diarrhea, and septicemia from water consumption [1, 2, 3]. Especially, Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) represented as Gram-positive and Gram-negative bacteria are concerned a case of creating many human diseases above [4, 5], and E. coli is part of fecal coliform bacteria which is one of the indicators of water quality assessment [6]. Therefore, contaminated water by bacteria should be recommended to treat below water quality standards before use.

The commonly disinfectant methods in wastewater systems are ozone, ultraviolet light (UV), and chlorine [7]. Among these methods, chlorination is popularly used because of its cost-effective and easy operation; however, this method also leaves unwanted odor and residual chlorine which may affect organisms in the water. Therefore, many studies have investigated alternative choices instead of the use of chlorine. Various metal oxides of zinc oxide (ZnO), titanium dioxide (TiO₂), copper (II) oxide (CuO), and magnesium oxide (MgO) including silver nanoparticles [8, 9, 10, 11, 12] were obtained much attention with high antibacterial efficiencies in several Gram-positive and Gram-negative bacteria; however, positive and negative effects of uses of these materials should be weighted. Metal oxides may accidently release effluents that may affect the environment, so the applications of these metal oxides need careful use for a safe purpose. As a result, another option of uses of extracted plants against bacteria is an appropriate idea to deal with bacteria removal in water after wastewater treatment.

Mentha cordifolia, Sechium edule Sm, Piper betle, Limnophila aromatic, Oscimum sanctum, Pithecellobium dulce, Cratoxylum formosum, Polygonum odoratum, Garcinia cowa, and Piper sarmentosum are attractive plants because of their proper chemical properties such as rutin, catechin, hydroxychavicol, and vitexin [13, 14, 15, 16]. As a result, they may inhibit many types of bacteria such as Staphylococcus epidermidis, Bacillus cereus, Klebsiella pneumonia, Pseudomonas aeruginosa, Acetobacter aceti, Enterobacter aerogenes, S. aureus, and E. coli [17, 18, 19, 20, 21]. Since Garcinia cowa (G. cowa) and Piper sarmentosum (P. sarmentosum) consists of phenols, flavonoids, alkaloids, xanthones, vitexin, terpenoids, and tannin [20, 22, 23, 24, 25], they were appropriate selected for examining the abilities to inhibit S. aureus and E. coli. Therefore, the current study attempted to synthesize bead materials of the extracted G. cowa and P. sarmentosum for investigating their antibacterial against S. aureus and E. coli by batch tests and to investigate the feasibility of application in the wastewater treatment system.

The current research aimed to extract G. cowa and P. sarmentosum with ethanol and synthesize G. cowa beads (GCB) and P. sarmentosum beads (PSB) for the antibacterial studies against S. aureus and E. coli. Their characterizations on particle size, size of surface area, pore size, pore volume, surface morphology, chemical element, and the functional group were identified by particle size analysis (PSA), Brunauer-Emmett-Teller (BET), Field Emission Scanning Electron Microscopy and Focus Ion Beam (FESEM-FIB), Energy Dispersive X-Ray Spectrometer (EDX), and Fourier Transform Infrared Spectroscopy (FTIR), respectively. Antibacterial efficiencies were investigated by a disc diffusion assay and batch experiments. Furthermore, the adsorption pattern and mechanism of the material were investigated to understand which models good explained by studying adsorption isotherms and kinetics. Finally, the desorption experiments explored whether the material could reuse.

2. Materials and methods

2.1. Raw materials

Raw materials were leaves of Garcinia cowa (G. cowa) in Clusiaceae and Piper sarmentosum (P. sarmentosum) in Piperaceae which were bought at a local market in Khon Kaen province, Thailand.

2.2. Chemicals

All chemical reagents were analytical grades (AR) and used without purification. The culture media were nutrient agar (NA) and nutrient broth (NB) (HiMedia Laboratories, India). 99.9% Ethanol (C₂H₅OH) (RCI Labscan, Thailand) was used as a solvent for plant extraction. 99.5% Dimethyl sulfoxide (C₂H₆OS) or DMSO (SDFCL, India), streptomycin (Sigma-Aldrich, Germany), and gentamicin (Sigma-Aldrich, Germany) were used for a disc diffusion assay. For the bead formation, sodium alginate (Na-alginate) and calcium chloride (CaCl₂) (KEMAUS, Australia) were used. 0.65% Nitric acid (HNO₃) (Merck, Germany) and 1% sodium hydroxide (NaOH) (RCI Labscan, Thailand) were used for pH adjustments.

2.3. Microorganism

Staphylococcus aureus (DMST 562) and Escherichia coli (DMST 4212) were used as microorganisms and DMST represents the Department of Medical Sciences, Ministry of Public Health, Bangkok, Thailand.

2.4. Bacteria water sample preparation

The dilute concentrations in the range of 10⁴–10⁷ CFU/mL from 10⁸ CFU/mL of S. aureus and E. coli by sterile deionized water were used for preparing water samples.

2.5. Material synthesis

The material synthesis based on a method by P. Ngamsurach and P. Praipipat [26], and a flow chart of synthesis methods of six materials (GCP, PSP, EGC, EPS, GCB, and PSB) was demonstrated in Figure 1(a) and (b).

Figure 1.

A flow chart of synthesis methods of six materials (a) GCP, EGC, and GCB (b) PSP, EPS, and PSB.

Firstly, G. cowa powder (GCP) and P. sarmentosum powder (PSP) were prepared by washing G. cowa and P. sarmentosum leaves with tap water, drying them in a hot air oven (Binder, FED 53, Germany) at 50 °C for 12 h, grounding to powder by the blender, and then sieving size of 125 μm. Next, the extracted G. cowa (EGC) and P. sarmentosum (EPS) were obtained from the mixing of 10 g of GCP or PSP with 100 mL of ethanol in a 250 mL Erlenmeyer flask by an orbital shaker (GFL, 3020, Germany) of 200 rpm for 24 h at ambient temperature. Next, the samples were filtered by a vacuum pump, and then they were evaporated by a rotary evaporator (BUCHI, RE-111 Rotavapor, Switzerland) at 50 °C. After that, they were freeze-dried (LaboGene, Scanvac, Denmark) and kept at 4 °C. For the synthesis of G. cowa beads (GCB) and P. sarmentosum beads (PSB), EGC or EPS was added into 2% w/v Na-alginate solution, and they were homogenously mixed by a hot plate (Ingenieurbüro CAT, M. Zipperer GmbH, M 6, Germany) of 60 °C with constant stirring of 250 rpm for 30 min. Then, they were added into a 50 mL glass syringe of 3 mm diameter, dropped by drop into 0.1 M CaCl₂ solution, and soaked in the CaCl₂ solution for 24 h. After that, bead materials were filtrated and rinsed with deionized water (DI). Then, they were air-dried and kept in a desiccator before use.

Note that: The concentration ratio for preparing plant extract is indicated by how many grams of EGC or EPS is added to 2% w/v Na-alginate. For preparing of 200 mg/mL concentration, 0.2 g of EGC or EPS was added to 1 mL of 2% w/v Na-alginate [26].

2.6. Characterization of materials

Materials in powder and beads were characterized to identify the particle size, size of surface area, pore volume, pore size, surface morphology, chemical composition, and chemical functional group. For the particle size analysis (PSA), a Zetasizer Nano (Malvern, Zetasizer Nano ZS, UK) was used which 0.1 g of sample was added to 30 mL of polyethylene bottle containing 10 mL DI water and sonicated at ambient temperature. Then, they were transferred to a cuvette for a particle size analysis. Brunauer-Emmett-Teller (BET) (Bel, Bel Sorp mini II, Japan) by isothermal nitrogen gas (N₂) adsorption-desorption at 77.3 K and degas temperature of 80 °C for 24 h was used to analyze the size of surface area, pore volume, and pore size. For the investigations of surface morphology and chemical composition, Field Emission Scanning Electron Microscopy and Focus Ion Beam (FESEM-FIB) with Energy Dispersive X-Ray Spectrometer (EDX) (FEI, Helios NanoLab G3 CX, USA) was used which the samples were mounted on aluminum stubs by double-side carbon tapes and coated by gold-coater for 4 min by a 108 auto Sputter Coater with thickness controller MTM-20 model (Cressington, Ted Pella Inc, USA) with analyzing at a 10 kV accelerating voltage. Fourier Transform Infrared Spectroscopy (FTIR) (Bruker, TENSOR27, Hong Kong) in a range of 4000–600 cm⁻¹ with a resolution of 4 cm⁻¹ and 16 scans over the entire covered range was used to identify chemical functional group [26].

2.7. Disc diffusion assay

The method of disc diffusion assay is based on the study of Ngamsurach, P and Praipipat, P [27]. Paper disc, GCB, and PSB were used for a disc diffusion assay shown in Figure 2 including three steps, and the details were explained below. For GCB and PSB, step 2 was skipped.

Step 1

Preparing of bacteria concentration, a 0.5 McFarland standard was used for preparing of bacteria concentration of 10⁸ CFU/mL of S. aureus and E. coli.

Step 2

Preparing of test solution, 10% dimethyl sulfoxide (DMSO) was used as a solvent. EGC or EPS was added to the solvent in concentrations of 100–400 mg/mL.

Step 3

The antibacterial tests, paper disc, GCB, and PSB were used, and the details are explained below:

Step 3.1

Paper disc

For each plate test, a three-dimension swab technique was used for applying bacteria into nutrient agar and putting four pieces of paper disc soaked the test solution in step 2 in a plate test.

Step 3.2

GCB and PSB

For each plate test, a three-dimension swab technique was used for applying bacteria into nutrient agar and putting four pieces of GCB or PSB in a plate test.

After that, the plates were taken in an incubation oven (Termaks, KBP 6087, Norway) at 37 °C for 24 h. The results were reported by measuring the inhibition zone. The average diameters of the inhibition zones (mm) (mean ± SD) were reported, and the results were confirmed by triplicate experiments. For the controls, paper discs soaked with 30 μg/mL of streptomycin and gentamicin were used as positive controls of S. aureus and E. coli, respectively whereas paper discs soaked with 50 μL of 10% DMSO were used as negative controls of S. aureus and E. coli. Finally, the bead material with higher antibacterial activity was selected for investigating batch experiments [26].

Figure 2.

A disc diffusion assay of the paper disc (EGC and EPS) and bead materials (GCB and PSB) on S. aureus and E. coli.

2.8. Batch experiments

The bead materials (GCB or PSB) which demonstrated high antibacterial activity in a disc diffusion assay were used for batch tests, adsorption isotherms, adsorption kinetics, and desorption experiments.

For batch tests, the differences in dosage, contact time, pH, and concentration of GCB or PSB were investigated for bacteria removal efficiencies on S. aureus and E. coli, and the details were clearly explained below:

2.8.1. Effect of dose

The effect of dose was investigated which dose displayed the highest bacteria removal efficiency of GCB or PSB on S. aureus and E. coli, and 0.1, 0.2, 0.3, and 0.4 g of GCB or PSB were used to explore the optimum dose. A sample volume of 100 mL, a bacteria concentration of 10⁶ CFU/mL, a shaking speed of 200 rpm, a contact time of 6 h, and pH 7 were applied as the control condition.

2.8.2. Effect of contact time

The optimum dose of GCB or PSB from 2.8.1 was used to investigate the bacteria removal efficiency of GCB or PSB for the effect of contact time which 1, 2, 3, 4, 5, 6, 7, and 8 h were used to find the optimum contact time of GCB or PSB. A sample volume of 100 mL, a bacteria concentration of 10⁶ CFU/mL, a shaking speed of 200 rpm, and pH 7 were applied as the control condition.

2.8.3. Effect of pH

The optimum dose and contact time of GCB or PSB from 2.8.1 and 2.8.2 were used to investigate the bacteria removal efficiency of GCB or PSB for the effect of pH which pHs of 5, 7 and 9 were used to investigate the optimum pH of GCB or PSB. A sample volume of 100 mL, a bacteria concentration of 10⁶ CFU/mL, and a shaking speed of 200 rpm were applied as the control condition.

2.8.4. Effect of concentration

The optimum dose, contact time, and pH of GCB or PSB from 2.8.1, 2.8.2, and 2.8.3 were examined for the bacteria removal efficiency of GCB or PSB for the effect of concentration which bacteria concentrations of 10⁴, 10⁵, 10⁶, and 10⁷ CFU/mL were used to study the optimum concentration of GCB or PSB. The control condition was a sample volume of 100 mL and a shaking speed of 200 rpm.

The optimum condition was selected from the highest bacteria removal efficiency of the lowest value of each factor. Triplicate experiments were conducted and reported the average values. The plate count technique was used for data analysis, and the percentage of bacteria removal efficiency is calculated following Eq. (1):

Bacteria removal efficiency (%) = ((C₀ − C_e)/C₀) × 100 (1)

where C_e is the final bacteria concentration (CFU/mL), and C₀ is the initial bacteria concentration (CFU/mL).

2.9. Adsorption isotherms

The interaction of bacteria in the solution with GCB or PSB can be described by using linear and nonlinear adsorption isotherms of Langmuir, Freundlich, and Temkin following to equations in Table 1.

Table 1.

The equations of linear and nonlinear adsorption isotherms and kinetics.

	Linear	Nonlinear	Reference
Adsorption isotherms
Langmuir isotherm	Ce/qe= 1/qmKL+ Ce/qm	qe= qmKLCe/1 + KLCe	[29]
Freundlich isotherm	log qe= log KF+ 1/n log Ce	qe= KFCe1/n	[30]
Temkin isotherm	qe= RT/bT ln AT + RT/bT ln Ce	qe= RT/bT ln ATCe	[31]
Adsorption kinetics
Pseudo-first-order kinetic model	ln(qe − qt) = ln qe – k1t	qt= qe (1 − e−k1t)	[32]
Pseudo-second-order kinetic model	t/qt= 1/k2qe2+ t/qe	qt= k2qe2t/(1 + qek2t)	[33]
Intra-particle diffusion model	qt = kit0.5 + Ci		[34]

where q_e is the adsorption capacity of bacteria on GCB or PSB at equilibrium (CFU/g), and q_m is the maximum number of adsorbed bacteria on GCB or PSB (CFU/g). C_e is the final bacteria concentration (CFU/mL), and K_L is the Langmuir adsorption constant (L/CFU). K_F is Freundlich adsorption constant (CFU/g) (L/CFU)^1/n, and n is the constant depicting the adsorption intensity [26]. R is the universal gas constant (8.314 J/mol K), and T is the absolute temperature (K). b_T is the constant related to the heat of adsorption (J/mol), and A_T is the equilibrium binding constant corresponding to maximum binding energy (L/CFU) [28]. Graphs of Langmuir, Freundlich, and Temkin isotherms were plotted by linear plotting graphs of C_e/q_e versus C_e, log q_e versus log C_e, and q_e versus ln C_e, respectively whereas their nonlinear plotting graphs were plotted by q_e versus C_e.

For isotherm tests, the optimum dose of GCB or PSB was applied with various concentrations of S. aureus and E. coli from 10⁴ to 10⁷ CFU/mL. Moreover, a sample volume of 100 mL, a contact time of 6 h, pH 7, a temperature of 25 °C, and a shaking speed of 200 rpm were applied as the control condition [26].

2.10. Adsorption kinetics

The adsorption kinetic is used to determine the rate of adsorption to time or explain the adsorption mechanism of the adsorbent. Linear and nonlinear of a pseudo-first-order kinetic model, pseudo-second-order kinetic model, and intra-particle diffusion model were investigated characteristic constants of sorption following to equations in Table 1.

where q_e and q_t (CFU/g) are the adsorption capacities of bacteria on GCB or PSB at equilibrium and at the time (t), respectively. k₁ (min⁻¹), k₂ (g/(CFU·min)), and k_i ((CFU/(g·min^0.5)) are the reaction of rate constants of pseudo-first-order kinetic model, pseudo-second-order kinetic model [26], and intra-particle diffusion model, respectively. C_i is the constant value that gives an idea about the thickness of the boundary layer (mg/g) [26]. Graphs of the linear pseudo-first-order kinetic model, pseudo-second-order kinetic model, and intra-particle diffusion model were plotted by ln (q_e-q_t) versus time (t), t/q_t versus time (t), and q_t versus time (t^0.5) at the difference of initial bacteria concentrations, respectively. Finally, their nonlinear plotting graphs were plotted by q_t versus time (t).

For kinetic tests, the optimum dose of GCB or PSB was used with the control condition of S. aureus and E. coli concentration of 10⁶ CFU/mL, a sample volume of 1000 mL, a contact time of 8 h, pH 7, a temperature of 25 °C, and a shaking speed of 200 rpm [26].

2.11. Desorption experiment

Desorption experiments of three adsorption-desorption cycles based on a method of P. Ngamsurach and P. Praipipat [26, 27] were designed to investigate the possible reusability of GCB or PSB. Used GCB or PSB was added to a 250 mL Erlenmeyer flask containing 100 mL of 0.01 M HNO₃ solutions, and then they were shaken at 200 rpm for 2 h. Then, they were washed with DI water, air-dried at ambient temperature, and ready for next cycle use. Eq. (2) is used for calculating the percentage of desorption efficiency.

Desorption (%) = (q_d/q_a) × 100 (2)

where q_d is the number of bacteria desorbed (CFU/mL) and q_a is the number of bacteria adsorbed (CFU/mL).

3. Results and discussion

3.1. The physical characteristic of raw materials

The physical characteristics of G. cowa and P. sarmentosum in leaf, powder, and beads were illustrated in Figure 3(a, b, c, d, e, f). For G. cowa, a leaf is a simple, opposite, oblong-shape, acute or obtuse-apex shape, acute, cuneate or attenuate-base shape, and entire-leaf margin. Normally, G. cowa leaves are deep-green color whereas young leaves are light-green or reddish-purple green color [35] illustrated in Figure 3(a). G. cowa powder (GCP) was brown color shown in Figure 3(b) while G. cowa beads (GCB) were black color beads demonstrated in Figure 3(c). For P. sarmentosum, a leaf is a simple, alternate, heart-shaped, acuminate-apex shape, cordate-base shape, and entire-leaf margin. Leaves have a waxy surface and light to dark green color [35, 36] presented in Figure 3(d). P. sarmentosum powder (PSP) was olive-green color displayed in Figure 3(e) while P. sarmentosum beads (PSB) were black color beads shown in Figure 3(f).

Figure 3.

The physical characteristics of G. cowa and P. sarmentosum in (a, d) leaf (b, e) powder (c, f) beads, respectively.

3.2. Characterizations of antibacterial materials

3.2.1. Physicochemical properties of GCB and PSB by PSA and BET analysis

The physicochemical properties of GCB and PSB by PSA and BET analysis were reported in Table 2. For the particle size analysis (PSA), the diameters of GCB and PSB were 2402 ± 144.7 and 3353 ± 215.2 nm, respectively which PSB demonstrated a higher particle size than GCB. For BET Analysis, sizes of the surface area of GCB and PSB were 0.7129 and 0.6798 m²/g, respectively which GSB presented a higher surface area than PSB. For pore volumes, they were 0.2045 and 0.2331 cm³/g for GCB and PSB, respectively which PSB demonstrated a higher pore volume than GCB. The pore sizes of GCB and PSB were 0.0196 and 0.0233 nm, respectively which PSB represented a higher pore size than GCB corresponded to the results of particle size. Moreover, their pore sizes were classified as microporous size (<2 nm) following the classification of IUPAC [37]. Normally, a material that has a higher surface area and smaller pore size will highly adsorb the pollutant than another [28]. Therefore, since GCB had a higher surface area and smaller pore size than PSB, GCB might better adsorb or remove S. aureus and E. coli than PSB.

Table 2.

The particle sizes, sizes of surface area, pore volumes, and pore sizes of GCB and PSB by PSA and BET analysis.

	GCB	PSB
Diameter (nm) (±SD)	2402 ± 144.7	3353 ± 215.2
PDI	0.726	0.579
Surface area (m2/g)∗	0.7129	0.6798
Pore volume (cm3/g)∗∗	0.2045	0.2331
Pore size (nm)∗∗∗	0.0196	0.0233

^∗MultiPoint BET.

^∗∗Total pore volume for pores with Radius.

^∗∗∗Average pore radius.

3.2.2. FESEM-FIB and EDX analysis

FESEM-FIB with 5,000X magnification was used to investigate the morphology structures of GCP and PSP, and the morphology structures of GCB and PSB were explored through FESEM-FIB with 10,000X magnification. GCP and PSP were heterogeneous cracking or flake surfaces shown in Figure 4(a) and (c) whereas GCB and PSB had uneven and coarse surfaces shown in Figure 4(e) and (h). Moreover, the shapes of GCB and PSB were spherical shape with 65X magnification shown in Figure 4(f) and (i).

Figure 4.

The morphology structures of G. cowa and P. sarmentosum in (a, c) powder (e, h) beads, (f, i) shape, and chemical compositions of G. cowa and P. sarmentosum in (b, d) powder, (g, j) beads.

Figure 4(b, d, g, j) represented the chemical elements of GCP, GCB, PSP, and PSB by EDX analysis. Carbon (C), oxygen (O), and calcium (Ca) were the main chemical elements observed in all materials while chloride (Cl) was detected in all of them except GCP. Potassium (K) and copper (Cu) were found in GCP and PSP while sodium (Na) was observed in GCB and PSB which might be from Na-alginate in a bead formation. In addition, only PSP detected magnesium (Mg). The percentages by weight of each chemical element of GCP, GCB, PSP, and PSB were also demonstrated in Figure 4(b, d, g, j), and their three main compositions ranged from high to low as C > O > Ca. For K, GCB and PSB had 0.6 wt% and 7.6 wt%. For Cu, GCB and PSB had 1.1 wt% and 1.8 wt%. For Na, GCB and PSB had 0.5 wt% and 0.4 wt%. Finally, PSP had 0.7 wt% of Mg. Therefore, the changing material from a powder to a bead form affects the mass percentages of chemical elements by the increase of O, Ca, and Cl and decrease of C. In addition, the use of CaCl₂ in a bead formation might result in the increase of Ca and Cl in bead materials.

3.2.3. FTIR analysis

Figure 5(a, b, c, d) illustrated chemical functional groups of GCP, GCB, PSP, and PSB in a range of 4000−600 cm⁻¹ by FTIR analysis. Five main functional groups of O–H (carboxylic acid), C–H (alkane or aldehyde or methyl or alkene or benzene), N–H (amine I or amide II), C–O (ether and ester), and C–Cl (chloride compound) were realized in all materials whereas C=O (ester) was found in GCP, PSP, and PSB except GCB. Although they shared main functional groups mentioned above, their wavenumbers of each functional group of O–H (3600–3200 cm⁻¹), C–H (3000–2800 or 2900–2700 or 1450–1375 or 830–800 cm⁻¹), C–O (1300–1000 cm⁻¹), N–H (1650–1590 or 1550–1510 cm⁻¹), C–Cl (800–600 cm⁻¹), and C=O (1750–1730 cm⁻¹) were little different values [38].

Figure 5.

FTIR spectrum of G. cowa and P. sarmentosum in (a, c) powder (b, d) beads, respectively.

For GCP, the chemical functional groups observed through O–H (carboxylic acid) at 3286.04 cm⁻¹, C–H (alkane) at 2918.41 cm⁻¹, C–H (aldehyde) at 2856.47 cm⁻¹, C–H (methyl) at 1441.67 cm⁻¹, C–H (benzene) at 821.76 cm⁻¹, C=O (ester) at 1733.12 cm⁻¹, N–H (amine I) at 1620.15 cm⁻¹, C–O (ether and ester) at 1030.30 cm⁻¹, and C–Cl (chloride compound) at 762.81 and 665.72 cm⁻¹ [39] shown in Figure 5(a). For GCB, the chemical functional groups illustrated through O–H (carboxylic acid) at 3320.44 cm⁻¹, C–H (alkane) at 2921.60 cm⁻¹, C–H (aldehyde) at 2854.54 cm⁻¹, C–H (methyl) at 1421.70 cm⁻¹, C–H (alkene) at 987.18 cm⁻¹, N–H (amine I) at 1595.85 cm⁻¹, C–O (ether and ester) at 1116.74 and 1088.06 cm⁻¹, and C–Cl (chloride compound) at 622.26 cm⁻¹ shown in Figure 5(b). For PSP, the chemical functional groups examined through O–H (carboxylic acid) at 3285.36 cm⁻¹, C–H (alkane) at 2918.64 cm⁻¹, C–H (aldehyde) at 2852.61 cm⁻¹, C–H (methyl) at 1442.15 cm⁻¹, C–H (benzene) at 829.98 cm⁻¹, C=O (ester) at 1737.28 cm⁻¹, N–H (amine I) at 1620.60 cm⁻¹, C–O (ether and ester) at 1318.88, 1195.10, 1058.99, and 1025.11 cm⁻¹, and C–Cl (chloride compound) at 782.16 cm⁻¹ [40] shown in Figure 5(c). Finally, the chemical functional groups of PSB determined through O–H (carboxylic acid) at 3344.84 cm⁻¹, C–H (alkane) at 2923.95 cm⁻¹, C–H (aldehyde) at 2896.98 cm⁻¹, C–H (methyl) at 1432.91 cm⁻¹, C–H (benzene) at 819.01 cm⁻¹, C=O (ester) at 1733.60 cm⁻¹, N–H (amine I) at 1602.74 cm⁻¹, N–H (amide II) at 1511.33 cm⁻¹, C–O (ether and ester) at 1023.94 cm⁻¹, and C–Cl (chloride compound) at 740.86 cm⁻¹ shown in Figure 5(d).

3.3. Results of disc diffusion assay

A general method for the antibacterial examination by extracted plants is a disc diffusion assay. This study attempted to investigate how many of the antibacterial efficiencies of the paper disc (EGC and EPS) and bead materials (GCB and PSB) on S. aureus and E. coli were. The disc diffusion assay results are reported in Table 3.

Table 3.

Disc diffusion assay on S. aureus and E. coli by paper disc (EGC and EPS) and bead materials (GCB and PSB).

Bacteria	Positive control	Negative control		The average diameters of the inhibition zones (mm) (mean ± SD)
				Paper disc		Bead materials
				EGC	EPS	GCB	PSB
S. aureus	16.1 ± 0.2	0	100 mg/mL	7.3 ± 0.1	7.0 ± 0.1	3.3 ± 0.5	3.5 ± 0.1
			200 mg/mL	7.5 ± 0.1	7.2 ± 0.1	3.5 ± 0.3	3.6 ± 0.1
			300 mg/mL	8.2 ± 0.3	7.8 ± 0.3	4.6 ± 0.1	3.7 ± 0.3
			400 mg/mL	8.5 ± 0.3	7.9 ± 0.3	5.1 ± 0.8	3.8 ± 0.3
E. coli	15.5 ± 0.3	0	100 mg/mL	7.3 ± 0.3	7.0 ± 0.4	3.0 ± 0.0	3.4 ± 0.3
			200 mg/mL	7.5 ± 0.1	7.1 ± 0.1	3.4 ± 0.1	3.5 ± 0.0
			300 mg/mL	7.6 ± 0.3	7.5 ± 0.5	4.6 ± 0.3	3.6 ± 0.1
			400 mg/mL	7.8 ± 0.3	7.7 ± 0.2	5.0 ± 0.0	3.7 ± 0.1

For controls, the inhibition zones of positive controls were 16.1 ± 0.2 mm for S. aureus and 15.5 ± 0.3 mm for E. coli, respectively whereas their negative controls were 0 mm.

For the paper disc, the inhibition zones in various EGC concentrations from 100-400 mg/mL on S. aureus and E. coli were 7.3 ± 0.1, 7.5 ± 0.1, 8.2 ± 0.3, 8.5 ± 0.3 mm for S. aureus, and 7.3 ± 0.3, 7.5 ± 0.1, 7.6 ± 0.3, 7.8 ± 0.3 mm for E. coli, and the average of four concentrations of EGC on S. aureus and E. coli were 7.9 ± 0.6 and 7.6 ± 0.2 mm, respectively. Moreover, the inhibition zones in various EPS concentrations from 100-400 mg/mL on S. aureus and E. coli were 7.0 ± 0.1, 7.2 ± 0.1, 7.8 ± 0.3, 7.9 ± 0.3 mm for S. aureus, and 7.0 ± 0.4, 7.1 ± 0.1, 7.5 ± 0.5, 7.7 ± 0.2 mm for E. coli, and the average of four concentrations of EGC on S. aureus and E. coli were 7.5 ± 0.4 and 7.3 ± 0.3 mm, respectively. Therefore, the average inhibition zones of EGC and EPS represented a similar trend by the increase of antibacterial efficiency with the increase of EGC and EPS concentration, and EGC presented higher antibacterial activity on S. aureus and E. coli than EPS.

For bead materials, the inhibition zones in various GCB concentrations from 100-400 mg/mL on S. aureus and E. coli were 3.3 ± 0.5, 3.5 ± 0.3, 4.6 ± 0.1, 5.1 ± 0.8 mm for S. aureus, and 3.0 ± 0.0, 3.4 ± 0.1, 4.6 ± 0.3, 5.0 ± 0.0 mm for E. coli, and the average of four concentrations of GCB on S. aureus and E. coli were 4.1 ± 0.9 and 4.0 ± 1.0 mm, respectively. In addition, the inhibition zones in various PSB concentrations from 100-400 mg/mL on S. aureus and E. coli were 3.5 ± 0.1, 3.6 ± 0.1, 3.7 ± 0.3, 3.8 ± 0.3 mm for S. aureus, and 3.4 ± 0.3, 3.5 ± 0.0, 3.6 ± 0.1, 3.7 ± 0.1 mm for E. coli, and the average of four concentrations of GCB on S. aureus and E. coli were 3.7 ± 0.1 and 3.6 ± 0.1 mm, respectively. Therefore, the average inhibition zones on GCB and PSB also represented a similar trend by the increase of antibacterial efficiency with the increase of GCB and PSB concentration, and GCB presented higher antibacterial activity on S. aureus and E. coli than PSB.

For the comparison of disc diffusion assay results by paper disc (EGC and EPS), and bead materials (GCB and PSB), they demonstrated a similar trend of the increase of antibacterial efficiency with the increase of concentration. Because their average inhibition zones were almost 300 mg/mL, it was a suitable concentration to inhibit S. aureus and E. coli which was the same optimum concentration of extract plants found in our previous studies [26]. Furthermore, both EGC and GCB represented a higher antibacterial activity on S. aureus and E. coli than EPS and PSB which were similarly found in other studies [13, 20, 22, 40], so G. cowa could inhibit both bacteria types than P. sarmentosum. Moreover, these results corresponded to the results of PSA and BET analysis that GCB might highly inhibit S. aureus and E. coli than PSB. In our previous study [26], we used different plant types of Cratoxylum formosum and Polygonum odoratum in bead materials (CFB and POB) and found both bead materials could high be against E. coli than S. aureus whereas CFB illustrated a higher inhibition zone than POB. For comparing the highest antibacterial activity by bead materials (CFB and GCB) of both studies, CFB illustrated the higher antibacterial activity than GCB on S. aureus and E. coli which might result from the different main chemical extractions or chemical functional groups of both plants.

The possible mechanisms of the paper disc (EGC and EPS) and bead materials (GCB and PSB) to inhibit S. aureus and E. coli might be from the main substances of extracted G. cowa (phenolic acid, flavonoids, and chamuangone) and P. sarmentosum (phenolic acid, flavonoids, tannic acid, gallic acid, quercetin, naringin, and alkaloids) [41, 42] in paper discs and bead materials penetrated cell wall of bacteria, then it results in malfunction of bacteria cells [26]. Consequently, the cell dies.

However, the average antibacterial efficiency from paper disc (EGC) to GCB tests on S. aureus was decreased to 41.25% and 47.24% for E. coli which might be the material test effect. Two factors which were the size of test materials and Na-alginate in bead materials might result in the higher antibacterial of the paper disc than bead materials. Since the paper disc of 6 mm had a larger diameter than the glass syringe of 3 mm, the extracted plant solutions might be absorbed into the paper disc more than the bead materials similar reported by another study [27]. In addition, the bead formation by sodium alginate might affect the releasing the extracted plant to antibacterial activity. Therefore, batch experiments were designed to confirm the bacteria removal efficiencies of GCB on S. aureus and E. coli in this study.

3.4. Batch experiments

3.4.1. Effect of dose

Figure 6(a) illustrated the effect of GCB dose on bacteria removal efficiencies. Bacteria removal efficiencies of GCB on S. aureus and E. coli were increased from 96.50 to 99.99% and 96.50–100%, respectively with the increase of dosages from 0.1 to 0.4 g. Moreover, bacteria removal efficiencies of GCB on S. aureus and E. coli were constant after 0.3 g, so 0.3 g was the optimum dose of GCB against S. aureus and E. coli for the next experiment of contact time effect.

Figure 6.

The results of batch tests of GCB in (a) dose, (b) contact time, (c) pH, and (d) concentration on S. aureus and E. coli.

3.4.2. Effect of contact time

Figure 6(b) represented the effect of contact time from 1 to 8 h of GCB on bacteria removal efficiencies. For S. aureus, bacteria removal efficiencies of GCB were higher than 97% in all contact times, and they found that bacteria removal efficiencies of GCB were 100% from 6 to 8 h. As a result, 6 h was a suitable contact time because it was the lowest value for 100% bacteria removal efficiency of GCB. For E. coli, bacteria removal efficiencies of GCB were increased from 97.10 to 100% when the contact times were increased from 1 to 8 h. Then, they demonstrated a constant value of 100% after 3h. Thus, 3h was the optimum contact time of GCB on E. coli. Therefore, the conditions of GCB on S. aureus and E. coli were 0.3 g, 6 h and 0.3 g, 3 h, respectively for studying of pH effect.

3.4.3. Effect of pH

The changing of pH conditions might be another affecting factor to bacteria removal efficiencies of GCB on S. aureus and E. coli, so pH values of 5, 7, and 9 are good choices for representing acidic, neutral, and alkaline conditions. Figure 6(c) presented the effect of pH on bacteria removal efficiencies of GCB on S. aureus and E. coli. For S. aureus, the results illustrated all pH values represented almost 100% bacteria removal efficiencies, and they also found a little increase in bacteria removal efficiencies from pH 5 to 9 in the range of 99.82–99.96%. For E. coli, the results demonstrated pH 7 demonstrated 100% bacteria removal efficiencies whereas pHs 5 and 9 represented the same bacteria removal efficiencies of 99.99%. As a result, since they showed little difference in bacteria removal efficiencies in pH values of 5, 7, and 9, the changing of pH values did not affect to bacteria removal efficiencies of GCB on both bacteria types. In the case of safe water quality, pH 7 was a preferred value. Therefore, the conditions of GCB on S. aureus and E. coli were 0.3 g, 6 h, pH 7 and 0.3 g, 3 h, pH 7 for investigating of concentration effect.

3.4.4. Effect of concentration

Figure 6(d) demonstrated the effect of concentration from 10⁴ to 10⁷ CFU/mL of GCB on bacteria removal efficiencies. The result represented high bacteria removal efficiencies of GCB on both bacteria types almost 100 % at concentrations from 10⁴ to 10⁶ CFU/mL, and then they were a little decreased. Therefore, the concentration of 10⁶ CFU/mL was confirmed as the suitable concentration against S. aureus and E. coli.

In summary, all factors of dose, contact time, and concentration affected bacteria removal efficiencies in S. aureus and E. coli whereas pH did not affect GCB efficiencies. Finally, the optimum conditions of GCB were 0.3 g, 6 h, pH 7, 10⁶ CFU/mL for S. aureus and 0.3 g, 3 h, pH 7, 10⁶ CFU/mL for E. coli, and they could be treated almost 100% in 100 mL of contaminated water.

3.5. Isotherm study

Figure 7(a, b, c, d, e, f, g, h) demonstrated the results of linear and nonlinear Langmuir, Freundlich, and Temkin isotherms of GCB on S. aureus and E. coli, and Table 4 also reported their equilibrium isotherm parameters.

Figure 7.

The plotting graphs of (a, b, c, d, e, f) linear and (g, h) nonlinear adsorption isotherms of GCB on S. aureus and E. coli.

Table 4.

The equilibrium isotherm parameters of GCB on S. aureus and E. coli.

	Linear		Nonlinear
	S. aureus	E. coli	S. aureus	E. coli
Langmuir isotherm
qm (×109 CFU/g)	5.00	5.00	5.04	5.04
KL (L/CFU)	0.0002	0.0003	0.0001	0.0002
R2	0.70	0.80	0.94	0.96
Freundlich isotherm
KF (×106 (CFU/g) (L/CFU)1/n)	4.27	6.23	4.77	6.96
1/n	0.66	0.68	0.68	0.67
R2	0.99	0.99	1	1
Temkin isotherm
bT (×10−6 J/mol)	8.26	8.26	8.89	7.76
AT (L/CFU)	0.26	0.37	0.21	0.28
R2	0.59	0.66	0.58	0.66

For linear isotherms, the Langmuir maximum adsorption capacity (q_m) of GCB on S. aureus and E. coli had a similar value of 5.00 × 10⁹ CFU/g which meant GCB had the same inhibition on both bacteria types, and Langmuir adsorption constants (K_L) were 0.0002 and 0.0003 L/CFU, respectively. Freundlich adsorption constants (K_F) GCB on S. aureus and E. coli were 4.27 × 10⁶ and 6.23 × 10⁶ (CFU/g) (L/CFU)^1/n, respectively, and their 1/n values were 0.66 and 0.68, respectively. 1/n < 1 means the increase in concentration results in a decrease in adsorption capacity [26]. For Temkin isotherm, b_T value of GCB on S. aureus and E. coli had a similar value of 8.26 × 10⁻⁶ J/mol, and their A_T values were 0.26 and 0.37 L/CFU, respectively meant GCB had a stronger energy equilibrium on E. coli than S. aureus [28]. Since the higher R² closely to 1 is used to decide the best fit of the isotherm model, Freundlich corresponded to these studies with R² of 0.99 higher than Langmuir and Temkin isotherms in both bacteria types.

For nonlinear isotherms, the Langmuir maximum adsorption capacity (q_m) of GCB on S. aureus and E. coli had a similar value of 5.04 × 10⁹ CFU/g which meant GCB had the same inhibition on both bacteria types, and Langmuir adsorption constants (K_L) were 0.0001 and 0.0002 L/CFU, respectively. Freundlich adsorption constants (K_F) GCB on S. aureus and E. coli were 4.77 × 10⁶ and 6.96 × 10⁶ (CFU/g) (L/CFU)^1/n, respectively, and their 1/n values were 0.68 and 0.67, respectively. 1/n < 1 means the increase in concentration results in a decrease in adsorption capacity [43]. For Temkin isotherm, b_T values of GCB on S. aureus and E. coli were 8.89 × 10⁻⁶ and 7.76 × 10⁻⁶ J/mol, respectively, and their A_T values were 0.21 and 0.28 L/CFU, respectively meant GCB had a stronger energy equilibrium on E. coli than S. aureus [44]. Since the higher R² closely to 1 is used to decide the best fit of the isotherm model, Freundlich corresponded to these studies with R² of 1 higher than Langmuir and Temkin isotherms in both bacteria types.

The plotting graphs of both linear and nonlinear isotherms with various models could guarantee the best fit model of actual experiments without error analysis [[45], [46], [48], [49]], and the above results of linear and nonlinear isotherms were agreed with Freundlich isotherm because of higher R² than others which meant the adsorptions of GCB on S. aureus and E. coli were heterogeneous adsorptions relating to a process of physiochemical adsorption [27].

3.6. Kinetic study

The adsorption kinetic is normally used to describe the rate of adsorption to time or the adsorption mechanism of the adsorbent [47]. Figure 8(a, b, c, d, e, f, g, h) demonstrated the results of the linear and nonlinear pseudo-first-order kinetic model, pseudo-second-order kinetic model, and intra-particle diffusion model of GCB on S. aureus and E. coli. Their adsorption kinetic parameters are reported in Table 5.

Figure 8.

The plotting graphs of (a, b, c, d, e, f) linear and (g, h) nonlinear adsorption kinetic models of GCB on S. aureus and E. coli.

Table 5.

The adsorption kinetic parameters of GCB on S. aureus and E. coli.

	Linear		Nonlinear
	S. aureus	E. coli	S. aureus	E. coli
Pseudo-first-order kinetic model
qe (×106 CFU/g)	38.17	0.26	41.18	0.28
k1 (min−1)	0.02	0.01	0.22	0.10
R2	0.92	0.20	0.92	0.22
Pseudo-second-order kinetic model
qe (×106 CFU/g)	333.33	333.33	332.25	337.09
k2 (×106 g/(CFU·min))	0.002	0.09	0.004	0.10
R2	1	1	1	1
Intra-particle diffusion model
ki (×106 CFU/(g·min0.5))	6.04	5.69	6.98	6.57
Ci (×106 CFU/g)	237.89	245.43	225.59	233.85
R2	0.27	0.24	0.26	0.23

For linear kinetics, the adsorption capacities (q_e) of a pseudo-first-order kinetic model for GCB on S. aureus and E. coli were 38.17 × 10⁶ and 0.26 × 10⁶ CFU/g, respectively which meant GCB had strong inhibition on S. aureus than E. coli, and k₁ were 0.02 and 0.01 min⁻¹, respectively. The adsorption capacities (q_e) of a pseudo-second-order kinetic model for GCB on S. aureus and E. coli had the same value of 333.33 × 10⁶ CFU/g, and their k₂ were 0.002 ×10⁶ and 0.09 × 10⁶ g/(CFU·min), respectively. For the intra-particle diffusion model, k_i of GCB on S. aureus and E. coli were 6.04 ×10⁶ and 5.69 × 10⁶ CFU/(g·min^0.5), respectively, and their C_i values were 237.89 × 10⁶ and 245.43 × 10⁶ CFU/g, respectively. Since the higher R² closely to 1 is used to decide the best fit of the kinetic model, a pseudo-second-order kinetic model corresponded to these studies with R² of 1 higher than other kinetic models in both bacteria types.

For the nonlinear kinetic model, the adsorption capacities of a pseudo-first-order kinetic model (q_e) for GCB on S. aureus and E. coli were 41.18 × 10⁶ and 0.28 × 10⁶ CFU/g, respectively which meant GCB had strong inhibition on S. aureus than E. coli, and k₁ were 0.22 and 0.10 min⁻¹, respectively. The adsorption capacities (q_e) of a pseudo-second-order kinetic model for GCB on S. aureus and E. coli were 332.25 × 10⁶ and 337.09 × 10⁶ CFU/g, respectively, and their k₂ were 0.004 ×10⁶ and 0.10 × 10⁶ g/(CFU·min), respectively. For the intra-particle diffusion model, the k_i value of GCB on S. aureus and E. coli were 6.98 ×10⁶ and 6.57 × 10⁶ CFU/(g·min^0.5), respectively, and their C_i values were 225.59 × 10⁶ and 233.85 × 10⁶ CFU/g, respectively. Since the higher R² closely to 1 is used to decide the best fit of the kinetic model, a pseudo-second-order kinetic model corresponded to these studies with R² of 1 higher than other kinetic models in both bacteria types.

Comparing graphs of linear and nonlinear kinetics is used to confirm data analysis to protect data mistranslation similarly to adsorption isotherm [[45], [46], [48], [49]]. The results of linear and nonlinear kinetics agreed with a pseudo-second-order kinetic model because of higher R² than others which meant the adsorptions of GCB on S. aureus and E. coli were chemical adsorption with physicochemical interactions [27].

3.7. Desorption experiment

In the case of industrial applications, cost-effective, potential, and reuse materials are preferred, so adsorption-desorption experiments are normally required. This study designed the desorption experiment to confirm material reusability by running of adsorption-desorption of GCB on S. aureus and E. coli in 3 cycles which bacteria on GCB were removed by 0.01 M HNO₃ solution. The results of adsorption-desorption of GCB on both bacteria types are demonstrated in Figure 9(a) and (b) which confirmed the high reusability of GCB on S. aureus and E. coli in a range of 89 ± 0.4–100 ± 0.0% for the bacteria adsorption and 86 ± 0.2–100 ± 0.0% for the bacteria desorption, respectively. Therefore, GCB was a high potential material against S. aureus and E. coli for more than 3 cycles, and it is possible to further applications in the wastewater treatment system.

Figure 9.

Adsorption and desorption of GCB on (a) S. aureus and (b) E. coli.

4. Conclusions

G. cowa powder (GCP) and P. sarmentosum powder (PSP) were extracted and used to synthesize antibacterial materials of G. cowa beads (GCB) and P. sarmentosum beads (PSB). The surface area of GCB had higher than PSB whereas particle size and pore size of GCB were smaller than PSB. GCP and PSP were heterogeneous surfaces whereas GCB and PSB had spherical shapes with coarse surfaces. The three main chemical elements of GCP, GCB, PSP, and PSB were carbon (C), oxygen (O), and calcium (Ca). Five main functional groups of O–H, C–H, N–H, C–O, and C–Cl were found in all materials. Results of the paper disc (EGC and EPS) and bead materials (GCB and PSB) confirmed that the extracted G. cowa (EGC) and P. sarmentosum (EPS) had potential inhibitions on S. aureus and E. coli; however, EGC and GCB had higher antibacterial activities against both bacteria types than EPS and PSB. Thus, GCB was chosen to investigate bacteria removal efficiencies on both bacteria types by batch experiments. In batch experiments, the optimum conditions of GCB on S. aureus and E. coli with almost 100% bacteria removals were 0.3 g, 6 h, pH 7, 10⁶ CFU/mL and 0.3 g, 3 h, pH 7, 10⁶ CFU/mL, respectively. Freundlich isotherm and pseudo-second-order kinetic model well explained the adsorption pattern and rate of adsorption to time of GCB on S. aureus and E. coli. Furthermore, GCB could reuse for more than 3 cycles, so it is possible to apply GCB for the disinfection of a wastewater treatment system.

Various bacteria types need to investigate for appropriate application in real wastewater and confirm whether GCB is a potential material to deal with a variety of conditions of bacteria-contaminated wastewater. Moreover, several extract plants are recommended to investigate and compare which one has the high potential to inhibit S. aureus or E. coli.

Declarations

Author contribution statement

Pimploy Ngamsurach: Performed the experiments; Wrote the paper.

Pornsawai Praipipat: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Funding statement

This work was supported by Research and Technology Transfer Affairs of Khon Kaen University, Thailand; The Office of the Higher Education Commission and Thailand Research Fund grant (MRG6080114), Thailand; Coordinating Center for Thai Government Science and Technology Scholarship Students (CSTS) and National Science and Technology Development Agency (NSTDA) Fund grant (SCHNR2016-122), Thailand.

Data availability statement

Data will be made available on request.

Declaration of interests statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.