Preparation and antibacterial properties of an activated carbon sphere–quaternary phosphonium salt composite

Abstract

Synthesis and activation of nanosized colloidal carbon spheres (CS) for adsorption of a quaternary phosphonium salt as a new antibacterial material is reported. CS (400–500 nm in diameter) were synthesized via simple hydrothermal treatment of glucose solution. The surface of nonporous CS after being activated by steam possessed a high surface area (2325 m² g⁻¹). The activated CS (ACS) exhibited a high adsorption capacity for the quaternary phosphonium salt (QPS) and have demonstrated excellent antibacterial properties. The QPS on ACS were more stable than the QPS itself, resulting in long-term antibacterial effects. The excellent adsorption performance and reusability of the steam activated CS reported here could represent a new type of low-cost and efficient adsorbent nanomaterial for antibacterial materials.

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Introduction

Microbial pollution caused by microorganisms is one of the major problems related to human health and life quality.¹ It is an urgent challenge to develop new antibacterial materials to solve this issue. Quaternary phosphonium salt (QPS) is a new generation of efficient, broad-spectrum organic antibacterial agent and has been extensively studied due to a number of advantages such as low foam, strong capability of sludge stripping and wide range of pH values.² However, it is not safe to use a quaternary phosphonium salt directly because it easily loses its antibacterial efficiency in a short time.³ Loading this organic antibacterial QPS into a carrier is one of the best ways to prolong its antibacterial properties.⁴

A number of nanomaterial, such as clay, zirconium phosphate, activated carbon have been used as antibacterial carrier.⁵ Among them, carbon nanomaterials have desirable chemical and physical properties such as high surface areas, excellent biocompatibility, excellent adsorption capacity and have been widely used in drug delivery systems, catalyst carriers and absorbents.⁶ Carbon spheres (CS), as a novel carbon nanomaterial, have broad potential in biomedical applications, including the detection of biomolecules and drug delivery owing to their good biocompatibility, convenience and low cost preparation methods.⁷ It is expected that carbon spheres obtain certain specific surface area (BET) could present promising adsorption performance towards organic antibacterial material. The antimicrobial activities of nanocomposites against micro-organism will improve a lot by this method.

It has been widely reported that activation is an effective way to obtain high surface area of carbon nanomaterial and the adsorption capacities can be enhanced by chemical activation with acids and bases.⁸ However, the use of a large number of strong acids and strong alkali caused the pollution of the environment. Steam activation is a green and novel post-treatment step and used often for preparing activated carbons and other microporous materials with desired pore structures. Activation by this method produces activated carbon spheres with high surface areas and microporous structures.⁹ To the best of our knowledge, there is few literature reporting on the post treatment of carbon spheres by steam activation for adsorption applications.

In this paper, a surfactant of quaternary phosphonium salt (dodecyl tributyl phosphonium bromide) with excellent bactericidal activity has been selected and loaded into activated carbon spheres to obtain phosphonium carbon material (QPS–ACS). The effects of QPS–ACS on the characteristics, microstructure, release and thermal properties, cytotoxicity and long-acting antibacterial activity mechanism have been investigated. The novelty of the present study was that the use of ACS as QPS carrier would make the use of QPS safer, and the specific benefits of this novel nanocomposite included bacteria-adsorbed capability, dose control to achieve desired antibacterial effects, long-acting and specific-targeting capability. These new composites have great potential as antibacterial materials.

Experimental

Reagents

Glucose with analytical grade were purchased from Guangzhou Chemical Reagent Factory and used directly without further purification. Dodecyl tributyl phosphonium bromide (DDTBPBr) of C.R. grade was supplied by Qingte Chemical Industry Co., Ltd. (Shanghai, China). E. coli ATCC25922 and S. aureus ATCC 6538 were supplied by Guangdong Institute of Microbiology (Guangzhou, China). Luria–Bertani (LB) broth and nutrient agar culture medium were obtained from Huankai Microorganism Co., Ltd. (Guangzhou, China). Bovine serum was purchased from Aladdin Reagent Inc. (Shanghai, China). All other reagents and solvents were obtained from commercial suppliers. All aqueous solutions were prepared with ultrapure water (>18 MΩ) from a Milli-Q Plus system (Millipore).

Characterization techniques

The morphological features of the obtained samples were examined using a Philips TECNAI 10 transmission electron microscope (TEM). The Fourier transform infrared spectroscopy (FTIR) spectra were measured by an EQUINOX 55 (Bruker) spectrometer with the KBr pellet technique ranging from 500 to 4000 cm⁻¹. Thermal gravity analysis (TGA) was conducted with a thermal analyzer (NETZSCH TG 209) under air flow; the temperature range of the measurements was 40–900 °C and the scanning rate was 10 °C min⁻¹. The Brunauer, Emett, Teller (BET) equation was used to calculate the surface area. Before measuring specific surface area and pore structure parameters, the samples were out-gassed at 300 °C in a vacuum oven for 3 h.

Synthesis of activated carbon spheres

In a typical procedure, carbon spheres were synthesized as follows: 2 g glucose was dissolved in water (30 mL) to form a clear solution and then the mixture was placed into a Teflon equipped stainless steel autoclave and maintained in a muffle furnace at 180 °C for 12 h. After the reaction, the autoclave was cooled down naturally. The obtained samples were isolated by centrifugation, washed with deionized water and then vacuum dried for further use.

The carbon spheres were subsequently activated via steam activation in a stainless steel tubular furnace in flowing ultrahigh pure N₂, in order to remove all the gases evolved during the pyrolysis process. Then the water steam was added to the hot flowing N₂ stream to generate a (steam/N₂) mixture with a molar ratio of 2 : 1. A programmed temperature profile was adopted to control the heating process. The carbonization protocol involved heating (2 °C min⁻¹) the carbon spheres to 500 °C and holding it at that temperature for 0.5 h. The temperature was then raised (1 °C min⁻¹) to 800 °C, and the carbon spheres were held there for an additional 3–12 h. Subsequently, the samples were cooled down to room temperature, thus obtaining the final activated carbon spheres.

Preparation of activated carbon spheres loaded with quaternary phosphonium salt

Activated carbon spheres were dispersed in aqueous solution, then, as a kind of quaternary phosphonium salt, DDTBPBr was slowly added under continuous stirring. The reaction was allowed to proceed at 70 °C for 3 h. The resulting precipitate was collected by centrifugation with deionized water until it was free from bromonium ions. Finally, the sample was vacuum dried at 65 °C for 24 h to obtain a solid product. The 0.25, 0.5, 1.0 and 1.5 weight times QPS of ACS were designated as QPS–ACS1, QPS–ACS2, QPS–ACS3 and QPS–ACS4, respectively.

Antimicrobial tests

To determine the antimicrobial properties of these samples, all materials were autoclaved at 121 °C for 15 min to ensure sterility before each experiment.

Dilution methods were used to determine the minimum inhibitory concentrations (MICs) of antimicrobial agents ACS and ACS–QPS against E. coli and S. aureus.¹⁰ In a typical procedure, ACS and ACS–QPS were suspended into Mueller-Hinton broth medium to form homogeneous suspensions and then two-fold diluted into different concentrations. Each 1 mL of culture medium containing various concentrations of the test sample was inoculated with 0.1 mL of 10⁶ cfu mL⁻¹ bacterial suspension, cultured at 37 °C with shaking for 24 h, and then the growth of bacteria was observed. The MICs was defined as the lowest concentration of an antimicrobial agent that inhibits the growth of a microorganism was observed.

The optical density (OD₆₀₀) was also used to determine the antibacterial of the samples. E. coli K12 cells were grown in 50 mL liquid LB medium (Luria–Bertani broth) supplemented with 25, 50, 100 and 200 μg mL⁻¹ of QPS, ACS and ACS–QPS, respectively. Growth rates and bacterial concentrations were measured by a spectrophotometer at 600 nm wavelength to obtain the optical density (OD₆₀₀) for every 2 hours. Bacterial broth without any samples was taken as control. High OD₆₀₀ value represents high concentration of bacteria, indicating poor antimicrobial property of the sample. Each experiment was performed in triplicates.

Release of QPS from ACS–QPS

The released amount of adsorbed QPS into phosphate buffered saline (PBS) was examined. The dialysis bags were taken out at various intervals (6, 12, 24, 36, 48, 60 and 72 h) and the concentration of QPS released from ACS–QPS was quantified using an inductive coupled plasma (ICP) emission spectrometer.

Results and discussion

Transmission electron microscopy (TEM) images clearly revealed that the CS were spherical in shape and had a size distribution of 400–500 nm in diameter (Fig. 1a). After post-treatment with water steam, the spheres became transparent and had a smooth surface and the diameter decreased to 350–400 nm (Fig. 1b). The TEM image in Fig. 1c showed that the QPS–ACS became dark and displayed a notable difference in morphology towards ACS. The inner shadow and rough surface revealed some other materials which were found on the ACS and this might be due to the presence of QPS.

Fig. 1 TEM images of CS (a), ACS (b) and QPS–ACS (c).

In the FTIR spectrum of QPS–ACS (Fig. 2), the bands of oxygen functionalities become weak, the new peaks at 2931 and 2857 cm⁻¹ were ascribed to the vibration of methylene groups of the QPS. This suggests that QPS has been loaded to ACS. According to the literature, the QPS molecules stack non-covalently onto the pore space framework of ACS by electrostatic interactions. The zeta-potential measurements of ACS were negative of −3.5 mV, suggesting that the surface of the ACS has the carboxyl groups and providing them excellent dispersivity.

Fig. 2 FTIR spectrum of ACS and QPS–ACS.

The N₂ adsorption isotherm of CS exhibited that a large amount of N₂ was adsorbed remarkably at low relative pressure (<0.1 p/p₀) and further there was very little N₂ adsorption of CS at high relative pressure (Fig. 3). This isotherm could be considered as Type II for multilayer adsorption and have slit pore structure and a H4 hysteresis loop. Whereas N₂ adsorption isotherms of samples ACS showed adsorption significantly take at high relative pressure, indicating change of N₂ adsorption isotherms from Type II to mixture of Type I and IV. These isotherms reflected that ACS have micro- and meso-porous nature. When loaded with quaternary phosphonium salt, the N₂ adsorption isotherms was changed to Type II, confirming the absence of some pores structure and suggested that the porous were filled with QPS. Table 1 summarizes the adsorption properties including S_BET, V_meso, V_micro and D_BJH of the prepared CS, ACS and QPS–ACS, respectively. The results showed that the S_BET values of ACS were much higher than CS and gradually increased from 656 to 2325 m² g⁻¹ with the gasification time increase from 3 to 12 h. The pore volume and BET surface area of QPS–ACS were significantly lower than those of ACS as a result of the adsorption process. From the pore distribution results given in Table 1, changes in the internal structure of QPS–ACS were clearly indicated. It is possible that many of the pores of QPS–ACS have been filled by QPS.

Fig. 3 N₂ adsorption–desorption isotherms at 77 K for CS (a), QPS–ACS (b), ACS (c).

Table 1 The surface area and pore-structure parameters of the samples

Samples	SBET^a (m^2 g^−1)	Smicro^b (m^2 g^−1)	Smeso^c (m^2 g^−1)	Vt^d (cm^3 g^−1)	Vmicro^e (cm^3 g^−1)	Vmeso^f (cm^3 g^−1)	DBJH^g (nm)
a Total BET.b Microporous surface area.c Mesoporous surface area.d Total pore volume calculated at the relative pressure of p/p0 (0.995).e Microporous pore volume.f Mesoporous pore volume.g Pore sizes have been calculated by using the Barrett–Joyner–Halenda (BJH) method.
CS	8.80	2.78	6.02	0.016	0.004	0.012	13.0
ACS3	656	485	171	0.246	0.202	0.042	5.61
ACS6	1477	1016	461	0.268	0.209	0.059	4.87
ACS12	2325	1228	1097	1.150	0.584	0.566	3.52
QPS–ACS	137.0	65.04	71.96	0.147	0.068	0.079	5.60

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The TGA curves of ACS, QPS–ACS were shown in Fig. 4. According to the weight loss in TGA curve, the decomposition of ACS clearly occured in two general regions below 800 °C: (1) evolution of free (absorbed) and interlayer water residing between the porous structure of ACS between 50 °C and 150 °C; (2) dehydroxylation of the ACS between 400 °C and 650 °C. At the same time, the TGA curves of QPS–ACS may be divided into three regions: (1) evolution of absorbed water and gases below 200 °C; (2) evolution of the organic substances QPS between 200 °C and 500 °C; (3) dehydroxylation of the ACS between 500 °C and 650 °C. In addition, the organic content of QPS–ACS can be determined in range 200–500 °C, and the quaternary phosphonium content of QPS–ACS1, QPS–ACS2, QPS–ACS3 and QPS–ACS4 is 13.21, 20.42, 36.94 wt% and 38.02 wt%, respectively.

Fig. 4 TGA curves of ACS, QPS–ACS1, QPS–ACS2, QPS–ACS3 and QPS–ACS4.

The antimicrobial properties of ACS and QPS–ACS against E. coli and S. aureus were characterized by minimum inhibitory concentration (MIC). The MIC results were shown in Table 2. ACS showed poor antibacterial activity and the MIC values against the two kinds of microorganisms were higher than 10 000 mg L⁻¹. For QPS–ACS, they showed relatively high antibacterial activity against E. coli and S. aureus, and the antibacterial activity was enhanced along the enhancement of the QPS content of QPS–ACS. The QPS–ACS3 showed good antibacterial activity and the MIC against E. coli and S. aureus was 150 and 100 mg L⁻¹, respectively.

Table 2 MIC of the ACS and QPS–ACS

Samples	MIC/(mg L^−1)
	E. coli	S. aureus
ACS	>10 000	>10 000
QPS–ACS1	550	500
QPS–ACS2	300	225
QPS–ACS3	150	100

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The ability of the materials to kill bacteria was also tested by OD600 method as described in the experimental section. Fig. 5 showed the OD600 curves of ACS with or without QPS in the presence of E. coli and S. aureus, respectively. In addition, a control experiment was run in which only bacterial broth was present. The OD600 curve of ACS was high, demonstrating poor antimicrobial ability. Nevertheless, the ACS with QPS exhibited high antimicrobial activity and enhanced significantly while increasing the QPS contents.

Fig. 5 Normalized E. coli growth curves of different materials. (A) ACS; (B) QPS–ACS3 and (C) QPS.

The antibacterial effect of QPS–ACS determined in this study was found to be similar to that described in the earlier reports.^4a,11 From the results we can conclude that the content of the QPS in the ACS played a key role in the antimicrobial abilities of the spheres and the effect increased with higher content.

Both of these experiments reveal that QPS–ACS have great potential to be used as an antimicrobial agent. The mechanism of antibacterial action of QPS has already been reported in the literature.^11,12 It was believed that the QPS–ACS nanocomposite in contact with bacterial cells and the carbon nanomaterial damaged the cytoplasmic membrane of the bacterial cell and the QPS entered into the cell and the cell died.¹³

The long-term stability of the QPS–ACS was also assessed using the PBS abrasion test. The released quantity of QPS from QPS–ACS1, QPS–ACS2, QPS–ACS3 and QPS–ACS4 at different soaking time was shown in Fig. 6. During the first 36 h, QPS was released quickly with the lapse of soaking time, and then released slowly as time passed. The released quantity of the QPS from QPS–ACS1, QPS–ACS2, QPS–ACS3 and QPS–ACS4 were only 4.02, 4.68, 5.22 and 5.05 mass% until 72 h, respectively. The results showed that QPS–ACS exhibited long-acting antibacterial activity and will make the use of quaternary phosphonium salt safer.

Fig. 6 Released amount of QPS in PBS for different soaking time.

Conclusions

In conclusion, colloidal carbon spheres obtained by a green synthesis method can be easily activated with steam. The surface of the activated carbon spheres was enriched with functional groups and porous structure which induced the adsorption of the organic agents to the carbon spheres. The pore structure of the carbon spheres was full of dodecyl tributyl phosphonium bromide and the maximum adsorption capacity obtained with carbon spheres activated for 12 h at 800 °C was 36.94 wt%. The phosphonium carbon spheres exhibited excellent thermal stability. Moreover, QPS–ACS showed good antibacterial activity against E. coli and S. aureus with the MIC of 150 and 100 mg L⁻¹, respectively. The results indicated that phosphonium carbon spheres were potentially useful materials for long-acting antibacterial field and could be used in repeated applications.

Acknowledgements

This work was financially supported by the Youth Fund of Guangdong Provincial Academy of Sciences (qnjj201406) and the National Science Foundation of China (21401028).

Notes and references

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