Manganese ferrite graphene nanocomposite synthesis and the investigation of its antibacterial properties for water treatment purposes

The main objective of this study was to synthesize a nanocomposite using graphene and manganese ferrite nanoparticles (MnFe2O4-G) and to evaluate its antibacterial activity for water treatment purposes. Its morphological characteristics were evaluated by instrumental techniques, such as scanning electron microscopy and transmission electron microscopy. The characterization results indicated that the nanocomposite presented nanoparticles of approximately 25 nm well dispersed in transparent and large (14 μm) graphene nanosheets. The antibacterial activity was evaluated in a batch experiment using a concentration of 40 μg mL of nanocomposite (MnFe2O4-G, bare MnFe2O4 nanoparticles or graphene oxide), 1x10 5 CFU mL of Escherichia coli, and 8 h of contact time at room temperature. The highest antibacterial capacity was observed for the hybrid nanocomposite (91.91%), due to the synergic effect of graphene and MnFe2O4 nanoparticles. Various mechanisms were proposed to explain the effective antibacterial activity of MnFe2O4-G, such as wrapping, oxidative stress, sharp-edge cutting effect, among others. The results showed that MnFe2O4-G is a potential alternative in water treatment processes as an antibacterial agent.


INTRODUCTION
Water is an essential resource for all beings. However, unsafe drinking water still constitutes a major burden on public health in developing countries. Diseases related to drinking water contamination lead to millions of deaths every year and diarrhea remains a major cause of child deaths. The main health risk is ingestion of water contaminated with feces that contains pathogens that cause infectious diseases such as cholera and other diarrheal diseases, dysenteries and enteric fevers (Liu et al., 2012a). Therefore, the development of novel and efficient antibacterial agents to control and prevent contamination by pathogenic microorganisms in water is vital for human health and well-being.
Antibacterial nanomaterials provide great opportunities to develop next-generation sustainable water-disinfection technologies. Among all the antibacterial nanomaterials, graphene-based nanomaterials have emerged recently as a novel green broad-spectrum antibacterial material, with a severe cytotoxic effect on bacteria, fungi, and plant pathogens, with little resistance and tolerable cytotoxic effect on mammalian cells (Hegab et al., 2016;Ji et al., 2016) Graphene is a two-dimensional monolayer of sp 2 -hybridized carbon atoms that form a honeycomb structure with unique properties. Because of its peculiar configuration, it has unique properties, such as high mechanical strength and elasticity, and excellent conduction of electric current and heat, in addition to having good dispersion and remarkable thermal stability (Syama et al., 2016).
The degree of antibacterial effect of nanomaterials is determined by their shape, surface functionalization, size, stability and size distribution. Single-component graphene-based materials present slow antibacterial activity; generally, it takes several hours to totally inactivate bacterial cells in diluted suspensions. In practical applications, it is essential to optimize and accelerate the process minimizing the disinfection time (Zhou et al., 2016b).
Another major problem related to the use of graphene in water treatment is that graphene nanosheets tend to aggregate and re-stack, forming graphite when used during process operations and when used in larger quantities due to strong interplanar interactions (Cheng et al., 2012). As for GO, they have poor affinity for binding with anionic compounds due to their strong electrostatic repulsion.
These disadvantages can be overcome by covalent or non-covalent functionalization of different molecules and other nanomaterials (Xu et al., 2009). The surface functionalization of graphene materials with nanoparticles or other functional groups increases their sensitivity, selectivity and detection limit, and also may improve their antibacterial effect and stability (Hegab et al., 2016;Tu et al., 2016). The use of nanoparticles can be beneficial both to facilitate separation in the water treatment process and to confer antibacterial properties, which opens new opportunities to further explore their potential for water-and wastewater-treatment applications (Farghali et al., 2013;Gutes et al., 2012).
Thus, graphene derivatives are rapidly emerging as an extremely promising class of nanomaterials due to the combination of graphene derivatives and currently utilized antibacterial metal and metal-oxide nanostructures, in order to obtain the synergistic antibacterial effect, achieving exceptional bactericidal activity (Hegab et al., 2016;Rojas-Andrade et al., 2017).
Spinel ferrites represent an important family of iron-based heterostructured oxide materials and display great potential. Among these materials, MnFe2O4 has been considered a very attractive nanomaterial due to its high capacity, excellent chemical stability, easy fabrication, low cost and non-toxicity (Sakho et al., 2019).
Few studies were found in the literature with research focusing on the use of MnFe2O4 or composites of graphene and MnFe2O4 for bactericidal activity in water treatment (Chella et al., 2015). Most research focused on their antibacterial activity for the development of novel nanomaterials for biomedical applications (Esmaeili amd Ghobadianpour, 2016;Sakho et al., 2019), drug-delivery (Wang et al., 2016) and photocatalytic activity (Zhou et al., 2016a).
This paper therefore reports on the synthesis of a nanocomposite of manganese ferrite graphene (MnFe2O4-G), followed by an investigation of its antibacterial properties, evaluating its efficiency at removing Escherichia coli, to verify its potential use in water-and wastewatertreatment processes.

Manganese ferrite graphene nanocomposite synthesis
GO was synthesized according to the modified Hummers method (Hummers and Offeman, 1958;Kovtyukhova et al., 1999). The preparation of MnFe2O4-G was based on a simple onepot solvothermal method reported in our previous work (Yamaguchi et al., 2016). In short, anhydrous ethylene glycol (HOCH2CH2OH, ≥99.8%), GO, ferric chloride (FeCl3·6H2O, ≥97%), manganese chloride (MnCl2·4H2O, ≥99%) were dispersed under ultrasonication. Later, anhydrous sodium acetate (CH3COONa, ≥99%) was added and stirred for 30 min. The mixture was then autoclaved at 200°C for 10 h. The obtained mixture was then washed several times with deionized water and ethanol and dried in a hot air oven at 60°C. All chemicals were purchased from Sigma Aldrich. Bare MnFe2O4 nanoparticles were prepared using a similar approach, but in the absence of GO.

Nanocomposite characterization
The surface morphology of the as-synthesized nanocomposite was verified by scanning electron microscopy (SEM) under Shimadzu SS-550 -Scanning Electron Microscope and transmission electron microscopy (MET) under JEM-1400 -JEOL microscope. An extensive chemical characterization of the nanocomposites was performed in our previous work (YAMAGUCHI et al., 2016) and will be used for further discussion of antibacterial results in Section 3.2.

Antibacterial properties evaluation
Assays for the evaluation of E. coli removal were based on the Standard Methods for the Examination for Water and Wastewater (APHA et al., 2017). A stationary phase culture of E. coli ATCC 11229 was incubated at 35°C for 24 h in trypticasein soy broth (TSB). From the culture obtained, a bacterial suspension of 1.5x10 8 CFU mL -1 (colony forming unit) was prepared in a saline tube and determined by comparison with the turbidity of the McFarland scale # 0.5 tube.
To determine the antibacterial effect of the nanomaterials, a batch experiment was performed. Typically, 2 L of distilled water was contaminated with 1 mL of the previously prepared E. coli ATCC 11229 suspension to give a concentration of approximately 1x10 5 CFU mL -1 . Then, 100 mL of contaminated water was dispensed into 250 mL vials containing 10 mg of nanocomposite, resulting in 40 μg mL -1 nanocomposite concentration. After inoculation, they were shaken at 200 rpm and 35°C for 8 h. After 8 h, the antibacterial effect was evaluated using an adapted filter membrane technique of the Standard Methods for the Examination for Water and Wastewater (APHA et al., 2017), as illustrated in Figure 1. The filter membrane technique used can be summarized by: (1) First the sample was homogenized; then (2) 20 mL of the sample was diluted in 180 mL of 0.85% saline in a Schott ® vial, resulting in a 10 -1 dilution; (3) the vial was shaken and then 20 mL of the 10 -1 dilution vial was collected and added to another vial containing 180 mL of 0.85% saline resulting in a 10 -2 dilution; (4) this procedure was repeated until a 10 -5 dilution was obtained, as shown in Figure 1; next, (5) in a laminar-flow chamber previously sterilized with ultraviolet radiation, 100 mL of each 10 -5 dilution was vacuum filtered in a previously autoclaved Manifold Microfil ® Millipore using a membrane of 0.45 µm pore and 47 mm diameter; (6) the membranes were placed in Petri dishes containing M-Endo LES agar and then placed in an oven at 35°C; (7) after 24 h, the Petri dish readings were taken by counting the number of CFU. The E. coli viability loss was calculated using Equation 1.

Nanocomposite characterization
In the micrographs of the hybrid composite of MnFe2O4-G shown in Figure 2, it is possible to observe the graphene nanosheets, which look like crumpled sheets. Similar structures were found in micrographs obtained in our previous work (Yamaguchi et al., 2016) and by Yao et al. (2014), who used a similar methodology to that employed in this work. It was also noted that MnFe2O4 nanoparticles were uniformly anchored on the transparent graphene nanosheets (Figure 2b). The size of MnFe2O4 nanoparticles can be confirmed by TEM and SEM micrographs (Figure 2), showing an average particle size of 25 nm. It is noteworthy that MnFe2O4 particles are still strongly anchored to the graphene surface even after sample preparation for MET analysis (agitation and sonication), suggesting that there is a strong interaction between MnFe2O4 nanoparticles and graphene nanosheets, also showing mechanical stability (Yao et al., 2012).
In Figure 2b, the graphene nanosheet observed is approximately 14 nm and is much bigger than a bacteria cell (~ 1 μm), indicating that the mechanism of cell wrapping (discussed in Section 3.2) is able to contribute to the antibacterial activity when using MnFe2O4-G for antibacterial suspension tests. Also, large-size graphene nanosheets enhance the adhesion ability of bacteria, which means more chance to be in contact with and inactivate bacteria (Han et al., 2019). Additionally, the transparent nanosheets presented in Figure 2b indicate a fewlayer graphene. It is known that the number of layers that the graphene has significantly affects its antimicrobial activities, as graphene dispersibility in biological media displays a remarkable decrease with the increase in its thicknesses, resulting in agglomeration, which may affect the interactions between graphene and bacteria. Therefore, it was expected that our few-layer graphene nanocomposite would exhibit high antibacterial activity (Zheng et al., 2018).
Hybrid nanomaterials with graphene structure are known to help promote a smaller agglomeration of nanoparticles, ensuring a large specific area due to the close interaction between the nanoparticles and graphene sheets (Liu et al., 2013). Also in Figure 2, functionalized surfaces with considerable roughness and frequent ridges can be seen, which is also a favorable aspect for antibacterial activity, as it can cause cracking of bacterial cell walls during contact. The physical surface morphology resulting from graphene functionalization with MnFe2O4 is a crucial factor in affecting interaction with bacterial cells and can display a powerful antibacterial action by generating increased surface roughness, improving bacterial cell adhesion. Higher surface area and deep terrains in the surface will result in more contact with bacteria cells, which can easily destroy them (Hegab et al., 2016).

Antibacterial properties evaluation
The results of the evaluation of the antibacterial properties of the GO, MnFe2O4 and MnFe2O4-G are presented in Figure 3. Bacterial cell viability loss is the percentage of E. coli bacteria that were inhibited by the developed materials. GO exhibited 62.63% of cell inactivation presenting moderate cytotoxicity, while the nanohybrids showed 91.91% of cell inactivation. Bare MnFe2O4 nanoparticles showed lower removal in comparison to the nanohybrids, presenting 89.50% of bacterial viability loss.  Liu et al. (2011) who evaluated the antibacterial GO efficiency for E. coli and reported ~69.3% performance using 85 μg mL -1 for 2 h and they explained their results by the high density of oxygen-containing groups present in GO, which induce membrane stress leading to cell death. Similarly, experimental data from other groups obtained better results (> 80%) using higher material concentrations (Akhavan et al., 2010;Begum et al., 2020;Nine et al., 2017).
The antibacterial activity of graphene has been confirmed to be dependent on its carbon radical density, as the oxygen content of the nanosheets plays an important role in bacterial killing through the induction of oxidative stress, which will be further explained below. Thus, a higher carbon-radical density implies a stronger antibacterial effect (Han et al., 2019). GO is widely heterogeneous in their physicochemical properties resulting from its oxidation in the Hummers method. More oxidative content can generate more reactive oxygen species (ROS), which contribute to the higher bactericidal ability. GO possesses a high oxidation level, with oxidized groups such as C-OH, C-O and C-O-C, and can result in high oxidation performance for antibacterial capacity (Han et al., 2019). In our previous work (Yamaguchi et al., 2016), GO was characterized by FTIR analysis and the presence of these functional groups in GO surface was proved. Also, the ROS functional groups were observed in MnFe2O4-G nanohybrid samples when performing the XPS analysis (Yamaguchi et al., 2016), indicating that the complete reduction of GO was not achieved, and that these functional groups may have contributed to the better efficiency of the nanohybrid nanomaterial compared to bare MnFe2O4.
However, the slight difference in antibacterial efficiency may be related to the charge of the nanohybrids, which could have contributed to the improved adsorption of bare MnFe2O4 by the attraction of negatively charged bacteria cells through Coulombic interaction. The potential zeta characterization analysis (Yamaguchi et al., 2016) showed that MnFe2O4 is more positively charged compared to GO and MnFe2O4-G. Thus, our nanohybrids have a negative global charge, specially graphene, while bare MnFe2O4 nanoparticles have a positive charge, which may have contributed to the electrostatic attraction and later ion release, and thus presented a higher E. coli removal (Hegab et al., 2016;Ji et al., 2016). Chella et al. (2015) found a similar behavior when using MnFe2O4-G nanocomposite for antibacterial investigations. They obtained lower cell inactivation percentages (82%) than those obtained in the present study using 100 μg mL -1 of MnFe2O4-G for 2h of contact time. Another recent study, Sakho et al. (2019) reported 90% of E. coli death rate using 15 μg mL -1 of nanohybrid MnFe2O4-G after 2 h of contact time. In both studies, they attributed the antibacterial activity to the oxidation stress and membrane interaction (Liu et al., 2011). They explained that the antibacterial activity mechanism functioned by binding the hybrid composite with the E.coli cell wall by adsorption and electrostatic attraction and the cell wall rupture occurred by direct contact with the graphene nanosheets and also ROS generation, which induced oxidative stress to the membrane, disturbing the cell structure, leading to the death of bacterial cells. Graphene-based materials, which contain a great number of functional groups, are likely to interact with bacterial cell structures, resulting in damage and their death (Chella et al., 2015;Sakho et al., 2019). These mechanisms are further explained below, and presented in Figure 4, together with the major antibacterial mechanisms proposed for MnFe2O4-G according to the several mechanisms reported in the literature for graphene and graphene metaloxide based nanocomposites. Sharp-edge cutting effect: The physical damage in the bacteria membrane by graphene sharp edges is one of the main mechanisms of the antibacterial activity. The destruction of the integrity of the bacteria lipid bilayer upon direct contact with the atomically sharp edges of graphene nanosheets leads to bacterial inactivation, as they mediate the release of functional intracellular contents and form pores that could lead to osmotic imbalance, resulting in the reduction of the energy barrier required for membrane penetration (Hegab et al., 2016;Hu et al., 2010;Ji et al., 2016).
Wrapping: The cell wrapping antibacterial physical activity mechanism is caused by the graphene sheet area, which hinders the bacterial growth in cell suspensions. Isolated from the external environment, the bactericidal activity of graphene is amplified as the sheet area increases. The bacteria is trapped in a large graphene nanosheet, becoming isolated and inactive, affecting their interaction with cells in the environment, which has the consequence of preventing nutrient acquisition and, subsequently, cellular growth and proliferation (Liu et al., 2012b). This mechanism is more bacteriostatic, as it inhibits the growth of bacteria temporarily and normally does not kill them (Xia et al., 2019).
Insertion and extraction: The destruction mechanisms of graphene insertion and lipid extraction can cause bacterial membrane stress leading to a reduction in cell viability. When graphene is in contact with the bacteria, the graphene nanosheet starts to vibrate around the bacteria cell (swing mode); then the insertion mode starts, when the sheet edge moves in and pierces the cell membranes, due to the strong van der Waals interactions. Finally, the extraction step is when the nanosheet extracts the phospholipids from the lipid bilayers of the bacteria cell membrane causing damage to the cell and cytoplasm leakage (Hegab et al., 2016).
Oxidative stress: Oxidative stress leads to an imbalance between oxidation and antioxidation, which interferes with the bacterial metabolism, disrupts essential cellular functions, destroys cell structure, oxidized lipids, DNA and proteins that can ultimately lead to cell destruction and/or cellular growth inhibition. The large production of ROS, direct oxidation and charge transfer by MnFe2O4-G is believed to be the primary mechanism of cytotoxicity causing the oxidative stress of bacteria (Rojas-Andrade et al., 2017). Also, when the ROSmediated oxidation of lipid molecules takes place, the result is the formation of lipid peroxide radicals which initiate a chain reaction that leads to cell membrane oxidative destruction (Begum et al., 2020;Hegab et al., 2016).
Electronic transport disruption: The electron transfer from the bacterial membrane to the graphene surface is also a mechanism of the antibacterial activity of MnFe2O4-G. The negative charges of the bacterial cells interact with the graphene nanosheet edges which act as good electron acceptors and, therefore, contribute toward bacterial cell membrane damage. Respiratory chain electrons are extracted from the electron transport chain by graphene through a charge transfer mechanism, as graphene has high electronic conductivity. The electron transference can damage the membrane integrity, cause a depletion of the intracellular respiratory chain to extracellular molecular oxygen and provoke oxidative stress on bacteria metabolism, eventually causing its inactivation. Also, electron transfer from antioxidant biomolecules to graphene can also directly cause damage to bacterial antioxidant systems. In addition, the electron transfer occurring on MnFe2O4-G surface can generate free oxidative radicals, which are toxic to bacteria (Han et al., 2019;Hegab et al., 2016;JI et al., 2016;Rojas-Andrade et al., 2017).
Synergic effect: The synergic effect is composed of the cytotoxicity effect of metal oxide nanoparticles enhanced by the graphene substrate, due to reduced agglomeration of metal oxide nanoparticles. The BET analysis performed previously (Yamaguchi et al., 2016) for MnFe2O4-G and MnFe2O4 samples indicated that BET surface area presented a more than 4-fold increase. With this increase in surface area, the active surface area for adsorption properties towards bacteria cells is also increased, providing enhanced stability to immobilized metal oxide nanoparticles, and more effective release of cytotoxic nanoparticles in proximity to the bacterial cell, increasing bacterial inactivation (Begum et al., 2020). Additionally, the graphene substrate affords additional membrane damaging effects, such as wrapping of bacteria and lipid extraction, which increase the overall antibacterial performance. Also, there is a close relationship between the cytotoxic effects of ROS generated by MnFe2O4-G and its destructive effects on bacterial membranes, which enables the metal cations (Mn 2+ and Fe 3+ ) to enter the bacteria more easily acting synergistically with ROS to destroy DNA and proteins (Begum et Rev. Ambient. Água vol. 15 n. 4, e2515 -Taubaté 2020 al., 2020; Rojas-Andrade et al., 2017). Furthermore, the positive ions released are easily absorbed by the bacterial membrane surface, with negative charge, leading to cell wall damage by pit formation. This electrostatic interaction between positive charge of metal ions and negative charge of membrane cells causes changes in the efflux and influx of biomaterials from bacteria. Moreover, in the surface or inner part of bacteria, small nanoparticles (~10 nm) can bind to sulfur-containing amino acids with thiol functional groups as they have more affinity to attach metal ions; this attachment may lead to the enzyme malfunction (Alavi and Rai, 2019).
Other mechanisms were also reported in the literature, such as photothermal reaction and photocatalytic activity for microorganism inactivation. However, our experiments were performed without light and/or infrared irradiation. An enhanced antibacterial activity can be achieved by sunlight irradiation provoked by high oxidative stress, as sunlight produces ROS, and accelerates the electron transfer from bacteria to graphene, by this means destroying bacterial antioxidant systems and causing great membrane disruption (Han et al., 2019). Thus, a possibility to improve the antibacterial performance of MnFe2O4-G obtained in this work could be achieved using visible light irradiation in antibacterial assays.

CONCLUSIONS
The MnFe2O4-G nanohybrid was successfully synthesized in this research, as verified by SEM and TEM analysis. MnFe2O4 nanoparticles of an average particle size of 25 nm were anchored in crumpled large and transparent graphene nanosheets. The nanohybrid presented higher antibacterial activity when compared to GO and bare MnFe2O4 nanoparticles. The bactericidal activity of MnFe2O4-G was thoroughly discussed and it was concluded that graphene exerts its antibacterial action via physical damage such as direct contact of its sharp edges with bacterial membranes, destructive extraction of lipid molecules and wrapping mechanisms. Further, the chemical damage of bacteria is caused by oxidative stress with the generation of ROS and charge transfer. Furthermore, the synergic effect is observed when graphene and MnFe2O4 are together in the hybrid nanomaterial, obtaining improved antibacterial efficiency due to the reduced agglomeration of metal oxide nanoparticles, adsorption of bacterial cells, effective release of cytotoxic ions with greater proximity and the simultaneous damaging effects of graphene and metal cations on bacteria. Thus, due to the superior antibacterial properties obtained and to its good biocompatibility, MnFe2O4-G has been shown to have important potential for antibacterial purposes in water and wastewater treatment processes.