Evaluation of TiO₂ Nanoparticles Physicochemical Parameters Associated with their Antimicrobial Applications

Abstract

Titanium dioxide nanoparticles (TiO₂NPs) usage is increasing in everyday consumer products, hence, assessing their toxic impacts on living organisms and environment is essential. Various studies have revealed the significant role of TiO₂NPs physicochemical properties on their toxicity. However, TiO₂NPs are still poorly characterized with respect to their physicochemical properties, and environmental factors influencing their toxicity are either ignored or are too complex to be assessed under laboratory conditions. The outcomes of these studies are diverse and inconsistent due to lack of standard protocols. TiO₂NPs toxicity also differs for in vivo and in vitro systems, which must also be considered during standardization of protocols to maintain uniformity and reproducibility of results. This review critically evaluates impact of different physicochemical parameters of TiO₂NPs and other experimental conditions, employed in different laboratories in determining their toxicity towards bacteria. These important observations may be helpful in evaluation of environmental risks posed by these nanoparticles and this can further assist regulatory bodies in policymaking.

Introduction

Significant biotechnological advancements have been made in environmental and industrial sectors to make human life sustainable [1–4]. However, the recent pandemic attack has demonstrated its impact on human health and increased economic load worldwide. This disastrous activity suggests that more attention needs to be paid to the management of infectious diseases [5–7]. Nanotechnology, make use of nanoparticles (NPs) with unique properties, has the potential to develop new materials having applications in wide areas like biomedicine, food industries, textile manufacturing, enzyme immobilization, bioenergy production, biosensors, agriculture, electronics and telecommunication etc. [8–14]. The synthesis of nanomaterials by various methods i.e. physical, chemical, and biological have led to diverse alteration in their properties and applications [15–20]. According to their size, shape and physicochemical properties, NPs are classified into several categories like metal oxide NPs, carbon-based NPs, polymeric NPs, lipid-based NPs, semiconductor NPs, and ceramic NPs etc. Metal oxide nanoparticles (TiO₂, CuO, Fe₂O₃, ZnO, and others) have gained importance due to their uniform size distribution (10–100 nm) and large surface area. Titanium dioxide nanoparticles (TiO₂NPs) are one of the most produced and utilised NPs in commercial products due to their photocatalytic properties and inert nature of TiO₂ [21, 22]. Overall, a significant development in nanotechnology is playing a key role in various biotechnological applications for sustainable progress [23–32]. The manufacturing of NPs has expanded dramatically over the last decade, owing to their wide range of uses in everyday items. As a result, these NPs have been released into the environment uncontrollably. The toxic effects of NPs, as well as their fate and transport behavior, govern the probability of NPs being a source of emerging pollutants in the environment. The residues of the products incorporating TiO₂NPs are released in the environment at different stages i.e., production, usage and disposal, by different routes like air, rain and sewage [33–35]. When TiO₂NPs enter the environment, they undergo various types of physical, chemical and biological transformations. These transformations alter TiO₂NPs physicochemical properties due to changes in their surface chemistry which modify their toxicity. TiO₂NPs make aggregates in presence of natural organic matter (NOM) that range from nanometres to micrometres and have been reported to adsorb other ligands on their surface further magnifying their toxicity [36]. Also, interactions with macromolecules of flora and fauna tissues form a layer on their surface, known as bio-corona, which gives a biological identity to them [37, 38].

Numerous studies have reported the toxic and adverse effects of TiO₂NPs in different organisms like algae, Drosophila, Daphnia, mice, zebrafish, humans etc. [39–47]. Of all organisms, bacteria are ubiquitous and can adapt easily to adverse environmental conditions and pollutants; as a result, NPs interact with them more readily than any other higher organisms. Bacterial contact with NPs can either be toxic, or they can be resistant to them. Bacterial metabolites like enzymes, organic acids, and extracellular polymeric substances (EPS) have been shown to modify the hydrophobicity, charge, and surface properties of nanoparticles. Despite the fact that bacteria lack internalisation mechanisms, as found in eukaryotes, TiO₂NPs have been scanned inside the bacterial cells [48]. There are numerous studies which have been conducted to examine toxic impact of TiO₂NPs on bacteria, but their ecotoxicological effects are poorly understood due to their limited characterization [38, 49, 50]. Hence, it is crucial to examine how TiO₂NPs and bacteria interact in the environment with respect to their physicochemical properties (like stability, size, aggregation, surface charge, optical properties, catalytic behavior, and adsorption capacity) and enviornmental factors. The indiscriminate and widespread use of TiO₂NPs has resulted in higher human and environmental exposure but their adverse impact is largely not determined, necessitating the assessment of their ecotoxicological effects [38, 51]. This review evaluates TiO₂NPs toxicity towards bacteria and assesses various physicochemical parameters employed during laboratory studies to gain insight into nanoparticle-bacterial interactions in the environment.

Physicochemical Parameters of TiO₂NPs

The major physicochemical parameters that influence the toxicity of TiO₂NPs include pH, ionic strength (IS), size, surface charge, crystal structure, components of the media, aggregation, optical properties, catalytic behavior, zeta potential, and solar radiations (UVA region).

Ionic Strength and pH

Nanoparticles never remain in their native state, they form aggregates that modify their size, surface area and other physicochemical properties [52]. Toxicity of TiO₂NPs in a solution is highly influenced by IS i.e. total ion concentration and pH. According to Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, the aggregation of NPs depends on the isoelectric point and pH of the solution [53]. The attractive van der Waals forces between TiO₂NPs and an increased IS of solution, decreases the zeta potential and thickness of the electric double layer (electrostatic forces), resulting in enhanced aggregation [54]. The surface charge of TiO₂NPs in aqueous suspension depends upon the surface ionization, which changes with the pH of solution. TiO₂NPs can be positively or negatively charged at lower and higher pH, respectively, whereas, they have no net charge at the isoelectric point [53]. TiO₂NPs have been reported to form aggregates of several hundred nanometers to micrometers depending on the nature of electrolytes and ionic strength of aqueous solution in acidic pH range, for example, TiO₂NPs of 4–5 nm size are reported to readily form stable aggregates of diameter 50–60 nm at pH 4.5 in 4.5 M NaCl. But raising the ionic strength to 16.5 M while keeping the pH constant, results in 20–1000 nm micro-size aggregates, which have been shown to be toxic to bacteria [55]. In surface waters and soil, the presence of low concentrations of divalent cations (e.g., Ca²⁺, Mg²⁺) magnify this effect and speeds up the production of these micro-sized aggregates. At high NaCl concentrations, NPs adsorb on the bacterial cell surface more efficiently leading to cell death. The mechanisms involved have been studied through various molecular approaches like transcriptomics, and proteomics along with biochemical analysis. It has been shown that during these enhanced interactions, depolarization of cell membrane occurs, which in turn increases the efflux of potassium and magnesium ions with simultaneous influx of sodium ions and ATP depletion [56]. It has been reported in a recent study that increasing pH to 7, increases the antibacterial activity of TiO₂NPs modified with dendrimers in case of Staphylococcus aureus and Escherichia coli [57]. In most toxicological studies, for optimum bacterial growth, pH is maintained between 5 and 7; however, homogenous TiO₂NPs suspensions, with strong repulsive forces can be achieved only at acidic pH to prevent aggregation [58]. As a result, standardising the pH of the solutions used in toxicological studies is critical.

Salts and Natural Organic Matter

In addition to NPs, many monovalent and divalent salts play an important role in bacterial aggregation, e.g., Ca²⁺ causes bacterial aggregation in mouth resulting in dental plaque formation [59]. Also, in an aqueous solution, salts like Al³⁺ have been reported to dissociate and adhere to the surfaces of NPs and bacteria, neutralising their surface charges, resulting in the formation of large flocs as seen in sewage treatment plants [50]. At alkaline pH, TiO₂NPs have a negative charge, however, if the salt concentration is high, such as 0.1 M NaCl, TiO₂NPs and bacteria interact well [58]. The divalent cations (like Ca²⁺) lead to higher TiO₂NPs aggregation, for example, 0.0128 M CaCl₂ in solution changes the zeta potential from positive to negative and causes TiO₂NPs to aggregate more efficiently than 0.0125 M NaCl [55]. Therefore, during TiO₂NPs toxicology studies, it is critical to examine different types of salts and their concentrations. NOM has a high hydrophobicity and carries several functional groups with a strong affinity for metal oxide surfaces in water. The presence of NOM, like fulvic and humic acids, in water reservoirs like sea, lagoons, rivers and groundwater, also affect the aggregation, steric repulsion and electrophoretic mobility (EM) of TiO₂NPs [36]. There are higher chances of formation of large-sized aggregates and less EM of TiO₂NPs in seawater with high IS and low NOM [53]. As a result, TiO₂NPs are not as reactive in the environment as they are in laboratory experiments, due to the existence of NOM, which operates as a protective shell surrounding these NPs. NOM prevent direct contact of NPs with living organisms and also reduce the adverse effects caused by reactive oxygen species (ROS) [60]. Therefore, it is important to examine both organic and inorganic environmental factors that influence the impact of these NPs on organisms in the laboratory conditions to make accurate risk assessments.

Sonication

Probe sonication is a commonly used method for dispersion of TiO₂NPs in an aqueous solution and for dissociation of their agglomerates. Due to probe sonication, initially the hydrodynamic diameter decreases but then increases with time. The hydrodynamic diameter of TiO₂NPs is higher in solution (90 nm) as compared to the normal particle size (25 nm), leading to their increased toxicity [61]. Probe sonication has been reported to not only disperse TiO₂NP agglomerates in solution, but it also induces agglomeration due to increased particle-particle interaction and thus influences their toxicity [61, 62].

Since TiO₂NPs aggregation is primarily determined by pH and IS of the solution, probe sonication never creates uniformly dispersed nanoparticles. When sonicated TiO₂NPs suspension is transferred from a stock solution to a working solution, it leads to variations in pH, IS and other physicochemical properties of TiO₂NPs such as size, surface area and zeta potential, creating their heterogeneous distribution [63]. A major reason of variation in TiO₂NPs suspension properties is the lack of consistency in the standardization of sonication in different laboratories. Hence, it is critical to either use pulsed probe sonication or directly add powdered TiO₂NPs along with bacteria to carry out toxicological analysis in laboratories.

Size, Optical Properties and Crystal Structure

NPs possess optical properties such as transmission, absorption, reflection and light emission, which depend on their electron configuration. Although, TiO₂NPs show phototoxicity via generation of ROS, they are also used for photodegrading organic pollutants and toxic dyes in the environmental samples by ultraviolet (UV) light [64]. They can be doped with several metals/non-metals such as Fe (III), Co (II), Ni (II), Ag (I), C, N and S etc., to shift the band edge for better absorption efficiency [65–67]. For example, CdS@TiO₂ (CT) nanocomposites are better photocatalysts than TiO₂NPs alone, since in composites, the absorption band edge is shifted towards visible region, which makes photocatalytic degradation of pollutants in industrial waste samples more efficient [68]. Similarly, Fe-doping of TiO₂NPs at concentrations as low as 0.1% has been reported to enhance their photocatalytic activities by inducing photogenerated pores and photoexcited electrons. These photogenerated pores produce highly oxidizing species which are toxic to bacteria [69]. The coating of silica (SiO₂) leads to dipole formation at rutile TiO₂NPs: SiO₂ junction, leading to an increased photoexcited electrons positive charge of rutile TiO₂NPs and is found to be coat thickness dependendent. This enhances the interactions between the bacterial cell surface and TiO₂NPs resulting in an increased photocatalytic and antibacterial properties [70]. While SiO₂ modified TiO₂NPs have reduced toxic effects at lower concentrations on the higher organisms but an increase in their ratio (TiO₂:SiO₂ 3:1) can damage these cells due to ROS production leading to cell necrosis, cell cycle arrest and DNA damage etc. [71]. Upon activation by UV and visible light, TiO₂NPs show increased cytotoxicity but, in a size dependent manner i.e., smaller the size of NPs, higher is the toxicity. Higher toxicity of small TiO₂NPs is due to their large surface area per ml and a very strong correlations (R² = 0.9 and above) between the surface area and toxicity has been reported. Thus, overall toxicity, cell membrane damage and ROS production of TiO₂NPs can be attributed to a large extent to their surface area and size [72].

There are four major crystal forms of TiO₂NPs: rutile (stable; tetragonal), anatase (metastable; tetragonal), brookite (rhombohedral) and TiO₂-B (monoclinic), which exhibit different properties. For example, the anatase and brookite forms are more stable in nanoscale dimensions and rutile and anatase forms of TiO₂NPs with the same size have the same toxicity towards E. coli [73, 74]. However, the anatase form of TiO₂NPs is more photocatalytically active and shows higher toxicity when exposed to UV light due to induced oxidative stress to the cells [75]. Furthermore, rod-shaped TiO₂NPs are more toxic than spherical particles of the same size and surface area, demonstrating that the shape of NPs plays an important role in toxicity [46]. As a result, while investigating the ecotoxicity of TiO₂NPs, a detailed and careful evaluation of their crystal structure must be carried out.

Adsorption Capacity

NPs with higher adsorption capacity have the potential to be used for recovering pollutants from waste waters and to enhance the adsorption capacity of NPs their surface modifications are carried out by using different types of dopants. Dopants create charge space carrier regions on the surface of TiO₂NPs leading to accelerated production of -OH radicals and increased rate of photocatalysis. They also act as active sites for adsorption of pollutants on their surface, and thus enhance the rate of photodegradation [76]. The adsorption capacity of TiO₂NPs may be improved by synthesizing TiO₂ nanopowder at low temperatures (nearly 100 °C) and alkaline pH resulting in porous TiO₂NPs with high surface area and high adsorption capacity [77]. Photoactivation of TiO₂NPs leads to production of ROS and adsorption of biomolecules on their surface, the two major reasons for their toxicity. Proteins and other macromolecules easily get adsorbed onto the TiO₂NPs surface, thereby giving them a new biological identity, which may be toxic. More protein molecules get adsorbed on smaller sized NPs because of their large surface area. For example, chitosan-TiO₂ composite film was formed by TiO₂ nanopowder which resulted in reduced transmission of visible light and enhanced photocatalytic antimicrobial properties of these composites, leading to improved shelf life of packaged food [78]. However, coating TiO₂NPs with compounds like poly (ethylene-alt-maleic anhydride) (PEMA) lowers their adsorption capability as it hinders biomolecule adsorption and leads to the generation of hydroxyl radical (OH⁻) during photoactivation [72].

Surface Charge

Surface of TiO₂NPs shows a strong positive charge at pH 3.0 and the point of zero charge (PZC) is attained at pH 5.8−6.2 (pHPZC). At pH above pHPZC, the overall charge on TiO₂NPs becomes negative [79]. The positively charged TiO₂NPs show more toxicity than negatively charged NPs. Bacteria in aqueous solution have negative charge on their surface due to membrane phospholipids, whereas TiO₂NPs have a positive charge, resulting in strong electrostatic interactions among them. Hence, toxicity of TiO₂NPs decreases with increasing pH (5−10) [75]. Therefore, during the laboratory studies, the agglomeration of NPs should also be considered as an important factor contributing to the levels of complexity [61].

Catalytic Behavior

The most significant catalysis shown by TiO₂NPs is photocatalysis, whereby they degrade dyes, phenolic compounds and other pollutants upon photoactivation. Factors such as concentration of pollutants, concentration of catalysts and pH influences photocatalysis. pH influences aggregation of catalysts, generation of OH⁻ radicals, charge on catalysts etc. [80]. TiO₂ nanotubes are more effective pollutant scavengers as compared to TiO₂NPs, as the former shows dual activity i.e., better adsorption efficiency and photocatalysis [81]. There have been recent advances in the TiO₂NPs based photocatalysis, where strategies such as doping with cations and anions; codoping with cations and anions; self-doping with Ti³⁺ and doping with metals has been carried out, leading to enhanced photocatalysis potential of TiO₂NPs [82]. For example, Fe–Ag co-doped TiO₂NPs are outstanding in their performance for removal of flumioxazin pesticide residues [83]. TiO₂NPs are also used as catalysts for production of pharmacologically important substances such as pyrimido [4,5-d] pyrimidines [84].

The physicochemical parameters of TiO₂NPs control their reactivity and bioavailability, which has a significant impact on their environmental toxicity. Therefore, taking into account these parameters is pivotal while studying their adverse effects on organisms in laboratories and to understand ecotoxicological impacts of these NPs (Fig. 2).

Fig. 1.

Schematic representation to depict differences between laboratory and environmental conditions to study nanoparticles (NPs) and bacterial interactions. A Under laboratory conditions, NPs are used in pure form and high concentrations with optimum pH and ionic strength. Bacteria are also devoid of any stress or interspecies competition. B In the environment, NPs combine with inorganic and organic material as well as experience fluctuating pH and ionic strength. Bacteria also experience competitive inhibition from other organisms

Fig. 2.

Various physicochemical properties affecting nanoparticle toxicity on bacteria and other organisms

Bacterial Characteristics and Behavior in Aqueous Media

To understand the toxic effects of TiO₂NPs on bacteria, it is important to study the behavior of bacteria in aqueous media. Gram-positive bacteria have a thicker cell wall and can withstand higher concentrations of TiO₂NPs than Gram-negative ones. The standard procedure used to check the toxicity of TiO₂NPs towards bacteria in laboratories is to mix both in physiological water, incubate at optimum temperature for specific time intervals and then determine percentage survival of the cells.

It is imperative to examine bacterial survival in a given IS solution as they behave like charged particles in solution due to ionizing macromolecules present on their cell wall and membrane and follow the extended DLVO theory like NPs [85, 86]. As IS rises, the electric double layer shrinks and van der Waals forces take over, leading to cell aggregation which is influenced by the valence of cations in the solution. However, one significant distinction between TiO₂NPs and bacteria is that the latter are living organisms with extremely dynamic surface properties, mostly resistant to external factors and can deviate from the DLVO principle [85]. Also, bacterial surfaces are more complex than non-biological colloids due to EPS like polysaccharides, glycoprotein, and lipopolysaccharides released by bacteria which provide an extra layer of defence [87]. The physicochemical properties like pH and IS of the medium have a substantial effect on the adsorption of EPS on TiO₂NPs surfaces, which in turn influences the aggregation behavior of TiO₂NPs. Also, aggregation rate of TiO₂NPs decreases in the presence of EPS due to steric repulsion which prevents the adsorption of EPS onto NPs and it has been shown that the aggregation can be completely stopped in high IS concentrations e.g., 50 mM NaCl along with 10 mg L⁻¹ EPS in Bacillus substilis [52]. Thus, presence of EPS in the bacterial biofilms provides protection from the toxic effects of NPs.

TiO₂NPs-Bacterial Interactions

As discussed above, pH, IS, NOM, and valence of cations, all affect the stability of TiO₂NPs and bacteria. What happens if both TiO₂NPs and bacteria are present in the same solution? They can either attract or repel each other or stay independent. Several investigations have shown toxicity of TiO₂NPs towards E. coli, due to cell lysis, as demonstrated through TEM (Transmission electron microscopy) and SEM (Scanning electron microscopy) studies [21, 88–90]. Surprisingly, very few studies in literature illustrate the effects of these physicochemical parameters on TiO₂NPs-bacterial interactions. NPs exhibit change in their toxicity towards bacteria in culture media, owing to the presence of media particles, changes in bacterial surface characteristics during growth, and media components affecting bacterial physicochemical properties [91]. For example, the presence of peptone and yeast extract have a considerable influence on the Lactobacillus acidophilus cell wall. Changes in concentrations of these media components have significant effects on surface charges, hydrophobicity, and nitrogen-to-carbon ratio of cell walls [92]. Zeta potential of the bacterial surface depends upon the number of ionisable groups on their surface and factors like pH and IS. Enterococcus faecalis strains show pH-dependent culture heterogeneity in cell surface charges, which is independent of aggregation substances (Aggs), enterococcal surface protein (encoded by the esp gene), growth medium or growth phase [93, 94]. Thus, characterization of bacterial surface physicochemical properties is significant during in vitro TiO₂NPs toxicological studies.

Only few reports focused on E. coli are available that compare the surface properties of various bacteria in relation to physicochemical properties of TiO₂NPs. Therefore, there is a pressing need to investigate the impact of these NPs on other environmental bacterial strains. For example, C. metallidurans, a heavy metal-resistant environmental strain with unique cell wall compositions and a powerful efflux mechanism (gene duplication/horizontal gene transfer) is observed to be resistant to TiO₂NPs [95].

Toxicity of TiO₂NPs towards bacteria is assessed in control and test samples by comparing the number of viable to dead cells but other effects like bacterial behavior, their physiological activities are generally ignored. As surface characteristics and zeta potential fluctuate with the growth kinetics of bacteria, it is critical to relate the toxicity of TiO₂NPs with different bacterial growth phases. Furthermore, most studies have used high concentrations of TiO₂NPs (100−500 mg/L) against bacteria but these high concentrations are rarely found in the environment [21, 96]. The long-term effects of low concentrations of TiO₂NPs on bacteria are generally overlooked due to relatively short incubation periods (24−48 h) used in laboratories, ageing effects of NPs due to surrounding environment and altered absorption capacity and photosensitivity [97]. Hence, new methodologies or approaches must be developed to assess the toxicological impacts of NPs to draw clear inferences.

Mechanism of TiO₂NPs Toxicity

The mechanisms of TiO₂NPs toxicity against bacteria are still debated. Nonetheless, it is well-established that TiO₂NPs produce ROS like hydrogen peroxide (H₂O₂), hydroxyl free radical (OH^•−) and superoxide anions (O₂^•−) in the presence of UV light in an aqueous environment, which leads to bacterial cell surface modification and mortality [8, 98]. The mechanism of ROS generation is influenced by size, shape, surface area, and other physicochemical properties of TiO₂NPs. Due to their large surface area, small NPs produce more ROS and are more toxic. Excess ROS generation, decreased antioxidants and glutathione (GSH) levels, and increased lipid peroxidation, are the major signs of oxidative stress. The bacterial cell wall and membrane have been shown to be damaged by oxidative stress, which alters their permeability through membrane depolarization and loss of integrity [21, 99]. When exposed to ROS, two oxidative stress genes (Kat A and Ahp C) and a general stress response gene (Dna K) have been shown to increase their expression levels by 52, 7, and 17 times, respectively [100]. Bacterial cell walls and membranes are the first line of defence, they provide a protective barrier, assisting bacteria in maintaining their morphology and physiological activities. ROS cause damage to the bacterial cell wall by oxidising organic components such as lipopolysaccharide, phosphatidylethanolamine and peptidoglycan, due to their vulnerability to TiO₂NPs induced peroxidation [101]. As a consequence of cell membrane damage by TiO₂NPs, the mitochondrial respiratory chain is also disturbed [98]. Although, mitochondria produce superoxide anions as natural by-product of the electron transfer respiratory chain, mitochondrial enzymes such as superoxide dismutase (SOD), glutathione peroxidase, and catalase convert them to H₂O₂ and finally into water. But due to TiO₂NPs induced large production of ROS, these enzymes are unable to counteract the damage, like triggering protein denaturation and cytoplasm leakage [102]. DNA is highly susceptible to ROS radicals, which attack the sugar phosphate bonds, causing DNA strand breaks. For bacteria, this molecular level damage is extremely detrimental since it impacts all critical cellular functions, jeopardising the cellular integrity and ultimately leading to cell death [101]. The above-mentioned physicochemical properties of TiO₂NPs influence ROS production and hence their toxicological effects.

Apart from ROS, TiO₂NPs impart their toxicological effects by attaching to bacteria. The exact mechanism of toxicity is unknown; however, it is assumed that multilayers of NPs on the bacterial surface may block the ion channels, resulting in osmotic imbalance [21]. In a recent study, E. coli has been shown to produce membrane vesicles and repair damaged cell membrane parts to overcome this osmotic imbalance [21]. These new insights into bacterial defence mechanisms need to be further investigated in order to better understand, not just the interactions between TiO₂NPs and microbes in the environment, but also to combat antimicrobial resistance (AMR).

Methods to Assess TiO₂NPs Toxicity

Toxicity of a particular compound towards bacteria can be checked by CFU count, however, this method is not suitable to assess TiO₂NPs toxicity as they make aggregates with bacteria, which cannot be broken down by simple vortexing. Hence, colonies arising on media plates, after a particular incubation time, are not derived from a single cell but from a collection of cells, which can mislead the results. Commonly used methods for assessment of bacterial susceptibility or resistance to antimicrobials such as TiO₂NPs are growth inhibition assay, disc diffusion assay, BacLight assay, SEM and TEM [96, 97]. TiO₂NPs induced ROS production, (hydroxyl radicals, hydrogen peroxide and peroxyl radicals) can be measured by using the probe 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA). This is a non-fluorescent dye but when it diffuses through bacterial plasma membrane, it is hydrolysed by cellular esterases and converts into 2,7-dichlorodihydrofluorescein (DCFH). DCFH is further converted into fluorescent 2,7- dicholorofluorescein (DCF) which can be detected by absorption or fluorescence spectroscopy [103, 104]. Also, bacterial cell membrane damage and cell morphology changes caused by ROS can be measured by lipid peroxidation assay and field emission scanning electron microscopy (FESEM), respectively [98]. A powerful method for evaluating spatially resolved TiO₂NPs effects on bacterial biosurface nanomechanics at single-cell level is done by multiparametric atomic force microscopy/spectroscopy (AFM). It also incorporates electrophoretic cell fingerprinting into the context of TiO₂NPs toxicity assessment at a level that goes beyond the usual zeta-potential approach, which is ineffective for deciphering the electrokinetic features of soft (ion and flow-permeable) bacterial surfaces [21]. Various methods and parameters used in different toxicology studies of TiO₂NPs vary in different laboratories, therefore, it is essential to standardize physicochemical parameters and methods, to understand the actual behavior of NPs towards bacteria under native conditions for more effective and conclusive ecotoxicological studies (Table.1).

Table 1.

Different methods used to assess the toxicity of some commonly used NPs towards bacteria

NPs	Size(nm)/morphology	Model bacteria	Experimental conditions	Toxicity	References
CuO	> 50/na	Nitrifying bacteria in soil	Sonicated NPs used to assess NEA and DEA in soil microcosms. Microbial abundance measured by qPCR	NEA and DEA reduced by ~ 50% and ~ 40%, respectively, after 90 days exposure to upto 100 mg Kg−1 NPs	[105]
Ag	10–50/spherical	Azotobacter vinelandii	Bacteria exposed to NPs on solid media and flow cytometry, nitrogenase activity, ROS production used to assess toxicity	0.1 mg L−1 NPs inhibited bacterial growth and fourfold increase in ROS production after 4 h treatment and upto 60% reduction in nitrogenase activity	[106]
	10–80/spherical 30–60/spherical	Escherichia coli Pseudomonas aeruginosa Escherichia coli Streptococcus aureus	Resting cells exposed to NPs. LIVE/DEAD BacLight test, ATP production assay and flow cytometry used to check toxicity Bacterial cells exposed to NPs and growth inhibition checked by Kirby-Bauer method	Dose-dependent toxicity, upto 2.5 mg L−1 NPs inhibited P. aeruginosa growth, 80 mg L−1 NPs inhibited E. coli K12 growth and delayed exponential phase NPs showed zone of inhibition in the range of 2.2 cm for S. aureus and 2.1 cm against E. coli. MIC was found to be ~ 30 µM	[107–109]
CuO, NiO, ZnO and Sb2O3	10–210/na	Escherichia coli Bacillus subtilis Streptococcus aureus	Bacteria exposed to sonicated NPs dispersed in agar medium and toxicity checked by CFU method	Maximum growth inhibition of upto 90% was seen with CuO NPs (125 mg L−1) and minimum was seen with ZnO NPs (upto 60%)	[110]
Al2O3	< 50/na	Pseudomonas putida	Bacteria exposed to sonicated NPs and growth inhibition checked by turbidity method	200 mg L−1 NPs showed ~ 80% growth inhibition	[111]
	< 50/spherical	Soil Microorganisms	Microcalorimetric measurements and urease activity	Low toxicity below 100 µg mL−1; toxicity increased with increased concentration	[112]
Au	16/spherical	Salmonella typhimuriumCHO cells	Growth inhibition and mutagenic test was conducted in the presence of light and in the dark	No toxicity in S. typhimurium. NPs were photo-mutagenic due to the presence of Au+ citrate ions. Concentration upto 50 µg mL−1 could induce substantial DNA damage (~ 20%) in CHO cells	[113]
TiO2	6/spherical	Escherichia coli Bacillus subtilis	Resting cells exposed to sonicated NPs (pH and ionic strength measured). Toxicity checked by CFU method Disc diffusion method, ROS generated DCF, PI and SYTO9 fluorescent dyes for viable cell count	Growth inhibition of upto 80% was seen with TiO2NPs (100 mg L−1) MIC was found to be ~ 200 µg mL−1	[96, 98]
Fe3O4	50/spherical	Vibrio fischeri	Acute toxicity levels were analysed using Microtox bioassay	Pure Fe3O4 showed higher acute toxicity (EC50 ~ 250 µg mL−1)	[114, 115]
	30–50/spherical, and 1–3 µm/yolk-shell			Yolk-shell (EC50 ~ 650 µg mL−1) less toxic than pure Fe3O4 (EC50 ~ 350 µg mL−1)
Au-peptide	25/spherical	Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis	Agar diffusion assay	Au-peptide showed zone of inhibition in the range of 22–25 mm	[116]
CELE-Ag	15/spherical	Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Candida tropicalis, Candida krusei, Candida lusitaniae, Candida guilliemondii, Penicillium chrysogenum	Agar diffusion assay	CELE-AgNPs showed zone of inhibition in the range 10–24 mm	[117]
Ag-composite	15/spherical	Staphylococcus aureus, Escherichia coli	CFU method	MIC of Ag-GSiO2 NPs was 0.3 mg mL−1 for S. aureus and 0.35 mg mL−1 for E. coli	[118]
Fe-composite	100/spherical	Vibrio fischeri	Acute toxicity levels were analysed using Microtox bioassay	EC50 values upto 415 μg/mL after 15 min	[119]

na Data or information is not available

Conclusions and Future Directions

In twenty-first century, TiO₂NPs have widespread applications, but they also pose tremendous challenges for scientists, owing to their ecotoxicological effects. Bacteria are ideal model systems for assessing the toxicity of novel antimicrobials and other eco-toxic compounds. Although, TiO₂NPs toxicity has been extensively documented from laboratory studies on E. coli and other bacteria, their harmful effects in the environment are not well understood. As TiO₂NPs toxicity depends on their physicochemical properties and bacterial surface characteristics, it is critical to consider bacterial physiology in different aqueous media of specific IS and pH; however, these parameters are often neglected during in vitro studies. Gaps in toxicological studies must be addressed, and new standardised methodologies for investigating the harmful effects of TiO₂NPs on bacteria must be developed and designed. All these efforts are warranted in order to do a risk assessment of TiO₂NPs ecotoxicity. Nanoparticles are promising antimicrobial alternative to combat AMR. They can act synergistically with a wide variety of antibiotics, to augment the antibacterial activity of traditional antibiotics. However, their toxicological effects must be thoroughly investigated before they can be used as an alternative antimicrobials. Green synthesis of TiO₂NPs, an environment friendly approach for the production of non-toxic NPs, can be helpful in the production of safer and more stable NPs and tap their full potential for various ecological engineering and commercial applications. Hence, characterization of TiO₂NPs and bacteria is important to better understand their interactions in a holistic manner.

Abbreviations

NPs

Nanoparticles

TiO₂NPs

Titanium dioxide nanoparticles

NOM

Natural organic matter

EPS

Extracellular polymeric substances

IS

Ionic strength

DLVO

Derjaguin, Landau, Verwey, and Overbeek

EM

Electrophoretic mobility

ROS

Reactive oxygen species

PZC

Point of zero charge

GSH

Glutathione

TEM

Transmission electron microscopy

SEM

Scanning electron microscopy

SOD

Superoxide dismutase

AMR

Antimicrobial resistance

CFU

Colony forming units

H2DCF-DA

2',7'-Dichlorodihydrofluorescein diacetate

DCFH

2,7-Dichlorodihydrofluorescein

DCF

2,7- Dicholorofluorescein

FESEM

Field emission scanning electron microscopy

AFM

Atomic force microscopy/spectroscopy

NEA

Nitrification enzyme activity

DEA

Denitrification enzyme activity

CHO

Chinese hamster ovary cells

MIC

Minimum inhibitory concentration

EC₅₀

Half maximal effective concentration

Author Contributions

PS and RK designed and wrote the manuscript. MY contributed in writing and proof reading.

Funding

Not Applicable.

Declarations

Conflict of interest

The authors declare that there is no conflict of interest.