At the point of zero charge (pzc) at pH = 6.2, TiO₂ NPs have negative charges on their surface, which shows a less bactericidal effect in neutral and alkaline solutions. This is so because, at these conditions, TiO₂ NPs repel bacteria with a minus charge in the absence of light (Zhang et al., 2017; Sharma et al., 2022). During acidic pH conditions, the TiO₂ NPs are positively charged and interact strongly with the bacterial cells, resulting in the penetration of the bacterial membrane and inducing oxidative damage accordingly (Pagnout et al., 2012). Figure 2 shows a detailed sequence involved in the toxicity of TiO₂ NPs for microorganisms. TiO₂ inhibits or kills microorganisms by adsorbing TiO₂ NPs on the surface of the microorganism. There is a formation of reactive oxygen species [ROS] (•O2-,•OH) which first interacts with the lipids present on the surface of the membrane of the microorganisms (Khan et al., 2022). The interaction between lipids on the membrane and ROS leads to lipid peroxidation. In addition to this, the permeability of the lipid membrane increases. The ROS, after damaging the membrane lipids, gains entry into the cytoplasm of the microorganism, where it damages the various cellular organelles such as the mitochondria, nucleus, and their DNA and ribosomal proteins. The oxidation of proteins and DNA by ROS leads to their denaturation. Finally, all these damaged cytoplasmic contents flow out from the damaged lipid membrane, leading to the killing of microorganisms.

Earlier, several investigators have also shown the toxicity of TiO₂ NPs on various microorganisms; for instance, Kiwi et al. exhibited that TiO₂ NPs have a bactericidal effect on Escherichia coli by direct contact in dark conditions. During this process, the cell wall gets damaged because of the electrostatic attraction between the TiO₂ NPs and the minus-charged bacterial cell wall at a pH close to but below pzc (Kiwi et al., 2014). The antibacterial activity of TiO₂ NPs is mainly due to the production of ROS in the presence of UV light, suggesting that the bactericidal effect is due to UV light and not due to TiO₂ NPs (Vatansever et al., 2013). TiO₂ NPs have also shown potential for the killing of multidrug-resistant bacteria through the reactive radicals produced by electron–hole pairs upon UV irradiation (Kubacka et al., 2014). The inactivation of drug-resistant bacteria by a photocatalytic material such as TiO₂ NPs relies on the power and irradiation time of UV-A light (Tsai et al., 2010). Hence, the disinfection method needs a high-power UV source to excite TiO₂ NPs, and in visible light, there are fewer bactericidal uses owing to their ineffective photoexcitation. Due to this, in an indoor environment where there is a small amount of UV light, the efficiency of TiO₂ NPs against microorganisms is limited. As a result, the development of such TiO₂ NPs that may be activated with visible light in addition to their excellent antibacterial properties is one of the most urgent needs in the medical and industrial sectors.

Microbes have various enzymes, metabolites, and pigments that are responsible for transforming metal ions into metal oxides or metallic NPs (Choudhary et al., 2023). To date, metallic NPs have been synthesized by bacteria, fungi, actinomycetes, algae, and viruses (Choudhary et al., 2023; Dadhwal et al., 2023). Several investigators have synthesized TiO₂ NPs by using all these types of microorganisms. The TiO₂ NPs synthesized from the microbes have several advantages and features, such as biocompatibility, being eco-friendly, and being non-toxic (Ahmad and Kalra, 2020; Yadav et al., 2020b; Tripathy et al., 2023). The formation of TiO₂ NPs by microorganisms is explained below in detail.

To date, bacteria have been used most extensively for the synthesis of TiO₂ NPs. Bacteria are rich in several biomolecules that transform the Ti salts into TiO₂ NPs (Yadav et al., 2020b). There are several bacteria that could synthesize TiO₂ NPs, either intracellularly or depending on the nature of the bacteria (Farag et al., 2021). Due to changes in the environment, the bacteria become resistant to some of the metals that help them in the synthesis of NPs. The natural defense system is present in bacteria, which makes them resistant to harsh conditions. Bacterial-mediated synthesis can take place by all three means, i.e., by whole cells, supernatants, and extracts. Supernatants can be taken after the centrifugation of the bacterial culture, which has enzymes, microbial proteins, and metabolites secreted by the bacteria or released after centrifugation. Moreover, TiO₂ NPs can also be synthesized by using bacterial pellets after dispersing them with distilled water and providing a titanium precursor (Liou and Chang, 2012). The transformation of Ti³⁺ ions into TiO₂ NPs by microorganisms involves three basic steps, i.e., trapping, bioreduction, and capping. First, the Ti³⁺ ions get trapped by the bacteria in the aqueous solution or surrounding medium. Furthermore, with the help of enzymes and proteins, the trapped Ti³⁺ ions get reduced into TiO₂ NPs. From the investigations, it has been proven that the microbial proteins having functional groups –NH₂, –SH, –COOH, and –OH help in stabilizing the synthesized TiO₂ NPs (Wang B. et al., 2021; Hazem Najem et al., 2023). These functional groups generally provide a site for the binding of metallic ions such as Ti³⁺ ions in addition to a capping agent or stabilizing agent. Immediate to this, there is the reduction of Ti³⁺ ions into its NPs. The reduction of Ti³⁺ ions takes place either on the cell wall or in the periplasmic space. During this step, electrons move from reduced compounds to inorganic compounds. This promotes the bioreduction process in bacteria for NPs. Finally, the reduced TiO₂ NPs get capped by the various biomolecules present in the bacteria, acting as a natural capping agent for the synthesized TiO₂ NPs (Baig et al., 2020). Capping helps in maintaining the stability of NPs, which is an important factor. There are a few examples where these microbial proteins acted as the major reducing or capping agents at the time of formation and stabilization of TiO₂ NPs (Lahiri et al., 2021). Jha et al. (2009) and Jayaseelan et al. (2013) described a similar mechanism in Lactobacillus and Aeromonas hydrophila, respectively.

Jha et al. (2009) hypothesized a series of chemical reactions involved in the formation of TiO₂ NPs by using Lactobacillus bacteria.

Moreover, Jha et al. also deduced a schematic for the biosynthesis of TiO₂ NPs, which is shown below in Figure 3. Lactobacillus is said to have pH-dependent membrane-bound oxidoreductases, which exhibit oxidase activity at lower pH. Due to this, the titanium hydroxide gets converted into TiO₂ NPs by producing H₂O as a by-product (Jha et al., 2009).

Jayaseelen et al. suggested another possible mechanism for the synthesis of TiO₂ NPs by using A. hydrophila, which is shown in Figure 4. According to the investigators, the secondary metabolites produced by A. hydrophila, especially glycyl-L-proline and compounds having –COOH and –C=O as a functional group, have a major function in the synthesis of TiO₂ NPs. Furthermore, investigators suggested that the Ti precursor (titanyl hydroxide) can be dehydrated by the glycyl-L-proline to give TiO₂ NPs once the broth of A. hydrophila interacts with the precursor at ~30°C for 24 h. Furthermore, the investigators suggested that the synthesis of TiO₂ NPs by A. hydrophila could be accomplished in a series of steps. In the first step, one of the lone pairs of electrons present in O₂ picks up an H⁺ ion from the glycyl-L-proline. In the second step, there is protonation of TiO(OH)₂, while in the third step, the protonated TiO(OH)₂ loses an H₂O molecule, resulting in the formation of Ti³⁺ ions. Finally, an intermediate compound (5) pulls off a H⁺ ion from the Ti³⁺. In this study, the investigator concluded that the stability of the synthesized TiO₂ NPs is mainly due to the -COOH group containing water-soluble compounds. All these hypotheses and suggestions were based on the gas chromatography-mass spectroscopy (GCMS) analysis of the A. hydrophila broth culture. The GCMS showed four major compounds, namely uric acid (2.95%), glycyl-L-glutamic acid (6.90%), glycyl-L-proline (74.41%), and l-Leucyl-d-leucine (15.74%). Moreover, investigators concluded the presence of mainly two functional groups in the sample, namely –COOH and –C=O. So, the investigators finally concluded that these two compounds may play a significant role in the synthesis of TiO₂ NPs as stabilizing and capping agents (Jayaseelan et al., 2013).

From the various pieces of literature, it has been found that to date, 9–10 different bacteria have been used for the synthesis of TiO₂ NPs, for instance, three species of Bacillus (B. subtilis, Bacillus mycoides, and Bacillus amyloliquefaciens), two Lactobacillus (Lactobacillus johnsonii and Lactobacillus sps), one Propionibacterium jensenii, S. aureus, Planomicrobium sps, Halomonas elongata IBRC-M 10214, and A. hydrophila. Table 2 shows the various previous attempts by investigators to synthesize TiO₂ NPs. To date, various investigators have synthesized TiO₂ NPs by using bacteria and fungi; for instance, Jayaseelan et al. (2013) synthesized 28–84 nm-sized TiO₂ NPs using A. hydrophila. Landage et al. synthesized spherical-shaped 20 nm TiO₂ NPs by using S. aureus and later applied them for antibacterial activity. Al-Zahrani et al. (2018) used L. johnsonii for the synthesis of 4–9 nm-sized TiO₂ NPs. Among bacteria, Bacillus species have been used earlier for the synthesis of TiO₂ NPs (Khan and Fulekar, 2016). Taran et al. (2018) synthesized 104.63 ± 27.75 nm TiO₂ NPs by using H. elongata IBRC-M 10214. As far as Bacillus species are concerned, it was earlier used by Khan and Fulekar (2016) and Kirthi et al. for the synthesis of TiO₂ NPs. Kirthi et al. synthesized oval to spherical shapes of size 67–77 nm by using B. subtilis. Bacillus mycoides has been recently utilized for the synthesis of TiO₂ NPs at low temperatures according to Yamauchi et al. (2011) and Órdenes-Aenishanslins et al. (2014). Khan and Fulekar (2016) synthesized spherical TiO₂ NPs by using B. amyloliquefaciens, where the size of the TiO₂ NPs varied from 22.11 to 97.28 nm. The authors further revealed that alpha-amylase is accountable for the synthesis of TiO₂ NPs (Khan and Fulekar, 2016).

From the bacterial synthesis of TiO₂ NPs, it has been concluded that the majority of Gram-positive bacteria have been used for the synthesis of TiO₂ NPs. Among all the approaches, Ti(OH)₂ has been used maximally by investigators for the synthesis of TiO₂ NPs.

Actinomycetes are higher-GC-containing fungi that are mainly used for the production of antibiotics. In addition to this, several investigators synthesized TiO₂ NPs by using actinomycetes. Some of the most recent examples are highlighted below. Agceli et al. synthesized spherical-shaped, 30 to 70 nm TiO₂ NPs by utilizing Streptomyces sp. HCl. The developed TiO₂ NPs were evaluated for their antimicrobial activity against pathogenic bacteria S. aureus ATCC 29213, E. coli ATCC 35218, Candida albicans ATCC 10231, and fungi A. niger ATCC 6275. Investigators concluded that the TiO₂ NPs showed higher antimicrobial properties against bacteria than fungi (Agçeli et al., 2020).

An investigation led by Priyaragini synthesized TiO₂ NPs from the precursor's titanium hydroxide by using marine actinobacteria, i.e., Streptomyces bluensis. This particular strain was collected from the coastal area of Tamil Nadu, India. The spherical-shaped TiO₂ NP average size was 37.54 nm, which was further used for the photocatalytic degradation of Acid Red 79 (AR-79) and Acid Red 80 (AR-80) azo dyes with an efficiency of 84 and 85%, respectively (Priyaragini et al., 2014).

In comparison with bacteria, fungi are most preferred by scientists for the biosynthesis of TiO₂ NPs, as most of the fungi are extracellular, allowing easy recovery of the NPs. In addition to this, large-scale production and economic feasibility are the other factors for the fungi-mediated synthesis of TiO₂ NPs (Irshad et al., 2021). Various enzymes present in the fungus make them adaptable to different environmental conditions. Enzymes are accountable for the reduction of Ti³⁺ ions into oxide, and NADPH acts as a co-factor in this mechanism. There are various species for the synthesis of NPs, such as A. niger, Aspergillus flavus, and Fusarium oxysporum. Jha and their team synthesized 12.57 ± 0.22 nm, spherical-shaped TiO₂ NPs by using Saccharomyces cerevisiae (yeast) by using TiO(OH)₂, 0.025 M as a precursor. In this study, the investigators first heated the 24-h-old yeast culture on a water bath heater at 60°C for 10–20 min, which was further incubated at 28–30°C for the formation of TiO₂ NPs. Furthermore, the culture was harvested after 48–72 h by centrifugation. The characterization of the yeast-mediated synthesized TiO₂ NPs by Fourier transform infrared (FTIR) bands exhibited typical bands in the region of 400–3,600 cm⁻¹. In addition to this, X-ray diffraction (XRD) analysis revealed the two major intensity peaks at two thetas of 25 and 28°, which were assigned to anatase (101) and rutile, respectively. In this study, the investigators obtained an average particle size of ~18 nm by XRD, which was in close agreement with the result obtained by TEM. Investigators parallelly used Lactobacillus for the TiO₂ NPs and concluded that under similar conditions, yeast produced smaller-sized TiO₂ NPs, i.e., 18 nm, in comparison with Lactobacillus (30 nm). This is so because yeast is eukaryotic in nature and has a better level of organization at the cellular level. Furthermore, the investigator suggested a series of chemical reactions involved in the biotransformation of titanyl hydroxide to TiO₂ NPs by yeast. During the TiO₂ NPs synthesis by yeast, the glucose sugar molecules are converted to ethanol, acetaldehyde, and finally to acetic acid. Furthermore, this acetic acid gets ionized into acetate ions and H⁺ ions. Furthermore, sodium hydrogen carbonate present in the medium on ionization produces Na⁺ ions and carbonate ions. Furthermore, these hydrogen HCO3- ions split to produce hydroxyl ions and carbon dioxide gas. Finally, the OH ions generated in the previous reactions react with Ti³⁺ ions to form TiO₂ NPs by releasing a water molecule.

The investigators also explained the mechanism of the formation of TiO₂ NPs by yeast, which was almost similar to the mechanism reported in the case of Lactobacillus. As per the investigations, it was found that yeast has oxidoreductase and quinones, which are present on the membrane surface and in the cytosol too. Being pH-sensitive, oxidoreductase gets activated at lower pHs, while at higher pH values, it activates the reductase. While another molecule, quinone, facilitates the redox reaction due to tautomerization. When TiO(OH)₂ is added to an aqueous medium containing yeast, tautomerization of quinones and oxidases (sensitive at low pH) occurs, making molecular O₂ available for biotransformation. Once TiO(OH)₂ enters the cytosol, it will trigger the family of oxygenases present in the endoplasmic reticulum (ER). ER is known for detoxification at the cellular level by the phenomenon of oxidation/oxygenation (Jha et al., 2009).

Bansal et al. reported the biosynthesis of titania from A. niger by using K₂TiF₆ as a precursor. In this study, first, the fungus was grown in malt, glucose, yeast, and peptone (MGYP) medium, and later on, mycelia were separated from the medium, washed with deionized water, followed by an addition of 20 g of wet mycelia of fungi and 100 ml of aqueous solutions of K₂TiF₆, and kept under shaking conditions for 1 day. The investigators further characterized the sample by transmission electron microscopy (TEM) and scattering area electron diffraction (SAED) and found that the synthesized titania was spherical-shaped with sizes varying from 6 to 13 nm with an average size of 10.2 ± 0.1 nm. The SAED pattern showed a sharp ring for the calcinated TiO₂ NPs. This indicated the formation of brookite structures for titania. From the XRD and TEM, it was found that the d-values obtained (3.47 A°, 2.24 A°, 1.97 A°, and 1.28 A°) match reasonably well with the standard d-values (3.46 A°, 2.24 A°, 1.97 A°, and 1.28 A°) for the 111, 022, 032, and 004 planes, respectively, of the brookite polymorph of TiO₂ (Bansal et al., 2005).

Tarafdar et al. (2013) synthesized TiO₂ NPs of size <100 nm by using Aspergillus tubingensis. Aspergillus niger-mediated synthesis of TiO₂ NPs of size 73.58–106.9 nm was reported by Durairaj et al. (2014). Rajakumar et al. synthesized oval-shaped TiO₂ NPs of size 62–74 nm from A. flavus and assessed their antimicrobial activity against E. coli. The FTIR investigations by Rajakumar et al. (2012) observed a band at 590 cm⁻¹, which is attributed to the Ti-O bonds.

Raliya et al. synthesized spherical TiO₂ NPs whose size varied from 12 to 15 nm by using A. flavus TFR 7 with a purity of ~94% (atomic wt.%). Furthermore, the investigators assessed its physiological activity on mung bean (Vigna radiata L.). Investigators found that the plants treated with TiO₂ NPs in comparison with micron-sized TiO₂ were comparatively taller. In this study, the authors proposed a mechanism for the fungal-mediated synthesis of TiO₂ NPs, which is shown below (Figure 5).

In this study, A. flavus TFR 7 secretes enzymes outside and synthesizes the TiO₂ NPs extracellularly. The synthesized TiO₂ NPs obtained over here were highly pure, monodisperse nanoparticles, and free from cellular debris. Moreover, the downstream processing of TiO₂ NPs from A. flavus TFR 7 was very easy. Furthermore, investigators suggested a mechanism for the biotransformation of TiO₂ NPs from titanium precursors. The first step involves the secretion of extracellular enzymes by the fungi, which encapsulate the TiO₂ NPs by capping protein, which increases the stability of TiO₂ NPs. In addition to this, the associated proteins may help in the biotransformation of precursor salt (Raliya et al., 2015).

Chinnaperumal et al. (2018) extracellularly synthesized regular spherical-shaped 60–86.67 nm TiO₂ NPs by using Trichoderma viride and assessed their larvicidal, antifeedant, and pupicidal activity against Helicoverpa armigera. For the synthesis, the investigators used 10, 50, and 100% of fungal supernatant and mixed it with 20 ml of 5 mM TiO(OH)₂, followed by incubation along with shaking at 120 rpm for 24 h at 30°C. From the XRD investigation, peaks were obtained at peaks at 27.41°, 32.34°, 44.27°, 54.29°, and 64.55° which corresponded to (1 1 0), (1 0 0), (1 1 1), (2 1 1), and (3 0 1), indicating rutile form. From the FTIR analysis, investigators found bands at 3,430.48 cm⁻¹ (O–H stretch), 2,923.87 cm⁻¹ (O–H stretch; carboxylic acids), 2,148.48 cm⁻¹ (–C=C– stretch; alkynes), 1,729.88 cm⁻¹ (C=O stretch; carboxylic acids), 1,648.25 cm⁻¹ (–C=C– stretch; alkenes), 1,424.81 cm⁻¹ (C–C stretch), 1,375.87 cm⁻¹ (C–H rock; alkanes), 1,317.13 cm⁻¹ (C–N stretch; aromatic amines), 1,252.45 cm⁻¹ (C–N stretch), 1,038.87 cm⁻¹ (C–O stretch; alcohols, carboxylic acids, esters, and ethers), and 563.83 cm⁻¹ (C–Cl stretch; alkyl halides) (Chinnaperumal et al., 2018).

Heitzschold et al. stated that the synthesis of NPs takes place through the action of the NADPH factor, but studies have found that changes in pH, temperature, incubation time, type of fungi, and the source used can definitely affect the morphology of NPs. Rehman et al. reported the synthesis of TiO₂ and Ag NPs by using the wild mushroom Fomitopsis pinicola. Furthermore, the investigator evaluated the potential of both synthesized NPs against E. coli and S. aureus. Moreover, NPs were also evaluated on the human colon cancer cell line (HCT) by minimum inhibitory concentration/minimum bactericidal concentration (MIC/MBC) and MTT assays (Rehman et al., 2020).

Sathiyaseelen et al. synthesized TiO₂ NPs by using the endophytic fungus Paraconiothyrium Brasiliense, which was further assessed for its antibacterial activities. The synthesized TiO₂ NPs were spherical in shape, whose size was ~57.39 ± 13.65 nm by TEM, whereas the average hydrodynamic size was (68.43 ± 1.49 d. nm), and the zeta potential was found to be (−19.6 ± 1.49 mV) (Sathiyaseelan et al., 2022).

In one of the most recent attempts, Survase and Kanase (2023) synthesized TiO₂ nanospheres from precursor titanium chloride (TiCl₃) by using Aspergillus eucalypticola SLF1. Furthermore, the investigators used TiO₂ for the antimicrobial activity and dye removal (reactive blue 194) (Survase and Kanase, 2023).

One of the most important groups of photosynthetic organisms is algae. Various pieces of literature have shown that algae have a tendency to accumulate higher levels of heavy metals, so this property can be exploited for the biosynthesis of metallic and metal oxide NPs (Priyadarshini et al., 2019; You et al., 2021). Algae have been widely used for the synthesis of TiO₂ NPs due to their easy access and efficacy. In addition to enzymes and proteins, algae also have carotenoids and various photosynthetic pigments, which play an important role in the phyco-assisted synthesis of TiO₂ NPs. However, the algal-mediated synthesis of TiO₂ NPs is not as developed as for bacteria. Moreover, NPs could also be synthesized by algae by using their extracts/supernatant, which contain secondary metabolites. Hifney et al. (2022) synthesized TiO₂ NPs by using the algae Spirulina platensis. Vasanth et al. (2022) synthesized spherical-shaped TiO₂ NPs of 90 to 150 nm size by using S. platensis extract. Figure 6 shows the various approaches for the physio-assisted synthesis of TiO₂ NPs.

In one of the most recent investigations, Mathivanan et al. (2023) synthesized TiO₂ NPs from Sargassum wightii (seaweed) and evaluated their potential for killing the larvae of vectors responsible for causing malaria and filariasis. In another attempt, Balaraman et al. (2022) synthesized negatively charged cubic, square, and spherical-shaped, ~50–90 nm-sized TiO₂ NPs by using Sargassum myriocystum. Furthermore, the investigator assessed the antimicrobial activity of the synthesized TiO₂ NPs.

Viruses are a significant example of the synthesis of nanosized particles. To date, viruses have been used for the synthesis of nanotubes, nanorods, etc. (Koudelka et al., 2015). It has been shown by studies and experiments that plant viruses and some bacteriophages are easy to isolate and process further. All the parts of the virus cannot be used to formulate nanoparticles, and the reason still needs to be studied perfectly. Zhou et al. (2014) reported the formation of chalcogenide nanocrystals inside the genetically modified virus-like particles. Órdenes-Aenishanslins et al. also emphasized the importance of viruses in the synthesis of various types of NPs (Órdenes-Aenishanslins et al., 2014).

In general, UV-Vis analysis of the TiO2 NPs is not relevant for the synthesis, but it is very important for calculating the band gap, especially when the synthesized TiO₂ NPs are used in electronics (Abouhaswa, 2020). As a semiconductor, TiO₂ NPs, in order to be used in electronics, must have an optimal band gap which can be calculated by UV-diffraction reflectance spectroscopy (UV-DRS). UV-Vis analysis provides a peak in the range of 300–400 nm depending on the morphology and route used for the synthesis of TiO₂ NPs. Previously, several investigators have analyzed the bacterially synthesized TiO₂ NPs by UV-Vis spectrophotometer and suggested the formation of TiO₂ NPs by different types of bacteria, which is shown in Table 3.

From Table 3, it could be concluded that the UV-Vis peak for the TiO₂ NPs synthesized by bacteria mainly falls above 350 nm and maximum to 400 nm wavelength, while in a few cases, the peak was also obtained below 350 nm. The variations in the peak for TiO₂ NPs synthesized by bacteria could be mainly due to the presence of biologically different biomolecules, shapes, and sizes of the TiO₂ NPs as size and morphology affect the surface plasmon resonance.

When TiO₂ NPs are synthesized by bacteria or any other microorganism, it becomes very important to know the biomolecules associated with the synthesized TiO₂ NPs. FTIR analysis will reveal which functional groups are responsible for the stabilization and capping of the synthesized TiO₂ NPs. Moreover, some of the organic compounds, such as glycyl-L-proline in the case of A. hydrophila, are responsible for the biotransformation of titanyl hydroxide into TiO₂ NPs, which can be analyzed with FTIR in addition to other analytical tools. Jayaseelen et al. obtained bands in the range of 400–4,000 cm⁻¹ for the bacterially synthesized TiO₂ NPs. The major outcome from all the FTIR investigations was that the major and prominent bands were ~3,430, 2,937, 1,643, 1,403, 1,079 cm⁻¹, and 700–500 cm⁻¹. The bands of ~3,200–3,600 cm⁻¹ could be attributed to the -OH stretching from an alcoholic group present in the enzymes or microbial proteins of the bacteria. The band at ~1,578 cm⁻¹ indicates the presence of C–C ring stretching. The investigators reported that the bands at ~2,923 cm⁻¹, 1,649 cm⁻¹, and 679 cm⁻¹ could be attributed to the lipids and proteins associated with the synthesis of TiO₂ NPs (Landage et al., 2020). One major revelation made by FTIR by a group of investigators was that the band ~1,235 cm⁻¹ indicates amide linkage between bacterial proteins and TiO₂ NPs (Babitha and Korrapati, 2013), while the band for Ti-O stretching vibration could be obtained near 518 cm⁻¹ (Chelladurai et al., 2013). Priyaragini et al. (2014) obtained four major bands for the TiO₂ NPs synthesized by S. bluensis at 2,065.76 cm⁻¹, 1,637.56 cm⁻¹, 1,384.89 cm⁻¹, and 644.22 cm⁻¹ which were attributed to the C–H aldehyde stretching, C=C conjugate, NO₂ conjugate, and alkynes, respectively.

Chakravarty et al. obtained the FTIR bands in the region of 3,400–2,400 cm⁻¹ corresponding to the stretching vibration of terminating hydroxyl groups in samples, while the band at 1,350–1,000 cm⁻¹ attributed to the C–N stretching of the amine group, and 1,550–1,350 cm⁻¹ corresponds to nitro (N– – O) groups. In addition to this, the investigator also obtained intense bands at 500–700 cm⁻¹ attributed to the Ti–O stretching band and Ti–O–Ti bridging stretching modes (Chakravarty et al., 2023).

Since TiO₂ NPs exist in three different phases in nature, it becomes very important to analyze the synthesized TiO₂ NPs by XRD. The different phases of TiO₂ NPs are characterized by different peaks; for instance, a sharp peak near two theta 25–26° indicates the crystalline anatase phase, while the peak near two theta 27–28° is due to the rutile phase. Previously, numerous investigators synthesized TiO₂ NPs by different bacteria under different temperatures, and other parameters are shown in Table 4.

Electron microscopy (scanning and transmission) could be used to reveal the shape and size of the bacterially synthesized TiO₂ NPs (Yang R. et al., 2022; Yang D. et al., 2023). The range of size becomes very important when it has to be applied in the fields of electronics and medicine. The electron microscopy could reveal the carbon molecules associated with the TiO₂ NPs, as the non-metal area will be electron deficient and will appear darker in color in scanning electron microscopy, while in transmission electron microscopy that area will appear brighter in comparison with the dark Ti element. So, the data from electron microscopy in addition to FTIR and XRD could help in revealing the association of biomolecules with the TiO₂ NPs (Liu et al., 2018; Zhang et al., 2023). Moreover, the elemental analyzer attached to the electron microscopy helps in revealing the chemical composition and purity of the synthesized TiO₂ NPs. Various investigators have reported different sizes and shapes of the bacterially synthesized TiO₂ NPs, which are already shown in Table 2. From the electron microscopic investigation of the previously reported study, it was found that in the majority of the cases, the bacterially synthesized TiO₂ NPs were spherical in shape, while a few have also obtained an oval to irregular shape (Wang Z. et al., 2022; Xia et al., 2022). Some of them have also reported the aggregation of the TiO₂ NPs synthesized by bacteria. The size of the synthesized TiO₂ NPs by bacteria varied from 10 nm to above 100 nm, where the smallest size, i.e., 10–30 nm, was obtained by using B. subtilis by Vishnu Kirthi et al. (2011) and Dhandapani et al. (2012), and the largest size, i.e., 104.63 ± 27.75 nm, was obtained by Taran et al. (2018) by using H. elongata IBRC-M 10214.

Wastewater is one of the foremost concerns nowadays. Sources of wastewater include industries, homes, factories, and transportation (Lito et al., 2012; Caprarescu et al., 2017). Water is an essential requirement for all living beings, so, due to the limited quantity of freshwater on the earth, it is of utmost importance to conserve water and recycle the wastewater (Chahar et al., 2023). The wastewater released by industries, domestics, factories, and transportation sources contains many contaminants such as heavy metals, toxic compounds, chemicals, and detrimental microorganisms (Yadav et al., 2020a). The pollutants inhaled by living beings will move from one tropic level to another, causing more absorption (Modi et al., 2022b; Yadav et al., 2023a). TiO₂ NPs have shown the potential to eradicate all these contaminants much more efficiently. The photodegradation of the pollutants (dyes) present in wastewater is due to the photocatalytic effect of nanosized TiO₂ (Agarwal et al., 2022). TiO₂ NPs have been used in wastewater because they provide complete mineralization of pollutants (Panahi et al., 2018). The TiO₂ NPs have been utilized in the laboratory as well as on-site for their property to clean wastewater. TiO₂ helps to remove xenobiotic compounds from wastewater (Qamar and Ahmad, 2021). Photodegradation is a property of NPs that helps in the complete mineralization of organic pollutants without leaving behind any harmful by-products (Chenab et al., 2020).

TiO₂ NPs have been proven to be effective in cleaning wastewater by reducing contaminants. The special antimicrobial activity makes TiO₂ and other metal oxides suitable for the elimination of pathogenic microorganisms from wastewater. TiO₂ NPs are able to produce ROS in a very short time, making them very effective for water treatment. TiO₂ NPs remediate the toxic dyes from the wastewater by exhibiting a photocatalytic effect on the dyes in the presence of UV light (Pare et al., 2022).

Khan and Fulekar (2016) used TiO₂ NPs synthesized by B. amyloliquifaciens for the removal of reactive red 31 in the presence of UV light. In this study, the investigators doped the TiO₂ NPs with dopants such as Ag, Pt, La, and Zn and applied them for reactive red 31 dye removal. Furthermore, the authors concluded that Pt-doped TiO₂ NPs were the most efficient in removing the dye at ~90.98%, while the as-synthesized TiO₂ NPs removed the dye at only 75.83% (Khan and Fulekar, 2016). Table 5 shows the applications of TiO₂ NPs and modified TiO₂ nanocomposite in controlling the growth of pathogenic and non-pathogenic microorganisms.

Priyaragini et al. photocatalytically degraded the two azo dyes (Acid Red 79 and Acid Red 80) by using crude extracts of S. bluensis, immobilized bacteria cells, and TiO₂ NPs synthesized from them. The maximum degradation of both dyes with immobilized cells was 88% for AR-79 and 81% for AR-80, whereas with TiO₂ NPs, AR-79, and AR-80 were found to be 84 and 85%, respectively. Moreover, investigators also remediated these dyes with crude extracts, with an efficiency of ~81% for AR-79 and 83% for AR-80. The investigators further conclude that free radicals of TiO₂ NPs bind with the positively charged azo dyes and decolorize them. So, the maximum degradation of azo dyes was achieved with immobilized bacterial cells (Priyaragini et al., 2014).

Among all the investigations where TiO₂ NPs and their nanocomposites were used for the remediation of pollutants from wastewater, the highest removal percentage of dye was noticed with crystal violet, which was 99.95% at 6.5 pH. In this study, the initial dye concentration was ~25 mg/L along with a catalyst dose of ~25 mg/L, and the photocatalyst dose was 29 mg. The nanocomposite used over here was Ag-TiO₂/graphene aerogel (Ag–TiO₂/GA–ATG). In one more attempt to photocatalytically degrade the Indigo Carmine dye by using nanocomposite nitrogen-doped TiO₂ NPs [N-doped TiO₂ NPs (NT₃M₄)], an efficiency of 99% was obtained within 70 min (Divya et al., 2022). As far as the dye removal efficiency of pure TiO₂ NPs is concerned, the highest efficiency achieved was 87% for MB dye. In this study, the size of the synthesized anatase phase of TiO₂ NPs was 12–18 nm, along with a spherical shape. There were only two attempts at the photocatalytic degradation of dye by using bacterial-mediated synthesized TiO₂ NPs, one by Priyaragini et al. (2014) and another by Khan and Fulekar (2016). The size of the B. amyloliquefaciens mediates synthesized TiO₂ NPs was 22.11–97.28 (by TEM), whose reactive red 31 dye degradation efficiency was 75% in comparison with the Pt-doped TiO₂ NPs, whose efficiency was 90.98%. So, it could be concluded that TiO₂ NPs alone cannot mineralize the dyes, so they must be used either in the nanocomposite form or in the metal-doped form for enhanced dye removal efficiency from the wastewater. When TiO₂ NPs were used alone for dye removal, smaller-sized TiO₂ NPs exhibited higher dye removal percentages; for instance, 87% of MB dye was removed with 12–18 nm-sized TiO₂ NPs, whereas remediation of reactive red 31 dye was ~75% with TiO₂ NPs of size 22.1–97.28 nm (Khan and Fulekar, 2016; Rathi and Jeice, 2023). Balaraman et al. (2022) attempted to degrade ~92.92% MB dye from wastewater by using 50–90 nm-sized TiO₂ NPs from S. myriocystum.

TiO₂ NPs not only find application in wastewater treatment, solar cells, and energy but also find application in the medical field, especially as an antimicrobial agent (Chen and Selloni, 2014). TiO₂ NPs are also being used in drug delivery, where the nanosize helps them cross the blood–brain barrier, ultimately making them powerful against neurological disorders. TiO₂ NPs themselves have antimicrobial properties, which makes them more useful in these areas. All the cells easily uptake the nanoparticle-based drugs, and they have very few side effects. Nanomedicines have been developed by using TiO₂ NPs to be used in organ transplants, cosmetics treatments, and skin treatments (Sagadevan et al., 2022). The porous structure of titanium helps in the regeneration of bones and some muscles (Ouyang et al., 2019).

Dental disinfectants based on the nanostructures of metal dioxide have been shown to be effective in relieving tooth- and gum-related problems. TiO₂ NPs have been used in root canal treatment and as fillers because of their antibacterial properties, as reported by a group of investigators. Titanium and its oxides are used in dental care due to their non-corrosive properties (Jowkar et al., 2020; Raura et al., 2020; Liu et al., 2022). TiO₂ NPs provide assistance in oral cancer therapy, but further research is needed to prove their functions in vivo (Sargazi et al., 2022). Figure 7 shows a possible phenomenon for the antimicrobial properties of the different metal oxide NPs and photocatalytic semiconductors (Regmi et al., 2018).

The left-hand side of the figure exhibits the activation of the photocatalytic semiconductor by visible light. ROS formation by various semiconductors destroys bacterial cell components, as shown by the red arrows. Ag, Cu, and Au NPs also generate ROS for a bactericidal effect. The green arrow represents targets of Ag. Reproduced with permission from Regmi et al. (2018).

Matsunaga et al. developed a Pt-loaded TiO₂ (TiO₂/Pt) and assessed its potential for antimicrobial and photoelectrochemical activities. The antibacterial effect of TiO₂/Pt was assessed against Lactobacillus acidophilus, S. cerevisiae, and E. coli (Matsunaga et al., 1985). Another group of investigators exhibited that under UV irradiation, TiO₂ NPs show photocatalytic antibacterial activity against viruses and multiple drug-resistant (MDR) bacteria (Bogdan et al., 2015). To date, several investigators have tried to increase the photocatalytic antibacterial properties of TiO₂ NPs. To date, several attempts have been made for the development of visible light-responsive metal and non–metal-doped TiO₂ NPs. Moreover, the antibacterial effect of these NPs was evaluated against Gram-negative bacteria such as E. coli, Acinetobacter baumannii, Shigella flexneri, and Gram-positive bacteria, for instance, S. aureus, B. subtilis, Listeria monocytogenes, and spores of Bacillus anthracis (Liou and Chang, 2012). This nano-TiO₂ photocatalyst has been used for the eradication of pathogenic bacteria, thereby minimizing the spread of microbial-related illnesses. Markov and Vidaković (2014) performed the antimicrobial activity of TiO₂ photocatalysts in a thin-film technique, petri-dish, and PTFE membrane-separated system. In this study, the investigators embedded the TiO₂ NPs in the polymeric matrices in order to obtain nanocomposites (Charpentier et al., 2012; Hegedus et al., 2017). The major advantage of embedding the TiO₂ NPs in the matrices was that there was no mechanical damage to the TiO₂ NPs (Liao et al., 2013).

In addition to the antimicrobial activity/bactericidal activity of TiO₂ NPs, they are also used as a photodynamic therapeutic (PDT) agent for killing cancerous cells in biomedical fields. It destroys cancer cells from the skin to the internal organs, both under UV and visible light sources (Liu et al., 2016). The PDT effect is exhibited due to the generation of ROS by TiO₂. Moreover, such NPs can damage cellular respiration in mitochondria, which will release electron transfer proteins and eventually lead to cell death. When the TiO₂ NPs are activated by the light, there is DNA fragmentation as a result of the electron transfer process. Such approaches exhibit the potential for reprograming gene coding either by deleting or inserting gene codons. Several investigators have also shown that the TiO₂ nanotubes can be applied for the light-controlled delivery of drugs for the treatment of diseased tissues upon UV light illumination (Liu et al., 2016).

Ngoepe et al. reported the synthesis of TiO₂ NPs by using Monsonia burkeana plant extract and used them for the photocatalytic degradation of MB dye and inhibition of E. coli. In this study, the author degraded the pollutants by ~85.5% of the simulated wastewater (Ngoepe et al., 2020). Table 6 shows the antimicrobial performance of TiO₂ and modified TiO₂ NPs under visible light.

From Table 6, it was found that either the TiO₂ NPs or their nanocomposite were used against both Gram-positive and Gram-negative bacteria in addition to yeast such as C. albicans and S. cerevisiae. Out of the tested pathogens, Gram-negative bacteria E. coli were used most widely against the synthesized TiO₂ NPs, followed by Klebsiella pneumoniae, E. faecalis, and P. aeruginosa. Among Gram-positive bacteria, S. aureus was extensively used for the evaluation of the synthesized TiO₂ NPs (Yu et al., 2022; Wang et al., 2023). The antimicrobial activity of the TiO₂ NPs synthesized by bacteria, along with their zone of inhibition (ZOI), is shown in Table 7.

From Table 7, it was found that the TiO₂ NPs synthesized by bacteria and fungi were tested against Gram-positive, Gram-negative, and certain yeast. Among all the studies, it was found that the maximum ZOI obtained was 33 mm against S. aureus, while the minimum ZOI obtained was 9 mm against B. subtilis. The highest ZOI was obtained by the agar well diffusion method, while the lowest ZOI was obtained by the disk diffusion method. In addition to this, bacterially synthesized TiO₂ NPs were also assessed against some of the common fungi, such as A. niger, which was inhibited at a concentration of 100–400 μl.