Environ Res. 2014 Nov;135:333-45.

Cytotoxicity of TiO2 nanoparticles towards freshwater sediment microorganisms at low exposure concentrations.

Kumari J1, Kumar D1, Mathur A1, Naseer A1, Kumar RR1, Thanjavur Chandrasekaran P2, Chaudhuri G1, Pulimi M1, Raichur AM3, Babu S4, Chandrasekaran N1, Nagarajan R5, Mukherjee A6.
  • 1Centre for Nanobiotechnology, VIT University, Vellore 632014, India.
  • 2Department of Materials Engineering, Indian Institute of Science, Bangalore, India.
  • 3Department of Materials Engineering, Indian Institute of Science, Bangalore, India; Department of Chemical Technology, University of Johannesburg, South Africa.
  • 4School of Bio Sciences and Technology, VIT University, Vellore, India.
  • 5Department of Chemical Engineering, IIT Madras, Chennai, India.
  • 6Centre for Nanobiotechnology, VIT University, Vellore 632014, India. Electronic address: amit.mookerjea@gmail.com.



There is a persistent need to assess the effects of TiO2 nanoparticles on the aquatic ecosystem owing to their increasing usage in consumer products and risk of environmental release. The current study is focused on TiO2 nanoparticle-induced acute toxicity at sub-ppm level (≤1ppm) on the three different freshwater sediment bacterial isolates and their consortium under two different irradiation (visible light and dark) conditions. The consortium of the bacterial isolates was found to be less affected by the exposure to the nanoparticles compared to the individual cells. The oxidative stress contributed considerably towards the cytotoxicity under both light and dark conditions. A statistically significant increase in membrane permeability was noted under the dark conditions as compared to the light conditions. The optical and fluorescence microscopic images showed aggregation and chain formation of the bacterial cells, when exposed to the nanoparticles. The electron microscopic (SEM, TEM) observations suggested considerable damage of cells and bio-uptake of nanoparticles. The exopolysaccrides (EPS) production and biofilm formation were noted to increase in the presence of the nanoparticles, and expression of the key genes involved in biofilm formation was studied by RT-PCR. Copyright © 2014 Elsevier Inc.

KEYWORDS: Bacteria; Consortium; TiO(2) NPs; Visible light and dark conditions

PMID: 25462683



Titanium dioxide nanoparticles have the maximum manufacture rate anticipated approximately 5000 tons before 2010 and are expected to enhance up to 2.5 million tons by 2025. The International Agency for Research on Cancer (IARC) has rated Titanium dioxide as the most cancer causing agent (Group 2B) for the human being. The existence of bacteria is necessary for the preservation of a sustainable ecological unit. Reports suggested that TiO2 NPs can go into the aquatic ecosystem through direct or indirect discharge from food additives, nano-paints, medical use, sunscreen lotions, groundwater remediation and recycling of plastic/metal/glass with nano-coating. (O’Brien and Cummins, 2010), which in sequence may grounds toxicity to the alive organisms in the surroundings. Bacteria are being increasingly used as possible test systems for evaluating nanoparticles toxicity. Microbes can act as simple experimental subjects to facilitate miniaturized toxicological evaluation system for speedy risk detection. Prior information indicated that the adsorption of TiO2 NPs on the bacterial cell wall, disruption of the transmembrane electron shift, alteration of the membrane, bodily injure ensuing in leakage of the cell filling. Reactive oxygen species production was among the major contributor in the cell TiO2 nanoparticles cytotoxic interactions (Klaine et al., 2008; Li et al., 2008). The present study was done to explore the phototoxic property of TiO2 NPs to diverse bacterial strains as well as their consortium (a mixture cultures) which was isolated from the freshwater sediments at exposure concentrations (0.25, 0.50, 0.75 and 1.00 μg/mL) in a freshwater matrix devoid of nutrient supplements (to imitate the elemental medium of a freshwater environment) in visible light and dark environment. The majority of the preceding information on the subject of ecological toxicity of TiO2 NPs contract was done with individual bacterial isolates, which might not permanent be the case in the usual surroundings. It can be assumed that the bacterial consortium being diverse in nature can encounter a lesser amount of toxic impact of the nanoparticles in the environment than the single bacterial isolate, which is homogenous. In the current study, the Titanium dioxide nanoparticles in lake water matrix were found to be stable against aggregation at the concentration (1 μg/mL). The professed stability of the nanoparticles could aggravate their toxic effects.

This study explained that under both light and dark conditions, there is a notable exposure and dose-dependent lessening in cell viability. For the consortium, the decline in the cell viability was found to be less than that of single isolates (B. altitudinis, B. subtilis, and P. aeruginosa for 24 h period at 1 mg/mL), which signify a resistance capability of a bacterial consortium towards the nanoparticles. Though, the percentage bacterial cell loss for the consortium was noted to be lesser for every single isolates (B. altitudinis, B. subtilis, and P. aeruginosa) at 1 mg/mL for 24 h, but it was not statistically significant (p>0.05). The stress caused by TiO2 NPs is usually determined by the production of reactive oxygen species. The ROS level of the B. altitudinis, B. subtilis, and P. Aeruginosa cells and the consortium after treatment of TiO2 NPs (1mg/mL) was measured. The ROS release was statically higher (p<0.05) under treated light conditions as compared to dark treated conditions in every case. Nevertheless, there was substantial ROS produced yet under dark conditions. Prominently, the difference in ROS production in the bacterial consortium when compared to the single bacterial isolates was statistically insignificant (p>0.05). The oxidative stress outcomes recommended that the consortium were more defiant to the titania NPs toxicity when compared to the single isolates, which explicate the higher viability observed for bacterial consortium cells. The membrane permeability of TiO2 NPs (1 mg/mL, 24 h) for single bacteria and their consortium were analyzed for by the lactate dehydrogenase assay. Bacterial cell membrane injury and raised membrane permeability lastly lead to the cell demise consequently damage by TiO2 NPs. The dissimilarity in LDH release under light and dark conditions were found to be noteworthy (p<0.05), and extra membrane injury was found in dark conditions.

Usually, exopolysaccharides were considered to be the structural support for biofilm and are a combination of macromolecules, together with secreted proteins and polysaccharides. The extracted EPS from the TiO2 NPs (1 mg/mL, 24 h) from single bacteria and the consortium were measured to learn the bacterial confrontation against the toxicant. Higher amount of EPS production was assessed in the TiO2 NPs-treated consortium when compared with the single bacterial isolates; however the dissimilarity was not statistically significant. The raise in EPS production was found to be statistically significant for every the treated bacterial samples when compared to the control. Scanning electron micrographs observations pointed out the modification in the exterior morphology of bacterial consortium before and after the interaction. The untreated cells found to be an even arrangement. The cell wall disruption and the loss of intracellular materials were observed in both light and dark conditions representing that TiO2 NPs were toxic to the bacterial cells yet at low exposure concentration (1 μg/mL NP concentration, 2 h). The lifeless cells and the lysed cell membrane were noted, which indicate that the TiO2 NPs-induced toxic outcome leads to the leakage of the polysaccharides, which leads to cell death. Scanning electron microscopic images were taken to comprehend the early organization of biofilm and its changes following exposure to TiO2 NPs. The structural biofilm design of the consortium with and with no TiO2 NPs was inspected under light and dark conditions after 24 h interaction period. The control consortium biofilm found as even isolated cells devoid of any injury, and no structural aberration was noticed. An extracellular matrix, mostly composed of the collective cells, was found in light and dark-treated consortium. These pictures verified that TiO2 NPs could quickly enter through the biofilm due to the adsorption of chemical components within EPS resulting in cellular injury.

Additionally, the cytotoxic property of TiO2 NPs, changes in morphology of cells, and biodistribution of NPs under visible light and dark surroundings were evaluated by transmission electron microscopy. The distinctive look of the bacterial consortium previous to TiO2 NPs treatment, which is even and damage-free. The distinct cell wall and evenly stained within of the cell were noticeably visible. Extreme morphology changes were observed after TiO2 NPs interaction (1 μg/mL, 2 h). The distorted cells from the light experiment, representing a loss of cell integrity leading to the outflow of an interior element, and consequently, the inactivation of the bacteria was seen. Also, the morphology of the consortium altered from normal form to uneven distorted shape. Creation of small vacuole in the cell giving a irregular look to cells was noticed in the light as well as dark-treated samples.

Importance of the study:

On the whole, this learning conveyed enhanced understanding of ecotoxicological effect of TiO2 nanoparticles. The assessment brought ahead the link between the toxicity reaction with the physicochemical actions of the nanoparticles under environmentally applicable conditions. The toxicological impact on the representative test species provided the effect of TiO2 NPs into the environment. Nevertheless, considering the vibrant nature of nanoecotoxicity and input of particle size, shape and surface composition along with the nature of experimental matrix, large scale of parameters need to be considered. Application of high throughput screening is recommended which might be the only way to keep pace with growing trend of nanotechnology. The void in long term effects of nanoparticles exposure is another major concern presently. Adequate efforts to bring in fresh inputs in this aspect are the need of the hour.


Figure 1_Mukherjee_VIT

Figure 2_Mukherjee_VIT

Figure 3_Mukherjee_VIT


A. Transmission electron micrograph of bacterial consortium showing the characteristic look of bacterial consortium prior to TiO2 NPs treatment,

B. Transmission electron micrograph of bacterial consortium treated withTiO2 NPs (1μg/mL, 2 h) interacted bacterial consortium showing disrupted morphology under light conditions and appearance of small vacuole like structures,

C. Transmission electron micrograph of bacterial consortium treated withTiO2 NPs (1μg/mL, 2 h) treated bacterial consortium in dark conditions showing distorted morphology and internalization of TiO2

Notations used: Blue arrow indicates vacuole formation in the cell, yellow arrow marks membrane damage and cell distortion; green arrow demarcates internalization of NPs into cell.



  1. O’Brien, N., Cummins, E., 2010. Ranking initial environmental and human health risk resulting from environmentally relevant nanomaterials. J. Environ. Sci. Health A 45 (8), 992–1007.
  2. Klaine, S.J., Alvarez, P.J., Batley, G.E., Fernandes, T.F., Handy, R.D., Lyon, D.Y.,     Lead, J.R., 2008. Nanomaterials in the environment: behaviour, fate, bioavailability, and effects. Environ. Toxicol. Chem. 27 (9), 1825–1851.
  3. Li, Y., MA, M., Wang, X., Wang, X., 2008. Inactivated properties of activated carbonsupported TiO2 nanoparticles for bacteria and kinetic study. J. Environ. Sci. 20 (12), 1527–1533.



Dr. Amitava Mukherjee

Sr. Professor & Deputy Director

Centre for Nanobiotechnology, VIT University, Vellore-632014

Email: amit.mookerjea@gmail.com, amitav@vit.ac.in

Tel.: +91 416 220 2620




Multiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier SchönmannMultiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier Schönmann