Assessment of carbon nanotubes and silver nanoparticles loaded clays as adsorbents for removal of bacterial contaminants from water sources

There is no doubt that drinking water sources receive heavy loads of microorganisms through several means such as industrial, agricultural, and domestic wastes (Annual Drinking Water Quality Report 2005). A wide spectrum of pathogenic agents can be found in water and monitoring of their presence on a routine basis is impractical. Traditionally, microbial safety of drinking water has been confirmed by monitoring for the absence of microorganisms of fecal origin (Le-Chevallier & Au 2004).

To reduce the incidence of waterborne diseases and to make water suitable for drinking, removal of pathogenic organisms, fecal matters, suspended solids, algae, organic matters, and harmful chemicals from polluted water is mandatory (Gupta & Chaudhuri 1995). The removal capacity of a multiple-barrier water disinfection device for protozoa, bacteria, and viruses was studied and the efficiency of the multiple-barrier device in removing Escherichia coli was close to 100%, and more than 87% of Cryptosporidium oocysts and more than 98% of Giardia cysts were removed. Close to 100% of coliphages were removed and 99.6% of the adenovirus was removed (Espinosa-García et al. 2014). Moreover, for wastewater, stabilization ponds and constructed wetlands are efficient for the reduction of Cryptosporidium in wastewater, especially when the retention time is longer than 20 days with suitable sunlight and temperature while high rate filtration and chlorine disinfection are inefficient for the reduction of Cryptosporidium in effluents (Nasser 2016).

This study focused on nanotechnology and its applications which is considered as one of the rapidly developing sciences. As demand for fresh drinking water is increasing, nanotechnology can contribute notable development and improvement to water treatment processes. The disinfection process is the last and most important step in water and wastewater treatment processes. Some nanomaterials can be used as disinfectants due to their antimicrobial properties (Hossain et al. 2014). Carbon-based nanomaterials such as carbon nanotubes (CNTs), activated carbons, fullerene and graphene are widely used as the currently most promising functional materials due to their high adsorption capacities. Therefore, graphene nanosheets will challenge the current existing adsorbents, including other types of carbon-based nanomaterials (Yu et al. 2015).

Nano-sorbent materials such as CNTs and poly materials are applied to remove heavy metals, organics and biological impurities (Savage & Diallo 2005). CNTs have been gradually applied to the removal of organic contaminants (e.g., dyes, pesticides, and pharmaceuticals/drugs) from wastewater through adsorption processes (Yu et al. 2014), besides this, CNTs as adsorbent media have been proved to be able to remove a wide range of contaminants including bacteria (Akasaka & Watari 2009). CNTs with different diameters would have different specific surfaces, which would affect their adsorptive properties. However, the effects of different outer diameters of CNTs on adsorption of organic contaminates has not been investigated up to now (Yao et al. 2014).

Meanwhile, application of silver nanoparticles (AgNPs) has been extensively studied in the food industry for drinking water treatment (Konopka et al. 2009; Kumar & Raza 2009; Zhao et al. 2010). The antimicrobial activity of silver can be mainly attributed to interactions of silver ions with thiol groups of cellular proteins, leading to their inactivation. Processes such as cell respiration and ion transport across membranes, are also employed (Marambio-Jones & Van Hoek 2010). However, the mechanisms of toxic action for AgNPs are still not very well defined (Fabrega et al. 2011). The use of AgNPs in drinking water treatment has extensively become of interest (Feng et al. 2000; Jain & Pradeep 2005). However, addition of silver to water in such concentration that shows bactericidal activity does not impair the taste, color, odor, and other physicochemical characteristics of water (Klueh et al. 2000).

The present study aimed to evaluate the antimicrobial efficacy of natural kaolin clay and nano-adsorbent materials (CNTs and silver nitrate-loaded clay) against isolated bacteria from different water supplies (tap, underground and surface water) commonly used for human and animal drinking in addition to wastewater.

A cross-sectional study was carried out in nine different rural areas in Beni-Suef Province (coordinates: 29°04'N 31°05'E), Egypt during the period from September 2014 to July 2015. These areas are suffering from health problems related to water quality on both human and animal levels.

A total of 160 water samples were collected from main water sources intended for animal and human drinking. Samples represented three different water supplies (tap, underground [hand pumps], surface water [water channel] source) beside wastewater (n = 40 of each) in investigated districts. The tap and hand pump water samples were collected in 250 ml sterilized Schott Duran Bottles. For this purpose, the outlets of the taps and hand pumps were thoroughly disinfected by ethyl alcohol 70%, water was allowed to flow and then water samples were taken. Surface water and wastewater samples were collected 20 cm below the surface of the water using sterile plastic syringes (50 ml capacity) according to the method described previously (Azam et al. 2012). Sampling bottles were tightly capped, properly labeled and identified according to their source and site and immediately sent to the laboratory in an ice box for further microbiological examination according to standard guidelines (American Public Health Association (APHA) 2012).

E. coli was isolated on MacConkey agar (Oxoid; CM 0115) and Eosin Methylene Blue agar (EMB; Oxoid; CM 69) plates meanwhile, Salmonella and Shigella species were isolated by pre-enrichment of water samples on buffer peptone water, then enrichment of samples on Selenite F broth, followed by isolation of the typical organism on selective medium, Xylose Lysine Deoxycholate agar (XLD) (Collee et al. 1996).

All colonies with different characteristics on EMB and XLD were sub-cultured onto nutrient agar for purification. Enteric bacteria isolated on respective selective or differential media were identified on the basis of their colony and morphological characteristics and then using API 20E (Biomerieux, Crappone France); final verification based on serological identification was carried out at the Ministry of Health, Cairo, Egypt, using techniques adopted by Sojka (1965). Serotyping was carried out by the slide agglutination technique using diagnostic polyvalent and monovalent E. coli O & K antisera.

The antimicrobial effects of natural kaolin clay and its loaded forms were evaluated against a total of 50 isolated bacterial pathogens from three different water supplies in addition to wastewater in the investigated districts.

The antibacterial efficiency of kaolin clay and nano-adsorbent materials was determined using the broth macro dilution technique according to Li et al. (2008) with some modifications. Five sterile test tubes for each tested material (each test tube containing 1.2 ml homogeneous bacterial suspensions) were prepared in Brain Heart Infusion (BHI), then 0.8 ml of the tested material suspension were aseptically added to four test tubes to obtain final concentrations of 0.01, 0.03, 0.05 and 0.1 mg/l. Each of the natural kaolin, CNTs-loaded clay and AgNPs-loaded clay treatments were thoroughly mixed separately and allowed to interact with bacteria at different contact times (5, 15, 30 min and 2 h). The remaining test tube (the fifth one) was used as the negative control (1.2 ml bacterial suspension without any adsorbent material). Then, 1 ml of inocula was transferred to BHI-containing tubes, for incubation at 37 °C for 24 h. A sample was considered positive if it was exhibiting medium turbidity and the formation of a thin surface skin or of a precipitate in the bottom of the tube. After incubation, the suspension was spiked in solid medium (trypticase soya agar) to confirm the presence or absence of microorganisms tested against various tested materials and exposure times. The absence of bacterial growth in plates indicates the effectiveness of the adsorbent.

The data collected and recorded on specifically designed forms were entered in the Microsoft Excel spreadsheet then prepared for analysis. The frequent distribution of bacterial isolates from different water sources was calculated by using percentage values and analyzed using a Chi-square test (SPSS, version, 22).

The frequent distribution of pathogenic bacteria isolated from different water sources in (Table 2) clarified that there were in total 121 bacterial isolates out of 160 examined samples. The highest percentages of bacterial contaminants were isolated from waste and surface water sources (100% of each) at X² = 9.52 (P < 0.05), while the lowest percentages were presented in underground and tap water (20% and 5% respectively). Moreover, the predominant bacterial isolates were E. coli and Shigella flexneri followed by Salmonella spp., Klebsiella aerogenes and Klebsiella pneumoniae (34.7, 24.7, 16.5, 14.0 and 9.9%, respectively).

Concerning serological identification of E. coli isolated from different water sources (Table 3), it has been found that the predominant E. coli serotypes in different water sources were O128:k67, O157:k-, O111:k58 and O55:k59. The highest serotype percentages in wastewater were O157:k- and O111:k58 (33.3% and 25.9%, respectively) followed by O128:k67 and O55:k59 (18.55% of each). However, in surface water, the highest percentage of E. coli serotype was O157:k- followed by O128:k67, O55:k59 and O111:k58 (30.7, 23.07, 23.07 and 15.3%, respectively) while in underground water, O128:k67 and O111:k58 accounted for 50% each.

A comparison of the antimicrobial efficacy of natural kaolin clay with nano-adsorbent materials against bacterial isolates from different water sources is presented in Table 4. It has been revealed that AgNPs-loaded clay at a concentration of 0.1 mg/l had the highest antimicrobial activity against Salmonella spp. (90%) followed by E. coli, Klebsiella pneumoniae and Shigella flexneri (80% each) and Klebsiella aerogenes (70%) after 2 h of exposure time. The antimicrobial activity of CNTs-loaded clay at the same concentration was 70% for each of Salmonella spp. and Klebsiella pneumoniae, while it was 60% for E. coli isolates.

The results in Table 5 show the antimicrobial efficacy of AgNPs-loaded clay at different exposure times. It was found that the highest antimicrobial activity was achieved at the concentration dose of 0.1 mg/l and 2 h exposure time for all tested bacterial isolates (Salmonella spp. was 90%, followed by E. coli, Klebsiella pneumoniae and Shigella flexneri at 80% each). Meanwhile all bacterial isolates showed a highly resistant profile to other concentrations (0.01, 0.03 and 0.05 mg/l) at different exposure times (5 min, 15 min, and 30 min, respectively). The presented data in Table 6 clarify that the antimicrobial efficacy of CNTs-clay was higher against Salmonella spp. and Klebsiella pneumoniae (70% for each) compared to E. coli, Klebsiella aerogenes and Shigella flexneri (60, 60 and 40%, respectively). Moreover, the same bacterial isolates revealed that highly resistant patterns were present at low tested concentrations of CNTs at different exposure times.

Drinking water has the potentiality to transport microbial pathogens to a great number of individuals, causing subsequent illness, which is well documented in countries at all levels of economic development (Payment 1997). In this study, results revealed that the highest percentages of bacterial isolates (E. coli and Salmonella spp.) were observed in wastewater followed by surface water supply as compared to other water sources in the investigated districts. These results could be attributed to the impact of unsupervised anthropogenic activities including excessive usage of fertilizers and pesticide, waste disposal and industrial waste, seepage from septic tanks and construction of water pipes. The surface water presented significant acute toxicity, and could cause diseases if used in livestock farms without being adequately treated. This represents a dangerous public health risk, which needs future evaluation and control. This was in agreement with Melegy et al. (2014) who showed that pathogenic bacteria can occur in surface water in large numbers, either being excreted in feces or occurring naturally in the environment. Moreover, Mohammed (2016) revealed that in surface water, E. coli was isolated in the highest percentage (17/56; 56.7%) followed by Staph. aureus (11/56; 36.7%), Salmonella spp. (8/56; 26.7%), Strept. faecalis (7/39; 23.3%), Shigella flexneri (5/56; 16.7%), Proteus spp. (5/56; 16.7%) and Klebsiella pneumoniae (3/56; 10.0%) at X² = 9, P ≤ 0.01. Sources of fecal contamination include humans, livestock and wild animals; in general, human fecal waste gives rise to the highest risk of waterborne disease (Craun 1996). The most sporadic cases of waterborne intestinal illness will not be detected, or if detected may not be recognized as water-related, while it is estimated that water, sanitation, and hygiene are responsible for 40% of all deaths and 5.7% of the total disease burden occurring worldwide (Prȕss et al. 2002).

E. coli is a widely used bio-indicator of fecal contamination in water sources. External contact and subsequent ingestion of bacteria from fecal contamination can cause detrimental health effects (Money et al. 2009). In this study, it has been found that the predominant E. coli serotypes in different water sources were O128:k67, O157:k-, O111:k58 and O55:k59. Environmental Protection Agency and epidemiological investigations, suggested that cultural fecal indicator counts are valid predictors of disease risk (Sinton et al. 1994) and sewage can serve as a vehicle for bacteria to enter into human and non-human hosts either by direct contact or through contamination of drinking water supplies (Boczek et al. 2007).

The current increased attention is directed to health problems associated with drinking water quality at both human and animal level. In particular, communities who use surface water supplies are of special concern, since their drinking water is commonly not treated or monitored for contamination on a routine basis. The present study evaluated the efficiency of natural kaolin and nano-adsorbent materials for the removal of water contaminants, and subsequently risk hazards to human and animal health. The present study revealed that different types of microorganisms vary in their response to the tested material of kaolin clay, CNTs-loaded clay, and AgNPs-loaded clay. Natural kaolin clay was the least effective against all bacterial pathogens. The results also suggested that the antimicrobial effects of kaolin and nano-adsorbent materials are not only dependent on the types of material but also on their concentrations and exposure times. A previous study showed that AgNPs supported on granular activated carbon gave good results against Escherichia coli (Acevedo et al. 2014), instead, in the present work we used kaolin as a support material which is highly available and cost effective. The efficiency of AgNPs-clay was highly effective against bacterial pathogens at concentrations of 0.05 and 0.1 mg/l. These results are in agreement with Sondi & Salopek-Sondi (2004) who suggested that the antimicrobial effects of the AgNPs on bacteria were dependent on the concentration of Ag in the nanoparticles (NPs); where increasing the concentration of NPs delayed the growth of bacteria. The treated bacterial cells were significantly changed and showed major damage, which was characterized by the formation of ‘pits’ in their cell walls. Previous studies have shown that antimicrobial formulations in the form of NPs could be used as effective bactericidal materials due to their enhanced reactivity, resulting from their high surface/volume ratio (Pal et al. 2007; Choi et al. 2008; Park et al. 2009). Particularly, silver in the form of NP (AgNPs) is known to exhibit strong biocidal effects on different bacterial species (Sondi & Salopek-Sondi 2004; Rai et al. 2009). Moreover, silver ions and silver-based compounds are highly toxic to Gram-negative and Gram-positive microorganisms (Bae et al. 2010; Fayaz et al. 2010; You et al. 2011; Maillard & Hartemann 2013).

CNTs are graphene sheets rolled into a tube and possibly capped by half a fullerene. They can be either single-walled (SWNTs), a single pipe with a diameter from 1 to 5 nm, or multi-walled (MWNTs), with several nested tubes, at lengths varying from 100 nm up to several tens of micrometers (Li et al. 2008). In the present study, the antimicrobial activity of CNTs against bacterial isolates from different water sources revealed that the sensitivities (%) of almost all bacterial pathogens to CNTs were 70% after 2 h exposure time.

However, the bacterial isolates which showed resistant to CNTs were E. coli, Klebsiella aerogenes (30% each), Salmonella spp., Shigella flexneri (20% each) and Klebsiella pneumoniae (10%) after 2 h exposure time. The results could be interpreted to suggest that the main factors which controlled their sensitivity were nanomaterial (CNTs) concentration and contact times. These results are supported by Kang et al. (2007) who clarified that antimicrobial activity of CNTs requires direct contact between CNTs and target microorganisms. Suspension of non-functionalized CNTs in water is extremely difficult and does not provide enough CNT-microbe contact for disinfection. Accordingly, the antibacterial activity of CNTs could be exploited by coating CNTs on a reactor surface in contact with the pathogen-laden water, also it has been found that using immobilized SWNTs on a membrane filter surface led to 87% killing of E. coli in a 2 h contact time.

Based on the present results, it can be concluded that the application of AgNPs-loaded clay for removal of water bacterial contaminants resulted in highly effective removals at a concentration of 0.1 ppm and 2 h contact time, and that CNTs-loaded clay was also effective, but to a lesser extent, at the same concentration and exposure time.

The authors would like to acknowledge the Projects Funding and Granting Unit in Beni-Suef University, Egypt for funding the project and supporting the research.