134

# Handbook of Engineering Hydrology

Print publication date:  March  2014
Online publication date:  March  2014

Print ISBN: 9781466552494
eBook ISBN: 9781466552500

10.1201/b16766-4

#### Abstract

Waterborne diseases are the main indication of deterioration of water quality. The greatest waterborne risk, which poses a significant problem to human health, is microbial contamination of water sources, contributing to disease outbreaks [24]. Also it is estimated that the population living in water-stressed areas of the world will reach 44% by 2050 [18]. In terms of escalating demands and pollution of limited water sources, particularly in rural and developing communities, the condition will become even worse. To prevent or reduce the risk of waterborne diseases, many water utilities use disinfection processes to remove pathogens in water and wastewater. Nanotechnology is recognized as a new-generation technology that will influence economies through new commercial products, materials usage, and manufacturing methods [1]. Under this new technology, the use of nanoparticles for water disinfection is being explored. This chapter reviews water pathogens and common methods for disinfection of water and highlights recent progresses toward the development of nanotechnology in disinfection of water and wastewater.

#### 3.1  Introduction

Waterborne diseases are the main indication of deterioration of water quality. The greatest waterborne risk, which poses a significant problem to human health, is microbial contamination of water sources, contributing to disease outbreaks [24]. Also it is estimated that the population living in water-stressed areas of the world will reach 44% by 2050 [18]. In terms of escalating demands and pollution of limited water sources, particularly in rural and developing communities, the condition will become even worse. To prevent or reduce the risk of waterborne diseases, many water utilities use disinfection processes to remove pathogens in water and wastewater. Nanotechnology is recognized as a new-generation technology that will influence economies through new commercial products, materials usage, and manufacturing methods [1]. Under this new technology, the use of nanoparticles for water disinfection is being explored. This chapter reviews water pathogens and common methods for disinfection of water and highlights recent progresses toward the development of nanotechnology in disinfection of water and wastewater.

#### 3.2  Water and Wastewater Pathogens

Untreated and secondary treated effluent contains a range of pathogenic microorganisms that pose a significant risk to the health of humans. In the late nineteenth century, acute waterborne diseases, like typhoid fever and cholera, became common. It is essential to note that the host must have been in contact with required numbers of pathogenic organisms to cause a disease. These required numbers could differ for different types of organisms. Pathogens, which can be potentially presented in wastewater, are divided into four separate groups. These groups are the bacteria, viruses, protozoa, and helminths.

#### 3.2.1  Bacteria

Bacteria are the most common microorganisms found in wastewater ranging from 0.5 to 5 μm in size. A wide range of bacterial pathogens and opportunistic pathogens can be detected in wastewaters, and some of them are used as indicator organisms of human wastes. The principal bacteria in wastewaters are the following:

• Fecal coliforms are naturally found in the human intestine and their presence can demonstrate fecal contamination of a water source.
• Shigella is a vast group of bacteria. The majority of waterborne infections are caused by Shigella sonnei species, and contaminated waters are the main source of Shigella-related diseases.
• Salmonella, of which there are more than 2000 known species, pose a risk to human health and mostly are causing gastroenteritis.
• Campylobacter jejuni is known to cause gastroenteritis for up to 1 week in humans.
• Yersinia enterocolitica causes gastroenteritis and can grow at low temperatures as low as 4°C. This bacterium is mostly resistant to chlorine and can be carried by both animals and humans.
• Escherichia coli is the most commonly used bacterial indicator of fecal contamination of water, which is 0.5 μm wide and 1–3 μm long. E. coli is transmitted through water and can cause gastroenteritis in humans and diarrhea in infants.
• Vibrio cholerae causes cholera, dehydration, vomiting, and diarrhea, and if no medical treatment is provided, death might happen after a few hours. This bacterium has led to major epidemics in the world.
• Pseudomonas is one of the premier causes of infections in swimming pools and is almost resistant to chlorination.
• Leptospira is transmitted by sewer rats and can be infected through ingesting contaminated water while swimming in lakes and rivers.

#### 3.2.2  Viruses

Viruses are among the most important and potentially most hazardous of the pathogens found in wastewater. Viruses are not cells but are infectious particles ranging from 10 to 25 nm in diameter; therefore, they pass through filters and are hard to remove in water treatment. Viruses live only inside the host cell and they are inactive outside the host. All of the prevalent pathogenic viruses found in wastewater enter the environment through fecal contamination from infected hosts. The most important viruses in wastewater are as follows:

• Hepatitis A virus (HAV) affects the liver and causes infectious hepatitis. Symptoms are fever, vomiting, and diarrhea and, in acute cases, might lead to jaundice. HAV can be removed by flocculation, coagulation, and filtration.
• Norwalk-type viruses can lead to acute epidemic gastroenteritis.
• Rotaviruses can lead to acute gastroenteritis mostly in children and can cause mortality of infants in developing countries. Rotaviruses can be removed through flocculation, coagulation, and filtration.
• Enteroviruses, Adenoviruses, and Reoviruses are three different types of viruses that infect the enteric and upper part of respiration system in humans.

#### 3.2.3  Protozoan

Protozoa are unicellular organisms, which are detected more prevalently in wastewater than in other environmental sources. Many types of protozoan can survive methods of disinfection like chlorination. The chief waterborne protozoa include the following:

• Giardia lamblia is a flagellated protozoan that can exist for up to three months as a cyst and can cause gastrointestinal disease. Giardia cysts are more resistant to chlorine than other organisms and can be removed by granular media filtration.
• Cryptosporidium, two species of Cryptosporidium, can cause infection in mammals. The symptoms in humans are acute diarrhea, abdominal pain, vomiting, and fever. The oocysts are resistant to chlorine but ultraviolet (UV) irradiation and ozonation can destroy them.
• Entamoeba histolytica is a protozoan and the cysts can be spread by the use of polluted water for irrigation or by using sludge as a fertilizer. E. histolytica causes amoebic dysentery, and this organism can invade the bloodstream and is able to reach other organs like the liver. The cysts are resistant to chlorine, but the large size of them helps their removal by conventional filtration.

#### 3.2.4  Helminths

Helminths (rotifers and nematodes) are multicellular microscopic animals, which are common intestinal parasites and are usually transmitted by fecal–oral route. Small nonparasitic worms are present globally, even in finished drinking water at the faucet [3]. Ascaris lumbricoides, Enterobius vermicularis, Fasciola hepatica, Hymenolepis nana, Taenia saginata, Taenia solium, and Trichuris trichiura are the main helminths. Helminth eggs are resistant to chlorination, and studies have shown that eggs of Ascaris can survive in the sediments of oxidation ponds for up to 10 years [15].

#### 3.3  Disinfection of Water

Disinfection is the partial inactivation or destruction of disease-causing pathogens. These pathogens and diseases caused by them were mentioned briefly in the last section. Disinfection of wastewater is currently achieved by the use of various methods, which are discussed in the following.

#### 3.3.1  Chemical Agents

There are various chemical agents used as disinfectants, but chlorination, chloramination, usage of chlorine dioxide, and ozonation are the most common methods universally.

#### 3.3.1.1  Chlorination

Chlorine is the most common strategy of disinfection because it is economical, effective, and helpful in control of infection. Chlorine is a greenish-yellow gas that is 2.48 times as heavy as air and inactivates pathogens by reacting with their enzymes. Chlorine can be easily compressed and transformed into liquid; 450 mL of gas forms 1 mL of liquid.

Chlorine forms hydrochloric acid and hypochlorous acid in water and will lower the pH of water:

3.1 $H 2 O + C L 2 → HCL + HOCL$

At pH above 7.5, hypochlorous acid ionizes into hypochlorite ion and hydrogen and it becomes less and less effective:

3.2 $H O C l + H 2 O → H 3 O + + O C l −$

Chlorine gas is highly toxic and potentially poses health risks to treatment plant operators and the general public health if released by accident. Though hypochlorite salts, which are solid forms of chlorine, are used as an alternative for chlorine gas. Unlike chlorine gas, hypochlorite salts raise the pH of the water.

The effectiveness of chlorine is dependent on pH, chlorine type, temperature, contact time period, and concentration. As pH of water increases, the chlorine will become less effective, while the effectiveness of chlorine varies directly with the temperature. As temperature rises, the disinfection would become quicker and the required contact time will become shorter. Longer contact time would contribute to more effective disinfection, and this required contact time varies at different temperatures to react with pathogens.

Higher concentration of chlorine would help the effectiveness of disinfection. According to the Safe Drinking Water Act (SDWA), the maximum permitted chlorine residual in water distribution systems is 4 mg/L, and the minimum required is 0.2 mg/L. 0.5–1 mg/L of free residual chlorine effectively disinfects water. Exposure to 1000 ppm of chlorine in the air will be rapidly fatal and the maximum allowed amount of chlorine in the air is 1 ppm.

#### 3.3.1.2  Chloramination

Chloramination is the use of chloramines, which are formed by reacting ammonia with chlorine, for disinfection. Chloramines are less effective and slower than free residual chlorine. Therefore, to achieve to same degree of disinfection, higher dosage and longer contact time for chloramines are required relative to free residual chlorine. There are three types of chloramines: monochloramines, dichloramines, and trichloramines. Monochloramine is the most prevalent species among the others because it is more effective and less odorous. Overall, chloramination is more effective above pH 8.

#### 3.3.1.3  Chlorine Dioxide Application

Chlorine dioxide, a yellow to red gas, is a very powerful disinfectant formed by reacting sodium chlorite with chlorine or an acid:

3.3 $2NaCl O 2 +C l 2 ↔ 2NaCl+2Cl O 2 ↑$
3.4 $5 N a C l O 2 + 4 H C l ↔ 5 N a C l + 4 C l O 2 ↑ + 2 H 2 O$

The efficiency of this process should be more than 95% and the efficiency is pH dependent with an optimum at a pH of 3–5. Chlorine dioxide is more effective when it is followed by chlorine or chloramines. Some disadvantages like being relatively expensive to produce, being explosive at a concentration above 10% in the air, and short stability have limited chlorine dioxide application.

#### 3.3.1.4  Ozonation

Ozone was first used to disinfect water supplies in 1905 at Nice, France. Ozone, a blue gas with a distinct pungent odor, is a triatomic form of oxygen, O3. Ozone can be produced by electrolysis, photochemical reaction, or radiochemical reaction by electrical discharge. Ozone is mostly produced by UV light and lightning during a thunderstorm. The electrical discharge method is implemented for the production of ozone in water and wastewater disinfection processes. Ozone is generated from either air or oxygen when a high voltage is applied between two electrodes. Ozone is 20 times more soluble in water than oxygen and the maximum reported solubility of ozone in water is 40 mg/L. Ozone is chemically unstable and decomposes to oxygen very quickly after production and thus must be produced onsite. Its half-life at room temperature is only 15–20 min:

3.5 $O 3 → O 2 + O$

Ozonation treatment of water can be separated into three distinct sections: preparation of feed gas, production, and contact.

In the production process, the feed gas at a low pressure is passed through two electrodes, which are separated by a gap to produce ozone. At this stage, a large amount of heat is released into the cooling water.

### Table 3.1   By-Products of Chemical Agents

Disinfectant

Significant Organohalogen Products

Significant Inorganic Product

Significant Nonhalogenated Products

Chlorine

THMs

Chlorophenols

Aldehydes

Hypochlorous acid

Haloacetic acids

Halofuranones

Chlorate (mostly from hypochlorite use)

Cyanoalkanoic acids

Haloacetonitriles

N-Chloramines

Alkanoic acids

Chloral hydrate

Bromohydrins

Benzene

Chloropicrin

Carboxylic acids

Chlorine dioxide

Haloacetonitriles

Chloramino acids

Nitrate

Chloramine

Cyanogen chloride

Chloral hydrate

Nitrite

Aldehydes

Organic chloramines

Haloketones

Chlorate

Ketones

Hydrazine

Ozone

Bromoform

Chlorate

Aldehydes

Monobromoacetic acid

Iodate

Ketoacids

Dibromoacetic acid

Bromate

Ketones

Dibromoacetone cyanogens

Hydrogen peroxide

Carboxylic acids

Bromide

Hypobromous acid

Epoxides

Ozonates

In the contact process, ozone and oxygen mixture is dispersed through the water by diffusers at the bottom of a contact chamber to contact the pathogens and destroy them.

Ozone is the strongest disinfectant, which can also be used in wastewater treatment to control tastes and odors, color, and algae. Biocidal properties of ozone are not influenced by pH.

Despite these benefits, there are some disadvantages of ozonation, including high capital cost of production and lack of residual influence. These obstacles have made chlorination a more prevalent method in the world. Overall, ozonation is mostly followed with other disinfection methods like chlorination and chloramination, or it is combined with other disinfectants such as hydrogen peroxide, which can quicken the oxidation of some organics by two to six times relative to ozone by itself.

By-products are produced during the treatment of water by chemical agents. These by-products are summarized in Table 3.1 [25].

#### 3.3.2  Physical Agents

Heat, light, and sound are physical disinfectants that can be used. Heat is currently used to destroy many bacteria in food and dairy industry. Heating water to the boiling point can be an effective way to disinfect water, but it is not a feasible strategy of disinfecting large quantities of water due to the high cost.

Sunlight is also a good disinfectant because of the UV radiation portion of the electromagnetic spectrum. Sunlight can decay microorganisms effectively because of exposure to the UV component and thermal heating of solar light.

The disinfection of treated wastewater by implementation of UV radiation is a physical process, which mainly involves passing a film of wastewater within close proximity of a UV source. UV light penetrates the cell wall of the microorganism and is absorbed by the nucleic acid and enzymes of pathogens and inactivates them. Unlike other disinfectants, UV disinfection does not produce harmful by-products. UV disinfection does not result in a lasting residual in the wastewater, which is a disadvantage when water must be stored or piped over long distances or time. One of the problems of the UV treatment is high required costs, which can limit its application in some developing countries. The most commonly accessible wavelength of the UV light is 254 nm that is generated by a mercury vapor lamp, but the most effective wavelength is 265 nm. Effective dosage of UV differs from system to system with regard to the type of microbes and the quality of water. Currently, drinking water is mostly disinfected with UV dosage of 38–40 mJ/cm2. Higher dose of UV would contribute to better disinfection of water, but the treatment will become more expensive. The required UV doses to reach a 90% of inactivation with different pathogens are shown in Table 3.2 [2].

### Table 3.2   Required UV-C Dose to Reach 90% of Inactivation for Different Microorganisms

Microorganism

[UV-C Dose] 90% (mW s c/m2)

Protozoa cysts

Giardia muris

82

Cryptosporidium parvum

80

Giardia lamblia

63

Viruses

Rotavirus SA 11

8

Poliovirus

15

Hepatitis A virus

3.7

Bacteria

Pseudomonas aeruginosa

5.5

Escherichia coli

3

Salmonella typhi

2.5

Shigella dysenteriae

1.7

Legionella pneumophila

0.38

Some factors influence the effect of UV treatment. These factors are discussed in the following:

• Turbidity. UV treatment is more effective in clean water because turbidity shields pathogens against radiation. Therefore, water should be as clear as possible to make the transmittance of UV rays easier and water treatment more effective.
• Dissolved organic matter. As the dissolved organic matter is less in water, the treatment will be more effective because dissolved organic matter absorbs the UV rays and shields pathogens from radiation.
• Total dissolved solids. Solids deposit and foul the UV lamp; therefore, treatment will be more effective if dissolved solids are less in water.
• Depth of water. UV light can penetrate better through shallow water and the treatment would be more effective. Suggested depth of clear water for UV treatment is about 3–5 in.

#### 3.3.3  Mechanical Tools

Pathogens are also partially removed by mechanical tools in water and wastewater treatment.

#### 3.3.3.1  Membrane Filtration

Filtration is the mechanical removal of specific sized particles from water by passing it through a porous medium. A membrane is a very thin paperlike structure that is capable of removing most of the contaminants. Membranes are divided into five groups:

1. Microfiltration membranes remove the particles bigger than 1 μm, including Cryptosporidium oocysts, Giardia cysts, and all bacteria. Microfiltration membranes are the most numerous on the market and less expensive than other types of membranes.
2. Ultrafiltration membranes are similar to microfiltration membranes and function like a sieve. Their pore size ranges from 0.003 to 0.1 μm and can remove all particles bigger than this pore size by 99.99%. Ultrafiltration membranes cannot remove salt or sugar.
3. Nanofiltration membranes have nanometer pore size and can remove all the particles above nanometer size such as viruses, cysts, and bacteria.
4. Reverse osmosis membranes are primarily used for desalination throughout the world. Currently, there are more than 100 reverse osmosis drinking water plants in the United States, and it is a very prevalent process in Middle Eastern countries to treat salty water for drinking purposes. These membranes exclude ions, but high pressure is needed to produce the deionized water.
5. Electrodialysis membranes use direct electric current to separate ionic components of a solution. The electric current will cause the collection of anions at the anode and cations at the cathode, after passing through the solution.

Electromagnetic, acoustic, and particle are the main types of radiation. In this method, gamma rays are emitted from radioisotopes, like cobalt-60, and these rays are very strong to disinfect water and wastewater. Despite extensive studies in this field, there are no commercial devices or installations in operation yet [13].

#### 3.4  Nanotechnology and Water Disinfection

Conventional methods of disinfection are dependent on chemical agents that are ineffective against cyst-forming protozoa such as Giardia and Cryptosporidium, and also these methods often produce harmful by-products. Therefore, there is a need to consider innovative strategies that boost the reliability of disinfection while preventing unintended adverse health effects. The advent of nano-engineered materials has spurred noticeable interest in the environmental applications that will improve technologies to protect the public health, including water treatment, groundwater remediation, and air quality control. Large specific surface area and high reactivity of nanomaterials are the main reason of their great application in different fields. The use of nanoparticles exhibiting the antimicrobial activity offers the possibility of an efficient removal of pathogens from wastewater. The main nanoparticles exhibiting antimicrobial activities are as follows:

#### 3.4.1  Silver

Silver was used as a disinfectant for water in ancient Greek period [20]. In recent years with the developments in nanotechnology, silver has found the application in different fields like biomedical [10], catalysis [7], and water purification [6]. Several antimicrobial mechanisms of nanosilver are postulated, such as adhesion to cell surface changing the membrane properties, penetrating inside bacterial cell to damage DNA, and the release of antimicrobial Ag+ ions as a result of nanosilver dissolution. The inactivation of bacteria and viruses is also enhanced by silver nanoparticles in the presence of UV-A and UV-C irradiation. In general, silver nanoparticles ranging from 1 to 10 nm are more toxic to bacteria like E. coli. Nowadays, there are a lot of commercial products that use nanosilver as an antimicrobial agent including refrigerators, textiles, faucets, laundry additives, and nutrition supplements. Also some companies like Aqua Pure and Kinetico that manufacture home water purification systems are utilizing nanosilver to remove 99.99% of pathogens in water [12].

#### 3.4.2  Chitosan

Chitosan is a type of natural polyaminosaccharide, which is a polysaccharide constituting mainly of unbranched chains of β-(1→4)-2-acetoamido-2-deoxy-d-glucose. After cellulose, chitin is the most abundant polymer in nature. It can be obtained from crustacean shell such as prawns, crabs, fungi, and insects [23]. Chitosan nanoparticles are implemented in cosmetics, agriculture, and medical applications. It has been found that nanochitosan is effective against viruses, bacteria, and fungi. Nanochitosan exhibits more effective antimicrobial activity against viruses and fungi than bacteria [16]; within bacteria, chitosan exhibits a higher antimicrobial activity toward Gram-positive bacteria than Gram-negative bacteria [4]. Various antimicrobial mechanisms are suggested for chitosan. One mechanism includes positively charged chitosan particles interacting with negatively charged cell membranes, leading to more permeability of membrane and finally fracture and leakage of intracellular elements. In the other suggested mechanism, chitosan chelates trace metals, contributing to inhibition of activities of enzyme. Nanoscale chitosan is used in membranes and water storage tanks as an antimicrobial agent that is more effective than many other disinfectants. It is also used in water and wastewater treatment systems as a coagulant. Nanochitosan is less toxic toward humans and animals than the other nanoparticles.

#### 3.4.3  Titanium Dioxide

Forty thousand tons of titanium dioxide nanoparticles were manufactured in the United States during 2006, and the annual production of nano titanium dioxide is estimated to reach 2.5 million tons in 2025 (Figure 3.1) [17].

Titanium dioxide nanoparticle is a widely used nanomaterial; only cosmetics and sunscreen products constitute 50% of titanium nanoparticle usage. TiO2 is the most prevalent semiconductor photocatalyst and is activated by UV-A (320–400 nm) irradiation [14]. TiO2 is effective against both Gram-negative and Gram-positive bacteria. Depending on the size of the particles and the intensity and wavelength of the light used, a concentration between 100 and 1000 ppm can kill bacteria. The most promising property of TiO2-based disinfection is basically its photoactivation by sunlight. A conducted experiment showed an initial bacterial concentration of 3000 cfu/100 mL was inactivated in 15 min by storing water in a plastic container that was coated inside with TiO2 and exposed to sunlight [5]. Doping TiO2 with other metals can improve its photocatalytic inactivation of bacteria and viruses. Ag/TiO2 is found to be one of the most promising photocatalytic materials due to its photoreactivity and visible light response [19].

Figure 3.1   Prediction of titanium nanoparticles production in the United States (MT, metric tons).

#### 3.4.4  Zinc Oxide

As an environment-friendly material, ZnO is used in catalyst industry, solar cells, gas sensors, and so on. Many characteristics of nano-sized ZnO are similar to TiO2. ZnO is used in sunscreens, coatings, and paints because of its high UV absorption efficiency and photocatalytic activity. The ZnO nanoparticles have exhibited a strong antibacterial effect on a vast range of bacteria [8]. The antibacterial mechanism of ZnO is still under study. One of the main mechanisms of photocatalytic degradation by ZnO is attributed to the generation of hydrogen peroxide within the cells, which oxidize the cell components. The membrane disruption is attributed to peroxidation of the unsaturated phospholipids. The predomination of the polysaccharides over the amide has charged the bacterial surface negatively. Therefore, bacteria are adsorbed easily on the ZnO powder and the nanoparticles might penetrate the cell. The bacterial growth will be inhibited as a result of cell membrane and the wall destruction in contact with ZnO nanoparticles [11]. In addition, binding Zn2+ ion to the membranes of microorganisms will result in extended lag phase of the microbial growth cycle. ZnO is also used as an antimicrobial agent in some lotions and creams.

#### 3.4.5  Fullerenes

Fullerenes are a sort of molecules composed totally of carbon. Fullerenes (C60, C70, etc.) have a low solubility in water, and therefore, they should be modified on the surface or combined with a stabilization agent to become soluble [21]. Fullerenes are known for their antimicrobial properties to inactivate viruses and bacteria and kill tumor cells. Fullerenes are not still used in commercial products as disinfectants because the knowledge of their properties and characteristics is limited. One of the main obstacles in the use of fullerol in water treatment is the difficulty in separating and recycling fullerol nanoparticles. Currently, there is no method to remove these small, light nanoparticles easily and cost-efficiently.

#### 3.4.6  Carbon Nanotubes

Carbon nanotubes (CNTs) are made of graphene sheets that are rolled into a tube and probably capped by half a fullerene. CNTs are considered as the most extensively analyzed allotrope of carbon in this millennium. They have very dramatic and particular physicochemical properties that own immeasurable applications in the industry and affect society noticeably. CNTs are basically divided into two types: single-walled nanotubes (SWNTs) that are a single pipe with a diameter from 1 to 5 nm and multiwalled nanotubes (MWNTs) that are constituted of several nested tubes at lengths varying from 100 nm up to several tens of micrometers. CNTs are utilized in several ways for disinfection applications. First, SWNTs can be coated on filters and MWNTs can be made into hollow fibers [22]. One of the main problems in the use of CNTs is the difficulty of dispersing them in water. This obstacle is maybe the reason that has limited the research on antimicrobial activity of CNTs. In an experiment, SWNTs were immobilized on a membrane filter surface and 87% E. coli bacteria were killed in 2 h [9].

Antimicrobial nanoparticles can be used and combined with other disinfection methods to improve their abilities. For example, some pathogens are highly resistant to UV disinfection. Therefore, the combination of UV disinfection with photocatalytic nanoparticles can be helpful to overcome this problem.

Despite all numerous advantages of antimicrobial nanoparticles in disinfection of water, some challenges can limit their applications. One of the main barriers is dispersion and retention of nanomaterials. TiO2 nanoparticles aggregate severely when they are added to the water. On the other hand, nanoparticles that are well dispersed in water cannot be separated easily because of their small sizes. The retention of nanoparticles can be expensive, and also these nanoparticles can be potentially harmful to human health and environment. Also the extensive use of nanoparticles can lead to leakage and accumulation of them in the environment. These nanoparticles can destroy beneficial microbes that play an important role in bioremediation and nitrogen fixation for plant growth.

#### 3.5  Case Study

The aim of this study that was conducted at the Microbiology Laboratory of Islamic Azad University of Shahreza is to observe the antibacterial activity of ZnO nanoparticles with different sizes against E. coli (ATCC 25922). All chemicals are analytical grade and are used as received without further purification. Double distilled water was used throughout the whole synthetic process. In a typical synthesis, (x) mL NH3•H2O (28%) was added into 15.0 mL 0.5 M zinc nitrate aqueous solution to form a transparent solution under stirring. Then x (g) CTAB and 15.0 mL of absolute ethanol were added to the previously formed solution. After stirring at 60°C for 30 min, the mixture was then transferred into a 60.0 mL Teflon-lined autoclave and maintained at 140°C for 15 h. Subsequently, after the autoclave was cooled, the as-formed white precipitate was filtered, washed with absolute ethanol and double distilled water, and then dried at 80°C. Detailed conditions for other samples are summarized in Table 3.3.

To determine the crystal phase composition of the prepared nanoparticles, x-ray diffraction (XRD) measurement was carried on a Bruker D8 ADVANCE XRD spectrometer with a Cu Kα line at 1.5406 Å and a Ni filter for an angle range of 2θ = 20°–80°. The XRD patterns of ZnO nanoparticles are shown in Figure 3.2.

All peaks of the obtained products were corresponding to the hexagonal wurtzite structure of ZnO with lattice parameters. No peak from either ZnO in other phases or impurities was observed. These results showed the ZnO nanoparticles were successfully synthesized by solvothermal reaction in all solvents.

Philips XL30 scanning electron microscope (SEM) measurements were also used to investigate the morphology of the samples with an accelerating voltage of 17 kV. SEM micrographs of three samples with different mixtures of solvents are shown in Figure 3.3. The average particle sizes were about 85, 70, and 57 nm for A, B, and C samples, respectively.

### Table 3.3   Starting Chemicals Used in the Solvothermal Syntheses

Expt. No.

0.5 M Zn(NO3)2 (mL)

CTAB (g)

28% NH3•H2O (mL)

H2O (mL)

EtOH (mL)

A

15

0

1.5

7.5

15

B

15

0.5469

1.5

7.5

15

C

15

0

6

3

15

Figure 3.2   XRD patterns of ZnO samples.

Figure 3.3   SEM images of ZnO nanoparticles.

The antibacterial efficiency of the synthesized ZnO nanoparticles is assessed using the macro-dilution (tube) broth method, determining the minimum inhibitory concentration (MIC) leading to inhibition of bacterial growth (National Committee for Clinical Laboratory Standards, NCCLS document M100-S12:NCCLS, Wayne, PA, 2000). Different concentrations of ZnO nanoparticles were prepared in sterilized Mueller–Hinton broth at final concentrations of 0.032, 0.016, 0.008, 0.004, 0.002, and 0.001 g/mL. Suspension of E. coli in sterile peptone water was adjusted to 0.5 McFarland. Then, 1 mL of E. coli suspension (0.5 McFarland) was added to each sample. The samples were kept in incubation at 37°C for 48 h. After incubation, samples were taken from tubes with no visible growth of bacteria and then were applied on nutrient agar (NA). After 24 h incubation, if one or two colonies were observed, this concentration was considered as MIC, but if no bacterial growth was observed, this concentration was recognized as minimum bactericidal concentration (MBC). The results of the antibacterial activity performance studies are collected in Table 3.4. It was found that the antibacterial activity of ZnO nanoparticles increased with decreasing particle size.

### Table 3.4   Results of the Antibacterial Activity Performance Studies

Expt. No.

MIC (g/mL)

MBC (g/mL)

A

0.016

0.032

B

0.008

0.016

C

0.002

0.004

#### 3.6  Summary and Conclusions

The quantity and quality of water is considered to be one of the critical issues for the coming decades. The control of microbial pathogens in water is one of the main problems that can limit available water resources. Different methods are implemented to disinfect water. Current methods of disinfection can form by-products that are harmful to the ecosystem. Also some pathogens are resistant to these methods. In recent years, the development of nanotechnology and antimicrobial agents that have little or no negative effect on the environment has become significant. Some nanoparticles can effectively inactivate and destroy these pathogens that are harmful to the health of humans. The action of nanoparticles is different against various microorganisms.

Despite high ability of some nanoparticles to inactivate pathogens, retention and suspension of nanoparticles can limit their application. Also some nanoparticles like silver can destroy beneficial microorganisms that are important for the environment. Overall, there is still a long way to go in order to study detailed characteristics of nanoparticles for the disinfection of water.

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