1825 © IWA Publishing 2013 Water Science & Technology | 68.8 | 2013

Inactivation of bacteria and helminth in wastewater

treatment plant effluent using oxidation processes

Regiane Aparecida Guadagnini, Luciana Urbano dos Santos,

Regina Maura Bueno Franco and José Roberto Guimarães

ABSTRACT

The contamination of bodies of water by raw and even treated sewage is worrying because

pathogens that affect public health and the environment are not fully eliminated in wastewater

treatment systems. The disinfection step is an important barrier to adopt to reduce this

contamination. However, widely used disinfectants such as chlorine do not guarantee the

inactivation of resistant organisms such as spore-forming bacteria and helminth eggs. This study

evaluated the effectiveness of processes of peroxidation (H2O2), ultraviolet radiation (UV) and

peroxidation assisted by ultraviolet radiation (H2O2/UV) in terms of reduction and inactivation of total

coliform bacteria, Escherichia coli, helminth eggs and larvae present in a treated sewage. Doses of

UV radiation of 70 mJ cm�2 and hydrogen peroxide concentration of 30 mg L�1 were used. The

number of bacteria reduced after UV and H2O2/UV processes was 3 and 4 log, respectively. An

average reduction of 59% in the number of eggs was verified when using H2O2, UV, and H2O2/UV

processes. Helminth larvae were reduced by 24% after H2O2 and UV; the process H2O2/UV did not

reduce the number of larvae. Statistically significant differences between the processes for both

organisms were not observed.

doi: 10.2166/wst.2013.431

Regiane Aparecida GuadagniniLuciana Urbano dos SantosJosé Roberto Guimarães (corresponding author)Laboratory of Oxidation Processes,Department of Sanitation and Environmental,University of Campinas (UNICAMP),Campinas, SP,BrazilE-mail: jorober@fec.unicamp.br

Regina Maura Bueno FrancoLaboratory of Protozoology, Department of Animal

Biology,University of Campinas (UNICAMP),Campinas, SP,Brazil

Key words | advanced oxidation process, disinfection, helminth eggs

INTRODUCTION

In recent decades, the world’s population has increased sig-nificantly with consequent growth in waste generation and

demand for water that is suitable for consumption. Chemi-cal compounds and microorganisms emerging and re-emerging in bodies of water increase the cost of makingwater drinkable. The World Health Organization (WHO

) estimated that 1.1 billion people did not have accessto drinking water and 2.6 billion people lacked adequatesanitation. This scenario leads to a serious public health pro-

blem because water quality affects people’s health,especially children. The concern with respect to drinkingwater quality is growing because many worldwide outbreaks

of waterborne protozoa were registered between January2004 and December 2010 (Baldursson & Karanis ).Among possible waterborne pathogens, helminths werelisted as being responsible for 6% of infections (WHO

). The helminth was also the most common of thehuman parasites, causing a series of health complications,such as chronic infections and reduction in productivity

and decline in physical and cognitive development inchildren.

These parasites affect over 2 billion people, of which 1billion people are infected with Ascaris lumbricoides, aparasite of global distribution. Ascaridiasis is a neglected dis-ease frequently associated with another species of helminth,

Trichuris trichiura, and other diseases such as malaria andHIV (Scott ; Holland ). Infection has been reportedin 5-month-old children and the prevalence and intensity

increase with age (Scott ).Due to its high prevalence and close association with

poor infrastructure, helminths are used as a social indicator

of a country. The lack of sanitation and behavior such aspoor hygiene habits are the forms of disease transmission.

Raw and treated sewage are among the main causes ofcontamination of water sources. They may contain the infec-

tious forms of helminths and various other pathogenicspecies (Cantusio Neto et al. ). In some Brazilianstates, legislation establishes parameters for the quality of

1826 R. A. Guadagnini et al. | Microorganism inactivation using oxidation processes Water Science & Technology | 68.8 | 2013

treated wastewater which depend on the quality level of the

receptor. An 80% reduction in biochemical oxygen demand(BOD) is required. Only bacteria of the coliform group areevaluated because they are considered a major environ-

mental indicator of fecal contamination. Escherichia coli isthe main thermotolerant coliform and the most efficientindicator of contamination by fecal matter, since it isrestricted to the intestinal tract of warm-blooded animals

(Soller et al. ). Parasites are much more resistant tochemical processes than bacteria, and resistant forms mayremain infectious for several months in the environment.

An aggravating factor is that parasitological assessment isnot routinely performed in water and wastewater treatmentplants. Therefore, the control of bacteria as an indicator of

water quality cannot be used as an indicator of parasites.The Atibaia River is the main water source for treatment

in the city of Campinas in the state of São Paulo in south-eastern Brazil. This river crosses a large metropolitan and

industrialized area and receives a high load of fecal materialdischarged as domestic effluent as demonstrated by themean values of thermotolerant coliforms of the order of

104MPN/100 mL (Cantusio et al. ). Toxoplasmagondii and Cyclospora cayetanensis protozoa were recentlyresponsible for a waterborne outbreak of disease in the

southeast and south of Brazil (Zini et al. ; Mouraet al. ). These records indicate the fragility of the sani-tation system and the importance of the disinfection

process as a barrier to reducing these pathogens.The water chlorination process ensures safety in relation

to viruses and bacteria, but does not ensure the inactivationof helminths (Mun et al. ) and there are few conclusive

studies on inactivation of helminth eggs.For example, helminth eggs of the genus Ascaris are

extremely resistant and survive numerous treatment con-

ditions. Aladawi et al. () evaluated a disinfectionprocess using ultraviolet radiation and observed a 2 log inac-tivation of A. lumbricoides with a UV dose of 400 mJ cm�2;

however, in a study by Brownell & Nelson (), this inac-tivation level was not achieved using UV doses of up to15,000 mJ cm�2. Mun et al. () reduced 2.5 log helminth

eggs after 60 s (700 W) using microwave radiation. Thesevariations observed in existing results confirm the need forstudies on the effect of different water disinfection processeson the helminths.

Advanced oxidation processes (AOP) have a high oxi-dation potential mainly due to the generation of hydroxylradicals (•OH) (E¼ 2.80 V). These free radicals are not

selective and interact with a wide range of recalcitrant com-pounds and cell components. AOP are usually considered

clean technologies; they can mineralize organic compounds,

generating products that aren’t as harmful to health (Cor-deiro et al. ; Cho & Yoon ). They have beenshown to be effective at inactivating Clostridium perfringensand coliphages (Guimarães & Barretto ), Escherichiacoli, Bacillus subtilis, MS2 virus (Mamane et al. ; Alrou-san et al. ) and protozoa (Cho & Yoon ; Guimarãeset al. submitted).

There are several combinations used in AOP to obtainthe hydroxyl radical, among them ultraviolet light-assistedperoxidation (H2O2/UV). This has the advantage of a high

oxidizing potential, and hydrogen peroxide is commerciallyavailable, thermally stable, and can be stored at the point ofuse (Domènech et al. ). Theoretically, a molecule of

H2O2 produces two hydroxyl radicals (Equation (1)) whensubjected to a source of energy sufficient to break thebond between the two oxygen atoms with quantum yield(Φ) of 0.98 at 254 nm:

H2O2 þ hν ! 2�OH (1)

When in excess, the hydrogen peroxide can produceother types of reactions. They are competitive and canhave the effect of reducing degradation efficiency (Domè-

nech et al. ). Studies are needed to provide anoptimum concentration of oxidant and UV radiation dosefor the inactivation of different target species.

The aim of this study was to evaluate the efficiency ofprocesses of peroxidation (H2O2), ultraviolet radiation(UV) and peroxidation assisted by ultraviolet radiation

(H2O2/UV) in the reduction and inactivation of total coli-form bacteria and Escherichia coli and helminth eggs andlarvae present in a treated domestic sewage.

MATERIALS AND METHODS

The samples (n¼ 4 for bacteria and n¼ 5 for helminths) werecollected at Samambaia Wastewater Treatment Plant

(Samambaia WWTP) in Campinas, Brazil. The plant receivesonly domestic sewage and has the capacity to treat 150 L s�1.Samambaia WWTP uses a conventional sewage treatmentsystem based on activated sludge and secondary clarification.

In order to evaluate the efficiency of bacteria and hel-minth removal by the activated sludge process, twosampling points were selected: (1) influent and (2) effluent.

To evaluate the oxidative process, five samples of the efflu-ent were taken in jugs that had been previously

Table 1 | Variation in number of total coliform bacteria in different samples

Samples Mean SD Max. Min.

Influent 7.20 × 107 1.9 × 107 9.20 × 107 4.90 × 107

Effluent 2.45 × 105 9.88 × 104 3.70 × 105 1.40 × 105

Effluentþperoxidation

1.08 × 105 2.39 × 104 1.40 × 105 8.30 × 104

EffluentþUV radiation

6.20 × 101 4.97 × 101 1.10 × 102 <1

EffluentþAOP 1.01 × 101 1.50 × 101 3.20 × 101 <1

Table 2 | Variation in number of E. coli bacteria in different samples

Samples Mean SD Max. Min.

Influent 223 × 107 6.08 × 106 2.10 × 107 1.70 × 107

Effluent 3.05 × 104 9.88 × 103 4.50 × 104 2.30 × 104

Effluentþperoxidation

5.55 × 104 7.65 × 104 1.70 × 105 1.30 × 104

EffluentþUV radiation

8.55 7.29 1.60 × 101 <1

EffluentþAOP 2.03 2.49 5.1 <1

1827 R. A. Guadagnini et al. | Microorganism inactivation using oxidation processes Water Science & Technology | 68.8 | 2013

conditioned with 0.1% Tween 80 eluting solution. Samples

were refrigerated at 10 WC and taken to the Laboratory ofOxidative Processes (LABPOX) for the tests and analysis.LABPOX is part of the Civil Engineering, Architecture

and Urban Design School of the University of CampinasDepartment of Sanitation and Environment.

Disinfection process: reactor

The experimental system consisted of a photochemical reac-tor designed by Guimarães et al. (submitted): a 42.5 cm long

borosilicate glass cylinder with an internal diameter of3.8 cm and a germicidal lamp (15W, λmax¼ 254 nm, and2.5 cm internal diameter) inserted in the center in direct con-

tact with the sample. The internal volume of the internalreactor is 190± 0.05 mL, and the system also includes a reser-voir (2 L) and a peristaltic pump to the solution inmovement.

The assays were carried out using 70 mJ cm�2 doses ofUV radiation. Initial hydrogen peroxide concentration was30 mg L�1. The following conditions were used: (a) only inthe presence of hydrogen peroxide (H2O2; (b) only in pres-

ence of ultraviolet radiation (UV); and (c) in the presenceof hydrogen peroxide and ultraviolet radiation (H2O2/UV).The UV and peroxidation processes were used as a control

to evaluate the synergistic effect of AOP.The system was decontaminated before and after each

test to remove possible residues and microorganisms that

may be retained in the system. The experimental conditionsand procedures proposed followed the protocols establishedby Guimarães et al. (submitted).

Bacterium and helminth detection and inactivation

Quanty-Tray®/2000 test (IDEXX Laboratories, Incorpor-

ation) based on IDEXX Enzymatic Defined SubstrateTechnology® was used to evaluate the inactivation of bac-teria by oxidative processes. The test had a detection limit

of 1 MPN/100 mL and maximum limit of 2,419.6 MPN/100 mL. The samples were incubated for 24 h at 35 WC.

Helminth analyses fromprocessed or unprocessedwaste-

water samples were performed according to the Victoria &Galván () protocol. One liter of effluent sample was cen-trifuged at 480 × g for 5 min in 50 mL centrifuge tubes. Afterremoving the supernatant, the pellet was washed with dis-

tilled water and centrifuged at 480 × g for 5 min. After twowashes, the pellet was resuspended by zinc flotation and cen-trifuged at 480 × g for 5 min. The supernatant was filtered

through a 47 mm diameter 5.0 μm membrane. The mem-brane was submitted to sample elution by alternately

scraping the membrane with a smooth-edged plastic loop

and rinsing with elution solution. The final 1 mL pellet wastransferred to a microcentrifuge tube after a homogenizationprotocol (Redlinger et al. ). Aliquots of 15 μL of the

samples were tested with Trypan Blue (1%) for quantificationand verification of possible egg inactivation.

Statistical analysis

Student’s t-test with a p-value< 0.05 was used to analyze the

data.

RESULTS AND DISCUSSION

The evaluation of the disinfection processes’ efficiency was

based on the inactivation and/or the number of microorgan-isms reduced. Total coliform (TC), E. coli (EC) bacteria, andhelminth eggs and/or larvae were found in every influentand effluent sample. The TC quantification varies between

samples (Table 1) with an average of 7.2 × 107MPN/100 mLin influent samples and 2.5 × 105MPN/100 mL in effluentsamples. The EC quantification varies between samples

(Table 2) with an average of 2.2 × 107MPN/100 mL in influ-ent samples and 3.1 × 104MPN/100 mL in effluent samples.

Table 4 | Number of helminth larvae in the effluent and after disinfection processes

Samples Mean SD Max. Min.

Influent 1,157.6 1,236.8 2,777.8 0

Effluent 197.3 235.6 600.0 0

Effluentþ peroxidation 124.1 159.6 370.4 0

EffluentþUV radiation 176.4 247.0 600.0 0

EffluentþAOP 219.8 247.5 555.6 0

Table 3 | Number of helminth eggs in the effluent and after disinfection processes

Samples Mean SD Max. Min.

Influent 300.3 305.2 740.8 0

Effluent 80.0 86.9 200.0 0

Effluentþ peroxidation 42.0 88.4 200.0 0

EffluentþUV radiation 30.7 33.9 66.7 0

EffluentþAOP 26.7 59.6 133.3 0

1828 R. A. Guadagnini et al. | Microorganism inactivation using oxidation processes Water Science & Technology | 68.8 | 2013

The activated sludge treatment system applied in the

WWTP was seen to be responsible for a reduction ofapproximately 2 log of total coliform bacteria and E. coli.These data agree with the reduction of bacteria expected

for the activated sludge system, ranging from 1 to 2 log inac-tivation (Wen et al. ).

Among the disinfection processes evaluated, peroxi-dation was the least efficient at bacteria inactivation. Other

authors found no inactivation of E. coli exposed to hydrogenperoxide concentrations between 25 and 40 mg L�1 with con-tact times of 60 min (Mamane et al. ; Labas et al. ).The UV process resulted in a 3 log reduction for both bacteriastudied, TC and EC. UV disinfection systems have been usedin many wastewater treatment plants in North America and

Europe. UV applications are based on several studies whichreport a minimum UV dose of 1.2 mJ cm�2 to reach a 6 logreduction of E. coli and more than 30 mJ cm�2 to preventphotoreactivation (Guo et al. ). Bacteria inactivation

data shows that H2O2 assisted by UV is very efficient at inac-tivation, reaching 4 log reduction for both bacteria. Theseresults show an increase of 1 log in inactivating the bacteria

when H2O2 and UV are used together.An average of 300 helminth eggs and 1,157 larvae were

found per liter of influent samples, while 80 eggs and 197

larvae were found in each liter of effluent. The helminth eggsobservedwere of the generaAscaris and hookworm.However,all eggs were fertilized (viable) when analyzed by Trypan Blue

staining. Embryonated eggs of hookworms were seen.The activated sludge system also had an average reduction

of helminth eggs and larvae of less than 1 log.Wen et al. ()reported a reduction of almost 3 log in the eggs ofAscaris spp.in an activated sludge laboratory system.However, the authorsclaimed that this high reduction might have been affected bythe hydraulic conditions in the laboratory system.

A great variation in the number of organisms can be seenin the samples in this study, both of eggs and larvae. This factshows the difficulty ofworkingwith a real sample for detection

and study of helminths in order to quantify the organisms,since there are many variables that have a direct influence,mainly seasonality, the amount of suspended solids and

solids that settle, and wastewater treatment plant influentflow. The average helminth eggs and/or larvae observed insamples of effluent (disinfected or not) are shown in Tables 3and 4. The standard deviation values confirm that there is a

high variability in quantification of helminths.The detention time of raw sewage in the activated sludge

process at Samambaia WWTP is about 48 hours. This could

explain why a great number of helminth larvae are seen. Hel-minth eggs at this point can adhere to particulate matter and

have been sedimented. This prevents them from beingcounted accurately since the supernatant was evaluated.

Even considering these variations, the peroxidationprocess assisted by ultraviolet radiation was more efficientthan the UV and peroxidation processes for helminth eggs.

Although a larger number of helminth larvae were observedin samples disinfected by H2O2/UV than by the H2O2 or UVprocesses, no statistically significant difference was found(p¼ 0.05) between the data obtained from the three processes.

CONCLUSIONS

Using an activated sludge system, the SamambaiaWWTPhas

reached the expected results in terms of reduction of numbersof pathogens, considering the control data from treated efflu-ent. H2O2/UV was more effective at reducing helminth eggsand inactivating bacteria. Thus, the H2O2/UV advanced oxi-

dation process has a great potential as a step disinfectiontechnique to control total coliform, Escherichia coli bacteria,helminth eggs, and helminth larvae present in treated sewage

and to minimize contamination of the receiving waters inorder to protect public health.

ACKNOWLEDGEMENT

This work was funded by the State of São Paulo ResearchFoundation – FAPESP (2010/15550-8).

1829 R. A. Guadagnini et al. | Microorganism inactivation using oxidation processes Water Science & Technology | 68.8 | 2013

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