Study of sequential disinfection for the inactivation of protozoa and indicator microorganisms in wastewater

Sewage disinfection has the primary objective of inactivating pathogenic organisms to prevent the dissemination of waterborne diseases. This study analyzed individual disinfection, with chlorine and ultraviolet radiation, and sequential disinfection (chlorine-UV radiation). The tests were conducted with anaerobic effluent in batch, in laboratory scale, with two dosages of chlorine (10 and 20 mg L) and UV (2.5 and 6.1 Wh m). In addition, to guarantee the presence of cysts in the tests, 10 cysts per liter of Giardia spp. were inoculated. The resistance order was as follows: E. coli = Total Coliforms < Clostridium perfringens < Giardia spp.. Furthermore, synergistic effects reached 0.06 to 1.42 log of inactivation in sequential disinfection for both the most resistant microorganisms.


Introduction
Giardia lamblia (synonyms: G. duodenalis and G. intestinalis) is a flagellated protozoan that was discovered by Antonie van Leeuwenhoek in 1681.This microorganism raises concerns due to its low infective dose and survival in unfavorable environments, being released in great numbers by ill and asymptomatic individuals.It is an obligatory parasite and the most associated with diarrheic diseases in the world, presented as a cyst in its resistant form (OKOH et al., 2007;PLUTZER et al., 2010).
Sewage, depending on the type of treatment process, presents high concentration of Giardia cysts.In raw sewage, 10² to 10 5 cysts per liter are normally found (ROBERTSON et al., 2006;CANTUSIO NETO et al., 2006).After secondary treatment of sewage, such as activated sludge, approximately 5.9 to 79 cysts per liter are usually reported (CASTRO-HERMIDA et al., 2008;MOULIN et al., 2010).
Persistence in the aquatic environment, resistance to disinfection by chlorine and infectivity are characteristics that make Giardia spp. a high-risk pathogen for public health (BETANCOURT; ROSE, 2004).Therefore, an efficient sewage treatment process with high removal rate and microorganism inactivation decreases the amount of pathogens discharged in water sources (CASTRO-HERMIDA et al., 2008;LEVANTESI et al., 2010).
Chlorine is the most widely used disinfectant in the world for treating water and wastewater.With the discovery of chlorine byproducts, ultraviolet radiation became more popular (BV, 2010).The disinfection mechanism through UV light is based on changes in the DNA and the RNA of microorganisms.UV radiation has the advantage of not leaving any residual in the treated sewage, and its effectiveness is not affected by pH or temperature (HIJNEN et al., 2006;BV, 2010).
In sequential disinfection, the effect of the combined action of disinfectants, which promotes an inactivation of microorganisms that is higher than the sum of the inactivation of the same disinfectants applied separately, is called synergism (USEPA, 1999;CHO et al., 2010).The probable reasons for the increased interest in these processes are the use of smaller concentrations of disinfectants and the possible decrease in the formation of disinfection byproducts, the control of the formation of biofilms in the water distribution system and the high inactivation levels of pathogenic microorganisms, including the most resistant types, such as Giardia spp.and Cryptosporidium spp.(USEPA, 1999).
Thus, the goal of this study was to evaluate and compare the resistance of Giardia spp.and indicator microorganisms -total coliforms, Escherichia coli and Clostridium perfringens -to disinfection with chlorine, ultraviolet radiation, and chlorine followed by ultraviolet radiation, with the evaluation of synergistic effects.

Material and methods
The sewage used in the study experiments was obtained from the São Paulo University Wastewater Treatment Plant (WWTP) in São Carlos, which has preliminary treatment composed of screening, sand and a grease removal tank and an Upflow Anaerobic Sludge Blanket (UASB) reactor with 18.8 m³ of operating volume.Table 1 presents the characteristics of the treated wastewater used in the study.
The disinfection tests were conducted in bench units, in batches, with inoculation of approximately 10 4 Giardia spp.cysts per liter.Aliquots of 600 mL were retrieved as samples for the analysis.

Disinfection with chlorine
Borosilicate glass beakers with capacity of two L were disinfected and washed with a Tween 80 (0.1%) solution.After the sewage was added, the containers were placed in a jar test apparatus and submitted to a speed gradient of 100 s -1 .A solution of Sodium hypochlorite (NaOCl -analytical grade) was prepared on the day of the tests from a stock solution of 4 to 6% P.A. (Vetec Química Fina, Ltda).The final free chlorine concentration used was analyzed by the DPD colorimetric method.The dosages used were: 10 mg L -1 for 10 min. of contact time; 10 mg L -1 for 20 min.and 20 mg L -1 for 10 min.

Disinfection with ultraviolet radiation
Prior to the tests, the average intensity of radiation emitted by the lamps inside the reactor was determined through radiometry (UVX Radiometer -sensor for 254 nm -UVP, CA, USA) in nine evenly distributed points, with the reactor empty.The correction of average intensity on the liquid was carried out considering wastewater radiation absorbance (average in a DR 4000 spectrophotometer) and depth of 3 cm; the applied dosage (Wh m -3 ) was estimated using the corrected average intensity that had been previously calculated.A volume of 5.4 L was prepared in a UV radiation reactor that had been previously disinfected and washed with a Tween 80 (0.1%) solution, and six 15W low pressure mercury lamps that were emerged from the liquid.After the absorption reading reached 254 nm (DR 4000 -Hach), the times to obtain received doses of 2.5 and 6.1 Wh m -³ were established.

Sequential disinfection
After testing with chlorine, an intermediary sample was removed (chlorinated sewage).The remaining sewage was sent to the UV radiation reactor.Figure 1 shows the disinfection test scheme.Determination of total and free residual Chlorine The total and free residual chlorine analyses were conducted by the DPD colorimetric method (N, N-diethyl-p-phenylenediamine) using the spectrophotometer DR 2010 (Hach).The reagents used were DPD Total Chlorine Reagent-CAT 14064-99 and DPD Reactive for free Chlorine -CAT 14070-99 (Permachem ® Reagentes, Hach).The DPD was oxidized by the chlorine present in the sample with the formation of a pink solution, which had an intensity that was proportional to chlorine concentration.
At the end of each test, a 3% sodium metabisulfite solution (Na 2 S 2 O 5 ) was used in a proportion of 0.1 mL for each 100 mL of the sample to remove the combined and free residual chlorine.
The methodology was adapted from McCuin and Clancy (2005) and Robertson et al. (2000).For concentration of cysts, two centrifugation steps were used at 1500 g for 15 min.from a 75 mL sewage sample with 25 mL Tween 80 (0.1%).Supernatant was removed keeping a volume of 5 mL and no more than 500 μL of sediment.At the stage of purification by immunomagnetic separation (IMS), the Invitrogen Dynal AS kit (Norway) was used following the manufacturer's protocol with the use of dynabeads specific for Giardia (Dynal CG -Combo anti-Cryptosporidium and Giardia).The acid dissociation of cysts was performed with HCl (0.1 mol L -1 ).The detection with immunofluorescence assay (IFA) performed with the Merifluor kit (Meridien Bioscience, Cincinnati, Ohio) and DAPI solution (4',6-diamidino-2phenyl-indol) (DAPI -Sigma ® ), with a 1:4000 dilution.
The viability was tested through vital dye assay with propidium iodide (Sigma-Aldrich, USA).The reagent responsible for the emission of red fluorescence (λ = 510 to 550 nm) penetrates only the microorganisms with a damaged membrane (dead cells).The recovery of cysts reached a value of 53.1 (± 29.7%), which was within the values predicted in the 1623.1 method (USEPA, 2012).The samples were examined with an immunofluorescence microscope (Olympus BX51) with an increase in magnification from 400 to 800 X.The result was expressed as the number of cysts per liter.

E. coli and total coliforms
For the quantification of these microorganisms, the pour plate technique was used with Chromocult ® Coliform Agar medium (Merk Cat.No.1.10426).According to Olstadt et al. (2007), Chromocult ® Coliform Agar medium has the ability to detect the presence/absence of total coliforms and E. coli and to enumerate organisms.The Petri dishes were subsequently incubated at 36 ± 1ºC for 24 ± 1 hours.This methodology was used due to the amount of solids in the membrane that make the identification of colonies difficult.The colonies that show a dark purplish blue color were identified as E. coli, whereas those that revealed a reddish salmon color added to the number of E. coli colonies, were identified as total coliforms.The result was given in CFU by mL.
A comparison of this technique with the membrane filter technique (APHA et al., 2005) was performed.The statistical results from the ANOVA test indicated that there was no significant difference between the two methods for E. coli and total coliforms counts (P-value = 0.87 and 0.14 for total coliforms and E. coli, respectively) (Statistica 7.0, StatSoft, Inc).

Clostridium perfringens
For the detection of this pathogen, the membrane filter technique was performed next, with a mCP agar.It is also the method used by Massé et al. (2011).Muller-Spitz et al. (2010) demonstrated that the mCP method is a robust approach to enumerate and isolate C. perfringens from samples receiving faecal pollution.First, the sample in decimal dilutions was heated in a water bath at 60 ± 0.5ºC for 15 min.to eliminate any nonspore-forming organisms and vegetative forms.Subsequently, the sample was immediately cooled in an ice bath.
Subsequently, filtering of 100 mL of the sample was performed through a sterile cellulose nitrate membrane with 0.45 μm porosity (Sartorius Stedim Biotech GmbH, Germany), and the sample was then transferred to a disposable sterile Petri dish containing a selective and differential culture medium m-CP agar (Oxoid Ltda, England).Subsequently, the dishes were incubated in an anaerobiosis jar (Permution, Brazil; Gas Pak ® System, BBL) for 24 hours at 44.5±0.2ºC.Maintenance of an anaerobic atmosphere was achieved by inserting an anaerobic system envelope (BD BBLTM GasPakTM Plus, ref. 271040; Becton, Dickinson and Company, USA).
The checking of the colonies was made in a culture medium containing a Thioglycolate Broth (CM 0173, Oxoid Ltd, England), which was incubated at 35±0.5ºC for 24 hours.From the positive tubes with thioglycolate broth, 1 mL was inoculated in tubes containing a milk medium with modified iron, and the tubes were incubated at 44.5 ± 0.2ºC for 2 hours.The reading of the confirmatory test was conducted with positive tubes, which showed the production of turbulent fermentation in a medium with milk and modified iron, with formation of clot.The result was expressed in CFU per 100 mL.

Result and discussion
Table 2 presents the results of the tests conducted in this study.It is worth emphasizing that to quantify the inactivation in the cases where the number of organisms was below the detection limits (100% of inactivation), the occurrence of one colony forming unit (CFU) was considered for the calculations.Disinfection with chlorine, considering the concentrations used and repetitions applied, obtained 100% effectiveness in inactivating E. coli in all the tests and total coliforms in tests III, V and VI.The spore forming bacteria -Clostridium perfringensproved to be very resistant to chlorine, as also shown by Tyrrel et al. (1995), because only when CT (concentration x contact time) values were applied to 200 mg min -1 L -1 , above 1 log of inactivation was obtained (tests IV and VI).Nevertheless, in test V, where the same CT value was used, the same effectiveness was not obtained.This may have occurred due to the characteristics of the sewage.
Concerning Giardia spp., chlorine was not very effective, with a maximum of 0.16 log of inactivation (test VI).Rice et al. (1982) found, in a study conducted by in vitro excystation, great resistance of Giardia ssp.cysts, where CT of 75 mg min -1 L -1 was needed for a 2 log inactivation in pH 7.0 and at 5ºC.Li et al. (2004) found CT values similar to those used in this study, between 0.9 and 1.2 log of Giardia spp.inactivation using free chlorine.They also reported that chlorine first acts at the cyst wall resulting in an increase in cyst permeability followed by plasma membrane disintegration and cytoplasm damage, which is highly valuable for ultraviolet radiation sequential application.
In the tests that applied two doses of UV radiation, the same resistance standard was observed with E. coli and the coliforms presenting the higher inactivation.The use of a second dose of UV radiation provided a complete inactivation of E. coli in the three tests (II, IV and VI), and complete inactivation of total coliforms in the sixth test, leading to an inactivation increase of up to 1.7 logarithmic units when compared to the first dose.Clostridium perfringens again proved to be resistant to a second dose of UV radiation, implying in the inactivation of a maximum of 0.78 log more than the first dose in the fourth test.For Giardia it reached a maximum of 0.15 log of inactivation.Liberti et al. (2002) used doses from 100 to 160 mWs cm -2 to obtain approximately 60% of Giardia ssp.removal.Cantusio Neto et al. (2006) found infective cysts after doses of 25 to 30 mJ cm -2 in 33% of the animals submitted to in vivo assays.Li et al. (2009) performing in vivo infectivity could not obtain Giardia ssp.cysts infectivity to decrease more than 1 log after the application of ultraviolet radiation with doses from 18 to 57 mJ cm -2 .
As for the tests with sequential disinfection with chlorine and UV (2.5 Wh m -³), E. coli, which was inactivated below the detection limit with the application of chlorine only, maintained the same inactivation (100%) in the sequential tests.Total coliforms were 100% inactivated, except for test IV, where the inactivation was of 3.6 log.Clostridium perfringens showed an increase in inactivation in the sequential tests reaching up to 3.43 log in test V.For the Giardia spp., up to 0.23 log of inactivation was reached in test I.
The second doses of UV in the chlorine sequence provided a more complete total coliform inactivation.Tests II and IV maintained the same inactivation of Clostridium perfringens promoted by the first UV dose; however, in test VI, this bacteria was 100 % inactivated above 4.43 log.Despite presenting the same CT at 200 mg min. - L -1 , test VI resulted in a higher inactivation rate than test IV, and in this case, the dose seemed to be more important than time contact.
Sequential disinfection studies are still incipient for wastewater effluents; however, many articles for sequential disinfection of water may be found in literature.Synergism only occurred for more resistant microorganisms-Clostridium perfringens and Giardia spp, as shown in Table 3, since the E. coli bacteria and total coliforms were often completely inactivated with the application of only one of the used disinfectants.Caretti and Lubello (2003) found synergic effects using peracetic acid followed by UV radiation in the disinfection of fecal and total coliforms, E. coli, fecal streptococcus and Pseudomonas aeruginosa in wastewater.Ryu et al. (2007), after analyzing samples from tertiary effluents of seven WWTPs, concluded that WWTPs with combined disinfection of chlorine followed by UV radiation reached the target for annual acceptable risk for Giardia ssp.infection -one infection for each ten thousand inhabitants.Souza and Daniel (2011) found synergistic effects when low doses of chlorine were applied combined with ozone for E. coli disinfection.
Wang et al. ( 2012) reported the occurrence of synergistic effects for heterotrophic bacteria count, total bacterial count and total coliforms in sequential disinfection composed of ultraviolet radiation followed by chlorination of effluent for reuse.According to Koivunen and Heinonen-Tanski (2005), the synergic action can be explained by the mechanism of multiple damage, two different disinfectants can cause damage to different types of microorganisms and, therefore, promote a more effective inactivation of them.
The lower level of inactivation shown by Giardia spp.may be explained by an underestimation of the vital dye method, as reported by Campbell and Wallis (2002), in comparison to in vivo infectivity.These authors, along with Shin et al. (2009), noticed a greater effectiveness of UV radiation in the inactivation of this protozoan -above 2 log of inactivation -for doses close to the ones used in this study.
However, in the study of Li et al. (2009), even with in vivo assays, the decrease in the infectivity of Giardia ssp.cysts was lower than 1 log.The same authors report that these results may be due to the cysts protection from UV radiation provided by particles present in the wastewater, cysts reactivation potential after exposure to UV light and bacteria attack and adsorption of colloidal material from cyst surface, partially not permitting UV radiation to penetrate the cysts.
Heaselgrave and Kilvington (2011) reported that although the vital dye PI underestimate inactivation especially for disinfection assays, this problem highlights the requirement for robust and reproducible in vitro tests for determination of viability.Current cell culture-based methods are technically difficult to perform and the gold standard for determining the viability of cysts requires the use of in vivo animal infectivity studies which are complex, expensive, time demanding and involve ethical questions (SCHETS et al., 2005).

Conclusion
It is possible to use smaller CT for primary disinfection with chlorine, since sequential disinfection may be present inactivation similar to the one obtained with the use of only one disinfectant at higher CT.
Giardia spp.proved to be insusceptible even to sequential disinfection and synergistic effect was noticeable for C. perfringens and Giardia spp. in some of the tests.
E. coli and total coliforms were not particularly resistant to the disinfectants used, implicating in the questioning of the actual range reached by the traditional indicators.

Figure 1 .
Figure 1.Scheme of the disinfection tests.

Table 1 .
Anaerobically treated wastewater used in the disinfection tests.

Table 2 .
Inactivation of microorganisms with chlorine, UV radiation and sequential (chlorine-UV radiation).