Journal of Photochemistry and Photobiology B: Biology 117 (2012) 278–285

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Journal of Photochemistry and Photobiology B: Biology

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New kinetic model for predicting the photoreactivation of bacteria with sunlight

J.J. Vélez-Colmenares ⇑, A. Acevedo, I. Salcedo, E. NebotDepartment of Environmental Technologies, Faculty of Marine and Environmental Sciences, University of Cádiz, Cadiz, Spain

a r t i c l e i n f o a b s t r a c t

Article history:Received 17 January 2012Received in revised form 3 September 2012Accepted 11 September 2012Available online 16 October 2012

Keywords:PhotoreactivationUltraviolet irradiationKineticEscherichia coliWastewater

1011-1344/$ - see front matter � 2012 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.jphotobiol.2012.09.005

⇑ Corresponding author. Address: Polígono Rio SanCádiz, Spain. Tel.: +34 956016587.

E-mail address: julia.velez@uca.es (J.J. Vélez-Colm

Exposure to ultraviolet radiation is a disinfection process that is used worldwide for the treatment ofwastewater in order to minimize microbial contamination caused by wastewater discharge to naturalwaters. Once organisms have been exposed to ultraviolet radiation, they are able to repair the damagethrough two processes – dark repair and photoreactivation. In the work described here, the photoreacti-vation process after ultraviolet disinfection has been studied in pure culture of Escherichia coli ATCC11229, ATCC 15597 and in real wastewater, using both a laboratory plant and a pilot plant. A new kineticmodel is proposed that is a modification of the model proposed by Kashimada et al. [15] including a firstorder decay phase. This model was applied to the photoreactivation process with sunlight. The newmodel incorporates a decay rate constant (Ms) for solar reactivation in order to explain correctly thedecay phase detected in the experimental data for photoreactivation with sunlight. The new model fitsthe data obtained in reactivation experiments, thus allowing the interpretation of the kinetic parametersSm, Sm � So, ks, and Ms and their relationship with UV dose.

� 2012 Elsevier B.V. All rights reserved.

1. Introduction

UV irradiation has become one of the most important alterna-tives to chlorination for wastewater disinfection throughout theworld [31]. The reuse of reclaimed wastewater for agricultural pur-poses and golf course irrigation is increasing, and the advantages ofapplying UV disinfection to enable wastewater to be reused arewidely recognized [15].

Light at wavelengths between 250 nm and 270 nm shows themaximum effectiveness for inactivating microorganisms, a situa-tion that is consistent with the maximum absorbance of nucleotidebases of the genome including thymine, cytosine and uracil[11,24]. For this reason, UV-C radiation at 254 nm causes damagein DNA and RNA with inhibition of cell transcription and replica-tion. The alteration of DNA is caused by the induction of the forma-tion of photoproducts such as thymine–thymine cis-syncyclobutane pyrimidine dimer (CPD), thymine–thymine pyrimi-dine (6-4) pyrimidone photoproducts [TT (6-4) photoproducts][3] and pyrimidine photohydrates, whose significance in biologicalinactivation is low. CPDs have biological consequences in the inac-tivation of cells and the photoproducts play an important role inUV-induced cytotoxic damage [8]. Some microorganisms, particu-larly bacteria, are known to be capable of repairing their damaged

ll rights reserved.

Pedro s/n 11510, Puerto Real,

enares).

DNA in the presence or absence of visible light by dark repair orphotoreactivation mechanisms [28].

Dark repair or nucleotide excision repair is a multi-enzyme re-pair process that involves the excision of dimers [38] and requiresthe coordination of over a dozen proteins to excise and repair thedamaged DNA segment [6]. In contrast to dark repair, photoreacti-vation requires light to activate the repair mechanism. Light be-tween 330 and 480 nm (UV-A) activates repair enzymes thatsplit the pyrimidine dimers to recover the damaged DNA[36,16,22]. Photolyase is the light enzyme responsible for photore-activation processes [31]. This enzyme contains two co-factors:5,10-methenyltetrahydrofolate (MTHF) absorbs about 90% of visi-ble light, while the double-electron reduced form of Flavin AdenineDinucleotide (FADH�) catalytically reverses DNA damage [10].

Photoreactivation is considered to be the most important of thetwo mechanisms and it follows a two-step reaction scheme [11]:

Step 1: Formation of a complex between a photoreactivationenzyme (PRE) and the dimer to be repaired. This step doesnot require light, but is dependent on temperature, pH andionic strength [17].

Step 2: Release of PRE and repaired DNA. The restoration of thedimer to its original monomeric form is absolutely depen-dent on the intensity of light energy [27]. The PRE-dimercomplex is formed in the process. An extended period ofexposure to photoreactivating light would enable therelease of PRE and this would then be available to formnew complexes (Step 1) [20].

J.J. Vélez-Colmenares et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 278–285 279

Wastewaters after UV disinfection are often exposed to visiblelight and the ability of microorganisms to be reactivated musttherefore be considered to ensure conditions for hygienically safewater disinfection [30].

Reported exposure times that result in maximum photoreacti-vation have ranged from minutes [11,27,20,10], to hours[21,37,12,5,39,9] to days [19]. The differences in these time scales,although not fully understood, may be attributable to several fac-tors, including the particular experimental set up, the initial num-ber of pyrimidine dimers formed, the number of photoreactivatingenzymes present in the organism, the temperature during PRE-dimer complexing, and the dose rate of photoreactivating radiation.

According to Lindenauer and Darby [17], the extent of photore-activation is dependent upon the number of PRE-pyrimidine dimercomplexes formed. The number of complexes formed is limited bythe number and availability of the PREs in each cell.

Most studies on photoreactivation involve the use of visiblelight from artificial sources such as fluorescent lamps that emitlight at 360 nm [15,24,38,18,25,28,4] and halogen lamps emittingbetween 400 nm and 800 nm [10]. However, very few studies ofthis type concern the use of natural sunlight. UV solar radiationis able to inactivate microorganisms due to the synergistic effectof the UV light and heating of water by infrared radiation.The UV wavelengths that reach the earth‘s surface are classifiedas UV-A (320–400 nm) and UV-B (290–320 nm). UV-B radiationcan cause direct DNA damage by inducing the formation of DNAphotoproducts [26] and UV-A radiation has a bacteriostatic effectand, in some water matrixes, sunlight could produce dissolvedoxygen, leading to highly reactive oxygen species in the presenceof dissolved oxygen [7,29]. UV-A also has a repair effect on DNAand RNA damaged by the action of UV-B radiation on exposureto natural sunlight and the effect of UV-C upon artificial exposure.

Likewise, the majority of photoreactivation studies involve theuse of collimated beam tests with low fluences and optimal condi-tions for light exposure for repair, such as a thin layer of fluid [14].

Very few studies have focused on modelling the reactivationprocesses and in most cases the process is simply described[12,17,13,39,23,25,10]. Other authors have tried to model jointlythe inactivation and reactivation phases [15,31,2,20]. In 1996Kashimada et al. developed a photoreactivation model to predictthe reactivation phase for Escherichia coli with a saturation-typefirst order reaction. In 1999 Tosa and Hirata, used the Kashimadamodel to study the photoreactivation of E. coli exposed atfluorescent lamps by comparing experiments with and withoutreactivation and established a relation between microorganismconcentration and UV dose. In 2002, Beggs used the Tosa and Hira-ta experimental data to describe a theoretical model for quantify-ing the photoreactivation process and the photolysis rates for E. colistrains. In 2007, Nebot et al. studied the photoreactivation anddark repair of bacteria – total coliforms, faecal coliforms and faecalstreptococci – in order to develop a kinetic model that would allowthe prediction of the reactivation after UV disinfection dependingon the UV-C dose applied.

In the majority cases, the photoreactivation had been studiedin controlled conditions using only UV-A, but the photoreactiva-tion with real conditions of sunlight (including UV-A and UV-Bradiation) had not been studied and the decay phase after reacti-vation phase had not been observed. Therefore, it is necessary toinvestigate and to apply new kinetic models that can predict reac-tivation processes and provide a better understanding of the fac-tors that affect this interesting phenomenon. For this reason, theprincipal objective of the work described here was to propose anew kinetic model to explain the photoreactivation of microor-ganisms after UV disinfection in real conditions of sunlightexposition.

2. Materials and methods

2.1. UV irradiation

2.1.1. Laboratory plantUV disinfection experiments were carried out at laboratory

scale in the system described by Vélez-Colmenares et al. [35]. Thissystem has a UV germicidal emission from a low pressure lamp of2.6 W and the time of exposure to radiation can be assumed to bethe hydraulic residence time because the dispersion coefficient ob-tained is 0.05 and this is considered the optimal design for an ultra-violet disinfection reactor [32,33,34]. Disinfection experimentswere performed with pure cultures of two strains of E. coli in syn-thetic wastewater.

2.1.2. Pilot plantThe disinfection experiments with real wastewater were per-

formed in a Pilot Plant made by Trojan Technologies S.L., Canada.The UV channel received the water from the unfiltered secondaryeffluent of the Municipal Wastewater Treatment Plant of Jerez dela Frontera (Spain).

The UV Pilot Plant treatment was performed with two horizon-tal lamps directed into an open-channel with a flow rate in therange 4–15 m3/h. The unit consisted of a single stainless steel UVlamp bank (22 cm � 7.5 cm � 122 cm) and a double transitionbox (49 cm � 23 cm � 47 cm). The bank contained two low pres-sure lamps, each with a length of 914 mm and a production of78 W UV-C at 254 nm. Lamps were positioned parallel to the fluidflow direction and encased in a 60 mm diameter quartz sleeve.

2.2. Microorganisms, culture media and culture conditions

E. coli was selected due to its strictly fecal character and as it isone of the most common biological indicators of efficient waterdisinfection [39] and is governed by all water quality regulations.The following commercial strains of E. coli were employed in a lab-oratory plant: ATCC 15597, derived from E. coli K-12, and ATCC11229, a host bacteriophage, both of which are commonly usedas indicators in disinfection studies [25]. These strains were ob-tained from the American Collection of Microorganisms and CellCultures provided by LGC Standards. An initial volume of 1 mL ofbacteria from frozen stock was shaken for 10 s, resuspended in30 mL of Tryptic Soy Broth (TSB) and incubated for a period of24 h at 37 �C. The cells were centrifuged at 3000 rpm for 10 min[25] and the resulting pellet was washed in a 10% peptone solutionto ensure complete removal of the culture medium. Finally, eachpellet was resuspended in 50 mL of sterile milli-Q water and,depending on the requirements of the study, the bacteria solutionwas diluted or concentrated. This process guaranteed an initialbacterial concentration between 105 and 1011 colony forming unitsper 100 mL (CFU/100 mL).

The initial concentration of commercial strains of E. coli beforeultraviolet radiation exposure was estimated by the constructionof an experimental relationship between turbidity and microbio-logical concentration. This estimation subsequently was verifiedby membrane filtration. Turbidity measurements (nephelometricunits, NTU) were carried out with a Eutech TN100 portable turbi-dimeter (range 0.1–1000 NTU).

Each test started with 10 L of buffered and sterilized milli-Qwater, to which was added a determined volume of a concentratednutrient medium to achieve a synthetic wastewater of 35 mg/L O2

of COD (Table 1) and a specific concentration of enriched microbi-ological culture.

The membrane filtration method specified by Standard Meth-ods for the Examination of Water and Wastewater [1] was always

Table 1Synthetic wastewater composition used in the laboratory plant experiments.

Component Concentration (mg/L)

Peptone 160Urea 30Meat extract 110Sodium chloride (NaCl) 7Calcium chloride monohydrate (CaCl2�H2O) 4Magnesium sulfate heptahydrate (MgSO4�7H2O) 2Monoacid potassium phosphate K2HPO4 28

280 J.J. Vélez-Colmenares et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 278–285

used to determine the microbiological indicator E. coli in the disin-fection and reactivation experiments.

E. coli was identified with Colinstant Chromogenic Mediumfrom Sharlab. The growth conditions for these microorganismswere 37 �C for 24 h. The result was expressed as colony formingunits (CFU) per 100 mL of sample and each sample was measuredby four replicates, using as acceptance criteria a variation coeffi-cient of less than 0.3. In cases where this coefficient was higherthe data were rejected.

2.3. Photorepair experiments

Several samples of water inoculated with a microbiological con-centration of E. coli or real wastewater were exposed to ultravioletradiation with different UV doses at the scales studied and then leftexposed to sunlight. In the UV treatment several different flowrates were tested according to the previously measured UV trans-mittance of the water in order to obtain the target UV doses (from50 to 200 mJ/cm2). Before exposure to UV radiation, the sampleswere analyzed to ascertain the efficiency of the disinfection pro-cess and survival rate of the microorganisms.

The photoreactivation was assessed in an open reactor of 32 Lcapacity exposed to sunlight by taking samples from time zero to180 min, immediately after UV irradiation. The plastic reactor witha white bottom only was stirred when the samples were taken and

Table 2Kinetic parameter of Velez-Colmenares model for pure culture and environmental E. coli i

Microorganisms Sunlight radiation(mJ/m2)

Minimum temperature and maximumtemperature (�C)

UV(m

ATCC 11229 24.6 23.7–24.3 1524 24.0–25.0 1325.7 22.7–23.5 1125.4 25.4–26.0 1023.8 27.2–28.0 9424.5 26.5–27.1 7723.6 26.0–26.7 6225.3 25.8–26.5 50

ATCC 15597 23.5 24.3–25.1 1524.2 25.0–25.7 1225.4 23.5–24.6 1025.5 25.0–26.1 9524.2 23.2–24.0 8424.8 26.3–27.0 6725.3 25.0–26.1 6225.8 25.2–26.3 56

E. coli WWPP 24.5 24.8–25.7 5724.5 24.1–24.9 6825.1 24.5–25.7 7424.6 24.1–25.3 7725.5 25.3–26.4 8325.2 25.0–25.9 9525.3 25.1–26.2 1025.6 25.6–26.4 1125.9 25.1–26.3 1324.4 23.5–24.8 15

no contamination was observed during experimental time ofphotorepair.

The 3D-size of the reactor was 34 cm of depth, 38 cm of lengthand 22 cm of wide. The water depth inside reactor was 15 cm.

The concentration of bacteria (E. coli) and the temperature wasmeasured in situ at different time intervals (from 0 to 180 min) inorder to characterize the photoreactivation process.

In each trial, the temperature was monitored with a digital ther-mometer and maximum and minimum values and the daily aver-age were recorded (Table 2). The sunlight radiation was taken fromthe website Agroclimatic Information Network of Andalucía. Thisstation is approximately 15 km of distance from our laboratoryand has a pyranometer to measure broadband solar irradiance be-tween 350 and 1100 nm.

The experiments were carried out in the months of June, Julyand August and the exposure to sunlight was always at the samehour and without clouds (around midday). In Table 2 is observedthat the sunlight radiation was very similar in all experiments.

A microbiological control sample was taken for each photoreac-tivation test. This sample was subjected to dark conditions duringthe test period with sampling every 30 min to verify the optimalstate of the organism under investigation. A second control samplewas also taken before ultraviolet disinfection, and this was exposedto sunlight to check the natural effect of solar radiation on micro-organisms. This sample is denoted as the solar control sample.

In general, the reactivation is frequently expressed as a functionof the survival ratio with respect to the initial microorganism con-centration prior to UV disinfection [20] in order to understand andmodel this process. The survival rate of microorganisms was calcu-lated using following equation:

St ¼Nt

No� 100 ð1Þ

where St is the survival ratio at time t, Nt (CFU/100 mL) is the bac-terial concentration obtained at each time t after irradiation, No

n different UV doses.

doseJ/cm2)

Kinetic parameters

Sm

(experimental)Sm

(model)ks Ms Error r2

0 0.02 0.03 5.00 1.08E�02 9.18E�06 0.9838 0.03 0.04 4.84 1.21E�02 3.63E�05 0.9640 0.18 0.19 4.19 8.60E�03 5.84E�04 0.9650 0.25 0.30 3.60 6.64E�03 9.97E�04 0.956

0.30 0.32 3.25 7.48E�03 1.36E�03 0.9660.41 0.52 3.00 1.68E�02 1.30E�03 0.9920.62 0.86 2.59 2.09E�02 1.27E�03 0.9970.98 1.14 2.00 1.44E�02 1.09E�02 0.985

0 0.02 0.03 10.52 1.63E�02 2.70E�05 0.9225 0.04 0.06 8.17 3.46E�02 1.24E�04 0.9292 0.11 0.11 5.00 1.25E�02 6.72E�04 0.906

0.22 0.27 2.98 1.91E�02 1.91E�03 0.9560.23 0.30 2.40 1.10E�02 1.00E�03 0.9140.35 0.69 2.33 3.17E�02 3.94E�03 0.9720.68 0.79 1.30 1.19E�02 6.71E�03 0.9780.61 0.95 1.28 1.69E�02 3.73E�03 0.9901.16 1.70 0.03 5.89E�03 2.63E�03 0.9910.91 1.05 0.09 9.63E�03 1.79E�02 0.9760.59 1.01 0.09 1.38E�02 4.91E�03 0.9820.60 0.97 0.08 1.36E�02 4.82E�03 0.9810.64 0.81 0.19 8.69E�03 7.70E�03 0.9690.49 0.53 0.29 4.35E�03 5.30E�03 0.944

0 0.47 0.41 0.49 9.88E�03 8.49E�02 0.4531 0.14 0.16 0.91 5.28E�03 3.26E�04 0.9485 0.14 0.17 1.26 1.22E�02 1.18E�04 0.9910 0.08 0.11 1.79 1.11E�02 9.56E�05 0.975

J.J. Vélez-Colmenares et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 278–285 281

(CFU/100 mL) is the concentration of microorganisms before UVdisinfection.

The experimental data were standardized (using Eq. (2)) toevaluate the effect of repair and to compare different experiments:

Sn ¼St

Soð2Þ

where Sn is the normalized survival, St is the survival obtained ateach time t after UV disinfection and So is the initial survival at timezero after UV exposure.

2.4. Modelling of photoreactivation kinetic

In 1996 Kashimada et al. proposed a saturation-type first orderreaction model to predict the microbiological survival in the pho-toreactivation process. The model is expressed as:

dsdt¼ k � ðSm � SÞ ð3Þ

where S is the survival at time t, Sm is the maximum survival and kis the first order reaction rate constant (min�1).

Considering So as the initial survival after ultraviolet disinfec-tion and integrating Eq. (1), the expression of S as a function oftime is:

S ¼ ðSm � SoÞ � ð1� e�k�tÞ þ So ð4Þ

where the term (Sm � So) is defined as maximum net photoreactiva-tion, which represents the fraction of microorganisms that can bereactivated with respect to the initial concentration.

According to this model, the microorganisms exposed to lightafter UV-C disinfection have an increasing exponential behavior,i.e. a reactivation phase. Over a prolonged period, a maximum va-lue Sm is reached and the curve shows asymptotic behavior in thismaximum and describes a stationary phase.

The Kashimada model was applied to the photoreactivationprocess with sunlight, but this model does not adequately fit theexperimental data because there is a decay phase after the reacti-vation phase, a finding that is not explained by the Kashimadamodel (Fig. 1).

For this reason, a new model is proposed and this was applied toexplain the photoreactivation of E. coli with sunlight. In this modela new term is introduced to the Kashimada model in an effort toaccount for the observed first order decay phase, whereby the rateof decay of reactivation (also defined in terms of survival after dis-infection) is given by equation:

� dSdt¼ MS � St ð5Þ

Fig. 1. Photoreactivation curves of experimental data with Kashimada model andVelez-Colmenares model.

By integrating Eq. (5), the survival is expressed as:

St ¼ Si � e�MS �t ð6Þ

where t is the time in minutes, St is the survival after ultraviolet dis-infection at time t, Si is the survival of the decay phase at an initialtime t and Ms is the solar decay rate constant (min�1) that only de-pends on the solar radiation to which the sample is exposed.

During the solar reactivation phase, a decay survival begins attime t where the survival Si coincides with the existing survival Sof the Kashimada model, grouping the corresponding expressionsfor the growth and decay phases, the equation that defines the so-lar reactivation kinetic model (referred to as the Velez-Colmenaresmodel) is:

St ¼ ðSm � e�MS �tÞ � ðSm � SoÞ � e�ðksþMsÞ�t ð7Þ

where ks is the growth rate constant of solar reactivation (min�1)

3. Discussion and results

3.1. Photoreactivation with sunlight

In the photoreactivation studies the samples were previouslyirradiated with UV doses between 50 and 200 mJ/cm2 and the sam-ples were then exposed to sunlight during 180 min. The photoreac-tivation curves for E. coli (survival ratio versus time) obtained foreach experimental plant are shown in Fig. 2.

Similar behavior is observed in all experiments; the reactivationphase begins at So (initial survival) and reaches a maximumvalue at Sm (maximal survival). The stationary phase describedby Kashimada et al. [15] disappears in this case, but a decay phaseappears immediately after the reactivation phase. This type ofphase is described here for first time because the majority ofstudies carried out to date e.g. [15,24,20,38,18,25,28,4,10] involvethe use of lamps with UV-A only, whereas in the present paperthe reactivation was studied under real sunlight conditions.

In general, the microbiological behavior has two phases – reac-tivation and decay – because the sunlight contents UV-A and UV-Band these types of radiations act simultaneously. UV-A radiation isresponsible for the reactivation phase, whereas UV-A, UV-B andheat favor the inactivation of microorganisms in the decay phase.

The natural disinfectant effect of sunlight with the temporalevolution of a solar control sample (sample exposure to solar radi-ation without prior UV disinfection) can be seen in Fig. 3 and acomparison is made with two samples exposed to sunlight afterUV disinfection in a pilot plant (i.e. samples with photoreactiva-tion). The normalized survival is used to compare these samples.The solar control sample follows a decreasing exponential behaviorwith a fast decay and after a few minutes the inactivation is prac-tically total. In contrast, the sample with photoreactivation showsan initial increase in reactivation and then the decay phase followsa similar behavior to that in the control sample, but in this case themicrobiological inactivation is minor. The exponential functionsobtained with the experimental data from the decay phase havethe following form:

Sn ¼ A � e�kd �t ð8Þ

where A is a constant that coincides with the survival of the decayinitial, t is the time of exposure to sunlight (minutes) and kd is theinactivation constant in min�1.

The value of kd in the control sample (i.e. without reactivation)is greater (0.037 min�1) than the values for the disinfected samplesexposed to sunlight (0.009 and 0.014 min�1) since the reactivationprocess reduces the inactivating effect of UV solar radiation. The kvalues for the samples with UV doses of 89.3 and 75.22 mJ/cm2

coincide with the parameter Ms obtained by applying the newmodel to the experimental data (Table 2), thus demonstrating

0,000

0,200

0,400

0,600

0,800

1,000

0 30 60 90 120 150 180

Surv

ival

(%)

Time (minutes)

UV dose (mJ/cm2)

56

62

67

84

95

102

125

150

0,000

0,200

0,400

0,600

0,800

1,000

0 30 60 90 120 150 180

Surv

ival

(%)

Time (minutes)

UV dose(mJ/cm2)

50

62

77

94

110

120

138

150

0,000

0,200

0,400

0,600

0,800

1,000

1,200

0 30 60 90 120 150 180

Surv

ival

(%)

Time (minutes)

UV dose (mJ/cm2)

576874838999111135150

(a)

(b)

(c)Fig. 2. Photoreactivation curves of E. coli ATCC 11229 (a), ATCC 15597 (b) and E. coli in a pilot plant (c).

282 J.J. Vélez-Colmenares et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 278–285

the reliability of the model and the necessity to include this newterm to explain the decay phase.

The above discussion shows that Ms does not depend on the UV-C dose previously applied, but it does depend on the solar radiationto which the sample is exposed. The newly proposed model wasapplied to the experimental data for pure strains of E. coli andthe same behavior for Ms was found (data not shown).

3.1.1. Photoreactivation of pure strains of E. coli ATCC 11229 and ATCC15597

The model described in Eq. (7) was applied to experimental datausing non-linear regression. The values of the estimated kineticparameters Sm, ks and Ms are given in Table 2 with the r2 and theerror function. A good fit between the model and the experimentaldata can be observed.

y = 1.966e-0.009x

R² = 0.9708

y = 1.3244e-0.013x

R² = 0.9179

y = 0.762e-0.034x

R² = 0.93610,0000,2000,4000,6000,8001,0001,2001,4001,6001,8002,000

0 30 60 90 120 150 180

S n

Time (minutes)

Fig. 3. Survival curves of photoreactivation at two different UV doses (89.3 mJ/cm2

�, 75.22 mJ/cm2 s) compared with a solar control sample (4) in a pilot plant. Ithave been included the exponential fits of the decay phases.

J.J. Vélez-Colmenares et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 278–285 283

The behavior of Sm, ks and Ms versus UV dose is shown in Fig. 4.The parameter Sm is related to the potential reactivation and thisshows a negative exponential trend with the UV-C dose, since agreater UV dose diminishes the reactivation capacity of the bacte-ria. The net capacity for reactivation can be estimated from the dif-ference Sm � So and, as one would expect, also depends on the UVdose, as shown in Fig. 4.

The fundamental photoreactivation parameter in Eq. (7) is themaximum net photoreactivation Sm � So, so the relationship foundwith the dose indicates that high UV-C doses lead to marked alter-ation of the bacteria, making their reactivation minor, while lowdoses permit the photoreactivation process to take place. Althoughthe strain ATCC 11229 has a higher photoreactivation percentage(Sm) than ATCC 15597, comparison of the maximum net photoreac-tivation (Sm � So) shows that both strains have practically the samesurvival after exposure to sunlight.

On the other hand, ks shows an inverse trend with UV-C. Thisfinding means that the reactivation phenomenon is more rapid

0,000

0,200

0,400

0,600

0,800

1,000

1,200

1,400

1,600

1,800

0 30 60 90 120 150 180

S m(%

)

UV dose (mJ/cm2)

0,0000,2000,4000,6000,8001,0001,2001,4001,6001,800

0 30 60 90 120 150 180

S m-S

o(%

)

UV dose (mJ/cm2)

Ks

(min

-1)

-1

(a)

(b)Fig. 4. Evolution of kinetic parameters Sm (a), Sm � So (b), ks (c) and Ms (d) with UV

with high UV doses, although a lower survival percentage isreached. At high UV doses, the reactivation was more rapid, likelybecause the number of survival microorganisms is minor than atlow UV doses, and then when the sample is exposed to sunlightthe microorganism have more availability of nutrients to be ableto reactivate more rapid. In this case, the values of k for bothE. coli strains are different, with the value for ATCC 11229 beinggreater than that for ATCC 15597. Finally, Ms does not show anyrelation to applied UV dose, as observed in Fig. 4, and it can there-fore be considered to have a constant value for the experimentalconditions defined in the present study, but this parameter maydepend on solar light intensity.

The equations for ks, Sm, Sm � So with respect to UV dose are gi-ven in Table 3 along with the constant value of Ms and the corre-sponding standard deviations.

The reactivation curve for pure E. coli cells can also be predictedfrom the equations proposed in this work. The model obtainedwith these equations is adequately adjusted to the experimentaldata – as shown in Fig. 5, which represents the observed data ver-sus the predicted data.

3.1.2. Photoreactivation of E. coli in a pilot plantMicrobiological analysis in the laboratory under controlled con-

ditions is essential to understand the processes of disinfection andreactivation. It can be seen from Fig. 1 that the reactivation phasefor the two pure strains of E. coli appears in the first 15 min ofexposure to sunlight and subsequently the microbial concentrationdecreases at 45 min, with values below the original survival. In theexperimental conditions exposed in the article after this time thewater could be reused according normative.

It should be noted, however, that these results cannot beextrapolated to real wastewater and these studies were thereforeextended to include a pilot plant using real wastewater. Althoughthe behavior of E. coli is similar in the laboratory and pilot plant,

0,000

2,000

4,000

6,000

8,000

10,000

12,000

0 30 60 90 120 150 180

UV dose (mJ/cm2)

0,000

0,005

0,010

0,015

0,020

0,025

0,030

0,035

0,040

0 30 60 90 120 150 180

Ms

(min

)

UV dose (mJ/cm2)

(c)

(d)dose in E. coli ATCC 11229, ATCC 15597 and E. coli in a pilot plant (WWPP).

Table 3Equations of kinetic parameters Sm, Sm � So, ks and Ms of Velez�Colmenares model where x is the UV dose.

Microorganism Sm � So Sm ks Ms Standard deviation

ATCC 11229 4.9691.e�0.042x 6.8284.e�0.037x 0.1006x � 5.0949 0.0192 0.0091r2 = 0.9341 r2 = 0.9764 r2 = 0.9411

ATCC 15597 3.6224.e�0.037x 8.9941.e�0.037x 0.03x + 0.6176 0.0119 0.0052r2 = 0.9532 r2 = 0.9841 r2 = 0.9835

E. coli WWPP 4.5378.e�0.031 9.0205.e�0.031x 0.0196x � 1.3433 0.0094 0.0033r2 = 0.7464 r2 = 0.9343 r2 = 0.9369

0,000

0,200

0,400

0,600

0,800

1,000

1,200

0,000 0,200 0,400 0,600 0,800 1,000 1,200

Pred

icte

d va

lue

Observed value

Fig. 5. Goodness-of-fit for the Velez-Colmenares model for E. coli.

284 J.J. Vélez-Colmenares et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 278–285

microbial survival reaches higher values in the pilot plant than inthe laboratory (Fig. 2). In both pure cultures, the maximum sur-vival in the reactivation phase appears at 15 min, whereas in realwastewater the time varies between 15 and 45 min and its effectis longer in terms of time. The behavior of E. coli in the pilot plantwith sunlight is adjusted to the characteristics of the new modeland Eq. (7) was therefore applied to the experimental data (Table 2)and the kinetic parameters obtained were analyzed. It can be seenfrom Fig. 4 that Sm and Sm � So have a decreasing exponential rela-tionship with the UV dose and the values are higher than those ob-tained for pure strains. The difference in the net rate ofphotoreactivation could be due to the nutrient content in thewastewater. The photoreactivation occurs with or without nutri-ents by enzymatic processes, but the availability of nutrients en-sures that the growth bacteria could be observed alongphotoreactivation process and the maximum survival value willtherefore be higher. This hypothesis could be interesting for fur-ther studies.

According to Fig. 4, the ks values are always lower compared tothe previous case but the dependence on the UV-C dose is main-tained. Ms is constant, it is similar to the values obtained for thepure strains and is independent of the UV dose applied. The kineticmodel can be predicted using the equations found and with aknowledge of the UV dose applied (Table 3).

According to the results exposed and with similar experimentalconditions to those adopted in this study a minimum time of 2 h isproposed before the reuse of wastewater disinfected with UV-Clight and the water should be stored in sunlight prior to use in or-der to avoid the survival maximum of the photoreactivation pro-cess. For this reason the residence time of the water in reservoirsmust be greater than this time for similar conditions (2 h).

4. Conclusions

Two pure strains of E. coli (ATCC 11229, ATCC 15597) and realwastewater showed photoreactivation when UV irradiated sam-ples were exposed to sunlight. The bacteria survival curves show

an initial reactivation phase, which reached a maximum and thenimmediately underwent a decay phase. This decay phase is re-ported for first time in this work. This decreasing survival is dueto the sunlight acting simultaneously as a cause of reactivation(UV-A) and inactivation (UV-B and UV-C). The model proposedby Kashimada only is able to explain the photoreactivation phasebecause the experimental conditions involved the use of fluores-cent light at 360 nm. However, in sunlight this model does not fitthe experimental data. For this reason, in the work described herethe kinetic model of Kashimada was modified by including a firstorder decay term, which is able to fit the decreasing survival phasedetected when UV irradiated bacteria is exposed to sunlight. Thekinetic parameters of the Kashimada model, Sm (maximum sur-vival) and ks (rate constant), have a dependence on the UV-C dose,while the new decay constant Ms does not show any dependenceon the previous UV dose and depends on the UV solar radiation.In our case, the solar radiation intensity was the same for all theexperiments and Ms therefore has a constant value. A relation be-tween Ms and the inactivation constant of the exponential functionfor the decay phase was established and the Ms values are indepen-dent of sunlight exposure time. Exponential equations were devel-oped that enable the parameters of the model to be estimated as afunction of UV-C dose and it is then possible to predict the reacti-vation curve versus time. Some differences were found betweenpure E. coli strains and real wastewater. In the latter case, Sm issmaller than that found for pure E. coli but ks is greater and, as aconsequence, the repair rate is faster. The net rate of photoreacti-vation under real conditions is higher than that obtained for purestrains; this difference could be due to the nutrients contained inthe wastewater. The nutrients favor the photoreactivation processand therefore the maximum survival value would be higher.

The knowledge of the photoreactivation phenomenon enablesmake recommendations concerning the time before reuse of awastewater irradiated with UV. In the experimental conditions ofour work such water must be left for at least 2 h with exposureto solar radiation to ensure that the decay phase is reached andthe maximum reactivation is avoided.

Acknowledgements

The authors wish to express their gratitude to Mr. José AntonioAndrades Balao, at the company Aqualia and AJEMSA, for supplyingwastewater samples from the Wastewater Treatment Station ofJerez de la Frontera.

This work was partially funded by the Spanish Ministry of Edu-cation and Science through I+D Project CTM2009-09527/TECNOand the CONSOLIDER-INGENIO 2010 Project CSD2007-00055.

References

[1] American Public Health Association (APHA). American Water WorksAssociation (AWWA) and Water Pollution Control Federation (WPCF), 2005.Standard Methods for the Examination of Water and Wastewater. 21st ed.Washington, DC.

[2] C.V. Beggs, A quantitative method for evaluating the photoreactivation ofultraviolet damaged microorganisms, Photochemical and PhotobiologicalSciences 1 (2002) 431–437.

J.J. Vélez-Colmenares et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 278–285 285

[3] O.V. Belov, E.A. Krasavin, A.Y. Parkhomenko, Model of SOS-inducedmutagenesis in bacteria Escherichia coli under ultraviolet irradiation, Journalof Theoretical Biology 261 (2009) 388–395.

[4] M. Bucheli-Witschel, C. Bassin, T. Egli, UV-C inactivation in Escherichia coli isaffected by growth conditions preceding irradiation, in particular by thespecific growth rate, Journal of Applied Microbiology 109 (5) (2010) 1733–1744.

[5] S. Chrtek, W. Popp, UV disinfection of secondary effluents from sewagetreatment plants, Water Science and Technology 24 (1991) 343–346.

[6] E.R. Friedberg, G.C. Walker, W. Siede, DNA Repair and Mutagenesis, ASM Press,Washington, DC, 1995. 92–107.

[7] S. Gelover, L.A. Gómez, K. Reyes, M.T. Leal, A practical demonstration of waterdisinfection using TiO2 films and sunlight, Water Research 40 (2006) 3274–3280.

[8] H. Görner, Photochemistry of DNA and related biomolecules: quantum yieldsand consequences of photoionization, Journal of Photochemistry andPhotobiology B: Biology 26 (1994) 117–139.

[9] M. Guo, H. Hu, J.R. Bolton, M.G. El-Din, Comparison of low and mediumpressure ultraviolet lamps: photoreactivation of Escherichia coli and totalcoliforms in secondary effluents of municipal wastewater treatment plants,Water Research 43 (3) (2009) 815–821.

[10] C. Hallmich, R. Gehr, Effect of pre- and post-UV disinfection conditions onphotoreactivation of fecal coliforms in wastewater effluents, Water Research44 (9) (2010) 2885–2893.

[11] W. Harm, Biological Effect of Ultraviolet Radiation, Cambridge UniversityPress, New York Chapter, 1980. 8.

[12] D.G. Harris, V.D. Dean Adams, D.L. Sorensen, M.S. Curtis, Ultravioletinactivation of selected bacteria and viruses with photo-reativation ofbacteria, Water Research 21 (1987) 687–692.

[13] A. Hassen, M. Mahrouk, H. Ouzari, M. Cherif, A. Boudabous, J.J. Damelincourt,UV disinfection of treated wastewater in a large-scale pilot plant andinactivation of selected bacteria in a laboratory UV device, BioresourceTechnology. 74 (2) (2000) 141–150.

[14] W.A.M. Hijnen, E.F. Beerendonk, G.J. Medema, Inactivation credit of UVradiation for viruses, bacteria and protozoan (oo)cysts in water: a review,Water Research 40 (1) (2006) 3–22.

[15] K. Kashimada, N. Kamiko, K. Yamamoto, S. Ohgaki, Assessment ofphotoreactivation following ultraviolet light disinfection, Water Science andTechnology 33 (10) (1996) 261–269.

[16] H. Liltved, B. Landfald, Influence of liquid holding recovery andphotoreactivation on survival of ultraviolet-irradiated fish pathogenicbacteria, Water research 30 (1996) 1109–1114.

[17] K.G. Lindenauer, J.L. Darby, Ultraviolet disinfection of wastewater: effect ofdose on subsequent photoreactivation, Water Research 28 (1994) 805–817.

[18] A. Locas, J. Demers, P. Payment, Evaluation of photoreactivation of Escherichiacoli and enterococci after ultraviolet disinfection of municipal wastewater,Canadian Journal of Microbiology 54 (2008) 971–975.

[19] K.L. Mechsner, T. Fleischmann, C.A. Mason, G. Hamer, UV disinfection: shortterm inactivation and revival, Water Science and Technology 24 (1991) 339–342.

[20] E. Nebot Sanz, I. Salcedo Dávila, J.A. Andrade Balao, J.M. Quiroga Alonso,Modelling of reactivation after UV disinfection: effect of UV-C dose onsubsequent photoreactivation and dark repair, Water Research 41 (14) (2007)3141–3151.

[21] A. Novick, L. Szilard, Experiments on light reactivation of ultraviolet-inactivated bacteria, Proceedings of the National Academy of Sciences USA35 (1949) 591–600.

[22] K. Oguma, H. Katayama, H. Mitani, S. Morita, T. Hirata, S. Ohgaki,Determination of pyrimidine dimers in Escherichia coli and Cryptosporidiumparvum during UV light inactivation, photoreactivation and dark repair,Applied and Environmental microbiology 67 (10) (2001) 4630–4637.

[23] K. Oguma, H. Katayama, S. Ohgaki, Photoreactivation of Legionella pneumophilaafter inactivation by low- or medium-pressure ultraviolet lamp, WaterResearch 38 (2004) 2757–2763.

[24] K. Oguma, H. Katayama, S. Ohgaki, Spectral impact of inactivating light onphotoreactivation of Escherichia coli, Journal of Environmental Engineeringand Science 4 (S1) (2005) S1–S6.

[25] P.H. Quek, J. Hu, Indicators for photoreactivation and dark repair studiesfollowing ultraviolet disinfection, Journal of Industrial Microbiology andBiotechnology 35 (6) (2008) 533–541.

[26] A.-G. Rincón, C. Pulgarín, Field solar E. coli inactivation in the absence andpresence of TiO2: is UV solar dose an appropriate parameter forstandardization of water solar disinfection?, Solar Energy 77 (2004) 635–648

[27] I. Salcedo, J.A. Andrades, J.M. Quiroga, E. Nebot, Photoreactivation and darkrepair in UV treated microoganisms: effect of temperature, Applied andEnvironmental Microbiology 73 (5) (2007) 1594–1600.

[28] C. Shang, L.M. Cheung, C.-M. Ho, M. Zeng, Repression of photoreactivation anddark repair of coliform bacteria by TiO2-modified UV-C disinfection, AppliedCatalysis B: Environmental 89 (2009) 536–542.

[29] C. Sichel, J. Blanco, S. Malato, P. Fernández-Ibáñez, Effects of experimentalconditions on E. coli survival during solar photocatalytic water disinfection,Journal of Photochemistry and Photobiology A: Chemistry 189 (2007) 239–246.

[30] R. Sommer, M. Lhotsky, T. Heides, A. Cabaj, UV inactivation liquid holdingrecovery, and photoreactivation of E. coli O157 and other pathogenic E. colistrains in water, Journal of Food Protection 63 (2000) 1015–1020.

[31] K. Tosa, T. Hirata, Photoreactivation of enterohemorrhagic Escherichia colifollowing UV disinfection, Water Research 33 (2) (1999) 361–366.

[32] United States Environmental Protection Agency (USEPA), 1986. Designmanual: Municipal Wastewater Disinfection. EPA/625/1-86/021. Office ofResearch and Development, Water Engineering Research Laboratory. Centerfor Environmental Research Information, Cincinnati, OH.

[33] United States Environmental Protection Agency (USEPA), 1996. UltravioletLight Disinfection Technology in Drinking Water Application: An Overview.EPA 811-R-96-002, Office of Ground Water and Drinking Water, Washington,DC.

[34] United States Environmental Protection Agency. USEPA, 2006. UltravioletDisinfection Guidance Manual for the Final Long Term 2 Enhanced SurfaceWater Treatment Rule. EPA 815-R-06-007. Office of water (4601). Washington, DC.

[35] J.J. Vélez-Colmenares, A. Acevedo, E. Nebot, Effect of recirculation and initialconcentration of microorganisms on the disinfection kinetics of Escherichiacoli, Desalination 280 (2011) 20–26.

[36] G.C. Walker, Mutagenesis and inducible responses to deoxyribonucleic aciddamage in Escherichia coli, Microbiology Research 48 (1984) 60–93.

[37] G.E. Whitby, G. Paimateer, W.G. Cook, J. Maarschalkerweerd, D. Huber, K.Flood, Ultraviolet disinfection of secondary effluent, Journal of water pollutioncontrol federation 56 (1984) 844–850.

[38] C.G. Yoon, K.-W. Jung, J.-H. Jang, Microorganism repair after UV-disinfection ofsecondary-level effluent for agricultural irrigation, Paddy Water Environmental5 (2007) 57–62.

[39] J.L. Zimmer, R.M. Slawson, Potential repair of Escherichia coli DNA followingexposure to UV radiation from both medium- and low-pressure UV sourcesused in drinking water treatment, Applied and Environmental Microbiology 68(7) (2002) 3293–3299.