Fecal Coliform Removal in Wastewater Treatment Plants Studied by Plate

Water Research 36 (2002) 2607–2617

Fecal coliform removal in wastewater treatment plants studied byplate counts and enzymaticmethods Isabelle George*, Philippe Crop, Pierre Servais

Ecologie des Systemes" Aquatiques, Universite! Libre de Bruxelles, Campus de la Plaine CP221, Boulevard du Triomphe, 1050 Bruxelles, Belgium

Received 17 November 2000; received in revised form 12 October 2001; accepted 25 October 2001

Abstract

Twelve wastewater treatment plants (WWTPs) were sampled in France and Belgium in 1999 and 2000 in order to estimate the fecal coliform (FC) removal efficiencyof various typesof treatment. Onlyone of these WWTPs was equipped with a specific step to eliminate microorganisms (UV disinfection preceded bysand filtration). FC abundance was measured in raw and treated sewage byplate counts on selective medium and rapid b-d-glucuronidase (GLUase)- based assays. Removal of culturable FC was the most efficient in treatments with high retention time (activated sludge process with nitrification and denitrification, lagooning), in biofiltration and in the treatment with a tertiarydisinfection step. GLUase activitymeasurements showed the same removal pattern as plate counts except for UV disinfection, where no reduction of GLUase activitywas measured. Specific loads of culturable FC and GLUase activity,i.e . daily amounts of culturable FC or GLUase activityin sewage per inhabitant-equivalent, were calculated in raw and treated wastewater for the different WWTPs. r 2002 Elsevier Science Ltd. All rights reserved.

Keywords: Fecal coliforms; Wastewater treatment; Removal efﬁciency; b-d-glucuronidase activity

1. Introduction water. However, the direct and indirect exposure of populations to sewage is of primaryconcern. The Wastewater effluents are a major source of fecal increasing demographyand the growing water demand contamination of aquatic ecosystems and cause severe has lead to a global deterioration of surface waters disturbance in their ecological functioning. Wastewater qualityand, in areas facing a water shortage, more and treatment plants (WWTPs) have been primarilyde- more reclaimed water will be used in the future for signed to reduce pollution of natural waters by irrigation of parks and crops. Therefore increasing suspended solids and organic matter, and in some efforts are devoted at present to assessing the treatment WWTPs, treatment for nitrogen and phosphorus efficiencyof wastewater treatment facilities for removal removal have been introduced. Despite the fact that of fecal micro-organisms. raw wastewater also carries large quantities and a wide Bacterial indicators of fecal contamination and enteric varietyof fecal micro-organisms (including pathogens viruses are present in high concentrations in raw for humans), the reduction of bacteriological pollution wastewater. For example, typical abundance of total in wastewater has not been a priorityso far in Europe and fecal coliforms (FC) in raw sewage are, respectively, and, at present, there are no European directives 107–109 and 106–108 100 ml1 [1–4]. Classical treat- regarding the bacteriological qualityof treated waste- ments, which do not include anyspecific disinfection step, reduce fecal micro-organisms densities by1–3 *Corresponding author. Tel.: +322-650-5715; fax: +322- orders of magnitude [3,2,5,6], but because of their high 650-5993. abundance in raw sewage, theyare still discharged in E-mail address: [email protected] (I. George). large numbers with treated effluents in the environment.

Most studies on the microbiological qualityof waste- 2.2. Bacteriological analysis of the effluents water have been aimed to comparing the removal efficiencyof various micro-organisms in one or a few 2.2.1. Enumeration of culturable FC by plate count WWTPs and were based onlyon culturable counts on FC were enumerated after membrane filtration selective media [1,2,5–7,4,8–10]. (0.45 mm-pore-size, 47-mm-diameter sterile cellulose In the present work, we chose to compare the nitrate filters, Sartorius) or spread plating on lactose efficiencyof 12 WWTPs in France and Belgium to agar with Tergitol (0.095% wt vol1 final concentration) remove FC from wastewater. These plants were chosen and triphenyl 2,3,5-tetrazolium chloride (TTC) for their difference in size (from 90 to 7 106 inhabitant- (0.024% wt vol1 final concentration) according to the equivalents), and various biological treatments (acti- French standards AFNOR [17]. Prior to filtration or vated sludge process, activated sludge with nitrification spread plating, wastewater samples were sonicated for and/or denitrification, biofiltration, lagooning). The 45 s in an ultrasonication cleaner bath (Branson 1210E- Sainte Marie La Mer WWTP, located on the Mediter- MT, 60 W, 47 kHz) to detach bacteria from particles ranean coast, was the onlystudied treatment plant with and/or break up cellular aggregates, as described by a specific step to remove bacteria (sand filtration George et al. [15]. Orange colonies producing a yellow followed byUV raysdisinfection). Raw and treated halo under the membrane after incubation at 441C for wastewater were collected in each plant and analysed for 24 h were considered FC colonies. FC counts were their FC content byclassical enumeration on agar expressed as colony-forming units (CFU) per 100 ml of medium and bya rapid enzymaticmethod recently sample. In wastewater samples, the coefficient of developed [11–15]. Our objectives were to compare the variation in plate count replicates equaled 20–50% [15]. various treatment types with both methods and to determine the most effective steps in the treatment lines 2.2.2. b-d-glucuronidase activity measurement to remove FC from sewage. Another goal of this study This method to detect FC in aquatic environments is was to establish a specific load of FC per inhabitant and based on the production of the b-d-glucuronidase per day(expressed in culturable FC or GLUase activity enzyme by the main FC, E. coli, and has been shown inh1 day1) for raw and treated wastewater, in order to to be stronglycorrelated to FC plate counts in natural assess the impact of raw or variouslytreated sewage and waste waters [12,14,15]. The protocol was fully discharged in aquatic environments. described byGeorge et al. [14]. Briefly,a volume ranging from 1 to 20 ml of wastewater was filtered through a 0.2 mm-pore sized polycarbonate filter. The b-d-glucuronidase activityretained on the filter was measured by 2. Material and methods the production over 15–20 min of a fluorescent product after the addition of the fluorogenic substrate 2.1. WWTPs characteristics and sampling 4-methylumbelliferyl-b-d-glucuronide MuGlu (final concentration, 165 mg l1) (Biosynth, Switzerland). The The type and location of the sampled WWTPs are production rate over time of fluorescent 4-methylum- summarised in Table 1. Theywere grouped, indepen- belliferone (MUF) bybacterial enzymaticactivitywas dentlyof their size, in categories of typeof treatment for measured byspectrofluorometry. b-d-glucuronidase the convenience of the subsequent discussion. In the activitywas expressed as picomoles of MUF liberated different WWTPs, raw water, primarysettled water and per minute for 100 ml of wastewater sample. For water treated water were collected. As our goal was to assess and wastewater samples, the coefficient of variation of the removal efficiencyof different treatments, onlyfully activitymeasurement replicates equaled 10–15% [15]. treated water was sampled, even when there were by- Both plate counts and GLUase activitymeasurements passes of the treatment line (at Acheres" in May1999 and were multiplied when necessarybya correction factor to 2000 and at Couillyin June 2000). take into account the increase of FC abundance during In order to integrate the dailyfluctuations of FC the mean dailysampling. The calculation of this factor is abundance in sewage due to human activities [6,16], explained in Section 3. mean dailysamples were collected at each plant (except at the Sainte Marie la Mer WWTP). Subsamples of 2.3. Physico-chemical analysis of the effluents identical volume were collected over a 24 h period with refrigerated (ca. 61C) automatic samplers and mixed 2.3.1. Suspended matter (SM) together. Samples from WWTPs with several treatment SM was estimated as the weight of material retained lines working in parallel were reconstituted from on a pre-weighted Whatman GF/F membrane after fractions pumped in the various lines proportional to filtrating a known volume of sample and drying the the lines discharge. Samples were returned refrigerated membrane at 1051C until a constant weight was reached. to the laboratoryand analysedwithin 6 h of collection. Data were expressed in mg l1. I. George et al. / Water Research 36 (2002) 2607–2617 2609

Table 1 Location, date of sampling and characteristics of the sewage treatment plants

WWTP Localisation Date of sampling Inhabitant- Nominal Type of Categoryof (country, province/ equivalents discharge treatmenta treatment department) capacity (m3 day1)

Wavre Belgium, Brabant February1999 (3 times), 165,000 40,000 PT, D, AS A Wallon April 1999 Acheres" France, Ile-de-France May1999, 7,000,000 2,100,000 PT, D, AS A December 1999, April 2000, May2000, June 2000 Guerard! France, Ile-de-France June 2000 2000 216 ASnit B CouillyFrance, Ile-de-France June 2000 15,000 3000 PT, D, ASnit B Rixensart Belgium, Brabant March 1999 125,000 35,000 PT, D, ASnit B Wallon Troyes France, Champagne- May1999 300,000 46,000 PT, D, ASnit B Ardenne Acheres," France, Ile-de-France May1999, 18,000 PT, D, AS, Bnit C Biofors pilote December 1999, April 2000, May2000, June 2000 Waterloo Belgium, Brabant May1999 20,000 3600 PT, D, ASnit+denit D Wallon Rouen France, Haute- April 1999 450,000 70,000 PT, D, ASnit+denit D Normandie Valenton France, Ile-de-France April 1999 1,200,000 300,000 PT, D, ASnit+denit D Colombes France, Ile-de-France April 2000, 800,000 240,000 PT, D E May2000, (Densadegs), June 2000 Bc, Bnit, Bdenit AulnoyFrance, Ile-de-France June 2000 90 14 L F Sainte Marie France, Languedoc- July2000 20,000 3000 PT, D, AS, SF b,UVb G La Mer Rousillon

a PT=pretreatment (screening, grease collection), D=primarydecantation (primarysettling), AS=activated sludge process followed bydecantation, ASnit=activated sludge process with nitrification followed bydecantation, ASnit+denit=activated sludge process with nitrification and denitrification, followed bydecantation, Bc=up-flow biofilter for carbon depollution (Biofor s), Bnit=up-flow biofilter for nitrification (Biofors/Biosytrs), Bdenit=up-flow biofilter for denitrification (Biofors), L=lagoon (with 2 ponds), SF=filtration on sand bed, UV=disinfection byUV rays. b These treatment steps are functioning in the summer period only. Plants are grouped byincreasing retention time or complexityof treatment (categories from A to G).

2.3.2. Biochemical oxygen demand (BOD) 2.5. Specific load of FC BOD was defined as the amount of oxygen consumed in the sample over 5 days at 201C and in the dark, In order to compare the different treatment plants, according to AFNOR [18]. Data were expressed in mg FC and GLUase activitymeasured in the raw and of oxygen l1. treated wastewater were expressed in terms of specific load per inhabitant and per day, i.e. the daily numbers 2.4. Temporal follow-up of refrigerated and non- of culturable FC or GLUase activitydischarged through refrigerated wastewater samples wastewater byone inhabitant-equivalent. Specific loads were calculated according to Servais et al. [19] by Three raw and one treated sewage samples (from the considering a dailycharge in BOD of 54 g per inhabitant Wavre WWTP, Belgium) were incubated in sterilised 2 l- and per dayas proposed bythe WHO [20]. For each Duran bottles at 201C and 61C with slow stirring in the WWTP, the dailywastewater volume per inhabitant (m 3 dark over 24 h, and FC counts and GLUase activity inh1 day1) was calculated bydividing the value of 54 measurements were performed on subsamples in tripli- (g inh1 day1) bythe average BOD concentration in cates every3 or 6 h. raw wastewater (mg l1). The specific load of FC or 2610 I. George et al. / Water Research 36 (2002) 2607–2617

GLUase activityin raw or treated wastewater was FC, GLUase activity, removal efficiency and specific calculated bymultiplyingthe FC abundance or GLUase loads on the basis of the different temporal values for activity(CFU or GLUase act. per 100 ml of raw or each variable. These mean values were subsequently treated sewage) by10,000 and bythe dailywastewater used in calculations of removal efficiencyand specific volume per inhabitant (m3 inh1 day1). For the Sainte loads in raw and treated wastewater for each categoryof Marie la Mer WWTP, specific loads were calculated on treatment (Fig. 5 and Table 3), in order to avoid that, in the basis of a dailywastewater volume per inhabitant of one given category, the most sampled WWTPs strongly 180 l inh1 day1 as no data of BOD were available. influenced both calculations in comparison with WWTPs sampled once. The range from minimum to 2.6. Calculations maximum value was given for each geometric mean as information on the dispersion of values. For each WWTP and each sampling campaign, removal efficiencies byprimarysettling and whole treatment as well as specific loads in raw and treated wastewater were calculated on the basis of FC plate 3. Results counts and GLUase activitymeasurements. Removal efficiencies were calculated as follows: 3.1. Evolution of refrigerated and non-refrigerated wastewater samples % of removal byprimarysettling or complete treatment

¼ðXraw2Xdec=treatedÞ=Xraw100 Wastewater samples analysed at each WWTP were mean dailyones, which implied that the subsamples Log removal byprimarysettling or complete treatment collected over a 24 h period waited various times in the refrigerated sampler before the mean dailysample was ¼ log Xraw2log Xdec=treated reconstituted. Therefore several experiments were con- where Xraw is the culturable FC or GLUase activityin ducted in the laboratoryto evaluate the ﬂuctuations of raw wastewater, Xdec the culturable FC or GLUase the FC abundance in raw and treated sewage over 24 h. activityin primarysettled (decanted) wastewater, Xtreated A same sewage sample was incubated in refrigerated the culturable FC or GLUase activityin treated waste- (61C) and non-refrigerated (201C) incubators, and water (i.e. at the outlet of the WWTP). regularlyanalysedbyplate counts and GLUase activity In WWTPs that were sampled several times, a measurements. Fig. 1 shows a typical result of one of geometric mean value was calculated for culturable these experiments.

4 4 o 3 3

2 2

1 1 value ti / value t value ti / value to 0 0 0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 24 (a) Time (h) (b) Time (h)

4 4 o o 3 3

2 2

1 1 value ti / value t value ti / value t 0 0 0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 24 (c) Time (h) (d) Time (h) Fig. 1. Example of ﬂuctuations of FC abundance in raw and treated wastewater incubated in refrigerated (61C) and non-refrigerated (201C) conditions (samples from the Wavre WWTP, June 23, 2000). FC abundance (or b-d-glucuronidase activity) measured at time ti was normalised to FC abundance (or GLUase activity) at the initial time to. Error bars refer to triplicates. (a) raw wastewater at 201C, (b) raw wastewater at 61C, (c) treated wastewater at 201C, (d) treated wastewater at 61C. I. George et al. / Water Research 36 (2002) 2607–2617 2611

In all experiments on non-refrigerated raw sewage, the of 0.62 log10 for GLUase activityand 0.74 log 10 for FC combination of growth and mortalityprocesses resulted (Table 2). These fluctuations were of the same order of in a net increase of the FC abundance. FC densityand magnitude as differences measured in raw wastewater GLUase activitywere multiplied over 24 h up to seven between WWTPs (Fig. 2a and b). Geometric mean times and twice respectively. A slight increase of FC specific loads of FC in raw wastewater were 1.18 108 abundance was observed in most experiments on GLUase activityunits inh 1 day1 and 1.39 1011 refrigerated raw sewage, that never exceeded a doubling culturable FC inh1 day1 (Table 3). Specific loads of of the abundance after 24 h of incubation (0.6–1.9 times FC in raw wastewater entering the WWTPs showed a the initial FC abundance, and 1–1.3 times the initial general tendencyto increase with the size of the sewered GLUase activity). In treated wastewater, the FC population (Fig. 3). abundance showed no fluctuation over time, even in non-refrigerated conditions (Fig. 1c and d). These results underlined the combined effects of temperature 3.3. Effect of primary treatment and wastewater qualityon the increase of FC in sewage, and the importance of refrigerating raw or poorly Fig. 4 presents the log10 removal byprimarysettling treated wastewater samples before their analysis. of culturable FC and GLUase activityplotted against In the light of these results, culturable FC abundance the SM removal. SM was appreciablyreduced by and enzymatic activity measured in each reconstituted primarytreatment (geometric mean removal of 0.38 mean dailysample were corrected to reflect what this log10 (58%)). FC were generallyless removed than SM sample would have been if none of its subsamples had (Fig. 4). GLUase activitywas reduced on average by changed over time. Calculations were based on the 0.20 log10 (37%) and culturable FC by0.15 log 10 (29%) hypothesis that the increase of FC abundance in (Fig. 6). No significant linear relationship (in log–log refrigerated conditions was linear over 24 h, which in units) was observed between the removal of SM and the the case of subsamples with identical volume amounted removal of culturable counts or GLUase activity to considering that the whole sample evolved globally (Fig. 4). for 12 h. Results on refrigerated raw sewage presented in Fig. 1b were plotted with results of two similar experiments, and a regression line with the intercept 3.4. Additional effect of secondary and tertiary set at 1 was calculated separatelyfor plate counts treatments (slope ¼ 0:047; r2 ¼ 0:35) and enzymatic activity (slope ¼ 0:015; r2 ¼ 0:65). The regression lines allowed Unlike primarysettling alone, most complete treat- to calculate what would have been the mean culturable ment systems eliminated FC more efficiently than SM or FC abundance and GLUase activityin each whole BOD (Fig. 5a and b). In particular, FC were much more refrigerated sample if it had not globallyevolved over removed than SM or BOD bythe lagooning (treatment 12 h. Mean dailyvalues for both variables were therefore F) (Fig. 5a and b). Secondaryand tertiarytreatments multiplied bya correction factor of 0.73 and 0.87, were responsible for the major reduction of the respectively, resulting in a ‘real value’ of FC density and microbiological pollution in wastewater. To compare activitylower than the measured one. These correcting the removal efficiencies of the different treatments, factors were applied to samples with a BOD value higher WWTPs were grouped bycategories of treatment in than 50 mg l1 (the limit of 50 mg l1 was fixed Fig. 6. The mean removal rate of each type of treatment considering the results of the various experiments). is presented in Fig. 6, and the resulting FC abundance and GLUase activityin the treated effluents are detailed 3.2. FC abundance and specific load in raw wastewater in Fig. 2a and b. Removal efficiencyof culturable FC depended Culturable FC abundance and GLUase activityin on the type of secondary and tertiary treatment raw wastewater of the different WWTPs are presented in and the order of increasing efficiencywas: activated Fig. 2a and b. GLUase activities varied from 6992 to sludge process (A) oactivated sludge process with 72007 pmol MUF min1 100 ml1 of sample (i.e. a range nitrification (B) or activated sludge followed by 7 of 1.01 log10), and FC densities from 1.61 10 to biofiltration (C) oactivated sludge with nitrification 8.78 107 culturable FC per 100 ml (i.e. a range of 0.74 and denitrification (D) oseries of 3 biofilters (E) log10). These values were 3–5 log10 greater than FC olagooning (F) oactivated sludge, sand filtration and abundance and activitymeasured in the Seine river disinfection (G) (Fig. 6). Removal of culturable FC was upstream from Paris byGeorge et al. [21]. Raw globallyrelated to higher retention times (in activated wastewater entering the largest of the studied WWTPs, sludge processes with nitrification and denitrification, or the Acheres" epuration plant (7 106 inhabitant-equiva- in lagooning) or to the presence of efficient systems of lents), showed fluctuations over 5 sampling campaigns SM, carbon and nitrogen depollution (biofiltration) as 2612 I. George et al. / Water Research 36 (2002) 2607–2617

in out 1.E+06

1.E+05

1.E+04

1.E+03

(pmoles MUF min-1) 1.E+02 GLUase act. per 100 ml

1.E+01

rard (B) Wavre (A) è AchèresCouilly (A)Gu (B) Troyes (B) Rouen (D) Aulnoy (F) Rixensart (B)ères piloteWaterloo (C) (D)ValentonColombes (D) (E) (a) Ach Ste Marie la mer (G)

in out 1.E+09 1.E+08

1.E+07 1.E+06 1.E+05 1.E+04 CFU per 100 ml 1.E+03 1.E+02 B) C) ) d (B) s (B) (D) ères (A) rar nton (D) Wavre (A) Couilly è(B) erlooRouen (D Ach Gu Troye Vale Aulnoy (F) Rixensart (ères piloteWat ( Colombes (E)arie la mer (G) (b) Ach Ste M

Fig. 2. (a) and (b) b-d-glucuronidase activity(Fig. 2a) and culturable FC counts (Fig. 2b) per 100 ml of raw (‘in’) and treated (‘out’) wastewater in the different WWTPs. For WWTPs that were sampled several times, the geometric mean value and the range between maximum and minimum values (vertical bar) were presented on the ﬁgures.

Table 2 b-d-glucuronidase activityand culturable fecal coliforms in raw and treated wastewater at the Ach eres" WWTP

Date of sampling Raw wastewater Treated wastewater

GLUase act. 100 ml1 Culturable FC GLUase act. 100 ml1 Culturable (pmol MUF min1) 100 ml1 (pmol MUF min1) FC 100 ml1

May18, 1999 141,886 1.54 108 12,045 4.11 107 December 16, 1999 48,806 3.67 107 5589 3.50 106 April 6, 2000 33,700 2.79 107 2624 1.90 106 May11, 2000 39,677 1.19 108 14,420 6.60 107 June 15, 2000 56,221 4.11 107 9475 1.10 107 well as disinfection units. Treatment types F and G Removal efficiencyof the GLUase activityfollowed showed a spectacular decrease of culturable FC levels of the same pattern, except for treatments F and G, where 4 and 5.4 log10, respectively. In the treatment G, sand the reduction of enzymatic activity was only about 2 filtration was responsible for 0.78 log10 of this decrease, log10. In categoryG, GLUase activitywas reduced by and UV rays for 2.91 log10 (data not shown). 0.55 log10 bysand filtration and did not change after UV I. George et al. / Water Research 36 (2002) 2607–2617 2613

Table 3 Speciﬁc load of culturable fecal coliforms and b-d-glucuronidase activityper inhabitant and per dayin raw and variouslytreated wastewater

Treatment type GLUase act. (units inh1 day 1) Culturable FC (inh1 day1)

Geometric mean Range Geometric mean Range

Raw 8.94 107 (1.66–22.2) 107 8.13 1010 (2.49–25.0) 1010 Decantation 3.87 107 (0.66–12.1) 107 5.04 1010 (1.34–28.0) 1010 A 6.64 106 (2.15–20.5) 106 5.13 109 (0.84–31.2) 109 B 1.89 106 (0.78–5.60) 106 1.28 109 (0.83–2.23) 109 C 3.29 106 1.31 109 D 8.07 105 (2.08–13.0) 105 1.31 108 (1.05–1.89) 108 E 4.11 105 3.27 108 F 1.77 106 1.93 107 G 1.88 105 2.19 105

When different WWTPs were included in one category, the geometric mean value was calculated and the range between maximum and minimum values was reported.

6.E+11 1.2 5.E+11

) 1.0 4.E+11 ) 0.8 3.E+11 2.E+11 0.6 FC inh-1 day-1 1.E+11 0.4 0.E+00 or GLUase act. ( Log removal of FC ( 0.2 1.E+01 1.E+03 1.E+05 1.E+07 Inhabitant-equivalents 0.0 Fig. 3. Semi-log plot of the specific load of culturable FC in 0.0 0.2 0.4 0.6 0.8 1.0 1.2 raw wastewater entering the various WWTPs as a function of Log removal of SM their inhabitant-equivalents capacity(in log units). For each sampling campaign, the capacityof the WWTP was calculated Fig. 4. Log removal efficiencybyprimarysettling of b-d- as the product of the BOD in raw sewage (mg l1) multiplied by glucuronidase activityor culturable FC plotted against log the dailywastewater flow entering the plant (m 3 day1) and removal of SM. The dotted line represents the equivalence line. divided by54 (g BOD inh 1 day1). disinfection (data not shown). These contrasting results will be discussed subsequently. the 5 sampling campaigns slightlyexceeded the range in raw wastewater (Table 2 and Fig. 2a and b). As expected, the various treatments resulted in specific 3.5. FC abundance and specific load in treated effluents loads of GLUase activityand culturable FC in treated sewage differing byseveral orders of magnitude (Table Due to the variabilityof raw sewage qualityand of 3). Primarysettling reduced both specific loads of 0.36 removal efficiencyof FC, the GLUase activityand log10 and 0.21 log10, respectively. Activated sludge culturable FC in treated effluents from the different process following primarysettling (treatment A) was WWTPs varied from 99 to 7526 activityunits per 100 ml the less efficient complete treatment to reduce specific 2 7 (range of 1.88 log10) and 1.21 10 –1.15 10 CFU per loads (1.13 log10 and 1.20 log10, respectively) and, 100 ml (range of 4.98 log10) (Fig. 2a and b). Treated treatments F and G excepted, activated sludge with high effluents from the Acheres" plant were the most retention time and biofiltration (both preceded by contaminated, and the range of values for GLUase primarysettling) were the most efficient as theyreduced activity(0.74 log 10) and culturable FC (1.54 log10) over specific loads up to 2.8 log10. In treatments G and F, the 2614 I. George et al. / Water Research 36 (2002) 2607–2617

6 6 G 5 5

) 4 ) 4 F 3

3 Log removal 1 2 F G

or GLUase act. ( 0

Log removal of FC ( dec A B C D E F G 1 GLUase act. FC

0 Fig. 6. Removal efﬁciencies of b-d-glucuronidase activityand 0123456 culturable FC bydifferent wastewater treatments. Results are expressed in log removal compared to the raw wastewater. (a) Log removal of SM 10 When different WWTPs were included in one category, the geometric mean value was calculated and the range between 5 maximum and minimum values (vertical bar) was presented in the ﬁgure. Dec=primarydecantation, A–G refer to treatment F types (Table 1).

) 4 ) 3 seemed plausible that FC could multiplyin the sewer networks. This had been previouslydemonstrated in the F 2 sewer network of the Acheres" WWTP for heterotrophic

or GLUase act. ( bacteria bySeidl et al. [22] and for nitrifyingbacteria by

Log removal of FC ( 1 Brion and Billen [23]. For coliforms, it was proposed by Miescer and Cabelli [2] and recentlyconfirmed by Ashleyand Dabrowski [24] in the sewer network of 0 Dundee (Scotland). In dryweather conditions, these 01 2345 latter authors measured indeed higher coliforms abun- (b) Log removal of BOD dance in wastewater from the main sewer trunks than Fig. 5. (a) and (b) Log removal bythe whole various from the upper collectors. treatments of b-d-glucuronidase activityand FC abundance To test the hypothesis of bacterial growth with our plotted against log removal of SM (Fig. 5a) and BOD (Fig. 5b). results on various WWTPs, we supposed that the extent The dotted line represents the equivalence line. No BOD values of multiplication of bacteria in raw sewage depended on were available for treatment G. the mean residence time of wastewater in the sewer network and thus approximatelyof its size. Moreover, specific load of culturable FC was drasticallyreduced the size of the sewer network was supposed to be related compared to the specific load of GLUase activity, as a to the size (capacity) of the connected WWTP. In Fig. 3, consequence of contrasting removal efficiencies for both specific loads of FC in raw sewage were plotted against variables. the sewered population for the various sampled WWTPs. Results showed that both variables were only roughlyrelated, probablybecause our deductive reason- 4. Discussion ing was too simplified and the multiplication of FC was dependent of parameters that were not directlymea- 4.1. Quality of raw wastewater sured or documented, such as the wastewater temperature [24] or the hydraulics (slope, mean residence time of Our data on FC levels in raw wastewater were in water) characterising each catchment network. accordance with other studies [1,3,6,4]. The temporal follow-up at the Acheres" WWTP showed that the microbiological qualityof raw sewage entering a 4.2. Effect of primary, secondary and tertiary treatments treatment plant could be highlyvariable. Following our results from laboratoryexperiments on The primarytreatment of settling (decantation) is the net increase of FC abundance in raw sewage, it designed to reduce the SM in raw sewage. In comparison I. George et al. / Water Research 36 (2002) 2607–2617 2615 to secondaryand tertiarytreatments, it was not very Bahlaoui et al. [8] observed maximum removals of efficient in removing the microbiological pollution. Our pollution-indicator bacteria in spring and summer data confirmed the percentages of FC removal of 10– compared to the fall and winter period. Moreover, the 60% cited byAudic [3] and 20–50% byDuprayet al. [6]. lagooning reduced the GLUase activityof only2 log 10 Primarysettling probablyremoved FC associated to (Fig. 6). This difference between FC and enzymatic SM. George et al. [21] reported indeed that 30–60% of activityremovals could be explained bythe presence in the FC population in raw wastewater was retained by sunnyshallow ponds of FC in an active but non filtration on 5 mm-pore-sized membranes (the authors culturable state. The transition of coliforms to a considered the size of 5 mm as the limit between free metabolicallyactive but non culturable state due to bacteria and those associated to particles or clumped light stress is well documented in the literature [31– together). However, this proportion of coliforms asso- 33,14]. Another explanation could be the interference of ciated to ‘decantable’ particles varied from one raw algae with the GLUase activitymeasurement. Some sewage to the other, as no significant relationship was freshwater algae and plants produce the b-d-glucuroni- observed between SM and FC removals byprimary dase enzyme and, in closed systems such as oxidation settling in Fig. 4. ponds, their high biomass can influence the bacterial The elimination of FC was much more efficient in GLUase activitymeasurements [34]. secondaryand tertiarytreatments. It was generally Ultraviolet disinfection is considered as one of the greater than the reduction of SM and BOD (Fig. 5), two best alternatives to chlorination to improve the micro- standard parameters used to assess the efficiencyof biological qualityof treated wastewater. The coliforms wastewater treatment. However, the FC abundance in abundance in UV-disinfected effluents depends on the raw wastewater was so high that most treatments applied UV dose (in mW s cm2), the wastewater produced treated effluents of poor microbiological suspended solids content, its UV transmittance, its quality, which represent a major source of FC to rivers initial coliforms concentration, and the degree of and coastal waters. For example, a large increase of FC association of bacteria with particles [35,36]. In this abundance was observed in the Seine river downstream study, UV disinfection (following activated sludge and from the Valenton and Acheres" outfalls in the Parisian sand filtration) was the most efficient treatment to area [21]. About 25% of coliforms discharged with reduce culturable FC abundance. The removal rate by treated wastewater are linked to particles >3–5 mm UV rays was 2.91 log10, which was close to data from (George, unpublished data; [6]). The FC load of treated Perrot and Baron [37] and Moreno et al. [38] (3.2 and effluents depended both on the qualityof raw waste- 2.59 log10 reduction, respectively). However, GLUase water and the efficiencyof the treatment. In the Ach eres" activitywas not reduced byUV disinfection, which WWTP, neither the raw wastewater FC content nor the challenges the effectiveness of this treatment to really FC removal efficiencywere constant, which resulted in eliminate micro-organisms in wastewater. UV B radia- highlyvariable FC abundance in treated wastewater. tions inactivate microorganisms bycausing thymine The various removal efficiencies reported in this work dimerization in microbial DNA [3,25,30], and many (Fig. 6) were in accordance with those mentioned in studies are currentlydevoted to assess the abilityof other studies [5,6,25,10]. Treatments with high retention micro-organisms to repair from UV damage by time such as the activated sludge process with (de)ni- enzymatic photoreactivation [39–41]. The extent to trification favoured the biological elimination of FC, which these ‘inactivated’ micro-organisms are uncultur- mainlybyprotozoan grazing, competition with the able but still metabolicallyactive Fand virulent in the reactors microflora and sedimentation with flocs. case of pathogensFremains an open field of investiga- Biofilters in series also reduced considerablyFC tion (for a review on viable but nonculturable bacteria, densities in sewage, partlybecause of their great see [42]). efficiencyin eliminating small-sized particles. Lagooning was particularlyefficient to eliminate FC 4.3. Specific load of FC in raw and treated effluents compared to other wastewater treatment systems, as a result of long retention time (60 days for treatment F) In raw treated wastewater, specific loads were high [8,26], efficient grazing byheterotrophic nanoflagellates and in accordance with data cited byMiescer and protozoa [27], high pH generated byphotosynthesis Cabelli [2]. In treated wastewater, specific loads were [28,25,26] and inactivating effect of sunlight [29,25,30,8]. stronglydependent of the typeof wastewater treatment, In this study, a 4 log10 decrease of culturable FC and variations in the culturable FC specific load of more abundance was observed, that largelyexceeded annual than 5 log10 were observed between treated wastewaters. removal rates of 1–2 log10 reported byother studies Specific loads can be used to estimate the dailyamount [25,8,9]. But the high removal rate we observed in the of FC discharged in the environment byanystep, by Aulnoylagoon in June 2000 was probablynot repre- multiplying the specific loads in treated wastewater sentative of the mean annual removal efficiency, as corresponding to the treatment in use in the step, byits 2616 I. George et al. / Water Research 36 (2002) 2607–2617 sewered population. These estimations can then be References applied as input data representing point sources to models on the dynamics of FC in rivers and coastal [1] Hirn J. Indicator bacteria and Salmonella in food waters. processing and domestic effluent. J Water Pollut Control Fed 1980;52(1):48–52. [2] Miescer JJ, Cabelli VJ. Enterococci and other microbial indicators in municipal wastewater effluents. J Water 5. Conclusion Pollut Control Fed 1982;54(12):1599–606. [3] Audic JM. Evolution des technologies d’elimination! des microorganismes. 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