Effects of bacterivorous ciliated protozoans on degradation efficiency of a petrochemical activated sludge process

PII: S0043-1354(99)00378-4

Wat. Res. Vol. 34, No. 7, pp. 2051±2060, 2000 # 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/00/$ - see front matter

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EFFECTS OF BACTERIVOROUS CILIATED PROTOZOANS ON DEGRADATION EFFICIENCY OF A PETROCHEMICAL ACTIVATED SLUDGE PROCESS PETER HOLUBAR*M, TANJA GRUDKE, ANDREAS MOSER, BIRGIT STRENN and RUDOLF BRAUNM Institute of Applied Microbiology, UniversitaÈt fuÈr Bodenkultur, Muthgasse 18, A-1190 Vienna, Austria (First received 1 September 1998; accepted in revised form 1 July 1999) AbstractÐThe aim of this work was to adapt protozoans to a saline and crude-oil contaminated petrochemical sewage. Wild type protozoans were isolated from di€erent marine locations and from a municipal-re®nery mixed sewage treatment plant. In batch culture the micro-organisms were adapted by slowly increasing concentrations of sodium chloride and hydrocarbons. No wild type strain survived in the saline sewage. Using selected type strains of protozoans as inocula, Cohnilembus rheniformis and Uronema marinum were found to adopt to the brine. In a 20 l-batch culture U. marinum grow up to a number of 14.000 cells
mlÿ1. Since the yield was recognized to be to small for further scale-up, additional experiments were done in continuous culture. In a lab-scale 2-step continuous culture, inoculated with natural activated sludge mixed populations of protozoans, one single species of a ciliated protozoan could be enriched and identi®ed as Uronema nigricans. The e‚uent turbidity, measured as optical density (OD600), of the lab-scale activated sludge plant decreased dependent on the increase of U. nigricans count. In addition, chemical oxygen demand degradation eciency was found to be 45.8%, compared to 35.4% in activated sludge mixed cultures missing protozoans. # 2000 Elsevier Science Ltd. All rights reserved Key wordsÐprotozoa, ciliates, activated sludge, petrochemical sewage, suspended growth system, crude oil

INTRODUCTION

A pilot-plant for the biological treatment of sewage of a petrochemical plant had been initially inoculated with primary activated sludge from a municipal sewage treatment plant. At the beginning this sludge had shown a wide range of typical species of ciliated protozoans (Curds, 1982; Foissner et al., 1995). These genera were not able to adapt to the petrochemical sewage, and after a few days the activated sludge did not contain any protozoans. However one day, a sudden decrease in e‚uent turbidity after the settle tank was measured, and a microscopic observation of the activated sludge showed an abundance of a single species of an unknown ciliated protozoan species. After one day the protozoans disappeared again. Obviously, the decrease in turbidity was caused by the presence of these protozoans. The aim of this work was to adapt protozoans to the crude-oil contaminated brine to decrease the e‚uent turbidity, and to *Author to whom all correspondence should be addressed. Tel.: 43-1-360066212; fax: 43-1-3697615; e-mail: [email protected]

increase the degradation eciency of the speci®c activated sludge process. The impact of ciliated protozoans on the overall puri®cation performance of the activated sludge process had been shown earlier. Especially biological oxygen demand (BOD), concentration of suspended solids (SS) and the number of viable bacteria in the e‚uent of the observed sewage treatment plants were reduced signi®cantly due to the bacterivorous activity of ciliated protozoans (Curds et al., 1968; Salvado et al., 1995). Surplus-sludge production could be reduced that way also (Lee and Welander, 1996). In chemostat culture, bacterial biomass was reduced by about 12±43% by the grazing of protozoans in comparison to a culture lacking in protozoans (Ratsak et al., 1994). In the special case of the petrochemical sewage, the unfavorable chemical composition has to be considered. The known demands of ciliated protozoans on environmental conditions vary widely: Euplotes anis (Dujardin, 1841) Kahl, 1932 can adapt to a salinity of 3.5%, but will tolerate only 0.2 mg
lÿ1 ammonium (Foissner et al., 1991); Colpidium colpoda (Losana, 1829) Stein, 1860 will tolerate up to 18 mg
lÿ1 H2S and can survive in

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Peter Holubar et al.

oxygen-free media, but will be limited at 5 mg
lÿ1 ammonium (Foissner et al., 1994). Dale (1987) found that hydrocarbon concentrations of 0.62 and 1.72 mg
lÿ1 caused declining densities of heterotrophic ciliates in closed marine environments. MATERIALS AND METHODS

Sewage composition The sewage showed a pH of 8.2 and temperature was constant at 208C. A salinity of 2.1% sodium chloride, crude-oil concentrations of 40±100 mg
lÿ1 and ammonium concentrations of 80 mg
lÿ1 were measured. Chemicals Sea-salt (sodium chloride) was provided by SeraAquaristik GmbH. (Heinsberg, Germany) and had to be used for all experiments, because it showed sucient low Iodine concentrations. Complan (milk powder) is a product of Heinz & Co Ltd. (UK). Sigma Chemical Co. was the supplier of cereal leaves and tricine. All other chemicals were of analytical grade and available by Merck (Darmstadt, Germany). Microorganisms Wild strains of protozoans were collected from various oil contaminated habitats. Sampling locations near the city of Hamburg (FRG) were Stadersand, Jork-Neuenschleuse, Yachthafen, Petroleumhafen (British Petroleum), oil collecting basin (Shell), St Pauli Hafenstraûe and the docks of Hamburg-Hafen. Austrian sampling locations were the ®lter device of a large marine aquarium (Haus des Meeres, Vienna) and a combined municipal-re®nery sewage treatment plant (AWV Schwechat, MannswoÈrth). Type strains of ciliated protozoa were provided by Culture Collection of Algae and Protozoa (CCAP, UK). These strains were chosen, either because of their habitats or their reported tolerance to salinity and/or high ammonium concentration (Table 1). Klebsiella pneumoniae susp. pneumoniae ATTC 27889 served as bacterial prey organism. Media and culture conditions Klebsiella pneumoniae susp. pneumoniae ATTC 27889 was cultivated on glucose agar (ATCC Media 1) at 378C. Protozoa culture media were made according to CCAP guidelines. The cultures were incubated in polystyrene ¯asks for tissue culture with a surface area of 150 cm2

(Corning, Cambridge, USA) at 208C or 378C respectively. The ¯asks were kept in the dark and illuminated for 12 h each day. For the cultivation of Colpoda steinii the arti®cial seawater for protozoa (ASWP) was supplemented with a few pieces of boiled rice. In arti®cal seawater (ASW) the vitamin solution was replaced by yeast-extract (Hanna and Lilly, 1974). The composition of soil/water biphasic media (S/W), Sigma Cereal Leaf-Prescott Liquid (SPL), Sonneborne's paramecium media (SPM) and uronema media (UM) was not changed. The composition of rice-media was varied by adding 0.25 vol% yeast-extract (RoÈttger, 1995). 20 l-batch culture 100 ml of a 6 week old CCAP-type strain Uronema marinum culture was mixed with 100 ml of a Klebsiella pneumoniae subsp. pneumonia culture, grown for 24 h at 308C in nutrient broth (containing 1% NaCl), and 200 ml Uronema-media (UM). This culture broth was diluted by adding 19.6 l of ¯ash-¯otation e‚uent (FFE; 2.1% w/v NaCl, hydrocarbon concentration 30 mg
lÿ1) in a 50 l plastic vessel and sparged with compressed air, using a diffuser. Protozoan enrichment by continuous culture A two-stage laboratory-scale activated sludge plant was operated in continuous culture. The volume of each aeration tank was 5 l, each settle tank had a volume of 6 l. Hydraulic retention time in each stage was 2.5 h. The sludge retention time was in the range of 7 to 43 days. Table 2 shows the experimental set-up. In phase 0 the feed was synthetic sewage (1 l distilled water contained: 160 mg peptone, 110 mg meat extract, 30 mg urea, 28 mg K2HPO4, 7 mg NaCl, 4 mg CaCl2
2H2O and 2 mg MgSO4
7H2O), which was supplemented with increasing concentrations of sea-salt. In phase I the composition of synthetic sewage was changed by adding e‚uent of the activated sludge pilot-plant (PPE). In this case the concentration of ammonium in the synthetic sewage formula had to be reduced to a maximum of 80 mg
lÿ1 in the overall mixture. During phases II±VII the e‚uent of a ¯ash-¯otation plant was added to the synthetic sewage. Using this ¯ash-¯otation plant the hydrocarbon concentration of the sewage could be stabilized at about 30 mg
lÿ1. This ¯ash¯otation e‚uent (FFE) was similar in composition to the sewage of the petrochemical plant, which will be treated biologically in the future. In case of bulking sludge 10 mg
lÿ1 of a 1 M FeCl3-solution was added into the aeration tanks. Initial inoculation was made using activated sludge from a sewage treatment plant where mixed

Table 1. List of chosen protozoan type strains, CCAP-number, natural habitats, environmental requirements and literaturea Type of strain

CCAPnumber

Media

Cohnilembus reniformis Kahl Colpoda steinii Maupas 1883

1610/1 1615/3

ASWP+rice ASWP+rice

Cyclidium glaucoma Muller 1773

1616/1

S/W

Paramecium putrinum ClapareÂde and Lachmann 1858

1660/14

SPL, S/W

Spirostomum intermedium Kahl 1930 Uronema marinum Dujardin 1841

1677/3

S/W

1686/2

ASW (+YE), ASWP, UM

a

Natural habitat and environmental requirements Marine Isolated from soil, a waste water treatment plant, tolerates ammonium up to 120 mg
lÿ1 Isolated from fresh water, a waste water treatment plant; was found at 50±588C in hot springs Isolated from fresh water, activated sludge, highly polluted water, and from the White Sea at a salinity of 1.5% Isolated from fresh water, tolerates a salinity of 1% Isolated from sea water, activated sludge and marine habitats

Literature Ax and Ax (1960). Rivera et al., 1988; Esteban et al., 1991. Rivera et al., 1988; Esteban et al., 1991. Curds, 1975; Foissner et al., 1994. Foissner et al., 1992. Esteban et al., 1991; Parker, 1976.

Abbreviations: CCAP=culture collection of algae and protozoa; ASW=arti®cial sea water; ASWP=arti®cial sea water for protozoa; S/ W=soil/water biphasic media; SPL=sigma cereal leaf-prescott liquid; YE=yeast extract, UM=uronema media.

Protozoan enrichment in brine petrochemical and municipal sewage is treated (AWV Schwechat, MannswoÈrth). Microscopic analyses Identi®cation and enumeration of the ciliated protozoans was made in bright-®eld mode using a ReichertUnivar microscope (Foissner et al., 1995; 1991). Exactly 1 ml of activated sludge was dropped on a glass slide using a 2 ml-syringe (Type 7002, Hamilton, Switzerland) and covered with a cover slide. At 100-fold magni®cation the total volume could be examined for enumeration of the organisms. This procedure was repeated ®ve-fold. Protozoans were identi®ed using the keys of Foissner et al. (1995; 1994; 1992; 1991). Chemical analyses Concentration of suspended solids (SS) was measured as weight per volume by air-drying 10 ml of activated sludge samples at 1058C (TV30u, Memmert, Germany). Afterwards those samples were incinerated in an oven (Naber, Lilienthal, Germany) at 6808C to determine the amount of volatile suspended solids (VSS). Due to the high salinity of the sewage, all samples were ®ltrated and diluted 1:4 with distilled water to measure chemical oxygen demand (COD) using a LKT-photometer (Dr Lange, Austria). Concentration of ammonium was quanti®ed using the Merckoquant 10024 test. Nitrate and nitrite were quanti®ed using Merckoquant 10020 and Merckoquant 10007 (Merck, Darmstadt, Germany). E‚uent quality was measured as optical density at 600 nm (OD600) in 1 cm cuvettes using a Novaspec-IIRapid spectro-photometer (Pharmacia, Vienna, Austria). Distilled water served as a blank. Concentration of dissolved oxygen was determined by using an Oxi90-device equipped with an EO90-electrode (WTW, Vienna, Austria). The value of pH was measured by using a pH 91 pH-meter and an E56 pH-electrode (WTW, Vienna, Austria). RESULTS AND DISCUSSION

Cultivation of wild type ciliates First cultivation experiments were made in Erlenmeyer-¯asks with SPM-media and 1- sodium chloride (Ax and Ax, 1960). Only ciliates sampled from oil containing locations like Stadersand

2053

and Petroleumhafen survived. Further experiments showed that the cultivation is more successful when aeration was made using porous aquarium stones, instead of using orbital shakers. Also rice-media was shown to be more e€ective than SPM-media. For cultivation of Stadersand-ciliates paper ®bres in rice-media served successfully as substratum for growth of Vorticella species. Sodium chloride concentration of the rice-media was slowly increased stepwise from 0.5% up to 1.25%. Only Vorticella sp. survived until 1.25% NaCl, but there was a complete lack in crawling ciliates. At concentrations of 1.5% NaCl only cysts were visible, and further cultivation of any wild type ciliates was not possible. Cultivation of CCAP-strain ciliates Each CCAP-type strain was cultivated in the recommended media in tissue-culture ¯asks. Due to the large surface area of the ¯asks, the providing of oxygen was possible without additional aeration or agitation. Instead of sea-salt, the crude-oil containing FFE (2.1% NaCl, hydrocarbon concentration 30 mg
lÿ1) from the petrochemical plant was added stepwise (0, 25, 50, 100 vol%) to each media. Paramecium putrinum and Spirostomum intermedium did not survive 25 vol% (0.52% NaCl, hydrocarbons 7.5 mg
lÿ1) of FFE in the media. Cyclidium glaucoma survived only 25% whereas Colpoda steinii survived at 50 vol% (1.05% NaCl, hydrocarbons 15 mg
lÿ1) of FFE. Only Cohnilembus reniformis and Uronema marinum showed good growth in 100 vol% FFE. Morphology of C. reniformis was changed drastically to a smaller and more oval form. A similar e€ect of ultra-structural modi®cation following chronic exposure to partially degraded crude oil for the ciliated protozoa Colpidium colpoda has been reported by Rogerson and Berger (1982). In contrast, the cell morphology of U. marinum remained unmodi®ed.

Table 2. The chosen parameters of the adaptation experiment using continuous culture are shown. Phase 0: adaptation of activated sludge and the protozoans to increasing NaCl-concentrations. Phase I: the arti®cial waste water was supplemented with e‚uent of the activated sludge pilot plant to adapt protozoans to the speci®c petrochemical sewage at low hydrocarbon concentrations. Phases II±VII: the protozoans had to adapt to increasing hydrocarbon concentrations by mixing arti®cial waste water and ¯ash ¯otation e‚uenta Phase

Time (days)

Feed

0

1±91

I

92±130

Synthetic sewage+25 vol% PPE; further increase of NaCl-concentration, up Holotrichia day 51±100 to 2.1% in increments of 0.25% and 0.1%

II III IV V VI VII

131±149 150±162 163±177 178±191 192±219 220±346

75 vol% AWW+25 vol% FFE 50 vol% AWW+50 vol% FFE 33.3 vol% AWW+66.7 vol% FFE 25 vol% AWW+75 vol% FFE 15 vol% AWW+85 vol% FFE 100 vol% FFE

a

Synthetic sewage with increasing NaCl-concentrations, up to 1.5% in increments of 0.25%

Dominant protozoan group Vorticellida day 1±50

Scuticociliata day 101±346

Abbreviations: AWW=arti®cial waste water (=synthetic sewage+2.1% NaCl); PPE=activated sludge pilot-plant e‚uent; FFE=crudeoil polluted ¯ash-¯otation e‚uent.

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Peter Holubar et al.

20-l batch culture of CCAP-type strain Uronema marinum

E€ects of protozoan growth on the activated sludge process

To achieve a sucient amount of inoculum for the pilot-plant, the ciliate showing best cell yield in the preliminary experiments, U. marinum Dujardin 1841, was cultivated in a 50-l batch vessel. In a small-scale experiment the ciliated protozoa U. marinum PW2 was reported to grow up to populations of 350,000 per ml after 7 days at 248C in axenic media in 20-ml batch cultures (Hanna and Lilly, 1974). In our own experiments U. marinum reached a maximum of only 14,000 cells
mlÿ1 after three days of incubation. From there on, the protozoan number decreased steadily, despite further weekly feeding of 100 ml of bacterial prey (data not shown). Since the achieved protozoan numbers were recognized to be too small for further experiments, these were done in continuous culture.

To start with a stable mixed culture of activated sludge, the plant was fed with synthetic sewage respectively AWW+PPE for the ®rst 130 days of operation. During the start period only microscopic observation was made. Afterwards, the concentration of dissolved oxygen, ammonium, nitrate, nitrite and pH was measured as routine parameters. The dissolved oxygen concentration was always above 7.0 ppm. The ammonium concentration was between 20±60 mg
lÿ1. Nitrate respectively nitrite concentrations were in the range of 50±150 mg
lÿ1 and 0±15 mg
lÿ1. The pH varied from 7.8 to 8.2. The number of protozoans as well as e‚uent turbidity of both stages of the two-step continuous culture showed high ¯uctuation during the 220 days of full-monitored operation (Figs 2 and 3). Especially in the ®rst 200 days of the experiment the exact microscopic enumeration of the organisms was dicult to achieve due to the presence of a high variety of protozoans. At this time the standard deviation was about 10±50%, even when the enumeration was made ®ve-fold. On the other hand, quite similar ¯uctuations of protozoan numbers were found in both stages of the lab-scale sewage plant. From our experience the high ¯uctuations were caused by sudden presence or absence of speci®c protozoan species. During steady-state operation, only a single protozoan species remained. In that case enumeration was much easier, and the standard deviation was only about 5% when counting was made ®vefold. In stage 1 a ®rst maximum of protozoans of 1.21
105 cells
mlÿ1 was reached on day 135 in phase I. E‚uent turbidity at the same day reached a minimal OD600 of 0.061, caused mainly by suspended bacterial cells and small sludge-¯ocs with incorporated crude-oil droplets. Five days later, on day 140, the number of protozoans decreased to 4.94
104 cells
mlÿ1, whereas e‚uent turbidity was increased to an OD600 of 0.147. A maximum of 2.48
105 cells
mlÿ1 was reached again, one week later on day 147. E‚uent turbidity showed a minimal OD600 of 0.011. In phases II±V the adaptation and change of protozoan species took place, and the number of protozoans was reduced to a mean of 1.00
105 cells
mlÿ1. E‚uent turbidity was rather low in phases III and IV, but nearly doubled in phase V to a mean value of OD600 of 0.076. During phases VI and VII both parameters stabilized. Since most of the COD was already oxidized in the aeration tank of stage 1, the second stage was carbon-limited. In stage 1 concentration of VSS was about 6.5 g
lÿ1, whereas in the second stage only about 2 g
lÿ1 was reached. Due to this lack of bacterial prey the protozoan number remained small. Luckinbill (1974) showed a typical predator±prey dynamic in batch cultures of two protozoan species,

Appearance of ciliated protozoa during enrichment by continuous culture Using natural activated sludge mixed culture protozoans, the variety of dominant protozoan species changed during the adaptation phase (Table 2). With increasing concentrations of FFE and NaCl a single species of the family Scuticociliata could survive exclusively. These organisms were moving very fast between activated sludge ¯ocs while grazing on the ¯oc's surface. The cells showed an oval form, 25±50 mm in length and 13±25 mm in diameter (Fig. 1). The morphology of the new isolate was very similar to the CCAP-type strain U. marinum. It could be identi®ed as Uronema nigricans (Muller, 1786) Florentein, 1901.

Fig. 1. Photograph of the newly isolated ciliated protozoan species identi®ed as Uronema nigricans (Muller, 1786) Florentein, 1901; bright-®eld microscopy, magni®cation 1000-fold; 10 units=25 mm.

Protozoan enrichment in brine

where Didinum nausatum was the predator and Paramecium aurelia served as prey. In the present continuous culture experiment, using the more complex mixed bacterial culture of activated sludge as prey, and Uronema nigricans as predator, a similar dynamic was not recognizable at ®rst sight. During adaptation of the protozoans (phase 0±VI) high ¯uctuations in both, protozoan number and e‚uent turbidity, made interpretation dicult (see Figs 2 and 3). But, using the data of phase VII in stage 1, and calculating the derivative of protozoan number and e‚uent quality it could be shown clearly, that with each increase in protozoan number the e‚uent turbidity decreased (Fig. 4). One could question, if a single predator-species like U. nigricans could reduce the plant's e‚uent turbidity by ingestion of bacteria like that. No data were found on the ingestion rates of U. nigricans, but for the similar species U. marinum a clearance rate of 49.0 nanoliters.cellÿ1
hÿ1, and an ingestion rate of 710 bacteria.cellÿ1
hÿ1 had been measured at 228C, and bacterial prey concentrations of about 106
mlÿ1 (Sherr et al., 1988). Using this ingestion rate value and the average protozoan number of 1.5
105 in the ®rst aeration tank a total number of 1.07
108 bacteria
hÿ1 was calculated to be ingested by the protozoans.

2055

E€ect of protozoan growth on COD-degradation eciency in continuous culture In the ®rst experiment the lab-scale sewage treatment plant was operated using activated sludge without any protozoans being present (Fig. 5). The values of COD for the raw sewage varied in a wide range, depending on the performance of the used ¯ash-¯otation. Degradation eciency varied also, the mean was 35.4% (n = 63, standard deviation 10.66, min=16.4, max=57.9). More than 90% of the possible biological COD-elimination took place in the ®rst stage of the continuous culture. In Fig. 6 the COD-course during the experimental phases of the protozoan enrichment procedure in continuous culture is shown. Only the results of the phases II± VII (days 131±268) are comparable to the experimental conditions of the ®rst experiment. Despite the higher COD of the raw sewage, the degradation eciency of the protozoa-containing activated sludge reached a mean of 45.8% (n = 32, standard deviation 10.82, min=14.5, max=67.0). A t-test 1 and a F-test, calculated by using Statgraphics , showed that the di€erence of the means is signi®cant at the 95.0% con®dence interval, and that the variances of the two samples are equal. This shows that by enrichment of protozoans the overall puri®-

Fig. 2. Number of protozoans in activated sludge and e‚uent turbidity (OD600) after settle tank of stage one of the two-step continuous culture (phases II±VII).

Fig. 3. Number of protozoans in activated sludge and e‚uent turbidity (OD600) after settle tank of stage two of the two-step continuous culture (phases II±VII).

2056 Peter Holubar et al.

Fig. 4. Derivative of the number of protozoans in activated sludge and the e‚uent turbidity (OD600) after settle tank of stage one of the two-step continuous culture (phase VIIÐsteady state operation).

Protozoan enrichment in brine 2057

Fig. 5. Chemical oxygen demand (COD) of the ¯ash-¯otation e‚uent (FFE) (=feed) and the biologically puri®ed sewage (=e‚uent) after settle tank of stage two of the two-step continuous culture. Activated sludge without protozoans being present.

2058 Peter Holubar et al.

Fig. 6. Chemical oxygen demand (COD) of the ¯ash-¯otation e‚uent (FFE) (=feed) and the biologically puri®ed sewage (=e‚uent) after settle tank of stage two of the continuous culture. Activated sludge with growth of the enriched ciliated protozoa Uronema nigricans (Muller, 1786) Florentein, 1901.

Protozoan enrichment in brine 2059

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Peter Holubar et al.

cation performance of a petrochemical activated sludge process could be increased signi®cantly. There are no clear data available, how grazing of protozoans on activated sludge can enhance the COD-elimination rate. It seems possible that some of the activated sludge bacteria might remain in the logarithmic growth phase due to the grazing activity of the protozoans (Ratsak et al., 1996). In summary it can be stated that it was possible to adapt the CCAP-type strain U. marinum Dujardin, 1841 and the new isolate U. nigricans (Muller, 1786) Florentein, 1901 to the high salinity and the crude oil content of the speci®c petrochemical sewage. An activated sludge process with this newly isolated ciliated protozoan species being present, showed signi®cantly higher COD degradation eciency than activated sludge without protozoan growth. AcknowledgementsÐThis research was supported by OMV AG. We want to thank J. Winter, H.P. Grosser, E. Panzer and N. Phillipovich for their support. We also gratefully acknowledge the help of John G. Day of CCAP Culture Collection of Algae and Protozoa, Institute of Freshwater Ecology, Windermere Laboratory, UK, for identifying the enriched ciliated protozoans. REFERENCES

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