p. 3322-3324Las condiciones físico-químicas de los estanques de algas de alta tasa fueron responsables de una reducción de más del 97% en la infectividad de ooquistes Cryptosporidium parvum en ratones recién nacidos. El uso de bolsas semipermeables de celulosamostró que el pH, amoníaco, y / o luz parece ser un factor importante para la inactivación de ooquistes en las aguas residuales, el apoyo a la importancia de los sistemas basados en algas para su reutilización segura de las aguas residuales tratadasS

Araki, S.

Bécares Mantecón, Eloy

Luis Calabuig, Estanislao de, 1949-

Martín Gómez, Sebastián

Rojo Vázquez, Francisco A.

English

Leon University (Spain)

  10.1128/AEM.67.7.3322-3324.2001. 2001, 67(7):3322. DOI:Appl. Environ. Microbiol. and F. Rojo-VazquezS. Araki, S. Marti?n-Gomez, E. Bécares, E. De Luis-Calabuig  OocystsCryptosporidium parvumofEffect of High-Rate Algal Ponds on Viabilityhttp://aem.asm.org/content/67/7/3322Updated information and services can be found at: These include:REFERENCEShttp://aem.asm.org/content/67/7/3322#ref-list-1This article cites 16 articles, 5 of which can be accessed free at:CONTENT ALERTS more»articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when newhttp://journals.asm.org/site/misc/reprints.xhtmlInformation about commercial reprint orders: http://journals.asm.org/site/subscriptions/To subscribe to to another ASM Journal go to:  on June 30, 2014 by UNIVERSIDAD DE LEONhttp://aem.asm.org/Downloaded from  on June 30, 2014 by UNIVERSIDAD DE LEONhttp://aem.asm.org/Downloaded from APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/01/$04.0010 DOI: 10.1128/AEM.67.7.3322–3324.2001July 2001, p. 3322–3324 Vol. 67, No. 7Copyright © 2001, American Society for Microbiology. All Rights Reserved.Effect of High-Rate Algal Ponds on Viability ofCryptosporidium parvum OocystsS. ARAKI,1* S. MARTÍN-GOMEZ,2 E. BÉCARES,1 E. DE LUIS-CALABUIG,1AND F. ROJO-VAZQUEZ2Área de Ecologı́a, Instituto del Medio Ambiente,1 and Departamento de Patologı́aAnimal (Sanidad Animal),2 Universidad de León, 24071 León, SpainReceived 8 December 2000/Accepted 2 May 2001The physicochemical conditions of high-rate algal ponds were responsible for a more than 97% reduction inthe infectivity of Cryptosporidium parvum oocysts in neonatal mice. The use of semipermeable bags of celluloseshowed that pH, ammonia, and/or light seems to be a major factor for the inactivation of oocysts in wastewater,supporting the importance of alga-based systems for safer reuse of treated wastewater.Present conventional wastewater treatment is very expensivefor application in rural areas and, moreover, does not guaran-tee the inactivation of Cryptosporidium parvum oocysts fromsewage (3, 12, 16, 18). The high-rate algal pond (HRAP) is alow-cost wastewater treatment system designed to achieve twogoals: secondary wastewater treatment and algal biomass pro-duction. The HRAP is a combination of intensified oxidationponds and an algal reactor. Algae supply the oxygen demandfor bacterial degradation of organic matter, and bacteria ex-crete mineral compounds that provide the algae with nutrition.HRAPs have proved effective in removing organic matter (13)and in reducing bacterial contamination (8) and the number ofnematode eggs (1), but no data are available on their role inremoving Cryptosporidium oocysts, a subject of special interestwhen dealing with rural wastewater. We will focus this study onthe effect of the HRAP physicochemical conditions on theviability of Cryptosporidium oocysts as measured with a neona-tal mouse infectivity model.HRAP pilot plants. Two identical pilot plants fed with urbanwastewater were used for this study (ponds A and B) (Fig. 1).The average physicochemical characteristics of the ponds dur-ing the study period are shown in Table 1.C. parvum oocysts. Oocysts were obtained from the feces ofan experimentally infected lamb, purified according to the pro-cedures of Arrowood and Sterling (2), and stored at 4°C in a2.5% (wt/vol) aqueous potassium dichromate solution untiluse.Treatment of oocysts in HRAP. Regenerated cellulose semi-permeable bags with 14,000-Da porosity were used for theexperiment. These bags allowed oocysts to be in contact withthe small ions present in the water while reducing the effect ofother mortality factors such as bacterial and/or fungal contam-ination or predation. The bags were filled with 50 ml of sterilewater and 108 oocysts. Two bags were placed in each pond for3 (pond A) and 10 (pond B) days. Two other bags withoutoocysts were also placed in the ponds to test the osmoticinterchange through the semipermeable membrane, and twobags with oocysts were stored at 4°C in sterile water and usedas a control. At the end of the hydraulic retention time (HRT),the oocysts were washed and centrifuged with distilled water.The sediment was resuspended and adjusted to obtain 5 3 105oocysts/25 ml.Bioassay for viability. Two hundred fifty-seven suckling micefrom 21 individual litters (7 to 16 mice/litter) of NMRI micewere divided in four groups of 3- to 4-day-old mice, day 0postinfection (p.i.). Mice in groups A (98 mice) and B (79mice) were inoculated intragastrically with 5 3 105 oocystsrecovered from pond A and B bags, respectively. Control mice(group C, 80 mice) also received the same infection dose butwith oocysts preserved under control conditions. The other twolitters (group D) (19 mice) were used as uninoculated mice tocheck that no accidental infection occurred in the litters duringthe experiment.Half of the mice in each group were sacrificed by inhalationof ether 6 days p.i., and the other half were sacrificed 10 daysp.i. to verify that oocysts were not able to recover their infec-tivity after longer times in the intestine.The whole intestinal tract of each mouse was removed,placed in an individual glass tube with 1 ml of phosphate-buffered saline, homogenized, and diluted 1/5 in a solution of0.16% green malachite and 1% sodium dodecyl sulfate in wa-ter. The number of oocysts per milliliter was counted at 4003using a Neubauer slide.Statistics. Chi-square tests were used to analyze differencesin percentages of infectivity between mouse groups, and Mann-Whitney U tests were used to compare physicochemical valuesand infection intensities between ponds.The percentage of infectivity was calculated as the ratio ofthe number of infected mice to that of inoculated mice (seeTable 2). The number of oocysts per milliliter of intestinal tractwas used to evaluate the infection intensity, and the percentageof infection reduction was calculated as the difference betweenthe infection intensity of group C and that of group A or B,divided by the infection intensity of group C.The infectivity of the oocysts from the HRAP was muchlower than that from the control (Table 2), this differencebeing highly significant (P , 0.001). Infectivity in group C(control) was 100% after 6 and 10 days p.i., whereas in groupA (3 days HRT) it was 40 and 5% for 6 and 10 days p.i.,respectively, and only 20.5 and 12.5% in group B, respectively* Corresponding author. Mailing address: Instituto del Medio Am-biente, C/ La Serna s/n, León University, 24071 León, Spain. Phone: 34987 291568. Fax: 34 987 291501. E-mail: degma@unileon.es.3322 on June 30, 2014 by UNIVERSIDAD DE LEONhttp://aem.asm.org/Downloaded from (10 days HRT). The intensity of infection significantly declined(P , 0.001) in mice inoculated with oocysts exposed to theHRAP conditions (groups A and B).Intestines from uninoculated mice (group D) did not containany C. parvum oocysts, confirming that no accidental infectionsoccurred during the experiment. Observed differences betweenpond A and pond B infectivities were not statistically signifi-cant (P . 0.05). This was in accordance with the lack ofdifferences in the measured physicochemical conditions be-tween ponds (Table 1). It means that the different HRTs as-sayed did not produce significant differences in the water char-acteristics of the ponds, and therefore, physicochemicalcharacteristics inside the semipermeable bags were not differ-ent between pond A and pond B. Only conductivity presentedsignificantly lower values inside the bags (Table 3).The semipermeable bags used in the experiments excludedpredation, bacterial or fungal infection, and the effect of largemolecules as potential factors for oocyst inactivation. Thelower conductivity values for the bags suggest that only smallmolecules were able to diffuse through the membrane, leavingtemperature, pH, small ions (ammonia, phosphates, etc.), andlight as the main factors potentially responsible for the signif-icant reduction in oocyst infectivity.With respect to temperature, the oocysts of C. parvum canretain their viability and infectivity after freezing (6) or attemperatures above 65°C (11). In our case, the water temper-ature was very consistent during the experiment (Table 1),suggesting that temperature was not an important factor forthe inactivation of oocysts in the HRAP.Using oocysts in semipermeable containers, Robertson et al.(15) showed that both high and low pH have a significantimpact on oocyst viability. The pH in the HRAP was alwayshigher than 8, reaching 9.5 at noon on sunny days as a conse-quence of the algal activity. The pH clearly influences thehydrophobic properties of the oocysts, allowing them to auto-flocculate or to increase their adhesion to other particles (5).Light also plays a significant role in the inactivation of Cryp-tosporidium oocysts, but its efficiency depends on the light type,intensity, and length of exposure. Exposure to UV light wasresponsible for 90 to 99% of oocyst inactivation (4, 11, 14).White light also seems to have a significant effect on Crypto-sporidium infectivity, achieving 90% reduction in 48 h (10), andFIG. 1. Plant view of the HRAP pilot system: depth, 30 cm; length,235 cm; area, 1.54 m2; width, 70 cm; flow rate, 30 cm/s. HRTs were 3days for pond A and 10 days for pond B.TABLE 1. Physicochemical characteristics of HRAPduring the study periodCharacteristicaValue (m 6 SE) at 3 daysPond A (n 5 3) Pond B (n 5 10)Ta (°C) 5.23 6 0.35 4.78 6 0.13pH 8.70 6 0.20 8.85 6 0.04DO (ppm) 14.5 6 0.55 14.83 6 0.21Cond. (mS/cm) 323.50 6 2.23 335.75 6 0.55CODd (mg of O/liter) 33.17 6 1.61 32.87 6 0.31N-NH4 (mg of N/liter) 3.81 6 0.54 3.91 6 0.08P-PO4 (mg of P/liter) 1.47 6 0.15 1.42 6 0.05a Ta, ambient temperature; DO, dissolved oxygen; Cond., conductivity; CODd,dissolved organic matter.TABLE 2. Infectivity in neonatal mice inoculated with 5 3 105C. parvum oocysts after treatment in an HRAP at an HRT of3 days (group A) and 10 days (group B)Time p.i.and mousegroupa% Infectivity (no. ofmice infected/no. ofmice inoculated)Intensity of infection(105 oocysts/ml,m 6 SE)% Reductionof infection6 daysA 40.0 (20/50)***b 0.31 6 0.12*** 97.1B 20.5 (8/39)*** 0.21 6 0.13*** 98.0C 100.0 (40/40) 10.56 6 1.28 0D 0 (0/9)10 daysA 5.0 (2/48)*** 0.01 6 0.01*** 99.9B 12.5 (5/40)*** 0.11 6 0.04*** 99.9C 100.0 (40/40) 15.59 6 1.31 0D 0 (0/10)a Group C, control group infected with oocysts that had been stored in arefrigerator at 4°C; group D, uninfected mice used to check for accidentalinfection during the experiment.b ***, P , 0.001.TABLE 3. Physicochemical characteristics of the water in theHRAP and in the cellulose semipermeable bagsCharacteristicaValue for water from:Pond A (3 days) Pond B (10 days)HRAP Liquid of semi-permeable bag HRAPLiquid of semi-permeable bagpH 8.96 8.88 9.02 9.0Temp (°C) 4.9 5.1 6.6 6.8DO (ppm) 25.2 26 26.4 26.4Cond. (mS/cm) 316 123b 361 187bCODd (mg of O/liter) 31.57 31.41 30.76 30.24N-NH41 (mg ofN/liter)1.46 1.23 1.68 1.49P-PO432 (mg of P/liter) 1.08 1.05 1.45 1.38a DO, dissolved oxygen; Cond., conductivity; CODd, dissolved organic matter.b P , 0.05 for HRAP value versus control liquid value, by Mann-Whitney Utest.VOL. 67, 2001 EFFECT OF HIGH-RATE ALGAL PONDS ON C. PARVUM OOCYSTS 3323 on June 30, 2014 by UNIVERSIDAD DE LEONhttp://aem.asm.org/Downloaded from sunlight clearly inactivates oocysts in comparison with darkconditions (9). In our experiment, the semipermeable bagswere dangling in the HRAP reactor and sagging as a conse-quence of the water agitation. This means that bags wereerratically moving from the surface to several centimeters un-der the surface during the study period and that therefore lightcould have affected oocyst viability.Another potential factor influencing oocyst inactivation isammonia (7). Due to the high-pH conditions of the HRAP, theammonium-ammonia equilibrium tends toward the gas form.This dissolved ammonia could also have a significant role ininactivating oocysts.Results show that inactivation of oocysts by HRAP is higherthan that by conventional wastewater treatment systems. Alloocysts present in the effluent of activated sludge plants arestill active despite removal of more than 80% of the influentoocysts (18). Using semipermeable bags, Robertson et al. (17)showed that, after 1 week of exposure to different conventionaltreatments (activated sludge and trickling filters), none of theenvironments tested had a deleterious effect on oocyst viabilityexcept the anoxic sludge storage tank.As with nematode eggs (1), this work shows that the physi-cochemical conditions commonly present in the HRAP seemto be responsible for the inactivation of more than 97% of theC. parvum oocysts. The absence of differences between the tworetention times tested suggests that oocysts lost their infectivityin a very short time after contact with the water environment(probably less than 3 days). Our results strongly support othergeneral data on the important role of alga-based systems as avery valuable treatment process in the reuse of treated waste-water.REFERENCES1. Araki, S., J. M. González, E. Luis, and E. Bécares. 2000. Viability of nem-atode eggs in high rate algae ponds. The effect of physico-chemical condi-tions. Water Sci. Technol. 42:10–11.2. Arrowood, M. J., and C. R. Sterling. 1987. Isolation of Cryptosporidiumoocysts and sporozoites using discontinuous sucrose and isopycnic percollgradients. J. Parasitol. 13:314–319.3. Bukhari, Z., H. V. Smith, N. Sykes, S. W. Humphreys, C. A. Paton, R. W. A.Girdwood, and C. R. Fricker. 1997. Occurrence of Cryptosporidium sppoocysts and Giardia spp cysts in sewage influents and effluents from treat-ment plants in England. Water Sci. Technol. 35:385–390.4. Campbell, A. T., L. J. Robertson, M. R. Snowball, and H. V. Smith. 1995.Inactivation of oocysts of C. parvum by ultraviolet irradiation. Water Res.29:2583–2586.5. Drozd, C., and J. Schwartzbrod. 1996. Hydrophobic and electrostatic cellsurface properties of Cryptosporidium parvum. Appl. Environ. Microbiol.62:1227–1232.6. Fayer, R. 1994. Effect of high temperature on infectivity of Cryptosporidiumparvum in water. Appl. Environ. Microbiol. 60:2732–2735.7. Fayer, R., T. Graczyk, M. R. Cranfield, and J. M. Trout. 1996. Gaseousdisinfection of Cryptosporidium parvum oocysts. Appl. Environ. Microbiol.62:3908–3909.8. Garcia, M., and E. Bécares. 1997. Bacterial removal in three pilot-scalewastewater treatment systems for rural areas. Water Sci. Technol. 35:197–200.9. Huffman, D. E., T. R. Slifco, K. Salisbory, and J. B. Rose. 2000. Inactivationof bacteria, virus and Cryptosporidium by a point-of-use device using pulsedbroad spectrum white light. Water Res. 34:2491–2498.10. Johnson, D. C., C. E. Enriquez, I. J. Pepper, T. L. Davis, C. P. Gebra, andJ. B. Rose. 1997. Giardia cysts and Cryptosporidium oocysts at sewage treat-ment works in Scotland. Water Sci. Technol. 35:261–268.11. Lorenzo-Lorenzo, M. J., M. E. Ares, I. Villacorta-Martinez de Maturana,and D. Duran-Oreiro. 1993. Effect of ultraviolet disinfection of drinkingwater on the viability of C. parvum oocysts. J. Parasitol. 79:67–70.12. Madore, M. S., J. B. Rose, C. P. Gerba, M. J. Arrowood, and C. R. Sterling.1987. Occurrence of Cryptosporidium sp. oocysts in sewage effluents andselected surface waters. J. Parasitol. 73:702–705.13. Oswald, W. J. 1988. Large scale culture systems. Engineering aspects, p.357–392. In L. Borowitzka (ed.), Microalgal biotechnology. Cambridge Uni-versity Press, Cambridge, United Kingdom.14. Ransome, M. E., T. N. Whitmore, and E. G. Carrington. 1993. Effect ofdisinfectants on the viability of Cryptosporidium parvum. Water Supply 11:75–89.15. Robertson, L. J., A. T. Campbell, and H. V. Smith. 1992. Survival of Cryp-tosporidium parvum oocysts under various environmental pressures. Appl.Environ. Microbiol. 58:3494–3500.16. Robertson, L. J., H. V. Smith, and J. E. Ongerth. 1994. Cryptosporidium andcryptosporidiosis. Part III: development of water treatment technologies toremove and inactivate oocysts. Microbiol. Eur. 2:18–26.17. Robertson, L. J., C. A. Paton, A. T. Campbell, P. G. Smith, M. H. Jackson,R. A. Gilmour, S. E. Black, D. A. Stevenson, and H. V. Smith. 2000. Giardiacysts and Cryptosporidium oocysts at sewage treatment in Scotland. WaterRes. 34:2310–2322.18. Villacorta-Martı́nez de Maturana, I., M. E. Ares-Mazás, D. Durán-Oreiro,and M. J. Lorenzo-Lorenzo. 1992. Efficacy of activated sludge in removingCryptosporidium parvum oocysts from sewage. Appl. Environ. Microbiol.58:3514–3516.3324 ARAKI ET AL. APPL. ENVIRON. MICROBIOL. on June 30, 2014 by UNIVERSIDAD DE LEONhttp://aem.asm.org/Downloaded from 

Effect of High-Rate Algal Ponds on Viability of Cryptosporidium parvum Oocysts

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Effect of High-Rate Algal Ponds on Viability of Cryptosporidium parvum Oocysts

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