Viruses in Wastewater Utilities: A Review Ziqiang Yin, Zhassulan Svambayev , Irene Xagoraraki

Abstract Viruses are the most abundant microorganisms on the earth, and ingestion of waterborne

viruses may cause various diseases. In conventional wastewater treatment plants, adsorption is

considered as the major mechanism to remove viruses in the activated sludge. Comparatively,

rejection by the gel layer and cake layer of bio-film, and adsorption on the surface of

membranes and bio-particles have been suggested as the main mechanisms of virus removal in

membrane bioreactors (MBR). In general, MBRs appear to have better performance in removal

of viruses than conventional wastewater treatment process. Data from our research group shows

that full-scale conventional activated sludge could only remove 2.2 logs of adenoviruses in

average; combined with chlorination, the removal efficiency could be enhanced by less than 1

log. In MBRs, the removal efficiencies for adenoviruses are usually greater than 4.0 logs, and

our research group has reported that the efficiency could reach up to 6.3 logs. Complete removal

of virus in either conventional or MBR wastewater treatment plant has not been achieved yet.

Our work has shown infectious viruses may be found in the treated effluents and biosolids,

which are released to the environment. Surface properties of viruses, such as size, isoelectric

point (IEP), and zeta potential, have been suggested as key factors that affected virus adsorption

and rejection in wastewater utilities. A beach-scale MBR system is currently under construction

in our lab and the objectives of this project are to identify the role of bio-film, soluble microbial

product and extracellular polymeric substances in virus removal.

Virus Detection Methods in Wastewater

Viruses as Waterborne Pathogens Comparison of Virus Removal in Full Scale Traditional WWTP and MBRs

Virus Removal in Bench and Pilot Scale MBRs

Sources of Human Viruses Associated with Wastewater

Figure 4 - Summary of virus removal efficiencies in

bench and pilot MBRs

As shown in Figure 4, MBRs can

achieve high removal of viruses.

For example, up to 8 logs of T4

phage and 7.3 logs of MS-2

coliphage could be removed by

bench scale MBRs. Membrane pore

size has been suggested as a key

factor for virus removal, and

membranes with small pore sizes

usually lead to high removal for

viruses. However, small pore size

can’t guarantee high virus removal.

Membranes with small pore size

are usually expensive and

energy-consuming during operation.

Table 1 - Virus in the EPA Contamination Candidate List

Virus Associated Diseases CCL 1 CCL 2 CCL 3

Enteroviruses Hand, foot and mouth disease; Gastroenteritis;

Heart anomalies; meningitis Yes

Coxsackieviruses Hand, foot and mouth disease; Myocarditis;

Pericarditis; Meningitis; Pancreatitis

Yes Yes

Echoviruses Yes Yes

Hepatitis A viruses Hepatitis A Yes

Caliciviruses Gastroenteritis Yes Yes Yes

Adenoviruses Gastroenteritis; Conjunctivitis; Respiratory

diseases Yes Yes Yes

Figure 3 - Summary of virus removal efficiencies in full scale WWTPs: (a) adenoviruses; (b) enteroviruses; (c) noroviruses

As shown in Figure 3, activated sludge without

disinfection can reduce adenoviruses and enteroviruses

by up to 4.1 logs and 3.9 logs, respectively. In contrast,

virus removal efficiency is generally higher in MBR

systems. For instance, data from our group showed that

human adenoviruses were inactivated from 4.1 to 6.3

logs in an MBR, while the removal efficiency for

enteroviruses was from 4.1 to 6.8 logs. Norovirus II

showed relatively less reduction in MBRs, which

ranged from 3.5 to 4.8 logs. Even though the removal

efficiencies are higher in MBR systems, the effluents

are not free of infectious viruses. Our work has

demonstrated that infectious enteric viruses in the final

effluent and biosolids of wastewater treatment plants

can be released in the environment .

Viruses in Sludge (Biosolids)

Virus(1) Genetic

Type

Virion Size

(nm)

Isoelectric

Point Envelope Structure(2) References

Enterovirus

ssRNA 22-30

4.0 - 6.4

Non-enveloped [15] – [19] Coxsackieviruses 4.75 - 6.75

Echoviruses 4.0 - 6.4

Hepatitis A viruses ssRNA 27-28 2.8 Non-enveloped [15], [20]

Caliciviruses ssRNA 30-40 5.5 - 6.0(3) Non-enveloped [21], [22]

Adenoviruses dsDNA 70-140 3.5 - 4.5 Non-enveloped [23] – [26]

Table 2 – Characteristics of viruses in Contamination Candidate List

(1) All types of viruses in CCL are Icosahedral in shape.

(2) In general, viruses with a lipid envelope are hydrophobic and viruses without a lipid

envelope are hydrophilic.

(3) For Norwalk virus (a member of norovirus).

References [1] Aulicino, F. A., Mastrantonio, A., Orsini, P., Bellucci, C., Muscillo, M., Larosa, G., 1996. Enteric viruses in a wastewater treatment plant in Rome. Water, Air, and Soil Pollution, Volume 91, Numbers 3-4, 327-334, DOI: 10.1007/BF00666267.

[2] Costán-Longares, A., Mocé-Llivina, L., Avellón, A., Jofre, J., Lucena, F., 2008. Occurrence and distribution of culturable enteroviruses in wastewater and surface waters of north-eastern Spain. Journal of Applied Microbiology. Volume 105, Issue 6, pages 1945–1955.

[3] Da Silva, A. K., Le Saux, J-C., Parnaudeau, S., Pommepuy, M., Elimelech, M., Le Guyader, F. S. Evaluation of removal of noroviruses during wastewater treatment, using real-time reverse transcription-PCR: different behaviors of genogroups I and II. Applied and

Environmental Microbiology, Vol. 73, No. 24, Dec. 2007, pp. 7891–7897

[4] Haramoto, E., Katayama, H., Oguma, K., Ohgaki, S., 2007a. Quantitative analysis of human enteric adenoviruses in aquatic environments. Journal of Applied Microbiology, Volume 103, Issue 6, pages 2153–2159.

[5] Hewitt, J., Leonard, M., Greening, G. E., Lewis, G. D., 2011. Influence of wastewater treatment process and the population size on human virus profiles in wastewater. Water Research, Volume 45, Issues 18, Pages 6267–6276.

[6] Katayama, H., Haramoto, E., Oguma, K., Yamashita, H., Tajima, A., Nakajima, H., Ohgaki, S., 2008. One-year monthly quantitative survey of noroviruses, enteroviruses, and adenoviruses in wastewater collected from six plants in Japan. Water Research, Volume 42,

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[7] Kuo, D. H. W., Simmons, F. J., Blair, S., Hart, E., Rose, J. B., Xagoraraki, I., 2010. Assessment of human adenovirus removal in a full-scale membrane bioreactor treating municipal wastewater. Water Research 44 (2010), pp. 1520-1530.

[8] Lodder, W. J., deRoda Husman, A. M., 2005. Presence of Noroviruses and Other Enteric Viruses in Sewage and Surface Waters in the Netherlands. Appl. Environ. Microbiol. vol. 71, no. 3 1453-1461.

[9] Nordgren, J., Matussek, A., Mattsson, A ., Svensson, L., Lindgren, P., 2009. Prevalence of norovirus and factors influencing virus concentrations during one year in a full-scale wastewater treatment plant. Water Research, Volume 43, Issue 4, Pages 1117–1125.

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[18] Grce, M., Pavelic, K., 2004. Antiviral properties of clinoptilolite. Microporous and Mesoporous Materials 79 (2005) 165–169.

[19] Zerda, K.S. and Gerba, C.P., 1984. Agarose isoelectrofocusing of intact virions. J Virol Methods 9, 1–6.

[20] Nasser, A.M., Battagelli, D. and Sobsey, M.D. (1992) Isoelectric focusing of hepatitis A virus in sucrose gradients. Isr. J. Med. Sci. 28 , 73.

[21] Carter, M. J. and Madeley, C. R., in Animal virus structure, Chapter 7 Caliciviridae, edited by Nermut, M. V. and Steven, A. C. Perspectives in Medical Virology, Vol. 3. Elsevier Science Publishers, Biomedical Division, 1987.

[22] Goodridge, L., Goodridge, C., Wu, J.Q., Griffiths, M. and Pawliszyn, J. (2004) Isoelectric point determination of norovirus virus-like particles by capillary Isoelectric focusing with whole column imaging detection. Anal Chem. 76, 48–52.

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[27] Wong, K., Onan, B. M., Xagoraraki, I., 2010. Quantification of Enteric Viruses, Pathogen Indicators, and Salmonella Bacteria in Class B Anaerobically Digested Biosolids by Culture and Molecular Methods. Applied and Environmental Microbiology, Vol. 76, No. 19, p.

6441-6448

[28] Wong, K., Harrigan, T., Xagoraraki, I., 2012. Leaching and ponding of viral contaminants following land application of biosolids on sandy-loam soil. Journal of Environmental Management 112 (2012) 79 e86.

(a) (b) (c)

Figure 1 – Sources of human viruses associated with wastewater Figure 2 – Summary of virus detection methods

Additionally, it has been suggested membrane attached biofilm is an important factor that

enhances virus removal. It has been reported that clean membranes without bio-film could barely

remove viruses. However, growth of bio-film will cause membrane fouling, which lead to decrease

of flux and increase of trans-membrane pressure. Adsorption on the surface of membranes and bio-

particles has suggested as the main mechanisms of virus removal in MBRs. Figure 5 – Level of viruses in mesophilic anaerobic

digestion (MAD) and dewatered biosolids [27]

Sludge is the solid waste byproduct of the municipal wastewater treatment plants and treated

sludge is called biosolids. There are two classes of biosolids. Class A biosolids are sold directly

to the public for lawn and garden use and should not contain any detectable concentrations of

pathogens. Class B biosolids are applied on agriculture and forest lands as fertilizers. Air, soil,

water, and animal vectors (such as flies) were suggested as potential transmission pathways of

human pathogens from biosolids. Currently, monitoring the occurrence of enteroviruses in

biosolids is suggested by the EPA.

Previous studies from our group

indicated human adenoviruses had

higher levels and frequencies than

other enteric viruses in biosolids, as

shown in Figure 5. Data from our

group also suggested sandy-loam

soils can effectively remove/adsorb

the indigenous viruses leached from

the land-applied biosolids, but there

is a potential of viral pollution from

runoff following significant rainfall

events when biosolids remain on

the soil surface.

4.1-5.6

0.26-3.04 0.6-3.2

1.37-2.83

1.02-4.08

4.1-6.3

3.4-4.5

0

1

2

3

4

5

6

7

Log re

moval

References

Adenovirus

4 5 4 6 7 12 13

0.1-2.3 0.7-1.8 0.51-2 1.9

5

1 - 3.9

-0.9 - 5.95

3.2

4 4.2

5.23

4.1-6.8

3.2-4.3

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4

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6

7

8

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g, R

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References

Enterovirus

8 2 5 1 10 6 2 2 2 11 2 12 13

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1.2-1.6

0.57-3.07

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1-1.4

0.71-3.21

2.3-4.9 3.5-4.8

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3

4

5

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emoval

References

Norovirus I and Norovirus II

Norovirus I Norovirus II

5 9 6 3 5 9 6 3 12