Dynamics of dsdna Viruses Hosted by Indigenous Microorganisms in Activated Sludge in the Treatment of Synthetic Wastewater

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1 Dynamics of dsdna Viruses Hosted by Indigenous Microorganisms in Activated Sludge in the Treatment of Synthetic Wastewater He YANG, Hiroyasu SATOH, Takashi MINO Department of Socio-Cultural Environmental Studies, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwanoha, Kashiwa, Chiba , Japan ABSTRACT Viruses hosted by microorganisms in activated sludge are of particular interest for researchers because they may affect the performance of wastewater treatment. In this study, the authors investigated at which condition of wastewater treatment viruses, especially double stranded DNA (dsdna) viruses, were released from activated sludge to the supernatant. To determine the concentrations of viruses, supernatant samples were filtered through membrane filter, viral capsid was digested by treatment with proteinase K, and the dsdna concentration was determined. The authors confirmed that most of dsdna in the filtered supernatant cannot be digested by DNase I, and thus treatment with DNase I was omitted. The behavior of dsdna viruses was monitored in twelve selected cycles from three laboratory-scale sequencing batch activated sludge reactors with sequencing anaerobic and aerobic conditions. The concentrations of dsdna viruses increased at different timings of treatment: during anaerobic phase, aerobic phase, and settling phase. The rate of increase was varied, sometimes rapid while other times gradual. Decrease of dsdna concentration was also observed. The behavior of dsdna viruses in activated sludge during wastewater treatment was found to be very diverse and complex. Keywords: activated sludge microorganisms, viral dynamics, wastewater treatment INTRODUCTION Viruses are ubiquitous and important components in natural bodies of water (Paul, 1999; Wommack and Colwell, 2000; Suttle, 2005). Virus-mediated disturbances of microbial communities have a significant effect on aquatic ecosystems (Wilhelm and Suttle, 1999). Lysis resulting from viral infections controls the sizes of specific host populations, and also promotes carbon and nutrient cycles in aquatic ecosystems (Fuhman, 1999; Wommack and Colwell, 2000). Viruses can also affect microbial communities through horizontal gene transfers (Paul, 1999; Miller, 2004; Breitbart and Rohwer, 2005; McDaniel et al., 2010). The activated sludge process is the most widely used method to treat household and industrial wastewaters. The performance of the process is dependent on the activity of microorganisms, including bacteria. The existence of viruses hosted by indigenous microorganisms in activated sludge has been reported (Ewert and Paynter, 1980; Ogata et al., 1980; Rabinovitch et al., 2003; Otawa et al., 2007; Wu and Liu, 2009). Barr et al. (2010) reported a decline of biological phosphorus removal in a laboratory-scale activated sludge reactor, which was caused by bacteriophages. On the other hand, there are studies to employ bacteriophages to control filamentous bulking and foaming in the activated sludge process (Kotay et al., 2011). Address correspondence to He Yang, Department of Socio-Cultural Environmental Studies, The University of Tokyo, yanghe @yahoo.co.jp Received May 31, 2013, Accepted October 31,

2 Wastewater treatment by the activated sludge process usually goes as follows: wastewater and return activated sludge are mixed, pollutants in wastewater are removed by microorganisms in activated sludge by reactions under different electron acceptor conditions, and finally, the effluent and activated sludge microorganisms are separated typically by gravimetric settling in a clarifier. The electron acceptor conditions employed for the reaction depend on the target of treatment: for example, in the case of the enhanced biological phosphorus removal (EBPR) process for the removal of organic pollutants and phosphorus, reactions are performed with sequencing anaerobic and aerobic phases. That is, wastewater treatment by activated sludge process involves different conditions. It is of interest at which timing of wastewater treatment are viruses released from activated sludge microorganisms to the supernatant. As far as the authors are concerned, at this moment, no study has been done on the detailed behavior of viruses in the course of wastewater treatment by activated sludge. In the present study, the authors attempted to clarify the detailed behavior of viruses in the course of wastewater treatment especially focusing on double stranded DNA (dsdna) viruses which are released from activated sludge to the supernatant. Three laboratory-scale sequencing batch activated sludge reactors (SBRs) fed with synthetic wastewater were operated with sequencing anaerobic-aerobic conditions, and the behavior of the viral dsdna in supernatant in the twelve selected cycles were monitored. Total viral dsdna that the authors monitored included free dsdna, which is degradable by DNase I. A preliminary experiment showed that the amount of free DNA was small in comparison to dsdna protected from degradation by DNase I, and at a negligible level. MATERIALS AND METHODS Laboratory-scale activated sludge processes Four laboratory-scale SBRs (Reactors X, A, B, and C) were operated. Reactor X was used to examine the level of free dsdna concentrations in the supernatant, and Reactors A, B, and C were used for studying the behavior of dsdna viruses in the course of wastewater treatment. All the four SBRs were operated in the same way. Each four-hour SBR cycle consisted of the feeding of synthetic wastewater followed by a one-hour anaerobic phase, a two-hour aerobic phase, 55-minute settling, and five-minute effluent discharge. During anaerobic and aerobic phases, the content of the reactor was mixed with a stirrer. The composition of the synthetic wastewater was CH 3 COONa 3H 2 O (113 mg), CH 3 CH 2 COONa (53.6 mg), peptone (100 mg), yeast extract (20 mg), K 2 HPO 4 (36 mg), CaCl 2 2H 2 O (13.2 mg), MgSO 4 7H 2 O (110 mg), and KCl (42 mg) for every liter of tap water. The concentration of COD was 250 mgc/l, and the COD:N:P ratio was 39:0:1. Studies with similar synthetic wastewater have been published by the authors group (Li et al., 2012; Satoh et al., 2013). The SBRs had a working volume of 10 L, and in each cycle, 5 L of effluent was discharged and the same volume of influent was supplied to the reactor. Thus, the hydraulic retention time (HRT) was eight hours. In each cycle, 240 ml of sludge mixed liquor was removed during the anaerobic and aerobic phases to maintain the sludge retention time (SRT) to be at around 7 days

3 Reactors X, B, and C were seeded with activated sludge from full-scale wastewater treatment plants treating domestic sewage, while Reactors A was seeded with activated sludge from other laboratory SBR which had been operating for more than 6 months. Mixed liquor suspended solids (MLSS) of these reactors were measured at the end of the aerobic phase according to Standard Methods (Eaton et al., 2005). The following cycles were monitored for virus behaviors: six cycles for Reactor A (A 112, A 113, A 114, A 116, A 118, and A 131 ), three cycles for Reactor B (B 18, B 19, and B 21 ), and three cycles for Reactor C (C 2, C 3, and C 7 ) (Note: subscripts for Reactors A, B and C indicate the number of days after the start of each reactor operation). Sampling periods in each cycle were as follows: 12, 32, 52, 72, 92, 112, 132, 152, 172 min from Reactors A and B, and 8, 20, 40, 59, 80, 100, 120, 140, 160, 179 min from Reactor C. Whole effluent from each reactor was collected to a bucket, mixed, and then the effluent sample was collected from it. Viral dsdna concentration profiles at the end of settling were examined on the 126 th day and the 131 st day in Reactor A. Supernatant samples were taken at different depths, with the purpose of knowing the vertical profile of viral dsdna concentrations. Quantification of viral dsdna concentrations The concentrations of viral dsdna were determined according to the method developed by Otawa et al. (2008) with a modification. The original procedure by Otawa et al. (2008) is briefly as follows activated sludge mixed liquor is centrifuged, the supernatant is filtered through a 0.2 μm membrane filter (Advantec, Tokyo, Japan), the filtrate is treated with DNase I (20 μg/ml, Funakoshi, Tokyo, Japan) for 30 min at 37 C, viral DNA is extracted from viral capsid by heating at 65 C for 60 min in the presence of 5 μg/ml proteinase K (MP Biomedicals LLC, USA) and 20 mm EDTA (Wako, Osaka, Japan), and finally, the concentrations of dsdna are determined using a Quant-iT PicoGreen dsdna reagent and kit (Invitrogen, USA) according to the manufacturer s instructions. The concentrations of dsdna including viral dsdna in the samples from Reactors A, B, and C were determined by this method without treatment with DNase I. The error of this method in dsdna concentration was estimated to be less than 5% through determining dsdna concentration in the same treated water 24 times. In order to know the contributions of free dsdna in the filtered supernatant of activated sludge, 22 supernatant samples from Reactor X were collected once every one or two days, and analyzed in a way as follows: to a sample of 210 μl, 2.1 μl of DNase I solution (2,000 μg/ml) or Milli-Q water were added, incubated for 30 min at 37 C, and the DNA concentrations were determined by using Quant-iT PicoGreen. dsdna reagent and kits. The DNA concentration after incubation with the addition of MilliQ was subtracted by the DNA concentration after incubation with the addition of DNase I to obtain free dsdna concentration

4 RESULTS Free dsdna concentrations Distribution of free dsdna in 22 supernatant samples from Reactor X was investigated. Free dsdna concentrations were in the range of 0 to 1.2 μg/l, with an average of 0.55 μg/l and a standard deviation of 0.36 μg/l. Results of the cycle monitoring The temporal profiles of the concentrations of viral dsdna in the SBR cycles are as shown in Fig. 1. The concentrations of MLSS ranged between 1,500 and 3,700 mg/ L and the concentrations of viral dsdna were in the range of 15 and 205 μg/l. In cycles A 112, A 114, and A 116, viral dsdna concentrations were stable during the anaerobic and aerobic phases, but significantly higher concentrations were observed in the effluent. That is, viral dsdna concentrations increased during the settling phase. A gradual increase of viral dsdna concentrations during the aerobic phase was observed in A 118. Sharp increases at the beginning of the aerobic phase and notable decreases during the settling phase of viral dsdna concentrations were observed in B 18 and B 19. Gradual increases of viral dsdna concentrations during the anaerobic phase were observed in B 19, B 21, and C 7. In C 7, the viral dsdna concentration decreased significantly during the aerobic phase. In A 113, A 131, C 2, and C 3, viral dsdna concentrations were stable throughout each SBR cycle

5 Fig. 1 - Dynamics of viral dsdna concentrations within a single cycle of the SBR process. An: anaerobic phase; Ox: aerobic phase; S: settling phase; E: effluent; Numbers in parentheses show MLSS concentrations of the reactor on monitored date

6 Profiles of viral dsdna concentrations at the end of settling Concentration profiles of dsdna virus in Reactor A were examined on days 126 and 131 at the end of settling phase. The profile obtained on day 126 showed that viral dsdna concentrations increased linearly in relation to water depth (Fig. 2a). This contrasts with the profile obtained on day 131 in which no significant difference was noted (Fig. 2b). Fig. 2 - Vertical profiles of viral dsdna concentrations at the end of settling on days 126 th (a) and 131 st (b). Numbers in parentheses show MLSS concentration of the reactor monitored

7 DISCUSSION In the original method to determine viral dsdna concentrations developed by Otawa et al. (2008), viral dsdna concentrations were determined after treatment with DNase I. In the present study, the authors determined viral dsdna concentrations without treatment with DNase I. This means that what the authors determined as viral dsdna concentrations included not only viral dsdna, which were coated with capsid and protected from DNase I, but also free dsdna which were prone to digestion with DNase I. So, what the authors determined as viral dsdna should be referred to as total dsdna outside bacterial cells. Yet, free DNA concentrations were in the range of 0 to 1.2 μg/l which were mostly less than 10% of viral dsdna concentrations shown in Fig. 1 (15 and 205 μg/l) that were determined without treatment with DNase I. Based on this observation, the authors refer to total dsdna outside bacterial cells as viral dsdna. As shown in Fig. 1, dsdna virus concentrations increased during different phases: anaerobic, aerobic, and settling. The rate of increase was sometimes sharp and sometimes gradual. A significant increase in viral dsdna concentration was observed during settling and at the beginning of aerobic phases in cycles C 18 and C 19. The increase of viral dsdna concentrations during settling was confirmed by the results shown in Fig. 2a. That is, in the profile of viral dsdna concentrations on the 126 th day (Fig. 2a), the concentration of viral dsdna was higher at deeper positions in the reactor vessel. The observed profile suggests that dsdna viruses were released from activated sludge biomass to the supernatant during settling. Yet, as can be seen in Fig. 2(b), the liberation of viral particles during settling did not always occur. The authors did not notice any significant differences in the characteristics of sludge between 126 th day and 131 st day. In the present study, not only increases but also decreases in viral dsdna concentrations were observed. The possible causes for the decrease are a rapid decay of viral particles (Rabinovitch et al., 2003) and adsorption to activated sludge flocs (Wellings et al., 1976). It is generally apparent that the behavior of viral dsdna concentrations was very diverse. The observation of the viral dsdna concentrations is thought to directly reflect the behavior of dsdna viruses especially bacteriophages. They were released from host cells at different stages of synthetic wastewater treatment. At present, this is the first study to report the behavior of viruses indigenous to activated sludge microorganisms in the course of wastewater treatment. Ewert and Paynter (1980) compared total concentrations of viruses in activated sludge mixed liquor and sewage influents and effluents; they observed a net production of viruses within the reactor. Otawa et al. (2007) observed the existence of significant concentrations of viruses in the effluent from an activated sludge reactor used for treating synthetic wastewater. Wu and Liu (2009) reported a high variety of virus morphotypes in sludge and a diverse and dynamic viral community in different stages of the system was observed. These studies could only be used as indicators of virus abundance during particular stages in the wastewater treatment process; they did not provide any information on the timing of increased virus concentrations

8 There have been very few studies on the detailed temporal behavior of viruses in aquatic environments. Bratbak et al. (1996) reported on the behavior of viruses within two hours, at around 10-min interval in coastal seawater. They did not apply any change to the conditions of the microbial system that they studied; nevertheless, they observed sudden and unexpected changes in virus concentrations. They did not discuss the possible mechanisms behind the phenomena. The present study clearly showed that viral particles can be liberated during the aerobic phase as well as during the anaerobic and settling phases. In general, viruses are dependent on energy and nutrients provided by their hosts. In other words, viruses and their hosts are in competition for energy and nutrients inside the host cells. Their competition may be under a subtle balance, and slight change in environmental conditions, such as shift from anaerobic condition to aerobic condition and from aerobic condition to settling, might affect the balance of competition. Yet, under anaerobic and settling conditions, oxygen is less available and host cells are thought to have more difficulty to obtain energy. There could mainly be two possible explanations for the release of viruses under anaerobic and settling phases. One of the explanations is that viruses perform reproductive activities mainly during aerobic conditions, and finally lyse host cells during anaerobic or settling phases with minimal amount of energy available. In the present study, the SRT was 7 days, and during the 7 days, microorganisms experienced 42 cycles during their life in the reactor. Viruses hosted by these microorganisms are thought to have had the chance to actively reproduce progeny viral elements in the host cells in aerobic phases in plural cycles. Another possible explanation is that certain group of microorganisms in activated sludge can utilize energy even under anaerobic conditions by utilizing stored energy source. The so-called polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) (Mino et al., 1998) are able to store energy in such forms as polyphosphate and glycogen, and can utilize the stored energy under anaerobic conditions (Satoh et al., 1994). The viruses hosted by these microorganisms can utilize polyphosphate or glycogen as the energy source under anaerobic conditions, if they can utilize the host s energy system. And it may also be possible to selectively study viruses on PAOs and GAOs by focusing on viruses that are released to the supernatant during the anaerobic phase. Unfortunately in the present study, the other parameters such as removal of carbon and phosphorus were not studied. In addition, only viruses with dsdna were focused on in this study, though there are single stranded DNA viruses and RNA viruses. In the future, activities of viruses with different nucleic acid types would be worth studying with treatment performances. CONCLUSIONS The behavior of dsdna viruses in activated sludge during the course of synthetic wastewater treatment was thus found to be very diverse and complicated. (1) An increase in dsdna viruses was observed during the aerobic phase and in the anaerobic and settling phases; (2) both steep and gradual increases were observed; (3) a decrease

9 in viruses was also observed; and (4) dsdna viruses were less active during the initial stages of reactor operation (Reactor C). These findings represent a first attempt to track the behavior over time associated with virus release during the wastewater treatment process. It should, however, be noted that only the behavior of dsdna viruses were investigated. Although the timing of virus release was known, the preceding processes (infection, induction, and progeny biosynthesis) were not known. We expect that similar findings will be generated from studies on various wastewater treatment processes. ACKNOWLEDGEMENTS This work was financially supported by Grant-in-Aid for Scientific Research (B) ( ). The authors would also like to extend their sincere appreciation to Tao SU, Wei SHI, Yang OU, and Yuki SATO for SBR operation; to Tiffany Chua and Therese Chua for their English check. REFERENCES Barr J. J., Slater F. R., Fukushima T. and Bond P. L. (2010) Evidence for bacteriophage activity causing community and performance changes in a phosphorus-removal activated sludge. FEMS Microbiol. Ecol., 74(3), Bratbak G., Heldal M., Thingstad T. F. and Tuomi P. (1996) Dynamics of virus abundance in coastal seawater. FEMS Microbiol. Ecol., 19(4), Breitbart M. and Rohwer F. (2005) Here a virus, there a virus, everywhere the same virus? Trends Microbiol., 13(6), Eaton A. D., Lenore L. S., Eice E. W. and Greenberg A. E. (2005) Standard methods for the examination of water and wastewater, 21st ed. American Public Health Association, American Water Works Association, Water Environment Federation. Washington, DC, USA. Ewert D. L. and Paynter M. J. B. (1980) Enumeration of bacteriophages and host bacteria in sewage and the activated-sludge treatment process. Appl. Environ. Microbiol., 39(3), Fuhman J. A. (1999) Marine viruses and their biogeochemical and ecological effects. Nature, 399, Kotay S. M., Datta T., Choi J. and Goel R. (2011) Biocontrol of biomass bulking caused by Haliscomenobacter hydrossis using a newly isolated lytic bacteriophage. Water Res., 45(2), Li N., Satoh H. and Mino T. (2012) Dynamics of dewaterability and bacterial populations in activated sludge. Water Sci. Technol., 66(8), McDaniel L. D., Young E., Delaney J., Ruhnau F., Ritchie K. B. and Paul J. H. (2010) High frequency of horizontal gene transfer in the oceans. Science, 330(6000), 50. Miller R. V. (2004) Bacteriophage-mediated transduction: an engine for change and evolution. In: Microbial Evolution: Gene establishment, survival, and exchange. Miller R. V. and Martin J. D. (ed.), ASM Press, Washington DC, USA, pp Mino T., van Loosdrecht M. C. M. and Heijnen J. J. (1998) Microbiology and biochemistry of the enhanced biological phosphate removal process. Water Res., 32(11), Ogata S., Miyamoto H. and Hayashida S. (1980) An investigation of the influence of bacteriophages in the bacterial flora and purification powers of activated sludge. J

10 Gen. Appl. Microbiol., 26, Otawa K., Lee S. H., Yamazoe A., Onuki M., Satoh H. and Mino T. (2007) Abundance, diversity, and dynamics of viruses on microorganisms in activated sludge processes. Microb. Ecol., 53(1), Otawa K., Satoh H., Kanai Y., Onuki M. and Mino T. (2008) Rapid quantification of total viral DNA in the supernatants of activated sludge samples with the fluorescent dye PicoGreen. Lett. Appl. Microbiol., 46(4), Paul J. H. (1999) Microbial gene transfer: an ecological perspective. J. Mol. Microbiol. Biotechnol., 1(1), Rabinovitch A., Aviram I. and Zaritsky A. (2003) Bacterial debris an ecological mechanism for coexistence of bacteria and their viruses. J. Theor. Biol., 224(3), Satoh H., Mino T. and Matsuo T. (1994) Deterioration of enhanced biological phosphorus removal by the dominance of microorganisms without polyphosphate accumulation. Water Sci. Technol., 30(6), Satoh H., Oshima K., Suda W., Ranasinghe P., Li N., Gunawardana E. G. W., Hattori M. and Mino T. (2013) Bacterial Population Dynamics in a Laboratory Activated Sludge Reactor Monitored by Pyrosequencing of 16S rrna. Microbes. Environ., 28, Suttle C. A. (2005) Viruses in the sea. Nature, 437, Wellings S. R., Alpers C. E., McCain B. B. and Miller B. S. (1976) Fin erosion disease of starry flounder (Platichthys stellatus) and English sole (Parophys vetulus) in the estuary of the Duwamish River, Seattle, Washington. J. Fish. Res. Board Can., 33 (11), Wilhelm S. W. and Suttle C. A. (1999) Viruses and nutrient cycles in the sea. BioScience, 49(10), Wommack K. E. and Colwell R. R. (2000) Virioplankton: viruses in aquatic ecosystems. Microbiol. Mol. Biol. Rev., 64(1), Wu Q. and Liu W. T. (2009) Determination of virus abundance, diversity and distribution in a municipal wastewater treatment plant. Water Res., 43(4),