key: cord-275565-xerr4vki
authors: Kumar, Manish; Kuroda, Keisuke; Patel, Arbind Kumar; Patel, Nidhi; Bhattacharya, Prosun; Joshi, Madhvi; Joshi, Chaitanya G.
title: Decay of SARS-CoV-2 RNA along the wastewater treatment outfitted with Upflow Anaerobic Sludge Blanket (UASB) system evaluated through two sample concentration techniques
date: 2020-09-15
journal: Sci Total Environ
DOI: 10.1016/j.scitotenv.2020.142329
sha: 
doc_id: 275565
cord_uid: xerr4vki

For the first time, we present, i) an account of decay in the genetic material loading of SARS-CoV-2 during Upflow Anaerobic Sludge Blanket (UASB) treatment of wastewater, and ii) comparative evaluation of polyethylene glycol (PEG), and filtration as virus concentration methods from wastewater for the quantification of SARS-CoV-2 genes. The objectives were achieved through tracking of SARS-CoV-2 genetic loadings i.e. ORF1ab, N and S protein genes on 8th and 27th May 2020 along the wastewater treatment plant (106 million liters per day) equipped with UASB system in Ahmedabad, India. PEG method performed better in removing materials inhibiting RT-qPCR for SARS-CoV-2 gene detection from the samples, as evident from constant and lower CT values of control (MS2). Using the PEG method, we found a reduction >1.3 log10 in SARS-CoV-2 RNA abundance during UASB treatment, and the RNA was not detected at all in the final effluent. The study implies that i) conventional wastewater treatment systems is effective in SARS-CoV-2 RNA removal, and ii) UASB system significantly reduces SARS-CoV-2 genetic loadings. Finally, PEG method is recommended for better sensitivity and inhibition removal during SARS-CoV-2 RNA quantification in wastewater.

Wastewater-based epidemiology (WBE) has already proved its capability as a tool of environmental surveillance of epidemic and pandemic in a given community through viral load detection in the wastewater, shredded both from symptomatic and asymptomatic COVID-19 patients (Bivnis et. al., 2020; Xagoraraki and O'Brien, 2020; Choi et al. 2018; Yang et al., 2015; Hellmér et al., 2014; Asghar et al., 2014; Ahmed et al., 2020a , Hata et al., 2020 Wölfel et al., 2020a; Zhang et al., 2020; Randazzo et al 2020) . The second quarter of 2020 has been exceptional in discovering several new knowledge pertaining to SARS-CoV-2 genetic material loading, its analytical methods, and various strong implications pouring around the world (Ahmed et al., 2020a; Bar-or et al., 2020; Haramoto et al., 2020; La Rosa et al., 2020; Medema et al., 2020; Nemudryi et al., 2020; Randazzo et al., 2020a; Rimoldi et al., 2020; Wurtzer et al., 2020a; Wurtzer et al., 2020b Kumar et al., 2020a . While most of the studies could explicitly prove the correlation of SARS-CoV-2 genetic loading with the covid-19 patients in the area, some compared the methods to improve SRS-CoV-2 RNA extraction (Ahmed et al., 2020b) and some studies traced back the SARS-CoV-2 genes in the wastewater long before any COVID-19 patient were declared, implying high early warning capability of WBE (Randazzo et al 2020a , Prevost,et al 2015 , Lodder and Husman 2020 , Prussin et al 2020 , Ahmed et al., 2020c , Sherchana et al., 2020 . However, the great majority of the existing studies are based on analysis of raw wastewater only, or just a comparison of influent and final treated effluent samples from wastewater treatment plants (WWTPs).

Thus, there still remains questions pertaining to: i) capability of conventional WWTPs to reduce the abundance of SARS-CoV-2 RNA, ii) better understanding of the protocol, virus J o u r n a l P r e -p r o o f Journal Pre-proof precipitation through PEG and filtration which one is better methods for concentrating the samples before RNA isolation. Further, while in WBE surveillance is being accelerated in India upon phasing out of lockdown, several questions are raised around its capability owing to incomplete sewer systems, significant wastewater leak, high ambient temperature, open defecation, strong seasonality component, and common sewer overflow (CSO) situations in India. Overall, there are several apprehensions about infectivity through genetic material present in the wastewater become a pertinent question. These all warrants a study that can track the genetic loading after each wastewater treatment stage in Indian settings and highlight the effects of wastewater treatment on RNA decay of the corona virus. Such study will help curing the commonly perceived fear of the commons pertaining to the effectiveness of WWTPs. Further, the number of confirmed cases in India has passed 0.5 million as of the last day of June, 2020 (Ministry of Health and Family Welfare, India) with >16000 official casualties. In Gujarat Province, which is a hotspot and our study site, the confirmed cases will soon reach to 25,000 in Ahmedabad city. (Ministry of Health and Family Welfare, India) indicating the need of immediate attention.

At this juncture keeping the pulse of WBE progression as mentioned above, we focused on three major objectives i.e.: i) Tracking the conventional treatment system for genetic loading decay of SARS-CoV-2 along the treatment process and evaluate its effectiveness, ii)

Appraising the genetic loading reduction through Upflow Anaerobic Sludge Blanket (UASB) systems, and iii) Comparing the performances between PEG and filtration as virus concentration methods in terms of SARS-CoV-2 RNA sensitivity and inhibition removal. We thus hereby present the first ever data pertaining to UASB performances in SARS-CoV-2 RNA J o u r n a l P r e -p r o o f Journal Pre-proof removal; and comparisons of two most frequently used techniques of virus concentration in wastewater samples for SARS-CoV-2 RNA detection.

We collected wastewater samples on 8 and 27 May, 2020 from Old Pirana WWTP at Ahmedabad, Gujarat that receives wastewater of 106 million liters per day (MLD), including the wastewater from hospitals treating COVID-19 patients. Confirmed cases of Covid-19 patients in Ahmedabad was 56,352 and 150,857 respectively on 7 th and 26 th May 2020 (Kumar et. al., 2020a) . The WWTP employed upflow anaerobic sludge blanket (UASB) after the primary treatment of raw sewage water ( Figure 1 ). Three separate streams join three inlet chambers (7.5 m x 5 m x 2.5 m) that uses six grit chambers (10.2 m × 10.2 m x 1.0m) i.e. 

Viral RNAs were isolated from sewage samples using the following steps: Precipitation of viral particle; Viral RNA isolation and quality checking of RNA; RT-PCR analysis of viral RNA for the presence of SARS-CoV-2.

The procedure was followed as described in an earlier report with minor modifications (Hjelmsø MH et al., 2017) . The sewage samples (50 mL) were centrifuged at 4500×g (Model:

Sorvall ST 40R, Thermo Scientific) for 30 min to remove the sludge particles. The supernatants were filtered with 0.22 micron filters (Mixed cellulose esters syringe filter, Himedia) to remove bacterial and eukaryotic cells. Further each sewage filtrate was concentrated (for viral precipitation) using two methods: 1) using 96 well filter plate and 2) J o u r n a l P r e -p r o o f poly ethylene glycol (PEG) method. For the first method filtrate was concentrated using the 96 well filter plate (AcroPrep TM Advance 350 10K Omega TM ; Pall Corporation) with a capacity to filter less than 10KD molecules and samples were concentrated 30 times for before RNA isolation.

For the second method, PEG 9000 (80 g/L) (Make: SRL) and NaCl (17.5 g/L) (Make: VETEC) were mixed in 25 ml filtrate and incubated at 10°C, 100 rpm (Model: Incu-Shaker TM 10LR, Benchmark) overnight. The next day the mixture was centrifuged at 13000×g (Model:

Kubota 6500, Kubota Corporation) for 90 mins. After centrifugation supernatant was discarded and the remaining pellet was resuspended in 300µL RNase free water. This was further used as a sample for RNA isolation.

RNA isolation was carried out using a commercially available kit ( 

Quantitation of SARS-CoV-2 viral RNA was carried out by real-time PCR with an instrument Control), one negative control (from extraction run spiked with MS2), and no template control (NTC) were included. The real-time PCR thermal profile was a primary UNG incubation step of 1 cycle of 25°C 2 minutes, 1 cycle of reverse transcription 53°C 10 minutes, 1 cycle of activation 95°C 2 minutes which was followed by 40 cycles of amplification including denaturation at 95°C for 03 seconds and extension 60°C for 30 seconds.

Interpretation of the result was performed by the Applied Biosystems Interpretive Software.

In the process, the probes anneal to three specific SARS-CoV-2 target gene sequences:

ORF1ab, N Protein, S Protein, MS2 (internal process control). All control wells must pass for the real-time RT-PCR plate to be considered valid. If all genes show amplification then the sample will be considered as the positive. Detailed procedures were carried out as described in the product manual and interpretations of results were analysed as instructed in manual. Although there is no direct correlation of the C T value to copy numbers as the kit J o u r n a l P r e -p r o o f used for the detection is absent present assay but considering 500 copies of SARS-CoV-2 genes taken as positive control with C T values of average 26 for all the three genes i.e.

ORF1ab, N and S, the same was extrapolated to compare it with sample C T values and derive approximate copies of genes in the wastewater sample, using the well-established principle of 3.3 C T change corresponding to a 10-fold gene concentration change. In this semiquantitative assay, the amount of RNA used as template was multiplied with the enrichment factor to derive copy numbers for each waste water sample i.e. the enrichment factor with PEG method i.e. 80x and filtration methods (30x) was taken into the account to maintain the equivalence.

We analyzed ORF1ab, N protein genes and S protein gene from the raw wastewater, influent of UASB, effluent of UASB, water in the aeration pond, and the final effluents from WWTP Old Pirana after treatment by polishing pond, sampled on May 8 and May 27, 2020.

The obtained amplification cycles (C T ) and concentrations of SARS-CoV-2 using PEG for virus concentration are shown in Table 1 , and those using filtration method in Table 2 . The positive control sample had C T values of the three SARS-CoV-2 genes ranging 27.92 to 29.52, while the SARS-CoV-2 genes were not detected from the negative control sample. The limit of quantification (LOQ) of the overall method was defined as sample concentration equivalent to 1 copy per reaction tube, which was 1.7×10 2 copies/L.

Our results showed that PEG was more suitable than filtration as a concentration method of SARS-CoV-2 RNA in terms of sensitivity and inhibition removal. MS2 was added in each superior performance over filtration method in terms of SARS-CoV-2 RNA sensitivity and inhibition removal. In the following sections, we evaluated SARS-CoV-2 RNA reduction based on results with PEG method.

We observed a reduction of SARS-CoV-2 RNA both during UASB treatment and during treatment at the aeration tank and the polishing pond. On May 8, all the samples were detected but inconclusive (only 1 out of 3 SARS-CoV-2 genes was positive) and/or not quantifiable (concentration below the LOQ of 1.7×10 2 copies/L). On May 27, on the other hand, raw wastewater and UASB inlet samples were detected above the LOQ at 1.8×10 3 copies/L and 3.5×10 3 copies/L, respectively. These SARS-CoV-2 RNA abundance of raw wastewater in Old Pirana WWTP were comparable to those of untreated wastewater samples in Istanbul, Turkey (Kocamemi et al 2020) infection density of surveyed catchment. On May 27, the SARS-CoV-2 RNA concentration was reduced to a level with inconclusive detection after UASB treatment. When the LOQ of 1.7×10 2 copies/L was used as a maximum concentration after UASB, the viral reduction during UASB treatment was more than 1.3 log 10 .

Comparison of SARS-CoV-2 RNA abundance during various wastewater treatment processes are provided in Table 3 . The reduction of SARS-CoV-2 RNA during wastewater treatment processes have been observed for treatments including secondary treatment (activated sludge/A2O/extended aeration) and tertiary treatment (decantation, coagulation, flocculation, sand filtration, disinfection and NaClO/UV; Randazzo et al 2020a, Balboa et al 2020) in Spain and an unspecified wastewater treatment process in Paris (Wurtzer et al 2020 , Borchardt et al 2007 . In those studies, the log 10 reduction of SARS-CoV-2 RNA was inferred to be 2 in Wurtzer et al (2020) and from >0.1 to >0.8 in Randazzo et al (2020) . In the present study, the resulting log 10 reduction of >1.3 during UASB was well within the ranges above. To our knowledge, this is the first report on SARS-CoV-2 RNA during UASB treatment.

In the case of sewage sludge digestion, anaerobic treatment has shown to be less effective in inactivation of Poliovirus 1, Echovirus 1 and Rotavirus SA-11 in sludge (Scheuerman et al 1991) . Anaerobic wastewater treatment has been employed for treating variety of wastewaters, such as industrial, agricultural and municipal wastewater (McCarty 1981; McCarty and Smith 1981) . Therefore, reduction of SARS-CoV-2 RNA during various anaerobic wastewater treatment, such as UASB, must be investigated in more details in the future.

In the case of treatment at the aeration tank and the polishing pond, the SARS-CoV-2 RNA was detected but inconclusive and/or not quantifiable in the aeration tank water, but the final effluents were negative with all three genes on both May 8 and May 27, potentially suggesting a reduction of SARS-CoV-2 RNA in the aeration tank and the polishing pond. In wastewater treatment ponds, viruses are removed through various mechanisms, including adsorption, predation and sunlight inactivation (Verbyla and Mihelcic, 2015) . In the study J o u r n a l P r e -p r o o f site, significant degradation by sunlight and constant high temperature (~40 o C in average) in the polishing pond is very likely, but further study needs to be substantiated through high data resolution.

In summary, we have successfully evaluated PEG and filtration as concentration methods for SARS-CoV-2 RNA detection. We also demonstrated the removal of SARS-CoV-2 during UASB treatment process. Note that our results are based on small number of samples and semiquantitative analytical method, therefore our results must be substantiated through more thorough investigation in the future.

We tracked the SARS-CoV-2 genetic loading i.e. ORF1ab, N protein genes and S protein genes along the conventional treatment system outfitted with UASB system. We have found a gradual decrease in RNA copies of SARS-CoV-2 from the raw wastewater to influent of UASB after primary treatment, effluent of UASB, aeration pond, and the final effluents after polishing pond at the study cite of 106 MLD WWTP of Pirana, Ahmedabad, India. Higher RNA loading detected in the influent of 27 th May, 2020 owing to higher COVID-19 active cases in Ahmedabad than that on 8 th May, 2020 directly translated into higher decay along the treatment. On May 27, the SARS-CoV-2 RNA concentration was reduced to a level with inconclusive detection after UASB treatment owing to a reduction >1.3 log 10 . To our knowledge, this is the first report on SARS-CoV-2 RNA during UASB treatment, yet a detailed research pertaining to the reduction of SARS-CoV-2 RNA during various anaerobic wastewater treatment, such as UASB, is further required. As we could not detect any genes J o u r n a l P r e -p r o o f in the final effluents on both May 8 and May 27, a remarkable reduction of SARS-CoV-2 RNA in the aeration pond followed by polishing pond is evident.

Among the concentration methods of wastewater sample, our results explicitly indicated that PEG has an advantage over filtration in terms of sensitivity and inhibition removal for RT-qPCR run and gene detection. We conclude this on the basis of C T values of MS2 which was nearly constant for PEG method but varying in nature with filtration method, particularly for the raw wastewater samples. It implies that filtration method was not capable of sufficiently removing sample matrix in the sample water, and thus resulted in greater inhibitory effect of RT-qPCR than PEG method. Overall, implications of our study can be expressed through three major findings i.e.: i) Conventional treatment system seems to be effective in reducing the SARS-CoV-2 genes, ii) UASB system enhances the decay of genetic loading, and iii) On this first-time comparison of PEG method and filtration method, PEG method has shown superior performance over filtration method in terms of SARS-CoV-2 RNA sensitivity and inhibition removal.

The authors declare no competing financial interest. J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f 

GBRC is highly appreciated. We acknowledge the financial assistance from Kiran C Patel Centre for Sustainable Development

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Acknowledgement: