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. 2022 Nov;214(Pt 4):114057.
doi: 10.1016/j.envres.2022.114057. Epub 2022 Aug 20.

Fate of SARS-CoV-2 coronavirus in wastewater treatment sludge during storage and thermophilic anaerobic digestion

Sabrina Guérin-Rechdaoui  1 , Ariane Bize  2 , Camille Levesque-Ninio  3 , Alice Janvier  3 , Carlyne Lacroix  4 , Florence Le Brizoual  3 , Jérôme Barbier  5 , Céline Roose Amsaleg  6 , Sam Azimi  4 , Vincent Rocher  4
Affiliations

Fate of SARS-CoV-2 coronavirus in wastewater treatment sludge during storage and thermophilic anaerobic digestion

Sabrina Guérin-Rechdaoui et al. Environ Res. 2022 Nov.

Abstract

Since the COVID-19 outbreak has started in late 2019, SARS-CoV-2 has been widely detected in human stools and in urban wastewater. No infectious SARS-CoV-2 particles have been detected in raw wastewater until now, but it has been reported occasionally in human stools. This has raised questions on the fate of SARS-CoV-2 during wastewater treatment and notably in its end-product, wastewater treatment sludge, which is classically valorized by land spreading for agricultural amendment. In the present work, we focused on SARS-CoV-2 stability in wastewater treatment sludge, either during storage (4 °C, room temperature) or thermophilic anaerobic digestion (50 °C). Anaerobic digestion is one of the possible processes for sludge valorization. Experiments were conducted in laboratory pilots; SARS-CoV-2 detection was based on RT-quantitative PCR or RT-digital droplet PCR. In addition to SARS-CoV-2, Bovine Coronavirus (BCoV) particles were used as surrogate virus. The RNA from SARS-CoV-2 particles, inactivated or not, was close to the detection limit but stable in wastewater treatment sludge, over the whole duration of the assays at 4 °C (55 days) and at ambient temperature (∼20 °C, 25 days). By contrast, the RNA levels of BCoV and inactivated SARS-CoV-2 particles decreased rapidly during the thermophilic anaerobic digestion of wastewater treatment sludge lasting for 5 days, with final levels that were close to the detection limit. Although the particles' infectivity was not assessed, these results suggest that thermophilic anaerobic digestion is a suitable process for sludge sanitation, consistent with previous knowledge on other coronaviruses.

Keywords: Bovine coronavirus; Droplet digital PCR; Max 6); RT-qPCR; SARS-CoV-2; Sludge; Stability.

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Conflict of interest statement

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
Experimental design. SAV: Seine Aval plant – SEG: Seine Grésillons plant – SEC: Seine Centre plant – SEM: Seine Morée plant – MAV: Marne Aval plant – SAM: Seine Valenton plant.
Fig. 2
Fig. 2
Persistence of BCoV and SARS-CoV-2 in spiked WWT sludge, at 4°C (55 days) and room temperature (12 days). Upper plot: CT values obtained for SARS-CoV-2 detection in the pool used for spiking (positive control). Middle plot: CT values obtained for SARS-CoV-2 detection in spiked sludge. Lower plot: CT values obtained for BCoV detection in spiked sludge. All CT values were obtained by RT-qPCR. The complete data corresponding to Fig. 2 are available in the Supplementary Table S1. In this assay, it was not possible to perform biological replicates due to the limited amount of SARS-CoV-2 pool available for spiking. Each curve therefore corresponds to the kinetics for a single pool or sludge batch.
Fig. 3
Fig. 3
Persistence of SARS-CoV-2 at 4°C and room temperature, in open (17 days) or closed (25 days) bottles, in unspiked fresh WWT sludge (Sludge_high). SARS-CoV-2 RNA levels were measured by RT-qPCR. Each curve corresponds to the kinetics of a single reactor.
Fig. 4
Fig. 4
Dynamics of SARS-CoV-2 and BCoV RNA levels during thermophilic anaerobic digestion of spiked or unspiked fresh WWT sludge. GU/g of total weight: genomic units per gram of total weight of samples from the anaerobic digestion batch reactors. The same batch of fresh WWT sludge was used for all assays, Sludge_low (Table 1, Table 2). Due to the limited amount of SARS-CoV-2 collected from the swab samples, it was not possible to include biological replicates. The points of same color correspond to technical duplicates of sludge preparation and RNA extraction. At each time point, 2 reactors were sacrificed for analysis (one with unspiked sludge, one with spiked sludge), hence a total of 8 reactors. A value of 0 is indicated when the quantification values were below the detection level. A. SARS-CoV-2 RNA levels in spiked and unspiked sludge, measured by RT-ddPCR targeting the regions N1 + N2 + IP2 (detected in the same channel). B. BCoV RNA levels in spiked sludge, measured by RT-qPCR targeting the N gene. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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