Highly efficient and sensitive membrane-based concentration process allows quantification, surveillance, and sequencing of viruses in large volumes of wastewater.

Wastewater-based epidemiology is experiencing exponential development. Despite undeniable advantages compared to patient-centered approaches (cost, anonymity, survey of large populations without bias, detection of asymptomatic infected peoples…), major technical limitations persist. Among them is the low sensitivity of the current methods used for quantifying and sequencing viral genomes from wastewater. In situations of low viral circulation, during initial stages of viral emergences, or in areas experiencing heavy rains, the extremely low concentrations of viruses in wastewater may fall below the limit of detection of the current methods. The availability during crisis and the cost of the commercial kits, as well as the requirement of expensive materials such as high-speed centrifuge, can also present major blocks to the development of wastewater-based epidemiological survey, specifically in low-income countries. Thereby, highly sensitive, low cost and standardized methods are still needed, to increase the predictability of the viral emergences, to survey low-circulating viruses and to make the results from different labs comparable. Here, we outline and characterize new protocols for concentrating and quantifying SARS-CoV-2 from large volumes (500 mL-1 L) of untreated wastewater. In addition, we report that the methods are applicable for monitoring and sequencing. Our nucleic acid extraction technique (the routine C: 5 mL method) does not require sophisticated equipment such as automatons and is not reliant on commercial kits, making it readily available to a broader range of laboratories for routine epidemiological survey. Furthermore, we demonstrate the efficiency, the repeatability, and the high sensitivity of a new membrane-based concentration method (MBC: 500 mL method) for enveloped (SARS-CoV-2) and non-enveloped (F-specific RNA phages of genogroup II / FRNAPH GGII) viruses. We show that the MBC method allows the quantification and the monitoring of viruses in wastewater with a significantly improved sensitivity compared to the routine C method. In contexts of low viral circulation, we report quantifications of SARS-CoV-2 in wastewater at concentrations as low as 40 genome copies per liter. In highly diluted samples collected in wastewater treatment plants of French Guiana, we confirmed the accuracy of the MBC method compared to the estimations done with the routine C method. Finally, we demonstrate that both the routine C method processing 5 mL and the MBC method processing 500 mL of untreated wastewater are both compatible with SARS-CoV-2 sequencing. We show that the quality of the sequence is correlated with the concentration of the extracted viral genome. Of note, the quality of the sequences obtained with some MBC processed wastewater was improved by dilutions or enzyme substitutions suggesting the presence of specific enzyme inhibitors in some wastewater. To the best of our knowledge, our MBC method is one of the first efficient, sensitive, and repeatable method characterized for SARS-CoV-2 quantification and sequencing from large volumes of wastewater.

Deep eutectic solvent pretreatment of biomass: Influence of hydrogen bond donor and temperature on lignin extraction with high ß-O-4 content.

Recovering lignin with high ß-O-4 content is of prime interest for further high yield depolymerization in low molecular weight phenolic compounds. Pretreatment of lignocellulosic biomass with deep eutectic solvents (DES) was studied to extract this type of tailored lignin from softwood and brewer's spent grains. In this work, choline chloride (ChCl) based DES with two different hydrogen bond donors (HBD) (lactic acid (LA) and Glycerol (Gly)), were investigated at mild temperatures (60 and 80 °C). The influence of DES pretreatment on extracted lignin molecular weight and structural characteristics was analysed. The acidity and density of DES were proved to affect lignin extraction yield and its features. The lignin characteristics (type of interunits, accessibility) were shown to impact their ability to be recovered. Acidic-DES ChCl:LA at 80 °C with woody biomass gave promising results with 78% of lignin extracted exclusively composed of G units with 61% of ß-O-4 linkages with narrow molecular weights distribution.

Fractionation of polyphenols from thermomechanical pulp mill process water by flotation and membrane integrated process.

Fractionation of phenolic compounds in thermomechanical pulp mills was performed with a coupling of a prior treatment realized by flotation and a ceramic membrane process. Two lines of membranes filtration were tested. After a common 150 kDa clarification, 1 kDa filtration was performed with or without previous 5 kDa filtration. Flotation was shown to be inevitable to retain lipophilic compounds which cause severe membrane fouling. 150 kDa permeate flux was 20% higher when process water was firstly floated and was around 260 L h-1 m-2. 1 kDa membrane was fouled with 31% of irreversible fouling without previous 5 kDa filtration and phenolic compounds purity reached only 26% in this 1 kDa permeate. Phenolic compounds as lignin-like substances which might be attached to hemicelluloses were recovered in 5 kDa retentate. Retentate of 1 kDa might contain a major fraction of lignin derivatives with molecular weights around 1 kDa free or linked with phenolic acids. Permeate of 1 kDa contained 14% of phenolic compounds such as lignans and free phenolic acids purified at 50%.

Stabilized landfill leachate treatment by combined physicochemical-nanofiltration processes.

Landfill leachate is a complex wastewater which the composition and concentration of contaminants are influenced by the type of waste deposited and the age of landfill. In the last years, several processes or process combinations were developed and tested to reach requirements for the discharge of leachate. Among the new processes, membrane processes are considered as promising: reverse osmosis is one of the most widely used treatment in the Northwestern European countries and nanofiltration is gained in popularity during the last 5 years. Successful application of membrane technology for the treatment of landfill leachates, requires efficient control of membrane fouling. Two organic membranes of nanofiltration were used for pilot-scale testing. Leachates were subject to several pretreatments (pH modification, prefiltration and coagulation with FeCl3) to remove potential foulants including dissolved organic and inorganic substances, colloidal and suspended particles. These pretreatments do not enhance the performances (retention and permeation flux) of membranes because the pH range and the presence of Fe3+ ions contribute greatly to change the characteristics of organic matter and the surface charges of membranes. However, the results show that nanofiltration is sufficient to eliminate refractory COD, the permeates have a COD lower than the requirements for discharge.