Microbiological and physicochemical quality enhancement of treated wastewater using raw and chemically modified clays from Sidi Bouzid region, Tunisia

1 Microbiological and physicochemical quality enhancement of treated 1 wastewater using raw and chemically modified clays from Sidi Bouzid 2 region, Tunisia 3 4 5 6 Hakima Gharbi-Khelifi1,2, Habib Jmii1, Mohamed Mosbahi3, Samiha Hamdi2,6 , Rawand 7 Hamdi1, Jihen Brahmi2 Mohamed Chamkha4, Sami Sayadi5, Ana Barreiro6 , Maria J. 8 Fernández-Sanjurjo6, Avelino Núñez-Delgado6 and Esperanza Álvarez Rodríguez6 9 10 11 1Laboratory of Transmissible Diseases and Biologically Active Substances, Faculty of 12 Pharmacy of Monastir, University of Monastir, Avenue Avicenne, 5000 Monastir, Tunisia 13 2Department of Biotechnology, Faculty of Science and Technology of Sidi Bouzid, University 14 of Kairouan, 9100 Sidi Bouzid, Tunisia 15 3Laboratory of Georessources CERTE, Technopole Borj Cedria, Tunisia 16 4Laboratory of Environmental Bioprocesses, Centre of Biotechnology of Sfax, Sfax University, 17 PO Box 1177, 3018 Sfax, Tunisia 18 5Biotechnology Program, Center for Sustainable Development, College of Arts and Sciences, 19 Qatar University, Doha 2713, Qatar 20 6Department of Soil Science and Agricultural Chemistry, Engineering Polytechnic School, 21 University of Santiago de Compostela, 27002 Lugo, Spain 22 23 24 Abstract 25 Environmental discharge of wastewater represents a source of chemical substances that may be 26 pollutants (such as phosphorus, chloride, heavy metals), as well as of microorganisms of 27 concern (such as Escherichia coli, Salmonella typhi), that are spread into water courses and 28 crop soils. These facts make clear the need for improving those treatment techniques currently 29 applied in wastewater treatment plants (WWTPs) being not effective. This study firstly 30 evaluates the microbiological, and physicochemical quality of treated wastewaters collected 31 from two WWTPs located in two different Tunisian cities namely Sidi Bouzid (SB) (Centre of 32 Tunisia) and Gafsa (G) (Southern Tunisia). Then, the capacity of three raw and acid/base-33 activated local clays to enhance the quality of wastewaters was assessed. The results indicate 34 2 that the quantities of enteric bacteria (oscillating from 1.381.103 to 1.4.108 CFU/100 mL), fungi 35 (between 1.331.103 and 1.781.104 CFU/100 mL), as well as SARS-CoV-2 (between 4.25.103 36 and 5.05.105 CFU/100 mL) and Hepatitis A virus RNA (form 4.25.103 to 7.4 104 CFU/100 mL 37 ) detected in effluent wastewaters were not in compliance with the Tunisian standards for both 38 studied WWTPs. Likewise for other indicators such as electrical conductivity (ranging 4.9-5.4 39 mS/cm), suspended matter (145-160 g/l), chemical oxygen demand (123-160 mg/l), biological 40 oxygen demand 5 (172-195 mg/l), chloride, Total Kjeldahl nitrogen and phosphorus contents 41 (710, 58-66 and 9.47-10.83 mg/l respectively), the registered values do not agree with the set 42 standards established for wastewater treatment. On the other hand, the pH values fitted 43 (oscillating from 6.86 (at G) to 7.24 (at SB) with the Tunisian standards for both WWTPs. After 44 treatment, wastewaters showed better values for the microbiological parameters, especially for 45 the clays designed as AM and HJ1, which eliminated 100% of viruses. In addition, when acid-46 activated AM clays were applied, a marked improvement in the quality of physicochemical 47 parameters was obtained, especially for suspended matter (2 and 4 g/l for SB and G, 48 respectively), Total Kjeldahl Nitrogen (5.2 (SB) and 6.40 (G) mg/l), phosphorus (1.01 (SB) and 49 0.81 (G) mg/l), where SB WWTP effluents presented a better physicochemical quality than G 50 WWTP effluents after being treated with clays. Our results open perspectives for the possibility 51 of efficiently using these specific clays (which are cheap and easily available) in the 52 enhancement of the quality of treated wastewaters. This can be seen as really relevant as regards 53 environmental and public health considerations. 54 55 Keywords 56 Wastewater; Clay modification; Wastewater clay treatment; Microbiological quality, 57 Physicochemical quality 58 59 Introduction 60 Currently, water scarcity is becoming a growing problem, aggravated by the rapid increase in 61 population, as well as massive pollution of rivers (Gao et al., 2008) and sea (Vikas and 62 Dwarakish, 2015), rapid urbanization, megacity development, and increasing competition 63 among water users, triggering concerns about both human and environmental health 64 (Schwarzenbach et al., 2010). Every year, Tunisia loses a significant part of its hydraulic 65 resources due to the over-evaporation in summer under the effect of high temperatures and the 66 overexploitation of drinking water in the various areas, as well as successive periods of drought 67 3 (Ben Boubaker, H. et al., 2010). Besides, food security depends on the availability and quality 68 of consumption water, and currently this is considered as one central problem in the world 69 (Chahed et al., 2010). To prevent the eventual severe lack of water, several solutions can be 70 implemented to improve the quality and the availability of the resource: (i) water basin transfer, 71 but it is not always possible to apply due to political or environmental issues within and between 72 countries; (ii) desalination, which is a high cost treatment and only countries who have surplus 73 of energy are able to use this process for water recovery; and (iii) wastewater recycling, which 74 would be the most affordable solution. The reuse of treated wastewater effluents in agriculture 75 has several benefits such as: (i) its production is unaffected by climate conditions; (ii) it is a 76 potential source of nutrients and therefore can improve crop yield; and (iii) the groundwater 77 reservoir is preserved (Molinos-Senante et al., 2010). 78 Despite the continuous development of new systems for wastewater treatment, since the late 79 nineteenth century, reused wastewater is still considered an important vector of organic and 80 inorganic pollutants, which endangers human health and environmental quality (Salgot and 81 Folch, 2018) due to the persistence of pathogens and contaminants in different environmental 82 compartments. Pathogens such as Escherichia coli, Salmonella typhi, and Vibrio cholera, 83 common in wastewaters, cause various diseases leading to high morbidity and even mortality, 84 especially in low-middle income countries. Jyoti and Pandit (2001) reported that 88% of 85 diarrhea disease cases are linked to unsafe water supply and hygiene, which results in the death 86 of millions of children every year (Lanata et al., 2013). A growing body of evidence points to 87 the presence of a wide range of pathogenic microorganisms in treated wastewater (El Ouali 88 Lalami et al., 2014). In addition to bacteria, viruses are responsible as well of several diseases 89 (Hassine et al., 2010; Brugha et al., 1999; Metcalf et al., 1995; Bosch et al., 1991). Human 90 pathogenic viruses such as HAV and SARS-COV-2 are commonly detected in urban 91 wastewater and some of them are responsible for a considerable proportion of waterborne 92 diseases (Rosa et al., 2020), although the presence of SARS-COV-2 in wastewater is rather 93 seen as indicative of the epidemiological situation of the specific airborne disease in the area. 94 According to the World Health Organization and several seroprevalence studies, the 95 Hepatovirus A, is considered endemic in Tunisia and its epidemiological pattern ranges 96 between high and intermediate endemicity (Ibrahim et al., 2020; Gharbi-Khelifi et al., 2006; 97 Letaief et al., 2005). Several studies carried out in 2020, during the pandemic, estimated the 98 presence and evolution of the SARS-CoV-2 virus in water resources such as rivers (Guerrero-99 Latorre et al., 2020), affected by wastewater discharge and sewers spreading sludge (Núñez-100 4 Delgado, 2021; Rimoldi et al., 2020; Malik, 2020). The detection of the new COVID19 101 coronavirus in treated wastewater effluents proved the deficiency of conventional treatments 102 applied in WWTPs to remove these microbes (Kitajima et al., 2020; Randazzo et al., 2020; Tran 103 et al., 2020; Jmii et al., 2021). 104 Wastewater treatment within a treatment plant follows several well-defined processes to 105 eliminate pollutants (Benneni and Bouarissa, 2020). Firstly, a pre-treatment step including 106 screening, de-sanding and oil removal is necessary to eliminate large components (Orssatto et 107 al., 2017), followed by a first treatment consisting of extraction of suspended solids and easily 108 decantable organic matter by physicochemical processes (Shewa and Dagnew, 2020; Arashiro 109 et al., 2019). Secondary treatments (biological treatments) are then carried out to degrade and 110 decompose biodegradable organic matter by the action of a wide range of microorganisms, 111 mainly bacteria (Neveux-Guilluy, 1993). Finally, tertiary treatments improve the quality of 112 residual wastewater and, in recent years, extraordinary efforts have been made to implement 113 these treatments to meet the global challenges of water shortage (Rashid et al., 2021), even 114 though they are still absent in a large number of WWTPs. These tertiary treatments include a 115 wide variety of techniques, such as chlorination (Yang et al., 2005; Mitch and Sedlak, 2002), 116 UV disinfection, photocatalysis (Iervolino et al., 2020), different filtration techniques (Gómez 117 et al., 2007; Altmann et al., 2016; Ramos et al., 2016), ozonation (Nawrocki and Kasprzyk-118 Hordern, 2010), oxidation and physical methods such as adsorption and membrane filtration 119 (Rashid et al,. 2021). 120 Adsorption is generally considered as a process of transfer of removable species from an 121 adsorbate (liquid phase) on the surface of the adsorbent (solid phase) through physicochemical 122 interactions (Manchisi et al., 2020). It has been recognized as one of the most effective 123 procedures for wastewater treatment (Slatni et al., 2020; Liu et al., 2020). The percentages of 124 pollutants removal from wastewater by adsorption can reach 99.9%, a very high level of 125 purification which ranks adsorption among the best wastewater treatment processes according 126 to the United States Environmental Protection Agency (US EPA) (Anil et al., 2020). The 127 adsorption method offers significant advantages such as low cost, flexibility, design simplicity, 128 profitability, sustainability, efficiency and preventing secondary pollutants formation (Barakan 129 and Aghazadeh, 2021; El Ouardi et al., 2019; Foroutan et al., 2019; Singh et al., 2018). 130 Currently, different adsorbents have been used to remove the most concentrated pollutants in 131 municipal and industrial wastewater, including domestic waste and industrial by-products 132 5 (Crini, 2006), clays (Pineda et al., 2020; Poetsch and Lippold, 2016; Tahar et al., 2014; Errais 133 et al., 2010), and zeolites (Tahar et al., 2014). 134 The treatment of WWTP effluents using natural clay as adsorbent has shown good results 135 favoring the reuse of the treated water for agricultural irrigation (Errais et al., 2010). When 136 compared with other physicochemical techniques such as the coagulation-flocculation method, 137 the clay treatment appears to be more effective. This physicochemical technique based on 138 coagulation-flocculation process transforms dissolved pollutants into a solid residue, which is 139 certainly of a lower volume, but with higher pollutant concentration. Moreover, this 140 conventional technique, which is generally expensive, does not always reach the standards for 141 the re-use of the resulting sludge in the environment, which supposed a risk for environmental 142 pollution since the sludge resulting from treatment plants is usually applied to agricultural fields 143 as fertilizer all over the world, including Tunisia. On the other hand, the clay used as adsorbent 144 may be re-used in brickyards, cement factories, pottery and ceramics or as an impermeable 145 barrier in the construction of discharge sites (Errais et al., 2010). 146 In recent years, wastewater treatment in Tunisia has been moving towards techniques that are 147 more economical and more environmentally friendly. In this context, this study focuses on the 148 use of clays to improve the microbiological and physicochemical characteristics of wastewater. 149 Therefore, the present study aimed, firstly, to survey the presence of microorganisms indicative 150 of pollution such as fungi, total and fecal coliforms, fecal Staphylococcus aureus, Escherichia 151 Coli and Salmonella, as well as hepatovirus A (HAV) and SARS-CoV-2 in effluent wastewaters 152 (WW) collected from the WWTPs of Sidi Bouzid (SB) and Gafsa (G). The quality of those 153 effluents was evaluated by analyzing the main physicochemical parameters: pH, electric 154 conductivity (EC), suspended matter (SM), chemical oxygen demand (COD), biological 155 oxygen demand (BOD5), Chloride, Total Kjeldahl nitrogen content (TKN) and phosphorus 156 content (P). Secondly, we assessed the effectiveness of three different types of local clays, both 157 raw and after performing activation treatments with an acid, HCl (AA), and with a base, Na2CO3 158 (AB), in the amelioration of the microbiological and physicochemical quality of treated 159 wastewaters. This alternative is included as a tertiary treatment with the aim of overcoming the 160 deficit of conventional secondary treatment processes that guarantee a microbiological and 161 physicochemical quality corresponding to the standards. We hypothesized that both raw and 162 activated natural clay will adsorb pathogenic bacteria and viruses and will improve other quality 163 parameters in wastewater, resulting in reduction of SM, turbidity, COD, BOD5, Chloride, TKN 164 6 and phosphorus. All this can be considered of high relevance from the human health and 165 environmental points of view. 166 167 Material and Methods 168 169 Clays description 170 All the studied sites are placed in the region of Sidi Bouzid, Tunisia (Figure 1). Three different 171 clay samples were collected from three natural formations. The sample HJ1 was taken from the 172 top of the El Haria formation situated in West of Jebel Jebbes El Meheri, Maknassy and the 173 sample HJ2 was taken from the base of the Chouabine formation of Ypresian age (Lower 174 Eocene) Maknassy. The third sample (AM) was collected from the Aleg formation at Jebel 175 Mazzouna. 176 The predominant mineralogy of the clays in these formations is calcite, dolomite, quartz, 177 gypsum, and clay minerals (smectite, illite, kaolinite, palygorskite, and sepiolite) (Mosbahi et 178 al., 2014). In addition, they are characterized by a relatively large specific surface area, up to 179 50.47 m2g-1 (Mosbahi et al., 2017). 180 Importantly, before any experiment, raw clay samples were suspended in distilled water, sieved 181 through a 40 µm sieve to separate the impurities, and then they were oven-dried at 60 °C. 182 Table 1 indicates the mineralogical composition of the clays that were used in this work, with 183 analysis carried out by means of RX diffraction (Philips PW1710 diffractometer, The 184 Netherlands). 185 186 Description of the wastewater treatment plants (WWTPs) 187 Effluent wastewaters (discharge point) were collected from two Tunisian WWTPs, namely Sidi 188 Bouzid (SB) and Gafsa (G), located in the southwest of Tunisia (Figure 2). Both WWTPs use 189 activated sludge sanitation methods. SB has an average daily flow rate = 7,300 m3 /day and 190 serves 50,000 inhabitants, while the G WWTP has an average daily flow rate = 14,000 m3 /day 191 and serves 105,000 inhabitants. 192 In these wastewater treatment plants, pre-treatment begins with screening (elimination of the 193 largest waste through a grid), then desilting (elimination of sand) and ends with de-194 oiling/degreasing (extraction of fats and floating matter found on the surface of the water). 195 Additionally, the primary treatment allows the elimination of suspended solids accumulated at 196 the bottom of the primary purification basins, by simple decantation. Subsequently, the 197 7 wastewater in treatment goes to secondary treatment where oxygen-consuming organisms 198 actuate by means of activated sludge (prolonged aeration at low load). Finally, an ultraviolet 199 disinfection process is performed as a tertiary treatment to eliminate bacteria and viruses and 200 even the most resistant forms such as bacterial spores or cysts, followed by micro-algae 201 sorption. The purified water derived from both SB and G WWTPs is finally discharged into the 202 neighboring natural environments. 203 204 Wastewater sampling 205 The wastewater (WW) sampling was performed 3 times in the period between March and May 206 2021 at the discharge point of the mentioned WWTPs. In total, the analyses were carried out 207 for two wastewater samples corresponding to both WWTPs. The wastewater samples were 208 collected in autoclaved (121°C for 30 minutes) glass bottles, and then conserved in plastic 209 containers that were sterilized beforehand and kept at 4 °C upon arrival until were subjected to 210 different treatments with clay in different experimental conditions (different types of clay tested 211 at different concentrations as detailed below). As a control, the same wastewater samples (but 212 without carrying out clay treatments), were used for each experiment. 213 214 Treatment of wastewater with activated clay 215 During the current study, we have tested the potential of both raw and activated natural clay for 216 the purification of wastewater previously treated with activated sludge. Two types of activation 217 have been tested: activation of the natural clay with hydrochloric acid (AA) and with sodium 218 carbonate (AB). 219 The acid activation was not carried out on HJ1 and HJ2 because its carbonate content is low 220 (HJ1) or zero (HJ2), while AM clay has a carbonate percentage of 50%. The aim of this acid 221 treatment is to destroy the carbonate structure on the one hand and increase their porosity on 222 the other hand. The preparation of the activated clays was performed as follow: 30 g of clay 223 type AM was mixed with 30.33 mL of 3N hydrochloric acid (AA) and 150 mL of distilled 224 water, and this mixture was stirred for 4 hours at 75 °C. After settling, the activated clay was 225 washed several times with distilled water until reaching the neutralization of its pH (which was, 226 every time, measured by using an electronic pH meter). Finally, the activated clay was dried in 227 the oven at 60 °C (Srasra, 1989; Jarray, 1996; Komadel and Madejová, 2006). 228 The alkaline clay activation consisted in the dissolution of 30 g of natural clay in 300 mL of 229 distilled water with 5 % of solid Na2CO3. This mixture was placed under continuous stirring 230 8 for one hour at 75 °C. After decantation, the supernatant was removed using a syringe, then the 231 resident clays were moved to the oven keeping it at 60 °C until they are dehydrated. 232 The treatment of wastewater samples with raw clay was performed per triplicate for each type 233 of clay (HJ1, HJ2, and AM), using three different amounts of clay (60 g, 80 g, and 100 g) per 234 liter of wastewater. Each clay sample was mixed with 1 L of wastewater in a beaker, stirred 235 gently for half an hour and let to settle overnight. After decantation, the supernatant was 236 removed with a syringe and the remaining clay was dehydrated in oven at 60 °C. Regarding 237 the treatment of wastewater with activated clay, it was performed similarly to that carried out 238 for the raw ones, but with only 30 g of activated clay. 239 240 Microbiological analysis 241 Mycological and bacteriological analysis 242 The microbiological analyses were performed with the aim of detecting possible contaminant 243 germs namely fungi, fecal and total coliforms, Staphylococcus aureus, Escherichia coli (E. 244 coli), and Salmonella in WW samples. Detection and quantification of fungi was performed 245 after cultivation and isolation of saprophytic or pathogenic yeasts and fungi on sabouraud agar, 246 which is a classic fungi isolation medium created and named by Raymond Sabouraud in 1892. 247 For isolation, a volume of 100 µL of each sample was inoculated on the surface of a Petri dish, 248 and the same was done for different dilutions (10-1 to 10-10), and then it was incubated at 25-30 249 °C for 3 to 5 days (Mendes et al., 1998). 250 Total aerobic mesophilic bacteria (TG) quantification was performed on Plate Count Agar 251 (PCA) medium after incubation at 30 °C for 72 hours by counting the whitish colonies pushed 252 in depth (Kacprzak et al., 2005). Furthermore, total (TC) and fecal coliforms (FC) were detected 253 in the wastewater samples using the Violet Red Bile Lactose (VRBL) medium by counting the 254 red colonies with a diameter greater than 0.5 mm. From the stock solutions (dilution in cascades 255 of 10-1 to 10-10), 1 mL of sample was transfer to an empty Petri dish using a sterile pipette, and 256 then proceeding to inoculate deeply with approximately 20 mL of melted Plate Count Agar 257 (PCA) yeast extract glucose agar, then making circular movements to allow the inoculum to 258 mix with the agar. Leaving to solidify on the bench, then adding a second layer of approximately 259 5 mL of the same PCA agar. This double layer has a protective role against various surface 260 contaminations. The dishes were incubated with the lid down at 30 °C for 72 hours and then 261 read by counting the whitish colonies of TG grown in depth. 262 9 To investigate the presence of Escherichia Coli in our samples, two steps were executed: firstly, 263 few colonies of fecal coliforms were pre-enriched in nutritive broth (peptone water) and then 264 the standard enrichment in bromocresol purple agar (BCP) at 37 °C for 24 hours was performed 265 (Dupray and Derrien, 1995). Similarly, Salmonella detection and quantification required 266 several steps: pre-enrichment in peptone water, enrichment using specific enrichment medium: 267 MSRV (Rappaport-Vassiliadis Semi-Solid Modified) and finally isolation of Salmonella 268 colonies on Salmonella-Shigella (SS) Agar (Dupray and Derrien, 1995). Concerning 269 Staphylococcus aureus (SA), a highly salty environment (Chapman) was used for its isolation 270 and quantification (Porrero et al., 2014). 271 272 Virological Analysis 273 Virus concentration 274 In the current study viruses were concentrated by the adsorption-elution method using 275 aluminum hydroxide and beef extract as described by (Jmii et al., 2021) with minor 276 modifications. Briefly, 2 L of treated wastewater were filtered through 0.45 µm membranes 277 (Millipore) which were latter cut and placed in 250 mL PPCO centrifuge tubes. Then 100 mL 278 of the obtained filtrate was added to the PPCO tubes, adjusting the pH to 6.0 with AlCl3 solution 279 (0.9 N) and generating Al(OH)3 precipitate (Metcalft et al., 1995), and vortexed to detach the 280 viral particles stuck to the membranes. Afterwards, PPCO tubes were centrifuged at 2000 rpm 281 for 5 min, and the supernatants were collected to measure virus concentration using the 282 adsorption-elution method. Finally, the sample was centrifuged again (1700 x g, 20 min) and 283 viruses were collected from the pellets, which were eluted using 3% alkaline beef extract buffer, 284 transferred in 50 mL PPCO centrifuge tubes and shaken for 10 min at 150 rpm. Afterwards, the 285 concentrate was recovered by centrifugation at 1900 x g for 30 min and the resulted pellet was 286 re-suspended in 1 mL of phosphate-buffered saline (PBS). The obtained concentrates were 287 aliquoted and conserved at -80 °C until use (Randazzo et al., 2020). 288 289 SARS-CoV-2 and HAV viral RNA extraction and quantification 290 RNA was extracted from 600 µl of the concentrate virus using the RNeas Power Water Kit 291 (Qiagen, Germany) according to the manufacturer’s instructions. The RNA was eluted in 100 292 µl of RNAse-free water, aliquoted and conserved at -80 °C until use for viral RNA detection 293 (Chomezynski and Sacchi, 1987). Analysis for SARS-CoV-2 RNA was performed using the 294 QantiTect virus Kits (Qiagen) enabling a one-step quantitative detection of viral RNA targets. 295 16 decrease in COD was recorded, especially when applying the treatments with raw clays HJ1 494 and AM and the HCl-activated clay. The following results: 79 mg L-1 (TWWSB) and 83 mg L495 1 (TWWG) were counted for the maximum COD value, which are very low compared to the 496 COD values of untreated water and compared to the COD limit value set by the standard NT 497 106-002 (125 mg L-1). 498 The BODs values in the untreated waters were 172 and 195 mg O2 L-1 for SB and G, 499 respectively, much higher than the Tunisian standard limit (30 mg O2 L-1). A strong decrease 500 in the amount of oxygen consumed by microorganisms is observed in all treated waters, parallel 501 to the increase in the amount of added clay, with values between 31 mg O2 L-1 (60 g HJ1G and 502 60 g HJ2G) and 9 mg O2 L-1 (30 g AMG (AA). 503 Basically, in untreated wastewater of both G and SB’s WWTPS, the rates of Phosphorus (10.83 504 mg L-1 for G and 9.47 mg L-1 for SB), Chloride (710 mg L-1 for both G and SB) and TKN (66 505 mg L-1 for G, and 58 mg L-1 for SB) exceeded the limit values set by the Tunisian standard (NT. 506 106.002) (Table 4). 507 The phosphorus concentrations, that were around 10 mg L-1 in the raw wastewater of both 508 WWTPs, decreased to 3.9-6.0 mg L-1 (for treatments with 60 g of clay), 1.9-1.2 mg L-1 (for 509 treatments with 80 g of clay), and 2.0-0.3 mg L-1 (for 100 g of clay). After treatment with 510 activated clay, the concentration of phosphorus varied between 1.9 -1.01 mg L-1 for the clays 511 subjected to basic activation, and between 1.0-0.81 mg L-1 for those acid-activated. With the 512 addition of 80 and 100 g of clay and with the acid and basic activation treatment, the values 513 achieved were generally lower than the Tunisian standards (NT. 106.002) (<2 mg L-1). 514 All the treatments applied reduced N concentration. When 60 g of clay are added, N 515 concentrations are between 29 and 21 mg L-1 for all the clays used, while for 80 g of clay the 516 values obtained are between 22 and 15 mg L-1 ,and adding 100 g the levels of TKN were 517 between 16 and 6 mg L-1. It should be noted the low values of TKN achieved when using clays 518 activated with acid (6.4-5.2 mg L-1). All the treatments applied decreased this parameter below 519 the limit values established by Tunisian legislation (30 mg L-1) (Table 4). 520 Regarding chloride, its concentration in the clay-treated wastewater has a wide range (16.4 to 521 994 mg L-1) and it was sometimes higher than that recorded in the untreated wastewater, 522 especially in activated clay treatments (Table 4). The lowest values and the most respectful of 523 the Tunisian standard (NT. 106.002) of chloride (700 mg L-1) are recorded for raw HJ1 clays, 524 especially with the doses of 80 and 100 g of clay, contrary to other types of clays with which 525 the treatment turns out to be ineffective in the elimination of chloride. 526 17 Globally, the results of the analyses of the physicochemical parameters of SB and G’s WW 527 treated with clays showed that the values of the physicochemical parameters recorded in the 528 samples treated with activated clays are very close to the limit values quoted by the standard, 529 compared to those measured in wastewater treated with 60 and 80 g of raw clay. However, 530 when using 100 g of raw clays, we obtained values close to those achieved with 80 g of clay. 531 532 Discussion 533 534 1. Microbiological properties 535 The results obtained for the microbiological analyses of raw wastewaters did not comply, 536 generally, with the values determined by the Tunisian standard NT 106-002. Therefore, the 537 wastewater treated in the Lessouda (Sidi Bouzid) and Gafsa WWTPs is not suitable to be 538 discharged into the environment or reused in irrigation due to its poor bacteriological quality. 539 Statistically, the concentrations measured for the microorganisms detected were significantly 540 higher (p<0.05) in the wastewater leaving the Gafsa station than those in the wastewater from 541 Sidi Bouzid. 542 Correspondingly, other fecal pollution indicator bacteria were detected in wastewater treated 543 in other WWTP located in South of France, as mentioned by Brienza et al. (2019), who 544 indicated that the numbers of E. coli in the influent was 105, higher than that in our study, while 545 E. faecalis were about 104 CFU/mL. Besides, Ben Salem et al. (2011) showed that E. coli was 546 detected at a rate of 76.6% at entrance points and 50% at the exit points, and Salmonella was 547 detected in percentages of 66.6% and 20% at the entry and exit points, respectively, in other 548 Tunisian WWTPs located in different regions (Sousse, Monastir, Kairouan and El kef); this 549 fact indicates that wastewater treatment did not remove all the pathogens but gave reductions 550 of 26.6 % and 40.6% for E. coli and Salmonella, respectively. 551 The amount of HAV and SARS-CoV-2 in the effluents of the SB and G WWTPs is also 552 noteworthy (Table 4). Previous studies conducted in Tunisia have revealed that the situation 553 regarding the epidemiology of Hepatovirus A is endemic, as demonstrated by the presence of 554 virus traces in wastewater collected from different sewage treatment plants throughout the 555 country (Ibrahim et al., 2020; Ouardani, 2016); this situation also occurs in other parts of the 556 world (İnat and Koluman, 2013; McCall et al., 2020; Yang et al., 2021). In a recent Tunisian 557 paper, Hepatovirus A was detected respectively in 62% and 66% of the collected wastewater 558 samples at El Menzeh I and Charguia I WWTPs (Ibrahim et al., 2020). Actually, HAV was 559 18 detected in 38% of the water samples in Saudi Arabia, with concentrations ranging from 5.0 560 101 to 1.9 104 RNA copies/L of surface water (Blanco et al., 2019). To note, the latter study, 561 performed by Blanco et al. (2019), has shown the non-effectiveness of the conventional 562 secondary treatments carried out in the majority of WWTPs where high loads of Hepatovirus 563 A RNA were detected in effluent wastewaters, which constitutes a potential public health issue. 564 As tertiary treatment, UV irradiation, has been proved to be inefficient in removing completely 565 hepatovirus A from wastewater, as reported by Ibrahim et al. (2020), which is in accordance 566 with our results demonstrating the presence of high levels of HAV RNA in effluent wastewater 567 where UV radiation is used. Even more, biological treatments and the tertiary treatment with 568 UV radiation proved to be insufficient to completely eliminate the HAV viruses. 569 Another current and urgent issue is the presence of SARS-CoV-2 in wastewater (Rimoldi et al., 570 2020). The presence of viable virus in the stools has been reported, suggesting that SARS-CoV-571 2 can be transmitted through the oral-fecal route (Arslan et al., 2020; Heller et al., 2020; Wu et 572 al., 2020; Tran et al., 2020) raising epidemiological and environmental concerns. 573 Traces of SARS-CoV-2 (its RNA) have been detected in clinical liquid discharges (hospitals) 574 (Zhang et al., 2020), in wastewaters of planes, navy and cargo ships (Ahmed et al., 2020), in 575 rivers (Rosa et al., 2020) and even in effluent wastewaters subjected to a secondary treatment 576 in sewage treatments plants (Medema et al., 2020; Randazzo et al., 2020; Romero et al., 2020). 577 Even biological treatments and the tertiary treatment with UV radiation were insufficient to 578 completely eliminate SARS-CoV-2 from wastewaters (Bhatt et al., 2020). Viral RNA was also 579 detected in the activated sludges used in sewage treatments plants (Arslan et al., 2020) which 580 drives us to rethink about the ways to treat wastewaters and WWTPs residues to prevent 581 potential virus spread. Similarly, to our results, Haramoto et al. (2020) detected 2.4.103 RNA 582 copies/L of SARS-CoV-2 in one out of 5 secondary-treated samples, while Randazzo et al. 583 (2020) reported 2.5 105 RNA copies/L. 584 Given the inadequacy of the treatment processes currently available at the WWTPs studied, it 585 is necessary to adopt more efficient processes. Previous studies have dealt with the assessment 586 of the performance of natural clays and their drifts in purifying water (wastewater or domestic 587 drinking water) as a new, more effective, and cost-effective treatment alternative (Meçabih et 588 al., 2006). In fact, natural clay was tested to eliminate microbiological pollutants (mainly 589 viruses and persistent bacteria), and chemical pollutants such as pharmaceuticals and personal 590 care products (Dijaani and Amer, 2020). 591 19 The results obtained in the present study indicate that the three types of clay cantnot remove 592 completely TG, even when a high concentration of clay was applied (100 g L-1), being only 593 100% effective the AM acid-activated clay (Fig. 2). For TC and FC, complete removal is 594 observed when an amount equal to or greater than 80 g of any of the clays used in this study is 595 added. The three types of clay HJ1, HJ2 and AM were able to adsorb efficiently fungi, 596 Salmonella and Staphylococcus even at a small amount of 60 g, while for E. coli AM clay is 597 only effective when added at high doses or activated with acid or base. To sum up, according 598 to the bacteriological analyses carried out for our wastewater samples, the activation with 599 Na2CO3 was more effective for HJ1 and AM than for HJ2, since the latter was able to adsorb 600 only five of the seven pathogens studied. Acid activated clay (AM) seems to be efficient and 601 adsorbed all of the studied microorganisms present in the wastewater. 602 The adsorption process is different depending on the clay. Several studies on the antibacterial 603 effect of green clays have shown that smectite-based clays, such as our clay samples, are 604 characterized by strong antibacterial capacity and by completely eliminate E. coli, S. Enterica 605 serotype Typhimurium, P. Aeruginosa, Stapylococcus and M. Marinum (Williams et al., 2004; 606 2008; Xia et al., 2005). Under normal conditions, illite and kaolinite adsorb organic molecules 607 onto their external surfaces, whereas swelling clays adsorb organic compounds mainly into the 608 interlayer space, with very little external adsorption. This is due to their relevant interior specific 609 surface area and adsorption sites, where hydrated exchangeable cations can be replaced by 610 organic molecules (Errais et al., 2010). In relation to carbonate minerals, several authors show 611 their ability to adsorb different pathogens as well as to inhibit their growth. Li et al. (2022) 612 indicate the ability of dolomite to adsorb coliform bacteria, especially when the pH is around 613 7.8. According to Lee et al. (2022), the presence of CaCO3 can interrupt the formation of the 614 cell walls of different pathogens, such as E. Coli, causing the lysis of these microorganisms. 615 Other authors indicate that calcite decomposition causes an increase in porosity and pore size, 616 so the presence of active sites for E. coli adsorption is also greater (Tong et al., 2013). All of 617 the above would justify the greater microbial adsorption obtained in the present work in the 618 three types of clay, since they present in their composition carbonate minerals (calcite and 619 dolomite), as well as sepiolite, palygorskite and/or montmorillonite (Table 1). 620 Moreover, the results of elimination of fungi, TC, FC, Salmonella, E. coli, Staphylococcus and 621 even TG by 30 g of HCl-activated AM indicate that it is more effective than raw AM, especially 622 when only 60 g are used. The use of 30 g of AM+HCl produces results comparable to the use 623 of 100 g of raw AM. 624 20 In relation to HAV and SARS-CoV, present in the effluents of both WWTPs, they decrease to 625 unquantifiable values for practically all clay treatments. A study by Lipson and Stotzky (1985) 626 showed that there are specific adsorption sites on clay for each virus population, which makes 627 it possible to predict the behavior of viruses on clay minerals. Thus, the results of this study can 628 explain the variation in the adsorption power of AM, HJ1 and HJ2 clays against HAV and 629 SARS-COV-2 viruses, where HJ1 and AM are the most effective among the raw clays, which 630 could be related to the presence of calcite and dolomite in these clays, while those minerals are 631 absent in HJ2 (Table 1). 632 Thus, the difference in microbiological parameters between treated wastewater compared to 633 untreated wastewater from G and SB was significant for all treatments, especially for samples 634 treated with 100 g of clay. According to Pineda et al. (2020), the relevant content of silica, 635 aluminum and ferric oxides in clays generates a biocidal environment, reducing the content of 636 viruses, bacteria and protozoa. 637 In the current study, the microbiological quality of wastewater collected at the outlet of G and 638 SB WWTPs was improved following clay treatment, to comply with the Tunisian standard NT 639 106 002, setting the acceptable bacterial loads in effluents wastewater, which was not the case 640 before the clay treatment. 641 Several authors indicate the variable effectiveness of different natural clays, raw or physically 642 or chemically activated, in adsorbing different types of micropollutants existing in wastewater 643 (clinical, industrial and domestic effluents) (Heidari et al., 2022; Mohamed Amin et al., 2016; 644 Mahouachi et al., 2020), which suggest its future use as an effective, affordable, eco-friendly 645 and safe alternative treatment. In the context of the strong emergence of infections caused by 646 antibiotic resistance, the use of natural clays as antibacterial and antiviral agents in municipal 647 wastewater rich in these microorganisms may constitute a practical and economical alternative 648 due to their adsorbent power (Benali et al., 2017). 649 650 2. Physicochemical properties 651 The various analyses of the physicochemical parameters of the wastewater treated in the two 652 treatment plants G and SB revealed values that did not comply with the Tunisian standards in 653 force NT 106-002 (CE < 50 dS m-1, MES < 30 mg/L, COD < 90 mg O2/L, BOD5 <30 mg O2/L, 654 phosphates < 2 mg/L, nitrates < 30 mg N/L) with the exception of pH values, which comply 655 with the Tunisian standards. These results seem to be consistent with those obtained in other 656 21 previous studies carried out in a pilot wastewater treatment plant (Ibrahim et al., 2020; 657 Letshwenyo and Veronicah, 2020). 658 The results obtained after the treatment with raw clay showed a decrease in suspended matter, 659 COD, BOD5, phosphate, nitrate and electrical conductivity, being overall slightly more 660 effective with AM clay, and especially with HJ1, relative to HJ2. Similar conclusions were 661 drawn from other studies conducted by Khamis et al. (2012) and Errais et al. (2010), which 662 were focused on the removal of organic substances and chemical elements, strongly detected in 663 wastewater, by means of clay materials of other origins. 664 The most efficient clays to improve the physicochemical parameters of the water were HJ1 and 665 AM (Table 4), which present in their composition montmorillonite, paligorskite, or sepiolite, 666 as well as calcite and dolomite, while in the HJ2 clay, of these minerals, only sepiolite is present. 667 The three previous phyllosilicates would clearly contribute to the greater efficiency of these 668 clays, since their adsorbent properties are well known. Regarding carbonated materials, several 669 authors point out their high efficiency in adsorbing different contaminants, being the main 670 adsorption mechanisms ionic exchange, surface complexation, physical adsorption and, above 671 all, precipitation processes (Khoshraftar et al., 2022; Shah et al., 2020; Ariffin et al., 2017). 672 According to Mosbahi et al. (2017), the specific surface of HJ1 clays is relatively high, around 673 50.47 m2 g-1, which also explains their high efficiency in the purification of wastewater 674 compared to raw AM and HJ2. 675 Electrical conductivity values recorded in the wastewater at the outlet of the stations of SB and 676 G, are close to the Tunisian standards. In any case, these values are high and can contribute to 677 soil degradation due to salinity (Hacini et al., 2013). The average conductivity decreased after 678 treatment (although not significantly), what can be related to adsorption of cations and anions 679 on the surface of the clay, or due to the constitution of oxides as previously shown by Al Bakri 680 et al. (2011). 681 Regarding COD and BOD5, we note a significant reduction in their values from G’s and SB’s 682 wastewaters after clay's treatment. Awad et al. (2013) found that two natural clays called Shendi 683 and Singa reduced COD by 26.3% and 28.1%, respectively, and their combination with 684 polyaluminium chloride (PAC) improved their COD removal efficiency by up to 70.7%. A 685 previous study has shown a strong reduction of COD and BOD, even higher than in our study, 686 after filtering wastewater through a micellar clay column (Khamis et al., 2012). According to 687 these authors, the initial high BOD and COD observed in untreated waters may be due to 688 22 residues of chemicals in the wastewater, which were not well removed by the secondary 689 biological treatment. 690 The large amounts of chlorides contained in domestic wastewater can significantly alter the 691 ecological balance (Apte et al., 2011). For this reason, in this study we focused on the removal 692 of these toxic compounds. The reduction in chloride concentration was particularly significant 693 with the HJ1 treatment. Apte et al. (2011) showed that the efficiency of a dried plant biomass 694 of the species Parthenium sp. in the reduction of chlorides in wastewater by biosorption was 695 very high, around 40%, but still lower than that obtained in our work using different doses of 696 the HJ1 clay. 697 Regarding suspended organic matter (SM), there was a significant reduction of this parameter 698 after treatment, thus meeting the Tunisian standard. Other authors also obtained a high 699 efficiency in the removal of SM from wastewater using raw clays (Young et al., 2021) or 700 montmorillonite clays modified with aluminum (Al) or ferric (Fe) polymeric species (Jiang et 701 al., 2004). 702 In addition to the effectiveness of raw clays in improving the physicochemical conditions of 703 these wastewaters, treatment with acid-activated or base-activated clays generally provided 704 better results, with acid activation of AM clay being the most effective. Thus, an excellent 705 physicochemical quality of these waters was obtained after the treatments. According to several 706 authors studying different activated clays from Tunisia (Krupskaya et al., 2017; Komadel and 707 Madejova, 2013), during acid activation protons are exchanged by exchange cations in the 708 interlayer and the crystalline structure of the clay is partially dissolved, releasing some of the 709 cations such as Mg, Al or Fe from the octahedral layer, leading to an increase in porosity, 710 surface acidity and specific surface area in these acid-activated minerals (Krupskaya et al., 711 2017; Komadel and Madejova, 2013). According to Mosbahi et al. (2017), the destruction of 712 AM carbonates following the acid attack is the origin of the improvement in the adsorbent 713 capacity by increasing the specific surface and the active sites within the crystalline lattice of 714 the clays. This may justify that acid activation is more effective when applied to clays with high 715 calcite/dolomite content (HJ1 and, especially AM) (Table 1). 716 Regarding the basic activation of clays with similar characteristics to those of this work, 717 Mosbahi et al. (2017) observed an increase in the specific surface area, the formation of sodium 718 smectite clays, as well as the destruction of dolomites and the growth of zeolites. This could 719 explain the higher adsorption obtained in these clays compared to the raw clays. 720 23 The improvement observed in the purification capacity after the activation of the clays, in 721 comparison with the raw natural ones, has been described in other studies where starch was 722 added (Mohamed Amin et al., 2016). 723 724 Conclusion 725 726 This work shows the results of the first study on the treatment of wastewater highly loaded with 727 micropollutants performed by means of clays from the Maknessy-Mazzouna basin, center west 728 of Tunisia. During this research, the effectiveness of these clays in the elimination of organic 729 and microbiological pollutants from wastewater, as well as in improving its quality as regards 730 physicochemical parameters, was assessed in detail. 731 Almost all of the high pollutant load concentrating in the effluents discharged by the two 732 wastewater treatment plants (WWTPs) studied, one of Lessouda, Sidi bouzid and the other of 733 Gafsa, was reduced after the treatment of the effluents using samples of natural and chemically 734 modified clays, with removal percentages reaching up to 100%. Among the raw clays, HJ1 is 735 the most effective in the wastewaters treatment, while AM activated by acid (HCl) proved to 736 be the most effective among the activated clays. We have found that the acid or base activation 737 of the clays made it possible to reduce the quantity of clays used (from 100 g to 30 g), achieving 738 the same purification quality. This can help in preserving clay resources, that may be limited, 739 during the implementation of a large-scale project. 740 In this respect, the method here used seems to be very promising, due to its simplicity and also 741 great results in improving the microbiological quality of treated wastewaters. 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Total Environ,741, 1149 140445. https://doi.org/10.1016/j.scitotenv.2020.140445. 1150 1151 1152 37 Statements and Declarations 1153 Acknowledgements 1154 The authors wish to express their gratitude to the director and staff of the National Sanitation 1155 Office (Sidi Bouzid and Gafsa), Tunisia, and the members of the Department of Biology in FST 1156 Sidi Bouzid, Tunisia. 1157 Author contributions 1158 Funding 1159 This work was supported by the Tunisian Ministry of Higher Education and Scientific 1160 Research. 1161 Competing Interests 1162 The authors declare that there are no conflicts of interest. 1163 Ethics approval and consent to participate 1164 Not applicable 1165 Consent for publication 1166 Not applicable. 1167 1168 38 1169 1170 1171 1172 1173 1174 1175 1176 Figure 1. Location of the Maknessy-Mazzouna basin, center west of Tunisia (a) Geologic 1177 outcrops at Jebel Meheri El Jebbes showing position of lithological section (Khlifi, 2004) and 1178 (b) Location of the Meknassy-Mezzouna basin, Centerwestern Tunisia (Mosbahi et al., 2007) 1179 1180 1181 a b N W -Middle to upper: Jebs Formation -Lower Eocene: Metlaoui group -Upper Maastrichtian-Lower Paleocene: El Haria Formation -Middle CompanianLower Maastrichtian: Abiod Formation -Lower ConiacianLower Companian: Aleg Formation S Study are a Scale Study area 39 1182 1183 1184 Figure 2. Geographic map of Sidi Bouzid and Gafsa (ref) 1185 1186 1187 40 1188 1189 1190 1191 0 500 1.000 1.500 2.000 2.500 WW 60g HJ1 80g HJ1 100g HJ1 60g HJ2 80g HJ2 100g HJ2 60g AM 80g AM 100g AM 30g HJ1 (AB) 30g HJ2 (AB) 30g AM (AB) 30g AM (AA) Total aerobic bacteria in Sidi Bouzid NT 0 500 1000 1500 2000 2500 3000 3500 4000 4500 WW 60g HJ1 80g HJ1 100g HJ1 60g HJ2 80g HJ2 100g HJ2 60g AM 80g AM 100g AM 30g HJ1 (AB) 30g HJ2 (AB) 30g AM (AB) 30g AM (AA) Total aerobic bacteria in Gafsa NT ====== 0 2000 4000 6000 8000 10000 WW 60g HJ1 80g HJ1 100g HJ1 60g HJ2 80g HJ2 100g HJ2 60g AM 80g AM 100g AM 30g HJ1 (AB) 30g HJ2 (AB) 30g AM (AB) 30g AM (AA) Total Coliforms in Sidi Bouzid NT x104 ====== ====== 100/L 41 1192 1193 1194 1195 Figure 3. Bacteriological parameters of treated wastewaters from Sidi Bouzid and Gafsa 1196 WWTPs. 1197 1198 0 2000 4000 6000 8000 10000 WW 60g HJ1 80g HJ1 100g HJ1 60g HJ2 80g HJ2 100g HJ2 60g AM 80g AM 100g AM 30g HJ1 (AB) 30g HJ2 (AB) 30g AM (AB) 30g AM (AA) Total Coliforms in Gafsa NT x104 0 4000 8000 12000 16000 WW 60g HJ1 80g HJ1 100g HJ1 60g HJ2 80g HJ2 100g HJ2 60g AM 80g AM 100g AM 30g HJ1 (AB) 30g HJ2 (AB) 30g AM (AB) 30g AM (AA) Fecal Coliforms in Sidi Bouzid NT x102 0 4000 8000 12000 16000 WW 60g HJ1 80g HJ1 100g HJ1 60g HJ2 80g HJ2 100g HJ2 60g AM 80g AM 100g AM 30g HJ1 (AB) 30g HJ2 (AB) 30g AM (AB) 30g AM (AA) Fecal Coliforms in Gafsa NT x102 ====== ===== ====== 100/L