Use of bacteriophage to inactivate pathogenic bacteria from wastewater.

The aim of this study was to enhance the rhizobacterium potential in horizontal subsurface flow constructed wetland (CW) system planted by Phragmites australis using specific and lytic phages. The bioinoculation of specific bacteriophage for target bacteria; Salmonella typhi, and the monitoring of bacterial inactivation under different conditions showed the effectiveness of this methodology to enhance bacteria reduction and consequentially ameliorate purification performance of this studied biological treatment system. The injection of the phage at a concentration equal to 103 UFP/mL within the rhizosphere of the inoculated filter (F) was allowed 1 U-Log10 of improvement of bacterial inactivation compared to the control filter (T) nearly 1 logarithmic unit thus, a 90% improvement of bacteria reduction. When we increased the phage titer (105 UFP/mL), the bacterial reduction equal to 2.75 U-Log10 (N/N0) was registered that corresponds to a decrease of nearly 99.9%. According to the first-order model, the inactivation coefficient is equal to 2.29 min-1 (0.88 min-1 for the first experiment) and the bacterial reduction rate is 5 times higher than that determined for the control filter. This results show the positive impact of the phage in the bacterial inactivation and the improvement of water treatment of the biofilter C.

First Investigation of Seasonal Concentration Behaviors and Sources Assessment of Aliphatic Hydrocarbon in Waters and Sediments from Wadi El Bey, Tunisia.

The contents, composition profiles, and sources of aliphatic hydrocarbons were examined in surface sediment and water samples collected from Wadi El Bey, in Tunisia, during different year seasons in 14 stations receiving domestic effluent, industrial discharge, and agricultural drainage wastes. The target substances were analyzed by gas chromatography coupled with mass spectrometric detection (GC/MS). Total concentrations of n-alkanes (n-C14-n-C38) ranged from 0.08 ± 0.01 to 18.14 ± 0.1 µg/L in waters and 0.22 ± 0.04 to 31.9 ± 24.6 µg/g in sediments, while total aliphatic fraction ranged from 0.08 ± 0.01 to 196 ± 140 µg/L in waters and 0.22 ± 0.04 to 1977 ± 1219 µg/g in sediments, which means that almost all sites were affected by hydrocarbon contents in sediments exceeding the recommended limit (100 µg/g). Various diagnostic indices (ADIs) were used to identify the hydrocarbon sources, namely the concentration ratios of individual compounds (n-C17/pristane, n-C18/phytane, pristane/phytane, n-C29/n-C17, n-C31/n-C19) as well as cumulative quantities (Carbon Preference Index, natural n-alkanes ratio, terrigenous/aquatic compounds ratio, unresolved complex mixture percentage, low molecular weight vs. high molecular weight homologues, Alkane Proxy and Terrestrial Marine Discriminants). In general, these indexes indicated that the origin of aliphatic hydrocarbons affecting sediments and waters of Wadi El Bey were linked to both biogenic and petrogenic inputs, attesting the impact of plankton and terrestrial plants and of oil contamination, respectively. The average carbon chain length computation (ACL), used to further index the chemical environment, ranged from 25.5 to 31.1 in sediments and 47.9-116 in waters. This finding could depend on the severe disturbances suffered by the ecosystem as a consequence of heavy anthropogenic inputs. Petroleum contamination associated with high eutrophication rates in Wadi El Bey must be strictly controlled, due to possible harmful effects induced on ecosystem and humans.

Detection of active pathogenic bacteria under stress conditions using lytic and specific phage.

In this study, we have monitored the potential activity of a foodborne and waterborne pathogenic bacterium, Salmonella typhi, under starvation conditions. The interaction between lytic phage and starved-VBNC pathogenic bacteria was studied to establish reliable methods for the detection of active cells before resuscitation. The analysis of phage kinetic parameters has demonstrated the flexibility of lytic with the quantity and mainly the quality of host cells. After 2 h of phage-starved-VBNC bacteria interaction, the reduction of phage amplification rate can reveal the ability of specific-lytic phage to recognize and to attach to their host cells with a probability of burst and release of infectious phages by active bacteria. After an extension of the latent period, the boost of the phage amplification rate was directly related to the positive interaction between potential intracellular 'engaged' phages and potential active bacteria. Furthermore, the modeling of the Salmonella-specific phage growth cycle in relationship with starved host cells can highlight the impact of the viability and the activity state of the host cells on the phage's growth cycle.

Enhancement of rhizocompetence in pathogenic bacteria removal of a constructed wetland system.

The main goal of the present study was to enhance the rhizobacterium potential in a horizontal subsurface flow constructed wetland system planted with Phragmites australis, through environmentally friendly biological approaches. The bioinoculation of antagonist bacteria has been used to promote higher rhizosphere competence and improve pathogenic bacteria removal from wastewater. The experiment was performed both with single and sequential bioinoculation. The results showed that strain PFH1 played an active role in pathogenic bacteria removal, remarkably improving inactivation kinetics of the pathogenic tested bacterium Salmonella typhi in the plant rhizosphere. The single bioinoculation of selected bacteria into the rhizosphere of P. australis improved the kinetics of S. typhi inactivation by approximately 1 U-Log10 (N/N0) (N is the number of viable cultured bacteria at time t, N0 is the number of viable and cultivable bacteria at time t0) compared to the control. By a series of multi-bioinoculations, the enhancement of pathogenic bacteria reduction compared to the inhibition rate in the pilot-scale control was of 2 U-Log10(N/N0). These findings suggested that this strain represents a promising candidate to enhance water purification in constructed wetlands.

Seasonal occurrence, source evaluation and ecological risk assessment of polycyclic aromatic hydrocarbons in industrial and agricultural effluents discharged in Wadi El Bey (Tunisia).

Polycyclic aromatic hydrocarbons are of great concern due to their persistence, bioaccumulation and toxic properties. The occurrence, source and ecological risk assessment of 26 polycyclic aromatic hydrocarbons in industrial and agricultural effluents affecting the Wadi El Bey watershed were investigated by means of gas chromatographic/mass spectrometric analysis (GC/MS). Total PAHs (∑ 26 PAH) ranged from 1.21 to 91.7 µg/L. The 4- and 5-ring compounds were the principal PAHs detected in most of 5 sites examined. Diagnostic concentration ratios and molecular indices were performed to identify the PAH sources. Results show that PAHs could originate from petrogenic, pyrolytic and mixed sources. According to the ecotoxicological assessment, the potential risk associated with PAHs affecting agricultural and industrial effluents ranged from moderate to high for both aquatic ecosystem and human health. The toxic equivalency factor (TEF) approach indicated that benzo[a]pyrene and benz[a]anthracene were the principal responsible for carcinogenic power of samples.

Seasonal Distribution, Source Identification, and Toxicological Risk Assessment of Polycyclic Aromatic Hydrocarbons (PAHs) in Sediments from Wadi El Bey Watershed in Tunisia.

Surface sediments were collected from the Watershed of Wadi El Bey in Tunisia to evaluate the degree of polycyclic aromatic hydrocarbons (PAHs) contamination. Sediments were collected during different seasons in 14 sites that received wastes from domestic effluent, industrial discharge, and agricultural drainage wastes. Twenty-six individual PAHs were analyzed. The total PAH contents (Σ PAHs) in surface sediments showed wide variability, ranging from 6.89 ± 0.05 to 340 ± 0.1 ng g-1. The 4-, 5-, and 6-ring compounds were the most abundant PAHs detected at the majority of sites. Diagnostic concentration ratios between pairs of PAHs and molecular indices, calculated with the purpose of drawing information about pollution sources, indicated that PAHs were of both petrogenic and pyrolytic origins. Toxic contaminants concentrations were determined according to the numerical effect-based sediment quality guidelines (SQGs). PAH levels did not exceed the SQGs, indicating that PAHs seem to pose low and occasional toxicity risks. Total carcinogenicity and mutagenicity (TEQBaP and MEQBaP) ranged from 0.08 to 65 ng and from 0.02 to 135.0 ng g-1 of dry weight, respectively. Among the seven carcinogenic PAHs, BaP accounted for the majority of the potency and could potentially be used as a unique indicator of PAH toxicity. This study provides a baseline to promote environmental protection programs and pollution monitoring/control in Watershed and coastal areas.

Phytotoxicity assessment of treated vegetable oily wastewater via environmentally coagulation/flocculation and membrane filtration technologies using lettuce (Lactuca sativa) seeds.

The present investigation highlights the necessity of monitoring some basic physico-chemical water quality indicators and their phytotoxic effect using ecotoxicological bioassays such as "seed germination tests." The phytotoxicity of raw and treated vegetable oil refinery wastewater (VORW) using different treatment processes was assessed through some physiological responses (relative seed germination (RSG), seedling elongation, and germination index (GI)) using Lactuca sativa cultivar. Biotest results of different raw water samples revealed a noticeable correlation between the organic matter content and water phytotoxicity. In fact, VORW showed a very low RSG (17 ± 0.7 to -47 ± 0.58%) and high phytotoxic effects (GI < 50%). The use of coagulation/flocculation (CF) allowed a satisfactory phytotoxicity removal where RSG obtained ranged from 83 ± 1.58 to 90 ± 1.2%. However, the effluent still presents high to moderate phytotoxicity since GI remained below 80% which indicates the presence of toxic elements remaining after CF treatment. When VORW were treated using membrane processes, their phytotoxicity was gradually decreased with the decrease in the membrane pore size. The use of microfiltration membranes (MF), with pore size of 5 µm, 1.2 µm, 0.45 µm, and 0.22 µm, showed RSG values ranged from 37 ± 1.15 to 77 ± 1.68% and GI of less than 80% indicating a moderate to high phytotoxicity. However, the use of ultrafiltration (UF) membranes with molecular weight cut-off (MWCO) of 100 kDa, 30 kDa, and 10 kDa made it possible to achieve an RSG of 100% and an IG exceeding 80% showing that the VORW-treated using UF does not exhibit any phytotoxicity effect. Hence, UF appears to be the most efficient and environmentally friendly technology that could be used for safely treated VORW irrigation purposes compared to CF and MF processes.

Development of a rapid method to detect and to monitor bacteriophage amplification in contact with viable but non-culturable bacteria.

We propose in this study to develop a rapid, reliable, and non-culture method to detect and estimate bacteriophage (phage) titre as an alternative to the routine use of the double agar overlay assay (DLA). The present method is based on the analysis of nanoparticle (NPs) dispersion/aggregation dynamic in interaction with the phage. Titanium dioxide nanoparticles (TiO2-NPs) were used as nanosensors to detect and monitor virions' titres in aqueous samples. Dispersion stability of TiO2-NPs in aqueous suspension was investigated using a UV-Visible spectrophotometer. The comparison of NP spectral profiles with and without phage elucidated the impact of phage's titre on NP dispersion/aggregation behaviour in an aqueous solution. Indeed, the increase of nanoparticle dispersion stability is correlated with the increase of phage titre. Thus, based on this result, the phage was considered as a bio-dispersant agent. The determination of area under spectral profiles limiting the UV region [200-400 nm] was allowed to quantify, and compare the NPs bio-dispersion rate, in relation with added phage at different titres. In this study, this method was applied to monitor the phage amplification cycle for the detection of bacteria in viable but non-culturable (VBNC) state after water treatment by photocatalysis. The analysis of NP bio-dispersion rate shows an increase of TiO2-NP dispersion stability correlated with an increase of free phage titration, mainly after the entry of target bacteria in VBNC state underestimated using a conventional method. Thus, this method could allow the establishment of new recommendations of wastewater treatment and assessment.

Constructed wetland as a low cost and sustainable solution for wastewater treatment adapted to rural settlements: the Chorfech wastewater treatment pilot plant.

The paper presents the detailed design and some preliminary results obtained from a study regarding a wastewater treatment pilot plant (WWTPP), serving as a multistage constructed wetland (CW) located at the rural settlement of 'Chorfech 24' (Tunisia). The WWTPP implemented at Chorfech 24 is mainly designed as a demonstration of sustainable water management solutions (low-cost wastewater treatment), in order to prove the efficiency of these solutions working under real Tunisian conditions and ultimately allow the further spreading of the demonstrated techniques. The pilot activity also aims to help gain experience with the implemented techniques and to improve them when necessary to be recommended for wide application in rural settlements in Tunisia and similar situations worldwide. The selected WWTPP at Chorfech 24 (rural settlement of 50 houses counting 350 inhabitants) consists of one Imhoff tank for pre-treatment, and three stages in series: as first stage a horizontal subsurface flow CW system, as second stage a subsurface vertical flow CW system, and a third horizontal flow CW. The sludge of the Imhoff tank is treated in a sludge composting bed. The performances of the different components as well as the whole treatment system were presented based on 3 months monitoring. The results shown in this paper are related to carbon, nitrogen and phosphorus removal as well as to reduction of micro-organisms. The mean overall removal rates of the Chorfech WWTPP during the monitored period have been, respectively, equal to 97% for total suspended solids and biochemical oxygen demand (BOD5), 95% for chemical oxygen demand, 71% for total nitrogen and 82% for P-PO4. The removal of E. coli by the whole system is 2.5 log units.

Monitoring of methylene blue monomers and dimers to control the bacterialogical water quality including application to photocatalysis.

In this study, we propose the development of a rapid and reliable method to control and to monitor microbial water quality. The methylene blue (MB) decolorization assay was based on the analysis of spectral profiles of dye in interaction with a different bacterial concentration. The determination of dye decolorization rate (DDR) shows a correlation between the MB reduction rate and the bacterial density. Moreover, the kinetic of the monomer and dimer equilibrium of MB in water mainly, the monitoring of bounded MB species in relationship with a knowed concentration of target bacteria, was allowed to establish a relationship between MB decolorization rate and bacterial density. Furthermore, this method was applied to evaluate the water quality after photocatalysis. Based on this method, the photocatalytic effects on bacterial density was highlighted by the decrease in DDR after photocatalytic treatment with fractioned times (0 to 5 h); this increase was followed by a decrease of bounded MB species and, an increase in free MB forms miming the reduction of bacterial density due to the biocide effects of photocatalysis process. However, the analysis of spectra profiles shows a weak but a continuous decrease in bounded MB dimer and monomer forms in the treated water samples exempt of culturable bacteria. Moreover, the MB spectra profiles were tended toward a negative control spectrum without superposition. Thus, the possibility of the presence of viable but non-culturable bacteria was expected; therefore, to optimize this tertiary water treatment process, an extending on proceeding time was recommended to avoid the bacterial resuscitation after photocatalysis.

Use of the catalytic complex TiO2/red cabbage anthocyanins to reduce the biofilm formation by planktonic bacteria.

The bacterial cells dwelling within the biofilm usually develop resistance against common disinfectants. In this current study, to improve the effectiveness of photocatalytic treatment, a natural sensitizer in combination with unsupported titanium dioxide nanoparticles (TiO2-NPs) was used to optimize the absorbance of NPs in the visible region and, to enhance the catalytic activity of the semiconductor. Different kinetic parameters were determined according to the first-order and the biphasic models to evaluate the ability of tested bacteria to form biofilm under different photocatalytic treatment conditions. As a result, the addition of red cabbage anthocyanins (RCA) as photosensitizer allows the enhancement of biocide activity of TiO2-NPs and the reduction of biofilm formation by tested bacteria.

The application of phage reactivation capacity to sens bacterial viability and activity after photocatalytic treatment.

We purpose in this study to develop a reliable and low-cost method for the detection of Viable but nonculturable (VBNC) bacteria. Indeed, after water disinfection, injured-VBNC bacteria can be underestimated using conventional assessment methods, causing false-negative results and, posing a significant and potential health risk. The VBNC bacterial survival strategy can hide the real microbial quality of treated water. To overcome this bacterial assessment limitation, we were used a specific and lytic phage to monitor the presence of active bacteria; Pseudomonas aeruginosa after photocatalytic treatment. Within 2 h of phage-target bacteria contact, the reduction of phage amplification rate (At) can reveal the ability of specific-lytic phage to recognize and to attach to their host cells with a probability of new infectious phages release despite their lose of cultivability in the usual media. The determination of phage reactivation coefficient (Rt) after 2 and 8 h of phage-target cell contact time reveals the ability of phages to reactive their infectivity and their amplification in positive correlation with their host cells viability and activity. The increase in phage reactivation coefficient (Rt) after an extension of the latent period was directly related to the positive interaction between infectious phages and potential active bacteria. The use of this method can improve the water disinfection process and avoid public health-hazardous especially related to the resuscitation of active-nonculturable bacteria mainly for pathogenic bacteria.

Effect of photocatalysis (TiO2/UVA) on the inactivation and inhibition of Pseudomonas aeruginosa virulence factors expression.

Water disinfection using visible light-active photocatalyst has recently attracted more attention due to its potential to inactivate microbes. In this study, we have investigated the efficiency of photocatalysis (TiO2/UVA) on the inactivation of Pseudomonas aeruginosa and the attenuation of its virulence factors. For this aim, the photocatalytic effects of TiO2/UVA on the cultivability and viability of P. aeruginosa were investigated. Furthermore, during the photocatalysis, the morphology of the bacterial cells was examined by atomic force microscopy (AFM) while the virulence factors were assessed by protease and lipase activities in addition to the mobility and communication of cells. The results revealed that during the photocatalysis the bacterial cells lost their cultivability and viability on agar under the action of the reactive oxygen species generated by the photocatalytic reaction. In addition, AFM observations have shown a damage of the bacterial membrane and a total disruption of the bacterial cells. Moreover, the major virulence factors such as biofilm, lipase and protease expression have been markedly inhibited by TiO2/UVA treatment. In addition, the bacteria lost their ability of communication 'quorum sensing' and mobility with twitching and swarming types after 60 min of photocatalytic treatment. Accordingly, TiO2/UVA is an effective method to reduce P. aeruginosa virulence and to prevent biofilm formation.

Process optimization via response surface methodology in the physico-chemical treatment of vegetable oil refinery wastewater.

The present paper investigates the efficiency of coagulation/flocculation process using aluminum sulfate as coagulant and CHT industrial flocculent as coagulant aid/flocculent in the treatment of vegetable oil refinery wastewater (VORW). The process optimization was conducted in two steps, jar test experiments for preliminary evaluation to identify the most influencing factors and response surface methodology using Box-Behnken design to investigate the effects of three major factors and their interactions. The variables involved were the coagulant concentration (X1), flocculent dosage (X2), and initial pH (X3) of water samples, while the responses were COD removal (Y1) and residual turbidity (Y2). The optimal conditions obtained by solving the quadratic regression models, as well as by analyzing the response surface contour plots, were as follows: 2.4 g/L of coagulant (aluminum sulfate), 60.05 mg/L of flocculent, and about 9.23 as initial pH. Under these conditions, the coagulation/flocculation treatment was able to achieve 99% of COD removal with total turbidity elimination (100% removal). Analysis of variance showed high variance coefficient (R2) values of 0.929 and 0.836 for COD and turbidity removals, respectively, thus ensuring a satisfactory adjustment of the second-order regression model with the experimental data. This statistical design methodology was demonstrated as an efficient and feasible approach for the optimization of coagulation/flocculation treatment.

Comparative study of Gram-negative bacteria response to solar photocatalytic inactivation.

Solar photocatalytic inactivation of Gram-negative bacteria with immobilized TiO2-P25 in a fixed-bed reactor was modeled with simplified kinetic equations. The kinetic parameters are the following: the photocatalytic inactivation coefficient (kd,QUV), the initial bacterial reduction rate (A) in the contact with the disinfecting agent, and the threshold level of damage (n) were determined to report the effect of QUV/TiO2-P25 on bacterial cultivability and viability and to compare the response of bacterial strains to photocatalytic treatment. In addition, the integration of the reactivation coefficient (Cr) in the photocatalytic inactivation equation allowed evaluating the ability of bacterial reactivation after photocatalytic stress. Results showed different responses of the bacteria strains to photocatalytic stress and the ability of certain bacterial strains such as Escherichia coli ATCC25922 and Pseudomonas aeruginosa ATCC4114 to resuscitate after photocatalytic treatment.

Stress response of mammalian cells incubated with landfill leachate.

Environmental contamination from landfill leachate has been linked to disturbances in human health. Often, however, only global parameters, such as dissolved organic content, chemical oxygen demand, and 5-d biological oxygen demand, are used to evaluate wastewater quality. In the present study, we determined leachate cytotoxicity and stress response of leachate-treated mammalian cells using in vitro bioassays and other molecular techniques. The modified E-screen assay using human breast cancer MCF-7 cells was used to determine the estrogenic potential and/or cytotoxicity of water samples from two solid-waste landfills in Tunisia. The cytotoxicity mechanism of the leachate was determined by DNA fragmentation and lactate dehydrogenase assays. The stress response of heat shock protein (HSP) 47-positive Chinese hamster ovary cells treated with leachate also was determined. Proteomics analyses and real-time reverse transcription-polymerase chain reaction were used to determine and confirm the enhanced expressions of certain stress-related proteins. Results showed that the leachate samples generally did not have estrogenic activity. Instead, they were cytotoxic toward MCF-7 cells, and the cytotoxicity was by necrosis during the early stages of incubation. Leachate also enhanced the expression of HSP and various stress-related proteins, such as heterogeneous nuclear ribonucleoprotein E1, phosphoglycerate mutase, and nuclear matrix protein 200, in MCF-7 cells. These can be considered as survival mechanisms against leachate-induced cytotoxicity.

ZnO Nanorods with High Photocatalytic and Antibacterial Activity under Solar Light Irradiation.

ZnO nanorods (NRs) with an average length and diameter of 186 and 20 nm, respectively, were prepared through a mild solvothermal route and used as photocatalysts either as dispersed powder or immobilized on glass slides. The ZnO NRs were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-ray diffraction (XRD). Dispersed ZnO NRs and, to a lesser extent, immobilized ZnO NRs were demonstrated to exhibit high photocatalytic activity under simulated sunlight of low intensity (5.5 mW/cm²) both for the degradation of the Orange II dye and for Escherichia coli bacterial decontamination (2.5-fold survival decrease after 180 min irradiation for immobilized NRs). SEM, atomic force microscopy (AFM), fluorescence spectroscopy, and epifluorescence microscopy demonstrate that cell surface damages are responsible of bacterial inactivation. The immobilized ZnO NRs could be reused up to five times for bacterial decontamination at comparable efficiency and therefore have great potential for real environmental applications.

Landfill leachate treatment with ozone and ozone/hydrogen peroxide systems.

In the search for an efficient and economical method to treat a leachate generated from a controlled municipal solid waste landfill site (Jebel Chakir) in the region of greater Tunis in Tunisia, ozone alone and ozone combined with hydrogen peroxide were studied. The leachate was characterised by high COD, low biodegradability and intense dark colour. A purpose-built reactor, to avoid foaming, was used for the study. It was found that ozone efficacy was almost doubled when combined with hydrogen peroxide at 2g/L but higher H(2)O(2) concentrations gave lower performances. Enhancement in the leachate biodegradability from about 0.1 to about 0.7 was achieved by the O(3)/H(2)O(2) system. Insignificant changes in pH that may due to buffering effect of bicarbonate was found. A small decrease in sulphate concentrations were also observed. In contrast, chloride concentration declined at the beginning of the experiment then increased to reach its initial value. Estimates of the operating costs were made for comparison purposes and it was found that the O(3)/H(2)O(2) system at 2g/L H(2)O(2) gave the lowest cost of about 3.1TND( approximately 2.3USD)/kgCOD removed.

Assessment of estrogenic activity in Tunisian water and wastewater by E-screen assay.

Wastewater and surface water samples from three wastewater treatment plants (WWTPs) and three rivers in Tunisia were assayed for estrogenic activity using the E-screen assay and enzyme-linked immunosorbent assay (ELISA). Results showed that all the Tunisian raw wastewater samples as well as the Roriche river water sample induced a strong proliferative response in human MCF-7 breast cancer cells. Tunisian raw wastewater had an average 17beta-estradiol content of 2,705.4 pg/ml, whereas that of the Roriche river was 36.7 pg/ml, which is sufficient for inducing endocrine-mediated responses in aquatic organisms. Results further showed that the Mornag WWTP, which uses the activated-sludge treatment system, has a higher estrogen removal efficiency than the stabilization ponds of the Gammart and pilot WWTPs. This study, which is the first of such studies in Tunisia, and probably the first in the North African region, underscores the need to detect and monitor the estrogenic activity of water and wastewater, given the scarcity of water in Tunisia and the detrimental impact of endocrine-disrupting compounds on the physiology of both animals and humans.

Impact of influent wastewater quality on nitrogen removal rates in multistage treatment wetlands.

Nitrogen removal in treatment wetlands is influenced by many factors, and the presence of electron donors (biodegradable organic matter) and electron acceptors (nitrate ions) is the main limiting one; for obtaining these conditions, multistage treatment wetlands (MTWs) are required, where an extensive nitrification can be obtained in the first stages under aerobic conditions leaving then to the following anoxic/anaerobic stages the duty of the denitrification. Most of the biodegradable organic matter is however oxidised in the first stages, and therefore, the inlet to the denitrification beds is usually poor of easily degradable carbon sources. This study is comparing the long-term performances obtained at several MTWs operating in Europe (North and South) and North Africa in order to understand if there is a significant avail in making use of the influent chemical oxygen demand (COD)/N ratio during the design phase for ensuring proper performances in terms of N overall removal. The statistic analysis performed in this study have shown that MTWs are capable to ensure sufficient removal of both organic and nutrients even in unfavourable proportions of macronutrients (C and N). The usual assumptions for conventional biological treatment systems concerning adequate C/N ratios seem to be dubious in case of wastewater treatment in MTWs.