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Special Issue Water and Human Settlements of the Future.-- 16 pages, 4 figures, 4 tables, supplementary materials https://www.mdpi.com/article/ 10.3390/w13192731/s1, Cork matrices biofunctionalized with Moringa oleifera seed extracts (MoSe) have potential for use as a biofilter with antibacterial properties to reduce waterborne pathogens. The aim of this study was to evaluate the effect of cork biofunctionalized with active antimicrobial compounds of MoSe (f-cork) on the inhibition of Escherichia coli (InhEc). The LacZ gene from a strain of E. coli was used as the target sequence using viability quantification Polymerase Chain Reaction (qPCR) and differentiation of viable and dead bacteria through selective cell viability PMA staining. To perform this, a 27−4 fractional factorial design and a biofiltration system were used to evaluate the effect of the active protein in MoSe immobilized in granulated cork on InhEc. We found that the potential for antimicrobial activity increased with f-cork for an effective maximal bacterial reduction (99.99%; p < 0.05). The effect of f-cork functionalized with MoSe on E. coli viability was of 0.024% and 0.005% for the cells exposed to PMA, respectively, being the relevant conditions in treatment 2: (0 L/min) without aeration, (5%) MoSe and (5 mm) cork particle. In conclusion, the f-cork functionalized with MoSe presented biosorbent and antibacterial properties that effectively reduced the E. coli growth, This research was funded by Mexico-CONACYT, grant number 3006060 and grant by the UNESCO Chair on Sustainability of the Polytechnic University of Catalonia (Spain), With the institutional support of the ‘Severo OchoaCentre of Excellence’ accreditation (CEX2019-000928-S).

In this study, the removal of cadmium (Cd) by free-living Oscillatoria sp. was studied. Our results showed that maximal Cd removal efficiency (~60%) by the cyanobacterial culture was achieved within 12-24 h in the presence of 5.0 or 25.0 mg/L of Cd. The mechanisms underlying this phenomenon were explored by elemental analysis and FTIR-ATR spectroscopy. It was found that metal adsorption by negatively charged functional groups in the cyanobacterial biomass was the main mechanism used by Oscillatoria sp. to remove metal from the aqueous medium, followed by Cd bioaccumulation into living cells. Additionally, Cd-exposed microalgae showed increased oxidative stress (MDA formation), a decreased dehydrogenase activity, a higher amount of soluble carbohydrates and a decreased total carotenoid concentration, as compared to the control cells. These results suggest that Oscillatoria sp. improved its antioxidative defense system under stressful conditions, through carotenoid-mediated ROS quenching and induction of carbohydrate catabolism, in order to counteract the oxidative damage and preserve the photosynthetic machinery and cellular energetics. In fact, no significant reduction in Oscillatoria sp. cell density, total protein amount, and chlorophyll a content was observed after 24-h Cd exposure, even at the highest metal concentration tested (i.e., 25.0 mg/L). Hence, the presented results are the first to describe some new insights about the metabolic and physiological behavior of living Oscillatoria sp. during Cd remediation, and open up the possibility of finding an equilibrium that maximizes metal removal performance with an active cyanobacterial metabolism, to achieve a rewarding and sustainable management of industrial metal-polluted wastewater. Fil: Carralero Bon, Iván. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Facultad de Química e Ingeniería del Rosario. Departamento de Investigación Institucional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina Fil: Salvatierra, Lucas Matías. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Facultad de Química e Ingeniería del Rosario. Departamento de Investigación Institucional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina Fil: Lario, Luciana Daniela. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Facultad de Química e Ingeniería del Rosario. Departamento de Investigación Institucional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina Fil: Morató Farreras, Jordi. Universidad Politecnica de Catalunya. Escuela Superior de Ingenierías Industrial, Aeroespacial y Audiovisual de Terrassa. Cátedra Unesco de Sostenibilidad.; España Fil: Perez, Leonardo Martin. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Facultad de Química e Ingeniería del Rosario. Departamento de Investigación Institucional; Argentina. Universidad Politecnica de Catalunya. Escuela Superior de Ingenierías Industrial, Aeroespacial y Audiovisual de Terrassa. Cátedra Unesco de Sostenibilidad.; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina

Fil: Aguilara, Lorena. Universitat Politécnica de Catalunya; España Fil: Aguilara, Lorena. UNESCO Chair on Sustainability; Francia Fil: Gallegos, Ángel. UNESCO Chair on Sustainability; Francia Fil: Gallegos, Ángel. Universitat Politécnica de Catalunya; España Fil: Pérez, Leonardo Martín. Pontificia Universidad Católica Argentina. Facultad de Química e Ingeniería del Rosario. Instituto de Investigaciones en Ingeniería Ambiental, Química y Biotecnología Aplicada; Argentina Fil: Pérez, Leonardo Martín. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentinas Fil: Pérez, Leonardo Martín. Politécnica ecnica de Catalunya; España Fil: Pérez, Leonardo Martín. NESCO Chair on Sustainability; Francia Fil: Arias, Carlos A. University of Aarhus. Department of Biological Sciences; Dinamarca Fil: Arias, Carlos A. Aarhus University. Centre for Water Technology; Dinamarca Fil: Rubio, Raquel. Universitat Politécnica de Catalunya; España Fil: Rubio, Raquel. UNESCO Chair on Sustainability; Francia Fil: Haulani, Leila. Universitat Politécnica de Catalunya; España Fil: Haulani, Leila. UNESCO Chair on Sustainability; Francia Fil: Garciá Raurich, Josep. Universitat Politécnica de Catalunya. Centro de Investigación en Seguridad y Control Alimentario; España Fil: Pallarés, Marc. Grupo de Desarrollo Empresarial; España Fil: de Pablo, Joan. Universitat Politécnica de Catalunya. Resource Recovery and Environmental Management; España Fil: Morató, Jordi. Universitat Politécnica de Catalunya; España Fil: Morató, Jordi. UNESCO Chair on Sustainability; Francia Abstract: In this work, we have evaluated the impact of intermittent induced aeration in total nitrogen (TN), ammonia (NH4-N) and nitrate-nitrogen (NO3-N) removal in four pilot-scale vertical flow constructed wetlands (VFCW) (two aerated two non-aerated) using cork by-product or gravel as the filter material and planted with Phragmites australis. Both aerated and non-aerated systems achieved high COD and BOD5 elimination rates ( 90%) at the end of the 5-month test period. However, the aerated systems presented maximal COD and BOD5 removal from the third month of operation onwards since air supply favored the oxidative bioprocesses occurring within the wetlands. Cork and gravel aerated VFCW also proved to be more efficient (p

A series of ionic complexes with a comb-like architecture and a nearly stoichiometric composition were prepared from bacterial poly(gamma,dl-glutamic acid) (PGGA) and alkyltrimethyl phosphonium bromides (nATMP·Br) bearing long linear alkyl chains with even numbers of carbon atoms (n) ranging from 12 to 22. The nATMP·PGGA complexes were non-water soluble but readily soluble in organic solvents, and they displayed a high thermal stability. Combined DSC and XRD studies revealed that these complexes adopted an amphiphilic layered structure with the polypeptide chain and the alkyl chain separated in two differentiated phases with a nanoperiodicity that increased steadily with the length of the alkyl chain. The paraffinic phase was found to be partially crystallized in an extent that decreased with n, so that complexes with n = 12 and 14 did not show any sign of crystallinity whereas those with n from 16 to 22 showed crystalline melting in the ~30–70 °C range. The structural transitions taking place by temperature effects were characterized by simultaneous SAXS/WAXS using synchrotron radiation at real time. In all cases, a shortening of the layer periodicity occurred upon heating with recovering of the initial structure after cooling. nATMP·PGGA with n = 16 showed strong antimicrobial activity against both E. coli and S. aureus, a property that could be related to the weak dissociation of the complexes happening upon incubation in water. The structure and properties of these complexes where comparatively discussed taking as reference their analog complexes made from PGGA and alkyltrimethylammonium bromides already studied by us.

Phosphorus (P) is a limited resource and can promote eutrophication of water streams and acidification of oceans when discharged. Crushed autoclaved aerated concrete (CAAC), a by-product from demolition, has shown great potential for recovering P. The potential of CAAC to be used in nature-based solutions as a P-reactive filter medium was evaluated by performing preliminary batch essays. Here, we evaluated the interactions and main effects of the initial concentration of P (Pi, 5, 10 or 20 mg L&minus, 1), particle size (PS, 4 or 5 mm) and contact time (CT, 60, 180, 360, 720 and 1440 min) upon the removal. We performed physical and chemical characterization to understand the removal processes. Data collected were fitted in adsorption kinetic models. The statistical analysis showed a significant interaction between CT and Pi, with the combination of its main effects stronger on P removal than each one separately. Intriguingly, we noticed that the higher the concentration of Pi, the faster and higher the removal of P. Contrary to expectations, PS 5 mm showed higher removal rates than PS 4 mm, indicating that besides adsorption, other unidentified chemical processes are in place. Further studies using columns/pilots with real wastewater are recommended for a future follow-up.

9 pages, 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

Ethyl alpha-N-lauroyl L-arginate hydrochloride (LAE) was coupled with hyaluronic acid (HyA) 2 to form ionic complexes with LAE to HyA ratios of 1:1 and 1:2. The complexes were extensively 3 characterized by FTIR and NMR spectroscopies and their thermal properties evaluated by 4 thermogravimetry and calorimetry. Thin films prepared from these complexes by casting 5 displayed a smectic-like structure based on an ordered arrangement of LAE and HyA layers 6 with a nanometric periodicity of 3.8-3.9 nm. Films immersed in water at pH 7.4 and 5.5 7 dissociated to deliver free LAE to the environment and reaching the equilibrium in few hours. 8 The biocide activity of these films against both Gram-positive and Gram-negative bacteria was 9 preliminary assessed by the liquid medium method, and shown to be notable in both cases. The 10 antibacterial property of the complexes was found to increase with the content of LAE and to be 11 particularly efficient against Gram-negative S. enterica bacteria.

Reusing by-products is an important strategy to ensure the preservation of natural capital and climate change mitigation. This study aimed at evaluating the potential of cork granulates, a by-product of winery industry, as an organic carbon (OC) source for the treatment of hydroponic wastewaters. First, chemical characterization was performed and discussed. Secondly, batch studies were performed using synthetic hydroponic wastewater to understand the role of particle size (PS), pH and contact time (CT) on the release of OC. The suberin is the major compound, representing >50%. It was noticed that a variance on the content of suberin across species, within the same species and depending on the extraction part (belly, cork and back) could be expected. >60% of the sample is composed by carbon while

12 pages, 14 figures, 3 tables, supplementary data https://doi.org/10.1016/j.scitotenv.2018.10.426, Agricultural practices have raised the level of nutrients reaching aquifers. In Europe, nitrate pollution is considered as one of the main threats for the quality of groundwater in agricultural areas. Treatment wetlands (TWs), also known as Constructed Wetlands, are used for groundwater treatment in areas with an important concentration of nitrogen compounds; total nitrogen removal depends on the type and operation scheme. Cork by-product from the industry has shown clear adsorbent properties to remove organic pollutants. The work is focused on the characterization of microbial communities involved in the nitrate‑nitrogen removal process in groundwater polluted from agricultural activities. The experimental design allowed the comparison of nitrate removal efficiency depending on the filter media material, cork by-product or gravel, used in two hybrid TWs (a vertical flow cell followed by a horizontal subsurface flow cell), installed in areas close to two irrigated agricultural plots at the Lleida plain area (Spain). Both physicochemical and microbial results were consistent and confirm the nitrate removal efficiency using cork as a filter media. A significant (p = 0.0025) higher removal in Bellvís TW using cork compared with the Vilanova de la Barca gravel system was observed, achieving a removal rate from 80 to 99% compared to the 5–46%, respectively. Regarding the community composition of the two different TWs, microorganisms were mainly related to the phylum Proteobacteria, and included members found to be key players in the nitrogen cycle, such as ammonia and nitrite oxidizers, as well as denitrifiers. Also, the group Bacteroidetes turns to be another abundant phylum from our bacterial dataset, whose members are suggested to be strongly involved in denitrification processes. Some groups showed to prevail depending on the type of media (cork or gravel); Firmicutes and Delta and Epsilonproteobacteria had a significant higher abundance in the TW with cork, while Acidobacteria and Planctomyces were prevalent in gravel. Therefore, cork could be an alternative material used by treatment wetlands to minimize the impact in the environment caused by nitrogen pollution in groundwater bodies, To LIFE Programme, the EU's financial instrument supporting environmental, nature conservation and climate action projects throughout the EU, that supports REAGRITECH LIFE 11 ENV/ES/579

Amphiphilic ionic complexes of hyaluronic acid and alkyltrimethylphosphonium soaps with alkyl chains containing even numbers of carbons from 12 to 22 have been produced. The complexes have a nearly stoichiometric composition, are non-water soluble, and are stable to heat up to temperatures above 200 °C. These complexes are amphiphilic and able to adopt a biphasic structure with the paraffinic and polysaccharide phases ordered arranged with a periodicity ranging between 3 and 5 nm depending on n. The paraffinic phase in complexes with n ≥ 18 was crystallized and showed melting at temperatures between 58 and 70 °C depending on the n value. The complexes decomposed upon incubation in water under physiological conditions, and undergone extensive biodegradation by the action of hyaluronidases. Biocide assays carried out in both solid and liquid media demonstrated a high antimicrobial activity of the complexes against Gram-positive S. aureus but moderate against Gram-negative E. coli and C. albicans fungi.

© 2017 by the author. The biocide agent LAE (ethyl aN-lauroyl l-arginate chloride) was coupled with poly(y-glutamic acid) (PGGA) to form stable ionic complexes with LAE:PGGA ratios of 1 and 0.5. The nanostructure adopted by these complexes and its response to thermal changes were examined in detail by Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) using synchrotron radiation in real time. A layered biphasic structure with LAE filling the space between the polypeptidic sheets was adopted in these complexes. The complexes were stable up to above 250 °C, non-water soluble, and were able to form consistent transparent films. The release of LAE from the complexes upon incubation in aqueous buffer was examined and found to depend on both pH and complex composition. The antibacterial activity of films made of these complexes against Gram-positive (L. monocytogenes and S. aureus) and Gram-negative (E. coli and S. enterica) bacteria was preliminary evaluated and was found to be very high against the formers and only moderate against the later. The bactericide activity displayed by the LAE·PGGA complexes was directly related with the amount of LAE that was released from the film to the environment

The change in nitrogen balance causes many environmental and socioeconomic impacts. In relation to food production and nitrogen release in wastewater systems, wastewater and sludge discharge and mineral fertilizer use intensify nitrogen imbalance of a region. The replacement of mineral fertilizer by nitrogen from treated wastewater, biosolids, and treated urine is a promising alternative. This work presents a model to support decision taking for the management of reactive nitrogen flows in wastewater systems based on system dynamics. Six scenarios were simulated for nitrogen flows in wastewater systems and related components.

Rural areas of developing countries require low-cost treatment systems to purify wastewater which is contaminated with pesticides and organic matter. This work evaluated for six months the simultaneous removal of chlorpyrifos and dissolved organic matter in water using four horizontal sub-surface flow constructed wetlands (SSFCW) at a pilot scale, that were planted with Phragmites australis at 20 ± 2 °C water temperature. In each wetland, three concentrations of chlorpyrifos and three of dissolved organic carbon (DOC) were tested by liquid chromatography and an organic carbon analyzer respectively. The pesticide and DOC were added to the wetlands in synthetic wastewater. For the experiments, four wetlands of equal dimensions were used, with granular material of igneous rocks, 3.9–6.4 mm in diameter and at a depth of 0.3 m with a layer of water 0.2 m deep. For each treatment, regular sampling was carried out for the influent and effluents. As a supporting feature NH4+, NO3− and PO43− were quantified and in situ measurements of dissolved oxygen (DO), pH, electrical conductivity, water temperature and redox potential were taken. The overall removal of the chlorpyrifos (92.6%) and DOC (93.2%) was high, as was DOC removal as a function of pesticide concentration in the influent. The minimum magnitude (92.0%) was reached with 425.6 μg L−1 of chlorpyrifos and, with the highest pesticide removal (96.8%). At lower concentrations of the agrochemical, DOC removal increased. The removals were possibly due to mineralization processes, biological decomposition and sorption in plants. These findings demonstrate that SSFCW are capable of simultaneously removing dissolved organic matter and organophosphate pesticides such as chlorpyrifos, which indicate that chlorpyrifos did not interfere with the removal of organic material.

Aims: It is difficult to determine the effects of bactericidal compounds against bacteria in a biofilm because classical procedures for determining cell viability require several working days, multiple complicated steps and are frequently only applicable to cells in suspension. We attempt to develop a compact, inexpensive and versatile system to measure directly the extent of biofilm formation from water systems and to determine the viability of respiring bacteria in high surface biofilms. Methods and Results: It has been reported that the reduction of tetrazolium sodium salts, such as XTT (sodium 3,3′-[1-[(phenylamino)carbonyl]-3,4-tetrazolium]Bis(4-methoxy)-6-nitro)benzene sulfonic acid hydrate), during active bacterial metabolism can be incorporated into a colorimetric method for quantifying cell viability. XTT is reduced to a soluble formazan compound during bacterial aerobic metabolism such that the amount of formazan generated is proportional to the bacterial biomass. Conclusions: We show here, for the first time, that this colorimetric approach can be used to determine the metabolic activity of adherent aerobic bacteria in a biofilm as a measure of cell viability. This technique has been used to estimate viability and proliferation of bacteria in suspension, but this is the first application to microbial communities in a real undisturbed biofilm. Significance and Impact of the Study: This simple new system can be used to evaluate the complex biofilm community without separating the bacteria from their support. Thus, the results obtained by this practice may be more representative of the circumstances in a natural system, opening the possibility to multiple potential applications.

The synthesis, characterization and comparative evaluation of properties of two series of cationic PBS copolyesters bearing respectively ammonium and tributylphosphonium side groups, are reported. The copolyesters with contents in ionic units up to 50 mole-% as well as the fully ionic homopolyesters were prepared by polycondensation in the melt catalyzed by CALB or TBT. Their Mn ranged between 20,000 and 5000 g mol-1 depending of composition and the type of ionic group that is involved. All the copolyesters were non-water soluble and showed good thermal stability. They were semicrystalline with melting temperatures and enthalpies decreasing with the ionic contents. The interactions interplayed by the ionic groups restricted largely the molecular mobility and caused a significant increase in the melt viscosity and glass transition temperature of PBS and a decrease in crystallization rate. Both ammonium and phosphonium containing PBS copolyesters were able to be coupled with sulfonated PBS to generate ionic polymer blends with modified crystallizability. The presence of both ammoniums and phosphoniums provided PBS with remarkable antimicrobial activity against gram-positive and gram-negative bacteria.

This study investigates the effects of chlorothalonil (CLT) on chemical oxygen demand (COD) and dissolved organic carbon (DOC) in pilot-scale horizontal subsurface flow constructed wetlands (HSSFCW) planted with Phragmites australis. Physicochemical parameters of influent and effluent water samples, microbial population counting methods and statistical analysis were used to evaluate the influence of CLT on organic matter removal efficiency. The experiments were conducted on four planted replicate wetlands (HSSFCW-Pa) and one unplanted control wetland (HSSFCW-NPa). The wetlands exhibited high average organic matter removal efficiencies (HSSFCW-Pa: 80.6% DOC, 98.0% COD; HSSFCW-NPa: 93.2% DOC, 98.4% COD). The addition of CLT did not influence organic removal parameters. In all cases CLT concentrations in the effluent occurred in concentrations lower than the detection limit of the analytical method. Microbial population counts from HSSFCW-Pa showed significant correlations among different microbial groups and with different physicochemical variables. The apparent independence of organic matter removal and CLT inputs, along with the CLT depletion observed in effluent samples demonstrated that HSSFCW are a viable technology for the treatment of agricultural effluents contaminated with organo-chloride pesticides like CLT.

IntroductionNowadays, as in the past, our knowledge of microbiology islimited because of the capacity of available technologytools. In recent decades, molecular techniques are playinga central role in the understanding of the microbial world. Inthis sense, a complete scientific evaluation of biologicalsamples is not possible without using molecular biology.The most influential technique has probably been the poly-merasechainreaction(PCR),whichallowsustoworkbeyondclassicalmicrobiology.WithPCR,insomecases,theneedforculture may be avoided. Nevertheless, until the developmentof real-time PCR, our evaluation of the microbial complexityhas been merely qualitative.Real-time PCR is an evolution of the conventional end-point PCR wherein the amplification of a target gene and itsfluorescence detection occurs simultaneously during eachcycle. Different strategies based on the use of nonspecificDNA intercalating dyes or specific fluorescent probes existfor linking target amplification to fluorescence detection.Regardless of the fluorescence emission mechanisms in-volved, real-time PCR enables both detection and quantifica-tion of a genetic target by a continuous fluorescencemonitoring of DNA amplification (Higuchi et al. 1993).An advantage in real-time PCR isthat target quantificationsensitivity is independent of the copy number within thesample (Freeman et al. 1999). Moreover, the key aspect thatdifferentiates real-time PCR from previous semi-quantitativemethodologies is that target DNA concentration can be deter-mined from the fractional cycle at which a threshold amountof target DNA is produced (i.e., measured signal exceeds abackground level), set at a point where amplicon DNA justbecomesdetectablebutisstillwithintheexponentialphaseoftheamplification(Higuchietal.1993;Rasmussen2001).Thisquantificationcycle(Cq)(orcyclethreshold,Ct)areinverselyproportionaltotheamountoftargetnucleicacidinthesample(i.e., the lower the Cq value, the greater the amount of targetnucleic acid in the sample). This approach ensures that inter-fering factors associated with later stages of the amplificationare minimized and considerably improves the measurementprecision (Rasmussen 2001).One of the most important points is that real-time PCRallows quantitative measurements. In order to estimate atarget concentration in environmental samples, absolutequantification can be performed using a standard curveconstructed by amplifying known amounts of target DNA(Rutledge and Cote 2003). Nowadays, this approach is themost commonly used in environmental microbiology.Real-time PCR has many advantages over conventionalendpoint PCR including speed, broad dynamic range ofquantification, reduced risk of contamination, and goodsensitivity. However, despite all these objective strengths,real-time PCR sensitivity is not better than conventionalPCR if a protocol validation is not performed (Bastien etal. 2008).

This work used subsurface flow constructed wetlands, planted with Phragmites australis, using 2 water depths and 2 sizes of granular material, in order to find the optimal conditions for the remova...

The advent of quantitative PCR has improved the detection of human viral pathogens in the environment. However, a serious limitation of this method may arise from the inability to discriminate between viruses that are infectious and viruses that have been inactivated and do not represent a human health hazard. To assess whether propidium monoazide (PMA) pre-treatment is a good approach to inhibiting DNA amplification from non-infectious viruses, bacteriophage T4 survival was measured using cell culture titration and real-time PCR with and without PMA pre-treatment. Heat (85 degrees C) and proteolysis methods were carried out. After these inactivation treatments, the results indicated that the PMA pre-treatment approach is not appropriate for differentiating infectious viruses. However, when a heat treatment at 110 degrees C was undertaken, PMA pre-treatment did allow differentiation of non-infectious from infectious viruses. In this case, effective binding of PMA to bacteriophage T4 DNA could be taken to indicate capsid damage. Therefore, PMA pre-treatment may be appropriate for assessing effective disinfection treatments and for a more reliable understanding of the factors that contribute to viral inactivation through capsid damage monitoring. The PMA-PCR approach could be useful as a rapid and inexpensive analytical tool for screening and evaluation of the efficacy of disinfectants.

Microbial quality in water distribution systems is strongly affected by the development of microbial biofilms. Production and release of microbial cells by the biofilm affect microbial levels in the water column and in some cases this fact constitutes a public health concern. In this study, we attempt to analyze in which way the existence of different episodes of chlorine depletion affects both biofilm formation and microbial load of an artificial laboratory system. The work was carried out using two parallel packed bed reactors both supplied with running tap water. One of the reactors was used as a control and was permanently exposed to the action of chlorine. In the other reactor, chlorine was neutralized at selected times during the experiment and for periods of variable length. During the experiment the concentration of total and viable cells from the effluent was monitored at the exit of each of the reactors. The data obtained were used to estimate microbial production from the biofilms. As an average, release of microbial cells to the water phase increased tenfold in the absence of chlorine. The results also indicate that disinfectant efficiency against the biofilm was not recovered when chlorine returned to normal levels after each event of chlorine neutralization. Cell viability in the water phase in the presence of chlorine was low at the beginning of the experiment but increased 4 orders of magnitude after five neutralization periods. Therefore, subsequent episodes of chlorine depletion may accelerate the development of microbial communities with reduced susceptibility to disinfection in real drinking water systems.

The disinfectant properties of chlorine have been known for centuries but in the last few years water chlorination has attracted some criticism due to its secondary effects and the increased resistance of bacterial strains to chlorine inactivation. In this paper the kinetics of inactivation by chlorine of different Gram-positive and Gram-negative bacterial strains isolated from chlorinated water is studied. The Gram-positive strains were more resistant to chlorine and the behaviour of some of them in the presence of chloramphenicol suggests either the synthesis of unique proteins or aggregation of the bacteria as mechanisms of resistance to inactivation. The concept of Ki, the inactivation rate constant, by comparison with Ks in Michaelis-Menten enzyme kinetics (considering enzymic saturation), or with Ks in Monod growth kinetics (considering limiting rates of transport and metabolism of substrates), may be an interesting parameter to define microbial resistance to disinfectants and toxics.

The influence of chlorine levels, the concentration of dissolved organic carbon and the abundance of bacteria in suspension, on the formation of biofilms on experimental glass surfaces were evaluated. Twelve reactors, packed with glass spheres, were continuously perfused with tap water. The properties of water were altered in different ways: chlorine was neutralized by the addition of thiosulfate, the levels of assimilable organic carbon were increased through the addition of acetate, and the bacterial load was modified by means of the continuous inoculation of a growing active culture of Pseudomonas aeruginosa. Continuous addition of bacteria to water containing 0.5 mg/l of free chlorine, did not result in the formation of detectable biofilms even after one month. When bacteria were added simultaneously with thiosulfate as a chlorine neutralizer, a community of attached bacteria appeared in less than 24 hours. Addition of acetate with the presence of 0.5 mg/l of chlorine did not stimulate the formation of biofilms. On the contrary, neutralization of chlorine with thiosulfate allowed the formation of biofilms with 10(6) cfu/cm(2) in about two weeks.