WSP Systems Research Articles

The Wsp chemosensory system modulates c-di-GMP-dependent biofilm formation by integrating DSF quorum sensing through the WspR-RpfG complex in Lysobacter

The ubiquitous Wsp (wrinkly spreader phenotype) chemosensory system and DSF (diffusible signal factor) quorum sensing are two important chemically associated signaling systems that mediate bacterial communications between the host and environment. Although these two systems individually control biofilm formation in pathogenic bacteria via the ubiquitous second messenger c-di-GMP, their crosstalk mechanisms remain elusive. Here we present a scenario from the plant-beneficial and antifungal bacterium Lysobacter enzymogenes OH11, where biofilm formation favors the colonization of this bacterium in fungal hyphae. We found that the Wsp system regulated biofilm formation via WspR-mediated c-di-GMP signaling, whereas DSF system did not depend on the enzymatic activity of RpfG to regulate biofilm formation. We further found that WspR, a diguanylate cyclase (DGC) responsible for c-di-GMP synthesis, could directly bind to one of the DSF signaling components, RpfG, an active phosphodiesterase (PDE) responsible for c-di-GMP degradation. Thus, the WspR-RpfG complex represents a previously undiscovered molecular linker connecting the Wsp and DSF systems. Mechanistically, RpfG could function as an adaptor protein to bind and inhibit the DGC activity of unphosphorylated WspR independent of its PDE activity. Phosphorylation of WspR impaired its binding affinity to RpfG and also blocked the ability of RpfG to act as an adaptor protein, which enabled the Wsp system to regulate biofilm formation in a c-di-GMP-dependent manner by dynamically integrating the DSF system. Our findings demonstrated a previously uncharacterized mechanism of crosstalk between Wsp and DSF systems in plant-beneficial and antifungal bacteria.

Characterization and evaluation of waste stabilization pond systems in Namibia

Abstract Waste stabilization ponds (WSP) exist worldwide to treat wastewater, especially in warm climates. They are characterized by simple operation and maintenance and over 50 years many WSP were built in urban communities in Namibia. This study characterized and evaluated nine of these WSP systems in terms of their influent and effluent water quality and compared them with the requirements for water reuse in agriculture. In their current state none of them adhered with the Namibian or the new European reuse standards, especially due to tCOD concentrations above 100 mg/L caused by high algal fractions in the pCOD. The algae related chlorophyll-a concentrations correlated linearly with the pCOD and this correlation can therefore be used to fractionate the tCOD for further judgement. Additionally, microbial community analyses determined the composition of pathogens in the WSP influent and effluent, this helped to assess potential risks and distinguish between potentially toxic and non-toxic cyanobacteria. The EU requirement of less than 1,000 E. coli per 100 mL for fodder crop irrigation was only achieved with one WSP system which was enhanced with additional pre- and post-treatment. This research delivers a first overview of the current situation and can be used as basis to establish possible enhancement measures for existing WSP as well as to investigate possible effluent application in agricultural irrigation.

Analysis of Pseudomonas aeruginosa c-di-GMP High and Low Subpopulations Using Flow-assisted Cell Sorting (FACS) and Quantitative Reverse Transcriptase PCR (qRT-PCR).

Cyclic diguanylate monophosphate (c-di-GMP) is a second messenger signaling molecule that drives the transition from planktonic to the biofilm mode of growth in many bacterial species. Pseudomonas aeruginosa has at least two surface sensing systems that produce c-di-GMP in response to surface attachment, the Wsp and Pil-Chp systems. We recently used a plasmid-based c-di-GMP reporter (pP cdrA::gfp ) to describe how the Wsp system generates heterogeneity in surface sensing, resulting in two physiologically distinct subpopulations of cells during early biofilm formation. One subpopulation has elevated c-di-GMP and produces biofilm matrix, serving as the founders of initial microcolonies. The other subpopulation has low c-di-GMP and engages in surface motility, allowing for exploration of the surface. Here, we describe the protocol for a key experiment to confirm our initial observation of c-di-GMP heterogeneity during surface sensing: the use of flow-assisted cell sorting (FACS) to isolate subpopulations of cells with high and low c-di-GMP reporter activity, followed by quantitative Reverse Transcriptase PCR (qRT-PCR) of genes that are known to be transcriptionally regulated in response to cellular c-di-GMP levels (pelA, pslA). This protocol can be adapted by others to isolate subpopulations of high- and low- c-di-GMP P. aeruginosa cells that are genetically identical, but phenotypically distinct for future experiments examining specific mRNA transcripts as we did or, presumably, for additional applications like RNAseq, proteomics, or TNseq. Graphical abstract.

The small, the big, and the beautiful: Emerging challenges and opportunities for waste stabilization ponds in Australia

AbstractWaste stabilization ponds (WSPs) are used extensively for the treatment of wastewater in Australia, mostly in regional and remote areas. Wastewater treatment plants (WWTPs) using pond technologies are also distributed over the full geographical extent of Australia, encompassing many climatic zones. Predominantly used to service small to medium‐sized communities, WSPs are also used to service large metropolitan Australian populations, up to 2.5 million people. When well‐maintained, WSPs are a sustainable and resilient treatment option, and treatment is achieved at significantly lower cost when compared with conventional WWTPs. Increasing population, changing regulations, and climate variability are placing increasing pressure on Australian WSP systems. Sludge accumulation over time presents a significant challenge to pond maintenance, along with increasing occurrence of toxic cyanobacterial bloom events. These challenges are only enhanced by the wide geographical distribution and by increasing operational and maintenance costs. Increased demand for recycled water is placing further pressure on Australian WSP systems, as higher value treated water is expected from WSP infrastructure that is often overloaded or under‐designed. This increased demand for high‐quality treatment presents an opportunity for operators and researchers to develop a better understanding of the coupling between hydraulics and microbial ecology of these systems. With more stringent guidelines for greenhouse gas emissions (GHGs), a better understanding of biophysicochemical processes in WSPs will lead to better estimates of GHG fluxes and variability. This information will become critical for the future planning, maintenance and operation of WSPs, and will result in a better understanding of WSP systems overall.This article is categorized under:Engineering Water > Water, Health, and SanitationEngineering Water > Sustainable Engineering of Water

Occurrence, removal and risk assessment of steroid hormones in two wastewater stabilization pond systems in Morogoro, Tanzania

The present study investigated the occurrence and removal of 10 steroid hormones (4 androgens, 3 progestagens and 3 estrogens) in two WSP systems, Mafisa and Mzumbe in Morogoro, Tanzania. All 10 steroid hormones were detected in the influent of both WSP systems in the dry as well as in the rainy season. The concentrations of steroids in influent wastewater ranged from 0.1 ng/L for 17-OH-pregnenolone to 445 ng/L for estrone and from below limit of detection for 17-OH-pregnenolone to 45 ng/L for estrone in effluent. During dry season, the overall mean ± standard deviation removal efficiency for the 10 steroids were 70 ± 21% for Mzumbe WSP and 97 ± 3% for Mafisa WSP. During the rainy season the overall mean removal efficiency for all the steroid hormones were 52 ± 32% for Mzumbe WSP and 94 ± 8% for Mafisa WSP. Risk was characterized by calculating the risk quotients (RQs) for fish and humans. 46% of the total RQs calculated were above one, indicating high risk. Low RQs were estimated for androgens and progestagens but the estrogen concentrations measured in the WSP systems and Morogoro River indicated a high risk for fish. However, estrogens appeared not to pose an appreciable risk to human health from water intake and fish consumption. The results indicated that WSP systems are quite effective in removing steroid hormones from wastewater. Thus, low technology systems such as WSP systems are suitable techniques in low income counties due to relatively low costs of building, operating and maintaining these systems.

Thermo-tolerant coliform bacteria decay rates in a full scale waste stabilization pond system in Northeast Brazil

This paper presents the results for thermo-tolerant coliform (TTC) decay rates (K(b)) in a full scale WSP system located in Natal-RN, northeast Brazil. The series comprises a primary facultative pond (2 m deep), followed by two maturation ponds (1.5 m deep) giving a total area of 11 ha. The influent sewage and the pond effluents were monitored weekly during a seven month period. The results showed that the K(b) values predicted by the Marais equation assuming a hydraulic regime of complete mixing overestimated TTC die-off rates. The K(b) value adopted in the project design was 6.20 d(-1) but the mean value found for the WSP system during the monitoring programme was only 0.85 d(-1). This value is low compared to the values cited in the literature for shallow ponds (<1.25 m deep) but similar to values for deeper ponds. The sub optimal TTC removal rate in this WSP system may be caused by the adoption of too high a K(b) value at the design stage and the negative influence of high wind conditions on the mixing regime in the water columns of the ponds. Thus values for K(b) adopted at the design stage of WSP systems should be coherent with the hydraulic flow model, the type of pond, pond depth, and with the surface organic loading.

Fate of coliforms and pathogenic parasite in four full-scale sewage treatment systems in India

The occurrence and removal of fecal indicators (total coliforms (TC), fecal coliforms (FC), fecal streptococci (FS)) and pathogens (helminthes eggs) were studied in various municipal wastewater treatment processes (UASB + FPU, ASP, EA, WSP). The reductions in TC and FC concentrations were usually between 2.0 and 2.5 log units in up-flow anaerobic sludge blanket reactor incorporated with final polishing unit (UASB + FPU). Almost similar reduction was observed in activated sludge process system (ASP) and waste stabilization ponds system (WSP), while it was log 3.0 in extended aeration system (EA). UASB + FPU and WSP systems were observed more efficient to reduce helminthes eggs at almost 100%, whereas only 97% removal was observed in case of ASP and EA system. In addition to monitoring of indicator organisms, turbidity, suspended solids (SS), and biochemical oxygen demand (BOD) were used as indirect measure of the potential presence of microorganisms. Interrelationship of BOD, SS, and turbidity with fecal indicator bacteria concentration in influent and effluent manifest that improvement of the microbiological quality of wastewater is strongly linked to the removal of BOD and SS.

Nitrification–denitrification in waste stabilisation ponds: a mechanism for permanent nitrogen removal in maturation ponds

A pilot-scale primary maturation pond was spiked with (15)N-labelled ammonia ((15)NH(4)Cl) and (15)N-labelled nitrite (Na(15)NO(2)), in order to improve current understanding of the dynamics of inorganic nitrogen transformations and removal in WSP systems. Stable isotope analysis of delta(15)N showed that nitrification could be considered as an intermediate step in WSP, which is masked by simultaneous denitrification, under conditions of low algal activity. Molecular microbiology analysis showed that denitrification can be considered a feasible mechanism for permanent nitrogen removal in WSP, which may be supported either by ammonia-oxidising bacteria (AOB) or by methanotrophs, in addition to nitrite-oxidising bacteria (NOB). However, the relative supremacy of the denitrification process over other nitrogen removal mechanisms (e.g., biological uptake) depends upon phytoplanktonic activity.

Morphological and molecular characterization of cyanobacteria from a Brazilian facultative wastewater stabilization pond and evaluation of microcystin production

The cyanobacterial population in the Cajati waste stabilization pond system (WSP) from Sao Paulo State, Brazil was assessed by cell isolation and direct microscope counting techniques. Ten strains, belonging to five genera (Synechococcus, Merismopedia, Leptolyngbya, Limnothrix, and Nostoc), were isolated and identified by morphological and molecular analyses. Morphological identification of the isolated strains was congruent with their phylogenetic analyses based on 16S rDNA gene sequences. Six cyanobacterial genera (Synechocystis, Aphanocapsa, Merismopedia, Lyngbya, Phormidium, and Pseudanabaena) were identified by direct microscope inspection. Both techniques were complementary, since, of the six genera identified by direct microscopic inspection, only Merismopedia was isolated, and the four other isolated genera were not detected by direct inspection. Direct microscope counting of preserved cells showed that cyanobacteria were the dominant members (>90%) of the phytoplankton community during both periods evaluated (summer and autumn). ELISA tests specific for hepatotoxic microcystins gave positive results for six strains (Synechococcus CENA108, Merismopedia CENA106, Leptolyngbya CENA103, Leptolyngbya CENA112, Limnothrix CENA109, and Limnothrix CENA110), and for wastewater samples collected from raw influent (3.70 μg microcystins/l) and treated effluent (3.74 μg microcystins/l) in summer. Our findings indicate that toxic cyanobacteria in WSP systems are of concern, since the treated effluent containing cyanotoxins will be discharged into rivers, irrigation channels, estuaries, or reservoirs, and can affect human and animal health.

HIL simulation of WSP systems on MI-6 test rig

In this article, a multi-purpose platform for Hardware In the Loop (HIL) testing of safety relevant railway subsystems, such as odometry boards or wheel slide protection systems, is shown. The rig, called MI-6, is a product of the cooperation of Trenitalia with researchers of Dip. Energetica Sergio Stecco (University of Florence). In this work, special attention has been paid to vehicle real-time model optimization according to customer specifications (Trenitalia) and to simulate artificially degraded adhesion between rolling surfaces where conventional adhesion models have poor performances or require unaffordable computational resources for real-time applications. In this article, a heuristic model based on energetic considerations and a wide archive of experimental test (courtesy of Trenitalia Società per Azioni) is presented. Also some simulation results and comparison with the experimental data are shown.

Twenty years' monitoring of Mèze stabilisation ponds: part II – removal of faecal indicators

The WSP system serving Mèze and Poussan (French Mediterranean coast) was constructed in 1980 and enlarged and upgraded from 1994 to 1998. Water quality along the waste stabilisation pond to (WSP) system has been monitored over the years, thus allowing us to assess the influence of enlargement and upgrading works. A significant enhancement of the average microbiological quality of the effluent was observed, with respective E. coli and streptococci average abatements of 4.1 and 3.4 log. units. Former seasonal variations of microbiological removal have vanished. The contribution of the different ponds to the disinfection performance of the WSP system was analysed. A microbiological quality model was proposed to evaluate the die-off kinetics related to the different ponds and as a tool for the design and management of WSP systems. Though the relationships between die-off coefficients and environmental factors appeared somewhat frail, this modelling is considered a promising approach for the prediction of WSP microbiological performance.

Waste stabilisation ponds in France: state of the art and recent trends

Waste stabilisation ponds represent 20% of the total number of wastewater treatment plants in France. Practical expertise acquired during these last 20 years has led to modification in the design of the first facultative basin of WSP systems. Its active surface area is now dimensioned at 6 m2(p.e.)(-1) in order to limit the risk of malfunctioning. The cumulated surface of the 2nd and 3rd basin is maintained at 5 m2(p.e.)(-1). Another practical point is also that WSPs must receive mainly diluted influents. Globally, the plants are on average far from their nominal loadings, which explains why the first sludge removals took place on average 13 years after being put in operation. Based on a representative sample of plants, i.e. 1 5% of the French WSPs, it has been possible to estimate the time, material means and cost needed for sludge removal as well as the amount of sludge accumulated. The sludge removed at the 1st yields on average 110 L (p.e.)(-1) which represents 12 kg DM (p.e.)(-1). The current trend of increasing the quality levels necessary for discharge into sensitive receiving bodies has led to adaptive solutions of polishing treatments by intermittent sand filter systems with or without the plantation of reeds.

Under-performance evaluation and rehabilitation strategy for waste stabilization ponds in Mexico

Fourteen high altitude (> 2,500 m amsl) small Waste Stabilisation Pond systems (WSPs) commissioned during the last 12 years in the State of Mexico in Mexico, were built to a common 3-stage design. Each system is comprised of 2 parallel series of bio-digesters, anaerobic and facultative ponds. All fourteen WSP systems produce poor quality effluents, and eight studied in more detail did not meet any of the national standards for discharge to rivers or the standards required for reuse. The under-performance of these WSPs is underlined by the anaerobic condition of the upper reaches of the Lerma river which receives the sewage from the towns served by these treatment plants. Preliminary surveillance diagnostics identified fundamental operational problems in all eight WSP systems located in the upper Lerma catchment. The results of an intensive secondary diagnostic performance evaluation on one system were used to identify the reasons for under-performance. Under-performance was caused by under-design, hydraulic short-circuiting, adverse environmental conditions and poor operation and maintenance. A strategy for improvement of design and operation to meet national standards is presented.

Waste stabilisation ponds in extreme continental climates: a comparison of design methods from the USA, Canada, northern Europe and the former Soviet Union

The paper presents a brief review of the application of WSPs in extreme climates where ice formation occurs during winter. Design standards and methods are compared and different systems are described. Design equations developed by the US EPA and normative standards from the former Soviet Union are compared in a simple example using typical wastewater and performance characteristics. The results are similar except at low temperatures, where the Soviet method can give pond depths outside the prescribed limits. The paper examines construction and operational aspects of extreme climate WSPs, comparing North American, Northern European and Russian standards. It considers why WSP systems have not been widely adopted in Russia and the NIS, and looks at the advantages these systems may have in countries in economic transition.

A comparative assessment of bacteriophages as tracers and models for virus removal waste stabilisation ponds

The suitability of five strains of bacteriophage as hydraulic tracers and as models of pathogenic virus removal in waste stabilisation ponds was determined in vitro. The bacteriophages investigated included Serratia marcescens, Pseudomonas syringae, Erwinia amanas, and two phages of Erwinia amylovora. The study modeled the survival of these bacteriophages under the physico-chemical conditions typically encountered in WSP systems. This demonstrated that the selected phages, were greatly affected by increased pH and sunlight, but survived for sufficient time at moderate pH levels (6.5-8.5) to justify their use as tracers in full-scale WSPs. Furthermore, their survival/removal in adjusted physico-chemical conditions, suspended solids concentration and temperature, was comparable to human pathogenic viruses. It was concluded that these phages are potentially useful tracers and indicators for pathogenic viruses in WSPs.

Efficiency of removal in stabilization ponds II. Statistical analysis of K values

The analytical values obtained from five WSP systems located in different ecoclimatic zones in the centre and South of Portugal, were subjected to a statistical analysis of K and K20 values (as used in the van Hoff-Arrhenius equation) utilizing an analysis of variance and a vectorial analysis of principal components. Significant differences between K and K20 values in facultative, maturation ponds and in the whole system were verified. The projection of the first principal components of eigenvectors made possible the classification of the stations, showing the existence of similarities and discrepancies in their removal BOD kinetics behaviour. The K20 value must be estimated and taken into account for the design of waste stabilization ponds.

Efficiency of removal in stabilization ponds II. Statistical analysis of K values

The analytical values obtained from five WSP systems located in different ecoclimatic zones in the centre and South of Portugal, were subjected to a statistical analysis of K and K20 values (as used in the van Hoff-Arrhenius equation) utilizing an analysis of variance and a vectorial analysis of principal components. Significant differences between K and K20 values in facultative, maturation ponds and in the whole system were verified. The projection of the first principal components of eigenvectors made possible the classification of the stations, showing the existence of similarities and discrepancies in their removal BOD kinetics behaviour. The K20 value must be estimated and taken into account for the design of waste stabilization ponds.