Occurrence of Giardia cysts and Cryptosporidium oocysts in activated sludge samples in Campinas, SP, Brazil

Giardia and Cryptosporidium have caused several outbreaks of gastroenteritis in humans associated with drinking water. Contaminated sewage effluents are recognized as a potential source of waterborne protozoa. Due to the lack of studies about the occurrence of these parasites in sewage samples in Brazil, we compared the efficiency of two procedures for concentrating cysts and oocysts in activated sludge samples of one sewage treatment plant. For this, the samples were submitted to i) concentration by the ether clarification procedure (ECP) and to ii) purification by sucrose flotation method (SFM) and aliquots of the pellets were examined by immunofluorescence. Giardia cysts were present in all samples (100.0%; n = 8) when using ECP and kit 1 reagents, while kit 2 resulted in six positive samples (85.7%; n = 7). As for SFM, cysts were detected in 75.0% and 100.0% of these samples (for kit 1 and 2, respectively). Regarding Cryptosporidium, two samples (25.0%; kit 1 and 28.5% for kit 2) were detected positive by using ECP, while for SFM, only one sample (examined by kit 1) was positive (12.5%). The results of the control trial revealed Giardia and Cryptosporidium recovery efficiency rates for ECP of 54.5% and 9.6%, while SFM was 10.5% and 3.2%, respectively. Considering the high concentration detected, a previous evaluation of the activated sludge before its application in agriculture is recommended and with some improvement, ECP would be an appropriate simple technique for protozoa detection in sewage samples.

Microbiology of membrane bioreactors for wastewater treatment: a molecular approach

Dissertation presented to obtain the Ph.D degree in Engineering and Technology Sciences, Biotechnology.

Modelling of a wastewater treatment plant using GPS-X

Dissertation to obtain the degree of Master in Chemical and Biochemical Engineering

Polymerase chain reaction and nested-PCR approaches for detecting Cryptosporidium in water catchments of water treatment plants in Curitiba, State of Paraná, Brazil

Introduction Cryptosporidium is an important protozoan cause of waterborne disease worldwide of concern to public health authorities. To prevent outbreaks of cryptosporidiosis, the monitoring of this parasite in drinking water is necessary. In the present work, the polymerase chain reaction (PCR) and nested-PCR techniques were used to detect Cryptosporidium in raw water from catchment points of four water treatment plants (WTP) in Curitiba, Paraná, Brazil. Methods First, DNA extraction techniques were tested in samples containing decreasing amount of oocysts in reagent water, and PCR and nested-PCR with specific primers for 18SSU rDNA of Cryptosporidium were conducted to determine their sensitivity. In reagent water, a commercial extraction kit provided the best analytical sensitivity, and PCR and nested-PCR allowed the detection of five and two oocysts, respectively, with the primers XIAOR/XIAOF and XIAO1F/XIAO2R. Results In the spiking experiments, only the PCR with the primers AWA995F/AWA1206R was successful at detecting concentrations of 0.1 oocysts/mL. Two catchments samples of raw water and/or water sludge from four WTPs were contaminated with Cryptosporidium. Conclusions The application of the techniques to monitor Cryptosporidium in water and detect contamination in water catchments of WTPs in Curitiba are discussed in the present work.

Optimization of the electrodialytic phosphorus recovery from sewage sludge ash

Phosphorus (P) is becoming a scarce element due to the decreasing availability of primary sources. Therefore, recover P from secondary sources, e.g. waste streams, have become extremely important. Sewage sludge ash (SSA) is a reliable secondary source of P. The use of SSAs as a direct fertilizer has very restricted legislation due to the presence of inorganic contaminants. Furthermore, the P present in SSAs is not in a plant-available form. The electrodialytic (ED) process is one of the methods under development to recover P and simultaneously remove heavy metals. The present work aimed to optimize the P recovery through a 2 compartment electrodialytic cell. The research was divided in three independent phases. In the first phase, ED experiments were carried out for two SSAs from different seasons, varying the duration of the ED process (2, 4, 6 and 9 days). During the ED treatment the SSA was suspended in distilled water in the anolyte, which was separated from the catholyte by a cation exchange membrane. From both ashes 90% of P was successfully extracted after 6 days of treatment. Regarding the heavy metals removal, one of the SSAs had a better removal than the other. Therefore, it was possible to conclude that SSAs from different seasons can be submitted to ED process under the same parameters. In the second phase, the two SSAs were exposed to humidity and air prior to ED, in order to carbonate them. Although this procedure was not successful, ED experiments were carried out varying the duration of the treatment (2 and 6 days) and the period of air exposure that SSAs were submitted to (7, 14 and 30 days). After 6 days of treatment and 30 days of air exposure, 90% of phosphorus was successfully extracted from both ashes. No differences were identified between carbonated and non-carbonated SSAs. Thus, SSAs that were exposed to the air and humidity, e.g. SSAs stored for 30 days in an open deposit, can be treated under the same parameters as the SSAs directly collected from the incineration process. In the third phase, ED experiments were carried out during 6 days varying the stirring time (0, 1, 2 and 4 h/day) in order to investigate if energy can be saved on the stirring process. After 6 days of treatment and 4 h/day stirring, 80% and 90% of P was successfully extracted from SSA-A and SSA-B, respectively. This value is very similar to the one obtained for 6 days of treatment stirring 24 h/day.

Electrokinetic treatment of environmental matrices. Contaminants removal and phosphorus recovery

There is a need to develop viable techniques for removal and recovery organic and inorganic compounds from environmental matrices, due to their ecotoxicity, regulatory obligations or potential supplies as secondary materials. In this dissertation, electro –removal and –recovery techniques were applied to five different contaminated environmental matrices aiming phosphorus (P) recovery and/or contaminants removal. In a first phase, the electrokinetic process (EK) was carried out in soils for (i) metalloids and (ii) organic contaminants (OCs) removal. In the case of As and Sb mine contaminated soil, the EK process was additionally coupled with phytotechnologies. In a second phase, the electrodialytic process (ED) was applied to wastes aiming P recovery and simultaneous removal of (iii) toxins from membrane concentrate, (iv) heavy metals from sewage sludge ash (SSA), and (v) OCs from sewage sludge (SS). EK enhanced phytoremediation showed to be viable for the remediation of soils contaminated with metalloids, as although remediation was low, it combines advantages of both technologies while allowing site management. EK also proved to be an effective remediation technology for the removal and degradation of emerging OCs from two types of soil. Aiming P recovery and contaminants removal, different ED cell set-ups were tested. For the membrane concentrates, the best P recovery was achieved in a three compartment (3c) cell, but the highest toxin removal was obtained in a two compartment (2c) cell, placing the matrix in the cathode end. In the case of SSA the best approach for simultaneous P recovery and heavy metals removal was to use a 2c-cell placing the matrix in the anode end. However, for simultaneous P recovery and OCs removal, SS should be placed in the cathode end, in a 2c-cell. Overall, the data support that the selection of the cell design should be done case-by-case.

Electrodialytic recovery of phosphorus and organic contaminants removal from sewage sludge

Phosphorus is a macronutrient essential to life which comes from phosphate rock, a non-renewable resource. Sewage sludge from wastewater treatment plants (WWTP) is a secondary resource rich in phosphorus that can be valorized. However, organic compounds are detected in sewage sludge, due to its non-polar and hydrophobic character, being considered an environmental risk. The present dissertation aims to study the efficiency of the electrodialytic process (ED) when applied to sewage sludge aiming phosphorus recovery and organic contaminants removal. Four organic compounds were analyzed: 17α-ethynylestradiol (EE2), bisphenol A (BPA), caffeine (Caf) and oxybenzone (MBPh). The experiments took place in an ED cell with two compartments and an anion exchange membrane, with the sludge in the cathode compartment. The experiments were carried out for three days with spiked sewage sludge (six assays). One control experiment was done without current, three experiments were carried out applying a constant current of 50, 75, and 100 mA and two experiments were carried out applying sequential currents: 50 mA, 75 mA and 100 mA and the opposite (100-75-50 mA). A qualitative and quantitative analysis of microorganisms existing in the samples was also done. At the end, the pH increased in the sewage sludge favoring phosphorus recovery. In terms of phosphorus, the highest recovery was achieved in the experiment run with 100 mA, where 70.3±2.0% of total phosphorus was recovered in the electrolyte. Generally, compounds degradation was favored by the current. Caf and MBPh achieved degradation percentages of 96.2±0.2% and 84.8±1.3%, respectively, in 100 mA assay. EE2 (83.1±1.7%) and BPA (91.8±4.6%) degradations were favored by 50 mA current. A total of 35 taxa from four different groups were identified, totalizing between 81,600-273,000 individuals per gram of initial sludges. After ED, microbial community population decreased between 47-98%. Arcella gibbosa represented 61% of the total observed organisms and revealed to be more tolerant to medium changes.

The production of vermicompost from dairy sludge and its value as a plant growth medium

Dairy sludge generated at Glanbia Ingredients Ltd., Kilkenny has up until now been landspread. This study investigated the feasibility of using earthworms to vermicompost the sludge as an alternative method of treatment. It was found that high levels of ammonia in the sludge led to earthworm fatality but that by manually aerating the sludge the ammonia could be volatilised or by adding zeolite the ammonia could be absorbed, thus solving the problem. In a medium scale trial, the earthworm species Dendrobaena veneta and Eisenia fetida dominated the polyculture. Earthworms grew and generated cocoons during vermicomposting. During vermicomposting no leachate was generated. Nutrient changes took place during vermicomposting. There were high levels of nitrate, increased calcium and sulphate in the vermicomposted dairy sludge. The amount of magnesium, potassium and chloride did not change, while phosphate was undetectable after vermicomposting. The levels of nitrate and phosphate were good indicators of the extent of vermicomposting. The vermicomposted dairy sludge provided improved growth and yields of radishes and barley compared to the dairy sludge and control. Compared to the vermicompost, the dairy sludge provided heavier ryegrass yields and more marigolds with larger flower diameters. Generally, it is the amount of phosphate in dairy sludge that dictates how much can be applied as a fertiliser on land. Vermicomposting reduced the amount of phosphate to an undetectable level but on the other hand created a problem of high nitrate levels. In a pot trial with grass grown in vermicompost the nitrate leached from the vermicompost. In field conditions the leaching of nitrate might occur and could cause an increased risk of contamination of groundwater and watercourses.

Molecular detection of human astrovirus in an urban sewage treatment plant in Rio de Janeiro, Brazil

The objective of this study was to evaluate the prevalence and dissemination of human astroviruses (HAstV) in the environment by analyzing urban sewage samples from a wastewater treatment plant in the city of Rio de Janeiro, Brazil. A one-year study was performed with a total of 48 raw and treated sewage composite samples, which were collected biweekly from an activated sludge plant. Virus particles were concentrated by the adsorption-elution method using negatively charged membranes associated to a Centriprep Concentrator® 50 (Nihon Millipore). HAstV were detected in 16.7% of the samples in raw and treated sewage by using both qualitative and quantitative reverse transcriptase-polymerase chain reactions (RT-PCR and qPCR, respectively). Positive untreated sewage sample exhibited mean values of 1.1 x 10(4) gEq/mL. The qPCR sensitivity was 18 gEq/reaction. Through utilization of qPCR, a HAstV recovery efficiency of 4.2% and 4.3% was demonstrated for raw and treated sewage samples, respectively. The presence of HAstV in both the raw and treated sewage samples demonstrated the dissemination of these viruses in the environment as well as viral permanence after sewage treatment. There was a reduction in the total and faecal coliform levels, indicating efficiency of the wastewater treatment plant.

The efficiency of concentration methods used to detect enteric viruses in anaerobically digested sludge

The presence of enteric viruses in biosolids can be underestimated due to the inefficient methods (mainly molecular methods) used to recover the viruses from these matrices. Therefore, the goal of this study was to evaluate the different methods used to recover adenoviruses (AdV), rotavirus species A (RVA), norovirus genogroup II (NoV GII) and the hepatitis A virus (HAV) from biosolid samples at a large urban wastewater treatment plant in Brazil after they had been treated by mesophilic anaerobic digestion. Quantitative polymerase chain reaction (PCR) was used for spiking experiments to compare the detection limits of feasible methods, such as beef extract elution and ultracentrifugation. Tests were performed to detect the inhibition levels and the bacteriophage PP7 was used as an internal control. The results showed that the inhibitors affected the efficiency of the PCR reaction and that beef extract elution is a suitable method for detecting enteric viruses, mainly AdV from biosolid samples. All of the viral groups were detected in the biosolid samples: AdV (90%), RVA, NoV GII (45%) and HAV (18%), indicating the viruses' resistance to the anaerobic treatment process. This is the first study in Brazil to detect the presence of RVA, AdV, NoV GII and HAV in anaerobically digested sludge, highlighting the importance of adequate waste management.

Prediction of the bulking phenomenon in wastewater treatment plants

The control and prediction of wastewater treatment plants poses an important goal: to avoid breaking the environmental balance by always keeping the system in stable operating conditions. It is known that qualitative information — coming from microscopic examinations and subjective remarks — has a deep influence on the activated sludge process. In particular, on the total amount of effluent suspended solids, one of the measures of overall plant performance. The search for an input–output model of this variable and the prediction of sudden increases (bulking episodes) is thus a central concern to ensure the fulfillment of current discharge limitations. Unfortunately, the strong interrelationbetween variables, their heterogeneity and the very high amount of missing information makes the use of traditional techniques difficult, or even impossible. Through the combined use of several methods — rough set theory and artificial neural networks, mainly — reasonable prediction models are found, which also serve to show the different importance of variables and provide insight into the process dynamics

Water-dispersible clay in soils treated with sewage sludge

A by-product of Wastewater Treatment Stations is sewage sludge. By treatment and processing, the sludge is made suitable for rational and environmentally safe use in agriculture. The aim of this study was to assess the influence of different doses of limed sewage sludge (50 %) on clay dispersion in soil samples with different textures (clayey and medium). The study was conducted with soil samples collected from native forest, on a Red Latosol (Brazilian classification: Latossolo Vermelho distroférrico) loamy soil in Londrina (PR) and a Red-Yellow Latosol (BC: Latossolo Vermelho-Amarelo distrófico) medium texture soil in Jaguapitã (PR). Pots were filled with 3 kg of air-dried fine earth and kept in greenhouse. The experiment was arranged in a randomized block design with six treatments: T1 control, and treatments with limed sewage sludge (50 %) as follows: T2 (3 t ha-1), T3 (6 t ha-1), T4 (12 t ha-1), T5 (24 t ha-1) and T6 (48 t ha-1) and five replications. The incubation time was 180 days. At the end of this period, the pots were opened and two sub-samples per treatment collected to determine pH-H2O, pH KCl (1 mol L-1), organic matter content, water-dispersible clay, ΔpH (pH KCl - pH-H2O) and estimated PZC (point of zero charge): PZC = 2 pH KCl - pH-H2O, as well as the mineralogy of the clay fraction, determined by X ray diffraction. The results showed no significant difference in the average values for water-dispersible clay between the control and the other treatments for the two soil samples studied and ΔpH was the variable that correlated best with water-dispersible clay in both soils.

Chemical and microbiological attributes of an oxisol treated with successive applications of sewage sludge¹

Studies on sewage sludge (SS) have confirmed the possibilities of using this waste as fertilizer and/or soil conditioner in crop production areas. Despite restrictions with regard to the levels of potentially toxic elements (PTE) and pathogens, it is believed that properly treated SS with low PTE levels, applied to soil at adequate rates, may improve the soil chemical and microbiological properties. This study consisted of a long-term field experiment conducted on a Typic Haplorthox (eutroferric Red Latosol) treated with SS for seven successive years for maize production, to evaluate changes in the soil chemical and microbiological properties. The treatments consisted of two SS rates (single and double dose of the crop N requirement) and a mineral fertilizer treatment. Soil was sampled in the 0-0.20 m layer and analyzed for chemical properties (organic C, pH, P, K, Ca, Mg, CEC, B, Cu, Fe, Mn, Zn, Cd, Ni, and Pb) and microbiological properties (basal respiration, microbial biomass activity, microbial biomass C, metabolic quotient, microbial quotient, and protease and dehydrogenase enzyme activities). Successive SS applications to soil increased the macro- and micronutrient availability, but the highest SS dose reduced the soil pH significantly, indicating a need for periodic corrections. The SS treatments also affected soil microbial activity and biomass negatively. There were no significant differences among treatments for maize grain yield. After seven annual applications of the recommended sludge rate, the heavy metal levels in the soil had not reached toxic levels.

Hydraulic conductivity and soil-sewage sludge interactions

One of the main problems faced by humanity is pollution caused by residues resulting from the production and use of goods, e.g, sewage sludge. Among the various alternatives for its disposal, the agricultural use seems promising. The purpose of this study was to evaluate the hydraulic conductivity and interaction of soil with sandy-silty texture, classified as Spodosols, from the Experimental Station Itapirema - IPA, in Goiana, state of Pernambuco, in mixtures with sewage sludge from the Mangueira Sewage Treatment Station, in the city of Recife, Pernambuco at rates of 25, 50 and 75 Mg ha-1. Tests were conducted to let water percolate the natural saturated soil and soil-sludge mixtures to characterize their physical, chemical, and microstructural properties as well as hydraulic conductivity. Statistical data analysis showed that the presence of sewage sludge in soils leads to an increase of the < 0.005 mm fraction, reduction in real specific weight and variation in optimum moisture content from 11.60 to 12.90 % and apparent specific dry weight from 17.10 and 17.50 kN m-3. In the sludge-soil mixture, the quartz grains were covered by sludge and filling of the empty soil macropores between grains. There were changes in the chemical characteristics of soil and effluent due to sewage sludge addition and a small decrease in hydraulic conductivity. The results indicate the possibility that soil acidity influenced the concentrations of the elements found in the leachate, showing higher levels at higher sludge doses. It can be concluded that the leaching degree of potentially toxic elements from the sewage sludge treatments does not harm the environment.

Carbon and Nitrogen Mineralization in Soil Combining Sewage Sludge and Straw

ABSTRACT The combined incorporation of sewage sludge (SS) and oat straw (OS) to the soil can increase straw carbon mineralization and microbial nitrogen immobilization. This hypothesis was tested in two laboratory experiments, in which SS was incorporated in the soil with and without OS. One treatment in which only straw was incorporated and a control with only soil were also evaluated. The release of CO2 and mineral N in the soil after organic material incorporation was evaluated for 110 days. The cumulative C mineralization reached 30.1 % for SS and 54.7 % for OS. When these organic materials were incorporated together in the soil, straw C mineralization was not altered. About 60 % of organic N in the SS was mineralized after 110 days. This N mineralization index was twice as high as that defined by Resolution 375/2006 of the National Environmental Council. The combined incorporation of SS and OS in the soil caused an immobilization of microbial N of 5.9 kg Mg-1 of OS (mean 3.5 kg Mg-1). The results of this study indicated that SS did not increase straw C mineralization, but the SS rate should be adjusted to compensate for the microbial N immobilization caused by straw.