Design parameters for sludge reduction in an aquatic worm reactor

Reduction and compaction of biological waste sludge from waste water treatment plants (WWTPs) can be achieved with the aquatic worm Lumbriculus variegatus. In our reactor concept for a worm reactor, the worms are immobilised in a carrier material. The size of a worm reactor will therefore mainly be determined by the sludge consumption rate per unit of surface area. This design parameter was determined in sequencing batch experiments using sludge from a municipal WWTP. Long-term experiments using carrier materials with 300 and 350 μm mesh sizes showed surface specific consumption rates of 45 and 58 g TSS/(m² d), respectively. Using a 350 μm mesh will therefore result in a 29% smaller reactor compared to using a 300 μm mesh. Large differences in consumption rates were found between different sludge types, although it was not clear what caused these differences. Worm biomass growth and decay rate were determined in sequencing batch experiments. The decay rate of 0.023 d⁻¹ for worms in a carrier material was considerably higher than the decay rate of 0.018 d⁻¹ for free worms. As a result, the net worm biomass growth rate for free worms of 0.026 d⁻¹ was much higher than the 0.009-0.011 d⁻¹ for immobilised worms. Finally, the specific oxygen uptake rate of the worms was determined at 4.9 mg O₂/(g ww d), which needs to be supplied to the worms by aeration of the water compartment in the worm reactor.     

Die-off of enteric bacterial pathogens during mesophilic anaerobic digestion

Conventionally treated sewage sludge may contain high concentrations of potentially pathogenic microorganisms and additional treatment is required to minimise the risks to health if it is to be recycled to agricultural land. Mesophilic anaerobic digestion (MAD) is the most widely used process in the UK for stabilising sludge prior to agricultural recycling, but little is known about the fate of a number of enteric pathogens as the sludge passes through the treatment processes. The aim of this study was to determine the efficiency of MAD in removing the bacterial enteric pathogens, Salmonella senftenberg, Listeria monocytogenes and Campylobacter jejuni which were added as a spike to the digester feedstock, together with the die-off of indigenous Escherichia coli already present in the sludge. The primary sludge digestion stage of MAD was found to achieve a log removal of 1.66 for E. coli, 2.23 for L. monocytogenes and 2.23 for S. senftenberg. However, the extent of die-off was a function of the numbers of pathogens in the feed and as these increased the log removal also increased. The numbers of C. jejuni were not affected by primary sludge digestion. Additional die-off was provided by secondary sludge digestion with log removals of 1.70 for E. coli, 2.10 for S. senftenberg and 0.36 for C. jejuni.     

Leaching techniques to remove metals and potentially hazardous nutrients from trout farm sludge

A fish farm sludge high in P (2-6% w/w as dry matter), Fe (5-7%), C (40-50%) and N (0.8-4%) was subjected to a series of acid leaching treatments using HCl, organic acids, and biologically mediated acid production. Additions of biodegradable organic acid solubilized heavy metals better than HCl, while additions of 1.5% w/v glucose followed by 7 day incubation stabilized the sludge releasing 92% P, 100% Fe. The use of homo-lactic Lactobacillus plantarum starter cultures were more effective than hetero-lactic Lactobacillus buchneri, solubilizing 81.9% P, 92.2% Fe, 93.0% Zn and 96.4% Ca in the sludge. The anaerobic sludge-glucose fermentation using L. plantarum produced a leached sludge that has low heavy metal and nutrient content while affording the recovery of nutrients. The potential of these methods for practical application are briefly discussed.     

Specific detection of viable Listeria monocytogenes in Spanish wastewater treatment plants by Fluorescent In Situ Hybridization and PCR

Listeria monocytogenes detection in wastewater can be difficult because of the large amount of background microbiota and the presence of viable but non-culturable forms in this environment. The aim of this study was to evaluate a Fluorescent In Situ Hybridization (FISH) assay combined with Direct Viable Count (DVC) method for detecting viable L. monocytogenes in wastewater samples, as an alternative to conventional culture methods. 16S rRNA sequence data were used to design a specific oligonucleotide probe. In order to assess the suitability of the method, the assays were performed on naturally (n = 87) and artificially (n = 14) contaminated samples and results were compared to those obtained with the isolation of cells on selective media and with a PCR method. The detection limit of FISH and PCR assays was 10⁴ cells/mL without enrichment and 10 cells/mL after enrichment. A total of 47 samples, including 3 samples from effluent sites, yielded FISH positive results for L. monocytogenes. Using DVC-FISH technique, the presence of viable L. monocytogenes cells was detected in 23 out of these 47 FISH positive wastewater samples. PCR and culture methods yielded 27 and 23 positive results, respectively. According to these results, FISH technique has the potential to be used as a sensitive method for the detection and enumeration of L. monocytogenes in environmental wastewater samples.     

Sorption of ionized and neutral emerging trace organic compounds onto activated sludge from different wastewater treatment configurations

The objective of this study was to examine sorption of a suite of 19 trace organic contaminants (TOrCs) to activated sludge. Compounds examined in this study included neutral, nonionized TOrCs as well as acidic TOrCs which may carry a negative charge and basic TOrCs which may carry a positive charge at the pH of wastewater. These TOrCs were evaluated to examine how sorptive behavior might differ for TOrCs in different states of charge. Additionally, multiple sludges from geographically and operationally different wastewater treatment plants were studied to elicit how solid-phase characteristics influence TOrC sorption. Characterization of sludge solids from 6 full scale treatment facilities and 3 bench-scale reactors showed no significant difference in fraction organic carbon (f_oc) and cation exchange capacity (CEC). Sorption experiments demonstrated that sorption of TOrCs also exhibits little variation between these different sludges. Organic carbon normalized partition coefficients (log K_oc) were determined as a measure of sorption, and were found to correlate well with octanol-water partition coefficients (log K_ow) for nonionized TOrCs, and log D_ow for anionic TOrCs where log D_ow is greater than 2. These data were used to construct a linear free energy relationship (LFER), which was comparable to existing LFERs for sorption onto sludge. No trend in sorption was apparent for the remaining anionic TOrCs or for the cationic TOrCs. These data suggest that predicting sorption to activated sludge based on K_ow values is a reasonable approach for neutral TOrCs using existing LFERs, but electrostatic (and likely other) interactions may govern the sorptive behavior of the charged organic chemicals to sludge.     

Stability and maturity of thickened wastewater sludge treated in pilot-scale sludge treatment wetlands

Thickened wastewater activated sludge was treated in 13 pilot-scale sludge treatment wetlands of various configurations that operated continuously for three years in North Greece. Sludge was loaded for approximately 2.5 years, and the beds were left to rest for the remaining period. Three different sludge loading rates were used that represented three different population equivalents. Residual sludge stability and maturity were monitored for the last year. Sludge was regularly sampled and microbial respiration activity indices were measured via a static respiration assay. The phytotoxicity of sludge was quantified via a seed germination bioassay. Measurements of total solids, organic matter, total coliforms, pH and electrical conductivity were also made. According to microbial respiration activity measurements, the sludge end-product was classified as stable. The germination index of the final product exceeded 100% in most wetland units, while final pH values were approximately 6.5. The presence of plants positively affected the stability and maturity of the residual sludge end-product. Passive aeration did not significantly affect the quality of the residual sludge, while the addition of chromium at high concentrations hindered the sludge decomposition process. Conclusively, sludge treatment wetlands can be successfully used, not only to dewater, but also to stabilize and mature wastewater sludge after approximately a four-month resting phase.     

The effects of temperature, pH, and ammonia concentration on the inactivation of Ascaris eggs in sewage sludge

The reported inactivation of Ascaris eggs during alkaline sludge stabilization is highly variable. The objective of our research was to better understand the sources of this variability by quantifying the effects of temperature, pH, and ammonia concentration on the inactivation of indigenous Ascaris eggs in wastewater sludge. Primary sludge was supplemented with ammonia (0, 1000, and 5000mg/l NH(3)-N) and Ca(OH)(2) and incubated in sealed bottles across the range of temperatures (20, 30, 40, and 50 degrees C) and pH (7 and 12) that may be encountered during treatment. Changes in egg viability over time were fit to a two-parameter kinetic model (shoulder and first-order region); to compare treatment conditions, the time for 99% inactivation (t(99)) was also calculated. Each 10 degrees C increase in temperature caused a significant decrease in t(99) at every pH and ammonia concentration tested. At 50 degrees C, the effect of temperature was dominant, such that no effect of pH or ammonia was observed. At 30 and 40 degrees C, raising the pH from 7 to 12 decreased t(99), but at 20 degrees C no pH effect was seen over 80 d (very little inactivation occurred). At 20, 30, and 40 degrees C, the addition of ammonia dramatically decreased t(99). The effect of pH could not be completely separated from that of ammonia, as the unamended sludge samples contained 100-200mg/l indigenous ammonia. Because temperature, pH, and ammonia all contributed to Ascaris egg inactivation, it is essential that these parameters are measured and accounted for when assessing the effectiveness of alkaline stabilization. Furthermore, inactivation by ammonia could be exploited to improve the effectiveness of alkaline sludge stabilization.     

Antibiotic resistance of E. coli in sewage and sludge

The aim of the study is the evaluation of resistance patterns of E. coli in wastewater treatment plants without an evaluation of basic antibiotic resistance mechanisms.Investigations have been done in sewage, sludge and receiving waters from three different sewage treatment plants in southern Austria. A total of 767 E. coli isolates were tested regarding their resistance to 24 different antibiotics. The highest resistance rates were found in E. coli strains of a sewage treatment plant which treats not only municipal sewage but also sewage from a hospital.Among the antimicrobial agents tested, the highest resistance rates in the penicillin group were found for Ampicillin (AM) (up to 18%) and Piperacillin (PIP) (up to 12%); in the cephalosporin group for Cefalothin (CF) (up to 35%) and Cefuroxime-Axetil (CXMAX) (up to 11%); in the group of quinolones for Nalidixic acid (NA) (up to 15%); and for Trimethoprime/Sulfamethoxazole (SXT) (up to 13%) and for Tetracycline (TE) (57%).Median values for E. coli in the inflow (crude sewage) of the plants were between 2.0 x 10(4) and 6.1 x 10(4)CFU/ml (Coli ID-agar, BioMerieux 42017) but showed a 200-fold reduction in all three plants in the effluent. Nevertheless, more than 10(2)CFU E. coli/ml reached the receiving water and thus sewage treatment processes contribute to the dissemination of resistant bacteria in the environment.     

Applying fluorescence based technology to the recovery and isolation of Cryptosporidium and Giardia from industrial wastewater streams

As increasing water shortages continue, water re-use is posing new challenges with treated wastewater becoming a significant source of non-potable water. Rapid detection strategies that target waterborne pathogens of concern to industry are gaining importance in the assessment of water quality. This study reports on the ability to recover spiked Cryptosporidium and Giardia from a variety of industrial wastewater streams of varied water quality. Incorporation of an internal quality control used commonly in finished water-enabled quantitative assessments of pathogen loads and we describe successful analysis of pre- and part-treated wastewater samples from four industrial sites. The method used combined calcium carbonate flocculation followed by flow cytometry and epifluorescence microscopy. Our focus will now aim at characterising the ambient parasites isolated from industrial wastewater with the objective of developing a suite of highly specific platform detection technologies targeted to industrial needs.     

Long-term analysis of a full-scale activated sludge wastewater treatment system exhibiting seasonal biological foaming

The seasonal accumulation of biological foam on the activated sludge system of the Urbana-Champaign Sanitary District Northeast (UCSD-NE) wastewater treatment plant was investigated over an 8-year period by statistical analyses including path analysis, multivariate regression, and principal component analysis. Results of these analyses suggested that variation in the activated sludge reactor temperature and the use of a stream bypassing the primary clarifier were the two main factors determining the observed temporal foam profile. Characterization of the primary clarifier influent and effluent suggested the involvement of high lipid loading rates from the bypass stream in foam accumulation. In light of these results, it is hypothesized that increasing temperatures and lipid loading rates are responsible for foam formation through the same mechanism: the foam-forming microbial population is specialized in consuming lipids, substrates classified as slowly degradable. When the temperature increases, the rate of lipid hydrolysis becomes sufficiently high for this population to become abundant, accumulate on the surfaces of the aeration basins, and cause biological foaming.     

Biocontrol of biomass bulking caused by Haliscomenobacter hydrossis using a newly isolated lytic bacteriophage

This research demonstrates the first ever application of lytic bacteriophage (virus) mediated biocontrol of biomass bulking in the activated sludge process using Haliscomenobacter hydrossis as a model filamentous bacterium. Bacteriophages are viruses that specifically infect bacteria only. The lytic phage specifically infecting H. hydrossis was isolated from the mixed liquor of a local wastewater treatment plant. The isolated bacteriophage belongs to the Myoviridae family with a contractile tail (length-126 nm; diameter-18 nm) and icosahedral head (diameter-81 nm). Titer of the isolated phage with H. hydrossis was calculated to be 5.2 ± 0.3 × 10⁵ PFU/mL and burst size was found to be 105 ± 7 PFU/infected cell. The phage was considerably stable after exposure to high temperature (42 °C) and pH between 5 and 8, emphasizing that it can withstand the seasonal/operational fluctuations under real-time applications. Phage to host (bacteria) ratio for the optimal infection was found to be 1:1000 with ∼54% host death. The isolated phage showed no cross infectivity with other bacteria most commonly found in activated sludge systems, thus validating its suitability for biocontrol of filamentous bulking caused by H. hydrossis. Following the phage application, successful reduction in sludge volume index (SVI) from 155 to 105 was achieved, indicating improved biomass settling. The application of phage did not affect nutrient removal efficiency of the biomass, suggesting no collateral damage. Similar to phage therapy in medical applications, phage-mediated biocontrol holds a great potentiality for large-scale applications as economic agent in the mitigation of several water, wastewater and environmental problems. Present study in this direction is a novel effort.     

Biodegradation of p-chlorophenol by a microalgae consortium

An aquatic community was recovered from a waste discharge container fed with several aromatic pollutants. After 3 months of selective enrichment with p-chlorophenol and p-nitrophenol, two microalgae species, Chlorella vulgaris and Coenochloris pyrenoidosa, were recovered from the microbial consortium. As an axenic culture, this microalgae consortium was able to remove p-chlorophenol under different photo-regimes. Cultures grown under a 24h light regime were capable of biodegrading 50mg l(-1) of p-chlorophenol within 5 days. Addition of zeolite, an adsorbing material, did not improve the p-chlorophenol removal. However, when p-chlorophenol at 150mgl(-1) was fed to the culture supplemented with zeolite, the growth rate of the consortium improved, but the lag phase was longer (16 against 14 days in the absence of zeolite).     

Infection risk assessment of diarrhea-related pathogens in a tropical canal network

A quantitative microbial risk assessment (QMRA) of Cryptosporidium, Giardia and diarrhegenic Escherichia coli (DEC) infection was performed using Monte Carlo simulations to estimate the human health risks associated with the use of canal water for recreational purposes, unrestricted and restricted irrigation in a tropical peri-urban area. Three canals receiving municipal, agricultural, and, predominantly, industrial wastewater were investigated. Identification of pathogenic protozoans revealed the major presence of Cryptosporidium hominis and both assemblages A and B of Giardia lamblia. The highest individual infection risk estimate was found to be for Giardia in an exposure scenario involving the accidental ingestion of water when swimming during the rainy season, particularly in the most polluted section, downstream of a large wholesale market. The estimated annual risks of diarrheal disease due to infection by the protozoan parasites were up to 120-fold greater than the reported disease incidence in the vicinity of the studied district and the entire Thailand, suggesting a significant host resistance to disease beyond our model's assumptions. In contrast, annual disease risk estimates for DEC were in agreement with actual cases of diarrhea in the study area.     

Study on Fe(VI) species as a disinfectant: quantitative evaluation and modeling for inactivating Escherichia coli

Ferrate (Fe(VI)) has high potentials as a multi-purpose water treatment chemical acting as an oxidant, coagulant, and disinfectant, but little detail has been reported concerning its biocidal ability. In this study, the inactivation efficiencies of three Fe(VI) species (H(x)FeO(4)(x-2), x=0, 1, 2) are quantified using Escherichia coli as a model microorganism. E. coli inactivation by Fe(VI) was performed in solutions buffered with 25 mM phosphate in the pH range of 5.6-8.2 and at 25 degrees C. Kinetics of E. coli inactivation were successfully fitted to the Modified Delayed Chick-Watson model in the tested pH range, indicating that log inactivation level of E. coli is linearly proportional to exposure amount of E. coli to Fe(VI). The rate constant of E. coli inactivation by Fe(VI) (k(obs)) that was obtained from the linear regression increased non-linearly from 0.33 to 6.25 l(mg min)(-1) with decreasing pH from 8.2 to 5.6. From the measured pH dependency of k(obs) and the known acidity constants of Fe(VI) species (pK(a), H(2)FeO(4)=3.50 and pK(a), HFeO(4)(-)=7.23), HFeO(4)(-) and H(2)FeO(4) were found to be 3 and 265 times as effective as FeO(4)(2-) in E. coli inactivation, respectively.     

Disinfection effectiveness of organic chloramines, investigating the effect of pH

The disinfection effectiveness of three organic N-chloramines (chlorinated amino acids and peptides) on the bacteria Escherichia coli (E. coli) was investigated, including a more detailed study into the pH dependency of the disinfection effectiveness of N-chloroglycine. The organic N-chloramines were prepared by combining sodium hypochlorite with each amino acid or peptide (glycine, Ala-Ala and Arg-Gly-Asp-Ser), at a N:Cl molar ratio of 1:0.4, and then used to treat E. coli suspensions for 180 min.No evidence of inactivation was observed at pH 8.1 for any of the tested organic N-chloramines. At pH 6.0 and 6.9, E. coli inactivation with N-chloroglycine was characterized by an initial lag phase, during which little or no measurable inactivation occurred, followed by a pseudo-first-order inactivation. This is in accordance with other results in the literature and supports the two step microbial inactivation mechanism proposed by some authors. Inactivation rate coefficients (Chick-Watson and lag coefficients) were calculated by fitting the experimental data with the Rennecker-Mariñas model. pH-dependent inactivation kinetics were observed, with faster inactivation rates occurring at lower pH values, when temperature and chlorine-to-nitrogen ratio where kept constant.N-chloroglycine was determined to be the only contributor to the inactivation process in these experiments. The free chlorine contribution was considered to be negligible in all experiments due to its very low concentration. As well, given that the anionic form of N-chloroglycine is expected to be the single predominant species over the tested pH range, changes in residual N-chloroglycine speciation could not be responsible for the observed pH-dependency of E. coli inactivation. However, while pH stress was considered as a possible synergistic factor, no significant effect of pH stress on E. coli viability was observed at the tested pH levels.     

Antimicrobial resistance of Listeria monocytogenes isolated from dairy-based food products

In this study Listeria monocytogenes (L. monocytogenes) was isolated from three traditionally consumed Lebanese dairy-based food products. One hundred and sixty four samples (45 samples of Baladi cheese, 36 samples of Shankleesh and 83 of Kishk) were collected from the Bekaa Valley in the Northeast region of Lebanon. Suspected Listeria colonies were selected and initially identified by using standard biochemical tests. Initial identification of the positive L. monocytogenes colonies was confirmed at the molecular level by Polymerase Chain Reaction (n = 30) and the confirmed isolates were evaluated for their susceptibility to 10 commonly used antimicrobials. All of the 30 isolates were confirmed to be L. monocytogenes yielding a PCR product of ∼ 660 base pairs (bp). L. monocytogenes was detected in 26.67%, 13.89% and 7.23% of the Baladi cheese, Shankleesh and Kishk samples, respectively. The highest resistance in L. monocytogenes isolates was noted against oxacillin (93.33%) followed by penicillin (90%). The results provide an indication of the contamination levels of dairy-based foods in Lebanon and highlight the emergence of multi-drug resistant Listeria in the environment.     

Development of a rapid DNA extraction method and one-step nested PCR for the detection of Naegleria fowleri from the environment

Naegleria fowleri is a small free-living amoebo-flagellate found in natural and manmade thermal aquatic habitats worldwide. The organism is pathogenic to man causing fatal primary amoebic meningoencephalitis (PAM). Infection typically results from bathing in contaminated water and is usually fatal. It is, therefore, important to identify sites containing N. fowleri in the interests of preventive public health microbiology. Culture of environmental material is the conventional method for the isolation of N. fowleri but requires several days incubation and subsequent biochemical or molecular tests to confirm identification. Here, a nested one-step PCR test, in conjunction with a direct DNA extraction from water or sediment material, was developed for the rapid and reliable detection of N. fowleri from the environment. Here, the assay detected N, fowleri in 18/109 river water samples associated with a nuclear power plant in South West France and 0/10 from a similar site in the UK. Although culture of samples yielded numerous thermophilic free-living amoebae, none were N. fowleri or other thermophilic Naegleria spp. The availability of a rapid, reliable and sensitive one-step nested PCR method for the direct detection of N. fowleri from the environment may aid ecological studies and enable intervention to prevent PAM cases.     

Ability of three DNA-based assays to identify presumptive Escherichia coli colonies isolated from water by the culture-based mFC agar method

We tested the ability of three PCR assays, targeting uidA and tuf genes to correctly identify Escherichia coli colonies isolated from water and we compared them to two β-glucuronidase-based culture methods (Colilert^® and Readycult^®), in terms of specificity and sensitivity. E. coli isolates recovered on mFC agar were first tested for the presence of the uidA positive colonies were presumed to be E. coli. For further characterization, uidA-negative colonies were subsequently identified using the Vitek 2 automated system. Colilert^® and Readycult^® detected 436 and 442 of 468 colonies identified as E. coli on mFC corresponding to sensitivities of 93.2 and 94.4%, respectively. None of the 59 non-E. coli isolates was detected by both methods for a specificity of 100%. Two (2) uidA and 1 tuf PCR assays were also tested. The uidA PCR assays yielded positive signals for 447 (95.5%) and 434 (92.7%) of 468 E. coli isolates tested respectively, whereas the tuf PCR assay showed a sensitivity of 100%. None of the 59 non-E. coli isolates was detected by both uidA PCR assays (100% specificity), whereas tuf PCR false-positive signals were obtained with Escherichia fergusonii and Escherichia albertii. However, since these 2 species are principally found in the feces of mammals and birds, their detection indicates a fecal contamination. Consequently, using a 1-h tuf rtPCR assay to confirm the identity of E. coli colonies on mFC agar is as specific, more sensitive, and potentially more cost-efficient than culture methods based on β-glucuronidase detection.     

An examination of the mechanisms for stable foam formation in activated sludge systems

Screening pure cultures of 65 mycolic acid producing bacteria (Mycolata) isolated mainly from activated sludge with a laboratory based foaming test revealed that not all foamed under the conditions used. However, for most, the data were generally consistent with the flotation theory as an explanation for foaming. Thus a stable foam required three components, air bubbles, surfactants and hydrophobic cells. With non-hydrophobic cells, an unstable foam was generated, and in the absence of surfactants, cells formed a greasy surface scum. Addition of surfactant converted a scumming population into one forming a stable foam. The ability to generate a foam depended on a threshold cell number, which varied between individual isolates and reduced markedly in the presence of surfactant. Consequently, the concept of a universal threshold applicable to all foaming Mycolata is not supported by these data. The role of surfactants in foaming is poorly understood, but evidence is presented for the first time that surfactin synthesised by Bacillus subtilis may be important.     

Development of an improved PCR-ICT hybrid assay for direct detection of Legionellae and Legionella pneumophila from cooling tower water specimens

A novelly improved polymerase chian reaction and immunochromatography test (PCR-ICT) hybrid assay comprising traditional multiplex-nested PCR and ICT, (a lateral-flow device) was developed for direct detection of Legionella bacteria from environmental cooling tower samples. The partial 16S rDNA (specific for Legionella spp.) and dnaJ (specific for Legionella pneumophila) genes from Legionella chromosome were first specifically amplified by multiplex-nested PCR, respectively, followed by detection using ICT strip. Reading of results was based on presence or absence of the two test lines on the strips. Presence of test line 1 indicated existence of Legionella spp. specific 16S rDNA and identified Legionella spp. Presence of test line 2 further indicated existence of dnaJ and thus specifically identified L. pneumophila. In contrast, for non-Legionellae bacteria no test line formation was observed. Results of direct detection of Legionella bacteria and L. pneumophila from water tower specimens by this assay showed 100% sensitivity, and 96.6% and 100% specificity, respectively compared with traditional culture, biochemical and serological identification methods. The PCR-ICT hybrid assay does not require sophisticated equipment and was proved to be practically useful in rapid and direct Legionellae detection from environmental water samples.