Use of image analysis in the study of competition between filamentous and non-filamentous bacteria

One of the most common problems in the operation of activated sludge plants is the inability to separate solids from treated effluents in the clarifiers caused by the overgrowth of filamentous micro-organisms (FM) with respect to floc-forming bacteria. In order to develop kinetic models that help to predict bulking events, growth kinetics of the FMs and non-filamentous bacteria (NFB) should be known. This paper addresses the competition of a FM and a NFB in a continuous stirred tank reactor. Experimental observations of the effect of the dilution rate on the composition of the mixed culture were compared to simulated results. Image analysis was used to measure NFB and FM fractions in order to evaluate the proposed mathematical model. Experimental results and numerical simulations showed that low D values favored the growth of FM; on the contrary, when high D values were applied a rapid overgrowth of the NFB were observed. Thus, high D values favored the growth of NFB minimizing the risk of filamentous bulking.     

Modeling of chlorine effect on floc forming and filamentous micro-organisms of activated sludges

Chlorination is the most economical, non-specific method to control the excessive growth of filamentous micro-organisms causing bulking in activated sludge systems in the treatment of food industrial wastewaters; it was one of the first methods used to control filamentous bulking and is still widely employed. Considering that chlorination affects both floc-forming and filamentous micro-organisms and leaves undesirable disinfection by-products, it is necessary to define the adequate doses to control bulking, minimizing the effect on floc-forming bacteria.In the present work the effect of biomass concentration and type of micro-organism on chlorine decay kinetics was evaluated; the inactivation of either a filamentous (Sphaerotilus natans) or a floc-forming (Acinetobacter anitratus) micro-organism due to chlorination was also analyzed.For chlorine decay assays, the samples were treated in a batch system with sodium hypochlorite ranging between 9.8 and 56.6 mg Cl(2) (gVSS)(-1). Respirometric assays were used to evaluate the effect of chlorine on micro-organisms respiratory activity; in these cases, sodium hypochlorite doses ranged between 2.5 and 18 mgCl(2) (gVSS)(-1).A model that allowed to predict simultaneously chlorine consumption and respiratory activity decay for both micro-organisms as a function of time was proposed. The model includes three coupled differential equations corresponding to respiratory inhibition, readily organic matter oxidation by chlorine and chlorine decay. The rate of chlorine decay depended on both, type and concentration of the micro-organisms in the system. Chlorine consumption rate due to S. natans was 2-4 times faster than A. anitratus. Using the proposed model initial critical chlorine doses (the lowest initial dose that leads to a total inhibition of the respiratory activity) were calculated for both micro-organisms and values of 11.9 mgCl(2) (gVSS)(-1) for S. natans and 4.5 mgCl(2) (gVSS)(-1) for A. anitratus were obtained. These critical doses indicated that in non flocculated pure cultures, floc-former bacteria A. anitratus was more susceptible to chlorine action than S. natans.     

Effect of chlorine on filamentous microorganisms present in activated sludge as evaluated by respirometry and INT-dehydrogenase activity

Activated sludge technology is more used than any other for biological treatment of wastewater. However, filamentous bulking is a very common problem in activated sludge plants, chlorine being the chemical agent normally used to control it. In this work the effect of chlorine on microorganisms present in activated sludge flocs was assessed by a respirometric technique (oxygen uptake rate, OUR) and by the INT-dehydrogenase activity test (DHA) measured by two techniques: spectrophotometry (DHA(a)) and image analysis (DHA(i)). Both DHA tests were optimized and correlated with the respirometric technique (OUR) using pure cultures of a filamentous microorganism (Sphaerotilus natans) under chlorine inhibition. Using these correlations the tested methods were applied to determine the action of chlorine on respiratory activity in activated sludge. The OUR and the DHA(a) quantifies the action of chlorine on the total respiratory activity (RA) of flocs (filamentous and floc-forming bacteria); in contrast, the DHA(i) test evaluates specific action of chlorine on the RA of filamentous microorganisms. In activated sludge flocs containing filamentous microorganisms, a chlorine dose of 4.75 mgCl(2) (gVSS)(-1) with a contact time of 20 min reduced about 80% of the RA of filamentous bacteria while affecting only 50-60% of the total RA of flocs. Besides, a chlorine dose of 7.9 mgCl(2) (gVSS)(-1) produced the total respiratory inactivation of filamentous microorganisms after 10 min contact, however, with this dose the total RA of activated sludge flocs was reduced only about 45-65%; controlling filamentous bulking without affecting too much floc-forming bacteria. At the tested chlorine concentrations the inhibition of filamentous microorganisms was higher than in the whole activated sludge. Although floc-forming microorganisms were demonstrated to be more susceptible to chlorine than filamentous in pure cultures, results obtained in the present work confirmed that it is the location of the filamentous microorganisms in the flocs and the presence of extracellular polymer substances which largely determines their higher susceptibility to chlorine; consequently this feature plays a critical role in bulking control.     

Impact of the repetition of oxygen deficiencies on the filamentous bacteria proliferation in activated sludge

Cases of low stress can frequently occur in a wastewater treatment plant (WWTP). As they have no visible impact on the plant operation, they generally go unnoticed. Nevertheless, it would appear that an accumulation of such cases can result in serious operational problems. The impact of the repeated application of oxygen deficiency on the proliferation of the filamentous bacteria Sphaerotilus natans, Haliscomenobacter hydrossis, Eikelboom type 021N and Thiothrix spp has been studied on WWTP pilots. Two different oxygen deficiencies series were tested. Filamentous bacteria evolution in activated sludge was monitored using fluorescent in situ hybridization. For the weak deficiencies, no significant variation in the filamentous bacteria response level was recorded, whereas with more severe deficiencies the filamentous bacteria response increased considerably with the increase in stress number. From the results obtained, it would appear that there is an intensity and frequency threshold beyond which the filamentous bacteria response levels increase when the stress is applied in series. This "oxygen deficiencies repetition" appears to be an important factor in the appearance of bulking.     

The effects of food waste disposers on the wastewater system: a practical study

Domestic food waste disposers (FWDs) have recently come to prominence as a possible alternative for disposal of organic waste, to reduce the quantities of this type of waste sent to landfill. There has been little research undertaken on the potential effects of food waste on the wastewater system, and it is believed no previous practical studies have been undertaken in the United Kingdom. In this study, food waste was ground in an FWD and analysed for chemical oxygen demand (COD), biological oxygen demand (BOD), ammonia, total nitrogen, total phosphorus, total suspended solids and rapidly settleable solids to determine their effects on the wastewater system. The largest impacts were on COD, BOD and suspended solids, compared with the amounts of these determinands that currently arrive at sewage treatment works (STW). Experiments using settled samples showed that a relatively high proportion of nitrogen, phosphorus, COD and BOD would pass through to secondary treatment at the STW.     

The growth of cladophora glomerata in a river receiving sewage effluent

A considerable improvement in the natural flora of the River Great Stour has occurred since the mid-1960s when dense growths of Cladophora glomerata dominated sections of the river with the disappearance of some rooted macrophytes. Cladophora growths in the river in response to changing river conditions and sewage treatment operations during a thirty year period are reviewed. A vegetation mapping programme undertaken from 1978 to 1982 to assess the influence of a sewage treatment works discharge on the growth of the filamentous alga has shown that previous recommendations to reduce dissolved phosphorus concentrations in the final effluent need not be implemented.     

Calibration of a model for an A + B acivated sludge pilot plant treating industrial wastewater

A mathematical model has been developed and calibrated for a two-stage (A + B) activated sludge pilot plant treating wastewater from a chemical-pharmaceutical industry. Two series of simulations have been performed: (a) by using typical values for the parameters of the model from the bibliography, which remain constant with time, and (b) by using, for some parameters, values which are variable with time, and are determined following a specific methodology. The calibration is constrained to be exactly consistent with daily observations of COD from the effluent and reactor suspended solids (MLSS). Three internal parameters of the system were thus obtained in their daily variation: MLSS growth rate, specific substrate removal rate and concentration factor in the settling tank. Mean values obtained for MLSS growth rate were 0.73 d⁻¹ and 0.022 d⁻¹ for reactors A and B respectively, and the average observed biomass yield coefficient was 0.38 (reactor A) and 0.21 (reactor B). The model infered the evolution of the COD and MLSS with a fairly good accuracy, proving that the method used to obtain the parameters does not have internal inconsistencies and may be used in other situations.     

Including the effects of filamentous bulking sludge during the simulation of wastewater treatment plants using a risk assessment model

The main objective of this paper is to demonstrate how including the occurrence of filamentous bulking sludge in a secondary clarifier model will affect the predicted process performance during the simulation of WWTPs. The IWA Benchmark Simulation Model No. 2 (BSM2) is hereby used as a simulation case study. Practically, the proposed approach includes a risk assessment model based on a knowledge-based decision tree to detect favourable conditions for the development of filamentous bulking sludge. Once such conditions are detected, the settling characteristics of the secondary clarifier model are automatically changed during the simulation by modifying the settling model parameters to mimic the effect of growth of filamentous bacteria. The simulation results demonstrate that including effects of filamentous bulking in the secondary clarifier model results in a more realistic plant performance. Particularly, during the periods when the conditions for the development of filamentous bulking sludge are favourable – leading to poor activated sludge compaction, low return and waste TSS concentrations and difficulties in maintaining the biomass in the aeration basins – a subsequent reduction in overall pollution removal efficiency is observed. Also, a scenario analysis is conducted to examine i) the influence of sludge retention time (SRT), the external recirculation flow rate (Q_r) and the air flow rate in the bioreactor (modelled as k_La) as factors promoting bulking sludge, and ii) the effect on the model predictions when the settling properties are changed due to a possible proliferation of filamentous microorganisms. Finally, the potentially adverse effects of certain operational procedures are highlighted, since such effects are normally not considered by state-of-the-art models that do not include microbiology-related solids separation problems.     

Life history responses of Daphnia magna feeding on toxic Microcystis aeruginosa alone and mixed with a mixotrophic Poterioochromonas species

The toxic effects of a mixotrophic golden alga (Poterioochromonas sp. strain ZX1) and a cyanobacterium Microcystis aeruginosa grazed by Poterioochromonas to a cladoceran were investigated through life history experiments using Daphnia magna. Poterioochromonas cultured in two ways (fed M. aeruginosa in an inorganic medium or grown in an organic medium) both induced starvation-like effects on D. magna, indicating that Poterioochromonas is neither acutely toxic nor a good food for D. magna. Despite a microcystin-LR content of 10⁻⁸ μg cell⁻¹ in M. aeruginosa, no toxins were accumulated in Poterioochromonas fed the cyanobacterium. The toxic effect of M. aeruginosa to D. magna was significantly reduced in the presence of Poterioochromonas, which may be performed in two ways: decrease M. aeruginosa cells ingestion of D. magna by grazing on M. aeruginosa; and decrease the toxicity of the medium by degrading the toxins released by M. aeruginosa. This study provides new information on the interactions between a cyanobacterium and its grazer under laboratory conditions and may increase our understanding of the ecological significance of such interactions in the aquatic food webs.     

The use of Bacillus thuringiensis var. israelensis to control the nuisance fly Sylvicola fenestralis (Anisopodidae) in sewage filter beds

The effectiveness of using the microbial insecticide Bacillus thuringiensis var. israelensis (Bti) to control the nuisance fly Sylvicola fenestralis at Rossendale Sewage Works, Lancashire, was investigated. Following a provisional trial in 1987, the whole Works (28 filter beds) was treated with a two-stage application of Bti in 1988; the first application in late April was separated from the second in late May by about half the length of the Sylvicola life-cycle. The Bti treatment was timed to reduce fly nuisance in June, the month when most complaints (71%) had been received. For each application, 0.161. of the Bti preparation, containing nearly 10¹⁰ spores·cm⁻³, was applied per square metre of bed surface. An 80 and 98% kill of Sylvicola larvae was recorded following the first and second applications respectively. The lower kill achieved by the first application was believed to be due to insufficient mixing of the Bti with the settled sewage prior to dosing. Nuisance caused by the flies was considerably reduced; there were no complaints from the public during June, although a few were received in late July, early August. Sewage purification efficiency was not affected by Bti treatment. The maximum concentration of Bti spores in the Works' effluent was 7 × 10⁴ spores·cm⁻³. This concentration did not appear to affect the already impoverished macro-invertebrate fauna of the receiving river.     

Tertiary treatment of wastewater with Phormidium bohneri (Schmidle) under various light and temperature conditions

Experiments were carried out on secondary effluents under different conditions of light and temperature with the aim of evaluating the potential and limits of the epilithic filamentous cyanobacterium Phormidium bohneri (Schmidle) for biotreatment of domestic wastewaters. Aerated cultures were incubated at 10 and 30°C in a chamber fitted with a photic gradient plate receiving 120 and 720 μE/m²s. Results indicate that the strain used prefers elevated temperatures, and can withstand considerable variation in light intensity. Response at 30°C with regard to NH₃ and PO₄⁻³ removal agrees with that reported for unicellular chlorophytes. A complete exhaustion of these nutrients was obtained after 3 and 5 days, respectively. It appears that NH₃ stripping contributed substantially (38–100%) to the overall NH₃ removal efficiency observed in the system. In addition, the protein and carbohydrate content of the biomass produced suggests potential feedstock use.     

An investigation into the occurrence of sewage fungus in rivers containing papermill effluents—IV. Growth in experimental channels dosed with effluents and pure compounds

Wastewaters from the manufacture of pulp and paper have given rise to problems of excessive microbial growth in rivers over a number of years. This paper is the first in a series of four articles describing research undertaken by PIRA at four U.K. paper/board mills (one integrated with pulp production) over the period 1978–1980. This first paper briefly reviews the published literature on sewage fungus growth from pulp and paper mill discharges up to 1978, but mainly describes previously unpublished work undertaken by PIRA over the period 1965–1975. This introductory paper thus provides a state-of-the-art review of methods to control sewage fungus growth from pulp and paper mill effluents prior to commencement of the research described in the following three articles.     

Responses of Cyperus involucratus Rottb. to nitrogen and phosphorus, with reference to wastewater reclamation

There is increasing use of aquatic plants in biosystems to ameliorate wastewaters. Cyperus involucratus Rottb. is a large emergent aquatic plant which may be useful in treating wastewater because it grows quickly, does not die back in winter, has little weed potential and grows in dryland situations. Many wastewaters contain high levels of N and P, and the capacity of C. involucratus to take up these nutrients was studied under glasshouse conditions. Maximum growth was attained between 84 and 168 mg NO₃-Nl⁻¹ and 1–5 mg PO₄-Pl⁻¹. At a low level of supplied NO₃-N, most of the N in plant organs was reduced-N; at levels of supplied NO₃-N above 84 mg l⁻¹, there was considerable accumulation of NO₃-N in plant organs. The accumulation of NO₃-N was greatest in culms where it made up over 50% of their N content when plants were grown on 350 mg NO₃-Nl⁻¹. Linear regression equations best described the relationships between dry matter and reduced-N contents in roots, culms and leaves. Over 60% of the total plant N and P accumulated in above ground organs, and concentrations of up to 0.53% P and 3.4% N (dry wt basis) were recorded in leaves. C. involucratus would be a suitable plant for wastewater reclamation on account of its high dry matter production, its ability to tolerate hyper-eutrophic levels of N and P, and the accumulation of most of the N and P in the harvestable portions of the plant.     

Nitrification of ammonia in wastewater: Field observations and laboratory studies

Complete nitrification of ammonia in partially treated domestic wastewater was demonstrated in a mixed culture chemostat. The optimal detention time of water in the chemostat for maximal nitrification was 6–7 days. In the field, a significant number of nitrifying bacteria was present in a wastewater reservoir, as well as in samples of inflowing water from the preceding anaerobic overloaded oxidation pond.     

Cryptosporidium and Giardia detection in water bodies of Galicia, Spain

The objective of this study was to determine the mean concentration (per litre) of Cryptosporidium oocysts and Giardia cysts in recreational river areas (n = 28), drinking water treatments plants (DWTPs; n = 52) and wastewater treatment plants (WWTPs; n = 50) in Galicia (NW Spain). Water samples from rivers and from the influent (50–100 l) and the treated effluent (100 l) of the water plants were filtered using Filta-Max filters (IDEXX Laboratories, Inc., Westbrook, ME, USA). A total of 232 samples were processed and the (oo)cysts were concentrated, clarified by IMS and then detected by IFAT. The viability was determined by applying fluorogenic vital dye (PI).In the recreational areas, infective forms of Cryptosporidium and Giardia were detected in 16 (57.1%; 1–60 oocysts per litre) and 17 (60.7%; 1–160 cysts per litre) samples, respectively. In the water flowing into the water treatment plants, oocysts were detected in 21 DWTPs (40.4%; 1–13 oocysts per litre) and cysts were observed in 22 DWTPs (42.3%; 1–7 cysts per litre). In the effluents from the treatment plants, Cryptosporidium oocysts and Giardia cysts were identified in 17 DWTPs (32.7%; 1–4 oocysts per litre) and in 19 DWTPs (36.5%; 1–5 cysts per litre), respectively. The highest concentrations of (oo)cysts were found in the WWTPs; specifically, oocysts were detected in 29 (58.0%; 1–80 oocysts per litre) and cysts in 49 (98.0%; 2–14.400 cysts per litre) WWTP effluents. Cryptosporidium and Giardia were detected in 32 (64.0%; 1–120 oocysts per litre) and 48 (96.0%; 2–6.000 cysts per litre) WWTP effluents, respectively. The percentage viability of the (oo)cysts ranged between 90.0% and 95.0%. In all samples analysed. Moreover, it was found that the effluents from coastal WWTPs were discharged directly into the sea, while inland WWTPs were discharged directly into rivers. The concentrations of both enteropathogens detected in effluents from WWTPs therefore represent a significant risk to human and animal health.These results demonstrate the wide distribution of Cryptosporidium and Giardia in the environment, the ineffectiveness of treatments in DWTPs and WWTPs in reducing/inactivating both protozoa and the need to monitor the presence, viability and infectivity of Cryptosporidium and Giardia in water bodies. In conclusion, the findings suggest the need for better monitoring of water quality and identification of sources of contamination.     

Identification of spatial agglomerations in the German food processing industry

We identify spatial agglomerations in the German food industry, using the Cluster Index by Sternberg and Litzenberger. Previous studies have analysed this industry utilizing aggregated data with the result of a lack of agglomerations. Given its heterogeneous character, the present study analyses subsectors of the German food industry at a disaggregated spatial and sectoral level. The results show spatial agglomerations in particular for the subsectors processing and preserving of meat, fish, fruit, wine, milk, mineral water as well as for breweries. This indicates that those subsectors are characterized by stronger cluster self‐enforcing processes likely in combination with a favourable proximity to natural resources.     

Polyphosphate accumulating abilities of acinetobacter isolates from a biological nutrient removal pilot plant

Of a total of 156 Acinetobacter isolated obtained from a biological nutrient removal pilot plant, 80 could accumulate polyphosphate from a medium with low phosphorus concentrations (constitutive accumulators), 61 accumulated polyphosphate only from medium with high phosphorus concentrations (conditional accumulators) and the remainder were unable to accumulate polyphosphate on either medium. There was no relationship between the ability to accumulate polyphosphate in pure culture and the taxonomic status of the isolates, in terms of their genospecies.     

Degradation of pharmaceuticals in non-sterile urban wastewater by Trametes versicolor in a fluidized bed bioreactor

The constant detection of pharmaceuticals (PhACs) in the environment demonstrates the inefficiency of conventional wastewater treatment plants to completely remove them from wastewaters. So far, many studies have shown the feasibility of using white rot fungi to remove these contaminants. However, none of them have studied the degradation of several PhACs in real urban wastewater under non-sterile conditions, where mixtures of contaminants presents at low concentrations (ng L⁻¹ to μg L⁻¹) as well as other active microorganisms are present. In this work, a batch fluidized bed bioreactor was used to study, for the first time, the degradation of PhACs present in urban wastewaters at their pre-existent concentrations under non-sterile conditions. Glucose and ammonium tartrate were continuously supplied as carbon and nitrogen source, respectively, and pH was maintained at 4.5. Complete removal of 7 out of the 10 initially detected PhACs was achieved in non-sterile treatment, while only 2 were partially removed and 1 of the PhACs analyzed increased its concentration. In addition, Microtox test showed an important reduction of toxicity in the wastewater after the treatment.     

The determination of 2,3,7,8-tetrachlorodibenzo-p-dioxin in treated wastewater

A procedure has been developed for the determination of low levels (parts-per-trillion) of 2,3,7,8-tetrachlorodibenzo-p-dioxin in treated wastewater. A combination of cleanup steps on silica, alumina, and reactant modified adsorbents yields a relatively interference-free residue when examined by gas chromatography/mass spectrometry in the selected ion monitor (SIM) mode of operation.