Microbiological aspects of a bioreactor with submerged membranes for aerobic treatment of municipal wastewater

An aerobic membrane bioreactor treating municipal wastewater at complete biomass retention was studied in respect of microbiological parameters over a period of 380 days. The results were compared to those obtained from a conventional activated sludge wastewater treatment plant (WWTP) treating the same wastewater. Microscopically, significant changes in the structure of the flocs and of the ratio between free suspended and aggregated cells could be observed. The presence of filamentous bacteria varied from almost not present to very high numbers. With the exception of short periods after changes in operating conditions, protozoa and metazoa were rarely present in the sludge community. The rate of oxygen consumption and the cell detectability by fluorescence in situ hybridizatio (FISH) with rRNA-targeted oligonucleotide probes were used to assess the physiological state of the bacterial cells Oxygen consumption rates of sludge samples obtained from both the conventional and membrane filtration plant wer determined without and after addition of different energy and carbon sources. In contrast to the conventional activate sludge, a pronounced increase in respiration activity upon the addition of organic substrates could be observed in th membrane filtration sludge. In situ probing with the Bacteria-specific probe EUB338 visualized 40-50% of all DAPI stainable bacteria in the membrane bioreactor, compared to 80% cells detectable by FISH in the conventional activate sludge. These results suggest that bacteria present in the highly concentrated biomass of the membrane reactor use the energy supplied for their maintenance metabolism and were not in a physiological state characteristic for growth This assumption could explain the zero net biomass production observed in the reactor.     

Direct quantification of bacterial biomass in influent, effluent and activated sludge of wastewater treatment plants by using flow cytometry

A rapid multi-step procedure, potentially amenable to automation, was proposed for quantifying viable and active bacterial cells, estimating their biovolume using flow cytometry (FCM) and to calculate their biomass within the main stages of a wastewater treatment plant: raw wastewater, settled wastewater, activated sludge and effluent. Fluorescent staining of bacteria using SYBR-Green I + Propidium Iodide (to discriminate cell integrity or permeabilisation) and BCECF-AM (to identify enzymatic activity) was applied to count bacterial cells by FCM. A recently developed specific procedure was applied to convert Forward Angle Light Scatter measured by FCM into the corresponding bacterial biovolume. This conversion permits the calculation of the viable and active bacterial biomass in wastewater, activated sludge and effluent, expressed as Volatile Suspended Solids (VSS) or particulate Chemical Oxygen Demand (COD). Viable bacterial biomass represented only a small part of particulate COD in raw wastewater (4.8 ± 2.4%), settled wastewater (10.7 ± 3.1%), activated sludge (11.1 ± 2.1%) and effluent (3.2 ± 2.2%). Active bacterial biomass counted for a percentage of 30-47% of the viable bacterial biomass within the stages of the wastewater treatment plant.     

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.     

Development and application of a resazurin-based biomass activity test for activated sludge plant management

A rapid, robust and cost-effective method of assaying the metabolic activity of the biomass of activated sludge plants would be a valuable process control tool in the wastewater treatment industry. We have developed and optimised a simple colorimetric test protocol, based on the redox dye resazurin, in which levels of reduction of the dye are proportional to cell biomass and respiration rate in both freshly sampled municipal sludges and in a surrogate activated sludge culture, Polytox. The method has been used to assess the impact of trade wastes on the activities of two municipal activated sludge populations of differing characteristics.     

高浓度消毒剂对活性污泥系统中微生物的影响

针对污水厂活性污泥易于发生丝状菌污泥膨胀问题,以西安市第二污水处理厂活性污泥为研究对象,在次氯酸钠投加量为15 mg/L的条件下,探索高浓度消毒剂对活性污泥中微生物的杀灭效果以及对胞外聚合物(EPS)含量和不同类型微生物活性的影响。结果表明,高浓度次氯酸钠能有效杀灭丝状菌,从而控制污泥的丝状膨胀现象,但在杀灭丝状菌的同时也会影响菌胶团絮体内的部分微生物,且对不同微生物的杀灭效果不同,亚硝酸盐氧化菌(NOB)是最易被杀灭的类型。当次氯酸钠投加量为15 mg/L时,反应3 h后污泥胞外聚合物总含量降低了15. 48%,硝化活性丧失殆尽且再未恢复,反硝化活性下降明显;恢复7 d后,絮体内部微生物可以得到有效恢复,而丝状菌大多依然处于死亡状态,EPS总含量继续下降。总之,高浓度消毒剂作用于活性污泥后,可以达到控制丝状菌的目的,但是污泥的硝化和反硝化活性也受到了影响。     

Microbial community structure in activated sludge floc analysed by fluorescence in situ hybridization and its relation to floc stability

The efficiency of activated sludge treatment plants is dependent on the solid-liquid separation properties of the activated sludge. A critical parameter is the stability of the microbial flocs. Weak flocs deflocculate easily leaving increased concentrations of suspended solids in the effluent. The knowledge about how different bacteria are attached to the flocs and their influence on the bioflocculation is limited. In this study, the deflocculation of different phylogenetic groups of bacteria in activated sludge from a full scale plant was investigated. The experiments were carried out by using a shear method where the sludge flocs are deflocculated under controlled shear conditions. The degree of deflocculation was measured as increase in turbidity of the supernatant. Identification and quantification of the microbial community structure of both total activated sludge and deflocculated bacteria were conducted with group-specific gene probes for broad groups of bacteria (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Actinobacteria) and fluorescence in situ hybridization (FISH). The microbial community structure of the deflocculated bacteria was different compared to the total activated sludge with a higher abundance of Gammaproteobacteria in the supernatant indicating that different groups of bacteria are bound with different strength to the floc surface. The results show that the bacteria in the outer parts of the flocs are relatively loosely bound to the floc matrix and can be easily eroded from the surface when exposed to shear.     

Effects of sonication on bacteria viability in wastewater treatment plants evaluated by flow cytometry--fecal indicators, wastewater and activated sludge

The application of sonication to wastewater or sludge contributes to the dispersion of aggregates, the solubilisation of particulate matter with an increase in its biodegradability, the damage of microorganisms due to the loss of cellular membrane integrity. This research is aimed at investigating the effects of sonication at 20kHz frequency on viability of microorganisms present in raw wastewater and activated sludge taken from a municipal wastewater treatment plant, as well as pure strains of Escherichia coli and E. faecalis. Flow cytometry was applied for the identification and quantification of viable and dead bacteria free in the bulk liquid, after the fluorescent staining of cellular nucleic acids. The main results showed that: (i) cells of E. coli were highly sensitive to sonication, even at low specific ultrasonic energy (E(s)), and disintegration of a large amount of cells was observed; (ii) on the contrary E. faecalis were more resistant than E. coli, even if high levels of E(s) were applied; (iii) bacteria in raw wastewater exhibited a dynamic of viable and dead bacteria similar to E. coli; (iv) in activated sludge samples, low levels of E(s) produced a prevalent disaggregation of flocs releasing single cells in the bulk liquid, while disruption of bacteria was induced only by very high levels of E(s).     

Measuring the activities of higher organisms in activated sludge by means of mechanical shearing pretreatment and oxygen uptake rate

A pretreatment method was developed to assess the activities of higher organisms. The method is based on mechanical shearing to damage the large cells of the protozoan and metazoan community in activated sludge. The procedure was confirmed through experimentation to be effective in determining the activities of higher organisms by comparing oxygen uptake rates (OURs) before and after the higher organisms were eradicated. Shearing led to disintegration of flocs, which could be effectively reconstituted by centrifugation. The reconstitution of the sludge flocs was essential since otherwise the activity of the floc mass would be too high due to lack of diffusion limitation. Mechanical shearing had no influence on the morphology, quantity and specific activity of yeasts, and it was inferred that bacteria smaller than yeasts in size would also not be influenced by the applied shearing procedure. Moreover, the effect of filamentous organisms on the measured activities of higher organisms was experimentally demonstrated and analyzed, and determined to be so weak that it could be ignored. Based on these tests, five typical activated sludge processes were selected to measure the contribution of higher organisms to the original OUR. The measured activities of higher organisms ranged from 9.4 to 25.0% of the original OURs.     

Aerobic granulation in a sequencing batch airlift reactor

Aerobic granular sludge was cultivated in an intensely mixed sequencing batch airlift reactor (SBAR). A COD loading of 2.5 kg Acetate-COD/(m3 d) was applied. Granules developed in the reactor within one week after inoculation with suspended activated sludge from a conventional wastewater treatment plant. Selection of the dense granules from the biomass mixture occurs because of the differences in settling velocities between granules (fast settling biomass), and filaments and flocs (slow settling biomass). At 'steady state' the granules had an average diameter of 2.5 mm, a biomass density of 60g VSS/I of granules, and a settling rate of > 10 m/h. The biomass consisted of both heterotrophic and nitrifying bacteria. The reactor was operated over a long period during which the granular sludge proved to remain stable. The performance of the intermittently fed SBAR was compared to that of the continuously fed biofilm airlift suspension reactor (BASR). The most importance difference was that the density of the granules in the SBAR was much higher than the density of the biofilms in the BASR. It is discussed that this could be due to the fact that the SBAR is intermittently fed, while the BASR is continuously fed.     

Micro-profiles of activated sludge floc determined using microelectrodes

The microbial activity within activated sludge floc is a key factor in the performance of the activated sludge process. In this study, the microenvironment of activated sludge flocs from two wastewater treatment plants (Mill Creek Wastewater Treatment Plant and Muddy Creek Wastewater Treatment Plant, with aeration tank influent CODs of 60-120 and 15-35 mg/L, respectively) were studied by using microelectrodes. Due to microbial oxygen utilization, the aerobic region in the activated sludge floc was limited to the surface layer (0.1-0.2mm) of the sludge aggregate at the Mill Creek plant. The presence of an anoxic zone inside the sludge floc under aerobic conditions was confirmed in this study. When the dissolved oxygen (DO) in the bulk liquid was higher than 4.0mg/L, the anoxic zone inside the activated sludge floc disappeared, which is helpful for biodegradation. At the Muddy Creek plant, with its lower wastewater pollutant concentrations, the redox potential and DO inside the sludge aggregates were higher than those at the Mill Creek plant. The contaminant concentration in the bulk wastewater correlates with the oxygen utilization rate, which directly influences the oxygen penetration inside the activated sludge floc, and results in redox potential changes within the floc. The measured microprofiles revealed the continuous decrease of nitrate concentration inside the activated sludge floc, even though significant nitrification was observed in the bulk wastewater. The oxygen consumption and nitrification rate analyses reveal that the increase of ammonia flux under aerobic conditions correlates with nitrification. Due to the metabolic mechanisms of the microorganisms in activated sludge floc, which varies from one treatment plant to another, the oxygen flux inside the sludge floc changes accordingly.     

The influence of key chemical constituents in activated sludge on surface and flocculating properties

This paper examines the influence of the chemical constituents of activated sludge and extracted extracellular polymeric substances (EPS) on the surface properties, hydrophobicity, surface charge (SC) and flocculating ability (FA) of activated sludge flocs. Activated sludge samples from 7 different full-scale wastewater treatment plants were examined. Protein and humic substances were found to be the dominant polymeric compounds in the activated sludges and the extracted EPS, and they significantly affected the FA and surface properties, hydrophobicity and SC, of the sludge flocs. The polymeric compounds proteins, humic substances and carbohydrates in the sludge flocs and the extracted EPS contributed to the negative SC, but correlated negatively to the hydrophobicity of sludge flocs. The quantity of protein and carbohydrate within the sludge and the extracted EPS was correlated positively to the FA of the sludge flocs, while increased amounts of humic substances resulted in lower FA. In contrast, increased amounts of total extracted EPS had a negative correlation to FA. The results reveal that the quality and quantity of the polymeric compounds within the sludge flocs is more informative, with respect to understanding the mechanisms involved in flocculation, than if only the extracted EPS are considered. This is an important finding as it indicates that extracting EPS may be insufficient to characterise the EPS. This is due to the low extraction efficiency and difficulties involved in the separation of EPS from other organic compounds. Correlations were observed between the surface properties and FA of the sludge flocs. This confirms that the surface properties of the sludge flocs play an important role in the bioflocculation process but that also other interactions like polymer entanglement are important.     

Suspended biofilm carrier and activated sludge removal of acidic pharmaceuticals

Removal of seven active pharmaceutical substances (ibuprofen, ketoprofen, naproxen, diclofenac, clofibric acid, mefenamic acid, and gemfibrozil) was assessed by batch experiments, with suspended biofilm carriers and activated sludge from several full-scale wastewater treatment plants. A distinct difference between nitrifying activated sludge and suspended biofilm carrier removal of several pharmaceuticals was demonstrated. Biofilm carriers from full-scale nitrifying wastewater treatment plants, demonstrated considerably higher removal rates per unit biomass (i.e. suspended solids for the sludges and attached solids for the carriers) of diclofenac, ketoprofen, gemfibrozil, clofibric acid and mefenamic acid compared to the sludges. Among the target pharmaceuticals, only ibuprofen and naproxen showed similar removal rates per unit biomass for the sludges and biofilm carriers. In contrast to the pharmaceutical removal, the nitrification capacity per unit biomass was lower for the carriers than the sludges, which suggests that neither the nitrite nor the ammonia oxidizing bacteria are primarily responsible for the observed differences in pharmaceutical removal. The low ability of ammonia oxidizing bacteria to degrade or transform the target pharmaceuticals was further demonstrated by the limited pharmaceutical removal in an experiment with continuous nitritation and biofilm carriers from a partial nitritation/anammox sludge liquor treatment process.     

Active biomass in activated sludge: Comparison of respirometry with catalase activity measured using an on-line monitor

On-line measurements of the activity of activated sludge biomass may allow more efficient operation of treatment plants. A novel monitor for activity which can be used on-line follows oxygen evolution from hydrogen peroxide by catalase activity in activated sludge. Results from this monitor were compared with assays of oxygen uptake rate (OUR) by respirometry. Samples from the five activated sludge plants were assayed and showed, as expected, that the mixed liquor suspended solids (MLSS) and volatile suspended solids (VSS) levels were a poor measure of the activity of the sample determined by OUR. However, the activity of the biomass determined by the catalase activity biomass monitor correlated well with the respiration rate. This monitor should provide a simple and robust alternative to OUR measurements in activated sludge plants.     

Effect of extended idle conditions on structure and activity of granular activated sludge

In industry and in tourist areas, periods exist during which no or only very little sewage is produced, and the wastewater treatment facilities have to be set into an idle phase over several days and even weeks. When wastewater is generated again and delivered to the treatment plant, the microorganisms in the activated sludge plant may have lost activity, and the activated sludge flocs may have disintegrated. From previous observation, it is assumed that granular activated sludge is more resistant against long-term storage than activated sludge flocs. Experiments using a laboratory-scale sequencing batch reactor (SBR) were conducted to study the impacts of a 7-weeks anaerobic idle time on structural integrity and metabolic activity of granular activated sludge, and the time required to regain the former operational status of the plant. Oxygen consumption rate (OCR) was used as an indicator to evaluate the metabolic activity of the sludge. The results revealed that the size, color and sedimentation characteristics of the granular sludge did hardly change during the storage period. Sludge activity, however, dropped to values as low as 0.17 mg min(-1)L(-1). After restarting the reactor, the OCR increased within 1 day to a level of 0.57 mg min(-1)L(-1), kept rising at a linear rate in the following days, and reached after 1 week, a value of 5.74 mg min(-1)L(-1) typical for the former activity status. These results imply that granular activated sludge can be stored for a considerably long period of time, and brought into service again relatively quickly. After an idle period of 7 weeks, it took less than a week to regain full capacity of the SBR.     

Estimation of viable biomass in wastewater and activated sludge by determination of ATP, oxygen utilization rate and FDA hydrolysis

ATP content, oxygen utilization rate (OUR) and fluorescein diacetate (FDA) hydrolysis were tested for the ability to express the amount of viable biomass in wastewater and activated sludge.The relationship between biomass and these activity parameters was established in growth cultures made by inoculating a nutrient medium with either wastewater or activated sludge. Biomass was then determined directly by measurement of dry weight of growth culture (dw), and compared to data obtained by using the previously mentioned methods. In the exponential growth phase, ATP content showed the best correlation with biomass, while FDA hydrolysis in the sludge failed to show any such correlation. Conversion factors of 3 mg ATP/g dw, 300 mg O₂/h g dw and 0.4 A/h (mg dw/ml) for ATP, OUR and FDA methods, respectively, were calculated.When the methods were applied for in situ determinations in four different wastewater plants, it was found that ATP content and respiration rate estimated viable biomass to range from 81 to 293 mg dw/g SS for raw wastewater and from 67 to 187 mg dw/g SS for activated sludge with a rather weak correlation between ATP and respiration measurements. The FDA hydrolysis estimated viable biomass to be higher than suspended solids, for which reason this parameter could not be recommended for determination of viable biomass in wastewater and activated sludge.     

Bacteriophages--potential for application in wastewater treatment processes

Bacteriophages are viruses that infect and lyse bacteria. Interest in the ability of phages to control bacterial populations has extended from medical applications into the fields of agriculture, aquaculture and the food industry. Here, the potential application of phage techniques in wastewater treatment systems to improve effluent and sludge emissions into the environment is discussed. Phage-mediated bacterial mortality has the potential to influence treatment performance by controlling the abundance of key functional groups. Phage treatments have the potential to control environmental wastewater process problems such as: foaming in activated sludge plants; sludge dewaterability and digestibility; pathogenic bacteria; and to reduce competition between nuisance bacteria and functionally important microbial populations. Successful application of phage therapy to wastewater treatment does though require a fuller understanding of wastewater microbial community dynamics and interactions. Strategies to counter host specificity and host cell resistance must also be developed, as should safety considerations regarding pathogen emergence through transduction.     

Test for assessing shear sensitivity of activated sludge flocs: a feasibility study

Sludge deflocculation can cause, like excessive growth of filamentous bacteria, activated sludge wastewater treatment failure. Yet, unlike the latter cause, there exists no widely accepted tool to assess the flocculation level of activated sludge and to predict sludge deflocculation. In this study, a test procedure is proposed to assess the sensitivity of activated sludge flocs to shear. The test consists in subjecting sludge sample to a shear treatment followed by a sludge volume measurement (SV(30)), with reference to a control. The ratio (%) obtained by dividing the SV(30) of the sheared sample by the SV(30) of the control is used to express the shear sensitivity. In a first series of experiments using two types of sludge, the test was shown to be able of ranging sludge samples in a correct order of shear sensitivity. Applying this so-called shear-SV test procedure to a series of sludge samples allowed to distinguish amorphous sludges (SV(30) ratio> or =100%) from normal sludges (SV(30) ratio<100%). The test was shown to be repeatable and simple. It requires only basic laboratory equipment and implies minimum cost. It can be used as framework for the assessment of activated sludge shear sensitivity in practice.     

Modelling nitrification, heterotrophic growth and predation in activated sludge

A mathematical model describing the interaction between nitrifiers, heterotrophs and predators in wastewater treatment has been developed. The inclusion of a predation mechanism is a new addition to the existing activated sludge models. The developed model considered multi-substrate consumption and multi-species growth, maintenance and decay in a culture where nitrifiers, heterotrophs and predators (protozoa and metazoa) are coexisting. Two laboratory-scale sequenced batch reactors (SBRs) operated at different sludge retention time (SRT) of 30 and 100 days for a period of 4 years were used to calibrate and validate the model. Moreover, to assess the predator activity, a simple procedure was developed, based on measuring the respiration rate with and without the presence of the predators. The model successfully described the performance of two SBRs systems. The fraction of active biomass (ammonia oxidisers, nitrite oxidisers and heterotrophs) predicted by the proposed model was only 33% and 14% at SRT of 30 and 100 days, respectively. The high fraction of inert biomass predicted by the model was in accordance with the microscopic investigations of biomass viability in both reactors. The presented model was used to investigate the effect of increasing sludge age and the role of predators on the biomass composition of the tested SBR system.     

Microbial processes associated to the decontamination and detoxification of a polluted activated sludge during its anaerobic stabilization

Xenobiotic compounds accumulate in activated sludge resulting from wastewater treatment plants serving both civil and industrial areas. The opportunity to use anaerobic digestion for the decontamination and beneficial disposal of a contaminated activated sludge was investigated in mesophilic and thermophilic microcosms monitored through an integrated chemical, microbiological and ecotoxicological procedure. The 10 months anaerobic sludge incubation at 35 degrees C resulted in an extensive production of a methane-rich biogas, a marked reduction of pathogenic cultivable bacteria and, importantly, a marked biodegradation of the sludge-carried organic pollutants, including some polychlorinated biphenyls and polycyclic aromatic hydrocarbons, along with a relevant sludge detoxification. The sludge decontamination seemed to occur mostly under methanogenic conditions and was not significantly affected by the addition of yeast extract or molasses. Lower bioremediation and biomethanization yields were observed under thermophilic conditions.     

The microbiology of an activated sludge waste-water treatment plant chemically treated for phosphorus removal

Microbiological research was conducted on a dual, secondary wastewater treatment system which was part of The Pennsylvania State University wastewater treatment plant. Each aeration basin received identical wastewater which was the effluent from a high rate trickling filter. One of the aeration basins was dosed with aluminum sulfate for the purpose of phosphorus removal. The other aeration basin (control) was operated in the conventional manner without alum addition. Plate counts performed on combined chemical-biological sludge and control activated sludge revealed that a higher number of viable micro-organisms was contained in the chemical-biological sludge, but the magnitude of difference between the two sludges was significant depending on the culture medium employed. Results suggest the aluminum flocs formed in the chemical-biological treatment enmesh dispersed wastewater micro-organisms, some of which are qualitatively unlike those indigenous to natural activated sludge. The combined chemical-biological sludge contained significantly higher numbers of lipolytic, gelatinolytic, and thiosulfate oxidizing micro-organisms and, possibly, fewer nitrite oxidizing micro-organisms than did control activated sludge. Alum did not appear to affect flagellated protozoa in mixed liquor; however, amoeboid and ciliated protozoa were found less frequently in alum dosed than in control mixed liquor. The settled effluent from the combined chemical-biological aeration basin generally contained fewer total coliforms, fecal coliforms, and fecal streptococci than did counterpart control effluents.