Identity, abundance and physiology of Aquaspirillum-related filamentous bacteria in activated sludge.

Microcolony-forming bacteria closely related to the genus Aquaspirillum in the Betaproteobacteria were recently observed to be abundant in many nutrient removal wastewater treatment plants. The developed oligonucleotide probe, Aqs997, however, occasionally also targeted some filamentous bacteria in activated sludge samples when fluorescence in situ hybridization was performed. In this study, the identity, abundance, and ecophysiology of these Aqs997-positive filamentous bacteria were studied in detail. Most of the Aqs997-positive filamentous bacteria could morphologically be identified as either Eikelboom Type 1701, Type 0041/0675 or possibly Type 1851, all characterized by epiphytic growth. They were found in almost all 21 wastewater treatment plants investigated. Two morphotypes were found. Type A filaments, which seemed to be the same genotype as the microcolony-forming bacteria targeted by probe Aqs997.Type B filaments also hybridized with probe GNS941, specific for the Chloroflexi phylum, so the true identity remains unclear. Aqs997-positive filaments usually stained Gram-negative, but Gram-positive filaments were also found, stressing the difficulties in identifying bacteria from morphology and simple staining results. Studies on the ecophysiology by microautoradiography showed that Aqs997-positive filamentous bacteria did not consume acetate and glucose, while some took up butyrate, mannose, and certain amino acids. Most likely, some Aqs997-positive filamentous bacteria were able to perform full denitrification such as the Aqs997-positive microcolony-forming bacteria, and some were able to store polyhydroxyalkanoates under anaerobic conditions, potentially being glycogen accumulating organisms.     

Description of filamentous bacteria present in industrial activated sludge WWTPs by conventional and molecular methods.

Conventional cultivation methods and molecular approaches were utilised to describe the filamentous bacterial population of industrial activated sludge WWTPs. In total 43 strains were isolated by micromanipulation and were affiliated with 12 different species, comprising two new species and a new genus. In particular, a new species of Microthrix, a new genus of a filamentous Alphaproteobacteria morphologically similar to Nostocoida limicola, and a new filamentous species closely related to the opportunistic pathogen Propionibacterium propionicum were obtained. Despite the high number of isolates, the cultivation approach was unable to describe the filamentous bacteria most common in industrial WWTP. A culture-independent approach, termed the cell sorting/RT-PCR method, was therefore applied to identify fastidious or non-culturable filamentous microrganisms from different industrial plants. By this method the relevant filaments were micromanipulated and their 16S rDNA genes were amplified by RT-PCR. This approach was highly efficient. In total 31 16S rRNA sequences were obtained and 16 of them were used for the design of new specific oligonucleotide probes that highlighted dominant filaments in industrial activated sludge plants.     

Modelling carbon oxidation in pulp mill activated sludge systems: calibration of Activated Sludge Model No 3.

Activated Sludge Model No 3 (ASM3) was chosen to model an activated sludge system treating effluents from a mechanical pulp and paper mill. The high COD concentration and the high content of readily biodegradable substrates of the wastewater make this model appropriate for this system. ASM3 was calibrated based on batch respirometric tests using fresh wastewater and sludge from the treatment plant, and on analytical measurements of COD, TSS and VSS. The model, developed for municipal wastewater, was found suitable for fitting a variety of respirometric batch tests, performed at different temperatures and food to microorganism ratios (F/M). Therefore, a set of calibrated parameters, as well as the wastewater COD fractions, was estimated for this industrial wastewater. The majority of the calibrated parameters were in the range of those found in the literature.     

Nitrogen removal and sludge reduction in a symbiotic activated sludge system between anaerobic archaea and bacteria.

The possible symbiosis between bacteria and anaerobic archaea was investigated in intermittent aeration (I/A) systems. Archaea solution added to I/A reactor might play an important role in biological activities as well as in improvement of mineralization of organic matter. I/A reactor with archaea solution (I/A-arch) could increase both nitrification and denitrification rate and also reduce the sludge yield remarkably. These results indicate the possibility of the symbiotic activated sludge system with anaerobic archaea by controlling the DO level in the aeration tank. In this study, DO was controlled by intermittent aeration schemes and a successful symbiotic activated sludge system was achieved to reach the following conclusions. 1) SOUR of I/A-arch system was 2.9 mg-O2/g-VSS x min. SOUR and nitrification rate of the sludge from I/A-arch was higher than those from the I/A and A/S reactors. 2) Removal efficiencies of organic matter (TCOD(Cr)) in I/A-arch, I/A and conventional activated sludge (A/S) reactors were 93, 90 and 87%, respectively. 3) Nitrification occurred successfully in each reactor, while denitrification rate was much higher in the I/A-arch reactor. Efficiencies of TN removal in A/I-arch, I/A and A/S reactors were 75, 63 and 33%, respectively. 4) Observed yield coefficients of I/A-arch, I/A and A/S reactors were 0.28, 0.41 and 0.37 g-VSS/g-COD.     

Quantification of ammonia-oxidizing bacteria populations in full-scale sewage activated sludge systems and assessment of system variables affecting their performance.

This study carried out quantification of ammonia-oxidizing bacteria (AOB) populations in 12 full-scale sewage activated sludge systems that were different in ammonia removals and treatment processes during three different seasons. Experiment was divided into 3 parts: 1) analysis of AOB communities by PCR-DGGE-cloning-sequencing of 16S rRNA genes; 2) development of four real-time PCR primer sets for quantification of the particular AOB of interest; and 3) quantification of AOB populations by using the newly developed real-time PCR primer sets. The results suggested that all the primer sets gave good reproducibility and specificity for PCR amplification with the detection limits of 10(2) copies/PCR reaction. Although the 12 systems were different in several aspects, one of the identified sequence types of Nitrosomonas oligotropha cluster was the dominant AOB in every system and every season studied. However, the other sequence type of this cluster was not significantly involved in ammonia removals in the systems. The occurrence of N. communis cluster in the systems seemed to depend on the remaining oxygen concentrations in the sludge floc and thus the activity of aerobic heterotrophs in the aeration tanks. N. europaea-Nitrosococcus. mobilis solely existed in one A20 system of which the influent contained twice the chloride concentrations than those of other systems.     

Anammox bacteria enrichment and characterization from municipal activated sludge

A sustainable option for nitrogen removal is the anaerobic ammonium-oxidizing (anammox) process in which ammonium is oxidized to nitrogen gas with nitrite as electron acceptor. Application of this process, however, is limited by the availability of anammox biomass. In this study, two Brocadia-like anammox phylotypes were successfully enriched, detected and identified from an activated sludge taken from a domestic wastewater treatment plant (Minas Gerais, Brazil) employing a Sequencing Batch Reactor (SBR). The dominant phylotype was closely related to 'Candidatus Brocadia sinica', but one clone seemed to represent a novel species for which we propose the name 'Candidatus Brocadia brasiliensis'. Based on Fluorescence in situ hybridization (FISH) analysis, this enrichment led to a relative population size of 52.7% (±15.6) anammox bacteria after 6 months of cultivation. The cultivation process can be divided into three phases: phase 1 (approximately 25 days) was characterized by heterotrophic denitrification metabolism, phase 2 was the propagation phase and phase 3 (from the 87th day onwards), in which significant anammox activity was detected. A long-term performance of the SBR showed a near perfect removal of nitrite based on the influent NO(2)(-)-N concentration of 61-95 mg L(-1). The average ammonia removal efficiency was 90% with the influent NH(4)(+)-N concentration of 55-82 mg L(-1). Therefore, anammox cultivation and enrichment from activated sludge was possible under a controlled environment within 3 months.     

Occurrence and control of filamentous bulking in aerated wastewater treatment plants of the French paper industry.

The occurrence of filamentous bacteria was investigated in 15 French pulp and paper activated sludge wastewater treatment plant (WWTP). Large filamentous populations were present in most of the plants. Identification carried out with conventional methods based on morphological features and staining techniques showed that the four main filamentous bacteria encountered in these industrial WWTP and responsible for bulking belong to the genera Thiothrix sp., Type 021 N, Haliscomenobacter hydrossis and Type 0092. During two years a specific survey was performed for three of these WWTP showing recurrent bulking phenomena. Data from WWTP performance, chemical data and filaments characterization were compared to correlate the presence of specific filaments with process operating conditions.     

Comparative study of MBR and activated sludge in the treatment of paper mill wastewater.

The study was based on a full scale activated sludge plant (AS) compared to a parallel operated pilot membrane bioreactor (MBR) with flat sheets membranes. Both systems received their influent from an anaerobic bioreactor treating paper mill wastewater. MBR produced an effluent of much better quality than AS in terms of suspended solids, containing 1 mg/L or less in 80% of the monitoring time, while the AS effluent contained 12 mg/L. This could save the necessity of further treatment by filtration in the case of MBR. Other effluent quality parameters, such as organic matter (COD and BOD), phosphorus and ammonia nitrogen, did not indicate substantial differences between AS and MBR. Calcium carbonate scaling and formation of a bacterial layer on the membrane caused severe flux reduction. The membrane blockage because of scaling and biofouling proved to be very serious, therefore, it required proper and more complicated maintenance than the AS system. This study leads to the conclusion that in the case of paper mill wastewater, after anaerobic biotreatment, if there is no need for excellent effluent quality in terms of suspended solids, the replacement of the AS by the MBR would not be strongly justified, mainly because of maintenance cost.     

Occurrence of EPS in activated sludge from a membrane bioreactor treating municipal wastewater.

EPS are supposed to be among the causes of membrane fouling in membrane bioreactors (MBR). In this work they are measured as total proteins and total polysaccharides. Theoretical and empirical considerations of biomass membrane filtration lead to the conclusion that EPS in the water phase is decisive for the filterability of activated sludge. In this study therefore different ways of separating the water phase from the biomass are investigated, where a simple filtration over a paper filter turned out to be sufficient. Subsequently, a simple batch test set up was used to investigate the influence of substrate conditions on the amount of EPS in the water phase. Dilution of the biomass does not result in changes. Dilution together with substrate addition leads to an increase both in proteins and polysaccharides. Replacement of the water phase leads to no significant changes in protein concentration, but polysaccharide concentration may vary considerably. This phenomenon is more pronounced after replacement of the water phase and substrate addition.     

A comparison of conventional activated sludge and low sludge production strategies for advanced treatment of kraft pulp mill effluent.

Parallel laboratory investigations were conducted to examine aspects of two distinct but related bioprocess strategies for low sludge production in the treatment of the same TCF kraft pulp mill effluent. The purpose of this article has been to compare the performance results from these two bench-scale trials with respect to nutrient demands, nutrient discharge, COD removal, and waste sludge characteristics. The LSP (Low Sludge Production) process can be used to significantly reduce sludge yield with excellent sludge characteristics. These sludge characteristics seemed to be related to elevated protozoan grazing pressures. The BAS (Biofilm-Activated Sludge) process achieves similar reduced sludge yields and sludge characteristics while at the same time significantly reducing the nutrient demands and discharge levels. For both LSP and BAS process optimization, the selector nutrient loading is critical to the overall process performance. Selector nutrient requirements are distinct from the overall process nutrient requirements.     

Isolation and characterization of thermophilic bacteria capable of lysing microbial cells in activated sludge.

To find an efficient biological method to solubilize waste activated sludge (WAS) from the biological wastewater treatment process, several strains of thermophilic bacteria capable of solubilizing WAS were isolated from sewage sludge compost. The culture supernatants of the isolates were able to lyse vegetable bacterial cells and the lytic activity mainly came from the exoenzyme produced by the isolates. The culture supernatants of the different isolates showed different lysis characteristics. The factors affecting bacterial cell lysis were investigated using E. coli as a model bacterium. The E. coli cells were lysed easily at higher temperature (60 degrees C or 70 degrees C) while little lytic activity by the supernatants of isolates was observed at lower temperature (50 degrees C). The level of pH also had great influence on the lysis of E. coli cells. The E. coli cells in the early stationary growth phase were easier to lyse than those in the late stationary growth phase or death phase.     

Fine-scale differences between Accumulibacter-like bacteria in enhanced biological phosphorus removal activated sludge.

A lab-scale sequencing batch reactor (SBR) and six full-scale wastewater treatment plants (WWTPs) performing enhanced biological phosphorus removal (EBPR) were surveyed. The abundance of Accumulibacter-related organisms in the full-scale plants was investigated using fluorescent in situ hybridization. Accumulibacter-related organisms were present in all of the full-scale EBPR plants, at levels ranging from 9% to 24% of total cells. The high percentage of Accumulibacter-related organisms seemed to be associated with configurations which minimize the nitrate recycling to the anaerobic zone and low influent BOD:TP ratios. PCR-based clone libraries were constructed from the community 16S rRNA gene plus the internally transcribed spacer region amplified from the SBR and five of the full-scale WWTPs. Comparative sequence analysis was carried out using Accumulibacter-related clones, providing higher phylogenetic resolution and revealing finer-scale clustering of the sequences retrieved from the SBR and full-scale EBPR     

Analysing sludge balance in activated sludge systems with a novel mass transport model

In activated sludge systems the mechanically treated wastewater is biologically cleaned by biomass (activated sludge). The basic requirement of an efficient biological wastewater treatment is to have as a high biomass concentration in the biological reactor (BR) as possible. The activated sludge balance in activated sludge systems is controlled by the settling, thickening, scraper mechanism in the secondary settling tank (SST) and sludge returning. These processes aim at keeping maximum sludge mass in the BR and minimum sludge mass in the SST even in peak flow events (storm water flow). It can be, however, only reached by a high SST performance. The main physical processes and boundary conditions such as inhomogeneous turbulent flow, geometrical features of the SST, wastewater treatment plant (WWTP) load, return sludge flow, sludge volume index etc. all influence settling thickening and sludge returning. In the paper a novel mass transport model of an activated sludge system is presented which involves a 2-dimensional SST model coupled with a mixed reactor model of the biological reactor. It makes possible to investigate different sludge returning strategies and their influence on the sludge balance of the investigated activated sludge system, furthermore, the processes determining the flow and concentration patterns in the SST. The paper gives an overview on the first promising model results of a prevailing peak flow event investigation at the WWTP of Graz.     

Comparison of linear alkylbenzene sulfonates removal in conventional activated sludge systems and membrane bioreactors.

The potential of a membrane bioreactor (MBR) and a conventional activated sludge (CAS) system to remove polar micropollutants was evaluated using linear alkylbenzene sulfonates (LAS) as model components. Removal efficiencies over 97% were achieved in both reactor systems. The appearance of biological breakdown metabolites and the respirometric response of the sludges to LAS addition indicated that LAS removal was due to biodegradation, rather than sorption phenomena. The effect of operational variables, such as hydraulic retention time, LAS composition and hydrophobicity of the membrane used in the MBR, was negligible in the range tested. A stepwise increase in LAS influent concentration resulted in higher residual effluent concentrations but did not change the procentual removal efficiency. Because an increase in LAS and SPC effluent concentration occurred to a larger extent in the CAS than in the MBR under similar operating conditions, MBRs may turn out to be be more robust with respect to biological degradation of micropollutants than CAS.     

Co-conditioning and dewatering of alum sludge and waste activated sludge.

Co-conditioning and dewatering behaviors of alum sludge and waste activated sludge were investigated. Two different sludges were mixed at various ratios (2:1; 1:1; 1:2; 1:4) for study. Capillary suction time (CST) and specific resistance to filtration (SRF) were utilized to assess sludge dewaterability. Relatively speaking, waste activated sludge, though of higher solid content, was more difficult to be dewatered than alum sludge. It was found that sludge dewaterability and settlability became better with increasing fraction of alum sludge in the mixed sludge. Dosage required of the cationic polyelectrolyte (KP-201C) for dewatering was reduced as well. It is proposed that alum sludge acts as skeleton builder in the mixed sludge, and renders the mixed sludge more incompressible which is beneficial for sludge dewatering. Implications of the results of the study to the sludge management plan for Taipei City that generates both alum sludge and waste activated sludge at significant amount are also discussed. The current sludge treatment and disposal plan in Metropolitan Taipei could be made more cost-effective.     

Multicriteria evaluation tools to support the conceptual design of activated sludge systems.

During the past decade the pressure of the whole spectrum of stakeholders has increased considerably leading the consideration of different types of objectives, i.e. economical, technical, legal and environmental, into the process design efforts. Thus, the traditional design approaches should turn into more complex assessment methods including different types of objectives in order to conduct integrated assessments. The objective of this paper is to present and discuss the usefulness of three evaluation tools, based on multicriteria decision analysis, to support the conceptual design of activated sludge systems These support tools consist of: i) preliminary multiobjective optimization, where the most promising options (those located near to the optimum) are compared based on the results of dynamic simulation, ii) identification of strong and weak points for each option by means of classification trees and the subsequent extraction of knowledge-based rules, and iii) evaluation of the trade-offs between a certain evaluation criteria and the overall process performance through the integrated application of mathematical modelling and qualitative knowledge extracted during the design process.     

Potential of activated sludge ozonation.

The disposal of sewage sludge and the agricultural use of stabilised sludge are decreasing due to more stringent regulations in Europe. An increasing fraction of sewage sludge must therefore be dewatered, dried, incinerated and the ashes disposed of in landfills. These processes are cost-intensive and also lead to the loss of the valuable phosphate resources incorporated in the sludge ash. The implementation of processes that could reduce excess sludge production and recycle phosphate is therefore recommended. Partial ozonation of the return sludge of an activated sludge system significantly reduces excess sludge production, improves the settling properties of the sludge and reduces bulking and scumming. The solubilised COD will also improve denitrification if the treated sludge is recycled to the anoxic zone. However, ozonation partly kills nitrifiers and could therefore lead to a decrease of the effective solid retention time of the nitrifier, thus reducing the safety of the nitrification. This paper discusses the effect of ozonation on sludge reduction, the operating stability of nitrification, the improvement of denitrification and also presents an energy and cost evaluation.     

Potential of activated sludge disintegration.

The disposal of sewage sludge and the agricultural use of stabilised sludge are decreasing due to more stringent regulations in Europe. An increasing fraction of sewage sludge must therefore be dewatered, dried, incinerated and the ashes disposed of in landfills. These processes are cost-intensive and also lead to the loss of valuable phosphate resources incorporated in the sludge ash. The implementation of processes that could reduce excess sludge production and recycle phosphate is therefore recommended. Disintegration of biological sludge by mechanical, thermal and physical methods could significantly reduce excess sludge production, improve the settling properties of the sludge and reduce bulking and scumming. The solubilised COD could also improve denitrification if the treated sludge is recycled to the anoxic zone. However, disintegration partly inhibits and kills nitrifiers and could therefore shorten their effective solid retention time, thus reducing the safety of the nitrification. This paper discusses the potential of disintegration on sludge reduction, the operating stability of nitrification, the improvement of denitrification and also presents an energy and cost evaluation.     

Protein extraction from activated sludge.

Two methods for the separation of protein originating from activated sludge were compared. In one method, the total protein was isolated out of the activated sludge (crude extract). These samples included all dissolved proteins originating from the bacterial cells and biofilm made up of extracellular polymeric substances (EPS). Every time polyacrylamide gel electrophoresis (PAGE) was done, the protein bands from samples of crude extract were covered by polymeric substances including carbohydrates, uronic acids or humic compounds. Using the immunoblot technique it was possible to demonstrate the presence of the heat shock protein HSP70 in crude extracts of activated sludge. The comparison of protein fingerprints required that clear and distinct bands appear on the PAGE analysis. To this end, a procedure to separates bacterial cells from the EPS was developed. Bacterial cells were separated by incubation with EDTA and subsequent filtration. The isolated cells were directly incubated in a sample buffer.     

A comparison of BNR activated sludge systems with membrane and settling tank solid-liquid separation.

Installing membranes for solid-liquid separation into biological nutrient removal (BNR) activated sludge (AS) systems makes a profound difference not only to the design of the membrane bio-reactor (MBR) BNR system itself, but also to the design approach for the whole wastewater treatment plant (WWTP). In multi-zone BNR systems with membranes in the aerobic reactor and fixed volumes for the anaerobic, anoxic and aerobic zones (i.e. fixed volume fractions), the mass fractions can be controlled (within a range) with the inter-reactor recycle ratios. This zone mass fraction flexibility is a significant advantage of MBR BNR systems over BNR systems with secondary settling tanks (SSTs), because it allows changing the mass fractions to optimise biological N and P removal in conformity with influent wastewater characteristics and the effluent N and P concentrations required. For PWWF/ADWF ratios (fq) in the upper range (fq approximately 2.0), aerobic mass fractions in the lower range (f(maer) < 0.60) and high (usually raw) wastewater strengths, the indicated mode of operation of MBR BNR systems is as extended aeration WWTPs (no primary settling and long sludge age). However, the volume reduction compared with equivalent BNR systems with SSTs will not be large (40-60%), but the cost of the membranes can be offset against sludge thickening and stabilisation costs. Moving from a flow unbalanced raw wastewater system to a flow balanced (fq = 1) low (usually settled) wastewater strength system can double the ADWF capacity of the biological reactor, but the design approach of the WWTP changes away from extended aeration to include primary sludge stabilisation. The cost of primary sludge treatment then has to be offset against the savings of the increased WWTP capacity.