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.     

Occurrence and significance of filamentous bacteria in pulp and paper activated sludge systems.

A microbial survey of 27 activated sludge (AS) systems included 16 conventional activated sludge (CAS) systems, five sequential batch reactors (SBR) and six oxygen-activated sludge (OAS) systems, all treating pulp and paper effluents. The most prevalent filaments observed were Thiothrix (26%) and Type 021N (22%). The designs of the activated sludge systems seemed to have an effect on the filament types. We found Thiothrix to be the most common filament associated with bulking. For CAS systems, a completely mixed mode of operation promoted Thiothrix and Type 021N growth. Type 021N was favoured in CAS systems with food to microorganism (F/M) ratios higher than 0.2, and with dissolved oxygen (DO) residuals higher than 2 ppm, while Thiothrix generally proliferated at lower F/M ratio and DO residuals. Nutrient deficiencies as well as nutrient dosage variations were suspected in most of the systems having Thiothrix and Type 021N as the most prevalent filaments. Thiothrix appeared to prefer polyphosphate and/or ammonia rather than urea/phosphoric acid as N and P sources. Systems with aerobic selectors showed the lowest filament counts, while systems with no selectors showed the highest filament counts.     

Evaluation of filamentous microorganism growth factors in an industrial wastewater activated sludge system

An investigation comprising four studies was undertaken to determine possible factors affecting the growth of several different types of filamentous microorganisms present in a bulking industrial wastewater activated sludge. Results from laboratory-scale continuous-flow and full-scale studies suggested that DO concentration and F:M ratio were the likely key factors affecting filamentous growth in the activated sludge. From the results of two laboratory studies isolating the effects of DO concentration and F:M ratio on filamentous growth, favorable growth ranges of DO concentration or F:M ratio were estimated for the following filaments: Microthrix parvicella, Nocardia spp., Nostocoida limicola II, and Types 0041, 1851, and 1863. Most of the bacteria causing filamentous bulking of the activated sludge were found to be filaments typically associated with low F:M, and increasing the F:M ratio appeared to cause N. limicola II to lose its competitive advantage in the activated sludge system. Type 1863, on the other hand, was found to be a low DO filament, as DO concentrations of 0.1 mg O₂/l or less appeared to be a necessary condition for its filamentous growth. Though Nocardia was found to be a low F:M filament, its growth also seemed to be affected by DO concentration, as its growth was stimulated by concentrations of 1.0 mg O₂/l or greater, with a near linear relationship up to at least 5 mg O₂/l.     

Detection of filamentous and nitrifying bacteria in activated sludge with 16S rRNA probes

Activated sludge samples from wastewater treatment plants from potato starch and starch derivatives factory and from a municipal sewage treatment plant were analyzed with DNA probes specific for several filamentous bacteria. It was found that Haliscomenobacter hydrossis, Sphaerotilus natans, Thiothrix sp. and Eikelboom Type 021N were common in the activated sludges. Fluorescent in situ Hybridization (FISH) analysis could detect more types of sheathed bacteria and yielded a more accurate quantification of bacteria than conventional microscopy.In a pilot and a full scale wastewater treatment plant (WWTP) clear correlations were found between the SVI and growth of a Sphaerotilus natans and a Thiothrix sp. Addition of chlorine to the bulking sludge resulted in an improved SVI of the sludge but only damaged filamentous cells outside the floc. Nitrification was measured with substrate depletion and FISH analysis. Signal interpretation of FISH analysis was demonstrated both manually and with automated image analysis.     

Interactions between filamentous sulfur bacteria,sulfate reducing bacteria and poly-P accumulating bacteria in anaerobic-oxic activated sludge from a municipal plant

The interactions between filamentous sulfur bacteria (FSB), sulfate reducing bacteria (SRB) and poly-P accumulating bacteria (PAB) in the activated sludge of a municipal plant operated under anaerobic-oxic conditions were examined in batch experiments using return sludge (RAS) and settled sewage. Phosphate release and sulfate reduction occurred simultaneously under anaerobic conditions. SRB were more sensitive to temperature changes than PAB. SRB played an important role in the decomposition of propionate to acetate. When the sulfate reduction rates were high, there was a tendency for the maximum release of phosphate also to be high. This was explained by the fact that PAB utilized the acetate produced by SRB. Sulfur oxidizing bacteria were sensitive to temperature change. When the sulfate reduction rate was high, the sulfide oxidizing rate was also high and filamentous bulking occurred. The results showed that sulfate reduction was a cause of filamentous bulking due to Type 021N that could utilize reduced sulfur.     

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.     

Biodegradation of phenolic compounds by an acclimated activated sludge and isolated bacteria

The degradation of a mixture of phenol, 4-chlorophenol (4CP), 2,4-dichlorophenol (24DCP) and 2,4,6-trichlorophenol (246TCP) by acclimated activated sludge and by isolated bacteria was studied. Activated sludge was acclimated for 70 days to 40 mg phenols/1 then the microorganisms responsible for the CP degradation were isolated and identified. Four types of Gram-negative bacteria (Aeromonas sp., Pseudomonas sp. Flavomonas oryzihabitans, and Chryseomonas luteola) were identified. Also, two acid-fast bacilli with distinct glycolipid patterns were isolated. From their chemical composition and their growth characteristics, both isolates appeared to be mycobacteria closely related to Mycobacterium peregrinum. The degradation kinetics of each phenol by Aeromonas sp., Pseudomonas sp. Flavomonas oryzihabitans, Chrvseomonas luteola and activated sludge were determined. The acclimated activated sludge degradation rates were from one to two orders of magnitude higher than those of pure strains when uptake rates were calculated in terms of the viable biomass (CFU). The specific substrate uptake rate for acclimated activated sludge varied between 8.2 and 15.8 ß 10⁻⁷ mg/CFU·d (407-784 mg/BVSS·d). Aeromonas sp. had the highest specific substrate uptake rate of the pure strains, based on a VSS basis (33–57 mg/gVSS·d) but, in terms of viable biomass (5.0–15.6 × 10⁻⁸ mg/CFU·d), the Pseudomonas sp. rate was the highest. Specific substrate uptake rates were 1.8 mg chlorinated phenoWg VSS-d for unacclimated activated sludge.     

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.     

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.     

Membrane bioreactor versus conventional activated sludge system: population dynamics of nitrifiers.

Although membrane bioreactors have attracted increasing attention in recent years, little research has been undertaken on the influence of the membrane separation on the microbial community composition. This paper compares the startup behaviour and the performance of the subsequent eight months of a membrane bioreactor with a conventional activated sludge pilot plant. Both plants were operated in parallel at the same sludge age and treated the same domestic wastewater. The identification of the nitrifying community composition using fluorescent in situ hybridization revealed only minor differences between the two reactors for both ammonia-oxidizing bacteria and nitrite-oxidizing bacteria. Accordingly, both systems exhibited the same maximum nitrification rates. Confocal laser scanning microscopy showed that the aggregates formed by nitrifying bacteria were located mostly in the inner part of the flocs and were overgrown by heterotrophic bacteria. It is concluded that the membrane separation itself does affect neither the nitrifying community composition nor the nitrification performance. However, impacts on kinetic parameters are emphasized.     

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.     

Phylogenetic assessment of five filamentous bacteria isolated from bulking activated sludges

Nearly complete 16S ribosomal RNA (TRNA) sequences were determined for fully characterised axenic strains of Thiothrix, Eikelboom type O21N, and Eikelboom type 1701 originally isolated from bulking activated sludges. Thiothrix strains formed a monophyletic group (100% bootstrap support) with previously described Thiothrix nivea strain JP2 and Thiothrix ramosa. Eikelboom type O21N strain AP3 revealed a sufficiently strong relationship to the Thiothrix group to suggest a common ancestry for the two organisms although it was not possible to designate type O21N as a species of Thiothrix. Eikelboom type 1701 contained within its sequence the target sequence of an oligonucleotide probe for the detection of Sphaerotilus natans.     

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     

Characterization of filamentous foaming in activated sludge systems using oligonucleotide hybridization probes and antibody probes

A quantitative method was developed for estimating Gordona mass in activated sludge foam and mixed liquor samples. The technique involves in situ hybridization with a genus-specific fluorescently labeled oligonucleotide probe calibrated on pure cultures of Gordona. The immunofluorescent technique of Hernandez et al. was modified to allow staining with fluorescently labeled antibody and hybridization probes. The results of this technique were compared to those from membrane hybridization studies using radioactively-labeled oligonuelcotide probes. Quantitative membrane hybridizations, in situ hybridizations, and antibody staining resulted in significantly different levels of Gordona in activated sludge foam, activated sludge mixed liquor, return activated sludge, and anaerobic digester sludge. Simultaneous staining with labeled antibodies and oligonucleolide probes provide a definitive identification for Gordona, and represents a new approach for in situ studies of this organism's role to foaming.     

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.     

Hydrogen sulphide removal by activated sludge diffusion.

Odours from wastewater treatment plants comprise a mixture of various gases, of which hydrogen sulphide (H2S) is the main constituent. Microorganisms commonly found in wastewater can degrade sulphurous compounds. Therefore, the use of activated sludge (AS) for odour control offers an alternative to traditional waste gas treatment processes, such as biofilters, bioscrubbers and biotrickling filters, both in practical terms (use of existing facilities) and economically (minimal capital cost). The performance of AS diffusion as a bioscrubber for removing H2S at concentrations at 25, 75 and 150 ppmv was evaluated. Pilot-scale trials were undertaken using parallel 60-L aeration tanks and 20-L clarifier reactors at the Bedford Sewage Treatment Works, Carington, UK. Olfactometry measurements were also carried out to determine whether there was any increase in odour concentration owing to H2S diffusion. Hydrogen sulphide removal rates of 100% were obtained, with no noticeable increase in odour concentration throughout the trials as measured by olfactometry. Odour concentration was highest at the beginning of the trials and lowest during the high H2S dosing period, with similar values being obtained for test and control. It was concluded that AS diffusion is an effective bioscrubber for the removal of H2S odour.     

Comparison of nitrification performance and microbial community between submerged membrane bioreactor and conventional activated sludge system.

A submerged membrane bioreactor (SMBR) and a conventional activated sludge system (CAS) were compared in parallel over a period of more than 260 days on treating synthetic ammonia-bearing inorganic wastewater without sludge purge under decreased hydraulic retention times (HRTs). Conversion of NH4(+)-N to NO3(-)-N was achieved with an efficiency of over 98% at an HRT > or = 10 h in the SMBR, while similar performance was obtained at an HRT > or = 20 h in the CAS. Denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) amplified 16S rDNA was used to monitor variations of community structures in the two systems. With the prolongation of operation, the number of DGGE bands in the SMBR gradually increased from the initial 11 bands to the final 22 bands, whereas that in the CAS varied in a range between 13 and 183 Sequence analysis indicates that Nitrosomonas sp. and Nitrospira sp. were the dominating nitrification species responsible for ammonia and nitrite oxidation, respectively. Heterotrophic bacteria like Pseudomonas sp. and Flavobacteria sp. existed in both of the systems although only inorganic wastewater was fed. Substantive accumulation of extracellular polymeric substances (EPS) in the SMBR was confirmed by scanning electron microscopy and EPS analysis.     

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.     

Treatment of winery wastewater in a conventional municipal activated sludge process: five years of experience.

A full-scale wastewater treatment plant where municipal and winery wastewaters were co-treated was studied for five years. The experimental results showed that suspended solids, COD, nitrogen and phosphorous were effectively removed both during the treatment of municipal wastewater and the cotreatment of municipal and winery wastewater. The sludge production increase from 4 tons to 5.5 tons per day during the harvesting and wine making period. In any case the specific sludge production was 0.2 kgMLVSS per kgCOD(removed) despite the organic loading increasing. About 70% of the COD was removed through respiration. Also the energy demand increased from 6,000 to 7,000 kWh per day. The estimated costs for the treatment of the winery wastewater was 0.2-0.3 Euros per m3 of treated wastewater. With reference to the process efficiency, the nitrogen removal was just 20%. The co-treatment of municipal and winery wastewater in conventional activated sludge processes can be a feasible solution for the treatment of these streams at relatively low costs.     

Beginning a molecular analysis of the eukaryal community in activated sludge

As a means to furthering our understanding of the eukaryal community to activated sludge, we have applied contemporary molecular techniques to an activated sludge community maintained in a laboratory-scale bioreactor. The initial inoculum was derived from a local wastewater treatment facility in East Lansing, MI and maintained with continuous aeration and a daily feeding regime that included glucose and peptone Samples were taken on a weekly basis for 5 weeks and community DNA was extracted from 2–5 ml of activated sludge. Using a variety of oligonucleotide primers specific to eukaryotic small subunit ribosomal DNA, we PCR amplified rDNA from the total community DNA. PCR products were analyzed by three techniques, denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP) and comparative sequence analysis of rDNA clones. Over the course of 5 weeks, analysis with DGGE revealed dramatic changes in the eukaryotic community based on differences in denaturation profiles. However, the analysis is limited to 5–6 bands corresponding to 5–6 different “ribotypes”. Analysis with T-RFLP also suggests changes to the eukaryotic community over time. Both increases and decreases in population size can be detected as a function of time. Up to 15 different terminal restriction fragments can be detected with T-RFLP indicating that this technique is considerably more sensitive than DGGE. Phylogenetic analysis of partial sequences from 1 I cloned rDNAs indicate that all 11 clones are from the Ciliophora phylum.