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.     

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.     

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.     

The in situ physiology of "Nostocoida limicola" II, a filamentous bacterial morphotype in bulking activated sludge, using fluorescence in situ hybridization and microautoradiography.

The in situ physiology of the actinobacterial bulking and foaming filamentous bacterium "Nostocoida limicola" II was studied by fluorescence in situ hybridization/microautoradiography. Substrate assimilation patterns of pure cultures of this bacterium were different to those seen in activated sludge biomass samples. There was no evidence to suggest that "N. limicola" II preferred hydrophobic substrates, but evidence was produced to support the view that it is metabolically active under anaerobic conditions in activated sludge.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Distribution of ammonia-oxidizing bacteria in sewage activated sludge: analysis based on 16S rDNA sequence.

This study carried out analysis of ammonia-oxidizing bacteria (AOB) communities in 12 sewage activated sludge systems standing in eight sewage treatment plants located in Tokyo. The systems were different in the treatment process configuration: anaerobic/anoxic/aerobic (A20), anaerobic/aerobic (AO), and conventional activated sludge (AS) processes. AOB communities were analyzed by sequences of 16S rDNA amplicons, which were separated by denaturing gradient gel eletrophoresis (DGGE) after specific polymerase chain reaction (PCR) amplification. The results demonstrated that low ammonium concentrations in the influents of the 12 sewage activated sludge systems resulted in the dominance of Nitrosomonas oligotropha-like sequences. Further, Nitrosomonas europaea- and Nitrosomonas cryotolerans-like sequences were recovered from only one A20 system of which the influent contained higher ammonium and chloride concentrations than those of other systems. Nitrosomonas communis-like sequences were found in every A20 and AO system, but mostly not found in every AS system. In summary, influent characteristics and treatment process configuration affected the AOB communities in the 12 sewage activated sludge systems.     

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.     

Microbial community change of sulfate reduction and sulfur oxidation bacteria in the activated sludge cultivated with acetate and peptone.

The growth of sulfate reducing bacteria (SRB) and filamentous sulfur bacteria was monitored on a laboratory scale in activated sludge reactors using acetate and peptone as the artificial wastewater. When the artificial wastewater contained acetate and peptone, filamentous bacteria increased in the sludge and the SVI values increased. There was a good correlation between sulfate reducing activity and sulfur oxidation activity in the produced sludge. The microbial community change of filamentous sulfur bacteria and sulfate reducing bacteria was analyzed using the fluorescent in situ hybridization (FISH) method. The tendency for the growth of filamentous sulfur bacteria Thiothrix eikelboomii following the growth of SRB was observed. The percentage of SRB385- hybridized cells and DNMA657-hybridized cells found in the total cell area increased from 2-3% to 7-10% when the filamentous bulking occurred.