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.     

Microbial community structure of activated sludge during aerobic granulation in an annular gap bioreactor.

A novel annular gap reactor was designed to create a controlled shear environment in which aerobic granular sludge could be developed. The bacterial and eukaryal community structures during two aerobic granular sludge experiments were tracked using denaturing gradient gel electrophoresis (DGGE). The first granule cultivation experiment, using an organic loading rate of 1.6 kg/m3d COD, resulted in biomass that was dominated by filamentous bacteria and Zoogloea ramigera colonies. A second experiment with a higher organic loading rate of 6 kg/m3d COD developed a granule-like morphology but was ultimately dominated by filamentous fungi. Species identification via DGGE band purification and DNA sequencing closely matched the observed sludge morphology and behavior.     

Microbial community analysis during continuous fermentation of thermally hydrolysed waste activated sludge

Acidogenic fermentation of thermally hydrolysed waste activated sludge was carried out at laboratory scale in two reactors operated under different hydraulic retention times (HRT). Process performance was assessed in terms of volatile fatty acid (VFA) composition and yield. The diversity of the microbial population was investigated by constructing a 16S rRNA gene library and subsequent phylogenetic analysis of clones. Fluorescence in situ hybridization (FISH) was used to assess the relative abundance of different bacterial groups. Bacteroidetes and Firmicutes were the dominant taxonomic groups representing 93% of the total sequences obtained in the reactor with 4 d HRT. A similar VFA yield (0.4-0.5 g VFA(COD) g SCOD(-1)) was obtained for the HRTs tested (1-4 d), indicating that extended retention times were not useful. Within Firmicutes, Clostridia was the major group detected in the clone sequences. These had close affiliation to Sporanaerobacter acetigenes, suggesting organisms of this group were important for hydrolysis of the protein fraction of the substrate. However, FISH analysis failed to detect the major portion of the bacteria, and this is most likely due to the lack of appropriate probes. This work emphasizes the diversity of fermentative communities, and indicates that more work is needed to identify and detect the important members.     

Microbial degradation of estrogens using activated sludge and night soil-composting microorganisms.

In order to investigate the potential for microbial degradation of estrogens, and the products formed, activated sludge collected from Korea (ASK) and night soil-composting microorganisms (NSCM) were used to degrade estrogens. Results showed that both ASK and NSCM degraded almost 100% of the natural estrogens estrone (E1), 17beta-estradiol (E2), and estriol (E3) from initial concentrations of 20-25 mg/L, while synthetic estrogen, ethynylestradiol (EE2), was not degraded. Analysis of degradation products of E2 by using HPLC-ECD and a consecutive first-order reaction calculation confirmed that E2 was sequentially degraded to E1, which was further degraded to other unknown compounds by ASK and NSCM. We then used the yeast two-hybrid assay to show that the unknown degradation products did not appear to possess estrogenic activity when E1, E2 or E3 were degraded to below the detection limit after 14 days of incubation, indicating that ASK and NSCM not only degrade natural estrogens, but also remove their estrogenic activities.     

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.     

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.     

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.     

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     

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.     

Polyhydroxyalkanoates production by activated sludge in a SBR using acetate and propionate as carbon sources.

In this work, sludge was submitted to aerobic dynamic substrate feeding. Two sequencing batch reactors were operated, with acetate or propionate as carbon substrates. When acetate was used the system only produced a homopolymer of polyhydroxybutyrate (PHB). In order to maximize the PHB production, tests with different concentrations of acetate and ammonia were preformed. The best results (67.2% of PHB by cell dry weight) were obtained for 0.7 Nmmol/l of ammonia and 180 Cmmol/l of acetate. The PHB cell content was further improved by pulse addition of substrate, three times 60 Cmmol/l of acetate, reaching a value of 78.5%. Propionate can be used as a precursor for hydroxyvalerate. In conjunction with other substrates, it allows for the formation of copolymers, which present better processing properties on commercial applications. Tests with different concentrations of propionate and ammonia were performed. Under the operating conditions used, the maximum PHA accumulated inside cells was 34.8%, with 30 Cmmol/l of propionate and no ammonia.     

Analysis of microbial community structure and diversity in the thermophilic anaerobic digestion of waste activated sludge.

The microbial community structures in the thermophilic anaerobic digestion (TAD) of waste activated sludge (WAS) and WAS were analyzed with molecular biological techniques including real-time polymerase chain reaction (PCR) and cloning. The microbial community of TAD had less diversity than that of WAS, and the sequences obtained in WAS were not present in TAD by the cloning analysis. In the TAD bacterial clone library, 97.5% of total clones were affiliated with the phylum Firmicutes and 73.1% with the genus Coprothermobacter. Real-time PCR and cloning analysis revealed that the number of Methanosarcina thermophila, which is an acetoclastic methanogen, is larger than that of Methanoculleus thermophilus, which is a hydrogenotrophic methanogen, in terms of the numbers of copies of 16S ribosomal DNA (rDNA).     

Evaluation of the impacts of model-based operation of SBRs on activated sludge microbial community.

Impact of model-based operation of nutrient removing SBRs on the stability of activated sludge population was studied in this contribution. The optimal operation scenario found by the systematic model-based optimisation protocol of Sin et al. (Wat. Sci. Tech., 2004, 50(10), 97-105) was applied to a pilot-scale SBR and observed to considerably improve the nutrient removal efficiency in the system. Further, the process dynamics was observed to change under the optimal operation scenario, e.g. the nitrite route prevailed and also filamentous bulking was provoked in the SBR system. At the microbial community level as monitored by DGGE, a transient shift was observed to gradually take place parallel to the shift into the optimal operation scenario. This implies that the model-based optimisation of a nutrient removing SBR causes changes at the microbial community level. This opens future perspectives to incorporate the valuable information from the molecular monitoring of activated sludge into the model-based optimisation methodologies. In this way, it is expected that model-based optimisation approaches will better cover complex and dynamic aspects of activated sludge systems.     

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.     

生物捕食污泥减量化系统的污泥特性

通过中试分析了以减量化为目的的微型动物捕食系统中活性污泥的特性.结果表明,细菌分散培养段的悬浮污泥浓度比原生动物捕食段约高15%,比大型微型动物捕食段约高40%;微型动物的捕食作用在减少剩余污泥产率的同时,可以提高悬浮污泥的沉降性能约36%,轮虫可作为该系统污泥沉降性能的指示生物;低底物浓度下,大型微型动物的捕食活动可以增强污泥的活性.     

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.     

Treatment of an ECF effluent by combined use of activated sludge and advanced oxidation process.

The combined biological and chemical treatments of the cellulose effluents have been studied aiming to promote a more significant degradation of their recalcitrant compounds and to reduce their toxicity, as compared with the isolated treatments. In this work the effluent from acid stages of the ECF bleaching of Eucalyptus urograndis pulp was treated by using separately activated sludge and UV radiation and its combination. The treatment efficiency was evaluated by colour, total phenol, COD, BOD, UV spectroscopy, molar weight distribution and toxicity. The untreated effluent presented 587 +/- 18 CU, 19.3 +/- 0.6 mg.L(-1) of total phenol, 2246 +/- 137 mgO2.L(-1) of COD and 904 +/- 48 mgO2.L(-1) of BOD. It did not show acute toxicity to Escherichia coli, but presented chronic toxicity to Selenastrum capricornutum (EC50 = 25%). The sludge treatment resulted in a colour increasing of 42% and decreasing of total phenol, COD and BOD of 33%, 64% and 92%, respectively. The UV radiation treatment for 120 min resulted in a decrease of colour, total phenol, BOD and COD of 70%, 43%, 62% and 43%, respectively. The combined treatment promoted an expressive decrease for colour and total phenol. The UV absorption indicated a degradation of the aromatic compounds. The biological treatment did not remove chronic toxicity and after UV radiation treatment, a 10 times improving toxicity was noticed.     

Microbial community of anammox bacteria immobilized in polyethylene glycol gel carrier

Anaerobic ammonium oxidation (anammox) is a recently discovered microbial pathway in the biological nitrogen cycle and a new cost-effective way to remove ammonium from wastewater. We have so far developed new immobilization technique that anammox bacteria entrapped in polyethylene glycol (PEG) gel carrier. However, fate and behavior of anammox bacteria in a gel carrier is not well understood. In the present study, we focused on the population changes of anammox bacteria in a gel carrier. Three specific primer sets were designed for real-time PCR. For quantification of anammox bacteria in a gel carrier, real-time PCR was performed. The anammox bacteria related to HPT-WU-N03 clone were increased the rate in anammox population, and found to be a major population of anammox bacteria in a gel carrier. Furthermore, from the results of nitrogen removal performance and quantification of anammox bacteria, the correlation coefficient between copy numbers of anammox bacteria and nitrogen conversion rate was calculated as 0.947 in total anammox population. This is the first report that population changes of anammox bacteria immobilized in a gel carrier were evaluated.