Eikelboom Type Research Articles

Artificial intelligence and multivariate statistics for comprehensive assessment of filamentous bacteria in wastewater treatment plants experiencing sludge bulking

Abstract Sludge bulking is an operational hurdle that affects the solid–liquid separation in wastewater treatment plants (WWTPs) worldwide. In this study, filamentous bulking issues were investigated in seven WWTPs located in South Africa using artificial neural network (ANN) and multivariate statistics. The microbial community belonging to sludge bulking was determined using staining and microscopic methods, with a further confirmed identification of selected species via fluorescent in situ hybridization (FISH). Based on a filament index scale from 1 (None filament) to 7 (Excessive filament), the developed ANN could predict the sludge volume index (SVI) using the abundances of ten inputs of filamentous species. Eikelboom Type 0041 attained the highest impact on SVI, followed by Gordonia spp., Nostocoida limicola, and Thiothrix spp. Principal component analysis (PCA) combined with FISH images showed that most WWTPs experienced inadequate sludge settling properties; however, the application of an efficient aeration system (i.e., diffusion) in the three-stage Phoredox process improved the settling characteristics of bio-flocs. Operational conditions that caused filament overgrowth in each WWTP were also determined. The study outputs would provide a scientific basis to control the proliferation of filamentous bacteria in other WWTPs located in similar environmental conditions to South Africa.

Exploring the operating factors controlling Kouleothrix (type 1851), the dominant filamentous bacterial population, in a full-scale A2O plant

This study reveals that the abundance of the filament Kouleothrix (Eikelboom type 1851) correlated positively with poor settleability of activated sludge biomass in a Japanese full-scale nutrient removal wastewater treatment plant sampled over a one-year period. 16S rRNA amplicon sequence data confirmed that Kouleothrix was the dominant filament in the plant, with a relative abundance of 3.06% positively correlated with sludge volume index (SVI) (R = 0.691). Moreover, Kouleothrix (type 1851) appeared to form interfloc bridges, typical of bulking sludge, regardless of season. Together with earlier studies that indicated the responsibility of Kouleothrix (type 1851) on bulking events, these data suggest that their high relative abundances alone may be responsible for sludge bulking. 16S rRNA qPCR data for this filament showed changes in its relative abundance correlated with changes in several operational parameters, including mixed liquor temperature, sludge retention time, and suspended solids concentration, and it may be that manipulating these may help control Kouleothrix bulking.

Decision tree for identification and prediction of filamentous bulking at full-scale activated sludge wastewater treatment plant

This study attempted to model sludge bulking in a full-scale wastewater treatment plant operated as a 3-stage Phoredox process. Principal component analysis and a regression tree model were employed to describe the correlations between influent wastewater characteristics and operational conditions (as inputs) and sludge volume index (SVI) as an output. A classification tree model was used to determine the environmental factors that affected the proliferation of filamentous microorganisms. Fluorescent in situ hybridisation analysis identified filamentous species of Microthrix parvicella, Thiothrix I & II, and Eikelboom Types 0041, 0092, and 021 N. It was found that SVI increased with an increment in sludge retention time, but it negatively correlated with soluble chemical oxygen demand (sCOD) and ammonium-nitrogen. The dominance of Microthrix parvicella was observed with a decline in temperature below 15.5 °C, causing an increase in SVI during the winter and spring seasons. The overgrowth of Thiothrix could be linked to the unbalanced ratio between readily biodegradable COD and nutrient species. The filament Type 0092 contributed to high SVI, and it prevailed with a decrease in food-to-microorganisms ratio below 0.08 1/d. Based on the satisfactory training, validation, and generalization procedures, the proposed models could be applied for the prediction of sludge bulking episodes.

Quantification of Chloroflexi Eikelboom morphotype 1851 for prediction and control of bulking events in municipal activated sludge plants in Japan.

The dominant filamentous bacteria associated with bulking incidents in Japanese activated sludge plants with nutrient removal were identified and their quantitative correlations with sludge settleability were assessed, with the aim of controlling bulking incidents by specifically suppressing bacterial growth. Fluorescence in situ hybridization (FISH) analyses using existing oligonucleotide FISH probes indicated that the presence of Eikelboom type 1851 filamentous bacteria belonging to the phylum Chloroflexi is correlated with biomass settleability in the municipal wastewater treatment plants examined. Real-time quantitative PCR (qPCR) assays developed in this study also showed a linear correlation between type 1851 filament members and sludge settleability, with the exception of some winter samples. The real-time qPCR assays and 16S ribosomal RNA gene amplicon sequencing to reveal the microbial community of activated sludge showed that the abundance of type 1851 at 200mLg-1 of sludge volume index was estimated to be about 1.9% of the total microbial cells. The abundance of type 1851 served as a bulking indicator in plants where type 1851 was dominant.

Effect of a static magnetic field on activated sludge community

ABSTRACTThe aim of this study was to determine the effect of a static magnetic field (SMF) on the composition of activated sludge biocenosis. The experiment was carried out in two parallel bench scale Sequencing Batch Reactors (SBRs). Both SBRs were treated with dairy wastewater. The activated sludge in the first SBR was exposed to an SMF via the induction of a 0.6 T magnetic field generated by four magnetic liquid activators. The second reactor (control reactor) was operated at the same operational parameters but the activated sludge was not exposed to the SMF. The mean length of the bacterium Eikelboom Type 0092 was lower in the SMF-exposed reactor than in the control reactor. Different activated sludge morphologies in SBRs were reflected in the values of the sludge volume index and sludge biotic index calculated on the basis of the microfauna composition.

A logistic model for the remediation of filamentous bulking in a biological nutrient removal wastewater treatment plant.

Biological nutrient removal (BNR) systems across the globe frequently experience bulking and foaming episodes, which present operational challenges such as poor sludge settling due to excessive filamentous bacteria. A full-scale BNR plant treating primarily domestic wastewater was monitored over a period of 1 year to investigate filamentous bacterial growth response under various plant operating parameters. Identification of filamentous bacteria by conventional microscopy and fluorescent in situ hybridisation indicated the dominance of Eikelboom Type021N, Thiothrix spp., Eikelboom Type 1851 and Eikelboom Type 0092. A cumulative logit model (CLM) was applied to elucidate significant relationships between the filamentous bacteria and plant operational parameters. The model could predict the potential abundance of dominant filamentous bacteria in relation to wastewater treatment plant operational parameters. Data obtained from the model corroborated with previous findings on the dominance of most filaments identified, except for Type 0092, which exhibited some unique traits. With further validation, the model could be successfully applied for identifying specific parameters which could contribute towards filamentous bulking, thus, providing a useful tool for regulating specific filamentous growth in full-scale wastewater treatment plants.

Experimental attempt at using Lecane inermis rotifers to control filamentous bacteria Eikelboom Type 0092 in activated sludge.

In laboratory experiments, the authors tested the efficacy of Lecane inermis rotifers in reducing the abundance of filamentous bacteria in activated sludge samples dominated by one filamentous bacterium, Eikelboom Type 0092. The experiment was conducted in three replicates and repeated three times at 1-month intervals. In all repetitions, rotifers were able to significantly reduce the density of bacterial filaments through predation. It was also shown that the influence of rotifers on filament abundance depends on the duration of grazing pressure. The data show that L. inermis, already reported to be effective in reducing the density of Microthrix parvicella, Nostocoida limicola, and Type 021N in activated sludge, can be equally helpful in limiting Type 0092, another troublesome filamentous bacterium found in low-loaded water resource recovery facilities.

Filament identification and dominance of Eikelboom Type 0092 in activated sludge from wastewater treatment facilities in Cape Town, South Africa

Routine characterisation of activated sludge and identification of the filamentous population by microscopic and/or other non-culture dependent techniques can assist in diagnosing the aetiology of poor performance of wastewater treatment works (WWTWs). In South Africa, most facilities rely solely on physicochemical indicators, treating reactors as ‘black-boxes’, with the result that process adjustments are often delayed, to the detriment of the environment. This study was performed in order to gain insight into the filamentous population found in activated sludge in Cape Town WWTWs, to compare these with other global and local literature findings, and to build capacity in this science. Physicochemical and plant performance parameters, in terms of nutrient removal and settling, were obtained from routine operational data and assessed in conjunction with the microscopic analyses of activated sludge samples taken over a 6-month period. Hypotheses on the links between filament types and/or plant configurations and/or operational parameters were formulated using existing literature. In order of prevalence, the five most common dominant filament species in 96 activated sludge samples were: Eikelboom Type 0092, Eikelboom Type 1851, nocardioforms, Microthrix parvicella and Eikelboom Type 021N. In order to compile a statistically significant database, it is recommended that an extensive nationwide study is conducted to link filament types with plant configurations, operational parameters and geographical locations. Keywords : activated sludge, bulking, identification, filament, Type 0092

Control filamentous bulking caused by chlorine-resistant Type 021N bacteria through adding a biocide CTAB

Filamentous bulking sludge due to excessive growth of filamentous bacteria is a serious operational problem in activated sludge plants. The addition of chemicals is one of widespread ways to control filamentous bulking. In this study, filamentous bulking in a continuous activated sludge system was found to be mainly caused by Eikelboom Type 021N filamentous bacteria likely due to low substrate concentration gradients. These Type 021N bacteria were found to be resistant to chlorination, maintaining cell integrity at a dosage of up to 80 mg Cl/gSS. An alternative biocidal agent, cetyltrimethyl ammonium bromide (CTAB), exhibited a much stronger biocidal effect on these filaments, which significantly improved sludge settleability. Type 021N with filamentous index of 5 was selectively killed, but floc-formers recovery their activity after CTAB termination. The study implied that CTAB might have more penetration capacity to cell wall of chlorine-resistant Type 021N bacteria. We therefore suggest the penetration property of filament cell wall should be considered or tested before the selection of biocide type in practice.

Molecular phylogeny and taxonomy of colorless, filamentous sulfur bacteria of the genus Thiothrix

Comprehensive investigation combining molecular genetic techniques and comparative studies of morphological and physiological properties made it possible to resolve the disputed issue of the taxonomic status of the groups “T. nivea” and “Eikelboom type 021N” of the genus Thiothrix. The phylogenetic trees constructed on the basis of 16S rRNA and gyrB gene sequences demonstrated that members of the genus Thiothrix formed a cluster within the order Thiotrichales. According to the “ribosomal” tree, the cluster of the genus Thiothrix was divided into two main groups, I and II, corresponding to the groups “T. nivea” and “Eikelboom type 021N”. The levels of similarity between the 16S rRNA gene sequences of Thiothrix species reached 88.9–100%. On the contrary, in the “gyrase” tree, these species were not divided into “T. nivea” and “Eikelboom type 021N” groups. The levels of similarity between the amino acid sequences of the gyrB gene fragments of Thiothrix species varied from 74.5 to 99.2%. Importantly, members of the groups “T. nivea” and “Eikelboom type 021N” formed very similar 16S rRNA secondary structures in the variable region V3, where a 30-nucleotide deletion characteristic of all Thiothrix species was detected. Phenotypic analysis of the studied bacteria revealed some morphological and physiological properties shared by the groups “T. nivea” and “Eikelboom type 021N”. The data obtained indicate that members of the groups “T. nivea” and “Eikelboom type 021N” are phenotypically and genetically heterogeneous species within the single monophyletic genus Thiothrix..

Stable limited filamentous bulking through keeping the competition between floc-formers and filaments in balance

Limited filamentous bulking (LFB) was proposed to save aeration energy consumption and enhance the capacity of filaments to degrade substrates with low concentrations in activated sludge systems. Operational parameters favorable for maintaining the LFB state were investigated in an anoxic–oxic reactor treating domestic wastewater. The experiments showed that the LFB state would deteriorate with sharply decreasing temperature, reducing substrate gradients or removing anoxic zones. The balance between filaments and floc-formers could be achieved by controlling dissolved oxygen and sludge loading rates to be in optimal ranges. Eikelboom Type 0041 and CandidatusMicrothrix parvicella were the filamentous bacteria responsible for the LFB state. However, the excess growth of Eikelboom Type 021N and Sphaerotilus natans were observed when serious bulking occurred under low substrate gradients. It was demonstrated that stable maintenance of LFB for energy saving was feasible by process control and optimization.

Eikelboom's morphotype 0803 in activated sludge belongs to the genus Caldilinea in the phylum Chloroflexi

Micromanipulated filamentous bacteria from bulking and foaming activated sludge morphologically identified as Eikelboom type 0803 were shown to be affiliated to the genus Caldilinea within the phylum Chloroflexi. Specific FISH probes were designed for their in situ detection and quantification in seven Danish wastewater treatment plants with biological nutrient removal. The survey applied all species-specific probes for Chloroflexi of relevance in activated sludge treatment plants as well as the phylum-specific probes. Type 0803 filaments constituted around 20% of the total Chloroflexi population. In four of the treatment plants, type 0803 and type 0092 co-occurred and were the dominating fraction of the Chloroflexi population. In the other plants, most Chloroflexi could not be identified beyond the phylum level, suggesting a yet far larger diversity. On average, for all plants, the total Chloroflexi population constituted 12% of the entire microbial population and seems to play an important structural role in the sludge floc formation. Ecophysiological characterization of type 0803 showed their potential role in macromolecule conversion as evident by high levels of exoenzyme expression. Acetate was not consumed. Glucose was consumed with oxygen, nitrite and nitrite as electron acceptors, suggesting that type 0803 may be a denitrifier. Their surfaces were hydrophobic, explaining their occasional occurrence in foaming incidents.

Filamentous bacteria in a foaming membrane bioreactor

The foam-forming filamentous bacteria can result in serious membrane fouling problems, not only in traditional activated sludge processes, but also in membrane bioreactors (MBR). In this study, we examine the influence of the hydrophobicity of the membrane on membrane fouling in a submerged MBR using two sets of polytetrafluoroethylene membranes with different surface hydrophobicity. We also identify and compare the foam-forming filamentous bacteria via the Eikelboom method, the combination of a polymerase chain reaction with DNA sequencing, and the fluorescent in situ hybridization (FISH) approach based on non-cultured and cultured methods. The experimental results showed that during foaming period, the transmembrane pressure of the high hydrophobicity membrane increased as the flux decreased; but no significant influence was observed for the low hydrophobicity membrane. Of the foam-forming bacteria identified by non-cultured method, only Actinomycetes and Nosotcoida limicola III were observed using Eikelboom identification keys I and II, while high G + C content Gram-positive (HGC) bacteria was the predominant filamentous bacteria identified via the FISH approach. On the other hand, the Microthrix parvicella, Eikelboom type 1851, Eikelboom type 0092, and Flexibacter were identified as the predominant type of filamentous bacteria using the culture method with Eikelboom identification key I. The HGC bacteria made up 77.1% of the total isolates. Finally, the results of 16S rRNA sequences screened by the Eikelboom method showed that the genera Gordonia and Cytophaga, which contributed 75.7% and 20.1%, respectively, were the predominant filamentous bacteria.

A study on filamentous bacteria in activated sludge process of sewage treatment plant in Dubai, United Arab Emirates

A study was conducted on filamentous bacteria implicated in bulking and foaming problems in activated sludge process of sewage treatment plant in Dubai, United Arab Emirates over a period of six months. To determine morphological characteristics of diverse filaments, foam and mixed liquor samples were collected and studied using various simple and differential staining techniques. Fluorescent in situ hybridization analysis was carried out in mixed liquor samples with nocardioform group specific probes using VIT kit (Vermicon Identification Technology, Vermicon, Munich). The dominant filamentous bacteria identified from mixed liquor and foam samples included: A long branched form of Gram varibale nocardioform actinomycetes species, Thiothrix, Eikelboom Type 021N, Sphaerotilus natans, Beggiatoa and Nostocoida limicola type I. Occasionally attached growth forms of Eikelboom type 0041/0675 like filaments were observed in mixed liquor and foam samples especially during warm weather. All filamentous bacteria identified were found in both the samples throughout the study period. FISH analysis successfully identified filamentous and non-filamentous morphotypes of nocardioform group members. It is concluded that specific filamentous bacterial population in mixed liquor and foaming activated sludge was constant and not dependent on variable wastewater characteristics.

Filamentous Bacterium Eikelboom Type 0092 in Activated Sludge Plants in Australia Is a Member of the Phylum Chloroflexi

Molecular data show that the filamentous bacterium Eikelboom type 0092, frequently seen in Australian activated sludge plants, is a member of the phylum Chloroflexi. Fluorescence in situ hybridization (FISH) probes designed against cloned 16S rRNA sequences from a full-scale enhanced biological phosphate removal-activated sludge plant community, where this was a dominant filament morphotype, suggest that it can exist as two variants, differing in their trichome diameter. When applied to samples from several treatment plants in eastern Australia, each FISH probe targeted only the type 0092 filament morphotype against which it was designed. The patterns of FISH signals generated with both were consistent with the ribosomes not being evenly distributed but arranged as intracellular aggregates. The FISH survey data showed that these two variants appeared together in most but not all of the plants examined. None stained positively for intracellular presence of either poly-beta-hydroxyalkanoates or polyphosphate.

Amyloid-Like Adhesins Produced by Floc-Forming and Filamentous Bacteria in Activated Sludge

Amyloid proteins (fimbriae or other microbial surface-associated structures) are expressed by many types of bacteria, not yet identified, in biofilms from various habitats, where they likely are of key importance to biofilm formation and biofilm properties. As these amyloids are potentially of great importance to the floc properties in activated sludge wastewater treatment plants (WWTP), the abundance of amyloid adhesins in activated sludge flocs from different WWTP and the identity of bacteria producing these were investigated. Amyloid adhesins were quantified using a combination of conformationally specific antibodies targeting amyloid fibrils, propidium iodide to target all fixed bacterial cells, confocal laser scanning microscopy, and digital image analysis. The biovolume fraction containing amyloid adhesins ranged from 10 to 40% in activated sludge from 10 different WWTP. The identity of bacteria producing amyloid adhesins was determined using fluorescence in situ hybridization with oligonucleotide probes in combination with antibodies or thioflavin T staining. Among the microcolony-forming bacteria, amyloids were primarily detected among Alpha- and Betaproteobacteria and Actinobacteria. A more detailed analysis revealed that many denitrifiers (from Thauera, Azoarcus, Zoogloea, and Aquaspirillum-related organisms) and Actinobacteria-related polyphosphate-accumulating organisms most likely produced amyloid adhesins, whereas nitrifiers did not. Many filamentous bacteria also expressed amyloid adhesins, including several Alphaproteobacteria (e.g., Meganema perideroedes), some Betaproteobacteria (e.g., Aquaspirillum-related filaments), Gammaproteobacteria (Thiothrix), Bacteroidetes, Chloroflexi (e.g., Eikelboom type 1851), and some foam-forming Actinobacteria (e.g., Gordonia amarae). The results show that amyloid adhesins were an abundant component of activated sludge extracellular polymeric substances and seem to have unexpected, divers functions.

Growth of filamentous bacteria in an enhanced biological phosphorus removal system

Activated sludge bulking caused by filamentous bacteria Eikelboom type 021N and Thiothrix spp. is reported for the first time in a multistage pilot-scale wastewater treatment plant for enhanced biological phosphorus removal (EBPR). Both types of microorganisms grew gradually reaching their maximum cumulative length within a period of 2 months. For identification of the strains, in addition to physiological observations under the microscope, Gram staining, Neisser staining and sulfur storage test took place. Results confirmed that biological nutrient removal systems may also suffer from sludge bulking. Plant configuration, wastewater composition, removal efficiency and filaments’ growth stages were examined in relation to the occurrence of the certain strains in an EBPR system.

Relationships among filamentous microorganisms in rotating biological contactors

The existing relationships were studied among the different types of filamentous microorganisms that appear in the biofilm of a biological contactor system. Using the hierarchical cluster analysis it was observed that, in all the stages, Beggiatoa sp. and the Eikelboom's types 0803 and 1863 always appeared associated, while Sphaerotilus natans was always associated with the morphological type 021N. The remaining microorganisms were associated in variable forms in the plants. In addition, different association models were obtained according to the season of the year and the stage-season interaction. It has also been observed that a significant correlation exists among the filamentous microorganisms we have studied and the different physical-chemical parameters.

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.

Substrate uptake tests and quantitative FISH show differences in kinetic growth of bulking and non‐bulking activated sludge

The competition between filaments and floc formers in activated sludge has been historically described using kinetic selection. However, recent studies have suggested that bacterial storage may also be an important factor in microbial selection, since the dynamic nature of substrate flows into wastewater treatment plants elicit transient responses from microorganisms. Respirometry-based kinetic selection should thus be reevaluated by considering cell storage, and a more reliable method should be developed to include bacterial storage in the analysis of growth of filaments and floc formers in activated sludge. In this study, we applied substrate uptake tests combined with metabolic modeling to determine the growth rates, yields and maintenance coefficients of bulking and non-bulking activated sludge developed in lab scale reactors under feast and famine conditions. The results of quantitative fluorescence in situ hybridization (FISH) showed that the filaments Eikelboom Type 1851, Type 021N, and Thiothrix nivea were dominant in bulking sludge, comprising 42.0 % of mixed liquor volatile suspended solids (MLVSS), with 61.6% of the total filament length extending from flocs into bulk solution. Only low levels of Type 1851 filament length (4.9% of MLVSS) occurred in non-bulking sludge, 83.0% of which grew inside the flocs. The kinetic parameters determined from the substrate uptake tests were consistent with those from respirometry and showed that filamentous bulking sludge had lower growth rates and maintenance coefficients than non-bulking sludge. These results provide support for growth kinetic differences in explaining the competitive strategy of filamentous bacteria.