Eikelboom Type 021N Research Articles

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.

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.

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.

Quantification of an Eikelboom type 021N bulking event with fluorescence in situ hybridization and real-time PCR

Primers targeting 16S rRNA genes were designed to detect and quantify Eikelboom type 021N organisms by real-time PCR. Eikelboom type 021N filamentous bulking was induced in a laboratory-scale sequencing batch reactor and the evolution of Eikelboom type 021N 16S rRNA and 16S rRNA genes was monitored. A significant correlation was found between the sludge volume index and the amount of these filamentous organisms present in the sludge (r (2)=94.6%, n=10, P<0.01), as measured by real-time PCR. The amount of Eikelboom type 021N 16S rRNA genes increased by a factor of 21 during the experiment, while the 16S rRNA increased by a factor of 33. Moreover, Eikelboom type 021N 16S rRNA increased with increased feeding frequency. It was observed that the RNA:DNA ratio peaked before the sludge volume index increased. In parallel, a fluorescence in situ hybridization study indicated a factor of four increase in the length of Eikelboom type 021N filaments, due to a factor of two increase in both length and number of Eikelboom type 021N filaments. Further, an increase in the fraction of filaments extending outside the activated sludge flocs was observed (19-55%). Monitoring of 16S rRNA genes and 16S rRNA of Eikelboom type 021N was shown to be valuable in evaluating activated sludge settling characteristics; and measuring RNA:DNA ratios may be used as an early warning tool for sludge bulking.

Filamentous Alphaproteobacteria Associated with Bulking in Industrial Wastewater Treatment Plants

The phylogeny and distribution of filamentous Alphaproteobacteria, morphologically similar to "Nostocoida limicola" and Eikelboom Type 021N that cause the solids separation problem of bulking in industrial activated sludge plants is described here. A combination of culture-dependent and culture-independent molecular methods has characterized 5 novel species. 16S rRNA targeted oligonucleotide probes were designed for their in situ identification by fluorescence in situ hybridisation (FISH) and used to monitor their presence in 86 WWTPs treating different industrial effluents in four European countries. The involvement of these bacteria in bulking in these plants was confirmed. Filaments hybridising with the ALF-968 probe for the Alphaproteobacteria were present in 65% of the WWTPs examined. They were dominant and therefore probably responsible for bulking in 25.5% of them. The heterogeneous filamentous alphaproteobacterial populations in these communities could be completely identified after application of the oligonucleotide probes used in this study in 91% of the plants containing them. The only filamentous Alphaproteobacteria retrieved in pure culture was isolated from three different industrial WWTPs plants. None of these isolates could grow anaerobically on glucose or denitrify, but all grew aerobically and heterotrophically on a range of carbon sources. Although morphologically similar to the Eikelboom Type 021N morphotype, they were not involved in sulphur metabolism. These bacteria accumulated lipidic storage granules that were associated with their presence under the unbalanced growth conditions existing in these plants.

Impact of the repetition of oxygen deficiencies on the filamentous bacteria proliferation in activated sludge

Cases of low stress can frequently occur in a wastewater treatment plant (WWTP). As they have no visible impact on the plant operation, they generally go unnoticed. Nevertheless, it would appear that an accumulation of such cases can result in serious operational problems. The impact of the repeated application of oxygen deficiency on the proliferation of the filamentous bacteria Sphaerotilus natans, Haliscomenobacter hydrossis, Eikelboom type 021N and Thiothrix spp has been studied on WWTP pilots. Two different oxygen deficiencies series were tested. Filamentous bacteria evolution in activated sludge was monitored using fluorescent in situ hybridization. For the weak deficiencies, no significant variation in the filamentous bacteria response level was recorded, whereas with more severe deficiencies the filamentous bacteria response increased considerably with the increase in stress number. From the results obtained, it would appear that there is an intensity and frequency threshold beyond which the filamentous bacteria response levels increase when the stress is applied in series. This “oxygen deficiencies repetition” appears to be an important factor in the appearance of bulking.

Phylogenetic relationships of filamentous sulfur bacteria (Thiothrix spp. and Eikelboom type 021N bacteria) isolated from wastewater-treatment plants and description of Thiothrix eikelboomii sp. nov., Thiothrix unzii sp. nov., Thiothrix fructosivorans sp. nov. and Thiothrix defluvii sp. nov.

The relationship of mixotrophic and autotrophic Thiothrix species to morphologically similar chemoorganotrophic bacteria (e.g. Leucothrix species, Eikelboom type 021N bacteria) has been a matter of debate for some years. These bacteria have alternatively been grouped together on the basis of shared morphological features or separated on the basis of their nutrition. Many of these bacteria are difficult to maintain in axenic culture and, until recently, few isolates were available to allow comprehensive phenotypic and genotypic characterization. Several isolates of Thiothrix spp. and Eikelboom type 021N strains were characterized by comparative 16S rRNA sequence analysis. This revealed that the Thiothrix spp. and Eikelboom type 021N isolates formed a monophyletic group. Furthermore, isolates of Eikelboom type 021N bacteria isolated independently from different continents were phylogenetically closely related. The 16S rRNA sequence-based phylogeny was congruent with the morphological similarities between Thiothrix and Eikelboom type 021N. However, one isolate examined in this study (Ben47) shared many morphological features with the Thiothrix spp. and Eikelboom type 021N isolates, but was not closely related to them phylogenetically. Consequently, morphology alone cannot be used to assign bacteria to the Thiothrix/type 021N group. Comparative 16S rRNA sequence analysis supports monophyly of the Thiothrix/type 021N group, and phenotypic differences between the Thiothrix spp. and Eikelboom type 021N bacteria are currently poorly defined. For example, both groups include heterotrophic organisms that deposit intracellular elemental sulfur. It is therefore proposed that the Eikelboom type 021N bacteria should be accommodated within the genus Thiothrix as a new species, Thiothrix eikelboomii sp. nov., and three further new Thiothrix species are described: Thiothrix unzii sp. nov., Thiothrix fructosivorans sp. nov. and Thiothrix defluvii sp. nov.

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.

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.

Survey on filamentous micro-organisms in activated sludge processes in Bangkok, Thailand

A survey to investigate the proliferation of filamentous micro-organisms, which cause filamentous bulking in activated sludge wastewater treatment processes, was conducted for the first time in the tropical zone. Activated sludge samples were collected from six wastewater treatment processes in Bangkok City, Thailand. The activated sludge plants investigated include those for 1) an industrial wastewater from an industrial estate, 2) a domestic wastewater from apartment houses, 3) a brewery wastewater, 4) a hotel wastewater, 5) a textile industry wastewater and 6) a milk processing wastewater. The identification of proliferating filamentous micro-organisms in the samples was attempted. Type 021N was exclusively dominant in the milk processing wastewater treatment plant. The characteristics of the 021N present were different from “typical” 021N. The most proliferating filament in the textile wastewater treatment plant was suspected to be Nostocoida limicola. There was a predominant filament in the brewery wastewater treatment plant, which is suspected to be a new type. Other major filaments proliferating include Eikelboom Type 021N, 1701, 0041 and 0092. Nocardia was also found in a few plants.

Survey on filamentous micro-organisms in activated sludge processes in Bangkok, Thailand

A survey to investigate the proliferation of filamentous micro-organisms, which cause filamentous bulking in activated sludge wastewater treatment processes, was conducted for the first time in the tropical zone. Activated sludge samples were collected from six wastewater treatment processes in Bangkok City, Thailand. The activated sludge plants investigated include those for 1) an industrial wastewater from an industrial estate, 2) a domestic wastewater from apartment houses, 3) a brewery wastewater, 4) a hotel wastewater, 5) a textile industry wastewater and 6) a milk processing wastewater. The identification of proliferating filamentous micro-organisms in the samples was attempted. Type 021N was exclusively dominant in the milk processing wastewater treatment plant. The characteristics of the 021N present were different from “typical” 021N. The most proliferating filament in the textile wastcwatcr treatment plant was suspected to be Nostocoida limicola. There was a predominant filament in the brewery wastewater treatment plant, which is suspected to be a new type. Other major filaments proliferating include Eikelboom Type 021N, 1701, 0041 and 0092. Nocardia was also found in a few plants.

Identification and in situ Detection of Gram-negative Filamentous Bacteria in Activated Sludge

Gram-negative filamentous bacteria are commonly observed in activated sludge and contribute to poor settlement of activated sludge flocs in secondary sedimentation tanks, a problem referred to as activated sludge bulking. However, the standard morphological identification system is of limited value for a high resolution, rapid monitoring of these bacteria. Therefore, specific 16S rRNA-targeted oligonucleotide probes were developed for Haliscomenobacter spp., Sphaerotilus spp., Leptothrix spp., Thiothrix spp., Leucothrix mucor and bacteria of the Eikelboom type 021N. Probe specificities were evaluated by nonisotopic dot blot hybridization to 145 reference strains representing a diverse collection of taxa. In situ hybridization with fluorescent probe derivatives was combined with scanning confocal laser microscopy (SCLM) for analyzing the three dimensional localization of the filaments inside the sludge flocs. Filaments could be localized even in the center of fixed flocs at a high resolution undisturbed by problems like autofluorescence.

The Effect of Controlled pH on the Development of Rosette-Forming Bacteria in Axenic Culture and Bulking Activated Sludge

An infestation of a rosette-forming microorganism, with morphological characteristics bearing some resemblance to Eikelboom type 021N and Thiothrix spp., occurred in the activated sludge process of a paper and pulp manufacturing facility during the autumn of 1986. The microorganism could not be eliminated by periodic acid-shocking of the mixed liquor with sulfuric acid, however, exposure of the mixed liquor solids for 10 days at controlled pH (circum pH 5.5) brought almost immediate lowering of the sludge volume index and effluent suspended solids to levels observed in normal operations. The bulking correction technique required no purchase of special chemicals in that the procedure was a mere modification of the liming step normally employed to bring the natural pH of the wastewater (pH 3) to a level suitable for the practice of biological treatment.