Filamentous Bacteria Identification by VIT Method

Water Practice & Technology

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 ...

FEMS Microbiology Reviews, 2009

Excessive growth of filamentous bacteria in activated sludge wastewater treatment plants (WWTPs) can cause serious operational problems. With some filaments there may be the problem of bulking, where inadequate flocculation and settling of the biomass in the secondary clarifier results in a carryover of solids with the final treated liquid effluent. Their proliferation often encourages the development of stable foams on the surface of the reactors, and these foams may impact negatively on plant performance and operation. The availability of culture-independent molecular methods now allows us to identify many of the more common filamentous organisms encountered in WWTPs, which are phylogenetically diverse, affiliating to seven separate bacterial phyla. Furthermore, the extensive data published in the past decade on their in situ behaviour from the application of these culture-independent methods have not been summarized or reviewed critically. Hence, here, we attempt to discuss what we now know about their identity, ecophysiology and ecological niches and its practical value in better managing activated sludge processes. Some of this knowledge is already being applied to control and manage full-scale WWTPs better, and the hope is that this review will contribute towards further developments in this field of environmental microbiology.

DESALINATION AND WATER TREATMENT, 2020

If not controlled, excessive sludge bulking can lead to a complete failure of the entire wastewater treatment process. Selection of an appropriate control measure requires information about the filaments type, level of dominance and the most probable causes. The main aim of this paper is to present the filamentous bacteria identified in Kuwait's wastewater treatment plants and to compare their seasonal levels of dominance. Wastewater samples were collected weekly from Riqqa and Um-Al-Haiman activated sludge systems in Kuwait for eight months. Vermicon identification technology, a molecular method, was used for the identification and quantification of the following six filamentous bacteria: Type 1851, H. hydrossis, Nocardioform, Type 021N/Thiothrix, N. Limicola and M. parvicella. The obtained results indicated that the rapid growth of the filaments was triggered by the sharp drop in water temperature during winter (January). Further, most of the filaments dominate the systems almost all through the year. Furthermore, Microthrix was found to have the highest rate of fluctuations.

International Journal of Environmental Science and Technology, 2017

In this study, Illumina sequencing was used for the identification of bulking and foaming bacteria in industrial wastewater treatment plants. The reliable identification of bulking and foaming bacteria represents the first step in developing effective and specific control strategies to avoid disturbances in activated sludge systems. Illumina sequencing revealed 432 16S rRNA operational taxonomic units, representing phylotypes and including 21 bulking and foaming bacteria in the two investigated industrial wastewater treatment plants. Foaming represents the most severe problem in the cascade biology system. Up to 22.5% of all sequencing reads are bulking and foaming bacteria, including Chryseobacterium, Candidatus Microthrix parvicella and Gordonia sp. as the dominant bulking and foaming bacteria which are known for foam formation. Moreover, Illumina sequencing revealed an increase in Candidatus Microthrix parvicella and Gordonia sp. reads from activated sludge to foam and scum samples, indicating a preferred flotation and/or growth advantages in the foam and scum layers. Analyses of the taxonomic assignment and distribution showed that the phylum Actinobacteria is the most dominant phylum, underlining the key role of Actinobacteria in bulking and foaming. Multivariate data analysis was applied, revealing that the dominant bulking and foaming bacteria are positively correlated with the sludge age and influent flow and negatively correlated with the dissolved oxygen level and the temperature. In terms of developing a specific control strategy, the positive linear relationships to the fatty acid and surfactant sludge loadings are highlighted and the removal of lipid compounds from the wastewater influent could avoid an overgrowth of bulking and foaming bacteria. Keywords Candidatus Microthrix parvicella Á Filamentous bacteria Á Filamentous foaming Á Illumina sequencing Á Next-generation sequencing Á Industrial activated sludge plants Editorial responsibility: BV Thomas.

Journal of applied & environmental microbiology, 2015

Filamentous bacteria are important in the activated sludge process as they contribute to proper solidsliquid separation in the secondary clarifier by providing a skeletal matrix for the formation of compact flocs. Morphological and molecular techniques were applied over a one-year period to comprehensively identify filamentous bacteria in municipal and industrial wastewater treatment plants (WWTPs) in Ghana. Several morphologically distinguishable filamentous microorganisms were observed and in most cases, microscopic characteristics of these organisms were in close agreement with those described in published keys. The presence of Haliscomenobacter hydrossis, Thiothrix nivea, Sphaerotilus natans, Nostocoida limicola II, Eikelboom Type 1851 and members of the Eikelboom Type 021N group II was subsequently confirmed based on hybridization with their respective oligonucleotide probes. Filaments were not unique to each plant, with higher filament diversity in industrial compared to municipal plants. Additionally, the results revealed that although geographic differences have no significant effect on filament morphology, they likely play a limiting role in the occurrence of specific filaments.

2012

This study aimed at isolating filamentous bacteria from the activated sludge samples of a full- scale municipal wastewater treatment plant in Dubai and studying their physiological and biochemical characteristics. Fluorescent in situ hybridization technique (FISH) was applied to identify filamentous bacteria pure culture isolates. In total, sixteen filamentous bacteria were successfully isolated and maintained on various culture media. Ten gram positive filamentous bacteria were successfully identified as nocardioform actinomycete using nocardioform group specific probes in FISH analysis. Most of the isolates were shown to utilize different carbon sources like glycerol, maltose, fructose etc. The optimum growth of most of the isolates was found to be at 27° C and neutral pH, however a few isolates were able to grow at 47° C and acidic pH. Overall results indicate that filamentous bacteria isolated in this study were quite diverse in their physiological and biochemical characteristic...

Microbiology-sgm, 2008

Filamentous members of the Bacteroidetes are commonly observed in activated sludge samples originating from both municipal and industrial wastewater treatment plants (WWTP), where they occasionally can cause bulking. Several oligonucleotide 16S rRNA-targeted probes were designed to target filaments with a needle-like appearance similar to Haliscomenobacter hydrossis. The design of these probes was based on an isolate and a sequence obtained from a micromanipulated filament. The abundance of filamentous Bacteroidetes was determined in 126 industrial samples applying already published and the newly developed probes. Small populations were found in 62 % of the WWTP investigated. However, only relatively few WWTP (13 %) contained large populations of filamentous Bacteroidetes potentially responsible for bulking incidences. The identity of the most abundant filamentous Bacteroidetes with H. hydrossis morphology could be detected by probes CFB719, SAP-309 and the newly designed probe HHY-654. A comprehensive study on the ecophysiology of probe-defined Bacteroidetes populations was conducted on Danish and Czech samples. The studies revealed that they were specialized bacteria involved in degradation of sugars, e.g. glucose and N-acetylglucosamine, and may participate in the conversion of lipopolysaccharides and peptidoglycan liberated by decaying cells. Many surface-associated exo-enzymes were excreted, e.g. chitinase, glucuronidase, esterase and phosphatase, supporting conversion of polysaccharides and possibly other released cell components. The role of filamentous bacteria with a H. hydrossis-like morphology in the activated sludge ecosystem is discussed.

2014

The study was carried out for identifying microorganisms present in the extended aeration activated sludge process at Common Effluent Treatment Plant (CETP), Thane. The stabilization of biological foams results from production of extra cellular materials like lipids, peptides, proteins and carbohydrates which have the properties of surface active agents and bulking phenomenon has been recognized for a long time and is known to be caused by at least 14 types of filamentous microorganisms. The microbial population from treated sludge was isolated on nutrient medium, MacConkey’s agar and Sabauroud’s agar using streak plate method. The isolated colonies were then Gram stained and isolates were used further for biochemical tests to confirm the organism upto probable genus level. Diverse microflora was observed in the water sample collected from aeration tank at CETP. The microscopic examination of water samples showed the presence of Diatoms (Bacillariophycophyta), Paramoecium species, V...

The activated sludge process is the most used biological wastewater treatment method in the world. Ever since the inception of the activated sludge process, several kinds of sedimentation problems have been observed; particularly filamentous bulking foaming which considerably compromise the efficiency of the process. Filamentous bulking has long been attributable to the extensive and uncontrollable growth of different types of filamentous bacteria. Several strategies to prevent bulking sludge have been applied but still fail as long-term control measures due to a lack of understanding of the biochemistry and physiology of the causative organisms. Effective means to long-term bulking control strategies rely on an intense understanding of filamentous bacteria in pure culture, on the basis of their biochemistry and physiology in view of corroborating these findings in situ. It was the aim of this study therefore to extrapolate on the biochemistry and physiology of previously isolated filamentous bacteria in pure culture particularly focusing on growth parameters like pH, temperature and salt tolerance, polymer hydrolyses and carbon source utilization profiles. Fifty two isolates from previous studies were investigated, however, herein are results for eight isolates. An affinity for a variety of substrates, some simple and some more complex, was displayed by the isolates. Some of the more complex substrates included starch, dextrin, peptides and lipids. This presented them with an added survival advantage over ordinary bacteria that are not as efficient with regards to hydrolyzing larger and slowly biodegradable substrates. They also preferred a variety of amino acids and sugars. The above information will add substantial value in understanding their substrate requirements and the contribution of filamentous bacteria to bulking conditions.

Systematic and Applied Microbiology - SYST APPL MICROBIOL, 2002

Materials and Methods Sampling Activated sludge foam was collected from the aerobic zone of the Centurion Wastewater treatment plant in Gauteng, South Africa. Microorganisms present The filaments in the foam sample from Centurion WWTP occurred as Gram positive, irregularly coiled filaments that were found coiled in and around the floc. Filaments were 0.6-0.8 µm in diameter and 100-400 µm in length (not illustrated). No sheath or attached growth was present and no branching occurred. Cellular inclusions were common which gave a "beaded" appearance. According to Jenkins et al. (1993) these are characteristics true to "Microthrix parvicella". Sample preparation Volumes of 100 ml foam were homogenised for 10 min using 70g glass beads. The foam was pelleted by centrifugation for 10 min at 7000 rpm in a Hermle 360 K centrifuge. DNA extraction and purification (Modified from EHLERS, 1995) After centrifugation, the supernatant was discarded. The pellet was resuspended in volumes of 40 ml Sodium-Tris