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

Vervaeren, Han; Wilde, Kurt De; Matthys, Jorg; Boon, Nico; Raskin, Lutgarde; Verstraete, W.

doi:10.1007/s00253-005-1963-9

Cited by 34 publications

(22 citation statements)

References 32 publications

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“…In contrast, real-time PCR is a specific, sensitive, precise, and high-throughput method for quantifying a gene or organism group in various types of environmental samples. For example, the method has been used to quantify nitrifiers 26) and filamentous microorganisms 27) in activated sludge, and has provided useful information on the sludge process. Accordingly, real-time PCR may become a powerful tool for the high-throughput analysis of PAO abundance, and is the best method for achieving a comprehensive understanding of the relationship between sludge phosphorus content and major PAO abundance.…”

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confidence: 99%

Quantification of Rhodocyclus-Related and Actinobacterial Polyphosphate-Accumulating Organisms in an Enhanced Biological Phosphorus Removal Process Using Quenching Probe PCR

et al. 2007

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Quenching probe (QProbe) PCR-based methods for quantifying the abundance of Rhodocyclus-related and actinobacterial polyphosphate-accumulating organisms (PAOs) in an enhanced biological phosphorus removal (EBPR) process are described. PCR primer-QProbe sets specific for the 16S rRNA genes of Rhodocyclus-related PAOs (RPAOs) and actinobacterial PAOs (APAOs) were newly designed, and their specificity tested using database searches, a selection of pure cultures, and sludge DNA. The sets were found to be specific for most of the target sequences. QProbe PCR with the newly designed sets was used to enumerate the 16S rRNA genes of RPAOs and APAOs in a laboratory-scale EBPR sludge. The copy number of 16S rRNA genes of RPAOs was larger than that of APAOs before the phosphorus removal performance of the reactor deteriorated, and the amount of both PAOs decreased in the deterioration period. The approaches developed in this study may become a powerful tool for a high-throughput analysis of PAO abundance in EBPR processes.

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Quantification of Rhodocyclus-Related and Actinobacterial Polyphosphate-Accumulating Organisms in an Enhanced Biological Phosphorus Removal Process Using Quenching Probe PCR

et al. 2007

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“…Moreover, using mulitplex PCR, simultaneous detection of total Bacteria or other problematic species such as Eikelboom's types (021N, 0675, 0041, 0961, 1701, 0914 and 0092), Thiothrix eikelboomii, Nostocoida limocola, and Gordonia amarae, etc. would be possible (Dumonceaux et al, 2006;Jenkins et al, 2004;Marrengane et al, 2011;Nielsen et al, 2009;Seviour et al, 1994;Vervaeren et al, 2005). However, the development of a multiplex qPCR using the same gene is difficult due to the competition for resources.…”

Section: Comparison Between Taqman Sybr Green and Microscopymentioning

confidence: 99%

Development and evaluation of a TaqMan duplex real-time PCR quantification method for reliable enumeration of Candidatus Microthrix

Vanysacker

Denis

Roels

et al. 2014

Journal of Microbiological Methods

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Candidatus Microtrhix parvicella is one of the most common filamentous bacteria reported to be involved in bulking and foaming problems in activated sludge plants worldwide. In order to detect and quantify both M. parvicella and Microthrix calida by quantitative PCR (qPCR), primers targeting 16S rDNA genes were designed. The qPCR reaction was optimized by using the TaqMan technology and an internal positive control was included to ensure the absence of PCR inhibitors. A total of 29 samples originating from different wastewater treatment plants were analyzed and the results were compared by using conventional microscopy, fluorescent in situ hybridization and an existing SYBR Green-based assay. Our assay showed a 100% specificity for both M. parvicella and M. calida, a sensitivity of 2.93 × 10 9 to 29 copy numbers/reaction, an amplification efficiency of 93% and no PCR inhibition. By performing a spiking experiment including different Microthrix concentrations, recovery rates ranging from 65 to 98% were obtained. A positive correlation with the SYBR Green assay (R 2 = 0.85) was found and most of the samples were in accordance with the microscopical observation. In comparison with SYBR Green assay, the probe-based TaqMan assay had a much lower detection limit. Compared with microscopy, some samples had a lower or higher enumeration when using qPCR. In conclusion, a qPCR method is forwarded here that could be useful as an early warning tool for fast and reliable detection of Microthrix in for instance sludge bulking events.

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“…FISH has been used extensively to study biofilms, activated sludge floc and even to relate species levels to bulking (Liao et al 2004). Real-time PCR has been used to correlate filament levels with bulking events (Vervaeren et al 2005) and to quantify Microthrix parvicella (Kaetzke et al 2005) but to date, no one has used molecular methods to measure the in-situ growth rate of filamentous bacteria in activated sludge.…”

Section: Introductionmentioning

confidence: 99%

Quantifying the In Situ Growth Rate of a Filament and a Floc-former in Activated Sludge

Nguyen¹,

Reyes²

2008

proc water environ fed

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The goal of this project was to develop a species-specific method to quantify the in-situ growth rate of one filament and one floc-former in activated sludge. Chemostats were used to study pure cultures of Sphaerotilus natans, as the representative filament, and Arthrobacter globiformis, as the representative floc-former, to determine the relationship between growth rate and RNA:DNA ratio for each species. Real-time qPCR and reverse transcription real-time qPCR were used to measure DNA and RNA levels respectively. The relationship between RNA:DNA ratio and growth rate was found to be positive and linear, with R 2 of 0.58 and 0.98 for S. natans and A. globiformis respectively. This relationship was used to determine in-situ growth rate of S. natans in activated sludge from the aeration basin of a full-scale WWTP and in samples collected from a previous study on the effect of different substrates on bulking. A. globiformis could not be detected in any of the WWTP samples. This study represents the first time qPCR and RT-qPCR have been used to quantify in-situ microbial growth rates in activated sludge.

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

Cited by 34 publications

References 32 publications

Quantification of Rhodocyclus-Related and Actinobacterial Polyphosphate-Accumulating Organisms in an Enhanced Biological Phosphorus Removal Process Using Quenching Probe PCR

Quantification of Rhodocyclus-Related and Actinobacterial Polyphosphate-Accumulating Organisms in an Enhanced Biological Phosphorus Removal Process Using Quenching Probe PCR

Development and evaluation of a TaqMan duplex real-time PCR quantification method for reliable enumeration of Candidatus Microthrix

Quantifying the In Situ Growth Rate of a Filament and a Floc-former in Activated Sludge

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