Development and Use of Fluorescent In Situ Hybridization Probes for the Detection and Identification of “Microthrix parvicella” in Activated Sludge

Erhart, Robert; Bradford, Debbie; Seviour, Robert J.; Amann, Rudolf; Blackall, Linda L.

doi:10.1016/s0723-2020(97)80078-1

Cited by 149 publications

(97 citation statements)

References 24 publications

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“…This was necessary because there are no pure cultures whose 16S rRNA would bind the PAO probes. A similar approach was used for "Microthrix parvicella" by Erhart et al (19). Generally all three designed PAO probes were applied to any one individual sample spotted on the slide.…”

Section: Microbiological Analyses (I) Microscopy Of Ebpr Mixed Cultumentioning

confidence: 99%

Identification of Polyphosphate-Accumulating Organisms and Design of 16S rRNA-Directed Probes for Their Detection and Quantitation

Crocetti

Hugenholtz

Bond

et al. 2000

Appl Environ Microbiol

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661

530

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Laboratory-scale sequencing batch reactors (SBRs) as models for activated sludge processes were used to study enhanced biological phosphorus removal (EBPR) from wastewater. Enrichment for polyphosphateaccumulating organisms (PAOs) was achieved essentially by increasing the phosphorus concentration in the influent to the SBRs. Fluorescence in situ hybridization (FISH) using domain-, division-, and subdivision-level probes was used to assess the proportions of microorganisms in the sludges. The A sludge, a high-performance P-removing sludge containing 15.1% P in the biomass, was comprised of large clusters of polyphosphate-containing coccobacilli. By FISH, >80% of the A sludge bacteria were ␤-2 Proteobacteria arranged in clusters of coccobacilli, strongly suggesting that this group contains a PAO responsible for EBPR. The second dominant group in the A sludge was the Actinobacteria. Clone libraries of PCR-amplified bacterial 16S rRNA genes from three high-performance P-removing sludges were prepared, and clones belonging to the ␤-2 Proteobacteria were fully sequenced. A distinctive group of clones (sharing >98% sequence identity) related to Rhodocyclus spp. (94 to 97% identity) and Propionibacter pelophilus (95 to 96% identity) was identified as the most likely candidate PAOs. Three probes specific for the highly related candidate PAO group were designed from the sequence data. All three probes specifically bound to the morphologically distinctive clusters of PAOs in the A sludge, exactly coinciding with the ␤-2 Proteobacteria probe. Sequential FISH and polyphosphate staining of EBPR sludges clearly demonstrated that PAO probe-binding cells contained polyphosphate. Subsequent PAO probe analyses of a number of sludges with various P removal capacities indicated a strong positive correlation between P removal from the wastewater as determined by sludge P content and number of PAO probe-binding cells. We conclude therefore that an important group of PAOs in EBPR sludges are bacteria closely related to Rhodocyclus and Propionibacter.

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Section: Microbiological Analyses (I) Microscopy Of Ebpr Mixed Cultumentioning

confidence: 99%

Identification of Polyphosphate-Accumulating Organisms and Design of 16S rRNA-Directed Probes for Their Detection and Quantitation

Crocetti

Hugenholtz

Bond

et al. 2000

Appl Environ Microbiol

Self Cite

661

530

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“…The microbial community, which is dominated by bacteria (Wagner et al 2002), plays an essential role in the biological treatment reactors and has been studied for several decades by both isolation (Neilson 1978) and molecular methods, such as polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (Muyzer et al 1993;Ye and Zhang 2010), terminal restriction fragment length polymorphism (Liu et al 1997), cloning (Schuppler et al 1995), and fluorescent in situ hybridization (Erhart et al 1997). The culturing methods have been a very direct and effective way to characterize the microbial community.…”

Section: Introductionmentioning

confidence: 99%

Bacterial Communities in Different Sections of a Municipal Wastewater Treatment Plant Revealed by 16S rDNA 454 Pyrosequencing

Ye¹,

ZHANG²

2015

Bioremediation of Wastewater

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In this study, we successfully demonstrated that 454 pyrosequencing was a powerful approach for investigating the bacterial communities in the activated sludge, digestion sludge, influent, and effluent samples of a full scale wastewater treatment plant treating saline sewage. For each sample, 18,808 effective sequences were selected and utilized to do the bacterial diversity and abundance analysis. In total, 2,455, 794, 1,667, and 1,932 operational taxonomic units were obtained at 3 % distance cutoff in the activated sludge, digestion sludge, influent, and effluent samples, respectively. The corresponding most dominant classes in the four samples are Alphaproteobacteria, Thermotogae, Deltaproteobacteria, and Gammaproteobacteria. About 67 % sequences in the digestion sludge sample were found to be affiliated with the Thermotogales order. Also, these sequences were assigned into a recently proposed genus Kosmotoga by the Ribosomal Database Project classifier. In the effluent sample, we found high abundance of Mycobacterium and Vibrio, which are genera containing pathogenic bacteria. Moreover, in this study, we proposed a method to differentiate the "gene percentage" and "cell percentage" by using Ribosomal RNA Operon Copy Number Database.

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“…The EUB338 probe was used for the hybridization of all bacteria (Amann et al, 1990) and the MPA-MIX probe for M. parvicella and M. calida (Erhart et al, 1997) ( Table 2). Slides were mounted in Citifluor (Citifluor Ltd.) and visualized with a BX51 epifluorescence microscope (Olympus) equipped with a UPlanApo 100x/1.35 oil objective, U-M51009 filterset and Cell P software (Olympus).…”

Section: Fluorescence In Situ Hybridizationmentioning

confidence: 99%

“…Its slow growth rate and difficulty in storing isolates and in generating phenotypic information have hindered attempts to elucidate the reasons for the proliferation of M. parvicella in WWTPs. Up to now the abundance of these bacteria is determined subjectively by observing Gram-and Neisser-stained filaments (Eikelboom and van Buijsen, 1983) or by in situ hybridization with probes targeting the 16S rDNA (Erhart et al, 1997). The first method is considered to be feasible, but requires experienced staff because of morphological variability resulting from different growth conditions (de los Reyes et al, 2002;Roels et al, 2002;Westlund et al, 1998).…”

Section: Introductionmentioning

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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Development and Use of Fluorescent In Situ Hybridization Probes for the Detection and Identification of “Microthrix parvicella” in Activated Sludge

Cited by 149 publications

References 24 publications

Identification of Polyphosphate-Accumulating Organisms and Design of 16S rRNA-Directed Probes for Their Detection and Quantitation

Identification of Polyphosphate-Accumulating Organisms and Design of 16S rRNA-Directed Probes for Their Detection and Quantitation

Bacterial Communities in Different Sections of a Municipal Wastewater Treatment Plant Revealed by 16S rDNA 454 Pyrosequencing

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

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