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

Abstract

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 200 mL g 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.

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

“…A 1 mL aliquot of mixed liquor from each sample was washed with Tris-EDTA (T 10 E 1 , pH 8.0) buffer on the sampling day and stored at −20 °C prior to DNA extraction. DNA was extracted using the ISOIL for Beads Beating kit (Soil DNA Extraction Kit, Nippon Gene, Tokyo, Japan) as detailed previously 11,12 . The 16S rRNA genes of Kouleothrix were amplified and quantified by real-time qPCR according to the methods used previously 11 .…”

“…DNA was extracted using the ISOIL for Beads Beating kit (Soil DNA Extraction Kit, Nippon Gene, Tokyo, Japan) as detailed previously 11,12 . The 16S rRNA genes of Kouleothrix were amplified and quantified by real-time qPCR according to the methods used previously 11 . The PCR mixture (20 μL) contained 10 μL of THUNDERBIRD ® SYBR qPCR Mix (TOYOBO, Osaka, Japan), 2 μL (0.5 pmol) of each primer (CHL1851_658f and CHL1851_815r), 1 μL (30 ng) of template DNA, and 5 μL of sterilized milliQ water.…”

“…The PCR mixture (20 μL) contained 10 μL of THUNDERBIRD ® SYBR qPCR Mix (TOYOBO, Osaka, Japan), 2 μL (0.5 pmol) of each primer (CHL1851_658f and CHL1851_815r), 1 μL (30 ng) of template DNA, and 5 μL of sterilized milliQ water. Each amplification was performed in triplicate using a Thermal Cycler Dice Real Time System TP800 (Takara Bio, Shiga, Japan) under the optimized conditions of Nittami et al (2017) 11 . Standard curves were constructed with plasmid DNA possessing the 16S rRNA gene of Kouleothrix B09 clone sequence as described previously 11 .…”

“…Each amplification was performed in triplicate using a Thermal Cycler Dice Real Time System TP800 (Takara Bio, Shiga, Japan) under the optimized conditions of Nittami et al (2017) 11 . Standard curves were constructed with plasmid DNA possessing the 16S rRNA gene of Kouleothrix B09 clone sequence as described previously 11 . The 16S rRNA gene copy numbers ranged from 2.28 × 10 2 copies ng-DNA −1 to 2.28 × 10 6 copies ng-DNA −1 where the average amplification efficiencies showed 97.7% (S.D.…”

“…We have shown that relative percentage abundances of type 1851/ Kouleothrix had a quantitative linear relationship with biomass settleablity in several municipal wastewater treatment plants (WWTPs) in Yokohama, especially during the summer months 11 . More recently this approach has been extended to quantify Kouleothrix in 11 municipal WWTPs across Japan, and the data generated confirmed that increases in their abundances corresponded to sludge bulking episodes 12 .…”