Quantification of ammonia-oxidizing bacteria populations in full-scale sewage activated sludge systems and assessment of system variables affecting their performance

2006 ◽  
Vol 54 (1) ◽  
pp. 91-99 ◽  
Author(s):  
T. Limpiyakorn ◽  
F. Kurisu ◽  
O. Yagi
Keyword(s):  
Activated Sludge ◽  
Real Time ◽  
Real Time Pcr ◽  
Full Scale ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Ammonia Oxidizing Bacteria ◽  
Primer Sets ◽  
Activated Sludge Systems ◽  
Pcr Primer

This study carried out quantification of ammonia-oxidizing bacteria (AOB) populations in 12 full-scale sewage activated sludge systems that were different in ammonia removals and treatment processes during three different seasons. Experiment was divided into 3 parts: 1) analysis of AOB communities by PCR-DGGE-cloning-sequencing of 16S rRNA genes; 2) development of four real-time PCR primer sets for quantification of the particular AOB of interest; and 3) quantification of AOB populations by using the newly developed real-time PCR primer sets. The results suggested that all the primer sets gave good reproducibility and specificity for PCR amplification with the detection limits of 102 copies/PCR reaction. Although the 12 systems were different in several aspects, one of the identified sequence types of Nitrosomonas oligotropha cluster was the dominant AOB in every system and every season studied. However, the other sequence type of this cluster was not significantly involved in ammonia removals in the systems. The occurrence of N. communis cluster in the systems seemed to depend on the remaining oxygen concentrations in the sludge floc and thus the activity of aerobic heterotrophs in the aeration tanks. N. europaea–Nitrosococcus. mobilis solely existed in one A2O system of which the influent contained twice the chloride concentrations than those of other systems.

scholarly journals Power Analysis for Real-Time PCR Quantification of Genes in Activated Sludge and Analysis of the Variability Introduced by DNA Extraction

2003 ◽  
Vol 69 (11) ◽  
pp. 6597-6604 ◽  
Author(s):  
Hebe M. Dionisi ◽  
Gerda Harms ◽  
Alice C. Layton ◽  
Igrid R. Gregory ◽  
Jack Parker ◽  
...  
Keyword(s):  
16S Rrna ◽  
Activated Sludge ◽  
Real Time ◽  
Real Time Pcr ◽  
Power Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Pcr Assays

ABSTRACT The aims of this study were to determine the power of discrimination of the real-time PCR assay for monitoring fluctuations in microbial populations within activated sludge and to identify sample processing points where methodological changes are needed to minimize the variability in target quantification. DNA was extracted using a commercially available kit from mixed liquor samples taken from the aeration tank of four bench-scale activated-sludge reactors operating at 2-, 5-, 10-, and 20-day solid retention times, with mixed-liquor volatile suspended solid (MLVSS) values ranging from 260 to 2,610 mg/liter. Real-time PCR assays for bacterial and Nitrospira 16S rRNA genes were chosen because they represent, respectively, a highly abundant and a less-abundant bacterial target subject to clustering within the activated sludge matrix. The mean coefficient of variation in DNA yields (measured as microgram of DNA per milligram of MLVSS) in triplicate extractions of 12 different samples was 12.2%. Based on power analyses, the variability associated with DNA extraction had a small impact on the overall variability of the real-time PCR assay. Instead, a larger variability was associated with the PCR assay. The less-abundant target (Nitrospira 16S rRNA gene) had more variability than the highly abundant target (bacterial 16S rRNA gene), and samples from the lower-biomass reactors had more variability than samples from the higher-biomass reactors. Power analysis of real-time PCR assays indicated that three to five samples were necessary to detect a twofold increase in bacterial 16S rRNA genes, whereas three to five samples were required to detect a fivefold increase in Nitrospira 16S rRNA genes.

Monitoring of Microbial Community Inhabiting a Low-Grade Copper Sulphide Ore by Quantitative Real-Time PCR Analysis of 16S rRNA Genes

2007 ◽  
Vol 20-21 ◽  
pp. 539-542 ◽  
Author(s):  
Francisco Remonsellez ◽  
F. Galleguillos ◽  
Sonestie Janse van Rensburg ◽  
G.F. Rautenbach ◽  
Pedro A. Galleguillos ◽  
...  
Keyword(s):  
16S Rrna ◽  
Real Time ◽  
Real Time Pcr ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Low Grade ◽  
Copper Sulphide ◽  
Quantitative Real Time Pcr ◽  
Sulphide Ore ◽  
Heap Bioleaching

Microbial heap bioleaching is being used as an industrial process to recover copper from low grade ores. It is known that a consortium of different microorganisms participates in this process. Therefore identification and quantification of communities inhabiting heap bioleaching operations is a key step for understanding the dynamics and role of these microorganisms in the process. A quantitative real-time PCR approach was used to investigate the microbial dynamics in this process. To study the microbial population inhabiting a low-grade copper sulphide ore bioleaching industrial heap process at Escondida Mine in Chile, 16S rRNA genetic libraries were constructed using bacterial and archaeal universal primers. Phylogenetic analyses of sequences retrieved from genetic libraries showed that the community is mainly composed by microoganisms related to Acidithiobacillus ferrooxidans (2 strains), Acidithiobacillus thiooxidans, Leptospirillum ferrooxidans, Leptospirillum ferriphilum and the archaea Ferroplasma. Specific primers for real-time PCR determination were designed and tested to amplify each of the sequences obtained by cloning. Standard curves for real time PCR were performed using plasmid DNA from selected clones. This methodology is actually being used to monitor relevant microorganisms inhabiting this low-grade copper sulphide ore bioleaching industrial heap.

scholarly journals Development of Candida-Specific Real-Time PCR Assays for the Detection and Identification of Eight Medically Important Candida Species

2016 ◽  
Vol 9 ◽  
pp. MBI.S38517 ◽  
Author(s):  
Jing Zhang ◽  
Guo-Chiuan Hung ◽  
Kenjiro Nagamine ◽  
Bingjie Li ◽  
Shien Tsai ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Candida Species ◽  
Rapid Detection ◽  
Corneal Transplantation ◽  
High Sensitivity ◽  
Turnaround Time ◽  
Pcr Assays ◽  
Primer Sets ◽  
Pcr Primer

Culture-based identification methods have been the gold standard for the diagnosis of fungal infection. Currently, molecular technologies such as real-time PCR assays with short turnaround time can provide desirable alternatives for the rapid detection of Candida microbes. However, most of the published PCR primer sets are not Candida specific and likely to amplify DNA from common environmental contaminants, such as Aspergillus microbes. In this study, we designed pan- Candida primer sets based on the ribosomal DNA-coding regions conserved within Candida but distinct from those of Aspergillus and Penicillium. We demonstrate that the final two selected pan- Candida primer sets would not amplify Aspergillus DNA and could be used to differentiate eight medically important Candida pathogens in real-time PCR assays based on their melting profiles, with a sensitivity of detection as low as 10 fg of Candida genomic DNA. Moreover, we further evaluated and selected species-specific primer sets covering Candida albicans, Candida glabrata, Candida tropicalis, and Candida dubliniensis and show that they had high sensitivity and specificity. These real-time PCR primer sets could potentially be assembled into a single PCR array for the rapid detection of Candida species in various clinical settings, such as corneal transplantation.

The development and use of real-time PCR for the quantification of nitrifiers in activated sludge

2002 ◽  
Vol 46 (1-2) ◽  
pp. 267-272 ◽  
Author(s):  
S.J. Hall ◽  
P. Hugenholtz ◽  
N. Siyambalapitiya ◽  
J. Keller ◽  
L.L. Blackall
Keyword(s):  
Wastewater Treatment ◽  
Activated Sludge ◽  
Real Time ◽  
Real Time Pcr ◽  
Wastewater Treatment Plants ◽  
Quantitative Polymerase Chain Reaction ◽  
Analytical Data ◽  
Full Scale ◽  
Rrna Gene ◽  
Dna Templates

Chemical analytical data has long been used to monitor the performance of activated sludge plants even though the process relies on the performance of microorganisms. It is now evident that a rapid and reliable quantitative method is required, to be able to monitor the organisms responsible for nutrient transformation and their activities, allowing avenues for more efficient nutrient removal. The development of real-time or quantitative polymerase chain reaction (PCR) also known as TaqMan® or 5′-nuclease assay has allowed the rapid, quantitative analysis of DNA templates, eliminating some of the variability traditionally associated with other quantitative techniques. In this study analysis of Nitrospira spp., one of the key organisms in nitrite oxidation in wastewater treatment, was used to validate real-time PCR for the their quantification in activated sludge. A probe and primer set, targeting the 16S rRNA gene of Nitrospira spp. was designed according to the constraints of the TaqMan® specifications. Samples used to evaluate the method included DNA from the sludge from full-scale wastewater treatment plants and laboratory scale systems. The reproducibility, quantitative efficiency and specificity were assessed in the evaluation. It was concluded that the method is sensitive and reproducible but has some constraints on the quantitative efficiency. A survey of full-scale systems for Nitrospira spp. was carried out and the results are presented here.

scholarly journals Real-Time PCR Quantitation of Clostridia in Feces of Autistic Children

2004 ◽  
Vol 70 (11) ◽  
pp. 6459-6465 ◽  
Author(s):  
Yuli Song ◽  
Chengxu Liu ◽  
Sydney M. Finegold
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Late Onset ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Pcr Analysis ◽  
Bacterial Cells ◽  
Autistic Children ◽  
Bacterial Strains ◽  
And Control

ABSTRACT Based on the hypothesis that intestinal clostridia play a role in late-onset autism, we have been characterizing clostridia from stools of autistic and control children. We applied the TaqMan real-time PCR procedure to detect and quantitate three Clostridium clusters and one Clostridium species, C. bolteae, in stool specimens. Group- and species-specific primers targeting the 16S rRNA genes were designed, and specificity of the primers was confirmed with DNA from related bacterial strains. In this procedure, a linear relationship exists between the threshold cycle (CT ) fluorescence value and the number of bacterial cells (CFU). The assay showed high sensitivity: as few as 2 cells of members of cluster I, 6 cells of cluster XI, 4 cells of cluster XIVab, and 0.6 cell of C. bolteae could be detected per PCR. Analysis of the real-time PCR data indicated that the cell count differences between autistic and control children for C. bolteae and the following Clostridium groups were statistically significant: mean counts of C. bolteae and clusters I and XI in autistic children were 46-fold (P = 0.01), 9.0-fold (P = 0.014), and 3.5-fold (P = 0.004) greater than those in control children, respectively, but not for cluster XIVab (2.6 � 108 CFU/g in autistic children and 4.8 � 108 CFU/g in controls; respectively). More subjects need to be studied. The assay is a rapid and reliable method, and it should have great potential for quantitation of other bacteria in the intestinal tract.

Linking nitrification characteristic and microbial community structures in integrated fixed film activated sludge reactor by high-throughput sequencing

2016 ◽  
Vol 74 (6) ◽  
pp. 1354-1364 ◽  
Author(s):  
Bin Dong ◽  
Jie Tan ◽  
Yang Yang ◽  
Zishan Pang ◽  
Zhongtian Li ◽  
...  
Keyword(s):  
Microbial Community ◽  
Activated Sludge ◽  
Ammonia Removal ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Nitrifying Bacteria ◽  
Ammonia Oxidizing Bacteria ◽  
Community Structures ◽  
Microbial Community Structures ◽  
Fixed Film

The primary goal of this study is to investigate ammonia removal, abundance of nitrifying bacteria and microbial community structures in a laboratory-scale integrated fixed film activated sludge (IFAS) reactor. The results of Illumina MiSeq sequencing based on 16S rRNA genes showed Proteobacteria and Bacteroidetes were the dominant phyla in both biofilm and suspended sludge samples in the IFAS reactor. The dominant ammonia-oxidizing bacteria (AOB) species was Nitrosomonas and the dominant nitrite-oxidizing bacteria species was Nitrospira. The contribution of biofilm to ammonia removal increased from 4.0 ± 0.9% to 37.0 ± 2% when the temperature decreased from 25 °C to 10 °C. The real-time polymerase chain reaction (PCR) result showed the abundance of AOB in suspended sludge was higher than that in biofilm at the same time. However, nitrification is more dependent on attached growth than on suspended growth in the IFAS reactor at 15 °C and 10 °C and the abundance of AOB in biofilm was also higher than that in suspended sludge. The more robust ammonia removal rate at low temperatures by biofilm contributed to the relatively stable ammonia removal, and biofilm attached on carriers in the IFAS reactor is advantageous for nitrification in low-temperature environment.

scholarly journals Real-Time PCR Screening for 16S rRNA Mutations Associated with Resistance to Tetracycline in Helicobacter pylori

2005 ◽  
Vol 49 (8) ◽  
pp. 3166-3170 ◽  
Author(s):  
Erik Glocker ◽  
Marco Berning ◽  
Monique M. Gerrits ◽  
Johannes G. Kusters ◽  
Manfred Kist
Keyword(s):  
Helicobacter Pylori ◽  
16S Rrna ◽  
Real Time ◽  
Real Time Pcr ◽  
Tetracycline Resistance ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Pcr Screening ◽  
H Pylori

ABSTRACT The effectiveness of recommended first-line therapies for Helicobacter pylori infections is decreasing due to the occurrence of resistance to metronidazole and/or clarithromycin. Quadruple therapies, which include tetracycline and a bismuth salt, are useful alternative regimens. However, resistance to tetracycline, mainly caused by mutations in the 16S rRNA genes (rrnA and rrnB) affecting nucleotides 926 to 928, are already emerging and can impair the efficacies of such second-line regimens. Here, we describe a novel real-time PCR for the detection of 16S rRNA gene mutations associated with tetracycline resistance. Our PCR method was able to distinguish between wild-type strains and resistant strains exhibiting single-, double, or triple-base-pair mutations. The method was applicable both to DNA extracted from pure cultures and to DNA extracted from fresh or frozen H. pylori-infected gastric biopsy samples. We therefore conclude that this real-time PCR is an excellent method for determination of H. pylori tetracycline resistance even when live bacteria are no longer available.

scholarly journals Novel Sensitive Real-Time PCR for Quantification of Bacterial 16S rRNA Genes in Plasma of HIV-Infected Patients as a Marker for Microbial Translocation

2011 ◽  
Vol 49 (10) ◽  
pp. 3691-3693 ◽  
Author(s):  
M. Kramski ◽  
A. J. Gaeguta ◽  
G. F. Lichtfuss ◽  
R. Rajasuriar ◽  
S. M. Crowe ◽  
...  
Keyword(s):  
16S Rrna ◽  
Real Time ◽  
Real Time Pcr ◽  
Microbial Translocation ◽  
16S Rrna Genes ◽  
Rrna Genes
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