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Comparison of PCR-DGGE and Nested-PCR-DGGE Approach for Ammonia Oxidizers Monitoring in Membrane Bioreactors’ Activated Sludge

Aleksandra Ziembińska-Buczyńska Jarosław Wiszniowski Sławomir Ciesielski

Archives of Environmental Protection | 2014 | vol. 40 | No 4 | DOI: 10.2478/aep-2014-0036

Keywords AOB 16S rRNA gene PCR-DGGE nested PCR

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Abstract

Nitritation, the first stage of ammonia removal process is known to be limiting for total process performance. Ammonia oxidizing bacteria (AOB) which perform this process are obligatory activated sludge habitants, a mixture consisting of Bacteria, Protozoa and Metazoa used for biological wastewater treatment. Due to this fact they are an interesting bacterial group, from both the technological and ecological point of view. AOB changeability and biodiversity analyses both in wastewater treatment plants and lab-scale reactors are performed on the basis of 16S rRNA gene sequences using PCR-DGGE (Polymerase Chain Reaction – Denaturing Gradient Gel Electrophoresis) as a molecular biology tool. AOB researches are usually led with nested PCR. Because the application of nested PCR is laborious and time consuming, we have attempted to check the possibility of using only first PCR round to obtain DGGE fingerprinting of microbial communities. In this work we are comparing the nested and non-nested PCR-DGGE monitoring of an AOB community and presenting advantages and disadvantages of both methods used. The experiment revealed that PCR technique is a very sensitive tool for the amplification of even a minute amount of DNA sample. But in the case of nested-PCR, the sensitivity is higher and the template amount could be even smaller. The nested PCR-DGGE seems to be a better tool for AOB community monitoring and complexity research in activated sludge, despite shorter fragments of DNA amplification which seems to be a disadvantage in the case of bacteria identification. It is recommended that the sort of analysis approach should be chosen according to the aim of the study: nested-PCR-DGGE for community complexity analysis, while PCR-DGGE for identification of the dominant bacteria.

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Authors and Affiliations

Aleksandra Ziembińska-Buczyńska

Jarosław Wiszniowski

Sławomir Ciesielski

PCR-based methods in detection and identification of dermatophytes in dogs and cats with suspected dermatophytosis in 2021 in Poland

Dawid Jańczak Piotr Górecki Aleksandra Kornelia Maj

Polish Journal of Veterinary Sciences | 2023 | vol. 26 | No 4 | 629-634 | DOI: 10.24425/pjvs.2023.148282

Keywords dermatophytes dogs cats Poland nested PCR multiplex PCR

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Abstract

Dermatophytes from Microsporum, Trichophyton and Epidermophyton genera are divided into geophilic, zoophilic and anthropophilic species which cause skin infection in humans and wide group of animals, mainly mammals. Main species causing dermatophytosis in dogs and cats are Microsporum and Trichophyton. Conventional mycological diagnostic technique includes Saburaud Dextrose Agar (SAD) and others medium cultures, 10% KOH mount and direct microscopy of hairs and scraping. Molecular diagnostic become more frequent in veterinary practice due to shortening of waiting time. In this study we based on two PCR methods. The nested PCR amplified CHS1 gene for dermatophytes detection, and multiplex PCR coding ITS1 and ITS2 fragments for species identification of detected derpatophytes. Most frequently detected species was Microsporum canis, mainly in young cats. Geophilic Microsporum gypseum and anthropophilic Trichophyton rubrum was found primarily in dogs. Molecular methods in dermatophytosis identification are rapid in contrast to routinely, long lasting culture.

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Authors and Affiliations

Dawid Jańczak

Piotr Górecki

Aleksandra Kornelia Maj

Animallab Veterinary Laboratory, Środkowa 2/4, 03-430 Warsaw, Poland

Molecular Analysis of Microorganisms Responsible for the First Phase of Nitrification in an Anoxic Environment

Aleksandra Ziembińska Sławomir Ciesielski Anna Raszka Korneliusz Miksch

Archives of Environmental Protection | 2011 | vol. 37 | No 1

Keywords 16S rRNA gene amoA gene beta-proteobacterial ammonia oxidizers DGGE nested-PCR Nitrosomonas-like bacteria

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Abstract

Ammonia-oxidizing bacteria communities were evaluated in a completely mixed, laboratory scale membrane reactor (MBR) working under anoxic conditions for 5 months. The microorganisms in activated sludge were fed a synthetic medium containing 66-150 mg NH4 +-N/l. The age of the activated sludge in MBR was 50 days and the hydraulic retention time (HRT) was 3.3 days. The estimation of the diversity and complexity of the AOB community together with the identification of the dominant bacteria in the activated sludge under anoxic conditions were performed using denaturing gradient gel electrophoresis (DGGE) and DNA sequencing. Molecular analysis of the microbial community carried out with two microbial molecular markers, 16S rRNA gene and amoA gene, suggested that nitrification was led by a Nitrosomonas-like species. In the biocenosis of the investigated bioreactor, oxygen was the crucial selective parameter. The results obtained in this work showed that amoA gene research is more suitable to study the stability and effectiveness of ammonia oxidation. This information emphasizes the necessity of the usage of molecular markers based on functional genes instead of ribosomal ones in order to present the actual state of the process performed in bioreactors. It was also stated that Nitrosomonas -like bacteria are able to perform nitritation even in anoxic environment, that is probably the reason why these bacteria are the most common AOB in different bioreactors.

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Authors and Affiliations

Aleksandra Ziembińska

Sławomir Ciesielski

Anna Raszka

Korneliusz Miksch

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Comparison of PCR-DGGE and Nested-PCR-DGGE Approach for Ammonia Oxidizers Monitoring in Membrane Bioreactors’ Activated Sludge

Abstract

Authors and Affiliations

PCR-based methods in detection and identification of dermatophytes in dogs and cats with suspected dermatophytosis in 2021 in Poland

Abstract

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Authors and Affiliations

Molecular Analysis of Microorganisms Responsible for the First Phase of Nitrification in an Anoxic Environment

Abstract

Authors and Affiliations