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

• Autorzy: Ziembińska-Buczyńska A. , Wiszniowski J. , Ciesielski S.

Identyfikatory

DOI

10.2478/aep-2014-0036

Warianty tytułu

PL

zalety i wady wykorzystywania techniki nested-PCR w monitoringu bakterii utleniających amoniak w osadzie czynnym bioreaktora membranowego

• Języki publikacji: EN

Abstrakty

EN

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.

PL

Nitiritacja – pierwszy etap nitryfikacji, jest uznawany za krok limitujący przebieg całości procesu utleniania amoniaku. Bakterie utleniające amoniak (ang. ammonia oxidizing bacteria, AOB), które prowadzą ten proces są stałymi mieszkańcami osadu czynnego – mieszaniny bakterii, Protozoa i Metazoa, wykorzystywanych do biologicznego oczyszczania ścieków. Z tego powodu są one interesujące zarówno z punktu widzenia technologii, jak i ekologii mikroorganizmów. Analizy zmienności i bioróżnorodności bakterii utleniających amoniak, zarówno w oczyszczalni ścieków, jak i w reaktorach w skali laboratoryjnej, są prowadzone w oparciu o sekwencje genu kodującego 16S rRNA z użyciem metody biologii molekularnej, jaką jest PCR-DGGE (Polymerase Chain Reaction – Denaturing Gradient Gel Electrophoresis). Analizy te są zazwyczaj prowadzone techniką tzw. nested-PCR. Ze względu na fakt, że metoda ta wymaga większego nakładu pracy i czasu, niż tradycyjny jednoetapowy PCR (ang. non-nested PCR) podjęto próbę sprawdzenia możliwości zastosowania techniki jednoetapowego PCR do uzyskania wzorów prążkowych DGGE bakterii utleniających amoniak. W tej pracy zaprezentowano wyniki analizy PCR-DGGE z użyciem technik nested i non-nested PCR oraz podjęto próbę wykazania ich wad i zalet.

Słowa kluczowe

EN

ammonia oxidizing bacteria; AOB; 16S rRNA gene; Polymerase Chain Reaction – Denaturing Gradient Gel Electrophoresis; PCR-DGGE; nested-PCR

PL

utlenianie amoniaku; AOB; bakteria utleniająca amoniak; gen kodujący 16S rRNA; PCR-DGGE; Polymerase Chain Reaction – Denaturing Gradient Gel Electrophoresis

• Wydawca: Institute of Environmental Engineering, Polish Academy of Sciences
• Czasopismo: Archives of Environmental Protection
• Rocznik: 2014
• Tom: Vol. 40, no. 4
• Strony: 31--38
• Opis fizyczny: Bibliogr. 15 poz., rys., tab.

Twórcy

autor

Ziembińska-Buczyńska A.

• The Silesian University of Technology Environmental Biotechnology Department Akademicka 2; 44-100 Gliwice

autor

Wiszniowski J.

• The Silesian University of Technology Environmental Biotechnology Department Akademicka 2; 44-100 Gliwice

autor

Ciesielski S.

• The Silesian University of Technology Environmental Biotechnology Department Akademicka 2; 44-100 Gliwice

Bibliografia

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