Microbial biodiversity and genotypic structure changes during start-up of anammox process in a pilot scale SBR reactor A. Banach, M. Tomaszewski, G. Cema, A. Ziembińska – Buczyńska The Silesian University of Technology, Faculty of Power and Environmental Engineering, Environmental Biotechnology Department, Akademicka 2, 44-100 Gliwice, Poland (E-mail:
[email protected])
INTRODUCTION Biological anaerobic ammonium oxidation (anammox) is a process of nitrite and ammonium conversion into dinitrogen gas. Process is catalysed by anammox bacteria belonging to the phylum Planctomycetes. Since anammox bacteria discovery nearly 20 years ago, they have been applied in this cost-effective and sustainable process of ammonium removal in many wastewater treatment plants worldwide. Anammox process consumes less oxygen, has lower biomass yield and involves no usage of external organic carbon in comparison to traditional denitrification – nitrification process. The characteristic of microorganisms involved in the anammox process and knowledge about their variability and biodiversity are required for better understanding of bioreactor functioning. Due to a fact that until this time pure culture of anammox bacteria has not been obtained, molecular biology methods application is needed to analyse both biocenosis composition and biodiversity of these microorganisms. The aim of this studies was to evaluate relationships between microbial biodiversity and physicochemical parameters during the start-up of anammox process in a SBR (Sequence Batch Reactor) pilot scale bioreactor. The experiment lasted 85 days.
MATERIALS AND METHODS Microbial biodiversity and genotypic structure during the bioreactor start-up were examined by using: PCR-DGGE (Polymerase Chain Reaction – Denaturing Gradient Gel Electrophoresis) - method based on studies of bacterial DNA, RT-PCR-DGGE (PCR-DGGE followed by reverse transcription) as the molecular method based on studies of bacterial RNA. Specific primers were used for the PCR reactions: 338f-GC/518r for total bacteria community, Pla46f-GC/518r for anammox bacteria, amoA (ammonia monooxygenaze α subunit) and CTO189f/654r for ammonia oxidizing bacteria (bacteria responsible for the first step of nitrification). On the basis of the obtained DGGE fingerprints, densitometric analysis with Quanitity One 1D Software (Biorad) was performed, then Species Richness and Shannon biodiversity index were calculated.
Analysis of bacterial biocenosis composition during anammox process start-up was made possible by using Fluorescence in Situ Hybridization (FISH). Selected probes which hybridize with the 16S rRNA have been used to collect data of bacteria in samples collected from the bioreactor. Images were taken after observations under confocal microscope.
RESULTS AND CONCLUSIONS Physicochemical parameters of the bioreactor 120,00
% of removal
100,00 80,00 60,00 40,00 20,00 0,00 3
10
15
27
34
41 50 59 day of experiment
% of NH4-N removal
64
71
% of NO2-N removal
78
pH level
8,1 8 7,9 7,8 7,7 7,6 7,5 7,4 7,3 85
pH
• Results obtained by molecular methods were compared to physicochemical parameters measured during the experiment Correlation was found between anammox bacteria biodiversity and the inorganic nitrogen removal in the bioreactor (p
