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  • Author: Aleksandra Ziembińska-Buczyńska x
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Comparison of PCR-DGGE and Nested-PCR-DGGE Approach for Ammonia Oxidizers Monitoring in Membrane Bioreactors’ Activated Sludge

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.

Open access
Impact of Flowback Water on Activated Sludge Biocenosis During Municipal Wastewater Treatment

Abstract

The aim of this study was to determine the effect of flowback water on an activated sludge biocenosis during municipal wastewater treatment in the sequencing batch reactors (SBRs). Two series were performed. In series 1, only municipal wastewater was treated, whereas in series 2, municipal wastewater with pre-treated flowback water was used. Flowback water constituted 3-5% of the influent and was introduced to the SBRs twice per week. Introducing flowback water did not decrease the quality of effluent from the SBRs. However, the composition of the activated sludge biocenosis differed between series, ie the biodiversity of protozoa and the relative abundance of microfauna in functional groups changed after flowback water addition. Polymerase chain reaction - denaturing gradient gel electrophoresis (PCR-DGGE) showed that the ammonia oxidizers community responded faster to flowback water addition than the total bacterial community and remained relatively stable during treatment. However, after 9 weeks of exposure to flowback water, ammonia oxidizing bacteria (AOB) biodiversity decreased. This suggests that prolonged exposure could cause nitrification problems, leading to deterioration in effluent quality

Open access
Detection of antibiotic resistance genes in wastewater treatment plant – molecular and classical approach

Abstract

Antibiotics are a group of substances potentially harmful to the environment. They can play a role in bacterial resistance transfer among pathogenic and non-pathogenic bacteria. In this experiment three representatives of medically important chemotherapeutics, confirmed to be present in high concentrations in wastewater treatment plants with HPLC analysis were used: erythromycin, sulfamethoxazole and trimethoprim. Erythromycin concentration in activated sludge was not higher than 20 ng L−1. N-acetylo-sulfamethoxazole concentration was 3349 ± 719 in winter and 2933 ± 429 ng L−1 in summer. Trimethoprim was present in wastewater at concentrations 400 ± 22 and 364 ± 60 ng L−1, respectively in winter and summer. Due to a wide variety of PCR-detectable resistance mechanisms towards these substances, the most common found in literature was chosen. For erythromycin: erm and mef genes, for sulfamethoxazole: sul1, sul2, sul3 genes, in the case of trimethoprim resistance dhfrA1 and dhfr14 were used in this study. The presence of resistance genes were analyzed in pure strains isolated from activated sludge and in the activated sludge sample itself. The research revealed that the value of minimal inhibitory concentration (MIC) did not correspond with the expected presence of more than one resistance mechanisms. Most of the isolates possessed only one of the genes responsible for a particular chemotherapeutic resistance. It was confirmed that it is possible to monitor the presence of resistance genes directly in activated sludge using PCR. Due to the limited isolates number used in the experiment these results should be regarded as preliminary.

Open access
Molecular Monitoring Of Bacterial And Microalgal Biocenoses’ Biodiversity In High Loaded Farming Ponds

Abstract

Eutrophication process is a serious problem in water ecosystems. There is a great need to study the relation between the physico-chemical condition of water and the influence of these parameters on the diversity of biological life, especially on changes in the structure of microbiocenoses. The most interesting are bacteria and microalgae, due to the important roles they play in maintaining the balance of the aquatic environment. In this study, biodiversity analysis of eukaryotic microalgae and bacteria in two artificial water ecosystems - fish farming ponds - was performed. Aquaculture was based on IMTA technology, in which every part of the trophic chain plays a significant role in maintaining the balance in the ecosystems. Experimental intensive - extensive systems differed in terms of nutrient loads, ponds were characterized by high loads of organic and inorganic nitrogen and phosphorus. During the experimental period, the physicochemical conditions, quantitative genotypic structure of the two biocenoses being studied and the relation between these factors were monitored and investigated. For the biodiversity analysis, the PCR - DGGE technique was used. The results of preliminary research showed that there is a correlation between nutrient loads, diversity expressed in the Shannon-Wiener Index and the overall condition of experimental systems. Higher loadings of nutrient promote the development of bacteria and microalgae without any influence on the balance in the artificial ecosystem being tested.

Open access