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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

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

Aleksandra Ziembińska-Buczyńska Ewa Felis Justyna Folkert Anna Meresta Dominika Stawicka Anna Gnida Joanna Surmacz-Górska
Archives of Environmental Protection | 2015 | vol. 41 | No 4 | DOI: 10.1515/aep-2015-0035
Keywords DGGE PCR sulfamethoxazole trimethoprim and erythromycin resistance WWTP
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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.

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Authors and Affiliations

Aleksandra Ziembińska-Buczyńska
Ewa Felis
Justyna Folkert
Anna Meresta
Dominika Stawicka
Anna Gnida
Joanna Surmacz-Górska

Adsorption of oxytetracycline and ciprofloxacin on carbon-based nanomaterials as affected by pH

Filip Gamoń Mariusz Tomaszewski Grzegorz Cema Aleksandra Ziembińska-Buczyńska
Archives of Environmental Protection | 2022 | 48 | 2 | 34-41 | DOI: 10.24425/aep.2022.140764
Keywords adsorption, ciprofloxacin, oxytetracycline, carbon-nanomaterials
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Abstract

With the increase in use and application of carbon nanomaterials and the frequent presence of fluoroquinolones and tetracyclines antibiotics in the aquatic environment, their interactions have attracted extensive attention. In this study, adsorption of two antibiotics: oxytetracycline (OTC) and ciprofloxacin (CIP) by four carbon-based nanomaterials (graphene oxide, reduced graphene oxide, multiwalled carbon-nanotubes, oxidized multiwalled carbon-nanotubes) affected by pH was investigated. The experiment was performed in two steps: (i) adsorption of OTC and CIP at different pH values, (ii) adsorption isotherm studies of both antibiotics on four carbon-based nanomaterials. Both steps were conducted using the batch equilibration technique. The results showed that the adsorption of both antibiotics on studied adsorbents was highly pH-dependent. The highest adsorption was obtained at pH 7.0, implying the importance of the zwitterionic antibiotics forms to adsorption. Antibiotics adsorption isotherms at three given pH values followed the order of pH 7.0 > 1.0 > 11.0, which confirmed zwitterionic species of OTC and CIP as having the greatest ability to adsorb on carbonaceous nanomaterials. Electrostatic interaction, π-π EDA interaction, hydrophobic interaction for both antibiotics, and additionally hydrogen bond for CIP were possible mechanisms responsible for OTC and CIP adsorption onto studied nanomaterials. These results should be important to understand and assess the fate and interaction of carbon-based nanomaterials in the aquatic environment. This study can also be important for the use of carbon nanomaterials to remove antibiotics from the environment.
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Authors and Affiliations

Filip Gamoń
1
Mariusz Tomaszewski
1
Grzegorz Cema
1
Aleksandra Ziembińska-Buczyńska
1
  1. Silesian University of Technology, Department of Environmental Biotechnology, Gliwice, Poland

DNA vs RNA based studies of nitrogen removal bacteria genes via qPCR

Anna Banach-Wiśniewska Filip Gamoń Aleksandra Ziembińska-Buczyńska
Archives of Environmental Protection | 2021 | vol. 47 | No 1 | 19-25 | DOI: 10.24425/aep.2021.136444
Keywords qPCR RT-qPCR nitrogen removal bacteria relative gene abundance
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Abstract

Improvements in water quality requires the removal of nitrogen compounds from wastewater. The most promising and cost-effective methods for this purpose are biological ones based on activated sludge microorganisms such as nitrifiers, denitrifiers, and anammox bacteria. Due to the most of the nitrogen removal bacteria are uncultivable in a laboratory, the application of the molecular tools is required to investigate microorganisms involved in the nitrogen removal. In case of this study for the analysis of relative genes abundance of nitrogen removal bacteria, quantitative PCR (qPCR) based on bacterial DNA and qPCR preceded by reverse transcription (RT-qPCR) based on bacterial mRNA as a template, were used with specific bacterial functional genes ( amoA, nrxA, nirS, nirK, hzo). Samples from four anammox sequencing batch reactors (SBRs) were analyzed, while the nitrogen removal process and bacteria growth were supported by biomass immobilization and nanoparticles addition. There were statistically significant differences between results obtained in the case of mRNA and DNA (p<0.05). Statistically significant positive correlations were found between results obtained with those two approaches. In case of mRNA analysis, positive results were obtained only for hzo, amoA and partly for nirS genes, despite additional purification and removal of inhibitors from samples prior to reaction.
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Authors and Affiliations

Anna Banach-Wiśniewska
1
Filip Gamoń
1
Aleksandra Ziembińska-Buczyńska
1
  1. Silesian University of Technology, Faculty of Power and Environmental Engineering, Environmental Biotechnology Department, Gliwice, Poland

Manganese dioxide (MnO2) nanoparticles influence on the nitrification and anammox activity

Mariusz Tomaszewski Filip Gamoń Grzegorz Cema Aleksandra Ziembińska-Buczyńska
Archives of Environmental Protection | 2020 | vol. 46 | No 4 | 54-58 | DOI: 10.24425/aep.2020.135764
Keywords wastewater nanoparticles nitrification anammox manganese dioxide
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Abstract

The anammox (anaerobic ammonia oxidation) process is one of the most efficient processes of nitrogen removal from wastewater. Although there are some applications of anammox-based technologies, it is still difficult to apply this process widely because of the high optimal temperature around 30–40°C. Thus, the main objective of this study was to evaluate the short-term effects of MnO 2 on the anammox and nitrification process activity at a wide range of temperatures between 10 and 30°C, using statistical methods based on the central composite design (CCD). The influence of MnO 2 on anammox and nitrification activity, suspended biomass from the laboratory-scale sequencing batch reactor (SBR), and activated sludge from WWTP, respectively, was used. MnO 2 concentration range was set between 15 and 85 mg/L, and the temperature range was set between 10 and 30°C. Anammox and nitrification process activity was measured based on the batch test and oxygen uptake rate (OUR), respectively. The results were statistically analyzed. Results revealed that nanoparticles can slightly improve anammox activity by several percent, by up to 10%, but in most cases MnO 2 influence was insignificant. The optimal concentration for the anammox stimulation at temperatures below 20°C was evaluated between 40 and 60 mg/L, corresponding to 36 and 56 mg/g VSS. Manganese oxides contribution in the nitrogen removal processes was proved and they should be considered in the field of the anammox process. Thus, further studies are suggested to investigate the long-term effects of MnO 2 on the low-temperature anammox process, overcoming possibility of inhibition
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Authors and Affiliations

Mariusz Tomaszewski
1
Filip Gamoń
1
Grzegorz Cema
1
Aleksandra Ziembińska-Buczyńska
1
  1. Silesian University of Technology, Gliwice, Poland

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