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.
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.
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.