The aim of the project is to establish a molecular biology laboratory, capable of answering the following questions arising during the bioremediation of polluted areas: whether the composition of the particular microflora suitable for conducting a successful bioremediation, namely whether it is enough to intensify the local microflora with the release of nutrients and other components. If so, which nutrients and components are needed to be released? If exogenous inoculum is needed to perform bioremediation then the toxicity and hazardousness of substances for the inoculum in the particular area must be investigated to reveal the possibility of bioremediation. How will the amount of saprophytic microorganisms consuming hazardous compounds change during bioremediation? Answering these questions is imperative when bioremediation processes are being designed; moreover the observation of the concentration of waste consuming microorganisms is crucial to follow up on the success of bioremediation. Parallel to this investment project a technological package – that can answer the above-mentioned questions – is going to be developed. 1. Metagenomic analysis (by Roche 454 and Illumina platforms): Based on direct sequencing of DNA extracted from environmental samples, the bacterial composition can be determined (including those microorganisms that can not be cultured in the laboratory, which is approximately 99% of the total species (hivatkozást jó lenne beszúrni ha ezt állítjuk)), furthermore the cell numbers of different species will be compared to each other. The technology – differently from the non metagenomic based methods – has the advantage to detect not only those species, which are expected to be present, but we are capable of the determination of all bacteria strains, which are present in the samples. Based on this information we can reveal the concrete picture of the composition of the microbial consortia in the polluted area, thus a decision can be made on whether it is enough to intensify the local microflora to remove the pollution, or additional exogenous inoculum is needed. Standardization of sampling technology to gain representative samples for the metagenom analysis is part of the development. 2. MFCDiagn: The device is a development of domestic patents. The instrument was developed in contribution of the project manager, who submits this project proposal. The device is operated based on the principle of microbial fuel cell (MFC). The test microorganisms are mixed with the substances to be tested and are found in the anode cell. The microbial metabolism creates potential difference between the anode and the cathode, which induces electrical current. The measured current is proportional to the concentration of the bacteria that can be found in the anode cell. If any substances reduce the metabolism of the bacteria, namely toxic to the tested microorganisms, the measured current intensity will be decreased, which is measured by MFCDiagn device. We can conclude the toxicity of the tested substances from the decrease of the current intensity. The advantage of the method is that only the activity of the living microorganisms is detected, as well as the speed of measurement (2-6 hours). During the environmental remediation process the toxic effects of soil and ground water on the bacteria community wanted to be applied can be measured by MFCDiagn. Thereby we can predict, whether the bioremediation will be successful on the particular area. 3. qPCR with ethidium-monoazide pre-treatment: The relative frequency of the genes coding the (key) enzymes responsible for the degradation of the unwanted waste materials can be cost effectively and quickly measured by this method. The data (along with the Metagenomic data) plays a crucial role to determine whether or not additional bacterial inoculation needed. During the monitoring of the remediation the measurement of gen frequency can reveal the success of bioremediation moreover the need for additional exogenous inoculation or intensification. In addition the presence and relative abundance of the inoculated bacteria can also be determined. The pretreatment ensures the detection of the DNA only from living bacteria. The effectiveness of the developed technological package will be evaluated in field tests. The laboratory will be located in the research institute of the project leader; therefore the establishment of it requires only the renewal of one existing building. Sample storing and the supercomputer needed for the evaluation of biological data, will be placed in this refurbished building. The project owner possesses almost all of the instruments needed for the project, but the Illumina MiSeq DNA sequencer and the supercomputer with the Geneious bioinformatics software, which will be purchased in the framework of this project. The outcome of the development is molecular biological laboratory applying a standardized kit-package.
Total project budget: 175 003 095 Ft
Amount of the support: 123 137 139 Ft
Project holder: Enviroinvest Environmental and Biotechnological Corp.
Project partner: Biological Research Center, Szeged
Project’s implementation period: 05/05/2014 – 30/04/2016