Quantitative monitoring of microorganisms in environmental samples using biomarkers

University dissertation from Stockholm : Stockholm University

Abstract: The use of microorganisms in environmental biotechnology is an area of growing interest. Microorganisms have been used for diverse applications, such as biological fertilizers, biological pesticides and bioremediation of toxic chemicals. In many cases, the bacteria of interest have been genetically alterred to improve certain traits. Before large scale environmental release of genetically modified microorganisms is attempted, careful risk assessment of the fate and survival of the inoculum and potential effects on the ecosystem must be evaluated. In order to monitor the inocula in complex environmental samples among a large population of indigenous microorganisms, the methods for detection of the microbe must be very specific and sensitive. To achieve any reliable estimations of fate, survival and efficacy of the inocula the methods for detection also need to be quantitative.Bacteria of interest can be tagged with specific biomarkers of eukaryotic origin, such as the firefly luciferase gene, luc, or the Aequoria victoria green fluorescent protein gene, gfp. These biomarkers confer novel genotypes (the marker gene itself) and phenotypes (bioluminescence or fluorescence, respectively) that are absent in the natural microbial flora. For example, PCR amplification, with primers targeting the marker gene, can be used for sensitive detection of biomarked bacteria. Alternatively, the bioluminescent reaction of the firefly luciferase enzyme can be used as: 1) an in vivo indicator of metabolic activity of the cell since luciferase activity is dependent on cellular energy reserves, or 2) an in vitro assay (supplemented with ATP and substrate). The in vitro assay of luciferase activity is independent of the energy status of the cell. The in vivo fluorescence of the green fluorescent protein is also independent of cellular energy reserves or co-factors.Methods were developed for specific detection of bacteria with luc or gfp biomarkers in soil and sediment samples. Complementary methods were used for quantitative monitoring of bacteria based on: DNA sequence, cellular activity, cellular protein content and total numbers of cells. All methods developed were independent of traditional cultivation techniques.Different quantitative approaches were developed and applied for monitoring of specific bacteria with environmental relevance. For example, luc-tagged cyanobacteria were quantitated in Baltic Sea sediment by competitive PCR amplification of the luc biomarker. Luciferase activity, expressed by the luc biomarker, was quantitated by luminometry. In addition, Arthrobacter chlorophenolicus A6 cells were tagged with gfp or luc biomarkers and quantitated during degradation of high concentrations of 4-chlorophenol in soil. As the cells bioremediated the soil, the metabolic activity of the Arthrobacter population was monitored as bioluminescence by luminometry and the total number of fluorescent Arthrobacter cells were enumerated by flow cytometry. In conclusion, these quantitative approaches, based on biomarkers, were demonstrated to be useful for accurate monitoring of specific cells in environmental samples.

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