Studies of the physiological status of bacteria used for environmental applications

Sammanfattning: Today, it is difficult to study the physiological status of inoculants in complex environments, such as soil. In this thesis, we describe the combined use of marker genes and viability stains to determine the physiological status of the bacterial cells in pure cultures and in soil. In the experiments, cells previously tagged with gfp and luxAB or luc marker genes (encoding the green fluorescent protein and bacterial or eukaryotic luciferase, respectively) were used. Viability stains, 5-cyano-2,3-ditolyitetrazolium chloride (CTC) and propidium iodide (M), were used to stain active and dead cells, respectively. The soil bacterial community was extracted using Nycodenz density gradients. The GFP fluorescent and stained cells were monitored by flow cytometry. Luciferase activity was used to monitor metabolic activity of the population. In addition, the number of culturable cells was determined by selective plate counting and compared to the results obtained by flow cytometry in order to estimate the number of dormant cells. The physiological status of Arthrobacter chlorophenolicus A6 (a phenol degrading bacterium) was investigated upon inoculation into natural (uncontaminated) soil microcosms at different temperatures, 5 and 28 'C. Luciferase activity showed that the majority of the A6 population remained metabolically active, after 20 days of incubation in soil at 5 'C. However, there was a fraction of the GFP-fluorescent A6 population that was stained neither with CTC nor PI, presumably indicating a state of dormancy. At 28 'C, a majority of the cells lost their cell integrity, metabolic activity and the ability to be cultured on agar-plates, indicating that the cells were dying. The ability of A. chlorophenolicus A6 to enter a state of dormancy, might be advantageous for the cells during stressful conditions in soil, and makes this strain a good candidate for treating chlorophenol contaminated soil in temperate climates. Pseudomonas fluorescens SBW25, a plant growth promoting bacterium, has been widely studied due to its potential as an inoculum for improving crop yields. Similar to A6, the physiological status was monitored in soil and in pure cultures incubated under different nutritional conditions. Most of the cells died after incubation for nine days in nutrient rich medium. By contrast when incubated under starvation conditions, most of the population was not stained with CTC or PI, indicating that most of the cells were presumably dormant. In soil, a large fraction of the SBW25 cell population became inactive and died, as determined activity and viability staining as well as by selective plate countings. However, approximately 60% of the population was unstained, presumably indicating that the cells entered a state of dormancy in soil similar to that observed under starvation conditions in pure cultures. These results demonstrate the applicability of this approach for monitoring the physiological status of specific cells under stress conditions, such as experienced by environmental inoculants in soil.