Fate of Pseudomonas putida after Release into Lake Water Mesocosms: Different Survival Mechanisms in Response to Environmental Conditions

To study the fate of Pseudomonas putida DSM 3931 in an aquatic environment, cultures of the strain were released into lake water mesocosms. P. putida, bearing the TOL-plasmid, was released as a representative xenobiotic-degrading microorganism. The release was carried out in mesocosms with unamended lake water and in lake water with added culture medium to compare the survival of the strain due to the influence of different organic load. As a comparison, the survival of P. putida was followed in microcosms with sterile lake water. Survival and fate of the strain were determined by means of immunofluorescence with highly specific monoclonal antibodies and growth on selective agar medium for up to ten weeks after release. Addition of medium had a pronounced influence on survival in mesocosms. In mesocosms without added medium, the number of P. putida cells decreased within ten days by over 2 orders of magnitude. In mesocosms with medium, cell numbers increased in the first two days by an order of magnitude and were, after ten days, in the same range as at the time of introduction. Over time, cell numbers decreased but remained detectable in both types of mesocosms for up to ten weeks after release. In mesocosms with unamended lake water, the major fraction of the cells was attached to particles after two days. In mesocosms with medium, large aggregates of P. putida cells formed which included algae. The observed decrease in cell numbers in mesocosms was attributed mainly to grazing. Sedimentation was an additional factor contributing to loss of cells out of the water column, which especially affected aggregate-forming cells in mesocosms with medium in the long run (beyond two weeks). These studies demonstrate that experimental tools on a mesoscale are crucial in order to understand the complex processes microorganisms are subjected to after release into a natural environment, and that single cell detection, such as immunofluorescence, is essential to understand mechanisms of survival and elimination.