We are investigating a methanotrophic filter strategy for the in situ bioremediation of low levels of chlorinated aliphatic, volatile organic chemicals (VOCs). It is based on the use of pregrown, resting cells, instead of growth-nutrient stimulations. The economic feasibility of such a filter is dependent on its operational longevity at groundwater temperatures. The latter, in turn, is dependent on several key parameters, such as the bacterial attachment densities reached during the injection of the microbial suspension and the subsequent detachment-removal of cells from the filter over time. Scaled attachment/ detachment experiments were carried out using a representative quartzitic sand in glass 1-cm x 10-cm columns to simulate a filter. A rosette-dominated form of Methylosinus trichosporium OB3b was isolated and used in these and the subsequent catalytic longevity experiments. Its initial attachment, employing Higgins’ medium phosphate buffer, pH 7.0 (HPB), was 7.0 to 8.0 x 10(8) bacteria/g of dry sand. This was elevated to similar to 1.5 x 10(9) cells/g by including 1.0 mM MgCl2, 100 mu M FeSO4, and 0.025% agar in the cell-suspension loading buffer. These loading additives also increased the time required to reach 50% cell detachment with HPB alone from 5 days to similar to 45 days. The functional longevity of a column biofilter, formed with resting-state rosette-enriched cells in the presence of the aforementioned additives, was determined at 21 degrees C by challenging it with weekly 12 h, similar to 250 ppb pulses of trichloroethylene (TCE). The column results indicate that for our attached-cell filter to biodegrade TCE levels of several hundred ppb sufficiently, to <5 ppb, it will likely need replenishment at similar to 8 week intervals, due to the instability of the endogenous whole-cell soluble methane monooxygenase specific activity beyond that time period. This study represents the first time that anyone has shown that a rosette-enriched substrain can be isolated from a well-known methanotrophic strain and then stably cultured and utilized advantageously for a specific application-namely its improved attachment-slowed detachment characteristics in a microbial filter.