Enumeration and factors influencing the relative abundance of a denitrifier, Pseudomonas sp. JR12, entrapped in alginate beads

The relative abundance of the denitrifier, Pseudomonas sp. JR12, was examined in an alginate-based entrapment complex under non-sterile, denitrifying conditions. Immuno-labeling of the Pseudomonas inoculant followed by flow cytometry (FCM) was used for determination of the relative abundance of this bacterium under the various incubation conditions. Additional information on the relative abundance of the inoculant was obtained by a quantitative enzyme-linked immunosorbent assay (ELISA) and results obtained by FCM and ELISA were compared. Ambient nitrate levels controlled the successful, long-term proliferation of the inoculant. At low ambient nitrate levels, Pseudomonas sp. remained the dominant microorganism during incubation. Higher ambient nitrate concentrations, attained by either decreasing the inoculum size of Pseudomonas sp. or raising inlet nitrate concentrations of the medium supplied to the incubation vessels, resulted in a gradual shift toward other, nitrite-accumulating denitrifiers. Thus far, most studies on the use of entrapped microorganisms for bioremediation purposes have been conducted under controlled laboratory conditions. Based on this study, conducted under non-sterile laboratory conditions, it is concluded that in-situ bioremediation using entrapped target microorganisms is bound to fail without a proper understanding of the factors that cause the target microorganism to out-compete undesired microbial invaders. Furthermore, based on the close agreement between the two detection methods used, it is concluded that flow cytometry provides a rapid and accurate tool for the detection of the relative abundance of immuno-labeled target organisms in heterogeneous microbial populations.