New method for assimilable organic carbon determination using flow-cytometric enumeration and a natural microbial consortium as inoculum

The concentration of easily assimilable organic carbon (AOC) largely determines the microbiological stability of drinking water. However, AOC determination is often neglected in practice due to the complex and tedious nature of the conventional bioassay. The three major drawbacks of the conventional method are (1) a long assay time of 9-12 days, (2) the use of a labor-intensive enumeration technique (plating on growth media), and (3) limited information supplied by the use of selected pure cultures (Pseudomonas fluorescens P-17 and Spirillum NOX) for measuring a complex pool of natural bioavailable carbon compounds. A new method is proposed here, in which plating was replaced with fluorescence staining of total nucleic acids combined with flow cytometry as a rapid and straightforward growth enumeration method. This approach also allowed for the detection of inactive and/or unculturable microorganisms. Hence, the conventionally used pure cultures were replaced in the new AOC assay with a natural microbial consortium. It was shown that the flow-cytometric enumeration method could be used to establish complete growth curves for a natural microbial consortium growing on AOC. Compared to the end-point measurements of the conventional method, such kinetic data provide much clearer insight into the actual growth potential of a water.