Abstract
BACKGROUND: Many issues concerning sample processing for intracellular metabolite studies in filamentous fungi still need to be solved, e.g. how to reduce the contact time of the biomass to the quenching solution in order to minimize metabolite leakage. Since the required time to separate the biomass from the quenching solution determines the contact time, speeding up this step is thus of utmost interest. Recently, separation approaches based on cold-filtration were introduced as promising alternative to cold-centrifugation, which exhibit considerably reduced contact times. In previous works we were unable to obtain a compact pellet from cold methanol quenched samples of the filamentous fungus Penicillium ochrochloron CBS 123.824 via centrifugation. Therefore our aim was to establish for this organism a separation technique based on cold-filtration to determine intracellular levels of a selected set of nucleotides. RESULTS: We developed a cold-filtration based technique as part of our effort to revise the entire sample processing method and analytical procedure. The Filtration-Resuspension (FiltRes) device combined in a single apparatus (1) a rapid cold-filtration and (2) a rapid resuspension of the biomass in hot extraction solution. Unique to this is the injection of the extraction solution from below the membrane filter (FiltRes-principle). This caused the mycelial cake to detach completely from the filter membrane and to float upwards so that the biomass could easily be transferred into preheated tubes for metabolite extraction. The total contact time of glucose-limited chemostat mycelium to the quenching solution could be reduced to 15.7 ± 2.5 s, whereby each washing step added another 10-15 s. We evaluated critical steps like filtration time, temperature profile, reproducibility of results, and using the energy charge (EC) as a criterion, effectiveness of enzyme destruction during the transition in sample temperature from cold to hot. As control we used total broth samples quenched in hot ethanol. Averaged over all samples an EC of 0.93 ± 0.020 was determined with the FiltRes-principle compared to 0.89 ± 0.049 with heat stopped total broth samples. CONCLUSIONS: We concluded that for P. ochrochloron this technique is a reliable sample processing method for intracellular metabolite analysis, which might offer also other possible applications.