Abstract
BackgroundMany 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.ResultsWe 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.ConclusionsWe concluded that for P. ochrochloron this technique is a reliable sample processing method for intracellular metabolite analysis, which might offer also other possible applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-2649-8) contains supplementary material, which is available to authorized users.
Highlights
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
Our efforts resulted in the development of the FiltRes-principle, which combines the cold-filtration step and the subsequent rapid resuspension of the mycelium in the hot extraction solution in one single device
The FiltRes-principle combines the cold-filtration step and the subsequent rapid resuspension of the resulting filter cake in the extraction solution in one single device. Unique to this technique is the injection of the extraction solution from below the membrane filter, which causes the entire mycelial filter cake to detach from the filter and float upwards
Summary
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. This methodological adaptation might even be necessary on a phenotypic level since changes in physiology, plasma membrane and cell wall composition can affect the organism’s response to the applied methods (da Luz et al 2014; van Gulik et al 2013; Zakhartsev et al 2015) These findings led to a series of critical evaluations and improvements of techniques for rapid sampling, quenching, separation of biomass, extraction of metabolites and evaporation (e.g. Bolten and Wittmann 2008; de Jonge et al 2012; Douma et al 2010; Schaub et al 2006; VillasBôas et al 2005; Zakhartsev et al 2015)
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