Organic Acid Excretion in Penicillium ochrochloron Increases with Ambient pH

Despite being of high biotechnological relevance, many aspects of organic acid excretion in filamentous fungi like the influence of ambient pH are still insufficiently understood. While the excretion of an individual organic acid may peak at a certain pH value, the few available studies investigating a broader range of organic acids indicate that total organic acid excretion rises with increasing external pH. We hypothesized that this phenomenon might be a general response of filamentous fungi to increased ambient pH. If this is the case, the observation should be widely independent of the organism, growth conditions, or experimental design and might therefore be a crucial key point in understanding the function and mechanisms of organic acid excretion in filamentous fungi. In this study we explored this hypothesis using ammonium-limited chemostat cultivations (pH 2–7), and ammonium or phosphate-limited bioreactor batch cultivations (pH 5 and 7). Two strains of Penicillium ochrochloron were investigated differing in the spectrum of excreted organic acids. Confirming our hypothesis, the main result demonstrated that organic acid excretion in P. ochrochloron was enhanced at high external pH levels compared to low pH levels independent of the tested strain, nutrient limitation, and cultivation method. We discuss these findings against the background of three hypotheses explaining organic acid excretion in filamentous fungi, i.e., overflow metabolism, charge balance, and aggressive acidification hypothesis

Adapting High-Resolution Respirometry to Glucose-Limited Steady State Mycelium of the Filamentous Fungus Penicillium ochrochloron: Method Development and Standardisation.

Fungal electron transport systems (ETS) are branched, involving alternative NADH dehydrogenases and an alternative terminal oxidase. These alternative respiratory enzymes were reported to play a role in pathogenesis, production of antibiotics and excretion of organic acids. The activity of these alternative respiratory enzymes strongly depends on environmental conditions. Functional analysis of fungal ETS under highly standardised conditions for cultivation, sample processing and respirometric assay are still lacking. We developed a highly standardised protocol to explore in vivo the ETS-and in particular the alternative oxidase-in Penicillium ochrochloron. This included cultivation in glucose-limited chemostat (to achieve a defined and reproducible physiological state), direct transfer without any manipulation of a broth sample to the respirometer (to maintain the physiological state in the respirometer as close as possible to that in the chemostat), and high-resolution respirometry (small sample volume and high measuring accuracy). This protocol was aimed at avoiding any changes in the physiological phenotype due to the high phenotypic plasticity of filamentous fungi. A stable oxygen consumption (< 5% change in 20 minutes) was only possible with glucose limited chemostat mycelium and a direct transfer of a broth sample into the respirometer. Steady state respiration was 29% below its maximum respiratory capacity. Additionally to a rotenone-sensitive complex I and most probably a functioning complex III, the ETS of P. ochrochloron also contained a cyanide-sensitive terminal oxidase (complex IV). Activity of alternative oxidase was present constitutively. The degree of inhibition strongly depended on the sequence of inhibitor addition. This suggested, as postulated for plants, that the alternative terminal oxidase was in dynamic equilibrium with complex IV-independent of the rate of electron flux. This means that the onset of activity does not depend on a complete saturation or inhibition of the cytochrome pathway

Hypothesized electron partitioning between COX and AOX.

<p>The safety valve hypothesis suggests two distinct states: (a) Electrons are not drained to AOX if the cytochrome pathway is not operating to full extent. (b) Electrons flow to AOX in situations of a fully saturated or blocked cytochrome pathway. (c) The dynamic inter-dependence hypothesis in contrast excludes a regulation of AOX activity solely by the degree of saturation or a blockage of the cytochrome pathway. Electron partitioning is regulated dynamically by different metabolic demands.</p

Representative original respirometer traces illustrating the standardised respirometric assay with glucose-limited steady state mycelium of <i>Penicillium ochrochloron</i>.

<p>For the assays steady state mycelium was used and resuspended in the respiration medium. The assay consisted in total of six single measurements in three runs with two chambers, respectively (blue line, y₁ axis: oxygen concentration; red line, y₂ axis: oxygen consumption rate). Marked sections (red boxes) indicate data range for the calculation of averaged fluxes. (a and b) Assay 1: effect of uncoupler and solvent. (c and d) Assay 2: effect of SHAM and cyanide, with either SHAM prior to or after cyanide. (e and f) Assay 3: identical to assay 2, but 24 hours later for validating reproducibility and monitoring the stability of chemostat culture.</p

Multiple convergent pathways for electrons into the quinone pool of fungal mitochondria.

<p>Electrons flowing downstream the quinone pool are drained into the cytochrome pathway (complex III and IV) or via an alternative route to AOX. Arrows indicate the direction of electron fluxes downstream the thermodynamic cascade of the ETS.</p

Oxygen consumption of glucose-limited steady state mycelium of <i>Penicillium ochrochloron</i>.

<p>Oxygen consumption in the presence of SHAM (AOX inhibitior), cyanide (COX inhibitior) and CCCP (uncoupler of mitochondrial proton gradient). The oxygen consumption rates were normalized to the steady state oxygen consumption rate without inhibitors. The bars represent mean and standard deviation of three separate steady state cultivations (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146878#pone.0146878.s003" target="_blank">S1 Table</a> Off-line respirometry). AOX was inhibited with 2.5 mM SHAM, COX with 1 mM cyanide, and uncoupling was done with 3 μM CCCP. The mean residual oxygen consumption was calculated with both inhibitors present.</p