Excretion of citrate and other TCA cycle intermediates by Penicillium simplicissimum: A continuous culture study

Filamentous fungi are an important group of micro-organisms in biotechnology. For instance, Penicillin, vitamin B and citric acid are produced with the aid of filamentous fungi. In the past few years new fields of application have been opened up for these fungi. One example is the non-polluting winning of metals from low-grade ores and industrial wastes by organic acids. In this project the regulation of the excretion of organic acids, especially citric acid, is investigated in the heavy metal tolerant fungus Penicillium simplicissimum. Citrate excretion by Penicillium simplicissimum was used for the non-polluting recycling of filter dusts from the metal Industry and combustion furnaces. Penicillium simplicissimum excretes also other organic acids besides citric acid. The spectrum of acids, which are actually excreted, and the excreted amount of each acid depends mainly on the composition of the medium and on other growth parameters such as oxygen supply, pH etc. In this project we investigate the influence of selected growth conditions on the excretion of organic acids by Penicillium simplicissimum. In order to identify the impact of a single component, the concentrations of the other components as well as other cultivation parameters must be held constant. This requirement is only met with the method of ,continuous cultivation". With all other cultivation methods several factors change simultaneously, which does not allow to judge the importance of a single factor. Therefore continuous cultivation is used in this project for investigating the excretion of organic acids. The results of this project will extend our knowledge about the regulation of primary metabolism in filamentous fungi (fundamental research). Additionally, criteria will be gained, which allow to judge the suitability of growth substrates and growth conditions for maximum excretion of organic acids by Penicillium simplicissimum (applied research).

Summary The excretion of citrate by the filamentous fungus Penicillium simplicissimum is increased in particular by a deficiency of phosphor and by additional stress factors, such as a high salt concentration. These findings are a further step towards the biotechnological application of this fungus in environmetal friendly, biohydrometallurgical metal leaching processes. Abridged version of the final report Moulds excrete high amounts of acids if they are cultivated under specific conditions. This phenomenon is exploited, for instance, to produce citric acid with the mould Aspergillus niger on an industrial scale. Another biotechnological application of this phenomenon is the use of acid-excreting fungi for the winning of metals from industrial residues and low grade ores ("biohydrometallurgical leaching processes"). To influence the metabolism of a mould in the direction of increased acid production, we must understand the reasons, which "force" a cell to excrete acids. The greater part of acid excretion is coupled to growth. Therefore, to study this phenomenon one needs a method which allows to vary the growth velocity of the fungus in a controlled manner. This method is the so-called "continuous culture". With this cultivation method the influence of environmental factors and of the growth velocity on acid excretion can be unambigously determined. Until to the start of this project no systematic study of acid excretion by a filamentous fungus using continuous culture has been carried out. This is all the more astonishing as citric acid has been produced with the mould Aspergillus niger since the beginning of the 20th century. Reasons for this are surely that this cultivation method needs a complex equipment and that there are special difficulties if filamentous fungi are grown in continuous culture. We investigated in this project acid excretion by the mould Penicillium simplicissimum. This fungus is common in soils all over the world and is used for metal leaching processes because of its tolerance against heavy metals. Acid excretion by Penicillium simplicissimum is characterized by the following points: (i) acids are excreted if only slow growth is possible and there is simultaneously an excessive supply of sugar; (ii) the amount of excreted acids depends on the specific nutrient which limits growth (a deficiency of phosphorus was most favourable); and (iii) additional stress factors - for instance a high salt concentration in the medium - increased the excretion of acids. The results gained in this project (which are, by the way, throughout transferable to other moulds) are important in two respects. On the one hand, our knowledge about the physiology of this biotechnologically important group of microorganisms is expanded. On the other hand, the metabolism of this fungus can now be influenced in a specific manner in the direction of increased acid production. With that we are a step closer to establish environmentally friendly metal leaching processes as a commercially viable method able to compete conventional metal winning processes.