Energetics of citrate transport through the plasma membrane of Penicillium simplicissmum. A study using plasma membrane vesicles

Penicillium simplicissimum is a heavy metal tolerant soil fungus, which can be used in biohydrometallurgical leaching processes to solubilize metals from industrial wastes and low-grade ores. Metal solubilization is brought about by excreted organic acids, mainly citric acid. Biohydrometallurgical processes for the winning or recycling of metals are less polluting and need less energy than traditional metal winning processes. Preliminary results gained with Penicillium simplicissimum indicated that the transport step across the plasma membrane may influence the excretion of citrate and thus the metal leaching rate. Neither with Penicillium simplicissimum nor with any other filamentous fungus has the citrate transport across the plasma membrane been investigated in detail up to now. Only indirect results have been gained about this transport because whole hyphae were used for the investigations. For this reason, this question is still an unsolved problem, not only with Penicillium simplicissimum, but also, in general, within fungal physiology, because the excretion of organic acids is a frequently occuring phenomenon - in terrestrial environments as well as in laboratory cultures. To investigate the mode and and energetics of citrate transport plasma membrane vesicles must be used. Only the vesicular system will lead to reliable results about the mode of citrate transport across the plasma membrane. Therefore it is our aim within this project to study for the first time with the filamentous fungus Penicillium simplicissimum the mode and energetics of citrate transport using purified plasma membrane vesicles. Penicillium simplicissimum is used because (i) we want to improve the metal leaching process with Penicillium simplicissimum, and (ii) we carried out a lot of preliminary research with this fungus (for instance, we established a method to purify plasma membrane vesicles). We expect a twofold benefit from this project. First, understanding the transport step across the plasma membrane may help to increase citrate excretion by Penicillium simplicissimum. This is a prerequisite to make the biohydrometallurgical leaching process competitive with traditional metal winning processes. Second, the knowledge of how citrate is transported through the plasma membrane of Penicillium simplicissimum will give us hints about this transport process also in other fungi, for instance in Aspergillus niger, which is used for the biotechnological production of citrate.

The most important achievement of this project is an improved method for the purification of plasma membranes from so-called "filamentous" fungi. Why is this important? "Filamentous" fungi - a colloquial term for fungi without sexual states of reproduction - have a unique life style, which discriminates them distinctly from both plants and animals. These fungi play a key role in forestry and agriculture, in human nutrition and health, and are indispensable model organisms in basic biological research. The impact of these fungi on ecological processes and human affairs can hardly be overestimated. Additionally, many fungi belonging to this group are used in biotechnological processes, for instance for the production of citric acid and enzymes. The plasma membrane is the most important barrier between the outside and the inside of a cell. Proteins embedded into the plasma membrane regulate the uptake of nutrients and the excretion of metabolic waste products. There is almost no function within a cell, which does not depend somehow on the transport of substances across this membrane. To understand how a fungal cell works during growth or during the biotechnological production of a metabolite, the study of the plasma membrane and of the functions of its proteins is therefore important. To be able to study the plasma membrane, one needs a purified preparation of this membrane, which is not contaminated with the other membrane types of the cell. To improve such a purification method was one result of this project. And using this purification method we began a characterization of the excretion of citrate by the filamentous fungus Penicillium simplicissimum. Via the excretion of citrate this fungus is able to solubilize metals from solid substrates and thus to contribute to the recycling of metal-containing industrial wastes or to the winning of metals from low-grade ores.