Elsevier

Journal of Biotechnology

Volume 112, Issue 3, 9 September 2004, Pages 289-297
Journal of Biotechnology

A drop of intracellular pH stimulates citric acid accumulation by some strains of Aspergillus niger

https://doi.org/10.1016/j.jbiotec.2004.05.002Get rights and content

Abstract

By comparing kinetic parameters of plasma membrane proton pumps from two Aspergillus niger strains, significant differences in specific activities were observed. In low citric acid producing A158 strain the H+-ATPase activity was about four-fold higher than in a high yielding A60 strain. Previously pH homeostasis was reported in A158 strain while in A60 strain spontaneous drop of intracellular pH was observed. During the growth in the medium with ammonium ions more rapid drop of extracellular pH was recorded with A158 strain and not so fast proton accumulation in the medium with A60 strain, indicating that proton pumps from later strain perhaps can not extrude all the protons that are released in the cytosol after the assimilation of ammonium ions. Vanadium ions were found to be potent inhibitors of both H+-ATPases. By adding sodium vanadate in millimolar concentrations to the chemically defined medium that induces citric acid accumulation by A. niger, reduced pHi and increased rate of acid production was observed in A158 strain while in A60 strain intracellular pH decreased below 6.5 and concomitantly citric acid overflow was suppressed. The presented results suggest that one of the mechanisms stimulating citric acid accumulation by A. niger could be also a slight cytoplasmic acidification.

Introduction

Filamentous fungus Aspergillus niger belongs to microorganisms of extreme biotechnological importance since it is used for production of various primary metabolites (organic acids) and enzymes. In fact citric acid production by this fungus is one of the most efficient bioprocesses in terms of productivity, since A. niger can convert up to 80% of the substrate to the final product. Therefore, understanding the regulation of metabolism under high citric acid yielding conditions can contribute to further strain improvement or even to transfer some properties to other commercial microorganisms.

Intracellular pH has often been neglected as an important parameter determining the activity of enzymes and overall metabolism in industrial strains, yet there are some reports about the relationship between intracellular pH and biosynthetic capacity in fungi. By 31P NMR and 13C NMR glucose and xylose metabolism with respect to ethanol production was studied in Saccharomyces cerevisiae and Pichia stipitis. In bakers yeast the intracellular pH and the rate of glucose metabolism and ethanol production were the same under aerobic and anaerobic conditions. In contrast, in P. stipitis cytoplasmic pH of well oxygenated cells was more alkaline than that of non-oxygenated cells, while ethanol production was slightly lower in aerobic suspension (Lohmeier-Vogel et al., 1996). In the fungal cells of Fusidium coccineum higher antibiotic biosynthesis was recorded at pH values of cytoplasm in the range of 7.0–7.5 then at slightly more alkaline values of 7.3–7.9 (Shipanova et al., 1995). Even in A. niger intracellular pH was measured under citric acid yielding conditions, however the results obtained by two different strains were conflicting. In A60 strain significant drop of pHi from 7.0 to about 6.5 during the initial phase of growth was recorded (Legiša and Kidrič, 1989) while in another acid accumulating strain N131, which is a derivative of N400 (CBS120.49) strain (Ruijter et al., 2000) constant cytoplasmic pH of 7.6 was observed under similar growth conditions (Hesse et al., 2002).

The enzyme that plays a key role in regulating intracellular pH homeostasis and maintenance of a proper ion balance in cells is H+-extruding plasma membrane P-ATPase (Serrano, 1988). For its normal activity the enzyme requires ATP, therefore, intracellular pH is directly linked to cellular energy levels and metabolism (Sureshkumar and Mutharasan, 1993).

In Aspergilus niger the activation of plasma membrane H+-ATPase by the addition of ammonium ions to the medium was observed. It was proposed that activation of proton extrusion is mediated via phosphatidyl-inositol signalling pathway, involving Ca2+/calmoduline-dependent protein kinase (Jernejc and Legiša, 2001).

Under citric acid accumulating conditions ammonium salts are the preferred source of nitrogen. A. niger consumed ammonium very rapidly and it was depleted from the medium within 24–36 h, well before the fungus stopped to grow (Dawson et al., 1988). Moreover, by the work carried out on the production of citric acid using A. foetidus, exhaustion of nitrogen in a form of ammonium nitrate, appeared to be a necessary prerequisite for the commencement of acid accumulation (Kristiansen and Sinclair, 1978).

Due to a very rapid ammonium consumption by A. niger cells in the early stages of growth in high citric acid yielding medium, highly active H+-ATPases seem to be responsible for maintaining pH homeostatis in the cells. Since different intracellular pH values have been reported in two strains of A. niger, basic kinetic properties of partially purified H+-ATPases isolated form both strains have been compared and eventual effect of decreased intracellular pH on the induction of citric acid excretion studied.

Section snippets

Microorganisms

A. niger strains A60 and A158 from the Culture Collection of the National Institute of Chemistry, Ljubljana (MZKI) were used throughout all experiments. A60 strain originates from NRRL 2270 strain, while A158 is identical to CBS 120.49 strain. Spores were harvested from 7 day old agar slants and suspended in sterile 0.1% Tween 80 solution. Approximately 5 × 107 spores were used for inoculation of 100 ml medium and incubated in 500 ml baffled erlenmeyer flasks on a rotary shaker (New Brunswick

Results

During the growth of two selected strains of A. niger in a chemically defined substrate with 15% of saccharose and ammonium ions as a sole nitrogen source differences in specific growth rate were observed. Ammonium was often reported to be consumed by A. niger from similar media very rapidly and after 24–36 h virtually no ammonium ions could be detected in the medium any more (Dawson et al., 1988). After 44 h of growth 5.30 g of dry weight biomass per litre was formed by A158 strain and only 2.93 g l

Discussion

H+-ATPases belong to P2 subfamily of P-type ATPases that couple ATP hydrolysis to the active transport of cations across cell membranes. In S. cerevisiae nine P2-type ATPases were determined that transport a wide array of monovalent and divalent cations including Na+, K+, Mg2+, Ca2+, but only two H+-ATPases were described in yeasts (Morsomme et al., 2000). H+-ATPases are involved in intracellular pH (pHi) regulation and generation of the electrochemical proton gradient across the cytoplasmic

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