Institute of Microbiology, Leopold-Franzens-University of Innsbruck, Technikerstrasse 25, 6020 Innsbruck, Austria1
Author for correspondence: Martin Gallmetzer. Tel: +43 512 507 6015. Fax: +43 512 507 2929. e-mail: martin.gallmetzer{at}uibk.ac.at
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ABSTRACT |
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Keywords: overflow metabolism, continuous culture, chemostat, nutrient limitation, biomass yield
Abbreviations: µ, specific growth rate (h-1); mCO2X, maintenance coefficient for carbon dioxide (molg-1 h-1); mGlcX, maintenance coefficient for glucose (gg-1 h-1); mO2X, maintenance coefficient for oxygen (molg-1 h-1); qCO2, rate of carbon dioxide production (mmolg-1 h-1); qGlc, rate of glucose consumption (mmolg-1 h-1); qO2, rate of oxygen consumption (mmolg-1 h-1); RQ, respiratory quotient (CO2/O2); SHAM, salicylhydroxamic acid; TCA, tricarboxylic acid; YCO2X, biomass yield on carbon dioxide (gmol-1); YGlcX, biomass yield on glucose (gg-1); YO2X, biomass yield on oxygen (gmol-1)
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INTRODUCTION |
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The most important biotechnological application of fungal overflow metabolism is the production of citric acid by Aspergillus niger (Kristiansen et al., 1999 ). However, the excretion of intermediates of the tricarboxylic acid (TCA) cycle takes place in every culture of a filamentous fungus growing in a medium with a high C:N ratio. Because the main part of these intermediates is excreted during the growth phase and not during the stationary phase [for A. niger see Wallrath et al. (1992)
, McIntyre & McNeil (1997)
and Krzystek et al. (1996)
; for Penicillium simplicissimum see Burgstaller et al. (1994)
], the method of choice to study the physiology of this excretion process is continuous cultivation.
We used continuous cultivation to investigate the excretion of TCA cycle intermediates by P. simplicissimum, a filamentous fungus which excretes citrate and which has been used in the leaching of heavy metals from industrial wastes and ores (Burgstaller et al., 1992 ). Because growth conditions are difficult to control in leaching processes, we aim to understand the overflow metabolism in P. simplicissimum in as much detail as possible, to increase the yield of the leaching agent.
In a previous article, we presented results concerning only citrate excretion by P. simplicissimum (Gallmetzer & Burgstaller, 2001 ). To understand the energetic situation as a whole, however, the total excreted carbon must be considered. In this article we report on the excretion of all TCA cycle intermediates by P. simplicissimum, on the effects of ammonium, nitrate and phosphate limitation on the excretion of TCA cycle intermediates, and on the effects of the presence of benzoic acid, salicylhydroxamic acid (SHAM) and 2,4-dinitrophenol on the excretion of TCA cycle intermediates. Additionally, we compare our results with overflow metabolism in other micro-organisms. Finally, we suggest a general strategy to increase the excretion of a primary metabolite.
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METHODS |
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Chemostat.
All chemostat experiments were performed in a Biostat M bioreactor (Braun) at 30 °C, 7001000 r.p.m. and 12 v.v.m. [vol. air (vol. fermentation liquid)-1 min-1], with a working volume of 1700 ml. The conditions for the bioreactor runs were as described previously (Gallmetzer & Burgstaller, 2001 ).
The chemostat was inoculated with 100 ml of the filamentous pre-culture and processed in the batch mode overnight. Then the chemostat mode was started by switching on the medium feed and the alkali pump. Between four and six generation times were allowed to pass for the culture to reach the steady state. Samples were taken every one to four generation times (four samples per steady state).
The results presented in this study are from single chemostat runs.
Analytical methods.
Sampling and dry weight determination of the mycelium were performed as described previously (Gallmetzer et al., 1998 ). Extracellular organic acids and glucose were determined by HPLC (Burgstaller et al., 1994
). The specific rate of O2 consumption was determined according to Rane & Sims (1994)
or measured with a respirometer (BioMetric Systems). CO2 production was also measured with the respirometer. The elemental composition of the biomass was determined by the Austrian Research Centers, Seibersdorf.
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RESULTS |
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Glucose-limited growth
The elemental composition of the biomass when grown under glucose limitation was CH1·79O0·44N0·11P0·004. Very low amounts of TCA cycle intermediates were excreted under glucose limitation. Neither a rise in the extracellular osmolarity nor in the extracellular pH increased the excretion of organic acids, as was observed with ammonium-limited hyphae (Gallmetzer & Burgstaller, 2001 ). The relationship between µ and qGlc, qO2 and the rate of CO2 production [qCO2 (mmol g-1 h-1)] is shown in Fig. 1
. With these data we calculated the maintenance coefficients (m) and the maximum yield coefficients (Y) for glucose, O2 and CO2 (Tables 1
and 2
) using the linear equation for substrate consumption (Pirt, 1965
). A comparison of P. simplicissimum Y and m values with those gained from other filamentous fungi, which were grown under glucose-limited conditions, shows that despite the different media used by the different authors the values are similar for all fungi tested (Tables 1
and 2
).
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In phosphate-limited hyphae (CH1·85,O0·37,N0·053,P0·002), the overall rate of metabolism was decreased (qGlc, qCO2 and qO2) but the excretion of TCA cycle intermediates was the highest under all tested conditions (Figs 2 and 3
).
The nature of the limiting nutrient determined the relationship between the productivity of TCA cycle intermediate excretion and the specific growth rate (Fig. 5). Under ammonium limitation this relationship corresponded to the Pirt model of catabolic product synthesis (Pirt, 1993
), whereas under phosphate limitation the LuedekingPiret model (Luedeking & Piret, 1959
) was more suitable. Overall, YGlcX seemed to be the most important factor, because it was inversely correlated with the degree of overflow metabolism in P. simplicissimum (data not shown).
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DISCUSSION |
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Ammonium-limited hyphae showed a higher qGlc, qCO2 and qO2 compared to glucose-limited hyphae: 0·69 mmol g-1 h-1 additional carbon was taken up, 0·64 mmol g-1 h-1 additional CO2 was produced, 0·29 mmol g-1 h-1 additional O2 was consumed, and 0·36 mmol g-1 h-1 carbon was excreted in the form of organic acids (at µ=0·1 h-1). The sum of CO2 plus organic acids is higher than the amount of additional consumed carbon. This can be explained as follows. Ammonium-limited hyphae had a lower nitrogen (see biomass formulae) and protein content [27·6% (w/w) versus 43·5% (w/w) in glucose-limited hyphae]. Because of this, less glucose was needed for protein synthesis (precursor formation and energy). In this situation, part of the surplus glucose was oxidized to CO2 and part was spilled via the excretion of intermediates.
The low O2 consumption under phosphate limitation was obviously caused by the limitation of phosphate and thus of ATP synthesis [the phosphorus content of the mycelium was 0·32% (w/w) compared to 0·58% (w/w) in ammonium-limited hyphae]. This bottle-neck, together with a high glycolytic flux (i.e. a high qGlc; Fig. 2), led to a high excretion of intermediates, mainly in the form of citrate. Phosphate limitation is also important for citric acid excretion by A. niger (Zhang, 1999
).
Nitrate-limited hyphae showed a qGlc and qO2 similar to phosphate-limited hyphae. However, nitrate-limited hyphae excreted only low levels of intermediates. Unlike phosphate limitation, the glycolytic flux must have been low because a lot of NADPH was needed for the reduction of nitrate to amino-nitrogen. Comparing ammonium- and nitrate-limited growth, we can conclude that a high glycolytic flux is a general prerequisite for a high excretion rate of TCA cycle intermediates.
If ammonium-limited hyphae were challenged with benzoic acid at pH 5, qGlc, qO2 and qCO2 increased. This increase must have been due to a higher energy demand, because benzoic acid uncouples the proton gradient at the plasma membrane (Verduyn et al., 1992 ). Unlike phosphate limitation, it was not a bottle-neck that increased the excretion of intermediates, but the unequal rates of glycolysis, the TCA cycle and the respiratory electron-transport chain. Of the additional consumed carbon, 33% was oxidized to CO2 and less than 5% was excreted in the form of organic acids. Another uncoupler, 2,4-dinitrophenol, decreased the excretion of intermediates (similar to the observation in A. niger; Netik et al., 1997
).
That SHAM did not change the qGlc and qO2 but increased YGlcX by 23% can be explained as follows. Because more biomass was formed per glucose consumed, more ATP must have been synthesized. Part of the flow of electrons must therefore have been redirected from non-proton-pumping respiration to proton-pumping respiration. This implies that (i) the synthesis of biomass was limited by ATP synthesis, and (ii) during glucose-sufficient growth non-proton-pumping, alternative respiration was a constitutive part of respiration.
Further conclusions can be drawn from the results gained from these experiments with P. simplicissimum. First, because citrate was always excreted in highest amounts (Fig. 3), we can term this an energy-spilling process, which avoids the synthesis of too much NADH or ATP, respectively. Second, overflow metabolism in P. simplicissimum was inversely correlated with YGlcX, but not with the biomass yield on O2 (YO2X) (Table 3
). This is in contradiction to Linton (1991)
, who stated that rapid metabolite production is inversely related to YO2X.
If we compare overflow metabolism in P. simplicissimum with overflow metabolism in other micro-organisms, we find common themes but different features. One common feature is that no intermediates are excreted if the energy source is the limiting nutrient. Another similarity is that an increase in the concentration of the carbon and energy source increases the excretion of intermediates [Liu (1998) ; Klebsiella aerogenes (Klebsiella pneumoniae), Hueting & Tempest (1979)
; A. niger, Papagianni et al. (1999)
; Methanobacterium thermoautotrophicum, Liu et al. (1999)
]. We observed the same with P. simplicissimum if we increased the glucose concentration in the medium reservoir from 100 to 400 mM (data not shown).
Nutrient limitations other than glucose increased qGlc and qO2 in K. aerogenes much more than in P. simplicissimum, the qO2 of K. aerogenes being highest under phosphate limitation (Neijssel & Tempest, 1975 ). This is a clear difference to the situation in P. simplicissimum. The catabolic pathways seem to be more strictly regulated in P. simplicissimum than in K. aerogenes. With both K. aerogenes (Neijssel & Tempest, 1975
) and Escherichia coli (Holms, 1996
), pyruvate and/or acetate are the major excreted metabolites, whereas in P. simplicissimum these intermediates did not play an important role.
The most interesting micro-organisms for comparison with P. simplicissimum are clearly other filamentous fungi. If we look at the sum of the excreted acids, the relationship between µ and the excretion of TCA cycle intermediates is similar in ammonium-limited hyphae of P. simplicissimum, Aspergillus foetidus (Kristiansen & Sinclair, 1979 ) and A. niger (Kristiansen & Charley, 1981
). However, looking at only citrate excretion under either ammonium limitation (Gallmetzer & Burgstaller, 2001
) or phosphate limitation (Fig. 5
) shows that other relationships between µ and productivity are possible. In A. niger, the respiratory quotient (RQ) during citrate production in the growth phase is always distinctly lower than 1 (Foster, 1949
; Kubicek et al., 1980
; McIntyre & McNeil, 1997
; all authors used batch cultivation). In P. simplicissimum, an RQ <1 was only observed under phosphate limitation (0·7, Table 3
). As is assumed for A. niger, a high activity of CO2-fixing anaplerotic reactions would explain this low RQ. The RQ of 1·3 found under ammonium limitation is obviously not an indication of a fermentative metabolism, but seems to be a consequence of the overflow metabolism. In both fungi, i.e. A. niger and P. simplicissimum, the so-called alternative respiration seems to be constitutive during growth (Kubicek et al., 1980
; Wallrath et al., 1992
; Kirimura et al., 1987
).
The rate of glucose consumption by P. simplicissimum depended strongly on the specific growth rate, but only weakly on the kind of nutrient limitation (Figs 1 and 2
). In contrast, qO2 and qCO2 varied much more with the kind of nutrient limitation. This means that the hyphae took up as much carbon as the growth rate allowed and then adjusted the activity of catabolic pathways to meet the different demands of the different nutrient limitations. Maintaining a high flux of the carbon and energy source is probably a characteristic feature of soil micro-organisms (Teixeira de Mattos & Neijssel, 1997
). High metabolic fluxes serve as an adaptation to low nutrient concentrations in order to maximize the growth rate (Westerhoff et al., 1982
). A high flux means that the energetic efficiency of the overall metabolism is lowered (Teixeira de Mattos & Neijssel, 1997
). Additionally, soil micro-organisms were never forced to develop a control of glucose uptake at high glucose concentrations, because of the low carbon concentration in soil. Both features result in the necessity to spill carbon and energy during growth at a high carbon concentration. The advantage of an overflow metabolism compared to other energy-spilling processes (such as alternative respiration, futile cycles) would be that the synthesis of too much NADH is avoided.
Thermodynamically viewed, a high flux is coupled with a lower efficiency of energy conversion (Teixeira de Mattos & Neijssel, 1997 ; Westerhoff et al., 1982
). This could, in principle, explain an uncoupling of catabolism and anabolism at high rates of carbon flux (Westerhoff et al., 1982
). However, it is not clear if this concept which has been developed from physically coupled flows, such as oxidative phosphorylation (Stucki, 1980
) can be applied to the uncoupling of catabolism and anabolism, where the mechanistic kind of coupling is unclear. It is probably more reasonable to look for a physiological explanation for overflow metabolism than for a thermodynamic explanation.
From the results presented, a general strategy to increase the excretion of intermediates of primary metabolism can be suggested: (i) look for the kind of nutrient limitation which gives the highest excretion rate; (ii) with this nutrient limitation determine the growth rate for maximum productivity; and (iii) maximize the non-growth energy consumption (e.g. by low potassium concentration, high pH, high osmolarity, addition of benzoic acid, etc.). Additionally, if glucose uptake is limiting (which is not the case with P. simplicissimum) an auxiliary carbon source can be added (Babel et al., 1993 ). This strategy should be valid in general for increasing an overflow metabolism in a filamentous fungus, although it will be modulated by the unique genotype of each strain.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 23 August 2001;
revised 28 November 2001;
accepted 29 November 2001.
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