Xylose Fermentation by Candida tropicalis - NCBI

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1989, p. 1974-1980 0099-2240/89/081974-07$02.00/0 Copyright © 1989, American Society for Microbiology

Vol. 55, No. 8

31P Nuclear Magnetic Resonance Study of the Effect of Azide on Xylose Fermentation by Candida tropicalis ELKE LOHMEIER-VOGEL,' KERSTIN SKOOG,2 HANS VOGEL,' AND BARBEL HAHN HAGERDAL2* Department of Biological Sciences, University of Calgary, Calgary, Albertca, Canad(a T2N 1N4,' and Department of Applied Microbiology, Lund Universitv, P.O. Box 124, S-221 00 Lulnd, Sweden2 Received 19 December 1988/Accepted 8 May 1989

Maximal ethanol production by Candida tropicalis grown on xylose was obtained at an oxygen transfer rate of 5 to 7 mmol/liter per h. Addition of 0.2 mM azide increased the ethanol yield by a factor of 3 to 4, based on the cell mass produced, and decreased the formation of the by-product xylitol by 80%. In the presence of azide, ethanol was reassimilated before the carbon source was depleted. At all oxygenation levels studied, azide caused 25 to 60% of the carbon to be lost, most probably as carbon dioxide. Identical spectra were obtained with 31P nuclear magnetic resonance spectroscopy performed on extracts of C. tropicalis grown on xylose in the absence and presence of azide. Azide lowered the levels of sugar phosphates. Enzymatic analysis showed extremely low levels of fructose 1,6-diphosphate compared with the levels obtained in the absence of azide, while the level of malate, a citric acid cycle intermediate, was not influenced by azide. 31P nuclear magnetic resonance spectroscopy performed on xylose-grown whole cells of C. tropicalis showed that azide lowered the intracellular pH, inhibited the uptake of external Pi, and decreased the buildup of polyphosphate in relation to results with untreated cells. Similar results were obtained with the uncoupler of oxidative phosphorylation carbonyl cyanide m-chlorophenylhydrazone (CCCP), except that CCCP treatment led to extremely high levels of internal Pi. The dual effect of azide as a respiratory inhibitor and as an uncoupler is discussed with respect to the metabolism and product formation in xylose-assimilating C. tropicalis. Candida tropicalis is one of a number of yeasts which can ferment xylose to ethanol. This requires oxygen, probably owing to a redox imbalance in the first two steps of xylose metabolism, an imbalance previously found with Candida iitilis and Pachysolen tainnophiliis (2). The best ethanol production has been observed under oxygen-limited conditions (8). In a previous study we found that the respiratory inhibitor azide, at 0.2 mM, increased the ethanol yield and decreased the formation of the by-product xylitol (11). The interpretation made was that azide, being a respiratory inhibitor, fine-tuned the oxygenation during xylose fermentation. In the present study we investigated ethanol and xylitol production from xylose by C. tropicalis as a function of the oxygen transfer rate (OTR). There have been many investigations of the oxygen limitation in xylose fermentations as a function of culture shaking rate, culture volume, or vessel volume, or in fermentors in which different aeration rates or stirring speeds were used (for an overview, see reference 19). This does not allow comparisons between different investigations. Use of the OTR to define the oxygen limitation offers a possibility to make such comparisons. The influence of azide on xylose metabolism was studied by 31P nuclear magnetic resonance spectroscopy (NMR) of cell extracts and whole cells of C. tropicalis. Concentrations of intermediary metabolites of the glycolytic pathway and the citric acid cycle were enzymatically determined in batch cultures at constant sugar uptake rate. To our knowledge, no 31P NMR studies have been performed on xylose metabolism in yeasts, although in a recent study, '3C NMR was used to study xylose uptake and product formation by Pichia stipitis (9).

*

MATERIALS AND METHODS Organism. C. tropicalis ATCC 32113 was maintained at 4°C on slants containing the following medium: 10 g of yeast extract per liter, 20 g of Bacto-Peptone (Difco Laboratories) per liter, 8 mM NaH2PO4, and 2 mM K2HPO4, supplemented with 50 g of xylose per liter and 15 g of agar per liter. Cultivation conditions. For OTR studies, cells were grown in the following medium: 5 g of yeast extract per liter, 3 g of Bacto-Peptone per liter, 5 g of hydrolyzed casein per liter, and 2.5 ml of vitamin solution per liter (containing 0.5 g of inositol per liter, 100 mg of riboflavin per liter, 0.1 g of calcium pantothenate per liter, 50 mg of pacra-aminobenzoic acid per liter, 0.1 g of pyridoxine per liter, and 25 mg of biotin per liter). The pH was adjusted to 5.5. Fermentations were performed in 11 baffled shake flasks containing 0.3, 0.4, 0.5, and 0.6 liter of medium. The temperature was kept at 30°C, with shaking at 140 rpm, corresponding to OTR values of 13, 7, 5, and 0.5 mmol/liter per h as estimated by the sodium sulfite oxidation method (4) with modifications as specified by the Annuial Book of the American Society for Testing and Materials (1). When used, azide was added to 0.2 mM. For NMR studies, cells were grown at 30°C on maintenance medium (without agar) supplemented with 50 g of carbon source per liter in unbaffled Erlenmeyer flasks filled to 50% capacity, with shaking at 140 rpm. When used, carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added to 0.029 mM. Analytical methods. Cell growth was estimated from the optical density at 620 nm. Glucose, xylose, xylitol, ethanol, glycerol, acetic acid, and succinic acid concentrations in the media were measured by high-pressure liquid chromatography on an Aminex HPX-87H organic acid analysis column (Bio-Rad Laboratories) as previously described (11). Preparation of perchloric acid extracts. Samples were taken from the C. tropic(alis xylose-fermenting cultures by

Corresponding author. 1974

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EFFECT OF AZIDE ON C. TROPICALIS XYLOSE FERMENTATION

rapid filtration (10) when the sugar uptake rates were constant. Perchloric acid extracts were made as described by Skoog and Hahn-Hagerdal (20). For NMR studies, for which more concentrated extracts were required, 3.5 g (wet weight) of xylose-grown cells of C. tropicalis were suspended in 7 ml (total volume) of morpholineethanesulfonic acid (MES)-salts buffer (pH 6.0) (14). The resulting cell paste was incubated at 30°C in 50-ml Erlenmeyer flasks with shaking at 140 rpm. At time zero, 1.4 ml of carbon source (or buffer) was added. After 25 min, the suspensions were transferred to precooled centrifuge tubes containing 1.4 ml of 70% perchloric acid and processed as above. The samples were then subjected to batch treatment with Chelex and filtration through membrane filters (Millipore Corp.). Finally, EDTA and an internal methylenediphosphonic acid standard were added to give final concentrations of 10 and 0.3 mM, respectively, in the extracts. The extracts were then adjusted to pH 8.4 by the addition of potassium hydroxide and stored frozen until analyzed by NMR. Assay of intermediary metabolites. Fructose 1,6-diphosphate (FDP), phosphoenolpyruvate, and pyruvate were analyzed on a Hitachi 3000 spectrofluorimeter by the method of Lowry and Passonneau (12). Citrate, malate, and fumarate levels were determined by the method of Williamson and Corkey (23). Each metabolite concentration was determined as the mean of six independently filtered, frozen, and extracted samples. The intracellular concentration of the metabolites was calculated from the emission measurements by using the factor of 0.334 g/ml for the ratio of dry weight to volume of the cells (3). The concentrations of intermediary metabolites were recalculated (normalized) with respect to the sugar uptake rate to compare flows through metabolic pathways under different cultivation conditions. Preparation of cell suspensions for NMR work. Mid-log-phase cells were cooled to 4°C on ice with agitation to keep the cells from becoming anaerobic. Once cold, the cells were harvested by centrifugation, washed twice, and suspended in ice-cold MES-salts buffer. The final cell pellet volume was 10% in the wash buffer, D20 was present at 7% (vol/vol), and NaH,P04 was added to give an external Pi (Pi ) concentration of 10 mM. SAG 471 silicon antifoam fluid (Ixarrison & Crosfield Ltd., Canada) was used to control foaming. Oxygen-limited conditions were achieved by inserting a capillary down to the bottom of the NMR tube and bubbling oxygen at a flow rate of 10 cm3/min, about 10 to 20 times lower than the rate used by den Hollander et al. (5) to maintain fully aerobic conditions with Saccharomyces cerei'isiae. No oxygen was detectable with an oxygen electrode when cells metabolized xylose. All experiments contained an external methylenediphosphonic acid standard, set to -18.59 ppm. Calibrations of peak areas by integration were compared with this standard, which was arbitrarily set to 100 integration units (IU). NMR spectroscopy. NMR spectra were obtained on a Bruker AM-400 wide-bore spectrometer, operating in the Fourier Transform mode, with either a 10-mm broad-band probe or a 15-mm 13C-31P switchable dual-tuned probe. 31P-NMR spectra were recorded at 161.8 MHz. The recycle time used for both nuclei was 2.0 s, and the flip angle used was 90°. In all 31p spectra, composite pulse decoupling in a bilevel scheme was used, the decoupler power during acquisition being 2 W. For the experiments involving cell suspensions, 31p spectra were acquired in 5-min blocks. The 31p spectra of the extracts were obtained in the 10-mm probe, 8 h being required to obtain a reasonable signal-to-noise ratio.

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Cell suspensions were run unlocked; extracts were locked on D,O.

RESULTS Product formation as a function of OTR in the absence and presence of azide. Xylose uptake, cell growth, ethanol, and xylitol production in four cultures of C. tropic li/s fermenting xylose at various degrees of oxygenation are summarized in Fig. la to d, respectively. Ethanol production (Fig. lc) reached its highest level (6 g/liter) at an OTR of 5 to 7 mmol/liter per h before being reassimilated. Comparing Fig. Ic with Fig. la shows that ethanol was not assimilated until the supply of xylose was depleted, which takes longer the less oxygen is available. Xylitol production was not influenced by the OTR, reaching a final concentration of 20 to 25

g/liter (Fig. ld). To gain a better understanding of how azide affects C.

tropic alis metabolism, we studied the effect of this inhibitor on product formation at OTRs of 5 to 13 mmol/liter per h. The lowest OTR was omitted because the cells did not assimilate xylose in the presence of 0.2 mM azide. The results are summarized in Fig. 2, which shows that xylose uptake in the presence of azide took almost twice as long to reach completion (Fig. 2a), compared with the situation in the absence of inhibitor (Fig. la). Cell growth was reduced dramatically (by 60%), reaching a final optical density at 620 nm of only 15 to 20 (Fig. 2b). The production of ethanol reached its highest level (6 g/liter) at the lowest OTR, 5 mmol/liter per h (Fig. 2c), as was the case in the absence of azide (Fig. lc). However, since the yield derived from only about one-third of the cell mass, this actually represented an increase in specific ethanol production by a factor of 3, in agreement with what had previously been found (11). Azide did not stop the reassimilation of ethanol (Fig. 2c). Contrary to the situation without azide present (Fig. la and c), it seemed to promote the reassimilation of ethanol, since this occurred already before the xylose was fully depleted (Fig. 2a and c). The inhibitor was, however, able to repress the formation of the by-product xylitol, keeping the level below 4 g/liter, a reduction of 80% (Fig. 2d). The carbon mass balance was made for the xylose fermentations at different oxygenation levels and in the presence of azide (Table 1). In the absence of azide, the mass balance was made for 24- to 48-h cultures when sugar consumption and product formation were constant (Fig. 1). With the same criteria, 50- to 100-h cultures were chosen for fermentations in the presence of azide (Fig. 2). Although it is difficult to measure evolved carbon dioxide levels in shake flask cultures when oxygenation is required, the mass balance was made with two different assumptions: (i) 1 mol of carbon dioxide is produced for every 1 mol of ethanol, assuming an actively working phosphoketolase (7); (ii) an additional 1 mol of carbon dioxide is produced for every 1 mol of xylose assimilated, assuming the absence of phosphoketolase activity and a recirculation of fructose 6-phosphate through the pentose phosphate pathway (PPS). In the absence of azide, the carbon mass balance was balanced in the OTR range of 0.5 to 7 mmol/liter per h when the second assumption was used, whereas it was balanced under the first assumption at an OTR of 13 mmol/liter per h (Table 1). This suggests that phosphoketolase is not active or induced at OTR levels below 13 mmol/h in C. tropicalis. In the presence of azide it was not possible to close the carbon mass balance by using either of the assumptions (Table 1). The higher the degree of oxygenation, the greater the loss of

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FIG. 1. (a) Xylose consumption, (b) growth (estimated as optical density at 620 nm OD6201). (c) ethanol production, and (d) xylitol production by C. tropicalis as a function of OTR. Symbols: *. 0.5 mmol/liter per h; A, 5 mmol/liter per h; O. 7 mmol/liter per h; *. 13 mmol/liter per h.

carbon, most probably as carbon dioxide, since no glycerol, acetic acid, or succinic acid was produced. Intracellular metabolite concentrations. The influence of azide on the xylose metabolism of C. tropifcalis was further investigated by studying the steady-state levels (at constant sugar uptake rate) of glycolytic, PPS, and citric acid cycle intermediates in neutralized perchloric acid extracts, analyzed by 31P NMR (Fig. 3) and enzymatically (Table 2). Figure 3 shows the expanded sugar phosphate (SP) region of perchloric acid extracts analyzed by 31P NMR. Figure 3a shows results for a xylose-grown sample of C. tropicalais fed xylose for 20 min before extraction. Starting from the left, there are two small peaks at 5.4 and 5.25 ppm due to 6-phosphogluconate and glucose 6-phosphate, respectively. The large peak in our extracts, at 5.2 ppm, is most probably sedoheptulose 7-phosphate, based on addition of standard sedoheptulose 7-phosphate to the extract. There is evidence that a xylulose 5-phosphate peak is visible at 5.07 ppm, between the sedoheptulose 7-phosphate and the ribulose 5-phosphate peak, appearing at 5.0 ppm. Another peak at 4.60 ppm may in part be composed of ribose 5-phosphate. However, fructose 6-phosphate, AMP, and IMP standards also fall in this region. In these xylose extracts, FDP levels are quite low but may still appear at 4.85 and 4.75 ppm. Finally, the peaks with chemical shifts at 4.5 and 4.17 ppm have not been positively identified. The phosphoethanolamine standard has a chemical shift at 4.5 ppm, and NADP may be the compound appearing at 4.17 ppm. Neither of

these peaks is erythrose-4-phosphate, since this compound decomposed too quickly in the extracts to be useful as a standard. An extract made of azide-treated, xylose-fed cells (Fig. 3b) had a spectrum that was in all respects identical to the xylose spectrum discussed above. There was no evidence of a change in any of the relative peak heights and no evidence of the appearance of a novel intermediate. However, the total integrated area of the SP region in the azide extract was about 70% of that in the xylose extract. Neutralized perchloric acid extracts from C. tropicalis cells in 48-h cultures with azide present at an OTR of 13 mmol/liter per h were assayed enzymatically to obtain accurate levels of the glycolytic intermediates FDP, phosphoenolpyruvate, and pyruvate, as well as the citric acid cycle intermediates citrate, fumarate, and malate. Neither phosphoenolpyruvate nor citrate was present in measurable concentrations. and the levels of pyruvate and fumarate were extremely low. The concentrations of FDP and malate, normalized to the sugar uptake rate, were compared with previously obtained data on intermediary metabolite concentrations in the absence of azide (Table 2) (19). Both FDP and malate levels decreased when oxygenation increased. In the presence of azide, the levels of FDP were extremely low compared with the levels in the absence of azide, whereas malate levels were not affected. '3P NMR of C. tropicalis whole cells. 31p NMR studies were performed on C. tropicalis whole cells to compare the effects

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FIG. 2. (a) Xylose consumption, (b) growth (estimated as optical density at 620 nm [OD620]). (c) ethanol production. and (d) xylitol production by C. tropicalis in the presence of 0.2 mM azide as a function of OTR. Symbols are the same as in Fig. 1.

of azide with those of CCCP, a known uncoupler of oxidative phosphorylation, on the regulation of metabolism. Figure 4 shows the offset 31P NMR spectra of whole cells of C. tropicalis which are the averages of four 5-min plots obtained between 20 and 40 min after the addition of the carbon source when the cells were at steady-state. Assignments for the peaks were made by comparison with previously published yeast spectra (5, 13-15). The peak farthest downfield in these spectra, labeled SP, is composed of all the sugar phosphomonoesters seen in our extracts. Individual peaks are not very well resolved owing to the greater viscosity of the intracellular environment.

Figure SC illustrates the fluctuation of SP levels as a function of time. It shows that C. tropicalis cells metabolizing xylose have a high initial level of SP, but settle down after 20 min to a steady-state level. Cells inhibited with azide or CCCP tended to have lower SP levels. Next to the SP region (Fig. 4) are three peaks labeled and P. All these peaks are Pi, but the chemical P, Pi shlfts are different owing to differences in environmental pH. The Pi levels fluctuate greatly during the course of the experiments (Fig. 5B). C. tropicalis xylose cells take up Pi from the medium when oxygen is available, and they nearly exhaust the supply after 1 h. The uptake process is stopped ,

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TABLE 1. Carbon mass balance in xylose fermentations with C. tropicalis at different levels of oxygenation Product formation (mol of C): Carbon dioxide" Carbon dioxide'

Xylose cosmtn (mol of C)

Cell mass"

Ethanol

Azide absent 0.5 5 7 13

0.661 0.611 0.993 1.131

0.124 0.242 0.277 0.406

0.061 0.085 0.115 0.096

0.03 0.042 0.058 0.048

0.132 0.122 0.199

Azide present 5 7 13

0.567 1.233 1.033

0.0698 0.116 0.0889

0.156 0.139 0.0478

0.0783 0.0435 0.0239

0.113 0.247

OTR (mmol/liter per h)"

0.226

Xylitol

With

% Carbon recovery Without pathway"

pathway'

0.351 0.089 0.318 0.543

86 75 77 96

106 95 97 117

0.0066 0.0238 0.0205

55 26 18

74 46 38

0.2(07 " Cultures with azide absent were run at different levels of oxygenation for 24 to 48 h with sugar consumption and product formation constant. Cultures with

azide (0.2 mM) present were run for 50 to 100 h under the same conditions as those run in the absence of azide. " Using the composition formula CHR 500s5No 2 (17). '*With a carbon-saving phosphoketolase pathway (7). "Without a phosphoketolase pathway (7).

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