Effect of Oxygenation on Xylose Fermentation by Pichia stipitis

APPLID AND NVIRONMNTAL MICROBIOLOGY, Nov. 1990, p. 3389-3394 0099-2240/90/113389-06$02.00/0 Copyright C) 1990, American Society for Microbiology Vol. 56, No. 11 ffect of Oxygenation on Xylose Fermentation by Pichia stipitis KRSTIN SKOOG* AND BARBL HAHN-HAGRDAL Applied Microbiology, Chemical Center, P.O. Box 124, S-221 00 Lund, Sweden Received 25 May 1990/Accepted 24 August 1990 The effect of oxygen limitation on xylose fermentation by Pichia stipitis (CBS 6054) was investigated in continuous culture. The maximum specific ethanol productivity (0.20 g of ethanol g dry weight-' h-') and ethanol yield (0.48 g/g) was reached at an oxygen transfer rate below 1 mmol/liter per h. In the studied range of oxygenation, the xylose reductase (C 1.1.1.21) and xylitol dehydrogenase (C 1.1.1.9) activities were constant as well as the ratio between the NADPH and NADH activities of xylose reductase. No xylitol production was found. The pyruvate decarboxylase (C 4.1.1.1) activity increased and the malate dehydrogenase (C 1.1.1.37) activity decreased with decreasing oxygenation. With decreasing oxygenation, the intracellular intermediary metabolites sedoheptulose 7-phosphate, glucose 6-phosphate, fructose 1,6-diphosphate, and malate accumulated slightly while pyruvate decreased. The ratio of the xylose uptake rate under aerobic conditions, in contrast to that under anaerobic assay conditions, increased with increasing oxygenation in the culture. The results are discussed in relation to the energy level in the cell, the redox balance, and the mitochondrial function. Lignocellulosic biomass represents one of the most abundant renewable energy sources. Between 10 and 40% of all lignocellulosic biomass consists of hemicellulose, the main component being xylose (20), which can be fermented to fuel ethanol. Pichia stipitis is one of the better xylose-fermenting yeasts (9, 33, 36). In the initial steps in the conversion of xylose to ethanol, xylose is transported by a proton symport (8, 17) into the cell where xylose is reduced to xylitol via a xylose reductase (XR) and thereafter is oxidized to xylulose with a xylitol dehydrogenase (XDH). The xylulose is phosphorylated, channeled into the pentose phosphate shunt, and converted to C6 and C3 compounds. These compounds then enter glycolysis (3). Oxygen plays an important role in the conversion of xylose to ethanol by yeasts, and there is a critical level of oxygenation at which the ethanol yield and productivity are high and the cell yield is low (21, 30, 34, 39). There have been several hypotheses of the role of oxygen in yeast metabolism. Under anaerobic conditions the difference in the cofactor requirement of XR (NADPH) and XDH (NAD+) causes a redox imbalance (5). However, P. stipitis can use either NADPH or NADH as the cofactor, but the Km for NADPH (0.009 mm) is lower than for NADH (0.021 mm) (38). In Kluyver-positive yeasts, oxygen is needed for sugar transport (32). A further hypothesis is that an unimpaired mitochondrial function distinct from a role in cofactor balancing is necessary for optimal fermentation of xylose with P. stipitis (22). To reach a better understanding of the role of oxygen in ethanol production from xylose by P. stipitis, xylose fermentation was investigated in an oxygen-limited continuous culture with an excess of xylose. nzyme activities and concentrations of intracellular intermediary metabolites deriving from pentose phosphate shunt, glycolysis, and the citric acid cycle were measured at different oxygen levels. The effect of oxygen on the initial transport of xylose was also investigated. * Corresponding author. MATRIALS AND MTHODS Strain. P. stipitis CBS 6054 was maintained at 4 C on slants containing (per liter) 10 g of yeast extract, 10 g of malt extract (Difco), 20 g of peptone (Difco), and 50 g of xylose. Cultivation conditions. The culture medium consisted of (per liter) 50 g of xylose, 6.7 g of yeast nitrogen base (Difco), 0.81 g of K2HPO4, and 12.90 g of KH2PO4. The fermentor used was a 1.5-liter glass vessel (Applicon) with an FC 24 control system (lectrolux). The culture conditions were as follows. The temperature was set at 30 C, and ph was controlled to 5.5. Agitation varied between 400 and 1,000 rpm, and the air flow varied from 0 to 0.9 liters min to get an oxygen transfer rate (OTR) between 0 and 411 mmol liter-' h-1. OTR was estimated by the sodium sulfite oxidation method (7) with modifications as described previously (2). Dissolved oxygen was measured with a polarographic electrode (Ingold). For batch fermentation, cells grown under oxygen-limited conditions were inoculated into an anaerobic fermentor. The culture was sparged with 1.0 liter of nitrogen per min. Analytical methods. Xylose, xylitol, ethanol, acetic acid, and glycerol were analyzed by high-performance liquid chromatography (14). Cell growth was estimated from the optical density at 620 nm. Cell dry weight was determined after 25 min of drying at full power in a microwave oven containing a bottle of water. Cell dry weights obtained under these conditions correlated well with those obtained by drying in a conventional oven at 120 C. Preparation of perchloric acid extracts for the determination of intermediary metabolite concentrations. Samples for the determination of intermediary metabolites were harvested from the fermentations by rapid filtration (24). Perchloric acid extracts were made with 1.2 M perchloric acid by using a 10-min extraction time and one freeze-thaw cycle (34). The samples were frozen and stored at -80 C until analyzed enzymatically. Assay of intermediary metabolites. Fructose 1,6-diphosphate and pyruvate were determined by the method of Lowry and Passonneau (26). Malate was determined by the method of Williamson and Corkey (40). Glucose 6-phosphate and sedoheptulose 7-phosphate were measured by the 3389

3390 SKOOG AND HAHN-HAGRDAL method of Racker (28) with 0.1 M imidazole buffer (ph 7.6). The production or consumption of NADH or NADPH was detected in a Hitachi 3000 spectrofluorometer. ach metabolite concentration was determined as the mean of six independently filtered, frozen, and extracted samples. Preparation of cell extract for intracellular enzyme activities. After centrifugation, the cells were washed in phosphate buffer (ph 7.0) and suspended in buffer containing 5 mm mercaptoethanol and 0.5 mm DTA. The cells were disrupted by being pressed twice in an X-Press (Nike, Domkrafts AB, skiltuna, Sweden). Cell debris was removed by centrifugation, and the supernatant was frozen and stored at -80 C until analyzed. Assay of enzyme activities. XR was measured by the method of Smiley and Bolen (35) with the following final concentrations: xylose, 0.33 M; mercaptoethanol, 5 mm; DTA, 0.5 mm; phosphate buffer, 0.1 M (ph 7). XDH was measured by the method of Rizzi et al. (29) with final concentrations of 1 M xylitol and 4 mm NAD. Pyruvate decarboxylase, malate dehydrogenase, and protein were measured as described earlier (4, 16, 40). The specific activity of the enzymes was expressed as millimoles of converted substrate per gram of protein per minute. Transport assay. Cells were harvested at steady state from cultures with different levels of oxygenation, centrifuged, and washed twice. The pellet was suspended in the cultivation medium without xylose. During the uptake experiments, the cell suspension was incubated at 30 C and stirred by bubbling either air or nitrogen gas. After 5 min of incubation, '4C-labeled xylose was added. The total concentration of xylose was 5 mm. The reaction was stopped by adding ice-cold water. The suspension was filtered, and the filter was washed with water and then put into scintillation vials (27). The radioactivity was measured in a Beckman LS1801 scintillation counter. RSULTS thanol productivity and yield. The specific ethanol productivity and ethanol yield of the continuous culture increased with decreasing oxygenation at a dilution rate of 0.12 h-' (Fig. 1A and B). When the oxygen was further limited, the dilution rate had to be decreased to 0.06 h-' to prevent wash-out. At these low oxygenation levels, the detection limit of the OTR measurement was exceeded. Therefore, the levels are reported as being below 1 mols liter-1 h-1. The specific ethanol productivity and yield still increased to a maximum of 0.20 g g (dry weight)-' h-' and 0.48 g/g, respectively, at the dilution rate of 0.06 h-'. P. stipitis was unable to grow anaerobically but did ferment xylose to ethanol in a batch culture with a productivity of 0.02 g g (dry weight)-1 h-' and a yield of 0.25 g/g. The specific ethanol productivity in this culture was obtained when there was a linear relation between both ethanol production and xylose consumption versus time. In contrast to the specific ethanol productivity and yield, the specific xylose consumption decreased when the oxygenation decreased (Fig. 1C). Carbon balance. For the conversion of cell dry weight to moles of carbon, the elementary composition formula reported for Saccharomyces cerevisiae, CH1 8300.56No.17 was used (31). It was assumed that 1 mol of carbon dioxide was formed for every mole of ethanol or acetic acid formed. The consumed carbon could be reasonably well accounted for in the products at low levels of oxygenation (Table 1), but at more aerated levels and in the anaerobic batch fermentation, 40 to 60% of the assimilated carbon was not accounted for. No xylitol formation was observed (Table 1). S Sm 0.3- A D-0.12 D 0.06 batch 0.2. 0.1 0.0 411 182 51 31 13 8 2 >1 >1 0 0.6- B D=0.12 D=0.06 batch 0.4. 0.2 0.0 411 182 51 31 13 8 2 >1 >1 0 1n.*. 0.8 0.6 0.4 0.2 0.0 APPL. NVIRON. MICROBIOL. C D-0.12 D=0.06 batch im 411 182 51 31 13 8 2 >1 >1 0 FIG. 1. Specific ethanol productivity (A), ethanol yield (B), and specific xylose consumption rate (C) at different OTRs. In vitro enzyme activities. The activities of XR and XDH (Table 2) were constant over the entire range of oxygenation. The ratio of the XR activity measured separately with NADPH and NADH was constant and approximately 1.6 over the entire range of oxygenation. The specific activity of XDH measured with NADP+ was very low (<0.05 mmol g protein-' min-'; data not shown). The activity of pyruvate decarboxylase increased with decreasing oxygenation, whereas the malate dehydrogenase activity decreased (Table 2). Intracellular concentrations of pentose phosphate shunt and glycolytic and tricarboxylic acid cycle metabolites. The concentrations of intracellular intermediary metabolites in cells fermenting xylose at different levels of oxygenation are summarized in Fig. 2A. The data were obtained from three independent experiments. The pyruvate concentrations were five to six times higher (60 to 70 nmol mg [dry weight]-') in cells from the two most aerated cultures than in cells at other levels of oxygenation. The concentrations of the other metabolites (sedoheptulose 7-phosphate, glucose 6-phosphate, fructose 1,6-phosphate, and malate) were in the range of 1 to 10 nmol mg (dry weight)-1, with a slight tendency to decrease with decreasing oxygenation. With decreasing oxygenation, the overall xylose consumption (in millimoles liter-' h-1) also decreased (Fig. 2B). In order to separate the effect of oxygenation from the effect of

VOL. 56, 1990 XYLOS FRMNTATION BY P. STIPITIS 3391 TABL 1. Carbon mass balance Product formation (mmol of carbon) OTR (mmol/liter Xylose consumption per h) (mmol of carbon) thanol Carbon dioxide Cell mass Glycerol Acetic acid Carbon recovery ( 411 120.0 3.4 8.9 22.3 2.6 14.4 43 182 108.0 10.7 6.9 24.9 2.0 3.0 44 51 62.8 13.3 8.1 15.4 1.8 2.9 66 31 57.2 11.3 6.2 18.9 2.1 1.2 69 13 96.0 22.2 11.2 22.9 0.3 0.6 60 8 62.6 18.7 9.7 21.3 0.3 0.6 81 2 48.0 23.7 12.0 18.8 0 0.2 114 <1 36.8 17.2 8.6 6.2 0 0 88 <la 43.5 26.4 13.2 7.1 0 0 108 ob 20.0 6.0 3.0 0 0 0 45 a D = 0.06. b Batch culture. xylose consumption, the intermediary metabolite concentra- the slope between 4 and 16 s. The ratio of the uptake rate tions were divided by the respective overall xylose con- under aerobic conditions to the uptake rate under anaerobic sumptions and were expressed as nanomoles gram (dry conditions was calculated (Table 3). Cells taken from the weight)-1 divided by millimoles of xylose liter-' h-1 (Fig. aerobic batch culture took up xylose 27 times faster when 2C) (25, 34). These calculations indicated a striking accumu- assayed under aerobic conditions than when assayed under lation of pyruvate at the two highest levels of aeration (Fig. anaerobic conditions. This ratio decreased when the cultures 2C). When the oxygenation was decreased, the metabolic became more oxygen limited, and the ratio was close to 1 for pattern changed so that while pyruvate decreased the other the anaerobic cells. metabolites (sedoheptulose 7-phosphate, glucose 6-phosphate, fructose 1,6-phosphate, and malate) accumulated. DISCUSSION This accumulation increased under anaerobic conditions (Fig. 2C). Oxygen was used in this investigation as the limiting Transport activity. The initial xylose uptake rate was parameter in continuous culture. The OTR was used to investigated in cells which had been assimilating xylose define the different levels of oxygenation in the fermentor under aerobic, oxygen-limited, and anaerobic conditions. (25). Previously, the redox potential has been used to control The oxygen-limited cells came from continuous cultures oxygen limitation (11). Apart from measuring free oxygen, it with OTR values of 8 and 2 mmol liter-1 h-', respectively. is also influenced by ph, temperature, and medium compo- The aerobic and anaerobic cells came from batch cultures. sition (18). Oxygenation can be directly measured by using a The aerobic cells were chosen from batch cultures to prevent polarographic electrode with a digital oxygen meter (10). The oxygen and carbon source limitation, and the cells were oxygenation levels of interest for ethanol production are, harvested when the xylose consumption and growth were however, very close to the detection limit. In the present linear versus time. Batch cultures were also used for the study the dissolved oxygen was 0 at all levels of oxygenation anaerobic cells since P. stipitis does not grow anaerobically, except for the two highest (OTR values 411 and 182 mmol and the cells were harvested when the ethanol production liter-' h-1). These cultures were still considered oxygen was linear. limited since the cells produced ethanol. There were excess The initial uptake rate was assayed under both aerobic and amounts of xylose under all cultivation conditions. anaerobic conditions. The uptake rate was calculated from The maximum specific ethanol productivity and the max- TABL 2. In vitro enzyme activities Sp act (mmol/g` min-') of: OTR (mmol/liter per h) XR (NADPH)a XR (NADH)b XDH (NAD)' Pyruvate de- Malate dehycarboxylase drogenase 411 1.61 1.25 0.51 0.07 27.2 182 1.76 1.02 0.40 0.29 18.3 51 1.55 1.11 0.42 0.40 18.1 31 1.48 1.11 0.38 0.45 20.3 13 1.89 1.11 0.38 0.38 5.5 8 1.89 1.15 0.38 0.51 14.9 2 2.33 1.36 0.51 0.29 10.7 <id 1.39 0.86 0.51 0.32 9.5 <id 1.51 0.94 0.49 0.35 6.5 oe 0.89 0.55 0.33 0.62 13.2 a XR was measured separately with NADPH. b XR was measured separately with NADH. C XDH was measured separately with NAD. d D = 0.06 h-1. e Batch culture.

3392 SKOOG AND HAHN-HAGRDAL APPL. NVIRON. MICROBIOL. 80 - A, D=0.12 D=0.06 batch i 0 60-40- 20- I/ISLSLISI2I 1 u 411 182 51 31 13 2 <1 '1 0 20 - B kv.0 D=0.1 2 D=0.06 batch Downloaded from http://aem.asm.org/ 10 0 411 182 51 31 13 2 imum ethanol yield were remarkably high, 0.20 g g (dry weight)-1 h-' and 0.48 g/g, respectively. This was achieved at a 0.06-h-' dilution rate and with a level of oxygenation below the detection limit of the sodium sulfite method. A specific ethanol productivity of 0.13 g g (dry weight)-1 h-1 and an ethanol yield of 0.25 g/g was reported earlier in continuous culture with a dilution rate of 0.165 h-1 and an oxygen uptake rate of 0.09 mol liter-' h-1 (13). On the other hand, in oxygen-limited batch cultures, a specific ethanol productivity and an ethanol yield of 0.20 g g (dry weight)` h-' and 0.47 g/g, respectively, have been obtained (21). The carbon mass balance was not closed at high OTR values. This could be the result of a production of unknown components, cell growth on the wall of the vessel, or excess carbon dioxide production. No components other than those reported in Table 1 were detected by high-performance liquid chromatography. Assuming that all of the missing FIG. 2.-Continued. 1 '1 0 carbon constitutes cells, the specific ethanol productivity will change and a more distinct maximum would be found at the lowest level of oxygenation. If, on the other hand, the missing carbon is carbon dioxide, this would suggest an increased recirculation of fructose 6-phosphate as a result of a higher requirement for NADPH for cell synthesis. When the carbon mass balance was not closed in the oxygen-limited cultures, the ethanol yields were below 0.3 g/g. The yield of ethanol from xylose has been calculated to be 0.51 g/g with no recirculation of fructose 6-phosphate and 0.31 g/g with recirculation (6, 12). The activities of both XR and XDH were independent of oxygen as was the cofactor requirement of XR. This has also been shown in batch culture (10) and in continuous culture with Candida shehatae (1). However, these reported activities were lower, which could be the result of differences in strains or assay conditions. In the present investigation, no on November 2, 2018 by guest

VOL. 56, 1990 XYLOS FRMNTATION BY P. STIPITIS 3393 4000 3000 411 182 51 31 13 2-1'1 0 FIG. 2. Intracellular intermediary metabolite concentrations (A), xylose consumption rate (B), and intracellular intermediary metabolite concentrations divided by the xylose consumption (C) at different OTRs. Symbols: *, sedoheptulose 7-phosphate; O, glucose 6-phosphate; l, fructose 1,6-diphosphate; M, pyruvate; l, malate. Bars show standard deviation. xylitol was detected, indicating that the regeneration of NAD+ was not limiting the ethanol formation in the strain used, P. stipitis CBS 6054, as has been shown for other xylose-fermenting yeasts (5). The xylose transport activity was dependent on the level of oxygenation during xylose assimilation as well as on the presence of oxygen in the transport assay. It has been suggested that xylose transport is a limiting step in xylose fermentation (17, 23). The present results suggest that oxygen induces or activates a transport system. These results may also be interpreted in terms of an adaptation of the transport system to oxygen limitation and anaerobic conditions, since the anaerobic cells showed the same transport rate under both aerobic and anaerobic assay conditions. Kluyver-positive yeasts can utilize certain disaccharides and galactose oxidatively but are unable to ferment them, even if they ferment glucose anaerobically (19). These yeasts were later shown to require oxygen for sugar transport (32). In Schizosaccharomyces pombe, the glucose uptake rate has been found to be inhibited by 30% under anaerobic conditions (15). It was suggested that this was the result of anaerobic cells being less energized. This might also be the case in P. stipitis since it has an active transport system for xylose (8, 17), which indicates that oxygen is only indirectly involved in the transport activity. TABL 3. Ratio of the initial uptake of xylose under aerobic and anaerobic assay conditions Oxygen level Ratio (air/nitrogen) Aerobic... 27 Oxygen limited (8 mmollliter per h)... 9 (2 mmol/liter per h)... 3 Anaerobic... 1 The increase in pyruvate decarboxylase activity and the decrease in malate dehydrogenase activity with increasing oxygen limitation point towards an increase in specific ethanol productivity, which was also observed in this study. In S. cerevisiae, the pyruvate decarboxylase activity has been shown to be crucial for ethanol production (37). The development of enzyme activities with oxygenation was supported by the levels of intermediary metabolites. Since the specific ethanol productivity increased up to the lowest level of oxygenation, an even higher productivity would have been expected in the anaerobic culture. However, when P. stipitis assimilated xylose anaerobically, the specific ethanol productivity and ethanol yield were dramatically reduced despite the fact that the pyruvate decarboxylase activity reached its highest value. Under anaerobic conditions, the cells did not grow and do not have functioning mitochondria. Therefore, in P. stipitis CBS 6054, oxygen seems to be necessary for ethanol production not mainly because of the redox imbalance but rather because it is required for growth (5) and/or the function of the mitochondria (22) in addition to generating energy necessary for the transport of xylose (15). ACKNOWLDGMNTS This study was supported by the Swedish National nergy Administration. We thank Sven Tornquist for excellent technical assistance and Christer Holmgren for linguistic advice. LITRATUR CITD 1. Alexander, M. A., V. W. Yang, and T. W. Jeffries. 1988. Levels of pentose phosphate pathway enzymes from Candida shehatae grown in continuous culture. Appl. Microbiol. Biotechnol. 29:282-288. 2. American Society for Testing and Materials. 1974. Annual book of American Society for Testing and Material standards, p.

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