NOTES
Effect of nitrogen sources on oxidoreductive enzymes and ethanol production during D-xylose fermentation by Candida shehatae'
Can. J. Microbiol. 1992.38:258-260. Downloaded from www.nrcresearchpress.com by Depository Services Program on 11/12/14. For personal use only.
SANJAY PALNITKAR AND ANILLACHKE Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, India Received June 28, 1991 Revision received September 25, 1991 Accepted October 1, 1991 PALNITKAR, S., and LACHKE,A. 1992. Effect of nitrogen sources on oxidoreductive enzymes and ethanol production during D-xylose fermentation by Candida shehatae. Can. J. Microbiol. 38: 258-260. S~ and the corresponding xylitol and ethanol production by Candida shehatae The effect on D - X Y ~ Outilization (ATCC 22984) were examined with different nitrogen sources. These included organic (urea, asparagine, and peptone) and inorganic (ammonium chloride, ammonium nitrate, ammonium sulphate, and potassium nitrate) sources. Candida shehatae did not grow on potassium nitrate. Improved ethanol production (Y(,,,,, yield coefficient (grams product/ grams substrate), 0.34) was observed when organic nitrogen sources were used. Correspondingly, the xylitol production was also higher with organic sources. Ammonium sulphate showed the highest ethano1:xylitol ratio (1 1.O) among all the nitrogen sources tested. The ratio of NADH- t o NADPH-linked D - X Y ~ O Sreductase ~ (EC 1.1.1.21) appeared to be rate limiting during ethanologenesis of D-xylose. The levels of xylitol dehydrogenase (EC 1.1.1.9) were also elevated in the presence of organic nitrogen sources. These results may be useful in the optimization of alcohol production by C. shehatae during continuous fermentation of D-xylose. Key words: xylose fermentation, Candida shehatae, nitrogen source, oxidoreductive enzymes. PALNITKAR, S., et LACHKE,A. 1992. Effect of nitrogen sources on oxidoreductive enzymes and ethanol production fermentation S~ by Candida shehatae. Can. J. Microbiol. 38 : 258-260. during D - X Y ~ O L'effet de differentes sources d'azote sur l'utilisation du D-xylose et sur la production correspondante de xylitol et d'ethanol a ete examine chez Candida shehatae (ATCC 22984). Ces sources d'azote comprenaient des composes organiques (uree, asparagine et peptone) et inorganiques (chlorure d'ammonium, nitrate d'ammonium, sulfate d'ammonium et nitrate de potassium). Candida shehatae n'a pas pousse en presence de nitrate de potassium. Une production accrue d'ethanol (Y(,,,,, 0'34) a ete observee lorsque des sources d'azote organique ont ete utilisees. La production de xylitol a ete egalement plus elevee avec des sources organiques. Le sulfate d'ammonium a montre le plus haut rapport ethano1:xylitol (1 1'0) parmi toutes les sources d'azote qui ont ete testees. La proportion de NADH sur NADPH lie a la D-xylose reductase (EC 1.1.1.21) a semble Etre un facteur limitatif du taux d'elaboration d'ethanol a partir du D-xylose. Les niveaux de xylitol deshydrogenase (EC 1.1.1.9) ont aussi ete eleves en presence de sources d'azote organique. Ces resultats peuvent Etre utilises en vue d'optimiser la production d'alcool par C. shehatae pendant la fermentation continue du D-xylose. Mots clks : fermentation du xylose, Candida shehatae, source d'azote, enzymes oxydo-reductrices. [Traduit par la redaction]
Conversion of hemicellulosic sugars to ethanol in the absence of oxygen is limited to only a small number of yeasts, although many yeasts can assimilate these sugars aerobically (Barnett 1976). A few yeasts, namely, Pachysolen tannophilus (Schneider et al. 1981; Jeffries 1985), Candida shehatae (Du Preez et al. 1984), Pichia stipitis (Dellweg et al. 1984), and Candida tropicalis (Jeffries 1982) have been found to produce ethanol from D-xylose, with appreciable yields, under well-defined aerobic or anaerobic conditions. D-Xylose is first reduced to xylitol by D-xylose reductase (usually NADPH linked), and xylitol is oxidized to D-xylulose by xylitol dehydrogenase (exclusively NAD linked). D-Xylulose is then phosphorylated to D-xylulose5-phosphate, which enters into the pentose phosphate pathway (Chakravorty et al. 1962; Evans and Ratledge 1984). During the fermentation, xylitol accumulates as a by-product (Ditzelmiiller et al. 1984; Slininger et al. 1985). Nitrate is known to enhance the enzyme levels involved in the pentose phosphate pathway of yeasts and also in certain cultivated ' ~ a t i o n a lChemical Laboratory Communication No. 5 153. Printed in Canada / Imprime au Canada
plant cells. This increase in the enzyme levels can be correlated to the requirement of NADP(H) by the nitrateassimilating enzymes (Osmond and Ap Rees 1969). The theoretical calculations of the NADPH requirement for yeast biomass formation reveal that this parameter is strongly dependent on the carbon and the nitrogen source (Bruinenberg et al. 1983). Candida shehatae is an efficient fermenter of D - X Y ~ O Sbut ~ is not able to utilize nitrate as the sole nitrogen source (Barnett et al. 1979). In the present communication, the influence of organic and inorganic nitrogen sources on the oxidoreductive enzymes involved in D-xylose metabolism, and the corresponding ethanol production by C. shehatae, are evaluated. Candida shehatae ATCC 22984 was maintained on MXYP (malt extract, 0.3%; D-xylose, 1%; yeast extract, 0.3%; peptone, 0.5%; agar, 2%) slants. The cells were grown in 250-mL Erlenmeyer flasks. The medium contained D-xylose, 2%, and yeast nitrogen base (YNB), 0.67%. The YNB was filter sterilized. Incubation was carried out at 30°C, 200 rpm, for 48 h. The cells were harvested by centrifugation (2000 x g) and washed with sterile distilled
NOTES
TABLE1. Effect of nitrogen sources on ethanol yields and corresponding activities of oxidoreductive
Can. J. Microbiol. 1992.38:258-260. Downloaded from www.nrcresearchpress.com by Depository Services Program on 11/12/14. For personal use only.
enzymes Xylose reductase (XR) (U/mg)
Nitrogen source
Y(,,,,
Ethano1:xylitol
NADPH
Urea Asparagine Peptone NH4C1 NH4N03 (NH4)2S04
0.35 0.33 0.34 0.29 0.27 0.30
5.8 5.1 5.9 5.2 7.9 11.0
0.34 0.32 0.32 0.26 0.26 0.29
water, twice, and then with 0.025 M phosphate buffer, pH 6.0. The medium for inoculum buildup consisted of 2% D-xylose, 0.67% yeast base (prepared according to the Difco manual), and 20 mM of appropriate nitrogen source. Seven different nitrogen sources, organic and inorganic, were used separately. The initial pH of the medium was 5.5. Each flask was inoculated with C. shehatae cells (5% wet weight). The flasks (500 mL) containing 200 mL medium were shaken at 30°C, 200 rpm, for 72 h. The cells were harvested as mentioned above, and washed with sterile distilled water and then with potassium phosphate buffer (0.025 M, pH 6.0). Equal amounts of cells were inoculated into the fermentation media. This medium was concentration same as that of used D-xylose for inoculum was 7.5% buildup, and that except of nitrogen that the
NADH
NADH-linked/ NADPHlinked XR
Xylitol dehydrogenase (U/mg)
0.07 0.05 0.06 0.04 0.04 0.05
0.21 0.16 0.18 0.15 0.15 0.17
0.18 0.17 0.16 0.14 0.14 0.15
>
3
-S w
4-
V,
;
source was 76 mM. The flasks were shaken at 200 rpm at 30°C for 192 h. Samples were collected periodically to determine the residual xylose, ethanol, xylitol, and intracellular enzymes. D-Xylose, xylitol, and ethanol were quantified by HPLC (Hewlett Packard model 1082B) using a Waters' Sugar-Pak column, with water at 75°C as the eluent. The activity of D-xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) was determined as described earlier (Lachke and Jeffries 1986). One unit is the amount of enzyme necessary to catalyze the oxidation or reduction of one micromole of coenzyme (NAD or NADP) per minute at room temperature under the given experimental conditions. specificactivities are expressed as units (U) per milligram protein. Protein was estimated by the method of Bradford (1976). The assimilation of D-xylose by C. shehatae with respect to time and the corresponding ethanol and xylitol formation were monitored with selected organic and inorganic nitrogen sources (Fig. 1). Rapid D-xylose metabolism in the presence of organic nitrogen sources was apparent within the initial 24 h, as shown in Fig. 1. D-Xylose was completely utilized within 4-5 days when organic nitrogen sources were used. In contrast, with inorganic nitrogen sources, D-xylose was not completely utilized even after 8 days. Potassium nitrate did not support the growth of C . shehatae. Ethanol formation with respect to D-xylose assimilation was observed periodically and is shown in Fig. 1. Detectable amounts-of ethanol were produced using organic the use nitrogen sources within inorganic nitrogen sources in the medium showed delayed production of ethanol, with detectable levels only appearing after 2-3 days of fermentation. Moreover, all the
6-
\
i o
n
$
2-
-S
-
A 0
a z I
I-
L
I
DAYS
FIG. 1. The production of ethanol and xylitol during utilization of D-xylose by C.shehatae under the influence of various nitrogen sources (urea, ; peptone, A; asparagine, A ; (NH4),S04, m; NH4C1, o;NH4N03, 0).
CAN. J. MICROBIOL. VOL. 38, 1992
Can. J. Microbiol. 1992.38:258-260. Downloaded from www.nrcresearchpress.com by Depository Services Program on 11/12/14. For personal use only.
260
examined organic nitrogen sources gave higher ethanol yield as compared with inorganic sources. Among the organic nitrogen sources urea gave maximum ethanol production, with a yield coefficient (grams product/grams substrate), Y(,,,), of 0.35, followed by peptone (0.34) and asparagine (0.33). Inorganic nitrogen sources could produce only 2% (w/v) ethanol, corresponding to an average Y(,/,) of 0.28. Ammonium sulphate was the preferred nitrogen source over ammonium chloride and ammonium nitrate (Table 1). Ethanol production in the present study was marginally higher than values reported by Du Preez and van der Walt (1983) and Du Preez et al. (1986) with C. shehatae (CSIR-Y 492) and P. tannophilus (NRRL Y-2460). One C. shehatae strain was reported to produced 20.6 and another 24.0 g L - The levels of xylitol produced during the fermentation were also comparable with those reported elsewhere (Du Preez and van der Walt 1983). It has also been demonstrated that the product ratio, ethanol/xylitol, during D-xylose fermentation by yeast strains was influenced by several factors. In the present studies the maximum production of xylitol (0.45% w/v) was observed after 5 days when organic nitrogen sources were used. The high ethanol/xylitol ratio is necessary in the D-xylose fermentation to ethanol, particularly when C. shehatae is used, because this culture cannot efficiently use xylitol for growth (Du Preez et al. 1986). In this respect, ammonium sulphate was found to be a suitable nitrogen source, considering the highest ratio of ethanol/xylitol noted among all the examined nitrogen sources (Table 1). Enzymes involved in D-xylose assimilation in different yeasts have different specificities for cofactors such as NAD(P)H and NAD (Lachke and Jeffries 1986; Alexander et al. 1988). In the case of C. shehatae, the NADPH-linked D-xylose reductase activity was threefold to fivefold higher than the NADH-linked activity as shown in Table 1. The ratio (0.2 1) of NADH-linked/NADPH-linked D-xylose reductase activities was high in urea- and peptone-grown C. shehatae cells. The ratio was lower when inorganic nitrogen and asparagine were incorporated as nitrogen sources. The organic nitrogen sources were found to increase the levels of xylitol dehydrogenase in contrast to inorganic nitrogen sources (Table 1). The results showed that high NADH-linked/NADPHlinked D-xylose reductase activities may play a pivotal role for enhanced ethanol production. This effect could be due to a closed-loop pathway for the oxidation and reduction of NADH in the formation of D-xylulose, thereby preventing accumulation of NADH, which suppresses this conversion (Jeffries 1985). The data of this study, indicating improved yields of ethanol with inclusion of organic nitrogen sources, could be useful for designing continuous controlled fermentation for optimizing ethanol yields from C. shehatae. Moreover, the efficiency of simultaneous saccharification and fermentation of hydrolysates of agricultural residues by C. shehatae (Palnitkar and Lachke 1990) can also be increased.
'.
Acknowledgement A Senior Research Fellowship from the Council for Scientific and Industrial Research (India) to S.S.P. is gratefully acknowledged.
Alexander, M.A., Yang, V.W., and Jeffries, T.W. 1988. Levels of pentose phosphate pathway enzymes from Candida shehatae grown in continuous culture. Appl. Microbiol. Biotechnol. 29: 282-288. Barnett, J.A. 1976. The utilization of sugars by yeasts. Adv. Carbohydr. Chem. Biochem. 32: 125-234. Barnett, J.A., Payne, R. W., and Yarrow, D. 1979. A guide to identifying and classifying yeasts. Cambridge University Press, London. Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. Bruinenberg, P.M., van Dijken, J.P., and Scheffers, W.A. 1983. A theoretical analysis of NADPH production and consumption of yeasts. J. Gen. Microbiol. 129: 953-964. Chakravorty, M., Veiga, A., Bacila, M., and Horecker, B.L. 1962. Pentose metabolism in Candida. J. Biol. Chem. 237: 1014-1020. Dellweg, H., Rizzi, M., Methner, H., and de Bus. 1984. Xylose fermentation by yeasts. Comparison of Pachysolen tannophilus and Pichia stipitis. Biotechnol. Lett. 6: 395-400. Ditzelmiiller, G., Kubicek, C.P., Wohrer, W., and Rohr, M. 1984. Xylitol dehydrogenase from Pachysolen tannophilus. FEMS Microbiol. Lett. 25: 195-198. du Preez, J.C., and van der Walt, J.P. 1983. Fermentation of D-xyloseto ethanol by a strain of Candida shehatae. Biotechnol. Lett. 5: 357-362. du Preez, J.C., Prior, B., and Monteiro, A. 1984. The effect of aeration on xylose fermentation by Candida shehatae and Pachysolen tannophilus. Appl. Microbiol. Biotechnol. 19: 261-266. du Preez, J.C., Bosch, M., and Prior, B.A. 1986. Xylose fermentation by Candida shehatae and Pichia stipitis: effects of pH, temperature, and substrate concentration. Enzyme Microb. Technol. 8: 360-364. Evans, C.T., and Ratledge, C. 1984. Induction of xylulose-5phosphate phosphoketolase in a variety of yeasts grown on D-xylose; the key to efficient xylose metabolism. Arch. Microbiol. 139: 42-52. Jeffries, T.W. 1982. A comparison of Candida tropicalis and Pachysolen tannophilus for conversion of D-xylose to ethanol. Biotechnol. Bioeng. Symp. 12: 103-1 10. Jeffries, T.W. 1985. Emerging technology for fermenting D-xylose. Trends Biotechnol. 3: 208-2 12. Lachke, A.H., and Jeffries, T.W. 1986. Levels of enzymes of the pentose phosphate pathway in Pachysolen tannophilus Y-2460 and selected mutants. Enzyme Microb. Technol. 8: 357-362. Osmond, C.B., and Ap Rees, T.A. 1969. Control of pentose phosphate pathway in yeasts. Biochim. Biophys. Acta, 184: 35-42. Palnitkar, S.S., and Lachke, A.H. 1990. Efficient simultaneous saccharification and fermentation of agricultural residues by Saccharomyces cerevisiae and Candida shehatae-the D-xylose fermenting yeast. Appl. Biochem. Biotechnol. 26: 151-158. Schneider, H., Wang, P.Y., Chan, Y.K., and Maleszka, R. 1981. Conversion of D-xylose into ethanol by the yeast Pachysolen tannophilus. Biotechnol. Lett. 3: 89-92. Slininger, P.J., Bothast, R.J., Okos, M.R., and Ladisch, M.R. 1985. Comparative evaluation of ethanol production by xylose fermenting yeasts presented with high xylose concentrations, Biotechnol. Lett. 7 : 43 1-436.
Effect of nitrogen sources on oxidoreductive enzymes and ethanol production during D-xylose fermentation by Candida shehatae'
Can. J. Microbiol. 1992.38:258-260. Downloaded from www.nrcresearchpress.com by Depository Services Program on 11/12/14. For personal use only.
SANJAY PALNITKAR AND ANILLACHKE Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, India Received June 28, 1991 Revision received September 25, 1991 Accepted October 1, 1991 PALNITKAR, S., and LACHKE,A. 1992. Effect of nitrogen sources on oxidoreductive enzymes and ethanol production during D-xylose fermentation by Candida shehatae. Can. J. Microbiol. 38: 258-260. S~ and the corresponding xylitol and ethanol production by Candida shehatae The effect on D - X Y ~ Outilization (ATCC 22984) were examined with different nitrogen sources. These included organic (urea, asparagine, and peptone) and inorganic (ammonium chloride, ammonium nitrate, ammonium sulphate, and potassium nitrate) sources. Candida shehatae did not grow on potassium nitrate. Improved ethanol production (Y(,,,,, yield coefficient (grams product/ grams substrate), 0.34) was observed when organic nitrogen sources were used. Correspondingly, the xylitol production was also higher with organic sources. Ammonium sulphate showed the highest ethano1:xylitol ratio (1 1.O) among all the nitrogen sources tested. The ratio of NADH- t o NADPH-linked D - X Y ~ O Sreductase ~ (EC 1.1.1.21) appeared to be rate limiting during ethanologenesis of D-xylose. The levels of xylitol dehydrogenase (EC 1.1.1.9) were also elevated in the presence of organic nitrogen sources. These results may be useful in the optimization of alcohol production by C. shehatae during continuous fermentation of D-xylose. Key words: xylose fermentation, Candida shehatae, nitrogen source, oxidoreductive enzymes. PALNITKAR, S., et LACHKE,A. 1992. Effect of nitrogen sources on oxidoreductive enzymes and ethanol production fermentation S~ by Candida shehatae. Can. J. Microbiol. 38 : 258-260. during D - X Y ~ O L'effet de differentes sources d'azote sur l'utilisation du D-xylose et sur la production correspondante de xylitol et d'ethanol a ete examine chez Candida shehatae (ATCC 22984). Ces sources d'azote comprenaient des composes organiques (uree, asparagine et peptone) et inorganiques (chlorure d'ammonium, nitrate d'ammonium, sulfate d'ammonium et nitrate de potassium). Candida shehatae n'a pas pousse en presence de nitrate de potassium. Une production accrue d'ethanol (Y(,,,,, 0'34) a ete observee lorsque des sources d'azote organique ont ete utilisees. La production de xylitol a ete egalement plus elevee avec des sources organiques. Le sulfate d'ammonium a montre le plus haut rapport ethano1:xylitol (1 1'0) parmi toutes les sources d'azote qui ont ete testees. La proportion de NADH sur NADPH lie a la D-xylose reductase (EC 1.1.1.21) a semble Etre un facteur limitatif du taux d'elaboration d'ethanol a partir du D-xylose. Les niveaux de xylitol deshydrogenase (EC 1.1.1.9) ont aussi ete eleves en presence de sources d'azote organique. Ces resultats peuvent Etre utilises en vue d'optimiser la production d'alcool par C. shehatae pendant la fermentation continue du D-xylose. Mots clks : fermentation du xylose, Candida shehatae, source d'azote, enzymes oxydo-reductrices. [Traduit par la redaction]
Conversion of hemicellulosic sugars to ethanol in the absence of oxygen is limited to only a small number of yeasts, although many yeasts can assimilate these sugars aerobically (Barnett 1976). A few yeasts, namely, Pachysolen tannophilus (Schneider et al. 1981; Jeffries 1985), Candida shehatae (Du Preez et al. 1984), Pichia stipitis (Dellweg et al. 1984), and Candida tropicalis (Jeffries 1982) have been found to produce ethanol from D-xylose, with appreciable yields, under well-defined aerobic or anaerobic conditions. D-Xylose is first reduced to xylitol by D-xylose reductase (usually NADPH linked), and xylitol is oxidized to D-xylulose by xylitol dehydrogenase (exclusively NAD linked). D-Xylulose is then phosphorylated to D-xylulose5-phosphate, which enters into the pentose phosphate pathway (Chakravorty et al. 1962; Evans and Ratledge 1984). During the fermentation, xylitol accumulates as a by-product (Ditzelmiiller et al. 1984; Slininger et al. 1985). Nitrate is known to enhance the enzyme levels involved in the pentose phosphate pathway of yeasts and also in certain cultivated ' ~ a t i o n a lChemical Laboratory Communication No. 5 153. Printed in Canada / Imprime au Canada
plant cells. This increase in the enzyme levels can be correlated to the requirement of NADP(H) by the nitrateassimilating enzymes (Osmond and Ap Rees 1969). The theoretical calculations of the NADPH requirement for yeast biomass formation reveal that this parameter is strongly dependent on the carbon and the nitrogen source (Bruinenberg et al. 1983). Candida shehatae is an efficient fermenter of D - X Y ~ O Sbut ~ is not able to utilize nitrate as the sole nitrogen source (Barnett et al. 1979). In the present communication, the influence of organic and inorganic nitrogen sources on the oxidoreductive enzymes involved in D-xylose metabolism, and the corresponding ethanol production by C. shehatae, are evaluated. Candida shehatae ATCC 22984 was maintained on MXYP (malt extract, 0.3%; D-xylose, 1%; yeast extract, 0.3%; peptone, 0.5%; agar, 2%) slants. The cells were grown in 250-mL Erlenmeyer flasks. The medium contained D-xylose, 2%, and yeast nitrogen base (YNB), 0.67%. The YNB was filter sterilized. Incubation was carried out at 30°C, 200 rpm, for 48 h. The cells were harvested by centrifugation (2000 x g) and washed with sterile distilled
NOTES
TABLE1. Effect of nitrogen sources on ethanol yields and corresponding activities of oxidoreductive
Can. J. Microbiol. 1992.38:258-260. Downloaded from www.nrcresearchpress.com by Depository Services Program on 11/12/14. For personal use only.
enzymes Xylose reductase (XR) (U/mg)
Nitrogen source
Y(,,,,
Ethano1:xylitol
NADPH
Urea Asparagine Peptone NH4C1 NH4N03 (NH4)2S04
0.35 0.33 0.34 0.29 0.27 0.30
5.8 5.1 5.9 5.2 7.9 11.0
0.34 0.32 0.32 0.26 0.26 0.29
water, twice, and then with 0.025 M phosphate buffer, pH 6.0. The medium for inoculum buildup consisted of 2% D-xylose, 0.67% yeast base (prepared according to the Difco manual), and 20 mM of appropriate nitrogen source. Seven different nitrogen sources, organic and inorganic, were used separately. The initial pH of the medium was 5.5. Each flask was inoculated with C. shehatae cells (5% wet weight). The flasks (500 mL) containing 200 mL medium were shaken at 30°C, 200 rpm, for 72 h. The cells were harvested as mentioned above, and washed with sterile distilled water and then with potassium phosphate buffer (0.025 M, pH 6.0). Equal amounts of cells were inoculated into the fermentation media. This medium was concentration same as that of used D-xylose for inoculum was 7.5% buildup, and that except of nitrogen that the
NADH
NADH-linked/ NADPHlinked XR
Xylitol dehydrogenase (U/mg)
0.07 0.05 0.06 0.04 0.04 0.05
0.21 0.16 0.18 0.15 0.15 0.17
0.18 0.17 0.16 0.14 0.14 0.15
>
3
-S w
4-
V,
;
source was 76 mM. The flasks were shaken at 200 rpm at 30°C for 192 h. Samples were collected periodically to determine the residual xylose, ethanol, xylitol, and intracellular enzymes. D-Xylose, xylitol, and ethanol were quantified by HPLC (Hewlett Packard model 1082B) using a Waters' Sugar-Pak column, with water at 75°C as the eluent. The activity of D-xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) was determined as described earlier (Lachke and Jeffries 1986). One unit is the amount of enzyme necessary to catalyze the oxidation or reduction of one micromole of coenzyme (NAD or NADP) per minute at room temperature under the given experimental conditions. specificactivities are expressed as units (U) per milligram protein. Protein was estimated by the method of Bradford (1976). The assimilation of D-xylose by C. shehatae with respect to time and the corresponding ethanol and xylitol formation were monitored with selected organic and inorganic nitrogen sources (Fig. 1). Rapid D-xylose metabolism in the presence of organic nitrogen sources was apparent within the initial 24 h, as shown in Fig. 1. D-Xylose was completely utilized within 4-5 days when organic nitrogen sources were used. In contrast, with inorganic nitrogen sources, D-xylose was not completely utilized even after 8 days. Potassium nitrate did not support the growth of C . shehatae. Ethanol formation with respect to D-xylose assimilation was observed periodically and is shown in Fig. 1. Detectable amounts-of ethanol were produced using organic the use nitrogen sources within inorganic nitrogen sources in the medium showed delayed production of ethanol, with detectable levels only appearing after 2-3 days of fermentation. Moreover, all the
6-
\
i o
n
$
2-
-S
-
A 0
a z I
I-
L
I
DAYS
FIG. 1. The production of ethanol and xylitol during utilization of D-xylose by C.shehatae under the influence of various nitrogen sources (urea, ; peptone, A; asparagine, A ; (NH4),S04, m; NH4C1, o;NH4N03, 0).
CAN. J. MICROBIOL. VOL. 38, 1992
Can. J. Microbiol. 1992.38:258-260. Downloaded from www.nrcresearchpress.com by Depository Services Program on 11/12/14. For personal use only.
260
examined organic nitrogen sources gave higher ethanol yield as compared with inorganic sources. Among the organic nitrogen sources urea gave maximum ethanol production, with a yield coefficient (grams product/grams substrate), Y(,,,), of 0.35, followed by peptone (0.34) and asparagine (0.33). Inorganic nitrogen sources could produce only 2% (w/v) ethanol, corresponding to an average Y(,/,) of 0.28. Ammonium sulphate was the preferred nitrogen source over ammonium chloride and ammonium nitrate (Table 1). Ethanol production in the present study was marginally higher than values reported by Du Preez and van der Walt (1983) and Du Preez et al. (1986) with C. shehatae (CSIR-Y 492) and P. tannophilus (NRRL Y-2460). One C. shehatae strain was reported to produced 20.6 and another 24.0 g L - The levels of xylitol produced during the fermentation were also comparable with those reported elsewhere (Du Preez and van der Walt 1983). It has also been demonstrated that the product ratio, ethanol/xylitol, during D-xylose fermentation by yeast strains was influenced by several factors. In the present studies the maximum production of xylitol (0.45% w/v) was observed after 5 days when organic nitrogen sources were used. The high ethanol/xylitol ratio is necessary in the D-xylose fermentation to ethanol, particularly when C. shehatae is used, because this culture cannot efficiently use xylitol for growth (Du Preez et al. 1986). In this respect, ammonium sulphate was found to be a suitable nitrogen source, considering the highest ratio of ethanol/xylitol noted among all the examined nitrogen sources (Table 1). Enzymes involved in D-xylose assimilation in different yeasts have different specificities for cofactors such as NAD(P)H and NAD (Lachke and Jeffries 1986; Alexander et al. 1988). In the case of C. shehatae, the NADPH-linked D-xylose reductase activity was threefold to fivefold higher than the NADH-linked activity as shown in Table 1. The ratio (0.2 1) of NADH-linked/NADPH-linked D-xylose reductase activities was high in urea- and peptone-grown C. shehatae cells. The ratio was lower when inorganic nitrogen and asparagine were incorporated as nitrogen sources. The organic nitrogen sources were found to increase the levels of xylitol dehydrogenase in contrast to inorganic nitrogen sources (Table 1). The results showed that high NADH-linked/NADPHlinked D-xylose reductase activities may play a pivotal role for enhanced ethanol production. This effect could be due to a closed-loop pathway for the oxidation and reduction of NADH in the formation of D-xylulose, thereby preventing accumulation of NADH, which suppresses this conversion (Jeffries 1985). The data of this study, indicating improved yields of ethanol with inclusion of organic nitrogen sources, could be useful for designing continuous controlled fermentation for optimizing ethanol yields from C. shehatae. Moreover, the efficiency of simultaneous saccharification and fermentation of hydrolysates of agricultural residues by C. shehatae (Palnitkar and Lachke 1990) can also be increased.
'.
Acknowledgement A Senior Research Fellowship from the Council for Scientific and Industrial Research (India) to S.S.P. is gratefully acknowledged.
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