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Fermentation of d -xylose-1-C14 by Fusarium lini Bolley
Fermentation Martin From
of D -Xylose-1 44 by Fusarium
Bolleyl
Zini
Gibbs, Vincent W. Cochrane, L. M. Paege and Harold the
Department York;
of Biology, Rrookhaven and from the Department University, Middletown,
Received November
National Laboratory, of Biology, Wesleyan Connecticut
Upton,
Wolh? New
9, 1953
The dissimilation of hexose by anaerobically growing cultures of the fungus Fusarium has been shown to correspond to that of yeast in which hexose yields 2 moles of ethanol and 2 moles of carbon dioxide (l-3). Cultures of Fusaria growing anaerobically on n-xylose and L-arabinose yield the same products; however, the ratio of alcohol to carbon dioxide is 1: 2 (l-3). According to Dietrich and Klammerth (4), during early growth Fusarium lini Bolley (FlB) produces 2 moles of ethyl alcohol per mole of xylose. Besides ethanol and COz , they reported the presence of acetaldehyde and acetic acid in the media of FlB growing on xylose. These authors postulate cleavage of the pentose chain into CZ and CZ fragments, the C3 fragment giving rise to carbon dioxide and ethyl alcohol via pyruvic acid while three C& fragments combine to yield hexose which is further metabolized to ethyl alcohol and carbon dioxide via the Embden-Meyerhof-Parnas pathway. Cleavage of pentose is also indicated by the isolation of pyruvic acid from cultures of FlB grown on various pentoses (5). Evidence for the existence of a C&--C& cleavage is provided by the manner in which Lactobacillus pentosus (6) and Lactobacillus pentoaceticus (7) ferment pentoses labeled in t,he aldehyde carbon with CY4. 1 Part of this research was carried out at the Brookhaven National Laboratory under the auspices of the U. S. Atomic Energy Commission, and part was performed with the aid of a research grant, No. G-3154 (C), from the National Institutes of Health. 2 Present address: Department of Bacteriology, Cornell University, Ithaca, New York. 237
238
GIBBS,
COCHRA4NE,
PAEGE
AND
WOLIN
These bacteria ferment the l-labeled pentose into unlabeled lactic acid and methyl-labeled acetic acid. The presence of the tracer exclusively in one carbon atom strongly suggests cleavage into C& and Cz fragments in the Lactobacilli. In order to test whether the fungus FIB carries out a similar cleavage, we have investigated its pathway of pentose dissimilation with D-XylOSel-CJ4. It was evident, however, that the stoichiometry of pentose dissimilation by FIB had to be established. Using n-xylose-grown resting cells of FIB in place of growing cultures, it was found that each mole of pentose was fermented to equimolar parts of carbon dioxide, ethyl alcohol, and acetic acid. METHODS The fungus used was Fusarium Zini Bolley kindly supplied by Dr. F. F. Nord of Fordham University. The stock strain of the organism was propagated at 30°C. in test tubes on slants containing, per 100 ml., 1 g. of glucose and 2.3 g. of Difco nutrient agar. The organism was grown in 250-ml. Erlenmeyer flasks containing 50 ml. of medium of the following composition (per liter): proteose peptone No. 3 (Difco) 20 g.; beef extract (Difco) 3 g.; yeast extract (Difco) 3 g.; malt extract (Difco) 3 g.; n-xylose (Pfanstiehl) 10 g. The sugar was sterilized separately and added aseptically to the growth medium. The flasks inoculated directly from the stock test tubes were placed on a rotary shaker (New Brunswick Scientific Co.). The shaker was located in a room maintained at 25-28°C. After 2-3 days, the cells were centrifuged off and washed thoroughly by filtration. The cell material was weighed, suspended in 4 ml. water/g., and blended in a Waring blendor for 30 sec. Following a 5-min. aeration with prepurified nitrogen, the cell material was centrifuged and weighed. It was suspended in 1 ml. water/g. cell material. Each flask yielded about 6-7 g. cell material. For the balance and tracer studies, 1 ml. of FIB suspension was added to the main compartment of a double side-arm Warburg vessel containing 0.5 ml. of 0.1 M phosphate buffer, pH 6.4. The side arm carrying the venting plug contained 0.1 ml. of 5 N HzSOr while the other arm contained xylose. For the balance studies, 0.2 or 0.3 ml. of 0.1 A4 xylose was used. In the tracer studies, 0.2 ml. of 0.21 $1 n-xylose-1-C” containing 73 millimicrocuries (mGc.) with a specific activity of 29 wc./mg. carbon was used. This sugar was kindly given to us by Dr. H. Isbell of the National Bureau of Standards. The flask was aerated for 20 min. with prepurified nitrogen, brought to equilibrium, and the reaction started by tipping in the xylose. The reaction was stopped by the addition of H&O4 . The bath temperature was 30°C. In the balance studies, after the volume of CO* produced was recorded, the cell suspension was removed by centrifugation and washed with water. The supernatant and washings were made to volume and analyzed for residual xylose (8) and the fermentation products of D-xylose. In addition to COZ , ethyl alcohol and acetic acid were found. The alcohol was determined enzymatically using crystalline yeast alcohol
D-XYLOSE
FERVENTATlON
BY FLB
239
dehydrogenase (9) prepared by the method of Racker (10) or titrimetrically according to Neish (11). The presence of acetic acid was indicated by a positive reaction with lanthanum nitrate. The supernatant solution of the fermentation was also chromatographed on a diatomaceous earth (Celite) column according to the method of Bueding and Yale (12), as modified by Kohlmiller and Gest (13), and found to contain only acetic acid. The acid was also identified by determination of the Duclaux numbers. The products of the xylose-1-W fermentation were separated and degraded in the following manner. The 0402 was collected and counted by a previously described method (14). After removal of the CO2 , carrier alcohol (555 rmoles) and sodium acetate (397 pmoles) were added, and the cell material was removed by centrifugation. The supernatant solution and washings were made to volume. Aliquots were analyzed for residual pentose. The remainder of the solution was made alkaline to phenol red with KOH and distilled to separate the alcohol. The alcohol was degraded by conversion to acetic acid by heating at 99°C. for 2 hr. with 0.5 g. of K&r201 in 4 N H&SO1 . The residue was adjusted to pH l-2 with HzSO, and steam distilled to isolate the acetic acid. The acetic acid formed from the alcohol and the preformed acetic acid were degraded by the method of Phares (15). All Cl4 assays were carried out with barium carbonate. Radioactivity was determined using a methane-flow beta proportional counter (16). RESULTS
The results tabulated in Table I give evidence that resting cells of FIB ferment xylose to correspond to the stoichiometry of: 1 xylose --+ 1 CO2 + 1 ethyl alcohol + 1 acetic acid. It is evident that the fermentation of xylose by the resting cell suspension is not the same as with growing cultures. It is also to be noted that when FlB is grown with pentose as a carbon source, only traces of acetic acid are found (4). As indicated, the endogenous values are high. Attempts to lower the endogenous values by using older cells were not successful since the older cells fermented the exogenous pentose poorly. Younger cells whose endogenous reserves were destroyed by starvation or prolonged aeration were not suitable, since a reduction in the endogenous rate caused a proportionate or more than proportionate decrease in the rate of xylose fermentation. From the degradation data tabulated in Table II, it is evident that the alcohol and COn contained comparatively little C14, while most of the recovered label appeared in the methyl carbon of the acetic acid. In the two recorded experiments, pentose containing approximately 54 mpc. of the added 73 mpc. was fermented. The isotope recovery in the products was low, about 63 %; also, the specific activity of the acetic acid methyl carbon was lower than expected. Since the specific activity of the added xylose was 29 mpc./mg. C, the specific activity of the methyl
240
GIBBS,
Fermentation
Expt. 1. Fermentation Exut. 2. Fermentation Expt.
PAEGE
Xylose
in Productr
Formed
used
-
_
Products
co2
Ethanol
pmoles
pmoles
!.moles
17.8 17.8 17.1 17.1
32.9 16.5 16.4 30.4 14.3 16.1
21.8 7.0 14.8 19.7 8.2 11.5
:
TABLE
of Label
Distribution
WOLIN
TABLE I D-Xylose by Fusarium lini Bolley time, 285 min. Alcohol determined enzymatically. time, 300 min. Alcohol determined titrimetricallv.
20 rmoles xylose added No xylose added Net change 30 rmoles xylose added No xylose added Net change
I
AND
of
Conditions
1
2
COCHRANE,
-I
-
4cetic
acid
pmoles
40.4 20.8 19.6 40.4 28.1 12.3
II
During lini Bolley
Fermentation
D-Xy10Se-l-Ct4
by
Fusarium Fermentation time was 300 minutes. Xylose
Products --___ Carbon
Expt.
Pentose
added
Pentose
dioxide
____.
Ethanol
Acetic
acid
used co2
CHa _____ ?lSfl‘.
____
1 2
formed
mpc.
/Lt??OlL?S
mpc.
54.0 55.0
25.8’ 25.0~
2.1 1.6
CHzOH mpc.
0 1.2 0 , 0.1
CHa _____ mpc.
COOH !
mpc.
28.1 1.4 26.9 , 1.1
D Corrected
for endogenous.
carbon of the acetic acid should have approached 145 mpc./mg. C. This value was 87 mpc./mg. C in Exp. 1 and 86 mpc./mg. C in Exp. 2. This low value is, in part, caused by the occurrence of tracer in other positions. Taking this into account, t,he expected specific activity would have been 125 mpc./mg. C. The low values could also be caused by the low recoveries of the tracer. DISCUSSION
The data presented show that the methyl carbon of acetic acid is derived from the aldehyde carbon (C-l) of xylose. Assuming that carbon atom 2 (C-2) of xylose gives rise to the carboxyl carbon of acetic acid, the remaining three carbon atoms would be the precursor of the carbon
WXYLOSE
FERMENTATION
BY FLB
241
dioxide and ethanol. These results are similar to those obtained with bacteria (6, 7) and would offer strong evidence that FlB dissimilates pentose initially into Cz and Ca fragments with the CZ fragment being the precursor of the acetic acid. Gest and Lampen (6) postulate that the compound cleaved is a 2-ketopentose phosphate which has a ribose configuration. In view of recent publications (17, 18) it would appear that the cleavage compound is ribulose 5-phosphate. Transketolase, the enzyme responsible for this fission, has been demonstrated in yeast (18) and spinach leaves (19). Assuming that ribulose 5-phosphate is the compound cleaved during xylose dissimilation by FlB, this splitting is probably not catalyzed by a transketolase similar to that isolated from yeast and spinach leaves since this’enzyme causes cleavage only in the presence of an “acceptor aldehyde.” If the CZ fragment were attached to another carbon fragment before its conversion to acetic acid, it is unlikely that the tracer would appear exclusively in the acetic acid methyl group (20). The yield of products in these experiments is not the same as that obtained with growing cultures. Both types of fermentations yield 1 mole of ethanol per mole of pentose; however, resting cell suspensions produce acetic acid equivalent to alcohol, while growing cultures produce only traces of acetic acid. The possibility exists that the growing cells metabolize the CZ fragment in a manner different from that of the resting cell, e.g., that growing cells convert the Cz fragment to lipide, while resting cells produce acetic acid. Another explanation is a further metabolism of acetic acid produced by the growing cultures. This possibility is supported by the work of Coleman and Nord (21) who demonstrated that, under special conditions, growing cultures of Fusaria could utilize acetate as a sole carbon source for growth. As growing Fusaria can also metabolize ethyl alcohol (22), this would suggest a preferential dissimilation of the acetic acid over the ethanol since the latter is produced in the same yield in both fermentations. This preferential dissimilation may be caused by the nonavailability of ethanol for growth by proliferating cells so long as pentose is present (5). The difference in yield data between pentose dissimilation by growing cultures and resting cultures of FIB is still not resolved. SUMMARY
Resting cells of the fungus Fusarium Zini Bolley (FlB) ferment one mole of n-xylose to equimolar quantities of carbon dioxide, ethyl alcohol,
242
GIBBS,
COCHRANE,
PAEGE
AND
WOLIN
and acetic acid. This is in contrast to growing cultures which have been reported to yield only ethyl alcohol and carbon dioxide in a ratio of 1:2. When FlB fermented n-xylose-1-C14, only the acetic acid methyl carbon was found to contain appreciable tracer. This is in agreement with pentose dissimilation by Lactobacillus pentosus and Lactobacillus pentoace&s and indicates that molds as well as bacteria cleave the pentose chain into Cz and C3 fragments. It is pointed out that if ribulose Sphosphate is the compound cleaved, then the enzyme catalyeing the cleavage is probably not transketolase. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 19. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
ANDERSON, A. K., Research Pubis. Univ. Minn. Biol. Sci. 6, 237 (1924). WHITE, M. G., AND WILLAMANN, J. J., Biochem. J. 22, 583 (1928). NORD, F. F., DAMMANN, E., AND HOFSTETTER, H., Biochem. 2.286,241 (1936). DIETRICH, K. R., AND KLAMMERTH, 0. 0. L., Chem.-Ztg. 63, 763 (1939). WIRTH, J. C., AND NORD, F. F., Arch. Biochem. 1, 143 (1942); J. Am. Chem. Sot. 63, 2855 (1941). GEST, H., AND LAMPEN, J. O., J. Biol. Chem. 194, 555 (1952). RAPPOPORT, D. A., BARKER, H. A., AND HASSID, W. Z., Arch. Biochem. and Biophys. 31, 326 (1951). MEJBAUM, W., Z. physiol. Chem. 268, 117 (1939). BONNICHSEN, R. K., AND THEORELL, H., Stand. J. Clin. & Lab. Invest. 3, 58 (1951). RACKER, E., J. Biol. Chem. 184, 313 (1950). Methods for Bacterial Fermentations.” Nat. Res. NEISH, A. C., “Analytical Council Canada No. 2952. Saskatoon, 1952. BUEDI;~G, E., AND YALE, H., J. Biol. Chem. 193, 411 (1951). KOHLMILLER, E. F., AND GEST, H., J. Bacterial. 61, 269 (1951). GIBBS, M., AND GASTEL, R., Arch. Biochem. and Biophys. 33,448 (1951). PHARES, E. F., Arch. Biochem. and Biophys. 33, 173 (1951). BERNSTEIN, W., AND BALLENTINE, R., Rev. Sci. Instr. 20,347 (1949). HORECKER, B. L., AND SNYRNIOTIS, P. Z., J. Am. Chem. Sot. 74, 2123 (1952). RACKER, E., DE LA HABA, G., AND LEDER, I. G., J. Am. Chem. Sot. 76, 1010 (1953). HORECKER, B. L., AND SMYRNIOTIS, P. Z., J. Am. Chem. Sot. ‘76, 1009 (1953). HORECKER, B. L., GIBBS, M., KLENOW, H., AND SMYRNIOTIS, P. Z., J. Biol. Chew 207, 393 (1954). COLEMAN, R. J., AND NORD, F. F., Arch. Biochem. and Biophys. 33, 346 (1951). ROTINI, 0. T., DAMMANN, E., AND NORD, F. F., Biochem. Z. 238,414 (1936).
of D -Xylose-1 44 by Fusarium
Bolleyl
Zini
Gibbs, Vincent W. Cochrane, L. M. Paege and Harold the
Department York;
of Biology, Rrookhaven and from the Department University, Middletown,
Received November
National Laboratory, of Biology, Wesleyan Connecticut
Upton,
Wolh? New
9, 1953
The dissimilation of hexose by anaerobically growing cultures of the fungus Fusarium has been shown to correspond to that of yeast in which hexose yields 2 moles of ethanol and 2 moles of carbon dioxide (l-3). Cultures of Fusaria growing anaerobically on n-xylose and L-arabinose yield the same products; however, the ratio of alcohol to carbon dioxide is 1: 2 (l-3). According to Dietrich and Klammerth (4), during early growth Fusarium lini Bolley (FlB) produces 2 moles of ethyl alcohol per mole of xylose. Besides ethanol and COz , they reported the presence of acetaldehyde and acetic acid in the media of FlB growing on xylose. These authors postulate cleavage of the pentose chain into CZ and CZ fragments, the C3 fragment giving rise to carbon dioxide and ethyl alcohol via pyruvic acid while three C& fragments combine to yield hexose which is further metabolized to ethyl alcohol and carbon dioxide via the Embden-Meyerhof-Parnas pathway. Cleavage of pentose is also indicated by the isolation of pyruvic acid from cultures of FlB grown on various pentoses (5). Evidence for the existence of a C&--C& cleavage is provided by the manner in which Lactobacillus pentosus (6) and Lactobacillus pentoaceticus (7) ferment pentoses labeled in t,he aldehyde carbon with CY4. 1 Part of this research was carried out at the Brookhaven National Laboratory under the auspices of the U. S. Atomic Energy Commission, and part was performed with the aid of a research grant, No. G-3154 (C), from the National Institutes of Health. 2 Present address: Department of Bacteriology, Cornell University, Ithaca, New York. 237
238
GIBBS,
COCHRA4NE,
PAEGE
AND
WOLIN
These bacteria ferment the l-labeled pentose into unlabeled lactic acid and methyl-labeled acetic acid. The presence of the tracer exclusively in one carbon atom strongly suggests cleavage into C& and Cz fragments in the Lactobacilli. In order to test whether the fungus FIB carries out a similar cleavage, we have investigated its pathway of pentose dissimilation with D-XylOSel-CJ4. It was evident, however, that the stoichiometry of pentose dissimilation by FIB had to be established. Using n-xylose-grown resting cells of FIB in place of growing cultures, it was found that each mole of pentose was fermented to equimolar parts of carbon dioxide, ethyl alcohol, and acetic acid. METHODS The fungus used was Fusarium Zini Bolley kindly supplied by Dr. F. F. Nord of Fordham University. The stock strain of the organism was propagated at 30°C. in test tubes on slants containing, per 100 ml., 1 g. of glucose and 2.3 g. of Difco nutrient agar. The organism was grown in 250-ml. Erlenmeyer flasks containing 50 ml. of medium of the following composition (per liter): proteose peptone No. 3 (Difco) 20 g.; beef extract (Difco) 3 g.; yeast extract (Difco) 3 g.; malt extract (Difco) 3 g.; n-xylose (Pfanstiehl) 10 g. The sugar was sterilized separately and added aseptically to the growth medium. The flasks inoculated directly from the stock test tubes were placed on a rotary shaker (New Brunswick Scientific Co.). The shaker was located in a room maintained at 25-28°C. After 2-3 days, the cells were centrifuged off and washed thoroughly by filtration. The cell material was weighed, suspended in 4 ml. water/g., and blended in a Waring blendor for 30 sec. Following a 5-min. aeration with prepurified nitrogen, the cell material was centrifuged and weighed. It was suspended in 1 ml. water/g. cell material. Each flask yielded about 6-7 g. cell material. For the balance and tracer studies, 1 ml. of FIB suspension was added to the main compartment of a double side-arm Warburg vessel containing 0.5 ml. of 0.1 M phosphate buffer, pH 6.4. The side arm carrying the venting plug contained 0.1 ml. of 5 N HzSOr while the other arm contained xylose. For the balance studies, 0.2 or 0.3 ml. of 0.1 A4 xylose was used. In the tracer studies, 0.2 ml. of 0.21 $1 n-xylose-1-C” containing 73 millimicrocuries (mGc.) with a specific activity of 29 wc./mg. carbon was used. This sugar was kindly given to us by Dr. H. Isbell of the National Bureau of Standards. The flask was aerated for 20 min. with prepurified nitrogen, brought to equilibrium, and the reaction started by tipping in the xylose. The reaction was stopped by the addition of H&O4 . The bath temperature was 30°C. In the balance studies, after the volume of CO* produced was recorded, the cell suspension was removed by centrifugation and washed with water. The supernatant and washings were made to volume and analyzed for residual xylose (8) and the fermentation products of D-xylose. In addition to COZ , ethyl alcohol and acetic acid were found. The alcohol was determined enzymatically using crystalline yeast alcohol
D-XYLOSE
FERVENTATlON
BY FLB
239
dehydrogenase (9) prepared by the method of Racker (10) or titrimetrically according to Neish (11). The presence of acetic acid was indicated by a positive reaction with lanthanum nitrate. The supernatant solution of the fermentation was also chromatographed on a diatomaceous earth (Celite) column according to the method of Bueding and Yale (12), as modified by Kohlmiller and Gest (13), and found to contain only acetic acid. The acid was also identified by determination of the Duclaux numbers. The products of the xylose-1-W fermentation were separated and degraded in the following manner. The 0402 was collected and counted by a previously described method (14). After removal of the CO2 , carrier alcohol (555 rmoles) and sodium acetate (397 pmoles) were added, and the cell material was removed by centrifugation. The supernatant solution and washings were made to volume. Aliquots were analyzed for residual pentose. The remainder of the solution was made alkaline to phenol red with KOH and distilled to separate the alcohol. The alcohol was degraded by conversion to acetic acid by heating at 99°C. for 2 hr. with 0.5 g. of K&r201 in 4 N H&SO1 . The residue was adjusted to pH l-2 with HzSO, and steam distilled to isolate the acetic acid. The acetic acid formed from the alcohol and the preformed acetic acid were degraded by the method of Phares (15). All Cl4 assays were carried out with barium carbonate. Radioactivity was determined using a methane-flow beta proportional counter (16). RESULTS
The results tabulated in Table I give evidence that resting cells of FIB ferment xylose to correspond to the stoichiometry of: 1 xylose --+ 1 CO2 + 1 ethyl alcohol + 1 acetic acid. It is evident that the fermentation of xylose by the resting cell suspension is not the same as with growing cultures. It is also to be noted that when FlB is grown with pentose as a carbon source, only traces of acetic acid are found (4). As indicated, the endogenous values are high. Attempts to lower the endogenous values by using older cells were not successful since the older cells fermented the exogenous pentose poorly. Younger cells whose endogenous reserves were destroyed by starvation or prolonged aeration were not suitable, since a reduction in the endogenous rate caused a proportionate or more than proportionate decrease in the rate of xylose fermentation. From the degradation data tabulated in Table II, it is evident that the alcohol and COn contained comparatively little C14, while most of the recovered label appeared in the methyl carbon of the acetic acid. In the two recorded experiments, pentose containing approximately 54 mpc. of the added 73 mpc. was fermented. The isotope recovery in the products was low, about 63 %; also, the specific activity of the acetic acid methyl carbon was lower than expected. Since the specific activity of the added xylose was 29 mpc./mg. C, the specific activity of the methyl
240
GIBBS,
Fermentation
Expt. 1. Fermentation Exut. 2. Fermentation Expt.
PAEGE
Xylose
in Productr
Formed
used
-
_
Products
co2
Ethanol
pmoles
pmoles
!.moles
17.8 17.8 17.1 17.1
32.9 16.5 16.4 30.4 14.3 16.1
21.8 7.0 14.8 19.7 8.2 11.5
:
TABLE
of Label
Distribution
WOLIN
TABLE I D-Xylose by Fusarium lini Bolley time, 285 min. Alcohol determined enzymatically. time, 300 min. Alcohol determined titrimetricallv.
20 rmoles xylose added No xylose added Net change 30 rmoles xylose added No xylose added Net change
I
AND
of
Conditions
1
2
COCHRANE,
-I
-
4cetic
acid
pmoles
40.4 20.8 19.6 40.4 28.1 12.3
II
During lini Bolley
Fermentation
D-Xy10Se-l-Ct4
by
Fusarium Fermentation time was 300 minutes. Xylose
Products --___ Carbon
Expt.
Pentose
added
Pentose
dioxide
____.
Ethanol
Acetic
acid
used co2
CHa _____ ?lSfl‘.
____
1 2
formed
mpc.
/Lt??OlL?S
mpc.
54.0 55.0
25.8’ 25.0~
2.1 1.6
CHzOH mpc.
0 1.2 0 , 0.1
CHa _____ mpc.
COOH !
mpc.
28.1 1.4 26.9 , 1.1
D Corrected
for endogenous.
carbon of the acetic acid should have approached 145 mpc./mg. C. This value was 87 mpc./mg. C in Exp. 1 and 86 mpc./mg. C in Exp. 2. This low value is, in part, caused by the occurrence of tracer in other positions. Taking this into account, t,he expected specific activity would have been 125 mpc./mg. C. The low values could also be caused by the low recoveries of the tracer. DISCUSSION
The data presented show that the methyl carbon of acetic acid is derived from the aldehyde carbon (C-l) of xylose. Assuming that carbon atom 2 (C-2) of xylose gives rise to the carboxyl carbon of acetic acid, the remaining three carbon atoms would be the precursor of the carbon
WXYLOSE
FERMENTATION
BY FLB
241
dioxide and ethanol. These results are similar to those obtained with bacteria (6, 7) and would offer strong evidence that FlB dissimilates pentose initially into Cz and Ca fragments with the CZ fragment being the precursor of the acetic acid. Gest and Lampen (6) postulate that the compound cleaved is a 2-ketopentose phosphate which has a ribose configuration. In view of recent publications (17, 18) it would appear that the cleavage compound is ribulose 5-phosphate. Transketolase, the enzyme responsible for this fission, has been demonstrated in yeast (18) and spinach leaves (19). Assuming that ribulose 5-phosphate is the compound cleaved during xylose dissimilation by FlB, this splitting is probably not catalyzed by a transketolase similar to that isolated from yeast and spinach leaves since this’enzyme causes cleavage only in the presence of an “acceptor aldehyde.” If the CZ fragment were attached to another carbon fragment before its conversion to acetic acid, it is unlikely that the tracer would appear exclusively in the acetic acid methyl group (20). The yield of products in these experiments is not the same as that obtained with growing cultures. Both types of fermentations yield 1 mole of ethanol per mole of pentose; however, resting cell suspensions produce acetic acid equivalent to alcohol, while growing cultures produce only traces of acetic acid. The possibility exists that the growing cells metabolize the CZ fragment in a manner different from that of the resting cell, e.g., that growing cells convert the Cz fragment to lipide, while resting cells produce acetic acid. Another explanation is a further metabolism of acetic acid produced by the growing cultures. This possibility is supported by the work of Coleman and Nord (21) who demonstrated that, under special conditions, growing cultures of Fusaria could utilize acetate as a sole carbon source for growth. As growing Fusaria can also metabolize ethyl alcohol (22), this would suggest a preferential dissimilation of the acetic acid over the ethanol since the latter is produced in the same yield in both fermentations. This preferential dissimilation may be caused by the nonavailability of ethanol for growth by proliferating cells so long as pentose is present (5). The difference in yield data between pentose dissimilation by growing cultures and resting cultures of FIB is still not resolved. SUMMARY
Resting cells of the fungus Fusarium Zini Bolley (FlB) ferment one mole of n-xylose to equimolar quantities of carbon dioxide, ethyl alcohol,
242
GIBBS,
COCHRANE,
PAEGE
AND
WOLIN
and acetic acid. This is in contrast to growing cultures which have been reported to yield only ethyl alcohol and carbon dioxide in a ratio of 1:2. When FlB fermented n-xylose-1-C14, only the acetic acid methyl carbon was found to contain appreciable tracer. This is in agreement with pentose dissimilation by Lactobacillus pentosus and Lactobacillus pentoace&s and indicates that molds as well as bacteria cleave the pentose chain into Cz and C3 fragments. It is pointed out that if ribulose Sphosphate is the compound cleaved, then the enzyme catalyeing the cleavage is probably not transketolase. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 19. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
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