Formation of Extracellular Sphingolipids by MicroorganismsIV. Pilot-Plant Production of Tetraacetylphytosphingosine by Hansenula ciferrii

HANNS G. MAISTER, S. PETER ROGOVIN, FRANK H. STODOLA, AND LYNFERD J. WICKERHAM

Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Illinois

Received for publication March 2, 1962

ABSTRACT

MAISTER, HANNS G. (Northern Regional ResearchLaboratory, U.S. Department of Agriculture, Peoria,I11.), S. PETER ROGOVIN, FRANK H. STODOLA, ANDLYNFERD J. WICKERHAM. Formation of extracellularsphingolipids by microorganisms. IV. Pilot-plant produc-tion of tetraacetylphytosphingosine by Hansenula ciferrii.Appl. Microbiol. 10:401-406. 1962.-Tetraacetylphyto-sphingosine (TAPS) formation by the F-60-10 mating typestrain of the yeast Hansenula ciferrii, previously observedon agar plates, has been shown to take place in submergedcultures. The optimal conditions for TAPS formation, andthe correlation of TAPS production and sugar utilizationunder aerobic conditions, were studied in 10-liter fermen-tors. For each gram of glucose consumed, 5 mg of TAPSwere formed; for each gram of yeast solids produced, 15mg of TAPS were synthesized. A 750-liter pilot-plant runyielded 175 g of crude TAPS, which were obtained byhexane extraction of centrifuged yeast cells.

As a member of the important class of compounds knownas the sphingolipids, phytosphingosine

OH OH NH2

CH3 (CH2)13-CH-CH-CH-CH20H

has been of interest to biochemists since its discovery byOda (1952). Its isolation in quantity, however, presentedcertain difficulties because the complicated compounds, ofwhich it is a part, had first to be isolated and then sub-jected to hydrolytic and fractionation procedures. Betterprospects of its availability appeared when Wickerhamand Stodola (1960) reported in the first paper of thisseries that the yeast Hansenula ciferrii secretes thecrystalline tetraacetyl derivative of phytosphingosine intothe culture medium, from which it can be isolated bysimple extraction with hexane. Their original work wasdone with solid media and shaken flasks; our work was toproduce tetraacetylphytosphingosine (TAPS) from aeratedcultures on a pilot-plant scale. If ample amounts of TAPSwere obtained for research purposes, the minor con-stituents shown by Stodola and Wickerham (1960) to bepresent in the crude product could be investigated.

This paper describes the development of a fermentationprocedure for producing on pilot-plant scale a larger

amount of TAPS for research purposes. It deals withstudies of (i) the correlation of TAPS formation andsugar utilization during the aerobic yeast growth, (ii) theproper harvesting time of the fermented medium, (iii)the yield of TAPS, and (iv) the effect of antifoam materialin four 10-liter experimental runs; it also reports (v)results of pilot-plant runs with 750 liters of medium, andthe crude TAPS recovery by hexane extraction of thecentrifuged yeast cells.

MATERIALS AND METHODS

The medium used for inoculum preparation and for themedia of the experimental and pilot-plant runs was theyeast maintenance (YM) medium of Wickerham (1951). Itcontained (per liter of tap water) 3 g each of yeast extractand malt extract, 5 g of peptone, and 30 g of technicalglucose monohydrate. The pH of the YM medium was 6.5to 6.8.The experimental runs were conducted with 10 liters of

medium in the 20-liter fermentors described byDworschack, Lagoda, and Jackson (1954). Corman et al.(1957) studied aeration effects in these fermentors fordifferent air-flow rates, agitator speeds, and air pressures.The aeration effect is expressed as the oxygen-absorptionrate (OAR) in millimoles of oxygen per liter per minuteas determined by the sodium sulfite method of Cooper,Fernstrom, and Miller (1944).The inoculum for setting the 10-liter experimental runs

was prepared in 2,800-ml Fernbach flasks, each containing500 ml of YM medium. YM slants were heavily inoculatedand incubated at 25 C for 48 hr. The entire growth fromtwo slants was used to inoculate one Fernbach flask,which was incubated on a reciprocal shaker at 25 C for 2days. The contents of two Fernbach flasks formed theinoculum for one fermentor. The inoculum used wastherefore 10 % for the 10-liter runs.The pilot-plant runs were made in a 600-gal stainless-

steel fermentor described by Pfeifer et al. (1958), with amedium volume of 757 liters (200 gal) and an OAR of 0.8.A 60-gal seed tank served as a sterilization and storagevessel for the concentrated glucose solution, which wasadded continuously to the fermenting medium in the600-gal fermentor. The inoculum for the pilot-plant runswas grown in the 20-liter fermentors as described pre-viously. Four fermentors, each with 10 liters of YM

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medium (4 g of glucose monohydrate/100 ml), were

inoculated with 500 ml of fermenting medium of the secondstep and fermented for 48 hr with an OAR = 0.8 at 25 C.The contents of the four fermentors were combined andtransferred aseptically to the 600-gal fermentor. Theinoculum was 5.3 %. Samples of fermenting medium takenat intervals were analyzed for dissolved solids concen-

tration, glucose content, TAPS formation, and pH. Thedissolved solids concentration was measured with theBalling (Bllg) saccharometer. Glucose was determined bythe method of Shaffer and Hartmann (1921). TAPS was

obtained by extracting the medium twice with one-tenthvolumes of petroleum ether (bp 40 to 60 C), centrifuging,and filtering the ether layer. Removal of the solvent left anoil that crystallized at 0 C. This procedure cannot beconsidered as specific for TAPS; however, Stodola andWickerham (1960) found that the material so obtainedcontained over 85 % TAPS. The TAPS values reported inthis paper refer to the crude product obtained on evapora-

tion of the petroleum ether from the extract.To determine the extracellular distribution of TAPS in

the fermented medium (FM), a portion was centrifuged for30 min at 500 X g. The sedimented yeast and the super-

natant liquid were analyzed for TAPS, and the amount ofTAPS determined.

RESULTS

The first attempt to produce TAPS was made in a

series of runs (run I) in the 20-liter baffled fermentorcharged with 10 liters of YM medium (2.69 g of glucose/100 ml). The air flow was one-half volume of air per

volume of medium per min; agitator speed was 120 rev/min and air pressure in the fermentor was 25 psi. Theseconditions gave an OAR of 0.75. The fermentation tem-perature was held at 25 C and the runs lasted for 10 days.This extended fermentation period was selected at thetime because it was believed that the extracellular forma-tion of TAPS took place after the yeast growth had ceased.In these runs, a heavy, very sticky foam developed at theend of the first fermentation day and lasted for 24 to 36hr. Some of the fermentors foamed so vigorously thatculture medium was lost. No antifoam was used becauseof possible difficulty in the ether extraction of TAPS. Theyield of TAPS in these runs ranged from 0 to 100 mg/literand was not reproducible.Such erratic results necessitated a more detailed study of

growth behavior of the yeast in order to obtain therequired information for pilot-plant production. For thispurpose, two 20-liter fermentors (run II) were set underthe conditions used in run I, but with an increased amountof glucose in the medium. The glucose content of run I

(3.0 g/100 ml) was apparently insufficient for optimalyeast growth under strong aeration. In run II, the glucosecontent was increased to 4.45 g/100 ml by adding 500 mlof a 36% (w/v) glucose solution to the medium 22 hr afterthe start of the fermentation. This timing was selected to

minimize foam formation. Samples were taken daily andanalyzed for pH, glucose, and TAPS (Fig. 1). Contrary toour original belief, the data indicate clearly that TAPS was

formed during the dissimilation of the glucose by the grow-

ing yeast. The peak of TAPS formation was reached in 3days fermentation time simultanecusly with exhaustion ofglucose. At this time, pH dropped to the lowest level, 4.2,and there were 6.6 mg of TAPS formed per g of glucoseconsumed. A sample taken then showed strong flocculationof the yeast. On continued aeration beyond the third day,the amount of TAPS declined from a peak of 270 mg/literon the 3rd day to 160 mg at the end of the 7th day. Mean-while, the pH remained fairly constant, presumably owingto the buffering action of yeast autolysis products. Therun was terminated after the 7th day, since the mainquestion, the mode of TAPS formation, had been answered.The close correlation between TAPS formation and

glucose dissimilation indicated that a still further increasein glucose content might improve the yield of TAPSaccordingly. To test this point, run III was made with two20-liter fermentors (A and B), each charged with 10 litersof YM medium of increased glucose content. Table 1 givesthe operational conditions of the run.

The inoculum was prepared as previously described,except that the glucose content of the inoculum medium

Days

FIG. 1. Glucose, pH, and tetraacetylphytosphingosine (TAPS)data of run II; 10 liters of YM medium (4.45 g of glucose/100 ml).OAR = 0.75; fermentation temperature: 25 C.

TABLE 1. Operational conditions of run III

Condition Fermentor A Fermentor B

Volume of set medium (liters) ........... 9 9Glucose in set medium (g) ............... 356 356Glucose added at 18 hr (g) ............... 200 200Glucose added at 25 hr (g) ............... 200 200Total volume of medium (liters) ......... 10 10Total glucose content (g/100 ml) ......... 7.6 7.6Air flow (volumes per min) .............. 1Y2 12Agitator speed (rev/min) ................ 120 200Air pressure in fermentor (psi) ........... 25 5OAR (mmoles 02 per liter per min) 0.75 1.0

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was increased from 2.7 to 3.0 g/100 ml. The course of thefermentations can be followed in Fig. 2 and 3.As in run II, the peak formation of TAPS occurred when

the sugar was exhausted, although in fermentor B therewas some consumption of glucose on the 4th day withoutadded TAPS formation. From the 25th to the 75th fer-mentation hour, the amount of TAPS formation per gram

of glucose fermented was 7.9 mg in A and 9.7 mg in B. Atharvest time (96 hr), 290 mg of TAPS/liter of FM were

formed in A and 324 mg in B.After 12 hr of aeration, the media of both fermentors

formed a very sticky foam which filled the entire freespace above the medium but did not cause an overflow.After 48 hr, vigorous flocculation of the yeast began andthe foam disappeared.An unexpected phenomenon occurred during the storage

of FM. After sampling, the harvested media were storedfor convenience (week end) at 2 C for 65 hr. When re-

analyzed, the apparent TAPS content in A had increasedfrom 290 mg to 395 mg/liter of FM and in B from 324 to485 mg. All of the TAPS was in the centrifuged yeastsludge, with none in the supernatant.

.C

8.4

07.4

0

3Ef 6.(

E= 5.10

c- 3.'o-W 2.1q

'D 1.1

TAPS0 ci-~~~~40i

0-~~~~~~~~3...... pH

0 -'A A. . 20

o , 101

0 Glucose

244n2 ,--j-u 24 48 72Hours

4 Fermentation

96 "<160

Storage

0

2-

1-

EoE

FIG. 2. Glucose, pH, and tetracetylphytosphingosine (TAPS)data of ruin III; fermentor A, 10 liters of YM medium (7.56 g ofglucose/100 ml); OAR = 0.75; fermentation temperature: 25 C.

TAPSW 500

.v8.0 _400 E

7.0

4.0XoooT\. ~---A|..A4. l200o13.0~~ ~ ~ ~~~~~0

E2.0 %. 100

41 .0 O20 24 48 721961600

Hours

Fermentation

FIG. 3. Glucose, pH, and TAPS data of run III;fermentor B; 10liters of YM medium (7.56 g of glucose/100 ml); OAR = 1.0;fermentation temperature: 25 C.

Although runs II and III appeared to supply theinformation needed for pilot-plant runs, our first attemptwith a 200-gal fermentor (run P1) failed; much of themedium was lost through excessive foaming. Since an

antifoam agent appeared necessary in the pilot-plant runs,

run IV was made to determine the effect of a silicone underthe conditions used with fermentor B, run III. A total of5 g of antifoam was used; half was added to the startingmedium and the rest to the glucose solution added duringthe fermentation. As in previous runs, the medium startedto foam after 12 hr of aeration; the foam, however, was

fluffy and broke up easily. The antifoam did not appear tointerfere with TAPS extraction.

Pilot-plant preparation. In a pilot-plant run withinadequate cooling facilities (run P2), the temperatureclimbed to 32 C and stayed there during the entire run.

This elevated temperature strongly influenced the TAPSformation and its extracellular distribution. After 48 hr,310 mg/liter were formed; 90 mg were in the centrifugatedyeast sludge, and 210 mg in the supernatant. Upon con-

tinued aeration, the TAPS decomposed and after 4 daysonly 120 mg/liter was in the FM, 65 mg in the yeastsludge, and 55 mg in the supernatant. The yeast did notflocculate. This low TAPS content did not warrantextraction and recovery.

Another pilot-plant run (P3) was made with bettercooling. The set medium (757 liters) contained (per 100ml): 6.5 g of technical grade glucose monohydrate, 0.3 g ofyeast extract, 0.3 g of malt extract, and 0.48 peptone(Difco). All the ingredients, with the exception of 46.3 %of the total amount of glucose, were dissolved in tap waterand steam sterilized continuously with the procedure andequipment described by Pfeifer and Vojnovich (1952).The medium was heated to 135 C, held at this temperaturefor 4 min, cooled at 25 C, and pumped into the fermentor.After adding 40 liters of inoculum, the aeration startedwith an OAR = 0.8. The remaining 46.3 % glucose was

dissolved in 110 liters of tap water, sterilized batchwise inthe seed tank, and cooled. All of the sterile glucose solutionwas added continuously to the fermentation between the18th and 24th hr. Antifoam (75 g) was added and sterilizedwith the starting medium, and 312 g additional were usedduring the fermentation, to combat strong foaming. It wascontemplated to make the run with an OAR = 0.8; how-ever, this aeration rate could not be maintained becauseof heavy foam, despite the use of an antifoam agent. TheOAR was therefore decreased after 18 hr of fermentationto an OAR = 0.3 and held at this level until the end of the3rd fermentation day. On the 4th day, the aeration ratewas increased to OAR = 0.8 and held until harvest (89hr). The data on glucose dissimilation, yeast solids, andTAPS formation are compiled in Table 2.Only 8.5 g of yeast solids and 123 mg of TAPS were

formed per liter of FM during 69 hr of fermentation with

OAR = 0.3. In the next 20 hr, when the OAR was in-creased to 0.8, the yeast solids went up to 21.8 g/liter and

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the TAPS content to 203 mg. With a lowered OAR, moreorganic acids were formed, since the pH dropped to 3.75compared to 4.2 to 4.4 at a higher OAR in 20-liter runs I,II, and III. The 21- and 45-hr-old samples showed thesame glucose content, which indicated no dissimilation ofglucose, although during this period the 110-liter glucosesolution had been added.The FM after harvest was transferred to stainless-steel

containers and stored for 72 hr in a cold room (2 C). As inrun III, the TAPS content of the FM increased duringcold storage, from 203 to 324 mg/liter. The extracellulardistribution was 312 mg in the yeast sludge and 12 mg inthe supernatant fluid. Total TAPS of the 753.3-liter FMwas 244 g; the yeast sludge contained 235 g and thesupernatant fluid contained 9 g. The amount of TAPS was5.99 mg/g of glucose fermented, and the yeast produced anaverage of 14.8 mg/g of yeast solids formed.For crude TAPS recovery, the FM was centrifuged in a

Sharples Super Centrifuge and 24.68 kg of yeast sludge,with a solids content of 66.5 %, were obtained. The effluentwas discarded. Redistilled hexane (bp 69 C) was employedin the extraction.The first trial extraction duplicated the laboratory

extraction. About 5 ke of yeast sludge were slurried-upwith water, 1 liter of hexane added, and the mixtureagitated. This method produced a very dense emulsion

TABLE 2. Levels of pH, glucose content, yeast solids, andTAPS of samples taken during pilot-plant run P3

Age of sample (hr)Type of analysis

0 21 45 69 89 161*

pH 6.15 4.8 4.1 3.95 3.75 3.75Concn degree Bllg. 5.4 3.4 3.2 2.7 2.3 2.3Glucose (g/100 ml) 3.75 2.52 2.68 1.45 0.79 0.77Yeast solids, MFB (g/ 0.23 1.95 6.3i) 8.50 21.8 21.8

liter)TAPS content of yeast 10 14 90 105 188 312

sludge (mg/liter)Supernatant (mg/liter) 10 27 20 18 15 12Total in FM (mg/liter) 20 41 110 123 203 324TAPS formed/g yeast 12.2 14.8 12.5 8.5 14.1

solids (mg)

*Fermented medium stored for 72 hr at 2 C.

TABLE 3. Formation of tetraacetylphytosphingosine (TAPS)in laboratory and pilot-plant runs

Run TAPS formed (mg)/g of glucosedissimilated

II* 6.58III, Fermentor At 5.30III, Fermentor Bt 6.83P3t 5.99

* After 70 hr of fermentation.t TAPS formed during fermentation and storage at 2 C.

with very poor layer formation. The emulsion did notyield to either centrifugation or filtration and was thereforediscarded. In the extraction procedure finally adopted,the yeast sludge, without added water, was dispersed inhexane in the ratio of 1 kg sludge to 2 liters of hexane,then gently agitated. The hexane layer containing someyeast emulsion was decanted and filtered. The extractionwas effected batchwise, and each batch was extractedtwice. A total of 69.5 liters of extract was accumulated.Removal of the solvent yielded 175 g of a pale yellow oilthat crystallized on cooling to 5 C. The infrared curve andelementary analysis of this product indicated that it wasessentially TAPS. Found: C, 64.9; H, 9.90; N, 2.76:Calculated for C26H407N: C, 64.3; H, 9.76; N, 2.89.Crude TAPS recovery from the yeast sludge was 74.5 %.Included in the recovery loss is the material lost in thefirst trial extraction.

DISCUSSION

In all runs, TAPS was formed only during the dissimila-tion of glucose. When the glucose content of the mediumwas exhausted, TAPS formation stopped. Run P3 demon-strated the close relationship between TAPS formatic-and yeast growth. For each gram of yeast solids produceea definite amount of TAPS was formed. Its formatiorate per gram of glucose dissimilated was quite uniformthe different runs (Table 3). The highest rate was obtainsin fermentor B, where the medium was aerated vaOAR = 1.0. Evidently the higher oxygen supply productmore yeast cells.The results of run III support the view that an incr

of the glucose content of the medium above 7.5 g/100 mland of the OAR above 1.0 should enlarge the yeast growthand therefore increase the TAPS formation in the fer-mented medium. However, the tendency of the yeast forheavy foaming made such an increase inadvisable. Thefoaming difficulties in the pilot-plant runs confirmed thisviewpoint.

Flocculation of yeast cells toward the end of a fermenta-tion is characteristic of Hansenula ciferrii mating typeF-60-10, providing the temperature of incubation issufficiently low (25 C) to cause crystallization of TAPS.In the early stages of propagation, the cells with their budsoccur singly in the medium. After some 12 hr, refractileglobules of sphingolipid appear on the surface of the yeastcells; there is an increase in TAPS content of the culture;and the cells begin to aggregate in clusters. After 48 hr, theglobules crystallize, the hydrophobic cells and crystalsflocculate in large masses, and foaming subsides. Figure 4shows the edge of two masses.At the higher temperature (32 C) of run P2, the yeast

produced globules that separated from the cells and wentinto the surrounding medium. Small clusters formed, butthey did not flocculate in large masses.

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The large increase in recoverable TAPS that occurs whilethe harvested cells are stored for 65 hr at 2 C is believedto be due to excretion of TAPS previously formed in thecells but not extractable until it has passed through thecell wall to the surface.

ACKNOWLEDGMENTS

The authors wish to express their gratitude to Kermit A.Burton, Adolph A. Lagoda, Dean E. Uhl, William J.Albrecht, and Irving M. Jett for their technical assistance.

FIG. 4. Rim of two large yeast clusters enlarged sufficiently to disclose the ribbonlike crystals of tetraacetylphytospingosine (X880)

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DWORSCHACK, R. G., A. A. LAGODA, AND R. W. JACKSON. 1954.Fermentor for small-scale submerged fermentations. Appl.Microbiol. 2:1009-1012.

ODA, T. 1952. Studies on the components of penicillin-producingmolds. II. On fungus cerebrim (I). Bull. Pharm. Soc. Japan72:136-138.

PFEIFER, V. F., AND C. VOJNOVICH. 1952. Continuous sterilizationof media in biochemical process. Ind. Eng. Chem. 44:1940-1946.

PFEIFER, V. F., C. VOJNOVICH, H. G. MAISTER, V. E. SOHNS, E. N.HEGER, AND W. M. BOGART. 1958. Pilot-plant production ofground Serratia marcescens. Ind. Eng. Chem. 60:1143-1148.

SHAFFER, P. A., AND A. F. HARTMANN. 1921. The iodometricdetermination of copper and its use in sugar analysis. II.Methods for the determination of reducing sugars in blood,urine, milk, and other solutions. J. Biol. Chem. 45:365-390.

STODOLA, F. H., AND L. J. WICKERHAM. 1960. Formation of extra-cellular sphingolipids by microorganisms. II. Structuralstudies on tetraacetylphytosphingosine from the yeast Han-senula ciferrii. J. Biol. Chem. 235:2584-2585.

WICKERHAM, L. J. 1951. Taxonomy of yeasts. 1. Techniques ofclassification. 2. A classification of the genus HansenulaU.S. Dept. Agr. Tech. Bull. 1029.

WICKERHAM, L. J., AND F. H. STODOLA. 1960. Formation of extra-cellular sphingolipids by microorganisms. I. Tetraacetyl-phytosphingosine from Hansenula ciferrii. J. Bacteriol.80:484-491.

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