I
S. P. ROGOVIN, V. E. SOHNS, a n d E. L. GRIFFIN, Jr. Northern Regional Research Laboratory, U. S. Department of Agriculture, Peoria, Ill.
A Fermentation Pilot Plant Study for Making Phosphomannan A newly discovered yeast is used to make a polysaccharide which forms viscous, sparkling clear solutions that are resistant to bacterial attack. The white polymer, which may replace many imported gums, can be used for
b
I
thickening
N D U S T R I A L L Y IMPORTAIT polysaccharide hydrocolloids are now obtained primarily from plant sources. These include starch and cellulose, seed gums. gum exudates, and sea\veed extractives. The one outstanding exception is the bacterial polysaccharide. dextran. produced by Leuconostoc mesenteroides a n d related species, which has been previously described by lvorkers in this laboratory ( 3 , 6. 70). Sanborn reported the possible usefulness of many gum polysaccharides produced by fungi from waste cellulose. starch, and starch-derived saccharides ( 7 7 , 72). Recently the laboratory-scale production of bacterial polysaccharides by species of Xanthomorias has been described ( 7 : 8 ) . ii survey at this laboratory some years ago revealed that exocellular polysaccharides could be synthesized by the action of microorganisms on starch-derived saccharides. T h e polysaccharides obtained differed tvidely in composition from each other. a n d exhibited properties similar to those possessed by natural gums: seaweed extractives: a n d synthetic polymers. Since many of the gums Lvith comparable properties are imported, production of microbial polysaccharides Lvould provide new outlets for agricultural products xvith a minimum amount of competition from exisring domestic markets. T h e first of the neiv microbial polysaccharides. a phosphorylated mannan designated as phosphomannan was described b y Jeanes a n d others (4). I t is produced from glucose by the activity of a netvly discovered yeast. Hunsenula holstl'i described by it'ickerham ( 7 3 , I n the Lvork described here. pliosphomannan was prepared from commercial dextrose by fermentation u i t h the yeast Hansenula holstii S R R L Y2448. A Lvhite, granular. stable product was recovered by a proper sequence of processing operations Lvith a yield of 4.3 pounds of polymer per 100 pounds of
b
stabilizing
b
dispersing
suspending
anhydrous dextrose supplied. Fermentations of media containing 6'3 dextrose and cultured aerobically a t 82' F. ivere complete in 96 hours. A crude polymer was precipitated with methanol in ihe presence of a n electrolyte a n d purified by one reprecipitation. Dry product was obtained by either dehydrating n i t h methanol or by d r u m drying. However: viscosity of solutions of drum-dried material was much lower than that of methanol-dehydrated pol) mer. Preliminary cost estimates shoiv a "cost to make'' of about 93 cents per pound (87c moisture) for tivice-piecipirated phosphomannan or 8' cents per pound for the once-precipitated polymer Lrith a possibility of materially reducing these costs by substituting a more economical nutrient source a n d cheaper materials of construction.
Air \vas sterilized b y passage thruugll a 12-inch column packed ivith 6 feet of 10- to 24-mesh carbon. Foaming of the medium during fermentation was controlled by the automatic addition of a silicone antifoam in a manner as described by Pfeifer and I Iegc:r [ Y).
Equipment and Procedures
Centrifuges for removing cells and recovering product
Equipment. Pilot-plant fermenrarions were conducted in tlvo stainlesssteel fermentors (illustrated) of 60- and 600-gallon capacity. T h e 60-gallon fermentor was 21 inches i n diameter. 48 inches high. and jacketed. Agitation \vas provided by a 3-blade, 8-inch propeller mounted a t the bottom of a top-enrering shaft and poxvered b!- a 3-hp. motor. Agitator speed varied from 90 to 290 r.p.m. Sterile air \vas introduced through a pipe cross sparger direetly below the agitator. T h e 600-gallon fermentor \(-as 48 inches in diameter, approximately 90 inches high, and jacketed. I t \vas agitated with a top-entering, flat-blade, 18-inch disk turbine that had eight 3inch blades and \vas driven by a 3-hp. motor. Agitator speed varied from 50 to 250 r.p.m. Sterile air was introduced through a sparger directly beloiv the disk turbine,
These Items Were Used in Pilot-Plant Production of Phosphornannan 1:quil)iiieiit
Fermentation vessels
Jet heater Spiral heal exchanger
Central Copper and Supply Co., Cincinnati, Ohio Pfaudler-Permutit Co., Elyria, Ohio Schutte and Koerting Co., Philadelphia, Pa. American Heat Reclaiming Corp., New York, N. Y. Sharples Corp., Philadelphia, Pa. Tolhurst Centrifugals, Div. of A. M. and M., Inc., East Moline, 111.
Vacuum tray dryer
Buflovak EquipI, ment Div. of , Blaw-Knox, Double drum dryer, Buffalo, N. Y. Rotary vacuum dryer The PattersonKelly Co., Inc., East Slroudsburg, Pa. Precipitation and dis- stainless and Steel solving tanks Products Co., St. Paul, Minn. Distillation column Vulcan-Cincinnati, for solvent recovery Inc., Cincinnati, Ohio C'oiiiiiierr.i:ill~. . I v a i l u i ~ l t ~ ('II(~IIII(~:~~~
Anhydrous dextrose Corn steep liquor ( 5 0 7 , solids) Tryptone
VOL. 53, NO. 1
Potassium phosphate, monobasic Potassium chloride Methanol
JANUARY 1961
37
This type of equipment was used for the pilot plant study
.4utomatic temperature controls maintained temperatures during fermentation within 1' of the desired temperature.
A dry product \vas rrcoirrreil either by dehydrating with methanol and drying in either a vacuum tray or rotary drier or b) d r u m drying an aqueous solution of the polymer. Methods of Analysis. T h e total reducing sugars, calculated as glucose. were determined by the Shaffer-Hartmann method ( 73). Viscosities were obtained at 35' C. with a Brookfield viscometer operating at 30 r.p.m. Phosphomannan concentration was determined by optical rotation in 0 . l M KCI-H20 solution using the specific rotationof f103' (5). Process, Major process steps were production of inoculum, fermentation.
82" F. T h e medium was sterilized continuously with a steam jet heater and a 3inch stainless-steel holding coil as a retention chamber. Cells were removed from the fermented media by centrifuging with either a No. 16 Sharples Supercentrifuge or a Sharples DV-2 continuous controlled solids-discharge centrifuge. Precipitation and purification were performed in Type 304 stainless steel tanks, equipped with propeller-type airdriven agitators.
I
"I
5
1000
5
900
4-
l s
%'.
- 800
%. % . . .
=e:0, 3 -
W
7
,' 4
"*+.,-Glucose ,*,
,,' \ ,,'
Viscosity
e * + ,
2-
,,$'
,% *,,',
- 700 5 -600.5i
z
- 500 > .I? - 400
"% ",,,
- 300 -200
% ",*,
,4
10
,' / 38
8
e
*,,'
*," "**.
INDUSTRIAL AND ENGINEERING CHEMISTRY
- 100
Figure 1. Changes that occurred in pH, glucose concentraiion, and viscosity during fermentation. Viscosity of the broth increased as the glucose disappeared and pH dropped
A.
The medium was sterilized continuously with a steam jet heater and a 3-inch stainless-steel holding coil as o retention chamber
E.
The sterile medium in its continuous passage to the fermentor was cooled with a spiral heat exchanger
C.
Sixty-gallon stainless steel fermentor
D.
Six-hundred-gallon stainless steel fermentor
E.
Cells were removed from the fermented media b y centrifuging with either a No. 16 Sharples Supercentrifuge or a Sharples DV-2 continuous controlled solids-discharge centrifuge
precipitation and purification, and dehydration. Production of Inoculum. All inocula were cultured aerobically at 82' F. Stock cultures of the yeast were carried on yeast-malt agar slants and transferred to yeast-malt broth; after incubation for 24 hours, 10% by volume of the yeast-malt broth culture was used to seed a flask containing pilot-plant production medium; after 24 hours, 5% by volume of inoculum was transferred to a fermentor. When large amounts of inoculum were required, the 60-gallon fermentor was used as a seed tank for the 600-gallon fermentor. T h e production medium contained 6% commercial anhydrous dextrose, O.lyotryptone, 0.1% corn steep (507, solids). 0.5% KH?P04, 0.0576 KCI, and 0.5% by volume of solution B: Speakman salts ( 7 4 all adjusted to pH 5.0. Fermentation. All equipment was steam-sterilized at 250" to 260" F. for 3 hours before use. T h e medium, at pH 5.0! was pumped a t constant rate to a steam jet heater where it was mixed with steam to 275' F. instantaneously. After 4 minutes' retention in a holding coil, the medium in its continuous passage to the fermentor was cooled in a spiral heat exchanger to the required fermentation temperature, 82 ' F. After inoculating the fermentor contents with 5'3 of its volume with liquid culture NRRL Y-2448, agitation and aeration were adjusted so that the oxygen-
PHOSPHOMANNAN absorption rate \cas 0.5 ininole of oxygen absorbed per liter per minute tvhen measured according to the sodium sulphite method of Cooper, Fernstrom, a n d Miller (2). T h e course of the fermentation \cas followed by determining the pH, the total reducing sugar! and the viscosity. Fermentation Results. T h e work described was carried out primarily to produce relatively large amounts of phosphomannan for industrial evaluation purposes, so fermentation variables \Yere not studied. except for the effect of adding KC1 to the medium. E'ermentarions were conducted substantially as outlined by Anderson a n d others, based on their laboratory work ( 7). T h e pH increased slightly in the first 30 hours and then slo\vly decreased to a pH of 4.5 by the end of the fermentation. TYhile the pH \vas increasing. very little polymer was synthesized although the glucose consumption was a t its peak. After 30 hours, the increase in viscosity isas approximately linear as \vas the disappearance of the glucose (Figure 1). By adding 0 . 0 5 ~KC1 to the medium! fermentation time \vas decreased from 118 to 96 hours. and the yield of phosphomannan was increased from 39 to 437, (Table I ) .
2 100 Ib. Dextrose (L Otber Ingredients
Seed l n n k -Laborntory Air
Culture filter
Jet Herder
Mixing Holding Coil
Cooler
-
KCI r H AdjustmentMcOH--
The process involved four major steps: making inoculum, fermentation, purification, and dehydraiion
the sticky gum precipitate were dehydrated \\ithout a prior dilution, only partial dehydration occurred and resulted in a semidry sticky product.
Both the dehydration and product recovery steps are suitable for a continuous process provided polymer concentration in 25cGmethanol is greater
Recovery of Phosphomannan I n reporting how phosphoxnannan \cas recovered. all methanol concentrations are expressed as per cent by bveight. Phosphornannan was recovered from the fermented broth in a manner substantially as outlined by Jearies (5). Irhich was as follo\vs: adding 2.257, KCl and adjusting to pH 6.0: adding methanol to 25yc; centrifuging to remove yeast cells and suspended impurities; precipitating a crude polymer by adding methanol; and dehydrating. T h e KCI. a n electrolyte! served a dual purpose: Viscosity of the solution \vas decreased, thereby facilitating the removal of cells, and an electrolyte was required in the subsequent precipitation of the polymer. Adjusting the pH and adding methanol to 257, facilitated cell removal in subsequent centrifuging. T h e polymer \vas precipitated from the clarified liquor by adding methanol to 427, concentration. T h e precipitated polymer, a n offwhite. semifirm, sticky gum. \cas dissolved to 11% concentration in 25yo methanol containing 0.05:& KC1; then it \cas dehydrated by adding this solution to seven parts of methanol. h white: coarse granular product \vas recovered by centrifuging or filtering and by washing with methanol. A yield of 43 pounds of polymer per 100 pounds of anhydrous dextrose \vas obtained. If
Table I.
Adding KCI to Medium Increased Yield of Phosphomannan and Decreased Fermentation Time Ferinentator, llediuiii KCI in l;ermentation Yield."
"
Gal,
1701.,Gal.
60 60 60 600
20
30 30 250
IIedium. %
Tiiiie, Hr.
.Iv.
118 118 96 96
37.5 39.5 42.5 43
0 0
0.05 0.05
Pounds of dry p o l ~ m i e rper 100 pounds of commercial dextrohe.
2000
1600
----
& 1400
1111111,1111,
I-,-,-
Concentration,
Figure 2.
% by
MeOH Dehydrated Voc. Drum Dried - pH 5.2 Vat. Drum Dried pH 6 . 9 Atmor. Drum Dried - pH 6 . 9
-
0 Wt.
Drum-drying the polymer adversely affects its viscosity VOL. 53, NO. 1
JANUARY 1961
39
vestment. and profit. kurthermore? it has been assumed that a neiv plant has been built for the process and that stainless steel or stainless clad steel \vas used for the material of construction for the major items of equipment. Fixed capital investment for such a n installation is estimated to be near $1.300,000 (Table IV). If the crude polymer from one precipitation is recovered: the "cost to make" is about 87 cents per pound. If a cheaper organic nitrogen source such as yeast hydrolyzate can be substituted for tryptone in the fermentation medium, the production cost \vould be reduced by approximately 13 cents per pound of polymer.
:."[ 1600
4001
1!0
15
,!5
*io Concentration,
Figure 3.
Table 11. SO. O f
Prrriliitatiorilh 2" 3h 3c
I
4.0
% by
5.0
Wt.
Purification was complete after two precipitations
Reprecipitation Improved Purity of the Phosphomannan Sitrogen. Ash 1'liosphornu.s. Yisrosity," c % /G CP.
cz
0.15 0.10 0.05 0.14
12.6 12.8 12.7 12.5
than 105;. T h e product from the centrifuge \vas vacuum dried at 28 inches of mercury and 104' F. in either a rotary or tray drier to remove residual methanol. Drb- product was also ob-
Table Ill. Estimated Plant Production Costs to Produce Phosphomannan (.hinual caliwity, 1 000,000 pounds product: twice 1 m ( : i p i t a t d ; 8 7 , inoisture) Centc' Lh.
Raw materials Utilities Miscellaneous factory supplies and expenses Labor and supervision Maintenance Fixed charges Working capital charge Total plant production cost
41.03 14.45 1.06 13.64 6.08 15.92 0.67
3.33 3.33 3.33 3.33
1,400 1,980 1,980 1,820
tained by both atmospheric and vacuum d r u m drying of aqueous solutions of the phosphomannan ; however. the viscosit>of solutions of this product ivere much lower than that of methanol-dehydrated polymer (Figure 2). \Vhen a product of higher purity \vas desired. the crude polymer \vas redissolved in water to a 57, concentration and was reprecipitated one or two more times by adding 1.5ycKC1 and methanol to 467,; but the product purity was not greatly improved with more than one reprecipitation (Table I 1 and Figure 3). hIethanol used in the dehydration and ivashing steps was recovered and reused, as is. in the precipitation step. Supernatants from precipitation were redistilled to recover methanol.
92.85
Table IV. Preliminary Estimated Fixed Capital Investment for Phosphomannan Plant (Annual capacity 1,000,000 pounds product: twice precipitated; 856 moisture) Land and improvements $ 25,000 Building, 300,000 cu. ft. 225,000 Equipment, delivered 489,000 Installation of equipment 146,000 Piping, wiring, and instrumentation 190,000 Contingencies, engineering and 225,000 contracting fees Total fixed capital investment $1,300,000
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INDUSTRIAL AND ENGINEERINGCHEMISTRY
Cost Estimate
'4 preliminary cost estimate for this process as described indicates that: in a plant which operates 24 hours per day. 300 days per year and bvhich produces 1,000.000 pounds of phosphomannan a t t?yc moisture annually, the "cost to make" for twice precipitated product is approximately 93 cents per pound (Table 111). This cost includes all expenses for land. buildings, raw materials, equipment. labor, supervision, utilities. factory supplies, and ivorking capital; but it excludes general. administrative. and selling expenses, interest on the in-
Acknowledgment T h e authors are indebted to Allene Jeanes and R . F. Anderson for valuable discussions, t o L4. C. Cadmus for preparing laboratory inoculum, and to J. E . Pittsley- and D. E . L-hl for their analytical determinations.
literature Cited (1) Anderson, R. F., Cadmus. hi. C.. Benedict, R. G.. Slodki, M. E.. Arch. Biochem. Biophys. 89, 289-92 (1960). (2) Cooper, C. M., Fernstrom, G. A , . Miller. S . .\.. IKD.ENG. CHEM.36, 504 (1 944). (3) Hellman, N. K.. Tsuchiya. H . M., Rogovin, S. P.. Lambert, B. L.:Tohin. R., Glass. C. .A,. Stringer. C. S., Jackson, R. LV., Senti, F. R.. Ibid.. 47, 1953-8 (1955). (4) Jeanes. A , . Benedict, R . G., Cadmus, M. C.. Watson, P. R.? Rogovin, S. P., Pittsley. J. E.. Dimler, R. .l.. .Jackson, R . I%'.; Senti. F. R., Abstract of Papers, p. 23D, 134th Meeting, Am. Chem. Soc., Chicago. Ill., 1958. (5) Jeanes, -4.. Pittsley, J. E.. Watson. P. R . , and Dimler. R. J.. . h h . Biochern. Biophys.. in press, (6) Jeanes, A,, Wilham, C. A , , Miers, J . C., J . Bioi. Chem. 176, 603-15 (1948). (7) Leach, J. G.. Lilly. V. G.. Wilson, 'H. A,: Purvis, M. R.'. Jr., Phyinpatholoey 47, 113-20 (1957). (8) Lilly. V. G.. Wilson. H. .4..Leach, J. G.. &pi. M i c r o b i d . 6, No. 2. 105-8 (1958). (9) Pfeifer, V. F.. Heger, E. N., Zbid., 5, No. 1, 44 (1957). (10) Rogovin, S. P.. Senti. F. R., Benedict, R . G., Tsuchiya, H. M.: Watson, P. R., Tobin, R., Sohns, V. E., Slodki, M. E.. Abstract of Papers, p. 16A, 134th Meeting, Am. Chem. Soc., Chicago, Ill., 1958. (11) Sanhorn, J . R.. J. Bacterid. 26, 373 (19 33) . (12) Sanborn. J. R.. IND.ENG.CHEM.28, 1189 (1936). (1 3) Shaffer, P. h., Hartmann. .A. F.: J. Biol. Chem. 45, 365 (1921). (14) Snell. E. E.. Strong, F. M.. IND.ENG. CHEM.,ANAL.ED. 11, 346-50 (1939). (15) Wickerham, L. ~ J . > .tZycoioqia. in press. RECEIVED for review May 6:1960 ACCEPTED September 15, 1960 hiention of firm names for commercial products does not constitute an endorsement by the U. s. Department of .4griculture.
