Vitamin B,, by Fermentation with Streptomyces olivaceus 79
V. F. PFEIFER, C. VOJNOVICH,
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AND
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N. HEGER
Nolthern b g i o n o l Roreorch Labomtor,, Burem of Agricultural and lndurfriol Chemistry, U. S. Depdment of Agriculture, Peoria, Ill,
P '
RODUCTION of vitamin Bn by fermentation with m i c m organunder aerobic or anrterobic submerged culture conditions has created considerable commercial interest. A variety of vitamin Ba concentrates of microbiological origin have been marketed and axe employed in the production of mixed animal feeds, principally for poultry, swine, and small furred animals. Of the nearly 35,000,000 tons of mixed feeds manufactured during 1952, about 23,000,000 tons nequired supplementation with vitsmin B L ~which , gave a market for this vitamin of approximately 200 kg. valued at about $1O,OOO,oM). The production of commercial vitaBu concentrates by biosynthesis with BaeiZZus megatmiurn has been described by Garibaldi et al. (4), who reported yields UP to 0.7 mg. per liter in an 8- to 12hour fermentation. Leviton and Wargrove (le) mnorted - - ~-~~ the oroduction of vitamin BIZin a two-ohase fermentation employing . h c t o W w m e i in the fimt p h m and PropimG bacWurn f r d m r e i c h i i in the second phase, with resulting yields of over 4.0 mg. per liter in a 312hour fermentation. Hodge, Wanson, and Ngeier (9) described the production of vitamin BIS in a mixed culture anaerobic fermentation employing a mccharolytic stage and a proteolytic atage, with yields to 0.5 mg. per liter in 6 4 t o 72honr fermentations. Production of vitamin B,, from fish press liquors hy fermentation with strains of Streptomyces grise= and S. aureofaciens has been reported by Tam ( i s ) , with yields t o 1.1 mg. per liter. Burton and Lochhead ( 1 ) reported the Production of Btractive materials by a variety of bacteria. and actinomycetes isolated from soils, manure, and lit,t,er. A verv comolete review covefing the nroduotion of vitamin B,n by various microorganisms, and its occurrence in plants, has been presented by Darken (8). The literature contains many other references to vitamin Bn production by micro(18, 16, m), Flavobacterium organisms, including S. &eus devorans (7), S.fradiae (10),and Ashbya gosnypzz (17) The commercial production of vitamin Ba by fermentation with S. o l has been ~ described ~ by Heater and Ward (8). Research on the production of vitamin Bm by fermentation of pmteinaceous substraks with S. olivaceua NRRL B-1125 has been conducted at the Northern Regional Research Laboratory. The we of this organism in the production of vitamin B,s ba9 been patented by Hall (6). Results of laboratory experiments in shake flasks and lC-liter fermentors have been described fully by Hall et al. (6),with yields in the range of 1.5 t o 3.0mg. ofvitamin Blaper liter. Dried concentrates of ahont 14 mg. per pouud were prepared and tested in chick feeding trials for potency and toxicity. Results showed that the concentrate waa a suitable sonrce of vitamin, with no toxicity when fed at normal or high levels. This namr dearibes oilot olant exoeriments in which vitamin -~~~~ -~~ Ba liquors and concentrates were produced hy fermentation of nut.rient aubstrates with S. olivaceus. This oilot Dlant evaluation was carried out in order t o determine optimum conditions for the process on a commercial scale. An approximate cost estimate ~
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May 1954
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is included, based an engineering data obtained from the fermentation and recovery investigations.
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pi& pbnt Equipment
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Epuipment. Fermentations were conducted in a 600-gallon fermentor constructed of Type 347 stainless steel, two 800-gallon fermentors constructed of copper, and a 4MWFgallon fermentor oonstrueted of steel. The m-gallon fer---.-7 1 r &-"A . +"I, ^..A FA^+ :"A:"-.-+-" ~ULTUWL), ~,*'_" "=', a.,." 1 lrr" ,"".c."Anm., ;+L a jacket and dished heads. Agitation was n/.u I h.7 t-%e-type agitator mounted on a vertical shaft witb a spegd~range of 77 to 230 r.p.m. The agitator turbine rotated inside a wid$3 circular deflecting shield. The Emperature of the contents of the fermentor could be m a m t m e d mtbm 0.5' F. of that desired. Air entering the fermentor through a circular sparger 16 inches in diameter, constructed of lllrincb pi e, waa directed against a target mounted below the turbine. ! I A fermentations in this v e l were d e w i t h 300, gallons of medium, correa onding to a liquid depth of about 42 mches. Each copper fermentor was 5 feet tall and 5'12 feet in dmmeter and was equipped with internal coils for temperature control. Agitation was provided by a topentering fan-type turbine with blades set a t a 45" angle, and rotating at a fixed speed of 90 r.p.m. Air entered each fermentor through a perforated pipe cross sparger. All fermentations in these vessels were made m t h oallnna of -- medium, corresponding to a liquid depth of &ut 26 inches. T h e steel fermentpr @h a p v m i t y of gallons ~.I was L . l l . 8 feet m (usmewr, w ~ z ncomucm w p auu UUGWIU. %h waceran$~81/1 rrom anfeetL rxzernal BpFay ring, was used for tem erature control. T h e tank was equipped m t h four 8-inch bades, and agitation wss provided by a conventional turbine consisting of a 24-inch disk containing six blades, each of which was 7 incbes by 511, inches, mounted a t the periphery of the diak. The agitator was the to entenng type, rotatmg a t a fixed speed of 70 r.p.m. Air e n t e r d i e fermentor through an open 1-inch pipe mounted below the turbine diak. All fermentations in this vessel were d e with loo0 galIons of medium, corresponding to a liquid de th of about 36 inches. $wo copper tanks, each with a cap@tyof 8Ogallons, were used for preparing seed for the fermentatrons. The tanks were 4 feet high and 2 feet in diameter, with dish+ tops and c o n i d bot,tow, and were equipped, with perforated pipe spargers for the mtroduction of sterile aw. The tem erst- of +e seed cultures was --..Aumeans of internay C O O ~ ~. Wcolls. The seed tanks W I I W V I I ~ " bv I &x-~ Tith agitators. were notPrYv.--., Air for the fermeritors and seed ta&s was s,terilbed by assing column c o n t a i m g 8 feet $10- to i t through a 16-incb 24-me*.activated F o a y o f the 1arbon. nedium during fermentation was controlled by the ad tlonof antifoam, manually, as needed.
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Culhue Preparation Stock cultures of S. olivliceus were carried on Bennett's agar (11). A loopful of spores was t r a n s ferred t o 100 ml. of broth in a 500-ml. flsskprovided with a special side transfer tube. Medium in this flask and in all subsequent seed cultures wa6 prepared as follows:
INDUSTRIAL AND EWGINEERING CHEMISTRY
843
ENGINEERING, QESIGN, AND PROCESS DEVELOPMENT Dext,rose; 0.5 Corn steep liquor solids, % 0.5 2.0 Cobalt chloride (CoC12.6H,O), p.p.m. Soybean oil, 70 0.1 Adjust pH to 7.0 Sterilize 30 to 60 minutes at'250" F. (15 1h.isq. inch gage)
Aft,er incubation of the flask on a reciprocating shaker for 48 hours at 80' to 85" F., the contents n-ere transferred ascpticalljto 4 liters of sterile medium in a 9-liter glass bottle. This culture was aerated by sterile air from a perforated tube at the bottom of t,he bottle for 48 hours at 80" to 85' F. The culture IYas transferred aseptically t o 45 gallons of eterile medium in t,he seed tank and aerated for 48 hours at 83" F. At this time the culture m-as used to inoculate a fermentor containing sterile production medium.
Table I.
Effect of Medium Sterilization on Yields of Vitamin B,2
i4,07a Distillers' solubles plus unheated extracted Boyhean meal, l.0F0 glucose, 0.5% CaCOz, 0.1% soybean oil, 10 p , p . m . CoCln.6HnO)
Tank
Vitamin Yield, IIg./Liter
PH Batchwise, 2 Hours a t 250' F.
Copper Copper Copper Stainless steel Copper Copper Copper Stainless eteel Stainless steel Steel
0.2 0 6 0.9 1 0
;:p7 . 2 4.5
Continuous, 13 X n . a t 330° F. 4 8 5,5 6 5 5 5 4.5 4.5
1.2 1.2 1.3 1.2 1.3 1.2
Fermentations. Medium was st'erilized eit'her batchwise or continuously. \Gth batch sterilizat,ion all ingredients of t,he medium were put into the fermentor and heat,ed to the desired temperature by the direct introduct'ion of steam. After the liquor had been ret,ained at this temperature for a definite time, it, was cooled. For cont,inuous sterilization and cooling, the complete medium mas pumped from a storage t,ank at a constant rate to a steam jet heater, where it was mixed with steam, heated instantaneously to the desired temperature, held in a retaining coil for the required time, cooled continuously, and passed to the fermentor. This continuous sterilization equipment has been described recently (14). When necessary, the medium was adjusted to p H 7.0 to 7.5 after sterilization, either by the aseptic addition of a solution of sodium hydroxide or by pumping caust,ic solution through the sterilizer until the desired p H ivas reached. Determinations of sugar content (16),vitamin Bl2 content, and t,he pH of the liquor were made throughout the course of the fermentation. Precaut,ions were taken at all times to maintain pure culture conditions. When contamination did occur, poor yields of vitamin B12 Irere obt,ained invariably. Vitamin Bl2 in the fermcnted beers and dried concentrates was determined by a modificat'ion of the procedure of Skeggs et al., as 'described by Hall et ol. (61, using the assay organism L . leichmannii ATCC 4797. Routine samples were aesayed in the pilot plant by the disk method employing Difco CS vitamin BI2 agar (dehydrated). I n this method, both samples and st>andards vw-e autoclared w i h sodium bisulfite-sodium citrate-potassium phosphate buffer at p H 4.5,with vitamin AI? standards in the range of 0.05 to 0.30 mg. per liter. Growth zones of L. leichnzannii ATCC 4797 were compared after 21 hours incubation at 37' C. Vitamin B12 concentrates prepared in large ecale fermentations were checked for potency and toxicity in chick feeding tests. Resuhs substantiated the potency as determined by
844
microbiological methods, with no toxicity when fed at normal levels. Fermentation Process Variables Are Investigated
The folloTving factors influencing the formation of vitamin B,? were investigated on a pilot plant scale, methods oi sterilization, medium composition, amount of inoculum, aeration and agitation! fermentation temperature, and p H adjust,ment. Sterilization Methods. 3lethods of medium sterilization which \\-ere studied were batch sterilization at high and lox- pH and continuous steriiization at high and loit- pH. Khen batch sterilization ITas employed, it \vas necessary to hold the mediuni for 11/, to 2 hours at 250" F. in order to ensure sterility. A t low pH, slJfficient copper was dissolved during batch sterilization in coppcr tanks to appress t,hc growth of the organism and the production oi the vitamin. Batch sterilization was satisfactory in copper tanks nhen the pH oi thc medium was raised to 7.0 before it i r a s heated. Continuous eterilieation at low pH, employing a sterilizing temperature OP 330" F. with a retention time of 13 minutes, TTas satisfactory for fermentations conducted in copper, stainless steel, or steel fermentors. When contamination occurred, yields a w e reduced to as loit- as 0.1 mg. per liter, and foaming troubles m r e usually encountered. About, 10% of the frrmentations ronducted during this study became contaminated, but most of these contaminations occurred in the early part of the \rork. Typical results are given in Table I. Medium Composition. Fermentation media used in these experiments usually contained crude proteinaceous materials, dextrose, calcium carbonate, cobaltous chloride, and a small amount of soybean oil employed as an antifoam. A11 variet,ics of distillers' solubles-corn, wheat, or sorghum-mre satisfactory for vitamin production, although one brand of corn soluhles consistently gave low yields. In general, wheat solubles were slightly superior to the other varieties of distillers' solublee. In the various fermentations in which soybeans, solvcntextracted soybean meal, or expeller soybean meal were used a8 the proteinaceous materials, highest yields and best growth of thc microorganism were obtained with extracted unheated soybean meal. hlt,hough this material caused the fermentation to foam, the foaming could be controlled by the addition of antifoam oils. Low yields of vitamin rvere obtained when dried penicillin mycelium or ground wheat lvere used. When t,he medium contained both distillers' solubles and extracted unheated soybean meal, the grox-th of the organism \vas improved, and satisfactory yields were obtained. Results of 49 fermentations in which the proteinaceous material \vas eit,her dist,illers' solubles or a mixture of solubles nit'h ext,racted unheat,ed soybean nieal showed an average vitamin B,, yield of 1.24 mg. per liter. Typical results obtained vith a variety of proteinaceous materials are iist,ed in Table 11. Fermentations in which no cobalt was added gave very low yields of vitamin BIZ. Consistent' yields were obtained when the level of CoCI..GH20 Tr-as kept in t,he range of 1 . 3 to 9.0 p.p.m. The use of cobalt to increase t,he yield of vit,amin B12 in fermented media has been patent'ed by K o o d and Hendlin (20). \Vhen calcium carbonat'e xras not used in the medium, the fermenhtion foamed excessively, and slightly lower yields of vitamin B12 vere obtained, especially when ext,racted unheated soybean meal was present, as all or part of the proteinaceous material. \Then dextrose \\-as omitted from the medium, t>hcpH of the beer rose quickly, and the final j-ield of vitamin was somewhat reduced. This \vas particularly true when extractcd unheated soybean meal was the proteinaceous material. Instead of a normal decline of 0.5 t o 0.8 pH during the first 21 hours of the fermentation, a reduction of only 0.1 to 0.2 \vas obtained, after which the pH increased rapidly and excecdd 8.0 in 48 hours or less.
INDUSTRIAL A N D ENGINEERING CHEMISTRY
Val. 48, No. 5
changed appreciably. I t was possible to correlate the rate of vitamin production in dif(Each medium supplemented with 1.0% dextrose, 0.2% CaCOa, 9 p.p.m. CoCIz 6HzO) ferent tanks by means of the oxygen absorption rates obtained according to the method of Cooper, Fernstrom, , . 1.0 .. .. .. 3.0 0.5 .. , . 2.0 .. , . .. 2.0 0.6 and Miller ( 2 ) . The two copper 4:o. , . .. .. .. , . 0.5 fermentors and the stainless .. 3' 0 .. .. .. .. .. , 0.9 .. ,. .. 4.0 , . .. .. 1.1 steel fermentor all differed con.. .. .. .. .. 4 0 .. .. 0.8 siderably in power input, de.. .. , . .. , . 5 0 .. 1.0 .. , . .. .. ., 4.b .. 0.8 sign, and aeration efficiency, .. .. 2' '0 .. .. ., 2.0 .. 0.9 .. .. 1.0 .. .. 2.0 .. 1.1 but when the three tanks were .. .. .. 2.0 ,. 2.0 .. 1.2 operated at the same oxygen .. .. .. 4.0 .. .. .. , . 1.1 ,. . .. 2.0 .. .. .. 0.9 2.0 absorption rate, as determined 2.0 .. .. 2.0 ,. .. 0.8 .. .. .. .. 4.0 .. , . .. 0.9 by the oxidation of sodium sul4.0 .. .. .. .. .. 1.2 fite, approximately equal vita.. 4.b .. .. .. , . .. 1.2 5 0 .. .. ,. ,. .. .. 1.2 min production rates and final .. 2.0 2.0 .. .. .. ,. 1.2 2.0 .. .. 2.0 .. .. , . 1.4 yields were obtained. In gen2.0a .. 2.0 .. .. .. .. 1.0 .. 2.0 .. 2.0 .. .. .. , . 1.2 eral, it was necessary to operate .. 3.0 .. 1.0 .. .. .. .. .. 1.3 the fermentors with an oxygen 2 0 .. 1.0 .. .. .. .. .. 1.1 .. 2.7 , . 1.3 .. .. .. .. 1.3 absorption rate of a t least 0.4 One brand consistently gave low yields. millimole of oxygen absorbed per liter per minute in order to obtain a satisfactory vitamin yield in an 89-hour fermentaResults of experiments in which the content of cobaltous tion. For a satisfactory yield in a 72-hour fermentation, an oxychloride, calcium carbonate, and dextrose was varied are listed in gen absorption rate of 0.7 or higher was required. Results of Table 111. typical experiments in which agitation and aeration were varied Inoculum. The amount of inoculum used for seeding a ferare listed in Table IV. Figure 2 shows the variation in rate of mentor containing sterile production medium was varied from vitamin production with agitation and aeration in the stainless 1 to 127, by volume of the medium. No significant differences in steel fermentor. vitamin yield were obtained, although the fermentation was somewhat slower when only 1% of inoculum was used. On the 1.6basis of these tests, 5% of inoculum was chosen as suitable for this fermentation. 1.4 Fermentation Temperature. Fermentations were conducted in the temperature range of 72' to 90' F. The medium used in these experiments contained 2.7% distillers' wheat solubles, 1.37, c8 1.2extracted unheated eoybean meal, 1.0% dextrose, 0.5% calcium 8 a carbonate, 9.0 p.p.m. CoC12.6Hz0, and 0.1% soybean oil. The 1.0progress of each fermentation was followed by assaying the broth for vitamin B12 content after 17, 41, 65, 89, and 113 hours of 5fermentation. Although the experiments were carried out in I0.8different tanks, all tanks were operated under conditions of z N agitation aeration such that the oxygen absorption rates were 0.6approximately equal. The best yields were obtained a t a temperr ature of 80" F. The results of these experiments are shown graphically in Figure 1. 0.4pH Adjustment. Since most of the vitamin Blz is produced before the pH reaches 8.0, attempts were made to maintain the pH at 7.0 and 7.5 by the addition of concentrated sulfuric acid Oe2 after the initial drop in pH had occurred. The medium for these experiments contained 2.0% extracted unheated soybean meal, 2.0% soybeans, 1.0% dextrose, 0.5% calcium carbonate, 9.0 ERMENTATION TIME, hours p.p.m. CoCIz.6Hz0, and 0.1% soybean oil. With no pH control, three runs gave an average yield of 1.16 mg. per liter. There Figure 1. Effect of Fermentation appeared to be no advantage to maintaining the pH a t 7.0. I n Temperature on Vitamin BI2 Production five experiments with this medium the pH was maintained a t 7.5, and the vitamin B12 yield increased to an average value of 1.40 Evaporation Temperature, Drying, and Additives mg. per liter. Affect Recovery of Vitamin B12 from Fermented Beer I n another experiment dextrose was omitted from the original medium, but was added in increments in an attempt to maintain One set of experiments was conducted to determine the effect the pH a t 7.5. Although pH control was obtained, the final of evaporation temperature on the recovery of vitamin Biz. A yiclds averaged only 1.07 mg. per liter. fermented beer was treated with sulfuric acid to lower its pH t o Aeration and Agitation. The rate of vitamin BIZ production 5.0, four aliquots were evaporated to sirups over a temperature increased with an increase in either aeration or agitation. When range of 125' to 215" F., and the sirups were drum dried with the rate of aeration or agitation was reduced, the total fermentasteam a t 40 pounds per square inch gage. The dried contion time was increased, but the final yield of vitamin was not centrates prepared at 180" F., or below, were of approximately the Table II. Yields of Vitamin
B12
in Media Containing Various Proteinaceous Materials
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May 1954
INDUSTRIAL AND ENGINEERING CHEMISTRY
845
ENGINEERING, DESIGN, AND PROCESS DEVELOPMENT vacuum drum drying. The experiments were conducted Cnheated Vitamin with sirup prepared by adE x t d . SoyCoC1s.Yield justing the pH of fermented Distillers' Solubles, % bean Ileal. Soybeans, Dextrose, CaC03, 6Hz0, Mg./ Corn Wheat Sorghum 7; 7% % 9% P.P.M. Liter beer to 5.0 and evaporating it, 2.0 2 0 .. 0 0.5 0 0.2 a t a temperature below 150" F'. .. 2.h 2 0 ,. 0 0,s 01 . 3 01 .. 3 to a solids content of approxi.. 2'. 0 2.0 .. 1.0 0.3 0 .. , . 2 0 2.0 .. 0 0 5 9 0 0.9 niately 20%. S o advantage 2.0 2 0 0 0 3 9.0 1.0 .. 2.b 2 0 . .. 1.0 0.5 9 0 1 2 resulted from conducting the .. .. 4 0 .. 1.0 0 2.0 1.0 drum drying under vacuum, .. , . .. 4 0 .. 1 .0 0.3 2.0 1.4 2 0 , . 1.0 0 2.0 1.2 even with very low steam pres2.0 .. 1.0 0.5 2 0 1 7 4 0 .. .. 1 0 0 2.0 1.2 sure in the drums. No ap4 0 , . .. 1.0 0.3 5 0 preciable differences in potency .. .. 4 0 .. 0 0 3 Y 0 01 . 72 .. .. 4 0 .. 1.0 0 3 Y O 1.2 LTere apparent in concentrate .. .. 3 0 0 0.5 9 0 1. 0 .. 5.0 1 0 0.5 9 0 1.1 prepared by atmospheric drum .. io 1 0 .. 0 0,: $4 1 drying using st,eam pressures .. 2.0 1.0 .. 0.: 0 5 9 .. 0 0 1 .. 12 .. 2.0 2 0 .. 0 0.5 9.0 1.0 in the drums varying from 2.0 2 0 .. 1.0 0 5 9.0 1.4 2.b. 2.0 .. 0 0.3 9.0 0.8 20 to 60 pounds per square 2 0a .. 2.0 1.0 0 3 $1 . 0 0.9 inch gage. Concentrates prea One brand consistently gave loa. Fields. pared by spray drying were of about the Pame potc1ric.y as those prepared by drum drying. b n incroase in posame potency, and the recovery of vitamin BI? was about 80% of tency of about 10% was realized when special prc'cautions that originally present in the beer. The potency of concentrate> were taken t o conduct the spr:i!--drying process TTith an euitprepared a t evaporation temperatures above 180" F. vias reduced gas temperature of 200" F. or lovi-er and to cool the dried prodabout 10%. uct immediately. Addition of Protective Agents to Beer. The customary procedure for preparing dried concentrate for use in feeds is to reduce the pH of the beer to 4.5 to 5.5 n-ith acid, evaporate to a sirup a t temperatures belon- 150" F., and drum dry the sirup on steam 1.4 heated, double-drum dryers, with steam a t 60 to 100 pounds per square inch gage. Eight fermented beers x-ere processed in this manner, with an average recovery in the conrentrates of g 1.2.78.2% of the vitamin B12 originally present in the beer. Sodium Lg sulfite !vas added t,o five fermented beers in an effort to protect g 1.0the vitamin during the evaporation and drying processes. The sulfite was added in a concent'ration of 100 p.p.m. of beer. Avera* age vitamin BI, recovery in these concentrates was 88.7%. 0.8One fermented beer was treated with potassium cyanide in a z concentration of 10 p.p.ni. of beer before evaporation and drying, N 5 0.6as previously described by TYolf ( 1 9 ) . Recovery of vitamin Bla in the dried concentrate amounted to 86.5% of the vitamin prepent in the beer. 1000-Gallon Fermentations. TKOfermentations of 1000 gallons each were conducted in the 4000-galion steel fermentor. The 0.2 BSORBEO PER LITER PER net power input with the agitator operating a t 70 r.p.m. was 2.S hp. per 1000 gallons. Sterile air was supplied a t the rate of 50 cubic feet per minute, or three eighths of a volume of air per 20 40 60 80 100 I20 140 0 minute per volume of medium, corresponding t o a superficial air FERMENTATION TIME, hours velocity of 53 feet per hour. The oxygen absorption rate, as Figure 2. Effect of Agitation and Aeration on determined by sodium sulfite oxidation measurements for this Vitamin B12 Production in Stainless Steel combination of aeration and agitation, is 1.0 millimole of oxygen Fermentor absorbed per liter per minute. In the first fermentat'ion the complete medium, at pII 4.5, was sterilized continuously a t 330' F., with a retention time of 13 Treatment of Sirup before Drying. Esperiments were carried minutes. Before inoculation it contained 3.6% distillers' wheat o u t t o determine the effect of pH adjustment on the sirup before solublee, 1.0% dextrose, 0.5% calcium carbonate, 9.0 p.p.m. drum drying. Sirups containing 20% solids were adjusted to CoCl~.G€i~O, and 0.1% soybean oil. The pH was adjusted to various pH values in the range 4.1 t'o 7.5 and drum dried n-ith 7.2 by the addition of 6.2 pounds of sodium hydroxide, 5% steam at 40 pounds per square inch gage. There was no signifiinoculum was added, and the temperature was maintained at cant difference in the potencies of the dried concentrates. 82" t o 84" F. After 89 hours of fermentation the yield of vitamin Sodium thioglycollate was added to sirups a t pH 5 . 2 , and dried Bluwas 1.28 mg. per liter, and the pI-1 had reached 8.0. The pI-I concentrates were prepared by drum drying wit,h steam a t 40 of the beer was reduced to 5.0 by adding 8700 ml. of technical pounds per square inch gage. These concentrates contained from grade sulfuric acid. The beer, which contained 2.57, solids, 10 to 20% more vitamin than did controls prepared without the was evaporated a t 1-10" F. to a sirup conhining 13.270 solids and reducing agent. A concentration of 100 p.p.m. of reducing agent assaying 4.5 mg. of vitamin Blzper kg. The Firup was dried on :L was sufficient to protect the vitamin during drum drying. double-drum dryer operated with steam at, GO pounds per square Drying of Sirups. Several experiments were made t o investiinch gage. The output of the dryer Tvaq 1.4 pounds of dried gate t,he merits of spray dq-ing, atmospheric drum drying, and Table 111.
Effect of Media Composition on Yields of Vitamin B12 Produced by
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846
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
Vol. 46,No. 5
PILOT PLANTS
Table
IV. Effect of Aeration and Agitation on Yields of Vitamin Net
Fermentor Stainless Stainless Stainless Stainless
steel steel steel steel
Superficial
B12
89-Hour
Agitation,
Hp./1000 Power,
Aeration
Velocity Air
Absorption Oxygen
Vitamin Yield
R.P.M.
Gal.
Ratea RIedium A
Ft./Hr.'
Rateb
Mg./Liker
100 125 150 180
0.45 0.76 1.25 1.70
a/,
71 71 71 95
0.14 0.43 0.70 1.35
0 78 1 38 1 50 1 51
76
0.32 0.47 0.40 0 43
0 1 1 1
0 10 0 32 0 30 0 40 0 47
0.83 0.87 0 94 1 31 1 31 Medium C
a/, 3 18 1:/2
Medium B Copper, N o . 3 Copper, No. 3 Copper, No. 4 Stainless steel
90 125
KO.3
90
90
90
1 04
1.03 1 60 0 76
1 '/Z '/8
100 50
71
65
04 12 20
RIedium C Copper, Copper, Copper, Copper, Copper,
No. 3 No. 4 No. 4 No. 3
90 90 90 90
1 05 1 04 1 65 1 60
a/,
1 03
1
1/Z
%/a 1/2
Medium A 2.7 Wheat solubles, % .. Soybeans, % Corn solubles, % Unheated extracted soybean meal, % 1'3 Dextrose. '?4 1 0 0.5 CaC03, % 0.1 Soybean oil, % 9 CoClz 6Hz0, p.p.m. z Volume of air/(min.) (volume of medium). 5 $'vIillimolesof oxygen absorbed/(liter) (min.).
50 76 38
50 100
Medium B . I
2.0 2 0 1 0
0.5 0.1 9
material per hour per square foot of dryer surface. The concentrate contained 35 micrograms of vitamin Blz per gram or 16 mg. per pound. Only 73% of the vitamin prescnt in the bcer was recovered in the dried concentrate. The second fermentation was carried out exactly as described above. The beer contained 2.5% solids and 1.10 mg. of vitamin BIZ per liter. The pH of the beer was adjusted to 4.9, and 1 pound of anhydrous sodium sulfite waa added to protect the vitamin during the recovery process. The sirup contained 20.3% solids and assayed 7.6 mg. of vitamin per kg. The dryer output was 1.7 pounde of dried material per hour per square foot of dryer surface. The concentrate contained 39 micrograms of vitamin B12 per gram or 18 mg. per pound, which accounted for a recovery of 93% of the vitamin originally present in the beer.
centrate contained 21 mg. of vitamin Bls per pound, with a recovery of 95% of the vitamin B12 present in the beer. A concentrate of much higher potency may be prepared from the mycelium separat,ed from whole fresh fermented beer. In this instance the mycelium is suspended in water, the pH is reduced to 5.0 with sulfuric acid, the suspension is heated to boiling to free the vitamin, and the slurry is centrifuged or filtered. The liquor is evaporated to a sirup under vacuum and dried. By this procedure, concentrate containing over 100 mg. of vitamin BIZ per pound may be prepared. Cost Estimales Are Based on Commercial Feasibility of Process
Analysis of the experimental results indicates that satisfactory yields of vitamin Bit may be 0 5 produced in aerated and agitated, mbmerged0 1 culture fermentations of suitable nutrient sub9 strates by S. olzvaceus. The medium may be sterilized batchwise, in steel or stainless steel vessels by maintaining it for 1 to 2 hours at a temperature of 250' F., or continuously a t 325; F. with a retention' time of 13 minutes. The sterile medium should contain 4.0% of a mixture of distillers' solubles and extracted unheated soybean meal, 1.0% dextrose, 0.5% calcium carbonate, 0.1% soybean oil or other suitable antifoam agent, and a source of cobalt such as CoC12.6HzO at a concentration of 2 to 10 p.p.m. 2 0 2 0
Special Procedures Allow Preparation of High Potency Concentrates
During the early part of the fermentation practically all of the vitamin is present in the mycelium, but during the latter stages a considerable portion of it passes into solution. This is shown in Figure 3, in which measurements of the vitamin content of the whole beer and of the filtrate are plotted against fermentation time. A high potency concentrate may be prepared by reducing the pH of the whole fermented beer to 5.0 with sulfuric acid, heating it to boiling to free the vitamin, filtering or centrifuging the suspension, evaporating the filtrate under vacuum, and drying the sirup. Concentrate containing from 25 to 30 mg. of vitamin BIZ per pound may be prepared by this procedure, since about half of the inert solids in the whole beer are eliminated by filtration or centrifugation. -4s shown in Figure 3, filtration or centrifugation of the whole beer during the period 70 to 90 hours results in the recovery of the major portion of the vitamin with the mycelium, which may then be dried directly to yield a high potency concentrate without the necessity of evaporating large quantities of beer. One test was carried out with a beer which, a t age 89 hours, assayed 0.93 mg. of vitamin per liter and contained 3.20% total solids and 1.79% suspended solids. The whole beer, a t pH 7.7, was centrifuged and the centrifugate was discarded. The residue was dried in an atmospheric-compartment dryer a t 120' F. The dried con-
May 1954
0.6
0.2
Y 20 40 60 80 100 120
0
FERMENTATION TIME, hours
Figure
3.
Distribution of Vitamin Fermented Beer
BIZ
in
Inoculum for the fermentation should be prepared from medium containing 0.5% steep liquor solids, 0.5% dextrose, and 0.1% soybean oil or other suitable antifoam agent. This medium may be batch sterilized a t 250" F. for 1 hour a t pH 7.0 and inoculated with 1 to 2% by volume of a culture of S. olivaeeus prepared in the laboratory. The seed culture should be aerated for 48 hours a t 80' to 85" F., after which time it should contain a good growth of the organism.
INDUSTRIAL AND ENGINEERING CHEMISTRY
a47
ENGINEERING, DESIGN, AND PROCESS DEVELOPMENT
Table V.
Estimated Investment Cost for Plant with Daily Capacity of 28.5 Grams of Vitamin Blu
Land and improvements Building, 40 X 50 X 36 Equipment, delivered Installation of equipment Piping, wiring Other construction costs Contingencies, engineering and contracting fees Total plant cost
Table VI.
5 10,000 50,000 120.000 18,000 82,000 40,000 100,000 S4S0,000
Estimated Production Cost for Plant with a Daily Capacity of 28.5 Grams of Vitamin Biz Daily Cost
Raw materials Utilities Labor and super>-ision Maintenance Fixed charges Total
S177,71 1.83.26
130.67 69.00
186.00 S765 64
c
_
Cost/h1g. of
Vitamin BL, $0.0063 0.0064 0,0053 0,0024
0.0065 _-$0,0269
--
0
Figure 4.
20 40 60 80 100 FERMENTATION TIME, hour8
120
niin yield in the beer of 1.2 ing. per liter, with a recovery of 83.3y0 in the dried concentrate. The total estimated plant investment is $460,000, including complete installation of all equipment. The estimated plant, production costs for this plant are givcn in Table VI. These amount t o $765.64 daily, or 2.69 ccnts per m g . of vitamin Bi2 contained in the (Sonccn-
Changes in Medium during 812 Fermentation
The sterile medium in the production fermentor should be inoculated with 5% by volume of seed culture. 1-igorous mechanical agitation should be employed, and sterile air should be passed through SOYBEAN t,he medium, The combination of agiDISTILLERS' SOYBEAN DEXTROSE OIL tation and aeration employed should SOLUBLES MEAL be such that the oxygen absorption rate, determined by the sulfite oxidation method, is at' least 1.0 millimole of oxygen absorbed per liter per minute. This value will probably require the expendihre of 1 to 3 hp. of energy per 1000 gallons of medium, a n d aeration a t the rate of */d to 112 volume of air per minute per volume of medium. The temperature of the fermenting medium Qhould be conWATER LABORATORY CULTURE trolled in the range of 78" to 82" F. STREPTOMYCES OLIVACEUS After 72 hours the vitamin Blz con'LED TANK tent of the fermented beer should htx MIXING --in the range of 1.3 to 1.5. The pH CAUSTIC TANK of the beer should be reduced to 5.0 with sulfuric acid, and 100 p.p.m. of sodium sulfite should be added. The beer should be evaporated to sirup CO~KER of 20 to 25% solids content at 150' F. PUMP or below, and the sirup drum dlicd to a concentrate. The final dried concentrate should contain about 15 to 17 mg. of vitamin per pound. Changes in pH, sugar concentration, PUMP AIR .and B12 content in a typical fernienCOMPRESSOR tation are shown in Figure 4. EVA PoRATOe A flow sheet for estimating production costs of the process under the conditions described is shown in Figure 5. Table T7 lists the land, huilding, and equipment needed for a plant producing 28.5 grams of vitamin B12 per day in the form of concentrate CONCENTRATE with a potency of 14.5 mg. per pound. Figure 5. Flow Sheet for Production of Vitamin 812 b y Fermentation 'The calculations are based on a vita-
* I!,
:Ma
1
INDUSTRIAL A N D ENGINEERING CHEMISTRY
Vol. 46, No. 5
PILOT PLANTS trate. These are plant production costs only and do not include administrative and selling expenses, or royalties t o be paid under the Wood and Hendlin patent (20). The strain of S. olivaceus employed in this investigation may be obtained from the Culture Collection Section of the Xorthern Regional Research Laboratory. Acknowledgment
The authors are grateful to Harlow H. Hall and to Carolyn Ilemp of the Fermentation Division of this laboratory for their assistance in planning the developmental work, in preparing laboratory inocula for use in the fermentations, and for vitamin BI2assays of the various liquors and concentrates. Literature Cited
Burton, M. O., and Lochhead, A. G., Can. J. Botanv, 29, 352 (1951). Cooper, C. M., Fernstrom, G. A., and Miller, S. A., IND. EXG. CHEM.,36, 504 (1944). Darken, M. A,, Botan. Rev.,19, 99 (1953). Garibaldi, J. A., Ijichi, K., Snell, N. S., and Lewis, J. C., IKD.ENG.CHEW,45, 838 (1953).
(5) Hall, H. H., U.S. Patent 2,643,213 (1953). (6) Hall, H. H., Benedict, R. G., Wiesen, C. F., Smith, C. E., and Jackson, R. W., A p p l . Microbial., 1, 124 (1953). (7) Hall, H. H., and Tauchiya, H. M., U. S.Patent 2,561,364 (1951). (5) Heater, A. S.,and Ward, G. E., IND.ERG. CHEX., 46, 238 (1954). (9) Hodge, H. PI.,Hanson. C. T.. and Allgeier, R. J., Ibid., 44, 132 (1952). (10) Jackson, W. G., Whitfield, G. B., DeVries, TV. H., Xelson, H. A., and Evans, J. S., J . Am. Chem. Soc., 73, 337 (1951). (11) Jones, K. L., J . Bacteriol., 57, 141 (1949). (12) Leuiton, il., and Hargrove. R. E., ISD. ERG. CHEX, 44, 2651 (1952). (13) Lleyer, C. E., and DeVries, W. H., U. S. Patent 2,595,159 (1952). (14) Pfeifer, V. F., and Vojnovich, C., ISD. ENG.C H E l r . , 44, 1940 (1952). (15) Rickes, E. L., Brink, E.G., Koniussy. F. R., Wood, T. R., and Folkers. K., Science, 108, 634 (1948). (16) Shaffer, P. A., and Hartmann, A. F., J . Biol. Chem., 45, 365 (1921). (17) Smiley, K. L., et aZ., IND. ENG.Cmar., 43, 1380 (1951). (18) Tarr, H. L. A , , Can. J . Technol., 29, 391 (1951). (19) Wolf, F. J., P. S.Patent 2,530,416 (1950). (20) Wood, T. R., and Hendlin, D., I b i d . , 2,595,499 (1952). RECEIVED for review December 11, 1953.
ACCEPTEDFebruary 8, 1934.
Activation of Carbons W.
K. LEWIS AND A.
B. METZNERl
Massachusetts lnstitufe o f Technology, Cumbridge
39,
Muss.
A
CTIVATION is the process by means of which greatly improved adsorptive properties are imparted to charcoals made from a variety of raw materials, such as coconut or other nut shells, hardwood, petroleum coke, and coal. The desired ends of an activat,ion process are: 1. A product vhich has :in adsorptive capacity several times as great as that of the r a a material. 2. B product which is mechanically strong and obtainable in good yield. 3. A product which is resistant to contamination or poisoning (as by process impurities) and which can be readily regenerated if its adsorptive properties degenerate during use. 4. h product which has a high preferential affinity for one compound or one class of compounds, if the product is to be used in processes where a mixture is to be separated by selective adsorption. The Hypersorption and Arosorb processes are examples of industrial eeparational processes requiring such selective adsorbents.
Most activation processes involve a slou. oxidation of the raw material; this oxidation is selective with respect to the hydrogenrich constituents originally present. A relationship between the activity-Le., adsorptive capacity-and the hydrogen content of the product w m observed by early workers (21, SO), the activity increasing with decreasing hydrogen content, Chaney and coworkers (8, 16) were the first to combine these facts into a clearly defined activation theory. This theory has been amply reproved and extended by the work of more recent investigators (2, 4,7 , 21), and the present interpretation of it may be stated as f olio\vs : Any process which selectively removes the hydrogen or hydrogen-rich fractions from a carbonaceous raw material in 1
Present address, University of Delaware, Kewark, Del.
May 1954
such a manner as to produce an open, porous residue will constitute a process for the activation of the raw material. This statement is supported by all known reliable data, including those presented in this paper. Some prior workers h a w added other qualifying clauses-for example, that a high concentration of Oxidizing agent is necessary for optimum activation ( 2 7 ) . Work carried out as a part of the present investigation ( 1 ) showed that this is not necessary. I n this country the most widely used activation process consists of exposing the charcoal to the action of oxidizing gases a t a high temperature level (700" to 1000' C ) The gases most usually used are carbon dioxide or steam, or a mixture of both. I n addition to gasifying most of the hydrogen originally present in the raw material, the oxidizing gases commonly used react ~ i t ah large part of the carbon itself The yields of adsorptive carbon suitable for gas adsorption which can be obtained by conventional steam activation are only from 15 to 50% of the aeight of the oiiginal charcoal, and usually fall between 25 and 30% ( 1 , I, 7 , 16, 24). For carbons used in certain liquid-phase adsorption applications, optimum activity is not a primary requisite and higher steam activation yields are satisfactory. For gas adsorption applications, the low yield obtained contributes both to a high product cost and to a material which is mechanically weak, since much of the carbon consumed is probably gasified from n ithin the cracks and capillaries in the particle and not merely on the surface. Furthermore, the relatively large pores produced in this manner sometimes reduce the selectivity of the carbon for one component of a mixture of gases. An extremely important economic factor is that to produce a product u i t h the necessary mechanical strength by steam activation a dense, expensive (imported) rakt material, such as coconut
INDUSTRIAL AND ENGINEERING CHEMISTRY
849
