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The effects of continuous propagation on the growth of microorganisms Butterworth, Earl McKenzie 1950

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hi* ft¥ THE EFFECTS OF CONTINUOU3 PROPAGATION ON THE  GROWTH OF MICROORGANISMS. -by-Earl MeKenzie Butterworth A Thesis Submitted In Partial Fulfilment of the Requirements For the Degree of MASTER OF SCIENCE IN AGRICULTURE in the DEPARTMENT OF DAIRYING The University of Br i t i s h Columbia October, 1950. ABSTRACT The method of continuous propagation has been employed successfully to culture four organisms; Lactobacillus plantarum  Pseudomonas aeruginosa. A&otobaoter vlnelan&i and Torula cre- morls. The varied cultural requirements of these organisms made i t necessary to adapt a basic apparatus to meet these Individual requirements before satisfactory growth or ferm-entation could be attained. An apparatus satisfactory for the continuous growth of the aerobic organism Ps. aeruginosa was constructed. It was found that there was a correlation within certain limits be-tween the rate of flow of sterile broth and the population level of the organism. The continuous propagation apparatus used with Ps. aeruginosa was modified to permit a satisfactory fermentation by an anaerobic organism L. plantarum. It was shown that a constant maximum yield of l a c t i c acid could be obtained from a lactose medium when the rate of flow of the medium through the apparatus was properly adjusted. Azotobacter vlnelandl was cultured continuously on a whey medium. It was shown that the l a c t i c acid In whey could be used by the organism as the energy source for the nitrogen fixing process. It was also shown that the protein and protein breakdown products In whey did not inhibit nitrogen fixation by the organism. Maximum uellds (31.^ mgma.AOO ml) of fixed nitrogen were obtained with Azotobaeter grown continuously on a nitrogen free medium. An apparatus which seperated growth and fermentation was constructed for the fermentation of whey by Torula cremorle. The amounts of ethyl alcohol obtained with this apparatus were greater than those reported for the batch method. An attempt was made to Increase the vitamin content of whey by growing the yeast Torula cremoris in i t under cond-itions of controlled fermentation. However, no increase in nutritive value of the whey was observed. In some cases the B vitamin content was decreased slightly. ACKNOTB^ BDGMENT I wish to express my sincere appreciation to Dr. J.J.R. Campbell f o r his encouragement and assistance during the course of t h i s study and also to Dr. B.A. Eagles and Miss N. Neilson f o r t h e i r h e l p f u l suggestions. TABLE OP CONTENTS Page No. INTRODUCTION 1 REVIEW OP LITERATURE 3 PART I THE CONTINUOUS GROWTH OP LACTOBACILLUS PLANTARUM AND PSEUDOMONAS AERUGINOSA. Pseudomonas aeruginosa Methods 15 Apparatus 17 Experimental 17 Summary 19 Lactobacillus plantarum Methods 19 Apparatus 21 Experimental 21 Age of c e l l s i n the propagator 25 Summary 28 PART I I THE CONTINUOUS GROWTH OP AZOTOBACTER VINELANDI Introduction 29 Review of Literature 29 Experimental 33 Apparatus 35 Summary 38 PART I I I THE PRODUCTION OP ETHYL ALCOHOL PROM WHEY Batch method -Methods 39 Experimental 39 Carbon balance 41 Summary 44 Continuous propagation Apparatus 45 Experimental 47 Summary 48 PART IV THE CULTIVATION OP TORULA UTILIS IN WHEY Introduction 49 Review of Literature 51 Summary 53 Experimental 52 BIBLIOGRAPHY 55 1. INTRODUCTION In recent years the technique of continuous propa-gation of microorganisms has attained a po s i t i o n of importance and has been used with considerable success i n the c u l t i v a t i o n of a great variety of microorganisms. This method permits the-laboratory propagation of large volumes of cultures and offers a great saving i n time, space, and labour over the batch system. Of interest from a commercial point of view i s the fact that i n certain fermentations much greater y i e l d s of products can be achieved by the continuous process than by the batch method. Moreover, i n the usual batch method a large amount of time i s consumed i n s t e r i l i z i n g equipment and developing the culture to i t s maximum strength and, when fermentation i s completed, emptying the tanks and preparing them for another run. I f a fermentation vat could be supplied continuously with fresh fermentable material and the spent medium removed at the same rate, i t should be possible to maintain the fermentation at i t s maximum speed f o r any desired period. This process has not been more widely accepted f o r several reasons, the f i r s t of which i s the cost of converting fermentation industries from the proven batch method to the r e l a t i v e l y new continuous system. Greater r i s k of contamination of the fermentation system i s another important fac t o r . In that the organisms are continuously cul t i v a t e d , any contami-nating organism woxild have a greater chance of multiplying and thus displacing or otherwise i n t e r f e r i n g with the pa r t i c u l a r fermentation being attempted. Cultures grown under the continuous method of fermentation are also more susceptible to d i s s o c i a t i o n and th i s would again, tend to destroy a t y p i c a l fermentation. I t i s therefore of paramount importance to choose an organism with stable s t r a i n c h a r a c t e r i s t i c s . The purpose of the present work was to aet up an apparatus f o r the continuous growth of bacteria and to study the effect of this system of c u l t i v a t i o n on the a c t i v i t i e s of various microorganisms. 3. REVIEW OP LITERATURE Perhaps the f i r s t step i n the development of continuous propagation was unknowingly taken by Graham-Smith (1) during studies on factors inducing the stationary phase of b a c t e r i a l growth. He observed that a culture of Staphylo-coccus aureus could be revived at any time after the stationary growth period and caused to increase i n numbers by adding s t e r i l e medium i n concentrated form, and that the logarithmic death phase could be postponed i n d e f i n i t e l y by small d a i l y additions of s t e r i l e medium. I t was not u n t i l 1929, however, that a more di r e c t contribution was reported. In that year H.V. Moyer (2) described an apparatus f o r a continuous method of culturing bacteria for chemical study. He found that Bacterium  aerogenes apparently remained pure throughout several months of culturing i n his apparatus. This method of propagation permitted excellent control over environmental conditions affec t i n g the growth of the bacteria. By neutraliging with 10% sodium hydroxide, the pH of the medium could be kept f o r an i n d e f i n i t e period of time within the l i m i t s most suitable f o r rapid m u l t i p l i c a t i o n of the organism. The flowing culture medium removed products of metabolism which would otherwise retard growth. The m u l t i p l i c a t i o n of bacteria i n the fresh medium that flowed continuously into the growth tube was at a rate which maintained the high concentration of organisms at a constant l e v e l . I t was suggested that the organisms could be l i t t l e more than 48 hours old after t h e i r growth i n the apparatus as the contents of the growth tube (18 l i t r e s ) were replaced every 24 hours. The flow of gas into the apparatus could be effec-t i v e l y controlled and, although no attempt was made to grow anaerobic organisms i t i s probable that i t could e a s i l y have been accomplished. An explanation of the behavior of bacteria i n such a continuous system was p a r t i a l l y achieved by the theories of Rogers and Whittier (3). These workers stated that the growth curve f o r a b a c t e r i a l culture i n a f l u i d medium follows very closely that of a colony of m u l t i c e l l u l a r organisms up to the time when the maximum population i s approached. When the normal population i s reached i n such a colony of m u l t i c e l l u l a r organisms, i t continues approximately l e v e l with only a slow increase or decrease, while the b a c t e r i a l colony soon passes into a period of rapid decline. In the case of a colony of m u l t i c e l l u l a r animals there i s a constant food supply and natural or a r t i f i c i a l provision f o r the removal of products of metabolism. In the b a c t e r i a l colony the quantity of food i s l i m i t e d and the products of growth accumulate. Rogers and Whittier wondered whether the b a c t e r i a l colony would r e t a i n i t s e l f i n d e f i n i t e l y as does the colony of mu l t i -c e l l u l a r organisms i f the b a c t e r i a l colony were given a continuous food supply and i f the products of metabolism were removed. In a preliminary experiment described by Roger s and Whittier (3), fresh medium was passed through a culture f l a s k at such a rate that the medium i n the f l a s k was displaced i n 24 to 30 hours. I t was found that when cultures of Streptococcus l a c t i s or Escherichia c o l i were grown i n f l a s k s through vi/hich broth was slowly passed a constant population l e v e l was maintained as long as the experiment was continued i n spite of the constant removal of c e l l s from the culture. In a second experiment the overflow was carried out through a Berkefeld f i l t e r suspended i n the culture. A revolving brush reduced the numbers of c e l l s accumulating on the surface of the f i l t e r . With the rate of flow used, the broth i n the culture f l a s k was changed from one to f i v e times every 24 hours, and under these conditions the population remained constant for a seven day period. When the flow of broth was stopped, three days elapsed before there was a material decrease i n the viable count. This, they thought, indicated that the c e l l s did not die as long as the flow continued. In a t h i r d experiment a mixed culture containing a Lactobacillus and a Mycoderm was used to produce l a c t i c acid. The fermenting material was an infusion broth containing tomato juice and 5.92$ lactose. Under favourable conditions an 800 ml. culture delivered every 24 hours from 500 to 900 ml. of effluent from which p r a c t i c a l l y a l l of the sugar had been fermented. This experiment, which was run f o r s i x t y days, showed that from 0.78 to 2.19 grams of t he carbohydrate was -->-fermented per hour. The v a r i a t i o n i n the fermentation rate was caused by f a i l u r e to keep the reaction within the l i m i t s of greatest a c t i v i t y . Had the culture fermented 2.19 grams of sugar per hour, 98.8$ of the sugar would have been fermented from the medium leaving the fermentor. In 1933 Bushwell and Boruff (4) described mechanical equipment f o r the continuous fermentation of fibrous materials. Cornstalks were added to a fermentation tank at the rate of 105 pounds per day per 1000 cubic feet of tank capacity. Prom this experiment which was continued f o r seventeen months, an average and a maximum gas production of 420 and 555 cubic feet of gas per day per 1000 cubic feet of tank capacity respectively was coll e c t e d . This gas contained an average of 52$ methane. In an attempt to determine the factors responsible fo r the cessation of growth In b a c t e r i a l cultures, Cleary, Beard and C l i f t o n (5) confirmed the data of Graham-Smith (1). They adopted the p r i n c i p l e of Rogers (3) apparatus as a means of extending their knowledge of the problem. Using^a culture °^ E. c o l i they determined the effect of varying concentrations of peptone, using glucose as an additional energy source, both i n buffered and i n unbuffered solutions, and of aeration by means of a motor-driven s t i r r e r i n the continuous flov/ apparatus. Cleary et a l , compared the growth of an organism with a constant amount of broth to the growth of an organism sxipplied with a continuous flow of broth. The population peak attained was approximately the same i n both cases. At 24 hours both cultures had a count of about 1800 m i l l i o n c e l l s per ml. In the constant supply the count had dropped to 140 m i l l i o n at the end of 168 hours whereas i n the continuous supply the count was 1400 m i l l i o n at the end of 192 hours. When glucose was added to the medium of the continuous flow apparatus at 192 hours the population reached almost 2 b i l l i o n c e l l s per ml. but a severe drop followed, probably due to a drop i n the pH to 5.3. The re s u l t s of t h i s and of other experiments led them to conclude t h a t — 1. the effect of metabolites i s variable, depending . !•-; on the p a r t i c u l a r metabolite and i t s effect on the enzymic mechanism of the c e l l . 2. growth w i l l not mount above a certain point even with the effect of metabolites reduced to a low l e v e l They believed this to be because of the reduction i n energy available per c e l l . 3. growth ceases because of changes i n the a v a i l a -b i l i t y of nutrient material and energy demand. Unger, Stark, Scalf and Kolachov (6) i n an attempt to conserve time and space i n commercial breweries, outlined the methods and apparatus f o r a continuous aerobic process of culturing d i s t i l l e r ' s yeast. They obtained 500 m i l l i o n c e l l s per ml. where 150 m i l l i o n c e l l s per ml. were obtained by the sour-mash anaerobic process. This yeast compared favourably with that from the old process i n fermentability. In p i l o t -plant runs they sprayed wort into a 300 gallon propagator at the rate of 75 gallons per hour and had an equal draw off of yeast wort. The o v e r - a l l processing time was reduced from 20 - 40 hours to 4 hours for an equivalent volume of product and at the same time the number of yeast c e l l s increased from 150 to 500 m i l l i o n per ml. B i l f o r d et a l (7) outlined an apparatus f o r the rapid continuous alcoholic fermentation of molasses. Using a s t r a i n of Saccharomyces cerevisiae, they obtained a fermentation of a wort containing 12 to 13 grams of sugar per 100 ml. i n a cycle of 5 to 7 hours. The alcohol y i e l d s obtained with the con-tinuous system were comparable to those obtained from standard 50 hour batch fermentations of the same material. Jordon and Jacobs (8) used a new approach to the problem of b a c t e r i a l growth by subjecting a population of E. c o l i c e l l s to a constant food supply over a r e l a t i v e l y long period. Their apparatus consisted of a 5 l i t e r f l a s k which contained a constant l e v e l (1450 ml.) of culture. Fresh s t e r i l e broth was added i n known amounts and the l e v e l i n the f l a s k was kept constant by a heated a i r current passing through the culture. The culture was huffered at pH 7.0 with phosphate buffer. Pood was supplied at the rate of 15.2 or 30.4 mgms. of dehydrated difco broth per hour. At each of the two rates of food supply used, an i n i t i a l period i n \¥hich both the t o t a l and the viable counts were increasing was followed by a steady phase i n which the viable counts remained constant or decreased s l i g h t l y while the t o t a l c e l l counts steadily increased. In order to determine the influence of increasing the food supply upon the c e l l m u l t i p l i c a t i o n an experiment was carried out with a culture which had reached the steady phase of c e l l m u l t i p l i c a t i o n . I t was found that when the food supply of t h i s culture was doubled a second phase of c e l l m u l t i p l i c a t i o n similar to that i n the i n i t i a l period began and was succeeded by a second steady phase. In another experiment the food supply was stopped and the t o t a l c e l l population remained constant. However, the viable count decreased to a constant low l e v e l . 9. They calculated the amount of. food used i n the forma-t i o n of a new E. c o l i c e l l and i n the maintenance of a c e l l apart from reproduction. The amounts were l . l / l 0 ~ 9 mgms. and -9 0.4 to 0.5 x 10 mgms. per 25 hours of difco dehydrated broth respectively. Moor (9) recently described an apparatus for the continuous c u l t i v a t i o n of P e n i c i l l i u n notaturn and a s t r a i n of yeast. For the c u l t i v a t i o n of the mold the fermentation tube was 4 x 200 cm. and contained 200 ml. of fermenting substrate. An inflow of 20 ml. per hour of s t e r i l e corn steep medium was maintained. The apparatus was so arranged that the mat and the p e n i c i l l i n containing substrate were caught i n separate con-tainers. The flow was regulated and the mat removed at such a rate that s e l f inoculation occurred with, a minimum lag period and adult organisms were removed before they could mutate. Daily c u l t i v a t i o n s from the removed mat showed that during the course of the run no morphological change occurred to the organism and no change i n p e n i c i l l i n production could be detected. For the yeast culture the apparatus used was much the same as that used f o r the mold. 50 ml. of s t e r i l e media per hour flowed into the fermentation f l a s k which contained 300 ml. of culture. The alcohol determination of the overflow was based*, on s p e c i f i c gravity. The percentage of alcohol formed on th i s basis was about 4$ and varied very l i t t l e from day to day. Daily sub-cultures of the yeast at the removal point did not show any change i n i t s c h a r a c t e r i s t i c s . In 1946, Feustel and Humfeld (10) described what they 10. termed 'a new yeast fermentor.' I t consisted of a v e r t i c a l tube (30 inches by 3 inches) which contained from 500 to 2000 ml. The pH was kept constant (5.0 to 5.5) by the addition of ammonia which also served as a nitrogen source. Yeast volumes of about 3.6 ml. per 15 ml. of culture l i q u i d were obtained. The count on t h i s 3.6 ml. was 4 b i l l i o n c e l l s per ml. The more nearly optimum cul t u r i n g conditions provided by the new fermentor, which makes possible the attainment of high c e l l concentrations and the maintenance of the normal propaga-t i o n rate f o r prolonged periods of cultur i n g , was ascribed to improved dispersion of a i r . Without diminishing the propaga-t i o n rate they obtained eight times as many c e l l s per ml. as was obtained by Unger et a l (6). In continuous culture studies on Brucella suis i n aerated broth culture, Gerhardt (11) outlined two methods for the propagation of a highly v i r u l e n t organism. 1. A c y c l i c system applied to one or a number of culture vessels i n series. This amounted to a s e r i a l transfer i n a closed system employing a large inoculum. 2. A device which maintained a constant flow of broti into a f l a s k which held a constant volume (400 ml.). The rate of inflow was 400 ml. every 7.7 hours. The effluent g culture from t h i s showed b a c t e r i a l counts of 44 x 10 whereas 9 the best c y c l i c system gave counts of 2S x 10 every eight hours. Stra i n mutation was minimal but no virulence tests were conducted. W'ith customary batch methods, i t had previously been 11. possible to produce a given large unit of Brucella 3uis i n approximately 48 hours o v e r - a l l time. Whereas i t i s believed that 8 hours would produce an equivalent product i n the c o n t i -nuous flow apparatus. Operated under negative pressure the system minimized the hazards of producing a highly v i r u l e n t organism i n quantity. The product contained a very high concen-t r a t i o n of c e l l s , was free from contamination and contained p r a c t i c a l l y no morphological s t r a i n variants. A recent paper of Jordon and Jacobs (12) dealt with the effect of temperature on the growth of Bacterium c o l i at pH 7 with a constant food supply. Total and viable c e l l counts were made frequently and growth curves constructed. At a l l stages of growth the t o t a l count greatly exceeded the viable count. Very early i n the i n i t i a l phase, c e l l d i v i s i o n lagged behind c e l l growth. At 15 p C. t h i s condition persisted through-out the whole experiment. The i n i t i a l phase was longest when the temperature was lowest, but the time taken to reach 300 m i l l i o n t o t a l c e l l s per ml. did not vary greatly, except at 15 degrees. In the following steady phase the calculated rates of increase i n both t o t a l and viable c e l l s were highest when the temperature was low, and bore a l i n e a r r e l a t i o n to the temperature. The rate of increase i n viable c e l l s at 35 degrees was almost zero. This was apparently a c r i t i c a l temperature for v i a b i l i t y . However, growth occurred at both 35 and at 40 degrees since the t o t a l counts continually increased, but approximately h a l f the c e l l s formed were non-viable. After cessation of the inflow of food the t o t a l count declined at both 15 and at 30 degrees, being s l i g h t l y f a s t e r at 12. the lower temperature. The viable count also declined during starvation at 15 degrees but the rate of decrease of the viable count was greater at 30 degrees. Where food concentration did not l i m i t the grov/th-rate the points obtained by p l o t t i n g the logarithms- of the numbers of viable c e l l s against time should f a l l on a straight l i n e . At 15 degrees t h i s was so f o r a considerable time (5 days). The lag i n t h i s case, according to the method of Lodge and Hinshelwood (13), was s l i g h t l y over one day. B a i l (20A) suggested that f o r any given culture and conditions of growth there i s a maximum population which can be supported. Evidently the numbers of t o t a l and of viable c e l l s formed from a fi x e d amount of food remained approximately constant irr e s p e c t i v e of the age of the culture (ie) of the number of c e l l s composing i t . The above experiment i s i n agreement with this view i n so f a r as the numbers of viable c e l l s are concerned at a temperature.of 37 degrees. However, i f the t o t a l amount of c e l l substance i s regarded as the 'population' i n these cultures, then there appears to be no ind i c a t i o n , at any of the temperatures used, of the existence of B a i l ' s maximum population. Kaplan and Elberg (14) demonstrated quite a d i f f e r e n t type of apparatus f o r continuous propagation. They used a f i l t e r system. The s t e r i l e broth flowed into the f i l t e r cup, which was the growth chamber. The spent broth passed through the f i l t e r leaving behind a concentrated b a c t e r i a l culture. They concentrated Brucella suis from broth culture i n this way with no loss i n virulence. Favorable r e s u l t s were also 13. obtained with, cultures of Brucella abortus' and Brucella melitensis. This apparatus could probably be used to great advantage f o r the production of exotoxins. A l z o l a (15) outlined a process whereby a continuous alcoholic fermentation of' blackstrap molasses mash, or other carbohydrate containing l i q u i d was carried out i n a single tower-like fermentor. A tower three times as high as i t s diameter was found to be best. Carbon dioxide was used f o r aeration throughout. The mash entered just above the conic bottom at which point the residue i s extracted. The flow i s upwards through several compartments and i s regulated by butter-f l y valves set i n the separatory trays, to enable v a r i a t i o n of holding time at the several decks i n the apparatus. Brockman and St e i r (16) demonstrated a steady state fermentation by yeast. They maintained a constant population f o r more than 200 hours that showed a stable rate of sugar u t i l i z a t i o n i n a glucose, yeast extract, potassium d i hydrogen phosphate medium. 0.18 - 0.25 grams of glucose per hour was used per 10"*"^  yeast c e l l s and there were 300 m i l l i o n c e l l s per ml. Using a s t r a i n of Lactobacillus , Whittier'and Rogers (17) studied the application of continuous fermentation to the production of l a c t i c acid from the lactose of sweet whey. They demonstrated that a volume of whey equal to the "working volume of the fermentation vat could be almost e n t i r e l y fermented i n 24 hours under conditions of continuous flow. Calculations showed that the u i e l d of l a c t i c acid ivas 90$ of the o r e t i c a l b ased on the lactose o r i g i n a l l y present. 14. Humfeld X<18)» reporting on a modification of the apparatus of Peusal and Humfeld (10) stated that the new continuous fermentor was found suitable f o r the submerged culturing of aerobic microorganisms. This fermentor had a larger capacity and a more ef f e c t i v e mechanical foam breaker. I t was employed i n the propagation of the yeast, Torulopsis  u t i l i s , as well as f o r the production of a n t i b i o t i c s by Ba c i l l u s s u b t i l u s . The y i e l d of yeast based on the sugar supplied was 55% at a rate of 1 l i t er every 2 hours. Owen (19) devised a continuous fermentation apparatus providing for- a downflow of mash through a column equipped ... of with a series/decks. A number of molasses mashes were fermented. The fermenting e f f i c i e n c y approached 85%, Although there has been an Increasing tendency to use the p r i n c i p l e of continuous propagation i n i n d u s t r i a l fermen-tations In recent years, i t would appear that the application of continuous propagation i s , as yet, a comparatively new f i e l d and one i n which no general preferences for p a r t i c u l a r types of equipment are evident. 15. PART I TEE'XyOWSTmStfOS^X^^TEOF"MCTOBAC'ILfcUS PTLAWT ARUM"'AFP" "^ OF" PSBUJ^MONAl5'~gERUGI NO SA The purpose of the following experiments was to determine whether or not L. plantarum and Ps. aeruginosa could be s a t i s f a c t o r i l y cultured by the methods of continuous propagation. The most important problem encountered i n the i n i t i a l stages of thi s project was the devising of a method or methods of following the action of the organism i n the continuous propagation apparatus. METHODS Since there has been considerable work done with Ps. aeruginosa (9027) i n t h i s laboratory with a resultant accumulation of detailed information on i t s idiosyncrasies and also because i t possesses stable s t r a i n c h a r a c t e r i s t i c s , the organism was selected f o r use i n the i n i t i a l stages of t h i s s tudy. Before t h i s work could be started a means of follow-ing the a c t i v i t y of the organism i n the propagation apparatus (referred to hereafter as the 'propagator') had to be found, lii/hen t h i s culture was grown i n the medium used, i t was not possible to employ as an index of a c t i v i t y , either a c i d i t y or the production of ammonia. I t was decided to use one of the methods which would give an account of the numbers of bacteria produced during the operation. Of these, the plate count appeared to be the most sa t i s f a c t o r y . These counts were made from peptone glycerol agar pour plates. The organism was 16. carried on Werkraan's media and on a t r y p t i c digest of casein. Trpptic digest broth was used as the medium f o r continuous growth i n the propagation apparatus. The formulae of these media are given below. Peptone gly c e r o l agar. peptone 2$ Glycerol 2% Beef Extract 0.3$ Adjust pH to 7. Agar 1.5$ Gelatin 1$ Workman1s Medium Beef Extract 0.4$ Peptone 0.4$ Yeast Extract 0.2$ Adjust to pH 7. NaCl 0.2$ Tap water 1000 ml. Tryptic digest of casein To 200 grams of commercial casein add 1 l i t r e of cold water ( d i s t i l l e d ) i n a 6 l i t r e florence f l a s k . Add 1 l i t r e of hot d i s t i l l e d water and shake vigorously. Adjust the pH to 8.1. Add 15 ml. of toluene and shake w e l l . Add 7 grams of pancreatin and shake again. Transfer the contents to a Winchester b o t t l e and incubate at 37°C fo r 4 days. Add another 6 grams of pancreatin, adjust the pH to 8 and incubate at 37°C for another 8 days. Transfer to a large f l a s k and add 150 ml. of a 1:10 d i l u t i o n of HC1. Steam 30 - 60 minutes and f i l t e r . Add 5 $ NaOH to f i l t r a t e u n t i l f a i n t l y a l k a l i n e to litmus. One part of t h i s f i l t r a t e i s mixed with 2 volumes of 0.4$ NaCl. Adjust pH and d i s t r i b u t e i n 1 l i t r e q uantities. Plug and s t e r i l i z e . LEGEND: 1. Stop-cock f o r inflow control 2. Growth f l a s k 3. Below surface entry device 4. S t i r r i n g apparatus 5 . Mercury seal 6. Growth f l a s k outlet 7. Stop-cock f o r outlet control 7a. Sample tube 8. Step-cock f o r outlet control 9. Effluent storage f l a s k 17. APPARATUS (Fig. 1) The s t e r i l e medium which was stored i n a 5 l i t r e f l a s k (A) passed through a ground-glass stop-cock (1) which was used to regulate the rate of flow. The s t e r i l e medium entered the growth f l a s k (500 ml. Wolle f l a s k ) (2) below the surface of the f l u i d (3) thus allowing a better dispersion of the fresh nutrients. The growing culture was agitated by a mechanical s t i r r i n g apparatus (4), the shaft of which passed through a mercury seal (5). The culture was carried out of the growth f l a s k by a syphon system (6) and the speed of flow at t h i s point was also governed by adjusting a ground glass stop-cock (7). Thus i t can be seen that i n order f o r the volume i n the growth f l a s k to remain constant the rate of s t e r i l e broth i n t o , and the rate of spent broth out ofthe f l a s k must be equal. S t e r i l e samples f o r plate counts were taken from one arm of the three way overflow stop-cock (7a). The other arm (7b) carried the effluent broth through another ground glass stop-cock (8) to the overflow f l a s k (9) from which the effluent culture was taken. EXPERIMENTAL The apparatus was assembled a s e p t i c a l l y after auto-claving at 15 pounds pressure f o r 15 minutes. About 250 ml. of s t e r i l e broth was run into the growth f l a s k and the contents were allowed to cool. The apparatus was then placed i n a 37°C water bath and the growth f l a s k was inoculated. 10 ml. of a vigourous young culture (12 hours) of P. aeruginosa was used as a source of inoculum. Before the flow of s t e r i l e broth was started the culture i n the growth f l a s k was incubated overnight 18. (12 hours) so that there would be a f a i r l y large number of young c e l l s present. A plate count was made to determine the number of c e l l s present at the beginning of the experiment, then the broth was allowed to flow through the f l a s k at a known rate and the plate count was used to check p e r i o d i c a l l y the population i n the growth f l a s k . I t has been reported (8)(12) that i n t h i s type of aa apparatus, using a constant flow of broth, the population reaches a certain l e v e l and remains constant. However, when the rate of flow.was increased or the broth was used i n a more concentrated form, the viable and t o t a l counts increased—accordingly. The experiment was carried out to determine whether or not such a cor r e l a t i o n could be shown. In the f i r s t experiment the rate of flow through the growth f l a s k was 142.56 ml. per hour. The average plate count on this run over a s i x day period was 139 m i l l i o n c e l l s per ml. In the second experiment the rate of flow was 118.8 ml. per hour. The average count here was 184 m i l l i o n c e l l s per ml. over a 5 day period. In the t h i r d experiment the rate of flow was 95.04 ml. per hour with an average count of 318 m i l l i o n c e l l s per ml. over a s i x day period. When the rate of flow i n t h i s experiment was increased to 143 ml. per hour the population dropped to a con-stant l e v e l having a count about the same as that i n experiment number one above. I t w i l l be noticed that comparing the f i r s t two rates of flow with the t h i r d that there appears to be a much greater increase i n c e l l count between the second and t h i r d than between 19. the f i r s t and second. This may be due to the fa c t that at t h i s p a r t i c u l a r rate of flow some stimulatory substance, perhaps a coenzyme, excreted by the organism i s l e f t i n the propagator at such concentration that more of the same substance can be excreted by the organisms whose growth i s enhanced by i t , thus making the population larger than would be expected. SUMMARY P. aeruginosa was successfully cultured by the method of continuous propagation. There was no d e f i n i t e c o r r e l a t i o n between the rate of flow of broth and the viable count as reported by Jordon and Jacobs. LACTOBACILLUS PLANTARUM The object of the following experiments was to deter-mine how ra p i d l y a s t e r i l e broth could be passed through the apparatus without effecting too greatly the production of acid by the organism and ultimately, to determine the rate of flow that would permit a maximum production of acid from the carbo-hydrate i n the medium to be used. METHODS The organism selected f o r the second part of this study was Lactobacillus plantarum ( C c . 53). This microorganism i s more fast i d i o u s i n i t s growth requirements than P. aeruginosa and, as such, presents a more d i f f i c u l t problem f o r continuous propagation studies. Lactobacillus plantarum was grown continuously using 20. the p r i n c i p l e s of the previous experiment. In this case, however, the a c t i v i t y of the organism was followed by t i t r a t i o n " of the acid produced, by pH determinations and by the plate count. The organism was carried on Orla Jensenls agar and was cultured continuously on corn steep broth. Plate counts were made on Orla Jensen's agar. Media Corn steep nutrient broth Tryptone 1$ Adjust pH to 7.3 K 2HP0 4 0.5$ Glucose 0.5$ Corn steep 8.3 ml./lOOO ml. of broth. Orla Jensen's media Peptone 1$ Adjust pH to 7.2 Yeast 1$ Glucose 0.0$ K 2HP0 4 0.5$ \ 7 d LEGEND 1. growth f l a s k a. stop-cock f o r inflow control b. below surface entry device c. s t i r r i n g apparatus d. mercury seal e. nitrogen tube f. sample tiibes 2. holding f l a s k g. stop-cock to prevent back pre 3. effluent storage f l a s k > o td > o H h-l C\ CO T) > > > > ft) > t-3 (—• *—1 o M: I' IV) 21. APPARATUS The apparatus employed i n t h i s experiment i s shown i n Pig. 2 and can be seen to consist of three f l a s k s . The s t e r i l e broth flowed through a stop-cock (a) into the growth f l a s k (1) i n such a way that the s t e r i l e broth entered under the l e v e l (b ) of the growth broth. The contents of t h i s f l a s k (1) were agitated by a motor-driven propellor (c), the shaft of which passed through a mercury seal (d). Nitrogen gas, which was used to maintain anaerobic conditions i n the growth f l a s k was passed into the f l a s k through a tube (e) drawn to a f i n e t i p . The effluent culture v/as drawn through the f l a s k s by suction. Flask 2 i s merely used as a safety measure to prevent contamination of the main f l a s k . The growth f l a s k was inocu-lated through a cotton plugged tube (h). Samples f o r observation could be taken a s c e p t i c a l l y from the sample tube (f) of f l a s k 1 or 2 but were preferably taken from f l a s k 2 to lessen the p o s s i b i l i t y of contamination i n the main f l a s k . Flask 3 contained the effluent culture, when i t was emptied the glass stop-cock (&) was closed to prevent a back pressure. EXPERIMENTAL The growth f l a s k was inoculated with a young culture of L. piantarum and incubated at 30°C for 12 hours before the of s t e r i l e broth was started. This procedure was followed i n a l l of the following experiments. 22. In the f i r s t experiment the inflow was one drop i n 4.6 seconds. This meant that the growth culture volume was renewed every 7.2 hours. TIME COUNT PH Titrat a b l e a c i d i t y (hours) ( c e l l s per ml.) mi l l i o n s (ml. 0.1 N NaOH) st a r t 232 4.65 2.5 24 58.5 6.65 1.1 48 83.2 5.75 1.3 68 96.7 5.75 1.5 76 109.5 5.85 1.8 84 106 5.95 1.8 112 96 5.8 1.8 126 83 5.8 1.7 132 82 5.7 2.1 144 # 75 5.5 2.3 168 # 80 5.42 2.5 192 # 77 5.4 2.6 224 # 82 5.4 2.8 I t can r e a d i l y be seen that t h i s rate of flow was too rapid. The culture was passed out of thefermentor too rapidly f o r a l l of the sugar to be u t i l i z e d . # This would appear to indicate the development, probably by selection, of a s t r a i n of the Lactobacillus which produced a high y i e l d of acid per organism In a shorter time than was possible by the o r i g i n a l culture. 23. In the second experiment the growth culture was changed every 11 hours which means that the culture volume v/as changed more slowly than during the f i r s t experiment. TIME COUNT PH Tit r a t a b l e a c i d i t y (hours) ( c e l l s per ml.) mi l l i o n s (ml. 0.1 N NaOH) 19.5 160 6.4 1.8 47 159.5 5.1 3.2 65 129.5 5.0 3.2 71 127 5.2 3.2 105 189 5.0 3.2 132 204 4.7 3.8 153.5 269 4.46 4.0 186 224 4.36 4.2 204 # 198 4.3 4.3 228.5# 182 4.4 4.1 This rate of flow gave a higher acid production but there seemed to be l i t t l e c o r r e l a t i o n between the size of the population, the pH and the amount of acid formed. In the t h i r d experiment the growth culture was replaced every 16 hours. The culture was allowed to remain i n the f e r -mentation vessel f or a longer period of time than i t was i n either of the two previous experiments. (TIME COUNT PH Ti t r a t a b l e a c i d i t y (hours) ( c e l l s per ml.) mi l l i o n s (ml. 0.1 N NaOH) 48 220 4.75 3.4 66 211 4.65 3.4 110 215 4.63 3.6 134 212 4.61 4.5 146 159 4.6 4.7 188* 197 4.1 4.8 236* 183 4.1 4.6 Prom the above chart i t can be seen that t h i s slower rate of flow permitted the organism to ferment an even larger percentage of sugar present i n the medium to l a c t i c acid. jf As i n the f i r s t experiment there appears to be a develop-ment of a rapid fermenting s t r a i n . The change i n the num-bers of viable bacteria between the times 204 and 228.5 would seem to indicate that the decrease i n numbers was caused by a l i m i t i n g pH. I t w i l l be shown l a t e r that this was not the case. 24. The f i n a l pH f o r t h i s s t r a i n was about 4.1 at which time the culture was forming 5.2 grams of l a c t i c acid per l i t r e from the sugar present i n the corn steep broth. The culture did not appear to weaken or die off at this pH so long as the propagator was running but i f the flow of s t e r i l e broth was stopped for about 10 hours the population diminished considerably. Even though the population was diminishing, there was sometimes an increase i n the t i t r a t a b l e a c i d i t y depending on how much sugar was l e f t unfermented. Stern and Frazeir ( 35) have reported that after growth stops the production:,of acid may continue. No apparent sign of di s s o c i a t i o n was observed during the 5 to 6 day periods i n which the experiments were conducted. I t was found that the apparatus used i n these experiments was more sat i s f a c t o r y both f o r growth and f o r acid production than was the one used f o r the experiment with Pseudomonas. A larger fermentation f l a s k and a slower rate of inflow of s t e r i l e medium was used so that the culture could be kept longer i n the propagator thus allowing more acid to be formed than was possible with the smaller sized fermen-ta t i o n f l a s k . The smaller size of the inflow j e t used here allowed a much greater control over the rate of addition of s t e r i l e media. The viable counts ranged from 17 m i l l i o n to 558 m i l l i o n per ml. depending on the rate of flow of s t e r i l e media used. The development of the rapid fermenting s t r a i n was also i n evidence i n t h i s experiment but was not as sharply defined as i t had been i n the two previous experiments. 25. Age of the Cells i n the Propagator. I t appeared that i f i n a continuous system the organisms continued to reproduce at a constant rate the majority of the c e l l s present would be active young c e l l s . In order to support t h i s presumption i t was necessary to f i n d a method that could be used to determine approximately the age of the c e l l s as they grew i n the propagator. The following methods were t r i e d and i t was found that acid agglutination was the most sa t i s f a c t o r y method. 1..Changes i n the e l e c t r o k i n e t i c potential of bacteria at various phases of the culture cycle. (20) 2. Adaptation - old c e l l s adapt more slowly, i f a l a l l , than do young c e l l s . Young growing cultures produce the new enzymes e a s i l y . (21)(22)(23)(24)(31) (32) 3. The resistance of subcultures to unfavourable conditions such as heat, s a l t , etc. (25)(32)(28) 4. The age of parent cultures i n r e l a t i o n to the rate of growth of subcultures. (26)(27) G i l l e s p i e (29) i n 1914 used acid agglutination to d i f f e r e n t i a t e between b a c t e r i a l species. He found that old broth cultures showed less resistance to acid agglutination than did young c e l l s . In the present work sulphuric acid was used to agglutinate washed suspensions of Streptobacterium plantarum. EXPERIMENTAL A known amount of the washed culture to be tested was put into a series of test tubes i n which the concentration of acid was gradually increased. A pH reading was taken on 26. the tubes that showed signs of agglutination. This test was incubated at 30°C f o r 3 hours before readings were taken. I t was found that between pH 3.55 and pH 4, a 48 hour culture showed p r e c i p i t a t i o n and the formation of granules whereas there was no such action at higher or lower pH values. There was no p r e c i p i t a t i o n of cultures younger than 24 hours. A plate count and an acid agglutination test were done on samples of the effluent culture from the propagator to check the above r e s u l t s . A growth curve was plotted f o r t h i s organism so that the results from the plate count could be f i t t e d approximately into part of the growth curve and then these results were correlated with the acid agglutination t e s t . Another method was that of Tolstoohov (30) using a combination of dyes sensitive enough to detect differences i n the i s o - e l e c t r i c point of a single microorganism. This technique was used to d i f f e r e n t i a t e the ages of the c e l l s but the test was too f i n e and in t e r p r e t a t i o n of the results was d i f f i c u l t . Streptobacterium plantarum showed a v a r i a t i o n of morphology at several times during t h i s experiment. A pure culture was used f o r the inoculation and p e r i o d i c a l l y the c e l l s i n the propagator were checked f o r contamination. Results of gram stains. 1. Gram po s i t i v e rods. 2. Gram po s i t i v e cocci. 3. A mixture of gram p o s i t i v e and gram negative cocci on the same s l i d e . Sometimes the cocci would appear to have gram negative outer edges and gram p o s i t i v e centers. Occasionally 27. chains of c e l l s were observed i n which some of the c e l l s i n the same chains were gram p o s i t i v e and some were gram negative. There are various theories as to how this phenomenon could occur according to Saulter (33). 1. Membrane permeability to dyes. 2. Relationship between the i s o - e l e c t r i c point of the c e l l protoplasm and i t s a f f i n i t y f or the basic dye. gram p o s i t i v e have a lower pH i s o - e l e c t r i c point, gram negative have a higher pH i s o - e l e c t r i c point. 3. Morphological i n t e r p r e t a t i o n (34) - around the gram p o s i t i v e organisms there i s a gram po s i t i v e electroplasmic layer which i s absent from the gram negative organisms while the endoplasm of a l l the organisms i s gram negative. Only one culture was i s o l a t e d which was dissociated. (36)(37)(38)(39) Twenty-six cultures of l a c t o b a c i l l i were i s o l a t e d from the propagator and a l l but one developed a f i n a l pH of from 4.2 to 4.4 aft e r 3 days growth i n test-tube cultures. Culture number 15 showed a pH of 7*15 whlcb/was the i n i t i a l pH of the broth. it Number 15. 1. Appeared on plates as a rough colony. 2. Growth i n broth culture was stringy and mucous-like. T i t r a t i o n r e s u l t s showed that no acid, was formed. L. plantarufe when i n the (R) dissociated form loses i t s sacchrolytic power and has greater proteolytic powers. (37) 28 . SUMMARY L. piantarum has been successfully propagated i n an apparatus designed so as to premit a constant flow of s t e r i l e media i n t o , and effluent culture out of a culture chamber. A method has been found that gives f a i r l y accurately the age of the majority of the c e l l s which are growing i n the apparatus. 29. PART I I CONTINUOUS GROWTH OF AZOTOBAGTER VINELANDI INTRODUCTION I t i s an established fact that Azotobacter can f i x free nitrogen from the atmosphere. This reaction i s and has been of great Importance to agriculture i n that the reaction as such increases the nitrogen content of non-acid s o i l s . The essential components of this s o i l nitrogen f i x a t i o n process are free nitrogen and an available energy source. The object of the present experiment i s to culture Azotobacter vinelandl i n whey. 'Whey contains two possible sources of energy, l a c t i c acid and the disaccharide lactose. Strong aeration w i l l provide the free nitrogen essential to the f i x a t i o n process. The growth of the organism should increase the nitrogen content and the vitamin content of the whey medium. REVIEW OF LITERATURE I t has been shown conclusively many times that f i x e d nitrogen i n h i b i t s or prevents the process of free nitrogen f i x a t i o n . Azotobacter w i l l u t i l i z e available combined nitrogen where possible instead of following the more laborious process of f i x i n g free nitrogen (40). Burk (42) states that "the view of previous workers i s supported and enlarged upon, that f i x a t i o n i s a function resorted to only i n the absence of s u f f i c i e n t l y available f i x e d nitrogen. Fixation i s , indeed, not an essential function." The decrease i n nitrogen f i x a t i o n by cultures, due to the addition of nitrogenous compounds, corresponds very c l o s e l y to the amount of f i x e d nitrogen u t i l i z e d (50). peptone has a marked e f f e c t , r e s u l t i n g i n an 30. increase of growth and a d e f i n i t e depression of nitrogen f i x a -t i o n (40)(42). Greaves et a l (41) state that casein and albumin materially increased nitrogen f i x a t i o n by Azotobacter whereas g e l a t i n greatly retarded i t . They reasoned that t h i s increase with casein and albumin was due to iron and vitamins carried thereon that the organism needed. Wilson (66) employed an acid hydroysate of casein to furnish a mixture/amino acids, but Azotobacter vinelandi did not obtain an appreciable amount of nitrogen from t h i s source. Azotobacter are able to u t i l i z e n i t r a t e s , ammonium sa l t s and amino acids i n preference to free nitrogen (42)(50). I t has been shown that of the amino acids, only the dicarboxylic acids are assimilated.(50) With the exception of uramil, any purines assimilated are those which occur naturally i n nucleic acid and contain nitrogen i n an amino group outside the r i n g . Ammonia and compounds re a d i l y converted to ammonia are used to the v i r t u a l exclusion of molecular nitrogen. Wilson et a l (66) believe that ammonia i s a key figure i n nitrogen f i x a t i o n . Greaves (41) proved that cystine and d l methionine increased the nitrogen fixatioriapproximately 20%. Horner (50) gave evidence which showed that arginine, p a r t i c u l a r l y i n low concentration, increased nitrogen f i x a t i o n by Azotobacter. Gainey (68) cultured Azotobacter r e a d i l y i n a modified Ashby's medium containing calcium and adjusting pH values between 6.1 and 6.5 but found that i t did not grow i n the same medium at pH values below 6. Increasing the calcium content of the medium by 300 p.p.m. by addition of calcium absorbed on 51. c o l l o i d a l clay, had no measureable effect on growth of the test organism at pH values below 6. In the past there has been r e l a t i v e l y poor growth of cultures i n media not known to be def i c i e n t i n one or more of the elements essential f o r nitrogen f i x a t i o n and growth, p a r t i c u l a r l y calcium, i r o n , and molybdenum (50,SV, 43, 51). Lewis (70) demonstrated an in t e r a c t i o n between copper and iodine i n the early growth stages of Azotobacter. The presence of certain concentrations of copper i n the medium lengthened the lag phase of the population cycle, but t h i s e f f e c t was diminished by the preSence of iodine. The ro l e of associated organisms may often by simply an a l t e r a t i o n of the medium so that i t becomes more favourable f o r the growth of Azotobacter. Lind (65) showed that an organism grown with Azotobacter increased the amount of iron i n the medium i n that i t put the i r o n i n a more available form. Iron i s important i n the growth of Azotobacter whether the nitrogen source be free or combined. Several investigators have claimed that calcium i s s p e c i f i c a l l y required f o r nitrogen f i x a t i o n by Azotobacter, but more recent studies (68) question whether calcium i s specif i c f o r this reaction. The growth of Azotobacter i n a stationary l i q u i d medium takes place very slowly unless the layer of the medium i s very t h i n . Thus i f a deep l i q u i d medium i s to be used, aeration i s ess e n t i a l . (51, &$) 'Vigourous aeration i s necessary to repress f l o c u l a t i o n of suspended bacteria. This f l o c u l a t i o n r e s u l t s i n almost complete cessation of growth after 24-48 hours.' (48) 32. Glucose i s fermented by Azotobacter as follows-C 6H 1 20 6 / 6 0 2 ~> 6 C 0 2 / 6 H ?0 -~> -674,000 c a l . (64) Vigourous aeration with small bubbles of gas i s not only e f f e c t i v e i n sweeping out COg but also furnishes an extremely large g a s - l i q u i d interface from which the gas exchange may take place (COg given off and nitrogen absorbed.) Since Azotobacter possesses the highest r e s p i r a t i o n rate of any known organism, i t might be expected to contain a high l e v e l of respiratory enzymes and vitamins. Lee (5ffi) assayed c e l l s f or the B complex and showed that Azotobacter c e l l s grown on a nitrogen free sucrose, mineral s a l t s medium have a vitamin content equal or superior to that found i n yeast. Hydrogen i n h i b i t s nitrogen f i x a t i o n byt has no s i g n i f i c a n t effect on the growth of Azotobacter supplied with usual quantities of r e a d i l y available f i x e d nitrogen. (50) Carbon monoxide (0.1 - 0.2$) i n h i b i t i o n of nitrogen f i x a t i o n by Azotobacter has also been established. (61) The organic compound serving as the growth substrate for Azotobacter has a marked influence on the subsequent respiratory a c t i v i t i e s of the r e s u l t i n g c e l l s . When placed under favourable conditions the c e l l s are capable of adapting t h e i r respiratory system to the u t i l i z a t i o n of new compounds i n r e l a t i v e l y short periods of time, usually less than two hours. (53) Sucrose and dextrose are the sugars most commonly used. There exists a close c o r r e l a t i o n between the rate of dextrose fermentation and the rate of nitrogen f i x a t i o n . (67) I t has been found generally that the e f f i c i e n c y of conversion of sugar to c e l l s i s about 15$. 3 3 . EXPERIMENTAL • There i s present i n the l i t e r a t u r e considerable data concerning the f i x a t i o n of atmospheric (free) nitrogen by Azotobacter. There i s , however, nothing d e f i n i t e regarding the f i x a t i o n process i n an organic medium such as whey. Whey contains the disaccharide lactose and l a c t i c acid as sources of carbon. The nitrogen sources are, however, more complex. Although the greatest part of the proteins of the milk have been removed there remains some protein and possibly protein breakdown products as sources of nitrogen. The problem was to determine whether lactose or l a c t i c acid was a sa t i s f a c t o r y carbon source, and i f the remain-ing protein breakdown products would prevent the f i x a t i o n of atmospheric nitrogen by Azotobacter. The organism ;used was a freshly i s o l a t e d culture which had been checked f o r p u r i t y . The following preliminary experiment was done to determine the action of the organism on various energy sources which have been shown to be sa t i s f a c t o r y f o r Azotobacter vinelandi. 200 ml. of modified Burk's medium i n each of 4 one l i t r e v a c o l i t r e flasks was inoculated with 24 hour cultures of Azotobacter (growth washed off s l a n t s ) . Aeration was by suction (water pressure). The cultures were incubated at 30°C f o r 4 days. 34 . Carbohydrate 2% Nitrogen mgms./lOO ml. sucrose 12.28 glucose 11.00 lactose 0.8 Ca lac t a t e 0.0 These results are not altogether i n agreement with reported values for Azotobacter but the low values obtained i n this work may be attributed to i n s u f f i c i e n t aeration, with the exception of the lactate which should support the growth of the organism. Perhaps the trouble l i e s with the anion. Generally the sodium or potassium s a l t i s used. Most of the protein i n the whey was precipitated (acid and heat) and f i l t e r e d o f f . There remained aft e r t h i s treatment, 39.40 mgms. of nitrogen per 100 ml. Several f l a s k s of t h i s whey were inoculated with lactose fermenting yeasts. The yeasts, i n the i r anaerobic fermentation should incorporate some or a l l of the remaining protein breakdown products into c e l l u l a r protein. After the growth of the yeasts (5 days), the flasks were s t e r i l i z e d and inoculated with Azotobacter along with one f l a s k of the normal deproteinized whey. These flasks were incubated and aerated for s i x days. Growth was slow i n a l l f l a s k s but there was pigment formation. The average nitrogen f i x e d was 2.6 mgms. per 100 ml. of whey. There was no s i g n i f i c a n t difference i n any of the samples. Although these results are low they indicate that the process of nitrogen f i x a t i o n by Azotobacter i s not completely i n h i b i t e d by the f i x e d nitrogen present i n the whey and also that there i s an energy source present i n whey which can be u t i l i z e d f or the process of nitrogen f i x a t i o n . 35. Contirmous propagation of Azotobacter vinelandi APPARATUS I t seemed advisable at this point to culture the organism continuously on a medium which has been proven to be satisf a c t o r y f o r the growth of the organism. An apparatus s i m i l a r to that used f o r the propagation of Lactobacillus plantarum was set up. I t di f f e r e d from the previous apparatus i n that growth conditions i n t h i s experiment were aerobic. Aeration was accomplished by drawing a i r through s t e r i l e cotton and from there through a porous carborundum a i r sparger. The growth f l a s k , which contained a constant volume (470 ml.) of Burk's modified nitrogen free medium, was kept at a temperature of 30°C i n a water bath. The growth f l a s k was inoculated with a 24 hour slant (Burk's) culture of Azotobacter vinelandi. The growth was washed off the slant with 10 ml. of Burk's l i q u i d medium. Aeration was commenced immediately after Inoculation but s t e r i l e broth was not passed through the growth chamber t i l l growth was evident, aboit 24 hours. Burk's Nitrogen Free Medium. K2HPO4 0.8 Grams Adjust to pH 7.3 KH 2P0 4 0.2 MgS04 0.2 NaCl 0.2 CaS0 4 0.1 F e 2 ( S 0 4 ) 3 0.01 Glucose 10 Grams D i s t i l l e d water 1000 ml. For s o l i d medium add 1.5$ Agar as free of nitrogen as possible. For modified Burk's medium (51) add Na2Mo04 0.1 p.p.m. PeS0 4 3.0 p.p.m. Sucrose i n place of glucose 2$ 36. Sucrose was the energy source f o r the f i r s t experiment, the r esults of which are shown below. Plow i n ml./24 hours Time a f t e r change of flow Hours Nitrogen mgms./lOO ml. pH i i 410 48 18.5 96 17.9 355.6 48 24.3 6.8 -• 7.0 96 24.65 216 48 31.4 6.8 -• 7.0 96 30.8 162 | 48 15.4 6.0 when the s t e r i l e medium was passed into the growth f l a s k at a rate of 216 ml. i n 24 hovirs the amount of nitrogen f i x e d by the organism was as high as any reported nalues. (51) I t has been shown (page 35) that there was an energy source i n whey which Azotobacter could u t i l i z e f o r the nitrogen f i x i n g process. There are two obvious energy sources available to this organism, namely, lactose and l a c t i c acid. The two following experiments were planned to determine whether Azotobacter could u t i l i z e either of these two as a source of energy and also to what extent they could be used (ie) the ef f i c i e n c y of conversion of the energy to f i x e d nitrogen. Azotobacter were grown i n the apparatus on Burk's modified medium at a rate of 355.6 ml. per 24 hours, the rate found most suitable f o r sucrose u t i l i z a t i o n . When 1500 ml. had passed through the apparatus, Burk's modified medium (lactose 2%) was passed through at the same rate. The green pigment soon l e f t the growth f l a s k and there was l i t t l e or no growth. Nitrogen determinations on the effluent showed l i t t l e f i x a t i o n of nitrogen. However, there was a p o s s i b i l i t y that some of the organisms may have been adapted to lactose. 37. Streak plates (on Burk's lactose agar) were made and Incubated but there was no v i s i b l e growth after 4 days incubation.j.at 30°C. The same procedure as above was used again except that Na lactate was used i n place of the lactose. During this experiment, the culture retained i t s properties of pigmentation but growth was not very heavy. Nitrogen determi-nations showed that 8.3 mgms. per 100 ml. of nitrogen had been f i x e d by the culture: The pH of the effluent was 7.1 - 7.4. I t i s obvious that t h i s high pH was caused almost completely by the freeing of the sodium ions as the lact a t e was being u t i l i z e d by the organism during the f i x a t i o n process. In an attempt to overcome th i s d i f f i c u l t y a t r i a l run was made i n which the growth was i n i t i a t e d i n sodium lactate broth, and s t e r i l e l a c t i c acid medium was passed into the growth f l a s k . I t appeared that the l a c t i c acid would be fermented as soon as i t was passed into the culture and thus would not affect the pH. This, however, was not the case. The pH soon dropped with a consequent lowering of f i x a t i o n by the organism. I t was impossible to control the pH as the d i v i s i o n between the points where growth could and could not be supported was too f i n e , Since there i s very l i t t l e buffer i n Burk's medium, the experiment was repeated using the same conditions and medium except that the buffer content was increased. 10.815$ KHpP04 9.0501$ Na2HP04 was added to Burk's media. 58. Flow i n ml./24 hours Nitrogen f i x e d i n mgms./lOO ml. pH 653.6 a - 5 " '""675 475.2 12 - 14 7 - 7.2 316.8 5 - 6 7.6 - 8.0 I t can be seen that the addition of buffer allowed a much greater control over pH and thus over the- growth of the organism. Although conditions f or f i x a t i o n appeared to be optimum, the highest y i e l d of f i x e d nitrogen was less than h a l f of the the o r e t i c a l y i e l d , 30 mgms. per 100 ml. per 20 grams of l a c t i c acid. SUMMARY The factors which are concerned In the process of nitrogen f i x a t i o n from whey constituents by Azotobacter vinelandi have been outlined. Although the a c t u a l continuous fermentation of the whey by Azotobacter was not attempted i t can be seen that before such a fermentation could be attempted, the lactose, which i s not available as a source of energy f o r Azotobacter, would have to be converted by fermentation to l a c t i c a c id. I t has been shown that l a c t i c acid i s by no means as sat i s f a c t o r y a carbon source as are some of the sugars. 39 PART I I I THE PRODUCTION OF ETHYL ALCOHOL FROM WHEY The object of the following experiments was to carry out an alcoholic fermentation of the 3sactose found i n whey by a continuous fermentation system. BATCH METHOD The f i r s t experiment was set up as a preliminary step i n that, before a continuous propagation system can be used successfully, the process to be attempted must be studied f i r s t by means of the batch method of progagation. METHODS The f i r s t step i n the approach to this problem was to f i n d a suitable lactose fermenting organism. A study was made of four lactose fermenting yeasts which appeared to be the most promising. (71, 72) Y 44 Torula cremoris Y 29 Saccharomyces f r a g i l u s Y 30 Saccharomyces f r a g i l u s Y 52 Zygosaccharomyces l a c t i s EXPERIMENTAL In order to f i n d which of these organisms was the most suitable f o r the production of et h y l alcohol from the lactose i n whey these four yeasts were inoculated (1% of a 24 hour culture) into 100 ml. of s t e r i l e whey i n 175 mL Erlernnyers and incubated at 30°C f o r 3 days. 40. The re s u l t s were as follows (average of duplicates): - t _ 44 1.46$ alcohol. Y 29 0.73$ alcohol. Y 30 0.52$ alcohol. Y 52 0.61$ alcohol. Although Y 44 yeast culture produced considerably more alcohol than did the others the y i e l d of 1.46$ alcohol i s far from being close to the theoret i c a l y i e l d . I t i s possible that the addition of a more r e a d i l y available nitrogen source or the addition of substances which are known to increase yeast growth and fermentation e f f i c i e n c y would increase the alcohol production. In order to determine what effect various activators would have on the action of the yeast the following experiment was set up. Culture Y 44 ALCOHOL $ MEDIUM Whey-as i s Whey-plus ye. extract 0.5$ Whey-plus ye. extract 1$ Whey-plus (NH 4) 3P0 4 0.7$ Whey-plus corn steep l i q u o r 0.8$ Whey-plus malt extract 1$ 1.491 1.551 1.512 1.596 1.575 1.428 "when additions were made to the whey, they were made before the whey medium was s t e r i l i z e d . A 1$ inoculum of the yeast (Y 44) was used. After a three day incubation period at 30°C aliquots of the culture were analysed f o r alcohol. The addition of (NH4)3P04 had an appreciable action In that the increase i n the f i n a l y i e l d of alcohol could be attributed to one facto r , the nitrogen containing radical/of the s a l t . Although the addition of the other activators did indicate some response t h e i r o v e r a l l action was negligable. I t was noted i n the l i t e r a t u r e (72, 73) that the 41. protein i n the whey was prec i p i t a t e d and f i l t e r e d off before any fermentation was attempted. There were no reasons given for carrying out this procedure so the following experiment was set up to determine what effect i t would have on the alcoholic fermentation of whey. The pH of the whey was adjusted to 4.8 with HC1» then the whey was steamed f o r two consecutive one hour periods and the protein p r e c i p i t a t e was f i l t e r e d off by suction through f i l t e r paper on a Buchner funnel. A series of fla s k s contain-ing normal whey, deproteinated whey and a suitable control was set up and inoculated with 10 ml. of a 12 hour culture of Torula cremoris. Substrate I n i t i a l Alcohol formed P i n a l pH (200 ml.) pH (per cent) 1. Whey 5.5 5.5 1.642 4.8 2. Whey 7.0 7.0 1.504 5.0 3. Deproteinated whey 7.0 1.596 4.7 Pr i o r to s t e r i l i z a t i o n o.l% (NH4)3P04 was added to these three f l a s k s . The removal of the protein caused no s i g n i f i c a n t difference i n the f i n a l y i e l d of alcohol but throughout the procedure i t was noted that the deproteinated whey was easier to work with. I t was noted also that the effect of the i n i t i a l pH was not so evident when the buffering action of the protein was removed (compare'the f i n a l pH values of nos. 2 and 3 above). CARBON BALANCE Although the conditions f o r fermentation were nearly optimum i t was evident that higher yi e l d s of alcohol could be obtained. Some of the available carbohydrate was being used by the organism for a reaction other than the desired alcoholic CARBON BALANCE CHART gms/lOO ml. gms/lOO gms of lactose mg./lOO mM of lactose mM/lOOmM of lactose Carbon Carbon Dioxide 1 .6 4 4 . 0 1 5 0 . 5 342 342 Ethyl Alcohol 1 .65 45 .3 1 5 4 . 9 344 688 Acetic Acid 0 . 0 2 0 . 3 3 1 .13 1.76 3 . 6 Glycerol trace Lactic Acid # 1033.6 # Lactose was 0 . 7 gms/lOO ml. before and after fermentation. From the 4.2$ lactose available to the organism 3.64$ was u t i l i z e d . Calculations: 12 C X 100 = 1200 carbons from lactose. 1033.6 1200.0 X 100 = 86.1$ recovery. 42 . fermentation. I t seemed advis&hlfie, therefore, to carry out a carbon balance on the fermentation products of the organism i n a lactose medium. Several semi-synthetic lactose media were used to culture the organism but neither the fermentation nor the growth were as sat i s f a c t o r y as that obtained on whey. As a r e s u l t of these observations, whey was used as the medium f o r the carbon balance studies. A 1$ inoculum of a 24 hour culture of Torula cremoris was used to i n i t i a t e growth i n 400 ml of whey medium. The whey culture was incubated at 30°C f o r three days then subjected to a chemical analysis. The procedures f o r the analysis w i l l be found on page (59). The r e s u l t s i n percentage of recovery from lactose appeared to be low but yeasts assimilate more carbohydrate than do bacteria f o r which only 5$ of the carbon i s attributable to assimilation. (79> 80, 81) The re s u l t s would seem to indicate that the yeast fermentation e s s e n t i a l l y constitutes the production of alcohol and carbon dioxide. The analysis of the uninoculated medium showed i t to contain 0.7 grams of l a c t i c acid per 100 ml. but no v o l a t i l e acids. The pH changed from 5 before fermentation to 4.5 at the end of the fermentation period. The amount of acetic acid formed by the yeast was equivalent to the amount of base which had been used to r a i s e the pH to 5. Another carbon balance was carried out using 1 ml. of 5 normal sodium hydroxide to raise the pH to 5.5 before fermen-tat i o n . The carbon balance for t h i s run was much the same as 43. the l a s t except that the v o l a t i l e acid was 0.069 grams per 100 ml. of fermented medium. The amount of a c i d i t y present over and above that given by l a c t i c acid was again almost equivalent to the amount of base which had been added to r a i s e the pH to 5.5. For example - 12.9 ml. of N/100 NaOH were required to neutralize 10 ml. of the neutral v o l a t i l e d i s t i l l a t e . This amount i s almost equal to the t i t r a t i o n of the fermented c u l -ture after subtracting the t i t r a t a b l e a c i d i t y of the l a c t i c acid. Acetic acid produced — 1.032 ml. of 5N NaOH i n 400 ml. of culture. Base added to st a r t — 1.0 ml. of 5N NaOH i n 400 ml. of culture. I f , as i t appears, acetic acid i s produced by t h i s yeast and this acetic acid i s responsible f o r the drop i n pH when alcohol i s produced from a carbohydrate then this yeast must be one which i s tolerant to acetic acid. This is a matter of importance since Gruess and Harcal (73) have reported that acetic acid i s toxic to yeast i n r e l a t i v e l y low concentration. They found that 0.3 grams of acetic acid per 100 ml. of culture greatly retarded yeast growth and a c t i v i t y . E a r l i e r i n t h i s work i t was shown that Torula cremoris deceased the pH to 4.7 - 5.0 even when the s t a r t i n g pH was 7.0. When the amount of acid required for t h i s change i n pH i s calculated out i t exceeds 0.3 gms. per 100 ml. Rogosa et a l (72) reported that the maximum pH drop during alcoholic fermentation by yeast was 0.6_ pH u n i t s . When they cultured Torula cremoris at pH 5.5 the pH dropped only to pH 5 at the end of the fermentation. An inspection of the results i n the presant work w i l l show much greater pH deviations 44. than those reported by Rogasa et a l . SUMMARY I t has been shown that of the yeast cultures used Torula cremoris i s the most suitable culture to be used f or the fermentation of lactose to ethyl alcohol. The optimum con-ditions f o r this fermentationjare as follows. The pH of the whey should be about 4 .7 - 5.0 before inoculation. I f the pH of the whey i s higher the organism w i l l produce the amount of acetic acid necessary to lower the pH to 4 .7 - 5.0. The deproteinated whey must be f o r t i f i e d with a r e a d i l y available nitrogen source such as (NH4)3PO4. The optimum temperature fo r the fermentation was found to be 30°C. 45. CONTINUOUS PROPAGATION The purpose of the present study was to construct an apparatus which would s a t i s f y the c u l t u r a l requirements of Torula cremoris and also to incorporate techniques which would best r e s u l t i n the construction of a sat i s f a c t o r y continuous fermentation apparatus. APPARATUS A continuous propagation apparatus which consisted of two separate systems was set up. The purpose of the two systems was to enable a heavy inoculum of yeast c e l l s grown aerobic-ally to be passed into an anaerobic fermentation chamber. Rogosa (72, 73) has shown that a heavy inoculum i s essential for a rapid and a complete fermentation of lactose to alcohol. The method to be used presents two problems - findi n g i d e a l conditions for growth and f i n d i n g i d e a l conditions f o r fermentation. However, the operation allows a more d e f i n i t e control over both of these factors than could be obtained i n a one stage anaerobic fermentation apparatus or i n the use of the batch method of fermentation. For example i t has been reported that NaN3 (0.0023M) i n h i b i t s the anaerobid as s i m i l a t i o n of glucose by yeast. I t reduces the accumulation of high energy phosphates i n the c e l l and thus greatly reduces the the o r e t i c a l 1/3 of the glucose that goes to b u i l d up glycogen i n the yeast c e l l . I f azide were added to the fermentation f l a s k i t should increase the y i e l d of alcohol and should have no effect on growth as the two, growth and fermentation are separate. Figure 3. Legend. 1. growth flask. 2 . fermentation flask. a. stop-cock for inflow control. D. corborundum sparger. c. settling flask. d. pinch-cock for yeast inflow control. e. stop-cock to control inflow of fermentation medium. f. stirring apparatus. g. mercury seal. h. leads to alcohol and carbon dioxide trap. 3 . effluent storage flask. 46. APPARATUS The apparatus employed i n th i s experiment i s shown i n Fi g . 3. Flask 1 i s the growth f l a s k , The rate of flow of s t e r i l e whey medium into t h i s f l a s k i s controlled at (a). The yeast culture i s strongly aerated by a corborundum a i r sparger (b). I t has been shown (76) that aerated cultures show better growth and u t i l i z a t i o n of glucose to alcohol when the c e l l s are used l a t e r f or fermentation. While yeast c e l l s w i l l bud for many generations i n the absence of oxygen, eventually th i s gas i s required f o r continued m u l t i p l i c a t i o n . Pure oxygen i s not as good a growth stimulant as a i r ($0). Dispersion of a i r throughout the medium induces yeast growth s p e c i f i c a l l y at the expense of fermentation. From the growth f l a s k the yeast c e l l s pass down into the fermentation f l a s k (2) v i a a funnel arrangement (c), the purpose of which i s to concentrate the culture by a s e t t l i n g e f f e c t . The rate of flow of yeast c e l l s into the fermentation f l a s k i s controlled by a stop-cock at (d). S t e r i l e whey broth enters the fermentation f l a s k at (e). The fermentation f l a s k then, i s fed by a continuous heavy yeast inoculum and also by a constant supply of s t e r i l e fermentation medium. The contents of the f l a s k are agitated by a mechanical s t i r r i n g device (f) the shaft of which passes through a mercury seal (8). I t De has been shown by/Breeze and Liebman (6'G) that a g i t a t i o n Increases both growth and fermentation rates through better contact of the yeast with the mash ingredients. From the fermentation cylinder the effluent culture flows into f l a s k 3 by way of a gravity-suction system which 47,. keeps the l e v e l of the fermentation culture at a constant volume. Flask 3 i s a holding f l a s k but i t also serves to lessen the danger of any possible contamination of the fermentation f l a s k . The growth medium was deproteinated whey to which was added 0 . 7 $ (NH 4)3PO4. No nitrogen was added to the deproteinated whey used for the fermentation medium. The pH of both media was adjusted to 4.8. EXPERIMENTAL After s t e r i l i z a t i o n and assembly of the apparatus s t e r i l e medium was run into flasks 1 and 2. The contents of the growth f l a s k were inoculated with a young culture of Torula cremoris and strongly aerated. After 24 hours incubation, s t e r i l e growth medium was passed into the f l a s k at a known rate. The flow of yeast inoculum into the fermentation f l a s k from the growth f l a s k was followed 12 hours l a t e r by a controlled flow of s t e r i l e fermentation medium. The purpose of the following experiments was to determine the most sat i s f a c t o r y rate of flow f o r the production of ethyl alcohol i n the fermentation cylinder. ml. of Y/hey fermented i n 24 hours 600 500 420 360 ml. of yeast i n 24 hours 60 60 60 60 alcohol y i e l d % 1.43 1.58 1.69 1.72 The highest y i e l d s of alcohol were obtained when rates of flow between 360 and 420 ml. of fermentation medium per 24 hours were used. The fermentation cylinder contained 580 ml. of continuously fermenting material. Therefore, the ove r a l l time of fermentation when calculated i n terms of the batch method was from 34 - 38 hours. 48. SUMMARY Satisfactory r e s u l t s were obtained with the continuous propagation system employed i n th i s experiment. A normal y i e l d of alcohol was obtained i n a shorter fermentation period than that used by Rogosa. Whey can not be used commercially as a material f o r ethyl alcohol production as the amount of alcohol produced during the fermentation does not warrant i t s recovery from the whey beer. I t was suggested that whey could be used as a source of activators f or the fermentation of some substance high i n carbohydrates such as molasses. In order to use whey as an activator an organism other than a lactose fermenting yeast must be used. In that the sugar used would be one other than lactose the organism employed f o r HUch a fermentation would probably be one used i n commercial brewing. These organisms do not appear to be fastidious i n th e i r growth requirements i n so f a r as whey i s concerned. 49 PART IV THE CULTIVATION OF MORULA UTILIS IN WHEY INTRODUCTION The object of the present investigation was to ascertain whether ot not the growth of Torula u t i l i s would increase the vitamin and the protein content of whey to such an extent that the fermentation product could be dried and used as a feed supplement. Whey i s r i c h i n lactose and vitamins. The former i s very valuable as a carbohydrate source for l i v e -stock r a i s i n g and i s p a r t i c u l a r l y valuable i n the d i e t of young chickens i n that i t helps to prevent c o c c i d i o s i s . In order to conserve the lactose content glucose, which i s more re a d i l y u t i l i z e d by this organism, was added to the whey. whey contains very l i t t l e protein so the major portion of the protein present i n the fermented whey w i l l be yeast protein. B i c k e l (84) has done metabolic experiments with various animals using yeast as a source of protein. He states that yeast contains protein which i s valuable both for human and f o r animal n u t r i t i o n . Nitrogen from yeast protein i s w e l l absorbed by the digestive tract and i s capable of increasing nitrogen metabolism. The value of yeast protein with respect to assimilation i s approximately equal to that of the most complete vegetable proteins but i s i n f e r i o r to that of meat, egg, and milk protein. Fischer (86) praises dried brewers yeast as a valuable poultry feed supplement. Klose and Fevold (87) confirmed 50. previous work that Torula yeast grown on molasses was de f i c i e n t i n methionine when fed at protein l e v e l s as high as 13$ crude protein. Dier and Decker (89) noted that large daily doses of dried yeast cause p o t e n t i a l l y dangerous increases i n blood u r i c acid l e v e l s . They attributed t h i s effect to the high purine content of yeast. Yeast Average Purine Nitrogen 0.75$ 8.7$ of t o t a l nitrogen Most Meats Purine Nitrogen 1.5$ of t o t a l nitrogen However, Hegsted et a l (7;8») studying the role of arginine and glycine i n chick n u t r i t i o n used a basal diet which was 10$ brewers yeast with casein being the p r i n c i p l e source of protein. This would indicate that i f yeast i s fed at proper concentrations (30$ of the t o t a l protein) that the high purine content of the yeast has a negligable e f f e c t . Boucher ($1) stated that dry yeast (non-fermentive) i s among the ri c h e s t and most dependable sources of a l l the members of the B complex vitamins. This c h a r a c t e r i s t i c accounts for i t s wide use i n animal n u t r i t i o n where i t i s p a r t i c u l a r l y valuable during n u t r i t i o n a l l y c r i t i c a l periods such as gestation, l a c t a t i o n , and early growth. 51. r REVIEW OF LITERATURE A survey by Burkholder, McVeigh and Moyer has revealed that p r a a t i c a l l y a l l yeasts can synthesize r i b o f l a v i n , but that they vary considerably i n t h e i r ab i l i t y to synthesize the other B vitamins. Rogosa by growing lactose fermenting yeasts i n a r i b o f l a v i n free medium showed conclusively that these organisms do synthesize ~E>r>, Stubbs and Noble (75) using waste f r u i t juice and NH4 s a l t s showed that Torulopsis u t i l l s yielded 42 - 58 grams of dry yeast per 100 grams of sugar fermented. The protein content was 53 - 58$ as calculated from the nitrogen content of the yeast. The values obtained for- the B vitamins corresponded to the published values f o r bakers and brewer's yeast. Lewis et a l CM!) cultured Torula yeast on a f r u i t juice substrate. Torula u t i l i s was used because of i t s high protein content and B complex vitamins. This yeast i s auto-trophic with regard to the B vitamins. They showed that when they used aeration there was a loss of thiamine from the culture, The following chart shows the yi e l d s obtained by Lewis et a l . N i c o t i n i c acid 375 per gram dry yeast. Pantothenic acid 830 per l i t r e . R i b o f l a v i n 441 per l i t r e . P.A.B . acid 430 per l i t r e . Thiamine 20 per gram dry yeast. Choline 3 per gram dry yeast. I n o s i t o l 2500 per gram dry yeast. Peterson et a l (88) studied the influence of aeration on the y i e l d , prot ein and vitamin content of food yeasts. They used a semi synthetic media. Aeration and a g i t a t i o n had 52. no appreciable effect on the protein content of the yeast but" as would be expected there was an increase i n c e l l synthesis. Vitamin content per gram of sugar u t i l i z e d increased markedly with an increase i n the y i e l d of c e l l s . Thiamine Ribo. Niacin F o l i c 3.7$ y i e l d 1.96 4 7.8 0.58 66.2$ y i e l d 11.5 41.5 31.4 5.2 They stated that a large quantity of sugar goes to maintain non-proliferating c e l l s . Overall e f f i c i e n c y should be increased i n a short period of fermention. Pavec et a l (83, 85) studying the y i e l d of Thiamine from brewer's yeast found that they could increase the y i e l d three f o l d by omitting aeration (40 I.U./gm. dry yeast). With Torula yeast there was also an increase i n anaerobic over aerobic conditions (2 times) however, i n aerated cultures there was more yeast produced so that the ov e r a l l production of Thiamine was about the same. Wilson (82) showed that Torula u t i l i s produced 0.06 of vitamin B c / m i l l i l i t r e . EXPERIMENTAL The following experiment carried out to determine whether or not the growth of Torula u t i l i s would increase the vitamin content of the reconstituted whey powder. Dried whey powder was reconstituted by adding 6 grams of powder to 100 ml. of water. 2 grams of glucose and 0.2 grams of NH^ HgPO^ . were added to act as sources of carbon and nitrogen respectively. 500 ml. quantities of the above medium i n 2 l i t r e 53. v a c o l i t r e f l a s k s were used f o r the growth of the organism. The cultures were a l l aerated through carborundum spargers which dispersed the a i r into f i n e bubbles. Foaming was checked by using octadecanol dissolved i n corn o i l . The pH of the medium was adjusted to 5. The medium was s t e r i l i z e d by autoclaving at 15 l b s . f o r 15 minutes. For inoculation, Torula u t i l i s was stib-cultured i n 10 ml. quantities of medium and the inoculation was made from t h i s . A 2% inoculum of a 24 hour was used. The inoculated culture was grown at 30°C for 5 days at the end of which time samples of the culture were taken f o r the various vitamin assay procedures. Five of the vitamins; Pyridoxine, n i c o t i n i c acid, pantothenic acid, b i o t i n , and f o l i c acid were assayed by the method of Landy and Dickens (56). Assays f o r thiamine and r i b o f l a v i n were carried out as by Sarett, and Cheldelin and Snell and Strong respectively. (58) (59), The results (average) are expressed as gammas per m i l l i l i t r e f o r both the whey powder and the fermented, reconstituted whey powder. Whey gammas/ml. Powder gammas/gm. Whey plus yeast gammas/ml. Ribof l a v i n 7.9 132 6.5 Pyridoxine 0.57 9.6 0.78 Ni c o t i n i c ac id0.63 10.6 1.26 Pantothenic acid 2.28 38. 1.96 B i o t i n 0.03 0.5 0.036 F o l i c acid 0.6 10. 0.5 Thiamin 0.22 4.2 0.38 B i o t i n appeared to be unavailable to the assay organism therefore the yeast growth was digested with 54. taka-diastase before assaying for B i o t i n content. The organism used f o r the assay of n i c o t i n i c acid exhib i t e d heavy growth i n the assay tubes but there was not a corresponding acid production so the assay tubes were read by using t u r b i d i t y rather than acid production. In general, there was no great increase i n the vitamin content of the whey after the growth of Torula u t i l i s . Rather, there was a s l i g h t decrease i n some cases, the most obvious of which was evident i n the res u l t s of the r i b o f l a v i n assays. Since vitamins have been shown to serve as sole carbon substrate (65) i t would not be unreasonable to f i n d that an excess of an "essen t i a l " vitamin could also be metabolized. Very l i t t l e glucose remained i n the culture medium after the trowth of Torula u t i l i s . The fermented product yMded 0.6 - 0.8 grams of dry yeast c e l l s per 100 ml. SUMMARY There was no s i g n i f i c a n t increase i n the vitamin content of the whey medium after the growth of Torula u t i l i s . 56. BIBLIOGRAPHY 1. Graham-Smith, G.S., J. Hyg. 19:133 (1920). 2. Moyer, H.V., J. Bact. 18(1):59 - 67 (1929). 3. Rogers, L.A. and Whittier, E.O., J. Bact. 20:127 (1930). 4. Bushwell, A.M. and Boruff, L.S., Ind. & Eng. Chem. 25:147 (1933) . 5. Cleary, J.P., Beard, P.J. and C l i f t o n , C.E., J. Bact. 29:205 (1934) . 6. Unger, E.D., Stark, W.H., Scalf, R.E. and Kolachov, P.J., Ind. & Eng. Chem. 34:402 (1942). 7. B i l f o r d , H.R., Scalf, R.E., Stark, W.H., and Kolachov, P.J., Ind. & Eng. 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Bot. 29:207 (1924J. 58 71. Browne, E.H., Eng. Chem. Hews Ed. 19:1271 (1941). 72. Rogosa, M., Browne, H.H. and Whittier, E.O,, J. Dairy Be. 30:263 (1938). 73. Cruess, W.V. and Hareel, R., F r u i t Products J. 3:20 (1924). 74. Lewis, J.C,, Arch. Bio. 4:389 (1944). 75. Stubbs, J . J . , Noble, W.M. and Lewis, J.O., Food Industries. . 16(9):68 (1944). 76. Brockman," M.C. and S t e i r , 2.J.B., J. C e l l , and Comp. Phys-iology 29:1 ( 1942). 77. Boucher, R.V., Yeasts i n Feeding Symposium. Mos. 8,9,10. Hotel P f i s t e r , Milwalciae, (1948). 78. Hegsted, U.M., Briggs, CM., Elvehjem, O.H. and Hart, E.B. , J. B i o l . Chem. 140:191 (1940). 79. Fales, F.W. and Baumberger, J.P., J. B i o l . Chem. 173:1 (1948). 80. Sussman, M., J. C e l l , and Comp. Physiology 29:149 (1942). 81. Winzler, R.J., Science 99:2573 (1942). 82. Wilson, K., Arch. Bio. 7:287 (1945). 83. Pavek, N., Peterson, W.H. and Elvehjem, CH., Ind. & Eng. Chem. 30:802 (1938). 84. B i c k e l , A., Biochem Z. 310(6):355 (1941). 85. Pavek, N., Peterson, «.H. and Elvehjem, CH., Ind. & Eng. Chem. 30:802 (1938). 86. Fischer, A.M., Brewers .Digest 19(6):74 (1944). 87. Klose, A.A. and Fevold, H.L., J. N u t r i t i o n 24:421 (1945). 88. Peterson, W.H., Arch. Bio. 18:181 (1948). 89. D i r r , K. and Decker, P., Biochem Z. 316(3):239 (1944). 59. ADDENDUM Determination of sugfcr(lactose) (44)(45). D i s t i l l a t i o n of V o l a t i l e Acids and V o l a t i l e Neutral Products (46). Determination of Carbon Dioxide (46) (47). Determination of Lactic Acid (49). Determination of Glycerol (52). Determination of Alcohol (54). Duclaux Constants for v o l a t i l e Acids (55). 

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