Microbiological Determination of Vitamins and Amino Acids Pro-duced by Microorganisms, Using the Dialysis Cell1

VEIKKO NURMIKKO

Laboratory of Valio, Biochemical Institute, Helsinki, Finland

Received for publication November 21, 1956

Investigations on the associative interrelationshipsbetween microorganisms have engaged increasing atten-tion in recent years. As is well known, these relationshipsplay an important role in nature and also in manybranches of industrial.microbiology. In view of thegreat significance of these associative phenomena inbiology, and especially in microbiological ecology, thisis undoubtedly a field in which great and important

a b c

1954, 1955, 1956). This method affords opportunitiesfor elucidating the biosynthetic pathways of microbialgrowth factors such as vitamins and amino acids. Morerecently, an approach to the study of the interrelation-ships among microorganisms has been made (Nur-mikko, 1955b). A simple apparatus has been constructedin which the organisms under investigation are sepa-rated from each other by one or more dialysis

EFIG. 1. Three-compartment dialysis cell; a, b, and c. A, dialysis membrane; B, rubber gaskets; C, aluminum hoops; D, set screws;

E, rubber packings.

discoveries have yet to be made. However, newtechniques will certainly be developed to solve thedifficult problems accompanying the study of thebiochemistry of mixed microbial populations. Theclassical pure culture methods are in most cases in-applicable to these studies, or yield only scant informa-tion on the chemical factors affecting associations ofmicroorganisms.The author has previously shown that in certain

cases the chemical factors affecting symbiosis amonglactic bacteria are vitamins of the B complex andamino acids (Nurmikko, 1952). On the basis of thesymbiotic interrelationships between different speciesof lactic acid bacteria, a method, designated the sym-biotic technique, has been developed (Nurmikko

1 This paper deals with those growth factors which aresecreted by microorganisms into the growth medium. Produc-tion of some growth factors with certain microorganisms, com-paring especially the secreted part of growth factors to theamounts of those reserved by the cells, will be presented in alater paper.

membranes in a cell divided into two or more com-partments. In these experimental conditions micro-organisms can also grow in associations composed ofmore than two species.

In this paper a method is described for assaying suchgrowth factors as vitamins and amino acids producedby microorganisms, using various dialysis cell systems.This method permits the assay directly in the growingcultures.

MATERIALS AND METHODSDescription of dialysis cell. The apparatus consists

of two or more compartments made of Pyrex glass,each furnished with a glass tube open at the top, whichcan be plugged with cotton wool. Figure 1 shows a three-compartment dialysis cell. Each compartment hasapproximately a 12- to 15-ml capacity (not includingglass tube). As shown in figure 1, dialysis membrane(A)2 is placed between the compartments, the surfaces

2 Cellulose No. 4465-A2, Arthur H. Thomas Co., Philadel-phia, Pennsylvania.

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DETERMINATION OF VITAMINS AND AMINO ACIDS 161

FIG. 5. Modification of dialysis cell, a and c, growth cham-bers. Middle compartment consists of a 50-ml Erlenmeyer flask.

FIG. 2. Six of the three-compartment dialysis cells as-sembled on a rack.

f

FIG. 3. Modification of dialysis cell with two compartments;f and c, growth chambers.

*:.

:..rA.

..;.

-aSS

diFIG. 4. Modification of dialysis cell with two compartments;

a and d, growth chambers.

between the glass sections being ground smoothly, andheld in place by rubber gaskets (B). The compartmentsare connected by aluminium hoops (C) equipped withset screws (D) and rubber packings (E). As the walls ofthe glass compartments and the rubber packings areslightly conical, the joints can be made water-tightby tightening the screws (D). Figure 2 shows six of the

...;E

:..... 0:

dFIG. 6. Modification of dialysis cell with two compartments;

d, growth chamber.

three-compartment dialysis cells assembled on therack for the experiment.o The three-compartment dialysis cell describedabove was employed mainly when lactic acid bacteriawere used as test organisms in microbiological de-terminations. Figure 5 shows a modification of thesame apparatus for the experiments in which a Neuro-spora mutant was used as test organism. This dialysiscell likewise consists of three compartments, but themiddle compartment consisted of a 50-ml Erlenmeyerflask. Figures 3, 4, and 6 show other modifications ofthe dialysis cell with two compartments. The use ofthese cells will be described later in this paper.

Cultures and inocula. The organisms used wereLactobacillus arabinosus strain 17-5, Streptococcusfaecalis strain R, Escherichia coli, Oospora lactis, andNeurospora sitophila (ATCC 9276). The lactic acidbacteria stock cultures were transferred by stab inoc-ulation into the glucose-citrate-tryptone-yeast extractagar medium of the following composition: 1 per centglucose, 1 per cent sodium citrate, 0.5 per cent Bacto-tryptone (Difco), 1.5 per cent agar and 20 per cent(by volume) yeast extract. The yeast extract wasprepared as follows: 1 kg of fresh yeast was suspendedin 1 L of water, and kept for approximately 24 hr at42 C. It was then centrifuged and the cell-free solution

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V. NURMIKKO

used. The pH of the agar medium was 6.7. The mediumwas sterilized by autoclaving for 15 min at 112 to115 C (Nurmikko, 1954). E. coli was maintained onBacto Nutrient Agar (Difco) slants with transfersat biweekly intervals, incubated at 37 C for 24 hr. 0.lactis and N. sitophila were maintained on Bacto Neuro-spora Culture Agar (Difco) slants by biweekly transfer,with incubation at 30 C for 24 to 48 hr.The inocula of lactic acid bacteria and of E. coli

were prepared by transferring the organisms from thestab culture to the glucose-citrate-tryptone-yeastextract medium. After incubation for 16 to 18 hr at37 C, the cells were centrifuged out and washed with0.9 per cent sterile saline. This process was repeated,the cells being washed 2 to 3 times. The cells werefinally suspended in saline. One drop of barely visiblesuspension was used as inoculum for approximately5 ml of the final medium in the dialysis cell and in thetest tubes. 0. lactis and N. sitophila were transferreddirectly with a sterile platinum loop from the agarslants to saline, care being taken not to transfer agarmedium together with the spores or mycelium.Assay procedure. The dialysis technique of micro-

biological assay for vitamins and amino acids producedby the microorganisms was as follows: Using lacticacid bacteria as test organism, the basal media ofHendersoni and Snell (1948) (in phenylalanine assay),and Anderson and Elliker (1953) (in nicotinic acidassay) in slightly modified form as described previously(Nurmikko 1954, 1955a) were used. In assays withNeurospora sitophila, Bacto Pyridoxine Assay Medium(Difco) was used. All basal media, free of the vitamin oramino acid to be determined, were prepared at twicetheir final concentration. The standard vitamin oramino acid solutions were added at increasing levels tothe test tubes (Pyrex 14 x 155 mm) when lactic acidbacteria were used as test organisms, or to the Erlen-meyer flasks (50 ml) when the assay organism wasN. sitophila. Duplicate tubes or Erlenmeyer flasks wereset up to obtain the values for the standard curves.Using lactic acid bacteria, the standard vitamin oramino acid solution was added in the following amounts:0, 0.5, 1.0, 1.5, 2.0, and 2.5 ml per tube. Quantities of2.5 ml of the basal medium were then placed in thetest tubes, and the total volume adjusted with dis-tilled water to 5 ml. In pyridoxine assays with Neuro-spora, 15-ml quantities of the basal medium wereplaced in Erlenmeyer flasks. Up to 15 ml of standardpyridoxine solution were added and the volume ad-justed with distilled water to 30 ml. In all cases, thebasal medium used was diluted with an equal volumeof distilled water and placed in the dialysis cells.

After autoclaving at 112 C for 7 min, the dialysis cellsand test tubes or Erlenmeyer flasks were cooled, then

as test organisms) or 30 C (Neurospora as test organ-

ism).The growth response of lactic acid bacteria was

followed turbidimetrically. The extent of growth was

recorded as galvanometer readings on a Klett-Summer-son photoelectric colorimeter with a 660-mA filter.In vitamin B6 assay, dry mycelial weights of Neuro-spora were determined. The mycelium was harvested byfiltering the culture medium through a filter paper

placed in a Buchner funnel. After washing with distilledwater, the mycelium was dried at 100 C for 3 to 4hr and weighed. Also, the extent of the growth ofE. coli and 0. lactis was measured by weighing the driedcells. The cells of E. coli were harvested by centri-fugation, and the cells of Oospora by filtering the culturefiltrates through a membrane filter. Both organismswere washed with distilled water and dried at 100 Covernight.A standard curve was prepared by plotting growth

(dry weight of the cells) against micrograms of vitaminor amino acid per ml in the test tubes or Erlenmeyerflasks of the standard series. From this standard curve

the amount of vitamin or amino acid per ml in thecompartment of the assay organism was determined.Since the volume of the growth medium in the com-

partment of the test organism was exactly known, itwas possible to calculate the amount of the vitamin or

amino acid which was produced by the microorganisminvestigated. An example of the calculation: In a

separate phenylalanine assay with a 3-compartmentdialysis cell (figure 1; in table 5, dialysis cell no. 1),it was calculated from the standard curve that theproduction of L-phenylalanine by E. coli was 1.33 ,ugper ml. The volume of the growth medium in themiddle compartment was 18.9 ml. Thus, the wholeproduction of L-phenylalanine was 1.33 X 18.9 =

25.1 ,ug. The dry cell weight of E. coli (obtained fromboth the end compartments) was 16.7 mg. Conse-quently the production of L-phenylalanine by E. coliper mg of dry weight was 1.5 ,ug.

RESULTS

Control experiments. To test the accuracy of thedialysis method, a series of control experiments was

carried out to determine the percentage recovery. Inthese experiments, nicotinic acid and phenylalanineassays were made with L. arabinosus 17-5 and folicacid assay with S. faecalis R as test organism, using a

3-compartment dialysis cell of the model shown infigure 1. In pyridoxine determinations, the test organ-

ism was N. sitophila (ATCC 9276) and the apparatus wasthe 3-compartment dialysis cell illustrated in figure 5.In all determinations, known amounts of amino acidor vitamin were added to the two end compartmentsafter sterilization and cooling. The middle compartment

inoculated and incubated at 37 C (lactic acid bacteria

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TABLE 1. Recovery of nicotinic acid*

Nicotinic AcidDeviation Recovery

Added Found

pg ,Ag pg per cent

0.20 0.21 +0.01 1050.20 0.19 -0.01 950.20 0.19 -0.01 950.20 0.25 +0.05 1250.20 0.23 +0.03 1150.40 0.39 -0.01 980.40 0.40 0.00 1000.40 0.41 +0.01 1020.40 0.41 +0.01 1020.40 0.42 +0.02 105

Average ...... 104.2

* Three-compartment dialysis cells. Lactobacillus arabinosusstrain 17-5 as test organism.

TABLE 3. Recovery of pyridoxine*

Pyridoxine

Added

pg

0.400.400.500.500.500.500.600.600.600.60

Found

pg

0.410.400.540.480.560.520.620.560.630.58

Deviation

pg

+0.010.00

+0.04-0.02+0.06+0.02+0.02-0.04+0.03-0.02

Recovery

per cent

102100108961121041039310597

Average ............. ......... 102.0

* Three-compartment dialysis cells. Neurospora sitophilaas test organism.

TABLE 2. Recovery of folic acid*

Folic AcidDeviation Recovery

Added Found

mpg mpg pg per cent

8.0 8.3 +0.3 1048.0 7.7 -0.3 968.0 8.3 +0.3 1048.0 8.4 +0.4 1058.0 9.2 +1.2 1158.0 8.3 +0.3 1048.0 7.8 -0.2 988.0 7.6 -0.4 95 .

Average .102.6

* Three-compartment dialysis cells. Streptococcus faecalisstrain R as test organism.

organism. In the experiments in which L. arabinosus17-5 was used, the time of incubation was 72 hr. Inthe Neurospora assays the time of incubation variedfrom 60 to 72 hr.An indication of the accuracy of the method may be

obtained from the results of the recovery experimentspresented in tables 1 to 4. The results show that thequantitative recovery of nicotinic acid, folic acid,pyridoxine and phenylalanine with mean average102.9 per cent (when all determinations of these com-pounds were taken into consideration) was obtainedwhen known amounts of these compounds were addeddirectly to the dialysis cell used for assaying by theprocedure described. The recoveries of the compoundstested were between 93 and 125 per cent. The greatestvariation was found in nicotinic acid assays (rangingfrom 95 to 125 per cent) and the lowest variation inphenylalanine assays (ranging from 100 to 108 percent). It was found that the variation was approxi-mately ± 5 per cent of the average (102.9 per cent)recovery.

TABLE 4. Recovery of L-phenylalanine*

L-PhenylalanineDeviation Recovery

Added Found

pg pg pg per cent

20.0 20.9 +0.9 10420.0 21.7 +1.7 10820.0 20.1 +0.1 10040.0 40.3 +0.3 10180.0 84.7 +4.7 10680.0 83.2 +3.2 10480.0 81.0 +1.0 10180.0 81.0 +1.0 101

Average .103.0

* Three-compartment dialysis cells. Lactobacillus arabinosusstrain 17-5 as test organism.

Experiments on the production of growth factors. Inorder to obtain information on the usefulness of thedialysis method, the production of nicotinic acid,folic acid, and phenylalanine by E. coli and the pro-duction of pyridoxine by 0. lactis were studied. The testorganisms and the basal media in these determinationswere the same as those used in the control experiments.Folic acid, phenylalanine and pyridoxine assays werecarried out with a 3-compartment dialysis cell andnicotinic acid assay with a 2-compartment dialysiscell. Using the 3-compartment dialysis cell, the two endcompartments were inoculated with the microorganismsto be investigated and the middle one with the assayorganism. Using the 2-compartment dialysis cell innicotinic acid assay, one compartment was inoculatedwith E. coli and the other with L. arabinosus 17-5.The results of these experiments are given in tables

5 to 8. As can be seen in table 5, the production of L-phenylalanine by E. coli in the experimental conditionsused was 1.4 Ag per mg of cell dry weight. This is amean value obtained from 5 individual determinations

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TABLE 5. Production of L-phenylalanine by Escherichia coli*

Dialysis Cell Growth of E. coli L-Phenylalanine L-PhenylalanineNo. (Dry Wt of Cells) Produced Produced per Mg(Dry Wt of Cells)

mg pg pg1 16.7 25.1 1.502 13.6 20.0 1.473 10.4 16.6 1.604 11.9 15.4 1.295 13.4 17.6 1.31

Average .1.43

* Three-compartment dialysis cells. Lactobacillus arabinosusstrain 17-5 as test organism.

TABLE 6. Production of nicotinic acid by Escherichia coli*

Dialysis Cell Growth of E. coli tinic Acid Nicotinic Acid Pro-No. (Dry Wt of NPcodced duced Per Mg (DryCells)Prdued Wt of Cells)

Mg pg pg

1 8.0 0.324 0.0402 9.2 0.376 0.0413 10.1 0.431 0.4314 7.1 0.270 0.0385 9.6 0.445 0.0466 10.0 0.340 0.034

Average .0.040

* Two-compartment dialysis cells. Lactobacilluts arabinosutsstrain 17-5 as test organism.

TABLE 7. Production of folic acid by Escherichia coli*

Dialysis Cell Growth of E. coli Folic Acid ProducedNo. (Dry Wt of Folic Acid Produced per Mg (Dry Wt ofNo. ~~~Cells) Cells)

mg mpg mpg.1 17.7 4.62 0.2602 17.1 4.65 0.2743 18.4 4.73 0.2574 18.1 4.99 0.2765 20.3 4.73 0.233

Average ............ 0.260

* Three-compartment dialysis cells. Streptococcuts faecalisstrain R as test organism.

TABLE 8. Production of vitamin B6 (pyridoxine) by Oospora*

Dialysis Cell Growth of Oospora Vitamin Bo Vitamin B6 Pro-No. (DryWtCof Produced duced per Mg (DryCells) rue Wt of Cells)

Mg pg mpAg1 67.7 0.63 9.32 39.3 0.39 9.93 52.8 0.56 10.64 53.5 0.54 10.15 60.0 0.60 10.0

Average .10.0

* Three-compartment dialysis cells. Neurospora sitophila(ATCC 9276) as test organism.

(using 5 replications). The variation of the individualdeterminations is approximately + 8 per cent of theaverage value. Tables 6 and 7 show that the productionof nicotinic acid and folic acid by the same organism was40.0 m,ug and 0.26 m,ug, respectively, per mg of thedry weight of the cells. In nicotinic acid assay (6 rep-lications), the variation is approximately 4- 10 percent and in folic acid assay (5 replications) i 5 percent of the average value.

In all these experiments with E. coli, the time ofincubation was 72 hr. The separate determinationsshowed that this organism can grow very rapidly inthe basal media used in this study. The organismreached its. maximum growth after approximately 10hr. When a nicotinic acid assay was made using anincubation time of 92 hr, practically the same nico-tinic acid production was observed as in the experi-ments with an incubation time of 72 hr.

In table 8 it can be seen that the production ofvitamin B6 (revealed as pyridoxine) by 0. lactis was 10.0m,ig per mg of dry weight of the cells. This mean valuewas obtained using 5 replicates. The variation of theindividual determinations was approximately -+f4 per cent of the average value. An incubation timeof 120 hr was used in this experiment.

DISCUSSION

The principal purpose of the work reported here wasto develop a simple microbiological method for thequantitative estimation of vitamins and amino acidsdirectly in growing microbial cultures. The dialysiscell technique described in this paper, which permitsthe determination of these compounds in the mannerdesired, is based on their known action as growthfactors in associations of microorganisms. The useof dialysis cell systems affords the following advantages.Firstly, the performance of the determinations withthe dialysis cell apparatus is easy and time-saving,because this method is designed to obviate the usualnecessity of making separate microbiological or chemicaldeterminations from the culture filtrates. Secondly,because the vitamin or amino acid investigated in thedialysis cell system passes through the dialysing mem-brane to the adjoining compartment containing thetest organism, which utilizes this compound immedi-ately for growth, the possibility of the growth factorproduced being to any extent inactivated remainsvery meager. In addition, the fact that the compoundtested cannot accumulate in the culture fluids in thedialysis cell system (because this compound will beutilized by the test organism as rapidly as the othermicroorganism in the adjoining compartment is capableof producing it) may have a considerable influence onthe amount of the growth factor produced.

Figures 1 to 6 illustrate 5 modifications of the dialysiscell used for the determination of the vitamins and

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amino acids produced by microorganisms. The dialysiscell to be used must be chosen according to the organ-ism employed. For example, with an organism whichgrows best on the surface of the medium, it would bepreferable to use compartments of type c or d (figures3 to 6). In these cases especially, shaking of the dialysiscells, and also the solutions of the standard series, maybe necessary in order to attain rapid dialysis of thegrowth factors. The modifications shown in figures 1 to4 were designed for use with lactic acid bacteria astest organisms. Because these organisms are micro-aerophilic, they must be inoculated into compartmentsa, b, or f. When Neurospora mutants are to be used asassay organisms, it is preferable to choose one of thedialysis cells shown in figures 3 to 6. In this situation,the mold inoculum would be inoculated into com-partments c or d.

In determinations of the growth factors described inthis paper, the variation of the average value using5 or 6 replications per growth factor tested was of theorder found in microbiological assays, that is, from 5to 10 per cent. It is of special interest that the averagerecoveries of the 3 added vitamins and 1 amino acidfrom the different dialysis cell systems was 102.9 percent. This suggests that the test organism can reallytake up the compound tested quantitatively from theadjoining compartment, through the dialysis mem-brane.

In the course of developing the dialysis method, itwas found to be necessary to take into special con-sideration the following precautions. In separateexperiments it should be verified that no inhibitorygrowth effect exists in the dialysis cell system betweenthe test organism and the organism whose growthfactor production is to be investigated. If the growth ofthis organism and its growth factor production wasespecially vigorous, the dialysis cell system was con-structed with a view to allowing sufficient nutrientmedium for the maximum growth of the test organism(at least at the level of the maximum growth value ofthe standard series). For example, in pyridoxine de-termination the compartment of the type a (figure 5)was used for Oospora. The small air area on the surfaceof the nutrient medium is the factor limiting the growthof this organism, and thus also indirectly the pro-duction of pyridoxine. In these growth conditions,the nutrient medium was sufficient for the assay organ-ism Neurospora. Further, it is preferable that thevolume of the nutrient solution in the tubes of thestandard series and in the compartment of the testorganism should be the same. If it seems probablethat the activity of the compound tested will decreaseduring autoclaving, the compound can be added asep-tically to the tubes of the standard series after auto-claving of the basal medium. In this case the sterili-

zation of this compound can be accomplished byfiltration.

Finally, it should be pointed out that the determi-nation described here is to be regarded as mainly illus-trative. Using as examples the determinations ofphenylalanine, folic acid, nicotinic acid and vitaminB6, an attempt has been made to give a picture of thegeneral procedure of the dialysis technique. Thus, theestimation of other vitamins and amino acids and theuse of other microorganisms require other basal mediaand modifications of the growth conditions describedin this paper.

ACKNOWLEDGMENTThe author wishes to thank Professor Artturi I.

Virtanen, Director of the Biochemical Institute,Helsinki, Finland, for his encouraging interest in thiswork and for his valuable discussions.

SUMMARYA rapid microbiological procedure is described for

the determination of vitamins and amino acids pro-duced by microorganisms. Primarily, this method wasdeveloped for the investigation of biochemical factorsof importance in microbial associations. Using adialysis cell system especially constructed for thispurpose, it was possible to make analyses directly inthe growing cultures. Various types of dialysis cellapparatus are described. Recovery experiments werecarried out with phenylalanine, folic acid, nicotinicacid, and vitamin B6. The production of these growthfactors with Escherichia coli and Oospora lactis bymeans of this method was also studied.

REFERENCES

ANDERSON, A. W. AND ELLIKER, R. P. 1953 The nutritionalrequirements of lactic acid streptococci isolated fromstarter cultures. I. Growth in a synthetic medium. J.Dairy Sci., 36, 161-167.

HENDERSON, L. M. AND SNELL, E. E. 1948 A uniform mediumfor determination of amino acids with various micro-organisms. J. Biol. Chem., 172, 15-29.

NURMIKKO, V. 1952 Chemical factors affecting associationsof lactic acid bacteria. Acta Chem. Scand., 6, 1258-1264.

NURMIKKO, V. 1954 Symbiosis experiments concerning theproduction and biosynthesis of certain amino acids andvitamins in associations of lactic acid bacteria. Ann.Acad. Sci. Fennicae Ser. A II, 54, 1-58.

NURMIKKO, V. 1955 Application of the symbiosis phenome-non among lactic acid bacteria to the study of the bio-synthetic pathways of growth factors. Ann. Acad. Sci.Fennicae Ser. A II, 60, 216-225.

NURMIKKO, V. 1955a Phenylalanine as a precursor in thebiosynthesis of folinic acid (citrovorum factor) in lacticacid bacteria. Suomen Kemistilehti B, 28, 62-66.

NURMIKKO, V. 1955b The dialysis technique in the study ofthe vitamins and amino acids affecting associations ofmicroorganisms. Acta Chem. Scand., 9, 1317-1322.

NURMIEKO, V. 1956 Biochemical factors affecting symbiosisamong bacteria. Experientia 12, 24-249.

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