and certain - journal of bacteriology · combination alone. thus, pantothenic acid and riboflavin,...

THE INTERRELATIONSHIP OF IRON AND CERTAINACCESSORY FACTORS IN THE GROWTH OF

RHIZOBIUM TRIFOLII, STRAIN 2051

VIRGIL GREENE LILLY AND LEON H. LEONIAN

Department of Plant Pathology and Bacteriology, West Virginia Agricultural ExperimentStation, Morgantown, W. Va.

Received for publication June 2, 1945

The influence of environment upon vitamins as accessory factors for micro-organisms has not received the attention it deserves. Robbins and Kavanagh(1938) observed that the need for thiamine for Pythium butleri was the functionof the concentration of the basal medium. K6gl and Fries (1937) found thiamineto be an accessory factor for Nematospora gossypii. Fries (1938) showed inositolto be such a factor for Valsa pini. Leonian and Lilly (1942) studied the effectof five vitamins on ten strains of Saccharomyces cerevisiae and demonstratedmany instances in which certain of the B vitamins acted as accessory growthfactors.

Rhizobium trifolii, strain 205 (Wisconsin), has been used by West and Wilson(1939, 1940) in studies on growth factor requirements, and by Wilson andWilson (1942) on the essentiality of an external source of biotin. The organ-ism grows poorly (Wilson and Wilson, 1942) in the absence of added biotin, andSteinberg (1938) abandoned attempts to grow it without organic growth factors.It has been used by West and Wilson (1940), West and Woglom (1942), andLeonian and Lilly (1945) as a test organism for the microbiological assay ofbiotin. West and Wilson (1939, 1940) found thiamine, riboflavin, and panto-thenic acid (or ,B-alanine) to be accessory growth factors, although both riboflavinand thiamine are synthesized by this strain (West and Wilson, 1938a, 1938b,1939).

Steinberg (1938) concluded that R. trifolii, strain 205, required added Fe,Mn, Mo, and Ca for optimum growth in highly purified nutrient solutions. Al-though the necessity of trace elements fbr microorganisms is generally recognized,often it is not emphasized that special methods are required to demonstratethis need.The term "accessory growth factors" is used in. this paper to denote certain

B vitamins which induce increased growth of strain 205 in the presence of biotin.Although thiamine, riboflavin, pantothenic acid, etc., are synthesized by thisorganism, when the rate of synthesis is less than that needed for the optimumrate of growth, the addition of these factors to the medium permits a greatergrowth than that induced by biotin alone. Since the response is not of the"all or none" type, a significant increase in growth above that of the controlis the criterion of the presence and effectiveness of an accessory growth factor.

1 Published with the approval of the Director of the West Virginia Agricultural Experi-ment Station as Scientific Paper No. 339.

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Whether or not a given compound will act as an accessory factor dependsupon the choice of experimental conditions. If these be such as to enable theorganism to synthesize all the compounds needed by it, and if these be synthe-sized at a rate sufficient for optimum growth, no factor will be "accessory";but if conditions somewhat less favorable be used, various substances will be-come accessory. If the experimental conditions, such as lack of iron, be suchas to prevent the proper function of certain essential activities of the cell, thenthose factors the synthesis of which is directly or indirectly mediated by thepresence of iron become essential. The addition of such factors to the nutrientsolution, preformed, will enable the organism to grow. However, the role ofiron in the bacterial cell is undoubtedly complex, and not limited to the synthesisof accessory growth factors. Indeed, were it possible to free media entirely ofiron, they would not support growth at all. At present this goal of perfectiondoes not seem possible to attain; but by decreasing the iron content to sufficientlylow levels it should be possible to demonstrate the intimate connection betweeniron and the need of certain growth factors.

It is the purpose of this paper to show that there is an intimate connectionbetween the iron content of the medium and the need of "accessory" factors forR. t7ifolii, strain 205.

MATERIALS AND METHODS

The culture of Rhizobium trifolii, strain 205, used in this work was obtainedfrom the Department of Agricultural Bacteriology, University of Wiscon.The basal medium consisted of the following:

E P0 .. . . .. . . . .. . . .. . . . .. . . .. . . . .. . . .. . . . gKHE2PO04.1 gNa.. 0.2 gMgSO,.740..0.2gCaSO.2H40.0.1gSucrose....................................................... 10 gDistiled water.......................................................1,000 ml

Since the final composition of the medium depends upon the method of prepa-ration, it will be described here in detail. The inorganic constituents weredissolved in 250 ml of distilled water by heating, the solution was cooled, and thepH adjusted to 8.0 with sodium hydroxide. The solution was then boiled andfiltered hot through a folded filter paper. This procedure removed sufficientresidual iron to make the medium deficient. The sucrose was added to 250 mlof water, boiled with 10 grams of norit A, and filtered while hot; the charcoal waswashed with 250 ml of hot distilled water, the two solutions were combined,cooled, and the pH was adjusted to 6.3. by the addition of hydrochloric acid(the onset of clumping was delayed when the pH was 6.3 rather than 6.9).Pyrex Erlemmeyer flasks, of 125-ml capacity, were used as culture vesels.

They were always kept in sulfuric acid dichromate solution for 24 hours beforeusing. The cotton plugs were extracted with hot ethyl alcohol before use. All

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ACCESSORY GROWTH FACTORS FOR RHIZOBIUM TRIFOLII

media employed in this investigation contained 0.1 jug of biotin (free acid, Mercksynthetic) per liter. The accessory growth factors and iron were added asdescribed later. Minor elements other than iron were not added because theyfailed to induce significant increases in growth. Ten ml of medium, with theappropriate organic and mineral factors added, were transferred by pipette tothe flasks. Sterilization was accomplished by autoclaving 15 minutes at 15pounds' steam pressure. The stock inocula were grown on the foregoing basalmedium to which had been added 0.01 ,ug of biotin and 200 ,g of iron per liter.The cultures used for inoculum were grown at 25 C, and were subjected toagitation 10 minutes out of every hour. Inocula less than 5 days or more than7 days old were not used. One loopful of cell suspension was transferred to eachflask. The platinum loop used was made of B. & S. 24-gauge, double coil, withan inside diameter of 2.5 mm.

Cultures were made in quadruplicate for each treatment. For presentationin the tables the results were averaged. In general the agreement in values wasgood as evidenced by low standard deviation.The amount of growth was estimated by determining the turbidity with an

A.C. Fisher electrophotometer. A 13-mm tube was used with a 525 filter.Heavy suspensions were diluted with distilled water to such an extent as torender the response of the instrument essentially linear. The readings obtainedwith such diluted suspensions were multiplied by the appropriate factor.

All pH measurements were made with a Leeds and Northrup glass electrodeassembly. The results presented are taken from single experiments in whichthe same medium and inoculum were used. However, most of these experimentshave been repeated many times over the course of 9 months with the same es-sential findings. It is emphasized that the results presented here are valid onlyinsofar as they apply to the conditions under which the work was done. Theneed for this caution will be seen when the results obtained under differentexperimental conditions are compared.

EXPERIMENTS

The following variables were tested for their effect on growth: time, tempera-ture, agitation of cultures, iron content of the medium, vitamins singly and incombinations, and the interaction of some of the variables.Two temperatures, 30 C and 25 C, were used in all of the experiments. Agita-

tion was used only at 25 C because of lack of agitation facilities at 30 C. Agi-tation was accomplished by placing the flasks in a flat-bed shaking apparatusadjusted to operate ten minutes every hour. The stroke of the shaking appa-ratus was six inches, with fifty strokes per minute.

The effect of varying amounts of iron on growth. During the preliminary workit was observed that growth in the control flasks showed wide variations. Thisled to a consideration of the iron content of the medium as the responsible vari-able. It was found that growth was proportional to the amount of iron in themedium over a considerable range of concentrations. A series of dilutions ofiron in the basal medium was prepared and distributed into 125-ml Erlenmeyer

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VIRGI GREENE LILY AND LEON H. LEONAN

flasks.. These were divided into three sets, inoculated, and incubated for sixdayys at 30 C, 25 C, and 25 C with agitation. The standard-iron solution wasprepared by dissolving iron wire in dilute sulfuric acid. Figure 1 shows thecondensed iron curve of R. trifolii, strain 205, under the three different conditions.

Concentrations of iron greater than 2.5 ,g per flask of 10 ml of medium failedto induce additional growth.

FIG. 1. GROWTH OF RHIZOBIUM TRIFOLI, STRAIN 205, AS A FUNCTION OF TH3 IRONCONCENTRATION OF THE MEDIUM

Effect of culturing at 30 C and at 25 C with and without agitation. Six days' incuba.tion.

The effect of increased iron content upon the pH of the medium. In view ofthe results of Pappenheimer and Shaskan (1944) on the production of lacticacid by Clostridium welchii, it was decided to test the pH of R. trifolii culturemedium after six days of growth. It was noted that the pH of cultures incubatedat 25 C was at first upward as the concentration of iron increased; at a certainconcentration of iron (about 2.5 ,g per 10 ml) the pH of the cultures began tofall rapidly. On the other hand, the pH of cultures incubated at 30C decreasedfrom the start, and continued until the iron concentration was 5.12 ,ug per 10 ml.

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The effect of time on pH change in the cultures containing no added iron andin those with 500 mg of iron was studied at 30 C, 25 C, and 25 C with agitation.Inasmuch as the results were essentially the same under the three conditions,

2. 3 4 SF 6 a

FIG. 2.GROWTH OFRHIZOBIUmTRIFOLII, STRAIN 205,ANDCHANGE IN THE PH OF THE CULTUREMEDIUM AS A FUNCTION OF THE IRON CONCENTRATION OF THE MEDIUM AND

THE TIME OF INCUBATION25 C without agitation

only the data for 25 C without agitation will be presented (figure 2). With no

added iron the pH rose slightly by the sixth day of incubation, fell somewhaton the eighth day, and rose considerably on the tenth. Cultures containing500 ,g of iron increased in pH slightly until the third day of incubation. The

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VIRGI GREENE LILLY AND LEON H. LEONIAN

pH then fell linearly until the eighth day, and rose considerably by the tenthday of incubation.

The interrelationships of 8even vitamins as accessory factors in the absence andin the presence of added iron. Seven B vtamins were used singly and in variouscombinations. The amount of inositol was 5 mg per liter; thiamine, pantothenicacid, pyridoxine, nicotinic acid, riboflavin, and p-aminobenzoic acid were used atthe rate of 100 jug per liter. Each series was distributed into 12 flasks, 4 flasksfor each of the 3 environments, 30 C, 25 C without agitation, and 25 C withagitation. A second series of flaks were prepared in exactly the same wayexcept that 500 pg of iron per liter was added to the medium. Two other con-centrations of iron, 5 and 50 ,ug per liter, were also tested, but it was found that5 pg iron gave essentially the same results as no iron, and 50 ug of iron presenteda picture like that of 500 pg, except that the actual amount of growthwasnotquite so great. Photometer readings were made after an incubation period of6 days. Table 1 gives the results.The data in table 1 are presented in two sections in order to simplify their

interpretation. The first section contains results obtained without the additionof iron. It can be seen that pantothenic acid induced the greatest increase ingrowth, riboflavin and p-aminobenzoic acid the least. In fact, these two vtaminsin nonagitated cultures at 25 C caused an actual decrease in growth. The con-trols grew better at 25 C than at 30 C.The effect of combining with thiamine, and with pantothenic acid, all of the

other vitamins taken one at a time is presented next. At 30 C the most effectivecombination was pantothenic acid and nicotinic acid, followed closely by thiamineand pantothenic acid, and pantothenic acid and riboflavin. At 25 C the mosteffective combination was thiamine and pantothenic acid. A synergistic effectof these two vitamins was apparent only in the agitated cultures. This effectis seen also in pantothenic acid and riboflavin, and in pantothenic acid and p-aminobenzoic acid at 30 C. Certain combinations of two vitamins, on the otherhand, seemed to be antagonistic, inducing less growth than one member of thecombination alone. Thus, pantothenic acid and riboflavin, and pantothenicacid and p-aminobenzoic acid induced less growth in cultures incubated at25 C than did pantothenic acid alone. These are the same combinations thatacted synergistically in cultures incubated at 30 C.The results of combinations of three or more vitamins are listed next. The

most striking increases occurred in cultures incubated at 25 C. ffighly signifi-cant increases were induced only when the other vitamins were added to panto-thenic acid, and to thiamine. The omission of thiamine from any of thesecombinations greatly lowered the percentage increase. The same situationoccurred in the agitated cultures. However, no combination of vitamins ex-celled thiamine and pantothenic acid. At 30 C combinations of three or morevitamins induced no more growth than did thiamine and pantothenic acid; orpantothenic acid and nicotinic acid.

Section 2 indicates that the effect of seven vitamins as accessory factors inthe medium containing added iron varies with the conditions of incubation.

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In cultures incubated at 30 C, all seven vitamins acted as accessory factorswhen used singly. At 25 C, with or without agitation, none of them causedsignificant increases over the controls.

TABLE 1The interrelationships of 7 vitamins as shown by the growth of Rhizobium trifolii, strain 205,

with and without iron added to the medium

GROWTH WITOUT GROWTH wITH 500 pGADDED IRON IRON PER LITER

T}IA THENIC INOSI- PYII- MICO- RUBO- tANO -MINE EAC TOL DOXINE TINC PAVIN BENZOIC 25 C 25 CACID ACMD ACID5 230 C 25 C (agi- 30 C 25 C (agi-

tated) tated)

Control (no vitamins)............... 19 25 28 37 108 123

++

+

++

+

+

+

+

++++

++++

+

+

+

+

+

+

+

+

+

+

+

+

±

* + indicates presence of vitamin.

+

+

29332631272221413128323328313344403841424744404436404543

39443240272224494544454241363036353470697148467163476772

364735432829281014353465451404144413791939348539096459296

60876257595960774945534950757874747158676365575864646470

119126114112112116113126121116116119111127120134127123108112118107111118111104110114

13313613213113112612414013313113(13A12t139143141141144154155156149141160157153162161

The effect of combining the different vitamins one at a time with thiamineand with pantothenic acid is presented next. In cultures incubated at 25 C,with or without agitation, only one combination induced more than a 20 percent increase; however, at 30 C all combinations caused definite increases.Nevertheless, none of them were so effective as pantothenic acid alone. Inositol,pyridoxine, nicotinic acid, riboflavin, and p-aminobenzoic acid exerted a de-

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pressive effect when used with thiamine. These same vitamins showed a slightdepressive effect when used with pantothenic acid.None of the combinations Tcontaining three or more vitamins induced so

great an increase in growth at 30 C as pantothenic acid alone, or of any one ofthe other vitamins in combination with pantothenic acid. At 25 C withoutagitation no increases occurred, whereas some fair increases resulted when thecultures were agitated.A comparison of sections 1 and 2 of table 1 leads to the following conclusions:

The need of R. trifolii 205 for some of the B vitamins as accessory factors dependson the iron content of the medium, and upon the conditions of incubation. Asthe iron content becomes optimal, the need for accessory factors largely dis-appears in cultures incubated at 25 C, whereas at 30 C this need is still apparent.

TABLE 2The effect of iron concentration and thiamine, pantothenic acid, and inositol as accessory

factors on the rate of growth of Rhizobium trifolii, strain 205

30 c 25 c 25 C (AGITATED)

DAYS NoNNoOgde0r No SOpg No So"adediron iro

n°cu- No "-'-Aadded SirOpgn a added Aopg i iron3ATxoN added .' irniron added iron iron acces- adod ionaces acces-ironronaccessorysor ironacesr sory sory

factors* fators factor s factors factors

2 6 18 8 22 7 10 7 7 7 9 9 73 15 33 10 48 15 35 28 39 19 38 33 334 17 27 24 65 19 54 46 62 27 68 63 745 25 42 27 68 30 83 72 91 31 106 100 1206 26 53 33 89 41 112 89 124 39 139 124 1648 34 56 42 99 55 130 113 168 44 181 170 21810 41 57 68 103 70 150 137 200 39 180 150 213

* Accessory factors, thiamine and pantothenic acid 100 ,g per liter, inositol 5 mg perliter.

Insofar as the actual amount of growth is concerned, the following conditionswere most favorable: the addition of thiamine, pantothenic acid, inositol, pyri-doxine, and nicotinic acid as accessory factors, together with 500 ;ig of iron,and incubation at 25 C with agitation.

The effect of iron and certain vitamins on growth as a function of time. Datapresented in table 1 are based on an incubation period of six days only. There-fore, it was believed that it would be of sufficient interest to study growth overa period of ten days. Thiamine, pantothenic acid, and inositol in combinationwere selected as the accessory factors. The data are presented in table 2. Itshould be noted in the cultures incubated at 30 C that the rate of growth declinedon the fourth or fifth day. The effect of the accessory factors in the presence of500 ,g of iron is shown on the third day of incubation and thereafter. In culturesincubated at 25 C, without agitation, the effect of the three accessory factorsbecame strikingly apparent from the t;hird day of growth on to the tenth in theabsence of added iron. However, when 500 ,g of iron were added to the medium,

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the effect of the three vitamins was not great until the sixth day, but on theeighth and tenth days their influence on growth became significant. Apparentlyan incubation period of ten days is not sufficient for maximum growth underthe conditions described. The agitated cultures at 25 C showed much the samesituation. Apparently growth had reached the maximum by the eighth day,whereas in the nonagitated cultures growth continued until the tenth day. Thus,extension of the time of incubation to ten days shows clearly the accessory actionof thiamine, pantothenic acid, and inositol even in the presence of 500 ,ug of iron.

TABLE 3The effect of varying volumes and constant surface upon the growth of Rhizobium trifolii,

strain 205(Time of incubation 6 days)

30 c 25 C 25 C (AGITATED)

3 ml per tubeControl, no iron or accessory factors............ 15 15 21Thiamine, pantothenic acid, and inositol.........34 30 47500 pg iron..................................... 36 39 52500 Ag iron, thiamine, pantothenic acid, and

inositol..................................... 44 51 62

5 ml per tubeControl, no iron or accessory factors............ 12 20 21Thiamine, pantothenic acid, and inositol........ 27 23 45500 Ag iron..................................... 24 33 36500 pg iron, thiamine, pantothenic acid, and

inositol..................................... 30 36 46

10 ml per tubeControl, no iron or accessory factors..... 10 13 14Thiamine, pantothenic acid, and inositol........ 11 13 14500 pg iron..................................... 13 14 23500 pg iron, thiamine, pantothenic acid, and

inositol......13........... 14 21

The effect of aeration in growth. Since agitation causes better aeration, growthbecomes accelerated in the agitated cultures, and reaches the declining pointafter a shorter period of incubation. Allison and Hoover (1934) observed thatR. trifolii, strain 205, made better growth when the cultures were shaken atleast once a day. Our work confirms this. Table 2 shows that agitated culturesattained maximum growth on the eighth day, whereas the nonagitated ones failedto do so even by the tenth day. Aeration caused by agitation not only increasedthe rate but also the total amount of growth.The effect of aeration was illustrated in another way. Test tubes (18 x 150

mm) were substituted for flasks; by varying the amount of nutrient solution thedegree of aeration was controlled by the depth of the medium. Three sets oftest tubes containing 3, 5, and 10 ml of nutrient solution were prepared. Four

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92VIRGIL GREENE I LLY AND LEON H. LEONIAN

kinds of solutions were used: the first, without added iron or accessory factors,was the control; the second contained thiamine, pantothenic acid, and inositol;the third contained 500 jag of added iron; and the fourth, 500 ,ug of iron, andthiamine, pantothenic acid, and inositol. The test tubes were inoculated inthe usual manner and incubated at 30 C, 25 C, and 25 C with agitation. Table3 gives the results.Table 3 shows that under these conditions growth was an inverse function of

the depth of the nutrient solution. The effect of the added iron and vitaminsdecreased as the depth of the nutrient solution increased. In tubes containing5 or 10 ml of medium, growth occurred mainly at the surface, as shown by theformation of a ring of bacteria near the upper part of the solution.

DISCUSSION

The foregoing data lead to the general conclusion that a given vitamin orcombination of vitamins may function as accessory growth factors for R. t7ifolii,strain 205, only under certain environmental conditions. For instance, panto-thenic acid induced significant increases in growth when the iron content of themedium was low. When 500 Mug of iron per liter were added to the medium, thisvitamin continued to serve as an accessory factor at 30 C only, causing no sub-stantial percentage increases at 25 C, whether agitated or not (table 1).

Iron is known to be an important constituent of many enzyme systems. War-ing and Werkman (1944) found that when Aerobacterindologenes was grown in aniron-deficient medium, many of the enzyme systems of its cells were depleted ormissing. Apparently, the organism used the smnall amount of iron in the mediumto complete the more vital enzyme systems, leaving the less vital ones more orless inoperative. Pappenheimer and Shaskan (1944) found that the course ofglucose metabolism by Cloatidium welchii was profoundly altered by varying theiron content of the medium. The enzyme systems responsible for the trans-formation of glucose into acetic and butyric acids and into carbon dioxide op-erated only in the presence of considerable iron. When the iron content waslow, the course of fermentation took the low energy-producing route and yieldedlactic acid. Tanner, Vojnovich, and- Van Lanen (1945) studied riboflavinproduction by three species of Candida and found that the iron content of themedium was the controlling factor. Amounts of added iron greater than 10Mg per liter markedly lowered the yield. According to the, same authors, ironconcentration was one-of the controlling factors regulating riboflavin productionby Clostridium acetobutylicum, although' the iron requirement of this organismis about 100 times as great as that of Candida species. Feeney, Mueller, andMiller (1943) found control of the iron content of the medium to be requisitefor maximum toxin production by Clostridium tetani. Thus, the control ofthe iron content of the medium makes it possible to increase, in some instancesat least, the synthesis of certain desirable products; also, it leads to a furtherinsight into the life processes of microorganisms.Many of the B vitamins, such as thiamine, riboflavin, and nicotinic acid,

are known to be active constituents of enzyme systems. If environmental

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conditions are favorable, the organism synthesizes them at a rate sufficient forrapid growth; otherwise, it devotes most of its energies to the syntheses of sub-stances, and the maintenance of reactions, which are most vital for its survival.According to Van Niel (1944) "it may readily be assumed that an organism whichshows such a response [to growth-promoting or accessory growth factors] isintrinsically capable of performing the synthesis of related cell constituents fromother nutrients, but that this occurs at so low a rate that the overall phenomenonof growth becomes limited by this particular synthetic process." (Italics are ours.)It must always be born in mind that we are not dealing with an all or none re-sponse, but with a difference in rates which is manifested in cell multiplication.In the absence of accessory factors a longer incubation period is necessary toachieve a given amount of growth (table 2). But when these factors are addedto the medium their low rate of synthesis by the organism is no longer a limitingfactor; consequently more growth in a shorter period of time is the result. Thisis particularly true when the environmental conditions are such as to imposesevere handicaps on growth.When cultures of R. trifolii, strain 205, were given a suitable environment

with respect to iron supply and temperature, and were allowed to grow for morethan six days, the beneficial effect of added accessory factors emerged once more.Apparently, in such cases a rapid multiplication of cells depletes the iron reserveof the medium; therefore, the enzyme systems responsible for the synthesis ofthese vitamins either fail to produce them at a rate sufficient for optimumgrowth, or divert the limited iron supply to enzyme systems more vital to thelife of the cell. It is not too unreasonable to assume that the rapid synthesis ofthese accessory factors is a "luxury" function and consequently may be dispensedwith when conditions become critical. The behavior of cultures grown at30 C illustrates the effect of imposing unfavorable conditions on the organism.Here the addition of 500 ,ug of iron per liter resulted only in a 100 per cent increasein growth, whereas at 25 C the percentage increase was three or four times asgreat. It may be assumed that the higher temperature is unfavorable for arapid synthesis of vitamins insofar as the conditions described in this work areconcerned. But it should be remembered that the nutrient medium used hereis by no means complete, either qualitatively or quantitatively; that othersources of nitrogen and sugar, other vitamins, and substances other than thosementioned here may bring about profound changes in the response of the organ-ism.The foregoing discussion is devoted largely to the interrelationship of iron

concentration and various vitamins as accessory factors. Insofar as the maxi-mum amount of growth is concerned, 25 C, agitation, 500 Mug of iron, 100 ,g eachof thiamine, pantothenic acid, pyridoxine, and nicotinic acid, and 5 mg of inositolper liter induced the greatest increase. In the absence of added iron, thiamineand pantothenic acid caused the most growth. The interrelationships of thesevitamins seem to be very delicately balanced, so much so that even the absence ofone vitamin may bring about significant changes for which we can offer nosatisfactory explanation as yet. Apparently all seven of the vitamins used in

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3 VIRGI GREENE LILLY AND LEON H. LEONIAN

this work have a definite place in the enzyme systems of the organism, and anyshift, no matter how small, may alter the working system of the cell. Furtherstudies now in progress at this laboratory may shed more light on this complicatedsituation.

SUMMARY

Thiamine, pantothenic acid, inositol, pyridoxine, nicotinic acid, riboflavin,and p-aminobenzoic acid, alone and in combinations, were found to be accessorygrowth factors for Rhiwobium trifolii, strain 205, under certain environmentalconditions. In media containing no added iron and incubated at 30 C, thefollowing vitamins singly induced significant increases (20 per cent or more):thiamine, pantothenic acid, inositol, pyridoxine, and nicotinic acid. At 25-C,with or without agitation, thiamine, pantothenic acid, inositol, and pyridoxinecaused significant increases. Although ineffective when used singly, p-amino-benzoic acid and riboflavin markedly increased the yield when used in com-bination with thiamine or pantothenic acid. This was particularly noticeablein the agitated cultures at 25 C. All the combinations of vitamins induced signi-ficant increases in growth under all three conditions of incubation. The combina-tions producing maximum growth were different for each condition of incubation.

In the presence of 500 ,g of added iron all seven vitamins, used singly, in-duced significant increases in growth when the cultures were incubated at 30 C.Only one combination of vitamins caused an increase of more than 20 per cent incultures grown at 25 C and not agitated. Although ten combinations of vitaminsinduced increases of 20 per cent or more in cultures incubated at 25 C with agita-tion, the greatest growth occurred at 25 C with agitation in a medium containingthiamine, pantothenic acid, inositol, pyridoxine, and nicotinic acid.

In the absence of accessory factors the amount of growth was proportionalto the iron content of the medium within certain concentrations. Amountsofiron greater than 250 ,g per liter failed to bring about additional growth.The pH of the culture medium was also a function of the iron concentration

of the medium, more iron being necessary to induce the lowest pH than to producemaximulm growth.

REFERENCESALLSON, G. E., AD HOOVER, S. R. 1934 An accessory factor for legume nodule bacteria.

J. Bact., 27, 561-581.FEENEY, R. E., MUELLER, J. H., AND MILLER, P. A. 1943 Growth requirements of Clos-

tridium tetani. II. Factors exhausted by growth of the organism. J. Bact., 46,559-562.

Fiues, N. 1938 VUber den Bedeutung von Wuchsstoffen fWr das Wachstum verschiedenerPilze. Uppsala. 188 p.

KOGL, F., AND FRIES, N. 1937 tYber den Einfluss von Biotin, Aneurin, und Meso-inositauf das Wachstum verschiedener Pilzarten. Z. Physiol. Chem., 249, 93-110.

LEON1AN, L. H., AND LILLY, V. G. 1942 The effect of vitamins on ten strains of Sac-charomyces cerevi8iae. Am. J. Botany, 29, 456-464.

LEONIAN, L. H., AND LILLY, V. G. 1945 The comparative value of different test organismin the microbiological assay of B vitamins. West Va. Agr. Expt. Sta., Bull. 319.

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PAPPENHEIMER, A. M., JR., AND SHAsx1RN, E. 1944 Effect of iron on carbohydrate metab-olism of Clostridium welchii. J. Biol. Chem., 155, 265-275.

ROBBINS, W. J., AND KAVANAGH, F. 1938 Thiamin and growth of Pythium butleri. Bull.Torrey Botan. Club, 65, 453-461.

STEINBERG, R. A. 1938 Applicability of nutrient solution purification to the study oftrace-element requirements of Rhizobium and Azotobacter. J. Agr. Research, 57,461-476.

TANNER, F. W., JR., VOJNOVICH, C., AND VAN LANEN, J. M. 1945 Riboflavin productionby Candida species. Science, 101, 180-181.

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