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Nitrogen-fixing microorganisms in paddysoils: VIAzuma Okuda a & Masuro Yamaguchi aa Faculty of Agriculture , Kyoto University , KyotoPublished online: 29 Mar 2012.

To cite this article: Azuma Okuda & Masuro Yamaguchi (1960) Nitrogen-fixing microorganisms inpaddy soils: VI, Soil Science and Plant Nutrition, 6:2, 76-85, DOI: 10.1080/00380768.1960.10430930

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[Soil and Plant Food, Vol. 6, No.2, 1980]

NITROGEN-FIXING MICROORGANISMS IN PADDY SOILS: VI

Vitamin B.2 Activity in Nitrogen-fixing Blue-green Algae

AZUMA OKUDA AND MASURO YAMAGUCHI

Faculty of Agriculture, Kyoto University

RECBIVEV SfJPTEMRER 21, 1960

During the experimental study of the role ofblue-green algae in the nitrogen economy ofpaddy fields which were hitherto carried out inour laboratory, it was observed that smallanimals in paddy fields such as water fleas, mudsnails, small freshwater fish, tadopoles or larvaeof small insects had a preference for feedingon blue-green algae.

Though this was a great obstacle in the ex­periment to ascertain the effect of inoculationof the nitrogen-fixing blue-green algae on ricecrop, it awaken us to the fact that blue-greenalgae might contain some substances effectivefor the growth of the small animals.

During the purification of the blue-green algaewe also experienced that they were persistentlyaccompanied with some bacteria. Indeed thismight be mainly because of the existence ofslimic substance around the cell membrane ofthe algae, but some part of it seemed to bedue to a growth-promoting substance containedin the algae.

In mid course of our search for the growth­promoting substances which was carried out in1954, we found vitamin BI2 activity of a con­siderable high level in nitrogen-fixing blue­green algae, Tolypothrix tenuis and Nostocmuscorum, and suspected that in the soil thesealgae might synthesize and supply the vitaminfor other microorganisms which have need forit.

Vitamin B1h which is well known as one ofthe growth factors indispensable for the growthof animals and a certain circle of microorgani­sms, had been believed to be produced only bysome kinds of bacteria, actinomyces, fungi andyeasts until ROBBINS et al. (1) and HASHIMOTO

et al. (2) found these compounds in algae.ROBBINS investigated the content of vitamin

B12 in various kinds of algae and found thecompounds in many of them. HASHIMOTO et al.also reported that vitamin BI2 was found invarieties of algae and its content in purplelaver came up to about 20 rr9o.

In this connection, ERICSON (3) presented anassumption that vitamin B'2 found in the algalbody was of bacterial origin and, came fromthe surrounding water or mud based upon theabsorption experiments using radioactive cobalt.But among many species which ROBBINS inves­tigated, at least three species of blue-greenalgae should be considered to be able to syn­thesize vitamin B12 because they were culturedin bacteria·free condition. FOGG (4) also repor­ted that Anataena cylindrica contained 1.1j.Lg ofvitamin B12 activity per g dry weight.

The synthesis of vitamin B12 active compoundsby blue-green algae is very interesting not onlyfrom the physiological, but also from the eco­logical point of view, and its investigation seemsto open a new way for the solution of thesituation of blue-green algae among varietiesof soil microoganisms.

For this reason, we made a series of experi­ments to see the synthesis of vitamin BI2 activecompounds by Tolypothrix tenuis under variousconditions. The possibility that the vitamin Billactivity might have any connection with thenitrogen fixation was, at the same time inves­tigated.

Experimental Methods and Results

Experiment I Occurrence of vitamin B I2 ac­tivity in nitrogen-fixing blue-green alga, Toly­pothrix tenuis.

As mentioned above, in mid course of thesearch for the growth-promoting substances wesuspected that vitamin B12 might be one of the

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NITROGEN-FIXING MICROORGANISMS IN PADDY SOILS: VI

pH of the solution was adjusted to 7.0 with sodium

hydroxide.

Fig. 1. Diagram of the flask used forculturing blue-green algae.

Table 1. Composition of Nutrient Solutionfor Tolypothrix tenuis

gil

7.03.00.50.11.0

2.0

6.8

Salt

pH

K.IIPO.KH2PO.

Na Citrate·3lhOMgSO,,7H.O

(NH,)2S0.

Glucose

For the preparation of a sample solution forvitamin B12 assay, acetate buffer extractingmethod was employed. This extracting proce­dure has been universally used for the prepa­ration of sample solution in vitamin BIZ assay.The procedure was follows:

To 1 g of the sample powder, 50 ml of distil­led water and 10 to 20 drops of acetate buffer(pH 4.5) were added and boiled for 30 min. llitreof this buffer solution contained 207 g of sodiumacetate and 126 g of acetic acid. After that, pHvalue of the solution was adjusted to 6.0 withsodium hydroxide and filtered into a flask ofadequate volume and filled up to the mark withdistilled water.

Along with this, an attempt on the extrac­tion i. e. a trial of extracting BI2 active com­pounds with boiling water from frozen-driedcells, was also made.

For the determination of B'2 activity, bioas­say by Lactobacillus leichmannii method as wellas by Escherichia coli mutant method wereemployed. The strain of the test organismsused in vitamin B.2 determination were L. lei­chmannii ATCC 7830 and E. coli 113-3 respec­tively, and the basal medium for lactobacilluswas the same as mentioned in U.S.P. xv, whilethe one for E. coli had the following composi­tion (Table 2).

Incubation periods, temperatures and wavelengths used for measuring turbidity at thebioassay of B12 were 18 hr, 35°C, 625 milo forlactobacillus and 22 hr, 37°C, 560 milo for E. colirespectively. Of course consideration was givento eliminate the error brought by desoxyribo­sides or methionine in these assay procedures.

The content of vitamin B,! activity in thealgal cells was of comparatively high level as

Table 2. Composition of Basal Nutrient SolutionUsed for Vitamin B.2 Assay by E. coli

11.6

1.40.39oA·!2.30.25

air fromcompressor

FeCla

MnCh·4H.OCuSO,·5H.OZnSO.·7H.O

HaBOsNa~MoO,·2H20

Micro-nutrient (mg/l)

0.50.50.20.05

NH.NOaK.HPO.MgSO.·7H.O

CaCI.

Macro-nutrient (gil)

... fl' -.- .-light •• 0 light. , .,- ' " ... ..',

components, and to ascertain this conjecturethe following experiment was undertaken.

Tolypothrix tenuis was inoculated into 150 mlof nutrient solution of the following composi­tion (Table 1) in 300 ml ERLENMEYER flasks,after autoclaving. These flasks were culturedfor 10 days at 30°C under the light of fluores­cent lamps. The illumination of the surface ofthe flasks was about 3500 lux.

The solution was constantly aerated throughcotton filters by a compressor (Fig. 1).

After 10 days' culture, the culture solutionwas filtered off through 1 G 3 glass filter andthe algal cells, which remained on the filterplate, were vacuum-dried and ground in amortar.

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A. OKUDA AND M. YAMAGUCIII

Table 3. Vitamin Bl~ Activity in Tolypothrixtenuis Determined by Various Combinationof Extracting Methods and Assay Organisms

shown in Table 3.Experiment II Vitamin R1' activity of To­

lypothrix tenuis under various nutritive condi­tions.

As already pointed out in the previous ex­periment, Tolypothrix tenuis contained a con­siderable amount of vitamin B1, active com­pounds. Though the contaminated microor·ganisms proved to be unable to synthesize vita­min BI2 active compounds, it seemed to be stillimprudent to decide only from the data thatthe alga could synthesize vitamin B12 activecompounds by itself. And another experimentwas designed to extend our knowledge aboutthe vitamin BI2 active compounds synthesis bythe alga.

The alga was inoculated into 200 ml of nut·rient solution in 300mI ERLENMEYER flask equip.ped with an aerating nozzle after autoclaving,

and incubated for 10 days at 30'C under thelight of fluorescent lamps. The illumination ofthe lamps was about 2500 lux. The flasks wereaerated from 9 in the morning till 5 in the even­ing every day by a compressor.

The experimental design and the compositionof the basal nutrient solution used are as follows(Table 4).

After 10 days' incubation, the culture solu~

tions were filtered off by IG3 glass filters andthe algal cells on the filter plates were weighedafter vacuum-drying.

Sample solutions for vitamin Bl2 activitydetermination were prepared just the same asdescribed in experiment I.

The results obtained are shown in the follo­wing (Table 5).

Because the inoculum used in the above ex­periments was not yet bacteria-free, some bac­teria grew to the extent that their effect onthe alga could not be neglected in treatment 7and 8. And we had to give up the continua.tion of their cultivation.

Bacteria penetrate and live in the gelatinoussheaths which surround the cells and filamentsof blue.green algae, so that it is almost impos­sible to remove the bacteria from the algae bytreatments normally used for purification inthe field of bacteriology. And this has been a

'Y%84

128

Boiling waterextraction offrozen-driedsample

'Y%76

110

Extracting'method i Acetate buffer

: extractionAssayorganisms

L. leichmanniiATCC 7830E. coli 113-3

Table 4. Experimental Design and Composition of Nutrient Solution Used (gil)

0.05

0.05

0.05

0.05

0.050.050.05

eaCh

0.20.5

0.2

0.2

2

2

0.5

0.5

Nutrient salts \- - l' . I . I INH.NOa Glucose CoCh·6H,O I KlIHPO. _ _MgS_O"7H~O 1_

-I -- ------- ~ -- - ------~--

: mg/l I 0.5 0.20.2 I 0.5 0.2

'0.5 0.2

0.5 0.2

0.5 0.2

0.5 0.2

Treatment------1. Basal medium only

2. Cobalt added3. Nitrogen source added4. Nitrogen source, cobalt

added5. Carbon source added6. Carbon source, cobalt

added7. Carbon source, nitrogen 0.5 2 :

source added i8. Carbon source, nitrogen 2 0.2 I

__. __source an~~balt added 0.5 i I I 0.5 0.2 0.05

pH of the solution was adjusted to 7.0 with sodi~m -hydroxide. To supply micronut~ients, ! m! of thefollowing solution was added to 1 ! of the nutrient solution.

Fe FeCI. 11.6Mn MnCIa.4IhO 1.8Mo NazMoO••2H.O 0.16

Zn ZnSO••7HzOCu CuSO••5H.OB HaBOa

mg/I0.220.082.9

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NITROGEN-FIXING MICROORGANISMS IN PADDY SOILS: VI

Table 5. Vitamin BI2 Activity in Tolypothrix tenuis under Various Nutritive Conditions

: Experiment 1. Contaminated culture Experiment 2. Bacteria-free culture

Weig-ht·ofaIga I Vitamin BI2 Weight of alga Vitamin BI~(Dry wei~hU>a!>is_) activity .cDry .weight ~asis>- activitY

83.9 mg 465 y% 96.4 mg 400 y%

103.6 530 111.3 470

92.4 700 100.6 580

Treatment

1. Basal medium only

2. Cobalt added

3. Nitrogen source added

4. Nitrogen source, cobaltadde~

5. Carbon source added

6. Carbon source, cobaltadded

7. Carbon source, nitrogensource added

8. Carbon source, nitrogensource and cobalt added

95.4

97.8

134.2

590

400

530

108.3

99.1

125.0

107.8

104.5

600

410

500

410

420

great hindrance in the physiological study ofthe algae.

As the result of the investigation of contami­nating microorganisms, we found that some spe­cies of bacteria and imperfect fungi harbouredin the gelatinous mass surrounding the alga.

Although it was proved that they were una­ble to fix atmospheric nitrogen or synthesizevitamin Bn active substances, yet it seemednecessary to eliminate these contaminants tostudy the physiological properties of the algaand almost every possible means was tried toobtain the pure culture.

Among the biological isolation techniques,successive transplanting on silica gel plates con­taining nitrogen-free inorganic nutrient solutionwas considered most promising and tried toge­ther with the isolation method by phototacticmovement of the alga, but was not effective.

Various kinds of chemical substances werealso used to remove the contaminating micro­organisms from culture of alga. As the che­mical agents, oxidative reagents such as blea­ching powder, bromine water, or hydrogen per­oxide, antibiotics such as penicillin, streptomy­cin, aureomycin or chloromycetin, and manyother kinds of bactericides or fungicides wereused. These attempts, however, all failed inthe purification process, because of the narrowdifferences in the resistibility against thesechemicals between the alga and contaminants;in most of them, both the alga and contami­nants were killed in high concentrations, while

in low concentrations they were all left alive.Of course some mechanical trials were ap­

plied, for instance, we tried to pick up a singlecell of the alga by capillary pipette or scatterthe cells on the surface of the plates by sprayerafter dispersed the algal cells in nutrient solu­tion by homogeneous blender. These were alsonot successful.

And we went at last to the ultra-violet irra­diation which was employed by ALLISON andMORRIS (1930) (5), ALLISON et al, (1937) (6) andGERLOFF, FITZGERALD and SKOOG (1950) (7).

The alga was dispersed homogeneously in thenutrient solution by use of homogeneous blen­der and diluted with the nutrient solution toan adequate concentration. This algal suspen­sion was irradiated by an ultra-violet lamp atthe distance of 15 cm for 5 min with constantagitation, and small volumes were sampled at1 min intervals and inoculated on the nutrientagar slants.

We could thus obtain the bacteria free cultureof Tolypothrix tenuis, which was isolated fromTokiwamura soil, from the slant inoculated bythe adequately irradiated sample solution. Thealga was checked for purity on CZAPEK'S me­dium, yeast-ctextrose medium, ASHBY'S nitrogen­free medium, sodium caseinate medium and T.C. G. medium.

We again carried out the above experimentsusing this bacteria-free alga.

The result was of the same tendency as thepreceding one (Table 5).

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A. OKUDA AND M. YAMAGUCHI

As are shown in Table 5, the addition ofcobalt promoted the growth of alga as well asvitamin BI? activity in the cells, but the effectof the addition of nitrogen or carbon source onthe growth and vitamin BI? activity was farless than was expected.

Experiment III Comparative study on thepossibility of suspectable relationship betweenvitamin B" synthesizing ability and nitrogenfixation.

From the result of the above experiment, itseemed that there exists no relationship betweenvitamin B12 synthesizing ability and nitrogenfixation. Vitamin B" synthesizing ability hasbeen proved to be widespread in various kindsof microorganisms.

But many of the organisms which were kno­wn to have the nitrogen-fixing ability had beenreported to have fairly good vitamin B12 syn­thesizing ability, and we intended to carry outfurther study to clear up the possibility of theexistence of any specific relationship betweenvitamin BI2 synthesizing ability and nitrogenfixation by determining vitamin BI2 activity invarious kinds of nitrogen fixers.

In the first place, vitamin B12 activity in legu­minous plants was determined. These plantswere carefully washed by distilled water anddevided into three parts i. e. aerial part, rootpart and nodule part. After vacuum-dried andground in a mortar, each was extracted withboiling water containing potassium cyanide atthe rate of 1 mg per "1 of vitamin B12 for 30 min.

The extracted solution was used for determiningtotal vitamin Ble activity. A part of the solu­tion was again boiled for 30 min at pH of over12.0 and used for the determination of desoxyri­bosides.

The vitamin B12 activity was calculated bysubtracting the activity due to desoxyribosidesfrom the total vitamin B'2 activity.

The results are as shown in Table 6.As indicated in Table 6, vitamin BI2 activity

was concentrated in the root nodule part, andit seemed reasonable to consider that root no­dule bacteria might have some relation withthe vitamin B" activity from this data.

And in the second place, we made a seriesof experiments to investigate the possible rela­tionship between root nodule bacteria and vita­min BI2 activity.

To begin with, turbidimetric experimentswere carried out to see the effect of the addi­tion of cyanocobalamin and cobalt on the grow­th of pea nodule bacteria. The experimentaldesign and the composition of the nutrientsolution used in this experiment are mentionedin Table 7.

10 ml portions of the basal solution with tre­atments as in Table 8 were poured into testtubes of the same diameter with cotton Woolplugs. After autoclaving, in each tube a dropof the suspension of pea nodule bacteria, whichwas precultured in vitamin B1dree medium,was inoculated and incubated on a shaking ma­chine for 3 day at 30°C.

Table 6. Locality of Vitamin B12 Activity in Various Kinds of Leguminous Plants

Vitamin B12 activity ("1%)

(1)

(2)

(3)

(1)

(2)

Plants

Broad bean

Pea

Aerial part ! Root part I Nodule part

-Dr~-~.ba~i~lre~h w. baSisl D~;~~ b-;:sis \F;e~h ~.-basisl-D-;~~.-b~sis-IF;esh w~aSis- --------------------- ----------- ,-- ---- - ----1--- --- --- 1--------

0.4 0.07 1.0 1 0.24 72.0 14.20.1 0.02 1.2 0.13 214.5 33.70.01 0.002 0.42 0.057 156.7 30.8

0.0 0.00 3.7 0.26 164.7 25.20.23 0.053! 0.02 0.003 39.4 5.67

Red clover 0.2 0.05! 0.7 0.29 99.2 26.2White clover 0.21 0.029! 0.51 0.19 68.9 18.7Alfalfa 0.3 0.06 I 2.0 0.76 78.1 m.3Kidney bean 0.3 I 0.6 25.8--- ------ ----' I I J --'- _

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NITROGEN-FIXING MICROORGANISMS IN PADDY SOILS: VI

Table 7. Experimental Design and Composition of Nutrient Solution for Pea Nodule Bacteria (gil)

tr.tr.tr.

tr.tr.tr.tr.

tr.tr.tr.

tr.tr.

tr.tr.

0.050.050.050.050.050.050.05

0.2 '0.20.20.20.20.20.2

I

MgSO.' CaCI MnSO.1 FeCl3.7HzO z

0.20.20.20.20.20.20.2

NaCI

0.50.50.50.50.50.50.50.5

0.5

0.50.50.50.50.5

10.010.010.010.010.010.010.0

0.010.11

10

1001000

I I I I ~:

Tre~~~ent~ ~J~~~~!~_~ ~6~~?JG~I:~o~eLK_N~3_'~H~~.Cyanocobalamin series I -r /1 -rII1. Control i

2. 0.01 m-r/ml3. 0.1 /I

4. 1 /I

5. 0.01 -rlml6. 0.1 /I

7. 1 /I

Cobalt series8. Control9. 0.04 m-r/m1 I

10. 0.4 /I

11. 4 II

12. 0.04 -rlml13. 0.4 /I

14. 4 II

0.040.44

40400

4000

10.010.010.010.010.010.010.0

0.50.50.50.50.50.50.5

0.50.50.50.50.50.50.5

0.20.20.20.20.20.20.2

0.20.20.20.20.20.20.2

0.050.050.050.050.05

0.050.05

tr.tr.tr.tr.tr.

tr.

tr.

tr.tr.tr.tr.

tr.

tr.

tr.

pH of the solutIOn was adjusted to 6.8.

Growth of the bacteria was determined bymeasuring turbidity of the culture solution byspectrophotometer. Wave length used in thisdetermination was 550 mJL. The results areshown in Fig. 2.

0.700

•

0.500

0.400

•

•••••

•····•

0.600 O.:l()()

D D

0.500 • • 0.:200

•

0.400• •

••

•• ••

0.100

• ••

•.400040040o 0.04 0.4 4

my CobaltB. Growth response of pu nodule bacteria

to the addition of cobalt.Fig. 1.

0.300 '-----~--...........---_-_......-o 0.01 0.1 1 10 100 1000

ffi'y Cyanocobalamin

A. Growth response of pea nodule bacteriato the addition of cyanocobalamin.

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A. OKUDA AND M. YAMAGUCIlt

Table 9. Composition of Nutrient SolutionUsed for Azotobacter vinelandii

Azotobacter vinelandii. At first to see the ef­fect of the addition of cyanocobalamin on thegrowth of the azotobacter, turbidimetric testswere made. For the nutrient solution the fol­lowing composition was adopted (Table 9).

In this preliminary experiment, the growthof the bacteria was slightly promoted withincreasing addition of cyanocobalamin, but notby the addition of cobalt except 0.04 my/mllevel and was rather depressed at over O.4'Y/mllevel.

Further experiment was carried out to seehow the vitamin B12 activity in pea nodulebacteria was affected by the addition of cyano­cobalamin and cobalt.

In this experiment pea nodule bacteria wereincubated in 100 ml of the nutrient solution ofthe same composition as shown in the prelimi­nary test in 300 ml ERLENMEYER flasks. Treat­ments are as shown in Table 8.

Salt

K2HPO.

MgSO.NaCI

CaC!!

FeSO.Glucose

gil

1.0

0.20.20.05tr­

IO

,:Vitamin BI2 Activity

Table 8. Effect of the Addition of Cyanocobala·min or Cobalt salt on Vitamin BI~ Activity inthe Culture Solution of Pea Nodule Bacteria

After incubated on a shaking machine for 9<lays at 30·C, the culture solutions were usedfor vitamin B12 assay. The results are alsoshown in Table 8.

Table 8 indicates that the cyanocobalaminadded to the nutrient solution decreased from10 my to 7.7 my per ml during the incubationperiod even when the bacteria were not ina­<:ulated (Treatment 2). Probably this would be<lue to the decomposition of the cyanocobalamin.As the similar decrease in the added cyanoco­balamin was also expected, the net increase inthe vitamin Bla activity in Treatment 1 wouldbe about 7 m'Y/ml. Therefore, it wowld be con­duded that the bacteria synthesize vitamin BI~

active compounds quite regardless to the addi­tion of cyanocobalamin. Addition of cobalt atthe rate of 0.4 'Y per flask stimulated the syn­thesis of vitamin BII active compounds.

We also made some investigations about thesynthesis of vitamin Bu active compounds by

pH of the solution was adjusted to 7.0.

The levels of cyanocobalamin concentrationset up in this tests were 0, 0.1, 1, 10 and 100ppm. 10 ml of the nutrient solution was pou­red into each of test tubes of the same diameterand after receiving the cyanocobalamin treat­ments they were autoclaved at 15 lb for 10 min.A drop of the suspension of azotobacter, whichhad been precultured in vitamin BIa·free medi­um, was inoculated into the nutrient solutionin each of the test tubes· These tubes wereincubated on the shaking machine at 30·C for7 days and the turbidity of the culture solutionswas measured by spectrophotometer. The wavelength used in this measurement was 550 mIL.

As is shown in the results (Fig. 3), little dif.:ferences were observed in the growth of azoto­bacter in the cyanocobalamin levels.

As it was considered from the above resultsthat azotobacter has no need for cyanocobalaminor that it has an ability to synthesize vitaminB12 active compounds, we planned a furtherexperiment to see which was the reason.

A drop of the suspension of Azotobacter vi­nelandii precultured in vitamin Bwfree medi­um was inoculated into 100 ml portions of thenutrient solutions in 300 ml ERLENMEYER flasksrecived various treatments as described in Ta­ble 10 and cultured on the shaking machinefor 2 days at 30°C.

The bacterial cells were separated from theculture solutions by centrifugation and weighed.Vitamin BI~ activity was determined by the

0.641.110.60

1.48

0.777.7

6.411.16.0

..... ~:. ~'Y/m!J'Y/flask! 14.8

Treatment

1. Cyanocobalamin 10 m'Y/ml

2. " 10 m'Y/ml(Not inoculated)

3. If 0.1 m'Y/ml

4. Cobalt 4 m'Y/ml5. Basal solution only

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NITROGEN-FIXING MICROORGANISMS IN PADDY SOILS: VI

D

0.700

O.lppm

0.60010ppm DIppmOppm

100ppm 0.5000.500I ppm

0.400 100 ppm0.400 10ppm

Oppm0.300 0.1 ppm

0.300

0.200

0.2000.100

0.100

0 :? 3 4 5 6 7Days

0 2 3 4 5 6 7Days (m

(1)

Fig. J. Growth response of Azotobacter vinelandii to the addition of cyanocobalamin.

method mentioned before. The results are alsoshown in Table 10. Though little, the growthof Azotobacter vinelandii was affected by theaddition of cyanocobalamin and soil extract,whereas there were great differences in vitaminBl~ activity in the cells. In the cyanocobalaminadded series, vitamin BIZ activity in the cells

came up to 188.8 to 405.8 m'Y, but in the otherseries the activity was only 0.7 to 2.9 m'Y.

From this data we had to consider that thebacteria did not synthesize nor need vitaminBIZ active compounds even when it was fixingatmospheric nitrogen contrary to our first con­jecture.

Table 10. Effect of Some Nutritional Treatments on the Vitamin B12 Activity.in the Culture of Azotobacter vinelandii

Weight of azotobacter Vitamin BI2 Activity m'Y/flaskTreatment (Dry w. basis) I

in Cells in Medium Total----------

1. Basal medium only 68.6 mg/flask 0.7 1.6 2.3

2. Cyanocobalamin* added 71.9 188.8 923.2 1112.03. Soil extract** added 64.6 0.7 2.3 3.D4. Cyanocobalamin and soil extract 85.8 4D5.8 819.5 1225.3added

5. Nitrate**'" added 97.2 2.9 2.1 5.06. Nitrate and cyanocobalamin 105.8 354.4 1288.5 1642.9added

7. Nitrate and soil extract added 105.0 1.1 1.4 2.58. Nitrate, cyanocobalamin and 108.4 298.1 997.9 1296.0soil extract added

pH of the solution was adjusted to 7. O.*) Cyanocobalamin was added to the nutrient solution at the rate of 3')'/100 ml.

**) Soil extract was added to the nutrient solution at the rate of 1 ml;lOO ml to the nutrient ~olutlOn.

This soil extract was made by autoclavmg I kg of soil with I 1 of water at 15 lb for I hr.*••) As the nItrogen source, 1 g of KNOB was added to I I of the nutnent solution.

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A. OKUDA AND M. YAMAGUCHI

Discussion

The vitamin B12 activity of Tolypothrix ten­uis by E. coli mutant method was higher thanthat by L. leichmannii method.

This will be probably due to the differenceof response to vitamin BI2 analogs between thetwo types of test organisms.

As for the extracting method, we could con­firm that the extraction by acetate buffer,which has been universally used for this pur­pose, was a satisfactory one.

In any way, it was evident that a nitrogen­fixing blue-green alga Tolypothrix tenuis con­tained a considerable large quantity of vitaminBI , active compounds which did not come fromother origins, but were synthesized in the algaas shown in Table 3 and 5.

We would give attention to the fact thatsuch kinds of blue·green algae as Tolypothrixtenuis are one of the producers of vitamin B12

active compounds in soil and consider that oneof the reasons why small animals in paddy soilhave a preference for feeding on blue·greenalgae may be this high content of vitamin B12

active compounds in the algae.Moreover, there lives a number of microor·

ganisms which have need for vitamin BiZ activecompounds as growth factor in soil as reportedby LaCKHEAD et al. (8). Probably the blue-greenalgae will also contribute to the growth of othermicroorganisms, which need vitamin B12 inpaddy soil, by supplying the vitamin.

It will be a very interesting problem to inve­stigate further the situation of the algae amongsoil microorganisms from this point of view.

Furthermore, the role of the considerablelarge quantity of vitamin B12 active compoundsin the algae is a matter of interest. It hasbeen believed that vitamin B12 active com­pounds play roles in various biological processes,for instance, in the metabolisms of amino acids,methyl groups, nitrogenous bases or nucleicacids. This compound is also reported by someworkers to have a close connection with thecontrol of reductive condition of living bodies,but it seems fully probable that this compoundmay bear other important roles in some un.known fields.

When the list of the species of microorgani­sms, which have proved, to have the ability ofnitrogen fixation was surveyed, we could findthat many of them also had the fairly goodability of synthesizing vitamin B12 active com­pounds. Anabaena cylindrica, Clostridium bu­tyricum, Aerobacter aerogenes, and some ofRhizobium species, all these have been reportedto have both abilities. In addition to these.many genera which have been reported to haveboth abilities could be also found.

Of course, it is clear that this ability of vi­tamin B12 active compounds synthesis is widelydistributed in a variety of microorganisms andis not the specific character of the nitrogenfixers, but it occurred to us to try an experi­ment to confirm whether there was any rela­tionship between the ability of vitamin BIZ ac­tive compounds synthesis and the nitrogen fixa­tion, by using a nitrogen-fixing blue-green algaTolypothrix tenuis.

The results of the experiments given in Table5 show that there was no relationship betweenthe vitamin B12 synthesizing ability and thenutritive conditions, and so we were compelledto conclude that vitamin B12 active compoundswould play no direct act in the course of nit­rogen fixation. In this connection, further ex­periments were carried out using several kindsof nitrogen-fixing microorganisms. In root no­dules of various leguminous plants, high vita­min Bn activity was found, while the activityin aerial part or root part was neglegible.

And the root nodule bacteria seemed to beresponsible for the high activity.

In fact. it was proved that root nodule bac­teria could synthesize a considerable quantityof vitamin BI2 active compounds in nitrate-Nmedium. From these results, it may be alsodeduced that root nodule bacteria could synthe­size vitamin B12 active compounds regardlessof the nitrogen sources. This problem has alsobeen studied and reported by BURTON andLOCKHEAD (9) and some others.

The results obtained about vitamin Bu syn­thesizing ability of Azotobacter vinelandii wasmore unexpected. The azotobacter was littleaffected in its growth by the addition of cyano­cobalamin, nor did azotobacter produce vitamin

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NITROGEN·FIXING MICROORGANISMS IN PADDY SOILS: VI

BI2 active compounds. If vitamin B12 activecompounds bear important roles indispensablefor the growth of many forms of life as havebeen reported in many papers, then the com­pounds should be synthesized by themselvesor otherwise be absorbed into them to be uti­lized for their growth from the environments.

But actually these compounds have been re­ported riot to be contained in some organisms.Higher plants have been known as one of them(10). And in the present experiment the azoto­bacter was also found to be another organismof such kind. To elucidate these facts withoutany contradiction, it would be most reasonableto consider that there may be some substanceswhich have the same functions as vitamin BI2

active compounds in higer plants or Azotobactervinelandii, but the substances can not be de­termined because of the fact that the test or­ganisms used in the vitamin B12 assay do notrespond to the compounds.

Accordingly further investigations about theassay methods are necessary before we carryout the comparative studies on the physiolo­gical functions of vitamin BI2 active compounds.

From our experiments, however, it may beconcluded that there is no relationship betweenvitamin BI2 activity and nitrogen fixation.

Summary

1. A nitrogen-fixing blue-green alga, Tolypo­thrix tenuis could synthesize vitamin Bl2 active

compounds. the yield of which came up to about700 'Y96.

2. The growth of the alga and its produc­tion of vitamin BI2 active compounds was pro­moted by the addition of a proper amount ofcobalt salt. and not affected much by the addi­tion of nitrogen or carbon sources.

3. The possibility of the relationship betweenthe abilities of synthesizing vitamin BI2 activecompounds and nitrogen fixation of Tolypothrixtenuis was tested. The relationship betweenthem seemed to be negative.

4. In this connection, further studies were car­ried out with some other nitrogen fixers. Inthem, also, no relationship was found.

Literature Cited

(1) ROBBINS. W. J. et al .• Bull. Torrey Bot. Club,78, 363 (1951).

(2) HASHIMOTO. Y. and SATO, A., Jap. Soc. Sci.Fish., 19. 987 (1954).

(3) ERICSON, L.E., Chem. & Ind.• No. 34. 829 (1952).(4) FOGG, G.E., Nature, 177. 188 (1956).(5) ALLISON, F.E. and MORRIS. H. J.• Science, 71,

221 (1930).(6) ALLISON, F. E .• HOOVER, S. R. and MORRIS. H.

J., Bot. Gaz., 98. 433 (1937).

(7) GERLOFF. G.C., FITZGERALD, G.P. and SKOOG.F., Amer. ]. Botany, 37. 216 (1950).

(8) LOCKHEAD, A. G. and THEXTON, R.II., SoilSci.• 63. 219 (1952).

(9) BURTON. M. O. and LOCKIlEAD, A. G.• Can. ].Botany. 30. 521 (1952).

(10) DARKEN. M.A., Bot. Rev.• 19,99 (195..1).

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