THE CHEMISTRY OF THE OXIDATION OF SULFUR BY MICROORGANISMS TO SULFURIC ACID AND

TRANSFORMATION OF INSOLUBLE PHOSPHATES INTO SOLU-

BLE FORMS.*

BY SELMAN A. WAKSMAN AND JACOB S. JOFFE.

(From the Department of Soil Chemistry and Bacteriology, New Jersey Agricultural Experiment Station, New Brunswick.)

(Received for publication, October 13, 1921.)

Oxidation of Sulfur by Microorganisms.

When sulfur is added to unsterile soil, it is slowly oxidized to sulfuric acid; when the soil is previously sterilized, oxidation of the sulfur takes place only to a very limited extent depending upon the other chemical substances present. But when a sulfur- oxidizing organism is introduced, the sulfur is rapidly oxidized to sulfuric acid. This acid acts upon insoluble soil constituents such as calcium and magnesium carbonates, calcium silicates, and tricalcium phosphate, and brings them into solution. This process has been utilized by Lipman and associates (1916) for the transformation of the insoluble tricalcium phosphate into soluble forms by cornposting rock phosphate, sulfur, and soil to which the sulfur-oxidizing bacteria have been added. A few principles involved in these transformations, both by crude and pure cul- tures of the sulfur-oxidizing organisms, are set forth in this paper.

As a result of a series of studies, several organisms have been isolated, which are able to oxidize sulfur under various condi- tions. The oxidation of sulfur under acid and alkaline condi- tions seems to be affected by different groups of microorganisms. A detailed study of occurrence, morpholo,gy, and physiology of

* Technical Paper No. 54 of the New Jersey Agricultural Experiment Station, Department of Soil Chemistry and Bacteriology.

35

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36 Oxidation of Sulfur

these organisms is found elsewhere (Waksman and Joffe, 1920- 22). One of the most interesting organisms isolated by the authors is an aerobic, autotrophic, minute bacterium, Thiobacillus thiooxidans Waksman and Joffe, which is able to oxidize sulfur to such an extent as to reduce the hydrogen ion concentration of the medium to a pH of less than 1.0, even in the presence of buffering materials. It derives its energy from the oxidation of the sulfur and the carbon from the CO2 of the atmosphere. The nitrogen can be supplied in the form of inorganic or organic materials.

In taking up the chemistry of the sulfur oxidation, attention must be called to the aerobic nature of the phenomenon.

25 + 2H,O + 302 = 2Hz SO4

64 96 196

Thus, for 64 units of sulfur, 96 units of oxygen are required to produce 196 units of sulfuric acid. The effect of oxygen is, therefore, of prime importance in the oxidation of sulfur.

Experiments with crude cultures of the organism reported elsewhere (Joffe, 1922) substantiate the theoretical suppositions based on the empirical equations involved in the chemistry of sulfuric acid. Mixtures of sulfur, rock phosphate, and soil inocu- lated with crude cultures of sulfur-oxidizing organisms were prepared; one set was aerated and the other left unaerated. The amount of phosphates brought into solution and the change in the hydrogen ion concentration, as expressed by the exponent pH of Sorensen, were used as criteria. The aerated mixtures were leading and, after 100 days, the percentage increase of sol- uble phosphates in the aerated over the non-aerated was 6 per cent, with a similar correlation in the increase of the hydrogen ion con- centration. It is interesting to record here the fact that this biological process follows the laws of inorganic reactions. Accord- ing to the mass law, the velocity of any reaction depends on the mass of the active ingredients involved and is at any moment proportional to the molecular concentration of the reacting com- ponents and a constant, which is characteristic of the chemical nature of the reacting substances. Whatever transformations the oxygen undergoes in the metabolism of the organism, the end-product is sulfuric acid; an increase in oxygen tension increases

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S. A. Waksman and J. S. Joffe 37

the mechanism of oxidation of sulfur by the organisms. It is aIso possibIe that the oxygen from the air is not the only source; as pointed out above this particular organism derives its energy not from carbohydrates but from the oxidation of sulfur and is autotrophic in nature. Like green plants, the autotrophic organ- isms use the carbon dioxide from the air for structural purposes, but, unlike plants, these organisms accomplish it without the intervention of the photochemical reactions. The process of assimilation of carbon dioxide is accompanied by the splitting of? of oxygen, which may also be used by the sulfur organisms in the process of oxidation.

Oxidation of XI&~ in the Ordinary Cultivated Soil.

Several typical experiments will be reported here to illustrate the mechanism of sulfur oxidation in the soil, both in the absence and in the presence of small and large amounts of rock phosphate. The sulfur and phosphate were added to the soil and well mixed. A crude well developed culture was used for inoculation. The moisture content of the soil was kept at an optimum by the addi- tion of water at weekly intervals. The cultures were incubated at 25-27°C. The pH values were determined calorimetrically, according to the method of Clark and Lubs (1917); the phos- phates and sulfates according to the method of the Official Agri- cultural Chemists (1916).

The results tabulated in Table I represent the oxidation of small amounts of sulfur in the soil. In this case 22.5 mg. of sulfur and 90 mg. of rock phosphate were added to 600 gm. of soil. The results tabulated in Table II represent the oxidation of large amounts of sulfur when introduced with large amounts of rock phosphate into the soil. In this case 30 gm. of sulfur and 90 gm. of rock phosphate were mixed with 480 gm. of soil in small pots.

When the course of change in reaction due to the sulfur oxida- tion, in the presence of tricaleium phosphate is studied, we find that the curve is regular till the pH reaches 2.8, then it becomes flat. This is a crucial point and, as long as there will be any phosphate left undissolved, the reaction will not go down very much, since at that point all the acid formed from the oxidation of the sulfur is used not in increasing the reaction of the medium, but in transforming the rock phosphate into soluble form. Once

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38 Oxidation of Sulfur

the phosphates have been made soluble, the acidity begins to increase. This will be made clearer in the discussion of the sulfur oxidation by pure cultures in solution.

TABLE I.

The Oxidation of Small Amounts of Sulfur in the Soil.

Period of incubation.

days

0 3 9

15 22 29

39 56 70

102

PI3 value.

6.2

6.2 6.2 6.0 5.8

5.6 5.6 5.2 5.2 5.2

TABLE II.

The Oxidation of Large Amounts of Sulfur in the Soil in the Presence of Large Amounts of Rock Phosphate.

Period of incubation pH value.

days

0 3 9

15 22 29

39 56 85

102

6.2 6.2

5.0 3.4 3.2 3.0

3.0 2.2 2.0 1.8

-

-

Soluble sulfates in 1 gm. of soil.

mg. of so4

0.95 0.96

Citrate-soluble phosphates

in 1 gm. of soil.

mg.ofP

2.83

2.83

3.60 4.28 20.80 7.13

30.80 14.76 35.25 20.67

19.58

Oxidation of Suljur in Solution by Thiobacillus thiooxidans.

When a proper medium is used, with sulfur as the only source of energy, the pure culture of the organism rapidly oxidizes the sulfur to sulfuric acid. To prevent a rapid change in reaction

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S. A. Waksman and J. S. Joffe 39

buffering substances are used. The course of reaction depends chiefly upon the nature of the buffering agents. When soluble phosphates are used, the curve is more or less continuous; when insoluble phosphates are used the curve has a definite flat portion at a pH of 2.6 to 2.8, the point at which the insoluble phosphates become soluble, and, only after all the phosphate has gone into solution, the curve rises again. When more insoluble phosphate is added at this point, the curve reaction will be kept at the pH of 2.8 to 2.6, till all the insoluble phosphate has disappeared. The medium used for this experiment consisted of sulfur, 10 gm.; (NH& SOJ, 2 gm.; MgS04, 0.5 gm.; FeS04, 0.01 gm.; KH~POA,

TABLE III.

The Oxidation ofSulfur by Pure Culture of Thiobacillus thiooxidans.

Period of incubation No C~J(PO&.

-

days

0

3

7 11 17

23 33 52

PH PfI Pfl

4.4 5.0 4.4 4.4 5.0 4.2

3.2 4.4 3.2” 2.2 2.8 2.8 1.6 2.6 2.6*

1.6 2.4 3.2 1.4 2.1 3.0 1.2 1.8 2.8

3 per cent ch(POd2. Gradual addition of CaS(P04)2.*

* 0.5 gm. of Ca3(POJ2 has been added per 100 cc. of medium.

5 gm.; distilled water to make 1,000 cc. When Ca,(PO& is not used, 0.25 gm. of CaClz has been added per 1,000 cc. of medium. The medium was placed, in 100 cc. portions, in 250 cc. Erlenmeyer flasks and sterilized on 3 consecutive days in flowing steam. The flasks were then inoculated with a pure culture of Thiobacillus thiooxidans by means of a loop, and incubated at 25-27°C. The results are presented in Table III.

It has been pointed out elsewhere (Waksman and Joffe, 1921) that the optimum reaction for the activities of Thiobacillus thiooxidans lies at a pH of 3.0 to 4.0. If the reaction of the medium is less acid, the reaction changes in the beginning only very slowly, but, once the optimum is attained, the curve rises rapidly.

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40 Oxidation of Sulfur

A detailed experiment is next reported which will show definitely the relation between the sulfur oxidation, as demonstrated by change in reaction, accumulation of sulfates, and the transforma- tion of phosphates, as shown by the amounts of soluble phosphates and calcium.

A medium containing 2 gm. of (NH&SO+ 0.5 gm. of MgSOa, 5 gm. of HH2P04, and a trace of FeSOc per liter, was placed, in 400 cc. portions into fifteen 1 liter Erlenmeyer flasks. 3 gm. of Ca3(PO& and 4 gm. of powdered sulfur were added to each flask. The flasks were inoculated with 1 cc. portions of a pure culture of

TABLE IV.

Course of Sulfur Oxidation as Indicated by Change in Reaction and Amount of Soluble Sulfates, Phosphates, and Calcium in the Culture

Age of culture. PH

Control. 6.0 20 hours. 6.0 70 “ 5.4 88 “ 4.9

110 “ 3.5 134 “ 3.0

6.5 days. 2.6 8.5 “ 2.6

10.5 “ 2.5 13.5 “ 2.3

19.5 “ 2.1 34 “ 1.3

m _-

Solution.

‘hosphatea in 100 cc. of solution.

?nQ. Of P

123

125.26

123.20 200.06 171.64

210.04 255.46 350.00

-

E

-

Sulfates in 100 DC of solution.

mg . of so4

230 230.4

248.0

260.15 322.2

366.4 498.8 511.4 450.6

Mcium in 100 cc. of solution.

mg. Of Ca

17.4

17.44

24.74

26.85 31.0 64.2

118.8 104.9 101.4

81.6

Thiobacillus thiooxiidans and incubated at 25-27°C. At various intervals, small amounts of the liquid were taken out from four to six flasks, and determinations made of the pH value, of the sulfates, phosphates, and calcium in solution. The results based on the average of four to six determinations are tabulated in Table IV.

The course of sulfur oxidation is best followed by the change in the hydrogen ion concentration of the medium (pH value). Of course, where there are large amounts of buffering agents or insol- uble carbonates or insoluble calcium phosphate, a much larger

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S. A. Waksman and J. S. Joffe 41

amount of sulfur will have to be oxidized to bring about a definite change in the pH value. The amount of sulfur oxidized has been reported in Table IV as sulfates; it may be pointed out here that in practically all cases the sulfur oxidized, as indicated by the amount of residual sulfur in solution, has been almost quantita- tively transformed into sulfates. The phosphates and calcium columns in Table IV will be discussed below.

Transformation of Insoluble Phosphate into Soluble Forms.

The reactions involved in the conversion of rock phosphate (insoluble tricalcium phosphate) into soluble forms (di- and monocalcium phosphate and phosphoric acid) by means of acids belong to the type of reactions of heterogeneous systems. The rock phosphate minerals have no definite composition and the products formed are not always definite. In such heterogeneous systems the speed of the reaction is a function of a greater number of variables than in the case of a homogeneous system. According to Kazakov (1913), there are some factors which are common to both systems and these are: (1) concentration of the reacting mass; (2) temperature of the reacting medium; (3) the amount of contact of the reacting substances; (4) the speed of diffusion of the reacting substances; and (5) catalytic agents.

Besides these factors we have others in a heterogeneous system where solid solution phases occur. These are: (1) the size of contact surface;l (2) chemical composition of the solid phase; (3) the physical properties of the solid phase; and (4) the influence of formation of a solid phase as a result of the reactions.

The factors; chemical composition of the solid phase, and the physical properties of the solid phase; have a tremendous influence on the speed of the reaction and they are the least known, since the chemical make-up of t,he rock phosphate is still obscure.

1 The size of the particles of the rock in the manufacture of acid phos- phate has an important influence. Theoretically, all other conditions being equal, the speed of solution of a solid in a liquid is proportional to the contact surface and in circular bodies (as we would suppose in finely powdered rock phosphate) the surface is proportional to the square of

the radius; then, particles with a radius of 0.1 mm. will dissolve twenty- five times faster than particles with a radius of 0.5 mm.

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42 Oxidation of Sulfur

The process on the transformation of insoluble phosphates has been the subject of study by a number of investigators in this country and in Europe. We may merely refer to the work of Cameron and Bell (1907) of the Bureau of Soils, of Schucht (1909), Meyer (1905), Stoklasa (1911), Kazakov (1913), and others.

According to Kaeakov (1913), the scheme of reactions involved in t,he formation of soluble phosphates are:

NO. HPSO~

1 When added in ex cess.

2 Close to optimum

3 Optimum.

4 Not enough acid.

i Resultants obtained.

Liquid phases. Solid phases.

H,PO, + H2S04 + sulfates of Ca, Al, and Fe.

H3P04 + sulfates of Ca, Al, and Fe.

CaS04

CaS04

H3P04 + sulfate of Ca +

phosphates of Al and Fe. H3P04 + sulfate of Ca +

phosphates of Ca, Al, and

Fe.

CaSOI

CaS04 + part of undissolved

phosphate.

Before we go into a discussion of the scheme, we shall take up the experimental results of the transformation of the tricalcium phosphate into soluble phosphate t,hrough the oxidation of sul- fur by Thiobacillus thiooxidans.

The culture medium given above has been used, with a slight modification: the KHzP04 was reduced to 1 gm. per liter and I gm. of Ca3(P0& was added to each flask containing 100 cc. of -medium. The medium was sterilized in flowing steam on 3 con- secutive days, 30 minutes each day, then the flasks were inocu- lated with Thiobacillus thiooxidans and incubated at 27°C. Only the pH values and water-soluble phosphates (in solution) are reported in Table V and Fig. 1. The results are based on aver- ages of four to six flasks.

The column of soluble sulfates is of extreme interest. At a pH of 2.6, a sudden rise in the amount of soluble phosphates takes place after the soluble sulfates have reached a maximum. This is in accordance with the scheme suggested by Kazakov. Up tc the point of pH = 2.6 to 2.7, hhe liquid phase consists of mono- calcium phosphate and gypsum, we therefore have a large amount

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S. A. Waksman and J. S. Joffe 43

of soluble sulfates. However, as soon as more sulfuric acid is formed through the oxidation of sulfur, the monocalcium phos- phate is attacked first, since in any reaction the liquid phase comes in first; the products formed are phosphoric acid and gypsum.

CaH4(P0& + HzSOP + 2Hz0=CaS04*2H20 + ZH,POd

The CaXC4~2H20 is soluble to a certain extent in phosphoric acid, but is forced out from solution because of the fact that the phos- phoric acid reacts with the remaining tricalcium phosphat#e, form- ing again monocalcium phosphate until the reaction comes to an equilibrium forming gypsum and phosphoric acid. With the

TABLE V.

Course of Sulfur Oxidation and Transformation of Insoluble Phosphates. -

Age of culture. PH

Control. 5.4

1 5.4 2 5.3 4 4.6 6 3.5 8 2.6

10 2.6 12 2.6 15 2.4

mg. of s

68.39

67.64 69.70 73.79

152.53 109.7

78.54

87.6

T

SolubleIp&o;~hates in

mg. of P

45.57

42.61 47.20 55.00

103.56 93.00

186.30 207.28

accumulation of the phosphoric acid, more gypsum goes in solution and the soluble sulfates increase again. The continuous increase of the insoluble sulfates after all of the tricalcium phosphate goes in solution is then due to the further oxidation of sulfuric acid.

The column of soluble phosphates also proves the mechanism of the process suggested by Kaaakov. Here also we find a gradual increase of the soluble phosphates, since the amounts of sulfuric acid in the early part of the incubation period is small. As soon, however, as the pH reaches 2.6 to 2.7, which is the crucial point of the react,ion, the soluble phosphates increase rapidly. Prac- tically all of the tricalcium phosphate goes into solution in 2 days after the crucial point is reached.

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44 Oxidation of Sulfur

The course of conversion of insoluble phosphates in composts of rock phosphate and sulfur has been taken up by Joffe (1922).

Soluble Sulfates inlOOCC M&ofSO+

150

PH 200 2.4

120 160

3.4

5 10 Incubation period

FIG. 1. Change in reaction and increase of soluble sulfates and phos- phates by pure culture of Thiobacillus thiooxidans.

In this case the reactions are not so apparent, since the indefinite chemical make-up of the raw phosphates introduces a great num- ber of side reactions.

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S. A. Waksman and J. S. Joffe

SUMMARY.

1. The curve of sulfur oxidation both in the soil and in solu- tion by pure and impure cultures of Thiobacillus thiooxiclans is a growth curve.

2. The mechanism of sulfur oxidation to sulfuric acid by Thio- bacillus thiooxidans obeys the laws of inorganic catalysis.

3. The transformation of insoluble rock phosphate to soluble phosphates by the sulfuric acid formed from the oxidation of sulfur by Thiobacillus thiooxidans is similar to the process taking place in inorganic reactions.

BIBLIOGRAPHY.

Cameron, F. K., and Bell, J. M., U. S. Dept. Agric., Bureau of Soils, Bull. 42, 1907.

Clark, W. M., and Lubs, H. A., J. Back, 1917, ii, 1. Joffe, J. S., Soil SC., 1922, xiii, in press. Kasakov, A. V., Moskau Inst. Agron., 1913, ix, 21-45, 57-65. Lipman, J. G., McLean, H. C., and Lint, C., Soil SC., 1916, ii, 499. Meyer, T., 2. angew. Chem., 1906, xviii, 1382.

Official Agricultural Chemists, J. Assn. Ogicial Agric. Chem., 1916, i, 1. Schucht, L., Die Fabrikation des Superphosphats, 1909, l-460. Stoklasa, J., Biochemischer Kreislauf des Phosphat-Ions im Boden, Jena,

1911. Waksman, S. A., and Joffe, J. S., Proc. Sot. Esp. Biol. and Med., 1920-21,

xviii, 1; Science, 1921, liii, 216; J. Back, 1922, in press.

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Selman A. Waksman and Jacob S. JoffePHOSPHATES INTO SOLUBLE FORMS

TRANSFORMATION OF INSOLUBLESULFURIC ACID AND

TOOF SULFUR BY MICROORGANISMS THE CHEMISTRY OF THE OXIDATION

1922, 50:35-45.J. Biol. Chem. 

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