promoting effect of molybdate on the growth of a …

J. Gen. App!. MicrobioL, 23,163-173 (1977)

PROMOTING EFFECT OF MOLYBDATE ON THE

GROWTH OF A SULFUR-OXIDIZING BACTERIUM,

THIOBAGILL US THIOOXIDANS'

SUSUMU TAKAKUWA, TSUNEO NISHIWAKI,2 KAYAKO HOSODA, NORIKO TOMINAGA, AND HIDEKAZU IWASAKI

Biological Institute, Faculty of Science, Nagoya University, Aichi 464

(Received March 17, 1977)

In order to analyze the growth of Thiobacillus thiooxidans in the basal Starkey's medium supplemented with solid particles of sulfur, CS, method was developed to measure the cells adhering to the sulfur particles; the cells were totally released from the sulfur particles into the liquid phase by shaking of the culture with carbon disulfide, so that the total amount of cells existing in the culture could be measured quantitatively. The growth of Thiobacillus thiooxidans was remarkably enhanced by the addition of a trace amount of heavy metal ions to the basal Starkey's medium. It was found that, at the initial phase of growth, practically all the cells were attached on the surface of sulfur particles and, with progress of the culture, an increasing number of cells appeared in the liquid

phase. At the stationary phase of growth, about equal amounts of cells were distributed on the sulfur particles and in the liquid phase. Among the trace elements added, molybdate was found to be the very

substance to accelerate the growth of Thiobacillus thiooxidans. In the

presence of molybdate (below 0.3 ppm as molybdenum) the specific growth rate and the final cell yield increased 2- and 4-fold, respectively, compared to those in its absence. This stimulative effect could not be substituted by tungstate, chromate, or vanadate.

The growth physiology of the sulfur-oxidizing bacterium, Thiobacillus thio-oxidans, which was first discovered by NATHANSOHN (1) and isolated by WAKSMAN and JOFFE (2), has been the subject of repeated studies in the past (3-10). A salient feature of this bacterium is that it thrives chemoautotrophically by oxidizing elementary sulfur to sulfuric acid, and it has been observed (4,11) that during the

1 A preliminary report of a portion of this work was presented at the Annual Meeting

of the Japanese Society of Botany, Toyama, October 6-8,1976, 2 Present address : Yamagata High School , Nakabora, Miyama-cho, Yamagata-gun,

Gifu 501, Japan.

163

164 TAKAKUWA, NISHIWAKI, HOSOnA, TOMINAGA, and IWASAKI VOL. 23

culture the sulfur granules added to the medium were surrounded by bacterial cells, a finding which was later confirmed by several investigators using modern techniques; by SCHAEFFER et al. (12), using a transmitting electron microscope, and by BALDENSPERGER et al. (13) using a scanning electron microscope. In such studies, however, the growth of this bacterium was followed only by observing the cells liberated into the medium, without giving due considerations to the cells adhering to sulfur particles. Satisfactory method for enumeration of total cell

growth of Thiobacillus thiooxidans in the medium supplemented with solid particles of sulfur has not been made yet.

In the experiments to be described here, a method of applying carbon disulfide was used for releasing the adhering cells from the particles, so that the quantitative measurement could be made of total number of cells existing in the culture. It will be shown that the growth of this bacterium is markedly accelerated specially by the addition of a trace amount of molybdate to the medium.

MATERIALS AND METHODS

Basic method of culture. The organism used was a strain of Thiobacillus thiooxidans which had been isolated in our laboratory by IWATSUKA and Morn (14). The culture was performed using Starkey's solution as the basal medium with ad-dition of an excess (about 30 g/liter) of elemental or orthorhombic sulfur. Starkey's solution contained per liter of distilled water: 3.0 g of KH2P04, 0.2 g of (NH4)2504, 0.5 g of MgSO4.7H2O, 0.3 g of CaC12.2H2O, 0.01 g of FeSO4.7H2O, pH 4.5. The sulfur particles added were previously purified by washing with 1 N NaOH and 1 N HCl repeatedly, followed by water and acetone, and then dried in air at room temperature. It was filtered through a sieve of 0.14 mm mesh. The com-

position of agar slant used was as follows (14) : 3.0 g of KH2P04, 0.1 g of NH4C1, 0.25 g of CaCl2, 0.1 g of MgC12, 5.0 g of Na2S203.5H20, 0.01 g of FeS04.7H20, and 20.0 g of agar per liter of distilled water. The compositions of the medium used in different experiments will be described in respective cases. Series A experiments. The culture solutions were dispensed in 150 ml quan-tities in petri dishes, about 20 cm in diameter, and after inoculation of the bacterium the culture was incubated at 30°. For the inoculation, the bacterium was grown at 30° using Starkey's medium modified by KoDAMA and MORI (10), the details of which will be given later. On the 4th day of culture the cells were harvested and after washing 3 times with distilled water a definite amount (corresponding to 9.6 mg dry weight of cells per liter of the culture) was used as the inoculum. Series B experiments. In this series of experiments potassium phosphate solution was treated with H2S according to the method of NICHOLAS (15) to remove the contaminated heavy metals. The pH of the treated solution, after complete removal of H2S, was adjusted to 4.5 with conc.KOH solution. All other mineral

1977 Effect of Molybdate on T. thiooxidans 165

salts used for cultivation were the highest purity available from commercial sources. The culture medium containing 0.45 g sulfur powder was used in 15-ml

portions in the usual size of petri dish (about 8.5 cm in diameter) and incubated at 30°. To avoid the effect of molybdate present in excess in the bacterium, the cells for inoculation were precultured for 2 or 3 generations in the basal Starkey's medium, in which 1 ml of EDTA-Fe504 complex (1.0 g of FeSO4.7H2O and 1.34 g of Nat-EDTA per 100 ml) per liter medium was used in place of FeSO4 7H20, and then washed 3 times with distilled water before inoculation.

Measurement of growth. At appropriate intervals during the culture, the amount of bacterial cells present in the liquid medium as well as those attached to the sulfur particles was measured. The sulfur particles loaded with bacterial cells were separated from the liquid medium by filtration of the culture through Toyo filter paper No. 5A. To estimate the cells firmly adhering to sulfur particles, the whole culture (for example, 150 ml in series A experiments) was poured into a conical flask and, after addition of a sufficient amount of CS2 (usually 15 ml was enough to dissolve completely the sulfur particles contained in 150 ml of culture), the mixture was stirred for a few minutes with a magnetic stirrer at room temperature. By this treatment the bacterial cells that had been attached to the sulfur particles were completely released in the culture medium, while sulfur was transferred to the CS2 layer.

The quantity of bacterial calls in th culture medium was determined by measuring the optical density of the medium using a Klett-type colorimeter (Ito Chotanpa Co., Tokyo) fitted with a red filter (660 nm) in series A experiments. The optical density of the medium was not affected by the presence of CS2. In our experiments it was found that the optical density of 50 Klett units was equivalent to 0.4 mg dry weight of cells per ml. Based on this relationship the quantity of bacterial cells in the culture was expressed in terms of dry weight of cells per dish

(mg/ 150 ml medium). In the B series experiments, absorbance of cell suspension in the medium was measured at 660 nm, using the Hitachi-Perkin Elmer spectro-

photometer (Type 139) equipped with a 1-cm light path cuvette. All the culture experiments to be described below were performed in duplicate.

The figures indicating the quantities of bacterial cells to be given below are the average of the values obtained in two parallel experiments.

In this series of experiments the quantity of protein present in the culture was measured in parallel by the measurement of optical density as described above. The method used was that of LowRY et al. (16), with bovine serum albumin as the standard. Since CS2 was found to disturb the method markedly, the cells which had been in contact with the chemical were thoro ghly freed from it by repeated washing with distilled water. The results of protein measurement showed a good

parallelism with that obtained by the optical method, indicating reliability of the optical method for determination of the amount of bacterial cells in the culture.

Check of contamination. Since the cultures of bacterium in our studies were

166 TAKAKUWA, NISHIWAKI, HOSOAA, TOMINAGA, and IWASAKI VOL. 23

all performed under non-aseptic conditions, contamination of cultures by other microorganisms had to be checked. This was carried out not only by microscopic observations but also by culture tests using an organic culture medium composed of meat extract (0.5 %), yeast extract powder (0.1 %), Polypepton (0.75 %), sodium acetate (0.5 %), and NaC1(0.05 %), with the pH adjusted to 7.0 with KOH. The agar slant made of this medium was smeared with a loopful of the culture to be tested and inoculated for 2 days at 30° to detect any contamination. In some cases, a portion of the culture to be tested was taken out and the cells (6.8 mg in dry weight) were suspended in a solution of 50 mM phosphate buffer (pH 7.0) or citrate buffer (pH 4.5) containing 20 mM sodium lactate, sodium succinate, or sodium malate, and occurrence of aerobic oxidation of organic substances was tested by using the conventional manometric technique at 30°. The tests were made with a number of cultures run under various conditions, but in no case contaminations of het : rotrophic bacteria could be detected by these methods.

Measurement of pH. The pH-values of the cultures were measured using a Hitachi-Horiba pH-meter (Type M-7), after removing the sulfur particles by filtration through Toyo filter paper No. 5A.

RESULTS

Series A experiments (I) Effect of addition of various micro-elements to the Starkey's medium.

In growing photosynthetic microorganisms (such as Chlorella and Scenedesmus) under photo-autotrophic conditions, the culture medium of classical prescriptions, such as Knop's solution, is usually supplemented with a solution containing various elements which are required in minute quantities for the growth of the organisms. To test the effect of addition of such substances, a solution of the following composition was prepared; Dissolved in 1 liter of distilled water; 0.25 g of ZnCl2, 2.10 g of MnSO4.4H2O, 0.20 g of CuCl2 2H2O, 0.20 g of CoC12.6H2O, 2.80 g of H3B03 and 0.75 g of Na2MoO4 2H2O. One milliliter of this solution, which will be called the solution of trace elements in the following, was added to 1 liter of original Starkey's medium and growth of the bacterium in the presence and absence of the trace elements was compared. The results obtained are presented in Fig. 1.

As is apparent from this graph, the addition of trace elements to Starkey's medium caused a considerable acceleration of growth rate and a remarkable en-hancement of the final stationary level of population density.

(II) Effect of addition of molybdate to Starkey's medium. To clarify the substance(s) responsible for the marked growth-promoting effect of trace elements, the effect of each salt was examined (data not presented here), and it was clearly revealed that molybdate was the very substance that was responsible for the effect in question.


In the three sets of culture, the specific

population density attained were as follows:

Addition

None

Na2Mo04.2H2O

K2Mo04

Concn. (mg/liter)

0.75

0.75

growth rates

Specific growth rates (hr-1)

0

0

0

011

038

032

and the maximum

Final population density (mg dry weight/liter)

83 340 330

It should be noted that the cells cultivated in the molybdate-containing media

(0.3 ppm as molybdenum) were used as the inoculum, and the stimulative effect of molybdate could not be observed, indicating that the inoculated cells carried a

quantity of molybdate that was large enough for accelerating the subsequent pro-liferation of the cells in the culture. The development of colonies in the presence of molybdate (0.03 ppm as molybdenum) was also remarkable compared with that in its absence in the slant medium. With addition of 0.03 ppm molybdenum in the solid medium, large colonies with brown-yellow color in the center were observed within a few days of incubation at 30°, although, in the original com-

position without addition of molybdate, very tiny colorless colonies appeared on the surface of the slant after about 1 week of incubation as described by IWATSUKA and M0R1(14).

(III) Apparent growth promoting effect of barium carbonate applied to the

Fig. 1. Effect of trace elements in Starkey's medium on the growth of the bacterium.

growth in the presence of trace elements; free, cells suspended in the liquid phase; bound, cells adhering to the sulfur particles; total, total amount of cells in the culture. ------, total amount of cells in the culture without addition of trace elements .


Starkey's medium. In their study on the physiology of Thiobacillus thiooxidans, KODAMA and Moil (10) used the following method with the purpose of promoting the bacterial growth by supplying BaC03 which is not contained in the Starkey's medium. BaC03 powder was placed in a small cellophane bag, and two such bags were placed in the medium (ca. 10 g of BaC03/300 ml medium). This culture method was used in several studies by MORI and his co-workers (17 -20) . It was thought that H2SO4 produced during the culture reacted with BaC03 to give rise to C02, and that this stimulated the growth by serving as the carbon source necessary for the chemoautotrophic growth of the bacterium.

In the present study, re-examination of the experimental results reported by MORI and his co-workers was made using two kinds of BaC03 preparations ob-tained from different manufacturers. For the sake of convenience, these two pre-

parations will be referred to as BC1 and BC2 in the following.3 The experiments followed the methods devised by KODAMA and MORI (10), using cellophane bags as the container of BaC03 (10 g of BaC03/150 ml of culture). Addition of BaC03 to Starkey's medium had more or less a marked growth-promot-ing effect, the effect of BCl being much stronger than that of BC2. In another experiment not described here, it was observed that, when the quantity of BC2 applied was increased 3 times, the growth was promoted to approximately the same degree as it with BC1. The decrease of pH which took place during the culture by the addition of BaC03 was faster than that in its absence. Figure 2 shows the results of experiments in which the bacterium was grown in Starkey's medium to which BC2 was added together with molybdate, and the result was compared with those obtained by the addition of either one of the two carbonate preparations. As may be seen, the addition of molybdate together with BC2 enhanced the bacterial growth to almost the same level as that obtained with BC1. This fact indicates that BC1 contained some element which was lacking or not present in a sufficient amount in BC2. When each salt in trace elements, except molybdate, was supplied together with BC2, no enhancement of the growth was observed. Based on the results obtained in the preceding experiments, it may not be unreasonable to infer that the growth-promoting substance contained in BC1 would most probably be molybdate.

Series B experiments

(I) Effect of molybdate concentration on the growth. To evaluate what con-centration of molybdate is required for the growth, effect of molybdate concen-tration on the growth was examined as shown in Fig. 3. It was found that a very minute amount of molybdate, that is, only 1 ppb molybdenum (2.5,ug sodium molyb-date per liter), was sufficient to sustain the maximum growth and its quantity in 300 ppb was slightly suppressive to the growth. Relatively good growth appearing

3 BC1 (Nakarai Chemicals , Ltd., Osaka) and BC2 (E. Merk, Darmstadt) were labeled as of extra pure grade and guaranteed grade, respectively.


in the control seemed to be due to the molybdate contaminated in other mineral salts, except for potassium phosphate. The rate of pH decrease in the medium supplied with molybdate was more rapid than that of the control.

(II) Effect of tungstate, chromate, and vanadate on the growth. As shown in Fig. 4A, the promoting effect of molybdate on the growth could be neither substituted nor inhibited by the addition of tungstate into the medium. The

rig. ~. rig. J.

Fig. 2. Comparison of bacterial growth in Starkey's medium supplied with differ-ent preparations of barium carbonate in the presence and absence of molybdate (0.3 ppm molybdenum). Growth curves show the total amount of the bacterium in the culture. a (0-0), BC2 added with molybdate; b (0-0), BCl added without molybdate; c (•-•), BC2 added without molybdate.

Fig. 3. Effect of molybdate concentration on the growth of the bacterium. Total amount of bacterial cells in the cultures is shown in the lower part of the graph. To the original Starkey's medium were added various amounts of molybdate: 0, none; •, 1 ppb of molybdenum; x, 30 ppb of molybdenum; o, 300 ppb of molybdenum. The upper part of the graph shows the time course of pH change during the culture.

pH change during the culture with supply of various amounts of molybdenum; ------, pH change of the control.


decline of pH in the medium supplied with tungstate was identical with that

control. Neither chromate nor vanadate exhibited the promoting effect on

growth as shown in Fig. 4B.

of

the

DISCUSSION

Among a number of organic solvents which is able to more or less dissolve sulfur, such as acetone, benzene, CS2, CCl4, and some oils, CS2 was selected for its high solubility of sulfur (100 g of CS2 dissolves 41.8 g of elementary sulfur at 20°) (21) and for its low solubility in water. From the following reasons this method using carbon disulfide has been revealed to be valid and reliable for the estimation of true cell population of Thiobacillus thiooxidans in the sulfur-supple-

Fig. 4. Effect of tungstate, chromate, and vanadate on the growth of the bacterium. Curves in the lower part show the total amount of bacterial cells in the cultures. The pH change during the culture is shown in the upper part of the graph. A. Lower graph: To the basal Starkey's medium was added 30 ppb of one of the following metals: 0, none; x, Na2WO42H2O (0.91 mg per liter); •, Na,MoO4 2H2O

(0.75 mg per liter); o, tungstate plus molybdate. Upper graph: , pH change in the presence of molybdate or molybdate plus tungs-tate in the culture medium; ------, pH change in the presence of tungstate or absence of these heavy metals in the culture medium. B. Lower graph: To the basal Starkey's medium was added: 0, none; ~, Na2VO3

(0.72 mg per liter) or K2CrO4 (0.75 mg per liter); •, Na2MoO4 2H2O (0.75 mg per liter). Upper graph : , time course of pH change in the presence of molybdate in the cul-ture; ------, time course of pH change in the presence of vanadate or chromate or absence of these heavy metals in the culture.


mented Starkey's medium. First, CS2 treatment had practically no effect on the optical density of the cell suspension at 660 nm. Second, the dry weight of cells calculated from the optical density of the cell suspension obtained from culture medium with CS2 treatment is consistent with that calculated from the

protein content measured by Lowry's method. Both sulfur- and sulfite-oxidizing activities of intact cells were completely abolished by CS2 treatment but, against expectations, the mobility and form of the cell, when observed under a microscope, were not changed at all. However, there are some evidences that, besides other substances, a portion of the proteins

probably present in the cell envelope became solubilized in water by CS2 treatment, as detected by polyacrylamide gel disc electrophoresis.

The sulfur particles removed by filtration from the culture medium after 4 days of incubation were rinsed thoroughly with distilled water on the filter paper till the filtrate became clear and then the washed sulfur particles were inoculated in the fresh medium without additional inocula. Within a few days of incubation at 30° in still culture, growth occurred as observed by COOK (8). These results also clearly indicate the presence of a relatively firm and direct contact between the bacterium and sulfur particles.

As presented in Fig. 1, at the initial phase of growth, the cells proliferate apparently on the surface of the sulfur particles. This result supports the view that the uptake of sulfur by T, thiooxidans may take place by the direct contact between sulfur and bacterial cells, as suggested by UMBREIT et al. (4,12). In fact, there was no production of oxidizable sulfur oxides in the medium, because oxygen uptake was not found by the ordinary warburg manometer when the culture medium freed from sulfur particles after several days' culture was used as a sub-strate, so that the cells must be unable to grow in a free state. Present study clearly demonstrated that the trace amount of molybdate is specially required for the proliferation of this chemolithotroph. From the results shown in Fig. 2, it is concluded that the apparent growth-stimulating effect of BaCO3 reported early (10) is mainly due to molybdate contaminated in the BaCO3 used.

Among some micro-nutrient essential for the growth of microorganisms, molybdate is distinguished as the constituent of two enzymes involved in the nitrogen metabolism, nitrate reductase (22, 23) and nitrogenase (24, 25). There are many reports that molybdate requirement for the growth of N2-fixing bacteria can be replaced with tungstate or vanadate, though nitrogenase activity is lower compared with that in the cell grown normally in the presence of molybdate (26-28). Substitution of tungsten for molybdenum in nitrate reductase from different sources always led to inactive enzymes (29, 30). However, these enzymes are apparently not concerned with the present case because this bacterium uses only ammonia as a nitrogen source and could not proliferate without ammonia in the medium, although a brief observation on the activity of acetylene reduction by some strains

172 TAKAKUWA, NISHIWAK[, HOSODA, TOMINAGA, and IWASAKI VOL. 23

of Thiobacillus ferrooxidans sp. has been reported (31). Mo as a cofactor of enzymes is also known to exist in hepatic sulfite oxidase (32), xanthine oxidase (33), aldehyde oxidase (34), and in formate dehydrogenase in Escherichia toll (35, 36). The physi-ological and biochemical mechanisms involved in Mo-Cu-S interaction have been the subject of intensive study in animal nutrition (37). Moreover, there is an interesting interaction between Mo and Fe in tomato plants (38); addition of an adequate Mo enhances absorption and translocation of Fe and also decreases the availability of iron compounds in the root media. The role of molybdenum on the growth of this chemolithotroph, however, must await future investigations.

One of the authors (ST.) was granted a postdoctorate fellowship from Japan Society for the Promotion of Sciences.

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