superimposed layers of aluminum and silicon, bound together

Transcription

1 PHYSICO-CHEMICAL BEHAVIOR OF SOIL BACTERIA IN RELATION TO THE SOIL COLLOID' T. M. McCALLA Kansas Agricultural Experiment Station Received for publication October 12, 1939 The natural environment of soil bacteria is highly complex from a nutritional standpoint. The colloidal clay fraction is the most active constituent of the soil in controlling a balance of the mineral nutrients, since it is this portion of the soil which adsorbs the various essential ions (Albrecht and McCalla, 1938). The colloidal clay is a hydrated aluminum silicate composed of superimposed layers of aluminum and silicon, bound together through oxygen bridges (Ford, Loomis, and Fidion, 1940). When the sheets of the clay crystal become hydrated, they expand and the clouds of adsorbed ions vibrate around and between these sheets. The nature and the interpenetration of the adsorbed ion have a marked influence on the character of the colloidal clay. Since bacteria are in the upper range of colloidal limits, it seems perfectly logical to expect them to assume some of the physical characteristics of colloids. Furthermore, in a previous paper (McCalla, 1940) it has been shown that they do behave as colloids in so far as cation adsorption is concerned. McGeorge, 1932, also stated from his work on the adsorption of minerals by alfalfa that the adsorbed mono and di-valent ions were exchanged in stoichiometric proportions. Hence, in a study of the influence of the natural environment on the soil bacteria the relationship between these two colloidal substances-one a living organized entity and the other an inorganic colloid-should be considered. It is the purpose of this paper to show that the mineral needs of Contribution No. 186, Department of Bacteriology. 33

2 34 T. M. MCCALLA some common soil bacteria are probably satisfied by either solution or contact exchange of adsorbed ions between the bacterial cell and soil colloid, the degree of exchange of adsorbed ions being governed by the relative and differential adsorbability of the essential ions by these two colloidal systems. It is a well known fact that ions vary considerably in their adsorbability by colloids (Docking and Heymann, 1939). In a colloidal system containing two ions the play of forces determining which of the two ions will occupy the adsorption sites of the colloid is represented by the following equation, (Jenny, 1936). W= [S+Nw+Nb(-1)] 4 [S+Nw+Nb (1 -V) -4SNw 1 YV 2-1V-) Where W = number of electrolyte ion w particles adsorbed or electrolyte ion b released at equilibrium. S = saturation capacity. VW, Vb = oscillating spaces of the adsorbed ions. NW = number of w particles added initially (electrolyte ion w). Nb = number of b particles added initially (electrolyte ion b). As other ions are added to the system the relationships become increasingly more complex. If, as in the case of a living bacterial cell, the adsorbed ion was removed by growth, it would be necessary for a new set of reactions to occur until equilibrium was again established. Knowledge of the laws governing the interrelationship of the ions in the bacterial cell and soil colloid should furnish a logical basis for evaluating bacterial utilization of mineral nutrients in the soil. Without the means of some force for attracting the ions adsorbed by the soil colloid, it seems probable that a bacterial cell would be unable to grow. If, however, bacteria do behave as

3 PHYSICO-CHEMICAL BEHAVIOR OF SOIL BACTERIA colloids and exhibit base adsorption phenomena, it would be expected that when the soil bacteria grow in the soil, they can then compete with the soil colloid for the solution ions. If there were no excess of ions in solution, then the colloid having the greatest amount of adsorbed ions would be expected to yield these to the other until an equilibrium was established. Normally, the flow of ions would be to the bacterial cell; but, conceivably, in a soil deficient in bases the process might be reversed. In such a system not only would growth not be expected to occur but death would ensue. If the bacterial cell utilized these adsorbed ions in growth and they became an intimate part of the cell through the growth process, this would remove them from the adsorption sphere of the bacteria and there would be a further shift of ions from the soil colloid to the bacterial cell. Assuming the presence of sufficient amounts of all other nutrients and proper elimination of waste products, growth might be expected to proceed until there was an ultimate equilibrium in ions between the two systems. EXPERIMENTAL The influence of a colloidal clay complex upon soil bacteria was studied experimentally as follows. The base adsorption capacity of Putnam colloidal clay2 was measured by converting the clay colloid with different adsorbed bases into an H-clay by dialyzing under an electrical potential of 110 volts and then titrating the H-clay with a base. The adsorption capacity was 67 m.e. (milligram equivalents) per 100 grams of clay. The method of measuring the cation adsorption capacity of bacteria has been given in a previous paper (McCalla, 1940). The Ca(OH)2 titration curves of H-legume bacteria and H-clay are presented in figure 1. Some of the common soil bacteria-bacterium globiforme, Corynebacterium simplex, Bacillus elegars, Bacillus alpinus,s Rhizobium meliloti, Azotobacter chroococcum-were used 2 The Putnam clay colloid was supplied through the courtesy of W. A. Albrecht, Chairman, Department of Soils, University of Missouri, Columbia, Missouri. ' The first four cultures named were furnished through the kindness of Francis E. Clark, Associate Bacteriologist, Bureau of Plant Industry, U. S. Department of Agriculture. 35

4 36 T. M. McCALLA to determine whether soil bacteria would adsorb cations. Gibson, 1939, has emphasized the numerical importance of the Corynebacterium complex and B. globiforme in the soil. Conn et al, 1928, 1940, have frequently used B. globiforme as a test organism in soil microbiology studies. FIG. 1. PR n.e. of base per 100 gms. Bacteria CALCIUM HYDROXIDE CURVES FOR COLLOIDAL CLAY AND LEume BACTERIA Hydrogen and methylene blue adsorption The hydrogen adsorption capacity of the soil bacteria was determined by growing them on nutrient agar in large Blake bottles for 2 to 4 days at room temperature, suspending the cells in distilled water, thoroughly washing, and then converting them into H-bacteria by adding enough 0.01 N HCl to saturate the displacing capacity of the bacterial system. The adsorbed H+ was measured by titrating the washed H-bacteria with Ca(OH)2. In order to determine the viability of the H-bacteria,

5 PHYSICO-CHEMICAL BEHAVIOR OF SOIL BACTERIA plating in quadruplicate was made with untreated and H-bacteria. Almost complete viability of the H-bacteria was evident. Adsorption of methylene blue by the soil bacteria was determined by adding a known concentration of methylene blue4 solution of 88 per cent purity, medicinal, to a washed bacterial suspension, throwing the cells out of suspension by means of an angle centrifuge and measuring the unadsorbed methylene blue by colorimetric method. The adsorption value was expressed as m.e. per 100 grams of bacteria dried at 105'C. The adsorp- TABLE 1 The adsorption of hydrogen and methylene blue by 8oil bacteria M.E. ADSORPTION PER 100 GRAMS. ~~~~~PRRBCEINT blue Hydrogen BACTZRIA ORGANISM TRIALS VIA3LR OF H- 1 Bacterium globiforme.{ Corynebacterium simplex I~ Bacillus Baclls eegm elegant at Bacillus alpinus Azotobacter chroococcum tion value of H+ was similar to that obtained with methylene blue as indicated in table 1. In all instances there was some adsorption of H+ or methylene blue+. The replacement of adsorbed methylene blue Unth different metallic ions If bacteria adsorb cations, then a relative adsorbability of different ions varying as their adsorption energy should be evident. In order to get a comparative view of the energy of 4Certified by commission on standardization of biological stains. 37

6 38 T. M. McCALLA adsorption of the different essential ions, determinations were made to see how much of some single adsorbed ion would be removed when equivalent ion concentrations of the various minerals were added. Methylene blue was used as the adsorbed ion because its quantitative removal may be measured colorimetrically. Gieseking and Jenny, 1936, showed that methylene blue could be adsorbed by a soil colloid and then replaced with some ion. Methylene blue was added at the rate of 20 m.e. per 100 grams to washed cells of B. globiforme grown on calcium gluconate agar (Albrecht and McCalla, 1937a). The cells were TABLE 2 Replacement of adsorbed methylene blue with different ions IONIC BADIS PER CENT MUTHYLUND BLUM REPLACED ION OF CATION A' - UNIT Na NH K Mg Ca Ba Mn Al... o Fe H * Data from Baver and Hall, Emelaus and Anderson, washed to remove the unadsorbed methylene blue. To determine the amount of adsorbed methylene blue a given ion would replace, the exchanging ion was added in symmetry concentration (1S)5 and the amount of methylene blue released from the bacterial cell was measured by comparing with a standard in a colorimeter. The symmetry concentrations of the different ions used were 0.5S, is and 5S. The results are listed as symmetry values. For example, if one-half of the adsorbed methylene blue is displaced by an ion, the symmetry value is recorded as c Symmetry concentration is obtained when the number of replacing ions added is equal to the number of adsorbed ions.

7 PHYSICO-CHEMICAL BEHAVIOR OF SOIL BACTERIA per cent. The displacing ions used were Na+, K+, NH4+, Ca++, Ba++, Mg++, Mn++, Fe+++, Al+++, and H+. All were prepared as 0.01 N solutions as chlorides except Al, Mn and Mg, which were prepared in the form of sulfates, and added to a suspension of B. globiforme. A typical set of data given in table 2 place the adsorption series as follows in replacing power: Na <NH4 <K <Mg <Ca <Ba <Mn <Al <Fe <H. This experiment was repeated using different organisms. The replacement value, however, varied with the type and age of organism and kind of media upon which the culture was grown. The adsorp- TABLE 3 The relative growth of soil bacteria in solution and colloidal media VUAIA(MEANOF DACTURA IN MILLONS PER ML. ORGAN11M TRIATM) 01 Calcium gluconate Colloidlal clay ,980 Rhizobium meliloti ,250 Mean 387 3,115 Bacterium globiforme Mean Corynebacterium simplex ,433 Bacillus alpinus Mean 9 72 tion series, nevertheless, retained the same general sequence as might be expected from the charge and size of the ion. Biochemical relation of soil bacteria to soil colloid The effect of the colloidal clay upon the growth of the soil bacteria was measured quantitatively by growing the organisms in an ordinary calcium gluconate medium and in this same medium plus H-clay colloid in 2.5 per cent concentration. The acidity of the calcium gluconate medium, caused by the addition of the H-clay, was neutralized by the addition of equal quantities

8 40 T. M. MOCALLA of KOH and Ca(OH)2. The composition of the calcium gluconate medium was as follows: Na as NaCl 3.34 m.e.; Mg as MgSO m.e.; K 2.66 m.e., and P 4.00 m.e. as K2HPO4 calcium gluconate 1.5 gram; sucrose 10 grams; NH4NOs 0.1 gram; and distilled water to make 1000 ml. Each trial carried out consisted of triplicate tubes containing 25 ml. of the medium inoculated with a measured suspension of the actively growing soil organism used. In order to make the conditions in such media as favorable as possible, a current of sterile air was forced through the cultures for three to four days after which quantitative plate counts were made. An example of the data collected in this manner is presented in table 3. From these data it is evident that the clay-containing medium was very much more favorable for growth than the ordinary mineral liquid medium. Attention has already been called to the influence of a colloidal clay complex upon various biochemical phenomena (Albrecht and McCalla, 1937b; McCalla, 1937; McCalla, 1939; Conn and Conn, 1940). DISCUSSION The fact that the various soil bacteria made better growth in the presence of colloidal clay indicates that there is a marked difference in the effect of the two media. Conn and Conn, 1940, also have demonstrated the value of colloidal clays as growth promoters. The addition of the clay, apparently inert as a nutritive material, has in some way stimulated the growth of the soil bacteria. Broughton, 1940, verified previous findings that clays were of value as commercial catalysts. Since the addition of the colloidal clay to the mineral medium increased bacterial growth, it would not seem impossible that the clay functioned catalytically in speeding up biochemical reactions, either by providing for a more efficient utilization of nutritive material or by decreasing toxic effects of waste products by adsorbing them. In the soil the colloidal material intermingled with coarser minerals forms definite structures. Some soil scientists suggest that soil structure is a result of an edge-to-edge contact of the colloidal platelets. Under these conditions, if a bacterial cell

9 PHYSICO-CHEMICAL BEHAVIOR OF SOIL BACTERIA were located at one side of the soil colloidal structure, the ions adsorbed at different positions on the colloidal particles could migrate on the colloidal surfaces (Jenny and Overstreet, 1939) to the cell as they were utilized. In ordinary laboratory media used to cultivate bacteria, water is the major constituent and the nutritive material is about 1 to 5 per cent; but under normal conditions in the soil the solid material constitutes about 80 per cent whereas the water is present in about 20 per cent concentration around and between the particles of the soil. Under Hg mg H H E H CaH Ca H CH 7 Ca FIG. 2. K bacterium Ca Ca H H K H EH mlg Ca m x ~~~~~~~~Ca ay + bacterium H H Ca R H H H MECHANISM OF ION ExCHANGs BETWEEN SOIL BACTERIA AND COLLOIDAL CLAY these circumstances the bacteria probably live in the water films which adhere to the surface of the colloid containing the adsorbed ions. The bacterial cell is undoubtedly in close proximity to the soil particles and assuming that the bacteria may adsorb ions and hold some of them in the outer surface of the cell, this would permit contact exchange of adsorbed ions. Ions with large oscillating volumes would overlap, and exchange between systems could readily take place. Other ions which are strongly adsorbed would not be expected to wander far from the surface 41

10 42 T. M. McCALLA of the colloid. On the basis of the displacement of adsorbed methylene blue the ions would be expected to be adsorbed by the bacteria from the soil colloids in the following series: H>Al> Fe >Mn >Ba >Ca >Mg >K >NH4 >Na. From the foregoing facts and theoretical considerations it is suggested that in the adsorption of nutrients from the soil by the bacteria, and possibly by living cells in general, an exchange of adsorbed bases takes place between the bacteria and soil colloid as depicted in figure 2. In bacterial metabolism large amounts of carbon dioxide and water are formed. In the presence of H20 H-ions are produced from the carbon dioxide which may be adsorbed at the cell's surface. When a colloidal clay particle, saturated with adsorbed bases contacts a bacterium saturated with H-ions, an exchange of ions takes place until an equilibrium is reached. As this equilibrium is upset by the more complete utilization of the adsorbed basic ions in bacterial metabolism a further exchange may take place, the colloidal clay functioning as a constant reservoir for basic ions utilized in the growth of such organisms. CONCLUSIONS 1. Soil bacteria adsorb cations. 2. Adsorbed methylene blue may be replaced by other cations. The degree of adsorption of the replacing ion determines how much methylene blue is replaced. 3. The order of the adsorption series is as follows: Na <NH4 < K <Mg <Ca <Ba <Mn <Al <Fe <H. 4. Colloidal clay stimulates the growth of soil bacteria. 5. It is proposed that soil bacteria obtain their mineral needs by contact exchange of adsorbed ions between the bacterium and clay particles. 6. A series of equilibrium reactions occur between soil colloid and bacteria depending upon relative adsorbability of the ions by these two systems. REFERENCES ALBRECET, W. A., AND MCCALLA, T. M. 1937a A new culture medium for Rhizobia. J. Bact., 34,

11 PHYSICO-CHEMICAL BEHAVIOR OR 9O!L BACTERIA 43 ALBRECHT, W. A., AND MCCALLA, T. M. 1937b Nitrification of ammonia adsorbed on colloidal clay. Proc. Soil Sci. Soc. Am., 2, ALBRECHT, W. A., AND MCCALLA, T. M Colloidal clay as a cultural medium. Am. J. Botany 25, BAyrER, L. D., AND HALL, N. S Colloidal properties of soil organic matter. Missouri Agr. Expt. Sta. Research Bull., 267, BROUGHTON, G Catalysis by metallized bentonites. J. Phys. Chem., 44, CONN, H. J A type of bacteria abundant in productive soils, but apparently lacking in certain soils of low productivity. N. Y. Agr. Expt. Sta. Tech. Bull., 138, CONN, H. J., AND CONN, J. E The stimulative effect of colloids upon the growth of certain bacteria. J. Bact., 39, DOCKING, A. R., AND HEYMANN, E Studies on the lyotropic series. II. The adsorption of salts on gelatin. J. Phys. Chem., 43, EMELEIBU, H. J., AND ANDERSON, J. S Modern Aspects of Inorganic Chemistry. D. Van Nostrand Company, Inc., New York, New York. FORD, T. F., LooMIs, A. G., AND FIDION, J. F The colloidal behavior of clays as related to their crystal structures. J. Phys. Chem., 44, GIBSON, T Investigations of the bacterial flora characteristics of soils. Intern. Congr. Microbiol. Abst. Commun., 1, GIESEKING, J. E., AND JENNY, H Behavior of polyvalent cations in base exchange. Soil Sci., 42, JENNY, H Simple kinetic theory of ionic exchange. I. Ions of equal valency. J. Phys. Chem., 40, JENNY, H., AND OVERSTREET, R Surface migration of ions and contact exchange. J. Phys. Chem., 48, MCCALLA, T. M Behavior of legume bacteria in relation to exchangeable calcium and hydrogen ion concentration of the colloidal fraction of the soil. Missouri Agr. Expt. Sta. Research Bull., 256, MCCALLA, T. M The adsorbed ions of colloidal clay as a factor in nitrogen fixation by Azotobacter. Soil Sci., 48, MCCALLA, T. M Cation adsorption by bacteria. J. Bact., 40, McGEORGE, W. T Chemical equivalent base exchange reactions in plants. Plant Physiol., 7,