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Part II, continued

  Soil is known to adsorb various substances. There are a number of forms of soil adsorption: mechanical, physical, chemical, biological, and physicochemical.

  Mechanical adsorption. The soil, as any other porous body, retains particles present in the filterable liquid. In other words the soil acts as a filter. Ordinarily, the size of retained particles exceeds the size of the soil pores but even smaller particles can be retained.

  Physical adsorption is linked to the phenomenon of surface tension and is manifested by the fact that increase or decrease in the molecular concentration of compounds in the solution takes place an the surface of particles.

  Physicochemical or exchange adsorption--consists in cation exchange. The cations from the solid phase of the soil are being exchanged for an equivalent quantity of cations present in the surrounding soil solution.

  Chemical adsoerption expresses itself in adsorption of certain ions from the soil solution, which form insoluble salts in soil. Consequently, a precipitate is formed which enters into the solid phase. Thus, for example, the ion of phosphoric acid precipitates in the presence of calcium salt (carbonic, hydrofluoric. or sulfuric acid). An insoluble salt of tricalcium phosphate is obtained. The latter precipitates and enters into the composition of the solid phase of the soil.

  Biological adsorption according to Gedrolts is characterized by the adsorption of compounds from the soil solution, by microbial cells and green plants.

  The adsorption of microbial cells by the soil also belongs here.

  According to Gedroits, the physicochemical adsorption is of the greatest importance. In his opinion it is conditioned by the soil-adsorbing complex, consisting of chemical substances capable of exchange reactions. These substances may be organic and inorganic compounds or colloids undissolved in the soil solution. The latter represent the smallest particles, in size less than 1µ more often from 1-100m µ which do not precipitate in water and pass through fine filters. They are only visible in the ultramicroscope.

  Organic compounds of the soil such as the humic acids, organomineral and inorganic compounds such as aluminum-silicates, iron hydroxide, argillaceous minerals and others may exist in the colloidal state. They represent a finely dispersed system, the particles of which possess high surface-reaction capacity for adsorbing substances present in solution. The soil colloids can be divided into hydrophiles and hydrophobes. The first adsorb water molecules and hydrated ions of the soil solution on their surface. The latter do not absorb molecules of the liquid phase of the solution.

  The soil colloids adsorb cations. This adsorption is an exchange process, since with the adsorption of these cations, other cations are being released in equivalent quantity.

  The sum of all adsorbed or exchanged cations which can be eliminated from the soil is a constant value for a given soil (Gedroits). It varies only with the acquisition of new properties by the soil and with the change of its essential nature.

  The sum of adsorbed bases comprises the volume capacity of adsorption. It in expressed in milliequivalents per 100g of soil. The adsorption capacity varies in different soils. It is smallest in podsol soils and largest in chernozems. The former is conditioned essentially by a mineral adsorbing complex and the latter by the organomineral part of the soil.

  Soils saturated with bases (chernozem, etc), contain magnesium and calcium in the adsorbing complex. Saline soils. besides these two elements also contain sodium. There are soils which are not saturated with bases. To these belong the podsol soils which contain hydrogen.

  The adsorption capacity is conditioned by the composition, properties and degree of dispersion of the soil. The greater the number of small particles in the soil, the higher the specific adsorption surface. Soils having a large percentage of highly dispersed organic humus compounds possess higher adsorption capacity than soils poor in organic compounds. The adsorption capacity of humus is 150-250 milliequivalents and humic acid, 300 milliequivalents per 100g.

  Soils possess exchange capacity not only in regard to cations but also anions. The adsorption of anions takes place in the soil in the presence of iron and aluminum hydroxides.

  The adsorption of microbial cells by soil particles is also of great importance. This phenomenon has been inadequately studied and much of the data requires experimental verification; certain data are contradictory. Nevertheless, the little data available are of great interest.

  It has long been noted, in the laboratory practice, that bacterial cells are adsorbed by various materials in powder form. Kruger, (1889) demonstrated the adsorption of bacterial cells from their aqueous solutions by coke, clay, brickflour, magnesium oxide and other substances. Later Eisenberg (1918) showed that bacterial cells can be adsorbed by animal charcoal. Michaelis (1909) noticed that different bacterial genera are adsorbed to a varying degree.

  Bacterial adsorption by soil particles was demonstrated by Chudiakov, N. N. (1926) and his collaborators Dianova, Voroshilova (1925), Karpinskaya (1925) and others. These investigators have shown that the soil adsorbs considerable quantities of bacterial cells. According to Dianova and Voroshilova (1925), between 252 and 4,350 million bacterial cells par hectare are adsorbed depending on the kind of soil and generic peculiarities of the bacteria (Table 14).

  The loam soils of the experimental fields of the Agricultural Academy im. Timiryazev adsorb Bac. mycoides 95.5%; Bac. ellenbachensis 57.5%; Bac. mesentericus, 40.5%; Ps. fluorescens liquefaciens 79.7 %; Staph. pyogenes, 80%; Bact. prodigiosum,98%; Bact.coli, 10-20%.

  Novogrudskii (1936c) studied the adsorption of podsol soils of the experimental fields of the Agricultural Academy im. Timiryazev, the soils of the Moscow Botanical Garden and chernozem of the Voronezh Oblast'. We have compiled the result of these studies in the tables. Table 15 shows the data on bacterial adsorption and Table 16 the adsorption of fungi and actinomycetes.

  According to our data the Moldavian chernozem (medium loams, carbonate) adsorbs two to three times more cells of Azotobacter than the upper layer of the podsol soil which was previously under forest (Experimental Station of the Moscow State University, Chashnikovo, Moscow Oblast'). In the former soil, 4,000 million Az. chroococcum and 600 million Az. vinelandii were adsorbed per gram and in the latter soils 2,400 million and 200 million per gram respectively.

  The adsorption capacity of soil varies according to the depth. The upper layers of the soil are characterized by higher adsorption capacity than the lower, The poorly cultivated soil of the Experimental Station of Chasnikov adsorbed Az. chroococcum, according to the different horizons, as follows:

  a) Layer A-A₁; 80 % in May and 92 % in August
  b) Layer A2 (10-20 cm); 50% in May and 53% in August
  c) Layer B₁ (30-40 cm); 35 % in May and 25% in August
  d) Layer B₂ (50-70 cm); 60% in May and 75% in August

  Under the same conditions soil of the same type. but well cultivated, adsorbed cells of Azotobacter as follows:

  In the layer 0- 10 cm; 85 % in May and 93 % in August
  In the layer 10-20 cm; 80% in May and 83% in August
  In the layer 30-40 cm; 65% in May and 65% in August
  In the layer 50-70 cm; 78% in May and 87% in August

  The adsorption capacity of soils is closely connected with their mechanical composition. Sand contains particles 1.0 to 0.25 mm in diameter and sand dust with particles 0.25-0.05 mm in diameter, adsorb bacterial cells weakly. Dust containing particles 0.05-0.01 mm 0.01-0.005 mm and 0.005-0.0015 mm in diameter adsorbs microbial cells most actively. The slimes of rivers and lakes having particles of 0.0015 mm and less in diameter are devoid of adsorbing capacity. since the size of the particles of slimes (1-1. 5 µ and less) does not exceed that of the ordinary bacterial cell. In such an environment bacterial cells are themselves adsorbants. The greater the adsorption of bacteria, the fewer the cells found afterward in the suspension.

  The character and degree of adsorption of microbial cells by the soil is conditioned to a large extent by the qualitative features of the organisms proper, and their generic properties. The degree of adsorption depends on their metabolic state and their vital potential. Some bacterial genera are adsorbed more vigorously and in a larger quantity than others. According to some authors, many nonsporiferous bacteria are adsorbed considerably weaker, by the same adsorbent, than the sporiferous bacteria or micrococci.

  For example, podsol soil adsorbs bacterial cells as follows:
  Bac. mycoides . . . . 71%
  Bac. megatherium . . . . 61%
  Az. chroococcum . . . . 64%
  Ps. fluorescens . . . . 18%
  Bact. coli . . . . 10%
  Bact. denitrificans . . . . 36%
  Rhizob. leguminosarum . . . . 44%

  Gram-positive bacteria are adsorbed by the soil in larger quantitites than the gram -negative bacteria. Bogopol'skii (1933) gives the following data. Peat of medium decomposition adsorbs 74% of Bac. mycoides, 81% of Urobact. pasteurianum, 21-22% of Bact. coli and Ps. fluorescens. The same results were obtained by Eisenberg (1918) while studying the adsorption of bacteria by charcoal and other adsorbants. According to his data, the adsorption of gram-positive bacteria, Micr. pyogenes, Micr. candicans Sarcina lutea and others was 500 times larger than that of the gram-negative bacteria, Bact. coli, Bact. typhi, Ps. pyocyanea, Vibrio cholera and others.

  The degree of adsorption of microbial cells by the same soils depends upon the pH of the suspension from which the cells are being adsorbed. The spores of Bac. mycoides were maximally adsorbed at PH 4.5, with the increase of pH to 5.8-6.7 the percentage of spore adsorption decreases. When the pH of the medium is raised to the neutral (pH 7.0), and higher into the alkali zone (pH 7.8), the degree of bacterial adsorption remains on the same level or even decreases slightly (Table 17).

  The adsorption of Bact. coli under the same conditions is different. The highest adsorption takes place at a pH of 6.3-7.5; in an acid or strongly alkaline medium the cells are adsorbed much more weakly.

  The adsorption capacity of the soil varies in relation to its moisture. Very wet soil adsorbs less bacteria. Upon rinsing with water a considerable quantity of adsorbed bacterial cells is desorbed and washed out, A single washing of podsol soils, according to our experiments, releases about 11% of Az. chroococcum. The larger the volume of water, and the more prolonged the elution of the soil, the more bacteria are washed out. Of the 2,900 million adsorbed cells of Azotobacter the following quantities were eluted:

  First elute, after one minute, 330 million (11.4%)
  Second elute, after two minutes, 54.0 million (0.18%)
  Third elute, after two minutes, 35.2 million (0.12%)
  Fourth elute, after two minutes, 0.2 million (0.007%)

  The eluted bacteria comprised 14% of the total. One hundred ml of water were used for each washing per 5g of soil.

  The elution of the bacteria is apparently, limited. Above this limit the bacteria are not desorbed even after prolonged washing. The quantity of desorbed cells in different soils varies. The desorption of bacteria by water from their natural surroundings is observed after rain or irrigation. This is closely connected with the seasons.

  The adsorption capacity of soils varies during the vegetation cycle. According to the data of Novogrudskii (1937), less bacteria are being adsorbed in spring and autumn than in summer,

  In April and September, in the podsol soils of the fields of the Agricultural Academy im. Timiryazev, 40-66% of bacteria were adsorbed and in the summer 60-90%. The seasonal variations of the adsorption capacity of the soil are conditioned not only by moisture but also by temperature. The less moisture in the soil, and the higher its temperature, the stronger the adsorbing capacity for microbial cells.

  Samples of soils at 25% moisture per dry weight maintained at 0° and at 25°, adsorbed 57% and 68% Bac. mycoides respectively.

  The adsorption of bacterial cells by the soil is a reversible process. Upon change of pH, temperature, moisture and other factors. the bacteria are desorbed.

  An exchange adsorption is observed, similar to that of mineral substances. if the soil is saturated with one type of bacteria and is then saturated with cells of another more adsorbable kind, then an exchange of bacterial cells will take place. The formerly adsorbed cells will be released or displaced and will reappear in the suspension.

  When two or more kinds of bacteria are simultaneously adsorbed, the more adsorbable ones will be preferentially adsorbed (Novogrudskii, 1936).

  According to Chudyakov and collaborators, the adsorbed cells preserve their viability but their metabolism slows down or stops altogether.

  Dianova and Voroshilova (1925) determined the biological activity of bacteria in strongly adsorbing soils and in sand. The substrates were wetted with nutrient mediums (such as peptone, glucose and others), were sterilized and inoculated with Bac. mycoides, Bact. prodigiosum and Sarcina flava. Their biological activity was determined by the CO₂ released.

  Under all experimental conditions the authors noticed a strongly diminished liberation of CO₂ from the soil.

  For example, in experiments with Bac. mycoides the following amounts of CO₂ were released: in sand, 106.3-126.8 mg, in soil, 0-7.8 mg; with Bact. prodigiosum 24-52 mg of CO₂ was released in sand and 0 in soil; Bact. megatherium released 66 mg of CO₂ in sand and 2.6 mg in soil; Bact. coli released 41-69 mg of CO₂ in sand and 10-23 mg in soil.

  The stronger the adsorption of bacteria, the less their activity. The activity of Bact. coli in sand is 2-4 times higher than in soil, while that of Bact. mycoides and Sarcina flava is 10-30 times higher than in soil.

  The activity of adsorbed bacteria increases with the rise of soil moisture. At 60% of total moisture capacity, 5.2 mg of CO₂ was released from the soil, at 100% of moisture capacity, 28.6 mg (experiments with Bac. mesentericus).

  According to Lipman (1912), the nitrification process by bacteria in a clay soil proceeds slower than in sand. Bact. proteus releases 40% more ammonia nitrogen in sand than in clay; Sarcina lutea 80% and Bac. mycoides 87%.

  According to our observations, cells in the adsorbed state reproduce quite actively. Thus, for example, after careful washing of the soil Azotobacter remained in the adsorbed state at a level of 55 millions per gram of a total 100 million per gram introduced. We washed the soil samples daily for a month. About 300 million cells per gram were washed out. However, after the last elution, a considerable quantity of cells remained in the adsorbed state. Thus, they increased daily by 10 million bacterial cells for each gram of soil.

  Apparently, the process of adsorption of microbial cells by soil particles is also of a biological, and not on ly a physiochemical character, and it must not be considered only from the point of view of physical or chemical forces. Krishnamurti and Soman (1951), analyzing the literature data and their own investigations, reached the conclusion that the phenomenon of adsorption of bacterial cells is of a specific character. The percentage cell adsorption is conditioned by the adsorbent properties as well as by the generic properties of the microbe. The adsorption coefficient is strictly constant under given conditions. The authors even recommend the differentiation of bacterial species on this basis.

  There are almost no data in the literature on the adsorption by the soil of products of microbial metabolism although this problem is of considerable theoretical and practical interest. Microbes, as was pointed out above, grow abundantly in soils, proliferate, display high biological activity and synthesize and release various metabolic products into the milieu. Among these products there are many biologically active substances: enzymes, vitamins, auxins, amino acids and other biotic substances. Antibiotic metabolites, toxins, etc can also be found, Once these substances are excreted from the cell into the soil, part of them undergo decomposition and inactivation, another part is adsorbed by soil elements. The degree of adsorption of such active metabolites is unknown.

  It should be noted that the literature contains very little data on the adsorption of organic compounds by the soil. The adsorption capacity of the soils, as was stressed above, was studied mainly in reference to mineral elements: cations and anions. No attention was paid to organic compounds. However, these processes of interaction between the soil and organic compounds should provide an explanation for the formation of organomineral compounds which determine the essence of soil fertility or the formation of soils as such.

  The available data on adsorption of organic compounds by the soil mainly refer to the problem of humus formation.

  Kravkov (1937) introduced aqueous extracts of grasses and straw into the soil and observed their fixation. According to his data, the water-soluble plant compounds are adsorbed by soil particles to a varying extent which depends on the type and properties of the soil. A different adsorption capacity was recorded for each soil. The organomineral compounds so formed are considered by the author to be the humus of the soil.

  Persin (1944) introduced aqueous extracts of fresh straw and hay of various grasses as well as extracts of straw and hay, after they had been subjected to decomposition by microorganisms. It was found that the water-soluble extracts of fresh straw and hay are not adsorbed by the soil and that extracts of decomposed straw and hay are adsorbed to a varying degree, depending on the stage of decomposition. The greatest adsorption of water-soluble compost substances was observed after 75 days of decomposition at the optimal temperature for microbial activity.

  According to the observations of the author, chernozems adsorb organic compounds in greater quantities than podsol soils. The adsorption capacity of soils for organic substances is conditioned by their mechanical composition. The larger the clay fraction in the soil, the greater its adsorption capacity, and, consequently, it retains the adsorbed substances more tenaciously.

  It should be noted that Kravkov, Persin and some other authors (Chizhevskii and Makarov, 1939) carried out their experiments in nonsterile soil. Consequently, a great part of the introduced organic compounds (if not all of them) was decomposed by microorganisms and was lost to the investigators. The real magnitude of adsorption in these experiments cannot be precisely determined.

  It should be noted that in the investigations of Persin, the soil adsorbed only those water-soluble organic substances which are obtained from decomposed plant residues, i. e., substances formed by bacterial activity.

  Simakov (1938) carried out experiments with tannin and xylan. These substances were differentially adsorbed by the soil, xylan less than tannin. The author in his work during 1944 carried out experiments on adsorption of amino acids and sugars by lignin, which represented one of the components of the soil complex. The experments have shown that the afore-mentioned substances were strongly adsorbed by lignin and equally strongly retained. During this process their properties changed; they became more stable.

  According to Simakov (1944), the amino acids asparagine and glycine adsorbed by lignin are decomposed slowly by microorganisms.

  A considerable number of papers have been devoted to the adsorption of humic substances by the soil (see Zyrin, 1945; Khan, 1950-51; Aleksandrova, 1944; Tyurin, Gutkina, 1940, and others). These investigations have shown that humic substances form stable organomineral compounds with the mineral parts of the soil, the bond between the mineral particles and organic substances of the humus may be of a physical or a chemical nature.

  We (Krasil'nikov, 1954c) have tested antibiotics of actinomycetes, bacteria and fungi.

  Antibiotics, due to their specific antibacterial action are easily detectable, and can be found in various natural substrates, as for example in the soil. They are, therefore, convenient objects for the determination of the adsorption capacity of soil particles. Antibiotics were introduced into various soils and their adsorption determined. We tested penicillin, streptomycin, globisporin, aureomycin, terramycin, subtilin, gramicidin, and other antibiotics, and have shown that they are adsorbed in considerable quantities. For example, we introduced streptomycin at a concentration of 2,000 units/ g; after some time 1,120 units/g were adsorbed by the chernozem, 1,800 units/g by podsol, 1,080 units/g by the serozern and 1,540 units/g by krasnozem. Similar quantities of globisporin were adsorbed by the different soils. Penicillin was adsorbed as follows: 380 units/g by chernozem, 280 units/g by podsol soils, 380 units/g by serozem, and 200 units/g by krasnozem. Aureomycin and terramycin as well as antibiotics of bacterial origin such as subtilin and gramicidin were adsorbed by the afore-mentioned soils in varying quantities. The antibiotic 1609 was only adsorbed by the podsol soils, and then only in a very small quantity, 20-30 units/g. This antibiotic was not held by any other soil.

   Thus, the various soils adsorb different quantities of antibiotics, however, the nature of their adsorption is different from that of mineral compounds. Soils poor in humus (podsols, krasnozems) adsorbed antibiotics in considerably larger quantities than soils rich in humus. Different layers of the same soil possess different adsorption capacities. We have studied the streptomycin adsorption capacity of the podsol soils (Experimental Station Chashnikovo, Moscow Oblast') of cultivated and noncultivated soils. The results are given in Table 18.

  The B₁-B₂ layer has the strongest adsorption capacity and the layer A₀-A₂ the smallest. The degree of adsorption of antibiotics by the soil does not depend solely on the soil properties but also upon the properties of the antibiotics themselves. In one and the same soil, for example in chernozem, we have observed the following adsorption:

  The antibiotics in the adsorbed state preserve their antibacterial activity for some time. The period for which a given antibiotic preserves its activity depends on the soil and the properties of the antibiotic itself. For example, in same soils penicillin remains active for 20-30 hours, in others 2-3 hours; terramycin remains active for 3-5 days in podsol and 1-2 days in chernozem. Some antibiotics (preparation 1600) lose their activity immediately.

  Organic compounds adsorbed by the soil undergo various transformations; they are decomposed, inactivated and disappear, They are replaced by other compounds.

  The adsorbed fraction of the antibiotics retains its antibacterial properties for a more prolonged period than the antibiotics in the free state present in the soil solution.

  For example, free streptomycin disappears from the podsol soil after 10-12 hours, while adsorbed streptomycin is preserved for more than 30 hours. In the serozem, the nonadsorbed streptomycin is inactivated after 20-25 hours; its absorbed fraction can be detected even after two days. A still greater difference was observed in the experiment with aureomycin. In podsol it can be detected in the free state after 20 hours, in the adsorbed state after 5 days In the serozem the free antibiotic is preserved for no more than two days, while in the adsorbed state it retains its activity for more than seven days.

  The antibiotics in the soil are partially inactivated by the soil solution and by microorganisms.

  Not only antibiotics but also other microbial metabolites, as well as intermediate decomposition products of plant residues, and various compounds of humus, are adsorbed by the soil.

  The biologically active metabolites present on the surface of soil particles exert a great influence on their physicochemical state. The soil particles holding the substance on their surface gain new properties.

  The presence of living microbial cells adsorbed on the soil particles should be regarded as a complex system of biotic-mineral complex. Each soil particle carries elements of living organisms, the study of which is of the utmost importance to soil biologists.

  Microorganisms are an integral part of the soil. If the soil should lose these organisms it would lose its main property--fertility--and it would turn into a dead, barren, geological body.

  The soils are inhabited by numerous representatives of the microflora--bacteria, actinomycetes, yeasts, fungi, algae, protozoa, insects, worms, and others. Besides, there are in the soils various ultramicroscopic organisms: bacteriophages and actinophages.

  No accurate data on the numbers of microorganisms in the soil are available. Methods for the detection of the entire soil population are not available. The existing methods give only a relative idea of the density of the microbial population.

  Two essentially different methods are employed for the quantitative estimation of microbes in the soil: a) determination by means of soil inoculation of artificial media-liquid and solid, b) direct count of cells.

  These two methods give different data on the quantitative aspect of the microbial population of the soil.

  In practice, the inoculation method is more extensively used. There are various methods of inoculation and media.

  The amounts of micoorganisms detectable in the soil vary, depending on whether they are inoculated into a solid or liquid medium, or inoculated by sowing the surface of agar medium, or dispersed in aqueous suspension by serial dilutions. Inoculation with soil is often carried out by placing small soil particles on agar medium.

  In all instances the number of bacteria grown on agar media is smaller than upon growth in liquid media inoculated by the method of serial dilutions.

  Data from the literature on the amount of bacteria, actinomycetes and fungi in the soil are obtained, in the majority of cases, from growth on agar media. According to these data, the number of bacteria per gram fluctuates within the range of from several tens or hundred thousands to many millions depending upon the soil composition and the medium (Starkey, 1929, 1931, 1955; Gray and Thornton, 1928; Clark, 1940; Timonin, 1940-41, Waksman, 1952; Jensen, 1934-36; Mishustin, 1956, and others).

  Thom (1938), summing up data from the literature and the results of his own investigations on the quantitative determination of the bacterial population of the soil, considers that the total number of bacteria in one gram of soil reaches 50 millions. Since the greatest number of bacteria is concentrated in the plant rhizosphere many authors give data on microbial composition of this zone. Starkey showed, by the method of counting on agar media, the presence of 199 to 3,470 million bacterial cells per gram, depending upon the species of plant.

  Humfeld and Smith (1932), counted 5-8 billion bacteria in one gram of soil with green fertilizer. Clark (1949) found 5 billion bacteria per gram of well-fertilized soil and also in soils under a mixture of grasses. Rippel (1939), analyzing the soils of Germany, and Feher (1933) analyzing the soils of Hungary and Austria, counted from one hundred thousand to 5 billion bacteria per gram of soil, depending upon soil composition and climatic conditions.

  Nonfertilized soils have a smaller microbial population; ranging between hundred thousands and millions, but on the average 3-7 million per gram. Bunt and Rovina (1955) counted from 400,000 to 15 million bacteria per gram in the subartic soils of Iceland.

  We have obtained similar figures. We have counted from several hundred thousands up to 15 million bacteria per gram, in the soils of the Kola Peninsula, the Islands of the Arctic Ocean and in mountainous soils of Pamir and Caucasus. The podsols of noncultivated or poorly cultivated soils contain, according to our investigations, 300 thousand to 10 million cells per gram of soil. Chernozems rich in humus contain 10-1,000 million cells per gram. Similar data were obtained by many other investigators studying various soils.

  Higher counts (ten and hundred times higher) are obtained by the method of inocculation into liquid media and by the serial dilution method. For example, soils poor in organic compounds (podsols) gave from one to 100 million cells per gram, and fertile soils (chernozem) from one hundred to 1, 000 million bacteria per gram estimated by the solid-media method, meat-peptone agar (MPA). The method of inoculations on liquid media, meat-pe ptone broth (MPB) revealed to 10-500 million and 1,000-10,000 million bacteria per gram of soil respectively.

  While studying the rhizosphere soil of lucerne in Central Asia (serozems) we have found (by the method of serial dilutions 50-100 billion bacteria per gram, and Raznitsina (1947) and Korenyako (1942) obtained even higher numbers. Such high figures are constantly obtained during the investigation of plant rhizosphere under given conditions.

  Such high indices of microbial population throws doubt on the accuracy of the methods employed. Experiments especially designed to check this method were carried out. We have assumed that the high figures obtained by this method can be explained by the adsorption of bacterial cells on the walls of pipettes and with their subsequent elution (desorption). Experiments have shown that adsorpton of cells does indeed take place. The number of bacteria is 2-5 times, and sometimes even 10 times less if the pipettes are changed upon each dilution. The numbers obtained, when the pipettes are changed. are of an order of 1-10 billion per gram of soil, upon dilution of the soil with one pipette the numbers increase to 5-100 billion per gram of soil.

  We checked the trustworthiness of this method by using pure cultures of Bact. prodigiosum, Ps. fluorescens, Mycob. rubrum, Az. vinelandii and Bac. subtilis. Aqueous suspensions of these bacteria were diluted with and without change of pipettes.

  The number of bacteria in billions per 1 m/ obtained in such an experiment are as follows:

  In this experiment the change of pipettes lowered the number of bacteria 1.5-4 times depending upon the bacterial species. Similar lowering of bacterial numbers was observed on studying soil samples. The difference in the numbers is more pronounced if the number of bacteria in the soil is large. Hundred billions and more of bacteria were detected in the rhizosphere of lucerne grown in the Vakhsh valley when the soil suspension was diluted with one pipette; the number was five times less. when the pipettes were changed. The control soil contained 100-500 millions per gram upon dilution with one pipette, 50-150 millions were counted upon dilution, when the pipettes were changed, i.e., 30-50% less.

  According to Vinogradskii, the method of direct counting of the soil bacteria also gives higher numbers than the method of growth on agar media, i.e., approximately the same are obtained upon serial dilution, or even higher (Table 19).

  The difficulty of the method of direct counting is that the smears contain living cells and dead particles of the same soil. and they cannot be differentiated with certainty. The soil always contains a large amount of small particles which can be stained and thus become indistinguishable from the bacteria themselves. It is especially difficult to tell the coccoid cells from the small globular bodies and granules.

  In recent years some investigators attempted to use fluorescent dyes for the differentiation of bacteria from the dead soil particles. Burrichter (1953). employed acridine-orange for the staining of soil smears and studied them under a fluorescent microscope in ultraviolet light. In soils rich in humus (9. 98 %)the author counted 9,453 million bacteria per gram of soil and the total number of microbes was 18,331 million bacteria per gram of soil; in soil poor in humus (1.80%) 1,230 million bacteria per gram were found. The number of colonies of slimy bacteria in the first soil amount ed to 157 million per gram. Soil, fertilized with compost. contained a total number of microbial cells of about 16,132 million per gram, and soils poor in organic substances 25 million per gram. Strugger (1948, 1949) found from 1,038 to 8,640 million bacterial cells per gram of soil employing the same method of fluorochrome staining.

  It should be noted that the method of fluorescent staining also has shortcom -ings. The green color of living cells or the red color of dead cells and other particles

  may often depend not on the cell viability but upon many other factors, such as the concentration of the dye, pH of the medium, temperature and others (Krasil'nikov and Bekhtereva, 1956). It is sometimes difficult to say what are the green or especially the red-stained bodies; are they living bacterial cells or dead, or soil particles.

  The method of direct microscopy of soil smears (Kubiena, 1932) is also of little use for the quantitative estimation of cells.

  As can be seen from the given data, the existing methods of microscopic analysis are inadequate for quantitative determination of microbial numbers in the soil and for the determination of their forms. Therefore, when comparing data obtained by employing one of the aforementioned methods, the investigators limit themselves to relative figures.

  The bacterial numbers vary in different soils, according to their fertility and nutritional qualities. The more fertile the soil, the richer it is in humus, the denser its microbial population. The podsol soils (Moscow Oblast) contain, in well-cultivated fields, 3-10 millions per gram, and the chernozem soil of the Kuban, contains (similar method of counting) 15-50 million bacterial cells per gram of soil.

  One and the same type of soil also varies in the amount of microbes it contains. The podsol soils, not well cultivated and poor in humus, contain 500,000 to 1.5 million cells per gram and in some cases only a few thousand per gram (the soils of the Kola Peninsula). Well-cultivated, systematically fertilized soils contain 3-25 million cells per gram. Garden soils, as a rule, are richer in microbes than the soil of fields.

  Virgin soils contain less microbes than cultivated soils. (Table 20).

  The upper layer of the soil s richer in microbes than the deeper layers. For example, we have found the following amount of bacterial cells in podsol soils of the experimental fields of the Academy of Agriculture im. Timiryazev:

  in the layer 0-20 cm deep, 5.7 million/g
  in the layer 20-35 cm deep, 2.4 million/g
  in the layer 40-60 cm deep, 0.5 million/g
  in the layer 80-100 cm deep, 0.001 million/g

  In the root zone of the vegetating plants, in other words, in the rhizosphere, the soil is saturated with bacteria to a greater extent than in the zone outside the roots. The vegetative cover, as will be shown later, exerts a strong influence on the concentration of microbes in the soil.

  The number of microorganisms in the soil varies with the season. According to the literature and our own data, their total number in winter is smaller than in summer. This is especially noticeable in the soils of the north.

  The analysis of soils of Severnaya Zemlya and other islands of the Northern Ocean showed that in May, when the soil was still in the frozen state, it contained tens of thousands of organisms per gram and in August many millions of bacteria per gram (Table 21).

  The number of microbes in the soil of the temperate zone is greatest in spring, smaller in summer, it increases somewhat in autumn.

  The data on numbers of bacteria in winter are few and contradictory. The majority of investigators think that life in the soil stops altogether during the winter. A considerable number of microbes die from cold and their total number decreases.

  According to our observations, the microbial activity does not always cease in winter. Under a deep snow cover the earth is not always frozen and in such a soil microbiological processes take place. This can be found by studying the growth dynamics of individual species of actinomycetes. Korenyako has shown that during the winter months of 1952-1954 certain species of actinomycetes (A. globisporus) grew more abundantly, in Moscow Oblast' soils, than during the summer and autumn.

  Besides, certain biochemical processes, leading to detoxification of the soil take place in winter (Krasil'nikov, Korenyako and Mirchink, 1955).

  The vigorous growth of microbes in spring is, according to our opinion, not only caused by the warm temperature and by moisture, but also by other factors, First, the toxins are inactivated or decomposed in winter due to low temperature. Second, low temperatures, as was noted above, stimulate the growth and activity of microbes. in addition, many soil nutrients under the action of low temperature, change and become more available to microbes.

  It was pointed out above that microbial growth in the soil depends on the presence of organic substances of humus. This is not always true. The amount of organic substances in the soil may be very high (peats, marshy soils) while the growth of microorganisms is rare. Not infrequently a reverse picture may be observed. Certain primitive soils of mountainous regions are poor in organic substances and at the same time rich in bacteria.

  The concentration of microorganisms in the soil depends mainly on the presence of such organic substances as can be easily utilized by bacteria. There are fresh plant and animal residues and products of their primary decomposition which have not yet been transformed into humus, as well as a number of products of synthesis, etc.

  Of great importance for the life of microbes are organic growth factors; vitamins, auxins, various biotic elements and substances which suppress their growth and multiplication.

  Small doses of these substances markedly enhance the growth and multiplication of microbial cells as well as that of plants, by promoting various biochemical and physiological processes.

  This part of the organic compounds, or soil humus, is, in our opinion, of the greatest importance, and a correlation should be found between their quantity and the total number of the microbial population. Unfortunately, such a correlation is very difficult to study and has not as yet been methodically worked out.

  Adsorption should be taken into account in the determination of microbial numbers in the soil. The data of observations and experiments given above showed the degree of bacterial adsorption by soil particles. Bacteria in the adsorbed state can be found in tens, hundreds, millions and billions in one gram of soil. The method employed by us (inoculation on media) in most cases accounts only for microbes in the free state as well as for a fraction of those adsorbed. The majority of adsorbed cells remains unaccounted for; the number differing from case to case.

  The majority of investigators give data obtained by analysis of dry soil samples. Naturally, these data are far from the real figures. It is known that the number of microbes is decreasing in dry soil. During prolonged storage a large number of microbial cells die. Sometimes, upon drying, the total number of bacteria decrease by a factor of 2-3 and not infrequently 5-10 times (Table 22).

  * Samples of the soil of Severnaya Zemlya (taken in August) were analyzed the same day and then after one month.

  Upon storage of samples in the dry state the qualitative composition of the bacteria also changes. Some bacterial genera disappear almost completely, others remain in small quantities, still others do not decrease in numbers at all.

  Actinomycetes and then mycobacteria are the most stable in this respect. The highest percentage of destruction is noted among the bacteria (Table 23).

Table 23
The survival of various groups of microbes upon storage in dry soil
(in thousands/g)

Soils	Sporiferous bacteria	Nonsporiferous bacteria	Mycobacteria	Actinomycetes
Chestnuts of the Trans-Volga region
Fresh	1,500	56,000	1,000	1,500
Dry	450	5,000	900	1,000
Podsol of Moscow Oblast'
Fresh	650	5,500	850	1,250
Dry	325	1,400	600	980
Chernozem of Kuibyshev Oblast'
Fresh	2,500	400,000	25,000	25,000
Dry	280	46,000	16,000	26,000

  In those cases where the soil dries up slowly, an increase in number of actinomycetes and certain species of mycobacteria is observed. These organisms can grow in soil of minimal moisture, when the growth of other microorganisms ceases (Krasil'nikov, 1940c).

  Differences in survival capacity have been observed not only in different groups but also in different species, and even different strains of the same microbe show different ability to survive. According to our observation, cultures of Bact. herbicola, Az. vinelandii, nodule bacteria of soya and Ps. aurantiaca, die out rapidly in the dry soils of podsol (Moscow Oblast') and in virgin serozern soils. Of some hundred million cells only a few (10-100 cells/g) remained viable after two weeks storage. Mycobacteria such as Mycob. rubrum and some other species remain viable in considerable quantities (100,000 cells/g and more.)

  Not all strains of Azotobacter, in dry samples of soil, die out at the same rate. Of twenty cultures of Az. chroococcum studied, eight strains of Azotobacter survived in considerable numbers--up to 10% and more of the cells. Of 10 strains of root nodule bacteria of lucerne only about 10% of 4 strains survived in dry samples of serozem soils; in the podsol soil only one strain survived and then only in a negligible amount (0.5% and less).

  The sporiferous bacteria show the same diversity as far as their survival capacity is concerned. About 80-90% of Bac. megatherium dies out in dry podsol soils of the Moscow Oblast', and 30-40% of Bac. subtilis and Bac. mesentericus. Only 5 strains out of 20 of the latter, when isolated from various podsol soils, were resistant to storage in dry soil samples.

  No complete drying out of bacteria in dry soils was observed. Even in the cultures most sensitive to drying, there are single cells which are stable and survive for long periods in the dry state. Thanks to such cells the species does not die out under conditions of prolonged drought.

  Great variations in the composition of the microflora also take place when the soil samples are kept moist.

  It is clear that the soil as a whole, and the separate soil aggregates possess different physicochemical conditions for the life of microbes than those present in isolated soil samples. Some bacterial species grow quicker in natural surroundings, others, slower.

  Table 24 shows data from an analysis of samples of a frozen soil, taken from the archipelago of Severnaya Zemlya in May.

  The total number of bacteria In the samples after 10 days increased 2 to 4 times, and the number of mycobacteria 16 to 50 times. Actinomycetes in fresh samples, were almost nonexistent, and after 10 days their numbers reached 500-1,500 per 1 gram of soil.

  Similar changes in the composition of the microflora is noted in other soils during their storage in the moist state. In samples of podsol soil no Azotobacter could be detected by us after 2-3 days. The reason for this is the abundant growth of its antagonists--Bac. subtilis and Bac. mesentericum. In samples of chestnut soil mucolytic bacteria grew abundantly, and fungi of the genus Fusarium disappeared almost entirely.

  For the determination of the microflora of the soil one has to take into account the composition of the medium into which the microorganisms are being inoculated. Experiments show that organisms from many soils grow better on synthetic media of Chapek, CPI, etc, than on media containing protein. On starvation media (water agar) and semistarvation media (Ashby agar) the number of bacteria is often 2-5 times greater than on rich nutrient media (Table 25).

  It should be pointed out that it is easier to isolate bacteria from primitive soils ,or mountainous soils (mountain summits, islands of the Arctic Ocean, etc) on starvation, semistarvation or synthetic media which do not contain protein. The bacterial colonies on such media are very small and can often be seen only with the aid of a a magnifying glass, or even only under a microscope. Such microcolonies usually consist of a few cells only.

  The microflora detected after inoculation on starvation and semistarvation media differs, from that found in peptone media.

  The predominant organisms capable of growing on the synthetic medium of Chapek, CPI, etc are the auxotrophs (prototrophs), which do not require any growth factors or organic nitrogen. They can synthesize all the necessary biotic substances such as vitamins, auxins, etc.

  On water agar and on the Ashby medium microorganisms usually grow at the expense of their food reserves. Among these organisms auxoautotrophs and auxoheterotrophe can be detected likewise. Often the so-called oligonitrophils grow on the nitrogen-less medium of Ashby. These are unique forms of bacteria and mycobacteria which are capable of nitrogen fixation in small quantities, satisfying their growth requirements (Mishustina, 1953).

  On peptone media, and generally on media rich in organic substances, predominantly auxoheterotrophs (metatrophs) grow. Auxoautotrophs also grow on these media. The quantitative ratio of prototrophs to metatrophs varies from soil to soil. In soils rich in humus, and well-fertilized with organic fertilizers, the amount of the former and the latter is approximately the same.

  Organic, protein-containing media, are toxic for many soil microorganisms.

  Apart from the afore-mentioned microorganisms, there are great numbers of organisms in the soil possessing specific functions. Such organisms can be detected on special, so-called selective media. To such bacteria belong the nitrifiers, sulfur bacteria, iron bacteria, Azotobacter, cellulose-decomposing bacteria and others. Special media are required for the cultivation of such bacteria.

  The principle underlying the use of selective media (Vinogradskii, 1952) is as follows: in the selective medium, favorable conditions exist for the detection of a given function.

  It should be noticed, that the selective media are of relative importance. Investigations have shown, that many if not all prototroph bacteria have the capacity of growing on complex nonselective media. For example, Azotobacter can grow on nitrogen-less media due to the capacity of nitrogen fixation, but it can also grow on media containing inorganic and organic nitrogen.

  Experiments have shown, that selective media are not strictly selective. No matter what the composition of the selective medium and how carefully it is prepared, other bacterial forms grow on it in addition to the desired bacteria.

  On the nitrogen-less medium of Ashby or Beijerinck, apart from Azotobacter, many oligonitrophils and metatrophs grow well. On the medium of Vinogradskii, used for the nitrifiers, bacterial satellites also grow well. On media containing cellulose, not only bacteria capable of decomposing cellulose grow, but also other forms of bacteria.

  Selective media are not optimal for the growth of bacteria. In. many cases, bacteria grow better upon addition to these media of ready sources of nutrition. Nitrogen compounds may be added for the growth of Azotobacter, sugar and other organic compounds for the growth of the cellulose-decomposing organisms, protein and nonprotein substances for others, etc (Rotmistrov, 1950).

  According to Kalinenko (1953a, b) iron bacteria and nitrifiers grow well on ordinary organic and even protein-containing media.

  It is evident that there are no strictly selective media. Universal media do not exist either.

  The data presented in this chapter provide the basis for the assumption that the data on bacterial numbers in the soil are rather lower than in reality. Knowing their numbers, their total mass can be determined, or in other words, the soil productivity.

  Cocci are 0.7 µ in diameter, their volume is 0.18 µ³, and their weight 7 x 10^-10 mg. About 5 x 10 ⁹ cells are present in 1 ml. The size of nonsporiferous bacteria is on the average 3 µ x O.7 µ, the cell volume 1.15 µ³ , and the weight 10 ^-9 mg. About 900-1,000 million cells may be present in 1 ml. Cells of larger size (5 µ x 1 µ) have a volume of 3. 9 µ³, their weight is 10-8 mg. In 1 ml there are about 350 million cells.

  According to the data of Tanson (1948), in 1 ml there are 1,000 cocci of 1 µ in diameter; 330 million sporiferous bacteria of the size of 3 µ x 1 µ, and 1 million spores of fungi, 10 µ in diameter. According to Van Niel (1936),there can be 1,400 million cells of Bact. coli per 1 ml; according to Butkevich (1938), 10 ⁹ cells weigh 0.5 mg; Jensen (1940) found that 10 ⁹ cells of Azotobacter weigh about 5 mg. Similar data are given by Kendall (1928), Strugger (1948) and some other authors.

  We have obtained the following data on the total microflora of the rhizosphere of vegetative plants. There are 2-2.5 kg of cells in a soil under lucerne in Central Asia, per 120 kg of soil; i.e., 6,000-7,000 kg of cells per hectare. Outside the root zone there are, according to our calculations, 1,500-2,000 kg bacterial cells per hectare of the upper (plow) layer. Consequently, there are about 7-9 tons of bacterial mass per hectare (Krasil'nikov, 1944).

  In soils of medium fertility the total mass is considerably smaller. For example, in podsol soils under two-year clover and frequently fertilized we have found 1,000-3,000 millions of organisms per gram of soil in the rhizosphere and in the zone outside the roots, 300-800 million organisms per gram of soil. The total bacterial mass in the root zone amounted to 1,200-3,000 kg and outside the root zone about 350-1,000 kg. The total bacterial mass per hectare was 1,500-4,000 kg.

  In the same soil under wheat, there were 800-1,200 million organisms per kg in the rhizosphere, and 100-200 million outside the roots. The total mass of bacteria was 1,100 kg per hectare.

  In a poor. lightly cultivated soil (podsol) we have found under wheat, only 100-150 kg of bacterial mass per hectare in the upper (plow) layer. Eighty per cent of this mass was found in the rhizosphere.

  Strugger (1948). on the basis of his investigations and those of Kendall, calculated that the total bacterial mass comprises 0.03-0.28% of the weight of the soil. Clark (1949) has shown that the bacteria constitutes 300-3,000 parts per million by weight of the soil. These data agree with our own.

  Similar numbers are given by Khudyakov (1953c), Mishustin (1954), Berezova (1953) and others.

  It should be recalled that our calculation takes into account only bacteria, whereas other organisms living in the soil, such an actinomycetes, fungi, algae, and protozoa are unaccounted for. They comprise a considerable mass of living substance.

  The total number of fungi and actinomycetes per gram of soil runs to tens and hundred thousands, and not infrequently millions of organisms per gram of soil. The number of algae reaches thousands and hundred thousands and the diatomaceous algae 100 million per gram of soil (Brendemuhl 1949). The total mass of these organisms cannot be calculated owing to the peculiarities of their structure and growth. Nevertheless. according to the investigators, it is only slightly less than the total bacterial mass.

  The total mass of protozoa and insects per hectare is 2-3 tons (Gilyarov, 1949, 1953).

  The total mass of the living organisms does not represent merely a static reserve of organic substances, but a living active mass with a large potential, This mass is in constant growth. The individual cells of this mass grow, reproduce, grow old, and die. A constant change and regeneration of the whole living mass takes place.

  Under natural soil conditions bacteria give on the average no less than two generations per month during the whole vegetation period, which lasts 7-9 months in the south and 3-5 months in the moderate belt. Consequently, the entire bacterial mass undergoes regeneration 14-18 times during the summer (in the southern belt), and 6-10 times in the moderate belt. The total bacterial production in the upper (plow) layer reaches tens of tons of living mass for one vegetative period.

  The intensity of bacterial growth in the soil was determined by the time required for the doubling of their numbers. Three organisms were used in the experiment. Az. chroococcum, Ps. aurantiaca and Bact. prodigiosum. A sample of a garden soil was placed in an asbestos bag inoculated with the above-listed organisms , carefully mixed and immediately subjected to a microbiological analysis. The soil in the bag was washed with water, until all the desorbed bacteria were removed, and then the bag was buried in the same garden soil from which the samples were taken. After 1-2 days the bags were taken out and the soil was subjected to the same procedure as before. This was repeated for a month. The experiments were carried out in May, July-August and September-October, three series in all. In each series 100 million organiams,were introduced into the soil. In the first analysis (immediately after mixing) 26 million Azotobacter cells were washed out. 18 million cells of Ps. aurantiaca and 34 million cells of Bact. prodigiosum (all these numbers are per gram of soil). The rest of the cells were in the adsorbed state, but they did not lose their capacity to reproduce.

  Upon subsequent analyses the following amount of cells was washed out (the May experiment), in millions/ g:

  As can be seen from the above data, the doubling of Azotobacter cells took place every 5 days, Ps. aurantiaca every 4 days. and Bact. prodigiosum every 10 days. In other words the number of generations of the first was 6, of the second, 7 and the third 3. In July-August the number of generations was 4, 4 and 2 respectively, and in September-October, 4, 3, 1 respectively.

  The results of these experiments served as a basis for the calculation of the speed of growth of the bacterial maps in the soil.

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