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Part II, continued

 

The Adsorption Capacity of Soils

  Soil is known to adsorb various substances. There are a number offorms 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 actsas a filter. Ordinarily, the size of retained particles exceeds the size of the soilpores but even smaller particles can be retained.

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

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

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

  Biological adsorption according to Gedrolts is characterizedby the adsorption of compounds from the soil solution, by microbial cells and greenplants.

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

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

  Organic compounds of the soil such as the humic acids, organomineraland inorganic compounds such as aluminum-silicates, iron hydroxide, argillaceousminerals and others may exist in the colloidal state. They represent a finely dispersedsystem, the particles of which possess high surface-reaction capacity for adsorbingsubstances present in solution. The soil colloids can be divided into hydrophilesand hydrophobes. The first adsorb water molecules and hydrated ions of the soil solutionon 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 equivalentquantity.

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

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

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

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

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

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

 

Adsorption of bacteria by soil

  It has long been noted, in the laboratory practice, that bacterialcells are adsorbed by various materials in powder form. Kruger, (1889) demonstratedthe adsorption of bacterial cells from their aqueous solutions by coke, clay, brickflour,magnesium oxide and other substances. Later Eisenberg (1918) showed that bacterialcells can be adsorbed by animal charcoal. Michaelis (1909) noticed that differentbacterial 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 quantitiesof bacterial cells. According to Dianova and Voroshilova (1925), between 252 and4,350 million bacterial cells par hectare are adsorbed depending on the kind of soiland generic peculiarities of the bacteria (Table 14).

Table 14
The adsorption of Bact. prodigiosum by different soils
(number of cells in millions, per 5 g of soil)
.

Cells introduced

Total adsorbed

% adsorbed

Podsol exp. fields of Agr. Acad. im Timiryazev

 

 

 

 

58,600

4,470

58.8

 

5,860

4,988

85.9

 

58.6

52

90.4

Chernozem of the Voronnezh Oblast'

 

 

 

 

32,800

28,000

87.5

 

16,400

16,200

98.8

 

3,280

3,230

98.5

 

328

327

99.7

  The loam soils of the experimental fields of the Agricultural Academyim. 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 theexperimental fields of the Agricultural Academy im. Timiryazev, the soils of theMoscow Botanical Garden and chernozem of the Voronezh Oblast'. We have compiled theresult of these studies in the tables. Table 15 shows the data on bacterial adsorptionand Table 16 the adsorption of fungi and actinomycetes.

Table 15
Adsorption of bacteria by different soils (in %)
Soils

% Bac.Mycoides

% Bac. Mesentericus

% Bac. megatherium

% Bac. chroococcum

% Bac. fluorescens

% Bac. denitrificans

% Bac. leguminosarum

The timiryazev Agricult. St. (podsol)

71

10

61

64

8

36

44

Botanical Garden

82

76

62

44

20

20

45

Voronnezh Oblast'

99

99

93

95

50

82

88


Table 16
Adsorption of spores of fungi and actinomycetes by different soils (in %)
Soils

% Asperg. niger

% Penic. glaucum

% Mucor mucedo

% Fus. sp.

% Botrytis cinerea

% Act. 154

% Act 105

% Act. 110

Podsol

14

43

57

99

93

8

10

13

Botanical Garden

20

43

27

97

97

15

31

28

Voronezh chernozem

97

94

97

99

99

99

75

94

  According to our data the Moldavian chernozem (medium loams, carbonate)adsorbs two to three times more cells of Azotobacter than the upper layerof the podsol soil which was previously under forest (Experimental Station of theMoscow State University, Chashnikovo, Moscow Oblast'). In the former soil, 4,000million Az. chroococcum and 600 million Az. vinelandii were adsorbedper 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. Theupper layers of the soil are characterized by higher adsorption capacity than thelower, The poorly cultivated soil of the Experimental Station of Chasnikov adsorbedAz. 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 Mayand 25% in August
  d) Layer B2 (50-70 cm); 60% in May and75% 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 mechanicalcomposition. Sand contains particles 1.0 to 0.25 mm in diameter and sand dust withparticles 0.25-0.05 mm in diameter, adsorb bacterial cells weakly. Dust containingparticles 0.05-0.01 mm 0.01-0.005 mm and 0.005-0.0015 mm in diameter adsorbs microbialcells most actively. The slimes of rivers and lakes having particles of 0.0015 mmand less in diameter are devoid of adsorbing capacity. since the size of the particlesof slimes (1-1. 5 µ and less) does not exceed that of the ordinary bacterialcell. In such an environment bacterial cells are themselves adsorbants. The greaterthe adsorption of bacteria, the fewer the cells found afterward in the suspension.

  The character and degree of adsorption of microbial cells by the soilis conditioned to a large extent by the qualitative features of the organisms proper,and their generic properties. The degree of adsorption depends on their metabolicstate and their vital potential. Some bacterial genera are adsorbed more vigorouslyand in a larger quantity than others. According to some authors, many nonsporiferousbacteria are adsorbed considerably weaker, by the same adsorbent, than the sporiferousbacteria 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 quantititesthan the gram -negative bacteria. Bogopol'skii (1933) gives the following data. Peatof medium decomposition adsorbs 74% of Bac. mycoides, 81% of Urobact. pasteurianum,21-22% of Bact. coli and Ps. fluorescens. The same results were obtainedby Eisenberg (1918) while studying the adsorption of bacteria by charcoal and otheradsorbants. According to his data, the adsorption of gram-positive bacteria, Micr.pyogenes, Micr. candicans Sarcina lutea and others was 500 times larger thanthat of the gram-negative bacteria, Bact. coli, Bact. typhi, Ps. pyocyanea, Vibriocholera and others.

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

Table 17
Bacterial adsorption by podsol soil at different pH
(number of cells grown in the Petri dish)
(according to Eisenberg, 1918)
 

pH of the suspension

suspension

soil

adsorbed %

Bac. mycoides

 

 

 

 

 

4.6

82

8

90

 

5.8

66

33

50

 

6.7

67

59

12

 

7.4

63

46.5

27

 

7.8

66

45.5

28

Bact. coli

 

 

 

 

 

4.4

384

311

19

 

5.6

361

384

0

 

6.3

384

178

35

 

6.9

360

199

45

 

7.5

358

294

20

  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 alkalinemedium 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 quantityof adsorbed bacterial cells is desorbed and washed out, A single washing of podsolsoils, 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, themore bacteria are washed out. Of the 2,900 million adsorbed cells of Azotobacterthe 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 ofwater were used for each washing per 5g of soil.

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

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

  In April and September, in the podsol soils of the fields of the AgriculturalAcademy 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 notonly by moisture but also by temperature. The less moisture in the soil, and thehigher 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 cellsof 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 thesuspension.

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

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

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

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

  For example, in experiments with Bac. mycoides the followingamounts of CO2 were released: in sand, 106.3-126.8mg, in soil, 0-7.8 mg; with Bact. prodigiosum 24-52 mg of CO2was released in sand and 0 in soil; Bact. megatherium released 66 mg of CO2 in sand and 2.6 mg in soil; Bact. coli released41-69 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, whilethat of Bact. mycoides and Sarcina flava is 10-30 times higher thanin soil.

  The activity of adsorbed bacteria increases with the rise of soilmoisture. At 60% of total moisture capacity, 5.2 mg of CO2was released from the soil, at 100% of moisture capacity, 28.6 mg (experiments withBac. mesentericus).

  According to Lipman (1912), the nitrification process by bacteriain a clay soil proceeds slower than in sand. Bact. proteus releases 40% moreammonia nitrogen in sand than in clay; Sarcina lutea 80% and Bac. mycoides87%.

  According to our observations, cells in the adsorbed state reproducequite actively. Thus, for example, after careful washing of the soil Azotobacterremained in the adsorbed state at a level of 55 millions per gram of a total 100million per gram introduced. We washed the soil samples daily for a month. About300 million cells per gram were washed out. However, after the last elution, a considerablequantity of cells remained in the adsorbed state. Thus, they increased daily by 10million bacterial cells for each gram of soil.

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

 

Adsorption of products of microbial metabolism by soil

  There are almost no data in the literature on the adsorption by thesoil of products of microbial metabolism although this problem is of considerabletheoretical and practical interest. Microbes, as was pointed out above, grow abundantlyin soils, proliferate, display high biological activity and synthesize and releasevarious metabolic products into the milieu. Among these products there are many biologicallyactive substances: enzymes, vitamins, auxins, amino acids and other biotic substances.Antibiotic metabolites, toxins, etc can also be found, Once these substances areexcreted 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 activemetabolites is unknown.

  It should be noted that the literature contains very little data onthe 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: cationsand anions. No attention was paid to organic compounds. However, these processesof interaction between the soil and organic compounds should provide an explanationfor the formation of organomineral compounds which determine the essence of soilfertility or the formation of soils as such.

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

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

  Persin (1944) introduced aqueous extracts of fresh straw and hay ofvarious grasses as well as extracts of straw and hay, after they had been subjectedto decomposition by microorganisms. It was found that the water-soluble extractsof fresh straw and hay are not adsorbed by the soil and that extracts of decomposedstraw 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 75days of decomposition at the optimal temperature for microbial activity.

  According to the observations of the author, chernozems adsorb organiccompounds in greater quantities than podsol soils. The adsorption capacity of soilsfor organic substances is conditioned by their mechanical composition. The largerthe 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 (Chizhevskiiand Makarov, 1939) carried out their experiments in nonsterile soil. Consequently,a great part of the introduced organic compounds (if not all of them) was decomposedby microorganisms and was lost to the investigators. The real magnitude of adsorptionin these experiments cannot be precisely determined.

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

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

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

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

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

  Antibiotics, due to their specific antibacterial action are easilydetectable, and can be found in various natural substrates, as for example in thesoil. They are, therefore, convenient objects for the determination of the adsorptioncapacity of soil particles. Antibiotics were introduced into various soils and theiradsorption determined. We tested penicillin, streptomycin, globisporin, aureomycin,terramycin, subtilin, gramicidin, and other antibiotics, and have shown that theyare adsorbed in considerable quantities. For example, we introduced streptomycinat a concentration of 2,000 units/ g; after some time 1,120 units/g were adsorbedby the chernozem, 1,800 units/g by podsol, 1,080 units/g by the serozern and 1,540units/g by krasnozem. Similar quantities of globisporin were adsorbed by the differentsoils. Penicillin was adsorbed as follows: 380 units/g by chernozem, 280 units/gby podsol soils, 380 units/g by serozem, and 200 units/g by krasnozem. Aureomycinand terramycin as well as antibiotics of bacterial origin such as subtilin and gramicidinwere adsorbed by the afore-mentioned soils in varying quantities. The antibiotic1609 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 largerquantities than soils rich in humus. Different layers of the same soil possess differentadsorption capacities. We have studied the streptomycin adsorption capacity of thepodsol soils (Experimental Station Chashnikovo, Moscow Oblast') of cultivated andnoncultivated soils. The results are given in Table 18.

Table 18
Adsorption of streptomycin by the podsol soils at various layers (units/ g)
Soil

A0 Layer

A2 layer

B1-B2 layers

Forest mixed

1,300

700

5,400

Meadow

1,800

1,200

7,400

Roima* of the river Klyaz'ma

3,000

2,400

3,100

Glade

1,500

300

3,000

*[Russian term designating a very low and broad river terace,which may be floodedat the highest water mark. Flood basin but very extensive.]

  The B1-B2layer has the strongest adsorption capacity and the layer A0-A2 the smallest. The degree of adsorption of antibioticsby the soil does not depend solely on the soil properties but also upon the propertiesof the antibiotics themselves. In one and the same soil, for example in chernozem,we have observed the following adsorption:

 

Units/g

µ g

Streptomycin

1,120

2.2

Globiosporin

1,080

1.8

Terramycin

900

1.0

Pennicillin

380

0.1

Preparation 1609

0

--

  The antibiotics in the adsorbed state preserve their antibacterialactivity for some time. The period for which a given antibiotic preserves its activitydepends on the soil and the properties of the antibiotic itself. For example, insame soils penicillin remains active for 20-30 hours, in others 2-3 hours; terramycinremains 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 antibacterialproperties for a more prolonged period than the antibiotics in the free state presentin the soil solution.

  For example, free streptomycin disappears from the podsol soil after10-12 hours, while adsorbed streptomycin is preserved for more than 30 hours. Inthe serozem, the nonadsorbed streptomycin is inactivated after 20-25 hours; its absorbedfraction can be detected even after two days. A still greater difference was observedin the experiment with aureomycin. In podsol it can be detected in the free stateafter 20 hours, in the adsorbed state after 5 days In the serozem the free antibioticis preserved for no more than two days, while in the adsorbed state it retains itsactivity for more than seven days.

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

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

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

  The presence of living microbial cells adsorbed on the soil particlesshould be regarded as a complex system of biotic-mineral complex. Each soil particlecarries elements of living organisms, the study of which is of the utmost importanceto soil biologists.

 

The microflora of the soil

  Microorganisms are an integral part of the soil. If the soil shouldlose these organisms it would lose its main property--fertility--and it would turninto 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 areavailable. 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 quantitativeestimation of microbes in the soil: a) determination by means of soil inoculationof artificial media-liquid and solid, b) direct count of cells.

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

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

  The amounts of micoorganisms detectable in the soil vary, dependingon whether they are inoculated into a solid or liquid medium, or inoculated by sowingthe 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 agarmedium.

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

  Data from the literature on the amount of bacteria, actinomycetesand fungi in the soil are obtained, in the majority of cases, from growth on agarmedia. According to these data, the number of bacteria per gram fluctuates withinthe range of from several tens or hundred thousands to many millions depending uponthe 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 ofhis own investigations on the quantitative determination of the bacterial populationof the soil, considers that the total number of bacteria in one gram of soil reaches50 millions. Since the greatest number of bacteria is concentrated in the plant rhizospheremany authors give data on microbial composition of this zone. Starkey showed, bythe method of counting on agar media, the presence of 199 to 3,470 million bacterialcells per gram, depending upon the species of plant.

  Humfeld and Smith (1932), counted 5-8 billion bacteria in one gramof soil with green fertilizer. Clark (1949) found 5 billion bacteria per gram ofwell-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 andAustria, 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 betweenhundred thousands and millions, but on the average 3-7 million per gram. Bunt andRovina (1955) counted from 400,000 to 15 million bacteria per gram in the subarticsoils of Iceland.

  We have obtained similar figures. We have counted from several hundredthousands 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. Thepodsols 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 contain10-1,000 million cells per gram. Similar data were obtained by many other investigatorsstudying various soils.

  Higher counts (ten and hundred times higher) are obtained by the methodof 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 pergram, and fertile soils (chernozem) from one hundred to 1, 000 million bacteria pergram estimated by the solid-media method, meat-peptone agar (MPA). The method ofinoculations on liquid media, meat-pe ptone broth (MPB) revealed to 10-500 millionand 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 highfigures are constantly obtained during the investigation of plant rhizosphere undergiven conditions.

  Such high indices of microbial population throws doubt on the accuracyof the methods employed. Experiments especially designed to check this method werecarried out. We have assumed that the high figures obtained by this method can beexplained by the adsorption of bacterial cells on the walls of pipettes and withtheir subsequent elution (desorption). Experiments have shown that adsorpton of cellsdoes indeed take place. The number of bacteria is 2-5 times, and sometimes even 10times 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 billionper gram of soil.

  We checked the trustworthiness of this method by using pure culturesof Bact. prodigiosum, Ps. fluorescens, Mycob. rubrum, Az. vinelandii and Bac.subtilis. Aqueous suspensions of these bacteria were diluted with and withoutchange of pipettes.

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

 

With change of pipettes

Without change

Bact. prodigiosum

35.5

38.1

Ps. flourescens

22.8

100

Mycob. rubrum

1.0

4.5

Az. vinelandii

2.1

2.5

Bac. subtilis

3.9

7.3

  In this experiment the change of pipettes lowered the number of bacteria1.5-4 times depending upon the bacterial species. Similar lowering of bacterial numberswas observed on studying soil samples. The difference in the numbers is more pronouncedif the number of bacteria in the soil is large. Hundred billions and more of bacteriawere detected in the rhizosphere of lucerne grown in the Vakhsh valley when the soilsuspension was diluted with one pipette; the number was five times less. when thepipettes were changed. The control soil contained 100-500 millions per gram upondilution with one pipette, 50-150 millions were counted upon dilution, when the pipetteswere changed, i.e., 30-50% less.

  According to Vinogradskii, the method of direct counting of the soilbacteria 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).

Table 19
The number of bacteria detected by various methods in the plow layer of soils under perennial grasses
(in thousands/ g)

Soil

Direct count method

Liquid inoculation

Solid inoculation

Podsol soil, field

560,000

500,000

7,500

Turf-podsol soil, garden, Moscow Oblast'

6,800,000

5,600,000

15,600

Chernozem, Moldavia

8,700,000

7,200,000

25,000

Chestnut soil, Trans-Volga region

3,500,000

1,000,000

9,500

Serozem, Central Asia

9,300,000

7,500,000

90,000

  The difficulty of the method of direct counting is that the smearscontain living cells and dead particles of the same soil. and they cannot be differentiatedwith certainty. The soil always contains a large amount of small particles whichcan be stained and thus become indistinguishable from the bacteria themselves. Itis especially difficult to tell the coccoid cells from the small globular bodiesand granules.

  In recent years some investigators attempted to use fluorescent dyesfor the differentiation of bacteria from the dead soil particles. Burrichter (1953).employed acridine-orange for the staining of soil smears and studied them under afluorescent microscope in ultraviolet light. In soils rich in humus (9. 98 %)theauthor counted 9,453 million bacteria per gram of soil and the total number of microbeswas 18,331 million bacteria per gram of soil; in soil poor in humus (1.80%) 1,230million bacteria per gram were found. The number of colonies of slimy bacteria inthe 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, andsoils poor in organic substances 25 million per gram. Strugger (1948, 1949) foundfrom 1,038 to 8,640 million bacterial cells per gram of soil employing the same methodof fluorochrome staining.

  It should be noted that the method of fluorescent staining also hasshortcom -ings. The green color of living cells or the red color of dead cells andother 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'nikovand Bekhtereva, 1956). It is sometimes difficult to say what are the green or especiallythe red-stained bodies; are they living bacterial cells or dead, or soil particles.

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

  As can be seen from the given data, the existing methods of microscopicanalysis are inadequate for quantitative determination of microbial numbers in thesoil and for the determination of their forms. Therefore, when comparing data obtainedby employing one of the aforementioned methods, the investigators limit themselvesto relative figures.

  The bacterial numbers vary in different soils, according to theirfertility and nutritional qualities. The more fertile the soil, the richer it isin 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 theKuban, contains (similar method of counting) 15-50 million bacterial cells per gramof soil.

  One and the same type of soil also varies in the amount of microbesit contains. The podsol soils, not well cultivated and poor in humus, contain 500,000to 1.5 million cells per gram and in some cases only a few thousand per gram (thesoils of the Kola Peninsula). Well-cultivated, systematically fertilized soils contain3-25 million cells per gram. Garden soils, as a rule, are richer in microbes thanthe soil of fields.

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

Table 20
The number of bacteria and actinomycetes in various soils
(in thousands / g on meat-peptone agar, in Petri dishes)

Soils

Bacteria

Actinomycetes

Podsol containing iron, Kola Peninsula

10-30

5-25

Podsol of Moscow Oblast' from under forest

100-300

70-100

Podsol of Moscow Oblast', garden

1,000-10,000

500-1,000

Chernozem, Kuban', under wheat

5,000-15,000

400-800

Serozem, Central Asia, virgin soil

850-1,500

600-1,000

Serozem, Central Asia, under lucerne

3,000-10,000

500-800

Chestnut soils, Trans-Volga region, virgin soil

400-1,500

450-860

Chestnut soils, Trans Volga region, under lucerne

5,000-15,000

500-1,000

  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 soilsof 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 therhizosphere, the soil is saturated with bacteria to a greater extent than in thezone outside the roots. The vegetative cover, as will be shown later, exerts a stronginfluence on the concentration of microbes in the soil.

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

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

Table 21
Seasonal variations of microbial numbers in the soil of the Severnaya Zemlya
(in thousands/g, counted on meat -peptone agar MPA)

Soil sample

May

August

Sector I, loam

23

1,340

Sector II, loam

40

4,380

Sector III, loose calcareous soil

91

16,600

Sector IV, loam

14

3,600

Sector V, loose calcareous soil

112

6,600

  The number of microbes in the soil of the temperate zone is greatestin 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 duringthe winter. A considerable number of microbes die from cold and their total numberdecreases.

  According to our observations, the microbial activity does not alwayscease in winter. Under a deep snow cover the earth is not always frozen and in sucha soil microbiological processes take place. This can be found by studying the growthdynamics of individual species of actinomycetes. Korenyako has shown that duringthe 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 detoxificationof 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 ofmicrobes. in addition, many soil nutrients under the action of low temperature, changeand become more available to microbes.

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

  The concentration of microorganisms in the soil depends mainly onthe presence of such organic substances as can be easily utilized by bacteria. Thereare fresh plant and animal residues and products of their primary decomposition whichhave 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 growthand multiplication.

  Small doses of these substances markedly enhance the growth and multiplicationof microbial cells as well as that of plants, by promoting various biochemical andphysiological 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 quantityand the total number of the microbial population. Unfortunately, such a correlationis very difficult to study and has not as yet been methodically worked out.

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

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

Table 22
The decrease in bacterial cells during dry storage in the laboratory
(in thousands per gram)

Soils

Fresh samples

Samples after 10 days storage

Chernozem of the Kuibyshev Oblast'

500,000

50,000

Serozem of the Uzbek SSR

150,000

45,000

Podsol of the Moscow Oblast'

3,500

1,500

Chestnut, Trans-Volga region

60,000

10,000

Severnaya Zemlya*

9,300

1,300

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

  Upon storage of samples in the dry state the qualitative compositionof 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 numberof actinomycetes and certain species of mycobacteria is observed. These organismscan 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 differentgroups but also in different species, and even different strains of the same microbeshow 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 serozernsoils. Of some hundred million cells only a few (10-100 cells/g) remained viableafter two weeks storage. Mycobacteria such as Mycob. rubrum and some otherspecies remain viable in considerable quantities (100,000 cells/g and more.)

  Not all strains of Azotobacter, in dry samples of soil, dieout at the same rate. Of twenty cultures of Az. chroococcum studied, eightstrains of Azotobacter survived in considerable numbers--up to 10% and moreof the cells. Of 10 strains of root nodule bacteria of lucerne only about 10% of4 strains survived in dry samples of serozem soils; in the podsol soil only one strainsurvived and then only in a negligible amount (0.5% and less).

  The sporiferous bacteria show the same diversity as far as their survivalcapacity is concerned. About 80-90% of Bac. megatherium dies out in dry podsolsoils 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. Evenin the cultures most sensitive to drying, there are single cells which are stableand survive for long periods in the dry state. Thanks to such cells the species doesnot die out under conditions of prolonged drought.

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

  It is clear that the soil as a whole, and the separate soil aggregatespossess different physicochemical conditions for the life of microbes than thosepresent in isolated soil samples. Some bacterial species grow quicker in naturalsurroundings, others, slower.

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

Table 24
Variations in the microflora in various samples of soil during moist storage
(in thousands/g; counted on meat-peptone agar)

Soils

Bacteria, fresh

Bacteria, after 10 days

Myco- bacteria fresh

Mycob. after 10 days

Actino- mycetes fresh

Actino- mycetes after 10 days

Sector 1, loam

23

40

0.5

8

0

0.5

Sector II, loam

40

56

0.8

22

0

0

Sector III, loose calcareous soil

91

150

1.5

65

0

1.0

Sector IV, loam

13

62

1.0

37

0

0.8

Sector V, loose calcareous soil

112

346

2.5

120

0

1.5

  The total number of bacteria In the samples after 10 days increased2 to 4 times, and the number of mycobacteria 16 to 50 times. Actinomycetes in freshsamples, were almost nonexistent, and after 10 days their numbers reached 500-1,500per 1 gram of soil.

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

  For the determination of the microflora of the soil one has to takeinto account the composition of the medium into which the microorganisms are beinginoculated. Experiments show that organisms from many soils grow better on syntheticmedia of Chapek, CPI, etc, than on media containing protein. On starvation media(water agar) and semistarvation media (Ashby agar) the number of bacteria is often2-5 times greater than on rich nutrient media (Table 25).

Table 25
The number of soil bacteria after inoculation of various media
(in thousands/g)

Medium

Garden soil

Primitive soil, mountainous, 3,800 m

Primitive soil, Severnaya Zemlya

Meat-peptone agar (MPA)

3,500

54

23

Synthetic medium of Chapek

3,800

270

154

Synthetic medium CPI

4,400

850

--

Ashby medium

4,200

680

187

Water agar

3,800

800

--

  It should be pointed out that it is easier to isolate bacteria fromprimitive 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 withthe aid of a a magnifying glass, or even only under a microscope. Such microcoloniesusually consist of a few cells only.

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

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

  On water agar and on the Ashby medium microorganisms usually growat the expense of their food reserves. Among these organisms auxoautotrophs and auxoheterotrophecan be detected likewise. Often the so-called oligonitrophils grow on the nitrogen-lessmedium of Ashby. These are unique forms of bacteria and mycobacteria which are capableof 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 thesemedia. The quantitative ratio of prototrophs to metatrophs varies from soil to soil.In soils rich in humus, and well-fertilized with organic fertilizers, the amountof 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 numbersof organisms in the soil possessing specific functions. Such organisms can be detectedon special, so-called selective media. To such bacteria belong the nitrifiers, sulfurbacteria, 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 thedetection 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 capacityof growing on complex nonselective media. For example, Azotobacter can growon nitrogen-less media due to the capacity of nitrogen fixation, but it can alsogrow 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, usedfor the nitrifiers, bacterial satellites also grow well. On media containing cellulose,not only bacteria capable of decomposing cellulose grow, but also other forms ofbacteria.

  Selective media are not optimal for the growth of bacteria. In. manycases, 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 otherorganic compounds for the growth of the cellulose-decomposing organisms, proteinand nonprotein substances for others, etc (Rotmistrov, 1950).

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

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

  The data presented in this chapter provide the basis for the assumptionthat 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, thesoil 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 presentin 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. About900-1,000 million cells may be present in 1 ml. Cells of larger size (5 µ x1 µ) have a volume of 3. 9 µ3, their weight is 10-8 mg. In 1ml there are about 350 million cells.

  According to the data of Tanson (1948), in 1 ml there are 1,000 cocciof 1 µ in diameter; 330 million sporiferous bacteria of the size of 3 µx 1 µ, and 1 million spores of fungi, 10 µ in diameter. According to VanNiel (1936),there can be 1,400 million cells of Bact. coli per 1 ml; accordingto Butkevich (1938), 10 9 cells weigh 0.5 mg; Jensen (1940) found that10 9 cells of Azotobacter weigh about 5 mg. Similar data are givenby Kendall (1928), Strugger (1948) and some other authors.

  We have obtained the following data on the total microflora of therhizosphere of vegetative plants. There are 2-2.5 kg of cells in a soil under lucernein Central Asia, per 120 kg of soil; i.e., 6,000-7,000 kg of cells per hectare. Outsidethe root zone there are, according to our calculations, 1,500-2,000 kg bacterialcells per hectare of the upper (plow) layer. Consequently, there are about 7-9 tonsof 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 havefound 1,000-3,000 millions of organisms per gram of soil in the rhizosphere and inthe zone outside the roots, 300-800 million organisms per gram of soil. The totalbacterial mass in the root zone amounted to 1,200-3,000 kg and outside the root zoneabout 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 organismsper kg in the rhizosphere, and 100-200 million outside the roots. The total massof 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 percent 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 thesoil. Clark (1949) has shown that the bacteria constitutes 300-3,000 parts per millionby 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 onlybacteria, whereas other organisms living in the soil, such an actinomycetes, fungi,algae, and protozoa are unaccounted for. They comprise a considerable mass of livingsubstance.

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

  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 astatic 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 takesplace.

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

  The intensity of bacterial growth in the soil was determined by thetime required for the doubling of their numbers. Three organisms were used in theexperiment. Az. chroococcum, Ps. aurantiaca and Bact. prodigiosum.A sample of a garden soil was placed in an asbestos bag inoculated with the above-listedorganisms , 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 procedureas 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 millionorganiams,were introduced into the soil. In the first analysis (immediately aftermixing) 26 million Azotobacter cells were washed out. 18 million cells ofPs. aurantiaca and 34 million cells of Bact. prodigiosum (all thesenumbers 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 washedout (the May experiment), in millions/ g:

Analysis

Az. chroococcum

Ps. aurantiaca

Bact. prodigiosum

Second

16

22

14

Third

23

26

10

Fourth

20

28

8

Fifth

24

34

10

  As can be seen from the above data, the doubling of Azotobactercells took place every 5 days, Ps. aurantiaca every 4 days. and Bact. prodigiosumevery 10 days. In other words the number of generations of the first was 6, of thesecond, 7 and the third 3. In July-August the number of generations was 4, 4 and2 respectively, and in September-October, 4, 3, 1 respectively.

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




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