ISSN 0012�4966, Doklady Biological Sciences, 2014, Vol. 455, pp. 87–90. © Pleiades Publishing, Ltd., 2014.Original Russian Text © G.N. Fedotov, L.V. Lysak, 2014, published in Doklady Akademii Nauk, 2014, Vol. 455, No. 1, pp. 114–117.

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Formation of humic substances is known to occurunder the influence of microorganisms that reside insoils or get there along with plant and animal remains[1]. However, there is no unambiguous understandingof the mechanism of this process so far. One of themost widespread theories on the humic substance(HS) formation suggests that when the plant and ani�mal remains (waste) are processed in soil, microor�ganisms secrete enzymes that degrade waste biopoly�mers to the low�molecular�weight components thatserve as nutrients. The researchers have proposed sev�eral mechanisms underlying formation of the poly�meric HS molecule from the products of biopolymerenzymatic degradation. One of them is the oxidativetransformation of these rather simple organic mole�cules and their interaction with each other to producepolymeric HS molecules. Another mechanism sug�gests that HS macromolecules are formed due to poly�merization of the biopolymer destructive productsunder the influence of enzymes (synthetases) that areproduced by microorganisms.1 However, some evi�dence [4] suggests other mechanisms of this process.

It is well known that an excess of waste in soils pro�motes growth of microorganisms the abundance ofwhich is markedly reduced after nutrient exhaustion.Under these conditions, HS may not form immedi�ately after formation of the low molecular weight sub�stances2 in the course of biopolymer decomposition;they may appear only after the death of microorgan�

1 These approaches suggest that microorganism activity and HSformation occur simultaneously and can be referred to as “par�allel” ones. They assume the existence of only HS macromole�cules [1–3].

2 Substances with molecular weights lower than 1000 Da arereferred to as low molecular weight substances.

isms. HS macromolecules and HS supramolecularcompounds can also exist [4].3

Soil study demonstrated that HS form the basis ofsoil gels that bind mineral soil particles into a singlestructure. The humus matrix of soil gel has a multilevelorganization, where 100�nm fractal clusters (F�clus�ters) of the primary HS particles, several nanometersin size, are the main elements [5]. It is known that bac�terial cells in soil are no larger than several microme�ters [6]. Their diameter ranges from 0.2 to 2.5 µm; thelength, from 0.5 to 7.3 µm, an average bacterial cellbeing 0.8–1.5 µm in size. Smaller so�called nanobac�teria (nanoforms of common bacteria), 0.2–0.3 µm insize, are also found in soil [7]. Since F�clusters aremostly about 100 nm in size (100–200 nm), i.e., theyare much smaller than common soil bacteria and bac�terial nanoforms, there is an opportunity to compareand classify the objects observed in soil. This allowedus to determine morphologically the time and locationof HS appearance, as well as to answer the questionwhat is the mechanism of HS formation (“parallel” or“sequential”).

In this study, we aimed at verifying the role ofmicroorganisms in HS formation in soil. We used thefollowing zonal soil samples from the collection of theFaculty of Soil Science, Moscow State University:illuvial�iron podzol; podzolic, sod�podzolic, and greyforest soils; chernozem of different types; light� anddark�chestnut soil; brown semidesert soil; grey and redsoils.

We have also conducted model experiments withvarious bacterial species (Bacillus subtilis, Arthrobacterglobiformis, and Pseudomonas fluorescens) from thecollection of soil microorganisms of the Faculty of SoilScience, Moscow State University.

Bacterial cultures were grown for 4 months on slantagar (Czapek’s agar medium with sucrose). During

3 This approach can be referred to as “sequential” one, i.e., it isbelieved that microorganisms are initially reproduced, which isfollowed by their death and HS formation.

The Possible Role of Microorganisms in Humus Formation in Soils

G. N. Fedotova and L. V. Lysakb

Presented by Academician G.V. Dobrovol'skii March 29, 2013

Received June 24, 2013

DOI: 10.1134/S001249661402001X

a Institute of Ecological Soil Sciences, Moscow State University, Moscow, 119992 Russiab Faculty of Soil Sciences, Moscow State University, Moscow, 119992 Russiae�mail: gennadiy.fedotov@gmail.com

GENERAL BIOLOGY

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this period of time, a portion of the bacterial cells wasexpected to perish, and the opportunity would appearto examine the results of the process by means of ascanning electron microscope.

The samples were prepared for microscopic exam�ination using bacterial biomass removed from agar andplaced into sterile tap water. A drop of suspension wasplaced on the atomically smooth surface of freshly�split mica, and the samples were dried at 40°C.

Microscopic examination was conducted using aJEOL�6060A scanning electron microscope (JEOL,Japan) with a tungsten cathode. Before examination,the samples were covered with platinum using aJFC�1600 device (JEOL, Japan).

Analysis of plant waste in the samples of zonal soilsdemonstrated that, in most cases, they include eitherparticles of F�cluster size or microorganisms along

with the particles of F�cluster size. However, therewere mostly microorganisms on the plant wastes. Amicrophotograph of a material sampled from the hori�zon of podzolic soil serves as an example (Fig. 1). Theparticles of F�cluster size, but not microorganisms,can be observed (Fig. 1a); in another case, microor�ganisms are clearly seen, while the number of particleswith the size of F�clusters is insignificant (Fig. 1b).

Let us analyze the “parallel” and “sequential” pro�cesses in dynamics by means of electron microscopy.In the course of the “parallel” process, when HS areformed from the degradation products of biopolymersdue to the activity of microorganism synthetases, bothmicroorganisms and F�clusters are expected to beobserved at all stages. At the last stage, after exhaustionof nutrients and microorganism death, only F�clustersshould be observed. The probability that only micro�organisms will be seen on the plant surface in theabsence of F�clusters is extremely small; it is possibleonly at the earliest stage of waste decomposition. Ourresults (Fig. 1b) suggest that a significant probability toobserve mostly microorganisms is expected when HSformation is a result of the “sequential” but not the“parallel” pathway. Nevertheless, the results of soilstudy do not allow us to conclude clearly whether the“parallel” or “sequential” mechanism of HS forma�tion occurs in nature.

Analysis of microorganism transformation at thelast stage of their growth provided more unambiguousinformation. It turned out that the death of somemicroorganism populations was accompanied at thelatest stages by breaking down of microorganisms intoparticles having the size of F�clusters. This is charac�teristic of both Gram�positive (Arthrobacter globifor�mis) and Gram�negative (Pseudomonas fluorescens)bacteria (Fig. 2). Spore�forming Gram�positive bacte�ria (Bacillus subtilis) formed spores. Autolysis of somecells of these bacteria resulted in the formation of par�ticles similar in size to F�clusters (Fig. 3).

Summarizing all the experimental data, we caninfer the following:

(1) Microorganisms were observed on the plantwaste, while F�clusters were almost absent, whichwould be impossible if HS formation occurred due tothe “parallel” mechanism.

(2) Microorganism dying was accompanied by for�mation of particles similar in size to F�clusters, whichtestifies to the “sequential” mechanism of HS forma�tion.

Thus, our results based on micromorphologicaldata suggest indirectly that HS formation in soilsoccurs through the “sequential” mechanism after thedeath of microorganisms and hence, our results rejectthe “parallel” mechanism.

Analysis of the process of HS formation supportsthis conclusion additionally. Soil�dwelling microor�

0.5 µm(a)

0.5 µm(b)

Fig. 1. Electron microscope photography of plant waste ina sample of the podzolic soil horizon. (a) Microorganismsare absent on plant waste, but particles similar in size to F�clusters are clearly seen. (b) Microorganisms are presenton plant waste, but there are no particles the size of F�clus�ters.

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THE POSSIBLE ROLE OF MICROORGANISMS IN HUMUS FORMATION IN SOILS 89

ganisms are expected to adapt the ambience to supporttheir existence; hence, the products of cell degrada�tion have to fulfill an important function after reuti�lization, and they cannot be regarded as accidental by�products that remain in soil due to their high resis�tance [3]. Perhaps, HS are used by microorganisms asa store of nutrients to support soil homeostasis. But the“parallel” mechanism of HS formation hardly under�lies soil homeostasis, because it implies secretion of

enzymes for biopolymer decomposition, enzymes forpolymerization of organic molecules that are productsof biopolymer degradation, and enzymes that causeHS degradation. Enzyme formation is an energy�con�suming process, and it is difficult to expect that micro�organisms save energy in this case [1]. Thus, in termsof the “parallel” mechanism, it is difficult to explainthat HS serves as a store of nutrients for microorgan�isms which consume too much of energy for the syn�

0.5 µmFragment 1 Fragment 2 Fragment 3

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Fig. 2. Electronmicroscope photography of Arthrobacter globiformis. (1) Microorganisms; (2) particles are the size of F�clusters.

Fragment 31 µm

1 2 3

Fragment 2Fragment 1

Fig. 3. Electron microscope photography of Bacillus subtilis. (1) Particles the size of F�clusters; (2) microorganism envelopes;(3) spores of microorganisms.

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thesis of enzymes involved in the formation and degra�dation of HS biopolymers (macromolecules).

Our experiments and analysis of HS formation insoil suggest that, when microorganisms perish in thedying cells, the “sequential” mechanism underlies theformation of the fractal clusters from HS supermole�cules, probably, with involvement of a certain primermolecule.

REFERENCES

1. Tate, R.L., III, Soil Organic Matter: Biological and Eco�logical Effects, New York: John Wiley and Sons, 1987.

2. Kononova, M.M., Organicheskoe veshchestvo pochv(Soil Organic Matter), Moscow: Akad. Nauk SSSR,1963.

3. Orlov, D.S., Gumusovye kisloty pochv i obshchaya teor�iya gumifikatsii (Soil Humic Acids and the GeneralTheory of Humification), Moscow: Mosk. Gos. Univ.,1990.

4. Piccolo, A., Soil Sci., 2001, vol. 166, no. 11, p. 166.

5. Fedotov, G.N. and Dobrovol’skii, G.V., Pochvovedenie,2012, no. 8, pp. 908–920.

6. Guzev, V.S. and Zvyagintsev, D.G., Mikrobiologiya,2003, vol. 72, no. 2, pp. 221–227.

7. Lysak, L.V., Lapygina, E.V., Konova, I.A., and Zvyag�intsev, D.G., Pochvovedenie, 2010, no. 7, pp. 819–824.

Translated by A. Nikolaeva