ISSN 00124966, 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.
GENERAL BIOLOGY
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
Formation of humic substances is known to occur under the influence of microorganisms that reside in soils or get there along with plant and animal remains [1]. However, there is no unambiguous understanding of the mechanism of this process so far. One of the most 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 lowmolecularweight components that serve as nutrients. The researchers have proposed sev eral mechanisms underlying formation of the poly meric HS molecule from the products of biopolymer enzymatic degradation. One of them is the oxidative transformation of these rather simple organic mole cules and their interaction with each other to produce polymeric HS molecules. Another mechanism sug gests that HS macromolecules are formed due to poly merization of the biopolymer destructive products under the influence of enzymes (synthetases) that are produced 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 of which 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
isms. HS macromolecules and HS supramolecular compounds can also exist [4].3 Soil study demonstrated that HS form the basis of soil gels that bind mineral soil particles into a single structure. The humus matrix of soil gel has a multilevel organization, where 100nm fractal clusters (Fclus ters) of the primary HS particles, several nanometers in 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; the length, from 0.5 to 7.3 µm, an average bacterial cell being 0.8–1.5 µm in size. Smaller socalled nanobac teria (nanoforms of common bacteria), 0.2–0.3 µm in size, are also found in soil [7]. Since Fclusters are mostly about 100 nm in size (100–200 nm), i.e., they are much smaller than common soil bacteria and bac terial nanoforms, there is an opportunity to compare and classify the objects observed in soil. This allowed us to determine morphologically the time and location of HS appearance, as well as to answer the question what is the mechanism of HS formation (“parallel” or “sequential”). In this study, we aimed at verifying the role of microorganisms in HS formation in soil. We used the following zonal soil samples from the collection of the Faculty of Soil Science, Moscow State University: illuvialiron podzol; podzolic, sodpodzolic, and grey forest soils; chernozem of different types; light and darkchestnut soil; brown semidesert soil; grey and red soils. We have also conducted model experiments with various bacterial species (Bacillus subtilis, Arthrobacter globiformis, and Pseudomonas fluorescens) from the collection of soil microorganisms of the Faculty of Soil Science, Moscow State University. Bacterial cultures were grown for 4 months on slant agar (Czapek’s agar medium with sucrose). During
1 These
approaches suggest that microorganism activity and HS formation 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 are referred to as low molecular weight substances. a
Institute of Ecological Soil Sciences, Moscow State University, Moscow, 119992 Russia b Faculty of Soil Sciences, Moscow State University, Moscow, 119992 Russia email: [email protected]
3 This
approach can be referred to as “sequential” one, i.e., it is believed that microorganisms are initially reproduced, which is followed by their death and HS formation.
87
88
FEDOTOV, LYSAK
(a)
0.5 µm
(b)
0.5 µm Fig. 1. Electron microscope photography of plant waste in a sample of the podzolic soil horizon. (a) Microorganisms are absent on plant waste, but particles similar in size to F clusters are clearly seen. (b) Microorganisms are present on plant waste, but there are no particles the size of Fclus ters.
this period of time, a portion of the bacterial cells was expected to perish, and the opportunity would appear to examine the results of the process by means of a scanning electron microscope. The samples were prepared for microscopic exam ination using bacterial biomass removed from agar and placed into sterile tap water. A drop of suspension was placed on the atomically smooth surface of freshly split mica, and the samples were dried at 40°C. Microscopic examination was conducted using a JEOL6060A scanning electron microscope (JEOL, Japan) with a tungsten cathode. Before examination, the samples were covered with platinum using a JFC1600 device (JEOL, Japan). Analysis of plant waste in the samples of zonal soils demonstrated that, in most cases, they include either particles of Fcluster size or microorganisms along
with the particles of Fcluster size. However, there were mostly microorganisms on the plant wastes. A microphotograph of a material sampled from the hori zon of podzolic soil serves as an example (Fig. 1). The particles of Fcluster size, but not microorganisms, can be observed (Fig. 1a); in another case, microor ganisms are clearly seen, while the number of particles with the size of Fclusters 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 are formed from the degradation products of biopolymers due to the activity of microorganism synthetases, both microorganisms and Fclusters are expected to be observed at all stages. At the last stage, after exhaustion of nutrients and microorganism death, only Fclusters should be observed. The probability that only micro organisms will be seen on the plant surface in the absence of Fclusters is extremely small; it is possible only at the earliest stage of waste decomposition. Our results (Fig. 1b) suggest that a significant probability to observe mostly microorganisms is expected when HS formation is a result of the “sequential” but not the “parallel” pathway. Nevertheless, the results of soil study 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 the last stage of their growth provided more unambiguous information. It turned out that the death of some microorganism populations was accompanied at the latest stages by breaking down of microorganisms into particles having the size of Fclusters. This is charac teristic of both Grampositive (Arthrobacter globifor mis) and Gramnegative (Pseudomonas fluorescens) bacteria (Fig. 2). Sporeforming Grampositive bacte ria (Bacillus subtilis) formed spores. Autolysis of some cells of these bacteria resulted in the formation of par ticles similar in size to Fclusters (Fig. 3). Summarizing all the experimental data, we can infer the following: (1) Microorganisms were observed on the plant waste, while Fclusters were almost absent, which would be impossible if HS formation occurred due to the “parallel” mechanism. (2) Microorganism dying was accompanied by for mation of particles similar in size to Fclusters, which testifies to the “sequential” mechanism of HS forma tion. Thus, our results based on micromorphological data suggest indirectly that HS formation in soils occurs through the “sequential” mechanism after the death of microorganisms and hence, our results reject the “parallel” mechanism. Analysis of the process of HS formation supports this conclusion additionally. Soildwelling microor DOKLADY BIOLOGICAL SCIENCES
Vol. 455
2014
THE POSSIBLE ROLE OF MICROORGANISMS IN HUMUS FORMATION IN SOILS 1
Fragment 1
0.5 µm
89
2
Fragment 2
Fragment 3
Fig. 2. Electronmicroscope photography of Arthrobacter globiformis. (1) Microorganisms; (2) particles are the size of Fclusters.
1
2
Fragment 1
3
Fragment 2
1 µm
Fragment 3
Fig. 3. Electron microscope photography of Bacillus subtilis. (1) Particles the size of Fclusters; (2) microorganism envelopes; (3) spores of microorganisms.
ganisms are expected to adapt the ambience to support their 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 as a store of nutrients to support soil homeostasis. But the “parallel” mechanism of HS formation hardly under lies soil homeostasis, because it implies secretion of DOKLADY BIOLOGICAL SCIENCES
Vol. 455
2014
enzymes for biopolymer decomposition, enzymes for polymerization of organic molecules that are products of biopolymer degradation, and enzymes that cause HS degradation. Enzyme formation is an energycon suming process, and it is difficult to expect that micro organisms save energy in this case [1]. Thus, in terms of the “parallel” mechanism, it is difficult to explain that HS serves as a store of nutrients for microorgan isms which consume too much of energy for the syn
90
FEDOTOV, LYSAK
thesis of enzymes involved in the formation and degra dation of HS biopolymers (macromolecules). Our experiments and analysis of HS formation in soil suggest that, when microorganisms perish in the dying cells, the “sequential” mechanism underlies the formation of the fractal clusters from HS supermole cules, probably, with involvement of a certain primer molecule.
3. Orlov, D.S., Gumusovye kisloty pochv i obshchaya teor iya gumifikatsii (Soil Humic Acids and the General Theory 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.
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.
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
DOKLADY BIOLOGICAL SCIENCES
Vol. 455
2014
GENERAL BIOLOGY
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
Formation of humic substances is known to occur under the influence of microorganisms that reside in soils or get there along with plant and animal remains [1]. However, there is no unambiguous understanding of the mechanism of this process so far. One of the most 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 lowmolecularweight components that serve as nutrients. The researchers have proposed sev eral mechanisms underlying formation of the poly meric HS molecule from the products of biopolymer enzymatic degradation. One of them is the oxidative transformation of these rather simple organic mole cules and their interaction with each other to produce polymeric HS molecules. Another mechanism sug gests that HS macromolecules are formed due to poly merization of the biopolymer destructive products under the influence of enzymes (synthetases) that are produced 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 of which 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
isms. HS macromolecules and HS supramolecular compounds can also exist [4].3 Soil study demonstrated that HS form the basis of soil gels that bind mineral soil particles into a single structure. The humus matrix of soil gel has a multilevel organization, where 100nm fractal clusters (Fclus ters) of the primary HS particles, several nanometers in 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; the length, from 0.5 to 7.3 µm, an average bacterial cell being 0.8–1.5 µm in size. Smaller socalled nanobac teria (nanoforms of common bacteria), 0.2–0.3 µm in size, are also found in soil [7]. Since Fclusters are mostly about 100 nm in size (100–200 nm), i.e., they are much smaller than common soil bacteria and bac terial nanoforms, there is an opportunity to compare and classify the objects observed in soil. This allowed us to determine morphologically the time and location of HS appearance, as well as to answer the question what is the mechanism of HS formation (“parallel” or “sequential”). In this study, we aimed at verifying the role of microorganisms in HS formation in soil. We used the following zonal soil samples from the collection of the Faculty of Soil Science, Moscow State University: illuvialiron podzol; podzolic, sodpodzolic, and grey forest soils; chernozem of different types; light and darkchestnut soil; brown semidesert soil; grey and red soils. We have also conducted model experiments with various bacterial species (Bacillus subtilis, Arthrobacter globiformis, and Pseudomonas fluorescens) from the collection of soil microorganisms of the Faculty of Soil Science, Moscow State University. Bacterial cultures were grown for 4 months on slant agar (Czapek’s agar medium with sucrose). During
1 These
approaches suggest that microorganism activity and HS formation 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 are referred to as low molecular weight substances. a
Institute of Ecological Soil Sciences, Moscow State University, Moscow, 119992 Russia b Faculty of Soil Sciences, Moscow State University, Moscow, 119992 Russia email: [email protected]
3 This
approach can be referred to as “sequential” one, i.e., it is believed that microorganisms are initially reproduced, which is followed by their death and HS formation.
87
88
FEDOTOV, LYSAK
(a)
0.5 µm
(b)
0.5 µm Fig. 1. Electron microscope photography of plant waste in a sample of the podzolic soil horizon. (a) Microorganisms are absent on plant waste, but particles similar in size to F clusters are clearly seen. (b) Microorganisms are present on plant waste, but there are no particles the size of Fclus ters.
this period of time, a portion of the bacterial cells was expected to perish, and the opportunity would appear to examine the results of the process by means of a scanning electron microscope. The samples were prepared for microscopic exam ination using bacterial biomass removed from agar and placed into sterile tap water. A drop of suspension was placed on the atomically smooth surface of freshly split mica, and the samples were dried at 40°C. Microscopic examination was conducted using a JEOL6060A scanning electron microscope (JEOL, Japan) with a tungsten cathode. Before examination, the samples were covered with platinum using a JFC1600 device (JEOL, Japan). Analysis of plant waste in the samples of zonal soils demonstrated that, in most cases, they include either particles of Fcluster size or microorganisms along
with the particles of Fcluster size. However, there were mostly microorganisms on the plant wastes. A microphotograph of a material sampled from the hori zon of podzolic soil serves as an example (Fig. 1). The particles of Fcluster size, but not microorganisms, can be observed (Fig. 1a); in another case, microor ganisms are clearly seen, while the number of particles with the size of Fclusters 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 are formed from the degradation products of biopolymers due to the activity of microorganism synthetases, both microorganisms and Fclusters are expected to be observed at all stages. At the last stage, after exhaustion of nutrients and microorganism death, only Fclusters should be observed. The probability that only micro organisms will be seen on the plant surface in the absence of Fclusters is extremely small; it is possible only at the earliest stage of waste decomposition. Our results (Fig. 1b) suggest that a significant probability to observe mostly microorganisms is expected when HS formation is a result of the “sequential” but not the “parallel” pathway. Nevertheless, the results of soil study 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 the last stage of their growth provided more unambiguous information. It turned out that the death of some microorganism populations was accompanied at the latest stages by breaking down of microorganisms into particles having the size of Fclusters. This is charac teristic of both Grampositive (Arthrobacter globifor mis) and Gramnegative (Pseudomonas fluorescens) bacteria (Fig. 2). Sporeforming Grampositive bacte ria (Bacillus subtilis) formed spores. Autolysis of some cells of these bacteria resulted in the formation of par ticles similar in size to Fclusters (Fig. 3). Summarizing all the experimental data, we can infer the following: (1) Microorganisms were observed on the plant waste, while Fclusters were almost absent, which would be impossible if HS formation occurred due to the “parallel” mechanism. (2) Microorganism dying was accompanied by for mation of particles similar in size to Fclusters, which testifies to the “sequential” mechanism of HS forma tion. Thus, our results based on micromorphological data suggest indirectly that HS formation in soils occurs through the “sequential” mechanism after the death of microorganisms and hence, our results reject the “parallel” mechanism. Analysis of the process of HS formation supports this conclusion additionally. Soildwelling microor DOKLADY BIOLOGICAL SCIENCES
Vol. 455
2014
THE POSSIBLE ROLE OF MICROORGANISMS IN HUMUS FORMATION IN SOILS 1
Fragment 1
0.5 µm
89
2
Fragment 2
Fragment 3
Fig. 2. Electronmicroscope photography of Arthrobacter globiformis. (1) Microorganisms; (2) particles are the size of Fclusters.
1
2
Fragment 1
3
Fragment 2
1 µm
Fragment 3
Fig. 3. Electron microscope photography of Bacillus subtilis. (1) Particles the size of Fclusters; (2) microorganism envelopes; (3) spores of microorganisms.
ganisms are expected to adapt the ambience to support their 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 as a store of nutrients to support soil homeostasis. But the “parallel” mechanism of HS formation hardly under lies soil homeostasis, because it implies secretion of DOKLADY BIOLOGICAL SCIENCES
Vol. 455
2014
enzymes for biopolymer decomposition, enzymes for polymerization of organic molecules that are products of biopolymer degradation, and enzymes that cause HS degradation. Enzyme formation is an energycon suming process, and it is difficult to expect that micro organisms save energy in this case [1]. Thus, in terms of the “parallel” mechanism, it is difficult to explain that HS serves as a store of nutrients for microorgan isms which consume too much of energy for the syn
90
FEDOTOV, LYSAK
thesis of enzymes involved in the formation and degra dation of HS biopolymers (macromolecules). Our experiments and analysis of HS formation in soil suggest that, when microorganisms perish in the dying cells, the “sequential” mechanism underlies the formation of the fractal clusters from HS supermole cules, probably, with involvement of a certain primer molecule.
3. Orlov, D.S., Gumusovye kisloty pochv i obshchaya teor iya gumifikatsii (Soil Humic Acids and the General Theory 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.
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.
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
DOKLADY BIOLOGICAL SCIENCES
Vol. 455
2014
