ISSN 1064�2293, Eurasian Soil Science, 2011, Vol. 44, No. 8, pp. 893–896. © Pleiades Publishing, Ltd., 2011.Original Russian Text © J. Frouz, X. Li, A. Brune, V. Pizl, E.V. Abakumov, 2011, published in Pochvovedenie, 2011, No. 8, pp. 973–977.

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INTRODUCTION

Humic substances play an important role in manysoil processes; they favor the regulation of the acid–base properties of soils, their water�retention capacity,and their nutrient�exchange capacity [21], which isespecially important at the early pedogenesis stage fordeveloping zoocenoses and phytocenoses. Althoughthe formation mechanisms of humic substances areextensively covered in the literature, there are stillproblems to be solved.

Humic acids are formed during the decompositionand further transformation of dead organic matter.Kononova [7], Flaig [13], and Komissarov [6] held tothe hypothesis of the condensation�polymerizationmechanism of humification, which was to a certaindegree confirmed by the EPR spectroscopy study ofhumic acids [8]. Orlov believed that the condensationhypothesis has some shortcomings [5]; in particular, itprovides no explanation for the appearance of car�boxyl groups in humic substances. Nonetheless, Orlovconsidered this hypothesis as one of the main humifi�cation concepts, along with his own view (the kineticconcept) and Aleksandrova’s theory [2].

In this context, the study of different condensationmechanisms is also of current importance, especially

the aspects of soil fauna’s participation in the humifi�cation process. The complete polymerization of phe�nols and proteins (or amino acid fragments) is one ofthe possible humification processes [13, 21]. Thisreaction is used in laboratory experiments for the syn�thesis of artificial humic substances, whose propertiesare usually similar to those of natural analogues [19].The implementation of this reaction under laboratoryconditions requires the following conditions: an alka�line medium, anaerobiosis, and peroxidase activity[19]. These conditions are met in the digestive tracts ofmost saprophagous invertebrates [3, 15]. Earthwormsfavor an increase in the polyphenol oxidase activity inthe soil, which presumably has a positive effect on thehumification [4].

In this context, a question arises as to whether thepassage of plant residues through the digestive tractcan favor the formation of humic substances duringthe interaction of proteins and phenols. Seeing thatsome soil invertebrates contribute not only to thetransformation of the consumed organic material butalso to the interaction of organic matter with clay min�erals, an experiment was performed for the determina�tion of the role of clay minerals in the reaction betweenphenols and proteins.

Effect of Soil Invertebrates on the Formation of Humic Substances under Laboratory Conditions

J. Frouza, X. Lib, A. Bruneb, V. Pizla, and E. V. Abakumovc

a Institute of Soil Biology, Academy of Sciences of the Czech Republic, Na Sádkách 7, CZ�37005, Ceské Budéjovice, Czech Republic

b University of Konstanz, 78457 Konstanz, Germanyc St. Petersburg State University, 16 Liniya Vasil’evskogo o�va 29, St. Petersburg, 199178 Russia

Received August 18, 2010; in final form, February 7, 2011

Abstract—The complete polymerization of phenols and proteins (one of the processes involved in the forma�tion of humic substances) was explained. It was shown that fly (Bibio marci) larvae and earthworms (Aporrec�todea caliginosa) participate in the complete polymerization of phenols and proteins. In a laboratory experi�ment, invertebrates participated in the degradation of organic matter and the synthesis of humic substances,which was proved in experiments with 14C�labeled phenols and proteins. The same organic substances (phe�nols and proteins) without the impact of invertebrates were used as the control substances. The distributionsof the 14C isotope in alkaline extracts separated by solubility in acids (humic and fulvic acids) was comparedto those of the control substances. The portion of the 14C isotope in the humic acids in the excrements of Bibiomarci was higher than that in the control substances. The content of 14C�labeled humic substances in theexcrements of the earthworm Aporrectodea caliginosa exceeded the control values only in the experiment withproteins. When clay material was added to the organic substances, the portion of the 14C isotope in the humicacids increased in both experiments with phenols and proteins. When these substrates passed through thedigestive tracts of the invertebrates, the polymerization of organic substances and the inclusion of proteinsand phenols into humic acids occurred.

DOI: 10.1134/S1064229311080047

SOIL BIOLOGY

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EXPERIMENTAL

Two species of soil invertebrates were used in theexperiment: the larva of St. Mark’s fly (Bibio marci L.,Diptera, Bibionidae) inhabiting forest litter on thesurface of soils at the early decomposition stages ofplant material, and the earthworm Aporrectoria caligi�nosa (Lumbricidae) usually consuming more decom�posing organic material together with mineral soilmaterial.

Both invertebrate species were collected in alderplantations of posttechnogenic ecosystems of coalquarry dumps in the region of the town of Sokolov[1, 12]. Plant material (alder leaves) and clay sub�strates were collected at the same place. The clay sub�strates were sampled from tertiary clay dumps at theearly stages of self�overgrowth. From the X�ray dif�fraction analysis [16], the clay dump material con�sisted of a mixture of kaolinite, illite, and finely dis�persed calcium carbonate.

Before the beginning of the experiment, the inver�tebrates were kept in the dark at 15°C. The alder plantmaterial was sorted manually in sterilized water; theseparate leaves were dried on filter paper and cut intopieces 1 × 2 cm in size. The excrements of B. marciwere obtained by placing the larvae in litter.

The experiment was performed in Erlenmeyerflasks of 500 ml. The experimental flasks containedone of three sources of organic matter: alder litter,larva excrements, or a mixture of larva excrementswith clay. 14�C�labeled proteins were added to half ofthe flasks, and 14�C�labeled phenols were added to theother flasks. Thus, each type of organic material con�tained labeled phenols or proteins.

The labeled proteins were obtained from a labora�tory culture of Bacillus megaterium according to themethod described in [17], and the labeled phenolswere supplied by Fluka. The specific activity of theproteins was 2.25 × 106 dpm/mg, and that of the phe�nols was 2.43 × 106 dpm/mg.

Half of the samples of the proteins and phenolsadded to the leaves, excrements, and the mixture ofclay and excrements were used as the control group,and the invertebrates were placed in the other samples.Bibio marci larvae were placed in flasks with aldermaterial, and earthworms (Aporrectodea caliginosa)were placed in excrements of Bibio marci or in themixture of excrements and clay. The flasks were closedwith rubber valves, and the carbon dioxide released bythe invertebrates and microorganisms was trappedwith a sodium hydroxide solution to prevent the con�tamination of the laboratory with radioactive carbondioxide. The flasks were ventilated every two days. Oneweek later, the experiment was stopped and theorganic matter was sampled. The earthworms wereextracted from the flasks, and all the material remain�ing in the flasks and the organic matter was sampled.From the flasks with the alder material, samples weretaken from the treatments with and without larvae.

The larvae were manually sorted; their digestive tractswere separated, and the residual leaves and excrementswere collected and sieved through a 0.5�mm sieve. Theresidual organic matter from the treatments with andwithout the earthworms, as well as the residual mate�rial from the treatments with the larvae, was subjectedto alkaline extraction (by the IHSS method using0.25�g samples); then, the humic and fulvic acids wereisolated by the conventional method based on theirsolubility in sulfuric acid [22]. Because the fulvic acidfraction was obtained by the dissolution of the organicmaterial in sulfuric acid, it contained, along with ful�vic acids, amino acids and phenols. It was supposedthat the increase in the portion of phenols and proteinsincluded in the humic acids, compared to the fulvicacids, indicated an increase in the rate of the synthesisof humic substances by complete polymerization.

RESULTS AND DISCUSSION

In the experiment with B. marci larvae, the portionof phenols and proteins included in the humic acidsfrom the excrements was significantly higher than inthe initial material (reliable differences at the level P <0.05). The content of phenols and proteins decreasedin the fulvic acids isolated from the larva excrements(Fig. 1), which well agreed with an increase in theircontent in the humic acids.

In the materials extracted from the digestive tracts,the portion of phenols and proteins included in thefulvic acid fraction was reliably higher than in thehumic acids and lower than in the insoluble residue.This indicated the inclusion of phenols in the fulvicacid fraction of the organic matter during its occur�rence in the digestive tract of the larvae.

The reworking of the litter by the earthworm Apor�rectodea caliginosa increased the portion of proteinsincluded in the humic acids and decreased the portionof proteins included in the fulvic acid fraction (Fig. 2).No effect of earthworms on the content of phenols wasobserved in the materials studied. These differenceswere reliable (at P < 0.05 for the portion of proteinsincluded in the humic acids and at P < 0.01 for theportion of proteins included in the fulvic acid frac�tion).

The addition of clay significantly affected the dis�tribution of the phenols (Fig. 3). In the control treat�ment (without earthworms), it was found that theaddition of clay significantly (P < 0.05) increased thecontent of phenols in the humic acid fraction, whilethe portion of phenols in the insoluble fraction wasreliably lower (P < 0.05). The effect of the clay addi�tion was also manifested in the treatment with theearthworms. The portion of phenols included in thehumic acids reliably increased (P < 0.05), and the por�tion of phenols in the fulvic acids decreased (P < 0.05).

In accordance with the earlier data [10, 14], thecontent of humic acids in the organic or organomin�eral material increases during the passage through the

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EFFECT OF SOIL INVERTEBRATES ON THE FORMATION OF HUMIC 895

digestive tract of the invertebrates. Our data show thatthe portion of proteins and phenols in the humic acidsincreases under the conditions of the digestive tract.This process is significantly affected by the followingfactors: (1) the alkaline conditions in the digestivetract, which increases the solubility of phenols andamino acids [18]; (2) the shift toward anaerobic(microaerophilic) conditions in the digestive tract ofthe soil invertebrates [18]; (3) the presence of proteasein the digestive tract of the soil invertebrates, whichcan result in the decomposition of proteins and an

increase in the concentration of free amino acids fromlitter [11, 12]; (4) the presence of peroxidases widelydistributed in the digestive tracts of the invertebrates[15], which favors the humification process [20]; and(5) the increase in the polyphenol oxidase activity inthe earthworm excrements [4].

The mechanisms of these interactions can varydepending on the composition of the soil invertebrategroups, as was shown with the Bibio marci larvae andthe earthworms Aporrectodea caliginosa as an example.

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Fig. 1. Distributions of 14C�labeled proteins and phenols among the humic acids, fulvic acids, and insoluble residue in an exper�iment with larvae: (1) plant material; (2) material from the digestive tract of flies; (3) fly excrements.

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Fig. 2. Distributions of 14C�labeled proteins and phenols among the humic acids, fulvic acids, and insoluble residue in experi�ments with (I) earthworms and (II) earthworms and clay: (1) control treatment; (2) treatment with earthworms.

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In addition to the above mechanisms, earthworms canincrease the role of clay material in humification [9].

CONCLUSIONS

On the basis of a laboratory experiment, the com�plete polymerization of phenols and proteins (one ofthe processes involved in the formation of humic sub�stances) was explained. It was shown that fly (Bibiomarci) larvae and earthworms (Aporrectodea caligi�nosa) favor the polymerization of phenols and pro�teins.

The performed experiments revealed significantchanges in the organic matter during the passage oforganic substances through the digestive tracts ofinvertebrates, which are manifested in the polymeriza�tion of components forming humic acids.

ACKNOWLEDGMENTS

This work was supported by the Czech ScienceFoundation (grant no. 526/01/1055), the Ministry ofYouth and Science (grant no. 2B08023), the SlovenianCoal Company, and by the target federal program“Studies on the Priority Directions of the Develop�ment of Science and Technology in Russia for 2007–2012” (state contract no. 16.512.11.2161).

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