A. G. Zavarzina

Accelerated fractionation of heavy metals in contaminated soils and sediments using rotating coiled columns

A new approach to performing an accelerated sequential extraction of trace elements from solid samples has been proposed. It has been shown that rotating coiled columns (RCC) earlier used in counter-current chromatography can be successfully applied to the dynamic leaching of heavy metals from soils and sediments. A solid sample was retained in the rotating column as the stationary phase under the action of centrifugal forces while different eluents (aqueous solutions of complexing reagents, mineral salts and acids) were continuously pumped through. The procedure developed is time saving and requires only 4-5 h instead of the several days needed for traditional sequential extraction (TSE), complete automation being possible. Losses of solid sample are minimal. In most cases the recoveries of readily bioavailable and leachable forms of Pb, Zn, and Cd are higher, if a dynamic extraction in RCC is used. Since naturally occurring processes are always dynamic, continuous extraction in RCC may help to estimate the contents of leachable forms and their potential risk for the environment more correctly than batch TSE. The Kersten-Foerstner and McLaren-Crawford leaching schemes have been compared, the former has been found to be preferable.

Laccase and tyrosinase activities in lichens

Phenoloxidase activity was found in lichenized ascomycetes belonging to different taxonomic groups. Most of the epigeic and epilithic lichens of the order Peltigerales were found to possess both laccase and tyrosinase activities; the lichens of the order Lecanorales possessed only laccase activity, which was an order of magnitude lower than that of Peltigerales. Water-soluble phenoloxidases were present only in peltigerous lichens: activity that could be washed out from intact thalli comprised 10% of that released from disrupted thalli. The activity of the peltigerous lichens and the release of soluble phenoloxidases into the medium increased when the thalli were rehydrated quickly. In some of the lichens tested, the phenoloxidase activity was stimulated by desiccation-rehydration cycles. The oxidases discovered may play an important role in the phenolic metabolism of lichens and be involved in the biochemical reaction of humus synthesis during primary soil formation, which may be a previously unknown geochemical function of these symbiotic microorganisms.

Extraction of humic acids and their fractions in poly(ethylene glycol)-based aqueous biphasic systems

The possibility of extraction and fractionation of humic acid (HA) in the aqueous biphasic systems (ABS) was shown for the first time. The 10% PEG–10% (NH 4)2SO4–H2O and 5% PEG–7.5% dextran (or dextran sulphate, sodium salt)–H2O systems were used. HA originated from peat, soddy-podzolic soil and chernozem were studied. The distribution coefficients were measured for HA of different origin, size of the molecules, molecular weights (MW) of the polymers and pH of the system. The PEG–(NH4)2SO4–H2O system was found to be better for preconcentration and isolation of HA, whereas the PEG–dextran–H2O system is preferable for HA fractionation. The extractability of HA in ABS increases with increasing the MW of HA molecules. Peat HA are extracted in ABS with higher distribution coefficients compared with chernozem and soddy-podzolic soil HA. It is consistent with higher hydrophobicity of peat HA revealed by hydrophobic interaction chromatography. ABS are promising for HA separation into fractions that differ in their hydrophobic/hydrophilic properties as well as for comparing HA of different origin by their relative hydrophobicity.

Water-soluble phenolic compounds in lichens

The quantity and the qualitative composition (for some species) of phenolic compounds (PC) washed out of the intact thalli of lichens of the orders Peltigerales (the genera Peltigera, Solorina, and Nephroma) and Lecanorales (the genera Cladonia, Alectoria, and Cetraria) were studied. It was shown that the quantity of leachable PCs in Peltigerales was on average 2–3 times higher than in Lecanorales. At the same time, the extractability of PC from intact thalli by water was higher in Lecanorales than in Peltigerales: 48–88% and 34–70%, respectively, of the PC content in ethanol extracts from crushed thalli (i.e., of the total content of soluble PC). Water-soluble PC in the lichens Peltigera aphthosa, Solorina crocea, Cetraria islandica, Flavocetraria nivalis, Cladonia uncialis, and Cladonia arbuscula were represented by 7–12 phenolic compounds with similar qualitative composition in the species of the same order. The most part of water soluble PC were phenylpropanoids. All of the studied species showed the presence of p-hydroxybenzoic acid derivatives; vanillic and protocatechuic acid derivatives were found in Cetraria and Cladonia species, respectively.

A mineral support and biotic catalyst are essential in the formation of highly polymeric soil humic substances

The hypothesis was proposed that highly polymeric humic substances in the mineral horizons of soils in a temperate humid climate originate from polymerization of water-soluble structural precursors directly on mineral surfaces under the catalytic effect of immobilized phenoloxidases (heterophasic biocatalysis). This hypothesis was confirmed by a laboratory experiment using a mixture of monomeric phenols and nitrogenous compounds as structural precursors, fungal laccase as a biotic catalyst, and a hydroxyaluminum-kaolinite complex as a mineral support. Enzymic oxidation of phenolic precursors on the mineral surface was substantially more rapid than abiotic oxidation and led to synthesis of a highly polymeric fraction with a molecular weight over 75 kDa. These products were not produced on the mineral with an absence of laccase (abiotic catalysis) or in solution without the mineral matrix (homogeneous catalysis).

Fungal Oxidoreductases and Humification in Forest Soils

Humification is aerobic, largely oxidative process of non-living organic matter biotransformation into recalcitrant humic substances (HS). HS comprise up to 90% of soil organic matter and represent a long-time sink for atmospheric CO2 with mean residence time of 102–103 years. Wood- and soil-colonizing fungi are the major driving force in humification, being involved in transformation of plant residues, synthesis, and degradation of HS. The chapter is focused on production of ligninolytic oxidoreductases by different groups of fungi and their role in humus synthesis and transformation in forest soils. White-rot fungi and litter-decomposing basidiomycetes producing acidic laccases and ligninolytic peroxidases are mainly involved in delignification and HS degradation, leading to release of small soluble fragments (fulvic acids, monomers) and CO2. Brown-rot fungi producing non-enzymatic oxidative agents and probably laccase are responsible for synthesis of high molecular weight humic acids from partially oxidized lignin. Ascomycetes produce non-ligninolytic peroxidases, neutral laccases, and tyrosinases and are mainly involved in synthesis of HS by partial lignin oxidation or extracellular polymerization of low molecular weight polyphenols. Laccases of ectomycorrhizae and lichens may participate in humus formation via polymerization of phenols, while tyrosinases may contribute to humic acid fraction via melanization.

Heterophase Synthesis of Humic Acids in Soils by Immobilized Phenol Oxidases

Adsorption complexes of humic substances with soil minerals comprise the bulk of organic matter in humus horizons of cold and temperate soils. They represent the most stable Corg fraction in soils with mean residence time of 102–103 years. A considerable fraction of adsorbed organic matter is represented by high molecular weight (50–100 kDa) humic acid–like polymers. The concept of sorptive preservation cannot explain the origin of such polymers on mineral surfaces, because their migration to adsorption sites should be limited by low solubility. It can be suggested that high molecular weight humic acid–like polymers are formed in situ in mineral soil horizons. A possible mechanism is heterophase polymerization of low molecular weight (and thus soluble) precursor material in presence of catalytically active solid phases. The chapter summarizes available data supporting the concept of surface polymerization of humic acids and provides an evidence for the key role of immobilized phenol oxidases and solid matrix in accelerating this process.

Soils on Hard Rocks in the Northwest of Russia: Chemical and Mineralogical Properties, Genesis, and Classification Problems

Soil formation on hard rocks—nepheline syenite, amphibolite, metamorphized gabbro diabase, and their derivatives—was studied in the mountainous tundra and in the northern and middle taiga zones of the Kola Peninsula and Karelia (in the Kivach Reserve). It was found that the soils developing from these rocks could be classified into three groups: (1) petrozems with the O-M profile (the most common variant), (2) podzols and podzolized podburs on the substrates with an admixture of morainic derivatives of acid rocks, and (3) shallow (<5–10 cm) pebbly soils on the substrates without an admixture of allochthonous material (the rarest variant). In soils of the third group, the pedogenic alteration of the mineral matrix does not result in the appearance of phyllosilicates in the fine fractions if these phyllosilicates are initially absent in the rock. In these soils, the protion of the organic matter, and binding of iron released from the weathered silicate minerals into iron-organic complexes) are virtually undifferentiated by the separate soil horizons because of the very low thickness of the soil profiles. These soils have the Oao-BHFao-M profile; it is suggested that they can be classified as leptic podburs. An admixture of morainic material containing phyllosilicate minerals favors a more pronounced differentiation of the modern pedogenic processes by separate soil horizons even in the case of shallow soil profiles; the intense transformation of phyllosilicates takes place in the soils.

Fractionation of Humic Acids according to Their Hydrophobicity, Size, and Charge-Dependent Mobility by the Salting-out Method

Humic acids (HAs) represent heterogeneous and polydisperse mixture of molecules that differ in their chemical structure, composition, and functional properties. Fractionation of HAs is of key importance for understanding their interactions with various organic and inorganic compounds, for studying their physiological activity, and for predicting their behavior in natural environments and agroecosystems. Existing fractionation methods are rather laborious and time consuming, which limits their application in fundamental science and industry. It is shown that fractionation of humic acids with ammonium sulfate ensures their preparative separation with respect to (a) hydrophobicity, (b) molecular size, and (c) charge dependent on the amount of functional groups. Salting out at the lowest and highest degrees of saturation with ammonium sulfate, upon which precipitation of the molecules occurs, makes it possible to separate humic acids into functionally different high-molecular-weight/hydrophobic and low-molecular-weight/hydrophilic fractions. The first fraction is characterized by a lower electrophoretic mobility than the second fraction. The weight percentage of the components coagulated at the lowest degree of salt saturation can be used as a quantitative parameter for comparing hydrophobic properties of humic acids. Salting out is recommended as a fast, simple, and cheap alternative to chromatographic methods for preparative separation of humic acids if large amounts of functionally different fractions need to be obtained.

Xylotrophic and Mycophilic Bacteria in Formation of Dystrophic Waters

The microbial communities developing in ultrafresh stagnant water originating from rainfall comprise the group of ombrophiles. The microorganisms of the myco-bacterial community developing on coarse woody debris are involved in formation of humus-enriched dystrophic waters in the watersheds of forested wetlands. Oligotrophic acidophilic dissipotrophs participate in the transformation of organic matter in such waters. The scheme of trophic interactions in the microbial community is proposed.