V. V. Demin

Effects of humic acids on the growth of bacteria

The influence of humic acids of different origins on the growth of bacterial cultures of different taxa isolated from the soil and the digestive tracts of earthworms (Aporrectodea caliginosa)—habitats with contrasting conditions—was studied. More than half of the soil and intestinal isolates from the 170 tested strains grew on the humic acid of brown coal as the only carbon source. The specific growth rate of the bacteria isolated from the intestines of the earthworms was higher than that of the soil bacteria. The use of humic acids by intestinal bacteria confirms the possibility of symbiotic digestion by earthworms with the participation of bacterial symbionts. Humic acids at a concentration of 0.1 g/l stimulated the growth of the soil and intestinal bacteria strains (66 strains out of 161) on Czapek’s medium with glucose (1 g/l), probably, acting as a regulator of the cell metabolism. On the medium with the humic acid, the intestinal bacteria grew faster than the soil isolates did. The most active growth of the intestinal isolates was observed by Paenibacillus sp., Pseudomonas putida, Delftia acidovorans, Microbacterium terregens, and Aeromonas sp.; among the soil ones were the representatives of the Pseudomonas genus. A response of the bacteria to the influence of humic acids was shown at the strain level using the example of Pseudomonas representatives. The Flexom humin preparation stimulated the growth of the hydrocarbon-oxidizing Acinetobacter sp. bacteria. This effect can be used for creating a new compound with the elevated activity of bacteria that are destroyers of oil and oil products.

Biochemical degradation of soil humic acids and fungal melanins

Studies were conducted to compare properties and biodegradation of fungal melanins from Aspergillus niger and Cladosporium cladosporiodes with those of humic acids from soils and brown coal. Compared to the humic acids the fungal melanins contained more functional groups, were less hydrophilic and had relatively high molecular weights. Under the conditions of incubation the melanins were found to be more readily degradable than the humic acids studied. The changes in elemental composition, optical parameters and the decrease of molecular weight, observed for both fungal melanins during degradation, made them more similar to soil humic acids.

Stimulating effect of earthworm excreta on the mineralization of nitrogen compounds in soil

The effect of excreta of earthworm species Aporrectodea caliginosa and Eisenia fetida on the mineralization of nitrogen compounds in soils has been studied. A single application of excreta obtained from three earthworms in one day increased the formation of nitrate nitrogen compounds in the soil by 10–50%. The application of ammonium nitrogen (in the form of NH4Cl) in amounts equivalent to the ammonium nitrogen content in the daily excreta of three earthworms had the same effect on the mineralization of nitrogen compounds. The effect of earthworm excreta, as well as the effect of ammonium nitrogen, on the nitrification process was an order of magnitude higher than their contribution to the formation of nitrates due to the oxidation of the introduced ammonium. Hence, ammonium—an important component of the earthworm excreta—can exert a stimulating effect on nitrification processes in the soil and produce long-term cumulative effects that are much more significant than the direct effect of this nitrogen compound.

Quantitative determination of indole-3-acetic acid in yeasts using high performance liquid chromatography—tandem mass spectrometry

High performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) was applied to the comprehensive analysis of the ability of yeast strains to synthesize a plant hormone indole-3-acetic acid (IAA). A total of 124 strains (37 species) of yeasts isolated from various regions and substrates were studied. Testing of IAA production showed that 92% strains were capable of IAA synthesis. The results indicated that, in general, ascomycetous yeasts were more active auxin producers than basidiomycetous ones. Geographically, strains from tropical regions were the most active IAA producers. Analysis of the substrate variability of the strains showed higher auxin production (on average) by the yeasts isolated from plants compared to the soil isolates, indicating a specific regulatory role of the plant yeast population.

Sorption of humic acids by bacteria

Capacity for sorption of humic acid (HA) from water solutions was shown for 38 bacterial strains. Isotherms of HA sorption were determined for the cells of 10 strains. The bonding strength between the cells and HA (k) and the terminal adsorption (Qmax) determined from the Langmuir equation for gram-positive and gram-negative bacteria were reliably different. Gram-positive bacteria sorbed greater amounts of HA than gram-negative ones (Qmax = 23 ± 10 and 5.6 ± 1.2 mg/m2, respectively). The bonding strength between HA and the cells was higher in gram-negative bacteria than in gram-positive: k = 9 ± 5 and 3.3 ± 1.1 mL/mg, respectively.

Earthworms as modifiers of the structure and biological activity of humic acids

Passage of humic acids (HAs) through the digestive tract of the earthworm, Eisenia fetida andrei, resulted in a decrease in molecular masses of the HAs. The effect of earthworm-modified HAs on individual bacteria and on bacterial communities as a whole is different from the effect of native HAs. Modified HA probably induces and regulates microbial successions in soils and composts in a different manner than the native HA, suppressing or stimulating different groups of microorganisms. These results may explain why the positive effects of commercial humates in real soil ecosystems, unlike model communities, attenuate rapidly.

Taxonomic composition and physiological and biochemical properties of bacteria in the digestive tracts of earthworms

Several hundred bacterial strains belonging to different taxa were isolated and identified from the digestive tracts of soil and compost earthworms. Some physiological and biochemical properties of the bacteria were characterized. The majority of intestinal bacteria in the earthworms were found to be facultative anaerobes. The intestinal isolates as compared to the soil ones had elevated activity of proteases and dehydrogenases. In addition, bacteria associated with earthworms’ intestines are capable of growth on humic acids as a sole carbon source. Humic acid stimulated the growth of the intestinal bacteria to a greater extent than those of the soil ones. In the digestive tracts, polyphenol oxidase activity was found. Along with the data on the taxonomic separation of the intestinal bacteria, the features described testified to the presence of a group of bacteria in the earthworms intestines that is functionally characteristic and is different from the soil bacteria.

Quantitation of Ergosterol as a Mycorrhizal Symbiosis Biomarker

Several procedures for the detection of mycorrhizal fungi in plant roots are available. Direct microscopic assessment is labor-consuming and subjective [1], and therefore indirect methods for estimating the rate of mycorrhization have been proposed. These methods are based on assays for various components of fungal cells, such as chitin [2] and phospholipid fatty acids [3]. The first approach is relatively simple but takes into account both living and dead fungal biomass, whereas the second method is labour-consuming and requires expensive equipment. Ergosterol is another biomarker of fungal biomass [4, 5], which undergoes rapid decay after the death of fungi and can therefore be regarded as an indicator of living fungal biomass [6]. Ergosterol is not a component of plant cells [7, 8], which has allowed its use as a mycorrhiza biomarker for assessing mycorrhization of roots with arbuscular mycorrhiza [9], ectomycorrhiza [10–12], and ericoid mycorrhiza [13, 14]. Conventional procedures for ergosterol quantitation in roots and soil are labour-consuming, as they involve thermal processing, long-term shaking, centrifugation, and a liquid–liquid extraction. The procedure proposed by Beni et al. [15] does not require thermal processing, employs solid-phase extraction (SPE), and provides for rapid analysis. The authors of the procedure used it for ergosterol quantitation in poorly humified forest litter. The organic component of these samples is similar to plant tissues with regard to susceptibility to chemical processing. The aim of this study was to adapt the procedure [15] to ergosterol quantitation in fine plant roots and to assess this parameter in a series of plant species from mountain tundra ecosystems. Sample collection for analysis. Fine roots (<1 mm) of various plants were collected in the mountain tundra of the Avrorin Polar Alpine Botanical Garden– Institute (the Khibiny mountain range, Murmansk oblast). Ergosterol was determined in roots of several plant species with different mycorrhiza types and some species characterized by the absence of mycorrhizal symbiosis (Table 1). Plants were excavated together with a soil monolith, from which the fine roots were washed out, weighted, and frozen for transporting to the laboratory. Roots of each plant species (0.5–2 g wet weight) were collected from different soil monoliths in three to five replicates. Procedure modification. The original procedure [15] was modified taking into account the availability of analytical equipment (chromatographic system and column, and SPE column) and specific features of the substrate to be analyzed (plant roots). Parameters of the original procedure and modifications are listed in Table 2. Reduction of sample weight to 0.5–2 g fresh (frozen) roots was explained by the complication of collecting roots from individual plant species in multidominant natural plant communities. The substrate used in the original procedure (poorly humified forest litter) was less resistant to processing, and therefore КОН concentration in the methanol solution used for extraction was increased from 0.07 to 0.2 M in order to achieve a higher degree of cell wall destruction and to extract maximum amounts of ergosterol. As reported in [16], this concentration is sufficient for ergosterol extraction from plant tissue-associated fungi. Since the buffering capacity of the extracts was low, their neutralization was performed under control of pH in order to prevent degradation of the SPE column. According to [17], the retention of hydrophobic components on C-18 columns is most efficient at pH 6–8. An acetonitrile–water mixture (90 : 10 vol/vol) was used as the eluent in HPLC system, instead of 100% methanol used in the original procedure. The calibration curve of optical density versus ergosterol concentration (Sigma-Aldrich, >95% purity) was verified in the range of 0 to 10 μg. Spiking was used for procedure validation: a non-spiked mixed sample of fine roots from cereal plants and similar samples spiked with ergosterol (4.0 and 8.0 μg/g) were analyzed. Analytical procedure. A sample (0.5–2 g) of frozen fine roots was placed in a 100-mL conical glass f lask, suspended in 50 mL of 0.2 M КОН solution in СН3ОН, put onto a rotary shaker for 1 h, and then

Compositions of n -Alkanes and n -Methyl Ketones in Soils of the Forest-Park Zone of Moscow

The seasonal changes in the contents and compositions of n-alkanes and n-methyl ketones have been studied in typical soddy-podzolic soils (Albic Retisols (Ochric)) under lime forests in the Losiny Ostrov National Park, Moscow. In the humus horizons, the reserves (about 370 mg/m2) of odd n-alkanes with the chain length of C25–C35—the biomarkers of terrestrial vegetation—are 4–5 times below their amount entering with the leaf falloff in autumn. A noticeable contribution of microbial biomass hydrocarbons to the lipid fraction in the AY and AYel horizons manifests itself in the increased fraction of odd and medium-chain (<C25) homologues (OEP = 4–6, LSR = 5–7) in the spectrum of n-alkanes. The lowest content of methyl ketones and odd n-alkanes was observed immediately after the winter period (OEP = 22, A/K = 21–170). In the EL and BT horizons, the n-alkane fraction of organic matter and the relative content of high molecularweight n-alkanes more resistant to microbial destruction (C33, C35) increases by 3–4 times in comparison with that in the abovelying horizons, and the type of distribution of n-alkanes by the carbon chain length changes: the dominance of odd homologues is absent; in the EL horizon, OEP = 1.

Interaction of the Mixture of Phenolic Acids with Modified Kaolinite under Batch and Dynamic Conditions

Phenolic acids play an important role in the formation of soil profiles, however their cooperative sorption by mineral phases under environmentally relevant concentrations is poorly studied. In the present work the sorption of an equimolar mixture of phenolic acids by kaolinite modified with amorphous aluminum hydroxide has been studied under both batch and continuous-flow conditions. It has been found that the sorption of gallic and protocatechuic acids containing OH groups in the ortho position exceeds the sorption of p-hydroxybenzoic and methoxylated acids (vanillic, syringic, and ferulic) by an order of magnitude. The study of sorption under dynamic (continuous-flow) conditions has shown the competition of acids for binding sites, while the active centers of kaolinite-Al(OH)x are being occupied. The sorbed gallic acid displaces the other acids, which pass into solution in the following order: p-hydroxybenzoic acid > vanillic acid > syringic acid ≫ ferulic acid > protocatechuic acid. The revealed regularities indicate potentially important role of ortho-substituted hydroxybenzoic acids in the formation of soil organic matter, while p-hydroxybenzoic, vanillic, and syringic acids can be more important for the composition of soil solutions and natural waters.