The structural and functional state of microbial communities of different-aged buried ancient soloids (soil-like bodies lacking genetically distinct horizons) have been comprehensively assessed in open archaeological pits of ancient human sites in the middle reaches of the Mzymta River on the Sochi Black Sea coast in the Akhtsu grotto and Akhshtyrskaya cave. A number of changes in the functional biodiversity of ancient soloids as compared with the background soils (alluvial soils formed immediately near the studied soloids with similar mesomorphological properties) are observed. They include a 1.2-fold decrease in the specific metabolic activity of microbial communities assessed by multisubstrate testing and an increased instability of paleomicrobial systems according to the coefficient of rank distribution of the range of substrate consumption d > 1, which is typical of irreversibly damaged systems. The microbial community of ancient soloids has undergone a number of changes associated with anthropogenic activity. According to the analysis of the main range of consumed substrates (multisubstrate testing), the microbial community of the Akhtsu grotto soloid displays a more intensive consumption of alcohols and amino acids, which suggests the supply of the organic matter of an animal origin to the cultural layer of ancient human site. Lactococci and bifidobacteria are identified in the Akhshtyrskaya cave soloid; they are extremely rare in soil and develop under conditions of excess carbohydrates on rich complex media, such as fermented meat and plant residues. In addition, an increased content of keratinolytic fungi capable of decomposing the keratin of hair, wool, feathers, and so on has been found in the cultural layer of the cave site. A comparative analysis of the prokaryotic taxonomy demonstrates the prevalence of the members of the Thermoleophilia class, family Gaiellaceae, order Solirubrobacterales in all the studied soils and soloids. These microorganisms require only positive temperatures for their development, suggesting a mild climate during the soil formation. The Akhtsu grotto paleosols are a promising source of bacteria (genera Janthinobacterium, Lysobacter, and Chitinophaga ) that may possess biotechnological potential and useful properties.

A comprehensive assessment of the structural and functional state of microbial communities of multi-temporal buried ancient soloids (a soil-like formation similar to soil but without genetically formed horizons) in open archaeological pits of ancient human sites located in the middle reaches of the Mzymta River on the Sochi Black Sea coast in the Akhtsu Grotto and Akhshtyrskaya Cave was carried out. Changes in the functional biodiversity of ancient soloids compared to the background (alluvial soils that form in close proximity to the studied soloids and have similar mesomorphological properties) are noted: a decrease in the specific metabolic work (W) of microbial communities by 1.2 times according to the method of multisubstrate testing and an increase in the instability of paleomicrobial systems according to the index of the rank distribution of the spectra of consumption of substrates d 1, which is typical for irreversibly damaged systems. The microbial community of ancient soloids has undergone a number of changes associated with anthropogenic activity. The analysis of the main absorption spectrum of substrates (MST method) showed that the microbial community of the soloid of the Akhtsu Grotto is oriented towards more intensive consumption of alcohols and amino acids, which suggests that organic matter of animal origin entered the cultural layer of the ancient human site. In the microbiome of the soloid of the Akhshtyrskaya cave the presence of lactotococci and bifidobacteria was found, which are extremely rare in soil and develop in conditions of excess carbohydrates on rich complex media, such as fermented meat and plant residues. Also, an increase in the content of keratinolytic fungi capable of decomposing keratin of hair, wool, feathers, etc. was found in the cultural layer of the cave site. Prokaryotic taxonomy in all the studied soils, with or without anthropogenic impact, was characterized by the predominance of the members of the Thermoleophilia clade and the families Gaiellaceae and Solirubrobacterales. Considering that these species require positive temperatures to develop, it suggests that the soils were formed in a mild climate. The Akhtsu grotto paleosols are a promising source of bacteria (genera Janthinobacterium, Lysobacter, Chitinophaga) that may have biotechnological potential.

In a model laboratory experiment on infertile arable soil with low biological activity, it was found that the introduction of glyphosate leads to a short-term change in the intensity of the main processes of microbial transformation of nitrogen in the soil. When incubating soil with glyphosate at the maximum recommended dose of 8 l/ha for 22 days, there is an increase in nitrogen-fixing and denitrifying activity by 30–80% and 300% and a decrease in the nitrification process by 20–40%. The effects are of a short-term nature and do not reflect the entire complex of ongoing microbiological processes: no effect of glyphosate was detected on the emission of CO2, which is an integral indicator of biological activity. At the end of incubation in the soil with the introduced glyphosate, there was an increase in the number of bacteria by 40% and a decrease in the number of micromycetes by 70%. In general, under the selected conditions, the introduction of glyphosate led to a marked deterioration in the biological activity of the soil. By the method of multisubstrate testing, it was shown that under the action of the herbicide there is an increase in the value of the coefficient of rank diversity of the consumption spectra of substrates d, accompanied by a decrease in the specific metabolic work W and the integral vitality index G. It was shown for the first time that when glyphosate is introduced into soil with low biological activity and availability of phosphorus and the herbicide is degraded along the sarcosine pathway with a break in the C–P bond, excluding the formation of toxic metabolites, there is a pronounced negative effect of glyphosate on soil microorganisms, which leads to inhibition of wheat plant growth.

The response of the microbial community (microbial biomass carbon (Cmic), basal respiration (BR), and functional diversity (FD)) of agrosoddy-podzolic soil (Albic Glossic Retisols (Loamic, Aric Cutanic, Ochric)) to pollution by heavy metals (HMs: Cu 660, Zn 1100, Pb 650 mg/kg) and carbon-containing preparations (5% of biochar and 0.25% of lignohumate) was studied in model experiment (30 days). Soils with different organic carbon contents (Corg 3.86 and 1.30%) were sampled at two sites (Chashnikovo, Moscow oblast). We determined Cmic by the substrate-induced respiration method and FD by multisubstrate testing (47 substrates). It was found that HMs application reduced Cmic on average by 49–57%, BR by 23–52%, and FD by 45%, but, on the contrary, increased the microbial metabolic quotient (qCO2 = BR/Cmic) by 9–46%. The changes of these properties were most significant in the soil with low Corg content (1.30%). Carbon-containing preparations did not contribute to variations in Cmic, BR, and qCO2 in both soils with HMs, but increased their FD. It is concluded that the studied microbiological parameters may be used as indicators for optimal assessment of soil quality: FD and Cmic are the more sensitive to HMs than BR and qCO2.

Ionizing radiation is an important environmental factor affecting the dynamics of biospheric processes in the past and present, as well as limiting the spread of life outside the Earth. The effect of radiation on microorganisms has been studied for decades, but studies of the response of natural microbial ecosystems are still scarce. We have studied the effect of 100 kGy gamma irradiation under low pressure (1 Torr) and low temperature (–50°C) on microbial community of the ancient Antarctic permafrost sedimentary rock. After irradiation, the total number of prokaryotic cells determined by epifluorescence microscopy, as well as the number of metabolically active bacterial and archaeal cells detected by fluorescence in situ hybridization remained at the control level, while the number of cultured heterotrophic bacteria decreased by an order of magnitude. Using the multisubstrate testing method, it has been found that the microbial complex retained a high potential metabolic activity and functional diversity after exposure to a combination of extreme physical factors. The resistance demonstrated by the microbial community significantly exceeded the generally accepted estimates of the prokaryotes’ radioresistance and indicated an underestimation of the microorganisms' radioresistance in natural habitats and the important role of mineral heterophase environment and irradiation conditions (pressure, temperature). The study confirmed the potential for long-term cryopreservation of viable terrestrial-like microorganisms in the Martian regolith, as well as the possibility of transferring anabiotic life forms as a part of small bodies in the space environment.

93 Effects of lignohumate and biochar on microbial communities in agricultural soils differing in organic matter content Terekhova V.A., Fedoseeva E., Pukalchik M.A., Ivanova A.E., Verkhovtseva N.V., Pozdnyakov L.А., Kulachkova S.A., Gorlenko M.V., Karpukhin M.M., Yakimenko O.S. Lomonosov Moscow State University, Moscow, Russia, vterekhova@gmail.com Severtsov Institute of Ecology and Evolution, Moscow, Russia Pirogov Russian National Research Medical University, Moscow, Russia Skolkovo Institute of Science and Technology, Moscow, Russia

The effect of crane fly (Diptera, Tipulidae) larvae on the functional diversity of soil microorganism communities and the intensity of nitrogen and carbon transformation processes in soil has been investigated. The vital activity of larvae in the soil has been shown to significantly accelerate nitrogen fixation, denitrification, and methane production and elevate the functional diversity (the number of substrates consumed and metabolic work) and stability of the microbial complex.

The descriptive ability of two integral biotic indices obtained from laboratory experimental data focused on estimating the effects of humic products (HPs) on the microbiota under conditions of copper contamination in a model soil substrate is evaluated. The “Standard Soil” prepared from sand, kaolin, and peat (ISO 11268-1) as recommended for the assessment of biological effects was used. To summarize the results of ecotoxicological studies of Cu-polluted samples (660 mg Cu/kg) and to provide the integral estimation of HPs impact on the microbiota, two statistical models were used: the Harrington’s desirability function D and the integrated parameter of the soil microbial system sustainability G (ratio of biodiversity to instability) using multisubstrate testing. The indices obtained based on two sets of multidimensional data were compared in their descriptive ability when estimating the detoxifying efficiency of HPs. It was found that HPs mainly stimulate multispecies bacterial complexes, while the test cultures representing higher plants and algae are less sensitive to their impact. Three of the five studied HPs showed a pronounced detoxifying effect and improved the state of the studied system. Both integral biotic indices—the generalized Harrington desirability function D and the General sustainability parameter of the system G—demonstrated a similar effect vector. For the purpose of environmental regulation, the use of the integrated approach based on a generalized desirability function could be recommended due to its higher completeness and reliability of the system of biotic indicators.

The amount of chemicals used for plant protection is growing due to the intensification of agriculture. Glyphosate is one of the most widely used herbicides; consequently, its influence on the microbial communities of agricultural soils is of interest. Structural and functional changes in the prokaryotic community in soddy-podzolic soil related to glyphosate treatment have been studied. No influence of the herbicide on the total number of prokaryotes or on the indices of substrate utilization intensity by the soil microbial community was observed. An increase in CO2 emissions was a short-term effect of glyphosate application. The numbers of metabolically active Archaea and Acidobacteria decreased, while the number of metabolically active Actinobacteria increased after long-term exposure of the soil to glyphosate.

It is supposed that the biosphere could be formed under conditions of early Mars, and it is cryo-conserved up to now. The period of its preservation is limited by the effect of ionizing radiation. The viability of a soil microbial community thtat underwent gamma radiation (100 kGy) under simulated conditions (‒50°C, 1 Torr) of the surface layer of the Martian regolith is studied. Irradiation did not result in the death of the microbial community: the number of living cells, metabolic activity, and functional diversity remained high. The data obtained suggest that microorganisms could be preserved in regolith of Mars for no less than 1.3 m.y. and in general contribute to the modern concepts concerning radiation resistance of the Earth’s life form.

We report the results of experiments on laser printing (wavelength λ=1064 nm) with gel microdrops acting as carriers of living microbial and cellular objects. The dynamics of transport processes with the help of high-speed optical video was studied, which allows to determine characteristics of the formed gel jets and to optimize the operating mode of the laser. It is shown that laser pulses of 4 to 20 ns duration and energy E ≤ 20 μJ should be used to minimize the negative effect on living systems. The results can be used to optimize the technologies of cellular printing and laser engineering of microbial systems (LEMS). LEMS technology is used to isolate hard-cultivated and non-cultivated by classical methods of microorganisms that can act as producers of new biologically active substances and antibiotics.

The report presents the Laser Engineering Microbial System (LEMS) technology for direct isolation of pure microbial cultures and microbial consortia from soil by laser 3d printing technology. We study thermal and transport processes involved in the transfer of gel microdroplets under the conditions of microprinting by LIFT method. The specific features of the interaction of pulsed laser radiation (l = 1.064 mm, pulse duration 4 - 200 ns, energy 2 mJ - 1 mJ) with the absorbing gold film deposited on the glass donor substrate are determined.

Recently, it was shown that laser‐induced forward transfer (LIFT) technology and the laser engineering of microbial systems (LEMS) technique (based on LIFT method) are effective for isolation of micro‐organisms from different complex substrates. These techniques frequently utilize Au as an absorbing layer material. The purpose of this study was to investigate the influence of absorbing film materials (Au, Ti and Cr) on the effectiveness of laser printing of micro‐organisms to improve LEMS and LIFT techniques. It was shown that application of Ti and Cr absorbing layers activates bacterial growth after laser printing and is significantly more effective in comparison to Au films, which actually show a suppressing effect on bacterial cells. Results of this study can be applied for LEMS and LIFT protocols for improving bacterial isolation and microbial growth.

As the key parameters of the Martian regolith, we have studied the combined effect of gamma radiation (1 and 10 kGy), low temperature (–50°C), and low pressure (1 Torr) on the microbial communities of extreme ecotopes of Earth to estimate the duration of cryopreservation of hypothetical Martian ecosystems in a viable state. The obtained data suggest that cryopreservation of viable microorganisms in the surface layer of the regolith is possible for at least 130000 years; at a depth of 30 cm, for 170000 years; at a depth of 2 m (the depth which the ExoMars 2020 mission must reach), for 330000 years; and at a depth of 5 m, for 2 million years.

Standard microorganism isolating technology applied for complex multiphase environmental samples such as soil or sediment needs pre-treatment steps to remove living cells from their mixed-phase microniche, by creating a liquid-phase sample. This process removes synergetic relationships, which help to maintain viability of yet-to-be-cultured and hard-to-culture bacteria. In this paper we demonstrate a high throughput Laser Micro-Sampling (LMS) technology for direct isolation of pure microbial cultures and microbial consortia from soil. This technology is based on laser printing of soil microparticles by focusing near-infrared laser pulses on specially prepared samples of a soil/gel mixture spread onto a gold-coated glass plate. Microsamples of soil are printed on glucose-peptone-yeast agar plates, to estimate the LMS process influence on functional and taxonomic microbial diversity, and on «Eco-log» sole carbon sources microplates, to investigate functional diversity by “metabolic fingerprinting”. The obtained results are compared with traditionally treated soil samples. It was shown that LMS treatment leads to increasing of cultured biodiversity and modifies the functional diversity. The strain of rare genus Nonomuraea was isolated by LMS from complex natural environment without using media selective for this genus.

We study thermal and transport processes involved in the transfer of gel microdroplets under the conditions of laser cell microprinting. The specific features of the interaction of pulsed laser radiation ( λ = 1.064 µm, pulse duration 4 – 200 ns, energy 2 µJ – 1 mJ) with the absorbing gold film deposited on the glass donor substrate are determined. The investigation of the dynamics of transport processes by means of fast optical video recording and optoacoustic methods makes it possible to determine the characteristics of the produced gel jets as functions of the laser operation regimes. The hydrodynamic process of interaction between the laser radiation and the gold coating with the hydrogel layer on it is considered and the temperature in the region of the laser pulse action is estimated. It is shown that in the mechanism of laser-induced transfer a significant role is played by the processes of explosive boiling of water (in gel) and gold. The amount of gold nanoparticles arriving at the acceptor plate in the process of the laser transfer is determined. For the laser pulse duration 8 ns and small energies (less than 10 µJ), the fraction of gold nanoparticles in the gel microdroplets is negligibly small, and their quantity linearly grows with increasing pulse energy. The performed studies offer a base for optimising the processes of laser transfer of gel microdroplets in the rapidly developing technologies of cell microprinting.

It was found that ants significantly affect the physiological activity and functional diversity of soil microbial communities, and redistribution of biophilic elements (C and N) down through the profile occurs in anthills compared to the control soil, as well as their accumulation in the underground part of the ant nests. A high urease activity was revealed in ant nests and ants. Functional dissimilarities of bacterial communities in all studied objects were determined by the multisubstrate test.