Response of microbial communities of Lake Baikal to extreme temperatures

The survival rate, metabolic activity, and ability for growth of microbial communities of Lake Baikal have been first studied after exposure to extremely low temperatures (freeze-thawing) for different lengths of time. It has been shown that short-term freezing (1–3 days) inhibits the growth and activity of microbial communities. The quantity of microorganisms increased after 7-and 15-day freezing. In the periods of maximums, the total number of microorganisms in the test samples was twice as high as in the control. It was established that after more prolonged freezing the microorganisms required more time after thawing to adapt to new conditions. In the variants with 7-and 15-day freezing, the activities of defrosted microbial communities were three or more times higher than in the control. The survival rate and activity of Baikal microorganisms after freeze-thawing confirms the fact that the Baikal microbial communities are highly resistant to this type of stress impact.     

Generation time of pure cultures of heterotrophic microorganisms isolated from Lake Ba?kal]

Generation time was determined in pure cultures of heterotrophic microorganisms in the conditions similar to those of Baikal in June--July of 1972. Generation time was found to be 37+/-7, 16+/-2.5, 16+/-3.2, and 10+/-2.5 hours, respectively, when the cultures had been diluted with Baikal water in the following rations: 1 : 0,1 : 5,1 : 10, and 1 : 20. No differences in the growth rate were found among 11 cultures of heterotrophic microorganisms isolated from Baikal. Conditions limiting the microbial growth improve from the dilution of 1 : 0 to the dilution of 1 : 5. The mean time of generation is 27 hours for June--July. Generation time determined for pure cultures of heterotrophic microorganisms in the conditions similar to natural can be used to calculate production of the bacterial biomass for a definite period of the year.     

The significance of culture for successful cryopreservation of isolated pancreatic islets of Langerhans

It was the aim of the present study to investigate the significance of culture before and after freeze-thawing of isolated mouse pancreatic islets. To evaluate the impact of culture before freezing (5 degrees C/min; 2 M dimethyl sulfoxide), islets were frozen either directly after isolation or after 2, 4, or 7 days of culture in medium RPMI 1640. The culture period after thawing was 7 days. Islets immediately frozen exhibited virtually no (pro)insulin biosynthesis and also a severe inhibition of glucose-stimulated insulin release. The precultured (2-7 days), frozen islets synthesized and released insulin at rates comparable to those of nonfrozen, cultured islets. Studies of the effects of culture after freeze-thawing were performed after a 3-day culture period prior to freezing. The (pro)insulin biosynthetic rates did not differ between islets cultured for 0-7 days after thawing. There was an apparent increase of glucose-stimulated insulin release when the islets were cultured for more than 2 days after thawing. It may be that the decreased viability of islets frozen immediately after isolation was due to minor cell damage induced by the collagenase incubation. During culture the islets may recover and become more resistant to freeze-damage. The beneficial effect of culture after thawing may reflect the loss of damaged cells, which otherwise would influence the results of the viability tests.     

添加葡萄糖和淀粉对盆栽甜樱桃根区土壤碳代谢及根功能的影响

在1年生盆栽甜樱桃土壤中添加葡萄糖和淀粉(4 g·kg^-1),以不添加外源碳为对照,处理后0~60 d内定期采根区土样测定土壤微生物生物量碳、蔗糖酶和淀粉酶活性以及微生物群落功能多样性,处理后第30天测定根系呼吸速率、呼吸途径和根系活力.结果表明： 添加葡萄糖后,土壤蔗糖酶活性及微生物生物量碳均表现为先升高再降低,峰值分别出现在处理后第15天及第7天,分别高于对照14.0%和13.1%,土壤有机质含量表现为先升高再降低再缓慢回升;添加淀粉后显著提高了土壤淀粉酶活性,第15天时为对照的8.5倍,土壤微生物生物量碳除在第7天低于对照外,其余时期均高于对照,土壤有机质含量表现为先升高再下降,处理后第60天高于对照19.8%.BIOLOG分析表明,处理后第15天平均吸光度（AWCD）值及微生物活性均达到最大值,表现为淀粉>葡萄糖>对照.处理后第30天,葡萄糖处理显著增加了土壤微生物对碳水化合物类、羧酸类、氨基酸类、酚酸类和胺类碳源的利用,淀粉处理显著增加了土壤微生物对碳水化合物类、羧酸类、聚合物类和酚酸类碳源的利用.处理后第30天,葡萄糖处理甜樱桃根系总呼吸速率分别较对照及淀粉处理提高21.4%和19.4%,根系活力分别提高65.5%和37.0%.添加葡萄糖和淀粉影响了甜樱桃根区土壤稳定碳源及不稳定碳源的代谢过程,整体上提高了土壤微生物活性,增强了甜樱桃根系呼吸速率及根系活力.     

The classification and the monitoring of the state of mouth riverine and lacustrine ecosystems in lake Baikal based on the composition of local microbiocenoses and their activity

The paper presents the results of the long-term investigation of microbial communities in the technogenically vulnerable mouth riverine and lacustrine ecosystems of Lake Baikal. The structural and functional parameters of the microbial communities were analyzed from the standpoint of developing destructive processes. The analysis showed that the total number of microorganisms (TNM), the number of saprophytic bacteria (NSB), and bacterial production (BP) were greater in the river-mouth water than in the near-mouth lake water. In the offshore direction, TNM and NSB decreased by a factor of 1.5 to 2, and BP decreased by a factor of 4 to 7. Based on TNM, NSB, and BP data, we classified the Lake Baikal rivers with respect to the degree of the impact of human activities on them. The degrading capability of the riverine microbial communities was found to be such that they degrade daily from tenths of a percent to 3.5% of the total amount of organic compounds polluted the river waters.     

Taxonomic composition of Lake Baikal bacterioneuston communities

The taxonomic composition of microbial communities of Lake Baikal surface microlayer was studied by pyrosequencing of the 16S rDNA amplicons. Statistically reliable differences were found between bacterioneuston of the shallow and deep-water stations. The shallow station community was characterized by higher diversity than the deep-water one. While bacterioneuston communities were shown to be less diverse than the water column communities, their diversity was comparable to that of other biofilm associations. Microbial communities of Lake Baikal surface microlayer were shown to be similar to those of the water column in the composition of predominant phyla, while differing considerably at the genus level. Bacterioneuston of Lake Baikal was comparable to microbial communities of the surface microlayer of other freshwater basins, although it was characterized by high abundance of the Alphaproteobacteria and Verrucomicrobia. High abundance of photoheterotrophs compared to the water column communities of other freshwater basins was another distinctive feature of Lake Baikal bacterioneuston. Our results showed the Lake Baikal surface microlayer to be a specific microbial community with low species diversity and relatively high abundance of photoheterotrophic microorganisms.     

The Classification and the Monitoring of the State of Mouth Riverine and Lacustrine Ecosystems in Lake Baikal Based on the Composition of Local Microbiocenoses and Their Activity

The paper presents the results of the long-term investigation of microbial communities in the technogenically vulnerable mouth riverine and lacustrine ecosystems of Lake Baikal. The structural and functional parameters of the microbial communities were analyzed from the standpoint of developing destructive processes. The analysis showed that the total number of microorganisms (TNM), the number of saprophytic bacteria (NSB), and bacterial production (BP) were greater in the river-mouth water than in the near-mouth lake water. In the offshore direction, TNM and NSB decreased by a factor of 1.5 to 2, and BP decreased by a factor of 4 to 7. Based on TNM, NSB, and BP data, we classified the Lake Baikal rivers with respect to the degree of the impact of human activities on them. The degrading capability of the riverine microbial communities was found to be such that they degrade daily from tenths of a percent to 3.5% of the total amount of organic compounds polluting the river waters.     

The structure and biogeochemical activity of the phototrophic communities from the Bol'sherechenskii alkaline hot spring

Microbial communities growing in the bed of the alkaline, sulfide hot spring Bol'sherechenskii (the Baikal rift area) were studied over many years (1986-2001). The effluent water temperature ranged from 72 to 74 degrees C, pH was from 9.25 to 9.8, and sulfide content was from 12 to 13.4 mg/ml. Simultaneous effects of several extreme factors restrict the spread of phototrophic microorganisms. Visible microbial fouling appears with a decrease in the temperature to 62 degrees C and in the sulfide content to 5.9 mg/l. Cyanobacteria predominated in all biological zones of the microbial mat. The filamentous cyanobacteria of the genus Phormidium are the major mat-forming organisms, whereas unicellular cyanobacteria and the filamentous green bacterium Chloroflexus aurantiacus are minor components of the phototrophic communities. No cyanobacteria of the species Mastigocladus laminosus, typical of neutral and subacid springs, were identified. Seventeen species of both anoxygenic phototrophic bacteria and cyanobacteria were isolated from the microbial mats, most of which exhibited optimum growth at 20 to 45 degrees C. The anoxygenic phototrophs were neutrophiles with pH optimum at about 7. The cyanobacteria were the most adapted to the alkaline conditions in the spring. Their optimum growth was observed at pH 8.5-9.0. As determined by the in situ radioisotope method, the optimal growth and decomposition rates were observed at 40-32 degrees C, which is 10 to 15 degrees C lower than the same parameter in the sulfide-deficient Octopus Spring (Yellowstone, United States). The maximum chlorophyll a concentration was 555 mg/m2 at 40 degrees C. Total rate of photosynthesis in the mats reached 1.3 g C/m2 per day. The maximum rate of dark fixation of carbon dioxide in the microbial mats was 0.806 g C/m2 per day. The maximum rate of sulfate reduction comprised 0.367 g S/m2 per day at 40 degrees C. The rate of methanogenesis did not exceed 1.188 micrograms C/m2 per day. The role of methanogenesis in the terminal decomposition of the organic matter was insignificant. Methane formation consumed 100 times less organic matter than sulfate reduction.     

Transformation of Organic Matter by a Microbial Community in Sediments of Lake Baikal under Experimental Thermobaric Conditions of Protocatagenesis

Early diagenesis of organic matter in bottom sediments of Lake Baikal is a focus of many geochemical studies, because it is one of the few sites of petroleum formation in a nonmarine environment. Although Baikal is a rift lake and considered one of the prospective fields for deep biosphere investigations, the transformation processes of organic matter by microbial communities from deep bottom sediments and likely entering of the microorganisms from deep sediments into the near-surface sediments were not previously studied in Lake Baikal. The natural microbial community from near-surface sediments of the cold methane seep Goloustnoe (Southern Baikal Basin) was incubated with methane and the diatom Synedra acus at 80°C and 49.5 atm to simulate catagenesis. The 11-month incubation yielded the enrichment culture of viable thermophilic microorganisms. Their presence in low-temperature sediment layers may be indicative of their migration through fault zones together with gas-bearing fluids. After culturing, molecular biological methods allowed for the detection of both widespread microorganisms and unique clones whose phylogenetic status is currently unknown. The sediment after the experiment showed the formation of polycyclic aromatic hydrocarbon, retene. Retene can be either a conifer or algal biomarker, thus, interpretation of paleoclimate data is tenuous.     

The effect of pyrogenically modified substrates on mineralizing activity and growth strategies of microorganisms of grey forest soil

The rates of the mineralization processes initiated by the input of plant residues and pyrogenically modified plant material into gray forest soil under forests and meadows were assayed. While meadow plant residues was mineralized more rapidly than the forest floor, decomposition of the pyrogenic material resulted in disproportional changes in CO₂ emission from soils. Statistical treatment showed that the respiratory activity of CO₂ emission by heterotrophic microorganisms, which is a physiological characteristic of microbial communities, is 89% determined by the substrate quality. The maximal specific growth rate, which reflects the functional changes in microbial communities, was affected by the cenosis (36%) and the substrate (30%). Most of the carbon of the original plant material (up to 90%) was removed during the burning of plant substrates. The remaining compounds in the pyrogenically transformed material changed the process of mineralization in soil compared both to the control variant and to soil enriched with plant residues. Input of plant residues and ash into the soil resulted in increased total and active biomass, while the maximal specific growth rate decreased and the generation time for the active biomass increased. In the case of soils with plant residues, these changes in the state of microbial communities were brief and occurred during the period of intense mineralization (0–5 days), while, in soils with plant ash, stable changes were revealed after more prolonged incubation. Experimental determination of the microbial biomass turnover time (MTT) by means of two methods (from the ratio between the microbial biomass and respiration and from microbial specific growth rates) made it possible to determine the economical coefficient Y for microbial communities metabolizing the substrates of different availability. Depending on the experimental variant, the Y values varied from 0.22 to 0.51. Decreased maximal specific growth rate and increased values of Y (the coefficient of efficiency of substrate utilization) showed the predominant contribution of K-strategists in the mineralization of low available substrates in soil. The balance calculations and physiological characteristics of the microbial community suggested that the priming effect was most probable in soils enriched with plant ash.     

Microbial community of the water column of the Selenga River-Lake Baikal biogeochemical barrier

The microbial communities of the estuarine zone and the mixing zone of river and lake waters in the Selenga River estuary were studied using the fluorescence in situ hybridization (FISH) method. The microorganisms belonging to the phylogenetic group Gammaproteobacteria were found to predominate in the river estuary, constituting up to 17% of the total bacterial community. Among cultivable microorganisms, organotrophic bacteria were predominant (2040 CFU/ml) in this zone, which results in high rates of microbial production (6.0 μg C/(l day). The microbial community structure changed with distance from the river estuary; representatives of the Alpha-, Beta-, and Gammaproteobacteria were present in equal proportions; psychrotolerant and oligotrophic bacteria were numerous. The rate of heterotrophic carbon dioxide assimilation decreased to 3.8 μg C/(l day). At 5–7 km from the river estuary, where the hydrologic, physical, and chemical conditions are similar to those of lake waters, members of the Betaproteobacteria, which are typical of the open waters of Lake Baikal, are the major representatives of planktonic microorganisms.     

Effect of low temperature and culture media on the growth and freeze-thawing tolerance of Exiguobacterium strains

Bacteria of the genus Exiguobacterium have been repeatedly isolated from ancient permafrost sediments of the Kolyma lowland of Northeast Eurasia. Here we report that the Siberian permafrost isolates Exiguobacterium sibiricum 255-15, E. sibiricum 7-3, Exiguobacterium undae 190-11 and E. sp. 5138, as well as Exiguobacterium antarcticum DSM 14480, isolated from a microbial mat sample of Lake Fryxell (McMurdo Dry Valleys, Antarctica), were able to grow at temperatures ranging from -6 to 40 degrees C. In comparison to cells grown at 24 degrees C, the cold-grown cells of these strains tended to be longer and wider. We also investigated the effect of growth conditions (broth or surface growth, and temperature) on cryotolerance of the Exiguobacterium strains. Bacteria grown in broth at 4 degrees C showed markedly greater survival following freeze-thawing treatments (20 repeated cycles) than bacteria grown in broth at 24 degrees C. Surprisingly, significant protection to repeated freeze-thawing was also observed when bacteria were grown on agar at either 4 or 24 degrees C.     

Ecological Strategies of Soil Microbial Communities under Plants of Meadow Ecosystems

Dominant growth strategies of soil microbial communities of mown and unmown meadows were assessed with respect to the constants of saturation and maximal specific growth rate of microorganisms. The microbial community of mown-meadow soil was characterized by a greater biomass and activity due to prevalence of microorganisms with the r strategy, compared to the microbial community of unmown-meadow soil. In contrast to nonrhizosphere soil, rhizosphere soil was dominated by rapidly growing microorganisms with the r strategy. The dependence of the dominant ecological strategy of the rhizosphere microbial community on the vegetation stage of plants has been traced. Study of the effect of plant species on the growth strategies of rhizosphere microorganisms showed that the features of the K strategy are more pronounced in the following rhizosphere microbial communities of grasses at the same growth stage: r strategy–Bromopsis inermis L.–Poa pratensis L., P. compressa L.–Dactylis glomerata L.–Festuca pratensisL.–K strategy. In the absence of limitation by climatic factors, the growth strategies of rhizosphere microorganisms are determined by the competition between microorganisms and plants for nutrients.     

Antagonistic activity of actinomycetes of Lake Baikal

It was demonstrated that actinomycetes of Lake Baikal are strong antagonists of other microorganisms. Representatives of the genera Streptomyces and Micromonospora inhibit the growth of bacteria isolated from the lake, as well as of antibiotic-resistant microorganisms causing various human diseases. Baikal actinomycetes display a wide range of antagonistic activity and are potential producers of new biologically active substances.     

Microbial community structure in methane hydrate-bearing sediments of freshwater Lake Baikal

Gas hydrates in marine sediments have been known for many years but recently hydrates were found in the sediments of Lake Baikal, the largest freshwater basin in the world. Marine gas hydrates are associated with complex microbial communities involved in methanogenesis, methane oxidation, sulfate reduction and other biotransformations. However, the contribution of microorganisms to the formation of gas hydrates remains poorly understood. We examined the microbial communities in the hydrate-bearing sediments and water column of Lake Baikal using pyrosequencing of 16S rRNA genes. Aerobic methanotrophic bacteria dominated the water sample collected at the lake floor in the hydrate-bearing site. The shallow sediments were dominated by Archaea. Methanogens of the orders Methanomicrobiales and Methanosarcinales were abundant, whereas representatives of archaeal lineages known to perform anaerobic oxidation of methane, as well as sulfate-reducing bacteria, were not found. Affiliation of archaea to methanogenic rather than methane-oxidizing lineages was supported by analysis of the sequences of the methyl coenzyme M reductase gene. The deeper sediments located at 85-90 cm depth close to the hydrate were dominated by Bacteria, mostly assigned to Chloroflexi, candidate division JS1 and Caldiserica. Overall, our results are consistent with the biological origin of methane hydrates in Lake Baikal.     

Preservation of gastrointestinal bacteria and their microenvironmental associations in rats by freezing.

The use of frozen rat gastrointestinal tissue samples for both the recovery of viable bacteria and for observation of microbial communities associated with the tissue was investigated. A decrease of 1 log in lactobacilli, bifidobacteria, and anaerobes was observed when the numbers of bacteria recoverable from frozen tissue (stored 7 to 9 days) were compared to those recoverable from fresh nonfrozen tissue (zero time control). However, freezing did not appear to decrease the numbers of recoverable coliforms. Tissues, cleaved with razor blades after being frozen and stored for 7 to 9 days, showed bacterial communities situated on the mucosa and in the lumen of gastrointestinal specimens. This freezing technique preserved structures not previously observed in the gastrointestinal tract. This indicates that freezing is a good method to use to study such fragile microenvironments.     

Degradation of bis(2-ethylhexyl)phthalate by microorganisms of water and sediments of the Selenga river and Baikal Lake under experimental conditions

Degradation of bis-(2-ethylhexyl)phthalate (BEHP) by microbial associations of water and bottom sediments of the Selenga River and Lake Baikal and by pure cultures of microbial species belonging to various taxa isolated from the sediments under discussion has been studied. It has been shown that intense biological degradation occurs in both water and sediments. The degrees of conversion in experimental closed systems on minimal media are 46 and 24%, respectively. The most active of the organisms studied is a Micromironospora actinomycete. It degraded BEHP by 36% of its initial concentration. Spore-forming bacteria and microorganisms of the genus Pseudomonas were less active (17-23% and 7-11%).     

Long-term storage of industrial microbial strains

Morphofunctional characteristics of industrial microorganisms belonging to different genera, species, and strains were investigated after 15 to 20 years of storage in liquid nitrogen. The taxonomic position of microorganisms, the cell physiological state prior to storage, and the cryoconservation regime were found to affect microbial cryoresistance. Protective media, density of cell suspensions, freezing rate, and heating temperature are the parameters important for development of efficient technologies for cryoconservation of industrial microorganisms at ?196°C.     

Effects of salicylic acid and cold on freezing tolerance in winter wheat leaves

The effects of salicylic acid (SA) (0.01, 0.1 and 1 mM) and cold on freezing tolerance (freezing injury and ice nucleation activity) were investigated in winter wheat (Triticum aestivum cv. Dogu-88) grown under control (20/18 °C for 15, 30 and 45-day) and cold (15/10 °C for 15-day, 10/5 °C for 30-day and 5/3 °C for 45-day) conditions. Cold acclimatisation caused a decrease of injury to leaf segments removed from the plants and subjected to freezing conditions. Exogenous SA also decreased freezing injury in the leaves grown under cold (15/10 °C) and control (15 and 30-day) conditions. Cold conditions (10/5 and 5/3 °C) caused an increase in ice nucleation activity by apoplastic proteins, which were isolated from the leaves. For the first time, it was shown that exogenous SA caused an increase in ice nucleation activity under cold (15/10 and 10/5 °C) and control conditions. These results show that salicylic acid can increase freezing tolerance in winter wheat leaves by affecting apoplastic proteins.