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Vol 86, No 6 (2017)

Reviews

Methane formation and oxidation by prokaryotes

Kallistova A.Y., Merkel A.Y., Tarnovetskii I.Y., Pimenov N.V.

Abstract

The review deals with systematization and generalization of new information concerning the phylogenetic and functional diversity of prokaryotes involved in the methane cycle. Methane is mostly produced by methanogenic archaea, which are responsible for the terminal stage of organic matter decomposition in a number of anoxic ecotopes. Although phylogeny, physiology, and biochemistry of methanogens have been extensively studied, important discoveries were made recently. Thus, members of deep phylogenetic lineages within the Euryarchaeota phylum (Methanomassiliicoccales, “Candidatus Methanofastidiosa,” “Methanonatronarchaeia”) and even outside it (“Ca. Verstraetearchaeota” and “Ca. Bathyarchaeota”) were reported to carry out methyl-reducing methanogenesis. Moreover, evidence was obtained on aerobic methane production by marine heterotrophic bacteria, which demethylate polysaccharide esters of methylphosphonic acid. Methanotrophic microorganisms oxidize methane both aerobically and anaerobically, decreasing significantly the release of this greenhouse gas into the atmosphere. In the presence of oxygen methane is oxidized by methanotrophic members of Alpha- and Gammaproteobacteria, as well as by Verrucomicrobia. Methanotrophic gammaproteobacteria have been recently revealed in hypoxic and even anoxic environments, where they probably oxidize methane either in a trophic consortium with oxygenic phototrophs and/or methylotrophs or using electron acceptors other than oxygen. Anaerobic methane oxidation has been known for a long time. Sulfat- and nitrate-dependent anaerobic methane oxidation carried out by the ANME archaea via reverse methanogenesis are the best studied processes. While metal-dependent anaerobic methane oxidation is considered possible, the mechanisms and agents responsible for this process have not been reliably identified. Intracellular oxygen production during nitrite-dependent anaerobic methane oxidation was shown for bacteria “Ca. Methylomirabilis oxyfera.” These findings stimulate interest in the processes and microorganisms of the methane cycle.

Microbiology. 2017;86(6):671-691
pages 671-691 views

Mesosomes associated with hydrogen peroxide in bacteria

Li X., Tang Z., Pang X., Zhang M., Liu Y.

Abstract

Mesosomes are unique membranous structures in bacteria. It is recognized that the mesosomes should be involved in several fundamental processes. The structure and behaviour of mesosomes have been studied and largely identified, while new evidences of mesosome function have been strikingly obtained. Our previous studies confirmed that hydrogen peroxide is involved in mesosomes formation during cell injury and cell division processes. Mesosome formation is always accompanied by excessive H2O2 accumulation. Furthermore, our recent data showed that mesosomes could not only enrich the excess H2O2, but also bring the H2O2 outside of the cells injured by antibiotics. It is a possibility that the enrichment of H2O2 in mesosomes might be a mechanism of drug resistance of bacteria. This article describes the bacterial mesosome and its functions as well as the involvement of hydrogen peroxide in mediating these functions.

Microbiology. 2017;86(6):692-697
pages 692-697 views

Experimental Articles

Characterization and stress-protective action of Saccharomyces cerevisiae extracellular peptide factors on propionic acid bacteria

Vorob’eva L.I., Rogozhin E.A., Khodzhaev E.Y., Volodyashkin R.A., Samoilenko V.A.

Abstract

Protective effect of Saccharomyces cerevisiae exometabolites (RF1 and RF2) on seven strains of propionic acid bacteria under exposure to bile salts and acid stress was studied. RF2 had a stronger protective effect than RF1. Comparison of the analytical profiles of RF1 and RF2, separated by RP-HPLC, showed that nearly 80% of RF1 components were present in RF2. Separation of S. cerevisiae RF1 into individual components by means of semi-preparative RP-HPLC was used for structural characterization of the active component of the RF1 fraction. It was found to contain two pentapeptides (<1 kDa) with identical amino acid sequences (1Gly-Pro-Tre-Gly-Pro5), differing in one hexose residue. The RF2 separation method based on three-stage liquid chromatography at low and high pressure was developed, which involved analytical high-pressure gel chromatography. The use of the latter method made it possible to separate high-molecular components and perform enrichment of low-molecular peptides. Peptide origin of the RF2 active substances with molecular masses allegedly below 300 Da was confirmed.

Microbiology. 2017;86(6):698-707
pages 698-707 views

Antagonistic activity of lactobacilli isolated from natural ecotopes

Anisimova E.A., Yarullina D.R., Ilinskaya O.N.

Abstract

Lactobacilli are widely used in silage production, for fermentation of foodstuffs, and as probiotics. Their therapeutic effect in preparations is based on their antagonistic activity against pathogens. In this work, antagonistic activity of Lactobacillus strains isolated from silage and fermented plant-derived foodstuffs was studied in order to select the strains promising for industry, agriculture, and medicine. Twenty Lactobacillus strains were ranked according to the intensity and rate of acid production and antibiotic resistance. Lactobacillus sp. Cа9L was selected as a promising starter culture strain for biotechnology based on the optimal combination of acid production, rate of acidification, and antibiotic resistance.

Microbiology. 2017;86(6):708-713
pages 708-713 views

Biocrystalline structures in the nucleoids of the stationary and dormant prokaryotic cells

Loiko N.G., Suzina N.E., Soina V.S., Smirnova T.A., Zubasheva M.V., Azizbekyan R.R., Sinitsyn D.O., Tereshkina K.B., Nikolaev Y.A., Krupyanskii Y.F., El’-Registan G.I.

Abstract

Compaction and biocrystallization of the nucleoid are presently considered as a necessary and important stage in the transformation of the cell ultrastructure during change of microbial cultures strategies from growth to survival. Nucleoid biocrystallization in the stationary phase cells is achieved due to structural regularity of the DNA complexes with the histone-like Dps protein. Our experiments with Escherichia coli mutants, overproducers of the Dps protein, confirmed nucleoid biocrystallization in the late stationary phase cells. Since nucleoid biocrystallization was revealed in E. сoli cells without Dps overproduction at late stages of starvation, it is constitutive in the cycle of development of microbial populations. The present work concentrated on detection of the nucleoid biocrystalline structure in (1) long-starved (21 day in the chemostat mode) bacterial cells (genera Arthrobacter and Pseudomonas), (2) dormant ametabolic (anabiotic) cells of such prokaryotes as archaea and non-spore-forming bacteria, (3) endospores of bacilli, (4) streptomycete exospores, and (5) in the cells surviving in permafrost for (2‒3 Ma). The topics discussed include nucleoid biocrystallization as a necessary stage of maturation of the dormant microbial cells providing for survival and preservation of the species, dynamics of nucleoid biocrystallization during maturation of the dormant cells, and its possible role for the preservation of genetic information in the case of autolysis of most of the cells in a developing culture.

Microbiology. 2017;86(6):714-727
pages 714-727 views

Toxic metals accumulation in Trichoderma asperellum and T. harzianum

Hoseinzadeh S., Shahabivand S., Aliloo A.A.

Abstract

Heavy metal contamination represents an important environmental issue due to the toxic effects of metals on different organisms. Filamentous fungi play an important impact in the bioremediation of heavy metal-contaminated wastewater and soil. The purpose of this investigation was to observe fungal uptake behavior toward heavy metal. For this aim Trichoderma asperellum TS141 and T. harzianum TS103 at growth period were screened for their tolerance and uptake capability of cadmium (Cd), lead (Pb) and nickel (Ni) at different concentrations (0, 25, 50, 100, and 200 mg/L) in PDB media (potato dextrose broth as a complex medium). Results showed that both fungi were able to survive at the maximum concentration of 200 mg/L of the heavy metals, and remove them. T. asperellum had a better uptake capacity for Cd compared to Pb and Ni in the highest metal concentration in media. Maximum removal efficiency of Pb (68.4%) at 100 mg/L and Ni (78%) at 200 mg/L was performed by T. asperellum. For Cd, the highest removal efficiency (82.1%) was recorded by T. harzianum at 200 mg/L Cd in aqueous solution. The uptake of Cd was highly dependent on pH of solution than Pb and Ni so that the optimal pH of Cd uptake was 9 for T. asperellum and 4 for T. harzianum. Also, optimal temperature was 35°C for Cd and Pb uptake in both fungi, whereas for Ni uptake was 30 and 35°C in T. harzianum and T. asperellum, respectively. We propose that T. asperellum TS141 and T. harzianum TS103 can be used as a bioremediation agent for metal remediation from wastewater and heavy metal-contaminated soils.

Microbiology. 2017;86(6):728-736
pages 728-736 views

Methylobacillus caricis sp. nov., an obligate methylotroph isolated from roots of sedge (Carex sp.)

Agafonova N.V., Kaparullina E.N., Doronina N.V., Trotsenko Y.A.

Abstract

A new obligately methylotrophic bacterium, strain OVT, was isolated from roots of sedge (Carex sp.). The isolate was represented by aerobic gram-negative motile, non-spore-forming rods, which divided by binary fission. Optimal growth occurred at 22−29°C and pH 7.5−8.5 in the presence of 0.5−2% NaCl; growth was inhibited by 3.5% NaCl. Strain OVT utilized methanol as the only carbon and energy source. The organism used the KDPG variant of the ribulose monophosphate pathway (RuMP) of С1 metabolism. Ammonium was assimilated by reductive amination of α-ketoglutarate. The major cellular fatty acids were C16:0 (45.5%), C16:1ω7c (40.7%), and C17cyc (8.0%). The major ubiquinone was Q8. The dominant phospholipids were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The DNA G + C content of strain OVT was 51.4 mol % (Tm). While the 16S rRNA gene sequence of strain OVT exhibited high similarity to those of Methylobacillus species: M. gramineus LapT (99.6%) and M. glycogenes TK 0113T (98.7%), the DNA-DNA hybridization level between strain OVT and M. gramineus LapT was only 52%. Based on the data obtained, strain OVT was assigned to the new species Methylobacillus caricis sp. nov. (=VKM B-3158T = JCM 32031T).

Microbiology. 2017;86(6):737-744
pages 737-744 views

Anaerobic cellulolytic microbial communities decomposing the biomass of Anabaena variabilis

Petrova E.V., Egorova M.A., Piskunkova N.F., Kozhevin P.A., Netrusov A.I., Tsavkelova E.A.

Abstract

Four previously isolated methanogenic anaerobic consortia, which were originally cultivated on a cellulose-containing substrate (filter paper), were used as inocula for the anaerobic conversion of the biomass of Anabaena variabilis into biogas at 55°C. The cumulative methane yield in the biogas produced by the most active consortia reached 64%. However, the biotransformation was only efficient in the course of the single inoculation and pretreatment of the cyanobacterial biomass by its concentration and freeze-thawing. The DGGE analysis of the structure of the selected microbial consortia, cultivated on the filter paper, revealed qualitative variations in the biodiversity of predominant Bacteria, showing differences in band number and intensity. The composition of methanogenic Archaea in these consortia was similar, with the presence of the genera Methanoculleus and Methanosarcina. The efficiency of the microbial consortia selection, and the role of the various microbial trophic groups in bioconversion of the substrates, such as cellulose and the biomass of phototrophic microorganisms are discussed.

Microbiology. 2017;86(6):745-752
pages 745-752 views

Isolation, characterization, and identification of two methomyl-degrading bacteria from a pesticide-treated crop field in West Bengal, India

Roy T., Das N.

Abstract

Two methomyl-degrading bacteria (initially named Disha A and Disha B) were isolated from a pesticide-treated crop field in Baruipur, 24 Parganas (South), West Bengal, India. Both strains could not grow in mineral salt (MS) medium but showed efficient growth in the presence of methomyl. The highest growth was observed in the MS medium containing 0.16% methomyl. When methomyl was supplemented with glucose, no further enhancement of growth was observed, whereas supplementation with yeast extract had a positive effect on growth of both strains, indicating that methomyl could be utilized as the sole source of carbon but not that of nitrogen. In Nutrient Broth and Luria Bertani medium, these strains could tolerate 0.4% methomyl. Optimum pH and temperature for growth of both bacteria in the methomyl-containing MS medium were 7.0 and 30°C, respectively. Protein concentration in the cell-free extracts of bacterial cultures was directly proportional to methomyl concentration in the medium. Disha A was more resistant to the antibiotics amoxicillin and penicillin, as indicated by minimum inhibitory concentration (600 and 500 μg/mL, respectively), which were higher than those obtained for Disha B (350 and 300 μg/mL, respectively). Both Disha A and Disha B were plasmid-bearing, gram-positive, endospore-producing, rod-shaped bacteria. Biochemical studies, 16S rDNA sequencing, and phylogenetic analysis indicated maximum similarity of Disha A to Bacillus cereus ATCC 14579, whereas Disha B showed maximum similarity to Bacillus safensis F0-36b ATCC BAA-1126. The HPLC analysis clearly indicated that B. cereus and B. safensis showed 88.25 and 77.5% of methomyl (Sigma) degradation, respectively within 96 h of growth. This is the first report of Bacillus species that can degrade the carbamate pesticide methomyl and thrive in presence of its high concentrations.

Microbiology. 2017;86(6):753-764
pages 753-764 views

Variability of the composition of the microbial community of the deep subsurface thermal aquifer in Western Siberia

Kadnikov V.V., Frank Y.A., Mardanov A.V., Beletsky A.V., Ivasenko D.A., Pimenov N.V., Karnachuk O.V., Ravin N.V.

Abstract

The deep subsurface biosphere is one of the least studied ecosystems on Earth, containing communities of extremophilic microorganisms. The present work was aimed at molecular genetic characterization of microbial communities of underground thermal waters in Western Siberia, lying at depths of 2–3 km. Water samples were collected from the 5P oil-exploration well, drilled to a depth of 2.8 km near the village Chazhemto (Tomsk region). The water had a temperature of about 20°C, a neutral pH and a low redox potential (–304 mV). Underground aquifers have a complex structure and may contain both planktonic microorganisms and those immobilized on the surface of rocks in the form of biofilms, which may be washed out and detected in the water flowing out of the well. Community composition was analyzed by amplification and pyrosequencing of the 16S rRNA gene fragments in seven water samples taken at different times during 26 hours. Bacteria, which constituted about half of the community, were represented mainly by uncultured lineages of the phyla Firmicutes, Ignavibacteria, Chloroflexi, Bacteroidetes, and Proteobacteria. Archaea belonged mainly to known methanogens of the genera Methanothermobacter, Methanosaeta, and Methanomassiliicoccus. Analysis of the samples taken at different times revealed large variations in the content of most groups of bacteria, with a decrease in Firmicutes abundance accompanied by an increase in the shares of Ignavibacteria and Chloroflexi. The share of archaea of the genus Methanothermobacter varied slightly during the day, while significant variations were observed for the phylotypes assigned to Methanosaeta and Methanomassiliicoccus. Hydrogenotrophic archaea of the genus Methanothermobacter are probably a permanent component of the microbial community occurring in the planktonic state, while most of the identified groups of bacteria are present in biofilms or spatially localized parts of the underground water reservoir, the material of which accidentally enters the well.

Microbiology. 2017;86(6):765-772
pages 765-772 views

Microorganisms of low-temperature heavy oil reservoirs (Russia) and their possible application for enhanced oil recovery

Nazina T.N., Sokolova D.S., Babich T.L., Semenova E.M., Ershov A.P., Bidzhieva S.K., Borzenkov I.A., Poltaraus A.B., Khisametdinov M.R., Tourova T.P.

Abstract

Physicochemical and microbiological characteristics of formation waters low-temperature heavy oil reservoirs (Russia) were investigated. The Chernoozerskoe, Yuzhno-Suncheleevskoe, and Severo-Bogemskoe oilfields, which were exploited without water-flooding, were shown to harbor scant microbial communities, while microbial numbers in the water-flooded strata of the Vostochno-Anzirskoe and Cheremukhovskoe oilfields was as high as 106 cells/mL. The rates of sulfate reduction and methanogenesis were low, not exceeding 1982 ng S2–/(L day) and 9045 nL СН4/(L day), respectively, in the samples from water-flooded strata. High-throughput sequencing of microbial 16S rRNA gene fragments in the community of injection water revealed the sequences of the Proteobacteria (74.7%), including Betaproteobacteria (40.2%), Alphaproteobacteria (20.7%), Gammaproteobacteria (10.1%), Deltaproteobacteria (2.0%), and Epsilonproteobacteria (1.6%), as well as Firmicutes (7.9%), Bacteroidetes (4.1%), and Archaea (0.2%). DGGE analysis of microbial mcrA genes in the community of injection water revealed methanogens of the genera Methanothrix, Methanospirillum, Methanobacterium, Methanoregula, Methanosarcina, and Methanoculleus, as well as unidentified Thermoplasmata. Pure cultures of bacteria of the genera Rhodococcus, Pseudomonas, Gordonia, Cellulomonas, etc., capable of biosurfactant production when grown on heavy oil, were isolated. Enrichment cultures of fermentative bacteria producing significant amounts of volatile organic acids (acetic, propionic, and butyric) from sacchariferous substrates were obtained. These acids dissolve the carbonates of oil-bearing rock efficiently. Selection of the efficient microbial technology for enhanced recovery of heavy oil from terrigenous and carbonate strata requires model experiments with microbial isolates and the cores of oil-bearing rocks.

Microbiology. 2017;86(6):773-785
pages 773-785 views

Yeast population of the Kindo Peninsula lichens

Kachalkin A.V., Glushakova A.M., Pankratov T.A.

Abstract

Yeast abundance and species diversity in the lichens collected at the Kindo Peninsula (Karelia) were studied. A total of 14 lichen species analyzed belonged to the genera Bryoria, Cladonia, Hypogymnia, Icmadophila, Nephroma, Peltigera, and Ramalina. Abundance of cultured yeasts in lichens was intermediate between soil and phyllosphere. The average yeast number on lichens was ~2.5 × 103 CFU/g, while it exceeded 8 × 103 CFU/g on plants and reached only 1 × 103 CFU/g in soil. Yeast population of different parts of Cladonia lichens was found to vary significantly in abundance, species diversity, and community structure. The highest yeast abundance and diversity were revealed in the growth zone. Fifteen yeast species were isolated from lichens, including 6 basidiomycetous and 9 ascomycetous ones. Unlike soils and plants, yeast population of lichens consisted mainly of ascomycetous species, with predominance of Candida sphagnicola and anamorphous yeasts of the genus Dothiora. These results show that yeasts from different taxonomic and ecological groups are a necessary component of lichens; conditions favoring the preservation and development of specific yeast communities differing from the typical soil and phyllosphere yeast complexes are formed in the lichens of northern taiga forests.

Microbiology. 2017;86(6):786-792
pages 786-792 views

Determination of cell concentrations in stationary growing Lactobacillus salivarius cultures in relation to formation of biofilms and cell aggregates

Afonyushkin V.N., Kechin A.A., Tromenshleger I.N., Filipenko M.L., Smetanina M.A.

Abstract

Lactobacillus salivarius belongs to the microbiota of human oral cavity and gastrointestinal tract, as well as of bird and pig intestines. Probiotic activity of various L. salivarius strains has been recently extensively investigated. Production of exopolysaccharides and formation of biofilms as a mechanism providing for resistance to unfavorable factors are also of interest. The goal of this work was to assess the efficiency of microbiological methods for analysis of bacterial concentrations in the cultures of L. salivarius strain NBR2. Samples of lactobacteria grown in liquid medium were collected at equal intervals. The parameters determined were the number of colony-forming units (CFU/mL), share of dead cells by the membrane permeabilization test (LIVE/DEAD) using flow cytometry and fluorescence microscopy, optical density at 595 nm, and pH. After 10 h of cultivation, formation of aggregates commenced, which consisted mainly of living cells and were detected throughout the experiment (30 h). This resulted in underestimation of bacterial abundance determined by plating (CFU/mL). Optical density of the culture and the share of dead cells determined by the LIVE/DEAD method are more reliable criteria of growth of the statically developing L. salivarius culture, which is prone to formation of biofilms and cell aggregates.

Microbiology. 2017;86(6):793-798
pages 793-798 views