Succession of Chitinolytic Microorganisms in Chernozem Soil

Microbiology - The chitinolytic prokaryotic and eukaryotic microbial complex of chernozem soil has been investigated in the course of a succession initiated by the introduction of chitin and...     

Temperature as an autoecological factor of chitinolytic microbial complex formation in soils

The dynamics of carbon dioxide emission from soil was studied during chitinolytic succession induced by humidification and chitin introduction at different temperatures (5, 27, and 50°C) using gas chromatography. The abundance and biomass of the chitinolytic bacterial and actinomycete complex in soil were evaluated by luminescent microscopy. Active development of the chitinolytic microbial complexes was observed at all studied temperatures. The most active growth of chitinolytic microorganisms was observed at high temperature during early succession and at low temperature during late succession. High and low temperatures provided for active development of the chitinolytic microbial complex in soils confined to warm climatic zones (brown desert-steppe soil) and soils of temporary zones (gray forest soil). Actinomycetes demonstrated the most active growth among chitinolytic microorganisms in the studied soil samples both at low and high temperatures.     

Microbial transformation of chitin in soil under anaerobic conditions

Anaerobic chitinolytic complex was studied in three soil types: chernozem, gray forest soil, and chestnut soil. The abundance and biomass of anaerobic chitinolytic microbial complex of fungi, bacteria, and actinomycetes were evaluated by luminescent microscopy. The dynamics of methane emission from soil during chitinolytic succession was studied by gas chromatography. All three studied microbial groups proved to participate in chitin transformation in soil under anaerobic conditions. The highest biomass growth was observed among prokaryotes, particularly actinomycetes, whose biomass doubled. The increase in methane emission during chitinolytic succession was most pronounced in soils with low organic matter content.     

Description of the phylogenetic structure of hydrolytic prokaryotic complex in the soils

With the help of the molecular-biological method of cell hybridization in situ (FISH), the abundance of a physiologically active hydrolytic prokaryotic complex in chernozem and gley-podzolic soils is determined. The total proportion of metabolically active cells, which were detected by hybridization with universal probes as representatives of the domains Bacteria and Archaea, in samples of the studied soil, was from 38% for chernozem up to 78% for gley-podzolic soil of the total number of cells. The differences in the structure of chitinolytic and pectinolytic prokaryotic soil complexes are detected. Along with the high abundance of Actinobacteria and Firmicutes in the soils with chitin, an increase in phylogenetic groups such as Alphaproteobacteria and Bacteroidetes is observed.     

Thermophilic chitinolytic microorganisms of brown semidesert soil

In brown semidesert soil, thermophilic prokaryotic organisms identified as Streptomyces roseolilacinus and Silanimonas lenta were shown to play the main role in chitin transformation at 50°C. The phylogenetic positions of the isolated dominant chitinolytic microorganisms were determined on the basis of 16S rRNA gene sequencing. The consumption of chitin as a source of carbon and nitrogen by both the bacterium and the actinomycete was evident from considerable biomass accumulation, high emission of carbon dioxide, and presence in the medium of the chitinase exoenzyme.     

Methodological aspects of assessing chitin utilization by soil microorganisms

A computational method for estimating specific activity of chitin decomposition by microorganisms is proposed. Spectrophotometric and gas chromatographic methods have been used to determine the rates of chitinase production, biomass accumulation, and carbon dioxide emission by pure cultures of microorganisms grown on a chitin-containing medium. Among dominants of the chitinolytic community of chernozem (Trichoderma viride, Stretomyces albolongus, Alcaligenes, and Arthrobacter), the highest chitinolytic activity is characteristic of prokaryotes. In brown desert-steppe soil, the main destructor prokaryotes are actinomycetes (S. roseolilacinus). The biomass of the fungus T. viride growth on the chitin-containing medium markedly exceeds that of prokaryotes, but the specific activity of respiration and chitinase production in actinomycetes S. roseolilacinus and S. albolongus is an order of magnitude higher than in T. viride.     

A chitinolytic actinomycete complex in black soil

A chitinolytic actinomycete complex in chernozem soil has a specific taxonomic composition, which differs from that of the actinomycete complex which is typically isolated on standard nutrient media containing sugars and organic acids as carbon sources. The actinomycete complex that was isolated by using nutrient media with chitin as the source of carbon and nitrogen was dominated by representatives of the genus Streptosporangium, and the actinomycete complex that was isolated by using nutrient media with sugars and organic acids as the carbon sources was dominated by representatives of the genus Streptomyces. The confirmation to the ability of actinomycetes to utilize chitin as a sole source of carbon and nitrogen came from the augmented length and biomass of the mycelium, the increased number and biomass of the actinomycete spores, the production of carbon dioxide, and the accumulation of NH4+ ions in the culture liquid of the actinomycetes that are grown in the nutrient media with chitin.     

Specificity of the chitinolytic microbial complex of soils incubated at different temperatures

The structural and functional specificity of the chitinolytic microbial complex changes dramatically depending on the incubation temperature of soil microcosms. It was shown that the highest rates of chitin degradation occurred in desert soils at high temperatures (50°C); in the moderate and northern zones, these rates peaked at lower temperatures (5°C). The role of prokaryotes as the main chitin degraders in soils incubated at high temperatures, with fungi more actively participating in chitin decomposition at low temperatures, was shown for the first time. Fluorescent in situ hybridization (FISH) revealed the predominance of actinomycetes in the metabolically active chitinolytic prokaryotic complex of desert soils (high temperatures); in the soils of the northern latitudes (low temperatures), proteobacteria prevailed. The relationship between the taxonomic position of the dominant members of the chitinolytic complex of soil microorganisms, isolated in pure cultures with the dominant phylogenetic groups and the sequence types obtained by using molecular biological techniques (FISH) was revealed.     

A Chitinolytic Actinomycete Complex in Chernozem Soil

A chitinolytic actinomycete complex in chernozem soil has a specific taxonomic composition, which differs from that of the actinomycete complex typically isolated on standard nutrient media containing sugars and organic acids as carbon sources. The actinomycete complex that was isolated by using nutrient media with chitin as the source of carbon and nitrogen was dominated by representatives of the genus Streptosporangium, and the actinomycete complex that was isolated by using nutrient media with sugars and organic acids as the carbon sources was dominated by representatives of the genus Streptomyces. The confirmation of the ability of actinomycetes to utilize chitin as a sole source of carbon and nitrogen came from the augmented length and biomass of the mycelium, the increased number and biomass of the actinomycete spores, the production of carbon dioxide, and the accumulation of NH₄ ⁺ ions in the culture liquid of the actinomycetes grown in the nutrient media with chitin.     

Chitinase and changes of microbial community in soil

Enrichment of soil with chitin (0.6%) significantly stimulated growth of chitinolytic microorganisms (the relative proportion was increased from 1.7 to 26.5%) and the formation of chitinase in soil. In a soil enriched with chitin and glucose (0.6%), the proportion of chitinolytic microorganisms remained similar to that in the nonenriched soil (1.4%), the enzyme formation was negatively affected.     

Mycophagous mites and their internal associated bacteria cooperate to digest chitin in soil

The greater bulk of soil nitrogen is immobilized in chitinous cell walls of fungi. Mycophagous soil mites participate in chitin decomposition and, hence, in the subsequent mobilization of nitrogen. The source of the chitinolytic enzymes was searched in this study. A multimethodical approach was designed for these studies. Histology, plating and identification of bacteria from mite homogenate and, finally, homogenate and bacterial treatment of the soil fungi were applied. Here the presence and activity of chitinolytic bacteria inside mycophagous mites are reported. These bacteria form an extraintestinal group within the mite’s body and pass their enzymes into the mite’s gut. Our results demonstrate that true mycophagous mites, defined by their ability to digest chitin (i.e. the fungal cell wall), achieve this through internal “cooperation” with chitinolytic bacteria that provide the necessary chitinolytic enzymes. The nitrogen from chitin is thus made available to other soil organisms and plants.     

The composition of the chitinolytic microbial complex and its effect on chitin decomposition at various humidity levels

The dynamics of assimilation of chitin by soil microorganisms (primarily prokaryotes) as a source of carbon and nitrogen has been determined by gas chromatography and fluorescence microscopy. The highest rates of chitin decomposition in chernozem were detected at humidity levels corresponding to the pressure of soil moisture (P) of -1.4 atm. The rate of microbial consumption of chitin is three times higher than that of the carbon of soil organic matter. Fluorescence microscopy revealed that an increase in the pressure of soil moisture from P = -10 atm to P = -0.7 atm resulted in a considerable increase in the proportion of the specific surface of mycelial bacteria (actinomycetes).     

Assessment of chitin decomposer diversity within an upland grassland

The breakdown of chitin within an acidic upland grassland was studied. The aim was to provide a molecular characterisation of microorganisms involved in chitin degradation in the soil using soil microcosms and buried litter bags containing chitin. The investigation involved an examination of the effects of liming on the microbial communities within the soil and their chitinolytic activity. Microcosm experiments were designed to study the influence of lime and chitin enrichment on the grassland soil bacterial community ex situ under controlled environmental conditions. Bacterial and actinomycete counts were determined and total community DNA was extracted from the microcosms and from chitin bags buried at the experimental site. PCR based on specific 16S rRNA target sequences provided products for DGGE analysis to determine the structure of bacterial and actinomycete communities. Chitinase activity was assessed spectrophotometrically using chitin labelled with remazol brilliant violet. Both liming and chitin amendment increased bacterial and actinomycete viable counts and the chitinase activity. DGGE band patterns confirmed changes in bacterial populations under the influence of both treatments. PCR products amplified from DNA isolated from chitin bags were cloned and sequenced. Only a few matched known species but a prominent coloniser of chitin proved to be Stenotrophomonas maltophilia.     

Chitin-supplemented formulations improve biocontrol and plant growth promoting efficiency of Bacillus subtilis AF 1

Formulations of a chitinolytic biocontrol and a plant growth promoting Bacillus subtilis AF 1 were prepared in peat, in peat supplemented with either 0.5% chitin or Aspergillus niger mycelium, or in spent compost obtained from Agaricus bisporus cultivation and were evaluated for biocontrol of two fungal pathogens and plant growth promoting activities on pigeon pea and groundnut. A steady increase in cell numbers of introduced B. subtilis AF 1 was observed in all the formulations at 30 degrees C. The increase in cell numbers was about 5.0 log units. Peat or spent compost inoculated with physiologically active and dormant states of B. subtilis AF 1 showed different time period requirements to attain maximum cell numbers. The presence of chitin or A. niger (in peat) or A. bisporus (in spent compost) as supplement in the carrier material improved the multiplication of B. subtilis AF 1. When used as seed treatments, formulations of AF 1 in peat supplemented with chitin or chitin-containing materials showed better control of A. niger (causing crown rot of groundnut) and Fusarium udum (causing wilt of pigeon pea) than AF 1 culture alone, in both groundnut and pigeon pea. Bacillus subtilis AF 1 formulations promoted seed germination and biomass of both groundnut and pigeon pea even under pathogen pressure. Survival of AF 1 on fresh culture-treated and formulation product-treated plants was similar in pathogen-infested soil.     

Molecular analysis of the hydrolytic component of petroleum-contaminated soils and of soils remediated with chitin

Molecular genetic techniques (FISH and metagenomic analysis) were used to investigate prokaryotic complexes in native soils (gray forest soil and urbostratozema typical), soils contaminated by petroleum products (gasoline or diesel fuel), and soils subject to remediation by addition of a nitrogen-containing polysaccharide biopolymer chitin. The share of metabolically active prokaryotic cells in the hydrolytic complex of soil microcosms was determined, as well as their biomass and biodiversity. Compared to the control, in the pollutant-containing experimental microcosms, a decrease in the share of metabolically active prokaryotic cells was observed, as well as changes of the hydrolytic complex structure, such as an increase in the share of the phylum Actinobacteria (specifically of the genera Galiella and Nocardioides in the samples contaminated with gasoline and diesel fuel, respectively). Supplementing the hydrocarbon-contaminated system the biopolymer chitin resulted in processing of mixed-minerals with an increase in the number of layers of the smectite type and, as a result, in formation of aggregates and improved aeration. An increase in the number of metabolically active prokaryotic cells and decreased diversity of the soil prokaryotic complex were observed, which were probably associated with the development of a selective group of the hydrolytic complex of chitindegrading microorganisms.     

Estimate of the degradation potentials of cellulose,xylan, and chitin across global prokaryotic communities

Complex polysaccharides (e.g. cellulose, xylan, and chitin), the most abundant renewable biomass resources available on Earth, are mainly degraded by microorganisms in nature. However, little is known about the global distribution of the enzymes and microorganisms responsible for the degradation of cellulose, xylan, and chitin in natural environments. Through large-scale alignments between the sequences released by the Earth Microbiome Project and sequenced prokaryotic genomes, we determined that almost all prokaryotic communities have the functional potentials to degrade cellulose, xylan, and chitin. The median abundances of genes encoding putative cellulases, xylanases, and chitinases in global prokaryotic communities are 0.51 (0.17–1.01), 0.24 (0.05–0.57), and 0.33 (0.11–0.71) genes/cell, respectively, and the composition and abundance of these enzyme systems are environmentally varied. The taxonomic sources of the three enzymes are highly diverse within prokaryotic communities, and the main factor influencing the diversity is the community's alpha diversity index rather than gene abundance. Moreover, there are obvious differences in taxonomic sources among different communities, and most genera with degradation potentials are narrowly distributed. In conclusion, our analysis preliminarily depicts a panorama of cellulose-, xylan-, and chitin-degrading enzymatic systems across global prokaryotic communities.     

Monitoring of the chitinolytic microbial complex of the phylloplane

A comprehensive study of chitinolytic microbial complexes of the phylloplane from cultured and forest plants has been conducted. An increase of the number and biomass of metabolically active cells of the representatives of the domain Bacteria and a decrease in fungal biomass in the experimental microcosms have been shown to occur after the introduction of chitin. The characteristic features of the taxonomic structure of metabolically active chitinolytic complexes of the phylloplane of the plants studied have been elucidated. Representatives of the phyla Proteobacteria, Bacteroidetes, and Verrucomicrobia have been shown to play important roles in the chitinolytic complexes of green leaf samples, while mycelial actinobacteria of the phylum Actinobacyteria played a similar role in needles of coniferous trees. A collection of chitinolytic microorganism cultures isolated from the phylloplane of different plant species has been created.     

The composition of the chitinolytic microbial complex and its effect on chitin decomposition at various humidity levels

The dynamics of assimilation of chitin by soil microorganisms (primarily prokaryotes) as a source of carbon and nitrogen has been determined by gas chromatography and fluorescence microscopy. The highest rates of chitin decomposition in chernozem were detected at humidity levels corresponding to the pressure of soil moisture (P) of ?1.4 atm. The rate of microbial consumption of chitin is three times higher than that of the carbon of soil organic matter. Fluorescence microscopy revealed that an increase in the pressure of soil moisture from P = ?10 atm to P = ?0.7 atm resulted in a considerable increase in the proportion of the specific surface of mycelial bacteria (actinomycetes).     

Microbial biomass and growth kinetics of microorganisms in chernozem soils under different farm land use modes

The carbon content of microbial biomass and the kinetic characteristics of microbial respiration response to substrate introduction have been estimated for chernozem soils of different farm lands: arable lands used for 10, 46, and 76 years, mowed fallow land, non-mowed fallow land, and woodland. Microbial biomass and the content of microbial carbon in humus (Cmic/Corg) decreased in the following order: soils under forest cenoses-mowed fallow land-10-year arable land-46- and 75-year arable land. The amount of microbial carbon in the long-plowed horizon was 40% of its content in the upper horizon of non-mowed fallow land. Arable soils were characterized by a lower metabolic diversity of microbial community and by the highest portion of microorganisms able to grow directly on glucose introduced into soil. The effects of different scenarios of carbon sequestration in soil on the reserves and activity of microbial biomass are discussed.     

Mutual relationships between soils and biological carrier systems

Improved viability and antagonistic activity of biocontrol agents during soil inoculation is of crucial importance to their effective application. The chitinolytic bacterium Serratia marcescens was used as a model organism to study the efficacy of freeze-dried alginate beads (in comparison to their non-dried counterparts) as possible carriers for immobilized biocontrol agents. The release of bacteria and chitinolytic enzyme from alginate beads, before and during their application in soil, was examined, and the beads' physical properties characterized. Dispersal of the alginate bead-entrapped S. marcescens in the soil resulted in high soil cell densities throughout the 35 days of the experiment. Chitin inclusion in the beads resulted in significantly higher chitinolytic activity of S. marcescens, increased dry-bead porosity and decreased stiffness. Rehydration of the dried beads (after immersion in soil) resulted in a sixfold increase in weight due to water absorption. No significant differences were found in bacterial count inside the non-dried (gel) versus dried beads. However, higher cell densities and chitinase activity were detected in soil containing dried beads with chitin than in that containing their non-dried counterparts. The biological performance of S. marcescens was examined in the greenhouse: a free cell suspension reduced bean (Phaseolus vulgaris L.) disease by 10%, while immobilized bacteria found in the dried, chitin-containing beads reduced disease by 60%.