B. A. Byzov

Predation on fungal and bacterial biomass in a soddy-podzolic soil amended with starch, wheat straw and alfalfa meal

The variation in bacterial, fungal and total microbial biomass and activity was studied together with the abundance of soil nematodes and microarthropods after the addition of substrates differing in nitrogen availability to a soddy-podzolic soil. The experiments were carried out in microcosms with native and defaunated soil to evaluate stimulatory and suppressive effects of the microfauna on soil micro-organisms. Predation by microfauna (nematodes) and mesofauna (microarthropods) reduced the microbial biomass and microbial respiration by approximately 25% after addition of nitrogen rich alfalfa meal. When starch and wheat straw were supplied, the microbial biomass and activity were stimulated by up to 30% by grazing. Thus, the effect of predation on the microbiota depended on the composition of the available substrates and available nitrogen seems to be an important factor controlling stimulation or suppression of soil micro-organisms by the soil fauna when fresh organic compounds are accessible. The presence of soil fauna stimulated bacteria and, thus, reduced the fungal/bacterial ratio during the course of decomposition. In contrast, the fungal/bacterial ratio declined due to decreasing fungal biomass in defaunated soil.

Effect of the Earthworms Lumbricus terrestris and Aporrectodea caliginosa on Bacterial Diversity in Soil

Earthworms ingest large amounts of soil and have the potential to radically alter the biomass, activity, and structure of the soil microbial community. In this study, the diversity of eight bacterial groups from fresh soil, gut, and casts of the earthworms Lumbricus terrestris and Aporrectodea caliginosa were studied by single-strand conformation polymorphism (SSCP) analysis using both newly designed 16S rRNA gene-specific primer sets targeting Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Bacteroidetes, Verrucomicrobia, Planctomycetes, and Firmicutes and a conventional universal primer set for SSCP, with RNA and DNA as templates. In parallel, the study of the relative abundance of these taxonomic groups in the same samples was performed using fluorescence in situ hybridization. Bacteroidetes, Alphaproteobacteria, and Betaproteobacteria were predominant in communities from the soil and worm cast samples. Representatives of classes Flavobacteria and Sphingobacteria (Bacteroidetes) and Pseudomonas spp. (low-abundant Gammaproteobacteria) were detected in soil and worm cast samples with conventional and taxon-targeting SSCP and through the sequence analysis of 16S rRNA clone libraries. Physiologically active unclassified Sphingomonadaceae (Alphaproteobacteria) and Alcaligenes spp. (Betaproteobacteria) also maintained their diversities during transit through the earthworm intestine and were found on taxon-targeting SSCP profiles from the soil and worm cast samples. In conclusion, our results suggest that some specific bacterial taxonomic groups maintain their diversity and even increase their relative numbers during transit through the gastrointestinal tract of earthworms.

Reaction of microorganisms to the digestive fluid of earthworms

The reaction of soil bacteria and fungi to the digestive fluid of the earthworm Aporrectodea caliginosa was studied. The fluid was obtained by centrifugation of the native enzymes of the digestive tract. The inhibition of growth of certain bacteria, spores, and fungal hyphae under the effect of extracts from the anterior and middle sections of the digestive tract of A. caliginosa was discovered for the first time. In bacteria, microcolony formation was inhibited as early as 20–30 s after the application of the gut extracts, which may indicate the nonenzymatic nature of the effect. The digestive fluid exhibited the same microbicidal activity whether the earthworms were feeding on soil or sterile sand. This indicates that the microbicidal agents are formed within the earthworm’s body, rather than by soil microorganisms. The effect of the digestive fluid from the anterior and middle divisions is selective in relation to different microorganisms. Of 42 strains of soil bacteria, seven were susceptible to the microbicidal action of the fluid (Alcaligenes faecalis 345-1, Microbacterium sp. 423-1, Arthrobacter sp. 430-1, Bacillus megaterium 401-1, B. megaterium 413-1, Kluyvera ascorbata 301-1, Pseudomonas reactans 387-2). The remaining bacteria did not die in the digestive fluid. Of 13 micromycetes, the digestive fluid inhibited spore germination in Aspergillus terreus and Paecilomyces lilacinus and the growth of hyphae in Trichoderma harzianum and Penicillium decumbens. The digestive fluid stimulated spore germination in Alternaria alternata and the growth of hyphae in Penicillium chrysogenum. The reaction of the remaining micromycetes was neutral. The gut fluid from the posterior division of the abdominal tract did not possess microbicidal activity. No relation was found between the reaction of microorganisms to the effects of the digestive fluid and the taxonomic position of the microorganisms. The effects revealed are similar to those shown earlier for millipedes and wood lice in the following parameters: quick action of the digestive fluid on microorganisms, and the selectivity of the action on microorganisms revealed at the strain level. The selective effect of the digestive gut fluid of the earthworms on soil microorganisms is important for animal feeding, maintaining the homeostasis of the gut microbial community, and the formation of microbial communities in soils.

Principles of the digestion of microorganisms in the gut of soil millipedes: specificity and possible mechanisms

Abstract The digestion of different groups of microorganisms in the gut of the soil millipede Pachyiulus flavipes was studied. Gramnegative aerobic bacteria, bacilli, streptomycetes, coryneform bacteria and fungi of the genus Penicillium , isolated from leaf litter, resisted digestion, whereas the majority of predominating yeasts and fungi, including species of family Dematiaceae, were considerably decreased by gut passage. Microorganisms showed species-dependent sensitivities to the midgut fluid of the diplopod. Those that were found to be sensitive to the digestion were killed after only a few minutes of incubation in the fluid. The separation of the fluid by LH-20 Sephadex chromatography reduced its killing activity markedly. The compounds responsible for the mortality were extracted from the Sephadex with 50% methanol. The microbolytic activity of the midgut fluid related to its protein fraction. When applied separately, neither killing nor protein fractions caused the destruction of the cells. Complete destruction of the cells was observed only when the mixture of the two fractions or the native fluid were applied. The mechanism of the induced autolysis is suggested to play a role in the digestion process.

Culturable microorganisms from the earthworm digestive tract

The cultured aerobic copiotrophic bacteria and fungi from food-free digestive tracts of Aporrectodea caliginosa, Lumbricus terrestris, and Eisenia fetida earthworms, soil (compost), and fresh earthworm excrements were investigated. The microorganisms were isolated on nutrient media and identified by sequencing the fragments of bacterial 16S rRNA and fungal 28S rRNA (D1/D2 domain) gene sequences with subsequent phylogenetic analysis. Bacteria isolated from the digestive tracts of earthworms belonged to the families Aeromonadaceae, Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae, Moraxellaceae, Pseudomonadaceae, and Sphingobacteriaceae (Bacteroidetes), as well as Actinobacteria. For five strains, namely Ochrobactrum sp. 341-2 (α-Proteobacteria), Massilia sp. 557-1 (β-Proteobacteria), Sphingobacterium sp. 611-2 (Bacteroidetes), Leifsonia sp. 555-1, and a bacterium from the family Microbacteriaceae, isolate 521-1 (Actinobacteria), the similarity to known 16S rRNA sequences was 93–97%; they therefore, probably belong to new species and genera. Bacterial groups isolated from the digestive tracts of earthworms were significantly different from those isolated from soil and excrements. Some bacterial taxa occurred in different sections of A. caliginosa intestine and in intestines of different earthworm species; however, the overall composition of bacterial communities in these objects is different. Existence of bacterial groupings symbiotically associated with intestines is proposed. Among the fungi, Bjerkandera adusta and Syspastospora parasitica were isolated from the cleaned digestive tracts as light-colored, sterile mycelium, as well as Geotrichum candidum, Acremonium murorum (A. murorum var. felina), Alternaria alternata, Aspergillus candidus, A. versicolor, Cladosporium cladosporioides, Rhizomucor racemosus, Mucor hiemalis, Fusarium (F. oxysporum, Fusarium sp.), and Penicillium spp. These fungi survive for a long time in the earthworm’s digestive environment. Investigation of the functional characteristics and role in the host organism is required to confirm the symbiotic status of the microorganisms associated with the earthworm digestive tract.

Interrelationships between yeasts and soil diplopods

Abstract Some functional interrelationships have been established between yeasts and the soil diplopods Pachyiulus flavipes C.L. Koch and Glomeris connexa C.L. Koch. Yeasts can serve as a food for diplopods when these animals are feeding on leaf litter. The digestion of yeast cells takes place mainly in the midgut as a result of enzymatic activity of the gut fluid. The midgut is a well-protected zone in which enzymatic activity prevents exogenic infection and thereby provides a steady-state environment for the yeast community. The hindgut is a region where specific yeasts can utilize readily-available carbon sources from leaf litter and hydrolyze uric acid—the end-product of nitrogen metabolism of the host animals. A model for yeast distribution in the gut of diplopods is described.

Response of bacteria to earthworm surface excreta

Response of bacteria to the surface excreta of the Aporrectodea caliginosa earthworm was studied. The excreta were obtained by a 1 h incubation of the earthworms in petri dishes with subsequent collection of the slime. Both inhibition and stimulation of growth were revealed, as well as suppression of the respiratory activity of some bacterial species treated with A. caliginosa surface excreta. The organisms studied included various taxa of soil bacteria (19 strains), bacteria isolated from A. caliginosa intestine and excrements (82 strain), and 48 Bacillus thuringiensis strains. For the cultures of soil bacteria, the respiratory activity was determined using the formazan color reaction due to the activity of the respiratory cycle enzymes. Earthworm excreta caused a consistent 30–50% decrease of dehydrogenase activity in 13 out of the 19 cultures. Determination of the growth rates (derived from OD620 of cell suspensions) after 10 h of incubation revealed growth stimulation in 48 out of the 82 strains isolated from intestines and excrement. Other strains exhibited no reaction to the excreta. For 29 out of 45 B. thuringiensis strains, growth stimulation was observed, while growth of two strains was suppressed; other strains exhibited no reaction to the excreta. No relation was found between bacterial reaction to the excreta and their taxonomic position. These results correlate with the research, demonstrating antibacterial and antifungal activity of the extracts from the earthworm body and digestive tract. Thus, earthworms, apart from their medium-forming function, affect the formation of soil microbial communities by direct stimulation or suppression of specific microbial populations.

Regulation of the biomass and activity of soil microorganisms by microfauna

Microcosm experiments showed that the microbial biomass and the respiration activity in soil were regulated by nematodes. Depending on nematode number and plant residue composition, the trophic activity of nematodes can either stimulate or inhibit microbial growth and respiration as compared to soil containing no nematodes. The stimulating effect was observed when nitrogen-free (starch) or low-nitrogen (wheat straw, C : N = 87) organic substrates were applied. Inhibition occurred when a substrate rich in nitrogen (alfalfa meal, C : N = 28) was decomposed and the nematode population exceeded the naturally occurring level. A conceptual model was developed to describe trophic regulation by microfauna (nematodes) of the microbial productivity and respiration ctivity and decomposition of not readily decomposable organic matter in soil. The stimulating and inhibiting influence of microfauna on soil microorganisms was not a linear function of the rate of microbial consumption by nematodes. These effects are largely associated with the induced change in the physiological state of microorganisms rather than with the mobilization of biogenic elements from the decomposed microbial biomass.

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.

Interrelationships between yeast fungi and collembolans in soil

The possibility of feeding on green and newly fallen leaves of the small-leaved lime Tilia cordata was studied for the collembolans Protaphorura armata and Vertagopus pseudocinereus. Young leaves grown under sterile conditions and almost free of yeast fungi were found to be toxic to the collembolan V. pseudocinereus: feeding on them led to the death of the animals. Leaves grown under natural conditions were nontoxic: when used by the collembolans as feed, they provided for collembolan growth and fecundity. Feeding preferences of the collembolans in relation to the yeasts attributed to different ecomorphs—epiphytes, litter saprophytes, pedobionts, and saccharobionts—were studied. Of the 24 yeast strains isolated from plant green parts, litter, and soil and assigned to eight species, no strain was revealed that was not used by the collembolans. However, certain yeast strains were preferable for the collembolans. The population of the V. pseudocinereus collembolans feeding on the yeast Rhodotorula glutinis (nss 31–4) exceeded that grown on Cryptococcus terricola (2044) 1.5-fold. Thus, the collembolans have feeding preferences in relation to yeast fungi, as was shown earlier with mycelial micromycetes. The possible mechanisms of the feeding preferences of the collembolans in relation to yeasts are discussed.