I. K. Kravchenko

A hydrocarbon-oxidizing acidophilic thermotolerant bacterial association from sulfur blocks

A stable bacterial association isolated from a sulfur block sample of the Astrakhan gas processing complex was able to utilize n-alkanes as the sole carbon and energy source at low pH. Hydrocarbon-dependent growth occurred at pH 1.6–5.5 (optimum at pH 2.5) and 20–50°C (optimum at 30–35°C). Analysis of the 16S rRNA gene fragments isolated from the total DNA of the enrichment by PCR-DGGE revealed the nucleotide sequences most closely related to extreme acidophilic chemolithotrophs Acidithiobacillus thiooxidans and Sulfobacillus sp. (98–99% similarity) and the sequences exhibiting high similarity to those of slowly growing actinobacteria Mycobacterium europaeum and M. parascrofulaceum (98%). Capacity of any of these organisms for hydrocarbon oxidation has not been reported previously. The taxonomic position of the 16S rRNA gene fragments from the enrichment culture suggests that this bacterial association is a unique microbial community, in which development of acidophilic hydrocarbon-oxidizing bacteria is mediated by a localized pH decrease in the sulfur blocks resulting from elemental sulfur oxidation due to massive development of chemolithotrophic sulfur-oxidizing bacteria.

Hydrocarbon-oxidizing potential and the genes for n-alkane biodegradation in a new acidophilic mycobacterial association from sulfur blocks

The capacity of AGS10, a new aerobic acidophilic (growing within the pH range from 1.3 to 4.5 with the optimum at 2.0–2.5) bacterial association from sulfur blocks of the Astrakhan gas-processing complex (AGC), for oxidation of hydrocarbons of various chemical structure was investigated. A broad spectrum of normal (C10-C21) and iso-alkanes, toluene, naphthalene, and phenanthrene, as well as isoprenoids resistant to microbial degradation, pristane and phytane (components of paraffin oil), and 2,2,4,4,6,8,8,-heptamethylnonane, a branched hydrocarbon, were biodegraded under acidic conditions. Microbiological investigation revealed the dominance of mycobacteria in the AGS10 association, which was confirmed by analysis of the 16S rRNA gene clone library. In the phylogenetic tree, the 16S rRNA sequences formed a branch within the cluster of slow-growing mycobacteria, with 98% homology to the closest species Mycobacterium florentinum. Genomic DNA of AGS10 culture grown on C14-C17n-alkanes at pH 2.5 was found to contain the genes of two hydroxylase families, alkB and Cyp153, indicating their combined involvement in hydrocarbon biodegradation. The high hydrocarbon-oxidizing potential of the AGS10 bacterial association indicated that further search for the genes responsible for degradation of various hydrocarbons in acidophilic mycobacteria could be promising.

Evaluation of the diversity of nitrogen-fixing bacteria in soybean rhizosphere by nifH gene analysis

Biological nitrogen fixation plays an important role in the nitrogen balance of agricultural ecosystems and provides an essential part of nitrogen nutrition for plants, even in conditions of intensive fertilization. The main agrobiotechnological method for soybean cultivation (Glycine max (L.) Merril) is an application of microbial preparations based on Bradyrhizobium japonicum. Successful inoculation strongly depends on the interactions between the introduced microorganism and the aboriginal rhizosphere microorganisms. To evaluate the composition of diazotrophic communities, a study of the diversity of the molecular marker for nitrogen fixation, the nifH gene, in the samples of soybean rhizosphere soil was carried out. Experiments were performed in the variants when soybean was cultivated without inoculation and after adding bacterial preparations, as well as in enrichment cultures of diazotrophs. The revealed diazotrophic microorganisms demonstrated low level of similarity to the known microorganisms (74–95% identity by nucleotides), and were identified as species of the phyla Firmicutes and Proteobacteria. In the composition of nitrogen-fixing communities in the rhizosphere soil, the microorganisms of the genera Clostridium, Paenibacillus, and Spirochaeta were shown to prevail.

Investigation of the methanotrophic communities of the hot springs of the Uzon caldera, Kamchatka, by molecular ecological techniques

The methane-oxidizing microbial communities inhabiting the bottom sediments of 36 hot springs of the Uzon caldera (Kamchatka, Russia) located in the thermal fields Vostochnoe, Oranzhevoe, and Severnoe, as well as near the lakes Fumarol’noe and Khloridnoe and the Izvilistyi stream, were studied. Methanotrophic bacteria were detected by PCR and FISH in only 8 hot springs. The highest numbers of copies of the pmoA gene (molecular marker of methanotrophy) (2.8 × 107 and 1.1 × 107 copies/mL sediment) were detected in the Kul’turnyi and Kvadrat springs; however, in other springs, the numbers of the pmoA gene copies were significantly lower (5.4 × 103–2.8 × 106 copies/mL sediment). By using the FISH method, only type I methanotrophs were detected in these springs, with their percentage ranging from 0.3 to 20.5% of the total number of eubacteria. PCR-DGGE analysis of the pmoA gene showed that the diversity of methanotrophs was extremely low (no more that two components). Analysis of the deduced PmoA amino acid sequences demonstrated that methanotrophic bacteria of the genus Methylothermus, closely related to representatives of two valid species, widely occurred in the thermal springs near Lake Fumarol’noe. Other bacteria differing considerably from the detected Methylothermus species were detected as well. In the springs with low pH values (2.6–3.8), methanotrophic Gammaproteobacteria most closely related to the genera Methylomonas and Methylobacter were detected for the first time.

Diversity of diazotrophic gut inhabitants of pikas (Ochotonidae) revealed by PCR-DGGE analysis

Diazotrophic gut symbionts are considered to act as nitrogen providers for their hosts, as was shown for various termite species. Although the diet of lagomorphs, like pikas or rabbits, is very poor in nitrogen and energy, their fecal matter contains 30–40% of protein. Since our hypothesis was that pikas maintained a diazotrophic consortium in their gastrointestinal tract, we conducted the first investigation of microbial diversity in pika guts. We obtained gut samples from animals of several Ochotona species, O. hyperborea (Northern pika), O. mantchurica (Manchurian pika), and O. dauurica (Daurian pika), in order to retrieve and compare the nitrogen-fixing communities of different pika species. The age and gender of the animals were taken into consideration. We amplified 320-bp long fragments of the nifH gene using the DNA extracted directly from the colon and cecum samples of pika’s gut, resolved them by DGGE, and performed phylogenetic reconstruction of 51 sequences obtained from excised bands. No significant difference was detected between the nitrogen-fixing gut inhabitants of different pika species. NifH sequences fell into two clusters. The first cluster contained the sequences affiliated with NifH Cluster I (Zehr et al., 2003) with similarity to Sphingomonas sp., Bradyrhizobium sp., and various uncultured bacteria from soil and rhizosphere. Sequences from the second group were related to Treponema sp., Fibrobacter succinogenes, and uncultured clones from the guts of various termites and belonged to NifH Cluster III. We suggest that diazotrophic organisms from the second cluster are genuine endosymbionts of pikas and provide nitrogen for further synthesis processes thus allowing these animals not to be short of protein.