Anelia Kenarova

The global methane budget is largely driven by biogenic sources, many of which remain insufficiently characterized. Here, we investigated the community composition and seasonal dynamics of methanogenic and methanotrophic assemblages to elucidate the key contributors to methane cycling and the environmental factors shaping these processes in lake sediments of the Rila Mountains (Bulgaria). Methanogenic communities are primarily composed of Methanothrix, Methanosarcina, Methanobacterium and Methanoregula with summer peaks in Methanothrix and Methanoregula, and cold-season proliferation of Methanobacterium. Methanotrophic communities are dominated by representatives of the Pseudomonadota, including Crenothrix, Methylobacter, and Methylocystis with summer maxima observed for Crenothrix and Methylobacter. Methanosarcina and Methylocystis showed relatively stable abundances throughout the ice-free season. Ordination and correlation analyses revealed that temperature, pH, and carbon (organic and inorganic) concentration and lability emerged as the environmental drivers influencing on microbial communities, with seasonally variable effects on methane-cycling microorganisms. These findings provide a foundation for future research on methane cycling in alpine lake ecosystems of the Rila Mountains and contribute to improving predictions of methane emissions under changing climatic conditions.

High-altitude lakes are sensitive sentinels of climate change, and yet the microbial processes driving biogeochemical cycles under warming remain poorly understood. Seasonal dynamics of sediment microbial communities in four glacial lakes within the cirques of the Seven Rila Lakes (Rila Mountains, Bulgaria) were investigated during June, August, and October 2024. Environmental monitoring showed pronounced seasonal variability with summer peaks in temperature, dissolved organic carbon and nitrogen, and increased inputs of labile organic matter. Multivariate analyses revealed strong seasonal structuring of microbial assemblages, with the largest shifts occurring between the early ice-free season and summer. Archaeal communities were dominated by Halobacteriota over time, whereas lake warming corresponded to a shift from early-season dominance of Actinomycetota and Bacillota to the summer prevalence of Pseudomonadota and Cyanobacteriota, as well as a proliferation of minor phyla Gemmatimonadota. At the genus level, summer enrichment of archaeal methanogen Methanosaeta and bacterial genera Paucibacter, Ca. Accumulibacter, Methylibium, Crenothrix, and Pseudanabaena was observed. Canonical correspondence analysis identified temperature and nutrient availability as the primary drivers of microbial community restructuring. Our results provide empirical evidence that warming and associated changes in organic matter inputs drive shifts in sediment microbial communities, with direct implications for carbon cycling and methane production. Additionally, the results highlight the sensitivity of high-altitude lakes to global warming and emphasize the critical role of microbial communities in modulating ecosystem responses to climate change.

The aim of the study was to evaluate the long-term impact of heavy metals on soil bacterial metabolism by assessing their capacity to utilize a set of carbon sources. The study area is in the vicinity of the Pb/Zn smelter KCM 2000, South-Central Bulgaria, with considerable Pb, Zn, Cd, Cu, and As pollution. The Biolog Ecoplate approach was used to evaluate the bacterial metabolic activity, expressed by the average well colour development; the bacterial metabolic fingerprints expressed by community-level physiological profiling, and Shannon bacterial functional diversity. The results revealed reduced metabolic activity recorded for bacterial communities inhabiting the heavily polluted soils. The most preferred carbon groups utilized by the bacterial communities were the polymers, followed by carbohydrates and amino acids. Local variability of soil properties, such as inorganic ions, total organic carbon, pH, soil moisture, and soil texture, could modify the effects of heavy metals. This study highlighted that Biolog EcoplateTM is a suitable approach for assessing bacterial functional shifts in relation to long-term soil pollution stress.

High-mountain lakes are biodiversity hotspots sensitive to increasing regional and global climate warming. However, their microbial communities remain insufficiently characterized due to their remoteness and limited accessibility. This study aimed to determine how seasonal environmental parameters shape the composition, structure and diversity of littoral bacterial communities in three glacial lakes in Rila Mountains (Bulgaria). Water samples were collected during ice-free periods in 2023 and 2024, and bacterial taxonomic composition was analysed by Next-generation sequencing. A total of 1158 bacterial OTUs were identified encompassing 18 phyla and 165 families. Actinomycetota, Pseudomonadota, and Bacteroidota were dominant at the phylum level, and Sporichthyaceae, Comamonadaceae, Chitinophagaceae and Mycobacteriaceae were most abundant among the families. Community richness and diversity peaked in June, immediately after ice melting, particularly in the highest-altitude lake (Sulzata Lake), and declined during the warm season (August), when the relative abundances of Sporichthyaceae and Mycobacteriaceae (Actinomycetota) increased. Seasonal restructuring occurred across phyla and families even in a single taxon, with water temperature and organic carbon availability identified as the main environmental drivers. The findings have improved our understanding of temperature-driven bacterial responses. They have also highlighted the vulnerability of cold-adapted taxa to regional climate warming which may contribute to more effective biodiversity conservation strategies for these unique ecosystems.

Soil pollution by heavy metals (HMs) poses a major threat to soil quality and human health, with mining and smelting industries identified as key sources. Soils around smelters are often considered polluted hotspots, being generally unsuitable for agricultural activities. Although many studies have identified microbial taxa able to survive in such environments, most have focused on relatively low HM concentrations. The purpose of the study was to assess the ecological risk and to evaluate the diversity and structural shifts in microbial communities, as well as to predict key metabolic pathways associated with HM resistance in soils near Pb–Zn smelter in Bulgaria. The soils ranged from low-risk to disastrous, with cadmium (Cd) identified as the primary contributor to soil toxicity. High-throughput sequencing of 16S rRNA and ITS amplicons revealed widespread dominance of the phyla Proteobacteria, Actinobacteriota and Acidobacteriota, and Ascomycota, with the prevailing classes Acidobacteriae, Chloroflexia, and Eurotiomycetes, indicating their high tolerance to HMs. Functional predictions suggested enrichment of key pathways in the most polluted soils related to HM resistance, including efflux systems and detoxifying enzymes. These results highlight the necessity of integrating soil microbial indicators into agricultural management strategies to ensure safe food production.

Glacial lakes are harsh ecosystems harbouring unique microbiota. In this study, we investigated the distribution of bacterial families in the water and sediment of three glacial lakes located in Rila Mountain, Bulgaria. Our results revealed high bacterial diversity and pronounced habitat preferences, as evidenced by the presence of 310 bacterial families with distinct distribution patterns between lake water and sediments. Taxonomic dissimilarities between water and sediment bacterial communities ranged from 74% to 100%. In general, water communities exhibited a common set of bacterial families with relatively consistent abundances across lakes, whereas sediment communities were more lake specific. Dominant families in sediments included Bacteroidaceae, Veillonellaceae, and Nitrosomonadaceae, whereas Comamonadaceae, Mycobacteriaceae, and Sporichthyaceae were prevalent in water samples. These findings highlight a clear bacterial partitioning between lake habitats and provide new insights for glacial lake conservation in a changing environment, particularly in the context of global warming.

Global warming poses a significant threat to lake ecosystems, with high-mountain lakes being among the earliest and most severely impacted. However, the processes affecting water ecology under climate change remain poorly understood. This study investigates, for the first time, the effects of regional warming on three high-mountain lakes, Sulzata, Okoto and Bubreka, located in the Rila Mountains, Bulgaria, by examining shifts in bacterial metabolic capacity in relation to the rate and range of utilizable carbon sources using the Biolog EcoPlate™ assay. Over the last decade, ice-free water temperatures in the lakes have risen by an average of 2.6 °C, leading to increased nutrient concentrations and enhanced primary productivity, particularly in the shallowest lake. Bacterial communities responded to these changes by increasing their metabolic rates and shifting substrate preferences from carbohydrates to carboxylic acids. While the utilization rates of some carbon sources remained stable, others showed significant changes—some increased (e.g., D-galactonic acid γ-lactone and itaconic acid), while others decreased (e.g., α-D-lactose and D-xylose). The most pronounced effects of warming were observed in June, coinciding with the onset of the growing season. These findings suggest that rising temperatures may substantially alter bacterial metabolic potential, contributing to a long-term positive feedback loop between lake nutrient cycling and climate change.

The goals of the study were to assess the diversity and structure of the bacterial communities within the soil depth along a gradient of heavy metal (HM) contamination and to identify indigenous bacterial species in the agricultural area of a non-ferrous metal plant KCM 2000 Group (Plovdiv) by using 16S rRNA gene retrieval. 16S rRNA gene clone libraries were constructed for nine samples, which were collected from two soil depths in June 2020. From each library, up to 100 clones were analysed and grouped into operational taxonomic units (OTUs) by restriction fragment length polymorphism (RFLP). The representatives of the OTUs were sequenced, followed by phylogenetic analysis. The results revealed that phyla Proteobacteria (11.11–71.43%) and Actinomycetota (3.57–33%) were the most abundant. Surface soils (12 phyla and 15 classes) were more diverse than subsurface ones (7 phyla and 12 classes). The lowest diversity at both phylum and class levels was calculated for the moderately contaminated soils from the two studied depths. Thirteen 16S rDNA sequences were identified to a species level, and they belonged to Proteobacteria, Actinomycetota and Firmicutes. This study highlighted that both HM contamination and soil depth caused shifts in diversity and structure of soil bacterial communities.

Macrophyte overgrowth in freshwater ecosystems is a worldwide problem, and a common management practice for these ecosystems is to remove the aquatic vegetation. In the study, the short-term effects of macrophyte harvesting in a mountain lake (Trevnoto Lake, Bulgaria) on both bacterial metabolic activity and fingerprints were assessed using Biolog Ecoplate™ assay. Bacterial responses to human activity were manifested by: 1) increased bacterial metabolic activity by 15%, and 2) shifts in bacterial metabolic fingerprints. The most affected carbon sources were those with low availability for aquatic bacteria – 85% of them were influenced and the rates of their utilizations changed from a decrease of 96% to an increase of 681%. In general, a clear trend was formed that the greater the bioavailability of the carbon sources, the less susceptible is their utilization to the environmental change. This preliminary study indicates an immediate impact of macrophyte harvesting on lake bacteria. In this aspect, Biolog Ecoplate™ assay could be useful in lake monitoring programmes in order to develop better sustainable management practices for restoration.

Soil enzyme activities are potentially valuable indicators of soil health in cases of heavy metal and metalloid (HM) pollution. However, the extent to which HM pollution affects specific enzyme activities remains unclear. In this study we assessed the level of HM pollution and its effects on soil enzyme activities to select the most reliable biochemical indicators of soil health under HM stress. The initial hypothesis was that enzyme responses would be site-specific, depending not only on the level of pollution but also on soil abiotic and biotic properties. The Nemerow pollution index (NPI) and the enzyme activity index (EAI) were calculated. EAIs were based on the activities of dehydrogenase (Dha), β-glucosidase (BGl), acid (AcP) and alkaline (AlP) phosphatases, arylsulphatase (Ars) and urease (Ur). NPI indicated slight (1.0) to serious (67.46) soil pollution. The EAI varied from 4.50 to 11.54. The overall functional dissimilarity between soils was around 26%. Cluster and SIMPER analyses both indicated that the activities of Dha, BGl and AlP grouped together with Cu, Zn and Pb, and accounted for 85% of the between-soil functional dissimilarity. The results indicated that there was enzyme functional redundancy between soils. Soil enzymes showed a relatively high capacity to tolerate long-term HM pollution. Dha, BGl and AlP were highly sensitive to environmental fluctuations, including HM concentrations. The long-term HM stress on soil enzyme activities calls for further studies of the soil properties and the time scale involved in the evolution of metal tolerant enzyme producers in HM-impacted soils.