The ability of MALDI TOF MS (matrix-assisted laser desorption ionisation time-of-flight mass spectrometry) to identify cultivable microflora from two waste disposal sites from non-ferrous metal industry was analysed. Despite the harsh conditions (extreme pH values and heavy metal content in red mud disposal site from aluminium production or high heavy metal content in nickel sludge), relatively high numbers of bacteria were recovered. In both environments, the bacterial community was dominated by Gram-positive bacteria, especially by actinobacteria. High-quality MALDI TOF mass spectra were obtained but most of the bacteria isolates could be not identified using MALDI Biotyper software. The overall identification rate was lower than 20 %; in two of the environments tested identification rates were lower than 10 %. As a dominant bacterial species, Microbacterium spp. in drainage water from an aluminium red mud disposal site near Žiar nad Hronom, Bacillus spp. in red mud samples from the same site, and Arthrobacter spp. from nickel smelter sludge near Sereď were identified by a combination of the Biolog system and 16S rRNA sequence analysis. As the primary focus of the MALDI TOF MS-based methodology is directed towards medically important bacteria, reference database spectra expansion and refinement are needed to improve the ability of MALDI TOF MS to identify environmental bacteria, especially those from extreme environments.

Waste disposal sites from non-ferrous metal industry constitute environments very hostile for life due to the presence of very specialized abiotic factors (pH, salt concentration, heavy metals content). In our experiments microflora of two waste disposal sites in Slovakia – brown mud disposal site from aluminium production near Ziar nad Hronom and nickel sludge disposal site near Sered - was analyzed for cultivable bacteria. Isolated bacteria were characterized by a combination of classical microbiological approaches and molecular methods and the most of isolated bacteria shown a poly-extremotolerant phenotype. The most frequently halotolerant (resistant to the high level of salt concentrations) and alkalitolerant (resistant to the high pH level) bacteria belonging to the Actinobacteria class were detected. The most of bacteria shown very high level of heavy metal resistance e.g. more than 500 μg/ml for Zn2+ or Cu2+. Based on our data, waste disposal sites thus on one side represents an important environmental burden but on other side they are a source of new poly-extremotolerant bacterial strains and species possibly used in many biotechnology and bioremediation applications.

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Brown mud, as a waste product of the industrial process of aluminum production, represents a great environmental burden due to its toxicity to living organisms. However, some microorganisms are able to survive in this habitat, and they can be used in bioremediation processes. Traditional cultivation methods have a limited capacity to characterize bacterial composition in environmental samples. Recently, next-generation sequencing methods have provided new perspectives on microbial community studies. The aim of this study was to analyze the bacterial community in the drainage water of brown mud disposal site near Žiar nad Hronom (Banská Bystrica region, Slovakia) using 454 pyrosequencing. We obtained 9964 sequences assigned to 163 operational taxonomic units belonging to 10 bacterial phyla. The phylum Proteobacteria showed the highest abundance (80.39%) within the bacterial community, followed by Firmicutes (13.05%) and Bacteroidetes (5.64%). Other bacterial phyla showed an abundance lower than 1%. The classification yielded 85 genera. Sulfurospirillum spp. (45.19%) dominated the bacterial population, followed by Pseudomonas spp. (13.76%) and Exiguobacterium spp. (13.02%). These results indicate that high heavy metals content, high pH, and lack of essential nutrients are the drivers of a dramatic reduction of diversity in the bacterial population in this environment.

a department of general and Quarantine diagnostics, central control and testing institute of Agriculture, haniska, slovakia; b institute of Animal Physiology, slovak Academy of sciences, Košice, slovakia; c faculty of natural sciences, department of chemistry, ABSTRACT Genetic variability of 17 wild strains of γ-proteobacteria isolated from different host plants, locations and seasons, identified via routine diagnostics as Erwinia amylovora, was analysed. The ERIC-PCR confirmed the genetic homogeneity among 15 virulent strains while 2 avirulent strains were genetically distinct. These two avirulent strains differed in their antibiotic susceptibility from all virulent strains. The only avirulent Ra1051/98 strain showed significant ampicillin resistance. All 15 virulent strains were confirmed via the ERIC-PCR and MALDI-TOF MS, as closely related to the reference strain NCPPB 683 of E. amylovora. Avirulent strains were identified as Rahnella aquatilis and L. quercina subsp. britannica (formerly Brenneria quercina), by MALDI-TOF MS and the identification was confirmed by 16S rRNA gene sequence analysis. For the first time, L. quercina subsp britannica was identified in the fire blight-symptomatic samples in Central Europe, and for the first time this bacterium was isolated from the host different than oak.

Brown mud is a waste by-product of alumina production by Bayer process. Due to extensive sodium hydroxide use in the process, brown mud disposal site near Ziar nad Hronom (Banska Bystrica region, Slovakia) and drainage water are ones of the greatest environmental burdens in Slovakia. Drainage water from this landfills has pH value higher than 13, and it contains many heavy metals and elevated salt content. In our experiments, relatively numerous bacterial population was detected in the drainage water with frequency of about 80 cfu/ml using cultivation approach. The alkalitolerant heterotrophic isolates were identified by combination of MALDI-TOF and 16S rDNA analysis. Drainage water population was dominated by Actinobacteria (Microbacterium spp. and Micrococcus spp.) followed by low G + C-content gram-positive bacteria (Bacillus spp.). Two isolates belonged to gram-negative bacteria only, identified as Brevundimonas spp. Phylogenetic and biochemical analyses indicate that nearly half of the bacteria isolated are probably representatives of a new species. Brown mud disposal site is proposed as a source of new bacterial taxa possibly used in bioremediation processes.