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Isolation and Identification of Bacterial Communities in Neutral Mine Drainage in Central Slovakian Neovolcanites (Slovakia)

Veronika Prepilková
Zuzana Perháčová
Marián Schwarz
1 and
Katarína Trnková
Department of Environmental Engineering, Faculty of Ecology and Environmental Sciences, Technical University in Zvolen, 960 01 Zvolen, Slovakia
Department of Biology and General Ecology, Faculty of Ecology and Environmental Sciences, Technical University in Zvolen, 960 01 Zvolen, Slovakia
Department of the Environment, Faculty of Natural Sciences, Matej Bel University, 974 01 Banská Bystrica, Slovakia
Author to whom correspondence should be addressed.
Water 2023, 15(5), 951;
Submission received: 20 December 2022 / Revised: 20 February 2023 / Accepted: 24 February 2023 / Published: 1 March 2023


There are several sources of mine drainage left over from past mining sites in the Central Slovakian neovolcanites. The neutral pH and high concentrations of sulphates and multiple potentially hazardous elements, such as zinc or manganese, are typical in this region. However, this environment could be home to specific microbiota. The aim of the study was to characterize bacterial populations in mine drainage in the Central Slovakian neovolcanites. Direct microscopic observations, cultivation methods, MALDI TOF mass spectrometry, and 16S rRNA gene sequencing of isolates were used for identification. Gram-positive and Gram-negative bacteria were almost equally represented in the mine water samples. The most abundant bacterium was the genus Bacillus spp. (43.48%). Another large group of bacteria consisted of Proteobacteria (34.78%), represented by Pseudomonas spp. (17.39%), Serratia spp. (13.04%), and Providencia spp. (4.35%). Our data confirm the presence of Bacillus spp. and Pseudomonas spp. as bacterial species occurring in an environment polluted by potentially hazardous elements, which may indicate their bioremediation potential.

1. Introduction

Mine water is surface or groundwater flowing from an active or abandoned mine. These discharges may have similar characteristics to natural water or altered compositions; acidic discharges with high concentrations of heavy metals are very common. From an environmental perspective, it is vital to determine the characteristics of mine water as it can often contaminate water resources and significantly affect aquatic life [1].
Although mine water is a significant source of environmental contamination (particularly of surface water), even long after mining at the site has ceased, they also provide a habitat for specific microorganisms that have a highly adaptable metabolism, and these microorganisms can degrade a wide range of substrates.
There are several significant sources of mine water in the Central Slovakian neovolcanites that have received insufficient attention.
The Central Slovakian neovolcanites are situated on the inner side of the Carpathian arc and cover approximately 5000 km2 [2]. This area contains several known Ag-Au epithermal vein-type deposits, which formed mining sites for both precious and base metals in the past [3] and can be divided into two ore districts.
The Voznická inheritance adit is important for hydroenergetic systems of most mining facilities in the Hodruša-Štiavnica ore district. Mine water from the adit has a pH of approximately 7.5 and a total mineralisation of 1311.7 mg·L−1 [4,5]. According to Bajtoš et al. [6], approximately 45 t Zn, 30 t Fe and Mn, 6 t Al, 1 t Pb, and about 150 kg Cd enter the Hron River annually through the Voznická inheritance adit. Zn (4–5 mg·L−1), Pb (0.031 mg·L−1), Cd (0.02 mg·L−1 and Mn (more than 2 mg·L−1) contents especially pose an environmental risk [5]. Mine water from the Zlatý stôl adit has a pH of approximately 7.3 and a mineralisation of 853.6 mg·L−1 [4]. Increased amounts of Mn (0.366 mg·L−1) and Ca (130 mg·L−1) were detected in 2020. Drainage water from the tailings pound has a pH in the range of 7–8 and total mineralization of 695.0 mg·L−1 [4,5]. Monitoring results indicate that the drainage water contains elevated concentrations of sulphate (610 mg·L−1), Mn (1.331 mg·L−1), Zn (0.495 mg·L−1), and Ca (213 mg·L−1) [5].
A large part of the mine in the Kremnica ore district is drained by the main inheritance adit. Based on hydrometeorological discharge measurements for the years 2008–2010, an average flow rate of 658 L·s−1 was determined [4]. The mine water has a neutral pH (approximately 7.5) and increased amounts of manganese (1.0 mg·L−1) [5]. The deep inheritance adit discharges an average of 1.65 L·s−1 mine water with a neutral pH, which may be a source of manganese contamination (a measured concentration of 0.37 mg·L−1 in 2020) [4,5]. In 2008–2010, the average flow rate of the Horná Ves tunnel was measured as 1.75 L·s−1. The water has a slightly acidic pH (5–6.5). Fine sediments of ochre are deposited on the walls of the channel, through which the sediments drain into the Lučanský brook. In 2020, elevated manganese (0.37 mg·L−1) and zinc (0.052 mg·L−1) contents were detected in the mine water [4,5].
As confirmed by several studies, mine waters are richly populated by a wide range of bacteria, e.g., Rhizobium, Microbacterium, Pseudomonas, and Acinetobacter in the mine water of the Rozália gold mine (Hodruša-Hámre), and Azotobacter, Pseudomonas, Dechloromonas, and Methyloversatilis in the mine water of the Elizabeth shaft (Slovinky) [7,8]. The dominant species in drainage water from various tailing ponds in Slovakia is Acinetobacter spp. [9]. The aim of this study was to isolate bacteria, that are suitable for use in biotechnology, from contaminated environments.

2. Materials and Methods

2.1. Sampling and Storage

In September 2021, six samples of mine water, one sample from each localised site in the Kremnica and Hodruša-Štiavnica mining districts (Figure 1 and Figure 2; geographic coordinates are provided in Table 1), were collected from the shoreline. On-site pH was measured using inoLab®Multi 9310 IDS (WTW Weilheim Xylem Analytics Germany) (Table 1). Samples were collected in sterile glass bottles, stored in dark and cold (4 °C) conditions, and transported to the laboratory in accordance with the requirements of EN ISO 19458: Water quality. Samples were subjected to microbiological analysis.

2.2. Cultivation, Gram Staining

For cultivation analyses, 100 microliters of water from each sample was inoculated onto a nonselective medium, nutrient agar (BioLife, Italy), and incubated under aerobic conditions at 25 °C for 24–72 h. After cultivation, the total number of cultivable heterotrophic bacteria was determined as the number of colony-forming units (CFU) per mL of water. Subsequently, morphologically different and separated bordered colonies were selected from Petri dishes containing nutrient agar and inoculated onto a new plate. This procedure was followed until a pure culture was obtained [10,11].
Gram staining was performed according to Aryal [12]. Oil immersion light microscopy at 1000× magnification on an Olympus BX 40 (Japan) equipped with a digital camera was used to observe Gram-stained isolates.

2.3. Preparation of Samples for MALDI TOF MS Profiling

Two methods recommended by the manufacturer (Bruker Daltonics) were used for sample preparation in MALDI TOF MS analysis: (1) direct transfer (intact cell profiling— ICP) and (2) protein extraction in ethanol and formic acid (protein extract profiling—PEP). All isolates obtained by cultivation were analysed using MALDI TOF MS, direct transfer (ICP) was performed for 22 pure culture colonies, and 23 isolates were analysed using protein extraction (PEP) (Table 2). A small amount of material was taken from freshly grown colonies and transferred onto a single spot on the 96-well stainless-steel target plate (Bruker Daltonics GmbH) using a wooden toothpick. The bacteria were overlaid with 1 µL of matrix solution containing 75 mg/mL 2,5-dihydroxybenzoic acid (DHB) in acetonitrile/ethanol/water (1:1:1) supplemented with 3% trifluoroacetic acid, and the suspension was left to dry at room temperature until the DHB crystals became visible.
For the formic acid extraction method, cells from a single colony were suspended in 300 mL of HPLC grade water by pipetting, pure ethanol was added to the final concentration of 75% (v/v), and the sample was mixed via pipetting and spun down at 13,000× g. The supernatant was discarded, and the pellet was resuspended in 50 µL of formic acid and 50 µL of acetonitrile. The sample was centrifuged at 13,000× g for 1 min, and 1 µL of supernatant was transferred onto a spot on a MALDI target plate. After drying, the samples were overlaid with 1 µL of matrix solution, and the suspension was left to dry at room temperature.
The measurements were performed with a Microflex mass spectrometer (Bruker Daltonics, Bremen, Germany) using FlexControl version 3.0, and the spectra were imported and analysed using MALDI Biotyper (version 2.0; Bruker Daltonics). The MALDI TOF MS software FlexControl was calibrated with the Bruker Bacterial Test Standard. MALDI TOF MS profile spectra were generated, and bacterial species were identified using the MALDI Biotyper database. The results are represented as log-score values ranging from 0 to 3, where values higher than 1.7 and 2.0 are generally used for reliable genus identification and species identification, respectively [13,14].
DNA-based tools: 16S rRNA gene sequence identification—DNA isolation, bacterial 16S rDNA PCR amplification, and Sanger sequencing.
The isolates were identified based on their 16S rRNA gene sequence (Table 3).
DNA isolation: total DNA was isolated from overnight cultures grown on nutrient agar using the Maxwell® 16 DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions.
The amplification of 16S rDNA fragments was performed using fD1 (5′-AGAGTTTGATCCTGGCTCAG-3′) and rP2 (5′-ACGGCTACCTTGTTACGACTT-3′) universal bacterial primers in a T100 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA). PCR was performed in 40 μL reaction mixtures using HOT FIREPol® Blend Master Mix (Solis Biodyne, Tartu, Estonia), containing 0.8 µL/10 μM of each primer, 8 µL HOT FIREPol® Blend Master Mix (Solis Biodyne, Tartu, Estonia), 26.4 µL of PCR water, and 4 μL of DNA template.
Initial denaturation at 95 °C for 3 min was followed by 35 cycles of denaturation at 95 °C for 20 s, primer annealing at 54 °C for 1 min, and extension at 72 °C for 1.5 min, with the final elongation step at 72 °C for 5 min. The sizes of the PCR products were verified using a 1.5% agarose gel and purified using EPPiC Fast mixture (A&A Biotechnology, Gdansk, Poland).
Sequencing: Sanger sequencing was performed using the SEQme service (Dobříš, Czechia).
BLAST: The quality of sequencing data was analysed using ChromasPro software (Technelysium Pty Ltd., South Brisbane, Australia), and then Basic Local Alignment Search Tool ( (accessed on 22 September 2022) was used to identify closely related sequences available in the GenBank® database.

3. Results

Following the cultivation on nutrient agar, four morphologically different isolates were obtained: one from the Voznická inheritance adit (1.1; 1.3; 1.4; and 1.5), one from tailings pond drainage water (2.2; 2.3; 2.4; and 2.5) and two isolates from the Zlatý Stôl adit (3.2 and 3.3). From the main hereditary adit, six different isolates were obtained (4.1; 4.2; 4.3; 4.4; 4.5; and 4.6); four isolates were obtained from the Horná Ves tunnel (5.1; 5.2; 5.5; and 5.6) and only three isolates from the deep hereditary adit (6.2; 6.3; and 6.4). The Gram staining results indicate a relatively even distribution of both Gram-positive and Gram-negative bacteria in the mine water samples. In the sample from the deep hereditary adit, only Gram-positive bacteria were identified, and in the sample from the Voznická inheritance adit, Gram-negative bacteria were identified.
Under a microscope, most bacteria appeared in short or long bacillus shapes, sometimes as streptobacillus. Only a few bacteria appeared as coccus or cocci bacilli (Figure 3a,b).
Fresh cultures (24 h) of 22 isolates were analysed using the direct transfer (ICP) MALDI TOF method. However, a comparison of the measured spectra with the database did not yield a satisfactory match. Most of the isolates were unreliably identified. The extraction method (PEP) resulted in more accurate identification (i.e., higher scores) for all isolates, although six isolates (1.3, 2.2, 4.4, 4.6, 5.5, 6.2, and 6.3) could not be reliably identified (results shown in Supplementary Materials). According to MALDI TOF analysis, Bacillus spp. seemed to be the most abundant (Table 2).
In most cases, sequencing analysis confirmed the MALDI TOF MS results, at least at a genus level. Some isolates were identified using sequencing, even if they were not identified using mass spectrometry (Table 3). However, some isolates were identified quite differently, e.g., isolate 1.4 was identified as Morganella morgani by MALDI TOF with a high score. The BLAST analysis of the sequence of isolate 1.4 was assigned to the Microbacterium genus, for which all members were Gram-positive. In this case, the result of Gram staining correlated more with the MALDI TOF identifications. The same situation was found for isolate 1.5. According to Gram staining, the isolate contained Gram-negative bacteria, which correlated with the MALDI TOF result for Pseudomonas monteilii.
Nevertheless, different Gram stain results are not necessarily incorrect. Even though there is a standard routine for Gram staining, there are some variables that can affect this stain, e.g., the age of the culture, the amount of decolourizer used, the time of decolorization, the type of organism (acid-fast bacteria and spores do not stain well), the thickness of the smear, and the general care of the stainer. Moreover, some bacterial species can be Gram-variable [15].
From all of the obtained isolates, 43.48% belonged to the genus Bacillus spp. (phylum Bacillota), and 34.78% belonged to the phylum Proteobacteria: Pseudomonas spp. (17.39%), Serratia spp. (13.04%), and Providencia spp. (4.35%). There were also representatives of Actinobacteria (Microbacterium spp.) and Enterobacteria (Proteus spp.) between the isolates.

4. Discussion

Although most of the mine drainage in the Central Slovakian neovolcanites had a neutral pH between 7 and 8, the pH of Horná Ves tunnel water was 5.9 on average. The concentrations of sulphates (typical for acid mine drainage) were significantly different between the sampling sites from the lowest in the Horná Ves tunnel (48 mg·L−1) to the highest in the Voznická hereditary adit (365 mg·L−1) [16].
Generally, Proteobacteria, Nitrospirae, Actinobacteria, and Firmicutes are most frequently detected in acid mine drainage [17,18,19]. On the other hand, Proteobacteria, Deinococcus/Thermus, Gemmatimonadetes, Acidobacteria, and Actinobacteria are frequently detected in neutral mine drainage. Multiple studies confirm the presence of Pseudomonas spp., Bacillus spp., and Stenotrophomonas spp. in neutral mine drainage [20,21]. This correlates with our results (Table 4).
The cultivable microflora from sampling sites has not yet been described. As the closest comparable results, we contemplated research on the cultivable microflora of gold-bearing ore samples from the Rozália mine (Hodruša-Hámre). The samples mainly consisted of the Proteobacteria strain. The identification of 473 isolates revealed the presence of four dominant genera—Rhizobium, Microbacterium, Pseudomonas, and Acinetobacter—which together accounted for 89% of the cultured bacteria associated with gold ore. These results are consistent with previous studies on gold mine microbiota and suggest that these genera form the core of the gold ore microbiota [7]. Sedláková-Kaduková et al. [27] confirmed the presence of a homogeneous population of Gram-negative rods in samples of weathered ores from the Hodruša-Hámre mine, which was proven by Gram staining. Molecular analyses revealed that the population of sulphur-oxidizing bacteria in the gold mine was dominated by a single species of the genus Aciditiobacillus, identified as A. albertensis, indicating a low level of diversity of autotrophic bacteria in deep deposits.
Kisková et al. [8] investigated the microbial composition of mine drainage water from the Elizabeth shaft (Slovinky, Slovakia). This bacterial community was dominated by Proteobacteria (69.55%), followed by Chloroflexi (10.31%) and Actinobacteria (4.24%). Within the bacterial community, the most abundant genera were Azotobacter (24.52%) and Pseudomonas (14.15%), followed by the iron-oxidizing Proteobacteria Dechloromonas (11%) and Methyloversatilis (8.53%). Typical sulphur bacteria were detected with a lower frequency, e.g., Desulfobacteraceae (0.25%), Desulfovibrionaceae (0.16%), or Desulfobulbaceae (0.11%). The composition of the bacterial community of the Elizabeth shaft’s drainage water reflects the observed neutral pH and high levels of iron and sulphur ions in this water environment.
Perháčová et al. [28] investigated the microbial composition of drainage water (neutral pH) from the Slovinky and Markušovce tailing ponds. Direct microscopic observations showed the presence of iron-oxidizing bacteria such as Gallionella spp. and Leptothrix spp. These bacteria represent an important part of the microbial communities in the tailings Slovinka and Markušovce, which significantly differ in several physicochemical parameters (including EC, TDS, and especially iron content).
In the drainage water from a brown sludge landfill (Žiar nad Hronom, Slovakia), the highest abundance (80.39%) of deposits belonged to the strain Proteobacteria, followed by Firmicutes (13.05%) and Bacteroidetes (5.64%). Other bacterial strains had an abundance of less than 1%. This classification yielded 85 genera. Sulfurospirillum spp. (45.19%) dominated the bacterial population, followed by Pseudomonas spp. (13.76%) and Exiguobacterium spp. (13.02%) [17]. Liu et al. [29] identified representatives of Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes in alkaline copper mine drainage. In neutral copper mine drainage (Sossego Mine, Brazil), three genera were isolated and identified: Bacillus, Pseudomonas, and Stenotrophomonas [30]. Bacteria, which are known to be natural scavengers, transform heavy metals into less harmful or nontoxic species or immobilize them into stable structures [31,32]. Bacteria isolated from polluted mine environments have adapted to higher concentrations of heavy metals, and thus can be exploited in pollution removal [33]. Bacteria represent an environmentally friendly, cost-effective approach to remediate polluted environments [34]. Several bacteria identified by our study group have already been confirmed to have bioremediation ability and potential biotechnological applications.
Colak et al. [32] investigated a Bacillus cereus isolate that was highly resistant to copper and lead compared to the studied control strain. It was found that the biosurfactants produced by Bacillus cereus were able to remove metals from contaminated soil. This also confirms the findings of Ayangbenro and Babalola [35], in which the biosurfactant had metal removal efficiencies of 69%, 54%, and 43%, respectively, for Pb, Cd, and Cr. Raja and Omine [36] identified several representatives of Bacillus spp. that have a high resistance to As, Cu, Ni, Pb, and Zn. Njoku et al. also confirmed the bioremediation potential of B. megaterium for Pb, Cd and Ni [37].
The genus Pseudomonas is ubiquitous in soil and aquatic ecosystems, and is capable of metabolising a wide range of organic and inorganic compounds. In addition, Pseudomonas spp. has been well studied and exhibits high resistance to antibiotics, heavy metals, detergents, and organic solvents [38]. From surface water in the most important mine sites in Mexico, Pseudomonas koreensis was identified as heavy-metal-multiresistant bacteria with potential use in biosorption systems [39].
In total, we were able to obtain 21 isolates of neutral mine water bacteria confirmed by sequencing. The identification of these bacteria yielded the first data on microbial communities in the mine water of the Central Slovakian neovolcanites. The bioremediation potential of some isolates was confirmed. We suggest that future research focuses on testing these isolates for their heavy metal resistance or biosorption ability.

5. Conclusions

Mine drainage represents a specific environment in which divergent microbiota may thrive. Although predominantly acid mine drainages have previously been studied, the microbiota of neutral mine drainage could also form a source of sustainable bioremediation for microorganisms. Therefore, we should pay attention to the microbial composition of mine water. The microbiological analysis of mine water and drainage water revealed that microbial communities were relatively abundant. The greatest diversity of bacteria was found in the water from the main inheritance adit. In total, we were able to obtain 23 morphologically distinct isolates. In mine water samples, we identified many different species of Bacillus spp., Pseudomonas spp., Providencia rettgeri, and Serratia liquefaciens. According to previous studies, representatives of Proteobacteria and Actinobacteria are predominantly found in specific environments of mine drainage. This statement is also confirmed by our results. Many bacteria species used in bioremediation were isolated from contaminated environments such as mine drainage or tailings ponds. Therefore, future research should aim to explore the bioremediation potential of obtained isolates.

Supplementary Materials

The following are available online at, Figure S1: Results of MALDI TOF analysis.

Author Contributions

V.P.: investigation (sampling, isolation, Gram staining), manuscript preparation. Z.P.: conceptualization, investigation (Gram staining, microscope observation), manuscript preparation. M.S.: conceptualization, project administration. K.T.: methodology (cultivation, Gram staining, MALDI TOF, PCR, BLAST), manuscript preparation, correspondence. All authors have read and agreed to the published version of the manuscript.


This research was funded by the Grant Agency KEGA, No. 029UMB-4/2021 and No. 006UMB-4/2020, project IPA 17/2021 of Technical University in Zvolen and Comprehensive research of determinants for ensuring environmental health (ENVIHEALTH), ITMS 313011T721,supported by the Operational Programme Integrated Infrastructure(OPII) funded by the European Regional Development Fund.

Data Availability Statement

The data supporting the reported results are published in the article, with the exception of sequences of isolates, which are available upon request.

Conflicts of Interest

The authors declare no conflict of interest.


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Figure 1. The localization of Kremnica and Hodruša-Štiavnica mine districts.
Figure 1. The localization of Kremnica and Hodruša-Štiavnica mine districts.
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Figure 2. Tailings pond in Hodruša-Hámre (Hodruša-Štiavnica ore district) and the main inheritance adit (Kremnica ore district).
Figure 2. Tailings pond in Hodruša-Hámre (Hodruša-Štiavnica ore district) and the main inheritance adit (Kremnica ore district).
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Figure 3. Microscopic observation (×1000 magnification) of (a) isolate 5.6 G− and (b) isolate 5.1 G+ observed in mine drainage water of Horná Ves tunnel (Central Slovakian neovolcanites).
Figure 3. Microscopic observation (×1000 magnification) of (a) isolate 5.6 G− and (b) isolate 5.1 G+ observed in mine drainage water of Horná Ves tunnel (Central Slovakian neovolcanites).
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Table 1. Geographic coordinates of sampling sites and on-site pH values.
Table 1. Geographic coordinates of sampling sites and on-site pH values.
Kremnica Ore DistrictpHHodruša-Štiavnica Ore DistrictpH
Main inheritance adit
48°35′4.2″ N  18°51′46.44″ E
7.35Voznická inheritance adit
48° 27′ 51.87″ N  18°42′ 28.38″ E
Horná Ves adit
48°40′40.8″ N  18°54’23.04″ E
5.63Tailings pond Hodruša-Hámre
48°27′52.01″ N  18°45′17.84″ E
Deep inheritance adit
48°41′9.24″ N  18°55′5.16″ E
6.92Zlatý stôl adit
48°28′3.1″ N  18°50′24.38″ E
Table 2. Identification results by MALDI TOF mass spectrometry for direct transfer (intact cell profiling—ICP) and protein extraction in ethanol and formic acid (protein extract profiling—PEP).
Table 2. Identification results by MALDI TOF mass spectrometry for direct transfer (intact cell profiling—ICP) and protein extraction in ethanol and formic acid (protein extract profiling—PEP).
IsolateDirect Transfer (ICP)ScoreExtraction (PEP)Score
1.1.not reliable identification1.596Providencia rettgeri1.860
1.3not reliable identification1.412not reliable identification1.697
1.4not reliable identification1.478Morganella morganii2.175
1.5Pseudomonas monteilii1.710Pseudomonas monteilii1.980
2.2not reliable identification1.550not reliable identification1.505
2.3Aeromonas hydrophila1.719Aeromonas hydrophila1.809
2.4Lysinibacillus fusiformis1.998Lysinibacillus fusiformis2.395
2.5Bacillus cereus1.847Bacillus cereus1.951
3.2not reliable identification1.558Bacillus cereus2.076
3.3Bacillus cereus1.712Bacillus cereus1.985
4.1not reliable identification1.271Bacillus pumilus1.843
4.2not reliable identification1.460Serratia liquefaciens1.719
4.3not reliable identification1.113Serratia liquefaciens1.903
4.4not reliable identification1.292not reliable identification1.435
4.5not reliable identification1.544Bacillus altitudinis1.956
4.6not reliable identification1.170not reliable identification1.364
5.1not reliable identification1.222Bacillus cereus2.151
5.2not reliable identification1.485Bacillus cereus2.700
5.5Pseudomonas fragi1.805not reliable identification1.391
5.6not reliable identification1.568Pseudomonas fluorescens1.774
6.2not reliable identification1.387not reliable identification1.496
6.3not reliable identification1.263not reliable identification1.414
6.4not reliable identification1.178Bacillus pumilus1.946
Table 3. Comparison of identification: results of Gram staining, MALDI TOF mass spectrometry, and BLAST analysis of 16s rRNA sequences.
Table 3. Comparison of identification: results of Gram staining, MALDI TOF mass spectrometry, and BLAST analysis of 16s rRNA sequences.
IsolateMALDI TOFGram StainingBLAST
1.1Providencia rettgeriG−Providencia rettgeri
1.3not reliable identification
G−Serratia plymuthica, S. quinivorans, S. grimesii
1.4Morganella morganiiG−Microbacterim oxydans, M. maritypicum, M. algeriense
1.5Pseudomonas monteiliiG−Bacillus pumilus, B. zhangzhouensis
2.3Aeromonas hydrophilaG−Proteus hauseri, P. vulgaris
2.5Bacillus cereusG+Bacillus cereus, Bacillus thuringiensis
3.2Bacillus cereusG+Bacillus cereus
3.3Bacillus cereusG+Bacillus cereus, Bacillus thuringiensis
4.1Bacillus pumilusG+Bacillus safensis, Bacillus pumilus
4.2Serratia liquefaciensG−Serratia liquefaciens
4.3Serratia liquefaciensG−Serratia liquefaciens
4.4Pseudomonas cedrina, P. fluorescens
G−Pseudomonas fluorescens,
P. gessardii
4.5Bacillus altitudinisG+Bacillus pumilus, B. zhangzhouensis
Staphylococcus arlettae
G+Pseudomonas fluorescens,
P. gessardii
5.1Bacillus cereusG+Bacillus cereus, B. thuringiensis
5.2Bacillus cereusG+Bacillus velezensis, B. cereus
5.5Pseudomonas fragiG−Pseudomonas fragi
5.6Pseudomonas fluorescensG−Pseudomonas veronii, P. fluorescens
6.2not reliable identification
G+Proteus hauseri, P. vulgaris
6.3not reliable identification
G+Bacillus safensis, B. pumilus
6.4Bacillus pumilusG+Bacillus pumilus
Note: For isolates 2.2 and 2.4, we did not obtain reliable PCR products for sustainable sequencing.
Table 4. Comparison of microbial communities in mine drainage at different sites.
Table 4. Comparison of microbial communities in mine drainage at different sites.
SitepHBacterial CommunitiesReferences
Slovakia neovolcanites7–8ProteobacteriaBacteroidetesActinobacteriaour results
Žiar nad Hronom,
13.2Proteobacteria FirmicutesBacteroidetes[22]
Slovinky, Slovakia6.8Proteobacteria ChloroflexiActinobacteria[8]
Pezinok, Slovakia6.5–8.0ProteobacteriaActinobacteriaChloroflexi[23]
Neves-Corvo, Portugal4.6–8.7ThiobacillusHalothiobacillusThiomicrospira[24]
Lancaster, South Africa2.3–2.9Acidobacteria ActinobacteriaChloroflexi[25]
Onyeama, Nigeria3.1ProteobacteriaBacteroidetesAcidobacteria[26]
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Prepilková, V.; Perháčová, Z.; Schwarz, M.; Trnková, K. Isolation and Identification of Bacterial Communities in Neutral Mine Drainage in Central Slovakian Neovolcanites (Slovakia). Water 2023, 15, 951.

AMA Style

Prepilková V, Perháčová Z, Schwarz M, Trnková K. Isolation and Identification of Bacterial Communities in Neutral Mine Drainage in Central Slovakian Neovolcanites (Slovakia). Water. 2023; 15(5):951.

Chicago/Turabian Style

Prepilková, Veronika, Zuzana Perháčová, Marián Schwarz, and Katarína Trnková. 2023. "Isolation and Identification of Bacterial Communities in Neutral Mine Drainage in Central Slovakian Neovolcanites (Slovakia)" Water 15, no. 5: 951.

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