Solid samples were harvested from fermenting hay (1 g) (Zgierz, Poland, 51°51'24.8"N 19°23'42.9"E) and extracted during 60 min of mixing, in 100 mL of phosphate-buffered saline (PBS; Chempur, Gliwice, Poland) with the addition of 1 mL of polysorbate 80 (Biomaxima, Lublin, Poland). Liquid samples, containing rotting plant fragments, derived from geothermal karst spring (64°18'34.7"N 20°18'12.6"W), hot spring (64°00'34.3"N 21°11'04.9"W) and vicinity of geothermal pond Blue Lagoon (63°52'50.5"N 22°26'52.4"W) (all originating from Iceland) were used directly. Screening of thermophilic bacteria was performed using serial dilution and spread plate technique on nutrient Gelzan™ medium. It was prepared by solidification of nutrient broth (BTL, Lodz, Poland) with the addition of gellan gum 8 g/L (Gelzan™; Sigma-Aldrich, Merck, St. Louis, MO, USA) and MgSO₄ 1 g/L (Chempur, Piekary Slaskie, Poland). Single colonies were picked after 48 h of incubation at 50 or 60 °C. Pure cultures of isolated bacteria were kept frozen at -80 °C in glycerol stocks.

Bacteria were cultured for 24 h on nutrient Gelzan^TM medium at the temperature of their isolation. The morphology was observed after Gram staining under the light microscope (BX63; Olympus, Tokyo, Japan) at the magnification of 1000×.

Bacterial strains were activated at 50 °C for 24 h in 20 mL of nutrient broth (BTL). The precultures were centrifuged for 10 min at 1731×g and 22 °C (3-18KS; Sigma-Aldrich, Merck, Osterode am Harz, Germany), and the biomass was suspended in 9 mL of sterile saline to obtain a cell amount corresponding to 0.50±0.05 on the McFarland scale or approx. 1.5·10⁸ CFU/mL (DEN-1B densitometer, Biosan, Riga, Latvia). The next step was performed in microplates and triplicated. The wells containing 170 µL of nutrient broth were inoculated with 30 µL of standardised suspension of microorganisms. The cultures were incubated at 4, 20, 25, 30, 37, 45, 50, 55, 60, 65 and 70 °C. After 24 h, the changes in the absorbance (λ=600 nm) were measured regarding the control sample which contained non-inoculated nutrient broth (Multiscan GO spectrophotometer; Thermo Fisher Scientific, Waltham, MA, USA).

To determine the ability to grow at 50 °C the BioLector^® microbioreactor system (m2p-labs GmbH, Baesweiler, Germany) was used. Strains were activated overnight in 10 mL of nutrient broth at the temperature of isolation. The microplate wells containing 1250 µL nutrient broth were inoculated by 125 µL of precultures prepared as described previously. Incubation was carried out at 50 °C with shaking frequency 1000 rpm. The changes in biomass concentration expressed as scattered light intensity (λ=620 nm) were measured online every 15 min for 48 h starting from the moment of inoculation, using a built-in BioLector® detector. Growth curves were prepared using Microsoft Excel 2010 (27).

Cellulase-producing bacteria were screened on carboxymethyl cellulose (CMC) agar plates containing: low-viscosity carboxymethyl cellulose sodium salt 2 g/L (Glentham Life Sciences, Corsham, UK), NaNO₃ 2 g/L, K₂HPO₄ 1 g/L, MgSO₄^.7H₂O 0.5 g/L, KCl 0.5 g/L (all from Chempur, Piekary Slaskie), peptone 0.2 g/L (BTL) and agar 2 g/L (VWR; Avantor, Radnor, PA, USA) (28). Strains were activated during 24 h of cultivation at 50 °C in nutrient broth. Then, the microorganisms were plated onto CMC agar and incubated at 50 °C for 2 days. Resulting colonies were picked up and transferred onto a new CMC agar to ensure that bacterial growth was a result of CMC degradation. After 3 days of incubation at 50 °C, the zones surrounding the colonies were visualised by Congo red (Carl-Roth GmbH & Co. KG, Karlsruhe, Germany) staining (29). To compare the capability of CMC degradation, hydrolysis capacity was calculated. The hydrolysis capacity is defined as the ratio of the diameter of clearing zone around the colony and the colony diameter (30).

Bacteria were activated for 24 h at 50 °C in nutrient broth. The cultures were centrifuged for 10 min at 1731×g and 22 °C (3-18KS; Sigma-Aldrich, Merck), and the biomass was suspended in sterile saline to obtain a cell amount corresponding to 5-6 on the McFarland scale (approx. 1.5-1.8·10⁹ CFU/mL; DEN-1B Densitometer). API® ZYM tests (BioMerieux, Marcy-l'Étoile, France) were performed following the manufacturer's instructions. However, due to the specificity of the microorganisms, the incubation temperature was raised to 50 °C.

Microorganisms were activated on the nutrient Gelzan^TM medium as described earlier. The activities of the cytochrome oxidase and l-alanine aminopeptidase were detected with Bactident® Oxidase (Merck, Darmstadt, Germany) and Bactident® Aminopeptidase (Merck) test strips respectively. The tests were performed according to the manufacturer’s recommendations. The catalase activity was determined as follows: the bacterial biomass was placed in the microscope slide and a drop of 5% hydrogen peroxide (Chempur, Piekary Slaskie) was applied on the biomass. Catalase activity was confirmed based on O₂ formation.

Total genomic DNA was extracted and purified using NucleoSpin® microbial DNA kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) according to manufacturer instructions. DNA yield and purity were assessed by spectrophotometric method (NanoVue Plus^TM, Biochrom, Cambridge, UK). The 16S rRNA gene was amplified with the universal primers 27F: 5’-AGAGTTTGATCCTGGCTCAG-3’, 1492R: 5’-TACGGTACCTTGTTACGACTT-3’. The PCR reaction mixture contained 4 µL of the DNA template, 1 µL of each primer at a concentration of 10 µM, 25 µL PrimeSTAR® Max DNA Polymerase (Takara, Kyoto, Japan) and 25 µL water. The polymerase chain reaction (PCR) was carried out in 30 cycles according to the following program: 10 s denaturation at 98 °C, 10 s annealing at 55 °C, and 10 s extension at 72 °C. The presence of the appropriate-size amplicons of each isolate was confirmed by agarose gel electrophoresis. The PCR products were sequenced by external service (Genomed, Warsaw, Poland). The sequences were analysed with Chromas (31) and Decipher (32) open source software, aligned and compared with the deposited sequences in GenBank (33).

Thermophilic bacteria described in this study were isolated from geothermal karst spring, hot spring, geothermal pond and fermenting hay. Most of the strains were obtained after incubation at 50 °C and only seven grew well at a higher temperature (Table 1). The vast majority of isolated bacteria were Gram-positive and rod-shaped. The relatively small number of them (BSO4, BSO6 and BSO8) exhibited alternative construction of the cell wall. Examples of the morphological differences are presented in Fig. 1. Similar properties are often shown by other microorganisms isolated from high-temperature environments (34, 35).

The bacteria showed different capability of carboxymethyl cellulose utilization (Table 1). Strains BSO2, BSO4 and BSO5 were not able to grow under the described cultivation conditions, whereas BWO2, BWO3, BWO4, BWO6, BWO8, BGR8, BGR9, BBLN7, BSO3, BSO8 and BSO12 formed colonies, but the clearing zones surrounding them were not observed. Nevertheless, most of the isolated strains utilized carboxymethyl cellulose. In the cultures of BWO1, BWO5, BGR3, BGR4, BGR5, BSO11 and BSO15, the clearing zones were larger than 40 mm in diameter. However, the most important parameter is the hydrolysis capacity. The ratio of the diameter of the clearing zone around the colony and the colony diameter allowed us to compare the cellulolytic activities and to single out the most appropriate strains. The hydrolysis capacity in most cases was approx. 1. The following strains showed favourable properties: BBLN1, BSO6, BSO10 and BSO13, with the hydrolysis capacities of 2.74, 1.62, 1.30 and 1.38 respectively (Table 1). However, its highest value was observed in the culture of BSO14 and reached 8.02.

The bacterial growth was evaluated in the temperature range of 4-70 °C (Table 2). For most of the isolated strains, the minimum was 25 °C and the maximum did not exceed 60 °C. Only BWO3, BWO4 and BGR8 grew at a higher minimum temperature, i.e. 30, 37 and 45 °C, respectively. Moreover, similar to strain BGR9, BGR8 was able to grow even at 65 °C. Although the maximum growth temperatures are high, only a few strains can be classified as moderate thermophiles. Assuming that the optimal growth temperature of thermophilic bacteria cannot be lower than 50 °C, only BWO3, BWO4, BWO6, BWO7, BWO8, BGR1, BGR2, BGR8, BGR9, BBLN1, BBLN7, BSO6, BSO8 and BSO11 can be included in this group. These strains are particularly interesting for our further studies.

The results presented previously show that temperature is an important factor affecting bacterial cell growth. The BioLector® microbioreactor allows us to compare the cell densities in bacterial cultures after a specified incubation time and to analyse the growth phases. Based on the changes in light scattering, we tried to find the strains capable of fast and stable growth at 50 °C (Fig. 2).

The vast majority of isolated strains grew at 50 °C. However, BWO6, BSO2, BSO5, BSO6 and BSO12 did not proliferate under the described cultivation conditions (Fig. 2a, Fig. 2d and Fig. 2e). This fact is surprising since the previously determined maximum growth temperature was lower than 50 °C only for BSO5 (Table 2). We observed differences also in the length of the lag phases. In a significant number of the bacterial cultures, it lasted less than 8 h. Quite different were the cultures of strains BWO2, BWO3, BWO7, BWO8, BSO4, BSO8 and BSO14 (Fig. 2a, Fig. 2d and Fig. 2e). Their adaptation phase lasted at least 8 h, and even up to 20 h, as in the case of BWO3 strain (Fig. 2a). The scattered light intensities show that the exponential phases were usually short and intensive. However, in a few cases: BWO2, BWO3, BBLN7, BSO3 and BSO9, the logarithmic growth curve was prolonged, showing first a gentle and then a low increase in biomass concentration (Fig. 2a, Fig. 2c, Fig. 2d and Fig. 2e). A significant number of tested microorganisms did not maintain stationary phase at 50 °C. The scattered light intensities decreased rapidly immediately after reaching the maximum. Moreover, the experiment carried out in the BioLector® bioreactor allowed us to observe the phenomenon of diauxie in the cultures of the BWO4 and BWO7 strains (Fig. 2a). This may indicate adaptations of that microorganism to the demanding environmental conditions. The classic examples of a stationary growth phase can be observed only in the cultures of BWO2, BWO3 and BSO1 (Fig. 2a and Fig. 2d).

The bacterial strains isolated from extreme environments demonstrated a large variety of enzymatic activities (Table 3).

Analysis of basic biochemical traits, i.e. the ability to produce oxidase and catalase, showed that only BSO2 did not exhibit both of these activities. Thus, the mentioned strain could be anaerobic bacteria. This conclusion finds its confirmation in previous observations, which indicated that BSO2 is not able to grow in aerated cultures. Another enzymatic activity, l-alanine aminopeptidase, occurs almost exclusively in the cell walls of Gram-negative bacteria. Among the isolated strains, only BSO4, BSO6 and BSO8 exhibited this feature. The Gram-positive behaviour, also presented in these studies, is analogous. All enzyme activities evaluated by the API® ZYM test occurred with a similar frequency among microorganisms, regardless of their origin. Alkaline phosphatase, esterase (C4), esterase lipase (C8), acid phosphatase and β-glucosidase activities were found often, whereas leucine arylamidase, α-chymotrypsin, naphthol-AS-BI-phosphohydrolase, β-galactosidase and α-glucosidase occurred moderately often (Table 3). Particularly interesting are activities detected rarely or very rarely. Up to 15 strains show the activities of valine arylamidase, cystine arylamidase and α-galactosidase. Very rare lipase (C14), trypsin and α-mannosidase activities were found. The lipase (C14) was detected in BWO6, BGR8 and BSO16. Furthermore, BGR8 also contained trypsin. The presence of α-mannosidase was found in BWO3, BWO4 and BBLN7 cells. None of the tested strains contained β-glucuronidase, N-acetyl-β-glucosaminidase or α-fructosidase.

Cultures of bacteria selected for this part of the study were capable of growing at least at 37 °C and exhibited hydrolysis capacity values greater than 1.3. Strains that meet the selection criteria are BBLN1, BSO10, BSO13 and BSO14. The results of the analyses of their 16S rDNA sequences are presented in Table 4. According to the obtained results BBLN1, BSO10 and BSO13 were classified as Bacillus licheniformis, and BSO14 as Paenibacillus lactis.

Paenibacillus was distinguished from Bacillus group 3 based on 16S rRNA analysis (36). Bacteria belonging to Paenibacillus sp. have been repeatedly isolated from a variety of environments including fresh and salty water, soil, bentonite, plants, rhizosphere, compost, sewage, sediments and caves. Its presence was reported also in food, insect larvae and human clinical samples (37-39). The main sources of Paenibacillus lactis that have been described so far are soil, water, raw and ultra-high temperature (UHT) milk and a dairy farm environment (37, 40-42). It was reported that the endospores of P. lactis can survive UHT processing of milk as well as industrial sterilisation (37). Therefore, it is not surprising that P. lactis BSO14 that we isolated was able to outlast inside the fermenting hay, where the highest recorded temperature reached 52 °C. However, the optimal growth temperature of BSO14 reached 45 °C and was higher than usually described for P. lactis, which is about 30-40 °C. Moreover, the BSO14 strain showed the ability to proliferate even at 60 °C, which has not yet been reported (37, 41). P. lactis can utilize various carbon sources, for example, arabinose, cellobiose, fructose, d-glucose, glycogen, lactose, maltose, mannitol, mannose, melibiose, raffinose, ribose, starch, sucrose, trehalose and xylose (37, 41). Although many representatives of Paenibacillus can degrade cellulose, so far it has not been confirmed for P. lactis (43). New strain P. lactis BSO14 exhibited high cellulose hydrolysis capacity. With a few exceptions, the value of this parameter was nearly 8 times higher than for other isolated strains. Based on the API® ZYM test, strain BSO14 showed also the activities of esterase (C4), esterase lipase (C8), naphthol-AS-BI-phosphohydrolase, α-galactosidase and β-galactosidase. BSO14 is a new strain with unique properties, interesting for industrial applications.

BBLN1, BSO10 and BSO13 described in this manuscript also exhibited valuable properties. The strains were identified as Bacillus licheniformis. Their presence is not surprising since B. licheniformis is one of the most prevalent Bacillus species that inhabits a wide variety of environments and its cellulolytic properties are well known. Among them are hot springs (44), compost (45) and mangrove soil (46). The microorganisms we described originate from Blue Lagoon (Iceland) and fermenting hay (Poland). The sources of their isolation are similar to those previously described. B. licheniformis is classified as mesophilic microorganism. B. licheniformis BSO10 and BSO13 grow well at 37 °C similar to the type strain B. licheniformis (Weigmann) Chester (41). Nevertheless, a significant number of the species can be assigned to the thermophile group. The optimal incubation temperature for BBLN1 is 50 °C. B. licheniformis BL1 (47) and Ad978 (48) have comparable properties. A broad spectrum of B. licheniformis enzymatic activities makes this microorganism an exceptional tool for industrial applications. Proteases, α-amylases and lipases from B. licheniformis have a commercial application (49-51). The cellulases discussed here are still the subject of research. Nonetheless, many publications indicate that the enzymes produced by B. licheniformis exhibit unique properties such as thermostability and operation at a wide range of pH (44, 45, 51). Strains BBLN1, BSO10 and BSO13 that we isolated can be a source of cellulases with equally interesting properties. However, to confirm their industrial potential, further studies are required. Other enzymatic activities of the strains are worth mentioning. B. licheniformis BBLN1, BSO1 and BSO13 produced specific enzymes, rare in comparison with the other examined bacteria, such as arylamidase and α-galactosidase. The strains also showed activities of alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, α-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, β-galactosidase, α-glucosidase and β-glucosidase.