Thermophilic cellulase: screening of novel cellulolytic bacteria from Algerian hot springs for CMCase production

Thermophilic cellulase: screening of novel cellulolytic bacteria from Algerian hot springs for CMCase production | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Thermophilic cellulase: screening of novel cellulolytic bacteria from Algerian hot springs for CMCase production Souad SOUALAH, Jihen MISSAOUI, Mohamed LATAOUI, Hafsa CHERIF SILLINI, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8574179/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Hot spring microbiological studies in underexplored regions like Algeria are important because they contain extremophiles that may yield unique and robust biomolecules. So that, this current study aimed for the screening of enzymatic potentiality through the production of cellulase, in order to identify the diversity of cultivable bacteria isolated from two thermal spas of Hammam Ouled Tebben and the Zdim hot spring, in south of Setif (Algeria). The obtained results showed that cellulolytic activity was prevalent, according to the enzymatic index values. The valuation of the cellulase production on 1% of CMC liquid medium, eight strains were selected for their great activity. Indeed, the physicochemical parameters (NaCl; pH; temperature; PEG6000) that affected enzymatic activity and stability, the bacterial growth and cellulase production were assessed. Then, the majority of strains were found to be thermo-tolerant, neutrophilic, halotolerant, and tolerant to 30% PEG 6000. Withal, the screening of the effects of physicochemical parameters on the activity and stability of the cellulase enzyme allowed to select four strains among the most effective, their CMCase active and stable over a wide pH range, temperature, NaCl and they were slightly affected at 30% PEG6000. The phenotyping and genotyping of the selected bacteria were belonging to Bacillus sp. group. Overall, this work highlighted those Algerian hot springs represents an important reservoir of potentially bacterial producer of cellulase. Obviously, future studies should be addressed to confirm and promise the production potential and industrial and biotechnological applications because of these bacterial strains newly isolated and identified. Thermal spring Bacillus genus extracellular enzymes cellulase activity enzyme stability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1 Introduction Geothermal springs are thought to be particular hotspots for significant of thermophilic microbial communities, primarily from the bacterial and archaeal domains ( Sahay et al. 2017 ). Few studies have been done on the microbial diversity of Algerian hydrothermal springs, despite the fact that terrestrial thermal springs are the subject of extensive research worldwide. Despite the recent emergence of research on thermophilic bacteria, it still limited to specific aspects and few numbers of hot springs ( Gomri et al. 2018 ; Arab et al. 2019 ; Benammar et al. 2020 ). Thermophilic microorganisms associated with these ecosystems have garnered a lot of attention lately, despite the fact that these resources are typically used for therapeutic and curative purposes ( Thebti et al. 2016 ; Bouacem et al. 2018 ; Bouacem et al. 2022 ). Due to their specific benefit for the synthesis of thermostable enzymes alike protease, amylase, xylanase, cellulose and many others, these enzymes maintain their function at temperatures significantly higher than those optimal conditions for the proliferation of the microorganisms ( Saboto et al. 1999 ; Bouacem et al. 2022 ). Lignocellulosic biomass (LCB) is the most renewable and underappreciated biomass sources that are abundant on our planet, cellulose, hemicelluloses, and lignin are the primary components of LCB, the proportion of which varies from 35 to 50%, 25 to 30%, and 25 to 30%, respectively, depending on the biomass source ( Al-Battashi et al. 2019 ; Malik et al. 2021) . However, a significant obstacle to the conversion and exploitation of cellulosic biomass has been the persistent nature of lignocellulosic biomass. Otherwise, using solvents, acids and/or bases to chemically treat lignocelluloses has several drawbacks, including high operating pressure, high costs, low glucose yield, and the creation of hazardous by-products like furfural ( Maurya et al. 2015 ; Yousef & Mawad. 2023). Therefore, there are three methods for pre-treating lignocellulose: physical, chemical, and biological ( Balla et al. 2022 ). Hemicellulose and lignin are strongly bound by cellulose (C 6 H 10 O 5 ), ( Sethi et al. 2013 ; Abada et al. 2021 ). The glucose units are joined by β-1,4 bonds to form a polymer chain, and the photosynthetic process produces cellulose waste, as a vast renewable bioresource ( Hussain et al. 2017 ). Cellulolytic and hemi-cellulolytic enzymes are necessary for the biological conversion of lignocellulosic waste into industrial product ( Jourdier et al. 2012 ). Thus, the industry has shown a great deal of interest in the intriguing pre-treatment of lignocellulosic materials using microorganisms or microbial enzymes ( Balla et al. 2022 ). However, enzymatic (biological) hydrolysis on the other hand, is a profitable process that effectively removes lignocellulose and is environmentally friendly due to its high specificity and selectivity as well as its mild operating conditions ( Yousef et al. 2020 ). Several studies have reported that cellulose degradation involves a group of enzymes β-glucosidases (EC 3.2.1.21), endoglucanases (EC 3.2.1.4), and exoglucanases (EC 3.2.1.91) called cellulase, which could breakdown β-1,4-glycosidic bonds synergistically while releasing glucose molecules ( Jayasekara & Ratnayake 2019 ) . According to the global enzyme market reports, cellulase is the most claimed enzyme, thus covering about 20% of the global market products ( Singh et al. 2021 ). Otherwise, for effective breakdown of cellulosic biomass, the synergistic action of all three forms of cellulases is fundamental ( Sharma et al. 2019 ). Furthermore, cellulase is increasingly used in variety of industries, including the food industry (fruit juice production and aroma and flavour valorisation), the paper industry (bio-bleaching and ink removal), biotechnology (bioethanol production and lignocellulosic material valorisation), and the textile industry (bio-washing, bio-polishing, bio-stonewashing, and denim bleaching ) ( Singh et al. 2019 ). Detergents and other cleaning and laundry products are also made with cellulase ( Singh et al. 2019 ; Balla et al. 2022 ). These enzymes are produced by a huge variety of microorganisms, such as bacteria, fungi, and actinomycetes. Several publications have classified the various categories of microorganisms that contribute to cellulose degradation ( Sharma et al. 2016 ; Singh et al. 2019 ; Balla et al. 2022 ; Masmoudi et al. 2025 ). Moreover, due to their diversity, the presence of multi-enzyme complexes that provide synergy their high growth rate, and stability under extreme conditions there is an increasing demand for bacteria as a source of novel cellulases as effective catalysts ( Balasubramanian & Simões 2014 ; Manfredi et al. 2016 ). Among the bacterial genera that have been found to exhibit cellulolytic activity: Bacillus, Clostridium, Cellulomonas, Rumminococcus, Alteromonas, Acetivibrio, etc… ( Rawat & Tewari 2012 ; Balla et al., 2022 ; Masmoudi et al. 2025 ), but the genus most studied and cited remains Bacillus (Sadhu & Maiti. 2013; Balla et al. 2022 ; Masmoudi et al. 2025 ). Furthermore, Bacillus spp . are thought to be among the most interesting bacteria in industrial biotechnology, especially when it comes to cellulase production. They are typically distinguished by their ease and speed of culture, low nutritional requirement and their high enzyme production ( Balla et al. 2022 ). There is growing interest in the production of cellulase by these organisms, which are isolated from various environments, such as Bacillus sp. ( Acharya & Chaudhary 2012 ), Bacillus siamensis YC-9 ( Wang et al. 2021 ), Bacillus subtilis subsp. inaquosorum ( Regmi et al. 2020 ) and Bacillus cabrialesii , which is reported as cellulase producer that degraded the cell walls ( Masmoudi et al. 2025 ). Due to its versatile metabolism and extremely effective protein secretion system, B. subtilis has been extensively employed as a cellular factory for microbial enzyme synthesis ( Su et al. 2020 ; Malik et al. 2021) . Recently, numerous reports have been realized to identify and separate microorganisms that degrade cellulose in diverse environments, which are of crucial importance for the development of novel cellulases with distinctive properties including the identification of different strains of extremophile Bacillus sp. : halophiles, thermophiles, psychrophiles, acidophiles and alkalophiles, xerophiles, and their genes involved in the production of cellulases (Sharma et al. 2016 ; Chen & Jiang 2018 ; Balla et al. 2022 ). Indeed, various factors, such as pH, temperature, aeration, incubation time, inoculum size and carbon source could affect and influence bacterial growth and enzyme production ( Hussain et al. 2017 ). This present study, aimed to investigate the efficient hydrolytic activities and the generation of hydrolase enzymes (cellulase) of Bacillus strains isolated for the first time from various thermal networks: Hammam Oulad Tebben and the thermal spring Zdim in Setif, Algeria. Studies were conducted on the selected cellulolytic bacteria with the goal of assessing their cellulase activity and optimizing their production under the effect of certain physico-chemical factors. However, it was necessary to evaluate the enzyme's stability in the presence of numerous physicochemical variables as well as its capacity to break-down specific cellulosic substrates, in order to involve them in the future as industrial and biotechnological innovative tools. 2 Materials & Methods 2.1 Isolation of bacterial strains Thermal water samples were collected from two distinct locations in Hammam Oulad Tebban (geographic coordinates 35°48'33.1"N and 5°06'27.1"E) and Zdim thermal spring (Geographic coordinates 36.035210 "N, 5.244283 "E) south of Setif-Algeria. Depending on the volume of water analysed, strains were isolated using the membrane filtration method on solid culture media Tryptic soy agar (TSA) (Liofilchem, TE, Italy) ( Arya et al. 2015 ). A 0.45 µm cellulose acetate membrane (THOMAPOR, RCT, Germany) was used to filter sterilizing volumes of 1 ml, 10 ml, 20 ml, and 30 ml of each sample. Thereafter, this membrane was put on the top of TSA medium (Liofilchem, TE, Italy) and it was incubated at 30 ° C and 45 ° C for 24 h to 48 h. Colonies of various appearance were removed from each Petri dish, and purified by successive subcultures on nutrient agar (NA) (Liofilchem, TE, Italy). The pure strains were kept (stored) at 4°C. 2.2 Screening of strains exhibiting extracellular hydrolytic activities and Cellulase production All bacterial strains were inoculated in Lauria broth medium (LB) (Accumix, Malaga, Spain) for 24 h at 30°C and their hydrolytic activities were conducted out by spot inoculation of 2µL of the bacterial culture on various culture media according to the specific substrate for each enzyme. The enzymatic index (Ei) was determined for all the enzymes according to ( Ferbiyanto et al. 2015 ) using the following formula: Ei = (Halo diameter - colony diameter) / colony diameter In order to demonstrate the cellulosic activity of the isolates, the bacterial culture was spot inoculated onto a slightly modified carboxymethylcellulose based medium (CMC 1%; Sigma-Aldrich, St. Louis, USA) and incubated at 30 °-37° C for 5 to 6 days ( Sahay et al. 2017 ). This activity is observed after flooding the dishes with Red Congo (C.I. 22120 France) in 1% solution for 15 min then decolorized with a NaCl solution (1M) ( Teather & Wood 1982 ) . 2.3 Characterization of cellulase activity 2.3.1 Enzyme activity test The selected bacterial strains (21 strains) were tested for their capacity to produce the enzyme in a liquid medium. Firstly, 100µL of each bacterial suspension cultured in LB broth, was inoculated into tubes containing 10 mL of CMC broth (1%) with a small adjustment, then incubated at 30°C for 72 h with shaking. After incubation, the cultures were centrifuged at 5000 rpm/10 min/4°C. The supernatants containing the crude enzyme were gathered and kept at 4 ◦ C for further analysis ( Balla et al. 2022 ). 2.3.2 Enzyme dosage The 3,5-dinitrosalicylic acid (DNS) method was used to measure the amount of reducing sugar released by CMC in order to determine CMCase activity ( Miller 1959 ) . 1 mL of the enzyme solution supernatant was mixed with 0.5 mL of acetate buffer (50 mM, pH 4) containing CMC (1%). After incubation for 30 min at 50 ◦ C, the reaction was stopped by adding 1 mL of DNS reagent (SUVCHEM, MH, India) and the mixture was boiled for 5 min and cooled to stabilize the colour. The absorbance was measured at 540 nm ( Balla et al. 2022 ; Malik & al. 2022). CMCase activity was measured using a glucose standard curve as a guide. All samples were examined in triplicate, and one unit (U) of CMCase activity was defined as the quantity of enzyme needed to release one micromole of reducing sugars (measured as glucose) per minute ( Ho et al. 2017 ; Hussain et al. 2017 ; Balla et al. 2022 ). The corresponding control (blanks) of the enzyme-free substrate were performed concurrently with all enzyme assays. 2.4 Impact of physicochemical factors on bacterial growth and cellulase production Eight bacterial strains were chosen based on the results to assess their ideal bacterial growth and cellulase activity under various physico-chimical factors. Cellulase production was estimated by measuring the enzymatic activity using the DNS method previously mentioned, and bacterial growth was assessed by measuring the absorbance at 600 nm using a UV-visible spectrophotomer (JENWAY-7205, France). 2.4.1 pH value To determine the ideal and optimal pH for bacterial growth and cellulase production capacity in 1% CMC liquid medium, three pH values ​​were tested: 5.0, 7.0 and 9.0. The cultures were incubated at 30°C for 72 h ( Balla et al. 2022 ) using the pHmeter (ADWA-Ad1000, Szeged - Hungary). 2.4.2 Temperature To assess the impact of ttemperature on the growth of the selected isolates and enzyme production, the cultures were incubated in 1% CMC medium at temperatures of 30 ◦ C, 45 ◦ C and 50 ◦ C for 72 hours ( Balla et al. 2022 ). 2.4.3 Salinity and water stress Valuation of the impact of the salinity on bacterial growth and cellulase production was carried out using 1% CMC medium with increasing NaCl concentrations of 0, 400, 800 and 1000 mM, then the cultures were incubated at a temperature of 30°C/72 hours. For the water stress impact, polyethylene glycol (PEG6000) was assessed at different concentrations from 0 and 30%. As previously, cultures were incubated at 30°C for 72 hours ( Balla et al. 2022 ). 2.5 Influence of physicochemical parameters on cellulase activity and stability of cellulases Four bacterial strains were selected to investigate their enzymatic activity and stability under various physicochemical stress parameters. The crude enzyme extract was produced after 72 hours of growth under optimal circumstances. 2.5.1 Influence of pH on cellulase activity and stability A range of buffers with pH values ​​from 3 to 11 were used to assess the impact of pH variation on CMCase activity and stability; acetate buffers (pH 3), citrate buffers (pH 4–6), phosphate buffers (pH 7–11) and glycine buffers (pH 9–10), and the enzyme solutions were incubated with the appropriate pH buffer (3–11) for one hour at room temperature. Then, DNS standard method was performed to measure the residual activity ( Balla et al. 2022 ). 2.5.2 Influence of temperature on cellulase activity and stability The thermal stability and activity of the enzyme were determined at respective temperatures (45°, 50°, 70° and 80°) with pre-incubation of the enzyme in the same acetate buffer (pH 4) without the substrate for 1 hour. The standard DNS method was then used to quantify each sample's residual activity for CMC hydrolysis under the enzyme assay's optimal conditions ( Shankar & Isaiarasu 2011 ; Balla et al., 2022 ). 2.5.3 Influence of salinity on cellulase activity and stability The reaction salinity that maximized CMCase activity with 1% (w/v) CMC was measured in 50mM acetate buffer (pH4) containing various concentrations of NaCl (Supelco, Denmark) (0mM, 500mM, 1000mM,1500mM, 2000mM). To determine the salinity stability, the enzyme was incubated with 50mM acetate buffer (pH4) without 1% CMC with the same salinity concentrations for one hour at room temperature. The residual CMCase activity was tested using the DNS standard method, after addition of 1% CMC ( Wang et al. 2009 ; Balla et al. 2022 ). 2.5.4 Influence of water stress on Cellulase Activity and Stability According to the standard assay conditions, CMCase activity was measured using a buffer substrate at two concentrations of polyethylene glycol ( SPECILAB, Tlemcen, Algeria) (PEG6000) at 0 and 30%. By pre-incubating the enzyme (without substrate) in 50mM acetate buffer pH4 containing varying concentrations of PEG6000 for 30 minutes at 50°C, the impact of water stress on enzyme stability was as certain. The standard DNS method was used to measure residual activity ( Manfredi et al. 2016 ; Balla et al. 2022 ). 2.6 Hydrolysis power of different substrates The activity of crude cellulase was measured against different substrates. According to Balla et al. ( 2022 ), the mixtures of 2 mL of each bacterial supernatant with 1 mL of acetate buffer pH 4 containing 1% of each of the following substrates: cellulose powder (Sigma-Aldrich, St. Louis, USA) and wheat bran were incubated at 50°C for 30 min. Pieces of filter paper Whatmann No. 3 (GE, HealthCare Life Sciences, Cleveland, UK), of size (1×5 cm) equivalent to 50 mg were added to 1 mL of buffer supplemented with 2 mL of supernatant and were incubated at 50°C for 60 min. Using glucose (Fisher Sientific, UK) as a reference, the DNS method was used to determine the reducing sugars ( Miller 1959 ; Balla et al. 2022 ) 2.7 Combined effects of physico-chemic factors on microbial growth and cellulase production in solid media The impact of various parameters on cellulase activity on solid medium of four selected strains was studied using 1% CMC medium adjusted to pH 5, with a NaCl concentration of 800 mM, incubated at 50°C. The bacterial culture was inoculated onto CMC agar in a 2µL spot, then Red Congo solution (1%) was used to reveal the CMC hydrolysis zone appeared around the colony for 15 min before being decoloured by NaCl solution (1M). The enzymatic index was calculated as previously described. The enzymatic indexes for the single stress factor and the combination of the three factors types were compared with a control ( Balla et al. 2022 ). 2.8 Characterization of the four selected bacterial strains 2.8.1 Phenotypic characterization Macroscopic observation of colonies was reported after 24 hours on TSA culture medium, in order to characterize the main components: Colony shape, colony size, colony color, elevation, opacity and surface ( Guiraud, 2012 ). Moreover, the purification of cellulase-producing strains is carried out by successive subculturing. To characterize microscopically these isolates, Gram staining (Gram 1884) was performed to identify their morphology and to distinguish between Gram (+) and Gram (-) bacteria ( Bhagat & Kokitkar 2021 ). 2.8.2 Molecular Biotyping and Bacterial Identification Phylogenetic analysis was used to identify the four bacterial isolates were performed in collaboration with Macrogen (Amsterdam, Netherlands) according to the following steps: i DNA extraction was performed using the salting out procedure by a standard extraction protocol. Selected isolates were grown overnight on TSA agar. A culture loop was re-suspended in 100 µl of sterile distilled water and vortexed well, then incubated at 95°C for 20 min, rigorously vortexed, and incubated once time in ice for 10 min. finally the DNA medusa was recuperated by centrifugation for 10 min at 10,000 rpm at 4°C (Hussain et al. 2017 ). ii the purified DNA was amplified by standard PCR using universal primers 27F' (5' AGAGTTTGATCMTGGCTCAG-3') and 1492 R (5'GGTTACCTTGTTACGACTT-3') following standard instructions. Then, iii the bacterial identification was obtained upon partial sequencing of 16S rRNA gene, using the primers primers 907R (5'-CCGTCAATTCMTTTRAGTTT-3') and 785F (5'-GGATTAGATACCCTGGTA-3') based on Sanger sequencing protocol, in order to discriminate among species belonging to the Bacillus group. The DNA sequence homology was determined using bioinformatics tools through pair-wise sequence alignments, using BLAST within the NCBI nucleotide collection database. The evolutionary analysis was inferred by using the Maximum Likelihood method and Kimura 2-parameter model ( Kimura 1980 ) and the evolutionary analyses of the final dataset were conducted in MEGA X (Kumar et al., 2018 ). 2.9 Statistical analysis All experiments were performed in triplicate, with results expressed as mean ± standard deviation (SD). Unifactorial ANOVA and multifactorial ANOVA was used to identify variance among different treatments. Results were deemed significant when p -values ​​were less than 0.05. When a significant difference was detected, post-hoc comparison tests of the Tukey test were conducted. Different letters are included at the top of each column to indicate differences between the different groups. All statistics are performed using Origine Lab Pro 2022 developed by Origine lab Corporation. MEGA X was used for the molecular evolutionary genetics analyses (sequence alignment, phylogenetic tree) of the final dataset. 3 Results 3.1 Screenings of extracellular hydrolytic activities: cellulose production As known that cellulose could be broken down by the complicated enzyme “cellulase”, which could be produced by bacteria, and its importance to be used in many different industries, including as food processing, textiles, and biofuel production. In this present research, forty-five strains isolated and purified on solid culture medium (TSA) from both Algerian hot springs (Hammam Oulad Tebben and Zdim thermal spring, Algeria) were characterized for their hydrolytic activity at 30–37°C. For this reason, the enzymatic productive capacity of cellulase was carried out and using carboxymethylcellulose (CMC) as specific substrate. The results obtained are displayed in the figures and graphs according to the values ​​of the Enzyme Index (Ei). According to the analysis of cellulase activity mentioned in ( Fig. 1 ) , among the 45 selected strains just 34 strains were competent for cellulase producing, which means that 75,55% (n = 34/45) of all the strains under investigation. They expressed good cellulase activity with enzymatic indexes varied from 0.2 to 2.6 ( Fig. 2 (A&B)) . 3.2 Characterization of cellulase activity Figure 3 displayed the cellulase activity (in U/ml) of 21 various bacterial strains with the highest enzymatic indexes and cellulase production which was assessed in liquid medium. The enzymatic activity across all tested strains was ranged from 1.03 to 2.18 U/mL for about 15 isolates from both hot springs (S4, S8, S10, S11, S12, S13, S16, S17, S20**, S22, S24*, S25, Z1, Z4, Z7, Z9*, Z9**, Z12, and Z17). However, the highest cellulase activity was obtained by Z1, Z17 and S11 with exhibition of approx. 2.15 U/ml, the other isolates there is either very low enzymatic activity or no activity was shown (S22). According these results, 8 strains were selected as the most promising candidates for cellulase production based on their high and significant activity. These strains would be the focus for further analysis, such as optimization of enzyme production or genetic characterization. 3.3 Effect of physicochemical factors on bacterial growth and cellulase production Analysis of the impact of various parameters on bacterial growth and cellulase production was carried out for 8 isolates from both hot springs (S8, S11, S24*, S25, Z1, Z7, Z9* & Z17) selected according to their enzymatic performances in liquid medium. 3.3.1 Effect of various pH value on bacterial growth and on cellulase production The findings of this experiment presented in ( Fig. 4 -A ) reported the pH dependence of bacterial growth for eight distinct bacterial strains as determined by Optical Density at 600 nm (DO 600 nm) at three distinct pH levels (pH 5, pH 7, and pH 10). The data unequivocally demonstrates that nearly all strains develop best at pH 7. Thus, strain Z17 is the most resilient strain, showing the highest growth at ideal pH as well as the highest relative tolerance to acidic (pH 5) and alkaline (pH 10) environments. With strain Z17 exhibiting the widest resistance to pH extremes, this suggests that the majority of strains are neutrophilic (pH = 7.0), however the bacterial growth is moderate at acidic medium pH (= 5.0), but it is lower at alkaline culture medium pH (= 10.0). Otherwise, the cellulase activity (U/mL) of the selected eight strains was examined also at different pH values (pH 5, pH 7, and pH 10) is shown in Fig. 5 -A. The data clearly indicated that the pH value significantly impacted the enzymatic activity for all tested strains: Four isolates (S8, S24*, Z1, and Z17) produced significant CMCase at pH 5, while the lowest CMCase activity was recorded at pH 10. The optimal pH for maximum cellulase activity was reached at pH 7, with important CMCase activity ranging from 1.4 U/mL (S25) to 2.35 U/mL (Z1). The findings implied that the ideal pH range for the cellulase production under measurement is ranged between pH 5 and pH 7 with detection of the the highest enzymatic activity for the strains: S11 and Z1 with ca. 2.2 U/mL and 2.0 U/mL at the acidic pH, moreover, Z1 and Z17 exhibited approx. 2.4 U/mL and 2.1 U/mL respectively at neutral pH. Then, the sharp decreased in activity at pH 10 strongly implies that the high alkalinity denaturized or significantly inhibited the active site of the enzyme, inhibiting efficient catalysis ( Fig. 5 -A ) . 3.3.2 Effect of variable temperature value on bacterial growth and on cellulase production Temperature profiles were determined by assessment of the bacterial growth and cellulase production at three different temperatures: 30°, 45° and 50°C. Figure 4 -B showed the selective effect of heat treatment. In contrast to the ideal temperature of 45°C, the majority of isolates showed a notable reduction in bacterial growth at 30°C and 50°C. But strain S8, which was isolated from Hammam Ouled Tebben's thermal waters, showed that 50 ◦ C was the ideal growth temperature. Indeed, the highest overall growth (highest OD values) for most strains is observed at 45°C, with Z1, Z7 and Z17strains that revealed the greatest growth. However, the lowest growth is generally seen at 30°C and 50°C, suggesting 45°C is the optimum temperature for growth for the majority of these strains. The physiology and enzymatic activity of microorganisms are significantly influenced by temperature. In order to investigate the impact of temperature on cellulase production, the tested isolates were incubated for 72 hours at various temperatures ranging from 30°C to 50°C. The majority of the tested isolates were found to be able to produce cellulase, with the highest cellulase activity occurring at temperatures as high as 45°C and the enzymatic activities varied from approximately 1.35 U/mL to almost 3.0 U/mL. The activities at 30°, and 50°C are typically lower indicating that 45°C is the ideal temperature for this enzyme's function and stability across all tested strains ( Fig. 5 -B ) . 3.3.3 Effect of salinity on bacterial growth and cellulase production The Fig. 4 -C illustrated the salinity impact of the bacterial growth (DO 600 nm) of the eight tested strains across four different sodium chloride (NaCl) concentrations: 0 mM, 400 mM, 800 mM, and 1000 mM. The majority of bacteria were found to be able to grow at salinity levels between 400 and 1000 mM. In contrast to their contributions at 400 and 1000 mM, isolates S24*, S25, Z1, Z7, and Z17 were better shown to be able to proliferate at 800 mM NaCl concentration, and among them strains Z1 and Z17 remained the best performer maintaining their salt tolerance and high relative growth. After 72 hours of incubation at 30°C. Among the concentrations tested, all isolates tested were able to synthesize cellulase at high salinity levels of 400, 800 and 1000 mM NaCl ( Fig. 5 -C ) . Furthermore, the findings reported that almost all the strains were halotolerant and have to be the best performer of enzyme production/use in high-salt environments, despite not being the fastest grower and this is strain-dependant. 3.3.4 Effect of water activity on bacterial growth and cellulase production PEG 6000 is frequently used in microbial research to create osmotic stress or water potential stress, simulating drought-like conditions. The histograms Fig. 4 -D illustrated the impact of water activity on the bacterial growth of the eight isolates, after 72 hours of incubation at 30°C. This was done by adding increasing concentrations of PEG 6000 to the CMC liquid medium at different concentration. A trade-off is evident in the graph: under ideal circumstances, strain Z17 was a high-yield strain, but it is extremely susceptible to osmotic stress caused by PEG 6000 . The strains S8 and Z7 exhibited the maximum tolerance to PEG 6000 at 30%, suggesting that they are better suited to low water availability. Nevertheless, the impact of osmotic stress caused by PEG 6000 on cellulase production was determined by measuring enzyme activity. Cellulase production was significantly decreased at 30% PEG6000 for most isolates tested except S11, which produced the enzyme abundantly ( Fig. 5 -D ) . In terms of growth and enzymatic activity, strains S8 and Z7 consistently exhibit the highest tolerance to PEG 6000 -induced osmotic stress, which makes them ideal for use in settings with limited water availability. Despite being the best overall performance under control conditions, strain Z17 is significantly reduced in both growth and enzyme activity at PEG 6000 (30%). 3.4 Physicochemical characterization of cellulases: activity and stress resistance Withstand the combined effects of pH, temperature, salinity, and PEG 6000 , four strains Z1, Z7, Z17, and S11 were selected for the investigation of their enzymatic activity and stability under stress conditions. 3.4.1 Influence of pH on the activity and stability of cellulases The effect of pH on the stability and activity of cellulase was examined at pH values ranging from 3.0 to 11. The pH range in which this enzyme functioned was 4.0 to 11.0, however its optimal pic was recorded between pH 6.0 and pH 10.0 and it is substantially lower at pH 3.0 for all tested bacteria. Furthermore, depending on the strains tested or the same parameter, the residual activity remained largely unchanged. There seems to be an ideal pH range where the activity of is at its peak. Due to pH-induced modifications in the three-dimensional structure of the enzyme (denaturation), extreme pH values (both extremely acidic and highly alkaline) typically result in a decrease in enzyme activity, a frequent feature of enzyme function. The enzymatic activity is often higher in Graph A ( Fig. 6 -A ) for all samples. Optimal pH: All samples appear to maintain or reach their maximum activity in the alkaline range (pH 8.0 to pH 11.0). With values around 1.2 U/mL, samples Z1 and Z17 exhibit the highest activities, peaking between pH 10.0 and pH 11.0. Samples Z7 and S11 have lower activity than Z1 and Z17, but in the pH 7.0 to pH 11.0 range, their activity is also fairly stable or slightly increases. According to cellulase stability, strain Z1's activity was completely maintained at pH 4.0, whereas strain S11's activity was maintained between pH 4.0 and 11. However, strains Z1, Z7, and Z17 demonstrated remarkably stable enzymatic activity at over 60% in the same pH range ( Fig. 7 -A ) . 3.4.2 Influence of temperature on the activity and stability of cellulases Temperature effects on enzyme stability and activity were also assessed at a range of temperatures, from 45 to 80°C. Both graphs ( Fig. 6 -B and Fig. 7 -B ) , a clear trend showed that increasing the temperature from 45°C to 80°C generally leads to a decrease in the enzymatic activity for most of the samples, especially when moving from the optimum temperature to 80°C. At all temperatures examined, CMCase activity levels were extremely high, peaking for all strains at 50 ◦C and 70 ◦C. The stability, as measured by residual enzyme activity, was at its best at 50°C and 70°C after 1 hour of pre-incubation at various temperatures. For all strains, the enzyme exhibited above 40% residual activity up to 80°C. This is typical for enzymes, as excessive heat causes denaturation, reducing their efficiency. The experiments unequivocally demonstrated that, the common optimal temperature for the highest activity of the enzyme sample Z1 is 50° C. For other samples, such Z17, the stability and ideal temperature, with higher activity at 70°C and 80°C. 3.4.3 Influence of salinity on the activity and stability of cellulases In both graphs ( Fig. 6 -C and Fig. 7 -C ) , the cellulase typically showed halotolerance or haloactivity, which means that they continue to function even at extremely high NaCl concentrations (up to 2000mM). This implies that the enzymes either originate from or function in highly salinized settings. In details, the experiments ( Fig. 6 -C ) highlighted that the tested enzyme from all strains (Z1, Z7, Z17, and S11) are halotolerant because they continue to exhibit notable enzymatic activity at 2000mM. Z1 exhibited the highest activity overall reaching approx. 2.6 U/mL) at normal condition (0 mM of NaCl). Its activity decreased slightly but remains high even for high concentration of NaCl up to 2000mM, demonstrating good salt tolerance for this strain. Z7 consistently showed the lowest activity across all NaCl concentrations in this condition, maintaining stable activity around 1.8 U/mL. However, both isolates Z17 and S11 maintained relatively stable activity, presenting a small initial drop from 0 mM to 500 mM, but then sustaining activity up to 2000mM. Figure 7 -C demonstrated a lower overall enzymatic activity range. Despite, for especially the strains Z1 and Z17, the enzymatic activity was almost stable and remained maximal at 500 mM and/or 2000 mM NaCl achieving the highest activity at approx. 1.4 U/mL. Generally, the four bacterial isolates maintained a high degree of activity up to the highest tested concentration, 2000 mM, further confirming their ability, their haloactivity and their halostability. 3.4.4 Influence of water stress on the activity and stability of cellulases The effect of PEG 6000 concentration on the enzymatic activity (U/mL) on the tested cellulase from four strains (Z1, Z7, Z17, and S11) at two concentrations (0% and 30%) under two different conditions, was demonstrated in Fig. 6 -D and Fig. 7 -D. The findings on Fig. 6 -D reported that under 0% of PEG 6000 condition, Z1 had the highest activity reaching 2.4 U/mL followed by Z7, Z17, and S11, that all showed high activity (around 2.0 U/mL). However, the addition of PEG 6000 at 30% caused a significant drop in activity (1.6 U/mL) for all samples except for Z1 that maintained the highest activity (approx. 2.3 U/mL), showing high stability/tolerance to PEG 6000 . PEG 6000 at a 30% concentration proved ineffective in analyzing the effects of the medium's absence of water on the enzyme's initial and residual activity. However, regardless of whether PEG 6000 was present or not, the enzymatic stability was maintained and varied by 40% based on the isolates. The results mentioned in Fig. 7 -D demonstrated a lower overall enzymatic activity range, reaching an optimum about 1.15 U/Ml when enzyme stability was assessed. Although, Z1 showed also the highest activity (ca. 1.15 U/mL) with PEG 6000 (0%). The Z7, Z17, and S11 exhibited very similar activities (around 0.9 U/mL). Using PEG 6000 30%, the obtained results were less drastic than, but still showing low enzymatic activity reaching 1.05 U/mL for Z1, and demonstrated significant decrease in activity for the other strains. This experiment revealed that Z1 maintained the highest enzymatic activity while being the most robust, resilient and tolerant to the addition of PEG 6000 30%. The high concentration of PEG 6000 often affected the enzymatic activity and stability as strain-dependent manner. 3.5 Hydrolysis power of different substrates The graph ( Fig. 8 ) illustrated the enzymatic activity (U/mL) of crude enzyme from four strains (Z1, Z7, Z17, and S11), when acting upon four different cellulose substrates: CMC, powdered cellulose, wheat bran, and filter paper in order to ascertain the cellulose substrate's specificity. The outcomes of this experiment strongly confirmed that according to the substantial substrate-dependent variations in enzyme activity that the tested enzyme was probably cellulase with varying specificities toward beta-glucan linkages and cellulose's physical structures. Indeed, Z1 was the best enzyme producer for breaking down soluble cellulose (CMC) with a significant and maximum activity (ca. 2.8 U/mL), suggesting high endoglucanase activity, Z17 was considered as the best enzyme for breaking down crystalline cellulose (powdered cellulose and filter paper), suggesting strong and significant exoglucanase activity reaching ca. 2.4 U/mL and ca. 2.1 U/ mL respectively. All four enzymes are equally effective on the complex natural substrate, Wheat Bran showing very similar and high activity (ca. 2.2 U/mL). 3.6 Combined effects of parameters physico-chemical on microbial growth and cellulase production in solid media The graph ( Fig. 9 ) demonstrated the enzymatic index for the four enzyme samples (Z1, Z7, Z17, and S11) under various single and combined stress factors: T 50°C (Temperature), pH 5, 800 mM NaCl. The findings reported that cellulase production in solid media was significantly impacted by both single and multiple stress parameters (p < 0.05). Indeed, taken strain by strain; Z1 was notably robust under the combined stress of high temperature (50°C), acidic pH (= 5.0), and high salinity (800 Mm NaCl), highlighting the grater activity/stability under these extreme conditions, Z7 was the most active in the control condition and most stable under single pH and NaCl stress. Although, Z17 and S11 generally performed better under control and single stress factors compared to the combined stress. 3.7 Characterization of the four selected bacterial strains 3.7.4 Phenotypic characterization The selected four strains (Z1, Z7, Z17, and S11) from both hot springs Hammam Oulad Tebban and the Zdim thermal spring were characterized macroscopically. The resulting colonies are typically spherical, white, and beige in color, with a diameter of 2 to 5 mm. They typically had a rough, and viscous aspect. The majority of strains have regular or uneven borders and are flat. Following Gram staining, microscopic analysis exhibited that all four strains were discernible as Gram + sporulated rods. Thus, the four tested strains most likely belong to the genus Bacillus relying on the Gram + Bacillus criteria and the presence of spores. 3.7.5 Molecular identification of selected isolates The phylogenetic tree based on the comparison of the 16S rRNA sequences of the strains studied with similar sequences of strains available in databases (GenBank) confirmed that all strains belonged to the Bacillus genus ( Fig. 10 ). The isolates (Z1, Z7, Z17, S11) from both hot springs Hammam Oulad Tebban and the Zdim thermal spring showed different standard PCR profiles (data not shown). Therefore, all the isolates were subjected to molecular identification and gene alignment based on bioinformatics tools and databases on GenBank (Blast, NCBI). The phylogenetic tree built upon the combination of the profiles (Fig. 10 ) showed that all the identified strains were belonging to Bacillus genus however strains were allotted to: Z1 as Bacillus siamensis (PX112786.1), Z7 as Bacillus sp . (in: firmicutes) (MG470713.1) and strains Z17 and S11 , showed the highest sequence similarity Bacillus cabrialesii ( ON287153.1) and Bacillus inaquosorum (MN581182.1) respectively (Table 1) . Table 1 16S rRNA gene sequencing-based identification of the four selected strains 4 Discussions The use of enzymes, also known or biological catalysts has grown significantly in recent years, and the discovery of new enzymatic catalysts offers new applications opportunities. However, thermos-tolerant or thermophilic microorganisms those that can grow and survive in hot environments with temperatures above 50°C—have attracted attention and lead to the discovery of a wide variety of original enzymes with distinctive properties (Wilson et King. 2022) . In this present study, the investigation of cellulase production, was carried out using the specific substrate carboxymethylcellulose using bacterial strains isolated from two thermal springs “Hammam Oulad Tebben and Zdim hot spring, in Setif, Algeria for the first time in the worldwide. According to the obtained results, among the 45 strains selected and purified on TSA culture medium at 30°C in aerobic conditions for 72 hours of incubation, 34 strains were qualified as cellulase producers with a rate of 75%. The outcomes of this study indicated that the strains isolated from Zdim hot spring (Z) had greater cellulosic activity (n = 17) than those of Hammem Oulad Tebben (S). The obtained results confirm the high capacity of strains isolated from thermal springs to produce extracellular hydrolytic enzymes. The molecular identification of the selected strains demonstrated that they belonged all to Bacillus genus but they were different species affiliated to Bacillus subtilis sp. which improve the cellulase production by our isolates as known that Bacillus species are very important producer of extracellular enzymes and particularly cellulase production ( Schallmey et al. 2004 ; Balla et al. 2022 ; Malik et al. 2022 ). Otherwise, the availability of nutrients in the environment has a direct impact on the enzymatic baggage of microorganisms, which is important to their natural habitat’s adaptation. In fact, these enzymes play a crucial role for the metabolism of environmental nutrients, to ensure the bacterial survival. Furthermore, these innate bacterial skills are used for biotechnology across various industrial sectors ( Zhao et al. 2010 ). Moreover, many thermophilic bacteria synthesize various hydrolytic enzymes (such as protease, cellulase, amylase, glucosidase, glucoamylase, pullulans, and cyclodextrin glycosyltransferase) when they are subjected to natural substrates. Other polysaccharides, like glycogen supplied by animal cells or microorganisms, can also be broken down by them ( Benkahoul et al. 2017 ). Indeed, Bacillus species are highly interesting for the development of industrial applications. Industrialists are interested in them because of their quick growth, which leads to short fermentation cycles, their capacity to produce proteins in the extracellular medium, and the fact that some species are listed on the FDA (Food and Drug Administration) and GRAS (Generally Regarded as Safe) list ( Schallmey et al. 2004 ). Although, the capacity of thermophilic bacteria to generate extracellular enzymes has been the subject of numerous investigations. Recently, a study was carried out, on the characterization of thermophilic bacteria related to members of the different genera such as Anoxybacillus , Geobacillus , Bacillus , Meiothermus , etc… that had the efficiency to produce hydrolytic enzymes from the Hammam Righa thermal spring, in Aîn Defla, Algeria Thus, in this study 14 strains were isolated, and qualified as strong producers of amylase, protease, xylanase and cellulase, which was in concordance with the findings of this present study (Bouacem et al. 2022 ). Further study on 2017, was conducted on the characterization of bacterial strains from Algerian hot environments, in which 13 strains were isolated, and the latter exhibited extracellular hydrolytic activities (protease, amylase, pectinase and cellulase) (Benkahoul et al. 2017 ). Interestingly, cellulase well-known as a complex enzyme group produced by different microorganisms (fungi, bacteria, and protozoa) which could break-down cellulose into simpler sugars, have garnered global attention due to their multiples industrial uses, especially in the paper and pulp sector, textiles, and biofuels. Furthermore, cellulase is the subject of both academic and industrial research, and they have a commercial validity of over 30 years. The thermal springs were the site of the sampling. According similar study done in Algeria, only 16% of bacteria isolated from hot springs in West-Algeria were found to contain cellulase, this suggests that this underutilized biotope may be a promising source of thermophilic enzymes with significant industrial value (Khelil et Cheba 2014) . Nevertheless, as a potential source of cellulolytic enzymes, thermophilic bacteria have garnered a lot of interest lately ( Shyaula et al. 2023 ). Different species have different levels of enzyme activity on liquid media; which is matching with the findings of this research that showed that isolates from Zdim thermal spring outperformed the others, with CMCase activity ranging from 1.033 to 2.18 U/mL. As a result, these bacteria's cellulase could break down CMC as specific substrate. Since CMC is a cellulose-soluble material, cellulase are hydrolytic enzyme holding both endoglucanases and β-glucosidases, ( Sharma et al. 2016 ). Research conducted by Sarangthem et al. ( 2023 ) screened a thermophilic cellulase-producing bacterium, identified as Bacillus velezensis MRC 5958, from the natural hot springs of Bakra, India. However, these current results were more performant with those obtained by ( Balla et al. 2022 ), who reported that the CMCasic activity for bacteria from thermal waters ranges from 0.7 to 2.2 U/mL. Yet, according to another research by ( Maryam et al. 2018 ), B. cellulosillyticus had a CMCasic activity of 1.99 U/mL. Nonetheless, other Bacilli strains have been demonstrated to have higher endoglucanase activity, ranging from 7.3 U/mL to 9.1 U/mL ( Mawadza et al. 2000 ) which is contrasted with the present findings. Maximum cellulase has been achieved by optimizing a number of physicochemical growth condition parameters. Having the ideal value for each variable is essential to achieve successful enzymatic processes because it boosted outputs and performance ( Nnaemeka et al. 2021 ; Shyaula et al. 2023 ). Among the physicochemical parameters; temperature, medium pH, salinity, and osmotic activity (water activity) are all important factors in microorganisms growth enzyme synthesis modulation. The effects of these parameters differ from microorganism to another ( Sarsan and Merugu 2019 ) . In fact, cellulase production is largely controlled by the pH of the culture medium, which has a significant impact on the ability of bacteria to produce it ( Sarsan & Merugu 2019 ) . Every bacterial species has a specific optimal pH for its enzyme production needed to breakdown cellulose, according to many previous research ( Hussain et al. 2017 ; Balla et al. 2022 ; Malik et al. 2022 ; Sarangthem et al. 2023 ). To increase the cellulose hydrolysis process's efficiency, it is crucial to comprise these ideal circumstances. All strains' bacterial growth (OD600 nm) was found to be maximal at pH 7.0 and moderate at pH 5.0, indicating that they are either neutrophils or moderately acid-tolerant. Nonetheless, the ideal pH of 7.0 indicated a maximum cellulase activity that ranged from 1.46 to 2.35U/mL. At pH 5, the CMCase levels of the tested bacteria S8, S24*, Z1, and Z7 were 1.54, 2.18, 1.98, and 1.70 (U/mL), respectively. Conversely, these bacteria produced very little of this enzyme and grew very poorly at alkaline pH. These findings are consistent with previous research on various strains of Bacillus species, including B. megagaterium, B. subtilis, B. amyloliquefaciens , and A. flavithermus ( Hussain et al. 2017 ) and Bacillus velezensis strain MRC 5958 ( Sarangthem et al. 2023 ). Additional research by Balla et al. ( 2022 ) demonstrated that cellulase activity was ranged between 0.3 and 1.8 U/mL and its peak was obtained at neutral pH (= 7.0). In contrast to antecedent study, other investigations have reported that strains such as Bacillus species exhibit optimal cellulase production at acidic-pH values approximately pH = 3.0 ( Islam et al. 2019 ), which improve that Bacillus bacteria are a major source of cellulase enzymes, which are capable of breaking down the cellulose polymers into smaller sugar units. An important factor in fermentation is temperature, which varies from a microorganism to another and regulated the growth and biosynthesis of enzymes by different microorganisms ( Malik et al. 2022 ). Based on the physical properties of the cell membrane modification, temperature variation affects growth and the production of extracellular enzymes ( Bakare et al. 2005 ). Furthermore, the strain ideal growth and cellulase activity could be affected by temperature and the isolation site. Thus, in the present study, the ideal temperature for the bacterial growth was 45°C, and at that temperature, their optimal CMCase activity ranges from 1.34to 2.88 U/mL. Nonetheless, the majority of strains have cellulase activities ranging from 1.08 to 2.35U/mL and grow best at temperatures between 30° and 50°C. Numerous studies showed that 45°C is the ideal temperature for cellulolytic bacteria to grow and produce their enzyme ( Nargotra et al. 2016 ; Balla et al. 2022 ; Malik et al. 2022 ). With an ideal cellulase capacity of 1.2 U/mL and 1.44 U/mL at 45° and 50° C, respectively, the results obtained are comparable to those of B. velezensis (HOT1) and B. subtilis (HOT2) cellulase production ( Balla et al. 2022 ). However, for other Bacillus strains, like B. halodurans CAS 1, the ideal temperatures for growth and cellulase production are 55◦C and 60◦C, respectively (Annamalai et al. 2011 ). Generally, bacterial strains differ in their ability to tolerate salt. On culture media with 400 mM, 800 mM, and 1000 mM NaCl concentrations, all strains could grow-up. According to research reported by Shankar et al . 2011 , B. pumilus exhibited the highest cellulotic power at 2.5% NaCl (~ 400 mM) with CMCase activity of 0.085 U/mL. All strains produced cellulase enzyme at high salinity levels, ranging from 400 to 1000 mM NaCl leading to an enzyme activity ranging from 0.81 to 2.71 U/mL. According to another study of [13] 50% of the tested strains were able to synthesize cellulase enzyme at high salinity levels, between 400 and 800 mM NaCl. Furthermore, cellulase with the highest enzymatic activity was produced by the bacterial strain Brevibacterium halotolerans (CDB2) in the range of 6–15% (~ 1000 to 2500 mM) salt concentration which is very high salinity ( Yousef et al. 2020 ). Osmotic stress affected the growth of bacteria and the cellulase production; so that, all strains examined exhibit osmo-tolerance and residual enzyme production in the presence of 30% of PEG 6000 , with S11 producing the most at 1.56 U/mL. This outcome was in concordance with prior report that demonstrated B. velezensis (HOT1) exhibiting cellulase production with an enzymatic activity of 1.22 U/mL when 30% PEG 8000 was used ( Balla et al. 2022 ). Some strains from the soil belonging to the genus Bacillus spp. had the ability to develop and grow well in media containing 25% PEG 6000 ( Vardharajula et al. 2011 ). According to Shankar et al ., 2011 , to ensure optimum enzyme production, it is important to assess the amount of water available and monitor the “aw”. Since enzymes are the building blocks of all biological processes (metabolism), biotechnology, and industrial applications, their stability and activity are crucial. In fact, enzyme activity and stability must be regulated in living microorganisms in order to preserve homeostasis and enable effective life activities. For these reasons, in this present study, the activity and stability of cellulase were evaluated for their tolerance to the impact of different stress parameters. The findings revealed that cellulase activity and stability were significantly affected by factors that could modify their functioning, including pH, temperature, salinity, and water (osmotic) stress. Using a range of pH values from 3 to 11, the effect of pH on the activity of the cellulases generated was examined. For strains Z1 and Z7, the ideal pH range for endoglucanase activity was 9 to 10 (1.19 U/mL, 0.71 U/mL), while for strains Z17 and S11, the ideal pH range was 6 to 7, with 1.22 U/mL and 0.71 U/mL, respectively. The enzyme's active site changed in response to any change in pH ( Irfan et al. 2012 ). According to the same metric, residual activity also stayed relatively constant. Depending on the strains that were tested, cellulase activity was fairly stable and had more than 60% to 90% residual activity in the pH range 4.0–11.0. For strain S11, 100% (0.84 to 0.88 U/mL) of cellulase activity was maintained over the various pH ranges from 4.0 to 11.0 and pH 4.0 (0.84 U/mL) for strain Z1. Prior studies have demonstrated that cellulase is active and stable across a range of pH values, from 4.0 to 11.0 for Bacillus strains (Balla et al. 2022 ) and between pH 6.0 and 6.5 for Bacillus subtilis YJ1, while it was stable between pH 6.5 and 7.5 according to ( Yin et al. 2010 ). Although CMCase of Virgibacillus salarius BM-02 showed great activity over all pH ranges from 4.0 to 9.0, but the optimum pH was found at 6.5 and stable in the pH range of 5.0–9.0 [12] ( Yousef & Mawad 2023 ) . Furthermore, cellulase from Bacillus velezensis MRC 5958, from Bakra natural hot springs, India was stable at pH 6 to 9 (Sarangthem et al. 2023 ) and the cellulase from Bacillus smithii QT03 was stable at 70% of enzymatic activity at pH 6 for 24 hours (Villanueva et al. 2025) . However, the cellulase of Cellulomonas sp. ASN2 was stable at pH 6.5–9.5 and more active at pH 7.5 (~ 0.45 IU/ml/min). This enzyme, which could be utilized in alkaline settings like pulp processing, requires a pH value of 7.5 ( Irfan et al. 2012 ). Cellulase typically exhibit stability across a broad pH range of 5 to 10. Because of their significant industrial properties, a number of researchers have recently attempted to isolate alkaline thermostable enzymes using microbes ( Samiullah et al. 2009 ). Enzyme used in industry depends critically on the optimal temperature range of the enzyme activity and enhanced stability at high temperatures, particularly at temperatures of 50°C or upon 50°C. Temperature was found to have an impact on enzyme activity over a broad temperature range between (45 to 80°C), with all strains exhibiting optimal activity at 50°C and 70°C. At 50°C, strain Z1 exhibits an optimal CMCase activity of 2.89U/mL ( Bhatti et al. 2007 ; Yeoman et al. 2010 ; Yousef and Mawad 2023 ) . Similar results indicate that cellulases from certain Bacillus species, such as Bacillus subtilis YJ1, B. velezensis Z2.6, and Bacillus sp. strain RH68 have an optimum temperature of 50°C (Yin et al. 2010 ; Cai et al. 2024 ) and Bacillus . sp RH68 strain, have an optimal temperature of 50°C ( Yin et al., 2010 ) and 70°C (RH68) (Mawadza et al . 2000) . Stability, measured as residual enzyme activity, was optimal at 50◦C and 70◦C with an activity of 0.65 to 1.28 U/mL. According to another study, Bacillus sp. AR03's cellulolytic cocktail reaches 60°C (Manfredi et al. 2016 ) and the strain Cellulomonas sp . ASN2 reached its optimum at 60°C ( Irfan et al. 2012 ). Furthermore, strain Bacillus sp . Z2.6 demonstrated significant activity, maintained above 75% between 40°C and 60°C (Cai et al. 2024 ). According to the reported results of Sarangthem et al. ( 2023 ), the enzyme from Bacillus velezensis strain MRC 5958 is stable over a wide temperature range, from 30°C to 70°C and cellulase from Bacillus smithii QT03 was maintained 100% and 58% of its activity at 55°C for 3 hours and at 60°C for 24 hours respectively (Villanueva et al. 2025) . Regarding the impact of salinity on enzyme properties, the activity and stability of the cellulase by the different strains under investigation were not significantly impacted by rising NaCl levels (0–2000 mM). In fact, at all salinity levels, the strains retained a high enzymatic potency (1.5–2.5 U/mL). With strains Z1 and Z7, crude cellulases maintained 50–60% of their initial capacity even at 2000 mM and remained stable between 40 and 50% up to 500 mM. Although, purified CMCase from Bacillus licheniformis strain AU01 was found to retain only 34% of its activity at 30% NaCl (ca. 5000mM) ( Annamalai et al. 2011 ), whereas Bacillus sp . AR03's cellulolytic activity exhibited high resistance to concentrations up to 2.0 M NaCl (Manfredi et al. 2016 ). These cellulases' high salt tolerance is of great biotechnological interest; it will be essential for future uses in a variety of processes that rely on high salinity or osmotic pressures (Wang and al. 2009; Annamalai et al. 2011 ). Interestingly, in the presence of 30% PEG 6000 , the enzyme's initial and residual activity (i.e., 2.39 U/mL) was unaffected by water deficiency; however, the stability of the enzyme was maintained up to more than 40% (1.02 to 0.72 U/mL). Balla et al. ( 2022 ), also reported similar findings. They demonstrated that at 30% PEG 8000 , CMCase stability and activity were only marginally impacted. The specificity of the crude enzyme extract was assessed by incubating it with various soluble and insoluble substrates (CMC, powdered cellulose, wheat bran, and filter paper). In fact, Z17 was thought to be the best enzyme producer for breaking down crystalline cellulose (powdered cellulose and filter paper), indicating strong and significant exoglucanase activity reaching ca. 2.1 to 2.4 U/mL, while Z1 was the best enzyme producer for breaking down soluble cellulose (CMC), with a significant and maximum activity (ca. 2.8 U/mL), suggesting high endoglucanase activity. All four enzymes are similarly effective on the complex natural substrate, wheat bran demonstrating extremely similar and high activity (ca 2.2 U/mL). Exoglucanases were typically linked to the capacity to break down crystalline cellulose, while endoglucanases from fungi and Bacillus species that possessed both Avicelase (cellulase) and filter paper (FPase) hydrolytic capacities in addition to their CMCase activity, have demonstrated the ability to accomplish this through the assistance of an exo-activity that resides within the same molecule ( Li et al. 1998; Sharma et al. 2016 ). Soluble substrates, particularly celluloses with a low degree of polymerization (DP), could be dissolved in water because of their chemical substitutions. Thus, ionically substituted CMC was typically used to measure the activity of endoglucanase, also referred to as CMCase. The DP of CMC is significantly reduced as a result of endoglucanases' random cleavage of intramolecular β-1,4-glucosidic bonds and previous report had shown that a portion of the C-terminus of the full endoglucanase gene product is essential for the interaction between this enzyme and the insoluble substrate ( Zhang et al. 2006 ). It was predicted that the FPase activity ranged from 1.45 (Z7) to 2.13 (Z17) U/mL and the enzyme activity on powdered cellulose ranged from 1.86 (S11) to 2.03 (Z17) U/mL. These findings allow for the classification of this enzyme as both an endo- and exo-type of cellulase, as it was previously described ( Sharma et al. 2016 ). The impacts of several stressors on cellulase production on solid medium were investigated at pH5, 50°C, and 800 mM NaCl. The results of the enzymatic index indicated that strain Z1 genetically identified as Bacillus siamensis had an optimal cellulase production of 2.73 at 50°C by providing a control, which is comparable with, Bacillus subtilis subsp. inaquosorum isolate CGR-1 that exhibited amylolytic and cellulolytic activities at 50°C ( Geraldi et al. 2022 ) Withal, Hassan et al. ( 2020 ), have proven that abiotic stressors, especially high temperatures, stimulate the production of high-value products such as antioxidants and increase enzymatic activity. The effect of pH and the presence of 800 mM NaCl was remarkable, with enzymatic indexes ranging from 0.3 to 2 for the selected four strains belonged to Bacillus species. The combination of the three factors—pH 5, T 50°C, and 800 mM NaCl—was ideal for cellulase production for strain Z1, which had an Ei of 3, while the other strains produced cellulase with an Ei varied between 0.8and1.7. Those outcomes were correlated with prior report that substantiated notable multi-stress resistance (pH 9, T50°C, and 400Mm NaCl) of the cellulase on solid media ( Balla et al., 2022 ). Using both macro and microscopic morphological standard tests, the four selected cellulolytic bacterial strains were most likely belong to the Bacillus genera, which is characterized as a rod-shaped, Gram-positive bacterium, and sporulated strains which is confirmed by Alcaraz et al. ( 2010 ), whose reported that in reaction to environmental or nutritional stressors, bacillus species generated extremely resilient dormant endospores and is facultatively aerobic or anaerobic. According to a previous study suggesting that 16S rRNA sequencing is an accurate method for species identification and distinction between closely related bacterial species (Hussein et al. 2017). So that, Comparison of 16S rRNA gene sequences revealed that strain Z1 showed the highest similarity of 99. 72% with B. siamensis (PX112786.1) which was firstly isolated from salted crab (poo-khem) in Thailand in 2010 ( Sumpavapol et al. 2010 ). The Bacillus siamensis YC-9 strain, originating from termite nests, synthesizes various enzymes of industrial interest, including cellulases, reaching an optimal production of 1.54 IU/g when cultivated on wheat bran pretreated in an alkaline medium (Wang et al. 2021 ). Rathod et al. ( 2025 ) used a microbial co-culture of Bacillus siamensis F2 (as a bacterium that produces multiple enzymes such as cellulase) and Candida albicans GP1 to produce bioethanol from fruit waste. Strain Z7 was identified through 16S rRNA gene sequencing, and the sequence obtained was identified according to the GENBANK (NCBI) under accession number MG470713.1, showing high homology (99.65%) with different strains of Bacillus sp. (in: Firmicutes). Lugani et al. ( 2015 ) optimized cellulase production from a newly isolated strain of Bacillus sp. Y3 , achieving a maximum FPase activity of 6.84 IU/mL and a CMCase activity of 7.82 IU/mL. The strain Z17 showed the highest similarity of 99.65% with B. cabrialesii (ON287153.1) newly isolated and identified for the first time by Santos et al. (2019) . Then, Bacillus cabrialesii , has been isolated from wheat plants in the Yaqui Valley, Mexico. This bacterium is known for its plant growth-promoting properties and as a biological control agent (Figueroa et al. 2023). Furthermore, Masmoudi et al. ( 2025 ), studied the ability of the new thermo-halotolerant bacterium Bacillus cabrialesii , native to the Qatar desert, to produce cell wall-degrading enzymes, such as cellulase. In addition, bacterial strain S11 showed the closest sequence similarity to B. inaquosorum , (MN581182.1) with percentage of 99. 73%. However, research conducted by Srivastava et al. ( 2024 ) indicated that B. inaquosorum KCTC 13429 could be developed as a high-yielding commercial EG endoglucanase strain for the degradation of cellulose-rich biomass. In previous studies, B. subtilis and Bacillus subtilis subsp. inaquosorum have been reported to produce cellulase enzymes, including B. subtilis K-18 ( Irfan et al. 2017 ), B. subtilis CD001 (Malik et al. 2021) , and B. subtilis subsp. inaquosorum CGR-1 ( Geraldi et al. 2022 ). Indeed, a lot of extracellular enzymes can be produced and secreted by Bacillus species ( Kazemi et al., 2014 ). They are regarded as one of the most adaptable and prevalent microbial fermentations because they typically showed rapid growth rates and brief fermentation times ( Sadhu et al. 2011 ; Kazemi et al. 2014 ; Manfredi et al. 2016 ). The current work successfully gathered novel, stress-tolerant thermophilic Bacillus strains with high-activity, and stable cellulase enzymes. Eventual research into maximizing the synthesis of extracellular enzymes and assessing the commercial and biotechnological worth of these bacterial strains and their enzymes should be conducted. 5 Conclusions The aim of the current study was to isolate and identify thermophilic bacteria from Algerian hot springs. According to the findings of the bacterial hydrolytic activity screening, most of the isolates exhibited strong cellulasic activity, confirming the presence of promising extracellular hydrolytic enzymes like cellulase. After valuating different stress parameters, four among the 45 bacterial strains tested and assessed for their cellulase activity were picked for further analysis and identified to be belonging to Bacillus group: Z1 as B. siamensis , Z7 as Bacillus sp . (in firmicutes), Z17 as B. cabrialesii and S11 as B. inaquosorum . The crude enzyme was active over a broad pH range of 4 to 11, with residual activities decreasing from 100% to 60%, demonstrating the impact of physicochemical parameters on cellulase. Their higher activity was mentioned at 50°C and 70°C and their stability at 50°C and 80°C. Their residual activity remained between 40 and 60% across a wide range of salt concentrations from 0 to 2000 mM and weakly affected by PEG 6000 . The cellulase-rich crude enzyme extract, which can be described as a blend (combination) of endo and exo-cellulase types, effectively degraded both soluble and insoluble substrates. Furthermore, this particular bacterial genus ( Bacillus ) exhibited intriguing traits of resilience to wide stress-conditions on solid media. To conclude, this current work lays the groundwork for future investigations into the optimization of extracellular enzyme production, and further analysis should be conducted in order to determine the value of those bacterial strains and their enzymes in industrial and biotechnological scales. Abbreviations BLAST Basic Local Alignment Search Tool CMC Carboxymethyl cellulose DNA Deoxyribonucleic acid DNS 3,5 dinitrosalicylic acid DP Degree of polymerization Ei Enzymatic index FDA Food and Drug Administration GRAS Generally Regarded as Safe LB Luria broth medium LCB Lignocellulosic biomass NA Nutrient agar NaCl Sodium chloride NCBI National Center for Biotechnology Information OD Optical density PEG6000 Polyethylene glycol 6000 16S rRNA 16S ribosomal ribonucleic acid TSA Tryptic soy agar Declarations Supplementary Materials See Appendix 1 Conflicts of Interest: The authors declare no conflict of interest. Funding: This research received no external funding. Author Contribution Conceptualization, L.A., and S.S; Formal analysis, H.C.S.; Investigation, S.S., J.M.; Writing-original draft, S.S., J.M.; Writing-review & editing, J.M., M.L, H.C.S.; Supervision, J.M & L.A. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8574179","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":581072545,"identity":"6b9ef3cf-7ee9-494d-9207-75e32bc2a27f","order_by":0,"name":"Souad SOUALAH","email":"","orcid":"","institution":"University of Monastir","correspondingAuthor":false,"prefix":"","firstName":"Souad","middleName":"","lastName":"SOUALAH","suffix":""},{"id":581072546,"identity":"ebc5dd9c-1c92-467a-905c-871c2fd69673","order_by":1,"name":"Jihen MISSAOUI","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYDACZgjFw9jMA6QqgJidgYGxgUGOWC1ngCxmsBZjYuwDamFsI0KLbjv74w8/Kg7LMLfzHnxcOM8mcT8z88GHMxgM8nFpMTvMYybZc+Yw0GF8ycYzt6Ul9jCzJRtuYDCwbMCthY2Btw2khcdMmnfbYaAWoCEPGP4Y4LaF/fHHvxAt5r9558C1GODRwmAgDbOFmbcBqmUDXi1A98icSQdpMZbmOZZm3HMY6JcZBni0nD/++OObCmt7w/4zhp95amxk29ubDz7sqcCtBQ4MG1C4hDUwMMgToWYUjIJRMApGKAAAVaZL9BLGMsQAAAAASUVORK5CYII=","orcid":"","institution":"University of Monastir","correspondingAuthor":true,"prefix":"","firstName":"Jihen","middleName":"","lastName":"MISSAOUI","suffix":""},{"id":581072547,"identity":"8278076a-c418-44f2-8c43-02c4ccb3ce56","order_by":2,"name":"Mohamed LATAOUI","email":"","orcid":"","institution":"University of Monastir","correspondingAuthor":false,"prefix":"","firstName":"Mohamed","middleName":"","lastName":"LATAOUI","suffix":""},{"id":581072548,"identity":"4b39b005-8c51-4000-a1ac-622793675475","order_by":3,"name":"Hafsa CHERIF SILLINI","email":"","orcid":"","institution":"Ferhat Abbas University","correspondingAuthor":false,"prefix":"","firstName":"Hafsa","middleName":"CHERIF","lastName":"SILLINI","suffix":""},{"id":581072549,"identity":"7916d3d7-8d72-42fe-99e4-c026659d22b3","order_by":4,"name":"Lotfi ACHOUR","email":"","orcid":"","institution":"University of Monastir","correspondingAuthor":false,"prefix":"","firstName":"Lotfi","middleName":"","lastName":"ACHOUR","suffix":""}],"badges":[],"createdAt":"2026-01-11 14:38:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8574179/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8574179/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101419582,"identity":"f1530369-f3ad-40a2-8d47-9c0f4ebf02f9","added_by":"auto","created_at":"2026-01-29 13:20:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":669880,"visible":true,"origin":"","legend":"\u003cp\u003eScreening of enzyme production by different bacterial strains isolated from Algerian hot springs “S” from Hammam Oulad Tebben and “Z” from Zdim thermal spring respectively for cellulase production on specific solid medium at 30°C for 5 days in aerobic conditions.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8574179/v1/1ed98b0e19d3225e9532c751.png"},{"id":101419590,"identity":"1b420d52-72e3-41bd-b2df-948c76657247","added_by":"auto","created_at":"2026-01-29 13:20:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":613080,"visible":true,"origin":"","legend":"\u003cp\u003eEnzymatic index of isolated bacterial strains “S” from Hammam Oulad Tebben \u003cstrong\u003e(A) \u003c/strong\u003eand “Z” from Zdim thermal spring \u003cstrong\u003e(B)\u003c/strong\u003e after the screening of cellulose production. Results are expressed as mean-standard error of triplicate Enzyme Index values. Protease production corresponding to each treatment and not sharing the same letters are significantly different according to Tukey's HSD post-hoc test.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8574179/v1/9bd0e79962d4850cc4da0318.png"},{"id":101751198,"identity":"d1704771-7329-4efd-9efe-f0457e60d20a","added_by":"auto","created_at":"2026-02-03 10:18:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":159545,"visible":true,"origin":"","legend":"\u003cp\u003eCellulase activity on CMC broth 1% of the 21 strains tested. Results are expressed as mean-standard error of OD values in triplicate. Cellulase activities corresponding to each treatment and not sharing the same letters are significantly different according to Tukey's HSD post-hoc test.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8574179/v1/e2313ec58253cafa4006d1ed.png"},{"id":101419584,"identity":"2fd48a48-e13f-4e6d-949b-1ad1aeb6c2b5","added_by":"auto","created_at":"2026-01-29 13:20:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1088670,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of physicochemical stress parameters on bacterial growth (OD 600) on 1% CMC broth of the selected 8 strains. \u003cstrong\u003e(A) \u003c/strong\u003eeffect of various pH,\u003cstrong\u003e (B) \u003c/strong\u003eeffect of various temperatures\u003cstrong\u003e(C) \u003c/strong\u003eeffect of salinity using different concentration of NaCl and\u003cstrong\u003e (D)\u003c/strong\u003eeffect of water activity using various concentration of PEG6000.\u003cstrong\u003e \u003c/strong\u003eThe results are expressed as the mean-standard error of OD values in triplicate. The effects of the different stress parameters corresponding to each treatment and not sharing the same letters are significantly different according to Tukey's HSD post hoc test.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8574179/v1/1cb4f452bc40d056b2533933.png"},{"id":101419591,"identity":"d0f0e9fb-0ec6-4555-b0a0-664ba811ff32","added_by":"auto","created_at":"2026-01-29 13:20:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":746048,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of physicochemical stress parameters on cellulase production on 1% CMC broth of the selected 8 strains. \u003cstrong\u003e(A) \u003c/strong\u003eeffect of various pH,\u003cstrong\u003e (B) \u003c/strong\u003eeffect of various temperatures\u003cstrong\u003e(C) \u003c/strong\u003eeffect of salinity using different concentration of NaCl and\u003cstrong\u003e (D)\u003c/strong\u003eeffect of water activity using various concentration of PEG6000.\u003cstrong\u003e \u003c/strong\u003eThe results are expressed as the mean-standard error of OD values in triplicate. The effects of the different stress parameters corresponding to each treatment and not sharing the same letters are significantly different according to Tukey's HSD post hoc test.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8574179/v1/c017f25f13faf5c3499ecc82.png"},{"id":101751185,"identity":"a2af06e0-9a99-4483-8cde-857820f6e3af","added_by":"auto","created_at":"2026-02-03 10:17:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":803624,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of physicochemical stress parameters on cellulase activity (U/ml) of the selected 4 strains. \u003cstrong\u003e(A) \u003c/strong\u003eeffect of various pH,\u003cstrong\u003e (B) \u003c/strong\u003eeffect of various temperatures\u003cstrong\u003e(C) \u003c/strong\u003eeffect of salinity using different concentration of NaCl and\u003cstrong\u003e (D)\u003c/strong\u003eeffect of water activity using various concentration of PEG600.\u003cstrong\u003e \u003c/strong\u003eThe results are expressed as the mean-standard error of OD values in triplicate. The effects of the different stress parameters corresponding to each treatment and not sharing the same letters are significantly different according to Tukey's HSD post hoc test.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8574179/v1/8f10bd6680c2b818a6bbb3b7.png"},{"id":101419587,"identity":"4b7394a0-5c56-4957-acd9-70620a04a6b9","added_by":"auto","created_at":"2026-01-29 13:20:27","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":806479,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of physicochemical stress parameters on cellulase Stability (U/ml) of the selected 4 strains. \u003cstrong\u003e(A) \u003c/strong\u003eeffect of various pH,\u003cstrong\u003e (B) \u003c/strong\u003eeffect of various temperatures\u003cstrong\u003e(C) \u003c/strong\u003eeffect of salinity using different concentration of NaCl and\u003cstrong\u003e (D)\u003c/strong\u003eeffect of water activity using various concentration of PEG600.\u003cstrong\u003e \u003c/strong\u003eThe results are expressed as the mean-standard error of OD values in triplicate. The effects of the different stress parameters corresponding to each treatment and not sharing the same letters are significantly different according to Tukey's HSD post hoc test.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8574179/v1/6d6822ba35a039a1b1a77240.png"},{"id":101751431,"identity":"76285072-bef4-4b7b-9d0e-94bbded76863","added_by":"auto","created_at":"2026-02-03 10:20:12","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1370786,"visible":true,"origin":"","legend":"\u003cp\u003eCellulase activity (U/ml) on different substrates of the 4 strains tested. The results are expressed as the mean and standard error of enzyme unit values in triplicate. The cellulase activities on different substrates corresponding to each treatment and not sharing the same letters are significantly different according to Tukey’s post hoc HSD test.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8574179/v1/cc6d7d2bcc9984329834cb22.png"},{"id":101751532,"identity":"90fbdad1-a01b-4317-a332-a636a2a3e7f9","added_by":"auto","created_at":"2026-02-03 10:21:07","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":210998,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of multiple stress on the production of cellulose in solid medium. The results are expressed as theme a standard error of the values of the enzymatic indexes in triplicate. The effects of the stresses corresponding to each treatment and not sharing the same letters are significantly different according to Tukey's post hoc HSD test.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8574179/v1/f3098e47adc8ad1b40240827.png"},{"id":101419592,"identity":"60061da5-c4d9-45a5-ae47-a8ba2f7427f4","added_by":"auto","created_at":"2026-01-29 13:20:27","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":218415,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree (Maximum Likelihood method) obtained by combined standard amplified polymorphic DNA-polymerase chain reaction and 16S rRNA gene sequencing patterns of the isolates from Algerian thermal springs. Branch supports were assessed by bootstrap resampling of the dataset with 1000 replications and the branches represented the Operational Taxonomic Units (OTUs), which are different strains or species of the bacterial genus \u003cem\u003eBacillus\u003c/em\u003e, with different Bootstraps value ranged between 0 and 100 indicating the confidence in the grouping of the clade.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-8574179/v1/e88b04139f91f5e50bc998f1.png"},{"id":107396221,"identity":"97246129-c82a-49f1-9d49-ec0674d988d2","added_by":"auto","created_at":"2026-04-21 06:40:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6379432,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8574179/v1/c52ea616-e712-4474-98a4-3a402ee78fec.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Thermophilic cellulase: screening of novel cellulolytic bacteria from Algerian hot springs for CMCase production","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eGeothermal springs are thought to be particular hotspots for significant of thermophilic microbial communities, primarily from the bacterial and archaeal domains \u003cb\u003e(\u003c/b\u003eSahay et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Few studies have been done on the microbial diversity of Algerian hydrothermal springs, despite the fact that terrestrial thermal springs are the subject of extensive research worldwide. Despite the recent emergence of research on thermophilic bacteria, it still limited to specific aspects and few numbers of hot springs \u003cb\u003e(\u003c/b\u003eGomri et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Arab et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Benammar et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Thermophilic microorganisms associated with these ecosystems have garnered a lot of attention lately, despite the fact that these resources are typically used for therapeutic and curative purposes \u003cb\u003e(\u003c/b\u003eThebti et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Bouacem et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Bouacem et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Due to their specific benefit for the synthesis of thermostable enzymes alike protease, amylase, xylanase, cellulose and many others, these enzymes maintain their function at temperatures significantly higher than those optimal conditions for the proliferation of the microorganisms \u003cb\u003e(\u003c/b\u003eSaboto et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Bouacem et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Lignocellulosic biomass (LCB) is the most renewable and underappreciated biomass sources that are abundant on our planet, cellulose, hemicelluloses, and lignin are the primary components of LCB, the proportion of which varies from 35 to 50%, 25 to 30%, and 25 to 30%, respectively, depending on the biomass source \u003cb\u003e(\u003c/b\u003eAl-Battashi et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; \u003cb\u003eMalik et al. 2021)\u003c/b\u003e. However, a significant obstacle to the conversion and exploitation of cellulosic biomass has been the persistent nature of lignocellulosic biomass. Otherwise, using solvents, acids and/or bases to chemically treat lignocelluloses has several drawbacks, including high operating pressure, high costs, low glucose yield, and the creation of hazardous by-products like furfural \u003cb\u003e(\u003c/b\u003eMaurya et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; \u003cb\u003eYousef \u0026amp; Mawad. 2023).\u003c/b\u003e Therefore, there are three methods for pre-treating lignocellulose: physical, chemical, and biological \u003cb\u003e(\u003c/b\u003eBalla et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHemicellulose and lignin are strongly bound by cellulose (C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e), \u003cb\u003e(\u003c/b\u003eSethi et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Abada et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The glucose units are joined by β-1,4 bonds to form a polymer chain, and the photosynthetic process produces cellulose waste, as a vast renewable bioresource \u003cb\u003e(\u003c/b\u003eHussain et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Cellulolytic and hemi-cellulolytic enzymes are necessary for the biological conversion of lignocellulosic waste into industrial product \u003cb\u003e(\u003c/b\u003eJourdier et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Thus, the industry has shown a great deal of interest in the intriguing pre-treatment of lignocellulosic materials using microorganisms or microbial enzymes \u003cb\u003e(\u003c/b\u003eBalla et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, enzymatic (biological) hydrolysis on the other hand, is a profitable process that effectively removes lignocellulose and is environmentally friendly due to its high specificity and selectivity as well as its mild operating conditions \u003cb\u003e(\u003c/b\u003eYousef et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Several studies have reported that cellulose degradation involves a group of enzymes β-glucosidases (EC 3.2.1.21), endoglucanases (EC 3.2.1.4), and exoglucanases (EC 3.2.1.91) called cellulase, which could breakdown β-1,4-glycosidic bonds synergistically while releasing glucose molecules \u003cb\u003e(\u003c/b\u003eJayasekara \u0026amp; Ratnayake \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. According to the global enzyme market reports, cellulase is the most claimed enzyme, thus covering about 20% of the global market products \u003cb\u003e(\u003c/b\u003eSingh et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Otherwise, for effective breakdown of cellulosic biomass, the synergistic action of all three forms of cellulases is fundamental \u003cb\u003e(\u003c/b\u003eSharma et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Furthermore, cellulase is increasingly used in variety of industries, including the food industry (fruit juice production and aroma and flavour valorisation), the paper industry (bio-bleaching and ink removal), biotechnology (bioethanol production and lignocellulosic material valorisation), and the textile industry (bio-washing, bio-polishing, bio-stonewashing, and denim bleaching\u003cb\u003e) (\u003c/b\u003eSingh et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Detergents and other cleaning and laundry products are also made with cellulase \u003cb\u003e(\u003c/b\u003eSingh et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Balla et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These enzymes are produced by a huge variety of microorganisms, such as bacteria, fungi, and actinomycetes. Several publications have classified the various categories of microorganisms that contribute to cellulose degradation \u003cb\u003e(\u003c/b\u003eSharma et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Singh et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Balla et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Masmoudi et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Moreover, due to their diversity, the presence of multi-enzyme complexes that provide synergy their high growth rate, and stability under extreme conditions there is an increasing demand for bacteria as a source of novel cellulases as effective catalysts \u003cb\u003e(\u003c/b\u003eBalasubramanian \u0026amp; Sim\u0026otilde;es \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Manfredi et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Among the bacterial genera that have been found to exhibit cellulolytic activity: \u003cem\u003eBacillus, Clostridium, Cellulomonas, Rumminococcus, Alteromonas, Acetivibrio, etc\u0026hellip;\u003c/em\u003e \u003cb\u003e(\u003c/b\u003eRawat \u0026amp; Tewari \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Balla et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Masmoudi et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), but the genus most studied and cited remains \u003cem\u003eBacillus\u003c/em\u003e \u003cb\u003e(Sadhu \u0026amp; Maiti. 2013;\u003c/b\u003e Balla et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Masmoudi et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Furthermore, \u003cem\u003eBacillus spp\u003c/em\u003e. are thought to be among the most interesting bacteria in industrial biotechnology, especially when it comes to cellulase production. They are typically distinguished by their ease and speed of culture, low nutritional requirement and their high enzyme production \u003cb\u003e(\u003c/b\u003eBalla et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). There is growing interest in the production of cellulase by these organisms, which are isolated from various environments, such as \u003cem\u003eBacillus sp.\u003c/em\u003e \u003cb\u003e(\u003c/b\u003eAcharya \u0026amp; Chaudhary \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), \u003cem\u003eBacillus siamensis\u003c/em\u003e YC-9 \u003cb\u003e(\u003c/b\u003eWang et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), \u003cem\u003eBacillus subtilis\u003c/em\u003e subsp. \u003cem\u003einaquosorum\u003c/em\u003e \u003cb\u003e(\u003c/b\u003eRegmi et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and \u003cem\u003eBacillus cabrialesii\u003c/em\u003e, which is reported as cellulase producer that degraded the cell walls \u003cb\u003e(\u003c/b\u003eMasmoudi et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Due to its versatile metabolism and extremely effective protein secretion system, \u003cem\u003eB. subtilis\u003c/em\u003e has been extensively employed as a cellular factory for microbial enzyme synthesis \u003cb\u003e(\u003c/b\u003eSu et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; \u003cb\u003eMalik et al. 2021)\u003c/b\u003e. Recently, numerous reports have been realized to identify and separate microorganisms that degrade cellulose in diverse environments, which are of crucial importance for the development of novel cellulases with distinctive properties including the identification of different strains of extremophile \u003cem\u003eBacillus sp.\u003c/em\u003e: halophiles, thermophiles, psychrophiles, acidophiles and alkalophiles, xerophiles, and their genes involved in the production of cellulases (Sharma et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Chen \u0026amp; Jiang \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Balla et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Indeed, various factors, such as pH, temperature, aeration, incubation time, inoculum size and carbon source could affect and influence bacterial growth and enzyme production \u003cb\u003e(\u003c/b\u003eHussain et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis present study, aimed to investigate the efficient hydrolytic activities and the generation of hydrolase enzymes (cellulase) of \u003cem\u003eBacillus\u003c/em\u003e strains isolated for the first time from various thermal networks: Hammam Oulad Tebben and the thermal spring Zdim in Setif, Algeria. Studies were conducted on the selected cellulolytic bacteria with the goal of assessing their cellulase activity and optimizing their production under the effect of certain physico-chemical factors. However, it was necessary to evaluate the enzyme's stability in the presence of numerous physicochemical variables as well as its capacity to break-down specific cellulosic substrates, in order to involve them in the future as industrial and biotechnological innovative tools.\u003c/p\u003e"},{"header":"2 Materials \u0026 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.1 Isolation of bacterial strains\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThermal water samples were collected from two distinct locations in Hammam Oulad Tebban (geographic coordinates 35\u0026deg;48'33.1\"N and 5\u0026deg;06'27.1\"E) and Zdim thermal spring (Geographic coordinates 36.035210 \"N, 5.244283 \"E) south of Setif-Algeria. Depending on the volume of water analysed, strains were isolated using the membrane filtration method on solid culture media Tryptic soy agar (TSA) (Liofilchem, TE, Italy) \u003cb\u003e(\u003c/b\u003eArya et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). A 0.45 \u0026micro;m cellulose acetate membrane (THOMAPOR, RCT, Germany) was used to filter sterilizing volumes of 1 ml, 10 ml, 20 ml, and 30 ml of each sample. Thereafter, this membrane was put on the top of TSA medium (Liofilchem, TE, Italy) and it was incubated at 30 \u0026deg; C and 45 \u0026deg; C for 24 h to 48 h. Colonies of various appearance were removed from each Petri dish, and purified by successive subcultures on nutrient agar (NA) (Liofilchem, TE, Italy). The pure strains were kept (stored) at 4\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Screening of strains exhibiting extracellular hydrolytic activities and Cellulase production\u003c/h2\u003e \u003cp\u003eAll bacterial strains were inoculated in Lauria broth medium (LB) (Accumix, Malaga, Spain) for 24 h at 30\u0026deg;C and their hydrolytic activities were conducted out by spot inoculation of 2\u0026micro;L of the bacterial culture on various culture media according to the specific substrate for each enzyme. The enzymatic index (Ei) was determined for all the enzymes according to \u003cb\u003e(\u003c/b\u003eFerbiyanto et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) using the following formula:\u003c/p\u003e \u003cp\u003eEi = (Halo diameter - colony diameter) / colony diameter\u003c/p\u003e \u003cp\u003eIn order to demonstrate the cellulosic activity of the isolates, the bacterial culture was spot inoculated onto a slightly modified carboxymethylcellulose based medium (CMC 1%; Sigma-Aldrich, St. Louis, USA) and incubated at 30 \u0026deg;-37\u0026deg; C for 5 to 6 days \u003cb\u003e(\u003c/b\u003eSahay et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This activity is observed after flooding the dishes with Red Congo (C.I. 22120 France) in 1% solution for 15 min then decolorized with a NaCl solution (1M) \u003cb\u003e(\u003c/b\u003eTeather \u0026amp; Wood \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e1982\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Characterization of cellulase activity\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Enzyme activity test\u003c/h2\u003e \u003cp\u003eThe selected bacterial strains (21 strains) were tested for their capacity to produce the enzyme in a liquid medium. Firstly, 100\u0026micro;L of each bacterial suspension cultured in LB broth, was inoculated into tubes containing 10 mL of CMC broth (1%) with a small adjustment, then incubated at 30\u0026deg;C for 72 h with shaking. After incubation, the cultures were centrifuged at 5000 rpm/10 min/4\u0026deg;C. The supernatants containing the crude enzyme were gathered and kept at 4\u003csup\u003e◦\u003c/sup\u003eC for further analysis \u003cb\u003e(\u003c/b\u003eBalla et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Enzyme dosage\u003c/h2\u003e \u003cp\u003eThe 3,5-dinitrosalicylic acid (DNS) method was used to measure the amount of reducing sugar released by CMC in order to determine CMCase activity \u003cb\u003e(\u003c/b\u003eMiller \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1959\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. 1 mL of the enzyme solution supernatant was mixed with 0.5 mL of acetate buffer (50 mM, pH 4) containing CMC (1%). After incubation for 30 min at 50\u003csup\u003e◦\u003c/sup\u003eC, the reaction was stopped by adding 1 mL of DNS reagent (SUVCHEM, MH, India) and the mixture was boiled for 5 min and cooled to stabilize the colour. The absorbance was measured at 540 nm \u003cb\u003e(\u003c/b\u003eBalla et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; \u003cb\u003eMalik \u0026amp; al. 2022).\u003c/b\u003e CMCase activity was measured using a glucose standard curve as a guide. All samples were examined in triplicate, and one unit (U) of CMCase activity was defined as the quantity of enzyme needed to release one micromole of reducing sugars (measured as glucose) per minute \u003cb\u003e(\u003c/b\u003eHo et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Hussain et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Balla et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The corresponding control (blanks) of the enzyme-free substrate were performed concurrently with all enzyme assays.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Impact of physicochemical factors on bacterial growth and cellulase production\u003c/h2\u003e \u003cp\u003eEight bacterial strains were chosen based on the results to assess their ideal bacterial growth and cellulase activity under various physico-chimical factors. Cellulase production was estimated by measuring the enzymatic activity using the DNS method previously mentioned, and bacterial growth was assessed by measuring the absorbance at 600 nm using a UV-visible spectrophotomer (JENWAY-7205, France).\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 pH value\u003c/h2\u003e \u003cp\u003eTo determine the ideal and optimal pH for bacterial growth and cellulase production capacity in 1% CMC liquid medium, three pH values ​​were tested: 5.0, 7.0 and 9.0. The cultures were incubated at 30\u0026deg;C for 72 h \u003cb\u003e(\u003c/b\u003eBalla et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) using the pHmeter (ADWA-Ad1000, Szeged - Hungary).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Temperature\u003c/h2\u003e \u003cp\u003eTo assess the impact of ttemperature on the growth of the selected isolates and enzyme production, the cultures were incubated in 1% CMC medium at temperatures of 30\u003csup\u003e◦\u003c/sup\u003eC, 45\u003csup\u003e◦\u003c/sup\u003eC and 50\u003csup\u003e◦\u003c/sup\u003eC for 72 hours \u003cb\u003e(\u003c/b\u003eBalla et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3 Salinity and water stress\u003c/h2\u003e \u003cp\u003eValuation of the impact of the salinity on bacterial growth and cellulase production was carried out using 1% CMC medium with increasing NaCl concentrations of 0, 400, 800 and 1000 mM, then the cultures were incubated at a temperature of 30\u0026deg;C/72 hours. For the water stress impact, polyethylene glycol (PEG6000) was assessed at different concentrations from 0 and 30%. As previously, cultures were incubated at 30\u0026deg;C for 72 hours \u003cb\u003e(\u003c/b\u003eBalla et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Influence of physicochemical parameters on cellulase activity and stability of cellulases\u003c/h2\u003e \u003cp\u003eFour bacterial strains were selected to investigate their enzymatic activity and stability under various physicochemical stress parameters. The crude enzyme extract was produced after 72 hours of growth under optimal circumstances.\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1 Influence of pH on cellulase activity and stability\u003c/h2\u003e \u003cp\u003eA range of buffers with pH values ​​from 3 to 11 were used to assess the impact of pH variation on CMCase activity and stability; acetate buffers (pH 3), citrate buffers (pH 4\u0026ndash;6), phosphate buffers (pH 7\u0026ndash;11) and glycine buffers (pH 9\u0026ndash;10), and the enzyme solutions were incubated with the appropriate pH buffer (3\u0026ndash;11) for one hour at room temperature. Then, DNS standard method was performed to measure the residual activity \u003cb\u003e(\u003c/b\u003eBalla et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2 Influence of temperature on cellulase activity and stability\u003c/h2\u003e \u003cp\u003eThe thermal stability and activity of the enzyme were determined at respective temperatures (45\u0026deg;, 50\u0026deg;, 70\u0026deg; and 80\u0026deg;) with pre-incubation of the enzyme in the same acetate buffer (pH 4) without the substrate for 1 hour. The standard DNS method was then used to quantify each sample's residual activity for CMC hydrolysis under the enzyme assay's optimal conditions \u003cb\u003e(\u003c/b\u003eShankar \u0026amp; Isaiarasu \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Balla et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.5.3 Influence of salinity on cellulase activity and stability\u003c/h2\u003e \u003cp\u003eThe reaction salinity that maximized CMCase activity with 1% (w/v) CMC was measured in 50mM acetate buffer (pH4) containing various concentrations of NaCl (Supelco, Denmark) (0mM, 500mM, 1000mM,1500mM, 2000mM). To determine the salinity stability, the enzyme was incubated with 50mM acetate buffer (pH4) without 1% CMC with the same salinity concentrations for one hour at room temperature. The residual CMCase activity was tested using the DNS standard method, after addition of 1% CMC \u003cb\u003e(\u003c/b\u003eWang et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Balla et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.5.4 Influence of water stress on Cellulase Activity and Stability\u003c/h2\u003e \u003cp\u003eAccording to the standard assay conditions, CMCase activity was measured using a buffer substrate at two concentrations of polyethylene glycol \u003cb\u003e(\u003c/b\u003eSPECILAB, Tlemcen, Algeria) (PEG6000) at 0 and 30%. By pre-incubating the enzyme (without substrate) in 50mM acetate buffer pH4 containing varying concentrations of PEG6000 for 30 minutes at 50\u0026deg;C, the impact of water stress on enzyme stability was as certain. The standard DNS method was used to measure residual activity \u003cb\u003e(\u003c/b\u003eManfredi et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Balla et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Hydrolysis power of different substrates\u003c/h2\u003e \u003cp\u003eThe activity of crude cellulase was measured against different substrates. According to Balla et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), the mixtures of 2 mL of each bacterial supernatant with 1 mL of acetate buffer pH 4 containing 1% of each of the following substrates: cellulose powder (Sigma-Aldrich, St. Louis, USA) and wheat bran were incubated at 50\u0026deg;C for 30 min. Pieces of filter paper Whatmann No. 3 (GE, HealthCare Life Sciences, Cleveland, UK), of size (1\u0026times;5 cm) equivalent to 50 mg were added to 1 mL of buffer supplemented with 2 mL of supernatant and were incubated at 50\u0026deg;C for 60 min. Using glucose (Fisher Sientific, UK) as a reference, the DNS method was used to determine the reducing sugars \u003cb\u003e(\u003c/b\u003eMiller \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1959\u003c/span\u003e; Balla et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.7 Combined effects of physico-chemic factors on microbial growth and cellulase production in solid media\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe impact of various parameters on cellulase activity on solid medium of four selected strains was studied using 1% CMC medium adjusted to pH 5, with a NaCl concentration of 800 mM, incubated at 50\u0026deg;C. The bacterial culture was inoculated onto CMC agar in a 2\u0026micro;L spot, then Red Congo solution (1%) was used to reveal the CMC hydrolysis zone appeared around the colony for 15 min before being decoloured by NaCl solution (1M). The enzymatic index was calculated as previously described. The enzymatic indexes for the single stress factor and the combination of the three factors types were compared with a control \u003cb\u003e(\u003c/b\u003eBalla et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Characterization of the four selected bacterial strains\u003c/h2\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e2.8.1 Phenotypic characterization\u003c/h2\u003e \u003cp\u003eMacroscopic observation of colonies was reported after 24 hours on TSA culture medium, in order to characterize the main components: Colony shape, colony size, colony color, elevation, opacity and surface \u003cb\u003e(\u003c/b\u003eGuiraud, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2012\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e Moreover, the purification of cellulase-producing strains is carried out by successive subculturing. To characterize microscopically these isolates, Gram staining \u003cb\u003e(Gram 1884)\u003c/b\u003e was performed to identify their morphology and to distinguish between Gram (+) and Gram (-) bacteria \u003cb\u003e(\u003c/b\u003eBhagat \u0026amp; Kokitkar \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e2.8.2 Molecular Biotyping and Bacterial Identification\u003c/h2\u003e \u003cp\u003ePhylogenetic analysis was used to identify the four bacterial isolates were performed in collaboration with Macrogen (Amsterdam, Netherlands) according to the following steps: \u003cb\u003ei\u003c/b\u003e DNA extraction was performed using the salting out procedure by a standard extraction protocol. Selected isolates were grown overnight on TSA agar. A culture loop was re-suspended in 100 \u0026micro;l of sterile distilled water and vortexed well, then incubated at 95\u0026deg;C for 20 min, rigorously vortexed, and incubated once time in ice for 10 min. finally the DNA medusa was recuperated by centrifugation for 10 min at 10,000 rpm at 4\u0026deg;C (Hussain et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). \u003cb\u003eii\u003c/b\u003e the purified DNA was amplified by standard PCR using universal primers 27F' (5' AGAGTTTGATCMTGGCTCAG-3') and 1492 R (5'GGTTACCTTGTTACGACTT-3') following standard instructions. Then, \u003cb\u003eiii\u003c/b\u003e the bacterial identification was obtained upon partial sequencing of 16S rRNA gene, using the primers primers 907R (5'-CCGTCAATTCMTTTRAGTTT-3') and 785F (5'-GGATTAGATACCCTGGTA-3') based on Sanger sequencing protocol, in order to discriminate among species belonging to the \u003cem\u003eBacillus\u003c/em\u003e group. The DNA sequence homology was determined using bioinformatics tools through pair-wise sequence alignments, using BLAST within the NCBI nucleotide collection database. The evolutionary analysis was inferred by using the Maximum Likelihood method and Kimura 2-parameter model \u003cb\u003e(\u003c/b\u003eKimura \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1980\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e and the evolutionary analyses of the final dataset were conducted in MEGA X (Kumar et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were performed in triplicate, with results expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Unifactorial ANOVA and multifactorial ANOVA was used to identify variance among different treatments. Results were deemed significant when p -values ​​were less than 0.05. When a significant difference was detected, post-hoc comparison tests of the Tukey test were conducted. Different letters are included at the top of each column to indicate differences between the different groups. All statistics are performed using Origine Lab Pro 2022 developed by Origine lab Corporation. MEGA X was used for the molecular evolutionary genetics analyses (sequence alignment, phylogenetic tree) of the final dataset.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e3.1 Screenings of extracellular hydrolytic activities: cellulose production\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eAs known that cellulose could be broken down by the complicated enzyme \u0026ldquo;cellulase\u0026rdquo;, which could be produced by bacteria, and its importance to be used in many different industries, including as food processing, textiles, and biofuel production. In this present research, forty-five strains isolated and purified on solid culture medium (TSA) from both Algerian hot springs (Hammam Oulad Tebben and Zdim thermal spring, Algeria) were characterized for their hydrolytic activity at 30\u0026ndash;37\u0026deg;C. For this reason, the enzymatic productive capacity of cellulase was carried out and using carboxymethylcellulose (CMC) as specific substrate. The results obtained are displayed in the figures and graphs according to the values ​​of the Enzyme Index (Ei). According to the analysis of cellulase activity mentioned in \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e, among the 45 selected strains just 34 strains were competent for cellulase producing, which means that 75,55% (n\u0026thinsp;=\u0026thinsp;34/45) of all the strains under investigation. They expressed good cellulase activity with enzymatic indexes varied from 0.2 to 2.6 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u003cb\u003e(A\u0026amp;B))\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Characterization of cellulase activity\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e displayed the cellulase activity (in U/ml) of 21 various bacterial strains with the highest enzymatic indexes and cellulase production which was assessed in liquid medium. The enzymatic activity across all tested strains was ranged from 1.03 to 2.18 U/mL for about 15 isolates from both hot springs (S4, S8, S10, S11, S12, S13, S16, S17, S20**, S22, S24*, S25, Z1, Z4, Z7, Z9*, Z9**, Z12, and Z17). However, the highest cellulase activity was obtained by Z1, Z17 and S11 with exhibition of approx. 2.15 U/ml, the other isolates there is either very low enzymatic activity or no activity was shown (S22). According these results, 8 strains were selected as the most promising candidates for cellulase production based on their high and significant activity. These strains would be the focus for further analysis, such as optimization of enzyme production or genetic characterization.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Effect of physicochemical factors on bacterial growth and cellulase production\u003c/h2\u003e \u003cp\u003eAnalysis of the impact of various parameters on bacterial growth and cellulase production was carried out for 8 isolates from both hot springs (S8, S11, S24*, S25, Z1, Z7, Z9* \u0026amp; Z17) selected according to their enzymatic performances in liquid medium.\u003c/p\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1 Effect of various pH value on bacterial growth and on cellulase production\u003c/h2\u003e \u003cp\u003eThe findings of this experiment presented in \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-A\u003cb\u003e)\u003c/b\u003e reported the pH dependence of bacterial growth for eight distinct bacterial strains as determined by Optical Density at 600 nm (DO 600 nm) at three distinct pH levels (pH 5, pH 7, and pH 10). The data unequivocally demonstrates that nearly all strains develop best at pH 7. Thus, strain Z17 is the most resilient strain, showing the highest growth at ideal pH as well as the highest relative tolerance to acidic (pH 5) and alkaline (pH 10) environments. With strain Z17 exhibiting the widest resistance to pH extremes, this suggests that the majority of strains are neutrophilic (pH\u0026thinsp;=\u0026thinsp;7.0), however the bacterial growth is moderate at acidic medium pH (=\u0026thinsp;5.0), but it is lower at alkaline culture medium pH (=\u0026thinsp;10.0). Otherwise, the cellulase activity (U/mL) of the selected eight strains was examined also at different pH values (pH 5, pH 7, and pH 10) is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e-A. The data clearly indicated that the pH value significantly impacted the enzymatic activity for all tested strains: Four isolates (S8, S24*, Z1, and Z17) produced significant CMCase at pH 5, while the lowest CMCase activity was recorded at pH 10. The optimal pH for maximum cellulase activity was reached at pH 7, with important CMCase activity ranging from 1.4 U/mL (S25) to 2.35 U/mL (Z1). The findings implied that the ideal pH range for the cellulase production under measurement is ranged between pH 5 and pH 7 with detection of the the highest enzymatic activity for the strains: S11 and Z1 with ca. 2.2 U/mL and 2.0 U/mL at the acidic pH, moreover, Z1 and Z17 exhibited approx. 2.4 U/mL and 2.1 U/mL respectively at neutral pH. Then, the sharp decreased in activity at pH 10 strongly implies that the high alkalinity denaturized or significantly inhibited the active site of the enzyme, inhibiting efficient catalysis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e-A\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2 Effect of variable temperature value on bacterial growth and on cellulase production\u003c/h2\u003e \u003cp\u003eTemperature profiles were determined by assessment of the bacterial growth and cellulase production at three different temperatures: 30\u0026deg;, 45\u0026deg; and 50\u0026deg;C. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-B showed the selective effect of heat treatment. In contrast to the ideal temperature of 45\u0026deg;C, the majority of isolates showed a notable reduction in bacterial growth at 30\u0026deg;C and 50\u0026deg;C. But strain S8, which was isolated from Hammam Ouled Tebben's thermal waters, showed that 50\u003csup\u003e◦\u003c/sup\u003eC was the ideal growth temperature. Indeed, the highest overall growth (highest OD values) for most strains is observed at 45\u0026deg;C, with Z1, Z7 and Z17strains that revealed the greatest growth. However, the lowest growth is generally seen at 30\u0026deg;C and 50\u0026deg;C, suggesting 45\u0026deg;C is the optimum temperature for growth for the majority of these strains. The physiology and enzymatic activity of microorganisms are significantly influenced by temperature. In order to investigate the impact of temperature on cellulase production, the tested isolates were incubated for 72 hours at various temperatures ranging from 30\u0026deg;C to 50\u0026deg;C. The majority of the tested isolates were found to be able to produce cellulase, with the highest cellulase activity occurring at temperatures as high as 45\u0026deg;C and the enzymatic activities varied from approximately 1.35 U/mL to almost 3.0 U/mL. The activities at 30\u0026deg;, and 50\u0026deg;C are typically lower indicating that 45\u0026deg;C is the ideal temperature for this enzyme's function and stability across all tested strains \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e-B\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e \u003ch2\u003e3.3.3 Effect of salinity on bacterial growth and cellulase production\u003c/h2\u003e \u003cp\u003eThe Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-C illustrated the salinity impact of the bacterial growth (DO 600 nm) of the eight tested strains across four different sodium chloride (NaCl) concentrations: 0 mM, 400 mM, 800 mM, and 1000 mM. The majority of bacteria were found to be able to grow at salinity levels between 400 and 1000 mM. In contrast to their contributions at 400 and 1000 mM, isolates S24*, S25, Z1, Z7, and Z17 were better shown to be able to proliferate at 800 mM NaCl concentration, and among them strains Z1 and Z17 remained the best performer maintaining their salt tolerance and high relative growth. After 72 hours of incubation at 30\u0026deg;C. Among the concentrations tested, all isolates tested were able to synthesize cellulase at high salinity levels of 400, 800 and 1000 mM NaCl \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e-C\u003cb\u003e)\u003c/b\u003e. Furthermore, the findings reported that almost all the strains were halotolerant and have to be the best performer of enzyme production/use in high-salt environments, despite not being the fastest grower and this is strain-dependant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section3\"\u003e \u003ch2\u003e3.3.4 Effect of water activity on bacterial growth and cellulase production\u003c/h2\u003e \u003cp\u003ePEG\u003csub\u003e6000\u003c/sub\u003e is frequently used in microbial research to create osmotic stress or water potential stress, simulating drought-like conditions. The histograms Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-D illustrated the impact of water activity on the bacterial growth of the eight isolates, after 72 hours of incubation at 30\u0026deg;C. This was done by adding increasing concentrations of PEG\u003csub\u003e6000\u003c/sub\u003e to the CMC liquid medium at different concentration. A trade-off is evident in the graph: under ideal circumstances, strain Z17 was a high-yield strain, but it is extremely susceptible to osmotic stress caused by PEG\u003csub\u003e6000\u003c/sub\u003e. The strains S8 and Z7 exhibited the maximum tolerance to PEG\u003csub\u003e6000\u003c/sub\u003e at 30%, suggesting that they are better suited to low water availability. Nevertheless, the impact of osmotic stress caused by PEG\u003csub\u003e6000\u003c/sub\u003e on cellulase production was determined by measuring enzyme activity. Cellulase production was significantly decreased at 30% PEG6000 for most isolates tested except S11, which produced the enzyme abundantly \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e-D\u003cb\u003e)\u003c/b\u003e. In terms of growth and enzymatic activity, strains S8 and Z7 consistently exhibit the highest tolerance to PEG\u003csub\u003e6000\u003c/sub\u003e-induced osmotic stress, which makes them ideal for use in settings with limited water availability. Despite being the best overall performance under control conditions, strain Z17 is significantly reduced in both growth and enzyme activity at PEG\u003csub\u003e6000\u003c/sub\u003e (30%).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Physicochemical characterization of cellulases: activity and stress resistance\u003c/h2\u003e \u003cp\u003eWithstand the combined effects of pH, temperature, salinity, and PEG\u003csub\u003e6000\u003c/sub\u003e, four strains Z1, Z7, Z17, and S11 were selected for the investigation of their enzymatic activity and stability under stress conditions.\u003c/p\u003e \u003cdiv id=\"Sec32\" class=\"Section3\"\u003e \u003ch2\u003e3.4.1 Influence of pH on the activity and stability of cellulases\u003c/h2\u003e \u003cp\u003eThe effect of pH on the stability and activity of cellulase was examined at pH values ranging from 3.0 to 11. The pH range in which this enzyme functioned was 4.0 to 11.0, however its optimal pic was recorded between pH 6.0 and pH 10.0 and it is substantially lower at pH 3.0 for all tested bacteria. Furthermore, depending on the strains tested or the same parameter, the residual activity remained largely unchanged. There seems to be an ideal pH range where the activity of is at its peak. Due to pH-induced modifications in the three-dimensional structure of the enzyme (denaturation), extreme pH values (both extremely acidic and highly alkaline) typically result in a decrease in enzyme activity, a frequent feature of enzyme function.\u003c/p\u003e \u003cp\u003eThe enzymatic activity is often higher in Graph A \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e-A\u003cb\u003e)\u003c/b\u003e for all samples. Optimal pH: All samples appear to maintain or reach their maximum activity in the alkaline range (pH 8.0 to pH 11.0). With values around 1.2 U/mL, samples Z1 and Z17 exhibit the highest activities, peaking between pH 10.0 and pH 11.0. Samples Z7 and S11 have lower activity than Z1 and Z17, but in the pH 7.0 to pH 11.0 range, their activity is also fairly stable or slightly increases. According to cellulase stability, strain Z1's activity was completely maintained at pH 4.0, whereas strain S11's activity was maintained between pH 4.0 and 11. However, strains Z1, Z7, and Z17 demonstrated remarkably stable enzymatic activity at over 60% in the same pH range \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e-A\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003e3.4.2 Influence of temperature on the activity and stability of cellulases\u003c/h2\u003e \u003cp\u003eTemperature effects on enzyme stability and activity were also assessed at a range of temperatures, from 45 to 80\u0026deg;C. Both graphs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e-B \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e-B\u003cb\u003e)\u003c/b\u003e, a clear trend showed that increasing the temperature from 45\u0026deg;C to 80\u0026deg;C generally leads to a decrease in the enzymatic activity for most of the samples, especially when moving from the optimum temperature to 80\u0026deg;C. At all temperatures examined, CMCase activity levels were extremely high, peaking for all strains at 50 ◦C and 70 ◦C. The stability, as measured by residual enzyme activity, was at its best at 50\u0026deg;C and 70\u0026deg;C after 1 hour of pre-incubation at various temperatures. For all strains, the enzyme exhibited above 40% residual activity up to 80\u0026deg;C. This is typical for enzymes, as excessive heat causes denaturation, reducing their efficiency. The experiments unequivocally demonstrated that, the common optimal temperature for the highest activity of the enzyme sample Z1 is 50\u0026deg; C. For other samples, such Z17, the stability and ideal temperature, with higher activity at 70\u0026deg;C and 80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003e3.4.3 Influence of salinity on the activity and stability of cellulases\u003c/h2\u003e \u003cp\u003eIn both graphs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e-C \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e-C\u003cb\u003e)\u003c/b\u003e, the cellulase typically showed halotolerance or haloactivity, which means that they continue to function even at extremely high NaCl concentrations (up to 2000mM). This implies that the enzymes either originate from or function in highly salinized settings. In details, the experiments \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e-C\u003cb\u003e)\u003c/b\u003e highlighted that the tested enzyme from all strains (Z1, Z7, Z17, and S11) are halotolerant because they continue to exhibit notable enzymatic activity at 2000mM. Z1 exhibited the highest activity overall reaching approx. 2.6 U/mL) at normal condition (0 mM of NaCl). Its activity decreased slightly but remains high even for high concentration of NaCl up to 2000mM, demonstrating good salt tolerance for this strain. Z7 consistently showed the lowest activity across all NaCl concentrations in this condition, maintaining stable activity around 1.8 U/mL. However, both isolates Z17 and S11 maintained relatively stable activity, presenting a small initial drop from 0 mM to 500 mM, but then sustaining activity up to 2000mM. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e-C demonstrated a lower overall enzymatic activity range. Despite, for especially the strains Z1 and Z17, the enzymatic activity was almost stable and remained maximal at 500 mM and/or 2000 mM NaCl achieving the highest activity at approx. 1.4 U/mL. Generally, the four bacterial isolates maintained a high degree of activity up to the highest tested concentration, 2000 mM, further confirming their ability, their haloactivity and their halostability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec35\" class=\"Section3\"\u003e \u003ch2\u003e3.4.4 Influence of water stress on the activity and stability of cellulases\u003c/h2\u003e \u003cp\u003eThe effect of PEG\u003csub\u003e6000\u003c/sub\u003e concentration on the enzymatic activity (U/mL) on the tested cellulase from four strains (Z1, Z7, Z17, and S11) at two concentrations (0% and 30%) under two different conditions, was demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e-D \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e-D. The findings on Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e-D reported that under 0% of PEG\u003csub\u003e6000\u003c/sub\u003e condition, Z1 had the highest activity reaching 2.4 U/mL followed by Z7, Z17, and S11, that all showed high activity (around 2.0 U/mL). However, the addition of PEG\u003csub\u003e6000\u003c/sub\u003e at 30% caused a significant drop in activity (1.6 U/mL) for all samples except for Z1 that maintained the highest activity (approx. 2.3 U/mL), showing high stability/tolerance to PEG\u003csub\u003e6000\u003c/sub\u003e. PEG\u003csub\u003e6000\u003c/sub\u003e at a 30% concentration proved ineffective in analyzing the effects of the medium's absence of water on the enzyme's initial and residual activity. However, regardless of whether PEG\u003csub\u003e6000\u003c/sub\u003e was present or not, the enzymatic stability was maintained and varied by 40% based on the isolates.\u003c/p\u003e \u003cp\u003eThe results mentioned in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e-D demonstrated a lower overall enzymatic activity range, reaching an optimum about 1.15 U/Ml when enzyme stability was assessed. Although, Z1 showed also the highest activity (ca. 1.15 U/mL) with PEG\u003csub\u003e6000\u003c/sub\u003e (0%). The Z7, Z17, and S11 exhibited very similar activities (around 0.9 U/mL). Using PEG\u003csub\u003e6000\u003c/sub\u003e 30%, the obtained results were less drastic than, but still showing low enzymatic activity reaching 1.05 U/mL for Z1, and demonstrated significant decrease in activity for the other strains.\u003c/p\u003e \u003cp\u003eThis experiment revealed that Z1 maintained the highest enzymatic activity while being the most robust, resilient and tolerant to the addition of PEG\u003csub\u003e6000\u003c/sub\u003e 30%. The high concentration of PEG\u003csub\u003e6000\u003c/sub\u003e often affected the enzymatic activity and stability as strain-dependent manner.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec36\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Hydrolysis power of different substrates\u003c/h2\u003e \u003cp\u003eThe graph \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e illustrated the enzymatic activity (U/mL) of crude enzyme from four strains (Z1, Z7, Z17, and S11), when acting upon four different cellulose substrates: CMC, powdered cellulose, wheat bran, and filter paper in order to ascertain the cellulose substrate's specificity. The outcomes of this experiment strongly confirmed that according to the substantial substrate-dependent variations in enzyme activity that the tested enzyme was probably cellulase with varying specificities toward beta-glucan linkages and cellulose's physical structures. Indeed, Z1 was the best enzyme producer for breaking down soluble cellulose (CMC) with a significant and maximum activity (ca. 2.8 U/mL), suggesting high endoglucanase activity, Z17 was considered as the best enzyme for breaking down crystalline cellulose (powdered cellulose and filter paper), suggesting strong and significant exoglucanase activity reaching ca. 2.4 U/mL and ca. 2.1 U/ mL respectively. All four enzymes are equally effective on the complex natural substrate, Wheat Bran showing very similar and high activity (ca. 2.2 U/mL).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec37\" class=\"Section2\"\u003e \u003ch2\u003e3.6 \u003cb\u003eCombined effects of parameters physico-chemical on microbial growth and cellulase production in solid media\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe graph \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e demonstrated the enzymatic index for the four enzyme samples (Z1, Z7, Z17, and S11) under various single and combined stress factors: T 50\u0026deg;C (Temperature), pH 5, 800 mM NaCl. The findings reported that cellulase production in solid media was significantly impacted by both single and multiple stress parameters (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Indeed, taken strain by strain; Z1 was notably robust under the combined stress of high temperature (50\u0026deg;C), acidic pH (=\u0026thinsp;5.0), and high salinity (800 Mm NaCl), highlighting the grater activity/stability under these extreme conditions, Z7 was the most active in the control condition and most stable under single pH and NaCl stress. Although, Z17 and S11 generally performed better under control and single stress factors compared to the combined stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec38\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Characterization of the four selected bacterial strains\u003c/h2\u003e \u003cdiv id=\"Sec39\" class=\"Section3\"\u003e \u003ch2\u003e3.7.4 Phenotypic characterization\u003c/h2\u003e \u003cp\u003eThe selected four strains (Z1, Z7, Z17, and S11) from both hot springs Hammam Oulad Tebban and the Zdim thermal spring were characterized macroscopically. The resulting colonies are typically spherical, white, and beige in color, with a diameter of 2 to 5 mm. They typically had a rough, and viscous aspect. The majority of strains have regular or uneven borders and are flat. Following Gram staining, microscopic analysis exhibited that all four strains were discernible as Gram\u0026thinsp;+\u0026thinsp;sporulated rods. Thus, the four tested strains most likely belong to the genus \u003cem\u003eBacillus\u003c/em\u003e relying on the Gram\u0026thinsp;+\u0026thinsp;\u003cem\u003eBacillus\u003c/em\u003e criteria and the presence of spores.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec40\" class=\"Section3\"\u003e \u003ch2\u003e3.7.5 Molecular identification of selected isolates\u003c/h2\u003e \u003cp\u003eThe phylogenetic tree based on the comparison of the 16S rRNA sequences of the strains studied with similar sequences of strains available in databases (GenBank) confirmed that all strains belonged to the \u003cem\u003eBacillus\u003c/em\u003e genus \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e The isolates (Z1, Z7, Z17, S11) from both hot springs Hammam Oulad Tebban and the Zdim thermal spring showed different standard PCR profiles (data not shown). Therefore, all the isolates were subjected to molecular identification and gene alignment based on bioinformatics tools and databases on GenBank (Blast, NCBI). The phylogenetic tree built upon the combination of the profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e) showed that all the identified strains were belonging to Bacillus genus however strains were allotted to: \u003cb\u003eZ1\u003c/b\u003e as \u003cem\u003eBacillus siamensis\u003c/em\u003e (PX112786.1), \u003cb\u003eZ7\u003c/b\u003e as \u003cem\u003eBacillus sp\u003c/em\u003e. (in: firmicutes) (MG470713.1) and strains \u003cb\u003eZ17\u003c/b\u003e and \u003cb\u003eS11\u003c/b\u003e, showed the highest sequence similarity \u003cem\u003eBacillus cabrialesii (\u003c/em\u003eON287153.1) and \u003cem\u003eBacillus inaquosorum\u003c/em\u003e (MN581182.1) respectively \u003cb\u003e(Table\u0026nbsp;1)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eTable\u0026nbsp;1\u003c/strong\u003e \u003cp\u003e16S rRNA gene sequencing-based identification of the four selected strains\u003c/p\u003e \u003c/p\u003e \u003cp\u003e\u003cimg 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CX3jK/RDh5HhnhnQZBx66vNGZjLcGS5kFLn9X9kPRgseJIfJBLRBBphbC4ROS8raEdcWuYknv5zlyQNzjefy8TQXxxYaonfRH+eXvjNQtIN+5FG3ttDfu7IW9IL29RHP0w30DLlDhpFb+sjYRrv6wXh3/8osTx68NkNjQM/woD60mdGNU4K8+WI+MPYYhRY39A9eou/0R/o7Mg9cZlDXDGGAUU1msXHoKPTFG46OezfZKHSZkxucv/QZHrCzRV5yYLJlrt1ao8uks5vQOFphjKMT8pT9wwa14GO4a4/9iIfYeegE7+ER/UBH5KujrY5RimdHkKN0Ifqy2KAL0TceJyvJe05kdhH9RoZq36KB3GWTyM/GYHf2sW7RkLDfmA4YlJWTVTnFiMlNhFW/bQ71MfbUOb6n/D0LKSKKjJeDgCIYeKMhzZacyafIGP7y93LqBIxB8YSmyaDcCAnCEmIRCAHC00ZZQroXNZXdEkBI8ozylCIoDAB/PKo8bvpkjEJEZkzGAE+IFkFa9SJqAhuRwRevroULQc7IQ0ViFQAAEABJREFUYwgjXIKTQOZZZbCot+MMruDWahgDMMgIUnWaS8/wyttnxQp/DJdPZiXKZjBcGJgCgHcR4zTPoMcJ1WmMwLPxOYuKedRjXMoAhqa5xHgYSFsYD13puzzmXwjcq1fIWIE/bUizwMHw7mcF9O8j2RmLOsqPYDAfPLy8DoQbDy1Boe92I3i5CC8LOx54Xg1zTpmac3XCUx83XMCpMJsaXlTvNOAZoCv0ow1KVBsMTe13XlbeHJsLdQP1mh9zwZhhHFusmyP0zUBm5KIbRiqe5JnjtbMToKyFo/L6sVJgHPBuUY03GELGyaiBd8aI8aElfXIvNF54QFvo1j18UzTGLATyKitd6HkapKmHzLMQUh/HiPlG30J5lBeaU3nsIDCMenzvmzI9zr0yjDPGP2XIMDLH5IdFFpky3ad6XhwGzBujmOK3iCZn0TePM88fPQP/+EyNnW7c9zlCJ2Q8hw1j3DtDvIAcG/iZTJefkW2Brj5lxQH36kfLeFK6e7yED6XLNw3yWGxYxPI8MkgYMfQQvqdDlFG30FjJbLKZ8aNuQEcwsvEwPBg7r7q6GHA8s7zoaNY4GT/sBQsDvNLrR7/Gx8tJ3jPI7NZyUtD/6mAP0ONkhoU4OUVW6l/BbGAAnXJksBeA9zPIHwtVi74+33QN49qcc8ihDUYzHWiRhv7oQPoevbEL8RgbSRzbxs4q3UR2k9loDv+Re+wdediUjsqgb22wvdiRFgFoi1zEg8p0DOINY9Bn8pGcJN/RMFqkb40Db+E35aWjYUDPoc1e3xYNdXKxHTApmJmHkUDiMbZqwnQYE+NBlEEuVKfJIQghhNCATKspyof3EYHIs746xnVDMOhxxsPIQQhW9SacUeu4grSeb6VCOKNQESmvGWPCAghR75Wd0K8MFryMjaLACIS2McAbQmXUE6rGi2h7ujLwiyDlna5cHfIjUmlWtbzw5hLBI2hbuBSAvvO82lbq+ZVB1Lwt5sqcqU98BwRuwaAv5lteaZ71jbBWVj3ipAH3jkYw9ikQnh7MbsvaogytyLdYmKajxZZbznwWaYQPvPOm8ljz9sETnuKdM5dwaq7wCKHHCEQ/PA0MP4rQ+OBsU/qLprShvHnQBs+IxR3vobYXqle70vt2ZM9nTglFxgAa0Edp6JByf3E+GCNnADrIxxW77CxQMniRdxLebZ/qh4UJwW1cYGM7BYeLLcdIZ5jwAGkfjuBtvjYpR/iUzoBbbBsMRTiWn7FJYanL83ztVNz6MUD2MbbJYrhlfNp9YqjiAbqLjNoQfulOvIY/zKm5JXvNj50ycevvycKp62vbYpUTgIHO80lvcDBxBmjXgh4/j2tH0x0sTvAzWc/IEU+G0/30EMcEZx+ZzwZQjzxkFz1kzNP9U6eFgbx4kBFnd5qMs9Nk94vBR+ZbNDDqORzUq0zBlseARRw7xkLRopRzAh3R2+gKveglfcaxY+6AODqB0Y1G6CJ5xEsXR15ZBIpDc9LxIT3JwYjm8KF8+JDDg8EvjgMVbTnSwxZU1kKW85K8RW/qBdrTT3nwJXoV30GaMXX6lR/09B7fn7doqKOL7QDvIEFAMChDMRIOVj+Q4CwShEvrA54eLGUmrzzARDIgGDa2qR0vgFRpmwoEBYMXkVkx8tIiNCupTa1zU8shMMKeIaEORg3Ct1WJEHnOxHc8CRGzeYFLAp6gI4gJUHj1xRrCjXHEAKcUGPWYg+C2A6BOoB7gHihvDvSDgcCoxAxWtnBFiBLIto0tPGybMh4dzzEWdQCMxrC0MMEgaILAlwYIdt4c94Sz+rTt2RjnE/DSzB1DhzfXERHeHQLe1jUvqjT5VjOYc9uPFnvGwbtg+xGu4UY6HsEPBI85sVNinmw18oTwiBOIcA6vyqlrDOLHz+hqnI9xAZ/aYGCiAYtfPKjt6fLjuqShI31HX+Yan/U84tCjRYJ2GUF9DI4WoLtxX3q55QwZXIxbdKxtfXfMyCKGHCK/9Htj+6AusJhy8lFsZB4Z4B0jish8z1cej+K1+dJ6nLlQL+hxypATFuc8Y4xR8nqcp+etcEEMzCWQxZxZjpagEfPC0LSrZoufoQ8UMB9CuAbuO5iXsX6zyCTHHWlhzJKTPe9Shmhb3xlg+oQWeEgdiTEe8cYoTbtCII6MMV40ip6UJXscnaC39d8OEh3FwMf3ZIHdZ3WrT6gu9wCO1MnRox3ygMOLHCTjHG/Ck3gUr9gZYX/QQx3P6inYchgwP3b0Gdd0h3kj04R6hV94vd2jCTYKXWDnmYEuHtD5dBH7hvEsXXwHdONIozrwDzsF3bE9+3FIthRD3uISHfLSs7nYJvrEwKeD8IBdJHX2+tGfvrGjtM92QZ8MfHnYX3SkOM8zDRhtsR0kiEwgpPUXcJSnIE0GD6o8AAJMOA+g1RpkmExIGhvWymJSSGS4YFagT+qA7B4qP473rC1Gr3jPhATGd86P0WOS9c2EM0DkW0kg+ODMtiovDDwgJuNGKI4A6I94oW0k+JIOJ44mMb4JSkaXfLaOCGDEapuKwGX8Oo/Gq23xwrBX3jypFw57CEeYkHD2/gKFr16ecEZmxx/G4Km3Ip7Gn/rMt22xXg+jXj0MFh5Pz5QdD785UMYcoRmrZ3n1SZx5dq9v0hwzUa+xMzr1Rf+MT76ev5dVtzjP6KHPtfh+r9wsgD7a8fDyKUWrT+aQEqTYbBcScLzkFj7miPcY3synOYITec2VBYC5Rg/qAnDRce4e38Gf0DO8UMyUsgWcc4KOdJkTxp9+ySOv+uAQr3kGxiDOOVu06eiK87DGgXbRuvZ5iZWXpu/G4GgB+nUvbaVAf3mTbNlagGiXAId3Cw87euMxG6Oxoid5+3PP09PgD3jWhhAo41moLNzCIfmGP8wdniYj+9z1uuVXhzT8oA7Q46W5V5/+aV9esoPclRf9kC94ihxhkIkv2HgMwDXaxrMMTPymFnOJ3hkL+NG8wDcZz2ih68ytOZLGOdLnR3k7tICxQh+oS7z25Af9Hi2hDyCP0PwL5euhtGmQbrfaAsQiQrp+oEFtOyqnfjQK8L++oydgbIwqvC4vOewIoP6SKZxIcEInoDv6xztAaBfe6ALt6aO29QeeHM3TPh1pN5hxZ+HNEFTG+3l0j8UHR4b7Xod6CrYMBshuBrsdE4a1eaQLHCPjuLEQo0/sYuEDtO0dH/NH99FneIQuQIPm1vtJRsPoRqPoylEsdMO+YR+yjzhI8RS6VB4PikM3nF70J74A4hn3dsH0D63pAxpCg9rDXxwr7Cf0agdd/UJ61kJbHqCMUN3q6OUn9562MJiYxXYBgnklMCyvqVUzz6mz1rxcvMWEAuanSBwrYFhTmhgekzL2bJWYGO1S+oQhZcQo4PElMAgHkyW/83TSPWN6AtBkYnjHRIBJZYg4q0u5ISKEweDUH1svjCRtriQQZl6ish1JGcCZ40rG4Q1reBr3B2FQGLaJGNJ2P6we4clKE3HZysJExsfoNS6C1lh55byHIN2RAsQHF+ZCX3wpxwtdVra8/LaLMaBzlIQzA1ofELR5dUaSt9augv6M+8rY1I4z0xhWn3mZeRwZTo418LDzwmBeAgDTadOcdRpg/InXT1u/4ikLzOiMp61hi0heAF9gcJYOw6MreDQmCwDKkgDQHx4v9Tm+QpFpf9z3LXlPYVJ8DCxfNkEPvFNo2rYgw5IBgJ/wFcFijoydwsMjhFtfKJk7X0ExRgKnjw3dEWIMfjtr+E/bPBcWceryAhKvGN6hNLVvEWoxbLFoXvAgWvDZLooeXh2vI4gJQfNjceDYkDoIeXUAghQNaVs7Frb6xzggK9yvFBivXSV9dX7d2HmTyB2GDqPXeJ3vN2Y0RSnBHdyiOTRJQVgEwKNnfEa+oUvzwDOJprVnMcYjZPxwz8hBt+aPPMUT+MrLZIwkz+LRtUUPGUdukIfqM0/ymVO0jb/7i476gXc6PskMYyRfzZO8Pa3CjcOAuaPEvfiHT9F051symOwjg+EYjYkjw8wnWjJX5o18824XWawHdpLNP3oy3/hVO2jIM7ohr8ljx1odPaHj0BA9QD56T4IhYtGMLtQ7Dfpv8cjQwsfogqeTHMALXsYdl2FE2c2io8h2uoIcZ4TbxUNznADqwAMMJXrIWOCCo4AxRxdwQKFdfSRT8JIxcTCQg+QGmqe/yDILCTxKlsmLh8gNdZN5DLJxX+t+ZTFgTtCiueKYIDvRpV6Ql05G0BNoiC5mt3hGC/jG/LIN7A6hcXoQ7aAxdGlxx06iV/CO+WbDOS7D8HYMCx3gLfYKfmHzkY1oiDxnl9jtQnccYBaG+AbtsmHJVTyHJ42BjeV9PjqAntI/9ZK9ZDOeJ8eNyVjxEP5Dt+wLuwrwAgdbFDbGoKeEnPk0kQZsgpzrdTQDoigQg3Euj2GA4RnSVl8QxRNPgDDOTKC8tjnUSRgxGp2lhyyKkTEAqQw5BERhWalRwox6EyEPIpHuGIN2rKoQC6GjXwSO1R8Boc2VBAIUYeoPo0zfGT2I0lEURvG4P/J705/xxohioFMS+m7scI2QHEmxGHDeXDojnNed8IR3xMk7SpnDsYUB3FtgwQ/8w6s4gtuPQTkzj7kIcHMEt/oKf75cop1xX92bH0rInGEm44Nz/UAnjE99V9a5eIY2g0//0Ik6jMEixByiF8wLB4w+zNwB81CIhANFo++MLYsl9IhhKVG7AoSM+uxWUJra19YsgHmkoOweMeThzBair93YujaX+mkevGxunOiF9wN9myN4Rz/4hgJFC3YxKG5lAaMcHzK+0Z424cRii8K1aLBYE4+mKFbzzPijoC3czRe86gdeVxfeU588+orPfL0A/zFy8Jo5NO/6YNHCK4PP0TM601d9XEmwiGW8oD00Cu+EN/o3BjiFD4YInKMxhoXQohEt8TLBA5wwzs2XRTZ5he/MCf7C88rArfnGd4w6PIKu4ZoiQO/kqnkyN+qy0NEfCwfza5FMTqBh8b1tCz5ziI8ZPXjcYlA+CwD8wshEY2hpJXG9tbWFzskk80k2mjf0455MZizDu3HjU7SBn/EQYwG/mEs6C6+aY3nxg3hyjHHrOBo6YQjRm3aO8CDacDwMrXD4oE994RlFr9L1pctUdY+BjCQnyBx90Xchx4qxMNR7fuNAN2gRneJ5+gF/yMNQMjb0TsYbIxluAY/n0aB6lfNlK3Kfc8EiB50bH6PPAsERCl5Vcl2d2iF/5PXSuvYtNtC4MuSfNvSjYMtgAH0wrMlSco8dSM/rjXmzyKMPzLN5ZVyzN/ABu4X+N9f0ND1EF5hTtI0+yE27qOwXPEHGoT12hnrpeDKNPMcf9CH9bxFgwY0/HANDW+ItKLTFkYz+2RPqQp9kqXv8yNDHy2iu62Pl8L5xka/4zIJcO5yV+uqdMDxhDHCwRWFjDHoKnIIzQBNqsJge440H457SIrggCQGYcMYC77stEZPYBy4vIwCDq1teeUwwRM4OsoAAABAASURBVBMGygGGgmeKlTC0ehJKQxzKECwEmz6arC2JbMRGwRsH4xq+KGJ9EtdxIGSMUcIEIqUgD3xI64Ap4JEgJ0Sn0xkkcKG8c4jSGQPmQTy8qUN98EQRMUrUyfAXj3ngjxIgTNVjTqXNB9IYbgQ3gYwujHWsJOQxv/oAMGNfAJp/ccB82q614sV8FBdgRFqhW5ETDnAkv/GgCUaiZ94xQkB8f5au/fn6viXi8BF8mX+LMEaa+SSc8Mq4T+YCTswnnHnu6e7xgvmj/Hu8sQLP2oEH7chjjuAGbRGU8hB80s01OlEWjOcLfeBndQGKlWBUHo3hO3yoDvwrvoN+ym+MlL/8PW0lQzSNduAdvXRatYju/eAFhB9jREcEuXF5BowNY5FHPP4xB+6BetWhLJwBeDdu93CIH8lRc6Ftc86AIvPILPjB29oA5gSvyiddnL7gS3nNnz6RoWgLv3COUFY8tuod82Ifa4WLxwDcWxShHbg0l/iW0YHHxjXJQ/6ZI/OMz8yheaOj8J55U0bYaYpOlBft4Ef0qm50pS5ygC4h4zzrA5nQn9FYl6mtNdXPAT2ELvG1vqF9egid9UzS3NND7i1EyR2hfkrroG/oVx3qk7+nuSfT0bp0/CFOH/RRv9Fq10PwxYYgx4y1yxV0jV/JRbJjjLfeVoUrjwFzSV51eibz2R16gl7NIzo3z0L5Ol3iHbwgD7lErinXAQ+gK3QnH37oaUIOX3XiO7ygL2gTneA15ZRHO/KT3+wadIdn0KD89JVnfUN7+Ft+dKsOdqO2erw68GQvoz5ljVHILul51bPFYGMM+i3Wya28YUdQbFUx5m3324mwxbqVD3vB4WE6zOmIlV0Knl8eZIah3ZixElqwkjWUYFuSl882vW0/9+hoDaGghjrCAHpwBIgniUKk1EbJdVsYmBcDjtCRG2wC9MO76ojBvJkrsjBQGJg9DGDeTepVFVoyDDBgbfHYpvUlFwYrRbxkDayying0fWfW+xm8V4Dn0zYxzxLDfpUNaVm7y8PFm2Gb3ja484U8aMvaaFU+sxjgYXI8yJEKHlxesZntbHVsZjDAC+kIjaMI5C/PJW/mzHSwOlIYKAysHwNl0K8fPyuRyqC3dWObx3EV2znTW1Er0Y9ZacPWle1tW2e2tZ3lt63M0F/LC52F5gdObMfb3kZDtkBtiS6Uf0vEV5srhwHbzQx5W9pky8q1XC2tZgw47uAoBBlCDzn6Q7as5jFV3wsDawoDZdCvqemuwRYGCgOFgcJAYWCWMVB9KwwUBjYFA2XQbwrWqkxhoDBQGCgMFAYKA4WBwkBhYEYwsCYN+hnBfXWjMFAYKAwUBgoDhYHCQGGgMLDZGCiDfrNRWBUUBgoDWzEGamiFgcJAYaAwUBiYeQyUQT/zU1QdLAxsEAN+edLnT4HPn/qWdOdt99J9gs79BiurDHMYgDc49RlZv1btF6n94qXQr3X6JcK5zBt5Y35qPjYSaZW9MDDbGFjTvfPZZLKSfCQv6ZwxQnxSlzyVfuZMIAMzWPKLDtQ+OMOS1z7DFS4XQmd4yNW1wsBWhYHz52j8EulhGfpVVr8s6XvSl8tnBiehJt0v5RGkGV3XIjBAOe2S+fy6oU9A+nVZP27mB8/84usTM23vhHMmbOxF4Zgfc7OxZSt/YaAwUBiYRQz4LYMHZ8eOSCAv/TqxH7fLx+Hyi+HkqV899gurnCNDwhL+o/P8Sq3frHlR1usH9sjyvN36rzLoV9kcV3cLAyMMMAgZmVfJuJcmPCvhSgmvTCDU8Pf58t4PdN0hwwsk1LU4DPgGt99AeHNm3zHB97m/nyFF4ROh18j7Zycw7M+T4WIvXvmrZua9EnisMqirMFAYKAysegz4EbtX5yj8Vgz59tC8f0TC2RNcflVVuh+O9Ou/fvtA/FICua3+S2alt0m4TAI9mMHWf62ZgW79U1kjXIMYuHWOefcE3xz3E+t+0pqByeD0s9iZ1Ai31+TNaxP8RDVvha3QDo6V8BiLzyzDRS6Il8f9ELkG/1E4jHjfc/cDXhSW30Pw400vS3zwBlFcPPkM9YwaLvFwB4fuh8j8B5cXzXD/BIsEz+MtYXUoA6RltrlLPnWaJ2nyziUucFPRhYHCQGFgpTDgdwt+mo19LuHrCWTUPTPcN8ERm99mSE99MUO6yG/OkHWAfCPb3NNHXW72OPHi+rP86icLpfVneu/PWb/f48igaaPnEYobgzhyVR3qkybUB3HalEf9QukzCzPfwZnFXHWsMLDlMcD7btvyJtkVXnqC6Y9571d2GfeEG489D8mvM176dTJ0/OZBGT4s4YAE26RXzJA8IMQYqH55VjpPv3KZvCYvOOkDJ+jdU1y/yxvKgqA3Bz3t3Bl/wwTHnPbL0M6IOOnb5fMhCebr0hneL+FaCdLg+Op5z6P1gAyvm2AupClvl0W8PObUWdXMUldhoDBQGFgKDCxJHeQlY91xm49njXYhH5jhrRK6PJOHXHNc8f4ZTwfxpvOq8+pzeNwi4+W/WIb0kLjb5r2dUfWRnZfIZ3KUrKWv5FVvRrce2sW2u0rn3SgT1JnBcHkPipzVpvJ0qAUDeX7HzKHNm2V4qQQLE0dZ83Z2L4id3d5VzwoDhYH1YeDYTOQ1ZiA6/rFPPl844RsJn0mw/XiODAkrBryjIQxCBuLXMt525GMydBacQUkIErzOiRO2jNaDMn23BIIugzV92UqGAN6m7fOG9+bbGcI3456RzYi/c8b9MoEX/ikZUlrSePq/m8+8/X/IkBfrFxnyBtlteWbenyvBguEhGVJeFgzm5y75/OOEyyd4pnTytq7CQGGgMDBTGKBHvpw9olvIxovk/SMTGMzkJEdTPraT8p8dSwY9h0U+tsvmP+XoIXKRzKWzOEIY3gz0q2UeOurxGdJpQP10HDmb0XOXc/3kLscJ+XrjTKHL6DtGuveh3Cv3hEzj2NI/iwWLgDtlHEfM3TO0G5vB7F5l0M/u3Gx1PasBLTkGPp81esno+Ax3SiCcHAXhdbDlSHj+JuNtQRJSjEOLAGftHR0hqOR7VeaxDeqM/cPznoBzDt9LRbwYvMxr3YCEO4rE4olxzevj6A18wV2irVEChD5PkTjnRO2OUCYUBiOforPQYsi/PwtReObhPnnvCxA8W2/Ley8wW6Bpd4d89nKZXZY35T3P118zrKswUBgoDMwaBhj09A055QXZ32cHGcp2HxnfHBYZ1ewmc4iQh3QVBxKHh7Ke5eGw4shw/9X897EEIduVt53j6isZp00LBvI2H+euH+bdhxI+mECGdudK74+2GPpvyHTOkrtmSLbaZdAv9f0s416S8NmEmb4gZaY7WJ0rDBQG1ouB52YqA5Pxzet7zXxmsDMsGYMEXUYNW5CE5CfzwTlHXl6LAF7i92XciQmeGaW8xNJ5iS+Y8bwoBHHertkLLm25ekfhyMQCL/3BGX4koSsouOSR976CHQ4LAN4giyJbz5m1dZlrXtwDW7o8/up03hQ4TqU9Rv5xWZAyssjiKTKHFgcZvdVeNbDCQGFgdWOAQUxm0U0cR3QSzzrHEUPa6MhBYYfpZ/E9jpwFygI7ndqwEODJl3capMvLgSL/xTMDI51sFQKOEzqUjKcDyW5lMmvjDCNr6VkfSBA3s0CZzGznqmOFgcLAghjAu9fOVJ5znhDej3vk8ysSGOTd48uIz6i5SzlCy5YmYSe/8/YyMCh5PXjobW0SanYALBB4h+VZq0CwPycHz6C2M+K4kh0RC6CMHi5K5R15B+c8+RSEe3jM6HkvyoqxLy+8OyLlZVwK5ElZQru+DOHTmerxaVJb1D5Xmsl1FQYKA4WBmcUAQ57+eGP2kOd7zww5iMiyvF2SK1obHFaLqYz+42Ch69yTvXQoo51sfUZW4igQuZy3zXEdz+5nHgxo5jtZHSwMFAbmxYAv3PCk8wLbvuRp9z16X73hBWGcjwUn45Iwc16bQWor8nVZMznAE2xrk5efEHPswycanc1/V+ZhZGawZi8CnsFtu/f5iQULHPh3btMCKqOa9xec67Rt+4mMcLwmg3Uu9QDzYj6EtpXh1wLLF4rgnRJ8e5a0SOhK0TnSH2WcF8a8GGve8rGuwkBhoDAwMxjocq13yPEVn1f+aI8YhXQXeShKOR5494sF8rPDfGV45aU7j6+db2YmX35zjEZbdqYtNvSPw8YxSAsPeTNrI3vLoIeJgsLA1oKBGRwHIcWr66VVL2Ey1HWT0cnAdNaQkUigEZSMRc/OGfqBD+fuGY7ON14vCzoewvPs2+s8KN7u571Qn++mO9PdhVxmXzOXxQ1cewGWUmBg88IztuED7u+V2HBG3nEnX2uAb8eaeNHNkWf4z2xNeeXUBb9e2vpWJjhLat7ulvfqcu+lWN4jR3DsqnjXwQLMnFI6mbWuwkBhoDAwUxhgpIPeKfKTI+TAjLAbLI0MzMfmuIx0cpFM9L6QeM/iQZd1PW4ckoXq4txgoPe85LV4RxXV570mstgRIDrP2XuGvSOl3oeiPx1z9IU3ZdWjbbKYY0wdMw+QMPOdrA4WBgoDp8EAYUXo8J4zuA/NHI7HOMPtxSL3jFG/SMoo5IH3cqujOL5yQ1j5VVlGvSMcBBpvhPLOaPMy8xJfP+u1HdkFbz6uiYts9DKrM5887wxyZy19Mk0avFEK5sEn1HjqeX0Y53DprKYdEy8fe7ZIUs45ey8meynLYoBR73ynrV5efXPiJS2LLArHjokFgXn0lQdfHPJZUosv8WtiMlbZIKu7hYG1iAFOH0cSvXtFbjp22PFAVn0qHxwN9aIquZmP7ZjWmh+c8kIqeciR4ZihHUifsXSkcefMQw469+6oKOeT+jin7DaLI2O9i0RuWjDQgS/Ocj5r+cIMOUfsnpKdynovyRfJyGtHVR1v9FUyu9QWFvJbODjWesssvyqMegom+1pXYaAwsMowwCD38iVDkqBihNtOPCrHQWi6z9vG8PNmv3x+8MPb+j7X5Ru70sTZbvQGP0HnJU+fuWS4Mjz9sJK6eELUt5bAIobCYUjD2eE5eMrIQup7eQ9P4n0yjbKCTwsmnztjnDvSRMFZbFEgWaRRGL6MQ+nZgoZfiwVKTVl4txBg+ANt8Sq9PAubB4Z//3JERtVVGCgMFAZmAgN0RDekfeGLI2i6Y74244iLoy7SOEHIQzLSrjK5yMB+aiY61kgG+wgBOcuJ4viOX0X37Aii8l5W5VRRjx0Au890GrlNN5LhPmbgnSde/Ky6kblvzRvymUNL2z7lTMbq2+szzfft1WF3gW7MqOW4lq7OMuiXDpdVU2FgJTHAoCd8eNV5eAlJgk7Is6sv8hBGhJP4D2QkgeoTXY5u+MSlBYDz8gSj/ASXeuWXTshlsTV3wQODGg7gD868nApXfvGQQvCrh+LhEm7tiDDs7ZpQPBQcZeKIDoNencqpF34pkTFixWvLQs19z29+LQSU8dlKZ+7H5eq+MFABKH0HAAAQAElEQVQYKAxsaQzQRT7l62ggBwRn0XSfyETHEceGNVlHjnJwuCfnAJlHjpKJ0hnuPgBBvnomJzlOOEB43slhThXvk3G48PTbRdUXeThHpvvj88LqUdb7SdIdTXWWXhvAMdTeX+kzC2XQz+zUVMcKA0uDgaqlMFAYKAwUBgoDhYGtGwNl0G/d81ujKwwUBgoDhYHCwGIxUPkKA4WBVYoBBr2vLHj56zo5hoLWCgeFg6KBooGigaKBooGigaKBooFZpwEv7vpMdTDofUvZOVrnb5cfWqs2CgdFA0UDRQNFA0UDRQNFA0UDRQObTwM3Sof8Ntvstddexx9wwAEXPuiggy6eYcEBBxQOCgdFAzNCAyWTSh4VDRQNFA0UDRQNnJYGDjzwwIsnbL///vuf5fWvf33b5sgjj/zZPvvs86+73vWuraBwUDRQNFA0UDRQNFA0sAppoGyYsuPWHA2k/d723Xfff932trdt2/ziF7843Z/+9Ke23PDnP/+5rQ+Wu/2Vqn9jxyj/X/7ylwE3vY+eQX9eilB9YCnqWqk69BesVHtrsR30BxYauzRzIFwoz9YUb5wdFhrXhtLH5XrecThOd9/T3M8HG0qfr0zFLV6ndfxOh8uBQ230et2feOKJbcxf7sVJ6/mWO1zJtpZ6LKu570uNi6WsD17ng+k2pvP09IXie/rmhOO656tnnD6+n867vrSF8k7Hb+nnP/7xjw3oR8s/Z+gzWN4rIto222wzQES0iAmM49roLyKGvNIjos3M3wY6EhFDvyNiGGPLv4iYi2tTf8b3+9//vh177LHte9/73lDmn//8Z/voRz86xP39738f4qaKbfSjej75yU+297///e2vf/3r0J+NriQLRJw6rnxctisi2j/+8Y92zDHHtHe+851Dn5etsRmsOCKGOUIfEdHW9ycPWChPRAx1RaxbT8Sp8RExL51FRPvOd77T3v3ud7df//rX8+ZpW8lfRMzhKWKCj4ho47+IOE2etp4/87IQ9GIRMdTZ80Wcts2IGHAfEW2+v4j54+fLW3HzY2CM/4gY8C1u/tybFhsxqTcihjn/8pe/3F7wghe0t73tbYNS/te//tXe+973the+8IXtc5/7XKMLIqIt919EDONd7naWun7zExFLXW3VlxiIiIFGO44j1n1u+RcRA93IAyIiY0+9xIGIU+MjYq7eNs9fRMylK9um/iJinXR5Ik6tv+WfuPkgk+Yu6RGTchEx1Nnm+YuIIS1ikret5y8ihrzjutsK/y27QR8R7Vvf+lY75JBD2n//93+3BzzgAQM86EEPag984AMHOOyww9oPf/jDgTgiYkCKVcdRRx3VPvCBD7TTne50bdb/TOKPfvSj9vCHP3wYn3EaIzDOpz/96YMnJmJCGMbESHrmM5/ZnvWsZw04UgchzsD/8Ic/3P72t78NONmcsUdEU9dTnvKU9o53vKOddNJJm13n5vRnMWUjolmEfPCDH2xvfetbB4M+YoK3xZRf7Xly16y9+c1vHpT9L3/5y3npPyIGPvm///u/9upXv3qY1zb1h8YsGPHR1772tbl5j4iBtj70oQ+1Rz3qUe3JT35yQ7vy9yrco5uDDz64vfa1r22/+tWv5sr3PFtLGBGD8UTWwMdDH/rQAf8WlRETuoOP3/3ud+3Zz352w9Nk1ve///12hjOcYV40nP70p29vetOb2v3vf/91gCwwHxbW6vzud787yEb5nvGMZ7Qf//jHw7yqVN3a+J//+Z/22Mc+dpAR//Zv/yZpDhiBn/3sZ9vXv/71uXJziXWzQQyQt6985Svbgx/84Dm5bY6e9KQntc4zERMa2GBl68lgLi2M8VrnpXOc4xzDnH3xi18cHBgR0c5znvO0n/70pwMdREQzv+updrOS6Btt0U3oJ2Lzx7lZHdqIwnjn6KOPbvTnH/7whwGPG1G8si6AgYhoZNKhhx468ANeAOyZhz3sYe1d73rXoGvYES9/+cuHPNLkefSjH92+8Y1vNDtMz3/+8wc5yeb73//934GmzRl9ZgH7spe9rP3mN79ZR7dFxKD3v/SlL7UXvehFjX5CoxETunTPLjDvD3nIQ9pDEt7+9rcP/ZEWMSl/+OGHryNz9UFe/KwPhv6Zz3ymPeYxjxn6qH8/PMX+lNah14k/n/CEJ7T9999/wA1e7nl6GBGNrqVDLMjhMGLS755nJcIVMegpQt6If//3f2/3vve9B+F5netcp0HUJz7xieG4z1nOcpaBKRm5lOa+++7bnvOc5wyGBsSuBDI2tY2IGAwCyviMZzzjQEyUPsNgp512agg0Ipq0LqApkm233bbd8IY3bGc605kGga59hsD97ne/YaHzH//xH0O94jcHrnSlK7XLXe5yQ10RsdGGWUQMioXQN1+dKTanT+srC0dwggkZM//5n/85tL++MltDGjr/7W9/27zc8qpXvWpYiBGc4qfHFxEDbxxxxBGDN+/kk09eJ0tEDPP9vve9bzDI8aAMEdHkJZgZGHe84x3bpS996fbSl760ffvb354zUNHnVa961XaDG9xgELrmRPmtERjuFo7vec972p3udKfmLOJHPvKRxtCDB/TO8GGMucfbF7zgBRshj7fx7Bgv5ouystNGsNt9A+6/8pWvDPinFCg/CwO7Z3Bv3h//+Mc3RjzDnTx52tOe1siJC1zgAoM8/MlPfjLMh/a0q9xxxx3Xznve864JHjHupQRzdc1rXnOoEu/d7W53G3SUBdd+++3XvvnNbw7yMiKGPJv6LyKGRbG5VXdEtItd7GIDLZDNZBzauuhFL9rOetaztkte8pKDPtzU9hZTLiIGZ8kPfvCDQQevNh7/+c9/PjgCGXmLGe+M55mJ7qGBC13oQoMc3HbbbQedcJvb3GawabbbbrtG3zBYycwb3/jGjX3DLsAn+OgSl7hEY+dd61rXanSOxdb1r3/9dr7znW+gf4sADijylW6LmPBVRAzyi3OCLuIMwSv4E2KE9BbnksUA2cl+tBh9wxveMBj15CFaZqyTtWQukJfcZH/hsU996lNNGf1l7FtEW0BwpEVM+qM9C++nPvWp7SUveUm77GUv2+5+97s3cl8/9KlDRDTj1LfXvOY1jc419ohJXT3fSoTbLHcjCISxfotb3KI94hGPGAiAcqKECLZzn/vc7eY3v3k75znPORggVkrONjkThGhM0nL3cSnqN8nbJgPc5z73aVe84hXbDjvs0AhpBHqFK1yh3frWt259LBExjNUzAqG84Uk/IqKd61znGiAiTqNM5AMRIfvABJ6HhwX+8QSd/exnH/LKwkhRJmJSh7iIGNqSBnqcUF4e3Ne97nXDKrSnSxtDxGRc8vf4iEm9/VkaiIghKiKGdoeHU/5Jx1BogyDAhBGTfBEx5JLHTcTkeXyvf9IjYlCKEafmaTP8p89o6C53uUvbbbfdBrzAw3SXIybGAU/HCSecMCwUx3kiYpjrz3/+88OxJYKs1yMk5D7+8Y+3W97ylu3qV7/6sKhEH2984xsH74o8+oIn8apn9Xe8uo+IoX8R4XEA6W4iTo3zPMsQEYPXhYKhuK52tas1yuhWt7rVIJjh0E4ZY588ItQZYLe73e3aRS5ykUa5MN47jvpYKYMb3ehGjQIj6AHFYAFvbikzBv8d7nCHYeHw4he/uLmniHimyBPG+vHHH9923333tscee7Sf/exnzbzhB0BJ6d9VrnKVhlfMWW+/wsVhICLa+c9//kHeXvnKVx70EyNlr732GmT0scceO3gN1RYRA83DMxA3hoiF09GQhSJvoPbMLyPAAs+zBZ66zKl4vBcRohYF6+O9iIk80OeIU+tEz2jYInLHHXecaydikqfXOZeQN+oAeTtc43sR08/iIibt9/oi5q9fWaBMh4hJWfERk3LS9B0vHpqeZLiSLr634b5g0zBw5jOfuV3qUpdqFpdnO9vZGiMdfdzznvccdIWju4x4dOvFaQ4ORjwHBFtGqzzx+OmJT3xi23nnnQdHkbrucY97DHX0fPKCPn9kmXbYRWOZGhGD958XHA8xnDlYLn7xiw/H1LQdEU27dgwsGuQR8sST7WidXckmc2+hwVa72c1uNvC6MWkTsEPtmNptsDOx5557DkY93EzTmL6z9e585zu329/+9sOCxpi2BCy7QW9QDFQeeaHVNI9UV1q77rpro0TlgxiEw2uIWMStBtDviIliYAARNrw9r3jFK4YVLmMeAcmHWCh720aO2hDoxhgxEVbKMdQwDSXgJSlHHxytoNyVs51l1Wnhw9OqHopnmtDUCyiPf/7zn4Nxy6vB88iAsKhiGERMhKZVtvgjjzxy8OoyVCw6GBmMEf2w+mTYT69AjYvBcdRRRw1lGRqYwpjNt/GoX9tW+JgnIoazo47WSP/qV7/anve857VPf/rTw5ltx04YmYwfY7WdxbBi6Fhhw4M2tB0Rg7dJ//SftxOeLRrRHDzMOsCV+SD4CI6ICU1M9xs+nLHlxbNwNP7pPPACxzzsFtTq7nl4NpRRFn60pS60SDCac3nRDVCWoWHXAG61LZ6wO+aYY4Z3M77whS8MZ3/NiQVDxPx9V+8sgbHpL0HPM4V3xfHIwNvHPvaxRonAjXRx8M+LussuuwzedHwEn31c+NBC9NrXvvZgLFosWVQz1MhAHiHzTPaZH8+8tZQBOWFO4NdijaKkLMwJeYAnIyYeIbg3bxRuRAzGZu9DhYvHAJlIdtE98A3/5LAazKO5jQiPw7tOZCH5whuIH3j8JMqnHos3nkwyOyIG7zdZ9tznPrfZqUZf5h9NmVeGk2dzbsdHm4xU843+yED9UT/6pDvJ14iJzCM7eRjxID7FlxGT/uoX+rTwpDvIBXIRjVrE0h3O7fcy2nDc1e6dNDuy5D6ZT/8IjUM/0bM6veekr56N3TMcajtiYmQZP1zxvqJhuPP+gF14+kV5C1x1k1sRE51kAUumwIO+0wP4ost/OkU7eJLsV16fyTF1RpyKB/kKNowBtEFnCtGqe7gk+8gb+EcLaBHdMow5HehmtMmzTqZy4m633XbD6QN1yUsW0jcR888L/ts2HaN9gdt7izfITgvty1/+8o08JV85XtCsPuqPNIsCDg48RJfqO9l9pjOdaZCR6san5Lp6gXz4Tnt0AfpCa/vuu++cV14+45BnGvRBv+HI/XT6Sj0vu0GPIEzE9ttvP3ioCRPnnqykTLYVHkTLZ9AEHIRHzD/h8swimETEimhNOqHC2Kawr3vd6w6EFBEDcSMWBvH1rne9RtjCiXKIksCzMlRePZjEtrsyBJ8VJaHtTDxBziiDM4sHQlI/5sOPeFtK8jhi4WVHioIgl0b5UAp2E3htte8cKSFtceX4hSNA2peOKfuc6TsGsRChnOQhgHn1bX8dfvjhg6fSM+8Xge5YlWc4Mj4C3+KFsKBoKCDnIwkIjEVYe7YgINgZMZQKBWMMcGUxoV1tqNu52Ic85CENDqVHzD5Nwam+NaEpkQAAEABJREFUAvfTc2muCEzCiyeZp2OcLyKGdzUY7de4xjUaD8Y4nVBCA2jGfPY0AhD94cvpNtGqo3AEpTyUsPoZAuiQJwSddAOX4nZEJWL28R0Rg1xCe4yCPna4QeN4reOoK4CIGBbH5BraQ889TzvljxGuDvNkLuGN4XeZy1xm8OCQeWjYfPY8QgqNd0xIQaB9PGHe7LRQUr0uc4YX5Rn37ZQubFawlgozPowXPsliRiSZwaPOWCHf4J8M4hWk/Bk23klivJofc8NAxw8R0chBdZBVFn/0H1lITsqLXshcizl6Q/tokPNDWTTDwMXr+Oy4444bjlqhNf3o9aiTvKQHLBY4R/RXfRExvCvjOBnaYuzwWNID8qAz9MuR4hkYP/4mWxlM0vG3POhRHYxluoiMZfwbJxySHYwnziU4oce1xSD3jFb13RgZT3a6GO+MQ+OAV7RsDPCtTQsGdgL5Aw/6iLcufOELN7KebNIOfcXhwyusTU4hOIaHgs3DAF1Bz6BP70O6RwsRMRwZMx+clubUIg1PcOCSb+hhPA/mFW2P48a9Ey+PcBzv3rFCnnvpIGKyoMUveFcZhj56QO/yMM4t2JVTh76zZfAT+8a5erSCf/RXHs5SvERWc2I5dcEmwxvoT575wLiAfsyXvhJxy27QGwSlBQyWsMS8FBaBCdFjBLgHyq1GiIjhhSaGJkJzJrMLcIRG2BGEN73pTYetKEIOQyA+6fIziOEKHigUhhJFztiWxmCVTnhRLAw7uEK48Ox+GuRH7La/1EcwE8IIniBmWKvX4gMz2j7i/XjLW97SrDoJVf0TUgTG1NuIiKZtSsBKGMPoN6ZXJ0+ktvVfn62qGSAY6SY3uUmT1/h5KR/5yEcO7w84fmBs+o0JeQC8C8DIUge8wQVvvTFQfBSLeMca1EmJ8BJ4hhf47H1ejaExUOjGCscMxj6miBgWjfBFSDEULNw8G2tEDAYBBU0JozmCWnlgbuFL/Z7b6M88Enjw7jiQcrYtLUjRC4OEcraA2HvvvRsFjRam6xlVOVO3Fqz6yjDiWYRnhoJFCaMd/eNlxgcjQzq8WhhRWPATEacZkzp7JHrnZWXYwZ80dUiPiGGhj9+0g/8oJPNnQWZRyxCitPBTp3n5zLUtaHzDa9z7r96CDWPAPJhnRiCPtOMDHBto2/sU+IRsYnxzJohD42QPGmFkuGfoKE++kl3ktfkC5ttck4FkmGfGAT5WlkyT7pksI/fQld0a9ILe5GFMkNVGxfjwzLi2wCNb7XTjQ/LSuOSTZlGKxrWNfhhe6sS32hOPxi3KGWTKOPaAvowPTVnMo3MLUbJfSF67Vwf8kTkRMXyCWRlyhuMGD9l1pxvkh4eOV/2UV/8YUMbNcNR3ixY8Y7dMv+FKu8aHP9QnLSKaMcKhPPKqi0xTv7oKNg0DaIGcs/tjMUgOOUq44447Dsc61XryyScP74M4j44n0KiTCtKWEswlUCfa15YFM/2Oz6R1kCciBl2E1vFjT0PLPO+Mdi/z0nv0GLqSh5xHg3jJmPCCvBYA5Dj+Vf8swooY9AYeEcMLLM6UYlqMx0jDmJDYQV4QcVoFKX6WAeEwmOxAmHjnxQg5CkGacfMkEEqEmmeE5tn4jY2QJQAjJluOFj4AESE4Qosng9eHcFQ3w25ch3rmA3UQcurQH8ab/hLGPCAUSUQMq25Gu77wKspjntQpBL2/4txbmKnfSyS2XSkIRiDG0z9pQH8pFMLYwsaYKCttudc/+T3DnbojYs6rqd/yGIP64BnzETz61UF59VAW8BSx+ugJbjtETLwRjllQrObfuOETdO8yzwNgDKAv8XAFDwDO4C/iVHxExPBSkXzoKOLUNO3DuXJwjPbQBqFtbs2dEGiPQUqYU8bmTvlZB8aRF7x4+Bw54wnkeGDgMBiM0QIcTnkv7Wwx8CxIGTzoeUNj5dHEI/h+Oq9nc2YhxBg0B3CtXgam9uGX0Y6mKVdKCV/aDTEf97rXvYazpPqtvoh153DW52BL9C9iYnyaG04GBjkPPOXOq02Jo33zwlmABqSJQ+v4abvtthscHrySeMORgIhoXrgzd+bLPNnRMvc8y/iVAYoPzZ25xXs8g+QimlIXXlKOsYunlLOAYwyLQyMMeXKczmHEMurHuCT7LArQtF1Zxhj9oj0LSAsCC0fPjtZYLKJBfSBLjdEYLCz1Ux0MILRMxstvXPpK5lrIwwm9BofyW+gbm11kDheOAXLJjob6yTOLAzjp5eGGQUUn2J2WhyPK2Mg9PABXdIS+wwOZw5HGuPNsnuQv2DQMwJ95csTSronFlfPp++yzT0OfvdaICR+hR4tDc85pG7E8Mihioq/s0KB5C7jel3EYEcMRN7TJhupy0UKTPrM4J0fxtpdw0RUes9hWxuKcnkV33jdzzI3Mh5dxO7N0vyIGfUQML9vZuqO4MGd/ERYyIJG3mOCLiOEFCoicZcTp9xgiYtiG57nkUdttt92GF6yMrZ3yR3jxZozjJEXE4F2NmBjT4jogwg4El3jP49D9YkC5aVBOv2x1qr+nYwDCshO5fAuBcvJazWIwBr1jNISzOZTe61UHZqJoepr0iMkCxn0HecG//vWv4aUV8f25h+oVT+F4cRDDURSYj1JgrGlHPmVWK0RMvA0WhIQr48M5XQqVYnbciELkXWDU8SaaA8qQ8U25OtYE75Q1gwTeIia0Z/4pbmnz4ajjDy7RBuUuTh3yuxfCOYNUPs+zDvpt3Awxn5xl/Og/ry3Dh1A3RvTlq1XyMWDIKvhmQDG85VlorHDNcKIktDXOC08W0+Sitiwa1BMxkQWeKVAGOx6zi6Av+sngtCi2EGcY4ivKFz9HhGoK1oMBNMwrztPHeDQ3cMwzTe4xSsydXRlzJl4Z9zyDPJeMUwY1vnPPyJWOD4DmLQzpN/NGrzF8GOYMYLQjD72gPQtI/UAX+kWO6Rudaa4tJBn36qFHGK5e6qNzHGVhAKsPRETzzBjRL4tVdfYxkJUMM/WhQUa/MaIt5Y2d4aw/5AIZ4RlfkPMWOvCgPEDDjCHeW2U5Hxj/FsoWTXY90LL65NO+etEv2oVn5YzJ+C0MOMXsfuibPkXE8LlDuIBPdYnnILR76BiqhYk5EF+w6RiAQ4tHi12GvCOsdrwt1shNNUdM5BRZRGaSU2jajr8FAFqTb6kgYtIenRcRzQK708C4jYgY3s8jdy2i0VdEDC+4oxFy0rh8yYrdgL4sEPQX0HH416JWiAfpTjTfxz5ub1but1mpjvA0YDTtYT4ro4gYjGBMD8EdUZgZQiMm6RGzr5z02WR7YYqQImQJU2MClD9PuDjCjFBv+adcBoNBj5AiYriPmIQ9LWLdZ+UiQvJcfuWHiKl/Eevmi4i5MhExeJjUR+lgYveUBQFOwZiLXmXEqWV7nPEhfF4WZ9Z9r1VZRwAoM/3qoAyDw8qfgYNZxE1DRAy00fIv4tQ2IyJjJldEDOPwRPBQGPrKi0rx+KY4JWtM2meoYWz5VwNEnDo+YyAoeWkJTcoLUHrw7gwjzxQcGLd3UyhPHjkCmNACETGcq+elAH2uKXSLHwakue/4MbcRMcxFz2tXhOGDlqWL78DQRdsUvLi2Cv6MgReccU5hMZbwgqMVcC49IoaXo7wTw+PIC8UY+q//+q/BaDJM+AAR4XGAiBg+38ZQ4dUc4wRN4gNGJaOGIaM8HMqvAm3LJ2TMoV80LU4+fIeHPKtbObSi7FqFxY4bLjlg0CuPN7qHQ7iGw4iJAcB4ZOTiL2mMfUaqPDzx5sE9eSZUh3lgpLtn2JB5dJ428Q/66fOtfcYw/cDbaE4Bo18d5lvIuCffyGT1krHqw9f41i6BfNIiYniXRt3SLEQtOozXGPTPM5q3sHBkkrHPQFKnOnjVjRNt4gPGEQcCL6wFhvJ0GQ8mXtA/YyOPyH0LHbzCE4qvLBb0BzDI0D7+IZ/gVBwZBNfGBpfqBowtMgqdq9cc2wHWT2X1mayTl50B5xGn8qH8BRuPgYiYk/1wjXZAyz/P5BU6tehy4sJCzxEYdMKJZL7MTWZf54rY8Nyof1woIoa+sKXQB7qnh/QH3QvbKX/K6hfnC/oSHTHZSbBwJu8tcqWT83iTzUCWonW8yC5VTy+L5tl2EROnMwM/4rTjiIg5u0TZlYRlN+gjYvhiiS0NTAYpe+65Z+sKH+M7p02pIQ4IJMwwqQlidHgWv5KI2Zi2IiaEwitK+CMQQkmfEbNx81YwOglsHlMLHOOTRpAiMluGETGsLAk96fpBGcAHAeeZIIQXoTaE0tXjWZ4O6lCOAFcPpSVN/crBLUODciEIeVzUwfukLOY0BuBZGW1TYuoBmFY5OzD64Xw34a2MtIhoxolJIiZHryKiaVO6vqlHu8KIySrcePRP/4H+y9vH4N449EvdXspl4N7+9rdvjlBgekpHft4D7y8QBtqYVYiIQWjBBVwbv/uWf/iDQLHVDMybRQolTwgRMISUeOk8E4wQ80Co8azBFYMgIoZfgIV/uKG8eYjlgetsbriUhUdKVDxDxLwQgIRfxGRuzW9EDD9+pAyP8VDBKvgXMRHAjBG7H3aYGDaOWOg+nJkD4yenvLCNz32GF72hL7TIoKEU0GREKDoA2uRFonzUITJiIhe9BM9TyaDjOeIp8iKgOYmY1KF9hhi5YUFhztAFx4g6GVrmB1/yWqIDZbRTMD8G0Ch8OlLIYEX3cGZ+HduA31133XV474SOItfUBMdkNYOFoYz/1CWNDDS/DAEOKs/S5LVIRAPmCajPIoJ8wucMIjoxIobflyDr0AX6ojPJY30Sr6/6yWFiDIwTPK8ttKEv7i0AGeRok3xguIuPiOHrTOidUcxQZhCpW1/QM486eYmW1Q03aFRbvJXi0Tt5BH/6xoCi99CfPjPy9VubZBfe0Ce6g7xRj8WKuvEIPHm3i7yxOIE/Mkb72lWPvHBFruFJ5XiD8aM2tG9OIia8AxcFG4eBiBh0EFpGC6DlH9xnMFwRMZy6sNBCB04kmHN56X76gZwkS/FMxGQ+zFlEDF+kU5/nocLRv07DeKSnR8RgJJOD7Cw066VxMpNtyXZRRUQIhneS0KPjZHSitiSgDXxGj7G3ttlmm+G4KdoXL98uu+zS0BtZbDz6gefVZQGPn9Av/sQvEZM2I2LAG97AD73vbQX/lt2gRxSMV8LAhDNqHcXgyabMnAHFvISAcTsu4KwdhSY/getNYwy7JRCkTxsCY0TUhBUhxtNgfMAYbXUSkIgEsQPHHxA7odUJDpF66QSxwReCtRBQFrhHSIgXMcKbfLbhlUXchN0YTxHR9E15wh8DUjaIHTHaqpXfuUnGgc9GeilMW7ZqCXzjR+wMQWPEVBggYkLI0gleRKwvxmy3glFtvuEHU6tTmjq8yMvgZEDZauUhcg5Unwh8Y6QMGIry6KexUkJowtETyhDDecZ02oDrgw8+uD32sY9thxxySPMii7FjPj9S5U8D7d0AABAASURBVJOZ6o84te/6PwsQEcNXKYzXFqbxEFbmCw4JG6ExA/cEFN5xLx1IA+YjYvL+gTmWB5hL3+WFX1+gQIvmloeLMFIHfMgrHviMHdzZ1rf7ZFtdHmCO0J4X6swF+rag6PXIM6sQMTnK5GgSBYQ/eJr2TKdDNyDg0bjQLmMPbu973/sOOx3wFRHDIhztqQMtwrcxS+fF5L01V3AaEcOxAXm1y5OFXoHvNpN1PEfKR0z6hz/svjAK1aEPjDTzSN6QmRZRFiK938oXnBYD6JLMQa/kL9nphT/zBxi35penmXFh4WTR5P0vfGkOzA88K4ufeLo5FJQnt8l6BgDlL49eoANyU/1oxELBnJGdFsnoTH5GvD6inYho9AG9QjYqR47Lp290qX5ZJJDXZIGyaES92sK76MyCnXddujr0n4yRhyHMMEffDGT94S3Xb22Ru+QtA954pdNBjB9xxsi4xvdo0wLGRy/ckzH6oE2ywbiMnfdeH/RVWXLZYkWIf+gydA3nFldwoKy2lBHCpz46Zomf6DWLXraDcUorWDwGIiYfuEAveATNcbi67/iMmLyEjNa9kC/N3JprLeEpedEiG8hxUPaAdHRAn+EFjgv35jIiBoOd7GMHoDd2i37Iq17P7BN2hC/e0e/AwhZt4AftRsTwy7TkLRpGS+K1w/lFz0lj5+ifNiwQ7SKhLwt1tIvefQYVXVmke7HbohgPwgm6Fo+G8S69bQGNtp3Lp0uMOSJ0f0Vg2Q16o7ANw5vFoNpvv/2a1RvhAazsxPFqmxCI9eKM86zy+9VU5QlIk6K+WQRC+IADDmiOOxCaxkZYIyhKlteYgEUQ++yzT/OWNW+aFZ8jKo6qyE/xw5VxE7KIi3KBD+kEJSVO+fusGAIlJA888MDh193gaYwfOCNkfXoJntXBW4s4H/e4xzXe9IgY3lL3EhelQBHxzBOMETGcX1fGG+HaNib9ULe2MEo3LoxdeVu85hGhy6cPhL80BqH6MbzxOSdpvAxHzAf005gcMVGGctU/+PVs8eGLQIx3xiXaUb85YBT5sRQhHDGGKA9pFDGm1e8VgY1sxNh5pAgU84M+KKeIdYUCnEZE8zUiizECaropwgQNoS081NO1YafIkR04ZygSZubUXPZ82jA/5sbWuLzmTX747vnQrPqky+cLQ7xlPX3WQzwET2gEX9rK1X/j13f4Ipd4FL37g+6MueNKPotyPOaLCAy+nqYsryN50OPkR/vwiE7HcNhhhzXzYiEsn/bxNGNMm/hJHIiI5itReBavOufK62vepRcsjAG0Ti77YSLH1cgb87vnnns2PyRDpqLxiBh+aIyM4twgsxn45ggdcEqgH3LROxZ4QD3m3NybQ/SEL5QnR80jOiF7lZUPX6kTHTIQ6Ao6ww+YaZN8JcvIYDub5KF5d6898kJ9ZJs20QCeZLyQ18ZDTtA/6NAYvPNk3PrDKPeMvrWnTvWRmfCEvvG1o3xoF+yTegzuIqKheXrKOOEkIoavl5Hhxq+/6F37xkzfc+roC9BHeg8P0hXKsBOUI5v0PyIGo8/OmfPPxkAPyEsH4DF4UIe5W3j2K2V9GDA/8O6olB9l6jqol0FfXSaZMzRqIdbT6Vhzxkahn5VXZ0QMP8bHNmDzmCf0EBG96OAZN+/O6+M5+odu06a59mNi7MKxzFSX+ZenV4S/2ZbqR1893j2aZ0uwQ9AduwH/onF14Edxjrbqi7rRK7vDAhLty48XLSTIZGXIXs6ggw46qHFm0hkR0dTZ21/ucNkN+ohoGJRxt0tuZQATjJmBe8qKEIAUhCROvg4ECyJZScRsDOIjJudrjUOf9d/YOnhmUBkfgmKgIRTjQqTSCPqIaBSBOhA7wpPWceee4JUObFciWIrCs3zStTHuv8WGNHnUiUkIe8+EIuNFGQQoTn8JR4oFzkHEhBnVw0MTcSoTSlcHJjLX6sAoETH8iql0oB9wYhyYQh8xibEC+NAmkEc92qNcKEr3QN3qcg/0lUefl4oCw1SEvMWFcWqDUDA+bRBG+qP9WQJ9ooiMFT6MH+CNiFPx3fscEc3Y4UO5Ht9Dc0pIoQ80p35pPVSvuZZO4PZ4ecaANtGruYP3iBgUqznswgxdqUtfGCML1TWudxbu9ZNQRhcAP+qXeCHAtwwtc9JpCW6lAXmN2bxJR7/ipCmLJxk8njugQe2Z3zGgZ/ypnLzqYdyrw724Dp61hVeUQ9/ienqF82MgYiKvO97NK9pGv+h8bJyowdyRk/KjD3ON78y3ezhHQ7z06mIgR4SiA5CJeMycoxsym5FgTmWIiOG79eK0pT7zz7Amv+TD3+pBM3hZu3hbmrmnN9U1BuloRz/Vo4/al4c+NVZGiPYiJnpa/9ETea4d5YxZ+3i7t4P34UT76pTf4kKf9V2dwi4X1KnP8ioLf/SFfPoDJ8YG98rpN7ljbPqvfnkjYjAK6UkyPSKaeugk86M9fZVXvQUbhwF4QyfmAi3AKeBkijiVps0Xe0ceIdrUkvLyilMOsFPUaV7xgTLi8RsdFDExepVVrzmXLp+yaEHd6hUnbQz0PFpXXj4hmrAI1qZn8R0ioqFp7eB7fKd/Pb3n1zd9NBb50a486Is+QIOdLrVD9suvb/rZeUGZlYJtVqIhCIKM9YE8+iKcL5946bMK+jdfv8dxve/jvP1eKF3Yy7gH42d5+rNw+ll+cWMQJy9wD9wD9z2ve3HAfY/vobiF0ubLw9iwPWYL2vlIZzJtV/W8wl6neoE4MI53D6QD98A9YFQS7raffTbTVrpjWrbzGKoMHVt4vFcYTb+0MaswHpvxeV6orxtKV26hPOqVBtzLOx9Ikwe4j5gcDbI16iiKoztCW/PS56tjluP02diA+/n6Kn596crIA9wvBtQ3H8xXx3xxvY1ex/ry9LwVTjAAVx1v06G0Sa7Jf889j3uxQnHuQX8W515cB8/ip5/FT8f1Z6F05YTzPS8UJ34Myo/rkbbYuHE5ZYDyYJw233OPm84nflxPf5bP/RjELSavPPPlHddV94vDQMclfHYQNy7tuacJPfd09+LGIE76OM59j5fWQXyHcbr7Hj8dSuvlezhf3DhtXMd8ecX1PO57WaFnae479DjxwHNPW6bwNNWuiEF/mlYrYk1gICKGl0RsP9k+482xFRxx6kq/LcEfj4/tZ9t0vGMYCfA+8QQx4MVbUWtOmrBg0zFAYPFO2Ip0VIA3DV4jlnZuN72HVbIwUBgoDBQGCgNrBwNl0K+duV7xkfKc24LqZ0GdoWQEMgaXsjMMSVtetmad52NgOsvpeIi2pAuB+6Vse6uua4HBwaHtc8eb4Nt5QtuStiKlLVCsogsDhYHCQGGgMFAYWCYMlEG/TIitaicY4JF31AUw8Cexy/N/ui0G/PK0VLXCgPk0rwDuxRUUBgoDaxMDNerCQGFgy2KgDPoti/9qvTBQGCgMFAYKA4WBwkBhoDCwWRhYRQb9Zo2zChcGCgOFgcJAYaAwUBgoDBQGtkoMlEG/lUxrRAyfiPSC6PSQnG32tRefTXOGfTp9c57V7Qszws2pZ31lvdSqDefk15ev0goDcxiom8JAYaAwUBgoDKwhDGyzhsY6c0P1YqHvpwKGuOdNMYwjovnBAz9V7Ff+nGmOmHxthDHsV3f9wppPOvq8IERETNLdrw8Y0RYD6pnOp69+LdAvJPp1uIjF1Tldz/qete+Tl34x1XfmPa8vf6UVBgoDhYHCQGFgYzBQeQsDWwMGyqDfArMYMTF8P/7xj7dHP/rRza/pMcY/+clPth/96EfDj/ZsTLcYuQz6t771rY3h63vgyjPC1cfgjoj2hS98ofmVNd9rl74hUO/vfve79opXvKJ94xvfWKdfFh/f+c53ml8SPOKII9r3vve9ddI3VPdi0yOife1rX2uvfvWr269//evFFqt8hYHCQGGgMFAYKAwUBtYMBsqgX5GpXrcRX1/54Ac/2I466qjm2+g3uMEN2s9//vP2tKc9rX3/+99vPN/rllj3KSIar/vHPvaxIUF9jtM8/OEPHxYIPOoS1PPud7+7/fKXv2w+HXnooYc2v9T25z//uS3m84IMekb7S17yksazP/6SiXu/zueX1rQnrzaXGoztute9bjvssMOaX25bTL+Xug9VX2GgMFAYKAwUBgoDhYFZxkAZ9Cs8OxHR/vSnPzUGvV8w9Q1v32q/2c1u1vy0NgN23CVedsdxAKMZ/PGPf2zvfOc7m2Mu3ZhmvPvZaz8/7F45R294zpXx7Lvsj3jEI5qfdR63Md+9MieeeGL7zGc+05xf5yX3HDHZXdBPP9Ps58P9NPPY0FbWWX195smPmJTp7URE6+nCiEm6fnvWV6Gy6oUnC5/+s+gR0aSpH7iPmNTR26iwMLBqMVAdLwwUBgoDhYHCwEZiYJuNzF/ZlwADjGHf8P7qV7/avvvd7w6/pnquc52rXf3qV29nPetZG+93xMRodRRn//33b095ylPaT37yk+YIzLOf/ez2+te/vr397W9vhxxyyHAkhdf9Na95TXvd617XHLn55je/2Xjkv/zlL7dPf/rTw7Eei4BznOMcg4G+mGFYMPzqV79qfqTpF7/4RTv++OOHvvayxgEiTjWmGfPOur/gBS8Ydgve8Y53DEdxGOPSgPTnPOc5bb/99msve9nLmr6LdxTola98ZXP06EUvetEwxohon/vc54bdDDsNjH64Uy+8HHTQQe0DH/jAHM563yosDBQGCgOFgcLA1o6BGl9hoGOgDPqOiRUKeZwZ7de+9rXbH/7wh8HQfstb3jJ47R0tucxlLtPkYbQeffTRjfF7vetdbzhic/DBBzfeeYb/+c9//nbxi1+87b777oO3+4UvfGF7+tOf3j784Q83nnlebeXOe97ztgte8IJDPjsA6lX/+oYbEUMdFgK8/nYQeM0tCCJiMNB7+XFdjHJjevGLX9z+8pe/NOWc37/HPe4xGPd2C/T/Pe95z3B85hrXuEYz9pe+9KXt2GOPbU960pOahYCXYJW3i2E8j3vc49prX/vaAV/aeOMb39i8LwCHjuE4X+/9gIjo3aqwMFAYKAwUBgoDhYHCwJrBwDZrZqQzNFBGqZ/Nv9e97jX06hnPeEZ74hOf2H784x8P3nNe6C9+8YuDZ91xFkdbLnnJSzbn2T/ykY8M5+559Bn0u+6663BUR30XuchFGsNbpdIZ9Oc5z3kGg36PPfZo6uD9l74h+O1vfzuc599jjz2aRcE5z3nO9olPfGJYYCxU1tEX3nQv0F7/+tdvt73tbZtFCuPcwkP/eNbtNPiyj0XHxS52seblYMd6HD9St7b23XffZgHjSI8xekcgIpodAcdxLGpufOMbNwsgR5jgLqIMevgrKAwUBgoDhYHCQGFgbWGgDPotMN+82o6g7L333oOHnrHLG37ooYcOL59KZ/Q67uJl2a985SuDoX7HO96xXfSiFx2O1DDMedtPOumk4RgMY/lkABHvAAAQAElEQVR85zvfYPAaEsNXmnzAvfwRGzZ6te+ojgUEb7iv3Dhu49gNg92CRBvTEDF5P0A76hDqlx0JCwzn/R0z8rUadTuXbwfA+wMXutCFml0Hhv3Zzna2Bj9eumXcb7/99sMuRG9P/itf+crDcR0v62onYsPj6uUrLAwUBpYJA1VtYaAwUBgoDGwRDJRBv8Joj4jhzDgPvKMxV7ziFduDH/zg9oAHPGA4duP4ibPivNo80be73e3aPe95z+bYijw83hExHMvpXWc8M9r78+aEEdFOOOGE9q1vfavd/OY3H3YDLnvZy7ab3vSmQ7V2CBYyoI2H53y77bZrn//854dxOg/PSGfQ66MjOYz4u93tbu3ud797s0vh3rGgXm9EDAsTzxYmxhcxMdgtJtTpHQILGIuhbbfddsg/dLD+FQYKA4WBwkBhoDAw8xioDi4tBsqgX1p8Lqo2L616GZYHnoHKG33LW96y7bbbbk2cb8oz5r/+9a8PhjVDOSKGz086x87QjZgYuBqMmNxHxDrn29UdEbLMQUQMHv2IdePb6M/xFS/P6pNjLTe5yU2a+ytd6UpDf+weREzKa6MXZbA74sOo/+xnPzssUrwDYCHC+JZPfi+/WrQw1JXxDB8Rkzrlizj13jNQ1vEan+J0FImnngdfWkSsM3ZxBYWBwkBhoDBQGCgMFAbWAgbKoN8Cs+ycu+/N+xEox08Y6L///e/b7xMud7nLNR7nHXfcsTly8uQnP7m96lWvas6e80o7d+5Tjc7Z8+L7Qgyvt0WAc+8WCxYAhsXTDnxuUhyD2IupXkB1hCYiZFsH1OkYC4PZokL94rTpnL7v3/OQaycihhdVGdnaNy6efS+oXuEKV2i+Ue9ojAUCiIjmm/vHHXdcO/LII4ev9PhSD4PeokY7PPKAsa9jQmOQpg1gPM7rCy0upOuDF24tEJSbQP0vDBQGCgOFgcJAYaAwsPVjoAz6LTDHjFTGOi/4u971ruYTjgxcL4re9a53bYxpX6RxHIWhK92POzmD3g1+x2Ac2/FZS8a5eiwSGLjHHHNM49336ccf/OAHg1fdUR7pvitvkeBLM/oRMTHqIybHXHxN5n3ve9/woirjP2KSrg1edwazL8zwkjsP74y9XQWLBPU7XsPbftRRRzXtG5dfp1WvhYeXZW9xi1s0ffdVG/13zp7BbwzO+mvfGXsLEPl8ltL5fV/EYbTvtNNOw6c6fd3GYgjefKZTP5TZAlNaTRYGCgOrHQPV/8JAYaAwsIoxUAb9Ck8eI9oXWxxhcT7eF1ycA7/1rW/d7nvf+zY/DCUP8JUaX7/xSUrfofdSLKOet1xZ32HfeeedmxdK5WX4P+EJT2gMXl+m2XPPPdvhhx/eGNSOzfhajiM0yvH089r34WsvIpr++La9hYXjM+KBF1u9xOuTlL797uszzsXf//73b8997nObc/AWKDznxvbMZz6zPe95z2vPf/7zhy/4SGOUW6yo+4gjjhg+UfmYxzxm8OT72s29733v5hv7d7jDHVpv22cp/RiW+L322qupB64OPPDA4bOYxvrYxz52OIvvWE8Z9H1GKywMFAYKA4WBwkBhYDkwMIt1lkG/BWbF0RReZSFj3IuxjqgwuBnPvUuMU+k77LDD8M15+Xu6vJe//OWH77nzivuEpXyAx1u6Oj33+rfddtvhazGMZQY047vXp82IGD5tKb9yvjYjHliEaK/X6Ws7jOueV5ojOm94wxuGH8diaDtzrx6flvRNeu3y8FuU+ISmNF+2Ub8FhmegLn3VN2fkext2JexY6Is61WFxY7zG714Z9RUUBgoDhYHCQGGgMFAYWCsYKIN+C8w0o7OfE2fg8pQDcePuyOdIiTThOE1e8cqP8/W4nu65g/KOpTh/70iP52nQTs+v3p7uvscL5Ru34dn5douLY445pjki5EiMs//HHXfc4Fm3iFFGXfKrR/+1Id5zB8/T8cooC+Trz0IgXpmtA2oUhYHCQGGgMFAYKAwUBhaHgTLoF4enrSIXI5k3e/vttx++a7+Ug1K34zR3utOdhh+U8hlKz4x4x2YczymDeykxXnUVBgoDhYFTMFBBYaAwsOYxUAb9mieBpUMAg92RGF/o8TUb5/V96cbxmaVrpWoqDBQGCgOFgcJAYaAwUBgYY2CxBv24TN0XBtaLAd76Maw3cyUWBgoDhYHCQGGgMFAYKAxsFga2yb9IOJ0XJL10WXD64ThK4aHwUDSwqTRQ5Yp2igaKBooGigaKBpaTBtjtCZF/g3N+m5NPPvmv+fDNXBZ8I8OCiMJB4aBooGigaKBooGhgJWig2ig6KxrYZBpI2/2b//jHP3539NFHt21OOOGE40488cQbJOz+17/+taBwUDRQNFA0UDRQNFA0UDRQNFA0MMM0cNJJJ+2eBv3u6Yx/w957733yNrvttts/dtlll1/svPPOx2+lUOOquS0aKBooGigaKBooGigaKBrYqmhghx12+MVOO+30lzTs23Duxk1BYaAwUBgoDBQGCgOFgcJAYaAwsPowUAb9lp+z02cXLp5w7oRIOF2C50tm6D6Dzb7Ue+Gs5XIJZ0hYDZexXyo7ukPCaulzdnWru7bNEV0+4T8T6ioMbK0YOE8OzPb1dTJE8xk08udGeXPFhDMmrKYLv/ZxrES/z5uN7JFAVmSwRq7W/i1HepYEOjaDugoDWw4DK2XQny+HeOuEeyY8KOEhCQ9IuFvCXRP+K4FAyKAxOsVNwx0z8WIJs3ydIztnTA/O0BjBf+e9uFtkeKaE8XW2fJD+7AwJQ8b9v+f9wxP2SzhzwuZep8sKrpXwvITDEs6asBouY39sdlS/4Slv18zFeLhCjvZ6CRsa+0Uzzy4JymRwmovCkX7B06S0ZuF4n4zHf/O1c9VMOyLhyAQLzK1daTHg7ptjvX/ClRLmk48MPPx9u0xHoxkseF05U6bl2F0yzrz2+TI/6nxgxt85YbpOhtneGX/7hPkMNH3cLtPQgfu8XdXXVbL390jYN4GOIEPhzPiWkv4ukvXfKqHrnQvkPTyTxZfJexdngrl+ZD7Mxx8ZPZMXmtkxe2ZsdA7dYkzLQR/m5LLZlnkyb0/IeyF5sdTtkYk3y/pnaS7Onv3ZOYHuzmBVX/QE2cdeuVqOZKEx/Uemoam7Z0hemuu8HS5zblHH1kMT+BjvnitTQQanuci8a2esRXUGw4VuzfW0/GQHXnrIMflHfurL/fIRv2awzrVbPk3XcaeMQ7P6mrfDSRV9VA+5IG4MbKZbZgRZoOxC48gscxe+UKf2OVM9zyUu100f0HLVr14DIVyelg+PSaA0IY0n5Ln5fFTCQxPOmWASGbMvy/tpOCTjCKgMZvbaKXv26AQLD5NpInl9XpRxmMT48nbu4nm2CJAXYf4rU/6W8LaEN7XWTspwcy/4xzCEICNCG5tap4WZBdemlt+Ycn/NzK9OeE7CnxPWyoUeCMkX5oCfknD+hIUuivvpmfioBHOcwTqXub9uxrwggXGewXDhe/EH5xPheIMMCfJpQcXzpD8EuLoy21Z5wcc1c2RkDPnEqLSYhJc+bvzziMxDiTGgGdhPyueF5gfOyDuLIYuiDhaod8hyeB8/UkSUhDotEp6caeRGBo2xcFDeMGgpsgPz3pxkMHdp/6b51Hf48nZVX8ZsrPDGEbF9joahDW8WSH0+MnqzLnVroxsBX8zayJv/y/DHCS4y+C1587GEPySshotDiM5BW9/PDjNGHpchgwTN5e2SXowwzieOI84pMuR/soUbJyxkEGbSJl3ogLxjP2xSBfMUUtd8zo55ss4b5ewyvW5BuFS0OW9DKxBJxjHqn59tPSPBmDJY5zJGMpLso5/IKrJOJjoIDZBTbCF2zSUygaNCXjtd+bjORfYyetlIjGyJ2qCXnpUPXW4KLbYPzzhyIINhd4T8VL/85DPHaac7skQd+Fx54P6ZWdiCRTv4ZM98Vi9b1Pjzce4ypmvkk8UkWqGb6U22UEbPeylDlrN5X5o5lNdW3i7vBZnL28KkdsLwPXlLMfG6UFIfz+eTE36X8IqE7yRQTojAZzQJWPCVjJfngxm6z2AmL0SEuXnarOQsUg7NnhKqX8rQ4uWEDMfXr/PhxQm/TCAQM2gMerh6Zz64z2Czrn9k6Y8mwLc2NtWgZ9hZbVt9Z3XLfjHo35GtvCrhxIS1cpnzL+dg8YD5+nvez3cx7HjCeBUs/P45lQlvE6Z2xOSFz56FkbRPPnwu4WEJvGq89TwZFpYZNVzH5H8L6152U2knq5npi6C2U8GYgw84M3aGCu8TnNw8R8BIYejjbQt3aXidAM/kdS64/2zGUDi8VeBe+fyuBIajOaNIeIkpO44MsuNCmW5eLdYorr3ymTFLOdww76+f0C88yZhHK19qrZGnPW21hvBOCR6bA6Cg4Reu4Rj+uvGQyZt1mWv1kc0qwmuU8C/y4TcJLm2aHzJ8IT6Ub1aADkIf6PLr2Sl9pnvhz+LEc0Yv2QVnt8naGHVomuOFzrt3xpHddE/eLtlFF3A8/HCJaiTzLH7w96ZWSeeTj3BO5m5qPbNQ7r3ZCXKIrcCAJVsyap0LrjiHGPvvzhTGOx4ii9h1t8241ySQo+Qk4/cD+Uw2WVzm7ToX5ycniYUg3SfRwhNNMb7JzQ5PzETOTryL9shPcpl8tBvwxkwnL66eoblgtKMZ9Njr0K/XZvpHEvSJ3jwu7z+TwOjufcjH4dI/fGMxADfGZPHASa2NIdPUP/V+L+O0oZ+eV0R3LtSh7MuSXr/N2t6Q8MkESLOdzWNNYCKet2Y85mfQfyrvCVrGI6BcP59xjA+Mk7cze/00e8Z4zqAhSh4Fq8ejM8LYEU/eNuMmSCxwCIIeL41QtpKFI/cIQh6rVyvom2QmhMwzApdWtxTR+rZU1aE/cKwOTGA7a3oLH1NhSHPjWADm1QbvIUaxOrWzojwGzq6sczEcHa0iCBiN+ojGGEzGxFjRd0qSR1FhfdN3DKKMdMaQsTNorG6tovUDDuQjTBhXVuPGo54OyjBM4Vf/e/xqCf+UHSUIKOS8nfeCGx5lIaPRvMLPOLO5NMdfzUgL4p6uDIFn7vFdJref5D/8ZV6mvVW8T4SRHTTpaNp8ZpHhMle8nGiU59mczEcbQ+YZ/Ic+eZPQ5vtP6R8lzfBmVDOojQcuLbS+cUqeb2dIuaB19JuP61xknsUQQ4fsA5/OHBwX5hbdUmTqM0eZ1CgWhhfvMZ6j1P7YWvtRgjkkN/oOGU8srysjisykdDLbqr/QG9kHF3jBgIwRTBvzaNouCpnFEeTezhOZohzgcSPPyC0yEP2bb1v86uxOFh43NPCFLNT1DLpWlzkjo9G941HkWmYbLvOkPv0WoR/6w+gxx2Si+A76oC/kpHrwobnUH/xj7nkWLd7sVOCvXnZDIWcL4wV9cRLhSfSLbtAPmWtniYzF5/CnDDrqz9ogO/RH3zxPg7GSPxarHHTwhf6MazKtlAAAEABJREFUXVnP8Ap/ZDGc4SHtGzsjiSxx1A9OpZm7TtvqwFf6pm045LhAF/hq7OAxbxYVQDn58S089vLiGJN0JTmmPfj/30yAc+W6rlCWfqOb6azMMlx4Ee7oRfOKFuGPbJQB7Zhb96sD5u+lMbG3OFPR8TQNOCWBbo/P4t34NW70Cmecs2QnGZpZBicDuceh+QMRI0CXcG2Bpj5t92ROCjvL5Cagq8hcfft5ZtIH86Gf4jKqcYSiDfSGp8hNxv7rMlEdgFOYHNdeRjd0qzz7zL24MaiDfO2yyAL5V5kBjWcw78VZY4HAtlXneFzzFliqSMpsqepaqB4EbyJtWyIADMMAtQLkCbGFBrkGzSC23SOviQKMfOUYLpCzUDtbOp5RZfL7RBMGPCOI5eXZOcojg2GbiDDhISXAMAIhozwc8NJhCh5DgpMhYSvISpdxa7vLmU7n4aURNBSWZ4auNuYDColAVQejl/eRsMWM8uuLFTZjjxJ8fEbaPtMnc4aBMLf2CG5GdmaZuyghAprQVD9jhVIDmPmpmZORbXVrcWBlT7BSOMarbeX6KrjjYf8sp10KgOeON1MbhLutXXjsfSEcHD/Rf54cnmfjwOB9nFndzF/4kiGxUEcpRIsfnga8I/84L+VE8RCAb88Ec5/BcEmzKEKPvx9iWpPumIE5pJhOiR4C/CveMRS0R4GjNYYG+jN3tl8pUAr8kCzFs0pJ5u3MX+ibYqEAxjgntCkafIx2KPFxuoExnPAoo8TzGH6WDyCDuYtRogylwLCEV3WO5++7mRs941WKwb05Mw+egfy8T/r9vsxvLjPYKi44QWc89ZQow4niNjgLpG4oMOQcJeH8MUcMQMqb3OlzSoYAZ5zteqB7uEPjPHoMUvhXN6NcaH7g2r3FKR7CY/rEiUCeaEs95pD8Iav1U5/QvnQGqjTGqLo68KCTq/qi7wxc80t2iycXlbVI5NDCa2ihl18oRJ9kngUg44RXkNzEo2SrMaNxepeTDM7ISXTEEGO86YdxSWPs8mDP1x6cGavFExluB0M57VkcGIPx0Gu7ZgWe6QHHyeh39WuXx5XMB5xFcAePxm5hYjxZfHCO0RvK053mVzxdRQfQB+LRCTzAqTEat/rkN7+OkdBfeMqCRDm75GSV/lucwTddY4EAB/DGBtE/uKR36E3j0wdAP+ozWeB5NQNc2aWyw8KBBq99PNLEWRyydeBMmnmWj/3Gy4/2xHfAT2y57rjo8XS/tsiwbjdJY++hKfzvGbA/4JhxrT5zhZa/londLuzlzDHZiHctADPLcOFXNPutfBq3l4/NWIzP/RjI6i5fLRDJEWNkj9Kb47zT9/TGdNyyPhvEsjZwSuUQDYEQRpARHibF1syHT8kDORQoQpEmmoDBXCbAokDcLEMnZAKY4WNCKQAryt5v46coXpIRByQgdOPM2+FiBMAToSOCpwPu5GF0OVPO409QWgQwqGzHM/IY5cosBHAMlwxdR5gsJnguMKT+apNQZXCbG8Y1IYYRKVNGjiNCzrKNx6Q9go4wZ3ATnJjJPFLAGIJgpSx7up0GAlWdhCqhaeuOcrSiVr8xA/0mRIxXfzGxetAOI17/jZ8xCZ8UgdU45U6hMlz1RT9XOxgPXBMoDA00Nh4Tnqb84M2xBcoXPfU8hBpljKbgtcejM0JwrKh6Grrm2bD4Rh8UMLwzHvrc8khQ3LZJKUPGgr70OmY1hAO0RilTVr3PeAEu0BzFQvEwRBgtxgKn6I4BIY+4Mah3THPqp4DQJwUEd7zQFkHmo5dVPxpXHp3LyyDVtnbMO0NLOZ4w82tO4J2y8tzrWo0hZUxZM3Yt4Mkd+OCAQM9wCl8W+jzQHB2OeaBrNO/Mu7nD+wxrNOscLXzKQxd9IhFjR8puKvkkvwWrnSryN5MbA4LRB9/aNNfkF14wl/LoF17jTcQ/ZLu5ckZXX9XLUy5vB0aMRRtj0dyiLUbDmzMD/YefheS8BYuFMgM1k9d7kQlktXbJSpnxpIURjyQaR2+cQ4wlCye069iEMXLSaNtYyXt4lU8904Dn7SSpmxHnOAL5oD3HIchx7X0oC3I6uDenZA58S7e7pR3PjC5jRteAvKanGNOMZOlkPTwz6ODNAoHxbYeL4a59YwLStYmXsgvNvKMVc+soBLlp0aM+Oo5nF03Ri2jIsbpXZkGLFXOsT3iXLhevPvMGV/iN7rLD4T6LrfrLuNARe4M+JgcNCj45cuAaP4kDcI5+4HWa3tEuWsbLaM9iiC1Ahwh53tEFeaquDuP6pcEvvkPL8rCxPKMVzx04ie0i0GXjOqQrgw7ZJtNp0hcCdMAm4piFj74AXCj/FovvymulOmDieZcxj21ML0Ksr21MRMliPgpwfXlnJY1AYLBTBgxbyqP3Db6tZG0LESYYh3Bj1BISnilpZRAcQrZdReAyejEYhULxELaIWxlETOhRIL2t6VDbBD2BJo0CwqgEFmMYnglpaZQegatOW5LqBQQWJpRnGtSN6Rh1FhaOL5g382zrCaMTqMZFIFBqlCgBbUdGujooTUpB+xSEfisjn3L63euBE8xL0PCSap/S0jd54Uk5OGQMiV/NAP8UPAEIT+YebjoYG0OEsWfMlKVxywekA3SFBpXzDChzc4sOPXeQt+Mc7VGg5oVgZ5yiVfRn7rRh7swBQ2e6rl7nLIX6zLCAL7TL4OL5YxyjKXRE9uANBpDdCAtRxhtPKmMMX25oTPCBzxlA8vK0WxzjP8Ypzy2+EZo3OKV4GFb4RJsMDoYOJYn30b9FLHnKIFIPRWnOtLGKYK6rdhCNhfK3KO+0RInKhE45DozZS8fkaB+vRRf6443lrODRZbgz7hj27uU1F8qTT+qklxjsns2LODIRn6ELBiJaxx+APMY7dsnIHDIGn5FL5BVPvrnzYrt49XUgD9EX3Wb+tSnNHDPK0Zn6lUMHDMfeJ/nmA3yG5/GgRUrPw+Chi9BwN3zskpIRZLy86N/iG+6M0zNZzOGjvl7XdMixgA7Jcfmlo2WLCjoNX5gri3v1csgxvO1aMPwY5ObC4gBfwCN5DeeANxStK0vuaUs83Oi/BR++tViDIws59oS51id6wRjIOX1Vn0UBXvZsDvQbwBND0w4Go91CAv1YZNGX9CC9TheaC7SJ94zZGBmz8Aun4lY7mBe0bGcdLtgy5oBTgaHM9pCnjxPeLLzgAp56vNC8o4dehxML5g/988ybH/nQnbl2Pw3qZ4ib755Hf9z38r2MvPoy7p80z3aF8OX66FreacCHjsFa6Hm30Xl8x9aMdzrvFn0mfFaqA4SOM4GUIAa02sGw2ocYq1wT4RmYAF5oyMfEJlz8rANPgAmnPHgnxv1GhAiTMiCwjcX9OI/7cRw8YBJz5R4wrOSBN3X0Z2U9LwTK6oN09fV6GcSMFcJKGoB3hgrFNp4XafMBRrHlTVlSZLaP1anNvhjobROqBCIFgC6MY1oYiu840p48QH36Lk5oDOplBMA5DxmhQ0FgbopF3q0BeC9tw1PAdiF4l+DbYgzNGTOe4Zm0cJRuG5oAdRwKoBVKDH7hr+MFDRCQFGKP6yGcw7HQnMjDsDEfQHyvC74Zo5Rmj+v1zGrIwGCovz47SOhT9gwk/OCFLmPmbXSEDl8wLhhftoAtbii4LLrgBXcUGOMTfmTEvzyc5kidzixTmBZKFBfjBk/woDIyAG8W+qZgGGnyO8bDMOM5xE+MXTyhjdUGaAYeGNGMKYt6ThH0RXcwvBhqFj0cBox9YxRP7sIbBS8dThkP0hlb0uATvVqMMQ7hWDqjhVHP2PZsvhjY6AKt4xlAiZMzdk7IRDyGNiyulTO/jCBGrQUYOSd+DHSCenn61KG/eBHNkaFe7NOWPljc4Mner3E943u8yIusX4zZnmZhQ2Yo38dO/6IPfWZMkx/Gb4GorV52Q6HFLlpF/52m0SY9rj11WfAYl2fvJpD55oF8ltf8MfYs1vQT/uDXs3kyx3DBQ2o+GIdkC94k89CHedSWsubKOBnY6IQBbxz41GKZY8mzxZsjVBZ/+NBYGPDoyM4jGWpcvMrqZ9R7udJi3ztE8Gbe1WUs6mYAa1vcagf8ZgwWSRwQcGJ+6B1zAmfSO6Bz/AbvcNnjhXiMfKJz4J/Nh8fJUCc1yD8hHYXHLZrYC8p2YEeol1yYxLVmkUUOwn+PE6oHjeArzx3YAvrPAWVOe/xiQnqALHF8lYPFGBxpxb+LKb9ieVZS4WJ+Rp7J4uWzzdURS5ATgARTHzwCInycV8LIPX6WQxPsbCYCdFYQEesvBhHHK+SZ8iBU3XcYM4n8PX6+cDp9+nm+MvPF9QUAgqVUeOx7PmkEJaE7zRw9zziUh9Bz9pFSwpjmm2AmlMd5jR0dqJtgXIgOFzMu/VQ3ReDTUwwt/SAoHEeysu555FvNAMc8BJQH3hkrEHg0Tgqd4JRXnj5ec2A+lSGQ8Jy56emeKd4NGafminBklKjLszq0LcQDaEkf9UncrAPeo7R5+5zJtUvGCIBrhrP+Ux48frb5eXvhltHgSIgdDHkWAvxuwaQNbfV86rBgcJbYOybmh2FKkXYjCQ7xCRnIQGOI6Id0eckVyq3PLx4ez2tvazWEdhfwL/1AJukz3KI1Rh65wcFAdtjVgy8ygveVsubxM3YLWzhDn+oYg7Lwpg2OJbRqsWV+0b+85osHmfdc++LgVRsWWAw6ZbSLPiwY5NGeXQXGoHPptvjFj4GThONDeYaSNAarsTMYeKvFMbJ9zlR95l7cQsAQpVvkQ1M9H68o3FkoiEM7FoF0LzmBZz3zzjOIx7Qp//qAsY6mjb+XY6gx4NStrDHR6QxrNCrOfCnL+GOQm2f9Mg94QV2Mdwsaxr8yjDS8Zs712aLBfDE2pY9BPcZk3PBBVlkYmTPP5BXehjN48MzJZjFs/hmmjpvia3hRN5xa7Jsfx9uUFw/0C+Bt9ChuawDzRMbQoYxv+lychd/0+Ng5nGnwCNfT6cp1MH/oUX46Cs+Yc2WkwaHQM1DO/JODfT7E0/fmX5uegbwMfHTT+VY8wE9CNDCuX9zGAPpGz53nN6bssudFzMveSDbAWPAyAUYmRBlcBGomNQKW4OE16RNrYghUjEJ59Xj5ZxUIEitNXlC7D5RBJxyCj9cIvhEbYWbFaZzKEXoIjvARJ5+wj9W9uPEzgSdenFA68DwfyAPGaT0/JYNBbBUTdPJQoDwZjAtMJ055oM/aF9eBh8h2rncgGCc8hgQ1BWv+5FdOeQzq3qpXeXHCji/3QBnh+qCXJZAZAwQQ+vJegfcU1CkP3GpXuL76pG1p0Geg3+O+EGhoi+HpzLSzujxtlKrte0a03SxnFqUDiolys5VPgRKMvGUMQQai+tEnA8PRAfMnroN+mAcgDk2gX20SyOLMpXl2z/ARMhAYme5XAxgnOoUXi1JeHoY2fPX+S8czDBfeJMY9I0S6eIYgcC+uA+cEHsJnPa6H2pWfQ4MzwGBZ+3gAABAASURBVLEaXjDxPQ86MD+OAfFcM/qk88jyeuJVdZgHSo5S7GVXS4i+4ACeKHzjoxt4P3l50SzdwaA3Jww6Y2bckbviGNLGi+48q0Me+GeMq4/xDF+O0EgnDxiA2iTDpJHN5AlZbcfGQoI+QuMMejSOB8R7xj/6rixvpYWF8ZDr+gMYgTy/4s2b9vVLmvaMw0Lbs/rsxOF3Tgpx6Mpi2f006Jc5H/MuncswtrhHI/DAC+3eC4/wbOwMMONklKEz4+46YLqd/mxcPOzkirGIxzfkL28s4x0t0gf0PC88XMunH3QdGlYWDvWToURmeOb886yP8sMToxoNSMeHxmue1am/5pd8Z9CRa+SgPOZN3+ga9ekXGmKsMso8W1QYO+8tZxQ5iib0y5xZAKrLF6u0aZ61q4wPfNgZQHv6JX41gzEYlzGYMzrDPPvwhh1F45cGej7GM73ESCfDLADMkzwAzQNziS952jk86SfvX1gsaYOusoA1T8oBtI0eLNDxvDhAlqJtNIae9MWujcU822ucV7tok86yYFN+GoxVnHqEHdAWnacf4jzjEQsb/ZQfzZEraEmeDtO47PHLFhroslV+SsXawGzO8hGoJh9z+PIID4W3x738gxkhQDHCiCDFJASfuFkGYyQ4vOTKE4Bo7C4YH+CtcQxCGgOZ0vUSKhwQPgQ4wqS0CVj4QpwWOeq1xSgkRCkS2+zivFjKM4OB5LeYoBzGuMJIPEkAY8CrvAgc8VtMEcDOHxJcXj6zZWxrkXebt1J9GI7QJ+D0DwGL74DILQiMCRCiDBPCEn7UTfGq29Y1QW9ujYsnR1+MgyCguPQLEOZwYuzw41l+9/CDboyFQqSwnNN8dHaKMcYDw+hVt5erCScvt6DDzDKTF8EBJ7bCzSWcWhhNCwudN7dwBZQTNw14CU57eYIL7hkilBG+ZJwqZxHWedAz4LE3hzw0cKgM5WhupcuPnhz1QOvohoFAWWtLnlkHAptBhk+do6cMvLTIoNJ3uEVvcOWrKc4FW0BRPsYvD28sWrOYHfOGsmiWQmLsyQvMHaMBL1CCcItmGQ3kozyAwuDYICcoTJ5A8YCCsoOJHxmA+mJeGT/SVwswkMgutMXQY/zCNRlp0UROwAs65HggL+GLTGB0iWdoMNTgjrIlb9EieUVOWKBJs0DAE+QVmYR3ePkYt+S2OSJTKW50wbCER3NITjLmyRq0zZA4a2sNf2oDDeFX80F3GYuygJzSF4syvGJeHfkgG9WNZsgxab7QAidkmf6QA3aFyH11TYOFNZ3CWOppFif6p89kP5xZGJHp8spHl+g3w9Z4yQnvuPn6T5cX8k2DxQVc81T2uuASbuBQe3BKTqNHC7FeB9lLd/HiizcX+sGYxnfmxTNdA2f4x5wxnoxJG+QXIwrf0Cf0Bn6CK/Oof/CqffaGZ+DZuNSPR8hYc20xR6Y5l48OzZPxGZuxwDvdguYYizz5xkOfmmuGKANT3GoGdMxuoX+MF/2TgWwWizILTnSEhs0v+UXXwyGZ/9AcPJlkXryUjocBvmA/kIGZZZ0LH6AJgAbXScwHdoQ24Rz/ZtRw4XdHoiymvJdhbsyfvpoj/DlkzH/mHlgQKpdRcxc6QQdkiXY4Xo0fHclE1pM96oYT6fia/kOTeEb7jtqhK2XQGLpBG/qPnsgp8dKXDQxm2So/pWJtGDikEM4UIcRRkABD28qySiOYTynWCESMomyPm9UQUZosY+QVRjzG1sEYCT8GPabwIhviQgA8rBS5Fy4wBUGOKHn+MBRDQVnnZ+GNwKNktKNdTEAQekb02h7jCf55VLy17vyme8qD0rNCRpDqYfxakFAOiJHitHomENWHGZ3jJbgYDj1eGuAdMrc8H4S71TN8yKsPyvMOYR5GiW/MyktoW7EbLwVEMMOlcVr1++IN/MEF5aBOQp6Rw8AxBkyqLkqBgWMR4rgEb4vxMYb02xgpBfXp8yyC8VPw8MXTbn7hCA6n+0toMSp5bce8M85nHr3TAOc9niK1k6EcZaksgcRD1vP0kKLz8qF6GFryyGsu5dEvaRQkw96c8L4wJKSvBmAcUGZ4A48wzOEffvXfGCkt40PnvrCExsayCd8wlhwLgE/lgLKUIn5Dp+IAnrPQxCvmRp1od2yUyQfUQU6o23MH7VhAqFtfLUbgv6evhhAe4J/cQ8vkAB4lg4wbrRkXA9AY6QXOB2UAnJgLxyTIVjjB93QHeUJOkC+22skitCo/Iw7e5SdX0DkZYU6F8ljMMhjMiTrIFAsAclZb2pSXgcnrbY4YeeK8OMnA7HOgPTuH+JtMJ9fQC0OGgSud8icf1Y8e1Kc8fuX5Vq/nadCOPowNHvxJVqJXdRsD3GoXraqDPiIH6Ab8ij7pJUYZXMszDebLAoPBQ2/DqTwMcO3RTdqjk8hvuFWvPEC96hdPZiiP7uEW7oF7ceZKWXNj/OpVP7zAjzbxLY86ujcuuOK4YXQZO7qBZ3RjbHBEn/I2S2doKu8dGjrWKQL9hCv1OyHAQERL8ExXM/TJCkei9JVxB7/KrWagZ+gezhg2gjHCF3xycBi/ecer4sw/mYhuzaN54jCj29E53SIdLXphH57mww8+ozPmo28ywHybq+myeNoRSXNIjqIp9bAFxnn1hYxcqH7jlp/cVxf6ZmOIQzfoBT6MRTpbFc2gZfRJJ6In+FEGj1j0kxW+CEj+ox/9aE2OZQLIWqaq56pF6IQugmAQ2lKGuA4UkjgMjJkVhBjE4giA51kHk6qvvqpgLAzhPj6h4x9dkMIHwcIQJfQJIoIPjoybUoMngoNSgYdeL4FDGPLWIGQGNKLmWVWGkLMoGOMLQ/IESVOGAGJs66NnZRnnhCuhqa/6xthDvL0u6ZSWdMrNc08TUgjaNg5HQixKeGukAQIewyFwuCCYCQF1og3jtSihYCka5eFSPAXtmdAF6jGmTjvSebcIBt+hhwugPPxTDBhT25QXwa9Pswjwap7NuTHoP4XSeWPcZ/gzdnO10JgIMQKWYhuXRWvwiA7QANypb5zHvflA2wwrfVGmz2vPT4BaLKEbR3vQDWGn/GoAtIgXLXwo/z6+3ne4RzdwRZgzLMT1dCF8qoOBhH7FAfNp+78rAHGAHGA0qBPPq5MckTYGeIRPcmJcb8/D4MRP5gbv4feethpC40OjZBl6R/fGAuCG/KMQO60x2Hy218uy5sRckVuMfHyuPjjxTE7iD4YZfMMH5SuNDCaT1Mf4R79oXXkGDcOAPPOsbTygT9rk0WZA6wNZiP7xlzg0hAYYIMpqExiDPIwDcogBgGct2C2ULQ6MF6AH5bWrLOOUjtAfz9Og3xag43hGp7P69Cr5jj7VwdPa88nTx4Ce4Yihhi7HtGjRYeeA15WhY9Fi3HDZ6zKHZASjTVvmgNwxJz2PUF/JfM4Xbdg5sdBRF3zRI/oJP+ZHn/Ak3jJG/KQO7ZD/2oQXcerv8wmf2sYz5CO+1Rb+ULbXZ9z6oX3yC/61La/+WLgxBslINKEPDDZeezwrTpvaXu1Az6Bx+pgORx9o0ALTLpm5QMf0NL2MX9GYdGOHL+noB4/BJZzCPaPcfMo3BmXoHnxDDo/T3KN9tIamPE8DPkEv2rH4mm8u1EH/o4Xp8saEFvEBe4GdxEbq7eFpcgStmWsyFv/rt7qURy/Gqx1xaIyccFyOjaVO8gXtSl82WAmDXucN2mA6EGQdMKJ4TCUvgCxlxnHiZxn01zhAH1sPjdEk9/4bH0EirzFKwzjSe7w098A9cC+/+jwDz+LdA8/qGYP6pQF5x30Vpz/y64O69VsZcWNQt/zKj+PdqwNIU0cvb/XLi2I3wjam9qXJq5xn+dUrFA/EiwPugXugPHAPKBzHcpzz8/a51bF2ebsoIMKaErJFiFnnEyz6MgvQxw4XxiaE04X6Bg/j9Ol85lQe4XSa+TTXcKvd6fT+rKy+yNvbIjt4sxgjtsF5HeXRVi+3WsI+Pv13P91vuDEucyH0vFCe+dLheb4y8q6vzt6GPqmjP0+HvR75ptNWw7N+wz2AD3QGPM83bjQon9D45BnTMFx3nIzj5dWWOGU8y+u51yWu5xF6BvLrj3o9S/Ms3rN6xOmX+jyL7+AZaEceeXk6benjH0aC+qRpQ95xWXHK9LhxKC/jleGuTmni9EO58fO4Dvfy9DHIp3/AfQdeWbKUl9IxArLVomNsPPX2etn+LOz1CD3rU2+zP+vLfOni5NVPeT0DcXAF3IsD8qh/nN8zkCaPPkrvbYqTJk590sUB8fKZG2D3mMyjQzgv0Kl8WwMYt/F3MG7jEoJ+Dw8d5B3jXx7P0uEGwCs8SpsP1C2PcDpdnPqm4/uzepWdrx/jPOuro49bn9WjPvUqr33P4sF89Ygb05dynuUf1yl+WYFSXtYGqvI1jwHn5whBK13nNSmHpUaKlTdPjZW6M5XOgXo3g/eLZ5NQsb1uZ4JHrzPrUvdjLdVnO9H5b4YITxWF73jTWsJBjbUwsKkYIBOdL3YckJdR6JjhptbHu8lT7ZjERtSxqKy8lTyTjg04X8yQtTO8FuWo45rsJt5sBtuiEFiZCgMrgQGEuRLtVBtrFwM8OV4kZGB7iYVnY6mxYRVti9c2q/cAnLm1bWibrQtdK2WG/VK3vVbrg0tbkD5N6uVFSp4nY63io8ZdGNgYDPDgOSrlRUI8ZMve0Z2NqWOclwx0pMcxiXH8UtzzQDr24F0ax2gcO9HeUtS92uowR5xTnEirre/V360cA8tu0G/l+KvhbRgDDHgeHuB+Ob06lKRtZ15joecN97BybAoGzKMtd/MqZOCL25S6qkxhYC1igDwkp/AQA3FzjWTlGd/LgUvHEno/1zKfwzFcLAeOq87CwGZhoAz6zUJfFS4MFAaWEANVVWGgMFAYKAwUBgoDm4CBMug3AWlVpDBQGCgMFAYKA4WBLYmBarswUBgYY6AM+jE26r4wUBgoDBQGCgOFgcJAYaAwsMowUAb9eiZslSX50QI/fuMTaNPz6ssHPrXlhx6mf3hqc4fps5Da9CMvm1vXfOV989eXBXxhob6iMh+GKq4wUBgoDBQGCgOFgTWNgWnDb00jY4UHD/eMVJ9ZvGm27ZfOfD/dr67l46IudfSMfkXWD5b4gQYG8Dj+dvnwuATfYPejEH6JkaGcURu8fJ7Q99v7943HBXx73K//+uEO338f92ecb3PuLVR8HtGPU/ie/ebUVWULA8uFgaq3MFAYKAwUBgoDWwwDy2GAbbHBrKKG4f3K2V+/IuYX+Hzb1w+M+HU23yPOpA1efuRjh1EuX0zw2Ua/Utg/1chov1XmuXrCIxLun2DRwECXlo8bvHxf3CfVbp45fTs5g7nLwsEPRumLMc0lLOGNrwr4lToLFb/4uIRVV1WFgcJAYaAwUBhYaQxUe4WBpcfAchlhS9/TratG3vS755DOmLB/whMS3pFwzoTFeOj9OBOv/jUyf798NtA331+bEb4XSWmmAAAQAElEQVS5nsFw8Zz7zJjPo70/Yxj1fhRDXD6u90Ife2SOSyT4hUDe+rydu76fdwxtP9a02AVCFtmoi0H/lSzhG/Zl0Cci6ioMFAYKA4WBwkBhoDAwxgCDbfxc9yuDAb9gul025Ty77wYDv/Lnx3l86zeT5i5HcXjHr5MxFgCM6tvm/aMSrp1wkwRn2M2lIynOyp8u486TcIsEHnZHbBztUdfnM86PMC3GoHcm/7KZ3yJBqL/5uM6l777LOzboLTjsCmhfm+sUyAfn7a+VoXS7Bfqej42n//J5c8EEOxXazNsh/gp5412ADJr8FhnwYvx2Ccbty1NQGCgMFAYKA4WBwkBhYE1ggGG0JgY6Y4Pk0f5s9olB/vgMGateLv1I3vu1vwyas+NXy5s7Jtw54RkJhyacN8FxHWfaGcO3zmeG7z4ZviWBt5/BzMh39tw5/Ytl/F4JV0pY7IU2ds3Mv0l4ZYLnG2e4oYvRrc9+AVGfnpoFHpZwlwTHffRNPbfP530THBFy3MhYHDl6Y8btnfDwBO8EXCVDv0j6sgyNw7EfuwbqvVvGPSThyQlwUEZ9IqKuwsBGYqCyFwYKA4WBwsAqxwAjbZUPYVV2nxeegcp4ZWi/IEdxj4RfJ/wgwcVAdSyHR90LrUdn5H0SHH05PMPPJfCc3zvDTyfwkvPgO6KSj82583u11hyv+WCG6pc/bxd1nTVz8bI7l/+9vGfY3ylD3vcMFry8D8CgNyaG+tcz5/0SeNHR2w3ynpGu3oPy3gLmgRlun3B8gp0Fv/Rq8fLMfFbGboIFjrFZ+Oyc8XYZjOngvFeWR1+efKyrMFAYKAwUBgoDhYGlx0DVOKsYYCzNat+29n45f35gDvKQBJ7l/TJ8SoKjJBk03vQd88ZLrQxen5z8Qj4zeBmugLc6o5oFgjP4ju2IFwcYv57lE4pbLGjbcZ2LZAHHY9ThSI/dgYw6zcXoFunIj5dlf5sP4o7N8McJzu//LEPeeYsS3nXjYqT/NON/lOArPH/K8FcJ30x4a4KFifg/5z08eT/AjoEz9d4hsGiw+NA/6ZmtrsJAYaAwUBgoDBQGCgNrBwNl0G+ZuXYsxVEYHumjsgs8zYxW59wZ9s6qO1rDAD4g0xn9vlLjKIrPTjKUp41Xz7z0mX3uEjf3sBE3+seI9+Uc5/Ud3VHcC7u+yuN+GnpbPPKOFHlHwCLCMRlpDHX9dj7+vVmYZ924vKTLg28x4v2Af2ba3xLGl3IMf3Hu5WPIWxw4uuRlWW1JLygMFAYKA4WBwkBhoDCwpjBQBv2WmW4vf+6WTTOQ/54hI5i3/t15v3sCL3wGzfEVeXisAU+8s+YLGa+MXeU6eO7Q4xYTXviUTBYad8174P7beX/NBN+fz2C41O+mh44CvT0jlHlihnYYXpXhcQkuxrgXX42bIe6YkZeELWB6HRYA8k4Do94Z/AdkggWDYzkfy3vxGdRVGCgMrDEM1HALA4WBwkBhIDFQBn0iYQtcvgzjOIsXWhmzusAb7pz6D/OBN5uR7xiJ8/O+cOPcOa+0l10ZvoDhaw4ZuZ4Zy1m8uRcCx27kcw8Yzv1YjOdp8Guszqj3fvR0R2henA++WsOo124+Dl+c0c/ephdfHYH5VCY6C//yDD+eIE8G7RP5z8vAXpi1YHH8xiKmjyGTG/wIO2hLefHG6FiSxYXjN77EI066vi+02Ol1VVgYKAwUBgoDhYHCwJrDwNY9YIbS1j3C2R0dQ9tXbLwUy5vtPLljOM/LLvNcfzRDP+j0/+zdCbxvU/k/8BUpVGRKpUyFBuXSRKJIhkJzUhpIIVIpFRKFDEVFSGnQYCop81ByKfOcmUjKUESESsP/ee//Xfe37/d+z3Huued7zvee73Ne+zlr7zWvz3rWs571rLX3lzLvBVPk6y6UY4qyc+e+Me/YiWMtvkvvfDvr95qR1qcqvURLObcY8FUZ5+GVJ9+3Rhx1CGf6RRl2xMYRGC+Zqk8NdAyIZd7XdbYNTy+3KsMXeCjY64Qfy77dBcdyNo1nn6XcPlzn3X3RxlEe91eFH3/Hh7w3YFFjJ0LduBR8FngLES8Hy8f5fQsg5/O9SOsLPD5baYGhbrC02KHYR/Z5JQKJQCKQCCQCiUAiMBgIpEI/Mf3sBU+KrWM2fvCJtZ5S65djj48qOY7CSu9zjD7L6As3rOPiO3fvjDrFn9IvnWMrvj7jPL64/CjGFF1f0zkt8qSIU4Yp076cw1LfqdBToP241TkR3/Ee+cRtc1HGWcjV6eLwkbcfyLKroC4WIepA2Zb+7Ijj6zpehvUVHoq5BYWjNztG2AFBjubsH646+kLP3XF/aBDLu7rGbVEH1nifrlQ/dTgoAk4I0oZfhLt7kDrBpfP8fQTllQgMCgLZzkQgEUgEEoFBRCAV+onpdUq8r7hQxH07nqJOKXVMpa2QOobj1193jWpSdH2q0dEWZ8adVfdi6bERxt8vtsoHHRN+lHZ5+iVa5Lz5NeFP8aUQs/R3vkTr+cCI48VcZfqF1nhsLoq44z/yRz6BqQ6fjlAv7vr+/U1xz5p+Rri+Pe+zlJWODD+LFPWn9CtHmAUMf9/g9ywvdVW2uI7oqD//fSIPX8Oh4FtY+G69dwt8AhM+8JQmouWVCCQCiUAikAgkAkMikAGTCoFU6CdVd46oMY7JsKw7o28nYESJRhjJ2Xlfx3Fsh2LviJDz9sr06Uq7CyPMKqMlAolAIpAIJAKJQCKQCIwEgVToR4LS5IrDwk2Z95LrWFuzWdodpfHteGfanYX34iv/PwaMdgDCySsRSAQSgUQgEUgEEoFEYKwQSIV+rJCcc/Jxfh+NtTIPARZ/R4m87Op4DHLvl2bbR4nETUoEEoFEoIVA3iYCiUAikAiMFoFU6EeLXKYbCgELBS+1ssp7sdbiwYusQ8VP/0QgEUgEEoFEIBFIBEaOQMacCYFU6GeCJD0SgUQgEUgEEoFEIBFIBBKBOQeBVOjnnL7Kmo4vAllaIpAIJAKJQCKQCCQCcwQCqdDPEd2UlUwEEoFEIBHoXwSyZolAIpAITCwCc11wwQXLXX755VcH3XjppZcmJQbJA8kDyQPJA8kDyQPJA8kDyQO94IExyvOyyy678Yorrrj+4osvfu+xxx4791z/+c9/nvi4xz1uhcc//vHLzTPPPEmJQfJA8kDyQPJA8kDyQPJA8kDyQB/zAL19rrnmWj5o4be//e1lrvnmm++/8ffvRx99tPzrX/9KGmcM4P7vf/+7cCv+nlF9HgtXfmgs8hqvPNQXjVd5k6icMRvH+FIfcBOflI/JA7PHA8ZRGNGKMVWxdM9PWPWbaLfWiTsedVEODLjjUV6WMXt8nPj1D34xZv4bRvnmS4I9OUMfmZdYOZR55plnSBKnfdrI89xzz11Q29/9UHnxl06cfiB1GarN6tpZR/HRP//5z0ahd/+///2v/OMf/2jIfWea0TzH6q1R8B555JEyVnmOph6zkkY91ffhhx+eY+o8K+0bLi4+MA6Q++Hi6lvxhouD98TrjMNPWtQZ5ll4LPaLPjDZ8pvMpL11/HbDRF/AssbxPFI8xK1pldNO57nmyW2X3U7nvp2u3kuP6nO6wyMAx9oX8K7Eb6xx1JdImYhMu++++5oxRcahBx98sNx///2NjB6+5r0KnTnfhx56qKkTlwyYOcbQPtoJS+7QsWYMUU4blxlDR/6kTGW3+9E9v5HnkjErAhXPOkYqL9dwzzUMztW/unCv4e7lJ0zc6i+P6i8MeeZf40jLv5LwGtbpCmvHkw+qfp1uuy7y6ozrmT8StzN9t2fx1JnbLbxXfnP1ImNC66abbiq//e1vy5VXXlmuuOKKxvV89dVXl+uuu64QYrXs2ug777yzoOrPJUx+//vfF+k6SRlWiu0OlGaiSLvV8YpoL9J2pN2/+93vSlspUudYWZVzzz237LHHHmXq1KkFDuIcfPDB5atf/WqRH7/ZaY9ybr/99rLffvuVPffcs8Edg44mT3mh0aSdlTTabJHzla98pey8887l73//exmPcksf/Gknnr7rrrvKH/7whwIHft2qxt8k+Kc//akYJ51xhOMn/d8ebzWeSVSYsjrTS3vPPfeU73znO00f3HzzzQ1/1rSTzdXee++9t5FZV111VYEJv9pO98brLbfc0siy66+/vll049UaZyhXWov0W2+9tcj7z3/+8/Sowv7617+Wa665pimbrLjjjjua/pQ3GXDDDTc0MtPCit/0xNNuKIMPPPDAwIyRac0etcMSrh9hrT/IaITH4a1PRp15K6G+wkfGsTIpBL/4xS/KRz7ykXLQQQc1Y9v4/ta3vlU233zz8qtf/arpw7Eqv1WVEd8qG5+fcsopZeutty6f/exnG8We/0gzIVfMd9zHSiecvDvhhBPKNttsU/bff/9mYcN/pOW148FT2cYDf/kY178PHYIs5Jc0MgTwr/Fw4403Ttfh/vjHPza7S8LkYu4xdsgvcqxTt4C7cONMHPlJK65n+pI8jQ/5IX1moWvsSIfMU8KQcLLw2muvnS4zjWXET5g44uKtu+++u3SWIQyJhz/UQ3qk3PZ8qI3qIMx8K420Q5G64z9tN4cOFa8X/mOu0Gushnzuc58ru+yySzFQCbEf//jHZbvttitbbLFFo1gCWMcCXIcfddRRRZojjzyyEWoaizmA+4UvfKEReIRemwhCE6V8xJ8o0mZkoUJxPv3008uZZ55ZfvnLX5YjjjiibLvttuXb3/52MxDEU08uxsMkmBCj89eWF77wheVFL3pRs8uBOfiPlpQDa4ufv/3tb6O2dsvHIJSX+9HWZyTptFnfv/jFLy4vf/nLm12ekaSb0+Poezxx1llnNQuwffbZp1D+rPQ72yauCfPrX/96+eY3v9ks/tpx9BEMjS3jh0DiV+Pcdttt5dhjjy277bZb45oI2+HuKSLSEXhtAVfzmEwuBf273/1uMXZ//vOfN5hefvnljSyCtfF58sknlx/96EeFPDsixvUPf/jDQsAL74YFDPEyuUC+HX300U3+5CN/4eSffqDMfOhDHyof+9jHmsW9MLwgzKLqe9/7XjnmmGOaRYQw5elffXPOOec0hpDqLyypOwIwgvkFF1zQzE/mqJNOOqkgBgRyWp+K1z2HkfvOO++85fjjjy+HHHJIIXspyuuuu2553eteV5Zddtky33zzFXHe+MY3llVXXbUsueSSzQ413hh5KWMbU9lPeMITinpOmTKlPOMZzyiLLLJI4T+SkuBmAfrlL3+5UKzx6HDp5AuD9dZbr5H1cJl//vlHXF47b2WRl8quY1e4hRIjmT4YaqyKl/T/EdCH+oVuQs795Cc/afSZ0047rfzgBz8odDk4i03Zp7N98pOfLOYixgX9UPMw/5BfBx54YLNgZVgi/yxoq8xjtDv77LMbg6d0Fl6XXXZZozORucYm+Xf++ec3RiVxLr300mZhTGZ+8IMfLNwtt9yy7Lrrro0s83TPsAAAEABJREFUVAfykzzHD8qjv/BXbyQfsoBc/fCHP9zkQf4ysgqDAb3p17/+daPLHX744UU76EBD8VFNY274zGc+U2Bm7pSfMntNY67QqzAFZLnllitbbbVV+cQnPtEo8i996UsbhVb4y172srL00ku7Lawixx13XGMJvOSSS5o4tfHyYdUyaT3pSU8qT3nKUxoiAAid1VdfvSy00EINIzSZTeA/TKj49773veWjH/1oMzFjMgJx4YUXLgSWeutw7cMQT3va08oGG2xQnvrUpzYWOen589twww2LNoqPCZF0rDxIPM8w8ixc+k6SfqWVViorrrhiEySd+Mh94zntn2f+ypWv/AXJm0JjcOgv4TVMeCV+0ksrL8/C3MuDKxy5F4aEIX7CpFPGRhttVDbbbLNm4tMOcZB4yhDXvTwqCZdWuHyq/5zgaqM6L7744gW/D6VYiMOiceqppzaCxqKWX7uNcGHpJWwp7+0w95SJZZZZphlvhCy/NhFCL3nJS5qJ/YlPfGITBG8E48Yj/rlHyhOG3EfQaK9xTQe3v/zlL4UMetazntUsvllQKVcmETuG+sVkQpH34pHx/Z73vKextpvs9IV8OisOh4svvrh8//vfbyaid7zjHU3+cBVfuEUT2fHxj3+8MWh8+tOfLq95zWsaRY9iZEJbf/31y5ve9KZmQpQf3paWgnjeeeeVxRZbrFEQ9VlnHfJ5RgT05ZOf/ORG7upjxhMGF/MURduY0tdtLGGNr1G959ac3Qsjd4wF/vpXHhTjTTbZpChT2fwoBHhNOvHwn3vzgTjyQPJB4tQ+bz8rUzrh/DuJvzionV+Nx6+Gyd+9NIiFkUJu3jZvCecvbU1X46uDcGHuzf0Mb89+9rNnmJvFkUb6ei9PRNkmp6ZMmdIsouXFXzy4cvkh/vJB7lH1f/rTn94Y/573vOfxaoxir3zlK8s73/nO6X3QBOS/IRHAoxRmCrq+ev/731+23377hvAzGURJt3P+6le/utF36GKUcgq/+USfoNe//vXlrW99axGPwm0cMHTqp0996lONEm3+spCm6ynPeDjxxBPL8ssv3yjt5C0DJ8VbmPiUc7oWBd6inDFY3dZcc80ib+NI+eZSZeLnzgYrS5ni7rDDDtPl7xprrNEYEcl+1vl11lmn0WHFYWAmc8ls+Xfm6dncvcQSSzSLeAuGoeKJO9bUE4V+3rBMvOpVryqI4gAYW3hWTEsvvXTZeOONi1U4xnnOc57TDEBCQMNRbSSgbVlgCB2OWax8dDAF1aBtx6/pJsLVlgUXXLCxtBA0mOU3v/lNwZgYwiJGvdRXuzAKyx0rKyFYw9xb5WJY8eRjgBB4FAcMiCqjmBysIoXLWz6dxB/JSzrbzYSne+WJX8PkZceAMigNkjfFgnJIAZFeWukqyQeTU/itwA12A48g1u+UU201Sag/i7A0yGDTXq68tVe+/FgCYCueevCTj/ojmKgjUpbFn/rDV5vwH3/59Tvpb/W1AKNsa1O3OovHAgWLFVZYoZm0+NW4sIKlXSLCrZuFzUKTVdCCuJ225sFVfiX9pe/0r/5UBnz1lb7VNxRQfa9PhMtjTiDbrcaRiZ/sWmCBBYqJAe9R4rXRJKRPyB2YkD3wO+OMM4o2w6LdVs94+dBDDy12mkyKz3/+8xtFXXr4yNcuozz05corr9womvrMWLLNTJkhMyl7MGYRkx5RPvWddHMKj7cxmqh72JMT+nfKlCmFQcUkvNRSSzUKKPkLVxjDlRzD9+QWuUfu6Dv118/4XRh5ro/kb7zoK+mXiYWzfhRXWvlR6JWhn6U1RtVDnupm3hPuWRyKDJmqTvwpFvLnZ2FHRopbSVni4U3jUpukFc5VL3namVIWUjdlyUudpF865mtxzOGepYWbsvGntjP2SC/c8Qb3sGSA4yeN8oTJS7iyzSXt8sSjB/CDITIPkefKV3ekvepqfD366KPNER3lyF+dLCTINfmIp47aoQ/Ek0dSdwT0AazpWfSYTTfdtNmlwU+eV1lllcY4yfhKJsIU3/In3372s58VugLs5aUPyVRp8YC525z17ne/u1D2uQy/+pQMVitjCH+Yo+hUFPLnPve5TT/jNX1IplooM3a84Q1vKK997WubHS7jWRrl0zEtSI0/9Zd3JfUyb9ndJuOND4YW+ZG10jOUrLbaao3RWP2f+cxnNvfqqg41r7arzfiPTDaXDxWvnWYs78dcodcAqyJAEJIEoC0MkyYQ3/zmNzfAi6chGk2YVSsgPwQYioOO1PG24nQwsHW8TiIUAS/+RJK2YFZ11A51p/yYrNXfAgZTiwcDFqADDjigOUd5dmw1Ef78CVLb57ZqvvGNbzTHKAgzZ+xt9dgCswW/4447Nkd5nEGzUHLO0XaxgdANB+XyNxj0g9U1SyBFgj9mJwwdC7A9rG5WvhhdWwhO9SKIWenVwWAQJj1XP4tja5PiL45tM/mq2xe/+MUi3KLMitrRBfVxpMOxD9tiVr477bRTY9GcOnVq0c699tqrOfevrZ///OeLZwslZVjYqY/2qYM4X/va15pzmPvuu2/56U9/WhxfMRmLo679TurZps76aqcJ9bbbbmuUv0UXXXSG7WnhBI6tTuPKzlBnHp5rGe6HInGE6Sd8o+/wDSUVL5ic8aajQXA+7LDDiq1XVmtp1EX6fiZtpJwZg7We/OBKMBvHJiFx+ItjnBozJib3eJx/Je2mtDFCuGcJJgPxtHyMdXEturgmLMd9WMFMlPwQmaIvyTj5KJOfe3LBJMQyRnGSJ5IuaXgE9CPlWyyTOFwpjhbA+pwSAGd4UhDtsDjbTeaQtyz6F154YbOQJnPJGXKHbNLneJ+/4wqskhQcZVIKpMNbdm2VLy6lm8IpnKwin40pir26UbA8s4x6VncymjIib/7kL76Qp3qTz477eBfrS1/6UjNf4D3pxTUHkcvmFcdiyWdjWxgFmFymGDHwyINcvuiiixrLpbRkNjlODqmvo6b4WRi5LY1xozxji8xWnqMPjlB4n0ue+Fs4WWK3hM6gHcYP2S4fspwF1liEkTmTn/6wqFVH+DpGpQ277757IZ/kbR7SX3bg6jiCUdLQCJjvEdlCNpFx8EX6hjGDv6Mo+kk4vcdu+mtid5Eeob/xoVKkqy4lWb76WX+IY56i19Eb5U/PY9TQv+YdvKyP6ZTGCV2K/oe35CFvY0g9+LfLc4/EaRM//Kp8OqjjNI7bePdRHYRbrCjLszpYrKirxQM+FKedZ73nry71eTzdMVfoVV5jrY6AZRVO6FDOdYijJADSaHG5Gs/1XMkzocoiZrVtcPIjeAk0VkxlSFvT9MKd1Ty1mTAnQAi0973vfcXCQz21h8AmqCj5hJUwVhaYiGOSprARrPw8s8aw4GE8TMfiLw/xTBgf+MAHCoZHmLxbneVl8MHS9rIdA4JRXfWN7Sx9RRgSvKyPlHtC3GqThdFEtPnmmzeKtsGnP5SlTGVbIFht20ozYSnToFFPSo80JgEra/WnvAhHJi3WGdtaBLt2SwcH5YgjD1gY7I5FqJcyhRHgJlWraC7BwhUuL/Wc0wme8MD/dra01bio7RLuXj9qMwzwFL/ZIWUQsiZgSicFAu/gSX0Ee8qtSdZ5YGfQLT5np8zxSmu8mmSMAWMThsYppUb7tJ2cwc/CtZWgx/P4DnViLE9WUUoRq5U4trCNdwtefIynYUpRo3DhZ5MTxcpYUgcGArJUPvIzCb7gBS8o0uMBEyOZAG+LLZjLVxvGC785rRzYkINVAWC0sEA1oVNSGEds9WuXftBfFE7+zvzifXKMoYbstBAz3hyZotDgD/1tYWBnmQJCrlF4KNT6Ur97VhfzmTTyNF9YQFBuzAnGOl4SBz9aGOpfiit+xDvqLJ38hMnT2KdU4RF8RWbLy+IBr2qzRYhdKAsDPKoMO+p4TD0pdHjPUQfKO0UGn8HCHO54g3bYCSWTlQ8zxjc7XfK0IFCuhZK0FCHlwY68d7TB2NJ+GJIh6qeelCdGAvOOM9bGmzTCLY7V3TzDz1hBLLXyVLZ5Tt7mGroCJVQcGKlnUncEyCCLNLzN8o6f2jHhT8klA8WDPx6VTn/TR+zOGBeUcTzUmYc+QPKVH/7BZ/ibvGWgYPgVxrDo6KMyjCd+0iLpET/jRjpzorrwH46kp3tZiJK/ztDjO/IX7ytPHHnYDbfwZbDyjNdrmOd+op4o9LWBhJAXKKysDTDnTwkMYACs3dH8arq2yx/xwxwmRukI3eovrB9IvdSJpZmlwrlXwg3D1TDCTV0prYSUCdrAEcezLR/bSeKLx4KE2eFnksBMGJ8SQgBKQ7AS7JSSmk7aNqmXAWcFK678DCSTAWHO4uecmzgEIYEtbxYpQrwzr/aze31jUBHc8ltrrbWac/sUT4q3CchgIwimTJlSLA5Y1FnDtFG56uQl2LXXXrtYtMBGvQliW3r6XN3hwIUBhVIbCBYDcenYIpaXhaAylI3wm3rOyaRvCUn9AkPKnHuEL7gmc4qnSYySoF+0He5oNO2Htb6TF940IcMaxrDFv7CWP373TBnSd6MpbzzTqCPFGbGo2kUiryhe+Nh4cQSHskPxYBU1VmBMuTO5UcCm1blx5GmyI//gQdFgJXSOlzJFUTHpiKdPYUs2Ur4sIsgPSiecKYl2uljDnMGnrJAtZIBzmurj+IOJ0CSq7vq7qUj+64oAhZsiWmUPOcIaTonW18YRhQaenvWBPjLeZIgnyDPHKVmW9QvlgNLoXK+xoO8ZLKTHW/oET1BwyTFjSd/rL/HJcH7GLcVCfuSYeYFcFc6QoXzy2fhzLIasfMUrXjH93LlwPKws9SZ/1fctb3lL866FcemoBPmKjFnl4mP5qCdslEsZJmuVh8e1STgZY0FELmufhY6PXRj3FpvIIka48vC7+eQ1Yb1VHn9YKg8G4pNf5jNjwhxvbKk/Hsf72k/e4HP1NXeY79SRAqndxid/8eQnL/gaa8aLOElDI6Av9Lv5nquvh45dmk9tiyudePgDv1hA6ienDPCh/hbeScaZcYef6AvmGX2mXGOE3MQndCZ8ZFzUsmpens3/8jH34y951PDhXPGkx2P4iIymT9hF0/6a1mKWbmQOIL8thvGetDVOv7g9U+h1Cuuut3wBZ0Cb/AHlmfDEDIAAjAHI9dyNhEknTwKKsuK5W9yJ8sO4lADWeYxtRQkH9cS8hDkG1xZCCRbSeBZHvbn86r3nTpKXONXfs3KkGY5qfK548CfsDChkQPEXjolNdCYO8fjVsLbrXh4EpuMdhK9dBLsy6lXbJn0lK2n5G4SEgDwMRHFhgtzDSJh0/Nwjz1wEU8LDpAhzAtyzOiNlyaudRro5jfSvic/2ONxsadp94GcxA3fbgiZDyigc7IKYGCknlEKk3fDgzgrBTx8YpxQLVjzYt/MQblJH+Emadng/3qsjWbL11lsXC0uWTbtVFAP8a4EJexZBCreznY5V2KGoVkXKl0Mus+oAABAASURBVLbX9rmHOWXHJCMfvGx8UAz1D4VdPyhffK6yvJipH2ErHaWLJRLZ1ZNWfEqLSY7yTwn0AjnMfdED38u71ifd/0MALvoN/8LWrjFckfEEP/LDuCH7yHCySv9YCJjwzT/6iEJvJ1Mf6xOKAUWEbDZGWfb0t/EgvWd8gN/Ug9w0JinLlGYykJ/dGIsDsssCT58rg9ItH8qF+BZznuWvhfiUckOhtxhUT/JV/hYV4qqzhQJew1/qTRFmaFMvcSxWpaFgqw9e1HbjnoIFP4sJbWV00W5Ktfwc9RNfXGUrD352hIXLzxjSNmUoH87ao17SOpqmThYWjl3A3GLYIkob7RBIw4rPCKRO6u24hH41DyjLOLCoUlbFCE79Q/1VExjiXf2C1/ASv85a8kMwpSPowxqHP3nkZILFLqVef+uPGoerH8lAu0142wJQWmH8GRLpSnbZLRbNb3Z3KNLSilfJWKNP4nk8V/1H4ipT/Y1HxheLTvmRAdILx092fhyFNg8YX3hSGnH6iXqi0GsogcfCYWIj1AghQkPjgWUVZAISl5Az6Ds7XdxKwgg21grgEgDAruET7WIyAoQyT8litcHwmEXdMCKh6N5Kk7+2e+ZKz/WM3CP3yD2q99xKQ/l3htfn6tZ08Fcf/VExFUbBJ0QN8pqmusLrvTQEgIG39957N5Z5fU8YE+DiVpKGHx7AF8qWnv9QJG23sOovPxMW6xgesU1OKasWOPFQJ8bd8uwnP3Vu1wd/mfRMUs6OUkAo84iQMfkbJ/hMGKFonHmm6KPOPNv5D3df0+krk64FFOXeczudfuVnUqhp2uH9ek8hcRTNeWCWGpOQiYliZWxQtliRTDDOAVNmLN4p4PpEHO1Fxgt5pr9MNLXNeJ3yQwERj391YWYMUcJgqzx+eJbcJEtY4il7lEjPFm3GrLLkLS7Zow9qvspImhEBu1zwgjVehhsMYW7XUp9RtOEsjn4gY1jn4Gt31YRO4aGIyL2Nt3vKKKWUDNLf5kHjzw6LsSFPcSzKLM6MJfmY3yil+Es+lFTj26LR2BbHPSuoRWenjMW3yiJbxW2TMO1yVIuSK3+ygezAlzBQNoVeu+ABG20mW9RTfsL5wwdvwkZ7+Fm0UIAsDmBJHrNsmkuUh18pZwxAlHHly095cIIrXPSLtBZczsVbXChDHnYnHe3TdrJQucLc25HQdn76h1Jp0aF8cdQ/aWgE4EYxxqOMRvqBX02BB/WrMYKP6AdtXN3rIzqaXSGLM+8x6Av+8pEfGWVuwid2aoxH/gg/eM/CyQT5O37ji4HGCgMG+SqfSvhT/+In5Vf/x3LVB4lHfpO/9AjyV32EqU8Nx+sUeuOEH6pxuJ4nmnqi0OssCoUOIKicnTMIK0gGmfOeBIbOwSRVKADWM2qDI09bhSZPQrYdNtH3OlO9WXcoWqw9mFT7tN+gsLNAkSfIvNSLCbXdipI/oW1lyg8W2oSZKg7y94yEiaPc+iycX40vTqUax7N7caSVxjNripU2QWugqjPBa0KypUvQim+wcKVTlvyQPLTHUQATlvOmFB1nOOWnPMoNHPCAycEzIa0sechXWL2Xpz737F4eym8/u+cnTH0JGpYg8eXt04IGqfrCtpYrXb8SHPAAUm9tQ+7xjsnNuT8v6XGNK8eRnDM1Low1iyrnAp3ZppxSAJxt9L1eeCEYwVt5sFBevffcJjyqL/Az6wWcTZye5aVf8YO+FCYuq8tQ+bXz7od79YQxnmT5Y6WHB7y0UR2Fi4fPHCGwU2LRbnxou/6hSFFQtJ/Sx0JvUjS+YU1ZEYeSByuLfGHKEk4GwNJOprKUi9yzVpEhrEgmQfHwg4lMndSBn8lGftIlzYgAjGFOLhkTJm79qs8oF/qQ0qr/GKSEkx9wtQignBhj0ho/CNbyMEbMdxRyY8KRFEoR5Uh6/aefLBDJSmXYBcAjlGvjSh/yo5DqY4oNntHPjpyIw6KJT8lZsk6ZnmtL1aWSummTsvEWhV5c7RKm3c6js7iTl3hSHG2n2Gi7e0cePFPM4WeMWHDw025l41FpLXLVDTbaqe14Up2UZ/FAYa/liW9MwApm+khexgCZZtdMHMfcLGYtYuRVlUlt00ayHfYWGcqGpfzgrK6e1TNpeATwLn6juNLTjAt9TA4iOgH5hz/tlMAeL+kzuOs/fnjHF2zsSuo7vC8OsmikK+EH84S+0494A3/qKyRcffAleYpHhLdboCz8xCCjfPHb4eqG+HPxTg23yKAX8Ef0MvmZU8Uhm405PKTtXDxu8cmgIo76GCNcz0gZcNBWz/Ku9557ST1R6A08Kywdp2GUcFtxFEbbZCxcOlBDDUKdi3l0ongYBnAVBB1lULNWGMiYCfC9BGZW88a0rHs61kqRJUR7Tegmf22CAwWAEPflBKtN7cbQBDnc5GPyIDRNPIhlrvrDwcTOT97SKguejgGYACputQ0seeITcPLhKpfAhbd62bI3gaiXiYsQZ9mwswJ/bbJCVbY0BlvNX3kGrH40QbBEGUAGhsnKvQlF/YRbmJkUCXX10UbhrDsGDQHCOiPMIHMPE20weNXdRCadyQBu+EW95S0fwsFXJ3w9x6BUroWGdzqUoc61/v3iqpO6aZe+gAkeMeHid4S/EEHEJXRMsp6FS8+/En9xuCZXbVUOvtGXFHBl4SF9qq/FQfrNYo+fBToeZqUkhCmW/JExDHv9i28IV4qMPOYE0m7WPcfEjGF4WQxqBwzIJeNRGyn7Fu0sUCxH5Js2ysPL5F6eIvdgJA9jBc/Bjku5IMPghkftJsHUmMG3lCFKn3Llq69MGMLlKVx9kPHj6IQwMkYahgQTIF6QfhZo0kclS1gLyRL4MECQF16IJatZFOEJP/LO+NBH4pMvxow5i8yhYFOMvXeB78lv45VF0Xg0hsk+8lg6YwSPGJ8mfH1lPsMH5F1VGMg4ZdQxLy95yFsdyHy8Sp4Jo9RTnuSnTerEOOOoK3mMp5QtDiWEy994J1fJBPwuLy4lRz7mGWX4YhVec/TIPKEe5hpzg3ZUptEOOFH41EU8GGpXLc9Y0FblKI+M0ifS8Fc3yr9FlXr7ITaudDBRrvbDWt+RTcaKepgP4Mz4BDvtEs/4YVQSVuua7tAI6DvzPGOFY9JOHDgzDnd8rk/MJY7UWHjiF3xknkLmZjyOZ/Abo4hdFv2AyDKfxPSNe7LUos1XkczNdbFmocg6T1f0pSZyl4y0i2Tekb8W6Hd8io8sQtSdfyV1UDe84YiYNuA34cLkqx5kgPGAz/AeGV35Wf28lGshbTzgVTqcsSCONH64UXrP8qZXGdvkBH/3cBLWaxpzhR6oBpAJ3Q8kWekB1Fk6Sj5hRBiaEA12yhhhavIUnwXEYDUo24034K2+dSjglNMOn8h7zEFpJFwwrw6s7TXRYioWTROFSZjVFEOYLAglL2Q4w2vlBx8u5oWbtAYOP8KeUMPAJm7KLaJMs2QYMJR3jN7GA5bycISAMg9zlj3KOkYnYK2k1YuCR2GhqPh6jnpqn/pQUAwmCov0tQwTgInEzoR+cnab8mESEKemd2+iIbh939aA1z5l2KY1gWobgaGtXsBUZ0LCYIchy41BSdmXnnKkzo5gGYxW8ZXwmbgmBYsoC0EDEH+qSz8S/PAATLVNu7XVs/7trDMFBOmvzjC46xcCCJ+1w2FmoiU89S3sCVtjq8ZTJvx9AYCfvqOw2O438RqD4sgbT+AbfOZlNn0jXLp+JpgauyYrPOdYje3d9gQBR8qKSYViYZIyXoxn7ddO/tLgY+OPvwlRXnjQgki4cQ4b5ZJ1+F0/kBmwtggWVjFzj1/xNNli/CkPUV68kKgvjHHj126Nsmv6dEvzwihM9CGZCmcy0wv8JnN95xO4jgzqU7j6oAFe1jf6U7+Ys7gUVbLFOxWwxzvyN14pQyX+KAXGhXjiGDfkE9eY1H8WZ2QaBZzyr6/lT1aZG82J6mDOwDvkl48mUA4oJ+ZWi0plR5HN52vVnWyVzng1pin96mycG7vaboecUg4L9Ta/aDuetMtHLhvPDC9299RFnZF5WD1rucp2r212n2pZ0vhKjfFFhmk/ma48+gGZZSywvmobXGBAmaQLGBNkN54WHw7Gp3FqTqBUytOYUKay9SVMyTFzkLzhD2P1THpsBPSxvvTL/vjF4ow+Q4bhK186mzJlSjOuyCZKKx3CwtE8oj/0I5fcIqP0jZLxAv7Tb3itjhH9awfIWKNbGIt4wKIMH+JrshS/4DV56WNKM97lr978US3fIsOcil8ZPug+4uGTpZdeuvk0uDGO3+kW+FN6ZMzhHzqTOlDmfbwAH8oDGbfawZWGHxzwrnkZX9Jn1VOdxOkljblCr9I6xifsbPkjlqtKnr3cQOgQII5m8KvkjKpvWxNCwNF4HUjxwEhAtvrm3y+EsQhBRyDUv7aVW9tFCcBElAPM5VNgPuFIuGF4Cjoh7l4e0jkTjrEdr/AsD8LUcQpxrB4xnE+mCWeNlg8GrtjAkKItTBpC0KfFWK+l8d1wCjWMLZbEU56BjBn5y0PdKOjaSKhqS7sMQtMEQdHR9+pOyRYHT0ivHvL2aUsDmT+Brx7IJ6qkMSlqK/wQTDzjG2Qrz4SrLsLlR6Exgdj9YSXVVi4lx0ShbfDST+qiTerWT6ROBJe2wkMb9DVM4S281tc9voOfhZZFUg2rLj4wblhAKA/SCOPqK5/oUoayfJKSgJNGnEoELJ7zHWhY4wHjVh7KF48w89KavsVfJgPhwvqd1JMCYCziP7KrzR/CKR6st8aGvqG080fax6WE4TUKO/41box1ClP9FKyFu7z5S6MPHI+CKx6mcPGXZyX5kHnqZbxUf64wY9RE5xOGZAn/zjz4DTLBAx+Te/gd4XkEe+ODTIaRuFzGGZY3Y8RET755MVn/k5fiUTTJI31IJhoP/I0PSql3MiiqFBtjSF4ML/I3no0VL9rpd32pjowoxqL6iGPnVLl4QF+zXFJ0lEkGdvKE8ilL5hft86UQ9ZG/ci0IKh/LjzKE98lHeZE/jlKoq/HsnmyRr3bgfRioDz95IvlThvxWiIVGxQLm8jGPUbrgqjzKjnTmK8/a5VmesMbT+kZZlHH5U5woU+Ym37HXL+2xpM1edIYj0naWX4sreSeNHAH9gB/wK37ES3idDKNwC4e9vqGX1TFlLsJDwpWm36qy7l5f6LsaX76IzoDnpUN4813velcRhl+NFWXJQ76IHki+4iUyml8leeDbqhspDz/hD+0Sjl+Vq134zGKFvzzwOh3F+JSWfDWmjf1aB+3Ht47Bqjt/edNdjR91R+pgjNe85d8rGnOFXkUBzWJni3EosqLTQBaqzjjSdioWwGOZ4AJNOf1Ej9VmW4zqq+7aZmWLpBPGT7j7igeMUH0WJh58+EmP8eDiGQlXhrwqsXgLQ/LzXPOQVj+IK604SFkYlD8Sp6ZTZ35tEldd9Ke8lSOcJdgK1yrf0Q6AuDqkAAAQAElEQVSrc2HiC3evPKQ9ykHK54fcI/dImloXZbFCseTY8bDbw8Jtxc1yAAtChMLP2m3gWTQoux+ps+3aC9Oh6goLJF1nHG03XuCqb9rhnmEnfySOuNK047nHF8LFg7uy9J/dIkdRHAnQx6wQ+kmYdHMKabf2IW3Vvs66a7f2a5/4neHaLAy108NZvtJ29pOw2gfSdctXOfy7jTlhtVz5qDu/pJkR0CfGkX7oJDJQX7RTeYYpgiuc9aN+ci8uf3mJ09k/+EW+4hpT4orHlVZ98IM89S8/VP2U71k+NY68+GuHvISJ00niSKNu4iqrxvEsbW1HjVvrL658xemWXl2l5dY8q6tt0tW8+CtPPtLU+qubuMLFbT/zE0/8djr+ylRfYcqpeQjjLx/laQNyz0+YOEmzjgCMYagvEP6Ebc1JX+FzYagzXDz46+d6r+/EbRM/ZYmD5CuvGsc9P2GV6riSrl2nGs6vXTd54YkaLp1y+eMp/FXD5K282nbxutVBHsrg1rTuxZcvEt7Ou8brhdsThb4XFc085ywEDAhWEkq1Ac2y4ziQQSFsrFpj0Fqd2+lhTaDUO/Nni8xgYtVhMWA1Zom16h6rsgc1H/1HQNvOtDhifbBgIrSEDSou2e4JQyALTgQSgURg4BFIhX7gWaA3AFC0KfK2XG2xOR5jy6q9/TtWJVMiKZXytz3tGJEjDG1rvO23ulU4VuUOaj761tERR59sRTq+pZ8t4IQNKi7Z7kQgEUgEEoF+R2Dy1i8V+snbt33RMgqerSvkvleVknctw71yuMg9at97Tpo9BOAJc+R+9nLL1IlAIpAIJAKJQCIwWgRSoR8tcpkuERgCgfROBBKBRCARSAQSgURgPBFIhX480c6yEoFEIBFIBBKB/0Mg7xKBRCARGBMEUqEfExj7IxNnyVG32vCv1C18dvzkOzvpR5J2PMoYST0yTiKQCCQCiUAikAgkAuOPwPAlpkI/PD49DaWk+l4vcj87hTnD7HNjqJ2PfJ1x9ukkn2DydZJ2+Ejuh6ufvJXpCycjyWs0cbxc6+s4yhpN+kyTCCQCiUAikAgkAonAZEYgFfoJ6l2Ktp/yvvDCCwvyi69+npriPdIqyUNcCjeF1w8g+BEG98L4+76qX0I77LDDiq+/+JEP3w0XLu1wJI4FgM9N+sZ4O668+fkZZz865JfR+LXjjMW9L6f4VUQ/WOIXN3tRxljUc7Lkke1IBBKBRCARSAQSgTkPgVToJ6DPKMqUZEr22WefXW666aZy+umnF79k5p4S+1jVoqjLo8bzq4J+tcyvn9X0Pi1IGfaTx37p0i8LUryvvvrqmmxYVz533HFHsUi44IILil2AmkAbKPvqe/311xf1qWFj6SrTt+N9Y94vcI5l3plXIpAIJAKJwKgRyISJQCLQRwikQj/OnUERZkE//vjji18T22677cp73vOestpqqxW/tnnPPfeUthVafIo1P/dIlSnpSJhn33ffaKONCsWdIi++X0Xzy6l+uewZz3hGWX/99ctuu+1WKP4UZemGIuU46nLxxRcXv8J65ZVXFvnwl8YRGz8WtfHGG5cFF1ywdOanfHXjit9J/Gt4zZPLv7ruHbN5/vOfXzbddNOy6KKLFs/yEkd65J5fUiKQCCQCiUAikAgkAoOIQH8r9JO0Ryj0f/jDHwpFtCqkLOsbbLBBecpTntIorcLQAw88UBzHufvuuwuLOIWWgv2DH/yg8b/zzjuL/CjY9957b7EgoFw7137bbbc1z87OO65iAeHHlli8HwtaZd93333N7sFaa61Vbr755nL//fc3da5plaP+dgeqH1dd1EOZNQ3lXJ413HEj4eosvjDtqMeOpFe++I4hid9eUDjuIz1stEu8pEQgEUgEEoFEIBFIBAYRgVToJ6DX55tvvrLccsuV888/v3z7298ulHtK7corr1ye+cxnNgo9ZdlxlpNPPrl8//vfL1/84hfLUUcdVSi20lFmKfY//vGPy4033limTp1adtppp/L1r3+9sMw7KnPccccV8Sjj0l522WVFOWi4ZlOu68LBr6uywrPWX3LJJTMo9PJQTy6STryzzjqrfOtb32radtBBB5UTTjihnHPOOYWCLt+LLrqo/OQnPynf+c53yu67797gYPFx6KGHNm0444wzmrSO+tx6661Nm8SjvFtAODIk3yOOOKIJO+SQQ4r2Kl89kgYTgWx1IpAIJAKJQCIwqAikQj/OPU8BptBTkl/+8peXH/3oR2WPPfYoJ510UllqqaXKkksu2SjNjt+cdtppZfnlly9bbrllecUrXtEo9Nddd13ZcMMNy7LLLltWXXXVstVWWzWLA9b722+/vbGiU5qXWWaZ8oEPfKA897nPLS94wQvKNttsU1ZfffVC4R5Jk1nLnbdfe+21m/wp9qeeemqzSzCU4sxS7zy9Rcgaa6xRPvGJTxTn3o888shmN0G5lHkLFbsR2mVH4uijj24WHsJvuOEGTnnjG99Y7AzAi9X+rrvuahYjAp3nV7+PfOQj5W1ve1tR5lVXXTXDUSXxkhKBRCARSAQSgS4IpFciMOkQSIV+grqUwr3rrrs2Cjnlfb/99mtePqW8skKzqrNqs1rvvPPO5ZRTTmmU6WuvvbZRXMWhQDs7T9l+5Stf2SjeFGBNcsRl3nnnLTWeRYT4wkZCymdRd26dRZ/LWk4Z76bQ85O/3QLp7DQsvPDC5aUvfWlzjMiiYvHFFy/nnntus3jZa6+9mvP84mvz/PPP37xHoM6LLbZYcW5+nXXWaRY02iZcvbXvzW9+c/nwhz/cvINw5plnFsd2LGiEJyUCiUAikAgkAolAIjBoCKRC36seHyZfVnJnwBdYYIHmhdgDDjigOG7DIu9svDPhzpCz1nuJlbJ/4IEHlp///Oflgx/8YKPQs8IrgoKL3FOo671n96jec0dCzqp7CdZRIEddLDxYwSnqXrLtlodyKP5LLLFEsXhwPEYdtcVLuhRy7Xbmf911122OEO2zzz7N0ZzDDz+8rLTSSo0Fvi5ApEXytThByq0LB5/iPPjgg4uFkcWGuMKTEoFEIBFIBBKBRCARGDQEUqEf5x6nkHpJ1bERL4zOM888ZcUVV2zOjjuCwyrPYq1aLPfis2wjSrFjNZRX/uKg4e7bYTUupbnTXxji73iLl2q/8IUvFGfVLSb23nvv4qs2zuGrt3g1Phexkr/sZS9rLO1Tp04txxxzTLEAeNWrXtWkrXG84OuojTaxxjvzb4EjHFHiuZ2kTIsCx5TsVDjS4+tAMBRXODcpEZjTEMj6JgKJQCKQCCQCs4NAKvSzg94o01I8fUPeD0pRXinYLNuI1d4xGsoz67aXZp2bpwT7pjwlv7NYSi4LNleYe6Qcyj8/ZSBnzynk4goXVskzUp4v4aiDIzAs7u7f8IY3FMduWOvlhaRVhvIo1nYW3Dv7rwxn8J2Ft3vA38vAPoXpCJGFg10AzxY5wuVX47qvfspQnniO6cDKsR7tsaMgXHnwlC4pEUgEEoFEIBGYhAhkkxKBrgikQt8Vlt57Uqp9/cUZcAq0L7twfUve2XMvwVKGhXvx1bfqf/jDH5YXvvCFzXffKf5eIPXFF4sDSjarta+9UPp97tLXcK655ppyyy23FPFY3X3/fuutty6OrFDea0vdU4YdtVEv9fNjUY7RUJYpzazqdg9Y3inVrOpecrVr4Fl89yeeeGJzlMZixJdqfGvfcSF5vfOd7yyLLLJI+drXvlbe9a53NWfhndenuHuxlcJOyVd/dVKeXQFf69EWdfHde3GR+mqz8pWtntLVdqWbCCQCiUAikAgkAonAZEcgFfoJ6GGWZl+q+fznP998htKLr34pdosttmh+GIpy69z59ttvXyjfz3ve85ovvvgajh+I8hKs9PLx6UpHcVj7V1hhhcKy7vOSFHhfpWERp/z7hKUFA8v2Qgst1HzqslP5ZeG2sJhrrrkKRbkq2iByJp7y7ogLpfq8884rvoJjEeHI0G233dbkqa5+uOo5z3lO85Ku8p1xV08K97Oe9azymc98pqy33npNOAV/8803b9Iqz1l69XcPB2V46dVLssqn4PuRKdZ5Cw/HeTbZZJMy77zzFjsbsLAwUeekRCARSAQSgUQgEUgEBgGBVOjHuZcpmz7lSOml6FJo999//7LvvvuWNddcs6mNOJRmyinLvG/L77jjjsWZc1ZuFmhffmH93myzzZpv12+77bbNN9lZvqVxll2e0iIvoPrxKgrwpz71qeK8vnyaAuOfMh11+eQnP9nko05TpkyZ/k18n8mURz1TX8uQN9pzzz2bL9M4M68dFim+RX/YYYeVL33pS8WLsBYg2mXhscsuuxQvtWq/Iz1rrbVWUab8lUOx11Z5OcOvDAugpZdeuinHy7rO0FvA+DznDjvsUJ72tKeN+LOc0eS8EoFEYAwQyCwSgUQgEUgEJh6BVOgnqA8otpRorirUe65n5F54Jc/8Uduv+nf68W/7UeDrcRbfp6dgiyO/Sp6l4VY/rmckDPFD1c+9M/92Chz1cUzGLoGjMKz7vthD4aakS98meVTi715+yD1q+3tuUztMmqREIBFIBBKBRCAR6DsEskI9RCAV+h6CO9Ksq3I6VPyhwvnXNO4rdfMTRvH1cqvjK0996lMLvxq37fJHbT/3/NrU9qOoO9rj+IwjPc77s7Y7b2/h4KiNIzTSoJqP+0rVj1v9uJ4reUaeucg9cp+UCCQCiUAikAgkAonAoCGQCv2A9TjlGvWi2ZRqv3br7P8hhxxSHLfxDXtfvGkr870oe3qeeZMIJAKJQCKQCCQCicCAIZAK/YB1eK+baxfAd+V9UQf5Nj2/Xpeb+ScCiUAiMKsIZPxEIBFIBCYLAqnQT5ae7LN2sNZX6rOqZXUSgUQgEUgEEoFEIBGYFQT6Pu5cYT2de7755nuib3snLdh84z1xSBySB5IHkgeSB5IHkgeSB5IH+pkHnvzkJ8/9hCc84fFnn3324+Z6+OGH73rkkUd2fvDBB3dNSgySByaQB3IMpgxKHkgeSB5IHkgeSB4YIQ889NBDn3300Ud/PXXq1P/Oteaaa/5llVVW2WfKlCl7hpu0yiqJQWKQPJA8kDyQPNDXPJDzdc7VyQPJAyuvvPJeQRfvvvvu/80z9H1/KiormAgkAolAIpAIJAKJQCKQCAyNwDAK/dCJMiQRSAQSgUQgEUgEEoFEIBFIBPoDgV4r9E+MZi4S9LSgBYMeFzRPULdyxX1KhA11SStcvKHiTJS/us0fhS8WtOg0Wjhc7RSm3jWMC4u5I3xQrsdHQxcKwgfaDw/YhNeIL3m8IGLvHrRekOdwRnTBesmIuV3Q64M8h9O3F2zwE5weq67Gw5OjJfgsnOaaN/4bd23ClwuEf73g99R4EAc/eo7HGS51WCt8PhW0dFC7jHhsXZPjFm5wwqPa3tlez7DCx8b3Y/WNPODbSfpVXlCDu3B56p/qLwwpmdz+tQAAEABJREFUw9hRnrj82iQ+HnhC23PA7/UjTGtfwtYz/7GERn/om4o9GbN/FPCVoJWC9I3xc1jc7xikf8MZ02u+yE2++CRu80oERo3AkyIl2WfcuMe/4TXTZX7Cc+IZW0g6Mop+104grjEiHInT5lXy1NiUF9eYaqcnK6UTjtfbYe7V0bgWr7Ns4Uh55sih2qRMddQO9evMRxvkr37i1fEu76FIvchlOI0k/lD5zJK/is5SglmIDJwPRPy9gj4f9IkgytRbw108qF4AfEU8bBu0c1DnpTOWC891g74WtHZQv13quEZUao+gnwQdEfTxIMyqM98c918PEnZguO8KokyFM+kvg3HjaOVng74QhBd2CFd/UnjidkTXMyPWTkFwNVnOCu/CevNIu1vQlCCDLZy+vAit1aJmnwnCT0uEO9SF77aIQHgSOHFbCCNj7Nh4+GkHwS+8Ctw3jJvvBh0X9I2gDYIIxnCmX6+MO5htEi6BGs6kvZ4RLftg0OeCvhj00SCLmHCayzh+edyRUfiYXHtDPMMynJkuk4Q8OvuADPhIxNbP8NbXeHrf8NsvCG+H01wmBONEffYOH/JT/8bt9MtE9bp4stjFD3E70BcMXhoIfDkIb5szjCPPcKZ0R9CYXM+JXL4T9Oog1/3x75Kgpwfp33DK70spfwnCX72QO8b6npH/cHIigvvrytr0JQKviVqRQT8Ol5yjv8TtTBdeI5OOjhCy0Pg6NO6PCiITycq4bS7z0tZx980gOtBW4ZqPwymU42/FTZWR5iN5h1chP18UN+Ys+ZN/0tbwCGrivDhu6I7kMRkYjzNcyiA35bFNhLTrFo/NXCic3NcW+WiDegsnT8hocoQ8+WF4vidIvuF0vcjoFSPEfHJAuNL3YuxH1jNes6IUzZhy+CcrIQoUxZVioSN/E0neH0So1kmQkrZ9+GEGCt8qcd++gEBYApnSsVkE9qNi8Z+o16VB3w4iuO8J98igvwc9GvTLoHODMNwF4Z4U9EDQZL9MbBTTjaKhvwg6KEhf/yHcjwUZ6JSWuH3M688R4/tBsP1vuP8LGukF6zMi8n1B0qG47csLHs+NmlEe1wzXczhdL4vbXSKEUCN44rawCDwvbv4YdPU0ujlcY4kfQWnx+Zbwg+lN4VrkUAr4x+P06/K4+1nQw0H9jFlUb7YumDE+LBW5mHgoaWQTIwTBDjsYmVyujDj4+NfhUsxNBsLjcYaL4qgf4Vv74ZqIQTbeFS48LZjk/4N4PiTIpT8ZOdwvH//IRf2nL8hKfR3e0y99pv5/Ch/jIpyBvmBwbSAAr2XDPSGIEeWUcCm/Fr/wisfZvv4ROdwQ9LcgFznDj3y7LTz0MWX+X3GvPo+EO9YXeWjR8M+xzjjzGzgELooWk0VkH0ORhXF4zXCZZ8hCSi/9kSxElF3pdo3YFFhhcVsein+MGFeFS2YeE65xEk5ZJ/5ZBJOpv4174cZL3BaymDHp3nggGyn79CcylwwN76IuZCwjCD2x27i2KHl+RDaXWiDUeoVXc6krg9XZ8aQddLM3xf3qQfJXJgVeOvKboZqMfnuEdy4Owqu5KPSMQeQ3QxmjZhPQ638q2YsylolMWaV1nE4CBIWOFaoKuohSqvA16ekMijH/NlGKL5zmAahucaYFV2fcXYKbYL01SsaAt4erTZR5bTTZmpQJXWFIWESbtJeBRCEyOCjiFGp8YOBayXv+ZLSepXgkfGiihC8BMJL4kfX0C9aUV/hP9+zTG2PGIvisqB++wj9xO9NFSMHuwQihKIgbt8UCQFrWYUdlEGX9xAiknLMKE7yUVvjrIwKKcJJfG1u8jK/VAdUyIqtJc1HGGRJeFS36XhDl26Ibj7J8W4zi5U0jDB6sV/iYS0ljoCDvIniGi+Ucxhau+gB9OmIwbFjgV550Tym8OMLkaSKzEI7H8sL4Ry5ODff8INYpVrS4bS51tngTrp8mY/80DZ2FfzAwZ/w70lCsTw9Xf502zYWZSTkeZ/six+04XjYtJ2PnZXFPwccrcVv0n4WFuPqc31gS/qHwVEVoLPPOvAYLATxkfib/8Oo7ovl4OpzpF1nI+kxumo+NLTLT+KLUPytiknN2r+K2GIfi0fuMx1vCU95kG4OeMLsC5CMrOOORMhkujBu7ABYD5N+ZkZasrgYPC+WTw4/ctHAw9uNxhsu4o3+oo3BUI1DYyVPy4lfhqS1kKcPXCvHM+KWelH1yXh3tBmgLRZ2Mj2gzXeZj+o05ZFx1DsDNVJsx8qAAEJ5AqCsUHaPTdbJiWKooGfzbQAtD/KwagVMFJP9+JXhidDRUHcURZnFidfijeGA9Yt2vK0iTukFhl8MK0ZlMK9uVI67J3vaRlaQdC0xpxWoBZcFkIFhdC8eEsGeZskpmqTLgIptG8bPKtt2lfIOJVVH9KAm2oCjjL4nI6kY5McAM1K+Gn/zWD1f8cGa6DAQ7NJRLykp7IUY5NxApr3ZtlGEHxqKPUknp5E9IsOhTQBXQWRblVRsowKdGhNq+ir8zb7Ay4N8W4bDCU3Hbt5dxA6vOtrYrTKiyIPw8PO0MhVNqmy1cLKItfAg5wgUPEFrw9mzMUSwtBvgR4ISZsE585KsueO/gKAhfsU7oI8rKh8PPogCvEL74lNWYMIygvr/mLqWw8jgiASMVhsGNcfO7IDjD25iArckovIvJ5Jy4wWPSx+0M1/XxdF6QPPUDouyReZRvMtAkQ+DDd+GIy2JP1rG4xmNRJ+HGi2eLLn76xFhldVIHBgN1FiepFFv6ZCu+titn3Ft0kUkWYfqxxB9/OFrwkmf41m7wlyJMv4ZTyFty1JghoywGyB2y1yKZ3KxHSOWlHEoR/pCeNdO4oszov1XDkyXQ7pryETlqDOEz8wKZZl7YMeKaE54dbudFJjqvrw7krCMA4pOZFujqRPGi7GuTessD78DnffGgTaygLJR2lMn28G74ztl/mDgy4MiksuQnjnvKnrrKz26ifNRJemNfPHM/eUGhYkHlDwNzlfIYHbTPQlU6WMCQocG8RJ7gceWYBx3PMFe9NyIbc7A3X0ivzXaz6u6/nXLzgbo7HiKdOYAstAhjSbY7Bqd9Ij/vZplH43agL7KI7DK3vDaQYP0OZ/rF8kweXRc+4obTXMZEVc7pGPqwjiE8gmp8PIDHWbDxiPnb0cIqw7h3RK7yNK/oU/f6zpG2On7FM85qvpFkpstcSmYrvzNQegsMyjs+xJ/KqrJZOvL4+EhIjpDhxra5k1w3V0fQTJd8ye1uZc4UeSw9hgNidsqxKnLEBEAUU2fKKZWOyxBAd7Yy12gd3PKa6VY9DfaZAvrQQ2dSfG3FEzaVCD14CFdtwpFgtxqkiGMkEwehbivK+Ssvf1KaMDxhQ0BRCgg6Fn+DxgRiYFD+WRExpkUQZYKAt6KkmFBqDSITkXpYbCmXQiBfAtKxA6tOg+hDUUmTDqalpGF6CwuTi205k5T89WlEneHSXyzI6m7giztDhHig1LAIWICIb5C4t5gxSCml0lGoYBNJZrpqG0zcFhnexaBgUnooSM7ESUTwG2D8Kv7850TSd5QE+Olj48IYqm0hjAii+mwy9A4KJRGeBBzh1I5jgqfcs0R04uNZuHJMqLCmfBDA+MO5RMd3CHn8wTppctSX7XrV+vSbi7/hUSf3Wj84qb9JSdsIcGMNnjUOnsKbxkz1qy4ehl19lg6/Gpv6ofpLy9pDcSIjKDqsVMJZguRvTFDY+Bv3+oPyKS8LMVvjZEZVqKQdZKKEk1NwJn8pn+8OQMw9FvgWseYcPEw5xft4l7JCOdbv+o9cpezPG2kPD6Js6kNjjLzCN5QMi+cILvqB4mzHxbMxQzEW76/hwbooD4tDcY1ligpllwKK59T9nRG3GkIYNuJxpouBy3EifIG3tIGxjCJE7ppf5EWRtRgnA7QLLzk6xs9RWGOeQi8u/jLPmAdYT807lHpzmXmF/CR75IH/1F3ZFk/mDzLFs/iUZOODDGZ0MLfpE8oexdrCg+JIOddueekrR96MG1Zbiy+LEbhZEFlQ4X0YmqP0KzmjLRRzu2nw0E7tZ321gLZLoz+NJX3qnrJqPMMddvqDDJgJ6E6PAXjWj3Z+yEb6A57QbOPAwopcotzCmT9yj5/xGyMceeSIIF4U3ibjhw5BJ7QIM//jF3wjH/1PrtFzlM+4aBEoHqMSvmrnN9p77dNOBhHjnG5kcc3ATLYKN27b5eFv+oXxT76MtuyepANQLzImMFl0vd1P8Ni6JjANUsIMw/Si3H7Jk3AhUNpEeBDwtY4YBVN7yYLAJ0xMKAYNJds5UCtRzMXCTXkj9AhHzM4ibaCZxFkTTQAGGQVaWtZugko5LIG2q0w0LCfysagiPE0CBDlBSSgbbOI6MmRhpizbXYSr+mN098ozeRGgtU3VNbAp+uLLzwCtYW2XICdkTa7aaWIU30AiNKyGDaah0msDJdPEhqcoRxVDA9PCinXI4gDhy17xfLtdvby3sDIxOoYGG+2BD+pWLr7SR4QWbDvjEFBwYvnQz53hBKx+0T/OOtqtsRCzeBLfVqXFBZ5TJ4oPvjTJ6v/O/PrtGa/hdWPH5IGnYUoBQnCzSLGQMelTRvA3vqXIuNf+x2oXK6KJQFnKrPGNRWHKZL1ltaT0CIc3S5mFugW7yQfmLPkWGepEqaEUWVRZYJM90g4q4TkyAN8aExRQyh9cKKcUQ9gwjFAy8S9F0QQNU2FkJ1lCkdC35CTc9QXZR24hygrrHd7Rj3YAGE/ILvkYd5RfMlQcxhvjhEKJr+RB3lMqyXXly8ezBSRZy1JtvMmvTca+Ohqb+ECe+FR+ypKX9Ma8PLnkrDPIZAKliwxXHzLAvXnIguWNURAjjrLNHXhTu1kzyQFlmRvIXOWZ24Sbbyh8viZGdpO96kD2mg/ILvG1T11hCj9tsJhl5YcD3UGbzHEW1OZRc5R6KltbyCv9o97qyF/91MGYVA91VQ9YKg/e+ocihjfUCbba5zmanVcgYOzAzi66RRG+Du/mXLuxRS6Jw69N/Mg24wzvMEo6f47/+Ne47s3deMN8gV+MSwZKvCmePmPwtECwwCSbyU7jRvhYkTHJ4Cc/+qlFPn7CR/w6iY5EJrDc47nO8Al9xsi9qIDBQcm0+mIZoXTpVJYA1mBHLHpRbj/kiakp3xYwGKUSxZkQFq6eGBguFIQtw4MQ+l8pcVeaF0nEJYAITXgSegQ1f5EwvDD58RNmoGAyLqFFQHsmAAl4Cq34iHXECpi1lTIgT+Ug8aSTP1d+8pIHqnHUT17Stkl5VYB2CxdXHsKUYfB08mLnszSdpA2sSAQHBR4O4pi48Ji63s0jSH1qeDzOkRdFmcChIJiI8AwMEUWmW6MIYOEEV7dwFisKpclTv3eLo//1qTA4UgIsKvQ/vhMmjnA7ASZ75ZqA+fU74SPHLCxsjFMyCs7qb6cRH7Gc2+Vi3eTaCWIdtEi21fxYbRTXhFT5sel1ldsAABAASURBVMa34LaD6WVNVioykqVSnwnjZxFFOXJUAA+oJ8WHsmOHxASrzhYYlLGheKGWOZldY99i0hiBiZ0PO5GUSIoDhZKibZFkTFAe8TRM4Gmy1p+shSZvyjyjFPnnxTz8TTbpTwsvSom0LPWUSH1R5QxFiNJPiSTnlGfn0rilMBk3DCx2ZdXXM4uyM/mUGPWk9OI/ZbTJ4pAV1DygvsJY0ck9fIsvjWdHToxRlnu7BeQkXjY/G7PyIYuNafVgUbcLxKijPsY5fqMYq5t2WhxRqM0NlGI4XBEV0G7lwZhSB7Pwbo7w8HNPIaMwmfPoBBR39WVdh5H+qunwuoUzOaOOxqR8GK0YD7zzwtiF3+FqIWYuYZxyVMTugzwtCPStOsKCUZGxTDvhZ0GiTvBSx0EnskdfsobrG2PB4tHxJ3wFM3G64cTfGPJVKfg7oaHf+OvDzjTKwe/GIb6yABZHucrDc2ScfrMLz6ovfKzI+DSWLerxPqOKL+qYFzvLMBbsplHmLdq7taczzbg+E0y9KJBlS8MNJpYPSqOJygCyfUYQEWrKBgrlBJN47kZW55iEAOoW3m9+cCUMKdWVCKV2PWFEMSC0MCqmlg4emB+149fn6naGVf/qtsPdd/oT/MqnfBm4hGSNw60kbZv4e66u+07ST/IzURogtmo74/AjZE2ABkcN1/56X91ufsLwkkWJ1b42WCQRBHDkIvfi1voOlZc4/U6UZMLVYtGEi1hQjCc7PRSIdhu0nVJigqQctsPcU3JMfJRWispIsBHHuNa3eFwZ8qqEz030wt1X/3521ddxDAtbijoFGn9SwExK6o7HHCWy9ev8LQWMxZESQUkRZyiilDgmQKGHXTsePE1q5BuFkaJEScKvwuDIisw6T2Y6hkcJwuurlVJMMiyTFBSKnzFB6WmXMUj3ZIpxwGKM780dFGpkh0W/mqwp0hRbmMHHzoy+pWhTClmMpaEICyfT8Ik+gS8Lv3DY6ytKovFU+cW4MDYZYvS7PMRzvErfyVc+jtQYJ/pPHPMg6zmecNzEAoV/J1mE62sKv/oqzzEYihdesQjRJnEoveLY5cFLsNEO9VFHVlHt0GbKuZ0I8SnKjmnCjCKnnuZ1ypVFBH6kYAk3PiwOWPjJJfJEnclgCw1pLCLws6M+2kWBUm/8rk/IMPWWTtnyMl4sNrTFzpRy8L95VZh+IeOUbezITzso8RYhMLbAk4c6WyQZi+6NOe113MjCrPKC8ged8Ab+Jt/ML46+worF/LGwwVsMF/iXbrdzJNAn/ON2pksfwp9+iO+Ura/twOBVi8NqoadLGmszZTIKD+UwOFtwWCDiS4sH7XT0Dk/VbMkVdcJ7FobGQg3rGxfz96IyBg0F3oQHNIOPIGGt9myC4ypbJ9eB6bkb6XDKQ7ewfvJTT/XRJuS+k/hrO+Zg0bE1Rbi344nTps6wzucal7/74VxhyPlnlgwWSYJNnfhLj+p9p1vbyB/VuO7bROFxdtEqm0AnoGs4y4xjWCYEVjSTXuUBk4t4tT4Ef+WXWnZ1baWbDKyYKayVnw021hYDn0VJfkj4AnHTrks89t0FU6Ri1XVPMXDOzxELigAiYOqimUAUrxJsWJ8srtr5wMGkRgFgnTZZw4SS08ar5lP7wrO+oEwoywQoDAlDBJ9JVPicMGbVGcGHUqXeMCPUvUyHN4UjShZFgJLoDC7sKeGVH+GKxG2TY3Ewai9chcO8Hd+zPlaHmqd4CK6sn8JNcsIpScYNpUk+SJ9oizSDSMY83qagkgNwNx/ha8qgPqR460MyCkaUYIorKy9ZYr4id8hl+IqD7y2o5UdGWUhRXPG4MIozJVYalm5jhB9lWnoyD19RNB1B0UfCxTV+ySvh6oJPKLeOtTH8SK9vle2ey5LJz5ETfviVAkyeK1P+5hftwKfknjQWAPhGWlhRXChuDDvKNxfgcbxoYWSRw3ptEQJHBhqKs/rCS56UIekoQOovL4sf9YKZHSdtFA4rfcMQQ0mEI3lhLuAvjfZRwr1L4KVa8kndtNF8aZyKh+DKOGVcKFcceoeFmf6w2MAP2m1saBM/ix79zDqrDyzejSl5TloaQcPwDRIVHuQb2WN3y3jBO8JQjVfvPSPPyCKKUq5/nUTQx/z1L/5zj9ybq+w6WtCJpw+NM2NWHDLRLqoxTZ7yq1TLrG71ry7/StpU/Y0NY9AYru1i3GLotIAUrq5kh7FobON1fsY3OSIv9Tde+HuuVMtSdvXrqasivSoAAFbgXIPcth2BQ3gYpEDUcQaTQc9SbcuFUmGQAgdhBuAiz1bjBAsAe1X30eSr8wlN7aAoI/VFBJk6i4NJPau/iRhTYA6CG1MTcBiW0IEZfOTJH4kLH0JKHEJbuLSeCT7lwIvlQTxCFLae5SmuvlcX5ctXOuHukfrCWl21Sz5IfPm5lyfLirZ0YmZgeuHMytsLVhYw2qKOJgmrYlZ1Cz0Mb0IwSVgAKIPgxQsmDF/t4SqTwIatOmgD/tAe9VBv94hwgL2VPcy0T/m2nCljeK+zzv3wrM7apI+1gzDRn/xNkiwfJleTPpeCYqIibNzXNohvXHEp7dUfZrA1NlkjHFeyqHJe0RcHlFfjcllM4O0sMte2vTwoEsawvsMnFBX4suawMlICqkCTTz8T/jWGjCWfmaSA2M6nsKk3DPUH3mVhtY2sjV6kgrs42o7X8Lrxwg/BCv+xVLUVEfGdZ7YjIFzerO/ieYdG2kp4neyEdcVdmMmF1VO9LZrxC2UOPwgfJNKHeNcuC56FtT61A6VPjQXndVmIxSXfyBP9wNqOf6UzxvQ3RYLCaayQpe7JR/1JluhjCiirv3sKCKVDHOOVnNWnFEVlSUfxlDfFUx7Ghzool1yj9OABddLXeIuhgpw1Vim5+pR8MB6NfwoO2Uj+GZ/OpFNOtJGMtUtBLuBfaY1VChpFFjZkrTLkafFqTsJH8hNuQYOnyHNptY+CTdZqk/TK42+RRInDj3hafNjD0if8LEgZksw72q59ZL46womuoG4wdySHhZ1ckj/s4OQ4K2y0Bam3uVOdlAlH/a4t8tGv2iBcOriRoRYA6g9Tc4/5AMlzUAnf6ht9gHfxkD6w62QMkDf8xKHT4Usu3sJ7eBrvyAeG8Gbh14f6WVr+8jJOjR3ppcMr3ikkV80rrOBcu0rGiHzxgLGj7+SDlE1HUb588YMxJgzpY/JTPHqVsVX7GV+QtXidTiCPykPKJ0fxteOQfk/Hl6G0BVmo0E/kZZx590n5ykTKUy6CU2e9xBlz0tgxzzQyJBRZGAhQ1kTb2c6AKo9FwPESnU15s+L2YhehQ0iYSClwgACKz1FROIRhCvl5NlDbHRfFTthFCPqUn/bqPEqj3QgDQ0eauCkBGJqAcw6Xwmk1qH22PVkwDBQKE8srBjfRYCaWCkqGQSY+pam+bILJvKvg7Cy8lGUb1K+qmWicTaRAiydPE48+oBiYgKx6CWCC0sCSN/wJRsc7KCjKstUqnNKHgVnHtc2b7JSJUsoM+OtflmErewLBVpmtXNtnlHvlwsugkZAi4+iRfrXlTEirH2skbAw0g0gbKVUs/D7tZlKFiYmF8MAnLEfaZxDC1laa/EwaLHAUXBOhcvuNCDjtccbXvUWR/iK0OuuK/02iqDNMWgKQdc6EWMPxJB7BT/jLeMJvXJMsPGtcLh41dvGA/mPRcMaQtUY40od4zlimBPhUHiufsH4nGOJfZ6WdtYYBF5+QY+pPQTCe8K52et/A+KZQCUfklXFhYjAu+CH9Rpk0CdX8+JtM8DRZ4D0Q44EMNFlQgMRB6mfcGfteUmv3tTGDVygptrfJXH2kDdIOEpFrrInkoLFOhjAcGfv6wwuwvqxCSWBhxufmEfKJYqkv4aZfKI4UUPfO9jpqRekki8g1Y4tiQEGQD5lkXJnQ9QkFURwLDK4jWcaIyR8vmffkpy5kvnjmNmNGPOfIjX87RHhBWeQdPtKn5huyXXnKlhelnrFM28TBdxQhVmjnkBkAjGOLBkf2pHfcUR1qu/G8hTg5a0GiLfIyV4ln/oKlnT3YUJopQeY8mOFfY0ebKWfKoUg7okaWq5/88Lh5yYLYYkI5+onstiDhr27mGpgok2JlZ4PVXR5tgocFl3qQ7TDWdvUztynDAk+bPVNM9Yl4+lB/wQgO7XwH7R7PGUNkXJ2LKdiO2TiGaCFkbJH/cCTrzcGUWX0mvRdLnc7QZ/DDE3Zj9C/e5ocYKJxbJ1PxtnkKjxhfyFjzkqy53Avajsfhd+WYx+WBLHTN8xR+45SeYbwIQ/jAHGqxSM57X0nZdFH9jd+0gZ7j4xp41tzGX3pyn+5k3tO+OlfiR/xsTFgc00lqucaerwSSyXiT0UZbzBHy7BlpVC8yt6oz4ZmAKLI6mKD1oo+tsKpMUSIJGIoFgUQoW7kRBiYuzODlCoqlMAJNx2AglgAd34v6z2qehB0G0E7bmNpDaSJIMDHGNdFTqLnaBCOrT8xs8sDwBgirtTNnJmnKrQGC4QhRQhbj2S6k5JnoYYNxMJRwdTCpY0TPFFp1w9QYWp4EKqsLpVy/+B4xiy0F0IKKpdGAtYKmdJscWTL0gS8RmBilU77FmAHQDTP9Q3BSlgwEcdXPANT3JpuaDk/AQR3wCeXQ4LUV7p6ln6DBAxYdeAsPqIeBZjKCmzpbQLLE2TozebL2aId0Fj8mCXWrZfeTS4ASmIQOC4A+0o42VrW+MCPQjAftrf5cSjzhZTHluRJLm6+hEMwWbZRKZGKj9ODXGpcrPiGnHsaqPjFpEobCKZysJnjawtMC1ISpbsL7nfABPrIwJbgpU5QQY7rWHZYmOrwrnvFFiZK2xjHZab+FEQWh+pNjJiQTVMVMGH/KuPEIWwIfn1Nu2vm6Vx8TGqVE2krCKHCUUgoSZQr/1PD+dse2dhZMZAw8Tb7mEfKGfDA+KIN1QUW5JZ/JFrLQLgcZbcyRbZRV8plsZZwhV/Sf+UgfGlPytxDT1xYDFrzmOs/6hTxyPIXsNnb5GceMGcYKxZQVkHymPFEoyWnlG0PawiihPIqOs74WKFCjPHtZ2nikbBv7yiHnhImDZ80VeBI22u6rc+QhoxL5i68YkLxsje/UwRggT5VtDjCHVfluzpAeHmSAduNd8hymta6UKrJaXuYzixJyxXEgi2H9QcYz1MCFrLCQwMfaxKDkgxH4Xbi2WNxYGIirfZW03VwnP/2ivuYyz/pM+epDjsGGcmXxrI+NFRgZ0/pD/Wu+g+hSrvUbvjV2GOTgXRV6fYgX4V11CTxn7FBgnZM33sgxY6ViqI8snvU9P/3KkGFsGTcWmpRjZQlH+kd8MtfODv7Xr2Q1nhAH4RM6VLWyG88Wx8IQ5V+f0yvs2OBJx69qX4urbvgY7xl3ZL3xYMzQm6Rrz5XmS2PEeNFOui6jKWyUCSd5qLc5nDFUHDJfeM+oVwq9jkEqbhDpQAARbPwqabgwg9Bq2iQU3GecAAAQAElEQVQpnoGm0xBm4CdMHANTfB1e85loVz11Vq2n+iFMIwzTEPS1/toEH+3XHmkpEPwJL/G1F1U/99JzpXOPPHe68uOH1MOzfhAXuVc3/sqXH+YVpo+U6V5acfWHZyRMHPfy5z5WX2iruPoaRsrt1mewEk+d1Ee+4vJ3z1+4MtWBv3D1rPGFG2Q1f1hKp43aAeca1o+uNsFYnbUF3+AJ/t3qq/2oM7z2L7edzrP8YSj/NsGoHbfet/tPWfwp8ixxLCOsjPxqXd3PSYR34I2P8Fln3WFrHOBdPOS5Mw5c9RNyX8PFhWvbr4YpVz/IlwvnGtZ28Syellfb3z0/dVL/odKLN9lJv+E/OOpHsgbBBXad7Sc/xCcfYCiOcQHrGldfimOMtLEVBz/oV3H1rWfxPSP1ka7GafvpL2nko0x1Ea4MpA381Yu/uPLGL575SyOO+PyUU8emZ3GUg9zzE1fe8FG2fLVfXjWOfBBFxI4TpVvZ0kujTGnkJX1td00vnvzwtHLE4yfcPdIn8uEnrJJy9Jl0yqr+4slTvapfdYVJB2tpPMNBvdxL59l9ra++lt4zfKQXzm+QCQ76Tf/g+8pv+gzBBsb6Bx8h8fEDv5pOPp14SmdMyEOYflGG9PqHfzeSlzh/icBu/a+f5aP/ueqiXyN6cylXvdSv1rf2fxMh/mmb9J28p57yl054m9RZeCQv8sNHtVz+ntVbGuWrF3/xe0a9Uuh7VuHMOBFIBPoCAduKLDV2kmw/skLUs5N9UcGsRCKQCMwyAsYwa6PdIzvKjjywfs5yRpkgEUgExheBVOjHF+8srW8RyIrNIgKsJY702Kp0dMF2bbXozGJWGT0RSAT6BAFj2LEfx2gcx3HMxRHFPqleViMRSASGQiAV+qGQSf9EIBEYDgHbi84QOkfrDL0tzZ5vKQ5XoQxLBMYNgclbkDHsiIAz8ca1d6Acj5i8Lc6WJQKTBIFU6CdJR2YzEoFEIBFIBBKBRCARSAT6C4Hxqk0q9OOFdJaTCCQCiUAikAgkAolAIpAI9ACBVOh7AGpmmQiMLwJZWiKQCCQCiUAikAgMMgKp0M95ve/HSfyYiM8Gdqu9rxSgocK7pel3P232QxXd+JW/zydyJ1Ob+71Psn6JQCIwJyKQdU4EEoFJiUA3BWlSNrRPGrVQ1MMnwPxghx+g8q1fP9bkmeuX0IbqE8qqH//xQyF+eMMvR0Z20y+/SOYzY35EwWfHfFZweuA431DA/RCDT5759T8LkNEq3PDwgxV+HMSvwdam8Pfsh3z8Qpwf5vGjUzU83UQgEUgEEoFEIBFIBAYCAUrRWDc08+uOACXXr699JYL9SuFu4X47yK/0ed4z7rcJoviGM9M1d/j4VcGPh+vX2OqP+cRjc7FS+5U3ea0RPpTocMb90k7fJPfZM78I6JfZ/BrcOlETi45whr0sXLSt4uB5wUixbFD1i9ticQML5fnVQp9P3DwC4BROXolAIpAIJAKJQCKQCAwGAqnQj18/V2X26CiSMu9nvlmwfz7t+ZBw/ZrYUJZ1v2Z2YsTx89/1F9ficfrll/f8DLKfE9evPj82PXAcb+wS+OlyPxfup5r93LmfUF8v6lAxiNshL+0Xl2VfJO32M+Ty9PPc/LRv9bixwDknXD8dvVe4fsJ9otodxec16whkikQgEUgEEoFEIBGYXQQoRrObR6YfGQIszWdGVD/A4yeG608H+9lgz1MjjLJOgY3bwvLMMu2YDiWX30Pxr4ZTjp8azwsEieu74MKqQstCLy3Lfbuf1UN+wuRfLdrizB95yVdaYeKF1yxdr4rYiwVdEnRH0A1BXwvq/HES5ai/erC8q9eTIh5LuyND2qU+4VW0zU84a5t4/JeIAPE9S39CPP8wSNxw8koEEoFEIBGYVAhkYxKBRGBIBChxQwZmwJgi4Jc1/VDHUD/SQRFlgX4wSqWwrhauc+GO4nws7imw4TQXRfslcbdj0P5B6wZRasNpLso46/Xn4mnvoFWCKP3hFPm8PW4+GyTt+uFSninidg42jGe/ELhLuM6tU7rjdsSXHyVZJGK/LcgxmXCKHx+6Mm4o5eEUirjyPhUP2ufcvyM0G8WzMpXv6NAG8fz0IEd2dg5XHO3cJO43DhL26XA/GGQREU5eiUAikAgkAolAIpAIDBYCqdDP2N+9fKKwdzsq0y5TOIvzquFJGT413JOD1g6i1FJm5UNhZ61nlWbpd159pYjjYsWmUPuFv+PCY8kg4Y6weOmW9dvRHkdh7AxQqCn/r4t4WwY5ynJPuJcFUZydx4/bEV/nRcxfBG0aZMFASVfXi+L5X0Es88qgnDtCdFr4iaMsx4W02cLHbsWlEeYIj3P43hvQbnmcG/4XBP01yI7H6eE6chROXolAIpAIJAKJQCKQCAwWAqnQ919/s847dsI67SVX58kXj2pSaFmhHau5K56vDrow6BtBFgErT3Pd3xL3wn8d7uFBywT5og6rPus8qzwLuq/COMdvAUG5vjvi3RR0eZC0Fgus+7PCJxYJ+0T6HwVZiDjXb6dh0Xh2sc5r1xviQV28LFvbR0G3EPl7hKm/HQsLi6PimV84Rfu174/xYNfDkR5xh9r5iGh5JQK9QCDzTAQSgUQgEUgE+gOBWVHU+qPGk78WviHPmn5/NJXSSnk9KO5Z0u8Lt555Z4mPx0K5dz59qXioYXE7/bo27qRz1IZiT6GWL/pNhDmuwpJf8wuv4t7CQH6s/fxGSizwLOy+3uMTmxYHW0ViuwTOywv3SUvKu3ja58s/+0Ycx42Uqex4bC4WeRZ+uxeNx7R/4qDk4WmApJMIJAKJQCLQpwhktRKBHiOQylCPAR5F9vqE0u1YyYGR3gulrOznxz3lVnjcTr8o386mO0bjOM70gGk3/CjDLNxekBX3BxH21aCDg1i/nXGP20JB5rZJ+vbzY92/LCL4xr6XfR2fcRZeec75V2u/dwB+GfG0DWmfIzms7+E909WtXjNFSo9EIBFIBBKBRCARSAQGEYFO5XAQMZioNlPEu5VN4aZEO5biHL04FHHHYrygKgzV9H5gCt0cEflTfrk1nFVePzvK4kiNZ0d6fOs9khRn7KuiXdPVtFwk3kjJOf1XRGR1Dqf8Lv79NMiOg6/nUNrVx3EbOxERVJyN1z4LGc+oXW63elU/rvhJiUAikAgkAolAIpAIDCQCFKuBbHgfNLoeLWkrrqrl2AkF2FdunD//ZniyYFPuhbF8e1HUERvKu7Pwjq94GZUlnhV/xUjjXDoF2QulF8ezH1/y8uhVce/rOH7USr52ASjbvoLDck4RVyfP7in+6moR4Gy8M/eRxbDXhyL0LUGODzlioz4+YakOdhK0z9l55dc2OhLkHLydBIq/cl8ceeBRbXH0h394FS8H+5VYCwFt5Kde+8WNYz7C4zavRCAR6I5A+iYCiUAikAhMJgQoS5OpPXNCWyillGxKNWu8zy76xVPWd/WnlDvT7igKpd3LsRR5X4Pxkqqv01DM/WiTL8FQmPeIhCzwXhI9IO6dj/ey7BFx76iO4zWUduftvaDq6zG+XrNmhPuhK+m2jXt183Wad8e9OlGWfV7SF2heHn6+tOMIDWU/Hrte6khRtxj4bsT4WZCXcZ2R95KrBYcy/ZDWchHmBd2/hKtNFite9KXUK9+CwFEdeFHod414dhN8xlOdhMPJpy4tcnxy09n95SNeXolAIpAIJAKJQCIwuwhk+jkCgVTox7+bKOw+67hdFE1Z3TpcSi8lNm6by7fcvQjrCzc+J0kJ9/UZlvPbIgalVXoKLsXYS7HhXRw/8YNOO8QDhZjS/7249xnKcJqLhd6LsL71Lm8/xuS4jrjq8/6I9asg5VtMOPrjvLsXVyn010XYcHzjRVuKvK/cUOLV0Tfv+TtuE8mLL+H4pGVt307hSdnXvhvjnoVffXy7noK/Wfi9Nkhe6npk3KsXPwsRFn8/zOXb9IdFmEVDOHklAolAIpAIJAKJQCIw+REYTjGb/K2fmBZSulnTKavXRBV8e51Czlofj9MvX3ah3DqmwrpeAyi9vrnuU40+6egLNjWMK5zCLH/KO+s7/0rKFy5fiwP+yvKsPsjxGAsI94gF3fEW8SxGLEqk60YWJtqizsq3G+BrNlWZr2nsOvjkpHIdw6n+4qmXMOUoWz7qgcSFV/Wr+LHWq6PPbcK35pduIpAIJAKJQCKQCCQCkxqBVOgndfeOWeMsAijSPjFJ0bZoGLPMxygjx3UsVHwm06JijLLNbBKBRKC/EMjaJAKJQCKQCHQikAp9JyL53A0BCj1lmbXcfbc4E+3H4m8ngFW/HxccE41Plp8IJAKJQCKQCAwWAgPU2lToB6izs6mJQCKQCCQCiUAikAgkApMPgVToJ1+fZovGF4EsLRFIBBKBRCARSAQSgQlFIBX6CYU/C08EEoFEIBEYHASypYlAIpAI9AaBVOh7g2vmmggkAolAIpAIJAKJQCKQCIwOgVlMlQr9LAI2SaL7MSo/IvW4HrVHvvNG3soIJ69EIBFIBBKBRCARSAQSgV4hkAp9r5Cd2Hwp0n6plWLdWZNnhYcfltoi3PmDenH51dsPR8bKSB4LIPr0ymolAolAIpAIJAKJwCRAIJWtSdCJHU1gfX97+Pml1QXC7bzeFR5+cfU14foxpnDG/Hp85LhU0LODui0qwjuvRCARSAQSgTkHgaxpIpAI9DMCqdD3c++Mrm4s86+LpOsFLRPUeX0jPE4J6qWi7Xv1H40ydg7yy6/h5JUIJAKJQCKQCCQCiUAi0AsE+kqh70UDBzDPF0ab5wvSt6zwcTvD9e94+mtQp6ItzTPD/+lB3Sz3Twr/JYKeEeR8fDjNZQFRafHwcdxH2U+Je/514cBq/7Twc+RnsXA9h5NXIpAIJAKJQCKQCCQCicDsIEDxmp30mba/EKBorxpVOjvo3KC1g5xnD2fYa8EIfVvQ54L2CHpT0JSg5wS5hG8QNzsF7Rv03qCFgl4UxO9D4doRODjc1wetHLRbkHh4jJL/6njePYj/58N9ZVAq9QFCXqNCIBMlAolAIpAIJAKJwDQEKFvTbtOZBAg4YrNktOOsIAr9auGuEzTc5cw9ZV68r0bE44KcsT88XOftvThLWX80nvcOOjpoq6CNgzYK2ixo/aB7g24IYoGX3+Zxv2KQa4X450z/7eHuGXR/kBdzLQriNq9EIBFIBBKBRKBXCGS+icDkRyAV+snTxxTzl0ZzHgj6UxDl2fGaNeOehTycrpejNOtGyG1Bvw86L+iiIIr9fuE6YvPGcFnduSz38rNYODD8Tw76X5C0u4R7UNAhQecHzR3kui/+WQicGK648nT8xjGf8MorEUgEEoFEIBFIBBKBRGC0CKRCP1rkOtL1waNz6SzpL4i6fCaIldw5eUdw6tGZ8J7p+m/4PBS0cJAjMBRu6TxTyB23YXVnqUf/iHiHBlH4xZOGws4/vJvLQkKezUP8uyPocUnYuwAABOlJREFUhCDHbt4T7qJBylVW3OaVCCQCiUAikAgkAolAIjBaBFKhHy1y/ZWO4u1lWMdeWMh/FdU7PuiIIMq8oy9D9TXF+5iIR8neJFwLARZ0lvp/xrNz+az/8vtyPB8QdFjQFUGU8nCKF1+Re+QeuUc+YekYjxdufWFnanhK244TXnklAgOBQDYyEUgEEoFEIBEYUwSGUvLGtJDMrOcIePHVUZijoiTK/C/C/WWQ57vD9QKqL87EbXOxrFOoPXD/GDe/CXreNKK4U7zFezj8WP83DLcekfG8RjxT9KWP2xkuftJyWeFXitDnB/0s6OqgR4L4ixO3eSUCiUAikAgkAonAzAikTyIwMgRSoR8ZTv0cyycmXxIVpDDfFW67T38Xz78O8rUbCr+4PifpB5+4zs87RuOHqBzN+XPElcfq4b47yHGbW8Ol3G8TrrPxXwzX13Ao+hR6i4nwKvJmcUeLhIcz8n7YykKCn3sLAS/CqosdAZ/J9Cw8kuSVCCQCiUAikAgkAolAIjCrCLSVv1lNm/H7A4HlohpbBi0b9M4gSno4xTGcteLGcRdn2j8Q9y8O8vnJpcN1Rt7xmnni/s4gx3Io8e+L+3cEbR/kE5QPhutzlI7lSC9PL9x6CXaLCJO/78+/Ne5Z8JGv31go+OIO/2sizA6Az1t6SffyeP5XkM9eppU+gMgrEUgEEoFEIBFIBBKB0SKQCv1okeufdL5os09U5xNBJwXVl1Mdd/ltPPvyDGXeJykp4eeE30eCKOynhuvoC6v8jnHvU5Pvn+b6Hr2v2eCRm8LP9+OFy8vXbVjupd8uwpTNik9JR87bbxv+HwtSnri+Pc+6f0H4fT+Ixf/0cH2VRx3iNq9EIBHocwSyeolAIpAIJAJ9iABlrQ+rlVWaBQR80/2yiH9pkPPpvhcft4WSzJJ+STywiKN74v6WIPGR78b71KUfgBLmRVdEgfcZS+fwWdDl5TjOVZH22qC/B3lh1r1y0fXhZycAyVf+/B37oeTbBVAX5/WlZ7W3GIlkeSUCiUAikAgkAonA5EIgWzOeCKRCP55o92dZFGxK/u5RPZZzPyjl6A6lmxJOmY+gvBKBRCARSAQSgUQgEUgE+hEBCr2XGn3y0BnqpFIGDQMvth4bzHlkkBdoLwyXks/fV2/6Go+oa9Zv8Hg2+zz7PHkgeSB5IHkgeeD/84APkTyOQu8lxe+EYvS9oO8mlUHE4OvR787V+9EnL8U6L48nBhGLbHMZyDGQ/T75+z37OPs4eSB5YDLywOtCh5uLQu9s9PnxgLywmFTKoGFwUfS/M+/O2SNn3QcNg2zv4PF99nn2efJA8kDyQPJAFx6YY/RAursPm/yPQk+Z+2QodB9PKolBSQxyHCQPJA8kDyQPJA8kDyQPzAE8sEPU0dcE/0uh93lDXyZJKiUxSAzGjQdiEGZZyW/JA8kDyQPJA8kDyQOzwwP0+EKhD70ir0QgEUgEEoFEIBHoUwSyWolAIpAIDItAKvTDwpOBiUAikAgkAolAIpAIJAKJQH8j8H8KfX/XM2uXCCQCiUAikAgkAolAIpAIJAJdEEiFvgso6ZUIJALDI5ChiUAikAgkAolAItA/CKRC3z99kTVJBBKBRCARSAQmGwLZnkQgERgHBFKhHweQs4hEIBFIBBKBRCARSAQSgUSgVwj8PwAAAP//18Mx1QAAAAZJREFUAwDRA+Ubg6DFjgAAAABJRU5ErkJggg==\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4 Discussions","content":"\u003cp\u003eThe use of enzymes, also known or biological catalysts has grown significantly in recent years, and the discovery of new enzymatic catalysts offers new applications opportunities. However, thermos-tolerant or thermophilic microorganisms those that can grow and survive in hot environments with temperatures above 50\u0026deg;C\u0026mdash;have attracted attention and lead to the discovery of a wide variety of original enzymes with distinctive properties \u003cb\u003e(Wilson et King. 2022)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eIn this present study, the investigation of cellulase production, was carried out using the specific substrate carboxymethylcellulose using bacterial strains isolated from two thermal springs \u0026ldquo;Hammam Oulad Tebben and Zdim hot spring, in Setif, Algeria for the first time in the worldwide. According to the obtained results, among the 45 strains selected and purified on TSA culture medium at 30\u0026deg;C in aerobic conditions for 72 hours of incubation, 34 strains were qualified as cellulase producers with a rate of 75%. The outcomes of this study indicated that the strains isolated from Zdim hot spring (Z) had greater cellulosic activity (n\u0026thinsp;=\u0026thinsp;17) than those of Hammem Oulad Tebben (S). The obtained results confirm the high capacity of strains isolated from thermal springs to produce extracellular hydrolytic enzymes. The molecular identification of the selected strains demonstrated that they belonged all to \u003cem\u003eBacillus\u003c/em\u003e genus but they were different species affiliated to \u003cem\u003eBacillus subtilis sp.\u003c/em\u003e which improve the cellulase production by our isolates as known that \u003cem\u003eBacillus\u003c/em\u003e species are very important producer of extracellular enzymes and particularly cellulase production \u003cb\u003e(\u003c/b\u003eSchallmey et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Balla et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Malik et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Otherwise, the availability of nutrients in the environment has a direct impact on the enzymatic baggage of microorganisms, which is important to their natural habitat\u0026rsquo;s adaptation. In fact, these enzymes play a crucial role for the metabolism of environmental nutrients, to ensure the bacterial survival. Furthermore, these innate bacterial skills are used for biotechnology across various industrial sectors \u003cb\u003e(\u003c/b\u003eZhao et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Moreover, many thermophilic bacteria synthesize various hydrolytic enzymes (such as protease, cellulase, amylase, glucosidase, glucoamylase, pullulans, and cyclodextrin glycosyltransferase) when they are subjected to natural substrates. Other polysaccharides, like glycogen supplied by animal cells or microorganisms, can also be broken down by them \u003cb\u003e(\u003c/b\u003eBenkahoul et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Indeed, \u003cem\u003eBacillus\u003c/em\u003e species are highly interesting for the development of industrial applications. Industrialists are interested in them because of their quick growth, which leads to short fermentation cycles, their capacity to produce proteins in the extracellular medium, and the fact that some species are listed on the FDA (Food and Drug Administration) and GRAS (Generally Regarded as Safe) list \u003cb\u003e(\u003c/b\u003eSchallmey et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough, the capacity of thermophilic bacteria to generate extracellular enzymes has been the subject of numerous investigations. Recently, a study was carried out, on the characterization of thermophilic bacteria related to members of the different genera such as \u003cem\u003eAnoxybacillus\u003c/em\u003e, \u003cem\u003eGeobacillus\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eMeiothermus\u003c/em\u003e, etc\u0026hellip; that had the efficiency to produce hydrolytic enzymes from the Hammam Righa thermal spring, in A\u0026icirc;n Defla, Algeria Thus, in this study 14 strains were isolated, and qualified as strong producers of amylase, protease, xylanase and cellulase, which was in concordance with the findings of this present study (Bouacem et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Further study on 2017, was conducted on the characterization of bacterial strains from Algerian hot environments, in which 13 strains were isolated, and the latter exhibited extracellular hydrolytic activities (protease, amylase, pectinase and cellulase) (Benkahoul et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Interestingly, cellulase well-known as a complex enzyme group produced by different microorganisms (fungi, bacteria, and protozoa) which could break-down cellulose into simpler sugars, have garnered global attention due to their multiples industrial uses, especially in the paper and pulp sector, textiles, and biofuels. Furthermore, cellulase is the subject of both academic and industrial research, and they have a commercial validity of over 30 years. The thermal springs were the site of the sampling. According similar study done in Algeria, only 16% of bacteria isolated from hot springs in West-Algeria were found to contain cellulase, this suggests that this underutilized biotope may be a promising source of thermophilic enzymes with significant industrial value \u003cb\u003e(Khelil et Cheba 2014)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eNevertheless, as a potential source of cellulolytic enzymes, thermophilic bacteria have garnered a lot of interest lately \u003cb\u003e(\u003c/b\u003eShyaula et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Different species have different levels of enzyme activity on liquid media; which is matching with the findings of this research that showed that isolates from Zdim thermal spring outperformed the others, with CMCase activity ranging from 1.033 to 2.18 U/mL. As a result, these bacteria's cellulase could break down CMC as specific substrate. Since CMC is a cellulose-soluble material, cellulase are hydrolytic enzyme holding both endoglucanases and β-glucosidases, \u003cb\u003e(\u003c/b\u003eSharma et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Research conducted by Sarangthem et al. (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) screened a thermophilic cellulase-producing bacterium, identified as \u003cem\u003eBacillus velezensis\u003c/em\u003e MRC 5958, from the natural hot springs of Bakra, India.\u003c/p\u003e \u003cp\u003eHowever, these current results were more performant with those obtained by \u003cb\u003e(\u003c/b\u003eBalla et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), who reported that the CMCasic activity for bacteria from thermal waters ranges from 0.7 to 2.2 U/mL. Yet, according to another research by \u003cb\u003e(\u003c/b\u003eMaryam et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), B. \u003cem\u003ecellulosillyticus\u003c/em\u003e had a CMCasic activity of 1.99 U/mL. Nonetheless, other \u003cem\u003eBacilli\u003c/em\u003e strains have been demonstrated to have higher endoglucanase activity, ranging from 7.3 U/mL to 9.1 U/mL \u003cb\u003e(\u003c/b\u003eMawadza et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) which is contrasted with the present findings. Maximum cellulase has been achieved by optimizing a number of physicochemical growth condition parameters. Having the ideal value for each variable is essential to achieve successful enzymatic processes because it boosted outputs and performance \u003cb\u003e(\u003c/b\u003eNnaemeka et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Shyaula et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Among the physicochemical parameters; temperature, medium pH, salinity, and osmotic activity (water activity) are all important factors in microorganisms growth enzyme synthesis modulation. The effects of these parameters differ from microorganism to another \u003cb\u003e(\u003c/b\u003eSarsan and Merugu \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2019\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eIn fact, cellulase production is largely controlled by the pH of the culture medium, which has a significant impact on the ability of bacteria to produce it \u003cb\u003e(\u003c/b\u003eSarsan \u0026amp; Merugu \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2019\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Every bacterial species has a specific optimal pH for its enzyme production needed to breakdown cellulose, according to many previous research \u003cb\u003e(\u003c/b\u003eHussain et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Balla et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Malik et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Sarangthem et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). To increase the cellulose hydrolysis process's efficiency, it is crucial to comprise these ideal circumstances. All strains' bacterial growth (OD600 nm) was found to be maximal at pH 7.0 and moderate at pH 5.0, indicating that they are either neutrophils or moderately acid-tolerant. Nonetheless, the ideal pH of 7.0 indicated a maximum cellulase activity that ranged from 1.46 to 2.35U/mL. At pH 5, the CMCase levels of the tested bacteria S8, S24*, Z1, and Z7 were 1.54, 2.18, 1.98, and 1.70 (U/mL), respectively. Conversely, these bacteria produced very little of this enzyme and grew very poorly at alkaline pH. These findings are consistent with previous research on various strains of \u003cem\u003eBacillus\u003c/em\u003e species, including \u003cem\u003eB. megagaterium, B. subtilis, B. amyloliquefaciens\u003c/em\u003e, and \u003cem\u003eA. flavithermus\u003c/em\u003e \u003cb\u003e(\u003c/b\u003eHussain et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and \u003cem\u003eBacillus velezensis\u003c/em\u003e strain MRC 5958 \u003cb\u003e(\u003c/b\u003eSarangthem et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Additional research by Balla et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) demonstrated that cellulase activity was ranged between 0.3 and 1.8 U/mL and its peak was obtained at neutral pH (=\u0026thinsp;7.0). In contrast to antecedent study, other investigations have reported that strains such as \u003cem\u003eBacillus\u003c/em\u003e species exhibit optimal cellulase production at acidic-pH values approximately pH\u0026thinsp;=\u0026thinsp;3.0 \u003cb\u003e(\u003c/b\u003eIslam et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), which improve that \u003cem\u003eBacillus\u003c/em\u003e bacteria are a major source of cellulase enzymes, which are capable of breaking down the cellulose polymers into smaller sugar units. An important factor in fermentation is temperature, which varies from a microorganism to another and regulated the growth and biosynthesis of enzymes by different microorganisms \u003cb\u003e(\u003c/b\u003eMalik et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Based on the physical properties of the cell membrane modification, temperature variation affects growth and the production of extracellular enzymes \u003cb\u003e(\u003c/b\u003eBakare et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Furthermore, the strain ideal growth and cellulase activity could be affected by temperature and the isolation site. Thus, in the present study, the ideal temperature for the bacterial growth was 45\u0026deg;C, and at that temperature, their optimal CMCase activity ranges from 1.34to 2.88 U/mL. Nonetheless, the majority of strains have cellulase activities ranging from 1.08 to 2.35U/mL and grow best at temperatures between 30\u0026deg; and 50\u0026deg;C. Numerous studies showed that 45\u0026deg;C is the ideal temperature for cellulolytic bacteria to grow and produce their enzyme \u003cb\u003e(\u003c/b\u003eNargotra et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Balla et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Malik et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). With an ideal cellulase capacity of 1.2 U/mL and 1.44 U/mL at 45\u0026deg; and 50\u0026deg; C, respectively, the results obtained are comparable to those of \u003cem\u003eB. velezensis\u003c/em\u003e (HOT1) and \u003cem\u003eB. subtilis\u003c/em\u003e (HOT2) cellulase production \u003cb\u003e(\u003c/b\u003eBalla et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, for other \u003cem\u003eBacillus\u003c/em\u003e strains, like \u003cem\u003eB. halodurans\u003c/em\u003e CAS 1, the ideal temperatures for growth and cellulase production are 55◦C and 60◦C, respectively (Annamalai et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGenerally, bacterial strains differ in their ability to tolerate salt. On culture media with 400 mM, 800 mM, and 1000 mM NaCl concentrations, all strains could grow-up. According to research reported by \u003cb\u003eShankar et al\u003c/b\u003e. \u003cb\u003e2011\u003c/b\u003e, \u003cem\u003eB. pumilus\u003c/em\u003e exhibited the highest cellulotic power at 2.5% NaCl (~\u0026thinsp;400 mM) with CMCase activity of 0.085 U/mL. All strains produced cellulase enzyme at high salinity levels, ranging from 400 to 1000 mM NaCl leading to an enzyme activity ranging from 0.81 to 2.71 U/mL. According to another study of \u003cb\u003e[13]\u003c/b\u003e 50% of the tested strains were able to synthesize cellulase enzyme at high salinity levels, between 400 and 800 mM NaCl. Furthermore, cellulase with the highest enzymatic activity was produced by the bacterial strain \u003cem\u003eBrevibacterium halotolerans\u003c/em\u003e (CDB2) in the range of 6\u0026ndash;15% (~\u0026thinsp;1000 to 2500 mM) salt concentration which is very high salinity \u003cb\u003e(\u003c/b\u003eYousef et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOsmotic stress affected the growth of bacteria and the cellulase production; so that, all strains examined exhibit osmo-tolerance and residual enzyme production in the presence of 30% of PEG\u003csub\u003e6000\u003c/sub\u003e, with S11 producing the most at 1.56 U/mL. This outcome was in concordance with prior report that demonstrated \u003cem\u003eB. velezensis\u003c/em\u003e (HOT1) exhibiting cellulase production with an enzymatic activity of 1.22 U/mL when 30% PEG\u003csub\u003e8000\u003c/sub\u003e was used \u003cb\u003e(\u003c/b\u003eBalla et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Some strains from the soil belonging to the genus \u003cem\u003eBacillus spp.\u003c/em\u003e had the ability to develop and grow well in media containing 25% PEG\u003csub\u003e6000\u003c/sub\u003e \u003cb\u003e(\u003c/b\u003eVardharajula et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). According to \u003cb\u003eShankar et al\u003c/b\u003e., \u003cb\u003e2011\u003c/b\u003e, to ensure optimum enzyme production, it is important to assess the amount of water available and monitor the \u0026ldquo;aw\u0026rdquo;.\u003c/p\u003e \u003cp\u003eSince enzymes are the building blocks of all biological processes (metabolism), biotechnology, and industrial applications, their stability and activity are crucial. In fact, enzyme activity and stability must be regulated in living microorganisms in order to preserve homeostasis and enable effective life activities. For these reasons, in this present study, the activity and stability of cellulase were evaluated for their tolerance to the impact of different stress parameters. The findings revealed that cellulase activity and stability were significantly affected by factors that could modify their functioning, including pH, temperature, salinity, and water (osmotic) stress. Using a range of pH values from 3 to 11, the effect of pH on the activity of the cellulases generated was examined. For strains Z1 and Z7, the ideal pH range for endoglucanase activity was 9 to 10 (1.19 U/mL, 0.71 U/mL), while for strains Z17 and S11, the ideal pH range was 6 to 7, with 1.22 U/mL and 0.71 U/mL, respectively. The enzyme's active site changed in response to any change in pH \u003cb\u003e(\u003c/b\u003eIrfan et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccording to the same metric, residual activity also stayed relatively constant. Depending on the strains that were tested, cellulase activity was fairly stable and had more than 60% to 90% residual activity in the pH range 4.0\u0026ndash;11.0. For strain S11, 100% (0.84 to 0.88 U/mL) of cellulase activity was maintained over the various pH ranges from 4.0 to 11.0 and pH 4.0 (0.84 U/mL) for strain Z1. Prior studies have demonstrated that cellulase is active and stable across a range of pH values, from 4.0 to 11.0 for \u003cem\u003eBacillus\u003c/em\u003e strains (Balla et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and between pH 6.0 and 6.5 for \u003cem\u003eBacillus subtilis\u003c/em\u003e YJ1, while it was stable between pH 6.5 and 7.5 according to \u003cb\u003e(\u003c/b\u003eYin et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Although CMCase of \u003cem\u003eVirgibacillus salarius\u003c/em\u003e BM-02 showed great activity over all pH ranges from 4.0 to 9.0, but the optimum pH was found at 6.5 and stable in the pH range of 5.0\u0026ndash;9.0\u003cb\u003e[12] (\u003c/b\u003eYousef \u0026amp; Mawad \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2023\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Furthermore, cellulase from \u003cem\u003eBacillus velezensis\u003c/em\u003e MRC 5958, from Bakra natural hot springs, India was stable at pH 6 to 9 (Sarangthem et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and the cellulase from \u003cem\u003eBacillus smithii\u003c/em\u003e QT03 was stable at 70% of enzymatic activity at pH 6 for 24 hours \u003cb\u003e(Villanueva et al. 2025)\u003c/b\u003e. However, the cellulase of \u003cem\u003eCellulomonas\u003c/em\u003e sp. ASN2 was stable at pH 6.5\u0026ndash;9.5 and more active at pH 7.5 (~\u0026thinsp;0.45 IU/ml/min). This enzyme, which could be utilized in alkaline settings like pulp processing, requires a pH value of 7.5 \u003cb\u003e(\u003c/b\u003eIrfan et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Cellulase typically exhibit stability across a broad pH range of 5 to 10. Because of their significant industrial properties, a number of researchers have recently attempted to isolate alkaline thermostable enzymes using microbes \u003cb\u003e(\u003c/b\u003eSamiullah et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEnzyme used in industry depends critically on the optimal temperature range of the enzyme activity and enhanced stability at high temperatures, particularly at temperatures of 50\u0026deg;C or upon 50\u0026deg;C. Temperature was found to have an impact on enzyme activity over a broad temperature range between (45 to 80\u0026deg;C), with all strains exhibiting optimal activity at 50\u0026deg;C and 70\u0026deg;C. At 50\u0026deg;C, strain Z1 exhibits an optimal CMCase activity of 2.89U/mL \u003cb\u003e(\u003c/b\u003eBhatti et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Yeoman et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Yousef and Mawad \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2023\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Similar results indicate that cellulases from certain \u003cem\u003eBacillus\u003c/em\u003e species, such as \u003cem\u003eBacillus subtilis\u003c/em\u003e YJ1, \u003cem\u003eB. velezensis\u003c/em\u003e Z2.6, and \u003cem\u003eBacillus sp.\u003c/em\u003e strain RH68 have an optimum temperature of 50\u0026deg;C (Yin et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Cai et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and \u003cem\u003eBacillus\u003c/em\u003e. sp RH68 strain, have an optimal temperature of 50\u0026deg;C \u003cb\u003e(\u003c/b\u003eYin et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and 70\u0026deg;C (RH68) \u003cb\u003e(Mawadza et al\u003c/b\u003e.\u003cb\u003e2000)\u003c/b\u003e. Stability, measured as residual enzyme activity, was optimal at 50◦C and 70◦C with an activity of 0.65 to 1.28 U/mL. According to another study, \u003cem\u003eBacillus\u003c/em\u003e sp. AR03's cellulolytic cocktail reaches 60\u0026deg;C (Manfredi et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and the strain \u003cem\u003eCellulomonas sp\u003c/em\u003e. ASN2 reached its optimum at 60\u0026deg;C \u003cb\u003e(\u003c/b\u003eIrfan et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Furthermore, strain \u003cem\u003eBacillus sp\u003c/em\u003e. Z2.6 demonstrated significant activity, maintained above 75% between 40\u0026deg;C and 60\u0026deg;C (Cai et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). According to the reported results of Sarangthem et al. (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), the enzyme from \u003cem\u003eBacillus velezensis\u003c/em\u003e strain MRC 5958 is stable over a wide temperature range, from 30\u0026deg;C to 70\u0026deg;C and cellulase from \u003cem\u003eBacillus smithii\u003c/em\u003e QT03 was maintained 100% and 58% of its activity at 55\u0026deg;C for 3 hours and at 60\u0026deg;C for 24 hours respectively \u003cb\u003e(Villanueva et al. 2025)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eRegarding the impact of salinity on enzyme properties, the activity and stability of the cellulase by the different strains under investigation were not significantly impacted by rising NaCl levels (0\u0026ndash;2000 mM). In fact, at all salinity levels, the strains retained a high enzymatic potency (1.5\u0026ndash;2.5 U/mL). With strains Z1 and Z7, crude cellulases maintained 50\u0026ndash;60% of their initial capacity even at 2000 mM and remained stable between 40 and 50% up to 500 mM. Although, purified CMCase from \u003cem\u003eBacillus licheniformis\u003c/em\u003e strain AU01 was found to retain only 34% of its activity at 30% NaCl (ca. 5000mM) \u003cb\u003e(\u003c/b\u003eAnnamalai et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), whereas \u003cem\u003eBacillus sp\u003c/em\u003e. AR03's cellulolytic activity exhibited high resistance to concentrations up to 2.0 M NaCl (Manfredi et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). These cellulases' high salt tolerance is of great biotechnological interest; it will be essential for future uses in a variety of processes that rely on high salinity or osmotic pressures \u003cb\u003e(Wang and al. 2009;\u003c/b\u003e Annamalai et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInterestingly, in the presence of 30% PEG\u003csub\u003e6000\u003c/sub\u003e, the enzyme's initial and residual activity (i.e., 2.39 U/mL) was unaffected by water deficiency; however, the stability of the enzyme was maintained up to more than 40% (1.02 to 0.72 U/mL). Balla et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), also reported similar findings. They demonstrated that at 30% PEG\u003csub\u003e8000\u003c/sub\u003e, CMCase stability and activity were only marginally impacted. The specificity of the crude enzyme extract was assessed by incubating it with various soluble and insoluble substrates (CMC, powdered cellulose, wheat bran, and filter paper). In fact, Z17 was thought to be the best enzyme producer for breaking down crystalline cellulose (powdered cellulose and filter paper), indicating strong and significant exoglucanase activity reaching ca. 2.1 to 2.4 U/mL, while Z1 was the best enzyme producer for breaking down soluble cellulose (CMC), with a significant and maximum activity (ca. 2.8 U/mL), suggesting high endoglucanase activity. All four enzymes are similarly effective on the complex natural substrate, wheat bran demonstrating extremely similar and high activity (ca 2.2 U/mL).\u003c/p\u003e \u003cp\u003eExoglucanases were typically linked to the capacity to break down crystalline cellulose, while endoglucanases from fungi and \u003cem\u003eBacillus\u003c/em\u003e species that possessed both Avicelase (cellulase) and filter paper (FPase) hydrolytic capacities in addition to their CMCase activity, have demonstrated the ability to accomplish this through the assistance of an exo-activity that resides within the same molecule (\u003cb\u003eLi et al. 1998;\u003c/b\u003e Sharma et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Soluble substrates, particularly celluloses with a low degree of polymerization (DP), could be dissolved in water because of their chemical substitutions. Thus, ionically substituted CMC was typically used to measure the activity of endoglucanase, also referred to as CMCase. The DP of CMC is significantly reduced as a result of endoglucanases' random cleavage of intramolecular β-1,4-glucosidic bonds and previous report had shown that a portion of the C-terminus of the full endoglucanase gene product is essential for the interaction between this enzyme and the insoluble substrate \u003cb\u003e(\u003c/b\u003eZhang et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). It was predicted that the FPase activity ranged from 1.45 (Z7) to 2.13 (Z17) U/mL and the enzyme activity on powdered cellulose ranged from 1.86 (S11) to 2.03 (Z17) U/mL. These findings allow for the classification of this enzyme as both an endo- and exo-type of cellulase, as it was previously described \u003cb\u003e(\u003c/b\u003eSharma et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe impacts of several stressors on cellulase production on solid medium were investigated at pH5, 50\u0026deg;C, and 800 mM NaCl. The results of the enzymatic index indicated that strain Z1 genetically identified as \u003cem\u003eBacillus siamensis\u003c/em\u003e had an optimal cellulase production of 2.73 at 50\u0026deg;C by providing a control, which is comparable with, \u003cem\u003eBacillus subtilis subsp. inaquosorum\u003c/em\u003e isolate CGR-1 that exhibited amylolytic and cellulolytic activities at 50\u0026deg;C \u003cb\u003e(\u003c/b\u003eGeraldi et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) Withal, Hassan et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), have proven that abiotic stressors, especially high temperatures, stimulate the production of high-value products such as antioxidants and increase enzymatic activity. The effect of pH and the presence of 800 mM NaCl was remarkable, with enzymatic indexes ranging from 0.3 to 2 for the selected four strains belonged to \u003cem\u003eBacillus\u003c/em\u003e species. The combination of the three factors\u0026mdash;pH 5, T 50\u0026deg;C, and 800 mM NaCl\u0026mdash;was ideal for cellulase production for strain Z1, which had an Ei of 3, while the other strains produced cellulase with an Ei varied between 0.8and1.7. Those outcomes were correlated with prior report that substantiated notable multi-stress resistance (pH 9, T50\u0026deg;C, and 400Mm NaCl) of the cellulase on solid media \u003cb\u003e(\u003c/b\u003eBalla et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Using both macro and microscopic morphological standard tests, the four selected cellulolytic bacterial strains were most likely belong to the \u003cem\u003eBacillus\u003c/em\u003e genera, which is characterized as a rod-shaped, Gram-positive bacterium, and sporulated strains which is confirmed by Alcaraz et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), whose reported that in reaction to environmental or nutritional stressors, bacillus species generated extremely resilient dormant endospores and is facultatively aerobic or anaerobic.\u003c/p\u003e \u003cp\u003eAccording to a previous study suggesting that 16S rRNA sequencing is an accurate method for species identification and distinction between closely related bacterial species \u003cb\u003e(Hussein et al. 2017).\u003c/b\u003e So that, Comparison of 16S rRNA gene sequences revealed that strain Z1 showed the highest similarity of 99. 72% with \u003cem\u003eB. siamensis\u003c/em\u003e (PX112786.1) which was firstly isolated from salted crab (poo-khem) in Thailand in 2010 \u003cb\u003e(\u003c/b\u003eSumpavapol et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The \u003cem\u003eBacillus siamensis\u003c/em\u003e YC-9 strain, originating from termite nests, synthesizes various enzymes of industrial interest, including cellulases, reaching an optimal production of 1.54 IU/g when cultivated on wheat bran pretreated in an alkaline medium (Wang et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Rathod et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) used a microbial co-culture of \u003cem\u003eBacillus siamensis\u003c/em\u003e F2 (as a bacterium that produces multiple enzymes such as cellulase) and \u003cem\u003eCandida albicans\u003c/em\u003e GP1 to produce bioethanol from fruit waste.\u003c/p\u003e \u003cp\u003eStrain Z7 was identified through 16S rRNA gene sequencing, and the sequence obtained was identified according to the GENBANK (NCBI) under accession number MG470713.1, showing high homology (99.65%) with different strains of Bacillus \u003cem\u003esp.\u003c/em\u003e (in: Firmicutes). Lugani et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) optimized cellulase production from a newly isolated strain of \u003cem\u003eBacillus sp. Y3\u003c/em\u003e, achieving a maximum FPase activity of 6.84 IU/mL and a CMCase activity of 7.82 IU/mL. The strain Z17 showed the highest similarity of 99.65% with \u003cem\u003eB. cabrialesii\u003c/em\u003e (ON287153.1) newly isolated and identified for the first time by \u003cb\u003eSantos et al. (2019)\u003c/b\u003e. Then, \u003cem\u003eBacillus cabrialesii\u003c/em\u003e, has been isolated from wheat plants in the Yaqui Valley, Mexico. This bacterium is known for its plant growth-promoting properties and as a biological control agent \u003cb\u003e(Figueroa et al. 2023).\u003c/b\u003e Furthermore, Masmoudi et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), studied the ability of the new thermo-halotolerant bacterium \u003cem\u003eBacillus cabrialesii\u003c/em\u003e, native to the Qatar desert, to produce cell wall-degrading enzymes, such as cellulase. In addition, bacterial strain S11 showed the closest sequence similarity to \u003cem\u003eB. inaquosorum\u003c/em\u003e, (MN581182.1) with percentage of 99. 73%. However, research conducted by Srivastava et al. (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) indicated that \u003cem\u003eB. inaquosorum\u003c/em\u003e KCTC 13429 could be developed as a high-yielding commercial EG endoglucanase strain for the degradation of cellulose-rich biomass. In previous studies, \u003cem\u003eB. subtilis\u003c/em\u003e and \u003cem\u003eBacillus subtilis\u003c/em\u003e subsp. \u003cem\u003einaquosorum\u003c/em\u003e have been reported to produce cellulase enzymes, including \u003cem\u003eB. subtilis\u003c/em\u003e K-18 \u003cb\u003e(\u003c/b\u003eIrfan et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), B. \u003cem\u003esubtilis\u003c/em\u003e CD001 \u003cb\u003e(Malik et al. 2021)\u003c/b\u003e, and \u003cem\u003eB. subtilis\u003c/em\u003e subsp. \u003cem\u003einaquosorum\u003c/em\u003e CGR-1\u003cb\u003e(\u003c/b\u003eGeraldi et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Indeed, a lot of extracellular enzymes can be produced and secreted by \u003cem\u003eBacillus\u003c/em\u003e species \u003cb\u003e(\u003c/b\u003eKazemi et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). They are regarded as one of the most adaptable and prevalent microbial fermentations because they typically showed rapid growth rates and brief fermentation times \u003cb\u003e(\u003c/b\u003eSadhu et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Kazemi et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Manfredi et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe current work successfully gathered novel, stress-tolerant thermophilic \u003cem\u003eBacillus\u003c/em\u003e strains with high-activity, and stable cellulase enzymes. Eventual research into maximizing the synthesis of extracellular enzymes and assessing the commercial and biotechnological worth of these bacterial strains and their enzymes should be conducted.\u003c/p\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eThe aim of the current study was to isolate and identify thermophilic bacteria from Algerian hot springs. According to the findings of the bacterial hydrolytic activity screening, most of the isolates exhibited strong cellulasic activity, confirming the presence of promising extracellular hydrolytic enzymes like cellulase. After valuating different stress parameters, four among the 45 bacterial strains tested and assessed for their cellulase activity were picked for further analysis and identified to be belonging to \u003cem\u003eBacillus\u003c/em\u003e group: Z1 as \u003cem\u003eB. siamensis\u003c/em\u003e, Z7 as \u003cem\u003eBacillus sp\u003c/em\u003e. (in firmicutes), Z17 as \u003cem\u003eB. cabrialesii\u003c/em\u003e and S11 as \u003cem\u003eB. inaquosorum\u003c/em\u003e. The crude enzyme was active over a broad pH range of 4 to 11, with residual activities decreasing from 100% to 60%, demonstrating the impact of physicochemical parameters on cellulase. Their higher activity was mentioned at 50\u0026deg;C and 70\u0026deg;C and their stability at 50\u0026deg;C and 80\u0026deg;C. Their residual activity remained between 40 and 60% across a wide range of salt concentrations from 0 to 2000 mM and weakly affected by PEG\u003csub\u003e6000\u003c/sub\u003e. The cellulase-rich crude enzyme extract, which can be described as a blend (combination) of endo and exo-cellulase types, effectively degraded both soluble and insoluble substrates. Furthermore, this particular bacterial genus (\u003cem\u003eBacillus\u003c/em\u003e) exhibited intriguing traits of resilience to wide stress-conditions on solid media. To conclude, this current work lays the groundwork for future investigations into the optimization of extracellular enzyme production, and further analysis should be conducted in order to determine the value of those bacterial strains and their enzymes in industrial and biotechnological scales.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBLAST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBasic Local Alignment Search Tool\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCMC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCarboxymethyl cellulose\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDeoxyribonucleic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDNS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e3,5 dinitrosalicylic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDegree of polymerization\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEi\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEnzymatic index\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFood and Drug Administration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGRAS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGenerally Regarded as Safe\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLuria broth medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLCB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLignocellulosic biomass\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNutrient agar\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNaCl\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSodium chloride\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNCBI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNational Center for Biotechnology Information\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOptical density\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePEG6000\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolyethylene glycol 6000\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e16S rRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e16S ribosomal ribonucleic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTSA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTryptic soy agar\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eSupplementary Materials\u003c/h2\u003e \u003cp\u003eSee Appendix 1\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConflicts of Interest:\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis research received no external funding.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, L.A., and S.S; Formal analysis, H.C.S.; Investigation, S.S., J.M.; Writing-original draft, S.S., J.M.; Writing-review \u0026amp; editing, J.M., M.L, H.C.S.; Supervision, J.M \u0026amp; L.A.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbada EA, Elbaz RM, Sonbol H, Korany SM (2021) Optimization of cellulase production from Bacillus albus (MN755587) and its involvement in bioethanol production. 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Bioresour Technol 101:292\u0026ndash;297\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Thermal spring, Bacillus genus, extracellular enzymes, cellulase activity, enzyme stability","lastPublishedDoi":"10.21203/rs.3.rs-8574179/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8574179/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHot spring microbiological studies in underexplored regions like Algeria are important because they contain extremophiles that may yield unique and robust biomolecules. So that, this current study aimed for the screening of enzymatic potentiality through the production of cellulase, in order to identify the diversity of cultivable bacteria isolated from two thermal spas of Hammam Ouled Tebben and the Zdim hot spring, in south of Setif (Algeria). The obtained results showed that cellulolytic activity was prevalent, according to the enzymatic index values. The valuation of the cellulase production on 1% of CMC liquid medium, eight strains were selected for their great activity. Indeed, the physicochemical parameters (NaCl; pH; temperature; PEG6000) that affected enzymatic activity and stability, the bacterial growth and cellulase production were assessed. Then, the majority of strains were found to be thermo-tolerant, neutrophilic, halotolerant, and tolerant to 30% PEG 6000. Withal, the screening of the effects of physicochemical parameters on the activity and stability of the cellulase enzyme allowed to select four strains among the most effective, their CMCase active and stable over a wide pH range, temperature, NaCl and they were slightly affected at 30% PEG6000. The phenotyping and genotyping of the selected bacteria were belonging to Bacillus sp. group. Overall, this work highlighted those Algerian hot springs represents an important reservoir of potentially bacterial producer of cellulase. Obviously, future studies should be addressed to confirm and promise the production potential and industrial and biotechnological applications because of these bacterial strains newly isolated and identified.\u003c/p\u003e","manuscriptTitle":"Thermophilic cellulase: screening of novel cellulolytic bacteria from Algerian hot springs for CMCase production","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-29 13:20:22","doi":"10.21203/rs.3.rs-8574179/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1e09c09b-e882-4268-9265-a604240c11a2","owner":[],"postedDate":"January 29th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-21T06:38:19+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-29 13:20:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8574179","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8574179","identity":"rs-8574179","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}