Screening of a Soil Bacteria Collection for the Production of Alkali Thermostable Xylanases

This work aimed to evaluate a collection of common and rare soil bacteria regarding to extracellular xylanases production and to characterize the stability in contrasting conditions of temperature and pH of these enzymes. This collection consists of 120 isolates belonging to six phyla that were subjected to screening for xylanase activity in pure cultures and in the extracellular proteic extract (EPE). The ratio between the halos diameters of xylan hydrolysis and in the colonies on solid medium (ratio H:C) was used for the evaluation of cultures as selection criteria. EPEs of isolates with highest ratios H:C were evaluated for the specific xylanases activity at 50 °C for 1 h. EPE of the three isolates with the highest potential for activity under this condition were evaluated for optimum activity, stability at 60 °C and different pH values. Twenty-two isolates showed xylanase activity under these conditions. Xylanases from TC21 and TC119 showed high relative activity at temperatures up to 70 °C and were less sensitive to changes in pH. Soil bacteria show high potential as a source of extracellular xylanases adapted to extreme pH and temperature conditions, which are required in agroindustrial processes.


Introduction
Despite the importance of microorganisms in the maintenance of the biosphere, it is difficult to estimate the number of organisms in microbial communities due to their great genetic diversity (Haegeman et al., 2013), and just over 7,000 bacterial species have been described (Achtman & Wagner, 2008).It is known that more than 99% of the bacteria from environmental samples remain uncultivated and most of these microorganisms found in the soil are insufficiently studied because of the rarity of their isolation in cultivation studies (Pham & Kim, 2012).
The expected scientific benefits of greater knowledge about microbial diversity are related to the discovery of potentially exploitable microorganisms for the most diverse biotechnological processes, such as the production of enzymes for industrial and technological applications.Among the microorganisms that are not well explored are those belonging to the soil microbiota, which have immense biotechnological potential since they produce several enzymes that have been commercially exploited over the years (Jayani, Shivalika, & Gupta, 2005).
There are about 200 enzymes produced by microorganisms available on the market from the 4000 enzyme types that are known at the moment, yet just 20 of these are produced in large quantities (Li, X. Yang, S. Yang, Zhu, & Wang, 2012).Regarding the global market for industrial enzymes, in 2014 this was around $4.2 billion and is expected to reach $6.2 billion from 2015 to 2020, with an annual growth rate (CAGR) of 7% (BCC Research, 2014).Therefore, due to the high price of microbial enzymes, technology for improving their quality is developing each day (Gurung, Ray, Bose, & Rai, 2013).such as rice straw or bagasse, which constitutes low-cost substrate for fermentation (Bon, Ferrara, & Corvo, 2008).
Xylanases are among the most widely used industrial enzymes on the market (Cowan, 1996;Li et al., 2012b) and represent 20% of the global enzyme market together with cellulases and pectinases.Xylanases are produced by various organisms and most of the bacteria and fungi secrete these enzymes in the extracellular medium where they will act on hemicellulose material to release xylose as a directly assimilable final product.Among the bacteria, Actinobacteria and Firmicutes of the Bacillaceae family are reported to be the main producers of industrially important enzymes involved in lignocellulose degradation (Nagar, Mittal, Kumar, & Gupta, 2012).
In several industrial applications, such as the bleaching stage of the kraft pulping process in paper production, the inlet pulp has a high temperature and alkaline pH, making it necessary to search for alkali thermostable xylanases (Sharma & Bajaj, 2005).In addition, cellulose-free xylanase is crucial to avoid degradation of the cellulose fibers (Techapun, Poosaram, & Watanabe, 2003).These enzymes promote the removal of xylan, facilitating the leaching of lignin (Niehaus, Bertoldo, Kahler, & Antranikian, 1999).Therefore, in the paper industry, the use of xylanases represents an important technological improvement because they promote an increase in the bleaching effect without chemical reagents and also decrease the production of pollutants during these processes (Walia, Guleria, Mehta, Chauhan, & Parkash, 2017).
Another outstanding industrial process that makes use of enzymes is the production of second generation ethanol, where xylanases are used in the degradation of lignocellulosic biomass.Bioconversion of biomass, such as sugarcane bagasse and straw, has been widely studied for the purpose of producing biofuel from a renewable source, representing an important alternative for increasing second-generation ethanol production (Canilha et al., 2012).It is recognized that a large amount of sugar bagasse is inappropriately used, contributing to environmental (Schettino & Holanda, 2015).As both bagasse and sugarcane straw are lignocellulose materials, they could be better utilized for biofuel generation, which would contribute even more to Brazil's leadership in the sugar-alcohol sector.
Bacterial xylanases are known to be stable at alkaline pH and high temperature and have faster growth than fungi propterties that are suitable for industrial requirements (Chakdar et al., 2016).Thus, the characterization of the enzymes is a crucial step to understanding their properties of optimal activity and stability at different values of pH and temperature, which enables an evaluation of their potential application in these different processes (Kashyap, Vohra, Chopra, & Tewari, 2001).Several of these processes are performed using the cells themselves as a source of enzymes, but efficiency of the processes can be increased through the use of isolated and purified enzymes (Mohana, Shah, Divecha, & Madamwar, 2008).
Considering the importance of xylanases in diverse agro-industrial uses, the present study involved evaluating a soil bacteria collection with the objective of selecting which produce alkaline thermostable extracellular xylanases.

Isolates and Growth Media
A total of 120 bacterial isolates obtained from the Soil Microbiology Laboratory of Embrapa Tabuleiros Costeiros (Aracaju-SE) were evaluated for in vitro xylanolytic activity.The isolates were obtained from soil samples collected in a secondary forest or in cultivated land planted with maize and pigeon pea and were cultured using VL55 culture media (Sait, Hugenholtz, & Janssen, 2002) with xylan as the sole source of carbon and nutrient agar in the original concentration or as a 10-fold, 100-fold and 1000-fold dilution.The taxonomic affiliation of the isolates was based on 16S rRNA gene partial sequencing.The 16S rRNA gene partial sequences obtained were compared with the sequences of bacteria of culture type deposited in the GenBank database (http://www.ncbi.nlm.nih.gov/) using the BLASTN software (Cavalcante, 2012).
The isolates were stored at -20 °C in 50% glycerol, reactivated in liquid culture media of the same composition to the media used in the isolation and incubated (32 °C) in the dark until initial media turbidity.Thereafter, the reactivated cultures were inoculated in Petri dishes containing the solid media to verify purity.

Selection of Extracellular Xylanase Producers in Solid Media
To select the xylanase-producing bacteria, the isolates were inoculated into 50-mL Erlenmeyer flasks containing 20 mL of mineral saline solution (MSS) containing xylan as the sole carbon source (Bajaj & Singh, 2010).The MSS solution is composed of 1% NH 4 NO 3 , 0.5% KH 2 PO 4 , 0.1% MgSO 4 •7H 2 O, 0.01% CaCl 2 •2H 2 O, 0.01% NaCl and 0.01% MnSO 4 •H 2 O, with the addition of 0.5% xylan.Once the isolates were at the beginning of the exponential phase, indicated by initial turbidity of the medium, xylanase production tests were carried out on solid MSS medium containing xylan as the sole carbon source (0.5%).For this, a 2-mL volume of the liquid culture was centrifuged (14000 rpm for 5 min) and washed twice with saline (0.85% NaCl, w/v).Thereafter, the optical densities (OD) of the cell suspensions were adjusted to 0.7 in a spectrophotometer using wavelength of 500 nm to give 1 × 10 6 UFC/mL.A 10-μL aliquot was deposited in the center of each Petri dish containing MSS solidified with agar (2%) or gellan (1%).Triplicate plates were used for each isolate.Plates were incubated in the dark at 30 °C (±2 °C) for seven days, and for an additional seven days if colony formation did not occur in the first period.Isolates whose colonies were not visible within 14 days were excluded from further procedures.Xylanase in vitro activity was evaluated by measuring the diameter of pale halos observed in the culture media, which are formed by xylan hydrolytic enzymes excreted by the colony and diffuse through the solid medium.To increase the contrast between the pale zone of the halo and the medium with xylan, 0.1% Congo red was added to the surface of the medium (Sharma & Bajaj, 2005).Two perpendicular measurements were made for each halo.These measurements were taken on the day of the test with Congo red and seven days after the test.The diameter of the colonies was also measured on the same evaluation dates.Selection of the isolates with the highest activity was performed using the relationship between the diameters of the activity halos and the colonies (H:C ratio) considering both evaluation dates.
To test the hypothesis that the methods used in the isolation of bacteria (Cavalcante, 2012) result in cultures with different xylanase activities, the effect of various aspects of bacterial isolation on the H:C ratios of the isolates was evaluated as described below.The t-test (p < 0.05) was used to compare the mean values of isolates obtained from different soils (agriculture versus forestry), solidifying agents in the culture medium (agar versus gellan), dilution of the inoculum used in plating (10 -6 versus 10 -7 ) and pour-plate versus spread-plate methods.The Bonferroni procedure (p < 0.05) was used to compare the mean H:C ratios of the isolates with different incubation durations until colony appearance and among the culture media.The t-test was also used to test the hypothesis that was a difference in xylanase activity between the bacteria in the collection classified as rare and common isolate.

Preparation of Extracellular Protein Extracts (EPE)
The isolates with higher H:C ratios were selected and grown in the MSS medium with xylan (0.5%) and incubated at 32 °C/150 RPM until the initial media turbidity was observed.This medium was filtered on a Millipore nitrocellulose filter (0.22-μm porosity), which was maintained at a temperature of -20 °C until the time of analysis.

Determination of Extracellular Xylanase Activity
The enzymatic activity was determined using the modified method of Schinner and Von Mersi (1990), which was used to evaluate the activity of xylanases in soil extracts.According to the original method, the reducing sugars formed during the incubation of extracts in the presence of xylan (1.2% in 2 M acetate buffer, pH 5.5) at 50 °C for 24 h were quantified colorimetrically after reaction with ferric potassium hexacyane under specific conditions, using glucose as the standard.In this study, the incubation period was reduced from 24 h to 1 h; other conditions were the same as in the original method.A low protein content of the cell extracts under the conditions used for the culture of bacteria was reported.Therefore, this method was chosen instead of the traditionally employed dinitrosalicylic acid method (Miller, 1959), since it has a sensitivity which is approximately 200 times higher for detection of reducing sugars (Schinner & Von Mersi, 1990).For these analyses, 100-μL aliquots of each EPE were added to 3 mL of acetate buffer and 3 mL of xylan buffer in test tubes, which were incubated at 50 °C for 1 h in triplicate.For the controls, EPE addition took place after the incubation period.For quantification of the reducing sugars, 100-μL aliquots were pipetted into test tubes and the volume made up to 1 mL with distilled water; 1 mL of reagent A (16 g L -1 of anhydrous sodium carbonate and 0.9 g L -1 of potassium cyanide) and 1 mL of reagent B (0.5 g L -1 of ferric hexacyane potassium) were then added.The test tubes were sealed and placed in a water bath at 100 °C for 15 min.After this period, the contents of the tubes were cooled rapidly in a cold water bath for approximately 5 min and 5 mL of reagent C (1.5 g L -1 ferric ammonium sulfate, 1.0 g L -1 of sodium dodecyl sulfate and 4.2 mL of L -1 of concentrated sulfuric acid) was then added.After color stabilization for 1 h at room temperature, the absorption was measured at 690 nm to determine the formation of reducing sugars.A reducing sugar standard curve based on glucose (0 to 18 μg glucose mL -1 ) was used to convert the absorbance into the amount of product formed by the reaction.Reactions for the detection of reducing sugars were performed in duplicate.
The total protein content in these extracts was determined by the method of Bradford (1976) as recommended by the supplier for the detection of proteins in the range of 8 to 80 μg mL -1 (Bio-Rad).Bovine serum albumin (BSA) solutions with known concentrations were used as the standard.
The specific activity of the extracellular xylanases was determined by the ratio between the rate of formation of reducing sugars and the concentration of total proteins in the EPE.The units were expressed as μg of reductive sugar min -1 mg -1 protein.

Enzyme Characterization
The three isolates with the highest specific activity in the previous assay were selected for the determination of the optimum temperature of activity and thermostability of the xylanases.Quantification of the activity in the two determinations was performed using the method of Schinner and Von Mersi (1990) in the same manner as previously described.For the optimum temperature of activity test, the incubation of the protein extracts was carried out at temperatures ranging from 30 to 80 °C, with increments of 10 °C.The results were presented as relative activity, expressed as the percentage of specific activity at each temperature in relation to the maximum specific activity observed at the six incubation temperatures.For determination of the enzymatic stability at temperature, 100 μL of extract was added to test tubes containing 3 mL of acetate buffer without xylan and incubated for 2, 4 and 6 h at 60 °C.After each of these incubation periods, 3 mL of acetate buffer with xylan (1.2%) was added to the contents of the vials and the incubation was carried out to determine the residual activity of xylanases at 50 °C for 1 h.Determination of the reducing sugars formed was performed as previously described.The specific activity of xylanases at 50 °C, without pre-incubation at 60 °C, was used as the control, and the activities after 2, 4 and 6 h of pre-incubation were expressed as a percentage of the control value.
The three isolates selected were also tested for xylanase stability at pH values of 4.0, 5.5 and 8.0.To this end, 250 μL of each extract was incubated for 24 h at 4 °C in 750 μL of McIlvaine buffer (McIlvaine, 1921) adjusted to each pH value.After this period, 100-μL aliquots were incubated at 50 °C in 3 mL of acetate buffer and 3 mL of acetate buffer with xylan (1.2%) for 1 h and the reducing sugar content formed by the residual activity of the xylanases quantified as previously described.For the background solution, 100-μL aliquots of the pre-incubated EPEs with different pH values were added immediately before the reactions for the determination of reducing sugars.The maximum residual specific activity among the three pH values tested was used as reference and the results expressed as relative activity (percentage of the reference activity).

Determination of Cellulase Activity
For the isolate with the highest xylanase specific activity, cellulase activity was determined in the protein extract obtained after incubation in medium containing xylan as the sole carbon source.This activity was determined by the method of Schinner and von Mersi (1990), replacing xylan with disodium CM-cellulose (7 g L -1 of 2M acetate buffer, pH 5.5).The procedures for reading the samples and the standard curve followed the methodology described in the previous sections.

Results and Discussion
From 120 isolates evaluated, 114 (95% of the total) presented growth in medium with xylan as the only carbon source.Despite this high frequency, only 22 isolates (25% of the total) showed xylanase activity, detected by the formation of pale halos around the colonies under the conditions tested (Table 1).This discrepancy may be associated with free glucose and arabinose contamination in the beechwood xylan used, which would act as a carbon source and allow initial growth of the colonies without xylanase activity.Although 50 bacteria from the collection were isolated in VL55 medium, which has several nutritional factors, especially vitamins, most of these bacteria are metabolically versatile in nutritional terms, displaying growth in MSS medium whose composition has only mineral salts and xilan as a source of carbon.From 22 bacteria that showed halo xylanase activity under the conditions tested, 20 showed growth at up to seven days of incubation and only two (TC14C and TC9D) between seven and 14 days.As for the date of detection of xylanase activity, all bacteria, except TC97, presented halos on the first date that activity was evaluated (Figure 1a).High variability among isolates was observed in xylanase expression capacity under the conditions tested, regardless of the dates of evaluation of enzyme activity (Figure 1).In the first evaluation date the H:C ratio ranged from 1.44 to 4.26, between TC89 and TC21 isolates, respectively.This variation corresponded to 3.7 times the standard deviation (SD) observed among the isolates of the collection and a 290% increase among the isolates of the minimum and maximum H:C value.For the second evaluation date, the H:C values ranged from 1.44 to 4.73 for TC89 and TC92 (Figure 1b), which corresponded to 3.4 times the SD between isolates and a 230% increase between the isolates of the minimum and maximum H:C value.Note. 1 Quoted names indicate families with which the isolates had the highest similarity in the 16S rDNA sequence, although they did not affiliate with them; 2 According to the methodology of Joseph et al. (2003); 3 FS: soil under secondary forest; AS: only under agricultural cultivation; 4 Plating of 100 μl of dilution 10-6 (D6) and 10-7 (D7); 5 VL: VL55 (Sait et al., 2002); AN, AN1: 10 and AN1: 100: nutrient agar without dilution, diluted 10 and 100 fold, respectively; 6 PP: "pour-plate", PE: spread plate.
The sequence of isolates with regard to the H:C ratio differed between the first and second evaluation dates (Figure 1).Several studies have previously observed late gene expression in bacterial isolates, such as studies with nodC and nodW nodulation genes and the nopP gene in Bradyrhizobium japonicum, which probably act on the infection of soybean roots (Bortolan, Barcellos, Marcelino, & Hungria, 2009), or expression of GDH gene in Lactobacillus plantarum DPPMA49 in response to different environmental conditions (De Angelis et al., 2010), which revealed differences in the activation of the gene expression machinery in response to the medium.
These changes were characterized by halo formation by TC97 only on the second evaluation date and by the increase in the H:C ratio between the two dates for some bacteria, especially TC119, TC66, TC92, TC13B and TC99.The gap between colony formation and expression of xylanase activity may be related to mechanisms of catabolic repression, in which sugars of more easy assimilation represses the expression of genes involved in the use of polymers of more complex structure, as already demonstrated with the fungus Trichoderma reesei (Mach, Strauss, Zeilinger, Schindler, & Kubicek, 1996) Figure 4. isolates

Table 1 .
Characterization of bacterial isolates with xylanase halos formation activity for taxonomic affiliation based on 16r DNAr, culture rarity, soil type and techniques used in isolation (Cho & Choi, 1999)earothermophilus and Bacillus subtilis(Cho & Choi, 1999).