Bioprocess and biotecnology: effect of xylanase from Aspergillus niger and Aspergillus flavus on pulp biobleaching and enzyme production using agroindustrial residues as substract

This study compares two xylanases produced by filamentous fungi such as A. niger and A. flavus using agroindustrial residues as substract and evaluated the effect of these enzymes on cellulose pulp biobleaching process. Wheat bran was the best carbon source for xylanase production by A. niger and A. flavus. The production of xylanase was 18 and 21% higher on wheat bran when we compare the xylanase production with xylan. At 50°C, the xylanase of A. niger retained over 85% activity with 2 h of incubation, and A. flavus had a half-life of more than 75 minutes. At 55°C, the xylanase produced by A. niger showed more stable than from A. flavus showing a half-life of more than 45 minutes. The xylanase activity of A. niger and A. flavus were somehow protected in the presence of glycerol 5% when compared to the control (without additives). On the biobleaching assay it was observed that the xylanase from A. flavus was more effective in comparison to A. niger. The kappa efficiency corresponded to 36.32 and 25.93, respectively. That is important to emphasize that the cellulase activity was either analyzed and significant levels were not detected, which explain why the viscosity was not significantly modified.


Introduction
Annually, large quantities of lignocellulosic wastes (agricultural and agroindustrial residues, like wheat bran, sugarcane bagasse, soybean, rice straw, corncob and orange peel) are generated through industrial processes such as breweries, paper-pulp, textile and timber industries. And their disposal is becoming a problem regarding space, causing environmental pollution because most of the wastes are disposed by burning. However, the plant biomass regarded as "wastes" are biodegradable and represent an inexpensive alternative source for microbial growth or enzymes production (Sepahy et al., 2011;Facchini et al., 2011;Okafor et al., 2007).
In the last decades, an increasing number of studies aimed to develop environmentally clean and non-toxic methods for industrial processes Peixoto-Nogueira et al., 2009). The possibility of using a variety of agricultural residues is an attractive practice for the production of plant cell wall degrading hydrolytic enzymes, especially xylanase and cellulase, because this system could simulate the natural environment (Facchini et al., 2011(Facchini et al., , 2012. The xylanase production has been reported for bacteria (Sepahy et al., 2011), actinomycetes (Garg et al., 2011) and fungi (Das et al., 2013;Sorgatto et al., 2012;Facchini et al., 2011Facchini et al., , 2012. Filamentous fungi which produce xylanases are attracting greater attention than bacteria and yeast because they are particularly interesting from an industrial point of view, due to the fact they secrete much higher xylanolytic enzymes into the medium (Okafor et al., 2007;Polizeli et al., 2005).
Among the filamentous fungi employed to produce xylanase, the Aspergillus genus is one of the most explored (Sandrim et al., 2005;Betini et al., 2009;Peixoto-Nogueira et al., 2009;Michelin et al., 2010). To date, however, there have been few reports of xylanases produced by Aspergillus flavus.
Xylanases produced by microorganisms has attracted a great deal of attention during the past few decades because of its potential biotechnological applications in various industries including food, feed, fuel, textile, and paper and pulp industries and in waste treatment (Yeasmin et al., 2011;Facchini et al., 2011;Michelin et al., 2010;Jiang et al., 2010).
An increasing awareness on environmental pollution has enforced the pulp and paper industries to strive for an alternate greener technology which will replace the use of harsh chemicals in their processes with microbial enzymes. The application of biocatalysts not only makes the process less toxic but also decreases costs associated with the production and consumption of resources (water, electricity, fuels) (Birijlall et al., 2011).
At present, bleaching process for kraft pulp uses large amounts of chlorine-based chemicals and sodium hydrosulfite, which are toxic, mutagenic, and persistent; they also cause numerous harmful disturbances in biological systems (Yeasmin et al., 2011). The enzymatic step in the process of cellulose pulp bleaching contributes to reduce the use of chlorine-containing reagents . Xylanases aid in catalyze the hydrolysis of glycosidic bonds in the xylan backbone, reducing the degree of polymerization of the substrate, enhancing the brightness of pulp (facilitating the chemical extraction of lignin from pulp in subsequent alkaline extraction) and diminish impurities. Cellulose-free, alkali and thermo-stable microbial xylanases are mostly ideal for biopulping and bleaching processes (Birijlall et al., 2011;Yeasmin et al., 2011;Peixoto-Nogueira et al., 2009). This paper shows an important comparing of the xylanase production by A. niger and A. flavus using agroindustrial residues as carbon sources and tested their suitability for cellulose pulp biobleaching. These fungi produced xylanases with special characteristics, such as high pH stability and high optimum temperature, in comparison to others reported in the literature (Facchini et al., 2011;Sepahy et al., 2011). And the xylanase activity from A. niger was the most thermostable of three enzyme samples (A. niger, A. flavus and A. japonicus var aculeatus, studied before in our laboratory).

Microorganisms
Aspergillus niger and Aspergillus flavus strains were isolated by us from soil samples, identified in the Federal University of Pernambuco -UFPE (PE, Brazil) and deposited in our laboratory fungi collection. Stock cultures were propagated at 30°C on slants of solid potato dextrose agar (PDA) media and stored at 4°C.

Xylanase production and enzyme extraction
Spores were inoculated into 125 ml Erlenmeyer flasks containing 25 ml media (Rizzatti et al., 2001) using 1% (w/v) of the desired carbon sources (xylan or agroindustrial residues such as wheat bran, rice bran, sugarcane bagasse or corncob). The cultures were incubated under orbital agitation (110 rpm) condition, at 30°C, during five (A. niger) or two days (A. flavus), already standardized in our laboratory. The media was subsequently vacuum-filtered using filter paper (Whatman nº 1) and the crude filtrate was used for the study of the extracellular enzyme.

Enzymatic assays and protein determination
The reaction mixture consisted in 500 μl of citratephosphate buffer (McIlvaine, 1921) pH 5.0 for both fungi containing 1% (w/v) of xylan (birchwood), and 500 μl of enzymatic extract appropriately diluted. Identical conditions of assay were employed for cellulase determination, using as substrate 1% (w/v) carboxymethylcellulose. The samples were incubated at 60°C to determine xylanase and cellulase activity. The amount of reducing sugar released was determined using the 3,5-dinitrosalicylic acid (DNS) method (Miller, 1959), employing xylose (Sigma) (xylanase) and glucose (cellulase) as the standards. One unit of enzyme activity was defined as the amount of enzyme which releases 1 μmol of reducing sugar per minute under assay condition. Specific activities were expressed as U/mg of protein. Protein concentrations were determined by the Lowry et al. (1951), using bovine serum albumin (BSA) as the standard.

Effect of temperature and pH on enzyme activity
The effects of temperature and pH on xylanase activity were analyzed using crude extracts from A. niger and A. flavus. The optimum temperature and pH for A. niger and A. flavus were 60°C and 5.0, respectively (results not shown). The thermal stability was determined with enzymes incubated between 45 and 55°C for different periods (5 to 120 min). The influence of protectors on xylanolytic activities of A. niger and A. flavus was tested at 55 and 50°C, respectively, during 120 minutes in the presence of 5% glycerol or polyethyleneglycol. The pH stability was analyzed using McIlvaine buffer in the pH range 3.0 -8.0 for 1 hour.

Biobleaching
The amount of enzyme used from A. niger or A. flavus for this treatment was 10 units of enzyme per gram of dried cellulose pulp from Eucalyptus grandis. All calculations and procedures were determined according to the standard methods of Technical Association of the Pulp and Paper Industry (TAPPI test methods 1996). The consistency was determined on a percent dry weight basis. The volume of enzyme or distilled water was added until it reached a 10% pulp consistency. Crude xylanase extracts from A. niger and A. flavus were added to the treated pulp and the control was prepared by adding distilled water instead of enzyme. The samples were incubated inside sealed polyethylene bags at 55°C for 2 hours and after that, the treated cellulose pulps were filtered on a Büchner funnel, rinsed with 200 ml of distilled water and used for determination of kappa number and viscosity. The filtrate was used to analyze the liberation of aromatic compounds monitored by absorbance values at 237 and 465 nm.

Reproducibility of results
All results are expressed as the means of at least three independent experiments.

Results and discussion
Xylanase production using different agroindustrial residues and substrates When different carbon sources were tested (Table 1) it was observed that the wheat bran 1% was the best carbon source for xylanase production by A. niger (12.76 U/mg of protein), followed by fine sugarcane bagasse 0.5% (10.64 U/mg of protein) and corncob 1% (10.00 U/ mg of protein). The production of xylanase was 18 (oat spelt xylan 1%) and 21% (birchwood xylan 1%) higher on wheat bran 1% when we compare the xylanase production with xylan (10.43 and 10.10 U/mg of protein, respectively), which means that it can substitute the xylan efficiently. In studies with A. fumigatus RP04, the production of xylanase was only between 5 and 6% higher on agroindustrial residues (powdered corncob, wheat bran and crushed corncob) when compared with media containing birchwood xylan as carbon source . Although the xylanase production was elevated in birchwood xylan 0.5% (15.72 U/mg of protein), the utilization of an alternative carbon source, like wheat bran or sugarcane bagasse, still is viable due A. niger and A. flavus were grown on SR liquid media (Rizzatti et al., 2001) and different carbon sources, which were incubated for five and two days, respectively, at 30°C under orbital agitation (110 rpm) condition. The assays were performed at 60°C and McIlvaine buffer pH 5.0.
In the tests of influence of mixtures (1:1) of substrates, the xylanase production by A. niger was 22% higher on the mixture of wheat bran and corncob when compared to media containing xylan as carbon source. When we compare the xylanase production by A. flavus in this mixture, it was almost 45% lower than from A. niger. And comparing the xylanase production between the used fungi in the other mixtures, A. flavus presented a very lower production than A. niger.
In literature, different combinations of agroindustrial residues as carbon sources for xylanase production, has been reported for several fungi (Khonzue et al., 2011;Facchini et al., 2011;Dhillon et al., 2011;Pal and Khanum, 2010;Betini et al., 2009;Peixoto-Nogueira et al., 2009). Aspergillus niger DFR-5 had his highest xylanase production in media containing a mixture of wheat bran and soybean powder in a ratio 7:3 (Pal and Khanum, 2010), while Aspergillus niger BC-1 mixed with T. reesei Rut C-30 (ATCC 56765) had the highest activity with rice straw and wheat bran (3:2) (Dhillon et al., 2011). Facchini et al. (2011) found that a mixture of soybean meal and crushed corncob increased xylanolytic activity by only 8.5%. And in work using the fungus A. niveus and some agroindustrial residues, wheat bran mixed with corncob not improved the production of xylanase, but A. niger and A. ochraceus had an increase of approximately 20% .
For the enzyme characterization and the biobleaching study, there were separated the filtrates that more produced enzymes. And the cellulase activity of theses filtrates was determined and did not detect significant levels (data not shown).

Effect of temperature and pH on xylanase stability
The xylanases produced by A. niger and A. flavus remained totally stable at 45°C. At 50°C both xylanases were stable for the first twenty minutes, where the xylanase of A. niger ( Figure 1A) still retained 85.72% activity with 2 hours of incubation, and A. flavus ( Figure 1B) had a half-life of more than 75 minutes. At 55°C, the xylanase produced by A. niger showed more stable than from A. flavus showing a half-life of approximately 45 minutes. In studies with A. casielus (Kronbauer et al., 2007), A. phoenicis (Chipeta et al., 2005) and A. giganteus (Coelho and Carmona, 2003) the xylanases had half-life of only 50, 25 and 13 minutes at 50°C, respectively. The xylanase produced by A. terreus FSS129 had a residual activity of only 70% approximately with 2 hours of incubation at 50°C, and just retained 25.4% activity at 55°C after 1 hour exposure, while our A. niger still retained 38.2% activity (Bakri et al., 2010). And at 55°C the xylanase of A. casielus had a half-life of only 17 minutes (Kronbauer et al., 2007).
The influence of glycerol and polyethyleneglycol as enzyme protectors was also tested ( Figure 1C,D). The addition of 5% glycerol somehow protected significantly the xylanase produced by A. niger from thermal inactivation at 55°C during all the incubation period. The xylanase activity increased 20.66% with 2 hours of incubation when compared to the control (without additives), and it was observed a half-life of approximately 32 minutes to the control and 60 minutes with glycerol ( Figure 1C). And the xylanase activity of A. flavus increased 30% with 2 hours of incubation at 50°C in the presence of glycerol, which was not sufficient time to get the enzyme half-life ( Figure 1D). The polyethyleneglycol was not effective as protector for both enzymes.
Regarding pH stability, it was verified that the xylanase from A. niger ( Figure 1E) was totally stable at all the pHs tested, retaining more than 95% activity during 1 hour, while the xylanase from A. flavus ( Figure 1F) had a small decrease of 10% at pH between 3.0 -5.5, maintaining stable (100%) at higher pHs. The stability of the xylanases of A. niger and A. flavus under alkaline pH is very interesting when we remember that the biobleaching process with xylanases is done under alkaline conditions. Michelin et al. (2010) related a xylanase produced by A. terricola Marchal and A. ochraceus that retained more than 70% of its original activity in a pH range of 2.5 -8.0 for 1 hour. The xylanase produced by A. fumigatus was stable just in pH from 6.0 to 8.0, while the xylanase from A. niveus was more stable from 4.5 to 6.0 . The xylanases of A. niger, A. niveus and A. ochraceus were stable in the range of pH 2 -7 retaining more than 70 -80% activity .

Assays of cellulose biobleaching using xylanase
To analyze the xylanases efficiency for cellulose pulp biobleaching assay, the cellulose pulp was clarified by A. flavus or A. niger crude extract. The procedures occurred  at 55°C for 2 h and it was observed that the xylanase crude extract from A. flavus was more effective in comparison to A. niger. The kappa number reduced 5.07 and 3.62 points, respectively, which corresponds to 36.32 and 25.93 kappa efficiency, respectively (Table 2). This efficiency can be also proved by cromophores liberation, which was redden at 237 and 465 nm and again was bigger when the cellulose pulp was clarified by A. flavus crude extract. That is important to emphasize that the cellulase activity was either analyzed and significant levels were not detected (data not shown), which explain why the viscosity was not significantly modified.
Comparing the used fungi that is clear that the xylanase from A. flavus is more effective in comparison to xylanase from A. niger. These advantages can also be seen when you compare the obtained results for A. flavus and A. niger to some data from the literature. The xylanase of A. caespitosus (10 U/g dry pulp/ 2 hours) reduced kappa number only in 12.6% (xyl II) and 1.7% (xyl I) (Sandrim et al., 2005), while the A. flavus and A. niger xylanases kappa efficiency corresponded to 36.32 and 25.93%, respectively. Medeiros et al. (2007) reported xylanases from T. longibrachiatum, P. corylophilum and A. niger that reduced only 1.1, 0.5 and 0.6 points the kappa number with 5 U/g dry pulp/ 4 hours; and Aspergillus fumigatus ABK9 reduced only 0.7, 1.2, 2.7, 3.3 and 4 points the kappa number, using 20, 40, 60, 80 and 100 U/g dry pulp/ 6 hours, respectively (Das et al., 2013). And Kumar et al. (2009) reported a xylanase (50 U/g dry pulp/ 3 hours) from T. lanuginosus MC 134 that reduced only 3.2 points the kappa number.

Conclusions
A. niger and A. flavus showed that are good xylanase producers and these enzymes can be obtained using alternative carbon sources such as wheat bran, corncob and fine sugarcane bagasse, that is important to emphasize that the xylanase production increased when a mix of residues was used, showing a better production than on media supplemented only by xylan, proving that they can substitute the specific substrate and consequently reduce the enzyme production cost, since the commercial xylan is extremely expensive for widespread use. The xylanase from A. niger showed to be stable for temperature and pH, while the A. flavus xylanase was not so stable for temperature but showed excellent stability at high pH (6-8), characteristics that are very important for the biobleaching of cellulose pulp process. On biobleaching assay the xylanases from A. flavus were more effective in comparison to xylanases from A. niger.