BACKGROUND
in nature, lignocellulosic materials are degraded by a consortium of microorganisms that synthesize many hydrolytic enzymes able to loosen and degrade these substrates.
improvement in the efficiency of hydrolysis of lignocellulosic materials has traditionally focused on cellulose, which is the most abundant plant polysaccharide  <cit> .
however, the presence of hemicellulose and lignin can restrict cellulose hydrolysis.
the hemicellulases, such as pectinases and xylanases, stimulate cellulose hydrolysis by removal of the non-cellulosic polysaccharides that coat the cellulose fibers  <cit> .
cellulolytic and hemicellulolytic enzymes have been extensively investigated as tools to achieve viable second-generation ethanol production.
the hemicellulases include accessory enzymes, which are a group of enzymes capable of increasing the yield of reducing sugars during enzymatic hydrolysis of lignocellulosic substrates.
the definition of the accessory enzymes has evolved over time.
enzymes such as the β-glucosidases were originally classified as accessories, but today are considered essential in enzymatic cocktails, following elucidation of their mechanisms of action during substrate degradation  <cit> .
the main accessory enzymes are currently considered to be α-l-arabinofuronosidase, hemicellulolytic esterases, β-mannanases, α-glucoronidases, β-xylosidases, pectinases, and xylanases.
several studies have shown that cellulase enzymes supplementation can improve the enzymatic hydrolysis of lignocellulosic biomass, in terms of speed and hydrolysis yield.
an issue is that crude multi-enzyme blends obtained from a single fungus strain are not ideal in biotechnological applications.
this is because cellulase activities are not expressed at sufficient levels, or the enzyme complexes are not well balanced in terms of the individual enzymes  <cit> .
for this reason, fungi strains isolated from unusual environments have been sought as alternative sources of hydrolytic enzymes  <cit> .
endophytic fungi are potentially amongst the most interesting microorganisms for screening for the production of industrial biocompounds.
these microorganisms are ubiquitous in plants, inhabiting plant tissues without inducing any apparent symptoms in their hosts  <cit> .
the fact that these microorganisms are present within plant tissues could explain their capacity to produce substances that could have useful industrial, agricultural, and medicinal applications  <cit> .
the endophytic fungi that have been reported to be xylanase producers include alternaria alternata <cit> , hymenoscyphus ericae <cit> , and aspergillus terreus <cit> .
de almeida et al.
<cit>  selected strains from the acremonium endophyte species for hemicellulases and cellulases production.
from  <dig> plant species, suto et al.
<cit>  isolated  <dig> strains of fungi that produced xylanases.
harnpicharnchai et al.
<cit>  purified a thermotolerant β-glucosidase from an endophytic periconia sp.
other studies have involved the selection of new isolates using extracellular enzymes as selection parameters for plant growth promotion.
silva et al.
<cit>  investigated fungi isolated from annona spp., while luz et al.
<cit>  employed isolates from passiflora edulis.
endophytic fungal strains may therefore constitute a valuable source of biological material that deserves to be studied and explored for the production of cellulolytic and hemicellulolytic enzymes.
in this context, the present work concerns the selection of endophytic fungi as producers of hemicellulases and related enzymes with different enzymatic profiles, for use in the deconstruction of lignocellulosic biomass.
RESULTS
agro-industrial waste material composition
the sugar cane hydrothermal pretreatment liquor showed the following composition : xylo-oligosaccharides , xylose , glucose , arabinose , cellobiose , furfural , hydroxymethylfurfural , acetic acid , formic acid , and total soluble lignin .
despite the presence of inhibitors, this liquor demonstrated to be a potential carbon source for the screening of enzyme producers and the production of hemicellulases.
the deb was composed of  <dig> % cellulose,  <dig> % hemicellulose, and  <dig> % lignin.
the sb consisted of 34% cellulose,  <dig> % hemicellulose,  <dig> % lignin, and  <dig> % protein.
the media prepared using these waste materials were therefore able to provide a suitable ratio of cellulose and hemicellulose for the synthesis of glycohydrolases, as well as a good source of nitrogen.
plate screening
a total of  <dig> fungal strains were bioprospected and used for calculation of hydrolysis rates .
the media containing liquor were stained with congo red, revealing the yellow hydrolysis halos .
a total of  <dig> strains were unable to grow on the medium, while only  <dig> were able to both grow and produce halos.
on the other hand, in the case of the medium with xylan, only two strains, one aspergillus sp.
and one diaphorte sp.
were unable to grow, while  <dig> strains grew and produced halos.
it was therefore demonstrated that the xylose/xylo-oligomers liquor produced by a simple pretreatment was able to sustain the growth of a significant number of the fungi tested.
selection of β-glucosidase producers employed the egda to determine β-glucosidase in the fungal culture extracts, with positive extracts forming dark-colored halos.
of the  <dig> extracts tested,  <dig> produced measurable halos,  <dig> showed dark precipitates although measurement was not possible, and  <dig> strains were negative for β-glucosidase production.
the plate screening and egda results were used to select  <dig> strains for a second screening employing shake flask cultivations.
some of these strains were negative in the hemicellulolytic and β-glucosidase tests, and were used as controls to ensure selection consistency.
shake flask screening
the strains were grown using deb + sb  at 29°c on a rotary shaker at 200 rpm for 96 h. the results obtained for some of the strains are presented in figure  <dig>  low β-glucosidase activities were detected up to 48 h of fermentation, while high activity levels were observed at 96 h. this was expected, since several filamentous fungi are known to begin to produce detectable amounts of this enzyme after 72 h of growth  <cit> .
the cmcase and fpase activities were low for all the strains, as expected because selection was performed using materials rich in hemicelluloses.
high xylanase production was detected at 48 h for many strains, but the largest peaks occurred at 96 h. pectinase production showed little variation between  <dig> and 96 h, although amounts of the enzyme nonetheless increased over the course of the fermentation.
strains morphologically similar to aspergillus fumigatus  were excluded due to possible pathogenicity, which could preclude their use in industrial applications.
glycohydrolase profile
in order to identify fungi that produced enzyme with different profiles, and hence obtain a more efficient enzymatic extract,  <dig> strains were selected according to their morphology and enzymatic profiles.
a new fermentation with deb + sb was performed, and samples were taken daily for measurement of xylanase, β-glucosidase, and pectinase activities.
the samples that showed the highest glycohydrolase activity were tested using different substrates .
the strains dr <dig> and dr <dig> , and dr <dig>  presented the highest xylanolytic activities for birchwood xylan, beechwood xylan, and rye arabinoxylan.
despite the fact that the strains dr <dig> and dr <dig> belong to the same genus, and have similar morphologies, they presented different enzymatic profiles .
selection was made of six strains  that showed enzymatic activities for a wider range of substrates, were morphologically different, and presented distinct enzymatic profiles.
these strains were cultured in shake flasks containing xylan and pectin as inducer carbon sources.
samples were taken daily for measurements of xylanase, β-glucosidase, and pectinase activities.
the fungal extracts that showed highest glycohydrolase activities were tested using different substrates .
a. niger
t. atroviride
t. atroviride
a. stygium
alternaria
t. wortmannii
the fungi xylanolytic profiles differed among the strains and the carbon sources used.
the dr <dig> strain produced xylanases with the same affinity for birchwood xylan, beechwood xylan, and rye arabinoxylan, when cultivated in the presence of beechwood xylan.
however, this was not observed when the same trichoderma sp.
was grown using deb + sb.
some strains showed higher activity for beechwood xylan than for birchwood xylan , and vice versa .
the dr <dig> strain showed the highest activity for rye arabinoxylan.
the dr <dig> strain only produced xylanase when the fungus was grown in the presence of xylan, in contrast to other strains such as dr <dig>  dr <dig>  and dr <dig>  for which deb and sb also induced the production of xylanases.
the production of β-glucanases was high for dr <dig> and dr <dig> strains when cultivated on xylan, for dr <dig> when grown on deb + sb, and for dr <dig> on pectin.
however, when these extracts were tested using xyloglucan, all the activities decreased, indicating less affinity for the hydrolysis of β-glucan with branched xylose residues.
a similar phenomenon occurred in the testing of lichenan, which is a linear glucan with more β- <dig>  bonds than β-glucan.
this indicates that the β-glucanases present in these extracts had lower lichenanase activity.
furthermore, when the dr <dig> and dr <dig> strains were grown on xylan, they showed activity against laminarin, indicating the presence of enzymes able to hydrolyze the β-d-glucosyl  β-d-glucose bond.
for almost all fungi, with the exception of dr <dig> and dr <dig>  the production of polygalacturonase was only induced in the presence of pectin.
the best producers were the strains dr <dig>  and dr <dig> .
the production of β-glucosidase showed no consistent induction pattern for the three carbon sources tested.
dr <dig> and dr <dig> produced more β-glucosidase on deb + sb, while dr <dig> produced more on pectin, and dr <dig> on xylan.
none of the fungi showed measurable activities for β- <dig> -d-glucosaminidase or α-mannosidase.
when the talaromyces sp.
dr <dig> strain was grown on xylan, it was able to produce multiple accessory proteins such as xylosidase, arabinofuranosidase, cellobiohydrolase ii, and β-glucosidase.
this strain might therefore be promising for the production of hemicellulases.
high cmcase activity was measured when this fungus was cultivated on xylan, but it did not present high activities against β-glucan.
however, opposite result was found when this strain was grown on deb + sb.
the hydrolytic action of the fungal extracts against mannan polymers was low for all the strains.
nevertheless, activities for heteromannans  were higher than for β- <dig> -mannan.
this could be explained by the presence of β--glucanase activity in the extracts in the case of glucomannan, and the presence of α- <dig> -galactosidase in the case of galactomannan.
fungal identification
strain identification was performed using morphological characteristics as well as sequencing of the its regions of the ribosomal dna gene and  the partial β-tubulin gene.
the best xylanase producer strain, dr <dig>  previously isolated from platanus orientalis, was identified according to morphology  as aspergillus section nigri.
the its regions and partial bt <dig> sequencing were performed and submitted to genbank .
the phylogenetic trees, built with reference strains of aspergillus nigri section species, showed that the dr <dig> isolate clustered with a. niger .
higher value of a. niger bt <dig> clustering confirm the its result, the strain dr <dig> belongs to the aspergillus niger species.
the dr <dig> strain, which is a good pectinase and β-glucosidase producer, did not present reproductive structures under the microculture technique.
as the classical methods did not lead to conclusive results, sequencing of the rdna its regions was performed .
the blast alignment suggested that the dr <dig> isolate belonged to the annulohypoxylon stygium species .
a separation of two groups in the its tree constructed with annulohypoxylon and related species was found.
one group revealed that the dr <dig> isolated clustered with a. stygium and annulohypoxylon urceolatum, but was closer to a. stygium.
the second group consisted on annuhypoxylon spp.
and hypoxylon investiens .
sánchez-ballesteros et al.
<cit>  analyzed the its1- <dig> s-its region, and found that annulohypoxylon spp.
cluster inter-mingled with species of the genus hypoxylon section hypoxylon.
therefore, sequencing of partial bt <dig> was also performed  as suggested by hsieh et al.
<cit> .
the phylogenetic tree was built and the dr <dig> isolated was clustered with a. stygium species, with a high bootstrap value, and was closer to a. stygium than to annulohypoxylon stygium var.
annulatum .
besides, h. investiens was consistently separated from annulohypoxylon.
the dr <dig> strain, previously isolated from spoiled books, was identified as talaromyces sp.
the blast alignment of the its regions  and partial bt <dig>  sequences suggest similarity with to talaromyces wortmanni.
the trees based on its and bt <dig> sequencing built with close related talaromyces spp.
corroborated with the blast aligned.
the talaromyces sp.
dr <dig> strain was clustered with talaromyces wortmannii with high bootstrap values in both trees .
the dr <dig> strain, isolated from e. benthamii, was previous identified by macro and micro morphology as alternaria sp.
the sequencing of rdna its , suggested that the dr <dig> isolate belonged to the alternaria alternata species  while no amplicon of the bt <dig> gene was obtained for this strain.
the tree based on rdna its sequencing built with correlated species showed no resolution among the strains of the alternata species group .
previous work has also found no genetic variation between the small-spored alternaria species in its sequences  <cit> .
according to andrew et al.
<cit> , taxonomical differentiation of the small-spored species within the alternata group is difficult, not only because there are few distinguishing morphological characteristics, but also because these characteristics are strongly influenced by the environment.
moreover, the same authors could not solve alternaria spp.
that belongs to the alternata group using a phylogenic multilocus approach.
the dr <dig> and dr <dig> strains were also endophytic isolates from e. benthamii, and were morphologically identified as trichoderma sp.
the its1- <dig> s-its <dig> sequences for trichoderma sp.
dr <dig> and trichoderma sp.
dr <dig>  aligned with the database trichoderma atroviride strain daom  <dig> with 100% similarity .
the tree based on rdna its sequencing  formed two groups, and the dr <dig> and dr <dig> isolates were clustered with the viride clade , and were closer to the t. atroviride species.
discussion
high activity, good stability, and low cost are key requirements of enzymes employed for large-scale hydrolysis of lignocellulosic biomass into sugar.
agro-industrial wastes can be useful materials for enzyme development, improvement, and production.
the liquor derived from sugar cane bagasse hydrothermal pretreatment is a low cost feedstock  <cit>  rich in xylose and xylo-oligosaccharides which are capable of inducing the expression of xylanases and accessory proteins in fungi such as a. niger <cit> .
other materials, such as steam-exploded delignified bagasse and soybean bran, have also been used as inexpensive culture media to achieve high xylanase, cellulase, and β-glucosidase activities employing trichoderma harzianum p49p <dig>  <cit> .
the full hydrolysis of lignocellulosic biomass requires several types of glycohydrolases that enable the release of saccharides and other compounds from the recalcitrant substrate.
however, plant species are highly diverse in terms of cell wall structure and composition, which increases the attraction of formulating specific biomass-degrading enzymatic cocktails.
the sugar cane cell wall polysaccharide is mainly composed of xyloglucan and arabinoxylan, closely associated with cellulose, as well as pectin, β-glucan and less branched xylan strongly bound to cellulose  <cit> .
several studies have shown that supplementing cellulases with other enzymes can assist in the enzymatic hydrolysis of lignocellulosic biomass.
xylanases and β-xylosidases improved the hydrolysis yield when combined with cellulases and β-glucosidades  <cit> .
the addition of pectinase to celluclast  <dig>  l increased the hydrolysis of pretreated corn stover  <cit> .
supplementation of cellulolytic cocktails with α-l-arabinofuranosidase and xylanase also showed a synergistic effect in the hydrolysis of wheat straw  <cit> .
the production of glycohydrolases is closely related to the nature of the carbon source, since fungal metabolism is greatly influenced by the composition of the medium .
each strain has a distinct metabolic profile, while the enzymatic profile is also distinct and depends on the medium and the cultivation time .
physiological variations are the result of the adaptation and evolution of fungi, considering their hosts, original habitats, and other factors.
the strain a. stigyum dr <dig> belongs to the xylariaceae family, members of which are frequently encountered as endophytes and saprophytes  <cit> .
gazis & chaverri  <cit>  isolated several endophytic xylariaceae strains and one strain of annulohypoxylon sp.
from hevea brasiliensis.
wei et al.
<cit>  cultivated an a. stigyum strain on avicel and confirmed the production of β-glucosidase, although only low levels of cellulases were detected.
most alternaria species are saprophytes commonly found in soil or on decaying plant tissues, and some species are opportunistic plant pathogens  <cit> .
however, endophytic strains of alternaria spp.
have been isolated from eucalyptus plants such as eucalyptus globulus <cit>  and eucalyptus citriodora <cit> .
strains of a. alternata are able to produce endopolygalactunorase  <cit>  in the presence of pectin, and β-glucosidase in the presence of saccharose  <cit> .
a. niger is known worldwide for its ability to produce an extensive range of extracellular glucohydrolases, including xylanases, pectinases, and β-glucosidase  <cit> .
this characteristic is associated with the ability of the fungus to propagate and colonize a variety of environments, principally those rich in decomposing plant materials  <cit> .
the fungus was recently reported to be endophytic in several plant species  <cit> .
however, this work is the first report of a. niger as an endophytic fungus in p. orientalis.
there have been no previous reports in brazil concerning t. wortmannii isolated from decaying materials.
jang et al.
<cit>  first described β-xylosidase activity in a t. wortmannii strain previously isolated from japanese red pine and larch woods in korea  <cit> .
jang et al.
<cit>  obtained a β-xylosidase production of  <dig>  u/ml for cultivation on xylan, in good agreement with the β-xylosidase activity  found in the present work for the dr <dig> strain grown on xylan.
trichoderma spp.
are present in soil as saprophytes, and have also been found as endophytic organisms  <cit> .
many species from this genus are good cellulase and xylanase producers, such as t. harzianum <cit>  and t. reesei <cit> .
t. atroviride strains are good producers of these glycohydrolases, and can produce high amounts of β-glucosidase  <cit> .
CONCLUSIONS
microorganisms play an essential role in the degradation of cellulose and hemicellulose standing out the endophytic fungi which are excellent sources of hydrolytic enzymes.
evidently, during the endophytic phase, the use of these enzymes must be related to the mutualistic relationship with the host plant  <cit> .
however, although the association between plants and endophytic fungi is ecologically important, little is known about the physiological characteristics of the interaction.
an important aspect of enzymatic studies involving endophytic fungi is the involvement of these microorganisms in the decomposition of plant material  <cit> .
since the fungi are already present in the senescent plant tissues, they may be able to initiate the decomposition process before it becomes dominated by saprophytic species.
this could suggest not only that the production of hydrolytic enzymes by endophytic species might be important for the nutrition of the fungus during the endophytic stage, but also that these enzymes are produced and secreted at the surface of the tissues, where they can compete for the substrate during the saprophytic stage.
kumaresan & suryanarayanan  <cit>  investigated the ability of endophytic fungi from mangrove leaves of different ages to produce hydrolytic enzymes.
it was found that endophytic species occurring at relatively low levels in living leaves were more prevalent after leaf fall, increasing the involvement of these fungi in decomposition of the plant material.
an important consideration is the range of substrates that can be utilized by endophytic microorganisms.
studies have shown that endophytes are capable of metabolizing in vitro most substrates found in plants, and produce enzymes including proteases, amylases, phenol oxidases, lipases, laccases, polyphenol oxidases, cellulases, mannanases, xylanases, and pectin lyase  <cit> .
the balanced use of microbial enzymes in biomass deconstruction required the understanding of the role played by these glycohydrolases, and also depends on an economic process development.
therefore, biochemical characterization of new reported glycohydrolases producer strains, as well as a bioprocess development of the selected strains in large scale, must be conducted to evaluate the enzyme applicability on the biomass deconstruction, principally on sugar cane bagasse.
the present work demonstrated that it is possible to select endophytic fungal strains that can produce glycohydrolases with activities against a wide range of target substrates.
this will enable the future formulation of specific enzymatic cocktails for an efficient biomass deconstruction.

 in nature, lignocellulosic materials are degraded by a consortium of microorganisms that synthesize many hydrolytic enzymes able to loosen and degrade these substrates. improvement in the efficiency of hydrolysis of lignocellulosic materials has traditionally focused on cellulose, which is the most abundant plant polysaccharide. however, the presence of hemicellulose and lignin can restrict cellulose hydrolysis. the hemicellulases, such as pectinases and xylanases, stimulate cellulose hydrolysis by removal of the non-cellulosic polysaccharides that coat the cellulose fibers. cellulolytic and hemicellulolytic enzymes have been extensively investigated as tools to achieve viable second-generation ethanol production. the hemicellulases include accessory enzymes, which are a group of enzymes capable of increasing the yield of reducing sugars during enzymatic hydrolysis of lignocellulosic substrates. the definition of the accessory enzymes has evolved over time. enzymes such as the β-glucosidases were originally classified as accessories, but today are considered essential in enzymatic cocktails, following elucidation of their mechanisms of action during substrate degradation. the main accessory enzymes are currently considered to be α-l-arabinofuronosidase, hemicellulolytic esterases, β-mannanases, α-glucoronidases, β-xylosidases, pectinases, and xylanases. several studies have shown that cellulase enzymes supplementation can improve the enzymatic hydrolysis of lignocellulosic biomass, in terms of speed and hydrolysis yield. an issue is that crude multi-enzyme blends obtained from a single fungus strain are not ideal in biotechnological applications. this is because cellulase activities are not expressed at sufficient levels, or the enzyme complexes are not well balanced in terms of the individual enzymes. for this reason, fungi strains isolated from unusual environments have been sought as alternative sources of hydrolytic enzymes. endophytic fungi are potentially amongst the most interesting microorganisms for screening for the production of industrial biocompounds. these microorganisms are ubiquitous in plants, inhabiting plant tissues without inducing any apparent symptoms in their hosts. the fact that these microorganisms are present within plant tissues could explain their capacity to produce substances that could have useful industrial, agricultural, and medicinal applications. the endophytic fungi that have been reported to be xylanase producers include alternaria alternata, hymenoscyphus ericae, and aspergillus terreus. de almeida et al. selected strains from the acremonium endophyte species for hemicellulases and cellulases production. from plant species, suto et al. isolated strains of fungi that produced xylanases. harnpicharnchai et al. purified a thermotolerant β-glucosidase from an endophytic periconia sp. other studies have involved the selection of new isolates using extracellular enzymes as selection parameters for plant growth promotion. silva et al. investigated fungi isolated from annona spp., while luz et al. employed isolates from passiflora edulis. endophytic fungal strains may therefore constitute a valuable source of biological material that deserves to be studied and explored for the production of cellulolytic and hemicellulolytic enzymes. in this context, the present work concerns the selection of endophytic fungi as producers of hemicellulases and related enzymes with different enzymatic profiles, for use in the deconstruction of lignocellulosic biomass.  agro-industrial waste material composition the sugar cane hydrothermal pretreatment liquor showed the following composition: xylo-oligosaccharides, xylose, glucose, arabinose, cellobiose, furfural, hydroxymethylfurfural, acetic acid, formic acid, and total soluble lignin. despite the presence of inhibitors, this liquor demonstrated to be a potential carbon source for the screening of enzyme producers and the production of hemicellulases. the deb was composed of % cellulose, % hemicellulose, and % lignin. the sb consisted of 34% cellulose, % hemicellulose, % lignin, and % protein. the media prepared using these waste materials were therefore able to provide a suitable ratio of cellulose and hemicellulose for the synthesis of glycohydrolases, as well as a good source of nitrogen. plate screening a total of fungal strains were bioprospected and used for calculation of hydrolysis rates. the media containing liquor were stained with congo red, revealing the yellow hydrolysis halos. a total of strains were unable to grow on the medium, while only were able to both grow and produce halos. on the other hand, in the case of the medium with xylan, only two strains, one aspergillus sp. and one diaphorte sp. were unable to grow, while strains grew and produced halos. it was therefore demonstrated that the xylose/xylo-oligomers liquor produced by a simple pretreatment was able to sustain the growth of a significant number of the fungi tested. selection of β-glucosidase producers employed the egda to determine β-glucosidase in the fungal culture extracts, with positive extracts forming dark-colored halos. of the extracts tested, produced measurable halos, showed dark precipitates although measurement was not possible, and strains were negative for β-glucosidase production. the plate screening and egda results were used to select strains for a second screening employing shake flask cultivations. some of these strains were negative in the hemicellulolytic and β-glucosidase tests, and were used as controls to ensure selection consistency. shake flask screening the strains were grown using deb + sb at 29°c on a rotary shaker at 200 rpm for 96 h. the results obtained for some of the strains are presented in figure  low β-glucosidase activities were detected up to 48 h of fermentation, while high activity levels were observed at 96 h. this was expected, since several filamentous fungi are known to begin to produce detectable amounts of this enzyme after 72 h of growth. the cmcase and fpase activities were low for all the strains, as expected because selection was performed using materials rich in hemicelluloses. high xylanase production was detected at 48 h for many strains, but the largest peaks occurred at 96 h. pectinase production showed little variation between and 96 h, although amounts of the enzyme nonetheless increased over the course of the fermentation. strains morphologically similar to aspergillus fumigatus were excluded due to possible pathogenicity, which could preclude their use in industrial applications. glycohydrolase profile in order to identify fungi that produced enzyme with different profiles, and hence obtain a more efficient enzymatic extract, strains were selected according to their morphology and enzymatic profiles. a new fermentation with deb + sb was performed, and samples were taken daily for measurement of xylanase, β-glucosidase, and pectinase activities. the samples that showed the highest glycohydrolase activity were tested using different substrates. the strains dr and dr, and dr presented the highest xylanolytic activities for birchwood xylan, beechwood xylan, and rye arabinoxylan. despite the fact that the strains dr and dr belong to the same genus, and have similar morphologies, they presented different enzymatic profiles. selection was made of six strains that showed enzymatic activities for a wider range of substrates, were morphologically different, and presented distinct enzymatic profiles. these strains were cultured in shake flasks containing xylan and pectin as inducer carbon sources. samples were taken daily for measurements of xylanase, β-glucosidase, and pectinase activities. the fungal extracts that showed highest glycohydrolase activities were tested using different substrates. a. niger t. atroviride t. atroviride a. stygium alternaria t. wortmannii the fungi xylanolytic profiles differed among the strains and the carbon sources used. the dr strain produced xylanases with the same affinity for birchwood xylan, beechwood xylan, and rye arabinoxylan, when cultivated in the presence of beechwood xylan. however, this was not observed when the same trichoderma sp. was grown using deb + sb. some strains showed higher activity for beechwood xylan than for birchwood xylan, and vice versa. the dr strain showed the highest activity for rye arabinoxylan. the dr strain only produced xylanase when the fungus was grown in the presence of xylan, in contrast to other strains such as dr dr and dr for which deb and sb also induced the production of xylanases. the production of β-glucanases was high for dr and dr strains when cultivated on xylan, for dr when grown on deb + sb, and for dr on pectin. however, when these extracts were tested using xyloglucan, all the activities decreased, indicating less affinity for the hydrolysis of β-glucan with branched xylose residues. a similar phenomenon occurred in the testing of lichenan, which is a linear glucan with more β- bonds than β-glucan. this indicates that the β-glucanases present in these extracts had lower lichenanase activity. furthermore, when the dr and dr strains were grown on xylan, they showed activity against laminarin, indicating the presence of enzymes able to hydrolyze the β-d-glucosyl β-d-glucose bond. for almost all fungi, with the exception of dr and dr the production of polygalacturonase was only induced in the presence of pectin. the best producers were the strains dr and dr. the production of β-glucosidase showed no consistent induction pattern for the three carbon sources tested. dr and dr produced more β-glucosidase on deb + sb, while dr produced more on pectin, and dr on xylan. none of the fungi showed measurable activities for β- -d-glucosaminidase or α-mannosidase. when the talaromyces sp. dr strain was grown on xylan, it was able to produce multiple accessory proteins such as xylosidase, arabinofuranosidase, cellobiohydrolase ii, and β-glucosidase. this strain might therefore be promising for the production of hemicellulases. high cmcase activity was measured when this fungus was cultivated on xylan, but it did not present high activities against β-glucan. however, opposite result was found when this strain was grown on deb + sb. the hydrolytic action of the fungal extracts against mannan polymers was low for all the strains. nevertheless, activities for heteromannans were higher than for β- -mannan. this could be explained by the presence of β--glucanase activity in the extracts in the case of glucomannan, and the presence of α- -galactosidase in the case of galactomannan. fungal identification strain identification was performed using morphological characteristics as well as sequencing of the its regions of the ribosomal dna gene and the partial β-tubulin gene. the best xylanase producer strain, dr previously isolated from platanus orientalis, was identified according to morphology as aspergillus section nigri. the its regions and partial bt sequencing were performed and submitted to genbank. the phylogenetic trees, built with reference strains of aspergillus nigri section species, showed that the dr isolate clustered with a. niger. higher value of a. niger bt clustering confirm the its result, the strain dr belongs to the aspergillus niger species. the dr strain, which is a good pectinase and β-glucosidase producer, did not present reproductive structures under the microculture technique. as the classical methods did not lead to conclusive results, sequencing of the rdna its regions was performed. the blast alignment suggested that the dr isolate belonged to the annulohypoxylon stygium species. a separation of two groups in the its tree constructed with annulohypoxylon and related species was found. one group revealed that the dr isolated clustered with a. stygium and annulohypoxylon urceolatum, but was closer to a. stygium. the second group consisted on annuhypoxylon spp. and hypoxylon investiens. sánchez-ballesteros et al. analyzed the its1- s-its region, and found that annulohypoxylon spp. cluster inter-mingled with species of the genus hypoxylon section hypoxylon. therefore, sequencing of partial bt was also performed as suggested by hsieh et al.. the phylogenetic tree was built and the dr isolated was clustered with a. stygium species, with a high bootstrap value, and was closer to a. stygium than to annulohypoxylon stygium var. annulatum. besides, h. investiens was consistently separated from annulohypoxylon. the dr strain, previously isolated from spoiled books, was identified as talaromyces sp. the blast alignment of the its regions and partial bt sequences suggest similarity with to talaromyces wortmanni. the trees based on its and bt sequencing built with close related talaromyces spp. corroborated with the blast aligned. the talaromyces sp. dr strain was clustered with talaromyces wortmannii with high bootstrap values in both trees. the dr strain, isolated from e. benthamii, was previous identified by macro and micro morphology as alternaria sp. the sequencing of rdna its, suggested that the dr isolate belonged to the alternaria alternata species while no amplicon of the bt gene was obtained for this strain. the tree based on rdna its sequencing built with correlated species showed no resolution among the strains of the alternata species group. previous work has also found no genetic variation between the small-spored alternaria species in its sequences. according to andrew et al., taxonomical differentiation of the small-spored species within the alternata group is difficult, not only because there are few distinguishing morphological characteristics, but also because these characteristics are strongly influenced by the environment. moreover, the same authors could not solve alternaria spp. that belongs to the alternata group using a phylogenic multilocus approach. the dr and dr strains were also endophytic isolates from e. benthamii, and were morphologically identified as trichoderma sp. the its1- s-its sequences for trichoderma sp. dr and trichoderma sp. dr aligned with the database trichoderma atroviride strain daom with 100% similarity. the tree based on rdna its sequencing formed two groups, and the dr and dr isolates were clustered with the viride clade, and were closer to the t. atroviride species. discussion high activity, good stability, and low cost are key requirements of enzymes employed for large-scale hydrolysis of lignocellulosic biomass into sugar. agro-industrial wastes can be useful materials for enzyme development, improvement, and production. the liquor derived from sugar cane bagasse hydrothermal pretreatment is a low cost feedstock rich in xylose and xylo-oligosaccharides which are capable of inducing the expression of xylanases and accessory proteins in fungi such as a. niger. other materials, such as steam-exploded delignified bagasse and soybean bran, have also been used as inexpensive culture media to achieve high xylanase, cellulase, and β-glucosidase activities employing trichoderma harzianum p49p. the full hydrolysis of lignocellulosic biomass requires several types of glycohydrolases that enable the release of saccharides and other compounds from the recalcitrant substrate. however, plant species are highly diverse in terms of cell wall structure and composition, which increases the attraction of formulating specific biomass-degrading enzymatic cocktails. the sugar cane cell wall polysaccharide is mainly composed of xyloglucan and arabinoxylan, closely associated with cellulose, as well as pectin, β-glucan and less branched xylan strongly bound to cellulose. several studies have shown that supplementing cellulases with other enzymes can assist in the enzymatic hydrolysis of lignocellulosic biomass. xylanases and β-xylosidases improved the hydrolysis yield when combined with cellulases and β-glucosidades. the addition of pectinase to celluclast  l increased the hydrolysis of pretreated corn stover. supplementation of cellulolytic cocktails with α-l-arabinofuranosidase and xylanase also showed a synergistic effect in the hydrolysis of wheat straw. the production of glycohydrolases is closely related to the nature of the carbon source, since fungal metabolism is greatly influenced by the composition of the medium. each strain has a distinct metabolic profile, while the enzymatic profile is also distinct and depends on the medium and the cultivation time. physiological variations are the result of the adaptation and evolution of fungi, considering their hosts, original habitats, and other factors. the strain a. stigyum dr belongs to the xylariaceae family, members of which are frequently encountered as endophytes and saprophytes. gazis & chaverri isolated several endophytic xylariaceae strains and one strain of annulohypoxylon sp. from hevea brasiliensis. wei et al. cultivated an a. stigyum strain on avicel and confirmed the production of β-glucosidase, although only low levels of cellulases were detected. most alternaria species are saprophytes commonly found in soil or on decaying plant tissues, and some species are opportunistic plant pathogens. however, endophytic strains of alternaria spp. have been isolated from eucalyptus plants such as eucalyptus globulus and eucalyptus citriodora. strains of a. alternata are able to produce endopolygalactunorase in the presence of pectin, and β-glucosidase in the presence of saccharose. a. niger is known worldwide for its ability to produce an extensive range of extracellular glucohydrolases, including xylanases, pectinases, and β-glucosidase. this characteristic is associated with the ability of the fungus to propagate and colonize a variety of environments, principally those rich in decomposing plant materials. the fungus was recently reported to be endophytic in several plant species. however, this work is the first report of a. niger as an endophytic fungus in p. orientalis. there have been no previous reports in brazil concerning t. wortmannii isolated from decaying materials. jang et al. first described β-xylosidase activity in a t. wortmannii strain previously isolated from japanese red pine and larch woods in korea. jang et al. obtained a β-xylosidase production of u/ml for cultivation on xylan, in good agreement with the β-xylosidase activity found in the present work for the dr strain grown on xylan. trichoderma spp. are present in soil as saprophytes, and have also been found as endophytic organisms. many species from this genus are good cellulase and xylanase producers, such as t. harzianum and t. reesei. t. atroviride strains are good producers of these glycohydrolases, and can produce high amounts of β-glucosidase.  microorganisms play an essential role in the degradation of cellulose and hemicellulose standing out the endophytic fungi which are excellent sources of hydrolytic enzymes. evidently, during the endophytic phase, the use of these enzymes must be related to the mutualistic relationship with the host plant. however, although the association between plants and endophytic fungi is ecologically important, little is known about the physiological characteristics of the interaction. an important aspect of enzymatic studies involving endophytic fungi is the involvement of these microorganisms in the decomposition of plant material. since the fungi are already present in the senescent plant tissues, they may be able to initiate the decomposition process before it becomes dominated by saprophytic species. this could suggest not only that the production of hydrolytic enzymes by endophytic species might be important for the nutrition of the fungus during the endophytic stage, but also that these enzymes are produced and secreted at the surface of the tissues, where they can compete for the substrate during the saprophytic stage. kumaresan & suryanarayanan investigated the ability of endophytic fungi from mangrove leaves of different ages to produce hydrolytic enzymes. it was found that endophytic species occurring at relatively low levels in living leaves were more prevalent after leaf fall, increasing the involvement of these fungi in decomposition of the plant material. an important consideration is the range of substrates that can be utilized by endophytic microorganisms. studies have shown that endophytes are capable of metabolizing in vitro most substrates found in plants, and produce enzymes including proteases, amylases, phenol oxidases, lipases, laccases, polyphenol oxidases, cellulases, mannanases, xylanases, and pectin lyase. the balanced use of microbial enzymes in biomass deconstruction required the understanding of the role played by these glycohydrolases, and also depends on an economic process development. therefore, biochemical characterization of new reported glycohydrolases producer strains, as well as a bioprocess development of the selected strains in large scale, must be conducted to evaluate the enzyme applicability on the biomass deconstruction, principally on sugar cane bagasse. the present work demonstrated that it is possible to select endophytic fungal strains that can produce glycohydrolases with activities against a wide range of target substrates. this will enable the future formulation of specific enzymatic cocktails for an efficient biomass deconstruction. 
these strains were identified morphologically and by sequencing the internal transcribed spacer regions and the partial β-tubulin gene. there is an imperative necessity for alternative sources of energy able to reduce the world dependence of fossil oil. dr annulohypoxylon stigyum dr and talaromyces wortmannii dr are excellent producers of hydrolytic enzymes to be used as part of blends to decompose sugarcane biomass at industrial level. dr annulohypoxylon stigyum dr and talaromyces wortmannii dr these strains produced glycohydrolases with different profiles, and production was highly influenced by the carbon sources in the media. two initial selections were performed, one employing the esculin gel diffusion assay, and the other by culturing on agar plate media with beechwood xylan and liquor from the hydrothermal pretreatment of sugar cane bagasse. a total of isolates were then grown at 29°c on steam-exploded delignified sugar cane bagasse plus soybean bran, with measurement of the xylanase, pectinase, β-glucosidase, cmcase, and fpase activities. twelve strains were selected, and their enzyme extracts were assessed using different substrates.
 there is an imperative necessity for alternative sources of energy able to reduce the world dependence of fossil oil. one of the most successful options is ethanol obtained mainly from sugarcane and corn fermentation. the foremost residue from sugarcane industry is the bagasse, a rich lignocellulosic raw material uses for the production of ethanol second generation. new cellulolytic and hemicellulytic enzymes are needed, in order to optimize the degradation of bagasse and production of ethanol 2g.  the ability to produce hemicellulases and related enzymes, suitable for lignocellulosic biomass deconstruction, was explored using endophytic fungi and fungi isolated from spoiled books in brazil. two initial selections were performed, one employing the esculin gel diffusion assay, and the other by culturing on agar plate media with beechwood xylan and liquor from the hydrothermal pretreatment of sugar cane bagasse. a total of isolates were then grown at 29°c on steam-exploded delignified sugar cane bagasse plus soybean bran, with measurement of the xylanase, pectinase, β-glucosidase, cmcase, and fpase activities. twelve strains were selected, and their enzyme extracts were assessed using different substrates. finally, the best six strains were grown under xylan and pectin, and several glycohydrolases activities were also assessed. these strains were identified morphologically and by sequencing the internal transcribed spacer regions and the partial β-tubulin gene. the best six strains were identified as aspergillus niger dr trichoderma atroviride dr and dr alternaria sp. dr annulohypoxylon stigyum dr and talaromyces wortmannii dr these strains produced glycohydrolases with different profiles, and production was highly influenced by the carbon sources in the media.  the selected endophytic fungi aspergillus niger dr trichoderma atroviride dr and dr alternaria sp. dr annulohypoxylon stigyum dr and talaromyces wortmannii dr are excellent producers of hydrolytic enzymes to be used as part of blends to decompose sugarcane biomass at industrial level. endophytic fungixylanasehemicellulasesaccessory enzymes 