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The genomes of two Penicillium strains were sequenced and studied in this study: strain 2HH was isolated from the digestive tract of Anobium punctatum beetle larva in 1979 and the cellulase hypersecretory strain S1M29, derived from strain 2HH by a long-term mutagenesis process. With these data, the strains were reclassified and insight is obtained on molecular features related to cellulase hyperproduction and the albino phenotype of the mutant. Both strains were previously identified as Penicillium echinulatum and this investigation indicated that these should be reclassified. Phylogenetic and phenotype data showed that these strains represent a new Penicillium species in series Oxalica, for which the name Penicillium ucsense is proposed here. Six additional strains (SFC101850, SFCP10873, SFCP10886, SFCP10931, SFCP10932 and SFCP10933) collected from the marine environment in the Republic of Korea were also classified as this species, indicating a worldwide distribution of this new taxon. Compared to the closely related strain Penicillium oxalicum 114-2, the composition of cell wall-associated proteins of P. ucsense 2HH shows five fewer chitinases, considerable differences in the number of proteins related to β-D-glucan metabolism. The genomic comparison of 2HH and S1M29 highlighted single amino-acid substitutions in two major proteins (BGL2 and FlbA) that can be associated with the hyperproduction of cellulases. The study of melanin pathways shows that the S1M29 albino phenotype resulted from a single amino-acid substitution in the enzyme ALB1, a precursor of the 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis. Our study provides important knowledge towards understanding species distribution, molecular mechanisms, melanin production and cell wall biosynthesis of this new Penicillium species.

Penicillium echinulatum 2HH and Penicillium oxalicum 114-2 are well-known cellulase fungal producers. However, few studies addressing global mechanisms for gene regulation of these two important organisms are available so far. A recent finding that the 2HH wild-type is closely related to P. oxalicum leads to a combined study of these two species. Firstly, we provide a global gene regulatory network for P. echinulatum 2HH and P. oxalicum 114-2, based on TF-TG orthology relationships, considering three related species with well-known regulatory interactions combined with TFBSs prediction. The network was then analyzed in terms of topology, identifying TFs as hubs, and modules. Based on this approach, we explore numerous identified modules, such as the expression of cellulolytic and xylanolytic systems, where XlnR plays a key role in positive regulation of the xylanolytic system. It also regulates positively the cellulolytic system by acting indirectly through the cellodextrin induction system. This remarkable finding suggests that the XlnR-dependent cellulolytic and xylanolytic regulatory systems are probably conserved in both P. echinulatum and P. oxalicum. Finally, we explore the functional congruency on the genes clustered in terms of communities, where the genes related to cellular nitrogen, compound metabolic process and macromolecule metabolic process were the most abundant. Therefore, our approach allows us to confer a degree of accuracy regarding the existence of each inferred interaction.

Considering the growing importance of biofuels in Brazil and throughout the world, and taking into account that glycerol is a surplus byproduct of biodiesel production, the present study aimed to evaluate the production of cellulases and xylanases by Peniciliium echinulatum, mutant strain S1M29, using different concentrations of glycerol as a carbon source in combination or not with cellulose. Experimental use of 50% glycerol and 50% cellulose showed promising FPA activity, however, this was only approximately 50% of that obtained by previous studies employing only cellulose as a carbon source. More relevant was the fact that the enzyme broth prepared with 50% glycerol and 50% cellulose proved to be efficient in the hydrolysis of lignocellulosic biomasses, especially for elephant grass pretreated by steam explosion.

One of the current challenges to produce second-generation ethanol is to obtain an efficient enzyme complex for hydrolysis of lignocellulosic biomass. Genetic improvement of lignocellulolytic fungi has helped to obtain strains that secrete higher enzymatic titers. This article analyzes the enzymatic profile of two strains of Penicillium echinulatum, wild type 2HH and mutant S1M29, grown on different carbon sources (sugarcane bagasse pretreated by steam explosion, cellulose, glucose, and glycerol) at different cultivation times. To determine enzyme activities, 23 substrates were used. P. echinulatum showed potential to produce cellulases, hemicellulases, esterases, and in minor proportions, pectinases. The mutant strain showed the highest enzymatic titers to most enzymes considered and its enzymatic broth was used in the enzymatic hydrolysis of vegetable biomass. This study broadened the understanding of the enzyme complex of this fungus and the importance of future studies with the mutant strain for use in the production of second-generation ethanol.

Biological pre-treatment seems to be promising being an eco-friendly process, with no inhibitor generated during the process. The potential for elephant grass pre-treatment with white degradation fungi Pleurotus ostreatus, Agaricus blazei, Lentinula edodes, Pleurotus citrinopileatus and Pleurotus djamor, in isolated or mixed cultures of these strains, was evaluated. The highest activities of enzymes involved in the degradation of lignocellulosic biomass (laccases, endoglucanases, xylanases and β-glucosidases) were observed for A. blazei, L. edodes and the combination of P. ostreatus and A. blazei. In the enzymatic hydrolysis, there was greater release of reducing sugars in the pre-treated elephant grass samples by A. blazei during 10 days (338.91±7.39 mg/g of biomass). For this sample, higher lignin reductions, 24.81 and 57.45%, after 15 and 35 days of incubation, respectively, were also verified. These data indicate the potential of macromycetes such as A. blazei to perform biological pre-treatments. This article is protected by copyright. All rights reserved.

One of the bottlenecks in the production of second-generation ethanol is the high cost of the enzymes used for the hydrolysis of lignocellulosic biomass. One way to reduce this cost is to use local biomass, such as elephant grass ( Pennisetum purpureum ), but for this use the lignocellulosic material must be pretreated. Among the pretreatments that have been studied, steam explosion has been the most efficient. In this study, it was evaluated the production of cellulases and xylanases by Penicillium echinulatum S1M29 in solid-state cultivation using elephant grass pretreated by steam explosion at different temperatures (180, 190 and 200 °C) and residence times (6, 8, 10 min). In culture media, it was used 100% elephant grass that was pretreated by steam explosion (washed and unwashed after the process) or in combination with wheat bran (50%). It was found that the untreated elephant grass is superior to wheat bran for Filter Paper Activity (FPA) with values of 23.38 ± 0.10 IU g −1 . The replacement of 50% wheat bran with elephant grass that was pretreated by steam explosion and washed favoured the production of enzymes with maximal activities of 32.93 ± 0.58 IU g −1 for FPA, 205.83 6.85 ± IU g −1 for endoglucanase, and 148.96 ± 0.00 IU g −1 for β-glucosidases. For xylanase production, untreated elephant grass proved more effective because the combination of substrates provided the greatest enzymatic activity in a sample that was not subjected to washing, reaching values of 571.74 ± 27.14 IU g −1 .

The cost of hydrolytic enzymes is one bottleneck for economic production of second generation ethanol and the use of low cost substrates is required. The use of elephant grass for the production of hydrolytic enzymes is a promising alternative due its high productivity, but it requires pretreatment. In this context, the steam explosion method appears to be advantageous for producing structural changes in the lignocellulosic material, making the biomass more accessible to enzymatic action. The elephant grass was pretreated via steam explosion – at 180, 190 and 200 °C, with residence times of 6, 8 and 10 min – for the production of cellulases and xylanases by Penicillium echinulatum S1M29 in submerged cultivation was evaluated. The biomass that was pretreated at 190 °C for 8 and 6 min showed a positive result for filter paper activity (FPA), endoglucanases and β-glucosidases, since the biomass is washed with water after the pretreatment. All of the pretreatments showed a negative result for xylanase. The results indicate that elephant grass that has been pretreated using steam explosion can be used as a substrate for the production of enzymes for the hydrolysis of lignocellulosic biomass.

Penicillium echinulatum S1M29 is a mutant with cellulase and xylanase production comparable to the most studied microorganisms in the literature. However, its potential to produce these enzymes has not been fully investigated. This study aimed at optimizing salt and urea concentrations in the mineral solution, employing the response surface methodology. A 25-1 Fractional Factorial Design and a 23 Central Composite Design were applied to elucidate the effect of salts and urea in enzyme production. Lower concentrations of KH2PO4 (2.0 g.L-1), (NH4)2SO4 (1.4 g.L-1), MgSO4.7H2O (0.375 g.L-1) and CaCl2 (0.375 g.L-1) were most suitable for the production of all enzymes evaluated. Nevertheless, higher concentrations of urea (0.525 g.L-1) gave the best results for cellulase and xylanase production. The maximum FPase (1,5 U.m.L-1), endoglucanase (7,2 U.m.L-1), xylanase (30,5 U.m.L-1) and β-glucosidase (4,0 U.m.L-1) activities obtained with the planned medium were, respectively, 87, 16, 17 and 21% higher when compared to standard medium. The experimental design contributed to adjust the concentrations of minerals and urea of the culture media for cellulase and xylanase production by P. echinulatum, avoiding waste of components in the medium.

In the search for renewable energy sources, elephant grass is an alternative substrate for ethanol production, but this substrate must be hydrolyzed by cellulases and xylanases to liberate fermentable sugars. During enzymatic hydrolysis, cellulase activity is reduced by the irreversible adsorption of cellulase onto cellulose, decreasing the rate of hydrolysis. Adding surfactants during hydrolysis can improve the process. The effects of Tween® and Triton® surfactants on the enzymatic hydrolysis of elephant grass were evaluated in this context. The data indicate that pretreatment with sodium hydroxide, along with a smaller particle size (0.075–0.152 mm) and the use of Tween 80®, increased the efficiency of releasing reducing sugars from pretreated elephant grass biomass. Thus, it is possible to reduce grinding costs in second-generation ethanol production through the use of surfactants, as they allow efficient hydrolysis of larger biomass particles.

Pennisetum purpureum (elephant grass) is a tropical species presenting C4 metabolism and high productivity, yielding approximately 45 t of dry matter/ha/year. Such productivity makes elephant grass an interesting biomass source to produce second-generation ethanol. However, the high cost of enzymes for enzymatic hydrolysis is one bottleneck on the way to making the production of this biofuel economically viable. The production of cellulases and xylanases by Penicillium echinulatum 9A02S1 was evaluated using 85 different non-treated elephant grass accessions in submerged culture, aiming to identify genotypes with potential biomass for the production of enzymes for the second-generation ethanol industry. The data clearly indicate that it is possible to replace cellulose with elephant grass biomass for the production of cellulases, β-glucosidases and xylanases. The accession IJ 7127 produced 4.7 times more than cellulose for endoglucanases and 4 times for Filter Paper Activity. Ten accessions (BAGCE 69, Napierzinho, IJ 7125, IJ 7126, IJ 7127, IJ 7136, IJ 7141, CAC-262, Ibitinema, 13 AD) produced at least 4 times more β-glucosidases than cellulose, and for xylanases, three accessions (Mercker 86 Mexico, Taiwan A-144, Napier S.E.A.) produced twice as much as the control. The data also indicate that there is no direct relationship between the amount of lignin and cellulose in the substrate, but the mineral salts present in the sample significantly influenced the enzyme production.

The high cost of the enzymes used for the hydrolysis of lignocellulosic biomass is one of the bottlenecks in the production of second-generation ethanol and the use of local biomass, such elephant grass, can help to reduce this cost. In this investigation elephant grass biomass was evaluated as a carbon and inducer source of production of cellulases and xylanases by the fungus Penicillium echinulatum strain 9A02S1 in solid-state cultivation. The highest filter paper activity (13.26 U.g-1 dry matter) and β-glucosidase activity (138.34 U.g-1 dry matter) were obtained on the fifth day of cultivation, in medium containing biomass pre-treated with sulfuric acid and untreated, respectively. The highest endoglucanase activity was 158.44 U.g-1 dry matter in the presence of elephant grass pre-treated with sulfuric acid. The xylanase activity was highest with medium that was formulated with 75% untreated elephant grass and 25% wheat bran (372.62 U.g-1 dry matter). In conclusion, the results showed that it is possible to obtain large amounts of cellulases and xylanases using a cosmopolitan and very low cost substrate such as elephant grass.

Penicillium echinulatum 9A02S1 is a filamentous fungus with a cellulolytic system that may be used in the processes involved in the degradation of lignocellulosic materials for ethanol production. In this study, the 9A02S1 strain of P. echinulatum was grown in submerged cultures with various carbon sources to assess the activities of cellulases and β-glucosidases and their enzymatic activities were assessed utilizing polyacrylamide gel electrophoresis. Cellulose was used as the inducer. Carboxymethyl cellulose proved to be an efficient carbon source for the production of endoglucanase and filter paper activity. Cellobiose, glucose and sorbitol stimulated the production of β-glucosidases. The presence of multimeric forms of the enzymes or their post-translational modifications were assessed.

The development of more productive strains of microorganisms and processes that increase enzyme levels can contribute to the economically efficient production of second generation ethanol. To this end, cellulases and xylanases were produced with the S1M29 mutant strain of Penicillium echinulatum , using different concentrations of cellulose (20, 40, and 60 g L −1 ) in batch and fed-batch processes. The highest activities of FPase (8.3 U mL −1 ), endoglucanases (37.3 U mL −1 ), and xylanases (177 U mL −1 ) were obtained in fed-batch cultivation with 40 g L −1 of cellulose. The P. echinulatum enzymatic broth and the commercial enzyme Cellic CTec2 were tested for hydrolysis of pretreated sugar cane bagasse. Maximum concentrations of glucose and xylose were achieved after 72 h of hydrolysis. Glucose yields of 28.0% and 27.0% were obtained using the P. echinulatum enzymatic extract and Cellic CTec2, respectively.

The effect of pH on the production of cellulases and xylanases by Penicillium echinulatum S1M29 was evaluated in a shake flask and in a bioreactor. To control the pH in a shake flask, a buffer made with citric acid and disodium phosphate was used. The buffer was capable of maintaining the culture pH values for the first 48 h. In the bioreactor, the pH was controlled automatically by the addition of NaOH and H2SO4. In the shake flask, the highest activities of xylanases (18.5 IU/mL) and endoglucanases (8.2 IU/mL), as well as the highest filter paper activity (FPA) (0.9 IU/mL), were obtained at initial pH values of between 6.0 and 7.0. In the bioreactor, the highest activities of these enzymes were obtained in a pH range of 5.5 to 6.5. Different isoforms of the endoglucanases were found in the various cultures depending on the pH. More acidic pH ranges favored the production of β-glucosidases in both the shake flask and the bioreactor.

This study evaluated the potential use of elephant grass biomass, a highly productive species, for cellulase and xylanase production by the cellulolytic mutant Penicillium echinulatum 9A02S1 in submerged cultivation, using untreated biomass, biomass pretreated with different concentrations of NaOH, H2SO4 or NH4OH, or biomass pretreated with H2O at 121 °C. For filter paper activity, all cultivation carried out with pretreated elephant grass under the evaluated conditions showed superior activity when compared with the control (untreated elephant grass). The activities of endoglucanases and β-glucosidases were higher in the cultivation prepared from pretreated samples than the control made with cellulose (Celuflok(®)). Without pretreatment, elephant grass can be used for xylanase production, enabling similar activities to those obtained in the cultivation with cellulose, reducing the enzyme production cost. These results indicate that the pretreatment of elephant grass, especially when pretreated with H2SO4, may be used as a partial or total replacement for cellulose to cellulase production, and untreated elephant grass may be used for xylanase production.

Background The enzymatic degradation of lignocellulosic materials by fungal enzyme systems has been extensively studied due to its effectiveness in the liberation of fermentable sugars for bioethanol production. Recently, variants of the fungus Penicillium echinulatum have been described as a great producer of cellulases and considered a promising strain for the bioethanol industry. Results Penicillium echinulatum, wild-type 2HH and its mutant strain S1M29, were grown on four different carbon sources: cellulose, sugar cane bagasse pretreated by steam explosion (SCB), glucose, and glycerol for 120 h. Samples collected at 24, 96, and 120 h were used for enzymatic measurement, and the 96-h one was also used for secretome analysis by 1D-PAGE LC–MS/MS. A total of 165 proteins were identified, and more than one-third of these proteins belong to CAZy families. Glycosyl hydrolases (GH) are the most abundant group, being represented in larger quantities by GH3, 5, 17, 43, and 72. Cellobiohydrolases, endoglucanases, β-glycosidases, xylanases, β-xylosidases, and mannanases were found, and in minor quantities, pectinases, ligninases, and amylases were also found. Swollenin and esterases were also identified. Conclusions Our study revealed differences in the two strains of P. echinulatum in several aspects in which the mutation improved the production of enzymes related to lignocellulosic biomass deconstruction. Considering the spectral counting analysis, the mutant strain S1M29 was more efficient in the production of enzymes involved in cellulose and hemicellulose degradation, despite having a nearly identical CAZy enzymatic repertoire. Moreover, S1M29 secretes more quantities of protein on SCB than on cellulose, relevant information when considering the production of cellulases using raw materials at low cost. Glucose, and especially glycerol, were used mainly for the production of amylases and ligninases. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0476-3) contains supplementary material, which is available to authorized users.

The high cost of cellulases and xylanases for the hydrolysis of lignocellulosic wastes is a bottleneck in the production of second-generation ethanol. The use of a low-cost carbon source, such as steam-exploded sugar cane bagasse (seSCB), is an alternative to reduce the costs in cellulase production. Enzyme supernatants produced by the mutant Penicillium echinulatum 9A02S1 on seSCB as a substrate have good filter paper activity (0.48 IU/mL) values combined with endoglucanases (3.24 IU/mL), β-glucosidase (0.48 IU/mL), and xylanases (0.64 IU/mL) activities. It was found that the enzymes produced on seSCB showed activity at the same hydrolysis potential of the enzymes produced with cellulose as a carbon source. In conclusion, the results presented in this paper showed that it is possible to obtain efficient enzymes by P. echinulatum using very low-cost substrates, such as seSCB, and, therefore, contributing to the development of on-site enzyme production to produce second-generation ethanol.

Aims: The aim of this study is to improve cellulase production and secretion by Penicillium echinulatum using mutagenesis and selection in association with microfermentation and microanalysis methods. Methods and Results: A new genetic variant was isolated from strain 9A02S1 and named S1M29. It was obtained by mutagenesis with H2O2 and two screening steps, which involved selection in Petri dishes using the medium supplemented with 2-deoxyglucose and microfermentations in submerged culture. The mutant showed higher cellulase productivity than 9A02S1 based on the Filter Paper Activity assay and endoglucanase; the peak activities for these enzymes were reached significantly faster than for the parent strain. Conclusions: The mutant obtained after mutagenesis and selection could produce and secrete cellulase faster than the parent strain. Significance and Impact of the Study: Mutagenesis followed by selection is a useful tool for rapidly generating new cellulase-producing phenotypes in fungi. Faster production and higher titers of cellulases in mutant strains contribute to reduce the production costs for enzymatic complexes that hydrolyse lignocellulose residues and form fermented syrups, thus contributing to the economic production of bioethanol.

To investigate the production of cellulases and xylanases from Penicillium echinulatum 9A02S1, solid-state fermentation (SSF) was performed by using different ratios of sugar cane bagasse (SCB) and wheat bran (WB). The greatest filter paper activity obtained was 45.82 ± 1.88 U gdm(-1) in a culture containing 6SCB/4WB on the third day. The greatest β-glucosidase activities were 40.13 ± 5.10 U gdm(-1) obtained on the third day for the 0SCB/10WB culture and 29.17 ± 1.06 U gdm(-1) for the 2SCB/8WB culture. For endoglucanase, the greatest activities were 290.47 ± 43.57 and 276.84 ± 15.47 U gdm(-1), for the culture 6SCB/4WB on the fourth and fifth days of cultivation, respectively. The greatest xylanase activities were found on the third day for the cultures 6SCB/4WB (36.38 ± 5.38 U gdm(-1)) and 4SCB/6WB (37.87 ± 2.26 U gdm(-1)). In conclusion, the results presented in this article showed that it was possible to obtain large amounts of cellulases and xylanases enzymes using low-cost substrates, such as SCB and WB.

Nowadays, cellulase importance is growing mainly due to its potential utilization in developing second-generation ethanol technology. Thus, considerable effort has been made to obtain new genotypes with improved capacity to produce efficient and cost-effective enzyme complexes for cellulose hydrolysis. The majority of the genetically manipulated strains that produce higher amounts of cellulases come from of the wild Trichoderma reesei QM6A. Such as T. reesei cellulolytic mutants, Penicillium echinulatum mutants also produce high titles of cellulases (filter paper activities—FPA), and its enzymatic complex effectively hydrolyses cellulose. In the present work, we obtained genetic variability with the purpose of enhancing cellulase production by protoplast fusion between P. echinulatum and T. harzianum . The resulting fusants, who were all morphologically like the P. echinulatum parental, were initially characterized by RAPD, and the results showed that almost all amplified DNA bands showed correlation with P. echinulatum DNA bands. Some of new selected strains presented higher FPA and β-glucosidases activities when compared to the parental strains. One particular clone – BP2 – showed values of FPA activities around of 2 UI ml −1 , in submerged cultures and all selected strains showed faster and higher secretion of cellulases in solid-state cultures, in comparison to the parental strain of P. echinulatum . Altogether, at the same time that our results show increased cellulase secretion by P. echinulatum strains, they also show that protoplast fusion techniques associated with an efficient selection process could be a useful tool in genetic improvement to generate better cellulose-producing strains aimed at economically hydrolyzing cellulose with a view to second-generation ethanol.

Penicillium echinulatum has been identified as a potential cellulase producer for bioconversion processes but its cellulase system has never been investigated in detail. In this work, the volumetric activities of P. echinulatum cellulases were determined against filter paper (0.27 U/mL), carboxymethylcellulose (1.53 U/mL), hydroxyethylcellulose (4.68 U/mL), birchwood xylan (3.16 U/mL), oat spelt xylan (3.29 U/mL), Sigmacell type 50 (0.10 U/mL), cellobiose (0.19 U/mL), and p-nitrophenyl-glucopiranoside (0.31 U/mL). These values were then expressed in relation to the amount of protein and compared those of Trichoderma reesei cellulases (Celluclast 1.5L FG, Novozymes). Both enzyme complexes were shown to have similar total cellulase and xylanase activities. Analysis of substrate hydrolysates demonstrated that P. echinulatum enzymes have higher beta-glucosidase activity than Celluclast 1.5L FG, while the latter appears to have greater cellobiohydrolase activity. Unlike Celluclast 1.5L FG, P. echinulatum cellulases had enough beta-glucosidase activity to remove most of the cellobiose produced in hydrolysis experiments. However, Celluclast 1.5L FG became more powerful than P. echinulatum cellulases when supplemented with exogenous beta-glucosidase activity (Novozym 188). Both cellulase complexes displayed the same influence over the degree of polymerization of cellulose, revealing that hydrolyzes were carried out under the typical endo-exo synergism of fungal enzymes.

New cellulases from the fungi Acrophialophora nainiana and Penicillium echinulatum were used in the finishing of knitted cotton fabrics (biopolishing) and compared with the well established enzymes from Trichoderma reesei. Both cellulases reduced the pilling tendency with a lower weight loss than T. reesei cellulases. Cellulases from P. echinulatum were also studied in stonewashing of denim fabrics to obtain the fashionable aged look in indigo dyed jeans ware and were found to remove more colour from denim fabrics and produce less indigo dye redeposition (back-staining) than commercial acid or neutral cellulases under the test conditions. Efficiency was found to be influenced by pH during textile processing and the substrate used for the production of cellulases. Cellulases produced by P. echinulatum grown on cellulose showed better stonewashing results (higher colour removal and less back-staining) than cellulases produced on sugar cane bagasse. The substrate used during enzyme production of P. echinulatum cellulases seems to have a significant influence on cellulose composition, which affects textile processing results.

Aim: To evaluate the solid-state fermentation (SSF) production of cellulase and hemicellulases (xylanases), by Penicillium echinulatum 9A02S1, in experiments carried out with different concentrations of the pretreated sugar cane bagasse (PSCB) and wheat bran (WB). Methods and Results: This study reports the production of xylanolytic and cellulolytic enzymes by P. echinulatum 9A02S1 using a cheap medium containing PSCB and WB under SSF. The highest amounts of filter paper activity (FPA) could be measured on mixtures of PSCB and WB (32·89 ± 1·90 U gdm−1). The highest β-glucosidase activity was 58·95 ± 2·58 U gdm−1 on the fourth day. The highest activity for endoglucanases was 282·36 ± 1·23 U gdm−1 on the fourth day, and for xylanases the activity was around 10 U gdm−1 from the second to the fourth day. Conclusions: The present work has established the potential of P. echinulatum for FPA, endoglucanase, β-glucosidase and xylanase productions in SSF, indicating that WB may be partially substituted by PSCB. Significance and Impact of the Study: The incorporation of cheap sources, such as sugar cane bagasse, into media for the production of lignocellulose enzymes should help decrease the production costs of enzymatic complexes that can hydrolyse lignocellulose residues for the formation of fermented syrups, thus contributing to the economic production of bioethanol.

In this work, steam explosion was used a pretreatment method to improve the conversion of elephant grass (Pennisetum purpureum) to cellulosic ethanol. This way, enzymatic hydrolysis of vaccum-drained and water-washed steam-treated substrates was carried out with Penicillium echinulatum enzymes while Saccharomyces cerevisiae CAT-1 was used for fermentation. After 48 h of hydrolysis, the highest yield of reducing sugars was obtained from vaccum-drained steam-treated substrates that were produced after 10 min at 200 °C (863.42 ± 62.52 mg/g). However, the highest glucose yield was derived from water-washed steam-treated substrates that were produced after 10 min at 190 °C (248.34 ± 6.27 mg/g) and 200 °C (246.00 ± 9.60 mg/g). Nevertheless, the highest ethanol production was obtained from water-washed steam-treated substrates that were produced after 6 min at 200 °C. These data revealed that water washing is a critical step for ethanol production from steam-treated elephant grass and that pretreatment generates a great deal of water soluble inhibitory compounds for hydrolysis and fermentation, which were partly characterized as part of this study.

Mutagenesis and selection were applied to a strain of Penicillium echinulatum by treating conidia with hydrogen peroxide or 1,2,7,8-diepoxyoctane and then by incubating the conidia for 48 h in broth containing microcrystalline cellulose washed in 0.5% (w/v) aqueous 2-deoxyglucose before plating them onto cellulose agar containing 1.5% (w/v) glucose from which colonies showing the fastest production of halos of cellulose hydrolysis were selected. This process resulted in the isolation of two new cellulase-secreting P. echinulatum mutants: strain 9A02S1 showing increased cellulase secretion (2 IU ml-1, measured as filter paper activity) in submerged culture in agitated flasks containing a mineral salts medium and 1% of cellulose, and strain 9A02D1, which proved more suitable for the production of cellulases in semisolid bran culture where it produced 23 IU of beta-glucosidase per gram of wheat bran.

The inducer effect of lactose on cellulase activity in Penicillium echinulatum 9A02S1 was studied. Submerged cultivation was performed using different concentrations of lactose and cellulose, in which the pH, mycelial mass, soluble proteins, filter paper activity (FPA), and activity of beta-glucosidases were measured. The cultures containing lactose only presented low FPAs (0.1 FPU/ml). The cultures with associated cellulose and lactose and those containing cellulose only presented similar enzymatic activities (1.5 FPU/ml), suggesting the possibility of up to 75% reduction in the cellulose concentration. In relation to the beta-glucosidases, increasing the lactose/cellulose ratio results in a proportional increase of enzymatic activity. In the cultures using both inducers, there is a longer duration of the acid phase in relation to treatments using only cellulose or lactose, indicating diauxia and catabolic repression.

New cellulases from a strain of Penicillium echinulatum were characterized for their filter paper activity and β-glucosidase activity. Both activities showed maximum values between pH 4 and 5. With citrate buffer, activities were slightly higher than in acetate buffer of the same pH. Thermal stability of both activities was good up to 55°C. Filter paper activity was significantly reduced at higher temperatures.

The main limitation to the industrial scale hydrolysis of cellulose is the cost of cellulase production. This study evaluated cellulase and xylanase enzyme production by the cellulolytic mutantPenicillium echinulatum9A02S1 using pretreated sugar cane bagasse as a carbon source. Most cultures grown with pretreated bagasse showed similar enzymatic activities to or higher enzymatic activities than cultures grown with cellulose or untreated sugar cane bagasse. Higher filter paper activity (1.253 ± 0.147 U·mL−1) was detected in the medium on the sixth day of cultivation when bagasse samples were pretreated with sodium hydroxide, hydrogen peroxide, and anthraquinone. Endoglucanase enzyme production was also enhanced by pretreatment of the bagasse. Nine cultures grown with bagasse possessed higherβ-glucosidase activities on the sixth day than the culture grown with cellulose. The highest xylanase activity was observed in cultures with cellulose and with untreated sugar cane bagasse. These results indicate that pretreated sugar cane bagasse may be able to serve as a partial or total replacement for cellulose in submerged fermentation for cellulase production usingP. echinulatum,which could potentially reduce future production costs of enzymatic complexes capable of hydrolyzing lignocellulosic residues to form fermented syrups.

The effect of different carbon sources on morphology and cellulase and xylanase production ofPenicillium echinulatumwas evaluated in this work. Among the six carbon sources studied, cellulose and sugar cane bagasse were the most suitable for the production of filter paper activity, endoglucanases, xylanases, andβ-glucosidases. However, sucrose and glucose showedβ-glucosidase activities similar to those obtained with the insoluble sources. The polyacrylamide gels proved the enzymatic activity, since different standards bands were detected in the media mentioned above. Regarding morphology, it was observed that the mycelium in a dispersed form provided the greatest enzymatic activity, possibly due to greater interaction between the substrate and hyphae. These data are important in understanding the physiology of fungi and could contribute to obtaining enzyme with potential application in the technology of second generation ethanol.

The production of cellulases and xylanases by Penicillium echinulatum in an airlift bioreactor was evaluated. In batch production, we tested media with isolated or associated cellulose and sorbitol. In fed-batch production, we tested cellulose addition at two different times, 30 h and 48 h. Higher liquid circulation velocities in the downcomer were observed in sorbitol 10 g L(-1) medium. In batch production, higher FPA (filter paper activity) and endoglucanase activities were obtained with cellulose (7.5 g L(-1)) and sorbitol (2.5 g L(-1)), 1.0 U mL(-1) (120 h) and 6.4 U m L(-1) (100 h), respectively. For xylanases, the best production condition was cellulose 10 g L(-1), which achieved 5.5 U mL(-1) in 64 h. The fed-batch process was favorable for obtaining xylanases, but not for FPA and endoglucanases, suggesting that in the case of cellulases, the inducer must be added early in the process.

The present work investigated the use of sorbitol as a soluble carbon source, in association with cellulose, to produce cellulases and xylanases in submerged cultures of Penicillium echinulatum 9A02S1. Because cellulose is an insoluble carbon source, in cellulase production, there are some problems with rheology and oxygen transfer. The submerged fermentations containing media composed of 0, 0.25, 0.5, 0.75, and 1% (w/v) sorbitol and cellulose that were added at different times during the cultivation; 0.2% (w/v) soy bran; 0.1% (w/v) wheat bran; and a solution of salts. The highest filter paper activity (FPA) ( IU·mL−1) was obtained on the seventh day in the medium containing 0.5% (w/v) sorbitol and 0.5% (w/v) cellulose added 24 h after the start of cultivation. However, the CMCases showed an activity peak on the sixth day ( IU·mL−1) in the medium containing 0.75% (w/v) sorbitol and 0.75% (w/v) cellulose added after 12 h of cultivation. The xylanases showed the highest activity in the medium with 0.75% (w/v) sorbitol and 0.25% (w/v) cellulose added 36 h after the start of cultivation. This strategy enables the reduction of the cellulose concentration, which in high concentrations can cause rheological and oxygen transfer problems.

Plant feedstocks are at the leading front of the biofuel industry based on the potential to promote economical, social and environmental development worldwide through sustainable scenarios related to energy production. Penicillium echinulatum is a promising strain for the bioethanol industry based on its capacity to produce large amounts of cellulases at low cost. The secretome profile of P. echinulatum after grown on integral sugarcane bagasse, microcrystalline cellulose and three types of pretreated sugarcane bagasse was evaluated using shotgun proteomics. The comprehensive chemical characterization of the biomass used as the source of fungal nutrition, as well as biochemical activity assays using a collection of natural polysaccharides, were also performed. Our study revealed that the enzymatic repertoire of P. echinulatum is geared mainly toward producing enzymes from the cellulose complex (endogluganases, cellobiohydrolases and β-glucosidases). Glycoside hydrolase (GH) family members, important to biomass-to-biofuels conversion strategies, were identified, including endoglucanases GH5, 7, 6, 12, 17 and 61, β-glycosidase GH3, xylanases GH10 and GH11, as well as debranching hemicellulases from GH43, GH62 and CE2 and pectinanes from GH28. Collectively, the approach conducted in this study gave new insights on the better comprehension of the composition and degradation capability of an industrial cellulolytic strain, from which a number of applied technologies, such as biofuel production, can be generated.

There has been a notable increase in interest about cellulases in recent years due to their numerous potential applications, including the hydrolysis of cellulose in lignocellulosic biomass to produce reducing sugars for the development of second-generation ethanol. However, the traditional cellulase assays are tedious, time-consuming and labor intensive. What is more, they require large amounts of reagents and produce large quantities of toxic effluents? The Filter Paper Assay (FPA) is the standard measure of cellulase activity, according to the International Union of Pure and Applied Chemistry. Although it can be reproduced in most laboratories, with some practice, this method has long been known for its complexity and many manual manipulations. In this study, adaptations were made to the FPA to create a low-cost, easier, faster, less labor intensive and less polluting alternative, while maintaining similar results to the standard method.

Aim: In this work, the effect of supplementing liquid cellulase production media (CPM) with methylxanthines (aminophylline, caffeine and theophylline), with and without the addition of glucose, on the secretion of cellulases by Penicillium echinulatum strain 2HH (wild-type) and the derived mutant strain 9A02S1 was studied. Methods and Results: When compared with unsupplemented CPM, both strains produced higher b-glucosidase and filter paper activities (FPAs) in CPM supplemented with 1 lmol l )1 of caffeine but lower activities with 5 lmol l )1 of caffeine. With theophylline only, strain 9A02S1 produced higher b-glucosidase and FPAs, while aminophylline produced no effect on the cellulase activity of either strain. Supplementation of CPM with 0AE5% (w/v) of glucose plus caffeine resulted in higher b-glucosidase and FPAs being produced by strain 2HH, but not strain 9A02S1, than in CPM supplemented with 0AE5% (w/v) of glucose only. Conclusions: These results indicate that different concentrations of caffeine and theophylline can increase the b-glucosidase and FPAs produced by P. echinulatum strains 2HH and 9A02S1. Significance and Impact of the Study: The results suggest that some methylxanthines, in adequate concentration, can be used as media components to increase cellulase production.

Considering that the cost of cellulases and hemicellulases substantially contributes to the price of 2nd-generation ethanol, reducing the cost of the substances used as inducers of cellulose and xylose is essential to reducing the cost of these enzymes. Penicillium echinulatum is able to secrete most hemicellulolytic and cellulolytic enzymes. In this context, the aim of this work was to evaluate the use of xylose for the production of cellulases and xylanases by two mutant strains of P. echinulatum (9A02S1 and S1M29) and the parental 2HH. Xylose acts as an inducer for the production of xylanases and cellulases, especially endoglucanases. These data indicate the possibility of using the pentose xylose, which is not used by the majority of yeast for ethanol production, as a total or partial substitute in cellulose-based processes for the production of enzymes for enzymatic hydrolysis.

Fodder radish (FR) is an oilseed crop with a high potential for biodiesel production due to its high productivity and the quality of its seed oil. FR oil extraction results in a residue that is rich in protein and fiber. In this study, FR cake (FRC) was evaluated as carbon and nitrogen source for the production of cellulases and xylanases using Penicillium echinulatum S1M29 during solid-state cultivation. It was determined that it is possible to partially replace wheat bran (WB) by FRC, resulting in 24.22 ± 0.25U/g Filter Paper Activity (144 hours), 210.5 ± 5.8U/g endoglucanase activity (144 hours), 22.62 ± 0.01U/g (-glucosidase activity (96 hours) and 784.7 ± 70.19U/g xylanase activity (120 hours). These values are equal or higher than the enzymatic activity obtained using WB. These results may contribute to the reduction of the cost of enzymes used in the production of cellulosic ethanol or other biotechnological applications.