Cellulase On-Site Production from Sugar Cane Bagasse Using Penicillium echinulatum

Penicillium echinulatum was evaluated as a cellulolytic enzyme producer in shaking flasks and bioreactor submerged culture using sugarcane bagasse as carbon source. Sodium hydroxide delignified steam-exploded pretreated bagasse (SDB) and hydrothermal pretreated bagasse had a maximum filter paper activity (FPase) of 2.4 and 2.6 FPU/mL, respectively. Delignified acid pretreated bagasse and Celufloc 200TM (CE) carbon sources displayed maximum FPase of 1.3 and 1.6 FPU/mL while in natura bagasse (INB) provided the lowest enzyme activity, ca. 0.4 FPU/mL. Measurement of surface specific area of lignocellulosic material and scanning electron microscopic images showed a possible correlation between fungal mycelia accessibility to lignocellulosic particles and obtained cellulolytic enzyme activity of fermentation broth. Fed-batch experiments performed in a controlled bioreactor attained the highest value of FPase of 3.7 FPU/mL, enzyme productivity of 25.7 FPU/L h, and enzyme yield from cellulose equal to 134 FPU/g with SDB. Enzyme hydrolysis of steam-pretreated bagasse accomplished with the obtained supernatant of fermentation broth (10 FPU/g of biomass and 5 % w/v) performed better than commercial cellulose complex. The results showed that P. echinulatum has potential to be used as an on-site enzyme platform aiming second bioethanol production from sugarcane lignocellulosic residue.     

Xylanase production by endophytic Aspergillus niger using pentose-rich hydrothermal liquor from sugarcane bagasse

Fungal xylanases have been widely studied and various production methods have been proposed using submerged and solid-state fermentation. This class of enzyme is used to supplement cellulolytic enzyme cocktails in order to enhance the enzymatic hydrolysis of plant cell walls. The present work investigates the production of xylanase and other accessory enzymes by a recently isolated endophytic Aspergillus niger DR02 strain, using the pentose-rich liquor from hydrothermal pretreatment of sugarcane bagasse as carbon source. Batch and fed-batch submerged cultivation approaches were developed in order to minimize the toxicity of the liquor and increase enzyme production. Maximum xylanase activities obtained were 458.1 U/mL for constant fed-batch, 428.1 U/mL for exponential fed-batch, and 264.37 U/mL for pulsed fed-batch modes. The results indicated that carbon-limited fed-batch cultivation can reduce fungal catabolite repression, as well as overcome possible negative effects of toxic compounds present in the pentose-rich liquor. Enzymatic panel and mass spectrometric analyses of the fed-batch A. niger secretome showed high levels of xylanolytic enzymes (GH10, GH11, and GH62 Cazy families), together with cellobiohydrolase (G6 and GH7), β-glucosidase, β-xylosidase (GH3), and feruloyl esterase (CE1) accessory enzyme activities. The yields of glucose and xylose from enzymatic hydrolysis of hydrothermally pretreated sugarcane bagasse increased by 43.7 and 65.3%, respectively, when a commercial cellulase preparation was supplemented with the A. niger DR02 constant fed-batch enzyme complex.     

Biosynthesis,optimization and potential textile application of fungal cellulases/xylanase multifunctional enzyme preparation from Penicillium sp. SAF6

The main task of the present work is to search for fungal strains isolated from agricultural soil with the potential to produce cellulases/xylanase enzyme preparation for bio-finishing of textiles. The most potent fungal strain (SAF6) was subjected to molecular identification using 18 SrRNA and was identified as Penicillium sp. SAF6 with the novel accession number of KM222497. Factors affecting the produced mixed enzyme activity were investigated. The optimum conditions for achieving maximum activity of the cellulases (FPase, CMCase and β-glucosidase) in addition to xylanase were the initial culture pH media 5, yeast extract (1.5gN/L), medium-to-air ratio (1:5) for FPase and CMCase and (1:10) for β-glucosidase, at 30?°C for 8 days incubation period. Potential application of the prepared crude enzyme in bio-finishing of cellulosic substrates, namely, bleached cotton, linen and indigo dyed fabrics were explored. Using the multi-component enzyme at appropriate dosage and conditions brought about a significant improvement and surface modification of the treated cotton substrates.     

Enhanced cellulase production by <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> by cultivation on glycerol followed by induction on cellulosic substrates

The use of glycerol obtained as an intermediate of the biodiesel manufacturing process as carbon source for microbial growth is a potential alternative strategy for the production of enzymes and other high-value bioproducts. This work evaluates the production of cellulase enzymes using glycerol for high cell density growth of Trichoderma harzianum followed by induction with a cellulosic material. Firstly, the influence of the carbon source used in the pre-culture step was investigated in terms of total protein secretion and fungal morphology. Enzymatic productivity was then determined for cultivation strategies using different types and concentrations of carbon source, as well as different feeding procedures (batch and fed-batch). The best strategy for cellulase production was then further studied on a larger scale using a stirred tank bioreactor. The proposed strategy for cellulase production, using glycerol to achieve high cell density growth followed by induction with pretreated sugarcane bagasse, achieved enzymatic activities up to 2.27 ± 0.37 FPU/mL, 106.40 ± 8.87 IU/mL, and 9.04 ± 0.39 IU/mL of cellulase, xylanase, and β-glucosidase, respectively. These values were 2 times higher when compared to the control experiments using glucose instead of glycerol. This novel strategy proved to be a promising approach for improving cellulolytic enzymes production, and could potentially contribute to adding value to biomass within the biofuels sector.     

Concomitant production of cellulase and xylanase by thermophilic mould <Emphasis Type="Italic">Sporotrichum thermophile</Emphasis> in solid state fermentation and their applicability in bread making

Sporotrichum thermophile BJAMDU5 secreted high titres of xylanolytic and cellulolytic enzymes in solid state fermentation using mixture of wheat straw and cotton oil cake (ratio 1:1) at 45?°C, pH 5.0 after 72 h inoculated with 2.9?×?10⁷ CFU/mL conidiospores. Supplementation of solid medium with lactose and ammonium sulphate further enhanced the production of hydrolytic enzymes. Among different surfactants studied, Tween 80 enhanced the production of all enzymes [3455 U/g DMR (dry mouldy residue), 879.26 U/g DMR, 976.28 U/g DMR and 35.10 U/g DMR for xylanase, CMCase (Carboxymethylcellulase), FPase (Filter paper activity) and β-glucosidase, respectively] as compared to other surfactants. Recycling of solid substrate reduced the production of all these enzymes after second cycle. End products analysis by TLC showed the ability of hydrolytic enzymes of S. thermophile to liberate monomeric (xylose and glucose) as well as oligomeric (xylobiose, cellobiose and higher ones) sugars. Supplementation of enzyme resulted in improved nutritional properties of the bread. Formation of oligomeric sugars by xylanase enzyme of S. thermophile BJAMDU5 make it a good candidate in food industry.     

Production of cellulases and xylanase by Aspergillus fumigatus SK1 using untreated oil palm trunk through solid state fermentation

Direct utilization of untreated oil palm trunk (OPT) for cellulases and xylanase production by Aspergillus fumigatus SK1 was conducted under solid-state fermentation (SSF). The highest activities of extracellular cellulases and xylanases were produced at 80% moisture level, initial pH 5.0, 1 × 10⁸ spore/g (inoculum) with 125 μm of OPT as sole carbon source. The cellulases and xylanase activities obtained were 54.27, 3.36, 4.54 and 418.70 U/g substrates for endoglucanase (CMCase), exoglucanase (FPase), β-glucosidase and xylanase respectively. The crude cellulases and xylanase required acidic condition to retain their optimum activities (pH 4.0). Crude cellulases and xylanase were more stable at 40 °C compared to their optimum activities conditions (60 °C for FPase and 70 °C for CMCase, β-glucosidase and xylanase). SDS-PAGE and zymogram analysis showed that Aspergillus fumigatus SK1 could secrete cellulases (endoglucanase, exoglucanase and β-glucosidase), xylanase and protease. Enzymatic degradation of alkaline treated OPT with concentrated crude cellulases and xylanases resulted in producing polyoses.     

Cost-effective production of cellulose hydrolysing enzymes from <Emphasis Type="Italic">Trichoderma</Emphasis> sp. RCK65 under SSF and its evaluation in saccharification of cellulosic substrates

Trichoderma sp. is a potential cellulase producing mesophilic fungi which grow under mild acidic condition. In this study, growth and nutritional conditions were manipulated for the maximum and cost-effective production of cellulase using lab strain Trichoderma sp. RCK65 and checked for its efficiency in hydrolysis of Prosopis juliflora (a woody substrate). Preliminary studies suggested that when 48 h old secondary fungal culture (20 % v/w) was inoculated in wheat bran moistened with mineral salt solution (pH 4.5 and 1:3 solid to moisture ratio), incubated at 30 °C and after 72 h, it produced maximum cellulase (CMCase 145 U/gds, FPase 38 U/gds and β-glucosidase 105 U/gds). However, using statistical approach a S:L ratio (1:1) was surprisingly found to be optimum that improved cellulase that is CMCase activity by 6.21 %, FPase activity by 23.68 % and β-glucosidase activity by 37.28 %. The estimated cost of crude enzyme (Rs. 5.311/1000 FPase units) seems to be economically feasible which may be due to high enzyme titre, less cultivation time and low media cost. Moreover, when the crude enzyme was used to saccharify pretreated Prosopis juliflora (a woody substrate), it resulted up to 83 % (w/w) saccharification.     

Enzymatic saccharification of sugar cane bagasse by continuous xylanase and cellulase production from cellulomonas flavigena PR‐22

Cellulase (CMCase) and xylanase enzyme production and saccharification of sugar cane bagasse were coupled into two stages and named enzyme production and sugar cane bagasse saccharification. The performance of Cellulomonas flavigena (Cf) PR‐22 cultured in a bubble column reactor (BCR) was compared to that in a stirred tank reactor (STR). Cells cultured in the BCR presented higher yields and productivity of both CMCase and xylanase activities than those grown in the STR configuration. A continuous culture with Cf PR‐22 was run in the BCR using 1% alkali‐pretreated sugar cane bagasse and mineral media, at dilution rates ranging from 0.04 to 0.22 1/h. The highest enzymatic productivity values were found at 0.08 1/h with 1846.4 ± 126.4 and 101.6 ± 5.6 U/L·h for xylanase and CMCase, respectively. Effluent from the BCR in steady state was transferred to an enzymatic reactor operated in fed‐batch mode with an initial load of 75 g of pretreated sugar cane bagasse; saccharification was then performed in an STR at 55°C and 300 rpm for 90 h. The constant addition of fresh enzyme as well as the increase in time of contact with the substrate increased the total soluble sugar concentration 83% compared to the value obtained in a batch enzymatic reactor. This advantageous strategy may be used for industrial enzyme pretreatment and saccharification of lignocellulosic wastes to be used in bioethanol and chemicals production from lignocellulose. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:321–326, 2016     

Improvement of β-glucosidase production by co-culture of Aspergillus niger and A. oryzae under solid state fermentation through feeding process

Eleven different Aspergillus strains were evaluated for their ability to produce β-glucosidase using sugar cane bagasse as a sole carbon source under solid state fermentation (SSF). The most potent strains, A. niger NRC 7 (674.6 U/g ds) and A. oryzae NRRL 447 (83 U/g ds), were used in a mixed culture to enhance β-glucosidase production by co-culturing under SSF. In mixed culture, β-glucosidase of the two strains (814 U/g ds) was nearly 1.2- and 9.8-fold than that of monocultures of A. niger NRC 7A and A. oryzae NRRL 447, respectively. Optimization of the culture parameters, initial pH value, moisture content, inoculum size and ratios of the two strains. and incubation time exhibited a significant increase in β-glucosidase production (1,893 U/g ds) than before optimization. Single feeding with citrate-phosphate buffer, succinate buffer, casein. and soybean flour individually after the third day of the fermentation time and controlling the moisture content at 90 % (w/w) induced β-glucosidase production. Maximum enzyme production increased up to 2.1-fold compared to 2,188 U/g ds during normal batch culture. Among nitrogen sources, soybean flour gave the highest β-glucosidase (4,578 U/g ds). while urea reduced β-glucosidase production (1,693 U/g ds). However, the combination of buffers with soybean flour through two fed cycles resulted in a decrease of the enzyme than single fed with buffers or soybean flour alone.     

Production of thermostable hydrolases (cellulases and xylanase) from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> RCKK: a potential fungus

Thermophilic fungi are potential sources of thermostable enzymes and other value added products. Present study has focused on optimization of different physicochemical parameters for production of thermostable cellulases and xylanase by Thermoascus aurantiacus RCKK under SSF. Enzyme production was supported maximally on wheat bran fed with 20 % inoculum, at initial pH 5, temperature 45 °C and moisture ratio 1:3. The supplementation of wheat bran with yeast extract, Tween-80 and glycine further improved enzyme titres (CMCase 88 IU/g, FPase 15.8 IU/g, β-glucosidase 25.3 IU/g and xylanase 6,543 IU/g). The crude enzymes hydrolyzed phosphoric acid-swollen wheat straw, avicel and untreated xylan up to 74, 71 and 90 %, respectively. In addition, T. aurantiacus RCKK produced antioxidants as fermentation by-products with significant %DPPH^? scavenging, FRAP and in vivo antioxidant capacity against H₂O₂-treated Saccharomyces cerevisiae. These capabilities show that it holds potential to exploit crop by-products for providing various commodities.     

Optimization of cellulase-free xylanase production by alkalophilic Cellulosimicrobium sp. CKMX1 in solid-state fermentation of apple pomace using central composite design and response surface methodology

Microbial xylanases and associated enzymes degrade the xylans present in lignocellulose in nature. Xylanase production by Cellulosimicrobium sp. CKMX1, isolated from mushroom compost, produced a cellulase-free extracellular endo-1, 4-β-xylanase (EC 3.2.1.8) at 35 °C and pH 8.0. Apple pomace—an inexpensive and abundant source of carbon—supported maximal xylanase activity of 500.10 U/g dry bacterial pomace (DBP) under solid state fermentation. Culture conditions, e.g., type of medium, particle size of carbon source, incubation period, temperature, initial pH, and inoculum size, were optimized and xylanase activity was increased to 535.6 U/g DBP. CMCase, avicelase, FPase and β-glucosidase activities were not detected, highlighting the novelty of the xylanase enzyme produced by CKMX1. Further optimization of enzyme production was carried out using central composite design following response surface methodology with four independent variables (yeast extract, urea, Tween 20 and carboxymethyl cellulose), which resulted in very high levels of xylanase (861.90 U/g DBP). Preliminary identification of the bacterial isolate was made on the basis of morphological and biochemical characters and confirmed by partial 16Sr RNA gene sequencing, which identified CKMX1 as Cellulosimicrobium sp. CKMX1. A phylogenetic analysis based on the 16Sr RNA gene sequence placed the isolate within the genus Cellulosimicrobium, being related most closely to Cellulosimicrobium cellulans strain AMP-11 (97% similarity). The ability of this strain to produce cost-effective xylanase from apple pomace on a large scale will help in the waste management of apple pomace.     

Integrated Strategies to Enhance Cellulolytic Enzyme Production Using an Instrumented Bioreactor for Solid-State Fermentation of Sugarcane Bagasse

The conversion of agro-industrial residues, such as sugarcane bagasse, into high-value products and renewable energy, within the biorefinery concept, is a potential alternative towards the sustainable management of these resources. This work evaluates the production of cellulolytic enzymes by a selected strain of Aspergillus niger cultivated in sugarcane bagasse under solid-state fermentation using an instrumented lab-scale bioreactor. The effects of environmental factors including the type of substrate and medium composition, as well as the operational conditions (air flow rate, inlet air relative humidity, and initial substrate moisture content) on the production of the enzymatic complex were evaluated using statistical design tools. Significant increases in FPase, endoglucanase, and xylanase activities were achieved under the optimized conditions predicted by the models, with values of 0.88, 21.77, and 143.85 IU/g of dry solid substrate, respectively, representing around ten-, four-, and twofold increases compared to the activities obtained under the initial growth conditions. This demonstrates the importance of evaluating environmental and operational criteria in order to achieve efficient enzyme production. The crude enzymatic extract obtained under optimized conditions was employed for enzymatic hydrolysis of pretreated sugarcane bagasse. Approximately 13 % of total reducing sugars, and a glucose concentration of 2.54 g/L, were obtained after 22 h of hydrolysis of steam exploded sugarcane bagasse, indicating that the enzymatic cocktail produced has good potential for use in the conversion of biomass.     

Characterization of commercial cellulases and their use in the saccharification of a sugarcane bagasse sample pretreated with dilute sulfuric acid

This study aimed to correlate the efficiency of enzymatic hydrolysis of the cellulose contained in a sugarcane bagasse sample pretreated with dilute H₂SO₄ with the levels of independent variables such as initial content of solids and loadings of enzymes and surfactant (Tween 20), for two cellulolytic commercial preparations. The preparations, designated cellulase I and cellulase II, were characterized regarding the activities of total cellulases, endoglucanase, cellobiohydrolase, cellobiase, β-glucosidase, xylanase, and phenoloxidases (laccase, manganese and lignin peroxidases), as well as protein contents. Both extracts showed complete cellulolytic complexes and considerable activities of xylanases, without activities of phenoloxidases. For the enzymatic hydrolyses, two 2³ central composite full factorial designs were employed to evaluate the effects caused by the initial content of solids (1.19–4.81%, w/w) and loadings of enzymes (1.9–38.1 FPU/g bagasse) and Tween 20 (0.0–0.1 g/g bagasse) on the cellulose digestibility. Within 24 h of enzymatic hydrolysis, all three independent variables influenced the conversion of cellulose by cellulase I. Using cellulase II, only enzyme and surfactant loadings showed significant effects on cellulose conversion. An additional experiment demonstrated the possibility of increasing the initial content of solids to values much higher than 4.81% (w/w) without compromising the efficiency of cellulose conversion, consequently improving the glucose concentration in the hydrolysate.     

Enhanced production of cellulase from a novel strain Trichoderma gamsii M501 through response surface methodology and its application in biomass saccharification

Cellulolytic enzymes produced by Trichoderma sp. have attracted interest in converting the biomass to simple sugars in the production of cellulosic ethanol. In this work, a novel cellulolytic strain M501 was isolated and identified as T. gamsii by sequencing the ITS rDNA region. The production of cellulase (CMCase) by T. gamsii M501 was enhanced by employing statistical methods. The strain grown in the optimized production medium composed of mineral salts, microcrystalline cellulose (13.7 g/l), tryptone (4.8 g/l) and trace elements (2 mL/l) at pH 5.5 and 28 °C for 72 h produced a maximum CMCase of 61.3 U/mL. The optimized production medium also showed the other enzyme activity of FPU (2.6 U/mL), β-glucosidase (2.1 U/mL), xylanase (681 U/mL) and β- xylosidase (0.6 U/mL). The crude cellulase cocktail produced by T. gamsii M501 efficiently hydrolyzed alkali pretreated sugarcane bagasse with glucose and xylose yield of 78 % and 74 % respectively at 10 % solid loading. This study is the first of its kind research on biomass saccharification using T. gamsii cellulase cocktail. Therefore, the novel strain T. gamsii M501 would be useful for further development of an enzyme cocktail for cellulosic ethanol production.     

Ethanol production from sunflower meal biomass by simultaneous saccharification and fermentation (SSF) with Kluyveromyces marxianus ATCC 36907

The lignocellulosic materials are considered promising renewable resources for ethanol production, but improvements in the processes should be studied to reduce operating costs. Thus, the appropriate enzyme loading for cellulose saccharification is critical for process economics. This study aimed at evaluating the concentration of cellulase and β-glucosidase in the production of bioethanol by simultaneous saccharification and fermentation (SSF) of sunflower meal biomass. The sunflower biomass was pretreated with 6 % H₂SO₄ (w/v), at 121 °C, for 20 min, for hemicellulose removal and delignificated with 1 % NaOH. SSF was performed with Kluyveromyces marxianus ATCC 36907, at 38 °C, 150 rpm, for 72 h, with different enzyme concentrations (Cellulase Complex NS22086-10, 15 and 20 FPU/g_substrate and β-Glucosidase NS22118, with a cellulase to β-glucosidase ratio of 1.5:1; 2:1 and 3:1). The best condition for ethanol production was cellulase 20 FPU/g_substrate and β-glucosidase 13.3 CBU/g_substrate, resulting in 27.88 g/L ethanol, yield of 0.47 g/g and productivity of 0.38 g/L h. Under this condition the highest enzymatic conversion of cellulose to glucose was attained (87.06 %).     

Impact of protein blocking on enzymatic saccharification of bagasse from sugarcane clones

Lignin plays an important functional and structural role in plants, but also contributes to the recalcitrance of lignocellulosic biomass to hydrolysis. This study addresses the influence of lignin in hydrolysis of sugarcane bagasse from conventional bred lines (UFV260 and UFV204) that were selected from 432 field-grown clones. In addition to higher sugar production, bagasse clone UFV204 had a small, but statistically significant, lower insoluble lignin content compared with clone UFV260 (15.5% vs, 16.6%) and also exhibited a significantly higher cellulose conversion to glucose (81.3% vs. 63.3%) at a cellulase loading of 5 (filter paper unit) FPU/g of glucan or 3 FPU/g total solids for liquid hot water pretreated bagasse (200°C, 10 min). The enzyme loading was further decreased by 50% to 2.5 FPU/g glucan and resulted in a similar glucan conversion (88.5%) for clone UFV204 when the bagasse was preincubated with bovine serum albumin at pH 4.8 and nonproductive binding of cellulase components was blocked. Comparison of Langmuir adsorption isotherms and differential adsorption of the three major cellulolytic enzyme components endoglucanase, cellobiohydrolase, and β-glucosidase help to explain differences due to lignin content.     

Production of cellulase and xylanase by a wild strain of Trichoderma viride

Summary The production of cellulase and xylanase was investigated with a newly isolated strain of Trichoderma viride BT 2169. The medium composition was optimized on a shake-flask scale using the Graeco-Latin square technique. The temperature and time for optimal growth and production of the enzymes in shake cultures were optimized using a central composite design. The temperature optima for maximal production of filter paper cellulase (FPase), xylanase and -gluosidase were 32.8°, 34.7° and 31.1° C, respectively, and the optimum times for production of these enzymes were found to be 144, 158 and 170 h, respectively. The optimized culture medium and conditions (33° C) gave 0.55 unit of FPase, 188.1 units of xylanase and 3.37 units of -glucosidase per milliliter of culture filtrate at 144 h of shake culture. Among different carbon sources tested, the maximum enzyme activities were produced with sulphite pulp and all three enzymes were produced irrespective of the carbon sources used. Batch fermentation in a laboratory fermentor using 2% sulphite pulp allowed the production of 0.61 unit of FPase, 145.0 units of xylanase and 2.72 units of -glucosidase. In a fed-batch fermentation on 6% final Avicel concentration FPase and -glucosidase were 3.0 and 2.4 times higher respectively than those in batch fermentation on 2% Avicel. The pH and temperature optima as well as pH and temperature stabilities of T. viride enzymes were found to be comparable to T. reesei and some other fungal enzymes.     

Enhanced Bioethanol Production from Blue Agave Bagasse in a Combined Extrusion–Saccharification Process

This paper describes an improved process for bioethanol production using a recently developed combined extrusion–saccharification technology. Blue agave bagasse (BAB) was pretreated via a thermo-mechano-chemical process (co-rotational twin-screw, reactive extrusion) to increase the availability of cellulose and hemicellulose for enzymatic saccharification. Then, several commercial enzyme preparations, boosted with accessory enzymes (exoglucanase, endoglucanase, hemicellulase, xylanase, and β-glucosidase), were tested with extruded BAB at 5 % consistency in a stirred vessel. The enzyme blend that produced the highest saccharification yield was evaluated at different BAB consistencies. The obtained concentration of sugars increased up to 69.5 g/L (73 % yield) when a 20 % BAB mixture was used. When the enzyme blend was fed into the extruder and with a residence time of 2 min, the yield reached 15 % of the maximum theoretical of C6 sugars along this step. This extruded and pre-saccharified BAB was further hydrolyzed and used for fermentation. The pre-saccharification step significantly enhanced cellulose degradation and ethanol production. Our results indicate that the enzymatic saccharification of BAB, coupled with reactive extrusion, produces an excellent substrate for bioethanol production.     

Fungi isolated from olive ecosystems and screening of their potential biotechnological use

This study investigated the fungi diversity of fresh olive (Olea europaea L.) fruits, olive paste (crushed olives) and olive pomace (solid waste) and screened and quantified enzymatic activities with biotechnological applications. Fungi were randomly isolated from olive cultivars from Castilla La Mancha region (Spain). Identification included comparison of their polymerase chain reaction (PCR) amplicons of the ITS1-5.8S-ITS2 ribosomal DNA region, followed by nucleotide sequence analysis. Fourteen different species with DNA sequences of different similarities were identified, belonging to seven different genera (Aspergillus, Penicillium, Rhizomucor, Mucor, Rhizopus, Lichtheimia and Galactomyces). Aspergillus fumigatus, followed by Galactomyces geotrichum, Penicillium commune and Rhizomucor variabilis var. regularior were the most frequent species. Specific enzyme screening was assayed on agar plates, using cellobiose, carboxymethylcellulose (CMC), polygalacturonic acid and CaCl(2)/Tween 80 as substrates for β-glucosidase, carboxymethylcellulase (CMCase), polygalacturonase and lipase, respectively. Species exhibiting the best activities were: Aspergillus fumigatus (for β-glucosidase, CMCase and lipase); Rhizopus oryzae (for β-glucosidase and lipase); Rhizomucor variabilis (for β-glucosidase, CMCase and polygalacturonase); Mucor fragilis (β-glucosidase, CMCase and lipase); Galactomyces geotrichum (for β-glucosidase, polygalacturonase and lipase) and Penicillium commune and Penicillium crustosum (for lipase). The species that had shown the best enzymatic activities were grown on hemicellulose, cellulose and pectin and some activities were quantified (xylanase, cellulase, β-glucosidase and pectinase). An isolate of A. fumigatus and one of A. niger showed the best cellulase and xylanase activities, while no species presented good pectinase and β-glucosidase activities. The selected species with potential enzymatic activities could be used for future applications of industrial interest.