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1.  Production of cellulose by Aspergillus niger under submerged and solid state fermentation using coir waste as a substrate 
Brazilian Journal of Microbiology  2011;42(3):1119-1127.
Aspergillus niger was used for cellulase production in submerged (SmF) and solid state fermentation (SSF). The maximum production of cellulase was obtained after 72 h of incubation in SSF and 96 h in Smf. The CMCase and FPase activities recorded in SSF were 8.89 and 3.56 U per g of dry mycelial bran (DBM), respectively. Where as in Smf the CMase & FPase activities were found to be 3.29 and 2.3 U per ml culture broth, respectively. The productivity of extracellular cellulase in SSF was 14.6 fold higher than in SmF. The physical and nutritional parameters of fermentation like pH, temperature, substrate, carbon and nitrogen sources were optimized. The optimal conditions for maximum biosynthesis of cellulase by A. niger were shown to be at pH 6, temperature 30 °C. The additives like lactose, peptone and coir waste as substrate increased the productivity both in SmF and SSF. The moisture ratio of 1:2 (w/v) was observed for optimum production of cellulase in SSF.
PMCID: PMC3768773  PMID: 24031730
Aspergillus niger; coir waste; cellulase; submerged fermentation; solid-state fermentation
2.  Production of Biomass-Degrading Multienzyme Complexes under Solid-State Fermentation of Soybean Meal Using a Bioreactor 
Enzyme Research  2012;2012:248983.
Biomass-degrading enzymes are one of the most costly inputs affecting the economic viability of the biochemical route for biomass conversion into biofuels. This work evaluates the effects of operational conditions on biomass-degrading multienzyme production by a selected strain of Aspergillus niger. The fungus was cultivated under solid-state fermentation (SSF) of soybean meal, using an instrumented lab-scale bioreactor equipped with an on-line automated monitoring and control system. The effects of air flow rate, inlet air relative humidity, and initial substrate moisture content on multienzyme (FPase, endoglucanase, and xylanase) production were evaluated using a statistical design methodology. Highest production of FPase (0.55 IU/g), endoglucanase (35.1 IU/g), and xylanase (47.7 IU/g) was achieved using an initial substrate moisture content of 84%, an inlet air humidity of 70%, and a flow rate of 24 mL/min. The enzymatic complex was then used to hydrolyze a lignocellulosic biomass, releasing 4.4 g/L of glucose after 36 hours of saccharification of 50 g/L pretreated sugar cane bagasse. These results demonstrate the potential application of enzymes produced under SSF, thus contributing to generate the necessary technological advances to increase the efficiency of the use of biomass as a renewable energy source.
PMCID: PMC3544265  PMID: 23365723
3.  Characterization of Cellulolytic Bacterial Cultures Grown in Different Substrates 
The Scientific World Journal  2013;2013:689235.
Nine aerobic cellulolytic bacterial cultures were obtained from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture (DSMZ) and the American Type Culture Collection (ATCC). The objectives of this study were to characterize the cellulolytic bacteria and to determine the optimum moisture ratio required for solid state fermentation (SSF) of palm kernel cake (PKC). The bacteria cultures were grown on reconstituted nutrient broth, incubated at 30°C and agitated at 200 rpm. Carboxymethyl cellulase, xylanase, and mannanase activities were determined using different substrates and after SSF of PKC. The SSF was conducted for 4 and 7 days with inoculum size of 10% (v/w) on different PKC concentration-to-moisture ratios: 1 : 0.2, 1 : 0.3, 1 : 0.4, and 1 : 0.5. Results showed that Bacillus amyloliquefaciens 1067 DSMZ, Bacillus megaterium 9885 ATCC, Paenibacillus curdlanolyticus 10248 DSMZ, and Paenibacillus polymyxa 842 ATCC produced higher enzyme activities as compared to other bacterial cultures grown on different substrates. The cultures mentioned above also produced higher enzyme activities when they were incubated under SSF using PKC as a substrate in different PKC-to-moisture ratios after 4 days of incubation, indicating that these cellulolytic bacteria can be used to degrade and improve the nutrient quality of PKC.
PMCID: PMC3844246  PMID: 24319380
4.  Correlation between Agar Plate Screening and Solid-State Fermentation for the Prediction of Cellulase Production by Trichoderma Strains 
Enzyme Research  2012;2012:793708.
The viability of converting biomass into biofuels and chemicals still requires further development towards the reduction of the enzyme production costs. Thus, there is a growing demand for the development of efficient procedures for selection of cellulase-producing microorganisms. This work correlates qualitative screening using agar plate assays with quantitative measurements of cellulase production during cultivation under solid-state fermentation (SSF). The initial screening step consisted of observation of the growth of 78 preselected strains of the genus Trichoderma on plates, using microcrystalline cellulose as carbon source. The 49 strains that were able to grow on this substrate were then subjected to a second screening step using the Congo red test. From this test it was possible to select 10 strains that presented the highest enzymatic indices (EI), with values ranging from 1.51 to 1.90. SSF cultivations using sugarcane bagasse and wheat bran as substrates were performed using selected strains. The CG 104NH strain presented the highest EGase activity (25.93 UI·g−1). The EI results obtained in the screening procedure using plates were compared with cellulase production under SSF. A correlation coefficient (R2) of 0.977 was obtained between the Congo red test and SSF, demonstrating that the two methodologies were in good agreement.
PMCID: PMC3514834  PMID: 23227312
5.  Cellulolytic enzymes on lignocellulosic substrates in solid state fermentation by Aspergillus niger 
Indian Journal of Microbiology  2008;47(4):323-328.
The production of cellulolytic enzymes by Aspergillus niger on lignocellulosic substrates groundnut fodder, wheat bran, rice bran and sawdust in solid state fermentation in a laboratory scale was compared. Czapek Dox liquid broth amended with cellulose (0.5%) was used to moisten lignocellulosic solid supports for cultivation of Aspergillus niger. The production of filter paperase, carboxymethyl cellulase and -glucosidase were monitored at daily intervals for 5 days. The peak production of the enzymes occurred within 3 days of incubation. Among solid supports used in the study, wheat bran was the best solid matrix followed by groundnut fodder in production of cellulolytic enzymes in solid state fermentation. Groundnut fodder supported significant production of FPase (2.09 FPU/g), CMCase (1.36 U/g) and -glucosidase activity (0.0117 U/g) in solid state fermentation. Considerable secretion of protein (5.10 mg/g) on groundnut fodder at peak time interval 1st day of incubation was recorded.
PMCID: PMC3450032  PMID: 23100685
Cellulolytic enzymes; Lignocellulosic substrates; Wheat bran; Groundnut fodder; Rice bran; Sawdust; Solid state fermentation; Aspergillus niger
6.  Filamentous fungi and media for cellulase production in solid state cultures 
Brazilian Journal of Microbiology  2014;45(1):279-286.
Cellulase production was evaluated in two reference strains (T. reesei Rut-C30 and T. reesei QM9414), two strains isolated from a sugarcane cultivation area (Trichoderma sp. IPT778 and T. harzianum rifai IPT821) and one strain isolated in a program for biodiversity preservation in São Paulo state (Myceliophthora thermophila M77). Solid state cultures were performed using sugarcane bagasse (C), wheat bran (W) and/or soybean bran (S). The highest FPA was 10.6 U/gdm for M77 in SC (10:90) at 80% moisture, which was 4.4 times higher than production in pure W. C was a strong inducer of cellulase production, given that the production level of 6.1 U/gdm in WC (40:60) was 2.5 times higher than in pure W for strain M77; T. reesei Rut-C30 did not respond as strongly with about 1.6-fold surplus production. S advantageously replaced W, as the surplus production on SC (20:80) was 2.3 times relative to WC (20:80) for M77.
PMCID: PMC4059312  PMID: 24948946
cellulase; productivity; solid media; sugarcane bagasse; Myceliophtora sp
7.  High-Yield Endoglucanase Production by Trichoderma harzianum IOC-3844 Cultivated in Pretreated Sugarcane Mill Byproduct 
Enzyme Research  2010;2010:854526.
The low-cost production of cellulolytic complexes presenting high action at mild conditions and well-balanced cellulase activities is one of the major bottlenecks for the economical viability of the production of cellulosic ethanol. In the present paper, the filamentous fungus Trichoderma harzianum IOC-3844 was used for the production of cellulases from a pretreated sugarcane bagasse (namely, cellulignin), by submerged fermentation. This fungal strain produced high contents of endoglucanase activity (6,358 U·L−1) after 72 hours of process, and further relevant β-glucosidase and FPase activities (742 and 445 U·L−1, resp.). The crude enzyme extract demonstrated appropriate characteristics for its application in cellulose hydrolysis, such as high thermal stability at up to 50°C, accessory xylanase activity, and absence of proteolytic activity towards azocasein. This strain showed, therefore, potential for the production of complete cellulolytic complexes aiming at the saccharification of lignocellulosic materials.
PMCID: PMC2962913  PMID: 21048871
8.  A biodegradation study of forest biomass by Aspergillus niger F7: correlation between enzymatic activity, hydrolytic percentage and biodegradation index 
Brazilian Journal of Microbiology  2012;43(2):467-475.
Aspergillus niger F7 isolated from soil was found to be the potent producer of cellulase and xylanase. The residue of forest species Toona ciliata, Celtris australis, Cedrus deodara and Pinus roxburghii was selected as substrate for biodegradation study due to its easy availability and wide use in industry. It was subjected to alkali (sodium hydroxide) treatment for enhancing its degradation. Biodegradation of forest waste by hydrolytic enzymes (cellulase and xylanase) secreted by A. niger under solid state fermentation (SSF) was explored. SSF of pretreated forest biomass was found to be superior over untreated forest biomass. Highest extracellular enzyme activity of 2201±23.91 U/g by A. niger was shown in pretreated C. australis wood resulting in 6.72±0.20 percent hydrolysis and 6.99±0.23 biodegradation index (BI). The lowest BI of 1.40±0.08 was observed in untreated saw dust of C. deodara having the least enzyme activity of 238±1.36 U/g of dry matter. Biodegradation of forest biomass under SSF was increased many folds when moistening agent i.e. tap water had been replaced with modified basal salt media (BSM). In BSM mediated degradation of forest waste with A. niger, extracellular enzyme activity was increased up to 4089±67.11 U/g of dry matter in turn resulting in higher BI of 15.4±0.41 and percent hydrolysis of 19.38±0.81 in pretreated C. australis wood. A. niger exhibited higher enzyme activity on pretreated biomass when moistened with modified BSM in this study. Statistically a positive correlation has been drawn between these three factors i.e. enzyme activity, BI and percent hydrolysis of forest biomass thus proving their direct relationship with each other.
PMCID: PMC3768808  PMID: 24031853
Aspergillus niger; SSF; enzyme activity; percent hydrolysis; biodegradation index
9.  Optimization for the Production of Cellulase Enzyme from Municipal Solid Waste Residue by Two Novel Cellulolytic Fungi 
The main purpose of this study is to reduce the production cost of cellulase by optimizing the production medium and using an alternative carbon source such as municipal solid waste residue. In the present investigation, we aim to isolate the two novel cellulase producing fungi (Aspergillus niger and Trichoderma sp.) from municipal solid waste. Municipal solid waste residue (4-5% (w/v)) and peptone and yeast extract (1.0% (w/v)) were found to be the best combination of carbon and nitrogen sources for the production of cellulase by A. niger and Trichoderma sp. Optimum temperature and pH of the medium for the cellulase production by A. niger were 40°C and 6-7, whereas those for the production of cellulase by Trichoderma sp. were 45°C and 6.5. Cellulase production from A. niger and Trichoderma sp. can be an advantage as the enzyme production rate is normally higher as compared to other fungi.
PMCID: PMC3042683  PMID: 21350668
10.  Canola Cake as a Potential Substrate for Proteolytic Enzymes Production by a Selected Strain of Aspergillus oryzae: Selection of Process Conditions and Product Characterization 
ISRN Microbiology  2013;2013:369082.
Oil cakes have excellent nutritional value and offer considerable potential for use in biotechnological processes that employ solid-state fermentation (SSF) for the production of high value products. This work evaluates the feasibility of using canola cake as a substrate for protease production by a selected strain of Aspergillus oryzae cultivated under SSF. The influences of the following process parameters were considered: initial substrate moisture content, incubation temperature, inoculum size, and pH of the buffer used for protease extraction and activity analysis. Maximum protease activity was obtained after cultivating Aspergillus oryzae CCBP 001 at 20°C, using an inoculum size of 107 spores/g in canola cake medium moistened with 40 mL of water to 100 g of cake. Cultivation and extraction under selected conditions increased protease activity 5.8-fold, compared to the initial conditions. Zymogram analysis of the enzymatic extract showed that the protease molecular weights varied between 31 and 200 kDa. The concentrated protease extract induced clotting of casein in 5 min. The results demonstrate the potential application of canola cake for protease production under SSF and contribute to the technological advances needed to increase the efficiency of processes designed to add value to agroindustrial wastes.
PMCID: PMC3886222  PMID: 24455400
11.  Solid-State Fermentation with Trichoderma reesei for Cellulase Production 
Cellulase yields of 250 to 430 IU/g of cellulose were recorded in a new approach to solid-state fermentation of wheat straw with Trichoderma reesei QMY-1. This is an increase of ca. 72% compared with the yields (160 to 250 IU/g of cellulose) in liquid-state fermentation reported in the literature. High cellulase activity (16 to 17 IU/ml) per unit volume of enzyme broth and high yields of cellulases were attributed to the growth of T. reesei on a hemicellulose fraction during its first phase and then on a cellulose fraction of wheat straw during its later phase for cellulase production, as well as to the close contact of hyphae with the substrate in solid-state fermentation. The cellulase system obtained by the solid-state fermentation of wheat straw contained cellulases (17.2 IU/ml), β-glucosidase (21.2 IU/ml), and xylanases (540 IU/ml). This cellulase system was capable of hydrolyzing 78 to 90% of delignified wheat straw (10% concentration) in 96 h, without the addition of complementary enzymes, β-glucosidase, and xylanases.
PMCID: PMC238371  PMID: 16346697
12.  A solid state fungal fermentation-based strategy for the hydrolysis of wheat straw☆ 
Bioresource Technology  2013;149(100):261-267.
•A solid state fungal fermentation strategy converting wheat straw to hydrolysate.•A biological pre-treatment of wheat straw by culturing A. niger on wheat straw.•24.0 ± 1.76 U/g cellulase was produced using wheat straw as the main substrate.•The fungal extract was used to hydrolyze the fermented wheat straw.•19% higher hydrolysis efficiency using freshly-prepared fungal extract than Ctec2.
This paper reports a solid-state fungal fermentation-based pre-treatment strategy to convert wheat straw into a fermentable hydrolysate. Aspergillus niger was firstly cultured on wheat straw for production of cellulolytic enzymes and then the wheat straw was hydrolyzed by the enzyme solution into a fermentable hydrolysate. The optimum moisture content and three wheat straw modification methods were explored to improve cellulase production. At a moisture content of 89.5%, 10.2 ± 0.13 U/g cellulase activity was obtained using dilute acid modified wheat straw. The addition of yeast extract (0.5% w/v) and minerals significantly improved the cellulase production, to 24.0 ± 1.76 U/g. The hydrolysis of the fermented wheat straw using the fungal culture filtrate or commercial cellulase Ctec2 was performed, resulting in 4.34 and 3.13 g/L glucose respectively. It indicated that the fungal filtrate harvested from the fungal fermentation of wheat straw contained a more suitable enzyme mixture than the commercial cellulase.
PMCID: PMC3824065  PMID: 24121367
Solid state fermentation (SSF); Wheat straw; Aspergillus niger; Cellulase; Hydrolysis
13.  Simultaneous Cellulase Production, Saccharification and Detoxification Using Dilute Acid Hydrolysate of S. spontaneum with Trichoderma reesei NCIM 992 and Aspergillus niger 
Indian Journal of Microbiology  2011;52(2):258-262.
Bioethanol production from lignocellulosic materials has several limitations. One aspect is the high production cost of cellulases used for saccharification of substrate and inhibition of fermenting yeast due to inhibitors released in acid hydrolysis. In the present work we have made an attempt to achieve simultaneous cellulases production, saccharification and detoxification using dilute acid hydrolysate of Saccharum spontaneum with and without addition of nutrients, supplemented with acid hydrolyzed biomass prior to inoculation in one set and after 3 days of inoculation in another set. Organisms used were T. reesei NCIM 992, and Aspergillus niger isolated in our laboratory. Cellulase yield obtained was 0.8 IU/ml on fourth day with T. reesei. Sugars were found to increase from fourth to fifth day, when hydrolysate was supplemented with nutrients and acid hydrolyzed biomass followed by inoculation with T. reesei. Phenolics were also found to decrease by 67%.
PMCID: PMC3386449  PMID: 23729891
Acid hydrolysate; Cellulase; Detoxification; Saccharum spontaneum; Saccharification
14.  Bioethanol production from rice straw residues 
Brazilian Journal of Microbiology  2013;44(1):225-234.
A rice straw - cellulose utilizing mold was isolated from rotted rice straw residues. The efficient rice straw degrading microorganism was identified as Trichoderma reesei. The results showed that different carbon sources in liquid culture such as rice straw, carboxymethyl cellulose, filter paper, sugar cane bagasse, cotton stalk and banana stalk induced T. reesei cellulase production whereas glucose or Potato Dextrose repressed the synthesis of cellulase. T. reesei cellulase was produced by the solid state culture on rice straw medium. The optimal pH and temperature for T. reesei cellulase production were 6 and 25 °C, respectively. Rice straw exhibited different susceptibilities towards cellulase to their conversion to reducing sugars. The present study showed also that, the general trend of rice straw bioconversion with cellulase was more than the general trend by T. reesei. This enzyme effectively led to enzymatic conversion of acid, alkali and ultrasonic pretreated cellulose from rice straw into glucose, followed by fermentation into ethanol. The combined method of acid pretreatment with ultrasound and subsequent enzyme treatment resulted the highest conversion of lignocellulose in rice straw to sugar and consequently, highest ethanol concentration after 7 days fermentation with S. cerevisae yeast. The ethanol yield in this study was about 10 and 11 g.L−1.
PMCID: PMC3804203  PMID: 24159309
cellulase; Trichoderma reesei; rice straw residues; enzymatic hydrolysis; bioethanol
15.  Microbial renewable feedstock utilization 
Bioengineered Bugs  2010;1(5):359-366.
Increasingly lignocellulosic biomass hydrolysates are used as the feedstock for industrial fermentations. These biomass hydrolysates consist of complex mixtures of different fermentable sugars, but also contain inhibitors and salts that affect the performance of the product-generating microbes. The performance of six industrially relevant microorganisms, i.e., two bacteria (Escherichia coli and Corynebacterium glutamicum), two yeasts (Saccharomyces cerevisiae and Pichia stipitis) and two fungi (Aspergillus niger and Trichoderma reesei) were compared for their ability to utilize and grow on different feedstock hydrolysates (corn stover, wheat straw, sugar cane bagasse and willow wood). Moreover, the ability of the selected hosts to utilize waste glycerol from the biodiesel industry was evaluated. P. stipitis and A. niger were found to be the most versatile and C. glutamicum, and S. cerevisiae were shown to be the least adapted to renewable feedstocks. Clear differences in the utilization of the more abundant carbon sources in these feedstocks were observed between the different species. Moreover, in a species-specific way the production of various metabolites, in particular polyols, alcohols and organic acids was observed during fermentation. Based on the results obtained we conclude that a substrate-oriented instead of the more commonly used product oriented approach towards the selection of a microbial production host will avoid the requirement for extensive metabolic engineering. Instead of introducing multiple substrate utilization and detoxification routes to efficiently utilize lignocellulosic hydrolysates only one biosynthesis route forming the product of interest has to be engineered.
PMCID: PMC3037588  PMID: 21326838
second generation feedstock; bioprocessing; filamentous fungi; industrial biotechnology
16.  Use of Laccase as a Novel, Versatile Reporter System in Filamentous Fungi 
Laccases are copper-containing enzymes which oxidize phenolic substrates and transfer the electrons to oxygen. Many filamentous fungi contain several laccase-encoding genes, but their biological roles are mostly not well understood. The main interest in laccases in biotechnology is their potential to be used to detoxify phenolic substances. We report here on a novel application of laccases as a reporter system in fungi. We purified a laccase enzyme from the ligno-cellulolytic ascomycete Stachybotrys chartarum. It oxidized the artificial substrate 2,2′-azino-di-(3-ethylbenzthiazolinsulfonate) (ABTS). The corresponding gene was isolated and expressed in Aspergillus nidulans, Aspergillus niger, and Trichoderma reesei. Heterologously expressed laccase activity was monitored in colorimetric enzyme assays and on agar plates with ABTS as a substrate. The use of laccase as a reporter was shown in a genetic screen for the isolation of improved T. reesei cellulase production strains. In addition to the laccase from S. charatarum, we tested the application of three laccases from A. nidulans (LccB, LccC, and LccD) as reporters. Whereas LccC oxidized ABTS (Km = 0.3 mM), LccD did not react with ABTS but with DMA/ADBP (3,5-dimethylaniline/4-amino-2,6-dibromophenol). LccB reacted with DMA/ADBP and showed weak activity with ABTS. The different catalytic properties of LccC and LccD allow simultaneous use of these two laccases as reporters in one fungal strain.
PMCID: PMC1489370  PMID: 16820501
17.  Phytase Production by Aspergillus niger CFR 335 and Aspergillus ficuum SGA 01 through Submerged and Solid-State Fermentation 
The Scientific World Journal  2014;2014:392615.
Fermentation is one of the industrially important processes for the development of microbial metabolites that has immense applications in various fields. This has prompted to employ fermentation as a major technique in the production of phytase from microbial source. In this study, a comparison was made between submerged (SmF) and solid-state fermentations (SSF) for the production of phytase from Aspergillus niger CFR 335 and Aspergillus ficuum SGA 01. It was found that both the fungi were capable of producing maximum phytase on 5th day of incubation in both submerged and solid-state fermentation media. Aspergillus niger CFR 335 and A. ficuum produced a maximum of 60.6 U/gds and 38 U/gds of the enzyme, respectively, in wheat bran solid substrate medium. Enhancement in the enzyme level (76 and 50.7 U/gds) was found when grown in a combined solid substrate medium comprising wheat bran, rice bran, and groundnut cake in the ratio of 2 : 1 : 1. A maximum of 9.6 and 8.2 U/mL of enzyme activity was observed in SmF by A. niger CFR 335 and A.ficuum, respectively, when grown in potato dextrose broth.
PMCID: PMC3928852  PMID: 24688383
18.  Strain improvement of Trichoderma reesei Rut C-30 for increased cellulase production 
Indian Journal of Microbiology  2009;49(2):188-195.
The strain of Trichoderma reesei Rut C-30 was subjected to mutation after treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NG) for 6 h followed by UV irradiation for 15 min. Successive mutants showed enhanced cellulase production, clear hydrolysis zone and rapid growth on Avicel-containing plate. Particularly, the mutant NU-6 showed approximately two-fold increases in activity of both FPA and CMCase in shake flask culture when grown on basal medium containing peptone (1%) and wheat bran (1%). The enzyme production was further optimized using eight different media. When a mixture of lactose and yeast cream was used as cellulase inducer, the mutant NU-6 yielded the highest enzyme and cell production with a FPase activity of 6.2 U ml−1, a CMCase activity of 54.2 U ml−1, a β-glucosidase activity of 0.39 U ml−1, and a fungal biomass of 12.6 mg ml−1. It deserved noting that the mutant NU-6 also secreted large amounts of xylanases (291.3 U ml−1). These results suggested that NU-6 should be an attractive producer for both cellulose and xylanase production.
PMCID: PMC3450144  PMID: 23100767
Cellulase; Trichoderma reesei; NTG and ultraviolet mutation; Fermentation
19.  Transcriptome analysis of Aspergillus niger grown on sugarcane bagasse 
Considering that the costs of cellulases and hemicellulases contribute substantially to the price of bioethanol, new studies aimed at understanding and improving cellulase efficiency and productivity are of paramount importance. Aspergillus niger has been shown to produce a wide spectrum of polysaccharide hydrolytic enzymes. To understand how to improve enzymatic cocktails that can hydrolyze pretreated sugarcane bagasse, we used a genomics approach to investigate which genes and pathways are transcriptionally modulated during growth of A. niger on steam-exploded sugarcane bagasse (SEB).
Herein we report the main cellulase- and hemicellulase-encoding genes with increased expression during growth on SEB. We also sought to determine whether the mRNA accumulation of several SEB-induced genes encoding putative transporters is induced by xylose and dependent on glucose. We identified 18 (58% of A. niger predicted cellulases) and 21 (58% of A. niger predicted hemicellulases) cellulase- and hemicellulase-encoding genes, respectively, that were highly expressed during growth on SEB.
Degradation of sugarcane bagasse requires production of many different enzymes which are regulated by the type and complexity of the available substrate. Our presently reported work opens new possibilities for understanding sugarcane biomass saccharification by A. niger hydrolases and for the construction of more efficient enzymatic cocktails for second-generation bioethanol.
PMCID: PMC3219568  PMID: 22008461
20.  Implications of cellobiohydrolase glycosylation for use in biomass conversion 
The cellulase producing ascomycete, Trichoderma reesei (Hypocrea jecorina), is known to secrete a range of enzymes important for ethanol production from lignocellulosic biomass. It is also widely used for the commercial scale production of industrial enzymes because of its ability to produce high titers of heterologous proteins. During the secretion process, a number of post-translational events can occur, however, that impact protein function and stability. Another ascomycete, Aspergillus niger var. awamori, is also known to produce large quantities of heterologous proteins for industry. In this study, T. reesei Cel7A, a cellobiohydrolase, was expressed in A. niger var. awamori and subjected to detailed biophysical characterization. The purified recombinant enzyme contains six times the amount of N-linked glycan than the enzyme purified from a commercial T. reesei enzyme preparation. The activities of the two enzyme forms were compared using bacterial (microcrystalline) and phosphoric acid swollen (amorphous) cellulose as substrates. This comparison suggested that the increased level of N-glycosylation of the recombinant Cel7A (rCel7A) resulted in reduced activity and increased non-productive binding on cellulose. When treated with the N-glycosidase PNGaseF, the molecular weight of the recombinant enzyme approached that of the commercial enzyme and the activity on cellulose was improved.
PMCID: PMC2427024  PMID: 18471276
21.  Microbial production host selection for converting second-generation feedstocks into bioproducts 
Increasingly lignocellulosic biomass hydrolysates are used as the feedstock for industrial fermentations. These biomass hydrolysates are complex mixtures of different fermentable sugars, but also inhibitors and salts that affect the performance of the microbial production host. The performance of six industrially relevant microorganisms, i.e. two bacteria (Escherichia coli and Corynebacterium glutamicum), two yeasts (Saccharomyces cerevisiae and Pichia stipitis) and two fungi (Aspergillus niger and Trichoderma reesei) were compared for their (i) ability to utilize monosaccharides present in lignocellulosic hydrolysates, (ii) resistance against inhibitors present in lignocellulosic hydrolysates, (iii) their ability to utilize and grow on different feedstock hydrolysates (corn stover, wheat straw, sugar cane bagasse and willow wood). The feedstock hydrolysates were generated in two manners: (i) thermal pretreatment under mild acid conditions followed by enzymatic hydrolysis and (ii) a non-enzymatic method in which the lignocellulosic biomass is pretreated and hydrolyzed by concentrated sulfuric acid. Moreover, the ability of the selected hosts to utilize waste glycerol from the biodiesel industry was evaluated.
Large differences in the performance of the six tested microbial production hosts were observed. Carbon source versatility and inhibitor resistance were the major discriminators between the performances of these microorganisms. Surprisingly all 6 organisms performed relatively well on pretreated crude feedstocks. P. stipitis and A. niger were found to give the overall best performance C. glutamicum and S. cerevisiae were shown to be the least adapted to renewable feedstocks.
Based on the results obtained we conclude that a substrate oriented instead of the more commonly used product oriented approach towards the selection of a microbial production host will avoid the requirement for extensive metabolic engineering. Instead of introducing multiple substrate utilization and detoxification routes to efficiently utilize lignocellulosic hydrolysates only one biosynthesis route forming the product of interest has to be engineered.
PMCID: PMC2795742  PMID: 19958560
22.  Comparative kinetic analysis of two fungal β-glucosidases 
The enzymatic hydrolysis of cellulose is still considered as one of the main limiting steps of the biological production of biofuels from lignocellulosic biomass. It is a complex multistep process, and various kinetic models have been proposed. The cellulase enzymatic cocktail secreted by Trichoderma reesei has been intensively investigated. β-glucosidases are one of a number of cellulolytic enzymes, and catalyze the last step releasing glucose from the inhibitory cellobiose. β-glucosidase (BGL1) is very poorly secreted by Trichoderma reesei strains, and complete hydrolysis of cellulose often requires supplementation with a commercial β-glucosidase preparation such as that from Aspergillus niger (Novozymes SP188). Surprisingly, kinetic modeling of β-glucosidases lacks reliable data, and the possible differences between native T. reesei and supplemented β-glucosidases are not taken into consideration, possibly because of the difficulty of purifying BGL1.
A comparative kinetic analysis of β-glucosidase from Aspergillus niger and BGL1 from Trichoderma reesei, purified using a new and efficient fast protein liquid chromatography protocol, was performed. This purification is characterized by two major steps, including the adsorption of the major cellulases onto crystalline cellulose, and a final purification factor of 53. Quantitative analysis of the resulting β-glucosidase fraction from T. reesei showed it to be 95% pure. Kinetic parameters were determined using cellobiose and a chromogenic artificial substrate. A new method allowing easy and rapid determination of the kinetic parameters was also developed. β-Glucosidase SP188 (Km = 0.57 mM; Kp = 2.70 mM) has a lower specific activity than BGL1 (Km = 0.38 mM; Kp = 3.25 mM) and is also more sensitive to glucose inhibition. A Michaelis-Menten model integrating competitive inhibition by the product (glucose) has been validated and is able to predict the β-glucosidase activity of both enzymes.
This article provides a useful comparison between the activity of β-glucosidases from two different fungi, and shows the importance of fully characterizing both enzymes. A Michaelis-Menten model was developed, including glucose inhibition and kinetic parameters, which were accurately determined and compared. This model can be further integrated into a cellulose hydrolysis model dissociating β-glucosidase activity from that of other cellulases. It can also help to define the optimal enzymatic cocktails for new β-glucosidase activities.
PMCID: PMC2847552  PMID: 20181208
23.  Genome-wide transcriptional response of Trichoderma reesei to lignocellulose using RNA sequencing and comparison with Aspergillus niger 
BMC Genomics  2013;14:541.
A major part of second generation biofuel production is the enzymatic saccharification of lignocellulosic biomass into fermentable sugars. Many fungi produce enzymes that can saccarify lignocellulose and cocktails from several fungi, including well-studied species such as Trichoderma reesei and Aspergillus niger, are available commercially for this process. Such commercially-available enzyme cocktails are not necessarily representative of the array of enzymes used by the fungi themselves when faced with a complex lignocellulosic material. The global induction of genes in response to exposure of T. reesei to wheat straw was explored using RNA-seq and compared to published RNA-seq data and model of how A. niger senses and responds to wheat straw.
In T. reesei, levels of transcript that encode known and predicted cell-wall degrading enzymes were very high after 24 h exposure to straw (approximately 13% of the total mRNA) but were less than recorded in A. niger (approximately 19% of the total mRNA). Closer analysis revealed that enzymes from the same glycoside hydrolase families but different carbohydrate esterase and polysaccharide lyase families were up-regulated in both organisms. Accessory proteins which have been hypothesised to possibly have a role in enhancing carbohydrate deconstruction in A. niger were also uncovered in T. reesei and categories of enzymes induced were in general similar to those in A. niger. Similarly to A. niger, antisense transcripts are present in T. reesei and their expression is regulated by the growth condition.
T. reesei uses a similar array of enzymes, for the deconstruction of a solid lignocellulosic substrate, to A. niger. This suggests a conserved strategy towards lignocellulose degradation in both saprobic fungi. This study provides a basis for further analysis and characterisation of genes shown to be highly induced in the presence of a lignocellulosic substrate. The data will help to elucidate the mechanism of solid substrate recognition and subsequent degradation by T. reesei and provide information which could prove useful for efficient production of second generation biofuels.
PMCID: PMC3750697  PMID: 24060058
Trichoderma reesei; Aspergillus niger; Glycoside hydrolases; Carbohydrate esterases; Antisense RNA; RNA-sequencing
24.  Lovastatin Production by Aspergillus terreus Using Agro-Biomass as Substrate in Solid State Fermentation 
Ability of two strains of Aspergillus terreus (ATCC 74135 and ATCC 20542) for production of lovastatin in solid state fermentation (SSF) using rice straw (RS) and oil palm frond (OPF) was investigated. Results showed that RS is a better substrate for production of lovastatin in SSF. Maximum production of lovastatin has been obtained using A. terreus ATCC 74135 and RS as substrate without additional nitrogen source (157.07 mg/kg dry matter (DM)). Although additional nitrogen source has no benefit effect on enhancing the lovastatin production using RS substrate, it improved the lovastatin production using OPF with maximum production of 70.17 and 63.76 mg/kg DM for A. terreus ATCC 20542 and A. terreus ATCC 74135, respectively (soybean meal as nitrogen source). Incubation temperature, moisture content, and particle size had shown significant effect on lovastatin production (P < 0.01) and inoculums size and pH had no significant effect on lovastatin production (P > 0.05). Results also have shown that pH 6, 25°C incubation temperature, 1.4 to 2 mm particle size, 50% initial moisture content, and 8 days fermentation time are the best conditions for lovastatin production in SSF. Maximum production of lovastatin using optimized condition was 175.85 and 260.85 mg/kg DM for A. terreus ATCC 20542 and ATCC 74135, respectively, using RS as substrate.
PMCID: PMC3478940  PMID: 23118499
25.  Production of Pectate Lyase by Penicillium viridicatum RFC3 in Solid-State and Submerged Fermentation 
Pectate lyase (PL) was produced by the filamentous fungus Penicillium viridicatum RFC3 in solid-state cultures of a mixture of orange bagasse and wheat bran (1 : 1 w/w), or orange bagasse, wheat bran and sugarcane bagasse (1 : 1 : 0.5 w/w), and in a submerged liquid culture with orange bagasse and wheat bran (3%) as the carbon source. PL production was highest (1,500 U  mL−1 or 300 Ug−1 of substrate) in solid-state fermentation (SSF) on wheat bran and orange bagasse at 96 hours. PL production in submerged fermentation (SmF) was influenced by the initial pH of the medium. With the initial pH adjusted to 4.5, 5.0, and 5.5, the peak activity was observed after 72, 48, and 24 hours of fermentation, respectively, when the pH of the medium reached the value 5.0. PL from SSF and SmF were loaded on Sephadex-G75 columns and six activity peaks were obtained from crude enzyme from SSF and designated PL I, II, III, IV, V, and VI, while five peaks were obtained from crude enzyme from SmF and labeled PL  I′, II′, III′, IV′, and VII′. Crude enzyme and fraction III from each fermentative process were tested further. The optimum pH for crude PL from either process was 5.5, while that for PL III was 8.0. The maximum activity of enzymes from SSF was observed at 35°C, but crude enzyme was more thermotolerant than PL III, maintaining its maximum activity up to 45°C. Crude enzyme from SmF and PL III′ showed thermophilic profiles of activity, with maximum activity at 60 and 55°C, respectively. In the absence of substrate, the crude enzyme from SSF was stable over the pH range 3.0–10.0 and PL III was most stable in the pH range 4.0–7.0. Crude enzyme from SmF retained 70%–80% of its maximum activity in the acid-neutral pH range (4.0–7.0), but PIII showed high stability at alkaline pH (7.5–9.5). PL from SSF was more thermolabile than that from SmF. The latter maintained 60% of its initial activity after 1 h at 55°C. The differing behavior of the enzymes with respect to pH and temperature suggests that they are different isozymes.
PMCID: PMC2913117  PMID: 20689719

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