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1
Yang, Jungwoo, Ji Eun Kim, Jae Kyun Kim, Sung ho Lee, Ju-Hyun Yu, and Kyoung Heon Kim. "Evaluation of commercial cellulase preparations for the efficient hydrolysis of hydrothermally pretreated empty fruit bunches." BioResources 12, no. 4 (September 7, 2017): 7834–40. http://dx.doi.org/10.15376/biores.12.4.7834-7840.
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The performance of cellulase in the enzymatic saccharification of lignocellulose depends on the characteristics of lignocellulosic biomass feedstocks and the pretreatment method used. Efficient hydrolysis of specifically pretreated lignocellulose necessitates the knowledge of the characteristics of the optimal commercial cellulases. In this study, commercial cellulase preparations (Accellerase™ 1000, Accellerase® 1500, and Spezyme® CP from DuPont and Cellic® CTec2 from Novozymes) were evaluated for their hydrolysis efficiency of hydrothermally pretreated empty fruit bunches (EFBs). The highest glucose yields of 91.3% and 84.7% were achieved for 30 FPU of Cellic® CTec2/g glucan with and without Cellic® HTec2, respectively. Of the four cellulases tested, Cellic® CTec2, which showed the highest cellobiohydrolase, xylanase, and β-glucosidase activities, showed the highest glucose yield in the enzymatic hydrolysis of hydrothermally pretreated EFBs. The results of this study are valuable for those who plan to enzymatically hydrolyze hydrothermally pretreated EFBs.
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Marchwicka, Monika. "Influence of pH and Cellic® CTec2 enzymes dose on the glucose yield after enzymatic hydrolysis of cellulose at 50 °C." Annals of WULS, Forestry and Wood Technology 114 (June 28, 2021): 53–58. http://dx.doi.org/10.5604/01.3001.0015.2373.
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Influence of pH and Cellic® CTec2 enzymes dose on the glucose yield after enzymatic hydrolysis of cellulose at 50 °C. Cellulose obtained by the Kürschner-Hoffer method from the wood of 3-year-old poplar (Populus trichocarpa) was subjected to enzymatic hydrolysis. Cellic® CTec2 enzymes (Novozymes, Denmark) were used. The enzymatic hydrolysis was tested within the conditions recommended by the manufacturer and the literature. The process was carried out at 50 °C at various pH – 4.8, 5.0, 5.5 and enzymes doses - 25, 50 and 100 mg per 100 mg of the dry mass of cellulose. The process was ended after 24 h. The hydrolysates were analysed by high-performance liquid chromatography (HPLC) to determine the glucose content, and then the highest glucose yield. The highest glucose yield was obtained for pH 4.8 and 100 mg of enzymes per 100 mg of the dry mass of cellulose – 72 %.
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Lucarini, Adriana Célia, Ana Carolina Tezotto Delquiaro, Ludmila Carolina Prado Tavares Vidoca, Rafaela Braz, Roberta Morelli Martins, and Taís Pereira Alves. "ESTUDO DA HIDRÓLISE ENZIMÁTICA DA PALHA DA CANA DE AÇÚCAR PARA PRODUÇÃO DE ETANOL DE SEGUNDA GERAÇÃO." Journal of Engineering and Exact Sciences 3, no. 2 (March 19, 2017): 242–53. http://dx.doi.org/10.18540/jcecvl3iss2pp242-253.
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Estudou-se a hidrólise enzimática da palha da cana para a obtenção de açúcares fermentescíveis. A palha passou por processos físicos (moagem, lavagem e secagem), foi submetida à uma deslignificação com H2O2 em meio alcalino e hidrólise. Após caracterização da biomassa lignocelulósica in natura e deslignificada observou-se remoção de 50% da lignina. Foi quantificada a atividade enzimática da celulase de T. reesei e comercial Cellic® CTec2. Foram estudados pH da reação, concentração de enzima e tempo de reação, obtendo-se pH ótimo 4,0 e 5,5 para a T. reesei e Cellic® CTec2, respectivamente. O maior rendimento em termos de concentração de enzima foi de 10 FPU/g de palha para ambas as enzimas e para o tempo, ambas enzimas apresentaram queda de conversão após 24 horas de hidrólise, possivelmente por contaminação microbiana. Por fim realizou-se o balanço material para a celulase comercial, projetando-se um rendimento do processo de hidrólise enzimática da palha de 23,8%.
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Woon, James Sy-Keen, Mukram M. Mackeen, Rosli M. Illias, Nor M. Mahadi, William J. Broughton, Abdul Munir Abdul Murad, and Farah Diba Abu Bakar. "Cellobiohydrolase B ofAspergillus nigerover-expressed inPichia pastorisstimulates hydrolysis of oil palm empty fruit bunches." PeerJ 5 (October 12, 2017): e3909. http://dx.doi.org/10.7717/peerj.3909.
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BackgroundAspergillus niger, along with many other lignocellulolytic fungi, has been widely used as a commercial workhorse for cellulase production. A fungal cellulase system generally includes three major classes of enzymes i.e., β-glucosidases, endoglucanases and cellobiohydrolases. Cellobiohydrolases (CBH) are vital to the degradation of crystalline cellulose present in lignocellulosic biomass. However,A. nigernaturally secretes low levels of CBH. Hence, recombinant production ofA. nigerCBH is desirable to increase CBH production yield and also to allow biochemical characterisation of the recombinant CBH fromA. niger.MethodsIn this study, the gene encoding a cellobiohydrolase B (cbhB) fromA. nigerATCC 10574 was cloned and expressed in the methylotrophic yeastPichia pastorisX-33. The recombinant CBHB was purified and characterised to study its biochemical and kinetic characteristics. To evaluate the potential of CBHB in assisting biomass conversion, CBHB was supplemented into a commercial cellulase preparation (Cellic®CTec2) and was used to hydrolyse oil palm empty fruit bunch (OPEFB), one of the most abundant lignocellulosic waste from the palm oil industry. To attain maximum saccharification, enzyme loadings were optimised by response surface methodology and the optimum point was validated experimentally. Hydrolysed OPEFB samples were analysed using attenuated total reflectance FTIR spectroscopy (ATR-FTIR) to screen for any compositional changes upon enzymatic treatment.ResultsRecombinant CBHB was over-expressed as a hyperglycosylated protein attached toN-glycans. CBHB was enzymatically active towards soluble substrates such as 4-methylumbelliferyl-β-D-cellobioside (MUC),p-nitrophenyl-cellobioside (pNPC) andp-nitrophenyl-cellobiotrioside (pNPG3) but was not active towards crystalline substrates like Avicel®and Sigmacell cellulose. Characterisation of purified CBHB using MUC as the model substrate revealed that optimum catalysis occurred at 50 °C and pH 4 but the enzyme was stable between pH 3 to 10 and 30 to 80 °C. Although CBHB on its own was unable to digest crystalline substrates, supplementation of CBHB (0.37%) with Cellic®CTec2 (30%) increased saccharification of OPEFB by 27%. Compositional analyses of the treated OPEFB samples revealed that CBHB supplementation reduced peak intensities of both crystalline cellulose Iαand Iβ in the treated OPEFB samples.DiscussionSince CBHB alone was inactive against crystalline cellulose, these data suggested that it might work synergistically with other components of Cellic®CTec2. CBHB supplements were desirable as they further increased hydrolysis of OPEFB when the performance of Cellic®CTec2 was theoretically capped at an enzyme loading of 34% in this study. Hence,A. nigerCBHB was identified as a potential supplementary enzyme for the enzymatic hydrolysis of OPEFB.
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Marchwicka, Monika. "Influence of pH and Cellic® CTec2 enzymes dose on the glucose yield after enzymatic hydrolysis of cellulose at 45 °C." Annals of WULS, Forestry and Wood Technology 112 (December 31, 2020): 85–91. http://dx.doi.org/10.5604/01.3001.0014.9045.
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Influence of pH and Cellic® CTec2 enzymes dose on the glucose yield after enzymatic hydrolysis of cellulose at 45 °C. The enzymatic hydrolysis with the use of industrial enzymes Cellic® CTec2 (Novozymes, Denmark) was carried out within the conditions recommended by the manufacturer and literature. Cellulose obtained by the Kürschner-Hoffer method from a wood of 3-year-old poplar (Populus trichocarpa) was used for the study. Three pH values of 4.8, 5.0 and 5.5 were applied. Also, three amounts of enzymes were used: 25, 50 and 100 mg per 100 mg of the dry mass of cellulose for each pH used. The temperature was 45 °C. Samples were taken after 24 h and subjected to chromatographic analysis to determine the glucose content in the hydrolysates, and then the process parameters allowing for the highest glucose yield after the enzymatic hydrolysis process. The highest glucose yield was obtained for pH 5.0 and 100 mg of enzymes per 100 mg of the dry mass of cellulose – 79 %.
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Megyeri, Gábor, Nándor Nemestóthy, Milan Polakovic, and Katalin Bélafi-Bakó. "Application of Ionic Liquids in the Utilization of the Agricultural Wastes: Towards the One-Step Pre-Treatment and Cellulose Hydrolysis." Hungarian Journal of Industry and Chemistry 43, no. 2 (October 1, 2015): 85–89. http://dx.doi.org/10.1515/hjic-2015-0014.
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Abstract Cheap, renewable lignocellulosic materials are relevant to the future of biofuel production. Wood and agricultural wastes (e.g. straw, corn stover) provide a raw material source that cannot be used for human consumption, thus biofuels from such sources do not threaten the food supply. The aim of the work was to carry out the pre-treatment and hydrolysis of lignocellulosic material in the same ionic liquid solvent (1-n-butyl-3- methyl-imidazolium-chloride, [Bmim]Cl), using ground wheat straw and a mixture of corn (Zea mays) leaf and stover, as substrates. Our measurements show that it is possible to achieve an acceptable glucose content from the cellulose by applying Cellic® CTec2 and Cellic® HTec2 enzyme complexes.
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Kancelista, Anna, Joanna Chmielewska, Paweł Korzeniowski, and Wojciech Łaba. "Bioconversion of Sweet Sorghum Residues by Trichoderma citrinoviride C1 Enzymes Cocktail for Effective Bioethanol Production." Catalysts 10, no. 11 (November 8, 2020): 1292. http://dx.doi.org/10.3390/catal10111292.
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Improved cost-effective bioethanol production using inexpensive enzymes preparation was investigated. Three types of waste lignocellulosic materials were converted—for the production of enzyme preparation, a mixture of sugar beet pulp and wheat bran, while the source of sugars in hydrolysates was sweet sorghum biomass. A novel enzyme cocktail of Trichoderma citrinoviride C1 is presented. The one-step ultrafiltration process of crude enzyme extract resulted in a threefold increase of cellulolytic and xylanolytic activities. The effectiveness of enzyme preparation, compared to Cellic® CTec2, was tested in an optimized enzymatic hydrolysis process. Depending on the test conditions, hydrolysates with different glucose concentrations were obtained—from 6.3 g L−1 to 14.6 g L−1 (representing from 90% to 79% of the CTec2 enzyme yield, respectively). Furthermore, ethanol production by Saccharomyces cerevisiae SIHA Active Yeast 6 strain DF 639 in optimal conditions reached about 120 mL kg d.m.−1 (75% compared with the CTec2 process). The achieved yields suggested that the produced enzyme cocktail C1 could be potentially used to reduce the cost of bioethanol production from sweet sorghum biomass.
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Jung, Woochul, Ratna Sharma-Shivappa, and Praveen Kolar. "Effect of Enzyme Interaction with Lignin Isolated from Pretreated Miscanthus x gigantues on Cellulolytic Efficiency." Processes 7, no. 10 (October 16, 2019): 755. http://dx.doi.org/10.3390/pr7100755.
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The effect of binding between the lignin isolates from an alkali (NaOH)– and an acid (H2SO4)– pretreated Miscanthus and cellulolytic enzymes in Cellic® CTec2 was investigated. Additonally, cellobiose and Avicel were enzymatically hydrolyzed with and without lignin isolates to study how enzyme binding onto lignin affects its conversion to glucose. Three carbohydrate–lignin loadings (0.5:0.25, 0.5:0.5, and 0.5:1.0% (w/v)) were employed. The results indicated that β-glucosidase (BG) had a strong tendency to bind to all lignin isolates. The overall tendency of enzyme binding onto lignin isolate was similar regardless of pretreatment chemical concentration. Though enzyme binding onto lignin isolates was observed, hydrolysis in the presence of these isolates did not have a significant (p > 0.05) impact on glucose production from cellobiose and Avicel. Cellobiose to glucose conversion of 99% was achieved via hydrolysis at both 5 and 10 FPU/g carbohydrate. Hydrolysis of Avicel with 5 and 10 FPU/g CTec2 resulted in 29.3 and 47.7% conversion to glucose, respectively.
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Bohn, Letícia Renata, Aline Perin Dresch, Matheus Cavali, Ana Carolina Giacomelli Vargas, Jaíne Flach Führ, Siumar Pedro Tironi, Odinei Fogolari, Guilherme Martinez Mibielli, Sérgio Luiz Alves Jr., and João Paulo Bender. "Alkaline pretreatment and enzymatic hydrolysis of corn stover for bioethanol production." Research, Society and Development 10, no. 11 (August 25, 2021): e149101118914. http://dx.doi.org/10.33448/rsd-v10i11.18914.
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The demand for ethanol in Brazil is growing. However, although the country is one of the largest producers of this fuel, it is still necessary to diversify the production matrix. In that regard, studies with different raw materials are needed, mainly the use of low cost and high available wastes such as lignocellulosic residues from agriculture. Therefore, this study aimed to analyze the bioethanol production from corn stover. An alkaline pretreatment (CaO) was carried out, followed by enzymatic hydrolysis (Cellic Ctec2 and Cellic Htec2) to obtain fermentable sugars. The best experimental condition for the pretreatment and hydrolysis steps resulted in a solution with 0.31 gsugar∙gbiomass-1. Then, the fermentation was performed by the industrial strain of Saccharomyces cerevisiae (PE-2) and by the wild yeast strain Wickerhamomyces sp. (UFFS-CE-3.1.2). The yield obtained was 0.38 gethanol∙gdry biomass-1 was, demonstrating the potential of this process for bioethanol production.
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Dörschmann, Philipp, Maria Dalgaard Mikkelsen, Thuan Nguyen Thi, Johann Roider, Anne S. Meyer, and Alexa Klettner. "Effects of a Newly Developed Enzyme-Assisted Extraction Method on the Biological Activities of Fucoidans in Ocular Cells." Marine Drugs 18, no. 6 (May 26, 2020): 282. http://dx.doi.org/10.3390/md18060282.
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Fucoidans from brown seaweeds are promising substances as potential drugs against age-related macular degeneration (AMD). The heterogeneity of fucoidans requires intensive research in order to find suitable species and extraction methods. Ten different fucoidan samples extracted enzymatically from Laminaria digitata (LD), Saccharina latissima (SL) and Fucus distichus subsp. evanescens (FE) were tested for toxicity, oxidative stress protection and VEGF (vascular endothelial growth factor) inhibition. For this study crude fucoidans were extracted from seaweeds using different enzymes and SL fucoidans were further separated into three fractions (SL_F1-F3) by ion-exchange chromatography (IEX). Fucoidan composition was analyzed by high performance anion exchange chromatography (HPAEC) after acid hydrolysis. The crude extracts contained alginate, while two of the fractionated SL fucoidans SL_F2 and SL_F3 were highly pure. Cell viability was assessed with an 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay in OMM-1 and ARPE-19. Protective effects were investigated after 24 h of stress insult in OMM-1 and ARPE-19. Secreted VEGF was analyzed via ELISA (enzyme-linked immunosorbent assay) in ARPE-19 cells. Fucoidans showed no toxic effects. In OMM-1 SL_F2 and several FE fucoidans were protective. LD_SiAT2 (Cellic®CTec2 + Sigma-Aldrich alginate lyase), FE_SiAT3 (Cellic® CTec3 + Sigma-Aldrich alginate lyase), SL_F2 and SL_F3 inhibited VEGF with the latter two as the most effective. We could show that enzyme treated fucoidans in general and the fractionated SL fucoidans SL_F2 and SL_F3 are very promising for beneficial AMD relevant biological activities.
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Kuglarz, Mariusz, and Klaudiusz Grübel. "Integrated Production of Biofuels and Succinic Acid from Biomass after Thermochemical Pretreatments." Ecological Chemistry and Engineering S 25, no. 4 (December 1, 2018): 521–36. http://dx.doi.org/10.1515/eces-2018-0034.
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Abstract The aim of this study was to develop an effective thermochemical method for treatment of industrial hemp, in order to increase its bioconversion to biofuels and bio-products. Industrial hemp was subjected to various thermochemical pretreatments using: alkaline (3 % NaOH), oxidative (3 % H2O2 at pH 11.5) and glycerol-based methods (70-90 % of glycerol, 1-3 % NaOH), prior to enzymatic hydrolysis with Cellic® CTec2/Cellic® HTec2 (15 FPU∙g−1 glucan). Innovative pretreatment with glycerol fraction (80 % glycerol content, 2 % NaOH, 12.5 % biomass loading) showed to be superior over commonly used alkaline and oxidative methods with respect to by-products generation and sugar losses. Integrated process of ethanol production from enriched cellulose fraction (172 kg EtOH∙Mg−1 of dry hemp) and succinic production from xylose-rich residue after ethanol fermentation (59 kg∙Mg−1 of dry hemp) allowed to convert about 97 % of sugars released (glucose and xylose) during enzymatic hydrolysis of pre-treated biomass. The present study showed that it is possible to replace 50 % of the costly yeast extract, used during succinic fermentation as nitrogen source, by alternative nitrogen source (rapeseed cakes) without significant deterioration of succinic yield. Pretreatment liquor after lignin precipitation (52 kg∙Mg−1 of biomass treated) exhibited a high biodegradability (92 %) and allowed to produce 420 m3 CH4/Mg VS). Results obtained in this study clearly document the possibility of biofuels (bioethanol, biogas) and bio-chemicals production from industrial hemp, in a biorefinery approach.
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Harahap, Budi Mandra, Robby Sudarman, Fildzah Sajidah, Diana Murti Indra Wahyuni, and Dea Tesalonika Sitorus. "Effect of Microwave Pretreatment on Production of Reducing Sugar from Oil Palm Empty Fruit Bunches." Jurnal Teknik Kimia dan Lingkungan 4, no. 2 (October 27, 2020): 141. http://dx.doi.org/10.33795/jtkl.v4i2.159.
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Perlakuan pendahulan biomassa merupakan tahapan terpenting dalam memproduksi produk-produk berbasis bio (bio-based products) secara biologis. Pada penelitian ini, energi gelombang mikro (microwave) digunakan selama perlakuan pendahuluan untuk meningkatkan kinerja proses sakarifikasi tandan kosong kelapa sawit (TKKS) menjadi gula-gula pereduksi. Faktor-faktor yang mempengaruhi perlakuan pendahuluan seperti daya (180-360 watt), waktu iradiasi (5-30 menit), dan solid loading (2,5%-7,5%) dievaluasi. Kinerja hidrolisis TKKS yang telah diberi perlakuan pendahuluan selanjutnya dianalisis dengan Cellic CTec2. Hasil penelitian menunjukkan bahwa cairan residu yang diperoleh setelah perlakuan pendahuluan menghasilkan gula pereduksi dalam jumlah yang rendah, yaitu antara 1,39 dan 3,92 mg/g-TKKS. Akan tetapi, setelah padatan residu dihidrolisis secara enzimatis, rendemen gula pereduksi meningkat secara signifikan. Menariknya, hanya pada level daya terendah (180 watt), gula pereduksi meningkat seiring dengan perpanjangan waktu iradiasi untuk semua solid loading. Sebaliknya, pada 360 watt, semakin lama waktu iradiasi diterapkan, semakin rendah gula pereduksi yang diperoleh untuk semua solid loading. Gula pereduksi tertinggi dihasilkan hingga 151 mg/g-TKKS, yaitu menggunakan 5% padatan pada 180 watt selama 25 menit. Berdasarkan hasil-hasil ini, perlakuan pendahuluan menggunakan gelombang mikro yang diikuti dengan hidrolisis enzimatis merupakan salah satu metode yang potensial untuk memproduksi gula dari TKKS.Pretreatment of biomass is the most crucial step in the biological production of bio-based products. In this study, microwave energy was used during the pretreatment process to enhance the saccharification performance of oil palm empty fruit bunches (OPEFB) into reducing sugar. The influential factors of pretreatment such as power level (180-360 watt), irradiation time (5-30 min), and solid loading (2.5%-7.5%) were evaluated. The performance of pretreated OPEFB hydrolysis was subsequently assessed by Cellic CTec2. The result showed that spent liquor produced after pretreatment only released a low amount of reducing sugar in the range between 1.39 and 3.92 mg/g-OPEFB. After residual solid was enzymatically hydrolyzed, a significant increase in the reducing sugar yield occurred. Interestingly, only at the lowest power level (180 watts), the reducing sugar rose along with the extension of irradiation time for all solid loadings. On the contrary, the longer irradiation time was applied, the lower reducing sugar was acquired at 360 watts for all solid loadings. The highest reducing sugar was produced up to 151 mg/g-OPEFB using 5% solid at 180 watts for 25 min. This indicated that microwave pretreatment followed by enzymatic hydrolysis was one of the potential methods to recover sugars in OPEFB.
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Ventrone, Michela, Chiara Schiraldi, Giuseppe Squillaci, Alessandra Morana, and Donatella Cimini. "Chestnut Shells as Waste Material for Succinic Acid Production from Actinobacillus succinogenes 130Z." Fermentation 6, no. 4 (November 6, 2020): 105. http://dx.doi.org/10.3390/fermentation6040105.
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Currently, the full exploitation of waste materials for the production of value-added compounds is one of the potential solutions to lower costs and increase the sustainability of industrial processes. In this respect, the aim of this work was to evaluate the potential of chestnut shells (CSH) as substrate for the growth of Actinobacillus succinogenes 130Z, a natural producer of succinic acid that is a precursor of several bulk chemicals with diverse applications, such as bioplastics production. Hydrolysis of ammonia pretreated CSH in citrate buffer with the Cellic CTec2 enzyme mix was optimized and strain performance was studied in bottle experiments. Data showed co-consumption of citrate, glucose and xylose, which resulted in a change of the relative ratio of produced acids, providing an insight into the metabolism of A. succinogenes that was never described to date. Furthermore, high C:N ratios seems to have a favorable impact on succinic acid production by decreasing byproduct formation. Finally, yield and volumetric production rate of succinic acid were studied in controlled 2 L bioreactors demonstrating the potential use of CSH as renewable raw material.
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Dąbkowska, Katarzyna, Monika Mech, Kamil Kopeć, and Maciej Pilarek. "Enzymatic Activity of Some Industrially-Applied Cellulolytic Enzyme Preparations." Ecological Chemistry and Engineering S 24, no. 1 (March 1, 2017): 9–18. http://dx.doi.org/10.1515/eces-2017-0001.
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Abstract Enzymatic hydrolysis is the essential step in the production of 2nd generation biofuels made from lignocellulosic biomass, i.e. agricultural or forestry solid wastes. The enzyme-catalysed robust degradation of cellulose and hemicellulose to monosaccharides requires the synergistic action of the independent types of highly-specific enzymes, usually offered as ready-to-use preparations. The basic aim of the study was to experimentally determine the enzymatic activity of two widely industrially-applied, commercially available cellulolytic enzyme preparations: (i) Cellic® CTec2 and (ii) the mixture of Celluclast® 1.5L and Novozyme 188, in the hydrolysis of pre-treated lignocellulosic biomass, i.e. (a) energetic willow and (b) rye straw, or untreated (c) cellulose paper as well, used as feedstocks. Before the hydrolysis, every kind of utilized lignocellulosic biomass was subjected to alkaline-based (10% NaOH) pre-treatment at high-temperature (121°C) and overpressure (0.1 MPa) conditions. The influence of the type of applied enzymes, as well as their concentration, on the effectiveness of hydrolysis was quantitatively evaluated, and finally the enzyme activities were determined for each of tested cellulolytic enzyme preparations.
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Kim, Tae, Dongjoong Im, Kyeong Oh, and Tae Kim. "Effects of Organosolv Pretreatment Using Temperature-Controlled Bench-Scale Ball Milling on Enzymatic Saccharification of Miscanthus × giganteus." Energies 11, no. 10 (October 5, 2018): 2657. http://dx.doi.org/10.3390/en11102657.
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The effect of organosolv pretreatment was investigated using a 30 L bench-scale ball mill reactor that was capable of simultaneously performing physical and chemical pretreatment. Various reaction conditions were tried in order to discover the optimal conditions for the minimal cellulose loss and enhanced enzymatic digestibility of Miscanthus × giganteus (MG), with conditions varying from room temperature to 170 °C for reaction temperature, from 30 to 120 min of reaction time, from 30% to 60% ethanol concentration, and a liquid/solid ratio (L/S) of 10–20 under non-catalyst conditions. The pretreatment effects were evaluated by chemical compositional analysis, enzymatic digestibility test and X-ray diffraction of the treated samples. The pretreatment conditions for the highest glucan digestibility yield were determined as 170 °C, reaction time of 90 min, ethanol concentration of 40% and L/S = 10. With these pretreatment conditions, the XMG (xylan + mannan + galactan) fractionation yield and delignification were 84.4% and 53.2%, respectively. The glucan digestibility of treated MG after the aforementioned pretreatment conditions was 86.0% with 15 filter paper units (FPU) of cellulase (Cellic® CTec2) per g-glucan enzyme loading.
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Rodrigues, Ana Cristina, Mai Østergaard Haven, Jane Lindedam, Claus Felby, and Miguel Gama. "Celluclast and Cellic® CTec2: Saccharification/fermentation of wheat straw, solid–liquid partition and potential of enzyme recycling by alkaline washing." Enzyme and Microbial Technology 79-80 (November 2015): 70–77. http://dx.doi.org/10.1016/j.enzmictec.2015.06.019.
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Baral, Pratibha, Lavika Jain, Akhilesh Kumar Kurmi, Vinod Kumar, and Deepti Agrawal. "Augmented hydrolysis of acid pretreated sugarcane bagasse by PEG 6000 addition: a case study of Cellic CTec2 with recycling and reuse." Bioprocess and Biosystems Engineering 43, no. 3 (November 8, 2019): 473–82. http://dx.doi.org/10.1007/s00449-019-02241-3.
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Qu, Xiao-Su, Bin-Bin Hu, and Ming-Jun Zhu. "Enhanced saccharification of cellulose and sugarcane bagasse by Clostridium thermocellum cultures with Triton X-100 and β-glucosidase/Cellic®CTec2 supplementation." RSC Advances 7, no. 35 (2017): 21360–65. http://dx.doi.org/10.1039/c7ra02477k.
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Schroedter, Linda, Roland Schneider, Lisa Remus, and Joachim Venus. "L-(+)-Lactic Acid from Reed: Comparing Various Resources for the Nutrient Provision of B. coagulans." Resources 9, no. 7 (July 20, 2020): 89. http://dx.doi.org/10.3390/resources9070089.
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Biotechnological production of lactic acid (LA) is based on the so-called first generation feedstocks, meaning sugars derived from food and feed crops such as corn, sugarcane and cassava. The aim of this study was to exploit the potential of a second generation resource: Common reed (Phragmites australis) is a powerfully reproducing sweet grass which grows in wetlands and creates vast monocultural populations. This lignocellulose biomass bears the possibility to be refined to value-added products, without competing with agro industrial land. Besides utilizing reed as a renewable and inexpensive substrate, low-cost nutritional supplementation was analyzed for the fermentation of thermophilic Bacillus coagulans. Various nutritional sources such as baker’s and brewer’s yeast, lucerne green juice and tryptone were investigated for the replacement of yeast extract. The structure of the lignocellulosic material was tackled by chemical treatment (1% NaOH) and enzymatic hydrolysis (Cellic® CTec2). B. coagulans DSM ID 14-300 was employed for the homofermentative conversion of the released hexose and pentose sugars to polymerizable L-(+)-LA of over 99.5% optical purity. The addition of autolyzed baker’s yeast led to the best results of fermentation, enabling an LA titer of 28.3 g L−1 and a yield of 91.6%.
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Paula, Leidiane de. "Estimativa da produção de etanol a partir de biomassas lignocelulósicas pré-tratadas com peróxido de hidrogênio." Revista Agraria Academica 4, no. 3 (May 1, 2021): 72–83. http://dx.doi.org/10.32406/v4n3/2021/72-83/agrariacad.
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The objective of this work was to compare pre-treatments with hydrogen peroxide at 1 and 2% (v/v) and pH at 11.5 used in elephant-grass (EC), mulatto-grass (HD364) lignocellulosic biomass, bark of rice (CA) and leaves of the pineapple (AB) plant to estimate the production of second-generation ethanol (2G). The experimental design was completely randomized in a 4x2 factorial scheme with four replicates per treatment. Enzymatic hydrolysis was performed with the pretreated biomasses using Cellic/CTec2 enzyme. 7 ml of the hydrolyzed material and 0.14 g of non-isolated Saccharomyces cerevisiae yeast were used. After this dilution the material was taken to a styrofoam oven and measured at the temperature and volume of the gas produced at the time of 0,04; 0,08; 0,2; 0,63; 0,92; 1,19; 1,93; 2,19; 2,82; 3,15; 3,94 hours post-incubation. The results showed that the observed differences between the pretreatments influenced the yield in 2G ethanol differently between the biomasses, being the CE that presented the best yield, however, when evaluating the yield by the peroxide content within each biomass, both AB and EC showed no differences between pre-treatments. HD364 had better yield with 1% peroxide and CA with 2% peroxide.
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Krafft, Malte Jörn, Olga Frey, Katrin U. Schwarz, and Bodo Saake. "Maize Silage Pretreatment via Steam Refining and Subsequent Enzymatic Hydrolysis for the Production of Fermentable Carbohydrates." Molecules 25, no. 24 (December 19, 2020): 6022. http://dx.doi.org/10.3390/molecules25246022.
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Maize, also called corn, is one of the most available feedstocks worldwide for lignocellulosic biorefineries. However, a permanent biomass supply over the year is essential for industrial biorefinery application. In that context, ensiling is a well-known agricultural application to produce durable animal feed for the whole year. In this study, ensiled maize was used for steam refining experiments with subsequent enzymatic hydrolysis using the Cellic® CTec2 to test the application possibilities of an ensiled material for the biorefinery purpose of fermentable carbohydrate production. Steam refining was conducted from mild (log R0 = 1.59) to severe conditions (log R0 = 4.12). The yields were determined, and the resulting fractions were characterized. Hereafter, enzymatic hydrolysis of the solid fiber fraction was conducted, and the carbohydrate recovery was calculated. A conversion to monomers of around 50% was found for the mildest pretreatment (log R0 = 1.59). After pretreatment at the highest severity of 4.12, it was possible to achieve a conversion of 100% of the theoretical available carbohydrates. From these results, it is clear that a sufficient pretreatment is necessary to achieve sufficient recovery rates. Thus, it can be concluded that ensiled maize pretreated by steam refining is a suitable and highly available feedstock for lignocellulosic biorefineries. Ultimately, it can be assumed that ensiling is a promising storage method to pave the way for a full-year biomass supply for lignocellulosic biorefinery concepts.
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Kim, Seonghun. "Evaluation of Alkali-Pretreated Soybean Straw for Lignocellulosic Bioethanol Production." International Journal of Polymer Science 2018 (2018): 1–7. http://dx.doi.org/10.1155/2018/5241748.
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Soybean straw is a renewable resource in agricultural residues that can be used for lignocellulosic bioethanol production. To enhance enzymatic digestibility and fermentability, the biomass was prepared with an alkali-thermal pretreatment (sodium hydroxide, 121°C, 60 min). The delignification yield was 34.1~53%, in proportion to the amount of sodium hydroxide, from 0.5 to 3.0 M. The lignin and hemicellulose contents of the pretreated biomass were reduced by the pretreatment process, whereas the proportion of cellulose was increased. Under optimal condition, the pretreated biomass consisted of 74.0±0.1% cellulose, 10.3±0.1% hemicellulose, and 10.1±0.6% lignin. During enzymatic saccharification using Cellic® CTec2 cellulase, 10% (w/v) of pretreated soybean straw was hydrolyzed completely and converted to 67.3±2.1 g/L glucose and 9.4±0.5 g/L xylose with a 90.9% yield efficiency. Simultaneous saccharification and fermentation of the pretreated biomass by Saccharomyces cerevisiae W303-1A produced 30.5±1.2 g/L ethanol in 0.5 L fermented medium containing 10% (w/v) pretreated biomass after 72 h. The ethanol productivity was 0.305 g ethanol/g dry biomass and 0.45 g ethanol/g glucose after fermentation, with a low concentration of organic acid metabolites. Also, 82% of fermentable sugar was used by the yeast for ethanol fermentation. These results show that the combination of alkaline pretreatment and biomass hydrolysate is useful for enhancing bioethanol productivity using delignified soybean straw.
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Mutrakulcharoen, Parita, Peerapong Pornwongthong, S. T. Anne Sahithi, Theerawut Phusantisampan, Atthasit Tawai, and Malinee Sriariyanun. "Improvement of potassium permanganate pretreatment by enzymatic saccharification of rice straw for production of biofuels." E3S Web of Conferences 302 (2021): 02013. http://dx.doi.org/10.1051/e3sconf/202130202013.
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Commonly, the agricultural waste, i.e. lignocellulosic biomass is disposed through combustion causing air pollution with production of PM2.5 and PM10 particles. However, it has been found that these biomasses can be used as source for the production of biofuels and other valuable biochemicals. Though deconstruction of lignocellulosic biomass is challenging due to its complex structure. In this study, rice straw (RS) was pretreated using potassium permanganate (KMnO4) to enhance the enzymatic saccharification efficiency. The study was carried out by varying the operational factors in pretreatment, including temperature (30-90°C), time (30-360 min) and concentration of KMnO4 (0.5-3.0, % w/v), respectively, based on Box-Behnken design (BBD). Through multi-regression analysis of the experimental data obtained after pretreatment, the optimum conditions were determined. The optimum conditions for temperature, time and potassium permanganate concentration were 48.09°C, 360 min, and 1.36% w/v, respectively. The saccharifications of pretreatment and untreated rice straw were carried out using Cellic Ctec2. The reducing sugar was determined by using DNS method and the yields of the untreated and pretreated RS were 32.38 and 49.011 mg/mL, respectively. The results showed that the sugar for pretreated RS were 1.51 fold times higher compared to untreated RS. Therefore, this work illustrates the pretreatment efficiency for KMnO4 to enhance the reducing sugar yield during saccharification, which can be used for biofuel and value-added product productions.
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Procentese, Alessandra, Maria Elena Russo, Ilaria Di Somma, and Antonio Marzocchella. "Kinetic Characterization of Enzymatic Hydrolysis of Apple Pomace as Feedstock for a Sugar-Based Biorefinery." Energies 13, no. 5 (February 26, 2020): 1051. http://dx.doi.org/10.3390/en13051051.
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The enzymatic hydrolysis of cellulose from biomass feedstock in the sugar-based biorefinery chain is penalized by enzyme cost and difficulty to approach the theoretical maximum cellulose conversion degree. As a consequence, the process is currently investigated to identify the best operating conditions with reference to each biomass feedstock. The present work reports an investigation regarding the enzymatic hydrolysis of apple pomace (AP). AP is an agro-food waste largely available in Europe that might be exploited as a sugar source for biorefinery purposes. A biomass pre-treatment step was required before the enzymatic hydrolysis to make available polysaccharides chains to the biocatalyst. The AP samples were pre-treated through alkaline (NaOH), acid (HCl), and enzymatic (laccase) delignification processes to investigate the effect of lignin content and polysaccharides composition on enzymatic hydrolysis. Enzymatic hydrolysis tests were carried out using a commercial cocktail (Cellic®CTec2) of cellulolytic enzymes. The effect of mixing speed and biomass concentration on the experimental overall glucose production rate was assessed. The characterization of the glucose production rate by the assessment of pseudo-homogeneous kinetic models was proposed. Data were analysed to assess kinetic parameters of pseudo-mechanistic models able to describe the glucose production rate during AP enzymatic hydrolysis. In particular, pseudo-homogeneous Michaelis and Menten, as well as Chrastil’s models were used. The effect of lignin content on the enzymatic hydrolysis rate was evaluated. Chrastil’s model provided the best description of the glucose production rate.
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Kongkeitkajorn, Mallika Boonmee, Chanpim Sae-Kuay, and Alissara Reungsang. "Evaluation of Napier Grass for Bioethanol Production through a Fermentation Process." Processes 8, no. 5 (May 11, 2020): 567. http://dx.doi.org/10.3390/pr8050567.
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Ethanol is one of the widely used liquid biofuels in the world. The move from sugar-based production into the second-generation, lignocellulosic-based production has been of interest due to an abundance of these non-edible raw materials. This study interested in the use of Napier grass (Pennisetum purpureum Schumach), a common fodder in tropical regions and is considered an energy crop, for ethanol production. In this study, we aim to evaluate the ethanol production potential from the grass and to suggest a production process based on the results obtained from the study. Pretreatments of the grass by alkali, dilute acid, and their combination prepared the grass for further hydrolysis by commercial cellulase (Cellic® CTec2). Separate hydrolysis and fermentation (SHF), and simultaneous saccharification and fermentation (SSF) techniques were investigated in ethanol production using Saccharomyces cerevisiae and Scheffersomyces shehatae, a xylose-fermenting yeast. Pretreating 15% w/v Napier grass with 1.99 M NaOH at 95.7 °C for 116 min was the best condition to prepare the grass for further enzymatic hydrolysis using the enzyme dosage of 40 Filter Paper Unit (FPU)/g for 117 h. Fermentation of enzymatic hydrolysate by S. cerevisiae via SHF resulted in the best ethanol production of 187.4 g/kg of Napier grass at 44.7 g/L ethanol concentration. The results indicated that Napier grass is a promising lignocellulosic raw material that could serve a fermentation with high ethanol concentration.
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Malgas, Samkelo, Shaunita H. Rose, Willem H. van Zyl, and Brett I. Pletschke. "Enzymatic Hydrolysis of Softwood Derived Paper Sludge by an In Vitro Recombinant Cellulase Cocktail for the Production of Fermentable Sugars." Catalysts 10, no. 7 (July 11, 2020): 775. http://dx.doi.org/10.3390/catal10070775.
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Paper sludge is an attractive biomass feedstock for bioconversion to ethanol due to its low cost and the lack of pretreatment required for its bioprocessing. This study assessed the use of a recombinant cellulase cocktail (mono-components: S. cerevisiae-derived PcBGL1B (BGL), TeCel7A (CBHI), ClCel6A (CBHII) and TrCel5A (EGII) mono-component cellulase enzymes) for the efficient saccharification of softwood-derived paper sludge to produce fermentable sugars. The paper sludge mainly contained 74.3% moisture and 89.7% (per dry mass (DM)) glucan with a crystallinity index of 91.5%. The optimal protein ratio for paper sludge hydrolysis was observed at 9.4: 30.2: 30.2: 30.2% for BGL: CBHI: CBHII: EGII. At a protein loading of 7.5 mg/g DW paper sludge, the yield from hydrolysis was approximately 80%, based on glucan, with scanning electron microscopy micrographs indicating a significant alteration in the microfibril size (length reduced from ≥ 2 mm to 93 µm) of the paper sludge. The paper sludge hydrolysis potential of the Opt CelMix (formulated cellulase cocktail) was similar to the commercial Cellic CTec2® and Celluclast® 1.5 L cellulase preparations and better than Viscozyme® L. Low enzyme loadings (15 mg/g paper sludge) of the Opt CelMix and solid loadings ranging between 1 to 10% (w/v) rendered over 80% glucan conversion. The high glucose yields attained on the paper sludge by the low enzyme loading of the Opt CelMix demonstrated the value of enzyme cocktail optimisation on specific substrates for efficient cellulose conversion to fermentable sugars.
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Fehér, Anikó, Soma Bedő, and Csaba Fehér. "Comparison of Enzymatic and Acidic Fractionation of Corn Fiber for Glucose-rich Hydrolysate and Bioethanol Production by Candida boidinii." Periodica Polytechnica Chemical Engineering 65, no. 3 (May 18, 2021): 320–30. http://dx.doi.org/10.3311/ppch.17431.
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Corn fiber is a by-product of the corn wet milling process and a promising raw material to produce bioethanol in a bio-refinery process. In this study, enzymatic and acidic fractionations of corn fiber were compared with particular attention to produce glucose-rich hydrolyzates. The acidic fractionation contained two, sequential, sulphuric acid-catalyzed, hydrolysis steps based on our previous study. In the enzymatic fractionation process, corn fiber was pre-treated by soaking in aqueous ammonia (18.5 % (w/w) dry matter, 15 % (w/w) ammonia solution, 24 hours) and then hydrolyzed by using Hemicellulase (NS 22002) enzyme cocktail. The cellulose part of the solid residues obtained after the acidic and enzymatic fractionation processes was enzymatically hydrolyzed by using Cellic Ctec2 and Novozymes 188 (12.5 % (w/w) dry matter, 50 °C, 72 hours). Cellulose hydrolysis after the acidic and enzymatic fractionation resulted in a supernatant containing 64 g/L and 25 g/L glucose, respectively. Therefore, ethanol fermentation experiments were performed in Separated Hydrolysis and Fermentation (SHF) and Simultaneous Saccharification and Fermentation (SSF) configurations after the acidic fractionation of corn fiber. SHF configuration was found to be more advantageous regarding the achievable ethanol yield. Although the fermentation with Candida boidinii NCAIM Y.01308 was accomplished within longer time (43 hours) compared to Saccharomyces cerevisiae (5 hours), the achieved ethanol yields were similar (79%) during the SHF process. It was concluded that acidic fractionation is more efficient to produce glucose-rich hydrolyzate from corn fiber compared to enzymatic fractionation, and Candida boidinii is suitable for ethanol fermentation on the glucose-rich hydrolyzate.
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Nguyen, Thuan Thi, Maria Dalgaard Mikkelsen, Vy Ha Nguyen Tran, Vo Thi Dieu Trang, Nanna Rhein-Knudsen, Jesper Holck, Anton B. Rasin, Hang Thi Thuy Cao, Tran Thi Thanh Van, and Anne S. Meyer. "Enzyme-Assisted Fucoidan Extraction from Brown Macroalgae Fucus distichus subsp. evanescens and Saccharina latissima." Marine Drugs 18, no. 6 (June 2, 2020): 296. http://dx.doi.org/10.3390/md18060296.
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Fucoidans from brown macroalgae (brown seaweeds) have different structures and many interesting bioactivities. Fucoidans are classically extracted from brown seaweeds by hot acidic extraction. Here, we report a new targeted enzyme-assisted methodology for fucoidan extraction from brown seaweeds. This enzyme-assisted extraction protocol involves a one-step combined use of a commercial cellulase preparation (Cellic®CTec2) and an alginate lyase from Sphingomonas sp. (SALy), reaction at pH 6.0, 40 °C, removal of non-fucoidan polysaccharides by Ca2+ precipitation, and ethanol-precipitation of crude fucoidan. The workability of this method is demonstrated for fucoidan extraction from Fucus distichus subsp. evanescens (basionym Fucus evanescens) and Saccharina latissima as compared with mild acidic extraction. The crude fucoidans resulting directly from the enzyme-assisted method contained considerable amounts of low molecular weight alginate, but this residual alginate was effectively removed by an additional ion-exchange chromatographic step to yield pure fucoidans (as confirmed by 1H NMR). The fucoidan yields that were obtained by the enzymatic method were comparable to the chemically extracted yields for both F. evanescens and S. latissima, but the molecular sizes of the fucoidans were significantly larger with enzyme-assisted extraction. The molecular weight distribution of the fucoidan fractions was 400 to 800 kDa for F. evanescens and 300 to 800 kDa for S. latissima, whereas the molecular weights of the corresponding chemically extracted fucoidans from these seaweeds were 10–100 kDa and 50–100 kDa, respectively. Enzyme-assisted extraction represents a new gentle strategy for fucoidan extraction and it provides new opportunities for obtaining high yields of native fucoidan structures from brown macroalgae.
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Smuga-Kogut, Małgorzata, Leszek Bychto, Bartosz Walendzik, Judyta Cielecka-Piontek, Roman Marecik, Joanna Kobus-Cisowska, Katarzyna Grajek, and Daria Szymanowska-Powałowska. "Use of Buckwheat Straw to Produce Ethyl Alcohol Using Ionic Liquids." Energies 12, no. 10 (May 26, 2019): 2014. http://dx.doi.org/10.3390/en12102014.
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Background: Common buckwheat (Fagopyrum esculentum Moench) is an annual spring-emerging crop that is classified among the dicotyledons, due to the manner of its cultivation, use, and chemical composition of seeds. The use of buckwheat straw for energy purposes—for example, for the production of second generation bioethanol—might enable its wider application and increase the cost-effectiveness of tillage. Methods: In this study, we examined the usability of buckwheat straw for the production of bioethanol. We pretreated the raw material with ionic liquids and subsequently performed enzymatic hydrolysis and alcoholic fermentation. The obtained chemometric data were analyzed using the Partial Least Squares (PLS) regression model. PLS regression in combination with spectral analysis within the near-infrared (NIR) spectrum allowed for the rapid determination of the amount of cellulose in the raw material and also provided information on the changes taking place in its structure. Results: We obtained good results for the combination of 1-ethyl-3-methylimidazolium acetate as the ionic liquid and Cellic CTec2 as the enzymatic preparation for the pretreatment of buckwheat straw. The highest concentration of glucose following 72 h of enzymatic hydrolysis was found to be around 5.5 g/dm3. The highest concentration of ethanol (3.31 g/dm3) was obtained with the combination of 1-butyl-3-methylimidazolium acetate for the pretreatment and cellulase from Trichoderma reesei for enzymatic hydrolysis. Conclusions: In summary, the efficiency of the fermentation process is strictly associated with the pool of available fermenting sugars, and it depends on the type of ionic liquid used during the pretreatment and on the enzymatic preparation. It is possible to obtain bioethanol from buckwheat straw using ionic liquid for pretreatment of the raw material prior to the enzymatic hydrolysis and alcoholic fermentation of the material.
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Krafft, Malte Jörn, Marie Bendler, Andreas Schreiber, and Bodo Saake. "Steam Refining with Subsequent Alkaline Lignin Extraction as an Alternative Pretreatment Method to Enhance the Enzymatic Digestibility of Corn Stover." Agronomy 10, no. 6 (June 8, 2020): 811. http://dx.doi.org/10.3390/agronomy10060811.
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Agricultural residues are promising and abundant feedstocks for the production of monomeric carbohydrates, which can be gained after pretreatment and enzymatic hydrolysis. These monomeric carbohydrates can be fermented to platform chemicals, like ethanol or succinic acid. Due to its high availability, corn stover is a feedstock of special interest in such considerations. The natural recalcitrance of lignocellulosic material against degradation necessitates a pretreatment before the enzymatic hydrolysis. In the present study, a novel combination of steam refining and alkaline lignin extraction was tested as a pretreatment process for corn stover. This combination combines the enhancement of the enzymatic hydrolysis and steam refining lignin can be gained for further utilization. Afterward, the obtained yields after enzymatic hydrolysis were compared with those after steam refining without alkaline extraction. After steam refining at temperatures between 160 °C and 210 °C and subsequent enzymatic hydrolysis with Cellic® CTec2, it was possible to enhance the digestibility of corn stover and to achieve 65.4% of the available carbohydrates at the lowest up to 89% at the highest conditions as monomers after enzymatic hydrolysis. Furthermore, the enzymatic degradation could be optimized with a subsequent alkaline lignin extraction, especially at low severities under three. After this combined pretreatment, it was possible to enhance the enzymatic digestibility and to achieve up to 106.4% of the available carbohydrates at the lowest conditions and up to 102.2% at the highest temperature as monomers after following enzymatic hydrolysis, compared to analytical acid hydrolysis. Regarding the utilization of the arising lignin after extraction, the lignin was characterized with regard to the molar mass and carbohydrate impurities. In this context, it was found that higher amounts and higher purities of lignin can be attained after pretreatment at severities higher than four.
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Sørensen, Annette, Peter Stephensen Lübeck, Mette Lübeck, Philip Johan Teller, and Birgitte Kiær Ahring. "β-Glucosidases from a new Aspergillus species can substitute commercial β-glucosidases for saccharification of lignocellulosic biomass." Canadian Journal of Microbiology 57, no. 8 (August 2011): 638–50. http://dx.doi.org/10.1139/w11-052.
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β-Glucosidase activity plays an essential role for efficient and complete hydrolysis of lignocellulosic biomass. Direct use of fungal fermentation broths can be cost saving relative to using commercial enzymes for production of biofuels and bioproducts. Through a fungal screening program for β-glucosidase activity, strain AP (CBS 127449, Aspergillus saccharolyticus ) showed 10 times greater β-glucosidase activity than the average of all other fungi screened, with Aspergillus niger showing second greatest activity. The potential of a fermentation broth of strain AP was compared with the commercial β-glucosidase-containing enzyme preparations Novozym 188 and Cellic CTec. The fermentation broth was found to be a valid substitute for Novozym 188 in cellobiose hydrolysis. The Michaelis–Menten kinetics affinity constant as well as performance in cellobiose hydrolysis with regard to product inhibition were found to be the same for Novozym 188 and the broth of strain AP. Compared with Novozym 188, the fermentation broth had higher specific activity (11.3 U/mg total protein compared with 7.5 U/mg total protein) and also increased thermostability, identified by the thermal activity number of 66.8 vs. 63.4 °C for Novozym 188. The significant thermostability of strain AP β-glucosidases was further confirmed when compared with Cellic CTec. The β-glucosidases of strain AP were able to degrade cellodextrins with an exo-acting approach and could hydrolyse pretreated bagasse to monomeric sugars when combined with Celluclast 1.5L. The fungus therefore showed great potential as an onsite producer for β-glucosidase activity.
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Junior, Nei Pereira, Anelize de Oliveira Moraes, Luiz Felipe Modesto, and Ninoska Isabel Bojorge Ramirez. "Reuse of Residual Biomass of Cellulose Industry for Second Generation Bioethanol Production." JOURNAL OF ADVANCES IN BIOTECHNOLOGY 6, no. 1 (January 30, 2016): 768–72. http://dx.doi.org/10.24297/jbt.v6i1.4805.
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This study aimed at evaluating the potential of pulp mill residue (PMR) as a feedstock for ethanol production. The simultaneous saccharification and fermentation (SSF) process was operated using 8 gL -1 of a commercial strain of Saccharomyces cerevisiae JP1 under optimal proportions of cellulase cocktail (24.8 FPU/g cellulose of Cellic® CTec2) and cellulosic residue (200 gL -1 ). After 48 hours of pre-hydrolysis at 50ºC and 200 rpm, the fermentation was carried out at 37 ºC, generating 48.5 gL -1 of ethanol in 10 hours and reaching a conversion efficiency of 53.3% from cellulose to ethanol and a volumetric productivity of 4.8 gL -1 h -1 that is within the range of values of first generation ethanol production (5-8 gL -1 h -1 ). These results showed that the pulp mill residue is an interesting and effective feedstock for the production of ethanol, which can be used for fuel purposes in the own pulp mills.
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Cheiwpanich, Sawitree, Navadol Laosiripojana, and Verawat Champreda. "Optimization of Organosolv Based Fractionation Process for Separation of High Purity Lignin from Bagasse." Materials Science Forum 883 (January 2017): 92–96. http://dx.doi.org/10.4028/www.scientific.net/msf.883.92.
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Lignocellulosic biomass is a renewable source for sustainable production of fuels, chemicals, and other materials with the advantages on its carbon-neutral nature. Fractionation of lignocellulosic materials is a pre-requisite in the biorefinery process in order to convert the cellulose, hemicellulose, and lignin to valuable products with maximized economics prospective. In this work, a modified clean fractionation (CF) process using ternary mixture system of ethyl acetate/methanol/water was studied with the use of acid promoters. H2SO4 was found to be the efficient promoter due to low cost compare to other acid promoters. The optimal fractionation conditions operated in the solvent mixture containing 0.025 M H2SO4 at 160°C for 50 min led to 63.72% recovery of the cellulose in the solid pulp while 90% and 59.94% of hemicellulose-derived products and lignin were recovered in the aqueous-alcohol and organic fractions, respectively. The enzymatic digestibility of the cellulose-enriched pulp was increased, resulting increasing glucose yield from 38.32% of the native biomass to 70.04% using the hydrolysis reaction with Cellic Ctech2® at 15 FPU/g. The work demonstrated the applicability of the modified CF process for fractionation of lignocellulose components for integrated biorefinery process.
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Morales-Martínez, Thelma K., Deniss I. Díaz-Blanco, José A. Rodríguez-de la Garza, Jesús Morlett-Chávez, Agustín J. Castro-Montoya, Julián Quintero, Germán Aroca, and Leopoldo J. Rios-González. "Assessment of different saccharification and fermentation configurations for ethanol production from Agave lechuguilla." BioResources 12, no. 4 (September 15, 2017): 8093–105. http://dx.doi.org/10.15376/biores.12.4.8093-8105.
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Different strategies were assessed for the production of ethanol from Agave lechuguilla that was pretreated by autohydrolysis. Separate hydrolysis and fermentation (SHF) was compared against simultaneous processes including simultaneous saccharification and fermentation (SSF) and prehydrolysis and simultaneous saccharification and fermentation (PSSF) using different solids (15%, 20%, and 25% w/w) and enzyme loadings (15 FPU/g, 20 FPU/g, and 25 FPU/g glucan). The results showed that the maximum ethanol concentration (53.7 g/L) and productivity (1.49 g/L h-1) was obtained at 36 h in the SHF configuration at the highest solids and enzyme loadings (25% w/v and 25 FPU/g glucan, respectively). The ethanol concentration and productivity obtained in the PSSF configuration at the same time were 45 g/L and 1.25 g/L h-1, respectively. The SSF configuration exhibited the lowest ethanol concentration and productivity (10.4 g/L and 0.29 g/L h-1, respectively) at 36 h. The enzyme used, Cellic CTec3, allowed for high glucose yields at the lower enzyme dosage assessed. The SHF configuration exhibited the best results. However, the PSSF configuration can be considered an attractive alternative because it eliminated the need for solid-liquid separation devices, which simplifies the industrial implementation of the process.
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De Bari, Isabella, Federico Liuzzi, Alfredo Ambrico, and Mario Trupo. "Arundo donax Refining to Second Generation Bioethanol and Furfural." Processes 8, no. 12 (December 3, 2020): 1591. http://dx.doi.org/10.3390/pr8121591.
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Biomass-derived sugars are platform molecules that can be converted into a variety of final products. Non-food, lignocellulosic feedstocks, such as agroforest residues and low inputs, high yield crops, are attractive bioresources for the production of second-generation sugars. Biorefining schemes based on the use of versatile technologies that operate at mild conditions contribute to the sustainability of the bio-based products. The present work describes the conversion of giant reed (Arundo donax), a non-food crop, to ethanol and furfural (FA). A sulphuric-acid-catalyzed steam explosion was used for the biomass pretreatment and fractionation. A hybrid process was optimized for the hydrolysis and fermentation (HSSF) of C6 sugars at high gravity conditions consisting of a biomass pre-liquefaction followed by simultaneous saccharification and fermentation with a step-wise temperature program and multiple inoculations. Hemicellulose derived xylose was dehydrated to furfural on the solid acid catalyst in biphasic media irradiated by microwave energy. The results indicate that the optimized HSSF process produced ethanol titers in the range 43–51 g/L depending on the enzymatic dosage, about 13–21 g/L higher than unoptimized conditions. An optimal liquefaction time before saccharification and fermentation tests (SSF) was 10 h by using 34 filter paper unit (FPU)/g glucan of Cellic® CTec3. C5 streams yielded 33.5% FA of the theoretical value after 10 min of microwave heating at 157 °C and a catalyst concentration of 14 meq per g of xylose.
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Reyna-Martínez, Ricardo, Thelma K. Martínez-Morales, David Castillo Quroz, Juan C. Contreras-Esquivel, and Leopoldo J. Ríos-González. "Pretratamiento fúngico de biomasa de Agave lechuguilla Torr. para la producción de etanol." Revista Mexicana de Ciencias Forestales 10, no. 51 (January 22, 2019): 86–106. http://dx.doi.org/10.29298/rmcf.v10i51.336.
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Recientemente la biomasa del cogollo de Agave lechuguilla ha sido establecida como materia prima con potencial para la producción de etanol, sin embargo, el alto gasto energético en el pretratamiento requiere la búsqueda de métodos que lo minimicen y propicie una mejora en la factibilidad económica del proceso. En comparación con las tecnologías tradicionales, el pretratamiento biológico ofrece una alternativa en la que las enzimas de remoción de la lignina son capaces de desdoblar las estructuras complejas de la misma, sin el uso de químicos y con menor gasto energético. En este trabajo se probó el uso de Phanerochaete chrysosporium H-298 en la biomasa de Agave lechuguilla. Los dos factores ensayados para la optimización del proceso fueron el tiempo de incubación y la concentración de la fuente de nitrógeno. Los resultados mostraron una máxima deslignificación (36.15 %), se preservó la celulosa sin cambios significativos. Las condiciones óptimas de pretratamiento fueron: 60 días de incubación y una concentración de nitrógeno de 1M. La hidrólisis enzimática del material pretratado con el complejo enzimático Cellic® CTec3 mostró una máxima liberación de glucosa de 44.9 g L-1 a las 92 horas, con rendimiento de hidrólisis de 93.09 %, mayor al obtenido en la hidrólisis de la muestra sin pretratar (37.92 %). La concentración de etanol a las 10 horas de fermentación fue de 16.53 g L-1 (equivalente a una concentración >2 % v/v de etanol) con 5.7 g L-1 de glucosa remanente a ese tiempo de incubación.
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Johansen, Katja S. "Discovery and industrial applications of lytic polysaccharide mono-oxygenases." Biochemical Society Transactions 44, no. 1 (February 9, 2016): 143–49. http://dx.doi.org/10.1042/bst20150204.
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The recent discovery of copper-dependent lytic polysaccharide mono-oxygenases (LPMOs) has opened up a vast area of research covering several fields of application. The biotech company Novozymes A/S holds patents on the use of these enzymes for the conversion of steam-pre-treated plant residues such as straw to free sugars. These patents predate the correct classification of LPMOs and the striking synergistic effect of fungal LPMOs when combined with canonical cellulases was discovered when fractions of fungal secretomes were evaluated in industrially relevant enzyme performance assays. Today, LPMOs are a central component in the Cellic CTec enzyme products which are used in several large-scale plants for the industrial production of lignocellulosic ethanol. LPMOs are characterized by an N-terminal histidine residue which, together with an internal histidine and a tyrosine residue, co-ordinates a single copper atom in a so-called histidine brace. The mechanism by which oxygen binds to the reduced copper atom has been reported and the general mechanism of copper–oxygen-mediated activation of carbon is being investigated in the light of these discoveries. LPMOs are widespread in both the fungal and the bacterial kingdoms, although the range of action of these enzymes remains to be elucidated. However, based on the high abundance of LPMOs expressed by microbes involved in the decomposition of organic matter, the importance of LPMOs in the natural carbon-cycle is predicted to be significant. In addition, it has been suggested that LPMOs play a role in the pathology of infectious diseases such as cholera and to thus be relevant in the field of medicine.
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Cheunsuk, Saijai, Zhe-Xiong Lian, Guo-Xiang Yang, M. Eric Gershwin, Jeffrey R. Gruen, and Christopher L. Bowlus. "Prss16 Is Not Required for T-Cell Development." Molecular and Cellular Biology 25, no. 2 (January 15, 2005): 789–96. http://dx.doi.org/10.1128/mcb.25.2.789-796.2005.
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ABSTRACT PRSS16 is a serine protease expressed exclusively in cortical thymic epithelial cells (cTEC) of the thymus, suggesting that it plays a role in the processing of peptide antigens during the positive selection of T cells. Moreover, the human PRSS16 gene is encoded in a region near the class I major histocompatibility complex (MHC) that has been linked to type 1 diabetes mellitus susceptibility. The mouse orthologue Prss16 is conserved in genetic structure, sequence, and pattern of expression. To study the role of Prss16 in thymic development, we generated a deletion mutant of Prss16 and characterized T-lymphocyte populations and MHC class II expression on cortical thymic epithelial cells. Prss16-deficient mice develop normally, are fertile, and show normal thymic morphology, cellularity, and anatomy. The total numbers and frequencies of thymocytes and splenic T-cell populations did not differ from those of wild-type controls. Surface expression of MHC class II on cTEC was also similar in homozygous mutant and wild-type animals, and invariant chain degradation was not impaired by deletion of Prss16. These findings suggest that Prss16 is not required for quantitatively normal T-cell development.
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Patil, Ashwini M., Stefanie Kesper, Vishal Khairnar, Marco Luciani, Michael Möllmann, Ulrich Dührsen, and Joachim R. Göthert. "A CXCL10/CXCR3 Driven Thymic Epithelium-Leukemia Cell Crosstalk Augments T Cell Acute Lymphoblastic Leukemia Notch1 Signalling." Blood 134, Supplement_1 (November 13, 2019): 2537. http://dx.doi.org/10.1182/blood-2019-125331.
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Introduction: The thymus is a specialized hematopoietic organ, which is responsible for the generation of T cells. The central thymic cell type controlling T cell development are thymic epithelial cells (TECs). Based on their specific function and anatomic location TECs are separated into cortical and medullary subsets (cTECs and mTECs). cTECs express pivotal NOTCH-ligands such as DLL4 controlling T cell lineage commitment while mTECs play a central role in negative selection of developing T cells. Acquisition of NOTCH1 gain-of-function mutations play a central role in acute T cell lymphoblastic leukemia (T-ALL) development. During T-ALL leukemogenesis aberrant expression of transcription factors such as SCL/TAL1 and LMO1 block T cell differentiation and increase self-renewal while NOTCH1 mutations promote survival and proliferation. Since most acquired NOTCH1 mutations still require ligand binding to exert augmented signaling we propose DLL4-expressing TECs playing a critical role during T-ALL leukemogenesis. Methods: In the present study, we used a Scl/Lmo1 T-ALL transgenic mouse model, murine ANV and TE71 TEC cell lines and human T-ALL cell lines (Jurkat, ALL-SIL, DND-41, and HPB-ALL) to investigate TEC dynamics and function in the T-ALL context. Results: First, we demonstrated T-ALL supporting potential of TEC cell lines in vitro, which was comparable to the mesenchymal cell line OP9. Next, we showed in the Scl/Lmo1 T-ALL mouse model which had a mean survival rate of 90 days that preleukemic thymocytes displayed a striking upregulation of Notch1 target genes. Interestingly, fluorescence microscopy revealed a relative expansion of cortical and a relative reduction of the medullary thymic areas in Scl/Lmo1 thymi (Fig. 1A). Correspondingly, absolute numbers of cTECs expanded while mTEC numbers declined (Fig. 1B). Gene expression profiling of sorted preleukemic Scl/Lmo1 cTECs revealed upregulation of the chemokine CXCL10 (Fig. 1C). Moreover, increased CXCL10 chemokine concentrations were detected in Scl/Lmo1 thymic interstitial fluid (Fig.1D). Strikingly, we demonstrated T-ALL dependence of TEC Cxcl10 upregulation. We showed that Cxcl10 upregulation in TEC cell lines was only induced by direct cellular contact with Scl/Lmo1 thymocytes while wild-type control thymocytes did not alter TEC cell line Cxcl10 expression (Fig. 1E). Next, a high proportion of the CXCL10 receptor CXCR3 expressing cells was revealed in Scl/Lmo1 thymi (Fig. 1F) and by human T-ALL cell lines. Finally, we demonstrated a CXCL10 dependent pro-survival effect within cultured SCL/LMO1 thymocytes (Fig. 1G), which was associated with the activation of NOTCH1 signaling (Fig. 1H). Conclusions: In summary, the data support a novel T-ALL-promoting regulatory circuit in which emerging T-ALL lymphoblasts induce CXCL10 in expanding TECs which positively feeds back to T-ALL cells via the CXCL10 receptor CXCR3. Disclosures Dührsen: Celgene: Research Funding; Takeda: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Teva: Honoraria; Novartis: Consultancy, Honoraria; Alexion: Honoraria; Roche: Honoraria, Research Funding; CPT: Consultancy, Honoraria; Janssen: Honoraria. Göthert:Proteros Biostructures: Consultancy; Novartis: Consultancy, Honoraria, Other: Travel support; Pfizer: Consultancy, Honoraria; Incyte: Consultancy, Honoraria, Other: Travel support; Bristol-Myers Squibb: Consultancy, Honoraria, Other: Travel support; AOP Orphan Pharmaceuticals: Honoraria, Other: Travel support.
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Dudakov, Jarrod A., Alan M. Hanash, Lauren F. Young, Natalie V. Singer, Mallory L. West, Robert R. Jenq, Odette M. Smith, Amanda M. Holland, Richard L. Boyd, and Marcel R. M. van den Brink. "Innate Lymphoid Cell-Derived IL-22 Mediates Endogenous Thymic Repair Under the Control of IL-23." Blood 118, no. 21 (November 18, 2011): 143. http://dx.doi.org/10.1182/blood.v118.21.143.143.
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Abstract Abstract 143 Despite being exquisitely sensitive to insult, the thymus is remarkably resilient in young healthy animals. Endogenous regeneration of the thymus is a crucial function that allows for renewal of immune competence following infection or immunodepletion caused by cytoreductive chemotherapy or radiation. However, the mechanisms governing this regeneration remain poorly understood. Thymopoiesis is a highly complex process involving cross-talk between developing thymocytes and their supporting non-hematopoietic stromal microenvironment, which includes highly specialized thymic epithelial cells (TECs) that are crucial for T cell development. IL-22 is a recently identified cytokine predominantly associated with maintenance of barrier function at mucosal surfaces. Here we demonstrate for the first time a critical role for IL-22 in endogenous thymic repair. Comparing IL-22 KO and WT mice we observed that while IL-22 deficiency was redundant for steady-state thymopoiesis, it led to a pronounced and prolonged loss of thymus cellularity following sublethal total body irradiation (SL-TBI), which included depletion of both thymocytes (p=0.0001) and TECs (p=0.003). Strikingly, absolute levels of IL-22 were markedly increased following thymic insult (p<0.0001) despite the significant depletion of thymus cellularity. This resulted in a profound increase in the production of IL-22 on a per cell basis (p<0.0001). These enhanced levels of IL-22 peaked at days 5 to 7 after SL-TBI, immediately following the nadir of thymic cellularity. This was demonstrated by a strong negative correlation between thymic cellularity and absolute levels of IL-22 (Fig 1a). In mucosal tissues the regulation of IL-22 production has been closely associated with IL-23 produced by dendritic cells (DCs) and ex vivo incubation of cells with IL-23 stimulates the production of IL-22. Following thymic insult there was a significant increase in the amount of IL-23 produced by DCs (Fig 1b) resulting in similar kinetics of intrathymic levels of IL-22 and IL-23. We identified a population of radio-resistant CD3−CD4+IL7Ra+RORg(t)+ thymic innate lymphoid cells (tILCs) that upregulate both their production of IL-22 (Fig 1c) and expression of the IL-23R (p=0.0006) upon exposure to TBI. This suggests that they are responsive to IL-23 produced by DCs in vivo following TBI and, in fact, in vitro stimulation of tILCs by IL-23 led to upregulation of Il-22 production by these cells (Fig 1d). We found expression of the IL-22Ra on cortical and medullary TECs (cTECs and mTECs, respectively), and uniform expression across both mature MHCIIhi mTEC (mTEChi) and immature MHCIIlo mTECs (mTEClo). However, in vitro stimulation of TECs with recombinant IL-22 led to enhanced TEC proliferation primarily in cTEC and mTEClo subsets (p=0.002 and 0.004 respectively). It is currently unclear if IL-22 acts as a maturation signal for mTECs, however, the uniform expression of IL-22Ra between immature mTEClo and mature Aire-expressing mTEChi, together with the preferential promotion of proliferation amongst mTEClo and cTEC seem to argue against IL-22 as a maturational signal but rather as promoter of proliferation, which ultimately leads to terminal differentiation of TECs. Of major clinical importance, administration of exogenous IL-22 led to enhanced thymic recovery (Fig. 1e) following TBI, primarily by promoting the proliferation of TECs. Consistent with this, the administration of IL-22 also led to significantly enhanced thymopoiesis following syngeneic BMT. Taken together these findings suggest that following thymic insult, and specifically the depletion of developing thymocytes, upregulation of IL-23 by DCs induces the production of IL-22 by tILCs and regeneration of the supporting microenvironment. This cascade of events ultimately leads to rejuvenation of the thymocyte pool (Fig. 1f). These studies not only reveal a novel pathway underlying endogenous thymic regeneration, but also identify a novel regenerative strategy for improving immune competence in patients whose thymus has been damaged from infection, age or cytoreductive conditioning required for successful hematopoietic stem cell transplantation. Finally, these findings may also provide an avenue of study to further understand the repair and regeneration of other epithelial tissues such as skin, lung and breast. Disclosures: No relevant conflicts of interest to declare.
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Klyuchnikov, Evgeny, Ute-Marie von Pein, Francis A. Ayuk, Maximilian Christopeit, Raissa Adjalle, Andre van Randenborgh, Christine Wolschke, and Nicolaus Kröger. "Daratumumab Is an Effective and Safe Salvage Therapy in Relapsed/Refractory Patients with Multiple Myeloma after Allogeneic Stem Cell Transplantation." Blood 128, no. 22 (December 2, 2016): 3437. http://dx.doi.org/10.1182/blood.v128.22.3437.3437.
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Abstract Daratumumab is a human monoclonal antibody that targets CD38, a cell surface protein that is overexpressed on multiple myeloma cells. Some clinical studies have shown encouraging efficacy and acceptable safety profile of daratumumab, so that the drug became the first monoclonal antibody as single agent therapy approved by the FDA for the treatment of multiple myeloma. The role of allo-SCT in myeloma patients remains unclear; nevertheless, the registry study of European Society for Blood and Marrow Transplantation (EBMT) suggests an increasing rate of allografts in Europe in last years. Despite the potentially curative potential of this approach, the increased relapse rate and low PFS remain a central clinical problem. In this single center retrospective analysis, we report on our experience on the use of daratumumab in relapsed/refractory myeloma patients after allo-SCT. A total of 10 patients (male, n=5) with median age of 59 years (range, 37-69) relapsing after allo-SCTs that had been performed during a period 2008-2015 at the University of Hamburg and received daratumumab as single agent salvage therapy. Before allografting 8 patients received one and 2 patients ≥2 autografts, respectively. All but one patient received at least 1 salvage therapy line prior to the allo-SCT. The allografts were performed from unrelated donors (MUD, n=5; MMUD, n=3) or matched related donors (MRD, n=2). Five patients experienced early relapses (≤12 months) after allo-SCT. The median number of salvage lines post-transplant and prior to first daratumumab infusion was 3 (range, 1-4). The salvage regimens included bortezomib, lenalidomide, pomalidomide and carfilzomib. Daratumumab infusions were started at a median of 21 months (range, 2-30) after relapse/progress. The median number of infusions was 6 (range, 2-12). A total of 10 and 9 patients were available to safety and efficacy evaluation, respectively. The safety was assessed according to the Common Toxicity Criteria (CTC). A total of 14 adverse events were observed in 9 patients: dyspnea (CTC1, n=2; CTC2, n=1), bronchospasm (CTC2, n=1) shivering (CTC1, n=3), cough (CTC1, n=1; CTC2, n=1), musculoskeletal pain (CTC1, n=2), acute coronary syndrome (CTC3, n=1), skin rush (CTC2, n=1), pressure on eyes (n=1). There were no cases of hematologic toxicity. There were no cases of GvHD. The adverse events appeared in all patients after the first infusion, with improved tolerance of following infusions. There were no cases, where the therapy had to be stopped due to adverse events. Within a median follow-up of 25 months (range, 3-38) from the relapse/progression 9 of 10 patients remain alive. One patient died due to severe infection after progress of myeloma. A total of 5 of 9 evaluable patients responded (56%), of those 3 of 5 patients with early relapses (PR, n=2; vgPR, n=1). The responses (decrease of paraprotein and/or free light chains; reduction of extramedullary tumor in 1 patient) occurred at a median of 7 days (range, 7-22) after the first administration of daratumumab. The median response duration is 35 days (range, 7-84). All responding and 2 non-responding patients showed clinical improvement of constitutional symptoms. No patients required blood or platelets transfusions during and after the therapy. All responding patients maintain their responses 7, 14, 35, 54 and 84 days after the first administration of daratumumab. Daratumumab demonstrated encouraging efficacy in relapsed/refractory patients with myeloma after allo-SCT. The administrations of the drug in these heavily pre-treated patients were associated with good safety profile and development in majority of cases non-severe adverse events mostly after the first infusion. Further studies on the use of daratumumab in post-transplant setting are warranted. Disclosures Kröger: Neovii: Honoraria, Research Funding; Riemser: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding.
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Kinsella, Sinéad, Cindy Evandy, Kayla S. Hopwo, Kirsten Cooper, Lorenzo Iovino, Colton W. Smith, Reema Jain, Paul DeRoos, and Jarrod A. Dudakov. "Damage-Induced Pyroptotic Cell Death Facilitates Regeneration of the Thymus." Blood 136, Supplement 1 (November 5, 2020): 28. http://dx.doi.org/10.1182/blood-2020-143051.
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T cell reconstitution after transplant is critically dependent on the thymus; an inverse relationship between a transplant recipient's age and their capacity to generate T lymphocytes (in particular CD4+T cells) has been found in several studies, and thymic function pre-transplant can have a significant impact on clinical outcomes. Although the thymus has a remarkable ability to repair following damage, the mechanisms underlying this endogenous regeneration remain poorly understood. Despite this regenerative capacity, delayed T cell reconstitution is associated with an increased risk of infections, relapse of malignancy and the development of secondary malignancies. Therefore, there is a clinical demand for therapeutics that restore immune function after damage. Our recent studies have identified two key pathways driving thymic regeneration; centered on the secretion of BMP4 by endothelial cells (ECs) and IL-22 by innate lymphoid cells (Dudakov 2012 Science 336:91; Dudakov 2017 Blood130:933; Wertheimer 2018 Sci Immunol3:19). However, the specific regulatory mechanisms that trigger these regeneration-associated factors after damage remain unclear. Our previous work identified that the presence of homeostatic apoptotic CD4+CD8+ (DP) thymocytes, as apoptotic thymocytes form the bulk of developing T cells, suppress the production of IL-23 in dendritic cells (DCs), a key downstream mediator for IL-22, and BMP4 in ECs (Fig. 1A), and that the depletion of apoptotic thymocytes after damage precedes the production of these regenerative factors. Therefore, together with our findings that the metabolic needs of key thymus populations alter drastically following injury due to damage-induced metabolic remodeling, we hypothesized that further to the loss of DP-specific suppression, metabolic dysfunction in DPs after damage triggers mitochondrial-induced pyroptotic cell death, which can directly promote regeneration of the thymus. Consistent with this hypothesis, our preliminary data shows increased levels of cl-caspase 1 (pyroptotic caspase) and a decrease in cl-caspase 3 (apoptotic caspase) in DPs after SL-TBI (550 cGy), demonstrating a preferential induction of pyroptotic cell death in DPs after damage (Fig. 1B). Furthermore, we demonstrated an increase in extracellular lactate dehydrogenase (LDH) levels, HMGB-1 and TNF⍺[canonical damage-associated molecular patterns (DAMPs) released during ICD] acutely after damage caused by SL-TBI (Fig. 1C).Given our previous findings that stromal cells are more radio-resistant than DP thymocytes (Wertheimer 2018 Sci Immunol3:19), and evidence for mitochondrial-induced pyroptosis, we identified hyperpolarization of the mitochondrial membrane potential accompanied by increased levels of ROS in DPs, an effect not observed in TECs, suggesting metabolic stability confers protection against acute damage (Fig. 1D). Furthermore, co-culture of pyroptotic thymocytes results in increased IL12p40+ DCs and increased Foxn1 expression in TECs (Fig. 1E), strengthening our hypothesis that cell-cell communication drives thymic regeneration after damage by inducing regenerative factors as well as directly promoting TEC function via secreted factors from pyroptotic DPs. One way in which DAMPs, such as ATP, can initiate cell signaling is by the activation of cell surface purinergic receptors, including P2Y2 which is widely expressed on TECs, and here we demonstrate that in vitro treatment with ATP or P2Y2 agonist increases Foxn1 in cTECs, and P2Y2 antagonism reverses this effect (Fig 1F). As P2Y2 activation promotes Ca2+efflux from the ER, we have further demonstrated that stimulating the intracellular release of Ca2+, using tunicamycin, induced Foxn1 expression in cTECs, which was reversed upon inhibition of Ca2+release (Fig. 1G). Importantly, we demonstrate here that this pathway can be therapeutically targeted by activating P2Y2 signaling in vivo with MRS2568 or ATP enhances thymus cellularity and expands cTECs in models of acute injury (Fig. 1H&I). These findings not only reveal a novel metabolic-mediated molecular mechanism governing tissue regeneration; but also by targeting FOXN1 directly offers a potentially superior therapeutic strategy for boosting thymic regeneration and T cell reconstitution after damage such as that caused by HCT, infection or cytoreductive therapy. Disclosures No relevant conflicts of interest to declare.
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Cuddihy, Andrew R., Shundi Ge, Judy Zhu, Julie Jang, Ann Chidgey, Gavin Thurston, Richard Boyd, and Gay M. Crooks. "VEGF-mediated cross-talk within the neonatal murine thymus." Blood 113, no. 12 (March 19, 2009): 2723–31. http://dx.doi.org/10.1182/blood-2008-06-162040.
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Abstract Although the mechanisms of cross-talk that regulate the hematopoietic and epithelial compartments of the thymus are well established, the interactions of these compartments with the thymic endothelium have been largely ignored. Current understanding of the thymic vasculature is based on studies of adult thymus. We show that the neonatal period represents a unique phase of thymic growth and differentiation, marked by endothelium that is organized as primitive, dense networks of capillaries dependent on vascular endothelial growth factor (VEGF). VEGF dependence in neonates is mediated by significantly higher levels of both VEGF production and endothelial VEGF receptor 2 (VEGF-R2) expression than in the adult thymus. VEGF is expressed locally in the neonatal thymus by immature, CD4−CD8− “double negative” (DN) thymocytes and thymic epithelium. Relative to adult thymus, the neonatal thymus has greater thymocyte proliferation, and a predominance of immature thymocytes and cortical thymic epithelial cells (cTECs). Inhibition of VEGF signaling during the neonatal period results in rapid loss of the dense capillaries in the thymus and a marked reduction in the number of thymocytes. These data demonstrate that, during the early postnatal period, VEGF mediates cross-talk between the thymocyte and endothelial compartments of the thymus.
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Thanapimmetha, Anusith, Suwatipat Tiyanusorn, Penjit Srinophakun, and Maythee Saisriyoot. "Reducing Sugar Production from Empty Fruit Bunches with Enzyme Cellic Ctec2®." Journal of King Mongkut's University of Technology North Bangkok, March 23, 2018. http://dx.doi.org/10.14416/j.kmutnb.2018.03.009.
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Jocquel, Chloé, Murielle Muzard, Richard Plantier-Royon, and Caroline Rémond. "An Integrated Enzymatic Approach to Produce Pentyl Xylosides and Glucose/Xylose Laurate Esters From Wheat Bran." Frontiers in Bioengineering and Biotechnology 9 (April 7, 2021). http://dx.doi.org/10.3389/fbioe.2021.647442.
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Alkyl glycosides and sugars esters are non-ionic surfactants of interest for various applications (cosmetics, food, detergency,…). In the present study, xylans and cellulose from wheat bran were enzymatically converted into pentyl xylosides and glucose and xylose laurate monoesters. Transglycosylation reaction catalyzed by the commercial enzymatic cocktail Cellic Ctec2 in the presence of pentanol led to the synthesis of pentyl β-D-xylosides from DP1 to 3 with an overall yield of 520 mg/g of xylans present in wheat bran. Enzymatic hydrolysis of wheat bran with Cellic Ctec2 and subsequent acylation of the recovered D-glucose and D-xylose catalyzed by the commercial lipase N435 in the presence of lauric acid or methyl laurate produced one D-glucose laurate monoester and one D-xylose laurate monoester. An integrated approach combining transglycosylation and (trans)esterification reactions was successfully developed to produce both pentyl xylosides and D-glucose and D-xylose laurate esters from the same batch of wheat bran.
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Ribes, Débora Duarte, Paula Zanatta, Darci Alberto Gatto, Washington Luiz Esteves Magalães, and Rafael Beltrame. "Produção de suspensões nanofibrilares de celulose vegetal por meio de processo combinado – Avaliação do gasto energético." Matéria (Rio de Janeiro) 23, no. 4 (2018). http://dx.doi.org/10.1590/s1517-707620180004.0603.
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RESUMO O presente estudo tem por objetivo produzir nanofibrilas de celulose vegetal com polpa Kraft marrom e branqueada por meio de um pré-tratamento enzimático e posteriormente um mecânico, denominado assim como processo combinado, com o intuito de diminuição de gasto energético. Para isso, as amostras de polpas de celulose foram previamente desestruturadas e encharcadas em água destilada com concentração de 3% de sólidos, afim de homogeneizá-las. Na hidrólise enzimática utilizou-se a enzima comercial Cellic CTec2, em porcentagens que variaram de 0,01 a 0,1% por diferentes períodos (1 e 2 horas). Após o tempo de hidrólise, a polpa foi passada em um moinho de discos, onde nesse processo ocorreu a medida do consumo do gasto energético com auxílio de um medidor de energia. Para a produção do gel, as polpas foram processadas pelo moinho de disco em ciclos de passagens. Para a inibição enzimática o conteúdo foi aquecido à 85°C. Os géis foram armazenados em resfriamento de 5°C. O processo foi caracterizado pelo gasto energético medido a cada amostra, além de ser medido o rendimento de cada gel produzido. De acordo com os resultados o processo combinado, apresentou-se viável para a produção de nanofibrilas de celulose vegetal, pois este diminui o gasto energético comparado com o processo mecânico e com a menor carga enzimática (0,01%). A polpa marrom mesmo sem ter passado por processos de deslignificação mostrou-se promissora na produção das nanofibrilas de celulose vegetal.
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Abdelazim, Mohamed. "A Simplified Procedure for Cellulase Filter Paper Assay." International Research Journal of Pure and Applied Chemistry, July 14, 2021, 60–64. http://dx.doi.org/10.9734/irjpac/2021/v22i530410.
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A new procedures to minimize labor intensiveness and complexity that has long been recognized in cellulase filter paper activity measurement (FPAase) described by the international union of pure and applied chemistry (IUPAC) was developed. It follows the main idea of IUPAC finding at least two cellulase dilutions have optical densities slightly more and less than a reference optical density of an arbitrary fixed 2 mg absolute glucose amount after a red-ox color reaction and due to cellulase-filter paper hydrolysis. The yielding glucose amount difference as compared to this reference is expressed in terms of absorbance difference percent determined by 3,5-dinitrosalicylic acid (DNS), in case of cellulase is cellubiase rich. If not, an external supplemental portion should be added. The intersection of the line of these two cellulase dilutions with abscissa intersect a vertical at a hypothetical 0% absorbance difference percent corresponding to a critical cellulase dilution exactly release this fixed arbitrary 2 mg glucose amount value. The factor 0.37 of this critical cellulase dilution equals to its filter paper units expressed in FPU per ml. A cellulase Cellic Ctech2 from novozymes has been tested with this methodology giving a satisfactory results with IUPAC procedures.
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Peciulyte, Ausra, Nikolaos Xafenias, Mats Galbe, Brian R. Scott, Lisbeth Olsson, and Katja S. Johansen. "Storage and handling of pretreated lignocellulose affects the redox chemistry during subsequent enzymatic saccharification." Bioresources and Bioprocessing 7, no. 1 (December 2020). http://dx.doi.org/10.1186/s40643-020-00353-3.
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AbstractThe decomposition of lignocellulose in nature, as well as when used as feedstock in industrial settings, takes place in a dynamic system of biotic and abiotic reactions. In the present study, the impact of abiotic reactions during the storage of pretreated lignocellulose on the efficiency of subsequent saccharification was investigated. Abiotic decarboxylation was higher in steam-pretreated wheat straw (SWS, up till 1.5% CO2) than in dilute-acid-catalysed steam-pretreated forestry residue (SFR, up till 3.2% CO2) which could be due to higher iron content in SFR and there was no significant CO2 production in warm-water-washed slurries. Unwashed slurries rapidly consumed O2 during incubation at 50 °C; the behaviour was more dependent on storage conditions in case of SWS than SFR slurries. There was a pH drop in the slurries which did not correlate with acetic acid release. Storage of SWS under aerobic conditions led to oxidation of the substrate and reduced the extent of enzymatic saccharification by Cellic® CTec3. Catalase had no effect on the fractional conversion of the aerobically stored substrate, suggesting that the lower fractional conversion was due to reduced activity of the lytic polysaccharide monooxygenase component during saccharification. The fractional conversion of SFR was low in all cases, and cellulose hydrolysis ceased before the first sampling point. This was possibly due to excessive pretreatment of the forest residues. The conditions at which pretreated lignocellulose are stored after pretreatment significantly influenced the extent and kind of abiotic reactions that take place during storage. This in turn influenced the efficiency of subsequent saccharification. Pretreated substrates for laboratory testing must, therefore, be stored in a manner that minimizes abiotic oxidation to ensure that the properties of the substrate resemble those in an industrial setting, where pretreated lignocellulose is fed almost directly into the saccharification vessel.
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Kadić, Adnan, Anikó Várnai, Vincent G. H. Eijsink, Svein Jarle Horn, and Gunnar Lidén. "In situ measurements of oxidation–reduction potential and hydrogen peroxide concentration as tools for revealing LPMO inactivation during enzymatic saccharification of cellulose." Biotechnology for Biofuels 14, no. 1 (February 18, 2021). http://dx.doi.org/10.1186/s13068-021-01894-1.
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Abstract Background Biochemical conversion of lignocellulosic biomass to simple sugars at commercial scale is hampered by the high cost of saccharifying enzymes. Lytic polysaccharide monooxygenases (LPMOs) may hold the key to overcome economic barriers. Recent studies have shown that controlled activation of LPMOs by a continuous H2O2 supply can boost saccharification yields, while overdosing H2O2 may lead to enzyme inactivation and reduce overall sugar yields. While following LPMO action by ex situ analysis of LPMO products confirms enzyme inactivation, currently no preventive measures are available to intervene before complete inactivation. Results Here, we carried out enzymatic saccharification of the model cellulose Avicel with an LPMO-containing enzyme preparation (Cellic CTec3) and H2O2 feed at 1 L bioreactor scale and followed the oxidation–reduction potential and H2O2 concentration in situ with corresponding electrode probes. The rate of oxidation of the reductant as well as the estimation of the amount of H2O2 consumed by LPMOs indicate that, in addition to oxidative depolymerization of cellulose, LPMOs consume H2O2 in a futile non-catalytic cycle, and that inactivation of LPMOs happens gradually and starts long before the accumulation of LPMO-generated oxidative products comes to a halt. Conclusion Our results indicate that, in this model system, the collapse of the LPMO-catalyzed reaction may be predicted by the rate of oxidation of the reductant, the accumulation of H2O2 in the reactor or, indirectly, by a clear increase in the oxidation–reduction potential. Being able to monitor the state of the LPMO activity in situ may help maximizing the benefit of LPMO action during saccharification. Overcoming enzyme inactivation could allow improving overall saccharification yields beyond the state of the art while lowering LPMO and, potentially, cellulase loads, both of which would have beneficial consequences on process economics.
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Montero-Herradón, Sara, and Agustín G. Zapata. "Delayed maturation of thymic epithelium in mice with specific deletion of β-catenin gene in FoxN1 positive cells." Histochemistry and Cell Biology, July 12, 2021. http://dx.doi.org/10.1007/s00418-021-02012-w.
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AbstractWnt signalling pathways have been reported to be involved in thymus development but their precise role in the development of both thymic epithelium (TE) and thymocytes is controversial. Herein, we examined embryonic, postnatal and adult thymi of mice with a specific deletion of β-catenin gene in FoxN1+ thymic epithelial cells (TECs). Together with a high postnatal mouse mortality, the analysis showed severe thymic hypocellularity, largely due an important reduction in numbers of developing thymocytes, and delayed, partially blocked maturation of mutant TECs. Affected TECs included largely cortical (c) TEC subsets, such as immature MTS20+ TECs, Ly51+ cTECs and a remarkable, rare Ly51+MTS20+MHCIIhi cell subpopulation previously reported to contain thymic epithelial progenitor cells (TEPCs) (Ulyanchenko et al., Cell Rep 14:2819–2832, 2016). In addition, altered postnatal organization of mutant thymic medulla failed to organize a unique, central epithelial area. This delayed maturation of TE cell components correlated with low transcript production of some molecules reported to be masters for TEC maturation, such as EphB2, EphB3 and RANK. Changes in the thymic lymphoid component became particularly evident after birth, when molecules expressed by TECs and involved in early T-cell maturation, such as CCL25, CXCL12 and Dll4, exhibited minimal values. This represented a partial blockade of the progression of DN to DP cells and reduced proportions of this last thymocyte subset. At 1 month, in correlation with a significant increase in transcript production, the DP cell percentage increased in correlation with a significant fall in the number of mature TCRαβhi thymocytes and peripheral T lymphocytes.