Biotechnology for the Eucalyptus Biorefinery Ana Gutiérrez, José C del Río IRNAS, CSIC, Seville, Spain Susana Camarero, Angel T Martínez CIB, CSIC, Madrid, Spain 7ICEP-May15
Biotechnology for the Eucalyptus Biorefinery Content: 1. Introduction 2. Eucalypt decaying fungi and their genomes Lignin vs polysaccharides decay (biopulping) Biodegradation of (pitch forming) wood lipids Why sequence basidiomycete genomes? 3. Oxidative enzymes for the eucalypt mill Ligninolytic peroxidases The laccase-mediator system Towards industrial feasibility Biobleaching + pitch biocontrol 4. Recent studies: Enzymes for eucalypt biorefinery Wood delignification for biofuel production The delignification process as shown by 2D-NMR A new generation of tailor-made enzymes 7ICEP-May15
1. Introduction ( Biotechnology for the Eucalyptus Biorefinery) H2020 (Horizon 2020 RTD programme) BioBased Industries Consortium KET-based Industrial Leadership BBI Public-Private Partnership total: € 77 billion 7ICEP-May15
An overview of results obtained at IRNAS (Seville) and CIB (Madrid) during over 15 years working in biotechnology for eucalypt biorefinery (cellulose and bioethanol production) is presented Sitosterol The aim was to develop Lignin model biocatalysts for removal of lignin (bottleneck for using wood in biorefineries) and lipophilic extractives (causing pitch troubles in the Cl-free production of cellulose) 7ICEP-May15
These studies were performed in collaboration with cellulose and biotechnology companies, and also included other research/academic partners Teresa Vidal Cristina Valls Javier Romero Blanca Roncero José L. Francisco José E. Colom Tiina Liitia Augusto Milanez Tarja Tamminen Jorge L. Colodette Henrik Lund Fernando J. Gomes Lisbeth Kalum Kim Borch 7ICEP-May15
Biotechnology for the Eucalyptus Biorefinery Content: 1. Introduction 2. Eucalypt decaying fungi and their genomes Lignin vs polysaccharides decay (biopulping) Biodegradation of (pitch forming) wood lipids Why sequence basidiomycete genomes? 3. Oxidative enzymes for the eucalypt mill Ligninolytic peroxidases The laccase-mediator system Towards industrial feasibility Biobleaching + pitch biocontrol 4. Recent studies: Enzymes for eucalypt biorefinery Wood delignification for biofuel production The delignification process as shown by 2D-NMR A new generation of tailor-made enzymes 7ICEP-May15
2. Eucalypt decaying fungi and their genomes The so-called white-rot fungi are responsible for the natural degradation of lignin in forests providing access to cellulose to other (micro)organisms Ganoderma australe Ceriporiopsis subvermispora These fungi (and their enzymes) are the biocatalysts Selective delignification by Ganoderma australe of choice in wood biorefinery 7ICEP-May15
Eucalyptus wood treatment with fungi: Py-GC/MS analysis Lignin peaks Control wood ( Eucalyptus globulus ) 22 S G S OMe S MeO OMe MeO OMe G OH OH OH 10 MeO OMe Carbohydrate peaks S OMe 16 18 OH OH MeO OMe OH 11 c g e 5 7 23 f G 6 20 OMe G 24 25 d 21 19 l OH 14 26 i OMe h j k b 2 13 OH 3 15 8 9 1 4 12 a m min 5 10 15 20 25 Paecilomyces sp . 22 Ceriporiopsis subvermispora 16 22 Carbohydrate peaks d 16 g Carbohydrate peaks 23 e 10 23 10 21 7 f 11 18 5 l l 6 11 24 i 18 5 25 g 20 7 k 21 j k 6 25 26 20 b d 13 14 24 26 b e 1 14 1 15 h 2 13 3 9 12 j 4 3 m 4 9 h 2 m a 12 min min 5 10 15 20 25 5 10 15 20 25 del Río J.C., A. Gutiérrez, M. J. Martínez, and A. T. Martínez. Py-GC-MS study of Eucalyptus globulus wood treated with different fungi. J.Anal.Appl.Pyrolysis 58/59:441-453, 2001. 7ICEP-May15
Eucalyptus wood treatment with fungi: Patterns observed Basidiomycetes caused the 7 Ascomycetes most selective removal of lignin OPI* MOL OVA (low lignin/carbohydrate ratio) Basidiomycetes PPU Control with simultaneous decrease of BAD OPI 6 its S/G ratio S/G CPU KEU PCH FOX PRA CVA MHE PAE Ceriporiopsis subvermispora FTR Deuteromycetes (CSU) showed the highest 5 biotechnological potential for CSU biological delignification of Eucalyptus globulus wood 4 0.5 1.0 1.5 2.0 Lignin/Carbohydrate Some of the basidiomycetes also removed the recalcitrant lipids respondible for pitch deposits in Eucalyptus TCF pulps and were assayed for simultaneous depitching and delignification 7ICEP-May15
Pitch-forming lipid classes in O Eucalyptus wood and pulps OH palmitic acid O stigmasta-3,5-dien-7-one OH n -octacosanol CH 2 OH O OH O HO HO OH campesterol sitosteryl 3- β -D-glucopyranoside CH 2 OH O OH O O HO HO sitosterol OH 7-oxositosteryl 3- β -D-glucopyranoside HO stigmastanol O O sitosteryl linoleate CO-O-CH 2 CO-O-CH O HO CO-O-CH 2 7-oxositosterol trilinolein Gutiérrez, A., J. C. del Río, M. J. Martínez, and A. T. Martínez. 1999b. Fungal degradation of lipophilic extractives in Eucalyptus globulus wood. Appl. Environ. Microbiol. 65:1367-1371. 7ICEP-May15
Lipid removal from Eucalyptus globulus chips by Ceriporiopsis subvermispora and other white-rot fungi (and bio-kraft pulping) 80 80 Ceriporiopsis Bjerkandera adusta Phlebia 70 70 subvermispora 60 60 mg/100 g wood mg/100 g wood Ceriporiopsis Pleurotus 50 50 Sterol removal (%) 40 40 Bjerkandera 30 30 20 20 10 10 0 0 0 7 14 28 49 days 0 7 14 28 49 days 80 Phlebia radiata Pleurotus 80 70 pulmonarius 70 mg/100 g wood 60 mg/100 g wood 60 50 50 Weight loss (%) 40 40 Up to 60% of wood steroids 30 30 20 20 can be removed in 1-2 week 10 10 treatments with only 1% 0 0 0 7 14 28 49 days 0 7 14 28 49 days loss of chips weight Fatty acids Squalene Steroidal hydrocarbons Sterols Steroidal ketones Sterol esters Triglycerides Gutiérrez, A., M. J. Martínez, J. C. del Río, J. Romero, J. Canaval, G. Martínez-Íñigo, M. J., A. Gutiérrez, J. C. del Río, M. J. Martínez, and A. T. Lenon, and A. T. Martínez. 2000. Fungal pretreatment of Eucalyptus Martínez. 2000. Time course of fungal removal of lipophilic extractives wood can strongly decreases the amount of lipophilic extractives from Eucalyptus globulus Labill. wood. J. Biotechnol. 84:119-126. during chlorine-free kraft pulping. Environ. Sci. Technol. 34:3705-3709. 7ICEP-May15
Scale-up of Eucalyptus pretreatment (biopulping) with Ceriporiopsis subvermispora (Ferraz & coworkers) Pretreatment of Eucalyptus grandis chips with Ceriporiopsis subvermispora for bio-TMP and bio-CTMP (bio-kraft, bio-sulfite and bio-organosolv also evaluated) Biopulping enzymatic/chemical mechanisms analyzed ↓ 18% ↓ 27% Enzymes and pH during treatment of Eucalyptus Energy consumption during mill-scale refining of Eucalyptus chips from chips (30 d) with Ceriporiopsis subvermispora (5 a 50-tonne pile treated with Ceriporiopsis subvermispora (60 d) ppm inoculum with CSL) See review of Ferraz et al. on "Technological advances and mechanistic basis for fungal biopulping" (Enzyme Microb. Technol. 43: 178, 2008) and subsequent papers 7ICEP-May15
Ceriporiopsis subvermispora also yielded good (selectivity) results in pilot-scale treatment of other types of wood However, the known lignin-degrading enzymes (lignin peroxidase and versatile peroxidase ) were never identified in this fungus USDA biopulping trial Why sequence Ceriporiopsis subvermispora ? To solve this paradox, and contribute to delignification applications, the genome of Ceriporiopsis subvermispora was sequenced at JGI (US DOE) and compared with Phanerochaete chrysosporium (the first basidiomycete sequenced as the model lignin-degrading fungus) 7ICEP-May15
Why sequence Ceriporiopsis subvermispora ? www.jgi.doe.gov Dr Dan Cullen Prof Rafael Vicuña FPL, USDA Universidad Católica Madison, WI, USA Santiago de Chile 7ICEP-May15
The Ceriporiopsis subvermispora genome
The missing enzymes were identified from the genome Degradation of synthetic Lignin peroxidase (LiP) Degradation of lignin model lignin by Ceriporiopsis LiP from Ceriporiopsis genome dimer by Ceriporiopsis LiP 7ICEP-May15
Biotechnology for the Eucalyptus Biorefinery Content: 1. Introduction 2. Eucalypt decaying fungi and their genomes Lignin vs polysaccharides decay (biopulping) Biodegradation of (pitch forming) wood lipids Why sequence basidiomycete genomes? 3. Oxidative enzymes for the eucalypt mill Ligninolytic peroxidases The laccase-mediator system Towards industrial feasibility Biobleaching + pitch biocontrol 4. Recent studies: Enzymes for eucalypt biorefinery Wood delignification for biofuel production The delignification process as shown by 2D-NMR A new generation of tailor-made enzymes 7ICEP-May15
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