RECENT ADVANCES IN THE TARGETED FUNCTIONALIZATION OF LIGNINS
RECENT ADVANCES IN THE TARGETEDFUNCTIONALIZATION OF LIGNINSFRONTIERS IN BIOREFINING 2014October 23, 2014Heiko LangeCrestini Group - University of Rome ‘Tor Vergata‘
AgendaIntroductionCharacterization of ligninQuantitative NMR – QQ-HSQC vs. HSQC0Quantitative 31P NMRSize exclusion chromatographyValorization of ligninChemicallyBiotechnologicallyLignin tailoring via ultrafiltrationConclusions2
Introduction
IntroductionLignin – the under-utilized biomass component- intelligent polymersapplications in pharmaceuticalsciences / industriesapplications in cosmeticsapplications in consumer careM. Aresta, A. Dibenedetto, F. Dumeignil (editors); Biorefinery: From biomass to chemicals and fuels. de Gruyter (Berlin/Boston) 2012.4
Characterization of lignin
Characterization of ligninquantitative NMR – QQ-HSQC vs. HSQC0
Structural aspects of ligninLignin – biosynthetic originR. Whetten, R. Sederoff; Lignin biosynthesis. Plant Cell 1995, 7, 1001.W. Boerjan, J. Ralph, M. Baucher; Lignin biosynthesis. Annu Rev Plant Biol 2003, 54, 519.7
Structural aspects of ligninLignin – structural diversity Polymeric or oligomeric? Random 3D network? No repetitive units!? No repetitive bonding patterns!?S.E.Lebo Jr., J.D. Gargulak, T.J. McNally (editors); Lignin. Kirk-Othmer Encyclopedia of Chemical Technology, 4th Edition. John Wiley & Sons, Inc. (New York) 2001.8
Structural aspects of ligninLignin – structural OOHOHOHOMeHOOHOMeHOOHHO MWL: Linear, oligomeric chains!C. Crestini , F. Melone, M. Sette, R. Saladino; Milled wood lignin: A linear oligomer. Biomacromolecules 2011, 12, 3928.9
Structural aspects of ligninLignin – structural diversity that is also depending on the isolation OSO3MOHOMeOOHOOHMO3SSO3MOOMeMeOOOOMeHOOMeOHkraft ligninJ. Gierer; Chemical aspects of kraft pulping. Wood Sci. Tech. 1980, 14, 241.F.S. Chakar, A.J. Ragauskas; Review of current and future softwood kraft lignin process chemistry. Industrial Crops Prod 2004, 20, 131.lignosulfonate10
Characterizing ligninLignin – Established methods for characterizing and analyzing lignin and lignin models (?)11
Structural aspects of ligninLignin – structural features delineated via NMR spectroscopy Heteronuclear 2D NMR spectroscopy Better resolution: spread in heteronucleus dimension Lower sensitivity: low giromagnetic ratio of heteronucleus Not quantitative S/N γexcγobs3/2{1 exp( trecycle/T1exc)} 1/2JCHINEPTReverse INEPTHSQC[4] S.E.Lebo Jr., J.D. Gargulak, T.J. McNally (editors); Lignin. Kirk-Othmer Encyclopedia of Chemical Technology, 4th Edition. John Wiley & Sons, Inc. (New York) 2001.[5] C. Crestini , F. Melone, M. Sette, R. Saladino; Milled wood lignin: A linear oligomer. Biomacromolecules 2011, 12, 3928.12
Quantitative bi-dimensional NMRLignin – structural features delineated via NMR spectroscopy Heteronuclear 2D NMR spectroscopy quantitative after slight changes to pulse-sequencesQuick quantitative HSQC (QQ-HSQC) Selection of the 3:1 D vales is done combining gradients and adiabatic pulsesD. J. Peterson, N. M. Loening; QQ-HSQC: a quick, quantitative heteronuclear correlation experiment for NMR spectroscopy. Magn. Reson. Chem. 2007, 45, 937–941.13
Quantitative bi-dimensional NMRLignin – structural features delineated via NMR spectroscopy Heteronuclear 2D NMR spectroscopy quantitative after slight changes to pulse-sequences HSQC0Experimental volumeHSQC numberln(Ai,n) ln(A0,n) i x ln(fA,n)Virtual HSQC0K. Hu, W. M. Westler, J. L. Markley; Simultaneous Quantification and Identification of Individual Chemicals in Metabolite Mixtures by Two-Dimensional ExtrapolatedTime-Zero 1H-13C HSQC (HSQC0). J. Am. Chem. Soc. 2011, 133, 1662–1665 .14
Quantitative bi-dimensional NMRQQ-HSQC / HSQC0 Heteronuclear 2D NMR spectroscopy with alterations: quantitative!intrinsic standardse.g., G C2-H on and quantification of interunit bonding motifs present in lignin samplesQQ-HSQC: D. J. Peterson, N. M. Loening; QQ-HSQC: a quick, quantitative heteronuclear correlation experiment for NMR spectroscopy. Magn. Reson. Chem. 2007, 45, 937–941.HSQC0: K. Hu, W. M. Westler, J. L. Markley; Simultaneous Quantification and Identification of Individual Chemicals in Metabolite Mixtures by Two-Dimensional ExtrapolatedTime-Zero 1H-13C HSQC (HSQC0). J. Am. Chem. Soc. 2011, 133, 1662–1665 .15
Structural aspects of ligninLignin – structural features delineated via NMR spectroscopy Different heteronuclear 2D NMR spectroscopy with alterations are quantitative! Which of the quantitative versions should I go for?β-O-4’β-5’β-ββ’ No difference! Drawback: Without a cryoprobe, all these efforts are useless .β-1’ 5,5’-αα,ββ-O-4’M. Sette, H. Lange, C. Crestini; Quantitative HSQC Analyses of Lignin: A Practical Comparison. Comput. Struct. Biotechnol. J. 2013, 6, e201303016.16
Characterization of ligninQuantitative 31P NMR & SEC
Quantitative 31P NMRQuantitative 31P NMR spectroscopy Phosphitylated lignin samples measured under standardized conditions using a suitable pulsesequence and internal standards.OOinternal standardPOPOOR1OLIGNINOR2R1R2OPOO-O-4' - P( -ether)G,S,HIdentification and quantification of interunit bonding motifs present in lignin samplesA. Granata, D. S. Argyropoulos; , a Reagent for the Accurate Determination of the Uncondensed and Condensed Phenolic Moieties in Lignins. J. Agric. FoodCbem. 1995, 43, 1538-1544.18
Gel permeation chromatographyGPC – our proposed standardized ,weightingMn 1300 DaMw 5700 DaPDI 4.3H. Lange, F. Rulli, C. Crestini, manuscript in prepration.19
Valorization of lignin20
Valorization of lignin I
Methods for valorizing ligninLignin – selectively OOMeOOOHOMeHOOHOMeOHHOHOOxidation of side-chainOxidative coupling(depolymerization process) (polymerization process)» decrease of aliphatic OHHydrolysis of alkyl aryl ethers(depolymerization process)» decrease of phenolic OH » increase of phenolic OH» increase of aliphatic OHDehydroxylation of side-chain(depolymerization process)» decrease of aliphatic OH22
Valorization of lignin- chemically & biomimetically -23
Methods for valorizing ligninOrganometal catalyzed oxidation methods – Methyltrioxo rhenium (MTO)OOOReOO (S)Me(SO)‡'(SO)k3 (S)k-3‡OOOReOO (S)Me OHHOO Re OOMek-4k4 Substrate(S)k-2k2 H2O2k-1 H2Ok1 H2O2MeReOOOMethyl rhenium trioxide(MTO)OOOReOOMe OHHW.A. Hermann, R.W. Fischer, W. Scherer, M.U.Rauch; Methyltrioxorhenium(VII) as catalyst for epoxidations: Structure of the active species and mechanism of catalysis. Angew. Chem. Int. Ed. Engl. 1993, 32,1157.24
Methods for valorizing ligninOrganometal catalyzed oxidation methods – Methyltrioxo rhenium (MTO)e.g.: C. Crestini, P. Pro, V. Neri, R. Saladino; Methyltrioxorhenium: a new catalyst for the activation of hydrogen peroxide to the oxidation of lignin and lignin model compounds. Bioorg. Med. Chem. 2005,13, 2569.25
Methods for valorizing ligninOrganometal catalyzed oxidation methods – polymer-supported Methyltrioxo rhenium (MTO)e.g.: C. Crestini, M.C. Caponi, D.S. Argyropoulos, R. Saladino; Immobilized methyltrioxo rhenium (MTO)/H2O2 systems for the oxidation of lignin and lignin model compounds. Bioorg. Med. Chem. 2006, 14,5292.26
Methods for valorizing ligninOrganometal catalyzed oxidation methods – porphyrin complexes of transition metalse.g.: C. Crestini, R. Saladino, P. Tagliatesta, T. Boschi; Biomimetic degradation of lignin and lignin model compounds by synthetic anionic and cationic water soluble manganese and iron porphyrins. Bioorg.Med. Chem. 1999, 7, 1897.27
Methods for valorizing ligninOrganometal catalyzed oxidation methods – polymer-supported porphyrin complexesooooooMeNNMeNNMn NNMeNNMeoooe.g.: C. Crestini, A. Pastorini, P. Tagliatesta; Immobilized metalloporphyrins as biomimetic catalysts in the oxidation of lignin model compounds. J .Mol. Catal. A: Chem. 2004, 208, 195.28
Methods for valorizing ligninOrganometal catalyzed oxidation methods – catalyst-mediator systems Mediating the organometal-catalyzed valorization of lignin Altered oxidation potentials of reactive species, attacking different functional groups Reducing reactivity problems originating from steric hindrances29
Valorisation of lignin- enzymatically -30
Methods for valorizing ligninEnzyme-based oxidation methods – general aspects Most active lignin-degrading enzymes are found in white-rot fungi:Laccase (E 0.5 - 0.8 V)(multi-copper enzyme)Manganese peroxidase (E 1.5 V)(Mn-heme core fragment)Lignin peroxidase (E 1.4 V)(Fe-heme core fragment)M.G. Paice, R. Bourbonnaise, I.D. Reid, F.S. Archibald, L. Jurasek; Oxidative bleaching enzymes: A review. J. Pulp Paper Sci. 1995, 21, J280.31
Methods for valorizing ligninEnzyme-based oxidation methods – laccase and horseradish peroxidase (HRP) Depolymerization at the lignin terminal units Increase of the residual lignin Mn due to oxidative coupling processes Only low molecular weight fractions in solution » exo-depolymerizing enzymesC. Crestini, F. Melone, R. Saladino; Novel multienzyme oxidative biocatalyst for lignin bioprocessing. Bioorg. Med. Chem. 2011, 19, 5071, and literature cited therein.32
Methods for valorizing ligninLaccase-mediator -based oxidation methodsC. Crestini, L. Jurasek, D.S. Argyropoulos; On the mechanism of the laccase–mediator system in the oxidation of lignin. Chem. Eur. J. 2003, 9, 5371.33
Methods for valorizing ligninEnzyme-based oxidation methods – Immobilized enzymes (example: laccase) Coating EncapsulatingC. Crestini, F. Melone, R. Saladino; Novel multienzyme oxidative biocatalyst for lignin bioprocessing. Bioorg. Med. Chem. 2011, 19, 5071, and literature cited therein.C. Crestini, R. Perazzini, R. Saladino; Oxidative functionalisation of lignin by layer-by-layer immobilised laccases and laccase microcapsules. Appl. Catal. A: Gen. 2010, 372, 115. Corrigendum: Appl. Catal. A:Gen. 2010, 382, 138.34
Methods for valorizing ligninEnzyme-based oxidation methods – Immobilized enzymes Immobilised laccases: more than 80% residual activity after 10 batch cycles Immobilised HRP: more than 70% residual activity after 10 batch cyclesC. Crestini, F. Melone, R. Saladino; Novel multienzyme oxidative biocatalyst for lignin bioprocessing. Bioorg. Med. Chem. 2011, 19, 5071, and literature cited therein.C. Crestini, R. Perazzini, R. Saladino; Oxidative functionalisation of lignin by layer-by-layer immobilised laccases and laccase microcapsules. Appl. Catal. A: Gen. 2010, 372, 115. Corrigendum: Appl. Catal. A:Gen. 2010, 382, 138.35
Methods for valorizing ligninEnzyme-based oxidation methods – Immobilized enzymes Mn decreases of the residual lignin due to hydrolysis. Increase in aliphatic and phenolic OH groups: hydrolytic processes Soluble fractions contain significant amounts of lower molecular weight polymeric material and notonly by oligomers. » endo-depolymerizing enzymes in the hydrolytic processEndo-depolymerization(alkyl-aryl ether hydrolysis)C. Crestini, F. Melone, R. Saladino; Novel multienzyme oxidative biocatalyst for lignin bioprocessing. Bioorg. Med. Chem. 2011, 19, 5071, and literature cited therein.C. Crestini, R. Perazzini, R. Saladino; Oxidative functionalisation of lignin by layer-by-layer immobilised laccases and laccase microcapsules. Appl. Catal. A: Gen. 2010, 372, 115. Corrigendum: Appl. Catal. A:Gen. 2010, 382, 138.36
Methods for valorizing ligninEnzyme-based oxidation methods – Co-immobilized enzymes higher lignin conversion Lower Mn of the lignin residual after treatment Mainly oligomers in solution » endo- and exo-depolymerizing activities within hydrolytic and oxidative pathways, respectivelyENDOEXOC. Crestini, F. Melone, R. Saladino; Novel multienzyme oxidative biocatalyst for lignin bioprocessing. Bioorg. Med. Chem. 2011, 19, 5071, and literature cited therein.C. Crestini, R. Perazzini, R. Saladino; Oxidative functionalisation of lignin by layer-by-layer immobilised laccases and laccase microcapsules. Appl. Catal. A: Gen. 2010, 372, 115. Corrigendum: Appl. Catal. A:Gen. 2010, 382, 138.37
Valorization of lignin- optional: fractionation of lignin -38
Fractionation of ligninLignin – structural features tailored via lignin fractionationMw cut-off 5 kDaMw cut-off 10kDaMw cut-off 1kDaO. Sevastyanova, M. Helander, S. Chowdhury, H. Lange, H. Wedin, L. Chang, M. Ek, J.F. Kadla, C. Crestini, M.E. Lindström; Tailoring the Molecular and Thermo–Mechanical Properties of KraftLignin by Ultrafiltration. J. Appl. Polym.Sci. 2014, 131, 40799.39
Fractionation of ligninLignin – structural features tailored via lignin fractionation Tailored /gPKL2.253.500.36 10 3.703.614.110.520.490.490.350.460.38O. Sevastyanova, M. Helander, S. Chowdhury, H. Lange, H. Wedin, L. Chang, M. Ek, J.F. Kadla, C. Crestini, M.E. Lindström; Tailoring the Molecular and Thermo–Mechanical Properties of KraftLignin by Ultrafiltration. J. Appl. Polym.Sci. 2014, 131, 40799.H. Lange, O. Sevastyanova, C. Crestini; unpublished results; University of Rome ‘Tor Vergata’ & KTH - Royal Institute of Technology40
Conclusions41
Conclusions Lignin is a valuable component of plant biomass. Lignin is oxidatively degradable using .chemical approaches . biomimetic approaches . biotechnological approaches However, high(er) value applications possible without degradation. Structural elucidation is key. Once the structure is elucidated, targeted chemical & biotechnological functionalisation is possible.42
AcknowledgementsThanks to Prof. Dr Claudia Crestini Dr Marco Sette Crestini groupCOST Action FP1105Principles of Cell Wall Assembly –Understanding and Implementation43
A. Granata, D. S. Argyropoulos; , a Reagent for the Accurate Determination of the Uncondensed and Condensed Phenolic Moieties in Lignins. J. Agric. Food Cbem. 1995 , 43, 1538-1544. O R2 O O O R2 LIGNIN-O-4' - P ( -ether) G,S,H R1 R1 P O P O O P O
May 02, 2018 · D. Program Evaluation ͟The organization has provided a description of the framework for how each program will be evaluated. The framework should include all the elements below: ͟The evaluation methods are cost-effective for the organization ͟Quantitative and qualitative data is being collected (at Basics tier, data collection must have begun)
Silat is a combative art of self-defense and survival rooted from Matay archipelago. It was traced at thé early of Langkasuka Kingdom (2nd century CE) till thé reign of Melaka (Malaysia) Sultanate era (13th century). Silat has now evolved to become part of social culture and tradition with thé appearance of a fine physical and spiritual .
On an exceptional basis, Member States may request UNESCO to provide thé candidates with access to thé platform so they can complète thé form by themselves. Thèse requests must be addressed to esd rize unesco. or by 15 A ril 2021 UNESCO will provide thé nomineewith accessto thé platform via their émail address.
̶The leading indicator of employee engagement is based on the quality of the relationship between employee and supervisor Empower your managers! ̶Help them understand the impact on the organization ̶Share important changes, plan options, tasks, and deadlines ̶Provide key messages and talking points ̶Prepare them to answer employee questions
Dr. Sunita Bharatwal** Dr. Pawan Garga*** Abstract Customer satisfaction is derived from thè functionalities and values, a product or Service can provide. The current study aims to segregate thè dimensions of ordine Service quality and gather insights on its impact on web shopping. The trends of purchases have
Chính Văn.- Còn đức Thế tôn thì tuệ giác cực kỳ trong sạch 8: hiện hành bất nhị 9, đạt đến vô tướng 10, đứng vào chỗ đứng của các đức Thế tôn 11, thể hiện tính bình đẳng của các Ngài, đến chỗ không còn chướng ngại 12, giáo pháp không thể khuynh đảo, tâm thức không bị cản trở, cái được
advances in agronomy adv anat em advances in anatomy embryology and cell biology adv anat pa advances in anatomic pathology . advances in organometallic chemistry adv parasit advances in parasitology adv physics advances in physics adv physl e advances in physiology education adv poly t advances in polymer technology
Le genou de Lucy. Odile Jacob. 1999. Coppens Y. Pré-textes. L’homme préhistorique en morceaux. Eds Odile Jacob. 2011. Costentin J., Delaveau P. Café, thé, chocolat, les bons effets sur le cerveau et pour le corps. Editions Odile Jacob. 2010. Crawford M., Marsh D. The driving force : food in human evolution and the future.
