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WOW Project Review

WOW Project Review. Friday 2 nd October 2009 Chemistry. Introduction. Degradation Bacterial degradation of lignin. Assay and bio-prospecting Extractions Improvements and alternative methods Analysis Identification of compounds Materials Uses and potential markets of breakdown products

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WOW Project Review

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  1. WOW Project Review Friday 2nd October 2009 Chemistry

  2. Introduction • Degradation • Bacterial degradation of lignin. Assay and bio-prospecting • Extractions • Improvements and alternative methods • Analysis • Identification of compounds • Materials • Uses and potential markets of breakdown products • Other • Links to electrospinning, biocomposites 2

  3. Summary-last review • Determine suitable methods for extraction of degrading straw, using both aqueous and organic solvents. • Identify (from literature search) and subsequent training / obtaining of suitable equipment for analysis of extracts. • Develop characterisation methods for extracts based on literature protocols, in particular, looking at MALDI, GC-MS & LC-MS. • Develop synthetic methods for materials from potential / model breakdown products. • Use crude breakdown mixture to produce material based on 4. • Set up large scale (20 L) reactor

  4. Degradation

  5. Lignin is a major component of plant cell walls peroxidases laccases Lignin-degrading microbes Bacterial aromatic degraders

  6. Assay can distinguish degraders from non-degraders: Time dependence (0-2 hr) Non-degraders Fluorescent Assay for Lignin Degradation Paper Submitted to Molecular Biosystems

  7. Time dependence (0-20 min) Specificity of bacterial lignin degraders towards MWL from pine, wheat straw & miscanthus: Rhodococcus RHA1 not selective Distinguishes lignin degraders from non-degraders Nocardia autotrophica shows selectivity for pine lignin Continuous UV-VIS Assay using Nitrated Lignin Paper Submitted to Molecular Biosystems

  8. Extractions

  9. Large Scale Extraction • 1.5 kg (wet) of P.chrysosporium-degraded straw was extracted using 20 L reactor • 12 L of water and 8 L of THF used to extract straw • THF was used due to combination of interesting peaks from LTQ analysis and mass recovered in previous trials

  10. Hexane Extraction • Recent research suggests that hexane can be used to extract triglycerides and fatty acids from straw.1 • Straw placed in soxhlet and extracted with hexane (200 mL) for 24 h. • Fatty acid and triglyceride mixture is collected in the distillation flask away from the straw 1 I. M. G. Lopes, M. G. Bernado-Gil, Eur. J. Lipid. Sci. Technol., 2005, 107, 12-19

  11. Hexane Extraction - Results It would appear that a higher content is made available by degradation, but it is unknown to the origin of the material.

  12. Analysis

  13. HPLC traces with time Non-degrader Bacillus subtilis shows no change Degrader Pseudomonas putida

  14. GC-MS data for small scale lignocellulose degradation trials GC-MS total ion chromatogram with EI ionisation for Rhodococcus RHA1incubated with wheat straw lignocellulose for 7 days at 30 oC. Mass spectrum of peak at RT 7.02 min, assigned to monosilylated derivative of ketone (1), m/z 268 (M-SiMe3)+, 253 (M-SiMe3-CH3)+.

  15. Analysis • Extracts have been analysed using LTQ-MS at HRI • Separates and detects using UV and MS 15

  16. Comparison of LTQ data - standards Vanillic Acid

  17. Aromatic metabolites identified (so far)

  18. Ferulic acid. 379 papers in 2008-9 on biological activity alone £1 per 1g Anti-oxidant Active breast cancer, liver cancer Active ingredient in anti-ageing creams / plumping creams Carboxy vanillic acid. 0 papers in 2008-9 Potential use as fine chemical building block. Vanillic acid precursor. Diacid for use in polyesters and polyamides

  19. Other potential major degradation products-yet to be fully identified from wheat straw No current market. Potential in poly-ethers, -ester or -urethanes Vanillic acid precursor? Diacid for use in polyesters and polyamides Derivative of Gallic acid. Anti-fungal, anti-viral, anti-oxidant. Gallic acid is used in dyes and inks.

  20. Hexane Extraction - Analysis 20

  21. Materials

  22. Hexane Extraction - Potential • Must be carried out before the water extraction • Fatty acids have potential applications in: • Soaps, personal care, perfumes • Polymeric species (e.g. plastics, rubber) • Lubricants, cleaners, coatings • Fatty acid derivatives (e.g. biofuel) • Food and related supplements (e.g. bio oils) • Around 7 – 8 % by weight of the dry mass is a significant portion of material 22

  23. Tungstan mediated fatty acid functionalisation: J. Appl. Poly. Science, In Prep

  24. Products from Extractions Vanillin derivative. Used in fragrances, flavouring. Annual demand for vanillin = 12,000 tons. Natural source = 1200 tons, synthesis = 10,800 tons Beneficial effects in atherosclerosis, osteoporosis, diabetes mellitus and certain cancers. Use as dietary supplements / plant extracts has been steadily increasing. Anti-oxidants. Conversion of ‘model feedstocks’ into polyurethane materials Two initial materials were identified 24

  25. Chrysin: a naturally occurring flavone Polyurethanes from Flavone derivatives: J. Appl. Poly. Science, In Prep

  26. Polyurethanes from Vanillin derivatives: J. Appl. Poly. Science, In Prep

  27. Other

  28. Alternative uses of lignin • Filler in biocomposite structures • May promote resin / matrix adhesion between for natural fibres • Use in electrospun nanofibres • Solutions not ideal for electrospinning • Potential to be co-spun with other polymers (e.g. PVOH) • Degradation products may have beneficial anti-oxidant properties which can be incorporated 28 A (DoE) approach to material properties of electrospun nanofibres. SR Coles, AJ Clark, K Kirwan et al. J. Appl. Poly. Science, 2009 Accepted

  29. Future work • Biodegradation • Isolation and purification of degradation enzymes from bacteria. • Analysis • Continued identification of novel lignin degradation products. • Preparation of LC-MS standards for unambiguous identification. • Materials • Identification of molecules for further study. • Scale up of chosen molecules (synthesis) • Identification of potential industrial partners (medical / cosmetic ?). • Evaluation of estolides as lubricants (Fuchs). • Evaluation as novel fatty amides as additives in paints (Akzo Nobel). • Evaluation of vanillin and flavone polymers for anti-oxidant / UV stability. • Preparation of materials from gallic acid, ferulic acid derivatives. • Other • Evaluation of lignin incorporation in electrospun fibres and composites.

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