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 Other –Links to electrospinning, biocomposites 2

Summary-last review 1.Determine suitable methods for extraction of degrading straw, using both aqueous and organic solvents. 2.Identify (from literature search) and subsequent training / obtaining of suitable equipment for analysis of extracts. 3.Develop characterisation methods for extracts based on literature protocols, in particular, looking at MALDI, GC-MS & LC-MS. 4.Develop synthetic methods for materials from potential / model breakdown products. 5.Use crude breakdown mixture to produce material based on 4. 6.Set up large scale (20 L) reactor 3

Degradation

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

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

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

Extractions

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 9 ExtractMass (g)Percentage of total (wet) Percentage of total (dry) Aqueous %38.6% Organic %3.57% Dry Straw %57.9% Water content-72.7%-

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 10 1 I. M. G. Lopes, M. G. Bernado-Gil, Eur. J. Lipid. Sci. Technol., 2005, 107, 12-19

Hexane Extraction - Results 11 It would appear that a higher content is made available by degradation, but it is unknown to the origin of the material. Straw TypeProcessingExtracted mass / mg% dry mass extracted UntreatedNone UntreatedWater UntreatedChopped P. ChrysosporiumNone P. ChrysosporiumWater P. ChrysosporiumChopped

Analysis

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

GC-MS data for small scale lignocellulose degradation trials GC-MS total ion chromatogram with EI ionisation for Rhodococcus RHA1 incubated with wheat straw lignocellulose for 7 days at 30 o C. Mass spectrum of peak at RT 7.02 min, assigned to monosilylated derivative of ketone (1), m/z 268 (M- SiMe 3 )+, 253 (M-SiMe 3 - CH 3 )+.

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

Comparison of LTQ data - standards 16 Vanillic Acid

Aromatic metabolites identified (so far) CompoundLC-MS Retention time (min) LC-MS m/z GC-MS Retention time (min) GC-MS m/z (silylated) Observed with MK M CH 3 P. Putida 6hr, 1d, 3d Rhodococcus RHA1 2hr, 4hr Miscanthus & wheat straw MNa MK M CH 3 P. Putida (straw) 7d Rhodococcus RHA1 Miscanthus 1d, straw 2d MH M-CH 3 P. Putida 6hr Rhodococcus RHA1 2hr, 6hr Miscanthus only MK M-CH 3 P. Putida 6hr Rhodococcus RHA1 4hr, 6hr Miscanthus only MH + Rhodococcus RHA1 6hr Miscanthus only

Ferulic acid. 379 papers in 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 Potential use as fine chemical building block. Vanillic acid precursor. Diacid for use in polyesters and polyamides

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

Hexane Extraction - Analysis 20 ProcessDegraded, WaterDegradedUntreated, ChoppedUntreatedDegraded, Chopped FA2a2b3a3b5a5b6a6b7a7b 14: : : : : : : : :0/22: : :

Materials

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

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

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

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

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

Other

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

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.