WOW Project Review
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Transcript of WOW Project Review
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WOW Project Review
Friday 2nd October 2009
Chemistry
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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
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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
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Degradation
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OH OMe
OMe
O
ROOH
O
OH
OMe
OMe
HO
OHOMe
HO O
OH
R
MeO
OH
OMeO
O
OMeOH
OHOMe
O
HOOMe
OMe
HO
MeO CO2H
OH
HO2COH
OMe
HO
OH
OH
OMe
OHOMe
CHOHO
MeOOH
OMe
R
OH
OOMe
OHMeO
OH
O
OMe
OMe
HO
MeO CO2H
HO2C
O
CO2HHO
HO OMe
O
O
OMeOH
HO
lyase
CO2HHO
MeO CO2H
OHOMe
CO2H
OHOH
CO2H
peroxidase
-aryl ether phenylcoumarane biphenyldiarylpropanepinoresinol
1) demethylase2) extradioldioxygenase
aldehyde dehydrogenase
CO2H
OHCO2Hdemethylase
decarboxylase
C-C hydrolase
lignostilbenedioxygenase
HCHO
OHCOH
CO2H
HO2C CO2H
CO2H
OHO CO2H
CO2H
OO
extradioldioxygenase
aldehydedehydrogenase
CO2H
CO2H O
HO
CO2H
O2 x
hydratase
aldolase
decarboxylase
CO2
Lignin is a major component of plant cell walls
peroxidaseslaccases
Lignin-degradingmicrobes
Bacterialaromaticdegraders
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Fluorescent Assay for Lignin Degradation
OHOMe
HO
LIGNIN
attach fluorophore
OOMe
HO
LIGNIN
OFl
lignindegrader
Fluorescence change
Could be performed in 96-wellmicrotitre plate reader
Fluoresence Vs Time For P. Putida Supernatant
16000
16500
17000
17500
18000
18500
0 20 40 60 80 100 120
Time (min)
Fluo
rsen
ce
30 ul
Time dependence (0-2 hr) Change in fluoresecence in the first 10 min
-2000
-1500
-1000
-500
0
500
1000
Fluo
resc
ence
Streptomyces ViridosporusB. SubtillisP.PutidaRhodococcus RHA1Rhodococcus spNocardia autotrophicaLeuconostoc Mesentoides
Non-degraders
Assay can distinguish degraders from non-degraders:
Paper Submitted to Molecular Biosystems
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RHA1
-0.002
-0.001
0
0.001
0.002
0.003
0.004
Abso
rban
ceNo ligninWheatMyscantusPine
N. Autotrophica
-0.001
-0.0005
0
0.0005
0.001
0.0015
0.002
0.0025
Abs
orba
nce
No ligninWheatMyscanthusPine
Specificity of bacterial lignin degraders towardsMWL from pine, wheat straw & miscanthus:
Rhodococcus RHA1 not selective
Nocardia autotrophicashows selectivityfor pine lignin
OHOMe
HO O
O
LIGNIN
OHOMe
HO O
O
LIGNIN
OHOMe
Oligninbreakdown increase in A400
can be performed inmicrotitre plate format
tetranitromethane
O2N O2N
Continuous UV-VIS Assay using Nitrated Lignin
Time dependance
0.0558
0.056
0.0562
0.0564
0.0566
0.0568
0.057
0.0572
0.0574
0.0576
0 5 10 15 20 25
Time (min)
Abso
rban
ce
Time dependence (0-20 min)
Bacterial degraders
-0.0015
-0.001
-0.0005
0
0.0005
0.001
0.0015
0.002
0.0025
0.003
0.0035
Abs
orba
nce
P.PutidaR. RHA1R. Sp.S. ViridosporusN. autotrophicaL.mesenteroidesB. Subtillis
Distinguishes lignin degradersfrom non-degraders
Paper Submitted to Molecular Biosystems
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Extractions
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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
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Extract Mass (g) Percentage of total (wet)
Percentage of total (dry)
Aqueous 156.88 10.6% 38.6%
Organic 14.5 0.98% 3.57%
Dry Straw 235.3 15.9% 57.9%
Water content - 72.7% -
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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
101 I. M. G. Lopes, M. G. Bernado-Gil, Eur. J. Lipid. Sci. Technol., 2005, 107, 12-19
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Hexane Extraction - Results
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It would appear that a higher content is made available by degradation, but it is unknown to the origin of the material.
Straw Type Processing Extracted mass / mg % dry mass extractedUntreated None 80 1.84
Untreated Water 24 0.55
Untreated Chopped 100 1.77
P. Chrysosporium None 310 7.40
P. Chrysosporium Water 40 0.96
P. Chrysosporium Chopped 230 5.35
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Analysis
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HPLC traces with time
Degrader Pseudomonas putida
Non-degrader Bacillus subtilisshows no change
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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 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)+.
O OH
OOH
1
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Analysis• Extracts have been analysed using LTQ-MS at HRI
– Separates and detects using UV and MS
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Comparison of LTQ data - standards
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Vanillic AcidHO
OOH
O
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Aromatic metabolites identified (so far)Compound LC-MS
Retention time (min)
LC-MSm/z
GC-MSRetention time (min)
GC-MSm/z(silylated)
Observed with..
1 4.29 235 MK+ 7.02 268 M+
253 -CH3
P. Putida 6hr, 1d, 3dRhodococcus RHA1 2hr, 4hrMiscanthus & wheat straw
2 4.56 209 MNa+
225 MK+7.71 243 M+
228 -CH3
P. Putida (straw) 7dRhodococcus RHA1Miscanthus 1d, straw 2d
3 5.25 195 MH+ 5.27 251 M-CH3
P. Putida 6hrRhodococcus RHA1 2hr, 6hrMiscanthus only
4 5.76 251 MK+ 6.03 341 M-CH3 P. Putida 6hrRhodococcus RHA1 4hr, 6hrMiscanthus only
5 9.09 169 MH+ Rhodococcus RHA1 6hrMiscanthus only
O OH
OCH3OH
COOH
COOHHO O
CO2H
OCH3OH
COOH
COOHOH
H3CO
COOH
OHOCH3
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OH
OMe
CO2H
Ferulic acid.379 papers in 2008-9 on biological activity alone£1 per 1gAnti-oxidantActive breast cancer, liver cancerActive ingredient in anti-ageing creams / plumping creams
CO2H
OH
OMeHO2C
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
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Other potential major degradation products-yet to be fully identified from wheat straw
O
OMe
OH
OHO OH
MeO
OH
OH
O
HO
OMe
OH
HO
OMe
OH
O
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
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Hexane Extraction - Analysis
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Process Degraded, Water Degraded Untreated, Chopped
Untreated Degraded, Chopped
FA 2a 2b 3a 3b 5a 5b 6a 6b 7a 7b
14:0 2.19 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
16:0 20.49 24.70 12.52 100.00 42.88 33.98 16.09 46.10 38.19 27.85
18:0 3.46 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
18:1 10.98 14.99 0.00 0.00 0.00 0.00 0.00 11.68 0.00 0.00
18:2 5.33 7.51 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
20:3 8.17 10.05 0.00 0.00 24.29 46.10 13.87 27.38 61.81 46.72
20:4 0.00 0.00 0.00 0.00 0.00 19.91 0.00 14.84 0.00 16.20
22:0 6.42 8.89 0.00 0.00 16.70 0.00 0.00 0.00 0.00 0.00
23:0/22:2 0.00 0.00 0.00 0.00 0.00 0.00 18.87 0.00 0.00 0.00
24:0 5.12 6.79 0.00 0.00 16.12 0.00 0.00 0.00 0.00 0.00
22:6 37.84 27.07 87.48 0.00 0.00 0.00 0.00 0.00 0.00 0.00
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Materials
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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
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Fatty acidsNa2WO4.2H2O
H2O2, aliquat, 100 °C, 5hr
HO2C CO2HAzelaic acid
Rosacea treatmentAcne treatment
Hair growth stimulantNylon-6,9
+ Estolides
LubricantsRust inhibitor
N
OHO
OH
Latex additives, Akzo Nobel
n=4
n=5
n=3n=2
O C
O
HOOH
HO
NaOOC
n-1
Tungstan mediated fatty acid functionalisation: J. Appl. Poly. Science, In Prep
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Products from Extractions• Conversion of ‘model feedstocks’ into polyurethane materials• Two initial materials were identified
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HO
OH
O
O
HO
OH
OMe
Chrysin Vanillyl Alcohol
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
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O
O
HO
OH
O
O
RO
OH
derivitizePolyethers, polyesters,poly(methacrylate) with
antioxidant/UV absorbing properties
Chrysin: a naturally occurring flavone
Polyurethanes from Flavone derivatives: J. Appl. Poly. Science, In Prep
25 75 125 175 225 275 325 37550
60
70
80
90
100
TGA Curve for Chrysin derivatives - N2
Temperature / oC
% M
ass C
hang
e
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NCOOCNMDI
OCNNCO
HDI
NCOOCNTDI
OH
HOOMe
Polyurethanes
Polyurethanes from Vanillin derivatives: J. Appl. Poly. Science, In Prep
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Other
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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
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A (DoE) approach to material properties of electrospun nanofibres. SR Coles, AJ Clark, K Kirwan et al. J. Appl. Poly. Science, 2009 Accepted
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Future work
BiodegradationIsolation and purification of degradation enzymes from bacteria.
AnalysisContinued identification of novel lignin degradation products.Preparation of LC-MS standards for unambiguous identification.
MaterialsIdentification 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.
OtherEvaluation of lignin incorporation in electrospun fibres and composites.