WOW Project Review
description
Transcript of WOW Project Review
WOW Project Review
Friday 2nd 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
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
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
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
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
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% -
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
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 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
Analysis
HPLC traces with time
Degrader Pseudomonas putida
Non-degrader Bacillus subtilisshows 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 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
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 AcidHO
OOH
O
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
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
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
Hexane Extraction - Analysis
20
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
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
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
Products from Extractions• Conversion of ‘model feedstocks’ into polyurethane materials• Two initial materials were identified
24
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
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
NCOOCNMDI
OCNNCO
HDI
NCOOCNTDI
OH
HOOMe
Polyurethanes
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
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.