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
Lignin is a major component of plant cell walls
OMe
OMe
OH
HO
O
MeO
RO
OH
OH
OH
OMe
O
O
peroxidases
laccases
O
OMe
OH
OMe
OMe
phenylcoumarane
diarylpropane
biphenyl
R
OH
OH
OH
CO2H
MeO
R
O
HO
OMe
pinoresinol
OMe
OH
OMe
HO
-aryl ether
OH
O
HO
O
OMe
O
HO
OH
HO
OMe
OMe
MeO
OH
O
OMe
OMe
OMe
OMe
OH
OH
peroxidase
lyase
lignostilbene
dioxygenase
CHO
1) demethylase
2) extradiol
dioxygenase
HCHO
OH
HO
MeO
OMe
OH
C-C hydrolase
HO2C
MeO
decarboxylase
OMe
CO2H
HO
CO2H
CO2H OH
CO2H
decarboxylase
OH
extradiol
dioxygenase
Bacterial
aromatic
degraders
CO2H
HO
demethylase
OH
O
CO2H
OH
CO2H
CO2H
MeO
HO
OMe
aldehyde
dehydrogenase
OMe
HO2C
OH
OH
Lignin-degrading
microbes
HO
O
CO2H
OHC
HO2C
CO2H
aldehyde
dehydrogenase
CO2H O
hydratase
HO
CO2H
aldolase
CO2H
CO2
O
OH
HO
O
CO2H
O
O
CO2H
2x
CO2H
Fluorescent Assay for Lignin Degradation
LIGNIN
LIGNIN
HO
HO
lignin
degrader
attach fluorophore
Fluorescence change
OMe
OMe
OH
O
Could be performed in 96-well
microtitre plate reader
O
Fl
Assay can distinguish degraders from non-degraders:
Time dependence (0-2 hr)
Change in fluore se ce nce in the firs t 10 m in
1000
Fluore s e nce Vs Tim e For P. Putida Supe rnatant
18500
500
0
17500
30 ul
17000
Fluorescence
Fluorsence
18000
B. Subtillis
P.Putida
-500
Rhodococcus RHA1
Rhodococcus sp
Nocardia autotrophica
-1000
16500
Streptomyces Viridosporus
Leuconostoc Mesentoides
-1500
16000
0
20
40
60
80
100
120
-2000
Tim e (m in)
Paper Submitted to Molecular Biosystems
Non-degraders
Continuous UV-VIS Assay using Nitrated Lignin
LIGNIN
LIGNIN
O
O
O
HO
tetranitromethane
OMe
O
HO
OMe
O2N
OH
lignin
breakdown
O
increase in A400
OMe
O2N
OH
can be performed in
microtitre plate format
OH
Time dependence (0-20 min)
Specificity of bacterial lignin degraders towards
MWL from pine, wheat straw & miscanthus:
Tim e de pendance
0.0576
0.0574
0.0572
Absorbance
0.057
0.0568
0.0566
0.0564
RHA1
0.0562
0.004
0.056
0.0558
0
5
10
15
20
25
0.003
Time (min)
No lignin
Wheat
Myscantus
Pine
Absorbance
0.002
Distinguishes lignin degraders
from non-degraders
0.001
0
Rhodococcus RHA1
not selective
-0.001
-0.002
Bacterial degraders
0.0035
N. Autotrophica
0.003
0.0025
0.0025
0.001
0.0005
0
-0.0005
0.002
P.Put ida
R. RHA1
0.0015
R. Sp.
S. Viridosporus
N. aut ot rophica
L.mesent eroides
B. Subt illis
Absorbance
Absorbance
0.002
0.0015
0.001
0.0005
0
No lignin
Wheat
Myscanthus
Pine
Nocardia autotrophica
shows selectivity
for pine lignin
-0.0005
-0.001
-0.001
-0.0015
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
Extract
Mass (g)
Percentage of total Percentage of total
(wet)
(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%
-
9
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
10
Hexane Extraction - Results
Straw Type
Processing
Extracted mass / mg % dry mass extracted
Untreated
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
It would appear that a higher content is made available by degradation, but it is
unknown to the origin of the material.
11
Analysis
HPLC traces with time
Non-degrader Bacillus subtilis
shows no change
Degrader Pseudomonas putida
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.
O
OH
O
OH
1
Mass spectrum of peak at
RT 7.02 min, assigned to
monosilylated derivative of
ketone (1), m/z 268 (MSiMe3)+, 253 (M-SiMe3CH3)+.
Analysis
• Extracts have been analysed using LTQ-MS at HRI
– Separates and detects using UV and MS
15
Comparison of LTQ data - standards
Vanillic Acid
16
Aromatic metabolites identified (so far)
Compound
1
O
OH
LC-MS
Retention
time (min)
LC-MS
m/z
GC-MS
Retention
time (min)
GC-MS
m/z
(silylated)
Observed with..
4.29
235 MK+
7.02
268 M+
253 -CH3
P. Putida 6hr, 1d, 3d
Rhodococcus RHA1 2hr, 4hr
Miscanthus & wheat straw
4.56
209 MNa+
225 MK+
7.71
243 M+
228 -CH3
P. Putida (straw) 7d
Rhodococcus RHA1
Miscanthus 1d, straw 2d
5.25
195 MH+
5.27
251
M-CH3
P. Putida 6hr
Rhodococcus RHA1 2hr, 6hr
Miscanthus only
5.76
251 MK+
6.03
341 M-CH3
P. Putida 6hr
Rhodococcus RHA1 4hr, 6hr
Miscanthus only
9.09
169 MH+
OCH3
OH
2
COOH
HO
3
O
COOH
CO2H
OCH3
OH
COOH
4
H3CO
5
COOH
OH
COOH
OCH3
OH
Rhodococcus RHA1 6hr
Miscanthus only
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
Other potential major degradation productsyet 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
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
20
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
n=3
n=2
n=4
n=5
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
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
24
Chrysin: a naturally occurring
flavone
TGA Curve for Chrysin derivatives - N2
100
% Mass Change
90
80
70
60
50
25
125
225
325
Temperature / oC
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.