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.