Dr. Mark CrockerAssociate Director, Center for Applied Energy ResearchUniversity of Kentucky

2

Mark Crocker Center for Applied Energy Research, University of Kentucky

2013 SEC Symposium

Lignin Deconstruction for the Production of Fuels and

Chemicals

3

Overview

• Introduction & Background

• Our Approach• In planta modification to modify lignin fraction• Lignin dissolution in ionic liquids• Oxidative cleavage of lignin and lignin model compounds

• Summary & Conclusions

4

Introduction: Lignocellulosic biomass

- Plants made of lignin, cellulose and hemicellulose

- Amount of each polymer differs depending on plant species, the part of the plant, age and

environmental factors

- Structure of lignin polymer also varies

Sinapyl alcohol (S)

Coumaryl alcohol (H)

Coniferyl alcohol (G)

J. Zakzeski, P.C.A. Bruijnincx, A.L. Jongerius, B.M. Weckhuysen, Chem. Rev. 2010, 110, 3552

5

Background: Why Lignin?

J. Zakzeski et al., Chem. Rev. 2010, 110, 3552

O

O

O

OH

OCH3

O

OCH3

O

O

OH

HO

CH3O

O

CHO

HO

O

O

OCH3

OH

OHO

CH3O

O

HO

CH3O

O

OCH3

O

OH

O

OCH3O

OCH3

O

O

HO

OCH3

HO

OH

OCH3

O

OH

OCH3OH

O

HO

O

OH

O

Y.H.P. Zhang, J. Ind. Microbiol. Biotechnol. 2008, 35, 367

Polymers, resins, dyes,pharmaceuticals

6

Our ApproachDevelop new processes for the direct conversion of lignin to

liquid fuels and chemicals:

In planta modification thereby accentuating lignin

fraction and facilitating downstream processing.

S. DeBolt

Catalytic oxidative cleavage of the lignin at benzylic position

M. Meier, M. Crocker

Dissolution and controlled thermolysis in ionic liquids

S. Morton

Catalytic upgrading of lignin derived oxygenates

M. CrockerLignin Product Stream

7

In planta Modification to Increase Lignin Content and Facilitate Downstream

Processing

8

Chemical secretions from endophytic microbes elicit ectopic lignificationin model plant arabidopsis:

• Isolated fungal and bacterial endophytes from switchgrass: 1080 identified and categorized into their taxonomic diversity

• Grow cells, spin and separate media from cells - extract DNA from cells and sequence identify by rDNA

• Freeze dry and extract secretion as bulk

• Add to sterile plant growth media

• Test for ectopic lignin (via a dye reaction)

Application of Endophyte Secretions to Bioenergy Grasses

9

The red giant was selected from a mutagenized population of Sorghum plants (2 year project in selection alone)

• Aim: sugars contained in stem can be converted to ethanol/ butanol and leaves can be converted to phenolics-based fuel

• Selected based on high phenolics in leaves• Little phenotype (loss of growth) relative to wild type plant• Grown over several generations (1 acre grown this year)

Application of Forward Genetics to Bioenergy Crops

10

Py-GC/MS Analysis of Mutant SorghumIn

ten

sit

y (

a.u

.)

Wild Sorghum Leaf

1.6 4.6 7.5 10.513.416.419.322.325.228.231.134.137.040.042.945.948.851.7

Retention Time (min)

Inte

nsit

y (

a.u

.)

Mutant Sorghum Leaf

Total lignin (area %)

S:G (area %)

Wild type leaf

36.2 (+/- 2.8) 0.37 (+/- 0.01)

Mutant leaf

41.5 (+/- 1.3) 0.51 (+/- 0.03)

Mostly holocellulose pyrolysates

Mostly lignin pyrolysates

- Pyrolysis-GC/MS: analytical scale thermal decomposition directly coupled to GC/MS- Efficient method for analysis of polymers

11

Lignin Dissolution in Ionic Liquids

12

Ionic Liquids for Lignin Dissolution and ManipulationSynthetic Routes Toward Dialkyl Imidazolium Halides

+ R-X + R’-XX

X

• 1-alkyl imidazoles with alkyl groups larger than butyl are not commercially available

• Properties of long chain di-alkyl imidazolium halides are not well understood

1-Hexyl-3-Dodecyl Imidazolium Halide

Imidazole 1-Alkyl Imidazole 1-Alkyl-3-Alkyl’ Imidazolium Halide

13

Butyl

Hexyl

Octyl

Decyl

Dodecyl

Lignin Dissolution in Alkyl-methyl Imidazolium Chlorides at 100 °C

0 min 30 min 60 min

• Lignin solubility decreases as alkyl chain length increases

• Butyl and hexyl show complete dissolution before 30 minutes

• Octyl and decyl show complete dissolution before 60 minutes

14

Butyl

Hexyl

Octyl

Decyl

Dodecyl

Cellulose Dissolution in Alkyl-methyl Imidazolium Chlorides at 100 °C

0 min 30 min 60 min

• Cellulose solubility is much lower, but still decreases as alkyl chain length increases

• All failed to dissolve cellulose before 60 minutes

• Decyl and dodecyl demonstrated no impact on cellulose

15

Dissolution of Biomass Constituents After 3 h at 100 °C

Cellulose Hemicellulose Lignin

Butyl

Hexyl

Octyl

Decyl

Dodecyl

0%

<50%

>50%

100%

16

Oxidative Cleavage of Lignin and Lignin Model Compounds

17

Linkage B-O-4 B-5 5-5

Structure

Abundance in Hardwood 60% 6% 9%

Linkage 4-O-5 B-B B-1

Structure

Abundance in Hardwood

6.5% 3% 7%

O

OH

R

OO

O

R

1

23

4

O

54

HO

OR

O

R

O

R

O

R

O

R

O

R

5

5

1

2

34

43

2

1

O

4

5

O OH

O

R

1

2

3

4

R 12

3

OO

O

R

O

O

R

O

OH

OH

1

O

R

O

O

O

R

J. Zakzeski et al., Chem. Rev. 2010, 110(6), 3552

Common Linkages Present in Hardwood Lignin (Spruce)

18

Our Approach for Lignin Deconstruction

Oxidation Sequence for Simple Model Compounds

LDH, O2 (1 atm)

Diphenyl ether90oC, 24 h

1.3 eq. m-CPBA1,2-dichloroethane

70oC, 24 h

LDH = Ni/Al layered double hydroxide,B.M. Choudary et al., Angew. Chem. 2001, 113, 785.

58% yield

85% yield

20

Compounds Modeling the β-O-4 Linkage in Lignin

Cmp Substituent

1 R1, R2, R3, R4, R5, R6 = H

2 R1, R2, R4, R6 = H, R3 = OH, R5 = n-Pr

3 R1, R2, R6 = CH3O, R3 = OH, R4 = CH3, R5 = n-Pr

4 R1, R6, = CH3O, R2, R3 = OH, R4 = CH3, R5 = n-Pr

5 R1, R2, R6 = CH3O, R3 = OH, R4 = CH2OH, R5 = n-Pr

6 R1, R6, = CH3O, R2, R3 = OH, R4 = CH2OH, R5 = n-Pr

21

Selective Aerobic Oxidation of Benzylic Alcohol Groups Using TEMPO, NaNO2

Cmpd Conv (%) A (% yield)

1 0 -

2 100 100

3 100 81

4 polymer -

5 100 80

6 polymer -

A

TEMPO, NaNO2,

HCl, NaCl, CH2Cl2,

O2 balloon, 25°C

Wang, L.; Li, J.; Yang, H.; Lv, Y.; Gao, S. J. Org. Chem. 2012, 77, 790

Excellent conversion and selectivity!

22

Cα-Cβ Bond Cleavage via Baeyer-Villiger Oxidation and Hydrolysis

Cmpd Conv (%) C (% yield) D (% yield)

2’ 90 10 -

3’ 100 - 81

C D

Liou, L., Huang, C.; Int. J. Appl. Sci. Eng. 2006, 4, 3

Note – Compounds 2’ and 3’ are benzylic alcohol group oxidation products of 2 and 3

30% H2O2

HCOOH, 1,2-DCE

Most of the oxidized material hydrolyzes in the reaction mixture Clearly, the ketones undergo B-V oxidation as expected

7 9 11 13 15 17 19 21

Retention Time (Minutes)

Rela

tive

Abso

rban

ce

MW 173: m-CPBA

OL + (i) benzylic oxidn.+ (ii) Baeyer-Villiger oxidn.+ (iii) transesterification

M.W. 256:

Organosolv lignin (OL)

OL + benzylic oxidation

O

OH

First Attempts at Lignin Deconstruction

Mobile phase: DMSO/THF; Column: PSS Suprema, Linear S Diode array detector (320 nm)

OCH3H3CO

OHOCH3

CHO

OHOCH3H3CO

CHO

OH

Retention time (min)

+ dimers

GPC analysis:

24

Summary & Conclusions

• In planta approaches have been successfully applied to increase lignin/phenolic content in plants

• Mutations in sorghum result in changes in lignin structure and composition as seen by Py-GC/MS

• Ionic liquids have been developed which permit selective dissolution of lignin in biomass

• Oxidative cleavage of lignin β-O-4 model compounds has been demonstrated by a sequence of benzylic alcohol oxidation, Baeyer-Villiger oxidation and hydrolysis

• This methodology is being applied to lignin

• Efforts to manipulate the structure of lignin and to effect its deconstruction are still in their infancy

25

Acknowledgements

• NSF – Dr. George Antos

• Dr. Rodney Andrews (PI), Dr. Seth DeBolt (co-I), Dr. Mark Meier (co-I), Dr. Samuel Morton (co-I)

• Students, postdocs and staff:

Anne Harman-Ware Nikhil PatilJustin Mobley Aman KaurRobby Pace Soledad YaoAndy Placido Dr. Venu MenduCharles Thacker Dr. Carloalberto PettiDr. James Hower Brian Williams

This work supported by a grant from the National Science FoundationNSF AWARD: EFRI-0937657

Dr. Mark CrockerAssociate Director, Center for Applied Energy ResearchUniversity of Kentucky