Bioconversion of Lignocellulosic Biomass into Bacterial ......School of Chemistry & Biochemistry ... 2012. 30(12): p. 627-637 3 ... Franz Wolf "Wood" in Ullmann's Encyclopedia of Industrial

Bioconversion of Lignocellulosic Biomass into Bacterial Bio-Oils

1

Tyrone Wells, Jr.

Georgia Institute of Technology School of Chemistry & Biochemistry Institute of Paper Science Technology

“Trees into Fat”

Potential Domestic Supplemental Fuel Platform

2

BIOCONVERSION

Using biological systems to convert abundant starting materials into more valuable compounds

Organism

Substrate Product Lignin Bio-oil

Wells, T. and A.J. Ragauskas, Biotechnological opportunities with the beta-ketoadipate pathway. Trends in Biotechnology, 2012. 30(12): p. 627-637

Lignin Recovery boiler limited mills BIODIESEL

3 Wells, T. and A.J. Ragauskas, Biotechnological opportunities with the beta-ketoadipate pathway. Trends in Biotechnology, 2012. 30(12): p. 627-637

Fundamental Review of “Trees into Fat”

• Background of Bacterial Strains

• Rhodoccocus opacus

• Experimental Work

• Characterize How Cells Grow

• Results and Discussion

• Model Compounds Based on Lignin Units

• Kraft Lignin

4

Rhodococcus opacus • Soil Bacteria

• Oleaginous

• >20% of cell dry weight in oil

• Two Strains

• DSM 1069

• PD 630

• “High affinity” towards the digestion of lignocellulosic aromatics

• β-ketoadipate pathway (β-KAP)

HSCoA

Glycerol


(β-KAP)

5

Experimental

• Model Compounds • Vanillic acid (G-type)

• p-Hydroxybenzoic acid (PHA/4-HBA) (H-type)

Fundamental Overview of Tracking Cell Growth

• Verifying that the cells are growing well on lignin as sole carbon source • Decreasing Concentration of the Substrate

• Increasing Cell Dry Weight (CDW)

• Track the number of “healthy cells” (aka Colony Forming Units, CFU)

• Characterize the Generated Fats (Fatty Acid Methyl Esters, FAME)

• Rupture and Transesterification GC/MS

6

[substrate] [cell dry weight]

7

Kosa M, Ragauskas AJ (2011) Lipids from heterotrophic microbes: advances in metabolism research. Trends Biotechnol 29:53-61

Lipid Composition Within Bio-Oil

8

Comparison of FAME compositions at maximum specific yields and

productivities

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

DSM

1069-Glu-

12

DSM

1069-4-

HBA-12

DSM

1069-

VanA-24

PD630-

Glu-12

PD630-4-

HBA-36

PD630-

VanA-48

strain-substrate-time [h] till maximum productivity

(and yield)

FA

ME

linoleate

c-oleate

10-me-stearate

stearate

c-heptadecenoate

10-me-heptadecanoate

heptadecanoate

t-palmitoleate

c-palmitoleate

palmitate

pentadecanoate

myristate

palmitic acid

c-oleic acid

c-palmitoleate acid



productivities

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

DSM

1069-Glu-

12

DSM

1069-4-

HBA-12

DSM

1069-

VanA-24

PD630-

Glu-12

PD630-4-

HBA-36

PD630-

VanA-48


(and yield)

FA

ME

linoleate

c-oleate

10-me-stearate

stearate

c-heptadecenoate


heptadecanoate

t-palmitoleate

c-palmitoleate

palmitate

pentadecanoate

myristate


productivities

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

DSM

1069-Glu-

12

DSM

1069-4-

HBA-12

DSM

1069-

VanA-24

PD630-

Glu-12

PD630-4-

HBA-36

PD630-

VanA-48


(and yield)

FA

ME

linoleate

c-oleate

10-me-stearate

stearate

c-heptadecenoate


heptadecanoate

t-palmitoleate

c-palmitoleate

palmitate

pentadecanoate

myristate


productivities

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

DSM

1069-Glu-

12

DSM

1069-4-

HBA-12

DSM

1069-

VanA-24

PD630-

Glu-12

PD630-4-

HBA-36

PD630-

VanA-48


(and yield)

FA

ME

linoleate

c-oleate

10-me-stearate

stearate

c-heptadecenoate


heptadecanoate

t-palmitoleate

c-palmitoleate

palmitate

pentadecanoate

myristate


productivities

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

DSM

1069-Glu-

12

DSM

1069-4-

HBA-12

DSM

1069-

VanA-24

PD630-

Glu-12

PD630-4-

HBA-36

PD630-

VanA-48


(and yield)

FA

ME

linoleate

c-oleate

10-me-stearate

stearate

c-heptadecenoate


heptadecanoate

t-palmitoleate

c-palmitoleate

palmitate

pentadecanoate

myristate

9

Sub

stra

te R

esi

du

e (

Lign

in m

g/m

l)


10

72 h 72 h

Kosa M, Ragauskas AJ (2011) Lipids from heterotrophic microbes: advances in metabolism research. Trends Biotechnol 29:53-61 Wells, T. and A.J. Ragauskas, Biotechnological opportunities with the beta-ketoadipate pathway. Trends in Biotechnology, 2012. 30(12): p. 627-637

11 Kosa M, Ragauskas AJ (2011) Lipids from heterotrophic microbes: advances in metabolism research. Trends Biotechnol 29:53-61 Wells, T. and A.J. Ragauskas, Biotechnological opportunities with the beta-ketoadipate pathway. Trends in Biotechnology, 2012. 30(12): p. 627-637

12

TBA

palmitic acid

Conclusions

• Oil-producing bacteria can metabolize aromatic biomass and produce bio-oil

• This process has potential as a supplemental energy platform

Current Accomplishments

• Successfully generated bacterial bio-oils from:

• Model compounds (G and H-type monolignol analogs)

• Low Mw Softwood Kraft lignin

Future Work

• Adapt the bacteria to higher Mw Kraft lignin

• Alternative lignocellulosic derivatives

13

Trees into Fat

14

• Acknowledgement • Arthur Ragauskas

• Matyas Kosa

• DOE Biorefinery Project

15

16

Review of Lignocellulosic Biomass • Cellulose

• Hardwood 40-44%

• Softwood 40-44%

• Hemicellulose • Hardwood 25-35%

• Softwood 20-32%

• Lignin • Hardwood 20-25%

• Softwood 25-35%

• Complex biomacromolecule

• Monolignols

• p-Hydroxyphenyl (H), Guaiacyl (G), Syringyl (S)

• 3D polyaromatic macromolecule

• Recalcitrant

• Significantly less applications

17 Horst H. Nimz, Uwe Schmitt, Eckart Schwab, Otto Wittmann, Franz Wolf "Wood" in Ullmann's Encyclopedia of Industrial Chemistry 2005, Wiley-VCH, Weinheim.

18

Optimizing the Use of Lignin

• Lignin

• (>90%) Kraft lignin burnt as a non-optimized fuel

• Opportunity for recovery-boiler limited mills

• Alternative Uses of Lignin

• Lignin Biodiesel

• Bacterial treatment

• Sustainable supplemental fuel platform

Pu Y, Kosa M, Kalluri UC, Tuskan GA, Ragauskas AJ (2011) Challenges of the utilization of wood polymers: how can they be overcome? Appl Microbiol Biotechnol, 91:1525-1536


Areskogh, J. Li, G. Gellerstedt, G. Henriksson, Investigation of the molecular weight increase of commercial lignosulphonates by laccase catalysis, Biomacromol. 11 (2010) 904-910.

19

• Fermentation Growth with PD 630

• Calculated via OD at 600 nm, 1 w/V%


20

• Fermentation Growth with PD 630

• Calculated via OD at 600 nm, 1 w/V%, 0.5 w/V%

PD 630 - OD [600]

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0 20 40 60 80 100 120 140 160 180

hours

OD

[60

0]

Glucose PHA VanA UE Gluc-0.5 VanA-0.5 UE-0.5


21

• Fermentation Growth with DSM 1069

• Calculated via OD at 600 nm, 1 w/V%


22

• Fermentation Growth with DSM 1069

• Calculated via OD at 600 nm, 1 w/V%, 0.5 w/V%

DSM 1069 - OD [600]

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 50 100 150 200 250 300

hours

OD

[60

0]

Glucose PHA VanA UE Gluc-0.5 VanA-0.5 UE-0.5


23


24

palmitic acid


Lipid Composition Within Bio-Oil

25


productivities

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

DSM

1069-Glu-

12

DSM

1069-4-

HBA-12

DSM

1069-

VanA-24

PD630-

Glu-12

PD630-4-

HBA-36

PD630-

VanA-48


(and yield)

FA

ME

linoleate

c-oleate

10-me-stearate

stearate

c-heptadecenoate


heptadecanoate

t-palmitoleate

c-palmitoleate

palmitate

pentadecanoate

myristate

palmitic acid

c-oleic acid

c-palmitoleate acid

linoleic acid

stearic acid

myristate acid


26

The β-KAP

Protocatechuate β -Carboxymuconate β -Ketoadipate

β –Ketoadipate enol-lactone

γ -Carboxymuconolactone

Protocatechuate 3,4-dioxygenase

β-carboxy-cis,cis-muconate lactonizing

enzyme

γ -Carboxymuconolactone decarboxylase

enol-lactone hydrolase

1 2 3 4

Precursory Compounds


R3

27

Experimental • Adaptation Process

• Cell proliferation (full media)

• Centrifugation

• Cell proliferation (minimal media + new carbon source)

• Lipid accumulation (reducing nitrogen content of minimal media)

28 Kosa M, Ragauskas AJ (2011) Lipids from heterotrophic microbes: advances in metabolism research. Trends Biotechnol 29:53-61

29


Documents

Bioconversion of Lignocellulosic Biomass into Bacterial ......School of Chemistry & Biochemistry ... 2012. 30(12): p. 627-637 3 ... Franz Wolf "Wood" in Ullmann's Encyclopedia of Industrial