Tyrone Wells, Jr - rbi1.gatech.edu

Bioconversion of Lignocellulosic Biomass into Bacterial Bio-Oils

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Tyrone Wells, Jr. Georgia Institute of Technology School of Chemistry & Biochemistry Institute of Paper Science and Technology

Potential Domestic Supplemental Fuel Platform

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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

Outline • Background of Bacteria

• Rhodoccocus opacus

• Experimental Progress

• Kraft Lignin

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Rhodococcus opacus • Both are soil bacteria • Both are 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)

Model Compounds vs. Lignin

• Microbes digested nearly all of substrate during adaptation tests

• Oleaginous amounts of lipid production • 30% palmitic acid

• Substantially less complex than actual lignin

• Lower molecular weight • High homogeneity

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Model Compounds

Lignin (basic representation) palmitic acid

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Subs

trat

e Re

sidu

e (L

igni

n m

g/m

l)

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

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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

8 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

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TBA

palmitic acid

Summary • Microbes can consume both aliphatic and aromatic functional groups in Kraft

lignin • Kraft lignin polymerizes during adaptation, more challenging for bacterial

digestion • Goal: Train microbes to digest progressively higher concentrations of high MW

lignin • Improved yields • FA composition is promising

Future Work • New sources for bioconversion opportunities • Adaptation to pretreatment waste streams

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Bioconversion

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• Acknowledgement • Arthur Ragauskas • Matyas Kosa • Zhen Wei • DOE Biorefinery Project

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palmitic acid


Summary • 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 • Ultrasonicated Ethanol Organosolv Lignin

• Successfully adapted bacteria to: • Tannin-derived pyrolysis oils • Ethanol Organosolv Hemicellulose

Future Work • Optimize adaptation to dilute acid pretreatment waste (contains lignin and hemicellulose • Adapt the bacteria to higher Mw Kraft lignin • Alternative lignocellulosic derivatives

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Experimental • Adaptation Process

• Cell proliferation (full media)

• Centrifugation • Cell proliferation (minimal media + new carbon source) PHASE I

• Lipid accumulation (reducing nitrogen content of minimal media) PHASE II

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

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, can also be tracked by UV-Vis absorbance @600 nm) • 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

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[substrate] [cell dry weight]

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Lipid Composition Within Bio-Oil

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0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

1

FAM

E

palmitic acid

c-oleic acid

c-palmitoleate acid


DSM1069-4-HBA-12

DSM1069-

VanA-24

PD630-4-HBA-36

PD630-VanA-48

linoleatec-oleate10-me-stearatestearatec-heptadecenoate10-me-heptadecanoateheptadecanoatet-palmitoleatec-palmitoleatepalmitatepentadecanoatemyristate

Comparison of FAME compositions at maximum specific yields and productivities

strain-substrate-time [h] till maximum productivity (and yield)

Pyrolysis

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Pyrolysis is the thermal degradation of biomass bio-oil

Light Oil Fraction

Heavy Oil

Fraction

Lignin

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600oC, 1 hr 500 ml/min N2 Gas Flow

M Kosa, H. Ben, H. Theliander, A. J. Ragauskas, Pyrolysis oils from CO2 precipitated Kraft lignin, Green Chemistry. 13 (2011) 3196-3202.

Future Work: Pyrolysis

Light Oil Fraction

Heavy Oil

Fraction

Lignin

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600oC, 1 hr 500 ml/min N2 Gas Flow

Heavy Oil Fraction Non-water soluble Hydrophobic globules Highly acidic Not a viable substrate for

bacteria

Comparison of EOL vs. EOL Oil Growth

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EOL Growth EOL Oil Growth

• Growth of DSM1069 on EOL and EOL Pyrolysis Oil • 68 h and at 0.3 w/V% substrate concentration

Kraft Lignin Pyrolysis Heavy Oil Adaptation

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Rhodococcus strains on Kraft lignin pyrolysis oilat 0.5 w/V%

0.0E+00

2.0E+04

4.0E+04

6.0E+04

8.0E+04

1.0E+05

1.2E+05

0 50 100 150 200time [h]

livin

g ce

ll nu

mbe

rs [C

FU/m

l]

Rhodococcus opacus DSM 1069 Rhodococcus opacus PD630

Could pyrolysis of Tannin Oils work?

Figure C.3.2.1. 13C-NMR of tannin pyrolysis oil (in DMSO-d6, 40.45 ppm) provided by Haoxi Ben. The major peaks are assignable to catechol (147.3, 122.7, 117.3 ppm), acetic acid (176.0 and 22.9 ppm). Catechol is a major

component of the Beta-ketoadipate pathway...

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GPC results of Tannin Oil reveals Samples are Predominantly Catechol

Figure C.3.2.2. GPC analysis of generated TAN LO. The largest population of material at ~110 g/mol, corresponds to catechol (110.1 g/mol).

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Hemicellulose

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Hemicellulose: Future work • GPC, NMR,HPLC for the substrate • Transesterification, GC/MS for the bacterials

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

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

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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

Kosa M, Ragauskas AJ (2011) Lipids from heterotrophic microbes: advances in metabolism research. Trends Biotechnol 29:53-61 Areskogh, J. Li, G. Gellerstedt, G. Henriksson, Investigation of the molecular weight increase of commercial lignosulphonates by laccase catalysis, Biomacromol. 11 (2010) 904-910.

Lipid Composition Within Bio-Oil

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Comparison of FAME compositions at maximum specific yields and productivities

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

DSM1069-Glu-

12

DSM1069-4-HBA-12

DSM1069-

VanA-24

PD630-Glu-12

PD630-4-HBA-36

PD630-VanA-48

strain-substrate-time [h] till maximum productivity (and yield)

FAM

E

linoleatec-oleate10-me-stearatestearatec-heptadecenoate10-me-heptadecanoateheptadecanoatet-palmitoleatec-palmitoleatepalmitatepentadecanoatemyristate

palmitic acid

c-oleic acid

c-palmitoleate acid

linoleic acid

stearic acid

myristate acid


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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

Tyrone Wells, Jr - rbi1.gatech.edu

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