Bioconversion of Lignocellulosic Biomass into Bacterial Bio-Oils

Transcription

1 Bioconversion of Lignocellulosic Biomass into Bacterial Bio-Oils Tyrone Wells, Jr. Georgia Institute of Technology School of Chemistry & Biochemistry Institute of Paper Science and Technology 1

2 Substrate Lignin Product Bio-oil BIOCONVERSION Organism Using biological systems to convert abundant starting materials Lignin into Recovery more valuable boiler BIODIESEL compounds limited mills Potential Domestic Supplemental Fuel Platform Wells, T. and A.J. Ragauskas, Biotechnological opportunities with the beta-ketoadipate pathway. Trends in Biotechnology, (12): p

3 Outline Background of Bacteria Rhodoccocus opacus Experimental Progress Kraft Lignin Wells, T. and A.J. Ragauskas, Biotechnological opportunities with the beta-ketoadipate pathway. Trends in Biotechnology, (12): p

4 Rhodococcus opacus Both are soil bacteria Both are oleaginous >20% of cell dry weight in oil (β-kap) Two Strains DSM 1069 PD 630 HSCoA High affinity towards the digestion of lignocellulosic aromatics β-ketoadipate pathway (β-kap) Glycerol Wells, T. and A.J. Ragauskas, Biotechnological opportunities with the beta-ketoadipate pathway. Trends in Biotechnology, (12): p

5 Model Compounds vs. Lignin Model Compounds Microbes digested nearly all of substrate during adaptation tests Oleaginous amounts of lipid production 30% palmitic acid Substantially less complex than actual lignin palmitic acid Lower molecular weight High homogeneity Lignin (basic representation) 5

6 Substrate Residue (Lignin mg/ml) Kosa M, Ragauskas AJ (2011) Lipids from heterotrophic microbes: advances in metabolism research. Trends Biotechnol 29:

7 72 h 72 h Wells, T. and A.J. Ragauskas, Biotechnological opportunities with the beta-ketoadipate pathway. Trends in Biotechnology, (12): p Kosa M, Ragauskas AJ (2011) Lipids from heterotrophic microbes: advances in metabolism research. Trends Biotechnol 29:

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

9 palmitic acid TBA 9

10 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 10

11 Bioconversion Acknowledgement Arthur Ragauskas Matyas Kosa Zhen Wei DOE Biorefinery Project 11

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

19 palmitic acid Kosa M, Ragauskas AJ (2011) Lipids from heterotrophic microbes: advances in metabolism research. Trends Biotechnol 29:

20 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 M w 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 M w Kraft lignin Alternative lignocellulosic derivatives 20

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

23 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 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 [substrate] [cell dry weight] 23

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

25 Lipid Composition Within Bio-Oil FAME Comparison of FAME compositions at maximum specific yields and productivities 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% DSM HBA-12 DSM VanA-24 PD HBA-36 PD630- VanA-48 linoleate c-oleate 10-me-stearate stearate c-heptadecenoate 10-me-heptadecanoate heptadecanoate t-palmitoleate c-palmitoleate palmitate pentadecanoate myristate strain-substrate-time [h] till maximum productivity (and yield) Kosa M, Ragauskas AJ (2011) Lipids from heterotrophic microbes: advances in metabolism research. Trends Biotechnol 29:53-61 palmitic acid c-oleic acid c-palmitoleate acid 25

26 Pyrolysis 26

27 Pyrolysis is the thermal degradation of biomass bio-oil Heavy Oil Fraction Lignin 600 o C, 1 hr 500 ml/min N 2 Gas Flow Light Oil Fraction 27 M Kosa, H. Ben, H. Theliander, A. J. Ragauskas, Pyrolysis oils from CO2 precipitated Kraft lignin, Green Chemistry. 13 (2011)

28 Future Work: Pyrolysis Heavy Oil Fraction Non-water soluble Hydrophobic globules Highly acidic Not a viable substrate for bacteria Lignin 600 o C, 1 hr 500 ml/min N 2 Gas Flow Heavy Oil Fraction Light Oil Fraction 28

29 Comparison of EOL vs. EOL Oil Growth Growth of DSM1069 on EOL and EOL Pyrolysis Oil 68 h and at 0.3 w/v% substrate concentration EOL Growth EOL Oil Growth 29

30 Kraft Lignin Pyrolysis Heavy Oil Adaptation 1.2E+05 Rhodococcus strains on Kraft lignin pyrolysis oil at 0.5 w/v% living cell numbers [CFU/ml] 1.0E E E E E E time [h] Rhodococcus opacus DSM 1069 Rhodococcus opacus PD630 30

31 Could pyrolysis of Tannin Oils work? Figure C C-NMR of tannin pyrolysis oil (in DMSO-d6, ppm) provided by Haoxi Ben. The major peaks are assignable to catechol (147.3, 122.7, ppm), acetic acid (176.0 and 22.9 ppm). Catechol is a major component of the Beta-ketoadipate pathway... 31

32 GPC results of Tannin Oil reveals Samples are Predominantly Catechol Figure C GPC analysis of generated TAN LO. The largest population of material at ~110 g/mol, corresponds to catechol (110.1 g/mol). 32

33 Hemicellulose 33

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

35 Review of Lignocellulosic Biomass Cellulose Hardwood 40-44% Softwood 40-44% Hemicellulose Lignin Hardwood 25-35% Softwood 20-32% Hardwood 20-25% Softwood 25-35% Complex biomacromolecule Monolignols p-hydroxyphenyl (H), Guaiacyl (G), Syringyl (S) 3D polyaromatic macromolecule Recalcitrant Significantly less applications Horst H. Nimz, Uwe Schmitt, Eckart Schwab, Otto Wittmann, Franz Wolf "Wood" in Ullmann's Encyclopedia of Industrial Chemistry 2005, Wiley-VCH, Weinheim. 35

36 Optimizing the Use of Lignin Lignin (>90%) Kraft lignin burnt as a nonoptimized fuel Opportunity for recovery-boiler limited mills Alternative Uses of Lignin Lignin Biodiesel Bacterial treatment Sustainable supplemental fuel platform 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) 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:

37 Lipid Composition Within Bio-Oil FAME 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Comparison of FAME compositions at maximum specific yields and productivities DSM 1069-Glu- 12 DSM HBA-12 DSM VanA-24 PD630- Glu-12 PD HBA-36 PD630- VanA-48 strain-substrate-time [h] till maximum productivity (and yield) linoleate c-oleate 10-me-stearate stearate c-heptadecenoate 10-me-heptadecanoate heptadecanoate t-palmitoleate c-palmitoleate palmitate pentadecanoate myristate Kosa M, Ragauskas AJ (2011) Lipids from heterotrophic microbes: advances in metabolism research. Trends Biotechnol 29:53-61 linoleic acid stearic acid palmitic acid myristate acid c-oleic acid c-palmitoleate acid 37

38 The β-kap R 3 Precursory Compounds Protocatechuate β -Carboxymuconate γ -Carboxymuconolactone β Ketoadipate enol-lactone β -Ketoadipate Protocatechuate 3,4- dioxygenase β-carboxy-cis,cismuconate lactonizing enzyme γ -Carboxymuconolactone decarboxylase enol-lactone hydrolase Wells, T. and A.J. Ragauskas, Biotechnological opportunities with the beta-ketoadipate pathway. Trends in Biotechnology, (12): p