Supporting Information

Catalytic biorefining of plant biomass to non-pyrolytic lignin bio-oil and carbohydrates through hydrogen transfer reactions

Paola Ferrini, Roberto Rinaldi*

Max-Planck-Institut für Kohlenforschung

Kaiser-Wilhelm-Platz 1, D-45470 Mülheim an der Ruhr, Germany

*Corresponding author: rinaldi@kofo.mpg.de

1. Experimental

1.1. Chemicals

Raney Ni 2800 slurry, 2-propanol (2-PrOH, 99.9 %), methanol (MeOH, 99.5 %), sulfuric acid

(95-97 %) and a commercial cellulase preparation (Celluclast® from Trichoderma reesei) were

used as purchased from Sigma Aldrich. Poplar wood (2 mm pellets, J. Rettenmaier & Söhne) was

used as received.

1.2. Organosolv process

Poplar wood (16-17 g) was suspended in a solution of 2-PrOH-water (140 mL, 7:3 v/v) in a

250 mL autoclave and heated to 180 °C within 1 h under mechanical stirring. The suspension was

processed at 180 °C for 3 h. In sequence, the mixture was left to cool down to room temperature.

A reddish-brown solution (liquor) was obtained after filtering out the pulp fibers. The pulp was

washed several times with 2-propanol portions (20 mL) and then dried under vacuum.

Organosolv lignin (containing hemicellulose impurities) was isolated from the liquor by

removing the solvent mixture at 60 °C under vacuum using a rotoevaporator. Organosolv lignin

was obtained as a reddish-brown solid residue.

1.3. Catalytic biorefining method

Poplar wood (16-17 g), Raney Ni (10 g wet) and solvent (140 mL; 2-PrOH:water 7:3 v/v, 2-

PrOH or 2-PrOH:MeOH 10:1 v/v) were placed in a 250 mL autoclave and heated to the desired

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temperature (160 – 220 °C) within 1 h under mechanical stirring. The reaction proceeded under

autogeneous pressure for 3 h. In sequence, the mixture was left to cool down to room

temperature. The liquor was separated from the solids (Raney Ni plus pulp) by filtration through

a glass fiber filter (GF6, Ø 90 mm, Whatman). Very important: as Raney Ni is a pyrophoric

material in its dried form, the wet solids (catalyst plus pulp) were immediately poured into a

round-flask (250 mL) containing 2-PrOH (100 mL). In sequence, under overhead mechanical

stirring, the solids were resuspended in 2-PrOH. Raney Ni was attracted to the flask bottom by a

magnet externally placed on the bottom of the round flask. This procedure was repeated another

four times in order to remove the entire catalyst content from the pulp fibers. The spent catalyst

was washed and stored in 2-PrOH. The pulp remained in suspension, and was recovered by

filtration. The liquor and the filtrates (from the catalyst separation procedure) were combined.

The non-pyrolytic lignin bio-oil was isolated by removing the solvent at 60 °C under vacuum

with a rotoevaporator.

1.4. Hydrogenation of organosolv lignin or bio-oil

Organosolv lignin or non-pyrolytic lignin bio-oil (0.2 g) and Raney Ni (1 g) were suspended in 2-

propanol (10 mL) in an autoclave equipped with mechanical stirrer. The autoclave was flushed

with Argon and the suspension was heated to 160 °C. After 18 h at 160 °C, the autoclave was

quenched in an ice-bath. The suspension was filtered on a Teflon filter previously weighted. The

filtrate was collected and the solvent evaporated with rotatory evaporator at 40 °C. The oil was

analyzed by GC×GC and TGA. Raney Ni was digested with a 5-mol L -1 HCl solution, thus

enabling the determination of the amount of unconverted solid lignin.

1.5. Enzymatic hydrolysis

The enzymatic hydrolysis was performed in a jacketed reactor (150 mL) containing 1-wt % (dry

basis) suspension of the substrate dispersed in 0.1 mol L-1 acetate buffer (100 mL, pH 4.5). The

mixture was stirred at 45 °C. The reaction was initiated by adding Celluclast® into the

suspension (0.5 mL, 350 U). At defined intervals, aliquots (ca. 1 mL) of the reaction mixture

were taken. The samples were immediately heated at 100 °C for 10 min to inactivate the

enzymatic preparation. Next, they were centrifuged and filtered. The formation of glucose and

xylose was determined by HPLC. The filtered sample was then analyzed on an HPLC (Perkin

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Elmer Series 200) equipped with a Nucleogel Ion 300 OA column (Macherey-Nagel). Analysis

conditions: mobile phase: H2SO4 5 mM; flow: 0.5 mL min-1; temperature: 80 °C.

2. Analytics

2.1. Moisture and ash

The humidity in the substrate and pulp was determined by weight loss at 105 °C for 10 min.

Typically, 2-3 grams of sample were analyzed in a thermal balance (Ohaus MB25). For each

sample, this analysis was repeated at least four times.

For the determination of the ash content, ca. 100 mg of pulp or starting material were placed in a

quartz crucible. The samples were then burned in a ventilated muffle oven under a controlled

temperature program: room temperature to 450 °C at 7 °C min-1; 450 to 750°C at 2.5 °C min-1;

and 750°C for 2 h. In sequence, the crucibles were quenched to room temperature in a desiccator.

The ash was weighted. For each sample, this analysis was repeated at least four times.

2.2. Determination of pulp composition

The content of glucans, xylans and lignin were determined by acid saccharification. Typically,

50.0 mg of ground and sieved sample (500 µm) was suspended in a 72 % sulfuric acid solution

(0.5 mL) under stirring at 38 °C for 5 min. In sequence, 10 mL of distilled water was added into

the suspension. The saccharification was then conducted under stirring at 130 °C for 1.5 h. The

suspension was left to cool down to room temperature. The suspension was filtered. The filtrate

was analyzed by HPLC. The determination of the sugar content was performed on an HPLC

Perkin Elmer Series 200 equipped with a Nucleogel Ion 300 OA column (Macherey-Nagel). The

analyses were carried out at 80 °C using a 5 mM H2SO4 solution as eluent (0.5 mL/min). For the

determination of the lignin content, the same saccharification procedure was used; however, the

determination initiates with 500 mg of sample and scaled up the volume of sulfuric acid solution

and water to 5 mL and 100 mL, respectively. After the saccharification at 130 °C for 1.5 h, the

reaction mixture was filtered on a 1 μm Millipore filter previously weighted. The solid was

washed with distilled water until neutral pH. The solid was dried in a ventilated oven at 60 °C for

1-2 days. The determination of the glucan, xylan and lignin contents was performed at least in

four replicates for each sample. The weight of this dried solid was considered as the residual

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lignin in the carbohydrate fraction. The average values (± 2 %) for the isolated pulp and its

composition (Table 1) are ‘dry and ash-free’ (daf).

2.3. Gel Permeation Chromatography

To analyze the apparent molecular weight distribution of Organosolv lignin and non-pyrolytic

lignin bio-oil samples, the sample (10 to 30 mg) and di-n-butyl ether were dissolved in THF (2

mL) and filtered prior to injection. The GPC analyses were performed at 60 °C on a Perkin–

Elmer HPLC 200 equipped with 4 columns (2×TSKgel Super HZ1000, TSKgel Super HZ2000

and TSKgel Super HZ3000, 4.6 mm × ID 15.0 cm, Tosoh Bioscience), and using inhibitor-free

THF as eluent (0.4 mL min-1, Aldrich). For detection, a diode-array detector was used. The

reported results show the chromatogram at 216 nm. The DAD response was normalized by the

sample weight. The apparent molecular weight is given relative to polystyrene standards (200 to

60,000 Da, Aldrich), and thus is only for a relative assessment of the overall changes in the

apparent molecular weight distributions.

2.4. GC GC Analysis

In 2.00 mL MeOH, an aliquot of bio-oil (50.0 mg) and the external standard (di-n-butyl ether,

STD, 20.0 mg) were dissolved. The sample was filtered (membrane filter 0.45 µm). The sample

solutions were analyzed using 2D GCGC-MS (1st column: Rxi-1ms 30 m, 0.25 mm ID, df 0.25

μm; 2nd column: BPX50, 1 m, 0.15 mm ID, df 0.15 μm) in a GC-MS 2010 Plus (Shimadzu)

equipped with a ZX1 thermal modulation system (Zoex). The injector temperature was 300 °C.

The temperature program started with an isothermal step at 40 °C for 5 min. Next, the

temperature was increased from 40 to 300 °C by 5.2 °C min-1. The program finished with an

isothermal step at 300 °C for 5 min. The modulation applied for the comprehensive GC×GC

analysis was a hot jet pulse (400 ms) every 9000 ms. The 2D chromatograms were processed

with GC Image software (Zoex). The products were identified by a search of the MS spectrum

with the MS libraries NIST 08, NIST 08s, and Wiley 9. In some cases, the structure was proposed

by the analysis of the EI-fragmentation pattern and by comparison of retention times with other

samples. The semi-quantification of the products was performed using the GCGC-FID images.

2.5. HSQC NMR Analysis

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All spectra were acquired at 25 °C in DMSO-d6 with a Bruker AV spectrometer (400 or

500 MHz 1H frequency) equipped with a BBFO probe head with z-gradient. The 2D

HSQC NMR (Bruker standard pulse sequence “hsqcetgpsi” with delay optimized for 1JCH

of 145 Hz) were set up with spectral widths of 20 ppm and 180 ppm for 1H- and 13C-

dimensions, respectively. The number of collected complex points was 2,048 for 1H-

dimension with a recycle delay of 3.13 s (3.0 s relaxation delay and 0.13 s acquisition

time). The number of transients for the HSQC spectra was between 12 and 24, and 512

time increments were recorded in 13C-dimension resulting for in an overall experiment

time of 6 to 12 h. For HSQC experiments, a squared cosine-bell apodization function was

applied in both dimensions, followed by zero-filling to 1,024 points in the 13C-dimension

prior to Fourier transform. The 1D 1H NMR and 2D HSQC NMR spectra were processed

using MestReNova 8.1.1 software. Note: HSQC spectrum data must be interpreted with

caution, since the 1JCH dependence of polarization transfer in HSQC experiments is not

suppressed in regular HSQC pulse sequences.[1] As a result, the absolute intensity of cross

peaks are not quantitative in the entire spectral range.[1-2] Regular HSQC NMR

experiments still offer extremely valuable (direct) semiquantitative information for

characterization and comparison of lignins as well as whole plant cell compositions. [3]

Semiquantitative determination of volume integral ratios is possible for 1H—13C pairs in a

similar chemical environment (e.g. Cα—Hα signals for the side-chain of lignin units), due

to the fact that the 1JCH values for the specific entities are reasonably similar.[3a, 3b]

Accordingly, for the different regions of the HSQC spectra, semiquantitative analysis was

performed separately by integration of 1H—13C pairs of interest.[3a, 3b]

2.6. Thermogravimetric analysis

The weight loss profile of the indicated samples was measured on a Mettler Toledo

TGA/DSC 1 Star System operating from 25 to 700 °C at 5 °C min-1 under argon.

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Scheme S1. Solvolysis of lignin occurring in the Organosolv process. a) and b) Reactions proposed for the solvolysis of α-O-4 (dominant in the Organosolv process) and β-O-4 ether linkages, respectively.

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Fig. S1. GC×GC images of the non-pyrolytic lignin bio-oil obtained by the catalytic biorefining method in 2-PrOH/H 2O (7:3, v/v) at 160 °C. SI stands for similarity index obtained from the comparison with the MS libraries NIST 08, NIST 08s, and Wiley 9. STD stands for external standard (di-n-butyl ether).

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Fig. S2. GC×GC images of the non-pyrolytic lignin bio-oil obtained by the catalytic biorefining method in 2-PrOH/H 2O (7:3, v/v) at 180 °C. SI stands for similarity index obtained from the comparison with the MS libraries NIST 08, NIST 08s, and Wiley 9. STD stands for external standard (di-n-butyl ether).

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Fig. S3. GC×GC images of the non-pyrolytic lignin bio-oil obtained by the catalytic biorefining method in 2-PrOH/H 2O (7:3, v/v) at 200 °C. SI stands for similarity index obtained from the comparison with the MS libraries NIST 08, NIST 08s, and Wiley 9. STD stands for external standard (di-n-butyl ether).

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Fig. S4. GC×GC images of the non-pyrolytic lignin bio-oil obtained by the catalytic biorefining method in 2-PrOH/H 2O (7:3, v/v) at 220 °C. SI stands for similarity index obtained from the comparison with the MS libraries NIST 08, NIST 08s, and Wiley 9. STD stands for external standard (di-n-butyl ether).

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Fig. S5. GC×GC images of the non-pyrolytic lignin bio-oil obtained by the catalytic biorefining method in 2-PrOH at 180 °C. SI stands for similarity index obtained from the comparison with the MS libraries NIST 08, NIST 08s, and Wiley 9. STD stands for external standard (di-n-butyl ether).

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Fig. S6. GC×GC images of the non-pyrolytic lignin bio-oil obtained by the catalytic biorefining method in 2-PrOH/MeOH (10:1, v/v) at 180 °C. SI stands for similarity index from the comparison with the MS libraries NIST 08, NIST 08s, and Wiley 9. STD stands for external standard (di-n-butyl ether).

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Fig. S7. GC×GC images of products obtained by further processing of Organosolv lignin through catalytic transfer hydrogenation in the presence of Raney Ni at 160°C for 18 h. SI stands for similarity index from the comparison with the MS libraries NIST 08, NIST 08s, and Wiley 9. STD stands for external standard (di-n-butyl ether).

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Fig. S8. GC×GC images of products obtained by further processing of non-pyrolytic lignin bio-oil (180 °) through catalytic transfer hydrogenation in the presence of Raney Ni at 160°C for 18 h. SI stands for similarity index from the comparison with the MS libraries NIST 08, NIST 08s, and Wiley 9. STD stands for external standard (di-n-butyl ether).

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Fig. S9. TG curves for Organosolv lignin, Organosolv lignin subjected to Raney Ni/2-PrOH at 160 °C for 18 h, non-pyrolytic lignin bio-oil obtained from catalytic biorefining in 2-PrOH/H2O (7:3, v/v) at 180 °C, and the lignin bio-oil subjected to Raney Ni/2-PrOH at 160 °C for 18 h. Note: Organosolv lignin suffer a loss of about 27 % weight upon heating this material from 30 to 300 °C (at 5 °C min-1 under argon). Considering the polymeric nature of this material, this weight loss is associated with the release of volatile fragments generated by lignin thermolysis rather than vaporization of low Mw components eventually occurring in the solid lignin.

References

[1] S. Heikkinen, M. M. Toikka, P. T. Karhunen, I. A. Kilpeläinen, J. Am. Chem. Soc. 2003, 125, 4362-4367.

[2] a) K. Hu, W. M. Westler, J. L. Markley, J. Am. Chem. Soc. 2011, 133, 1662-1665; b) K. Cheng, H. Sorek, H. Zimmermann, D. E. Wemmer, M. Pauly, Anal. Chem. 2013, 85, 3213-3221.

[3] a) S. D. Mansfield, H. Kim, F. Lu, J. Ralph, Nat. Protocols 2012, 7, 1579-1589; b) J. Ralph, Landucci, L. L., NMR of lignins, CRC Press, 2010; c) R. Samuel, M. Foston, N. Jaing, S. Cao, L. Allison, M. Studer, C. Wyman, A. J. Ragauskas, Fuel 2011, 90, 2836-2842.

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