Supporting Information. Lignin Extraction and Catalytic Upgrading from Genetically. Modified Poplar

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Priscilla Wade
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1 Electronic Supplementry Mteril (ESI) for Green Chemistry. This journl is The Royl Society of Chemistry 2017 Supporting Informtion Lignin Extrction nd Ctlytic Upgrding from Geneticlly Modified Poplr Ho Luo nd Mhdi M. Abu-Omr * Deprtment of Chemistry nd Biochemistry, University of Cliforni, Snt Brbr, CA 93106, USA. * Corresponding uthor: M. M. Abu-Omr. E-mil: buomr@chem.ucsb.edu S1

2 1. Instrumenttion nd Chrcteriztion Conditions HPLC-MS nlysis All HPLC seprtions for mss spectrometry nlysis were performed on Surveyor Plus HPLC system from Thermo Scientific consisting of quternry pump, n utosmpler, photodiode rry (PDA) detector, nd Zorbx SB-C18 column. A non-liner grdient of wter (A) nd cetonitrile (B) ws used s follows: 0.00 min, 95% A nd 5% B; min, 95% A nd 5% B; min, 40% A nd 60% B; min, 5% A nd 95 %; 38.00min, 5% A nd 95% B; min, 95% A nd 5% B; min, 95% A nd 5% B. Flowrte of the mobile phse ws kept t 500 μl/min. PDA detector ws set t the wvelength of 254 nm. Mss spectrometric nlysis (MS, MS 2 nd MS 3 ) of HPLC eluent ws performed using Thermo Scientific liner qudrupole ion trp (LQIT) mss spectrometer equipped with n electrospry ioniztion (ESI) source. All mss spectrometry experiments were performed under negtive ion mode. HPLC eluents were mixed vi T-connector with 1% sodium hydroxide wter solution t flow rte of 0.1 μl/min before entering the ESI source. Addition of sodium hydroxide fcilittes deprotontion of the nlytes. ESI source conditions were set s: 3.5 kv spry voltge; 50 (rbitrry units) sheth gs (N 2 ) flow nd 20 (rbitrry units) uxillry gs (N 2 ) flow. MS 3 nlysis ws performed using the dt dependent scn function of the Thermo Xclibur softwre. The most bundnt ion formed upon ESI ws isolted nd subjected to collision-ctivted dissocition (CAD). The most bundnt frgment ion ws further selected for isoltion nd frgmenttion. For ll MS 3 experiments, n isoltion window of 2 m/z units ws used long with normlized collision energy of 30 (rbitrry units). HPLC-UV nlysis The liquid phse from lignin depolymeriztion rections with methnol s solvent ws nlyzed with Agilent 1260 Infinity Quternry High-Performnce Liquid Chromtogrphy (HPLC) system, using Zorbx Eclipse XDB-C18 Column (250 x 74.6mm) set t 30 C. The chromtogrphy pprtus is equipped with G1315D Diode Arry Detector (DAD). A mixture of H 2 O (A) nd cetonitrile (B) were used s the mobile phse t flow rte of 0.5 ml/min. Nonliner grdient ws used (80% A nd 20% B from beginning to 5% A nd 95 % B t 55.0 min). A fixed mount (400μL) of internl stndrd benzyl phenyl ether (10 mm) ws dded into ech smple for the quntifiction purposes. Stndrd curves for ll the romtic products were mde by comprison of the products to internl stndrd. All results were nlyzed nd quntified ccording to stndrd curves. Before nlyzing by HPLC, the liquid smples were filtered through 0.22μm cutoff syringe filter (2 5mm dimeter). Nucler Mgnetic Resonnce (NMR) All NMR spectr were collected on Bruker Avnce DMX500 spectrometer with Tesl stndrd-bore superconducting mgnet operting t nd MHz for 1 H nd 13 C nuclei, respectively. C-H correltion spectr were recorded through phse sensitive grdient enhnced 2D HSQC using echo-ntiecho experiment (HSQCETGP experiment). Isolted lignin smple (20mg) ws dissolved in 700μL mixture of 5:1 v/v DMSOd 6 /pyridine-d 5 solvent for ech experiment. S2

3 Scnning Electron Microscopy (SEM) SEM imges were tken on n FEI Nov NnoSEM 650 high resolution instrument, equipped with high stbility Schottky field emission gun nd lrge specimen chmber. Oxford Inc x-ry EDX system ws used s detector, bck scttering detector for Z-imging. Lignin smples were first coted with plldium metl t plsm dischrge current 10mA for 100s under rgon tmosphere. The instrument ws vented to pressure less thn torr before use. Electron bem used for imge tking ws set to voltge t 7 kev. Gel Permetion Chromtogrphy (GPC) Gel permetion chromtogrphy (GPC) ws crried out on Wters (Millford, MA) chromtogrph equipped with Wters Allince high performnce liquid chromtogrphy (HPLC) system pump (2695 Seprtion Module) nd two Tosoh TSKgel Super HM-M columns. Detection ws provided by Wters 2414 differentil refrctometer, nd N,Ndimethyl formmide with 0.1% LiBr ws used s the mobile phse. Number verge moleculr weights (Mn) nd weight verge moleculr weights (Mw) were clculted reltive to liner polystyrene stndrds. S3

species by different solvents. Figure S2.

4 2. Supplementry Figures nd Tbles Figure S1. SEM imges of orgnosolv lignin extrcted from gene modified poplr species by different solvents. Figure S2. Aromtic products (HPLC-UV) fter ctlytic depolymeriztion of () orgnosolv lignin nd (b) ntive lignin. b S4

5 Figure S3. Two dimensionl HSQC-NMR spectr on the side chin region (δ C /δ H / ppm) of orgnosolv lignin from different tretments. S5

6 Figure S4. Two dimensionl HSQC-NMR spectr on the romtic region (δ C /δ H / ppm) of orgnosolv lignin from different tretments. S6

Figure S5. Two dimensionl HSQC-NMR spectr of unconverted lignin fter ctlytic depolymeriztion of lignin (CDL) rection, () side chin region nd (b) romtic region.

7 Figure S5. Two dimensionl HSQC-NMR spectr of unconverted lignin fter ctlytic depolymeriztion of lignin (CDL) rection, () side chin region nd (b) romtic region. Unconverted lignin from MeOH - wild type poplr orgnosolv lignin is used s n exmple. Tble S1. Composition nlysis of gene modified poplr species. 1,2 Substrtes Glucn % Xyln % Acid Acid Totl Others c Mss Soluble Insoluble Lignin b % % Blnce% Lignin b % Lignin b % Wild Type Poplr High-S Poplr Low-S Poplr Cellulose content ws reported s glucn, hemicellulose content ws reported s xyln. b Acid soluble lignin (ASL), cid insoluble lignin (AIL) nd totl lignin content were determined by stndrd NREL procedures 1. c Including cetyl, sh nd other wter nd ethnol extrctives. Tble S2. Reltive rtio of S-lignin in poplr substrtes. Substrte % S-lignin Wild-Type 64 High-S 82 Low-S 34 Lignin composition s determined by DFRC (derivtiztion followed by reductive clevge) nlysis. Mss blnce nlysis for ctlytic depolymeriztion of orgnosolv lignin. To perform mss blnce nlysis, methnol solvent in the finl product mixture ws fully evported under vcuum. 5 ml of H 2 O ws used to precipitte the recovered lignin. The H 2 O phse ws then extrcted with 5 ml of toluene three times. After extrction, ll monomeric phenolic compounds went to orgnic phse nd oligomers s well s recovered orgnosolv lignin remined in queous phse. After seprtion of two lyers, solvent ws removed under vcuum in order to nlyze the desired components. S7

8 Tble S3. Mss blnce clcultion fter ctlytic depolymeriztion of orgnosolv lignin. Substrtes b Toluene soluble Wter precipitted Totl% c romtics% oligomer & unconverted orgnosolv lignin% AA-Wild type AA-High-S AA-Low-S Acetone-Wild type Acetone-High-S Acetone-Low-S Methnol-Wild type Methnol-High-S Methnol-Low-S Orgnosolv lingin (50 mg) with 10wt% Ni/C ctlyst in 20 ml of MeOH, t 225 C nd 35 br H 2 for 12 h. b Orgnosolv lignin smples were nmed s the solvent used for extrction followed by the specific poplr species. For exmple, AA-Wild type mens wild type poplr being extrcted by cetic cid/formic cid solvent, Methnol-Wild type mens wild type poplr being extrcted by methnol, etc. c Totl mss blnce is clculted bsed on initil orgnosolv lignin substrte. Tble S4. Assignments on the common crbon-proton correltions of orgnosolv lignin in HSQC-NMR spectr () side chin region nd (b) romtic region. Crbon-proton correltions Assignments I / A α / A β (H,G) II / A β (S) III / A γ / B α / B β /3.8; /4.2 B γ / C α / C β / C γ I For the ssignments on crbon-proton correltions for the side chin region, A is used to represent β- O-4 linkge, B is used to represent β-β linkge, C is used to represent β-5 linkge. A α is for the α position of β-o-4 linkge, B α for the α position of β-β linkge, C α for the α position of β-5 linkge, etc. II β position of β-o-4 linkge in H nd G type lignin. III β position of β-o-4 linkge in S type lignin. S8

9 b Crbon-proton correltions Assignments I / H 2, / H 3, / G / G / G / / / S 2,6 H II 2,6 S III 2,6 I For the ssignments on crbon-proton correltions for the romtic region, H is used to represent H type lignin, G is used to represent G type lignin, S is used to represent S type lignin. H 2,6 for the 2 nd 6 positions of H lignin, G 2 for the 2 position of G lignin, S 2,6 for the 2 nd 6 positions of S lignin, etc. II Oxidized H unit (p-hydroxybenzoic Acid). III Oxidized S unit (Syringic Acid) 3. References 1. Sluiter, A.; Hmes, B.; Ruiz, R.; Scrlt, C.; Sluiter, J.; Templeton, D.; Crocker, D. Determintion of Structurl Crbohydrtes nd Lignin in Biomss; Technicl Report: NREL/TP , Issue Dte, 08/03/2013; Ntionl Renewble Energy Lbortory: Golden, CO, Sluiter, A.; Hmes, B.; Ruiz, R.; Scrlt, C.; Sluiter, J.; Templeton, D. Determintion of sugrs, byproducts, nd degrdtion products in liquid frction process smples; Technicl Report: NREL/TP , Issue Dte, 12/08/2006; Ntionl Renewble Energy Lbortory: Golden, CO, S9

Morphology characterization. (a), SEM image of PS template nanospheres showing

Morphology characterization. (a), SEM image of PS template nanospheres showing b Supplementry Figure 1 Morphology chrcteriztion. (), SEM imge of PS templte nnospheres showing mono-dispersed sphericl shpe with pproximtely 200 nm in dimeter. The scle br is 500 nm. (b), SEM imge of