This work was carried out as part of the Cluster of Excellence &#34;Tailor-Made Fuels from Biomass&#34; and &#34;Fuel Science Center&#34;, which are funded by the Excellence Initiative of the German Research Foundation to promote science and research at German universities, as well as part of the Bioeconomy Science Center (BioSC), supported in the project AP&#179; Focus Lab. The scientific activities of the Bioeconomy Science Center were financially supported by the Ministry of Innovation, Science, and Research within the framework of the NRW Strategieprojekt BioSC (no. 313/323-400-002 13).

NOTE: The process may be paused at any point by leaving the samples at room temperature (for a few days) or in the refrigerator (for longer periods). See the Table of Materials for details on the materials used in this protocol.
1. Beech wood particles
<ol>
	<li>Generate the desired particle size of beech wood (Fagus sp.) using a cutting mill with a 10 mm sieve, and dry the particles at 50 &#176;C to constant mass (~24 h), leaving a residual moisture content of ~10% water.</li>
</ol>
2. Lignocellulosic fractionation and workup
<ol>
	<li>Lignocellulose pretreatment and fractionation
	<ol>
		<li>Suspend 500 mg of beech wood (Fagus sp.) particles and 78.0 mg (0.5 mmol, 0.1 M) of FDCA in 5 mL of ultrapure water at room temperature in a 25 mL stainless steel high-pressure reactor. Add 5 mL of 2-MTHF and a stirring bar to the suspension, and close the reactor. Heat the reactor to 160 &#176;C on a heating plate at a stirring speed of 1500 rpm for 1 h.</li>
		<li>Let the reaction cool down to room temperature in ice-water over a period of ~10 min. Open the reactor, add 52.5 &#181;L of NaOH solution (50 wt% NaOH in distilled water), and stir for 15 min at room temperature and 500 rpm on a stirring plate.</li>
	</ol>
	</li>
	<li>Isolation of organic phase and lignin quantification
	<ol>
		<li>Centrifuge the mixture (room temperature, 5 min, 1880 &#215; g). Use a pipette to transfer the organic phase (2-MTHF) to a 50 mL round-bottom flask.</li>
		<li>Evaporate the organic phase in a rotary evaporator (40 &#176;C, 200 rpm, 180 mbar) until a solid and dry lignin fraction is obtained. Determine the lignin yield by weighing with an analytical balance. Store the solid lignin at room temperature for further analysis.</li>
	</ol>
	</li>
	<li>Separation of solid cellulose-enriched pulp and aqueous phase
	<ol>
		<li>Filter the aqueous phase using a cellulose filter paper (17-30 &#181;m pore size) in a funnel to isolate the cellulose-enriched pulp, and transfer the aqueous phase to a 5 mL vial. Wash the pulp until neutral pH with 3 x 25 mL water, and store the washing solution separately in a 100 mL beaker. Dry the pulp at 80 &#176;C to constant mass (~24 h).</li>
		<li>Determine the dried pulp yield by weighing with an analytical balance.</li>
	</ol>
	</li>
	<li>FDCA recovery and isolation of aqueous phase
	<ol>
		<li>Adjust the pH of the aqueous phase and the washing solution from step 2.3 separately under constant stirring to pH 1 using concentrated HCl while cooling the solution in an ice bath. Control the pH using universal indicator paper.</li>
		<li>Filter the precipitated solid (FDCA) from both solutions, combine the residues, and dry at 80 &#176;C to constant mass (~24 h). Discard the washes. Determine the FDCA yield by weighing with an analytical balance.</li>
		<li>Transfer the aqueous phase to a 25 mL flask, and store it at 4 &#176;C for analysis.</li>
	</ol>
	</li>
	<li>Sample preparation for furfural quantification
	<ol>
		<li>Perform a separate experiment to determine the quantity of furfural. Repeat steps 2.1.1-2.2.1.</li>
		<li>Add 40 mg of n-decane as an internal standard to the collected organic solvent fraction, and store for analysis.</li>
	</ol>
	</li>
</ol>
3. Analysis
<ol>
	<li>Analysis of sugar in the aqueous phase by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD)
	<ol>
		<li>Dilute 10 &#181;L of the aqueous phase collected in step 2.4.3 with 190 &#181;L of distilled water. Add 10 &#181;L of 2 mM 2-deoxy-D-glucose to the diluted sample.</li>
		<li>Perform the separation of monosaccharides on a monosaccharide separator column with a flow rate of 0.5 mL&#8729;min-1, and inject the sample after equilibration with 2 mM NaOH for 10 min. Separate the neutral sugars with 2 mM NaOH over 18 min. Afterwards, use 550 mM NaOH for 10 min to separate the uronic acids. Rinse the column with 800 mM NaOH for 10 min. 
		NOTE: The software normalizes the amounts of monosaccharides to the amount of the internal standard and quantifies them by using standard calibration curves of the different monosaccharides.</li>
	</ol>
	</li>
	<li>Lignin analysis via 1H-13C heteronuclear single quantum correlation nuclear magnetic resonance (1H-13C-HSQC NMR)
	<ol>
		<li>Dissolve ~50 mg of lignin in 0.5 mL of deuterated dimethylsulfoxide ([d6] DMSO), and transfer the mixture to an NMR tube. Conduct 1H-13C HSQC (measurement time 220 min) NMR measurements using a 400 MHz spectrometer.</li>
		<li>Determine the types of linkages present in the lignin using the spectrum.
		<ol>
			<li>Reference the chemical shift of the spectrum to the DMSO signal (&#948;(1H) = 2.500 ppm; &#948;(13C) = 39.52 ppm).</li>
			<li>Perform a manual phase correction on both axes until all the signals are positive, then perform a baseline correction.</li>
			<li>Integrate the signals of the aromatic units and the linkages of the lignin; see Table 1 for the chemical shifts.</li>
		</ol>
		</li>
		<li>Calculate the sum of the aromatic units (arom.) using the following formula: 
		&#931;(arom.) = (S2,6 / 2) + ((G2 + G5) / 2) + (H2,6 / 2) (1) 
		&#8203;With Si being the integral over the signal corresponding to the 2 and 6 syringyl protons, Gi being the integrals over the signals corresponding to the 2 and 5 guaiacyl protons, and Hi being the integral over the signal corresponding to the 2 and 6 p-hydroxyphenyl protons.</li>
		<li>Calculate the percentage of each unit using the following formulas: 
		S = (S2,6/ 2) / &#931;(arom.) &#215; 100% (2) 
		G = ((G2 + G5) / 2) / &#931;(arom.) &#215; 100% (3) 
		H = (H2,6 / 2) / &#931;(arom.) &#215; 100% (4) 
		&#8203;With S, G, and H being the percentages of respective monomers-syringyl- (S), guaiacyl- (G), and p-hydroxyphenyl (H)-monomer units per 100 monomer units.</li>
		<li>Calculate the number of linkages per 100 units using the following formulas: 
		&#946;-O-4 linkages = &#945; &#946;-O-4 / &#931;(arom.) &#215; 100% (5) 
		linkages = (&#945; &#946;-&#946; + &#946; &#946;-&#946; + &#947; &#946;-&#946;) / &#931;(arom.) &#215; 100% (6) 
		linkages = (&#945; &#946;-5 + &#946; &#946;-5 + &#947; &#946;-5) / &#931;(arom.) &#215; 100% (7) 
		With &#945;, &#946;, and &#947; being the integral over the signal corresponding to the &#945;-, &#946;- and &#947;-proton signals of the corresponding &#946;-O-4-, &#946;-&#946;- and &#946;-5 linkages. 
		NOTE: Linkages are given as linkage per 100 monomer units. Due to overlapping of peaks, &#946;-O-4 is calculated using only the &#945; proton signal. &#946;-&#946; and &#946;-5 linkages are calculated using all signals of the corresponding linkage.</li>
	</ol>
	</li>
	<li>Gel permeation chromatography (GPC) analysis
	<ol>
		<li>Dissolve 10 mg of dried lignin and 1 mg of glucose (as internal standard) in 1 mL of a 0.1 M NaOH and 0.01 wt% NaN3 aqueous solution in a 1.5 mL gas chromatography (GC)-vial. Close the GC-vial using a cap with septum.</li>
		<li>Inject 100 &#181;L of the sample into a high-performance liquid chromatography (HPLC) system equipped with an ultraviolet detector and monitoring a wavelength of &#955; = 280 nm. Use a system consisting of a precolumn programmed-temperature split/splitless injector system with polar silica (8 mm x 50 mm) and three gel columns (8 mm x 300 mm, particle diameter: 5 &#181;m, nominal pore width: 1000 &#197;) at a flow rate of 1 mL min-1. Reference the obtained data to the signal of the internal standard (glucose). Calculate the mass distribution using the software, referenced to an external calibration with poly(styrene sulfonate) in a range from 266 to 65000 Da.</li>
	</ol>
	</li>
	<li>Furfural quantification via GC
	<ol>
		<li>Add 20 mg n-decane as internal standard to the organic phase of the OrganoCat pretreatment. Transfer 1 mL of the organic phase into a 1.5 mL GC-vial.</li>
		<li>Inject 1 &#181;L of this solution into a gas chromatograph using a 30 m column with a polar polyurethane glycol stationary phase and helium as the carrier gas with a flow rate of 1.5 mL min-1 and a flame-ionization detector. Set the initial temperature to 50 &#176;C, then raise by 8 &#176;C min-1 to 250 &#176;C and maintain at 250 &#176;C for 5 min.</li>
		<li>Quantify furfural using the integrals (Int) given by the software and an externally calculated correction factor (cf).
		<ol>
			<li>Prepare a sample of 1 mg of furfural and 5 mg of n-decane in 1 mL of 2-MTHF, and inject it into the GC using the aforementioned procedure. Calculate the correction factor as follows: 
			cf = (Int(n-decane) / m(n-decane)) / (Int(furfural) / Int(furfural)) (8)</li>
			<li>Use the correction factor to calculate the amount of furfural in the unknown sample with the following formula: 
			m(furfural) = m(n-decane) / Int(n-decane) &#215; cf &#215; Int(furfural) (9)</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Cellulose-enriched pulp hydrolysis
	<ol>
		<li>Carry out pulp hydrolysis of cellulose-enriched residue obtained from OrganoCat pretreatment in a heating block with mixing (see the Table of Materials) using 1.5 mL vials.</li>
		<li>Add 20 mg of cellulose-enriched pulp and 10 &#181;L of cellulase (60 filter paper units (FPU) mL-1 and 82 cellobiase units (CBU) mL-1) to 1 mL of citrate buffer (pH = 4.5) in a 1.5 mL vial, and shake at 50 &#176;C for 0 h, 1 h, or 72 h. Afterwards, heat the samples to 99 &#176;C for 10 min to denature the enzymes.</li>
		<li>Determine the glucose concentration using a glucose (hexokinase) assay kit.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The described fractionation of lignocellulose shows a tradeoff between hemicellulose hydrolysis efficiency and selectivity to avoid sugar degradation to furans, depending on reaction time and temperature (Figure 1). Lignin extraction was similarly affected by the harsher conditions. Especially the reduction of &#946;-O-4-linkages and enhancement of mass average molecular weight due to recondensation at higher temperature and reaction time underlines this compromise that must be made. The selection of reaction time and temperature is therefore a critical step in this lignocellulose fractionation process. As the efficiency of enzymatic hydrolysis appears to be mostly determined by delignification in the FDCA-catalyzed OrganoCat process, the harshest processing conditions afford the most accessible pulp. Other variations of the process9,11,18,22, e.g., using different catalysts, show that the strength of the catalyst and the final pH in the reactive solution have the strongest effect on process efficiency. Modifications of the procedure, e.g., preswelling with phosphoric acid, have been shown to have a beneficial effect as well22. Due to the variety in composition, however, the process needs optimization, depending on the different feedstocks21. Considering the overall process performance, downstream purification of the separated fractions must be considered, which is why selectivity plays a major role. Compared to other organosolv-like processes, OrganoCat uses a biphasic water/2-MTHF system, which affords the major components in three relatively straightforward, separate streams. This way, further downstream and resulting energy and equipment costs can be reduced13,18.

The use of fossil resources has brought great technological advances as they form the basis for numerous products that are essential for everyday life. However, the limitation of resources such as oil and gas on earth and the environmental damages connected with their exploitation create an urgent need for alternatives. Lignocellulosic biomass is a promising source for carbon-based chemicals, as it is renewable, versatile, and carbon neutral1. Lignocellulose basically consists of three main fractions to make use of: hemicelluloses, cellulose, and lignin. Its industrial processing has a long history. However, established and widespread processes, such as the sulfite and Kraft processes from paper industry, mainly focus on cellulose for utilization in the pulp and paper industry2. A full valorization of all three lignocellulosic fractions is needed to make lignocellulose processing towards chemicals more profitable from economic and environmental perspectives.
In many lignocellulose valorization strategies, lignin is a mere byproduct that is often burned for energy recovery. Currently, only 1-2% of the industrially produced lignin is used to produce value-added products such as concrete additives, surfactants, and vanillin3. Nevertheless, it is the largest renewable source of aromatics and therefore has promising properties for application as a basis for polymers4, carbon fibers5, and fuel2. The challenges in the valorization of lignin lie in its complex structure and diversity, depending on the source material and extraction conditions. Moreover, due to their process conditions, the most prevalent lignocellulose fractionation processes deliver sulfonated lignin with a high number of C-C linkages between the monomer units. Therefore, commercially available lignin is challenging to depolymerize.
A range of different approaches, which focus on the holistic utilization of all three fractions, have been developed for lignocellulose fractionation. Most processes rely on the hydrolysis of hemicellulose, either with diluted acids and bases or by utilizing the autoprotolysis of water at elevated temperatures. As one of the most explored options, organosolv processes use low-boiling organic solvents, usually in combination with water. Well-known variants of this process include the Alcell process, which utilizes 50% ethanol, and the Organocell process, which uses methanol in the first step and adds NaOH in the second step. Acid organosolv processes that use formic or acetic acid are also described2. Owing to the recent focus on the valorization of lignin as a major biorefinery product, new approaches have been developed, which combine lignin extraction with subsequent or integrated conversion steps to yield smaller lignin compounds and more stable and valuable products6,7,8.
The OrganoCat lignocellulose fractionation process (OrganoCat) is based on a two-phase system of water and 2-methyltetrahydrofuran (2-MTHF)9. Additionally, a recyclable organic acid is used as catalyst, which selectively hydrolyzes hemicelluloses at mild temperatures. All process chemicals can be produced in a relatively inexpensive and biogenic manner, which lowers the environmental impact of the process in accordance to the principles of Green Chemistry10. The process delivers three separate product streams with lignin in the organic phase, depolymerized hemicellulose sugars in the aqueous phase, and cellulose-enriched pulp as a solid residue. As the product streams can be easily separated, downstream steps, energy demand, and material costs can be reduced significantly compared to, for example, monophasic approaches. The lignin has a relatively low molecular weight and a high number of &#946;-O-4 linkages11. The depolymerized hemicellulose sugars can be used for fermentation or conversion into fine chemicals12. The cellulose pulp is highly accessible for enzymatic depolymerization9.
The original OrganoCat process uses oxalic acid as catalyst to fractionate lignocellulose. Oxalic acid can then be recovered by crystallization9. However, this increases the process costs for cooling the reaction and the partial evaporation of water. The partial decomposition of oxalic acid would diminish the revenues further13. For this reason, the OrganoCat process was improved by introducing 2,5-furandicarboxylic acid (FDCA) as catalyst11. FDCA is not only sufficiently acidic to catalyze the reaction, but can also be derived from glucose via dehydration to 5-hydroxymethylfurfural and subsequent oxidation with metal-based catalysts or biocatalysts14,15,16,17. Although the acidity of FDCA is slightly lower, it has a higher thermal stability than oxalic acid. FDCA has a low solubility in water at room temperature, which allows its straightforward recovery from the aqueous phase after the reaction.
A scale-up of the OrganoCat process was successfully developed to a 3 L reactor18. Additional studies on OrganoCat lignin found that antisolvent precipitation with n-hexane or n-pentane allow an energy efficient lignin recovery19. It was possible to get lignin fractions with different molecular weights20. This paper presents the full preparative method for a scalable, one-step fractionation process of lignocellulosic biomass using FDCA as catalyst. This process yields extracted lignin, depolymerized hemicelluloses, and cellulose pulp in three easily separable product streams.

<table><tbody><tr><td>1200 HPLC system</td><td>Agilent</td><td>n.a.</td><td>was used for size exclusion chomatogaphy</td></tr><tr><td>2,5-furandicarboxylic acid</td><td>TCI Deutschland GmbH</td><td>F0710</td><td>Purity:&amp;nbsp;&gt;98.0%(T)(HPLC)</td></tr><tr><td>2-methyltetrahydrofuran</td><td>Carl Roth GmbH</td><td>6845.4</td><td>SOLVAGREEN&amp;nbsp;&amp;ge;99 %, extra pure</td></tr><tr><td>Accellerase 1500</td><td>Provided by Genencor (60&amp;nbsp;FPU&amp;nbsp;mL&lt;sup&gt;-1&lt;/sup&gt; and 82&amp;nbsp;CBU&amp;nbsp;mL&lt;sup&gt;-1&lt;/sup&gt;; 2300 AE Leiden, Netherlands)</td><td>n.a.</td><td>cellulase for pulp hydrolysis</td></tr><tr><td>beech wood (Fagus sp.)</td><td>local supplier</td><td>n.a.</td><td></td></tr><tr><td>BioTek Power Wave HT UV-Vis Spectrometer</td><td>BioTek Germany, 74177 Bad Friedrichshall, Germany</td><td>BT-RPRWI</td><td></td></tr><tr><td>Bruker AS400 (400&amp;nbsp;MHz) Spectrometer</td><td>Bruker, Billerica, MA 01821, USA</td><td>n.a.</td><td>HSQC-NMR analysis</td></tr><tr><td>CarboPac PA20 column</td><td>Dionex</td><td>302747</td><td>monosaccharide separator column for high-performance anion-exchange chromatography</td></tr><tr><td>centrifuge 5430 R</td><td>Eppendorf</td><td>5428000610</td><td></td></tr><tr><td>Focus GC</td><td>Thermo Fischer</td><td>n.a.</td><td>gas chromatograph</td></tr><tr><td>Glucose (hexokinase) assay kit</td><td>Sigma-Aldrich</td><td>GAHK20-1KT</td><td></td></tr><tr><td>GPC- precolumn PSS PolarSil in DMAc</td><td>PSS Polymer Strandards Service GmbH</td><td>PSA080505</td><td>precolumn with polar silica (8 x 50 mm)</td></tr><tr><td>HP-INNOwax column 30 m</td><td>Agilent J &amp;amp; W</td><td>19091N-213IE</td><td>GC column with a polar polyethylene glycol stationary phase</td></tr><tr><td>PSS MCX</td><td>PSS Polymer Strandards Service GmbH</td><td>&amp;nbsp;MCA0830051E3</td><td>gel columns (8 x 300 mm, particle diameter: 5 &amp;micro;m, nominal pore width: 1000 &amp;Aring;</td></tr><tr><td>ThermoMixer</td><td>Eppendorf</td><td>n.a.</td><td>mixing and heating block</td></tr><tr><td>tinyclave steel Typ 3 / 25 mL</td><td>B&amp;uuml;chi</td><td>49,33,45,10,000</td><td>100 bar, 200 &amp;deg;C</td></tr></tbody></table>

fractionation of lignocellulosic biomass using the organocat process

The shift from a petroleum-based to a more sustainable and bio-based economy requires the development of new refinery concepts to maintain the supply of raw materials and energy. For these novel and sustainable biorefinery concepts, it is important to use catalysts and solvents that are aligned with the principles of Green Chemistry. Therefore, the implementation of biogenic alternatives can be a promising solution. The lignocellulose pretreatment and fractionation process presented herein-OrganoCat-is an integrated fractionation of lignocellulose into its main components using biogenic acids such as 2,5-furandicarboxylic acid as catalyst. Hemicelluloses and other non-cellulosic polysaccharides are selectively depolymerized by the diluted acid and dissolved, while the crystalline cellulose remains in the solid pulp. In the presence of a second organic phase consisting of biogenic 2-methyltetrahydrofuran, disentangled lignin is extracted in situ. The process allows for the efficient fractionation of the three main components-lignin, cellulose, and non-cellulosic sugars. This helps to focus on the quality of the lignin, the improvement of enzymatic hydrolysis of the cellulose-enriched pulp, and the mild non-cellulosic sugar extraction with low degradation.

A typical set of conditions for the lignocellulose pretreatment and fractionation process OrganoCat (OrganoCat) uses 0.1 M FDCA as a catalyst, a biomass loading of 100 g L-1 (beech wood, compared to the aqueous phase), 1 h of reaction time and 160 &#176;C as the reaction temperature. The composition of beech wood has been published elsewhere21 (~48% cellulose, 27% hemicellulose, 26% lignin). Figure 1 shows the extracted hemicellulose hydrolysate with this set of conditions as well as longer reaction time (3 h) and lower temperature (140 &#176;C).
Using harsher conditions, e.g., higher temperature and longer reaction time, might lead to higher extraction yields, but also leads to more degradation of the products-furfural is a degradation product of xylose, whereas 5-(hydroxymethyl)furfural (5-HMF) is the corresponding degradation product of glucose. A higher amount of furfural was noted in the products (distributed between the aqueous and the organic phases) with a reaction time of 3 h at 160 &#176;C. As the sugar degradation products are highly reactive and tend to form humins-oligomers of furans and sugars-the shorter reaction time at higher temperature might be considered a good compromise between high extraction efficiency and low sugar degradation.
The amount of extracted lignin is directly related to the reaction temperature and time as well. Figure 2 displays the amount of lignin extracted, the &#946;-O-4-linkage content, and the mass-average molar masses of the extracted lignins. While the extracted lignin yield rises with longer reaction time, the number of intact &#946;-O-4-linkages diminishes by approximately half when reacting for 3 h instead of 1 h. Lowering the reaction temperature from 160 &#176;C to 140 &#176;C has a much lower impact on lignin, resulting in slightly less yield, smaller mass-average molar mass, and higher &#946;-O-4-content.
As enzymatic hydrolysis of (lingo-)cellulose is a common indicator for pulping efficiency, a commercial cellulose cocktail was applied to the different OrganoCat pulps resulting from the above mentioned OrganoCat reaction condition sets (Figure 3). As the cellulase is not optimized for the substrates, the overall cellulose conversion is not comparable to state-of-the-art performance; however, it allows the comparison of the different pulps to each other. The longer reaction time exhibits a significant impact on the initial reaction time and the glucose yield after 72 h, improving by a factor of ~2.5. Lowering the temperature appears to show a much smaller impact, implying that the main factor causing the differences in enzymatic digestibility within this treatment is the degree of delignification.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61933/61933fig1v2.png" /> 
Figure 1:&#160;Sugar extraction and furfural production in OrganoCat process with 0.1 M 2,5-furandicarboxylic acid as catalyst and 100 g L-1 beech wood (compared to the aqueous phase) at different reaction temperatures and times as indicated on the x-axis11. All experiments have been performed in triplicate. The average is shown with the standard deviation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61933/61933fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61933/61933fig2v2.png" /> 
Figure 2:&#160;Amount and analysis of lignin extracted by OrganoCat process with 0.1 M 2,5-furandicarboxylic acid as catalyst and 100 g L-1 beech wood (compared to the aqueous phase) at different reaction temperatures and times as indicated on the x-axis11. Lignin yields have been calculated in triplicate. The average is shown with the standard deviation. Molecular mass and linkages were derived from representative single experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61933/61933fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61933/61933fig3v2.png" /> 
Figure 3:&#160;Enzymatic hydrolysis of pulps. Glucose yields after 72 h (blue bars) and reaction rates within the first hour (gray bars) from the hydrolysis of untreated beech wood and cellulose-enriched pulps obtained from OrganoCat with 0.1 M 2,5-furandicarboxylic acid as catalyst and 100 g L-1 beech wood (compared to the aqueous phase) at different reaction temperatures and times as indicated on the x-axis. Cellulase was applied to the different substrates at 50 &#176;C for up to 72 h in contrite buffer (pH 4.5)11. All experiments have been performed in triplicate. The average is shown with the standard deviation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61933/61933fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Unit</td>
			<td>Shift &#948; (1H)(13C)</td>
			<td>Linkage</td>
			<td>Shift &#948; (1H)(13C)</td>
		</tr>
		<tr>
			<td></td>
			<td>[ppm]</td>
			<td></td>
			<td>[ppm]</td>
		</tr>
		<tr>
			<td>S2,6</td>
			<td>(6.95&#8211;6.46)(106.8&#8211;101.9)</td>
			<td>&#945; &#946;-O-4</td>
			<td>(5.08&#8211;4.69)(75.8&#8211;69.9)</td>
		</tr>
		<tr>
			<td>G2</td>
			<td>(7.12&#8211;6.72)(113.4&#8211;108.7)</td>
			<td>&#945; &#946;-&#946;</td>
			<td>(4.72&#8211;4.58)(87.46&#8211;84.0)</td>
		</tr>
		<tr>
			<td>G5</td>
			<td>(7.04&#8211;6.51)(117.8&#8211;113.4)</td>
			<td>&#946; &#946;-&#946;</td>
			<td>(3.35&#8211;3.11)(62.0&#8211;57.9)</td>
		</tr>
		<tr>
			<td>H2,6</td>
			<td>(7.01&#8211;6.8)(129.1&#8211;123.2)</td>
			<td>&#947; &#946;-&#946; 1</td>
			<td>(4.26&#8211;4.09)(73.0&#8211;70.0)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>&#947; &#946;-&#946; 2</td>
			<td>(3.87&#8211;3.71)(73.0&#8211;70.0)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>&#945; &#946;-5</td>
			<td>(5.51&#8211;5.41)(88.8&#8211;86.6)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>&#946; &#946;-5</td>
			<td>(3.52&#8211;3.42)(54.0&#8211;52.1)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>&#947; &#946;-5</td>
			<td>(3.80&#8211;3.67)(64.1&#8211;62.1)</td>
		</tr>
	</tbody>
</table>
Table 1: Chemical shifts determined by 1H-13C heteronuclear single quantum correlation nuclear magnetic resonance (1H-13C-HSQC NMR) for different linkages in lignin. Abbreviations: S = syringyl unit, G = guaiacyl unit, H = p-hydroxyphenyl unit.

Watch this Scientific Journal Video about Fractionation of Lignocellulosic Biomass using the OrganoCat Process at JoVE.com

Fractionation of Lignocellulosic Biomass using the OrganoCat Process

OrganoCat is a method for the pretreatment and fractionation of lignocellulose under mild conditions into lignin, fermentable sugars, and cellulose pulp. In a biogenic, biphasic solvent system of water and 2-methyltetrahydrofuran with 2,5-furancarboxylic acid as catalyst, the OrganoCat products are separated in situ for straightforward product recovery.

The shift from a petroleum-based to a more sustainable and bio-based economy requires the development of new refinery concepts to maintain the supply of ...

fractionation-of-lignocellulosic-biomass-using-the-organocat-process

Nanyang Technological University

Research

JoVE Journal

Chemistry

3.9K Views.  Pflanzenwissenschaften (IBG-2), Forschungszentrum Jülich. Lignine cellulose fractionering met behulp van de OrganoCat-technologie, maakt een eenvoudige scheiding van lignine, cellulose en fermenteerbare suikers mogelijk met behulp van een tweefasig éénpotreactiesysteem. Door de milde reactieomstandigheden kan een hoge productkwaliteit worden bereikt. Ons proces vergemakkelijkt eenvoudige productscheiding en katalysator- en oplosmiddelrecycling.Het is ook 100% biogeen, zodat de chemicaliën die in het proces worden gebruikt, kunnen worden ve...

Video: Fractionation of Lignocellulosic Biomass using the OrganoCat Process

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high quality paper. Black liquor is a pulping waste generated by the kraft process that has potential for downstream bioconversion. However, the recalcitrant nature of the lignocellulose resources, its chemical derivatives that constitute the majority of available organic carbon within black liquor, and its basic pH present challenges to microbial biodegradation of this waste material. Methods for the collection and modification of black liquor for microbial growth are aimed at utilization of this pulp waste to convert the lignin, organic acids, and polysaccharide degradation byproducts into valuable chemicals. The lignocellulose extraction techniques presented provide a reproducible method for preparation of lignocellulose growth substrates for understanding metabolic capacities of cultured microorganisms. Use of gas chromatography-mass spectrometry enables the identification and quantification of the fermentation products resulting from the growth of microorganisms on pulping waste. These methods when used together can facilitate the determination of the metabolic activity of microorganisms with potential to produce fermentation products that would provide greater value to the pulping system and reduce effluent waste, thereby increasing potential paper milling profits and offering additional uses for black liquor.

Industrial wastes can be collected and modified to analyze microbial growth. Lignocellulose extraction techniques provide components to analyze specific biodegradation ability. Gas chromatography-mass spectrometry identifies fermentation products of microorganisms grown on pulping waste. These methods determine the metabolic capacity of microorganisms to degrade pulping waste.

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high ...

Environment

Towards Biomimicking Wood: Fabricated Free-standing Films of Nanocellulose, Lignin, and a Synthetic Polycation

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and lignin. Layer-by-layer (LbL) assembly process which relies on the assembly of oppositely charged molecules from aqueous solutions was used to build a freestanding composite film of isolated wood polymers of lignin and oxidized nanofibril cellulose (NFC). To facilitate the assembly of these negatively charged polymers, a positively charged polyelectrolyte, poly(diallyldimethylammomium chloride) (PDDA), was used as a linking layer to create this simplified model cell wall. The layered adsorption process was studied quantitatively using quartz crystal microbalance with dissipation monitoring (QCM-D) and ellipsometry. The results showed that layer mass/thickness per adsorbed layer increased as a function of total number of layers. The surface coverage of the adsorbed layers was studied with atomic force microscopy (AFM). Complete coverage of the surface with lignin in all the deposition cycles was found for the system, however, surface coverage by NFC increased with the number of layers. The adsorption process was carried out for 250 cycles (500 bilayers) on a cellulose acetate (CA) substrate. Transparent free-standing LBL assembled nanocomposite films were obtained when the CA substrate was later dissolved in acetone. Scanning electron microscopy (SEM) of the fractured cross-sections showed a lamellar structure, and the thickness per adsorption cycle (PDDA-Lignin-PDDA-NC) was estimated to be 17 nm for two different lignin types used in the study. The data indicates a film with highly controlled architecture where nanocellulose and lignin are spatially deposited on the nanoscale (a polymer-polymer nanocomposites), similar to what is observed in the native cell wall.

The objective of this research was to form synthetic plant cell wall tissue using layer-by-layer assembly of nanocellulose fibrils and isolated lignin assembled from dilute aqueous suspensions.&#160; Surface measurement techniques of quartz crystal microbalance and atomic force microscopy were used to monitor the formation of the polymer-polymer nanocomposite material.

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and ...

High-throughput Screening of Recalcitrance Variations in Lignocellulosic Biomass: Total Lignin, Lignin Monomers, and Enzymatic Sugar Release

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the use of non-renewable energy sources. In order to identify which plants, out of a diverse pool, have the desired chemical traits for downstream applications, attributes, such as cellulose and lignin content, or monomeric sugar release following an enzymatic saccharification, must be compared. The experimental and data analysis protocols of the standard methods of analysis can be time-consuming, thereby limiting the number of samples that can be measured. High-throughput (HTP) methods alleviate the shortcomings of the standard methods, and permit the rapid screening of available samples to isolate those possessing the desired traits. This study illustrates the HTP sugar release and pyrolysis-molecular beam mass spectrometry pipelines employed at the National Renewable Energy Lab. These pipelines have enabled the efficient assessment of thousands of plants while decreasing experimental time and costs through reductions in labor and consumables.

Plant cell wall structure and chemistry traits are evaluated to identify ideal feedstocks for biofuels and bio-materials. Standard methods have limitations when applied to large data sets. These high-throughput pretreatment, enzyme saccharification, and pyrolysis-molecular beam mass spectrometry methods compare large numbers of biomass samples with decreased experimental time and cost.

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the ...

Pretreatment of Lignocellulosic Biomass with Low-cost Ionic Liquids

A number of ionic liquids (ILs) with economically attractive production costs have recently received growing interest as media for the delignification of a variety of lignocellulosic feedstocks. Here we demonstrate the use of these low-cost protic ILs in the deconstruction of lignocellulosic biomass (Ionosolv pretreatment), yielding cellulose and a purified lignin. In the most generic process, the protic ionic liquid is synthesized by accurate combination of aqueous acid and amine base. The water content is adjusted subsequently. For the delignification, the biomass is placed into a vessel with IL solution at elevated temperatures to dissolve the lignin and hemicellulose, leaving a cellulose-rich pulp ready for saccharification (hydrolysis to fermentable sugars). The lignin is later precipitated from the IL by the addition of water and recovered as a solid. The removal of the added water regenerates the ionic liquid, which can be reused multiple times. This protocol is useful to investigate the significant potential of protic ILs for use in commercial biomass pretreatment/lignin fractionation for producing biofuels or renewable chemicals and materials.

The pretreatment of lignocellulosic biomass with protic low-cost ionic liquids is shown, resulting in a delignified cellulose-rich pulp and a purified lignin. The pulp gives rise to high glucose yields after enzymatic saccharification.

A number of ionic liquids (ILs) with economically attractive production costs have recently received growing interest as media for the delignification of ...

A Novel Method for the Pentosan Analysis Present in Jute Biomass and Its Conversion into Sugar Monomers Using Acidic Ionic Liquid

Recently, ionic liquids (ILs) are used for biomass valorization into valuable chemicals because of their remarkable properties such as thermal stability, lower vapor pressure, non-flammability, higher heat capacity, and tunable solubility and acidity. Here, we demonstrate a method for the synthesis of C5 sugars (xylose and arabinose) from the pentosan present in jute biomass in a one-pot process by utilizing a catalytic amount of Br&#248;nsted acidic 1-methyl-3-(3-sulfopropyl)-imidazolium hydrogen sulfate IL. The acidic IL is synthesized in the lab and characterized using NMR spectroscopic techniques for understanding its purity. The various properties of BAIL are measured such as acid strength, thermal and hydrothermal stability, which showed that the catalyst is stable at a higher temperature (250 &#176;C) and possesses very high acid strength (Ho 1.57). The acidic IL converts over 90% of pentosan into sugars and furfural. Hence, the presenting method in this study can also be employed for the evaluation of pentosan concentration in other kinds of lignocellulosic biomass.

We present a protocol for the synthesis of C5 sugars (xylose and arabinose) from a renewable non-edible lignocellulosic biomass (i.e., jute) with the presence of Br&#248;nsted acidic ionic liquids (BAILs) as the catalyst in water. The BAILs catalyst exhibited better catalytic performance than conventional mineral acid catalysts (H2SO4 and HCl).

Recently, ionic liquids (ILs) are used for biomass valorization into valuable chemicals because of their remarkable properties such as thermal stability, ...

Extraction of Lignin with High &#946;-O-4 Content by Mild Ethanol Extraction and Its Effect on the Depolymerization Yield

Lignin valorization strategies are a key factor for achieving more economically competitive biorefineries based on lignocellulosic biomass. Most of the emerging elegant procedures to obtain specific aromatic products rely on the lignin substrate having a high content of the readily cleavable &#946;-O-4 linkage as present in the native lignin structure. This provides a miss-match with typical technical lignins that are highly degraded and therefore are low in &#946;-O-4 linkages. Therefore, the extraction yields, and the quality of the obtained lignin are of utmost importance to access new lignin valorization pathways. In this manuscript, a simple protocol is presented to obtain lignins with high &#946;-O-4 content by relatively mild ethanol extraction that can be applied to different lignocellulose sources. Furthermore, analysis procedures to determine the quality of the lignins are presented together with a depolymerization protocol that yields specific phenolic 2-arylmethyl-1,3-dioxolanes, which can be used to evaluate the obtained lignins. The presented results demonstrate the link between lignin quality and potential for the lignins to be depolymerized into specific monomeric aromatic chemicals. Overall, the extraction and depolymerization demonstrates a trade-off between the lignin extraction yield and the retention of the native aryl-ether structure and thus the potential of the lignin to be used as the substrate for the production of chemicals for higher-value applications.

Here, we present a protocol to perform ethanol extraction of lignin from several biomass sources. The effect of the extraction conditions on the lignin yield and &#946;-O-4 content are presented. Selective depolymerization is performed on the obtained lignins to obtain high aromatic monomer products.

Lignin valorization strategies are a key factor for achieving more economically competitive biorefineries based on lignocellulosic biomass. Most of the ...

Reducing Willow Wood Fuel Emission by Low Temperature Microwave Assisted Hydrothermal Carbonization

Biomass is a sustainable fuel, as its CO2 emissions are reintegrated in biomass growth. However, the inorganic precursors in the biomass cause a negative environmental impact and slag formation. The selected short rotation coppice (SRC) willow wood has a high ash content (<img alt="Equation 1" src="/files/ftp_upload/58970/58970eq1.jpg" /> = 1.96%) and, therefore, a high content of emission and slag precursors. Therefore, the reduction of minerals from SRC willow wood by low temperature microwave assisted hydrothermal carbonization (MAHC) at 150 &#176;C, 170 &#176;C, and 185 &#176;C is investigated. An advantage of MAHC over conventional reactors is an even temperature conductance in the reaction medium, as microwaves penetrate the whole reactor volume. This allows a better temperature control and a faster cooldown. Therefore, a succession of depolymerization, transformation and repolymerization reactions can be analyzed effectively. In this study, the analysis of the mass loss, ash content and composition, heating values and molar O/C and H/C ratios of the treated and untreated SCR willow wood showed that the mineral content of the MAHC coal was reduced and the heating value increased. The process water showed a decreasing pH and contained furfural and 5-methylfurfural. A process temperature of 170 &#176;C showed the best combination of energy input and ash component reduction. The MAHC allows a better understanding of the hydrothermal carbonization process, while a large-scale industrial application is unlikely because of the high investment costs.

A protocol for the emission precursor depletion from low quality biomass by low temperature microwave assisted hydrothermal carbonization treatment is presented. This protocol includes the microwave parameters and the analysis of the biocoal product and process water.

Biomass is a sustainable fuel, as its CO2 emissions are reintegrated in biomass growth. However, the inorganic precursors in the biomass cause a negative ...

Twin-Screw Extrusion Process to Produce Renewable Fiberboards

A versatile twin-screw extrusion process to provide an efficient thermo-mechano-chemical pre-treatment on lignocellulosic biomass before using it as source of mechanical reinforcement in fully bio-based fiberboards was developed. Various lignocellulosic crop by-products have already been successfully pre-treated through this process, e.g., cereal straws (especially rice), coriander straw, shives from oleaginous flax straw, and bark of both amaranth and sunflower stems.
The extrusion process results in a marked increase in the average fiber aspect ratio, leading to improved mechanical properties of fiberboards. The twin-screw extruder can also be fitted with a filtration module at the end of the barrel. The continuous extraction of various chemicals (e.g., free sugars, hemicelluloses, volatiles from essential oil fractions, etc.) from the lignocellulosic substrate, and the fiber refining can, therefore, be performed simultaneously.
The extruder can also be used for its mixing ability: a natural binder (e.g., Organosolv lignins, protein-based oilcakes, starch, etc.) can be added to the refined fibers at the end of the screw profile. The obtained premix is ready to be molded through hot pressing, with the natural binder contributing to fiberboard cohesion. Such a combined process in a single extruder pass improves the production time, production cost, and may lead to reduction in plant production size. Because all the operations are performed in a single step, fiber morphology is better preserved, thanks to a reduced residence time of the material inside the extruder, resulting in enhanced material performances. Such one-step extrusion operation may be at the origin of a valuable industrial process intensification.
Compared to commercial wood-based materials, these fully bio-based fiberboards do not emit any formaldehyde, and they could find various applications, e.g., intermediate containers, furniture, domestic flooring, shelving, general construction, etc.

A versatile twin-screw extrusion process to provide an efficient thermo-mechano-chemical pretreatment on lignocellulosic biomass was developed, which leads to an increased average fiber aspect ratio. A natural binder can also be added continuously after fiber refining, leading to bio-based fiberboards with improved mechanical properties after hot pressing of the obtained extruded material.

A versatile twin-screw extrusion process to provide an efficient thermo-mechano-chemical pre-treatment on lignocellulosic biomass before using it as ...

Engineering

Ultrafast Lignin Extraction from Unusual Mediterranean Lignocellulosic Residues

Pretreatment is still the most expensive step in lignocellulosic biorefinery processes. It must be made cost-effective by minimizing chemical requirements as well as power and heat consumption and by using environment-friendly solvents. Deep eutectic solvents (DESs) are key, green, and low-cost solvents in sustainable biorefineries. They are transparent mixtures characterized by low freezing points resulting from at least one hydrogen bond donor and one hydrogen bond acceptor. Although DESs are promising solvents, it is necessary to combine them with an economic heating technology, such as microwave irradiation, for competitive profitability. Microwave irradiation is a promising strategy to shorten the heating time and boost fractionation because it can rapidly attain the appropriate temperature. The aim of this study was to develop a one-step, rapid method for biomass fractionation and lignin extraction using a low-cost and biodegradable solvent. 
In this study, a microwave-assisted DES pretreatment was conducted for 60 s at 800 W, using three kinds of DESs. The DES mixtures were facilely prepared from choline chloride (ChCl) and three hydrogen-bond donors (HBDs): a monocarboxylic acid (lactic acid), a dicarboxylic acid (oxalic acid), and urea. This pretreatment was used for biomass fractionation and lignin recovery from marine residues (Posidonia leaves and aegagropile), agri-food byproducts (almond shells and olive pomace), forest residues (pinecones), and perennial lignocellulosic grasses (Stipa tenacissima). Further analyses were conducted to determine the yield, purity, and molecular weight distribution of the recovered lignin. In addition, the effect of DESs on the chemical functional groups in the extracted lignin was determined by Fourier-transform infrared (FTIR) spectroscopy. The results indicate that the ChCl-oxalic acid mixture affords the highest lignin purity and the lowest yield. The present study demonstrates that the DES-microwave process is an ultrafast, efficient, and cost-competitive technology for lignocellulosic biomass fractionation.

Deep eutectic solvent-based, microwave-assisted pretreatment is a green, fast, and efficient process for lignocellulosic fractionation and high-purity lignin recovery.

Pretreatment is still the most expensive step in lignocellulosic biorefinery processes. It must be made cost-effective by minimizing chemical requirements ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

Fractionation of Lignocellulosic Biomass using the OrganoCat Process

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