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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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:&#160;&#62;98.0%(T)(HPLC)</td>
		</tr>
		<tr>
			<td>2-methyltetrahydrofuran</td>
			<td>Carl Roth GmbH</td>
			<td>6845.4</td>
			<td>SOLVAGREEN&#160;&#8805;99 %, extra pure</td>
		</tr>
		<tr>
			<td>Accellerase 1500</td>
			<td>Provided by Genencor (60&#160;FPU&#160;mL-1 and 82&#160;CBU&#160;mL-1; 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&#160;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 &#38; 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>&#160;MCA0830051E3</td>
			<td>gel columns (8 x 300 mm, particle diameter: 5 &#181;m, nominal pore width: 1000 &#197;</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&#252;chi</td>
			<td>49,33,45,10,000</td>
			<td>100 bar, 200 &#176;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/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/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/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

Research

JoVE Journal

Chemistry

3.8K Views.  Pflanzenwissenschaften (IBG-2), Forschungszentrum Jülich. 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.

Video: Fractionation of Lignocellulosic Biomass using the OrganoCat Process

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

Synthesis of Zeolites Using the ADOR (Assembly-Disassembly-Organization-Reassembly) Route

Zeolites are an important class of materials that have wide ranging applications such as heterogeneous catalysts and adsorbents which are dependent on their framework topology. For new applications or improvements to existing ones, new zeolites with novel pore systems are desirable. We demonstrate a method for the synthesis of novel zeolites using the ADOR route. ADOR is an acronym for Assembly, Disassembly, Organization and Reassembly. This synthetic route takes advantage of the assembly of a relatively poorly stable that which can be selectively disassembled into a layered material. The resulting layered intermediate can then be organized in different manners by careful chemical manipulation and then reassembled into zeolites with new topologies. By carefully controlling the organization step of the synthetic pathway, new zeolites with never before seen topologies are capable of being synthesized. The structures of these new zeolites are confirmed using powder X-ray diffraction and further characterized by nitrogen adsorption and scanning electron microscopy. This new synthetic pathway for zeolites demonstrates its capability to produce novel frameworks that have never been prepared by traditional zeolite synthesis techniques.

Synthesis of Zeolites Using the ADOR Assembly-Disassembly-Organization-Reassembly Route

A protocol for the preparation of novel zeolites by the ADOR (Assembly-Disassembly-Organization-Reassembly) synthetic route is presented.

Zeolites are an important class of materials that have wide ranging applications such as heterogeneous catalysts and adsorbents which are dependent on ...

Synthesis and Exfoliation of Discotic Zirconium Phosphates to Obtain Colloidal Liquid Crystals

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and engineers. Growth of such anisotropic nanocrystals through a simple chemical method is a challenging task. In this study, we fabricate discotic nanodisks of zirconium phosphate [Zr(HPO4)2&#183;H2O] as a model material using hydrothermal, reflux and microwave-assisted methods. Growth of crystals is controlled by duration time, temperature, and concentration of reacting species. The novelty of the adopted methods is that discotic crystals of size ranging from hundred nanometers to few micrometers can be obtained while keeping the polydispersity well within control. The layered discotic crystals are converted to monolayers by exfoliation with tetra-(n)-butyl ammonium hydroxide [(C4H9)4NOH, TBAOH]. Exfoliated disks show isotropic and nematic liquid crystal phases. Size and polydispersity of disk suspensions is highly important in deciding their phase behavior.

A two dimensional model material of discotic zirconium phosphate was developed. The inorganic crystal with lamellar structure was synthesized by hydrothermal, reflux, and microwave-assisted methods. On exfoliation with organic molecules, layered crystals can be converted to monolayers, and nematic liquid crystal phase was formed at sufficient concentration of monolayers.

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and ...

Facile Synthesis of Worm-like Micelles by Visible Light Mediated Dispersion Polymerization Using Photoredox Catalyst

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins with the synthesis of a hydrophilic poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) homopolymer using reversible addition-fragmentation chain-transfer (RAFT) polymerization. Under mild visible light irradiation (&#955; = 460 nm, 0.7 mW/cm2), this macro-chain transfer agent (macro-CTA) in the presence of a ruthenium based photoredox catalyst, Ru(bpy)3Cl2 can be chain extended with a second monomer to form a well-defined block copolymer in a process known as Photoinduced Electron Transfer RAFT (PET-RAFT). When PET-RAFT is used to chain extend POEGMA with benzyl methacrylate (BzMA) in ethanol (EtOH), polymeric nanoparticles with different morphologies are formed in situ according to a polymerization-induced self-assembly (PISA) mechanism. Self-assembly into nanoparticles presenting POEGMA chains at the corona and poly(benzyl methacrylate) (PBzMA) chains in the core occurs in situ due to the growing insolubility of the PBzMA block in ethanol. Interestingly, the formation of highly pure worm-like micelles can be readily monitored by observing the onset of a highly viscous gel in situ due to nanoparticle entanglements occurring during the polymerization. This process thereby allows for a more reproducible synthesis of worm-like micelles simply by monitoring the solution viscosity during the course of the polymerization. In addition, the light stimulus can be intermittently applied in an ON/OFF manner demonstrating temporal control over the nanoparticle morphology.

This article describes a process for producing polymeric self-assembled nanoparticles using visible light mediated dispersion polymerization. Using low energy visible light to control the polymerization allows for the reproducible formation of self-assembled worm-like micelles at high solids content.

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins ...

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic acids containing three to six carbons), such as malate and citrate, are critical to the proper functioning of many biological systems, where they function in cellular respiration and can serve as indicators of cell health. In foods, organic acid content can have significant impact on taste, with increased SCCA levels resulting in a sour or &#34;acid&#34; taste. Because of this, methods for the rapid analysis of organic acid levels are of particular interest to the food and beverage industries. Unfortunately, however, most methods used for SCCA quantification are dependent on time-consuming protocols requiring the derivatization of samples with hazardous reagents, followed by costly chromatographic and/or mass spectrometric analyses. This method details an alternate method for the detection and quantification of organic acids from plant material and food samples using free zonal capillary electrophoresis (CZE), sometimes simply referred to as capillary electrophoresis (CE). CZE provides a cost-effective method for measuring SCCAs with a low limit of detection (0.005 mg/ml). This article details the extraction and quantification of SCCAs from plant samples. While the method provided focuses on measurement of SCCAs from coffee beans, the method provided can be applied to multiple plant-based food materials.

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

This article presents a method for the detection and quantification of organic acids from plant material using free zonal capillary electrophoresis. An example of the potential application of this method, determining the effects of a secondary fermentation on organic acid levels in coffee seeds, is provided.

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

Low Pressure Vapor-assisted Solution Process for Tunable Band Gap Pinhole-free Methylammonium Lead Halide Perovskite Films

Organo-lead halide perovskites have recently attracted great interest for potential applications in thin-film photovoltaics and optoelectronics. Herein, we present a protocol for the fabrication of this material via the low-pressure vapor assisted solution process (LP-VASP) method, which yields ~19% power conversion efficiency in planar heterojunction perovskite solar cells. First, we report the synthesis of methylammonium iodide (CH3NH3I) and methylammonium bromide (CH3NH3Br) from methylamine and the corresponding halide acid (HI or HBr). Then, we describe the fabrication of pinhole-free, continuous methylammonium-lead halide perovskite (CH3NH3PbX3 with X = I, Br, Cl and their mixture) films with the LP-VASP. This process is based on two steps: i) spin-coating of a homogenous layer of lead halide precursor onto a substrate, and ii) conversion of this layer to CH3NH3PbI3-xBrx by exposing the substrate to vapors of a mixture of CH3NH3I and CH3NH3Br at reduced pressure and 120 &#176;C. Through slow diffusion of the methylammonium halide vapor into the lead halide precursor, we achieve slow and controlled growth of a continuous, pinhole-free perovskite film. The LP-VASP allows synthetic access to the full halide composition space in CH3NH3PbI3-xBrx with 0&#160;&#8804; x&#160;&#8804; 3. Depending on the composition of the vapor phase, the bandgap can be tuned between 1.6 eV&#160;&#8804; Eg&#160;&#8804; 2.3 eV. In addition, by varying the composition of the halide precursor and of the vapor phase, we can also obtain CH3NH3PbI3-xClx. Films obtained from the LP-VASP are reproducible, phase pure as confirmed by X-ray diffraction measurements, and show high photoluminescence quantum yield. The process does not require the use of a glovebox.

Here, we present a protocol for the synthesis of CH3NH3I and CH3NH3Br precursors and the subsequent formation of pinhole-free, continuous CH3NH3PbI3-xBrx thin films for the application in high efficiency solar cells and other optoelectronic devices.

Organo-lead halide perovskites have recently attracted great interest for potential applications in thin-film photovoltaics and optoelectronics. Herein, ...

Essential Metal Uptake in Gram-negative Bacteria: X-ray Fluorescence, Radioisotopes, and Cell Fractionation

We demonstrate a scalable method for the separation of the bacterial periplasm from the cytoplasm. This method is used to purify periplasmic protein for the purpose of biophysical characterization, and measure substrate transfer between periplasmic and cytoplasmic compartments. By carefully limiting the time that the periplasm is separated from the cytoplasm, the experimenter can extract the protein of interest and assay each compartment individually for substrate without carry-over contamination between compartments. The extracted protein from fractionation can then be further analyzed for three-dimensional structure determination or substrate-binding profiles. Alternatively, this method can be performed after incubation with a radiotracer to determine total percent uptake, as well as distribution of the tracer (and hence metal transport) across different bacterial compartments. Experimentation with a radiotracer can help differentiate between a physiological substrate and artefactual substrate, such as those caused by mismetallation.
X-ray fluorescence can be used to discover the presence or absence of metal incorporation in a sample, as well as measure changes that may occur in metal incorporation as a product of growth conditions, purification conditions, and/or crystallization conditions. X-ray fluorescence also provides a relative measure of abundance for each metal, which can be used to determine the best metal energy absorption peak to use for anomalous X-ray scattering data collection. Radiometal uptake can be used as a method to validate the physiological nature of a substrate detected by X-ray fluorescence, as well as support the discovery of novel substrates.

A protocol for the extraction of a periplasmic transition metal chaperone in the context of its native binding partners, and biophysical characterization of its substrate contents by X-ray fluorescence and radiometal uptake is presented.

We demonstrate a scalable method for the separation of the bacterial periplasm from the cytoplasm. This method is used to purify periplasmic protein for ...

Synthesis of Soft Polysiloxane-urea Elastomers for Intraocular Lens Application

This study discusses a synthesis route for soft polysiloxane-based urea (PSU) elastomers for their applications as accommodating intraocular lenses (a-IOLs). Aminopropyl-terminated polydimethylsiloxanes (PDMS) were previously prepared via the ring-chain equilibration of the cyclic siloxane octamethylcyclotetrasiloxane (D4) and 1,3-bis(3-aminopropyl)-tetramethyldisiloxane (APTMDS). Phenyl groups were introduced into the siloxane backbone via the copolymerization of D4 and 2,4,6,8-tetramethyl-2,4,6,8-tetraphenyl-cyclotetrasiloxane (D4Me,Ph). These polydimethyl-methyl-phenyl-siloxane-block copolymers were synthesized for increasing the refractive indices of polysiloxanes. For applications as an a-IOL, the refractive index of the polysiloxanes must be equivalent to that of a young human eye lens. The polysiloxane molecular weight is controlled by the ratio of the cyclic siloxane to the endblocker APTMDS. The transparency of the PSU elastomers is examined by the transmittance measurement of films between 200 and 750 nm, using a UV-Vis spectrophotometer. Transmittance values at 750 nm (upper end of the visible spectrum) are plotted against the PDMS molecular weight, and &#62; 90% of the transmittance is observed until a molecular weight of 18,000 g&#183;mol&#8722;1. Mechanical properties of the PSU elastomers are investigated using stress-strain tests on die-cut dog-bone-shaped specimens. For evaluating mechanical stability, mechanical hysteresis is measured by repeatedly stretching (10x) the specimens to 5% and 100% elongation. Hysteresis considerably decreases with the increase in the PDMS molecular weight. In vitro cytotoxicity of some selected PSU elastomers is evaluated using an MTS cell viability assay. The methods described herein permit the synthesis of a soft, transparent, and noncytotoxic PSU elastomer with a refractive index approximately equal to that of a young human eye lens.

This study describes synthetic routes for aminopropyl-terminated polydimethylsiloxanes and polydimethyl-methyl-phenyl-siloxane-block copolymers and for soft polysiloxane-based urea (PSU) elastomers. It presents the application of PSUs as accommodating an intraocular lens. An evaluation method for in vitro cytotoxicity is also described.

This study discusses a synthesis route for soft polysiloxane-based urea (PSU) elastomers for their applications as accommodating intraocular lenses ...

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 ...