Name: ultrafast lignin extraction from unusual mediterranean lignocellulosic residues
Uploaded: 2021-03-09T12:30:00
Description: Watch this Scientific Journal Video about Ultrafast Lignin Extraction from Unusual Mediterranean Lignocellulosic Residues at JoVE.com

MK and TB thanks Haitham Ayeb for statistical analyses and figure preparation, Walloon Region (European Regional Development-VERDIR) and Minister of Higher Education and Scientific Research (Taoufik Bettaieb) for funding.

1. Preparation of biomasses
<ol>
	<li>Biomass drying
	<ol>
		<li>Place the Posidonia leaves and aegagropile balls (Posidonia oceanica), harvested from Mediterranean beaches, in an oven at 40 &#176;C for 72 h.</li>
		<li>Place the almond shells (Prunus dulcis), generated from food industries, and olive pomace (Olea europaea L.), obtained from olive oil mills, in an oven at 40 &#176;C for 72 h.</li>
		<li>Place the pinecones (Pinus halepensis), collected from a forest, and alfa leaves ...

<ol>
	<li>Kammoun, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrothermal+dehydration+of+monosaccharides+promoted+by+seawater+fundamentals+on+the+catalytic+role+of+inorganic+salts.">Hydrothermal dehydration of monosaccharides promoted by seawater fundamentals on the catalytic role of inorganic salts.</a> Frontiers in Chemistry. 7, 132 (2019).</li><li>Kammoun, M., Ayeb, H., Bettaieb, T., Richel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+characterisation+and+technical+assessment+of+agri-food+residues,+marine+matrices,+and+wild+grasses+in+the+South+Mediterranean+area:+A+considerable+inflow+for+biorefineries.">Chemical characterisation and technical assessment of agri-food residues, marine matrices, and wild grasses in the South Mediterranean area: A considerable inflow for biorefineries.</a> Waste Management. 118, 247-257 (2020).</li><li>Zhang, C. W., Xia, S. Q., Ma, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+pretreatment+of+lignocellulosic+biomass+using+deep+eutectic+solvents.">Facile pretreatment of lignocellulosic biomass using deep eutectic solvents.</a> Bioresource Technology. 219, 1-5 (2016).</li><li>Mora-Pale, M., Meli, L., Doherty, T. V., Linhardt, R. J., Dordick, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Room+temperature+ionic+liquids+as+emerging+solvents+for+the+pretreatment+of+lignocellulosic+biomass.">Room temperature ionic liquids as emerging solvents for the pretreatment of lignocellulosic biomass.</a> Biotechnology and Bioengineering. 108 (6), 1229-1245 (2011).</li><li>Chen, Z., Wan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+fractionation+of+lignocellulosic+biomass+by+microwave-assisted+deep+eutectic+solvent+pretreatment.">Ultrafast fractionation of lignocellulosic biomass by microwave-assisted deep eutectic solvent pretreatment.</a> Bioresource Technologie. 250, 532-537 (2018).</li><li>Francisco, M., Van Den Bruinhorst, A., Kroon, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+natural+and+renewable+low+transition+temperature+mixtures+(+LTTMs+):+screening+as+solvents+for+lignocellulosic+biomass+processing.">New natural and renewable low transition temperature mixtures ( LTTMs ): screening as solvents for lignocellulosic biomass processing.</a> Green Chemistry. 14 (8), 2153-2157 (2012).</li><li>Liu, Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+cleavage+of+lignin+-+carbohydrate+complexes+and+ultrafast+extraction+of+lignin+oligomers+from+wood+biomass+by+microwave-assisted+treatment+with+deep+eutectic+solvent.">Efficient cleavage of lignin - carbohydrate complexes and ultrafast extraction of lignin oligomers from wood biomass by microwave-assisted treatment with deep eutectic solvent.</a> Chem sus chem. 10, 1692-1700 (2017).</li><li>Xu, G. C., Ding, J. C., Han, R. Z., Dong, J. J., Ni, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+cellulose+accessibility+of+corn+stover+by+deep+eutectic+solvent+pretreatment+for+butanol+fermentation.">Enhancing cellulose accessibility of corn stover by deep eutectic solvent pretreatment for butanol fermentation.</a> Bioresource Technologie. 203, 364-369 (2016).</li><li>Jablonsk&#253;, M., Andrea, &. #. 3. 5. 2. ;., Kamensk&#225;, L., Vr&#353;ka, M., &#352;ima, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+eutectic+solvents+fractionation+of+wheat+straw+deep+eutectic+solvents+fractionation+of+wheat+straw.">Deep eutectic solvents fractionation of wheat straw deep eutectic solvents fractionation of wheat straw.</a> Bioresources. 10 (4), 8039-8047 (2015).</li><li>Shen, X. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+fractionation+of+lignocelluloses+by+biomass-derived+deep+eutectic+solvent+(DES)+pretreatment+for+cellulose+enzymatic+hydrolysis+and+lignin+valorization.">Facile fractionation of lignocelluloses by biomass-derived deep eutectic solvent (DES) pretreatment for cellulose enzymatic hydrolysis and lignin valorization.</a> Green Chemistry. 21, 275-283 (2019).</li><li>Alvarez-Vasco, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+low-molecular-weight+lignin+with+high+purity+extracted+from+wood+by+deep+eutectic+solvents+(DES):+a+source+of+lignin+for+valorization.">Unique low-molecular-weight lignin with high purity extracted from wood by deep eutectic solvents (DES): a source of lignin for valorization.</a> Green Chemistry. 18, 5133-5141 (2016).</li><li>Banu, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+on+biopolymer+production+via+lignin+valorization.">A review on biopolymer production via lignin valorization.</a> Bioresource Technologie. 290, 121790 (2019).</li><li>Gordobil, O., Olaizola, P., Banales, J. M., Labidi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lignins+from+agroindustrial+by-products+as+natural+ingredients+for+cosmetics+chemical+structure+and+in+vitro+sunscreen+and+cytotoxic+activities.">Lignins from agroindustrial by-products as natural ingredients for cosmetics chemical structure and in vitro sunscreen and cytotoxic activities.</a> Molecules. 25 (5), 1131 (2020).</li><li>Lee, C. S., Thu Tran, T. M., Weon Choi, J., Won, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lignin+for+white+natural+sunscreens.">Lignin for white natural sunscreens.</a> International Journal of Biological Macromolecules. 122, 549-554 (2019).</li><li>Widsten, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lignin-based+sunscreens-state-of-the-art,+prospects+and+challenges.">Lignin-based sunscreens-state-of-the-art, prospects and challenges.</a> Cosmetics. 7, 85 (2020).</li><li>Qian, Y., Qiu, X., Zhu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lignin:+a+nature-inspired+sun+blocker+for+broad-spectrum+sunscreens.">Lignin: a nature-inspired sun blocker for broad-spectrum sunscreens.</a> Royal Society of Chemistry. 17, 320-324 (2015).</li><li>Zijlstra, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+lignin+with+high+&#946;-O-4+content+by+mild+ethanol+extraction+and+its+effect+on+the+depolymerization+yield.">Extraction of lignin with high &#946;-O-4 content by mild ethanol extraction and its effect on the depolymerization yield.</a> Journal of Visualized Experiments. (143), e58575 (2019).</li></ol>

This study had many objectives; the first of which was to prepare and use low-cost green solvents with the characteristics of both ionic liquids and organic solvents. The second objective was to fractionate the biomass and extract lignin in a single step, without requiring preliminary steps such as the extraction of extractables using Soxhlet or hemicellulose using alkaline solvents, basic, or thermophysical techniques. The third aim was to recover lignin by simple filtration after the treatment, without adjustment of pH...

Sustainable biorefinery processes integrate biomass processing, its fractionation into molecules of interest, and their conversion to value-added products1. In second-generation biorefining, pretreatment is considered essential for fractionating biomass into its main components2. Traditional pretreatment methods utilizing chemical, physical, or biological strategies have been widely applied3. However, such pretreatment is considered the most expensive step in biorefining and has other disadvantages such as long processing time, high heat and power consumption, and solvent impurities4. Recently, DESs, whose properties are similar to those of ionic liquids3, have emerged as green solvents owing to advantages such as biodegradability, environmental-friendliness, ease of synthesis, and recovery after treatment5.
DESs are mixtures of at least one HBD, such as lactic acid, malic acid, or oxalic acid, and a hydrogen-bond acceptor (HBA) such as betaine or choline chloride (ChCl)6. HBA-HBD interactions enable a catalytic mechanism that permits cleavage of chemical bonds, causing biomass fractionation and lignin separation. Many researchers have reported the DES-based pretreatment of lignocellulosic feedstocks such as ChCl-glycerol on corn&#39;s cob and stover7,8, ChCl-urea, and ChCl-oxalic acid on wheat straw9, ChCl-lactic acid on Eucalyptus sawdust10, and ChCl-acetic acid11 and ChCl-ethylene glycol on wood11. To improve DES efficiency, the pretreatment should be combined with microwave treatment to accelerate biomass fractionation5. Many researchers have reported such a combined pretreatment (DES and microwave) of wood8 and of corn stover, switchgrass, and Miscanthus5, which provides new insight into the capacity of DESs for lignocellulosic fractionation and lignin extraction in one easy step over a short period.
Lignin is a phenolic macromolecule valorized as a raw material for the production of biopolymers and presents an alternative for the production of chemicals such as aromatic monomers and oligomers12. In addition, lignin has antioxidant and ultraviolet absorption activities13. Several studies have reported lignin applications in cosmetic products14,15. Its integration in commercial sunscreen products has improved the sun protection factor (SPF) of the product from SPF 15 to SPF 30 with the addition of only 2 wt % lignin and up to SPF 50 with the addition of 10 wt % lignin16. This paper describes an ultrafast approach for lignin-carbohydrate cleavage, assisted by combined DES-microwave pretreatment of Mediterranean biomasses. These biomasses consist of agri-food byproducts, particularly olive pomace and almond shells. Other biomasses that were investigated were those of a marine origin (Posidonia leaves and aegagropile) and those originating from a forest (pinecones and wild grasses). The focus of this study was to test low-cost green solvents to evaluate the effects of this combined pretreatment on feedstock fractionation, to investigate its influence on lignin purity and yield, and to study its effects on the molecular weights and chemical functional groups in the extracted lignin.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>HPLC Gel Permeation Chromatography</td>
			<td>Agilent 1200 series</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 methylimadazole</td>
			<td>Acros organics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-deoxy-D-glucose (internal standard)</td>
			<td>Sigma Aldrich (St. Louis, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma Aldrich (St. Louis, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic anhydride</td>
			<td>Sigma Aldrich (St. Louis, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adjustables pipettors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alkali</td>
			<td></td>
			<td></td>
			<td>alkali-extracted lignin</td>
		</tr>
		<tr>
			<td>Arabinose (99%)</td>
			<td>Sigma Aldrich (St. Louis, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>CERTO CLAV (Model CV-22-VAC-Pro)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water Bath at 70 &#176;C</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Sigma Aldrich (St. Louis, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bromocresol</td>
			<td>Sigma Aldrich (St. Louis, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Catalyst</td>
			<td>CTQ (coded A22) (1.5 g K2SO4 + 0.045 g CuSO4.5 H2O + 0.045 g TiO2)</td>
			<td>Merck</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugation container</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>BECKMAN COULTER</td>
			<td>Avanti J-E centrifuge</td>
			<td></td>
		</tr>
		<tr>
			<td>Ceramic crucibles</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Choline chloride 99%</td>
			<td>Acros organics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Column</td>
			<td>Agilent PLGel Mixed C (alpha 3,000 (4.6 &#215; 250 mm, 5 &#181;m) preceded by a guard column (TSK gel alpha guard column 4.6 mm &#215; 50 mm, 5 &#181;m)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Column</td>
			<td>HP1-methylsisoxane (30 m, 0.32 mm, 0.25 mm)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Crucible porosity N&#176;4 ( Filtering crucible)</td>
			<td>Shott Duran Germany</td>
			<td>boro 3.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Deonized water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dessicator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylformamide</td>
			<td>VWR BDH Chemicals</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide</td>
			<td>Acros organics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flask</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck (Darmstadtt, Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filtering crucibles, procelain</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filtration flasks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fourrier Transformed Inra- Red</td>
			<td>Vertex 70 Bruker apparatus 
			equipped with an attenuated total reflectance (ATR) module. 
			Spectra were recorded in the 4,000&#8211;400 cm&#8722;1 range with 32 scans 
			at a resolution of 4.0 cm&#8722;1</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Galactose (98%</td>
			<td>Sigma Aldrich (St. Louis, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gaz Chromatography</td>
			<td>Agilent (7890 series)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottle 100 mL</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass tubes ( borosilicate) with teflon caps 10 mL</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose (98%</td>
			<td>Sigma Aldrich (St. Louis, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Golves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Graduated cylinder 50 mL /100 mL</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>H2SO4 Titrisol (0.1 N)</td>
			<td>Merck (Darmstadtt, Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>H2SO4 (95-98%)</td>
			<td>Sigma Aldrich (St. Louis, USA)</td>
			<td>BUCHI R-114)</td>
			<td></td>
		</tr>
		<tr>
			<td>Hummer cutter equiped with 1 mm and 0.5 mm sieve</td>
			<td>Mill Ttecator (Sweden)</td>
			<td>Cyclotec 1093</td>
			<td></td>
		</tr>
		<tr>
			<td>Indulin</td>
			<td></td>
			<td></td>
			<td>Raw lignin control</td>
		</tr>
		<tr>
			<td>Kjeldahl distiller</td>
			<td>Kjeltec 2300 (Foss)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kjeldahl tube</td>
			<td>FOSS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kjeldhal rack</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kjeldhal digester</td>
			<td>Kjeltec 2300 (Foss)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kjeldhal suction system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Chem station Software</td>
			<td></td>
			<td></td>
			<td>GC data analysis</td>
		</tr>
		<tr>
			<td>Lactic acid</td>
			<td>Merck (Darmstadtt, Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium chloride LiCl</td>
			<td>Sigma Aldrich (St. Louis, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mannose (98%)</td>
			<td>Sigma Aldrich (St. Louis, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl red</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave</td>
			<td>START SYNTH MILESTONE Microwave laboratory system</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave temperature probe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave container</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Muffle Furnace</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Merck (Darmstadtt, Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen free- paper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Opus</td>
			<td></td>
			<td></td>
			<td>spectroscopy software</td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>GmbH Memmert SNB100</td>
			<td>Memmert SNB100</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxalic acid</td>
			<td>VWR BDH Chemicals</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P 1000</td>
			<td></td>
			<td></td>
			<td>Soda-processed lignin</td>
		</tr>
		<tr>
			<td>pH paper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>precision balance</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Infrared spectroscopy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Quatz cuvette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rhamnose (98%)</td>
			<td>Sigma Aldrich (St. Louis, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary vacuum evaporator</td>
			<td>Bucher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Round-bottom flask 500 mL</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sodium borohydride NaBH4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Schott bottle</td>
			<td></td>
			<td></td>
			<td>glass bottle</td>
		</tr>
		<tr>
			<td>Sovirel tubes</td>
			<td>sovirel</td>
			<td></td>
			<td>Borosilicate glass tubes</td>
		</tr>
		<tr>
			<td>Spatule</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Special tube</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectophotometer</td>
			<td>UV-1800 Shimadzu</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilization indicator tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stir bar in teflon</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stirring plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium borohydride</td>
			<td>Sigma Aldrich (St. Louis, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Titrisol</td>
			<td>Merck</td>
			<td>Merck 109984</td>
			<td>0.1 N H2SO4</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>VWR BDH Chemicals</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VolumetriC flask 2.5 L /5 L</td>
			<td>Bucher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylose (98%)</td>
			<td>Sigma Aldrich (St. Louis, USA)</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Figure 2A-C depict the lignin yield of extraction from the six feedstocks, shown in Figure 1A-F, after the combined microwave-DES pretreatment. The results show that the lignin yield obtained with DES1 (ChCl-oxalic acid) (Figure 2A) was lower than the yields obtained with DES2 (ChCl-lactic acid) and DES3 (ChCl-urea) (Figure 2B</...

Watch this Scientific Journal Video about Ultrafast Lignin Extraction from Unusual Mediterranean Lignocellulosic Residues at JoVE.com

Ultrafast Lignin Extraction from Unusual Mediterranean Lignocellulosic Residues

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 ...

The aim of our protocol is to fractionate the biomass and extract lignin in a single step. Using this method, lignin can be recovered by simple filtration after the treatment without adjustment of pH, but simply by adding distilled water. The focus of this study is to evaluate the effect of this combined treatment of feedstock fractionation, its influence on lignin purity and yield, and its effect on the molecular weights and chemical functional groups in the extracted lignin.Deep eutectic solution microwave process is an ultra fast, efficient, and cost competitive technology for lignocellulosic biomass fractionation and lignin recovery with high purity. Prepare the deep eutectic solution in a 500 milliliter round bottom flask as described in the text manuscript. Then place five grams of feedstock, 50 milliliters of deep eutectic solution, and a stirring bar in a microwave in a closed polytetrafluoroethylene reactor.Close the microwave container with an appropriate cap and attach a temperature cap. Place it on the edge of the turntable, ensuring that it is constantly agitated and run the microwave for one minute. Using suitable gloves, take the container out of the microwave and let the mixture cool.Prepare a homogenous anti-solvent solution and add 50 milliliters of this solution to the treated feedstock. Centrifuge the mixture for five minutes at 3, 000 times G.Filter the supernatant using a glass filter crucible. Collect the remaining cellulose residue and wash it by adding 25 milliliters of anti-solvent solution and centrifuging.Add the filtered lignin-rich fraction and the filtered washes to a 500 milliliter round bottom flask. Evaporate the ethanol using a rotary evaporator at 50 degrees Celsius and 110 millibar. Then add 150 milliliters of deionized water to the concentrated liquor.Precipitate the lignin by centrifugation. Collect lignin as a pellet and wash it four times with 25 milliliters of distilled water. Then lyophilize the lignin or dry it in an oven at 40 degrees Celsius.Place the filter crucible in a muffle furnace at 550 degrees Celsius for four hours. When the oven cools to 150 degrees Celsius, remove the crucible and place it in a desiccator to cool. Then weigh it.Add approximately 30 milligrams of lignin into a borosilicate glass tube. Then add one milliliter of 72%sulfuric to the sample and place it in a 30 degrees Celsius bath for 60 minutes. Remove the sample and transfer it to a 100 milliliter glass bottle.Then add 28 milliliters of distilled water to dilute the acid to a concentration of 4%Put the glass bottle in an autoclave at 121 degrees Celsius for 60 minutes. Then remove the bottle from the autoclave and allow it to cool. To analyze acid insoluble lignin, filter the hydrolysate using a crucible under vacuum and collect the remaining solids in the glass bottle with deionized water.Dry the crucible containing the solids by placing it in an oven at 105 degrees Celsius for 16 hours. Then cool it in a desiccator and weigh the sample. Place the crucible in a muffle furnace at 550 degrees Celsius for four hours.Weigh the sample after drying it in a desiccator. For analysis of acid soluble lignin, measure the absorbance of the hydrolysate filtrate with a spectrophotometer at 205 nanometers using quartz cuvettes. To analyze chemical function of the extracted lignin, process the background single channel without sample.Then adjust the parameters and place one milligram of the sample on the crystal. Press sample single channel and process the obtained spectra. To determine the molecular weight of the extracted lignin, dissolve three milligrams of the lignin sample in three milliliters of DMF with 0.5%lithium chloride.Put the dissolved lignin in a vial and install the column proceeded by a guard column. Inject the sample into a high-performance liquid chromatography ultraviolet system. After analyzing the data, calculate the molecular weight of the extracted lignin as described in the text manuscript.Weigh a 50 milligram sample of lignin in a borosilicate glass tube and add three milliliters of one molar sulfuric acid. Then heat the mixture for three hours at 100 degrees Celsius and cool it. Add one milliliter of 15 molar ammonium hydroxide and check the pH to ensure that it is neutral or alkaline.Add one milliliter of 2-deoxyglucose to each sample as internal standard. Then add 400 microliters of this solution into special tubes containing 400 microliters of mixed solution. Add two milliliters of sodium borohydride dimethyl sulfoxide solution.Close the tubes and incubate for 90 minutes at 40 degrees Celsius in a water bath. Remove the tubes from the water bath and add 0.6 milliliters of glacial acetic acid, 0.4 milliliters of 1-methylimidazole and approximately four milliliters of acetic anhydride. After 15 minutes, add 10 milliliters of distilled water.Cool.And add approximately three milliliters of dichloromethane. After two hours, collect approximately one milliliter of the lower organic phase and inject it into a gas chromatograph equipped with a flame ionization detector capillary column. The lignin yield obtained with deep eutectic solution one oxalic acid was lower than the yields obtained with deep eutectic solution two lactic acid and deep eutectic solution three urea.Lignin purity exceeded 70%for the three pre-treatments of the biomasses, except for deep eutectic solution three urea pretreatment of alpha leaves, Aegagropila, and almond shells, which gave a lignin purity of 65%The highest lignin purity exceeded 90%It was obtained with the deep eutectic solution one treatment. Lignin purity and yield data were subjected to principal component analysis, which showed the deep eutectic solution one treatment to be positively correlated with lignin purity and confirmed it to be the purest lignin with the lowest yield. The sugar content in lignin extracted using deep eutectic solution three was the highest and followed by that obtained from solutions two and one.Similarly, the nitrogen content of deep eutectic solution one lignin extract was lower than that obtained from solutions two and three. The type of sugars in the extracted lignin were also characterized, depicting D-xylose and D-glucose to be the most abundant monosaccharides. The chemical functional groups present in the extracted lignans were investigated by FTIR spectroscopy.Infrared spectra of the different lignans between 3, 500 and 800 reciprocal centimeters are presented here. The spectra of lignin extracted with deep eutectic solutions one and two show the stretching vibrations of unconjugated and conjugated carbonyl groups, which were absent in the three commercial lignans:raw, soda processed, and alkali extracted lignans. The carboxylic acid signal, C double bond O, indicates possibility of the conjugation of some lignin functions with acids during its extraction and solubilization with the microwave-assisted deep eutectic solvent treatment.These results demonstrate the possibility of extracting value-added lignin of high purity from Mediterranean biomasses, which is presently undervalued and can help determine the optimal deep eutectic solvent while ensuring the purity of lignin.

ultrafast-lignin-extraction-from-unusual-mediterranean

Research

JoVE Journal

Chemistry

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 ...

Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional units (e.g., domains). Traditional consensus-building models fail to account for interpositional dependencies &#8211; functionally required covariation of residues that tend to appear simultaneously throughout evolution and across the phylogentic tree. These relationships can reveal important clues about the processes of protein folding, thermostability, and the formation of functional sites, which in turn can be used to inform the engineering of synthetic proteins. Unfortunately, these relationships essentially form sub-motifs which cannot be predicted by simple &#8220;majority rule&#8221; or even HMM-based consensus models, and the result can be a biologically invalid &#8220;consensus&#8221; which is not only never seen in nature but is less viable than any extant protein. We have developed a visual analytics tool, StickWRLD, which creates an interactive 3D representation of a protein alignment and clearly displays covarying residues. The user has the ability to pan and zoom, as well as dynamically change the statistical threshold underlying the identification of covariants. StickWRLD has previously been successfully used to identify functionally-required covarying residues in proteins such as Adenylate Kinase and in DNA sequences such as endonuclease target sites.

Synthetic protein sequences based on consensus motifs typically ignore co-evolving residues, that imply interpositional dependencies (IPDs). IPDs can be essential to activity, and designs that disregard them may result in suboptimal results. This protocol uses StickWRLD to identify IPDs and help inform rational protein design, resulting in more efficient results.

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional ...

Direct Imaging of Laser-driven Ultrafast Molecular Rotation

We present a method for visualizing laser-induced, ultrafast molecular rotational wave packet dynamics. We have developed a new 2-dimensional Coulomb explosion imaging setup in which a hitherto-impractical camera angle is realized. In our imaging technique, diatomic molecules are irradiated with a circularly polarized strong laser pulse. The ejected atomic ions are accelerated perpendicularly to the laser propagation. The ions lying in the laser polarization plane are selected through the use of a mechanical slit and imaged with a high-throughput, 2-dimensional detector installed parallel to the polarization plane. Because a circularly polarized (isotropic) Coulomb exploding pulse is used, the observed angular distribution of the ejected ions directly corresponds to the squared rotational wave function at the time of the pulse irradiation. To create a real-time movie of molecular rotation, the present imaging technique is combined with a femtosecond pump-probe optical setup in which the pump pulses create unidirectionally rotating molecular ensembles. Due to the high image throughput of our detection system, the pump-probe experimental condition can be easily optimized by monitoring a real-time snapshot. As a result, the quality of the observed movie is sufficiently high for visualizing the detailed wave nature of motion. We also note that the present technique can be implemented in existing standard ion imaging setups, offering a new camera angle or viewpoint for the molecular systems without the need for extensive modification.

We present a protocol for creating a real-time movie of a molecular rotational wave packet using a high-resolution Coulomb explosion imaging setup.

We present a method for visualizing laser-induced, ultrafast molecular rotational wave packet dynamics. We have developed a new 2-dimensional Coulomb ...

Separation of Aldehydes and Reactive Ketones from Mixtures Using a Bisulfite Extraction Protocol

The purification of organic compounds is an essential component of routine synthetic operations. The ability to remove contaminants into an aqueous layer by generating a charged structure provides an opportunity to use extraction as a simple purification technique. By combining the use of a miscible organic solvent with saturated sodium bisulfite, aldehydes and reactive ketones can be successfully transformed into charged bisulfite adducts that can then be separated from other organic components of a mixture by the introduction of an immiscible organic layer. Here, we describe a simple protocol for the removal of aldehydes, including sterically-hindered neopentyl aldehydes and some ketones, from chemical mixtures. Ketones can be separated if they are sterically unhindered cyclic or methyl ketones. For aliphatic aldehydes and ketones, dimethylformamide is used as the miscible solvent to improve removal rates. The bisulfite addition reaction can be reversed by basification of the aqueous layer, allowing for the re-isolation of the reactive carbonyl component of a mixture.

Here, we present a protocol to remove aldehydes and reactive ketones from mixtures by a liquid-liquid extraction protocol directly with saturated sodium bisulfite in a miscible solvent. This combined protocol is rapid and facile to perform. The aldehyde or ketone can be re-isolated by the basification of the aqueous layer.

The purification of organic compounds is an essential component of routine synthetic operations. The ability to remove contaminants into an aqueous layer ...

Measurement of Ultrafast Vibrational Coherences in Polyatomic Radical Cations with Strong-Field Adiabatic Ionization

We present a pump-probe method for preparing vibrational coherences in polyatomic radical cations and probing their ultrafast dynamics. By shifting the wavelength of the strong-field ionizing pump pulse from the commonly used 800 nm into the near-infrared (1200-1600 nm), the contribution of adiabatic electron tunneling to the ionization process increases relative to multiphoton absorption. Adiabatic ionization results in predominant population of the ground electronic state of the ion upon electron removal, which effectively prepares a coherent vibrational state (&#34;wave packet&#34;) amenable to subsequent excitation. In our experiments, the coherent vibrational dynamics are probed with a weak-field 800 nm pulse and the time-dependent yields of dissociation products measured in a time-of-flight mass spectrometer. We present the measurements on the molecule dimethyl methylphosphonate (DMMP) to illustrate how using 1500 nm pulses for excitation enhances the amplitude of coherent oscillations in ion yields by a factor of 10 as compared to 800 nm pulses. This protocol may be implemented in existing pump-probe setups through the incorporation of an optical parametric amplifier (OPA) for wavelength conversion.

We present a protocol for probing ultrafast vibrational coherences in polyatomic radical cations that result in molecular dissociation.

We present a pump-probe method for preparing vibrational coherences in polyatomic radical cations and probing their ultrafast dynamics. By shifting the ...

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.

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

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 ...

Ultrafast Time-resolved Near-IR Stimulated Raman Measurements of Functional &#960;-conjugate Systems

Femtosecond time-resolved stimulated Raman spectroscopy is a promising method of observing the structural dynamics of short-lived transients with near infrared (near-IR) transitions, because it can overcome the low sensitivity of spontaneous Raman spectrometers in the near-IR region. Here, we describe technical details of a femtosecond time-resolved near-IR multiplex stimulated Raman spectrometer that we have recently developed. A description of signal generation and optimization, measurement, data acquisition, and calibration and correction of recorded data is provided as well. We present an application of our spectrometer to analyze the excited-state dynamics of &#946;-carotene in toluene solution. A C=C stretch band of &#946;-carotene in the second lowest excited singlet (S2) state and the lowest excited singlet (S1) state is clearly observed in the recorded time-resolved stimulated Raman spectra. The femtosecond time-resolved near-IR stimulated Raman spectrometer is applicable to the structural dynamics of &#960;-conjugate systems from simple molecules to complex materials.

Ultrafast Time-resolved Near-IR Stimulated Raman Measurements of Functional &amp;#960;-conjugate Systems

Details of signal generation and optimization, measurement, data acquisition, and data handling for a femtosecond time-resolved near-IR stimulated Raman spectrometer are described. A near infrared stimulated Raman study on the excited-state dynamics of &#946;-carotene in toluene is shown as a representative application.

Femtosecond time-resolved stimulated Raman spectroscopy is a promising method of observing the structural dynamics of short-lived transients with near ...

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.

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 ...

Designed for Molecular Recycling: A Lignin-Derived Semi-aromatic Biobased Polymer

The development of chemically recyclable biopolymers offers opportunities within the pursuit of a circular economy. Chemically recyclable biopolymers make a positive effort to solve the issue of polymer materials in the disposal phase after the use phase. In this paper, the production of biobased semi-aromatic polyesters, which can be extracted entirely from biomass such as lignin, is described and visualized. The polymer poly-S described in this paper has thermal properties similar to certain commonly used plastics, such as PET. We developed a Green Knoevenagel reaction, which can efficiently produce monomers from aromatic aldehydes and malonic acid. This reaction has been proven to be scalable and has a remarkably low calculated E-factor. These polyesters with ligno-phytochemicals as a starting point show an efficient molecular recycling with minimal losses. The polyester poly(dihydrosinapinic acid) (poly-S) is presented as an example of these semi-aromatic polyesters, and the polymerization, depolymerization, and re-polymerization are described.

An example of a closed-loop approach towards a circular materials economy is described here. A whole sustainable cycle is presented where biobased semi-aromatic polyesters are designed by polymerization, depolymerization, and then re-polymerized with only slight changes in their yields or final properties.

The development of chemically recyclable biopolymers offers opportunities within the pursuit of a circular economy. Chemically recyclable biopolymers make ...

Extraction of Non-Protein Amino Acids from Cyanobacteria for Liquid Chromatography-Tandem Mass Spectrometry Analysis

Non-protein amino acids (NPAAs) are a large class of amino acids (AAs) that are not genetically encoded for translation into proteins. The analysis of NPAAs can provide crucial information about cellular uptake and/or function, metabolic pathways, and potential toxicity. &#946;-methylamino-L-alanine (BMAA) is a neurotoxic NPAA produced by various algae species and is associated with an increased risk for neurodegenerative diseases, which has led to significant research interest. There are numerous ways to extract AAs for analysis, with liquid chromatography-tandem mass spectrometry being the most common, requiring protein precipitation followed by acid hydrolysis of the protein pellet. Studies on the presence of BMAA in algal species provide contradictory results, with the use of unvalidated sample preparation/extraction and analysis a primary cause. Like most NPAAs, protein precipitation in 10% aqueous TCA and hydrolysis with fuming HCl is the most appropriate form of extraction for BMAA and its isomers aminoethylglycine (AEG) and 2,4-diaminobutyric acid (2,4-DAB). The present protocol describes the steps in a validated NPAA extraction method commonly used in research and teaching laboratories.

The present protocol describes the extraction of non-protein amino acids from biological matrices via trichloroacetic acid (TCA) protein precipitation and acid hydrolysis before analysis using liquid chromatography-tandem mass spectrometry.

Non-protein amino acids (NPAAs) are a large class of amino acids (AAs) that are not genetically encoded for translation into proteins. The analysis of ...