Name: a novel method for the pentosan analysis present in jute biomass and its conversion into sugar monomers using acidic ionic liquid
Uploaded: 2018-06-01T14:30:00
Description: Watch this Scientific Journal Video about A Novel Method for the Pentosan Analysis Present in Jute Biomass and Its Conversion into Sugar Monomers Using Acidic Ionic Liquid at JoVE.com

We would like to thank the Ministry of Science and Technology (MOST) of Taiwan (104-2628-E-002-008-MY3; 105-2218-E-155-007; 105-2221-E-002-003-MY3; 105-2221-E-002-227-MY3; 105-2622-E-155-003-CC2) and the Aim for Top University Project at National Taiwan University (105R7706) for the funding support. We are thankful to the World Bank for partial funding of this work through a subproject of Higher Education Quality Enhancement Project (HEQEP), Complete Proposal #2071. This work was also partially supported by University of Wollongong&#39;s AIIM (Gold funding).

Several chemicals used in the presenting work are toxic and carcinogenic. Please use all appropriate safety practices when performing synthesis of IL and biomass processing.
1. Preparation of Acidic IL
<ol>
	<li>Add 7.625 mmol of 1,3-propane sultone in a 50 mL round bottom flask and then close the flask with a rubber septum.</li>
	<li>Add 7.625 mmol of 1-methylimidazole into 7.625 mmol of 1,3-propanesultone slowly (10 min) at 0 &#176;C using a syringe (1 mL).</li>
	<li>After the complete add...

<ol>
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V., 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=Pretreatment+of+Lignocellulosic+Biomass+with+Low-cost+Ionic+Liquids.">Pretreatment of Lignocellulosic Biomass with Low-cost Ionic Liquids.</a> J Vis Exp. (114), e54246 (2016).</li><li>Xu, F., 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=Transforming+biomass+conversion+with+ionic+liquids:+process+intensification+and+the+development+of+a+high-gravity,+one-pot+process+for+the+production+of+cellulosic+ethanol.">Transforming biomass conversion with ionic liquids: process intensification and the development of a high-gravity, one-pot process for the production of cellulosic ethanol.</a> Energy Environ. 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Technol. 169 (Supplement C), 336-343 (2014).</li><li>Shill, K., 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=Ionic+liquid+pretreatment+of+cellulosic+biomass:+Enzymatic+hydrolysis+and+ionic+liquid+recycle.">Ionic liquid pretreatment of cellulosic biomass: Enzymatic hydrolysis and ionic liquid recycle.</a> Biotechnol Bioeng. 108 (3), 511-520 (2011).</li><li>Tadesse, H., Luque, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+on+biomass+pretreatment+using+ionic+liquids:+An+overview.">Advances on biomass pretreatment using ionic liquids: An overview.</a> Energy Environ. Sci. 4 (10), 3913-3929 (2011).</li><li>Agirrezabal-Telleria, I., Gandarias, I., Arias, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+furfural+from+pentosan-rich+biomass:+Analysis+of+process+parameters+during+simultaneous+furfural+stripping.">Production of furfural from pentosan-rich biomass: Analysis of process parameters during simultaneous furfural stripping.</a> Bioresour. Technol. 143 (Supplement C), 258-264 (2013).</li><li>Yingying, L., 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=An+Improved+Method+for+Determination+of+Pentosans+in+Pulps+using+Dual-Wavelength+Spectroscopy.">An Improved Method for Determination of Pentosans in Pulps using Dual-Wavelength Spectroscopy.</a> BioResources. 11 (3), 6801-6807 (2016).</li><li>Kumar, A. K., Sharma, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+updates+on+different+methods+of+pretreatment+of+lignocellulosic+feedstocks:+a+review.">Recent updates on different methods of pretreatment of lignocellulosic feedstocks: a review.</a> Bioresour. Bioprocess. 4 (1), 7 (2017).</li><li>Kumar, P., Barrett, D. M., Delwiche, M. J., Stroeve, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+Pretreatment+of+Lignocellulosic+Biomass+for+Efficient+Hydrolysis+and+Biofuel+Production.">Methods for Pretreatment of Lignocellulosic Biomass for Efficient Hydrolysis and Biofuel Production.</a> Ind. Eng. Chem. Res. 48 (8), 3713-3729 (2009).</li></ol>

The pentosan present in jute biomass conversion into C5 sugar monomers is demonstrated using various homogeneous Br&#248;nsted acidic catalysts such as H2SO4, HCl, and acidic IL. Furthermore, the catalytic result of the acidic IL was compared with the IL without acidity (1-butyl-3-methylimidazolium chloride). All reactions were performed in a Parr autoclave at 160 &#176;C in water. The usage of acidic IL showed the highest pentosan conversion when compared to homogeneous acids used in this work (min...

Biomass has great potential as a renewable energy and chemical source because it is sustainable, inexpensive, and equally distributed unlike fossil resources, which makes it one of the promising candidates to replace fossil feedstocks. The estimated production of lignocellulosic biomass is 146 billion metric tons per year1. The lignocellulosic biomass is mainly comprised of lignin, cellulose, and hemicellulose as its three major constituents. Lignin is an aromatic polymer made from phenylpropanoid units; on the other hand, cellulose and hemicellulose are the polysaccharide parts of the lignocellulosic biomass. Cellulose is composed of glucose units connected by &#946;(1&#8594;4) glycosidic linkage, whereas hemicellulose is made up of C5 sugars, C6 sugars, and sugar acids linked together by &#946; (1&#8594;4), &#946; (1&#8594;3) and &#946; (1&#8594;6) glycosidic bonds2,3. Along with various lignocellulosic biomass (bagasse, rice husk, wheat straw, etc.), the jute lignocellulose biomass is also produced in very large quantities (ca. 98% in 2014) in Asia compared to the total jute production in the world. India produces 1.96 x 106 metric tons of jute biomass while Bangladesh produces 1.34 x 106 metric tons of jute biomass compared to the total production of jute biomass in the world (3.39 x 106 metric tons) in 20144. The utilization of this non-edible biomass will not conflict with food demand. Hence, it is beneficial to use it as a stock for synthesizing a variety of value added chemicals (xylose, arabinose, furfural, 5-hydroxymethylfurfural (HMF), etc.). According to the U.S. Department of Energy, furfural and HMF are considered as some of the top 30 building block chemicals derived from biomass5. Furfural is obtained from xylose or directly from hemicellulose and can be converted to many important chemicals. Furfuryl alcohol, methyl furan, and tetrahydrofuran are important chemicals obtained from furfural6. Hence, conversion of lignocellulosic biomass such as jute biomass into C5 sugars and other important chemicals is an important topic.
Extensive reports are available on the various catalytic methods for the conversion of lignocellulosic biomass into value added chemicals. The mineral acids (HCl and H2SO4) and heterogeneous catalysts (Amberlyst, HMOR, HUSY, SAPO-44, etc.) were used significantly for the conversion of hemicellulose and lignocellulosic biomass into sugars (pentose and hexose sugars) and furans (furfural and HMF)7,8. The reusability and corrosiveness of mineral acid is a major issue. However, with the solid acid catalyst, higher temperature and pressures are required because the reaction occurs at the surface of the catalyst. To overcome these issues, recently ILs are reported for the valorization of biomass as a catalyst or solvent9,10,11,12,13,14. The use of IL as a solvent is not a better method because of its higher cost and the lower vapor pressure of ILs that creates difficulty in product separation. Therefore, it is imperative to use recyclable IL as a catalyst (in small amounts) in a water solvent system for the biomass conversion to value added chemicals.
Here, we present a method to use 1-methyl-3-(3-sulfopropyl) imidazolium hydrogen sulfate acidic IL as the catalyst for the direct conversion of pentosan present in jute biomass into sugar monomers without any pretreatment. Commonly, ILs are reported for the pretreatment of lignocellulosic biomass10,15,16,17 whereas the very large quantity of ILs is used for the biomass pretreatment. Hence, it is always advantageous to use IL as the catalyst and to convert lignocellulosic biomass into chemicals without any additional treatment. Moreover, in the present work, the lignin concentration presented in jute biomass is calculated using Klason method which could be converted into various aromatic monomers18.

<table>
	<tbody>
		<tr>
			<td>1-Methylimidazole</td>
			<td>Sigma Aldrich</td>
			<td>M50834</td>
			<td></td>
		</tr>
		<tr>
			<td>1,3-Propanesultone</td>
			<td>Sigma Aldrich</td>
			<td>P50706</td>
			<td>Moisture sensitive</td>
		</tr>
		<tr>
			<td>p-nitroaniline</td>
			<td>Sigma Aldrich</td>
			<td>185310</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>J. T. Baker</td>
			<td>9460-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>Honeywell-Fluka</td>
			<td>30743</td>
			<td>Highly corrosive</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Honeywell-Fluka</td>
			<td>30719</td>
			<td>Highly corrosive</td>
		</tr>
		<tr>
			<td>1-butyl-3-methylimidazolium chloride</td>
			<td>Sigma Aldrich</td>
			<td>900856</td>
			<td>Highly hygroscopic</td>
		</tr>
		<tr>
			<td>D(+)-Xylose</td>
			<td>Acros Organics</td>
			<td>141001000</td>
			<td></td>
		</tr>
		<tr>
			<td>L(+)-Arabinose</td>
			<td>Acros Organics</td>
			<td>104981000</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Spectrometer</td>
			<td>JASCO</td>
			<td>V-670</td>
			<td></td>
		</tr>
		<tr>
			<td>Parr reactor</td>
			<td>Parr USA</td>
			<td>Seriese 4560</td>
			<td></td>
		</tr>
		<tr>
			<td>Parr reactor controller</td>
			<td>Parr USA</td>
			<td>Seriese 4848</td>
			<td></td>
		</tr>
		<tr>
			<td>High pressure liquid chromatography (HPLC)</td>
			<td>JASCO</td>
			<td>Seriese LC-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital hot plate stirrer</td>
			<td>Thermo Scientific</td>
			<td>SP142020-33Q Cimarec</td>
			<td></td>
		</tr>
		<tr>
			<td>Oven furnace</td>
			<td>Thermal Scientific</td>
			<td>FB1400 Thermolyne blast oven furnace</td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel method for the pentosan analysis present in jute biomass and its conversion into sugar monomers using acidic ionic liquid

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

The exact amount of pentosan and lignin recovered from the biomass depends on the type of lignocellulosic biomass. Similar types of lignocellulosic biomass collected from different places can have different concentration of pentosan and lignin. The jute biomass used in this study contains 20 wt% pentosan and 14 wt% lignin.
Figure 1 shows the comparison of the catalytic activity of ...

Watch this Scientific Journal Video about A Novel Method for the Pentosan Analysis Present in Jute Biomass and Its Conversion into Sugar Monomers Using Acidic Ionic Liquid at JoVE.com

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

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

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

The overall goal of this novel method is to convert pentosan present in jute biomass into C5 sugars using acidic ionic liquids.This method can also be used for the determination of pentosan concentrations present in the jute biomass.This measure enables the synthesis of C5 sugars from various low biomass in acerous media using a catalytic amount of acidic ionic liquid.The main advantage of this technique is that the acidic ionic liquid used for the conversion of jute biomass performs better compared to mineral acids.First have 7.625 millimoles of 1, 3-propane sultone to a cooled 50 milliliter round bottom flask and seal the flask with a rubber septum.Using a one milliliter syringe, slowly add 7.625 moles of 1-methylimidazole to the flask over 10 minutes at zero degrees Celsius.For the successful synthesis of this acidic ionic liquid the addition of 1, 3-propane sultone and 1-methylimidazole should be performed very slowly at zero degrees Celsius.Following this add 15 milliliters of dry toluene to the reaction mixture.After removing the ice water bath, reflux the reaction mixture or 16 hours at 120 degrees Celsius to obtain the solid zwitterion.After the reaction is complete separate the zwitterion from toluene using vacuum filtration.Then wash the zwitterion with 40 milliliters of toluene.For drying the zwitterion, set the temperature of an oven to 80 degrees celsius.Once the desired temperature is reached, transfer the zwitterion to a Petri dish and place in the oven to dry for four hours.After drying, transfer the dried zwitterion to a round bottom flask and add sulfuric acid using a 1, 000 microliter micropipet.Connect the round bottom flask to a reflux condenser.Then, heat the mixture under stirring at 110 degrees Celsius for 12 hours to obtain the desired ionic liquid.To prepare on liter of blank solution, add 10 milligrams of the para-nitroaniline indicator and distilled water to a 1 liter volumetric flask.Manually shake the solution thoroughly for two minutes, and set aside for one hour to fully mix the para-nitroaniline in water.To prepare the sample solution add 1.59 millimoles of H+ion of the acid catalyst to 50 milliliters of para-nitroaniline indicator solution and manual shake the mixture thoroughly for two minutes.Now perform a UV measurement of the blank and sample solutions and determine the maximum absorbance of para-nitroaniline.Add one gram of dried jute biomass and 15 milliliters of 72 weight percent sulfuric acid to a 50 milliliter vial.Stir the mixture on a hot plate at 30 degrees Celsius for two hours.Next add 150 milliliters of distilled water to a one liter round bottom flask and transfer the digested biomass sample to the flask.Wash the vial with 195 milliliters of water and transfer the water to the flask containing the digested biomass.Reflux the solution for four hours.Then cool the round bottom flask to room temperature.After 12 hours filter the solution using a G2 crucible to obtain the insoluble lignin and ash.Wash the insoluble solid with 150 milliliters of hot water to make it acid free.Dry the lignin and ash in the oven at 60 degrees Celsius for 16 hours, then transfer the solid to a Petri dish and further dry in the oven at 105 degrees Celsius for one hour.After drying place the sample in a desiccator to cool.Then weigh the cooled sample.Now perform the ash correction by heating the sample at 650 degrees Celsius for five hours in the presence of air.Determine the ash correction using the appropriate formula.Add two grams of oven dried jute biomass to a high pressure and high temperature batch reactor such as a 160 milliliter par reactor.Add 60 milliliters of water and 24 grams of acidic ionic liquid.Then close the reactor and increase the temperature to 160 degrees Celsius.Set the stirring speed to 200 RPM while the reactor is heating up to 160 degrees Celsius.Once the desired temperature is reached, increase the stirring speed to 600 RPM.After one hour, decrease the stirring speed to 200 RPM and stop the heating.Once the reactor is cool to room temperature stop the stirring and open the reactor.Separate the solid from the reaction mixture then analyze the reaction mixture using HPLC.Comparison of the catalytic activity of mineral acids and acidic ionic liquid for the conversion of jute biomass into C5 sugar is shown here.The catalytic activity is further compared with an ionic liquid without any Br

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Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

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

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

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

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

Pretreatment of Lignocellulosic Biomass with Low-cost Ionic Liquids

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

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

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

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial communities, a relatively precise but effort-intensive technique to assay microbial community composition is phospholipid fatty acid (PLFA) analysis. PLFA was developed to analyze phospholipid biomarkers, which can be used as indicators of microbial biomass and the composition of broad functional groups of fungi and bacteria. It has commonly been used to compare soils under alternative plant communities, ecology, and management regimes. The PLFA method has been shown to be sensitive to detecting shifts in microbial community composition.
An alternative method, fatty acid methyl ester extraction and analysis (MIDI-FA) was developed for rapid extraction of total lipids, without separation of the phospholipid fraction, from pure cultures as a microbial identification technique. This method is rapid but is less suited for soil samples because it lacks an initial step separating soil particles and begins instead with a saponification reaction that likely produces artifacts from the background organic matter in the soil. 
This article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. The method combines the accuracy achieved through PLFA profiling by extracting and concentrating soil lipids as a first step, and a reduction in effort by saponifying the organic material extracted and processing with the MIDI-FA method as a second step.

The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples.

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

Optimized Procedure for Determining the Adsorption of Phosphonates onto Granular Ferric Hydroxide using a Miniaturized Phosphorus Determination Method

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide (GFH), with little effort and high reliability. The phosphonate, e.g., nitrilotrimethylphosphonic acid (NTMP), is brought into contact with the GFH in a rotator in a solution buffered by an organic acid (e.g., acetic acid) or Good buffer (e.g., 2-(N-morpholino)ethanesulfonic acid) [MES] and N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid [CAPSO]) in a concentration of 10 mM for a specific time in 50 mL centrifuge tubes. Subsequently, after membrane filtration (0.45 &#181;m pore size), the total phosphorus (total P) concentration is measured using a specifically developed determination method (ISOmini). This method is a modification and simplification of the ISO 6878 method: a 4 mL sample is mixed with H2SO4 and K2S2O8 in a screw cap vial, heated to 148-150 &#176;C for 1 h and then mixed with NaOH, ascorbic acid and acidified molybdate with antimony(III) (final volume of 10 mL) to produce a blue complex. The color intensity, which is linearly proportional to the phosphorus concentration, is measured spectrophotometrically (880 nm). It is demonstrated that the buffer concentration used has no significant effect on the adsorption of phosphonate between pH 4 and 12. The buffers, therefore, do not compete with the phosphonate for adsorption sites. Furthermore, the relatively high concentration of the buffer requires a higher dosage concentration of oxidizing agent (K2S2O8) for digestion than that specified in ISO 6878, which, together with the NaOH dosage, is matched to each buffer. Despite the simplification, the ISOmini method does not lose any of its accuracy compared to the standardized method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide, with little effort and high reliability. In a buffered solution, the phosphonate is brought into contact with the adsorbent using a rotator and then analyzed via a miniaturized phosphorus determination method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric ...

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci

This paper reports the protocol for a rapid identification assay for Bemisia tabaci based on loop-mediated isothermal amplification (LAMP) technology. The protocol requires minimal laboratory training and can, therefore, be implemented on-site at points of entry for plant imports such as seaports and airports.

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many ...

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a significant increase and decrease in guard cell volume, respectively. Because shuttle transport of ions and water occurs between guard cells and larger neighboring epidermal cells during stomatal movement, the spaced distribution of plant stomata is considered an optimal distribution for stomatal movement. Experimental systems for perturbing the spaced pattern of stomata are useful to examine the spacing pattern's significance. Several key genes associated with the spaced stomatal distribution have been identified, and clustered stomata can be experimentally induced by altering these genes. Alternatively, clustered stomata can be also induced by exogenous treatments without genetic modification. In this article, we describe a simple induction system for clustered stomata in Arabidopsis thaliana seedlings by immersion treatment with a sucrose-containing medium solution. Our method is easy and directly applicable to transgenic or mutant lines. Larger chloroplasts are presented as a cell biological hallmark of sucrose-induced clustered guard cells. In addition, a representative confocal microscopic image of cortical microtubules is shown as an example of intracellular observation of clustered guard cells. The radial orientation of cortical microtubules is maintained in clustered guard cells as in spaced guard cells in control conditions.

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

The goal of this protocol is to demonstrate how to induce clustered stomata in cotyledons of Arabidopsis thaliana seedlings by immersion treatment with a sugar-containing medium solution and how to observe intracellular structures such as chloroplasts and microtubules in the clustered guard cells using confocal laser microscopy.

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a ...

Microalgae Cultivation and Biomass Quantification in a Bench-Scale Photobioreactor with Corrosive Flue Gases

Photobioreactors are illuminated cultivation systems for experiments on phototrophic microorganisms. These systems provide a sterile environment for microalgal cultivation with temperature, pH, and gas composition and flow rate control. At bench-scale, photobioreactors are advantageous to researchers studying microalgal properties, productivity, and growth optimization. At industrial scales, photobioreactors can maintain product purity and improve production efficiency. The video describes the preparation and use of a bench-scale photobioreactor for microalgal cultivation, including the safe use of corrosive gas inputs, and details relevant biomass measurements and biomass productivity calculations. Specifically, the video illustrates microalgal culture storage and preparation for inoculation, photobioreactor assembly and sterilization, biomass concentration measurements, and a logistic model for microalgal biomass productivity with rate calculations including maximum and overall biomass productivities. Additionally, since there is growing interest in experiments to cultivate microalgae using simulated or real waste gas emissions, the video will cover the photobioreactor equipment adaptations necessary to work with corrosive gases and discuss safe sampling in such scenarios.

Bench-scale, axenic cultivation facilitates microalgal characterization and productivity optimization before subsequent process scale-up. Photobioreactors provide the necessary control for reliable and reproducible microalgal experiments and can be adapted to safely cultivate microalgae with the corrosive gases (CO2, SO2, NO2) from municipal or industrial combustion emissions.

Photobioreactors are illuminated cultivation systems for experiments on phototrophic microorganisms. These systems provide a sterile environment for ...

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&ndash;Environment Interactions

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating a knowledge gap. To meet the challenge of improving crops to feed the growing global population, this gap must be bridged.
Physiological traits are considered key functional traits in the context of responsiveness or sensitivity to environmental conditions. Many recently introduced high-throughput (HTP) phenotyping techniques are based on remote sensing or imaging and are capable of directly measuring morphological traits, but measure physiological parameters mainly indirectly.
This paper describes a method for direct physiological phenotyping that has several advantages for the functional phenotyping of plant&#8211;environment interactions. It helps users overcome the many challenges encountered in the use of load-cell gravimetric systems and pot experiments. The suggested techniques will enable users to distinguish between soil weight, plant weight and soil water content, providing a method for the continuous and simultaneous measurement of dynamic soil, plant and atmosphere conditions, alongside the measurement of key physiological traits. This method allows researchers to closely mimic field stress scenarios while taking into consideration the environment&#8217;s effects on the plants&#8217; physiology. This method also minimizes pot effects, which are one of the major problems in pre-field phenotyping. It includes a feed-back fertigation system that enables a truly randomized experimental design at a field-like plant density. This system detects the soil-water-content limiting threshold (&#952;) and allows for the translation of data into knowledge through the use of a real-time analytic tool and an online statistical resource. This method for the rapid and direct measurement of the physiological responses of multiple plants to a dynamic environment has great potential for use in screening for beneficial traits associated with responses to abiotic stress, in the context of pre-field breeding and crop improvement.

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&amp;#8211;Environment Interactions

This high-throughput, telemetric, whole-plant water relations gravimetric phenotyping method enables direct and simultaneous real-time measurements, as well as the analysis of multiple yield-related physiological traits involved in dynamic plant&#8211;environment interactions.

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating ...

Estimation of Crystalline Cellulose Content of Plant Biomass using the Updegraff Method

Cellulose is the most abundant polymer on Earth generated by photosynthesis and the main load-bearing component of cell walls. The cell wall plays a significant role in plant growth and development by providing strength, rigidity, rate and direction of cell growth, cell shape maintenance, and protection from biotic and abiotic stressors. The cell wall is primarily composed of cellulose, lignin, hemicellulose and pectin. Recently plant cell walls have been targeted for the second-generation biofuel and bioenergy production. Specifically, the cellulose component of the plant cell wall is used for the production of cellulosic ethanol. Estimation of cellulose content of biomass is critical for fundamental and applied cell wall research. The Updegraff method is simple, robust, and the most widely used method for the estimation of crystalline cellulose content of plant biomass. The alcohol insoluble crude cell wall fraction upon treatment with Updegraff reagent eliminates the hemicellulose and lignin fractions. Later, the Updegraff reagent resistant cellulose fraction is subjected to sulfuric acid treatment to hydrolyze the cellulose homopolymer into monomeric glucose units. A regression line is developed using various concentrations of glucose and used to estimate the amount of the glucose released upon cellulose hydrolysis in the experimental samples. Finally, the cellulose content is estimated based on the amount of glucose monomers by colorimetric anthrone assay.

The Updegraff method is the most widely used method for the cellulose estimation. The main purpose of this demonstration is to provide a detailed Updegraff protocol for estimation of cellulose content in plant biomass samples.

Cellulose is the most abundant polymer on Earth generated by photosynthesis and the main load-bearing component of cell walls. The cell wall plays a ...