This work was supported by grants from DOE Great Lakes Bioenergy Research Center (DOE BER office of Science DE-FC02-07ER64494). We are grateful to Dr. Xudong Zhang and his group members for helpful technical discussions and invaluable comments on finalizing the protocol.

1. Sample Preparation and Acid Extraction
<ol>
	<li>Freeze-dry soil samples after field collection.</li>
	<li>Grind and homogenize soil samples using a ball mill, soil grinder, or a mortar and pestle.</li>
	<li>Weigh soil samples (containing &gt; 0.3 mg N) into a 25 mL hydrolysis flask.</li>
	<li>Add 10 mL 6M HCl into each hydrolysis flask, fill N2 gas in the flasks, and cap tightly.</li>
	<li>Hydrolyze at 105 &deg;C in an incubator for 8 hours using an auto timer switch.</li>
</ol>
2. Sample Purification
<ol>
	<li>Remove the flasks from the incubator; cool to room temperature.</li>
	<li>Add 100 &mu;L internal standard myo-inositol (1 mg mL-1 in water) to each flask; mix by swirling.</li>
	<li>Set up plastic funnels draining into 200 mL pear shaped flasks with ST/NS 24/40 joints, set on plastic cups for stability.</li>
	<li>Fold Whatman #2 Qualitative Circles (11 cm diameter) into quarters and set into funnels.</li>
	<li>Swirl each hydrolysis flask, and pour slurry into funnel to filter (you may further rinse each flask with ~3 mL of water).</li>
	<li>Dry the filtrate using a rotary evaporator at ~45 &deg;C, applying vacuum.</li>
	<li>Resuspend the dried residue from each pear flask with 3~5 mL water (use ultrasonic bath if desired), and pour into a 40 mL Teflon tube; rinse the flask with a second aliquot of water.</li>
	<li>Adjust the pH to 6.6 - 6.8 using 1M KOH solution to precipitate metal ions and other organic molecules.</li>
	<li>Remove the precipitates by centrifugation at 2000 rcf for 10 minutes.</li>
	<li>Pour the supernatant into a 40 mL glass tube, cover the tube opening with parafilm, freeze at -20 &deg;C, then poke holes in the parafilm.</li>
	<li>Freeze-dry the frozen supernatant to remove all liquid.</li>
	<li>Dissolve the residue with 3 mL dry methanol, vortexing thoroughly (use ultrasonic bath if desired); cap the tube, and then centrifuge at 2000 rcf for 10 minutes to settle out salts.</li>
	<li>Transfer the supernatant to a 3 mL conical reaction vial; evaporate to dryness by RapidVap machine at 45 &deg;C (or under a gentle stream of dry nitrogen gas if desired).</li>
	<li>To each vial, add 100 &mu;L recovery standard N-methylglucamine (1 mg mL-1 in water) and 1 mL H2O, cover the vial mouths with parafilm, freeze at -20 &deg;C, perforate the parafilm, and then freeze dry.</li>
	<li>Make standards: add 100 &mu;L muramic acid (0.5 mg mL-1 in methanol), 100 &mu;L glucosamine (1 mg mL-1 in water), 100 &mu;L myo-inositol (1 mg mL-1 in water), 100 &mu;L N-methylglucamine (1 mg mL-1 in water), and 1 mL H2O, parafilm each vial, freeze at -20 &deg;C, perforate the parafilm, then freeze-dry.</li>
</ol>
3. Derivatization
<ol>
	<li>Prepare derivatization reagent containing 32 mg mL-1 hydroxylamine hydrochloride and 40 mg mL-1 4-dimethylamino-pyridine in pyridine-methanol (4:1 v/v).</li>
	<li>Add 300 &mu;L of the derivatization reagent to each of the reactivials, cap tightly, and vortex thoroughly.</li>
	<li>Put the vials in 75-80 &deg;C water bath for 35 minutes (well sealed the vials to avoid allowing any water to enter the vials).</li>
	<li>Remove vials from the water bath, and cool to room temperature.</li>
	<li>Add 1 mL acetic anhydride to each of the 3 mL reactivials, cap tightly, and vortex thoroughly, then reheat in 75-80 &deg;C water bath for 25 minutes.</li>
	<li>Remove vials from the water bath, and cool to room temperature.</li>
</ol>
4. Separation and Measurement
<ol>
	<li>Add 1.5 mL dichloromethane to each vial, cap tightly, and vortex thoroughly.</li>
	<li>Add 1 mL 1M HCl to each vial, cap tightly and vortex thoroughly to allow the solutions to sit undisturbed until the two phrases separate, aspirate and discard the top (aqueous) phase using 1000 &mu;L pipettor.</li>
	<li>In the same fashion, extract the organic phase 3 times but with 1 mL H2O (with the last washing step, take special care to ensure that all of the aqueous top phase has been removed).</li>
	<li>Dry the final solution using RapidVap at 45 &deg;C (or dry using nitrogen gas if desired).</li>
	<li>Dissolve in 300 &mu;L ethyl acetate-hexane (1:1 v/v) and transfer to 2 mL amber screw-cap vials with a small-volume insert and cap tightly.</li>
	<li>For quantification, analyze by GC-FID using a fused silica nonpolar capillary column: 30 m long, 0.25 mm i.d., 0.25 um film thickness; stationary phase 5% phenyl-, 95% methyl-polysiloxane (DB-5 or equivalent) with hydrogen or helium as the carrier gas, at 0.5 mL min-1 constant flow. The chromatography is better on premium "inert" phases and hydrogen carrier gas, but is acceptable on the more common varieties and with helium. The detector settings are 300 &deg;C; 400 mL min-1 air and 30 mL min-1 for both the nitrogen and hydrogen makeup gases. The injection and oven parameters are as follows: 1 &mu;L split injection (30:1) with the GC inlet set at 250 &deg;C; initial oven temperature, 120 &deg;C; hold 1 min.; increase the oven temperature at 10 &deg;C min-1 to 250 &deg;C; hold 2.5 min.; ramp to 270 &deg;C at 20 &deg;C min-1; hold 2 minutes; ramp to 290 &deg;C at 20 &deg;C min-1, hold 5 min. Adjust the carrier gas flow rate so that the inositol, glucosamine and muramic acid derivatives elute at 250 &deg;C, and the N-methylglucamine elutes at 270 &deg;C.</li>
</ol>
5. Derivative Validation
<ol>
	<li>Use soft ionization LC-ESI-TOF-MS to identify the molecular ion and determine the molecular weight of the derivative.</li>
	<li>Use GC-EI-MS-SIM or GC-EI-MSMS for sensitivity enhancement to investigate targeted ions of the derivative.</li>
	<li>Use multiple isotopically labeled reagents for preparation of the derivatives, and then use the mass shift of those isotopically labeled derivatives in MS to investigate molecular ion and ion fragments.</li>
</ol>
6. Representative Results
The protocol of the method mainly comprises four steps: acid digestion, sample purification, derivatization and GC determination (Figure 1). An example of the analysis for glucosamine and muramic acid from standard stocks and from a soil sample is shown in Figure 2. Besides glucosamine and muramic acid, mannosamine and galactosamine (two isomers of glucosamine) can also be determined simultaneously using the method. Based on response factors of standards with respect to the internal standard myo-inositol, we can quantify these biomarkers in soil samples. The recovery standard has been used to qualitatively monitor the derivatization process. The schemes for formation of the aldononitrile acetate derivatized glucosamine and muramic acid are shown in Figure 3.
The proposed structures of the derivatives were determined by GC-EIMS-SIM for sensitivity enhancement, or soft ionization LC-ESI-TOF-MS8; the proposed structures of the ion fragments formed upon electron ionization were studied by comparing the ion spectra of the samples prepared with various isotope incorporations11. The Figure 4 shows the mass shift of the dominant ion m/z 187 of aldononitrile acetate derivatized glucosamine in three scenarios, prepared with non-labeled agents, D-acetic anhydrite, and U-13C-glucosamine. Other detailed information and explanations can be referred to our recent publications8, 11. This strategy could serve as a model to study derivative chemistry.
<img alt="Figure 1" src="/files/ftp_upload/3767/3767fig1.jpg" /> 
Figure 1. Measurement flow chart of the protocol for analysis of glucosamine and muramic acid in soil samples. The protocol mainly contains 4 steps: Acid digestion, Purification, Derivatization and GC determination.
<img alt="Figure 2" src="/files/ftp_upload/3767/3767fig2.jpg" /> 
Figure 2. GC-FID chromatograms of aldononitrile acetates of inositol, glucosamine, muramic acid, mannosamine, galactosamine and methylglucamine for standards and soil.
<img alt="Figure 3" src="/files/ftp_upload/3767/3767fig3.jpg" /> 
Figure 3. Schemes for formation of the aldononitrile acetate derivatized glucosamine and muramic acid. The number 1 represents nitrile reaction. The number 2 represents acetylation.
<img alt="Figure 4" src="/files/ftp_upload/3767/3767fig4.jpg" /> 
Figure 4. Mass spectra of aldononitrile acetate derivatized glucosamine associated with the dominant ion structures under EI mode prepared with (A) non-labeled agents, (B) D-acetic anhydrite, (C) U-13C-glucosamine. Star represents heavy isotope atom or isotope group. Please <a href="https://www.jove.com/files/ftp_upload/3767/3767fig4_lg.jpg">click here</a> to see a larger version of this figure.

<ol>
	<li>Zelles, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid+patterns+of+phospholipids+and+lipopolysaccharides+in+the+characterisation+of+microbial+communities+in+soil:+a+review.">Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review.</a> Biology and Fertility of Soils. 29, 111-129 (1999).</li><li>Kirk, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+studying+soil+microbial+diversity.">Methods of studying soil microbial diversity.</a> Journal of Microbiological Methods. 58, 169-188 (2004).</li><li>Joergensen, R. G., Emmerling, C. <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+evaluating+human+impact+on+soil+microorganisms+based+on+their+activity,+biomass,+and+diversity+in+agricultural+soils.">Methods for evaluating human impact on soil microorganisms based on their activity, biomass, and diversity in agricultural soils.</a> Journal of Plant Nutrition and Soil Science. 169, 295-309 (2006).</li><li>Guggenberger, G., Frey, S. D., Six, J., Paustian, K., Elliott, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+and+fungal+cell-wall+residues+in+conventional+and+no-tillage+agroecosystems.">Bacterial and fungal cell-wall residues in conventional and no-tillage agroecosystems.</a> Soil Science Society of America Journal. 63, 1188-1198 (1999).</li><li>Amelung, W., Lal, R., Kimble, J. M., Follett, R. F., Stewart, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Assessment Methods for Soil Carbon. , 233-270 (2001).</li><li>Guerrant, G. O., Moss, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+monosaccharides+as+aldononitrile,+O-methyloxime,+alditol,+and+cyclitol+acetate+derivatives+by+gas-chromatography.">Determination of monosaccharides as aldononitrile, O-methyloxime, alditol, and cyclitol acetate derivatives by gas-chromatography.</a> Analytical Chemistry. 56, 633-638 (1984).</li><li>Zhang, X., Amelung, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+chromatographic+determination+of+muramic+acid,+glucosamine,+mannosamine,+and+galactosamine+in+soils.">Gas chromatographic determination of muramic acid, glucosamine, mannosamine, and galactosamine in soils.</a> Soil Biology and Biochemistry. 28, 1201-1206 (1996).</li><li>Liang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+molecular+ion+structure+for+aldononitrile+acetate+derivatized+muramic+acid.">Investigation of the molecular ion structure for aldononitrile acetate derivatized muramic acid.</a> Journal of Microbiological Methods. 86, 224-230 (2011).</li><li>He, H., Xie, H., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+GC/MS+technique+to+assess+15N+and+13C+incorporation+into+soil+amino+sugars.">A novel GC/MS technique to assess 15N and 13C incorporation into soil amino sugars.</a> Soil Biology and Biochemistry. 38, 1083-1091 (2006).</li><li>Glaser, B., Gross, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compound-specific+delta+13C+analysis+of+individual+amino+sugars+-+a+tool+to+quantify+timing+and+amount+of+soil+microbial+residue+stabilization.">Compound-specific delta 13C analysis of individual amino sugars - a tool to quantify timing and amount of soil microbial residue stabilization.</a> Rapid Communications in Mass Spectrometry. 19, 1409-1416 (2005).</li><li>Liang, C., Balser, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric+characterization+of+amino+sugar+aldononitrile+acetate+derivatives+used+for+isotope+enrichment+assessment+of+microbial+residues.">Mass spectrometric characterization of amino sugar aldononitrile acetate derivatives used for isotope enrichment assessment of microbial residues.</a> Soil Biology and Biochemistry. 42, 904-909 (2010).</li><li>Liang, C., Pedersen, J. A., Balser, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aminoglycoside+antibiotics+may+interfere+with+microbial+amino+sugar+analysis.">Aminoglycoside antibiotics may interfere with microbial amino sugar analysis.</a> Journal of Chromatography A. 1216, 5296-5301 (2009).</li></ol>

We have nothing to disclose.

The presented GC-based method for the analysis of aldononitrile acetate derivatized glucosamine and muramic acid provides a relatively rapid method to quantify these amino sugars, extracted from soil. The derivatives are chemically stable, and can be determined in one analysis. The method is not restricted to soil samples, and can be simplified for samples from non-soil matrix.
The vacuum pump used in this method is built to be resistant to acid. We further suggest setting up a base trap to p...

<table><tbody><tr><td>Muramic acid</td><td>Sigma-Aldrich</td><td>M2503-25MG</td><td></td></tr><tr><td>D-(+)-glucosamine hydrochloride</td><td>Sigma-Aldrich</td><td>G1514-100G</td><td></td></tr><tr><td>N-methyl-D-glucamine</td><td>Sigma-Aldrich</td><td>M2004-500G</td><td></td></tr><tr><td>Myo-inositol</td><td>Fisher Scientific</td><td>A307003G025</td><td></td></tr><tr><td>Methanol (dry)</td><td>Acros Organics</td><td>AC326950010</td><td></td></tr><tr><td>4-dimethylamino-pyridine</td><td>Acros Organics</td><td>AC148270050</td><td></td></tr><tr><td>Ethyl acetate</td><td>VWR international</td><td>BJGC100-4</td><td></td></tr><tr><td>Hydroxlamine hydrochloride </td><td>Fisher Scientific</td><td>H330-100</td><td></td></tr><tr><td>Pyridine</td><td>Fisher Scientific</td><td>P368-500</td><td></td></tr><tr><td>Acetic anhydride</td><td>Fisher Scientific</td><td>A10-100</td><td></td></tr><tr><td>Dichloromethane (Methylene chloride)</td><td>Fisher Scientific</td><td>D37-500</td><td></td></tr><tr><td>Hexane</td><td>Fisher Scientific</td><td>H303-4</td><td></td></tr><tr><td>Hydrochloric acid (6M)</td><td>Fisher Scientific</td><td>S456-4</td><td></td></tr><tr><td>Hydroxylamine hydrochloride&lt;sup&gt;-15&lt;/sup&gt;N</td><td>Icon services</td><td>IN5280</td><td></td></tr><tr><td>Acetic anhydride&lt;sup&gt;-2&lt;/sup&gt;H (D&lt;sub&gt;6&lt;/sub&gt;C&lt;sub&gt;4&lt;/sub&gt;O&lt;sub&gt;3&lt;/sub&gt;)</td><td>Acros Organics</td><td>AC174670050</td><td></td></tr><tr><td>D-glucose-U&lt;sup&gt;-13&lt;/sup&gt;C</td><td>Cambridge Isotope Laboratories</td><td>CLM-1396-1</td><td></td></tr><tr><td>Ammonium sufate&lt;sup&gt;-15&lt;/sup&gt;N </td><td>Cambridge Isotope Laboratories</td><td>NLM-713-1</td><td></td></tr><tr><td>Rapid-Vap</td><td>Labconco Corp.</td><td>790002</td><td></td></tr><tr><td>Vacum pump</td><td>KNF Neuberger</td><td>D-79112</td><td></td></tr><tr><td>Hydrolysis flask</td><td>Fisher Scientific</td><td>06 423A</td><td></td></tr><tr><td>Derivatization microvial</td><td>Fisher Scientific</td><td>06-100E</td><td></td></tr><tr><td>GC</td><td>Hewlett-Packard</td><td>6890</td><td></td></tr><tr><td>MS</td><td>Hewlett-Packard</td><td>5972</td><td></td></tr><tr><td>LC-ESI-TOF-MS</td><td>Agilent Technologies</td><td>An Agilent 1200 series HPLC system coupled to an Agilent LC/MSD-TOF</td><td></td></tr></tbody></table>

gc-based detection of aldononitrile acetate derivatized glucosamine and muramic acid for microbial residue determination in soil

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. Current strategies for microbial analysis include both traditional laboratory culture-dependent techniques and those based on direct extraction and determination of certain biomarkers1, 2. Few among the diversity of microbial species inhabiting soil can be cultured, so culture-dependent methods introduce significant biases, a limitation absent in biomarker analysis.
The glucosamine, mannosamine, galactosamine and muramic acid have been well served as measures of both the living and dead microbial mass, of these the glucosamine (most abundant) and muramic acid (uniquely from bacterial cell) are most important constituents in the soil systems3, 4. However, the lack of knowledge on the analysis restricts the wide popularization among scientific peers. Among all existing analytical methods, derivatization to aldononitrile acetates followed by GC-based analysis has emerged as a good option with respect to optimally balancing precision, sensitivity, simplicity, good chromatographic separation, and stability upon sample storage5.
Here, we present a detailed protocol for a reliable and relatively simple analysis of glucosamine and muramic acid from soil after their conversion to aldononitrile acetates. The protocol mainly comprises four steps: acid digestion, sample purification, derivatization and GC determination. The step-by-step procedure is modified according to former publications6, 7. In addition, we present a strategy to structurally validate the molecular ion of the derivative and its ion fragments formed upon electron ionization. We applied GC-EI-MS-SIM, LC-ESI-TOF-MS and isotopically labeled reagents to determine the molecular weight of aldononitrile acetate derivatized glucosamine and muramic acid; we used the mass shift of isotope-labeled derivatives in the ion spectrum to investigate ion fragments of each derivatives8. In addition to the theoretical elucidation, the validation of molecular ion of the derivative and its ion fragments will be useful to researchers using &delta;13C or ion fragments of these biomarkers in biogeochemical studies9, 10.

Watch this Scientific Journal Video about GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil at JoVE.com

GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

We describe a method protocol for the GC-based analysis of the aldonitrile acetate derivatives of glucosamine and muramic acid extracted from soil. For elucidation of the chemical mechanism, we also present a strategy to confirm the structure of the derivative and the ion fragments formed upon electron ionization.

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. ...

gc-based-detection-aldononitrile-acetate-derivatized-glucosamine

Research

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Biology

16.8K Views.  University of Wisconsin, Madison. We describe a method protocol for the GC-based analysis of the aldonitrile acetate derivatives of glucosamine and muramic acid extracted from soil. For elucidation of the chemical mechanism, we also present a strategy to confirm the structure of the derivative and the ion fragments formed upon electron ionization.

Video: GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

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

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

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

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

Environment

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is not an appropriate measure for its availability; bioavailability rather depends on the dynamic interplay of potential mass transfer (flux) of a compound to a microbial cell and the capacity of the latter to degrade the compound. In water-unsaturated parts of the soil, mycelia have been shown to overcome bioavailability limitations by actively transporting and mobilizing organic compounds over the range of centimeters. Whereas the extent of mycelia-based transport can be quantified easily by chemical means, verification of the contaminant-bioavailability to bacterial cells requires a biological method. Addressing this constraint, we chose the PAH fluorene (FLU) as a model compound and developed a water unsaturated model microcosm linking a spatially separated FLU point source and the FLU degrading bioreporter bacterium Burkholderia sartisoli RP037-mChe by a mycelial network of Pythium ultimum. Since the bioreporter expresses eGFP in response of the PAH flux to the cell, bacterial FLU exposure and degradation could be monitored directly in the microcosms via confocal laser scanning microscopy (CLSM). CLSM and image analyses revealed a significant increase of the eGFP expression in the presence of P. ultimum compared to controls without mycelia or FLU thus indicating FLU bioavailability to bacteria after mycelia-mediated transport. CLSM results were supported by chemical analyses in identical microcosms. The developed microcosm proved suitable to investigate contaminant bioavailability and to concomitantly visualize the involved bacteria-mycelial interactions.

A whole cell bioreporter assay with Burkholderia sartisoli RP037-mChe was developed to detect fractions of an organic contaminant (i.e., fluorene) available for bacterial degradation after active transport by mycelia bridging air-filled pores in a water unsaturated model system.

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is ...

Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

In recent years, the carbohydrate moieties of plants have received considerable attention, as they are a potential source of cross-reactive, allergy-provoking immune responses. In addition, carbohydrate structures also play a critical role in plant metabolism. Here, we present a simple and rapid method for preparing and analyzing N-glycans from different cultivars of radish (Raphanus sativus) using an N-glycanase specific for the release of plant-derived carbohydrate structures. To achieve this, crude trichloroacetic acid precipitates of radish homogenates were treated with PNGase H+, and labeled using 2-aminobenzamide as a fluorescent tag. The labeled N-glycan samples were subsequently analyzed by ultra performance liquid chromatography (UPLC) separation and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for a detailed structural evaluation and to quantify relative abundancies of the radish-derived N-glycan structures. This protocol can also be used for the analysis of N-glycans from various other plant species, and may be useful for further investigation of the function and effects of N-glycans on human health.

Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

We describe a simple and rapid method for the preparation and analysis of N-glycans from different cultivars of radish (Raphanus sativus).

In recent years, the carbohydrate moieties of plants have received considerable attention, as they are a potential source of cross-reactive, ...

Biochemistry

Enhanced Oil Recovery using a Combination of Biosurfactants

Biosurfactants are surface-active compounds capable of reducing the surface tension between two phases of different polarities. Biosurfactants have been emerging as promising alternatives to chemical surfactants due to less toxicity, high biodegradability, environmental compatibility and tolerance to extreme environmental conditions. Here, we illustrate the methods used for screening of microbes capable of producing biosurfactants. The biosurfactant producing microbes were identified using drop collapse, oil spreading, and emulsion index assays. Biosurfactant production was validated by determining the reduction in surface tension of the media due to growth of the microbial members. We also describe the methods involved in characterization and identification of biosurfactants. Thin layer chromatography of the extracted biosurfactant followed by differential staining of the plates was performed to determine the nature of the biosurfactant. LCMS, 1H NMR, and FT-IR were used to chemically identify the biosurfactant. We further illustrate the methods to evaluate the application of the combination of produced biosurfactants for enhancing residual oil recovery in a simulated sand pack column.

We illustrate the methods involved in screening and identification of the biosurfactant producing microbes. Methods for chromatographic characterization and chemical identification of the biosurfactants, determining the industrial applicability of the biosurfactant&#160;in enhancing residual oil recovery are also presented.

Biosurfactants are surface-active compounds capable of reducing the surface tension between two phases of different polarities. Biosurfactants have been ...

The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides

Protein glycosylation is increasingly recognized as a common modification within bacterial organisms, contributing to prokaryotic physiology and optimal infectivity of pathogenic species. Due to this, there is increasing interest in characterizing bacterial glycosylation and a need for high-throughput analytical tools to identify these events. Although bottom-up proteomics readily enables the generation of rich glycopeptide data, the breadth and diversity of glycans observed in prokaryotic species make the identification of bacterial glycosylation events extremely challenging.
Traditionally, the manual determination of glycan compositions within bacterial proteomic datasets made this a largely bespoke analysis restricted to field-specific experts. Recently, open searching-based approaches have emerged as a powerful alternative for the identification of unknown modifications. By analyzing the frequency of unique modifications observed on peptide sequences, open searching techniques allow the identification of common glycans attached to peptides within complex samples. This article presents a streamlined workflow for the interpretation and analysis of glycoproteomic data, demonstrating how open searching techniques can be used to identify bacterial glycopeptides without prior knowledge of the glycan compositions.
Using this approach, glycopeptides within samples can rapidly be identified to understand glycosylation differences. Using Acinetobacter baumannii as a model, these approaches enable the comparison of glycan compositions between strains and the identification of novel glycoproteins. Taken together, this work demonstrates the versatility of open database-searching techniques for the identification of bacterial glycosylation, making the characterization of these highly diverse glycoproteomes easier than ever before.

The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides

Open searching enables the identification of glycopeptides decorated with previously unknown glycan compositions. Within this article, a streamlined approach for undertaking open searching and subsequent glycan-focused glycopeptide searches are presented for bacterial samples using Acinetobacter baumannii as a model.

Protein glycosylation is increasingly recognized as a common modification within bacterial organisms, contributing to prokaryotic physiology and optimal ...

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 ...

Chemistry

Trimethylamine Detection in Animal-Derived Medicine by Headspace GC-MS

An Improved Technique for Trimethylamine Detection in Animal-Derived Medicine by Headspace Gas Chromatography-Tandem Quadrupole Mass Spectrometry

Animal-derived medicines have distinctive characteristics and significant curative effects, but most of them have an obvious fishy odor, resulting in the poor compliance of clinical patients. Trimethylamine (TMA) is one of the key fishy odor components in animal-derived medicine. It is difficult to identify TMA accurately using the existing detection method due to the increased pressure in the headspace vial caused by the rapid acid-base reaction after the addition of lye, which causes TMA to escape from the headspace vial, stalling the research progress of the fishy odor of animal-derived medicine. In this study, we proposed a controlled detection method that introduced a paraffin layer as an isolation layer between acid and lye. The rate of TMA production could be effectively controlled by slowly liquefying the paraffin layer through thermostatic furnace heating. This method showed satisfactory linearity, precision experiments, and recoveries with good reproducibility and high sensitivity. It provided technical support for the deodorization of animal-derived medicine.

Author Spotlight: An Improved Technique for Trimethylamine Detection in Animal-Derived Medicine by Headspace Gas Chromatography-Tandem Quadrupole Mass Spectrometry  

Here, a headspace gas chromatography-tandem quadrupole mass spectrometry (HS-GC-MS/MS) method suitable for the determination of trimethylamine (TMA) in animal-derived medicines is described. The protocol includes sample pretreatment, headspace treatment, analysis conditions, methodological validation, and the determination of TMA in animal-derived medicines.

Animal-derived medicines have distinctive characteristics and significant curative effects, but most of them have an obvious fishy odor, resulting in the ...

Workflow Based on the Combination of Isotopic Tracer Experiments to Investigate Microbial Metabolism of Multiple Nutrient Sources

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods substantially contributes to providing extensive knowledge of the metabolism of microorganisms growing in chemically defined media containing unique nitrogen and carbon sources. In contrast, the management through metabolism of multiple nutrient sources, despite their broad presence in natural or industrial environments, remains virtually unexplored. This situation is mainly due to the lack of suitable methodologies, which hinders investigations.
We report an experimental strategy to quantitatively and comprehensively explore how metabolism operates when a nutrient is provided as a mixture of different molecules, i.e., a complex resource. Here, we describe its application for assessing the partitioning of multiple nitrogen sources through the yeast metabolic network. The workflow combines information obtained during stable isotope tracer experiments using selected 13C- or 15N-labeled substrates. It first consists of parallel and reproducible fermentations in the same medium, which includes a mixture of N-containing molecules; however,a selected nitrogen source is labeled each time. A combination of analytical procedures (HPLC, GC-MS) is implemented to assess the labeling patterns of targeted compounds and to quantify the consumption and recovery of substrates in other metabolites. An integrated analysis of the complete dataset provides an overview of the fate of consumed substrates within cells. This approach requires an accurate protocol for the collection of samples&#8211;facilitated by a robot-assisted system for online monitoring of fermentations&#8211;and the achievement of numerous time-consuming analyses. Despite these constraints, it allowed understanding, for the first time, the partitioning of multiple nitrogen sources throughout the yeast metabolic network. We elucidated the redistribution of nitrogen from more abundant sources toward other N-compounds and determined the metabolic origins of volatile molecules and proteinogenic amino acids.

This protocol describes an experimental procedure to quantitatively and comprehensively investigate the metabolism of multiple nutrient sources. This workflow, based on a combination of isotopic tracer experiments and an analytical procedure, allows the fate of consumed nutrients and the metabolic origin of molecules synthetized by microorganisms to be determined.

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods ...

Bioengineering

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

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