We would like to thank Jim McMurray for providing the black liquor and Dr. David Tilotta and August Meng for their help with GC-MS. Support for Stephanie L. Mathews was provided by a USDA National Needs Fellowship (award number 2010-38420-20399).

1. Collection of Black Liquor and Preparation of Black Liquor for Growth Cultures
<ol>
	<li>Collect a black liquor sample from the outlet valve attached to the kraft digester in a sterile glass bottle and allow it to cool to room temperature before proceeding with the following steps. 
	Neutralization</li>
	<li>Add 100 ml of black liquor sample to a 500 ml beaker equipped with a stir bar.</li>
	<li>Place the beaker on a stir plate and adjust the speed to medium.</li>
	<li>Slowly add drops of phosphoric acid until the black liquor becomes viscous (viscosity similar to glycerol).</li>
	<li>Aliquot all of the black liquor solution by slowly pouring into 50 ml conical tubes and centrifuge at 9,300 x g for 30 min.</li>
	<li>Place the supernatant into the beaker and measure the pH using pH test ribbons. The pellet of precipitated lignin can be discarded and treated as nonhazardous solid waste.</li>
	<li>Continue to add phosphoric acid and centrifuge the solution until the pH strips indicate a neutral pH of the supernatant. 
	Sterilization</li>
	<li>Pass the solution through a 0.22 &#181;m filter to remove any contaminating bacteria and place the filtered solution into a sterile container for storage. Store at room temperature.</li>
</ol>
2. Lignocellulose Extraction
Carbohydrate Separation
<ol>
	<li>Place 5 g of switchgrass, pine, or Bermuda grass (milled to 1 mm or smaller particle size) into a 500 ml Erlenmeyer flask. Then add 200 ml of distilled or deionized water, 7.5 g of NaClO2, 2.5 ml of glacial acetic acid,&#160;and a stir bar to the flask.</li>
	<li>Place the flask in an oil bath placed on top of a stir plate at 80 &#176;C for 1 hr. Adjust the speed of the stir plate to medium.</li>
	<li>Add an additional 7.5 g of NaClO2&#160;and 2.5 ml of glacial acetic acid to the flask. Continue stirring at 80 &#176;C in the oil bath for 30 min.</li>
	<li>Repeat the previous step 2x (Note: a total of 30 g of NaClO2 and 10 ml of glacial acetic acid is added to the flask containing the lignocellulosic biomass). Cool the solution to room temperature.</li>
	<li>Place a sheet of filter paper into a B&#252;chner funnel. Place the funnel on top of a side arm flask and attach it to a vacuum.</li>
	<li>Slowly pour the solution into the funnel.</li>
	<li>Wash the solid material 3x with 100 ml of distilled or deionized water. Discard the filtrate after washing the solid material.</li>
	<li>Reserve 1 g of the solid material and place it onto a clean sheet of filter paper.</li>
	<li>Dry this solid material overnight in a 50 &#176;C oven. After removing the sample from the oven, place into a storage vial labeled &#8216;holocellulose&#8217;.</li>
	<li>Add the remaining solid material to 250 ml of 10% NaOH (v/v) in a 500 ml Erlenmeyer flask. Cap the flask with a stopper.</li>
	<li>Place the solution in an incubator at 70 &#176;C for 1 hr shaking at 250 rpm.</li>
	<li>Cool the solution for 30 min&#160;at room temperature.</li>
	<li>Remove the solid material using filter paper and B&#252;chner funnel attached to a side arm flask.</li>
	<li>Transfer the solid material on a clean sheet of filter paper to a 50 &#176;C oven and allow it to dry overnight.</li>
	<li>After the residue has dried, place it in a storage vial and label it &#8216;cellulose&#8217;.</li>
	<li>Transfer the filtrate from step 2.13 to a 500 ml Erlenmeyer flask and add 30 ml of acetic acid and 250 ml of isopropyl alcohol. Cap the flask and place the solution on the bench top for at least 8 hr at room temperature.</li>
	<li>Transfer all of the solution to 50 ml conical tubes and centrifuge the solution at 9,300 x g for 30 min. Carefully pipette the solution out of the conical tube and discard.</li>
	<li>Resuspend the pellet in 30 ml of distilled or deionized water by vortexing.</li>
	<li>Repeat steps 2.17-2.18 15x to thoroughly wash the pellet.</li>
	<li>Remove the supernatant and save the pellet for freeze drying. Once freeze-dried, collect the sample and place it in a vial labeled &#8216;hemicellulose&#8217;. 
	Lignin separation 
	Lignin separation protocol based on Karaaslan et al.12</li>
	<li>Add 5 g of switchgrass, pine, or Bermuda grass (milled to 1 mm or smaller particle size) to 500 ml of a solution of 0.25 M NaOH and&#160;30% ethanol. Place the slurry in an incubator at 75 &#176;C with shaking at 250 rpm for 2 hr.</li>
	<li>Cool the solution to room temperature.</li>
	<li>Place a sheet of filter paper in a B&#252;chner funnel on top of a side arm flask. Attach to a vacuum and slowly pour the solution into the funnel.</li>
	<li>Discard the solid material.</li>
	<li>Add a stir bar to the flask containing the filtrate and place the flask on top of a stir plate adjusted to medium speed. Add concentrated hydrochloric acid dropwise to the solution until a pH of 2.0 is obtained (approximately 20 ml).</li>
	<li>Allow the acidified solution to sit overnight at room temperature.</li>
	<li>Centrifuge the solution at 9,300 x g for 30 min&#160;in conical tubes. Carefully pipette the solution out of the conical tube and discard.</li>
	<li>Resuspend the pellet in 30 ml of distilled or deionized water by vortexing.</li>
	<li>Repeat steps 2.27-2.28 15x to thoroughly wash the pellet.</li>
	<li>After centrifugation, remove the supernatant and save the pellet for freeze drying. Once freeze-dried, place the sample in a vial labeled &#8216;lignin&#8217;.</li>
</ol>
3. Preparation of Microbial Growth Cultures
Agar Plates
<ol>
	<li>Add 0.2% (w/v) sterile lignocellulose extraction products to M9 minimal media13 agar before pouring plates.</li>
	<li>Streak solidified plates with approximately 200 ul&#160;of a bacterial culture grown in M9 minimal media with an O.D. of approximately 2.0.</li>
	<li>Incubate plates at 37 &#176;C and monitor growth. 
	Liquid Cultures</li>
	<li>Add 50 ml of sterile Luria-Bertani (LB) media13 or M9 minimal media13 to a sterile 125 ml flask. Add black liquor at 10% (v/v) or 0.2% (w/v) of lignocellulose extraction products.</li>
	<li>Inoculate the media with 0.1% (v/v) of a bacterial culture at an O.D. of approximately 2.0.</li>
	<li>Incubate the cultures at 37 &#176;C with shaking at 200 rpm.</li>
	<li>Assess growth every 6 hr during the culturing period by measuring optical density (&#955; = 600 nm). Place a 1 ml aliquot of the culture into a cuvette. Determine optical density of culture blanked against uninoculated media.</li>
	<li>Transfer bacteria to an anaerobic serum bottle at mid log phase.
	<ol>
		<li>To prepare the anaerobic serum bottle: seal the serum bottle with a butyl rubber stopper and an aluminum crimp top under aerobic conditions and autoclave. Do not modify the headspace within the serum bottle after sterilization.</li>
		<li>Transfer the entire 50 ml culture from the 125 ml flask into the sealed sterile serum bottle using a syringe.</li>
		<li>Incubate at 37 &#176;C with shaking at 200 rpm.</li>
	</ol>
	</li>
</ol>
4. Sample Preparation for GC-MS
Prepare samples according to Raj, Reddy, and Chandra14. Negative control used for comparison consists of M9 minimal media with 10% black liquor and is also referred to as the uninoculated sample.
<ol>
	<li>Centrifuge uninoculated and inoculated bacterial cultures (50 ml) at 9,300 x g for 30 min&#160;after 450 hr of incubation.</li>
	<li>Transfer 10 ml of the supernatant into a glass test tube. (Note: Plastic is incompatible with the chemicals in the following steps.)</li>
	<li>Acidify the supernatant with concentrated HCl to pH 1-2.</li>
	<li>Add 3 volumes of ethyl acetate (30 ml), cap the tube and mix by inverting 4-6x.</li>
	<li>Place a small amount of anhydrous Na2SO4 (1-2 g) in a clean glass test tube.</li>
	<li>Carefully collect the organic layer (top) from the ethyl acetate solution by pipetting with a disposable, glass Pasteur pipette.</li>
	<li>Place the organic layer in the test tube with anhydrous Na2SO4.</li>
	<li>Dewater the organic layer over anhydrous Na2SO4 by gently tapping the test tube to mix. Gradually add small amounts of anhydrous Na2SO4and mix until there are no large clumps.</li>
	<li>Place a sheet of filter paper into a B&#252;chner funnel on top of a side arm flask attached to a vacuum.</li>
	<li>Pour the solution into the funnel. Discard the solid material.</li>
	<li>Transfer the solution to an evaporating flask and evaporate the filtrate using a rotary evaporator.</li>
	<li>Place 3 mg of ethyl acetate extraction residue into a 2 ml amber chromatography vial.</li>
	<li>Dissolve the residue with the addition of 100 &#181;l of 1,4-dioxane and 10 &#181;l pyridine.</li>
	<li>Add 50 &#181;l of N,O-Bis(trimethylsilyl) trifluoroacetamide with trimethylcholorsilane 99:1% (BSTFA). Then place in an incubator at 60 &#176;C with shaking for 15 min.</li>
	<li>Store samples at 4 &#176;C until use.</li>
</ol>
5. GC-MS
Equip the gas chromatograph (GC) interfaced with a mass spectrometer (MS) with a nonpolar capillary column and helium as the carrier gas with a flow rate of 1 ml/min.
<ol>
	<li>After the oven temperature is stabilized at 50 &#176;C, inject 1 &#181;l of the sample at a 1/50 split ratio.</li>
	<li>Hold the column at 50 &#176;C for 5 min&#160;and increase to 280 &#176;C at 5 &#176;C/min. Maintain the final temperature of 280 &#176;C for 20 min.</li>
	<li>Maintain the transfer line between the gas chromatograph and mass spectrometer at 300 &#176;C.</li>
	<li>Select a solvent delay of 15 min.</li>
	<li>Record electron ionization mass spectra between the range of 10-500 (m/z) at electron energy of 70 eV.</li>
	<li>Derivatize and chromatograph standard as above. Identify compounds by comparing retention times of purchased standards or data in the National Institute of Standards and Technology (NIST) mass spectral database.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulping+fundamentals.">Pulping fundamentals.</a> Handbook of pulping and papermaking. , 55-100 (1996).</li><li>Sj&#246;str&#246;m, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Wood chemistry: fundamentals and applications. , (1924).</li><li>Philbrook, A., Alissandratos, A., Easton, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+processes+for+generating+fuels+and+commodity+chemicals+from+lignocellulosic+biomass.">Biochemical processes for generating fuels and commodity chemicals from lignocellulosic biomass.</a> Environ. 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Biotech. 78, 548-554 (2003).</li><li>Zimmerman, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+lignin+by+bacteria.">Degradation of lignin by bacteria.</a> J. Biotechnol. 13, 119-130 (1990).</li><li>Ahamad, M., Taylor, C. R., Pink, D., Burton, K., Eastwood, D., Bending, G. R., Bugg, T. D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+novel+assay+for+lignin+degradation:+comparative+analysis+of+bacterial+and+fungal+lignin+degraders.">Development of novel assay for lignin degradation: comparative analysis of bacterial and fungal lignin degraders.</a> Mol. Biosystems. 6, 815-821 (2010).</li><li>Ramchandra, M., Crawford, D. 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The authors declare that they have no competing financial interests.

This protocol describes a combination of techniques that aims to identify microorganisms that can degrade pulping waste, the carbon sources utilized during growth on pulping waste, and the microbial metabolic products produced when grown on pulping waste. We have shown the success of this protocol with the microbial environmental isolate: a facultative anaerobe that can grow on 10% black liquor and use the lignocellulose extraction components as sole carbon sources for growth. This protocol could be used to determine the...

The pulping of wood is a chemically intensive process that has been optimized over many years to create a system with minimal waste. However, some outputs of this process could be used to produce higher value product(s). Black liquor is one such example. It is generated from the kraft process, which is the dominant chemical pulping method, representing 85% of world lignin production1. The kraft process (Figure 1) uses temperature (160-200 &#176;C), pressure (120 psig), and the chemicals contained in white liquor (sodium hydroxide and sodium sulfide) to dissolve the lignin from the wood fibers2,3. Black liquor contains lignin, organic acids, and polysaccharide degradation byproducts4. It is incinerated to produce steam and recover chemicals in the recovery boiler that provides thermal energy for downstream paper making and pulping processes. The volume of black liquor generated by pulping can exceed the amount that the recovery boiler can effectively process. Disposing of the black liquor as effluent negatively affects aquatic flora and fauna and thus is not an option. Application of microbial organisms that could use black liquor for growth would be beneficial in terms of increasing chemical recovery and generation of value-added product(s) that would improve the overall life cycle analysis of the pulping system. Chemical and biological conversion of lignin derived monomers has successfully produced vanillin and cinnamic acid for use as food sweeteners and fragrance additives, phenol used for plastic and resins, and cyclohexane, which could be used for fuel5.
Previous work on biodegradation of this pulp waste has been focused on lignin depolymerization. The International Lignin Institute (ILI) reports that between 40-50 million tons of lignin is produced each year (http://www.ili-lignin.com/aboutlignin.php). Only 1.5% of that lignin is used for commercial industrial processes6. Lignin depolymerization by laccase and peroxidase enzymes produced by white rot fungi of the Phanerochaete and Trametes genera has been studied at length7. Soil bacteria known to degrade aromatic compounds such as Nocardia&#160;and Rhodococcus8, Pseudomonas putida&#160;mt-29, and Streptomyces viridosporus&#160;T7A10 have also been shown to be capable of lignin degradation. Bacterial degradation of pulping waste is promising because some bacteria can thrive in the saline and alkaline (pH 10-14) conditions that characterize the pulping waste effluents11. While lignin is the main component of black liquor, microorganisms may also degrade the other components that make up black liquor. These techniques do not exclusively identify lignin degrading microorganisms, but serve to identify microorganisms that can be applied directly to the pulping waste black liquor instead of its further processed constituents.
Black liquor was collected and modified for microbial growth through neutralization and filter-sterilization. Microbial growth requirements were identified for an environmental microbial isolate by minimal media growth experiments on lignocellulosic components produced by a novel lignocellulose extraction protocol. Growth media were analyzed by gas chromatography-mass spectrometry (GC-MS) to determine the metabolic products of the environmental microbial isolate when grown on black liquor as the sole carbon source. The combination of these techniques provides an assessment tool to determine the metabolic capacity of a microorganism when grown on pulp mill wastes such as black liquor. Use of such techniques also offers insight into the value of application of specific microorganisms to pulp mill waste for the generation of byproducts.

<table border="0" cellpadding="0" cellspacing="0" width="578">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:117px;">1,4-Dioxane (Certified ACS)</td>
			<td style="width:153px;">Fisher</td>
			<td style="width:147px;">D111</td>
			<td style="width:161px;">Flammable, preoxidizable chemical</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:117px;">Black Liquor</td>
			<td style="width:153px;">Department of Forest Biomaterials at North Carolina State University&#160;</td>
			<td style="width:147px;">N/A</td>
			<td style="width:161px;">pH 12.72, 13.67% solids</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:117px;">Ethanol (microbiology grade)</td>
			<td style="width:153px;">Fisher</td>
			<td style="width:147px;">BP2818</td>
			<td style="width:161px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:117px;">Ethyl Acetate (ACS reagent grade)</td>
			<td style="width:153px;">EMD</td>
			<td style="width:147px;">EX0240-9</td>
			<td style="width:161px;">Flammable</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:117px;">Glacial acetic acid (Certified ACS)</td>
			<td style="width:153px;">EMD</td>
			<td style="width:147px;">AX0073</td>
			<td style="width:161px;">Corrosive</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:117px;">Guaiacol</td>
			<td style="width:153px;">Sigma Aldrich&#160;</td>
			<td style="width:147px;">PHR1136</td>
			<td style="width:161px;">Harmful by ingestion, corrosive</td>
		</tr>
		<tr height="162">
			<td height="162" style="height:162px;width:117px;">HP-5 capillary column</td>
			<td style="width:153px;">Agilent</td>
			<td>19091J-577</td>
			<td style="width:161px;">60 m x 0.18 mm internal diameter, 0.18 &#956;m thickness</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:117px;">Hydrochloric acid (certified ACS)</td>
			<td style="width:153px;">EMD</td>
			<td style="width:147px;">HX0603P</td>
			<td style="width:161px;"></td>
		</tr>
		<tr height="148">
			<td height="148" style="height:148px;width:117px;">N,O-Bis(trimethylsilyl)trifluoroacetamide with trimethylcholorsilane 99:1% (BSTFA)</td>
			<td style="width:153px;">Fluka</td>
			<td style="width:147px;">15238</td>
			<td style="width:161px;">Flammable, causes skin burns and eye damage with contact</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:117px;">Na2SO4 (FCC grade)</td>
			<td style="width:153px;">VWR</td>
			<td style="width:147px;">BDH8026</td>
			<td style="width:161px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:117px;">NaClO2 (80%)</td>
			<td style="width:153px;">Sigma Aldrich&#160;</td>
			<td style="width:147px;">244155</td>
			<td style="width:161px;">Flammable, toxic&#160;&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:117px;">NaOH (certified ACS)</td>
			<td style="width:153px;">EMD</td>
			<td style="width:147px;">1.06498.1000</td>
			<td style="width:161px;">Corrosive</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:117px;">Alkacid pH test ribbons</td>
			<td style="width:153px;">Fisher</td>
			<td>A979</td>
			<td style="width:161px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:117px;">Phosphoric Acid (certified ACS)</td>
			<td style="width:153px;">Fisher</td>
			<td style="width:147px;">A242</td>
			<td style="width:161px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:117px;">Polaris Q mass spectrometer&#160;</td>
			<td style="width:153px;">Thermo Electron Corporation</td>
			<td style="width:147px;"></td>
			<td style="width:161px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:117px;">Pyridine (99%)</td>
			<td style="width:153px;">Alfa Aesar</td>
			<td style="width:147px;">A12005</td>
			<td style="width:161px;">Flammable, toxic in contact with skin</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:117px;">Switchgrass</td>
			<td style="width:153px;">Cherry Research Farm Goldsboro, NC</td>
			<td style="width:147px;">N/A</td>
			<td style="width:161px;">Harvested August 2011</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:117px;">Thermo Finnigan trace gas chromatograph</td>
			<td style="width:153px;">Thermo Electron Corporation</td>
			<td style="width:147px;"></td>
			<td style="width:161px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:117px;">Whatman no. 1 filter paper</td>
			<td style="width:153px;">Whatman</td>
			<td style="width:147px;">1001-150</td>
			<td style="width:161px;"></td>
		</tr>
	</tbody>
</table>

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.

The collection and modification of black liquor generated in the kraft pulping process (Figure 1) will allow one to use this pulping waste to determine the biodegradation capacity of a single bacterial isolate or mixed culture. Figure 2 shows aerobic growth measured by optical density of the microbial environmental isolate cultured in LB media alone and LB media supplemented with 10% neutralized black liquor. These results indicate that this bacterium grows in the presence of neutralized...

Watch this Scientific Journal Video about Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes at JoVE.com

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

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 ...

methods-for-facilitating-microbial-growth-on-pulp-mill-waste-streams

Research

JoVE Journal

Environment

9.4K Views.  North Carolina State University. 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.

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

Waste Water Derived Electroactive Microbial Biofilms: Growth, Maintenance, and Basic Characterization

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup controlled by a potentiostat. Thereby the use of potentiostats allows an exact adjustment of the electrode potential and ensures reproducible microbial culturing conditions. During growth the current production is monitored using chronoamperometry (CA). Based on these data the maximum current density (jmax) and the coulombic efficiency (CE) are discussed as measures for characterization of the bioelectrocatalytic performance. Cyclic voltammetry (CV), a nondestructive, i.e. noninvasive, method, is used to study the extracellular electron transfer (EET) of electroactive bacteria. CV measurements are performed on anodic biofilm electrodes in the presence of the microbial substrate, i.e. turnover conditions, and in the absence of the substrate, i.e. nonturnover conditions, using different scan rates. Subsequently, data analysis is exemplified and fundamental thermodynamic parameters of the microbial EET are derived and explained: peak potential (Ep), peak current density (jp), formal potential (Ef) and peak separation (&#916;Ep). Additionally the limits of the method and the state-of the art data analysis are addressed. Thereby this video-article shall provide a guide for the basic experimental steps and the fundamental data analysis.

Chronoamperometric growth of anodic electroactive microbial biofilms in a fed-batch reactor using a three-electrode setup, controlled by a potentiostat, is demonstrated. The extracellular electron transfer is characterized using cyclic voltammetry in the presence of the electron donor and in the absence of the electron donor. Fundamental data analysis is demonstrated.

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup ...

Protocol for Assessing the Relative Effects of Environment and Genetics on Antler and Body Growth for a Long-lived Cervid

Cervid phenotype can be placed into one of two categories: efficiency, which promotes survival over extravagant morphometric growth, and luxury, which promotes growth of large weaponry and body size. Populations of the same species display each phenotype depending on environmental conditions. Although antler and body size of male white-tailed deer (Odocoileus virginianus) varies by physiographic region in Mississippi, USA and is strongly correlated with regional variation in nutritional quality, the effects of population-level genetics from native stocks and previous re-stocking efforts cannot be disregarded. This protocol describes how we designed a controlled study, where other factors that influence phenotype, such as age and nutrition, are controlled. We brought wild-caught pregnant females and six-month-old fawns from three distinct physiographic regions in Mississippi, USA to the Mississippi State University Rusty Dawkins Memorial Deer Unit. Deer from the same region were bred to produce a second generation of offspring, allowing us to assess generational responses and maternal effects. All deer ate the same high-quality (20% crude protein deer pellet) diet ad libitum. We uniquely marked each neonate and recorded body mass, hind foot, and total body length. Each subsequent fall, we sedated individuals via remote injection and sampled the same morphometrics plus antlers of adults. We found that all morphometrics increased in size from first to second generation, with full compensation of antler size (regional variation no longer present) and partial compensation of body mass (some evidence of regional variation) evident in the second generation. Second generation males that originated from our poorest quality soil region displayed about a 40% increase in antler size and about a 25% increase in body mass when compared to their wild harvested counterparts. Our results suggest phenotypic variation of wild male white-tailed deer in Mississippi are more related to differences in nutritional quality than population-level genetics.

Phenotypic differences among cervid populations may be related to population-level genetics or nutrition; discerning which is difficult in the wild. This protocol describes how we designed a controlled study where nutritional variation was eliminated. We found that phenotypic variation of male white-tailed deer was more limited by nutrition than genetics.

Cervid phenotype can be placed into one of two categories: efficiency, which promotes survival over extravagant morphometric growth, and luxury, which ...

The Effect of the Application of Thyme Essential Oil on Microbial Load During Meat Drying

Meat is a high protein meal that is used in the preparation of jerky, a popular food snack, where preservation and safety are important. To assure food safety and to extend the shelf life of meat and meat products, the use of either synthetic or natural preservatives have been applied to control and eliminate foodborne bacteria. A growing interest in the application of natural food additives for meat has increased. Microorganisms, such as Escherichia coli, contaminate meat and meat products, causing foodborne illnesses. Therefore, it is necessary to improve the meat conservation process. However, the use of essential oils when the meat is being dried has not been deeply studied. In this regard, there is an opportunity to increase the value of dried meat and reduce the risk of foodborne illnesses by applying essential oils during the drying process. In this protocol, we present a novel method of applying thyme essential oil (TEO) during meat drying, specifically in vapor form directly in a drying chamber. For evaluation, we use Minimal Inhibitory Concentration (MIC) to detect the number of harmful bacteria in the treated samples compared to raw samples. The preliminary results show that this method is a viable and alternative option to synthetic preservatives and that it significantly reduces microbial load in dried meat.

Microorganisms such as Escherichia coli that contaminate meat products cause foodborne illnesses. The use of essential oils in the meat drying process has not been deeply studied. Here, we present a novel method of applying thyme essential oil to meat during drying to reduce the microbial load in dried meat.

Meat is a high protein meal that is used in the preparation of jerky, a popular food snack, where preservation and safety are important. To assure food ...

Experimental Methods of Dust Charging and Mobilization on Surfaces with Exposure to Ultraviolet Radiation or Plasmas

Electrostatic dust transport has been hypothesized to explain a number of observations of unusual planetary phenomena. Here, it is demonstrated using three recently developed experiments in which dust particles are exposed to thermal plasma with beam electrons, beam electrons only, or ultraviolet (UV) radiation only. The UV light source has a narrow bandwidth in wavelength centered at 172 nm. The beam electrons with the energy of 120 eV are created with a negatively biased hot filament. When the vacuum chamber is filled with the argon gas, a thermal plasma is created in addition to the electron beam. Insulating dust particles of a few tens of microns in diameter are used in the experiments. Dust particles are recorded to be lofted to a height up to a few centimeters with a launch speed up to 1 m/s. These experiments demonstrate that photo and/or secondary electron emission from a dusty surface changes the charging mechanism of dust particles. According to the recently developed &#34;patched charge model&#34;, the emitted electrons can be re-absorbed inside microcavities between neighboring dust particles below the surface, causing the accumulation of enhanced negative charges on the surrounding dust particles. The repulsive forces between these negatively charged particles may be large enough to mobilize and lift them off the surface. These experiments present the advanced understanding of dust charging and transport on dusty surfaces, and laid a foundation for future investigations of its role in the surface evolution of airless planetary bodies.

Dust charging and mobilization is demonstrated in three experiments with exposure to thermal plasma with beam electrons, beam electrons only, or ultraviolet (UV) radiation only. These experiments present the advanced understanding of electrostatic dust transport and its role in shaping the surfaces of airless planetary bodies.

Electrostatic dust transport has been hypothesized to explain a number of observations of unusual planetary phenomena. Here, it is demonstrated using ...

Measuring the Flight Ability of the Ambrosia Beetle, Platypus Quercivorus (Murayama), Using a Low-Cost, Small, and Easily Constructed Flight Mill

The ambrosia beetle, Platypus quercivorus (Murayama), is the vector of a fungal pathogen that causes mass mortality of Fagaceae trees (Japanese oak wilt). Therefore, knowing the dispersal capacity may help inform trapping/tree removal efforts to prevent this disease more effectively. In this study, we measured the flight velocity and duration and estimated the flight distance of the beetle using a newly developed flight mill. The flight mill is low cost, small, and constructed using commonly available items. Both the flight mill arm and its vertical axis comprise a thin needle. A beetle specimen is glued to one tip of the arm using instant glue. The other tip is thick due to being covered with plastic, thus it facilitates the detection of rotations of the arm. The revolution of the arm is detected by a photo sensor mounted on an infrared LED, and is indicated by a change in the output voltage when the arm passed above the LED. The photo sensor is connected to a personal computer and the output voltage data are stored at a sampling rate of 1 kHz. By conducting experiments using this flight mill, we found that P. quercivorus can fly at least 27 km. Because our flight mill comprises cheap and small ordinary items, many flight mills can be prepared and used simultaneously in a small laboratory space. This enables experimenters to obtain a sufficient amount of data within a short period.

Measuring the Flight Ability of the Ambrosia Beetle, Platypus Quercivorus Murayama, Using a Low-Cost, Small, and Easily Constructed Flight Mill

We developed a low cost and small flight mill, constructed with commonly available items and easily used in experimentation. Using this apparatus, we measured the flight ability of an ambrosia beetle, Platypus quercivorus.

The ambrosia beetle, Platypus quercivorus (Murayama), is the vector of a fungal pathogen that causes mass mortality of Fagaceae trees (Japanese oak wilt). ...

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 ...

The Effect of Construction and Demolition Waste Plastic Fractions on Wood-Polymer Composite Properties

Construction and demolition waste (CDW), including valuable materials such as plastics, have a remarkable influence on the waste sector. In order for plastic materials to be re-utilized, they must be identified and separated according to their polymer composition. In this study, the identification of these materials was performed using near-infrared spectroscopy (NIR), which identified material based on their physical-chemical properties. Advantages of the NIR method are a low environmental impact and rapid measurement (within a few seconds) in the spectral range of 1600-2400 nm without special sample preparation. Limitations include its inability to analyze dark materials. The identified polymers were utilized as a component for wood-polymer composite (WPC) that consists of a polymer matrix, low cost fillers, and additives. The components were first compounded with an agglomeration apparatus, followed by production by extrusion. In the agglomeration process, the aim was to compound all materials to produce uniformly distributed and granulated materials as pellets. During the agglomeration process, the polymer (matrix) was melted and fillers and other additives were then mixed into the melted polymer, being ready for the extrusion process. In the extrusion method, heat and shear forces were applied to a material within the barrel of a conical counter-rotating twin-screw type extruder, which reduces the risk of burning the materials and lower shear mixing. The heated and sheared mixture was then conveyed through a die to give the product the desired shape. The above-described protocol proved the potential for re-utilization of CDW materials. Functional properties must be verified according to the standardized tests, such as flexural, tensile, and impact strength tests for the material.

Secondary material streams have been shown to include potential raw materials for production. Presented here is a protocol in which CDW-plastic waste as a raw material is identified, followed by various processing steps (agglomeration, extrusion). As a result, a composite material was produced, and mechanical properties were analyzed.

Construction and demolition waste (CDW), including valuable materials such as plastics, have a remarkable influence on the waste sector. In order for ...

Evaluating the Impact of Hydraulic Fracturing on Streams using Microbial Molecular Signatures

Hydraulic fracturing (HF), commonly called &#34;fracking&#34;, uses a mixture of high-pressure water, sand, and chemicals to fracture rocks, releasing oil and gas. This process revolutionized the U.S. energy industry, as it gives access to resources that were previously unobtainable and now produces two-thirds of the total natural gas in the United States. Although fracking has had a positive impact on the U.S. economy, several studies have highlighted its detrimental environmental effects. Of particular concern is the effect of fracking on headwater streams, which are especially important due to their disproportionately large impact on the health of the entire watershed. The bacteria within those streams can be used as indicators of stream health, as the bacteria present and their abundance in a disturbed stream would be expected to differ from those in an otherwise comparable but undisturbed stream. Therefore, this protocol aims to use the bacterial community to determine if streams have been impacted by fracking. To this end, sediment, and water samples, from streams near fracking (potentially impacted) and upstream or in a different watershed of fracking activity (unimpacted) must be collected. Those samples are then subjected to nucleic acid extraction, library preparation, and sequencing to investigate microbial community composition. Correlational analysis and machine learning models can subsequently be employed to identify which features are explanative of variation in the community, as well as identification of predictive biomarkers for fracking&#39;s impact. These methods can reveal a variety of differences in the microbial communities among headwater streams, based on the proximity to fracking, and serve as a foundation for future investigations on the environmental impact of fracking activities.

Here, we present a protocol to investigate the impacts of hydraulic fracturing on nearby streams by analyzing their water and sediment microbial communities.

Hydraulic fracturing (HF), commonly called &#34;fracking&#34;, uses a mixture of high-pressure water, sand, and chemicals to fracture rocks, releasing oil ...

Quantification of the Potential Impact of Glyphosate-Based Products on Microbiomes

Glyphosate-based products (GBP) are the most common broad-spectrum herbicides worldwide. The target of glyphosate is the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate pathway, which is virtually universal in plants. The inhibition of the enzyme stops the production of three essential amino acids: phenylalanine, tyrosine, and tryptophan. EPSPS is also present in fungi and prokaryotes, such as archaea and bacteria; thus, the use of GBP may have an impact on the microbiome composition of soils, plants, herbivores, and secondary consumers. This article aims to present general guidelines to assess the effect of GBP on microbiomes from field experiments to bioinformatics analyses and provide a few testable hypotheses. Two field experiments are presented to test the GBP on non-target organisms. First, plant-associated microbes from 10 replicated control and GBP treatment plots simulating no-till cropping are sampled and analyzed. In the second experiment, samples from experimental plots fertilized by either poultry manure containing glyphosate residues or non-treated control manure were obtained. Bioinformatics analysis of EPSPS protein sequences is utilized to determine the potential sensitivity of microbes to glyphosate. The first step in estimating the effect of GBP on microbiomes is to determine their potential sensitivity to the target enzyme (EPSPS). Microbial sequences can be obtained either from public repositories or by means of PCR amplification. However, in the majority of field studies, microbiome composition has been determined based on universal DNA markers such as the 16S rRNA and the internal transcribed spacer (ITS). In these cases, sensitivity to glyphosate can only be estimated through a probabilistic analysis of EPSPS sequences using closely related species. The quantification of the potential sensitivity of organisms to glyphosate, based on the EPSPS enzyme, provides a robust approach for further experiments to study target and non-target resistant mechanisms.

Glyphosate-based products (GBP) are the most common broad-spectrum herbicides worldwide. In this article, we introduce general guidelines to quantify the effect of GBP on microbiomes, from field experiments to bioinformatics analyses.

Glyphosate-based products (GBP) are the most common broad-spectrum herbicides worldwide. The target of glyphosate is the enzyme ...

Rhizobacterial Colonization Detection on &lt;i&gt;Arabidopsis thaliana&lt;/i&gt;

Measuring Bacterial Colonization on Arabidopsis thaliana Roots in Hydroponic Condition

Measuring bacterial colonization on Arabidopsis thaliana root is one of the most frequent experiments in plant-microbe interaction studies. A standardized method for measuring bacterial colonization in the rhizosphere is necessary to improve reproducibility. We first cultured sterile A.thaliana in hydroponic conditions and then inoculated the bacterial cells in the rhizosphere at a final concentration of OD600 of 0.01. At 2 days post-inoculation, the root tissue was harvested and washed three times in sterile water to remove the uncolonized bacterial cells. The roots were then weighed, and the bacterial cells colonized on the root were collected by vortex. The cell suspension was diluted in a gradient with a phosphate-buffered saline (PBS) buffer, followed by plating onto a Luria-Bertani (LB) agar medium. The plates were incubated at 37 &#176;C for 10 h, and then, the single colonies on LB plates were counted and normalized to indicate the bacterial cells colonized on roots. This method is used to detect bacterial colonization in the rhizosphere in mono-interaction conditions, with good reproducibility.

Author Spotlight: Enhancing Rhizobacteria Colonization on Plant Roots for Improved Microbial Fertilizer Efficiency

Colonization of plant growth-promoting rhizobacteria (PGPR) in the rhizosphere is essential for its growth-promoting effect. It is necessary to standardize the method of detection of bacterial rhizosphere colonization. Here, we describe a reproducible method for quantifying bacterial colonization on the root surface.

Measuring bacterial colonization on Arabidopsis thaliana root is one of the most frequent experiments in plant-microbe interaction studies. A standardized ...