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>

<ol>
	<li>Tejado, A., Pena, C., Labidi, J., Echeverria, J. M., Mondragon, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physico-chemical+characterization+of+lignin+from+different+sources+for+use+in+phenol-formaldehyde+resin+synthesis.">Physico-chemical characterization of lignin from different sources for use in phenol-formaldehyde resin synthesis.</a> Bioresource Technol. 98, 1655-1663 (2007).</li><li>Brannvall, E., Elk, M., Gellerstedt, G., Henriksson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+pulp+and+paper+processes.">Overview of pulp and paper processes.</a> Pulp and paper chemistry and technology. 2, 1-12 (2009).</li><li>Biermann, 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=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. Biotechnol. , 39-63 (2013).</li><li>Sanchez, R., Ferrer, A., Serrano, L., Toledano, A., Labidi, J., Rodriguez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hesperaloafunifera+as+a+raw+material+for+integral+utilization+of+its+components.">Hesperaloafunifera as a raw material for integral utilization of its components.</a> BioResources. 6 (1), 3-21 (2011).</li><li>Font, X., Caminal, G., Gabarrel, X., Romero, S., Vicent, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Black+liquor+detoxification+by+laccase+of+Trametesversicolorpellets.">Black liquor detoxification by laccase of Trametesversicolorpellets.</a> J. Chemical Tech. 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. L., Hertel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+an+extracellular+lignin+peroxidase+of+the+lignocellulolyticactinomycete+Streptomyces+viridosporus.">Characterization of an extracellular lignin peroxidase of the lignocellulolyticactinomycete Streptomyces viridosporus.</a> Appl. Environ. Micro. 54, 3057-3063 (1988).</li><li>Mishra, M., Thakur, I. S., Satyanarayana, T., Johri, B. N., Prakash, 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> Microorganisms in environmental management: microbes and the environment. , (2012).</li><li>Karaaslan, A. M., Tshabalala, M. A., Wood Bushcle-Diller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hemicellulose+chitosan-based+semi-interpenetrating+network+hydrogels:+mechanical,+swelling+and+controlled+drug+release+properties.">hemicellulose chitosan-based semi-interpenetrating network hydrogels: mechanical, swelling and controlled drug release properties.</a> BioResources. 5 (2), 1036-1054 (2010).</li><li>Lech, K., Brent, R., Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Kevin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Short protocols in molecular biology. , (1992).</li><li>Raj, A., Krishna Reddy, M. M., Chandra, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+low+molecular+weight+aromatic+compounds+by+gas+chromatography-mass+spectrometry+(GC-MS)+from+kraft+lignin+degradation+by+three+Bacillus+sp.">Identification of low molecular weight aromatic compounds by gas chromatography-mass spectrometry (GC-MS) from kraft lignin degradation by three Bacillus sp.</a> Int. Biodeterior. Biodegradation. 59, 292-296 (2007).</li><li>Chandra, R., Raj, A., Purohit, H. J., Kapley, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradation+of+kraft+lignin+by+three+pure+and+mixed+aerobic+bacterial+cultures+isolated+from+pulp+and+paper+sludge.">Biodegradation of kraft lignin by three pure and mixed aerobic bacterial cultures isolated from pulp and paper sludge.</a> , (2005).</li><li>Chen, Y., Chai, L., Tang, C., Yang, Z., Zheng, Y., Shi, Y., Zhang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kraft+lignin+biodegradation+by+Novosphingobium+sp.+B-7+and+analysis+of+the+degradation+process.">Kraft lignin biodegradation by Novosphingobium sp. B-7 and analysis of the degradation process.</a> Bioresource Technol. 123, 682-685 (2012).</li><li>Bandounas, L., Wierckx, N. J., de Winde, J. H., Ruijssenaars, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+novel+bacterial+strains+exhibiting+ligninolytic+potential.">Isolation and characterization of novel bacterial strains exhibiting ligninolytic potential.</a> BMC Biotechnol. 11 (94), (2011).</li><li>Chandra, R., Abhishek, A., Sankhwar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+decolorization+and+detoxification+of+black+liquor+from+rayon+grade+pulp+manufacturing+industry+and+detection+of+their+metabolic+products.">Bacterial decolorization and detoxification of black liquor from rayon grade pulp manufacturing industry and detection of their metabolic products.</a> Bioresource Technol. 102, 6429-6436 (2011).</li><li>Gellerdtedt, G., Ek, M., Gellerstedt, G., Henriksson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+of+Bleaching+of+Chemical+Pulp.">Chemistry of Bleaching of Chemical Pulp.</a> Pulp and Paper Chemistry and Technology. 2, 201-237 (2009).</li><li>Kringstad, K. P., Lindstrom, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spent+liquors+from+pulp+bleaching.">Spent liquors from pulp bleaching.</a> Environ. Sci. Tech. 18, 236-247 (1984).</li></ol>

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><tbody><tr><td>1,4-Dioxane (Certified ACS)</td><td>Fisher</td><td>D111</td><td>Flammable, preoxidizable chemical</td></tr><tr><td>Black Liquor</td><td>Department of Forest Biomaterials at North Carolina State University</td><td>N/A</td><td>pH 12.72, 13.67% solids</td></tr><tr><td>Ethanol (microbiology grade)</td><td>Fisher</td><td>BP2818</td><td></td></tr><tr><td>Ethyl Acetate (ACS reagent grade)</td><td>EMD</td><td>EX0240-9</td><td>Flammable</td></tr><tr><td>Glacial acetic acid (Certified ACS)</td><td>EMD</td><td>AX0073</td><td>Corrosive</td></tr><tr><td>Guaiacol</td><td>Sigma Aldrich</td><td>PHR1136</td><td>Harmful by ingestion, corrosive</td></tr><tr><td>HP-5 capillary column</td><td>Agilent</td><td>19091J-577</td><td>60 m x 0.18 mm internal diameter, 0.18 &amp;mu;m thickness</td></tr><tr><td>Hydrochloric acid (certified ACS)</td><td>EMD</td><td>HX0603P</td><td></td></tr><tr><td>N,O-Bis(trimethylsilyl)trifluoroacetamide with trimethylcholorsilane 99:1% (BSTFA)</td><td>Fluka</td><td>15238</td><td>Flammable, causes skin burns and eye damage with contact</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4 &lt;/sub&gt;(FCC grade)</td><td>VWR</td><td>BDH8026</td><td></td></tr><tr><td>NaClO&lt;sub&gt;2&lt;/sub&gt; (80%)</td><td>Sigma Aldrich</td><td>244155</td><td>Flammable, toxic&amp;nbsp;&amp;nbsp;</td></tr><tr><td>NaOH (certified ACS)</td><td>EMD</td><td>1.06498.1000</td><td>Corrosive</td></tr><tr><td>Alkacid pH test ribbons</td><td>Fisher</td><td>A979</td><td></td></tr><tr><td>Phosphoric Acid (certified ACS)</td><td>Fisher</td><td>A242</td><td></td></tr><tr><td>Polaris Q mass spectrometer&amp;nbsp;</td><td>Thermo Electron Corporation</td><td></td><td></td></tr><tr><td>Pyridine (99%)</td><td>Alfa Aesar</td><td>A12005</td><td>Flammable, toxic in contact with skin</td></tr><tr><td>Switchgrass</td><td>Cherry Research Farm Goldsboro, NC</td><td>N/A</td><td>Harvested August 2011</td></tr><tr><td>Thermo Finnigan trace gas chromatograph</td><td>Thermo Electron Corporation</td><td></td><td></td></tr><tr><td>Whatman no. 1 filter paper</td><td>Whatman</td><td>1001-150</td><td></td></tr></tbody></table>

methods for facilitating microbial growth on pulp mill waste streams and characterization of the biodegradation potential of cultured microbes

硫酸盐处理应用于木屑对木质素从多糖中分离木素纤维素纸浆，将产生一个高质量的纸张。黑液是由牛皮纸工艺，具有潜在的下游生物转化产生的制浆废水。然而，木质纤维素资源，这占了大部分内黑液可用的有机碳，以及其碱性pH存在挑战，这浪费材料的微生物降解及其化学衍生物的顽抗性质。方法黑液微生物生长的收集和修改的目的是利用这种纸浆废液中的木质素，有机酸和多糖降解副产物转化成有价值的化学物质。介绍了木质纤维素的提取技术制备木质纤维素底物的增长理解为培养微生物的代谢能力，提供了一个可重复的方法。使用燃气通道的romatography - 质谱法使之从微生物对制浆废水增长所带来的发酵产物的鉴定和定量。这些方法一起使用时可以促进微生物的潜在生产发酵产品，将提供更大的价值，制浆系统，减少污水垃圾，从而增加潜在纸铣利润，并提供用于黑液的其他用途的代谢活动的决心。

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.

方法对纸浆厂污水流和培养微生物的生物降解能力的表征促进微生物生长

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 次观看.  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.

视频: 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 ...

科研

环境科学

用于食物垃圾和污泥厌氧消化的呼吸测定方案

Measuring Biomethane Potential of Food Scrap Waste Anaerobically Co&#45;Digested with Waste&#45;Activated Sludge Using Respirometry

The use of respirometry to study the biokinetics of microbiota treating wastewater or digesting wastewater sludges has become more prevalent over the last few decades. The use of respirometry to examine the biokinetics of anaerobic microbiota co-digesting organic waste streams such as wastewater sludge and food scrap is an area of active research. To date, no visualized protocol has been published on the topic. Accordingly, in this protocol, we configured a respirometer to measure methane production and flow rate over time using three different food-to-microorganism (F:M) ratios and food scrap waste and waste-activated sludge as substrates. The resulting data, coupled with substrate utilization measurements, provides the basis for understanding how different substrate concentrations influence the rate at which anaerobic microbiota produce methane. Additionally, this protocol presents a method to develop biokinetic parameters (e.g., methane production rate constant and yield). Others can use this respirometry protocol to examine organic degradation under anaerobic conditions and develop microbial parameters.

作者聚焦：使用新型呼吸测定法方案推进厌氧微生物群研究

This protocol describes a best practice for determining methane production and microbial kinetic parameters using respirometry for anaerobic microbiota co-digesting food scrap waste and waste-activated sludge.

使用呼吸测定法测量厌氧共消化的食物残渣与废物活性污泥共消化的生物甲烷潜力

The use of respirometry to study the biokinetics of microbiota treating wastewater or digesting wastewater sludges has become more prevalent over the last ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

一种新型生物反应器为不同的微生物群落的高密度栽培

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

生物工程

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 that are able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified undetoxified hydrolyzates and to accumulate over 40 g/L ethanol.

鲁棒戊糖发酵酵母的木质纤维素生物转化的演进技术乙醇

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme ...

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

Bamboo is an important biomass, and bamboo hydrolysate is used by Klebsiella pneumoniae as a feedstock for chemical production. Here, bamboo powder was pretreated with NaOH and washed to a neutral pH. Cellulase was added to the pretreated bamboo powder to generate the hydrolysate, which contained 30 g/L glucose and 15 g/L xylose and was used as the carbon source to prepare a medium for chemical production. When cultured in microaerobic conditions, 12.7 g/L 2,3-butanediol was produced by wildtype K. pneumoniae. In aerobic conditions, 13.0 g/L R-acetoin was produced by the budC mutant of K. pneumoniae. A mixture of 25.5 g/L 2-ketogluconic acid and 13.6 g/L xylonic acid was produced by the budA mutant of K. pneumoniae in a two-stage, pH-controlled fermentation with high air supplementation. In the first stage of fermentation, the culture was maintained at a neutral pH; after cell growth, the fermentation proceeded to the second stage, during which the culture was allowed to become acidic.

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

Bamboo powder was pretreated with NaOH and enzymatically hydrolyzed. The hydrolysate of bamboo was used as the feedstock for 2,3-butanediol, R-acetoin, 2-ketogluconic acid, and xylonic acid production by Klebsiella pneumoniae.

化学品生产<em&gt;肺炎克雷伯菌</em使用竹水合物作为原料

Bamboo is an important biomass, and bamboo hydrolysate is used by Klebsiella pneumoniae as a feedstock for chemical production. Here, bamboo powder was ...

Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; agar-block). It is well-accepted that soil-block microcosms yield higher decay rates, fewer moisture issues, lower variability among studies, and higher thresholds of preservative toxicity. Soil-block testing is thus the more utilized technique and has been standardized by American Society for Testing and Materials (ASTM) (method D 1413-07). The soil-block design has drawbacks, however, using locally-variable soil sources and in limiting the control of nutrients external (exogenous) to the decaying tissues. These drawbacks have emerged as a problem in applying this method to other, increasingly popular research aims. These modern aims include degrading lignocellulosics for bioenergy research, testing bioremediation of co-metabolized toxics, evaluating oxidative mechanisms, and tracking translocated elements along hyphal networks. Soil-blocks do not lend enough control in these applications. A refined agar-block approach is necessary.
Here, we use the brown rot wood-degrading fungus Serpula lacrymans to degrade wood in agar-block microcosms, using deep Petri dishes with low-calcium agar. We test the role of exogenous gypsum on decay in a time-series, to demonstrate the utility and expected variability. Blocks from a single board rip (longitudinal cut) are conditioned, weighed, autoclaved, and introduced aseptically atop plastic mesh. Fungal inoculations are at each block face, with exogenous gypsum added at interfaces. Harvests are aseptic until the final destructive harvest. These microcosms are designed to avoid block contact with agar or Petri dish walls. Condensation is minimized during plate pours and during incubation. Finally, inoculum/gypsum/wood spacing is minimized but without allowing contact. These less technical aspects of agar-block design are also the most common causes of failure and the key source of variability among studies. Video publication is therefore useful in this case, and we demonstrate low-variability, high-quality results.

This video demonstrates a controlled environment approach to study degradation of lignocellulosic plant tissues by aerobic fungi. The ability to control nutrient sources and moisture is a key advantage of agar-block microcosms, but the approach often yields mixed success. We address critical pitfalls to yield reproducible, low-variability results.

为控制植物组织分解好氧菌的琼脂块缩影

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; ...

生物学

Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Globally, 4.5 billion people are economically dependent on the sea, where most of their livelihood is provided by coral reefs. Corals are of great importance and therefore their extinction leads to catastrophic consequences. There are several possible solutions to remediate marine pollutants and local contamination, including bioremediation. Bioremediation is the capacity of organisms to degrade contaminants. The approach presents several advantages, such as sustainability, relatively low cost, and the fact that it can be applied in different ecosystems, causing minimal impacts to the environment. As an extra advantage, the manipulation of endogenous microbiomes, including putative beneficial microorganisms for corals (pBMCs), may have probiotic effects for marine animals. In this context, the use of the two approaches, bioremediation and pBMC inoculation combined, could be promising. This strategy would promote the degradation of specific pollutants that can be harmful to corals and other metaorganisms while also increasing host resistance and resilience to deal with pollution and other threats. This method focuses on the selection of pBMCs to degrade two contaminants: the synthetic estrogen 17a-ethinylestradiol (EE2) and crude oil. Both have been reported to negatively impact marine animals, including corals, and humans. The protocol describes how to isolate and test bacteria capable of degrading the specific contaminants, followed by a description of how to detect some putative beneficial characteristics of these associated microbes to their coral host. The methodologies described here are relatively cheap, easy to perform, and highly adaptable. Almost any kind of soluble target compound can be used instead of EE2 and oil.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Bioremediation is the capacity of organisms to degrade contaminants. Here, we describe methodologies to isolate and test microbes presenting bioremediation ability and potential probiotic characteristics for corals.

生物修复和益生菌发育的微生物菌株的勘探，用于生物有机物的研究与保存

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. ...

Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to degrade plastics. Typically, when formulations of plastics are known and a source of the feedstock is available, powdered plastic can be suspended in agar-based media and degradation determined by visualization of clearing zones. However, this approach poorly mimics in situ degradation of BDMs. First, BDMs are not dispersed as small particles throughout the soil matrix. Secondly, BDMs are not sold commercially as pure polymers, but rather as films containing additives (e.g. fillers, plasticizers and dyes) that may affect microbial growth. The procedures described herein were used for isolates acquired from soil-buried mulch films. Fungal isolates acquired from excavated BDMs were tested individually for growth on pieces of new, disinfested BDMs laid atop defined medium containing no carbon source except agar. Isolates that grew on BDMs were further tested in liquid medium where BDMs were the sole added carbon source. After approximately ten weeks, fungal colonization and BDM degradation were assessed by scanning electron microscopy. Isolates were identified via analysis of ribosomal RNA gene sequences. This report describes methods for fungal isolation, but bacteria also were isolated using these methods by substituting media appropriate for bacteria. Our methodology should prove useful for studies investigating breakdown of intact plastic films or products for which plastic feedstocks are either unknown or not available. However our approach does not provide a quantitative method for comparing rates of BDM degradation.

Plastic films labeled "biodegradable" are commercially available for agricultural use as mulches. Tillage represents an attractive disposal method, but degradation under field conditions is poorly understood. The purpose of this study was to develop methods for isolating native soil fungi and bacteria that colonize plastic mulch films after field burial.

隔离原住民土壤微生物潜力打破生物降解塑料地膜应用于农业

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to ...

方法对纸浆厂污水流和培养微生物的生物降解能力的表征促进微生物生长

摘要

探索更多视频

此视频中的章节

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

使用呼吸测定法测量厌氧共消化的食物残渣与废物活性污泥共消化的生物甲烷潜力

使用呼吸测定法测量厌氧共消化的食物残渣与废物活性污泥共消化的生物甲烷潜力

使用呼吸测定法测量厌氧共消化的食物残渣与废物活性污泥共消化的生物甲烷潜力

一种新型生物反应器为不同的微生物群落的高密度栽培

鲁棒戊糖发酵酵母的木质纤维素生物转化的演进技术乙醇

化学品生产

为控制植物组织分解好氧菌的琼脂块缩影

生物修复和益生菌发育的微生物菌株的勘探，用于生物有机物的研究与保存

隔离原住民土壤微生物潜力打破生物降解塑料地膜应用于农业