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 phosp...

<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. 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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 border="0" cellpadding="0" cellspacing="0" width="578">
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		<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 ...

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9.3K vistas.  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.