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

Summary

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.

Abstract

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.

Introduction

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 °C), pressure (120 psig), and the chemicals contained in white liquor (sodium hydroxide and sodium sulfide) to dissolve the lignin from the wood fibers^2,3. 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 and Rhodococcus⁸, Pseudomonas putida mt-2⁹, and Streptomyces viridosporus 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.

Protocol

1. Collection of Black Liquor and Preparation of Black Liquor for Growth Cultures

1. 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
2. Add 100 ml of black liquor sample to a 500 ml beaker equipped with a stir bar.
3. Place the beaker on a stir plate and adjust the speed to medium.
4. Slowly add drops of phosphoric acid until the black liquor becomes viscous (viscosity similar to glycerol).
5. 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.
6. 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.
7. Continue to add phosphoric acid and centrifuge the solution until the pH strips indicate a neutral pH of the supernatant.
   Sterilization
8. Pass the solution through a 0.22 µm filter to remove any contaminating bacteria and place the filtered solution into a sterile container for storage. Store at room temperature.

2. Lignocellulose Extraction

Carbohydrate Separation

1. 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, and a stir bar to the flask.
2. Place the flask in an oil bath placed on top of a stir plate at 80 °C for 1 hr. Adjust the speed of the stir plate to medium.
3. Add an additional 7.5 g of NaClO₂ and 2.5 ml of glacial acetic acid to the flask. Continue stirring at 80 °C in the oil bath for 30 min.
4. 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.
5. Place a sheet of filter paper into a Büchner funnel. Place the funnel on top of a side arm flask and attach it to a vacuum.
6. Slowly pour the solution into the funnel.
7. Wash the solid material 3x with 100 ml of distilled or deionized water. Discard the filtrate after washing the solid material.
8. Reserve 1 g of the solid material and place it onto a clean sheet of filter paper.
9. Dry this solid material overnight in a 50 °C oven. After removing the sample from the oven, place into a storage vial labeled ‘holocellulose’.
10. 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.
11. Place the solution in an incubator at 70 °C for 1 hr shaking at 250 rpm.
12. Cool the solution for 30 min at room temperature.
13. Remove the solid material using filter paper and Büchner funnel attached to a side arm flask.
14. Transfer the solid material on a clean sheet of filter paper to a 50 °C oven and allow it to dry overnight.
15. After the residue has dried, place it in a storage vial and label it ‘cellulose’.
16. 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.
17. 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.
18. Resuspend the pellet in 30 ml of distilled or deionized water by vortexing.
19. Repeat steps 2.17-2.18 15x to thoroughly wash the pellet.
20. Remove the supernatant and save the pellet for freeze drying. Once freeze-dried, collect the sample and place it in a vial labeled ‘hemicellulose’.
    Lignin separation
    Lignin separation protocol based on Karaaslan et al.¹²
21. 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 30% ethanol. Place the slurry in an incubator at 75 °C with shaking at 250 rpm for 2 hr.
22. Cool the solution to room temperature.
23. Place a sheet of filter paper in a Büchner funnel on top of a side arm flask. Attach to a vacuum and slowly pour the solution into the funnel.
24. Discard the solid material.
25. 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).
26. Allow the acidified solution to sit overnight at room temperature.
27. Centrifuge the solution at 9,300 x g for 30 min in conical tubes. Carefully pipette the solution out of the conical tube and discard.
28. Resuspend the pellet in 30 ml of distilled or deionized water by vortexing.
29. Repeat steps 2.27-2.28 15x to thoroughly wash the pellet.
30. After centrifugation, remove the supernatant and save the pellet for freeze drying. Once freeze-dried, place the sample in a vial labeled ‘lignin’.

3. Preparation of Microbial Growth Cultures

Agar Plates

1. Add 0.2% (w/v) sterile lignocellulose extraction products to M9 minimal media¹³ agar before pouring plates.
2. Streak solidified plates with approximately 200 ul of a bacterial culture grown in M9 minimal media with an O.D. of approximately 2.0.
3. Incubate plates at 37 °C and monitor growth.
   Liquid Cultures
4. 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.
5. Inoculate the media with 0.1% (v/v) of a bacterial culture at an O.D. of approximately 2.0.
6. Incubate the cultures at 37 °C with shaking at 200 rpm.
7. Assess growth every 6 hr during the culturing period by measuring optical density (λ = 600 nm). Place a 1 ml aliquot of the culture into a cuvette. Determine optical density of culture blanked against uninoculated media.
8. Transfer bacteria to an anaerobic serum bottle at mid log phase.
   1. 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.
   2. Transfer the entire 50 ml culture from the 125 ml flask into the sealed sterile serum bottle using a syringe.
   3. Incubate at 37 °C with shaking at 200 rpm.

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.

1. Centrifuge uninoculated and inoculated bacterial cultures (50 ml) at 9,300 x g for 30 min after 450 hr of incubation.
2. Transfer 10 ml of the supernatant into a glass test tube. (Note: Plastic is incompatible with the chemicals in the following steps.)
3. Acidify the supernatant with concentrated HCl to pH 1-2.
4. Add 3 volumes of ethyl acetate (30 ml), cap the tube and mix by inverting 4-6x.
5. Place a small amount of anhydrous Na₂SO₄ (1-2 g) in a clean glass test tube.
6. Carefully collect the organic layer (top) from the ethyl acetate solution by pipetting with a disposable, glass Pasteur pipette.
7. Place the organic layer in the test tube with anhydrous Na₂SO₄.
8. 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.
9. Place a sheet of filter paper into a Büchner funnel on top of a side arm flask attached to a vacuum.
10. Pour the solution into the funnel. Discard the solid material.
11. Transfer the solution to an evaporating flask and evaporate the filtrate using a rotary evaporator.
12. Place 3 mg of ethyl acetate extraction residue into a 2 ml amber chromatography vial.
13. Dissolve the residue with the addition of 100 µl of 1,4-dioxane and 10 µl pyridine.
14. Add 50 µl of N,O-Bis(trimethylsilyl) trifluoroacetamide with trimethylcholorsilane 99:1% (BSTFA). Then place in an incubator at 60 °C with shaking for 15 min.
15. Store samples at 4 °C until use.

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.

1. After the oven temperature is stabilized at 50 °C, inject 1 µl of the sample at a 1/50 split ratio.
2. Hold the column at 50 °C for 5 min and increase to 280 °C at 5 °C/min. Maintain the final temperature of 280 °C for 20 min.
3. Maintain the transfer line between the gas chromatograph and mass spectrometer at 300 °C.
4. Select a solvent delay of 15 min.
5. Record electron ionization mass spectra between the range of 10-500 (m/z) at electron energy of 70 eV.
6. 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.

תוצאות

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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
1,4-Dioxane (Certified ACS)	Fisher	D111	Flammable, preoxidizable chemical
Black Liquor	Department of Forest Biomaterials at North Carolina State University	N/A	pH 12.72, 13.67% solids
Ethanol (microbiology grade)	Fisher	BP2818
Ethyl Acetate (ACS reagent grade)	EMD	EX0240-9	Flammable
Glacial acetic acid (Certified ACS)	EMD	AX0073	Corrosive
Guaiacol	Sigma Aldrich	PHR1136	Harmful by ingestion, corrosive
HP-5 capillary column	Agilent	19091J-577	60 m x 0.18 mm internal diameter, 0.18 μm thickness
Hydrochloric acid (certified ACS)	EMD	HX0603P
N,O-Bis(trimethylsilyl)trifluoroacetamide with trimethylcholorsilane 99:1% (BSTFA)	Fluka	15238	Flammable, causes skin burns and eye damage with contact
Na2SO4 (FCC grade)	VWR	BDH8026
NaClO2 (80%)	Sigma Aldrich	244155	Flammable, toxic
NaOH (certified ACS)	EMD	1.06498.1000	Corrosive
Alkacid pH test ribbons	Fisher	A979
Phosphoric Acid (certified ACS)	Fisher	A242
Polaris Q mass spectrometer	Thermo Electron Corporation
Pyridine (99%)	Alfa Aesar	A12005	Flammable, toxic in contact with skin
Switchgrass	Cherry Research Farm Goldsboro, NC	N/A	Harvested August 2011
Thermo Finnigan trace gas chromatograph	Thermo Electron Corporation
Whatman no. 1 filter paper	Whatman	1001-150