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, org....

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 pourin....

Results

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

List of materials used in this article

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