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

The overall goal of the following experiment is to determine the ability of a microorganism or consortia of microorganisms to utilize the pulping waste Black Liquor for Growth, the components of black liquor that are being used for growth and the products produced by microbial metabolism of this pulping waste. This is achieved by collecting the pulping waste Black liquor and preparing it for use as a culture medium component. As a second step, lignocellulose components are extracted, which are used to determine carbon source requirements of the microorganisms.

Next media are prepared, inoculated, and analyzed for microbial growth in order to determine the ability of the microorganisms to grow on black liquor and its components. The results show utilization of black liquor as the sole carbon source for growth and synthesis of metabolic products by the microbial environmental isolate based on its growth visualized on minimal media agar plates, and by GCMS. We first had the idea for this method when we were discussing the possibility of microbes being able to grow on pulp waste products and that we might be able to isolate these microbes directly from a pulp mill.

The main advantage of zignal cellose extraction technique over existing method today, like sodium hydroxide bleaching, is that the sodium chloride bleaching offer high and increased purity. Visual demonstration of this method is critical. As Theo cellulose and GCMS sample preparation steps are difficult to learn, and this is due to the number of steps required and because of the visual determination needed to verify process completion.

This method not only can provide insight into microbial degradation of black liquor, it also can be applied to industrial waste streams containing Magno cellulose materials such as crop residues, forest residues, and municipal solid waste. To begin this procedure, collect the black liquor sample from the outlet valve attached to the craft digester and a sterile glass bottle, and allow it to cool to room temperature before proceeding to the next step. Then add 100 milliliters of the black liquor sample to a 500 milliliter beaker equipped with a stir bar and stir the sample at medium speed.

Neutralize the black liquor by adding phosphoric acid dropwise until it becomes viscous. Following this aliquot all the black liquor solution into 50 milliliter conical tubes and centrifuge the samples at 9, 300 Gs for 30 minutes. To separate the carbohydrates, place five grams of one millimeter or smaller milled switchgrass and a stir bar into a 500 milliliter flask.

Then add 200 milliliters of distilled water, 7.5 grams of sodium chlorite, and 2.5 milliliters of glacial acetic acid. Place the flask in an oil bath placed on top of a stir plate at 80 degrees Celsius for one hour. Then add an additional 7.5 grams of sodium chloride and 2.5 milliliters of glacial acetic acid to the flask.

Continue stirring at 80 degrees Celsius for 30 minutes after repeating the previous step two times. Allow the solution to cool to room temperature. Then filter it through a buchner funnel under vacuum to isolate the solid material.

Wash the solid material three times with 100 milliliters of distilled water. Next, collect one gram of the solid material and place it onto a clean sheet of filter paper. Place the solid at 50 degrees Celsius to dry overnight.

After removing the sample from the oven the following day, transfer it to a storage vial labeled hollow cell. Add the remaining solid material to a 500 milliliter flask containing 250 milliliters of 10%sodium hydroxide, and cap the flask with the stopper. Place the solution in an incubator at 70 degrees Celsius for one hour with shaking at 250 RPM.

Following this, allow the solution to cool to room temperature After 30 minutes, isolate the solid material via vacuum filtration using a buchner funnel. Once the solid residue has been dried overnight in a 50 degrees Celsius oven, place it in a storage vial labeled cellulose. Transfer the filtrate to a 500 milliliter flask and add 30 milliliters of acetic acid and 250 milliliters of isopropyl alcohol cap and place the resulting solution on the benchtop for at least eight hours at room temperature.

When finished, transfer all of the solution to 50 milliliter conical tubes and centrifuge the sample at 9, 300 Gs for 30 minutes. Following centrifugation, carefully pipette the solution out of the conical tubes and discard. Next, wash the pellets with 30 milliliters of distilled water by vortexing and centrifuge at 9, 300 Gs for 30 minutes.

Repeat this step 15 times to thoroughly wash the pellet. After centrifugation, remove supernatant to isolate the pellet for freeze drying. Once the sample has been freeze dried, place it in a vial labeled hemi cellulose.

For lignin separation, add five grams of one millimeter or smaller milled switchgrass to a 500 milliliter solution of point 25 molar sodium hydroxide, and 30%ethanol. Place the slurry in an incubator at 75 degrees Celsius with shaking at 250 RPM for two hours. After filtering the cool solution, add a stir bar to the flask and place it on top of a stir plate adjusted to medium speed.

Add concentrated hydrochloric acid dropwise to the filtrate until a pH of two is obtained. Allow the solution to sit overnight at room temperature. Once the solution has been transferred to 50 milliliter conical tubes, centrifuge the samples at 9, 300 GS for 30 minutes.

When finished, carefully pipette the solution out of the conical tube and discard. Wash the pellet with 30 milliliters of distilled water by vortexing and centrifuge at 9, 300 Gs for 30 minutes. Repeat this step 15 times to thoroughly wash the pellet.

After centrifugation, remove the supine natant to isolate the pellet for freeze drying. Once freeze dried, place the sample in a vial labeled lignin. Add 50 milliliters of sterile M nine minimal media to a sterile 125 milliliter flask.

Then add 10%by volume of the black liquor to the media. For inoculation add 0.1%by volume of an overnight bacterial culture to the mixture, incubate the cultures at 37 degrees Celsius in an incubator with shaking at 200 RPM. At mid log phase, transfer bacteria to a sealed sterile anaerobic serum bottle using a syringe following centrifugation of the UN inoculated and inoculated bacterial cultures.

After 450 hours of growth, add 10 milliliters of each supernatant into separate glass Test tubes. Acidify each supernatant with concentrated hydrochloric acid added dropwise to pH one to two. For each sample, add 30 milliliters of ethyl acetate to the supra natant.

After capping the test tube, mix the solution by inverting the tube four to six times. Then place one to two grams of anhydrous sodium sulfate in a clean glass test tube. Carefully collect the top organic layer by pipetting with the disposable glass pasture pipette.

Place the organic layer in the test tube containing the anhydrous sodium sulfate. Dewater the organic layer over anhydrous sodium sulfate by gently tapping the test tube to mix Gradually. Add small amounts of anhydrous sodium sulfate and mix until there are no large clumps.

Following this, filter the solution through a buchner funnel under vacuum, then transfer the solution to an evaporating flask and evaporate the filtrate using a rotary evaporator. When finished, place three milligrams of the ethyl acetate extraction residues into a two milliliter amber chromatography vial. Dissolve the residue with 100 microliters of one four dioxane and 10 microliters of pyridine.

Then add 50 microliters of a solution of B-S-T-F-A with 1%trimethyl chlorine to the vial. Place the solution in an incubator at 60 degrees Celsius with shaking for 15 minutes. Analyze the sample by GCMS.

The collection and modification of black liquor generated in the craft pulping process will one, to use this pulping waste to determine the biodegradation capacity of a single bacterial isolate or mixed culture. Shown here is the aerobic growth measured by optical density of an environmental strain cultured in LB media and LB media supplemented with 10%neutralized black liquor. These results indicate that this bacterium grows in the presence of black liquor.

Growth of the environmental isolate in LB media is faster than when grown in the presence of black liquor, but optical density begins to decrease after 62 hours. Whereas growth of the microbial environmental isolate in the presence of black liquor continues to increase in optical density until after 230 hours of growth. The growth curve of the microbial environmental isolate in the presence of black liquor also depicts biphasic growth, which suggests that the black liquor contains more than one carbon source that can be used by the microbial environmental isolate, and that there is a preference of nutrient utilization exhibited by the bacterium.

Further experiments are necessary to determine its ability to utilize black liquor for growth requirements. One may also determine growth of a bacterium on non neutralized black liquor or determine the effect of increased concentrations of black liquor on growth. After demonstrating growth on black liquor, one may determine carbon source utilization using minimal media growth experiments.

Commercial substrates that make up the components of lignocellulose can be used as carbon sources in the minimal media. The LIGNOCELLULOSE extraction protocol presented here provides a model for determining the growth requirements of the bacterium pictured Here is growth of the environmental microbial isolate on M nine. Minimal media agar supplemented with each component of the lignocellulose extraction.

The presence of colonies on the minimal media agar plates indicates the ability of the environmental isolate to degrade holo cellulose, cellulose, hemi cellulose, and lignin colonies present on M nine. Minimal media may suggest the ability of the environmental isolate to degrade agar instead of plates. Minimal media growth curves for liquid cultures could also be used to determine the specific rate of growth on each lignocellulose fraction so that agar, which could be a potential carbon source, would not have to be added.

The analysis of extracted and deriv eyes, bacterial cultures by GCMS reveals the metabolic products produced. Shown here is the GCMS spectrum of TMS derivatives present in the un inoculated sample, the spectrum of TMS derivatives produced by the environmental microbial isolate when grown anaerobically and M nine, minimal media with 10%non neutralized black liquor is pictured here. Comparison of the two spectra reveals differences in the components present in the media suggesting the presence of fermentation products and the degradation of black liquor components.

A peak with a retention time of 39.34 minutes appeared in the inoculated sample. A peak with a retention time of 21 point 84 minutes increased in the inoculated sample. A peak with a retention time of 25.79 decreased in the inoculated sample.

The spectrum shown here is that of the guac standard, which was used to identify the peak with a retention time of 25.79 minutes. After watching this video, you should have a good understanding of how to determine if a microorganism or a consortium of microorganisms can degrade black liquor. Furthermore, following this protocol, you can use other methods such as growth curves to answer additional questions about the growth rate of a microorganism or microorganisms on various concentrations of black liquor or ligon cellulose extract components.