Decomposition of Lignin with Escherichia coli

2nd place Southern Korean Activities Conference (SKAC) Science Fair

-Passed competition at Korean Science & Engineering Fair (KSEF)

 

Background Research

Biomass has recently been in the attention of many manufacturers to its potential of becoming a widespread source of deriving the world’s energy. Biomass is an organic renewable energy source that uses agriculture and forest residues to convert to fuels such as gasoline, diesel, and jet fuel. It is especially valuable because natural sources such as plants, animals, and their byproducts can be valuable resources for the creation of bioenergy. Furthermore, bioenergy has the potential to decrease carbon footprint can improve the environment. Unfortunately, the cultivation of mass amounts of biofuel is a difficult, arduous, and expensive process than using traditional fossil fuels to obtain energy.

A prevalent type biofuel, ethanol, is produced from biomass mostly from a fermentation process in which feedstock is converted into glucose, then in which feedstock is converted into glucose, then the glucose is transformed by yeast into ethanol. In the process of converting corn into bioenergy, one of the biggest problems is lignin. Lignin is a polymer of aromatic subunits that serve as a matrix around polysaccharide components of plant cell walls. Lignin cats as a crucial support for plant growth. As one of the world’s most abundant natural polymers along with cellulose and chitin, Lignin acts as a crucial material for the formation and strength of cell walls. The material is especially crucial for corn. It forms the corn’s structures and accounts for 20 to 30% of the entire corn. However, In the process of converting corn to biofuel, lignin acts as a serious impediment.

There are limitations to lignin valorization in lignin conversion to useful chemicals and future biorefineries. The incompatible nature of converting hydrophobic materials into biomass propose challenges to scientists and engineers. In order to get rid of lignin, mass amounts of energy are used, and lignin becomes impractical waste that is difficult to get rid of. Scientists have been studying ways to get rid of lignin using genetic modification. However, the use of genetic modification in growing corns can inhibit growth and durability of corn.

By nature, lignin has an ether structure that is extremely complex and difficult to decompose. In this study, I tried to find a way to alter the ether structure in order to effectively disintegrate lignin so it naturally disseminates and does not become hindrance. If lignin can be efficiently be decomposed using microbes, it is possible to raise the production of bioethanol and have less organic substances go to waste and decrease energy costs used to rid lignin.

Biodegradable plastics made of degradable polymers have similar structures to lignin. The polymers largely consist of ester, amide, and ether groups, and they are often synthesized in various ways.

Escherichia coli are a very common bacteria. The main reservoir of E.coli strains is grass-feeding animals, and humans could acquire infection by consuming food or water contaminated with E.coli. Non-pathogenic strains of E.coli are normal inhabitants of humans and animals. Japanese researchers have discovered that the bacterium can feed on plastics. They found that E,coli’s secretion of terephthalic acid could make plastic have a insight to how microbes in nature have evolved to degrade plastics.

 

Hypothesis

My hypothesis is that Escherichia coli will be able to aid in the decomposition of lignin because it has seen efficient results in aiding the decomposition of biodegradable plastics, which have similar structures to lignin.

 

Problem & Purpose

There are limitations to lignin valorization in lignin conversion to useful chemicals and future biorefineries. The incompatible nature of converting hydrophobic materials into biomass propose challenges to scientists and engineers. In order to get rid of lignin, mass amounts of energy are used, and lignin becomes impractical waste that is difficult to get rid of. Scientists have only been trying to find ways to limit the production of lignin through genetic modification. In this study, I tried to find a way to alter the ether structure in order to effectively disintegrate lignin so it naturally disseminates and does not become hindrance. Using the notion that E.coli can effectively decompose biodegradable plastics, I wanted to see if the bacterium would be able to degrade lignin. I wanted to find an ecofriendly way to convert biomass into fuel with the least amount of lignin waste possible.

 

Materials

Lignin 5g, Water 50ml, KBr, beaker, rubber stopper, erlenmeyer flask, petri dish with E. coli, petri dish, Fourier Transform Infrared Spectrometry (FT-IR), Scanning Electron Microscope (SEM), High Performance Liquid Chromatography (HPLC), Gas Chromatography-Mass Spectrometry (GC/MS)

 

Procedure

FT-IR

Mix 5g of lignin and 50ml water in a beaker. Prepare a colony of E.coli in a petri dish. Mix the E.coli into the beaker with lignin at 60 degrees Celsius and pour the beaker onto a petri dish. Dry for 2hours. On the petri dish with E.coli, mix KBr. Use the FT-IR at scan range 400cm¯¹ ~ 4000cm¯¹ on the two petri dishes.

1

Morphology

Transfer the liquid with lignin and E.coli into an Erlenmeyer flask. Block the opening with a rubber stopper. After two weeks of observation, use the Scanning Electron Microscope on the liquid. Use the SEM with the beaker with only lignin and water for comparison.

23

Liquid Chromatography

Use liquid chromatography to separate the products of combination of lignin and E.coli for a period of 40minutes. Set the settings so that the pumping speed is 0.5ml/min.

4

Gas Chromatography

Turn the gas chromatography device on and make its setting so that the gas used is helium, temperature is between 100 to 250 degrees Celsius, and the analyzation time period is 40 minutes.

5

 

Data Analysis

FT-IR

According to the FT-IR graph, before E-coli was introduced to lignin as seen on the right side of the graph, it can be seen how E.coli is consuming KBr and secreting cellulose as the KBr is decomposing. After lignin is introduced as seen on the right, large amounts of cellulose are also produced, which shows that E.coli has an effect on decomposing lignin. The graph which shows absorbance at different wavelengths shows a peak in which the production of cellulose is the greatest. This large peak shows that E.coli is apt at the breakdown of lignin.

6

Morphology

At the end of two weeks, it has been shown how E.coli produced white circular waste and that the Erlenmeyer flask has become clouded with white waste. This can be seen as waste that E.coli produces as it decomposes lignin. When checked with the SEM pictures, it is visible that E.coli has an effect on lignin by attaching to the surface of lignin. The before and after pictures show that rigid and uniform. However, the picture with lignin and E.coli explains that over time, E.coli can effectively disintegrate the structure of lignin.

7

8

Liquid chromatography

In the HPLC, it was possible to analyze Styrene Monomers as products of the reaction with E.coli and lignin. Although there were a peaks of monomer p-coumaric, there was nearly an absence of Styrene Monomers 4-vinyl phenol, which is assumed to be have been due to disintegration from cell metabolism. These results tell that E.coli has effectively influences lignin structures.

9

Gas chromatography

The GC/MS graph shows that at a temperature range of 100 to 250 degrees Celsius, there is high indication of 4-vinylphenol at 10.04 minutes. From this reaction in which 4-vinylphenol is created, it is deducible that E.coli has effectively aided in the decomposition and structure alteration of lignin.

10

 

Conclusion

Biomass is held as the most compatible resource that can meet the needs of being eco-friendly, sustainable. Within biomass, corn is one of the most used natural resources for creating the world’s fuel. In this study, I researched a natural way in which researchers could get rid of lignin, which poses many environmental and energy problems when corn is converted to biofuel. The results show the reaction of lignin and E.coli through production of cellulose which is E.coli waste from feeding on lignin, change in lignin structure in lignin. These point to the conclusion that E.coli does aid in the decomposition of lignin. With this research, if studied further, we can hope for a future in which we can find a natural way using microbes to get rid of lignin, rather than using gene modification or chemical ways. If 4-vinylphenol is refined and altered in an eco friendly way for the production of biomass, it is possible that researchers can produce biomass, it is possible that researchers can produce biofuel without lignin. Although lignin has not been successfully altered due to its complex bonding structure, if this study is examined further and the monomers of lignin can be altered in more various ways, it will possible to decrease biomass waste.

 

 

Works Cited
Lignin Removal from Barley Straw by Ethanosolv Pretreatment. 김영란, 융안나, 정봉우, 한민희, 최기욱. KSBB Journal 제24권 제6호, 2005. 11, 981-985 (5pages)
Mechanical Properties of Natural Material Reinforced Polypropylene Bio-Composites: The Effects of Chemical Modification of Lignin. 여준석, 이상원, 황석호,. 폴리머 제39권 제6호, 20015. 11, 981-985(5pages)
Strassberger, zea, et al. “The pros and Cons of Lignin Valorisation in Integrated Biorefinery.” RSC Advances, Royal Society of Chemistry of Chemistry, 12 June 2014, pubs.rsc.org/en/content/articlehtml/2014/ra/c4ra04747h.
Alire, Rod. “The Reality Behind Biodegradable Plastic Packaging Material.” United States, FP International, www.foint.com/wp biodegradable plastic.aspx
Utilization of Lignin: Past, Present, Future.문성필
Delignification of Lignocellulosic Biomass with High Boilling Point Solvent and Acidic Catalyst. 김간재, 정진도, 정수은, 홍성범, 엄태진. 펄프종이기술 제 48권 제1호, 2016. 02, 119-126 (8Pages)
Synthesis and Adsorption Properties of Carboxymethyl Lignin with Irradiated Lignin by Electron Beam 이진영, 이영주 ,강필현, 전준자. 플리머 제40권 제1호, 2016. 1, 70-76(7Pages)
New Organosolv Pulping Method of Pinus densiflora by using Acid Catalyst 홍성범, 정수은, 김강재, 엄태진. 한국펄프종이공학회 2016년 춘계학술논문발표회 논문요약집 2016,4, 40-40(1pages)

 

 

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