Emily Hinkle

Dr. Collins
Advanced Biochemistry
24 April 2019

Biodiesel Production from Various Plants with an E. Coli Host Using Bovine Rumen Enzymes

Background and Significance

In 2009, over 172 billion gallons of fuel were consumed in the United States according to

the Office of Highway Policy Information.1 This number represents the destruction of

nonrenewable fossil fuels whose use emits greenhouse gasses such as carbon dioxide into the

atmosphere. A significant barrier to past proposals for mass biodiesel production with the

potential to displace petroleum fuel was a nonrenewable carbon source and high price.2 An

efficient and affordable alternative to these fuels is needed.3,4

Recently, many studies have been done involving the use of microorganisms to produce

biofuel.5,6,7,8 This process is known to emit fewer greenhouse gasses, and require little change in

the current fuel infrastructure.9,10 Biofuel has been derived from vegetable oil, fats, and lipids,

but without E. coli, this process has been too expensive for mass production. E. coli have the

ability to produce biofuel directly from lingo-cellulosic sugars when engineered to do so.11 This

allows them to bypass the transesterification process that is necessary to produce biofuel from

fats and decreases the cost associated with previous attempts at mass biofuel production.12,13

A study by Bokinsky, et al used endogenous promoters to mutate E. coli to grow on ionic

liquid pretreated switchgrass. Switchgrass was chosen due to its ready availability and lack of

defined purpose. The E. coli with cellulose-consuming enzymes, including the PcspD controlling

the osm Y-cel which had the highest cellulase activity, were engineered to include pathways

producing fatty-acid ethyl ester-derived biodiesel, butanol, and pinene directly from IL-

pretreated switchgrass. This study engineered E. coli with biodiesel-producing abilities, but the

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bacteria had low efficiency at breaking down cellulose in switchgrass, with only 8% of available

cellulose being broken down.14

Another study was done by Hess, et al which isolated rumen enzymes from cattle and

tested their effect on IL-pretreated switchgrass to glucose in an E. coli host. This study reported a

37% dry mass reduction of switchgrass after 72 hours, largely due to degradation of cellulose

and hemicellulose.15 Although switchgrass is a high cellulose plant, it can be used for hay to feed

cattle, so an alternative cellulose source is needed for larger scale production. Cirsium,

commonly known as the Canada thistle, is a plant which is a nuisance to many farmers, and

could provide a source of cellulose that does not have other uses. Cattle will not eat Cirsium due

to its coarseness, so currently the only option is to remove it from fields to prevent it from taking

nutrients that other plants could use. Using this plant for biodiesel would create a high cellulose

source that is readily available.

My study will compare the efficiency of the engineered biodiesel producing E. coli to

that of E. coli with the cow rumen enzymes expressed and the biodiesel pathway inserted. These

E. coli will be given IL-pretreated switchgrass as a cellulose source and their efficiency

measured by the volume of biodiesel that each type of bacteria produce. Additionally, the E. coli

with the expressed cow rumen enzymes will be tested with IL-pretreated Cirsium to determine

their ability to degrade the cellulose into glucose.

Hypothesis

Hypothesis: E. coli with induced bovine enzymes will produce more biodiesel from a

switchgrass carbon source than E. coli with promoted endogenous degradation enzymes and

produce more biodiesel when given pretreated Cirsium thistle

Experimental Aims

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Specific Aims:
1. Compare fatty-acid ethyl ester derived biodiesel yield from E. coli with activated

endogenous promoters and E. coli with inserted bovine rumen enzymes using switchgrass

as a cellulose source

2. Determine efficacy of bovine rumen enzymes MH-3, MH-20, MH-2, and MH-5 in

degradation of ionic-liquid pretreated Cirsium thistle to glucose in an E. coli host

Experimental Design

To address the first aim, the promoter-driven E. coli produced by Bokinsky, et al will be

replicated.14 The E. coli containing rumen enzymes will be made by isolating genes MH-2, MH-

3, MH-5, and MH-20 as discovered in the study by Hess, et al, from cow rumen tissue using a

polymerase chain reaction mechanism. These genes will each be cloned onto a separate

pALTER-Ex1 plasmid. Four sequential ligation reactions will be performed to transfect the E.

coli with the plasmids containing the rumen enzymes and successful transfection will be detected

by plating the E. coli with tetracycline. The biodiesel pathway will be added to a pES120

plasmid and transfected into both E. coli with endogenous promoters and those with the rumen

enzymes inserted. Switchgrass will be pretreated in 1-Ethyl-3-methy-limidazolium acetate to

expose the cellulose for the E. coli to digest. The IL-pretreated switchgrass will be placed in

minimal media at a concentration of 5.5% w/vol as the only carbon source. The E. coli will be

grown in this media for 72 hours and the amount of biodiesel produced by each type of E. coli

will be measured and compared.

To address the second aim, Cirsium thistle will be IL-pretreated and placed in minimal

media as the only carbon source. The E. coli with rumen enzymes will be grown in this media for

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72 hours. The amount of cellulose broken down at the end of the 72 hours will be measured to

determine the efficiency of these enzymes and the validity of Cirsium as a carbon source.

Expected Outcomes:

It is expected that the cow rumen enzymes will allow the engineered E. coli to more

efficiently break down the cellulose in switchgrass, resulting in a higher overall biodiesel

production. The higher production of biodiesel from less switchgrass would indicate a more

efficient mechanism for producing biodiesel which can be more easily integrated into the current

fuel infrastructure. If the rumen enzymes do not lead to more efficient production, this will show

that there must be another problem than the inefficiency of cellulose breakdown with the

endogenous promoters. This could guide the direction of research in this area toward figuring out

the mechanism by which the cellulose is converted into biodiesel and finding a way to improve

production. An alternative approach would be to clone all of the rumen enzyme genes onto the

same plasmid to see if this changes the efficiency of biodiesel production. It is also expected that

the E. coli with inserted rumen enzymes given Cirsium will be able to break down more

cellulose from this plant. If this is not true, this could cause more research into plants that are

currently wasted as a potential carbon source for future biodiesel production.

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