METU HS IGEM

# Project Description

### BIO-E: Bacterial Improvement of Ethanol

#### Our Motivation

Energy sources are divided into two: non-renewable and renewable. Non-renewable energy sources cannot replenish in a short time, thus are unsustainable (U.S. Energy Information Administration [eia], 2017a).

Fossil fuels; like petroleum and coal are examples to non-renewable energy resources. Governments around the world spare an iffy budget to acquire them even though they are not sustainable (Melikoğlu & Albostan, 2011; Patton, 2016; Summerton, 2016). Apart from their drastic effects on the economy and global schemes around the budget, all non-renewable energy sources, impose catastrophic effects on the environment from high greenhouse gas emissions to impairment of natural habitats (Hamilton, 1998).

Figure 1: Our representation of the economic setbacks and environmental disasters; such as oil spills and the increase in air pollutants fossil fuels’ use leads to.

Therefore, we have decided on solving this issue with the improvement in renewable energy sources and thereby decreasing fossil fuel consumption. In this path, we chose the most promising energy source, biofuels.

#### What are Biofuels?

Biofuels are fuels obtained from biomass, organic materials that come from plant wastes (EIA, 2017b, EIA, 2017c). One of the most important applications of biofuels is transportation since solar, wind and other alternative powers are not sufficient and cost-effective enough to produce the required amount of energy that can be used in this manner (Biofuels, n.d.).

#### Why Bioethanol?

In essence, bioethanol is no different from daily used ethanol; having the formula of C2H5OH (Zabed et al., 2014). Moreover, bioethanol has a higher octane number, evaporation enthalpy, flame speed and a wider range of flammability than the petroleum-based energy sources which increase its fuel consumption rate, making bioethanol more environmentally friendly (Zabed et al., 2014).

Bioethanol also contains 35% oxygen which is important because of the reducing effect on particulate and nitrogen oxides emission as well as other greenhouse gases such as carbon monoxide, during combustion (Zabed et al., 2014).

In brief, we chose bioethanol as our main focus due to its qualities of being eco-friendly, non-toxic, biodegradable, it reducing crude oil dependency from other countries and most importantly, emitting fewer greenhouse gases, thus creating less pollution (Oliveira et al., 2005; Tesfaw & Assefa, 2014; U.S. Energy Information Administration [eia], 2014; 2017; Melikoğlu & Albostan, 2011; Holzman, 2007).

In order to observe the advantages of ethanol in practice, we found the data below that compares engines that utilize ethanol, clean diesel, and electric-gasoline hybrid to conventional gasoline engines. Our research and observations lead to the table below:

Clean Diesel Electric hybrid Ethanol
Cost $3,000–3,500 per gallon$3,500–5,000 per battery \$1,000-1,500 per gallon
Efficiency Gain 20–30% more efficient 30–40% more efficient 20–30% more efficient
Emissions 25% lower CO2 emissions Up to 50% lower CO2 emissions NOx and PM reduction, compared with clean diesel

Less complex and easier to install than EH engine
Better engine performance

Larger battery means more safety and luxury electronic systems can be added one
Reduced engine weight, more space in the engine compartment, compared with electric hybrids.

Higher torque and horsepower, compared with clean diesel
Table: Comparison of ethanol, clean diesel, and electric-gasoline hybrid engines to gasoline engines by David C. Holzman (2007).

As we further investigated the ways to obtain bioethanol, we came across the concept of energy generations.

#### Energy Production Approaches

There are three energy generations that are currently in use: first, second, and third. These generations can be thought of as three different approaches to obtain energy, more specifically bioenergy.

The first generation energy evokes concerns in terms of the debate “food vs fuel”; since it is obtained from food crops such as corn, sugarcane, sugar beet and etc. (Aro, 2016).

On the other hand, the second-generation production uses non-edible lignocellulosic biomass, such as rice husks, wheat straws and so on, creating no such controversy as the first generation. In addition, production with second-generation energy requires less cultivation, fertilizers, and pesticides when compared to the first generation (Aro, 2016; EIA, 2017d).

Third generation biofuels are from algae cultures. Algae biofuels are common, provide a good alternative to fossil fuels and thought of as long-term energy sources (Hannon et al., 2014). However, this technology has many challenges; such as the need for strain identification and improvement, before it can compete in the fuel market (Hannon et al., 2014).

Microalgae, on the other hand, are photosynthetic organisms that also create contrast in this market (Brennan et al., 2010). Although they can provide an enormous amount of lipids, proteins, and carbohydrates in a short period of time, they are fragile and have numerous needs; from the percentage of the nutrients in their environment to the amount of sunlight they receive (Brennan et al., 2010). Moreover, another disadvantage is that algae, in newly introduced environments, are observed to act invasively; altering ecosystems, reducing biodiversity and causing overall economic losses (Demirbas, 2010).

In the light of our studies, we decided to improve second-generation bioethanol production where it can confidently compete with petroleum and other fossil fuels.

#### What are the Problems with Second Generation Bioethanol Production?

Figure 3: The chemical structures of lignin, cellulose and hemicellulose. Retrieved from: https://www.researchgate.net/figure/3-Structure-of-lignocellulosic-biomass-with-cellulose-hemicellulose-and-lignin_fig3_321075806

The main drawback of using lignocellulosic material is the structural components: lignin, cellulose, and hemicellulose.

Unlike cellulose and hemicellulose, lignin is highly resistant to chemical and physical processes (Yang & Wyman, 2008). Therefore, in order to hydrolyze the cellulose and hemicellulose into fermentable sugars, they must be separated from the lignin structure, leading to the need for a pretreatment process (Yang & Wyman, 2008).

Figure 4: Representation of the structure of the lignocellulosic material; how the pretreatment method separates lignin, cellulose, and hemicellulose; and the outputs: the solid residue and liquid hydrolyzate. Retrieved from: https://www.e-education.psu.edu/egee439/node/653

The problem is that the most common pretreatment method which is dilute acid pretreatment generates byproducts such as hydroxymethylfurfural (HMF), furfural and reactive oxygen species (ROS) that inhibit microbial metabolism, severely affect redox system and cause fatal disruptions in the cell system; ultimately leading to the death of the cell (Kumar & Sharma, 2017; Höck et al., 2013; Ask et al., 2013; Jönsson & Martín, 2016; Wang et al., 2013).

Figure 5: Our representation of how the furan output harms the bacteria and decreases the ethanol yield.

#### Our Solution

In order to enhance the ethanol fermentation process, we engineered ethanologenic E.coli strain KO11 to increase its tolerance to the toxic byproducts by integrating FucO and GSH genes.

Figure 6: Our representation of engineering the ethanologenic E.coli strain KO11 with our genes and creating Bio-E.
Figure 7: Our representation of Bio-E’s gene circuit.

The byproducts furfural and HMF act as thiol-reactive electrophiles and as a result, cellular glutathione levels get depleted in their presence, leading to the accumulation of reactive oxygen species (Kim et al., 2013).

Reactive oxygen species (ROS), a kind of free radicals are molecules that are unstable due to their unbalanced electron state. These radicals are an inseparable deadly byproduct of anaerobic life and many other reactions essential to organisms. (Free Radicals and Reactive Oxygen, n.d., “oxygen radicals”; Livingstone, 2001). Although easy to come by, ROS are responsible for mutagenesis, cancerogenesis and aging on multicellular organisms (Halliwell, 1993).

Cells utilize antioxidants to stabilize the distorted electron balance of free radicals by donating an excess electron (Pizzorno, 2014). Thus, overexpression of the master antioxidant, Glutathione, improves the tolerance of the bacteria to free radicals, reactive oxygen species and ultimately enhances cell growth (Pizzorno, 2014).

Figure 8: Our representation of free radicals, lacking an electron.

When furfural, another byproduct is present in the field, its reduction mechanism by NADPH-dependent oxidoreductases goes active (Zheng, 2013). In this mechanism, the expression of oxidoreductases that are NADPH-dependent, such as YqhD, are shown to inhibit the growth and fermentation in E.coli by competing with biosynthesis for NADPH (Zheng, 2013). The native conversion of NADH to NADPH in E. coli is already insufficient to revitalize the NADPH pool during fermentation, so the actions shouldn’t be interfering with NADPH metabolism as it leads to reduced cell mass followed by cell death (Wang et al., 2011).

Thus, the overexpression of plasmid-based NADH-dependent propanediol oxidoreductase (FucO) gene will reduce the need for oxidoreductases that are NADPH dependent, and convert furfural to furfuryl alcohol without interfering with the biosynthesis of NADPH (Wang et al., 2011).

In normal circumstances, both the detoxification of ROS and the conversion of HMF & furfural result in a more oxidized intracellular environment; by interfering with the NADPH metabolism thus deteriorating the antioxidant defense system of the cell, decreasing its mass and ultimately resulting in death. With these considered, we have come to the conclusion that the solution to this problem was in the dual overexpression of GSH and FucO (Ask et al., 2013).

#### Conclusion

For a more efficient production of bioethanol and bacteria with higher tolerance to toxicity and oxidative stress, our research has shown that the system is in need of a genomic improvement that can be provided with GSH and FucO (Kim & Hahn, 2013). This can be inferred from the fact that an increase in GSH enhances tolerance to furfural but not to 5-HMF, while FucO on the other hand is in need of GSH as it provides a longer lifespan and deals with the interactions of free radicals (Kim & Hahn, 2013).

So, we have come to the conclusion that the simultaneous overexpression of GSH and FucO would increase cellular growth rates, lifespan and ethanol yield in E. coli ethanologenic strain KO11 since GSH is the master antioxidant in living organisms coping with ROS; and FucO is the gene that converts HMF & furfural to non-toxic alcohols without interfering with the NADPH metabolism (Ask et al., 2013; Höck et al., 2013; Wang et al., 2011).

Figure 9: Our representation of using Bio-E in the fermentation process. The significance that Bio-E makes is higher ethanol yield, intact and tolerant bacteria and the conversion furfural to furfuryl alcohol.