Project Description | Nanjing-SDG

Abstract

With soaring global energy demand, conventional fossil fuels and nuclear power can no longer fulfill demands without jeopardizing human health and exacerbating greenhouse gas effect. Butyl butyrate’s low viscosity and low carbon emission makes it a biofuel with high potential of alleviating the above problems. In this project, we used Clostridium tyrobutyricum (C. tyrobutyricum) as an ideal chassis cell for its native high-yield synthesis pathway for butyric acid, also known as butyrate, tolerance to butyric acid, its potential high-yield butanol synthesis pathway and butanol tolerance. Its potential production capability of both butyrate and butanol can be further strengthened by directly improving certain enzymes in the pathway and inhibiting competing pathways via genetic engineering. The butyrate and butanol produced by such engineered strain can be used to rapidly produce immense amount of butyl butyrate through lipase catalyzed esterification. Therefore, genetically engineered C. tyrobutyricum can be used for large-scale microbial fermentation for biofuel butyl butyrate.

1 Problem: Energy shortage

Global energy sources have been classified into two types, traditional energy and new energy sources.

Traditional energies are mostly nonrenewable, such as petroleum, natural gas, hydrocarbon gas liquids, nuclear power, and coal. According to the data from United State Energy Information Administration, traditional energy sources provided 87.4% energies for residents in US in 2021[1]. Many serious problems are exposed by using traditional nonrenewable energies. The most serious one is pollution. Toxic particles released by burning fossil fuels are harmful to humans, and they could contribute to the greenhouse effect[2-4]. EESI (Environmental and Energy Study Institute) discovered that in 2019, burning fossil fuels contribute to 74% of greenhouse gas emissions in the United States, which leads to higher global temperature[2]. The leakage of nuclear power could result in radioactive pollution that affects the environment for a long period, like the Fukushima Daiichi nuclear accident happened in 2011[5].

New energy sources generally come from renewable natural resources, like wind, solar, hydropower, geothermal, and biomass energies [6]. Among these energy sources, biomass—organic material from plants or animals—can be converted through combustion, thermochemical conversion, chemical conversion, and biological conversion into biofuels [8-9]. Biofuels can replace traditional, nonrenewable fuels used in transportation, electricity generation, heating and cooking, and industrial and chemical projects [7]. Using biofuels reduces greenhouse gas emissions, lessens dependence on traditional fuels, benefits agricultural development, and promotes energy diversification.

Fig.1 A Pelican covered in oil in Grand Isle (by Louisiana GOHSEP, https://www.flickr.com/photos/lagohsep/4666757323/)

2 Potential biofuel: butyl butyrate

2.1 Introduction of butyl butyrate

Butyl butyrate is an ester of n-butanol and butyric acid (Fig.2). It is widely used as flavors, fragrances, plasticizers, additives, solvents, emulsifiers, electrolyte preparations, etc in various fields such as chemical manufacturing, food, pharmaceutics and agriculture [10].

Fig. 2 Ball-and-stick model of the butyl butyrate molecule

2.2 Butyl butyrate's potential as a biofuel

Butyl butyrate can be used as an excellent fuel additive and even a valuable alternative fuel source especially in the aviation industry due to its special features [10]. It has a high octane rating of 97.3 with excellent compatibility with fuels and similar great fuel properties as gasoline, aviation kerosene, diesel components and certain jet fuels [11]. It has even more favorable combustion properties such as lower viscosity and lower CO emission than conventional jet fuel such as Jet A-1 [12]. Butyl butyrate can be extracted from plants and can potentially be synthesized from lignocellulosic substrates via microorganism fermentation. Therefore, it is a valuable alternative biofuel.

Fig. 3 Sustainable Aviation Fuel advertisement on fuel storage at Frankfurt airport

2.3 Current approach of butyl butyrate production

The conventional synthesis approach for butyl butyrate for industrial purpose is the esterification catalyst method using concentrated sulfuric acid as catalyst (Fig. 4). With this method, butyl butyrate is condensed from butyric acid and butanol under high temperature. Other production methods include transesterification, butyric anhydride method and esterification catalyst method with other catalysts[13].

These methods have some common drawbacks, including high energy consumption for maintaining high temperature and pressure, environmental pollution with waste water/gas and harmful solvents/catalysts, and high production costs for equipment and expensive catalysts. In addition, these methods depend on the supply of raw materials such as butanol and butyric acid. Production of these raw materials adds economic and environmental burdens and instability.

Fig. 4 Chemical production of butyl butyrate using esterification catalyst method

3 Clostridium tyrobutyricum: Candidate microorganism for butyl butyrate synthesis from glucose

Clostridium tyrobutyricum (C. tyrobutyricum) is a Gram-positive and strictly anaerobic strain. Its size ranges from 1.9–13.3 × 1.1–1.6 μm. The optimum growth temperature is 30–37℃, with moderate growth at 25℃ and limited or no growth at 45℃. This bacteria has been isolated from raw milk and dairy products, chicken, fecal material, etc[14]. It is nonpathogenic to humans and animals. C. tyrobutyricum is a good candidate microorganism for the biosynthesis of butyl butyrate, because of two strengths it presents.

3.1 Strength 1: A native high-yield synthesis pathway for butyric acid and tolerance to butyric acid

C. tyrobutyricum is a species of Clostridium. Clostridium natively synthesize butyric acid, also known as butyrate, as a primary end product of their principal metabolic strategy[15-16]. C. tyrobutyricum is among the most promising hosts for the synthesis of butyric acid, and other carboxylic acids, because of its strong acidogenic metabolism and tolerance to high concentrations of acidic products (Fig.5)[17]. Studies have shown that with the supplement of butanol and lipase, fermentation of C. tyrobutyricum can yield large amounts of butyl butyrate, with 50g/L glucose yielding 34.7g/L butyl butyrate [18].

Fig.5 Beneficial traits inherent of and developed for Clostridium tyrobutyricum which make it an exemplary biocatalyst for the production of suites of fuel and chemical precursors from lignocellulosic biomass [17]

3.2 Strength 2: A potential high-yield butanol synthesis pathway and tolerance to butanol

Butyrate and acetate are previously the only products of C. tyrobutyricum, but development in metabolic engineering allowed the manufacturing of butanol, a key chemical precursor to the esterification of butyl butyrate, in C. tyrobutyricum (ebi.ac.uk, 2020; Fig.6). Through modification of C. tyrobutyricum, this butanol output could rise even more. C. tyrobutyricum is also known for its great tolerance for high concentration of butanol [18].

Fig.6 Clostrium tyrobutyricum central carbon metabolism and biosynthetic potential. (Black arrows: known biosynthetic pathways native metabolic intermediates. Green arrows: heterologous genes successfully expressed in C. tyrobutyricum for the production of non-native products. Red arrows: unexplored production routes in C. tyrobutyricum) [17]

4 Our project: microorganism fermentation for butyl butyrate synthesis

Here, we propose a butyl butyrate synthesis method based on microorganism fermentation (Fig.7). Bacteria strains are used to naturally produce the precursors. Our approach can converse lignocellulosic substrates to the biofuel butyl butyrate mainly through the fermentation of engineered bacteria strains. Microorganism fermentation offers a more energy efficient, and environmentally and economically friendly way.

In our proposal, butyl butyrate is rapidly produced via esterification of butyric acid and butanol by a lipase, a common type of enzymes in nature. This esterification process can be carried out at relatively low temperature, atmospheric pressure and mild pH conditions. It is both energy efficient and environmentally friendly. The enzymatic reactions are also usually highly specific and efficient. As a result, fewer byproducts or wastes are generated, reducing downstream processing costs.

To synthesize the precursors, butyric acid and butanol, for this esterification reaction, C. tyrobutyricum is selected as a good candidate strain for microorganism fermentation. With proper engineering, the strain has the potential to manufacture both precursors efficiently and rapidly from lignocellulosic biomass. Its potential production capability of both butyrate and butanol can be strengthened by directly improving certain enzymes in the pathway and inhibiting competing pathway of byproducts via genetic engineering. Since conventional industrial-scale production of the two precursors is largely done via chemical synthesis. Our proposed biological catalyst approach can immensely reduce the carbon footprint and energy cost. Therefore, it’s more environmentally friendly and sustainable in the long term.