Team:UPO-Sevilla/Integrated Practices


Glycerol (C3H8O3) is a polyalcohol, one of the main components of lipids, such as triglycerides. Triglycerides are made up of three fatty acid chains bonded by ester bonds to a glycerol molecule, through their three hydroxyl groups1.

Glycerol was the first organic molecule to be isolated by human beings, in 2800 BC. In 1846, glycerol was used for the production of nitroglycerin, the base for cordite (gunpowder). In 1886, nitroglycerin was mixed with “kieselguhr” (a siliceous sedimentary rock) to produce dynamite. Because of that, glycerol became an important strategic military resource. During the 1st World War, the demand of glycerol increased and it led to the first plants for synthetic glycerol manufacture. In 1943, glycerol production from petroleum began. For over 60 years, the glycerol demand was met by the synthesis from propylene and from soap manufacturing. However, since 2003, the glycerol market has been disrupted due to the surplus of the molecule obtained in the biodiesel and oleochemicals industries2.

Environmentally important properties of glycerol

- Gaseous emission: glycerol has a low vapor pressure and the pure material has no odour.

- Waste water: glycerol is completely biodegradable in sewage treatment plants. In some countries, it is not regarded as a danger to water supply. Nevertheless, the presence of glycerol in wastewater is liable to payment of duty because of its high oxygen demand. For glycerol the Chemical Oxygen Demand (amount of oxygen necessary to oxidize through chemic methods, COD) is 127 mg of O2 per gram. The Biochemical Oxygen Demand (the amount of oxygen that bacteria require to degrade the organic components, BOD) is 780 mg of O2 per gram.

- Storage: stable below 100ºC (non corrosive and little risk of ignition due to its high flash point (minimal temperature at which vapor pressure is highly enough to produce an inflammable mixture with air))

- Toxicology and occupational health: glycerol is not harmful to health. Ingestion even of large amounts causes no harm to humans. The use of glycerol as a food additive is permitted in most countries. Slight skin or mucours irritation is possible on contact with undiluted glycerol because of its strongly hygroscopicity (ability of a substance to attract and hold water molecules from the surrounding environment)3.


Triglycerides from both plant oils and animal fats are used in the industry to obtain biodiesel. This biofuel stands up as an energy fuel obtained from renewable sources, as an alternative to the use of petrol for generating energy. Despite the many problems associated to the use of these fats to obtain energy, such us the need of occupying large areas of crops, it is one of the most promising energy sources4. Biodiesel is obtained through a reaction known as transesterification, for which methanol is used. Methanol comes from methane, a fossil fuel. This reaction consists on the exchange of the acid groups of the esters of the triglycerides to another alcohol (methanol is the most common alcohol used due to its low cost), obtaining fatty acid methyl esters (FAME) (Fig. 1). Biodiesel demand is increasing worldwide, and as its production increases, also does the production of glycerol, by-product of the transesterification reaction4,5.

Figure 1. Transesterification reaction for the obtaining of FAME, having glycerol as by-product. NaOH acts as a catalyst6.

Biodiesel is made up of 10-20% of crude glycerol7. Because of that, the exponential increase in the biodiesel production also leads to high amounts of glycerol, which becomes a waste product with a low commercial value [Fig. 2]. Glycerol production derived from the biodiesel industry has increased from 200,000 tons in 2004, to 1.224 million tons in 20088. In 2012, it exceeded 2 million tones2. In 2007, the price of crude glycerol decreased from about $0.25 per pound to $0.05 per pound (it was $0.70 before the expansion of the biodiesel industry). For these reasons, many researches has focused on developing new ways to absorb the surplus of crude glycerol, so that biodiesel industry can be improved and its production cost can be lowered8. The number of scientific articles concerning new uses for crude glycerol doubled to more than 7000 between 2000 and 20072.

Figure 2. Global glycerol production per year and its price7.


Pure glycerol can be released to the environment. It is nonflammable, and it has low toxicity to fishes and other wildlife. Also, it is biodegradable9. However, glycerol obtained from biodiesel industry is not pure; it contains the toxic molecule methanol (more than 20% of crude glycerol total volume) among other components. For this reason, glycerol derived from the biodiesel industry cannot be directly released to the environment. Some cases of crude glycerol likeage from biodiesel plants have been reported. For example, in 2007 the Alabama Black Warrior river was contaminated by these remains and a biodiesel plant was sued10.

Methanol has health effects to humans: metabolic acidosis, neurologic sequels, irritation of the eyes, nose, mouth and throat, liver damage, headache, cardiac depression, nausea and it can even cause death, when ingested11,12. Methanol exposure may also elicit pain in legs, back and abdominals. Although it is a rapidly biodegradable molecule, it can be dangerous in large quantities. It may also affect birds and fishes, causing their death when released to the environment. Methanol also slows plant grow, reduces fertility, and alters appearance and behavior of the biota12.


Glycerol is a not harmful molecule, and it is used in diverse fields by human beings, such as in animal feedstock and soap making [Fig. 3].

Figure 3. Common uses of glycerol13.

However, glycerol generated in the biodiesel industry is impure and of little economic value. Specifically, it contains toxic methanol and high free fatty acids and salt content2. It is not pure enough to be used in high-tech applications, such as personal care products, cosmetics and food. For these reasons, many attempts to achieve an efficient glycerol purification process are being made, in order to “minimize industrial waste and maximize the utility of biodiesel industrial processes”14. Although efficient purification processes are being discovered, glycerol is still expensive to purify for the food, pharmaceutical or cosmetics industry. Crude glycerol is mainly sold to refineries, but as a consequence of the high amounts produced in recent years, it is sold for only 2,5-5 cents/lb.

As a consequence, many ways for using crude glycerol have been attempted, including combustion, composting and anaerobic digestion15. Combustion produces CO2, which increases greenhouse effect. As cattle can tolerate the poisonous methanol that contaminates crude glycerol, animal feed stock is one of the main industries that use it2. Currently, most efforts focus on using crude glycerol as a raw material for the production of value-added products. Numerous papers have been published on chemicals produced via biological conversions [Table 1]. One of the most important molecules produced by using glycerol as a sole carbon source is 1,3-propanediol, through anaerobic fermentation8. The list of the microorganisms used for this purpose includes Klebsiella pneumoniae16 and Clostridium butyricum17,8.

Table 1. Value-added molecules obtained by using crude glycerol as a sole carbon source8.

Glycerol may also be used as a substrate for chemical catalytic conversions. One example is the production of oxygenated chemicals, such as (2,2-dimethyl-1,3-dioxolan-4-yl) methyl acetate. This compound improves biodiesel properties according to the requirements established by the American and European Standards for flash point and oxidation stability. Other compounds obtained through conventional catalytic conversions are acrolein, hydrogen and monoglycerides8.


The iGEM UPO-Sevilla Team desires to highlight the importance of this environmental issue. Through the human practices of the team related to scientific divulgation, we have noticed that general public is not concerned about it. Although the biodiesel industry as a renewable energy source is well-known, there is a little concern about the large quantities of glycerol produced and its impact on the environment. As this problem affects worldwide, it should have a bigger social impact.

The team proposes a new way to utilize crude glycerol in high rates. In particular, it focuses on the use of biofilms employing glycerol as their carbon growth source. At the same time, these biofilms are susceptible to produce a molecule with added value (i.e. propionate) to increase the economic viability of the process [Fig. 4]. Biofilms have an accelerated metabolic activity that will improve the glycerol consumption (this method is expected eliminate more glycerol in the same time compared to other industries) and the useful molecule production.

Figure 4. Schema of the iGEM UPO-Sevilla Team Project. Crude glycerol obtained from the biodiesel industry is constantly added to a continuous bioreactor, where it is converted to an useful molecule by action of biofilms.


  1. Lehninger, Albert L., David L. Nelson, and Michael M. Cox. Lehninger Principles of biochemistry. Chapter 21 Lipid biosynthesis. New York: Worth publishers, 2000

  2. Ciriminna, R., Pina, C. Della, Rossi, M., & Pagliaro, M. (2014). Understanding the glycerol market. European Journal of Lipid Science and Technology, 116(10), 1432–1439.


  4. Jiang et al. (2016). Key enzymes catalyzing glycerol to 1,3-propanediol. Biotechnology for Biofuels, 9:57.

  5. Schuchardt, U., Sercheli, R., & Matheus, R. (1998). Transesterification of Vegetable Oils : a Review General Aspects of Transesterification Transesterification of Vegetable Oils Acid-Catalyzed Processes Base-Catalyzed Processes. J. Braz. Chem. Soc., 9(1), 199–210.

  6. Hannah Albers, Ben Guilfoyle, Melanie Thelen, and Cole Walker (2014). Team 14: GRE-cycle. ENGR 339--Senior Design Project

  7. Quispe, C. A. G., Coronado, C. J. R., & Carvalho, J. A. (2013). Glycerol: Production, consumption, prices, characterization and new trends in combustion. Renewable and Sustainable Energy Reviews, 27, 475–493.

  8. Yang, F., Hanna, M. a, & Sun, R. (2012). Value-added uses for crude glycerol--a byproduct of biodiesel production. Biotechnology for Biofuels, 5(13), 1–10.






  14. Wan Isahak, W. N. R., Che Ramli, Z. A., Ismail, M., Mohd Jahim, J., & Yarmo, M. A. (2014). Recovery and Purification of Crude Glycerol from Vegetable Oil Transesterification. Separation & Purification Reviews, 44(3), 250–267.


  16. Mu, Y., Teng, H., Zhang, D. J., Wang, W., & Xiu, Z. L. (2006). Microbial production of 1,3-propanediol by Klebsiella pneumoniae using crude glycerol from biodiesel preparations. Biotechnology Letters, 28(21), 1755–1759.

  17. González-Pajuelo, M., Andrade, J. C., & Vasconcelos, I. (2004). Production of 1,3-propanediol by Clostridium butyricum VPI 3266 using a synthetic medium and raw glycerol. Journal of Industrial Microbiology and Biotechnology, 31(9), 442–446.