Glycerol is a by-product of the biodiesel industry. The need to search for alternative renewable energy sources and the increase in the biodiesel production has led to the release of large quantities of glycerol. Glycerol produced in this industry is not pure; it is contamined by toxic methanol and other molecules that may affect the environment. For that reason, many researchers have focused on looking for new ways to use that crude glycerol and avoid its accumulation in the environment (for more information, see Integrated Human Practices).

To help solving that problem, the UPO Sevilla Team has developed a modified bacterium that is able to assimilate glycerol in a most efficient way. We make use of Pseudomonas putida, a bacterium that is able to degrade glycerol. However, glycerol is not a preferred carbon source, and grow on this substrate is not optimal. For that reason, we have genetically modified this bacterium in order to increase the assimilation of glycerol.

Metabolic pathway modeling performed by our team predicts that the limiting step for glycerol assimilation is its transport across the cell membrane. To solve that problem, we have integrated a gene coding for an inner membrane glycerol transporter in the genome of P. putida. This transporter is called GlpF; it is an aquaporine, a glycerol transporting channel. When GlpF was produced from a salicylate inducible expression system, this strain reached higher growth rates than the wild type when using glycerol as the sole carbon source.

On the other hand, we know that P. putida is able to form biofilms. Biofilms are more resistant to different forms of environmental stress such as pH changes or temperature. Also, biofilms have an accelerated metabolism compared to planktonic bacteria. In order to take advantage of this ability of the biofilms to degrade glycerol in higher rates, we have also genetically modified this bacterium to control the formation and dispersal of the biofilm.

The ultimate goal of this project is to have a bioreactor in which bacteria form biofilms on the surfaces of the tank when activated, consume glycerol (the input) and disperse at will when the bioreactor needs to be cleaned. Using glycerol as a carbon source, bacteria can produce another molecule with added value. We propose producing propionate, a small molecule with different uses for the human being. This way, we convert a waste to a useful molecule in a profitable manner.

Other conversions systems have been developed in order to use the crude glycerol from the biodiesel industry. One example is the production of 1,3-propanediol, using Klebsiella pneumoniae and Clostridium butyricum bacteria. However, we have increased the glycerol consumption by two complementary ways: by increasing the entrance of the molecule in the bacteria and by using biofilms. That way, I hope to eliminate glycerol from the environment in higher rates compared to other conversion systems. Is there a more elegant way to solve this environmental problem?

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.