PHAgave: a solution to a local problem from a global perspective
iGEM Tec GDA, is a team of Mexican students that are worried about the environmental situation around the world. As part of our iGEM project we decided to contribute with our knowledge in getting rid of wastes that cause contamination by using them to solve a global problematic.
Our natal state is Jalisco, which besides of having beautiful landscapes and gastronomy, is the state where a well known liquor is made, tequila. Tequila is a traditional alcoholic beverage obtained from Agave Tequilana Weber that has denomination of origin; this means, that it can only be produced in 6 states of Mexico. This industry, is one of the most important industries in our country and is known for its contribution to the economy by exporting to more than 90 countries. Unfortunately, the production of this memorable drink leaves a huge percentage of residues. One of the most significant one is a fibrous material called bagasse. Large volumes of this waste are generated every year by this important industry, and their disposal, has become an environmental and economic problem.
For every liter of tequila, 5 kilograms of bagasse are produced, which results in 400 thousand tons of bagasse in Jalisco every year. This, is equivalent to approximately 5 days of waste generation in New York, the unequivocal world capital of garbage.
The bagasse, which is the remaining matter left after the agave head juices extraction, is a residue of organic origin that can be considered biodegradable. However, when it is not properly managed it can take long time to naturally decompose and reintegrate its components into the environment. The main problem of the decomposition process is that it produces leachate (a toxic waste liquid) that contaminates soil and water changing their properties. Bad smells that pollute the air and proliferation of harmful organisms that can become vectors of health problems are some of the main things caused by the huge amount of bagasse produced by this industry. The environment is not the only one affected by the obtention of this waste during the production of tequila, but also the same tequila producers because it is extremely hard for them to get rid of it, and sometimes they even have to waste time and money in order to do so.
To corroborate this information, we visited several tequila factories, where we concluded that indeed bagasse management is a huge problem that is affecting our home state in a environmental and economical way. To eradicate this issue, we searched the components of bagasse, noticing that it is composed mostly of sugars, having a great potential as carbon source for bacteria. This means that bagasse can be effectively converted to glucose, and this sugar can be used to feed our recombinant bacteria, which has the ability of convert glucose into PHA bioplastic.
This is how iGEM Tec GDA, created PHAgave, a project that consists of using bagasse to create a bioplastic.
The team proposed different ways of production that allow us to obtain the PHA from the Agave Tequilana bagasse. The production of this compound has been detailed in two subprocesses, and agrees with the recommendations proposed by Gonzalez, Gonzalez and Nungaray in 2005.
The following diagram shows the proposal realized by our team to obtain PHA from bagasse:
The first system is aimed for pretreating the bagasse so that it can be processed by our bacteria. This process is characterized by physical, chemical and biological methods that will allow us to obtain a processable bagasse.
As mentioned before, the bagasse is characterized by containing high quantities of complex sugars, some of them are in form of lignin and cellulose which are rarely exploited (Iñiguez, Acosta, Martínez, Parra, González , 2004).
In order to be able to process those components it is important to dry in the sun until values of 2-5% of humidity are obtained, which will allow us to control competitive bacterial growth and will help us to triturate them. This process must be done, so that the particles are smaller and the microorganisms can have more access to the sugars in order to process them.
After the trituration process, the bagasse enters to an enzymatic treatment inside a bioreactor where Aspergillus niger will realize the first step by removing lignocellulose from the bagasse (Trejo, Oriol, López, Reoussos, Viniegra y Raimba, 1991). In this process there are used effluents from the dairy industry as a growth medium, nutrient solutions, and inductor components such as hydrolases and sucrose. At the end of the process, the treated bagasse is stabilized to obtain a neutral pH.
It is important to add that the effluents from the dairy industry are highly polluting, and in its majority they are fluids with high concentrations of carbohydrates and proteins (Munavalli y Saler, 2009., Kushwaha, Srivastava y Mall, 2011), that also contain a low concentration of phosphorus and nitrogen (Rocha, Tenorio y Cortes, 2015), which means that can be used as a growth medium for microorganisms, and for the enhancement of PHA production given that our recombinant bacteria produces more PHA in stress conditions. However, in order to use these compounds as a growth medium they must go through a pretreatment that includes a procedure to remove grease from the effluents by using a grease trap. After this process, the sedimentable solids have to be removed from the crude effluent the sedimentable solids, so that these can’t cause future problems with PHA extraction. To achieve the removal, sedimentation and filtration processes are needed to actually remove all the solids.
At the end of these proceedings, the crude effluent that has been pretreated can now be used inside an aerobic reactor for PHA production. Before entering the bioreactor for PHA production, the effluent is mixed with the pretreated bagasse. The variation in the chemical composition of the effluents of the dairy industry are not high enough, which means that we can actually have a concentration pattern of chemical compounds used in our processes.
By using the aerobic bioreactor with the previously processed effluent from the dairy industry and the bagasse, it is expected that our recombinant bacteria actually has the capacity to produce PHA. Escherichia coli BL21 was the bacteria selected to be transformed with PHA production genes from Pseudomonas putida KT2440 because it is not only cheaper and easier to grow and maintain, but also because higher yields of PHA can be obtained.
The next step after the production of PHA inside the reactor is the extraction of PHA. It was decided that the process should begin with the lysis of our bacteria. This process will facilitate the extraction, and will also help as a biosecurity method so that they are not released to the environment.
The wasted obtained in all the processes that were presented before, can actually be redirected to a biodigester for the production of methane. This energetic compound can be used after a combustion, into electric energy that can maintain from the triturator in the pretreatment of bagasse, to the bomb that adds oxygen to the bioreactor in which the PHA is being produced. Inside the effluents obtained from the digester it can also be found biol that at the end of the process can be turned into agricultural fertilizer.
González .Y, González O. y Nungaray J. (2005). Potencial del bagazo de Agave tequilero para la producción de biopolímeros y carbohidrasas por bacterias celulolíticas y para la obtención de compuestos fenólicos. e-Gnosis, 3: 0
Iñiguez C., Acosta T, Martínez C., Parra J, González O., (2004). Utilización de subproductos de la industria tequilera, Parte 7. Compostaje de bagazo de agave y vinazas tequileras. Rev. Int. Contam. Ambient. 17(2).
Kushwaha J., Srivastava V. y Mall I. (2011). An overview of various technologies for the treatment of dairy wastewaters. Critical Reviews in Food Science and Nutrition: 442-452.
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Trejo M., Oriol E., Lopez A., Reoussos G., Viniegra G. y Raimba M., (1991). Producción de pectinasas de Aspergillus niger por fermentación sólida sobre soporte. MICOL. NEOTROP. APL. 4 : 49-62.