Team:ITESM-Guadalajara/Technology

OUR TECHNOLOGY

Technology fundament

The current production process of Chitosan from shrimp shell takes place through three main steps: demineralization (removal of calcium carbonate), deproteinization (removal of proteins) and deacetylation (removal of N-acetyl group), using strong acids and alkalis at high temperatures for several hours as shown in the figure below (Raafat & Sahl, 2009) (Bristow, 2012) (Ortiz Rodríguez, 2013).

This means that large quantities of toxic chemicals (such as hydrochloric acid, acetic acid and sodium hydroxide) are released to the environment, damaging the aquatic flora and fauna; furthermore, this process has the inconveniences that cannot guarantee a standard quality of the product, it is expensive (since it requires chemical reagents in each Chitosan production cycle) and the access to it is restricted by the production seasonality of current producers.

Therefore, in order to solve these problems, Biophrame Technologies has proposed a novel method using the same three steps described previously to produce Chitosan from shrimp shell, but instead of using chemical substances, use two bacteria fermentations with two microorganisms: B. subtilis and a modified E. coli (see figure below)

B. subtilis is an ellipsoidal or cylindrical gram-positive bacterium that can grows over a wide range of temperatures and pH: from 5-15°C to 40-50°C, being its optimal temperature 28-30°C and an active pH range from 5.5-8.5. We choose this bacterium because it is a facultative anaerobic organism, the complete genome sequence has been reported (comprising 4100 protein coding sequences), it can produce and secrete big quantities per liter of extracellular proteins and it is able to produce acid from glucose, mannose, glycerol, glycogen, fructose, among others (Schallmey, Singh, & Ward, 2013).

On the other hand, E. coli is a very common rod shaped, gram-negative, facultative anaerobic and non-endospore forming bacterium, capable of using large quantities of inorganic carbon sources to grow (Förster & Gescher, 2014). Its optimal growth pH and temperature ranges are 6.4-7.2 and 37°C, respectively. We decided to use this bacterium as a recombinant protein producer because it can grows to high densities on inexpensive media and in the presence of chitin, its genome is completely sequenced and it has been used several times as a protein producer due to its capability of an easy transformation and fermentation, low cost and high protein yield (Manderson, Dempster, & Chisti, 2006), (Keyhani, Wang, Lee, & Roseman, 2000), (Roseman & Keyhani, 1997), (Plumbridge & Pellegrini, 2004).

Therefore, Biophrame Technologies’ innovative process involves the use of an anaerobic fermentation with B. subtilis to carry out the demineralization and deproteinization steps, since B. subtilis is capable of producing proteases and lactic acid. Proteases are enzymes capable to digest long protein chains into smaller fragments by breaking the peptide bonds that link amino acid residues; this means that proteases will be the responsible of eliminating the proteins (deproteinization process) present in shrimp shell. For the demineralization, we propose the use of lactic acid because it can react with the calcium carbonate component in the chitin fraction of the shrimp shell, leading to calcium lactate formation, which in turn precipitates and can be easily removed by washing. This approach leads to a liquor fraction rich in proteins, minerals, and asthaxanthin and to a solid chitin fraction.

The solid fraction is then submitted to a deacetylation process for Chitosan production using an Escherichia coli genetically modified with five different enzymes: two Chinitases (CH) and three Chitin Deacetylases (CDA) in two different expression vectors (one vector will contain the CH enzymes and the other the CDAs).

We suggest the use of two different vectors instead of one to control the expression of the enzymes at different times of the process, by modulating the temperature and by adding different activators (such as IPTG). However, we also propose a second strategy for the deacetylation step using an integration vector, that will contain the five enzymes for its incorporation to the E. coli genome. The second strategy arises to minimize the instability inherent of the replicative plasmids that limits their applied utility and to make easier the bacterial transformation process at pilot scale (Heap, et al., 2011), (Heap, Ehsaan, & Minton, Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker, 2012). The two strategies will be evaluated to choose the one that gives the better product quality.

The CHs (EC 3.2.1.14) are hydrolytic enzymes responsible to break down the glycosidic bond found in chitin. These proteins are going to be working in the first step of the deacetylation process by cutting the chitin obtained in the deproteinization and demineralization steps into smaller pieces. The CDAs (EC 3.5.1.41) are enzymes that will hydrolyze the acetoamido group in the GlcNAc units of chitin, to transform it into Chitosan. We decided to cut chitin into smaller pieces first, to allow more acetoamido groups to reach CDA’s active site and thus reach a higher conversion yield.

We are going to be expressing CHs from the beginning to certain time of the process, by using a repressible promoter Biobrick, which is negatively controlled by IPTG. These enzymes will be produced at optimal E. coli growth conditions (pH = 7, Temperature = 37ºC) until they reach the desired concentration. Then we are going to raise the temperature to 50°C for a certain period of time to let the CHs enzymes act upon the chitin and to let the expression of the CDAs begin (see figure below).

The CDAs gene expression is produced as a result of a Heat Shock Promoter coupled to LacI Regulated Promoter BioBrick action. This promoter is induced by an increase in temperature and also by the presence of IPTG. After allowing the CH enzymes to act upon chitin, we are going to decrease the temperature to 37°C to let E. coli express CDAs, and simultaneouly IPTG is going to be added to the aerobic fermentation tank to repress the expression of CHs and to optimize the expression of CDAs. Consequently, CDAs will be maintained at 50ºC to allow them hydrolyze the acetoamido group found in chitin, and thus make possible the transformation of chitin into Chitosan (Zhao, Park, & Muzzarelli, 2010) (see the figure below).

The selection strategy for the enzymes used in the process was based on their pH and temperature activity ranges. They needed to be close to each other, so they could have an optimal conversion yield without needing to change the conditions drastically. Also, they needed to be close to the optimal parameters for E. coli growth.

Pilot Plant

As we may see in figure 10, every month 1 metric ton of shrimp shell (without organic matter) will arrive from our supplier: “La Internacional Sinaloa”. First, this raw material will be chemically analyzed to obtain its moisture, protein, minerals, chitin and lipids content. Once analyzed, the raw material is going to be washed with water in an agitated tank for 1 hour. After that, the shrimp shell is going to be dried at a 250 Kg/h rate to reduce moisture content from 62% to 5%. Consequently, the dried shrimp shell is going to be grinded at a 100 Kg/h rate to obtain particles of 0.5mm of diameter to maximize contact surface and make the transformation process easier. Following, the shrimp shell powder will be stored at room temperature until required. Thenceforth, we are going to divide shrimp shell powder into 4 batch of 100 Kg before proceeding with the bacterial fermentations. Each batch will go through a sterilization process to eliminate all the microorganisms that could interfere with B. subtilis and E. coli growth. The sterilized shrimp shell powder will undergo an anaerobic fermentation with B. subtilis to eliminate 87% proteins and 72% calcium. Following, this mixture will experience a filtration process with help of a rotating drum to extract chitin and get rid of proteins, calcium and culture media. Thereafter, chitin will go through another sterilization process to ensure that no B. subtilis was left. Sterilized chitin will undergo another fermentation, this time with our genetically modified E. coli to transform chitin into Chitosan. This specific stage of the process represents our biggest innovation because we are eliminating the use of strong acids and bases by putting chitin through fermentation with an E. coli specifically designed for this purpose. The effluent will be centrifuged to remove E. coli from the mix. Following, this mixture will experience an ultrafiltration to extract the Chitosan, the rest is going to be discarded. Consequently, we will perform a size exclusion chromatography to purify our Chitosan. Finally, it will be spray dried. The obtained Chitosan will be tested for deacetylation degree, molecular weight, solubility, purity and viscosity. If it passes our quality standards it will be ready for bottling and packaging. We will obtain approximately 12 Kg of dried Chitosan at the end of the process.

Gantt Diamgram of the Process

The entire Chitosan production process, described above, will take approximately one month and a half. The times required for each unitary step are shown in the next Gantt diagram (see figure 11).

Fate of wastes generated with the Biophrame process. It is also important to mention that the wastes generated with the process, which are:

• Soluble calcium lactate and proteins produced after anaerobic fermentation
• Dirty water generated after shrimp shell wash pre-treatment
• Attenuated E. coli genetically modified obtained after aerobic fermentation
• Attenuated B. subtilis obtained after anaerobic fermentation

Will be recollected by the company SALUD AMBIENTAL INTEGRAL SA DE CV (Telephone number: 013336725990, address: Calle Miguel Bernal 3185, 44810 Guadalajara, Jalisco). This company is located in the same municipality than Biophrame Technologies (Zapopan), and is regulated by the Mexican norm “NOM-087-SEMARNAT-SSA1-2002 Protección ambiental-Salud ambiental- Residuos peligrosos biológico-infecciosos-Clasificación y especificaciones de manejo” (NOM-087-SEMARNAT-SSA1-2002 Environmental Health Protection environmentally-hazardous waste biological-infectious-Classification and management specifications) (SALUD AMBIENTAL INTEGRAL SA DE CV) The Mexican law “Ley General del Equilibrio Ecológico y la Protección al Ambiente” or General Law of Ecological Equilibrium and Environmental Protection, defines as hazardous waste every waste that, for its corrosive, reactive, explosive, toxic, inflammable and biologic-infectious, can represent a danger for the ecological equilibrium (Secretaría de Medio Ambiente y Recursos Naturales de los Estados Unidos Mexicanos, 2003). The Mexican law about genetically modified organism management is the “NORMA Oficial Mexicana NOM-164-SEMARNAT/SAGARPA-2013”. This norm establish the characteristics and information that has to be shown in the result report of genetically modified organisms liberation, in relation with the potential environment, biodiversity, animal, plant and aquatic health risks (Secretaría de Medio Ambiente y Recursos Naturales.- Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación de los Estados Unidos Mexicanos, 2014)