Each year, 700,000 tons of dyes are discharged in printing and dyeing mills, and a large proportion of them were discharged in waste stream without proper treatment. These emissions could cause human diseases or even lead to irreversible damage to the ecosystem (Guang etal., 2013). Our team sets the goal to build a system that could efficiently decompose synthetic dyes.

Azo dyes’ pollution (Guang etal., 2013)

There are three existing ways to decompose synthetic dyes: physicochemical method, photocatalytic method, and microbiological method.

Physicochemical method uses carbon-based sorbents to adsorb synthetic dyes. However, it is energy- consuming to make such substance. Consequently, this method is too expensive for smaller business owners, since lots of carbon sorbents is needed for removal of dyes. Synthetic dyes are usually designed to withstand photodegradation, so the photocatalytic method may require many catalysts and intensive irradiation, which makes the treating process to be very sophisticated. (Alessandra Piscitelli, C. P., Paola Giardina, Vincenza Faraco and Sannia Giovanni., 2010)

Microbiological decomposition is a relatively cheap solution, but in traditional biodegradation method, toxic gas like Hydrogen sulfide may be produced as a byproduct. Nevertheless, the decomposing rate of traditional biodegradation may potentially be lowered by a high flow rate which washes away bacteria floating in sewage. Thus we planed to build a bio-substrate that can be coated with bacteria or functional enzymes. In this case we decided to improve the on-going microbiological method, creating a system that holds all the cells in one piece and could decompose synthetic dyes. (Alessandra Piscitelli, C. P., Paola Giardina, Vincenza Faraco and Sannia Giovanni., 2010)

Focusing on our goal, we soon located the first key component of our system: the biofilm. Biofilm is a layer adhered by microorganisms and enzymes on the surface. It maximumly protect bacteria and enzyme in wide range pH environment, lowering the impact of acidic sewage water to laccase activity, as well as providing a large surface area for contact between laccase and synthetic dye molecules. In typical biofilm, such as biofilms formed by E. coli, cells were stuck together in curli fibers formed from the self-assembly of secreted CsgA protein. Researches have reported that different functional proteins can be fused to CsgA through functional peptides, and introduce diverse artificial functions to the biofilms.

At this point, we need a specific type of enzyme that could decompose synthetic dyes. Laccase, a blue multicopper oxidase, came into our sight. Laccase can oxidize a series of aromatic substrates. It is firstly discovered in Chinese or Japanese lacquer trees, and originally involve in plants lignification. CotA laccase, initially found in Bacillus subtilis, was selected as our basic decomposing enzyme. CotA have a very low expression rate in natural hosts, by constructing CotA gene with suitable promoter and RBS, we managed to get a relatively high expression rate of laccase from E. coli. Therefore, the idea of using Biofilm x Laccase system to decompose synthetic dye, came to our mind.

Figure of csgA - SpyTag

cotA - SpyCatcher

Signal peptides - CotA

Inspired by the autocatalytic formation of the isopeptide bond between a specific Lys and an Asp residue in Streptococcus pyogenes (Spy) fibronectin-binding protein FbaB, researchers split its autocatalytic domain, CnaB2, and obtained two peptides which they named SpyTag and SpyCatcher. It is widely use for connecting protein. In order to fix our powerful enzyme to the biofilm, we added a SpyCatcher sequence after the cotA. So that these proteins could fix onto the curli fibers of the biofilm using isopeptide bonds. These bonds are covalent bonds which is relatively stable.

Project Overview

References:

Synthetic dyes, which are widely used in many industries and researches, cause various environmental and health problems. Xintang, a village in China, where textile industries are located, have been heavily polluted with indigo blue dye. After enduring such bad environment, many villagers, unfortunately but inevitably, get heart disease as well as leukemia. (Guang etal., 2013)

Biological degradation of dyes is economic and environmentally friendly methods comparing to chemical degradation, so this year, our team aims to use CotA, a laccase coding gene, to deal with synthetic dyes containing effluents. CotA is a polyphenol oxidase which has capability to decolorize a wide range of dyes. Comparing to chemical process which has high expense and take tedious steps, CotA laccase is eco-friendly. It is proven that laccase decompose dye efficiently even in harsh condition with 3.5% salinity and pH 11.6 (Piscitelli etal., 2010, p. 252), which shows potential for dye decomposition.

However, bacteria are not capable to grow in harsh environment. We learn that previous researchers have reported that biofilm is able to be engineered to display proteins with different functions, which ameliorates the drawback. CotA laccase is fussed with Spycatcher to attach covalently to biofilm containing Spytag. Biofilm is a layer adhered by large amounts of microorganisms to the surface, and organic substances such as enzyme are trapped in it. It maximumly protect bacteria and enzyme in wide range pH environment, lowering the impact of acidic sewage water to laccase activity, enhancing reaction efficiency of laccase. We successfully attach CotA laccase to biofilm and prove the effectiveness of laccase to decompose dyes eventually.

To further improve the efficiency of discoloration, we come up with the idea to construct a filter which holds a large amount of laccase while letting water to get pass. We design to attach CotA laccase to biofilm which cover polyhydroxyalkanoates plastic beads to fill the filter. When processing sewages, the velocity of flow will be controlled to be appropriate, at which the treating efficiency as well as the stability of biofilm are both optimized.

Reference:

Alessandra Piscitelli, C. P., Paola Giardina, Vincenza Faraco and Sannia Giovanni. (2010). Heterologous laccase production and its role in industrial applications. Bioeng Bugs, 1(4), 252.

Guang, L. G. J. M. L. (2013). The denim capital of the world: so polluted you can’t give the houses away. Retrieved fromhttps://www.chinadialogue.net/article/show/single/en/6283-The-denim-capital-of-the-world-so-polluted-you-can-t-give-the-houses-away