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

Azo dyes’ pollution (Guang etal., 2013)

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

Chemical 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. In consideration of the synthetic dyes are usually designed to oppose photodegradation, so the decomposition required many catalysts and irradiation conditions which make the process tedious. (Alessandra Piscitelli, C. P., Paola Giardina, Vincenza Faraco and Sannia Giovanni., 2010)

Microbiological decomposition process is relatively cheap and this method does not generate any toxic end products when processing. Nevertheless, the decomposing rate are low when bacteria floating in sewage, thus we want to build a bio-substance that can fix bacteria or functional enzymes. In this case we decided to improve the on-going microbiological method. Therefore, we need to create 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 that, we soon located our 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 enhancing reaction efficiency of laccase. 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 to our mind. 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 protein, initially found in Bacillus subtilis which are accessible, are selected as the basic decomposing enzyme among all of laccase. CotA have a very low producible rate in natural hosts, and as we engineered cotA gene into E. coli it shown up a relatively high rate. Therefore, we build our Biofilm x Laccase system.

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 hard to broke.

Project Overview

References:

Synthetic dyes, which are widely used in many textile industries, cause various environmental and health hazards. Xintang, a village in Southern China has been heavily polluted with indigo blue dye used for jeans manufactory. After enduring such an environment in low condition, villagers face serious health crisis, which is an epitome of dye pollution problems (Guang etal., 2013)

Synthetic dyes pollutants are resistant to light, water, and various chemicals, and biological degradation of dyes is effective and environmentally-friendly methods comparing to chemical degradation. This year, our team aims to deal with synthetic dyes containing effluents. We choose CotA, polyphenol oxidase which has capability to decolorize a wide range of dyes, as the catalyst. It has been reported that CotA laccase decomposes dye efficiently even in harsh condition with 3.5% salinity and pH 11.6 (Piscitelli etal., 2010, p. 252), which makes it a great catalyzer for dye decomposition.

However, only CotA-expression bacteria are not enough, and through our human practices we have learnt that bacteria are not capable to grow in harsh environment such as sewage. Biofilm, which is capable to ameliorate the drawback, comes to our view. Biofilm is a layer adhered by microorganisms and enzymes on the surface. It maximally protects bacteria and enzyme in wide range pH environment, lowering the impact of acidic sewage water to laccase activity, as well as enhancing reaction efficiency of laccase. What’s more, previous researches have reported that biofilm can be engineered to display proteins with different functions. We have successfully fused CotA laccase with Spycatcher to attach covalently to biofilm containing Spytag. The effectiveness of decomposition by using attached CotA laccase has been demonstrated.

In order to apply biofilm in reality, we come up with the idea to construct a filter which holds a large amount of laccase while letting water to pass through, and the filter is filled with bio-degradable polyhydroxyalkanoates (PHA) plastic beads covered by biofilms containing CotA laccase. By operating data and modeling, the velocity of flow will be controlled to be appropriate when processing sewage, 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