Each year, seven lakh tons of dyes are produced in factories, and a large amount of them were discharged in waste stream without proper treatment. These emissions could cause diseases in human and even do irreversible harm to the ecosystem and environment. Our team sets our goal to come up with a system that could effectively decompose azo dyes.

Azo dyes’ pollution (Guang etal., 2013)

First, we researched about previous ways to decompose azo dyes. There are three existing ways to decompose azo dyes: Chemical method, photocatalytic method, and microbiological method. First, 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, so they would never use such method. Second, photocatalytic method involves several catalysis and requires special lighting equipment. The reaction tends to be very slow since most dyes are designed to tolerate strong lights. Thus, this method is time consuming. Third, microbiological decomposition process is relatively cheap and this method does generate any toxic by-products. Nevertheless, bacteria cell that could flow in the treatment apparatus is still an issue. We decided to improve the on-going microbiological method. We need to create a system that holds all the cells in one piece and could decompose azo dyes.

Focusing on that, we soon located our first key component of our system: The Biofilm. Biofilm is a network of individual cells which act not only as a net that holds everything in one piece, but also as a shield for individual cells to the harsh environment condition. As we all know, bacteria cannot grow as well in medias as in harsh environments like flowing sewage water.

The typical biofilm we researched on is formed by gene csgA on the genome of E. coli MG1655 wild type. Cells were stuck together by a kind of fiber protein known as curli fiber that is assembled by CsgA protein on the cell’s surface. By the way, csgA was a previous iGEM part and we mead improvements to it. We also read about adding the sequence of SpyTag after csgA gene could produce curli fiber with SpyTags on them.

Figure of csgA - SpyTag

We are now confident that we can fix anything we want to the surface of a biofilm. But we now need a substance that could decompose the azo dyes. After some more researching, we found laccase, a kind of blue multi-copper oxidases, has strong ability to oxidize and decompose azo dyes. Gene cotA, initially found in Bacillus subtilis, can produce protein with high laccase activity. Gene cotA has a very low translation rate in natural hosts. However, when cotA is produced in specialist strains like BL21 – DE3 (after T7 promoter), it shown up a relatively high rate. As mentioned, laccase belong to multi-copper oxidases, so that CotA protein still need to combine with Cu2+ to become active laccase. The solution is quiet simple, we can just ultrasonic cells and mix whatever is left with 0.1 mM/L of CuSO4.

Up till now, we have designed a biofilm with SpyTag and have located a kind of powerful enzyme. In order to fix our powerful enzyme to the biofilm, we added a SpyCatcher sequence after the cotA gene. So that these proteins could fix onto the curli fibers of the biofilm csgA - SpyTag using isopeptide bonds. These bonds are covalent bonds which is relatively hard to broke.

cotA - SpyCatcher

Signal peptides - CotA

We then came out with another solution of how to get CotA protein out of the cells. By adding signal peptide sequence ahead of cotA – SpyCatcher sequence, we could let the cells secrete CotA – SpyCatcher protein in to the media. Then we can let CotA – SpyCatcher combine with Cu2+.

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