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Turning Villains into Heroes with Wastewater Clean-Up

Phenol is widely used by industries to make everyday materials. That means it's also a major pollutant in nearby streams and rivers.

E coli tends to get a bad rap. That’s easy, of course, if you’re responsible for severe food poisoning after a sublime indulgence of cookie dough. But E. coli is a bacteria native to the human gut, and it’s also a very common workhorse in synthetic biology laboratories. In today’s paper, it’s used to clean wastewater from industrial runoff.

When it comes to fighting pollution, bioremediation (which uses biology as a clean-up crew ) is an attractive option. Previously, we’ve seen that plants that can be modified to help with TNT bioremediation. Here, we’re throwing an entire nine-step metabolic pathway into a cell to get the job done.

The Problem

The paper, Construction of an E. coli strain to degrade phenol completely with two modified metabolic modules, opens with a discussion of the dangers of phenol. It allows industries to make materials for the modern world, but it’s also carcinogenic and acts as a teratogen (impairing fetal development) and mutagen (causing mutations, which may of course result in cancer or impaired fetal development). We don’t need it in our rivers and streams.

The question in this paper is, can we engineer E. coli to degrade it?

The Prerequisites

It turns out, a degradation pathway for phenol has been characterized before. The species P. putida was found to degrade phenol completely via a nine-step metabolic pathway. First, phenol is converted to catechol. It is then, along a series of chemical steps, converted all the way down to succinyl-CoA and acetyl-CoA. If those last two molecules sound familiar, it’s because they’re both used by the Krebs Cycle in your favorite metabolic pathway, Cellular Respiration.

In other words, with this pathway, phenol can be detoxified. Its carbon is completely recycled into the cell’s energetics, never to harm another living thing again.

The Solution

With the enzymes for phenol degradation known, the experiment is fairly straightforward. The researchers put all nine genes onto a single plasmid and cloned it into an E. coli strain. They used a replicating plasmid that is induced whenever you add a chemical (called IPTG) into the mix. That means you can control for gene expression and phenol exposure; the experiment tested phenol degradation when cells were exposed to phenol (or not), and when the genes for phenol degradation were turned on (or not). This sort of set-up allows for controls, and thus, proper interpretation of the data.

Fig. 4 is a nice demonstration of the toxicity of phenol. Cells with the degradation pathway can eliminate phenol completely from 1 or 5 mM, but overload the cell with 10mM and you’ll kill it before it has a chance to break the molecule down. There’s also a nice increase in catechol observed as phenol decreases (Fig. 4B); recall catechol is the first product of phenol degradation. This catechol build-up before the rest of the degradation occurs is ok, however, because catechol wasn’t shown to be toxic to the cells (Fig. 5A).

Ultimately, the researchers grew their engineered E. coli in wastewater containing phenol, even boosting phenol content up to 1mM, which is a higher concentration than even severely polluted waters. They found that their bacteria were incredibly efficient at phenol destruction: it took 3 hours to completely degrade it into the harmless carbon products.

Conclusions

This is a nice experiment that neatly sums up how we can engineer microbes to really work for us. We have been depending on microbes like yeast and bacteria for thousands of years, using them to make bread, beer, and cheese. You might even add the microbes that live among the roots of our crops to this list. Unbeknownst to us throughout much of our history, symbiotic soil bacteria have worked to make our farmed crops more productive and more nutritious. Now, we are engineering microbes to diversify their skillsets. That includes skills like bioremediation, including the clean up of phenol in industrially polluted ecosystems.

This victory does not come without it’s own set of concerns, however. There is caution – wisely so – at inoculating a polluted ecosystem with a genetically engineered strain of bacteria. Even these E. coli, efficient as they are at phenol degradation, have unknown environmental effects. Sure, they’ll clean the phenol up. But what if they’re invasive and start overturning native microbial ecosystems? What effects will they have on the native soil microbiome and the plant, fungal, and animal communities it supports?

These environmental engineering questions are in need of some real research. A very handy E. coli strain has been constructed, but where and how can we use it to restore and protect our environments?

References

Wang, B., Xu, J., Gao, J., Fu, X., Han, H., Li, Z., Wang, L., Tian, Y., Peng, R., & Yao, Q. (2019). Construction of an Escherichia coli strain to degrade phenol completely with two modified metabolic modules. Journal of Hazardous Materials, 373(March), 29–38. https://doi.org/10.1016/j.jhazmat.2019.03.055

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