Description

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants in the urban environment, because of their hydrophobic and lipophilic character, PAHs is an easily enriched in sludge, which is by-product of sewage treatment and widely used in cultivated land (Chen, Yan, & Du, 2024). The accumulation of PAHs can pose huge threats to the water bodies. According to Guangzhou WWTP, the concentration of several PAHs in local effluent, even after wastewater treatment, still remained approximately 10 to 15 times above China’s acceptable risk levels(Liu, Shen, Sun, 2020). Due to the numerous drawbacks of the existing approaches, such as low efficiency and significant side effects, we come up with a more comprehensive solution to this problem: constructing an engineered E. coli which is integrated with plasmids containing diverse gene clusters, endowing it with higher degradation capability, enhanced environmental resistance, and safer switch control. The goal of the project is to provide a safer and more efficient mean of treating PAHs.

Hence, our team worked across Wetlab, Drylab, Human Practice, and Software, and together, we achieved the following:

1.Successfully created a quorum sensing system in E. coli which allows the genes to express only when reaching certain thresholds, balancing the growth and functioning of bacteria.

2.Successfully applying engineered E. coli for treatments of PAHs

3.Successfully added a suicide switch into the bacteria to ensure that no excessive amount of E. coli will cause accidental hazards on the environment.

4.Successfully established an AcrAB-TolC system that can effectively pump out harmful substances from the bacteria

5.Successfully expressed the NhaA transporter protein on the inner membrane of Escherichia coli to regulate intracellular pH.

The technology used in this project is very powerful and standardized, and is expected to be used in various iGEM projects in the future.

Initially, we planned to construct an engineered E. coli which can degrade PAHs effectively, survive longer in high salinity and toxicity ambient, and avoid bacteria to abscond by installing the physical interception and the suicide switch. Moreover, a quorum sensing system will linearly increase the expression of downstream genes as the concentration of E. coli rises, thereby expressing the genes that perform the aforementioned functions. We were able to achieve our aim and improve it from the following aspects:

1.Switching the degradation label SsrA into LAA-LAA to obtain better control of the membrane protein, so the membrane transport can be more precisely regulated in higher efficiency.

2.We shift the position of the pLuxR in front of the LuxI, forming a positive feedback circuit . Therefore, the originally linearly increasing expression pattern was transformed into a burst-like expression that occurs only when it reaches a certain threshold. Such changes make the regulation of the quorum sensing system more controllable and sensitive, and further reduce unnecessary consumption of protein.

Escherichia coli, as the host organism in our experiment, possesses numerous remarkable capabilities, making it the ideal choice for us. It has excellent platform characteristics, allowing us to integrate and replace various gene clusters on it. This enables us to create engineered bacteria with diverse functions. In addition, we already had some understanding of E. coli, which not only makes the experimental operation more smooth, but also facilitates our further improvement and enhancement of it in the future.

Background

1. Issue

Natural organic matter in water bodies primarily consists of humic acids, fulvic acids, and contaminants from domestic and industrial wastewater. Humic and fulvic acids are weakly functionalized aromatic macromolecular organic acids that account for over 95% of dissolved organic matter in natural waters (Metcal&Eddy, 2014). These compounds consume dissolved oxygen, leading to water quality deterioration, and under anaerobic conditions, promote putrefaction and fermentation, disrupting aquatic ecosystems.

PAHs, as typical persistent organic pollutants, originate from incomplete combustion of fossil fuels and industrial emissions. Their high lipophilicity, toxicity, and mutagenicity facilitate accumulation in sediments and water bodies. For instance, sediment samples from Meiliang Bay in Lake Taihu revealed PAH concentrations as high as 5,033.7 ng/g, over 90% of which consisted of highly toxic components such as benzo[a]pyrene and naphthalene(Zhu, 2021). Source analysis indicated that 35% originated from industrial coal combustion, with the remainder derived from vehicle emissions and daily activities.

The World Health Organization (WHO) classifies several PAHs as Group I carcinogens. Long-term exposure can lead to endocrine disruption, developmental abnormalities, and population decline in fish and invertebrates(WHO, 2017). Therefore, the development of efficient and safe wastewater treatment technologies is imperative.

2.Current Solution

Current biological wastewater treatment technologies mainly include aerobic and anaerobic processes, yet they exhibit significant drawbacks:

Aerobic Treatment:

1.Activated sludge systems are prone to sludge bulking, high energy consumption, substantial sludge production, and sensitivity to toxic shock loads(Tchobanoglous, Stensel, Tsuchihashi, 2020).

2.Biofilm systems face risks of media clogging, biofilm detachment affecting effluent quality, long start-up periods, and high initial investment costs(WEF,2018).

Anaerobic Treatment:

1.Effluent often exhibits high COD/BOD, requiring further aerobic polishing;

2.Process sensitivity to environmental conditions (temperature, pH) and strict requirement for anaerobic conditions;

3.Long start-up time, typically 8–12 weeks;

4.Generation of corrosive and malodorous byproducts (e.g., H₂S), generally lacking nutrient removal capabilities such as denitrification (Grady et al., 2011).

3.Our Solution

As conventional microbial treatment methods are limited by low degradation efficiency, high energy consumption, and insufficient safety mechanisms. In our study, we constructed an engineered E.coli platform integrating three core modules to overcome key limitations of conventional biological treatment. The system combines broad-spectrum degradation pathways (bph, dmp, nah, dbf, xyl), enhanced stress resistance through membrane transporters (NhaA, AcrAB-TolC), and dual biocontainment via immobilization and a thermosensitive suicide circuit, ensuring high efficacy and environmental safety. Furthermore to achieve a better balance between functional expression and population dynamics in the engineered strain, we designed a closed-loop genetic circuit based on the Lux quorum sensing system. This circuit incorporates LuxI and a mutant version of LuxR, under the control of the PLuxR promoter, forming a positive feedback loop. Once the bacterial density reaches a threshold, the system triggers strong activation of downstream gene expression, enabling a tight coupling of cellular growth and metabolic function and the inclusion of the PLuxR promoter allows more flexible and tunable regulation of target genes.For further details, please refer to design part

Our engineered system exhibits the following advantages over conventional microbial treatments:

- Comprehensive degradation capacity: simultaneous degradation of multiple aromatic hydrocarbons, overcoming the narrow substrate specificity of natural strains;

- Quorum sensing (QS) regulated enzyme expression balances cell growth and catalytic activity, enhancing degradation efficiency;

- Enhanced stress resistance and operational stability: membrane transporters improve resistance to ionic toxicity and organic pollutants, with greater adaptability to variations in temperature, pH, and salinity;

- Controlled environmental risk: dual safeguards via physical containment and genetic kill switches prevent engineered strain release; no need for exogenous inducers reduces chemical usage and operational costs;

- Reduced energy and maintenance demands: immobilized carriers allow long-term use without replacement or backwashing, facilitating integration into existing bioreactors or biochemical tanks.

To sum up, our system significantly improves the degradation capacity and operational stability for aromatic pollutants, demonstrating superior overall performance compared to traditional biological treatment methods and offering a new strategy for efficient, economical, and environmentally safe wastewater treatment. Future work will focus on pilot-scale validation, system optimization, and application in real industrial wastewater scenarios.

References

1.Chen, J., Yan, H., & Du, J. M. (2024). Concentration, sources and risk assessment of polycyclic aromatic hydrocarbons (PAHs) in sewage sludge from Xuchang city, China. Applied Ecology and Environmental Research, 22(4), 3629–3639.
2.Liu, Z., Shen, Y., Sun, H., & Li, S. (2020). Removal efficiency and risk assessment of polycyclic aromatic hydrocarbons (PAHs) in a representative wastewater treatment plant in Guangzhou, China. Environmental Science and Pollution Research, 27(30), 36576–36589.
3.Metcalf & Eddy, Inc. (2014). Wastewater Engineering: Treatment and Resource Recovery (5th ed.). McGraw-Hill.
4.Zhu, L., et al. (2021). Spatial Distribution, Sources and Risk Assessment of Polycyclic Aromatic Hydrocarbons in Surface Sediments of Taihu Lake. Journal of Lake Sciences, 33(4), 1205–1216.
5.World Health Organization. (2017). Guidelines for Drinking-water Quality, 4th ed., incorporating the 1st addendum.
6.World Health Organization. (2017). Guidelines for Drinking-water Quality, 4th ed., incorporating the 1st addendum.
7.McGraw-Hill Tchobanoglous, G., Stensel, H. D., Tsuchihashi, R., & Burton, F. (2020). Wastewater Engineering: Treatment and Resource Recovery (5th ed.).
8.Grady, C. P. L., Daigger, G. T., Love, N. G., & Filipe, C. D. M. (2011). Biological Wastewater Treatment (3rd ed.). CRC Press.