Every year, hundreds of thousands of tons of oil and its pollutants are discharged into the environment. The problem is equally severe in China. Oceans, water sources, and soil are severely harmed as a result. Petroleum contains alkanes, cycloalkanes, aromatic hydrocarbons, heavy metals, and other mixed pollutants, all of which are difficult to degrade.

Petroleum is characterized by high carbon content, diverse organic matter, strong hydrophobicity, high viscosity, and toxicity. It tends to bind with soil particles, clog soil pores, and thus disrupt soil ventilation and water permeability. These effects lead to soil salinization and increased toxicity. Petroleum pollution also harms biodiversity by increasing soil organic carbon, which causes an imbalance in the C/N ratio, negatively affecting the growth and reproduction of microorganisms.

Petroleum poses risks to human health, as humans and animals are highly sensitive to its aromatic hydrocarbons. These compounds can enter the body through various pathways, such as diet, respiration, and direct contact, and have toxic effects on internal organs like the kidneys and liver, with the potential to cause cancer. Polycyclic aromatic hydrocarbons (PAHs), in particular, can severely disrupt bodily functions. Petroleum also contains terpenes and benzenes, which, upon prolonged exposure, can cause symptoms such as vomiting, headaches, and confusion, and in severe cases, even be life-threatening.[3]

Aromatic hydrocarbons in petroleum pollutants can spread across water surfaces, forming thin films that reduce the water’s self-purification capacity and lead to a sharp decrease in oxygen levels. This oxygen deficiency, or hypoxia, drastically lowers the survival rate of aquatic organisms, causing significant damage to the water body’s microecological environment. Furthermore, if these pollutants enter groundwater systems, they pose a severe threat to the safety of human drinking water.[4][5]

The current major approaches for oil pollution remediation include physical, chemical, and biological methods. Physical methods rely on tools like booms, skimmers, and sorbent materials. While environmentally friendly, they are highly dependent on site conditions, labor-intensive, and costly. Chemical methods, such as surfactant washing, chemical oxidation, and nanoparticle foams, offer efficiency and ease of operation. However, controlling the toxic effects of these chemicals on the environment is challenging. In recent years, the biological approach has garnered more attention due to its lower costs and non-toxic impact on ecosystems. Although bioremediation holds great promise, it faces limitations like inefficacy in treating high-concentration petroleum pollutants and the risk of gene leakage, which hinder its widespread application.[6][7]

Bioremediation and phytoremediation (plant-based remediation) are highly promising due to their cost-effectiveness and minimal environmental impact. However, issues such as slow degradation rates, sensitivity to high levels of pollutants, and potential gene leakage prevent these methods from being used on a large scale. Currently, the mainstream approach for oil spill management focuses on removing the bulk of spilled oil while leaving behind micro-residuals. Unfortunately, these residuals, though minimal, can still pose serious long-term environmental risks.[9][10]

Our team, RDFZ-CHINA, has developed a novel, controllable method to address the problem of petroleum pollution, specifically focusing on the degradation of residual oil. We utilize B. subtilis to produce surfactants that enhance oil breakdown and E. coli to produce the plant hormone IAA, promoting cooperation between bacteria and plants in remediation efforts. This approach effectively addresses the challenge of residual oil pollution, avoids secondary contamination after treatment, and ensures biosafety by incorporating a suicide system to prevent gene leakage.

E. coli offers significant advantages, including ease of operation, rapid growth, and well-established gene manipulation techniques. We have designed two genetically engineered strains of E. coli for this project. The first strain expresses the petroleum-degrading system, which includes the AlkM enzyme and a cell surface display system for laccase and INP (ice nucleation protein). A suicide system has also been incorporated into this strain to ensure biosafety. The second strain of E. coli is designed to express the plant hormone IAA for seed treatment only. This IAA-producing strain does not carry the suicide system and will not be part of the final soil treatment or interact with the environment.

In this project, B. subtilis 168 is chosen for its high level of biosafety. Previous studies support the use of B. subtilis for the production of surfactin, a biosurfactant. We have engineered B. subtilis to exclusively produce surfactin, and a suicide system is also incorporated to prevent any unwanted environmental persistence.

In our project, the AlkM and laccase enzymes play crucial roles in degrading petroleum: AlkM breaks down long-chain alkanes, while Laccase targets aromatic hydrocarbons, with the latter's efficiency enhanced by cell surface display technology. To further boost degradation, B. subtilis produces surfactin, a biosurfactant that emulsifies oil, increasing its accessibility to microbes. Additionally, the plant-microbe cooperation system, aided by E. coli producing IAA (indole-3-acetic acid), promotes plant growth, enhancing remediation. Finally, our suicide system ensures biosafety by triggering bacterial self-destruction outside petroleum-contaminated areas, preventing any unintended environmental spread.

Alkane monooxygenase, used to break down long-chain alkanes in petroleum.Alkane hydroxylase, encoded by the alkM gene, is a key enzyme involved in the degradation of long-chain alkanes (C12-C36). AlkM is sourced from Acinetobacter sp. strain ADP1, where it plays an essential role in alkane metabolism. Alkane hydroxylases, including AlkM, belong to a family of bacterial integral-membrane hydrocarbon hydroxylases. These enzymes catalyze the initial oxidation of alkanes to alcohols, which are further metabolized into fatty acids and eventually into simpler compounds for cellular use .[14]

FIG. 9 Four aerobic degradation pathways of alkM

AlkM from Acinetobacter sp. is particularly effective in catalyzing the terminal hydroxylation of alkanes. This enzyme system has been shown to degrade long-chain alkanes that are resistant to breakdown by other means, making it especially useful for bioremediation efforts.

In our project, we utilized E. coli as the chassis microorganism and constructed a recombinant plasmid containing the alkM gene, regulated by a T7 promoter and terminated by a B0015 terminator. This engineered strain facilitates the breakdown of long-chain alkanes through AlkM activity, which initiates the degradation process by oxidizing inert alkanes into more reactive alcohols, setting the stage for further degradation.

Used to degrade aromatic hydrocarbons and enhance its ability to break down large-molecule oils through cell surface display technology.

To address the challenge of degrading polycyclic aromatic hydrocarbons (PAHs), which are difficult for AlkM to process, we incorporated laccase into our system. Laccase is a multicopper oxidase that catalyzes the oxidation of phenolic and aromatic compounds, making it highly effective for breaking down PAHs. Since the large molecular size of these compounds makes them difficult to penetrate the cell membrane, we employed the INP (Ice Nucleation Protein) surface display system. This system allows laccase to be displayed on the surface of the cell, enhancing direct interaction between the enzyme and the substrate.[15]

Cell surface display technology is a method used to present target proteins on the surface of microorganisms. It has applications in enzyme presentation, vaccine development, and environmental remediation. In our design, we combined INP with BsLac (laccase from B. subtilis ) using E. coli as the chassis, along with the T7 promoter and B0015 terminator. This setup enhances the attachment surface between the enzyme and PAHs, improving degradation efficiency.[16]

While enzymes like AlkM are effective for degrading some petroleum components, the degradation rate can be slow due to the limited contact area between E. coli and hydrophobic oil residues. To address this, we utilize surfactin, a biosurfactant produced by B. subtilis , to emulsify oil residues into an aqueous state, enhancing the interaction between the engineered bacteria and the oil, thereby significantly improving degradation rates.

Surfactin is a cyclic lipopeptide, composed of a seven-amino-acid peptide loop and a β-hydroxy fatty acid chain, typically 13 to 15 carbon atoms in length. Its amphiphilic nature allows it to interact with both hydrophilic and hydrophobic substances, making it an ideal agent for breaking down oil residues .

Producing surfactin at a scale suitable for environmental remediation requires optimization. Instead of manipulating the Psrf promoter (due to its complexity in genetic engineering), we focus on the following strategies to enhance surfactin production:

1. Enhancing Fatty Acid and Amino Acid Supply:

During the cultivation phase, we supplement the growth media with additional fatty acids and amino acids to ensure a sufficient supply of the necessary precursors for surfactin synthesis. This straightforward approach helps to drive higher production rates in the engineered B. subtilis .

2. Increasing Expression of Key Enzymes (fadd):

We utilize the fadd gene, which has been previously tested by our team, to enhance the synthesis of fatty acids. By upregulating this gene, we ensure a robust supply of fatty acids, which are critical precursors for surfactin production.

3. Improving the Secretion System:

Efficient secretion of surfactin is essential for its activity in degrading oil residues. We focus on enhancing the Sec-SRP pathway, which is responsible for protein translocation across the membrane. Specifically, we incorporate SecA, FtsY, and FtsE genes, which work together to boost surfactin secretion:

• SecA acts as a key component in the Sec pathway, driving the translocation of proteins across the cytoplasmic membrane.

• FtsY functions as a receptor for the signal recognition particle (SRP) and plays a critical role in the targeting and translocation of proteins to the membrane.

• FtsE, along with FtsX, forms an ATP-binding cassette (ABC) transporter complex, providing the energy needed for translocating proteins like surfactin.

Together, these three genes enhance the efficiency of protein secretion in B. subtilis , ensuring that surfactin is effectively transported out of the cell and into the environment where it can emulsify oil .

By combining enhanced precursor availability, upregulation of biosynthesis pathways (via fadd), and improved secretion systems, we aim to maximize surfactin production and increase its effectiveness in degrading oil residues in polluted environments.

Plant-coupled microbial remediation technology leverages the symbiotic relationship between plants and microbes to remediate petroleum hydrocarbon-contaminated soil. Plants improve the soil environment, provide habitat and nutrients for microbes, while microorganisms secrete amino acids, sugars, and other compounds that support plant growth. This reduces the stress and toxicity of petroleum hydrocarbons on plants and enhances overall soil remediation.

The ability of plants to remediate oil pollution depends on factors such as biomass, root development, genetic traits, and age. Plants with well-developed root systems are more effective at absorbing and degrading organic materials. Ideal plants for phytoremediation exhibit strong tolerance to pollutants, fast growth rates, and large biomass. There are three primary ways plants contribute to oil cleanup:

1.Absorption and Breakdown: Plants can absorb organic compounds from oil, breaking them down through lignification and mineralization, converting them into harmless substances like water and carbon dioxide.

2.Rhizosphere Microbial Interaction: Plant roots release organic acids, ethanol, and proteins that promote the growth of rhizosphere microorganisms. In return, these microbes enhance plant growth, improving the efficiency of oil degradation.

3.Soil Mineralization: Plants can alter the physical and chemical properties of the soil, stimulating the degradation of oil.

One of the primary challenges in using plants for petroleum remediation is the reduced seed germination rate under petroleum stress. To address this, we plan to engineer bacteria to produce indole-3-acetic acid (IAA), a plant hormone known to significantly improve seed germination efficiency.

IAA Production via Genetic Engineering

We employed two pathways to engineer E. coli for IAA production:

IAM Pathway: The IAM pathway is the conventional method of IAA synthesis in E. coli. Tryptophan is first converted into indole-3-acetamide (IAM) by the enzyme tryptophan-2-monooxygenase (encoded by the iaaM gene). IAM is then transformed into IAA by IAM hydrolase (encoded by the iaaH gene). We synthesized the iaaM and iaaH genes, arranged in a polycistronic structure with a B0034 ribosome binding site (RBS), and inserted them into the pET23b vector. The plasmid was successfully transformed into E. coli BL21 after sequencing verification.

IPA Pathway: The IPA pathway is a less-researched but promising method for IAA production. This pathway involves three key enzymes:

Tryptophan transaminase (aro8): Converts tryptophan into indole-3-pyruvate.

Indole-3-pyruvate decarboxylase (kdc): Converts indole-3-pyruvate into indole-3-acetaldehyde.

Aldehyde dehydrogenase (puuc): Converts indole-3-acetaldehyde into IAA.

The aro8 and kdc genes are derived from yeast, while puuc comes from E. coli. These genes were arranged in a polycistronic structure with B0034 RBS, inserted into the pET23b vector, and transformed into E. coli BL21 for IAA production.

Our suicide system is designed to ensure biosafety by preventing the engineered bacteria from surviving outside of petroleum-contaminated environments. It consists of an alkane-responsive promoter (PalkB) and a toxin-antitoxin system (mazF/mazE).

The PalkB promoter responds specifically to the presence of alkanes, which are common hydrocarbon compounds found in petroleum. In the presence of alkanes, the PalkB promoter activates the expression of mazE, the antitoxin. MazE neutralizes the toxic effects of mazF, allowing the engineered bacteria to survive and function in alkane-rich, petroleum-contaminated environments.[20]

MazF is a toxin that inhibits bacterial growth by cleaving mRNA, leading to cell death if not neutralized by MazE. In the absence of alkanes, the PalkB promoter becomes inactive, stopping the expression of mazE. Without MazE to neutralize it, MazF induces cell death. This ensures that the engineered strain self-destructs when it is no longer in a petroleum-contaminated environment, preventing it from surviving in clean, non-polluted areas and ensuring biosafety.

After an oil spill, physical methods are first employed to excavate and seal the contaminated soil. Our goal is to address the diffused and trace amounts of oil residues in the soil using our approach. Firstly, we broadcast E. coli that degrade oil and B. subtilis that produce surfactants into the contaminated soil, allowing them to work together to break down petroleum. It is important to note that considering the survival of the engineered bacteria, this process requires continuous sowing. At the same time, we culture IAA-producing E. coli and use their supernatant to treat seeds of plants that are beneficial for oil pollution remediation. After treatment, these seeds are then sown into the contaminated soil for phytoremediation. In combination with other methods, we may also need to apply nitrogen fertilizer or conduct multiple tillages to enhance the degradation effect. Ultimately, if the soil is fully remediated, our engineered B. subtilis 168 and E. coli will die off when being induced without engendering concerns for biosafety.

Firstly, our product is highly efficient. Through integrating the secretion system and adding precursors, we raised the production of surfactants in B. subtilis , which enables faster break down of petroleum.

We developed a new method to stimulate the degradation of petroleum through incorporating plants in the degradation system in which plants are stimulated to growth, forming a cooperation between plants and microbes to further break down and absorb petroleum, improving the efficiency and integrating with the environment better.

Besides, our product is safe and targeted. The product consists of bacteria that are not that poisonous to the environment. And we added a suicide system which enables the product to only function in polluted areas. Since it is targeted only on petroleum-polluted areas and would otherwise die as the suicide system activates, the product wouldn’t cause pollution or concerns in gene leakage to other areas.

Future research will focus on exploring microbial-plant-animal interactions to enhance petroleum degradation. Studies suggest that combining animals with plants and microbes can improve bioremediation effectiveness. Animal bioturbation increases soil oxygen and porosity, creating better conditions for microbial and plant growth, while the rhizosphere effect stimulates microbial activity. For example, a joint system involving Suaeda salsa, microbes, and sandworms showed significantly higher removal rates of petroleum hydrocarbons. In future work, we will investigate different animal-plant-microbe combinations to optimize this synergistic approach for more effective petroleum remediation.

Our project provides a cutting-edge solution to the global challenge of petroleum pollution, using synthetic biology to design an innovative, safe, and efficient method for environmental remediation. By integrating the strengths of microbial degradation and plant cooperation, our approach offers a targeted and sustainable way to clean up oil residues in contaminated soils while minimizing the risks of genetic leakage through a built-in suicide system.

This project aligns closely with several of the United Nations Sustainable Development Goals (SDGs):

Goal 3 (Good Health and Well-being): By mitigating petroleum contamination in the environment, our project reduces human exposure to harmful pollutants, contributing to healthier ecosystems and improved public health.

Goal 6 (Clean Water and Sanitation): Our technology helps prevent the seepage of oil pollutants into water sources, ensuring cleaner water for ecosystems and human use.

Goal 12 (Responsible Consumption and Production): The use of engineered microbes and plants as a natural remediation tool represents a sustainable and eco-friendly alternative to traditional chemical and physical cleanup methods, supporting more responsible environmental management.

Goal 14 (Life Below Water): Through preventing oil pollution from entering marine environments, we support the protection and restoration of aquatic ecosystems, safeguarding marine biodiversity.

Goal 15 (Life on Land): By remediating contaminated soils, our solution promotes healthier land ecosystems, restoring habitats and contributing to land-based biodiversity.

As we look to the future, this technology could be expanded to address other types of environmental pollutants, offering a scalable model for sustainable environmental management. By advancing biotechnological innovations that align with global sustainability goals, we aim to contribute to a healthier planet and a more sustainable future for all.

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