Water eutrophication is a widespread issue, mainly caused by excessive phosphate discharge from industries like detergent and fertilizer production. Phosphates, though essential, are limited resources and are becoming scarce. Effective phosphate management and recovery are crucial for sustainability. Current phosphate removal technologies are expensive, complex, and inefficient. To solve this, the WHHS-Pro-China team is developing a system for efficient phosphate recovery. The first-generation system used engineered bacteria with phosphate-binding proteins (PBP) on their surface to adsorb phosphate, but faced challenges such as high bacteria mortality and costs. The second-generation system improves on this by enabling PBP secretion and using NHS beads for better phosphate adsorption and recovery.
Phosphates are an important compound of phosphorus, one of the essential elements on Earth. They are widely present in almost all foods and are extensively used in food processing as an important ingredient and functional additive[1]. Additionally, phosphates are widely used in industrial production, often as a softening agent in detergents. In agriculture, phosphates are one of the three main nutrients essential for plant growth and are a key component of fertilizers, ensuring global food production. However, the excessive discharge of phosphates and their limited resource characteristics pose dual challenges globally.
The uncontrolled discharge of phosphorus-containing wastewater from households, agriculture, and industrial activities is leading to a sharp increase in phosphate content in water bodies, resulting in eutrophication. Eutrophication causes excessive nutrient accumulation in water, promoting abnormal growth of algae and other aquatic plants. The overgrowth of algae not only reduces water transparency and degrades water quality but also consumes large amounts of oxygen during decomposition, leading to hypoxia in water bodies, threatening the survival of other aquatic organisms. The balance of ecosystems is disrupted, biodiversity is reduced, and toxic algal blooms may occur, further endangering water safety and human health[2].
While excessive phosphate discharge causes environmental problems, from a global resource perspective, phosphates are a limited and non-renewable resource. Currently, the major reserves of phosphate rock globally are concentrated in a few countries, including Morocco, China, and the United States, and these reserves are rapidly depleting. As global agricultural demand grows, the continued depletion of phosphate resources could lead to a severe resource crisis in the coming decades, directly affecting agricultural production and food security. Therefore, the effective management, recovery, and reuse of phosphates are key to addressing both environmental issues and resource scarcity[3].
Existing phosphate removal and recovery technologies are diverse but have limitations, making it difficult to balance cost, efficiency, and sustainability. The challenges of the main technologies are as follows[5]:
• High Cost and Complexity: Many phosphate removal technologies, such as chemical precipitation and adsorption, are complex to operate and expensive, especially for large-scale treatment, where equipment and reagent costs are difficult to control.
• Unstable Efficiency: Biological treatment and membrane technologies are sensitive to environmental conditions, and their treatment efficiency is difficult to maintain due to variations in influent concentration and microbial growth environments.
• Secondary Pollution and Resource Waste: Chemical precipitation and adsorption methods generate large amounts of sludge and waste, making disposal difficult and increasing the risk of secondary pollution.
• Difficulty in Phosphate Recovery: While chemical and biological methods can effectively remove phosphates, they struggle to achieve efficient phosphate recovery, leading to resource waste.
Poor Scalability: Existing technologies perform well in the laboratory, but at an industrial scale, cost and efficiency are difficult to meet. In particular, the continuous operation of systems and efficient recovery of phosphate when treating large volumes of wastewater are major challenges.
In this context, the WHHS-Pro-China team envisions that collecting and recycling phosphorus from polluted water could have great prospects. Therefore, we are committed to developing an efficient, low-cost, and sustainable phosphate recovery and treatment system to mitigate water pollution and address potential future phosphate resource shortages.
Our project uses genetically modified Escherichia coli (E. coli ) as the chassis microorganism due to its ease of manipulation, fast growth, and low cost. Compared to other microorganisms, E. coli is flexible for genetic manipulation, making it suitable for large-scale industrial applications. Its modified capability to efficiently secrete phosphate-binding proteins (PBP) provides strong technical support for our system, ensuring the efficiency and sustainability of the phosphate recovery process.
Our first-generation system is based on cell surface display technology, which allows engineered bacteria to display target proteins on their outer membranes, directly interacting with phosphates in the environment. Through genetic engineering, we fused ice nucleation protein (INP) with a high-affinity phosphate-binding protein (PBP) to ensure PBP is stably anchored on the surface of E. coli , achieving efficient phosphate adsorption.
Cell surface display technology is a biotechnology that anchors proteins on the bacterial outer membrane. We used the anchoring function of INP, a protein derived from the outer membrane of Gram-negative bacteria, which binds to the bacterial outer membrane and remains stable. By fusing INP with the target protein—PBP in this project—engineered bacteria can display these high-affinity binding proteins on their cell surfaces. INP’s key characteristic is its stability under harsh environmental conditions, which allows engineered bacteria to continuously adsorb phosphates in the complex wastewater environment.
Phosphate-binding protein (PBP) is a key component of bacterial phosphate transport systems with a very high affinity for phosphate. PBP forms a deep cleft structure with 12 hydrogen bonds, selectively binding phosphate molecules while excluding structurally similar compounds like arsenate. In our engineered bacteria, PBP is expressed and anchored on the cell surface, enabling efficient phosphate capture from wastewater while avoiding nonspecific binding with other pollutants, thus improving adsorption efficiency.
Fermentation Production: In a fermenter, engineered bacteria are cultured in large quantities, expressing INP as an anchoring protein to ensure PBP is stably displayed on the bacterial outer membrane, achieving efficient phosphate adsorption.
Phosphate Adsorption: The engineered bacteria are added to wastewater, where PBP on the bacterial surface binds to phosphates in the wastewater through its high-affinity binding sites.
Phosphate Recovery: After phosphate adsorption, the engineered bacteria are collected by centrifugation or filtration, and the adsorbed phosphate is eluted from the bacterial surface by adjusting the pH for recycling.
While PhosLock 1.0 effectively demonstrated phosphate removal, several critical issues were encountered in practical application:
High Mortality of Engineered Bacteria: The cell surface display technology requires stable anchoring of proteins on the outer membrane, which imposes a burden on the bacterial membrane structure and metabolism. In complex and potentially harmful wastewater environments, the survival rate of engineered bacteria is low, affecting system efficiency.
High Cost: Maintaining the activity and stability of engineered bacteria requires high culture and maintenance costs, and the complex steps involved in phosphate recovery further increase overall costs. Additionally, the phosphate-binding protein displayed on the cell surface easily loses activity during elution, reducing the efficiency of reuse.
Limited Adsorption Capacity: Since phosphate-binding proteins are only displayed on the bacterial surface, the adsorption capacity and efficiency are limited by the number of bacteria and growth conditions, making it challenging to meet the demands of large-scale industrial wastewater treatment.
To overcome the challenges of PhosLock 1.0, we developed PhosRegen 2.0 by optimizing the secretion mechanism of phosphate-binding proteins (PBP), thereby enhancing the efficiency of phosphate adsorption and recovery. The key improvement in PhosRegen 2.0 lies in the secretion of PBP into the culture medium instead of being displayed on the cell surface. By combining this with NHS-activated agarose beads, the phosphate removal process is further optimized.
Secretion Mechanism of PBP: The second-generation system introduces the pectate lyase B (PelB) signal peptide, which guides PBP into the periplasmic space of E. coli and then secretes it into the culture medium, rather than anchoring it on the cell surface.
Bead Adsorption and Phosphate Treatment
In the microbial factory, the secreted PBP is collected from the culture medium and combined with NHS-activated agarose beads. These beads carry active groups that can form stable covalent bonds with amino acids in PBP. Through this bead-binding system, PBP is displayed on the bead surface and retains its high phosphate affinity. The beads are then used for wastewater treatment to achieve efficient phosphate adsorption. After the beads have adsorbed sufficient phosphate, the phosphate can be eluted and recovered by adjusting the pH.
Improved Cell Survival and Stability: Engineered bacteria avoid direct contact with wastewater, reducing environmental stress and significantly improving survival rates. Meanwhile, PBP is secreted into the culture medium, easing the burden on cells and further enhancing system stability.
Efficient Adsorption and Recovery: PBP binds to NHS-activated beads, forming an efficient adsorption platform that can quickly adsorb large amounts of phosphate from wastewater. Phosphate can be easily recovered by simple pH adjustments, making the system highly efficient and user-friendly.
Reusable Beads: Unlike PhosLock 1.0, where engineered bacteria could not be reused after severe pH changes, the NHS-activated beads in PhosRegen 2.0 can be reused multiple times after phosphate recovery, significantly reducing operating costs and improving the system’s economic and sustainable performance.
PhosRegen 2.0, through PBP secretion and bead-binding technology, overcomes the limitations of PhosLock 1.0, enhancing phosphate adsorption efficiency and bacterial stability. With this optimized system, we not only achieve efficient phosphate removal but also significantly reduce operating costs, making the system more suitable for large-scale industrial wastewater treatment.[11]
The implementation of our phosphate recovery system consists of two components: a microbial factory and a sewage treatment plant. First, in the microbial factory, genetically modified E. coli produce and secrete phosphate-binding proteins (PBP), which are secreted into the culture medium during fermentation and then combined with NHS-activated agarose beads. These PBP-loaded beads have a high capacity for phosphate adsorption.
Next, the beads are transported to the sewage treatment plant for actual wastewater treatment. In the sewage treatment plant, the beads are added to an adsorption tank where they come into full contact with the phosphate-containing wastewater. PBP quickly adsorbs the phosphate from the wastewater. After a certain reaction time, the phosphate-laden beads are separated from the treated wastewater by filtration or precipitation, and the treated wastewater can be directly discharged or further treated. The separated beads enter an elution pool where the phosphate is eluted by adjusting the pH, forming a concentrated phosphate solution for recycling, such as for agricultural fertilizers. The beads can be washed and reused in the adsorption tank to continue treating wastewater.
This system is designed to efficiently remove phosphate from wastewater while recycling phosphate. The reuse of beads significantly reduces operating costs. Through modular operation, the system can flexibly adapt to different scales of wastewater treatment and seamlessly integrate into existing processes, providing an economical and sustainable solution for industrial wastewater treatment.
1. Efficient Phosphate Recovery and Simplified Process
The second-generation system simplifies the phosphate recovery process through protein secretion and bead-binding technology. Compared to traditional chemical precipitation and membrane filtration technologies, our system can efficiently recover phosphate from wastewater in a shorter time while significantly reducing treatment costs. The reusable beads minimize material consumption, making the entire process more economical.
2. Sustainability and Resource Recycling
By implementing an innovative phosphate recovery mechanism, we achieve efficient phosphate reuse. The recovered phosphate can be used as agricultural fertilizer, reducing dependence on natural mineral phosphate resources and mitigating the global phosphate shortage crisis. This recycling model aligns with Sustainable Development Goals (SDGs), particularly Goal 6: Clean Water and Sanitation, and Goal 12: Responsible Consumption and Production, promoting effective resource management.
3. Biosafety
The engineered bacteria are only used in the fermentation tank, where the produced PBP proteins are extracted for wastewater treatment, preventing the engineered bacteria from entering the environment and avoiding the risk of biological leakage. This closed production and treatment process complies with strict biosafety standards, ensuring no adverse effects on ecosystems and further contributing to environmental protection and biosafety.
4. Social Benefits and Environmental Contribution
Our system reduces phosphate discharge, alleviating eutrophication and protecting aquatic ecosystems. Additionally, the recovered phosphate can be reintroduced into agricultural production, lowering fertilizer costs and supporting food security. This technology not only reduces overall wastewater treatment costs but also raises global awareness of environmental protection and promotes the sustainable use of natural resources.
5. Innovation and Uniqueness
Compared to existing phosphate treatment technologies, our second-generation system stands out for its innovative use of protein secretion and bead-binding technology, improving treatment efficiency and reducing costs. The simplified process, reusable materials, and highly controlled production make it uniquely competitive in the wastewater treatment field, showcasing the broad application prospects of biotechnology in environmental management.
In the future, we will continue to optimize the structure of the PBP protein to enhance its phosphate adsorption capacity and explore more efficient materials to further improve recovery efficiency and reduce costs. Additionally, we plan to expand the application scope of this technology beyond industrial wastewater to household wastewater and other types of wastewater treatment scenarios, developing customized solutions based on different environmental conditions.
We look forward to collaborating with countries and regions worldwide, sharing research results, and jointly addressing the global phosphate resource shortage. Furthermore, the project will be promoted in areas facing phosphate scarcity, helping improve local agricultural production conditions, supporting relevant policy development and implementation, and driving the standardization of phosphate recovery and reuse in the industry. We aim to ensure the technology’s safety and effectiveness are widely recognized. Through these initiatives, our project is expected to make a greater environmental and social impact globally.
References：
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