The coding sequence for phosphate-binding proteins (PBPs) PstS was synthesized, along with the truncated ice nucleation protein (INP) sequence, placed upstream of PstS. Codon optimization for Escherichia coli was performed, and the genes were cloned into the pET23b plasmid via NdeI and XhoI restriction sites, generating the recombinant plasmid (Genewiz, USA). The recombinant plasmid, driven by the T7 promoter for constitutive expression of downstream genes, was verified through sequencing (Qingke, China) and extracted using a plasmid extraction kit (TianGen, China). The plasmid was then transformed into E. coli DH5α and BL21 strains. E. coli DH5α was used for plasmid storage, while BL21 was used for plasmid expression. The engineered strains were cultured in LB medium containing ampicillin (Amp) (50 μg/mL) at 37°C.

Experimental Design Overview:

This experiment aimed to verify the phosphate adsorption capacity of different engineered strains. The experimental group used INP-PBP bacteria expressing PBP on the cell surface, while the control group consisted of bacteria expressing PBP intracellularly. The comparison of phosphate adsorption efficiency between these two groups was used to assess the necessity of displaying PBP on the cell surface.

• Experimental Group (INP-PBP bacteria): PBP was anchored to the cell surface by INP, enabling direct phosphate adsorption in the solution.

• Control Group (Intracellular PBP expression): PBP was expressed intracellularly, unable to directly contact the phosphate in the solution.

Procedure:

Both groups were cultured in LB medium containing 50 μg/mL ampicillin at 37°C, 250 rpm for 12 hours. After 12 hours, the cell density was adjusted to OD600 = 1, followed by the addition of 10 mg/L KH₂PO₄ as the phosphate source. The mixture was incubated at room temperature with shaking for 3 hours. The remaining phosphate concentration in the solution was measured using the Malachite Green Phosphate Detection Kit.

Principle of Malachite Green Phosphate Detection Kit:

Malachite Green Phosphate Detection Kit combines Malachite Green and Molybdate with free inorganic phosphate in solution. The green phosphomolybdic acid complex is formed. This complex has a characteristic absorbance in the wavelength range 620-640 nm, and the intensity of the absorbance is proportional to the phosphate concentration in the solution. By measuring the absorbance at 630 nm, the residual phosphate concentration in the solution can be quantitatively analyzed.

Results:

• Experimental Group (INP-PBP bacteria): The phosphate concentration in the solution significantly decreased, indicating that surface-displayed PBP efficiently adsorbed phosphate from the solution.

• Control Group (Intracellular PBP expression): Phosphate concentration remained unchanged, showing that intracellular PBP could not adsorb phosphate effectively.

Conclusion:

PBP must be displayed on the cell surface to efficiently adsorb phosphate. Intracellular expression alone is insufficient for effective phosphate removal. This result highlights the importance of surface-displayed PBP for developing a high-efficiency phosphate recovery system.

Experimental Setup:

This experiment analyzed how phosphate starvation affected the phosphate adsorption capacity of engineered bacteria compared to non-engineered strains. The comparison between strains with and without phosphate starvation was used to evaluate the performance of the engineered bacteria.

• Blue bars: Non-engineered strains without phosphate starvation.

• Red bars: INP-PBP engineered bacteria, expressing PBP.

The remaining phosphate concentration in the solution was measured using the Malachite Green Phosphate Detection Kit to calculate the adsorption capacity.

Results:

• Non-starved groups: The non-engineered strain exhibited low adsorption capacity, while INP-PBP engineered bacteria showed significantly higher adsorption even without starvation.

• Starved groups: After phosphate starvation, the adsorption capacity of engineered bacteria increased dramatically.

Conclusion:

Phosphate starvation further enhanced the adsorption capacity of INP-PBP engineered bacteria, demonstrating the effectiveness of genetic modification and starvation treatment in optimizing phosphate recovery.

Experimental Design:

To determine optimal conditions for phosphate adsorption by INP-PBP bacteria, the experiment adjusted two variables: pH and temperature. The pH conditions tested were pH 3, 5, 7, 8, and 10, and the temperatures were 25°C, 35°C, and 45°C.

Results:

• pH optimization: Phosphate adsorption was highest at pH 7, with lower efficiency under acidic (pH 3 and 5) and alkaline (pH 8 and 10) conditions.

• Temperature optimization: Adsorption was most effective at 35°C, with decreased efficiency at both lower (25°C) and higher (45°C) temperatures.

Conclusion:

Optimal phosphate adsorption by INP-PBP bacteria occurred at pH 7 and 35°C, making these ideal conditions for the phosphate recovery system.

Experimental Setup:

The phosphate desorption process is a key step in the whole phosphate recovery system. By adjusting the pH value and temperature of the environment, the engineered bacteria can release the phosphate that has been adsorbed, thus achieving the recovery of phosphate. Therefore, verifying the desorption capacity of engineered bacteria at different pH and temperature conditions is crucial for optimizing phosphate recovery systems.

This experiment examined the phosphate desorption capacity of INP-PBP bacteria under different pH and temperature conditions to optimize phosphate recovery.

Results:

Desorption was most efficient under acidic (pH 3) and alkaline (pH 10) conditions and at higher temperatures (45°C).

Conclusion:

Strong acidic and alkaline conditions, along with high temperatures, promote effective phosphate desorption, providing crucial parameters for optimizing phosphate recovery systems.

Experimental Setup:

To verify whether INP successfully anchored PBP on the cell membrane, cell membrane and cytoplasmic fractions were separated, and their phosphate adsorption capacities were measured.

Experimental steps for separating cell membrane and cytoplasmic components:

Bacterial culture: The engineered bacteria were first cultured overnight in LB medium (50 mL medium).

Cell collection and resuspension: After culture, centrifuge at 10,000 rpm for 1 min to collect bacterial precipitates. The precipitate was re-suspended with 10 mL PBS to ensure uniform cell distribution.

Cell lysis: Breaking up cells by ultrasound. The crushing condition was 150W power, ultrasound for 1 second, interval of 3 seconds, cycle for 20 minutes to ensure complete cell lysis.

Preliminary centrifugation: The broken cell mixture is centrifuged at 5,000 rpm for 10 minutes and the supernatant is collected for further separation.

Ultracentricentrifugation: The collected supernatant is centrifuged at 39,000 rpm for 1 hour to separate the cytoplasmic components (supernatant) and cell membrane components (precipitate).

Re-suspension of cell membrane components: The collected cell membrane precipitates were re-suspended with 2 mL PBS and gently mixed in preparation for adsorption experiments.

Results:

The experimental results showed that the phosphate adsorption capacity of the cell membrane component (red column) was significantly higher than that of the cytoplasmic component (blue column), indicating that PBP was mainly located on the cell membrane and demonstrated its phosphate adsorption function here. This result is consistent with our hypothesis that INP successfully anchors PBP to the cell membrane, resulting in stronger adsorption capacity of the cell membrane components.

If PBP is not successfully anchored to the cell membrane, all of the PBP will remain in the cytoplasm, resulting in higher adsorption capacity in the cytoplasmic portion and lower adsorption capacity in the cell membrane portion. Therefore, the results of the experiment verified that INP successfully located PBP on the cell membrane, and the significant improvement of the adsorption capacity of the cell membrane was the embodiment of this anchoring effect.

Conclusion:

The results clearly validate the function of INP, that is, INP successfully anchors PBP to the cell membrane. This makes the membrane components of the engineered bacteria exhibit higher phosphate adsorption capacity, while the cytoplasmic components exhibit lower adsorption capacity. If PBP is not successfully anchored to the membrane, only the cytoplasmic portion is expected to exhibit higher adsorption capacity. The results provide important support for the phosphate recovery system based on INP-PBP, indicating that this anchoring strategy is feasible and effective for improving the phosphate adsorption effect of engineered bacteria.

Although the PhosLock 1.0 system validates the effectiveness of phosphate removal, some problems have been encountered in practical applications. First of all, engineering bacteria have low survival rate in complex wastewater environment due to high outer membrane pressure, which affects system efficiency. Secondly, the cost of maintaining the activity and stability of the strain is high, and the complex operation and protein inactivation problems further increase the operating cost. Finally, phosphate-binding proteins can only be displayed on the cell surface, and the adsorption capacity is limited, which is difficult to meet the needs of large-scale wastewater treatment.

To address the limitations observed with the INP-PBP system, the phosphate-binding protein (PBP) PstS coding sequence was synthesized, along with a secretion signal (PelB), which was placed upstream of the PstS gene. The genes were codon-optimized for E. coli and cloned into the pET23b plasmid using NdeI and XhoI restriction sites, creating a recombinant plasmid (Genewiz, USA). This plasmid was constitutively expressed under the T7 promoter. After sequence verification (Qingke, China), the plasmid was extracted using a plasmid extraction kit (TianGen, China). The recombinant plasmid was transformed into E. coli DH5α for plasmid storage and E. coli BL21 for expression. The engineered strains were cultured in LB medium containing ampicillin (50 μg/mL) at 37°C.

After secretion, PBP was incubated with NHS beads. The NHS group forms stable peptide bonds with the primary amine groups of PBP, immobilizing it onto the magnetic beads, thus improving phosphate adsorption efficiency. This method is expected to enhance the operation efficiency and adsorption capacity of the PhosLock 1.0 system.

Experimental Design Overview:

To evaluate the phosphate adsorption capacity of different strains, the experiment involved three types of bacteria: non-engineered strains (blue bars), engineered strains expressing PBP intracellularly (red bars), and engineered strains secreting PBP with a PelB signal (green bars). Each strain was cultured in LB medium containing ampicillin at 250 rpm and 37°C for 12 hours. After culture, 1 mL of bacterial suspension was centrifuged to remove the supernatant and resuspended in Tris-HCl buffer. The cell density was adjusted to OD600 = 1, and 10 mg/L KH₂PO₄ was added to the solution. The reaction was conducted at room temperature under 250 rpm shaking for 3 hours, and phosphate concentration was measured using the Malachite Green Phosphate Detection Kit.

Results Explanation:

The figure compares the phosphate adsorption capacity of different strains:

• Blue bars: Non-engineered strains exhibited low phosphate adsorption capacity but still demonstrated some adsorption ability, indicating that even non-engineered strains can adsorb phosphate to a certain extent under appropriate conditions.

• Red bars: Strains expressing PBP intracellularly performed better than non-engineered strains, but their adsorption capacity was limited since PBP was primarily expressed inside the cells and could not adequately interact with external phosphate.

• Green bars: Strains secreting PBP with the PelB signal showed the strongest phosphate adsorption capacity, suggesting that the secretion of PBP outside the cell significantly enhanced phosphate binding and adsorption efficiency.

Conclusion:

The results indicate that while non-engineered strains possess some inherent phosphate adsorption capacity, engineered strains, particularly those secreting PBP via the PelB signal, demonstrate significantly enhanced adsorption performance. This confirms that the strategy of secreting PBP extracellularly is effective in improving phosphate adsorption, providing a solid foundation for the development of efficient phosphate recovery systems.

Experimental Design Overview:

To assess the impact of different active fractions on phosphate adsorption in PelB-PBP engineered bacteria, the experiment separated the periplasmic and soluble fractions of the engineered strains. The soluble fraction was obtained by sonicating overnight cultures and centrifuging the lysate to collect the supernatant. The periplasmic fraction was isolated by resuspending the bacteria in TES buffer and performing cold treatment and centrifugation. Phosphate adsorption in each fraction was measured using the Malachite Green Phosphate Detection Kit.

Results Explanation:

The total lysate exhibited the strongest phosphate adsorption capacity, as it contained all cellular components, including the membrane-bound and intracellular PBP. The soluble fraction had a higher adsorption capacity than the periplasmic fraction, suggesting that not all PBPs were successfully secreted outside the cell. While the periplasmic fraction showed some adsorption, it was significantly lower than that of the soluble fraction, indicating that PBP had been secreted into the periplasmic space but with limited efficiency.

Conclusion:

The results indicate that although the PelB signal was able to secrete some PBP into the periplasmic space, a large portion of the PBP remained in the intracellular soluble fraction. This suggests that the secretion efficiency was limited, likely due to short culture times or insufficient secretion capacity of the PelB signal. Enhancing secretion efficiency through extended culture times or optimizing the signal peptide could further improve overall phosphate adsorption performance. These findings offer valuable insights for improving phosphate recovery systems.

Experimental Design Overview:

To evaluate the adsorption and desorption efficiency of immobilized phosphate-binding proteins (PBP), crude enzyme extracts were obtained from engineered BL21 bacteria overexpressing PelB-PstS (with a C-terminal His tag). PBP was immobilized onto NHS-activated Sepharose 4 Fast Flow beads by incubating the enzyme with the NHS beads at 4°C for 16 hours. After multiple washes, the immobilized PBP beads were used in a reaction system with KH₂PO₄ to measure their phosphate adsorption capacity.

Results Explanation:

The figure shows significant differences in phosphate removal efficiency under different treatment conditions. The enzyme solution containing PelB-PstS demonstrated some phosphate adsorption capacity, but the immobilized PelB-PstS on NHS beads (PelB-PstS @ Bead) exhibited much higher phosphate removal efficiency. Immobilizing PBP onto the beads not only retained its function but also enhanced phosphate binding efficiency. In contrast, the control group (NHS beads alone) showed negligible phosphate adsorption.

Conclusion:

The results demonstrate that immobilizing PBP onto NHS-activated beads significantly enhances its phosphate adsorption capacity compared to the free enzyme solution. Immobilized PBP performs more efficiently under the same conditions. This finding validates the potential of immobilized PBP in phosphate recovery systems and lays a foundation for future applications in large-scale wastewater treatment.

Experimental Design Overview:

To investigate the effect of pH and temperature on phosphate desorption by immobilized phosphate-binding proteins (PBP), PelB-PstS was immobilized onto NHS-activated Sepharose beads. The beads were saturated with 150 mg/L KH₂PO₄, and after washing with Tris-HCl buffer, unbound phosphate was removed. Desorption was measured under different pH conditions (pH 3, 5, 7, 8, and 10) and temperatures (25°C, 35°C, and 45°C).

Results Explanation:

The figure shows that both pH and temperature significantly affect the desorption efficiency of immobilized PBP. Desorption was most efficient under strong acidic (pH 3) and strong alkaline (pH 10) conditions, where phosphate was effectively released from the beads. Desorption efficiency was lowest under neutral conditions (pH 7). Furthermore, higher temperatures (45°C) significantly enhanced desorption efficiency, while desorption was weaker at lower temperatures (25°C).

Conclusion:

The results indicate that strong acidic (pH 3), strong alkaline (pH 10), and higher temperatures (45°C) significantly improve phosphate desorption by immobilized PBP, while neutral pH and lower temperatures hinder phosphate release. By adjusting pH and temperature conditions, the desorption process can be optimized, offering an effective means to control phosphate release and recovery in wastewater treatment systems.

This study successfully constructed and optimized two engineered bacterial strains, verifying the roles of INP-PBP and PelB-PBP in phosphate adsorption. INP-PBP bacteria effectively anchored PBP to the cell membrane, exhibiting excellent adsorption performance under various conditions, especially enhanced by phosphate starvation. PelB-PBP bacteria, despite not fully secreting all PBP, still showed good phosphate adsorption efficiency. After immobilizing PBP onto NHS-activated beads, the phosphate desorption capacity was significantly improved, particularly under high temperatures and extreme pH conditions. Overall, the research confirms the feasibility of both INP-PBP and PelB-PBP strategies, providing a solid foundation for the further development of phosphate recovery systems.