Evaluating Sustainable Development Pathways for Protein- and Peptide-Based Bioadsorbents for Phosphorus Recovery from Wastewater.

Recovering phosphate (P) from point sources such as wastewater effluent is a priority in order to alleviate the impacts of eutrophication and implement a circular economy for an increasingly limited resource. Bioadsorbents featuring P-binding proteins and peptides offer exquisite P specificity and sensitivity for achieving ultralow P concentrations, i.e., <100 µg P L-1, a discharge limit that has been implemented in at least one treatment facility in nine U.S. states. To prioritize research objectives for P recovery in wastewater treatment, we compared the financial and environmental sustainability of protein/peptide bioadsorbents to those of LayneRT anion exchange resin. The baseline scenario (reflecting lab-demonstrated performance at a full-scale implementation) had costs that were 3 orders of magnitude higher than those for typical wastewater treatment. However, scenarios exploring bioadsorbent improvements, including increasing the P-binding capacity per unit volume by using smaller P-selective peptides and nanoparticle base materials and implementing reuse, dramatically decreased median impacts to $1.06 m-3 and 0.001 kg CO2 equiv m-3; these values are in line with current wastewater treatment impacts and lower than the median LayneRT impacts of $4.04 m-3 and 0.19 kg CO2 equiv m-3. While the financial viability of capturing low P concentrations is a challenge, incorporating the externalities of environmental impacts may provide a feasible path forward to motivate ultralow P capture.

Adsorption of recalcitrant phosphorus compounds using the phosphate-selective binding-protein PstS.

Currently available wastewater phosphorus (P) treatment technologies target removal of reactive forms of P. Selective adsorption of more recalcitrant soluble non-reactive phosphorus (sNRP) can improve P removal and recovery. A phosphate-selective phosphate-binding protein (PBP), PstS, was immobilized onto NHS-activated beads to assess the ability of this novel bioadsorbent to remove (adsorb) and subsequently recover (desorb) a range of sNRP compounds. Four sNRP compounds representative of wastewater sNRP were selected for use in this study: phytic acid (PA), sodium triphosphate (TrP), beta-glycerol phosphate (BGP), and sodium hexametaphosphate (HMP). Using PBP, adsorption of all sNRP compounds was thermodynamically favorable. The PBP had nearly equivalent binding affinity for PA compared to PBP's typical target, orthophosphate, although it had less affinity for the other sNRP compounds. Adsorption followed pseudo-second order reaction kinetics, with 95% of maximum adsorption occurring within 4 min. This was substantially faster sNRP adsorption compared to other adsorbents in the literature. Adsorption was modeled using the Langmuir isotherm, reflecting that one phosphate molecule attached to one PBP binding site. Notably, this selective 1:1 attachment resulted in higher total P removal for sNRP molecules with high P content. The binding site lost activity with increasing pH, and as such, highest desorption was achieved at pH 12, making the system amenable to sNRP removal as well as controlled recovery.

Fixed-bed column study of phosphate adsorption using immobilized phosphate-binding protein.

Bio-adsorption using high-affinity phosphate-binding proteins (PBP) has demonstrated effective phosphorus removal and recovery in batch-scale tests. Subsequent optimization of design and performance of fixed-bed column systems is essential for scaling up and implementation. Here, continuous-flow fixed-bed column tests were used to investigate the adsorption of inorganic phosphate (orthophosphate, Pi) using phosphate-binding proteins immobilized on resin (PBP-NHS) targeting Pi removal to ultra-low levels followed by recovery. Time to breakthrough decreased with higher influent Pi concentration, smaller bed volume, and higher influent flow rates. The Thomas and Yoon-Nelson breakthrough models adequately described PBP-NHS resin performance with a correlation coefficient of R2 > 0.95. The sharp S-shape of the breakthrough curves for both Pi-only solution and multi-ion solution indicated highly favorable and selective separation of Pi using PBP-NHS resin, beyond that achieved using LayneRT™, a commercial ion exchange resin. The Pi adsorption capacity of the PBP-NHS column was unaffected by competing anions, whereas capacity of the LayneRT™ column dropped by 20%. Tertiary wastewater effluent was also successfully treated in PBP-NHS column tests with a typical S-shaped breakthrough curve. Operating the fixed-bed column in multi-cycle mode evidenced the reusability of PBP-NHS resin with no significant decline in column performance. The results of this study contribute to efforts to scale up designs of PBP-NHS adsorption systems.

Cell surface-expression of the phosphate-binding protein PstS: System development, characterization, and evaluation for phosphorus removal and recovery.

Simultaneous overabundance and scarcity of inorganic phosphate (Pi) is a critical issue driving the development of innovative water/wastewater treatment technologies that not only facilitate Pi removal to prevent eutrophication, but also recover Pi for agricultural reuse. Here, a cell-surface expressed high-affinity phosphate binding protein (PstS) system was developed, and its Pi capture and release potential was evaluated. E. coli was genetically modified to express PstS on its outer membrane using the ice nucleation protein (INP) as an anchoring motif. Verification of protein expression and localization were performed utilizing SDS-polyacrylamide gel electrophoresis (SDS-PAGE), western blot, and outer membrane separation analyses. Cell surface characterization was investigated through acid-base titration, X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FTIR). These tests provided information on the macromolecular structure and composition of the bacteria surface as well as the proton-exchange properties of the surface functional groups (i.e., pKa values). Phosphate desorption and adsorption batch experiments were conducted to evaluate the effects of temperature, pH, and ionic strength on phosphate capture and release. The PstS surface-displayed cells demonstrated greater potential to release and capture phosphate compared to non-modified cells. Higher temperatures up to 40°C, basic pH conditions (pH = 10.5), and higher ionic strength up to 1.0 mol/L KCl promoted 20%-50% higher phosphate release.