Team:Nanjing-China/Results
Results
1、The best bacteria of Pi removal: CPP
Although PPKs from E. coli and C. freundii shares 96% amino acids’ identity, the C. freundii PPK has a glutamate and a lysine residue in positions 327 and 328, where in E. coli they are substituted by much less strongly charged alanine and glutamine residues, respectively. Although these natural occurred mutation sites found in C. freundii PPK are distant from the enzymes’ active site, they lie in the interfaces among monomers of the PPK tetramer. Benefit from this difference, a dramatic increase of intracellular polyP accumulation can be achieved with C. freundii.
As shown in Figure 1a, It is obvious that bacteria with different plasmids have disparate behavior in the SMW. Both CWT and DH5alpha served as controls. Associated with the synthesis of PolyP was the continuous uptake of exogenous Pi from the SMW, and for CPP, a maximal Pi removal of 19 mg of P/L was then achieved at 14 h. However, for the other three bacteria, the small amount of Pi removed could be attributed to stoichiometric incorporation into its cellular growth, which formed a background TP content.
Figure 1a
Considered that the synthesis of PolyP by ppk1 requires ATP as we mentioned above, synthesizing excessive PolyP may affect the growth of bacteria. We tested the influence of different-copies plasmid strategy in the E.coli and CF for the phosphorus removal(figure1b), M stands for medium copy strategy, and H stands for high copy strategy. As a result, Citrobacter freundii with medium copy CFPPK can remove the most phosphorus with the least COD consumption. Decreases in Pi removal approaching 60% of CF-HCPP relative to CF-MCPP suggested that the cooccurrence of a second high-copy plasmid impacted the genetically engineered enhancement of polyP biosynthesis.
Figure 1b
However, polyP degradation accompanied by Pi secretion occurred in both CPP and CWT between 15 and 16 h. Since then, they entered the stage of Pi release until their intracellular polyP pools were totally depleted at 92 h (Figure 1d). This result implied that we can recycle phosphorus from CPP in the form of Pi, which will be discussed below. We detected OD600 both in two stages, and it was obvious that CF, that had higher biomass, was more suitable for SMW than CWT(Figure1c).
Figure 1c
Figure 1d
At this point, we asked whether the polyP content of CPP could be further increased. The intracellular polyP of heterotrophic bacteria is essentially derived from exogenous Pi and the carbon source (represented as COD). In the transition phase of intracellular polyP synthesis and degradation, a certain amount of Pi (11 mg of P/L) remained in the supernatant, whereas COD was almost depleted (Figure 1b and Figure S1).
Therefore, a lack of available COD and not Pi likely accounted for further accumulation of intracellular polyP in CPP. To directly test this idea and avoid growth dilution effects, filtersterilized glucose was added to a final concentration of 10 mg of COD/L (a value calculated on the basis of Figure S1) per hour. Nevertheless, the polyP content of CPP was not further increased, and Pi release continued (data not shown).
2、Expression of ppk/ppx
In order to prove that it is the overexpression of ppk1 gene that CPP had high efficiency in Pi removal, we did qRT-PCR analysis to figure out the expression of ppk1 gene and ppx gene over time. As shown in the figures, both taken 16Sgene, a housekeeping gene, as a control and set as 1, the extra expression of ppk1 at different times in CPP was decided by the ppk1 on the plasmid(Figure 2a), and the expression of ppx remained stable and low(figure2b), that is why the Pi secretion occurred in 15-16 hours later.
Figure 2a
Figure 2b
3、MAP precipitation
Taken highest phosphorus removal rate as the standard, we did a series of experiments to figure out the best conditions for struvite precipitation. It was easy to find associated papers, which said the best pH for precipitation is 9, and varied in molar ratio, while temperature and reaction time is not that important in contrast to pH and the molar ratio. According to the Figure3a, When N and Mg mol ratios were slightly larger than P, the deposition rate of struvite was greatly increased. Considering the cost, we chose the Ph=9, the molar of N:Mg:P= 1.25:1.25:1 as the best reaction options, on that condition, the Pi in the SMW can be removed 98.14%(Figure3a). the precipitation (Figure3b) was proved to be MAP via EDS, SEM and XRD, the electron microscopy images and the contrast are as followed (figure3c,d).
Figure 3a
Figure 3b
Figure 3c
Figure 3d
4、Device
Figure 4a
Figure 4b
Figure 4c
As the most important part of this study, we sought to leverage the strengths of CPP to remove and concentrate Pi from SMW. Consistent with the results above the in-reactor Pi was completely depleted within 4 h. Thereafter, practically Pi-free water was consistently obtained through continuous withdrawal of the reactor supernatant until 0.02 mg of Pi/L was detected in the permeate stream at 16 h. During this period, the Pi from 85 L of SMW was fully taken up by CPP and mainly converted to a concentrated form (i.e., intracellular polyP). After membrane concentration, 9 L of an enriched CPP cell suspension was obtained and then subjected to Pi release. At the end of the Pi release phase, a cell suspension with supernatant Pi concentrations of ≤62 mg of P/L was formed; namely, Pi was effectively enriched up to 8-fold.
As confirmed by a recovery efficiency calculation, almost all the Pi was successfully concentrated apart from the portion assimilated by CPP. These results suggest that full-scale Pi removal and enrichment using CPP are technologically feasible.
5、Botanical experiments
In order to verify the fertilizer properties of struvite, we designed the positive control group of complete fertilizer, the negative control group without phosphorus and the experimental group with struvite. The complete fertilizer group used market compound fertilizer, the struvite group took struvite as the source of phosphate fertilizer, and the phosphorus free group was the fertilizer without phosphorus element.When the amount of fertilizer was the same, the growth of struvite group was the same as that of the whole fertilizer group, but the growth without phosphorus was a little worse. However, the total production of struvite group is the highest, which may be out of the low solubility of struvite makes it a high-quality slow-release fertilizer.
Figure 5a
Figure 5b
Figure 5c