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 naturally 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. Benefiting from this difference,C.freundii could achieve a dramatic increase of intracellular polyP accumulation.
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,the smaller amount of Pi removed in the other three strains could be attributed to their need for growth, which formed a background TP content.
Figure 1a
As mentioned above, the synthesis of PolyP by ppk1 requires ATP, which suggests that, 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), as M stands for medium copy strategy, and H stands for high copy strategy. As a result, Citrobacter freundii with medium copy CFPPK can remove most phosphorus with least COD consumption.An approximately 60% decrease in Pi removal of CF-HCPP relative to that of 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, 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 enables CPP 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, we take 16S gene, a housekeeping gene, as 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
Taking highest phosphorus removal rate as the standard, we did a series of experiments to figure out the best conditions for struvite precipitation. Associated papers have demonstrated that the best pH for precipitation is 9, and molar ratios of N, Mg and P are also important. In contrast, temperature and reaction time are relatively of less vitality, so we chose pH=9 and start to find out the best Molar ratios of N, Mg and P. According to the Figure3a, when N and Mg were of slightly larger molar ratios than P, the deposition rate of struvite was greatly increased. Considering the cost, we chose pH=9, and molar of N:Mg:P= 1.25:1.25:1 as the best reaction options, under this condition, the Pi removal rate in the SMW could achieve 98.14%(Figure3a). The precipitation (Figure3b) was proved to be MAP via EDS, SEM and XRD, the electron microscopy images and the contrast are presented. (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 a continuous withdrawal of the reactor supernatant until 0.02 mg Pi/L was detected in the permeate stream at 16 h. During this period, Pi from 85 L SMW was fully taken 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 Pi/L was formed; namely, Pi was effectively enriched up to 8-fold.
As confirmed by a recovery efficiency calculation, almost all 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 a 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 used fertilizer without phosphorus element.Having been applied the same amount of fertilizer, 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 was the highest, possibly because the low solubility of struvite makes it a high-quality slow-release fertilizer.
Figure 5a
Figure 5b
Figure 5c