Overview

Our team is trying to figure out an innovative way to process the sewage and therefore generate energy for the growth of crops. We develops a simple solo medium-copy plasmid-based polyphosphate kinase (PPK1) overexpression strategy for achieving maximum intracellular polyphosphate accumulation by environmental bacteria. In the inorganic experiments, we will find the suitable concentration of phosphate radical and ammonium, pH, and mole ratio of ions involved in the synthetic reaction of struvite. In the engineering experiments, we will design an integrated waste water processing device, which can remove phosphorus from sewage, and utilize the engineered bacterias to release phosphorus in other areas to produce precipitate with ammonium. In agricultural experiments, we will use our products as fertilizers, and compare them with chemical fertilizers.

The following list exhibits our key achievements in this project:

•Tested an existing part---K1807009

•Contributed 1 basic parts and 1 improved part.

•Constructed solo medium-copy plasmid-based polyphosphate kinase Citrobacter freundii which can overexpress polyphosphate kinase. 

•Designed a reaction device that caters for biosynthetic reaction of struvite.

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.

p.s.:SMW means synthetic waste water

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.

Reaction device

We designed a integrated waste water processing device, which can remove phosphorus and ammonium from sewage, and utilize the engineered bacteria to release phosphorus in other areas to produce precipitate. It cost less electric power than traditional sewage treatment process and can reutilize the phosphorus and ammonium. After a few test, we proved our device to be quite practical.(Have a look at Hardware for more details)