Team:Nanjing-China/Experiments
Experiments
In order to make phosphorus removal more effective, citrate frederik bacillus was selected instead of E.coli. Their different efficiency was due to the difference of the two bases at the position of ppk1 gene 327 and 328.
E.coli
C. freundi
Through the experiment, we verified that the phosphorus removal effect of the difference is real， We transferred the ppk1 gene of C. freundi(CF) and E.coil(DH5α) into C. freundi and E.coil by different gene copy number strategies, eventually identified only CF with medium copy of the CF ppk1 genes can achieve the best effect of phosphorus.
p.s.:M stands for medium copy strategy, and H stands for high copy strategy
We constructed pBBR1MCS2-ppk1 using the genomic DNA of bacillus citrate frumenii (CWT) as a template. Firstly, ppk1 was amplified by PPKF and PPKR primers. After sequencing confirmation, PCR products were digested by Kpn I and Hind III and inserted into pBBR1MCS2 (pBR322 ori) to obtain pBBR1MCS2-ppk1, and then obtain CWT transformed from pBBR1MCS2-ppk1, the strain was named CPP, it is the core engineered bacterium of our experiment.
The main function of this strain is to enrich PO43- in sewage by synthesizing Poly P in the cell. The engineered bacteria can overexpress ppk1 and reversibly catalyze the transfer of the terminal phosphate (Pi) from ATP to the growing poly P chain. After that, the expression product of PPX can released PO43- to the outside of the cell under certain conditions, so that we get high concentration of Pi to precipitate struvite.
After getting the engineered bacteria, we designed the following three experimental parts: struvite formation, design device, and plant experiment. In the first part of the experiment, we explored the conditions of struvite precipitation in advance and chose the conditions with the highest phosphorus removal rate.
In the second part of the experiment, we designed a device that contains five modules. The first module is filled with synthetic wastewater containing phosphorus. Then, the wastewater can be drawn into the second module to supply the nutrition of engineered bacteria, which could effectively remove phosphorous from the waste water. In addition, the second module includes aeration devices and the MBR biofilm reactor. The phosphorus-free water treated by the engineered bacteria will pass through the MBR membrane bioreactor and enter the third module, while the bacteria, trapped by the membrane, will continue to remove phosphorus in the second module. We use a small aeration device to supply oxygen and a large aeration device to avoid bacterial blockage on the membrane. After the engineered bacteria have absorbed enough phosphorus, they will be pumped to the fourth module to release phosphorus; thus, we can get a high concentration of phosphorous containing solution. It will be mixed with the high ammonia nitrogen wastewater containing magnesium ions and ammonium ions in proportion in the fifth module to generate struvite precipitation.
In the third part of the experiment, we used the obtained struvite as fertilizer to grow soybean and lettuce, and make comparison with the full-fertilizer group and the phosphor-free group to explore the utility of struvite as fertilizer.