The Construction Of Pseudomonas Aeruginosa Engineering Strains With High Bioelectricity Activity

Microbial fuel cells(MFCs)have a promising prospect in terms of biological production and wastewater treatment.However,the lower power output is the main bottleneck to the practical application of microbial fuel cells,and the acquisition of efficient microbes is a key way to solve these bottlenecks.Microbial performance of electricity production is a complex phenotype which controlled by multiple pathways and physiological systems,and it has shown some limitations in its performance improvement when individual functional genes was engineered.In this paper,a series of ? factor overexpression strains were constructed by using P.aeruginosa as the host.In order to screen the important target genes for electricity production,the content of pyocyanin(electronic shuttle)in these engineering strains and other engineering strains which were successfully constructed in the early stage were detected.And then,a potential high-electricity-yielding active engineering strains was constructed,which occurred closely following the expression vector and host screening.Finally,we inoculated this engineering strain into MFCs for the evaluation of power generation performance.The results will provide new strategies and applicable examples for the discovery of efficient electricigen in MFCs.The results showed that rpoF had a positive effect on the synthesis of pyocyanin in P.aeruginosa at a concentration of 64.6 mg/L,which was 4.6 times of that of the control strain.rpoD,rpoN,rpoHand rpoEhad a negative effects on the synthesis of pyocyanin in P.aeruginosa.The contents of pyocyanin in the culture medium were decreased to 12.6 mg/L,9.29 mg/L,8.07 mg/L,8.01 mg/L,relatively.And the negative regulatory intensity decreases in turn.At the same time,the ability of these engineering strains which were successfully constructed in the early stage to secrete pyocyanin was also tested.The results showed that pmpR had a negative effect on the synthesis of pyocyanin in P.aeruginosa,and the concentration of pyocyanin in PmpR was 39.04 mg/L,which was 2.85 times of that of the control strain.lasRrhlR,katA.phzS,bdlA and filCpilA had a positive effects on the synthesis of pyocyanin in P.aeruginosa,and the contents of pyocyanin in the culture medium were decreased to 10.12 mg/L,7.61 mg/L,6.17 mg/L,5.65 mg/L,2.91 mg/L,relatively.And the positive regulatory intensity decreases in turn.In addition,the detection of other phenazine substances in the rpoF-overexpressing strain and pmpR knockout strain showed that the total amount of phenazine was higher than that of the control strain.The expression vector pHERD20T and host P2-A-11 was screened by overexpression of green fluorescent protein and glyceraldehyde-3-phosphate dehydrogenase(GAPDH).P.aeruginosa P2-A-11/pHERD20T-rpoF a potential high-electricity-yielding active engineering strain,was constructed.And then,we inoculated this engineering strain into MFCs for the evaluation of power generation performance.The results showed that the stable time of P.aeruginosa P2-A-11/pHERD20T-rpoF-inoculated MFCs was reduced by 34.58%to 157 h,and the voltage was increased by 15%to 437 mV compared with the control strain.The results of cyclic voltammetry illustrated that the P.aeruginosa P2-A-11/pHERD20T-rpoF inoculated-MFCs had higher redox peaks,which indicated a higher content of phenazine(electron shuttle)in the MFCs.This result is consistent with the HPLC results.All these results suggest that overexpression of the rpoF gene can improve the electrical performance of the strain by increasing the amount of electron shuttle.