Team:TJU/Results


Results


1 Lactate producing system


In order to verify our ldhE part and pflB knockout strategy have works well, we get following results.



Figure 1. a:The growth curve in anaerobic condition (M9 medium) b: Glucose consumption curve in anaerobic condition (M9 medium)


Shown as figure 1a, under anaerobic conditions, wild-type MG1655 has the same growth rate as MG1655 ΔpflB, but MG1655 ΔpflB +ldhE keeps relatively lower growth rate than the two strains mentioned before. Shown as figure 1b,the anaerobic glucose consumption rate keeps the nearly same among MG1655, MG1655 ΔpflB and MG1655 ΔpflB+ldhE



Figure 2. a: The lactate production curve in anaerobic condition (M9 medium)b: The lactate production curve in anaerobic condition (M9 medium)


In anaerobic environment, lactate production of MG1655, MG1655 ΔpflB and MG1655 ΔpflB+ldhE has large differences between each other. Notably, knocking out of pflB together with ldhE insertion will increase lactate generation up to ~3 g/L and the amount produced by MG1655 ΔpflB is smaller than MG1655 ΔpflB+ldhE.


So, knocking out of pflB can dramatically improve the lactate production. Besides, ldhE also plays an important role in lactate increase. Engineered lactate producing strain (MG1655 ΔpflB+ldhE) has a greater utilization of carbon sources and a higher yield of lactate with M9 medium under the same concentrations of glucose.


2 Flavins producing system


To prove the EC10 and EC10* work as expected, we conduct several experiments and the results are as follows.



Figure 3. The yield of riboflavin in different strains :EC10, Rf02S (Δpgi+EC10), EC10*


As shown in figure 3, E. coli BL21 with EC10 reaches a final yield of 17 mg/L in tube cultivation. E. coli BL21 Δpgi+EC10 reaches 33 mg/L. E. coli BL21+EC10* reaches 90 mg/L.


3 Co-culture MFC -- Labor Division



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Figure 9： Among optimized shewanella, shewanella+MG1655 and Shewanella+ΔplfB ldhE+Rf02S, the three-strain coculture system reached a relatively higher potential that kept around 350 mV in 80 hours.


Conclusion: Shewanella+ΔplfB ldhE+Rf02S has been going through a preferable optimization. It led to the potential of 300 mV in 80 hours under the condition of 2 g/L glucose without any supplementary. So, it realized a higher and more endurable power generation when taking glucose as carbon sources in Shewanella.


Figure 10： The potential and time of duration in Shewanella+ΔplfB ldhE+B.Subtilis system can be raised into 511 mV and 80 h, respectively, resulted in a greater generation amount.


Figure 11：

The potential produce by Shewanella+ΔplfB ldhE+B.Subtilis is greater than Shewanella+ΔplfB ldhE+Rf02S for 176 mV. Cocultured Shewanella, E.coli and B.subtilis can achieve a better job division that can reduce the competition between two kinds of zymophyte since B,subtilis can anaerobically metabolize by using KNO3. It significantly improve the commensalism relations between three species.


Figure 12

(1) A series of voltage brought by different external resistance from Shewanella+ΔplfB ldhE+Rf02S

(2) The relations between current density and voltage is well suited to polarization curve while the power curve can represent the relations between output power density and current density. Besides, the highest power density can reach to 10 mW/m2.


Figure 13

(1) A series of voltage brought by different external resistance from Shewanella+ΔplfB ldhE+B.Subtilis

(2) The relations between current density and voltage is well suited to polarization curve while the power curve can represent the relations between output power density and current density. Besides, the highest power density can reach to 17 mW/m2.


Figure 14

The comparison of polarization curve and power curve among shewanella, shewanella+MG1655, shewanella+ΔplfB ldhE+Rf02S. It is obvious that the power output in shewanella+ΔplfB ldhE+Rf02S is far more bigger than the controled group.


Figure 15

The comparison of polarization curve and power curve among shewanella, shewanella+MG1655 and Shewanella+ΔplfB ldhE+B.Subtilis. We can apparently observed that the power output of Shewanella+ΔplfB ldhE+B.Subtilis is greater than the controled group.


Figure 16

The comparison of polarization curve and power curve among Shewanella+ΔplfB ldhE+B.Subtilis and Shewanella+ΔplfB ldhE+Rf02S. It has been shown in power curve that Shewanella+ΔplfB ldhE+B.Subtilis has a higher output than Shewanella+ΔplfB ldhE+Rf02S, which indicates that three-strain system can generate higher electricity and have a better MFC performance, in return, have a promising application.


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