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



Figure 4. As shown in figure，from left to right is EC10， RF02S and EC10* respectively.



Figure 5. The electrical output of MFCs of single strain Shewanella oneidensis MR-1 with different substrates .


From the diagram above, we demonstrate that：

1. Shewanella cannot use glucose as a mere carbon substrate to generate power and so the potential keeps no more than 50 mV.

2. Shewanella can produce the maximal potential up to 150 mV with supplement of sodium lactate.

3. Shewanella can produce even higher potential when we take sodium lactate as the carbon source and add little riboflavin into the system.

Conclusion: According to our experiment, our system is feasible in principle. Lactate can serve as the entry point in material flow and riboflavin as the major factor in energy and information flow, which lays a foundation in the later experiment design. We can use fermentation bacteria to provide carbon sources and electron shuttles, which subsequently, will increase the power generation capability in the co-culture system.



Figure 6. The electrical output of co-culture MFCs with preliminary labor division


Description：

The results show that dividing material, energy and information flow into separated fermentation bacteria is much better than combining the two tasks into one kind of bacteria. We suppose that the production of riboflavins and lactate cannot be consistent in single strain because lactate is the primary metabolites and riboflavin is the secondary.

However, the electricity output cannot sustain for long and decrease promptly (as shown in figure 6). Based on the measurement, samples from anode reaches an excessively low pH (lower than 6.2) which might not meet the survival requirement of Shewanella.

To optimize the system, we try to control the rate of lactate production and adjust the proportion of fermentation bacteria.


Figure 7. The electrical output of co-culture MFCs with optimized labor division and bacteria proportion


The maximal potential increased up to 322 mV and could maintain for 30 hours in the optimized three-strain system. (consist of Shewanella and two kinds of E. coli) 



Figure 8. The electrical output of co-culture system with fermentation bacteria added 8 h later into the system.


To reduce the competition of E. coli and Shewanella for electrode, we add fermentation bacteria 8 h later after the well developed adhesion of Shewanella in electrode. However, the later inoculation disturbs the stability of co-culture system.



Figure 9. The electrical output of three MFC systems with glucose as carbon source in 80 h


The results show Shewanella+ΔplfB ldhE+Rf02S has gone through a preferable optimization in both medium component and bacteria proportion. It led to the potential of 350 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 electrical output of single strain Shewanella compared to Shewanella+ΔplfB ldhE+B. Subtilis co-culture system with glucose as carbon source.


To achieve a more complete labor division, we introduce the B. subtilis with a higher yield of riboflavin into our co-culture system (The strain is from Dr. Tao Chen’s lab). After the introduction of riboflavin high-yield B. Subtilis strain, the potential and time of duration in Shewanella+ΔplfB ldhE+B. Subtilis system can be raised into 511 mV and 80 h, respectively, resulting in a greater generation amount.



Figure 11. The electrical output of Shewanella+ΔplfB ldhE+B. Subtilis compared to Shewanella+ΔplfB ldhE+Rf02S with glucose as substrate


We can learn from the diagram, the potential produced by Shewanella+ΔplfB ldhE+B.Subtilis is greater than Shewanella+ΔplfB ldhE+Rf02S for 176 mV. We suppose there are two reasons for higher electrical output. Firstly, the engineered B. subtilis has the greatest capability to produce riboflavins. Secondly, the growing competition between B. Subtilis and E. coli is much gentler than that of different E. coli because of the relatively slow growth rate of B. Subtilis. It significantly improves the commensalism relations between electricigens and fermentation bacteria.



Figure 12. a(1) A series of voltage brought by different external resistance from Shewanella+ΔplfB ldhE+Rf02S MFC system (2)The polarization curve and power density of Shewanella+ΔplfB ldhE+Rf02S MFC system Besides, the highest power density can reach to 10 mW/m2. b (1)A series of voltage brought by different external resistance from Shewanella+ΔplfB ldhE+B. Subtilis (2)The polarization curve and power density of Shewanella+ΔplfB ldhE+B. Subtilis MFC system


The relations between current density and voltage is referred as polarization curve while the power curve can represent the relations between output power density and current density. We can learn from the fugure ***, the highest power density of (1) reaches 10 mW/m2 and (2) reaches 17 mW/m2.


Figure 14 15

It is obvious that the power output in three-strain MFC systems with a more complete labor division are far greater than single-strain and two-strian MFCs.


Figure 16

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


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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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