Difference between revisions of "Team:TJU/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 MG1655ΔpflB, but MG1655ΔpflB + 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, MG1655ΔpflB and MG1655ΔpflB + ldhE.

Figure 2. a: The lactate production curve in anaerobic condition (M9 medium). b: The lactate production in anaerobic condition (M9 medium, 25h after inoculation)

In anaerobic environment, lactate production of MG1655, MG1655ΔpflB and MG1655ΔpflB+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 MG1655ΔpflB is smaller than MG1655ΔpflB+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 (MG1655ΔpflB+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 flask cultivation. E. coli BL21Δpgi + EC10 reaches 33 mg/L. E. coli BL21 + EC10* reaches 90 mg/L.

Figure 4. The yield of riboflavin in different strains :EC10, Rf02S (Δpgi+EC10), EC10*

3 Co-culture MFC -- Labor Division

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 150mV 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 5).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 8h 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 + ΔpflB 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 + ΔpflB 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 + ΔpflB 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 + ΔpflB ldhE + B. subtilis compared to Shewanella + ΔpflB ldhE + Rf02S with glucose as substrate.

We can learn from the diagram, the potential produced by Shewanella + ΔpflB ldhE + B. subtilis is greater than Shewanella + ΔpflB 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 + ΔpflB ldhE + Rf02S MFC system (2)The polarization curve and power density of Shewanella + ΔpflB ldhE + Rf02S MFC system. Besides, the highest power density can reach to 10mW/m². b (1)A series of voltage brought by different external resistance from Shewanella + ΔpflB ldhE + B. subtilis (2)The polarization curve and power density of Shewanella + ΔpflB 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 figure 13, the highest power density of (1) reaches 10 mW/m² and (2) reaches 17 mW/m².

Figure 13. (a) The comparison of polarization curve and power curve among Shewanella, Shewanella + MG1655, Shewanella + ΔpflB ldhE + Rf02S. (b)The comparison of polarization curve and power curve among Shewanella, Shewanella + MG1655 and Shewanella + ΔpflB ldhE + B. Subtilis.

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 14. The comparison of polarization curve and power curve among Shewanella + ΔpflB ldhE + B. Subtilis and Shewanella + ΔpflB ldhE + Rf02S.

It has been shown in power curve that Shewanella + ΔpflB ldhE + B. Subtilis has a significantly higher output than Shewanella + ΔpflB 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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