The Establishment of
Based on what we have illustrated above, it is our priority to both improve the yields of cellulose generated from G.xy though exploring and optimizing the various condition of co-culture system during the fermentation process and make sure “engineered bacterial”——E.coli have grown stably and normally.
Since the reproduction speed of E.coli have been much faster than the speed of G.xy, it is significant to calculate the ratio of inoculating both bacterial. Yields of cellulose will be compromised if we inoculate a number of E.coli while little volume of E.coli for which will fail to manipulate the pH.Therefore, it is necessary for us to to explore the best ratio of incubating E.coli and G.xy.
During the process that we utilized culture medium for G.xy to fermented both E.coli and G.xy, the speed of forming the biofilm has been slowed down, therefore we went through the study of growth and metabolism of G.xy and noticed that the glycerol has played a crucial role in metabolic route of G.xy. According to the researches, adequate amount of glycerol is able to stimulate G.xy to secrete BC in the later phase. Based on what we have in mind, we decided to adjust the combination of carbon source of culture medium, which is turn the former pattern that glucose as the single carbon source to integrated carbon source pattern which use the glucose and glycerol to co-provide carbon source.
In addition, we as well as did the trails to measure the impact of yields of BC from other impact factor as ethanol and citric acid etc..(Glu/Gly = Glucose/Glycerin)
We designed the table below and followed the steps to process our experiments, expiring different ratio of incubation and carbon source. Moreover, we dried the BC from day 7 culture medium and measured both the weight and thickness to obtain relative data.
The comparison of weight and thickness of BC showed that on the condition of equivalent carbon source and inoculated ratio of G.xy and E.coli is 4/1, the yields of BC will at least advance 11.5% compared to other groups. At the same time, we as well as discovered that the capacity of G.xy secrete BC has been significantly improved with the ratio of glucose and glycerol is 3/2~4/1.
After setting up the inoculation level and the range of carbon ratio, we moved forward to optimize the carbon source through RSM. During this process, we collected a series of data which based on the yield as main target and the pH residue as co-factor, the data is present below:
The finally optimize formula which has already reached the ideal ultimate pH and visibly improved the yields of BC is :
formula Glu(g) Gly(ml) 50%EtOH Predicting Final pH Target 15.11 9.53 0.05 4.36114
this optimized formula has similar carbon ratio to what we have measured in the culture medium of previous experiments, therefore we ultimately set up the carbon ratio as glucose/glycerol=3/2 of our co-culture medium.
There is what we have gathered after utilized 3/2 medium in the co-culture system based on the former experiments:
Compared the pH residue of single G.xy culture and co-culture during the fermentation, obviously approximately 15h after inoculation, pH of co-culture started to significantly drop while the yields of BC from co-culture clearly outnumber the yields of single G.xy culture.
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