## 3 Results
### Optimization of Fermentative Conditions for Lycopene Production in _\(\Delta\)crtC Mutant_  
To optimize the fermentative conditions, the effects of different temperatures, pH values, shaker speeds, inoculum amount, liquid volumes, and selected carbon sources on lycopene accumulation in the _\(\Delta\)crtC_ knockout mutant strain were investigated in a series of shake flask experiments. The yeast extract medium supplemented with 400 mg/L proline was used as the fermentation medium. The results show that both culture temperature and medium pH significantly affected the growth of and lycopene accumulation in the mutant strain. Optimal growth and lycopene yield were observed at 25-30 \({}^{\circ}\)C and at a  
Figure 6: Comparison of the _crtC_ knockout mutant and wildtype strains. (**A**) PCR identification results. (**B**) Growth and lycopene accumulation of wt and \(\Delta\)_crtC_ strains in the presence or absence of amino acid. The data with different lower-case letters in the figures are statistically different in the content of lycopene (\(p<0.05\)).  
pH of 7.0-7.5. Beyond the optimal temperature or pH range, both biomass and lycopene accumulation decreased significantly (Figure 7A,B).  
Bacterial biomass, expressed as \(OD_{660}\) of the \(\Delta crtC\) mutant strain, was not significantly influenced by rotating speed, although the highest lycopene yield was observed at 160 rpm (Figure 7C). The growth curve of the \(\Delta crtC\) mutant strain showed a gradually increasing trend, while the lycopene yield remained almost unchanged (17.11 \(\pm\) 0.35 mg/L to 18.41 \(\pm\) 0.28 mg/L) as the liquid volume increased from 50 mL to 400 mL (Figure 7D). This suggests that a relatively lower oxygen supply is sufficient to sustain the growth of the \(\Delta crtC\) mutant strain. Thus, the fermentation cost may be significantly reduced as bacterial growth and lycopene accumulation can be accomplished at relatively higher loading volume and microaerobic conditions.  
Within the inoculum volume range of 1-20%, both growth and lycopene yield increased with the increases in the inoculum size. The optimal inoculum volumes for bacterial growth and lycopene yield in the \(\Delta crtC\) mutant strain are 10 and 5%, respectively. Raising the inoculum volume up to 20% will be restricted by higher cost of seed preparation, although this is beneficial for promoting microbial growth and lycopene accumulation in the mutant strain (Figure 7E). Therefore, the recommended inoculum size is 5%, which is conducive to balance the operational cost and lycopene yield.  
Supplementation of several other carbon sources did not impact the growth of the \(\Delta crtC\) mutant strain but exerted a significant effect on lycopene accumulation. Among these, glucose, pyruvate, lactic acid, and glycerol promoted lycopene accumulation in  
Figure 7: The impact of different fermentation conditions on \(OD_{660}\) and lycopene accumulation in the strain DT.1. (**A**) Temperature, (**B**) pH, (**C**) shaker speed, (**D**) liquid volume, (**E**) inoculum amount, and (**F**) carbon source. The data with different lower-case letters in the figures are statistically different in terms of the content of lycopene (\(p<0.05\)).  
the \(\Delta\)_crtC_ mutant strain, with lycopene yield increased by 12.21 \(\pm\) 0.26%, 6.43 \(\pm\) 0.21%, 7.62 \(\pm\) 0.14%, and 12.64 \(\pm\) 0.26%, respectively, as compared with the control. The only exception is acetic acid, the addition of which resulted in a decrease in lycopene yield by 7.04 \(\pm\) 0.25% (Figure 7F).