# Isoprenoids Production from Lipid-Extracted Microalgal Biomass Residues Using Engineered E. coli
## 2 Results and Discussion
### Effect of Detoxification on Isoprenoids Production  
Figure 4 has presents the isoprene produced by the engineeried _E. coli_ YJM25 with seven different carbon sources, including pure glucose, raw hydrolysate, and hydrolysates detoxified by methods A-E. As can be seen, compared with the raw hydrolysate fermentation (160.26 mg/L), isoprene yield produced by using group E hydrolysate (223.23 mg/L) was increased by about 40% after the E detoxification. The concentration of isoprene produced by group E hydrolysate represented 86.02% of that produced using pure glucose (259.52 mg/L). This percentage is much higher than those obtained using the other five types of hydrolysate (treatments A-D and raw). Therefore, the calcium hydroxide/phosphoric acid detoxification method was proved to be better than the other four kinds of detoxification methods for isoprene production with _E. coli_. As is expected, Figures 5 and 6 have also demonstrated that, in comparision to raw hydrolysate fermentation, the yield of \(\alpha\)-pinene and \(\beta\)-pinene were increased about 35% and 52%, respectively, when using hydrolysate detoxified by method E and \(\alpha\)-pinene and \(\beta\)-pinene production accounted for about 90.16% and 88.32% of the yield produced on pure glucose. These results revealed that the _E. coli_ fermentability was improved greatly after using the calcium hydroxide/phosphoric acid detoxification strategy. Figure 3: Inhibitors concentration of raw and detoxification hydrolysates with A–E five different detoxification strategies. These five detoxification methods are corresponding to the five methods A–E described in the Materials and Methods section, respectively. The experiments were performed in triplicate. Figure 4: Isoprene production by the YJM25 strain using different carbon sources containing pure glucose, raw hydrolysate and five different kinds of hydrolysates detoxified by methods A–E. When OD\({}_{600}\) reached -0.6, cultures was induced at 30 \({}^{\circ}\)C for 24 h using 0.5 mM IPTG. The experiments were performed in triplicate. Figure 5: \(\alpha\)-Pinene production by the YJM29 strain using different carbon sources containing pure glucose, raw hydrolysate and hydrolysate detoxified by method E. When OD\({}_{600}\) reaches \(\sim\)0.6, cultures were induced at 30 \({}^{\circ}\)C for 24 h using 0.5 mM IPTG. The experiments were performed in triplicate. Figure 6: \(\beta\)-Pinene production by strain FHR-2 using different carbon sources containing pure glucose, raw and hydrolysate detoxified by method E. When OD\({}_{600}\) reaches \(\sim\)0.6, cultures were induced at 30 \({}^{\circ}\)C for 24 h using 0.5 mM IPTG. The experiments were performed in triplicate. Microorganism growth can be restricted by toxic inhibitive compounds such as furfurals, 5-HMF and organic acids [18]. Among those inhibitors, furfural was found to be able to inactivate the cell replication by breaking down the single-strand DNA [19; 20; 21; 22]. Organic acids (acetic acid) derived from hemicellulose could cross the cell membrane, which resulted in a lower cell pH than normal and consequently inhibited cell activity [21; 23]. Ultimately, the cell activity of _E. coli_ was inhibited and this directly reduced its fermentation ability. As shown in Figures 3 and 4, compared to other different carbon sources, the hydrolysate subjected to detoxification method E had the lowest concentration of inhibitors and consequently achieved the highest isoprene yield. [PERSON] et al. have reported that although the toxicity of acetic acid on microorganisms is lower thanthat of furans (furfural and 5-HMF), the synergistic toxicity is possibly more severe when furans are present in conjunction with acetic acid [18].