# Isoprenoids Production from Lipid-Extracted Microalgal Biomass Residues Using Engineered E. coli
###### Abstract  
Microalgae are recognized as a third generation feedstock for biofuel production due to their rapid growth rates and lignin-free characteristics. In this study, a lipid extracted microalgal biomass residues was used as the raw material to produce isoprene, \(\alpha\)-pinene and \(\beta\)-pinene with an engineered _E. coli_ strain. We adopted an optimal sulfuric acid hydrolysis method (1:7 ratio of solid to acid solution, 32% (_w/e_) concentration of sulfuric acid solution at 90 \({}^{\circ}\)C for 90 min) to efficiently convert holocellulose into glucose efficiently (6.37 g/L). Futhermore, we explored a novel detoxification strategy (phosphoric acid/calcium hydroxide) to remove inhibitors and notably acetic acid, furfural and 5-hydroxymethylfurfurfurfural (5-FHMF) were reduced by 5.32%, different number given later 99.19% and 98.22%, respectively. Finally, the fermentation concentrations of isoprene (223.23 mg/L), \(\alpha\)-pinene (382.21 mg/L) and \(\beta\)-pinene (17.4 mg/L) were achieved using the detoxified hydrolysate as the carbon source, equivalent to approximately 86.02%, 90.16% and 88.32% of those produced by the engineered _E. coli_ strain fermented on pure glucose, respectively.  
lipid extracted microalgae; isoprenoids; detoxification +
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compete with food supplies. These second generation materials are not applied to commercially due to their low yield and high cost resulting from the necessary hydrolysis process [6; 7]. Therefore, a third generation feedstock is required to both satisfy the demand for biofuel production on a large scale and maintain ecological balance at the same time. Currently, some researchers have proposed algae as an ideal alternative for biofuel production due to its rapid growth, a unicellular or simple multicellular structure, lignin-free properties and ready availability on Earth [8].  
Hence, in this paper, we utilized the lipid extracted microalgal biomass residues (LMBRs) as the feedstock to biosynthesize isoprene and its derivatives (\(\alpha\)-pinene and \(\beta\)-pinene) using the engineered _E. coli_. This began with the conversion of LMBRs into fermentable sugar, and then the microbial fermentation was performed to generate bio-based isoprenoids. However, as it is rather time- and money-consuming, the LMBRs were hydrolyzed with sulfuric acid rather than enzymatic hydrolysis. Finally, 6.37 g/L of glucose was achieved based on the following hydrolysis conditions: ratio of solid to acid (1:7), acid concentration (32%), hydrolysis temperature (90 \({}^{\circ}\)C), and hydrolysis time (90 min). Since fermentation inhibitors (weak acid, furfural, 5-HMF) were formed during the acid hydrolysis process [6], we adopted five different detoxification strategies with recombination to reduce the inhibition consequence on the fermentation [6; 7; 8; 9; 10]. Among five methods, the phosphoric acid/calcium hydroxide detoxification combination, the best detoxification approach, was applied to remove acetic acid, furfural and 5-HMF by about 55.32%, 99.19% and 98.22% respectively. Finally, 223.23 mg/L of isoprene, 382.21 mg/L of \(\alpha\)-pinene and 17.4 mg/L of \(\beta\)-pinene were obtained from the engineered _E. coli_ strain fermented on the detoxified hydrolysate of LMBRs, accounting for about 86.02%, 90.16% and 88.32%, respectively, of the isoprene, \(\alpha\)-pinene and \(\beta\)-pinene production by the _E. coli_ strain using pure glucose, respectively.