Exploiting the anaerobic fermentation of alfalfa as a renewable source of squalene

In response to the increasing demand for squalene and environmental challenges, exploiting and boosting the grass as a novel squalene source is promising. By comparing the squalene content in fresh alfalfa and silage, ensiling could enhance the squalene content. To investigate and exploit the anaerobic fermentation of forage as a new squalene source, alfalfa was ensiled without (CON) or with molasses (ML), and sunflower seed oil (SSL) for 10, 40, and 70 days.Natural ensiled alfalfa had poor quality but had higher squalene content (P < 0.001) than fresh alfalfa, up to 1.93 times. The squalene-producing bacteria was traced as cocci lactic acid bacteria (LAB). Adding ML and SSL decreased squalene content (P = 0.002 and P < 0.001) by 6.89% and 11.6%, respectively. According to multiple linear regression models and correlation analysis, the reasons were that squalene synthase was the key enzyme for squalene synthesis, and adding ML and SSL altered the structure of LAB communities, mainly decreasing the relative abundance of cocci LAB responsible for squalene synthesis, and changed fermentation products (lactic acid, propionic acid, and ammonia-N) influencing the squalene-related enzymes, thereby decreasing the squalene production. Compared with squalene production from the reference bacteria (Pediococcus acidilactici Ch-2, Rhodopseudomonas palustris, Bacillus subtilis, engineered Escherichia coli), alfalfa silage had the potential to be a new squalene source to explore and engineer novel high-yield squalene bacteria.Natural ensiled alfalfa was a promising source for squalene, and ensiling was a potential pathway to dig novel high-yield squalene bacteria. This article is protected by copyright. All rights reserved.