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

BACKGROUND: The use of alfalfa is a promising response to the increasing demand for squalene. Ensiling could enhance the squalene content of fresh alfalfa and silage. 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. RESULTS: Naturally ensiled alfalfa was of poor quality but had up to 1.93 times higher squalene content (P < 0.001) than fresh alfalfa. The squalene-producing bacteria were found to be 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. Multiple linear regression models and correlation analysis indicated that squalene synthase was the key enzyme for squalene synthesis. The addition of ML and SSL altered the structure of LAB communities, mainly decreasing the relative abundance of cocci LAB, which was responsible for squalene synthesis, and changing the fermentation products (lactic acid, propionic acid, and ammonia-N) influencing the squalene-related enzymes, thereby decreasing 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. CONCLUSION: Natural ensiled alfalfa was a promising source for squalene, and ensiling was a potential pathway to obtain novel high-yield squalene bacteria. © 2022 Society of Chemical Industry.