Combined action of larrea divaricata extract and monensin on lactic acid bacteria contaminating bioethanol fermentations

Abstract

Tucumán is the largest producer of bioethanol in the country. The production is carried out through the fermentation of sugar-rich substrates, such as molasses and sugarcane juice, using Saccharomyces cerevisiae. The fermented liquid is then distilled and dehydrated to obtain anhydrous ethanol. Lactic acid bacteria (LAB) are contaminants in this process. They reduce ethanol production by competing for available carbon and producing lactic acid, which inhibits yeast growth. The presence of LAB is controlled by antibiotics such as monensin (MO). However, antibiotics are expensive and have a negative environmental impact. This could be minimized by using extracts from readily available plant sources, such as Larrea divaricata. In this study, we investigated the combined action of the ethanolic extract of L. divaricata (EE-LaDi) and MO against four LAB species isolated from fermentation tanks. Microdilution assays were performed using MRS medium. 96-well plates were loaded with serial dilutions of EE-LaDi and MO using a checkerboard design, followed by the addition of bacterial inoculum. The bacterial density in each well was 5 x 105 CFU/ml. The plates were incubated at 30°C for 12 hours, after which the optical density at 630 nm was measured using a microplate reader. The fractional inhibitory concentration index (FICI) was calculated from the minimum inhibitory concentration values obtained for EE-LaDi and MO tested separately, as well as in combination. EE-LaDi demonstrated an additive effect in combination with MO against all evaluated LAB, with FICI values ranging from 0.54 to 1.03. Our results suggest that it is possible to develop a prototype that minimizes the use of MO, based on EE-LaDi/MO mixtures, for control of LAB contamination in the bioethanol production