Enhanced Sensitivity of PCR-based Detection of a Bacterial Contaminant in Ethanol Fermentation using a Functionalized Magnetic Nanoparticle
Lactic acid bacteria (LAB) are the primary contaminants in ethanol fermentation, which can lead to reduced fermentation efficiencies. This work aimed to develop a faster and more sensitive protocol for detecting LAB, using Limosilactobacillus fermentum as a model organism, using a functionalized magnetic nanoparticle (MNP-F1) in tandem with Polymerase Chain Reaction (PCR). Firstly, the percent cell capture efficiency (%CCE) by various concentrations of MNP-F1 of L. fermentum FM7 (108 CFU/mL), grown in MRS broth, was evaluated. Then, Transmission Electron Microscopy (TEM) was used to visualize the capture. Finally, thermal cycling of the MNP-F1 detection protocol was used to detect L. fermentum FM7 in MRS broth and when spiked in fermenting molasses without enrichment. The %CCE obtained was 99.70 ± 0.3 using 0.0125 mg/ml of MNP-F1. TEM validated the MNP-F1-L. fermentum FM7 conjugation. Successful detection of L. fermentum in MRS broth and fermenting molasses matrices with Saccharomyces cerevisiae BIOTECH 2030 was confirmed through PCR. Interestingly, detection was successful even at a cell concentration of 10 CFU/mL using the lowest MNP-F1 concentration of 0.0125 mg/mL. MNP-F1 facilitated the simple and rapid preparation of genomic DNA templates from captured cells, without the need for a lengthy enrichment step. The results demonstrated the applicability of MNP-F1 in tandem with PCR detection, although concentrations much lower were not further tested. This study showed the potential use of biocompatible functionalized MNPs or MNP-F1 to capture and extract bacterial contaminants in the ethanol fermentation process and rapidly detect using PCR with high sensitivity.