IR-ATR spectroscopy for in situ long-term monitoring of Lactobacillus parabuchneri biofilms

Microbial biofilms are a crucial problem in many areas including the food processing industry, biotechnology, water quality and medical scenarios. The complexity of biofilm formation and subsequent prevention strategies - requires a fundamental understanding of the involved molecular mechanisms and the possibility of long-term monitoring biofilm formation. Infrared attenuated total reflection (IR-ATR) spectroscopy is a versatile analytical technique for monitoring biofilm formation of bacteria isolates in situ, non-destructively, and close to real time as an innovative approach providing molecular insight into biofilm formation [1]. The utility of IR-ATR to investigate microorganism behavior within biofilms derives from the evanescent field penetrating few micrometers into the biofilm formed directly at the interface of a multi-reflection ATR waveguide and the sample. In the present study, isolates from food biogenic amine (BA)-producing bacteria, Lactobacillus parabuchneri DSM 5987 strains formed in cheese are analyzed for developing a deeper understanding on the formation of biofilms, which are significant contributors to the presence of histamine in dairy food products [2]. Infrared spectra were recorded using a custom flow-through ATR assembly for revealing the metabolism of microorganisms within such biofilms along with the effects of the substrate functionality and culture conditions on the extracellular biopolymeric matrices [3,4]. The appearance of key IR bands in the region of 1600-1200 cm^-1 indicates the production of lactic acid or lactate and the presence of amide groups, while most pronounced intensities in 1140-950 cm^-1 correspond to phospholipids, polysaccharides and nucleic acids. In this study, the spectral region between 1700 and 600 cm^-1 was determined to be the representative region for the identification of Lactobacillus parabuchneri biofilms enabling to study bioadhesion mechanisms and physico-chemical property changes during extended periods of biofilm growth. Real time monitoring has led to concrete steps for inhibition and disintegration via suitable antimicrobials by deposition on the IR inactive region of ATR waveguide. Multivariate data evaluation and classification strategies were applied to enable efficient multiparametric analysis for providing molecular information facilitating a better understanding of biofilm formation, maturation and changes in biofilm architecture via IR spectroscopic data. 
 
Keywords: IR-ATR spectroscopy, in situ monitoring, Lactobacillus parabuchneri, biofilm, ATR waveguide, flow-through ATR, lactic acid, multivariate data analysis. 

References: [1] Stenclova P, Freisinger S, et al. Appl. Spectro., 2019; Vol.73 (4) 424-432 [2] Yunda E, Quilès F, et al. Biofouling, 2019; Vol.35 (5) 494-507 [3] Diaz M, del Rio B, et al. Food Microbiol., 2016; Vol.7 (591) 85-91 [4] Lorite G, de Souza A, et al. Colloids Surfaces B. Biointerfaces, 2013; Vol. 102 519-525