Quantitative evaluation of biofilm composition using real-time PCR

Technical Session 18: Microbiology II Session
Robert Riedl, Research Center Weihenstephan for Brewing- and Food-Quality,TU Muenchen, Freising, Germany
Co-author(s): Jennifer Koob, Mathias Hutzler, and Fritz Jacob, Research Center Weihenstephan for Brewing and Food Quality, TU Muenchen, Freising, Germany

ABSTRACT: Biofilms are a serious problem in breweries and beverage bottling plants. Biofilms are associations of various species of bacteria, yeasts, and molds. In contrast to planktonic microorganisms, a layer of extracellular substances protects the cells in biofilms, which makes them much more resistant against cleaning and disinfection solutions. Most biofilm starter organisms, such as acetic acid bacteria (AAB) or Enterobacteriaceae, are considered to not be product spoiling. For this reason, most breweries do not use cultivation media that are designed to detect them. Therefore a biofilm will not be detected until product spoiling organisms colonize it. Additionally, established cultivation media methods such as the NBB-B-AM swab test, according to Prof. Back (1994), do not specify the associated organisms. The composition of the associated organisms is very important for evaluation of the level of maturity and potential product spoiling risk of biofilms in breweries or beverage plants. The rather long incubation time of 5–14 days for nutrition media tests is another disadvantage. With molecular biological screenings, the cultivation time can be reduced to 3 days using real time-PCR systems to detect different target fractions of microorganisms. Most commercial real time-PCR kits, established in brewing microbiology, focus on the detection of beer spoiling bacteria. In this study a modular PCR-screening assay was designed and evaluated to detect a wide spectrum of bacteria and yeasts involved in the growth of biofilms. The first screening step detects product specific, defined groups of organisms that can be used as indicator organism groups for the state of maturity in the biofilm development. The second step identifies the organisms within the groups. The identified organisms were linked with data about the organisms, containing metabolic products, product risk, and typical locations. Maturity, as well as the potential for product spoiling of the biofilm, can be measured by typical indicator organisms detected using the real time-PCR screening system.

Robert Riedl was born in 1983 in Munich. He studied brewing and beverage technology at the Technische Universität München and graduated with a Dipl.-Ing. degree in 2011. Since July 2011 he has been a scientific assistant at the Research Center Weihenstephan for Brewing and Food Technology and is working on biofilm development in beverage plants.