Quantification and characterization of microbial biofilm community attached on the surface of fermentation vessels used in green table olive processing Athena Grounta, Agapi I. Doulgeraki, Efstathios Z. Panagou ⁎ Agricultural University of Athens, Department of Food Science and Human Nutrition, Laboratory of Microbiology and Biotechnology of Foods, 75 Iera Odos str., Athens GR-11855, Greece abstract article info Article history: Received 8 October 2014 Received in revised form 10 February 2015 Accepted 1 March 2015 Available online 5 March 2015 Keywords: Table olives Microbial biofilm Fermentation vessels Lactic acid bacteria Yeasts The aim of the present study was the quantification of biofilm formed on the surface of plastic vessels used in Spanish-style green olive fermentation and the characterization of the biofilm community by means of molecular fingerprinting. Fermentation vessels previously used in green olive processing were subjected to sampling at three different locations, two on the side and one on the bottom of the vessel. Prior to sampling, two cleaning treatments were applied to the containers, including (a) washing with hot tap water (60 °C) and household detergent (treatment A) and (b) washing with hot tap water, household detergent and bleach (treatment B). Population (expressed as log CFU/cm 2 ) of total viable counts (TVC), lactic acid bacteria (LAB) and yeasts were enumerated by standard plating. Bulk cells (whole colonies) from agar plates were isolated for further character- ization by PCR-DGGE. Results showed that regardless of the cleaning treatment no significant differences were observed between the different sampling locations in the vessel. The initial microbial population before cleaning ranged between 3.0–4.5 log CFU/cm 2 for LAB and 4.0–4.6 log CFU/cm 2 for yeasts. Cleaning treatments exhibited the highest effect on LAB that were recovered at 1.5 log CFU/cm 2 after treatment A and 0.2 log CFU/cm 2 after treatment B, whereas yeasts were recovered at approximately 1.9 log CFU/cm 2 even after treatment B. High diversity of yeasts was observed between the different treatments and sampling spots. The most abundant species recovered belonged to Candida genus, while Wickerhamomyces anomalus, Debaryomyces hansenii and Pichia guilliermondii were frequently detected. Among LAB, Lactobacillus pentosus was the most abundant species present on the abiotic surface of the vessels. © 2015 Elsevier B.V. All rights reserved. 1. Introduction Table olives are one of the most important groups of vegetables marketed and consumed as fermented. A proper fermentation proce- dure results in a microbiologically safe final product with enhanced sensory attributes. This is achieved primarily by the growth of LAB that metabolize fermentable sugars diffused from the olive mesocarp into the brine and produce lactic acid which decreases the pH in the brine, resulting thus in the inhibition of undesirable microorganisms and the extended preservation of the end product even at ambient temperature (Corsetti et al., 2012; Heperkan, 2013). Development of some yeast species is also desirable as they contribute in the production of aroma compounds and maintenance or even stimulation of LAB populations (Arroyo López et al., 2008, 2012a; Viljoen, 2006). The pop- ulation dynamics of these diverse microbial groups throughout the fermentation process were, until recently, monitored in the cover brines. However, it has been shown that LAB and yeast communities colonize the surface of the naturally black and Spanish style table olives drupe forming mixed species aggregates characterized as biofilms (Domínguez Manzano et al., 2012; Grounta and Panagou, 2014; Nychas et al., 2002). Biofilm formation in food processing environments has been the focus of extensive scientific research, especially in the context of food hygiene, as many outbreaks have been associated with the presence of biofilms in food industries (Srey et al., 2013). Biofilms are defined as functional consortia of microorganisms attached to a surface which are embedded in the extracellular polymeric substances (EPS) produced by the microorganisms (Monds and O'Toole, 2009). It has been realized that biofilm formation is a natural phenomenon which occurs whenever there are microorganisms and surfaces, either biotic or abiotic, surrounded by a high or a low level of nutrients (Elhariry, 2011; Giaouris et al., 2014). In these environments, the accumulation of food nutrients at the solid/liquid interface on food surfaces leads to a higher concentration of nutrients compared to the fluid phase which as a process is known as “conditioning film” (Donlan, 2002). After film conditioning, microorganisms may adhere on the food contact surface, gradually form microcolonies and finally assemble themselves in biofilms exhibiting high microbial diversity in terms of genera, species and strain levels (Borucki et al., 2003; Burmølle et al., 2006). A similar International Journal of Food Microbiology 203 (2015) 41–48 ⁎ Corresponding author. Tel./fax: +30 210 5294693. E-mail address: stathispanagou@aua.gr (E.Z. Panagou). http://dx.doi.org/10.1016/j.ijfoodmicro.2015.03.001 0168-1605/© 2015 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro