Performance and microbial analysis during long-term anaerobic digestion of olive mill wastewater in a packed-bed biofilm reactor Ghizlane Enaime 1, ∗ , Edith Netmann 2 , Stephan Berzio 2 , Abdelaziz Baçaoui 1 , Abdelrani Yaacoubi 1 , Marc Wichern 2 , Tito Gehring 2 , Manfred Lübken 2 1 Laboratory of Applied Chemistry, Unity of Methodology and Environment, Faculty of Sciences Semlalia, Cadi Ayyad University, B.P 2390 Marrakech, Morocco, 2 Institute of Urban Water Management and Environmental Engineering, Ruhr-Universität Bochum, Universitätsstraße 150, 44780 Bochum, Germany, *Corresponding author: E-mail address: ghizenaime@gmail.com (G. Enaime) Abstract BACKGROUND: The treatment of olive mill wastewater (OMWW) in packed bed biofilm reactor (PBBR) and the identification of different microorganisms involved in the digestion process is an attractive field for research. RESULTS: A PBBR treating OMWW under different organic loading rates (OLRs) (0.94- 9.36 gCOD/(L.d)), showed high stability during an extensive time (723 days) without any sign of acidification. High volumetric biogas and CH 4 productions, 4.1 and 2.4 L N /L reactor /d, respectively were registered at the highest OLR. Meanwhile, high COD removal (62.5-79.7%) was obtained throughout the experiment. Phenolic compounds removal was much higher during the first steps of the experiment (75.9-84.2%), and decreased as the OLR increased to about 38%. High homogeneity of biofilm thickness and Bacteria and EPS distribution were demonstrated by confocal laser scanning microscopy. Results of real-time quantitative PCR and 16S rRNA amplicon analyses showed that Bacteria, mainly consisted of Firmicutes, Proteobacteria, Bacteroidetes and Chloroflexi phylum, was about eight times more abundant than Archaea in the reactor liquid phase, while Bacteria to Archaea ratio was almost 1 in the biofilm. This points to a major degradation of organic substances in the liquid, while methanogenesis occurred mainly within the biofilm. In the liquid, acetoclastic methanogens were more abundant than hydrogenotrophic ones, while both groups showed likely very similar contributions in the biofilm. CONCLUSION: Promising results have been presented throughout this work providing information about the long-term operation of PBBR-GAC treating OMWW and analyzing, in an efficient manner, the structure and the composition of the microbial community involved in OMWW digestion. This article is protected by copyright. All rights reserved. Accepted Article This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jctb.6275