Functionalised ceramic spawning tiles with probiotic Pseudoalteromonas biofilms designed for clownfish aquaculture Wiebke Wesseling a, ⁎, Sabine Wittka a , Stephen Kroll b , Christian Soltmann c , Pia Kegler d , Andreas Kunzmann d , Hans Wolfgang Riss e , Michael Lohmeyer a a Mikrobiologisches Labor Dr. Michael Lohmeyer GmbH, Mendelstraße 11, Muenster 48149, Germany b Advanced Ceramics, University of Bremen, Am Biologischen Garten 2, Bremen 28359, Germany c Novelpor UG, Huchtinger Heerstraße 47, Bremen 28259, Germany d Leibniz Center for Tropical Marine Ecology GmbH, Fahrenheitstraße 6, Bremen 28359, Germany e Department of Limnology, Institute for Evolution and Biodiversity, University of Muenster, Huefferstraße 1, Muenster 48149, Germany abstract article info Article history: Received 27 May 2014 Received in revised form 31 March 2015 Accepted 7 April 2015 Available online 18 April 2015 Keywords: Open porous ceramics Bacterial spawn infestation bioactive compound Anti-fungal Anti-bacterial To prevent marine fish egg clutches of substrate spawners from bacterial and fungal infestation, functionalised ceramic spawning tiles with probiotic bacterial biofilms were designed for clownfish aquaculture in this study. Therefore, mechanically stable ceramic spawning tiles made of alumina were fabricated with convenient sub- strate properties for Pseudoalteromonas immobilisation, i.e., hydrophobic matrix and hydrophilic surface proper- ties. An effective biofilm formation was achieved by equilibration of these ceramic tiles in marine liquid medium containing 1 wt% starch and subsequent inoculation with two different Pseudoalteromonas strains. Biofilm forma- tion was confirmed qualitatively by scanning electron microscopy (SEM) and quantitatively by photometrical measurements according to Safranin O staining. Furthermore, antagonistic effects originating from Pseudoalteromonas biofilms against Paecilomyces lilacinus were detected by SEM, which are possibly due to bio- active molecules. Consequently, these innovative microbiologically conditioned ceramic spawning tiles are promising candidates to prevent fish egg clutches from pathogenic infestation, which leads to an improved aqua- culture of substrate spawners such as clownfishes. © 2015 Elsevier B.V. All rights reserved. 1. Introduction Marine ornamental fishes gain more and more popularity in aquaristic fish keeping due to their variability in intensive colours and morphologies. However, breeding of these fishes is challenging owing to the fact that a considerable part of them is captured in the wild (Wabnitz et al., 2003; Moorhead and Zeng, 2010). Therefore, a sustain- able aquaculture of marine ornamental fish is required. Fish diseases or bacterial spawn infestation, decimating the yields of aquacultures, are mostly treated with antibiotics (Grave et al., 1999; Le and Munekage, 2004; Le et al., 2005). As it is known from other fields (e.g., human dis- eases), the non-specific targeting of antibiotics can create antibiotic- resistant germs, a critical issue in human and animal medicine (reviewed by Cabello, 2006). Therefore, the control of bacterial infec- tions with probiotics (bacteria with “antagonism toward pathogens” Kim, 2013) is a new approach tested for aquaculture (reviewed by Cruz et al., 2012). To protect the egg clutches of substrate spawners like clownfish from pathogenic infestation and thus increase yield in aquacultures, it is suggested that a ceramic substrate coated with a pro- biotic biofilm could serve as a remedy. Biofilms are cohabitations of multicellular complexes surrounded by an extracellular matrix sticking cells together and are formed in aque- ous systems as a boundary layer to a solid phase. Bacteria immobilised in a biofilm are in the stationary phase of growth and thus are assumed to produce secondary metabolites, also known as bioactive compounds (Schink, 2006). By these bioactive compounds, bacteria protect them- selves from hostile attacks, e.g., from outgrowth by other (pathogenic) bacteria. As biofilms represent a dynamic structure, motile cells are able to disperse from and coalesce to the biofilm (Stoodley et al., 2002). The colonisation of a material surface by bacteria depends on several parameters and may vary between different bacteria species (Dunne, 2002). Important material properties that have an impact on bacterial adhesion are related to porosity (open vs. closed porosity) as well as pore size, specific surface area, wetting characteristic (hydrophobic/hy- drophilic) and surface topography of the material's substrate. Aquaculture 446 (2015) 57–66 Abbreviations: BFM, biofilm medium with seawater; CV, crystal violet; DMSO, dimethyl sulfoxide; DSMZ, German Collection of Microorganisms and Cell Cultures; EDX, energy- dispersive X-ray spectroscopy; HAPES, high alkane phase emulsified suspensions; HDTMS, hexadecyltrimethoxysilane; MA, marine agar; MLM, marine liquid media; MLMS, marine liquid media containing 1 wt% starch; OD, optical density; PSU, practical salinity unit; rel. AU, relative arbitrary units; SEM, scanning electron microscopy; ZMT, Leibniz Center for Tropical Marine Ecology GmbH, Bremen, Germany. ⁎ Corresponding author. Tel.: +49 251 143765; fax: +49 251 143766. E-mail address: w.wesseling@mikrobiologisches-labor.de (W. Wesseling). http://dx.doi.org/10.1016/j.aquaculture.2015.04.017 0044-8486/© 2015 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aqua-online