**1. Introduction**

The first attempts to farm sponges date back to the 19th century, presumably as a consequence of periodical depletion of "bath-sponge" stocks [1,2], or—in more recent times—in pursuit of a safer and economically more attractive alternative to wild collection [3,4]. Overfishing and repeated outbreaks of mass mortality events halted the ancient tradition of Mediterranean fishing of commercially important "bath sponge" species, such as *Spongia officinalis* (Linnaeus) and *Hippospongia communis* (Lamarck) [3–6]. Sponge mariculture has received increased attention over the last two decades (e.g., [7–9]; see also reviews or comparative studies by [10–12]), particularly driven by the discovery of biologically active metabolites in many sponges (e.g., [13,14]). Sponge mariculture could potentially provide for a sustainable supply of sponge-derived bioactive compounds and biomaterials.

Sponges can be co-cultured with other organisms in so-called integrated mariculture systems, in which sponges take up metabolic wastes from other system components, including bacterioplankton growing on these metabolic wastes [15–19]. This way, sponges can effectively reduce waste streams from fish farms [2,5,20], since they have been shown to exhibit retention efficiencies of up to

99% for nano- and picoplankton (e.g., [21–23]), while processing large volumes of water, up to 0.6 cm<sup>3</sup> cm−<sup>3</sup> sponge s−<sup>1</sup> (e.g., [24–26]). Hence, a large-scale sponge culture facility that is constructed near a fish farm may positively affect the quality of the surrounding water. Conversely, the additional nutrition originating from the farmed fish may enhance the growth of the sponges in culture, thus providing a more efficient and profitable business.

In 2006–2007, an integrated mariculture approach using sponges was tested in the coastal waters around the Bodrum Peninsula, Turkey [27]. Two Mediterranean demosponge species with possible commercial interest, *Dysidea avara* (Schmidt, 1862) and *Chondrosia reniformis* Nardo, 1847 (Demospongiae, Chondrosiida, Chondrosiidae), were cultured at a pristine site (i.e., no fish farms within the nearest 30 km) and an organically polluted fish farm site, the latter sponges being directly cultured underneath an open cage fish farm. *D. avara* was chosen since it produces the bioactive compound avarol, a potential anti-psoriasis agent [14,28]. *Chondrosia reniformis* synthesizes large amounts of collagen, which is suitable for cosmetic and medical applications [29–31]. Type I & IV mammalian-like collagens can be effectively extracted from *C. reniformis* [32,33] and they can be used to promote the regeneration of human tissue and bone tissue engineering scaffolds [31]. *Chondrosia reniformis* showed better growth and survival rates at the pristine site, whereas *D. avara* grew and survived better at the polluted site [27]. The low survival rates of *C. reniformis* at the polluted site were largely due to the farming protocol used. *Chondrosia reniformis* is a highly plastic sponge, being able to de-attach and move around [34], a phenomenon that was frequently encountered using common culturing structures, such as pins, lines, plaques or metal/net grids [5,8,35,36]. To avoid displacement, explants of *C. reniformis* were put inside cages on the seafloor [27]. However, due to the high particle load in the water around the fish farm, the explants in these cages were suffocated by sediment.

This study describes progress towards the development of a raw collagen production pipeline using the sponge *C. reniformis* in an integrated multi-trophic aquaculture approach, i.e., by culturing the sponges in the vicinity of offshore floating fish cages. Using thin plastic plates as substratum, a series of consecutive trials were executed, aimed at developing an optimal, species-specific culture method. We monitored survival and growth rates of cultured explants of *C. reniformis,* thereby comparing a polluted fish farm site to a pristine site. Variables studied included methods for attaching explants to plates, plate materials and plate orientation. The culture methods (glue, cable-ties on plaques, net/mesh cover) were applied previously on other sponge species by several authors; for detailed information, see review by Duckworth et al. [10].
