Vol. 11: 375–391, 2019 AQUACULTURE ENVIRONMENT INTERACTIONS Published August 8 https://doi.org/10.3354/aei00319 Aquacult Environ Interact OPENPEN ACCESSCCESS Analysis of algal growth- and morphogenesis- promoting factors in an integrated multi-trophic aquaculture system for farming Ulva spp. Fatemeh Ghaderiardakani1, Gianmaria Califano2, Jan Frieder Mohr2, Maria Helena Abreu3, Juliet C. Coates1, Thomas Wichard2,* 1School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK 2Institute for Inorganic and Analytical Chemistry, Jena School for Microbial Communication, Friedrich Schiller University Jena, 07743 Jena, Germany 3Travessa Alexandre da Conceiçao, ALGAplus Lda, Ílhavo 3830-196, Portugal ABSTRACT: The marine green macroalgal genus Ulva (Chlorophyta) requires the presence of a combination of regulatory morphogenetic compounds released by their associated epiphytic bac- teria in addition to nutritional parameters. The activity of algal growth- and morphogenesis- promoting factors (AGMPFs) derived from bacteria was determined in a land-based integrated multitrophic aquaculture (IMTA) system of fish and macroalgae (located at the coastal lagoon Ria de Aveiro, Portugal) using a standardised bioassay with axenic cultures of Ulva mutabilis during a snapshot study. The study thus informs aspects of various potential aquaculture− environment interactions. It was observed that both the water from the lagoon (external to the farm system) and the water from the fish pond (input for algae cultures) could completely restore the normal growth and morphology of the macroalga under axenic conditions. The results high- light the presence of a sufficient chemical cocktail of AGMPFs in this IMTA system required for growth and morphogenesis of the sea lettuce Ulva spp. In addition, the water from fish farming increased the nutrient availability (nitrate and ammonium) needed for macroalgae production at low concentrations of metal contaminants. Interestingly, the abundances of known morphogene- sis-inducing bacteria (e.g. Maribacter mutabilis, Sulfitobacter spp.) were enriched in the Ulva aquaculture water compared to the water from the lagoon. We thus conclude that sustainable growth and development of Ulva spp. can benefit from multitrophic aquaculture systems and shallow-water systems, due to the naturally enriched AGMPFs and their in situ production by bacteria in intensive algal aquacultures. KEY WORDS: Algal growth- and morphogenesis-promoting factors · Aquaculture−environment interaction · Morphogens · Multitrophic aquaculture system · Seaweed · Waterborne compounds 1. INTRODUCTION waters. Therefore, the genus Ulva is regarded as a reliable bio-indicator that responds to eutrophic wa- Marine species of the green macroalgal genus ter pollution (Kozhenkova et al. 2006). On the other Ulva, belonging to the Ulvacean family, are common hand, Ulva spp. cultures can also be utilised for bio - throughout intertidal and subtidal habitats worldwide. remediation and extraction of nutrients from waste- Massive algal blooms, particularly in nutrient-rich water (Nielsen et al. 2012, Sode et al. 2013). Ulva spp. coastal waters, highlight the opportunistic nature of biomass sourced from green tides is used in high-vol- some Ulva species (Smetacek & Zingone 2013, Mineur ume markets (plant or animal care) (Fletcher 1996, et al. 2015) which benefit from highly eutrophic coastal Gao et al. 2017), but growing demand in human food © The authors 2019. Open Access under Creative Commons by *Corresponding author: [email protected] Attribution Licence. Use, distribution and reproduction are un - restricted. Authors and original publication must be credited. Publisher: Inter-Research · www.int-res.com 376 Aquacult Environ Interact 11: 375–391, 2019 and other high-value applications is promoting Ulva differentiation, respectively. The established tripar- production in aquaculture systems that can meet tite community was an essential step on the way to strict quality standards for e.g. the food industry standardised laboratory cultures, providing controlled, (Abreu et al. 2014, Santos et al. 2015). repeatable conditions to study different aspects of Similar to plant growth-promoting bacteria, which algal−bacterial interactions (Wichard et al. 2015, are microorganisms that naturally participate in com- Grueneberg et al. 2016). plex ecological interactions (Liu et al. 2017), for ex - Another designed community, introduced by ample, in the rhizosphere with land plants, certain Ghaderiardakani et al. (2017), resembled the tripar- bacteria also induce growth and morphogenesis in tite Ulva mutabilis−Roseovarius−Maribacter system macro algae (Singh & Reddy 2014, Wichard 2015). A but employed U. intestinalis axenic gametes and cocktail of compounds, harbouring different eco- pairs of bacterial strains isolated from species other physiological effects, is involved in macroalgal deve - than U. mutabilis and U. intestinalis. Although there lopment (Spoerner et al. 2012). Algal growth- and is specificity in the bacterial signals regulating algal morphogenesis-promoting factors derived from bac- development (i.e. a signal inducing cell division and teria (AGMPFs) induce cell division, cell wall for - another signal for inducing rhizoid formation), there mation, and holdfast development. AGMPFs are un- is no species-specificity among U. intestinalis, U. der investigation in several studies, particulary their linza, U. pertusa, and U. mutabilis, as a range of bac- ability to induce various morphogenetic changes teria was able to perform their eco-physiological across the prokaryote− eukaryote boundary (Wichard functions similarly in all tested species (Singh & & Beemel manns 2018). Reddy 2014, Ghaderiardakani et al. 2017). Members of Ulvaceae, like many other types of sea- Grueneberg et al. (2016) screened a collection of weed, harbour microbial biofilm communities on their sterile-filtered natural water samples collected from surfaces with functions related to host growth and 20 sample sites and tidal pools across the lagoon Ria morphological development. Recent research has Formosa (Algarve, Portugal). The authors applied a enabled a rapid expansion of knowledge focusing on bioassay-driven approach to examine the ecological many individual aspects of interactions between ma - relevance of waterborne bacterial morphogens by rine algae and their associated microbiota. Characteri- evaluating their morphogenetic impacts on the axenic sation of the chemical interactions between micro - gametes of U. mutabilis. The survey revealed that biome and host (Goecke et al. 2010), the structuring Ulva spp. could benefit from morphogenetic com- of microbial communities on the surface of algal host pounds with similar activity to the compounds released (Burke et al. 2011), bacterial diversity (Lachnit et al. by Roseovarius sp. and Maribacter sp. (or equivalents 2009, 2011, Barott et al. 2011), and algal pathogens of these strains) as diffusible waterborne morpho - and diseases (Gachon et al. 2017) have all been car- gens were present in the water body. Based on these ried out. findings, beyond the eco-physiological implications, In the absence of external delivery of growth- the morphogenetic inductive potential of filtered sea- promoting morphogenetic factors from an appropriate water has to be taken into account, particularly in the bacterial community, germ cells (spores and gametes) translation of this investigative research into practical of Ulva spp. develop into callus-like colonies consist- outcomes (Grueneberg et al. 2016). Using filtered sea- ing of undifferentiated cells with abnormal cell walls water in scientific investigations and even in commer- (Provasoli 1958, Spoerner et al. 2012, Grueneberg et cial-size aquaculture (e.g. land-based aquaculture al. 2016). Previous investigations have revealed the operations) may directly (positively) affect the suc- significant impact of the epiphytic bacterial commu- cess of biomass production due to its morphogenetic nity on the morphogenesis and have detailed the effect on the development of Ulva spp. (Gruene berg specificity of this interaction on the promotion of et al. 2016). Moreover, seawater composition can development in different Ulva species (Marshall et therefore also determine the growth, development, al. 2006, Spoerner et al. 2012, Wichard 2015, Ghade- and hence bloom-forming activity of naturally occur- riardakani et al. 2017). Spoerner et al. (2012) estab- ring Ulva spp. populations. lished a unique symbiotic tripartite system including In addition to the bacterial signals required for early 2 bacteria, Roseovarius sp. strain MS2 and Maribac- Ulva spp. development, mature blade cells of U. ter sp. strain MS6, working synergistically to induce mutabilis excrete regulatory factors into their cell complete morphogenesis in U. mutabilis through dif- walls and the environment. These factors, a glyco- fusible morphogens — Roseovarius-factor and Mari - protein (sporulation inhibitor [SI]-1) and a low molec- bacter-factor — which induce cell division and cell ular weight factor (SI-2), are essential for the mainte- Ghaderiardakani et al.: Algal growth- and morphogenesis-promoting factors in aquaculture 377 nance of the vegetative state (Stratmann et al. 1996, lagoon Ria de Aveiro, Portugal (Fig. 1A). The open- Vesty et al. 2015) and are of great interest in algal aquaculture system operates in the traditional way aquaculture management (Charrier et al. 2017). used in several locations throughout southern Europe,