The underexplored potential of green macroalgae in aquaculture

Green macroalgae (Chlorophyta) currently represent a residual fraction (<1%) of global seaweed biomass production landings. In turn, red (Rhodophyta) and brown (Ochrophyta) macroalgae dominate the remaining percentage of aquaculture production, exceeding 32 million tonnes per annum. However, the industry relies on a relatively low number of species, in which as few as seven macroalgal genera collectively represent the bulk of global production metrics. At present, innovation and increased sustainability of the industry calls for diversification of macroalgal species/strains in aquaculture to counteract potential adverse effects ensuing from


| INTRODUC TI ON
Macroalgae (or seaweeds) comprise a diverse group of photosynthetic, multicellular, eukaryotic organisms assigned to either the Plantae or Chromista kingdoms. Most are marine, where they play predominant roles as primary producers in coastal waters of the planet. 1 Despite being avascular and lacking true roots, stems, leaves and complex reproductive structures, some macroalgae display plant-like appearances, in that they present differentiated thalli with attachment organs (holdfasts), stem-like structures (stipes) and photosynthetic blades (fronds). 2 Marine macroalgae can be divided into three phyla: Chlorophyta (green), Rhodophyta (red) and Ochrophyta (brown), classified according to photosynthetic pigment content, carbohydrate reserves, cell wall components and flagella construction and orientation. 1 Collectively, the diversity of these groups exceeds 10,000 species, while new taxa are described every year. Currently, more than 1500 macroalgal species are assigned to Chlorophyta; approx. 2000 to Ochrophyta; and about 7300 to Rhodophyta. 3 Together, macroalgae constitute an important biological resource, providing a variety of ecosystem services and socioeconomic value. 4,5 Since prehistoric times, ca. 160 thousand years ago, early hominids have relied on intertidal habitats as foraging grounds for marine resources. 6,7 Eventually, the inclusion of macroalgae in human diet is thought to have prompted profound impacts upon the evolution of human civilisation. 8,9 The earliest evidence of the relationship between humans and marine macroalgae dates to the Neolithic (ca. 14,000 years ago) according to archaeological findings, when humans seemingly collected and transported a variety of seaweed species to be used as foodstuffs, trade items and ancient pharmacopeia. 10 In addition, written records support that seaweed harvesting has been going on for centuries in virtually all shoreline areas where coastal communities have established, namely in Asia, 11 Europe, 12 Americas, 10 Oceania 13,14 and Africa. 15 Such communities relied on marine macroalgae for a variety of different purposes including human and animal feed, soil fertilisation, medicinal, cultural activities and raw material for different applications. 14,16 Like other organisms included in human diet and well-being, some macroalgal species were domesticated and cultivated, once growing demand exceeded natural beds' holding capacities. 17,18 Modern cultivation technologies first emerged in the 20th century in China and in Japan, after the description of the reproductive cycle of bladed Bangiales (Pyropia sp., Rhodophyta). 11 Soon after, the aquaculture of macroalgae rapidly expanded into what currently represents approximately half of global marine aquaculture production landings (51%), comparing to molluscs (27%), fish (13%) and crustaceans (9%); while exploitation of natural stocks represents a fraction (~2.8%) of total production of seaweeds. 19 Macroalgal production is nowadays the fastest growing sector in global marine aquaculture generating an excess of US$ 13 billion per annum, 19 even though still holds a remarkable potential for innovation, particularly on the development of valuable products (eg functional foods, cosmeceuticals, nutraceuticals, pharmaceuticals) 20 and is expected to gain further traction given the increasing perception of algae as healthy and sustainable foodstuffs, particularly in developing markets of western cultures. [21][22][23] The bulk of macroalgae aquaculture takes place in South-East Asian and Pacific countries, of which China, the Philippines, Indonesia, Republic of Korea and Japan contribute a staggering ~98% of global seaweed biomass production, while the same industry is sustained by as few as seven macroalgal genera, all of which assigned to either Rhodophyta or Ochrophyta. 19 Some of these macroalgae are destined for the hydrocolloid industry, such as Eucheuma spp., Kappaphycus alvarezii and Gracilaria spp., whereas other taxa are used directly as human food, namely Saccharina japonica, Undaria pinnatifida, Sargassum fusiform and Pyropia spp. 19,24 In turn, the output of green macroalgae (Chlorophyta) aquaculture currently represents only a fraction of global landings (<1%), lagging well behind the most relevant taxa in terms of production metrics. Despite presenting comparably lower production figures, the aquaculture of green seaweeds has witnessed an increasing trend in productivity and commercial diversification over the last decades ( Figure 1). 19 Nevertheless, asymmetries deriving from the dominance of few taxa in macroalgal aquaculture intensify the need for increased innovation and sustainability of the industry. Particularly, the commoditisation of the industry is thought to be inconsistent with future demand of high-value seaweed products, by failing to provide standardised, high quality, traceable products. 25 Advances are therefore required to meet the challenges of an evolving industry, through species and/or cultivar diversification, standardisation of cultivation techniques and increased awareness of local genetic and environmental variability. 18,20,25,26 In this context, expanding green macroalgae aquaculture emerges as a compelling solution towards the diversification and improvement of the sector, by providing a diverse pool of largely untapped biological resources, with intrinsic potential to unlock an array of different biotechnological applications. Indeed, due to the evolutionary divergence between the major macroalgae phyla, the Chlorophyta, Rhodophyta and Ochrophyta differ in their elemental composition, 27 metabolomic 28,29 and fatty acid profiles, [30][31][32][33] nutritional properties, 34,35 polysaccharide types 36 and organoleptic properties. 37 Ultimately, distinctive features among macroalgal phyla will allow different applications, and therefore innovation in the industry. Accordingly, green macroalgae have been recently promoted for different applications, including biorefinery operations, 38 landbased integrated multitrophic aquaculture (IMTA) systems 39,40 and high-value food products in modern cuisine. 41 Examples of developing industrial applications using green macroalgae as raw material include the extraction of cellulose 42 or sulphated polysaccharides 43 and the production of biochar, 44 bioethanol 45 and bioplastics, 46 while an indefinite number of bioactive molecules will keep emerging from green macroalgae metabolite screening studies. 47,48 The present review aims to provide a complete overview of the status, ongoing developments and future perspectives of green macroalgae in aquaculture. A detailed description of the diversity and potential of major taxa are given alongside known applications, regional importance and global production estimates, as well as existing cultivation techniques. We further analyse existing literature on the prospection of bioactive compounds and conclude by outlining future perspectives of this mostly underexplored group of macroalgae in aquaculture.

| D IVER S IT Y AND P OTENTIAL OF G REEN S E AWEEDS IN AQUACULTURE
Most green seaweeds are currently assigned to Ulvophyceae, the most morphologically and ecologically diverse class of Chlorophyta, while few representatives occur in otherwise evolutionary distant groups, for example genus Prasiola (Trebouxiophyceae). 49  The variety of cytological architectures found in green macroalgae is explained by the independent evolution of macroscopic growth that took place in several ulvophycean lineages that diverged from ancestral unicelled green algae millions of years ago, following different evolutionary pathways. 52 The diversity of ulvophycean seaweeds presents itself in a pal-

| Ulvales and Ulotrichales
The Ulvales and Ulotrichales form an early branching clade among Ulvophyceae of predominantly marine macroalgae, presenting multicellular thalli that range from branched or unbranched filaments to blade or tubular morphologies. 50  spp. and Capsosiphon fulvescens (Ulotrichales). Today, these taxa collectively represent more than two thirds of global green seaweed aquaculture biomass production 19 (Figure 1).

F I G U R E 1
Global production of green macroalgae in marine aquaculture. (a) Production metrics are represented for the main taxa (tonnes fresh weight) from 1960-2018. (b) Five-year average relative percentage by weight of representative taxa by country (FAO, 2020). Note: aquaculture statistics for green macroalgal species in Japan are not listed separately in FAO databases and therefore not represented graphically. Production estimates of Monostroma, Ulva and Caulerpa in Japan can exceed 25,000 tonnes fresh weight annually, in which case global production figures are significantly underestimated (see text for references, Section 2) Taxa affiliated to the genera Ulva (syn. Enteromorpha) and Monostroma are registered in FAO databases under common trade names, for example 'bright green nori' and 'green laver', reflecting a certain degree of taxonomic uncertainty in those algal products. and Ulva flexuosa as biofilters of fish aquaculture effluents. 82,83 Overall, these studies showed promising results in using Ulva species in IMTA systems, by providing both increased water quality and added value when employed as feed for other trophic levels.
However, commercial scale application of Ulva in such systems has rarely been adopted. and Portugal 40 ( Figure 2).
Apart from its use in IMTA, aquaculture of Ulva for human consumption has been mostly assigned to a single species (U. prolifera).
Ulva prolifera (syn. Enteromorpha prolifera 72 ) is globally distributed, notorious for its bloom forming nature, and principal causative agent of green tides in China. 85 Dried specimens of U. prolifera are particularly dark green, with strong flavour compared with other congeners (eg U. linza and U. intestinalis) that confers high commercial value as 'green laver'. 86 In Japan, commercial aquaculture of U. prolifera is focused on niche, high-value food products, where mass cultivation of the species has been going on since the early 1980 s and relies on artificial seed production. 87,88 Today, U. prolifera aquaculture production in Japan turns out unnoticed in FAO aquaculture statistics, although annual production metrics have been reported elsewhere, despite with inconsistent values (eg 200, 1500, 3000 tonnes dry weight; equivalent to approximately ten times as much in fresh weight). 63,89,90 The species is also cultivated in the southern coast of Korea, where it is valued as an ingredient in salads, soup and cookies 62,91 and likely represents the bulk of 'green laver' production reported at 6800 tonnes in 2018 (as previously referred).

| Monostroma, Thuret
The genus Monostroma includes popular edible taxa consumed as

| Bryospidales
The Bryopsidales forms a diverse ulvophycean order that includes approx. 600 recognised species, 3 important primary producers in coral reefs, rocky shores, lagoons and seagrass beds, with representative species found from tropical to Arctic waters. 103  is commercially cultivated in Portugal by a company (ALGAplus Ltd.) dedicated to seaweed production in a land-based IMTA ( Figure 3).
The company optimised artificial propagation methods for C.
tomentosum production and grows juvenile thalli in outdoor tanks, fed by the effluent of a semi-intensive aquaculture system producing marine fish. Despite unreported annual production, C. tomentosum is sold to high-end restaurants, where it is highly appreciated as a gourmet ingredient for its flavour of barnacles with notes of peach; 41

| Cladophorales
The order Cladophorales is species-rich, comprising about 485 recognised species, 3  given comparatively higher nutrient uptake rates, higher tolerance to solar irradiance and lower propensity for seasonal changes in algal productivity. 134,135 Examples of studies that evaluated the usefulness of  (Table 1).  In Codium (green sponge fingers, velvet   Enteromorpha prolifera) artificial propagation in Japan, propagules were obtained by mincing vegetative thalli in a rotary blender; the resulting fragments would release swarmers, which would be directly dispersed in 'seeding tanks' holding culture nets for attachment. 166 Optimisations to the same method attempted to promote synchronous gamete/spore release by cutting thalli into 1.2 mm diameter discs and studying optimum salinity and irradiance conditions. 88

| Manipulation of reproductive structures
Using the same principles, variations to the same techniques Each step is artificially induced by manipulating osmotic, thermal and irradiance conditions. 94 Traditionally, propagule production starts with the collection of mature gametophyte fronds from the environment.  (Table 2). Desulphation significantly decreases bioactivity of ulvans, indicating that the sulphate residues are important for the stimulatory capacity of these molecules. [180][181][182] The presence of glucuronic acid in green macroalgae extracts has been related to skin hydration and protection capacities. Extracts from the green macroalgae Codium tomentosum are currently used as a moisturising agent in the cosmetic industry. 183,184 In addition, the bioactive properties displayed by sulphated polysaccharides extracted from Ulvophycean taxa are highly re- Antiviral and immunostimulatory activities were identified for sulphated polysaccharides extracted from several green macroalgal species 181,[189][190][191][192][193] (Table 2).

| Lipids
Lipids are molecules soluble in nonpolar solvents that act as main structural components of cell membranes but are also involved in energy storage and cell signalling pathways. Mass spectrometry has allowed detailed characterisation of the polar lipid profile of macroalgae (glycolipids, phospholipids and betaine lipids), some of which with proven nutritional and health benefits. 194 Of particular relevance for lipid bioactivity is the high content of polyunsaturated fatty acids (PUFA), considered as essential components in human and animal health and nutrition. 195 Macroalgae are a potential source for large-scale production of essential PUFA with wide applications in the nutraceutical and pharmacological industries. 178 High relative abundance of long-chain PUFA, namely n-3 fatty acids, was observed in Codium galeatum, Codium tomentosum and Ulva armoricana. 32,117,196 The  198 This sterol was also shown to reduce expression of pro-inflammatory proteins and could be used as a therapeutic agent against UVB-induced inflammatory and oxidative skin damage. 199  Siphonaxanthin is a more potent antitumoral agent than fucoxanthin, a carotenoid that was previously shown to inhibit the proliferation of cancer cells through the induction of apoptosis. 203,204 Chlorophyll a-derived pigments such as pheophytin and pheophorbide a have also been shown to possess bioactive properties.
Pheophytin a derived from Ulva prolifera has been reported to have strong anti-inflammatory and anticarcinogenic activities, 205,206 while antioxidant properties have been observed for pheophorbide a. 207 Although natural pigments have the technological disadvantage of having lower stability compared with their synthetic counterparts, different methods and compounds have been successfully used to improve stabilisation of pigment properties and bioactivities. 208 The development of efficient delivery systems is also an effective way to enhance pigment stability and bioavailability. 209

| Secondary metabolites
A diversity of other bioactive compounds have been described in green macroalgae, mainly secondary metabolites often playing important roles in defence against herbivory (  41 On the other hand, implementation of industrial operations using green seaweeds as raw material is expected to increase demand, given the growing interest in implementing biorefineries to produce various products (reviewed by Zollmann et al. 38 ).
Ultimately, sustainability of the entire production chain will define the viability of each operation.
Today, sustainability assessments are scarce but required to unlock green macroalgae (and other seaweeds) potentials.
Sustainability of products comprises three components: environment, economy and social aspects. 227 These three components must be properly assessed and balanced when products are designed or improved. Life-cycle assessment (LCA) is a structured, comprehensive and internationally standardised method to evaluate environmental impacts of the bioeconomy. 228 The LCA aims to assess the potential environmental impacts associated with a product, a process or a system throughout its life cycle.
With sustainability and green markets becoming more popular around the globe, the concept of life cycle sustainability assessment (LCSA) has been introduced. 227 230,231 with great promise to reduce the resource footprint of seaweed cultivation once energetic efficiency is improved (electricity and transport) and biomass productivity increases; 230,232 and/or improvements on infrastructural component materials (eg stainless steel chains, polypropylene ropes) and system design (eg number of cultivation strips in the water column) are employed. 233 Similarly, an explorative LCA study performed to evaluate the environmental impacts of producing bioplastics from green macroalgae (Ulva spp.) allowed to identify the main impacting hotspots in the production chain, those related to high energetic costs of cultivation in land-based systems and material efficiency. 46 In this context, performing LCAs at the initial stage of implementing green macroalgal aquaculture and conversion will become advantageous, as the recommended design improvements can be implemented without significant economic investments. 234 Overall, these studies suggest that an effective transition to renewable energies, and to more eco-friendly materials will greatly increase macroalgae aquaculture sustainability.
Synergies between the academic and private sectors will further allow developing multidisciplinary methodologies assessing macroalgae aquaculture activity to gain market and social trust.
Despite the existing opportunities for green macroalgae in aquaculture, it is not expectable that production of this select group of seaweeds reaches the order of magnitude of their red and brown macroalgal counterparts. Instead, it is likely that green macroalgae will remain to occupy specialised market niches, in which high-value products are favoured as opposed to large quantity production rendering low price biomass. This has been the case in the Asia-Pacific region, where green macroalgae have been cultivated to produce speciality food products, constituting relatively small, localised market niches, within specific cultural backgrounds. In turn, the aquaculture of hydrocolloid-bearing macroalgal species (red and brown) supplies a growing, mass scale production and increasingly commoditised industry. 235 To circumvent the competitive advantages of the seaweed-hydrocolloid industry thriving in Asia, the future of seaweed aquaculture in emerging markets (eg Europe) may not necessarily depend on producing large amounts of biomass, but rather focus on adding value to the seaweed production chain, through byproduct development (eg fodder, fertilisers, bioactive compounds) and capitalising on societal benefits (eg economic, environmental). 22 The unique features presented by Ulvophycean macroalgae (eg distinct biochemical profiles and organoleptic properties, high nutrient uptake and propagation capacity, diverse cultivation technologies, tolerance to extreme conditions) represent a remarkable potential for innovation in macroalgal aquaculture.