# Integrating Gammarus insensibilis in biofloc systems: A sustainable approach to nutrient enrichment and waste valorisation in aquaculture

Marta Castilla-Gavilán, José Manuel Guerra-García, Ismael Hachero-Cruzado

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Marta Castilla-Gavilán, José Manuel Guerra-García, Ismael Hachero-Cruzado. Integrating Gammarus insensibilis in biofloc systems: A sustainable approach to nutrient enrichment and waste valorisation in aquaculture. Aquaculture, 2025, 597, pp.741922. 10.1016/j.aquaculture.2024.741922. hal-04823138

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Submitted on 21 Jan 2025

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# Integrating *Gammarus insensibilis* in biofloc systems: A sustainable approach to nutrient enrichment and waste valorisation in aquaculture

Marta Castilla-Gavilán<sup>a,b,*</sup>, José Manuel Guerra-García<sup>c</sup>, Ismael Hachero-Cruzado<sup>b,d</sup>

<figure><img src="image_2.png" /></figure>

## ARTICLE INFO

### Keywords:

- Sustainable aquaculture

- Nutrition

- Low-trophic aquaculture

- Waste valorisation

- Circular economy

## ABSTRACT

This research addresses the cultivation of *Gammarus insensibilis* in biofloc systems. The aim of the work was to valorise and to bioremediate aquaculture effluents while enhancing the nutritional value of bioflocs through the introduction of amphipods, which can be grown in close aquaculture systems. Two experimental diets (aqua-culture waste detritus and commercial fish feed) were tested for amphipods in triplicated biofloc systems, against three control tanks without biofloc. The experiment was conducted over a six-week period at a temperature of 20 °C in the absence of light. The water quality, the survival, lipid profile and fatty acid composition of the amphipods and the bioflocs produced were studied. Significant differences were observed in the water quality between the treatment groups and the control, since nitrifying bacteria were present in the bioflocs formed in the treatment tanks. Indeed, 100 % mortality was reached in the control tanks from the second week of the experiment. The fish pellets diet and the pellet-based biofloc resulted in a significantly higher total lipid content than the detritus diet and biofloc. However, no differences were found in the essential long-chain poly-unsaturated fatty acids (LC-PUFAs) content (ARA, EPA and DHA) between the commercial pellets and the detritus-based biofloc, thus justifying the enrichment of the biofloc with amphipods. Indeed, the wild amphipods displayed higher levels of saturated fatty acids and omega-6 PUFAs compared to the experimental groups, which demonstrated higher omega-3 PUFAs content, particularly DHA. The study highlights the nutritional advantages of pellet-based diets for amphipods, including improved survival and lipid content, while also emphasising the enhanced nutritional profile of the enriched detritus-based biofloc. We suggest that the integration of aquaculture detritus with supplemental aquafeed in BFT systems will support both the bioflocculation process and the nutritional needs of the amphipods, thereby creating a sustainable and efficient cycle of waste valorisation and live food production. Further research is required in order to study the complete life cycle of amphipods and their reproductive capacity in these systems. A lower water temperature and the inoculation of microalgae should be considered in order to achieve higher survival rates and PUFAs content.

## 1. Introduction

Biofloc technology (BFT) was originated in the 1970s (Emerenciano et al., 2021) and has been widely adopted due to its environmental and economic benefits (Zimmermann et al., 2023). This technology is based on zero-water exchange systems with the formation of bioflocs in the culture medium where uneaten food, excess inorganic nutrients and faeces aggregate along with microorganisms (bacteria, microalgae such as diatoms, protozoa...) (Khanjani et al., 2024b). This aggregation is enabled by a matrix of extracellular polymeric substances secreted by

the microorganisms (Hargreaves, 2013). By maintaining a high carbon/nitrogen ratio (Hargreaves, 2013) through the addition of a carbon source (molasses, glycerol, flours; Zhao et al. (2016) to the culture medium, the colonies of chemoautotrophic bacteria present in the bioflocs assimilate the ammonium excreted by the cultured species during the nitrification process, transforming it into nitrate (Khanjani et al., 2022). Heterotrophic bacteria can also directly assimilate the ammonium into bacterial biomass. Additionally, microalgae contribute to nitrogen absorption during photosynthesis (Ebeling et al., 2006). BFT systems have been identified as a promising technology for sustainable

<footnote><sup>a</sup> Institut des Substances et Organismes de la Mer, ISOmer, UR 2160, Nantes Université, 44000 Nantes, France</footnote>

<footnote><sup>b</sup> IFAPA Centro El Toruño, Camino Tiro Pichón s/n, El Puerto de Santa María, 11500 Cádiz, Spain</footnote>

<footnote><sup>c</sup> Laboratorio de Biología Marina, Departamento de Zoología, Facultad de Biología, Universidad de Sevilla, Avda. Reina Mercedes 6, 41012 Sevilla, Spain</footnote>

<footnote><sup>d</sup> "Crecimiento Azul", Centro IFAPA el Toruño, Unidad Asociada al CSIC, El Puerto de Santa María, 11500 Cádiz, Spain</footnote>

<footnote>* Corresponding author at: Institut des Substances et Organismes de la Mer, ISOmer, UR 2160, Nantes Université, 44000 Nantes, France.
E-mail address: marta.castillagavilan@univ-nantes.fr (M. Castilla-Gavilán).</footnote>
food production, promoting low-trophic-level species cultivation, poly-culture, and the development of a circular economy model (Khanjani et al., 2024c). The feeding efficiency and waste management are enhanced in these systems, as they have the capacity to increase nutrient utilisation and minimize environmental impact, achieved through reduced water and fertilizer use (Knowler et al., 2020; Zimmermann et al., 2023). Thus, the need for water exchange is minimized, which in turn reduces pumping costs, prevents fluctuations in temperature and reduces the energy requirement for heating (McCusker et al., 2023). In BFT systems, food requirements are also reduced, and an increase in the survival and growth rates of cultivated species has been observed (Khanjani and Sharifinia, 2024). Bioflocs present a favourable nutritional profile (Crab et al., 2012) and their consumption have been demonstrated to enhance growth and fortify the immune system of cultivated species by increasing the activity of their digestive enzymes, resulting in an increase in the feed conversion rate (Xu and Pan, 2013). BFT is primarily employed in the cultivation of penaeids (Crab et al., 2010b) but numerous studies have applied this technology to the cultivation of filter feeders, detritivores and even some herbivorous or omnivorous finfish, which are able to feed directly on particulate organic matter (Crab et al., 2012; Das and Mandal, 2021; Dinda et al., 2020). These species are tolerant to changes in the concentrations of dissolved oxygen in the water and nitrogenous compounds and can withstand high culture densities and high concentrations of suspended solids (El-Sayed, 2021; Emerenciano et al., 2021).

Despite all these advantages, bioflocs have been observed to present low proportions of long-chain polyunsaturated fatty acids (LC-PUFAs) (Crab et al., 2010a), maybe linked with the absence of PUFAs in bacteria (Zhukova and Kharlamenko, 1999), and some essential amino acids (EAA) such as methionine (Abbaszadeh et al., 2022). In this sense, several studies have demonstrated that the incorporation of live food, such as diatoms, rotifers, *Artemia* or copepods, into BFT can enhance the nutritional value of the bioflocs, as well as the growth and survival rates of the cultivated species (Abbaszadeh et al., 2022; Brito et al., 2016; de Andrade et al., 2021). Biofloc enrichment with amphipods has been addressed in only one previous study (Promthale et al., 2021). These authors reported a high survival rate of amphipods in the BFT conditions and a high potential for the enriched bioflocs to replace fishmeal. Amphipods cultivation interest has increased in the last years due to their high-quality nutritional profile (Baeza-Rojano et al., 2014; García-García et al., 2016; Jiménez-Prada et al., 2018) adequate for feeding high market value fishes or cephalopods (Baeza-Rojano et al., 2010; Baeza-Rojano et al., 2013; Suontama et al., 2007). Indeed, the Spanish Institute of Oceanography (IEO) holds a patent for the methodology of cultivation of the common octopus paralarvae (Tur et al., 2020). This patent delineates particular zootechnical conditions and the utilisation of amphipods as an alternative live prey to crustacean zoae, and is currently being utilised by the company "Nueva Pescanova" for the cultivation of *Octopus vulgaris*. However, the upscaling of the systems requires increasing amounts of live food, being a bottleneck for the development of these species aquaculture (García-Fernández, 2022). The marine amphipod *Gammarus insensibilis* Stock, 1966 has been highlighted as a promising species for being intensively cultured due to its adequate nutritional profile, large body size and high natural densities (Jiménez-Prada et al., 2020). Moreover, as a detritivorous species, *G. insensibilis* can feed on aquaculture sludge and wastes (Castilla-Gavilán et al., 2023).

With this background, we suggest that *G. insensibilis* has the potential for utilisation as a live food source and could be cultivated intensively in BFT systems, thereby enriching bioflocs nutritional value. Moreover, their ability to feed on detritus would permit the valorisation of aquaculture wastes and the bioremediation of the effluents (Castilla-Gavilán et al., 2023). In the present study, *G. insensibilis*, fed with commercial aquafeed or detritus from aquaculture effluents, has been cultivated in BFT systems. The water quality, the survival rate and the nutritional profile of the amphipods as well as the biofloc produced were analysed.

## 2. Materials and methods

Specimens of *G. insensibilis* were sampled in March 2023 from a natural marsh pond at the IFAPA Centre "El Toruño" (El Puerto de Santa Maria, Cadiz Bay, Spain). Amphipods on their original subtract (*Ulva* sp. thalli, where they mainly inhabit) were transported to IFAPA facilities and transferred into a 400 L tank to allow their acclimation during a week prior experimentation. This tank was connected to a recirculation system (RAS) equipped with a cooling, mechanical filter, protein skimmer, ultraviolet lights and biofilter (Castilla-Gavilán et al., 2023). They were illuminated (1.5–1.7 µmol photons m⁻² s⁻¹) with fluorescent tubes in a 8:16 h (light:darkness) to acclimate amphipods to the darkness, according to Promthale et al. (2021). The seawater was maintained at the same conditions than in the natural environment, 17 °C and 38 g L⁻¹ salinity, and was renewed daily in continuous water flow.

Prior to the experiment, 2700 adults of *G. insensibilis* were sampled from the tank, devoid of algae and placed in 9 culture units (300 specimens per culture unit) filled with 25 L of aerated and mixed seawater. Three hundred additional "wild" specimens were sampled and stored at -80 °C for lipid profile analysis and comparison with the specimens used in the experiment. The culture units were maintained in darkness without any water exchange, following Promthale et al. (2021). An artificial substrate for amphipods was provided in all culture units (plastic mesh, following Castilla-Gavilán et al., 2023). Amphipods were starved for 24 h to standardize their nutritional conditions. Two experimental treatments were tested against a control, at 20 °C (Promthale et al., 2021), in triplicate culture units. In the first treatment, *G. insensibilis* specimens were fed with grounded sea bream commercial pellets (0.2 g day⁻¹). In the second treatment, the feed was waste detritus (1.2 g day⁻¹) obtained through the cleaning of the filters of a Senegalese sole RAS unit and consisted primarily of fish faeces and uneaten fish feed pellets. The quantity of food was determined by considering the organic matter percentage of each type, with detritus containing six times less organic matter than pellets. Three samples of each food type were also stored at -80 °C for lipid profile analyses.

Between three and four times a week (20 sampling days in total), three water samples were taken from the tanks and filtered on GF/C fiberglass filters that were treated following Castilla-Gavilán et al. (2023), in order to determine total suspended solids in the water and the organic matter content of these solids. Water was then analysed: dissolved nitrate and nitrite concentrations were sequentially quantified by the reduction of $NO_3^-$ to $NO_2^-$ with vanadium ($VCl_3$) following the method of García-Robledo et al. (2014). Ammonium concentration was determined by the indophenol-blue method (Aminot et al., 1997), and carbon concentration was then adjusted to a 16:1C:N ratio through the addition of corn flour (Promthale et al., 2021). The control treatment was also fed with detritus, and water was also sampled daily for monitoring purposes and ammonium concentration analyses, but no carbon source was added in order to avoid the bio-flocculation process. Water parameters (mean dissolved oxygen, temperature, salinity and pH) were checked daily in all tanks and were found to be 6.1 ± 0.04 mg L⁻¹ DO, 21.1 ± 0.33 °C, 40.1 ± 4.14 g L⁻¹ salinity (typical of marsh environments), and 8.1 ± 0.09 pH. The experiment lasted six weeks except for the control tanks, since 100 % mortality was observed from the second week. At the end of the experiment, surviving amphipods and the sludge (bioflocs) produced in the 6 experimental culture units were sampled, amphipods were counted and all samples were stored at -80 °C prior to biochemical analysis.

All the freeze samples were freeze-dried and grounded in to a fine powder. Ashes, moisture, total lipids and fatty acids profile were analysed. Total lipids (TL) and fatty acids (FA) profile methods were those previously described by Hachero-Cruzado et al. (2014). Briefly, TL were extracted from powder following the methodology of Folch et al. (1957) with slight modification by Christie and Han (2012). To extract FA, total lipid extracts were subjected to acid-catalysed transmethylation. Fatty
acids methyl esters (FAME) were separated and quantified by gas chromatography and the identified FA were compared to the authentic standards (FAME Mix C37) and well-characterized fish oil (Menhaden Oil, SUPELCO, USA) Christie and Han (2012).

Mean and standard deviation (SD) were calculated for all data. Statistical analyses and plots were performed using RStudio software. Survival was analysed through a one-way ANOVA test between amphipods in both treatments and the control. Differences in the inorganic nitrogen concentration of the different treatments were tested with two-way ANOVAs following an experimental design with orthogonal factors: treatment, a fixed factor with three levels (control vs pellets vs detritus) and time, a fixed factor with twenty levels (sampling days). Differences in TL between amphipods in the different treatments and between produced bioflocs and diets were also studied through one-way ANOVAs. Where ANOVAs indicated significant differences (*p* < 0.05), the source of the differences was identified using a Tukey test. Prior to ANOVAs, the normality was tested with Shapiro-Wilk test (*p* > 0.05) and the homogeneity of variances was tested with Levene test (*p* > 0.05). If variances remained heterogeneous even after data transformation, untransformed data were still analysed, as ANOVA is a robust statistical test and is relatively unaffected by the heterogeneity of variances (Underwood, 1997). In such cases, the level of significance was reduced to <0.01 to avoid type I error. Additionally, principal component analyses (PCA) were conducted to FA matrixes for the ordination of the samples.

## 3. Results

### 3.1. Survival rate

Survival was significantly higher in the tanks fed with pellets than in those fed with detritus (one-way ANOVA; df = 2; SS = 0.28; MS = 0.14; *F* value = 7.95; *p* value = 0.0206; Fig. 1). No amphipods were observed in the control tanks from the ninth day.

### 3.2. Water quality

Regarding inorganic nitrogen concentrations, statistics are summarized in the Table 1. Ammonium (N-NH₄) levels were significantly higher in the control tanks than in the other treatments (Fig. 2), with no differences observed between the latter (Table 1). Nitrite (N-NO₂) and nitrate (N-NO₃) concentrations were in general low (Fig. 3), and no differences were found between tanks fed with pellets or detritus (Table 1).

<figure><img src="image_3.png" /><figcaption>Fig. 1. Mean amphipods survival rates (%) in the different treatments. Bars represent the maximum and minimum survival rates.</figcaption></figure>

<table><caption>**Table 1**

Results of the two-way ANOVAs for ammonium, nitrite and nitrate concentrations.</caption><thead><tr><th>Source of variation</th><th>df</th><th>SS</th><th>MS</th><th><i>F</i> value</th><th><i>P</i> value</th></tr></thead><tbody><tr><td><strong>Ammonium</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Treatment (T)</td><td>2</td><td>11.48</td><td>5.74</td><td>15.33</td><td>0.000</td></tr><tr><td>Time (t)</td><td>19</td><td>18.40</td><td>0.97</td><td>2.59</td><td>0.002</td></tr><tr><td>T: t</td><td>21</td><td>1.80</td><td>0.09</td><td>0.23</td><td>0.999</td></tr><tr><td>Residuals</td><td>86</td><td>32.20</td><td>0.37</td><td></td><td></td></tr><tr><td>Levene Test</td><td>42</td><td></td><td></td><td>4.10</td><td>0.343</td></tr><tr><td>Tukey Test</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td>control > detritus</td><td></td><td></td><td>0.000</td></tr><tr><td></td><td></td><td>control > pellets</td><td></td><td></td><td>0.000</td></tr><tr><td></td><td></td><td>detritus = pellets</td><td></td><td></td><td>0.997</td></tr><tr><td><strong>Nitrite</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Treatment</td><td>1</td><td>0.00</td><td>0.00</td><td>0.06</td><td>0.805</td></tr><tr><td>time</td><td>4</td><td>0.00</td><td>0.00</td><td>5.07</td><td>0.006</td></tr><tr><td>T: t</td><td>4</td><td>0.00</td><td>0.00</td><td>1.04</td><td>0.410</td></tr><tr><td>Residuals</td><td>20</td><td>0.00</td><td>0.00</td><td></td><td></td></tr><tr><td>Levene Test</td><td>9</td><td></td><td></td><td>1.58</td><td>0.189</td></tr><tr><td><strong>Nitrate</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Treatment</td><td>1</td><td>0.01</td><td>0.01</td><td>3.12</td><td>0.093</td></tr><tr><td>time</td><td>4</td><td>0.14</td><td>0.04</td><td>9.73</td><td>0.000</td></tr><tr><td>T: t</td><td>4</td><td>0.04</td><td>0.01</td><td>2.38</td><td>0.086</td></tr><tr><td>Residuals</td><td>20</td><td>0.07</td><td>0.00</td><td></td><td></td></tr><tr><td>Levene Test</td><td>9</td><td></td><td></td><td>1.37</td><td>0.266</td></tr></tbody></table>

### 3.3. Nutritional profile

The Fig. 4 illustrates the organic matter (OM) and the total lipids (TL) content of the samples. Significant differences were observed in TL content between the experimental diets and the bioflocs (one-way ANOVA; df = 4, SS = 228.68, MS = 57.17, *F* value = 42.14, *p* value <0.001). The pellets diet exhibited the significantly highest TL content (Tukey test; *p* values <0.001) and the biofloc from the pellets treatment presented a significantly higher content than the biofloc from the detritus treatment (Tukey test; *p* value = 0.047). Concerning amphipods, no differences were found between them (one-way ANOVA; df = 2, SS = 14.38, MS = 7.19, *F* value = 1.65, *p* value = 0.268). Differences in the OM content were only found between the diets and the bioflocs (one-way ANOVA; df = 3; SS = 6447; MS = 2148.9; *F* value = 210.1; *p* value <0.001). The pellets presented the highest content (Tukey test; *p* values <0.001), followed by the pellet-based biofloc, which presented a significantly higher content than the two other treatments (Tukey test; *p* values <0.001). No differences were found between detritus-based diet and biofloc (Tukey test; *p* value = 0.6509).

The fatty acid profile (in percentage) of the diets and bioflocs analysed and the one-way ANOVAs are summarized in the Table 2. The isoC₁₆ fatty acid, which is characteristic of bacteria (Tanaka et al., 2014), was analysed among the SFA. While the pellets presented no evidence of this FA, the detritus diet and the different bioflocs did (Table 2), thereby corroborating the presence of biofloc-forming bacteria. Major FA were the SFA 16:0 (PA), the MUFAs 18:1n7 (cis-VA) and 18:1n9 (OA), the omega-6 PUFA 18:2n6 (LA) and the omega-3 PUFA 22:6n3 (DHA). The pellet-based diet presented the significantly highest level of OA (Tukey test; *p* value <0.001). However, the pellet-based biofloc did not show the same proportion of OA, and displayed a significantly lower content than the detritus-based biofloc (Tukey test; *p* value = 0.008) or no differences with the detritus-based diet (Tukey test; *p* value = 0.392). The pellet-based biofloc had the significantly highest level of cis-VA (Tukey test; *p* value ≤0.01), while the pellets presented the lower content (Table 2). These findings support the presence of biofloc-forming bacteria, as cis-VA is a product of many heterotrophic bacterial species (Gillan and Sandstrom, 1985).

In general, the detritus-based diet presented the significantly highest content of SFA and the lowest MUFAs content. The pellet-based diet exhibited the significantly highest content of omega-3 and omega-6
<figure><img src="image_4.png" /><figcaption>Fig. 2. Ammonium concentration (mean ± SD) in the different culture units during the experiment.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>Fig. 3. Nitrate and nitrite concentrations (mean ± SD) in the different culture units during the experiment.</figcaption></figure>

PUFAs (Table 2). However, the biofloc generated in this treatment presented the lowest proportion of PUFAs, showing no significant differences with the detritus-based diet (Tukey test; *p* value = 0.990) and a significantly lower content than the detritus-based biofloc (Tukey test; *p* value = 0.010).

The results of the PCA based on diets and bioflocs were also in

agreement with the previous results and identified OA, cis-VA, LA and PA as major contributors to the total variance. The first principal component explained 57 % of the total variance and the second principal component explained 34.5 %. PC1 correlated positively with the fatty acids OA (r = 0.950, n = 20, p < 0.005) and LA (r = 0.925, n = 20, p < 0.005), and negatively with cis-VA (r = -0.756, n = 20, p < 0.005). PC2
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Organic matter (OM) and total lipids (TL) content (%) in the two diets tested (pellets and detritus), in the bioflocs sampled from the different treatments at the end of the experiment, and in the amphipods in both treatments and those sampled from the wild (mean ± SD).</figcaption></figure>

correlated positively with PA (r = 0.902, n = 20, p < 0.005) and negatively with cis-VA (r = -0.603, n = 20, p < 0.005). The axes clearly separate the diets from the bioflocs, and differentiate the bioflocs based on their PA and cis-VA content (Fig. 5).

The fatty acid composition of the wild amphipods was compared with that of the amphipods used in the experiment (fed with pellets or detritus). Table 3 shows results (in %) and one-way ANOVAs. The major fatty acids were: the saturated fatty acids (SFA) 16:0 (palmitic acid; PA) and 18:0 (stearic acid; SA); the monounsaturated fatty acids (MUFAs) 16:1n7 (palmitoleic acid, POA), 18:1n9 (oleic acid, OA) and 18:1n7 (cis-vaccenic acid; cis-VA); and the polyunsaturated fatty acids (PUFAs) 18:2n6 (linoleic acid, LA), 20:4n6 (arachidonic acid, ARA), 20:5n3 (eicosapentaenoic acid, EPA) and 22:6n3 (docosahexaenoic acid, DHA). Only total SFA composition showed significant differences between the amphipods, with the wild animals displaying a higher concentration than those used in the experiment (Tukey test; *p* value <0.001). This was mainly attributed to a higher individual PA content (Table 2). Concerning MUFAs, wild amphipods presented a significantly higher content of POA while amphipods fed with pellets showed a higher content of OA (Table 2). Wild amphipods also showed the highest content of omega-6 PUFAs due to the higher LA and ARA levels, while the amphipods used in the experiment presented higher contents of omega-3 DHA. The principal differentiating factors between the two feeding regimes were the significantly higher levels of PA (Tukey test; *p* value = 0.012) and OA (Tukey test; *p* value <0.001) observed in the pellets group.

Principal components analyses (PCA) were consistent with the results shown above. The first principal component explained 57 % of the total variance and the second principal component explained 23 %.

Principal component 1 (PC1) correlated positively with the fatty acids DHA (r = 0.854, n = 9, p < 0.005) and cis-VA (r = 0.705, n = 9, p < 0.025), and negatively with PA (r = -0.958, n = 9, p < 0.005), EPA (r = -0.695, n = 9, p < 0.025), ARA (r = -0.934, n = 9, p < 0.025) and LA (r = -0.718, n = 9, p < 0.025). PC2 correlated positively with OA (r = 0.821, n = 9, p < 0.005). Axes clearly separated samples by treatments (wild, pellets or detritus) (Fig. 6).

# 4. Discussion

## 4.1. Survival rate

There are limited reports on the amphipods culture. Nevertheless, the survival rate observed in this study aligns with the findings of Jiménez-Prada et al. (2020), who reported values of approximately 40 % over a 21-days experiment. Similarly, Ribes-Navarro et al. (2022) observed comparable survival rates of *Gammarus locusta* when water at 20 °C and/or diets rich in short-chain PUFAs and SFAs were utilised in their experiment. However, their results indicated that lower temperatures (5–15 °C) and diets rich in LC-PUFAs were associated with enhanced survival rates.

## 4.2. Water quality

No statistically significant differences were found in water quality parameters among the treatments in this study. The results on nutrients concentration in the treatments tanks, when compared to the control, confirmed the effect of the carbon source on the colonisation of heterotrophic bacteria and the flocculation process (Crab, 2010; Hargreaves, 2013; Khanjani et al., 2024a): the concentration of ammonium was significantly lower, while nitrate cumulates in the treatment tanks, thus indicating that nitrifying bacteria were present in the biofloc and being consistent with previous works (Abbaszadeh et al., 2022; Brito et al., 2016).

## 4.3. Nutritional profile

The flocculation success was also corroborated by the lipid profile of the bioflocs, which exhibited major fatty acids including PA, cis-VA, OA and LA, as already observed in previous work (Anand et al., 2014). Essential LC-PUFAs were not among the major FA. However, the present study revealed clearly higher amounts of ARA (0.8 %), EPA (3.1 %) and DHA (5.5 %) than Anand et al. (2014), who found only 0.4 % ARA and did not detect DHA or EPA in their biofloc. These values were similar to the FA profile of the commercial aquafeed used in the present study (pellets), which is formulated to meet the nutritional needs of fish (see Table 2: no differences were found in the ARA, EPA and DHA content between the pellets and the detritus-based biofloc). This justifies the enrichment of the biofloc with amphipods. Indeed, amphipods showed a profile rich in EPA, DHA, LA and ARA, as also demonstrated by Jiménez-Prada et al. (2018). The gammarids from both treatments showed the same amount of TL as the wild specimens, yet demonstrated a reduction in ARA and EPA, which was counterbalanced by a higher amount of DHA. Interestingly, Ribes-Navarro et al. (2021) have demonstrated that gammarid amphipods are devoid of desaturase genes that are complementary to the elongases needed for the biosynthesis of LC-PUFAs. Consequently, they are unable to synthesise these compounds de novo. Similarly to the study conducted by Promthale et al. (2021), our experiment was performed in darkness. Thus, it's unlikely that these compounds were acquired through the diet (phytoplankton). It has been proposed that the high percentages of DHA detected in gammarids, even if fed with diets lacking LC-PUFAs, are the result of an accumulation of previous diets (Ribes-Navarro et al., 2022), which could explain the results observed in the present study. These selective retention mechanisms would enable them to survive during periods of limited or no bioavailability of these nutrients (Ribes-Navarro et al., 2022).
<caption>**Table 2**</caption>

Fatty acids composition (mean % ± SD) of the diets used in the experiment (i.e. pellets, detritus) and the bioflocs sampled from these two treatments at the end of the experiment.

<table><thead><tr><th>Fatty acid</th><th>pellets</th><th>detritus</th><th>Biofloc pellets</th><th>Biofloc detritus</th><th>P value</th><th>Tukey</th></tr></thead><tbody><tr><td><strong>SFA</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>14:0</td><td>1.44 ± 0.00</td><td>3.81 ± 0.13</td><td>1.57 ± 0.44</td><td>2.64 ± 0.16</td><td>0.000**</td><td>d > all; bd > bp; p = bp</td></tr><tr><td>15:0</td><td>0.24 ± 0.01</td><td>0.78 ± 0.01</td><td>0.66 ± 0.19</td><td>0.60 ± 0.05</td><td>0.017*</td><td>d > p; p = bp = bd; d = bp = bd</td></tr><tr><td>16:0</td><td>16.43 ± 0.03</td><td>38.21 ± 2.12</td><td>22.06 ± 0.92</td><td>26.78 ± 1.37</td><td>0.000***</td><td>d > bd > bp > p</td></tr><tr><td>iso-C<sub>16</sub></td><td>0.00 ± 0.00</td><td>0.84 ± 0.05</td><td>1.48 ± 0.32</td><td>0.79 ± 0.15</td><td>0.000**</td><td>bp > bd > d > p; d = bd</td></tr><tr><td>17:0</td><td>0.33 ± 0.00</td><td>1.09 ± 0.16</td><td>1.31 ± 0.26</td><td>0.48 ± 0.31</td><td>0.000**</td><td>d = bp > p; p = bd; bp > bd</td></tr><tr><td>18:0</td><td>4.87 ± 0.09</td><td>8.49 ± 0.06</td><td>8.22 ± 1.09</td><td>10.37 ± 1.03</td><td>0.000**</td><td>bd > bp > p; d = bd</td></tr><tr><td>20:0</td><td>0.36 ± 0.00</td><td>0.64 ± 0.06</td><td>0.82 ± 0.09</td><td>0.70 ± 0.03</td><td>0.000***</td><td>bp > bd = d > p</td></tr><tr><td>22:0</td><td>0.29 ± 0.02</td><td>0.65 ± 0.07</td><td>1.47 ± 0.36</td><td>1.19 ± 0.22</td><td>0.000***</td><td>p < bp; p = d = bd; bp = d = bd</td></tr><tr><td>24:0</td><td>0.19 ± 0.00</td><td>0.76 ± 0.08</td><td>1.08 ± 0.27</td><td>1.16 ± 0.16</td><td>0.000***</td><td>bd = bp = d > p</td></tr><tr><td>Σ SFA</td><td>24.14 ± 0.15</td><td>55.28 ± 2.46</td><td>38.68 ± 1.01</td><td>44.71 ± 2.05</td><td>0.000***</td><td>d > bd > bp > p</td></tr><tr><td><strong>MUFAs</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>16:1n5</td><td>0.03 ± 0.03</td><td>0.02 ± 0.02</td><td>0.36 ± 0.19</td><td>0.39 ± 0.23</td><td>0.044</td><td>bp > p = d; bp = bd</td></tr><tr><td>16:1n7</td><td>3.68 ± 0.04</td><td>3.21 ± 0.13</td><td>8.16 ± 2.14</td><td>5.83 ± 1.65</td><td>0.001**</td><td>bp = bd > d; d = p; bp > p; bd = p</td></tr><tr><td>18:1n5</td><td>0.16 ± 0.00</td><td>0.06 ± 0.01</td><td>0.58 ± 0.06</td><td>0.41 ± 0.23</td><td>0.000***</td><td>bp > p = d = bd</td></tr><tr><td>18:1n7</td><td>3.31 ± 0.09</td><td>4.73 ± 0.09</td><td>18.99 ± 5.55</td><td>8.01 ± 4.27</td><td>0.000***</td><td>p > all; bd > bp; d = bp; d = bd</td></tr><tr><td>18:1n9</td><td>30.67 ± 0.03</td><td>9.59 ± 0.71</td><td>8.30 ± 0.87</td><td>10.67 ± 1.64</td><td>0.000***</td><td>bp < p = d = bd</td></tr><tr><td>20:1n9</td><td>2.30 ± 0.01</td><td>2.38 ± 0.12</td><td>0.73 ± 0.31</td><td>2.42 ± 0.33</td><td>0.000***</td><td>bd > bp; bd = p = d; bp = p = d</td></tr><tr><td>22:1n11</td><td>1.22 ± 0.01</td><td>2.30 ± 0.06</td><td>0.87 ± 0.41</td><td>3.06 ± 0.45</td><td>0.003**</td><td>p = bp; d = bd; d > p; bd > bp</td></tr><tr><td>24:1</td><td>0.51 ± 0.01</td><td>1.88 ± 0.06</td><td>1.02 ± 0.23</td><td>2.39 ± 0.28</td><td>0.000***</td><td>p = bp > bd > d</td></tr><tr><td>Σ MUFAs</td><td>41.89 ± 0.09</td><td>24.16 ± 0.84</td><td>39.01 ± 2.72</td><td>33.19 ± 3.98</td><td>0.000***</td><td>p > bd > bp > d</td></tr><tr><td><strong>n-6 PUFAs</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>18:2n6</td><td>14.93 ± 0.01</td><td>4.15 ± 0.21</td><td>3.96 ± 0.33</td><td>5.04 ± 0.23</td><td>0.000***</td><td>p > bd > bp > d</td></tr><tr><td>20:4n6</td><td>0.85 ± 0.00</td><td>0.41 ± 0.12</td><td>0.68 ± 0.33</td><td>0.80 ± 0.24</td><td>0.440</td><td>p > all; d = bp; bd > d; bd > bp</td></tr><tr><td>Σ n-6 PUFAs</td><td>15.78 ± 0.02</td><td>4.56 ± 0.34</td><td>4.64 ± 0.24</td><td>5.84 ± 0.17</td><td>0.000***</td><td>p > all; d = bp; bd > d; bd > bp</td></tr><tr><td><strong>n-3 PUFAs</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>18:3n3</td><td>2.36 ± 0.01</td><td>0.48 ± 0.02</td><td>0.32 ± 0.25</td><td>0.37 ± 0.12</td><td>0.000***</td><td>p > d = bp = bd</td></tr><tr><td>20:5n3</td><td>3.68 ± 0.00</td><td>2.28 ± 0.15</td><td>1.87 ± 0.59</td><td>3.11 ± 0.90</td><td>0.002**</td><td>p > bp; p = d = bd; bp = d = bd</td></tr><tr><td>22:5n3</td><td>0.94 ± 0.00</td><td>0.46 ± 0.01</td><td>0.21 ± 0.03</td><td>0.48 ± 0.08</td><td>0.000**</td><td>p > all; bd > bp; bd = d</td></tr><tr><td>22:6n3</td><td>6.73 ± 0.03</td><td>3.76 ± 0.18</td><td>3.92 ± 1.12</td><td>5.54 ± 1.66</td><td>0.010*</td><td>p > d = bp; d = bp = bd; p = bd</td></tr><tr><td>Σ n-3 PUFAs</td><td>13.72 ± 0.04</td><td>6.99 ± 0.37</td><td>6.31 ± 1.73</td><td>9.50 ± 2.63</td><td>0.000***</td><td>p > all; bd > bp; d = bd; d = bp</td></tr><tr><td>Σ PUFAs</td><td>29.49 ± 0.03</td><td>11.56 ± 0.70</td><td>10.95 ± 1.87</td><td>15.34 ± 2.77</td><td>0.000***</td><td>p > all; bd > bp; d = bd; d = bp</td></tr></tbody></table>

p = pellet-based diet; d = detritus-based diet; bp = pellet-based biofloc; bd = detritus-based biofloc; SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acid.

<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Principal component analysis (PCA) plot based on FA composition (in % of FA) of experimental diets (i.e. pellets or detritus) and biofloc sampled from the experimental tanks. Only major FA are shown. 16:0 = PA, 18:1n7 = cis-VA, 18:1n9 = OA, 18:2n6 = LA. D = detritus; P = pellets; BD = detritus-based biofloc; BP = pellet-based biofloc.</figcaption></figure>

The detritus-based biofloc presented a higher TL content and a more nutritious FA profile than the detritus-based diet, promoted by the presence of the microorganism assemblage and the amphipods. Moreover, this biofloc showed higher percentages of PA, OA and LA than the pellet-based biofloc. This is probably due to the higher mortality rate in

this treatment, with fewer individuals being sampled for biochemical analyses and further enriching the biofloc. It is important to note that these are relative results and that absolute values of these FA were similar in both treatments (data not shown), since the pellet-based biofloc showed a significantly higher amount of TL. However, the
<caption>**Table 3**</caption>

Fatty acids composition (mean % ± SD) of *G. insensibilis* from salt marshes and those used in the experiment, fed with fish pellets or detritus.

<table><thead><tr><th>Fatty acid</th><th>wild</th><th>pellets</th><th>detritus</th><th>P value</th><th>Tukey</th></tr></thead><tbody><tr><td><strong>SFA</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>14:0</td><td>1.46 ±<br>0.12</td><td>0.75 ±<br>0.15</td><td>0.63 ±<br>0.17</td><td>0.000***</td><td>w > p = d</td></tr><tr><td>15:0</td><td>0.32 ±<br>0.27</td><td>0.41 ±<br>0.02</td><td>0.47 ±<br>0.02</td><td>0.538</td><td></td></tr><tr><td>16:0</td><td>20.40 ±<br>0.14</td><td>16.44 ±<br>0.37</td><td>15.47 ±<br>0.26</td><td>0.000***</td><td>w > p > d</td></tr><tr><td>16:0i</td><td>0.18 ±<br>0.03</td><td>0.90 ±<br>0.03</td><td>0.79 ±<br>0.39</td><td>0.057</td><td></td></tr><tr><td>17:0</td><td>0.12 ±<br>0.01</td><td>1.03 ±<br>0.04</td><td>1.22 ±<br>0.05</td><td>0.000***</td><td>w < p < d</td></tr><tr><td>18:0</td><td>3.46 ±<br>0.12</td><td>3.83 ±<br>0.15</td><td>4.39 ±<br>0.15</td><td>0.000***</td><td>w < p < d</td></tr><tr><td>22:0</td><td>0.30 ±<br>0.26</td><td>0.37 ±<br>0.16</td><td>0.70 ±<br>0.06</td><td>0.073</td><td></td></tr><tr><td>Σ SFA</td><td>26.23 ±<br>0.51</td><td>23.74 ±<br>0.42</td><td>23.67 ±<br>0.29</td><td>0.000</td><td>w > p = d</td></tr><tr><td><strong>MUFAs</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>16:1n7</td><td>5.97 ±<br>0.28</td><td>3.67 ±<br>0.29</td><td>4.16 ±<br>1.42</td><td>0.036*</td><td>w > p = d</td></tr><tr><td>18:1n9</td><td>15.89 ±<br>0.27</td><td>22.17 ±<br>0.85</td><td>16.14 ±<br>0.95</td><td>0.000***</td><td>p > w = d</td></tr><tr><td>18:1n7</td><td>4.30 ±<br>0.16</td><td>6.48 ±<br>1.25</td><td>8.35 ±<br>3.17</td><td>0.115</td><td></td></tr><tr><td>18:1n5</td><td>0.19 ±<br>0.01</td><td>0.65 ±<br>0.28</td><td>0.77 ±<br>0.39</td><td>0.092</td><td></td></tr><tr><td>20:1n9</td><td>0.80 ±<br>0.03</td><td>1.22 ±<br>0.03</td><td>0.79 ±<br>0.06</td><td>0.000***</td><td>p > w = d</td></tr><tr><td>20:1n7</td><td>0.33 ±<br>0.04</td><td>0.52 ±<br>0.11</td><td>0.57 ±<br>0.26</td><td>0.243</td><td></td></tr><tr><td>Σ MUFAs</td><td>27.47 ±<br>0.63</td><td>34.70 ±<br>2.55</td><td>30.77 ±<br>4.40</td><td>0.064</td><td></td></tr><tr><td><strong>n-6 PUFAs</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>18:2n6</td><td>7.43 ±<br>0.09</td><td>6.77 ±<br>0.63</td><td>5.69 ±<br>0.69</td><td>0.021*</td><td>w > d; w = p;<br>p = d</td></tr><tr><td>20:2n6</td><td>0.94 ±<br>0.02</td><td>0.85 ±<br>0.17</td><td>0.84 ±<br>0.18</td><td>0.636</td><td></td></tr><tr><td>20:4n6</td><td>7.59 ±<br>0.12</td><td>4.90 ±<br>0.52</td><td>5.49 ±<br>1.23</td><td>0.013*</td><td>w > p = d</td></tr><tr><td>22:5n6</td><td>0.55 ±<br>0.01</td><td>0.77 ±<br>0.15</td><td>0.64 ±<br>0.08</td><td>0.084</td><td></td></tr><tr><td>Σ n-6<br>PUFAs</td><td>15.51 ±<br>0.22</td><td>13.29 ±<br>1.44</td><td>12.66 ±<br>2.13</td><td>0.040*</td><td>w > d; w = p;<br>p = d</td></tr><tr><td><strong>n-3 PUFAs</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>18:3n3</td><td>0.75 ±<br>0.04</td><td>0.37 ±<br>0.32</td><td>0.19 ±<br>0.34</td><td>0.108</td><td></td></tr><tr><td>20:5n3</td><td>15.25 ±<br>0.28</td><td>11.37 ±<br>0.87</td><td>13.43 ±<br>3.34</td><td>0.136</td><td></td></tr><tr><td>22:5n3</td><td>0.97 ±<br>0.03</td><td>0.56 ±<br>0.09</td><td>0.76 ±<br>0.15</td><td>0.008**</td><td>w > p; w = d;<br>p = d</td></tr><tr><td>22:6n3</td><td>4.82 ±<br>0.00</td><td>9.47 ±<br>0.41</td><td>11.81 ±<br>2.17</td><td>0.002**</td><td>w < p = d</td></tr><tr><td>Σ n-3<br>PUFAs</td><td>21.79 ±<br>0.26</td><td>21.77 ±<br>1.21</td><td>26.19 ±<br>4.83</td><td>0.176</td><td></td></tr><tr><td>16:2n4</td><td>0.44 ±<br>0.48</td><td>0.05 ±<br>0.05</td><td>0.39 ±<br>0.07</td><td>0.261</td><td></td></tr><tr><td>16:4n1</td><td>1.08 ±<br>0.47</td><td>0.87 ±<br>0.60</td><td>1.33 ±<br>0.30</td><td>0.526</td><td></td></tr><tr><td>Σ PUFAs</td><td>39.82 ±<br>1.12</td><td>35.98 ±<br>2.66</td><td>40.57 ±<br>6.36</td><td>0.386</td><td></td></tr></tbody></table>

w = wild; p = pellets; d = detritus; SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acid.

pellet-based biofloc did not present a better profile than the pellet-based diet, highlighting the nutritional value of the diet, but also pointing the interest of valorising aquaculture wastes in BFT systems (Das et al., 2023; Kala et al., 2023). Furthermore, the pellet-based biofloc exhibited a significantly higher percentage of isoC₁₆ and cis-VA, commonly

associated with bacteria (Rontani et al., 2003). This is attributed to the significantly higher content of OM in the pellets and pellet-based biofloc, which provides essential nutrients and facilitates the establishment of heterotrophic bacteria and the conversion of the OM into bacterial biomass (Zhukova and Kharlamenko, 1999). This demonstrates the advantage of the pellets treatment in terms of bioflocculation capacity (Faust, 2014), in addition to the improved survival rates. It has been shown that the FA profile of amphipods as well as their survival rate are closely linked to the diet (Ribes-Navarro et al., 2022). Thus, the integration in BFT systems of aquaculture waste detritus with a supplementation of commercial aquafeed could potentially enhance the overall nutritional profile while enhancing the bioflocculation process for the bioremediation of the aquaculture effluents (Castilla-Gavilán et al., 2023). Moreover, it will be suitable to carry out the experiment under light conditions and with the addition of diatoms or chlorophytes, which have been highlighted by other authors to improve the LC-PUFAs and EAA profiles (Khatoon et al., 2009; Martins et al., 2016), and water quality (Brito et al., 2016; de Andrade et al., 2021).

The interest in integrated BFT systems (a combination of integrated multi-trophic aquaculture, IMTA, and BFT) has increased in recent years (Borges et al., 2020; Carvalho et al., 2023; Legarda et al., 2021; Lima et al., 2021). In these systems, the residues of one fed species serve as a source of OM for bioflocs, which can be enriched with a second species, which in turn is fed to a third species. This approach aims to minimize the production of waste and the use of aquafeed. The present study shows that the effluent of a fish RAS can be valorised to feed the medium of a BFT system enriched with amphipods. Both bioflocs and amphipods could be further used as food, as their fatty acids profile would satisfy the requirements of various species of interest in aquaculture as are salmonids (Carr et al., 2023), marine crustaceans (González-Félix et al., 2002), sea bream (Ibeas et al., 1996) or turbot (Castell et al., 1994). The use of bioflocs to feed fish has been demonstrated to be an effective approach, as evidenced by the findings of several studies (Borges et al., 2020; Holanda et al., 2023; Legarda et al., 2021; Pinho et al., 2021; Poersch et al., 2021; Silva et al., 2022). Furthermore, amphipods have also been successfully employed as a dietary source for fish (Moren et al., 2006; Suontama et al., 2007) and cephalopods (Baeza-Rojano et al., 2010; Baeza-Rojano et al., 2013). Finally, the present study supports the growing interest in the use of amphipods in aquaculture, with a particular focus on BFT systems. It aims to promote further research in this topic, given that to date, BFT systems have been predominantly studied in mono-specific or multi-specific decapod crustaceans aquaculture (Bajracharya et al., 2024; Ekasari et al., 2014; Emerenciano et al., 2021; Galasso et al., 2024; Huang et al., 2023; Lima et al., 2021; Nguyen et al., 2024; Pérez de Jesús et al., 2024; Pérez-Velasco et al., 2023; Pimentel et al., 2024; Qiu et al., 2023; Reis et al., 2023). Few studies have been conducted on echinoderms (Chen et al., 2018a, 2018b) and only a single study has been carried out on amphipod crustaceans (Promthale et al., 2021).

## 5. Conclusion

This study demonstrates the potential of using *G. insensibilis* in BFT systems, focusing on its survival and nutritional profile, when fed with commercial aquafeed or detritus from aquaculture effluents. The findings reveal that *G. insensibilis* can be effectively cultivated in BFT systems, leveraging its ability to feed on detritus, which promotes bioflocculation and enhances the nutrient profile of the biofloc. The presence of bioflocs enriched with amphipods significantly improves the overall fatty acid composition, particularly in essential LC-PUFAs such as EPA, DHA, and ARA. This enrichment addresses the inherent nutritional deficiencies of bioflocs, making them more suitable as food for high-value aquaculture species. The study highlights the dual benefits of this approach: enhancing the nutritional value of bioflocs while contributing to the bioremediation of aquaculture effluents. Furthermore, the use of close aquaculture systems reduces costs and maintain
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Principal component analysis (PCA) plot based on FA composition (in % of FA) of amphipods from the wild or fed with experimental diets (i.e. pellets or detritus). Only major FA are shown. 16:0 = PA, 20:4n6 = ARA, 20:5n3 = EPA, 18:1n9 = OA, 22:6n3 = DHA, 18:1n7 = cis-VA. D = detritus; P = pellets; W = wild. Three replicates were analysed, both in the wild and in each treatment (3 tanks per treatment). Each replicate consisted on a pool of amphipods.</figcaption></figure>

isolation from natural environment. The integration of aquaculture detritus with supplemental aquafeed in BFT systems will support both the bioflocculation process and the nutritional needs of the amphipods, thereby creating a sustainable and efficient cycle of waste valorisation and food production.

The use of *G. insensibilis* in BFT systems represents a promising strategy for improving the sustainability and productivity of aquaculture operations. By optimizing the use of organic waste and producing nutritionally rich bioflocs, this approach can significantly contribute to the development of a circular economy in aquaculture, reducing environmental impacts and enhancing the growth and health of cultivated species. Further research is needed in order to study the complete life cycle of amphipods and their reproductive capacity in these systems. A lower water temperature and the inoculation of microalgae should be considered in order to achieve higher survival rates and PUFAs content.

### CRediT authorship contribution statement

**Marta Castilla-Gavilán:** Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.
**José Manuel Guerra-García:** Validation, Supervision, Methodology, Data curation.
**Ismael Hachero-Cruzado:** Validation, Supervision, Methodology, Data curation, Conceptualization.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Data availability

Data will be made available on request.

### Acknowledgements

Authors wish to thank the European Maritime and Fisheries Fund (EMFF) for the funding during this research included in the project "Acuicultura y circularidad: valorización de residuos de la acuicultura mediante organismos de bajo nivel trófico (ACUICIRC)" under agreement EI.FEM.PPA202200.003. During this study MCG was supported by a "Margarita Salas" post-doctoral grant from the Spanish Ministry of Science, Innovation and Universities funded by the European Union – NextGeneration EU program. Thank you to Melissa Sant' Anna da Silva for her contribution during her master's internship.

### References

Abbaszadeh, A., Mozanzadeh, M.T., Qasemi, A., Oujifard, A., Nafisi Bahabadi, M., 2022. Effects of the addition of *Calanopia elliptica*, *Artemia franciscana*, and *Brachionus rotundiformis* in a nursery biofloc system on water quality, growth, gut morphology, health indices, and transcriptional response of immune and antioxidant-related genes in *Penaeus vannamei*. Aquac. Int. 30, 653–676. https://doi.org/10.1007/s10499-021-00823-1.

Aminot, A., Kirkwood, D.S., Kérouel, R., 1997. Determination of ammonia in seawater by the indophenol-blue method: evaluation of the ICES NUTS I/C 5 questionnaire. Mar. Chem. 56, 59–75. https://doi.org/10.1016/S0304-4203(96)00080-1.

Anand, P.S.S., Kohli, M.P.S., Kumar, S., Sundaray, J.K., Roy, S.D., Venkateshwarlu, G., Sinha, A., Pailan, G.H., 2014. Effect of dietary supplementation of biofloc on growth performance and digestive enzyme activities in *Penaeus monodon*. Aquaculture 418–419, 108–115. https://doi.org/10.1016/j.aquaculture.2013.09.051.

Baeza-Rojano, E., García, S., Garrido, D., Guerra-García, J.M., Domingues, P., 2010. Use of amphipods as alternative prey to culture cuttlefish (*Sepia officinalis*) hatchlings. Aquaculture 300, 243–246.

Baeza-Rojano, E., Domingues, P., Guerra-García, J.M., Capella, S., Noreña-Barroso, E., Caamal-Monsreal, C., Rosas, C., 2013. Marine gammarids (Crustacea: Amphipoda): a new live prey to culture *Octopus maya* hatchlings. Aquacult. Res. 44, 1602–1612.

Baeza-Rojano, E., Hachero-Cruzado, I., Guerra-García, J.M., 2014. Nutritional analysis of freshwater and marine amphipods from the strait of Gibraltar and potential aquaculture applications. J. Sea Res. 85, 29–36.

Bajracharya, S., Fisk, J.C., Fleckenstein, L.J., Ray, A.J., 2024. Salt type, sugar addition, and system type in intensive RAS for Pacific white shrimp (*Litopenaeus vannamei*) production. Aquaculture 586, 740755.

Borges, B.A.A., Rocha, J.L., Pinto, P.H.O., Zacheu, T., Chede, A.C., Magnotti, C.C.F., Cerqueira, V.R., Arana, L.A.V., 2020. Integrated culture of white shrimp *Litopenaeus*
vannamei and mullet *Mugil liza* on biofloc technology: Zootechnical performance, sludge generation, and Vibrio spp. reduction. Aquaculture 524, 735234. https://doi.org/10.1016/j.aquaculture.2020.07.023.

Brito, L.O., dos Santos, I.G.S., de Abreu, J.L., de Araújo, M.T., Severi, W., Gálvez, A.O., 2016. Effect of the addition of diatoms (*Navicula* spp.) and rotifers (*Brachionus plicatilis*) on water quality and growth of the *Litopenaeus vannamei* postlarvae reared in a biofloc system. Aquacult. Res. 47, 3990-3997.

Carr, I., Glencross, B., Santigosa, E., 2023. The importance of essential fatty acids and their ratios in aquafeeds to enhance salmonid production, welfare, and human health. Front. Anim. Sci. 4, 1140781.

Carvalho A., Costa, L.C.O., Holanda, M., Poersch, L.H., Turan, G., 2023. Influence of Total suspended solids on the growth of the sea lettuce Ulva lactuca integrated with the Pacific white shrimp *Litopenaeus vannamei* in a biofloc system. Fishes 8, 163. https://doi.org/10.3390/fishes8030163.

Castell, J.D., Bell, J.G., Tocher, D.R., Sargent, J.R., 1994. Effects of purified diets containing different combinations of arachidonic and docosahexaenoic acid on survival, growth and fatty acid composition of juvenile turbot (*Scophthalmus maximus*). Aquaculture 128, 315-333.

Castilla-Gavilán, M., Guerra-García, J., Moreno-Oliva, J., Hachero-Cruzado, I., 2023. How much waste can the amphipod *Gammarus insensibilis* remove from aquaculture effluents? A first step toward IMTA. Aquaculture 573, 739552.

Chen, J., Ren, Y., Li, Y., Xia, B., 2018a. Regulation of diet, intestinal microbiota, non-specific immune response and disease resistance of sea cucumber *Apostichopus japonicus* (Selenka) in biofloc systems. Fish Shellfish Immunol. 77, 175-186. https://doi.org/10.1016/j.fsi.2018.03.053.

Chen, J., Ren, Y., Wang, G., Xia, B., Li, Y., 2018b. Dietary supplementation of biofloc influences growth performance, physiological stress, antioxidant status and immune response of juvenile sea cucumber *Apostichopus japonicus* (Selenka). Fish Shellfish Immunol. 72, 143-152.

Christie, W.W., Han, X., 2012. Chapter 4 - analysis of simple lipid classes. In: Christie, W. W., Han, X. (Eds.), Lipid Analysis, Fourth edition. Oily Press Lipid Library Series. Woodhead Publishing, pp. 69-90. https://doi.org/10.1533/9780857097866.69.

Crab, R., 2010. Bioflocs Technology: An Integrated System for the Removal of Nutrients and Simultaneous Production of Feed in Aquaculture. Ghent University.

Crab, R., Chielens, B., Wille, M., Bossier, P., Verstraete, W., 2010a. The effect of different carbon sources on the nutritional value of bioflocs, a feed for *Macrobrachium rosenbergii* postlarvae. Aquacult. Res. 41, 559-567.

Crab, R., Lambert, A., Defoirdt, T., Bossier, P., Verstraete, W., 2010b. The application of bioflocs technology to protect brine shrimp (*Artemia franciscana*) from pathogenic Vibrio harveyi. J. Appl. Microbiol. 109, 1643-1649.

Crab, R., Defoirdt, T., Bossier, P., Verstraete, W., 2012. Biofloc technology in aquaculture: beneficial effects and future challenges. Aquaculture 356, 351-356.

Das, S.K., Mandal, A., 2021. Supplementation of biofloc in carp (*Cyprinus carpio* var. Communis) culture as a potential tool of resource management in aquaculture. Aquat. Living Resour. 34, 20. https://doi.org/10.1051/alr/2021019.

Das, S.K., Mondal, B., Sarkar, U.K., Das, B.K., Borah, S., 2023. Understanding and approaches towards circular bio-economy of wastewater reuse in fisheries and aquaculture in India: an overview. Rev. Aquac. 15, 1100-1114. https://doi.org/10.1111/raq.12758.

de Andrade, R.J.V., dos Santos, E.P., de Almeida Costa, G.K., da Silva Campos, C.V.F., da Silva, S.M.B.C., Gálvez, A.O., Brito, L.O., 2021. Effect of different frequencies of the addition of *Brachionus plicatilis* on the performance of *Litopenaeus vannamei* in a nursery biofloc system with rice bran (anaerobic and aerobic) as an organic carbon source. Aquaculture 540, 73666-73666.

Dinda, R., Mandal, A., Das, S.K., 2020. Neem (*Azadirachta indica* a. Juss.) supplemented biofloc medium as alternative feed in common carp (*Cyprinus carpio* var. communis Linnaeus) culture. J. Appl. Aquac. 32, 361-379. https://doi.org/10.1080/10454438.2019.1645076.

Ebeling, J.M., Timmons, M.B., Bisogni, J., 2006. Engineering analysis of the stoichiometry of photoautotrophic, autotrophic, and heterotrophic removal of ammonia-nitrogen in aquaculture systems. Aquaculture 257, 346-358.

Ekasari, J., Angela, D., Waluyo, S.H., Bachtiar, T., Surawidjaja, E.H., Bossier, P., De Schryver, P., 2014. The size of biofloc determines the nutritional composition and the nitrogen recovery by aquaculture animals. Aquaculture 426, 105-111.

El-Sayed, A.M., 2021. Use of biofloc technology in shrimp aquaculture: a comprehensive review, with emphasis on the last decade. Rev. Aquac. 13, 676-705.

Emerenciano, M.G., Miranda-Baeza, A., Martínez-Porchas, M., Poli, M.A., Vieira, F., 2021. Biofloc technology (BFT) in shrimp farming: past and present shaping the future. Front. Mar. Sci. 8, 813091.

Faust, L., 2014. Bioflocculation of Wastewater Organic Matter at Short Retention Times. Wageningen University and Research.

Folch, J., Lees, M., Sloane Stanley, G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497-509.

Galasso, H.L., Owatari, M.S., Arana, L.A.V., Lapa, K.R., 2024. Effects of scaled-down dissolved air flotation system on suspended solids removal from *Penaeus vannamei* culture under biofloc conditions. Aquac. Eng. 104, 102396.

García-Fernández, P., 2022. Estudio de la fase larvaria planctónica del pulpo común, *octopus vulgaris* cuvier. 1797 frente a distintas condiciones de cultivo mediante el empleo de tecnologías ómicas e identificación de genes biomarcadores.

García-Robledo, E., Corzo, A., Papaspyrou, S., 2014. A fast and direct spectrophotometric method for the sequential determination of nitrate and nitrite at low concentrations in small volumes. Mar. Chem. 162, 30-36. https://doi.org/10.1016/j.marchem.2014.03.002.

Gillan, F.T., Sandstrom, M.W., 1985. Microbial lipids from a nearshore sediment from Bowling Green Bay, North Queensland: the fatty acid composition of intact lipid fractions. Org. Geochem. 8, 321-328.

González-Félix, M.L., Gatlin III, D.M., Lawrence, A.L., Perez-Velazquez, M., 2002. Effect of dietary phospholipid on essential fatty acid requirements and tissue lipid composition of *Litopenaeus vannamei* juveniles. Aquaculture 207, 151-167.

Guerra-García, J.M., Hachero-Cruzado, I., González-Romero, P., Jiménez-Prada, P., Cassel, C., Ros, M., 2016. Towards integrated multi-trophic aquaculture: lessons from capreolids (Crustaceae: Amphipoda). PloS One 11, e0154776.

Hachero-Cruzado, I., Rodríguez-Rua, A., Román-Padilla, J., Ponce, M., Fernández-Díaz, C., Manchado, M., 2014. Characterization of the genomic responses in early Senegalese sole larvae fed diets with different dietary triacylglycerol and total lipids levels. Comp. Biochem. Physiol. Part D Genomics Proteomics 12, 61-73.

Hargreaves, J.A., 2013. Biofloc Production Systems for Aquaculture. Southern Regional Aquaculture Center Stoneville, MS.

Holanda, M., Ravagnan, E., Lara, G., Santana, G., Furtado, P., Cardozo, A., Wasieleksy Jr., W., Poersch, L.H., 2023. Integrated multitrophic culture of shrimp *Litopenaeus vannamei* and tilapia *Oreochromis niloticus* in biofloc system: a pilot scale study. Front. Mar. Sci. 10, 1060846.

Huang, H.-H., Li, C.-Y., Lei, Y.-J., Zhou, B.-L., Kuang, W.-Q., Zou, W.-S., Yang, P.-H., 2023. Effects of Bacillus strain added as initial indigenous species into the biofloc system rearing *Litopenaeus vannamei* juveniles on biofloc preformation, water quality and shrimp growth. Aquaculture 569, 739375.

Ibeas, C., Cejas, J., Gomez, T., Jerez, S., Lorenzo, A., 1996. Influence of dietary n-3 highly unsaturated fatty acids levels on juvenile gilthead seabream (*Sparus aurata*) growth and tissue fatty acid composition. Aquaculture 142, 221-235.

Jiménez-Prada, P., Hachero-Cruzado, I., Giráldez, I., Fernández-Díaz, C., Vilas, C., Canavate, J.P., Guerra-García, J.M., 2018. Crustacean amphipods from marsh ponds: a nutritious feed resource with potential for application in integrated multi-trophic aquaculture. PeerJ e, 4194.

Jiménez-Prada, P., Hachero-Cruzado, I., Guerra-García, J., 2020. Aquaculture waste as food for amphipods: the case of *Gammarus insensibilis* in marsh ponds from southern Spain. Aquac. Int. 29, 139-153.

Kala, A., Krishnan, S., Rani, A.M., 2023. Valorisation of aquaculture sludge into microbial protein using bioreactor with an optimised nutrients. Biochem. Eng. J. 198, 109014. https://doi.org/10.1016/j.bej.2023.109014.

Khanjani, M.H., Sharifinia, M., 2024. Feeding Nile tilapia with varying levels of biofloc: effect on growth performance, survival rate, digestive and liver enzyme activities, and mucus immunity. Aquac. Int. 32, 8171-8194. https://doi.org/10.1007/s10499-024-01561-w.

Khanjani, M.H., Mohammadi, A., Emerenciano, M.G.C., 2022. Microorganisms in biofloc aquaculture system. Aquac. Rep. 26, 101300. https://doi.org/10.1016/j.agrep.2022.101300.

Khanjani, M.H., Mohammadi, A., Emerenciano, M.G.C., 2024a. Water quality in biofloc technology (BFT): an applied review for an evolving aquaculture. Aquac. Int. https://doi.org/10.1007/s10499-024-01618-w.

Khanjani, M.H., Mozantzadeh, M.T., Sharifinia, M., Emerenciano, M.G.C., 2024b. Broodstock and seed production in biofloc technology (BFT): an updated review focused on fish and penaeid shrimp. Aquaculture 579, 740278. https://doi.org/10.1016/j.aquaculture.2023.740278.

Khanjani, M.H., Sharifinia, M., Emerenciano, M.G.C., 2024c. Biofloc technology (BFT) in aquaculture: what goes right, what goes wrong? A scientific-based snapshot. Aquac. Nutr. e7496572. https://doi.org/10.1155/2024/7496572.

Khatoon, H., Banerjee, S., Yusoff, F.M., Shariff, M., 2009. Evaluation of indigenous marine periphytic Amphora, *Navicula* and *Cymbella* grown on substrate as feed supplement in *Penaeus monodon* postlarval hatchery system. Aquacult. Nutr. 15, 186-193. https://doi.org/10.1111/j.1365-2095.2008.00582.x.

Knowler, D., Chopin, T., Martínez-Espíneira, R., Neori, A., Nobre, A., Noce, A., Reid, G., 2020. The economics of integrated multi-trophic aquaculture: where are we now and where do we need to go? Rev. Aquac. 12, 1579-1594. https://doi.org/10.1111/raq.12399.

Legarda, E.C., da Silva, D., Miranda, C.S., Pereira, P.K.M., Martins, M.A., Machado, C., de Lorenzo, M.A., Hayashi, L., Do Nascimento Vieira, F., 2021. Sea lettuce integrated with Pacific white shrimp and mullet cultivation in biofloc impact system performance and the sea lettuce nutritional composition. Aquaculture 534, 736265. https://doi.org/10.1016/j.aquaculture.2020.736265.

Lima, P.C.M., Silva, A.E.M., Silva, D.A., Silva, S.M.B.C., Brito, L.O., Gálvez, A.O., 2021. Effect of stocking density of Crassostrea sp. in a multitrophic biofloc system with *Litopenaeus vannamei* in nursery. Aquaculture 530, 735913. https://doi.org/10.1016/j.aquaculture.2020.735913.

Martins, T.G., Odebrecht, C., Jensen, L.V., D'Oca, M.G., Wasieleksy Jr., W., 2016. The contribution of diatoms to bioflocs lipid content and the performance of juvenile *Litopenaeus vannamei* (Boone, 1931) in a BFT culture system. Aquacult. Res. 47, 1315-1326.

McAusker, S., Warberg, M.B., Davies, S.J., Valente, C.S., Johnson, M.P., Cooney, R., Wan, A.H.L., 2023. Biofloc technology as part of a sustainable aquaculture system: a review on the status and innovations for its expansion. Aquac. Fish Fish. 3, 331-352. https://doi.org/10.1002/aff2.1028.

Moren, M., Suontama, J., Hemre, G.-I., Karlsen, Ø., Olsen, R., Mundheim, H., Julshamn, K., 2006. Element concentrations in meals from krill and amphipods,— possible alternative protein sources in complete diets for farmed fish. Aquaculture 261, 174-181.

Nguyen, T.T.T., Foysal, M.J., Gupta, S.K., Tay, A., Fotedar, R., Gagnon, M.M., 2024. Effects of carbon source addition in rearing water on sediment characteristics, growth and health of cultured marron (*Cherax cainii*). Sci. Rep. 14, 1349.

Pérez de Jesús, D., Hernández-Vergara, M.P., Pérez-Rostro, C.I., Frías-Quintana, C.A., 2024. Effect of fasting on compensatory growth and digestive enzymatic activity of freshwater prawn post larvae (*Macrobrachium rosenbergii*) during its culture in biofloc. Aquac. Int. 32, 119-135.
Pérez-Velasco, R., Hernández-Vergara, M.P., Pérez-Rostro, C.I., Frías-Quintana, C.A., 2023. Variation of dietary protein/lipid levels used in postlarvae of freshwater prawn *Macrobrachium rosenbergii* cultured in a biofloc system. Lat. Am. J. Aquat. Res. 51, 12-22.

Pimentel, O.A.L.F., Wasielesky Jr., W., da Silva, N.P., Do Valle Borges, L., Krummenauer, D., 2024. Fertilizing synbiotic system with different vegetable brands: effects on nitrification, plankton composition, and growth of *Penaeus vannamei* in the nursery phase. Aquac. Int. 1-23.

Pinho, S.M., David, L.H.C., Goddek, S., Emerenciano, M.G.C., Portella, M.C., 2021. Integrated production of Nile tilapia juveniles and lettuce using biofloc technology. Aquac. Int. 29, 37-56. https://doi.org/10.1007/s10499-020-00608-y.

Poersch, L., Brunson, J., Gaona, C.A.P., Stokes, A., Richardson, J., Pitts, K., Leffler, J., 2021. Pacific white shrimp, red drum, and tilapia integrated in a biofloc system: use of tilapia as a consumer of total suspended solids. J. World Aquac. Soc. 52, 1168-1177. https://doi.org/10.1111/jwas.12832.

Promthale, P., Withyachumnarnkul, B., Bossier, P., Wongprasert, K., 2021. Nutritional value of the amphipod *Bemlos quadrimanus* sp. grown in shrimp biofloc ponds as influenced by different carbon sources. Aquaculture 533, 736128. https://doi.org/10.1016/j.aquaculture.2020.736128.

Qiu, Z., Xu, Q., Li, S., Zheng, D., Zhang, R., Zhao, J., Wang, T., 2023. Effects of probiotics on the water quality, growth performance, immunity, digestion, and intestinal flora of giant freshwater prawn (*Macrobrachium rosenbergii*) in the biofloc culture system. Water 15, 1211.

Reis, W.G., Wasielesky Jr., W., Abreu, P.C., Brandão, H., Krummenauer, D., 2023. The influence of different light wavelengths in the culture of the Pacific white shrimp *Litopenaeus vannamei* reared in BFT using LED lights. Aquaculture 563, 738924.

Ribes-Navarro, A., Navarro, J.C., Hontoria, F., Kabeya, N., Standal, I.B., Evjemo, J.O., Monroig, Ò., 2021. Biosynthesis of long-chain polyunsaturated fatty acids in marine Gammarids: molecular cloning and functional characterisation of three fatty acyl Elongases. Mar. Drugs 19, 226. https://doi.org/10.3390/md19040226.

Ribes-Navarro, A., Alberts-Hubatsch, H., Monroig, Ò., Hontoria, F., Navarro, J.C., 2022. Effects of diet and temperature on the fatty acid composition of the gammarid *Gammarus locusta* fed alternative terrestrial feeds. Front. Mar. Sci. 9. https://doi.org/10.3389/fmars.2022.931991.

Rontani, J.-F., Koblížek, M., Beker, B., Bonin, P., Kolber, Z.S., 2003. On the origin of cis-vaccenic acid photodegradation products in the marine environment. Lipids 38, 1085-1092. https://doi.org/10.1007/s11745-006-1164-z.

Silva, V.F., Pereira, P.K.M., Martins, M.A., Lorenzo, M.A.D., Cella, H., Lopes, R.G., Derner, R.B., Magallón-Servín, P., Vieira, F.D.N., 2022. Effects of microalgae addition and fish feed supplementation in the integrated rearing of Pacific white shrimp and Nile Tilapia using biofloc technology. Animals 12, 1527. https://doi.org/10.3390/ani12121527.

Suontama, J., Kiessling, A., Melle, W., Waagbo, R., Olsen, R., 2007. Protein from northern krill (*Thysanoessa inermis*), Antarctic krill (*Euphausia superba*) and the Arctic amphipod (* Themisto libellula*) can partially replace fish meal in diets to Atlantic salmon (*Salmo salar*) without affecting product quality. Aquacult. Nutr. 13, 50-58.

Tanaka, K., Ishihara, A., Nakajima, H., 2014. Isolation of anteiso-C17, iso-C17, iso-C16, and iso-C15 Bacillomycin D from Bacillus amyloliquefaciens SD-32 and Their Antifungal Activities against Plant Pathogens. J. Agric. Food Chem. 62, 1469-1476. https://doi.org/10.1021/jf404531t.

Tur, R., Rodrigues, P., Almansa, E., Lago, M., García, P., Pérez, E., 2020. Procedimiento para el cultivo de paralarvas del pulpo común *Octopus vulgaris*. Pat. ES 2, 930.

Underwood, A., 1997. Experiments in Ecology: Their Logical Design and 977 Interpretation Using Analysis of Variance, ed. Cambridge University Press, Cambridge.

Xu, W.-J., Pan, L.-Q., 2013. Enhancement of immune response and antioxidant status of *Litopenaeus vannamei* juvenile in biofloc-based culture tanks manipulating high C/N ratio of feed input. Aquaculture 412, 117-124.

Zhao, D., Pan, L., Huang, F., Wang, C., Xu, W., 2016. Effects of different carbon sources on bioactive compound production of biofloc, immune response, antioxidant level, and growth performance of *Litopenaeus vannamei* in zero-water exchange culture tanks. J. World Aquac. Soc. 47, 566-576.

Zhukova, N.V., Kharlamenko, V.I., 1999. Sources of essential fatty acids in the marine microbial loop. Aquat. Microb. Ecol. 17, 153-157.

Zimmermann, S., Kiessling, A., Zhang, J., 2023. The future of intensive tilapia production and the circular bioeconomy without effluents: biofloc technology, recirculation aquaculture systems, bio-RAS, partitioned aquaculture systems and integrated multitrophic aquaculture. Rev. Aquac. 15, 22-31. https://doi.org/10.1111/raq.12744.