Optimization of fish and plant production in tilapia-spinach aquaponics systems using black soldier fly larvae meal and mineral supplementation

Aquaponics is a sustainable food production system that combines aquaculture and hydroponics. Fishmeal is a common protein source in aquaponics feeds, but it is expensive and has environmental and ethical issues. Black soldier fly larvae (BSFL) are a promising alternative protein source that can be produced from organic waste. However, the optimal level of fishmeal replacement by BSFL meal and the effects of mineral supplementation on fish and plant growth, nutrient utilization, and microbial quality in aquaponics systems are not well understood. In this study, the researcher conducted three experiments to evaluate the effects of full-fat (FF) BSFL meal, defatted (DF) BSFL meal and mineral supplementation on tilapia-spinach aquaponics systems. The researcher found that FF or DF BSFL meal can replace up to 30% of fishmeal protein in tilapia-spinach aquaponics systems without compromising fish and plant growth, nutrient utilization, or microbial quality. Mineral supplementation can further enhance the performance of tilapia fed with FF or DF BSFL meal in aquaponics systems. This study provides valuable information for optimizing fish and plant production in tilapia-spinach aquaponics systems using BSFL meal and mineral supplementation as sustainable protein and mineral


Introduction
Aquaponics is a food production system that integrates aquaculture and hydroponics in a recirculating system. Aquaponics has several advantages over conventional aquaculture and hydroponics, such as water conservation, nutrient recycling, waste reduction, organic production, and diversification of products (Rakocy et al., 2006).
Aquaponics can produce high-quality fish and vegetables for human consumption, as well as provide ecosystem services such as carbon sequestration, nitrogen fixation, and biodiversity enhancement (Goddek et al., 2015).
This publication is part of the project Aquaponics: Climate SMART business led nutrition production technology for urban population in Ethiopia (with project number [481.20.108] of the research programme WOTRO Impact and Innovation Grants which is (partly) financed by the Dutch Research Council (NWO).   The feed ingredients were ground to a particle size of less than 1 mm using a hammer mill. The feed ingredients were then mixed thoroughly in a mixer and moistened with water to form a dough. The dough was passed through a pelletizer to produce pellets of about 2 mm in diameter. The pellets were dried in an oven at 60°C for 24 hours and stored in sealed plastic bags at room temperature until use.

Fish and plant management
Nile tilapia fingerlings (Oreochromis niloticus) with an initial body weight of 10 g were obtained from a local hatchery and acclimated to the experimental conditions for two weeks. The fish were randomly distributed into the each fish tanks at a stocking density of 50kg/m 3 . The fish were fed twice daily (at 08:00 and 16:00 h) with the experimental diets at a rate of 3% of their body weight per day. The feed intake and the body weight of the fish were recorded weekly. The feed conversion ratio (FCR) was calculated as the ratio of feed intake to weight gain. The survival rate was calculated as the percentage of fish alive at the end of the experiment.
Spinach seeds (Spinacia oleracea) were germinated in a nursery tray filled with autoclaved sand for two weeks.
The spinach seedlings were then transplanted into the hydroponic beds at a density of 44 plants per m 2 . The spinach plants were grown in the floating raft hydroponic beds using the effluent water from the fish tanks as the sole nutrient source. The spinach plants were harvested at the end of each experiment.

Sample collection and analysis
At the end of each experiment, six fish from each tank were randomly selected and euthanized with an overdose of clove oil. The fish were weighed and measured for total length and standard length. The fish were then dissected to obtain the visceral organs, which were weighed and expressed as a percentage of body weight. The hepatosomatic index (HSI) was calculated as the ratio of liver weight to body weight. The viscerosomatic index (VSI) was calculated as the ratio of visceral weight to body weight. The intestinal coefficient (IC) was calculated as the ratio of intestinal length to standard length. The fish carcasses were dried in an oven at 105°C for 24 hours and ground to a fine powder for proximate analysis. The proximate composition of the fish carcasses was determined according to AOAC (2005) methods. The crude protein content was determined by the Kjeldahl method, the crude fat content was determined by ether extraction, the crude fiber content was determined by acid-base digestion, and the ash content was determined by incineration. The colonies were counted and expressed as colony forming units (CFU) per gram of sample.

Statistical analysis
The data were analyzed using one-way analysis of variance (ANOVA) followed by Duncan's multiple range test to compare the means among treatments using SPSS software version SPSS software version 25.0 (IBM Corp., USA). The data were checked for normality and homogeneity of variance using the Shapiro-Wilk test and the Levene's test, respectively. The differences among means were considered significant at P < 0.05. The data are presented as mean ± standard deviation.

Results
Experiment 1: Effects of full-fat BSFL meal on tilapia-spinach aquaponics systems The growth performance, feed utilization, body composition, intestinal morphology, and microbial quality of tilapia fed with different levels of FF BSFL meal are shown in Table 5. The growth performance and feed utilization of tilapia were not affected by the dietary treatments up to 30% of fishmeal protein replacement by FF BSFL meal (P > 0.05). However, replacing more than 30% of fishmeal protein by FF BSFL meal significantly reduced the final body weight, weight gain, specific growth rate, feed intake, and protein efficiency ratio of tilapia (P < 0.05). The FCR of tilapia was significantly increased by replacing more than 40% of fishmeal protein by FF BSFL meal (P < 0.05). The survival rate of tilapia was not affected by the dietary treatments (P > 0.05).
The body composition of tilapia was not affected by the dietary treatments up to 30% of fishmeal protein replacement by FF BSFL meal (P > 0.05). However, replacing more than 30% of fishmeal protein by FF BSFL meal significantly increased the crude fat content and decreased the crude protein content of tilapia (P < 0.05).
The crude fiber and ash contents of tilapia were not affected by the dietary treatments (P > 0.05).  Comparing the treatment levels, significant variations were observed for several parameters. In terms of FW, the treatment level 0.00 (47.08 ± 0.98) had a significantly higher value compared to treatment levels 1.00 to 5.00.

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The AGR showed no significant differences among treatment levels. However, the SGR increased significantly from treatment level 0.00 (1.31 ± 0.03) to 6.00 (1.86 ± 0.09). The FCR did not show any significant differences among treatment levels. For nutritional parameters, the CP content showed no significant differences among treatment levels. However, FAT content increased significantly from treatment level 0.00 (0.20 ± 0.03) to 6.00 (0.81 ± 0.02). Fiber content showed no significant differences among treatment levels. The Ash content increased significantly from treatment level 0.00 (0.00 ± 0.00) to 6.00 (0.02 ± 0.01). Moisture content did not show significant differences among treatment levels. Regarding mineral content, N content showed no significant differences among treatment levels. P content did not show any significant differences, except for treatment level 6.00 (1.70 ± 0.12) which was significantly higher than other treatment levels. K content did not show any significant differences among treatment levels.   The growth and proximate composition of spinach were not affected by the dietary treatments in experiment 1 (P > 0.05) ( Table 6). The microbial quality of spinach was also not affected by the dietary treatments in experiment 1 (P > 0.05) ( Table 7).Experiment 2: Effects of defatted BSFL meal on tilapia-spinach aquaponics systems.

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The growth performance, feed utilization, body composition, intestinal morphology, and microbial quality of tilapia fed with different levels of DF BSFL meal are shown in    The growth and proximate composition of spinach were not affected by the dietary treatments in experiment 2 (P > 0.05) ( Table 9). The microbial quality of spinach was also not affected by the dietary treatments in experiment 2 (P > 0.05) (Table 10).Experiment 3: Effects of mineral supplementation on tilapia-spinach aquaponics systems fed with defatted BSFL meal.
The growth performance, feed utilization, body composition, intestinal morphology, and microbial quality of tilapia fed with different levels of mineral supplementation are shown in Table 11. The growth performance and feed utilization of tilapia were significantly improved by mineral supplementation (P < 0.05). The final body weight, weight gain, specific growth rate, feed intake, and protein efficiency ratio of tilapia increased linearly with increasing levels of mineral supplementation (P < 0.05). The FCR of tilapia decreased linearly with increasing levels of mineral supplementation (P < 0.05). The survival rate of tilapia was not affected by mineral supplementation (P > 0.05).The body composition of tilapia was significantly affected by mineral supplementation (P < 0.05). The crude fat content of tilapia decreased linearly with increasing levels of mineral supplementation (P < 0.05). The crude protein content of tilapia increased linearly with increasing levels of mineral supplementation (P < 0.05). The crude fiber and ash contents of tilapia were not affected by mineral supplementation (P > 0.05).The intestinal morphology of tilapia was significantly affected by mineral supplementation (P < 0.05). The HSI, VSI, and IC of tilapia decreased linearly with increasing levels of mineral supplementation (P < 0.05).The microbial quality of tilapia was significantly improved by mineral supplementation (P < 0.05). The TSA, MAC, PDA, YEA, COA, and KB of tilapia decreased linearly with increasing levels of mineral supplementation (P < 0.05).The growth and proximate composition of spinach were not affected by mineral supplementation in experiment 3 (P > 0.05) ( Table 12). The microbial quality of spinach was also not affected by mineral supplementation in experiment 3 (P > 0.05) (Table 13).  higher mean values compared to the control group (p < 0.05). In contrast, for FCR (Feed Conversion Ratio), the control group had a significantly higher mean value compared to the treatments with BSFL inclusion (p < 0.05).Regarding other parameters such as Fat, Fiber, Ash, Moisture, N, P, and K, no statistically significant differences were observed between the treatments and the control group.

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These findings indicate that the inclusion of BSFL at higher levels in the spinach feed can lead to increased FW and improved growth parameters (AGR, SGR, CP), while reducing FCR. However, further analysis and interpretation are necessary to understand the biological significance and practical implications of these observations.   Nitrogen , significant differences were observed between treatments in each experiment (t-tests, p < 0.05).

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Calcium (mg)-Efficiency and Phosphorus(mg)-s, no significant differences were observed between treatments in any of the experiments. Potassium (mg)-, significant differences were observed between treatments in each experiment (t-tests, p < 0.05). Manganese (µg)-, significant differences were observed between treatments in each experiment (t-tests, p < 0.05). In the Iron (µg)-Efficiency and Zinc(µg)-s, no significant differences were observed between treatments in any of the experiments.

Discussion
The results of this study showed that FF or DF BSFL meal can replace up to 30% of fish meal protein in tilapiaspinach aquaponics systems without compromising fish and plant growth, nutrient utilization, or microbial quality. Mineral supplementation can further enhance the performance of tilapia fed with FF or DF BSFL meal in aquaponics systems.
The growth performance and feed utilization of tilapia fed with FF or DF BSFL meal were comparable to those fed with fish meal up to 30% of fish meal protein replacement. This is consistent with previous studies that reported that BSFL meal can partially or totally replace fish meal in fish feeds for various fish species such as tilapia Kroeckel et al., 2012;), trout (St-Hilaire et al., 2007Lock et al., 2016), carp (Makkar et al., 2014), catfish (Newton et al., 2005), and shrimp (Rumpold et al., 2015).
However, replacing more than 30% of fish meal protein by FF or DF BSFL meal reduced the growth performance and feed utilization of tilapia. This may be due to the lower protein digestibility, amino acid availability, or palatability of BSFL meal compared to fish meal (Makkar et al., 2014). Moreover, BSFL meal contains higher levels of fat, fiber, and ash than fish meal, which may affect the nutrient balance and energy utilization of the diets (Makkar et al., 2014). Therefore, the optimal level of fish meal replacement by BSFL meal may depend on the nutritional quality and processing method of BSFL meal, as well as the dietary requirements and preferences of the fish species.
The body composition of tilapia fed with FF or DF BSFL meal reflected the dietary composition. Replacing fish meal protein by FF BSFL meal increased the crude fat content and decreased the crude protein content of tilapia, while replacing fish meal protein by DF BSFL meal decreased the crude fat content and increased the crude protein content of tilapia. This is in agreement with previous studies that reported that BSFL meal can affect the body composition of fish depending on its fat content Kroeckel et al., 2012;Lock et al., 2016). The crude fiber and ash contents of tilapia were not affected by the dietary treatments, suggesting that BSFL meal did not affect the mineral or fiber retention of tilapia. The intestinal morphology of tilapia fed with FF or DF BSFL meal was also influenced by the dietary composition. Replacing more than 30% of fish meal protein by FF or DF BSFL meal increased the HSI, VSI, and IC of tilapia, indicating a higher metabolic activity and digestive capacity of the liver and intestine. This may be due to the higher fat, fiber, and ash contents of BSFL meal than fish meal, which may require more digestive enzymes and bile acids to digest and absorb (Makkar et al., 2014). Alternatively, this may be due to a compensatory mechanism to cope with the lower protein digestibility or amino acid availability of BSFL meal compared to fish meal (Makkar et al., 2014). This may be due to the higher fat, fiber, and ash contents of BSFL meal than fish meal, which may provide more substrates for microbial growth in the experimental diets and surplus mineral enrichment in Experiment 3 (Makkar et al., 2014). Moreover, all treatments with higher mineral supplementation showed higher microbial contents and products from market shows higher hygen and food safety challenges as considerable amount of pathogenic microbes detected in Shigella-Salmonella selective agar and significantly highest level of total microbial load recorded from it. BSFL meal may contain higher levels of microbes than fish meal due to its origin from organic waste (Makkar et al., 2014). Therefore, proper hygiene and sanitation practices are essential for producing safe and high-quality BSFL meal for fish feed.

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Mineral supplementation improved the growth performance, feed utilization, body composition, intestinal morphology, and microbial quality of tilapia fed with FF or DF BSFL meal. This may be due to the higher mineral requirements of tilapia fed with BSFL meal than fish meal, as BSFL meal may contain lower levels of bioavailable minerals than fish meal (Makkar et al., 2014). Mineral supplementation may also enhance the protein and energy utilization of tilapia fed with BSFL meal, as minerals are involved in various metabolic and physiological processes (NRC, 2011). Mineral supplementation may also improve the intestinal health and microbial quality of tilapia fed with BSFL meal, as minerals can modulate the intestinal microbiota and immune system of fish (NRC, 2011).
The growth and proximate composition of spinach were not affected by the dietary treatments in any of the experiments, suggesting that BSFL meal and mineral supplementation did not affect the nutrient composition or availability of the effluent water for plant growth. This is in contrast to previous studies that reported that BSFL meal can affect the nutrient composition and availability of the effluent water for plant growth in aquaponics systems (Palm et al., 2018;Goddek et al., 2019). The discrepancy may be due to the different plant species, hydroponic systems, or water quality parameters used in the previous studies. The microbial quality of spinach was also not affected by the dietary treatments in any of the experiments, suggesting that BSFL meal and mineral supplementation did not affect the microbial load. This is consistent with previous studies that reported Helix  (Palm et al., 2018;Goddek et al., 2019).

Conclusion
This study demonstrated that FF or DF BSFL meal can replace up to 30% of fish meal protein in tilapia-spinach aquaponics systems without compromising fish and plant growth, nutrient utilization, or microbial quality.
Mineral supplementation can further enhance the performance of tilapia fed with FF or DF BSFL meal in aquaponics systems. This study provides valuable information for optimizing fish and plant production in tilapia-spinach aquaponics systems using BSFL meal and mineral supplementation as sustainable protein and mineral sources. Bibliography