Recycling biofloc waste as novel protein source for crayfish with special reference to crayfish nutritional standards and growth trajectory

Freshwater crayfish account for a growing share of aquaculture production but remain nutritionally data-deficient compared to other crustaceans. There is interest in replacing fishmeal/oil with alternative proteins, including microbial biomass from biofloc technology (BFT) systems. Bioflocs are generated during intensive aquaculture under controlled C:N ratios and often require periodic removal, producing waste biomass. The study aims to: (a) formulate nutritional optima for freshwater crayfish through meta-analysis, (b) benchmark crayfish growth trajectory and thermal growth coefficient (TGC), (c) evaluate red swamp crayfish Procambarus clarkii responses to biofloc meal (BM) in diets, (d) assess heavy metal bioaccumulation or mineral stress risks, and (e) identify nutritional strengths and bottlenecks of BM for crayfish.

The authors synthesized dispersed literature (27 articles) to derive crayfish dietary standards, often compared with NRC (2011) penaeid shrimp guidelines historically used as proxies. Reported crayfish requirements include crude protein 29–34%, lipid 6.5–9%, ash 7.8–10.8%, non-protein energy:protein ratio 5.3–8.5 cal mg−1, and key digestible EAA requirements (e.g., arginine 2.1–2.7%). Prior work on biofloc/microbial meals in crustacean feeds typically used 10–30% inclusion with good growth; broader inclusion studies in Litopenaeus vannamei showed growth advantages diminishing above ~41–53% BM inclusion. Biofloc is known to vary widely in proximate composition and can be relatively low in arginine. NRC penaeid EAA standards may adequately cover many crayfish EAA needs except arginine.

The study comprised two components. (1) Meta-analyses: Peer-reviewed literature (English, or with English abstract) was searched in Web of Science, Scopus, and Google Scholar using targeted nutrition keywords. Data from 27 articles were extracted to formulate crayfish nutritional standards, describe growth trajectory, and analyze nutritional dependencies on growth and retention (details in supplementary material). (2) Experimental trial: Biofloc biomass was collected from an indoor freshwater BFT system stocked with Nile tilapia (35 kg m−3). The system maintained 25–50 mL L−1 suspended solids via daily vortex separation and sedimentation; biofloc was filtered (60 μm), dried at 80 °C, ground to meal (BM). Diets: A commercial fish feed (TILAPICO) served as basal diet; BM replaced basal feed on a weight basis to create isonitrogenous diets at 0% (control), 33% (BM33), 66% (BM66), and 100% (BM100) inclusion. Pellets (2 mm) were cold extruded, dried (45 °C, 12 h), vacuum sealed, and stored at 4 °C. Proximate composition, EAAs (except tryptophan), and heavy metals/minerals were analyzed by an accredited lab. Animals and rearing: 120 juvenile P. clarkii (stage 3; 7.8 ± 0.7 mg) were stocked (10 per tank) in 12 glass aquaria (46 L) in triplicate per treatment, with shelters provided. Rearing conditions: DO 7.9 ± 0.3 mg L−1, pH 7.6 ± 0.2, temperature 21.8 ± 0.3 °C, natural photoperiod (12L:12D). Feeding: twice daily to apparent satiation (~5–6% BW) for 9 weeks; wastes siphoned daily. Measurements: Body weight every 3 weeks before feeding; survivorship recorded; final body weight and length at trial end. Calculations: FCR, PER, survivability, live weight gain (LWG), coefficient of variation (CV). Statistical analysis: Normality (Shapiro–Wilk), then one-way ANOVA with Tukey HSD or Kruskal–Wallis with Dunn’s test (Bonferroni) at α=0.05; graphics generated in R (ggplot2). Heavy metal risk: Hg, Cd, Zn, Fe, Mn measured in muscle and hepatopancreas at trial end; compared with EU permissible limits (Cd, Hg 0.5 mg kg−1 wet weight) and Czech EPA fertilizer limits for BM.

- Crayfish nutritional standards and growth trajectory: From meta-analyses, crayfish TGC typically ranges 0.07–1.0 (IQR 0.32–0.64); TGC 0.5–0.64 qualifies as reasonably good growth. Calculated dietary standards include CP 29–34%, lipid 6.5–9%, ash 7.8–10.8%, non-protein energy:protein 5.3–8.5 cal mg−1, and digestible EAA requirements (arginine 2.1–2.7% of diet among others). - Growth trial (9 weeks, 21.8 °C): Survivability remained >70% for all groups with no significant differences among treatments. Size heterogeneity (CV) was significantly suppressed in BM100 due to many small runts; diet-driven size heterogeneity negatively correlated with BM inclusion (r = −0.63, p < 0.05). - Growth performance: Control, BM33, and BM66 exhibited higher and statistically similar growth (TGC and BW) than BM100. BM100 significantly depressed growth: terminal TGC approximately half of other diets; LWG in BM100 6 ± 1 mg day−1 vs 39 ± 15 (control), 44 ± 16 (BM33), 26 ± 9 (BM66). - Feed utilization: FCR increased linearly with BM inclusion: FCR = 1.156 + 0.006 × BM% (Adj. R² = 0.95, p < 0.05). PER decreased linearly: PER = 1.922 − 0.006 × BM% (Adj. R² = 0.95, p < 0.05). Thus, every +10% BM raised FCR by ~0.06 and lowered PER by ~0.066. - Diet composition constraints (BM100 vs standards): BM100 had ash 16.4% (>14% physiological threshold), lipid 4.5% (below 6.5–9% standard), non-protein energy:protein 3.7 cal mg−1 (below 5.3–8.5), and arginine 1.2% (below crayfish requirement 2.1–2.7% and penaeid 1.8%). Arginine emerged as the most limiting EAA in BM. - Heavy metals/minerals: BM heavy metal contents were below regulatory limits. No critical biomagnification occurred in crayfish tissues; however, hepatopancreas metal burdens increased with BM inclusion, notably Hg (significant increases with higher BM; BM100 > control). Elevated mineral loads (Fe, Mn, Zn, Cu) in BM100 suggest potential mineral stress. - Optimal BM inclusion: BM at 33–66% can elevate or maintain growth comparable to control, whereas 100% BM led to poor growth without increased mortality.

The study addressed three coupled unknowns—novel feedstuff, crayfish nutritional optima, and growth trajectory—by first establishing standards and then testing BM against them. Growth comparable to control at 33–66% BM indicates biofloc biomass can partially replace conventional feed protein. The growth collapse at 100% BM aligns with identified nutritional mismatches: excessive ash (and associated osmoregulatory/metabolic costs), insufficient non-protein energy relative to protein (leading to protein catabolism), and arginine deficiency. The linear deterioration in FCR and PER with higher BM reflects reduced protein utilization under these constraints. Heavy metal analyses show consumer safety is not compromised, yet tissue burdens (especially Hg in hepatopancreas) and high mineral intake may impair metabolism and growth. The EAA comparison suggests penaeid NRC standards are broadly applicable to crayfish except for higher arginine needs. Therefore, BM’s feasibility depends on inclusion level and nutritional balancing to meet crayfish-specific requirements.

The study provides updated crayfish nutritional standards and benchmarks a TGC range (0.5–0.64) indicative of good growth. Using these references, biofloc meal was evaluated as a protein source for Procambarus clarkii. Partial replacement (33–66% BM) maintained or improved growth relative to control, but full replacement (100% BM) significantly reduced growth without affecting survival. Growth limitations at high BM inclusion were attributed to high ash and mineral loads, low non-protein energy:protein ratio, and arginine deficiency. Heavy metal concentrations remained below critical limits, though hepatopancreas burdens increased with BM inclusion. Future work should focus on optimizing BM-based diets by correcting arginine shortfalls, increasing non-protein energy supply, and reducing ash/mineral burdens of BM, as well as validating long-term safety and performance across crayfish species and production conditions.

- Nutritional standards and growth-dependency models rely on meta-analyses of heterogeneous studies and species; applicability may vary by species, life stage, and conditions. - Experimental evaluation used a single crayfish species (P. clarkii), one BM source and processing method, and indoor conditions at a single temperature regime. - Tryptophan was not analyzed due to analytical error, limiting complete EAA assessment. - Potential mineral stress thresholds for crayfish are not well established; interpretations extrapolate from shrimp literature and modeled retention. - Sample size per treatment (n=3 tanks, 10 animals each) and observed size hierarchies may influence variability in growth outcomes.