## 4 Discussion
### Effects of Ex-Situ Biofloc as Feed Supplement to Improve Shrimp Growth Performance  
Interestingly, when supplementing ex-situ biofloc with 5% and 10% commercial pellet reduction, the survival rates and the shrimp body weights were significantly higher than the control, suggesting that ex-situ biofloc might play a role as a feed supplement to enhance shrimp growth performance. From proximate analysis, commercial pellet contained a high amount of macromolecule nutrients (e.g., carbohydrate, fat and protein), while ex-situ biofloc was comprised of the high amount of minerals (e.g., zinc, iron, calcium, magnesium and sodium). In addition, the amounts of fatty acids (stearic acid (18:0), oleic acid (18:1n9c), linoleic acid (18:2n6), linoleic acid (18:3n3), arachidonic acid (20:4n6), eicosapentaenoic acid (20:5n3; EPA) and DHA (22:6n3)) from the commercial pellet were significantly higher than those from the ex-situ biofloc. These fatty acids are essential nutrients for growth and development in animals and humans [36; 37; 38] and also play important roles in shrimp nutrition and shrimp growth and reproduction [39]. For amino acids, overall detectable amino acids (nine essential amino acids and ten non-essential amino acids) in commercial pellet were significantly higher than those in ex-situ biofloc. Proline, glutamic acid and glycine were predominant in the commercial pellet. They are involved in collagen synthesis for strengthening connective tissues such as bone, skin, cartilage and blood vessel in mammals, livestock and aquatic animals [40], maintaining digestive function and protecting the integrity of the intestinal mucosa of human [41]. This finding suggests that macromolecule nutrients from commercial pellet were the major source of nutrients feeding to shrimp; however, feed utilization efficiency may be improved by minerals obtained from ex-situ biofloc. According to the following supported evidence, the microbial community that mainly contributes to the water quality control process was possibly from microalgae. Microalgae biomass is a well-known source of essential vitamins and minerals (e.g., Na, K, Ca, Mg, Fe, and Zn) [42; 43]. Microalgae can also serve as \"prebiotic-like\" when interacting with heterotrophic bacteria in the biofloc system. This symbiosis can maintain the nutrient cycle and energy flow, resulting in balancing C and N compounds in aquaculture and supporting the aquaculture production yields [44]. Using a diatom, _Chaetocros calcirrans_ as feed replacement to protein, vitamins and minerals of commercial pellet in black tiger shrimp revealed increasing feed conversion efficiency, growth performance and survival [45]. Considering the roles of minerals, calcium (Ca) and magnesium (Mg) play an important role in molting and new shell formation [46]. Ca also support the growth of \(L\)_vannamei_ in low salinity conditions [47]. Sodium, potassium and Mg are essential for maintaining proper physiological function and osmoregulation in biological organisms [48]. Mg supplementation showed increasing survival of \(L\)_vannamei_ in low salinity conditions [48; 49]. Zinc (Zn) is an essential micronutrient required for plants, animals and humans for growth and reproduction [50]. Zinc also plays a pivotal role as a regulatory co-factor of different enzymes and proteins in many important biochemical pathways [51]. Zn supplementation to shrimp indicated enhancing the growth and induction of immune responses [52; 53]. Iron (Fe) plays an important role in energy metabolism, homeostasis and co-factor/activator of the enzyme system [54; 55]. It has been proven to support the growth and the survival of juvenile giant river prawns [56]. Improvement of shrimp growth performance and survival suggests that mineral contents (Zn, Fe, Ca, Mg and Na) from the ex-situ biofloc were essential for biological processes such as osmoregulation, structural components of shell, co-factors of enzyme system for nutrient absorption and energy production, leading to enhanced shrimp survival and growth performance.