## 4 Discussion
### Effects of Ex-Situ Biofloc as Feed Supplement to Enhance Shrimp Nutritional  
Ex-situ biofoc supplementation in shrimp culture also affected nutritional value in shrimp as observed from amino acids and fatty acids profile in shrimp muscle. Amino acid concentrations in shrimp fed with 5% and 10% commercial pellet reduction plus ex-situ biofoc supplements were mostly found to be similar to those in the shrimp fed with only the commercial pellet, except alanine, glycine, leucine, valine and isoleucine. This result indicated that amino acid concentrations did not increase in the shrimp muscle when supplementing with ex-situ biofoc. This might be caused by the fact that excessive amounts of proteins, for example, glycine, essential for muscle development, are turned to fat molecules for storage like in humans [67] and in juvenile Pacific white shrimp [68]. Another possibility may be from gut microbial and/or probiotics absorption and utilization of amino acids into health benefit molecules to the host, resulting in amino acid homeostasis in the host cells [69]. This agreed with our observation that most of the amino acid concentrations in shrimp muscle were still similar between control and supplementing with ex-situ biofoc, containing probiotic bacteria. In addition, this finding correlated with metabolomics study of whiteleg shrimp cultured in different culturing systems (recirculating aquaculture system (RAS), hybrid zero water discharge-recirculating aquaculture system (hybrid) and outdoor earthen pond), revealing that alanine concentrations decreased in RAS and hybrid systems because super-intensive shrimp culture caused oxidative stress induction [70]. This suggests that amino acids were affected by different factors, such as stress, from super-intensive culture, microbial utilization, and nutrient storage metabolism. Therefore,ex-situ biofloc supplementation may not affect amino acid content in shrimp muscle in terms of nutritional value. Interestingly, the ex-situ biofloc supplement affected fatty acid contents in shrimp muscle. Docosahexaenoic acid (DHA; 22:6n3) and eicosenoic acid (20:1) concentrations were significantly higher in shrimp fed with the ex-situ biofloc supplement with 5% and 10% commercial pellet reduction than feeding with commercial pellet alone. These fatty acids were found in both the commercial pellet and ex-situ biofloc; however, they were predominant in the commercial pellet. DHA is an omega 3 polyunsaturated fatty acid, a well-known nutrient source found in fish, shrimp, and marine organisms, that benefits visual and brain development in humans [71; 72; 73; 74]. Eicosenoic acid (20:1) is a monounsaturated fatty acid existing in three different forms: 9-eicosenoic acid (an omega-11 gadoleic acid), 11-eicosenoic acid (an omega-9 gondoic acid), and 13-eicosenoic acid (an omega-7 paullinic acid) [75]. An eicosenoic acid-rich marine oil diet showed decreasing risk factors for cardiovascular-related diseases and atherosclerosis in mice [76] and suppressing lipogenesis in mice [77]. These fatty acids were found to be beneficial to humans. _Bacillus megaterium_ and _B. cereus_ contained in the ex-situ biofloc are well-known heterotrophic bacteria that have been reported to be potential probiotic bacteria in other organisms. Previous studies reported that a probiotic _Lactobacillus acidophilus_ and _Lactobacillus plantarum_-supplemented diet provided benefits to overall health, immunity and gut microbial composition in crayfish [78]. Brevibacillus and Fermicutes communities showed abilities to increase nutrients absorption and digestion in gut mammal [79] and in gut shrimp \(p\). monodon [80]. The fact that genes related to lipid metabolism and energy metabolic process (e.g., _fatty acid synthase_, _triacylglycerol lipase_, _carnitine palmitoyltransferase_, _NADH dehydrogenase_, _ATPase_ and _cytochrome b oxidase_) were expressed higher in the ex-situ biofloc shrimp also supports that ex-situ biofloc-supplement enhanced lipids digestion and absorption in shrimp muscle to boost the nutritional value.