The schematic summary of the overall experiment is presented in
Fig. 1. A biofloc suspension was prepared in a 300 L fiber tank filled
with seawater and stockedwith 60 shrimp(5 g). The biofloc production
unit aswell as the experimental units (see below)were located indoors
at a light/dark regime of 12 h/12 h and light intensity of 450 lx. Feed pellets
containing 40% protein (Chuen Sin, PT. Grobest Indomakmur,
Indonesia) were administered three times daily at a total level of 8% of
the shrimp biomass day−1. To promote biofloc formation, 25 g ofmolasses
containing 44% of carbon (C)was added daily to obtain an estimated
C/Nratio of 20. The biofloc preparationwas performed for 3 weeks until
total suspended solids (TSS) reached a level of more than 500 mg L−1.
The shrimp and part of thewaterwere subsequently removed,whereas
the remaining 40 L of the shrimpculturemediumcontaining the majority
of the biofloc was enriched with 15N. The addition of (15NH4)2SO4
(20% atom excess (a.e.) 15N) was performed at a 15N enrichment level
of 12.5% on total N in biofloc biomass, coupled with the addition of molasses
(Avnimelech and Kochba, 2009) at an estimated C/N ratio of 20.
The suspensionwas gently aerated for 2 days until total ammonia nitrogen
(TAN) of the floc suspension was 0 mg L−1.