**2. Materials and Methods**

#### *2.1. Experimental Shrimp and Acclimation*

The *L. vannamei* juveniles used in this experiment were supplied by Baorong Aquaculture Corp., Qingdao, China, and the experiment was conducted at the Ruizi Marine Food Product Co., Ltd., Qingdao City, China. The shrimp were acclimated for 10 days in 11 polyethylene fiber tanks (400 L) from which 50% of the water was renewed daily. The shrimp were fed a commercial feed (crude protein, 40%; moisture, 12%; ash, 16%; crude fat, 4% and NaCl, 3%) produced by the Yuehai Feed Co., Ltd., Guangzhou, China. Feed was offered twice daily (7:00 a.m. and 6:00 p.m.) based on a feeding amount equivalent to 5% of shrimp body weight.

#### *2.2. Experimental Design*

A 34-day trial was conducted. Three kinds of culture systems, each replicated three times (400-L of water in each circular tank), were established and 60 healthy shrimp (2.70 ± 0.02 g) were stocked into each tank. The treatments were: a clear water system (CW) and two BFT systems with added molasses (M-BF) or PHBV (P-BF). No molasses or PHBV was added to the CW tanks. Molasses was applied to the M-BF treatment at the ideal theoretical application amounts suggested by Avnimelech [10]. The molasses had a total organic carbon concentration of 26.5%, and it was diluted with water from the tanks and splashed uniformly over the tank surfaces twice daily. As suggested by Zhang, et al. [19], after 7 days of activation in the acclimation tanks, 633 g of PHBV was placed in a PVC tube

with an inner diameter of 15 cm and an operating depth of 30 cm, and an air stone was placed inside the tube to sufficiently stir the PHBV. The PVC tube was wrapped with a filter screen, which was cleaned every five days.

The tanks were located within a greenhouse covered with clear plastic sheeting. Onefifth of the water in the CW treatment was replaced daily to ensure good water quality [13]. One-fifth of the water in the BFT tanks was replaced every 5 days [20]. The seawater used was pre-filtered using a sand filter. Each tank was continuously aerated with four air stones. The tanks were covered with shade netting to control sunlight. The feeding regime was the same as during the acclimation period.

#### *2.3. Water Quality*

Temperature, dissolved oxygen (DO), and pH were measured at 8:00 am daily using a portable DO meter (RDB20, Yuanmu, Shanghai, China) and a pH meter (pH-100, LICHEN, Shanghai, China). The salinity of the culture water was 31 g L−<sup>1</sup> which was monitored using an optical salinity meter (LYT-610, Yulin, Shanghai, China). Water samples were collected from the tanks every 5 days. Total ammonia nitrogen (TAN), nitrite (NO2 −-N), nitrate (NO3 −-N), total nitrogen (TN), total alkalinity (TA) and chlorophyll (Chl) a were analyzed following the standard methods of the Chinese State Environmental Protection Agency [21]. Particulate organic carbon (POC) and dissolved organic carbon (DOC) were analyzed according to the method of Gilcreas [22]. Total organic carbon (TOC) was calculated as the sum of DOC and POC. Water samples (1000 mL) were transferred to Imhoff cones (1000-0010, Nalgene) at 3:00 pm every five days to determine biofloc volume. The volume of the biofloc plug accumulating on the bottom of the cone was determined 30 min after sedimentation.

#### *2.4. Nitrification Rate*

Water samples (500 mL) were collected on days 14, 19, 24, 29 and 34 to measure the nitrification rate following the methodology of Bratvold and Browdy [23]. Briefly, duplicate samples were enriched with ammonium chloride (2.5 mg L−<sup>1</sup> of ammonia-N) and incubated in the absence or presence of 2.2 mg L−<sup>1</sup> of the nitrification inhibitor N-allylthiourea (Sigma-Aldrich, St. Louis, MO, USA). The nitrification process is expressed as NH4 <sup>+</sup> + 2O2 → NO3 <sup>−</sup> + 2H+ + H2O. The difference in the DO concentration of dissolved oxygen between the bottles with and without the inhibitor was the nitrification result. The nitrification rate (NR) (mg L−<sup>1</sup> h<sup>−</sup>1) was calculated using Equation (1):

$$\text{NR} = (\text{DO}\_1 - \text{DO}\_2) / \text{h},\tag{1}$$

where (DO1) = dissolved oxygen in the bottle with the inhibitor (mg L<sup>−</sup>1); DO2 = dissolved oxygen in the bottle without the inhibitor (mg L<sup>−</sup>1); h = incubation period (h).

#### *2.5. 16S rRNA Gene High Throughput Sequencing Analysis*

At the end of the study, 1-L water sample was filtered with a 50-mm membrane (pore size 0.20 μm) and used for bacterial DNA extraction and analysis. Three replicates were conducted for each treatment. Total DNAs from these samples were extracted by the CTAB/SDS method [24] and were diluted to 1 ng/μL with sterile water and used for PCR template. 16S rRNA genes of distinct regions (16S V4/V5) were amplified using specific primers (515F: 5 -GTGCCAGCMGCCGCGG-3 and 907R: 5 - CCGTCAATTCMTTTRAGTTT-3 ). These PCR products were purified with a GeneJET Gel Extraction Kit (Thermo Scientific, Waltham, MA, USA) and prepared to construct libraries. Sequencing libraries were generated using NEB Next®Ultra™DNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) following the manufacturer's instructions. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific, Waltham, MA, USA) and Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). Then the library was sequenced on an Illumina MiSeq platform and 250 bp pairedend reads were generated. The raw amplicon sequencing data were deposited to NCBI Sequence Read Archive (accession number: PRJNA738270).

Paired-end reads were merged using FLASH (v. 1.2.11, http://ccb.jhu.edu/software/ FLASH/, accessed on 29 April 2021), which was designed to merge paired-end reads when at least some of the reads overlap those generated from the opposite end of the same DNA fragments. The splicing sequences were called raw tags. Quality filtering on the raw tags were performed under specific filtering conditions to obtain the high-quality clean tags using Trimmomatic v. 0.33 [25]. The primer sequences were identified and removed using Cutadapt v. 1.9.1 [26]. The raw tags were compared with the reference database (Silva database, Release132, http://www.arb-silva.de/, 29 April 2021) using UCHIME algorithm v. 10 [27] to detect and remove chimera sequences [28]. The sequencing results were clustered into operation taxonomy units (OTUs) at a similarity threshold of 97% using USEARCH v. 10.0 [27]. The representative sequences for each OTU were made taxonomic annotations against the SILVA taxonomy database (Release132, http://www.arb-silva.de/, 29 April 2021) using Qiime 2 [29].

#### *2.6. Shrimp Growth Performance and Survival*

At the end of the experiment, all shrimp in the tanks were collected, counted, and weighed to calculate growth rate, feed conversion ratio (FCR), specific growth rate (SGR) and survival rate, which were determined as follows:

Growth rate (g/wk) = (average final weight of shrimp − average initial weight of shrimp)/culture weeks; (2)

Feed conversion ratio (FCR) = total weight of feed intake (g)/total shrimp weight gain (g); (3)

Specific growth rate (SGR) = (ln (initial wet weight) − ln (final wet weight))/culture days × 100; (4)

Survival rate (%) = (final shrimp number/initial stocking shrimp number) × 100. (5)
