Gc 7820a

The candidate strain (B2 strain) was inoculated at 1% (v/v) in TSB with or without ZEN (at a final concentration of 5 mg·L⁻¹), and incubated at 37 °C for 24 h. After 8 h and 24 h, samples were collected for checking bacterial numbers. At the end of incubation, the supernatants were collected and analyzed for the pH, NH₃-N, lactic acid, acetic acid, and total volatile fatty acids (VFAs). Bacterial count was done as previously. The pH value was measured with a pH meter (pH 22, Horiba, Kyoto, Japan). The NH₃-N concentration was determined as described by Chaney and Marbach [39 (link)]. The lactic acid concentration was determined by L-lactic acid assay kit (LC2653, Randox, Crumlin, UK). Acetic acid and total VFAs were analyzed using gas chromatography (GC7820A, Agilent, Santa Clara, CA, USA) with a flame ionization detector and the Nukol™ capillary GC column (size × I.D. 30 m × 0.25 mm, df 0.25 μm) (SUPELCO, Bellefonte, PA, USA). The carry gas was helium gas. The crotonic acid (25 g·L⁻¹) was used as an internal standard.

Pure gas permeation tests were carried out at a temperature of 35 °C with feed pressures up to 500 psi using a constant-volume variable-pressure apparatus. The mixed gas permeation properties were measured in the same membrane cell using the same constant-volume variable-pressure apparatus. The membrane was exposed to certified gas mixtures of CO₂/CH₄ (50/50 vol%) with feed pressures up to 500 psi at 35 °C. The gas compositions were analyzed by a gas chromatograph (GC-7820A, Agilent).

According to the AOCS Official Method Ch 3-91 [23 ], the fatty acid composition of animal fats before and after extraction was determined after transmethylation to fatty acid methyl esters (FAMEs). Gas chromatography (GC7820A; Agilent, Wilmington, DE, USA) equipped with a flame ionization detector (FID) was performed by a CP-sil88 capillary column (100 m × 0.25 mm × 0.2 mm) using nitrogen as the carrier gas under a constant flow pressure of 30.8 psi. About 0.8 μL of sample was injected with a split ratio of 40:1. The temperature for both injector and detector was 260 °C. The oven temperature was programmed to maintain at 120 °C for 3 min, then heated to 175 °C at 8 °C/min and held for 18 min. All data were collected with Agilent EZChrom Elite 3.2.0 software. The fatty acid composition was quantified as relative percentages of the total fatty acids after identification by comparison with 37 FAME standards (Supelco, Bellefonte, PA, USA).

The acid methanolysis samples were analyzed for their composition and yield of FAME by using gas chromatography (Agilent Technologies GC 7820A, USA), which was equipped with a flame ionization detector (FID) and SP-2380 capillary column (60 m × 0.25 mm × 0.5 µm film thickness; Supelco Analytical of Sigma-Aldrich Co., PA, USA). Nitrogen gas was used as carrier gas with a flow rate of 12 mL/min. The oven temperature was 70 °C (2 min) → 190 °C (3 min) with 4 °C/min → 210 °C (2 min) with 3 °C/min → 240 °C (2 min) with 3 °C/min → 270 °C (2 min) with 5 °C/min. The injector and detector temperatures were set at 250 and 260 °C, respectively. Sample injection volume was 1 µL and split ratio was 1:30. Calibration curves of the FAMEs were obtained by external standard method using the standard 37 Component FAME Mix (CRM47885, Supelco, USA) (the calibration curves’ correlation coefficients were >0.995). Based on the calibration curve of the FAME standard, the contents of the acid methanolysis samples were obtained in the unit of mg/mL. The total FAME yield (C12 to C22 of FAME) from the acid methanolysis samples was calculated by the following equation: $Total FAME yield (\%, \mathrm{w}/\mathrm{w})=\frac{\mathrm{F}\times \mathrm{H}}{\mathrm{S}}\times \frac{1 \mathrm{m}\mathrm{g}}{1000 \mathsf{\mu }\mathrm{g}}\times 100\%$

F: Total amount of the FAME from the acid methanolysis samples (µg/mL)

H: Amount of n-hexane used for each batch (75 mL)

S: Amount of dried sludge cake powder for each sampling (5000 mg)

At the end of feeding phase, shrimp were overnight fasted, shrimp from each tank were weighed and counted. Hemolymph from 10 shrimp per tank were taken from the pericardial cavity using a 1-ml sterile syringe (no anti-coagulant was used), pooled and mashed, then centrifuged (8000 rpm, 4°C, 10 min). The supernatant was collected and stored at -80°C. Intestine and hepatopancreas from two shrimp per tank were immediately frozen and stored at -80°C for RNA extraction.
Moisture, crude lipid, crude protein, and ash contents in the diets were determined using standard methods (AOAC, 1995 ). Fatty acid compositions of diets were determined by China National Analytical Center. Lipids were extracted by a mixture of chloroform and methanol (2:1, v/v), then solvents was evaporated and fatty acids was saponificated by potassium hydrate. Fatty acids methyl esters were separated and quanitified by a gas chromatograph (GC 7820A, Agilent, United States) equipmented with a detector flame ionization (FID) and a HP-88 column (long × inner diameter × film thickness: 30 m × 0.25 mm × 0.2 μm) with hydrogen as carrier gas. The temperature gradient was from 100°C to 180°C at 10°C/min and then to 200°C at 1°C/min and finally to 230°C at 4°C/min, each sample was running for 49.5 min.

The total solid (TS), total ammonia nitrogen (TAN), chemical oxygen demand (COD), C content, and N content were measured according to APHA Standard Protocols (Simmons et al., 1998 ). Volatile fatty acids (VFAs) were analyzed by a gas chromatography (GC7890, Agilent, United States) with a flame-ionization detector and a capillary column (DB-FFAP 0.25 × 0.5 × 30), the initial temperature of column was 80°C, raise it to 180°C at a speed of 28°C s⁻¹, and keeping 1 min, and then, raise it to 220°C at a speed of 40°C s⁻¹ and keeping 1 min, the temperature of injection port and detector were 210 and 230°C, respectively. Injection volume was 1 μl and split ratio was 1:10. Nitrogen was the carrier gas and the flow rate was 35 cm s⁻¹, the flow rate of hydrogen, air and tail blowing were 30, 300, and 38 ml min⁻¹, respectively. VFAs were carried out with external standard method. The biogas composition was analyzed by a gas chromatography (GC7820A, Agilent, United States) with a thermal-conductivity detector and stainless steel column (1/8 × 3 m) packed with supporter (Porapak Q), hydrogen as the carrier gas and the flow rate was 27 ml min⁻¹, and temperatures for the column, injection port, and detector were 65, 120, and 130°C, respectively. Gas content was quantified with the area normalization method. pH values were measured with pH meters (AS ONE AS600, Japan).