Strain, media and cell cultivations
A. oryzae was provided by Wilmar Biotechnology R&D Center Co., Ltd, Shanghai, China.
The seed medium (g/L) consisted of corn dextrin 20, corn steep powder 10, yeast extract powder 1, polyether defoamer 1, KH2PO4 5, MgSO4·7H2O 0.5, Na2HPO4·12H2O 1. The composition of the fermentation medium (g/L) was as follows: corn dextrin 40, peptone 30, yeast extract powder 4, polyether defoamer 1, KH2PO4 1.12, MgSO4·7H2O 0.5, Na2HPO4·12H2O 1.
A. oryzae spore suspension was inoculated into 250 mL shake flasks containing seed medium. After incubating at 28°C on a rotary shaker (150 rpm) for 28 h, the seed solution was transferred in 250 mL shake flasks containing fermentation medium with 10% inoculum. The flasks were incubated at 28℃ on a rotary shaker (200 rpm) for 168 h.
Screening of the surfactants and optimization of the sophorolipids addition amount
According to the addition amount of 0.2% (w/v), five surfactants including Tween-80, acacia, polyvinyl alcohol, Triton X-100 and sophorolipids (lactone type) were added to the fermentation medium respectively. Then, the flasks were incubated at 28℃ on a rotary shaker (200 rpm) for 168 h. In detail, the sophorolipids addition amount was set as 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35% and 0.5% respectively. Each set of experiments were repeated for three times.
Extracellular RML enzyme activity assay
RML activity in the diluted fermentation supernatant was determined by the p-nitrophenol (p-NP) colorimetric method (Wang 2012). One unit of RML activity was defined as the amount of enzyme, which liberated 1 μmol of p-NP from p-nitrophenol palmitate (p-NPP) per minute. The p-NPP solution and the substrate buffer containing phosphate buffer saline (PBS) were mixed in a ratio of 1:9 (v/v). 2.4 mL of the mixed solution was pipetted into a 5 mL EP tube, and all tubes were preheated at 37°C for 3 min. Next, 100 μL of the diluted fermentation supernatant was added to the test tube, and 100 μL of the boiled denatured sample solution was added to the control tube as a control. All the tubes were put in a constant temperature water bath oscillator at 37°C for 15 min. To stop the reaction, 2 mL of 95% ethanol was added immediately. Finally, all the tubes were centrifuged at 4000 rpm for 5 min. The absorbance of the sample and the control group were measured at 410 nm. In addition, p-NP standard solutions of different concentrations were prepared and measured by same method. And a p-NP standard curve was acquired. RML enzyme activity (U/mL) was calculated according to formula (1).
Where A1 is the absorbance of the sample, A0 is the absorbance of the corresponding control group, n is the dilution factor of the fermentation supernatant.
Intracellular RML enzyme activity assay
Pedersen's method was referred and modified slightly (Pedersen et al. 1999). About 0.07 g of wet mycelia was weighted in a 2 mL EP tube, and 1 mL of 0.2 M pH 8.0 PBS containing 1 mM ethylene diamine tetraacetic acid (EDTA) was added to resuspend the mycelia. Then, 500 μL of grinding beads (0.5 mm in diameter) were added in each EP tube, and all the tubes were put in a freezing grinder. The frequency and time were set as 65 Hz and 4 min, respectively. Finally, all the samples were centrifuged at 12000 rpm for 2 min. The supernatant was acquired and the enzyme activity was determined by colorimetry.
Specifically, to acquire accurate intracellular activity proportion, the units of intracellular and extracellular enzyme activity had been normalized into units in the whole fermentation broth.
Dry cell weight (DCW) assay
The biomass of A. oryzae was indicated by the DCW. 20 mL of fermentation broth was filtered with a piece of dried filter paper, followed by repeated washing for 3 times with deionized water. And then the wet biomass was transferred to the electric oven at 80℃ for 24 h until the constant weight was obtained, after that the dried biomass was weighed immediately.
Reducing sugar assay
1 mL of the diluted fermentation supernatant was put in a 10 mL colorimetric tube, then 1.5 mL of 3,5- dinitrosalicylic acid (DNS) reagent was added. After mixing well, all the colorimetric tubes were put in boiling water bath for 5 min. When cooled to room temperature, all the reaction systems were diluted to 10 mL. After mixing well, the absorbance of each sample was measured at 550 nm. Glucose of different concentrations were prepared and measured by same method. And a glucose standard curve was acquired. Reducing sugar content (g/L) in the fermentation supernatant was calculated according to formula (2).
Where A1 is the absorbance of the sample, A0 is the absorbance of the corresponding control group, n is the dilution factor of the fermentation supernatant.
Amino nitrogen assay
Pipette 1 mL of fermentation supernatant in a 150 mL conical flask. First, add 30 mL of distilled water and 2 drops of methyl red indicator, adjust to light red with 0.1 M hydrochloric acid, and leave for 3 min. Next, adjust to light yellow with 0.05 M sodium hydroxide solution. And then add 5 mL of 18% neutral formaldehyde, mix well and leave for 3~5 min. Last, add 4 drops of phenolphthalein indicator and titrate with 0.05 M sodium hydroxide solution to reddish (pH 8.0~10.0). The amino nitrogen content (g/L) in the fermentation supernatant was calculated according to formula (3).
Where c is the concentration of NaOH solution (mol/L), V1 is the volume of NaOH solution (mL) used for titration, V2 is the volume of sample supernatant (mL) used for determination.
Broth viscosity assay
About 15 mL of homogeneous fermentation broth was taken out, and then the viscosity was measured by LVDV-II + P viscometer with SC4-34 type rotor. And the specific parameters were as follows: the rotation speed was 200 rpm, and the temperature was room temperature.
Determination of extracellular amino acids
The S-433D amino acid analyzer (Jiesheng Yike Technology Development Co., Ltd, Beijing, China) was used to determine the extracellular amino acids concentrations in the fermentation supernatant. A total of 800 μL of the fermentation supernatant was pipetted into a 1.5 mL EP tube, and then 200 μL of 10% sulfosalicylic acid was added. After mixing well, all the EP tubes were transferred at 4°C for more than 30 min, then they were centrifuged at 13000 rpm for 10 min. Next, the supernatant was diluted 3 times with lithium salt diluent, filtered through a 0.22 μm filter membrane, and poured into a liquid phase vial for testing. After that, 100 μL of the mixed amino acid standard and 900 μL of the lithium salt diluent were mixed well, and poured into the liquid phase vial for testing. In addition, 1000 μL lithium salt diluent was prepared as a blank. Finally, the free amino acid analysis standard program was called, the mixed amino acid standard was measured twice, then the lithium salt diluent was measured once, last the samples were measured.
Determination of the pellet number and projected areas
The quantitative analysis method of Aspergillus niger established by Tang et al. (Tang et al. 2015) was referenced in this study. To fix pellets sample, 1 mL of fermentation broth was mixed with 1 mL of fixing agent consisting of 40% formaldehyde and 60% ethanol. After mixing, 300 μL of the mixture was pipetted into a 5 mL centrifuge tube, deionized water was added and mixed, then the centrifuge tube was kept stand for 5 min. Next, the upper suspended hyphae were wiped off, the bottom pellets were washed three times repeatedly with deionized water and diluted to 5 mL finally. Afterwards, the pellets sample was poured into a disposable plate of 8.5 cm and mixed with 20 mL of 60°C 0.05% agarose. After cooling, each sample was taken photos by the camera under black background. Last, Image J software was used to process the photos for parameter determination.