N hexadecane

The monomers of styrene (St, 99%, from Macklin, Shanghai, China) and methacrylic acid (MAA, 99%, from Aladdin, Shanghai, China) were first passed through an alumina column, respectively, to remove the inhibitors coming with the products, and then were stored at −20 °C prior to use in the study. 2, 2′-Azobisisobutyronitrile (AIBN, 98%, from Aladdin) was recrystallized from anhydrous ethanol before use. Dicyclohexylcarbodiimide (DCC, from Macklin), 4-dimethyaminopyridine (DMAP, from Macklin) and perfluoroalkyl polyethylene glycol (fPEG, from Du Pont, Wilmington, DE, USA) were used as received without further treatment. N, N-dimethyformamide (DMF, from Jiangsu Qiangsheng Chemical Co., Changshu, China) was dried by a 4A molecular sieve before use. Polyvinylidene fluoride (PVDF, FR904, supplied by Shanghai 3F New Material Co., Shanghai, China) was dried at 100 °C for 12 h before being used as the base material to prepare the membranes. Other materials, including N-methypl-2-pyrrolidone (NMP), polyvinylpyrrolidone (PVP, K30), bovine serum albumin (BSA), humic acid (HA, ≥90%), and n-hexadecane (≥98.5%) were supplied by Macklin Corp. Deionized water (DI, 18.2 MΩ cm) produced from a Millipore water purification system (Millipore ZRXQ015TO) was used as needed in the experiments.

To assess the degree of surface hydrophobicity, the microbial adhesion to hydrocarbon (MATH) method was adopted with minor modifications (Rosenberg, Judes & Weiss, 1983 (link); Slizewska, Chlebicz-Wojcik & Nowak, 2021 (link)). LAB strains were washed twice in PBS, harvested by centrifugation (5,000×g, 10 min, 4 °C), and re-suspended in PBS. We measured the optical density of bacterial suspension using spectrophotometry (Biochrom GeneQuant, Cambridge, UK) and adjusted the OD600_nm within the range of 1.0 ± 0.1. Each bacterial suspension (3 mL) was mixed with xylene, ethyl acetate, and n-hexadecane (1 mL, Macklin, Shanghai, China), vortexed for 2 min for emulsion formation, and incubated at 37 °C for 1 h. We then measured the OD600_nm of the aqueous layer. The hydrophobicity was calculated using the following equation:
$$\mathrm{H}\mathrm{y}\mathrm{d}\mathrm{r}\mathrm{o}\mathrm{p}\mathrm{h}\mathrm{o}\mathrm{b}\mathrm{i}\mathrm{c}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{\%}=[({\mathrm{A}}_0-{\mathrm{A}}_{\mathrm{t}})/{\mathrm{A}}_0]\times 100$$
where A₀ represented the absorbance at time t = 0 h, and A_t represented the absorbance taken at 1 h (Rastogi, Mittal & Singh, 2020 (link)).

Cell surface hydrophobicity was tested as previously described (Rosenberg et al., 1980 (link); Xie et al., 2019 (link); Mu et al., 2020 (link)). Briefly, 1 ml of bacteria (OD₄₀₀ = 0.6) was placed into glass tubes and 250 μl of n-hexadecane (Macklin, H810865) was added. The decrease in the OD₄₀₀ of the aqueous phase was taken as a measure of H%, which was calculated with the formula: H% = [(OD₀ - OD)/OD₀] × 100, where OD₀ and OD are the OD₄₀₀ before and after extraction with n-hexadecane, respectively. The experiments were performed using three independent cultures per condition.