Inoculum

Samples A, B, C and D were taken from Zhengzhou Sewage Treatment Co., Ltd. (112o42′-114o14′ E, 34o16′-34o58′ N), xinmi city Jinmen Sewage Treatment Co., Ltd. (110° 41′-113o14′ E, 34o16′-34o58′ N), Henan Chengsheng Sewage Treatment Co., Ltd. (113° 40′-115o14′ E, 34o16′-34o58′ N) and Zhengzhou Tongji Environmental Protection Engineering Co., Ltd. (110o42′-116o17′ E and 36o18′-36o58′ N) respectively. Their water sources are domestic sewage, landfill leachate, sewage from chemical industry park and sewage from food Industry Plant.

Media

Broth medium contains 5.0 g/L NaCl, 3.0 g/L beef paste, and 10.0 g/L peptone.

Heterotrophic nitrification liquid medium (HNLM) contains 0.5 g/L (NH4)2SO4, 50.0 mL/L Vickers salt solution, 5.62 g/L C4H4Na2O4. 6H2O and 10.0 mL/L trace elements.

Trace elements contains 5.5 g/L CaCl2, 5.06 g/L MnCl2, 1.1 g/L (NH4)2MoO4, 1.57 g/L CuSO4, 50.0 g/L EDTA, 2.2 g/L ZnSO4, 5.0 g/L FeSO4 and 1.61 g/L CoCl2.

The composition of heterotrophic nitrification solid medium (HNSM) was the same as that of HNLM with the addition of 15.0 g/L agar.

The inorganic salt medium contains 10.297 g/L CH3COONa.3H2O, 50.0 mL/L Vickers salt solution, 1.0 g/L (NH4) 2SO4 and 10.0 mL/L trace elements.

The Vickers salt solution comprises 0.05 g/L FeSO4, 0.05 g/LMnSO4, 5.0 g/L K2HPO4, 2.5 g/L MgSO4 and 2.5 g/L NaCl.

The simulated wastewater contains 500.0 mg/L glucose, 200.0 mg/L MgSO4,30.0 mg/L CaCl2, 30.0 mg/L KH2PO4, 500.0 mg/L KHCO3, 100.0 mg/L NaNO3, 30.0 mg/L (NH4)2SO4 and 100.0 mg FeCl3.

All above-mentioned solutions were adjusted to pH 7.0 and sterilized for 20 min at 121 °C.

Screening of PDBFs

Four samples, A, B, C, and D, were diluted 10 folds with 0.9% physiological saline and then shaken with glass beads for 1 h. Microorganisms in the samples were activated and acclimated by culturing for 24 h at 30 °C and 150 r/min in the broth medium and HNLM successively. The domesticated microorganisms were inoculated into simulated wastewater at a 0.1% ratio to evaluate their pollutant degradation capacity by determining the degradation ratios of NH4+-N, TN, TP, NO3-N, and COD after a 24 h treatment at 30 °C and 150 r/min.

Optimization of fermentation condition of PDBFs

Pollutant-degrading enzymes are induced enzymes. The composition and concentration of the culture medium affect the growth of microorganisms and their ability to reduce pollutants. However, the culture conditions mainly affect the growth of microorganisms. Therefore, the evaluation criteria were the concentration of viable bacteria and the ability to degrade NH4+-N to optimize culture medium components. The concentration of viable bacteria is the sole evaluation criterion to optimize culture conditions.

Medium composition optimization

Based on the composition of HNLM, the effects of the carbon and nitrogen sources on the viable bacterial concentration and NH4+-N degradation ability were studied using a single-factor experiment. These factors were studied successively, and the optimized results were used in the subsequent steps. Sodium citrate, sodium succinate, sodium acetate, and glucose were used as alternative carbon sources. Beef paste, soybean meal, corn steep liquor powder, yeast extract powder, fish meal, peptone, tryptone, and urea were used as alternative sources of organic nitrogen. Ammonium sulfate and ammonium chloride were used as alternative sources of inorganic nitrogen. Based on the single-factor experiment, an Taguchi L9 (33) orthogonal experiment was designed using SPSS software (version 20.0) to optimize the concentrations of the carbon and nitrogen sources.

Broths cultured in different media were centrifuged for 10 min at 5000 r/min. The precipitated PDBF cells were washed with PBS three times, inoculated into an inorganic salt medium at a concentration of 1.52 × 107 cfu/mL, and cultured at 150 r/min and 30℃ for 24 h to evaluate their ability to degrade NH4+-N.

Optimization of culture conditions

Based on the initial culture conditions (initial pH 7.0, culture temperature of 30 ℃, loading ratio of 40%, rotation speed of 150 r/min, inoculation ratio of 5%), the effects of temperature, pH, loading ratio, rotating speed, and inoculation ratio on the concentration of viable bacteria in culture broth were studied using a single factor experiment successively in a shake flask. All optimized results were used in the subsequent steps. Alternative temperatures were 20, 25, 30, 35, and 40 ℃. Alternative pH values of 5.0, 6.0, 7.0, and 8.0 were used. Alternative loading ratios of 2.5, 5.0, 10.0, 15.0, and 20.0% were used. Alternative rotating speeds of 90, 120, 150, 180, and 210 r/min were used. The alternative inoculation ratios were 1, 3, 5, 7, and 9%.

Optimization of feeding strategy for fed-batch fermentation

Before optimization of the feeding strategy (Table 1) for fed-batch fermentation, a pre-test was conducted in a 10 L fermentation tank. The test condition were: loading ratio 50%, ventilation ratio 1:1 VVM, stir speed 300 r/min, tank pressure 0.05 MPa, and temperature 30 ℃. The optimum medium for flask culture (15.0 g/L sodium acetate, 0.9 g/L ammonium sulfate, 0.7 g/L soybean meal powder, 50.0 mL/L Vickers salt solution, and 10.0 mL/L trace elements) was adopted as the initial medium. The feed time was designed according to the growth curves of the screened PDBF (Additional file 1: Fig. S1).

Evaluation of the efficiency of PDBF in zeolite trickling filter towers

The PDBF application verification experimental systems were constructed as 1200 mm × 150 mm diameter vertical Perspex tubes (Fig. 1), which were filled with zeolite (2–3 mm in diameter) as the main filter medium, with a 200 mm supporting layer filled with gravel (10–20 mm in diameter) at the bottom. There were five 150 mm interval sample outlets from the bottom to the top of the fillers and an air pump at the bottom of the system to create an aerobic environment. The experiments were performed in up-flow mode at 25 ℃.

Fig. 1

Schematic diagram of the experimental aerated zeolite trickling filter

Control and experimental groups were established. In the 10-d start-up procedure, a high-concentration nutrient solution (150 mg/L NH4+-N) mixed with mixed liquor volatile suspended solids (MLVSS, 3.723 g/L) was pumped into the system to stimulate microorganism growth and biofilm formation. The experimental conditions for the two groups were the same, except that the experimental group was inoculated with the studied microbial flora in addition to activated sludge. After the bacteria film was formed on the surface of the zeolite filler, simulated sewage containing 300.0 mg/L COD and 75.0 mg/L NH4+-N was pumped into the system to evaluate the pollutant degradation efficiency of the studied PDBF. The effluents were sampled from the sampling port E5 at 0 and 24 h to analyze the pollutant degradation ratio. The intermittent aeration mode was applied during the start-up and experimental processes, that is, aeration for 4 h to maintain the dissolved oxygen at 4–6 mg/L, then non-aerarion for 4 h. This process was repeated.

After the application effect evaluation experiment was completed, the biofilms on the surface of the zeolites located in E2 and E4 were soaked in sterilized saline solution. The microbial community composition was analyzed using second-generation sequencing.