Molasses fermentation to produce low-cost carbon source for denitrification

Figures & data

Table 1. Characteristics of methanol, ethanol and sodium acetate as carbon sources.

Carbon source	Methanol	Ethanol	Sodium acetate
Advantage	Low operating cost [4]; low sludge yield [5]	More readily available as a carbon source [6,7]	High denitrification rate [8] more readily available for denitrifiers [9]
Disadvantage	Toxicity [10] long hydraulic retention time [11]	Secondary pollution caused by the incomplete reaction of ethanol [12] remarkable nitrite accumulation [13]	Expensive [14] pH often increased to exceed 9.0 [15]

Figure 1. Schematic overview of the experimental setup.

Figure 2. Flow chart of the continuous fermentation system. 1: Culture medium tank. 2: peristaltic pump; 3: fermenter; 4: collection tank; 5: thermometer; 6: air bath temperature control box; 7: air pump and 8: pH control. Notes: The fermenter was a 200 mL Erlenmeyer flask and was completely closed except for the intermittent aeration (30 min every 12h). The temperature and pH of the fermentation broth were automatically controlled.

Table 2. Characteristics of three artificial diluted sewages.^a

Sewages	Diluted artificial brewing wastewater	Diluted artificial herbicide wastewater	Diluted artificial landfill leachate
Origin of natural sewage	Anoxic tank of a brewing wastewater treatment system in Anhui Province, China	Anoxic tank of picloram wastewater treatment system in Sichuan Province, China	Anoxic tank of a landfill leachate treatment system in Guangdong Province, China
TN (mg/L)	446	501	544
COD (mg/L)	547	813	1187
Salinity (g/L)	1.4	1.9	2.6
SV30(%)	41	37	42

^a Artificial diluted sewages were prepared by diluting natural sewages with carbon-free denitrifying medium to yield a final SV30 of 40%.

Figure 3. RTRE and C/N at different pH values (a); temperatures (b); dilution rates (c); sugar concentrations (d); ammonium chloride concentrations (e) and air-flow rates (f). Note: The fermentation parameters were stepwise optimized in the order of pH, temperature, dilution rate, sugar concentration, ammonium chloride concentration and aeration in the reactor. When pH was optimized, the other parameters were temperature, 30 °C; dilution rate, 0.25 h⁻¹; sugar concentration, 10%; ammonium chloride concentration, 2 g/L and air-flow rate in the bioreactor, 0.2 vvm for 30 min every 12 h. When temperature was optimized, the other parameters were pH, 4.0; dilution rate, 0.25 h⁻¹; sugar concentration, 10%; ammonium chloride concentration, 2 g/L and air-flow rate, 0.2 vvm for 30 min every 12 h. When the dilution rate was optimized, the other parameters were pH, 4.0; temperature, 33 °C; sugar concentration, 10%; ammonium chloride concentration, 2 g/L and air-flow rate, 0.2 vvm for 30 min every 12 h. When the sugar concentration was optimized, the other parameters were pH, 4.0; temperature, 33 °C; dilution rate, 0.15 h⁻¹; ammonium chloride concentration, 2 g/L and air-flow rate, 0.2 vvm for 30 min every 12 h. When the ammonium chloride concentration was optimized, the other parameters were pH, 4.0; temperature, 33 °C; dilution rate, 0.15 h⁻¹; sugar concentration, 13% and air-flow rate, 0.2 vvm for 30 min every 12 h. When the air-flow rate (aeration) was optimized, the other parameters were pH, 4.0; temperature, 33 °C; dilution rate, 0.15 h⁻¹; sugar concentration, 13% and ammonium chloride concentration, 0.5 g/L. After 60 h of fermentation, the cell-free fermentation broths, as the carbon sources for denitrification, were collected to measure the RTRE and the C/N in the undiluted artificial landfill leachate. In the control treatment, the unfermented molasses were used as the carbon source for denitrification.

Figure 4. Maximum RTRE and relative content of ethanol in the optimization experiment. The optimal conditions were 33 °C, 0.15 h⁻¹, 13%, 0.5 g/L and 0.1 vvm in the step to optimize temperature, dilution rate, sugar concentration, ammonium chloride concentration and air-flow rate, respectively.

Figure 5. LC-MS results for the fermentation broth and unfermented medium.

Figure 6. Kinetics of the total soluble solids content in triplicate fermentations.

Figure 7. RTRE (a) and C/N (b) of five types of carbon source in three sewages.

Figure 8. UPGMA (unweighted pair method with arithmetic mean) tree of three sewages.

Figure 9. OTU abundance clustering heat maps of the three sewages at the genus level. The distribution of the top 50 genera (in abundance) was plotted for each sample, and the abundance values corresponding to different colours of each square were the relative abundance values of the genus.

Figure 10. Relative abundance of Thauera and Acinetobacter in landfill leachate, herbicide wastewater and brewing wastewater and the RTRE when fermentation broth was used as the carbon source for denitrification.

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Data availability statement

The data that support the findings of this study (including the supplemental appendices) can be accessed at: https://data.mendeley.com/datasets/mp33mf9vz2