## 3 Results and Discussion
### Carbon and Nitrogen Removal Study
#### Nitrogen  
Total nitrogen removal occurs through microbial metabolic processes in classical wastewater treatment processes. Organic nitrogen is first mineralized to ammonia, which is then nitrified to nitrite and nitrate under aerobic conditions. Under anoxic conditions, the nitrate is denitrification to nitrogen gas [69]. Generally, pH decreases with nitrification and increases with denitrification [70]. Throughout this study, influent concentrations of total nitrogen varied, with average influent concentrations in Phases 1, 2, and 3 of 33.8 mg/L N, 37.2 mg/L N, and 27.7 mg/L N, respectively. Despite this variation, influent concentration did not greatly influence the system performance. An average of 72% of total nitrogen removal occurred within the first column of each system during Phase 1, 78% during Phase 2, and 85% during Phase 3. Except for Treatment 2b, each system exhibited >90% overall removal of total nitrogen across all phases; Treatment 2b had an average removal of 88% and 89% in  
Figure 4: Variation of the chemical oxygen demand (COD) removal efficiency during the start-up period with column inoculated and not inoculated prior to operation. Phases 2 and 3, respectively. Across all the systems and phases, 97.5% of final effluent values were below 5 mg/L, and treatment under cool conditions was not significantly different. Ammonia was removed entirely and immediately by the first column of every system to a concentration that was below the detection limit of 1 mg/L NH\({}_{3}\)-N. This high level of treatment was exhibited, regardless of the influent concentration, which averaged 14.6 mg/L NH\({}_{3}\)-N in Phase 1, 12.7 mg/L NH\({}_{3}\)-N in Phase 2, and 11.5 mg/L NH\({}_{3}\)-N in Phase 3, and spiked as high as 29 mg/L NH\({}_{3}\)-N. This complete removal with no observed lag period was also observed in the columns that had not been inoculated in the Start-Up test. In the Control, Treatment 1, and Treatment 2b systems, the second column exhibited detectable levels of ammonia in Phases 2 and 3. This release of ammonia was hypothesized to be a result of organic nitrogen ammonification due to heterotrophic degradation within the column. These levels were not concerning, as they were always below 4 mg/L and were always removed to concentrations below the detectable limit in the third column. As all effluent ammonia concentrations were below detectable limits, there was no statistical difference during reduced temperatures or between the treatment systems with and without clinoptilolite. These results are in contrast with prior work, showing that the addition of natural zeolite materials to an ammonia-rich solution (1000 mg NH\({}_{4}\)\({}^{+}\)-N/L) reduced free ammonia inhibition and increased nitrification rates [71]. The addition of adsorptive media might provide an advantage in systems operated at higher nitrogen loading rates. The high concentrations of potassium in winery wastewater may also have impacted ammonia removal, as clinoptilolite is known to have a higher affinity for potassium than ammonium when the two ions are present in equimolar concentrations. However, when ammonium concentrations are higher than potassium, this effect is reduced, as was observed in a 2017 study that demonstrated 80-90% ammonia removal efficiency by clinoptilolite when treating swine wastewater with a NH\({}_{4}\)\({}^{+}\) concentration of 54 M and a K+ concentration of 3.0 M [72]. Consequently, more research is needed to better understand the impact of potassium and other competing ions in winery wastewater. It is generally accepted that ammonia removal in constructed wetlands is a result of biological degradation, plant uptake, volatilization, and adsorption to bed media [57]. In this study, plants were not included, and volatilization can be largely neglected due to the acidic pH of the wastewater [73].