## 3 Results and Discussion
### Carbon and Nitrogen Removal Study
#### Nitrogen
Although gravel can serve as an adsorption media for ammonia [74], this removal mechanism can be discounted in this study due to detectable levels of ammonia in the effluents of the second column of the Control, Treatment 1, and Treatment 2b in Phase 2. It is unlikely that these detectable concentrations were a result of ammonia breakthrough, as there was never any detectable concentrations of ammonia going into the second columns. If able to be adsorbed, any ammonia mineralized in the column would have been adsorbed onto the gravel and would not have been present in the effluent, as observed. Influent concentrations of nitrate fluctuated widely throughout the study, which resulted in varying effluent concentrations. Effluent concentrations never exceeded 2.75 mg/L N, despite the variability. There was not a significant difference in nitrate removal between systems with and without tire chips for nitrate adsorption. The treatment performance was significantly better during Phase 3 than Phase 1 in the Control system. In the rest of the systems, there was not a significant difference in performance between phases. The second column in each system exhibited the highest removal across all systems and phases, reducing nitrate by an average of 92%, 93%, and 87% in Phases 1, 2, and 3, respectively. This phenomenon was expected due to the saturated conditions in the columns that promoted denitrification. Unexpectedly, nitrate was also immediately removed in the first column of each system, which indicated that both aerobic and anoxic zones were present in the columns or biofilms. This immediate removal was also observed in the Start-Up test, where the columns were not inoculated. After the Start-up period, a majority of the samples (76%) during Phase 1 were collected at the morning loading period after the system had rested for 15 h. The remaining samples were collected during the evening loading period to verify consistency. The observed concentrations were not substantially different between the morning and evening samples (data not shown). These resultsare in contrast to previous studies, where higher nitrate and lower ammonium concentrations were observed in the morning samples [50]. Figure 5 shows the average nitrogen transformations in the wastewater through each column of each system in Phase 1. Organic nitrogen was calculated by subtracting ammonia and nitrate from total nitrogen, assuming no other forms of inorganic nitrogen were present with the given operating conditions and influent ammonia concentrations [71; 75]. The largest differences in total nitrogen removal were observed in systems Treatment 2a and Treatment 2b, which was unexpected, as both systems had the same initial construction and operation. However, Treatment 2a consistently performed better than the other systems, which suggested that the nitrogen adsorption media might have enhanced performance. The high nitrogen removal of all the treatment systems, regardless of nitrogen adsorption media, is likely explained by the favorable COD:N:P ratio of winery wastewater of 270:1.5:1. Several review papers on biological treatment of winery wastewater have cited the imbalance of COD:N:P as an impediment for carbon removal, with nitrogen and phosphorus as the limiting nutrients [6; 66; 76]. The ratio of COD:N in winery wastewater is often 100, an order of magnitude larger than that in municipal wastewater [6; 77]. As such, nitrogen and phosphorus are expected to have higher removal efficiencies as microbes use up these nutrients for biosynthesis. A high C:N ratio is favorable for denitrification [78]; however, it might inhibit nitrification processes [76]. In a study of three constructed wetlands operating in Italy, it was found that total nitrogen removal was greater when the organic substrate concentrations were higher [79], emphasizing the vital role that the C:N ratio plays in the biological treatment of winery wastewater.