# Nitrate-Polluted Waterbodies Remediation: Global Insights into Treatments for Compliance
## 4 Nitrate Removal Technologies in Waterbodies
### Transformation-Based Technologies
#### 4.2.1 Biological Denitrification
Nitrate reduction proceeds stepwise in accordance to:  
\[\mathrm{NO_{3}}^{-}\left(\mathrm{aq}\right)\rightarrow\mathrm{NO_{2}}^{-} \left(\mathrm{aq}\right)\rightarrow\mathrm{NO}\left(\mathrm{g}\right) \rightarrow\frac{1}{2}\,\mathrm{N_{2}}\left(\mathrm{g}\right) \tag{1}\]  
Figure 6: Flowchart representing a typical biological denitrification process (adapted from [142]). Each stage is catalysed by an enzyme complex, the synthesis of which will occur under partially aerobic or anaerobic conditions [153]. The first step of denitrification is the reduction of NO3\({}^{-}\) to NO2\({}^{-}\), catalysed by nitrate reductase. The second step is the transformation of NO2\({}^{-}\) to nitric oxide (NO) by nitrite reductase. The reduction of NO to N2O is catalysed by nitric oxide reductase. Among the denitrification enzymes, this is the least characterized. The last stage of denitrification is the reduction of N2O to inert N2. It is carried out by the enzyme nitrous oxide reductase [154]. This enzyme is severely limited by the presence of O2. Because of this, it is important to avoid the entry of oxygen into the denitrifying process, so as not to release N2O, a gas that contributes to the global greenhouse effect. Some evidence revealed that the presence of Cu2+ in the raw water of a continuous denitrifying reactor is important in preventing the accumulation of N2O during the removal of high nitrate concentrations [155]. As a staged process, there is also the possibility of accumulation of intermediates (NO2\({}^{-}\), N2O, NO) depending on the characteristics of the feed water and the organic substrate used. Moreover, the operating conditions (temperature, pH, hydraulic retention time, and cell retention time) will be critical. Based on this, for the transformation to culminate in N2, environmental conditions such as dissolved O2 level, organic carbon source, nitrate concentration, C/N ratio, phosphorus availability, pH, temperature, and the possible presence of toxics must be controlled [156]. Groundwater has a low content of dissolved organic compounds. Therefore, it is necessary to add external carbon-containing substrates to facilitate nitrate removal by biological denitrification [157]. Liquid state electron donors (methanol, ethanol, sucrose, acetic acid, glucose syrup, starch, and molasses), as exogenous carbon source, have been successfully applied in different heterotrophic denitrification processes, showing an excellent efficiency [158]. Solid organic carbon donors as cellulose-based products (woodchip) [159] and different biodegradable polymers [160] are also commonly used. Numerous biological denitrification processes have been reported; in general, the results obtained are quite good, although they are affected by the specific conditions of each case. Denitrification efficiencies of 95-100% are reported using ethanol as an electron donor in waterbodies with elevated (>250 mg/L) nitrate concentration [161; 162]. Autotrophic denitrification is also generally considered to be cost-effective, scalable, targeted and highly efficient in a variety of situations. Using H2, S0 or S2\({}^{-}\) as electron donors, in packed-bed, fluidized-bed or membrane biofilm reactors and with contaminated groundwater or potable water, 98-100% denitrification efficiency was achieved [148; 154; 163]. Fluidized bed and fixed bed reactors are the preferred types of reactors for this biological treatment, with fluidized bed reactors providing the highest nitrate removal rates. However, with these reaction systems, greater control of the process is required. Although biological denitrification is very effective in nitrate removal, it has some drawbacks, such as a decrease in the denitrification rate as the temperature drops, leading to the need for additional treatment of the water [142; 158]. Thus, biological denitrification can lead to microbial contamination of treated water, and regulators require post-treatment using membrane technology as a method of protection.