## 2 Biological Wastewater Treatment and Emerging Research
### Total Nitrogen Removal  
Total nitrogen is one of the contaminants of concern, which is treated by nitrification and denitrification. Total nitrogen accumulates in the environment from different industrial activities. It enters the water streams through several pathways, such as in runoffs from contaminated soil and agricultural fields. If nitrogen is left untreated, it can result in environmental damage and can pose threats to human health [11; 12]. The aerobic nitrification and anaerobic denitrification approaches use microorganisms in a two-step process, whereby the first step involves the oxidation of ammonium to nitrate in aerobic conditions, and the second step involves the reduction of nitrate to molecular nitrogen under anaerobic conditions [13].  
Conventional nitrification and heterotrophic denitrification are carried out in separate reactors by two distinct groups of microorganisms, namely autotrophs and heterotrophs, respectively. Heterotrophic denitrification uses inorganic carbon as an electron donor [14]. The use of separate reactors increases the operational costs of traditional nitrification and heterotrophic denitrification processes. However, some researchers have proven that the microorganisms responsible for nitrification and denitrification can co-exist in the same reactor. This process is known as simultaneous nitrification and denitrification (SND) [15; 16]. In this process, the different microorganisms responsible for denitrification and nitrification synergistically interact with each other. Although the mechanism of SND has not yet been studied comprehensively, some studies have reported that SND results from the formation of biofilms, for which anaerobic conditions are provided in the middle zone of the biofilm, while the nitrifiers reside in the outer zone of the biofilm (Figure 1) [17]. SND has received attention because it addresses all of the shortfalls of traditional nitrification and heterotrophic denitrification and has high nitrogen removal efficiency, reducing up to 16% of the eutrophication potential of WWTPs and 1.72 kg of greenhouse gas (GHG) emissions, which is equivalent to USD 0.303/d\(\cdot\)m\({}^{3}\) savings in cost. Moreover, SND can also reduce the aeration costs by 50%. Therefore, SND has been deemed appropriate for the treatment of side steams and domestic wastewater feeds [18]. However, it has been reported that SND could induce competition between autotrophic anammox species and heterotrophic denitrifiers.  
The denitrification reaction is thermodynamically more feasible than nitrification due to its sensitivity, the low-growth yield of anammox species, and the standard free energy (\(\Delta\)G\({}^{\circ}\)) of the denitrification reaction compared to the anammox reaction [19]. Although SND is an important part of wastewater treatment, the microorganism responsible for these processes is slow-growing, meaning these processes are susceptible to inhibition by heavy metal loadings. Different studies have investigated methods of bypassing this inhibition, with different studies suggesting that the inhibition in biofilm systems is less severe compared to planktonic-based systems. This has led to many studies investigating different methods of enhancing biofilms for biological WWT [20]. While SND has been intensely investigated, there are still gaps such as a lack of studies investigating the enhancement of anammox bacteria to give them a competitive advantage in the SND system in order to achieve maximum nitrogen removal.  
Figure 1: Schematic diagram of a biofilm immobilized onto the surface.