**7. The fixation of carbon dioxide by autotrophic nitrogen families**

Carbon dioxide (CO2) is an essential element for living organisms; however, its concentration was calculated to be 421.00 ppm (parts per million) in March 2023 [1], contributing to the global warming. Microbes are the key contributors for biological CO2 elimination by utilizing six different pathways for cell or carbohydrate syntheses. Six known pathways of CO2 fixation are Wood-Ljungdahl pathway (W-L), 3-Hydroxypropionate 4-hydroxybutyrate cycle (3HP-4HB),

Calvin-Benson-Bassham cycle (CBB), 3-Hydroxypropionate cycle (3-HP), Reductive tricarboxylic acid cycle (rTCA) and Dicarboxylate 4-hydroxybutyrate cycle (DC-4HB) [77]. In the context of autotrophic CO2 assimilation, five distinct groups of nitrogen-related microbes are involved in the four above-mentioned pathways. First, aerobic ammonia-oxidizing archaea including mesophilic *Crenarchaeota* and thermophilic *Thaumarchaeota* prefer to use their respective modified versions of 3-Hydroxypropionate 4-hydroxybutyrate cycle (3HP-4HB) known as the Crenarchaeal HP/HB cycle and Thaumarchaeal HP/HB cycle [78]. These pathways enable them to assimilate CO2 and carry out NH4 + -N oxidation simultaneously. Second, anaerobic ammonia oxidizers (anammox bacteria) employ the Wood-Ljungdahl pathway for CO2 assimilation during their unique anaerobic ammonia oxidation process [64]. Third, the Calvin Benson Bassham cycle (CBB) is present in ammonia-oxidizing bacteria [79] as well as in the four genera of nitrite oxidizers, namely *Nitrobacter*, *Nitrococcus*, *Nitrotoga* and *Nitrolancea* [80]. These organisms utilize the CBB cycle to fix CO2 and perform their respective oxidation processes. Fourth, nitrite-oxidizing *Nitrospira* and autotrophic-denitrifying *Thiobacillus denitrificans* have been found to involve in Reductive tricarboxylic acid (rTCA) cycle [74]. This pathway allows these organisms to fix CO2 while carrying out NO2 − -N oxidation or denitrification. It is worth noting that nitrite-oxidizing *Nitrococcus* and *Nitrospina* have higher potential for CO2 utilization in marine environments compared to ammonia-oxidizing archaea and bacteria, particularly during the early exponential phase of microbial growth. Conversely, ammonia-oxidizing *Nitrosomonas* demonstrate rapid rate of cell synthesis in both late exponential and stationary phases [81]. In summary, autotrophic nitrogen-functional microbes possess the remarkable ability to utilize nitrogen compounds and CO2 as energy source and cell synthesis. This capability not only contributes to the reduction in global warming but also aids in the removal of nitrogenous pollutants from the environment.

## **8. Cultivation systems of nitrogen-functional microbes**

The cultivation of nitrogen-functional microbes relies on providing suitable energy sources for their growth. This includes nitrogen sources, alkalinity (typically NaHCO3), buffer (KH2PO4 and Na2HPO4), nutrients and trace elements [82]. Different nitrogen sources are utilized depending on the specific group of microbes being cultivated. For instance, NH4 + -N, NO2 − -N, NH4 + -N/NO2 − -N, NO2 − -N/NO3 − -N and N2O were, respectively, used for ammonia oxidizers (AOA and AOB), nitrite oxidizer, anammox, denitrifier and N2O-utilizing microbes. In addition to nitrogen sources, essential nutrients, such as calcium, magnesium and iron, are provided through CaCl2, MgSO4 and FeSO4. In the case of ammonia oxidizers carrying out partial nitrification, Na2SO4 is added as a supplement. Trace elements, which are crucial for microbial growth, consist of CuSO4, ZnSO4, MnCl2, NiCl2, CoCl2, NaMoO4, NaSeO4, NaWO4, Na2-EDTA and H3BO4. To avoid Na2-EDTA and H3BO4 from serving as carbon source for the growth of heterotrophic bacteria, they are excluded from the trace elements for cultivation of N2O-utilizing microbes. These cultivation systems aim to provide the necessary nutrients and conditions for the successful growth of nitrogen-functional microbes, enabling their study and potential application in various nitrogen cycling processes.

The cultivation system's design plays a crucial role in the successful enlargement of nitrogen-functional microbes. An important factor to consider is the choice of

### *The Contribution of Autotrophic Nitrogen Oxidizers to Global Nitrogen Conversion DOI: http://dx.doi.org/10.5772/intechopen.112709*

a suitable habitat for their growth. In this regard, the downflow hanging sponge (DHS) system utilizes a polyurethane sponge as a supporting material, as depicted in **Figure 1**. This sponge provides a three-dimensional (3D) space that facilitates the growth of microorganisms. When wastewater, containing microbes and foods, flows into the sponge, microbial cells are retained both inside and outside the sponge media. The unique microenvironment allows for the coexistence of aerobic and anaerobic nitrogen-functional microbes. Specifically, the surface of the sponge supports the growth of aerobic autotrophs responsible for nitritation, while deeper within the media, anaerobic microbes catalyze the anammox process [82]. Since its initial development in 1995, the DHS system has undergone several modifications, resulting in six different configurations of sponge setups [29]. The superiorities of the DHS are high biomass retention, long sludge retention, minimal sludge production and less energy consumption, particularly benefiting to cultivate the slow-growing autotrophic nitrogen-functional microbes. The combination of the polyurethane sponge as a support material and the unique microenvironment provided by the DHS system contributes to the successful cultivation of nitrogen-functional microbes, enabling their study and application in various nitrogen cycling processes.

The G1-type DHS reactor is a nonsubmerged fixed-bed reactor, illustrated in **Figure 2**. It consists of a closed rectangular column with a total volume of 2.5 L, while the working volume is 0.596 L, considering the 98.4% void ratio of the sponge material. Inside the reactor, 19 strips of triangular sponge (sized 2.8 × 2.8 × 4 cm) are arranged on two opposite inner walls, with a gap of 0.5 cm between each consecutive sponge. The height of the reactor column was 1 m, but the effective height was 2 m, as the sponge strips adhered on opposite walls were connected in series during the operation of the reactor. This design allows for enhanced contact between the wastewater and the sponge media, promoting efficient microbial growth and nutrient removal. Another cultivated system is the upflow submerged sponge (USS) reactor, shown in **Figure 3**. This reactor configuration provides an effective volume of 1.5 L within a 3-L column. The USS reactor employs a 1-L sponge volume as the attached media, creating a coexisted environment for suspended and biofilm-type microorganisms. The temperature control in the USS reactor is achieved through a cycled

### **Figure 1.**

*Conceptual cross-sectional view of the cube-type downflow hanging sponge (DHS sponge) (modified from [29]).*

### **Figure 2.**

*Schematic diagram of G1-type downflow hanging sponge (DHS) system.*

water system, ensuring optimal conditions for microbial activity and growth. These cultivation systems, namely the G1-type DHS reactor and the USS reactor, provide suitable environments for the growth of nitrogen-functional microbes. The welldesigned arrangement of sponge media in these reactors allows for efficient nutrient utilization and microbial interactions, enabling the study and application of nitrogen cycling processes in wastewater treatment and environmental biotechnology.

### **9. Test procedure and analytical methods used in NH4 + -N and N2O oxidation**

The G1-type DHS system was used for three processes that enhance NH4 + -N transformation. First, a stepwise increase of NH4 + -N concentration from 30 to 400 mg N/L was performed at eight different phases in the nitritation system, which corresponded to the nitrogen load of 0.47 to 6.42 kg N/m3 -day. Second, the potential of the anammox system was tested using four different parameters of cultivated temperature, inflow rate, substrate concentrations (including NH4 + -N and NO2 − -N) and effluent recirculation. Third, the operational conditions of the complete nitrogen transformation in a single-type DHS reactor (namely CnDHS) were similar to those of the nitritation system. All systems were placed in the 30–35°C incubator. In addition, the

*The Contribution of Autotrophic Nitrogen Oxidizers to Global Nitrogen Conversion DOI: http://dx.doi.org/10.5772/intechopen.112709*

**Figure 3.** *Schematic diagram of upflow submerged sponge (USS) system.*

low oxygen concentration inside both nitritation and CnDHS systems was controlled by adjusting the airflow rate from 1.5 to 16 L/day based on the partial pressure of oxygen in the reactor.

Two USS systems were used to improve the efficiency of anammox and N2O oxidation. In the anammox reactor, the increase of nitrogen load was observed to take place from 9.60 to 38.4 mgN/L-day, along with the concentration increase of chloride from 160 to 1200 mg/L under a fixed HRT of 4.2 days. For N2O oxidation, the rise of N2O load was performed by increasing the substrate flow rate from 0.04 to 0.32 L/ day (HRT shortened from 4 days to 0.5 days) under the fixed N2O substrate of 25 mM in the liquid based on the 100% gaseous N2O dissolved in the medium. The flow of

air inside the reactor was controlled to adjust the desired oxygen concentration, and the exhaust gas from the reactor was collected using a gas bag. The microbial activity was further tested in the batch assays with different N2O concentrations under the satisfactory oxygen conditions.

To monitor the performance of all systems, NH4 + -N, NO2 − -N and NO3 − -N in influent and effluent were regularly measured using a colorimetric method (HACH, USA) and ion chromatograph (SH-120A, SHINE, New Zealand). The composition of off-gas was determined using gas chromatography (Shimadzu GC-8APT for O2, GC-8AIT for N2O, CO2 and N2). Theoretical DO concentration in the bulk liquid flowing on the sponge surface was computed from oxygen content in the gas phase according to modified Henry's equation, and the actual DO concentration in effluent was measured directly by a DO meter (YSI/Nanotech Inc., Japan). The concentration of the retained biomass in the sponge material was measured at the end of the operation, and the biomass was stored in a −20°C freezer for microbial clarification. DNA was first isolated using MOBIO PowerSoil DNA extraction kit, and microbial community and functional genes catalyzed nitrogen transformation were further analyzed by TOPO cloning kit and SybrGreen quantitative PCR (QuantStudio 1, ThermoFisher Scientific, USA) with the specific primer pairs.

### **10. Use of autotrophic N-removal processes for NH4 + -N reduction**

This section aims to assess the efficiency of NH4 + -N conversion by comparing three autotrophic N-removal processes, including nitritation (also called partial nitrification), anammox and complete nitrogen transformation in a single-type reactor. The G1-type DHS is used for microbial enlargement and functional evaluation. The findings of the study are presented and discussed below.

In the operation of nitritation, the G1-type DHS reactor was subjected to a total of seven phases, with NH4 + -N inflow rates ranging from 0.47 to 1.60 kgN/m3 -day. The hydraulic retention time (HRT) was fixed at 1.5 h, and the temperature was maintained at 30°C. The airflow rate, ranging from 2 to 16 L/day, was adjusted to control the oxygen content in the system. Microbes cultivated in the DHS exhibited high capability for NH4 + -N oxidation, achieving rates of up to 1.92 kgN/m3 -day even at low oxygen condition. This performance surpasses those of a fixed-film bioreactor (0.58 kgN/m3 -day) [83] and submerged membrane bioreactor (1.30 kgN/m3 -day) [84] operating under sufficient oxygen supply. Furthermore, partial NH4 + -N oxidation of 58.6% was attained at NH4 + -N load of 3.24 kgN/m3 -day. This resulted in the production of 37.5% NO2 − -N and 4.0% NO3 − -N with an oxygen concentration of 0.40% O2 (0.16 mg/L of DO) (see **Figure 4**) [85]. Similarly, a biofilm system demonstrated that partial nitrification with 50% NH4 + -N conversion was attained at oxygen concentrations below 0.2 mg/L [86]. The growth rate of ammonium oxidizers was 2.56-fold faster than that of nitrite oxidizers under the DO concentration below 1.0 mg/L [87]. Similarly, the growth yield of *Nitrosomonas* sp. under oxygen stress as low as 1% was found to be 5 times higher compared to conditions with saturated DO conditions. The G1-type DHS reactor provided a high biomass concentration of 3.84 g volatile solids (VS)/L, enabling a nitrifying activity of 0.20 kg NH4 + -N/kg VS-day. The activity of ammonia oxidizer in the DHS was comparable to those of the suspended growth-type reactors (0.17–0.29 kg NH4 + -N/kg VS-day) [88], and higher than that of the biofilm-type system (0.08–0.10 kg NH4 + -N/kg VS-day) [89]. However, GHP-N2O production was detected at a level of 0.5% in the DHS, equivalent to 13% of the

*The Contribution of Autotrophic Nitrogen Oxidizers to Global Nitrogen Conversion DOI: http://dx.doi.org/10.5772/intechopen.112709*

### **Figure 4.**

*Effect of O2 in the downflow hanging sponge (DHS) for nitritation on the ratio of NO2 − -Nproduced/NH4 + -Nremoved. Wherein, P1 ~ P7 is the data taken from phase 1 to phase 7 of the operation in the DHS.*

oxidized NH4 + -N under a gas-phase oxygen content of 0.4% (0.16 mg/L of DO) (see **Figure 5**). Similarly, N2O production was observed from 10% of the oxidized NH4 + -N under the O2 concentration of 0.18 mg/L [30]. The analysis of microbial community revealed that 32.4% phylotypes closely related to *Nitrosomonas* sp. strain ENI-11 dominated in the DHS, while denitrifying genera of *Azoarcus* and *Bradyrhizobium* and nitrite-oxidizing *Nitrobacter* coexisted and participated in the nitrogen cycle [90]. The *amoA* gene encoded in the enzyme, which catalyzes ammonium oxidation, was used for determining the functional microbes, resulting in the phylotypes within the *Nitrosomonas europaea/Nitrosococcus mobilis* lineage being the key players in the nitritation in the DHS at low oxygen atmosphere. The images of fluorescence *in situ*

### **Figure 5.**

*Effect of O2 in the downflow hanging sponge (DHS) for nitritation on the ratio of N2Oproduced/NH4 + -Nremoved. Wherein, P6 ~ P7 is the data taken from phase 6 to phase 7 of the operation in the DHS.*

hybridization (FISH) demonstrated that 41% of *β-*proteobacterial ammonia oxidizers coexisted with 5.4% of *Nitrobacter* spp. within the bacterial community, accounting for 83% of the total population. Based on these findings, it can be concluded that the ammonia oxidizer as *Nitrosomonas* family was numerically dominant over nitrite oxidizer in the DHS reactor, facilitating the occurrence of nitritation at low oxygen supply. However, the presence of nitrite reductase, involved in N2O production through nitrifier denitrification, was induced at low oxygen partial pressures [39].

The optimal proportion of NH4 + -N and NO2 − -N for anammox reaction was achieved through first-stage nitritation. The DHS employed for the anammox process operated at a total nitrogen load ranging from 0.48 to 5.96 kgN/m3 -day with NH4 + -N and NO2 − -N maintained at an equal proportion. The HRT was set between 0.7 and 2 h, and the reactor was operated at a temperature range of 30 to 35°C. The highest nitrogen-removal rate achieved in the DHS was 2.27 kg N/m3 -day, which surpassed the performance of other biofilm systems with the removal rates of 0.2 to 2.0 kgN/ m3 -day [91, 92]. However, the nitrogen-removal rate in the DHS was lower than that reported for an upflow fixed-bed column reactor designed for highly enriched anammox [93]. The DHS exhibited a biomass concentration of 5.59 g VS/L within the sponge media, enabling anammox activity of 0.39 kgN/kg VS-day. Remarkably high removal efficiency of 95.4% was achieved at a loading rate of 1.94 kgN/m3 -day and HRT of 1.0 h, giving NO2 − -Nutilized/NH4 + -Nremoved of 1.25 ± 0.080 and NO3 − -Nproduction/ NH4 + -Nremoved of 0.25 ± 0.042 [94]. Notably, no N2O was detected in the DHS, highlighting the physiological capacity of anammox bacteria to suppress N2O production [95]. Moreover, based on theoretical calculations, approximately 76% of the removed NH4 + -N was converted to N2 through the anammox reaction, while the remaining 24% was suggested to be consumed via NO3 − -N reduction processes, including assimilation and dissimilation, as well as denitrification [96]. The anoxic microenvironment within the sponge media of the DHS, as depicted in **Figure 1**, likely provided a reducing environment and limited carbon sources. Additionally, cell lysis resulting from microbial mortality during resting periods further contributed to the availability of organic matter. As discussed earlier, the co-occurrence of nitrate reduction or denitrification alongside the anammox reaction in the DHS led to higher nitrogen removal (95%) than other systems [93, 96–98]. The key microbial players in this community included anammox genera *Kuenenia* and *Anammoxoglobus*, ammonium-oxidizing genus *Nitrosomonas*, as well as denitrifying capability of the genera *Comamonas* [99] and *Diaphorobacter* [100]. Together, these microbial groups worked synergistically to reduce nitrogenous compounds and facilitate efficient nitrogen removal in the system.

On the contrary, when it comes to the USS system designed for treating high salinity wastewater with a low C/N ratio, a remarkable removal efficiency of 93.3% was attained at a nitrogen load of 38.4 mgN/L-day, even under a chloride (Cl<sup>−</sup> ) concentration of 300 mg/L. However, it should be noted that the increase of Cl<sup>−</sup> concentration to 1200 mg/L resulted in an extended adaptation period of 1 month for the utilization of NH4 + -N and NO2 − -N. Comparing the USS system to the DHS system used for treating fresh water, the USS system enlarged the main groups of anammox *Brocadia*, ammonia-oxidizing *Nitrosomonas*, canonical nitrite-oxidizing or comammox-functional *Nitrospira*. Additionally, denitrifying genera, such as *Denitratisoma*, *Acinetobacter*, *Pseudomonas* and *Comamonas*, were also observed in the bacterial community of the USS system. The activities of microbes involved in ammonia oxidation, comammox, anammox and denitrification were assessed using the functional indicators of *amoA*, *crenamoA*, *Nts-amoA*, *hszA*, *nirS* and *nirK* genes. As shown in **Figure 6**, the abundance of the former four genes notably increased after *The Contribution of Autotrophic Nitrogen Oxidizers to Global Nitrogen Conversion DOI: http://dx.doi.org/10.5772/intechopen.112709*

### **Figure 6.**

*Functional genes of microbes are involved in the conversion of NH4 + -N and NO2 − -N in the. Upflow submerged sponge (USS) system for treating high-salinity wastewater. Wherein,* amoA *and* crenamoA *stand for ammonium oxidation,* Nts-amoA *for Comamonas,* hszA *for anaerobic ammonia oxidation (anammox) reaction, and* nirS *and nirK for nitrite oxidation. In addition, the value below the detected limits of 2.32x101 copy/kg VSS was used as 1.61x101 copy/kg VSS.*

693 days of the cultivation in the USS system. In contrast, the presence of denitrifying *nirS* and *nirK* genes decreased over time. These findings suggest that anammox bacteria replaced the denitrifying microbes in facilitating the reduction of nitrogen oxides such as NO2 − -N and NO3 − -N. Additionally, the USS system demonstrated the dominance of slow-growing autotrophic nitrifiers harboring the *amoA* gene.

To optimize the synergy between nitritation and anammox, a single reactor capable of completely converting NH4 + -N to N2 was implemented in the DHS. Initially, the slow-growing and environmentally sensitive anammox bacteria were cultivated within the DHS, followed by the colonization of enlarged aerobic ammonia oxidizers coated on the outer surface of the sponge media. The DHS operation involved varying NH4 + -N loads from 0.30 to 2.42 kg N/m3 -day, while maintaining limited oxygen levels controlled by airflow of 1.5 to 5.4 L/day. Remarkably, the maximum nitrogen-removal rate reached 1.53 kgN/m3 -day, surpassing the performance of suspended sludge system (0.2 kgN/m3 -day) [101] and biofilm-type reactor (1.5 kg N/m3 -day) [89]. Furthermore, this system demonstrated stable autotrophic nitrogen removal, even at an HRT as short as 2 h, in contrast to other processes requiring much longer HRTs (up to 10 h). Notably, an impressive efficiency of 84.8% was attained at NH4 + -N load of 1.51 kgN/m3 -day, giving 84.5% of N2 production alongside 8.0% NH4 + -N and 7.5% of nitrogen oxides. The precise adjustment of oxygen content in the DHS proved crucial in controlling the proportion of NO3 − -N and N2 production. In **Figure 7**, it is evident that an oxygen concentration of 1.0% serves as the critical threshold for distinguishing the dominant reaction pathway between anammox and nitrification, as indicated by the ratio of NO3 − -N/ (NO2 − -N + NO3 − -N). Anammox bacteria predominantly catalyze the production of NO3 − -N when the O2 content falls below 1%, whereas complete nitrification, driven by the faster growth rate of nitrite oxidizers compared to ammonia oxidizers, occurs at O2 concentrations above 1% in gas phase. Remarkably, a reactor operating with O2 levels below 0.5% air saturation efficiently cultivates microbes with varying oxygen requirements [102]. However, the restricted O2 concentration below 1% stimulates GHP-N2O production through the activity of NO2 − -N and NO reductases,

### **Figure 7.**

*Effect of O2 in the downflow hanging sponge (DHS) for N removal on the production of NO3 − -N/ (NO2 − -N + NO3 − -N).*

resulting in a loss of 7.2% nitrogen in the DHS. Considering the mass balance, it is observed that 55.7% of NH4 + -N is utilized for NO2 − -N production, 34.5% is further transformed to gaseous N2, but 9.5% is diverted toward the formation of N2O under an O2 concentration below 0.48% (**Figure 8**). In the DHS, a similar pattern of N2O production was observed during nitritation, with N2O accounting for 13% of the total nitrogen gas at an O2 content of 0.4%. Additionally, the DHS supported the coexistence of six different nitrogen-functional microbes, namely aerobic ammonia-oxidizing *Nitrosomonas*, anaerobic ammonia-oxidizing *Brocadia*, canonical nitrite-oxidizing or comammox-functional *Nitrospira*, denitrifying *Comamonas* and nitrogen-fixing *Bradyrhizobium*. This diverse microbial community, facilitated

### **Figure 8.**

*Effect of O2 in the downflow hanging sponge (DHS) for N removal on the ratio of N2O-Nproduced/NH4 + -Nremoved. Wherein, P1 ~ P7 is the data taken from phase 1 to phase 7 of the operation in the DHS.*

by the DHS's excellent biomass retention capacity, created favorable conditions for the complete transformation of NH4 + -N to N2.
