Makoto Shoda

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/68052

#### **Abstract**

*Alcaligenes faecalis* No. 4 (No. 4) has the ability to carry out the following heterotrophic nitrification and aerobic denitrification, NH<sup>4</sup> <sup>+</sup> → NH<sup>2</sup> OH → N2 O → N2 . Approximately, 40 and 60% of ammonium were converted to N<sup>2</sup> gas and cell mass, respectively. Only a few percent of NO2 − and NO<sup>3</sup> − were produced from ammonium. After brief explanation of significant properties of No. 4, several examples of application of No. 4 to treat ammonium, especially high‐strength ammonium in several wastewaters were presented. The ammo‐ nium removal rates in these examples showed several hundredfold higher than those in conventional ammonium treatment method. In wastewater treatment plants, the selection of handling of excess sludge after treatment is a problem to be solved. Some possibilities of utilization of the excess cells of No. 4 in agriculture or in cattle farming were also presented.

**Keywords:** heterotrophic nitrification, aerobic denitrification, high‐strength ammonium, ammonium removal rates, utilization of organic acids, *Alcaligenes faecalis* No. 4

### **1. Introduction**

#### **1.1. Brief review of conventional ammonium removal by autotrophic nitrification and anaerobic denitrification**

The oxidation of the ammonium to nitrogen gas is achieved with two step reactions, namely aerobic nitrification and anaerobic denitrification. The most common bacteria respon‐ sible for the aerobic nitrification are the autotrophic organisms, such as *Nitrosomonas* and *Nitrobacter*. They obtain energy from the oxidation of ammonia, obtain carbon from CO<sup>2</sup> and use oxygen as the electron acceptor. Many different heterotrophs are responsible for anaerobic denitrification. They use carbon from complex organic compounds, prefer low to zero dissolved oxygen, and use nitrate as the electron acceptor.

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Biological removal of ammonium in a conventional treatment system has been conducted using the two reactions. However, this system faces several problems including: (1) an extremely slow nitrification reaction, (2) deterioration of activity against overloading of ammonium and organic matter, (3) strong sensitivity to oxygen limitation, and (4) requirement of two sepa‐ rate reactors for an aerobic process in nitrification and an anaerobic process in denitrification. The low nitrification rates in this process result in the need for long hydraulic retention times or large reactor volumes to accomplish complete NH<sup>4</sup> + removal. Consequently, conventional treatment demands multiple and larger reactors and high capital and operation costs.

Over the past two decades, several new bioprocesses for ammonium removal from municipal and domestic wastewaters have been developed, including: simultaneous nitrification and denitrifi‐ cation, shortcut nitrification and denitrification, aerobic deammonification, complete autotrophic nitrogen removal over nitrite (CANON), oxygen‐limited nitrification and denitrification (OLAND), advanced treatments using combination of these process including membrane bioreactors, and cell‐immobilization systems. However, these processes also have some potential problems or limi‐ tations especially for high‐strength ammonium treatment [1].

#### **1.2. Brief review of anammox method**

Anammox (anaerobic ammonium oxidation) is a recent understanding on the nitrogen cycle. *Candidatus* "Brocadia anammoxidans" and *Candidatus "*Kuenenia stuttgartiensis*"* are representative anammox bacteria.

Anammox method consists of partial aerobic nitrification and anaerobic denitrification.

$$\text{NH}\_4^+ \rightarrow \text{O}\_2 \rightarrow \text{NO}\_2^- \text{ and } \text{NH}\_4^+ \rightarrow \text{NO}\_2^- \rightarrow \text{NO}\_3^- + \text{N}\_2$$

The advantages of this method are: (1) very little sludge production, (2) reduced oxygen supply, and (3) no need to supplement organic carbons, which are related with operating cost problems in conventional ammonium treatment.

Concerning the ammonium removal rates in anammox method, the relatively higher removal rates, 0.96 kg‐N/m<sup>3</sup> /day in SHARON‐anammox process [2], 2.3 kg‐N/m<sup>3</sup> /day in fluidized bed using synthetic medium [3], and more than 4 kg‐N/m<sup>3</sup> /day of gel pellets of anammox biomass [4], were reported. The problems of the method are that: (1) sufficient amount of biomass production is time‐consuming, (2) the long time for stabilization of the system, (3) difficult quick recovery of the system when the inefficient removal occurred, (4) NO<sup>3</sup> − accumulation, and (5) slow phosphate removal rate.

### **1.3. Heterotrophic nitrification and aerobic denitrification**

Recently, many microorganisms have been found to conduct heterotrophic nitrification and aerobic denitrification. **Table 1** shows representative microorganisms published previously and their removal abilities. These microorganisms have advantages such as (1) procedural simplicity, where nitrification and denitrification can take place simultaneously, (2) less acclimation problems, (3) lesser buffer quantity needed because alkalinity generated during denitrification can partly compensate for the alkalinity consumption in nitrification.


Biological removal of ammonium in a conventional treatment system has been conducted using the two reactions. However, this system faces several problems including: (1) an extremely slow nitrification reaction, (2) deterioration of activity against overloading of ammonium and organic matter, (3) strong sensitivity to oxygen limitation, and (4) requirement of two sepa‐ rate reactors for an aerobic process in nitrification and an anaerobic process in denitrification. The low nitrification rates in this process result in the need for long hydraulic retention times

treatment demands multiple and larger reactors and high capital and operation costs.

Over the past two decades, several new bioprocesses for ammonium removal from municipal and domestic wastewaters have been developed, including: simultaneous nitrification and denitrifi‐ cation, shortcut nitrification and denitrification, aerobic deammonification, complete autotrophic nitrogen removal over nitrite (CANON), oxygen‐limited nitrification and denitrification (OLAND), advanced treatments using combination of these process including membrane bioreactors, and cell‐immobilization systems. However, these processes also have some potential problems or limi‐

Anammox (anaerobic ammonium oxidation) is a recent understanding on the nitrogen cycle. *Candidatus* "Brocadia anammoxidans" and *Candidatus "*Kuenenia stuttgartiensis*"* are representative

Anammox method consists of partial aerobic nitrification and anaerobic denitrification.

<sup>−</sup> and NH<sup>4</sup>

The advantages of this method are: (1) very little sludge production, (2) reduced oxygen supply, and (3) no need to supplement organic carbons, which are related with operating cost

Concerning the ammonium removal rates in anammox method, the relatively higher removal

[4], were reported. The problems of the method are that: (1) sufficient amount of biomass production is time‐consuming, (2) the long time for stabilization of the system, (3) difficult

Recently, many microorganisms have been found to conduct heterotrophic nitrification and aerobic denitrification. **Table 1** shows representative microorganisms published previously and their removal abilities. These microorganisms have advantages such as (1) procedural simplicity, where nitrification and denitrification can take place simultaneously, (2) less acclimation problems, (3) lesser buffer quantity needed because alkalinity generated during

denitrification can partly compensate for the alkalinity consumption in nitrification.

/day in SHARON‐anammox process [2], 2.3 kg‐N/m<sup>3</sup>

quick recovery of the system when the inefficient removal occurred, (4) NO<sup>3</sup>

<sup>+</sup> → NO2

<sup>−</sup> → NO3

<sup>−</sup> + N2

/day of gel pellets of anammox biomass

−

/day in fluidized bed

accumulation,

+

removal. Consequently, conventional

or large reactor volumes to accomplish complete NH<sup>4</sup>

tations especially for high‐strength ammonium treatment [1].

<sup>+</sup> → O2 → NO2

problems in conventional ammonium treatment.

and (5) slow phosphate removal rate.

using synthetic medium [3], and more than 4 kg‐N/m<sup>3</sup>

**1.3. Heterotrophic nitrification and aerobic denitrification**

**1.2. Brief review of anammox method**

anammox bacteria.

32 Nitrification and Denitrification

NH<sup>4</sup>

rates, 0.96 kg‐N/m<sup>3</sup>

**Table 1.** Reported strains which have ability of heterotrophic nitrification and aerobic denitrification. The two mechanisms for heterotrophic nitrification and aerobic denitrification are reported.

$$\text{(1)}\\\text{NH}\_4^+ \rightarrow \text{NH}\_2\text{OH} \rightarrow \text{NO}\_2^- \rightarrow \text{NO}\_3^- \text{ and } \text{NO}\_3^- \rightarrow \text{NO}\_2^- \rightarrow \text{N}\_2\text{O} \rightarrow \text{N}\_2$$

Both reactions occur simultaneously [6, 16].

$$\text{(2)}\,\text{NH}\_4^+ \to \text{NH}\_2\text{OH} \to \text{N}\_2\text{O} \to \text{N}\_2\text{}$$

Almost no nitrite or nitrate was produced and neither nitrite nor nitrate was utilized as electron accepters [12, 18].

This kind of bacteria may have the potential to overcome the problems inherent in the con‐ ventional nitrogen removal process and to realize one‐stage nitrogen removal under aerobic conditions.

In **Table 1**, low‐strength ammonium in synthetic medium was used and main carbon sources are organic acids. The use of practical wastewater is scarcely reported. *Alcaligenes faecalis* No. 4 (No. 4) we isolated is one of these microorganisms, and No. 4 showed efficient removal ability for high‐strength ammonium and significantly higher removal rate. The following sections present the results when No. 4 was applied to practical wastewaters.

### **2. Characteristics of** *Alcaligenes faecalis* **No. 4 (No. 4)**

#### **2.1. Basic features of No. 4 [18]**

#### *2.1.1. Materials and methods*

Strain used: *A. faecalis* No. 4 (No. 4) was isolated from sewage sludge as an antagonistic microorganism to plant pathogens [19] .

Synthetic medium used: A synthetic medium containing (in units of g/L) 14 K<sup>2</sup> HPO<sup>4</sup> , 6 KH<sup>2</sup> PO<sup>4</sup> , 17 trisodium citrate dihydrate, 2 (NH<sup>4</sup> ) 2 SO<sup>4</sup> , 0.2 MgSO<sup>4</sup> ∙7H<sup>2</sup> O, and 2 mL of trace mineral solution was used for the preculture of No. 4. The trace mineral solution contained the following components (in g/L): 57.1 EDTA (2,2′,2″,2″′‐(ethane‐1,2‐diyldinitrilo)tetra acetic acid)∙2Na, 3.9 ZnSO<sup>4</sup> ∙7H<sup>2</sup> O, 7CaCl<sup>2</sup> ∙2H<sup>2</sup> O, 5.1 MnCl<sup>2</sup> ∙4H<sup>2</sup> O, 5.0 FeSO<sup>4</sup> ∙7H<sup>2</sup> O, 1.1 (NH<sup>4</sup> )6 Mo<sup>7</sup> O24∙4H<sup>2</sup> O, 1.6 CuSO<sup>4</sup> ∙5H<sup>2</sup> O, and 1.6 CoCl<sup>2</sup> ∙6H<sup>2</sup> O.

Method: Available carbon sources and available nitrogen sources were surveyed using various carbon and nitrogen materials. Then, the initial ammonium concentration of (NH<sup>4</sup> ) 2 SO<sup>4</sup> was fixed and carbon content of citrate was change from C/N ratio 5–20 and optimal C/N ratio was deter‐ mined. Optimal temperature and pH were determined using synthetic medium. Nitrogen balance was obtained using NO*<sup>x</sup>* analyzer to detect NO and NO<sup>2</sup> in exhaust gas. All experiments were conducted using shaking flasks (100 ml working volume in 500 ml nominal volume of flask).

#### *2.1.2. Results*

The following results were obtained.

Available carbon sources: Organic acids (oxalate, citrate, lactate, formate, acetate, propionate, *iso*‐butyrate, *n*‐butyrate), amino acids, and phenol. No sugars were available.

Available nitrogen sources: Inorganic ammonium salts, peptone, yeast extract, and hydroxylamine. Neither nitrate nor nitrite was utilized.

Optimal C/N ratio: Optimal C/N ratio was 10 when the NH<sup>4</sup> + ‐N removal rate was the highest and citrate and ammonium were exhausted simultaneously.

Temperature range: 15–37°C. Optimal temperature was 30°C

Initial pH: In the range of 6–8, ammonium removal rate was almost the same.

Nitrogen balance: Nitrogen balance at the initial 1122 mg‐N/l is shown in **Table 2**.

The emitted NO was less than 3% of removed NH<sup>4</sup> + ‐N.

The two mechanisms for heterotrophic nitrification and aerobic denitrification are reported.

Almost no nitrite or nitrate was produced and neither nitrite nor nitrate was utilized as

This kind of bacteria may have the potential to overcome the problems inherent in the con‐ ventional nitrogen removal process and to realize one‐stage nitrogen removal under aerobic

In **Table 1**, low‐strength ammonium in synthetic medium was used and main carbon sources are organic acids. The use of practical wastewater is scarcely reported. *Alcaligenes faecalis* No. 4 (No. 4) we isolated is one of these microorganisms, and No. 4 showed efficient removal ability for high‐strength ammonium and significantly higher removal rate. The following sections

Strain used: *A. faecalis* No. 4 (No. 4) was isolated from sewage sludge as an antagonistic

, 0.2 MgSO<sup>4</sup>

was used for the preculture of No. 4. The trace mineral solution contained the following components (in g/L): 57.1 EDTA (2,2′,2″,2″′‐(ethane‐1,2‐diyldinitrilo)tetra acetic acid)∙2Na,

Method: Available carbon sources and available nitrogen sources were surveyed using various

and carbon content of citrate was change from C/N ratio 5–20 and optimal C/N ratio was deter‐ mined. Optimal temperature and pH were determined using synthetic medium. Nitrogen balance

conducted using shaking flasks (100 ml working volume in 500 ml nominal volume of flask).

∙4H<sup>2</sup>

∙7H<sup>2</sup>

∙7H<sup>2</sup>

O, 1.1 (NH<sup>4</sup>

O, 5.0 FeSO<sup>4</sup>

HPO<sup>4</sup>

) 2 SO<sup>4</sup>

O, and 2 mL of trace mineral solution

)6 Mo<sup>7</sup>

in exhaust gas. All experiments were

, 6 KH<sup>2</sup>

O24∙4H<sup>2</sup>

PO<sup>4</sup> ,

O, 1.6

was fixed

. Synthetic medium used: A synthetic medium containing (in units of g/L) 14 K<sup>2</sup>

carbon and nitrogen materials. Then, the initial ammonium concentration of (NH<sup>4</sup>

analyzer to detect NO and NO<sup>2</sup>

)2 SO<sup>4</sup>

O, 5.1 MnCl<sup>2</sup>

<sup>−</sup> <sup>→</sup> N2 <sup>O</sup> <sup>→</sup> N2

<sup>−</sup> <sup>→</sup> NO2

(1) NH<sup>4</sup>

(2) NH<sup>4</sup>

conditions.

<sup>+</sup> <sup>→</sup> NH<sup>2</sup> OH <sup>→</sup> NO2

34 Nitrification and Denitrification

electron accepters [12, 18].

**2.1. Basic features of No. 4 [18]**

microorganism to plant pathogens [19]

17 trisodium citrate dihydrate, 2 (NH<sup>4</sup>

O, 7CaCl<sup>2</sup>

O, and 1.6 CoCl<sup>2</sup>

The following results were obtained.

∙2H<sup>2</sup>

∙6H<sup>2</sup> O.

*2.1.1. Materials and methods*

∙7H<sup>2</sup>

was obtained using NO*<sup>x</sup>*

∙5H<sup>2</sup>

3.9 ZnSO<sup>4</sup>

*2.1.2. Results*

CuSO<sup>4</sup>

<sup>+</sup> <sup>→</sup> NH<sup>2</sup> OH <sup>→</sup> N2 <sup>O</sup> <sup>→</sup> N2

<sup>−</sup> <sup>→</sup> NO3

Both reactions occur simultaneously [6, 16].

<sup>−</sup> and NO3

present the results when No. 4 was applied to practical wastewaters.

**2. Characteristics of** *Alcaligenes faecalis* **No. 4 (No. 4)** 

#### **2.2. Verification of N<sup>2</sup> production directly from ammonium by No. 4 [18]**

A 15N tracer experiment using (15NH<sup>4</sup> )2 SO<sup>4</sup> (50% by atomic fraction, Nippon Sanso Co., Ltd.) was carried out to confirm the production of N<sup>2</sup> by No. 4 in an aerated batch culture in the basic medium under C/N = 10 at 30°C. The exhaust gas was directly introduced into the GC/MS (GC 6850, Agilent Technologies, Japan, Ltd.). The change in nitrogen isotope ratio was measured and N<sup>2</sup> production by No. 4 was calculated from the difference between output <sup>29</sup>N2 and input 29N2 .

**Figure 1** shows temporal changes in N2 and N<sup>2</sup> O concentrations. It was confirmed that No. 4 can convert NH<sup>4</sup> +‐N to N2 gas and that N<sup>2</sup> production ratio among denitrified products was about 90%. In conventional denitrification, 20–30% of influent nitrogen was estimated to be emitted as N<sup>2</sup> O under high‐strength ammonium conditions. In this system, N<sup>2</sup> O production was less than 10% of removed ammonium.

#### **2.3. Ammonium removal under high salt condition by No. 4 [20]**

No. 4 exhibited the unique feature of removing ammonium under high salt conditions. **Figure 2** shows change in the ammonium concentration in the cultivation of No. 4 in synthetic medium containing 0, 3, and 6% NaCl in shaking flasks. Ammonium removal began after induction periods of 1 day at 3% NaCl and 5 days at 6% NaCl and the ammonium removal rates were similar to those found in the presence of 0% NaCl. Although No. 4 is not osmophilic, the cells


**Table 2.** Nitrogen balance (units: mg/l) of ammonium removal in shaken flask experiment by *A. faecalis* No. 4 after 93h cultivation [18].

**Figure 1.** Denitrification characteristics of No. 4 detected by using (15NH<sup>4</sup> ) 2 SO<sup>4</sup> . Symbols: NO (○), N<sup>2</sup> O (△) and N<sup>2</sup> (■) [18].

**Figure 2.** Ammonium removal by No. 4 under 0% NaCl (△), 3% NaCl (■) and 6% NaCl (●) conditions in shaking flasks containing 100 ml of synthetic medium at 30°C [20].

were able to achieve ammonium removal under high saline conditions. In our basic experi‐ ment, No. 4 was found to synthesize the osmoprotectant, hydroxyectoine during the lag time when the cells were exposed to high salt concentrations. Because most microorganisms are vulnerable to wastewater with high saline concentrations or high‐strength solvents due to the resulting high osmotic pressure, No. 4 is able to effectively remove ammonium under such conditions after a certain acclimation period. Thus, the No. 4 system can remove high‐ strength ammonium from marine aqua‐culture wastewater or fishery processing wastewater.

In the following sections, examples of removal of high‐strength ammonium from practical wastewaters are presented.
