**3. Application of No. 4 to removal of high strength ammonium in various wastewaters**

#### **3.1. Crude piggery wastewater [21]**

#### *3.1.1. Introduction*

Piggery wastewater contains not only high concentrations of nitrogen compounds but also high concentrations of carbon materials. The ammonium concentration reaches up to 1000–3000 mg/l, which is 50–100 times higher than in municipal wastewater. The C/N ratio of the mixture of urine and feces in piggery wastewater is usually in the range of 5–20. Therefore, conventional nitrification using autotrophic bacteria is difficult to apply to such wastewater because nitrification by autotrophic bacteria requires a long retention time of flowing wastewater in the reactor due to the slow growth rates of these autotrophic bacte‐ ria. Thus, No. 4 was applied to batch and continuous cultures using solids‐free wastewater (referred to as SFW) either alone or supplemented with additional carbon sources such as citrate or feces.

#### *3.1.2. Materials and methods*

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

**Figure 2.** Ammonium removal by No. 4 under 0% NaCl (△), 3% NaCl (■) and 6% NaCl (●) conditions in shaking flasks

) 2 SO<sup>4</sup>

. Symbols: NO (○), N<sup>2</sup>

O (△) and N<sup>2</sup>

(■) [18].

containing 100 ml of synthetic medium at 30°C [20].

**Figure 1.** Denitrification characteristics of No. 4 detected by using (15NH<sup>4</sup>

36 Nitrification and Denitrification

Microorganisms: The cells of No. 4 were stored in a 25% glycerol solution in vials at −80°C and each vial was used for preculture.

Medium: The synthetic medium described above was used as a preculture. In continuous culture, 500 ml of the preculture was prepared and put into the reactor.

Piggery wastewater: Piggery wastewater was provided by the Kanagawa Prefectural Livestock Industry Research, Kanagawa, Japan. Solids‐free wastewater (SFW) was obtained by separating the solids from the raw wastewater containing urine and washing water and feces by centrifugation at 1000 rpm. **Table 3** shows the characteristics of the SFW and mixed wastewater comprised of SFW supplemented with feces (3:1 on a weight basis) (referred to as MW).

Continuous experiments: Continuous treatment of SFW and MW was conducted in a 2.3 l aeration tank at room temperature at the airflow rate of 2.5 l/min. A total of 500 ml of No. 4 culture was mixed with wastewater and open continuous experiments were started by supplying SFW.


**Table 3.** Characteristics of piggery wastewaters used [21].

#### *3.1.3. Results*

#### *3.1.3.1. Batch experiments*

SFW or MW was treated with No. 4 in shaking flasks and the removal of NH<sup>4</sup> + ‐N was measured. In SFW, the addition of citrate was needed mainly due to small amount of carbon in SFW. In MW without addition of carbon, the ammonium removal proceeded smoothly and the maximum ammonium removal rates in SFW with supply of citrate and in MW were 0.7 and 0.66 kg‐N/m<sup>3</sup> /day, respectively.

#### *3.1.3.2. Continuous experiments*

**Figure 3** shows the results of SFW and MW treatment in continuous treatment in for 80 days. Initial 10 days, only SFW was supplied and the ammonium removal ratio declined mainly because of lacking of carbon source. Then, citric acid was added and the hydraulic retention time (HRT) was reduced. Consequently, ammonium removal was stabilized to about 80%. Then, the inlet ammo‐ nium concentration and citric acid increased gradually and the removal ratio reached almost 100%. From 52 days, instead of supplying citrate, influent NH<sup>4</sup> + ‐N concentration was increased to 2000 mg/l with addition of feces and HRT was set at 60 h. After 52 days, ammonium removal was high at 100% and outlet of ammonium was less than 20 mg/l. The pH was maintained at 7.4.8 after supply of MW and stripped ammonia from reactor was 2–5% of inlet ammonium concentration. The system was in a steady state. The cells number of No. 4 was measured with L agar plates. The data are summarized in **Table 4**. The nitrogen and carbon balances in the experimental periods are also shown in **Table 4**. After 52 days, feces containing MW were added directly. The denitri‐ fied N calculated from inlet ammonium minus the nitrogenous items was 73% and striking result was the high removal of COD. The estimated cell number of No. 4 reached up to 97% of total cells in the samples. The ammonium removal rate, 33 mg‐N/l/h corresponded to 0.79 kg‐N/m<sup>3</sup> /day, which was a few hundred times higher than conventional treatment methods. In diluted and digested piggery wastewater at C/N = 1, 64 mg/l/h removal rate was reported [22]. However, in the present study, No. 4 provided suitable to treat undiluted piggery wastewater with C/N ratio of 10, yielding removal rate of 33 mg‐N/l/h (0.79 kg‐N/m<sup>3</sup> /day).

#### **3.2. Anaerobically digested sludge [20]**

#### *3.2.1. Introduction*

Due to recent trends of limiting fossil energy consumption, sustainable methods of energy pro‐ duction including methane production in anaerobic digestion or bioethanol production have

*3.1.3. Results*

*3.1.3.1. Batch experiments*

38 Nitrification and Denitrification

**NH4 +**

**Table 3.** Characteristics of piggery wastewaters used [21].

\*SFW: Solid‐free wastewater (units, mg/l). \*\*MW: Mixed wastewater (units, mg/l).

**‐N NO2**

and 0.66 kg‐N/m<sup>3</sup>

*3.1.3.2. Continuous experiments*

/day, respectively.

100%. From 52 days, instead of supplying citrate, influent NH<sup>4</sup>

of 10, yielding removal rate of 33 mg‐N/l/h (0.79 kg‐N/m<sup>3</sup>

**3.2. Anaerobically digested sludge [20]**

*3.2.1. Introduction*

SFW or MW was treated with No. 4 in shaking flasks and the removal of NH<sup>4</sup>

**− ‐N, NO3 −**

\*SFW 830–1250 Less than 1 4150–5300 4–5 \*\*MW 1850–1960 Less than 1 13800–14650 7–9

measured. In SFW, the addition of citrate was needed mainly due to small amount of carbon in SFW. In MW without addition of carbon, the ammonium removal proceeded smoothly and the maximum ammonium removal rates in SFW with supply of citrate and in MW were 0.7

**Figure 3** shows the results of SFW and MW treatment in continuous treatment in for 80 days. Initial 10 days, only SFW was supplied and the ammonium removal ratio declined mainly because of lacking of carbon source. Then, citric acid was added and the hydraulic retention time (HRT) was reduced. Consequently, ammonium removal was stabilized to about 80%. Then, the inlet ammo‐ nium concentration and citric acid increased gradually and the removal ratio reached almost

2000 mg/l with addition of feces and HRT was set at 60 h. After 52 days, ammonium removal was high at 100% and outlet of ammonium was less than 20 mg/l. The pH was maintained at 7.4.8 after supply of MW and stripped ammonia from reactor was 2–5% of inlet ammonium concentration. The system was in a steady state. The cells number of No. 4 was measured with L agar plates. The data are summarized in **Table 4**. The nitrogen and carbon balances in the experimental periods are also shown in **Table 4**. After 52 days, feces containing MW were added directly. The denitri‐ fied N calculated from inlet ammonium minus the nitrogenous items was 73% and striking result was the high removal of COD. The estimated cell number of No. 4 reached up to 97% of total cells in the samples. The ammonium removal rate, 33 mg‐N/l/h corresponded to 0.79 kg‐N/m<sup>3</sup>

which was a few hundred times higher than conventional treatment methods. In diluted and digested piggery wastewater at C/N = 1, 64 mg/l/h removal rate was reported [22]. However, in the present study, No. 4 provided suitable to treat undiluted piggery wastewater with C/N ratio

Due to recent trends of limiting fossil energy consumption, sustainable methods of energy pro‐ duction including methane production in anaerobic digestion or bioethanol production have

+

**‐N CODCr C/N ratio**

/day).

+ ‐N was

/day,

‐N concentration was increased to

**Figure 3.** Ammonium concentration in influent (●) and effluent (○), stripped ammonia (◆), removal rate of ammonium (◇), removal ratio of ammonium (△) and hydraulic retention time (HRT) (solid line) in the continuous treatment of solid‐free piggery wastewater (before 52 days) and mixed wastewater (after 52 days) by No. 4 [21].


**Table 4.** The nitrogen balance and carbon change in continuous experiment using No. 4 for treatment of solid free piggery wastewater and mixed wastewater All data were average values in the operation periods [21].

been attracting increasing attention. In anaerobic digestion, livestock waste, municipal garbage, and waste from the food industry are used for the digestion, leading to the production of waste‐ water containing a high concentration of ammonium. Therefore, the development of an effec‐ tive method of the wastewater treatment is a crucial factor enabling the production of methane.

In this section, No. 4 was applied to remove high‐strength ammonium from digested sludge generated in a municipal anaerobic digestion plant to assess the possibility of efficient biological treatment of the wastewater.

#### *3.2.2. Materials and methods*

The reactor used the reactions that were carried out in a small‐scale jar fermenter (total volume 1 l, working volume 300 ml). Dissolved oxygen (DO) concentrations and pH values were monitored with a DO sensor and pH sensor inserted into the fermenter. The temperature was controlled at 20 or 30°C. The oxygen transfer coefficients, kLa, were varied by changing the agitation speed from 300 to 700 rpm at a constant air supply rate of 300 ml/min.

Experimental material: The digested sludge was supplied by Yokohama Municipal Sewage Center, Yokohama, Japan, where the excess municipal dehydrated activated sludge was digested at 37°C in a 6000 ton‐scale anaerobic digester. The main characteristics of the digested sludge are as follows: pH 7.3, 24 mg/l volatile fatty acids, 2700 mg/l total nitrogen, 1200 mg/l ammonium‐nitrogen, 150 mg/l soluble BOD, 1000 mg/l total BOD, 900 mg/l soluble COD, and 20,000 mg/l total COD.

Experimental procedure: The strain No. 4 cells were precultivated in 100 ml synthetic medium in a 500 ml shaking flask at 30°C. The ammonium removal was confirmed in the mixture of 250 ml of digested sludge, 50 ml of strain No. 4 preculture, and 20 g of trisodium citrate dehydrate in the jar fermenter operated at 30°C at an airflow rate of 300 ml/min and at an agitation speed of 700 rpm.

In repeated batch experiments, 50 ml of the preculture of No. 4, 250 ml of the digested sludge, and 20 g of trisodium citrate dihydrate were mixed in the fermenter, and the treatment of the ammonium was conducted. After the ammonium concentration was confirmed to be reduced by more than 90% of the initial concentration, 50 ml of the culture was used for the subsequence treatment by adding a fresh 250 ml of digested sludge and 20 g of trisodium citrate dihydrate.

The optimal C/N ratio for No. 4 was 10, indicating that at this ratio, nitrogen and carbon sources were simultaneously consumed. Based on the ratio, 1 g‐N and 10 g‐C was balanced and thus 10 g‐C corresponded to 38 g of trisodium citrate dihydrate. If no other carbon source existed in the sludge, 38 g of trisodium citrate should be added. In this experiment, 20 g of trisodium citrate dihydrate was arbitrarily chosen by expecting existence of some carbon sources in the sludge.

Analytical method: The ammonium concentration was determined using an ammonium sensor. To determine the number of cells No. 4, the sampled culture was diluted and plated on synthetic agar plates containing the synthetic medium and 1.5% agar, and then the plates were incubated at 30°C for 2 days. As No. 4 grew on the plates significantly faster than other cells indigenous to the digested sludge and that No. 4 exhibited characteristic morphological features, the colonies that appeared on the plates after 2 days were counted as No. 4 cells and the cell concentration was expressed as cells/ml.

#### *3.2.3. Results*

been attracting increasing attention. In anaerobic digestion, livestock waste, municipal garbage, and waste from the food industry are used for the digestion, leading to the production of waste‐ water containing a high concentration of ammonium. Therefore, the development of an effec‐ tive method of the wastewater treatment is a crucial factor enabling the production of methane. In this section, No. 4 was applied to remove high‐strength ammonium from digested sludge generated in a municipal anaerobic digestion plant to assess the possibility of efficient

The reactor used the reactions that were carried out in a small‐scale jar fermenter (total volume 1 l, working volume 300 ml). Dissolved oxygen (DO) concentrations and pH values were monitored with a DO sensor and pH sensor inserted into the fermenter. The temperature was controlled at 20 or 30°C. The oxygen transfer coefficients, kLa, were varied by changing the

Experimental material: The digested sludge was supplied by Yokohama Municipal Sewage Center, Yokohama, Japan, where the excess municipal dehydrated activated sludge was digested at 37°C in a 6000 ton‐scale anaerobic digester. The main characteristics of the digested sludge are as follows: pH 7.3, 24 mg/l volatile fatty acids, 2700 mg/l total nitrogen, 1200 mg/l ammonium‐nitrogen, 150 mg/l soluble BOD, 1000 mg/l total BOD, 900 mg/l soluble COD, and

Experimental procedure: The strain No. 4 cells were precultivated in 100 ml synthetic medium in a 500 ml shaking flask at 30°C. The ammonium removal was confirmed in the mixture of 250 ml of digested sludge, 50 ml of strain No. 4 preculture, and 20 g of trisodium citrate dehydrate in the jar fermenter operated at 30°C at an airflow rate of 300 ml/min and at an

In repeated batch experiments, 50 ml of the preculture of No. 4, 250 ml of the digested sludge, and 20 g of trisodium citrate dihydrate were mixed in the fermenter, and the treatment of the ammonium was conducted. After the ammonium concentration was confirmed to be reduced by more than 90% of the initial concentration, 50 ml of the culture was used for the subsequence treatment by adding a fresh 250 ml of digested sludge and 20 g of trisodium

The optimal C/N ratio for No. 4 was 10, indicating that at this ratio, nitrogen and carbon sources were simultaneously consumed. Based on the ratio, 1 g‐N and 10 g‐C was balanced and thus 10 g‐C corresponded to 38 g of trisodium citrate dihydrate. If no other carbon source existed in the sludge, 38 g of trisodium citrate should be added. In this experiment, 20 g of trisodium citrate dihydrate was arbitrarily chosen by expecting existence of some carbon

Analytical method: The ammonium concentration was determined using an ammonium sensor. To determine the number of cells No. 4, the sampled culture was diluted and plated

agitation speed from 300 to 700 rpm at a constant air supply rate of 300 ml/min.

biological treatment of the wastewater.

*3.2.2. Materials and methods*

40 Nitrification and Denitrification

20,000 mg/l total COD.

agitation speed of 700 rpm.

citrate dihydrate.

sources in the sludge.

#### *3.2.3.1. Ammonium removal in the repeated batch experiment*

**Figure 4** shows the change in ammonium concentration over times in a repeated batch experiment at 30°C, and **Figure 5** shows the change in the number of No. 4 cells during the same experiment. More than 90% of ammonium was removed within 10–20 h, and the number of No. 4 cells varied between 10<sup>8</sup> and 109 cells/ml. The average ammonium removal rate during the experimental period was 2.9 kg‐N/m<sup>3</sup> /day. This value is significantly higher than that in conventional nitrification‐denitrification processes and similar to that in an efficient anammox process [3, 4]. Between 169 and 221 h, the operation was stopped and the jar fermenter was left statically at room temperature. When the operation resumed, ammonium removal was observed without any delay, indicating that interrupted operation causes no adverse effect on the activity of No. 4. At 20°C, the average ammonium removal rate decreased to 1.5 kg‐N/m<sup>3</sup> /day.

**Figure 4.** Change in ammonium concentrations of the digested sludge in repeated batch treatment by No. 4. Operation was conducted at 30°C at agitation speed of 700 rpm in a jar fermenter [20].

**Figure 5.** Change in the cell number of No. 4 in the same experiment as shown in **Figure 4** [20].

#### *3.2.3.2. Effect of DO concentration on ammonium removal*

In practical operation, DO concentration is related to energy consumption, agitation, air sup‐ ply, and the activity of No. 4. The effects of changes in the oxygen supply rate on ammonium removal were studied by changing the agitation speed from 700 to 300 rpm. At 700 rpm, the DO concentration was maintained at more than 2 mg/l during the operation, and the ammonium was completely removed within 10 h. However, when the agitation speed was decreased to 400 or 300 rpm, the DO concentration decreased below 1 mg/l, reducing the ammonium removal rate, indicating that the oxygen supply is an important factor for efficient ammonium removal.

#### *3.2.3.3. Ammonium removal under high salt conditions*

Strain No. 4 exhibited the unique feature of removal ammonium under high salt conditions as shown in Sections 2 and 3. NaCl was added to the digested sludge to 3%, and repeated batch treatment was conducted at 30°C in a jar fermenter. The results of this experiment are shown in **Figure 6**. The ammonium removal rate reached 3 kg‐N/m<sup>3</sup> /day at the sixth repeated batch operation after the gradual acclimation of No. 4 to the saline medium.

#### *3.2.3.4. Carbon requirement by No. 4*

The experiment performed here included 20 g of trisodium citrate dihydrate. Generally, the C/N ratio of the intracellular components in microorganisms is 10, indicating that 10 units of carbon are used when 1 unit of N is consumed with the C mainly to synthesize cellular materials. In previous experiments, 30–40% of ammonium was reduced to nitrogen gas by No. 4. Assuming a similar level of denitrification in these experiments, 0.6–0.7 g‐N/l was used for cell synthesis, indicating that 6–7 g‐C/l was required. When 20–30% of carbon is available from the digested sludge, approximately 5 g‐C/l should be supplied from outside. As 20 g of trisodium citrate dihydrate contained 16 g‐C/l, 6 g of trisodium citrate dihydrate is sufficient to enable the complete removal of 1 g‐N/l.

**Figure 6.** Ammonium removal by No. 4 in the digested sludge containing 3% NaCl by repeated batch experiment. Symbols: 1st (□), 2nd (◆), 3rd (▲), 4th (■), 5th(△) and 6th (●) [20].

Concerning carbon requirement in strain No. 4, the conventional denitrification process using methanol is compared by using the following reaction.

$$\rm NO\_3^- + 1.08\,\rm CH\_3OH + H^+ \to 0.065\,\rm C\_5H\_7NO\_2 + 0.47\,\rm N\_2 + 0.76\,\rm CO\_2 + 2.44\,\rm H\_2O \tag{1}$$

The C/N ratio in this reaction is 2. No. 4 process demanded C/N ratio 10. In this point, strain No. 4 process is disadvantageous. However, as a total system, No. 4 process will be advan‐ tageous over the conventional process in that no dilution of high strength of wastewater is required, only single reactor with compact size is needed and significantly high removal rate is possible when less expensive carbon sources from waste or unused resources are available. Under these conditions, this system can achieve efficient ammonium removal.

Higher ammonium removal rates have been reported using the anammox method as described in Sections 1 and 2. On the other hand, it is easy to cultivate strain No. 4 in a synthetic medium with a doubling time of 2–3 h. When cultured strain No. 4 cells were stored at 4°C, high activity was maintained for several months, and the cells remained tolerant to high osmotic pressure. The comparison of three methods of ammonium treatment is shown in **Table 8** of Section 5.

In relatively small‐scale reactors like this jar fermenter, oxygen supply capacity is lower than those large‐scale reactors. The power requirement in wastewater treatment is one of the important factors considered to be in operation. Thus, DO level in large scale reactors can be maintained at lower agitation speeds and the power requirement for strain No. 4 for high‐ strength ammonium treatment will be almost equivalent to that for low‐strength ammonium treatment in conventional aerobic nitrification process.

#### **3.3. Wastewater from a chemical company [23]**

#### *3.3.1. Introduction*

*3.2.3.2. Effect of DO concentration on ammonium removal*

**Figure 5.** Change in the cell number of No. 4 in the same experiment as shown in **Figure 4** [20].

*3.2.3.3. Ammonium removal under high salt conditions*

in **Figure 6**. The ammonium removal rate reached 3 kg‐N/m<sup>3</sup>

operation after the gradual acclimation of No. 4 to the saline medium.

ammonium removal.

42 Nitrification and Denitrification

*3.2.3.4. Carbon requirement by No. 4*

to enable the complete removal of 1 g‐N/l.

In practical operation, DO concentration is related to energy consumption, agitation, air sup‐ ply, and the activity of No. 4. The effects of changes in the oxygen supply rate on ammonium removal were studied by changing the agitation speed from 700 to 300 rpm. At 700 rpm, the DO concentration was maintained at more than 2 mg/l during the operation, and the ammonium was completely removed within 10 h. However, when the agitation speed was decreased to 400 or 300 rpm, the DO concentration decreased below 1 mg/l, reducing the ammonium removal rate, indicating that the oxygen supply is an important factor for efficient

Strain No. 4 exhibited the unique feature of removal ammonium under high salt conditions as shown in Sections 2 and 3. NaCl was added to the digested sludge to 3%, and repeated batch treatment was conducted at 30°C in a jar fermenter. The results of this experiment are shown

The experiment performed here included 20 g of trisodium citrate dihydrate. Generally, the C/N ratio of the intracellular components in microorganisms is 10, indicating that 10 units of carbon are used when 1 unit of N is consumed with the C mainly to synthesize cellular materials. In previous experiments, 30–40% of ammonium was reduced to nitrogen gas by No. 4. Assuming a similar level of denitrification in these experiments, 0.6–0.7 g‐N/l was used for cell synthesis, indicating that 6–7 g‐C/l was required. When 20–30% of carbon is available from the digested sludge, approximately 5 g‐C/l should be supplied from outside. As 20 g of trisodium citrate dihydrate contained 16 g‐C/l, 6 g of trisodium citrate dihydrate is sufficient

/day at the sixth repeated batch

Some wastewaters from chemical companies or power‐generation plants contain a high concentration of ammonium and a small amount of BOD. In this section, No. 4 was applied to a wastewater from a chemical company to assess the possibility of the efficient biological treatment of high‐strength ammonium.

#### *3.3.2. Materials and methods*

#### *3.3.2.1. Wastewater used*

The wastewater (WC) was supplied by a Japanese chemical company. The main characteristics of the WC are as follows: pH 10.6, total COD concentration of 2280 mg/l, total BOD concentra‐ tion of less than 2 mg/l, total‐nitrogen concentration of 4840 mg/l, and ammonium‐nitrogen concentration of 4800 mg/l. In each experiment, the pH of the original WC was adjusted to approximately 7.5 by 5N H<sup>2</sup> SO<sup>4</sup> , and the ammonium concentrations of pH‐adjusted WC was diluted to approximately 1000 mg/l unless specifically described.

The experimental procedures in this section were similar to those in Section 3.2.

#### *3.3.3. Results*

#### *3.3.3.1. Ammonium removal in the repeated‐batch experiment*

**Figure 7** shows the change in the ammonium concentration over times in a repeated‐batch experiment at 30°C, and **Figure 8** shows the change in the number of No. 4 cells during the same experiment. More than 90% of ammonium was removed within 24–30 h, and the num‐ ber of No. 4 cells varied between 10<sup>8</sup> and 1010 cells/ml. The average ammonium removal rate during the experimental period was 1.1 kg‐N/m<sup>3</sup> /day. Between 620 and 800 h, the operation was stopped, and the jar fermenter was maintained static at room temperature (average 10°C). When the operation was resumed, ammonium removal was observed without any delay, indicating that the interruption in the operation exerted no adverse effect on the activ‐ ity of No. 4. In these experiments, the pH values were fluctuated between 7 and 8, which are within the optimal pH range of No. 4. Total amounts of nitrite, nitrate, and exhausted ammonium from the reactors were less than 2% of inlet nitrogen, and thus the majority of inlet ammonium was converted into N<sup>2</sup> gas and the cellular nitrogenous compounds.

**Figure 7.** Ammonium concentration in the wastewater from a chemical company during repeated‐batch treatment with No. 4 at 30°C [23].

#### *3.3.3.2. Ammonium removal at initial ammonium concentrations of 1000, 2000, and 5000 mg NH4 + ‐N/l*

*3.3.2. Materials and methods*

approximately 7.5 by 5N H<sup>2</sup>

ber of No. 4 cells varied between 10<sup>8</sup>

inlet ammonium was converted into N<sup>2</sup>

during the experimental period was 1.1 kg‐N/m<sup>3</sup>

*3.3.3. Results*

No. 4 at 30°C [23].

SO<sup>4</sup>

*3.3.3.1. Ammonium removal in the repeated‐batch experiment*

diluted to approximately 1000 mg/l unless specifically described.

The experimental procedures in this section were similar to those in Section 3.2.

The wastewater (WC) was supplied by a Japanese chemical company. The main characteristics of the WC are as follows: pH 10.6, total COD concentration of 2280 mg/l, total BOD concentra‐ tion of less than 2 mg/l, total‐nitrogen concentration of 4840 mg/l, and ammonium‐nitrogen concentration of 4800 mg/l. In each experiment, the pH of the original WC was adjusted to

**Figure 7** shows the change in the ammonium concentration over times in a repeated‐batch experiment at 30°C, and **Figure 8** shows the change in the number of No. 4 cells during the same experiment. More than 90% of ammonium was removed within 24–30 h, and the num‐

was stopped, and the jar fermenter was maintained static at room temperature (average 10°C). When the operation was resumed, ammonium removal was observed without any delay, indicating that the interruption in the operation exerted no adverse effect on the activ‐ ity of No. 4. In these experiments, the pH values were fluctuated between 7 and 8, which are within the optimal pH range of No. 4. Total amounts of nitrite, nitrate, and exhausted ammonium from the reactors were less than 2% of inlet nitrogen, and thus the majority of

**Figure 7.** Ammonium concentration in the wastewater from a chemical company during repeated‐batch treatment with

, and the ammonium concentrations of pH‐adjusted WC was

and 1010 cells/ml. The average ammonium removal rate

gas and the cellular nitrogenous compounds.

/day. Between 620 and 800 h, the operation

*3.3.2.1. Wastewater used*

44 Nitrification and Denitrification

**Figure 9** shows the ammonium removal obtained with initial ammonium concentrations of approximately 5000 mg NH<sup>4</sup> + ‐N/l, 2000 mg NH<sup>4</sup> + ‐N/l and 1000 mg NH<sup>4</sup> + ‐N/l. For concentrations of 5000 mg NH<sup>4</sup> + ‐N/l and 2000 mg NH<sup>4</sup> + ‐N/l, an intermittent supply of 20 g of trisodium citrate dihydrate was introduced, as indicated by the arrows in **Figure 9**. The average ammonium removal rates for 1000, 2000, and 5000 mg NH<sup>4</sup> + ‐N/l were 0.63, 0.96, and 0.92 kg‐N/m<sup>3</sup> / day, respectively. This indicates that even ammonium concentrations higher than 1000 mg NH<sup>4</sup> + ‐N/l were removed efficiently by supplying a sufficient amount of the carbon source.

**Figure 8.** Change in the number of No. 4 cells in the same experiment shown in **Figure 7** [23].

**Figure 9.** Change in the initial ammonium concentrations when 5000 mg‐NH<sup>4</sup> + ‐N/l (■), 2000 mg‐NH<sup>4</sup> + ‐N/l (▲), and 1000 mg‐NH<sup>4</sup> + ‐N/l (●) of wastewater from a chemical company were used in a batch culture. The arrows indicate the times at which citrate was added [23].

#### *3.3.3.3. Ammonium removal under high salt conditions*

NaCl was then added to the WC to a final concentration of 3%, and repeated‐batch treatment was conducted using protocol similar to that described in Section 3.2. The result was similar to that in Section 3.2. The ammonium removal rate reached 1.0 kg‐N/m<sup>3</sup> /day with the four‐batch operation after the gradual acclimation of No. 4 to the saline medium.

#### **3.4. Coking wastewater [24]**

#### *3.4.1. Introduction*

Coking wastewater (CW), which originates from the process of destructive distillation of coal at high temperatures in the absence of air, has been a severe problem. Phenols are the major constituents of the coking wastewater and seriously inhibit various biological reactions, especially the nitrification reaction. Conventional biological treatment for CW is difficult mainly due to refractory substances. When high‐strength ammonium is involved in CW, BOD in the wastewater is not sufficient to complete the removal of ammonium. In this section, first, phenol‐degradation ability by No. 4 was confirmed, and No. 4 was applied to a coking wastewater supplied by a chemical company to assess the effects of biological treatment of high‐strength ammonium and phenol using a 1‐l jar fermenter.

#### *3.4.2. Materials and methods*

Medium: A synthetic medium described above was used in a preculture, using lactate as a carbon source.

#### *3.4.2.1. Wastewater used*

The coking wastewater (CW) was supplied by a Japanese chemical company. The primary characteristics of the CW are as follows: pH 8.5, total COD concentration of 5200 mg/l, ammonium‐nitrogen concentration of 800 mg/l, and phenol concentration of 820 mg/l. In each experiment, the pH of the original CW was adjusted to approximately 7.5 by 5N H<sup>2</sup> SO<sup>4</sup> , and the pH‐adjusted CW was diluted arbitrarily.

#### *3.4.2.2. Experimental procedure*

The synthetic medium was prepared in the preculture of No. 4 containing phenol (0.2 g/l) only as a carbon source and No. 4 was precultivated for 3 days and the preculture was centrifuged at 10,000 rpm for 10 min. The collected cells of No. 4 were washed with 0.1 M phosphate buffer two times and the cells were inoculated into the synthetic medium, which was devoid of lactate and contained only phenol as a carbon source and utilization of phenol by No. 4 was tested.

In CW treatment, precultured No. 4 cells were introduced into different dilution CW and the growth of No. 4 was confirmed at 50% dilution in shaking flasks. The diluted CW was added with lactate and No. 4 culture in a jar fermenter and ammonium removal test was conducted.

#### *3.4.2.3. Analytical method*

*3.3.3.3. Ammonium removal under high salt conditions*

**3.4. Coking wastewater [24]**

*3.4.2. Materials and methods*

*3.4.2.1. Wastewater used*

*3.4.2.2. Experimental procedure*

the pH‐adjusted CW was diluted arbitrarily.

carbon source.

*3.4.1. Introduction*

46 Nitrification and Denitrification

that in Section 3.2. The ammonium removal rate reached 1.0 kg‐N/m<sup>3</sup>

high‐strength ammonium and phenol using a 1‐l jar fermenter.

operation after the gradual acclimation of No. 4 to the saline medium.

NaCl was then added to the WC to a final concentration of 3%, and repeated‐batch treatment was conducted using protocol similar to that described in Section 3.2. The result was similar to

Coking wastewater (CW), which originates from the process of destructive distillation of coal at high temperatures in the absence of air, has been a severe problem. Phenols are the major constituents of the coking wastewater and seriously inhibit various biological reactions, especially the nitrification reaction. Conventional biological treatment for CW is difficult mainly due to refractory substances. When high‐strength ammonium is involved in CW, BOD in the wastewater is not sufficient to complete the removal of ammonium. In this section, first, phenol‐degradation ability by No. 4 was confirmed, and No. 4 was applied to a coking wastewater supplied by a chemical company to assess the effects of biological treatment of

Medium: A synthetic medium described above was used in a preculture, using lactate as a

The coking wastewater (CW) was supplied by a Japanese chemical company. The primary characteristics of the CW are as follows: pH 8.5, total COD concentration of 5200 mg/l, ammonium‐nitrogen concentration of 800 mg/l, and phenol concentration of 820 mg/l. In each

The synthetic medium was prepared in the preculture of No. 4 containing phenol (0.2 g/l) only as a carbon source and No. 4 was precultivated for 3 days and the preculture was centrifuged at 10,000 rpm for 10 min. The collected cells of No. 4 were washed with 0.1 M phosphate buffer two times and the cells were inoculated into the synthetic medium, which was devoid of lactate and contained only phenol as a carbon source and utilization of phenol by No. 4 was tested.

In CW treatment, precultured No. 4 cells were introduced into different dilution CW and the growth of No. 4 was confirmed at 50% dilution in shaking flasks. The diluted CW was added with lactate and No. 4 culture in a jar fermenter and ammonium removal test was conducted.

experiment, the pH of the original CW was adjusted to approximately 7.5 by 5N H<sup>2</sup>

/day with the four‐batch

SO<sup>4</sup> , and For phenol concentration determination, the chemical analysis kit for phenol (LR‐PNL, Kyoritsu Chemical‐Check Lab., Corp., Tokyo, Japan) was used. The initial and final values of TOC in the prepared solution were determined. The crude coking wastewater was streaked on the LB medium and the synthetic agar medium and no colonies appeared after 3 days of incubation. Therefore, indigenous microorganisms in the crude coke‐production wastewater were negligible in number.

The air was supplied to the CW sample containing lactate in the jar fermenter for 4 days and neither the removal of lactate nor ammonium was observed, and thus the air‐borne microor‐ ganisms were neglected.

#### *3.4.3. Results*

#### *3.4.3.1. Availability of phenol by No. 4*

Complete removal of ammonium and phenol in the synthetic medium were confirmed when phenol was added as a sole carbon source and the growth of No. 4 (data not shown). The ammonium removal rates using phenol as a carbon source were 0.098–0.12 kg‐N/m<sup>3</sup> / day, which is approximately one‐tenth of the rate when organic acids were used as a car‐ bon source [20]. However, these data were approximately 10‐fold higher than the rate in conventional nitrification‐denitrification method. When the initial phenol concentration was 600–700 mg/l, this includes 459–535 mg/l of carbon. If the C/N ratio of cell synthesis was 10, consumption of 600–700 mg/l of phenol corresponded with the consumption of only approximately 50–60 mg/l of ammonium‐nitrogen. This suggests that for complete removal of high‐strength of ammonium in CW, addition of available carbon for No. 4 is needed.

The No. 4 culture was directly mixed with crude‐coking wastewater with fortified lactate in a jar fermenter, but removal rates of ammonium and lactate were significantly decreased, presumably toxic substances in coking wastewater inhibited the activity of No. 4. When CW was diluted, the normal growth of No. 4 was observed at 50% dilution. Then, 50% of dilu‐ tion CW wastewater was mixed with No. 4 preculture and 4 g/l of lactate. The result is shown in **Figure 10**. The initial ammonium‐nitrogen concentration, phenol concentration, and lactate concentration were 420 mg/l, 380 mg/l, and 4 g/l, respectively. The ammonium removal rate was 1.8 kg‐N/m<sup>3</sup> /day and phenol removal rate was 0.7 kg/m<sup>3</sup> /day. Phenol removal rate 0.7 kg/m<sup>3</sup> / day was two times larger than that in the previous report [25].

COD in the initial wastewater containing lactate was 12,000 mg/l, and after the treatment, this value decreased to 2830 mg/l. The COD of 50% diluted CW was 2130 mg/l. Thus, this ammonium treatment was primarily undertaken by No. 4 by consumption of added lactate and indigenous phenol. As coking wastewater contained some other carbon substances not available for No. 4, further treatment may be needed for complete treatment of COD after this system.

**Figure 10.** Removal of ammonium, phenol, and lactate in coking wastewater by No. 4. Symbols: NH<sup>4</sup> + ‐N concentration (●), phenol concentration (□), lactate concentration (▲), and dissolved oxygen (DO) concentration (○) [24].

#### **3.5. Preparation of organic acid solution for No. 4 [26]**

#### *3.5.1. Introduction*

As No. 4 utilizes primarily organic acids as a carbon source and no sugar is available, chemical agents of citrate or lactate were used as a carbon source in the previous sections. In practical treatment, inexpensive production and supply of organic acids is a key for the materialization of No. 4 in ammonium treatment. In this section, anaerobic fermentation was conducted and then a mixture of high organic acid solution was prepared and this mixture was supplemented with two high‐ammonium and low‐carbon wastewaters by balancing C/N ratio around to 10 and the effectiveness of the prepared organic acid solution was confirmed.

#### *3.5.2. Materials and methods*

#### *3.5.2.1. Wastewaters*

The leachate wastewater from a landfill area in B city where the city garbage was land filled was used for ammonium treatment. The total organic carbon (TOC) and ammonium concen‐ tration were 4310 mg/l and 880 mg NH<sup>4</sup> + ‐N/l, respectively.

For a sample containing high NH<sup>4</sup> + ‐N concentration and the least amount of carbon, anaerobi‐ cally digested sludge wastewater used in Section 3.2 was used. This contained approximately 900 mg NH<sup>4</sup> + ‐ , N/l and almost no BOD was used for ammonium treatment.

#### *3.5.2.2. Preparation of the organic acid solution*

Forty milliliter leachate wastewater and 20 g of glucose were mixed in a 1‐l plastic container and statically incubate at 30°C for 2 weeks. The TOC and concentrations of eight organic acids in the prepared solution were determined. The volume of organic acid solution was determined so as to be C/N 10.

Experiment 1: Ammonium treatment of the leachate wastewater using No. 4 culture and organic acid solution 230 ml of leachate wastewater, 30 ml of No. 4 culture, and 40 ml of organic acid solution were mixed and the ammonium treatment was conducted in a jar fermenter.

Experiment 2: Ammonium treatment of anaerobically digested sludge using No. 4 culture and organic acid solution 180 ml of the wastewater, 30 ml of No. 4 culture, and 90 ml of organic acid solution were mixed and the ammonium treatment was carried out.

#### *3.5.3. Results*

**3.5. Preparation of organic acid solution for No. 4 [26]**

As No. 4 utilizes primarily organic acids as a carbon source and no sugar is available, chemical agents of citrate or lactate were used as a carbon source in the previous sections. In practical treatment, inexpensive production and supply of organic acids is a key for the materialization of No. 4 in ammonium treatment. In this section, anaerobic fermentation was conducted and then a mixture of high organic acid solution was prepared and this mixture was supplemented with two high‐ammonium and low‐carbon wastewaters by balancing C/N ratio around to 10

The leachate wastewater from a landfill area in B city where the city garbage was land filled was used for ammonium treatment. The total organic carbon (TOC) and ammonium concen‐

cally digested sludge wastewater used in Section 3.2 was used. This contained approximately

Forty milliliter leachate wastewater and 20 g of glucose were mixed in a 1‐l plastic container and statically incubate at 30°C for 2 weeks. The TOC and concentrations of eight organic acids in the prepared solution were determined. The volume of organic acid solution was

N/l and almost no BOD was used for ammonium treatment.

‐N/l, respectively.

‐N concentration and the least amount of carbon, anaerobi‐

+

‐N concentration

and the effectiveness of the prepared organic acid solution was confirmed.

**Figure 10.** Removal of ammonium, phenol, and lactate in coking wastewater by No. 4. Symbols: NH<sup>4</sup>

(●), phenol concentration (□), lactate concentration (▲), and dissolved oxygen (DO) concentration (○) [24].

+

+

*3.5.1. Introduction*

48 Nitrification and Denitrification

*3.5.2. Materials and methods*

tration were 4310 mg/l and 880 mg NH<sup>4</sup>

*3.5.2.2. Preparation of the organic acid solution*

For a sample containing high NH<sup>4</sup>

determined so as to be C/N 10.

+ ‐,

*3.5.2.1. Wastewaters*

900 mg NH<sup>4</sup>

#### *3.5.3.1. Prepared highly concentrated organic acid solution*

After 2‐ week anaerobic incubation of the leachate wastewater, the resulting organic acid solution contained 20,049 mg/l of TOC and 52,103 mg/l of total organic acid content of eight types, as shown in **Table 5**. The estimated carbon content from the organic acid data was 20,754 mg/l, as shown in **Table 5**. As the carbon contents in the TOC and organic acid solution were almost similar, TOC was used as an indicator to adjust to the necessary carbon content required to treat ammonium completely by balancing C/N ratio 10.

#### *3.5.3.2. Experiment 1*

The result is shown in **Figure 11**. The initial TOC was 7017 mg/l and the initial NH<sup>4</sup> + ‐N concentration was 659 mg/l. The initial value of eight kinds of organic acids was 17,750 mg/l in which the estimated carbon content was 6500 mg/l (**Table 6**). The final value of TOC was 900 mg/l and the final carbon value of eight kinds of organic acids was 817 mg/l, as show in **Table 6**. Complete ammonium removal was observed and thus the effectiveness of the use of organic acid solution and the use of TOC as an index to determine C/N ratio was confirmed. The ammonium removal rate was 1.1 kg‐N/m<sup>3</sup> /day.


**Table 5.** Organic acid distribution and carbon content in the prepared organic acid solution [26].

#### *3.5.3.3. Experiment 2*

In Section 3.2, the high‐strength ammonium from anaerobically digested sludge was removed using No. 4 with addition of citrate. A similar sample that contained 900 mg NH<sup>4</sup> + ‐N/l and 20 mg/l of organic content indicated that the available carbon for No. 4 is scarce, and supple‐ mentation of the organic acid solution is essential for complete removal of ammonium. For the initial 180 ml sludge sample, 90 ml of organic acid solution and 30 ml of No. 4 culture were mixed, and the ammonium removal was measured. The results are shown in **Figure 12** and **Table 7**. Similarly, the initial NH<sup>4</sup> + ‐N concentration 635 mg/l was completely removed and the ammonium removal rate was 0.8 kg‐ N/m<sup>3</sup> /day.

**Figure 11.** Ammonium removal when leachate wastewater was treated with No. 4 culture and organic acid solution. Symbols: NH<sup>4</sup> + ‐N (●), dissolved oxygen concentration (DO) (□), and pH (△) [26].


**Table 6.** Change in the initial and final carbon contents of organic acids in **Figure 11** [26].

Heterotrophic Nitrification and Aerobic Denitrification by *Alcaligenes faecalis* No. 4 http://dx.doi.org/10.5772/68052 51


**Table 7.** Change in the initial and final carbon contents of organic acids in **Figure 12** [26].

*3.5.3.3. Experiment 2*

50 Nitrification and Denitrification

Symbols: NH<sup>4</sup>

+

**Table 7**. Similarly, the initial NH<sup>4</sup>

ammonium removal rate was 0.8 kg‐

In Section 3.2, the high‐strength ammonium from anaerobically digested sludge was removed

20 mg/l of organic content indicated that the available carbon for No. 4 is scarce, and supple‐ mentation of the organic acid solution is essential for complete removal of ammonium. For the initial 180 ml sludge sample, 90 ml of organic acid solution and 30 ml of No. 4 culture were mixed, and the ammonium removal was measured. The results are shown in **Figure 12** and

‐N concentration 635 mg/l was completely removed and the

**Initial carbon content (mg/l) Final carbon content (mg/l)**

**Figure 11.** Ammonium removal when leachate wastewater was treated with No. 4 culture and organic acid solution.

+

‐N/l and

using No. 4 with addition of citrate. A similar sample that contained 900 mg NH<sup>4</sup>

+

Oxalate 64 64 Citrate 94 94 Lactate 3600 100 Formate 65 65 Acetate 1680 100 Propionate 842 156 *iso*‐Butyrate 102 102 *n*‐Butyrate 136 136 Total 6500 817

‐N (●), dissolved oxygen concentration (DO) (□), and pH (△) [26].

**Table 6.** Change in the initial and final carbon contents of organic acids in **Figure 11** [26].

N/m<sup>3</sup>

/day.


**Table 8.** Comparison of No. 4, anammox, and conventional methods.

**Figure 12.** Ammonium removal when anaerobically digested sludge was treated with No. 4 culture and organic acid solution. Symbols: NH<sup>4</sup> + ‐N (●), dissolved oxygen concentration (DO) (□),and pH (△) [26].
