**4. Additional characteristics of No. 4**

In wastewater treatment, the excess sludge is inevitably produced, and new treatment possibilities of excess sludge containing No. 4 are presented in the following sections.

#### **4.1. Suppression of growth of plant pathogens by No. 4 [19]**

#### *4.1.1. Introduction*

In this section, it was presented that the *A. faecalis* No. 4 (No. 4) exhibited a suppressive effect on the damping‐off caused by the plant pathogen *Rhizoctonia solani* on soil [27].

#### *4.1.2. Materials and methods*

#### *4.1.2.1. Preparation of culture broth and cell suspension*

Fifty milliliters of culture broth of No. 4 after cultivation in L medium was mixed with 150 g of soil in a pot. For the treatment consisting of only the cell suspension, the cells of No. 4 were collected by centrifugation, and the sedimented cells were suspended in sterile distilled water and 50 ml of the cell suspension was mixed with 150 g of soil.

#### *4.1.2.2. Soil treatments and inoculation of soil with R. solani*

These procedures were described in detail in a previous section [19].

#### *4.1.2.3. Plant growth*

For each treatment, three pots were prepared. Tomato (*Lycopersicum esculentum*) seeds were germinated on 2% agar plates at 30°C for 2 days, and nine germinated seeds were planted in each pot and incubated in a grown chamber.

#### *4.1.3. Results*

The result of the effect of No. 4 culture broth on the damping‐off of tomato seedlings caused by *R. solani* in soils is shown in **Figure 13**. In a pot that was not infested with *R. solani*, all the seedlings grew normally and no disease was apparent. In a pot infested with only *R. solani*, the percentage of diseased plants was 78–82% and the shoot weight and leaf length were mark‐ edly decreased in soil. However when the culture broth of No. 4 was introduced into the soil, the percentage of diseased plants was reduced to 17%. When the cell suspension of No. 4 was applied to soil, the percentage of diseased plants in soil was similar to that soil treated with No. 4 culture broth (data not shown).

The finding suggests that the treatment with No. 4 cells is effective for plant disease control.

**Figure 13.** *A. feacalis* No. 4 exhibits suppressive effect on plant pathogens. Pot ③: Tomato seedlings with plant pathogens. Pot ①: Tomato seedling without plant pathogens. Pot ②: *A. feacalis* No. 4 was introduced to pot ③.

#### **4.2. Reduction of methane production from rumen of cows [28]**

#### *4.2.1. Introduction*

**4. Additional characteristics of No. 4**

*4.1.1. Introduction*

52 Nitrification and Denitrification

*4.1.2. Materials and methods*

*4.1.2.3. Plant growth*

*4.1.3. Results*

**4.1. Suppression of growth of plant pathogens by No. 4 [19]**

*4.1.2.1. Preparation of culture broth and cell suspension*

and 50 ml of the cell suspension was mixed with 150 g of soil.

These procedures were described in detail in a previous section [19].

*4.1.2.2. Soil treatments and inoculation of soil with R. solani*

each pot and incubated in a grown chamber.

No. 4 culture broth (data not shown).

In wastewater treatment, the excess sludge is inevitably produced, and new treatment

In this section, it was presented that the *A. faecalis* No. 4 (No. 4) exhibited a suppressive effect

Fifty milliliters of culture broth of No. 4 after cultivation in L medium was mixed with 150 g of soil in a pot. For the treatment consisting of only the cell suspension, the cells of No. 4 were collected by centrifugation, and the sedimented cells were suspended in sterile distilled water

For each treatment, three pots were prepared. Tomato (*Lycopersicum esculentum*) seeds were germinated on 2% agar plates at 30°C for 2 days, and nine germinated seeds were planted in

The result of the effect of No. 4 culture broth on the damping‐off of tomato seedlings caused by *R. solani* in soils is shown in **Figure 13**. In a pot that was not infested with *R. solani*, all the seedlings grew normally and no disease was apparent. In a pot infested with only *R. solani*, the percentage of diseased plants was 78–82% and the shoot weight and leaf length were mark‐ edly decreased in soil. However when the culture broth of No. 4 was introduced into the soil, the percentage of diseased plants was reduced to 17%. When the cell suspension of No. 4 was applied to soil, the percentage of diseased plants in soil was similar to that soil treated with

The finding suggests that the treatment with No. 4 cells is effective for plant disease control.

possibilities of excess sludge containing No. 4 are presented in the following sections.

on the damping‐off caused by the plant pathogen *Rhizoctonia solani* on soil [27].

Enteric methane (CH<sup>4</sup> ) production from livestock is a significant source of greenhouse gas. It is reported that nitrate (NO<sup>3</sup> ) suppresses enteric CH<sup>4</sup> production. However, the reduction of NO<sup>3</sup> to nitrite (NO2 ) in the rumen results in the accumulation in NO2 , which is toxic to livestock.

A denitrifying bacterium, *A. faecalis* No. 4, was coincubated with a low concentration of NO<sup>3</sup> (2 mmol/l) and the *in vitro* CH<sup>4</sup> production was tested.

#### *4.2.2. Materials and methods*

#### *4.2.2.1. Rumen liquid*

The rumen liquid which was collected from Holstein cows and No. 4 cells obtained after centrifu‐ gation of culture broth prepared in synthetic medium were mixed with 2 mmol/l NO<sup>3</sup> . The mixture was placed in a 1 l jar fermenter and CH<sup>4</sup> production was monitored under anaerobic condition.

#### *4.2.3. Result*

The methane production from rumens is shown in **Figure 14**. When No. 4 and 2 mmol/l NO<sup>3</sup> were mixed, methane production showed a significant decrease without causing an adverse impact on anaerobic fermentation in rumens. This suggests a possibility of re‐use of No. 4, which was produced as excess sludge after treatment of high‐strength ammonium.

**Figure 14.** Methane production from rumens when No. 4 was introduced [28].

### **5. Conclusions**

**Table 8** shows comparison among conventional ammonium treatment method, anammox method and No. 4 method. No. 4 has many advantages over other methods. No. 4 was effective to remove high‐strength of ammonium in several wastewaters when organic acids are supplied. The excess cells of No. 4 are produced during treatment of ammonium because the cell growth of No. 4 is associated with ammonium removal. Possibility of the re‐use of the excess cells in agricultural areas was presented. How to collect organic acids as carbon source for No. 4 is a problem to be solved. Production of high concentration of organic acids in anaerobic fermentation and the use of the produced organic acid solution to wastewaters were shown as one possible method.

### **Acknowledgements**

These researches were supported by many organizations of Japan; Able Cooperation (Tokyo), Kanagawa Prefectural Livestock Industry Research Institute, Yokohama Municipal Sewage Treatment Center, Several Chemical Companies, Obihiro University of Agriculture and Veterinary Medicine and Kanagawa Prefectural Agricultural Research Institute.

### **Author details**

Makoto Shoda

Address all correspondence to: mshoda@res.titech.ac.jp

Chemical Resources Laboratory, Tokyo Institute of Technology, Yokohama, Japan
