IJERD – International Journal of Environmental and Rural Development (2013) 4-2

Research articleerd

Changes in Surviving Microorganism in Cow Manure withAdding Lime Nitrogen
YUTA ISHIKAWA
Graduate School of Agriculture, Tokyo University of Agriculture, Tokyo, Japan

MACHITO MIHARA*
Faculty of Regional Environment Science, Tokyo University of Agriculture, Tokyo, Japan
Email: m-mihara@nodai.ac.jp
Received 14 December 2012

Accepted 30 January 2013(*Corresponding Author)

Abstract Pathogenic bacteria known as E. coli may survive in cow manure and may efflux
from upland fields where cow manure was applied. So, treating of E.coli throughfermentation process has been focused. It has been considered that air drying method was
effective method for decreasing E.coli. However, this treatment may possibly eliminate
not only pathogenic bacteria but also useful bacteria for decomposition. This study aimed
to investigate the changes in number of E.coli, coliform bacteria and general bacteria
during the fermentation of cow manure with adding lime nitrogen (nitrolime). Samples
used were fresh cow dung, 2 weeks and 12 weeks fermented manure. Lime nitrogen wasadded to all three types of dung samples then were kept in incubator within 14 days.
Sampling, stirring and supplying of physiological saline were conducted. Based on theexperimental results, there was a significant decrease in E.coli and coliform bacteria in
cow manure after adding of lime nitrogen; however there was no decrease in generalbacteria. It was observed that controlling to pH 9.0 in cow manure samples wasappropriate for decreasing E.coli and coli form bacteria during cow manure fermentation
with minimum damage to general bacteria. Therefore, it was concluded that treating of
cow manure by adding lime nitrogen, especially controlling to around pH 9.0, is aneffective strategy for sterilizing pathogenic bacteria such as E.coli and other coliform
bacteria with minimum damage to general bacteria.
Keywords Escherichia coli, general bacteria, lime nitrogen, cow dung, pHINTRODUCTION
Big amounts of cattle dung has been produced in farms. The point of view of organic agriculture,making manure was considered as proper treatment. However, bulk production of manure may
possibly contain immature fermented manure. In addition, the pathogenic bacteria known as
Escherichia coli (E.coli) or coliform bacteria may survive and remain in immature fermentedmanure (Chun-Ming et al., 2005; Indira et al., 1998). Especially, cow dung has an amount of E.coli
than other cattle dung (Nakazawa and Sameshima, 2002). These bacteria may survive in soil (Islam,M et al., 2005) and may be released from grazed land and upland field applied with immature
fermented manure (Mishra et al., 2007; Yagura et al., 2006; Ishikawa and Mihara., 2010). The
efflux of E.coli causes water pollution or contamination of potable water sources affecting humanhealth (Tamura et al., 2006; Mishina et al., 2007).
So, treatments to sterilize cow dung of E.coli during fermentation process were focused. Sometreatments as air drying treatment (Saito and Mihara, 2010) were carried out. However, air drying
treatment has possible affect to not only E.coli or coliform bacteria but also general bacteria(Ishikawa and Mihara, 2011). On the other hands, it was reported by Minato et al., (2001) that pH
control by lime nitrogen adding treatment, it decreases not only E.coli but also beneficial bacteria.
However, beneficial bacteria which affect fermentation of manure change in fermentation stage. So,
it is necessary to observe the surviving of microorganisms in lime nitrogen adding treatment.

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IJERD – International Journal of Environmental and Rural Development (2013) 4-2

This study aimed to observe the survival of several microorganisms such as E.coli, coli form
bacteria and general bacteria under lime nitrogen adding treatment.
METHODOLOGY
Cow dung and fermented manure used in this experiment were collected from Fuji Farm, a cattle
farm of Tokyo University of Agriculture located in Shizuoka Prefecture (Fig.1). There were three
types of cow dung collected for this experiment, fresh cow dung, 2 weeks, and 12 weeks fermented
manure with water contents at 76%, 75% and 68% respectively. In addition, cow dung and manurescontained 90%, 89% and 86% of organic matter. The colony forming units (cfu) of E.coli were
20×106 in cow dung, 1×103 in 2 weeks fermented manure. In 12 weeks fermented manure, E.coli
was not observed. Also, the colonies of coli form bacteria was 1×105 cfu/g in cow dung and 0 cfu/g
in 2 and 12 weeks fermented manures. The number of general bacteria was 128×10 6 in cow dung,
236×106 in 2 weeks fermented manure and 93×106 in 12 weeks fermented manure.

Cow dung
Manure fermented for 2 weeks

Manure fermented for 12 weeks

Fig.1 Situation of cow dung and manure
Table 1 Properties of cow dung and fermented manure

Cow dung
ManureManurePeriod offermentation

E.coli(cfu/g)0 days2 weeks
12 weeks

20×106
1×103
0

Coliform
bacteria(cfu/g)
1×105
00Generalbacteria(cfu/g)128×106
236×106
93×106

Fig. 2 Situation of incubator© ISERD
34Water
contents(%)
7675
68

Organicmatter(%)
908986

IJERD – International Journal of Environmental and Rural Development (2013) 4-2E.coli

General bacteria

Coliform bacteria

Fig. 3 Colonies of E.coli and coliform bacteria

Fig. 4 Colonies of general bacteria

Both of cow dung and manure at 3 kg were mixed after adding 60 g of lime nitorogen. Then,they were put in incubator at 37 degree temperature for 14 days (Fig. 2). At 4th, 7th, 11th, 14th day,sampling, stirring and supplying of physiological saline were conducted.
The number of E.coli, coliform bacteria and general bacteria in sample were measured
through laboratory experiments. The analysis of E.coli and coliform bacteria was carried out
through counts of colonies on XM-G agar medium as shown in Fig.3. The analysis of generalbacteria was carried out with general agar medium (Fig. 4). In addition, the pH in cow dung andmanure were measured through laboratory experiments.
RESULTS AND DISCUSSION
The changes in the number of E.coli in each sample were summarized in Figs. 5-7. In cow dung,
E.coli decreased after lime nitrogen was added then, E.coli was not observed. Based on the resultsof variance analysis, significant difference was observed. In 2 weeks fermented manure, E.coli wasdecreased after lime nitrogen was added as well as cow dung. However, significant difference was
not observed. In 12 weeks fermented manure, E.coli was not observed during experiment.
Changes in coliform bacteria were shown in Figs. 8-10. In cow dung, coliform bacteria weresterilized by lime nitrogen adding treatment as shown in Fig. 8. Moreover, it showed a significant
difference of 99% between before lime nitrogen adding and after. In addition, coliform bacteria
were not detected from 2 weeks and 12 weeks fermented manure. Therefore, it was considered thatthe adding of lime nitrogen is an effective treatment to decrease pathogenic bacteria as E.coli or
coliform bacteria. Figs. 11-13 show changes in general bacteria. In both of samples, it showed atendency that the number of general bacteria was not decreased by adding of lime nitrogen. Basedon results of variance analysis, significant difference was not observed during experiment.
Therefore, it was considered that general bacteria which contain beneficial bacteria are not affectedby lime nitrogen adding treatment.
1500

A**

E.coli (cfu/g)

E.coli (cfu/g)

3.E+066
3×10

2.E+066
2×10

1.E+066
1×10

0.E+00

0B**
00

B**
12adding lime nitrogen

344B**
77 8Time (day)56910B**11 121113B**
14141000500
0Significant difference at **99%

Fig. 5 Changes in E.coli (cow dung)

AA00

A
12adding lime nitrogen

344A
77 8Time (day)56910A11 121113A1414Fig. 6 Changes in E.coli(2 weeks fermented manure)

The pH in cow dung and manure are indicated in Fig. 14. In all of samples, pH was increased
to around 9.0. It was reported that E.coli or coli form bacteria can survive from pH 4.4 to 9.0(Nakanishi and Maruyama, 2009). Furthermore, based on a report of Fujita (1993), proper pH forfermentation of manure is 8 to 10. So, it was considered that pH controlling to around 9.0 is able todecrease E.coli and coliform bacteria without sterilize general bacteria which contain beneficialbacteria.

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Coliform bacteria (cfu/g)

E.coli (cfu/g)

10864ND

2000

123445677 8Time (day)adding lime nitrogen

91011 121113

2.E+055
2×10

1.E+055
1×10

0

14
14

6
4ND

22

34
4

5678

91011 12
1113

14
14

1.E+088AA

10

1.E+04410

1.E+000

1000

1234477 8Time (day)adding lime nitrogen5691011 121113101.E+101010B**

B**
141413Significant difference at **99%12443567891011 1211131414Time (day)B** *AAA*, B*

A*, B*

A*

1.E+08810

1.E+066

10

1.E+04410

1.E+02210

1.E+000

10141400

123445677 8Time (day)

91011 1211131414

Significant difference at *95%

Fig. 12 Changes in general bacteria(2 weeks fermented manure)
12A**

A**

9

1.E+066

10

1.E+044

10B**
A*a**b**

B**

A**

63

1.E+022

100Cow dung

1.E+000

1000adding lime nitrogenB**

A**
A**

B**
11 1211ND0pHGeneral bacteria (cfu/g)

10

1.E+088

102Fig. 11 Changes in general bacteria(cow dung)
1.E+10109Fig. 10 Changes in coliform bacteria(12 weeks fermented manure)AA64adding lime nitrogen

AA

56General bacteria (cfu/g)

General bacteria (cfu/g)

10B**
77 8Time (day)44

38Fig. 9 Changes in coliform bacteria(2 weeks fermented manure)
1.E+12122

10Time (day)adding lime nitrogen

B**
1Fig. 8 Changes in coliform bacteria(cow dung)
Coliform bacteria (cfu/g)

Coliform bacteria (cfu/g)

8100

adding lime nitrogen

1000

B**

0.E+00Fig. 7 Changes in E.coli(12 weeks fermented manure)0A**0012adding lime nitrogen

34477 8Time (day)5691011 12
1113

14
14

Significant difference at **99%

Fig. 13 Changes in general bacteria(12 weeks fermented manure)

Fermented for
2 weeks
BeforeAfter

Fermented for
12 weeks
Significant difference at **99%

Fig. 14 Changes in pH of manure

CONCLUSION
This study aimed to observe the survival of E. coli, coli form bacteria and general bacteria underlime nitrogen adding treatment in cow dung, 2 weeks and 12 weeks fermented manure.
Based on experimental results, E. coli was decreased after lime nitrogen addition treatment.
Moreover, there was a significant difference observed. Also, coli form bacteria were sterilized bylime nitrogen as well as E. coli. On the other hand, lime nitrogen has not affected the number ofgeneral bacteria which contain beneficial bacteria.

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IJERD – International Journal of Environmental and Rural Development (2013) 4-2In addition, as the results of pH, there were tendencies that pH was increased to around 9.0
which were proper for decreasing E. coli or coli form bacteria with minimum damage to generalbacteria.
Therefore, it was concluded that the pH control to around 9.0 using alkaline as lime nitrogen isan effective treatment for decreasing pathogenic bacteria in fermentation process.
ACKNOWLEDGEMENTS
We would like to express our gratitude to the member of the Lab. of Land and Water Use
Engineering at Tokyo University of Agriculture, Japan.
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