IJERD – International Journal of Environmental and Rural Development (2018) 9-1

Research articleerd

Enhancing Fermentation of Farmyard Manure UsingBacillus sp. in the Mid-hills of Nepal
MASKEY SARVESH
Graduate School of Agriculture, Tokyo University of Agriculture, Japan
Email: 45516008@nodai.ac.jp

MACHITO MIHARA*
Faculty of Regional Environment Science, Tokyo University of Agriculture, Japan
Email: m-mihara@nodai.ac.jp
Received 31 December 2017

Accepted 5 May 2018(*Corresponding Author)

Abstract Farmers in the mid-hills of Nepal have been using farmyard manure formaintaining soil fertility for their lands. However, there has been a decrease in productionof farmyard manure. Less application of farmyard manure has led to declined soil fertility
and reduced nutrient balance. Soil degradation through nutrient depletion is a serious issue.
Soils in mid hills have very low nutrients, particularly nitrogen and phosphorus
concentration. Sustainability of hill agriculture depends on how farmers use the available
resources. Application of incompletely fermented and decomposed farmyard manure could
have low nutrient contents, cause health risks as plants can uptake pathogenic bacteria
through soil. Also, imbalances in nutrient content of manure mainly nitrogen andphosphorus can pollute water sources through leaching and runoff. An experiment wasconducted to determine the action of Bacillus sp. as an inoculant for composting. Threedifferent concentrations of Bacillus sp. liquid were extracted from locally available rice
husk, which were 6.0×1010, 6.0×1012, 6.0×1014 cfu mL-1. An experiment was conducted
for 60 days comparing samples with and without Bacillus sp. liquid. Results showed thatBacillus sp. helped in enhancing the fermentation process and better decomposition oforganic matter, mineralization and the heat generated during the process helped in theelimination of pathogenic bacteria. Further, a growth experiment was conducted where
Brasicca rapa was grown on the compost prepared. The results of growth experimentshowed application of Bacillus sp. is good for development of plants.
Keywords Nepal, mid-hills, farmyard manure, Bacillus sp., soil fertility

INTRODUCTION
Farmers in the mid hills of Nepal (Fig. 1) have been using farmyard manure for maintaining soilfertility for their lands. However, there has been decreasing production of farmyard manure. Less
application of farmyard manure has led to decline soil fertility and reduce nutrient balance (Regmiet al., 2005). Soil degradation through nutrient depletion is also a serious issue (Lal, 2000). Soils in
mid-hills have very low nutrients, especially nitrogen and phosphorus concentration (Shah andSchreier 1991; Brown 1997; Westarp et al., 2004).
Application of farmyard manure, which is not completely fermented, raises health risks asplants can uptake pathogenic bacteria. Also, imbalance in nutrient contents of manure mainly from
nitrogen and phosphorus can pollute water sources through leaching and runoff.
Adding of microbial inoculants often referred as effective microorganisms (EM) have beenused in composting process. Li et al., (2013) suggested improvement of crop residue composting
using Bacillus sp. Also, Kuroda et al., (2009) stated better quality compost using the same. Studies
have been conducted addressing the use of microbial inoculants with the purpose of accelerating
composting and improving the final product (Zeng et al., 2009; Figueiredo et al., 2013). García etal., (2006) observed in their studies that the use of bacterial inoculants (Bacillus and actinobacteria)
in the composting of vegetable products increased the final humification of the compost andⒸ ISERD51

IJERD – International Journal of Environmental and Rural Development (2018) 9-1

consequentially improved the agricultural quality of the product. Nevertheless, further information
about the composting system is necessary.
Therefore, the aim of this study was to assess the effect of Bacillus sp. in composting processby evaluating the mineralization and its effect in eliminating pathogenic bacteria.

Fig. 1 Digital elevation model map of Nepal and study site
METHODOLOGYIsolation of Bacillus sp.
In this study, Bacillus sp. solution was extracted from rice straw and husk. Ten grams of rice huskand straw was added in 90 mL of sterile water and kept overnight in shaker. Serial dilutions weremade and inoculated in nutrient agar plates which was incubated for 24 h at 30 °C. Serial plate
count method was used to determine the number of colonies on nutrient agar. Three different
Bacillus sp. solutions were made which were 6.0×1010, 6.0×1012 and 6.0×1014 cfu mL-1 wasmade in nutrient broth. The Bacillus sp. solution was made 200 mL for each cfu mL-1.
Sample Preparation
Cow manure and leaves were used in this composting experiment, where 1 kg of cow manure wasmixed with 1 kg of leaves to make a total mass of 2 kg. Physical, chemical and microbial
experiments were conducted to analyze the initial condition of materials used. Four treatments weremade which were Control, Treatment 1, Treatment 2 and Treatment 3 (Table 1). In treatments otherthan Control, 200 mL of Bacillus sp. liquid was added and mixed thoroughly.
Table 1 Preparation of samples for composting experimentControl

Treatment 1Treatment 2Treatment 3

Cow manure 1kg

Cow manure 1kg

Cow manure 1kg

Cow manure 1kg

Leaves 1 kg

Leaves 1 kg

Leaves 1 kg

Leaves 1 kg

200 mL of

200 mL of

200 mL of

6.0×1010 cfu mL-1

6.0×1010 cfu mL-1

6.0×1014 cfu mL-1

Bacillus sp. added

Bacillus sp. added

Bacillus sp. added

No Bacillus sp. addedⒸ ISERD52

IJERD – International Journal of Environmental and Rural Development (2018) 9-1

Composting Setup and Experiment
An apparatus was designed where composting treatments were kept in glass containers. Thesecontainers were kept in a tank containing water and temperature was maintained at a range of 3035°C (Fig. 2) by heating water with an electric rod. Composting was performed for 60 days andsamples were taken for 0, 15, 30, 45 and 60 days. Moisture content was maintained at 50-55% withfrequent checking and testing. According to Gajalakshmi and Abbasi (2008), the ideal moisture
content for an efficient composting lies between 50-60%, which is ideal for microbial metabolism.
Carbon content, pH, nitrogen concentration, phosphorus concentration, C/N ratio, temperature andE. coli colony formed units were measured.

Fig. 2 Apparatus designed for composting

RESULTS AND DISCUSSION
Evaluating Fermentation with Bacillus sp.
The result of temperature (Fig. 3) shows that there is difference in highest temperature obtained
compared with control and Bacillus sp. added treatments. The highest temperature obtained byControl, Treatment 1, Treatment 2 and Treatment 3 were 55 °C, 59 °C, 58 °C, 60 °C respectively.
The high temperature can be corresponded to heat generated by microbial activity of added
Bacillus sp. compared to control. The temperature did not reach a very high level (above 65 °C),
which is considered harmful for microbial growth (Valente et al., 2009). Also, very low
temperature is unfavorable for composting as it delays decomposition (Bernal, Alburquerque andMoral, 2009).
Maintaining an appropriate pH in a compost pile is vital for the survival of beneficial
microbes. The pH of all the treatments was in between the range of 6.8 to 7.5 (Fig. 4), whichindicated that a favorable condition existed for growth and metabolism of microorganisms.
8705040

Control
Treatment 130pHTemperature (°C)

607Control
Treatment 1Treatment 2Treatment 32010Ambient temp.

0510 15 20 25 30 35 40 45 50 55 60

Treatment 2Treatment 36030

4560

No. of composting days

No. of composting days

Fig. 3 Changes in temperature with time

15Fig.4 Changes of pH with timeⒸ ISERD53IJERD – International Journal of Environmental and Rural Development (2018) 9-1

Table 2 shows the total numbers of colonies formed of E. coli for different treatments. After
15 days of composting, there were no colonies formed with Bacillus sp. added treatments. This canbe attributed to the heat generated during metabolism and competition of nutrients by microbes.
According to Haug et al., (1980) pathogenic microbes die if there is temperature above 55°C for 3
or more days. On the other hand, there were 5.1× 103cfu g-1 of E. coli colonies in control during thefirst 15 days. At 30 days of sampling colonies of E. coli were not found in control as well.
Table 2 E. coli (cfu g-1) in different treatments
Control
Treatment 1
Treatment 2
Treatment 30 days
9.3 × 103
9.3 × 103
9.3 × 103
9.3 × 103

15 days
5.1× 103
00030 days
000045 days
000060 days
0000

Of the many elements required for microbial decomposition, carbon and nitrogen are the mostimportant. Carbon provides both an energy source and the basic building block making up about 50
percent of the mass of microbial cells. As seen in Fig. 5 the carbon percent decreases with increasein composting days. After 60 days, carbon percent of Control was 40.2, Treatment 1 was 38.45, 2Treatment 2 was 38.45 and Treatment 3 was 36.18 percent. This result can be explained as numberof colonies of added Bacillus sp. increased there was higher decomposition of organic carbon.
Similarly C/N ratio (Fig. 6) observed, had significant difference in decrease between control andBacillus sp. added treatments.
6060

Length ofplant
CNratio(cm)302050Control
84040 Treatment
1Treatment
230430 Treatment 302020

Control
Control

Treatment
Treatment 1 1Treatment 2

Treatment 2A**

01530 No. of days4540 Control
A**

B**
B**
B**

B**
Treatment
1B**
B**

30 Treatment 2

Treatment 3

Treatment
330 15 4560 45
300 15 0days
0No. of composting
36 of
912days 15No.
composting

12A**

A**

B**
B**
B**

B**
B**
B**

60

12C N ratio

40(**) denotes significance difference p < 0.01

B**
B**

506050Carbon %C N ratio

5070
16B**

(**) denotes significance difference p < 0.01

Length ofplantCNratio(cm)

6070
1660

18 20A**50Control
840
Treatment 12Treatment 3 Treatment
304Treatment 32160

024 20No. of composting days

Control
Treatment 1Treatment 2Control

A**
A**

B**
B**
B**

B**
B**
B**

Treatment 3Treatment 1Treatment 2Treatment 330 15 45 3060 45
0 15 0days
0No. of composting
36 of
912days 15No.
composting60

1821No. of days

Fig. 5 Decrease in carbon percent with time

Fig. 6 Decrease in C/N ratio with time

According to Tumihairwe et al., (2009) large organic loss suggests pronounced microbialactivity. This supports the obtained result where added Bacillus sp. had resulted in increased
decomposition rate of carbon decreasing the carbon percentage and C/N ratio.
The nitrogen (Fig.7) and phosphorus concentration (Fig.8) of all the treatments showed
increase with increase in composting days. The final concentration of total nitrogen at 60 days forcontrol, treatment 1, treatment 2 and treatment 3 were 6825.07, 7728.02, 7835.02 and 8457.71 mg–kg
respectively. According to Viel et al., (2007) increase in total nitrogen value might increase due
to nitrogen fixing bacteria that commonly occurs at the end of composting. The results obtained
contradict to Tognetti et al., (2007) who showed that total nitrogen decreased during composting.
Decrease in nitrogen content may occur due to leaching and volatilization, but this experiment wasconducted in a closed container nullifying the effects of leaching.
Likewise, After 60 days of composting the total concentration of phosphorus for Control,
Treatment 1, Treatment 2, and Treatment 3 were 2728.1, 3178.29, 3168.52, 2920.52 mg kg–1

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IJERD – International Journal of Environmental and Rural Development (2018) 9-1

respectively. The result obtained matches with those of Felton et al., (2004) and Chandna et al.,(2013) where they found increase in total phosphorus.
8000

3000

mg/kg

mg/kg

6000
Control4000

Treatment 1
Treatment 220002000

Control
Treatment 1
Treatment 21000

Treatment 3Treatment 300015304560015304560

No. of composting days

No. of composting days

Fig. 7 Change of nitrogen concentration
with time

Fig. 8 Change of phosphorus concentration
with time

Plant Growth Experiment
A plant growth experiment was conducted to analyze the effects on growth of plants using thecompost that was made. The plant used was Brassica rapa (Komatsuna). Twelve pots were used;
three each for Control, Treatment 1, Treatment 2 and Treatment 3 (unit area is 1/10000 a, and aheight of 150 mm). Six hundred grams of soil was used in each pot with 350 grams of compostapplied. 100 ml of water was put in the pots after three days interval. Environmental conditions like
temperature, light were made constant for all the pots. The height of the plants was measured after3 days. The experiment was conducted for the period of 30 days from July 2017 to August 2017.
Table 3 Physical and chemical properties of soil used
Water content (%)
10.7±0.10
Organic content (%)
14.87±0.17
EC (mS/cm)
1.33±0.03pH
5.22±0.04
Total nitrogen Mg Kg-1
1590.36±0.72
Total phosphorus Mg Kg-1
907.65±0.03
Note: Values are mean ± SD (n=3).

In the result (Fig. 9) of plant growth experiment, the treatments with added Bacillus sp. had
higher crop height compared to control with significant difference at 99%. There was no significant
difference in height of plants with Bacillus sp. added treatments. The heights of plants forTreatment 1, Treatment 2 and Treatment 3 were 14.97, 14.56 and 14.3 cm respectively. With theobtained results it can be said that plants grown in compost made with added Bacillus sp. can help
in growth of plants which coincides with results obtained by Toyota and Watanabe (2013) whofound that Bacillus sp. helps in development of plant by suppressing disease and enhancing plant
growth.
6016(**) denotes significance difference p < 0.01

B**
B**
B**

Length of plant (cm)C N ratio

5040

12030450 150No. of composting
36 days 9Control

A**

Treatment 1B**
B**
B**

Treatment 24Treatment 320control

A**

Control
8Treatment 130Bacillus sp. addedtreatment

Treatment 2Treatment 3601215182124

No. of days

Fig. 9 Increase in plant growth with time

Fig. 10 Plant grown for two treatmentsⒸ ISERD55

IJERD – International Journal of Environmental and Rural Development (2018) 9-1

CONCLUSION
This research was conducted to examine if adding of Bacillus sp. is beneficial for enhancing
fermentation, better decomposition of farmyard manure and also elimination of pathogenic
microbes from it. The results showed that adding of Bacillus sp. was effective in increasing thetemperature, thus eliminating of pathogenic microbes. Additionally, increased microbial activity
helped in better decomposition of carbon matter and better C/N ratio. Further, there was increase innitrogen and phosphorus concentration in Bacillus sp. added treatments compared to control. The
plant growth experiment proved that compost prepared with added Bacillus sp. can help indevelopment of plants.
Thus, with the observed results we can conclude that adding Bacillus sp. is beneficial for
better farmyard manure. Further research is required to identify specific species of microorganisms
involved during composting. Also, field experiments should be conducted to determine the loss ofnutrients through leaching, runoff and volatilization.

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