IJERD – International Journal of Environmental and Rural Development (2022) 13-2

Research article
erd

Effect of Adding Agricultural and Organic Lime on Soil
Properties and Survival Rate of Pathogenic Bacteria(Coliform and E. coli) in Farmland Soils of Kampong Cham
Province, Cambodia
MUY LEANG KIM
Graduate School of Agriculture, Tokyo University of Agriculture, Japan
Email: muyleangkim70@gmail.com

MACHITO MIHARA*
Faculty of Regional Environment Science, Tokyo University of Agriculture, Japan/
Institute of Environmental Rehabilitation and Conservation, Tokyo, Japan
Email: m-mihara@nodai.ac.jp
Received 30 January 2022

Accepted 4 July 2022(*Corresponding Author)

Abstract Cow manure and compost has been applied as fertilizer for many years by
farmers in Kampong Cham province. Improperly processed of cow manure and compost
may contained pathogenic bacteria such coliform and E. coli that are harmful to humans.
Pathogenic bacteria coliform and E. coli are both recognized as hazardous microorganismin the environment and for public health. It is an important pathogen associated with several
foodborne and waterborne outbreaks of gastrointestinal illness, which has been widelyreported in Cambodia. Also, coliform and E. coli was detected in farmlands soils ofSamraong and Baray Communes, Kampong Cham Province. The contamination ofagricultural land, surface water, irrigation water, and fresh vegetables, can become areservoir of infections. Therefore, it is necessary to eliminate the pathogenic bacteria fromthe contaminated areas. Many studies have shown that inactivation of pathogens inbiosolids depends on several factors, from that the soil pH is one of the dominant factors
affecting the inactivation of pathogenic bacteria. Increasing of pH can be achieved byadding agricultural limestone calcium carbonate (CaCO3) or quick lime (CaO). The
objectives of this study are to investigate and evaluate the effects of adding different
agricultural and organic lime materials on soil chemical properties and the survival rates ofpathogenic bacteria coliform and E. coli. Pot experiments was conducted and different
CaCO3 and CaO material such CaCO3 powder, eggshell, clamshell, and wood ash are used
for the soil amendment. The CaCO3 and CaO added to soil at the ratio of 0 g, 5 g, and 15 g
to 750 g of soil to each pot. The CaO obtained by burning the CaCO3 powder, eggshell,clamshell, and wood ash at 800˚C. Soil chemical and biological parameters such as pH, EC,
K+, Na+, Ca2+, coliform and E. coli were determined. The results showed that increase insoil pH due to lime addition were observed. However, there’s no significant difference ofsoil pH responding to amounts of lime added 5 g, 10 g and 15 g. Similar trends were also
observed between the unburned and burned lime material. The survival rate of coliform andE. coli were decreased with unburned and burned lime added, the pathogenic bacteria canbe eliminated at 7 days after addition of lime. Since the performance of unburned andburned lime on the survival rates of pathogenic bacteria no different. So, it is not necessary
to burn the lime material of CaCO3, eggshell, clamshell, and wood ash. As burning material
need the temperature at 800 °C, farmers need a burner machine that can reach to thattemperature. Farmers in Cambodia usually burning wood or charco to get the heat.
Therefore, it is recommended that farmers can use the unburned lime instead of burnedmaterials in Kampong Cham Province.
Keywords: agricultural lime, calcium carbonate, fertilizers, shells, soil pH,
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INTRODUCTION
Organic amendments or fertilizers including livestock manure and compost are valuable sources ofnutrients for plant’s growth. The organic matter also contributing to improve the soil quality andfertility. Application of organic waste products to agricultural land without control of their hygienic
safety is one of the potential risks by which pathogens may enter the human food chain (Nicholson
et al., 2005). Pathogenic bacteria in raw vegetables and their consumption affects human health,resulting in diarrhea or serious disease (Beuchat, 1996; Summer and Peters, 1997). Muyleang and
Mihara (2019) reported that the coliform and E. coli was found in farmland soils in Samraong andBaray Commune, Prey Chhor District, Kampong Cham Province. The occurrence of coliform andE. coli maybe a potential of food-borne pathogens in the farmlands soils as well as vegetable. Also,
the application of fresh cow manure or immature compost that contained pathogenic bacteria canspread the pathogens to the farmland’s soils. The contamination of agricultural lands, surface water,
irrigation water, and fresh vegetable can become a reservoir of infection. To produce safety
agricultural production, it is necessary to eliminate pathogenic bacteria such as coliform and E. coli
in farmlands. The inactivation of pathogens in biosolids depends on several factors such astemperature, moisture content, competition from indigenous microflora, soil types and texture(Martin et al., 1990; Ward et al., 1981; Russ and Yanko, 1981). However, the survival rate of E.
coli became remarkably low with pH higher than 9.0. Increasing of pH can be achieved by addingof agricultural lime (CaCO3) and quick lime (CaO). Lime is commonly used to farmlands soil forneutralizing soil acidity for centuries and has been demonstrated to improve crop yield (Acosta etal., 2000). Calcium carbonate (CaCO3) is one of the basic inorganic and inexpensive materials thatwere applied in agriculture industries to stabilize the soil pH. About 95% of CaCO3 can be obtained
from shellfish and the rest is organic matter and other compounds (Hamester et al., 2012). An
increase in soil pH, with liming application help to increase in microbial activity, resulting inincreased decomposition of soil organic matter. Therefore, the study was proposed different lime
material application and its effect on soil properties and the microbial survival rates.
OBJECTIVE
The objectives of this study are to investigate and evaluate the effects of adding different
agricultural and organic lime materials on soil chemical properties and the survival rates ofpathogenic bacteria coliform and E. coli.
METHODOLOGY
Experimental Designed and Materials Used
Pot experiment was conducted in the experimental room at Tokyo University of Agriculture from
August to September 2021 during summer season. Fresh cow dungs were collected from Onoji
Farm and added to soil samples to get pathogenic bacteria contamination in Fig. 2.

CaCO3

EggshellClamshell

Fig. 1 Agricultural and organic lime materialsⒸ ISERD

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Wood ash

IJERD – International Journal of Environmental and Rural Development (2022) 13-2

Unburned and burned lime materials were used for the experiment. The unburned lime
materials such Agricultural lime CaCO3 powder, organic lime such eggshell, clamshell, and woodash (Fig. 1), and the burned lime materials were obtained from burned of CaCO3, eggshell,clamshell, and wood ash at 800̊ C.

Fig. 2 Cow manure collection in Onoji Farm, Tokyo, Japan (left)
and pot experiment design (right)

The pH of the soil samples used for the experiment was around 6.8 to 6.9. The
amounts of lime materials were added from 5 g, 10 g, and 15 g to 750 g of soil, thisamount added in the experiments (Fig. 3) was decided based on practical use in Cambodia(Chakraborty, 2014). For the actual field of lime added was from 33 kg a-1, 66 kg a-1, and
99 kg a-1.
Chemical Properties of Lime Materials Used
The lime materials of CaCO3, eggshell, clamshell, and wood ash that used for the experiment has
the calcium carbonate content (CCC%) of 99.9%, 97.8%, 98.4% and 40% respectively. The pH ofeach material ranged from 9.5 to 9.9 (Table 1).
Table 1 Chemical properties of lime materials used in the experiments
2+

Calcium carbonate
content (%)

pH

EC(mS/cm)Ca(mg/L)

CaCO3

98.9

9.5

0.45

23

Eggshell

97.8

9.5

0.26

20

Clamshell

98.4

9.5

1.03

14

Wood ash

40

9.9

14.6

170

Lime materials

Samples Preparation--

Soil samples from Samraong and Baray Communes were used for the experimental. The
samples were mixed and sieved with 2 mm sieve to remove the leaves, root and stone, and air
dried at room temperature before used for the experiment. The initial soil physical, chemical,
and biological properties were analyzed.
Eggshell and clamshell were collected from daily consumption in Cambodia, all the eggshelland clamshell were washed and air dried before crushed to powder. For CaCO3 and wood ash
were obtained from the commercial products. Also, for the CaO that was used for theexperiment was obtained from burned of the raw materials of CaCO3, eggshell, clamshell, andwood ash at 800 ̊C.

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Fig. 3 The experimental design of lime materials added treatments
Data Collection and Analysis
The soil samples were collection from the first day before and after added of different lime
materials and the following collection were done in every two days. The samples were analyzed on
the soil water contents, soil pH, EC, K+, Na+, Ca2+, coliform and E. coli. T-test was used to see thesignificant difference between the variables.
RESULTS AND DISCUSSION
Effect of Unburned and Burned Lime Materials on Soil Chemical Properties
Increases in soil pH due to lime addition were observed in this study. Similar tends of soil pH
increased were observed between the unburned lime (CaCO3) and burned lime (CaO) materials in(Fig. 4).

*: Significance difference at 95%, p<0.05, **: p<0.01

Fig. 4 The effect of unburned and burned lime materials on soil pH
Addition of CaCO3, the calcium ions (Ca2+) from the lime adsorb on soil particle surface andthen the carbonate (CO3) take place and reacts with the acidity to form water and carbon dioxide.
However, the effect of wood ash on soil pH became lower due to losses of K2O after burning at 800̊C. Moreover, the CaO materials provided more effectiveness to increase soil pH than CaCO 3
materials, except the burned wood ash which losses of K2O. Burned lime (CaO) reacts with water
to form calcium hydroxide (Ca (OH)2) and the solubility is higher than of CaCO3. The wood ashⒸ ISERD

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without extra burning should be applied to the farmlands directly. There was no significant
difference of soil pH responding to the amounts of lime added in this study. Therefore, the amountsof lime added less than 5 g should be considered.

**: Significance difference at 99%, p<0.01

Fig. 5 The effect of unburned and burned lime materials on soil EC, K+, Na+, and Ca2+
All the treatment with unburned and burned lime material application generally increased thesoil EC, K+, Na+, and Ca2+ when compared with control. However, the application of wood ash
increasing the soil EC higher than other unburned and burned lime treatments (Fig. 5). Increased in
EC was associated with increased concentration of K+, Na+ and Ca2+ in CaCO3, eggshell, clamshell,
and wood ash treatment. Similar trend was also found in the burned lime treatments, the soil ECⒸ ISERD

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IJERD – International Journal of Environmental and Rural Development (2022) 13-2

was also slightly increase with burned lime application (Fig. 5). Additionally, increased of woodash rates significantly effect to increase of soil EC compared to other lime treatment.
Wood ash and burned wood ash shown significantly increased higher of soil cation such as K+,+
Na and Ca2+ with increasing rates of wood ash application while other treatment the change ofapplication rate with the increasing of nutrients was not significantly different. Increase in K +, Na+
and Ca2+ reflected to the supply of these elements by wood ash and the high solubility of wood ash
one applied to the soil. It was observed that the unburned lime such CaCO3, eggshell, clamshell,
wood ash and burned CaO, eggshell, clamshell, wood ash effects the soil solution chemistry in twoways, all the material ack as liming agent and as a supplier of nutrients. There is evidence that thelimes application and wood ash could represent increased in availability of nutrients for plants.
However, large concentration of basic cations obtained with higher application rates could be aconcern because of potential solute transport to surface water and ground water. For maximum
effectiveness, limes should be uniformly spread and incorporated into the soil. However, farmers in
Cambodia tend to spread the lime by hands is therefore high laborious is need. Therefore, lime
application from CaCO3, eggshell, clamshell, and wood ash must be applied at the reasonable rates
to avoid any risk for the environment, soil quality and farmers laborious.
Effect of Unburned and Burned Lime Materials on Survival Rate of Coliform and E. coli
The survival rate of coliform decreased due to lime materials. Lime increased the soil pH, reducing
the survival rate of coliform, even the pH of soil didn’t reach to 9.0 (less than 8.0). Pathogenic
bacteria were eliminated at 7 days after the addition of lime materials (Fig. 6). However, the burned
wood ash had low potential in eliminating coliform compared with other materials as its low
increases in soi pH compared to other lime materials. Also, there is no significant difference ofsurvival rate of coliform to the amounts of lime added. So, the amounts of less than 5 g or 33 kg a-1
should be considered. Similar trends of CaO used were also observed compared with CaCO3
materials. Beside that the CaO from the burned material of CaCO3 working well on eliminating thepathogenic bacteria compared to other burned materials. Increase in soil pH, EC, andconcentrations of elements such K, Ca and Na have been shown to be main driver in shaping thebacterial communities in various soils. Both burned and unburned lime materials could eliminate E.
coli in 7 days. Therefore, farmers can apply lime materials directly without burning.

a**, a**, a**, a**a**, a**, a**, a****: Significance difference at 99%, p<0.01

Fig. 6 The effect of unburned and burned lime materials on the survival rates of E. coli
CONCLUSION
Different lime materials application increased soil pH, EC, K+, Na+, and Ca2+. Similar tends wereobserved on burned lime material. CaO materials has more effective to increase soil pH than
CaCO3 materials, however, the effect of burned wood ash on soil pH became lower due to losses of
K2O after burning at 800̊ C. The wood ash without extra burning should be applied to the farmlands
directly. There was no significant difference of soil pH responding to the amounts of lime added inthis study. However, Wood ash and burned wood ash shown significantly increased higher of soil
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cation such as K+, Na+ and Ca2+ than other lime treatment, increasing of wood ash application rates
lead to increase of soil cation while other treatment the change of application rates has not
significantly different to the increased of soil cation. It was observed that the unburned lime such
CaCO3, eggshell, clamshell, wood ash and burned CaO, eggshell, clamshell, wood ash effects thesoil solution chemistry in two ways, as liming agent and as a supplier of nutrients. There isevidence that the limes application and wood ash could represent increased in availability ofnutrients for plants. The survival rate of coliform decreased due to lime materials addition.
Pathogenic bacteria were eliminated at 7 days after the addition of lime materials. Both burned andunburned lime materials could eliminate E. coli in 7 days. Lime increased the soil pH, reducing thesurvival rate of coliform, even the pH of soil didn’t reach to 9.0 (less than 8.0). Since there is no
significant difference of soil pH, and the survival rates of coliform and E. coli to the amount oflime added, so the amounts of less 5 g of lime adding should be considered. Moreover, lime
application also effects to increase the soil cation large concentration of basic cations obtained withhigher application rates could be a concern because of potential solute transport to surface waterand ground water. For maximum effectiveness, limes should be uniformly spread and incorporated
into the soil.
However, farmers in Cambodia spread the lime by hands is therefore needed high laborious.
Therefore, lime application from CaCO3, eggshell, clamshell, and wood ash must be applied at thereasonable rates to avoid any risk for the environment as well as to avoid the labor shortage. Also,
farmers in Cambodia usually burning wood or charco to get the heat. To avoid extra laborious, it isrecommended that farmers can use the unburned lime instead of burned materials in Kampong Cham
Province. Inconclusion increases in soil pH, EC, K+, Na2+, and Ca2+ by adding of CaCO3, eggshell,clamshell and wood ash can eliminate pathogenic bacteria effectively within 7 days, therefore,
farmers can directly use eggshell, clamshell, and wood ash without burning for eliminating
pathogenic bacteria in the farmland’s soils. Additionally, local farmers should be trained on
production and application of these lime materials.
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