DEVELOPMENT AND EVALUATION OF ORGANIC BIOFERTILIZER USING OIL PALM EMPTY FRUIT

BUNCH AS COMPOSTED MEDIUM FOR VEGETABLE GARDENING

Eza Azuren Binti Yusuf

. S Master of Environmental Science 654.5 (Land Use and Water Resource Management) E99 2014 2014

Acknowledgement

" Alhamdulillah, my utmost thanks and gratitude to Allah S.W.T for given me the will and

strength to complete the CML6066 Research Project. This project would not have been a success

if not for the guidance and support of the people, to whom I am greatly thankful to and wish to

express many thanks and appreciation.

First and foremost I would like to express my sincerest gratitude to my supervisor, Assoc. Prof

Dr. Awang Ahmad Sallehin Awang Husaini for his constant guidance, persistent motivation and

support upon the completion of this project. My special appreciation goes to my dear friends ,

course mates as well as to the SLUSE's lecturers for their support, advice and encouragement all

this times. I also owe my deepest gratitude to the postgraduate students for their cooperation and

guidance throughout this study.

Last but not least, I am heartily thankful to my parents and siblings for their endless support,

devotion and understanding which keep me going and inspired me to complete this entire study.

/

Declaration

I hereby declare that this report is based on my original work except for quotations and citations

which have been properly acknowledged. I also declare that it has not been previously or

concurrently submitted in support of an application of another qualification of this or any other

university or institutions.

I

Eza Azuren binti Yusuf

Date:

ii

Pusst Khidmat Maklumat Akademil· UNlVERSm MALAVSIA SARAWA"

.'

Table of Contents

Page

Acknowledgement

Declaration II

Table of Contents III

List of Abbreviations vi

List of Tables and Figures VII

Abstract x

1.0 Introduction

1.1 Problem Statement 2

1.2 Objectives 3

2.0 Literature Review

2.1 Empty Fruit Bunch 4 I

2.2 Biofertilizer 6

2.3 Compost and Compo sting 7

2.4 Functional Microorganisms as Inoculums for Empty Fruit Bunch Composting 8

2.5 Burkholderia sp. 10

iii

2.6 Enterobacter sp.

3.0 Materials and Methods

3.1 Mature Compost and Bacterial Strains

3.2 Preparation of Bacterial Culture

3.3 Spiking of the Mature Compost ofEFB with Bacterial

3.4 Bacterial Count

3.5 Metabolite Preparation

3.6 Seed Sowing

3.7 On Field Evaluation

3.7 Data Analysis

4.0 Results and Discussion

4.1 Bacterial Count

4.2 On Field Result (First Evaluation)

4.2.1 Height of Vegetables

4.2.2 Width of Leaves

4.3 On Field Result (Second Evaluation)

4.3.1 Number of Leaves

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11 I

12

12

13

14

15

16

16

18

19

22

22

27

32

32

iv

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4.3.2 Height of Chili 35

4.3 .3 Length of Roots 38

4.3.4 Flowering and Fruiting 41

Conclusion 42

Reference 43

Appendix 47

v

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List of Abbreviations

EFB - empty fruit bunch

LB - Luria broth

MEA - Malt Extract Agar

CMS - Cahaya Mata Sarawak

CFU - colony forming unit

POME - palm oil mill effluent

NaCI - sodium chloride

TNTC - too numerous to count

ASTM - ASTM International (fonnerly the America\} Society for Testing and Materials)

ANOVA - Analysis of variance

SPSS - Statistical Package for the Social Sciences

PIO - Burkholderia unamae

PII - Enterobacter cloacae

°C - degree Celsius

vi

List of Tables and Figures ~ J

List of Tables Page

Table 1: Vegetable and treatments for on field first evaluation 15

Table 2: Vegetable and treatments for on field second evaluation 15

Table 3: One-Way ANOVA (height) of Brassica rapa var chinensis (Pak choy) 25

Table 4: One-Way ANOV A (height) of Capsicum annuum L. (Chili) 25

Table 5: Kruskal Wallis test (height) of Brassica rapa var chinensis (Pak choy) 26

Table 6: Kruskal Wallis test (height) of Capsicum annuum L. (Chili) 26

Table 7: One-Way ANOVA (width of leaves) of Brassica rapa var chinensis (Pak choy) 30

Table 8: One-Way ANOVA (width of leaves) of.Capsicum annuum L. (Chili) 30

Table 9: Kruskal Wallis test (width of leaves) of Brassica rapa var chinensis (Pak choy) 31

Table 10: Kruskal Wallis test (width of Leaves) of Capsicum annuum L. (Chili) 31

Table 11: One-Way ANOVA (number of leaves) of Capsicum annuum L. (Chili) 33

Table 12: Kruskal Wallis test (number of leaves) of Capsicum annuum L. (Chili) 34

Table 13: One-Way ANOVA (height) of Capsicum annuum L. (Chili) 37

Table 14: One-Way ANOVA (length of roots) of Capsicum annuum L. (Chili) 39

vii

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List of Figures Page

Figure 1: Process of spiking mature compost with bacterial 13

Figure 2: Serial dilution for bacterial count test 14

Figure 3: Filtering process to produce metabolites 15

Figure 4: Bacterial counting ofBurkh0 lderia unamae (PI 0) 20

Figure 5: Bacterial counting ofEnterebactor docae (P 11) 21

Figure 6: Height (cm) of Brassica rapa var chinensis (Pak choy) 22

Figure 7: Height (cm) of Capsicum annuum L. (Chili) 23

Figure 8: Mean Plot (height) of Brassica rapa var chinensis (Pak choy) 24

Figure 9: Mean Plot (height) of Capsicum annuum L. (Chili) 24

Figure 10: Width of leaves (cm) of Brassica rapa var chinensis (Pak choy) 27

Figure 11: Width ofleaves (cm) of Capsicum annuum L. (Chili) 27

Figure 12: Mean Plot (width ofleaves) ofBrassica rapa var chinensis (Pak choy) 28

Figure 13: Mean Plot (width ofleaves) of Capsicum annuum L. (Chili) 29

Figure 14: Number of leaves of Capsicum annuum L. (Chili) 32

Figure 15: Mean Plot (number ofleaves) of Capsicum annuum L. (Chili) 33

Figure 16: Height (cm) of Capsicum annuum L. (Chili) 35

viii

List of Figure Page

Figure 17: Mean Plot (height) of Capsicum annuum L. (Chili) 36

Figure 18: Mean Plot (length of roots) of Capsicum annuum L. (Chili) 39

Figure 19: Length of roots of Capsicum annuum L. (Chili) 40

Figure 20: The early flower buds of T2 (P I 1) treatment in the third week of application 41

Figure 21: The fruits of the T2 (P 11) treatment in the fifth week of application 41

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Development and Evaluation of Organic Biofertilizer Using Oil Palm Empt)l Fruit Bunch

as Composted Medium for Vegetable Gardening

Eza Azuren binti Yusuf

Master of Environmental Science Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

a n Malaysia, the oil palm industries generate approximately 90 million tonnes of renewable biomass each year. The l;;mpty fruit bunch (EFB) contributes the highest amount of solid waste from this industry. Various studies were conducted on managing the solid waste such as EFB by means of composting process. The objectives of this study were to develop organic biofertilizer using the oil palm empty fruit bunch as medium using effective microbes and to test and evaluate the use of biofertilizer developed on the vegetable gardening. The study revealed that vegetable of Capsicum annuum L. (Chili) that grew with the mature compost that were spIked with Burkholderia unamae PI 0 (Bioctiv-SFI) and Enlerobacter cloacae PII (Bioactiv-SF2) revealed to have positive impacts on the growth of the Chilies. The treatment with the mature compost cured with Enterobacter cloacae (P II) showed the best result among the other treatments. This included the highest values of height, greater growth of roots formation, and early induction of flowering. Further study should be conducted in longer period and bigger sample size in order to get more data on the application of these two types ofbacterial on the vegetable garden.

Key words: Empty fruit bunch, biomass, mature compos! and biofertilizer.

ABSTRAK

Industri lrelapa sawit di Malaysia menghasilkan kira-kira 90 juta tan biomas yang boleh dikitar semula pada setiap tahun. Tandan kelapa sawit kasong merupakan penyumbang terbesar kepada penghasilan sisa pepejal daripada indus,ri ini. Banyak kajian lelah dijalankan bagi menguruskan sisa pepejal seperti tandan kelapa sawit kosong iaitu melalui proses penghasilan kompos. Kajian ini dijalankan bagi menghasilkan baja bio menggunakan tandan kelapa sawil kosong sebagai media yang dicampur dengan bakteria dan unluk mengkaji serta menilai penggunaan baja bio lersebut Ire alas lanaman sayuran. Kajian ini membuklikan Capsicum annuum L. (cili) yang ditanam bersama kompos malang yang dicampur dengan Burkholderia unamae PI0 (Biocliv-SF 1) and Enlerobacler cloacae P 11 (Bioactiv..sF2) menunjukkan kesan positij lerhadap perlumbuhan pokok cili. Uji kaji y ang menggunakan kompos malang yang dirawat dengan Enterobacter cloacae (P 11) menunjukkan hasil yang lerbaik berbanding yang lain. Ini lermasukiah ukuran lretinggian yang tertinggi. pertumbuhan akar yang lebih banyak serla penghasilan bunga yang lebih (lWal. Kajian yang lebih mendalam hendaklah dijalankan unluk tempoh yang lebih panjang dan saiz sampel yang lebih besar bagi mendapatkan lebih banyak data ten tang penggunaan dua jenis bacteria ini lerhadap lanaman sayuran.

Kala kund: Tandan lrelapa sawit kosong. biomas. kompos matang dan baja bio.

x

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1.0 Introduction

The oil palm industry plays a major role in the economic development of several tropical

countries. In Malaysia, the oil palm industries generate approximately 90 million tonnes of

renewable biomass (empty fruit bunches, palm press fiber, trunks, fronds and shells) every year.

This includes about 1.3 million tonnes of oil palm trunks, 8 million tonnes of pruned and felled

fronds and 2.4 million tonnes of oil pa~m empty fruit bunches (EFB) (Malaysia Palm Oil Board,

2003). According to Tanaka et al., (2006), the annual production of EFB is 12.4 million tonnes,

which gives a great challenge to the solid waste management for its safe, scientific and

environment friendly disposal.

In the past years, EFB was being used as fuel to produce steam in the palm oil mills. The burning

ofEFB caused serious environmental concern and the authority imposed strict rules to control air

pollution from such activities. EFB is now used as mulch in agricultural and in the oil palm

plantation to control weeds, prevent erosion and preserve the soil moisture (Alam et al., 2009).

However, owing to increase cost of labor, transportation and its distribution, the utilization of

EFB as mulch is becoming more expensive.

Composting is a process of transforming organic material of plant and animal origin under

controlled conditions into a relatively stable product, hygienic and humus-like product called

compost (Saleh et al. , 2011). It is an alternative way to convert the bulky biomass into a

valuable, manageable product, for use on plantations or as a market product. Today, compo sting

is becoming very important in management of wastes through recycling and reducing the mass

and volume of the waste. This process reduces the bulk volume of organic materials, eliminates

the ri k of spreading of pathogens, weed seeds or parasites associated with direct land

1

"

application of manure and leads to final stabilized products, which can improve and maintain soil

fertility.

Agriculture is one of the main sources of income to this country. The concern over its direct and

indirect impacts to the environment has been increased as well. Thus, in considering to its

environmental issues, the agriculture industry has become more aware of the need to adopt more

environmental-friendly approach in providing plant nutrition and sustaining soil fertility. Over

the past few years, numerous researches have been carried out on managing the solid waste from

the oil palm production, which including the empty fruit bunch.

1.1 Problem Statement

Biofertilizer are seen as the most suitable for the small fanners, the large plantation, forestry and

recreational industries to meet their requirements and expectations. The development of organic

biofertilizer from EFB is an alternative way tc! replace the application of chemical fertilizers.

Chemical fertilizers are well known for its disadvantages, such as its impacts to the environment

and human health. The higher cost of chemical fertilizers than organic fertilizers has burdened

the users, including the smallholder fanners and gardeners.

The production of biofertilizer usmg the EFB will help to produce compost that is more

environmental friendly and cost effective. This not only offers better option of compost

application to the owners of oil palm industries, but also t9 the smaHholder fanners especially for

the application to their vegetable gardens. The process of converting the EFB to biofertilizer can

be accelerated in shorter time with the use of effective microbes that spiked on the mature

compost.

2

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1.2 Objectives

The pecific objectives of this study were:

1. to develop organic biofertilizer using the oil palm empty fruit bunch (OPEFB) as medium

using effective microbes.

ii. to test and evaluate the use of biofertilizer developed on the vegetable gardening.

3

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2.0 Literature Review " J

Malaysia is one of the countries in Asia that practiced agriculture as one of its major industries of

economic importance. Agriculture has played a vital role in the development of modern Malaysia

and continues to make a significant contribution to the national economy. The plantation sector,

especially oil palm, still leads the world in terms of vegetable oil production, research and

development.

Oil palm (Elaeis guineensis Jacq.) is a perennial tree crop and is an indigenous to Africa. It is

one of the major oil crops in the world, which is planted extensively in the humid tropical land

such as Malaysia. It has been domesticated from the wilderness and transformed to become a

plantation-based industry (Yusoff, 2006). Southeast Asia is the dominant region of production

with Malaysia being the leading producer and exporter of palm oil.

2.1 Empty Fruit Bunch (EFB)

The empty fruit bunches (EFB) is a lignocellulosic material which typically contains 25 percent

of lignin, 50 percent of cellulose and 25 percent of hemicellulose in their cell wall (Gu et al.,

2(00). EFB is the main solid waste from palm oil extraction. Together with other solid waste

such as the mesocarp fibres (from pressed fruits) and kernel shells (from fruit kernels), they are

usually used as boiler fuels for the steam turbines to propuce steam for sterilization of fruit and

to generate of electricity.

The disposal of agro-waste such as EFB through clean clearing technique, which includes

burning and re-burning, is found to pollute the air and is costly. In corresponding to these issues,

the government of Malaysia had imposed a ban on open burning in year 2000 under

4

'usat Khldmat MakJumat A](ademi~: UNlVERSm MALAYSIA SARAWAI\

'"

Environmental Quality Act (EQA, 1974). Sumathi et ai., (2009) stated that the EF,B were re-used

in the industry to produce bio-oil, biodiesel, chemical compounds, and microorganisms.

EFB is a suitable renewable raw material and selected as preferred source as composting

materials because it is easily accessible, abwndant locally and rich in lignocelluloses.

Furthermore, EFB has high porosity, water holding capacity and consequently high nutrient

holding capacity. At the oil palm, the EFB is used mostly as mulch. The practice of placing EFBs

on the oil surface has brought economic benefits as it enhances vegetative growth and increased

production. In addition, it also aids to prevent erosion, manage weeds and sustain soH moisture.

Owing to the current labor shortage, the transportation and distribution of EFB in the field is

getting more expensive. There is a growing interest in composting EFB, in order to add value,

and also to reduce the volume to make application easier. Other materials are often added,

particularly chicken manure and POME. Howe:,er, POME has a high nutrient content, and large

oil palm plantations prefer to use it directly as fertilizer. Composting of EFB has been extended

to farmers and the initial method adopted was to mix the EFB with chicken manure (Ferishman

et al., 201 2). It was later stacked in boxes that cover with plastic and this process took about

eleven to twelve months to reach its maturity.

5

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2.2 Biofertilizers

Biofertilizer can be defined as a natural product that carrying living microorganisms obtained

from the cultivated soil or root (Ramakrishnan & Selvakumar, 2012). In the simplest definition,

biofertilizer can be called as living fertilizers, because unlike the organic fertilizers, biofertilizers

are a mixture of beneficial microbes such as bacteria and fungi.

Biofertilizers have shown great potential as supplementary, renewable and environmental

friendl y ources of plant nutrients. They are non-bulky, low cost agricultural inputs playing a

vital role in improving the nutrient availability to the crop plants. Other benefits of biofertilizers

are to increase crop yield by 20 percent to 30 percent, replace chemical nitrogen (N) and

phosphorus (P) by 25 percent, stimulates plant growth, activate soil biologically (Toyota &

Kuninaga, 2006), restores natural fertility and provides protection against drought and some soil

borne diseases. Besides their role in atmospheric nitrogen fixation and phosphorous .

solubilisation, biofertilizers also facilitate in stimulating the plant growth hormones by enhancing

nutrient uptake and improved tolerance towards drought and moisture stress. A small amount of

biofertilizer is adequate to generate desirable resul ts. Anandaraj and Delapierre (2010) stated that

in each gram of carrier of biofertilizers, it contains approximately 10 million viable cells of a

specific strain.

There are several types of biofertilizers such as for (i) nitrogen supply: Rhizobium for legume

crops, Azotobacter or Azospirillum for non legume crops, Acetobacter for sugarcane only, (ii) for

phosphorous supply: Phosphatika for all crops to be applied with Rhizobium, Azotobacter,

Azospirillum and Acetobacter, and (iii) for enriched compost, cellulolytic fungal culture and

Phosphotika and Azotobacter culture (Singh & Amberger, 1991).

6

2.4 Functional Microorganisms as Inoculums for EFB Composting J

Composting is an extraordinarily complex process which involves microbiological degradation,

mass and energy transfer phenomena and coexistence of non-steady state conditions. The time

required for natural composting of EFB is simply too long and it is unable to satisfy the daily

waste produced by the palm oil mill. Previous researchers have conducted different methods of

EFB composting and these processes took at least two months.

The development of organic biofertilizer from EFB has gained much attention among the

researchers. A research by the Microbiology Research Group at the University Malaysia Sabah

(UMS) Biotechnology Research Institute has developed a simple and easy way to produce

biofertiliser from oil palm fibre empty fruit bunches (EFB) within 40 days (Borneo Post, 2013).

In shortening the time of composting, usage of additives has been a popular method to achieve

fast composting. According to Wei et aI., (2007), compost or biofertilizer could be produced

with the inoculation of appropriate functional microbes which increase the decomposition rate,

shorten the maturity period and improve the quality of compost. Some microorganisms have

been discovered as useful cultures in composting process as they act as biocatalysts in the

composting process in biological waste treatments.

Through microbial metabolic, the pollutants waste is transformed into environmental friendly

products. The microorganisms showed good performance when used as a starter culture in

shortening the length of composting period. For instance, the Bacillus sp. isolate improved the

rate of composting through the decrease of the concentration of polymer cellulose, hemicellulose

and lignin, which the produced composts showed increased of yields in tomato planting.

(Vargas-Garcia et ai., 2007).

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In addition, complex organic compounds of EFB like lignin are mostly degraded by thermophilic

microfungi and actinomycetes. The thermophilic cellulolytic degradation agents are included

Thermomonaspora sp., Thermoactinonmyces sp., Trichurus sp. and Chaetomium sp. (Suyanto et

ai., 2003). These thermophilic fungi have an optimum functioning temperature of around 40­

50°C, which is also the optimum temperature for lignin degradation in compost (Tuomela et aI.,

2000).

Some other microbes having cellulase and protease activities are Aspergillus sp., Cellulomonas

sp., Coprinus sp., Microbispora sp., Pseudomonas sp., Penicillium sp. and Trichoderma sp.

(Haddadin et al., 2009). Inoculation of these facultative bacteria and fungus may shorten the

composting time, improve the compost quality and inhibit the growth of some plant pathogens.

9

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2.5 Burkholderia sp.

" Burkholderia is a diverse genus, which occupy a wide array of ecological niches. It comprises

soil and rhizosphere bacteria including plant and human pathogens. The members of the

Burkholderia genus have significant ecological functions, together with plant-growth promotion,

symbiotic nitrogen fixation and degradation of xenobiotics (Suarez-Moreno et al., 2012).

Burkholderia sp. can be free-living in the rhizosphere, epiphytic, endophytic, obligate

endosymbionts and phytopathogen.

According to Coenye and Vandamme (2003), some strains have been identified to improve

disease resistance In plants, enhance nitrogen fixation and overall host adaptation to

environmental stresses, as well as contribute to better water management. Some members of

Burkholderia genus can excite growth in plant. The growth promotion caused by Burkholderia

sp. is possibly owing to the combination of ,several mechanisms that involve both plant and

bacterial partners. Besides, some Burkholderia sp. can also be used as biofertilizers, either by

fixing nitrogen or by releasing iron or phosphorus from rock phosphates and to benefit crops

cultivated in low-fertility soils.

Burkholderia unamae was found in an endophyte of plants grown in regions with climates

ranging from semi-hot sub humid to hot humid, B. unamae was isolated from rhizospheres and

plants growing in soils with pH values in the range 4.?-7.1, but not from soils with pH values

higher than 7.5 (Caballero-Mellado et al., 2004). Among the plant-associated Burkholderia, B.

unamae has relevant features, such as colonization of the rhizosphere and internal tissues of

taxonomically unrelated host plants, including maize, coffee, sugarcane, and tomato and exhibits

several potential activities involved bioremediation, or biological control.

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2.6 Enterobacter sp.

Bacteria of the Enterobacter genus are generally found in nature. Enterobacter sp. is gram­

negative bacteria that are categorized as facultative anaerobes, which denotes that they are able

to flourish in both aerobic and anaerobic environments. These microorganisms are saprophytic in

the environment and commensal in the enteric flora as they are originate in soil and sewage, as

well as in the human gastrointestinal tract (Maria et ai. , 2012). Alam et ai. , (2001) stated that

there are several rhizobacteria that have been identified as plant growth promoting rhizobacteria

(PGPR). These include Enterobacter, Burkholderia, Pseudomonas, Azospirilum, Azotobacter,

Bacillus and Serratia.

Enterobacter cloacae are belonging to the Enterobacteriaceae family. Within this family,

Enterobacter is most closely related to, and is grouped in a sub-clade with, Klebsiella. The E.

cloacae species consist of various group of bacteria that has been found in diverse environments,

ranging from plants to soil to humans. Plant pathogenic strains of E. cloacae have been reported

to cause Enterobacter bulb decay in onion plants and bacterial wilt in mulberry (Schroeder et ai.,

2010). E. cloacae are well recognized as a human opportunistic pathogen that is usually found in

hospitals. It causes a wide range of infections, such as lower respiratory tract infections, urinary

tract infections, bloodstream infection, sepsis and meningitis (Dijk et ai., 2002). A novel

microorganism belonging to the genus Enterobacter isolated from the soil in the rhizosphere of

cucumber, i.e., E. cloacae, speed up the growth of various types of agriculturally useful plants

including cucumber. A study conducted by Dewa et al., (2014) revealed that isolates of E.

cloacae improved the root system of rice, increased the macro nutrients content in the leaf,

increased the content of chlorophyll in the leaf, and increased the number of tillers per hill .

11

3.0 Materials and Methods .J

3.1 Mature Compost and Bacterial Strains

The mature compost was obtained from the Cahaya Mata Sarawak (CMS). As for the bacterial

strains, the Burkholderia unamae (PIO) and Enterobacter cloacae (Pll) were attained from the

Molecular Genetic Laboratory Universiti Malaysia Sarawak (UNIMAS).

3.2 Preparation of Bacterial Culture

The bacterial were cultured in the Luria broth (LB). According to Sambrook and RusseH (2001),

the standard recipe for preparing 1 liter of Luria broth involved the preparation of 109 of

tryptone, 5g of yeast extract and 109 ofNaCI.

The Luria broth powder was weighed and poured slowly into the conical flask that contained

distilled water. The Luria broth was sterilized by autoclaving at 121°C temperature with 15 psi

pressure. After autoclave, the Luria broth was left to be cooled at room temperature before

adding the bacterial. By using a micropipette, 100 !!l of Burkholderia unamae (P10) was added

into the Luria broth and then placed in the shaking incubator for 18-24 hours. The same

procedures were r epeated for the preparation of bacteria culture for Enterobacter cloacae (P 11).

12

3.3 Spiking of the Mature Compost of EFB with Bacterial

The process of spiking the mature compost of EFB with bacteria was as shown in Figure 1. The

Luria broth that incubated with bacterial for 18 to 24 hours was added into the mature compost

of EFB from the CMS. For a 100 g of mature compost, a 10 ml of Luria Broth was used. Both

the mature compost and the Luria broth with bacterial were mixed evenly with some addition of

distilled water. The compost was then left to cure for one week before used.

(a) The Luria broth that contained bacterial

(b) Mature compost from the CMS

(c) Cured mature compost after one week

Figure I: Process of spiking mature compost with bacterial

3.4 Bacterial count

UlmL Serial Dilt.1 ti on transrQl'/_." t,O 1'1'11 1.0mL 1,OmL 1,OmL 1.0 mL

trdn&a­ tran~r'er tfdl\sfer Ircu::ier tran,:;l'er/ \ ",--.\ " .~\ ......-....,( , /" - (---­

) ~

I ! -I

,

- .1

'= :\ .

Il0mL­/ __ c \ 1~

c_~

j J 10"'l c

count mlonies = N ec:w::h colony gJ"1!W from 3 singlp. CF.!1!

Figure 2: Serial dilution for bacterial count test (Source: Olson, 2014)

Serial dilutions (10·) to 10-6) as shown in the Figure 2 were prepared. 1.0 ml from the original

inoculums was transferred into the 10·) dilution tube. The bacterial suspension was mixed

thoroughly using the vortexes before proceeding to the next step. 0.1 ml of bacterial suspension

was later transferred in the center of a nutrient agar plate. The glass rod was sterilized by dipping

it into a 70% alcohol solution and then passing it quickly through the Bunsen burner flame. The

inoculum was spread evenly over the entire surface of tRe nutrient agar plate. The flaming and

spreading were repeated for each of the remaining dilutions . After the spread plates had been

permitted to absorb the inocula for several minutes, the plates were inverted and incubated at

37°C for 18 to 24 hours. The colony forming unit on each plate were compared and counted for

each serial dilution that was prepared.

14