Post on 13-Jun-2019
IMPROVING BANANA AND OIL PALM SEEDLINGS GROWTH USING
INDIGENOUS NITROGEN FIXING AND PHOSPHATE SOLUBILIZING
BACTERIA
THEN KEK HOE
UNIVERSITI TEKNOLOGI MALAYSIA
IMPROVING BANANA AND OIL PALM SEEDLINGS GROWTH USING
INDIGENOUS NITROGEN FIXING AND PHOSPHATE SOLUBILIZING
BACTERIA
THEN KEK HOE
A thesis submitted in fulfilment of the
requirements for the award of the degree of
Doctor of Philosophy (Bioprocess Engineering)
Faculty of Chemical and Energy Engineering
Universiti Teknologi Malaysia
SEPTEMBER 2016
ACKNOWLEDGEMENT
I would like to take this opportunity to express my deeply grateful and sincere
gratitude to my supervisor, Dr. Zainul Akmar Zakaria and my co-supervisor Prof. Dr.
Mohamad Roji Sarmidi from Institute of Bioproduct Development (IBD), Faculty of
Chemical and Energy, Universiti Teknologi Malaysia for their detailed and constructive
comments, valuable advice, friendly help, extensive discussions and encouraging
guidance around my study have been very significant for this thesis.
My sincere appreciation also extends to the management of Felda Global Ventures
Research & Development Sdn. Bhd., all my colleagues, sub-ordinates and friends in Felda
Global Ventures Research & Development Sdn. Bhd. and Felda Agricultural Services Sdn.
Bhd. for their full support, assistance and endless helps in this work.
Last but not least, deepest appreciation to my parent, wife, sons and family
members for their continuous supports and encouragements throughout the progress
of this study.
iii
ABSTRACT
Oil palm is the biggest commodity crop while banana is the second largest
fruit crop planted in Malaysia. Both are highly nutrient-demanding crops that
requires a large amount of fertilizer. This present an opportunity to find alternative
source of nutrient that is much cheaper than the imported inorganic fertilizer for oil
palm and banana plantation. Hence, the objective of this study was to develop a
newly formulated biofertilizer incorporating locally isolated indigenous nitrogen
fixing bacteria (NFB) and phosphate solubilising bacteria (PSB) into oil palm empty
fruit bunch (EFB) compost as alternative nutrient source for banana and oil palm to
improve their growth and nutrient uptake. Three NFB and PSB strains (Enterobacter
cloacae, Burkholderia cepacia and Serratia marcescens) were successfully isolated
from the root of oil palm and banana to be formulated as biofertilizer for evaluation
on oil palm and banana seedlings. The formulated biofertilizer showed high viable
cell count of NFB and PSB inoculants in the carrier (fine EFB compost) i.e 2.45 x
108 cfu/g to 4.31 x 10
9 cfu (colony forming unit)/g carrier was recorded at the second
day after inoculation. Biofertilizer formulation containing Enterobacter cloacae
showed promising and consistent effect on the growth of oil palm and banana
seedlings including increased plant height (13.7% for oil palm and 15.6% for
banana), improved total dry biomass production (27.4% and 33.8% respectively) and
high total nutrient uptake (nitrogen (N) 30.6-48.1%, phosphorous (P) 27.2-33.1%,
potassium (K) 30.5-37.7%, magnesium (Mg) 48.8%, sulfur (S) 18.1-28.5% and
boron (B) 24.5%). Enterobacter cloaceae also showed good root colonization ability
as shown from the Field emission scanning electron microscopy analysis. The use of
nutrient-rich EFB compost (3.06% N, 0.37% P, 4.74% K, 3.32% calcium (Ca),
0.79% Mg, 0.07% S, 70.3 mg/kg B, 102.1 mg/kg copper (Cu), 6600.9 mg/kg iron
(Fe), 160.2 mg/kg zinc (Zn), 455.2 mg/kg manganese (Mn) and 41.4% total carbon,
C) as carrier for biofertilizer was able to improve the soil properties notably soil pH,
organic C, total N, total P, available P and exchangeable Mg. As a conclusion, the
application of locally isolated NFB and PSB together with EFB as carrier (as well as
source of nutrient) was successful to improve the growth of oil palm and banana
seedlings together with the soil properties. Biofertilizer formulation containing
Enterobacter cloacae showed higher performance relative to the other formulations,
hence recommended to be applied as soil mixture or directly applied into the planting
hole of oil palm and banana seedlings during transplanting to the field to encourage
the growth and nutrient uptake during early planting phase.
iv
ABSTRAK
Kelapa sawit merupakan tanaman komoditi terbesar manakala pisang
merupakan tanaman buah-buahan yang kedua terbesar di Malaysia. Kedua-dua
tanaman ini memerlukan input nutrien yang tinggi. Ini membuka peluang untuk
mendapatkan sumber nutrien alternatif yang lebih murah daripada baja inorganik
import untuk ladang kelapa sawit dan pisang. Dengan ini, objektif kajian ini adalah
untuk membangunkan formulasi biobaja baru daripada gabungan bakteria pengikat
nitrogen (NFB) dan bakteria pelarut fosfat (PSB) ke dalam kompos tandan kosong
sawit (EFB) sebagai sumber nutrien alternatif kepada kelapa sawit dan pisang demi
menggalakkan pertumbuhan dan pengambilan nutrien. Tiga jenis NFB dan PSB,
iaitu Enterobacter cloacae, Burkholderia cepacia dan Serratia marcescens berjaya
diasingkan daripada akar kelapa sawit dan pisang untuk diformulasikan sebagai
biobaja untuk penilaian kepada anak benih kelapa sawit dan pisang. Biobaja ini
mencatatkan jumlah sel hidup inokulan NFB dan PSB yang tinggi dalam pembawa
(kompos EFB halus) sebanyak 2.45 x 108 hingga 4.31 x 10
9 cfu (unit pembentuk
koloni)/g pembawa. Formulasi biobaja yang mengandungi Enterobacter cloacae
menunjukkan prestasi yang menyakinkan dan konsisten kepada anak benih kelapa
sawit dan pisang dapat meningkatkan ketinggian sebanyak 13.7% dan 15.6%
masing-masing serta meningkatkan jumlah berat kering sebanyak 27.4% dan 33.8%
masing-masing. Formulasi Enterobacter cloacae ini juga menggalakkan jumlah
pengambilan nutrien oleh anak benih kelapa sawit dan pisang pada kepekatan 30.6-
48.1% (nitrogen, N), 27.2-33.1% (fosforus, P), 30.5-37.7% (potassium, K), 48.8%
(magnesium, Mg), 18.1-28.5% (sulfur, S) dan 24.5% (boron, B). Analisa Mikroskopi
pancaran medan imbasan elektron juga menunjukkan Enterobacter cloaceae
mempunyai keupayaan kolonisasi akar yang baik. Kompos EFB yang kaya dengan
nutrien (3.06% N, 0.37% P, 4.74% K, 3.32% kalsium (Ca), 0.79% Mg, 0.07% S, 70.3
mg/kg B, 102.1 mg/kg kuprum (Cu), 6600.9 mg/kg besi (Fe), 160.2 mg/kg zink (Zn),
455.2 mg/kg Mangan (Mn) dan 41.4% jumlah karbon, C) digunakan sebagai
pembawa dalam biobaja turut meningkatkan sifat-sifat tanah terutamanya pH, jumlah
organik C, jumlah N, jumlah P, jumlah P tersedia dan Mg tertukar-ganti. Sebagai
kesimpulan, aplikasi pencilan bakteria NFB dan PSB dari punca tempatan beserta
EFB sebagai bahan pembawa (juga sebagai sumber nutrien) telah berjaya
meningkatkan pertumbuhan anak benih kelapa sawit dan pisang beserta kandungan
tanah. Formulasi biobaja mengandungi Enterobacter cloaceae yang menunjukkan
kesan yang lebih tinggi berbanding formulasi baja lain adalah disyorkan untuk
digunakan sebagai campuran tanah atau ditaburkan terus ke dalam lubang tanam
anak benih kelapa sawit dan pisang semasa penanaman di ladang bagi menggalakkan
pertumbuhan dan pengambilan nutrien semasa fasa awal penanaman.
v
TABLE OF CONTENTS
CHAPTER TITLE PAGE
DECLARATION
ACKNOWLEDGEMENT
ii
iii
ABSTRACT
ABSTRAK
iv
v
TABLE OF CONTENTS vi
LIST OF TABLES xii
LIST OF FIGURES
LIST OF ABBREVIATIONS
LIST OF APPENDICES
xv
xxi
xxiii
1 INTRODUCTION 1
1.1 Background 1
1.2 Problem Statement 4
1.3 Aim and Objectives 5
1.4 Scopes of Study 5
1.5 Research Significance
6
2 LITERATURE REVIEW 7
2.1 Oil Palm Industry in Malaysia
2.2 Banana Plantation in Malaysia
2.3 Fertilizer
2.4 Fertilizer Usage in Malaysia
7
9
11
14
vi
2.5 Fertilizer Application Practices in Oil Palm
and Banana Plantation
2.6 Oil Palm Mill Biomass as Potential Plant
Nutrient
2.7 Biofertilizer
14
16
19
2.8 Plant Growth Promoting Bacteria 21
2.9 Root Colonization by Plant Growth
Promoting Bacteria
23
2.10 Nitrogen Fixing Bacteria
2.11 Nitrogen Fixing Bacteria Associated with
Oil Palm
2.12 Nitrogen Fixing Bacteria Associated with
Banana
2.13 Phosphate Solubilizing Bacteria
2.14 Phosphate Solubilizing Bacteria
Associated with Oil Palm
2.15 Phosphate Solubilizing Bacteria
Associated with Banana
2.16 Co-inoculation of Nitrogen Fixing and
Phosphate Solubilizing Bacteria
2.17 Multifarious Beneficial Traits of Plant
Growth Promoting Bacteria
2.18 Carrier Material for Biofertilizer
25
26
28
31
34
35
36
39
40
3 RESEARCH METHODOLOGY 43
3.1 Study Outline
3.2 Banana and Oil Palm Root Sampling
43
45
3.3 Isolation of Nitrogen Fixing and
Phosphate Solubilizing Bacteria
49
3.4 Screening of Nitrogen Fixing and
Phosphate Solubilizing Bacterial
Isolates
51
vii
3.4.1 Determination of Nitrogen
Fixation Capability
51
3.4.2 Determination of Phosphate
Solubilizing Activity
3.5 Growth Profile of Selected Bacteria
Strains
3.6 Biocompatibility of Mixed Cultures of
NFB and PSB
3.7 Processing of Oil Palm Empty Fruit
Bunch Compost
3.7.1 Temperature Monitoring of EFB
Compost
3.7.2 Compost Sampling for Chemical
Analysis
53
54
54
55
57
57
3.8 Identification of NFB and PSB
3.9 Biofertilizer Formulation Using NFB and
PSB Inoculated into EFB Compost
3.9.1 Carrier Preparation Using EFB
Compost
59
60
61
3.9.2 Preparation of NFB and PSB
Inoculant
3.9.3 Viable Cell Count of Bacterial
Inoculants in Biofertilizer
3.10 Efficiency of Formulated Biofertilizer on
Banana and Oil Palm Seedlings
3.10.1 Effect of Biofertilizer on Plant
Growth and Biomass Production
3.10.2 Effect of Biofertilizer on Total
Nutrient Uptake
3.10.3 Nutrient Analysis of
Biofertilizer Treated Soil
3.11 FESEM Study on Root Colonization by
Inoculated NFB and PSB
62
63
64
68
68
70
72
viii
3.12 Experimental Design and Data Analysis
on Biofertilizer Evaluation
72
4 RESULTS AND DISCUSSION 74
4.1 Isolation of Potential Nitrogen Fixing
Bacteria and Phosphate Solubilizing
Bacteria from Banana and Oil Palm
Roots
74
4.2 Nitrogen Fixing Capacity of Selected
Isolates
80
4.3 Phosphate Solubilizing Activity of
Selected Isolates
82
4.4 Selection of Potential Nitrogen Fixing
and Phosphate Solubilizing Bacteria
4.5 Mixed Cultures of Nitrogen Fixing and
Phosphate Solubilizing Bacteria
4.6 Identification of Selected Nitrogen
Fixing and Phosphate Solubilizing
Bacteria
4.6.1 Identification of Isolate N15
4.6.2 Identification of Isolate N12
4.6.3 Identification of Isolate P3
4.6.4 Identification of Isolate N7
4.7 Physical Characteristic during
Composting
4.7.1 Temperature Changes during
Composting
4.7.2 Changes in Nutrient Content
4.8 Formulation of Biofertilizer
4.9 Evaluation of Formulated Biofertilizer
on Oil Palm and Banana Seedling
84
88
90
91
94
97
99
101
102
104
109
117
ix
4.9.1 Effect of Biofertilizer
Application on Oil Palm
Seedling Growth
4.9.2 Effect of Biofertilizer
Application on Banana
Seedling Growth
4.9.3 Effect of Biofertilizer
Application on Soil Chemical
Properties
4.9.4 Effect of Biofertilizer
Application on Dry Biomass
Production of Oil Palm
Seedlings
4.9.5 Effect of Biofertilizer
Application on Dry Biomass
Production of Banana
Seedlings
4.9.6 Effect of Biofertilizer
Application on Improvement
of Total Nutrient Uptake by
Oil Palm Seedlings
4.9.7 Effect of Biofertilizer
Application on Improvement
of Total Nutrient Uptake by
Banana Seedlings
4.10 FESEM Study on NFB and PSB in
Biofertilizer Formulation
4.10.1 FESEM Study of NFB and
PSB Colonisation on Oil Palm
Roots
4.10.2 FESEM Study of NFB and
PSB Colonisation on Banana
Roots
118
125
132
139
144
149
154
157
161
165
x
4.11 Re-isolation of NFB and PSB Strains
from the Inoculated Banana and Oil
Palm Roots
169
5 CONCLUSION
5.1 Conclusion
5.2 Recommendation
174
174
175
REFERENCES 177
Appendices A – E 198 - 204
xi
LIST OF TABLES
TABLE NO. TITLE PAGE
2.1 The distribution of oil palm planted area in Malaysia (2012) 8
2.2 The distribution of banana planted area in Malaysia (2007) 10
2.3 The common PGPR strain for plant growth promoting
application 22
2.4 Example of NFB that was isolated from banana plant
tissue 29
2.5 Species of co-inoculation and improvement to banana, oil
palm and other crops 38
2.6 Various types of carrier for biofertilizer inoculants and
their efficiency 42
3.1 Banana and oil palm root sampling source, sampling sites,
agricultural practices of the sites and the total roots been
sampled 48
3.2 Full composition of the Burk’s nitrogen free (BNF)
agar and Pikovskaya (PKV) agar mediums 49
3.3 Formulations of biofertilizer from different species of NFB
and PSB in single culture and mixed culture 61
3.4 Treatments layout of biofertilizer evaluation for banana
seedling 66
3.5 Treatments layout of biofertilizer evaluation for oil palm
seedling 67
4.1 Potential NFB and PSB isolated from banana root
samples 77
xii
4.2 Potential NFB and PSB isolated from oil palm root
samples 78
4.3 Profile on the response of isolates on BNF and PKV agar
media 79
4.4 Nitrogen fixing capacity of potential NFB isolates 81
4.5 Phosphate solubilizing activity of potential PSB isolates 83
4.6 Ability of selected NFB and PSB isolates in nitrogen
fixing capacity and phosphate solubilizing 84
4.7 Spectrophotometer absorbance reading of various isolates
grown in NB for 48 hours to determine the growth profile 86
4.8 Viable cell count of various isolates grown in NB for
48 hours 87
4.9 Nitrogen fixing capacity, phosphate solubilizing index
and growth characteristic for the chosen isolates which
will be further evaluated for biofertilizer formulation 88
4.10 Viable cell count of isolates in mixed culture and
single culture 90
4.11 Top 10 hits blast results of isolate N15 against NCBI
16S rRNA sequences database 93
4.12 Top 10 hits blast results of isolate N12 against NCBI
16S rRNA sequences database 96
4.13 Top 10 hits blast results of isolate P3 against NCBI
16S rRNA sequences database 98
4.14 Top 10 hits blast results of isolate N7 against NCBI
16S rRNA sequences database 100
4.15 Physical and chemical characteristics of EFB compost 113
4.16 Characterization of macronutrients in EFB compost 114
4.17 Characterization of micronutrients and heavy metal
in EFB compost 115
4.18 Viable cell count of bacterial inoculants in carrier 117
4.19 Growth profile of oil palm seedling on biofertilizer treated
soil 123
xiii
4.20 Total primary roots length, total primary roots numbers,
stem girth size and total leaves numbers of oil palm
seedlings planted on biofertilizer treated soil 124
4.21 Growth profile of banana seedling on biofertilizer treated
soil 130
4.22 Total primary roots length, total primary roots numbers,
stem girth size and total leaves numbers of banana
seedlings planted on biofertilizer treated soil 131
4.23 Chemical properties of the soil that used as growing media
in biofertilizer evaluation 133
4.24 Soil fertility level for oil palm cultivation (Goh et al., 1997) 133
4.25 Soil nutrient level at 112 days after planted with oil palm
seedling 136
4.26 Soil nutrient level at 112 days after planted with banana
seedling 138
4.27 Dry biomass analysis of oil palm seedling after 112 days
planted on biofertilizer treated soil 142
4.28 Dry biomass analysis of banana seedling after 112 days
planted on biofertilizer treated soil 149
4.29 Total nutrient uptake of oil palm seedlings after 112 days
planted on biofertilizer treated soil 153
4.30 Total nutrient uptake of banana seedlings after 112 days
planted on biofertilizer treated soil 155
4.31 Top 10 hits blast results of isolate N12-B-S1 against NCBI
16S rRNA sequences database 173
xiv
LIST OF FIGURES
FIGURE NO.
TITLE PAGE
2.1
2.2
2.3
2.4
3.1
Types of fertilizers; (a) mixture (b) compound (granular)
(c) slow-release (d) liquid and (e) organic fertilizer
Example on the effect of the inoculation of PGPR on the
growth of roots and shoot of banana plantlets (Mia et al.,
2002)
Biochemistry of atmosphere nitrogen fixation
(Simarmata, 2013)
Schematic diagram of soil phosphorous mobilization and
immobilization by bacteria (Mohammadi and Sohrabi,
2012)
Overall research methodology for this study
12
24
25
33
44
3.2 Healthy and good yielding oil palm (a) and banana (b)
trees located in Sg. Tengi Selatan Research Station,
Selangor and Jengka 26 Research Station, Pahang were
selected for roots sampling
45
3.3 Primary (a) and secondary roots (b) of banana 46
3.4 Primary (a), secondary (b) and tertiary roots (c) of oil
palm
46
3.5 Roots sampling zone for banana (a) and oil palm (b) 47
3.6 Oil palm roots were sampled and placed into sterile bags
for storage in chiller before bacterial isolation
47
3.7
Three banana root sections were placed on BNF agar
medium for NFB isolation
50
xv
3.8
3.9
3.10
3.11
3.12
3.13
3.14
3.15
Assembly of apparatus for Kjeldahl digestion (Felda
Laboratory Test Method, 2014)
Processing of oil palm empty fruit bunch compost; the
shredded EFB fibres (a), POME drenching on EFB fibres
(b), mechanical turning of EFB composting windrow and
covered by air permeable canvas (c) and mature EFB
compost windrow (d)
Composting thermometer used to measure the
temperature of the composting windrow
Un-inoculated EFB compost carrier (a) and bacteria
inoculated EFB compost carrier with darker colour and
moist (b) immediately after inoculation
A 21 days old banana tissue culture seedlings (a) and
germinated oil palm seeds (b) were used as the planting
material to evaluate the efficiency of biofertilizer
Glass house (a), shade was provided for banana and oil
palm seedlings during early stage of planting (b) and the
pots arrangement (c) for banana and oil palm seedlings
for biofertilizer evaluation
Biofetilizer applied as soil mixture at 10% (b), 20% (c)
and 30% (d) were showed darker colour as compared to
soil only media (a)
Leaves, stem and roots samples of oil palm (a) and
banana (b) seedlings for determination of biomass and
nutrient content
53
56
57
63
64
65
65
70
4.1 Growth of nitrogen fixing bacterial isolated (red arrow)
on BNF agar (left) and halo zone (blue arrow) developed
by phosphate solubilizing bacterial isolates on PKV agar
media (right)
75
4.2
4.3
Various types of bacterial cultures isolated on BNF agar
media with potential as nitrogen-fixing bacteria
Colour morphology of major groups of NFB isolates;
cream (a), cream translucent (b), yellow (c), orange (d)
and red (e)
75
76
4.4
Growth of bacterial isolates in BNF media after 24 hours
of incubation
80
xvi
4.5 Formation of clear halo zone around the isolated bacterial
colonies (isolate P5, P6, P7 and P8 in replication R1, R2
and R3) on NBRIP agar media indicating phosphate-
solubilizing activity
82
4.6
4.7
4.8
4.9
4.10
4.11
4.12
4.13
4.14
4.15
4.16
4.17
Growth profile of various isolates grown in NB for 48
hours
Chromatography gel photo of isolates N15, N12, N7 and
P3
Colour changes of shredded EFB fibres during
composting for 51 days
Profile for changes in temperature during EFB
composting for 51 days
Changes of nitrogen content of EFB compost during
composting for 51 days
Changes of carbon content of EFB compost during 51
days of composting
Changes of C/N ratio of EFB compost during 51 days of
composting
Changes of K2O content of EFB compost during 51 days
of composting
Changes of P2O5 and MgO content of EFB compost
during composting period of 51days
Physical characteristic of (a) fine EFB compost of < 2
mm, after the (b) undecomposed coarse fibres and coarse
kernel shells > 2 mm were removed from the grinded
EFB compost and the physical characteristic of (c) fine
powder form EFB compost < 1 mm, after the (d) fine
kernel shells and undecomposed fine fibres > 1 mm were
removed from fine EFB compost < 2 mm
Diagram of the preparation of fine powder form of EFB
compost (< 1 mm) from ground EFB compost as the
carrier for biofertilizer inoculants
Isolated bacterial colonies from fine powdered EFB
compost after two days of inoculation (a) Enterobacter
cloacae – single culture, (b) Serratia marcescens – single
culture and (c) mixed cultures of Burkholderia cepacia
(blue arrow) and Serratia marcescens (red arrow)
86
91
102
104
105
105
106
108
108
110
111
116
xvii
4.18
4.19
4.20
4.21
4.22
4.23
4.24
4.25
Growth of oil palm seedlings treated with biofertilizer
Formulation N15, N12 and P3N15 at 10% soil mixture
(N15-10%, N12-10% and P3N15-10%) as compared to
un-inoculated carrier at 10% soil mixture (T2-10%) and
soil only media (T1-0%) at (a) 28 days, (b) 56 days, (c)
84 days and (d) 112 days after planting
Growth of oil palm seedlings treated with biofertilizer
Formulation N15, N12 and P3N15 at 20% soil mixture
(N15-20%, N12-20% and P3N15-20%) as compared to
un-inoculated carrier at 20% soil mixture (T3-20%) and
soil only media (T1-0%) at (a) 28 days, (b) 56 days, (c)
84 days and (d) 112 days after planting
Growth of oil palm seedlings treated with biofertilizer
Formulation N15, N12 and P3N15 at 30% soil mixture
(N15-30%, N12-30% and P3N15-30%) as compared to
un-inoculated carrier at 30% soil mixture (T3-30%) and
soil only media (T1-0%) at (a) 28 days, (b) 56 days, (c)
84 days and (d) 112 days after planting
Biofertilizer treated oil palm seedling (N12-10%) showed
bigger leaf (blue arrow) and heavy secondary roots (red
arrow) as compared to uninoculated seedling (T2-10%)
Growth of banana seedlings treated with biofertilizer
Formulation N15, N12 and P3N15 at 10% soil mixture
(N15-10%, N12-10% and P3N15-10%) as compared to
un-inoculated carrier at 10% soil mixture (T2-10%) and
soil only media (T1-0%) at (a) 28 days, (b) 56 days, (c)
84 days and (d) 112 days after planting
Growth of banana seedlings treated with biofertilizer
Formulation N15, N12 and P3N15 at 15% soil mixture
(N15-15%, N12-15% and P3N15-15%) as compared to
un-inoculated carrier at 15% soil mixture (T2-15%) and
soil only media (T1-0%) at (a) 28 days, (b) 56 days, (c)
84 days and (d) 112 days after planting
Biofertilizer treated banana seedling (N12-10%) showed
bigger leaf (blue arrow) and heavy secondary roots (red
arrow) as compared to uninoculated seedling (T2-10%)
Oil palm seedlings at 112 days after planted on various
biofertilizers treated growing media at (a) 10% (w/w), (b)
20% (w/w) and (c) 30% (w/w) as soil mixture were
harvested for destructive analysis
120
121
122
125
128
129
131
140
xviii
4.26
4.27
4.28
4.29
4.30
4.31
4.32
4.33
4.34
4.35
Banana seedlings at 112 days after planted on various
biofertilizers treated growing media at (a) 10% (w/w) and
(b) 15% (w/w) as soil mixture were harvested for
destructive analysis
Appearance of Serratia marcescens on SEM studies as
rod-shaped with smooth surface (red arrow) as reported
by (a) Abel et al., 2012 and (b) Castro et al., 2007 was
also observed in this study notably for the (c) N15
formulation (Serratia marcescens in 10% soil mixture)
SEM micrograph of Enterobacter cloacae (white arrow)
in the (d) N12 formulation showed similar morphology
with E. cloacae as reported by (a) Subudhi et al., 2013,
(b) Hood et al., 1998 and (c) Naik et al., 2012
SEM micrograph of (c) Burkholderia cepacia and
Serratia marcescens in Formulation P3N15 (red arrow)
showing similar morphology for Burkholderia cepacia
(white arrow) reported by (a) Cho et al., 2002 and (b)
Vidal-Quist et al., 2014 (b)
SEM micrograph of Enterobacter cloacae colonisation
(white arrow) on oil palm roots at 28 days (a & b) and
112 days (c & d) after inoculation in growing media
SEM micrograph of Burkholderia cepacia and Serratia
marcescens colonisation (white arrow) on oil palm roots
at (a) 28 days, (b) 56 days and (c) 84 days after
inoculation in growing media
SEM micrograph of Serratia marcescens colonisation
(white arrow) on oil palm roots at (a) 56 days and (b) 84
days after inoculation in growing media
SEM micrograph of oil palm roots at (a) 56 days, (b) 84
days and (c) 112 days after planting with un-inoculated
carrier in growing media has detected filamentous shaped
bacteria (white arrow)
SEM micrograph of oil palm roots at (a) 84 days and (b)
112 days after planted in soil only media has detected
filamentous shaped bacteria (white arrow)
SEM micrograph of Enterobacter cloacae colonisation
(white arrow) on banana roots at 56 days (a & b) and 84
days (c) after inoculation in growth media
148
158
159
160
162
163
163
164
164
166
xix
4.36
4.37
4.38
4.39
4.40
4.41
SEM micrograph of Burkholderia cepacia and Serratia
marcescens colonisation (white arrow) on banana roots at
(a) 56 days and (b) 84 days after inoculation in growing
media
SEM micrograph of Serratia marcescens colonisation
(white arrow) on banana roots at 84 days after
inoculation in growth media
SEM micrograph of banana roots at (a) 84 days and (b)
112 days after planting with un-inoculated carrier in
growth media has detected the presence of filamentous
shaped bacteria (white arrow)
SEM micrograph of banana roots at (a) 28 days and (b)
112 days after planting in soil only media has detected
the presence of filamentous shape bacteria (white arrow)
Oil palm (a) and banana (b) root samples placed on
nutrient agar for bacterial growth and the final pure strain
isolated from oil palm (c) and banana (d) roots
Chromatography gel photo of the bacteria strains re-
isolated from oil palm and banana roots
167
168
168
169
171
172
xx
LIST OF ABBREVIATIONS
ACC - 1-aminocyclopropane-1-carboxylate
ANOVA - Analysis of variance
ARA - Acetylene reduction assays
AS - Ammonium sulphate (AS)
Av. P - Available phosphorous
BNF - Biological nitrogen fixation
BNF - Burk’s nitrogen free
C - Carbon
C/N ratio - Carbon : nitrogen ratio
CEC - Cation exchange capacity
CPO - Crude palm oil
CRD - Complete Randomised Design
DMRT - Duncan’s Multiple Range Test
EFB - Empty fruit bunches
EM - Effective microbe
EX - Exchangeable
FESEM - Field Emission Scanning Electron Microscope
FESEM - Field Emission Scanning Electron Microscope
GML - Ground Magnesium Limestone
Ha - Hectares
IAA - Indole acetic acid
IRRI - International Rice Research Institute
K - Potassium
Kies - Kieserite
LCC - Leguminous cover crops
MC - Moisture content
xxi
Mg - Magnesium
MOP - Muriate of Potash
MPOB - Malaysia Palm Oil Board
MRP - Mussoorie Rock Phosphate
N - Nitrogen
NA - Nutrient agar
NB - Nutrient broth
NBRIP - National Botanical Research Institute’s Phosphate
NCBI - National Centre for Biotechnology Information
NFB - Nitrogen fixing bacteria
OC - Organic carbon
OM - Organic matter
P - Phosphorous
PGPR - Plant growth promoting rhizobacteria
PKV - Pikovskaya
POME - Palm oil mill effluent
PR - Phosphate Rock
PSB - Phosphate solubilizing bacteria
SAS - Statistical Analysis System
SEM - Scanning Electron Microscope
SI - Solubilization Index
SRF - Slow release fertilizer
TC - Total carbon
xxii
LIST OF APPENDICES
APPENDIX TITLE
PAGE
A
B
C
D
E
The list of chemical used for laboratory analysis
The Phylogenetic tree-neighbour joining
(unrooted tree) for isolate N15 by NCBI blast tree
method
The Phylogenetic tree-neighbour joining
(unrooted tree) for isolate N12 by NCBI blast tree
method
The Phylogenetic tree-neighbour joining
(unrooted tree) for isolate P3 by NCBI blast tree
method
The Phylogenetic tree-neighbour joining
(unrooted tree) for isolate P3 by NCBI blast tree
method
198
201
202
203
204
xxiii
1
CHAPTER 1
INTRODUCTION
1.1 Background
Palm oil is one of the most important edible oil in terms of world production
that contributed about 35% of the total vegetable oils production in 2012/2013.
Malaysia and Indonesia are the largest world palm oil producers with market
percentage of 39% and 48%, respectively in 2010/2011. Oil palm planted area in
Malaysia was drastically increased about 1.2 million hectares from 3.9 million
hectares in 2004 to 5.1 million hectares (ha) in 2012 where Peninsular Malaysia
remained as the largest planted area with 2.6 million ha, followed by Sabah with 1.4
million ha and Sarawak about 1.1 million ha in year 2010 (Malaysia Agribusiness
Directory 2013/2014, 2013).
Oil palm plantation in Malaysia is managed by government agencies, private
companies and smallholders. The Federal Land Development Authority of Malaysia
(FELDA) is among the largest oil palm plantation agency in Malaysia with total oil
palm planted area of 13% of the total oil palm planted area in Malaysia in year 2013.
Other government oil palm plantation agencies, independent smallholder estates and
private companies contributed about 11%, 14% and 62%, respectively of the total oil
palm planted area in Malaysia (Malaysia Palm Oil Board, 2015).
2
Banana is one of the most important fruit crops in Malaysia with the
estimated planted area around 33,495 ha in 2013. It is the second largest planted
fruit crop in Malaysia after durian (88,641 ha) (Malaysia Agribusiness Directory
2013/2014, 2013). Under the New Key Economy Area of Agricultural (NKEA
Agriculture) programme, banana was identified as one of the high-value non-
seasonal tropical fruit crops. Approximately 9,000 ha of oil palm plantation land
owned by a large government linked company (GLC) have been identified for
intercropping of banana during oil palm replanting period under the entry point
project number 7 (EPP 7) which is “Upgrading capabilities to produce fruit and
vegetables for premium markets” (Economy Transfer Programme, 2013).
Fertilizer is the major input in agriculture to sustain plant growth and
achieving high yield production. Oil palm and banana are among the highest nutrient
demanding crops as compared to other commodity and fruit crops. Typical nutrient
requirement for oil palm (136 palms/ha) are as follows (per hectare per year); 129 kg
of nitrogen (4.5 kg/palm of Ammonium Sulphate), 109 kg of phosphorous (P2O5)
(2.5 kg/palm of Rock Phosphate) and 367 kg of potassium (K2O) (4.5 kg/palm of
Muriate of Potash) (Izwanizam et al., 2013). Banana (1600 trees/ha) however,
requires a much higher nutrient input than oil palm with 566 kg of nitrogen, 566 kg
of phosphorous (P2O5) and 592 kg of potassium (K2O) per hectare for two years
(Department of Agriculture Malaysia, 2009).
The fertilizer consumption of oil palm in Malaysia in year 2010/2011 was
about 374,000 tonnes of nitrogen (N), 179,000 tonnes of phosphorous (P2O5) and
989,000 tonnes of potassium (K2O) which is 78.2% of the total fertilizer usage in
Malaysia (Heffer, 2013). Banana recorded the highest fertilizer consumption fruit
crops of Malaysia in 2008 with the estimated consumption of 6,425 tonnes of
nitrogen, 6,250 tonnes of phosphorous and 10,190 tonnes of potassium (Sabri, 2009).
Nitrogen and phosphorous are the two major macronutrients which is very
important for all the crops especially during early planting for vegetative growth.
3
Nitrogen is the most important element for banana plant growth where its deficiency
could occur even on the very fertile soil and often observed under the poor rooting
condition. Phosphorous uptake in banana is very rapid in the short and longer
periods of planting (between 2-5 months after planting) notably for vegetative
growth under tropics weather condition (Lahav and Turner, 1989).
From an industrial point of view, minimization of fertilizer cost is desirable,
as this would ultimately reduce the overall operational cost for oil palm and banana
plantations. For example, the estimated fertilizer cost for oil palm production in
Felda Agricultural Services Sdn. Bhd. (FASSB) was about 34.4% of the total
operation cost or RM 50.38/ton of fresh fruit bunch in year 2011 (FASSB, 2012).
Fertilization cost in oil palm and banana plantation was increased due to fluctuation
in fertilizer price in world market. The average imported fertilizer price in Malaysia
has increased about 87.0% from RM 739/ton in year 2005 to RM 1,383/ton in 2013.
Phosphorous fertilizer recorded the highest price increase at 135.4% followed by
nitrogen fertilizer and potassium fertilizer at 107.8% and 61.6% respectively
(Malaysia Agribusiness Directory 2013/2014, 2013). Therefore, an alternative
source of fertilizer is important to reduce the impact of high fertilizer cost to ensure
the competitiveness of banana and oil palm industry in Malaysia. In view of this, the
use of oil palm biomass which is cheap and present in abundance is a feasible and
commercially viable approach. One example is the effort by Felda Global Ventures
(FGV) to recycle the oil palm empty fruit bunch, EFB (obtained from oil palm mills)
in producing EFB compost, which can act as organic fertilizer to substitute the role
of synthetic chemicals in satisfying some part of the nutrient requirement for oil palm
and banana. Nevertheless, the application of EFB compost in the estate do pose
some limitations such as its bulky nature, high moisture content (50-60%) and large
volumes required to fulfill the plant requirement due to their low nutrient contents
(1.34 % N, 0.67 % P, 1.96 % K in dried weight; Kavitha et al., 2013) as compared to
inorganic fertilizer. Currently, FASSB has produced fortifier EFB compost by
incorporating the EFB with inorganic fertilizers such as Di-ammonium phosphate,
Christmas Island Rock Phosphate, Muriate of Potash and Kieserite to increase the
nutrient content of the compost. This fortifier EFB compost showed comparable
plant growth performance, nutrient uptake and dry biomass production for the newly
4
planted oil palm as compared to the effect from the use of conventional fertilizer.
The fortifier EFB not just able to save the fertilizer cost by reducing the nutrients
application concentration by 30%, but also maintaining the adequate nutrient
requirement for the plants (FASSB, 2013).
Nevertheless, there is always the possibility of further reducing the
dependency on these N and P-based inorganic fertilizer. One of the approaches that
can be taken is by the utilization of nitrogen fixing bacteria (NFB) and phosphate
solubilising bacteria (PSB) to fix the atmospheric nitrogen and solubilized the
insoluble phosphate in the soil to supply nitrogen and phosphorous for oil palm and
banana. Both NFB and PSB inoculation to the banana and oil palm plants has been
shown to have a synergistic effect in promoting faster plant growth (Rodriguez-
Romero et al., 2005, Mia et al., 2009 and Shamsudin, 1994). Based on this, it is
highly potential to incorporate NFB and PSB into EFB compost to produce an
effective biofertilizer that shall act as an alternative basal fertilizer for oil palm and
banana plantation. The biofertilizer is possible to be applied into the planting hole
during planting to supply the nitrogen and phosphorous for the plant and promote
vigorous plant growth in early stage of planting.
1.2 Problem Statement
Banana and oil palm are two nutrient-demanding crops that require a large
amount of fertilizer input during both vegetative and reproductive stage. However,
current high price of imported fertilizer in the world market (up to 87.1% increment
from RM 739/ tonne in 2005 to RM 1383/ tonne in 2013) has resulted in the drastic
increased of the production cost in oil palm and banana plantation. Therefore, an
alternative source of organic-based fertilizer is needed for oil palm and banana plants
to reduce the impact of high fertilizer price to ensure the competitiveness of oil palm
and banana industry in world market. The oil palm empty fruit bunch (EFB)
5
compost which consisted of high nutrient content of potassium (1.8% K), nitrogen
(2.2% N) and phosphorus (0.8% P) and magnesium (1.1% Mg)) has great potential to
be utilized as fertilizer for oil palm and banana (Pupathy and Radziah, 2013). This is
expected to substantially reduce the use of the relatively more expensive imported
inorganic chemicals as source for fertilizer. Apart from this, the application of N-
fixing and P-solubilizing bacteria into the EFB compost can also further reduce the
dependency on inorganic N and P fertilizers.
1.3 Aim and Objectives
To evaluate the effectiveness of a newly formulated biofertilizer, by
incorporating indigenous nitrogen fixing and phosphate solubilizing bacteria into oil
palm empty fruit bunch (EFB) compost, as carrier for an alternative nutrient source
to improve the growth and nutrient uptake of banana and oil palm seedling.
1.4 Scopes of Study
1. To isolate and screen the newly isolated indigenous nitrogen fixing and
phosphate solubilizing bacteria from banana and oil palm root for its nitrogen
fixing capacity, phosphate solubilizing activity, fastest growing strain and
most viable strain in mixed culture.
2. To produce EFB compost from oil palm empty fruit bunches with palm oil
mill effluent (POME) as the nutrient rich carrier for the attachment of
nitrogen fixing and phosphate solubilizing bacteria.
6
3. To formulate biofertilizer with newly isolated indigenous nitrogen fixing and
phosphate solubilizing bacteria into EFB compost as carrier.
4. To evaluate the effectiveness of biofertilizer formulation on banana and oil
palm seedlings growth, biomass production and total nutrient uptake by the
banana and oil palm seedling.
1.5 Research Significance
1. Evaluation on the potential of oil palm empty fruit bunch (EFB) compost
inoculated with indigenous newly isolated nitrogen fixing and phosphate
solubilizing bacteria as an effective nutrient source for banana and oil palm
seedling.
2. Development of a new biofertilizer with an immediate commercial-viability
to be used by Felda Agricultural Services Sdn Bhd (FASSB), as the R&D
commercialization company of Felda Global Ventures Holdings Berhad i.e.
the biggest oil palm plantation owner in Malaysia.
3. Reduction on the time period required for the growth of banana and oil palm
seedlings through the application of the newly formulated biofertilizer that
resulted in improved plant growth and nutrient uptake by the plants.
177
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