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Malaysian Journal of Microbiology, Vol 9(1) 2013, pp. 60-67
Malaysian Journal of Microbiology
Published by Malaysian Society for Microbiology
(In since 2011)
60 ISSN (print): 1823-8262, ISSN (online): 2231-7538
Agriculture wastes conversion for biofertilizer production using beneficial
microorganisms for sustainable agriculture applications
Siti Zulaiha Hanapi1, Hassan M. Awad1,2*, Sheikh Imranudin Sheikh Ali1, Siti Hajar Mat Sarip1, Mohamad Roji
Sarmidi1, Ramlan Aziz1
1Institute of Bioproduct Development, Universiti Teknologi Malaysia (UTM), 81310, UTM, Johor, Malaysia.
2Chemistry of Natural and Microbial Products Department, National Research Centre (NRC), Dokki, Cairo, Egypt.
E-mail: hassan@ibd.utm.my
Received 27 April 2012; Received revised form 15 August 2012; Acceptance 23 August 2012
ABSTRACT
Aims: The emphasis of this study is to generate new valuable bioproducts from non-toxic cleaning waste for
environmental healing technology.
Methodology and Results: Comparisons between different types of biofertilizer formulations and the field trial
effectiveness were done. Results indicated that biofertilizer C contained the highest N value (1.8%) when compared with
biofertilizers B and A, which only contained 1.7% and 1.4%, respectively. Biofertilizer A showed significant difference in
the total count of yeast, mould, ammonia oxidizing bacteria and nitrate oxidizing bacteria compared to biofertilizer B and
C. Meanwhile, biofertilizer C was found to be significantly different from others in Lactobacillus sp. and nitrogen-fixing
bacteria count. Photosynthetic total count and Actinomycetes sp. were not noticed in all formulations tested.
Conclusion, significance and impact of study: The findings of this study suggest that biofertilizer A is suitable to be
used as a promotional biofertilizer in flower and fruit production, biofertilizer B can be used for a leafy crop, while
biofertilizer C is good for the growth of roots and stem of plants.
Key words: Biofertilizer, Beneficial microorganisms, Microbiological analysis, Chemical analysis, Field trials.
INTRODUCTION
Currently, there are more than 4.3 million hectares of oil
palm plantation in Malaysia, which is equivalent to
approximately 67 percent of total agricultural land in the
country (DOA, 2010). Malaysia had generated in excess
of 15,000 tons of solid waste per day in the form of
biomass that consists of forest and mill residues, wood
wastes, agricultural wastes, and municipal waste.
Agricultural wastes from agro-based industries are also on
the increase. State of Johore, Selangor, and Perak
collectively accounted for 65.7% of the overall identified
pollution sources in the agro-based and manufacturing
sector (DOE, 2001).
These biomasses bear a huge potential to be
applied as an alternative and beneficial invention for
various applications such as in sustainable agriculture and
etc. because they are high in moisture, organic matter and
other minerals. Thus, they can actually be reproduced into
more useful and value-added products with safety and
profitability. Recently, many countries have made an effort
to recycle 15 – 50% of the wastes they generated (Diza et
al., 1993). Weeds, stalks, stems, fallen leaves, pruning,
and dead branches (Boraste et al., 2009); animal manure
(Bheki et al., 2010); vermicompost (Warman and
AngLopez, 2010); and agriculture wastes such as
cornstalks, sugarcane bagasse, drops and culls from fruits
and vegetables (Weber et al., 2007) have long been used
as the soil conditioner to fertilize the soil and plant with the
cooperation of beneficial microbes.
Biofertilizers are environmental friendly fertilizers that
not only prevent damages to natural sources but help, to
some extent, in cleaning the nature from precipitated
chemical fertilizers (Food and Agricultural Organization,
2008). The use of organic matter such as sawdust, rice
bran, rice husk and shredded paper in producing
biofertilizer is economical. They also act as the carrier
material for nutrient and microorganisms.
The role of plant nutrients in crop production is well-
established and 16 essential plant nutrients have to be
available to the crops in required quantities to achieve the
yield target. Many studies have also emphasized on the
importance of N, P and K in enhancing the natural ability
of plants to resist stress from drought and cold, pests and
diseases (Debosz et al., 2002; Tsai et al., 2007). Essential
plant nutrients such as N, P, K, Ca, Mg and S are called
macronutrients, while Fe, Zn, Cu, Mo, Mn, B and Cl are
called micronutrients. It is necessary to assess the
capacity of a soil to supply the lacking amounts of needed
plant nutrients (total crop requirement-soil supply) (Food
*Corresponding author
Mal. J. Microbiol. Vol 9(1) 2013, pp. 60-67
61 ISSN (print): 1823-8262, ISSN (online): 2231-7538
and Agricultural Organization 2008). This is also important
to produce a good biofertilizer formulation and to supply
nutrients that can improve soil health and fertility of plants.
Several authors such as Debosz et al. (2002); and
Chen et al. (2007) are concentrating on the potential
usage of nitrogen from animal manures. Nonetheless, the
effort to find another source instead of animal manure
needs further study. Granite powder has also been
studied as a good source of slow-release K fertilizer
(Chen et al., 2007). Generally, the addition of nitrogen to
high C: N ratio residues is capable of accelerating the
microorganism activity during the fermentation process
(Saratchandran et al., 2001).
The number of microorganisms and the level of
macro- and micronutrient obviously affect the growth of
plants (Coroneos et al., 1995). One of the benefits of
fertilizers is that they contribute to the availability of
microorganism population (Marrs, 1993). Having a higher
initial count of appropriate microbes in ready biofertilizer
right after the fermentation is essential. One of the ways
to increase the number of selected microorganisms is by
using the concept of an effective microorganism (EM) as
introduced by Higa and Wididana (1991).
Field experiments are needed to determine the
nutrient availability and efficacy of most organic fertilizers.
Such an experiment is important because the nutrient
content of organic fertilizers varies widely (Parr et al.,
1998). The quality is directly governed by the number of
selected microorganisms in the active form per gram and
their capability to promote plant growth and soil fertility.
The aim of the work is to investigate the
conversation and different formulation of the agriculture
wastes for biofertilizer production by beneficial
microorganisms.
MATERIALS AND METHODS
Raw materials
The raw materials used for the bioorganic fertilizer
production were obtained from a local manufacturer in
Kulai, Johor. The waste was in the form of granules with
chemical characteristics as shown in Table 1.
Table 1: Biofertilizer type combination
Ingredients (%)
Type of biofertilizers
A
B
C
1- Burned soil
41
46
39
2-Nitrogen source
meal
7
10
7
3- Saw dust
15
30
30
4- Burned rice husk.
15
-
-
5- EM
3%
3%
3%
6-Gibberelic Acid
10 ppm
10 ppm
10 ppm
(-) = without addition of burn rice husk, EM= Effective
microorganisms
Biofertilizer preparation
Generally, the ingredients of each biofertilizers differed in
or without following the addition of burned soil, nitrogen
source meal, saw dust and burned rice husk. The
formulation of these biofertilizers types is shown in Table
2. Each of biofertilizers was inoculated with 3% of
effective microorganisms (EM) before the fermentation
proceeds.
Table 2: Microorganisms and specific media used for
isolation and identification.
No.
Microorganism
Specific
medium
Reference
1
Lactobacillus sp.
Acidified
MRS
(Institute,2009)
2
Yeast and Mold
CGYE
(Leuschner et
al., 2003)
3
N2 fixing bacteria
Ashby‘s
medium
(Ashby, 1907,
Harunor et al.,
2008)
4
Photosynthetc
bacteria
Mineral
salts-
Succinate
Broth
(Prasertsan et
al., 1993)
5
Nitrifying
bacteria
AOB, NOB
(Bhuiya and
Walker, 1977)
6
Actinomycetes
Actinomyce
tes
isolation
agar
(Awad et al.,
2009, Shirling
and Gottlieb,
1966)
CGYE = Glucose Yeast Extract Agar, AOB: Ammonia-
oxidizing broth and Nitrogen-oxidizing broth (NOB)
Fermentation and temperature monitoring
The starting fermentation was performed for biofertilizer A,
B and C in different proportions of ingredients, which
differed in or without the addition of burned soil, nitrogen
source meal, saw dust and burned rice husk. The
formulations of these biofertilizer types are shown in Table
1. All biofertilizers were inoculated with 3% of effective
microorganisms (EM) before the fermentation proceeds.
The substrate temperature was measured daily from Day
1 until Day 7 at a depth of 50 cm with a thermometer.
Isolation and enumeration of microorganisms
The total microbial population of the sample was
determined using the following methods and the specific
media for each strain were according to the literatures as
shown in Table 2. The media composition and the
preparation methods which were used in this study are
also listed in Table 2.
Mal. J. Microbiol. Vol 9(1) 2013, pp. 60-67
62 ISSN (print): 1823-8262, ISSN (online): 2231-7538
Isolation and enumeration of Lactobacillus sp. by
dilution plate technique
Man Rogosa Sharpe (MRS) medium was used to
encourage the growth of lactic acid bacteria such as
Lactobacilli, Enterococci and Pediococci. Selection of
Lactobacilli was carried out using the pH selection method
(pH 5.5 to 6.2) with Enterocci and Pediococci growing
best in this range. For this purpose, acidified MRS agar
medium (Merk, Darmastdt, Germany) was used (Institute,
2009).
Standard method for determining number of yeasts
and molds
A pour plate method following (International Standards
Organization) ISO 7954 (ISO, 1987) using
chloramphenicol glucose yeast extract (CGYE) was used.
The CGYE agar medium contained (g/L): dextrose, 20;
yeast extract, 5.0; chloramphenicol, 0.1; and agar, 15.0.
The medium was adjusted to pH 6.6. ± 0.2 prior
autoclaving. Each substrate of 10 g was suspended in 90
ml sterile saline, shaken thoroughly and 0.1 mL of each
inoculum was inoculated with AMRS and incubated at
25 C ± 1 C for 48 hours. At the same time, the CGYE
medium was incubated at 25 C ± 1 C for five days
(Leuschner et al., 2003).
Determination of nitrogen-fixing bacteria by the
spread plate method
This method is based on the ability of nitrogen-fixing
bacteria to grow in a nitrogen-free medium. The total N2-
Fixing bacteria were counted using Ashby’s medium after
an incubation period (Ashby, 1907). Ashby’s medium
composed of (g/L): mannitol, 20; K2HPO4, 0.2;
MgSO4.7H2O, 0.2; NaCl, 0.2; K2SO3, 0.1; CaCO3, 5.0; and
agar, 15.0. One gram of sample was transferred into 50
ml Ashby’s medium and incubated at 30 C ± 1 C for 2-5
days. Then, the broth surface was examined and using
serial dilution, it was streaked to nitrogen-free medium
agar for enumeration. The colonies that grew on the
medium appeared as white, off white, gray and gray to
white. They were circular, flat, raised, serrate in elevation,
and small and pinpoint in size (Ashby, 1907; Harunor et
al., 2008).
Isolation and enumeration of photosynthetic bacteria
The current method is based on the ability of
photosynthetic bacteria to assimilate CO2 and use light as
their energy source during incubation under bright and
dark conditions. Determination of photosynthetic bacteria
was carried out by incubating 5 g of sample in succinate
broth. It consisted of three media as follows: Mineral
Salts-Succinate Broth medium (1) made up with (g/L):
K2HPO4, 0.33; MgSO4.7H2O, 0.33; NaCl, 0.33; NH4Cl,
0.50; CaCl2.2H2O, 0.05; sodium succinate, 1.0; yeast
extract, 0.02; and agar, 15 g, at pH 6.8-7.2 using 5M
NaOH.
Trace element's medium (2) with (mg/L):
ZnSO4.7H2O 10; MnCl2.7H2O, 3; H3BO3, 30; CoCl2.6H2O,
20; CuCl2.2H2O, 1; NiCl2.6H2O, 2; and Na2MoO4, 3 mg,
the solution was adjusted to pH 3-4 using 5M HCl.
Medium (3) composed of 0.02% FeSO4.7H2O. The
isolates were incubated for four to seven days at 30 °C ±
1 until the appearance of red pigment (bloom) which
indicated the presence of photosynthetic microorganisms.
Positive control (Rhodopseudomonas palustris NRRL B-
4267) was incubated under the same conditions
(Prasertsan et al., 1993).
Isolation and detecting of nitrifying bacteria (Multiple
Five Tube method)
Multiple Five Tube method (Bhuiya and Walker, 1977)
was used in detecting nitrifying bacteria using ammonia-
oxidizing broth (AOB) and nitrogen-oxidizing broth (NOB).
These media were composed of the following
constituents: the AOB-medium (g/L): MgSO4.7H2O, 0.04;
(NH4)2SO4, 0.50; KH2PO4, 0.20; CaCl2.2H2O, 0.04; and
phenol red, 0.001 and the NOB medium (g/L): KNO3,
0.30; MgSO4.7H2O, 0.1875; KHCO3, 1.5; K2HPO4, 0.5;
KH2PO4, 0.5; NaCl, 0.1875; CaCl2.2H2O, 0.0125; and
FeSO4.7H2O, 0.01. Each set of AOB and NOB tubes was
inoculated with 1 ml of sample suspended and incubated
at 25-30 C for 23-28 days in case of (AOB) and for 23
days or more for (NOB).
After the end of incubation, one drop of sulfanilic
acid and N, N-dimethyl-1-naphthylamine were added into
AOB and NOB media in tubes. Red color indicates the
presence of active AOB while an absence of any color
changes is a positive result for NOB. A confirmation test
for nitrite/nitrate was carried out by added one drop of
diphenylamine to a drop of sample on a clean spot plate.
Positive tubes or wells were identified by the development
of a blue color and the absence of color is scored
negatively. All results were computed into the MPN table.
Isolation and enumeration of actinomycete's colonies
by dilution plate technique
Isolation and enumeration of actinomycetes colonies were
performed by a soil dilution plate technique using two
different media: the first medium is an actinomycete's
isolation agar medium (Difco, NJ, USA) at pH 7.0. The
second medium, Streptomyces medium, consisted of
(g/L): glucose, 5; L-glutamic, 4; KH2PO4, 1.0;
MgSO4.7H2O, 0.7; NaCl, 1; FeSO4.7H2O, 3 mg; and agar,
25. This medium was supplemented with 50 µg/L
cycloheximide (Sigma-Aldrich Corp., MO, USA) (Awad et
al., 2009). The isolates were incubated at 28 ± 0.5 ºC for
7-10 days. The results obtained were expressed as the
colony forming unit (CFU).
Actinomycete colonies were characterized
morphologically and physiologically following the
directions given by the International Streptomyces project
(ISP) (Shirling and Gottlieb, 1966).
Mal. J. Microbiol. Vol 9(1) 2013, pp. 60-67
63 ISSN (print): 1823-8262, ISSN (online): 2231-7538
Chemical analysis
The total nitrogen, phosphorus and potassium (NPK)
content of samples were analyzed according to the
following methods. Nitrogen was determined by the acid
combustion elemental analysis method using the macro
kjeldahl system (Gerhadt, German) (Tandon 1993). The
phosphorus, potassium and other micronutrients were
digested using the acid digestion method and analyzed
spectrophotometrically (Spectroquant NOVA 60, Merck,
USA) using EPA method 3050B (Tandon, 1993). Moisture
content of the samples was determined using the moisture
analyzer (MX-50, A&D Company Ltd, Japan) to a constant
weight. The pH value was measured in a 5-fold dilution of
distilled water equilibrated with the sample for an hour
with a pH meter (Delta 320, Mettler Toledo, Germany).
Ash content in a dried sample was determined at 550°C
for 24 hours using (CWF 110, Carbolite, England). C %
was determined by APHA 5310 B method according to
APHA (2005). N % was determined by APHA 4500-N org
B (Mod) according to APHA (2005).
Efficacy of biofertilizers
Efficacies for the biofertilizers were carried out for six
months. Soil for this experiment was natural silt loam with
a pH of 7.3 and moisture content of 14.4%. A local variety
of ladyfingers was used as test plant. The experimental
design was the completely randomized design with three
replicates. Four different soil beds at a size of 15 feet x 4
feet (LxW) were prepared for each treatment as followed:
Plot 1 (Biofertilizer A), Plot 2 (Biofertilizer B), Plot 3
(Biofertilizer C) and Plot 4 Controls (without Biofertilizer).
Before the planting started, each plot was treated by
spreading a total of 200 g of the respective biofertilizers
into the loose soil. The plots were then watered regularly
for 14 days. After the soil treatment, a seeding inoculation
was performed. The seeds of ladyfingers were soaked in
water for 10 minutes, and good qualities seeds were taken
out for seeding; good seeds will sink underneath the water
and vice versa. For seeding, about 3-4 seeds were
pressed 1-2 cm into the soil bed. One tablespoon of the
respective biofertilizer (about 14 g) was then dispersed on
the soil surface surrounding the planted seed and water
was applied. This was done weekly and continued to twice
a month until the day of harvesting. During harvesting, the
plants were carefully uprooted from each plot and the
plant height, length of roots, diameter of leaves, fruits and
fruit weight were recorded.
Statistical Analysis
All experiments were carried out in triplicate. All data are
reported as means ± SD (standard deviation). The results
were analyzed statistically by a one-way ANOVA using
(SPSS Inc. 2006) (Levesque, 2007) with the results:
microbiological, chemical and field trial with biofertilizers
as the main factors. The mean of each measurement
parameter was separated statistically using Tukey’s and
Dunnet’s multiple range tests with the plants grown
without the aid of biofertilizer set as control. Significance
was defined as P<0.05, unless otherwise indicated.
RESULTS
Fermentation and temperature monitoring
The temperature was recorded from the first day of
production until the eighth day of biofertilizer fermentation.
The temperature increased rapidly during fermentation,
peaking at 71 C on Day 4 and then decreased gradually
until Day 8 when the biofertilizers achieved maturity. The
result is as shown in Figure 1.
0 2 4 6 8 10
20
30
40
50
60
70
Biofertilizer A
Biofertilizer B
Biofertilizer C
Temperature oC
Time [day]
Figure 1: Temperature profile of different types of
biofertilizers during fermentation period
Isolation and enumeration of microorganisms
During fermentation, the results of total microbial
population in different biofertilizers of A, B and C
measured using the CFU/g biofertilizer are as shown in
Table 3.
In all biofertilizers tested, Lactobacillus sp. was the
major population while the nitrogen-fixing bacteria were
the minority population. The results showed that the total
count of Lactobacillus sp was 3.3 x 105 CFU g/L, 4.9 x 105
CFU g/L, and 2.3 x 104 CFU g/L in biofertilizer A, B and C,
respectively. Meanwhile, biofertilizer B and C showed the
greatest growth of yeast total count, which were 3.0 x 107
CFU g/L and 3.5 x 107 CFU g/L and the lowest total count
of yeast, 2.4 x 105 CFU g/L, was recorded in biofertilizer
A.
On the other hand, the population of nitrifying
bacteria in terms of biofertilizer A was significantly
different from biofertilizer B and C and ranged between
Mal. J. Microbiol. Vol 9(1) 2013, pp. 60-67
64 ISSN (print): 1823-8262, ISSN (online): 2231-7538
3.20 x 102 to 3.60 x 102 CFU g/L for AOB and NOB,
respectively. Concurrently, the results showed that no
significant difference had been observed for all types of
biofertilizer formulation in the case of total nitrogen-fixing
bacteria count. Also, the results indicated that no growth
was detected in both photosynthetic bacteria and
Actinomycetes in all biofertilizers examined.
Chemical analysis
The results in Table 4 show that the pH value of all
formulated biofertilizers was slightly alkaline and ranged
between 8.2 - 8.5 due to the degradation of nitrogen-
containing materials to soluble organic nitrogen. The
moisture content for each formulated biofertilizer differed
from one another based on the formulation composition
and ranged from 16.60 to 22.30%.
In addition, the ash content of all biofertilizers ranged
between 0.42-0.61 percent, depending on the formulation
composition. The stability of ash content can be used as
the parameter of compost maturity. The total organic
carbon and nitrogen content was determined and the
results showed that biofertilizer B possessed the highest
content (19.2) followed by biofertilizer C (12.0) and lastly
biofertilizer A (6.0). The results also indicated that the
macro- and micronutrients content of biofertilizer B was
the highest followed by biofertilizer A and C.
Efficacy of biofertilizers
The field trail results in Table 5 show that the plants
treated with biofertilizer C grew more vigorously than the
other plants grown in different treatments. On the other
hand, the plants treated with biofertilizer A and contained
burned rice husk exhibited the largest fruit diameter as
well as the fruit weight. The plants treated with biofertilizer
B grew better than the plants treated with biofertilizer A
and C for the diameter of leaves recorded. Overall, it was
observed that the plants treated with biofertilizer A, B and
C were growing well and had better yields than the plants
which grew without any treatment (control).
DISCUSSION
During biofertilizer preparation, microbes decompose the
organic matter and release the fermentation heat (Yang,
2003). Temperature changes have to be recorded during
fermentation to monitor the activity of the microbes. The
results showed that the temperature increased from 41 C
to 71 C at Day 4 and gradually decreased to 50 C at
Day 8, indicating that the biofertilizers had achieved
maturity. The increasing temperature during fermentation
occurs due to the active microbial growth. The
temperature changing patterns in this work are similar to
the commercial composting process made by Pai et al.
(2003).
Proper fermentation also will effectively destroy
pathogens and weeds through the metabolic heat
generated by the microorganisms (Yang 2000; Nakasaki
et al., 1996). These results are in accordance to those
obtained by Tsai et al. (2007) who found that the
inoculation of appropriate microbes during fermentation
will shorten the period of maturity and thus improve the
quality of biofertilizers. Nevertheless, there is a lack of
reported studies in the number of actinomycetes and
photosynthetic bacteria present in biofertilizer samples.
Many of the literatures only showed that the isolation of
these microorganisms from environmental samples such
as oil was noticeable (Fuentes et al., 2010). In order to
prepare a multi-functional biofertilizer, thermo-tolerant
phosphate-solubilizing microbes, including bacteria,
actinomycetes and fungi have to be isolated from different
compost plants and biofertilizers (Chang and Yang, 2009).
Biofertilizers of three different formulations were
analyzed for their microbiological, chemical and physical
components. The presence of certain microorganisms and
the nutrient mineralization are favorable to support plant
growth and yields (Parthasarathi and Ranganathan,
1999). Another study was done by Edward and Fletcher
(1988); they stated that the increase of microbial
populations increases the performance of biofertilizer
microbiologically, chemically, and physically.
The large number of Lactobacillus sp. and yeast
isolated from the final product of biofertilizer indicated the
success of fermentation. The total number of Lactobacillus
sp. and yeast were in between 5.00 x 105-8 CFU g/L.
These results are consistent with the microbial analysis
results from liquid biofertilizers produced by several
authors such as Ngampinol and Kunathigan (2008); and
Department of Agriculture (2004).
Microorganism Total Count
Biofertilizers
A
B
C
Lactobacillus sp.
3.30 x 105 a
4.90 x 105 a
2.30 x 104 b
Yeast
2.40 x 105 b
3.00 x 107 a
3.50 x 107 a
Nitrifying
Bacteria
AOB
>1.60 x 103 b
3.3 x 102 a
3.5 x 102 a
NOB
6.20 x 102 b
3.2 x 102 a
3.6 x 102 a
Photosynthetic bacteria
NG
NG
NG
Nitrogen-fixing bacteria
4.5 x 101 b
5.2 x 101 b
1.4 x 101 b
Actinomycetes
NG
NG
NG
Table 3: The populations of Effective Microorganisms (EM) examined in Biofertilizer A, B and C in CFU/g.
Significance difference (P<0.05) NG= no growth, AOB=Ammonia-oxidizing bacteria, NOB= Nitrite-oxidizing bacteria
Mal. J. Microbiol. Vol 9(1) 2013, pp. 60-67
65 ISSN (print): 1823-8262, ISSN (online): 2231-7538
Table 4: Macro- and micronutrients and other chemical analysis for Biofertilizer A, B and C.
Biof.
Form
pH
Mois.
(%)
Ash
(%)
C
(%)
N
(%)
C:N
Ratio
Percentage (%)
Concentration (mg/L)
N
P
K
Mg
Ca
B
Fe
Mn
Na
Mo
A
8.5±0.01a
19.24±0.21a
0.61±0.11a
7.2
0.94
6.0
1.4±0.15a
<0.001a
4.7±0.11a
2.9±0.38a
<0.5a
0.6±0.05a
16.3±0.21a
0.8±0.04a
22.0±0.95a
0.1±0.11a
B
8.2±0.5a
16.60±0.01b
0.42±0.12b
20.5
0.81
19.2
1.7±0.25b
0.001a
6.6±0.20b
4.7±0.17b
0.8±0.02b
1.8±0.01b
<0.0001b
4.9±0.06b
8.5±0.12b
1.1±0.11b
C
8.2±0.12a
22.30±0.11c
0.45±0.19b
18.9
0.0001
12.0
1.8±0.11c
<0.001a
4.9±0.11a
8.5±0.10c
1.1±0.05b
0.5±0.05a
1.8±0.11c
0.2±0.03a
9.0±0.17b
1.0±0.00b
Significance difference (P<0.05)
Table 5: Physical analysis during field trial for ladyfingers fertilized with Biofertilizer A, B and C.
Physical analysis
Biofertilizer A
Biofertilizer B
Biofertilizer C
Control
Plant height (cm)
185.0±7.00a
217.5±6.93b
237.6±4.96c
79.9±3.53d
Root length (cm)
34.4±0.57a
36.7±0.47a
41.8±1.68b
17.1±0.85c
Leaves diameter (cm)
34.8±1.50a
44.4±0.50b
41.8±1.68a
17.3±0.32c
Fruits diameter (cm)
3.2±0.12a
2.8±0.15a
2.6±-0.47a
1.5±0.38d
Fruits weigh (g)
38.5±0.70a
36.2±3.63b
28.0±2.11c
11.4±0.95d
Significance difference (P<0.05)
Mal. J. Microbiol. Vol 9(1) 2013, pp. 60-67
ISSN (print):1823-8262, ISSN (online): 2231-7538
The nitrifying and nitrogen-fixing bacteria total counts
were low (< 1.00x 103 CFU/g) in all formulations prepared.
The chemical analysis results derived from the
composting process showed that the inoculated
biofertilizers with tested microbes had a significantly
higher temperature, ash content, pH, total nitrogen, and
soluble phosphorus content. Adding these microbes can
shorten the period of maturity, improve the quality,
increase the soluble phosphorus content, and enhance
the populations of phosphate-solubilizing and proteolytic
bacteria in the biofertilizers (Chang and Yang, 2009). The
pH value of each biofertilizer in these experiments was in
the range of 8.20 - 8.50 which is slightly alkaline than
other solid biofertilizers as reported by many authors such
as Debosz et al. (2002); and Tsai et al. (2007). The
slightly alkaline pH is beneficial because this will
contribute to the neutralization of acidic agricultural soil
(Fageria and Baligar, 2001).
The moisture content of compost decreased during
the incubation period because the inoculation of the
biofertilizer with EM increased the temperature and
decreased the moisture content of biofertilizer. The same
phenomena has also observed in open field composting
(Pai et al., 2003).
Total ash content in the biofertilizer samples was
determined. The stability of ash content can be used as a
parameter of compost maturity. The ash content
significantly increased during preparation since the
organic materials were decomposed to form the metabolic
gases (Yang, 2003; Chang and Yang, 2009). Total
organic carbon content (C:N ratio) decreased from 19.2 in
case of biofertilizer B to 12.0 and 6.0 for biofertilizer A and
C, respectively. Total organic carbon content significantly
decreased during composting due to the degradation of
organic matter. These results are in accordance with
those results obtained by Chang and Yang (2009). It has
been noted that the properties of the initial material, in
particular is affecting the C:N ratio of the biofertilizers.
Higher C:N ratio (>30%) contributed longer composting
process to occur (Tiquiaa and Tam, 2000). Other factors
such as aeration condition, moisture content, and
temperature are also affected by the degree of N loss.
The field trial study was conducted to monitor and
observe the differences in the biofertilizers’ effectiveness
regarding their abilities to encourage plant growth.
Significant reduction of all physical properties in the case
of non-treatment plant can be explained by lack of or low
soil fertility. The plant height, length of roots, diameter of
leaves and fruits as well as the ripe fruit weight increased
when the plants were treated with biofertilizers.
It has been noted that the addition of burned rice
husk in biofertilizer B provided a higher percentage of
potassium (6.6%), which contributed to extra growth in
fruit diameter and weight compared to other biofertilizers
(without burned rice husk). These results are in
agreement with Seripong (1989) who mentioned that the
dry weight of shoots and fruits significantly increased as
the burned rice husk was added. Similarly, with the
addition of more than 3% nitrogen source meal from a
total of 7% in biofertilizer A and C gave a good yield of
leaves diameter for plant treated with biofertilizer B.
On the other hand, the plants treated with
biofertilizer C recorded the highest root and stem lengths
(237.6 ± 4.96 cm) in comparison to plants treated with
biofertilizer A and B, which were 185.0 ± 7.00 cm and
217.5 ± 6.93 cm, respectively. Total nitrogen content for
all biofertilizers (> 1%) had no effect on populations of
total bacteria, yeast, as well as ammonia and nitrite
utilizing bacteria. These results are in accordance with
those results obtained by Sarathchandran et al. (2001)
who reported that the nitrogen content in biofertilizer
around 048 - 0.69% did not give any significant difference
to the total count of microbes studied.
CONCLUSION
In conclusion, the microbiological, chemical and physical
properties of biofertilizer A, B, and C were determined.
Based on these properties, we suggest that biofertilizer A
is the best in encouraging flower and fruit growth, while
biofertilizer B is superior in leaf production and biofertilizer
C is good for the strength development of roots and
stems. Furthermore, the formulated biofertilizers in this
study was prepared from an economical and low-cost raw
material with the inoculation of special microorganisms,
which is a feasible and potential market for the
commercialization as well as to promote environmental
friendly technology. This will reduce the country’s
reliability on chemical fertilizers in a way to produce,
increase and sustain food production. Therefore, the
utilization of agricultural waste converted to biofertilizer
can be one of the successful alternative ways of
optimizing the use of resources and to generate income.
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