12
Azotobacter sp
Azotobacter is a genus of usually motile, oval or spherical bacteria that form thick-walled cysts, and may produce large quantities of capsular slime,
elongated 1.4-2.0 µm diameter and rod-shaped cells. These bacteria being single and also couple, irregular colony, and sometimes a long chain with a variable.
Azotobacter does not produce endospora, but form cyst. This chemoorganotrophy bacteria, Gram negative, motility using flagella, or are not motil, aerobic, but can
also grow under low oxygen pressure. Azotobacter can be fixed N non symbiotic at least 10 mg N
2
per gram of carbohydrate usually in the form of glucose is consumed. In certain species, these bacteria use nitrate, ammonium salts and
certain amino acids as nitrogen sources, and able to grow in the pH range 4.8-8.5. While the pH optimum for nitrogen fixation and growth is 7.0-7.5. In the soil and
water, this species may be associated with the root of plant Holt et al. 1994.
Technology of Biofertilizer and Storage
a. Use Media Carrier Biofertilizer
In general there are 2 types of media that can be used in formulating bio- fertilizers, the liquid media and solid carriers. There are several alternative types
of solid media that can be used as carrier medium biofertilizers, including: rice flour, corn flour, seaweed and peat soil. Peat is more effective as a binding media
solid formulation of biofertilizers that contain bacteria Azotobcter sp., Azospirillum sp., Bacillus sp., and Pseudomonas sp. This media able to maintain
microbe’s viability until 6 months Hamim et al. 2007.
b. Preservation of Microorganisms by Drying
Preservation of micro-organisms by drying has been the preferred method for long term storage of microorganism. The method of freeze drying is mentioned in
relation to the preservation of microorganisms, it is nearly always with regards to long term storage of cell suspensions that contain greater than 10
8
cells ml
–1
.The reason for preserving high cell concentrations is on the premise that the majority
of cells die during long term storage, but a sufficient number survive to enable the continuation of the strain. Survival of 0.1 of the original cell population is a
“sufficient number” of survivors of freeze drying to allow continuation
Morgan
2006.
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13
Palmfeldt et al. 2003 sutdy the survival of pseudomonas chlororaphis
after preservation by frreez dryer . They reported, the highest freeze-drying survival values, 15–25, were obtained for initial cell concentrations between
1 × 10
9
and 1 × 10
10
CFUml. Moreover, Chotiah 2006 was studied the effect of freeze-drying process and preserving in a vacuum at room temperature against
viability and pathogenicity of veterinary microbe germ plasma of Pasteuerella multocida . He found that were viability decreases after exposed to freezdryer and
period of storage , one and two month.
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14
MATERIALS AND METHODS
Time and Place for Research
The experiment was conducted, in Laboratory of Plant Physiology and Laboratory of Microbiology Department of Biology Bogor Agricultural
University and in the Research Institute for Vegetables Balitsa in Lembang, West Java, from April- December 2009
Material and Equipment
The materials that were used in this experiment was tomato seed var.
Martha provided by Ballista and potato tuber variety Granola obtained from professional seed grower in Lembang, compost, anorganic fertilizers NPK and
biofertilizer containing isolate bacteria of Azotobacter 13, Azospirillium IDM3, Pseudomonas PD13 and Bacillus TG1. The equipments including freezdryer,
oven and centifuge were used to produce biofertilizer.
Experimental Design
Potato and tomato were grown in field conditions for two experiment. The first experiment was prepared using randomized block design with one factor
biofertilizer application including i.e B0 without biofertlizercontrol, B1 liquid biofertilizer ,B2 freezedried biofertilizer without storage, B3 freezedried
biofertilizer with 3 months of storage, B4 centrifuged biofertilizer without storage and B5 centrifuged biofertilizer with 3 months of storage. In this experiment 3
replications blockswere applied with total of 18 combination blocks. The second experiment was also prepared using randomized block design with two factors. The
first factor was anorganic NPK contained 2 dosage A1 50 NPK and A2 100NPK. The second factor was biofertilizer consist of 3 treatments i.e B0
without biofertlizer control, B1 liquid biofertilizer, B2 freezdryried biofertilizer without storage, B3 freezedried biofertilizer with 3 months storage for tomat,
whereas potato, B2 freezedried biofertilizer, B3 centrifuged biofertlizer. Besides that the plant were fertilized with manuare by about 20 tonHa. In this experiment 3
replications blocks were applied with total of 24 combination blocks. In every block for both experiments 10 samples were taken for measurement and analysis.
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15 N
W E S
a
B4 B3
B1 B5
B4 B3
B2 B5
B4 B0
B2 B5
B2 B1
B0 B0
B3 B1
I II III
b
B4 B3
B1 B5
B4 B3
B2 B5
B4 B0
B2 B5
B2 B1
B0 B0
B3 B1
I II III
Figure 1 expermental design 1 of plant a tomato b potato. Description: I, II, III = blockreplication.
a
A1B0 A1B1
A1B0 A1B1
A1B1 A2B1
A1B3 A2B0
A2B1 A1B2
A2B2 A1B3
A2B2 A2B3
A2B2 A1B3
A1B0 A2B3
A1B2 A2B1
A2B0 A2B3
A1B2 A2B0
I II III b
A1B0 A1B1
A1B0 A1B1
A1B1 A2B1
A1B4 A2B0
A2B1 A1B2
A2B2 A1B4
A2B2 A2B4
A2B2 A1B4
A1B0 A2B4
A1B2 A2B1
A2B0 A2B4
A1B2 A2B0
I II III
Figure 2 expermental design 2 of plant a tomato b potato.
Description
: I, II, III = blockreplication.
Preparation of Biofertilizer Rejuvenation of Bacteria
Biofertilizer were obtained from the existing isolate samples in the Laboratory of Microbiology, Bogor Agriculture University. Rejuvenation was
carried out before propagation by isolating Azotobacter sp in LGI solid medium
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16
and incubation for 48 hours, Azospirillium sp. in NFA solid medium and was incubated for 24 hours, Pseudomonas sp. in TSA medium and incubated for 24
hours, and Bacillus sp. in NA medium and was incubated for 18-24 hours. Before mix liqiuid media with peat to produce biofertilizer for each isolate, firstily
stander curve has been prepared to count number the cell population as in Figure 3. Secondly, prepared 1 lliter media from each bacteria, after that taken 1ml to
measurement by spectrophotometer. Finaly, counting the number of cell by accordance to stander curve until 10
8
ml. a. Bacillus Standard curve b. Azotobacter standard curve
c. Azospirilum standard curve d. Pseudomonas standard curve
Figure 3 Standard curves that used to count the number of cell bacteria.
On the other side, peat was sterilized in the oven at 100C for 24 hours, cooled
and stored in a suitable place. Cultur bacteria that have population 10
8
ml was mixed with peat using alternatively two methods: 1 with freeze dryer and 2
with concentrfugation mechanism. Before freeze dried, 1L of bacteria culture was mixed with 1kg of peat and were dried using freeze drier until moisture content
y = 0,378x - 2,347 R² = 0,912
-0,1 0,1
0,2 0,3
0,4 0,5
0,6
6 6,5
7 7,5
ab so
r b
an c
e
log cell
y = 0,020x - 0,137 R² = 0,890
-0,01 0,01
0,02 0,03
6,5 7
7,5 8
ab so
r b
an c
e
log cell
y = 0,047x - 0,300 R² = 0,925
-0,02 0,02
0,04 0,06
0,08
6 6,5
7 7,5
8
ab so
r b
an c
e
log cell
y = 0,792x - 5,613 R² = 0,980
0,2 0,4
0,6 0,8
1 1,2
7 8
9
ab so
r b
an c
e
log cell
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17
was about 10 to produce biofertilizer. In the second methods, 1liter of bacteria was centrifuged at 10000 rpm for 5 minutes and the supernatant was thrown. After
that the pellet were mixed with 1kg of peat to produce centrifuged biofertilizer. The products from both methods was packed into plastic bottles and stored in
suitable conditions at room temperature.
Observation of Viability Bacteria During Storage
Observation of bacterium viability consisting biofertilizer were observed periodical during storage 0, 1, 2 and 3 month. Observation of viability was
carried out by dissolved biofertilizer with 9 ml NACL 0.85 at various level starts from10
-3
-10
-6
, and observed at petri dishes which were given each bacterium medium and incubated for calculation colonies. Acountining of cell
population carried out according to methods petrii dish account Hadioetomo 1993.
Preparations of Soil and Planting The Tomato Seed and Potato Tuber
The land was prepared and blocks were made with the size 3 x 3 m. Before planting the soil and compost were analyzed to understand the nutrient and acidity
status. Seeds of tomato were germinated in the seed bed for 3 weeks. Tubers potato were planted directly to the plot with the distance of 50x30 cm
2
, while after 3 weeks, tomato seedling then were grown with the distance of 70x50 cm
2
.
Application of Compost NPK and Biofertilizer
Application of compost were made 1 week before planting. While, application NPK fertilizer was made during planting. Biofertilizer was applied
two times, in the early of planting and two weeks after planting. Biofertilizer was applied in the form of solid as well as form liquid. Application of solid
biofertilizer that contain fourth isolate was mixed until became one and application with dosage 40 grams per plant at the age of 1 week and 60 grams per
plant after two week from the first aplication. On the other hand, liquid biofertilizer has been applied by mixing of one litermedia of bacterium that
measured with concentration of 10
8
cellml and diluted 4 times by added distill water in order to easily apply to plant. After that the diluted medium was applied
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18
to each plant with the dosage of 40 ml per plant after two week of planting and with dosage 60 ml per plant after two week from the first application.
Parameters Observed
The following measurements were recorded:
a. Analysis of nutrient content of soil and compost samples.
