B I O D I V E R S I T A S ISSN: 1412-033X
Volume 6, Nomor 5 Juli 2005
Halaman: 175-177
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Isolation and Identification of Phosphate Solubilizing and
Nitrogen Fixing Bacteria from Soil in Wamena Biological
Garden, Jayawijaya, Papua
SULIASIH
♥
, SRI WIDAWATI
Microbiology Division, Research Center of Biology, Indonesian Institute of Sciences Bogor 16002.
Received: 28 October 2004. Accepted: 9 March 2005.
ABSTRACT
A study was undertaken to investigate the occurrence of phosphate solubilizing bacteria (PSB) and nitrogen-fixing bacteria (NFB) from soil
samples of Wamena Biological Garden (WbiG). Eleven soil samples were collected randomly to estimate microbial population which used
plate count method. The result showed that the microbial population ranged from 5.0x10
3
-7.5x10
6 cells
of bacteria/gram of soil and 5.0x10
3
-
1.5x10
7
cells of bacteria/gram of soil for PSB and NFB respectively. There were 17 isolates which have been identified till genus and
species. The isolated microorganism were identified as PSB i.e. Bacillus sp., B. pantothenticus, B. megatherium, Flavobacterium sp., F.
breve, Klebsiella sp., K. aerogenes, Chromobacterium lividum, Enterobacter alvei, E. agglomerans, Pseudomonas sp., Proteus sp. and as
NFB i.e. Azotobacter sp., A. chroococcum, A. paspalii, Rhizobium sp., and Azospirillum sp.
© 2005 Jurusan Biologi FMIPA UNS Surakarta
Key words: phosphate- solubilizing bacteria, Nitrogen-fixing bacteria, Wamena Biological Garden.
INTRODUCTION
Wamena Biological Garden (WBiG) is one of mountain
range-biota ex-situ conservation at eastern part of
Indonesia. The ex-situ conservation is the first conservation
built by Biological Research Center, Indonesian Institute of
Sciences. WBiG has unique vegetation and many variations
of soil colors. Soil is a unity of subsistence that includes the
varieties of microbe, because microbes’ community is one
of the important components of soil. Activity and species
composition of microbes are generally influenced by many
factors including physic-chemical properties of the soil,
temperature and vegetation (Jha et al., 1992).
The most important role of soil organism in ecosystem is
decomposing of organic matters, synthesize and release
them into inorganic forms that plant can use (Setiadi, 1989).
Most microbes in terrestrial ecosystem are in soil. Bacteria
are the most dominant group of soil microbes. In the fertile
soil, there are 10
6
-10
8
cells of bacteria/ gram of soil. Some
groups of soil bacteria (nitrogen-fixing bacteria and
phosphate-solubilizing bacteria) are useful as biofertilizer.
Some plants and microbes species have developed
symbiosis or mutually beneficial relationships. Rhizobium is
the root of legumes host nitrogen fixing bacteria which can
invade root and get sugars from the plant. In return, they
convert large amounts of dinitrogen (N
2
) from the
atmosphere into forms that the plants can use (Zahran,
1999). Another nitrogen fixing bacteria living in soil are
Azotobacter sp. and Azosprillum sp. Several species of
Azotobacter and Azosprillum are known to fix nitrogen
under field condition in association with roots of plant.
Nitrogen-fixing Azosprillum strains have been isolated from
tropical and some temperate grass root surface. Some
species of Azotobacter from root associations with seasonal
grasses are specific hosts, e.g. A. paspalii associated with
the root of Paspalum notatum (Berreiner et al., 1976,
Elmerich, 1984, Okon, 1985, Okon and Labandera-
Gonzales, 1994). The other biofertilizer bacteria are genus
of Pseudomonas and Bacillus. Those bacteria are able to
solubilize available forms of Fe, Ca, Mg, Al bound P. The
solubilization effect is generally due to the production of
organic acids (Kucey, 1983).
The present investigation was carried out to study the
occurrence of PSB and NFB from WBiG. The isolated
microbes were identified.
MATERIALS AND METHODS
Soil
Surface (0-15 cm) soil samples were collected randomly
from 11 different sites of Gunung Susu which was one of
WBiG sites. All samples were kept in plastic bags and
transported to the laboratory and stored in 4
o
C prior to be
analyzed. These samples were air-dried and ground to pass
2 mm sieve before the chemical analyses. The samples
were analyzed for pH, soil chemistry and texture, (Table 1
and 2).
Microbial count
Microbial population was estimated by plate count
method (Ravina et al, 1992; Thompson, 1989; Vincent,
1982). Ten grams soil was suspended in 90 mL sterile
distilled water in Erlenmeyer flask and mixed thoroughly for
30 minutes using a mechanical shaker at 110 rpm. Then 1
mL an aliquot transferred with sterile pipettes to 9 mL sterile
'2,ELRGLYG
BIODIVERSITAS Vol. 6, No. 3, Juli 2005, hal. 175-177
176
distilled water in test tube. This suspension was stir for 10
second. A subsequent serial dilution was prepared as
above to 10
7
. From each serial dilution, 0.2 mL of aliquot
was transferred to sterile putridity and over poured and
dispersed swirling with agar media (50
o
C).
PSB was grown on Pikovskaya agar media (Sundara
Rao and Sinha, 1963, Gaur, 1981), containing of 5g
Ca
3
(PO
4
)
2
; ten g glucose; 0.2g NaCl; 2.5mg MgSO
4
.7H
2
O;
2.5mg MnSO
4
.2H
2
O; 2.5mg FeSO
4
.7H
2
O; 5g yeast extract;
20g agar, diluted in 1 l distilled water. The plats were
incubated for 7 days at room temperature. Colonies of PSB
were detected by clear zones of solubilization around them.
For growing Rhizobium was used yeast extract mannitol
agar (YEMA) (Subba Rao,1994), containing 10g mannitol;
0.5g K
2
HPO
4
; 0.1g NaCl; 0.2g MgSO
4
.7H
2
O; 1g yeast
extract; 2.5 mL congo red 1%; 20g agar, diluted in 1 l
distilled water. The plats were incubated for 7 days at room
temperature. Rhizobia colonies have transparent white
color, shining.
Mannitol Ashby agar medium was used for isolating
Azotobacter containing of 20g mannitol; 0.2g K
2
HPO
4
; 0.2g
NaCl; 0.2g MgSO
4
.7H
2
O; 0.1g K
2
SO
4
; 5.0g CaCO
3;
20g
agar, diluted in 1 l distilled water. The plats were incubated
for 7 days at room temperature (Subba Rao, 1994).
The numbers of Azospirillum bacteria counted on a
Okon medium containing of 6.0g K
2
HPO
4
; 4.0g KH
2
PO
4;
0.2g MgSO
4
; 0.1g NaCl; 5g Sucrose; 0.02g CaCl
2;
1.0g NH
4
Cal; 5.0g NaOH; 2.1mg MnSO
4
; 10.0mg FeCl
3
; 2.0mg
Na
2
M
o
O
4
.; 0.1g yeast extract; 2.0 mg H
3
BO
3;
0.04mg
Cu(NO
3
)
2;
0.2 mg ZnSO
4;
2 mL bromthymol blue 0.5%; 20.0
agar, diluted in 1 L distilled water (Okon et al., 1977).
The number of bacterial colony was estimated after 7
days of incubation at room temperature. The isolates were
identified follows Bergey’s manual for bacteriology methods
systematic (Krieg and Hold, 1984).
RESULTS AND DISCUSSION
Table 1 and 2 showed that the result analyses of soil
chemical properties varied depending on the vegetations
and soil types. Soils that were dominated by Imperata
cylindrica indicated to be deficiency of soil nutrient. Setiadi
(1989) proposed that the land in area of Imperata cylindrica
has eroded, because the plants are less effective to avoid
the erosion. In the eroded land, organic matter and nutrient
leaching may generally occur, and results in fewer nutrients
available.
Table 1. Soil physic from 11 sites in Wamena Biological Garden
Soil Texture (%)
Soil
sam-
ples
Soil color Vegetation
Sand Clay Dust
1 Black
Pittosporum ramiflorum
5.79 51.37 42.84
2 Dark brown
Grevillea papuana
7.38 52.08 40.54
3 Brown
Castanopsis
accuminattisima
20.17 18.56 61.25
4 Brown
Vaccinium
varingiaefolium
16.73 28.01 55.26
5 Brown reddish
Imperata cylindrica
16.36 43.45 40.19
6 Red
Imperata cylindrica
11.51 65.75 22.69
7 Yellow
Imperata cylindrica
17.51 41.42 41.06
8 Gray
Imperata cylindrica
10.89 38.82 49.28
9 Black Imperata cylindrica 18.03 38.06 43.01
10 Gray
Imperata cylindrica
10.89 38.82 49.28
11 Brown
Imperata cylindrica
15.65 35.74 48.61
Table 2. Soil chemistry from 11 sites of Wamena Biological Garden.
Soil
sam-
ples
N
(%)
P
(ppm)
K
(me/100g)
C
(%)
C/N
Ca
(Me/100g)
PH
1 0.21
(m)
4.8 0.53
(m)
2.54
(m)
12.10
(m)
26.00
(v.h)
5.30
(acid)
2 0.30
(m)
3.9
(l)
0.36
(l)
3.05
(h)
10.17
(l)
19.23
(h)
4.80
(acid)
3 0.22
(m)
1.6
(v.l)
0.15
(l)
3.12
(h)
14.48
(m)
9.34
(m)
4.35
(acid)
4 0.23
(m)
3.3
(l)
0.26
(l)
3.85
(m)
16.74
(h)
8.82
(m)
4.90
(acid)
5 0.06
(v.l)
0.2
(v.l)
0.07
(v.l)
0.69
(v.l)
11.50
(m)
8.88
(m)
5.00
(acid)
6 0.06
(v.l)
0.3
(v.l)
0.07
(v.l)
0.62
(v.l)
10.33
(l)
9.55
(m)
6.05
(acid)
7 0.05
(v.l)
0.4
(v.l)
0.05
(v.l)
0.51
(l)
10.20
(l)
9.34
(m)
5.25
(acid)
8 0.04
(v.l)
1.6
(v.l)
0.13
(l)
0.36
(v.l)
9.00
(l)
9.73
(m)
4.60
(acid)
9 0.25
(m)
2.1
(l)
0.06
(v.l)
2.76
(m)
11.04
(m)
32.90
(v.h)
5.26
(acid)
10 0.23
(m)
2.7
(l)
0.10
(l)
2.47
(m)
10.74
(l)
19.23
(h)
4.25
(acid)
11 0.03
(v.l)
0.1
(v.l)
0.05
(v.l)
0.36
(v.l)
12.00
(m)
9.03
(m)
4.65
(acid)
Note: v.l = very low; l = low; m = moderate; h = high; v.h = very high
The estimates of total bacteria, PSB and NFB at each
sites ranging from 2.95x10
5
-2.5x10
8
cells of bacteria/ gram
soil. Soil samples number 2, 3, 4, 9, 10 reveal the number
of total bacteria which are higher than samples number 5, 6,
7, 8, 11 (Table 3).
Table 3. The population of microbes isolated from 11 sites of
Wamena Biological Garden.
NFB (cell/g soil)
Soil
sam-
ples
Total
bacteria
(cell/g
soil)
PSB
(cell/g
soil)
Rhizobium
sp.
Azospirillum
sp.
Azotobacter
sp.
1 2.5x10
8
5.5x10
4
2.5x10
6
5.8x10
5
1.5x10
7
2 5.0x10
7
1.5x10
6
1.5x10
6
1.0x10
6
1.5x10
6
3 2.5x10
7
1.1x10
5
2.0x10
6
5.0x10
6
1.5x10
6
4 2.0x10
8
1.5x10
6
2.0x10
6
1.5x10
6
2.5x10
6
5 3.5x10
6
2.6x10
6
4.4x10
5
6.0x10
4
2.0x10
4
6 2.4x10
6
3.5x10
4
1.9x10
5
1.0x10
5
1.0x10
6
7 2.4x10
6
5.0x10
3
2.2x10
5
- 1.5x10
5
8 3.0x10
5
5.5x10
4
2.1x10
5
2.0x10
4
5.0x10
3
9 5.0x10
6
7.5x10
6
1.5x10
6
8.5x10
4
5.5x10
4
10 5.0x10
7
2.5x10
6
5.0x10
6
1.0x10
6
2.5x10
6
11 5.5x10
6
5.4x10
5
2.5x10
6
1.2x10
5
1.5x10
5
Table 4 showed that the most dominant phosphate
solubilizing bacteria found were aerobic spore forming
bacteria. Identification of this group showed that Bacillus sp.
was the most predominant PSB was found in all of soils
tested, followed by B. panthothenticus and B. megatherium
were in soil samples numbers 1, 2, 3, 6 and 9 respectively.
Other PSB involved were Flavobacterium sp., F. breve,
Klebsiella sp., K. aerogenes, Chromobacterium lividum,
Enterobacter alvei, E. agglomerans, Pseudomonas sp. and
Proteus sp. The finding of predominance of spore formers
was in harmony with the work of Taha et al. (1969). He also
observed that sporeformer was well known to resist adverse
conditions such as high temperature and dryness. Thus the
important PSB can overcome such unfavorable conditions.
Swaby and Sperber cit. Taha et al. (1969) found that the
principal genera of PSB were Arthobacter, Pseudomonas,
Xanthomonas, Achromobacter and Flavobacterium. It is well
known that the interplay of so many factors such as physico-
chemical properties of soil, vegetation crop, rotation and envi-
ronmental conditions greatly influence soil microbial flora.
SULIASIH
and WIDAWATI – Phosphate solubilizing and nitrogen fixing bacteria
177
Table 4. Identified isolates from 11 sites of Wamena Biological Garden.
Soil
sam-
ples
Phosphate solubilizing
bacteria
Nitrogen-fixing
bacteria
1 Bacillus sp.
B. panthothenticus,
Enterobacter agglomerans
Rhizobium sp.
Azospirillum sp.
Azotobacter sp.
2 Bacillus sp.
B. panthothenticus,
Chromobacterium lividum,
Flavobacterium sp.
Rhizobium sp.
Azospirillum sp.
Azotobacter sp.
3 Bacillus sp.
B. panthothenticus,
Flavobacterium sp.
Klebsiella sp.
K. aerogenes
Rhizobium sp.
Azospirillum sp.
Azotobacter sp.
A. paspalii
4 Bacillus sp.
Enterobacter agglomerans
Rhizobium sp.
Azospirillum sp.
Azotobacter sp.
5 Bacillus sp.
Flavobacterium breve,
Pseudomonas sp.
Rhizobium sp.
Azospirillum sp.
Azotobacter sp.
A. paspalii
A. chroococcum
6 Bacillus sp.
B. panthothenticus,
Klebsiella aerogenes,
Enterobacter alvei
Rhizobium sp.
Azospirillum sp.
Azotobacter sp.
7 Bacillus sp.
Chromobacterium lividum,
Enterobacter agglomerans
Rhizobium sp.
Azotobacter sp.
8 Bacillus sp.
Flavobacterium sp.
Proteus sp.
Rhizobium sp.
Azospirillum sp.
Azotobacter sp.
A. paspalii,
A. chroococcum
9 Bacillus sp.
B. megatherium,
Enterobacter alvei
Rhizobium sp.
Azospirillum sp.
Azotobacter sp.
A. paspalii
10 Bacillus sp. Rhizobium sp.
Azospirillum sp.
Azotobacter sp.
11 Bacillus sp.
Pseudomonas sp.
Rhizobium sp.
Azospirillum sp.
Azotobacter sp.
A. paspalii
A. chroococcum
Nitrogen-fixing bacteria such as Rhizobium sp. and
Azotobacter sp were found in all of soil tested. Azospirillum
was found in almost soil samples except in soil number 7.
The isolates of Rhizobium and Azospirillum have already
been identified till genus level. Isolates of Azotobacter till
genus and species level. Both of Azotobacter paspali and
A. chroococum were obtained in soil samples numbers 5, 8,
11. Rhizobium, Azotobacter and Azospirillum are
heterotrophic bacteria which depend on outside carbon
sources to fix nitrogen. Soil microbes require energy and
essential nutrient to grow and reproduce, while plants
derive their energy from carbon acquired from the
atmosphere by means of photosynthesis. The carbon in
organic matter decomposing provides soil microbes with
their energy supply (John et al., 2001).
The result showed that different soil nutrient status and
vegetation type in the investigated sites resulted in the
different bacterial population and bacterial type. The
difference was caused by releasing organic and inorganic
root exudates that can be used by surrounding organism.
Jha et al. (1992) and Setiadi (1989) found that biological
activity and composition of soil microbes are generally
affected by many factors including physico-chemical
properties of soil, temperature and vegetation. C availability
in soil may affect the numbers and activities of microbes
directly. Setiadi (1989) reported that the roots of higher
plants might affect significantly the activity and the
development of soil microbes. (Katznelson cit. Mukerji and
Subba-Rao, 1982) reported that the influence of the root on
soil microbes starts immediately after seed germination
which increases as the plant grow and reach maximum
when plans have reaches the peak of the vegetative growth
Stenton cit. Mukerji and Subba-Rao (1982) proposed
that the roots of higher plants provide an ecological niche to
soil microbes within the soil. Mishara (1969) also reported
that different plant species grown in the same type of soil
could harbor different microbes in the rhizosphere.
CONCLUSSION
The microbial population range from 5.0x10
3
-7.5x10
6
cells of bacteria/g of soil and 5.0x10
3
-1.5x10
7
cells of
bacteria/g of soil for phosphate-solubilizing bacteria and
nitrogen-fixing bacteria respectively. There were 17 isolates
which have been identified till genus and species. The
isolated microorganism were identified as PSB i.e. Bacillus
sp., B. pantothenticus, B. megatherium, Flavobacterium sp.,
F. breve, Klebsiella sp., K. aerogenes, Chromobacterium
lividum, Enterobacter alvei, E. agglomerans, Pseudomonas
sp., Proteus sp. and as NFB i.e. Azotobacter sp., A.
chroococum, A. paspali, Rhizobium sp., and Azospirillum sp.
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