J. Trop. Agric. and Fd. Sc. 38(2)(2010): 211– 219
M. Stella and M. Suhaimi
Selection of suitable growth medium for free-living diazotrophs
isolated from compost
(Pemilihan medium pertumbuhan yang sesuai untuk bakteria pengikat nitrogen hidup
bebas yang dipencil daripada kompos)
M. Stella* and M. Suhaimi*
Keywords: growth medium, free-living diazotrophs
Abstract
Four free-living nitrogen fixing bacteria, namely NC 2, NC 4, NC 10 and NC
11 were tested for nitrogenase activity. NC 10 performed the highest nitrogenase
activity (3.01 x 10–3 mol/h/ml), followed by NC 4 (6.84 x 10– 4 mol/h/ml), NC 2
(2.36 x 10–5 mol/h/ml) and NC 11 (7.48 x 10–5 mol/h/ml). The nitrogenase activity
of mix culture was recorded as 7.48 x 10–5 mol/h/ml. Beijerinckia medium was
the most suitable growth medium for all four organisms, followed by Derxia
medium and Ashby’s medium. In these three mediums, high microbial density
was obtained in a short period. The optimal growth was achieved within 48 h.
Beijerinckia medium showed the highest O.D reading, 1.326 A at 24 h. This was
followed by Derxia medium, 1.135 A and Ashby’s medium, 0.811 A.
Introduction
Nitrogen is the most important and limiting
nutrient for plant growth. Biological
Nitrogen Fixation (BNF) is a natural
process whereby the atmospheric nitrogen
is converted to ammonia by a specific
enzyme known as nitrogenase. The process
makes the unavailable form of nitrogen
accessible to plants. The diazotrophs are
the mediator of this process. Diazotrophs
are the prokaryotic organisms, which have
the ability to fix atmospheric nitrogen.
They can be classified as symbiotic and
non-symbiotic or free-living forms (Dalton
1980). At least 90 genera of specialized
microorganisms are known to have the
enzyme nitrogenase and can fix atmospheric
N2 into NH3 (Murray and Jeff 2008).The
most important contribution of BNF comes
from the symbiotic association of certain
microorganisms with the roots of higher
plants. A classic example is the Rhizobium,
which infect the roots of leguminous plants
with a high degree of host specificity. Nonsymbiotic nitrogen fixation is restricted to
certain microorganisms, mostly free-living
bacteria and blue green algae.
BNF appears to be a very important
N source for paddy in the tropics,
where, Azolla-cynobacteria symbiosis is
estimated to fix a large amount of nitrogen.
Approximately 50% of the N requirement
of a flooded rice crop is met from the soil
N pool, which is believed to be maintained
through BNF by associate and free living
microorganisms (Bohlool et al. 1992). On
the other hand, Shuichi (1995) reported
that the nitrogen fixed by free-living
microorganisms is estimated less than 1 kg
N/ha/year.
Ravikumar et al. (2004) found that
nitrogen-fixing Azotobacters are used as
*Strategic Resources Research Centre, MARDI Headquarters, Serdang, P.O. Box 12301, 50774 Kuala Lumpur,
Malaysia
Authors’ full names: Stella Matthews and Suhaimi Masduki
E-mail: [email protected]
©Malaysian Agricultural Research and Development Institute 2010
211
Growth medium for free-living diazotrophs
marine biofertilizers. Three species of
Azotobacter, A. chroococcum, A. virelandii
and A. beijerinckii, exhibited high growth,
nitrogen fixation and in vitro production of
phytohormone. The Azotobacters, which
were inoculated with rhizophora seedlings,
increased significantly the average root
biomass up to 98.2%, the root length by
48.45%, the leaf area by 277.86%, the shoot
biomass by 29.49% as compared to controls
and they increased the levels of total
chlorophylls and carotenoids up to 151.0%
and 158.73% respectively.
However, it has been reported that the
potential of using free-living diazotrophs
as a source of nitrogen nutrition for crops
has not been widely practised because of
the inability of the organisms to multiply
effectively in temperate agricultural soils
(Keeling et al. 1998). There are many
factors affecting the nitrogenase activity of
free-living diazotrophs such as substrate
supply, mineral nutrition, oxygen supply,
pH and the presence of combined nitrogen.
It is assumed that in free-living N2 fixers,
the efficiency value of nitrogen fixation is
found to be 5–20 mg fixed N2 per gramme
of glucose consumed (Mulder 1975).
Molybdenum, iron, potassium, calcium and
magnesium are essential elements for freeliving N2-fixing bacteria.
In aerobic bacteria, addition of
combined nitrogen, particularly NH4+
declines nitrogenase activity (Anne and
William 2002). Oxygen, on the other hand
is required for energy supply and nitrogen
fixation. Most of the free-living N2-fixing
bacteria require a neutral or alkaline
reaction for growth (Mulder 1975). Not
much research has been conducted on the
suitable medium for nitrogen-fixing bacteria.
The common criteria for the selection of
N-fixing bacteria are N-free mediums with
a considerable amount of carbon source.
Aquilanti et al. (2004) described different
strategies to isolate Azotobacter from soil
samples. He concluded that soil pasteplate method combined with isolation of
mannitol agar proved to be the best strategy
212
in terms of reliability and selectivity.
Thorough research need to be done in order
to produce effective inoculant of free-living
diazotrophs. Growth medium is one of the
important criteria that must be looked into
especially in large scale production of the
bacterial inoculants.
In this study, four strains of
microorganisms namely, NC 2, NC 4, NC 10
and NC 11 were isolated from mature
compost. These strains were regarded
as diazotrophs based on their ability to
exhibit nitrogenase activity in an acetylene
reduction assay. Some published growth
mediums were compared to select the most
suitable N-free medium for the production
of free-living N2-fixing bacteria.
Materials and methods
Isolation of nitrogen-fixing bacteria
Nitrogen-fixing bacteria were isolated
from matured and stable compost made
of empty fruit bunches and chicken dung
mixture through an enrichment process.
After the completion of the composting
process, starch was mixed with the compost
to enhance the biological nitrogen fixation.
Ten grammes of the compost was collected
and transferred into a 250 ml Erlenmeyer
flask containing 90 ml sterile distilled
water, shaken for 20 min (120 rpm). Serial
dilutions were made and 0.1 ml of aliquots
from dilution 10–4 until 10 – 6were inoculated
on Burk’s N-free medium. The plates were
incubated for 7 days at 30 ºC. Pure colonies
were obtained by repeated streaking on
Burk’s medium. Morphologically different
colonies were isolated and subcultured for
further analysis.
Bacterial storage medium
The nitrogen-fixing bacteria were stored
in Burk’s N-free medium which contained
the following ingredients l–1: sucrose,
20.0 g; K2HPO4, 0.64 g; KH2PO4, 0.16 g;
MgSO4.7H2O, 0.20 g; NaCl, 0.20 g;
CaSO4.2H2O, 0.05 g; Na2MoO4.2H2O,
(0.05%) 5.0 ml; FeSO4.7H2O, (0.3%) 5.0 ml
and agar, 15 g. The pH was adjusted to
M. Stella and M. Suhaimi
7.3 and autoclaved at 121 ºC for 15 min.
Na2MoO4.2H2O and FeSO4.7H2O were
filtered and sterilized prior to adding into the
autoclaved medium.
Acetylene reduction assay (ARA) for
pure cultures
The acetylene reduction assay method
was adapted from the methods described
by Turner and Gibson (1980),Wright and
Weafer (1981) and Myoungsu et al. (2005).
Nitrogen-fixing bacteria were cultured
individually and as a mixed culture in 100
ml of Burk’s medium each (broth). All were
incubated at 30 ± 2 ºC in shaking incubator
for 3 days. After 3 days of incubation, 10 ml
of aliquots were transferred to 30 ml tubes
sealed with rubber stoppers. After 10% of
the atmosphere had been replaced by C2H2,
the tubes were held at 35 ± 2 ºC and 1.0 ml
gas samples were assayed for C2H4 after 2,
5, 24 and 48 h by injection into a PerkinElmer auto-system gas chromatography
fitted with a Propak T column and H-flame
ionization detector.
Inoculum preparation
Pure colony of each strain was used to
prepare 10% inoculum. Nutrient broth was
inoculated with a single, pure colony and
incubated at 35 ºC for 24 h in a rotary
shaker at 180 rpm. The optical density (O.D)
of the inoculum was adjusted to 0.1 A.
Growth medium
Mix cultures of NC 2, NC 4, NC 10 and
NC 11 were grown in nitrogen-free medium
namely, Diazotrophic medium (RBA)
(Peterson 1993), F2 medium (Dalton 1980),
Burk’s medium (BM) (Jack et al. 1953),
Burke’s modified nitrogen-free medium
(BMM) (Atlas 2004), Nitrogen-fixing
Hydrocarbon Oxidizer’s medium (HM)
(Atlas 2004), Norris medium (NM) (Atlas
2004), Winogradsky’s N-free medium (WM)
(Atlas 2004), Ashby’s medium (ASH)
(Subba Rao 1984), Jensen’s medium (JM)
(Subba Rao 1984), Beijerinckia medium
(BEJ) (Subba Rao 1984) and Derxia
medium (DM) (Subba Rao 1984) to
determine the best growth medium.
RBA medium contained l–1: mannitol,
2 g; glucose, 2 g; yeast extract, 0.05 g;
di-sodium succinate, 1 g; KH2PO4, 0.1 g;
K2HPO4, 0.9 g; NaCl, 0.1 g; CaCl2.2H2O,
0.1 g; Mg2SO4.7H2O, 0.1 g; Na2MoO4.
2H2O, 0.005 g; MnSO4.H2O, 0.005 g;
FeSO4.7H2O, 0.01 g and trace element
solution SL-6, 3 ml. F2 medium contained
l–1: Mg2SO4.7H2O, 2 mg; KH2PO4, 0.3 mg;
KCl, 0.7 mg; CaCl, 0.06 mg; Na2MoO4.
2H2O, 0.04 mg and FeSO4.7H2O, 180 mg.
Burk’s medium contained ingredients as
stated above (bacterial storage medium).
BMM contained l–1: Mg2SO4.7H2O,
2 g; Na2HPO4, 0.19 g; NaHCO3, 0.05
g; CaSO4.2H2O, 0.02 g; KH2PO4,0.011
g; NaCl, 0.1 g; FeSO4.7H2O, 6 mg
and Na2MoO3, 6 mg. HM contained
l–1: Na2HPO4, 0.3 g; KH2PO4, 0.2 g;
Mg2SO4.7H2O, 0.1 g; FeSO4.7H2O, 5
mg and Na2MoO4.2H2O, 2 mg. NM
contained l–1: glucose, 10 g; K2HPO4, 1 g;
Mg2SO4.7H2O, 0.2 g; CaCO3, 1 g; NaCl, 0.2
g; FeSO4.7H2O, 0.1 g and Na2MoO4.2H2O,
5 mg. WM contained l–1: glucose, 10 ml (10
g/100 ml); CaCO3, 5 mg; concentrated salt
solution, 5 ml which comprised of KH2PO4,
50 g/l; Mg2SO4.7H2O, 25 g/l; NaCl, 25 g/l;
FeSO4.7H2O, 1 g/l; MnSO4.4H2O, 1 g/l and
Na2MoO4.2H2O, 1 g/l.
ASH contained l-1: mannitol, 20 g;
K2HPO4, 0.2 g; Mg2SO4.7H2O, 0.2 g; NaCl,
0.2 g; K2SO4, 0.1 g and CaCO3, 0.5 g.
JM contained l-1: sucrose, 20 g; K2HPO4,
1 g; Mg2SO4.7H2O, 0.5 g; NaCl, 0.5 g;
FeSO4.7H2O, 0.1 g and CaCO3, 2 g. BEJ
contained l–1: sucrose, 20 g; K2HPO4, 0.2
g; KH2PO4, 0.8 g; Mg2SO4.7H2O, 0.5 g;
FeCl3, 0.1 g and Na2MoO3, 0.005 g. DM
contained l–1: starch, 20 g; K2HPO4, 0.05
g; KH2PO4, 0.15 g; Mg2SO4.7H2O, 0.2 g;
CaCl2, 0.02 g; FeCl3 (10% solution), 0.1 ml;
Na2MoO4.2H2O, 0.002 g; bromothymol blue
(5%), 5 ml and NaHCO3, 0.1 g.
213
Growth medium for free-living diazotrophs
Optical density determination
The density of inoculum was measured at
600 nm using biophotometer for 24, 48, 96,
120 and 144 h. About 1.5 ml of culture was
transferred into eppendorf tube. Then, it was
centrifuged at 10,000 x g rpm for 5 min.
The supernatant was discarded and replaced
by distilled water of the same amount. The
cells were washed at least twice. Prior to the
O.D measurement, the cultures in eppendorf
tubes were homogenized using vortex. The
growth profile of each bacterium in different
medium was plotted in a graph.
Biochemical characterization of bacterial
isolates
Physiological and biochemical characters
of the bacterial isolates were examined
according to methods described by John et
al. (1994). The isolates were characterized
for the following traits: colour pigment,
form, elevation, margin, diameter, surface,
opacity and texture.
The Gram reaction was performed as
per standard procedure. Motility, oxidase
reaction, catalase test, oxygen requirement,
carbohydrate and nitrogen source utilization,
oxidation and fermentation of glucose,
urease test, DNAse test, gelatinase test,
reduction of nitrate, were performed
according to standard methods (Sirockin and
Cullimore 1969).
BIOLOG assay
Metabolic fingerprint of the isolated
bacteria was performed using BIOLOG
Microplate. The carbon source utilization
was determined through this test. The
microplates performed 95 discrete tests
simultaneously and gave characteristic
reaction pattern called ‘metabolic
fingerprint’. All the metabolic fingerprints
were compared and identified using the
MicroLogTM database software. A pure
culture of a bacterium was grown on a
Biolog Universal Growth agar plate. A
homogenous suspension of inoculm was
made in GN/GP Inoculating Fluid and
diluted to an appropriate transmittance. The
214
inoculum turbidity is 61% and 20% for
Gram negative and Gram positive bacteria
respectively. The suspension was dispensed
into each well of the microplate, which
was incubated for 24 h at 35–37 ºC. The
Gram negative and Gram positive bacteria
were incubated in GN and GP Microplate
respectively. Microplates were read at
590 nm at 4 h and 24 h with a computercontrolled microplate reader. Each metabolic
profile was compared automatically with the
Microlog database.
Results and discussion
Four strains of microorganisms were isolated
from compost made of empty fruit bunches
and chicken dung and consequently labelled
as NC 2, NC 4, NC 10 and NC 11. The
four isolates were assumed to fix nitrogen
as they were isolated through an enrichment
process to enhance biological nitrogen
fixation. It is proven that when an energy
source such as starch was added to stable
compost, the expression to fix nitrogen is
enhanced (Mulder 1975). Keeling et al.
(1998) also stated that significant N-fixation
was stimulated by the glucose treatment
of compost. Based on these findings,
four bacterial isolates which survived
successfully in compost environment
after the enrichment process were grown
on N-free media. The four isolates were
regarded as free-living nitrogen-fixing
bacteria and were subjected to acetylene
reduction assay (ARA) to ensure their ability
to exhibit nitrogenase activity. In ARA, NC
10 exhibited the highest nitrogenase activity
(3.01 x 10–3 mol/h/ml) and followed by
NC 4 (6.84 x 10–4 mol/h/ml). NC 2 and
NC 11showed low nitrogenase activity.
The nitrogenase activity of NC 2 was
2.36 x 10–5 mol/h/ml when analysed after
2–5 h of incubation time. NC 11 showed
nitrogenase activity of 6.15 x 10–6 mol/h/ml
only after a long incubation time for 24 h.
The nitrogenase activity of mix culture was
better than NC 2 and NC 11, which was
recorded as 7.48 x 10–5 mol/h/ml.
M. Stella and M. Suhaimi
The nitrogenase activity of NC bacteria
was obviously higher than the result
reported by Myongsu et al. (2004), where
the highest nitrogenase activity was recorded
as 3677.81 nmol/h/mg protein or equivalent
to 3.67781 x 10–6 mol/h/mg protein. The
results obtained in this experiment were also
higher compared with the study done by
Rozycki et al. (1999). He reported that the
majority of the genera Pseudomonas and
Bacillus had nitrogenase activity within the
range from 4 to 20 nmoles C2H4 per culture
per hour.
Beijerinckia medium showed the
highest O.D reading, 1.326 A at 24 h
(Figure 1). This is followed by Derxia
medium, 1.135 A and Ashby’s medium,
0.811 A. These values were observed
to increase after 24 h and reached the
optimum growth at 48 h, where the O.D
of Beijerinckia medium, Derxia medium
and Ashby’s medium were 1.47 A, 1.066 A
and 0.836 A respectively. The mix culture
in Norris medium (NM) and Diazotrophic
medium (RBA) tend to multiply vigorously
after 48 h but the optical density were still
lower than Beijerinckia and Derxia medium.
Same kind of scenario was observed in F2
and Jensen’s medium, where the optical
density started to shoot up only after
96 h. The lowest bacterial population was
observed in BMM medium. The microbial
density decreased after 96 h in most of the
medium.
Based on the result obtained, it is
confirmed that Beijerinckia medium was
the most suitable growth medium for all the
four organisms, followed by Derxia medium
and Ashby’s medium. It was observed that
in these three mediums, high microbial
density was produced in a short period. The
optimal growth was achieved within 48 h.
The main difference of these mediums with
BMM, which has lowest optical density, is
the carbon source. BMM does not contain
any carbon source whereas BEIJ contained
sucrose as the main carbon source, while
DEX and ASH medium included starch
and mannitol, respectively. It is proven that
the development of free-living, nitrogenfixing bacteria is favoured by the presence
of considerable amount of available carbon
compounds.
This is supported by the finding of
Billings et al. (2003), which described
strong nitrogenase activity of free-living
heterotrophs as an effect of adding
dextrose-C in soil. Anne and William (2002)
also reported that glucose could enhance
nitrogenase activity. It addition to that, it is
1.8
1.6
Optical density (A)
1.4
◆
◆
◆
●
■
1.2
1
0.8
◆
■
■
■
◆
■
▼
▼
▼
0.2
0
▲
❋
▼
0.6
0.4
●
▼
●
▲
●
●
❖
▲
❖
▲
❋
❋
24
Time of incubation (h)
●
48
❖
▼
❖
❋
▲
❖
❋
◆
❖
▲
❋
96
120
■
BM
RBA
F2
BMM
HM
NM
WM
ASH
JEN
BEIJ
DER
144
Figure 1. Optical density of mix cultures in different growth medium
215
Growth medium for free-living diazotrophs
found that even in Anabaena sp. nitrogenase
activity was enhanced by the addition of
sucrose (Tonina and Shree Kumar 2000).
To differentiate isolates between Gram
positive and Gram negative, the KOH test
was used. NC 2 and NC 10 were identified
as Gram positive bacteria whereas NC 11
was identified as Gram negative. Cultures
were also subjected to Gram staining to
observe the cell morphology. NC 2 and NC
10 were rod shaped as shown in Plate 1
whereas NC 11 was observed in coccus
shape under microscope at a magnification
of x100. NC 4 was similar to yeast cells.
The cells were large and budding cells
were observed under the microscope. The
microscopic pictures at a magnification of
x100 are shown in Plate 2.
All four isolates were catalase positive
and oxydase negative. The biochemical
traits of the nitrogen-fixers are described in
Table 1.
Biolog’s technology uses each
microbe’s ability to use particular carbon
sources to produce a unique pattern
or ‘fingerprint’ for that microbe. As a
microorganism begins to use the carbon
sources in certain wells of the microplate,
it respires. For bacteria, this respiration
process reduces a tetrazolium redox dye and
those wells change colour to purple.
According to BIOLOG’s metabolics
assay results, NC 2 used up 71 out of 95
carbon sources tested. NC 10 utilized 79
carbon sources. The metabolic fingerprints
also clearly showed that NC 2 and NC 10
could have been originated from the same
genera as there was not much difference in
their morphology, biochemical attributes
and they shared most of the common carbon
sources.
NC 4 and NC 11 were definitely
different types of isolates although they have
shown similar biochemical characteristics
in common. The vast divergence in carbon
utilization was the main indication of their
differences. NC 4 was observed to use the
least number of carbon sources namely,
L-Aspartic acid and L-Glutamic acid. On the
contrary, 50 carbon sources were favoured
by NC 11 (Table 2). The microscopic
analysis also showed that NC 4 cells were
very much different from NC 11 cells. The
cells were big, ovoid in shape and showed
some budding cells. These characteristics
were very similar to yeast. Only yeast cell
reproduces by budding (Plate 2). Besides
that, yeast cells have very simple nutritional
Plate 1. Non-sporing Gram
positive rods of NC 2 (left) and
NC 10 (right)
Plate 2. Budding cells of NC
4(left) and ovoid shape cells of
NC 11(right)
216
M. Stella and M. Suhaimi
Table 1. Biochemical reaction result of the nitrogen-fixers
Gram stain
Oxidase test
Catalase test
Glucose fermentation
(TSI)
Urease
Gelatinase
Motility
DNAse
Nitrate test
D-Glucose acid
production
D-Glucose gas
production
Methyl red
Voges-Proskauer
Nutrient agar
NC 2
Positive
Negative
Positive
Acid/Acid
NC 4
Negative
Negative
Positive
Varies
NC 10
Positive
Negative
Positive
Alkaline/Acid
NC 11
Negative
Negative
Positive
Acid /Acid
Negative
Negative
Motile
Negative
Negative
Positive
Positive
Negative
Non-motile
Negative
Positive
Positive at 48 h but
changed to colorless
after 4 days
Small amount of gas
produced
Negative
Negative
Non-pigmented, smooth,
circular, very tiny,
opaque, entire (margin).
Translucent, 0.1 mm
Negative
Negative
Motile
Negative
Positive
Positive
Positive
Negative
Non-motile
Negative
Positive
Positive
Negative
Negative
Negative
Negative
Non-pigmented,
opaque, smooth,
undulate edges
(margin), round,
0.1– 0.2 mm
Negative
Negative
Non-pigmented,
translucent,
glistening, circular,
smooth, entire
(margin), 0.1 mm
Negative
Negative
Negative
Non-pigmented,
opaque, smooth,
undulate lobate
(margin), irregular,
0.1–0.2 mm
Table 2. Consumption of carbon sources by microorganisms
Carbon sources
Dextrin
Tween 40
Tween 80
Amygdalin
L-Arabinose
Arbutin
D-Cellobiose
D-fructose
Gentiobiose
α-D-Glucose
Maltotriose
D-Mannitol
D-Mannose
3-Methyl Glucose
β-Methyl D-Glucose
Palatinose
D-Psicose
D-Ribose
Salicin
Sucrose
D-Trehalose
Turanose
D-Xylose
L-Malic Acid
Pyruvic Acid
NC 2 NC 4 NC 10
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
NC 11
X
X
X
X
X
X
X
X
X
X
X
X
X
Carbon sources
L-Asparagine
Adenosine
2’-Deoxy Adenosine
Inosine
Thymidine
Uridine
Fructose-6- Phosphate
Glucose-6-Phosphate
N-Acetyl-D-glucosamine
D-Arabitol
L-Fucose
D-Galactose
m-Inositol
α-D-Lactose
Lactulose
Maltose
D-Melibiose
β-Methyl-D-Glucosidase
D-Raffinose
L-Rhamnose
D-Sorbitol
Methyl Pyruvate
Mono-Methyl-Succinate
Cis-Aconitic Acid
Citric Acid
NC 2 NC 4 NC 10
X
X
X
X
X
X
X
X
X
X
X
X
NC 11
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
(cont.)
217
Growth medium for free-living diazotrophs
Table 2. (cont.)
Carbon sources
D-Galacturonic Acid
D-Gluconic Acid
Quinic Acid
Bromo Succinic Acid
Glucuronamide
D-Alanine
L-Alanine
L-Alanyl-glycine
L-Histidine
NC 2 NC 4 NC 10
NC 11
X
X
X
X
X
X
X
X
X
needs and they require reduced carbon
source. This is also very prominent in
carbon utilization of NC 4 where they use
very few reduced carbon sources.
Conclusion
Suitable growth medium for NC 2, NC 4,
NC 10 and NC 11 are Beijerinckia medium
followed by Derxia medium and Ashby’s
medium. The carbon sources found in this
medium (sucrose, starch, mannitol) could
have promoted the growth of free-living
nitrogen-fixing bacteria. The metabolic
fingerprint of these four isolates also
revealed that sucrose, D-Mannose and
L-Arabinose were the most commonly
utilized carbon sources by the free-living
nitrogen-fixing bacteria. It can be concluded
that sucrose containg nitrogen-free medium
would be the most ideal medium for the
isolation, growth and the nitrogenase activity
of free-living nitrogen-fixing bacteria.
References
Anne, E.A. and William, H.S. (2002). Potential
environmental controls on nitrogen activity in
biological crusts of the northern Chihuahuan
Desert. Journal of Arid Environments 52:
293–304
Atlas, R.M. (2004). Handbook of microbiological
media, 3rd Edition, 1483 p. Washington DC:
CRC press
Aquilanti, L., Favilli, F. and Clementi, F. (2004).
Comparison of different strategies for
isolation and preliminary identification of
Azotobacter from soil samples. Soil Biology
& Biochemistry 36: 1475–1483
218
Carbon sources
NC 2 NC 4 NC 10
Hydroxy-L-Proline
D-Serine
L-Serine
Glycerol
D,L-α-Glycerol Phosphate
Glucose-1-Phosphate
L-Aspartic Acid
X
L-Glutamic Acid
X
NC 11
X
X
X
X
X
X
Billings, S.A., Schaeffer, S.M. and Evans, R.D.
(2003). Nitrogen fixation by biological soil
crusts and heterotrophic bacteria in an intact
Majove Desert ecosystem with elevated
CO2 and added soil carbon. Soil Biology &
Biochemistry 35: 643–649
Bohlool, B.B., Ladha, J.K., Garrity, D.P. and
George, T. (1992). Biological nitrogen
fixation for sustainable agriculture: A
perspective. Plant and Soil 141: 1–11
Dalton, H. (1980). The cultivation of diazotrophic
microorganisms. In: Methods for evaluating
biological nitrogen fixation, (Bergersen, F.J.,
ed.), p. 13–64. Chichester: John Wiley &
Sons
Jack, W.N., Wilson, P.W. and Burris, R.H.
(1953). Direct demonstration of ammonia
as an intermediate in nitrogen fixation by
Azotobacter. J. Biol. Chem. 204: 445–451
John, G.H., Noel, R.K., Peter, H.A.S., James,
T.S. and Stanley, T.W. (1994). Aerobic/
Microaerophilic,motile,helical/vibroid Gram
negative bacteria. In: Bergey’s Manual of
determinative bacteriology. p. 39– 45. USA:
Lippincott Williams & Wilkins
Keeling, A.A., Cook, J.A. and Wilcox, A.
(1998). Effects of carbohydrate application
on diazotroph populations and nitrogen
availability in grass swards established
in garden waste compost. Bioresource
Technology 66(2): 88–97
Mulder, E.G. (1975). Physiology and ecology
of free-living, nitrogen-fixing bacteria.
In: Nitrogen fixation by free-living microorganisms (Steward, W.D.P, ed.), p. 1–27.
London: Cambridge University Press
Murray, U. and Jeff, B. (2008). Measurement
of asymbiotic N2 fixation in Australian
agriculture. Soil Biology & Biochemistry
40(12): 2915–2921
M. Stella and M. Suhaimi
Myongsu, P., Chungwoo, K., Jinchul, Y.,
Hyoungseok, L., Wansik, S., Seunghwan,
K. and Tongmin, S. (2005). Isolation and
Characterization of diazotrophic growth
promoting bacteria from rhizosphere of
agricultural crops of Korea. Microbiological
Research 160: 127–133
Peterson, E. (1993). Catalogue of Strains 1993:
German collection of microorganisms and
cell cultures. Germany
Ravikumar, S., Kathiresan, K., Ignatiammal, S.T.M.,
Selvam, M.B. and Shanty, S. (2004). Journal
of Experimental Marine Biology and Ecology
312: 5–17
Rozycki, H., Dahm, H., Strzelczyk, E. And Li, C.Y.
(1999). Diazotrophic bacteria in root-free soil
and in the root zone of pine (Pinus sylvestris
L.) and oak (Quercus robur L.). Applied Soil
Ecology 12(3): 239–250
Shuichi, A. (1995). Biological nitrogen fixation.
Paper presented at the RDA-FFTC
International Training Course on Microbial
Fertilizers and Composting, 23–30 May
1995, Korea. Organiser: Rural Development
Administration and Food and Fertilizer
Technology Center for the Asian Pacific
Region
Sirockin, G. and Cullimore, S. (1969). Generalized
tests for factors influencing microbial growth.
In: Practical microbiology, p. 52-67. London:
McGraw-Hill
Subba Rao, N.S. (1984). Biofertilizers in
agriculture. New Delhi: Oxford & IBH
Publishing Co.
Tonina, A.F. and Shree Kumar, A. (2000).
Differential regulation of nitrogenase activity
by ionic and osmotic stresses and permeable
sugars in the Cyanabacterium Anabaena sp.
Strain L-31. Plant Science 150: 181–189
Turner, G.L. and Gibson, A.H. (1980).
Measurement of nitrogen fixation by indirect
means. In: Methods for evaluating biological
nitrogen fixation, (Bergersen, F.J. ed.), p.
111–138. Chichester: John Wiley & Sons
Wright, S.F. and Weafer, R.W. (1981). Enumeration
and identification of nitrogen fixing bacteria
from forage grass roots. Applied and
Environmental Microbiology 42(1): 97–101
Abstrak
Empat bakteria pengikat nitrogen yang hidup secara bebas, NC 2, NC 4, NC 10
dan NC 11 telah diuji untuk aktiviti nitrogenase. NC 10 menunjukkan aktiviti
nitrogenase yang paling tinggi (3.01 x 10–3 mol/jam/ml), diikuti oleh NC 4
(6.84 x 10–4 mol/jam/ml), NC 2 (2.36 x 10-5 mol/jam/ml) dan NC 11 (7.48 x
10–5 mol/jam/ml). Aktiviti nitrogenase kultur campuran ialah 7.48 x 10–5 mol/
jam/ml. Beijerinckia ialah medium yang paling sesuai untuk keempat-empat
bakteria tersebut, diikuti oleh medium Derxia dan medium Ashby. Ketigatiga medium ini menghasilkan populasi bakteria yang tinggi dalam masa yang
singkat. Pertumbuhan optimum dicapai dalam masa 48 jam. Medium Beijerinckia
menunjukkan bacaan O.D yang paling tinggi, iaitu 1.326 A dalam masa 24 jam.
Ini diikuti oleh medium Derxia, 1.135 A dan medium Ashby, 0.811 A.
Accepted for publication on 19 January 2010
219