Post on 06-Feb-2023
Nitrogen Fixation, Cytokinin and Exopolysachharide Production
by indigenous Azotobacter spp. from East Nusa Tenggara
Reginawanti Hindersah, Widiya Septiani Perdanawati, Dewi
Azizah Sulaksana, Hidiyah Ayu Ma’rufah
Fakultas Pertanian Universitas Padjadjaran
Jalan Raya Bandung-Sumedang Km. 21 Jatinangor 43565
Correspondence reginawanti@unpad.ac.id
ABSTRACT
Maize in some region in East Nusa Tenggara Indonesiabordering Republic Democratic of Timor Leste is importantlocal food crop and commonly cultivated using conventionalmethod without appropriate plant nutrition system so thatproductivity is still low. A way to enhance local corn yieldis adding biofertilizer containing nitrogen (N2) fixingbacteria such as Azotobacter. The purpose of this research wasto determine N2 fixation, cytokinin as well asexopolysachharide production capacity of six indigenousAzotobacter strains in pure culture. The N2 fixationcapacities of native 3 day old Azotobacter strains added toAshby Media varied from 0.01 to 0.39 µM/g/hour. Cytokininproduction of these strain in liquid culture of N-free Mediawas 0.11 to 40.04 ppm while exopolysachharide content inliquid culture of Vermani Media varied from 0.4 to 27.3 g/L.This results demonstrate that some local Azotobacter strainsmight be used as biofertilizer.
Key Words: Azotobacter, local isolate, N fixation,phythohormone, exopolysaccaride
INTRODUCTION
1
A significant change has taken place in plant nutrition
system in food production. In the past, the important
nutrition source to maximize plant productivity was
inorganic fertilizer. Nowaday, enhance the productivity was
drive by using natural fertilizer such as compost, biomass-
based organic fertilizer and biofertilizer. Nowadays,
biofertilizer widely used in agriculture is asymbiotic N2
fixing bacteria Azotobacter which colonize rhizophere of
important crops. Members of the genus Azotobacter are known to
be obligate aerobes, gram negative, pleomorphic in shape,
non-spore forming, mesophylic and heterotrophic bacteria
(Holt et al., 1994) which belong to the group of Plant
Growth Promoting Rhizobacteria, beneficial bacteria that
colonize rhizosphere and enhance plant growth.
The mechanisms by which Azotobacter increase plant growth
and yield of plant is asymbiotc dinitrogen (Sprent and
Sprent, 1990), phytohormone production, especially cytokinin
(Taller and Wong, 1989; Abbass and Okon, 1993; Hindersah et
al., 2003) and exopolysaccharides production (Vermani et al.,
1997; Emtiazi et al., 2004; Hindersah et al., 2006). Certain
research also reported a beneficial effect of phospate
solubilizing Azotobacter on agriculture (Kumar and Narula,
1999)
Increasing in growth and yield of important food crops
due to inoculation with Azotobacter have been reported
2
elsewhere. Pot as well as field trials carried out in
different environmental and soil showed that inoculation
important food crops with Azotobacter has positive effects on
plant growth and yields. The effect of Azotobacter on
vegetative growth and yields of maize has been studied by
numerous authors ((Martinez Toledo et al., 1988; Nieto and
Frankenberger, 1991; Setiawati et al., 2012). Azotobacter
chroococcum at concentration of 108 cfu ml-1 increased
germination of maize seeds in the N-free Hoagland’s medium
and treated seeds in soil pots; seedlings harvested in 25-35
days had significant increase in root length and plant height
and also in dry weight of root and shoot (Dhamangaonkar
Sachin and Pragati, 2009). Application Azotobacter at 3 l/ha
gave a same maize growth with application of inorganic
fertilizers (Katriani et al., 2011)
For a number of years, In Indonesia research of
Azotobacter as biofertilizer is increased significantly.
Nowadays some Azotobacter inoculum in form of liquid as well
as carrier-based biofertilizer are commercialized to support
sustainable production of important food crops. Maize in
some region in East Nusa Tenggara Indonesia bordering
Republic Democratic of Timor Leste is important local food
crop and commonly cultivated using conventional method
without appropriate plant nutrition system so that
productivity is still low. A way to enhance local corn yield
3
is adding biofertilizer containing nitrogen (N2) fixing
bacteria. The purpose of this research was to determine N2
fixation, cytokinin as well as exopolysachharide production
capacity of indigenous Azotobacter strains. This research will
be important to collect effective local strain of Azotobacter
in order to produce certain biofertilizer which has better
adaption to soil and climate condition of Melaka NTT.
MATERIAL AND METHOD
Isolate Source
Azotobacter were isolated from soil surrounding roots of local
maize grown in local farmer agricultural land in Alas Selatan
Village, Sub District of East Kobalima, District of Melaka,
in East Nusa Tenggara. Soil was collected in dry season of
2012. The soil is slighty alcaline Entisols (pH H2O 7.84)
with C-organic 0.67% (very low), total N 0,12 % (low),
P2O5 Potensial 92.01 mg/100 g (very high), K2O Potensial 24.19 mg/100
g.
Isolation Method
Azotobacter was isolated in two steps by using nitrogen-free
Ashby’s medium (manitol 10 g, KH2PO4, 0.2 g, MgSO4.7H2O 0,2
g, NaCl 0,2 g, CaCO3 0,1 g, Na2MoO4 10 mg, agar 15, aquadest
1000 mL; pH 7). At first step, 5 g soil was poured into 50 mL
autoclaved liquid medium. The culture was incubated in 30oC
4
for 5 days until biofilm appearance in the surface of medium.
The second step was isolation of Azotobacter from biofilm by
streaking one loop of liquid in Ashby’s plate agar. After a
period of 3 days of incubation at 30 C, colonies on the
plates were observed for presence of specific characteristics
appearance of Azotobacter. Strains were characterized by cell
morpholgy (Gram stain, presence of spores and capsule) as
well as colony morpholofy (form, elevation,margin,
appearance, optical property, pigmentation and texture).
Each strain showed morphological characteristics of Azotobacter
were then tested for its N2 Fixing ability, phytohormone
production and exopolysaccharide production.
N2 Fixation Capacity
Capacity of Azotobacter isolates were tested by determining
the concentration o nitrogen in liquid culture and by using
acetylene reduction assay in slant. Azotobacter were grown in
50 ml Ashby’s liquid medium on gyratory shaker (115 rpm) at
30 0C for 3 days. Concentration of nitrogen in culture was
measured by acid digestion and subsequent measurement by the
Kjeldahl method (Bremner, 1965). Azotobacter isolates were
streaked on slant with Ashby’s medium, incubated at 30 0C
for 3 days. For determination of acetylene reduction,
acetylene equivalent to 10C% of the net air volume of the
5
test tube injected (14, 21). Gas samples (0.5 ml) were
withdrawn every 30 min and Chromatographed immediately.
Cytokinin production
Pure culture in slant of Ashby were streaking onto plate
agar of modified N–free medium described Taller and Wong
(1989) which contained K2HPO4 0.5 g, MgSO4.7H2O 0.2 g, NaCl
0.2 g, CaCO3; 3.0 g, Mannitol 5.0 g, sucrose 5.0 g, 1 mL/L
n Fe-Mo solution, agar 15 g, aquadest 1000 mL. Plates were
incubated 3 days at 30oC. One mL of bacterial suspension
containing 108 CFU/ml was trasnferred into 50 ml of free-N
liquid media described above and incubated for 3 days at room
temperature on gyratory shaker. Cytokinin was analysis using
reverse phase High Permofrmance Liquid Chromatography
operated at room temperature by using -Bondapak C18/280 nm
and UV-VIS Spectrophotometer at 254 nm.
Exopolysaccharide production
Culture were streaking onto plate agar of basal medium (10 g
Sukrosa, 1,0 g KH2PO4, 1,0 g MgSO4.7H2O, 0,5 g NaCl, 0,1 g CaCO3
, 0,1 g NaNO3, 0,1 g FeSO4, 10 mg Na2MoO4 , 15 g agar , 1 L
aquadet) described by Vermani et al. (1997) to induce
exopolysaccharide (EPS) production. The bacteria were
maintained on the basal medium for 3 days at 30oC. One mL of
bacterial suspension containing 108 CFU/ml was trasnferred
6
into 50 ml of Vermani’s liquid media and incubated for 3
days at room temperature on gyratory shaker. Cultures were
centrifuged at 4000 rpm at 4 oC for 15 minutes. The
supernatant was removed, two volume of cold acetone (97%)
was added, and stand one night at 4 oC. The EPS was collected
on pre weight Whatman no 1 filter paper by centrifugation
at 4000 rpm at 4 oC for 15 minutes. The weight of EPS was
measured after heating at 35 oC for 1 hours and desiccating
for 20 minutes.
RESULTS AND DISCUSSION
After 5 days of enrichment in N2-free Ashby’s liquid medium,
the biofilm appeared in surface of liquid medium (Fig. 1
left). On N2 free Ashby’s Mannitol Agar medium the colonies
were grows up after three days of incubation, and showed dark
brown pigmentation (Fig. 2 cenetre). The shape of the
colonies was round, margins were entire, the surface of the
colonies were glistening, the density of colonies was
transparent. Most of colony was watery and mucilaginous. We
collected eight colonies which grows up to 1-3 mm in diameter
and passed to cell morphological test. Six colonies showed
charactersitics of Gram negatif, non spore forming and
7
showed capsular characteristics in negative staining, were
purified and maintained in slants with the same medium (Fig.
1 right). Microscopic cell shape of all isolate (AS1, AS2,
AS3, AS4, AS5 and AS6) was diplococcus, suggesting that all
isolates belong to Azotobacter chroococcum.
Figure 1. Biofilm appearance in the surface of liquid medium 5 days after incubation (left), morphology of Azotobacter isolates in Ashby agar medium (centre) and six pure culture of Azotobacter isolates.
All six isolates showed capacity to fix dinitrogen, produce
cytokinin and EPS (Table 1). Measurement nitrogen
concentration in liquid culture of isolate AS5 by using
Kjeldahl method, nitrogen was not detected but by using
chromatography, ethylene was detected which verified that
this isolate has a capacity to fix N2. All isolates has
different nitrogenase activity, isolate AS4 showed higher
nitrogenase activity.
8
Table 1. Nitrogen fixation, Cytokinin and Exopolysachharideproduction by six Azotobacter isolates from Melaka EastNusa Tenggara
Isolates
Total N(%)
N-aseactivityuM/g/hour
Cytokininconcentration
(ppm)
EPSconcentration
(g/L)AS1 0,008 0,01 0,11 0,4AS2 0,014 0,14 6,46 2,5AS3 0,006 0,26 16,21 4,0AS4 0,003 0,39 43,14 3,8AS5 nd* 0,25 40,04 27,3AS6 0,004 0,11 32,00 13,5
*nd: not detected
Cytokinin concentration in liquid culture was 0.11-43.14
ppm depend on the isolate. The highest capacity to produce
this phytohormone was showed by isolat AS4 (Table 1).
Capacity of Azotobacter to produce cytokinin was well
documented. Five cytokinin were identified in an A.
chroococcum culture filtrate (Nieto and Frankenberger 1989).
Cytokinins was detected in culture filtrate of A. vinelandii in
stationary phase (Taller and Wong, 1989). After 60 hours
incubation on the free N Page-Burk medium at 30 oC, A.
chrococcum isolated from corn rhizosphere excrete zeatin and
benzyladenine-9-glucoside (Hindersah et al. 2003).
The capacity of all isolates to produce cytokinin will
be an important factor if the bacteria will be used as
biofertilizer. Phytohomones are the primary substance
9
controlling the enhanced growth of important food crop such
as maize (Hindersah et al., 2003) and tomato (Azcorn and
Barea, 1975). In green house experiment, inoculum of A.
chroococcum in combination with certain cytokinins precursors
was effective to enhance vegetative growth of maiz up to
5.57- and 5.01-fold compared to that of control treatment
(Nieto and Frankenberger, 1991). The beneficial effect of
cytokinins excreted by A. choroococcum enhance root and shoot
growth in early vegetative phase of maize (Hindersah et al,
2003). In field experiment, productivity of hybrid maize was
enhance due to inoculation of Azotobacter with half dose of
conventional inorganic fertilizer (Setiawati et al., 2012).
To induce EPS production, each Azotobacter isolates was
grown on Vermani plate agar. After three days incubation,
colonies were grows up with intensive mucilage and brownish
black pigmentation (Fig. 2 left). The EPS was successfully
extracted by using acetone and collected on Whatman no 1
(Fig. 2 center and right). EPS concentration was varied
between 0,4-27.3 g/L depend on isolates.
10
Figure 2. Colonies appearance of Azotobacter in Vermani medium(left), EPS on one side of centrifuge tube after 7000rpm centrifugation at 4 0C (centre), EPS on theWhatman no 1 after heating at 35 0C (right)
EPS production in the cell surface of Azotobacter was well
documented (Vermani et al., 1997; Pal et al., 1999; Vargas-Garcia
et al., 2003; Emtiazi et al., 2004). The extent of EPS production
by Azotobacter was depend on isolate as well as carbon and
nitrogen source (Vermani et al., 1997; Emtiazi et al., 2004).
EPS synthesized by A. vinelandii isolated from Nelumbio nelumbo
was 1.1 g/L, and might be enhanced up to 6.7 g/L by
increasing sucrose and NaNO3 content in medium (Vermani et al.,
1997). Emtiazi et al. (2004) concluded that EPS production by
Azotobacter was 1,6 – 7,6 mg/mL depend on bacterial strain and
sukrosa content in N-free medium. Nine Azotobacter isolate
fenerates EPS in liquid medium with and without CdCl2 up to
11
1.1-1.7 g L-1 (Hindersah et al., 2006). EPS has a significant
role in soil aggregation and pore formation so that increase
nutrient uptake such as nitrogen (Alami et al., 2000).
CONCLUSION
Six isolates of Azotobacter were sucessfully isolated from
Entisol collected from Alas Selatan Village in District
Melaka, East Nusa Tenggara. The N2 fixation capacities of
native 3 day old Azotobacter strains added to Ashby Media
varied from 0.01 to 0.39 µM/g/hour. Cytokinin production of
these strain in liquid culture of N-free Media was 0.11 to
40.04 ppm while exopolysachharide content in liquid culture
of Vermani Media varied from 0.4 to 27.3 g/L. This results
demonstrate that some local Azotobacter strains might be used
as biofertilizer since they able to fix nitrogen as well
produce cytokinins and exopolysaccharide.
REFFERENCES
Abbass, Z., Y. Okon. 1993. Plant Growth Promotion byAzotobacter paspali in The Rhizosphere. Soil Biol. Biochem.8:1075-1083.
Alami, Y., W. Achouak, C. Moral, and T. Heulin, 2000.Rhizosphere soil aggregation and plant growth promotionof sunflowers by an exopolysaccharide-producing Rhizobiumsp. strain isolated from sunflower roots. Appl. Environ.Microbiol. 66:3393-3398
12
Azcorn R., Barea J.M. (1975). Synthesis of auxins,gibberellins and cytokinins by Azotobacter vinelandi andAzotobacter beijerinckii related to effects produced on tomatoplants. Plant Soil, 43: 609-619.
Dhamangaonkar Sachin, N and M. Pragati. Effect of Azotobacterchroococcum (PGPR) on the Growth of Bamboo (Bambusabamboo) and Maize (Zea mays) Plants. 2009. Biofrontiers,1 (1): 24-31
Emtiazi, G., Z. Ethemadifar, and M.H. Habibi. 2004.Production of extracellular polymer in Azotobacter andbiosorption of metal by exopolymer. Afr. J. Biotech.3:330-333
Hindersah, R, D.H. Arief, Y. Sumarni. Totowarsa. 2003.Produksi hormon sitokinin oleh Azotobacter. Abstract dalamBahasa Inggris. Prosiding Kongres dan Seminar NasionalHITI, Padang Juli 2003. Hal 549-555
Hindersah, R. , D. H Arief, S. Soemitro, L. Gunarto. 2006.Exopolysaccharide Extraction from RhizobacteriaAzotobacter sp. Proc. International Seminar IMTGT. Medan,22-23 Juni 2006. Pp 50-55
Pal, S., A. Manna, and A.K. Paul. 1999. Production of poly(β-hydroxybutyric acid) and exopolysaccharide byAzotobacter beijerinckii WDN-01. World J. of Microbiol andBiotechnol 15:11-16
Holt, J.G., N.R. Krieg, J.T. Shaley dan S.T. William. 1994.Bergey’s Manual of Determinative Bacteriology. William danWilkins. Baltimore.
Katriani, E. Syamun, D. Tandipada. 2011. Pertumbuhan danproduksi jagung pada berbagai konsentrasi azotobacter danpaket pemupukan N,P,K. Abstract in English. JurnalAgronomika. 1(2): 65-70
Kumar, V., N. Narula. 1999. Solubilization of InorganicPhosphate and Growth Emergence of Wheat as Affected byAzotobacter chroococcum Mutans. Biol. Fertil. Soil. 28:301-307.
13
Martinez Toledo M.V., Gozalez-Lopez J., De la Rubia T.,Moreno J., Ramos-Cormenzana A. 1988. Effect ofinoculation with Azotobacter chroococcum on nitrogenaseactivity of Zea mays roots grown in agricultural soilsunder aseptic and non-sterile conditions. Biol. Fert.Soils, 6: 170-173.
Martinez Toledo M.V., Moreno J., De la Rubia T., GozalezlopezJ. (1989). Root exudates of pea mays and production ofauxins, gibberellins and cytokinins by Azotobacterchroococcum. Plant Soil, 110: 149-152.
Nieto K.F., Frankenberger W.T. 1989. Biosynthesis ofcytokinins by Azotobacter chroococcum. Soil Biol Biochem, 21:967-972.
Nieto K.F., Frankenberger W.T. 1991. Influence of adenine,isopentyl alcohol and Azotobacter chroococcum on thevegetative growth of Zea mays. Plant Soil, 135: 213-221.
Setiawati, M.R., R. Hindersah, P. Suryatmana. 2012.Penggunaan Azotobacter sebagai pupuk hayati pada tanamanjagung. Laporan Penelitian kerjasama Unpad dengan PTPUSRI. Fakultas Pertanian Unpad. Bandung
Sprent, J.I., Sprent. P. 1990. Nitrogen Fixing Organisms,Pure and Applied Aspects. Chapman and Hall. London.
Taller, B.J. dan T.Y. Wong. 1989. Cytokinins in Azotobactervinelandii Culture Medium. Appl Environ Microbiol, 55:266-267.
Vargas-Garcia, M.C., M.J. Lopez, M.A. Elorrieta, F. Suarez,and J. Moreno. 2003. Properties of polysaccharidesproduced by Azotobacter vinelandii cultured on 4-hydroxybenzoicacid. J. Appl. Microbiol. 94:389-395
Vermani, M.V., S.M. Kelkar, M.Y. Kamat. 1997. Studies inPolisaccharide production and growth of Azotobacter vinelandiiMTCC 2459, a Plant Rhizosphere Isolate. Lettr. In ApplMicrob, 4:379-383.
14