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

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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

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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

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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

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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

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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

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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

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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.

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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

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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.

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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

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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

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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.

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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.

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