Acta Microbiologica et Immunologica Hungarica, 54 (2), pp.

167–177 (2007)
DOI: 10.1556/AMicr.54.2007.2.7

DEVELOPMENT AND STANDARDIZATION OF CYST

BASED LIQUID FORMULATION OF AZOSPIRILLUM
BIOINOCULANT

REGUPATHY THAMIZH VENDAN* and MUTHUSAMY THANGARAJU

Agricultural College and Research Institute, Tamil Nadu Agricultural University,
Tiruchirappalli-620 009, Tamil Nadu, India

(Received: 9 September 2006; revised: 11 November 2006; accepted: 26 November 2006)

Azospirillum bioinoculant is well known for its high nitrogen fixing and plant
growth promoting characters. The carrier based bioinoculants generally suffer from
shorter shelf life, poor quality, high contamination and low field performance. There-
fore, it is necessary to develop alternative new formulation of inoculants where cyst
based inoculants can play significant role. The cyst based liquid formulation was de-
veloped by inoculating Azospirillum into the cyst inducing minimal salts medium
(MSM). One hundred per cent conversion of vegetative cells into cyst cells was no-
ticed in 96 h. The survival of cyst cells in the MSM was observed up to one year and
two months and interestingly, the population level of 108 was maintained till the final
observation. The cyst cells of Azospirillum accumulated poly-b-hydroxybutyrate
(PHB) granules and exhibited desiccation tolerance up to 20 days and temperature tol-
erance up to 40 ºC. Thus the cyst based liquid formulation has twin advantage of lon-
ger shelf life and tolerance to harmful environmental conditions. Regeneration of cyst
cells into vegetative cells in different media viz., tap water, sterile water, rice gruel and
nutrient broth was studied. The changes started within 3 h and complete return of veg-
etative cells was observed at 24 h. Although all the media tested favoured regenera-
tion, comparatively quicker regeneration was observed in nutrient broth and followed
by rice gruel. Thus, cyst based liquid formulation of Azospirillum has all the survival
advantages and can be used as a potential bioinoculant.

Keywords: Azospirillum bioinoculant

* Corresponding author; E-mail: thamizhvendan@yahoo.co.in

1217-8950/$20.00 © 2007 Akadémiai Kiadó, Budapest

168 VENDAN and THANGARAJU

Introduction

Azospirillum species are probably the most studied and appeared to have
significant potential for commercial applications. These organisms characterized
by high nitrogen fixing ability, are found in abundant numbers in the rhizosphere
as well as in the intercellular spaces of the roots of certain cereals and other plants
[1]. Some of the suggested modes of action for Azospirillum are; secretion of
phytohormones, nitrogen fixation, production of undefined signal molecules that
can interfere with plant metabolism, nitrite production and the enhancement of
mineral uptake by plants [2]. Thus they exert beneficial effect on the growth of
plants [3] and increase the yield of many crops of agronomic importance.
Azospirillum are known to be highly pleomorphic and to change their metabolic
activities swiftly in the face of changing environmental conditions [4]. Production
of cyst may represent a mechanism by which azospirilla can persists in the
rhizosphere during unfavourable conditions [5], such as desiccation, temperature
and nutrient limitation and convert into enlarged cyst forms [6].
A frequent observation is that in carrier based inoculants, the number of vi-
able cells decrease from 109 to 107 colony forming units (cfu) per g after 90 days
of storage [2]. The most consistent feedback received from the farmers and
inoculant producers is the concern about the shelf life of carrier based inoculants,
since they have shorter shelf life, which hardly extends beyond three to four
months under normal storage conditions. The development of adequate formula-
tions, which would ensure survival, protection of the strain and the application
technology, which would allow timely, easy and precise delivery in the field could
be a major step towards this goal [7]. Hence techniques to increase the shelf life of
inoculants thus become necessary to propagate this technology in large scale. In
this study, experiments were conducted to induce the cyst forms in Azospirillum to
get a bioinoculant with longer shelf life and their tolerance to adverse conditions.

Materials and methods

Developing the cyst based liquid formulation of Azospirillum

The Azospirillum culture, Azospirillum lipoferum (Az-204) was grown in N

free malic acid (NFb) broth [8] up to OD value of 1.45 and 1.10 to get population
of 109 cfu/ml. The cells were harvested by centrifugation at 5000 rpm at 4 °C and
washed three times with 100 mM potassium phosphate buffer solution. Twenty-

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 AZOSPIRILLUM BIOINOCULANT 169

five milliliter of these cultures were inoculated into one litre cyst inducing me-
dium viz., Minimal Salts Medium (MSM) [9] and another set was maintained with
the addition of 10 ml of glycerol (10 mM). They were incubated at room tempera-
ture in an environmental shaker at 200 rpm to investigate cyst formation, viable
cell population, poly-b-hydroxy butyrate accumulation, desiccation and tempera-
ture tolerance of Azospirillum cultures.

Determination of conversion rate of vegetative cells of Azospirillum

into cyst cells

The conversion rate of vegetative cells into cyst cells of Azospirillum was
calculated by taking direct microscopic count using a Haemocytometer by observ-
ing morphological difference at 24 h interval. The number vegetative cells and
cyst cells per large square were counted and cyst conversion percent was worked
out.
Number of cyst cells in large square
Cyst conversion per cent = × 100
Total number of cells observed in the large square

Enumerating the viable cell population

The population was estimated using NFb medium through the drop plate
method [10] at monthly interval.

Estimation of poly-b-hydroxy butyrate content

The PHB content was measured [11] from the Azospirillum culture grown
in MSM sampled at 24 h interval of incubation. Bacterial cell suspension was pre-
pared by centrifuging the culture at 8000 rpm for 20 min at 4 °C. One ml of cell
suspension was added to one ml of 2 N hydrochloric acid and the mixture was di-
gested at 100 °C for 2 h. After digestion the mixture was cooled and extracted
twice with chloroform. The chloroform was then evaporated in a boiling water
bath and to the sediment, 5 ml of concentrated sulphuric acid was added. The sam-
ple was heated to 100 °C for 10 min in a water bath. After cooling, the absorbance
was measured at 235 nm in Systronics-UV-Vis Spectrophotometer-108.

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170 VENDAN and THANGARAJU

Desiccation tolerance of Azospirillum cyst cultures

One ml of fivefold diluted A. lipoferum culture grown in MSM was taken in

sterile 1.5 ml eppendorf tubes. The tubes were in turn kept open in a sterile petri
dish and this set up was incubated at 37 °C in an incubator. The dried cells from the
eppendorf tubes were washed with 100 µl of sterile distilled water with vigorous
agitation to remove them quantitatively and their viability was determined by
plating on NFb medium.

Temperature tolerance of Azospirillum cyst cultures

One ml of fivefold diluted A. lipoferum culture grown in MSM was taken

and kept in the water bath at temperatures 30 °C, 40 °C, 50 °C or 60 °C for 30 min.
Then the tubes were removed, rapidly cooled and the population was determined
by plating with one ml of culture on NFb medium.

Regeneration of cyst cells of Azospirillum in different media

Regeneration of cyst cells of Azospirillum was examined in different media

viz., tap water, sterile water, rice gruel and nutrient broth. Cyst inoculum (2.5 ×
108 cfu / ml) was added to the above media at 1, 2 and 5 per cent levels individu-
ally and incubated on a shaker with 120 rpm at room temperature. The regenera-
tion and multiplication of cells was examined by serial dilution and plating at var-
ied intervals from 0 h to 36 h.
All the data obtained from the above experiments were subjected to statisti-
cal analysis as per the method detailed by Panse and Sukhatme [12].

Results and discussion

A gradual increase in the conversion per cent of cyst formation from vege-
tative cells was observed with laps of time. In the present study, at 96 h, 100 per
cent conversion of vegetative cells in to cyst cells was recorded (Fig. 1), where
fructose and KNO3 were used as the carbon and nitrogen sources, respectively. It
was proved that media containing fructose and KNO3 favoured flocculation and
formation of cyst cells by providing nutrient stress to Azospirillum cells [6, 13,

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 AZOSPIRILLUM BIOINOCULANT 171

35

30

25
Cells (%)

20

15

10

0
0 24 48 72 96

Hours

Vegetative cells Cyst cells

Figure 1. Assessment of conversion of vegetative cells of Azospirillum into cyst cells in the minimal
salts medium (MSM)

14]. The reason behind the flocculation is believed to be connected with the pro-
duction of exocellular polymers, particularly b-linked polysaccharides [6]. Berg et
al. [4] reported that the capsular forms (C forms) of azospirilla were obtained on a
limiting carbon and nitrogen medium and these forms are responsible for cell ag-
gregation and synthesis of polysaccharides necessary for encystation.
The survival of cyst cells of Azospirillum was assessed in the minimal salts
medium (MSM) and MSM amended with glycerol (10 mM) up to the period of
420 days (Table I). The results revealed that the population level of 1010 was main-
tained up to 150 days of observation in both media. The population at 109 was no-
ticed from 180 days up to 330 days in both MSM and MSM + glycerol treatments.
Interestingly, the 420 days observation showed population level of 108 in both
treatments. This work is in line with Inamdar et al. [15] where they reported the
long life span of cyst based inoculant of Azotobacter and observed the survival for
over two years. MSM amended with glycerol (10 mM) supported higher number
of cells as compared to the MSM alone. It might be due to the fact that glycerol has
high water binding capacity and may protect cells from the effects of desiccation
by slowing the drying rate.
Production of reserve material of PHB granules in Azospirillum cultures
grown in MSM and MSM amended with glycerol was studied in this experiment.
The PHB accumulation increased constantly up to 108 h and thereafter a decline
was noticed (Table II). Suresh Babu et al. [16] found an increase in accumulation

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172 VENDAN and THANGARAJU

Table I

Survival of cyst cells of Azospirillum in the cyst based liquid formulation

Days Population of Azospirillum (log cfu/ml)
MSM* MSM + glycerol
30 11.01 11.02
60 10.99 11.00
90 10.95 10.98
120 10.93 10.95
150 10.92 10.94
180 10.01 10.85
210 10.00 10.04
240 10.00 10.04
270 9.98 10.02
300 9.96 10.02
330 9.94 9.97
360 8.98 9.02
390 8.96 9.00
420 8.90 8.99
Sed CD (0.05)
Days 0.007 0.014
Media 0.002 0.005
Interaction 0.010 0.020

* Minimal salts medium.

Table II

Poly-b-hydroxybutyrate (PHB) content of Azospirillum grown in MSM

Time (h) PHB (mg/g dry wt. of cells)
MSM MSM + glycerol
0 0.000 0.000
12 0.315 0.318
24 0.658 0.664
36 0.921 0.947
48 0.988 0.991
60 1.114 1.120
72 1.791 1.804
84 2.216 2.219
96 2.921 2.933
108 3.847 3.852
120 3.152 3.157
132 2.852 2.861
Sed 0.002 0.003
CD (0.05) 0.004 0.006

of PHB up to 96 h and a decline afterwards which is in conformity with the present

results. Under carbon and nitrogen limiting conditions, the Azospirillum cells tend
to form abundant PHB granules [6]. PHB production and its role in survival of var-

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 AZOSPIRILLUM BIOINOCULANT 173

ious stress and deleterious conditions in the A. brasilense were studied [17].
Kadouri et al. [18] reported the synthesis and utilization of PHB as a carbon and
energy source under stress conditions.
The cyst formation and its tolerance against stress conditions by
Azotobacter was reported earlier [19, 20]. The cyst cells of Azospirillum exhibited
tolerance to desiccation and survived for 20 days when subjected to desiccation
tolerance tests (Fig. 2). It was observed that the cyst cells of Azospirillum are resis-
tant to desiccation from a few hours to one month [5]. It is also apparent that the
PHB rich cyst cells confer more resistance to desiccation than the PHB deficient
vegetative cells. MSM+ glycerol treatment recorded higher number of viable cells
as compared to MSM alone. The inference of the present study is in agreement
with desiccation tolerance experiment conducted by Sadasivan and Neyra [21]
with cyst form of Azospirillum cells. Temperature tolerance study showed that
above 50 ºC, there were no viable cells of cyst based Azospirillum (Fig. 3),
whereas Berleman and Bauer [22] reported that 81.8 per cent survival of cyst cells
of Rhodospirillum centenum when incubated at 52 ºC for as long as 30 min. How-
ever in the present study, the Azospirillum cyst cells were tolerant up to 40 ºC. The
cyst formation process is generally accompanied by the production of a thick coat
or capsule [21] and Bleakley et al. [23] attributed the acquisition of resistance of
cysts to cyst coat against ultraviolet treatment, temperature and desiccation.
The capacity to form cyst endows Azospirillum with the ability to survive
under adverse environmental conditions. However, the success of this survival
strategy depends on the presence of an efficient mechanism for returning to the
vegetative state under favourable conditions. In this study, the regeneration of cyst

11 Vegetative cells

Cyst cells (MSM)

10

Cyst cells (MSM + glycerol)

9
log cfu /ml

6
0 2 4 6 8 10 12 14 16 18 20
Days
Figure 2. Desiccation tolerance of cyst cells of Azospirillum in the cyst based liquid formulation

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174 VENDAN and THANGARAJU

Vegetative cell
12
Cyst cells (MSM)
Cyst cells (MSM + glycerol)
10
log cfu/ml

4
30 40 50 60
Temperature (ºC)

Figure 3. Temperature tolerance of cyst cells of Azospirillum in the cyst based liquid formulation

cells was studied in different media and found that regeneration occurs more rap-
idly than the formation of cyst cells. Within 3 h of plating on nutrient medium, cyst
cells under phase contrast microscopy showed a decrease in the width and bright-
ness of the highly refractile outer coat (Table III). Fast regeneration was noticed in
nutrient broth followed by rice gruel and it might be due to its supply of suitable
carbon, nitrogen, vitamin and trace elements for the growth of Azospirillum. After
24 h, complete return of the vegetative cell was observed with a recurrence of
vibriod and spiral shapes and active cell motility. Similar results were obtained by
Berleman and Bauer [22] from germination of Rhodospirillum cyst cells. Interest-
ingly, sterile water and tap water also promoted regeneration of cysts which
clearly indicated that neither a carbon nor nitrogen source is specifically required
for regeneration. Among the cyst inoculum levels, 5 per cent level was found to be
optimum and produced higher population and quicker regeneration as compared
to other inoculum levels.
The cyst based liquid formulation of Azospirillum has twin advantage of
longer shelf life and tolerance to adverse conditions such as temperature and des-
iccation. Hence, it can be concluded that the accumulation of PHB and formation
of cyst cells are critically important for improving the shelf life, efficiency and re-
liability of commercial inoculants. Thus cyst based liquid inoculant of Azospi-
rillum therefore can be more reliable, potential bioinoculant which could safely be
distributed to the remote agricultural areas.

Acta Microbiologica et Immunologica Hungarica 54, 2007

 Table III

Regeneration of cyst cells of Azospirillum in different media

Inoculum level Population of Azospirillum (log cfu /ml)
0h 3h 6h 9h 12 h 24 h 36 h
Tap water
1% 3.01 5.06 5.12 7.15 8.00 8.12 7.10
2% 3.12 5.15 7.25 8.28 8.26 8.29 7.21
5% 3.24 5.30 7.39 8.36 8.33 9.23 8.17
Sterile water
1% 3.03 5.13 5.21 8.20 8.19 8.23 7.28
2% 3.15 5.29 7.32 7.28 8.29 9.21 8.24
5% 3.28 5.31 7.38 8.32 9.26 9.25 8.25
Rice gruel
1% 3.09 5.19 7.28 8.25 8.30 9.26 8.26
2% 3.24 5.30 7.36 8.38 9.33 9.34 8.29
5% 3.32 5.36 7.32 8.30 9.30 9.34 8.29
AZOSPIRILLUM BIOINOCULANT

Nutrient broth
1% 3.11 5.24 7.32 8.33 9.34 9.34 8.28
2% 3.26 5.34 7.39 8.44 9.36 9.37 8.29
5% 3.37 5.42 7.45 8.45 9.46 9.48 8.33
Sed CD (0.05)
Media 0.001 0.003
Concentration 0.001 0.002
Time 0.001 0.003
Interaction 0.005 0.010

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175
176 VENDAN and THANGARAJU

Acknowledgements

The financial support from ICAR through adhoc scheme on Liquid formu-
lation is gratefully acknowledge.

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