Arch Microbiol (2009) 191:275–281
DOI 10.1007/s00203-008-0444-9
S H O R T CO M MU N I C A T I O N
Encystment of Azotobacter nigricans grown diazotrophically
on kerosene as sole carbon source
Gabriela García-Esquivel · Graciano Calva-Calva · Ronald Ferrera-Cerrato ·
Luis Carlos Fernández-Linares · Refugio Rodríguez Vázquez ·
Fernando José Esparza-García
Received: 23 June 2008 / Revised: 9 September 2008 / Accepted: 27 October 2008 / Published online: 19 November 2008
© Springer-Verlag 2008
Abstract Encystment of Azotobacter nigricans was
induced by its diazotrophic cultivation on kerosene. Its
growth and nitrogenase activity were aVected by kerosene
in comparison to cultures grown on sucrose. Electron
microscopy of vegetative cells showed that when nitroge-
nase activity was higher and the poly-
-hydroxybutyrate
granules were not present to a signiWcant extent, peripheral
bodies were abundant. After 8 days of culture on kerosene,
the presence of cysts with intracellular bunches of poly-
-
hydroxybutyrate granules was observed. Germination of
cysts bears germinating multicelled yet unbroken capsule
cysts with up to three cells inside. This is the Wrst report of
encystment induction of Azotobacter species grown on
kerosene.
Keywords Cyst · Kerosene removal · Nitrogen Wxation ·
Multi-celled cysts · Nitrogenase activity ·
Poly-
-hydroxybutyrate · PHB · NFB
Communicated by Mary Allen.
G. García-Esquivel · G. Calva-Calva · R. R. Vázquez ·
F. J. Esparza-García (&)
Biotechnology and Bioengineering Department,
Centro de Investigación y de Estudios Avanzados del Instituto
Politécnico Nacional, C. P. 07360 Mexico D. F., Mexico
e-mail: fesparza@cinvestav.mx
G. Calva-Calva
e-mail: gcalva@cinvestav.mx
R. Ferrera-Cerrato
Edaphology Department, Colegio de Posgraduados,
Montecillo, Estado de México, Mexico
L. C. Fernández-Linares
Centro de Investigación en Calidad Ambiental,
Instituto Tecnológico y de Estudios Superiores de Monterrey,
Estado de México, Mexico
Introduction
In contrast to other genera of the family Azotobacteraceae,
which are nitrogen-Wxing bacteria (NFB), the genus Azoto-
bacter, as exempliWed by Azotobacter vinelandii and A.
chroococcum, bears resting cells with a resistant covering
known as cysts (SadoV 1975). This covering is composed
of organized in layers named intine and exine, both of
which contain alginate (Sabra et al. 2001), a co-polymer of

-D-mannuronic acid and
-L-guluronic acid (Fang et al.
2008). While exine is a rigid multilayered structure com-
posed of a lipoprotein–lipopolysaccharide complex, free
lipids, and calcium, intine consists of only two layers sepa-
rated by a membrane of carbohydrates, free lipids, some
proteins, and more calcium than in the case of exine (Vela
et al. 1970; Stevenson and Socolofsky 1972; Núñez et al.
1999). In association with the encystment process, mem-
bers of the Azotobacter genus also produce large quantities
of poly-
-hydroxybutyrate (PHB) as a carbon reserve as
well as alginate as a protector of the nitrogenase enzyme
(SadoV 1975; Sabra et al. 2001; Segura et al. 2003). How-
ever, like n-butyl alcohol,
-hydroxybutyrate also induces
the encystment of A. vinelandii and A. chroococcum when
added to exponential cultures grown diazotrophically on
traditional carbon sources (SadoV 1975; Reusch and SadoV
1981). Interestingly, germination of A. vinelandii cysts
bears multicelled bodies with up to three cells within an
unbroken capsule cyst (Cagle and Vela 1971), suggesting
that cell division may occur inside the cyst prior to the rup-
ture of the exine layer. Similar bacterial multicelled bodies
have hitherto only been reported for Rhodospirillum cente-
num cysts containing between four and ten cells per body
(Berleman and Bauer 2004). In this work, multicelled
bodies bearing up to three cells inside of a yet unbroken
capsule of germinating cysts of an NBF isolated from the
123
276
rhizosphere of Phaseolus vulgaris plants cultivated in
kerosene-contaminated soil, and partially identiWed as
A. nigricans, were observed. In contrast to previous reports
regarding the encystment of other Azotobacter species, in
which encystment inducers were used, in this work the
encystment process was observed in cultures grown diazo-
trophically on kerosene as the sole carbon source. Morpho-
logical and physiological changes during the encystment
process regarding the removal of kerosene, nitrogenase
activity, and the presence of PHB granules were monitored
by transmission electron microscopy (TEM). To our knowl-
edge, production of both PHB and cysts of A. nigricans has
not hitherto been reported for growth on any carbon source,
and, moreover, the encystment of Azotobacter species
using kerosene as the sole carbon source has not been
reported previously.
Materials and methods
Isolation and characterization of the bacterial strain
The bacterial strain used in this work was isolated from the
free living NFB group associated with the rhizosphere of
P. vulgaris cultivated on kerosene-contaminated soils, as
reported previously by Pérez et al. (2000, 2001)with some
modiWcations. Basically, 1 g of rhizospheric soil was
transferred into 50 mL of Rennie mineral culture medium
(RM) containing sucrose and mannitol (Rennie 1981).
Cultures were incubated for 3 days on an orbital shaker at
150 rpm, and a loopful was plated on solid medium and
incubated at 28°C. A single colony was isolated for partial
identiWcation and biochemical characterization by further
growth in liquid RM supplemented with several carbon
sources. Bacterial growth was assessed by monitoring the
optical density at 500 nm using a spectrophotometer
(Spectronic 20, Bausch and Lomb, USA), and the dry weight
was determined by Wltration through a 0.22 m pore size
membrane. Motility was evaluated by the hanging drop
slide technique.
Nitrogenase activity
Nitrogenase activity was evaluated by the method of acety-
lene reduction, as reported by Postgate (1971), with some
modiWcations. Vials of 144 mL with cotton plugs and con-
taining 25 mL of RM with kerosene 1% (v/v) as the
sole carbon source were incubated as described above for
the Xask cultures. After the incubation, the cotton plugs
were replaced by hermetic stoppers, and 10 mL of gas from
the headspace of each vial was replaced by 10 mL of acety-
lene by means of a sterile syringe. The vial cultures were
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Arch Microbiol (2009) 191:275–281
incubated for 24 h, and ethylene production was evaluated
by injecting 500 L of the headspace gas into a gas chro-
matograph (Tracor Model 570, Tracor Co., USA) equipped
with a Xame ionization detector and a Poropack N column.
The detector, injector, and column temperatures of the gas
chromatograph were 250, 200, and 55°C, respectively.
Kerosene assay
Kerosene was assayed as reported by Pérez et al. (2000,
2001) by extracting complete cultures with hexane
(3 £ 10 mL) and concentrating the combined extracts to
10 mL before GC analysis.
Encystment induction
A bacterial colony of the isolated strain was transferred to
50 mL of RM supplemented with 1% (w/v) sucrose and
incubated on an orbital shaker at 150 rpm and 28°C. Ali-
quots of 2.5 mL (109 cells/mL) were taken from these cul-
tures and transferred to 50 mL of fresh RM supplemented
with 1% (w/v) sucrose, and cultivated until the exponential
phase of growth was reached (usually 24 h). The whole cul-
ture was then centrifuged and the pellet obtained was
washed once with phosphate buVer (pH 7.4), suspended in
50 mL of RM containing n-butyl alcohol (0.2%, v/v), and
incubated for 120 h at 28°C on an orbital shaker at 150 rpm
for the encystment induction.
Encystment and germination assessment
The number of cysts in the cultures was estimated by count-
ing the number of cells resistant to desiccation, as reported
by Campos et al. (1996), but using RM supplemented with
sucrose. The cells resistant to desiccation were drawn from
stationary-phase bacterial cultures (192 h) grown on RM
supplemented with either kerosene (1%, v/v), sucrose (1%,
w/v), or n-butyl alcohol (0.2%, v/v). To count the number
of cysts, 2 mL aliquots of these cultures were Wltered
through 0.45 m pore size Millipore® cellulose membranes
(4 cm). The membranes containing the cells were washed
with sterile water and transferred to empty sterile plates
(4 cm) for desiccation by incubation for 5 days at 30°C.
The dry cells were released from the membrane by adding
1 mL of mineral RM (without carbon sources) to the plate
and agitating it on an orbital shaker at 80 rpm and 28°C for
24 h. The suspended cells were plated on solid RM with 1%
(w/v) sucrose and the number of colonies was counted after
3 days of incubation at 28°C. This number was considered
as the number of cells resistant to desiccation, which also
represents the number of cysts in the original culture prior
to desiccation. For germination studies, cysts in the mem-
Arch Microbiol (2009) 191:275–281
brane were transferred into fresh liquid RM supplemented
with 1% (w/v) sucrose.
Electron microscopy
The biomass from 50 mL bacterial cultures was harvested
by centrifugation at 10,000 rpm and transferred to 1.5 mL
centrifuge tubes maintained in an ice bath. Each pellet was
washed with refrigerated phosphate buVer solution (pH
7.4), and the medium-free pellet was incubated in an ice
bath for 2 h in the presence of glutaraldehyde (2%, v/v).
The glutaraldehyde was then eliminated by centrifugation,
and the biomass was washed with phosphate buVer. The
pellet was resuspended with 0.5–1 mL osmium tetroxide
(2%, w/v) and incubated at 4°C for 1 h with gentle shaking.
The osmium tetroxide was then eliminated by centrifuga-
tion and the pellet was washed with phosphate buVer and
sequentially resuspended in a series of ethanol solutions
(50, 60, 70, 80, 90, and 100% v/v) at intervals of 15 min.
The pellet was suspended in absolute ethanol for 15 min,
the suspension was centrifuged, and the pellet obtained was
resuspended in propylene oxide three times for 10 min
each. The pellet was then suspended in Spurr 60 cp resin
(Polysciences, Inc.) and incubated at 60°C for 3 days to
polymerize the resin. Ultra-thin sections were cut with a
Reichert-Jung ultramicrotome and contrasted for 30 min
with uranyl acetate. The sections were washed with dis-
tilled water, submerged for 5 min in lead citrate solution
(0.4 mM), washed with distilled water once more, and
observed by TEM with a JEM 2000 EX (JEOL Inc.) micro-
scope.
Statistics
Data represent the mean and standard deviation of three
replicates. Excel software was used for statistical analysis.
Fig. 1 Time course of Azoto-
bacter nigricans growth on 1%
sucrose (open circle) or 1% ker-
osene (Wlled circle) as sole car-
bon sources (a), and their
respective speciWc nitrogenase
activities (b). Cultures were
incubated at 28°C on a rotary
shaker (150 rpm). Residual ker-
osene (Wlled square) was ex-
tracted from the medium culture
and analyzed by GC. Bars repre-
sent the standard deviation of
three cultures
277
Results and discussion
Isolation and characterization of the bacterial strain
The strain isolated in this work was a Gram-negative rod-
shaped bacterium, and grew diazotrophically on sucrose
(Fig. 1), showing dark-brown colonies and diVerential char-
acteristics that matched those of A. nigricans (Table 1). As
part of the biochemical characterization, the isolated strain
was grown with several carbon sources (Table 1). In con-
trast to A. beijerinckii, it grew in starch, malonate, and man-
nitol, but was unable to grow in benzoate and inositol
(Garrity et al. 2005).
EVect of kerosene on growth and nitrogenase activity
Both, growth and speciWc nitrogenase activity were aVected
by kerosene as the sole carbon source, as compared to cul-
tures grown on sucrose (Fig. 1). The speciWc growth rate of
cultures on sucrose ( = 0.097 h¡1; Td = 10.3 h) was about
ten times higher than that of those on kerosene
( = 0.016 h¡1; Td = 63.8 h). The speciWc growth rate for
cultures on sucrose was considerably lower than those
reported by Mackenzie and Macrae (1972) and by SadoV
(1975) for A. vinelandii growing under nitrogen-Wxing con-
ditions on mannitol ( = 0.3 h¡1) or glucose ( = 0.3–
0.4 h¡1), respectively. However, it was similar to the value
reported for A. chroococum ( = 0.17 h¡1) growing under
nitrogen-Wxing and oxygen-limiting conditions with glu-
cose as sole carbon source (Quagliano and Miyazaki 1999).
On the other hand, the nitrogenase activity pattern was sim-
ilar between the two carbon sources until the latter stages of
the exponential phase of growth: the maximum nitrogenase
activity in the cultures was observed at the end of the expo-
nential phase of growth, showing a rapid increase at the
beginning of the exponential growth, and a drastic decline
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Table 1 DiVerential character-

istics of the Azotobacter strain
used in this work in comparison
with other Azotobacter species
Characteristics of Azotobacter
species other than the character-
istics of that used in this work
were taken from Garrity et al.
(2005)
D doubt
Characteristic Azotobacter strain
vinelandii beijerinckii nigricans This work
Colony Yellow-green Dark-brown Dark-brown Dark-brown
Gram ¡ ¡ ¡ ¡
Motility + ¡ ¡ ¡
Cysts + + + +
Oxidase activity + + + +
Growth in
Rhamnose + ¡ ¡ ¡
Caproate + ¡ ¡ ¡
Inositol + + ¡ ¡
Mannitol + d d +
Malonate + ¡ d +
Starch ¡ d d +
Benzoate + + ¡ ¡

at the end of the exponential phase. However, in cultures
with sucrose the cellular growth ended when no nitrogenase
activity was detected, whereas in cultures with kerosene the
biomass continued slowly growing throughout the time
(8 days) with a corresponding kerosene intake after the
nitrogenase activity was no longer detected. These nitroge-
nase activity results with cultures grown on kerosene are
consistent with previous studies on other free living NFB
grown on carbon sources other than carbohydrates, such as
Azomonas grown on kerosene (Pérez et al. 2001) and A.
vinelandii grown on olive mill wastes (Balis et al. 1996;
Papadelli et al. 1996). In contrast, Azotobacter sp. grown in
a mix-culture with Pseudomonas or Bacillus with light
crude oil as the sole carbon source showed a growth-associ-
ated nitrogenase activity pattern throughout the whole
kinetics of growth (Onwurah 1999). The nitrogenase activ-
ity pattern was also diVerent from those displayed by Kleb-
siella pneumoniae and Enterobacter sp. grown on sucrose
and malate, respectively (Haahtela et al. 1983), which were
not detected during the lag phase, increased during the
exponential growth, reaching the highest activity during
early stationary phase, and then declined drastically to
undetectable values during the stationary phase. Thus, cul-
tures grown on kerosene seem to Wx nitrogen but do not
accumulate biomass, leading the metabolic Xux to cell
maintenance and the biosynthesis of carbon storage
reserves, as will be discussed later.
Encystment on kerosene
In contrast to cultures of the isolated A. nigricans grown on
sucrose, those grown on kerosene showed gradual changes
in the cellular shapes (Fig. 2), indicating that the hydrocar-
bons of the kerosene induced the formation of polymorphic
cells in this bacterial strain. As reported for other species of
the genus Azotobacter, the polymorphic cells showed the
presence of PHB central granules as a prelude to the devel-
opment of cysts (Mulder and Brotonegoro 1974; Espín
2003; Copeland et al. 2006). On the other hand, during the
exponential and linear growth of cultures with sucrose, the
cell population showed a mixture of the typical vegetative
ovoid (1 m diameter) to large bacillary shape (1 m
wide £ 4.5 m long), some of which were in obvious divi-
sion, and without PHB granules, but showing some elec-
tron-lucent peripheral bodies (Fig. 2a). In contrast, in the
late exponential phase of cultures with kerosene, the vege-
tative cells were gradually transformed to encapsulated
ovoid shapes showing abundant peripheral bodies (Fig. 2b),
and during the linear phase of growth one or two central
PHB granules were typical in most of the cell population
(Fig. 2c,d). Similar morphological changes have been
reported to be induced by the replacement of glucose or
sucrose with butyl alcohol in A. vinelandii cultures (Wyss
et al. 1961; SadoV 1975). Interestingly, the peripheral bod-
ies were particularly abundant during the exponential phase
of growth, when the nitrogenase activity was high (5–6 h)
and the presence of PHB granules in the cells was relatively
insigniWcant (Fig. 2b). It should be noted that as the size of
the PHB granules and the space between the cell membrane
and the cytoplasm increased (Fig. 2d), the nitrogenase
activity decreased (Fig. 1b); this trend continued until the
PHB granules reached a maximum size (700–800 nm) and
the nitrogenase activity was no longer detected in the cul-
tures (after approximately 24 h). These observations are
consistent with the hypotheses that the peripheral bodies
might be related to the nitrogen Wxation process (Oppen-
heim and Marcus 1970) and that PHB is an energy and carbon
intracellular storage polymer (Kadouri et al. 2003). However,

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Arch Microbiol (2009) 191:275–281 279

Fig. 2 Microphotographs of
cells of A. nigricans after 24 h
(a), 9 h (b), 16 h (c), and 24 h (d)
of culture in RM with sucrose
(a) and kerosene (b–d) as the
sole carbon sources. Rod-shaped
vegetative cells grown in
sucrose (a), almost lacking PHB
and electron-lucent peripheral
bodies (pb), were transformed to
ovoid precystic cells with abun-
dant pb (b) and central PHB
granules during the exponential
(c) and linear (d) phases of
growth, leading to polymor-
phism when cultivated on kero-
sene. Bars represent 500 nm in
(a) and (c); 200 nm in (b) and (d)

Fig. 3 Mature cysts of A. nigri-

cans (a), showing the PHB cen-
tral granules, the intine (in), and
the exine (ex) layers of the cellu-
lar coat. The cells were isolated
at the end of the stationary phase
of growth from cultures grown
in RM with kerosene (a, b;
8 day) or sucrose, but induced
with 0.2% of n-butyl alcohol (c;
5 day). The bars represent 2 m
in (a), and 200 nm in (b) and (c).
Note that each cyst contains only
one central body

the suggestion that a biological function of PHB may be to source (Senior et al. 1972; Espín 2003; Copeland et al.
facilitate respiratory protection of nitrogenase in the spres- 2006) is not supported by these results, since a reverse rela-
ence of oxygen and the absence of an extracellular carbon tionship between the PHB accumulation and nitrogenase

123
280 Arch Microbiol (2009) 191:275–281

activity was evident and the PHB accumulation was higher

when the nitrogenase activity was undetectable. On the
contrary, the pattern for growth on kerosene is consistent
with that reported by Senior et al. (1972) for A. beijerinckii
cultures grown on excess glucose, which, like kerosene in
this study, was not completely consumed; 25% of the initial
kerosene remained after 8 days of culture. Also similar to
A. beijerinckii cultures, both growth and PHB accumulation
were sustained throughout the time without attaining a true
stationary phase. However, in the present study with
A. nigricans, extending the culture time to 8 days under
nitrogen-Wxing conditions resulted in the appearance of
cysts with intracellular bunches of PHB granules after
about 96 h, ultimately leading to the encystment of the
whole cell population after 192 h (Fig. 3a). However, from
the resistance to desiccation test, only 64.7% of the cell
population eventually became mature cysts. These mature
cysts were microscopically identical to those induced by
n-butyl alcohol in cells collected from cultures grown on
sucrose, which produced 74.4% of mature cysts (Fig. 3b).
This ratio of mature cysts is similar or lower than that
reported for A. vinelandii grown on glucose and induced
with n-butyl alcohol (Lin and SadoV 1968; Reusch and
SadoV 1983). Since it is well known that alcohols such as
n-butanol and isopropanol can induce the encystment pro-
cesses in several bacteria, and that alcohols are metabolic
intermediates in the catabolism of hydrocarbons (Watkin-
son and Morgan 1990), it is not surprising that extracellular
carbon sources other than carbohydrates can induce the
encystment process.
Furthermore, the microscopic features of the mature
cysts (Fig. 3) and their germinating cystic cells (Fig. 4)
showed by A. nigricans in this study were similar to those
reported by several researchers for A. vinelandii (Wyss
et al. 1961; SadoV 1975; Reusch and SadoV 1983; Segura
et al. 2003), and for A. chroococum (Pal et al. 1997). The
mature cysts (Fig. 3) were composed of a central body con-
sisting of the protoplasm, a cell membrane, a thin cell wall, Fig. 4 Germinating cysts with multiple central bodies of young vege-
and central PHB granules, surrounded by a coat with the tative cells prior to the rupture of the coat after cystic cells of A. nigri-
cans post-germination bore vegetative cells after 8 h of culture in RM
typical capsule-like structure reported for cysts of other supplemented with 1% sucrose. Typically, the multicelled germinating
species of the genus Azotobacter (Garrity et al. 2005). As cysts showed up to three central bodies surrounded by unbroken resid-
pointed out above, the coat is made up of intine and exine ual exine (ex) and intine (in) layers. Bars represent 200 nm
layers, and perhaps owing to the hardness and elasticity of
these polymers, they remained in the germinating cyst until and carbon source in facilitating the germination, enlargement,
it bore vegetative cells (Fig. 4), producing germinating and division of cells in multicelled cysts (Cagle and Vela
multicelled cysts with multiple central bodies, all sur- 1971). Similar germinating multicelled cysts originating
rounded by unbroken residual exine and intine layers. It from cysts with only one central body have been reported
should be noted that the cysts contain only one central body for A. vinelandii (Wyss et al. 1961; Cagle and Vela 1971)
before the germination process starts (Fig. 3a). After 8 h of and for R. centenum (Berleman and Bauer 2004). However,
culture in fresh medium, the intine layer in the multicelled in these studies, it was suggested that vegetative cells might
cysts was almost undetectable. This observation supports divide inside the cysts prior to rupture of the exine layer,
the view expressed in the literature that intine may function probably due to a failure of the cyst to delay chromosome
not only as a storage material in cysts, but also as an energy replication (Berleman and Bauer 2004). Actually, why and

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how cell division occurs inside the cysts remains uncertain
and deserves further investigation.
In summary, the mature cysts that developed in cultures
of A. nigricans with kerosene as the sole carbon source
under nitrogen-Wxing conditions were able to regenerate
vegetative cells with physiological characteristics identical
to those of their parent cells. To the best of our knowledge,
this is the Wrst report of the encystment of an Azotobacter
species in cultures using kerosene as the sole carbon
source.
Acknowledgments Special acknowledgment is given to Victoria
Teresita Velásquez Martínez and Ma. De Lourdes Rojas for their tech-
nical assistance in the microscopic studies. The Wrst author, Gabriela
Garcia Esquivel, acknowledges a doctoral fellowship (124460) from
CONACyT.
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