Arch Microbiol (2009) 191:275–281

DOI 10.1007/s00203-008-0444-9

SHORT COMMUNICATION

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 wasinduced by its diazotrophic cultivation on kerosene. Itsgrowth and nitrogenase activity were aVected by kerosenein comparison to cultures grown on sucrose. Electronmicroscopy of vegetative cells showed that when nitroge-nase activity was higher and the poly-�-hydroxybutyrategranules were not present to a signiWcant extent, peripheralbodies were abundant. After 8 days of culture on kerosene,the presence of cysts with intracellular bunches of poly-�-hydroxybutyrate granules was observed. Germination ofcysts bears germinating multicelled yet unbroken capsulecysts with up to three cells inside. This is the Wrst report ofencystment induction of Azotobacter species grown onkerosene.

Keywords Cyst · Kerosene removal · Nitrogen Wxation · Multi-celled cysts · Nitrogenase activity · Poly-�-hydroxybutyrate · PHB · NFB

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 coveringknown as cysts (SadoV 1975). This covering is composedof organized in layers named intine and exine, both ofwhich 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, freelipids, and calcium, intine consists of only two layers sepa-rated by a membrane of carbohydrates, free lipids, someproteins, and more calcium than in the case of exine (Velaet 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 quantitiesof poly-�-hydroxybutyrate (PHB) as a carbon reserve aswell 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 inducesthe encystment of A. vinelandii and A. chroococcum whenadded to exponential cultures grown diazotrophically ontraditional carbon sources (SadoV 1975; Reusch and SadoV1981). Interestingly, germination of A. vinelandii cystsbears multicelled bodies with up to three cells within anunbroken capsule cyst (Cagle and Vela 1971), suggestingthat cell division may occur inside the cyst prior to the rup-ture of the exine layer. Similar bacterial multicelled bodieshave hitherto only been reported for Rhodospirillum cente-num cysts containing between four and ten cells per body(Berleman and Bauer 2004). In this work, multicelledbodies bearing up to three cells inside of a yet unbrokencapsule of germinating cysts of an NBF isolated from the

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., Mexicoe-mail: fesparza@cinvestav.mx

G. Calva-Calvae-mail: gcalva@cinvestav.mx

R. Ferrera-CerratoEdaphology Department, Colegio de Posgraduados, Montecillo, Estado de México, Mexico

L. C. Fernández-LinaresCentro de Investigación en Calidad Ambiental, Instituto Tecnológico y de Estudios Superiores de Monterrey, Estado de México, Mexico

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rhizosphere of Phaseolus vulgaris plants cultivated inkerosene-contaminated soil, and partially identiWed asA. nigricans, were observed. In contrast to previous reportsregarding the encystment of other Azotobacter species, inwhich encystment inducers were used, in this work theencystment process was observed in cultures grown diazo-trophically on kerosene as the sole carbon source. Morpho-logical and physiological changes during the encystmentprocess regarding the removal of kerosene, nitrogenaseactivity, and the presence of PHB granules were monitoredby transmission electron microscopy (TEM). To our knowl-edge, production of both PHB and cysts of A. nigricans hasnot hitherto been reported for growth on any carbon source,and, moreover, the encystment of Azotobacter speciesusing kerosene as the sole carbon source has not beenreported previously.

Materials and methods

Isolation and characterization of the bacterial strain

The bacterial strain used in this work was isolated from thefree living NFB group associated with the rhizosphere ofP. vulgaris cultivated on kerosene-contaminated soils, asreported previously by Pérez et al. (2000, 2001)with somemodiWcations. Basically, 1 g of rhizospheric soil wastransferred 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 at150 rpm, and a loopful was plated on solid medium andincubated at 28°C. A single colony was isolated for partialidentiWcation and biochemical characterization by furthergrowth in liquid RM supplemented with several carbonsources. Bacterial growth was assessed by monitoring theoptical density at 500 nm using a spectrophotometer(Spectronic 20, Bausch and Lomb, USA), and the dry weightwas determined by Wltration through a 0.22 �m pore sizemembrane. Motility was evaluated by the hanging dropslide technique.

Nitrogenase activity

Nitrogenase activity was evaluated by the method of acety-lene reduction, as reported by Postgate (1971), with somemodiWcations. Vials of 144 mL with cotton plugs and con-taining 25 mL of RM with kerosene 1% (v/v) as thesole carbon source were incubated as described above forthe Xask cultures. After the incubation, the cotton plugswere replaced by hermetic stoppers, and 10 mL of gas fromthe headspace of each vial was replaced by 10 mL of acety-lene by means of a sterile syringe. The vial cultures were

incubated for 24 h, and ethylene production was evaluatedby injecting 500 �L of the headspace gas into a gas chro-matograph (Tracor Model 570, Tracor Co., USA) equippedwith a Xame ionization detector and a Poropack N column.The detector, injector, and column temperatures of the gaschromatograph 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 to10 mL before GC analysis.

Encystment induction

A bacterial colony of the isolated strain was transferred to50 mL of RM supplemented with 1% (w/v) sucrose andincubated 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 supplementedwith 1% (w/v) sucrose, and cultivated until the exponentialphase of growth was reached (usually 24 h). The whole cul-ture was then centrifuged and the pellet obtained waswashed once with phosphate buVer (pH 7.4), suspended in50 mL of RM containing n-butyl alcohol (0.2%, v/v), andincubated for 120 h at 28°C on an orbital shaker at 150 rpmfor 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 reportedby Campos et al. (1996), but using RM supplemented withsucrose. The cells resistant to desiccation were drawn fromstationary-phase bacterial cultures (192 h) grown on RMsupplemented with either kerosene (1%, v/v), sucrose (1%,w/v), or n-butyl alcohol (0.2%, v/v). To count the numberof cysts, 2 mL aliquots of these cultures were Wlteredthrough 0.45 �m pore size Millipore® cellulose membranes(4 cm). The membranes containing the cells were washedwith 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 adding1 mL of mineral RM (without carbon sources) to the plateand agitating it on an orbital shaker at 80 rpm and 28°C for24 h. The suspended cells were plated on solid RM with 1%(w/v) sucrose and the number of colonies was counted after3 days of incubation at 28°C. This number was consideredas the number of cells resistant to desiccation, which alsorepresents the number of cysts in the original culture priorto desiccation. For germination studies, cysts in the mem-

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brane were transferred into fresh liquid RM supplementedwith 1% (w/v) sucrose.

Electron microscopy

The biomass from 50 mL bacterial cultures was harvestedby centrifugation at 10,000 rpm and transferred to 1.5 mLcentrifuge tubes maintained in an ice bath. Each pellet waswashed with refrigerated phosphate buVer solution (pH7.4), and the medium-free pellet was incubated in an icebath 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. Thepellet 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 andsequentially 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 wasresuspended in propylene oxide three times for 10 mineach. The pellet was then suspended in Spurr 60 cp resin(Polysciences, Inc.) and incubated at 60°C for 3 days topolymerize the resin. Ultra-thin sections were cut with aReichert-Jung ultramicrotome and contrasted for 30 minwith 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, andobserved by TEM with a JEM 2000 EX (JEOL Inc.) micro-scope.

Statistics

Data represent the mean and standard deviation of threereplicates. Excel software was used for statistical analysis.

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). Aspart of the biochemical characterization, the isolated strainwas 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 aVectedby kerosene as the sole carbon source, as compared to cul-tures grown on sucrose (Fig. 1). The speciWc growth rate ofcultures on sucrose (� = 0.097 h¡1; Td = 10.3 h) was aboutten times higher than that of those on kerosene(� = 0.016 h¡1; Td = 63.8 h). The speciWc growth rate forcultures on sucrose was considerably lower than thosereported 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 valuereported for A. chroococum (� = 0.17 h¡1) growing undernitrogen-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 ofthe exponential phase of growth: the maximum nitrogenaseactivity in the cultures was observed at the end of the expo-nential phase of growth, showing a rapid increase at thebeginning of the exponential growth, and a drastic decline

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

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at the end of the exponential phase. However, in cultureswith sucrose the cellular growth ended when no nitrogenaseactivity was detected, whereas in cultures with kerosene thebiomass continued slowly growing throughout the time(8 days) with a corresponding kerosene intake after thenitrogenase activity was no longer detected. These nitroge-nase activity results with cultures grown on kerosene areconsistent with previous studies on other free living NFBgrown on carbon sources other than carbohydrates, such asAzomonas 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 ina mix-culture with Pseudomonas or Bacillus with lightcrude oil as the sole carbon source showed a growth-associ-ated nitrogenase activity pattern throughout the wholekinetics of growth (Onwurah 1999). The nitrogenase activ-ity pattern was also diVerent from those displayed by Kleb-siella pneumoniae and Enterobacter sp. grown on sucroseand malate, respectively (Haahtela et al. 1983), which werenot detected during the lag phase, increased during theexponential growth, reaching the highest activity duringearly stationary phase, and then declined drastically toundetectable values during the stationary phase. Thus, cul-tures grown on kerosene seem to Wx nitrogen but do notaccumulate biomass, leading the metabolic Xux to cellmaintenance and the biosynthesis of carbon storagereserves, as will be discussed later.

Encystment on kerosene

In contrast to cultures of the isolated A. nigricans grown onsucrose, those grown on kerosene showed gradual changesin 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 ofthe genus Azotobacter, the polymorphic cells showed thepresence of PHB central granules as a prelude to the devel-opment of cysts (Mulder and Brotonegoro 1974; Espín2003; Copeland et al. 2006). On the other hand, during theexponential and linear growth of cultures with sucrose, thecell population showed a mixture of the typical vegetativeovoid (1 �m diameter) to large bacillary shape (1 �mwide £ 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 thelate exponential phase of cultures with kerosene, the vege-tative cells were gradually transformed to encapsulatedovoid shapes showing abundant peripheral bodies (Fig. 2b),and during the linear phase of growth one or two centralPHB granules were typical in most of the cell population(Fig. 2c,d). Similar morphological changes have beenreported to be induced by the replacement of glucose orsucrose with butyl alcohol in A. vinelandii cultures (Wysset al. 1961; SadoV 1975). Interestingly, the peripheral bod-ies were particularly abundant during the exponential phaseof growth, when the nitrogenase activity was high (5–6 h)and the presence of PHB granules in the cells was relativelyinsigniWcant (Fig. 2b). It should be noted that as the size ofthe PHB granules and the space between the cell membraneand the cytoplasm increased (Fig. 2d), the nitrogenaseactivity decreased (Fig. 1b); this trend continued until thePHB granules reached a maximum size (700–800 nm) andthe nitrogenase activity was no longer detected in the cul-tures (after approximately 24 h). These observations areconsistent with the hypotheses that the peripheral bodiesmight be related to the nitrogen Wxation process (Oppen-heim and Marcus 1970) and that PHB is an energy and carbonintracellular storage polymer (Kadouri et al. 2003). However,

Table 1 DiVerential character-istics of the Azotobacter strain used in this work in comparison with other Azotobacter species

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 + + ¡ ¡

Characteristics of Azotobacter species other than the character-istics of that used in this work were taken from Garrity et al. (2005)

D doubt

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the suggestion that a biological function of PHB may be tofacilitate respiratory protection of nitrogenase in the spres-ence of oxygen and the absence of an extracellular carbon

source (Senior et al. 1972; Espín 2003; Copeland et al.2006) is not supported by these results, since a reverse rela-tionship between the PHB accumulation and nitrogenase

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

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activity was evident and the PHB accumulation was higherwhen the nitrogenase activity was undetectable. On thecontrary, the pattern for growth on kerosene is consistentwith that reported by Senior et al. (1972) for A. beijerinckiicultures grown on excess glucose, which, like kerosene inthis study, was not completely consumed; 25% of the initialkerosene remained after 8 days of culture. Also similar toA. beijerinckii cultures, both growth and PHB accumulationwere sustained throughout the time without attaining a truestationary phase. However, in the present study withA. nigricans, extending the culture time to 8 days undernitrogen-Wxing conditions resulted in the appearance ofcysts with intracellular bunches of PHB granules afterabout 96 h, ultimately leading to the encystment of thewhole cell population after 192 h (Fig. 3a). However, fromthe resistance to desiccation test, only 64.7% of the cellpopulation eventually became mature cysts. These maturecysts were microscopically identical to those induced byn-butyl alcohol in cells collected from cultures grown onsucrose, which produced 74.4% of mature cysts (Fig. 3b).This ratio of mature cysts is similar or lower than thatreported for A. vinelandii grown on glucose and inducedwith n-butyl alcohol (Lin and SadoV 1968; Reusch andSadoV 1983). Since it is well known that alcohols such asn-butanol and isopropanol can induce the encystment pro-cesses in several bacteria, and that alcohols are metabolicintermediates in the catabolism of hydrocarbons (Watkin-son and Morgan 1990), it is not surprising that extracellularcarbon sources other than carbohydrates can induce theencystment process.

Furthermore, the microscopic features of the maturecysts (Fig. 3) and their germinating cystic cells (Fig. 4)showed by A. nigricans in this study were similar to thosereported by several researchers for A. vinelandii (Wysset al. 1961; SadoV 1975; Reusch and SadoV 1983; Seguraet al. 2003), and for A. chroococum (Pal et al. 1997). Themature cysts (Fig. 3) were composed of a central body con-sisting of the protoplasm, a cell membrane, a thin cell wall,and central PHB granules, surrounded by a coat with thetypical capsule-like structure reported for cysts of otherspecies of the genus Azotobacter (Garrity et al. 2005). Aspointed out above, the coat is made up of intine and exinelayers, and perhaps owing to the hardness and elasticity ofthese polymers, they remained in the germinating cyst untilit bore vegetative cells (Fig. 4), producing germinatingmulticelled cysts with multiple central bodies, all sur-rounded by unbroken residual exine and intine layers. Itshould be noted that the cysts contain only one central bodybefore the germination process starts (Fig. 3a). After 8 h ofculture in fresh medium, the intine layer in the multicelledcysts was almost undetectable. This observation supportsthe view expressed in the literature that intine may functionnot only as a storage material in cysts, but also as an energy

and carbon source in facilitating the germination, enlargement,and division of cells in multicelled cysts (Cagle and Vela1971). Similar germinating multicelled cysts originatingfrom cysts with only one central body have been reportedfor A. vinelandii (Wyss et al. 1961; Cagle and Vela 1971)and for R. centenum (Berleman and Bauer 2004). However,in these studies, it was suggested that vegetative cells mightdivide inside the cysts prior to rupture of the exine layer,probably due to a failure of the cyst to delay chromosomereplication (Berleman and Bauer 2004). Actually, why and

Fig. 4 Germinating cysts with multiple central bodies of young vege-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 RMsupplemented with 1% sucrose. Typically, the multicelled germinatingcysts showed up to three central bodies surrounded by unbroken resid-ual exine (ex) and intine (in) layers. Bars represent 200 nm

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how cell division occurs inside the cysts remains uncertainand deserves further investigation.

In summary, the mature cysts that developed in culturesof A. nigricans with kerosene as the sole carbon sourceunder nitrogen-Wxing conditions were able to regeneratevegetative cells with physiological characteristics identicalto those of their parent cells. To the best of our knowledge,this is the Wrst report of the encystment of an Azotobacterspecies in cultures using kerosene as the sole carbonsource.

Acknowledgments Special acknowledgment is given to VictoriaTeresita Velásquez Martínez and Ma. De Lourdes Rojas for their tech-nical assistance in the microscopic studies. The Wrst author, GabrielaGarcia Esquivel, acknowledges a doctoral fellowship (124460) fromCONACyT.

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