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Vol. 163, No. 2
Copyright C 1985, American Society for Microbiology
Flocculation in Azospirillum brasilense and Azospirillum lipoferum: Exopolysaccharides and Cyst Formation
LAKSHMI SADASIVAN AND CARLOS A. NEYRA* and Microbiology, New Jersey Agricultural Experiment Station, Cook College, Rutgers Department of Biochemistry University, New Brunswick, New Jersey 08903 Received 25 February 1985/Accepted 22 May 1985
The phenomena of flocculation and floc formation by Azospirillum brasilense Sp7 (ATCC 29145) and Azospirillum lipoferum Sp59b (ATCC 29707) were studied in aerobic liquid cultures. Carbon sources representative of various entry pathways in combination with various nitrogen sources induced flocculation in both species of azospirilla. Noticeably, the combination of fructose and nitrate was the most effective in terms of floc yields. Phase-contrast microscopic observations revealed a transition in cell morphology from freely motile, vibrioid cells to nonmotile, highly refractile encysting forms during the formation of flocs. The nonmotile forms in flocs appeared to be entangled within a fibrillar matrix, and the cells were highly resistant to desiccation. Dried flocs kept for almost 6 months still tnaintained the highly refractile encysting forms, and their viability was confirmed by pellicle formation and acetylene reduction in semisolid malate medium. Electron microscopic observations of the desiccated flocs revealed the presence of cell forms containing abundant poly p-hydroxybutyrate granules within a central body and surrounded by a thick layer of exopolysaccharides. The latter were characterized by alkali and acid digestion, crude cellulase hydrolysis, and calcofluor staining. It was concluded that the overproduction of exocellular polymers induces the flocculent growth and is associated with the concomitant transformation of vegetative cells to the desiccation-resistant encysting forins under limiting cultural conditions.
Azospirillum spp. have been shown to be a common inhabitant of tropical and temperate soils and have also been shown to reduce acetylene in culture media and in association with the roots of forage grasses, cereals, and other plants (11, 19, 26). A. brasilense and A. lipoferum are known to grow well in culture media on a variety of carbon sources, including various sugars and organic acids (15, 24, 30, 32). These organisms, under aerobic conditions, also grow well on a variety of combined nitrogen sources, including ammonia, nitrate, or amino acids (20, 21, 22, 24). Recently, special attention has been given to the use and metabolism of fructose by strains of A. brasilense and A. lipoferum grown with N2 or NH4Cl as the nitrogen source (4, 9, 15, 32). During recent investigations in our laboratory on the various carbon sources for nitrate-dependent growth by azospirilla, we observed intense flocculation in media containing fructose and nitrate. A medium containing KNO3 and glucose has been reported to enhance exopolysaccharide synthesis in certain soil bacteria (14). Also, flocculation in liquid culture medium has been reported for several gramnegative bacteria, but azospirilla were not included in those studies (5, 8, 16). Thus, this research was undertaken to study the following: (i) the optimization of cultural conditions to achieve maximum flocculation, (ii) the interaction betWeen carbon and nitrogen sources to bring about flocculation, and (iii) the ultrastricture and chemical characterization of the flocs. (In this paper, "floc formation" refers to the aggregation of cells during growth, and "flocculation" refers to the clumping of cells in a turbid suspension.) MATERIALS AND METHODS Bacterial strains and culture media. A. brasilense Sp7 (ATCC 29145) and A. lipoferum Sp59b (ATCC 29707) were used throughout this study. These strains were maintained on
nutrient agar (Difco Laboratories, Detroit, Mich.) slants at
300C. Minimal salts medium, described as medium A by Neyra and Van Berkum (21), was prepared as follows to avoid precipitation. Salts other than organic carbon, inorganic nitrogen, or phosphate salts for 1 liter of medium were
dissolved in 10 ml of distilled water and autoclaved separately. After cooling, it was added to 940 ml of sterile KPO4 buffer (100 mM; pH 6.8). Organic acids and sugars (at a concentration range between 8 and 100 mM) as the carbon source and various inorganic nitrogen sources (viz., NH4Cl, KNO3, and glutamate) at concentration ranges between 0.5 and 20 mM were filter sterilized and added individually as required for various experiments. The final volume of the medium was then made up to 1 liter. Inoculum for all experiments was harvested from a logphase culture grown in nutrient broth (NB; Difco) by centrifugation at 5,000 rpm for 10 min at 40C, washed three times with equal volumes of 100 mM KPO4 buffer (pH 6.8), and inoculated into test medium to an initial optical density at 660 nm (OD6w) of '0.1 for floc formation experiments and >0.2 to 0.4 for flocculation experiments. (An OD6w of 0.8 corresponds to 5 x 109 CFU/ml for both strains.) Experiments were conducted either in 250-ml conical flasks containing 100 ml of media or in large batch cultures with 5-liter bottles containing 4 liters of media. Small flask cultures were incubated on a Gyrotory shaker (New Brunswick Scientific Co., Inc., New Brunswick, N.J.) at 200 rpm and kept in a chamber with temperature control at 34° C. The large batch cultures were incubated with vigorous air sparging at 34° C in a water bath. Phase-contrast and fluorescence microscopy. Flocs were allowed to develop by flocculation experiments in minimal salts medium containing 8 mM fructose and 0.5 mM KNO3. After 72 h, the supematant was decanted, and the settled flocs were washed several times with distilled water and
0 z 0r. 1. Later. to which was added 20 U of crude cellulase (EC 3.2. Optimization of media condition for floc formation and flocculation. 1985 EXOPOLYSACCHARIDES AND CYST FORMATION IN AZOSPIRILLA 717 21. The net weight of flocs was obtained by filtering the flocs through filter paper (Whatman no. 163. Flocs were never observed in NB cultures. or 8 mM fructose (A) (B).) and incubated at 45° C for 12 h.5 and 2 h. N. dehydrated in a series of graded ethyl alcohol solutions. A small piece of the dried floc was transferred to the semisolid nitrogen-free malate medium (6) and incubated at 34° C for 24 to 48 h to observe growth. the inoculum. (i) Floc formation. Louis. The NaOHinsoluble residue was suspended in 2 ml of 0.1. pellicle formation. 1) and weighing after the paper and flocs were air-dried for 30 min. 1A).. The dry flocs of A. Stained preparations were examined under a long-wavelength UV lamp and Nikon epifluorescence microscope with a UV excitation filter at 330 to 380 nm. the cells did not grow to the log phase in a medium containing fructose and nitrate. Instead. The residue (NaOH-insoluble fraction) was further treated with 5 ml of 0. Ultrastructural studies of desiccated flocs. Chemical characterization of floes. brasilense Sp7 (ATCC 29145) in NB cultures provided with (0) or without (0) 5 mM KNO3 (A) or minimal salts media containing 5 mM KNO3 and 40 mM malate (0). flocculated immediately within 2 to 4 h. RESULTS Cultural conditions for floc formation and flocculation. Sigma Chemical Co.4) from Trichoderma viridie (type V. The phenomenon of flocculation also occurs in the case of Azospirillum cultures.66 N HCl for 2 h at 100° C to eliminate noncellulosic glucans (5). Viability test and phase-contrast microscopy of desiccated flocs. 1B). Growth curves and floc formation of A. Similar growth patterns were also obtained after transferring washed NB-grown cells into a medium containing 40 mM malate as the sole carbon source and 5 mM KNO3 as the nitrogen source to an initial ODw0 of <0. 1B). Ultrathin sections were cut in an ultramicrotome and observed under a transmission electron microscope (Siemen model 1A Elmiscope). brasilense and A. 100 mM) for 10 min.. The remaining residue was treated with 2 ml of 6 N HCl for complete hydrolysis. Cultures of A. ItI <1. and finally embedded in Spurr plastic as described by Cole and Popkin (3). the inoculum grew only up to a period of 6 h and after reaching an OD6w of 0.2 / 0 12 2 4 6811012 TIME (h) FIG.2. When the washed cells of a log-phase culture grown in NB was transferred to a minimal medium containing (8 mM) fructose and (0.8. the flocs were dried in a desiccator oven at 80° C for 2 h. lipoferum were also photographed under a phase-contrast microscope after imbibition of the flocs in KPO4 buffer (pH 6. The reducing sugars released were measured by the colorimetric method of Nelson-Somogyi (18). 2A). In the case of malate and KNO3 medium.5 mM KNO3 were also recorded. dry weights of the insoluble fractions of 1 N NaOH and 0. For the dry weight measurements. followed by 1% osmium tetroxide fixation for 10 h. However. 27 mM fructose (A).66 N HCl hydrolysis of the flocs obtained from a 5-liter culture in 8 mM fructose-0.1 (Fig. Fresh flocs were stained with 0. The dried flocs obtained as described above were stored in a closed vial at room temperature (30° C) for up to 6 months. and acetylene reduction. showed normal growth kinetics. instead of growing. trace quantities of flocs began to appear after 10 to 20 h of incubation.Y. (ii) Flocculation. A 500-mg (fresh weight) sample of 72-h-old flocs was treated with 10 ml of 1 N NaOH for 1 h at 100° C to dissolve the cellular proteins and nucleic acids (5). (i) Alkali and acid treatment.5 mM) KNO3 to an initial turbidity of >0. leaving behind a clear supernatant (Fig. St. Floc formation was only observed in the fructose treatments.VOL. 024 681 photographed either directly on a petri dish or under a Nikon phase-contrast microscope. the cells started aggregating. Pearl River. The conditions for floc formation and flocculation were initially optimized by visual assessment of the flocs .2. (I) <0.025% calcofluor (American Cyanamid Co.5). The doubling time during the log phase of the cultures was between 1. The turbidity (absorbance at 660 nm) increased in a sigmoidal fashion as the time of incubation progressed (Fig.05 M sodium citrate buffer (pH 4. Quantification of flocs. (ii) Cellulase digestion of cellulosic material. The NB-grown cells were pelleted and stained with calcofluor to serve as the control.) which yields fluorescence staining of cellulose and other P-linked polysaccharides (16). brasilense (ATCC 29145). 4 mM malate (0). and no further increase in turbidity could be measured due to interference by the flocs (Fig. Mo. The dried flocs were fixed in 1% glutaraldehyde for 4 h. growing in NB with or without nitrate.
b Symbols: (-) no growth. Figure 2B is a close-up of the flocs. A medium containing KNO3 yielded more flocs than did any other nitrogen source. bacterial cells were inoculated into a large batch aerobic culture containing 8 mM fructose and 0. In the floc formation experiments. lipoferum yielded similar types of flocs. brasilense and A. a representative from the organic acids (malate) and one from sugars (fructose) were chosen to study the effects of various concentrations of carbon sources on floc formation and flocculation. and (+ + + +) excellent. and 20 mM. Floc formation and flocculations were also compared in a medium with 8 mM fructose and increasing concentrations of KNO3 ranging between 0. flocs began to appear after the OD660 or absorbance at 660 nm of the culture medium increased to ca. and the cells were vibrioid with impaired motility and receding cytoplasm. Increasing the fructose and KNO3 concentrations to 40 and 20 mM. Phase-contrast microscopy of developing flocs. Malate Fructose A. The massive flocculation of A. which is about the optimum pH at which azospirilla grow. _ ++++ ++++ +++ ++ 8 ++ +++ + +++ 20 ++ + +++ 40 ++ +++ +++ +++ 100 from the organic acids (malate) and one from sugars aA representative (fructose) were chosen for this study. Table 3 gives the effects of nitrogen sources on floc formation and flocculation. It was observed that all concentrations (. 15 mM.5 mM KNO3. Floc formation and flocculation in A. (+ + +) good. Effect of various concentrations of carbona sources in minimal salts medium on floc formation and flocculation in A. lipoferum - A. (+ +) fair.5 mM) as the nitrogen source Effect of: Concn (mM) A. (+) little floc. brasilense NB + + Malate +++ +++ Gluconate period of 6 to 8 h. lipoferum with KNO3 (0. in minimal salts medium. (+ +) fair. Flocs also appeared when the concentration of fructose was increased to 100 mM. appearing in the media containing various concentrations of carbon and nitrogen sources and by measuring the pH of the medium. respectively. lipoferum (ATCC 29707) in minimal salts medium containing fructose (8 mM) and KNO3 (0. BACTERIOL. indicating growth. The floc yield was greatly reduced when NH4Cl was used in combination with malate. 2A.4 (acidic) and yielded less flocs than did a medium with NH4NO3 whose pH remained between 6. whereas fructose and nitrate yielded maximum flocculation. and 20 mM) induced flocculation within 2 h of incubation at 34° C.s5 mM. The formation of flocs did not appear to be pH dependent. 6. 10 mM. a-ketoglutarate Fructose Glucose ++++ ++ ++++ +++ a Filter-sterilized carbon sources were added at 20 mM C. resulted in more turbidity in the supernatant.718 SADASIVAN AND NEYRA J. brasilense and A. Floc yields were later quantified by measuring the fresh and dry weights of the flocs obtained through flocculation with 8 mM malate and fructose as carbon sources and 5 mM NH4Cl lacking nitrate and KNO3 as nitrogen sources (Table 4). since a medium supplemented with NH4Cl decreased in pH to between 5. and (+ + + +) excellent. lipoferum A.5 mM) as the nitrogen source Floc formation and flocculation in b: Carbon source" A.4 in a ca. In a second experiment. Symbols: (-) no flocs. lipoferum in different carbon sources and KNO3 (0.5 mM). Both A. in a medium lacking carbon. .1. Sugars (fructose and glucose) were found to yield more flocs than the organic acids (Table 1). 2. The microscopic appearance of the vegetative cells was found to TABLE 2. (A) Flocculation of A.5 and 20 mM. 15. In a TABLE 1. brasilense 0 lipoferum brasilense A. however.9 and 6. although flocs were also present. A B FIG. the inoculum remained suspended freely. maximum settling of the flocs occurred at <5 mM. brasilense and A. (B) The macroscopic appearance of the flocs is more clearly presented after separation from the rest of culture contents and placement on a petri plate. showing the slimy and curdlike aggregation. 0. Carbon source concentrations of 8 mM yielded better flocs in fructose medium than in malate medium (Table 2). at 10.9 and 7. (+ + +) good. For this experiment.to 8-h period. There were no significant pattern differences in floc formation or flocculation by changing the concentrations of carbon sources. Macroscopic appearance of flocs. However. there was turbidity along with flocs. (+) little floc. lipoferum (ATCC 29707) and its tendency to settle down is clearly evident in Fig.
3D). brasilense and A. The typical subsurface pellicle formed was found to reduce acetylene to ethylene. Effect of carbona and nitrogen sources on floc yields after flocculation by A. The vegetative cells look entrapped within a fibrillar mesh.9) (8. (C) Forty-eight-hour-old flocs showing a more compact mesh containing polymorphic cells. b Numbers in parentheses are the pHs of the medium after 24 h at 34° C.5 4.18 TR TR 3. Thus.5) (6.4).9) + + + (7.4) 2.2 to 0. TABLE 3. 3). and patterns of aggregation also appear to change with culture age under conditions conducive to flocculation. Desiccated flocs maintained for up to 6 months were found to be viable after transfer into semisolid malate medium and incubation for 48 h at 37° C. FIG. brasilense + + + + + + (8. brasilense were also photographed. (D) One-week-old flocs showing highly refractile cells.2 6. 3. .2 7. and KNO3 or NH4C1 was the nitrogen source at 5 mM in minimal salts medium.1) + + (7. Flocs observed after 1 week of culture were arranged as a very compact aggregate of refractile cells. even after hard pressing between slide and cover slip.64 TR TR 0. Symbols: (+) little flock. 0. TR. change upon flocculation (Fig. 3A). 10 . lipoferum Fructose + NH4Cl Fructose + KNO3 TR 0.025 A. The dry flocs under a phase-contrast microscope revealed the presence of encysting forms which were still highly refractile (Fig.7 0. lipoferum + NH4Cl KNO3 NH4NO3 Glutamate a (5.0 TR TR Malate + NH4Cl Malate + KNO3 Nitrogen sources were added at 5 mM N.15 0. lost their characteristic motility and assumed a more oval to spherical shape within a period of 2 to 4 h (Fig. 163. Flocs of A. Traces. The cells (absorbance at 660 nm.2 7.0 6. (A) Vegetative cells from 24-h-old NB cultures. a Fructose or malate was added as the carbon source at 8 mM. Bar. lipoferum (ATCC 29707) were taken Similar observations in A. a very different configuration from that observed with fresh flocs 24 h after inoculation or transfer (Fig.6) + + (7.5 mM). 3B and 3C). cell types.2 8. (+ +) good. lipoferum Floc yield (g/liter)b Treatment Fresh wt Dry wt Culture pH A.5 7. 1985 EXOPOLYSACCHARIDES AND CYST FORMATION IN AZOSPIRILLA 719 at different stages during the flocculation process. metabolic activity.5 6. 4A and B). and (+ + +) excellent.5 mM).VOL. b Flocs were harvested for weight determinations ater 24 h. Effect of various nitrogen sources on floc formation and flocculation with fructose (8 mM) as the carbon source in minimal salts medium Nitrogen sourcea Floc formation and flocculation inb: A. brasilense Fructose +NH4Cl Fructose + KNO3 2.2) + (6. when transferred from NB to minimal medium containing fructose (8 mM) and KNO3 (0.2 1. (B) Aggregation of cells 24 h after transfer to minimal salts medium containing fructose (8 mM) and KNO3 (0.5 Malate + NH4Cl Malate + KNO3 A. Cells grown in NB appeared as actively motile curved rods and did not form any kind of aggregation (Fig. Microscopic characteristics of flocs as seen under a phase-contrast microscope.7 5.9) Viability and phase-contrast microscopy of desiccated flocs. These nonmotile cells appeared to be entangled in a mesh of fibrillar material which was not easily separable. The refractile forms were obTABLE 4.um.
were expected to contain the exocellular polysaccharides. We have provided evidence for the cultural conditions required to consistently bring about massive flocculation yielding exocellular polymers in liquid cultures of these bacteria. and the ultrastructures of A.66 N HCI at 100° C for 2 h possibly removed all a-glucans and left granular and fibrillar residues whose dry weights were 2 and 4 mg/5-liter culture of A. 5a. brasilense and A. 10 . d. The NB-grown cells did not fluoresce after staining with calcofluor (data not shown). (ii) Cellulase digestion of alkali-insoluble fraction. 5b. leaving behind reducing sugars and trace amounts of precipitates which could be completely hydrolyzed in 6 N HCl. brasilense (ATCC 29145) and A. brasilense and A. 21). served to be embedded inside a honeycomb-like network of extracellular matrix (Fig. 4C. (i) Alkali and acid hydrolysis. Table 5 gives the quantities of reducing sugars released during the cellulase digestion as measured by the Nelson-Somogyi method (18). when stained with 0. Ultrastructure of desiccated flocs. Chemical characteristics of flocs. with minimal quantities of refractile granules which are reported to be poly f-hydroxybutyrate (PHB) (23). lipoferum. 5e and d. respectively. (iii) Calcofluor staining of wet flocs. These forms are very different from vegetative cells in the properties of nonmotility and enlargement into a round. The treatment of NaOH-insoluble fractions with 0. The soluble fraction was presumed to contain proteins and nucleic acids. 4. The encysting forms of A. Highly refractile encysting forms of A. arrows). lipoferum (ATCC 29707). 13. [A. The central body is surrounded by thick capsular material. lipoferum are shown in Fig. This residue was completely hydrolyzed by treatment with 6 N HCI. Treatment of flocs with 1 N NaOH yielded two fractions. respectively. respectively. Ultrathin sections of the dry flocs (Fig. It was also observed that crude cellulase failed to disperse untreated flocs. exhibited fluorescence in a UV lamp. brasilense (A) and A. 72-h-old floc. c. Our attempts . BACTERIOL. 5a and b) are vibrioid. lipoferum. 5e and f) show a network of extracellular matrix tightly holding together the encysting cell forms. lipoferum (B). indicating that cellulose was not the sole component involved in flocculation but that other polysaccharides were involved. and e. one NaOH soluble and one NaOH insoluble. I FIG.025% calcofluor dye.720 SADASIVAN AND NEYRA J. and the insoluble fractions. brasilense are shown in Fig. Figure 4D shows the caste of the material after the central resting cell was removed by gentle tapping. and the same was later confirmed with an epifluorescence microscope. (D) Mold of the extracellular material after the encysting forms were gently tapped out. The vegetative cells (Fig. the insoluble NaOH residue did disperse significantly but not completely. which were 300 and 600 mg (dry weight)/5 liter of A.um. lipoferum revealed similar structures to those shown in (C) and (D)]. The ultrastructures of A. Bar. Malate has been the preferred carbon source for nitratedependent growth of these bacteria (2. To complement our chemical characterization of flocs. However. and f. The highly refractile central bodies and the transformed vegetative cells in the floc are shown in Fig. central body with abundant PHB granules. brasilense (C) are seen embedded within a matrix (arrows). Phase-contrast microscopic observation of the dried flocs after 6 months of desiccation. DISCUSSION The results presented throughout this report have shown conclusively the phenomena of floc formation and flocculation in cultures of A.
(1) were considered to be active. showing a change in cell morphology to round forms with abundant PHB granules and surrounded by a thick layer of polysaccharide.. Balston.). the cysts are the transformed vegetative cells containing abundant PHB granules with a high degree of resistance to desiccation (27. d. Bar. & R.It . c. we have shown that the flocs were resistant to desiccation for up to 6 months and are still viable. to use fructose instead of malate failed to produce a normal growth proffle in Azospirillurn cultures in a nitrate-containing medium (see above). 0. 31).. 28). (e and f) Ultrastructures of the desiccated flocs.5 p. The exines of the Azotobacter cysts are known to be composed of basic units of polysaccharides complexed with divalent cations (7.VTOL. (c and d) Single encysting cells from the desiccated flocs after 6 months. Like Azotobacter spp. A minimal medium containing fructose and KNO3 favors floc formation and flocculation in A. and e) and A..38 b After 12. Encysted forms of . hence. 5. 1985 EXOPOLYSACCHARIDES AND CYST FORMATION IN AZOSPIRILLA 721 I.5 mM KNO3) and ultrastructurally are similar to the dry flocs reported in this paper. showing the compact aggregations of encysting forms within a mesh of exopolysaccharides. lipoferum cultures and is better than malate and NH4Cl. The capsular forms (C forms) of azospirilla reported by Berg et al. 163. lipoferum (b. Hydrolysis of the NaOH (1 N)-insoluble fraction by crude cellulase Cellulose substrate (mg) enzymes (U) Amt of Reducing sugars produced (mg)a Pure cellulose (10)b Pure cellulose (10)b 1 N NaOH residue (500) 1 N NaOH residue (500) a 20 0 20 0 0 0. but they are believed to be connected with the production of exocellular polymers.. 28). azospirilla have also been isolated from 15-year-old dry soil samples (10. Whatman cellulose powder (W.- FIG. (a and b) Vibrioid rods with few PHB granules. The C forms were not believed to be resting structures due to the lack of characteristic exines and intines of a mature cyst as described in Azobacter spp.. particularly n-linked polysaccharides. Ultrastructure of vegetative cells and desiccated flocs of A. brasilense and A.h at 45C. brasilense (a. and f). nitrogen-fixing cells modified to protect the 02-sensitive nitrogenase enzyme to facilitate nitrogen fixation (1). By definition.m. no desiccation experiments were conducted (1). The capsule formation and the production of exocellular polysaccharides are the prerequisites for the development of a mature cyst (27).a mmb dwomm. TABLE 5. The differential effects on flocculation by diverse carbon and nitrogen sources are not fully understood at present. Ltd.5 0 1. However. These C forms were obtained on a limiting carbon and nitrogen medium (2 g/liter concentration of malate and 0.
Newton and C. 1980. 12. Magalhaes. S. which is supported by state and U. R. Catabolism of carbohydrates and organic acids and expression of nitrogenase by azospirilla. Mishra. and A.. Carotenoid composition and function in nitrogen-fixing bacteria of the genus Azospirillum. Microbiol. Y. 145:583-595. Chem. Nelson. 87:684-689. and J. K. and N. and D. M. 1984. N.. Phillips (ed. K. 159:86-92. N. 1981. M. p. This. for allowing us the use of microscopic facilities for the phase-contrast and epifluorescence microscopy. In L. Neyra.). on the induction of cyst formation. Tyler. Electron microscopy. and Y. 1981. . 1966.S. Martinez-Drets. Characterization and cyst production of azospirilla isolated from selected grasses growing in New Jersey and New York. Microbiol. Dazzo. In this report. ment of nitrogenase activity. Formation of cellulose fibrils by gram-negative bacteria and their role in bacterial flocculation. However. Steinitz. There are reports on Azotobacter spp. A photometric adaptation of the Somogyi method for the determination of glucose. S.C. and E. Thus. J. 25. P. Nitrate and nitrite reductase negative mutants of N2-fixing Azospirillum sp. N. Gen. 22. J. 1977. E. S. R. Das. Washington. 39:642-649. Department of Soils and Crops. Holm'es. Stephan. R. Friedman. J. C. Wyss. 11. M... C. Factors affecting growth and nitrogen fixation of Spirillum lipoferum. Rutgers University. P. and R. 1977. 21. the lack of development of exine and intine had been reported in Azotobacter spp. V. Utilization of fructose by Azospirillum brasilense.. Deinema. Manual of methods for general bacteriology. C. 14. We also thank R.. J. 2. M.. was supported by the New Jersey Agricultural Experiment Station. M. 20.. D-01204-01-85. K. A. Zh. Arch. and 0. N. Layne. V. and J. N. Microbiol. M. Knowles. E.. Johnson. J. D. A.. Burris (ed.. D.. thereby. Bacteriol. 1981. The physiology of Azospirillum in relation to its utilization as inoculum for promoting growth in plants. and C. E. Bacteriol. Agron. J. Mikrobiol. Microbiol. Synthesis and monosaccharide composition of exopolysaccharides in certain soil bacteria. New York. however. is a visiting scientist and postdoctoral fellow supported partially by funds provided by the New Jersey Agricultural Experiment Station. 9 10. Washington State University Press. H. Elsevier Science Publishing Inc. Microbiol. Pullman. 04400 on rhizosphere research.. Matthysse. Subba Rao. 17.. 122:27-32. Dobereiner. In W. Appl. Napoli. C. Van Berkum. 29:1213-1217. Cole. A. A. and D. Elaboration of cellulose fibrils by Agrobacterium tumefaciens during attachment to carrot cells. upon transfer from complete nutrient medium to a nutritionally minimal medium. Establishment and survival of Azospirillum lipoferum. 1978. Can. azospirilla obtained by various cultural conditions are found to be resistant to desiccation from several hours to 1 month (11. 1982. Neyra. J. Costilow. may be a matter of identifying the specific chemical agents and cultural conditions for their induction. Berg. Bothe. 23:300-305. H. M. C. K. the nature of aggregation and adsorption.. Can. 16. Laski. and L. 1984. Organic acid utilization. 78:42-52. Environ. J. E. R. 1983. 1969. Dobereiner. 44:33-36. R. 153:375-380. Sci.. 518-538. Structure of exocellular polymers and their relationship to bacterial flocculation. Day. W. T. 6. Y. Rutgers University. 1964. 1971. Okon. Bacteriol. A. B. Neyra. then the aggregated cells might shift their metabolic activity from encystation to germination and adsorption of the roots. S. 165-174. and the International Agriculture and Food Programs at Cook College. 1977. 92:1828-1830. In a liquid culture medium. Appl. L.. Bacteriol. M. Nester. R. J. American Society for Microbiology. Pfister. A. C. 25). Microbiol. Indian Acad. Bacteriol. and J. L. Nitrogen fixation in grasses. H. Microbiol. Burris. 27:1320-1325. B. depending upon the carbon and nitrogen source (27). J. 23. the induction of exine formation was achieved by the addition of the flocculating divalent cations such as CaNO3 (29). Proceedings of the First International Symposium on Nitrogen Fixation. Associative symbiosis in tropical grasses: characterization of microorganisms and dinitrogen-fixing sites. Burpee. J.. This paper also demonstrates that azospirilla can be induced to produce extracellular polysaccharides by manipulating the culture medium. Gallo. P. 30: 123-131. Tilak. Goebel. Vasil. V. Fructose catabolism in Azospirillum brasilense and Azospirillum lipoferum. Y. F. 117:247-252. Neyra. Okon. 5. We thank Alice Montana. and R. LITERATURE CITED 1. Okon.. Bacteriol. 15. Can. Remsen.. Werner. 1977. Albrecht. Hatch Act funds. G. Popkin. K. 1984. 1981. the lack of observation of a definite exine and intine in Azospirillum sp. Neyra. B. and Y. 4. and R. Partial support was also provided by the New Jersey Agricultural Experiment Station project no. 19. ACKNOWLEDGMENTS We thank Robert L. L. Lakshiui.g. In P. Kreig. G. and thus it will enable future researchers to identify and screen for strains varying in their exopolysaccharides and. and J. J. G. J. Natural factors involved in the induction of cyst formation in Azotobacter. 18.. a microaerophilic condition and presence of suitable carbon and nitrogen sources in the form of root exudates. E. immediately undergo metabolic changes and synthesize the polysaccharides which are necessary for encystation during desiccating conditions. Department of Biochemistry and Microbiology. M. Wood. Dugan. Pope. B. Can. Madison. R. C.. Microbiol. Hubbel. and G. Lam'm. The report here of the desiccation-resistant forms in flocs suggests that the bacteria. Y.. Microbiol. J. publication no. 1975. R. Gerhardt. Nitrate reduction and nitrogenase activity in Spirillum lipoferum. Satyanarayana Rao. H. Nur. Also. J.S. L. (12). is yet to be confirmed. J. 1976. 159:80-85.. I. Denitrification by N2-fixing Spirillum lipoferum. and P. Henis. Burris. J. Dobereiner. A. and R. Novick. 34-51. J. A. R. R. M. Gurlitz. Adv. Relationship of encapsulation and encystment in Azotobacter. Arch. Proceedings of the Fourteenth Steinbock Symposium at the University of Wisconsin. Dobereiner. H. B 86:397-404. Rutgers University. BACTERIOL. Klein. and I. V.722 SADASIVAN AND NEYRA J. V"ayalakshmi. Madeline Mendoza. p. H. 1981. M. J. Biol. Microbiol. M. 1976. A. 127:1248-1254. A. Bacteriol. 1982. Murray. Nyman (ed. Biology of Azospirillum-sugarcane association: enhance24. J. 1944. Proc. and N. Production of cellulose microfibrils by Rhizobium. Lalande. e. Zevenhuizen. Papen. 17). Sect. M. 13. Tate III. B. Maltseva. 8. 98:1328-1334. 29:1-38. E. and C. M. 7. 3. we have shown that the polysaccharides may also be playing a role in the survival mechanism of these bacteria under stress conditions such as desiccation and nutritional limitation. Transformations of inorganic nitrogen by Azospirillum spp. p. W. Ludden and J. and T. R.. G. 130:96-100.). 23:306-310. Dobereiner. It may be possible that if the conditions in nature in the rhizosphere were favorable for infection. Lakshmi Kumari. Eklund. extracellular polymers such as polysaccharides and cellulose microfibrils in flocs have been reported to play a possible role in the infection process in the rhizosphere and cell suspension cultures of carrots by causing aggregation of the infecting bacterial cells (16. Kreig. Vasil. and Sylvia Taylor for assistance in the preparation of this manuscript. Triemer and Laura Wood for their collaboration and technical assistance in the electron microscopic studies at Nelson Biological Laboratories. W. C. Arch. This work.).
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