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Brazilian Journal of Microbiology

Print version ISSN 1517-8382On-line version ISSN 1678-4405

Braz. J. Microbiol. vol.31 no.3 São Paulo July/Sept. 2000 



Heloiza R. Barbosa*; Daniela Strauss Thuler; Márcia Aiko Shirakawa; Natália R. S. Miyasaka

Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brasil.

Submitted: February 14, 2000; Returned to authors for corrections: May 30, 2000; Approved: August 30, 2000




The interactions between the nitrogen–fixing microorganism Beijerinckia derxii with two non-diazotrophic bacteria, either Escherichia coli or a facultative sulphur-oxidizing chemolitotroph, were studied in mixed cultures. Direct and indirect contact between B. derxii and E. coli were tested. B. derxii increased CFU numbers and/or maintained the viability of the non-diazotrophic bacteria, but neither growth nor nitrogenase activity of the nitrogen-fixing bacterium were affected by either partner.

Key words: Beijerinckia derxii, coculture, viability.




Mixed cultures of microorganisms are suitable systems for studying the interactions between organisms and their impact on the environment and may open up new perspectives that could lead to new discoveries. Some mixed cultures containing N2- fixing bacteria provide conditions more suitable for N2 fixation than pure cultures. For example, Halsall (10) showed that the nitrogenase activity of Beijerinckia indica B15 was stimulated by coinoculation with Cyathus stercoreus both in axenic culture and in native soil. An extremely unlikely association is the mixed culture of Azospirillum brasilense Cd with the non-N2-fixing marine mangrove rhizosphere bacterium Staplylococcus sp, which increased the N2 fixation of the former (11). Benefits to both partners were observed in a coculture of Bacillus and Azospirillum, where pectin degradation by Bacillus and N2 fixation by Azospirillum were enhanced (13). Studies on mixed cultures generally emphasize the effects of the non-diazothophic on N2-fixing bacteria, mainly with respect to nitrogenase activity. However, little is known about the effects of diazotrophs on the growth and maintenance of viability of compatible partners. Tsuchiya et al (22) and Trivedi and Tsuchiya (21) concluded that the association of a nitrogen-fixing organism from the genus Beijerinckia with the leaching bacterium Thiobacillus ferrooxidans enhanced the rate and extent of copper, nickel and ore leaching, indicating that nitrogen fixation can be important in bioextractive metallurgy.

In this study the N2-fixing microorganism Beijerinckia derxii, frequently found in Brazilian acid soils (8) was cocultured with two non-N2-fixing bacteria, Escherichia coli or a facultative sulphur-oxidizing chemolitotroph, in order to understand some of the ecological roles of free-living N2-fixing bacteria.




The bacterial strains used were: (a) Beijerinckia derxii ICB10, a free-living N2-fixing bacterium isolated from soil under cerrado (1) and identified by biochemical and morphological tests (4); (b) Escherichia coli ICB19, isolated from human feces; (c) a facultative chemolitotrophic sulphur-oxidizing bacterium isolated from garden soil in the city of São Paulo and named Bacterium C. Although E. coli is an unlikely natural partner of B. derxii, it was chosen because it is unable to fix N2 and is a non-fastidious bacterium. B. derxii grows in a simple medium composed of glucose and mineral salts. Bacterium C, unable to fix N2, was chosen because it is a soil microorganism with a metabolism quite different from that of the other two bacteria studied

Culture media

The following media were used: Nutrient Broth (NB), Nutrient Agar (NA) and mineral media which compositions are specified in Table 1.



Isolation of the facultative sulphur-oxidizing chemolitotroph bacterium

Medium S6 (12), containing Na2S2O3 as energy source and CO2 as carbon source, was used to isolate the facultative sulphur-oxidizing chemolitotroph bacterium. One gram of soil was suspended in 10 ml of sterile distilled water and shaken at 300 rpm for 30 minutes. 2ml of this suspension were inoculated in 20 ml of S6 liquid medium to which 0.0018 g.l–1 phenol red was added and incubated at 30°C with shaking (200 rpm). When the color of the medium changed to yellow, indicating a drop in the pH of the medium, 2 ml of the culture were transferred to fresh liquid medium. After 5 consecutive transferences, a sample was inoculated onto S6 (12) solid medium for isolation of colonies. The neutrophilic, autotrophic facultative sulphur bacterium recovered was not properly classified because ordinary tests were not sufficient to meet its complex identification requirements.

Cocultures assays

Bacterial interactions were tested in mixed cultures as specified in Table 2.



Preparation of cocultures

Coculture I was prepared as follows: - pure cultures of B. derxii (in LGb medium) and E. coli (in NB medium) were grown for 74h at 30°C in a rotary shaker (200 rpm). E. coli cells were centrifuged to discard the medium; the pellet was washed 3 times and ressuspended in LGb medium at a concentration of 1.8 x 108–1. 15 ml of the E. coli suspension was mixed with 150 ml of a B. derxii culture (contained in a 500 ml Erlenmeyer flask). In coculture II, pure cultures of B. derxii and E. coli were prepared as in coculture I. The suspension of E. coli was inoculated in a 500 ml Erlenmeyer flask containing 100 ml of LGb medium, so as to reach about 2.4 x 104 or 1.8 x 107–1. A sterile dialysis tubing containing 8 ml of the B. derxii pure culture was introduced into the E. coli suspension. This system was set according to Reporter (18). In coculture III, the B. derxii pure culture was prepared as in coculture I. The Bacterium C pure culture, grown in S6 liquid (12) medium for 120h at 30°C, with shaking (200 rpm) was centrifuged; the pellet was washed 3 times and ressuspended in LGc medium. About 5 x 108–1 (0.1 ml) of this suspension was mixed to 100 ml of B. derxii pure culture grown for 74 hours. Coculture III was incubated at 30°C with shaking (200 rpm). Still pure cultures of B. derxii and E. coli in LGb were used as controls of cocultures I and II; shaken pure cultures of B. derxii and Bacterium C in LGc were used as controls of coculture III.

Analytical Procedures

Samples of the three mixed cultures and controls were taken periodically for enumeration of Colony Forming Units (CFU) by the drop method (2). Diluted suspensions of pure B. derxii, E. coli and Bacterium C cultures were plated on solid LGa (16), NA and S6 medium, respectively. The dilutions of cocultures I and II were plated on both LGa and NA medium and those of coculture III on both LGa and S6 media. Six replicates of each dilution were plated.

The concentration of ammonia the supernatants of coculture I was determined according to Chaney and Marbach (5).

Nitrogenase activity of cocultures I and III was evaluated by the acetylene reduction method (23).

E. coli cell protein content in coculture II was measured by the method of Lowry et al. (15). Protein concentration was performed in samples collected periodically from the E. coli culture grown outside from the dialysis bag.

The presence of extracellular proteins in supernatants of cocultures I and II as well as in B. derxii and E. coli pure cultures was tested running the samples in a SDS polyacrylamide electrophoresis gel (PAGE) (14); the gels were silver stained (Merck AG. Darmstadt).



The growth curves in N-free medium (coculture I, Fig. 1b) show that E. coli in pure culture at 1.8x107–1 was practically unable to multiply during the first 24 hours. Following this period, a constant drop in CFU values was observed, with a 91.2% reduction to 340h. Conversely the association with B. derxii enabled E. coli to gradually increase in numbers (CFU values corresponding to 2 generations of growth) but mainly prevented loss of cell viability.


Figure 1 - Growth curves of B. derxii (a) and E. coli (b) in pure and mixed cultures in direct contact. (n B. derxii in pure culture; —oB. derxii in mixed culture; l E. coli in pure culture; —mE. coli in mixed culture)


When E. coli cells were cocultured with B. derxii separated by a dialysis membrane (coculture II) two different initial concentrations of E. coli were used . When the initial CFU value was 1.7 x 107 ml–1, i.e. similar to coculture I, this number reached, after 24 hours, 2.7 x 107 ml–1 in pure culture and 3.1 x 107 ml–1 in mixed culture; after 72 hours, the corresponding concentrations were 2.3 x 107 ml–1 and 3.0 x 107 ml–1. Following this time, the pure population entered in decline whereas cells in coculture kept their viability (data not shown). By using a lower initial CFU value (2.8 x 104 ml–1), it was possible to see that both mixed and pure cultures increased in number (Fig. 2) but the rate of E. coli growth was slightly enhanced in the mixed culture. This multiplication probably occurred because of contamination of the medium with traces of combined nitrogen, which could have been assimilated by the bacteria. After this period, while E. coli cells viability in pure culture began to decline, in the mixed culture it remained stable, indicating, again, that the presence of B. derxii enhanced E. coli survival. The data shown in Fig. 2 indicated that in coculture II the higher influence of B. derxii was in the maintenance of viability after exponential growth phase.


Figure 2 - Growth curves of E. coli in pure and mixed cultures in indirect contact.
(l CFU E. coli in pure culture; —m— CFU E. coli in mixed culture; u E. coli cell protein in pure culture; —o—-E. coli cell protein in mixed culture)


Fig. 2 indicates that E. coli’s cell protein content increased at the exponential phase, in parallel with increasing CFU values; it also shows that there was no difference in protein concentration between cells grown in pure culture and those in mixed culture. The similarity between results may be explained because the protein measurement does not discriminate between alive and dead cells present in the pure culture. The advantage of coculture II was that it enabled the determination of a single partner’s cell protein content in a mixed population. In coculture III (Fig. 3) the presence of B. derxii enhanced Bacterium C to multiply and reach a CFU value that corresponds to 12 generations of growth. In pure culture, the growth of Bacterium C by 6 generations, was, probably for the same reason that did E. coli in coculture II. The specific growth rate of Bacterium C in mixed culture (0.05514) was twice as high as that in pure culture (0.02559). The effect of B. derxii on Bacterium C multiplication was higher than that observed on E. coli multiplication (Fig. 1 and 3).


Figure 3 - Growth curves of B. derxii (a) and Bacterium C (b) in pure and mixed cultures in direct contact (n B. derxii in pure culture; —oB. derxii in mixed culture; Bacterium C in pure culture; —D— Bacterium C in mixed culture).


The results were negative regarding the presence of ammonia in culture supernatants, at least for concentrations above 0.5–1. SDS gel electrophoresis of supernatants from pure cultures and cocultures I and II revealed that no proteins were present. Neither E. coli nor Bacterium C did affect B. derxii growth (Fig. 1 and 3) or nitrogenase activity (Fig. 4).


Figure 4 - Specific nitrogenase activity curves of B. derxii in pure (n) and in mixed culture (o) with Bacterium C (coculture III), evaluated by acetylene reduction method.



In the present study the effects of B. derxii on its partners can not be ascribed to the excretion of both proteins and ammonia once the results were negative for these analysis. These results are in contrast to Narula et al. (17) who demonstrated the presence of ammonia in the culture media of several species of the free-living Azotobacter isolated from soils of India. Few papers in the literature deal with the increase of nitrogenated substances in co-cultures of different microoganisms. Cohjo et al. (6) showed that the total protein accumulation of Acetobacter diazotrophicus, an endophytic N2-fixing bacterium, was 25% higher when in mixed culture with Lipomyces konononkoae, as compared with pure cultures. However, the excretion of nitrogenated substances by this N2-fixing microorganism was not directly demonstrated.

Enhanced cell multiplication of the non-diazotrophic microorganism was observed in cocultures I and III (Fig. 1b and 3b) but in coculture II, the E.coli population was less stimulated (Fig. 2). This fact suggests that the dialysis tube retained substance(s) with MW higher than 50 KDa that could contribute to the non-diazotrophic multiplication. The similar E. coli cell protein concentration in both coculture II and pure culture indicates that no nitrogenated material, liberated by B. derxii was available to be assimilated by E. coli, increasing its biomass.

The observed maintenance in CFU numbers of non-diazotrophs in the cocultures may be explained by the diazotrophic bacterium secreting hormone-like substances into the medium. These may be nitrogenated substances, necessary in a very low concentration (7) so that this nitrogen does not influence biomass. A preliminary result showed that B. derxii is able to excrete indoleacetic acid (IAA) in the culture media supplemented with 0.5 g.l–1 tryptophan (20). In the present work this IAA precursor was always absent.

Substances like cytokinin (19) and indole-acetic acid (24) have been detected in the culture media of different diazotrophic bacteria. These substances are known to promote plant growth (9). The influence of phytohormone-like substances on microorganisms was studied by Barea et al. (3), who proposed a method by which the concentrations of auxins, kinetin and gibberelic acid could be determined using yeast cells. However, there is little information about their effects on bacteria.

In the present study, the effect of B. derxii on multiplication and maintenance of viability of both E. coli and Bacterium C co-cultures, indicated that the former produces substance(s) that promote proliferation and/or increased viability of bacteria. The extension of this effects may vary depending on the partners. While Bacterium C had its growth enhanced, E. coli had both its multiplication stimulated and its viability preserved in mixed cultures. A less marked effect on cell growth was that of Beijerinckia lacticogenes, which caused an increase of only 2 generations in Thiobacillus ferrooxidans, a strict sulphur-oxidizing chemolitotroph cocultured as reported by Tsuchiya (22).

In the present paper, it was observed that B. derxii in coculture with E. coli or a facultative sulphur-oxidizing chemolitotroph bacterium was not affected by the partners yet had a positive influence on these non-N2-fixing microorganisms. Even taking into account that the results were obtained under laboratory conditions, it can still be considered that B. derxii may also influence different soil microorganisms in the natural environment.



The authors would like to thank to Dr Beny Spira for his critical review of the manuscript, to Paula Rodrigues Abreu for the technical assistence and to CNPq (Conselho Nacional de Pesquisa) for the PIBIC fellowship.




Beijerinckia derxii estimula a viabilidade de bactérias não fixadoras de N2 em meio sem nitrogênio

Co-culturas com a bactéria fixadora de nitrogênio Beijerinckia derxii e duas bactérias não diazotróficas (Escherichia coli e uma bactéria quimiolitotrófica facultativa oxidante de enxofre) foram empregadas para o estudo de interações bacterianas. B. derxii foi colocada em contato direto e indireto com E. coli. Um aumento no número de UFC e a manutenção da viabilidade das não diazotróficas foi promovido por B. derxii, porém seu crescimento e atividade da nitrogenase não foram afetadas por nenhuma das parceiras.

Palavras-chave: Beijerinckia derxii, co-cultura, viabilidade.




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* Corresponding author. Mailing address: Departamento de Microbiologia, ICB – USP, Av. Prof. Lineu Prestes, 1374, Cidade Universitária, CEP 05508-900, São Paulo, SP, Brasil. E-mail:

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