Artigo Upload
Artigo Upload
Artigo Upload
ISSN 1517-8382
Submitted: November 16, 2000; Returned to authors for corrections: May 28, 2001; Approved: November 21, 2001
ABSTRACT
A strictly aerobic, thermophilic, spore-forming bacteria was isolated from a soil sample collected in Campos
dos Goytacazes City, Rio de Janeiro, Brazil. The cells of this organism were Gram-positive, catalase positive,
actively motile, 1.2 µm wide and 5.3 µm long. Growth occurred at pH values ranging from 6.5 to 9.0, and
optimum growth occurred at about pH 7.0. The optimum temperature for growth was around 55ºC, and the
upper temperature limit for growth was around 70ºC. Yeast extract stimulated growth on glucose and was
obligately required at supraoptimal temperatures (65ºC). The results of 16S rRNA sequence comparisons
indicated that the isolate was closely related to Bacillus caldoxylolyticus and Bacillus sp strain AK1, and
these three organisms exhibited levels of ribossomal DNA sequence homology of 94%.
* Corresponding author. Mailing address: Laboratório de Tecnologia de Alimentos/CCTA, Universidade Estadual do Norte Fluminense; Av. Alberto
Lamego, 2000. 28015-620, Campos dos Goytacazes, RJ, Brasil. FAX (+5524)726-3873. E-mail: meire@uenf.br
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16S rRNA sequence analysis 44ºC or above 70ºC. The strain had a broad pH range for growth
The genomic DNA was extracted from the isolate and the 6.5 – 9.0 with an optimum of 7.0 (Fig. 1).
amplification of the 16S rDNA was performed through PCR The following sugars were fermented with acid production:
technique. The purified PCR products were sequenced on an ribose, D-glucose, D-fructose, D-mannose, cellobiose, amygdalin,
automated DNA sequencer (ALFexpress, Pharmacia). The 16S esculin, salicin, N-acetyl-D-glucosaminemaltose, trehalose, starch,
rDNA sequence of isolate was aligned with the 16S rRNA gene glycogen, gentiobiose, D-Galactose, Lactose, sucrose, melibiose,
sequences of various members of the genus Bacillus obtained glycerol, L-xylose, β-m-xylosideo, β-gentiobiose, α-methyl-D-
from the Ribosomal RNA Database Project and from Genbank glucoside, D-turanose, D-mannitol and xylitol. It was not observed
(10). Matrices of evolutionary distances were computed from acid production in Erythritol, D- and L-arabinose, adonitol, dulcitol,
the sequences alignments by calculating a pair wise Jukes- rhamnose, inositol, sorbitol, α-methyl-D-mannoside, arbutin,
Cantor (9). From these distances, phylogenetic trees were inulin, D-raffinose, D-lyxose, D-tagatose, D- and L-fucose, D-
inferred by a neighbour-joining method (16). and L-arabitol, gluconate, 2-keto-gluconate and 5-keto-gluconate.
The strain grew in Nutrient Broth containing 5.0% NaCl
Growth conditions (m/v), but was inhibited by 7.0% NaCl. Urea was not attacked;
The organism was grown on TSY agar plates for 12h at 55ºC. catalase was positive. Indole was not formed and acetoin
Approximately 5 mL of the liquid medium containing (g/L of formation was positive. Nitrate was reduced to nitrite.
distilled water): NaH2PO4.2H2O, 1.56; NH4Cl, 5.35; KCl, 0.745; When the organism was grown at 55ºC in the absence of
Na2SO4.10H2O, 0.644; Citric acid, 0.42; MgCl2.6H2O, 0.25; CaCl2, glucose but in the presence of yeast extract in the liquid
2.2x10-3; ZnO, 2.5x10-3; FeCl3.6H2O, 2.7x10-2; MnCl2.4H2O, 1.0x medium, good growth was obtained, indicating that yeast
10-2; CuCl2.2H2O, 8.5x10-4; CoCl2.6H2O, 2.4x10-3 ; NiCl3.6H2O, extract can be used as one sole carbon and energy source.
2.5x10-4; H2BO3, 3.0x10-4; Na2MoO4, 1.0x10-3, pH 4.0 was pipetted Also, good growth was observed when gelatine or casein
into these agar plates and the cells scraped off using a sterile replaced yeast extract.
Pasteur pipette. The same liquid medium (50 mL contained in a
250 mL tightly closed Erlenmeyer flask) was inoculated with this Effect of temperature on growth
suspension to give an initial absorbance at 470 nm of at least 0.1 Growth obtained in the liquid medium supplemented with yeast
and the cultures were incubated at temperatures starting at 50ºC extract (0.5%) at 50ºC, 55ºC, 60ºC and 65ºC is shown in Fig. 2.
and going up to 65ºC, with vigorous aeration in a shaker at 250 It can be seen that the temperature determined the rate of
rpm. The pH was adjusted to 7.0 and the medium sterilised by growth. The shortest lag was observed at 55ºC and the longest
autoclaving at 121ºC for 30min. Glucose was sterilised separately at 50ºC. The highest turbidity was reached in cultures growing
and aseptically added to the flasks containing the liquid medium, at the temperature of 55ºC.
after cooling. At time intervals, the turbidity of the cultures was At 65ºC, addition of peptone (1%) to the medium caused a
determined by measuring the increase in optical density at 470 further increase in growth, inducing higher growth rate.
nm with a spectrophotometer Hitachi Model U-2000. However, a small amount of growth was observed in the absence
of yeast extract or peptone when glucose was used as an energy
RESULTS source (Fig. 3).
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Characteristics of thermophilic Bacillus
1.50 2.0
Optical density (470nm)
(a) (b)
1.5
1.00
1.0
0.50
0.5
0.00 0.0
0.0 2.0 4.0 6.0 0.0 2.0 4.0 6.0
Time (h) Time (h)
Optical density (470nm)
1.4 1.4
1.2 (c) 1.2 (d)
1.0 1.0
0.8 0.8
0.6 0.6
0.4 0.4
0.2 0.2
0.0 0.0
0.0 2.0 4.0 6.0 0.0 2.0 4.0 6.0
Time (h) Time (h)
Figure 2. Growth curve of the endospore forming thermophile cultivated in minimal medium supplemented with yeast extract
(0.5%) at 50ºC (a), 55ºC (b), 60ºC (c) and 65ºC.
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A.N. de Souza and M.L.L. Martins
Isolate
B. thermocatenulatus is a facultatively anaerobic bacterium 0.01
that does not hydrolyse starch and gelatine. It does not produce
acid from xylose and lactose; acetoin is not formed. The Figure 4. Phylogenetic dendrogram based on 16SrDNA
temperature range for growth of this organism is 35ºC – 78ºC. B. sequence data indicating the position of the thermophile isolate
flavothermus is also a facultatively anaerobic bacterium that among representatives of the genus Bacillus. All the sequences
does not hydrolyse gelatine and grows at 30 o C. B. used in the analysis were obtained from the Ribosomal Database
thermoleovorans does not form acetoin, degrades urea and Project and Genbank. Scale bar indicates evolutionary distance.
hydrolyses casein weakly. Therefore, the isolate could not be
identified as one of these species.
Another phylogenetically closely related species to the
isolate is B. thermodenitrificans, which belongs to the “B. thermophilic microorganisms have shown that some of them
stearothermophilus” complex. However, the isolate was had additional requirements as the incubation temperature
separated by a numerical study from typical strains of B. was increased and others showed no differences in growth
stearothermophilus (21). requirements, regardless of the incubation temperature
The strain Bacillus sp. AK1 was the bacteria most closely (6,24).
related to the isolate for which 16S rRNA sequence data is The thermophile isolate required yeast extract to grow at
available. On the other hand, the phylogenetically most closely supraoptimal temperatures (65ºC), suggesting that at higher
related to Bacillus sp AK1 species is B. thermoglucosidasius temperatures the enzymes responsible for the synthesis of a
(23). However, the isolate showed that levels of similarity to particular metabolite present in this compound underwent
this organism ranged from 90% to 91%. thermal inactivation and thus the organism required it as an
On the basis of morphological, biochemical, physiological exogenous source. An alternative explanation is that the
and phylogenetic studies we concluded that the isolate is complete inactivation of a gene responsible for the synthesis
closely related to the members of the Bacillus rRNA group 5. of an essential metabolite imposed upon the organism a
However, even taking all data into account, it is not possible requirement of an external source of the substance which it
to identify the isolate as being one of the above mentioned could no longer produce.
species. Indeed, this approach led to the classification of the
isolate as a thermophilic member of the genus Bacillus where ACKNOWLEDGEMENTS
all aerobic, endospore-forming organisms have been classified.
Reports in the literature dealing with the effect of We thank the FAPERJ for financial assistance and the André
temperature on the growth requirements of several Tosello Foundation for the 16S rRNA sequence studies.
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Characteristics of thermophilic Bacillus
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