Received 6 September 2005; received in revised form 31 October 2005; accepted 10 November 2005
Abstract
Four thermophilic strains were isolated by thermophilic treatment (2 days at 55 ◦ C) of the stevia-powder, and were identified by the sequence
analysis of the 16S rRNA gene; Ureibacillus thermosphaericus (FERM P-20039), Bacillus thermoamylovorans-1, B. thermoamylovorans-2 and
Thermoactinomyces candidus. Since all of them have nitrate-reducing and ammonium-forming ability, it is highly possible that they can first
produce nitrous acid from nitrate followed by the generation of ammonium. Only U. thermosphaericus had significantly large growing ability in
the medium contained 1000 ppm of “Lannate® -45 water lenitive” (carbamate insecticide) and 400 ppm of “Ortran® ” (organophosphorus insecticide)
compared with the same concentration-level in the contrast medium without adding the pesticide.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Thermophilic bacteria; Nitrate-reducing ability; Pesticide-tolerant; Lactic acid bacteria; Stevia-powder
0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2005.11.034
408 K. Okamoto, N. Satou / Enzyme and Microbial Technology 39 (2006) 407–413
to 9 ml of the same medium (the first dilution). 0.1 ml of each stepwise 2.5. Thermophilic bacteria’s degradation of substrates
diluted suspension from the first dilution was incubated at 55 ◦ C for 2 days
in the same medium included 0.5% skip jack extract and 1.5% agar. Repeated The degradation ability of substrates by the thermophilic nitrate-reducing
the incubation on the same medium, the thermophilic bacteria were iso- bacteria was examined by applying each substrate to the minimum nutrient
lated [3] and were identified by the sequence analysis of the 16S rRNA medium (contrast medium). Hydrolysis of gelatin, lipid and starch was measured
gene. in 3% tryptic soy broth agar supplemented with 1% (w/v) gelatin, 2% (w/v) egg
yolk, 1% (w/v) soluble starch, respectively. 1% (w/v) casein with agar was
2.1.2. Acid-producing bacteria used for the hydrolysis of casein. Digestion of meat-block was examined in the
One milliliter of the fermented hot-water extract of stevia was sus- cooked-meat (OXOID) agar [3].
pended in saline (the first dilution). 1.0 ml of each stepwise diluted suspen-
sion from the first dilution was incubated at 30 ◦ C for 3 days in the GYP 2.6. Pesticide tolerance
medium (glucose, 10 g; yeast extract, 5 g; peptone, 5 g; sodium acetate, 2 g;
tween80, 0.5 ml; MgSO4 ·7H2 O, 20 mg; MnSO4 ·4H2 O, 1 mg; FeSO4 ·7H2 O, The growth rate of the thermophilic or acid-producing bacteria were inves-
1 mg; FeSO4 ·4H2 O 1 mg, NaCl 1 mg; made up to 1 l with distilled water, tigated in the culture medium contained 0.1–1000 ppm of “Lannate® -45 water
pH 6.8) included 1.0% calcium carbonate and 1.5% agar covered with the lenitive” (Sankyo Co. Ltd., Japan (contained 45% methomyl), carbamate insec-
1.5% agar medium added a slight of sodium azide. Repeated the incuba- ticide), 0.4–4000 ppm of “Ortran® ” (Sumika-Takeda Co. Ltd., Japan (con-
tion on the same medium without sodium azide, the acid-producing bacteria tained 15% acephate), organophosphorus insecticide), 0.1–100 ppm of “Trebon”
were isolated and were identified by the sequence analysis of the 16S rRNA (Sankyo Co. Ltd., Japan (contained 20% ethofenprox), synthesized pyrethroid
gene. insecticide) and 0.1–100 ppm of “Kelthane” (Takeda Co. Ltd., Japan (con-
tained 40% kelthane) organochlorine insecticide) compared with the same
2.2. 16S rDNA sequence determination and phylogenetic analysis concentration-level in the contrast medium without adding the pesticide.
Genomic DNA extraction, PCR-mediated amplification of 16S rDNA and 2.7. Detection of the thermophilic nitrate-reducing bacteria in the
sequencing of the PCR products were carried out as described [4]. TESK
stevia-compost using the specific primers
buffer was used for genomic DNA extraction [5]. PCR-mediated amplifica-
tions were performed with common primers [6] to 16S rDNA of eubacteria
2.7.1. Design of specific primers
sequences of which were as follows: 16S27F, GAGTTTGATCCTGGCTCAG;
We examined whether four identified thermophilic nitrate-reducing
16S1544R, AGAAAGGAGGTGATC CAGCC, and Takara ExTaq (Takara Shu-
bacteria in the stevia-powder can be detected in the stevia-compost with
zou) as DNA polymerase. The PCR-products were purified with ExoSAP-IT
using the specific primers which were designed based on specific partial
(Amersham), determined the sequence with Amersham ThermoSequencing
sequences of 16S rDNA of four each bacterium. The sequences of the
kit (Amersham) and analyzed by an automatic sequencer; RISA-384 DNA
specific primers were as flows: Ureibacillus thermosphaericus (FERM
Sequencer (Shimadzu Corp. Kyoto). The BLAST programs were applied in
P-20039)—forward: 5 -ACATCAAAGTGCATGCT-3 , U. thermosphaericus
order to search the sequences similarity in 16S rDNA databases. Sequences were
(FERM P-20039)—reverse: 5 -GTGCAGCCAGTTACTACT-3 , Bacillus
then aligned with a CLASTAL W [7] and phylogenetic trees were drawn after
thermoamylovorans-1 and B. thermoamylovorans-2—forward: 5 -GCTTTTG-
distances had been determined by neighbor-joining algorithm [8] using the same
CCATCACTTACA-3 , B. thermoamylovorans-1 and B. thermoamylovorans-
software.
2—reverse: 5 -GTACCGGCATTTCCTCCGAT-3 , Thermoactinomyces candi-
dus—forward: 5 -ATGGGGAAAAGGGAAA-3 , T. candidus—reverse:
2.3. Biochemical and physiological properties 5 -TGAGTACCGTCAACCTT-3 .
was GC-341f and 534r, sequences of which were as follows: GC-341f, 5 - 3. Results and discussion
CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCTAC-
GGGAGGCAGCAG-3 ; 534r, 5 -ATTACCGCGGCTGCTGG-3 . The temper-
3.1. Phenotypic properties
ature-programs were same as the conditions described in Section 2.7.
Table 1
Growth temperature range of the thermophilic strains from the stevia-powder
Temperature (◦ C) Stain
SP1 SP2 SP3 SP4
30 − − + − + − −
35 + + + + + + +
40 + + 2+ 2+ 2+ 2+ +
45 + + 2+ 2+ 3+ 3+ 2+
50 2+ 2+ 2+ 2+ 2+ 2+ 2+
55 3+ 3+ 2+ 2+ 2+ 2+ 3+
60 2+ 2+ 3+ 3+ 2+ 2+ 3+
Fig. 1. Growth-ability of lactic acid bacteria isolated from the fermented hot-
water extract of stevia-powder with the concentration of glucose. Symbols:
Lactobacillus sp. FJAS 101 (closed circle in bold line); Lactobacillus sp. FJAS
102 (closed square in broken line); Lactobacillus sp. FJAS 201 (closed triangle
in dotted line). Fig. 3. Nitrate reducing ability of Lactobacillus sp. FJAS 101.
K. Okamoto, N. Satou / Enzyme and Microbial Technology 39 (2006) 407–413 411
Fig. 5. Growth rate of U. thermosphaericus (FERM P-20039) in the medium Fig. 7. Growth rate of Lactobacillus sp. FJAS 102 in the medium contained
contained 0.1–1000 ppm of “Lannate® -45 water lenitive”. 0.1–1000 ppm of “Ortran® ”.
412 K. Okamoto, N. Satou / Enzyme and Microbial Technology 39 (2006) 407–413
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