Enzyme and Microbial Technology 39 (2006) 407–413

Isolation, identification and characterization of effective bacteria on

bioremediation from the waste parts of Stevia rebaudiana Berutoni
Keiko Okamoto a,∗ , Naohiko Satou b
a Department of Applied Microbiology, Fukuoka Junior College of Agriculture, 250 Osano, Dazaifu City, Fukuoka 818-0134, Japan
b JBB Stevia Laboratory Ltd., 970-15 Nakao, Midori-ku, Saitama City, Saitama 336-0932, Japan

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

1. Introduction the farm field, there are few publications on biochemical or

The plant, Stevia rebaudiana Bertoni is an economically valu-
able source of sweetening agents because it contains ent-kaurene
diterpenoid glycosides, such as stevioside and rebaudioside [1].
Furthermore after the extraction of the sweetening agent from
S. rebaudiana Bertoni, the waste part such as the leaves and dis-
carded stems are gradually recognized as the important resource
in the farm field (Kagoshima Prefecture in Japan, unpublished
data). Sato et al. (JBB Stevia Laboratory Ltd., Japan) have
being developed various kinds of stevia-materials such as stevia-
powder (derivative fermented products mainly produced from
the stems of S. rebaudiana Bertoni), fermented hot-water extract
of this stevia-powder and the stevia-compost made from cat-
tle feces with this stevia-powder, and they have reported that
nitrate nitrogen and residual pesticides contained in crop plants
are reduced by applying these stevia-materials together (private
report). They have also clarified that the effect of the stevia
powder on the reduction of nitrate nitrogen content in Bras-
sica campestris L. var. Komatuna Makino is stronger than that
of the fermented hot-water extract of the stevia-powder [2].
In spite of many reports on the efficacy of stevia-materials in
∗ Corresponding author.
molecular-biological analysis of the efficacy of stevia-materials.
In the present study, with using the stevia-materials we have
isolated, identified and characterized the key bacteria which
can reduce nitrate nitrogen and residual pesticide containing in
crop plants. Since the temperature at the beginning of the fer-
mentation of the cattle feces with the stevia-powder mixed for
the compost reaches to more than 80 ◦ C, we have focused on
the isolation of the thermophilic nitrate-reducing bacteria from
stevia-powder. We have also tried to ascertain these thermophilic
nitrate-reducing bacteria in stevia-compost. Furthermore, since
the fermented hot waters is acidic (pH 4.3), we have tried to iso-
late acid-producing bacteria from the fermented hot-water. The
isolated thermophilic nitrate-reducing bacteria were character-
ized physiologically and biochemically, then we have examined
the growth rate in the medium of these bacteria after adding
pesticides.
2. Materials and methods
2.1. Isolation and identification of thermophilic or
acid-producing bacteria
2.1.1. Thermophilic bacteria
One gram of the stevia-powder was suspended in 9 ml of 3% tryptic
soy broth (the original solution). 1 ml of this original solution was added

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
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 incubation on the same medium, the thermophilic bacteria were iso-
lated [3] and were identified by the sequence analysis of the 16S rRNA
gene.
2.1.2. Acid-producing bacteria
One milliliter of the fermented hot-water extract of stevia was sus-
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
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,
1 mg; FeSO4 ·4H2 O 1 mg, NaCl 1 mg; made up to 1 l with distilled water,
pH 6.8) included 1.0% calcium carbonate and 1.5% agar covered with the
1.5% agar medium added a slight of sodium azide. Repeated the incuba-
tion on the same medium without sodium azide, the acid-producing bacteria
were isolated and were identified by the sequence analysis of the 16S rRNA
gene.
2.2. 16S rDNA sequence determination and phylogenetic analysis
Genomic DNA extraction, PCR-mediated amplification of 16S rDNA and
sequencing of the PCR products were carried out as described [4]. TESK
buffer was used for genomic DNA extraction [5]. PCR-mediated amplifica-
tions were performed with common primers [6] to 16S rDNA of eubacteria
sequences of which were as follows: 16S27F, GAGTTTGATCCTGGCTCAG;
16S1544R, AGAAAGGAGGTGATC CAGCC, and Takara ExTaq (Takara Shu-
zou) as DNA polymerase. The PCR-products were purified with ExoSAP-IT
(Amersham), determined the sequence with Amersham ThermoSequencing
kit (Amersham) and analyzed by an automatic sequencer; RISA-384 DNA
Sequencer (Shimadzu Corp. Kyoto). The BLAST programs were applied in
order to search the sequences similarity in 16S rDNA databases. Sequences were
then aligned with a CLASTAL W [7] and phylogenetic trees were drawn after
distances had been determined by neighbor-joining algorithm [8] using the same
software.
2.3. Biochemical and physiological properties
The morphological cell was photographed in phase contrast microscope.
Gram staining was done by the conventional method [9].
2.3.1. Thermophilic bacteria
After 24–48 h culture at 55 ◦ C, the presence of catalase and oxidase in the
bacterial colony was examined according to the conventional tests [10]. Changed
the temperature between 30 and 60 ◦ C, the growth ability was examined in 3%
tryptic soy broth included 0.5% skip jack extract and 1.5% agar. In the case for
liquid-type broth (without 1.5% agar), the temperature was changed between 55
and 70 ◦ C.
2.3.2. Acid-producing bacteria
After 24–72 h culture at 30 ◦ C, the presence of catalase in the bacterial
colony was examined [10]. Aerobic gas production from glucose, and pro-
duction of dextran from sucrose were qualitatively examined [11]. The growth
ability of the bacteria was examined with changing glucose between 0% and
20%.
2.4. Reduction of nitrate and nitrite
Being incubated the identified thermophilic or acid-producing bacteria in
the peptone medium including 0.1% KNO3 at 55 or 30 ◦ C for 4 days, nitrite
nitrogen content was measured at every 24 h interval of culture by using the
detection reagents of ␣-naphthylamine and sulfanyl acid [5]. Being incubated
the thermophilic or acid-producing bacteria in the same medium under the same
conditions as in the nitrate reduction experiments, producing ammonium was
detected by using Nessler’s reagent [12].
2.5. Thermophilic bacteria’s degradation of substrates
The degradation ability of substrates by the thermophilic nitrate-reducing
bacteria was examined by applying each substrate to the minimum nutrient
medium (contrast medium). Hydrolysis of gelatin, lipid and starch was measured
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
used for the hydrolysis of casein. Digestion of meat-block was examined in the
cooked-meat (OXOID) agar [3].
2.6. Pesticide tolerance
The growth rate of the thermophilic or acid-producing bacteria were inves-
tigated in the culture medium contained 0.1–1000 ppm of “Lannate® -45 water
lenitive” (Sankyo Co. Ltd., Japan (contained 45% methomyl), carbamate insec-
ticide), 0.4–4000 ppm of “Ortran® ” (Sumika-Takeda Co. Ltd., Japan (con-
tained 15% acephate), organophosphorus insecticide), 0.1–100 ppm of “Trebon”
(Sankyo Co. Ltd., Japan (contained 20% ethofenprox), synthesized pyrethroid
insecticide) and 0.1–100 ppm of “Kelthane” (Takeda Co. Ltd., Japan (con-
tained 40% kelthane) organochlorine insecticide) compared with the same
concentration-level in the contrast medium without adding the pesticide.
2.7. Detection of the thermophilic nitrate-reducing bacteria in the
stevia-compost using the specific primers
2.7.1. Design of specific primers
We examined whether four identified thermophilic nitrate-reducing
bacteria in the stevia-powder can be detected in the stevia-compost with
using the specific primers which were designed based on specific partial
sequences of 16S rDNA of four each bacterium. The sequences of the
specific primers were as flows: Ureibacillus thermosphaericus (FERM
P-20039)—forward: 5 -ACATCAAAGTGCATGCT-3 , U. thermosphaericus
(FERM P-20039)—reverse: 5 -GTGCAGCCAGTTACTACT-3 , Bacillus
thermoamylovorans-1 and B. thermoamylovorans-2—forward: 5 -GCTTTTG-
CCATCACTTACA-3 , B. thermoamylovorans-1 and B. thermoamylovorans-
2—reverse: 5 -GTACCGGCATTTCCTCCGAT-3 , Thermoactinomyces candi-
dus—forward: 5 -ATGGGGAAAAGGGAAA-3 , T. candidus—reverse:
5 -TGAGTACCGTCAACCTT-3 .
2.7.2. DNA extraction
According to Zhou’s method [13], total genomic DNA in the stevia-compost
was extracted from the different microorganisms.
2.7.3. PCR amplification
PCR amplifications were performed in the solution containing 1.5 or 3.0 mM
MgCl2 , 2.5–10 ng of template DNA which was extracted from the stevia-
compost with AmpliTaq Gold DNA polymerase (Applied Biosystems, CA,
USA). The temperature programs was as follows: 7 min at 94 ◦ C and 20 cycles
of 1 min at 94 ◦ C, 1 min at 56–65 ◦ C and 2 min at 72 ◦ C (the annealing tempera-
ture was reduced by 1 ◦ C every 2 cycles), and 15 cycles of 1 min at 94 ◦ C, 1 min
at 55 ◦ C and 72 ◦ C for 2 min, followed by 10 min at 72 ◦ C. The PCR-products
were confirmed by 2% agarose-gel electrophoresis.
2.8. Ascertainment of U. thermosphaericus in the stevia-compost
using denaturing gradient gel electrophoresis (DGGE)
2.8.1. DNA extraction
According to Zhou’s method [13], total genomic DNA in the stevia-compost
was extracted from the different microorganisms, and the extraction of DNA
from U. thermosphaericus (FERM P-20039) was performed by Nagashima’
method [14].
2.8.2. PCR amplification
Prepared DNA was used as temple DNA applying on AmpliTaq Gold
DNA polymerase (Applied Biosystems, CA, USA). The used primer set
 K. Okamoto, N. Satou / Enzyme and Microbial Technology 39 (2006) 407–413 409

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.

3.1.1. Isolation and identification of thermophilic bacteria

2.8.3. DGGE
Four thermophilic strains were isolated from thermophilic
PCR-products were analyzed by DGGE according to Muyzer’s protocol
[15]. DGGE was performed with D-code DGGE complete system (Bio Rad, treatment of the stevia-powder. After 2 days at 55 ◦ C incuba-
CA, USA). These two PCR-products were applied on the gels contained 10% tion on 3% tryptic soy broth included 0.5% skip jack extract
polyacrylamide and a linear gradient of the denaturants urea and formamide, and 1.5% agar plates, strain SP1 and SP2 were round, con-
increasing from 30% at the top of the gel to 60% at the bottom. The elec- vex, creamy-brownish colonies, SP3 was creamy-light brownish
trophoresis was performed in the conditions where 200 V was applied to the
colonies and SP4 had white mycelium. The strain SP1 showed
submerged gel for 3.5 h. After electrophoresis, the gels were stained in an aque-
ous ethidium bromide solution (Syber Green: Takara Bio) and photographed on an opaque brownish film over the plates, SP2 and SP3 presented
a UV (310 nm) transillumination table with CCD camera. transparent film over the plates. The cells of SP1, SP2 and SP3
were rod-shaped. Four strains were able to grow at the tem-
2.9. Measurement of number of U. thermosphaericus in the perature ranging from 35 to 60 ◦ C and the optimal temperature
stevia-compost using competitive- PCR method for growth was 55–60 ◦ C. No growth was observed at 70 ◦ C
(see Table 1). Physiological and biochemical properties of the
2.9.1. Sample DNA extraction strains are summarized in Table 2. The Gram reaction for all
According to Zhou’s method [13], total genomic DNA in the stevia-compost thermophilic strains was positive except for strain SP1. Four
was extracted from the different microorganisms. The extracted DNA was con-
centrated by ethanol and was purified by a GFXTM Genomic Blood Purification
strains showed all oxidase positive, catalase positive (except
Kit (Amersham Pharmacia Biotech, NJ, USA). for the strain SP4), motility and sporulation. Also they have
strictly aerobic metabolism. From the results of the physio-
2.9.2. Design of competitive DNA logical and biochemical characterizations, it was revealed that
Competitive DNA of U. thermosphaericus (FERM P-20039) was prepared three thermophilic strains are similar to the Bacillus, and one
with the specific primer of that bacterium by Competitive DNA Construction strain belongs to the Thermoactinomyces. By the whole or par-
Kit (Takara Bio) followed by PCR amplification. After the purification of PCR- tial sequence analysis of the 16S rRNA gene, it was revealed
product by Purification Kit (Qiagen, Hilden, Germany), the concentration was
that three thermophilic strains similar to the Bacillus (SP1,
adjusted to 102 to 1010 copies by stepwise dilution.
SP2, SP3) are identified to be U. thermosphaericus (FERM P-
2.9.3. Competitive-PCR
Table 2
The 1/100 dilution of the sample DNA and the each dilution (102 to 1010
Physiological and biochemical properties of the thermophilic strains
copies) of competitive DNA of U. thermosphaericus (FERM P-20039) were
used as template DNA applying on AmplliTaq Gold DNA polymerase (Applied Property Strain
Biosystems, CA, USA) with the specific primer of U. thermosphaericus (FERM
P-20039). The temperature-programs were same as the conditions described in SP1 SP2 SP3 SP4
Section 2.7. Amplification products and DNA Ladder (maker for size, Takara Form Bacillus Bacillus Bacillus Coccus
Bio) were analyzed by electrophoresis in 3% agarose gels. After electrophoresis, Gram reaction − + + +
the gels were stained in an aqueous ethidium bromide solution (Syber Green, Presence of spores + + + +
Takara Bio) and photographed on a UV (310 nm) transillumination table with Catalase + + + −
CCD camera. The photographs were scanned, and the image data were down- Oxidase + + + +
loaded into a computer in jpg form. The intensity of beam was measured by NIH Motility + + + +
image soft.

Table 1
Growth temperature range of the thermophilic strains from the stevia-powder
Temperature (◦ C) Stain
SP1 SP2 SP3 SP4

Growth Production Growth Production Growth Production

of film of film of film

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+

Numerals show the qualitative growth ability.

410 K. Okamoto, N. Satou / Enzyme and Microbial Technology 39 (2006) 407–413

20039), B. thermoamylovorans L27478, B. thermoamylovorans

gi|3228362, respectively. The last strain (SP4) belonging to the
Thermoactinomyces was identified to be AF138732 T. candidus
KCTC9557.

3.1.2. Isolation and identification of acid-producing

bacteria
Three acid-producing strains were isolated from the fer-
mented hot-water extract of stevia-powder. By the whole
sequence analysis of 16S rDNA, one strain was identified to
be Lactobacillus sp. FJAS 101 which had only one different
base from 16S rDNA of Lactobacillus pentosus, another strain Fig. 2. Nitrate-reducing ability of the thermophilic strains isolated from
was Lactobacillus sp. FJAS 102 which had 99.8% similarity to the stevia-powder. Symbols: Ureibacillus thermosphaericus (FERM P-20039)
Lactobacillus genomosp. C1, and the last one was Lactobacillus (closed diamond in bold line); Bacillus thermoamylovorans-1 (closed square
sp. FJAS 201 which had 98% similarity to Lactobacillus kefiri. in dotted line); B. thermoamylovorans-2 (closed triangle in broken line); Ther-
Lactobacillus sp. FJAS 201 was, however, closest relative to moactinomyces candidus (closed diamond in broken line).
Lactobacillus buchneri 175019 LBARR16SJ in a phylogenetic
tree. As for the type of lactate fermentation, Lactobacillus sp. Table 3
The ammonium forming ability from potassium nitrate
FJAS 101 was homo-type, Lactobacillus sp. FJAS 102 and Lac-
tobacillus sp. FJAS 201 were hetero-type. The Gram reaction for Strains Cultured day
three strains was positive, and all strains showed catalase neg- 1 2 3 4 5
ative. Fig. 1 represents the growth ability of Lactobacillus sp.
Non-strain − − − − −
FJAS 101 (closed circle in the figure), Lactobacillus sp. FJAS U.t − − 6+ 3+ 7+
102 (closed square in the figure) and Lactobacillus sp. FJAS 201 B.t1 − + 7+ 6+ 5+
(closed triangle in the figure) with the concentration of glucose. B.t2 − − 2+ + 8+
As shown in Fig. 1, since Lactobacillus sp. FJAS 201 (closed T.c + + 3+ + 10+
triangle in the figure) had the same ability of the proliferation in U.t: Ureibacillus thermosphaericus; B.t1: Bacillus thermoamylovorans-1;
20% glucose as that in 1% glucose, Lactobacillus sp. FJAS 201 B.t2: B. thermoamylovorans-2; T.c: Thermoactinomyces candidus; +: positive
seems to be most tolerant against glucose concentration. Both Nessler’s reagent, −: negative Nessler’s reagent.
Lactobacillus sp. FJAS 101 and Lactobacillus sp. FJAS 102 were
able to proliferate in the medium including 15% glucose. These
mophilic strains, whereas U. thermosphaericus (closed diamond
lactic acid bacteria isolated from the fermented hot-water extract
in the figure) showed the lowest. Table 3 shows the ammonium-
of stevia-powder including 10–15% sugar have the tolerance to
forming ability of four thermophilic bacteria with the cultured
high concentration of glucose allowedly.
day. From the third day of culture, 1 day before the phase of
logarithmic death, rapid increase in ammonium-forming ability
3.1.3. Reduction of nitrate and nitrite was observed in all of four thermophilic bacteria, which leads
Fig. 2 shows the nitrate-reducing ability of four thermophilic to our understanding that nitrite-N is produced from nitrate-N
strains. The concentration of produced nitrite-N from nitrate- during the first day of culture, followed by the generation of
N was measured at 540 nm. The nitrate-reducing ability of B. ammonium from the third day of culture which is continued for
thermoamylovorans-1 (closed square in the figure) shows the 3 days. As shown in Fig. 3, Lactobacillus sp. FJAS 101 had also
highest top-peak at the first day of culture among four ther- nitrate-reducing ability, however, which was not accompanied
by the ammonium-production.

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
Fig. 4. Growth rate of U. thermosphaericus (FERM P-20039) in the medium
containing 0.4–4000 ppm of “Ortran® ”.
3.1.4. Degradation of substrates
All strains except U. thermosphaericus (FERM P-20039)
were able to grow on tryptic soy broth and the block of
meat. U. thermosphaericus (FERM P-20039) could not digest
soy bean protein including tryptic soy broth and meat. Since
U. thermosphaericus (FERM P-20039) showed (3+) growth
on the agar medium of tryptic soy broth included 0.5% skip
jack extract (data not shown here), this bacterium can digest
fish meat protein. U. thermosphaericus (FERM P-20039) can
also digest casein protein because of the fact this bacterium
could proliferate on 1% casein medium. All thermophilic
strains except B. thermoamylovorans-2 could hydrolyze starch
weakly.
3.1.5. Pesticide tolerance
Figs. 4 and 5 represent the growth rate of U. thermosphaer-
icus (FERM P-20039) in the medium contained 0.4–4000 ppm
of “Ortran® ” and 0.1–1000 ppm of “Lannate® -45 water leni-
tive”, respectively. Compared with the contrast medium without
adding pesticide (the horizontal line at 100% in the figure),
U. thermosphaericus (FERM P-20039) had significantly larger
growth rate in 4.0–400 ppm of “Ortran® ” and 10–1000 ppm of
“Lannate® -45 water lenitive”. Especially as shown in Fig. 5, it
is notable that the growth rate in 10–1000 ppm of “Lannate® -45
water lenitive” continues to increase with the concentration of
this pesticide. This fact suggests the high possibility that U. ther-
mosphaericus (FERM P-20039) can analyze these pesticides.
Fig. 6 shows the growth rate of Lactobacillus sp. FJAS 201 in the
medium contained 0.1–100 ppm of “Trebon” (closed diamond
Fig. 5. Growth rate of U. thermosphaericus (FERM P-20039) in the medium
contained 0.1–1000 ppm of “Lannate® -45 water lenitive”.
411
Fig. 6. Growth rate of Lactobacillus sp. FJAS 201 in the medium contained
0.1–100 ppm of pesticide. Symbols: “Trebon” (closed diamond in bold line),
“Kelthane” (closed square in broken line), “Lannate® -45 water lenitive” (closed
circle in dotted line).
in the figure), “Kelthane” (closed square) and “Lannate® -45
water lenitive” (closed circle), respectively. The growth abil-
ity of this lactic acid bacterium increases in 0.1–100 ppm of
“Kelthane”, “Lannate® -45 water lenitive” and “Trebon”. Fig. 7
represents the growth rate of Lactobacillus sp. FJAS 102 in the
medium containing 0.1–1000 ppm of “Ortran® ”. The growth
rate of Lactobacillus sp. FJAS 102 increases in 0.1–1000 ppm
of “Ortran® ”. Since it was reported that residual pesticides con-
tained in crop plants are reduced by applying stevia-materials
(private report), it is highly speculated that the isolated U. ther-
mosphaericus (FERM P-20039), Lactobacillus sp. FJAS 201
and Lactobacillus sp. FJAS 102 from stevia-materials contribute
to this reduction.
3.2. Detection of the thermophilic nitrate-reducing bacteria
in the stevia-compost by using the specific primers
Fig. 8 shows the result of agarose gel electrophresis of 16S
rDNA amplification products from DNA of bacterial commu-
nity in stevia-compost by using the specific primers of three
thermophilic bacteria isolated from stevia-powder; U. thermo-
sphaericus (FERM P-20039), B. thermoamylovorans and T.
candidus. As shown in Fig. 8, only amplification product (No.
2 in the figure) by the specific primer of U. thermosphaer-
icus (FERM P-20039) was detected in the stevia-compost.
From this result, in the stevia-compost, the proliferative rate of
Fig. 7. Growth rate of Lactobacillus sp. FJAS 102 in the medium contained
0.1–1000 ppm of “Ortran® ”.
412 K. Okamoto, N. Satou / Enzyme and Microbial Technology 39 (2006) 407–413
Fig. 8. Agarose gel electrophoresis of 16S rDNA amplification products from
DNA of bacterial community in stevia-compost by using the specific primers
of three thermophilic bacteria; U. thermosphaericus (FERM P-20039), B. ther-
moamylovorans, T. candidus, from stevia-powder. Lanes: (1) size maker; (2)
amplification product by the specific primer of U. thermosphaericus (FERM
P-20039); (3) amplification product by the specific primer of B. thermoamylovo-
rans; (4) amplification product by specific primer of T. candidus.
U. thermosphaericus (FERM P-20039) might be higher than
those of other two thermophilic bacteria (B. thermoamylovo-
rans, T. candidus), which leads for U. thermosphaericus (FERM
P-20039) to be the most dominant thermophilic bacterial com-
munity in stevia-compost.
3.3. Ascertainment of U. thermosphaericus (FERM
P-20039) in the stevia-compost using denaturing gradient
gel electrophoresis (DGGE)
In order to validate the fact shown in Fig. 8 that U. ther-
mosphaericus (FERM P-20039) moved to the stevia-compost
from the stevia-powder, DGGE profiles of DNA amplicons
derived from the stevia-compost and from U. thermosphaeri-
cus (FERM P-20039) were compared, result of which is shown
in Fig. 9. The stevia-compost (SC in the figure) showed many
bands according to the migration distance of DNA amplicons
which correspond to the sequences amplified from 16S rRNA
gene of different strains or species. As shown in Fig. 9, the posi-
tion of one band in U. thermosphaericus (FERM P-20039) (U.t)
coincides with band “a” in the stevia-compost. This fact recon-
firms that U. thermosphaericus (FERM P-20039) isolated from
the stevia-powder moved to the stevia-compost. The bands “b”
and “c” in the stevia-compost (SC) in Fig. 9 were identified as
that of Bacillus sp. R-7413 with 100% similarity and that of
uncultured Chloroflexi bacterium with 92.3% similarity in 16S
rDNA sequence, respectively. From these results we can highly
expect that the stevia-compost has the same effect of stevia-
powder, which contributes to the nitrate-reducing ability and the
Fig. 9. DGGE profile (negative image) of PCR amplified 16S rDNA of bac-
terial community in stevia-compost and 16S rDNA of U. thermosphaericus.
SC: stevia-compost, U.t: U. thermosphaericus (FERM P-20039). The band “b”
and “c” in 16S rDNA of bacterial community in stevia-compost were identified
Bacillus sp. R-7413 and Chloroflexi bacterium, respectively.
reducing-ability of residual pesticides of U. thermosphaericus
(FERM P-20039).
3.4. Measurement of number of U. thermosphaericus
(FERM P-20039) in the stevia-compost using
competitive-PCR method
By the competitive-PCR method, it was revealed that the
number of copy of 16S rRNA gene of U. thermosphaericus
(FERM P-20039) in the stevia-compost is 1.71 × 107 copies/g
(data not shown here). Based on the fact that one Bacillus sub-
tillus has 10 copies, the stevia-compost is expected to include
1.71 × 106 cfu/g of U. thermosphaericus (FERM P-20039).
Since the number of U. thermosphaericus (FERM P-20039)
was 6.3 × 104 cfu/g in the stevia-powder, U. thermosphaericus
(FERM P-20039) in the stevia-compost increases 27 times as
much as in the stevia-powder. Additionally the total number of
thermophilic bacteria was 2.07 × 106 cfu/g at the first day after
distributing stevia-compost in the field (data not shown here),
which is almost the same as the number of U. thermosphaericus
(FERM P-20039) in the stevia-compost. From these facts we
can speculate that the nitrate-reduction ability and the reducing
ability of residual pesticides observed at the field with distribut-
ing the stevia-compost are attributed to U. thermosphaericus
(FERM P-20039) in the stevia-compost.
 K. Okamoto, N. Satou / Enzyme and Microbial Technology 39 (2006) 407–413
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