Waste Biomass Valor (2012) 3:409418

DOI 10.1007/s12649-012-9145-3

ORIGINAL PAPER

Isolation and Characterization of Bacteria Capable of Reducing

Tellurium Oxyanions to Insoluble Elemental Tellurium
for Tellurium Recovery from Wastewater
Tsubasa Kagami Akira Fudemoto Noriyuki Fujimoto
Emi Notaguchi Masaya Kanzaki Masashi Kuroda
Satoshi Soda Mitsuo Yamashita Michihiko Ike

Received: 2 December 2011 / Accepted: 25 June 2012 / Published online: 14 July 2012
Springer Science+Business Media B.V. 2012

Abstract The recovery of tellurium as an important rare

metal from wastes and wastewater must be promoted to
satisfy its increasing demand for use in many industries.
For development of a biological process for recovering
tellurium from wastewater, three bacterial strains capable
of reducing soluble tellurite into insoluble elemental
tellurium were isolated from the drainage wastewater of a
metal refinery plant. The bacterial strains, Stenotrophomonas maltophilia TI-1, Ochrobactrum anthropi TI-2 and
O. anthropi TI-3, resisted up to 30 mM tellurite, and
removed 8689 % of 1 mM tellurite from water phase
within 21 h in a reductive reaction. They showed a considerably higher tellurite removal rate than the previously
reported tellurite-reducing bacteria. Tellurite-reducing
activity was observed at 2040 C, pH 6.28.5 and
0.055 % NaCl (0.1 mM/h at maximum). The telluritereducing activities of strains TI-2 and TI-3 were not considerably different from each other, however strain TI-1
showed higher tellurite-reducing activity at low

T. Kagami  A. Fudemoto  E. Notaguchi  M. Kanzaki 

M. Kuroda  S. Soda  M. Ike (&)
Division of Sustainable Energy and Environmental Engineering,
Graduate School of Engineering, Osaka University,
Yamadaoka 2-1, Suita, Osaka 565-0871, Japan
e-mail: ike@see.eng.osaka-u.ac.jp
N. Fujimoto
Department of Biotechnology, Graduate School of Engineering,
Osaka University, Yamadaoka 2-1, Suita,
Osaka 565-0871, Japan
M. Yamashita
Department of Applied Chemistry, College of Engineering,
Shibaura Institute of Technology, 3-7-5 Toyosu, Koto-ku,
Tokyo 135-8548, Japan

temperatures, low pH and high NaCl concentrations than

the other strains. Because deposits of elemental tellurium
were formed on and free from the bacterial cells, it is
possible to recover tellurium from the water phase by
centrifugation, ultrafiltration, or coagulation following
tellurite reduction. All bacterial strains isolated in this
study are inferred to be great candidates to establish bacterial tellurium recovery processes from industrial wastewater containing high concentrations of tellurite.
Keywords Tellurite  Tellurium  Bacteria  Reduction 
Wastewater treatment

Introduction
Consumption of tellurium, which has been used widely in
metallurgy, electronics, and applied chemical industries, is
increasing particularly because of increased production of
cadmium-telluride (CdTe) solar cells [1]. A rapid increase
in the tellurium price is expected from the large-scale
development of the CdTe solar cells technology [2].
Because tellurium is present mainly along with copper ore,
more than 90 % of tellurium is produced from anode
slimes as a by-product of electrolytic copper refining [1].
The annual tellurium production from cupper mining is
estimated to grow from 550 tons in 2011 to 1,200 tons in
2030 [2]. Although tellurium can also be recovered from
industrial scrap such as photoreceptor drums or floor
sweepings, only 10 % of production derives from such
secondary sources [3]. Therefore the recycling of tellurium
from wastes and wastewater must be promoted to satisfy its
increasing demand. Nevertheless, few trials of processes to
recover or recycle tellurium especially from wastewater
have been reported to date.

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Tellurite (TeO32-), as telluriums major oxyanion in

water, is toxic to living organisms [4, 5], e.g. even 3.9 lM
is reported to be toxic for most microorganisms [6]. The
minor oxyanion tellurate (TeO42-) is less soluble and toxic
than tellurite [4, 6]. Ollivier et al. [7] described that
microbial tellurite resistance is a widespread phenomenon.
In most environmental samples, tellurite-resistant microorganisms comprise ca. 10 % of the total culturable
microbial population [3, 810]. To date, all tellurite-resistant microorganisms are known to be able to reduce tellurite to non-toxic elemental tellurium (Te0) as black
precipitates [3, 8]. Elemental tellurium can be readily
separated from water as a solid phase. Therefore, this
microbial reaction is inferred to be useful for recovering
tellurium from wastewater.
Many tellurite-reducing bacteria have been isolated and
characterized. However, bioreduction of tellurium has not
been fully studied with respect to tellurium recovery from
wastewater. Only one study [11] has reported that 99 %
of 10 mg/L soluble tellurium was recovered from wastewater eluted from the CdTe solar panel within 12 h
incubation with Pseudomonas mendocina MCM B-180, a
tellurite-reducing bacterium. However, tellurite-reducing
bacteria which can tolerate and reduce high concentrations of tellurite have been scarcely isolated until now,
therefore, further efforts to isolate bacteria suitable for
tellurium recovery from wastewater are required. This
report describes the isolation of three tellurite-reducing
bacteria from a metal refinery plant. Their telluritereducing abilities were then investigated under various
conditions.

Waste Biomass Valor (2012) 3:409418

Enrichment and Isolation of Tellurite-Reducing

Bacteria
A drainage water sample containing biofilm was taken
from a metal refinery plant operated by Shinko Chemical
Co. Ltd. in Amagasaki City, Hyogo Prefecture, Japan [12,
13]. To 50 mL vials, 200 lL aliquots of the sample and
20 mL of TSB containing 1 mM (Enrichment L) or 2 mM
(Enrichment H) tellurite were added. The vials plugged
with silicone sponge were incubated for about 2 days on a
rotary shaker (28 C, 120 rpm) in the dark. When the
enrichment cultures showed a black color and formed a
precipitate, indicating the formation of the elemental tellurium, 200 lL of the cultures were repeatedly transferred
to fresh TSB containing tellurite at a constant concentration
of 1 mM (Enrichment L), or at higher concentrations up to
20 mM (Enrichment H). Because high concentrations of
tellurite greater than 1 mM were used for the enrichment,
bacteria possessing high tolerance to tellurite and which
have reducing activity for tellurite were expected to be
selected.
Bacterial populations in the enrichment cultures were
enumerated using the plate count technique. Bacteria
counted on agar plates of TSB with and without 1 mM
tellurite were defined, respectively, as total heterotrophic
bacteria and tellurite-resistant bacteria. The plates were
incubated 28 C for 7 days to count viable bacteria.
Among typical black colonies indicating accumulation of
elemental tellurium, three different colony types were
isolated and designated as strains TI-1, TI-2, and TI-3.
Identification of Isolated Bacteria

Materials and Methods

Media
BactoTM Trypticase Soy Broth (TSBpancreatic digest of
casein, 17 g/L; papaic digest of soybean meal, 3 g/L;
sodium chloride, 5 g/L; dipotassium phosphate, 2.5 g/L;
dextrose 2.5 g/L; BectonDickinson, NJ, USA) was used
for the cultivation of bacteria. Unless otherwise stated, the
pH of TSB was adjusted to 7.2 with HCl. For isolation and
counting of the viable bacteria, agar plates were prepared
by adding 1.8 g/L agar to TSB. Tellurite as the sodium salt
was added to TSB for the bacterial enrichment and tellurite
removal tests. Tellurate as the sodium salt was added to
TSB for the tellurate removal tests and scanning electron
microscopy (SEM) observations. For the tellurite removal
tests, HCl or NaOH was added to the medium to adjust the
pH to 6.2, 7.2, 7.8, or 8.5. Sodium chloride was added to
the medium to adjust the salinity at 0.5, 1.0, 1.5, 3.0, or
5.0 %.

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Analyses of phenotypic traits (morphology, Gram staining,

motility, oxidation/fermentation (O/F) test, catalase activity, and oxidase activity) of strains TI-1, TI-2, and TI-3
were performed as described elsewhere [14].
Their Genomic DNA was extracted using an AquaPure
Genomic DNA Kit (Bio-Rad Laboratories, Inc., Richmond,
CA, USA). The fragments of 16S ribosomal RNA gene
(rDNA) were amplified using a PCR system (GeneAmp
9700; Applied Biosystems, Foster City, CA) as described
in a previous report [15]. F9 (50 -GAGTTTGATCCTGGC
TCAG-30 ) and R1541 (50 -AAGGAGGTGATCCAGCC-30 )
were used as primers. Nucleotide sequences were determined using a genetic analyzer (ABI Prism 3100; Applied
Biosystems, Foster City, USA). The 16S rDNA sequences
were compared to reference sequences using the Basic
Local Alignment Search Tool (BLAST) [16]. Closely
related sequences were obtained from GenBank. The
nucleotide sequences of the partial 16S rDNA fragment of
the isolated strains TI-1, TI-2, and TI-3 have been submitted respectively to the DNA Data Bank of Japan

Waste Biomass Valor (2012) 3:409418

(DDBJ) under accession numbers AB683956, AB683957,

and AB683958.
Carbon Utilization Tests
Carbon source utilization profiles of strains TI-1, TI-2, and
TI-3 were determined using Biolog GN2 microplates
(Biolog Inc., Hayward, CA, USA). One colony of each
strain TI-1, TI-2, and TI-3 was inoculated into 3 mL of
TSB, and incubated 12 h on a rotary shaker (30 C,
180 rpm). These cultures were transferred into 3 mL of
TSB as 1 % volume. After 10 h cultivation in the same
condition, the cells were harvested by centrifugation
(8,2009g, 10 min, 20 C); then they were washed and
resuspended in 0.85 % NaCl solution to an initial optical
density at 600 nm (OD600) of 0.02. Aliquots of 150 lL of
the suspensions were added to microplate wells. After 48 h
of static cultivation at 288C, OD595 of each well was
measured using a microplate reader (Vient XS, DS Pharma
Biomedical, Osaka, Japan) because utilization of the carbon source in each well causes the tetrazolium dye to turn
purple and absorb light at about 595 nm. Wells in which
OD595 was higher than 0.25 were judged as positive.
Tellurite-Resistance Tests
To investigate the tellurite resistance of strains TI-1, TI-2,
and TI-3, a loopful amount of the solid culture was inoculated into 3 mL of TSB, and cultivated for 12 h on a
rotary shaker (30 C, 180 rpm). These cultures in the latelog phase were transferred into 3 mL of fresh TSB as 1 %
volume, and cultivated for 10 h in the same conditions. The
cells were harvested by centrifugation (19,0009g, 10 min,
4 C); then they were washed with phosphate buffer (5 mg/L).
The cells were suspended to 30 mL TSB in 50 mL vials at
OD600 of 0.05, and up to 30 mM tellurite was added. These
were cultivated for 24 h on a rotary shaker (28 C,
120 rpm). Then OD600 of the culture containing bacterial
cells plus tellurium particles was measured periodically to
evaluate the growth.

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Tellurite removal tests were conducted using various

combinations of pH, temperature, and NaCl concentrations.
Tellurite-reducing rates of the bacterial strains were estimated using zero-order kinetics.
For tellurate removal tests, the cells obtained in the same
manner as in the tellurite removal tests were suspended to
an initial OD660 of 0.02 in 400 mL of TSB containing
1 mM tellurate in 500 mL Erlenmeyer flasks. The flasks
were incubated on a rotary shaker (30 C, 170 rpm).
SEM
Strains TI-1, TI-2, and TI-3 were cultivated in 3 mL of
TSB containing 1 mM of tellurate for 10 h. The liquid
cultures were spotted onto a glass plate that had been
presoaked with 0.1 % poly L-lysine and fixed in phosphate
buffer containing 2 % glutaric aldehyde for 1 h. The fixed
sample was washed three times with phosphate buffer and
dehydrated in tert-butyl alcohol, with subsequent freezedrying and carbon coating. Analyses conducted using SEM
(JSM-6390LA; JEOL, Tokyo, Japan) and energy dispersive
X-ray spectroscopy (EDX, Oxford Instruments, Oxfordshire, UK) were entrusted to JEOL Ltd. (Tokyo, Japan).
Identification of Volatile Tellurium Compounds
To identify volatile tellurium compounds, a loopful amount
of the solid culture of strains TI-1, TI-2, or TI-3 was
inoculated into 20 mL of TSB, and cultivated for 24 h on a
rotary shaker (28 C, 120 rpm). These cultures in the late
log phase were transferred into 20 mL of fresh TSB as 1 %
volume, and cultivated for 24 h in the identical conditions.
The cells were harvested by centrifugation (19,0009g,
10 min, 4 C); then they were washed with phosphate
buffer (5 mg/L). All cells were suspended to 20 mL TSB in
a 500 mL vial, and 1 mM tellurite was added. The vials
were sealed with Teflon-lined septa and cultivated for 24 h
on a rotary shaker (28 C, 120 rpm). Then 1 mL of the gas
phase in the headspace of the vials was collected and
analyzed immediately using GC/MS.
Analytical Procedures

Tellurite and Tellurate Removal Tests

Strains TI-1, TI-2, and TI-3 were cultivated in the same
manner as that used for the tellurite resistant tests. The cells
were washed using sodium tripolyphosphate buffer (5 mg/
L), and were suspended in 30 mL of TSB containing tellurite up to 10 mM in 50 mL vials. The vials were incubated on a rotary shaker (28 C, 120 rpm). The tellurite
concentration was measured periodically. The same tests
using Stenotrophomonas maltophilia NBRC14161T and
Ochrobactrum anthropi NBRC15819T were conducted.

Tellurite was analyzed by colorimetric assay method as

described previously by Turner et al. [17]. Aliquots of 600 lL
of cultures were centrifuged (19,000 9 g, 10 min, 4 C); then
supernatants were filtered (pore size 0.22 lm). The filtrate
was appropriately diluted, and 200lL aliquot was mixed with
200 lL of 10 mM diethyldithiocarbamate, and 600 lL of
0.5 M TrisHCl (pH7.2). After 1 min mixing, tellurite concentrations were determined by measuring absorbance at
340 nm of the mixtures. Soluble tellurium (tellurate ? tellurite) was analyzed using atomic absorption spectrometry

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Waste Biomass Valor (2012) 3:409418

(SAS7500A; Seiko Instruments Inc.). Tellurate concentrations in this study were defined as (soluble tellurium concentration)(tellurite concentration).
The GC/MS analysis of volatile tellurium compounds
was conducted on GC/MS 450-GC equipped with 220-MS
(Varian, Palo Alto, CA, USA). Helium was used as carrier
gas at a flow rate of 1 mL/min. Injector conditions were the
following: temperature of 290 C, with split mode at 1:10.
Transfer line, manifold, and ion trap temperatures were set
respectively to 280, 40 and 200 C. Separations were
conducted using 30 m x 0.25 mm (1.4 lm film thickness)
VF-624 ms column (Varian Inc.). The column temperature
was programmed as follows: 35 C held for 1 min, then
heated at 10 C/min to 250 C and 20 C/min to 280 C
held constant for 1 min. Scanning masses were
40450 amu. The compounds were identified by comparing the mass spectra with the previously reported ones
[18, 19].

Results
Construction of Enrichment Cultures
of Tellurite-Reducing Bacteria
Enrichment L was constructed by repeated transfer of
cultures into TSB containing 1 mM tellurite. The changes
of tellurite concentrations in water phase of Enrichment L
are presented in Fig. 1a. In the first batch, 1 mM tellurite
was removed almost completely in about 2 days. The
enrichment culture turned black, reflecting the accumulation of elemental tellurium and therefore the tellurite
reduction. Although tellurite removal rate decreased temporarily in the second batch, constant tellurite removal rate
was achieved after the third batch.
Enrichment H was constructed by repeated transfer of the
culture into TSB with higher tellurite concentrations (Fig. 1b).
Although Enrichment H removed 5 mM tellurite completely
in the third batch, it showed less removal for 10 mM tellurite
in the fourth batch incubation. In the final batch test, the tellurite concentration decreased from 20 mM to 12 mM within
3 days. The enrichment culture showed a black color, confirming the tellurite reduction. Furthermore, a garlic-like odor,
indicating the formation of volatile tellurium compounds, was
recognized during cultivation.
Viable counts of total heterotrophic and tellurite-resistant bacteria in the enrichment cultures are presented in
Table 1. Tellurite resistant bacteria were only 2.4 % of all
heterotrophs in the sample from the metal refinery plant.
However, the ratio increased considerably with progress of
the enrichment. At the end of the enrichment, almost
all bacteria resisted 1 mM tellurite in both Enrichment
L and H.

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Fig. 1 Time courses of tellurite concentrations in enrichment

cultures. Enrichment cultures L and H were constructed by transfer
of 1 % of the culture to the new medium containing tellurite at low
concentration (a) and high concentration (b), respectively. The
arrows indicate transfer of the cultures. The results represent a single
experiment

Physiological and Homological Properties of Isolated

Bacteria
At least two morphologically different colonies appeared
on the plates of Enrichment L on day 15. These bacteria
were isolated and designated as strains TI-1 and TI-2.
However, one bacterium was isolated from Enrichment
H and designated as strain TI-3. All strains were found to
be resistant to tellurite up to 30 mM. The OD600 of pure
cultures of all strains exceeded 1.0 within 24 h (data not
shown). The high OD600 values suggest growth of the
strains and accumulation of tellurium particles. Characteristics of the isolated strains are shown in Table 2. Strains
TI-2 and TI-3 showed mutually similar phenotypes. The
presumed genera of the bacterial strains were S. maltophilia for strain TI-1 and O. anthropi respectively for
strains TI-2 and TI-3, according to the phenotypic characterizations and 16S rDNA sequence analyses, which
exhibited that the 16S rDNA sequence of strain TI-1 was
99.3 % identical to that of Stenotrophomonas maltophilia
strain IAM 12423 (AB294553), and the sequences of strain
TI-2 and TI-3 were 99.9 % identical to that of Ochrobactrum anthropi strain STM 2148 (AY785314), respectively.

Waste Biomass Valor (2012) 3:409418

413

Table 1 Viable counts of

bacteria in enrichment cultures
for tellurite removal (CFU/ml)
Before enrichment (Drainage water sample)

Total heterotrophsa

Tellurite resistant
bacteriab

7.6 9 106

1.8 9 105

3.6 9 107

Enrichment culture L
a

Bacteria counted on TSB agar

Bacteria counted on TSB agar

containing 1 mM tellurite

c
d

Day 2 in Fig. 1
Day 15 in Fig. 1

After the first batch cultivationc

After the fifth batch cultivation

8.0 9 107

Enrichment culture H
After the first batch cultivationc
After the fifth batch cultivation

4.9 9 108

The carbon utilization profiles of strains TI-1, TI-2, and

TI-3 are depicted in Fig. 2. Only a few carbon sources can
be used efficiently by strain TI-1. The only carbon sources
showing an OD595 increase higher than 0.2 were Tween40
and L-fucose. However, strains TI-2 and TI-3 can use
various carbon sources.

9.6 9 10

9.5 9 108

tellurite in sealed vials, and the headspace gas was analyzed. Both dimethyl telluride and dimethyl ditelluride
were detected from the gas phase of all bacterial cultures
whereas dimethyl tellurenyl sulfide were detected from the
gas phase of strains TI-2 and TI-3.
Tellurite removal rates of strains TI-1, TI-2, and TI-3
increased with tellurite concentrations in the medium up to
1 mM. Strain TI-1 reduced tellurite with a higher rate than
that of either strain TI-2 or strain TI-3 at the initial tellurite
concentrations of 0.5 mM and 1.0 mM. Almost equal tellurite removal rates were obtained by strains TI-2 and TI-3
at all tested concentrations (Fig. 4). As described above,
since tellurite removal at the initial concentration of
10 mM was low. Zero-order kinetic constants could not be
determined at this high concentration.
The tellurite removal rates of strains TI-1, TI-2, and
TI-3 under various conditions are depicted in Fig. 5.
A higher tellurite removal rate by strain TI-1 was
observed at low pH of 6.2 than that by either strain TI-2
or strain TI-3 (Fig. 5a). The maximum tellurite removal
rate by strain TI-1 was obtained at 35 C, whereas at
28 C by strains TI-2 and TI-3 (Fig. 5b). Tellurite
removal did not occur at 15 C by strain TI-2 or strain TI3, although 24 % was removed by strain TI-1 (data not
shown). Tellurite removal rate by strains TI-2 and TI-3
drastically decreased concomitantly with increasing NaCl
concentration, although no significant decrease of tellurite

Tellurite reduction by Strains TI-1, TI-2, and TI-3

and Effects of Environmental Factors
The typical courses of the tellurite removal tests by strains
TI-1, TI-2, and TI-3 are portrayed in Fig. 3. All strains
showed 58 h lag time before rapid tellurite removal
occurred. The removal to 1 mM tellurite by strains TI-1,
TI-2, and TI-3 after 21-h incubation were, respectively 86,
87, and 89 % (Fig. 3a). Removal to 10 mM tellurite after
100 h incubation was 29 % by strain TI-1, 46 % by strain
TI-2, and 41 % by strain TI-3 (Fig. 3b).
Although the type strains S. maltophilia NBRC14161T
and O. anthropi NBRC15819T also removed 1 mM tellurite from the water phase within 30 h, strain NBRC14161T
showed a long lag time of 1621 h before rapid tellurite
reduction, and strain NBRC15819T showed slightly slower
reduction than strains TI-2 and TI-3, respectively (Fig. 3a).
Garlic-like odor was recognized from all cultures during
removal tests. To confirm the generation of gaseous tellurium, all strains were cultivated in TSB containing 1 mM
Table 2 Characteristics of
tellurite-resistant strains TI-1,
TI-2, and TI-3

1.2 9 10

Strain TI-1

Strain TI-2

Strain TI-3

Source

Enrichment culture L

Enrichment culture L

Enrichment culture H

Shape

Rod

Rod

Rod

Gram stain

Motility

Catalase activity

Oxidase activity

OF test

Homology of 16S rRNA gene

sequence

Stenotrophomonas
maltophilia

Ochrobactrum
anthropi

Ochrobactrum
anthropi

99.3 %

99.9 %

99.9 %

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Fig. 2 Carbon source

utilization of strains TI-1, TI-2,
and TI-3 detected as absorbance
of Biolog GN2 microplates after
48 h incubation. The results
represent data from a single
experiment

removal by strain TI-1 was observed up to 3 % NaCl

(Fig. 5c).
Tellurate Reduction and Elemental Tellurium
Formation by Strains TI-1, TI-2, and TI-3
The typical courses of the tellurate removal tests by the
three bacterial strains are portrayed in Fig. 6. Strains TI-1,
TI-2, and TI-3 strains were able to remove 1 mM tellurate
almost completely from the medium without significant
accumulation of tellurite within 100 h. S. maltophilia
NBRC14161T showed a long lag time of about 3672 h
before tellurite reduction. After the lag time, strain
NBRC14161T accumulated tellurite of about 0.1 mM and

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tellurate remained at 0.6 mM after 120 h. Although

O. anthropi NBRC15819T also showed a lag time of
1236 h, 1 mM tellurate was removed almost completely
from the medium within 100 h. All cultures showed a black
color, suggesting the accumulation of elemental tellurium.
Figure 7 portrays SEM and EDX observation results for
the cultures of strains TI-1, TI-2, and TI-3 after tellurate
removal. The particles of 200300 nm were observed
on and around the cells of strain TI-1. However, particles
of about 100 nm were observed outside of strains TI-2 and
TI-3. These particles were shown to be tellurium by EDX
analysis. Results confirmed that tellurate and tellurite were
removed from the medium by the reductive reaction to
form elemental tellurium.

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415

Fig. 3 Tellurite removal with batch cultivation of strains TI-1, TI-2,

and TI-3 at the initial concentration of 1 mM (a) and 10 mM
(b) tellurite at 30 C and pH 7.2. Open circles, strain TI-1; closed
squares, strain TI-2; open triangles, strain TI-3; open diamonds,
O. anthropi NBRC15819T; closed diamonds, S. maltophilia
NBRC14161T; crosses, control without bacterial cells. Vertical bars
represent the standard deviations of three independent experiments
with strains TI-1, TI-2, and TI-3. The results of control without
bacterial cells represent data from a single experiment

Fig. 5 Effect of pH, temperature, and NaCl concentrations on

tellurite reduction rates by strains TI-1, TI-2, and TI-3. Basic
conditions for the batch reduction test were the initial tellurite
concentration of 1 mM tellurite at 30 C and pH 7.2 without NaCl
addition: a pH, b temperature, and c NaCl concentration. Open
circles, strain TI-1; closed squares strain TI-2; open triangles strain
TI-3. Vertical bars represent the standard deviation of three
independent experiments

Fig. 4 Effect of the initial tellurite concentrations on tellurite

reduction rates of strains TI-1, TI-2, and TI-3 (30 C, pH 7.2). Open
circles strain TI-1; closed squares strain TI-2; open triangles strain
TI-3. Vertical bars represent the standard deviation of three
independent experiments

Discussion
In this study, we isolated bacterial strains with high tellurite-resistance capability for the final goal of tellurium

recovery from waste and wastewater because it is known

that tellurite-resistant bacteria commonly possess the
ability to reduce tellurite into elemental tellurium, which
can be removed easily from the water phase.
Tellurite-resistant bacteria were found as 2.4 % of the
total population in the drainage water samples used for
enrichment of tellurite-reducing bacteria in this study. This
ratio is similar to the values reported previously [3, 810].
By selective pressures of high concentrations of tellurite in
the Enrichment L and H, where almost 100 % of the bacteria showed resistance to 1 mM tellurite, were successfully obtained. Strains TI-1, TI-2, and TI-3 were isolated as
dominant bacteria in the enrichment cultures. All bacterial

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Fig. 6 Tellurate removal with batch cultivation of strains TI-1 (a),

TI-2, and TI-3 (b) at the initial concentration of 1 mM tellurate at
30 C and pH 7.2. Open circles tellurate concentration of strain TI-1;
closed squares tellurate concentration of strain TI-2; open triangles
tellurate concentration of strain TI-3; open diamonds O. anthropi
NBRC15819T; closed diamonds S. maltophilia NBRC14161T; closed
gray open circles, closed gray squares, closed gray triangles, and
crosses represent tellurite concentrations. The results represent data
from a single experiment

strains were grown in TSB containing concentrations of

tellurite as high as 30 mM, indicating they can tolerate
much higher concentrations of tellurite than any previously
reported bacterial strain [2024]. The three strains were
identified as S. maltophilia and O. anthropi. Type strains of
the species, S. maltophilia NBRC14161T and O. anthropi
NBRC 15819T were examined in this study for the references of the tellurite and tellurate reduction capability.
Although, type strains needed a longer lag time than strain
TI-1 for tellurite reduction, they can remove 1 mM tellurite
from the water phase, implying that tellurite reduction is a
common characteristics among these species. This speculation is encouraged by a report describing that S. maltophilia Sm777 can also grow under 25 mM tellurite, and can
reduce it into elemental tellurium [25]. Moreover, strains
TI-1, TI-2, and TI-3 showed higher tellurate removal
capacity than the type strains, although the bacterial tellurate reduction has not been specifically examined in type
strains because of its lower solubility and toxicity. Bacterial strains possessing higher reducing activities of tellurium oxyanions than the type strains were possibly selected
by the enrichment procedures. The lag time may be

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Fig. 7 Scanning electron microscopic images of strains TI-1, TI-2,

and TI-3. The arrows indicate a particle of elemental tellurium:
a strain TI-1, b strain TI-2, and c strain TI-3

explained by differences in the specific growth rate and the

induction time of enzymes related on reduction of tellurium
oxyanions among the strains.
Chien and Han [26] reported that reduced uptake and an
increased efflux mechanism are usually not the main
mechanisms of resistance to tellurite in microbes [2732].
Instead, bacteria usually convert tellurite to a less toxic
form by aerobic reduction, ultimately engendering the

Waste Biomass Valor (2012) 3:409418

formation of intracellular precipitations of black metallic

tellurium [27, 33]. Tellurite resistance by strains TI-1, TI-2,
and TI-3 might be attributable to such detoxifying mechanisms because all of them showed high tellurite reduction
activity. However, we have very little evidence about the
tellurite-reducing mechanisms by strains TI-1, TI-2, and
TI-3 within this report.
The elemental tellurium produced by bacterial strains
might be recovered easily using physicochemical solid
liquid separation techniques such as centrifugation, ultrafiltration, and coagulation, as is done for selenium particles
[34]. Furthermore, volatile tellurium compounds such as
dimethyl telluride, dimethyl ditelluride, and dimethyl tellurenyl telluride were detected from these bacterial cultures, although they were not quantified. If these volatile
tellurium compounds can be efficiently collected, separation of tellurium from bacterial cells without other chemicals can be accomplished, yielding extremely high-purity
tellurium.
The tellurite-reduction rates of strains TI-1, TI-2, and
TI-3 are higher than any value reported in the literature [7,
11, 18, 21, 23, 26, 3538]. Before this study Pseudomonas
strain TeU was considered to show the highest telluritereduction rate, which is ca. 0.06 mM/h (1,000400 lM
within 10 h) [36], however, this value is significantly lower
than those observed in this study for strains TI-1 to TI-3.
Furthermore, the high tellurite removal rate can be maintained at relatively wide ranges of pH, temperature, and
salinity. For application of these strains to tellurium
recovery from industrial wastewater, such characteristics
are advantageous because conditions of industrial wastewaters are generally varied. It is particularly noteworthy
that the tellurite-reducing rate by strain TI-1 was the
highest among all isolated strains, and that it did not
decrease much at lower pHs and higher NaCl concentrations, suggesting that this strain is an extremely attractive
agent for tellurium recovery from industrial wastewater.
However, strain TI-1 has the important limitation of being
capable of using only a few carbon sources for growth.
Although the tellurite reduction capabilities of strains TI-2
and TI-3 are slightly weaker than that of TI-1, they can
propagate by utilizing various carbon sources. These
results suggest that suitable strains for tellurium recovery
should be chosen depending on the wastewater
characteristics.
Recently, tellurium is regarded not only as a valuable
resource, but also as a potential pollutant because it has
high toxicity to living organisms [5, 39]. The rapid
increasing use of tellurium in industrial products may cause
future pollution problems. Tellurium can be found at high
concentrations in wastewaters discharged from industrial
plants (at a few mg/L levels) [13, 40]. Also, leachates from
landfill sites receiving industrial wastes have a high

417

probability of tellurium pollution [9, 11]. Tellurium should

be removed from those wastewaters leaving non-toxic low
levels in the water phase. In Japan, the effluent standard for
tellurium has not been established yet in the Water Pollution Control Law, but tellurium has been selected as a
chemical demanding additional investigation [41]. From
such a viewpoint, the dual benefits of resource recovery
and environmental protection can be achieved if a recovery
method of tellurium as a valuable resource from wastewater can be developed. The bacterial strains isolated in
this study are superior candidates for developing such a
process offering these dual benefits. However, our investigation found no isolated strains that formed substantial
flocs. Biofilm-formation, cell immobilization, and membrane separation are expected to be effective for maintaining high bacterial concentrations in bioreactors for
tellurium recovery. Our previous studies of selenium
removal from industrial wastewater found that acrylic
fringe and granular sludge, as biomass carriers, effectively
maintained selenium-reducing bacteria in the bioreactors
[13, 42]. Those results will facilitate for the appropriate
design and operation of bioreactors using tellurium
reducing bacteria.
Acknowledgments This study was partly supported by the Japan
Securities Scholarship Foundation. We thank Mr. Hisamitsu Takahashi and Mr. Nobuo Iwasaki (Shinko Chemical Co. Ltd.) for providing water samples, and Mr. Akio Isono (Kansai Application
Research Center, JEOL Ltd.) for his helpful advice related to SEM
EDX analysis.

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