Appl Microbiol Biotechnol (2013) 97:2173–2182
DOI 10.1007/s00253-012-4043-y
ENVIRONMENTAL BIOTECHNOLOGY
Iodine from bacterial iodide oxidization by Roseovarius spp.
inhibits the growth of other bacteria
Dan Zhao & Choon-Ping Lim & Kazuhiko Miyanaga &
Yasunori Tanji
Received: 13 February 2012 / Revised: 15 March 2012 / Accepted: 16 March 2012 / Published online: 19 April 2012
# Springer-Verlag 2012
Abstract Microbial activities in brine, seawater, or estua-
rine mud are involved in iodine cycle. To investigate the
effects of the microbiologically induced iodine on other
bacteria in the environment, a total of 13 bacteria that
potentially participated in the iodide-oxidizing process were
isolated from water or biofilm at a location containing
131 μg ml−1 iodide. Three distinct strains were further
identified as Roseovarius spp. based on 16 S rRNA gene
sequences after being distinguished by restriction fragment
length polymorphism analysis. Morphological characteris-
tics of these three Roseovarius spp. varied considerably
across and within strains. Iodine production increased with
Roseovarius spp. growth when cultured in Marine Broth
with 200 μg ml−1 iodide (I−). When 106 CFU/ml Escher-
ichia coli, Pseudomonas aeruginosa, and Bacillus pumilus
were exposed to various concentrations of molecular iodine
(I2), the minimum inhibitory concentrations (MICs) were
0.5, 1.0, and 1.0 μg ml−1, respectively. However, fivefold
increases in the MICs for Roseovarius spp. were obtained.
In co-cultured Roseovarius sp. IOB-7 and E. coli in Marine
Broth containing iodide (I−), the molecular iodine concen-
tration was estimated to be 0.76 μg ml−1 after 24 h and less
than 50 % of E. coli was viable compared to that co-cultured
without iodide. The growth inhibition of E. coli was also
observed in co-cultures with the two other Roseovarius spp.
D. Zhao : C.-P. Lim : K. Miyanaga : Y. Tanji (*)
Department of Bioengineering, Tokyo Institute of Technology,
4259 J2-15 Nagatsuta-cho, Midori-ku,
Yokohama 226-8501, Japan
e-mail: ytanji@bio.titech.ac.jp
D. Zhao
College of Materials Science and Chemical Engineering,
Harbin Engineering University,
No. 145 Nantong Street,
Harbin, Heilongjiang 150001, People’s Republic of China
strains when the molecular iodine concentration was as-
sumed to be 0.52 μg ml−1.
Keywords Iodide-oxidizing process . Molecular iodine .
Roseovarius spp. . Minimum inhibitory concentration .
Growth inhibition
Introduction
Iodine has been widely used as a disinfectant in surgery and
dentistry and water disinfection since 1839 (Belliot et al.
2008; Ellis and Van Vree 1989; Freney et al. 1988). Fur-
thermore, molecular iodine (I2) is effective on a broad range
of microorganisms due to its superior bactericidal, sporicid-
al, and fungicidal properties (Gottardi 1999, 2001; Gozlan
1968; Hickey et al. 1997). The chemical state of iodine
was detected not in elemental form but in ionic form in
seawater, brine, or in brackish waters from oil (Gottardi
2001; Whitehead 1984). However, the microbiologically
induced elemental iodine (especially I2) emissions in the
environment have been investigated within the last de-
cade (Amachi et al. 2005a, b; Fuse et al. 2003; Saiz-
Lopez and Boxe 2008). In addition, iodide oxidization,
accumulation, and methylation which are involved by
the microbial activities have been tracked out (Amachi
et al. 2005a, b; Amachi 2008; Fuse et al. 1989, 2003;
Li et al. 2011). During iodide oxidization, the inorganic
and organic iodines produced by iodide-oxidizing bac-
teria (hereafter, abbreviated as IOB), such as I2, CHI3,
CH2I2, and CH3I, are detected; however, the effect of
the iodine oxidized by IOB on other microorganisms in
situ still remains unknown.
Iodide oxidization by IOB was first reported in 1968
when Pseudomonas iodooxidans sp. involved in oxidizing
2174
iodide to iodine in the seawater aquaria caused the sudden
death of fish (Gozlan 1968; Gozlan and Margalith 1973,
1974). Amachi et al. (2005a, b) noted that IOB could be
isolated in an iodide-rich environment probably because
IOB occupy their ecological niche by taking advantage of
the toxic iodine and/or the molecular iodine they produced.
Japan is a country rich in iodine, and about 131 μg ml−1
iodide (Kunisue et al. 2002) was observed in brine from a
natural gas plant, in Chiba Prefecture. Iodide was recovered
from brine as another product after the natural gas recovery.
An on-site experiment conducted at the natural gas plant
(Lim et al. 2011) showed that the biomass accumulated after
iodide recovery with only 20 μg ml−1 iodide residue. Sub-
sequently, it caused bioclogging in pipelines. Severe bio-
clogging increased both the risk of bioclogging-associated
corrosion and the costs on required frequent backwashing.
The bioclogging in the low iodide environment was possi-
bly due to the loss of molecular iodine, which was produced
by IOB through an iodide-oxidizing process in an iodide-
rich environment (Lim et al. 2011).
As a substrate for microbial peroxidases and oxidases,
iodide could be oxidized to molecular iodine (Xu 1996;
Ihalin et al. 1998). Although the mechanisms of bacterial
iodide oxidization in marine or brine environment are still
kept unknown, bacterial haloperoxidases (Renirie et al.
2008) and oxidases (Amachi et al. 2005a, b) were supposed
to be responsible for this oxidizing process. Theoretically
speaking, the microbiologically induced iodine will inhibit
the growth of other microorganisms in the environment;
however, it has not been reported to date due to the uncer-
tainty of the metabolic activity of IOB and the complexity of
the natural environment. The aim of this study was to
examine the possible role of IOB-induced iodine as sup-
pressing the growth of other bacteria in an iodide-rich envi-
ronment. Furthermore, the initial investigation of the
competing strategy for IOB possibly benefits us on under-
standing the phenomena in the industry, such as the bioclog-
ging we mentioned above, which occurred at the natural gas
plant after iodine recovery.
Materials and methods
Sampling
Water samples and biofilms were collected on March 2010
from a natural gas plant located in Chiba Prefecture, Japan.
The natural gas at the plant, mainly methane, was recovered
as a product from the gas field water (GFW) using a liquid–
gas separation column. To recover the iodine, the gas-
depleted GFW was collected in a receiving tank and sub-
jected to an ion-exchange column after the pH was adjusted
with sulfuric acid. The recovered water (RW) following the
Appl Microbiol Biotechnol (2013) 97:2173–2182
iodine recovery was collected in an injecting tank and then
re-injected back into the aquifer. The physico-chemical
properties of GFW and RW were listed in Table 1. Both
the receiving and injecting tanks were under open air and
the biofilms were accumulated on the tank wall. GFW and
the biofilms in the inlet of the receiving and injecting tanks
were sampled, respectively. All samples were stored at 4 °C
in sterile polyethylene vessels and subjected to IOB-
screening within a week.
Isolation and cultivation
The biofilms were suspended in 10 ml gas-depleted GFW or
RW by vortexing and serially diluted (10−1 ~10−5). For each
diluted sample, 100 μl was spread on an agar plate contain-
ing 37.4 g Marine Broth (MB) 2216 (Difco), 1.2 g soluble
starch, 1 g potassium iodide, and 15 g agar in 1 l of distilled
water (Amachi et al. 2005a, b). The plates were incubated at
25 °C for over 1 week. After purified twice, the 13 isolates
with a positive phenotype on the agar plate were subcul-
tured in MB, and the cells were stored as glycerol stocks
at −80 °C. All the isolates were identified by 16 S rRNA
gene sequencing.
16 S rRNA gene sequencing and phylogenetic analysis
Cells for each isolate grown in MB were collected by
centrifugation, re-suspended in 200 μl Tris–EDTA (TE)
buffer (pH 07.5), and then subjected to beads beating
(4,800 rpm, 120 s). The DNA of each bacterium was
extracted by the phenol-chloroform method (Oda et al.
2004). Amplification was performed using 1 μl of the
extracted DNA as a template in 25 μl reaction mixtures
containing 2.5 μl 10× Ex Taq buffer, 27f primer (5′-
AGAGTTTGATCCTGGCTCAG-3′) (7.5 pmol), 1492r
primer (5′-GGCTACCTTGTTACGACTT-3′) (7.5 pmol),
deoxynucleoside triphosphates (250 μM each), and 0.625
U of Ex Taq DNA polymerase (TAKARA). The reactions
were amplified in a TP600 PCR thermal cycle dice
Table 1 Selected physico-chemical properties of gas field water, re-
covered water, and sea water
Gas field water Recovered watera Seawaterb
pH 7.9 6.9 8.2
Cl− (mg l−1) 19,450 19,350 19,353
Na+ (mg l−1) 11,400 11,300 10,781
I− (mg l−1)c 131 20.1 Trace
SO42− (mg l−1)c <25 213 2,712
a
Sulfuric acid was used for pH adjustment
b
Values were reported (Murray 2000)
c
Values were reported (Lim et al. 2011)
Appl Microbiol Biotechnol (2013) 97:2173–2182
(TAKARA) as follows: 94 °C for 2 min and 30 cycles of
94 °C 15 s, 49 °C for 30 s, and 72 °C for 2 min, followed by
a 20-min elongation step. The PCR products were visual-
ized on a 1 % agarose gel to ensure amplification of a single
product of the expected size. The 16 S rRNA gene ampli-
cons were digested with the restriction endonuclease
enzymes HhaI (Roche) and MspI (TAKARA) for restriction
fragment length polymorphism (RFLP) analysis. The RFLP
patterns were visualized on a 2 % agarose gel. The 16 S
rRNA gene amplicons with distinct patterns in the RFLP
analysis were purified by the phenol-chloroform method,
and the concentration of the purified products was adjusted
to ~2 ng μl−1 in TE buffer before sequencing.
The phylogenetic similarity of the 16 S rRNA gene
sequences was first determined with BLAST searches in
GenBank (http://ncbi.nlm.nih.gov/genbank/index.html) and
RDP classifier (http://rdp.cme.msu.edu/index.jsp). Then, a
phylogenetic tree was constructed using CLUSTAL X 1.83.
The reference sequences used in the tree construction were
obtained from GenBank and RDP. The sequences obtained
in this study were deposited in GenBank under accession
nos. JF417973 to JF 417975. The identified isolates were
deposited in NITE Biological Resource Center (Kisarazu,
Japan) under accession nos. NBRC 108865 to NBRC
108867.
Scanning electron microscopy (SEM) imaging of isolates
To enable scanning electron microscopic analysis of the dis-
tinct isolates, the cells were fixed in 2 % glutaraldehyde
buffered with 10 mM potassium phosphate (pH07.2) for 1 h
at 4 °C, postfixed in 1 % osmium tetroxide in the same buffer
for 1 h, dehydrated in a graded ethanol series (60 to 99 %), and
then subjected to a supercritical drying process (JCPD-5,
JEOL, Japan) with a supercritical CO2 fluid. The obtained
IOB were coated with Pt-Pd and then observed with a FE-
SEM (S-4700, Hitachi, Japan) at 6.0 kV accelerating voltage.
Iodine production by cultures of isolated strains
The growth media for iodine production were supplied
200 μg ml−1 iodide in MB (pH07.6). After the IOB were
inoculated in 15 ml sample vials containing 2 ml growth
medium for each, the vials were sealed with Teflon-lined
screw caps. All vials were incubated at 28 °C for 2 or 3 days.
IOB growth was monitored through an optical density at
660 nm (OD660). The supernatant was collected by centrifug-
ing the cell suspension and then used to determine the iodine
concentration. A calibration curve of triiodide concentration
was determined based on the absorbance at 352 nm using
serial diluted 0.527 mM triiodide solution which was prepared
with 0.483 g potassium iodide and 0.016 g molecular iodine
(Hosoya et al. 1962). All the experiments were performed at
2175
25 °C. The concentration of molecular iodine was determined
based on the triiodide concentration according to the follow-
ing equilibrium equation:
½I2  ¼ ½I3  =ðKc½I Þ
Where [I−], [I2], and [I3−] are the equilibrium concentration
of iodide, molecular iodine, and triiodide, respectively. The
equilibrium constant, Kc, is 7.10×102 dm3/mol at 298 K
(Barrow 1996).
Susceptibility test of IOB and other bacteria to molecular
iodine
The IOB, Escherichia coli W3110 K12 (ATCC 10798),
Bacillus pumilus T4 (NBRC 108859), and Pseudomonas
aeruginosa PAO1 were employed for the molecular iodine
susceptibility test. A molecular iodine stock solution was
prepared in 99.5 % ethanol and adjusted to the desired
concentration using phosphate buffered saline or marine
bacteria washing buffer containing 330 mM sodium chlo-
ride, 30 mM magnesium chloride hexahydrate, 6.8 mM
calcium chloride dehydrate, and 2.2 mM (Amachi et al.
2005a, b). E. coli, B. pumilus, and P. aeruginosa were
cultured in Luria–Bertani broth (LB) at 37 °C overnight,
while IOB were cultured in MB at 28 °C for 2 days. The
OD660 of each cell suspension was adjusted to 0.25. The
adjusted cell suspensions were added to 15 ml vials con-
taining various concentrations of molecular iodine. The
vials were sealed with Teflon-lined screw caps and the final
molecular iodine concentration in each vial was modified by
Henry’s law at 25 °C. All the bacteria were exposed to
various concentrations of molecular iodine (0, 0.2, 0.5, 1,
2, 5, or 10 μg ml−1) for 30 min. Then, the reaction in each
vial was stopped by adding excess sodium thiosulfate. Since
viable cell number could not be determined for the IOB, the
exposed IOB were re-inoculated in 96-well plates with I2-
free MB. The viable ratio corresponding to the I2 concen-
tration at which IOB was exposed in the susceptibility test
was determined based on the optical density in the well
divided by the optical density of the control (without I2
exposure). The minimum inhibitory concentrations (MICs)
for the IOB were the concentrations at which the viable ratio
fell below 50 % after a 2-day incubation in I2-free MB. The
MICs for other bacteria were determined based on the 50 %
survival ratio, which was worked out according to the viable
cell counts. The minimum bactericidal concentrations
(MBCs) for bacteria were estimated as no visible growth
in 96-well plates with I2-free MB or no colony on LB agar.
Co-culture of Roseovarius spp. and E. coli
IOB or E. coli were pre-cultured in MB or LB. The OD660 of
each culture was adjusted to 0.5, and 20 μl of IOB and the
2176
E. coli cell suspensions were inoculated into 2 ml MB with
or without iodide (200 μg ml−1), respectively. After the co-
cultures were performed for 7, 24, 30, 36, 48, 60, and 78 h,
the number of E. coli colonies was counted and the iodine
concentrations were determined as described as iodine pro-
duction by the IOB cultures.
Results
Identification of isolated iodide-oxidizing bacteria
A total of 13 bacteria that could be participated in iodide
oxidization were isolated from samples in an environment
containing 131 μg ml−1 iodide. Two of these strains were
from condensed GFW, while the others were obtained from
the biofilm in the receiving tank. No IOB candidates were
isolated from the biofilm in the injecting tank where the
iodide concentration was 20 μg ml−1. All of the 13 IOB
candidates exhibited a positive color (blue or purple) when
they were incubated on marine agar plates containing iodide
and soluble starch. None of the candidates could grow in LB
or form colony on LB agar plate. RFLP analysis showed
that they were three distinct strains, and a phylogenetic tree
(Fig. 1) of these strains indicated that they were Roseovarius
spp., and these strains were named as Roseovarius sp. IOB-
2, Roseovarius sp. IOB-7, and Roseovarius sp. IOB-12.
Fig. 1 Phylogenetic tree based
on16S rRNA gene sequences
(ca. 1,290 bp). The scale bar
corresponds to an approximate
0.01 change per nucleotide
position
Appl Microbiol Biotechnol (2013) 97:2173–2182
Morphological characteristics of IOB
After the strains were incubated for 1 week, colonies formed
on marine agar. The colonies were translucent, smooth,
circular, convex, and had an entire margin and a glistening
surface. After IOB-7 was incubated for more than 3 days, a
faint pink color appeared in the middle of the colony, and a
slimy consistency was observed. When the three strains
were incubated on marine agar containing iodide and solu-
ble starch, a positive color (blue or purple) appeared around
the IOB-7 and IOB-12 colonies but not in the IOB-2 colo-
nies. For further characterization, the three strains were
observed by a scanning electron microscope. They were
rod-shaped but varied in size, 5.0–10.0×0.5 μm for IOB-
2, 1.0–5.0×0.5 μm for IOB-12, and 1.0–3.0×0.5 μm for
IOB-7. All of these three strains had similar characteristics,
including appendages at one end of the cell and were prone
to form aggregates based on this end (Fig. 2).
Iodine production by IOB cultures
The three strains produced iodine in an iodide-supplemented
MB (Fig. 3) and iodine production increased with their
growth. When IOB-7 was cultured for 24 and 48 h in an
iodide-supplemented MB, the iodine concentration was de-
termined as 1.65 and 13.5 μg ml−1, while the optical density
was 0.5 and 1.30, respectively. The growth of IOB-2 or
0.01
Iodide-oxidizing bacterium C-3 (AB159205)
Iodide-oxidizing bacterium dMB-MAT33 (AB458526)
Iodide-oxidizing bacterium Mie-8 (AB159210)
Iodide-oxidizing bacterium Q-1 (AB159207)
Iodide-oxidizing bacterium WAI-2 (AB159208)
Marine b acterium ATAM407_61 (AF35952 5.1)
Roseovarius sp. CR-C06 (HM538455)
Roseobacter sp. TM1035 (A332660)
Iodide-oxidizing bacterium N213-3 (AB159204)
Roseovarius mucosus DFL-24 (AJ534215)
Roseovarius sp. S6V (AB114421)
Iodide-oxidizing bacterium YS-11 (AB159202)
Roseovarius sp. 2S5-2 (AB114422)
Iodide-oxidizing bacterium A-6 (AB159200)
Iodide-oxidizing bacterium dMB-MAT3 (AB458524)
IOB-2 (JF417975)
Iodide-oxidizing bacterium SE-1 (AB159209)
Iodide-oxidizing bacterium Ka-4 (AB159201)
IOB-7 (JF417973)
IOB-12 (JF417974)
Ro seovarius tolerans NBRC16695 (DQ915626 )
Marine bacterium ALUS253_4 3 (AF359526)
Appl Microbiol Biotechnol (2013) 97:2173–2182
Fig. 2 Images of appendages
and aggregation of a
(A)
Roseovarius sp. IOB-2, b
Roseovarius sp. IOB-7, and c
Roseovarius sp. IOB-12 as de-
termined by scanning electron
microscopy. Scale bar01.0, 10,
and 50 μm from the top to the
bottom of the images for each
bacterium
IOB-12 was slower than IOB-7 based on optical density.
Correspondingly, the iodine concentration in IOB-2 or IOB-
12 cultures at each time interval was lower than that of IOB-
7. After a 24-h incubation of IOB-2 or IOB-12 in MB
containing 200 μg ml−1 I−, the optical density was 0.16
and 0.12, respectively. No iodine was detected from their
24-h incubations. And then, after 48-h incubations, the
iodine concentrations were 8.35 μg ml−1 for IOB-2 and
7.62 μg ml−1 for IOB-12 and their corresponding optical
densities were 1.12 and 0.95, respectively.
Minimum inhibitory concentration and minimum
bactericidal concentration of molecular iodine for IOB
and other bacteria
E. coli, B. pumilus, and P. aeruginosa were exposed to serial
concentrations of molecular iodine for 30 min. The MICs
are shown in Fig. 4. The number of viable E. coli cells
(1.39×106 CFU/ml) was only half of the original cell numb-
ers (4.07×106 CFU/ml) after the cultures were exposed to
0.5 μg ml−1 molecular iodine. In addition, they decreased
approximately 100-fold when they were exposed to
1.0 μg ml − 1 molecular iodine. When exposed to
0.5 μg ml−1 molecular iodine, the number of viable P.
aeruginosa cells did not significantly differ from the origi-
nal cell numbers, and they decreased 1,000-fold after being
exposed to 1.0 μg ml−1 molecular iodine. When the con-
centration of molecular iodine was 1.0 μg ml−1, the number
of viable B. pumilus cells reduced 10-fold. Therefore, the
MIC of molecular iodine for E. coli, P. aeruginosa, and B.
pumilus was 0.5, 1.0, and 1.0 μg ml−1, respectively. The
MBCs for these bacteria are shown in Table 2.
2177
(B) (C)
The viable ratio of IOB after the susceptibility test is
shown in Fig. 4b. After a 2-day incubation at room temper-
ature, the OD660 of IOB-7 and IOB-12 showed that their
growth in I2-free medium was less than 50 % after being
exposed to 5 μg ml−1 iodine, while the growth of IOB-2 was
suppressed after it was exposed to 10 μg ml−1 iodine.
Therefore, the MICs of I2 for IOB were 10 μg ml−1for
IOB-2 and 5 μg ml−1 for IOB-7 and IOB-12. The MBCs
of I2 for IOB after a 1-week culture are shown in Table 2,
with more than 20 μg ml−1 for IOB-2 and 20 μg ml−1 for
IOB-7 and IOB-12.
Co-culture of IOB and E. coli
The number of viable E. coli cells was counted on LB agar,
which did not support IOB growth. The numbers of E. coli
cells from MB medium after 3 days of culture did not show
a big difference with those from LB medium (data not
shown). When E. coli and IOB were co-cultured in MB
without iodide, the number of E. coli cells (109 CFU/ml,
Fig. 5b–d) did not show a big difference with a pure E. coli
culture (109 CFU/ml, Fig. 5a). However, when these bacte-
ria were co-cultured in MB with iodide, the growth of E. coli
was significantly inhibited with increasing iodine concen-
tration (Fig. 5). For example, when in the co-culture with
IOB-2 and E. coli in MB containing iodide (Fig. 5b),
0.23 μg ml−1 iodine was detected after 24 h. However, the
growth of E. coli was inhibited after 36 h when the iodine
concentration was 0.52 μg ml−1. The inhibition continued
with the increase of iodine, and when the iodine concentra-
tion was 5.02 μg ml−1, there was no viable E. coli cell. Since
no E. coli could survive in the co-culture, the iodine
2178 Appl Microbiol Biotechnol (2013) 97:2173–2182

1.5 (A)
107
(A) 15

I2 (µg ml-1, )
1
OD660( )

106

Viable cells (CFU/ml)

10

0.5 105
5

0 0 104

(B) 15
103

I2 (µg ml-1, )
1
0.0 0.2 0.4 0.6 0.8 1.0
OD660( )

10 I2 (µg ml-1)

0.5 (B)
5 100 IOB-2
IOB-7
80
0 0 IOB-12

Viable ratio (%)

(C) 15 60
)

1
OD660( )

40
I2 (µg ml-1,

10
0.5
5
0 0
0 20 40 60 80
Time (h)
Fig. 3 Growth and iodine production by isolated iodide-oxidizing
bacteria in Marine Broth 2216 containing 200 μg ml−1 iodide. The
optical density at 660 nm (filled square) and iodine concentration
(filled triangle) in cultures of a Roseovarius sp. IOB-2, b Roseovarius
sp. IOB-7, and c Roseovarius sp. IOB-12
concentration increased drastically, and it was 8.89 μg ml−1
after a 78-h co-culture. Similarly, the growth of E. coli was
inhibited in co-cultures with IOB-7 or IOB-12 in MB
containing iodide.
Discussion
Roseovarius spp. and the iodide-oxidizing process
In this study, IOB were isolated before iodine recovery from
GFW and biofilms with an iodide concentration of
131 μg ml−1, whereas no IOB were isolated from biofilms
after iodine recovery when the concentration of iodide was
lower than 20 μg ml−1. Compared with other IOB reported
in the literatures (Fuse et al. 2003; Amachi et al. 2005a, b),
20
0
0 1 2 5 10 20
I2 (µg ml-1)
Fig. 4 Minimum inhibitory concentration of I2 for Roseovarius spp.
and other bacteria. After the bacteria were exposed to various concen-
trations of I2, the number of viable E. coli (empty triangle), P. aerugi-
nosa (empty square), and B. pumilus (empty circle) cells are shown in a
and the viable ratio of Roseovarius spp. is shown in b
our isolates are closely related to one group of IOB, which is
most closely related to Roseovarius tolerans with α-
subclass of Proteobacteria. Furthermore, other reports also
highlighted that IOB could be isolated from the cultures
enriched with an iodide supplement but not those without
(Amachi et al. 2005a, b). However, by culture-independent
methods, some DGGE clones of the samples from low
iodide concentration and location were identified as IOB
species in GenBank (Lim et al. 2011). In addition, iodide is
dispensable for the growth of Roseovarius spp. based on the
results of our studies. IOB could be isolated in an iodide-
rich environment possibly because the iodine they produced
gave them an advantage over other bacteria. Therefore,
when the isolation was performed on the same mass of
biofilms, there was a higher probability of obtaining IOB
from an iodide-rich environment than from a low iodide
environment.
All of the isolated IOB in this study were identified as
Roseovarius spp. according to a phylogenetic analysis.
Appl Microbiol Biotechnol (2013) 97:2173–2182 2179

Table 2 Minimum bactericidal concentrations of I2 for I Roseovarius spp., E. coli, P. aeruginosa, and B. pumilus

E. coli P. aeruginosa B. pumilus IOB-2 IOB-7 IOB-12

MBC of I2 (μg ml−1) 2 2 5 >20 20 20

Roseovarius spp. contributed to iodide-oxidizing process
in this study and other researchers also proposed that
these species are widely involved in iodide oxidization
in marine microbial environments (Amachi et al. 2005a,
b; Fuse et al. 2003). Like other Roseovarius spp. (Lai et
al. 2010; Labrenz et al. 1999), there was morphological
diversity among the three strains. In addition, their cell
size and shape varied considerably. All three Roseovarius
spp. were rosette-forming organisms, and appendages
were observed at the end or the middle of the cells.
There were high deviations (over 100-fold) obtained
when the number of viable Roseovarius cells was
counted by CFU counting possibly because the rosette-
forming structure could not be dispersed by serial dilu-
tion (Nair 2005). Even though the saline concentration in
the LB was increased to 3 %, none of the Roseovarius
spp. in this study could grow due to the absence of trace
elements compared to Marine Broth 2216.
The iodine concentration from each time interval was
estimated based on triiodide concentration, which was de-
termined by the absorbance at 352 nm, and the equilibrium
of iodide, iodine, and triiodide. The iodine species in MB
were probably more complicated than what we estimated
due to the presence of the organic nutrients (Amachi et al.
2001). However, the microbicidal activity of IOB-produced
iodine still indicates the possibility of inhibition in situ.
First, there is a general agreement that free molecular iodine
is a real active iodine species, which is based on the fre-
quently observed positive correlation between the iodine
concentration and the rate of microbial killing (Hickey et
al. 1997). Second, staining of biological material is attribut-
ed to the triiodide concentration (Gottardi 1999). Therefore,
the estimated concentration of molecular iodine based on the
triiodide concentration could allow us to predict the micro-
bicidal effect of iodine species in the environment.
The iodide-oxidizing process depended on IOB growth.
The growth of IOB-7 was faster than that of IOB-2 or IOB-
12 according to the OD660, and the molecular iodine con-
centration detected in the IOB-7 culture at 24 h was corre-
spondingly higher than that for IOB-2 and IOB-12. In the
co-culture with IOB-7, a higher concentration of molecular
iodine was obtained; E. coli growth could be significantly
inhibited. Therefore, the iodine concentration was strongly
correlated with IOB growth.

Fig. 5 The number of viable E. 1010 16

coli cells in pure cultures or co-
cultures with Roseovarius spp.
(A) (B) 14
during time course. The number 108
12
Viable cells (CFU/ml)

of viable E. coli cells in Marine

I2(µg ml-1, )
Broth with (filled square) or 10
106
without (empty square)
200 μg ml−1 iodide in a pure 8
cultures or co-cultures with b 104
6
Roseovarius sp. IOB-2, c Rose-
ovarius sp. IOB-7, and d Rose- 4
102
ovarius sp. IOB-12. The iodine
concentration produced in co- 2
cultures with Marine Broth 100 0
containing iodide is indicated
with a filled triangle (C) (D) 14
108
Viable cells (CFU/ml)

12

106 10
I2(µg ml-1, )

8
104
6

102 4
2
100 0
0 20 40 60 0 20 40 60 80
Time(h) Time(h)
2180
MICs of molecular iodine for Roseovarius spp.
and other bacteria
MIC and MBC are commonly used to determine the resis-
tance of bacteria for antibiotics (Andrew 2011). Broth dilu-
tion has been successfully applied for obtaining the MICs of
antibiotics for various bacteria (Kuang et al. 2009; Selvaraju
et al. 2005) but could not be performed in this study due to
the redox reaction of molecular iodine with peptone or yeast
extract (Gottardi 2001). Furthermore, the effective concen-
tration of a disinfectant was determined in buffer or distilled
water (Ellis and Van Vree 1989; Sauerbrei and Wutzler
2010). Thus, the susceptibility test of molecular iodine for
bacteria was performed in buffers. MICs of molecular iodine
for non-IOB bacteria were determined by viable cell counts,
while MICs for IOB were determined based on optical
density. The inoculums used were adjusted to the 0.5
McFarland standard, which allowed 105 to 106 CFU/ml to
be added to the broth. In this study, the cell density was
adjusted exactly to the same OD660, and 106 CFU/ml of E.
coli, P. aeruginosa, and B. pumilus were obtained by dilut-
ing the cell suspension with same OD660. However, the
viable cell counts for IOB could not be determined due to
the rosette-forming structures; thus, the same inoculums
based on the optical density and the following subsequent
dilutions were performed on IOB. The MICs for IOB were
determined based on the optical density of the subculture in
I2-free MB compared to IOB that was not exposed to
molecular iodine.
The MICs of molecular iodine for IOB were higher than
that for other bacteria. In addition, 20 μg ml−1 of molecular
iodine could be fatal to IOB-7 and IOB-12, whereas the fatal
concentration should be much higher for IOB-2 according to
the MBCs for IOB. Among the other three bacteria, E. coli
and P. aeruginosa were selected as model strains of Gram-
negative bacteria, while B. pumilus was the model strain of
Gram-positive bacterium. Moreover, B. pumilus was isolat-
ed from recovered water with an iodide concentration of
20 μg ml−1 in the iodine recovery plant. For 106 CFU/ml of
E. coli, P. aeruginosa, and B. pumilus, the MICs and MBCs
of molecular iodine were lower than those for IOB. How-
ever, if the cell numbers increased to 107 CFU/ml, the MICs
and MBCs of molecular iodine accordingly increased ten
times. However, in seawater or coastal sediments, the total
bacterial population is typically estimated to range from 103 to
106 count/ml by direct counts under a microscope (Simidu et
al. 1983). Furthermore, some specific groups of prokaryotes
were reported to have maximum concentrations of 106 cells/
ml (Austin 1988). Therefore, in brine or seawater, if the
molecular iodine concentration is higher than 0.5 μg ml−1,
the growth of sensitive bacteria probably will be inhibited by
IOB-produced iodine in the same niche. Furthermore, B.
pumilus was isolated from an environment with lower iodide,
Appl Microbiol Biotechnol (2013) 97:2173–2182
possibly because the population of B. pumilus population was
suppressed by the molecular iodine that was produced by IOB
in a high iodide environment since the MIC studies indicate
that B. pumilus is sensitive to molecular iodine. Hence, IOB
probably have an advantage over other species in the niche
since the MICs of iodine for IOB were much higher than those
for B. pumilus, E. coli, or P. aeruginosa. The role of IOB in the
iodide-rich environment will be further examined in the future
on the quantities of specific IOB genes by real-time PCR.
At a natural gas plant, the species and concentration of
iodine are thought to be stable since the iodide concentration
is 110 to 130 μg ml−1 during the year (http://www.gasukai.
co.jp/iodine/index3.html). To some extent, the MICs of
molecular iodine for bacteria will indicate the disinfecting
effects of IOB-produced molecular iodine on the other bac-
teria in the environment.
The inhibitory effect of IOB-produced iodine on the growth
of E. coli
To date, there have been no reports on the inhibitory effects
of iodine produced by microorganisms on other bacteria in
the environment. Iodide did not inhibit E. coli growth be-
cause E. coli growth curve was not significantly different in
MB with or without iodide. However, the growth of E. coli
was inhibited when the co-culture was performed in MB
with 200 μg ml−1 iodide. Furthermore, the inhibition was
not caused by nutrient competition since E. coli growth from
co-cultures was not suppressed in MB without iodide.
Meanwhile, this inhibition was reproduced when 108 CFU/
ml of E. coli was exposed to the supernatant from a 48-
h pure culture of IOB-7. After a 30-min exposure, only
105 CFU/ml viable E. coli cells could be counted. In addi-
tion, the growth inhibition of E. coli was intensified with the
iodine yield. Thus, it is supposed that iodine produced by
Roseovarius spp. inhibited E. coli growth.
The inhibitory effect of IOB-produced iodine
on other bacteria in situ
The inhibitory effects of iodine on the growth of other
bacteria could explain the phenomena at the iodine recovery
plant. The total biomass in the reinjection pipelines (after
iodine recovery) was much higher than that before iodine
recovery (Lim et al. 2011). Iodide oxidization participated
by microorganisms in brines or seawater was catalyzed by
peroxidase (Amachi et al. 2005a, b; Fweja et al. 2008).
Küpper et al. suggested that iodide was oxidized to HIO or
molecular iodine by haloperoxidases in kelp against oxida-
tive stress from the environment (Küpper et al. 1998, 2001,
2008). The bacterial iodide-oxidizing process in natural gas
brines was possibly mediated by iodoperoxidases as
reported (Amachi et al. 2005a, b). Another possibility is
Appl Microbiol Biotechnol (2013) 97:2173–2182
the oxidase generated from IOB which takes iodide ion as a
substrate during catalytic oxidation. As a product of this
catalytic oxidation, molecular iodine inhibits the growth of
sensitive bacteria in the environment.
The effective bactericidal iodine in situ is thought to be
molecular iodine. Toxic organic iodine has been reportedly
correlated with the components released or contained in the
media (Fuse et al. 2003). However, the oligotrophic charac-
teristics of GFW according to reports (Kunisue et al. 2002)
showed that organic iodine could not be as high as it was in
MB. Furthermore, Amachi et al. also reported that the iodine
species which were produced by IOB in seawater was
mainly molecular iodine but not organic iodine (Amachi et
al. 2005a, b). The inhibition occurred before iodine recovery
possibly because the iodine production depended on the
environmental iodide concentration. Therefore, the biomass
before iodine recovery was much less than that after recovery
because the sensitive bacteria were suppressed by IOB-
produced iodine in the environment. While in the pipelines
after iodine recovery, the heavy bioclogging occurred because
little molecular iodine was produced due to the lack of iodide.
Acknowledgments The authors are grateful to Japan Oil, Gas and
Metals National Corporation for financial support for this research and
to Mr. Jun Koki from the Centre for Advanced Materials, Tokyo
Institute of Technology for technical support with the SEM imaging.
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