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Journal of Bioscience and BioengineeringVOL. 117 No. 5, 576e581, 2014

Isolation of a selenite-reducing and cadmium-resistant bacteriumPseudomonas sp. strain RB for microbial synthesis of CdSe nanoparticles

Hiroyuki Ayano,1 Masaki Miyake,1 Kanako Terasawa,1 Masashi Kuroda,1 Satoshi Soda,1

Toshifumi Sakaguchi,2 and Michihiko Ike1,*

Division of Sustainable Energy and Environmental Engineering, Graduate School of Engineering, Osaka University, Yamadaoka 2-1, Suita, Osaka 565-0871, Japan1 and Department ofEnvironmental Sciences, Faculty of Life and Environmental Science, Prefectural University of Hiroshima, 562 Nanatsuka-cho, Shobara City, Hiroshima 727-0023, Japan2

Received 30 July 2013; accepted 8 October 2013Available online 9 November 2013

* CorrespondE-mail add

1389-1723/$http://dx.doi

Bacteria capable of synthesizing CdSe from selenite and cadmium ion were enriched from a soil sample. Afterrepeated transfer of the soil-derived bacterial cultures to a new medium containing selenite and cadmium ion 42 times(during 360 days), an enrichment culture that can simultaneously remove selenite and cadmium ion (1 mM each) fromthe liquid phase was obtained. The culture’s color became reddish-brown, indicating CdSe nanoparticle production, asconfirmed by energy-dispersive x-ray spectra (EDS). As a result of isolation operations, the bacterium that was the mostresponsible for synthesizing CdSe, named Pseudomonas sp. RB, was obtained. Transmission electron microscopy and EDSrevealed that this strain accumulated nanoparticles (10e20 nm) consisting of selenium and cadmium inside and on thecells when cultivated in the same medium for the enrichment culture. This report is the first describing isolation of aselenite-reducing and cadmium-resistant bacterium. It is useful for CdSe nanoparticle synthesis in the simple one-vesseloperation.

� 2013, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Cadmium selenide; Selenite; Elemental selenium; Enrichment culture; Pseudomonas sp. strain RB]

Quantum dots (QDs) are 1e10 nm semiconductor nanoparticlesthat possess size-dependent luminescence (1). One of the QDs,cadmium selenide (CdSe) attracts particular attention because it isuseful for light emitting diodes, solar cells, and biological imaging(2). However, conventional chemical synthesis of CdSe involveshighly toxic solvents and high temperatures (3,4). Additionally, it isnot easy to obtain nanoparticles of the desired size because of theirhigh growth rate (5).

To overcome the shortcomings of the chemical synthesismethod, microbial synthesis of CdSe at ambient temperature andpressure without toxic solvents has attracted much attentionrecently as an environmentally friendly procedure (6). Microbialmanufacturing will increase controllability of the formation andgrowth of the QDs (7). Kumar et al. synthesized CdSe nanoparticles(9e15 nm) using a fungus, Fusarium oxysporum, in a mixture ofCdCl2 and SeCl4 (1). Cui et al. synthesized CdSe using a yeast strain:Saccharomyces cerevisiae (8). Pearce et al. also synthesized CdSenanoparticles by adding CdCl2O8 to selenide (Se(-II)) producedfrom selenite (Se(IV)) by an anaerobic bacterium: Veillonella atypica(9). In the latter two studies (8,9), cadmium was added after mi-crobial formation of selenide for CdSe synthesis, probably becauseof its toxicity to the microbes. Consequently, they synthesized CdSein two-vessel processes consisting of reduction of selenite to sele-nide and subsequent synthesis of CdSe from selenide and cadmium

ing author. Tel.: þ81 (0) 6 6879 7672; fax: þ81 (0) 6 6879 7675.ress: ike@see.eng.osaka-u.ac.jp (M. Ike).

e see front matter � 2013, The Society for Biotechnology, Japan..org/10.1016/j.jbiosc.2013.10.010

ion. In contrast, only Kumar et al. (1) reported a one-vessel processin which the fungus generates CdSe in the co-presence of seleniteand cadmium ion, which might improve economic efficiencythrough its simple operation of fewer reaction vessels.

To date, only a few microbes have been applied to CdSe syn-thesis, as described above. It is therefore worthwhile to seek novel,effective CdSe-synthesizing microbes, especially for use in the one-vessel process. However, no report in the relevant literature to datedescribes a study conducted to screen microbes suitable for one-vessel CdSe synthesis. Such bacteria might be capable of being bothcadmium-resistant and of reducing selenite to selenide. This reportis the first describing isolation of bacteria with both cadmium-resistant and selenite-reducing capabilities as a promising micro-bial catalyst for producing CdSe nanoparticles using an economicaland environmentally friendly one-vessel process.

MATERIALS AND METHODS

Media A basal salt medium (BSM) used for this study contained K2HPO4

0.5 g, NH4Cl 1 g, NaCl 0.05 g, MgCl2$7H2O 0.2 g, FeCl3$6H2O 0.01 g, CaCl2 0.01 g,Na2SO4 0.05 g, H3BO3 0.06 mg, MnCl2$4H2O 0.1 mg, CoCl2$6H2O 0.12 mg, ZnCl20.07 mg, NiCl2$6H2O 0.025 mg, CuCl2$2H2O 0.015 mg L, Na2MoO4$2H2O 0.025 mg,Bacto yeast extract (Becton, Dickinson and Co., NJ, USA) 0.02 g, nitrilotriacetic acid(Dojindo Laboratories, Kumamoto, Japan) 0.216 g (as the chelating agent to preventthe coagulation of Cd2þ and PO4

3�) in 1 L of ultrapure water. The BSM containing20 mM sodium lactate as the carbon source, 1 mM sodium selenite, 1 mM cadmiumchloride, and 100 mg L�1 cycloheximide as a fungal suppresser was abbreviated asLeBSM. To prepare agar plates, 1.8 g agar was added to 1 L of LeBSM. Herein, LeBSMcontaining 1 mM of CdCl2$2.5H2O is designated as LCdeBSM. LeBSM medium with1 mM of selenite is designated as LSeeBSM. LCdeBSM containing 1 mM selenite is

All rights reserved.

FIG. 1. Time courses of the selenite and cadmium ion concentrations in the water phase of the enrichment culture. Symbols: closed diamonds, selenite; open squares, cadmium ion.Arrows indicate the transfer of culture into new medium.

VOL. 117, 2014 ISOLATION OF CdSe-SYNTHESIZING BACTERIUM 577

designated as LCdSeeBSM. In addition, LB medium (10) containing 1 mMCdCl2$2.5H2O is designated as CdeLB medium.

Enrichment culturing from soil samples A soil sample was collected froma location used for illegal dumping of electrical appliances. To 50ml vials, 0.5 g of thesoil sample and 20 mL of LCdSeeBSM were added. The vials were incubated stati-cally at 30�C. After the culture became reddish, 1 mL (5% v/v) of the culture wastransferred to the newmedium. The same operationwas repeated to enrich bacteriacapable of synthesizing CdSe.

Microbial community analysis The eubacterial community structure wasanalyzed using terminal restriction fragment length polymorphism (T-RFLP) tar-geting the 16S rRNA gene, as described in a previous report (11) with minormodifications. T-RFs were digested using HhaI. More than 1% of the total area ofT-RFs was defined as a positive peak. Terminal restriction fragment lengthpolymorphism (T-RFLP) method of 16Sr RNA gene (rDNA) was used to examinethe microbial community structure of the enrichment culture. DNA was extractedusing ISOIL for Beads Beating (Nippon Gene, Tokyo, Japan) from the 2 mL cultureafter transferring. The DNA was amplified with forward primer 27F (50-AGAGTTTGATCCTGGCTCAG-30) (12) and reverse primer 1392R (50-ACGGGCGGTGTGTAC-30) (13) where forward 27F was labeled at the 50 end withthe 6-FAM (phosphoramidite fluorochrome 5-carboxylfluorescein). PCRamplification was performed in a 50 mL PCR mixture containing 1.5U of Ex TaqDNA polymerase (TaKaRa Ex Taq, Takara Bio Inc., Otsu, Japan), 5 mL of 10 � Ex TaqBuffer (Takara), 200 mM dNTP (Takara), 1 mL of forward and reverse primer, 5 mL ofDNA template. The cycle programs used were initial denaturation at 95�C for10 min, 30 cycles of denaturation at 95�C for 1 min, annealing at 57�C for 1 minand extension at 72�C for 3 min, and a final extension step at 72�C for 10 min. AllPCR amplifications were carried out twice for each DNA templates with aMastercycler standard (Eppendorf, Germany). 5 mL of PCR products were subjectedto electrophoresis on 1.5% (w/v) agarose gels, stained with ethidium bromide(0.5 mL/mL) for 20 min and made sure of amplification of 16S rRNA by visualizingby UV excitation. The 96 mL residues of PCR products were purified usingSuperec PCR (Takara) according to the manufacturer’s protocol. Then the PCRproducts were digested for over 5 h at 37�C with Hha I restriction enzyme (cutsite: GCG/C) in a 20 mL solution (Hha I 20U, 10 � M Buffer 2 mL, purified DNA2 mL). Digested DNA (1 mL) was mixed with 12 mL of HiDi formamide (AppliedBiosystems, USA) and 0.5 mL of GeneScan 2500 ROXTM Size Standard (AppliedBiosystems), then denatured at 95�C for 3 min. Terminal restriction fragments (T-

FIG. 2. Time courses of selenite and cadmium ion concentrations in the 7th, 33rd, and 39tsquares, cadmium ion.

RFs) patterns were achieved with Laser-Induced Fluorescence Detection/CapillaryElectrophoresis using Genetic Analyzer (Applied Biosystems) with a GeneScanPOP-4TM capillary column (47 cm � 50 mm, Applied Biosystems). Thefluorescence intensity and the size of each T-RF in a given community fingerprintpattern were automatically calculated by GeneScan analysis software (AppliedBiosystems).

Isolation and identification of bacteria The spread plate method was usedto isolate individual bacterial cells from the enrichment culture. LCdeBSM plateswere used for this operation. Analysis of phenotypic traits (morphology, Gramstaining, motility, oxidation/fermentation (O/F) test, catalase activity, and oxidaseactivity) were performed as described elsewhere (14). In addition, the isolated strainwas characterized using API 20NE (Bio Mérieux, France). The bacterial strains wereidentified further by homology of the 16S rRNA gene sequences in a mannerreported elsewhere (15). The nucleotide sequence of the partial 16S rDNAfragment of the isolated strain has been submitted to the DNA Data Bank of Japan(DDBJ) under accession no. AB839949.

CdSe synthesis and selenite reduction test The isolated bacteria werecultivated aerobically in 20 mL of CdeLB medium in a 50 mL vial for 24 h. Subse-quently,1mL of the culturewas transferred to 20mL of LCdeBSM andwas cultivatedaerobically for 12 h (precultivation). Then, the cells were collected by centrifugation(20,000 �g, 4�C, 5 min) and washed with saline solution (0.85% NaCl). For the CdSesynthesis or the selenite reduction test, the cells were inoculated into 50 mL vialscontaining 20 mL LCdSeeBSM, LCdeBSM (as control), or LSeeBSM adjusting theoptical density at 600 nm (OD600) at 0.02. In addition, the CdSe synthesis test wasconducted with the isolated bacteria. The CdSe synthesis test was also conductedusing an authorized cadmium-resistant bacterium Ralstonia metallidurans CH34(formerly Ralstonia eutropha and Alcaligenes eutrophus) (16).

Cd-resistance test For the liquid culture, 100 ml of LCdeMSM medium wasused in a 300-ml Erlenmeyer flask. After aerobic cultivation of bacterial strains for12 h, 1 ml of the culture was transferred to the new medium containing cadmiumion at various concentrations. Flasks were incubated at 28�C on a rotary shaker(120 rpm). The specific growth rate was calculated using the bacterial cell concen-tration periodically measured as OD600.

Transmission electron microscopy Transmission electron microscopy(TEM) (H-7650; Hitachi Ltd., Tokyo, Japan) was used to examine the structure anddistribution of bacterial cells and particles. The cells in 1.0 mL of the fresh culturewere collected using centrifugation (21,600 �g, 4�C, 10 min) and were washed three

h batch cultures in the enrichment culture. Symbols: closed diamonds, selenite; open

FIG. 3. EDS spectra of the particles recovered from the 6th (A, B) and the 37th (C) batch cultures in the enrichment culture. Spectra A and B show different image sites of the samesample.

578 AYANO ET AL. J. BIOSCI. BIOENG.,

times with ultrapure water. Then 10 mL of the diluted sample was dried on carbon-supported copper grids (ELS-C10; Okenshoji Co. Ltd., Tokyo, Japan).

Particles in 1.0 mL of the enrichment culture were collected and washed usingthe method described above. The sample was dispersed using an ultrasonicator(130 W, 5 min, Vibracell VCX-130; Sonics and Materials Inc., CT, USA) and wascollected using centrifugation (21,600 �g, 4�C, 10 min). Subsequently, the collectedpellet was resuspended in a 1-mL solution containing 0.5% dodecyl sodium sulfateand 10 mg/mL protease K at 55�C for 24 h for cell lysis. The sample was centrifuged(21,600�g, 4�C,10min) and the resulting pellet was washed with ultrapure water. Itwas dispersed using ultrasonication (130 W, 5 min). The sample was dried on thecarbon-supported copper grids for observation of the extracted particles.

Energy-dispersive X-ray spectra Elemental analysis of the synthesizedparticles was conducted using an Energy-dispersive X-ray spectra (EDS) system (EX-24063 JGT; JEOL Ltd., Tokyo, Japan). The particles in 1.0 mL of the enrichment culturewere collected in the same manner as that used for TEM observations. The cell lysissolution was filtered through a 0.2 mm filter. Particles in the filtrates were collectedby centrifugation (21,600 �g, 4�C, 10 min), dried on the carbon-supported coppergrids, and AuePd coated for EDS.

Chemical analysis Before determination of chemical concentrations, sam-ples were centrifuged (15,000�g, 10 min, 4�C) and filtrated by 0.45 mm filter units toseparate the supernatant and precipitates. The selenite concentration in the su-pernatant was determined from ion-exchange chromatography (IC) as described in aprevious report (15). For soluble selenium and cadmium ion analysis, nitrate wasadded at 10% of the final concentration before determination by inductivelycoupled plasma atomic emission spectrometry (ICPeAES, SPS7800; SIINanoTechnology Inc., Chiba, Japan). For analysis of selenium in the solid phase,the precipitates were washed three times with sodium tripolyphosphate solution(5 mg/L). Then the precipitates were digested for 10 min in the mixture ofconcentrated nitric acid and sulfuric acid (10:0.5) at 100�C. The resulting solutionswere appropriately diluted and were analyzed using ICPeAES. The concentrationof soluble selenide was determined using the method described in Baesman et al.(17). In short, the supernatant was mixed with the equivalent 50 mM CuCl2$2H2Osolution to precipitate selenide as CuSe. Selenium in the resulting precipitates wasanalyzed in the same manner as that in the solid phase.

RESULTS

Enrichment of CdSe-synthesizing bacteria The time cour-ses of selenite and cadmium ion concentrations in the enrichmentculture derived from the soil sample are shown in Fig. 1. Theenrichment culture was transferred repeatedly to the newmedium 42 times during 360 days of cultivation. Selenite wasremoved stably from the liquid phase, whereas cadmium ion

FIG. 4. Hha Iedigested T-RF profiles of 16S rRNA gene in the 31st batch culture of the enrichme

removals at the early phase were unstable. After transfers of theenrichment culture had been repeated more than 10 times,0.65e0.7 mM of cadmium ion was removed stably andconcomitantly with selenite.

The detailed time courses of the selenite and cadmium ionconcentrations on the 7th (days 49e58), 33rd (days 278e288),and 39th (days 338e348) batch cultures of the enrichment cultureare shown in Fig. 2. In the 7th batch culture, selenite and cadmiumion were removed at almost equal rates from the liquid phase.Selenite had been completely removed from the liquid phase byday 55. In the 33rd batch culture, the selenite and cadmium ionremoval rates were lower than in the 7th batch culture. By day282, only 0.53 mM selenite and 0.11 mM cadmium ion had beenremoved, but cadmium ion was removed rapidly after that day. Inthe 39th batch culture, selenite was removed rapidly after twodays of lag time. The cadmium ion removal tended to consist of aslow-decrease period and the following rapid-decrease period(Fig. 2B and C).

Color changes of the enrichment culture were observed afteraround the 20th culture. The culture changed to dark red or red-dish-brown, suggesting the formation of CdSe nanoparticles (1).

Elements in the enrichment culture EDS spectra of the solidphases in the enrichment culture are shown in Fig. 3. Noticeablepeaks for selenium and cadmium were detected in the solidphase of the 6th batch culture in which 0.77 mM of both seleniteand cadmium ion were removed from the liquid phase (Fig. 3A).However, many microscopic sites were found in the same sampleshowing only a selenium peak (Fig. 3B). In the 37th batch culture,in which selenite and cadmium ion removals were stable, thesolid phase showed many obvious peaks for selenium andcadmium ubiquitously at microscopic sites (Fig. 3C).

Isolation of bacteria from enrichment culture The 31stbatch culture in the enrichment culture showed two T-RFs, a mainpeak at 150 bp, and a minor peak at 567 bp (Fig. 4). From this batchculture, colonies of two types appeared on the plates: one wasreddish-brown colonies of about 5 mm (designated as strain RB),showing a similar color to that of the enrichment culture. The

nt culture. Major peak A corresponds to strain RB. Minor peak B corresponds to strain M.

FIG. 5. Time courses of chemical species concentrations of the bacterial culture. Strain RB was cultured in the LCdSeeBSM and LCdeBSM (A), and in LSeeBSM (C). Strain RB andstrain M were mixed and cultured in LCdSeeBSM, each strain’s initial OD600 were adjusted at 0.01 (B). Symbols: closed diamonds, selenite; open squares, cadmium ion inLCdSeeBSM culture; crosses, cadmium ion in LCdeBSM culture; closed triangles, soluble selenium; open circles, solid phase selenium; open diamonds, soluble selenide.

TABLE 1. Maximum specific growth rates m (h�1) of strains RB and M at variouscadmium concentrations in LCdeBSM broth.

Strain Cadmium concentration (mM)

0 1 2 3 5

RB-R 0.46 0.43 0.44 0.41 0.33M 0.098 0.051 0.059 0.052 0.063

VOL. 117, 2014 ISOLATION OF CdSe-SYNTHESIZING BACTERIUM 579

other was reddish-yellow colonies of about 0.5 mm (designated asstrain M). After isolation, it was elucidated that the T-RFs at 150 bpand 567 bp respectively corresponded to those of strain RB andstrain M.

Identification of strain RB and strain M Results of pheno-typic analysis showed that both strain RB and strain M were Gramnegative, rod-shaped, motile, catalase-positive, and oxidase-posi-tive. Strain RB produced acid from D-glucose (oxidative in O/F test),whereas strain M produced alkaline. The respective API 20NE codesfor strain RB and strain M were 1024575 with 92.5% identificationto Pseudomonas aeruginosa and 100467 with 82.2% identification toAchromobacter denitrificans. The partial 16S rRNA gene sequence ofstrain RB (1301 bp) exhibited high similarity with Pseudomonasgenus, especially with P. aeruginosa DSM50071T (GenBank acces-sion number; NR026078) (98.8%). However, strain M exhibited highhomology with Achromobacter insolitus LMG6003T (AY170847)(99.6%).

CdSe synthesis, selenite reduction and cadmium-resistanceof strain RB and strain M Strain RB was cultivated inLCdSeeBSM, LCdeBSM, and LSeeBSM under the same conditionsas those of the enrichment culture. The time courses of seleniumand cadmium ion concentrations in the media are shown in Fig. 5.In LCdSeeBSM, selenite and cadmium ion were removed from thewater phase, but about 0.22 mM of cadmium ion remained(Fig. 5A). In LCdeBSM, cadmium ion was not removed from theliquid phase. The culture of strain M became reddish-brown, butit was not observed within 3 days cultivation (date not shown).No effect was apparent for the mixed cultivation of strain RB andstain M on selenite and cadmium ion removal (Fig. 5B). InLSeeBSM, selenite was removed within only 3 days withsimultaneous accumulation of soluble selenide and selenium inthe solid phase. Accumulated soluble selenide disappeared onday 2. Most of the selenium in the solid phase was also removedby day 6 (Fig. 5C).

Effects of the cadmium ion concentration on the growth rate ofthe isolated strains are presented in Table 1. The specific growthrates of strain RB were higher in each concentration of cadmiumion.

The maximum specific growth rates of strain RB and strain Mrespectively decreased to 0.28 h�1 and 0.12 h�1 at the cadmium ionconcentration of 2 mM. Neither selenite nor cadmium ion removalby R. metallidurans CH34 in LCdSeeBSMwas marked within 6 days.

Bacterial cells and synthesized particles TEM images of thestrain RB cells and extracted particles are shown in Fig. 6. Particlesof 10e20 nm diameter were distributed in/on the cells of0.4 � 1 mm (Fig. 6A). Such small particles were not observedoutside the cells, but particles larger than 100 nm weredistributed outside the cells. The ratio of the main elements ofthe extracted particles (Fig. 6B), except for Cu of the mesh grid

and C, P, and O of the cells are shown in Table 2. The Se/Cd ratioof the particles produced by strain RB was 1.18.

DISCUSSION

In this study, bacteria with both cadmium-resistant and sele-nite-reducing capabilities were screened intentionally for the firsttime for producing CdSe nanoparticles in the economical andenvironmentally friendly one-vessel process. Microbial CdSe syn-thesis has been studied using only a few microbes of authenticatedstrains. Only fungus, F. oxysporumwas used for the one-vessel CdSesynthesis (1). Bacteria can develop an efficient CdSe synthesisprocess because they have high growth rates attributable to theirlarge specific surface area. In addition, gene manipulation tech-niques for breeding their derivates with new features have beenbetter developed for bacteria than for eukaryotes. V. atypicawas theonly bacterium applied for CdSe synthesis (9). However, strictlyanaerobic bacteria are generally difficult to handle. Therefore, wespecifically examined the screening of aerobic or facultativeanaerobic bacteria for the CdSe synthesis in one vessel.

For this purpose, the enrichment culture of CdSe-synthesizingbacteria was constructed using a medium containing selenite,cadmium ion, and cycloheximide as a fungal suppressor in aerobicconditions. The culture’s color of reddish-brown was an indicatorof CdSe accumulation (1). Therefore, we inferred that the targetbacteria were enriched from a soil sample that was possiblypolluted with such metals. Although we attempted to enrich thetarget bacteria from unpolluted soil samples preliminarily, theyshowed no reddish-brown but rather a red color specific forelemental selenium, resulting in failures of the enrichment (datanot shown). Our research group has isolated Pseudomonas stutzeriNTeI capable of reducing selenite to selenide as methylatedcompounds (15,18). However, this bacterium cannot grow in thepresence of cadmium ion (19). R. metallidurans CH34, an authenticcadmium-resistant bacterium, can reduce selenite to elementalselenium (20), but not to selenide, which is requisite for CdSesynthesis. Those results suggest that the bacteria that were bothcadmium resistant and reducing selenite to selenide were notubiquitous.

The enrichment culture at the early phase showed red andreddish-brown colors, as well as lower removals of cadmium ionthan those of selenium (Fig. 2). Many microscopic sites in the

FIG. 6. Transmission electron micrographs of the strain RB cells (A), extracted biogenic CdSe particles (B), and EDS spectra of the extracted particles of TEM image (C).

580 AYANO ET AL. J. BIOSCI. BIOENG.,

sample in the early phase showed only selenium’s peak. These re-sults suggest that elemental selenium was the main component ofthe precipitation rather than CdSe. Cadmium ion in the enrichmentculture at the early phase was possibly removed mainly by ab-sorption to the bacterial cells or biopolymers. After the 30th batchculture, progress of the enrichment of the CdSe-synthesizing bac-teria was suggested by high cadmium ion removal rates (Fig. 2C)with the reddish-brown precipitation consisting of both Se and Cd(Fig. 3). It was inferred that the enrichment culture consisted ofonly twomajor populations, according to the detected T-RFs (Fig. 4)and colonies formed on the agar plate.

Strain RB contributed mainly to the CdSe synthesis in theenrichment culture with its capability of removing selenite andcadmium ionwith reddish-brown precipitation at the Se/Cd ratio of1.18 (Fig. 5A and Table 2). The Se/Cd ratio of the particles, whichwasslightly larger than 1.0, should be attributed to the minor produc-tion of elemental selenium aside from CdSe. Strain RB is the firstreported bacterium capable of synthesizing CdSe in one-vessel.This strain was designated as Pseudomonas sp. RB with highlygenotypic and phenotypic homology to P. aeruginosa. However,strain M showed a lower growth rate and lower cadmium resis-tance than strain RB (Table 1). Although it was able to synthesizeCdSe, it had no effect on selenite or cadmium ion removal in themixed culture with strain RB (Fig. 5B). The role of strain M in theenrichment culture remains unclear. Strain M might survive in the

TABLE 2. Elements of extracted biogenic parti-cles and the ratio of Se/Cd.

Element Atomic %

S 20.7Se 42.9Cd 36.5Se/Cd ratio 1.18

enrichment culture in which strain RB had lowered the cadmiumion concentration in the CdSe synthesis.

When strain RB was cultured in the absence of cadmium ion, alarge amount of selenitewas reduced rapidly to elemental seleniumwith temporal accumulation of soluble selenide (Fig. 5C). It isparticularly interesting that the accumulated elemental seleniumhad also disappeared by day 6. These results suggest that elementalselenium was reduced to volatile gas forms such as dimethyl sele-nide (21) and dimethyl diselenide (22). Presumably, strain RB re-duces selenite rapidly to soluble selenide. Subsequently, it isoxidized to elemental selenium. Eventually, elemental seleniumwould be reduced again to selenide as the volatile forms. Thedetailed mechanism of microbial volatilization of selenium has notbeen fully elucidated, but the volatilization pathway by strain RBmight resemble that of P. stutzeri NTeI (18). However, in the pres-ence of cadmium ion, selenite was reduced to selenite by strain RBand it chemically bound with cadmium to form CdSe, resulting inthe faster removal of selenite than cadmium ion from the waterphase. The small amount of production of elemental selenium inthe co-presence of selenite and cadmium ion (Fig. 5A) suggests thatthe rate of combination with cadmium ion for soluble selenide washigher than the chemical or enzymatic oxidation rate to elementalselenium. Hydrogen selenide (23) and selenocysteine (24) arecandidate CdSe precursors.

Strain RB accumulated small CdSe particles (10e20 nm) in andon their cells, and large particles (over 100 nm) on the cell surface(Fig. 6). This observation suggests that the CdSe particles weregrown inside or on the cells to a certain size, andwere subsequentlydetached from the cells. The biogenic CdSe particles produced inthis limited condition were too large to be used as QDs. Therefore,control of the size distribution and the purification of the CdSeparticles are expected to be necessary for practical use. For instance,the particles might be separated by lysing cells with heating orsurfactants or degrading the biological polymers around theparticles.

VOL. 117, 2014 ISOLATION OF CdSe-SYNTHESIZING BACTERIUM 581

In conclusion, as a promising microbial catalyst to synthesizeCdSe nanoparticles, we isolated a bacterium that has both cad-mium-resistant and selenite-reducing capabilities for the first timeever reported. Optimization of culture conditions of strain RB anddevelopment of nanoparticle recovery methods is necessary foradditional studies.

ACKNOWLEDGMENTS

This work was supported by a Grant-in-Aid for ChallengingExploratory Research (no. 21651040) from the Japan Society for thePromotion of Science and performed under the CooperativeResearch Program of Institute for Joining and Welding ResearchInstitute, Osaka University. This work was partly conducted at theKyoto-Advanced Nanotechnology Network, supported by Nano-technology Network in Japan Advanced Institute of Science andTechnology (JAIST) of the Ministry of Education, Culture, Sports,Science and Technology (MEXT), Japan (no. H22-JA034). We thankMr. Kenji Tohmoto and Dr. Hiroshi Nishikawa of Osaka Universityand Ms. Ai Ikeda and Ms. Madoka Katayama of JAIST for theirassistance with EDS analyses.

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