biotechnology for recovery of rare metals in wastewater · elevated concentration of rare metals...

Biotechnology for Recovery ofRare Metals in Wastewater

Satoshi SODADivision of Sustainable Energy and Environmental Engineering, Graduate School of Engineering, Osaka University

JUNBA 2009. January 13, 2009 1

Rare-metal Bioremediation and Recycling using Biotechnology

Mitsuo YAMASHITAApplied Chemistry, College of Engineering,Shibaura Institute of Technology

2

AGENDA

Selenium as a resource and a pollutant

Double benefit concept for selenium recovery from wastewater

Selenium-reducing bacterium (1) Bacillus selenatarsenatis SF-1

Selenium-reducing bacterium (2)Pseudomonas stutzeri NT-I

Possible applications of metal biology to other rare metals

Rare Metals

Consumption of metals is increasing in hi-tech Industries.

□ Rare Metal □ Rare Earth

3

Environmental Issues Related to Rare Metals

Rare metals are emitted into environment via wastes and wastewater

Environmental ContaminationElevated concentration of rare metals can cause toxic or adverse effects on the environment.Decay/Depletion as ResourceShortage as resource has started due to loss as wastewater and wastes, soaring market price of elements.

Recovery of rare metals from wastes/wastewater is necessary for sustainable society. 4

Selenium as Example

As Environmental Contaminant

Se-WastewaterSelenate (SeO4

2-) : Soluble SeSelenite (SeO3

2-) : Toxic

Industry Se conc.Oil refinery 50-300 µg/lCopper purification plant >20 mg/lChemical plant 250 mg/l

Environmental standard on water quality ≦ 0.01mg-Se/L Regulation of industrial wastewater ≦ 0.1mg-Se/LProvisional regulation for Se industry ≦ 0.3 mg-Se/L

5

Selenium as ExampleAs Resource

Selenium balance in Japan

Se in ore1000 t Cu refinery Se refinery

Pure selenium20-30 t Se production 600-700 t

Export as Se300-350 t

Se in food5 t

Se in coal37-50 t

Export as Products

Se in oil5.8-29 t

Suspended Se in air

Copier 80 tGlass 30 tChemicals 20 tPigment 15 tothers 70 t Stock

100-150 t

Urban activityDistribution, Consumption, Wastage

Recover

Air: 163-555 t Soil: 19-124 tWater: 371-5,044 tLOSS

Import

ExportSe in ore1000 t Cu refinery Se refinery

Pure selenium20-30 t Se production 600-700 t

Export as Se300-350 t

Se in food5 t

Se in coal37-50 t

Export as Products

Se in oil5.8-29 t

Suspended Se in air

Copier 80 tGlass 30 tChemicals 20 tPigment 15 tothers 70 t Stock

100-150 t

Urban activityDistribution, Consumption, Wastage

Recover

Air: 163-555 t Soil: 19-124 tWater: 371-5,044 tLOSS

Import

Export

Market Price: 2.01 $/Ib in 1996 47.4 $/Ib in 2005 6

Existing Se Wastewater Treatment

Physico-Chemical ProcessCoagulation

Fe3+

Effluent

Chemical sludge (Se <1%)

Waste disposal cost¥30,000/ton-sludge!

Catalytic electro-

reductionSe(VI)→Se(IV)

Influent

* Cannot be recycled due to low Se content

Dewatering<Disadvantages>

A large amount of coagulants and energy consumptionGeneration of a large amount of sludgeLow resource value of sludge due to low Se contentHigh-cost (including waste disposal cost)

7

Novel Concept for Se Recovery from Wastewater

Biological Process

Bio-sludge (Se max30%)

BioreactorC, N, P Membrane

Coagulationas post-treatment

EffluentInfluent

Se(VI)→Se(IV) →Se(0)

Ash (Se max75%)* Recyclable !

Due to high Se contentBenefit ¥ 650,000/ton-ash

Dewatering

Chemical Sludge Disposal

Burning

<Advantages>Low energy and reagent consumptionLow sludge generationHigh resource value of bio-sludge due to high Se contentDisposal cost turns a profit by Se recycle

8

Key Microbial ReactionSelenate/Selenite Reduction into Elemental Se

SeO42- SeO3

2- Se 0

Liquid / Solid Separation

Insoluble Se(Non-toxic)

Soluble Se (Toxic)

Physicochemicalmethods

Bacterial reduction

9

Selenate-Reducing Bacterium (1)Bacillus selenatarsenatis SF-1

Origin: Se-contaminated sedimentTaxonomy: gram positive, facultative anaerobeSelenate reduction: respiration with selenate as e- acceptor

Strain SF-1T (AB262082)

B. novalis LMG 21837T (AJ542512)B. vireti LMG 21834T (AJ542509)

B. drentensis LMG 21831T (AJ542506)B. soli LMG 21838T (AJ542513)

B. bataviensis LMG 21833T (AJ542508)B. niacini NBRC 15566T (AB021194)

B. fumarioli LMG 17489T (AJ250056)

B. koreensis BR030T (AY667496)

B. jeotgali YKJ-10T (AF221061)B. psychrosaccharolyticus ATCC 23296T (AB021195)

B. asahii MA001T (AB109209)B. flexus NBRC 15715T (AB021185)

B. megaterium IAM 13418T (D16273)

B. benzoevorans DSM 5391T (D78311)

B. indicus Sd/3T (AJ583158)B. herbersteinensis D-1,5aT (AJ781029)

B. halmapalus DSM 8723T (X76447)B. horikoshii DSM 8719T (X76443)

B. firmus IAM 12464T (D16268)B. lentus NBRC 16444T (AB271746)

B. circulans NBRC 13626T (AB271747)

Paenibacillus polymyxa DSM 36T (AJ320493)

Novel species of BacillusBacillus selenatarsenatis

10Fujita et al. (1997) J. Ferment. Bioeng., 83, 517-522;.Yamamura et al. (2007) Int. J. Syst. Evol. Microbiol., 57, 1060-1064.

Selenate Reduction by B. selenatarsenatisSF-1

Under anoxic condition (energy saving)Rate-determining step in selenite reduction

0

0.2

0.40.6

0.8

1.0

1.2

7.5

8.0

8.5

9.0

0 20 40 60 80 100

Cells

Selenate

Selenite

Sele

nate

, Sel

enite

(mM

)Log (C

ells/ml)

Time (h)

11

Deposition of Elemental Se by B. selenatarsenatisSF-1

Se deposition in cells (need for extraction from biomass)

C

O

Se

31 2Energy (keV)

0

10

20

30

40

cps

12

Lab-scale Se Recovery Process with B. selenatarsenatisSF-1

Se Recovery from Biomass

Se metal or SeO2 can be recovered from the biomass

Se-enriched Biomass

③ ③① ① ②

析出物のXRD分析結果

0 20 40 60 80

Se50%

Se75%

Se100%

0 20 40 60 80

(SeO 3 )4

2θ（ deg)

SeO 2

Se (赤）

Se(灰 )

X-Ray Diffraction (XRD) analysis

Burn at 500ºC (Acidic)

SeO2

Se

Se 50%

Se 75%

13

Selenate-Reducing Bacterium (2)Pseudomonas stutzeri NT- I

Origin: Biofilm from Se wastewater drainageTaxonomy: gram negative, aerobeSelenate reduction: in the presence of oxygen (detoxification?)

0.05 P. nitroreducens IAM 1439T (D84021)

P. syringae ATCC 19310T (AF094749)

P. chlororaphis IAM 12354T (D84011)

P. fluorescens IAM 12022T (D84013)

P. graminis DSM 11363T (Y11150)

P. koreensis Ps 9-14T (AF468452)

P. jessenii CIP 105274T (AF068259)

P. umsongensis Ps 3-10T (AF468450)

P. aeruginosa LMG 1242T (Z76651)

P. alcaligenes IAM 12411T (D84006)

P. nitroreducens IAM 1439T (D84021)

P. citronellolis ATCC 17594T (AB021396)

P. jinjuensis Pss 26T（AF468448）

P. stutzeri DSM 5190T (AJ288151)

Strain NT-IP. stutzeri ATCC 17589 (U25432)

P. stutzeri CCUG 11256T (U26262)P. stutzeri ATCC 17558 (AF094748)

14Soda et al. (2008) 5th SETAC World Congress.

Selenate Reduction Property by P. stutzeri NT- I

Under aerobic condition (energy consuming)Very effective reduction (the fastest reduction rate reported)

Time (h)

0

0.2

0.4

0.6

0.8

1

0 4 8 12 16 20 240

0.2

0.4

0.6

0.8

1

0 4 8 12 16 20 24Time (h)

Sel

enat

e, S

elen

ite (m

M)

P. stutzeri NT-I E. cloacae SLD1 a-1

● Selenate ■ Selenite

15

Deposition of Elemental Se by P. stutzeri NT- I

Se deposition outside cells (simple separation may be possible)16

Pilot-scale Se Recovery Process with P. stutzeri NT- I

Pilot-scale Reactor at Shinko Chemical Co Ltd.

UASBReactor Overview (0.2 million yen for 2 reactors, 200L×2)

17

0

10

20

30

40

50

60

70

0 5 10 15 20 25

Pilot-scale Se Recovery Process with P. stutzeri NT-I

Sequential batch trial using real industrial wastewaterwith NT-I attached onto biomass carrier

Efficient removal of soluble Se is achieved with weak aeration.

Time (day)

Se

conc

. (m

g/L)

Selenite

Selenite

Selenate

• pH : Neutralization• Salt conc. : 0.5% – 0.9%• TOC : 150 – 775 mg/L

18

1.0E+06

1.0E+07

1.0E+08

1.0E+09

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 20 40 60 80 100 120

DM

DS

e (P

eak

are

a of

GC

)

Sele

nat

e, S

eleni

te, S

e(0

) (

mM

)

Time (h)

Selenate SeleniteSe(0) DMDSe

Possibility of Se Recovery by Volatilization (DMDSe)

Observation of total Se removal from liquid and solid phase

More cost-effective and simple Se recovery ?19

CH3-Se-Se-CH3

Possible Application to Other Rare Metals

Transformation to Insoluble or Less-soluble Forms by Reduction of Metal Oxyanions

Cr(VI) Cr(III)

Soluble Insoluble/less-soluble

Se(VI) Se(IV) Se(0)

Te(IV) Te(0)

U(VI) U(IV)

Pd(II) Pd(0)

Pt(IV) Pt(0)

Tc(VII) Tc(IV)20

Possible Application to Other Rare Metals

Screening of Tellurate/Tellurite-Reducing Bacteria

Te 0

Insoluble Te(Non-toxic)

TeO66- TeO3

2-

Soluble Te (Toxic)

Stenotrophomonas maltophilia TI-1 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

keV

0

5

10

15

20

25

30

35

40

45

50

CP

S

CO

Al

Si

Te

TeTe

TeTe

TeBaBa

Ba Ba

keV

CP

S ( I

nten

sit y

)Te

21

Elemental Te

22

Bioremediationfor polluted wetlands

Wastewater treatment for metal-containing wastewater

Bioremediationfor polluted soils

Double Benefit Metal-Biotechnology

Metal recovery, recycling, processing

Low energy consumption, Speed up, New pathways, New compound (nano-particles), etc.

Possible Applications of Metal-Biotechnology

“Metal-Biotechnology” for Conservation of Environment and Resource

Bioprocess including reduction of metal-oxyanions has a great potential for concentration, extraction, and recover of rare metals from wastewater, wastes, and natural environment in cost-effective and eco-harmonizing manner.

Metal bioprocess not only in wastewater treatment and resource recovery (end-of-pipe) but also in mining, production, and processing (upper-stream application) can establish sustainable metal industries for future.

23

24

RESEARCH TEAM 2006-2008 (Osaka University)Prof. Michihiko IKEProf. Mitsuo YAMASHITA (Present affiliation, Shibaura Institute of Technology) Dr. Satoshi SODA Dr. Kazunari SEIDr. Masami KASHIWAProf. Toshihiro TANAKADr. Ken YOSHIKAWADr. Masahiro EGUCHI (Organo Co. Ltd)Mr. Kazuya UESUGI (Organo Co. Ltd)Mr. Nobuo IWASAKI (Shinko Chemical Co. Ltd)Mr. Hisamitsu TAKAHASHI (Shinko Chemical Co. Ltd)Ms. Emi NOTAGUCHI (Master course student), Mr. Kota NAGANO (Master course student)Mr. Tsubasa KAGAMI (Master course student)Mr. Masaki MIYAKE (Undergraduate student)

and many staffs

FOUNDATIONRegional New Consortium Project, the Ministry of Economy, Trade and Industry, Japan, the Kansai Bureau Economy, Trade and Industry (2007)Feasibility Study, Innovation Plaza Osaka, Japan Science and Technology Agency (2006)

25

■INVESTMENT FOR A TRIAL PRODUCT FOR A SPECIFIC MARKET

1.Selenium: treatment & recovery: Investment fee : About $ 0.4 Million

(Royalty, any cost for patent is not included) Period : 3 years

2. Other metals: treatment & recoveryInvestment fee : About $ 0.8 Million

(Royalty, any cost for patent is not included) Period : 5 years

Biotechnology for Recovery of Rare Metals in Wastewater


Sequential Batch Tests

Strict control of anoxic condition is required.

Selenate

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●●

●

●●

●

●●

●

●

●

●

0

0.2

0.4

0.6

0.8

0 1 2 3 4 5 6 7 8 9 10Time (day)

Sel

enat

e, S

elen

ite(m

M)

Selenate

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●●

●

●●

●

●●

●

●

●

●

0

0.2

0.4

0.6

0.8

0 1 2 3 4 5 6 7 8 9 10Time (day)

Sel

enat

e, S

elen

ite(m

M)

Selenite

●●

●

●

●●●●

●●●●

●●

●

●

●●

●

●

0

0.5

1.0

1.5

0 1 2 3 4 5 6 7 8 9 10Time (day)

Selenate

Selenite

Air

Sele

nate

, Sel

enite

(mM

)

26Kashiwa et al. (2000) J. Biosci. Bioeng., 89, 528-533;. Kashiwa et al. (2001) Biofactors, 14, 261-265;

0

10

20

30

40

50

0.1 1 10 100 1000123456789101112

細胞滞留時間 (h)

セレン濃度

(mg-

Se/L

)

細胞数

Log

(cel

ls/L

)


Continuous Flow Tests

27

Se6+

Se4+

Se0

X

ckYX

θ1in

61

1][Se][ −= +

cYk θ11

6 1][Se =+

)]Se[(1]Se[

][Sein

6121211

in6

114

cc

c

YkkkkYYk

θθ

θ+

++

+−

−=

)]Se[()1]Se[(

][Sein

61212111

2in

61120

cc

c

YkkkkYkYkk

θθ

θ+

+

+−

−=

θcmin

Kinetics of the reactor Cell retention tome θc(h)

Sele

nium

con

cent

ratio

n (m

g/L)

Cel

ls L

og(c

ells

/L)

Fujita et al. (2002) Biotechnol. Bioeng., 80, 755-761.

Rare-metal Bioremediation and Recycling using Biotechnology

Mitsuo YAMASHITAShibaura Institute of Technology

Faculty of Engineering

Raremetals○a few resource, ○ hard to mine and refine in

technical and economical, ○ a rapid increasing in use depending on the development of high technology such as IT, Space, Car, Energy Industry, ○ harmful contaminant

Possibility of Depleting ResourceExpensive in market, nation’s reservation：

Ga, Ge, Se, Te et al. 31 kinds；Ni, Cr, W, Mo, Co, Mn, VEnvironmental pollution in the century

Environmental quality standards for contamination and monitoring substances：

Cd, Pb, Cr, As, Hg, Se, B, Cu, Zn, Ni, Mo, Sb, Mn

Depleting resource a few resource, expensive in market

In：6.2-folds、Mo：4,1-folds、Sb：2.4-folds increased

for 5 years (2000～2005)

Environmental pollution new environmental quality standards for contamination

Remove and Recover from Environment

Problem of Rerametals

molybdenum（Mo） < 0.07 mg/l

antinomony（Sb） < 0.02 mg/l

uranium（U） < 0.002 mg/l

ex）

From 2006 in Japan

Physical and chemical treatment（coagulation-sedimentation, ion exchanger）merit：high efficiency from high conc. contaminant, short time

demerit：expensive

Biological treatment （bioremediation）

merit：high efficiency from low conc. contaminant, low cost

demerit：long time

Remediation・Recover works

Focus on Bacteria Metabolizing Metals

Soluble metal oxide anions

Bacteria

Respiration,Tolerance

Insoluble metals Recovery

Remove from waste water

Purpose• Screening and isolation bacteria available

metabolizing rerametals（Se,Te et al.) and recover them

• Analysis the genes and enzymes related to metabolite rerametals

• Construction of platform to enlarge rerametals recover process

Gas・Solidinsolublesoluble（toxic）

Selenium(Se)Selenium(Se)

Selenate（+Ⅵ）

Selenite（+Ⅳ）

Selenium（０）

Selenium compound（-Ⅱ）

・ >90％ in water・formation of stable ionization・selenite is strong toxic

・chemical compounds with Cd, Zn

Application for electronic part

Environmental pollution

H2SeO4 H2SeO3 Se ZnSe、CdSe

・ application for photocell and glass dyeing・ synthesis of amino acid containing Se in body

possibility of depleting resource

○

○

Essential element for organism

Bacillus sp. SF-1• Isolation from selenium-contaminated

sediment （M. Fujita et al., 1997）

• selenate ⇒ selenite （anaerobic）selenite ⇒ selenium

• High growth rate under aerobic condition• Enzyme complex of selenate reduction（S.

Yamamura et al., 2004）

• No Host-vector system Selenate reduction by SF-1Cause red color

Purpose Purpose ⇒ To construct Se remove process by SFTo construct Se remove process by SF--1,1,molecular mechanism of selenate reduction is examined.molecular mechanism of selenate reduction is examined.

membrane-bound enzymeMo containing enzymeinduction by selenate under anaerobic

Selenium（Se）

Phylogenetic trees of 16S rRNA betweenBacillus sp. SF-1 and related Bacillus species16S rDNA of SF-1 is 99.6% similar to that of Bacillus jeotgali JCM 10885T

Bacillus selenatarsenatis SF-1

Phenotype between strain SF-1 and JCM 10885T

Different phenotype in selenate and arsenate reduction

Strain SF-1 B. jeotgali JCM10885T

Selenate reduction ＋－

Arsenate reduction ＋－

Gram stain Positive Variable

７％ NaCl tolerance －＋

DNA-DNA reassociation value, 14％ between SF-1 and JCM 10885T

Novel species

Screening of selenium synthesis-defective mutants

Selenium synthesis-defective mutants

7 colonies（white）

Selenium synthesis-leakymutants

10 colonies （pink）

Wild-type（red）

target gene

Tn916 Tn916 insertion

Tn916

Conjugative transfer

Bacillus selenatarsenatis SF-1genome

WT（Smr, Tcs）

mutant（Smｒ, Tcr）

Enterococcus faecalis CG110（Sms, Tcr）genome

Cloning of target genes using a conjugating transposon Tn916

target gene

Tn916

WT（Smr, Tcs）

mutant（Smｒ, Tcr）

Hind III cutting

self ligation

inverse PCR

Tn916 insertion

Southern hybridization(copy number detection)

Tn916

Enterococcus faecalis CG110（Sms, Tcr）genome


Experimental flow

DNA sequencinghomology search

Bacillus selenatarsenatis SF-1genome

inverse PCR

Upstream Tn916 insertion Downstream Tn916 insertion

self ligation

inverse PCR

Homology search

Molybdopterin oxidoreductase

Generic name for oxidoreductase containing Mo, pterin, Fe-S cluster

Oxidoreductase related in anaerobic respiration(ex nitrate reductase） molybdopterin

・ Molybdopterin oxidoreductase

・ Diguanylate cyclase / phosphodiesterase（GGDEF domain）

From mutants carrying one copy of Tn916

○

○

Analysis of the Genes coding Molybdopterin Oxidoreductase

molybdopterin oxidoreductase, iron-sulfur binding subunit

molybdopterin oxidoreductase membrane subunit

molybdopterin dinucleotide-binding region

1kbmpoA mpoB mpoC

promoter &SD sequence Tn916

→ mpoA （0.9 kb）

→ mpoB （1.3 kb）

→ mpoC （3.2 kb）

terminator

Identification of Promoter region

pCVE1（4.4 kb）

choA

Ap

ColE1

3 stop codon

①Transform into E. coli

② Culture・Cell extracts

③ ChoA assay with supernatant

promoter sequence

pCVE1+promoter

Promoter proving vector with cholesterol oxidase gene （choA） as a reporter gene

Upstream region mpoA is promoter

LB broth

LB broth＋selenate

（U

/g

of

ｐro

tein

）

pCVE1+ promoterpCVE1

pCVE1

ChoA assay

0

4

8

12

14

Identification of mpoABC operon

cDNA synthesis

RNA extracts from SF-1 cells

DNA Amplification by PCR

mpoA mpoB mpoC

①

mpoABC is operon

M ②

0.5kb

①

② 1kb

Analysis of Transcripts by RT-PCR

①

②

③

Selenate reduction in E. coli carrying mpoABC

N.C.

mpoABC

LB LB +selenite

mpoA mpoB mpoC

promoter＆SD sequence

Red color

LB +selenate

Results

mpoABC are selenate reducing genes

N.C. mpoABC

Anaerobic condition ・・・ terminal electron acceptor

Aerobic condition ・・・ detoxification

easy works （culture・treatment）

high reaction rate and remove for short time

Few reports of aerobic selenate reducing bacteria

Construction of Aerobic waste water treatment?

Back ground Reduction process of selenate by microorganisms

Merit of aerobe

Se（+Ⅵ） Se（+Ⅳ） Se（0）

Toxic, soluble Atoxic, insoluble

selenate selenite elemental

isolation and identification of selenate reducing bacteria

physiology test

characterization of reduction（growing cell）

Purpose Construction of new selenate remediation system

Isolation of novel aerobic selenate high-reducing bacteria and characterization of reduction

Examination Items

Experiment Isolation

Selenate reducing bacteria

Isolation of selenate reducing bacteria, NT-I from an effluent drain of a selenium recycle factory

strain NT-I (Se is outside of cells) strain SF-1(Se is inside of cells)

Separation of Se element and NT-I cells by filtration Effective recovery of Se element

elemental Se

SEM and elemental analysis of selenate reducing NT-I and SF-1

Physiology test Identification

16S rDNA sequence analysis

100% similarity to that of Psedomonas stutzeri DSM 5190T

Physiology and Molecular biology testPseudomonas stutzeri NT-I

Physiology test

cell shape rod

Gram stain －

motile ＋

O-F test O

catalase reaction ＋

oxidase reaction ＋

0

0.1

0.2

0.3

0.4

4 5 6 7 8 9 10 11Spec

ific

grow

thra

te µ

（h-1

）

0

0.1

0.2

0.3

0.4

0 2 4 6 8 10

0

0.1

0.2

0.3

0.4

0 10 20 30 40 50

Carbon Assimilation test a variety of C source （80 kinds）(Biolog GN Microplates) (sugar,amino acids,organic acids）

Growth rate

pH Temperature（℃） NaCl concentration（%）

Growth:pH6.0-9.0, Temp.10-42℃, NaCl tolerance <5 % Optimum: pH 7.0, Temp. 38℃

pH 7.0 38 ℃ 0 ～ 5 %

Cell physiology Physiological characterization of NT-I

Medium ・・・ Glucose +mineral

Medium ： TSB + selenate or selenite

Conditions ： pH 7.2, Temp. 28℃, Aerobic

Comparison ： known aerobic selenate reducing bacteria

Pseudomonas stutzeri ATCC 51152 (Lortie et al., 1992)

Enterobacter cloacae subsp. cloacae SLD1 a-1 （ATCC 700258 ） (Losi et al., 1997)

Experiments Characterization of reduction（growing cells）

Pseudomonas stutzeriPseudomonas stutzeri NTNT--II

0

0.2

0.4

0.6

0.8

1

0 4 8 12 16 20

Con

cent

ratio

n (m

M)

Time（h）

： selenate

： selenite (control)： selenite

： selenate (control)○

selenate selenite

Reduce selenate to Se and Remove from culture

afterbefore

Results Characterization of reduction in NT-I

Time（h）

□

0

0.2

0.4

0.6

0.8

1

0 4 8 12 16 20

0

0.2

0.4

0.6

0.8

1

0 4 8 12 16 20 24

0

0.2

0.4

0.6

0.8

1

0 4 8 12 16 20 24Con

cent

ratio

n (m

M)

NT-I ATCC 51152 SLD1 a-1

NT-I ,ATCC 51152 ・・・100 % reduction(for 10-16h)

SLD1 a-1・・・25 % reduction(for 24h) ,45 % reduction(for 48h)

： selenate ： selenite

Results Selenate reduction between three strains

Time（h） Time（h） Time（h）

0

0.2

0.4

0.6

0.8

1

0 4 8 12 16 20 24

0

0.2

0.4

0.6

0.8

1

0 4 8 12 16 20 24

0

0.2

0.4

0.6

0.8

1

0 4 8 12 16 20 24

0

0.2

0.4

0.6

0.8

1

0 4 8 12 16 20 24

Con

cent

ratio

n (m

M)


NT-I , ATCC 51152 ・・・ 100 % reduction (for 14-20 h)

SLD1 a-1・・・40 % reduction(for 24h) ,100 % reduction(for 48h)

： selenite

Results Selenite reduction between three strains

Time（h） Time（h） Time（h）

Summary Comparison of reduction between three strains


1 mM selenatereduction rate （mol/h） 1.1×10-4 1.2×10-5 2.2×10-4

≧400 mM(visual

observation)

NT-I has the most selenate reducing activity

50 mM(Lortie et al., 1992)

1 mM(Losi et al., 1997)

1 mM selenitereduction rate （mol/h） 1.1×10-48.2×10-5 1.8×10-5

maximum selenate reducing

concentration

ＴＳＢ+selenate ＴＳＢ+selenite

Screening of selenate reductionScreening of selenate reduction--defective mutantsdefective mutants

Wild-type（Red）

target gene

Tet gene insertion


WT（Smr,Tcs）

mutant（Smｒ,Tcr）

E. coliE. coli DH5DH5αα（（SmSmss,Tc,Tcrr））pUTmini-Tn5 plasmid

tet

tet

Pseudomonas stutzeriPseudomonas stutzeri NTNT--IIgenome

Se synthesis-defective mutants（white）

pUTmini-Tn5 plasmid

TSB+H2SeO4 TSB+H2SeO3

H2SeO4 H2SeO3 H2SeO4 H2SeO3 H2SeO4 H2SeO3 H2SeO4 H2SeO3 H2SeO4 H2SeO3

Conversion to Se (％)

12h 99 33 3.2 34 2.3 40 0.0 37 2.9 39

24h 99 74 1.1 91 2.4 82 1.4 75 5.9 95

Control 1 32 4

Selenate and Selenite reduction of transposon mutagenesis of NT-Ⅰ

Definite conversion as 0 % from selenate or selenite at 5 mM in initial time to Se.

Tellurium (Te)・16 family element as Se and application of semiconductor material and glass-dyeing

・Soluble oxide and Toxic

・Environmental quality standards for monitoring substances (in Japan）

TeO66- TeO3

2- Te 0

Te recover・recycle

Insoluble Te（atoxic）soluble Te in waste water (toxic)

Solid-Liquid Separation

Tellurate reducing bacteria*tellurite reduction?

tellurate tellurite

ZnTe green Light Emitting Diode (LED) Te elemental mineral：native Te

Three strains were isolated from enrichments with tellurite.

Isolation of Tellurate Reducing bacteria

Tentative name as Ti-1,Ti-2,Ti-3

TSB+tellurite 1mM TSB+tellurite 20mMTSB+tellurite 5mMTSB+tellurite 2mM

Enrichment culture

TSB+tellurite 1 mM

agar plate

TSB+tellurite 1 mM

agar plate

2 strains 1 strain

Physiology and Molecular Biology Test in Ti-1, 2, 3

TSB TSB+tellurate1mM TSB+tellurite10mM

１：Ti-1, ２：Ti-2, ３：Ti-3、S： Stenotorophomonas maltophilia NBRC14161T, O：Ochrobactrum anthropi NBRC15819T

Ti-1 Ti-2 Ti-3

Cell shape rod rod rod

Gram stain Negative Negative Negative

16S rDNA

similarity

Stenotorophomonas maltophilia （１００％）

Ochrobactrum anthropi (１００％)

Ochrobactrum anthropi （１００％）

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

keV

0

5

10

15

20

25

30

35

40

45

50

CP

S

CO

Al

Si

Te

TeTe

TeTe

TeBaBa

Ba Ba

SEM (Scanning Electron Microscope)

and elemental analysis in Ti-1

Arrows indicate elemental Te.

Te

keV

CP

S（in

t ens i

ty）

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

keV

0

5

10

15

20

25

30

35

40

45

50

CP

S

CO

Al

Si

Te

TeTe

TeTe

Te

Ba

BaBa

Ba Ba

SEM and elemental analysis in Ti-2


Te

CP

S（in

t ens i

ty）

keV

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

keV

0

5

10

15

20

25

30

35

40

45

50

CP

S

C

O

Al

Si

Te

TeTe

Ba

BaBa

Ba

Te element synthesized from Ti-1 are bigger that that from Ti-2, 3.

SEM and elemental analysis in Ti-3

Te


CP

S（in

t ens i

ty）

keV

Measurement of Tellunate reduction

TSB＋telunate１ｍM

Atomic Absorption Spectrophotometry

Tellunate＋Tellunite

Colorimetric method

Tellunite

Tellunate

sampling

Tellunate reduction between Ti-1 and S. maltophilia NBRC14161T

テルル酸還元

0

0.2

0.4

0.6

0.8

1

1.2

0 50 100

時間（ｈ）

テル

ル酸

（ｍ

M）

Ti-1S. maltophilia

増殖曲線

0

1

2

3

4

5

6

7

8

9

10

0 50 100

時間（ｈ）

ＯＤ

６６

０

Ti-1

S. maltophilia

Ti-1 show high tellunate reducing activity.

Tellunate reduction between Ｔｉ-２，３ and O. anthropi NBRC15819T

増殖曲線

0

2

4

6

8

10

12

0 50 100

時間（ｈ）

ＯＤ

６６

０

Ti-2

Ti-3

O. anthropi

Ti-2, 3 show high tellunate reducing activity.

テルル酸還元

0

0.2

0.4

0.6

0.8

1

1.2

0 50 100

時間（ｈ）

テル

ル酸

（ｍ

Ｍ）

Ti-2Ti-3O. anthropi

SummarySummarySelenium（Se）

Identification of ＳＦ‐１Cloning of selenate reducing genesIsolation ・Identification as P. stutzeri NT-I High Selenate reducing activity

Tellunium（Te）Isolation of Ti-1, 2, 3High Tellunate reducing activity

→ Se and Te Remove and Recycle

biotechnology for recovery of rare metals in wastewater · elevated concentration of rare metals...

Documents