全 文 ：文章编号 :100028551 (2008) 022209204
BIOSORPTION OF RADIOCESIUM BY Deinococcus radiodurans( INFLUENCED BY CATIONS
GUO Jiang2feng1 ,2 　CHAI Li2hong2 　DING Xian2feng1 　ZHU Cong1
(1. Institute of Bioengineering , Zhejiang Sci2Tech University , Hangzhou , Zhejiang 　310018 ;
2. Institute of Nuclear Agricultural Sciences , Zhejiang University , Hangzhou , Zhejiang 　310029)
Abstract :Deinococcus radiodurans has a strong ability to withstand high doses of radiation , which makes it as an ideal
candidate for bioremediation of sites contaminated with radionuclides and toxic chemicals. However , no data is available on
whether D . radiodurans has a specific sorption capacity to radiocesium for bioremediation purpose. The radiocesium
biosorption capacity of live cells of D . radiodurans in the presence of other interfering cations was investigated. The maximum
biosorption capacity of radiocesium by D . radiodurans in equilibrium state was about 2 ,100 kBqΠkg (fresh weight basis) .
Among the tested monovalent cations , NH+4 had the strongest antagonism on 134 Cs biosorption for D . radiodurans. However ,
this antagonism could only be observed at a concentration as high as 100 mmolΠL. Divalent cations , such as Ca2 + and Pb2 +
could reduced the biosorption of radiocesium by D . radiodurans. Al3 + and Cr3 + were cytotoxic to D . radiodurans cells , the
growth of D . radiodurans cells was inhibited when the concentrations of these cations were greater than 1 mmolΠL.
Key words :radiocaesium ; Deinococcus radiodurans ; biosorption ; bioremediation
阳离子对耐辐射奇球菌对放射性铯生物吸收的影响
郭江峰1 ,2 　柴立红2 　丁先锋1 　朱　聪1
(1. 浙江理工大学生物工程研究所 ,浙江 杭州　310018 ;2. 浙江大学原子核农业科学所 ,浙江 杭州　310029)
摘 　要 :耐辐射奇球菌能够耐受高剂量的辐射 ,促使人们考虑利用此特性进行放射性核素和有毒化学品
的生物修复。但是 ,有关耐辐射奇球菌对放射性铯的生物吸收却未见报道。本文研究了耐辐射奇球菌
对放射性铯的生物吸收及其他阳离子对其生物吸收的影响。在平衡状态时 ,耐辐射奇球菌对放射性铯
的最大吸收量为 2100kBqΠkg(鲜重) 。在供试的一价阳离子中 ,NH+4 对其生物吸收的拮抗作用最强 ,但
是只有当 NH+4 的浓度超过 100mmolΠL 时 ,这种拮抗作用才比较明显。二价阳离子 ,如 Ca2 + 和 Pb2 + 也能
降低耐辐射奇球菌对放射性铯的吸收。Al3 + 和 Cr3 + 对耐辐射奇球菌具有较强的细胞毒性 ,当其浓度超
过 1mmolΠL 时 ,耐辐射奇球菌的生长就完全被抑制。
关键词 :放射性铯 ;耐辐射奇球菌 ;生物吸收 ;生物修复
收稿日期 :2007205223 　接受日期 :2007206229
基金项目 :国家自然科学基金资助 (项目批号 10405021)
作者简介 :郭江峰 (19692) ,男 ,山东聊城人 ,教授 ,博士 ,现从事生物芯片方面的研究工作。E2mail :jfguo @zstu. edu. cn　　137 Cs , a long remaining element in environment , is oneof important fission products with a high yield of 612 % anda decay half2life of 3012 years. A large quantity of 137 Cs hasbeen released into the environment throughout theatmosphere during the past five decades by nuclear testingand accidents. It has been estimated that about 119 ×1016Bq of 134 Cs and 317 ×1016 Bq of 137 Cs was released into the atmosphere in the Chernobyl reactor accident happened onApril 26 , 1986[1 ] . As such , 137 Cs distributes ubiquitouslyeven today in the environment .Under appropriate environmental conditions ,radiocesium is readily mobile and bioavailable[1 ] . And dueto the chemical and physiological similarity of radiocesiumand potassium , a bioessential element , their root uptake
902　核 农 学 报　2008 ,22 (2) :209～212Journal of Nuclear Agricultural Sciences
mechanisms appear to be closely related[2 ] . As radiocesium
can pass along food chains , the radiocesium has the highest
impact for humans among the radionuclides that were
released to the environment[3 ] .
Soil microorganisms , which exist widely in surface and
subsurface soil environment , exhibit a tremendous molecular
and metabolic diversity. They are surface charged and are
able to secrete various organic compounds such as low2
molecular organic acids , carbohydrates and enzymes[4 ] .
Thus , the microorganisms can react with soluble heavy
metals by binding and precipitating these metal ions on their
cell surfaces[5 ] . It has been proved that they are capable of
adsorbing heavy metals from aqueous solutions especially
with the metal concentrations below 50mgΠL [6 ] . Such
microbe2mediated metal2binding mechanisms are increasingly
being considered as the basis for developing bioremediation
technologies for treatment of radionuclidesΠmetal 2containing
nuclear wastes[7～9 ] .
Deinococcus radiodurans , a nonpathogenic ,
nonsporulating , obligate aerobic soil bacteria[10 ] , is the most
radiation2resistant organism discovered so far for its ability to
withstand doses of ionizing radiation as high as 15000Gy
without lethality[11 ,12 ] . Strains of D . radiodurans are also
tolerant to the solvent of toluene and trichloroethylene at
levels exceeding those of many radioactive waste sites[13 ] .
The remarkable ability to withstand high doses of radiation
and high concentrations of solvent makes it an ideal
candidate for bioremediation of sites contaminated with
radionuclides and toxic chemicals[13 ,14 ] . Holland and his co2
workers developed a morpholinepropanesulfonic acid based
medium solely containing a carbon source , salts , four
vitamins , and two amino acids to support a rapid growth of
D . radiodurans[15 ] . Several strains of D . radiodurans
expressing the cloned Hg ( II) resistance gene ( merA) from
Escherichia coli strain BL308 were generated , which were
shown to grow in the presence of both radiation and ionic
mercury at concentrations well above those found in
radioactive waste sites , and to effectively reduce Hg ( II) to
the less toxic volatile elemental mercury[16 ] . The further
study showed that the engineered bacteria were able to
oxidize toluene under both minimal and complex nutrient
conditions , and recombinant cells reduce Cr ( VI ) in
sediment microcosms[17 ] . With respect to radiocesium ,
however , no data is available on whether D . radiodurans
has a specific sorption capacity for bioremediation purpose.
The present study was undertaken to evaluate the
radiocesium biosorption capacity of live cells of D .
radiodurans. In view of the multimetallic composition of
genuine waste , radiocesium sorption in the presence of other
interfering cations was also investigated.
1 　Material and Methods
111 　Isotope
In this study , 134 Cs of which the decay half2life is 2106
year , was selected to replace 137 Cs as a tracer for waste
treatment purpose due to its relatively short physical decay
half2life. 134 CsCl with a specific activity of 317 ×109 kBqΠkg
was purchased from China Institute of Atomic Energy.
112 　Culture and medium
Deinococcus radiodurans R1 was kindly donated by
Professor Yuejin Hua , Institute of Nuclear Agricultural
Sciences. The bacteria were cultured in TGY medium
(pH7) containing 5g of trypone , 1g of glucose , and 3g of
yeast extract per liter of deionized water.
113 　Experiment Methods
11311 　Biosorption ability of D . radiodurans to 134 Cs 　
The sterilized liquid TGY medium was spiked with 134 CsCl
solution and was filtered with microfilter membrane (Millex·
GP filter unit , 0122 º m , Millipore) prior to use. The final
concentrations of 134 Cs in liquid TGY medium were brought
to 140 , 240 , 410 , 480 , 550 , 620 , 690 , 760 , and
830kBqΠL , respectively. Aliquot of 011ml of D .
radiodurans culture solution at a concentration of 1148 ×
108 cfuΠml was inoculated into each sample. The samples
were incubated at 30 ℃for 72h under darkness on a rotary
shaker. The biomass was then collected by centrifugation at
a speed of 5000rΠmin for 10min. The radioactivity in cells
was determined after measuring and recording its fresh
weight . Each concentration was treated in three replications.
All data were represented with the mean value of three
replications. All operations were performed under aseptic
conditions.
11312 　Interference of cations on 134 Cs biosorption 　134 Cs
biosorption in the simultaneous presence of other cations
were tested in bimetallic combinations , by adding an equal
concentration ( 690kBqΠL ) of 134 Cs and increasing
concentrations of test cations. The cations used were Na + ,
012 核　农　学　报 22 卷
K+ , NH+4 , Ca2 + , Zn2 + , Al3 + and Cr3 + . The final
concentrations of Na + , K+ , NH+4 and Zn2 + in TGY liquid
medium were 0 , 5 , 10 , 25 , 50 and 100 mmolΠL ; Ca2 +
were 0 , 10 , 20 , 30 , 40 and 50mmolΠL ; Pb2 + , Al3 + and
Cr3 + were 0 , 0125 , 0150 , 0175 and 1100mmolΠL. The
samples were incubated and treated as above after
inoculation. All data were represented with the mean value
of three replications. Standard deviations and error bars were
indicated wherever necessary.
114 　Measurement
The 134 Cs emitsβ andγ2rays when it decays , which
was measured with a multi2channel γ2spectrometer (model
BH1224 , Beijing Nuclear Instrumentation Factory) equipped
with a NaI scintillation detector (70mmolΠL in diameter)
installed in a lead2shielded chamber. The peak locating
between channel 340 and 450 was selected for radioactivity
determination. The counting error was controlled to be lower
than 5 %.
2 　Results and Discussion
211 　 Biosorption ability of D . radiodurans to
radiocesium
The biosorption of 134 Cs by D . radiodurans cells was
illustrated in Fig. 11 The biosorption of D . radiodurans
cells to 134 Cs increased with the increasing of the
concentrations of 134 Cs. It was apparent that the biosorption
increased rapidly when the 134 Cs concentration was lower
than 690 kBqΠL. The biosorption reached a dynamic
equilibrium state when the 134 Cs concentration was greater
than 690 kBqΠL. The relationship between the biosorption
( Y) and the concentration (X) of 134 Cs could be described
with the following regression equation : Y = - 010015X2 +
3156 X - 301891 According to this equation , the maximum
biosorption capacity of D . radiodurans was about 2 , 100
kBq Πkg (fresh weight basis) when it was in equilibrium
state.
212 　Interference of cations on radiocesium biosorption
The effect of cations of Na + , K+ , NH+4 , Zn2 + on 134
Cs biosorption by D . radiodurans was present in Fig. 21 It
was evident that the increase in the concentration of
interfering ions was accompanied by a concurrent decrease in
the 134 Cs biosorption. When the concentrations of cations
reached to 100mmolΠL , the inhibition to 134 Cs biosorption
Fig. 1 　134 Cs biosorption by D . radiodurans at
different concentrations of 134 Cs
图 1 　耐辐射奇球菌对不同浓度134 Cs 的生物吸收
Fig. 2 　134 Cs biosorption by D . radiodurans
in the presence of Na + , K+ ,
NH+4 and Zn2 + , respectively
图 2 　Na + 、K+ 、NH+4 和 Zn2 + 对耐辐射奇球菌
对134 Cs 生物吸收的影响
was 5315 % for Na + , and 35 % inhibition for K+ , 8813 %
inhibition for NH+4 , and 1414 % inhibition for Zn2 + ,
respectively. The rank of inhibition to 134 Cs biosorption by
cations was NH+4 > Na + > K+ > Zn2 + . Zhu and his
coworkers found that K+ was most important cation that
competes with Cs + uptake by plants among all alkaline
metals and NH+4 [18 ] . Our results showed that NH+4 had the
strongest antagonism on 134 Cs biosorption for D .
radiodurans. Zn2 + , a bivalent cation , did not show a
significant inhibition to 134 Cs biosorption.
The effect of Ca2 + was studied using a range of
concentrations between 0 and 50mmolΠL due to its solubility
in liquid TGY medium (Fig. 3) . 134 Cs biosorption decreased
112　2 期 BIOSORPTION OF RADIOCESIUM BY DEINOCOCCUS RADIODURANS( INFLUENCED BY CATIONS
Fig13 　134 Cs biosorption by
D . radiodurans in the presence of Ca2 +
图 3 　Ca2 + 对耐辐射奇球菌对134 Cs 生物吸收的影响
Fig. 4 　134 Cs biosorption by D . radiodurans in the
presence of Pb2 + , Al3 + and Cr3 +
图 4 　Pb2 + 、Al3 + 和 Cr3 + 对耐辐射奇球菌对
134 Cs 生物吸收的影响
with an increasing Ca2 + concentration. The inhibition to 134
Cs biosorption reached about 65 % when Ca2 + concentration
was only 10mmolΠL. 134 Cs biosorption changed slightly when
Ca2 + concentration was greater than 10mmolΠL.
Pb2 + , Al3 + and Cr3 + were cytotoxic to D .
radiodurans cells. Our preliminary studies showed that the
growth of D . radiodurans cells was inhibited when the
concentrations of these cations were greater than 1mmolΠL.
The effect of these three cations on 134 Cs biosorption within
the concentration range of 1mmolΠL was illustrated in Fig. 41
The inhibition to 134 Cs biosorption was about 30 % for Pb2 + ,
1718 % for Al3 + , and 1117 % for Cr3 + when the
concentration was 0125 mmolΠL. 134 Cs biosorption changed
slightly when the concentration was greater than 0125 mmolΠ
L. Among three tested cations , Pb2 + was the most toxic one
to D . radiodurans.
The results suggested that the biosorption of
radiocesium could be influenced by other cations. The
growth of D . radiodurans could be inhibited by heavy metal
ions , such as Pb2 + , Al3 + and Cr3 + , so that the biosorption
of radiocesium by D . radiodurans was reduced indirectly.
3 　Conclusions
As mentioned previously , D . radiodurans is
exceptionally resistant to ionizing radiation. Our results
suggested that the maximum biosorption capacity of D .
radiodurans could attain 2100kBqΠkg (fresh weight basis)
when being equilibrium state. The biosorption of radiocesium
by D . radiodurans could be influence by other cations.
Although NH+4 has the strongest antagonism on 134 Cs
biosorption for D . radiodurans , this antagonism can only be
observed at a high concentration. The biosorption of
radiocesium by D . radiodurans could also be inhibited by
divalent cations , such as Ca2 + and Pb2 + . The growth of D .
radiodurans cells was inhibited by Al3 + and Cr3 + due to
cytotoxicity when their concentrations were greater than
1mmolΠL.
The study suggests that D . radiodurans has a potential
as a biosorbent for radiocesium removal in radioactive
wastewater remediation , however , a prior dilution in toxic
heavy metal content is necessary. A clear insight into the
biomass radiocesium binding mechanism will improve its
feasibility for practical applications.
Acknowledgements : The work was carried out at Institute
of Nuclear Agricultural Sciences , Zhejiang University. We
thank Prof . Yuejin Hua , Institute of Nuclear Agricultural
Sciences , Zhejiang University , for his kind donation of D .
radiodurans.
参考文献 :
[ 1 ] 　Bell J N , Shaw G. Ecological lessons from the Chernobyl accident .
Environ Int , 2005 , 31 (6) :771～777
[ 2 ] 　White P J , Broadley M. Mechanisms of caesium uptake by plants. New
Phytol , 2000 , 147 (2) :241～256
[ 3 ] 　Rigol A , Vidal M , Rauret G. An overview of the effect of organic matter
on soil2radiocaesium interaction : implications in root uptake. Journal of
Environmental Radioactivity , 2002 , 58 :191～216
(下转第 222 页)
212 Journal of Nuclear Agricultural Sciences
2008 ,22 (2) :209～212
这有利于马铃薯幼苗生长 ,对其后期高产具有重要意
义。叶片含磷量对同化物的运输和分配有促进作用 ,
能延缓叶片和根系衰老 ,促进光合产物的形成及淀粉
的合成。叶片含磷量可用作衡量马铃薯产量的一个重
要生理指标。但是 ,CCC 在光合产物形成中的内在机
制有待进一步研究。
参考文献 :
[ 1 ] 　肖　凯 ,谷俊涛 ,Maria Harrison ,Wang Zeng2Yu. MtPAP1 表达特性
及异源表达对拟南芥有机态磷吸收的影响. 植物生理与分子生
物学学报 ,2006 ,32 (1) : 99～106
[ 2 ] 　Runcie J W , Ritchie R J , Larkum A W D. Uptake kinetics and
assimilation of phosphorus by Catenella nipae and Ulva lactuca can be
used to indicate ambient phosphate availability. Journal of Applied
Phycology ,2004 ,16 : 181～194
[ 3 ] 　Smith DL ,Hamel C著 ,王　璞 ,王志敏 ,周顺利 ,等译. 作物产量—
生理学及形成过程. 中国农业出版社 ,2001 ,10 : 261～277
[ 4 ] 　郭再华 ,贺立源 ,徐才国. 磷水平对不同耐低磷水稻苗根系生长
及氮、磷、钾吸收的影响. 应用与环境生物学报 ,2006 ,12 (4) : 449
～452
[ 5 ] 　Tekalign T ,Hammes P S. Response of potato grown under non2inductive
condition to paclobutrazol : shoot growth , chlorophyll content , net
photosynthesis ,assimilate partitioning ,tuber yield ,quality ,and dormancy.
Plant Growth Regulation ,2004 ,43 : 227～236
[ 6 ] 　Thamir S A N ,Michael L K,Timothy R M. Phosphorus uptake by bean
nodules. Plant and Soil ,1998 ,198 : 71～78
[ 7 ] 　高聚林 ,刘克礼 ,张永平 ,等. 春小麦磷素吸收、积累与分配规律
的研究. 麦类作物学报 ,2003 ,23 (3) : 107～112
[ 8 ] 　袁佐清 ,张怀渝 ,李晚忱. 应用32 P 和3 H示踪研究不同玉米自交系
的抗旱特性. 核农学报 ,2004 ,18 (6) : 468～473
[ 9 ] 　Macklon A E S ,Grayston S J , Shand C A ,et al . Uptake and transport of
phosphorus by Agrostis capillaris seedlings from rapidly hydrolysed
organic sources extracted from 32 P2labelled bacterial cultures. Plant and
Soil ,1997 ,190 : 163～167
[10 ] 　Lehmann J , Muraoka T ,Zech W. Root activity patterns in an Amazonian
agroforest with fruit trees determined by 32 P , 33 P and 15 N applications.
Agroforestry Systems ,2001 ,52 : 185～197
[11 ] 　高聚林 ,刘克礼 ,张宝林 ,盛晋华. 马铃薯磷素的吸收、积累和分
配规律. 中国马铃薯 ,2003 ,17 (4) : 199～203
(上接第 212 页)
[ 4 ] 　Xu D , Li G, Wu L , Zhou J , Xu Y. PRIMEGENS: robust and efficient
design of gene2specific probes for microarray analysis. Bioinformatics ,
2002 , 18 :1432～1437
[ 5 ] 　Kakiuchi H , Amano H , Ichimasa M. Chemical speciation of
radionuclides through the microbial process in soils. Journal of
Radioanalytical and Nuclear Chemistry , 2002 , 252 (2) :437～439
[ 6 ] 　Ghosal D , Omelchenko M V , Gaidamakova E K, et al . How radiation
kills cells : survival of Deinococcus radiodurans and Shewanella
oneidensis under oxidative stress. FEMS Microbiol . Rev. , 2005 , 29 :361
～375
[ 7 ] 　Sar P , D’Souza S F. Biosorptive uranium uptake by a pseudomonas
strain: characterization and equilibrium studies Journal of Chemical
Technology and Biotechnology , 2001 , 76 :1286～1294
[ 8 ] 　Lloyd J R , Lovley D R. Microbial detoxification of metals and
radionuclides. Current Opinion in Biotechnology , 2001 , 12 :248～253
[ 9 ] 　Barkay T , Schaefer J . Metal and radionuclide bioremediation : issues ,
considerations and potentials. Current Opinion in Microbiology , 2001 ,
4 :318～323
[10 ] 　White O , Eisen J A , Heidelberg J F , et al . Genome sequence of the
radioresistant bacterium Deinococcus radiodurans R11 Science , 1999 ,
286 (5444) :1571～1577
[11 ] 　Fredrickson J K, Kostandarithes H M , Li S W , et al . Reduction of Fe
( III) , Cr (VI) , U (VI) , and Tc (VII) by Deinococcus radiodurans
R11 Appl . Environ. Microbiol . , 2000 , 66 (5) :2006～2011
[12 ] 　Levin2Zaidman S , Englander J , Shimoni E , et al . Ringlike structure of
the Deinococcus radiodurans genome : a key to radioresistance ? Science ,
2003 , 299 (5604) :254～256
[13 ] 　Lange C C , Wackett L P , Minton K W , et al . Engineering a
recombinant Deinococcus radiodurans for organopollutant degradation in
radioactive mixed waste environments. Nat Biotechnol , 1998 , 16 (10) :
929～933
[14 ] 　Daly M J . Engineering radiation2resistant bacteria for environmental
biotechnology. Curr Opin Biotechnol , 2000 , 11 (3) :280～285
[15 ] 　Holland A D , Rothfuss H M , Lidstrom M E. Development of a defined
medium supporting rapid growth for Deinococcus radiodurans and analysis
of metabolic capacities. Appl Microbiol Biotechnol , 2006 , 72 (5) :1074
～1082
[16 ] 　Brim H , McFarlan S C , Fredrickson J K, et al . Engineering
Deinococcus radiodurans for metal remediation in radioactive mixed waste
environments. Nat Biotechnol , 2000 , 18 (1) :85～90
[17 ] 　Brim H , Osborne J P , Kostandarithes H M , et al . Deinococcus
radiodurans engineered for complete toluene degradation facilitates Cr
(VI) reduction. Microbiology , 2006 , 152 :2469～2477
[18 ] 　Zhu Y G, Smolders E. Plant uptake of radiocaesium : a review of
mechanisms , regulation and application. J Exp Bot , 20001 51 (351) :
1635～1645
222 Journal of Nuclear Agricultural Sciences
2008 ,22 (2) :218～221