全 文 ：Bioaccumulation of Nickel by Various Scenedesmus Species
in Culture Solution Containing Nickel
HU Zhang_Li2 , WONG Yuk_Shan1＊ , TAM Fung_Yee1
(1.Department of Biology and Chemistry and Centre for Coastal Pollution and Conservation , City University of Hong Kong ,
Hong Kong Special Administrative Region , China;
2.Col lege of Life Sciences , Shenzhen Universi ty , Shenzhen 518060 , China)
Abstract:　Sixteen Scenedesmus species or strains have been employed to investigate the maximum capacity of
nickel(Ni)accumulation in 10 mg/L Ni solution.The results showed that the capacity of accumulating Ni
from aqueous solution in 16 Scenedesmus species or strains showed the diversity.S.quadricauda freshwater
algae culture collection of the Institute of Hydrobiology (FACHB)44 and S .quadricauda FACHB 506 per-
formed much more capacity of Ni accumulation than other species such as Scenedesmus sp.FACHB 416 and
Scenedesmus sp.FACHB 489.Sequestration of Ni ions from aqueous solution was very efficient(26.7mg Ni/
g dry weight , in the 100 mg/L Ni solution)in S.quadricauda FACHB 44.The kinetics of Ni binding indi-
cated that Ni bioaccumulation , in algal cell of S.quadricauda FACHB 44 , possessed a rapid biosorption (5
min)and an slow bioaccumulation(2-3 h).More than 70%of Ni binding in algal cell were accumulated by
biosorption and the remaining 20%-30%were bioaccumulated by energy_consumed transportation.It is
much more higher ratio of energy_consumed transportation in S.quadricauda FACHB 44 than in other algae.
Both the transmission electron microscope(TEM)and the energy_dispersive X_ray (EDX)microanalyses also
revealed the different mechanisms of bioaccumulation in the various subcellular regions:a very fast adsorption
in the cell wall;and a time_dependent absorption in protoplasm , specially in starch and chromatin.
Key words:　bioaccumulation;Scenedesmus;nickel (Ni)
　　 Environmental contamination by heavy metals is
growing concern because of the health risks posed by hu-
man and animal exposure.Nickel (Ni)is mainly located
in core or mantle regions of earth , only at a concentration
of 0.22% is contained in the crust.On average , soils
contain Ni at a concentration of 16 mg/L to 40 mg/L.
The concentration of Ni in the oceans is 0.3 -0.6
mg/L[ 1] .However , somewhat high levels of Ni contami-
nation had been found in many sites such as the electro-
plating effluent which contained very high concentration of
Ni ion.In Hong Kong , there are 507 electroplating firms
operated , Ni with the discharging wastewater flowed into
aquatic environments , meanwhile , Ni also entered the en-
vironments indirectly from industrial and other air emis-
sions.
High concentration of Ni , in wastewater , are haz-
ardous to the environment , due to their toxicity , the exis-
tence of these ions in water may cause toxic and harmful
effects to living organisms in water and also to the con-
sumer of aquatic organism , even human being[ 2 ,3] , so it
is very important work to removal Ni from aqueous solu-
tion.
Microalgae has been recognized as a potential alter-
native for sequstering and recovering metal ions that offer
certain merits over current abiotic practices.Therefore , it
has been suggested as an efficient and cost_effective alter-
native in remediating heavy metal problem.However , Ni
ion was reported as a recalcitrant pollutant with very low
binding capacity in many metal tolerant microalgae[ 4] .
Therefore , the present study aims to focus on bioaccumu-
lation of Ni in strains of Scenedesmus , and measure the
capacity and characteristics of Ni removal from aqueous
solution in Scenedesmus cells , and further investigate the
related mechanisms.
1　Materials and Methods
1.1　Algal species and culture
The species of Scenedesmus were obtained from the
Institute of Hydrobiology , the Chinese Academy of Sci-
ences.Algal cells , grow in a modified Bristol medium
which containing NaNO3 250 mg/L , CaCl2 25 mg/L ,
NaCl 25 mg/L , K2HPO4 75 mg/L , KH2PO4 175 mg/L ,
MgSO4·7H2O 75 mg/L , Fe_EDTA solution 3 mL/L ,
Trace Elements Solution (A5)1 mL/L , pH 6.3 , were
harvested by centrifugation at 5 000 g for 15 min , washed
twice with sterilized distilled water and deionized water for
further experiments.
1.2　Nickel bioaccumulation assay
Stock solution(1 000 mg/L)of Ni was prepared in
deionized water using its chloride salt.Known amounts of
raw biomass was suspended in separate solution(pH 6.0)
containing different concentration of Ni ions , shaken on a
rotary shake at 100 r/min under 167μmol·m-2·s-1 illu-
mination and 25 ℃for 5min to 4 h.Nicekl concentration
was determined by atomic absorption spectroscopy(SHIMADZU AA_680/G V_5).
Received:2001-12-21　Accepted:2002-05-17
＊ Author for correspondence.
植　物　学　报　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　
Acta Botanica Sinica　2002 , 44(8):978-982
1.3 　Ultrastructural analysis of Scenedesmus cell
with Ni
Electron micrographs of Scenedesmus quadricauda
FACHB 44 cells were recorded on a Phillips CM 20 trans-
mission electron microscope according to the procedures of
preparing samples for TEM as described by Silverberg et
al[ 5] .
1.4　The energy_dispersive X_ray(EDX)microanal-
yses
The X_ray energy dispersion spectra were recorded
using the thin section previously prepared for transmission
electron microscopy and the X_ray energy dispersion anal-
ysis(EDAX)detection and data processing unit.For X_
ray diffraction analysis , a JEOL JSM_820 Scanning Micro-
scope and a diffractometer (Link , AN10000/85S)was
employed.
1.5　Algal biomass
Algal cell dry weight and Cell number in solution
were used for expressing the algal biomass.
2　Results and Discussion
2.1　The comparison of Ni accumulation among the
different species of Scenedesmus
Different Scenedesmus species had been measured for
the maximum values of Ni accumulation in 10 mg/L Ni
solution.The results showed that the capacity of accumu-
lating Ni from aqueous solution appeared to be more effi-
cient in S .quadricauda freshwater algae culture collec-
tion of the Institute of Hydrobiology(FACHB)44 and S .
quadricauda FACHB 506 than other strains like
Scenedesmus sp.FACHB 416 and Scenedesmus sp.
FACHB 489(Fig.1).Chong et al[ 6] reported that differ-
ent species had different ability to remove metals in 10
species of green algae , all species could be employed for
treating zinc with a retention time less than 90 min , but
only S .quadricauda FACHB 49 was effective for Ni re-
moval.Figure 1 shows that it was not equally effective in
all the species of Scenedesmus.As displayed in different
species of algae , these strains of Scenedesmus also showed
the diversity of capacity in Ni accumulation , and some re-
lationship were found between the algal morphology and
capacity of Ni accumulation by microscopy analysis.
2.2 　Removal of Ni from aqueous solution by S .
quadricauda FACHB 44
To determine the time required for reaching bioaccu-
mulation equilibrium , initial accumulation experiments
were carried out over 4 h.The reaction system contained
2.67 mg/mL of algal biomass , and 15 mg/L of Ni con-
centration at pH 6.0 , and samples were taken at regular
time intervals.As shown in Fig.2 , the accumulation of
Ni in algal cells was a biphasic bioprocess.The first
phase was a rapid reaction , which 70% of total Ni were
removed by algal cells from aqueous solution in 5 min.
However , the second phase was slower.It needed 2-4 h
to accumulate from about 70% to bioaccumulation equi-
librium(94%)in algal cells.This results is in agreement
with other reports[ 7] .
The Ni bioaccumulation by Scenedesmus cells was a
time_dependent process , indicating that the biosorption of
Ni in the first rapid phase was probably a physical binding
processes between Ni ions and the components of cell
wall.Meanwhile , an energy_consumed bioaccumulation
played an important role in Ni removal by S .quadricau-
da FACHB 44.The energy_consumed absorption of living
algal cell on metal have been described for Cu in Nostoc
calcicola
[ 8] , Ni in Anabena cylindrica[ 9] , and Cr in An-
abena doliolum[ 10] .However , this study showed the ratio
of Ni energy_consumed absorption in S.quadricauda
FACHB 44 was much higher than other algal species(Fig.2).
Fig.1.　The comparison of Ni accumulation among the different
strains of Scenedesmus.
FACHB , freshwater algae culture collection of the Institute of Hy-
drobiology.
Fig.2.　Removal of Ni by Scenedesmus quadricauda FACHB 44.
2.3　Equilibrium bioaccumulation isotherms of Ni by
S .quadricauda FACHB 44
Equilibrium bioaccumulation isotherms of Ni by
HU Zhang_Li et al:Bioaccumulation of Nickel by Various Scenedesmus Species in Culture Solution Containing Nickel 979　
S .quadricauda FACHB 44 showed in Fig.3.The accu-
mulation isotherm represents the equilibrium distribution
of Ni between in cells and in water versus Ni initial con-
centration.The results showed that the bioaccumulation
increased with the Ni initial concentration until reaching
100 mg/L of Ni in the reaction system containing algal
biomass(2.267 mg dry weight/mL).This indicated that
Ni accumulation by S .quadricauda is also an equilibrat-
ed and saturable mechanism.For S .quadricauda
FACHB 44 , the maximum values of bioaccumulation
reached 26.7 mg Ni/g dry weight algal biomass.It was
much more capacity in S .quadricauda FACHB 44 than
other microalgae[ 6 ,11] .These Ni , accumulating in algal
cells , mainly came from passive biosorption of algae to Ni
ions from solution , the metabolism of algal cell had been
broken over high Ni solution because of the toxicity of
Ni[ 12] .
Fig.3.　Equilibrium biosorption isotherms of Ni by Scenedesmus
quadricauda FACHB 44.
2.4　Instrumental analysis of Ni bioaccumulation in
S .quadricauda FACHB 44
The sections of Ni_accumulated cells of S .quadri-
cauda FACHB 44 were examined by transmission electron
microscopy to locate the sequestered Ni.The electron mi-
crographs of S.quadricauda FACHB 44 cells before and
after exposure to Ni_containing solutions are showed in
Fig.4 A and B respectively.As revealed in the micro-
graphs , most of the Ni from solution was accumulated in
the algal cell wall after exposure.It means that most of
the Ni ions sequestered from solution by a rapid bioaccu-
mulation process by algal cell located in cell wall.The
wall of algal cell contain major structural polysaccharide
and other polymers , which combined with heavy metal by
ion exchange , adsorption , coordination , complexation ,
chlation and microprecipitation[ 13] .For the section of al-
gal cell after 3 h bioaccumulation , some Ni ions was also
found interior of algal cells(Fig.4B).Usually , there are
some vacuoles , plastid , and other organelles in cell of S.
quadricauda FACHB 44 without metal treatment , little or
no accumulation of metal ions could be detected (Fig.
4A).However , at high level of Ni , the cytoplasm con-
tained abundant dense bodies which occupied by electron
opaque precipitates of Ni , and the electron_density re-
gions could be found in the starch and nuclear inclusion(Fig.4B).The formation of insoluble , intranuclear met-
al_protein complexes is characteristic of some forms of
heavy metal poisoning[ 14] , metallothinein and phy-
tochaletin were important components of binding heavy
metals in algal cell , so the cytoplasm could contain near
20%-30% of Ni ions in S .quadricauda FACHB 44(Fig.1).On the other hand , the nuclear inclusions were
Fig.4.　Electron micrograph of the Scenedesmus quadricauda FACHB 44 before and after exposed in Ni solution.
W , cell wall;R, chromatin;S , starch.
980　 植物学报　Acta Botanica Sinica　Vol.44　No.8　2002
Fig.5.　The EDAX spectrum of the algal cytoplasm in Scenedesmus
quadricauda FACHB 44 cell exposed in Ni solution for 0 min(A),
5 min(B)and 3 h(C).
gathered as some particles in algal cell exposed on Ni so-
lution , however , it was distributed randomly in the nucle-
oplasm in the normal cell of Scenedesmus (Fig.4B).It
also proved that Ni was incorporated into the nuclei of
Scenedesmus cell treated with Ni.　　The electron_dense areas observed on the electron
micrographs were further conformed as Ni by EDAX.The
EDAX probe , which connected to a scanning electron
microscope , was focused on the electron_dense areas of
algal cytoplasm.Figure 5 shows the typical EDAX spectra
of algal cell which exposed in Ni solution for 0 min(Fig.
5A), 5 min(Fig.5B), 3 h(Fig.5C).It further indicat-
ed that the most Ni absorbed by energy_consumed trans-
portation was accumulated mainly in starch and nuclear
inclusion.
Acknowledgements:The authors thank the Centre for
Coastal Pollution and Conservation of City University of
Hong Kong for Partial Funding of this Project.
References:
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[ 2] 　Stokes P.Nickel and Its Role in Biology.New York:Mar-
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New York:John Wiley and Sons Inc., 1992.201-210.[ 4] 　Wong Y S , Tam N F Y.Wastewater Treatment with Algae.
Berlin:Springer_Verlag , 1997.17-33.[ 5] 　Silverberg B A , Stokes P M , Ferstenberg L B.Intraunucle-
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ent microalgal species in removing nickel and zine from in-
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copper efflux as a mechanism of Cu2+ resistance in the
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HU Zhang_Li et al:Bioaccumulation of Nickel by Various Scenedesmus Species in Culture Solution Containing Nickel 981　
栅藻对水环境中镍的累积效应与机理分析
胡章立2 　黄玉山1＊　谭凤仪1
(1.香港城市大学生物及化学系海岸污染与保护中心 , 香港;2.深圳大学生命科学学院 , 深圳 518060)
摘要:　对不同 Scenedesmus 品种的藻细胞从含镍水溶液(10 mg/ L)中累积金属镍的能力进行了分析 , 结果表明:藻
细胞对镍的生物累积量表现出明显的品种差异性。 Scenedesmus quadricauda FACHB 44 和 Scenedesmus quadricauda
FACHB 506 表现出很强的累积能力(累积量达到 5 ～ 6 mg Ni /g 干重),而 Scenedesmus sp.FACHB 416 和 Scenedesmus
sp.FACHB 489 在相同条件对金属镍累积量要少得多(1～ 1.5 mg Ni /g 干重)。这种差异可能与不同品种藻细胞间
的形态结构和生理特性是相关的。 对 S.quadricauda FACHB 44 重金属抗性和累积能力进一步的分析表明 , S.
quadricauda FACHB 44 用于含镍重金属废水处理是非常有效的 ,在高浓度(100 mg/ L)的镍溶液中 , 藻细胞的最大累
积量能达到(26.7 mg Ni/ g 干重)。对该藻细胞镍累积动力学分析发现:藻细胞对镍的生物累积包括一个快速的被
动吸附过程(5 min , 结合 70%的镍)和一个缓慢的耗能累积过程(2 ～ 3 h 时间内的累积量占总量的 20%～ 30%)。
与其他藻类相比 , S.quadricauda FACHB 44 对水溶液中镍的耗能累积量明显高于其他藻类。透射电子显微镜
(TEM)和 X 射线能谱(EDX)分析结果均表明 ,藻细胞耗能累积的镍主要集中在原生质体中 ,尤以淀粉粒和染色质中
为多。
关键词:　生物累积;栅藻;镍
中图分类号:Q945.79　　　文献标识码:A　　　文章编号:0577-7496(2002)08-0978-05
收稿日期:2001-12-21　接收日期:2002-05-17
＊通讯作者。
(责任编辑:韩亚琴(实习))
982　 植物学报　Acta Botanica Sinica　Vol.44　No.8　2002