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lndian Journal of Experimental Biology

Vol. 42, May 2004, pp. 522-528

Biological process of arsenic removal using selected micro algae


A C Samal, G Bhar & S C Santra*
Department of Environmental Science, University of Kalyani, Ka lyani 741 235, India
Received J October 2003; revised 2 J January 2004

In the present invest igation, growth of the organisms was reduced due to presence of arsenic (III) and (V) in the culture
medium. In comparison to arsenic (V), arsenic (III) had more toxic effect on microalgae. Among the different algal strains,
blue green algal species Oscillatoria-LYllgbya mixed culture showed maximum efficiency in removing arsenic (64%) after
21 days of incubation and the same algal species could remove arsenic (III), but 60% after 21 days when incubated in 0.1
mg/l arsenic (III) containing medium. Maximum removal was observed at their exponential growth phase and also sometime
extended to the stationary phase.

Keywords: Arsenic remova l, Microalgae, Scenedeslllus bijltga, Allkiostrodes/llus cOllvolutus, Chlorellavulgaris, Euglella
gracilis, SpiruLillo platellsis, Osciliatoria-LYllgbia combination
IPC Code: Int.CI 7 CI2S

There are considerable studies made over few decades CH)AsO(OH)2 (MA), (CH))2AsO(OH) (DMA) and
on biological removal of arsenic I and their (CH)hAsO (TMA) 17. These transformations usually
biotransformation in the aquatic environment as occur biologically in the aquatic environment. First
arsenical compounds have been established as inorganic ar~;enic is incorporated by autotrophic
carcinogenic substances 2-4. Inorganic arsenic (As) organisms such as algae and then it is transported
ingested as As (III) or As(V) state. Before through food chain . During transfer methylation of
methylation, arsenate is reduced to arsenite. During arsenic progres,,~ within the body of organisms.
methylation two steps takes place-(1) arsenite Methylation of 'arsenic is probably the main
methylated to methyl arsenate (MA); and (2), it is detoxifying process for organisms. Both the fresh
further methylated to dimethyl arsenate (DMA). Both water and marine organisms are capable to bio-
MA and DMA are less toxic than inorganic arsenicals accumulate arsenic from their aquatic environment.
(arsenite and arsenate) and less tissue binding As the fresh water algae has enormous capacity to
affinit/. Methylation is a natural detoxification bio-accumulate and bio-transfonn inorganic arsenic,
process by sequent;al reduction/methy lation in so they can be used in removal process2 , 1 8, 1 ~. The
biotransformation of inorganic arsenical substances. present investigation was undertaken to study the
Many fungi can transform inorganic arsenic species, removal pattern of arsenic from contaminated water
sodium methylarsenate(CH 3AsO(ONah] and sodium using some algal cultures.
cacodylate (CH 3hAsO(ONa)] into trimethyl arsine,
whereas bacteria will produce anaerobically dimethyl Materials and Methods
arsine 6- IO . Determination of total arsenic-Total arsel1lc was
Based on severa] studies showing that the determined spectrophotometrically using silver
methylation of As (III) takes place mainly in the liver diethyl dithiocarbamate (Ag-DDTC) by following the
cystosols by enzymatic catalysis 11-13. Majority of Guitzeit method 2o . This method later modified by
arsenic accumu lates in the organisms as dimethyl other workers 21 ,22. In this method, 50 ml of sample
arsenic compounds and are mainly found in aJgal was taken in a conical flask (150 ml), and added conc.
bodyl4-16 and trimethyl arsenic compounds such as HCI (15 mI), 15% of KI (2 ml) and 40% of SnCh
arsenobetaine have been found in crustacean . (0.5 ml) (prepared in conc. HCl). The mixture was
Inorganic arsenic may undergo several biochemical kept for 5-10 min, added 4 g of granular zinc in the
transformations. The hydroxyl group of the arsenic flask and connected immediately t!1e absorbing tube
acid AsO(OHh is replaced by CH3 group to form to the flask by means of standard joint, that contains 7
ml of Ag-DDTC solution (0.5% Ag-DDTC and 3% of
*Correspol1d.;m author: E-mail : scsantra @yahoo.com hexamethylene tetrarnine dissolve in chloroform).
SAMAL et al. : ARSENIC REMOVAL USING MICROALGAE 523 .

Arsine gas generated in the conical flask passes consideration because the maximum ground water
through the glass wool soaked with saturated lead arsenic concentration in West Bengal is within the
acetate solution (place within the absorbing tube, just range of 1mg/!. However, higher concentration of
above the ground joint) to absorb any H2S gas arsenic showed adverse effect on the growth rate of
produced in it and then bubbled into the Ag-DDTC the studied algal strains. The inoculum den si ty was
solution which turn slowly red. After 30 min,S ml of maintained in equal amount. The density of inoculum
HCl 0:1) was again added through a funnel, and kept for Scenedes/llus bijuga, Ankiostrodesmus COIlVO!Utus,
for 10 min so as to pass evolved gas through absorber Chlorella vulgaris, and Euglena grac ilis was
tube. Finally , the absorbing solution was maintained at 2.4x104 cells/ml, and in Spirulina
quantitatively transformed to a volumetric flask 00 platensis and LYllgbia-Osciliatoria mixed culture, it
ml), the volume was made up to the mark with was about 9.3xl03 cells/m!. A control set also run
chloroform and the absorbance was measured at 520 parallel without arsenic. The cultures were kept under
nm against the reagent blank prepared in the same illumination at 2500 lux for 18 hr light and 6 hI' dark
procedure in spectrophotometer (model Spectronic phase at room temperature (282C). On each
200). Finally, the concentration of arsenic was alternate day, each set of algal culture in triplicate for
calculated from calibration curve prepared with each concentration was removed and the optical
known concentration of arsenic. density was measured at 635 nm using
Collection and culture of organisms-Six different spec trophotometer (model Spectronic 200) with
types of microalgae were collected and isolated from respect to a control set as blank (without arsenic).
different sources. Among the six microalgae strains This reading was taken upto 24 days.
three belonged to green algae category, one euglenoid Rate of removal of As(lIl) and As(V) using selected
type and rest two were blue green type. These algae-Cultures were prepared in conical flask (l00
organisms were Scenedesmus hijuga, ml) containing 50 ml of media supplemented with
Ankiostrodesmus convolutus, Chlorella vulgaris, different concentration of inorganic As(IIl) and
Euglena gracilis, Spirulina platens is and Oscillatoria- As(V). The culture were incubated under light (2500
Lyngbia mixed culture. Among the six algal strains lux) for 18 hr light and 6 hr dark at 282C. At 7
Spirulina platensis and Osciliatoria-Lyngbia mixed days interval, separate sets of cultures were
culture and Chlorella vulgaris were collected from centrifuged at 2000 rpm for 15 min, collected the
arsenic polluted areas and Scenedesmus bijuga, supernatant and analysed for arsenic. Each ex periment
Ankiostrodesmus COil volutes and Euglena gracilis was done in triplicate analysed spectrophotometrically
were collected from culture collection center (NFMC, using Ag-DDTC.
Bharathidasan University). Each group of algae was
grown in their respective medium. Green algae in Results and Discussion
Chu-lO medium 23 , euglenoid in tryptone-beef extract Growth response-Growth pattern of microalgae in
medium 23 and blue green algae in Zarrouk medium 24 . relation to different concentration of As (III) and
The collected algal strains were first made to pure As(V) has been shown in Figs 1, 2. Degree of
culture. Theil the strains were maintained by sub- inhibition varied widely in different algal species. The
culturing in respective medium under illurr.;nation at growth rate of algae reduced due to the presence of
2500 lux for 18 hr light and 6 hr dark phase at room arsenic in the culture rriedium.
temperature (282C). In As (III) and As(V) supplemented medium, all
Growth response testing-Algal strains were the six algae showed gradual decrease in growth rate
separately studied by growth estimation test with with increase of arsenic concentration. The growth
response to the effect of As(III) and As(V). The rate was inhibited more by As(III) than As(V) in all
respecti ve media (broth) was prepared for different cases (Figs 1, 2). Spin1-1 ina platensis reached
algal strains and dispersed in 100 ml conical t1ask @ stationary phase in 18-22 days in both As(III) and
50 mllflask, then autoclaved. These medium23.24 were As(V) enriched medium and then growth rate
fed with sterile As (III) and As(V) stock solution declined. Oscillatoria sp. and LYllgbya sp. in normal
separately, to get the concentration ranging from 0.1 culture medium as well as in As(III) and As(V)
to 1 mg/l (0.1 , 0.5 and 1 mg/l) and then inoculation enriched medium showed log phase up to 18 days,
was done with algae in thei r respective medium, after that growth rate ceased and gradually declined.
aseptically. These three concentrations were taken for The green algae, Scenedesmus bijuga, at normal as
524 INDIAN J EXP BIOL, MAY 2004

0.8 0.8
(A) -normal
0.7 0.7
--0.1 mg/l
0.6 0.6
0.5 0.5
0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1
0 0
2 4 6 8 10 12 14 16 18 20 22 24 2 4 6 8 10 12 14 16 18 20 22 24
0.7 0.7

0.6 0.6
S
~
V)
M
0.5 0.5
\0
...... 0.4 0.4
ro
v
u
~ 0.3 0.3
ro
.n
~
0 0.2 0.2
rJ)

.n
<r: J.l 0.1

0 0
2 4 6 8 10 12 14 16 18 20 22 24 2 4 6 8 10 12 14 16 18 20 22 24
0.7 . , - - - - - - - - - - - - - - - - - , 0.7 , - - - - - - - - - - - - - - - - ,

0.6 0.6

0.5 0.5

0.4 0.4

0.3 0.3

0.2 0.2

0.1 0.1

2 4 6 8 10 12 14 16 18 20 22 24 2 4 6 8 10 12 14 16 18 20 22 24
Days

Fig. i-Effect of dirferent concentration of arsenic (1lI) on growth rate of different selected algae-(A) Spirt/filla pialellsis; (B)
Oscillaloria and L)'Jlgb)'a mix culture; (C) SceJledcslllus bijllga; (D) AJlkislrodeslIIlIs cOllvollllllS; (E) Chlorella vulgaris; and (F) Euglella
gracilis .

well as in different concentration of As(III) and As(V) days and then gradually decreased. The euglenoid,
showed similar growth rate. Exponential growth Euglena gracili's showed growth pattern different
phase was there up to 22 days. Another green algae, from the others. At the initial stage, growth rate was
Allkiostrodeslllus convolutes, showed exponential high. From 4 to 10 days however, growth rate
growth phase lip to 20 days then growth rate ceased increased but not like the initial growth rate. After 10
gradually and then declined in medium enriched with days, the growth rate gradually decreased.
As(III) and As(V). Cldorella vulgaris another green Rate of reJlloval of As(III) and As(V) using the
alga, showed more or less same result like other two selected algae- Rate of removal of As(III) and As(V)
green algae, but its exponen tial phase stayed up to 22 by six test algae has been shown in Figs 3, 4. In the
SAMAL el al.: ARSENIC REMOVAL USING MICROALGA E 525

0.8 0.8
(A) (B)
0.7 0.7
-nomlal
0.6 -o- O.lmg/I 0.6
- - 0.5 mgt 1
0.5 0.5
---*-1.0 mg / 1
0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1
0 0
2 4 6 8 10 12 14 16 18 20 22 24 2 4 6 8 10 12 14 16 18 20 22 2d
0.7 0.7

0.6 0.6
E
~
VI 0.5 0.5
c-'l
\0
~

ro 0.4 0.4
<U
u
c 0.3 0.3
ro
,D
.....
0 0.2 0.2
r./J
..D
-< 0. 1 0.1

0 0
2 4 6 8 10 12 14 16 18 20 22 24 2 4 6 8 10 12 14 16 18 20 22 24
0.7 0.7
(E)

0.5
0.6

0.5
,l
0.4 0.4

0.3 0.3

0.2 0.2

0.1 0.1

0 0
2 4 6 8 10 12 14 16 18 20 22 24 2 4 6 8 10 12 14 16 18 20 22 24
Days
Fig. 2-Effect of different co nce ntrat ion of arse ni c (V) on growth rate of different selec ted algae-(A) Sp imlina plalensis: (B)
Oscil/a/oria and Ly ng bya mi x cu lture; (C) Sccned e.\ /II1I S bijllga; (D) AII/':islrode5111l1s cOII\'011II1I5; (E) Chlorel/a \'IIlga ris; and (F) Ellglella
gracilis
526 INDIAN J EXP BIOL, MAY 2004

50 ~--------------------------------~ 70,---------------------------------
45 130.1 mgll (A) (B)
40 IZI 0.5 mg /1
35 rn 1.0 mg! 1
30
25

20

15

10

5
O+-~~~UL-r_~~~llll-.-L~~Ull~

7 14 21 7 14 21
50 ,-----------------------------~ .50 , - - - - - - - - - - - - -- - - -- - - _
45 45
40 40
"@ 35 35
5 30 30
25
}..o
25
4-<
o 20 20
~ 15 15
10 10
5 5
o +-~~~~~~~~~~~~~~~~~ O+-~~~~._~~~UL._~~~~

7 14 21 7 14 21
45 ~--------------------------------- 60,----------------------------,
(E) (F)
40
35
30

25
20
15

10

5
O +-~~~li_~~~~WL._~~~~

7 14 21 7 14 21
Days

Fig 3-Per cent of removal of arsenic(llI) by selected algae at different duration-(A) Spirulina platensis; (B) Oscillatoria and LYllgbya
mix culture; (C) Scelledeslllus bijuga; (D) A Il kistrodes1I111s cOllvolutus; (E) Chlorella vulgaris; and (F) Euglena gracilis
SAMAL et al.: ARSENIC REMOV AL USING MICROALGAE 527

60 .---------------------______~ 70.--------------------------__~
DO.lmg/1 (A)
50 1210.5 mg /1
[] 1.0 mg / 1
40

30

20

10

7 14 21 7 14 21
60~----------------------------~ 60.-----------------------------.
(C) (D)
50

CiJ 40
o>
E
d)
30
....
4-.
o 20
'2f:-
10

7 14 21 7 14 21
50.-----------------------------~ 60 .------------------------------~

45
40
35
30
25
20
15
10
5
O+-~~UliL,~~~llll_.~~~~

7 14 21 7 14 21

Days
Fig 4--Per cent of removal of arseni c(V) by selected algae in different duration--(A) Spirulilla platensis; (B) Oscillatoria and LYllgbya
mi x culture; (C) Scelledesmus bijuga; (D) Allkistrodesmus cOI1VOlulus; (E) Chlorella vulgaris; and (F) Euglena gracilis

present study, removal of As(III) and As(V) through of growth. After 21 days, growth rate ceased, so th at
the selected algae was studied at an interval of 7 days. the removal percentage also decreased. Increasing of
Efficiency of removal of arsenic by these organisms concentration of arsenic In medium, was directly
was different, but their patterns of removal were more related to decrease of removal percentage. The growth
or less similar. The removal percentage increased rate decreased due to the increase in concentration of
with the time and was higher at the exponential phase arsenic (Figs 1, 2) . The growth rate was directly
528 INDIAN J EXP mOL, MAY 2004

relation to removal of arsenic. As the number of cells OrgmlOllletals alld orgallometalloids occurrallce alld fate ill
environlllent, edited by F E Brickman & J M Bel lama. Alii
increased due to growth , they may incorporate more
Chem Soc SYIIIP Ser, 82 (1978) 94.
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resul ted in decrease of arsenic concentration in the 286.
medi um. By comparing the removal of As(lII) and 12 Vahte r M & Morafante E, In tracellu lar interacti on and
As(V) by these organi sms , As(V) was removed more metabolic fate of arsenite and arsenate in mice and rabbit,
Chelll Biollnterac, 47 (1983) 29.
than As(lII) by the same species under the same
13 Buahet J P, Lanwerys R & Roels H, Compari son of the
culture co ndition s at same interval. It could be urinary excretion of arsenic metabolite< lfter a single oral
concl uded that removal percentage of As(V) was dose of sodium arsenite, monomethyl arsL. Ite, or dimethyl
h'gl-jer than As(lII) by the algal strains under study . arsinate in man , lilt A rch Occup En viroll HeeL" h, 48 (1981)
71.
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