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
on biological removal of arsenic I and their
biotransformation in the aquatic environment as
arsenical compounds have been established as
carcinogenic substances 2-4. Inorganic arsenic (As)
ingested as As (III) or As(V) state. Before
methylation, arsenate is reduced to arsenite. During
methylation two steps takes place-(1) arsenite
methylated to methyl arsenate (MA); and (2), it is
further methylated to dimethyl arsenate (DMA). Both
MA and DMA are less toxic than inorganic arsenicals
(arsenite and arsenate) and less tissue binding
affinit/. Methylation is a natural detoxification
process by sequent;al reduction/methy lation in
biotransformation of inorganic arsenical substances.
Many fungi can transform inorganic arsenic species,
sodium methylarsenate(CH 3AsO(ONah] and sodium
cacodylate (CH 3hAsO(ONa)] into trimethyl arsine,
whereas bacteria will produce anaerobically dimethyl
arsine 6- IO .
Based on severa] studies showing that the
methylation of As (III) takes place mainly in the liver
cystosols by enzymatic catalysis 11-13. Majority of
arsenic accumu lates in the organisms as dimethyl
arsenic compounds and are mainly found in aJgal
bodyl4-16 and trimethyl arsenic compounds such as
arsenobetaine have been found in crustacean .
Inorganic arsenic may undergo several biochemical
transformations. The hydroxyl group of the arsenic
acid AsO(OHh is replaced by CH3 group to form
*Correspol1d.;m author: E-mail : scsantra @yahoo.com
CH)AsO(OH)2 (MA), (CH))2AsO(OH) (DMA) and
(CH)hAsO (TMA) 17. These transformations usually
occur biologically in the aquatic environment. First
inorganic ar~;enic is incorporated by autotrophic
organisms such as algae and then it is transported
through food chain . During transfer methylation of
arsenic progres,,~ within the body of organisms.
Methylation of 'arsenic is probably the main
detoxifying process for organisms. Both the fresh
water and marine organisms are capable to bio-
accumulate arsenic from their aquatic environment.
As the fresh water algae has enormous capacity to
bio-accumulate and bio-transfonn inorganic arsenic,
so they can be used in removal process2 , 1 8, 1 ~. The
present investigation was undertaken to study the
removal pattern of arsenic from contaminated water
using some algal cultures.
Materials and Methods
Determination of total arsenic-Total arsel1lc was
determined spectrophotometrically using silver
diethyl dithiocarbamate (Ag-DDTC) by following the
Guitzeit method 2o . This method later modified by
other workers 21 ,22. In this method, 50 ml of sample
was taken in a conical flask (150 ml), and added conc.
HCI (15 mI), 15% of KI (2 ml) and 40% of SnCh
(0.5 ml) (prepared in conc. HCl). The mixture was
kept for 5-10 min, added 4 g of granular zinc in the
flask and connected immediately t!1e absorbing tube
to the flask by means of standard joint, that contains 7
ml of Ag-DDTC solution (0.5% Ag-DDTC and 3% of
hexamethylene tetrarnine dissolve in chloroform).
 SAMAL et al. : ARSENIC REMOVAL USING MICROALGAE
Arsine gas generated in the conical flask passes
through the glass wool soaked with saturated lead
acetate solution (place within the absorbing tube, just
above the ground joint) to absorb any H2S gas
produced in it and then bubbled into the Ag-DDTC
solution which turn slowly red. After 30 min,S ml of
HCl 0:1) was again added through a funnel, and kept
for 10 min so as to pass evolved gas through absorber
tube. Finally , the absorbing solution was
quantitatively transformed to a volumetric flask 00
ml), the volume was made up to the mark with
chloroform and the absorbance was measured at 520
nm against the reagent blank prepared in the same
procedure in spectrophotometer (model Spectronic
200). Finally, the concentration of arsenic was
calculated from calibration curve prepared with
known concentration of arsenic.
Collection and culture of organisms-Six different
types of microalgae were collected and isolated from
different sources. Among the six microalgae strains
three belonged to green algae category, one euglenoid
type and rest two were blue green type. These
organisms were Scenedesmus hijuga,
Ankiostrodesmus convolutus, Chlorella vulgaris,
Euglena gracilis, Spirulina platens is and Oscillatoria-
Lyngbia mixed culture. Among the six algal strains
Spirulina platensis and Osciliatoria-Lyngbia mixed
culture and Chlorella vulgaris were collected from
arsenic polluted areas and Scenedesmus bijuga,
Ankiostrodesmus COil volutes and Euglena gracilis
were collected from culture collection center (NFMC,
Bharathidasan University). Each group of algae was
grown in their respective medium. Green algae in
Chu-lO medium 23 , euglenoid in tryptone-beef extract
medium 23 and blue green algae in Zarrouk medium 24 .
The collected algal strains were first made to pure
culture. Theil the strains were maintained by sub-
culturing in respective medium under illurr.;nation at
2500 lux for 18 hr light and 6 hr dark phase at room
temperature (282C).
Growth response testing-Algal strains were
separately studied by growth estimation test with
response to the effect of As(III) and As(V). The
respecti ve media (broth) was prepared for different
algal strains and dispersed in 100 ml conical t1ask @
50 mllflask, then autoclaved. These medium23.24 were
fed with sterile As (III) and As(V) stock solution
separately, to get the concentration ranging from 0.1
to 1 mg/l (0.1 , 0.5 and 1 mg/l) and then inoculation
was done with algae in thei r respective medium,
aseptically. These three concentrations were taken for
523 .
consideration because the maximum ground water
arsenic concentration in West Bengal is within the
range of 1mg/!. However, higher concentration of
arsenic showed adverse effect on the growth rate of
the studied algal strains. The inoculum den si ty was
maintained in equal amount. The density of inoculum
for Scenedes/llus bijuga, Ankiostrodesmus COIlVO!Utus,
Chlorella vulgaris, and Euglena grac ilis was
maintained at 2.4x104 cells/ml, and in Spirulina
platensis and LYllgbia-Osciliatoria mixed culture, it
was about 9.3xl03 cells/m!. A control set also run
parallel without arsenic. The cultures were kept under
illumination at 2500 lux for 18 hr light and 6 hI' dark
phase at room temperature (282C). On each
alternate day, each set of algal culture in triplicate for
each concentration was removed and the optical
density was measured at 635 nm using
spec trophotometer (model Spectronic 200) with
respect to a control set as blank (without arsenic).
This reading was taken upto 24 days.
Rate of removal of As(lIl) and As(V) using selected
algae-Cultures were prepared in conical flask (l00
ml) containing 50 ml of media supplemented with
different concentration of inorganic As(IIl) and
As(V). The culture were incubated under light (2500
lux) for 18 hr light and 6 hr dark at 282C. At 7
days interval, separate sets of cultures were
centrifuged at 2000 rpm for 15 min, collected the
supernatant and analysed for arsenic. Each ex periment
was done in triplicate analysed spectrophotometrically
using Ag-DDTC.
Results and Discussion
Growth response-Growth pattern of microalgae in
relation to different concentration of As (III) and
As(V) has been shown in Figs 1, 2. Degree of
inhibition varied widely in different algal species. The
growth rate of algae reduced due to the presence of
arsenic in the culture rriedium.
In As (III) and As(V) supplemented medium, all
the six algae showed gradual decrease in growth rate
with increase of arsenic concentration. The growth
rate was inhibited more by As(III) than As(V) in all
cases (Figs 1, 2). Spin1-1 ina platensis reached
stationary phase in 18-22 days in both As(III) and
As(V) enriched medium and then growth rate
declined. Oscillatoria sp. and LYllgbya sp. in normal
culture medium as well as in As(III) and As(V)
enriched medium showed log phase up to 18 days,
after that growth rate ceased and gradually declined.
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)
showed similar growth rate. Exponential growth
phase was there up to 22 days. Another green algae,
Allkiostrodeslllus convolutes, showed exponential
growth phase lip to 20 days then growth rate ceased
gradually and then declined in medium enriched with
As(III) and As(V). Cldorella vulgaris another green
alga, showed more or less same result like other two
green algae, but its exponen tial phase stayed up to 22
days and then gradually decreased. The euglenoid,
Euglena gracili's showed growth pattern different
from the others. At the initial stage, growth rate was
high. From 4 to 10 days however, growth rate
increased but not like the initial growth rate. After 10
days, the growth rate gradually decreased.
Rate of reJlloval of As(III) and As(V) using the
selected algae- Rate of removal of As(III) and As(V)
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
the selected algae was studied at an interval of 7 days.
Efficiency of removal of arsenic by these organisms
was different, but their patterns of removal were more
or less similar. The removal percentage increased
with the time and was higher at the exponential phase
of growth. After 21 days, growth rate ceased, so th at
the removal percentage also decreased. Increasing of
concentration of arsenic In medium, was directly
related to decrease of removal percentage. The growth
rate decreased due to the increase in concentration of
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
increased due to growth , they may incorporate more
amount of arsenic fro m the medium and also
methylate arseni c within their own system that may
resul ted in decrease of arsenic concentration in the
medi um. By comparing the removal of As(lII) and
As(V) by these organi sms , As(V) was removed more
than As(lII) by the same species under the same
culture co ndition s at same interval. It could be
concl uded that removal percentage of As(V) was
h'gl-jer than As(lII) by the algal strains under study .
References
Katsoyianni s I A & Zouboul is A I, App lication of biological
processes for the removal of arsenic fro m ground waters,
Water Res, 38 ( I) (2004) 17.
2 Maeda S, Kumam oto M, Nakashima S, Takeshi ta T, Hi gashi
S & Ueno K, Bioaccumulation of arsenic by freshwa ter algae
and the app lication to the removal of inorganic arsenic from
an aq ueo us phase. Part I- Screenin g of freshwater al gae
havi ng hi gh resistance to inorga nic arsenic. Separation Sci
Tecllllol, 18 (1 %3) 375
3 Maeda S, Nakashima S, Takeshita T & Higashi S,
Bioaccumulation of arsenic by freshwate r algae and the
ap plication io the removal of inorganic arsenic from an
aqueo us phase. Part II-By Ch lorella viligaris isolated from
arsenic polluted environment, Separation Sci Technol, 20
(1985) 153.
4 Maeda S, Kumeda K. Maeda M, Hi gashi S & Takeshita T,
Bioaccu mlliati on of arsen ic in freshwater algae (Nostoc sp.)
and the applicati on to the re moval of inorga nic arsenic from
an aqueous phase, Appl Orgallolll etal Chelll , I (1987 ) 363.
5 US EPA, Speci al report on ingested inorganic arsenic: Skin
cancer, nutri tional essenti ality. (US : J:: nvironmenta l
Protecti on Agency, Washin gton, D.C.) 1988, [EPA 625/3-
87/01 3].
6 Cox D P & Alexander M, Production of trimeth yl ars ine gas
from various arsen ic compou nd s by three sewage fungi , Bull
EiI viroll Contalll Toxicol. 9 (1973 ) 84.
7 Maeda S, Kusadome K, Arima H, Ohki A & Naka K, Up take
and exc retion of total inorgani c arsenic by th e freshwater
algae Chlorella viligaris, Appl Orgall ollleal Chel11, 6 ( 1992)
399.
8 Bhar G, Samal A C & Santra S C, An in ves tigation on
arse nic remova l by soil fungal isolates, Sci Clllt, 69 (2003)
157.
9 Hu ysmans H D & Frankenberger W T, Evolution of
trimethyl ars ine by PellicciliulIl sp. isolated from agri cu ltural
evaporation pond water, Th e Science of total ell virolllllellf,'
Vol 105 (Elsevier Science Publi shers), 1991 , 13.
JO Mc Bride B C, Mesilee H, Cu llen W R & Pickett W,
Anaerobic and aerob ic alkylation of arsenic. 111
OrgmlOllletals alld orgallometalloids occurrallce alld fate ill
environlllent, edited by F E Brickman & J M Bel lama. Alii
Chem Soc SYIIIP Ser, 82 (1978) 94.
11 Vahter M, Biotransformati on of trivalent and pentava lent
inorganic arsenic in mice and rats, Ellviron Res , 25 ( 1981)
286.
12 Vahte r M & Morafante E, In tracellu lar interacti on and
metabolic fate of arsenite and arsenate in mice and rabbit,
Chelll Biollnterac, 47 (1983) 29.
13 Buahet J P, Lanwerys R & Roels H, Compari son of the
urinary excretion of arsenic metabolite< lfter a single oral
dose of sodium arsenite, monomethyl arsL. Ite, or dimethyl
arsinate in man , lilt A rch Occup En viroll HeeL" h, 48 (1981)
71.
14 Morita M & Shi bata Y, Chemical fo rm of arsen ic in marine
microalgae, Appl OrgwwlIletal Ch elll, 4 (1990) 181.
IS Ed monds J S & France~.c>l1i K A, Arsenosugars from Brow n
Kelp (Eklollia radiata) as intermedi ates in cycling of arsen ic
in a marine ecosystem, Nature (LOlldoll )' 289 (1981) 602,
16 Bottino N R, Cox E R, Irgolic K J, Maeda S, Mc. Shane W J,
Stockton R A & Zingaro R A, Arsenic uptake and
metabolism by the algae Tetrasellllis ch ui, in Organometals
and organometalloids Vol 82 (Ameri can Chemical Society,
USA) 1978, 116.
17 Lem mo N V, Faust S D, Belton T & Tucker R, Assess ment
of the chemical and biological significance of arseni cal
compounds in a heavil y contaminated watershed. Part 1-
The fate and speciation of arsenical compounds in aq uat ic
environments-A literature review, J Ellviron Sci Health , A l 8
( 1983) 335.
18 Maeda S, Wada H, Kumeda K, Onoue M, Ohki A, Higashi S
& Takeshita T, Methylation of inorganic arsenic by arseni c
to lerant freshwater algae, Appl Orgallolll efai Chelll. 1 ( 1987)
465.
19 Maeda S, Kumeda K, Ari ma H, Ohk i A & Naka K,
Biomethylation of arsenic and its excretion by an alga
Chlorella vlllga ris, Appi Organollletai Chell1, 6 (1992) 407 .
20 Vogel A E, A tex tbook of macro and semi macro qualitative
inorganic analys is, 4th edition (Longmans, London) 1955.
2 1 Koop J F, Ephid rin e in chloroform as a solvent for sil ver
di ethyl dithiocarbamate in th e determin at ion of arsenic, Allal
Chem , 45 (1973) 1786.
22 Sandhu S S & Nelson P, Ionic interference in the
determination of arsenic in water by the sil ver diethyl
dithiocarbamate method, Anal Chelll , SO (1978) 322.
23 Stein J R, Handbook of phys iological methods-culture
meth ods and grow th measurement, (Cambridge Universi ty
Press) 1973 , 448.
24 Zarruk C, Contributi on aJ' etude d'un e cyanophyceae.
Influence de divers facte urs physiqueI' et chemiques sur
lacroissance et al. photosynthese de Spirulina lII (uilll[l
(Setch. Et Gardner) Geitler, Ph .D. Thesis, Uni versity of
Pari s, France (1966).