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281
IRON REMOVAL FROM GROUNDWATER USING LEPTOTHRIX etc .....
clogged emitters (Smith
et al.,
1997; McNeill
and Edwards, 2001 and UNDP, 2010).
In groundwater, Fe (II) are being removed
by either physico-chemically or biologically
based-methods. The biological treatments are
more advantages than conventional physico-
chemical treatments because no use of chemi-
cals, have higher filtration rates, the possibili-
ty of using direct filtration and lower opera-
tion and maintenance costs (Mouchet, 1992).
Conventionally, iron is removed from ground-
water by the processes of aeration and rapid
filtration (Salvato, 1992). Different mecha-
nisms may contribute to the iron removal in
filters; flock filtration, adsorptive iron removal
INTRODUCTION
INTRODUCTION
Iron is a common trace element in soils
and groundwater and it is the fourth most
abundant mineral in the earth’s crust. The
bulk iron content of soils is typically in the
range of 0.5% to 5% (by volume), and it is de-
pendent upon the source rocks from which
the soil was derived, transport mechanisms,
and overall geochemical history. Iron occurs
naturally in water in soluble form as ferrous
iron (Fe+2) or non-soluble form as ferric iron
(Fe+3) (Prince
et al.,
2003; Vreeburg, 2007).
Iron presence at elevated levels can cause
aesthetic problems on ornamental plants,
buildings and structures, and its accumula-
tion on irrigation equipment can lead to
IRON REMOVAL FROM GROUNDWATER USING
IRON REMOVAL FROM GROUNDWATER USING
LEPTOTHRIX DISCOPHORA
LEPTOTHRIX DISCOPHORA
Doaa B. Darwish, Samy A. Shaaban and Sara M. Nafei
Doaa B. Darwish, Samy A. Shaaban and Sara M. Nafei
Botany Department, Faculty of Science, Mansoura University, Egypt
ABSTRACT
ABSTRACT
In the present study one strain of iron oxidizing bacteria was isolated from soil and water en-
riched sample with FeSO4.7H2O and identified as Leptothrix discophora (L. discophora). This
bacterium was used to remove iron from groundwater sample. The optimum temperatures for L.
discophora growth were 45 and 35 °C at pH of 3 and 5, respectively. Bioremoval of ferrous iron
by immobilized cells of L. discophora was investigated in repeated batch culture and continuous
operation. Effects of dilution rate and initial concentrations of Fe
+2 on bioremoval were also in-
vestigated. During repeated batch culture, the immobilized-cells were stable and showed high
constant iron-oxidizing activities. Our results showed that the immobilized cells of L. discophora
removed iron of the concentration of 1ppm from groundwater at natural condition in 25 min.
Key words :
Key words : Ferrous iron oxidation; Iron bioremoval; Immobilization; FeOB; Leptothrix disco-
phora; Groundwater.
Journal of Environmental Sciences, 2014; Vol. 43, No. 2 : 281-293
Doaa B. Darwish; et al...
282
Suzuki,
et al.,
2011). The genus
Leptothrix
forms microtubular sheaths that are distinct
in morphology from the twisted stalks pro-
duced by another Fe-oxidizing genus,
Gallio-
nella.
The study involved the examination of ex-
perimental conditions such as pH, tempera-
ture, contact time and iron concentration on
the removal of Fe+2 from aqueous solutions by
free and immobilized cells iron bacteria isolat-
ed. Moreover, we studied the feasibility of us-
ing the specific carrier for immobilization of
L.
discophora
, and established a procedure for
L. discophora
immobilization on a laboratory
scale, that is simple, fast, and easily reprodu-
cible which can be adapted to the industrial
scale.
MATERIALS AND METHODS
MATERIALS AND METHODS
Isolation and growth conditions of bacte-
Isolation and growth conditions of bacte-
ria
ria
Iron oxidizing bacteria (IOB) was isolated in
the laboratory from soil and water enriched
with iron (FeSO4.7H2O) as described by Smith
(1992). The medium used for isolation con-
sisted of: (NH4)2SO4 3.0 g/L, K2HPO4 0.5 g/
L, KCL 0.1 g/L, Ca(NO3)2 0.01 g/L,
MgSO4.7H2O 0.5 g/L, H2SO4 (10N) 1.0ml,
FeSO4.7H2O solution300ml. These cultures
were allowed to grow at 30 °C with shaking at
150 rpm and the organisms were tentatively
identified according to Krieg (1984) and Holt
et al.
(1994). The exact identification of bacte-
ria was carried out according to (Van Veen
et
al.
(1978); Czekalla
et al.
( 1985) ; and
Spring,( 2002).
Sampling and analytical methods
Sampling and analytical methods
The ferrous solution was prepared from
and biological iron removal. This mechanism
is dominant depends on the groundwater
quality and the process conditions (Hatva,
1989; Mouchet, 1992; Søgaard
et al.,
2000).
Nowadays biological processes to remove Fe
are widely used in Europe, and there are
some plants are also used in this treatment in
the United States and Canada (Mouchet,
1995; Gage and Williams, 2001).
However, iron removals by biological pro-
cesses are based on different stages of biofil-
tration where beds are colonized by Fe oxidiz-
ing bacteria. In nature, iron oxidizing bacteria
(IOB) is found in widespread. It is prevalent in
groundwater, swamps, ponds, in the hypolim-
nion of lakes, in sediments, soils, wells and
water-distribution systems. In the latter they
can cause significant clogging problems due
to biofilm formation (Ghiorse, 1984 a;b).
These bacteria which are present in raw water
can multiply in sand filters under appropriate
conditions and is able to oxidize divalent ions
Fe+2 and precipitate them under their oxided
forms Fe+3.
Iron fixing bacteria are belonging to the fil-
amentous genera such as
Gallionella
spp.,
Leptothrix
and
Sphaerotilus
and less from the
rod type, such as Psendomonas and Entero-
bacter. They react with soluble iron, Fe+2,
through an oxidation process that changes
the iron to an insoluble form, Fe+3 (Siering
and Ghiorse; 1996, Czekalla
et al.
, 1985 ;
Mouchet 1992). Moreover, The Fe/Mn-
oxidizing bacteria are known for their poten-
tial to form extracellular Fe- or Mn-encrusted
structures in aquatic environments (Ghiorse,
1984 a;b , Spring, 2006, Hashimoto,
et al.,
2007, Miot,
et al.,
2009, Sakai,
et al.,
2010,
283
IRON REMOVAL FROM GROUNDWATER USING LEPTOTHRIX etc .....
FeSo4.7H2O: One mg/L (ppm) aqueous solu-
tion (stock solution) with de-ionized water in
1% HNO3 solution and the stock solution was
diluted with de-ionized water to obtain the
working standard solutions (Oyedeji and
Osinfade, 2010).
Raw and treated groundwater and the ef-
fluent of each process were analyzed after in-
cubation time. The following parameters were
measured: temperature, pH (pH-meter HACH
sension1), growth turbidity (Spectrophotome-
ter HACH DR 4000). Ferrous iron was deter-
mined by the phenanthroline method (Spec-
trophotometer HACH DR 4000) as described
by Smith (1992).
Slides prepared for IOB were examined
with a binocular microscope (Olympus
BX100). Keys given in the ASTM Standard
Test Method for Iron Bacteria (ASTM,
1997), Standared Methods (APHA, 1995),
Methods for the Examination of Water and
Associated Materials (Environmental Agency,
1998).
Optimization of temperature and initial
Optimization of temperature and initial
pH for bacterial growth
pH for bacterial growth
The optimum temperature for growth of
isolated bacterium was obtained by its
growth in liquid medium at different tempera-
tures (25, 35, 45 °C) and optimum pH for
bacterial growth was determined at pH val-
ues ranging from 2.0 to 8.0 (Lee and Wil-
liam, 1985).
Immobilization of IOB
Immobilization of IOB
The purified IOB suspension was mixed
with sodium alginate solution (2%) 1:1 ratio.
The IOB-alginate mixture was added dropwise
into calcium chloride (0.2 M) solution with
continuous shaking at 4 °C. As soon as the
drop of IOB-alginate solution mixed with
CaCl2 solution, Na+ ions of Na-alginate were
replaced by the Ca+2 ions of CaCl2 solution,
which finally formed Ca-alginate beads. The
beads thus formed were washed 3-4 times
with deionized water and finally used for fur-
ther studies (Fraser and Bickerstaff, 1997).
Ca-alginate beads inoculated in water sam-
ple contain 1ppm of Fe+2 at different pH (3, 5,
7, 7.5 and 8) and temperature (25, 35 and 45
°C), 5 ml were taken at 5 min interval, to de-
termine iron concentration, and also placed in
groundwater sample to test the ability for re-
moving iron (Kim
et al.,
2005).
RESULTS
RESULTS
Isolation of iron bacteria:
Isolation of iron bacteria:
Iron bacteria were isolated from different
samples of soil and water polluted with iron
on the selective medium. The isolated bacteria
(Plate 1a) from all samples were of the same
type. The purified bacteria were examined un-
der light microscope as seen in Plate (1b).
The purified bacterial isolate was grown on
the selective medium containing different con-
centrations of iron and the best concentration
of iron for growth was 10g/l (Table 1).
Doaa B. Darwish; et al...
284
Table (1) :
Table (1) : The effect of FeSO4.7H2O concentration on bac-
terial growth.
Identification of iron bacteria:
Identification of iron bacteria:
The bacterial isolate formed flocculent and
filamentous growth smooth colony. Micro-
scopically, cells are smaller than those of the
other species of
Leptothrix
. They may occur in
narrow sheaths or be free-swimming; free
cells are motile by a thin polar flagellum.
On glucose-peptone agar, the trichomes
are thin and the colonies are small, often no
more than 0.1-0.3 mm in diameter, with
smooth edges. Increased supply of nutrients
such as glucose, peptone, methionine, purine
bases, vitamin B12, biotin and thiamine hard-
ly improve growth. When Mn+2 is supplied to
this agar medium, the black-brown colonies
will be somewhat larger (0.5-2 mm) and,
sometimes ,filamentous .If widely spaced,
they often are surrounded by a dark brown
halo of pinpoint granules or by a diffuse light
brown halo of oxidized manganese (Holt
et al.,
1994).
Sheath formation asserted via dilute crys-
tal staining solution (Siering and Ghiorse,
1996) that the isolate is Gram-negative and
chemoorganotrph. Glucose and peptone were
the optimum carbon and energy sourc-
(a) Different isolates of FeOB on solid selective me-
dium.
(b)
Leptothrix discophora
under light microscopy
(Gram stain)
Plate (1) :
Plate (1) : Iron bacteria.
285
IRON REMOVAL FROM GROUNDWATER USING LEPTOTHRIX etc .....
whereas at pH 5 the optimum temperature for
growth was 35 °C. Generally the growth curve
began to decline after the sixth day of incuba-
tion.
Optimization of iron bioremoval:
Optimization of iron bioremoval:
By free cells of
By free cells of
Leptothrix discophora
Leptothrix discophora
:
In this experiment the remaining iron was
measured in the media after incubation at dif-
ferent temperature (25, 35 and 45 °C) and pH
(3 and 5). The data clearly suggest that pH 3
was better than pH 5 at all tested tempera-
tures and at 35 °C removed iron effectively
than 25 and 45 °C (Figure (2 a,b and c).
es,respectively (Krieg, 1984). The species of
genus
Leptothrix
was identified using ei-
ther classical bacteriological tests and
automated identification system as a
disco-
phora
. Finally, the iron bioremoval isolate
was identified as
Leptothrix discophora
.
Effect of environmental factors on Lep-
Effect of environmental factors on Lep-
tothrix discophora growth:
tothrix discophora growth:
The measured growth of IOB at different
temperatures (25, 35 and 45 °C) and pH val-
ues (3 and 5)after 3 days of incubation are il-
lustrated in Figure (1a,b). At pH 3 increasing
temperature increased the rate of growth,
Fig. (1) :
Fig. (1) : Effect of temperature on bacterial growth at pH 3.0(a) and pH 5.0 (b).
(b)
(b)
(a)
(a)
(b)
(b)
(a)
(a)
Doaa B. Darwish; et al...
286
Plate (2) :
Plate (2) :
Leptothrix discophora
immobilized cells after and before removal of iron.
Fig. (3) :
Fig. (3) : Effect of pH on bio removal of iron by im-
mobilized cells of
Leptothrix discophora
.
Fig. (2) :
Fig. (2) : Effect of pH on bioremoval of iron at 25 °C(a), 35 °C (b)and 45 °C(c).
(c)
(c)
By immobilized cells of
By immobilized cells of
Leptothrix disco-
Leptothrix disco-
phora:
phora:
The immobilized
Leptothrix discophora
before and after removal of iron as shown in
Plate (2) and Figure (3).The best removal of
iron was recorded when bacterial growth at
pH 7.5 and 100% of iron was removed after
15 minutes. While it required 30 minutes at
pH 7 and 8 to reach the same percentage.
Also it was found that the 35 and 45 °C were
better than 25 °C to get rid of about 100% of
dissolved iron after 15 minutes Figure (4).
Fig. (4):
Fig. (4): Effect of temperature on iron removal by
immobilized cells of
Leptothrix discophora
.
287
IRON REMOVAL FROM GROUNDWATER USING LEPTOTHRIX etc .....
temperature greatly affected the removal
of iron , especially with the free bacteria.
At 35 °C free bacteria efficiently removed
iron from water that reached as low 0.3
ppm.
Free and immobilized Leptothrix disco-
Free and immobilized Leptothrix disco-
phora to remove iron :
phora to remove iron :
The immobilized cell removed iron al-
most completely under our experimental
conditions (Fig.5 a,b and c). Experimentally,
(b)
(b)
(a)
(a)
Fig. (5) :
Fig. (5) : Comparison between free and immobilized cells of
Leptothrix discophora
in bioremoval of iron at 25 °C(a), 35 °C(b) and 45 °C(c) .
(c)
(c)
100% of the iron from the used groundwa-
ter after 25 min., (Fig. 6). More than 90%
of iron was removed after 5 min of adding
the immobilized cells and in the rest 20
min the rest of contaminated iron was re-
moved.
Iron removal from groundwater:
Iron removal from groundwater:
The ability of immobilized cells was
tested to remove iron from groundwater
sample was taken from a well at Met abo
khaled; Meet Ghamr, Dakahlia. The results
showed that the immobilized cells removed
Doaa B. Darwish; et al...
288
study, the culture growth was affected by the
Fe2+ ion concentration.
The increase in iron concentration resulted
in an increase in the culture growth until
maximum value was achieved, after which a
decrease in bacterial growth occurred (Table
1). Continuous oxidation was affected by the
dilution rate and initial the Fe2+ ion concen-
tration (Ehsan, 2008).
The oxidation of iron takes place rapidly
by the bacterial cell. One species of
Leptothrix
was isolated from water enriched with iron
and it reflects a specific environmental condi-
tion. Regulation of Fe metal uptake would
seem reasonable in order to fulfill this need.
Temperatures inside well between 30 and
35°C was suitable for growth of
Leptothrix
species, particularly
Leptothrix
AT22. On the
other hand, these bacteria and other various
bacteria are able to acidify the medium, which
ensures a better solubility of ferric ions and
enables iron uptake (Peine,
et al.,
2000).
L. discophora
SS-1 is a gram-negative het-
erotrophic bacterium that has the unique
DISCUSSION
DISCUSSION
Iron is the most abundant transition ele-
ment in the Earth's crust, approximately one-
third of the Earth's mass is estimated to be
iron. Its concentration is relatively high in
most crustily rocks (lowest in limestone),
which is more or less pure calcium carbonate
(Farago, 1986).
The biological oxidation of ferrous iron
(Fe+2) by
L. discophora
is potentially a useful
industrial process for the bioremoval of iron
and in the regeneration of ferric iron (Fe+3) as
a leaching agent in oxidation processes (Lon-
ga
et al.,
2004).
There are several factors that play substan-
tial roles in the rate of oxidation of Fe+2 by
L.
discophora.
These factors include Fe+2/Fe+3
iron concentration, cell and oxygen concentra-
tions, pH, temperature and reactor type
(Daoud and Karamanev, 2006). Accordingly,
studies were carried out using batch experi-
ments for assessment of optimal environmen-
tal conditions, such as initial Fe+2 concentra-
tion and pH, for efficient bio-oxidation of Fe+2
to Fe+3 ions (Malhotra
et al.,
2002).In this
Fig. (6) :
Fig. (6) : Iron bioremoval by immobilized cells of
Leptothrix discophora
from groundwater sample.
289
IRON REMOVAL FROM GROUNDWATER USING LEPTOTHRIX etc .....
15 minute. Therefore, we conclude that the
decrease of pH and temperature caused de-
crease in iron oxidation in groundwater.
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Received on 24 / 10 / 2013
293
IRON REMOVAL FROM GROUNDWATER USING LEPTOTHRIX etc .....
Leptothrix discophora
- - - -
.
( )
, ( ) ( )
.
.
(
Leptothrix discophora :
,
.
.
Doaa B. Darwish; et al...
294
IRON REMOVAL FROM GROUNDWATER USING
IRON REMOVAL FROM GROUNDWATER USING
LEPTOTHRIX DISCOPHORA
LEPTOTHRIX DISCOPHORA
Doaa B. Darwish, Samy A. Shaaban and Sara M. Nafei
Doaa B. Darwish, Samy A. Shaaban and Sara M. Nafei
Botany Department, Faculty of Science, Mansoura University, Egypt
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Journal of Environmental Sciences, 2014; Vol. 43, No. 2 : 281- 293
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