Chemosphere 41 (2000) 283±287
Removal of heavy metals from anaerobically digested sewage
sludge by isolated indigenous iron-oxidizing bacteria
L. Xiang, L.C. Chan, J.W.C. Wong
*
Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, People's Republic of China
Abstract
The removal of heavy metals (Cr, Cu, Zn, Ni and Pb) from anaerobically digested sludge from the Yuen Long
wastewater treatment plant, Hong Kong, has been studied in a batch system using isolated indigenous iron-oxidizing
bacteria. The inoculation of indigenous iron-oxidizing bacteria and the addition of FeSO4 accelerated the solubilization
of Cr, Cu, Zn, Ni and Pb from the sludge. pH of the sludge decreased with an increase in Fe2‡ concentrations and
reached a low pH of 2±2.5 for treatments receiving both bacterial inoculation and FeSO4 . After 16 days of bioleaching,
the following heavy metal removal eciencies were obtained: Cr 55.3%, Cu 91.5%, Zn 83.3%, Ni 54.4%, and Pb 16.2%.
In contrast, only 2.6% of Cr, 42.9% of Cu, 72.1% of Zn, 22.8% of Ni and 0.56% of Pb were extracted from the control
without the bacterial inoculation and addition of FeSO4 . The residual heavy metal content in the leached sludge was
acceptable for unrestricted use for agriculture. The experimental results con®rmed the e€ectiveness of using the isolated
iron-oxidizing bacteria for the removal of heavy metals from sewage sludge. Ó 2000 Elsevier Science Ltd. All rights
reserved.
1. Introduction
Huge amounts of sewage sludge potentially contaminated with heavy metals are expected to be generated in South China due to the increasing number of
wastewater treatment facilities being constructed or to
be built in the next few decades (Tyagi and Couillard,
1991). Di€erent disposal options including incineration,
land®lling, marine discharge and land application, are
commonly used in most municipalities. Land application provides an economical alternative for the ®nal
disposal of sewage sludge but heavy metals in sewage
sludge is always an issue restricting its general use
(Davis, 1987). Reduction of heavy metals in sewage
sludge by source control is relatively expensive and the
sources are often dicult to identify. Therefore, removal
of heavy metals prior to land application is likely to be a
possible and practical means for reducing metal contents
*
Corresponding author. Fax: +852-2339-5995.
E-mail address: [email protected] (J.W.C. Wong).
in sewage sludge especially for sewage sludge derived
from mixed domestic and industrial sources (Sreekrishnan et al., 1993).
Several chemical methods, such as EDTA extraction
and acid treatment, have been suggested for solubilization of heavy metals from sewage sludge. Addition of
EDTA to sewage sludge showed high removal eciencies of Cu, Pb and Cd, but low eciencies for Fe, Ni and
Cr (Jenkins et al., 1981). In acid treatment, mineral and
organic acids have been commonly used to solubilize
heavy metals at low pH (Hayes et al., 1980; Lo and
Chen, 1990). In spite of the good extraction achieved in
the acid treatment method for most metals, Cu does not
show a high solubilization. The high cost, operational
diculties and large consumption of chemical agents
have made these chemical methods unattractive.
In the last decade, research work has been performed
mostly in Canada to investigate the feasibility of removing heavy metals such as Cu, Zn, Pb and Cd from
sewage sludge by means of a bioleaching method using
Thiobacillus ferrooxidans (an iron-oxidizing bacterium)
and T. thiooxidans (a sulfur-oxidizing bacterium)
0045-6535/00/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 4 5 - 6 5 3 5 ( 9 9 ) 0 0 4 2 2 - 1
284
L. Xiang et al. / Chemosphere 41 (2000) 283±287
(Jenkins et al., 1981; Tyagi et al., 1988; Tyagi and
Couillard, 1991; Chartier and Couilard, 1997). Bioleaching processes could reduce the cost by 80% as compared to the traditional chemical methods. It was
reported that the adapted indigenous iron and sulphur
oxidizing bacteria e€ectively reduced sludge pH to 2±3.
More than 80% of Cu, Zn, Mn, Cd and Ni could be
removed from the sludge in 8±10 days, while the removal
of Cr and Pb was about 30±40%. In addition, the pH of
sewage sludge needed to be adjusted to 6 4.5 for the
growth of the ATCC T. ferrooxidans strains. In view of
the shortcomings, adapted sulphur oxidizing bacteria
were used to leach metals from sewage sludge with the
addition of mineral sulphur as the major energy source
(Chartier and Couilard, 1997). However, the co-existence of T. thiooxidans and residual sulphur in the decontaminated sludge would lead to acidi®cation of soil.
Therefore, the aim of the present work is to investigate
the feasibility of removing heavy metals such as Cr, Cu,
Zn, Ni and Pb from the sludge using the isolated indigenous iron-oxidizing bacteria to render the sludge
suitable for agricultural application.
2. Materials and methods
2.1. Collection and properties of sewage sludge
The anaerobically digested sewage sludge was collected from the wastewater treatment plant in Yuen
Long district, Hong Kong. The pH of the sewage sludge
was determined immediately after collection while solid
contents were determined by oven-drying at 105°C. Subsamples were digested with di-acid digestion (conc.
HNO3 + conc. HClO4 ) and heavy metals in the digestates were determined by atomic absorption spectrometry (Page et al., 1982). Table 1 gives the
physicochemical properties of the sewage sludge.
2.2. Microorganisms and inoculum
The indigenous iron-oxidizing bacteria were isolated
from the native sewage sludge and kept at 4°C and pH
2.0 in the improved Leathen medium with 20 g/l
FeSO4 á 7H2 O as the energy source (Xiang, 1992).
The composition of the medium was as follows (g/l):
0.45,
K2 HPO4
0.15,
KCl
0.05,
(NH4 )2 SO4
MgSO4 á 7H2 O 0.5, Ca(NO3 )2 á 2H2 O 0.01. The bacteria
were activated by repeated inoculation (n ˆ 3) at 28°C
before being used in bioleaching experiments. The inoculum was prepared by growing the isolated indigenous iron-oxidizing bacteria in 250 ml Erlenymeyer
¯asks containing 100 ml of the improved Leathen medium at pH 2.0 and 28°C with 20 g/l FeSO4 á 7H2 O. The
¯asks were agitated on a horizontal shaker at 180 rpm
for 30 h, which was determined in a preliminary experiment to delineate the time period required for the
highest biological activity.
2.3. Bioleaching experiments
A sludge slurry of 250 ml contained in a 500 ml
Erlenmeyer ¯ask was pre-acidi®ed with sulphuric acid to
pH 3.0 and then agitated at 28°C, 180 rpm for 24 h to
increase the initial redox potential (ORP). An inoculum
of 15% volume was added to the acidi®ed sludge slurry
followed by the addition of Fe2‡ at a concentration of 0,
0.5, 1, 1.5, 2.0, 3.0 and 4.0 g/l (in the form of
FeSO4 á 7H2 O). The pH of the mixture was then readjusted to 3.0 with 5 N NaOH and the whole set up was
weighed. The water loss due to evaporation was replenished with distilled water. pH and ORP values were
monitored every two days and 8.0 g of sample was
drawn from each ¯ask for the determination of heavy
metal content in the liquid phase. All treatments were
done in duplicate.
2.4. Analytical methods and statistical analyses
Samples collected were centrifuged at 12,000 rpm for
15 min, and the supernatants were then ®ltered, acidi®ed
to pH 2 with 16 N HNO3 and stored at 4°C prior to the
determination of Cr, Cu, Zn, Ni, Pb and Fe by atomic
absorption spectrometry. All data were analyzed using
the SAS statistical package through an IBM personal
computer. One-way ANOVA was carried out to compare the means of di€erent treatments; where signi®cant
F values were obtained, di€erences between individual
means were tested using the Least Signi®cance Di€erence test (Little and Hills, 1978).
Table 1
Selected physico chemical properties of Yuen Long sludge
Selected characteristics
Solid (%)
Ash (%)
Total P (g/kg dry sludge)
Total N (g/kg dry sludge)
pH
EC (mS/cm)
Heavy metals (mg/kg)
2.21
31.5
15.4
51.6
7.1
3.8
Cr
Cu
Zn
Ni
Pb
Fe
663
255
2823
622
57.0
72,200
L. Xiang et al. / Chemosphere 41 (2000) 283±287
3. Results and discussion
3.1. Changes in pH and ORP with time
The changes in pH and ORP with bioleaching time
for sludge at a Fe2‡ concentration of 0±4 g/l are shown
in Fig. 1(a) and (b), respectively. pH of all treatments
decreased sharply to the lowest pH at day 5 and then
increased slightly till the end of the leaching period except for treatments receiving <2 g/l FeSO4 and the
control which showed a slight increase at day 2. Increasing the concentrations of FeSO4 caused an increase
in acidi®cation as indicated by the lower pH. After 16
days of bioleaching, the pH of the control was higher
than that of all other treatments. Inoculation alone
caused a decrease in pH to 2.7 indicating the possible
utilization of sulphide and iron in the sludge as the energy source for the growth of T. ferrooxidans. Opposite
to the trend of change in pH, the ORP of all treatments
decreased slightly in the ®rst two days, then increased to
the highest ORP at day 6 and remained at a fairly
constant value till the end of the bioleaching period. All
treatments had an ORP of about 580 mV except for the
control, which had a lower ORP of 497 mV. This should
provide a favourable condition for the oxidation of Fe2‡
to Fe3‡ in the presence of bacteria.
Fig. 1. Changes in pH (a) and ORP (b) during the bioleaching
of Yuen Long sewage sludge with isolated indigenous ironoxidizing bacteria.
285
Without the presence of bacteria and FeSO4 , pH
decreased only slightly and Fe2‡ was oxidized slowly to
Fe3‡ . A high ORP coupled with a low pH value was
considered as an indicator of the existence of a substantial population of iron-oxidizing bacteria. The increase in ORP can be attributed to the increase in the
Fe3‡ /Fe2‡ ratio, which may occur naturally in the
presence of air or through biological catalysis:
1
2Fe2‡ ‡ O2 ‡ 2H‡ ! 2Fe3‡ ‡ H2 O
2
E ˆ E° ‡ …2:3RT =nF † log …Fe3‡ =Fe2‡ †
where R is the gas constant, T represents temperature
(°K), F the Faraday constant, and n is the number of
electrons transferred in this process.
The observed acidi®cation of sludge can be explained
by the bio-oxidation of S° compounds to SO2ÿ
4 . The
precipitation of Fe3‡ in the form of ferric hydroxide and
jarosite also generates sulphuric acid:
Fe3‡ ‡3H2 O ! Fe…OH†3 ‡3H‡
‡
3Fe3‡ ‡K‡ ‡2HSOÿ
4 ‡ 6H2 O ! KFe3 …SO4 †2 …OH†6 ‡8H
3.2. Removal of heavy metals from sewage sludge
Fig. 2(a) shows the in¯uence of initial Fe2‡ concentrations on the removal of Cr from Yuen Long sludge.
Solubilization of Cr seems to be closely dependent on
the presence of bacteria and FeSO4 as indicated by the
low Cr solubilization of 2.6% for the control, throughout the bioleaching period. There was a lag period of 2±6
days prior to leaching of Cr from the sludge for all
treatments receiving the addition of FeSO4 . The lag
period decreased while the removal eciency increased
with an increase in Fe2‡ concentrations. After 10±16
days of bioleaching, 55.3±70.6% of Cr was extracted in
the presence of iron oxidizing bacteria and an initial
Fe2‡ concentration of 4.0 g/l. Therefore, both the biological and chemical factors contributed to the solubilization of Cr.
The pattern of Cu solubilization di€ered from that of
Cr with an initial increase to the maximum at days 6±8
followed by a gradual decrease till the end of the
leaching period. Inoculation of iron-oxidizing bacteria
increased the extraction of Cu from 70% to >90% while
the addition of FeSO4 showed little in¯uence on Cu
removal. After 6±10 days of bioleaching, the maximum
Cu removal ranged from 88% to 98%, depending on the
amount of FeSO4 added. The decrease in Cu removal
eciency with time may be due to the re-adsorption of
soluble Cu by solid sludge and/or the complexation between the soluble Cu and the organic compounds in the
sludge.
286
L. Xiang et al. / Chemosphere 41 (2000) 283±287
Fig. 3. Removal of Ni (a) and Pb (b) from Yuen Long sewage
sludge after the inoculation of the isolated indigenous ironoxidizing bacteria and addition of FeSO4 .
Fig. 2. Removal of Cr (a), Cu (b) and Zn (c) from Yuen Long
sewage sludge after the inoculation of the isolated indigenous
iron-oxidizing bacteria and addition of FeSO4 .
The variation of Zn concentration over time for each
treatment is shown in Fig. 2(c). pH appears to be the
major factor responsible for the solubilization of Zn.
There was only a slight increase in the solubilization of
Zn following the inoculation of the isolated iron-oxidizing bacteria and the addition of FeSO4 . Zinc removal
eciency ranged from 72% in the control to about 87%
in the treatment receiving 4.0 g/l of initial Fe2‡ .
The removal of Ni and Pb from sewage sludge was
also investigated and the results are given in Fig. 3(a)
and (b), respectively. Maximum removal was achieved
for treatment receiving 4.0 g/l Fe2‡ at day 10 for Ni and
Pb with 54% and 16% removal, respectively. Similar to
Cu, further extension of the bioleaching time to 16 days
signi®cantly reduced the removal eciency of Ni. This
may be due to the re-adsorption of soluble Ni by the
solid sludge. In spite of the extension of the leaching
period to 16 days, little Pb (less than 17%) was leached
into the solution phase, which may be due partly to the
insoluble property of PbSO4 in the pH range 2±3.
Nevertheless, the concentration of Pb in the sewage
sludge form Yuen Long sewage treatment plant was
below the permissible level for unrestricted use (Beavers,
1993).
Di€erence in metal solubilization eciencies can be
explained in terms of the inorganic species predominance in sewage sludge. Heavy metals associated with
the organic fraction of sludge are less available to the
disposal environment than inorganic precipitates. In
case of acid environments, inorganic precipitates could
be solubilized rapidly. For metals, which are organically
bound, a prolonged reaction time would be required
(Hayes and Thesis, 1978).
Table 2 shows the contents of heavy metals in the
original sludge and the sludge after 16 days of bioleaching. Compared to the original sludge, the contents of
heavy metals in sewage sludge decreased sharply after
the inoculation of 15% (v/v) bacteria of 4.0 g/l Fe2‡ ,
which were lower than the permitted levels for restricted
L. Xiang et al. / Chemosphere 41 (2000) 283±287
287
Table 2
Contents of heavy metals (mg/kg dry sludge) in sludge before and after bioleaching
Before bioleaching
After bioleaching
Permissible levelsa
a
Cr
Cu
Zn
Pb
Ni
663
143
200
255
75.7
140
2823
264
300
64.5
31.6
150
62.1
6.0
60
Limits on heavy metals for unrestricted use in the State of Queensland (Beavers et al., 1993).
use according to the sludge guidelines of Queensland,
Australia (Little and Hills, 1978).
4. Conclusion
The present study con®rmed that the isolated indigenous iron oxidizing bacteria were e€ective in removing
Cr, Cu, Zn and Ni from the anaerobically digested
sludge collected from the Yuen Long wastewater treatment plant to the solution phase. The inoculation of
isolated indigenous iron-oxidizing bacteria and addition
of 4 g/l of Fe2‡ (in the form of FeSO4 á 7H2 O) resulted in
the following heavy metal solubilization eciency after
10 days of bioleaching: Cr 55%, Cu 92%, Zn 83%, Ni
54%, Pb 16%. Extending the bioleaching time to 16 days
increased the removal of Cr to 71%, but reduced Cu
removal eciency to 67% due to the re-adsorption of
soluble Cu by the solid sludge. The removal of Cr and
Cu appeared to be dependent on the microbiological
activities of the sludge, especially for Cr. The relatively
low removal eciency for Cr indicates that the bioleaching system should be further improved through optimizing the bioleaching parameters and also the
development of more e€ective bioleaching microorganisms. Nevertheless, the heavy metal content of the leached sludge was within the permitted level for
agricultural use. Further experiments should be performed to shorten the time required for the bioleaching
of Cr in order to improve the overall removal eciency
for Cr.
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