Environ Earth Sci
DOI 10.1007/s12665-012-2110-4
ORIGINAL ARTICLE
Use of sulfur-oxidizing bacteria for
assessment of chromium-contaminated soil
Anup Gurung • Sang-Eun Oh
Received: 12 February 2012 / Accepted: 31 October 2012
© Springer-Verlag Berlin Heidelberg 2012
Abstract In recent years, biological toxicity tests have
been conducted for soil assessment of environmental pol-
lutants to evaluate the environmental risk due to heavy
metals. In this study, batch tests were conducted with soils
contaminated with hexavalent chromium (Cr6?) using
sulfur-oxidizing bacteria (SOB). For control soils (without
Cr6?), the electrical conductivity (EC) increased linearly
over time in all samples, indicating that no toxic substances
were present in the soil. The initial EC varied between 6
and 7.8 mS/cm, and the final EC varied between 22 and
27 mS/cm after incubation for 65 h. For batch tests per-
formed using Cr6?-contaminated soil, the EC increased
slightly or remained stable in all the test samples after a
few hours. Thus, the presence of toxic substance Cr 6?
inhibited the SOB, which leads to no sulfuric acid forma-
tion and therefore, no change in EC. These results indicated
that SOB can be employed as a test microorganism to
assess the quality of heavy metal-contaminated soils.
Keywords Biological test · Heavy metals ·
Sulfur-oxidizing bacteria · Toxicity
Introduction
Increasing soil pollution, especially heavy metal contami-
nation, is a common phenomenon throughout the world
(USDA 2000; Seregin and Ivanov 2001; Melekhova et al.
2006; Boluda et al. 2011). The most prevalent forms of soil
A. Gurung ·S.-E. Oh (&)
Department of Biological Environment,
Kangwon National University, Gangwon-do,
Chuncheon-si 200-701, South Korea
e-mail: ohsangeun@kangwon.ac.kr
contamination are due to mining, manufacturing, atmo-
spheric deposition, use of synthetic products, such as fer-
tilizer and pesticides, irrigation, and waste disposal (Raskin
et al. 1994; USDA 2000; Seregin and Ivanov 2001; Boluda
et al. 2011). Application of sewage sludge to agricultural
land as a fertilizer has many advantages as it contains a high
content of organic matter and nutrients (Albiach et al. 2001;
Selivanovskaya et al. 2003; Jamali et al. 2008). However,
the application of sewage sludge to agricultural land may
exert a negative effect on the soil surface and ground water
as sewage sludge contains pollutants including heavy metals
and organic compounds (Gao et al. 1997; Stevens et al.
2002; Oleszczuk 2006). Therefore, it is necessary to evalu-
ate sewage sludge prior to its use as a fertilizer on agricul-
tural land.
In recent years, biological tests have been conducted
for the assessment of environmental pollutants to evaluate
the environmental risk of heavy metals, since traditional
analytical methods alone do not provide information
regarding the biological activity (Plaza et al. 2005; Hoss
et al. 2009; Gonzalez et al. 2011; Oh et al. 2011; Van
Ginkel et al. 2011). Toxicity tests based on biological
approaches are often employed to detect the acute or
chronic effects of substances on a representative organ-
ism at different trophic levels such as fish, daphnia, algae,
plant tissue, animal cells, or several bacterial species
(Plaza et al. 2005; Palma et al. 2009; Boluda et al. 2011).
Biological toxicity tests can be achieved by monitoring
growth rate, biochemical properties, respiration (Tzoris
et al. 2005), electrical current (Shitanda et al. 2005),
bioluminescence (Gu and Geun 2001), or conductivity
(Liu et al. 2006). By monitoring the physiological
response of living organisms, bioassay provides valuable
information regarding environmental contamination,
especially by toxic chemicals (Farre et al. 2004).
13

Very recently, a novel biosensor based on sulfur-oxi-
dizing bacteria (SOB) has been developed to detect the
presence of toxic substances in wastewaters (Oh et al.
2011). As depicted in Eq. (1), this methodology exploits
the ability of SOB to oxidize sulfur particles under aerobic
condition to produce sulfate (SO42-) (Madigan et al. 2009).
○
S þ H2O þ 1:5O2 ! SO2— þ 2Hþ;
4
ð1Þ
MG ¼ —587 kJ=reaction:
SOB are chemolithotrophic bacteria that play an
important role in mineral cycling in the environment (Oh
et al. 2011; Shi et al. 2011). During the biological oxidation
of sulfur to sulfuric acid, protons are produced and the
reaction results in an increase in electrical conductivity
(EC) and a decrease in pH (Madigan et al. 2009; Oh et al.
2011). However, in the presence of toxic substances, the
activity of SOB is inhibited and thus the medium EC and
pH remain stagnant (Oh et al. 2011). The same principle
can be adopted for the assessment of contaminated soil
based on the inhibition of SOB in the presence of toxic
chemicals by measuring changes in pH and EC. This paper
presents a preliminary study of the use of SOB in batch
tests to assess the inhibition of SOB in chromium-
contaminated soil.
Materials and methods
Sulfur master-culture reactor
A sulfur master-culture reactor (SMCR) was set up con-
taining elemental sulfur (S°) particles. Aerobic return
activated sludge taken from the Chuncheon Wastewater
Treatment Plant in Chuncheon City, Kangwon-do,
Republic of Korea was used as the SOB inoculum in the
SMCR. S° (500 g, 2–4 mm in diameter) was placed in a
1 L beaker (Diamond, Korea), and synthetic stream water
(600 mL) was added. The synthetic stream water was
prepared by diluting nutrient mineral buffer (NMB) solu-
tion 100 times. The NMB solution consisted of: NaHCO 3
(3.13 g/L), NH4Cl (0.31 g/L), NaH2PO4. H2O (0.75 g/L),
KCl (0.13 g/L), NaH2PO4 (4.22 g/L), Na2HPO4 (2.75 g/L),
and trace metal and vitamin solutions as previously
described by Oh et al. (2004). Air was introduced into the
SMCR using a stone diffuser with a flow rate of 150–
250 mL/min, and the temperature of the reactor was held at
30 °C. The initial pH and EC of the SMCR were
7.11 and 0.07 mS/cm, respectively. Growth of the SOB
was determined by monitoring the changes in pH and EC.
An increase in EC and a decrease in pH indicated SOB
growth in the SMCR. The SMCR was operated for more
than 30 days in fed-batch mode.
13
Environ Earth Sci
Batch experiments
All studies were conducted using soil collected from the
premises of Kangwon National University, Republic of
Korea. The collected soil (200 g) was oven dried at 40 °C
for 24 h. Batch tests (without adding the toxic substance
hexavalent chromium (Cr6?)) were carried out in 100 mL
media bottles (Horex, Germany) that contained 40 mL of
1 % NMB solution. The soil concentrations used were 0,
2.5, 12.5, and 25 g/L in 1 % NMB solution. Sulfur parti-
cles (20 g) from the SMCR were used. The test bottles
were capped with aluminum foil to prevent evaporation
and provide air, and the tests were conducted at 30 °C with
agitation at 150 rpm (SI-600R, Korea) for 65 h.
The potential effects of the heavy metal Cr6? on SOB
were evaluated using potassium dichromate (K2Cr2O7) as
the model toxic chemical in the experiments. The test
concentrations of Cr6? were set at 0, 0.5, 1, 2, 4, 6 and
8 mg/L. Sulfur particles (20 g) were taken from the SMCR
and mixed together with Cr6?, soil (1 g), and 1 % NMB
solution (40 mL) in a 100 mL media bottle. The test bottles
were capped with aluminum foil and agitated at 150 rpm.
All the inhibition tests were conducted at 30 °C.
Chemical analyses
The Cr6? used in this experiment was purchased from
Sigma-Aldrich, St. Louis, MO, USA. The pH and EC were
measured manually over time using portable pH (Orion,
410A, Boston, MA, USA) and EC (Inlab 737 Mettler
Toledo, Schwerzenbach, Switzerland) meters. Samples for
the analysis of SO42- were collected during the pH and EC
measurements. The collected samples were centrifuged at
6000 rpm for 15 min prior to analysis using ion chroma-
tography (ICS-900, Dionex Corp AS40 Column, Sunny-
vale, CA, USA). The EC and pH of the soil were measured
as a 1:5 mixture of soil and distilled water. Prior to each EC
and pH measurement, the soil/water mixture was agitated
at 180 rpm for 1 h. The total content of select heavy metals
such as Cr, Cd, Zn, As, and Cu in the soil was measured. A
soil sample (3 g) was treated with aqua regia (75 % HCl,
25 % HNO3: 30 mL) in a long tube and the mixture was
left for 18 h. The soil sample was digested in a plastic
bottle for 2 h at 1208C in a fume hood. The digested
solution was filtered through filter paper (Whatman®
Schleicher & Schuell, 110 mm Ø, Maidstone, England)
and the volume of the filtrate was adjusted to 50 mL adding
distilled water. The total contents of Cr, Cd, Zn, As, and Cu
were analyzed using an inductively coupled plasma (ICP-
OES) spectrometer (Optima DV, Perkin-Elmer, Waltham,
MA, USA). Loss-on-ignition (LOI) procedure was used to
determine the soil organic matter (SOM) content (Dean
1974; Schulte and Hopkons 1996). For the LOI analyses,
Environ Earth Sci
Fig. 1 Changes of a EC and
b pH during the SOB batch tests
using soil with and without
sulfur
Fig. 2 Changes of SO
4
2-
concentrations over time with and
without sulfur
the soil sample was air dried and sieved through a 1 mm
sieve to remove bigger particles. The sample was dried at
105 °C for 12 h (OF-22GW, Jeio Tech, Korea), cooled in a
desiccator, and weighed before it was ignited at 550 °C for
2 h in a muffle furnace (MF21GS, Jeio Tech, Korea). The
organic matter in sludge was calculated according to the
standard methods (APHA 1998).
Results and discussion
The soil was slightly acidic with a pH value of 5.4. The EC
was 1.3 mS/cm. The heavy metal values measured indicated
that the soil contained relatively high concentrations of Zn
(64.0 mg/kg), Cr (56.6 mg/kg), and Cu (23.5 mg/kg). The
concentrations of Cd and As were 0.6 and 1.0 mg/kg,
respectively. The SOM value calculated by the LOI method
was 2 %. The soil sample contained high concentration of
SO42- (74 mg/L) than the sludge (24 mg/L) used as inoc-
ulum in the SMCR. Two sets of batch tests were conducted.
In the first set, batch tests were conducted using only 1 %
diluted NMB solution and different amounts of soil (0 g to
25 g/L) without SOB. In all these test samples, the pH
varied between 5.8 and 7.2. Similarly, the EC varied
between 82 and 225 lS/cm over an incubation period of
65 h.
In the second set of experiments, batch tests were con-
ducted with soil and SOB. The changes in EC and pH
during the SOB batch tests using different amounts of soil
(0–25 g/L) without Cr6? are shown in Fig. 1a, b. Prior to
the addition of SOB to the test samples, the pH was rela-
tively high. Before mixing with the sulfur particles/SOB
mixture, the pH values were 7.19, 5.68, 6.79, and 5.88 for
the 0, 2.5, 12.5, and 25 g of soil/L, respectively; and the EC
values ranged between 96 and 176 lS/cm. After mixing
with the sulfur particle/SOB mixture, the initial EC values
varied between 6 and 7.8 mS/cm in all the test samples.
The control (0 g of soil/L ? sulfur) EC value closely
resembled that of the test sample EC values. The final (at
65 h) EC values of the test samples were 27.5, 25.6, 21.9,
and 23.1 mS/cm, respectively, for the 0, 2.5, 12.5, and 25 g
of soil/L containing SOB. After the addition of sulfur
particles with SOB, the initial pH value in all the samples
was relatively low. This could be due to the presence of
sulfuric acid in the sulfur particles/SOB mixture taken from
the SMCR. The pH value further decreased and reached a
minimum of 1.14 in the control sample over the incubation
period of 65 h.
Figure 2 displays the changes in SO42- concentration.
The chemical analysis results showed a similar pattern of
SO42- generation among the test samples for 0, 2.5, 12.5,
and 25 g of soil/L. The highest SO42- concentration of
4,733 mg/L was found in the sample containing 2.5 g of
soil/L at 65 h. In order to determine whether the SO42- is
solely due to SOB or by the soil sample, only 1 % NMB
solution and different amounts of soil (0, 2.5, 12.5, and
25 g/L) were incubated for 65 h. The EC value of the
sample containing only soil and 1 % NMB solution did not
increase over the 65-h incubation period. Furthermore, the
SO42- concentration also remained constant during the
65-h incubation without sulfur particles and varied between
5 and 22 mg/L (Fig. 2).
13

Fig. 3 Changes of a EC and
b pH during the SOB batch tests
with Cr6?
In order to evaluate the toxicity of Cr6? on SOB, six
concentrations of Cr6? (0.5, 1, 2, 4, 6 and 8 mg/L) were
tested with one control using medium water consisting of
20 g of sulfur particles from the SMCR and 1 g of soil.
Figure 3a, b shows the changes in pH and EC of the SOB
batch tests at different concentrations of Cr6?. In the
control sample, the EC gradually increased and reached
10 mS/cm at 24 h. In contrast, in the test samples con-
taining Cr6?, the EC increased slightly and then stabilized.
However, the pH did not increase substantially in the
samples containing Cr6?, while the control showed a slight
decrease in pH.
In soil, heavy metals and metalloids can be found
in different forms such as dissolved, exchangeable,
and insoluble precipitates with other soil components
(Aydinalp and Marinova 2003). Thus, the toxicity of heavy
metals for soil microorganisms largely depends on several
factors such as pH, sorbent nature, temperature, inorganic
anions and cations, hydrous metal oxides, clay miner- als,
etc. (Bååth 1989; Giller et al. 1998; Aydinalp and
Marinova 2003; Violante et al. 2010). The pH plays an
important role in controlling the fate of heavy metals in soil
(Lindsay 1979). Immobilization of heavy metals in soil
increases when the soil pH rises, and it peaks under mildly
alkaline conditions (Aydinalp and Marinova 2003). On the
other hand, when the soil pH is low (\5) heavy metals are
largely soluble (Hutchinson and Collins 1978).
This preliminary study showed that soil contamination
can be examined by employing SOB as the test microor-
ganism. This SOB biosensor can detect the presence of
toxic substances at very low concentration in water (5 ppb
of Cr6?) (Oh et al. 2011). Furthermore, the SOB biosensor
is simple to use and construct, cost effective, and envi-
ronmentally friendly, since no toxic byproducts are pro-
duced (Hassan et al. 2010; Van Ginkel et al. 2010; Oh et al.
2011; Van Ginkel et al. 2011). The current study demon-
strates that the SOB biosensor is applicable for detecting
the presence of toxic substances in soil. The SOB batch test
with soil (0 g to 25 g/L) and sulfur particles with SOB
showed an increase in EC, whereas the SOB batch test with
13
Environ Earth Sci
Cr6?-contaminated soil (1 g) inhibited the activity of SOB,
and the EC value did not increase. Thus, the evaluation of
soil contaminated with heavy metals can be done by
employing SOB as the test microorganism.
Conclusion
This study consisted of the assessment of the heavy metal
Cr6? using SOB as the test microorganism. This method
exploits the simple mechanism that in the presence of toxic
substances, the SOB activity will be inhibited by reducing
the EC and increasing the pH. When the SOB batch test
was conducted with soil and 1 % NMB solution, the EC
increased linearly from *6 to 27 mS/cm. Conversely,
when the SOB batch test was conducted with Cr6?-con-
taminated soil, the EC increased slightly and then stabi-
lized. The decrease in EC is mainly due to the inhibition of
SOB activity by Cr6?, which stops the production of sul-
furic acid during the reaction. The current study demon-
strates that the SOB biosensor is applicable for detecting
the presence of toxic substances in soil.
Acknowledgments This research was supported by Basic Science
Research Program through the National Research Foundation of
Korea (NRF) funded by the Ministry of Education, Science and
Technology (2012R1A2041985) and the Institute of Environmental
Research at Kangwon National University.
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