RESEARCH ARTICLE

Nitrate-reducing, sul¢de-oxidizing bacteria as microbial oxidants

for rapid biological sul¢de removal
Bart De Gusseme1, Peter De Schryver1, Michaël De Cooman1, Kim Verbeken2, Pascal Boeckx3,
Willy Verstraete1 & Nico Boon1
1
Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Ghent, Belgium; 2Department of Metallurgy and Materials Science,
Ghent University, Ghent, Belgium; and 3Laboratory of Applied Physical Chemistry (ISOFYS), Ghent University, Ghent, Belgium

Correspondence: Nico Boon, Laboratory of Abstract

Microbial Ecology and Technology (LabMET),
Ghent University, Coupure Links 653, B-9000
Ghent, Belgium. Tel.: 132 9 264 59 76;
fax: 132 9 264 62 48;
e-mail: nico.boon@ugent.be
Received 17 April 2008; revised 25 August
2008; accepted 26 August 2008.
First published online 15 October 2008.
DOI:10.1111/j.1574-6941.2008.00598.x
Editor: Alfons Stams
Keywords
microbially induced concrete corrosion (MICC);
sulfate reduction; H2S production; biologically
produced sulfur; nitrate reduction; electron
sink.
The emission of hydrogen sulfide into the atmosphere of sewer systems induces the
biological production of sulfuric acid, causing severe concrete corrosion. As a
possible preventive solution, a microbial consortium of nitrate-reducing, sulfide-
oxidizing bacteria (NR-SOB) was enriched in a continuously stirred tank reactor in
order to develop a biological technique for the removal of dissolved sulfide. The
consortium, dominated by Arcobacter sp., was capable of removing 99% of sulfide.
Stable isotope fractioning of the sulfide indicated that the oxidation was a
biological process. The capacity of the NR-SOB consortium for rapid removal of
sulfide was demonstrated by using it as an inoculum in synthetic and real sewage.
Removal rates up to 52 mg sulfide-S g VSS
1 h
1 were achieved, to our knowledge
the highest removal rate reported so far for freshwater species in the absence
of molecular oxygen. Further long-term incubation experiments revealed the
capacity of the bacteria to oxidize sulfide without the presence of nitrate,
suggesting that an oxidized redox reserve is present in the culture.

dissolved sulfide by precipitation (McComas et al., 2001) or

Introduction
Hydrogen sulfide (H2S) formation by dissimilatory
sulfate-reducing bacteria (SRB) under strict anaerobic
circumstances is a common problem in sediments, sewer
systems, oil reservoirs and anaerobic effluents (Holmer &
Storkholm, 2001; McComas et al., 2001). The emission of
H2S into the atmosphere of sewer systems does not only
imply odor nuisances and possible health risks. It also
induces the biological production of sulfuric acid in the
aerobic zones, causing severe corrosion of the inner surface
of concrete sewer structures (Sand, 1987; Vincke et al.,
2002). Hence, preventive or curative actions are needed.
Physicochemical protective measures have been developed,
focusing on the decrease of sulfide production using bio-
cides (Jayaraman et al., 1999; Nemati et al., 2001), dosing
ozone (Elovitz et al., 2000) or injecting air to prevent
anaerobic conditions (Ochi et al., 1998). When sulfide is
already present in the water phase, curative measures can be
taken. Examples are the addition of iron salts to remove
application of chemical oxidants like hydrogen peroxide and
hypochlorite (Cadena & Peters, 1988).
The use of biological techniques is of recent interest.
Biological processes can operate at atmospheric pressure
and low temperatures without excessive costs for chemicals
(Zhang et al., 2008). Addition of thermodynamically more
favorable electron acceptors, such as nitrate, can increase the
microbial competition by altering the degradation pathways
in sewage (Bentzen et al., 1995). The increased redox
potential by dosing nitrate not only inhibits the growth
of many of the obligate anaerobic SRB (Widdel, 1988), it
promotes at the same time, the autotrophic nitrate-
reducing, sulfide-oxidizing bacteria (NR-SOB) (Garcia-de-
Lomas et al., 2006). The latter are capable of oxidizing
various reduced sulfur compounds (Nica et al., 2000). In
plant-scale tests, sulfide removal efficiencies up to 94% can
be obtained by the NR-SOB Thiomicrospira denitrificans
after nitrate dosing to the wastewater (Garcia-de-Lomas
et al., 2006). The biological production of elemental sulfur

FEMS Microbiol Ecol 67 (2009) 151–161 

c2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
152
(S0) is desired because the specific properties of biologically
produced sulfur (especially small particle size) enhance the
dissolution of H2S, resulting in an aqueous solution of
polysulfide ions (Kleinjan et al., 2005). This leads to a better
prevention of H2S emission out of the anaerobic sewage.
Gadekar et al. (2006) and Cardoso et al. (2006) already
observed that a molar S2
-S/NO
3 -N ratio between 1.6 and
2.5 is optimal for production of S0.
In marine environments, NR-SOB with large cells, such as
Beggiatoa sp. (diameter up to 200 mm) and Thiomargarita
sp. (diameter up to 750 mm), are able to accumulate nitrate
in huge vacuoles up to a concentration of 7 g NO
3 -N L

1
(Schulz et al., 1999; Mussmann et al., 2003). This oxidized
redox reserve of nitrate can be used as an electron acceptor
for anaerobic sulfide oxidation. Recently, Esteve-Núñez et al.
(2008) revealed by fluorescence analysis the capacity of
extracytoplasmic cytochromes to store electrons in the
freshwater species Geobacter sulfurreducens. This oxidized
redox reserve allows continued electron transfer across the
inner membrane sufficient to supply the maintenance
energy requirements when an external electron acceptor is
not present. In this way, bacteria with an oxidized redox
reserve can be seen as microbial oxidants.
The aim of this research was to study a rapid biological
sulfide-oxidation mechanism, producing biological S0
under anaerobic conditions, in order to develop a bioaug-
mentation technique for the removal of dissolved sulfide
in the absence of an external electron acceptor. Firstly, an
NR-SOB culture was grown in a continuously stirred tank
reactor (CSTR) and its mechanism of sulfide oxidation was
examined by means of elemental analysis and stable isotope
fractioning. Secondly, the microbial community structure
was determined by denaturing gradient gel electrophoresis
(DGGE) and 16S rRNA gene sequencing. Finally, the ability
to use this NR-SOB culture as a microbial oxidant for sulfide
removal from anaerobic wastewater was investigated in
both short- and long-term batch tests, with and without
the presence of nitrate.
Materials and methods
CSTR
The inoculum was grown in a CSTR, consisting of a 2-L
anoxic culture vessel (1.8 L active volume) and a control
system (Biostats B, B. Braun Biotech International,
Melsungen, Germany). Complete mixing was assured
using a turbine impeller rotating at 100 r.p.m. The reactor
temperature was controlled at 28 1C, and dissolved oxygen
(DO), pH and redox potential were continuously measured.
The CSTR was inoculated with 20 mL of an autotrophic
enrichment culture (Rombaut et al., 2003). The initial
hydraulic retention time (HRT) was set at 3 days and was

c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
B. De Gusseme et al.
gradually decreased to 1 day. The influent was pumped
continuously from a completely closed and anaerobic vessel
(headspace was filled with nitrogen gas) using a peristaltic
pump (Watson/Marlow Ltd, Cornwall, UK) into the reactor.
The pump tubing was made of norprene to minimize the
introduction of oxygen and thus the chemical oxidation of
sulfide.
The composition of this mineral medium was 2.2 g
NaH2PO4 L
1 and 15.0 g Na2HPO4 L
1 to buffer the pH of
the influent around 7.5; 1 g NaHCO3 L
1 and 10 mg L
1 of a
mixture of trace elements (Nutriflok 50 S, Vansever et al.,
1997). It was supplemented with nitrate (17.5–70 mg NO
3-
N mg L
1) as an electron acceptor and sulfide (100 mg
sulfide-S L
1) and acetate (88–350 mg CH3COOH L
1)
as electron donors. The latter two substrates represent
compounds present in septic sewage.
Batch incubation tests
All fed batch tests were performed in 120-mL serum bottles
protected against light with an aluminum foil, closed with
butyl rubber stoppers and sealed with metal caps. During
incubation, the serum bottles were placed on a shaker at
100 r.p.m. at room temperature (around 20 1C). ‘Synthetic
sewage’ refers to the sulfide mixture ‘S’ (see further). The pH
of the sulfide mixtures, used in all batch incubation tests,
was buffered around 7.5.
Short-term incubation test
The test was performed with four different sulfide mixtures
as a substrate. The synthetic sewage ( = mixture ‘S’)
contained 2.2 g NaH2PO4 L
1; 15.0 g Na2HPO4 L
1; 1 g
NaHCO3 L
1; and 25 mg sulfide-S L
1. The synthetic sewage
was used as such (‘S’) or in combination with nitrate
(‘S1N’), nitrate and acetate (‘S1N1A’) and acetate
(‘S1A’). 90 mL of each sulfide mixture and 30 mL of the
CSTR culture (sampled 148 days after startup) as inoculum
were introduced into the serum bottles. This resulted in a
final concentration of 25 mg sulfide-S L
1 in all cases,
17.5 mg NO

1
3 -N L in the case of ‘S1N’ and ‘S1N1A’ and
88 mg CH3COOH L
1 in the case of ‘S1A’ and ‘S1N1A’. A
final biomass concentration of 52 mg volatile suspended
solids (VSS) L
1 was used in all experiments. Each mixture
was tested in triplicate and one control bottle in which
30 mL of distilled water was dosed instead of inoculum.
Samples for the measurement of sulfide, nitrate, nitrite,
acetate and sulfate were taken through the rubber stopper
using a 10-mL syringe with a needle. During sampling, the
serum bottles were connected to a small bag with N2 gas in
order to prevent the introduction of air and thus the
chemical oxidation of sulfide. Samples were taken every
hour over a period of 4 h.
FEMS Microbiol Ecol 67 (2009) 151–161
NR-SOB as microbial oxidants for biological sulfide removal
Short-term incubation test after heat inactivation
A short-term incubation test was set up in triplicate using
30 mL of the CSTR culture (sampled 150 days after startup)
after heat inactivation (121 1C, 30 min) as inoculum.
A biomass-free control was prepared with 30 mL of distilled
water instead of the culture. The four bottles were
supplemented to 120 mL with the synthetic sewage.
Short-term incubation test with sewage
A short-term incubation test was performed with real
sewage as substrate. This was sampled at the inlet point of
the urban wastewater treatment plant Ossemeersen (Ghent,
Belgium), serving 175 000 inhabitant equivalents. The mean
levels of suspended solids, chemical oxygen demand (COD),
biological oxygen demand and total ammonia nitrogen
(TAN) were 189, 283, 112 and 18 mg L
1, respectively. After
heat inactivation (121 1C, 30 min), the sewage was enriched
with sulfide to a final concentration of 25 mg sulfide-S L
1.
As an inoculum, 30 mL of the CSTR culture (sampled 177
days after startup) was used. In the biomass-free control,
30 mL of distilled water was used instead of inoculum.
Long-term incubation test
The experiment was performed in six serum bottles
containing 90 mL of the synthetic sewage supplemented
with sulfide to a final concentration of 25 mg sulfide-S L
1.
In three bottles, nitrate was added in a final concentration of
17.5 mg NO

1
3 -N L . In the remaining three bottles, no
nitrate was dosed. As inoculum, 30 mL of the CSTR culture
(sampled 229 days after startup) was used, resulting in a
final biomass concentration of 52 mg VSS L
1. A biomass-
free control was prepared with 90 mL of the same synthetic
sewage and 30 mL of distilled water. Samples for the
chemical measurements were taken every day over a period
of 4 days as described above. At the end of each day (at 24, 48
and 72 h), the bottles were replenished with sulfide to the
initial concentration of 25 mg sulfide-S L
1.
Supernatant experiment
To examine whether the biomass was capable of sulfide
oxidation without its supernatant or an external compound
in the solution was indispensable for the biomass, four
samples of the CSTR mixed liquor (30 mL each, sampled
157 days after startup) were centrifuged (1476 g for 20 min)
in order to separate the biomass from the supernatant. The
separated biomass of three samples was introduced into
three serum bottles. The corresponding supernatant samples
were introduced into three other serum bottles. The
separated biomass of the fourth sample was resuspended in
FEMS Microbiol Ecol 67 (2009) 151–161
153
its own supernatant (from the initial CSTR mixed liquor
sample) and introduced into a seventh serum bottle. All
serum bottles were completely filled with the synthetic
sewage, resulting in the same final concentrations as those
used in the short-term incubation test with the ‘S’ mixture.
Samples for chemical analysis were taken every hour over a
period of 4 h as described above.
34
S/32S fractionation
A short-term incubation test was performed in four 120-mL
serum bottles containing 30 mL of CSTR inoculum
(sampled 209 days after startup) and 90 mL of the synthetic
sewage. At 0 h, all the sulfide in the solution of two
bottles was precipitated with a zinc acetate solution in
excess according to Greenberg et al. (1992). At 4 h, the
sulfide in the solution of the two remaining bottles was
again precipitated as ZnS. The precipitates were used
in duplicate.
Stable isotope analysis
34
S/32S analyses were carried out using an elemental analy-
sis-isotope ratio mass spectrometer (20-20, Europa Scienti-
fic Ltd, Crewe, UK). The isotope ratios d34SV-CDT were
expressed in per mill (%) units relative to the Canon Diablo
Troilite (V-CDT) standard. The d value is defined as
[(Rsp
Rst)/Rst]  1000, where R is the isotope ratio in
the sample (sp) and the standard (st). Analytical errors
associated with the overall process of these determinations
were 0.3%. The lab reference material used for sulfur
analysis was silver sulfide (d34SV-CDT = 13.96%).
Chemical analysis
Acetate concentrations were determined by analysis of the
volatile fatty acids. This fraction was extracted from the
samples using diethyl ether (Greenberg et al., 1992). The
samples were analyzed in triplicate using a capillary flame
ionization detector gas chromatograph (GC) (GC 8000
Carlo Erba Instruments, Wigan, UK) using an Alltech
(Deerfield) EC-1000 column (30 m, i.d. 0.32 mm, df
0.25 mm). Nitrate, nitrite, sulfite, thiosulfate and sulfate were
determined in triplicate using a Methrom 761 Compact Ion
Chromatograph (Methrom, Herisau, Switzerland) equipped
with a conductivity detector, and a metrosep A supp 5
column. Na2CO3 (1.06 g L
1) was used as an eluent
with a flow of 0.7 mL min
1 and a sample loop of 20 mL.
Sulfide was determined according to the methylene blue
method (Greenberg et al., 1992). The indication ‘sulfide’
describes all liquid species (H2S, HS
and S2
). COD and
TAN concentrations were measured as described by
Greenberg et al. (1992). The concentration of VSS was
measured based on the protocols described previously

c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
154 B. De Gusseme et al.

(Greenberg et al., 1992). The total Fe concentration in the
solution was measured using inductive coupled plasma
optical emission spectrometry (Varian Vista MPX, Varian,
Palo Alto). The detection limit was 0.025 mg Fe L
1. Liquid
CSTR samples were evaporated under air for elemental
analysis. The evaporation residues (white flocs) were ana-
lyzed on a double-sided carbon tape by means of a scanning
electron microscope (FEI SEM XL30, Eindhoven, the
Netherlands) equipped with an energy-dispersive X-ray
spectroscopy (EDX) detector (EDAX, Tilburg, the
Netherlands). This technique allows to obtain reasonable
quantitative results in a short period of time. The carbon
content was omitted from the elemental analysis as the
measurement is influenced by the carbon strip used during
the elemental analysis.
Microbial community analysis
Total DNA extraction from the biomass samples of the
CSTR was based on Boon et al. (2000). PCR was performed
as described previously (Boon et al., 2002) with primers
P388f and P518r, targeting all bacterial 16S rRNA genes
(Øvreas et al., 1997). According to Muyzer et al. (1993),
DGGE analyses were performed on a Bio-Rad D gene system
(Bio-Rad, Hercules, CA). For normalization and analyses of
the DGGE gel patterns, the BIONUMERICS software was used
(Applied Maths, Kortrijk, Belgium) (Boon et al., 2003).
DGGE bands of interest were excised off the gel and added
to 50 mL of DNAse- and RNAse-free filter-sterilized PCR
water (Sigma-Aldrich, Steinheim, Germany). After incuba-
tion for 7 h at 4 1C, 1 mL of the PCR water was reamplified
using the same primer set. DNA sequencing was carried
out by IIT Biotech-Bioservice (Bielefeld, Germany). These
sequences were compared with other rRNA gene sequences
of the BLAST server of the National Centre for Biotechnology
Information (http://www.ncbi.nlm.nih.gov) and the
RDP-II hierarchy classifier (http://rdp.cme.msu.edu/)
and expressed as percentage similarity. Sequences are
deposited in GenBank database under accession nos
EU236722–EU236724.
Microbial community diversity analysis was based on the
method of Lorenz (1905). Pareto–Lorenz (PL) evenness
distribution curves were constructed based on the DGGE
profiles, and Gini coefficients were calculated as described
previously (Mertens et al., 2005).
Results and discussion
Performance of the NR-SOB culture in the CSTR
A CSTR was operated for more than 250 days in order to
grow an enrichment culture of NR-SOB. The CSTR had, on
average, a pH of 7.5, a DO concentration of 0.0 mg O2 L
1
and a redox potential of
300  30 mV vs. standard hydro-
gen electrode. The average values of other effluent para-
meters, measured on specific days after startup, are given in
Table 1. Table 2 presents the soluble COD content of the
influent and the effluent of the CSTR. The effluent ammo-
nium concentration was beyond the detection level, indicat-
ing that no dissimilatory nitrate reduction to ammonium is
occurring in the CSTR. From the second day on, the effluent
sulfide concentration varied in the range of 0 to 4 mg
sulfide-S L
1, indicating that more than 95% sulfide removal
was achieved after 2 days of operation. After 29 days, the
removal efficiency was even 99%.
All nitrate was removed, but nitrite appeared to accumu-
late in the reactor up to a maximum concentration of 43 mg
NO
2 -N L

1
after 8 days of operation, indicating incomplete
denitrification. This may be explained by the fact that some
NR-SOB species do not have the ability to completely reduce
nitrate to N2 (Haveman et al., 2004). However, as the
influent composition was changed by lowering the acetate
concentration to 87.5 mg acetate L
1, a decrease of the nitrite
concentration to 24 mg NO
2 -N L

1
was observed in the
effluent on day 29. From day 50 on, as the HRT was 1 day,
the nitrite concentration in the effluent was 0 mg NO
2-
N L
1. Most likely, species with the capacity to completely
reduce nitrate to N2 were enriched in the culture by adapting
to the specific influent. Eventually, the combination of an
HRT of 1 day, a nitrate concentration of 17.5 mg NO
3 -N L

1

1
and an acetate concentration of 87.5 mg acetate L resulted
in complete nitrate and acetate removal (steady-state
performance from day 63 on). Moreover, no other volatile

Table 1. Hydraulic retention time and average values (n = 3) of influent and effluent parameters during the test period, measured at several days after
startup of the CSTR in filtered samples
CSTR influent (mg L
1) CSTR effluent (mg L
1)

Day HRT (days) S2
-S NO

3 -N Acetate S2
-S NO

3 -N NO

2 -N Acetate SO2

4 -S

2 3 100 70.0 350.0 41 0  0.5 6  0.5 43  3 45  0.5

8 2 100 70.0 350.0 21 1  0.5 43  0.5 132  9 40  0.5
29 2 100 70.0 87.5 11 0  0.5 24  0.5 72  5 27  0.5
50 1 100 70.0 350.0 12 0  0.5 0  0.5 73 20  0.5
63 1 100 17.5 87.5 11 0  0.5 2  0.5 01 19  0.5
251 1 100 17.5 87.5 11 0  0.5 0  0.5 01 21  0.5


c 2008 Federation of European Microbiological Societies FEMS Microbiol Ecol 67 (2009) 151–161
Published by Blackwell Publishing Ltd. All rights reserved
NR-SOB as microbial oxidants for biological sulfide removal 155

Table 2. Soluble COD content of the influent and the effluent of the Biological sulfide oxidation
CSTR, measured in triplicate at steady performance of the reactor
In order to examine whether the oxidation reaction in the
CSTR influent CSTR effluent CSTR was caused by a biological or a chemical effect, a
Time (days after startup) (mg O2 L
1) (mg O2 L
1)
short-term incubation test was performed after heat inacti-
169 201  1.7 28.3  2.8 vation of the CSTR culture. A total sulfide removal of
206 204.1  11.2 23.6  1.0
2.0  0.4 mg sulfide-S L
1 was measured after 4 h. However,
243 196.2  2.5 26.9  1.8
in the biomass-free control the sulfide concentration was
decreased from 24.8 mg sulfide-S L
1 to 22.4 mg sulfide-
S L
1 in 4 h. Thus, no significant removal of sulfide was
detected, demonstrating that no oxidation occurs when the
fatty acids were detected in the effluent at steady-state
biomass is inactive. An additional short-term incubation
performance.
test was set up to monitor stable isotope fractioning in the
The complete elimination of both nitrate and acetate
sulfide. In 4 h, a sulfide removal of 8.0  0.4 mg sulfide-S L
1
suggested that besides (facultative) chemolithotrophic
was obtained, resulting in a removal rate of 2.0 mg sulfide-
nitrate removal, heterotrophic denitrification also occurred.
S L
1 h
1. At 0 h, the d34SV-CDT measured in the ZnS
Nevertheless, the heterotrophic bacteria were not able to
precipitate was 5.15  0.32%. After 4 h of incubation, the
outcompete the NR-SOB. In 1979, Gottschal et al. (1979)
isotope ratio in the remaining sulfide was increased to
already demonstrated that (facultatively) chemolithotrophic
6.27  0.12%, showing an enrichment in 34S by 1.12%. This
bacteria are able to survive under appropriate limiting mixed
is a small although significant, isotope fractionation. Fry
substrate conditions in the presence of more ‘specialized’
(2006) also described the relatively small isotope effects in
heterotrophs. Probably, the NR-SOB may have an advantage
the oxidative side of the sulfur cycle. Isotopic enrichment is
over the heterotrophic bacteria because the presence of sulfide
indicative of a biological mechanism because the use of the
has an inhibitory effect on heterotrophic denitrifiers due to
lighter isotope in enzymatic processes requires a smaller
interaction with the iron-containing cytochromes responsible
activation energy. As a consequence, it is shown that the
for respiration (Sørensen et al., 1980).
presence of S0 flocs in the CSTR is due to a biological
Sulfate production decreased when the HRT was lowered
process.
(Table 1). From day 50 on, the sulfate concentration in the

1 These results fit well in the simultaneous removal of
effluent was on average 20 mg SO2
2

4 -S L . No sulfite (SO3 -
2
nitrogen, sulfide and acetate, as reported by Reyes-Avila
S) or thiosulfate (S2O3 -S) production was detected in the
et al. (2004). These authors described removal efficiencies
effluent. This implies that the major part (80 mg sulfide-
for nitrate, acetate and sulfide close to 100%, 69% and
S L
1) of the total amount of sulfide in the influent (100 mg
100%, respectively, in a continuous denitrifying sulfide-
sulfide-S L
1) was converted into other sulfur-containing
oxidizing reactor. Elemental sulfur seemed to accumulate in
components.
the reactor. In this CSTR, almost complete removal of the
In order to elucidate the final product of the sulfide-
three substrates is occurring at steady-state performance
oxidation reaction, elemental analysis was performed on the
(100 mg sulfide-S L
1, 17.5 mg NO
3 -N L

1
and 87.5 mg
white flocs present in the CSTR. The EDX spectrum
1
acetate L ). As stated above, it is most likely that hetero-
indicated that the flocs contained up to 51% sulfur on a
trophic denitrification is responsible for the removal of
molar base (data not shown). Based on the results, it was
acetate in the CSTR. Complete biodegradation of acetate to
calculated that maximum 19% of the sulfur could be present
CO2 is assumed, because no volatile fatty acids were
as sulfate (after evaporation of the CSTR liquid sample
present in the reactor effluent. Moreover, no substantial
under air). Yet, at least 81% of this sulfur was most probably
accumulation of the carbon storage compound poly-3-
present as elemental sulfur or polysulfides. The reaction of
hydroxybutyrate was detected in the biomass (data not
biologically produced sulfur particles with dissolved H2S is
shown). Supposing a yield coefficient of 0.5 in this CSTR,
known to result in the formation of polysulfide anions
43.8 mg acetate L
1 (0.729 mM) may be completely oxidized
(Kleinjan et al., 2005). As a conclusion, there is a conversion
to CO2 according to the following equation (Yamamoto-
in the CSTR of 99  1 mg sulfide-S L
1 of the total 100 mg
Ikemoto & Komori, 2003):
sulfide-S L
1 in the influent. In the effluent, 20  2 mg
sulfate-S L
1 is present in solution, whereas 80  2 mg 5CH3 COO
þ 8NO
þ
3 þ 13H
sulfide-S L
1 is retained in the reactor, possibly as elemental ! 10CO2 þ 4N2 þ 14H2 O ð1Þ
sulfur and polysulfide. The sulfur flocs also contained some
Na and P, small amounts of Al and Si and a negligible Assuming full conversion, 16.3 mg NO
3 -N L

1

amount of Ca but no metals, indicating that there is no (1.167 mM) is removed by heterotrophic denitrification.
precipitation of metal sulfides. The production of S0 was no surprise because from day 63

FEMS Microbiol Ecol 67 (2009) 151–161 

c2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
156
on, nitrate was dosed at a molar S2
-S/NO

3 -N ratio of 2.5 in
the CSTR influent, according to the following reaction
(Thauer et al., 1977):
1 1
7 þ 1 1 3
NO

3 þ HS þ H ! N2 þ S0 þ H2 O ð2Þ
5 2 10 10 2 5
Yet, a maximum of 6.6 mg sulfide-S L
1 can be removed by
reaction (2), using the remaining 1.2 mg NO
3 -N L

1
(0.082 mM). As a conclusion, a substantial part of the sulfide
(93.4 mg sulfide-S L
1) is most probably removed by an-
other mechanism, like polysulfide formation (Kleinjan et al.,
2005). The most common form of biologically produced
elemental sulfur is an S8 ring (Steudel, 1996). Formation of a
mixture of polysulfides by reaction of H2S with elemental
sulfur (S8-) rings is initiated by the opening of the S8 ring by
HS
, which reacts as a strong nucleophile. Subsequently,
the resulting long-chain polysulfide ion is rearranged
into shorter polysulfides by reaction with other HS
ions
(Steudel, 2000). These short-chain polysulfide ions seem to
be strong nucleophiles as well, capable of opening the S8
ring. As a result, formation of polysulfide ions has an
autocatalytic effect on the rate of sulfur dissolution in
aqueous sulfide solutions (Hartler et al., 1967). Kleinjan
et al. (2005) demonstrated the formation of polysulfides to
take place at the surface of the biologically produced sulfur
particles. After 63 days of producing elemental sulfur in this
CSTR, sufficient S0 flocs were most probably accumulated in
the reactor to serve as reaction sites between biologically
produced sulfur particles with dissolved H2S.
Characterization of the microbial community of
the CSTR
The composition of the CSTR culture was investigated by
means of molecular analysis. DGGE analysis showed that the
consortium clearly shifted in time (Fig. 1a). Species richness
was indicated by the number of bands in the DGGE gel. The
equality of species distribution within the culture is given by
the PL evenness distribution curves (Fig. 1b) and the Gini

c 2008 Federation of European Microbiological Societies
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B. De Gusseme et al.
coefficient. The latter is a number between 0 and 1, with 1
representing total evenness. At startup of the reactor,
under higher HRT (3 days), the microbial community
was dominated by three species in an even distribution,
indicated by a PL curve close to the 451 diagonal (the
theoretical perfect evenness line) and a high Gini coefficient
(0.93). Two of the bands were sequenced and showed the
highest similarity to the phylum Acidobacteria (Fig. 1a, band
2, accession no. EU236722, 96% similarity) and Thauera sp.
(Fig. 1a, band 3, accession no. EU236723, 100% similarity).
From day 13 on, some of the bands disappeared (band 3) or
became less intense (band 2), while other bands appeared
(band 1). At lower HRT (1 day), the culture became
dominated by the species Arcobacter sp. (Fig. 1a, band 1,
accession no. EU236724, 100% similarity) from day 151 on.
As a consequence, the PL curves deviated more from the 451
diagonal and the Gini coefficient decreased to 0.53  0.07
(day 13–151). Arcobacter sp. is of recent interest because it is
frequently isolated from products of animal origin and a
variety of water samples including those of drinking water
(Cervenka, 2007). Arcobacter sp. are NR-SOB (Gevertz et al.,
2000) that effectively compete with other sulfur-oxidizing
bacteria by being able to tolerate higher concentrations of
H2S and to grow at very low molecular oxygen concentra-
tions (Sievert et al., 2007). They excrete S0 and attach to the
white flocs and wool-like mats of produced filamentous
sulfur (Wirsen et al., 2002). Garcia-de-Lomas et al. (2007)
recently reported the stimulation of anaerobic sulfide oxida-
tion using nitrate as an electron acceptor by Arcobacter sp.
Rapid sulfide removal by the CSTR culture
At steady-state performance of the CSTR, samples of the
NR-SOB culture were taken out of the reactor and tested for
their ability to rapidly remove sulfide under various sub-
strate conditions during a 4 h short-term incubation
test (initial sulfide concentration of 25 mg sulfide-S L
1).
Removal rates are expressed as net removal (total sulfide
oxidation – both biological and chemical – minus chemical
Fig. 1. (a) DGGE analysis of the microbial
community of the CSTR as a function of time.
Lanes A and B show home-made markers. (b) PL
curves based on the DGGE fingerprint of the
microbial community as a function of time

(— — : startup; – –.– –: 13 days;

      : 41 days; – –’– –: 61 days; —
~—: 97 days; – –m– –: 111 days;     : 151
days; ——: evenness).
FEMS Microbiol Ecol 67 (2009) 151–161
NR-SOB as microbial oxidants for biological sulfide removal
sulfide oxidation in the control). The chemical oxidation in
the biomass-free control accounted for 0.5  0.2 mg sulfide-
S L
1 h
1 (ultimate decrease from 24.8  0.2 mg sulfide-S L
1
to 23.2  0.2 mg sulfide-S L
1). Figure 2 shows that in each
case, the introduction of the CSTR-culture had positive
effects on the sulfide oxidation. The short-term incubation
test with substrate ‘S’ yielded in an average net removal rate
of 1.9  0.2 mg sulfide-S L
1 h
1. Yet, the highest removal
efficiencies after 4 h were obtained using ‘S1N’ and ‘S1A’ as
a substrate, with an average net removal rate of 2.7  0.1 mg
sulfide-S L
1 h
1 and 2.5  0.2 mg sulfide-S L
1 h
1, respec-
tively. In other words, the presence of acetate did not affect
the sulfide removal rate in the latter experiment. Moreover,
no acetate removal was detected (data not shown). This is an
indication that the oxidation process by the consortium of
the CSTR is chemolithotrophic.
Nevertheless, acetate was completely removed in the
reactor (Table 1). This was most probably due to hetero-
trophic denitrification. Competition between heterotrophic
denitrifying bacteria and autotrophic NR-SOB seems to be
the reason why the sulfide removal in the short-term
incubation test was the lowest in mixture ‘S1A1N’. In this
test, both nitrate and acetate were present, which yielded an
average net removal rate of 1.2  0.2 mg sulfide-S L
1 h
1
(Fig. 2). However, it is stated by Cardoso et al. (2006) that
addition of acetate to the influent of chemolithotrophic
denitrification bioreactors could improve the reactor
stability compared with strict autotrophic denitrification.
The sulfide removal rates, up to 51.9 mg sulfide-
S g VSS
1 h
1 in the presence of nitrate and in the absence
of molecular oxygen, are higher than those reported pre-
viously for freshwater species (Cardoso et al., 2006; Gadekar
Fig. 2. Cumulative course of the net sulfide removal as a function of
time during the short-term incubation test over 4 h under anoxic
conditions. The test bottles contain CSTR inoculum and a sulfide mixture
 The initial removal of sulfide without a concomitant
as a substrate. Substrate ‘S’ (— —) contains 25 mg sulfide-S L
1;
substrate ‘S1N’ (– –.– –) 25 mg sulfide-S L
1 and 17.5 mg NO

1
3 -N L ;
substrate ‘S1A’ (   
    ) 25 mg sulfide-S L
1 and 87.5 mg acet-
ate L
1; and substrate ‘S1A1N’ (– –’– –) 25 mg sulfide-S L
1, 17.5 mg
NO
3 -N L

1
and 87.5 mg acetate L
1.
FEMS Microbiol Ecol 67 (2009) 151–161
157
et al., 2006; Mahmood et al., 2007; Manconi et al., 2007).
Moreover, the capacity of the CSTR culture to remove
sulfide in real sewage was also examined in a short-term
incubation test without adding nitrate to the sewage. An
average removal rate of 1.5  0.2 mg sulfide-S L
1 h
1 was
detected, equivalent to 28.8 mg sulfide-S g VSS
1 h
1. Hence,
an enriched NR-SOB-culture can be used as microbial
oxidant in a bioaugmentation technique for rapid sulfide
removal from anaerobic sewage.
The importance of this study lies in the fact that the
addition of nitrate to achieve sulfide removal was not
required in the 4-h testing period. For instance, in the
short-term incubation test with substrate ‘S,’ significant
sulfide removal was observed, without the presence of an
apparent electron acceptor. In fact, no acetate, nitrate or
nitrite removal was measurable in the four substrates, nor
did any sulfate production occur (data not shown). There
must be an alternative pathway of sulfide removal.
Alternative pathway of sulfide removal
A long-term incubation test was set up to compare the
sulfide removal in serum bottles fed with both sulfide and
nitrate and serum bottles fed with only sulfide (Fig. 3a).
After each day of testing, the sulfide concentration was
brought to its initial level, 25 mg sulfide-S L
1, in order to
examine the capacity of the CSTR culture to oxidize sulfide
over several days. Throughout 4 consecutive days, the sulfide
concentrations were measured. On day 1, sulfide was
removed by the biomass to a similar extent of 10.0  0.2 mg
sulfide-S L
1 with and without the addition of nitrate
(Fig. 3a). From day 2 on, smaller sulfide removal rates were
detected (probably because of some loss of biomass due to
sampling) and a stable level was reached. The inoculum
without nitrate showed lower sulfide removal rates and
reached lower levels of ultimate sulfide removal on day 2
and 3. On day 4, the inoculum without nitrate was no longer
able to remove sulfide (Fig. 3a).
In the presence of nitrate, however, the inoculum retained
its capacity to oxidize sulfide at day 4, resulting in a
continuous removal of sulfide (Fig. 3a). Starting on day 3, a
significant uptake of nitrate was detected (Fig. 3b). At the
end of the experiment (at 92 h), 1.8  0.8 mg NO
3 -N L

1
was reduced in the nitrate-containing bottles while concur-
rently a surplus of 7.9  0.6 mg sulfide-S L
1 was oxidized
compared with the nitrate-free bottles. As a consequence,
the molar S2
-S/NO
3 -N ratio was 2.0  0.5. Approximately,
this electron balance is in accordance with Eqn (2).
decrease of nitrate remains striking. As a hypothesis, it was
put forward that the CSTR culture accumulates or produces
a redox-active compound for the oxidation of sulfide.
Apparently, this compound is serving as a temporal electron

c2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
158
acceptor reserve for 2 days in the long-term experiment.
This is remarkable because the biomass is grown under
nitrate-limiting conditions. Probably, the bacteria are able
to oxidize substantial amounts of sulfide without the
need for an apparent electron acceptor such as nitrate.
Further research of the mechanism was performed by the
supernatant experiment. The biomass of the CSTR culture
was separated from its supernatant and both the biomass
and the supernatant were used as an inoculum in synthetic
sewage without nitrate. Neither in the three serum bottles
with the separated biomass nor in the three serum bottles
with the supernatant could any significant sulfide removal
different from the biomass-free control be detected. How-
ever, in the serum bottle incubated with the separated
biomass that was introduced into its supernatant again, a
sulfide removal of 7.2  0.4 mg sulfide-S L
1 was detected in
4 h. These findings strongly suggest that an external com-
pound for sulfide oxidation was present in the solution,
representing an indispensable oxidative redox reserve for the
biomass.
These findings are different from known internal oxida-
tive redox reserves in large sulfur marine bacteria storing
nitrate under temporary nitrate-surplus conditions such as

c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
B. De Gusseme et al.
Fig. 3. Long-term incubation test. (a) Daily
cumulative net sulfide removal as a function of
time in the serum bottles containing sulfide
(25 mg sulfide-S L
1) and nitrate (17.5 mg
NO

1
3 -N L ) (– –.– –) and the serum bottles with

only sulfide (25 mg sulfide-S L
1) (— —). At
the end of each day (at 24, 48 and 72 h), the
bottles were replenished with sulfide to the
initial concentration. (b) Cumulative course of
the nitrate removal as a function of the time in
the serum bottles containing sulfide and nitrate
(– –.– –). No nitrate removal could be detected in
the control bottle (not in figure).
Beggiatoa sp. and Thiomargarita sp. (Schulz et al., 1999;
Mussmann et al., 2003) and smaller freshwater bacteria such
as G. sulfurreducens (Esteve-Núñez et al., 2008). Apparently,
in this case, where nitrate is limited, another compound was
used by the CSTR culture for 2 days, before nitrate served
as an electron acceptor in the long-term incubation test
(Fig. 4). This strongly suggests that an external electron-
accepting substance (X) is preferentially used by the CSTR
culture in this batch experiment. It seems that denitrifica-
tion is not starting until the oxidized form of compound X is
completely reduced. Probably, the reduction of this com-
pound is thermodynamically more favorable (higher Gibbs
free energy production) for the bacteria than denitrification
(Fig. 4). In the first 2 days of the long-term incubation test
in the presence of nitrate, 15.2  0.2 mg sulfide-S L
1
was oxidized by the inoculum to probably elemental sulfur
(Fig. 3a). According to Eqn (3) (Buisman et al., 1990), this is
equivalent to 7.6  0.2 mg O2 L
1 (expressed as 7.6 mg O2-
eq L
1):
H2 S þ 0:5 O2 ! S0 þ H2 O ð3Þ
However, the CSTR culture is four times diluted in the
long-term incubation experiment and, therefore, it can be
FEMS Microbiol Ecol 67 (2009) 151–161
NR-SOB as microbial oxidants for biological sulfide removal
Fig. 4. Conceptual model for sulfide oxidation, coupled to denitrifica-
tion by a redox mediator (X).
calculated that an oxidative redox reserve of 30.4 mg O2-
eq L
1 is present in the inoculum itself. Up till now, it is
unclear whether this redox-active compound has an organic
or an inorganic chemical identity. Known organic redox
mediators with low molecular weight are 2,6-anthraquinone
disulfonate (Curtis & Reinhard, 1994), ferricyanide (Emde
& Schink, 1990) and the phenazine pyocyanin (Rabaey et al.,
2004). It has been reported that the latter can be produced
by Pseudomonas aeruginosa in amounts of several mg L
1 in
the supernatant after 10 h of growth (Dietrich et al., 2006).
Recently, von Canstein et al. (2008) reported the secretion of
flavin mononucleotide and riboflavin by a range of Schewa-
nella species as an extracellular electron shuttle. These
bacteria have an advantage over other microorganisms in
environments that lack exogenous redox mediators such as
humics. On the other hand, some of the redox mediators
mentioned above have a COD that is much higher than the
amount of O2-eq they can accept, and so it would require
substantial amounts of COD to accept 30.4 mg O2-eq L
1.
Because the average soluble COD content in the effluent of
the CSTR is 26.3  4.8 mg O2-eq L
1 (average value of the
three results given in Table 2), it seems that the oxidative
redox reserve has another chemical identity, potentially
inorganic. Further research is needed to chemically identify
this external molecule present in the solution of the CSTR
culture.
Conclusions
 To prevent the emission of H2S and deal with microbially
concrete corrosion, it is important to remove sulfide as soon
as possible from the anaerobic sewage. Therefore, incuba-
tion and biostimulation of NR-SOB in sewers may be useful.
Unfortunately, nitrate must be dosed. In this study, a
microbial consortium of NR-SOB was grown in a CSTR.
The consortium, dominated by Arcobacter sp., was capable
of removing 99% of sulfide in the absence of oxygen. Part of
the sulfide is oxidized into elemental sulfur, whereas a
considerable amount is removed by another mechanism,
probably formation of polysulfides. Stable isotope fraction-
ing of the sulfide confirmed the biological nature of the
oxidation.
FEMS Microbiol Ecol 67 (2009) 151–161
159
 In short-term batch experiments, removal rates up to
52 mg sulfide-S g VSS
1 h
1 were detected, to our knowledge
the highest removal rate reported so far for freshwater
species in the absence of molecular oxygen. It was most
striking that the sulfide oxidation occurred without the
presence of nitrate during these experiments.
 Further long-term incubation experiments revealed the
capacity of the microbial consortium to oxidize sulfide
without the presence of nitrate, suggesting that an oxidized
redox reserve is present in solution in the culture. This redox
reserve allowed the microorganisms to oxidize sulfide for 2
days in the absence of nitrate, representing 30.4 mg
O2-eq L
1. This new sulfide removal concept by adding
microbial oxidants to anaerobic sewage without extra
addition of nitrate provides promising opportunities for
prevention of sulfide emission.
Acknowledgements
This research was funded by Project Grant GOA 1205073
(2003–2008) from the ‘Ministerie van de Vlaamse Ge-
meenschap, Bestuur Wetenschappelijk Onderzoek’ (Bel-
gium). B.De.G. is supported by a Ph.D. grant (Aspirant)
from the Research Foundation–Flanders [Fonds voor
Wetenschappelijk Onderzoek (FWO) Vlaanderen]. K.V. is a
postdoctoral Fellow of the FWO. The authors are grateful to
Katja Van Nieuland, Petra Van Damme and Siska Maertens
for technical support, and Willem De Muynck and Tuba H.
Ergüder for the many critical and helpful suggestions.
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