water research 43 (2009) 4123–4132

Available at www.sciencedirect.com

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Inhibition of sulfate-reducing and methanogenic activities

of anaerobic sewer biofilms by ferric iron dosing

Lishan Zhang, Jürg Keller, Zhiguo Yuan*

Advanced Water Management Centre, Building 60, Research Road, The University of Queensland, St Lucia, Brisbane, QLD 4072, Australia

article info abstract

Article history: Ferric iron is commonly used for sulfide precipitation in sewers, thus achieving corrosion
Received 4 March 2009 and odour control. Its impact on the activities of sulfate-reducing bacteria and metha-
Received in revised form nogens in anaerobic sewer biofilms is investigated in this study. Two lab-scale rising main
27 May 2009 sewer systems fed with real sewage were operated for 8 months. One received Fe3þ dosage
Accepted 4 June 2009 (experimental system) and the other was used as a control. In addition to precipitating
Published online 12 June 2009 sulfide from bulk water, Fe3þ dosage was found to significantly inhibit sulfate reduction
and methane production by sewer biofilms. The experimental reactor discharged an
Keywords: effluent containing a higher concentration of sulfate and a lower concentration of methane
Ferric in comparison with the reference reactor. Batch experiments showed that the addition of
Methane ferric ions reduced the sulfate reduction and methane production rates of the sewer bio-
Inhibition films by 60% and 80%, respectively. The batch experiments further showed that Fe3þ
Methanogens dosage changed the final products of sulfate reduction with sulfide accounting for only 54%
Rising main of the sulfate reduced. The other products could not be confirmed, but were not dissolved
Sewer inorganic sulfur species such as sulfite or thiosulfate. The results suggest the addition of
Sulfate-reducing bacteria Fe3þ at upstream locations would minimize the ferric salts required for achieving the same
Sulfide level of sulfide removal. Fe3þ dosing could also substantially reduce the formation of
methane, a potent greenhouse gas, in sewers.
ª 2009 Elsevier Ltd. All rights reserved.

1. Introduction wastewater industry to minimize the adverse effect of

During the transportation of wastewater in a rising main over
a long distance, the wastewater becomes anaerobic due to the
high oxygen demand of the organic compounds and a lack of
oxygenation. Hydrogen sulfide is then generated by sulfate-
reducing bacteria (SRB) present primarily in the biofilms on
sewer walls (Thistlethwayte, 1972; USEPA, 1974; ASCE, 1989;
Nielsen et al., 1998; Hvitved-Jacobsen, 2002). The buildup of
sulfide and its subsequent transfer to the gas phase induce
odour and corrosion problems (Thistlethwayte, 1972; USEPA,
1974; USEPA, 1991). Many technologies are being used by the
hydrogen sulfide formed in sewer systems. The commonly
used liquid phase technologies include: injection/addition of
air, pure oxygen or nitrate salts to prevent sulfide formation
(ASCE, 1989; Tanaka and Takenaka, 1995; Ochi et al., 1998); the
addition of ozone, hydrogen peroxide, hypochlorites, chlorine,
and potassium permanganate to chemically oxidized sulfide
(Cadena and Peters, 1988; Millero et al., 1989; Tomar and
Abdullah, 1994; Charron et al., 2004); the addition of Mg(OH)2
to elevate pH thus minimizing the transfer of molecular H2S to
the gas phase (Rees et al., 2003) and the addition of metal salts
including iron, zinc, lead and copper salts to precipitate sulfide

* Corresponding author. Tel.: þ61 7 3365 4374; fax: þ61 7 3365 4726.
E-mail addresses: lishan@awmc.uq.edu.au (L. Zhang), j.keller@awmc.uq.edu.au (J. Keller), zhiguo@awmc.uq.edu.au (Z. Yuan).
0043-1354/$ – see front matter ª 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.watres.2009.06.013
4124 water research 43 (2009) 4123–4132
by forming highly insoluble metallic sulfide precipitates
(ASCE, 1989; Jameel, 1989; Padival et al., 1995; Poulton et al.,
2002).
Fe3þ is effective in reducing dissolved sulfide from waste-
water (Padival et al., 1995; Dewaters et al., 1999; Nielsen et al.,
2005). Fe3þ oxidizes sulfide to elemental sulfur while being
reduced into Fe2þ, which precipitates with sulfide to form
ferrous sulfide precipitants (Dohnalek and Fitzpatrick, 1983):
2Fe3þ þ S2 /2Fe2þ þ S0 (1)
Fe2þ þ HS /FeSY þ Hþ (2)
3þ
At typical pH of municipal sewage (6.5 < pH < 8.5), Fe may
hydrolyze rapidly to form amorphous Fe(OH)3(S). However,
ferric hydroxide can remove hydrogen sulfide from aqueous
systems through oxidation and precipitation reactions, in
a similar way to those described above (Davydov et al., 1998).
Ferric phosphate, which may also form when ferric ions are
added to wastewater, can also oxidize sulfide under anaerobic
conditions, resulting in phosphate release to bulk water and
the formation of FeS (Singer, 1972). In addition to sulfide
control in sewer systems (USEPA, 1974; ASCE, 1989; Jameel,
1989; USEPA, 1991; Padival et al., 1995; Nielsen et al., 2005),
ferric salts have also been used for the abatement of sulfide-
associated problems in groundwater supplies (Poulton et al.,
2002), high-strength pulp and paper manufacturing wastes
(Dewaters et al., 1999) and in anaerobic digesters (Dezham
et al., 1988).
Despite its proven effectiveness in controlling dissolved
sulfide in sewer systems, little is known about the effect of
Fe3þ on the activity of sewer biofilms. Most studies were
undertaken by adding Fe3þ to remove sulfide from raw
municipal sewage in the absence of sewer biofilms (Dohnalek
and Fitzpatrick, 1983). The interactions between Fe3þ addition
and sewer biofilm activities have not been investigated,
despite that it is well known that some metal ions inhibit the
activities of pure strains and mixed cultures of SRB (Utgikar
et al., 2003). This study was aimed at determining the effect of
Fe3þ addition on the activities of sulfate-reducing bacteria and
methane producing archea in anaerobic sewer biofilms.
Methanogens are present in sewer biofilms (Mohanakrishnan
et al., in press) and significant methane production in sewer
systems has been reported recently (Guisasola et al., 2008).
Two lab-scale rising main sewer systems, fed with real
sewage, were set up to develop anaerobic sewer biofilms. Fe3þ
was added to one line, the experimental system. The impact
of Fe3þ on the sulfate-reducing and methanogenic activities of
sewer biofilms was investigated by comparing the sulfate
reduction and methane production rates of the experimental
and control biofilms, and also the sulfate and methane
concentrations in the effluent of the two systems.
2. Materials and methods
2.1. Lab-scale sewer systems and their operation
Two reactor-based systems were set up to simulate rising
mains in laboratory conditions (Fig. 1). One was used as the
control line (RMC) and the other as the experimental line
(RME). Each system consisted of two completely sealed reac-
tors connected in series, named RMC1/RME1 and RMC2/RME2,
respectively, as shown in Fig. 1. Each reactor had a volume of
1.0 L and an internal diameter of 80 mm. Plastic Kaldnes K1
carriers (circular, 1 cm diameter/length; AnoxKaldnes,
Norway) were placed on rods inside the reactors to provide
additional biofilm growth area that could also be harvested
without disturbing the biofilm structure.
The system was intermittently fed with sewage using
a peristaltic pump following a typical pumping pattern
observed at the UC09 rising main (Gold Coast, Australia)
(Guisasola et al., 2008). Totally, 16 pumping events occurred
per day, which resulted in an average hydraulic retention time
(HRT) in the system of 3 h with a minimum and a maximum
HRT of 1 and 9 h, respectively (Fig. 1). Each pumping operation
lasted for 2 min at a flow rate of 0.5 L min1. The wastewater
was mixed by means of a magnetic stirrer in each reactor
during the pumping operation. The wastewater used for lab
reactor studies was collected weekly from a wet well located
at Robertson Park on Lambert Road, Indooroopilly (Brisbane).
The wastewater was of fresh, domestic origin and typically
contained sulfide at a concentration below 3 mg S L1, sulfate
at a concentration between 10 and 25 mg S L1, and VFA at
levels of 50–100 mg COD L1. Sulfite and thiosulfate were
detected at low concentrations of <1 mg S L1. The waste-
water was stored in a cold room at 4  C to minimize biological
transformations during storage. Before being pumped into the
reactors, the sewage was preheated by a water bath to 21  C.
Sulfide generation, methane production and VFA produc-
tion/consumption in the systems were monitored through
liquid phase sampling and off-line chemical analysis. Samples
were collected from each reactor before the start of nine
pumping events over a period of 9 h (daytime). As shown in
Fig. 1, the nine sampling points covered a wide range of HRT
(1–9 h). Previous studies showed that sulfide and methane
production in sewers are highly dependent on HRT. The
samples withdrawn from the reactors were measured for
dissolved sulfur species, methane, orthophosphate, iron and
volatile fatty acids (VFAs) using methods to be further
described later. Dissolved oxygen (DO) level in the first reactor
was measured online with a METTLER TOLEDO Inpro6050 DO
sensor connected to PLC via an O2 4100e transmitter. The
oxygen concentration was zero at all times.
Biofilms developed on sewer walls and biofilm carriers
within a few weeks. Baseline monitoring of the systems
showed consistent sulfide, sulfate, methane and VFA profiles
over days, four months after the startup, suggesting that
pseudo-steady state conditions were established in the
systems. These profiles were comparable to those measured
at the UC09 rising main (Guisasola et al., 2008), which was
simulated by the lab-systems.
The addition of Fe3þ to RME2 was initiated upon the
establishment of the pseudo-steady state. Fe3þ was added
from a stock solution prepared from reagent-grade ferric
chloride (FeCl3$6H2O) from Aldrich. The stock solution was
prepared weekly by dissolving appropriate amounts of ferric
salts in deoxygenated water containing 0.01 M HCl (to prevent
ferric hydroxide precipitation). The deoxygenated water was
prepared by bubbling purified N2 in MilliQ water for 1 h. 10 ml
 water research 43 (2009) 4123–4132 4125

Experimental Line

RME1 RME2
(Adapted to Ferric Dosing)

Control Line

RMC1 RMC2

Fe(III) Stock Solution Water Bath Cold Room 4 °C

10
HRT

hours
4

1 2 3 4 5 6 7 8 9 0
00:00:00 04:00:00 08:00:00 12:00:00 16:00:00 20:00:00 00:00:00
Time of the day

Fig. 1 – Simulated rising main sewer systems and their operation. The vertical lines in the plot indicate the pumping events,
each lasting for 2 min at 0.5 L minL1. HRT is the hydraulic retention time of the wastewater slug that is pumped into the
system during the corresponding event in the entire sewer system. ‘‘Y’’ indicates injection of Fe3D to the experimental
system during each pumping event (after Fe3D dosing commenced). Numbers 1, 2, 3, 4, 5, 6, 7, 8, 9 indicate sampling time
just before the corresponding pumping events.

of the Fe3þ stock solution was added into RME2 during each
pumping event using a dosing molar ratio around 0.6 (Fe) to 1
(S), resulting in a theoretical Fe3þ concentration of 21 mg L1 of
wastewater.
2.2. Batch experiments
Batch tests were conducted to assess the sulfate-reducing and
methanogenic activities of the sewer biofilms adapted and un-
adapted to Fe3þ, two months after Fe3þ addition to RME2 was
started. At this point, RME2 reached a new pseudo-steady
state as confirmed by the consistent sulfide, sulfate, methane
and VFA profiles over days. Three types of batch tests were
carried out. In the Type I tests, the sulfate-reducing and
methanogenic activities of RMC2 (un-adapted to Fe3þ) and
RME2 (adapted to Fe3þ) biofilms were measured and
compared. In these tests, RME2 received Fe3þ dosage. In the
Type II tests, the sulfate-reducing and methanogenic activities
of RMC2 and RME2 biofilms were measured and compared.
Neither RMC2 nor RME2 received Fe3þ addition. In both the
Type I and Type II batch tests, fresh sewage was pumped into
both the control and experimental systems for 10 min at
a flow rate of 0.5 L min1 to ensure the complete replacement
of the wastewater in RMC2 and RME2. In the Type I batch tests,
10 ml of the Fe3þ stock solution was added to RME2 immedi-
ately after the wastewater feeding. In the Type III tests, the
outlet of the reference line was connected to the inlet of the
experimental line at 9:00 am, when sulfate in all reactors had
been converted to sulfide. Then the pump was turned on for
3 min at a flow rate of 0.5 L min1. Thus, RME2 was filled with
4126 water research 43 (2009) 4123–4132
the sewage contained little sulfate, no Fe3þ, but a high level of
sulfide at the start of the batch tests. The Type III tests were
designed to investigate the potential removal of sulfide by
residual iron or elemental sulfur due to precipitation or
formation of polysulfide.
During each batch test, wastewater samples of 15 ml each
were removed from the reactor at 30 min intervals for 2 h. The
wastewater samples were filtered and preserved immediately
for the analysis of dissolved sulfur species, methane and
volatile fatty acids (VFAs) and in the case of Type I tests also of
Fe2þ and Fe3þ, using the methods to be further described
below. The magnetic stirrers in the reactors were switched on
1 min before sampling to ensure representative sampling and
switched off immediately after sampling.
2.3. Chemical analysis
Samples for analysis of soluble sulfur species were immedi-
ately preserved with a special sulfide anti-oxidant buffer
(SAOB) that allows the samples to be stored at 4  C for at least
four days without deterioration of any of the sulfur species
(Keller-lehmann et al., 2006). The soluble sulfur species were
measured on a compact Dionex ICS-2000 ion chromatograph
with an AD25 absorbance (230 nm) and a DS6 heated
conductivity detector (35  C). The injection volume was 25 mL
and the flow rate 1 ml min1. The separation was achieved
with a Dionex IonPac AG18 (4  50 mm) guard and an IonPac
AS18 (4  250 mm) separating column. Both columns were
heated to 35  C. VFAs were measured by gas chromatography
using Perkin Elmer Autosystem equipped with a polar capil-
lary column DB-FFAP at 140  C, while the injector and flame
ionization detector (FID) were operated at 220 and 250  C,
respectively. High purity helium was used as carrier gas at
a flow rate of 17 ml min1. Iron concentration was determined
by the MERCK Iron Test Kit (1.00796.001). Orthophosphate
concentration was determined by the MERCK Phosphate Test
Kit (1.14543.0001). Absorbance was measured with a Cary 50
spectrophotometer.
Dissolved methane analysis was performed using the
method described in Alberto et al. (2000). Sewage containing
dissolved methane was sampled by a 5 ml plastic syringe from
the reactors and transferred into freshly vacuumed BD vacu-
tainer tubes using a hypodermic needle. The tubes were
mixed overnight in a shaker in order to bring into equilibrium
between gas and liquid phases. Most of the methane (w97% at
25  C) was transferred to the gas phase in this process.
Methane concentrations in the gas phase of the tubes were
measured on a Shimadzu GC-9A Gas Chromatograph equip-
ped with a FID. The concentrations of methane in the initial
liquid phase (reactor) were calculated using mass balance and
Henry’s law.
3. Results
3.1. Sulfide production and methane formation
in lab-scale sewer systems prior to Fe3þ addition
Fig. 2 shows the sulfate and sulfide profiles in the four reactors
during the pseudo-steady state of the systems. The sulfide and
sulfate concentrations in the experimental line were almost
identical to those in the control line, both of which varied
considerably with the pumping pattern of the systems. Frequent
pumping events in the morning resulted in shorter HRTs and
lower sulfide production. Conversely, a lower frequency of
pumping events resulted in longer HRTs and higher sulfide
production. The concentrations of thiosulfate and sulfite in all
reactors were not significant (<0.5 mg S L1). The total dissolved
sulfur (sulfate þ sulfide þ sulfite þ thiosulfate) concentrations
in the systems were approximately constant indicating that all
the sulfate reduced was converted to sulfide, and there was no
loss of sulfur as H2S gas. The results also showed that the
methane production and VFA consumption were consistent
between the experimental and control lines (see Fig. 3). Acetate
and propionate accounted for 60–80% and 10–20%, respectively,
of the VFAs measured with the remaining being isobutyrate,
butyrate, isovalerate, valerate and hexanoic acid.
The behaviour of the experimental and control lines was
observed to be very similar before the start of the Fe3þ treat-
ment. Previous studies showed that the biotransformation
processes in sewers are performed primarily by sewer bio-
films. The contribution of planktonic biomass is negligible
(Gutierrez et al., 2009).
3.2. Effectiveness of Fe3þ on sulfide precipitation
in rising main reactors
Fig. 4 shows the dissolved sulfur species, soluble iron,
methane and VFA concentrations in RMC2 and RME2, 2
months after Fe3þ addition to RME2 was started. With the
addition of 21 mg Fe3þ in RME2 per liter of wastewater, the
sulfide concentration was controlled below 0.2 mg L1. In
comparison, the sulfide concentration in RMC2 varied
between 12.1 and 17.1 mg S L1. No Fe3þ was detected in RME2,
however, Fe2þ was found to accumulate in RME2. Fe2þ was
likely produced due to the reduction of Fe3þ.
The control of sulfide in the experimental line is as
expected. The amount of sulfide precipitated in the experi-
mental line can be estimated as the difference between the
total dissolved sulfur in the sewage and in RME2. The amount
of Fe precipitated is the difference between the amount of Fe3þ
added and the amount of dissolved Fe3þ and Fe2þ accumu-
lated in RME2. The calculation shows that the precipitation
ratio (molar) of Fe3þ and sulfide is approximately 0.6:1, which
is similar to the expected stoichiometric ratio of 0.67 (see Eq.
(1) & 2). Phosphate precipitation was also observed in periods
with shorter HRT (11:00–14:00). This is because less sulfide was
generated during these shorter HRT conditions, and iron
dosing was in excess to sulfide generation in these periods,
hence the Fe3þ ions precipitated some phosphate as FePO4.
Phosphate precipitation did not occur at longer HRTs (e.g.
during 9:00–11:00, 15:00–16:00).
Fig. 4 shows sulfate concentrations in RME2 were slightly
higher than those in RMC2 (average sulfate concentration was
2.5 mg S L1 in RME2 and 1.9 mg S L1 in RMC2). In contrast,
the sulfide and sulfate concentrations in RME1 and RMC1 were
very similar (data not shown, but similar to those presented in
Fig. 2). Addition of Fe3þ seemed to have retarded sulfate
reduction in RME2. This will be further discussed later.
 water research 43 (2009) 4123–4132 4127

S2--S SO42--S
2-
SO32--S S2O3 -S
Total Dissolved S

RME1 RMC1
Concentrations (mg/L) 20

15

10

RME2 RMC2
Concentrations (mg/L)

20

15

10

0
07:00:00 09:00:00 11:00:00 13:00:00 15:00:00 17:00:00 07:00:00 09:00:00 11:00:00 13:00:00 15:00:00 17:00:00
Time of the day Time of the day

Fig. 2 – Profiles of dissolved sulfur species in the sewer reactors prior to addition of Fe3D to RME2. Error bars refer to standard
deviations, n [ 3.

Fig. 4 also shows methane concentrations in RME2 were (average VFA concentration was 40.1 mg COD L1 in RME2 and
lower than those in RMC2 (average methane concentration 20.8 mg COD L1 in RMC2). Considering the fact that the
was 10.1 mg CH4 L1 in RME2 and 17.7 mg CH4 L1 in RMC2). methane and VFA concentrations in RME1 and RMC1 were
Conversely, the VFA concentrations in RME2 were higher very similar (as shown in Fig. 2), the addition of Fe3þ seemed to

RME 1 RMC1
40 CH4 40
VFA
15 15
VFA (mg COD/L)

VFA (mg COD/L)

mg CH4/L liquid

mg CH4/L liquid

30 30

10 10
20 20

5 5
10 10

0 0 0 0
RME2 40 RMC2 40

15 15
VFA (mg COD/L)

VFA (mg COD/L)

mg CH4/L liquid

mg CH4/L liquid

30 30

10 10
20 20

5 5
10 10

0 0 0 0
07:00:00 09:00:00 11:00:00 13:00:00 15:00:00 17:00:00 07:00:00 09:00:00 11:00:00 13:00:00 15:00:00 17:00:00
Time of the day Time of the day

Fig. 3 – Profiles of methane and VFA in the sewer reactors prior to addition of Fe3D to RME2.
4128 water research 43 (2009) 4123–4132

2-
S2--S SO3 -S Fe2+
2-
SO4 -S 2-
S2O3 -S PO43--P

25
RME2 RMC2
20
Concentrations (mg/l)
15

10

RME2 RMC2
25 CH4
mg CH4/L liquid

20

15

10

0
RME2 RMC2
VFA
50
VFA (mg COD/L)

40

30

20

10

0
08:00:00 10:00:00 12:00:00 14:00:00 16:00:00 08:00:00 10:00:00 12:00:00 14:00:00 16:00:00
Time of the day Time of the day

Fig. 4 – Comparison of sulfurous compounds, methane and VFA concentrations in RME2 and RMC2 after Fe3D addition to
RME2 (error bars refer to standard deviations, n [ 3).

have reduced methane production in RME2, which is also
consistent with the lower VFA consumption and hence higher
VFA concentrations in RME2.
3.3. Effect of long term ferric salt addition on sulfate-
reducing and methanogenic activities of anaerobic
sewer biofilms
3.3.1. Type I batch tests
The results obtained in the Type I batch tests are presented in
Fig. S1 (Supplementary data). In these tests, Fe3þ was added to
fresh sewage in RME2 resulting an initial theoretical Fe3þ
concentration of 21 mg/L.A good sulfur balance was observed
in RMC2. A sulfate reduction rate of 4.24  0.19 mg S L1 h1
was measured, which is very close to the measured sulfide
generation rate of 4.16  0.09 mg S L1 h1. In comparison, the
sulfate reduction rate of RME2 was 1.72  0.10 mg S L1 h1 (no
sulfide accumulation was observed due to the addition of
Fe3þ), which was approximately 60% lower than that in RMC2.
The methane production rate of RMC2 was 6.91  0.24 mg
CH4 L1 h1. In comparison, the RME2 methane production
rate was 1.38  0.07 mg CH4 L1 h1, 80% lower than that in
RMC2. The sulfate reduction and methane production rates of
RME2 and RMC2 measured in the Type I batch tests are
summarized and compared in Table 1.
Likely as a result of the higher SRB and methanogenic
activities in RMC2, there was a net VFA consumption observed
in RMC2. In comparison, the VFA concentration remained
almost constant in RME2, which indicates that the VFA
consumption for sulfate reduction and methane formation
was compensated for by fermentation.
3.3.2. Type II batch tests
The comparison of the sulfate reduction and methane produc-
tion in RME2 and RMC2 after fresh sewage addition is showed in
Fig. S2 (Supplementary data). No Fe3þ was injected to the reac-
tors in these tests. In RMC2, the sulfate reduction rate was
4.29  0.23 mg S L1 h1, which was very similar to the sulfide
generation rate of 4.10  0.11 mg S L1 h1. In contrast, the
sulfate reduction rate in RME2 was 2.63  0.52 mg S L1 h1,
 water research 43 (2009) 4123–4132 4129

which was 39% lower than that in RMC2. It is also noted that the

RME2/RMC2 (%)
sulfide production rate in RME2 (1.42  0.35 mg S L1 h1) was
46% lower than the corresponding sulfate reduction rate, sug-

61
34

52
gesting either that sulfide was not the sole product of sulfate

–
reduction, or that sulfide produced was simultaneously
removed by some mechanisms, as will be further discussed.
RMC2 had a methane production rate of 5.86  0.14 mg
CH4 L1 h1 and a VFA consumption rate of 15.60 mg

5.86  0.14 mg CH4 L1 h1

COD L1 h1. In comparison, the methane production rate in
4.29  0.23 mg S L1 h1
4.12  0.13 mg S L1 h1
RME2 was 3.05  0.22 mg CH4 L1 h1, 52% lower than that
Type II batch tests

in RMC2. The VFA consumption rate was 9.36 mg COD L1 h1
RMC2

in RME2, 40% lower than that in RMC2.

The sulfate reduction and methane production rates of
RME2 and RMC2 measured in the Type II batch tests are
96%

summarized and compared in Table 1.

3.3.3. Type III batch tests

3.05  0.22 mg CH4 L1 h1

The profiles of dissolved sulfur species in RME2 over a 2 h

2.63  0.52 mg S L1 h1
1.42  0.35 mg S L1 h1

period is presented in Fig. S3 (Supplementary data). A slight

decrease of sulfide concentration was observed, at approxi-
RME2

mately 0.5 mg S L1 h1. Sulfate, sulfite and thiosulfate

concentrations were not negligible, thus the reduction of
these sulfur species to form sulfide can be neglected. The
dissolved Fe3þ and Fe2þ concentration was less than
54%

0.2 mg L1, which was similar to the background level in the

wastewater. Phosphate concentrations were constant at
RME2/RMC2 (%)

11.8 mg P L1 during the entire experiment.

Table 1 – Comparison of sulfate reduction and methane production rates in RME2 and RMC2.

40

20
–
–

4. Discussion

4.1. Fe3þ addition reduces sulfate reduction and

methane production rates by sewer biofilms
6.91  0.24 mg CH4 L1 h1
4.24  0.19 mg S L1 h1
4.16  0.09 mg S L1 h1

Iron salts had been used to control hydrogen sulfide in sewers

Type I batch tests

for decades. Most previous studies have focused on the

RMC2

various chemical reactions involving Fe3þ, Fe2þ, S2 and other

anions in wastewater, and the optimal Fe3þ/Fe2þ to S2 ratios
(Dohnalek and Fitzpatrick, 1983; Padival et al., 1995; Nielsen
et al., 2005; Firer et al., 2008). Firer et al. (2008) found that
98%

higher Fe3þ to S2 ratios are required for wastewaters with

lower pH or when or a lower dissolved sulfide concentration is
1.38  0.07 mg CH4 L1 h1

to be achieved. Field tests by Padival et al. (1995) indicated that

1.72  0.10 mg S L1 h1

a mixture of Fe3þ and Fe2þ at a molar ratio of 2:1 was more

effective than either salt alone to remove sulfide from sewage.
RME2

It was postulated that the mixture iron salts can react with
Not measured

sulfide to form Fe2S3, which is unstable and further trans-

forms to FeS2 and Fe3S4.
In contrast, this study focused on the impact of Fe3þ
addition on the activities of sewer biofilms. The results
–

revealed, for the first time, that the long term addition of Fe3þ
Sulfide reduction to sulfate

considerably reduces the sulfate-reducing and methanogenic

Methane production rate

activities of sewer biofilms. This conclusion is supported by

Sulfide production rate
Sulfate reduction rate

the following evidence:

reduction ratio

C In both the Type I and Type II batch tests, the sulfate

reduction and methane production rates of the RME2
biofilms were substantially (39–80%) lower than those of
the RMC2 biofilms (Table 1). The reduction of these rates
was observed both in the presence and in the absence of
4130 water research 43 (2009) 4123–4132
Fe3þ addition during the batch tests, although higher
rate reductions were observed in the presence of Fe3þ
addition.
C Higher sulfate and lower methane concentrations were
observed in the effluent of the experimental line when
RME2 received Fe3þ addition, in comparison to the
reference line (2.5 mg S L1 in RME2 vs. 1.9 mg S L1 in
RMC2, and 10.1 mg CH4 L1 in RME2 vs. 17.7 mg CH4 L1
in RMC2), supporting the results obtained through batch
tests. These differences are less than those observed in
the batch tests. This is explained by the fact that Fe3þ
was added to the second reactor (RME2) rather than to
the first reactor, and therefore the biofilm activity of
RME1 was not affected (confirmed by data).
In addition to decreasing the sulfate reduction and meth-
anogenic activities, the long term Fe3þ addition also seemed
to alter the final product of sulfate reduction. Both the reactor
monitoring (Fig. 2) and batch tests data (Table 1) showed that
all sulfate reduced was converted to sulfide before Fe3þ
injection. However, in the Type II batch tests, sulfide
production by RME2 represented only 54% of sulfate reduc-
tion (Table 1 and Fig. S2). The slight sulfide decrease observed
in Fig. S3 suggests that the difference between sulfate
reduction and sulfide production observed in Fig. S2 was not
due to the reaction of sulfide with residual iron salts or
elemental sulfur deposited on the biofilms. Fe3þ oxidizes
sulfide species to solid elemental sulfur which may precipi-
tate on the sewer biofilm. Sulfide may react with solid
elemental sulfur to form polysulfide, thus reducing the dis-
solved sulfide in bulk water. Secondly, a portion of Fe3þ could
be precipitated on sewer biofilms in the form of ferric
hydroxide or ferric phosphate as residual iron. These ferric
precipitates may further react with sulfide, thus contributing
to the imbalance between sulfide and sulfate. However, the
Type III batch tests showed that the decrease of sulfide due to
polysulfide or iron sulfides formation was insignificant.
Therefore, it is likely that the end product of sulfate reduction
by biofilms adapted to Fe3þ was not only sulfide but also
included other sulfur species.
Our results are in agreement with previous reports that
Fe3þ reduced the sulfate reduction and methane production
rates in marine and freshwater sediments (Lovley and Phillips,
1986a; Lovley and Phillips, 1987; Achtnich et al., 1995; van
Bodegom and Stams, 1997; van Bodegom et al., 2004; Holmer
et al., 2005). Lovley and Phillips (1987) revealed that sulfate
reduction and methane production in the sediments were
reduced by 86–100% and 50–90%, respectively, through addi-
tion of ferric oxyhydroxide. Indeed, metallic ions including
Fe3þ have been widely reported to have an inhibitory effect on
pure strains and mixed cultures of SRB (Utgikar et al., 2001,
2002), and pure cultures of methanogens such as Meth-
anospirillum hungatei, Methanosarcina barkeri and Meth-
anosaeta concilii (van Bodegom et al., 2004). The sulfate
reduction activity of SRB was found to be completely inhibited
at 2–50 mg Cu L1, 13–40 mg Zn L1, 75–125 mg Pb L1, 4–
54 mg Cd L1, 10–20 mg Ni L1, 60 mg Cr L1 and 74 mg Hg L1
(Utgikar et al., 2002). This study revealed that SRB and
methanogens present in anaerobic sewer biofilms are sub-
jected similar inhibitory effects.
4.2. Mechanisms likely responsible for the inhibition
Lovley and Klug (1983) and van Bodegom et al. (2004) sug-
gested that the inhibition of sulfate reduction and methane
production by Fe3þ in sediments was caused by the competi-
tion of Fe3þ-reducing bacteria with sulfate-reducing bacteria
and methanogens for common electron donors, primarily
acetate and hydrogen (products of bacterial fermentation).
The concentrations of hydrogen and acetate were maintained
at very low level by Fe3þ-reducing organisms, thus preventing
hydrogen and acetate uptake by SRB and methanogens for
metabolism (Lovley and Phillips, 1986b,1987). This mechanism
is unlikely responsible for the inhibitory effect observed in
this study. Unlike in sediments, organic compounds in sewers
are abundant and therefore not expected to be a limiting
substrate for the growth of Fe3þ-reducing organisms, SRB and
methanogens. Indeed, the experimental data showed that
VFAs were always in excess in sewage in the lab-scale sewer
systems. In fact, higher VFA concentrations were observed in
RME2 than in RMC2, which directly contradicts the hypoth-
esis. Furthermore, only 21 mg L1 of Fe3þ was added to RME2,
which could be completely reduced to Fe2þ by 6 mg S L1 of
sulfide. The formation of amorphous iron(III) oxyhydroxide is
expected to be very limited in rising main sewers. Thus, the
competitive mechanism is not supported by the experimental
data.
Utgikar et al. (2002) hypothesized that the deposit of metal
sulfides on the surface of SRB or methanogenic cells could
cause the inhibition of the activity of these cells. During the
ferric treatment, the wastewater contained high concentra-
tions of ferrous sulfide precipitates because of high sulfide
production in rising main sewers. Ferrous sulfide precipitation
in vicinity of bacterial cell may reduce access of reactants
(sulfate and VFAs) to the necessary enzymes. Thus, the
metabolic rate of bacteria is reduced (Utgikar et al., 2002). Fe3þ,
likely many heavy metals, could also deactivate enzymes of
microorganisms by reacting with their functional groups,
denature proteins of microorganisms and compete with
essential cations utilized by microorganisms, which cause
adverse effects on the activities of microorganisms (Mazidji
et al., 1992; Atlas and Bartha, 1998). This deposit of metal
sulfides may also potentially alter the final products of sulfate
production, as observed in this study. However, fundamental
research at the cellular and enzymatic levels is needed in
order to further verify the above hypothesis.
4.3. Practical implications
In addition to precipitating sulfide, Fe3þ injection to anaerobic
sewers also results in reduced sulfate-reducing and meth-
anogenic activities of sewer biofilms. Batch experiments
showed that addition of Fe3þ to a rising main reactor inhibited
sulfate reduction by 60%. Even when Fe3þ addition was
stopped, the sulfate reduction was still inhibited by 39%,
which shows that SRB did not recover their activity instantly.
However, during continuous operation, the sulfate concen-
trations in the last reactor, where Fe3þ was injected, were only
slightly higher than those in the reference reactor. Fe3þ
addition did not inhibit sulfate reduction by 39–60%. Fig. 4
showed that, without Fe3þ addition, the majority of sulfate
 water research 43 (2009) 4123–4132
was reduced in the first rising main reactor particularly during
long HRT periods. The sulfate levels reaching the second
reactor were relatively low at most times. Consequently, the
effect of Fe3þ addition on sulfate reduction during continuous
operation was limited. It is postulated that inhibition of
sulfate reduction would be stronger when Fe3þ is added at
upstream locations. In this case, SRB activities can be limited
along the whole section of sewers, thus less sulfide will be
produced, requiring less chemical dosage. One potential issue
with adding Fe3þ upstream is that Fe3þ will be in excess
upstream due to the low availability of sulfide. In this case,
Fe3þ will first precipitate with other anions such as phosphate
and hydroxide. However, Fe3þ seems to become available for
sulfide precipitation when sulfide is produced downstream,
accompanied by phosphate release (Fig. 4). More research is
required to further elaborate the effect of upstream addition
of Fe3þ on sulfide control.
Guisasola et al. (2008) demonstrated that a significant
amount of methane could be produced in rising main sewers.
This is confirmed in this study. Up to 25 mg L1 of methane
was measured in the reference system (Fig. 3). Methane is
therefore a significant greenhouse gas emission from sewer
systems, and should be minimized. In batch experiments,
methanogenesis was suppressed by 80% and 52%, respec-
tively, with and without Fe3þ addition during the batch test.
During continuous operation, methane production was
reduced by 30–50% in comparison to the reference system,
depending on HRT. Fe3þ treatment is clearly effective in
reducing methane emissions from sewers. As in the case of
sulfide control, adding Fe3þ upstream is potentially more
effective for methane control than adding it downstream. This
hypothesis requires further validation.
5. Conclusions
This research produced the following findings:
C The addition of Fe3þ significantly (39–60%) inhibits the
SRB activity of anaerobic sewer biofilms. This inhibitory
effect is highly beneficial as it reduces the amount of
ferric salt required for sulfide control, particularly when
ferric salts are added to upstream of sewers.
C Addition of Fe3þ substantially inhibits the methanogenic
activity of anaerobic sewer biofilms (52%–80%). This
suggests that, while providing an effective method for
sulfide control, the addition of Fe3þ also considerably
reduces methane formation in sewers. This effect
should be considered when comparing Fe3þ addition
with other methods for sulfide control.
Acknowledgements
This work was supported financially by the Australian
Research Council through Project LP0454182 ‘‘Understanding
the Biotransformation Processes in a Sewer System to Achieve
Optimal Management’’, and Project LP0882016 ‘‘Optimal
Management of Corrosion and Odour Problems in Sewer
4131
Systems’’. The authors also want to acknowledge Dr. Beatrice
Keller-Lehmann and Ms. Kar Man Leung for their helpful
collaboration with the chemical analyses.
Appendix.
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.watres.2009.06.013.
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