Comparison of Anoxic Sulfide
Biooxidation Using Nitrate/
Nitrite as Electron Acceptor
Qaisar Mahmood,a Ping Zheng,aJing Cai,a Donglei Wu,a Baolan Hu,a Ejazul Islam,a
Muhammad Rashid Azimb
a
Department of Environmental Engineering, Zhejiang University, Hangzhou, China
b
Department of Botany, Federal Government Post Graduate College, Islamabad, Pakistan; [email protected] (for correspondence)
Published online 13 June 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/ep.10201
The performance of two lab-scale Anoxic Sulfide
Oxidizing Reactors (ASOR) utilizing diverse electron
acceptors was compared to evaluate the loading
potential for treating synthetic wastewaters. The nitrite utilizing reactor tolerated very high influent substrate concentrations compared with the nitrate using
reactor and displayed better performance at HRT of 2
days and shorter. Under steady-state denitrifying conditions, the maximal sulfide and nitrite removal rates
were 13.53 kg/(m3 days) and 16.31 kg/(m3 days),
respectively, while the maximal removal rates with nitrate as electron acceptor were 4.18 kg/(m3 days) and
1.73 kg/(m3 days), respectively. The reactor with nitrite as electron acceptor was able to tolerate high sulfide concentrations up to 1920 mg/L as compared
with one using nitrate with influent concentrations
up to 580 mg/L. ASORs could endure high nitrite concentrations of 2265 mg/L, while tolerating moderately
lower nitrate concentrations of 110 mg/L. The process
utilizing nitrite as electron acceptor could tolerate
shorter HRTs, as compared with one using nitrate.
Superior performance of the nitrite using ASO reactor
may be due to more nitrite reactivity. Nitrite might
have induced cytochrome production accepting electrons efficiently from sulfide that helped to overcome
nitrite toxicity to the denitrifiers involved. Nitrite as
an electron acceptor could be a better choice to treat
wastewaters containing sulfides. Ó 2007 American Institute of Chemical Engineers Environ Prog, 26: 169–177, 2007
Keywords: ASO reactor, biodesulfurization, biological nitrite reduction, optimum HRT, potential
assessment
Ó 2007 American Institute of Chemical Engineers
Environmental Progress (Vol.26, No.2) DOI 10.1002/ep
INTRODUCTION
Increasing levels of nitrogen and sulfur pollution
in wastewaters from industry, agriculture, and housing settlements have urged environmental engineers
to devise cost effective bioreactors. The hazardous
effects of sulfides upon human and ecosystem
include corrosion of metals, building materials, and
artistic works. Toxic effects of sulfide upon human
health include impaired lactate and oxygen uptake in
blood, persistent neurobehavioral dysfunction, headache following exposure, nausea, vomiting, depression, personality changes, nosebleeds, and breathing
difficulties [1–4]. It reacts with the iron from cytochromes inhibiting the cellular respiration [5]. Its toxicity and malodorous property (odor threshold conc.
0.4 mg/L) governs its removal from the environment.
The sources of nitrogenous species in wastewaters
include reject water from wastewater treatment plants
(WWTP), excessive use of swine manure, landfill
leachate, and industrial wastewaters [6]. The discharge of these nitrogen compounds into the receiving waters leads to several environmental and health
risks. If transferred from lactating mother to new
born baby, nitrate and nitrites can lead to an oxygen
shortage in newly born children (‘blue baby syndrome’). Also, during chlorination of drinking water,
carcinogenic nitrosamines may be formed by the
interaction of nitrite with compounds containing organic nitrogen.
Hulshoff et al. [7] suggested that the nitrogen and
sulfur metabolism interact at various levels of the
wastewater treatment process. Sulfide as electron donor can be oxidized to elemental sulfur or sulfate by
mixed communities of sulfide oxidizing bacteria
(SOB) using nitrate or nitrite as electron acceptor.
July 2007 169
each. The synthetic influent was pumped with a peristaltic pump from the 5-L influent vessel to the reactor (Figure 1). The flow rate varied between 0.6 to
12.5 L per day, which resulted in HRTs between 2
and 0.1 days. A recycling pump was used to mix the
influent (substrate) and sludge (biocatalyst) and to
decrease possible substrate inhibition. The ratio of
recycle flow to the influent flow was set at about
2.5–3. The temperature of the reactor could be controlled between 208C and 708C with a thermostat,
although the normal operational temperature was
308C, which is optimum for the ASO process.
Figure 1. The schematic presentation of experimental
set up: 1, influent tank; 2, feeding pump; 3, recycling
port; 4, bioreactor; 5, effluent container; 6, port to
gas collector; 7, gas collector containing water; 8,
recycling pump.
The overall biochemical reactions occurring during
sulfide oxidation under different electron acceptors
are shown in Eqs. 1 and 2 indicating that reactions
are thermodynamically favored and energy yielding.
3HS þ 8NO2 þ 5H þ ! 3SO4 þ 4N2 þ 4H2 O Gm
¼ 2944 KJ=mol
5HS þ
ð1Þ
þ
8NO3 þ 3H ! 5SO2
4
þ 4N2 þ 4H2 O Gm
¼
3848 KJ=mol
ð2Þ
Such denitrification reduces the overall carbon
requirements at a nutrient removal plant [8]. Because
autotrophic denitrifiers utilize inorganic carbon compounds (such as CO2, HCO3) as their carbon source
[9], no organic carbon is needed as in heterotrophic
denitrification. This leads to lower sludge production
and minimizes the need for excess sludge disposal
[10, 11].
Objectives
The literature review indicates that no research
work has been reported that compares the suitability
of electron acceptors for sulfide based autotrophic
denitrification by mixed communities of SOB. The
present study was designed to evaluate the performance of two Anoxic Sulfide Oxidizing (ASO) reactors
using either nitrate or nitrite as an electron acceptor.
The performance evaluation was based on the sulfide, nitrate, and nitrite removal at various loading
rates and HRT.
EXPERIMENTAL PROCEDURES
Anoxic Sulfide Oxidizing Reactor
Two lab scale Anoxic Sulfide Oxidizing Reactors
(ASOR) were operated in upflow mode with biomass
retention as shown in Figure 1. The reactors were
made of perspex with a working volume of 1.3 L
170 July 2007
Inoculum
Inoculum was taken from the anaerobic methanogenic reactor in Sibao wastewater treatment plant
located in Hangzhou city, China. Its total solids (TS)
and volatile suspended solids (VSS) were 145.03 g/L
and 68.68 g/L, respectively, with VS/TS ratio of 0.47.
Since the organisms used were not adapted to high
nitrogen (nitrate/nitrite) and sulfide concentrations,
special attention was given to the start-up with this
inoculum. The sludge was enriched for 1 month prior
to operation of reactors using sulfide and nitrate/nitrite as source of energy for the biomass.
Synthetic Wastewater
The reactors were fed with synthetic influent containing NaHCO3, MgCl2, KH2PO4, (1 g/L each),
(NH4)2SO4 (0.24 g/L), and trace element solution (1
mL/L). Trace element solution contained Na2-EDTA
(50 g/L), NaOH (11 g/L), CaCl22H2O (11 g/L),
FeCl24H2O (3.58 g/L), MnCl22H2O (2.5 g/L),
ZnCl2 (1.06 g/L), CoCl26H2O (0.5 g/L), (NH4+)6
Mo7O244H2O (0.5 g/L), and CuCl22H2O (0.14 g/L).
Sodium sulfide (Na2S9H2O), potassium nitrate
(KNO3) and sodium nitrite (NaNO2) were added as
per requirement. The nitrate–nitrogen, nitrite–nitrogen, and sulfide–sulfur concentrations varied according to the type of experiment conducted. During
HRT experiment, the sulfide–sulfur and nitrate–nitrogen concentrations for ASO reactor A were 460 mg
S/L and 110 mg N/L, respectively; while for ASO
reactor B, the sulfide-sulfur and nitrite-nitrogen concentration used were 1152 mg S/L and 1359 mg N/L,
respectively.
Analytical Procedures
Ammonium–nitrogen (NH4+-N) was measured by
the Phenate method, nitrite–nitrogen (NO2-N)
through the colorimetric method and nitrate–nitrogen
(NO3-N) was analyzed through the ultraviolet spectrophotometric screening method on daily basis using
a spectrophotometer (Unico UV-2102 PC and 722S,
China). The sulfide was determined by the iodometric
method and sulfate was measured through the turbimetric method. The pH was determined according to
Standard Method [12]. A three-point calibration of pH
meter was performed daily. TS (Total Solids) concentrations were determined according to the gravimetric
method at 1038C, and volatile solids were analyzed
through the gravimetric method at 5508C. All determiEnvironmental Progress (Vol.26, No.2) DOI 10.1002/ep
nations were carried out according to Standard Methods [12]. All determinations were performed in triplicate and mean values were presented in the results.
Operational Parameters
Operational and performance parameters used for
ASO reactor include volumetric loading rates, hydraulic residence time (HRT), and removal efficiency.
Mass loading rate defines the amount of contaminant
entering the ASO reactor per unit volume per unit
time. Hydraulic retention time is the time a unit
volume of wastewater will remain in ASO reactor. Removal efficiency (RE) is used to describe the performance of ASO reactor. As the loading rate is increased
at fixed HRT, a point of saturation or the maximum
removal efficiency corresponding to maximum microbial substrate utilization rate is observed. At fixed
substrate concentration with decreasing HRT determines the minimum time period for maximum treatment efficiency. RE is the fraction of contaminant
removed by bioreactor.
Statistical and Graphical Work
Means, standard deviation, and standard error
were calculated using Microsoft ExcelTM, while graphical work was carried out through Sigma Plot v.10TM.
RESULTS AND DISCUSSION
The present study demonstrated the treatment efficiency of two ASO reactors utilizing sulfide as electron donor and either nitrate or nitrite as electron
acceptor for simultaneous sulfur and nitrogen removal from synthetic wastewaters. Both ASO reactors
utilizing NO3 (A) and NO2- (B) as electron acceptors
were operated under lithoautotrophic conditions.
Start Up of ASO Reactors
During start up of ASOR A, the concentrations of
nitrate and sulfide in the influent were increased
from 41 to 69 mg/L and from 114 to 236 mg/L,
respectively, at HRT of 2 days (Table1). The removal
efficiencies of nitrate and sulfide in ASO reactor were
very high as can be seen in Table 1. The nitrate removal efficiency remained higher than 80%, while
that of sulfide was *99.5%. For the next 17–26 days,
the concentrations of nitrate and sulfide in the influent were increased up to 76.07 and 264.09 mg/L,
respectively (Table 1). The removal efficiencies of nitrate and sulfide were about 72.39% and 99.42%,
respectively. The loading rates of nitrate and sulfide
were 0.232 kg/(m3 days) and 0.76 kg/(m3 days),
respectively. The sulfide in the effluent was lower
than 2 mg/L (Table 1). The criteria for steady state
were sulfide and nitrate removal efficiencies over 95
and 70%, respectively, and stable operation for at
least 15 days. After the operation of 26 days the reactor reached steady state.
During start up of ASOR B, the concentration of
sulfide in the influent was increased from 32 to 448
mg/L and nitrite from 37.75 to 528 mg/L, respectively,
keeping HRT at 2 days (Table2). Previous investigation achieved the sulfide loading rate of 0.04–0.29
Environmental Progress (Vol.26, No.2) DOI 10.1002/ep
kg/m3 day [13] during start up, while no specific
loading for simultaneous removal of sulfide and nitrite was available in the literature. Earlier studies
achieved only moderate volumetric treatment capacities (<1 g NO3 N/L day) for combined hydrogen sulfide and autotrophic denitrification [14]. Thus, sulfide
loading of 0.2 kg/m3 day with removal efficiency
above 90% and nitrite loading of 0.05 kg/m3 day with
removal efficiency above 50% was regarded as the
criteria for successful start up of an ASO reactor. The
performance of ASOR B during start up has been
shown in Table 2.
The Maximum Loading Potential of the
ASO Reactors
Loading rate is an important index to assess the
loading potential of a bioreactor. Two kinds of sulfide
loading rates were applied to assess the potential of
an ASO reactor. Initially, HRT was kept fixed at 2
days, while the influent sulfide and nitrate or nitrite
concentrations were increased at regular increments.
In the second phase the substrate concentrations
were fixed and HRT was decreased step-wise to
judge the maximum loading potential of the reactors.
The maximum influent loading potential can be
judged by keeping HRT at fixed value by increasing
influent substrate concentrations incrementally. The
maximum volumetric loading potential is achieved by
keeping influent substrate at a fixed value while gradually decreasing HRT.
Keeping steady state HRT (2 days) unchanged in
nitrate utilizing reactor A, the concentration of the
influent nitrate and sulfide were increased from 80.2
to 140.70 mg/L and from 280–580 mg/L, respectively,
during the loading tests, as shown in Table3. The
nitrate removal efficiency was 63.15–85.35%, while
sulfide removal efficiency ranged from 91.3 to 99.8%
(Table 3).
The concentrations of nitrite and sulfide in the
influent of nitrite reducing reactor B were increased
up to 1920 mg/L and 2265 mg/L, respectively, at fixed
HRT of 2 days (Table4). The removal efficiencies of
nitrite and sulfide were about 78% and 99.8%, respectively. The loading rates for nitrite and sulfide
obtained were 1.13 kg/(m3 days) and 0.96 kg/(m3
days), respectively (Table 4). The sulfide in the effluent was lower than 2.0 mg/L until 120th day of operation, but increased to 211 mg/L due to substrate toxicity during final stages. Table 4 shows the performance of the ASO reactor B during start up and steady
state performance at fixed HRT.
Effect of HRT
During HRT tests at fixed influent sulfide concentration with decreasing HRT from 1 to 0.12 days, the
sulfide volumetric loading rate in ASO reactor A
ranged from 0.52 to 4.18 kg/(m3 days) (Figure 2A).
The removal efficiency was higher than 90% with
effluent sulfide concentration less than 2 mg/L during
HRT tests. While the nitrate volumetric loading rate
ranged from 0.12 to 1.73 Kg/(m3 days), with the reJuly 2007 171
172 July 2007
Environmental Progress (Vol.26, No.2) DOI 10.1002/ep
Influent
114.05 6 2.36
176.15 6 5.48
191.51 6 10.0
236.40 6 9.73
255.40 6 12.60
260.04 6 13.87
264.09 6 13.09
Effluent
1.00 6 0.32
1.36 6 0.14
0.83 6 0.21
0.96 6 0.07
1.45 6 0.09
1.12 6 0.13
1.56 6 0.62
Removal
efficiency*
99.24 6 0.32
99.02 6 0.31
99.54 6 0.15
99.59 6 0.05
99.46 6 0.03
99.57 6 0.09
99.42 6 0.24
Loading
rate†
0.127 6 0.004
0.159 6 0.005
0.325 6 0.008
0.406 6 0.008
0.568 6 0.010
0.679 6 0.012
0.767 6 0.011
Influent
Effluent
Removal
conc.
conc.
efficiency*
41.00 6 1.21 8.60 6 0.60 79.02 6 2.03
53.56 6 6.84 9.70 6 1.16 86.43 6 0.57
60.48 6 1.02 10.96 6 1.45 82.47 6 2.07
68.96 6 1.76 7.86 6 0.66 88.95 6 0.87
82.50 6 4.12 24.9 6 3.22 71.08 6 3.89
72.20 6 3.45 20.57 6 5.38 73.07 6 6.85
76.07 6 6.06 21.47 6 4.36 72.39 6 5.78
Loading
rate†
0.048 6 0.05
0.068 6 0.001
0.103 6 0.001
0.117 6 0.007
0.190 6 0.003
0.194 6 0.005
0.232 6 0.004
Q. Nitrate–nitrogen (mg/L)
Nitrogen
formed
32.40 6 2.11
43.86 6 4.64
49.52 6 2.73
61.10 6 3.22
57.60 6 5.29
51.63 6 4.39
54.60 6 6.21
Y-N2††
0.79 6 0.08
0.81 6 0.09
0.81 6 0.05
0.88 6 0.05
0.69 6 0.06
0.71 6 0.06
0.71 6 0.08
Influent
32 61.34
96 6 2.28
1.0 6 1.54
2.4 6 2.73
2.8 6 3.60
3.4 6 2.87
4.8 6 3.09
Removal
Loading
Influent
Effluent
Removal
Effluent
efficiency*
rate†
conc.
conc.
efficiency* Loading rate† Nitrogen formed
0.43 6 0.12 98.67 6 0.38 0.02 6 0.01
37.75 6 1.01 5.30 6 1.60 85.92 6 2.03 0.02 6 0.09
17 6 2.11
0.3 6 0.11 99.7 6 0.21 0.05 6 0.02 113.25 6 2.84 20.7 6 2.16 81.7 6 2.57 0.04 6 0.001
80.1 6 2.64
0.42 6 0.21 99.7 6 0.10 0.08 6 0.05
188.7 6 1.22 38.1 6 2.45 82.47 6 2.07 0.08 6 0.002
150.3 6 2.63
0.51 6 0.09 99.8 6 0.05 0.1 6 0.03
264.5 6 0.76 50.3 6 1.36 80.1 6 1.87
0.1 6 0.003
195.2 6 5.22
0.45 6 0.09 99.9 6 0.01 0.14 6 0.01
3.0 6 0.12 61.2 6 1.12
82 6 1.18 0.12 6 0.002
235.82 6 6.29
0.5 6 0.10 99.9 6 0.05 0.19 6 0.01
4.3 6 1.45 1.0 6 3.38 73.5 6 6.85 0.15 6 0.05
3.1 6 7.39
0.6 6 0.05 99.9 6 0.04 0.2 6 0.011
5.8 6 1.06 1.4 6 3.36
75 6 3.78
0.2 6 0.004
3.4 6 3.21
Q. Nitrite–nitrogen (mg/L)
Y-N2††
0.45 6 0.18
0.80 6 0.38
0.78 6 0.04
0.80 6 0.06
0.82 6 0.04
0.67 6 0.04
0.75 6 0.04
†
*Removal Efficiency % ¼ (cin cfin)*100/cin, where cin is the concentration of pollutant in the influent and cfin is its concentration in the effluent.
Loading Rate [kg/(m3 day)]. The amount of contaminant entering the ASO reactor per unit volume per unit time.
††
Denitrifying Yield (mgN2/mg NO3-N or mgN2/mg NO2-N). It is the amount of dinitrogen produced per mg nitrate/nitrite nitrogen utilized in the ASO reactor.
Time
(days)
3
9
16
24
30
39
45
Q. Sulfide–sulfur (mg/L)
Table 2. Performance of the ASO reactor B using nitrite as electron acceptor during start-up.
†
*Removal Efficiency % ¼ (cin cfin)*100/cin, where cin is the concentration of pollutant in the influent and cfin is its concentration in the effluent.
Loading Rate [kg/(m3 day)]. The amount of contaminant entering the ASO reactor per unit volume per unit time.
††
Denitrifying Yield (mgN2/mg NO3-N or mgN2/mg NO2-N). It is the amount of dinitrogen produced per mg nitrate/nitrite nitrogen utilized in the ASO reactor.
Time
(days)
1
5
9
16
20
23
26
Q. Sulfide–sulfur (mg/L)
Table 1. Performance of the ASO reactor A using nitrate as electron acceptor during start-up.
Environmental Progress (Vol.26, No.2) DOI 10.1002/ep
July 2007 173
280
340
400
460
520
580
1.46
1.93
2.4
2.62
2.96
3.29
6
6
6
6
6
6
6
6
6
6
6
6
0.98
7.45
9.20
4.48
7.10
9.67
Sulfate
formed
0.08 46.2
0.03 37.3
0.02 34.6
0.03 36.3
0.04 22.3
0.05 19.67
Loading
rate*
80.2
86.3
100.25
110.4
135.8
140.7
Influent
3.36
8.9
14.9
47.3
52.5
69.1
6
6
6
6
6
6
0.20
0.24
0.74
1.27
2.48
3.19
Effluent
608
768
1024
1408
1664
1920
2.73
0.69
1.02
1.63
1.42
211.77
6
6
6
6
6
6
2.19
0.51
0.17
0.29
0.29
77.8
Effluent
99.71
99.90
99.90
99.87
99.9
88.97
†
6
6
6
6
6
6
2.19
0.51
0.17
0.29
0.29
77.8
Sulfide
removal
efficiency
*[Kg/(m3 day)].
Denitrifying Yield (mgN2/mg NO3-N).
60
75
90
105
120
135
Time
(days) Influent
0.30
0.38
0.51
0.70
0.83
0.96
6
6
6
6
6
6
0.00
0.00
0.00
0.00
0.00
0.00
Loading
rate*
Q. Sulfide–sulfur, feed:(mg/L)
4.4
3.0
3.8
3.5
2.4
6 50.98
6 27.45
6 59.20
6 44.48
6 27.10
Zero
Sulfate
formed
6
6
6
6
6
6
25.20
18.45
30.75
2.78
53.67
49.75
Effluent
717.75 153.36
906.25 190.81
1208.25 266.56
1661.25
327.1
1963.25 412.83
2265.25 1122.06
Influent
6
6
6
6
6
6
0.20
0.45
0.75
2.78
3.67
9.75
0.42
0.49
0.57
0.65
0.77
0.81
6
6
6
6
6
6
78.62
78.94
77.93
72.39
78.96
50.46
6
6
6
6
6
6
25.20
18.45
30.75
2.78
53.67
49.75
Nitrite
removal
efficiency
6
6
6
6
6
6
0.01
0.01
0.02
0.00
0.03
0.02
Loading
rate*
0.359
0.453
0.604
0.831
0.982
1.13
Nitrogen
formed
559.47
700.87
929.45
1334.15
1550.41
619.50
6
6
6
6
6
6
17.67
15.86
43.05
2.78
53.67
52.92
Nitrogen
formed
0.04 77.1 6 1.67
0.03 77.4 6 1.86
0.02 85.35 6 3.05
0.05 63.15 6 2.78
0.02 83.3 6 3.67
0.06 71.6 6 2.92
Loading
rate*
Q. Nitrite–nitrogen, feed:(mg/L)
95.8
89.68
85.14
57.16
61.34
50.89
Nitrate
removal
efficiency
Q. Nitrate–nitrogen, feed:(mg/L)
Table 4. Steady state performance of the ASO reactor B at various loading rates (fixed HRT of 2 d).
†
0.09
0.31
0.77
0.49
0.34
3.8
6
6
6
6
6
6
99.81
99.76
94.6
93.13
91.3
91.37
6
6
6
6
6
6
0.05
0.22
0.07
0.09
0.19
0.37
Effluent
0.54
14.4
21.6
31.6
45.2
50.3
Sulfide
removal
efficiency
*[Kg/(m3 day)].
Denitrifying Yield (mgN2/mg NO3-N).
30
38
45
53
60
75
Time
(days) Influent
Q. Sulfide–sulfur, feed:(mg/L)
Table 3. Steady state performance of the ASO reactor A at various loading rates (fixed HRT of 2 d).
6
6
6
6
6
6
0.02
0.02
0.04
0.00
0.03
0.02
0.77
0.77
0.76
0.80
0.78
0.27
6
6
6
6
6
6
0.02
0.02
0.04
0.00
0.03
0.02
Denitrifying
yield Y-N2†
0.96
0.89
0.85
0.57
0.61
0.50
Denitrifying
yield Y-N2†
Figure 2. A: Effect of HRT on sulfide oxidation in nitrate reducing reactor A. B: Effect of HRT on nitrate reduc-
tion in ASO reactor A at influent sulfide and nitrate concentrations of 460 mg S/L and 110 mg N/L, respectively.
Note that effluent nitrate concentration for ASO reactor A was and can be neglected.
Figure 3. A: Effect of HRT on sulfide removal from ASO reactor. Note that sulfide removal was sensitive to HRT
of 0.08 days. B: Effect of HRT on nitrite removal from ASO reactor B at sulfide and nitrite concentrations of
1152 mg S/L and 1359 mg N/L, respectively. Note that the amount of effluent ammonia increased gradually
with decreasing HRT.
moval efficiency of 92–58.78%, and the nitrate volumetric removal rate was in the range of 0.12–1.02 kg/
(m3 days). (Figure 2B).
In the nitrite utilizing ASO reactor B, steady state
influent sulfide and nitrite concentrations were fixed at
1152 mg/L and 1359 mg/L, respectively, with decreasing
HRT from 1 to 0.08 day (Figure 3A). The sulfide loading
rate ranged from 1.15 to 13.82 kg/(m3 days), and the removal efficiency was higher than 99%. A sulfide removal
rate of 13.36 kg/(m3 days) was achieved at 0.08 day
HRT (Figure 3A). While investigating the effects of varying HRT, the nitrite volumetric loading rate ranged from
1.36 to 16.31 kg/(m3 days), with the removal efficiency
in the range of 88.68 to 54.56%, and the nitrite removal
rate ranged from 0.73 to 10.48 kg/(m3 days) (Figure 3B).
174 July 2007
During the experiment, HRT had little impact on
sulfide removal percentage in both ASO reactors. As
HRT was decreased from 1 to 0.12 days, the effluent
sulfide concentration remained lower than 3 mg/L
and removal efficiency was always higher than 99%.
The optimum HRT for sulfide conversion was as low
as 0.12 days.
HRT had a notable impact on either nitrate or nitrite removal efficiencies for the tested range in both
reactors. When HRT was decreased from 1 day to
0.13 day in ASO reactor A, nitrate removal efficiency
always remained higher than 92%. However, as HRT
was decreased from 0.13 to 0.12 day, effluent nitrate
concentration elevated to 22.3 mg/L, and removal efficiency dropped to 58.78%; at the same time the
Environmental Progress (Vol.26, No.2) DOI 10.1002/ep
effluent nitrite concentration increased from 8.79 mg/L
to 13.90 mg/L (Figure 2B). Thus the optimum HRT
for nitrate conversion was about 0.13 day.
At decreasing HRTs, nitrite removal percentage in
reactor B always remained around 80% until 0.1 day.
However, as HRT was decreased from 0.10 to 0.08
days, effluent nitrite concentration elevated to 617.6
mg/L and removal percentage dropped to 54.6%
(Figure 3B). Judging by these results, the optimum
HRT for nitrite removal might be 0.1 days.
Effect of Ammonium Accumulation
During the final 15 days of operation for Reactor
B, there was a gradual decrease in nitrite and sulfide
removal percentage accompanied by a rise in the
effluent ammonium, nitrite, and sulfide concentrations (Figure 3B). The reactor operation was terminated after 135 days due to self inhibition caused by
toxic substrates at very high influent nitrite and sulfide, i.e., 2265.25 and 1920 mg/L, respectively.
Comparison of ASO Reactor Performance
As indicated by the results, judged by the applied
influent concentrations, loading rates, and HRT, the
nitrite utilizing ASO reactor (B) showed better performance at both fixed and decreasing HRT (Tables 2
and 4, Figures 3A and 3B). This may be due to
greater NO2 reactivity as compared with NO3 [15].
The nitrogen loading rate for the nitrate using reactor
A was 0.41 kg/(m3 days), with a maximum of 100%
removal. However, the nitrite using reactor (B) displayed better performance for nitrogen loading, with
higher loading rate of 1.13 kg/(m3 days) that confirmed the better reactivity of nitrite. Moreover, reactor B tolerated very high influent sulfide and nitrite
concentrations, i.e., 1920 and 2265 mg/L, respectively
in comparison to 580 mg S/L and 140 mg N/L, sulfide
and nitrate, respectively, by ASO reactor A. Despite
decreasing HRT from 1 to 0.10 day in reactor B, nitrite removal percentage always remained higher than
80%. During the initial stages of the experiment, the
removal percentage was relatively low, but as microbial biomass acclimatized to new conditions, the nitrite removal percentage gradually stabilized above
80%. When HRT was decreased from 0.10 to 0.08 day
in reactor B, the effluent nitrite concentration
increased to 617.56 mg/L and the removal percentage
dropped to 54.56% (Figure 3B). The optimum HRT
for sulfide and nitrate conversion in reactor A (0.12
day) was lower than the optimum HRT for sulfide
and nitrite conversion in reactor B which was 0.10
day. Exploring the potential of ASO bioreactor with
decreasing HRT at fixed substrate concentration displayed better results as compared with increasing
substrate concentration at fixed HRT. Looking upon
the loading rates achieved; it is obvious that reactor B
utilizing very reactive NO2 as electron acceptor in
comparison to nitrate using ASO reactor A, bore very
high sulfide and nitrite loading rates i.e. 13.54 kg/m3
days and 16.32 kg/m3 days, respectively (Table 4, Figure 3A). Though, the effluent sulfide and nitrite of
ASO reactor B did not meet the effluent discharging
Environmental Progress (Vol.26, No.2) DOI 10.1002/ep
standards set by USEPA, it was capable of reducing
the sulfide and nitrite pollution loads, hence further
treatment under aerobic conditions is recommended
in order to meet discharge standards. Thus, the ASO
reactor B was capable of treating highly concentrated
wastewaters at shorter HRT of 0.1 days compared
with the ASO reactor A that failed to work below
HRT of 0.12 days.
Sulfide oxidation was partial during the present
investigation, producing both sulfate and sulfur as
shown in Eqs. 1 and 2. Factors like dissolved oxygen
in wastewater, pH and influent sulfide to nitrate/nitrite ratios can affect the fate of sulfide oxidation.
These findings are consistent with earlier research
[11, 14, 16–18].
At fixed HRT, toxic substrate increments did not inhibit the substrate utilization. The concentration of sulfide and nitrite did not increase in the reactor due to
efficient utilization by microbial biomass in the reactor.
At lower concentrations the reaction rate increases
with an increase in substrate [19]. However, when the
maximum rate is reached beyond which substrate concentration becomes inhibitory, a decrease in reaction
rate is observed. This is known as self-inhibition or
Haldane or Andrews’s kinetics [19]. A sudden drop in
the ASO reactor performance utilizing nitrate or nitrite
might be due to self-inhibition.
Reyes-Avila et al. [13] have demonstrated the simultaneous biological removal of nitrogen, carbon,
and sulfur by denitrification at a hydraulic retention
time of 2 days. Although simultaneous nitrogen, carbon, and sulfur removal was possible, carbon consumption efficiency decreased in their bioreactor. Partial anoxic sulfide oxidation occurred when nitrate
was used as electron acceptor. Heterotrophic denitrification using acetate as electron donor is well
described [20–22]. In contrast, the pathway of anoxic
sulfide oxidation under denitrifying lithoautotrophic
conditions is not yet well understood [13]. Hence, it
is possible to assume that both respiratory processes
are different. There is evidence in the literature of simultaneous oxidation of elemental sulfur or thiosulfate together with organic matter [22–25], but sulfide
oxidation in the presence of organic matter is rarely
observed [26].
The present investigation showed that an ASO reactor utilizing nitrite as electron acceptor showed
better performance when compared with a nitrate utilizing reactor. Compared with nitrate, nitrite is more
reactive substrate that can accept electrons efficiently
from sulfide, and seems to be an efficient electron
acceptor for sulfide based lithoautotrophic denitrification, as verified by the present study. It was demonstrated that under anaerobic or semi-aerobic conditions, nitrite induced cytochrome a2-c synthesis in a
denitrifying bacterium, Pseudomonas stutzeri [27].
This inductive effect of nitrite was counteracted by nitrate. Nitrate also repressed particulate cytochrome c552 synthesis to some extent but nitrite did not [27].
Such induction of cytochrome production in the presence of nitrite might be useful to promote resistance
to nitrite toxicity. Further investigation on the mechanism of substrate toxicity in the presence of higher
July 2007 175
amounts of sulfides and nitrites in sulfide oxidizing
bacteria (SOB) should be investigated to optimize the
process.
9.
CONCLUSIONS
The performance of Anoxic Sulfide Oxidizing
Reactors (ASOR) utilizing diverse electron acceptors
was compared using laboratory-scale bioreactors to
evaluate the loading potential of bioreactors. The nitrite utilizing reactor tolerated very high influent substrate concentrations, i.e., 1920 mg S/L sulfide and
2265 mg N/L nitrite, respectively, compared with 580
mg S/L sulfide and 140 mg N/L nitrate for nitrate
using reactor and displayed better performance both
at fixed and shorter HRTs. The display of superior
performance by the nitrite using ASO reactor B might
be due to more nitrite reactivity. Compared with
nitrate, nitrite may have induced cytochromes production along with accepting electrons from sulfide
efficiently that helped to overcome nitrite toxicity to
the denitrifiers involved. The ASO reactor B was capable of treating highly concentrated wastewaters at
shorter HRT of 0.1 days compared with the ASO reactor A which failed to work below HRT of 0.12 days.
Though the effluent sulfide and nitrite of ASO reactor
B did not meet the effluent discharging standards set
by USEPA, it was capable of reducing the sulfide and
nitrite pollution loads, hence further treatment under
aerobic conditions is recommended to meet discharge
standards.
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