Effect of Nitrite To Sulfide Ratios On The Performance of Anoxic Sulfide Oxidizing Reactor

Q. Mahmood, Ping Zheng, Y. Hayat, Jin Ren-cun, M. R. Azim, G. Jilani, E.
Islam, and Munir Ahmed
EFFECT OF NITRITE TO SULFIDE RATIOS ON THE

PERFORMANCE OF ANOXIC SULFIDE OXIDIZING
REACTOR
Qaisar Mahmood*
Department of Environmental Sciences COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
Ping Zheng
Department of Environmental Engineering, Zhejiang University, Hangzhou 310029, China
Yousaf Hayat
Institute of Statistical Genetics and Bioinformatics, Zhejiang University, Hangzhou 310029, China,
Department of Mathematics, Statistics and Computer Sciences, North Western Frontier Province Agricultural University
Peshawar Pakistan
Jin Ren-cun
Department of Environmental Sciences, Hangzhou Normal University, Hangzhou, China
Muhammad Rashid Azim

Department of Botany, Federal Government Post Graduate College Sector H-8 Islamabad, Pakistan
Ghulam Jilani
Department of Soil Science and Soil & Water Conservation, PMAS Arid Agriculture University, Rawalpindi 46300, Pakistan
Ejazul Islam
Nuclear Institute of Agriculture, Tandojam, Hyderabad, Pakistan
Munir Ahmed
Department of Mathematics, Federal Government Post Graduate College Sector H-8 Islamabad, Pakistan
:
( ASO)
%99 .
.
0.58 .
( 1400mg S/l)
. .(ASO) 71.89-86.86
. 1.75
*Corresponding Author
E-mail: pzheng@zju.edu.cn, mahmoodzju@gmail.com
Tel: +86-571-86971709
Paper Received: 1 September 2006; Accepted 4 June 2008
The Arabian Journal for Science and Engineering, Volume 34, Number 1A
January 2009
45
Q. Mahmood, Ping Zheng, Y. Hayat, Jin Ren-cun, M. R. Azim, G. Jilani, E. Islam, and Munir Ahmed
ABSTRACT
The present study reports the operation of a laboratory-scale anoxic sulfide oxidizing (ASO) reactor to
investigate the effects of influent nitrite to bisulfide molar ratios of 0.58, 1.45, and 1.75 upon the simultaneous
bisulfide and nitrite removal rates from the synthetic wastewater. For all tested ratios, very high bisulfide removal
percentages (above 99 %) were observed. The mass balance analysis suggested that the bisulfide oxidation was
incomplete. The sulfate formation decreased with the increasing influent sulfide concentrations. An influent bisulfide
concentration above 1400 mg S / l accompanied by high effluent nitrite might have some inhibitory effect on the
complete sulfide oxidation to sulfate. Nitrite removal percentage for the 0.58 ratio was in the range of 71.8986.86
%, showing some fluctuations in the working performance of the ASO reactor. Nitrite removal at the 1.45 ratio
ranged from 72.7087.30 %. Comparatively higher nitrogen removal (7894 %) was observed for the 1.75 ratio. The
application of an influent nitrite to sulfide molar ratio of 1.75 would be a better choice for higher nitrite and bisulfide
removal efficiencies in an ASO reactor.
Key words: anoxic bisulfide oxidation, anoxic sulfide oxidizing reactor, sulfide to nitrite ratio, anoxic nitrite
removal, lithoautotrophic denitrification
46
January 2009
EFFECT OF NITRITE TO SULFIDE RATIOS ON THE PERFORMANCE OF ANOXIC

SULFIDE OXIDIZING REACTOR
1. INTRODUCTION
Hydrogen sulfide is a toxic and corrosive gas that causes environmental and economic problems in a variety of
sectors such as sewerage, oil field, the petrochemical industry, etc. [1]. Among other strategies to control and remove
the sulfide is the use of nitrate, which has proved very effective, as shown by some researchers [24]. However,
nitrite was also found to be efficient to oxidize bisulfides in synthetic wastewater [5].
Anoxic sulfide oxidation (ASO) based lithoautotrophic denitrification requires an efficient supply of electron
donor for successful operation of tertiary treatment of wastewater. Such efficient supply of external electron donor is
desirable because most of the electron donors are depleted during secondary treatment of wastewater. Heterotrophic
denitrification is efficient for nitrate reduction when adequate amounts of organic carbon are available [6, 7].
However, when the organic carbon content in the wastewater is low compared to nitrogen, expensive chemicals such
as methanol must be added. The use of such compounds makes the application in full-scale reactors quite expensive.
For this reason, sulfur-based autotrophic denitrification has received more attention recently due to its two
advantages [710]: (a) no need for an external organic carbon source, thereby lowering the cost and risk of the
process; and (b) less amount of sludge produced, thereby minimizing the handling of sludge.
Lab-scale studies on simultaneous removal of bisulfide and nitrite from synthetic wastewaters as conducted by
our research group have shown promising results. The ASO reactor utilized bisulfide as electron donor and nitrite as
electron acceptor. The previous experiments achieved more than 95 % sulfide removal and 75 % nitrite removal
from synthetic wastewater [5]. Different molar ratios of nitrite to sulfide in the influent (Equations 1 and 2) show
that the nature of end products is dissimilar based on influent ratios [5].
3HS + 8NO2 + 5H+ 3 SO42 + 4N2 + 4H2O
Gm = 2944 KJ / mol
3HS + 2NO2 + 5H 3S + N2 + 4H2O
Gm = 917 KJ / mol
2
During ASO process reduced bisulfide was oxidized to SO4

dinitrogen.
(1)
(2)
or S while nitrite was converted into gaseous
1.1. Aim of the Study

The earlier studies on anoxic bisulfide biooxidation in our lab using nitrite as electron acceptor dealt with testing
the potential of an ASO reactor at influent molar ratio of 1.17 [5]. The present experiment was conducted to
investigate the behavior of bisulfide oxidation under different influent electron donor and acceptor molar ratios in an
ASO reactor to accomplish simultaneous bisulfide and nitrite removal from synthetic wastewater.
2. EXPERIMENTAL
2.1. Anoxic Sulfide Oxidizing Reactor
The anoxic sulfide oxidizing (ASO) reactor is an upflow reactor with biomass retention. The reactor is made of
perspex with a working volume of 1.3 liter. The synthetic influent was pumped with a peristaltic pump from the
influent vessel into the reactor. The flow rate can vary from 0.6 to 12.5 liters per day, which gives the possibility of
operating at HRTs between 2 and 0.1 days. For present study, HRT was kept constant (1 day). A recycling pump was
used in order to mix the influent (substrate) and sludge (biocatalyst) well and hence to enhance mass transfer and
decrease possible substrate inhibition. The ratio of recycling flow to the influent flow was set about 2.53. The
temperature of the reactor can be controlled between 20 C and 70 C with a thermostat, although the normal
operational temperature is 30 C, as is usual for the ASO process.
2.2. Inoculum
Inoculum was taken from the anaerobic methanogenic reactor in Sibao wastewater treatment plant located in
Hang Zhou city, China. Its total solids (TS) and volatile solids (VS) were 154.10 g/l and 50.89 g /l, respectively, with
VS/TS ratio of 0.322.
2.3. Synthetic Wastewater
The reactor was fed on daily basis with freshly prepared synthetic influent containing NaHCO3 (as a source of
inorganic carbon for the growth of bacteria), MgCl2, KH2PO4, (1 g/l each), (NH4)2SO4 (0.24 g/l) and trace element
solution (1 ml/l). The trace element solution contained Na2EDTA (5 g/l), NaOH (11g/l), CaCl2.2H2O (11 g/l),
FeCl24H2O (3.58 g/l), MnCl2.2H2O (2.5 g/l), ZnCl2 (1.06 g/l), CoCl2.6H2O (0.5 g/l), (NH+4)6 Mo7O24.4H2O (0.5 g/l)
and CuCl2.2H2O (0.14 g/l). The nitrite-nitrogen and sulfide-sulfur concentrations varied according to the type of
January 2009
47
experiment conducted. The influent concentrations used for ratios tests are shown in Table 1. The nitrite was used in
the form of NaNO2 while bisulfide as Na2S.9H2O. The influent pH was maintained in the range of 77.5 by 1 mol/l
HCl or 1 mol/l NaOH. The influent was flushed with argon gas for five minutes to create anoxic conditions. The
ASO reactor was operated at 30C operating at a HRT (hydraulic retention time) of 1 d because this was found to be
a suitable HRT for the operation of ASO reactor during its optimization. For a detailed account of the process
performance parameters, our previous work [5] may be consulted.
Table 1. The Nitrite and Sulfide Concentrations at Various Molar Ratios used During Experiments
Nitrite/Sulfide Ratio = 0.58
Sulfide
Nitrite
Sulfide
Nitrite
Sulfide
Nitrite
(mg S/l)
(mg N/l)
(mg S/l)
(mg N/l)
(mg S/l)
(mg N/l)
480
184
240
230
240
276
720
276
480
460
480
552
960
368
720
690
720
828
1200
460
960
920
960
1104
1440
552
1200
1150
1200
1380
1660
644
1440
1380
1440
1656
1900
736
1680
1610
1680
1932
1920
1840
1920
2208
2.4. Chemical Analyses

Ammonium nitrogen (NH4+-N), nitrite nitrogen (NO2-N) and nitrate nitrogen (NO3-N) were analyzed on a daily
basis using spectrophotometric methods (Unico UV-2102 PC and 722S, China). NO3-N was determined by
ultraviolet absorption spectroscopy. NO2-N was determined by N-(1-naphthyl)-1, 2-diaminoethane dihydrochloride
spectrophotometry. NH4+-N was determined by phenol-hypochlorites spectrophotometry. The S2 was determined by
ultraviolet absorption spectroscopy. SO42 was determined by barium chromate spectroscopy. The pH value was
measured by using a pH-meter. TS (Total Solids) concentrations were determined according to Standard Methods
[11].
2.5. Graphical Work
The computer program Sigma Plot version10 was use for graphical work.
3. RESULTS
3.1. ASO Process Performance at Influent Nitrite to Sulfide Molar Ratio Of 0.58
The sulfide oxidation in ASO reactor for influent sulfide to nitrite molar ratio of 0.58 has been presented in
Figure 1. The sulfide loading rates for nitrite to sulfide molar ratio of 0.58 were in the range of 0.51.9 kg S / m3 / d.
The sulfide removal efficiency was higher than 99 % for the applied range of influent sulfide concentration. The
results indicated that sulfide was oxidized to sulfate upon complete oxidation while nitrite might have reduced to
gaseous nitrogen as there was no dissimilatory ammonium formation. Sulfide oxidation resulted in almost constant
amounts of sulfate formation for tested sulfide and nitrite ratio of 0.58. As it is evident from Figure 1, the sulfate
production decreased with the increasing influent sulfide concentration.
48
January 2009
100.00
Influent sulfide
Sulfate formed
Removal Efficiency
99.95
1500
99.90
99.85
1000
99.80
99.75
500
99.70
Removal Efficiency
Influent sulfide (mgS/L) and

sulfate formed (mgN/L)
2000
99.65
0
99.60
0
10
15
20
25
30
Time (days)
Figure 1. Relation between influent sulfide, removal efficiency, and sulfate formed at influent molar ratio of 0.58
800
160
Nitrite Effluent
Removal efficiency
Influent Nitrite
140
700
120
600
100
500
80
400
60
300
40
Influent Nitrite (mg N/L)
Effluent Nitrite (mgN/L) and Removal Efficiency
An the sulfate produced at nitrite to sulfide molar ratio of 0.58 ranged between 49130 mg S / l . The influent
nitrite concentration ranged from 184 mg N / l to 736 mg N / l at influent ratio of 0.58 (Figure 1). At this ratio the
nitrite loading rates were in the range of 0.16-0.6 kg N / m3 / d. With the increasing influent nitrite concentration,
nitrite gradually built up in the ASO reactor (Figure 2). Its initial concentration was 26.6 mg N / l at the start of the
experiment that gradually increased to 136.98 mg N / l towards the end of the experiment (Figure 2). Overall nitrite
removal efficiency in ASO reactor remained in the range of 71.9 86.8 %, showing some fluctuations in the working
performance of the ASO reactor. The nitrite removal efficiency increased during the middle stages of the experiment
when it reached to %, but then dropped to 80 % as the influent nitrite concentration was increased above 736 mg N /
l resulting in the build up of effluent nitrite (Figure 2).
200
20
0
100
0
10
15
20
25
30
Time (days)
Figure 2. Relation between influent nitrite, effluent nitrite, and nitrite removal efficiency for influent molar ratio of 0.58
At a nitrite to sulfide molar ratio of 1.45, the sulfide loading rates were in the range of 0.251.9 kg S / m3 / d with
the sulfide removal percentage more than 99 %. Like other nitrite to sulfide ratios, sulfide oxidation into sulfate was
incomplete as shown in Figure 3. The sulfate formation was in the range of 122281 mg / l at various influent sulfide
concentrations. The amount of sulfate decreased with the increasing influent sulfide concentrations (Figure 3).
January 2009
49
100.0
Influent Sulfide
Sulfate formed
Removal Efficiency
2000
99.9
1500
99.8
1000
99.7
500
Removal Efficiency
Influent Sulfide and sulfate formed (mg S/L)
2500
99.6
99.5
0
10
Time (Days)
15
20
25
260
2000
Effluent Nitrite
Removal Efficiency
Influent Nitrite
240
220
1800
1600
200
1400
180
1200
160
1000
140
800
120
600
100
80
400
60
200
40
Influent nitrite (mgN/L)
Effluent nitrite (mgN/L) and Removal Efficiency
The nitrite loading rates for nitrite to sulfide molar ratio of 1.45 ranged 0.16-1.6 kg N / m3 / d. As far as nitrite
removal percentage is concerned, it ranged from 72.7 % to 87.3 % for nitrite to sulfide molar ratio of 1.45. The
relation between influent and effluent nitrite along with its removal percentage has been shown in Figure 4. The
effluent nitrite amount ranged from 62.8 to 240.3 mg N / l with nitrite-nitrogen removal efficiency in the range of
7187 % (Figure 4). There was a little amount of ammonium nitrogen at lower influent pH while later there was
absolutely no formation of ammonium in the reactor for the tested range of influent nitrite (data not shown).
0
0
10
15
Time (days)
20
25
Figure 4. Relation between influent nitrite, effluent nitrite, and nitrite removal efficiency at influent molar ratio of 1.45
Sulfide removal for this ratio observed was also pretty high i.e. more than 99%. At various applied influent
sulfide amounts i.e. 2401900 mg S/l, the effluent sulfide always remained less than 2 mg/l (Figure 5). The sulfide
loading rates for nitrite to sulfide molar ratio of 1.75 were in range of 0.2-1.9 KgS/m3.d.The amounts of sulfate
formed were in the range of 24256 mgS/l, almost similar to those formed at influent molar ratio of 1.45. However,
the amount of sulfate formed decreased with the increasing influent sulfidesulfur content. The amount of sulfate
formed (above 250 mg/l) above influent sulfide concentration of 1440 mg S/l which was accompanied by increase in
the effluent sulfide and nitrite concentrations (Figure 5 and 6).
50
January 2009
Comparatively higher nitrogen removal percentages were observed for present ratio i.e. 7894%, these nitrogen
removal percentages are highest among the all tested nitrite to sulfide molar ratios (0.58, 1.45, and 1.75). The nitrite
loadings for influent ratio 1.75 were in the range of 0.22.0 kgN/m3.d. Figure 6 presents the relation between influent
nitrite, effluent nitrite and nitrite removal efficiency for influent molar ratio of 1.75. No ammonium appeared in the
reactor during testing of present molar nitrite to sulfide ratios.
Influent Sulfide
SulfateFormed
Removal Efficiency
2500
100.0
99.9
2000
99.8
1500
99.7
99.6
1000
99.5
500
99.4
0
Removal Efficiency
Influent Sulfide and Sulfate Formed (mgS/L)
The results showed that the effluent sulfide concentration was less than 2% in all tested cases, while the
maximum nitrite removal percentage was observed for influent nitrite to sulfide molar ratio of 1.75 which was
statistically significant (p<0.05). In addition, relatively greater and statistically significant (p<0.05) amounts of
sulfate were formed due to complete oxidation of sulfide in the presence of abundant supply of electron acceptor
(nitrite) at higher nitrite to sulfide molar ratios viz .1.45 and 1.75.
99.3
99.2
0
10
15
Time (Days)
20
25
200
2500
Effluent Nitrite
Removal Efficiency
Influent Nitrite
180
2000
160
140
1500
120
1000
100
80
Influent Nitrite (mgN/L)
Effluent Nitrite (mgN/L) and Removal Efficiency
500
60
40
0
0
10
15
Time (days)
20
25
Figure 6. Relation between Influent, Effluent Nitrite and Nitrite removal efficiency at influent ratios of 1.75
3.4. Influence of Sulfide Oxidation Upon pH

The effluent pH is an important parameter used to judge stability of a reactor. Increase in pH results from the
OH ions produced from sulfide oxidation under anoxic conditions. The effluent pH was monitored throughout the
experiment and was presented in Figure 7. The results showed that the effluent pH was in the range of 89.5
(Figure 7).
January 2009
51
Influent and Effluent pH values
10.0
Influent pH
Effluent pH at 0.58
Effluent pH at 1.45
Effluent pH at 1.75
9.5
9.0
8.5
8.0
7.5
7.0
0
10
15
Time(days)
20
25
30
Figure 7. Relation between influent and effluent pH values at various molar ratios
4. DISCUSSION
For assessing the potential of a process, volumetric loading rate, removal percentage and the effluent
concentration are important performance indices. For all the tested ratios in the ASO reactor, very high sulfide
removal efficiency (above 99 %) was recorded. Very high sulfide removal (> 96 %) was also achieved in our
previous experiments with a ASO reactor at a nitrite to sulfide molar ratio of 1.17 [5]. Other workers [1215] also
obtained such higher sulfide removal efficiencies using different bioreactors from a variety of waste streams.
Mass balance showed that the sulfide oxidation for tested ratios seemed to be incomplete. Generally, the amount
of sulfate formed as a result of complete sulfide oxidation increased up to an influent sulfide concentration of 1440
mg S / l , and then decreased (Figures 1, 3, and 5). Actually, the sulfate formation did not correspond to the reaction
stoichiometry. Sulfides might have been oxidized to elemental sulfur [1617]. Stoichiometric analysis of data for
present study showed that around 1525 % of the fed sulfide was converted to sulfate for sulfide to nitrite ratio of
0.58. The sulfate formation was 30100 % and 40100 % for the ratios of 1.45 and 1.75, respectively. It was
assumed that rest of sulfide had been converted to elemental sulfur (not determined).
Incomplete oxidation of bisulfides can be explained in terms of substrate and product toxicity which can present
one of the technical barriers in the application of this technology to the treatment of sour wastes on a commercially
useful scale [18]. Bisulfide is toxic to the biotic communities present in the ASO reactor and, therefore, an inhibitory
substrate. Increasing bisulfides and nitrites in the influent, high effluent nitrites may have inhibited the complete
sulfide oxidation into sulfate. Some intermediates of bisulfide oxidation might have arisen in the reactor, which need
to be characterized through further research. The practical implication is that reactor systems employed to treat any
bisulfide laden wastes must be operated on a bisulfide-limited basis so that the steady-state concentration of bisulfide
in the bulk liquid is below inhibitory levels. Further, reactor systems must be well-mixed to avoid high gradients in
bisulfide concentrations and, therefore, isolated pockets of inhibitory bisulfide concentrations [18].
Sulfate is the end product of the aerobic or anoxic (with nitrate) oxidation of sulfides by Thiobacillus
denitrificans whose activity has been shown to be inhibited by sulfate concentrations in excess of 250 mg [19]. This
is likely not an inherent inhibition by sulfate but an effect of increasing ionic strength. In any regard, this product
inhibition places an operating restriction on both batch and continuous reactors. In batch systems, the accumulation
of sulfate can determine the cycle time between batches. In a continuous system the sulfate concentration at the
steady state is determined by the dilution rate; therefore, at any given sulfide feed rate, the reactor volume or
hydraulic throughput is controlled by the sulfate concentration to be maintained in the culture [18].
Our previous studies on simultaneous sulfide and nitrite removal at an influent molar ratio of 1.17 showed that
sulfide oxidation was incomplete and about 1011 % sulfide removal was due to auto-oxidation resulting from the
presence of small quantities of dissolved oxygen in the influent wastewater [5]. It may be logical to suppose that
most of the bisulfide oxidation was due to biotic action, and the possibility of autooxidation of bisulfide was the
minimum as the influent wastewater was flushed with argon gas to avoid oxygen. As far as the toxicity of sulfide
52
January 2009
and nitrite to bacterial communities present in the reactor is concerned, the effects of various nitrite and sulfide
concentrations were tested on the growth and physiology of two isolates from ASO reactor in batch cultures (data
not shown). The results showed that the isolates were more sensitive to sulfide compared with nitrite as IC 50 values
for sulfide were lower than those of nitrite.
Stability is an essential parameter to assess the usefulness of a process. The effluent pH is an important parameter
used to the judge stability of a reactor. Under all the tested influent nitrite to sulfide ratios, the effluent pH was in the
range of 89.5 (Figure 7). Increase in pH results from the OH ions produced from bisulfide oxidation under anoxic
conditions [20]. Clause and Kutzner [9] in their study on the kinetics of autotrophic denitrification with Thiobacillus
denitrificans have reported that a pH around 7.58.0 was optimum for denitrification. Further experiments on testing
different influent pH to investigate the performance of ASO reactor are required. We characterized the effects of
various influent pH values on the performance of anoxic sulfide oxidizing reactor [21]. It was found that the
bacterial communities in ASO reactors were more sensitive to acidic pH compared with alkaline pH, as nitrite and
sulfide removal rates dropped significantly when exposed to acidic pH 3.0. High dissolved bisulfide ions, nitrite and
excess of sulfate (> 300 mg / l) inhibited the sulfide oxidation under highly acidic and alkaline conditions in the ASO
reactor [21].
Judged by the results, a higher nitrite to sulfide molar ratio (1.75) was handy for simultaneous removal of sulfide
and nitrite from wastewater as shown by higher sulfide and nitrite removal efficiencies. Though the amount of
effluent nitrite was higher at higher sulfide to nitrite ratio, further aerobic treatment of the effluent may reduce the
nitrite concentration.
5. CONCLUSIONS
For all the tested sulfide to nitrite ratios in the ASO process, very high sulfide removal percentages (above 99 %)
were recorded. The sulfide and nitrite mass balance analysis suggested that the sulfide oxidation was incomplete.
The sulfate formation decreased with the increasing influent sulfide concentrations. An influent sulfide concentration
above 1400 mg S / l, accompanied by high effluent nitrite, may have an inhibitory effect on the complete sulfide
oxidation to sulfate. A higher ratio, viz. 1.75, may be a suitable ratio for better results to eliminate sulfide and nitrite
from the wastewater, simultaneously.
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Q. Mahmood, Ping Zheng, Y. Hayat, Jin Ren-cun, M. R. Azim, G. Jilani, E.

Islam, and Munir Ahmed

EFFECT OF NITRITE TO SULFIDE RATIOS ON THE

Muhammad Rashid Azim

EFFECT OF NITRITE TO SULFIDE RATIOS ON THE PERFORMANCE OF ANOXIC

3HS + 2NO2 + 5H 3S + N2 + 4H2O

During ASO process reduced bisulfide was oxidized to SO4

or S while nitrite was converted into gaseous

1.1. Aim of the Study

Nitrite/Sulfide Ratio = 1.45

Nitrite/Sulfide Ratio = 1.75

2.4. Chemical Analyses

Influent sulfide (mgS/L) and

Influent Nitrite (mg N/L)

Effluent Nitrite (mgN/L) and Removal Efficiency

Influent Sulfide and sulfate formed (mg S/L)

Influent nitrite (mgN/L)

Effluent nitrite (mgN/L) and Removal Efficiency

Influent Sulfide and Sulfate Formed (mgS/L)

Influent Nitrite (mgN/L)

Effluent Nitrite (mgN/L) and Removal Efficiency

3.4. Influence of Sulfide Oxidation Upon pH

Influent and Effluent pH values

A. J. Telang, S. Ebert, J. M. Foght, D. W. S. Westlake, G. E. Jenneman, D. Gevertz, and G. Voordouw, Effect of

J. L. Sund, C. J. Evenson, K. A. Strevett, R. W. Nairn, D. Athay, and E. Trawinski, Nutrient Conversion by

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