A novel two-module integrated aerobic-anaerobic bioreactor for denitrification
using H2S from biogas
L.H.P. Garbossa 1, J.A. Rodriguez 2, K.R. Lapa 1 and E. Foresti 1
1
Department of Hidráulica e Saneamento, Escola de Engenharia e São Carlos, Universidade de São Paulo, Av. do
trabalhador são carlense, 400 – CEP. 13560-970, São Carlos – SP – Brazil (E-mail: [email protected])
2
Escuela de ingeniería de recursos naturales y del ambiente, Universidad del Valle, Ciudad Universitaria Meléndez,
Calle 13 No. 100-00, A.A. 25360 – Cali – Colombia (E-mail: [email protected])
Abstract The biogas produced in an UASB reactor treating domestic sewage was utilized for the evaluation of its
effectiveness as alternative source of electron donor for denitrification. The experiment was carried out in a posttreatment unit consisting of a bench scale horizontal flow aerobic-anaerobic immobilized reactor fed with the UASB
reactor effluent and supplied with air (for nitrification) and biogas (for denitrification) in separate modules aiming the
removal of nitrogen. The experiment lasted five months. Complete denitrification was obtained and the results permit to
affirm that H2S instead of CH4 was preferable used as electron donor. Although the reasons for this preference are not
clear, some hypotheses are presented. More studies should be developed to provide a better understanding of the
mechanisms involved in the use of H2S and elemental sulfur as electron donor for denitrification. The results obtained
permit to consider the novel system configuration a promising reactor alternative for biological nitrogen removal and
also for the treatment of the biogas produced in anaerobic reactors utilized for domestic sewage treatment.
Keywords: Biogas; denitrification; nitrogen removal; sulfur; wastewater
Introduction
Biological nitrogen removal from wastewaters is normally carried out in two sequential steps,
nitrification and denitrification. Denitrification is generally the last step of wastewater treatment,
occurring when the major easily degradable organic matter has been already oxidized. Therefore,
denitrification depends on the availability of an efficient electron donor, and exogenous carbon
sources such as methanol, ethanol, and volatile fatty acids have been normally used. Recently, the
search for electron donors produced during the wastewater treatment processes has deserved special
attention of the researchers aiming to lower the costs of denitrification. The literature suggests that
methane or sulfur by its properties could be a possible and interesting alternative exogenous
electron donor (Islas-Lima et al., 2004; Santos et al., 2004, Costa et al., 2000; Thalasso et al., 1997;
Rajapakse and Scutt, 1999; Lampe and Zhang, 1996; Soares, 2002). On one hand, the anaerobic
treatment plant produces a biogas which contains significantly percentages of CH4 and H2S.
Consequently, there is a great source of low cost and efficient electron donors readily useable.
The anoxic methane oxidation coupled to heterotrophic denitrification could be described by the
following equation, which is thermodynamically favorable (Islas-Lima, 2004):
5CH4 + 8NO3- → 5CO2 + 4N2 + 8OH- + 6H2O
(1)
According to the literature (Thalasso et al.,1997), if methane is to be used as electron donor the
stoichiometry of the reaction indicates that each mole of nitrate needs 5/6 mole of methane
(equation (2a) and (2b)). However, this calculation does not take into account a wasteful methane
oxidation (equation (2c)). According to Werner and Kayser (1991), this can represent up to 90% of
the total methane oxidation. Consequently, the wasteful methane oxidation step presents a serious
bottleneck which must be overcome.
5CH4 + 5/2 O2 → 5CH3OH
5CH3OH + 6NO3- → 3N2 + 6OH- + 7H2O + 5CO2
5CH4 + 10O2 → 10H2O + 5CO2
(2a)
(2b)
(2c)
The autotrophic denitrification processes utilize autotrophic denitrifiers, such as Thiobacillus
denitrificans and Thiomicrospira denitrificans, to reduce nitrate to nitrogen gas. The energy source
of the autotrophic denitrifying microorganisms is derived from inorganic oxidation-reduction
reactions with elements such as hydrogen or various reduced-sulfur compounds (as electron
donors). Autotrophic denitrification has been divided into hydrogen-based and sulfur-based
processes (Lampe and Zhang, 1996).
Because it is difficult to generate hydrogen gas from low-strength wastewater, more attention has
been concentrated on sulfur-based autotrophic denitrification.
Under oxygen-limiting conditions, H2S is oxidized into elemental sulphur, according to the
following reaction (Kuenen, 1975, apud Janssen et al. 1999):
H2S + 0,5O2 → So↓ + H2O
(3a)
The elemental sulphur produced is suitable for use as electron donor in autotrophic denitrification.
The overall reaction for S-dependent denitrification can be summarized as (Soares, 2002):
5So + 6NO3- + 2H2O → 3N2 + 5SO42- + 4H+
and including the production of biomass
55S + 20CO2 + 50NO3- + 38H2O + 4NH4+ → 4C5H7O2N + 25N2 + 55SO42- + 64H+
(3b)
(3c)
Additionally the hydrogen sulfide can be directly used as an electron donor for denitrification.
5H2S + 8NO3- → 5SO42- + 4N2 + 4H2O + 2H+
(4)
The values of the standard Gibbs free energy clearly demonstrate the feasibility of the process
wherein nitrates, nitrites and oxygen accept electrons coming from reduced sulfurous compounds.
In the presence of oxygen, sulfur oxidation is preferentially coupled with oxygen reduction, as
reported by Sublette et al. (1998) indicating that denitrification using hydrogen sulfide as electron
donor only occurs for dissolved oxygen levels below 1 mg.l-1.
In addition, biological nutrient removal from wastewater may be performed by adopting various
systems configurations. Recently, a number of new processes and configurations were developed.
Immobilized biomass reactors have been increasingly used for nitrogen removal, achieving high
performance and stability due to their capacity of maintaining high cellular retention time.
The HAAIS (Horizontal flow aerobic anaerobic immobilized sludge) is a novel, promising, lowcost alternative to biological nitrogen removal, which may improve the biochemical process
occurrence and enhance the liquid-gas contact due to its hydraulic behavior. The conception of this
novel reactor was based in the previous studies of HAIS reactor developed by Zaiat et al. (1997).
The denitrification process using immobilized biomass reactor has been studied using different
electron donors including methane. However, the contribution of H2S from the biogas generated in
anaerobic treatment plants in denitrification has not been well studied so far.
The objective of this paper is to present the preliminary evaluation of the start-up, performance and
effectiveness of a novel two-module integrated aerobic-anaerobic bioreactor for denitrification. The
bench-scale treatment system used a biogas containing H2S as electron donor for denitrification.
Methods
Reactor description
The HAAIS bench-scale bioreactor is composed of 2 modules of 1.5 m long PVC tube and 0.15 m
diameter. Each module is provided with 5 openings, 2 on the side for sampling and 3 on the top for
air supplying tubes in the first module and biogas supplying tubes in the second module (Figure 1).
Three porous stones of 10 cm each uniformly distributed along the modules were used for air and
biogas distribution.
INTERNAL TUBE -> AIR SUPPLY
EXTERNAL TUBE -> EXCESS
AIR COLLECTION
INFLUENT
MODULE 1
SP1
SP2
EXTERNAL TUBE ->
EXCESS BIOGAS COLLECTION INTERM.
SP
INTERNAL TUBE -> BIOGAS SUPPLY
EFFLUENT
SP4
POROUS STONE
MODULE 2
SP3
SUPPORT MEDIA
Figure 1 Schematic diagram of the HAAIS reactor
The total reactor volume is 53.0 l and the void volume is 21.2 l. The HAAIS reactor was filled with
polyurethane foam cubic matrices (10 mm side) seeded with anaerobic and aerobic biomass. The
first module was meant to obtain nitrification and second module to obtain denitrification.
The reactor was inoculated with 20 l of sludge taken from full-scale up-flow anaerobic sludge
blanket reactor (UASB) treating domestic sewage and 8 l of sludge from full-scale activated sludge
nitrifying reactor treating domestic sewage.
The reactor was continuously fed with an effluent from an UASB reactor treating domestic
wastewater with a medium inflow of 1.8 l.h-1 corresponding to a total HRT of 11.8 h. The average
wastewater composition is summarized in Table 1.
Table 1 Average influent wastewater composition
COD
mg.l-1
142 + 33
CODf
mg.l-1
90 + 23
N-TKN
mg.l-1
40.82 + 8.45
N-Ammon
mg.l-1
36.74 + 7.48
Alkalinity
mg.l-1
107 + 16
pH
7.3 + 0.5
Temperature
°C
21.9 + 2.7
The air supplying for the first module was provided by an air pump. The air flow was 2 l.min-1
approximately. The biogas was continuously flushed to the second module thru a tube from the
UASB reactor directly connected to the HAAIS reactor with a mean flow of 1.25 l.min-1. The
biogas composition is summarized in Table 2.
Table 2 Mean biogas composition
CH4
376 mg.l-1
CO2
112 mg.l-1
H2S
493 mg.m-3
Sodium Bicarbonate was added to the feed tank to guarantee enough alkalinity for nitrification.
Analytical Methods
Monitoring consisted of collecting samples in different points of the reactor. The samples were
analyzed to determine COD (Chemical Oxygen Demand), CODf (Chemical Oxygen Demand of
filtered sample), total nitrogen, ammonium, nitrate, sulfate, sulphide and pH. The analyses were
carried out according to the Standard Methods for the Examination of Water and Wastewater
(1998), except for alkalinity. The alkalinity was determined by the Dilallo & Albertson’s (1961)
method, modified by Ripley et al. (1986).
Results and Discussion
The reactor was operated for five months. This experimental period was divided in 3 operational
conditions. The first condition was the reactor start-up for biomass adaptation and process
stabilization. In the second condition the reactor was operated with air supply and without biogas
supply aiming to obtain nitrate in the first and second module. Then rule out the possibility of
denitrification occurrence in the second module by the use of biomass or other unknown electron
donor. Once the nitrification process was established, the third condition started with biogas
supplying in the second module. The operational conditions are presented in Table 3.
Table 3 Operational conditions
Condition
Start-up
First
Second
Flow rate (l.h-1)
1.2
1.9
1.9
HRT (h)
18
11
11
Time (weeks)
2
10
8
Biogas flow (l.min-1)
0
0
1.25
Start-up. Once the reactor was seeded, the start-up readily occurred. This fact was attributed mainly
to the inoculum characteristic and the reactor configuration that allowed adequate biomass
retention.
Module 1. As can be seen in Figure 2 the nitrification had been established in the 7 th week of
operation and the nitrate mean concentration in the effluent was 17,8 mg.l-1 corresponding to about
75% of ammonium conversion. It may be argued that there was no more ammonium conversion due
to limitations in the air distribution system to supply and transfer oxygen to the biomass. On the
other hand, it is believed that a partial nitrate concentration was converted to nitrogen gas due to the
existence of anaerobic zones between the aeration points. This phenomenon may be related to the
organic matter consumption used as carbon source for denitrifying bacteria and observed by the
CODf decrease. Meanwhile, nitrite concentrations were below 1 mg.l-1.
Module 2. There were no significant changes in the reactor behavior concerning the nitrogen
compounds before starting the biogas supply. There was no air supply and consequently the
environmental conditions were not favorable for the growth of nitrifying bacteria. Therefore, the
ammonium nitrogen concentration remained constant along this module. At the same time,
denitrification did not occur due to the inexistence of appropriate electron donors.
As shown in Figure 2, the denitrification process occurred and the nitrate concentration rapidly
decayed just after biogas supply has been started (12nd week).
50
25
No biogas supplying
Biogas supplying
40
20
30
15
20
10
10
5
0
-1
N-NO3 [mg.l ]
N-Amon [mg.l-1]
Start-up
0
0
2
4
6
8
10
12
14
16
18
20
Time [weeks]
Figure 2 Variation of nitrogen compounds during the three operational conditions:  N-Ammon
influent, ▲ N-Ammon effluent and ○ N-NO3- effluent.
40
60
30
45
20
30
10
15
0
Sulfate [mg.l -1]
-1
N-Compounds [mg.l ]
In order to understand the reactor behavior, a profile of nitrogen compounds along the reactor was
performed, as shown in Figure 3.
0
Influent
SP 1
SP 2
Intermediate
SP
SP 3
SP 4
Effluent
Reactor length
Figure 3 Nitrogen profile along the reactor,  N-Ammon , ○ N-NO3-, – N-N2 and ▲Sulfate.
Simultaneously to the denitrification occurrence, an increase of sulfate concentration was observed.
Therefore, it is suggested that the electron donor used for denitrification was not methane, but the
hydrogen sulfide present in the biogas. In order to verify this supposition, a trap containing a NaOH
solution (30%) was prepared for removing the sulfide from the biogas.
As can be seen in Figure 4, the denitrification efficiency decreased. Besides that, the sulfate
production and nitrate conversion were according to the stoichiometry reactions reported by Soares
(2002), indicating that the electron donor was the elemental sulfur produced by the oxidation of the
hydrogen sulfide from biogas in this case. Additionally, it was observed an increase in the sulfate
effluent concentration over the influent, which was the sole source of sulfate. Consequently, this
excess sulfate was produced by the oxidation of the elemental sulfur accumulated in the support
media. This might have happened because the support media has given favorable conditions to the
contact between the elemental sulfur and the denitrifying bacteria, improving the denitrification as
reported by Soares (2002).
45
30
30
20
15
10
0
Sulfate [mg.l -1]
-1
N-Compounds [mg.l ]
40
0
Influent
SP 1
SP 2
Intermediate
SP
SP 3
SP 4
Effluent
Reactor length
Figure 4 Nitrogen profile along the reactor without hydrogen sulfide from biogas,  N-Ammon , ○
N-NO3-, – N-N2 and ▲Sulfate.
The accumulation of the elemental sulfur occurred during the biogas supplying with hydrogen
sulfide, which entered in contact with the remaining DO from the first module, as can be seen in
Figure 5.
Figure 5 Elemental sulfur adhered to the support medium
As observed in this research and reported in the literature (Houbron, et al., 1999), there are
indications that the denitrification occurs more easily by using sulfur compounds than methane. For
the denitrification using methane as electron donor to occur, an association of methanotrophic and
heterotrophic bacteria is necessary. The methanotrophic bacteria oxidize methane and produce a
carbon intermediate (methanol) used as carbon source by the denitrifying bacteria. Another
hypothesis is the existence of sulfate-reducing bacteria utilizing methanol, diminishing the
possibility of denitrification occurrence using this electron donor. Meanwhile, the denitrifying
bacteria may directly use sulfur compounds.
Conclusions
The use of biogas from anaerobic wastewater treatment plants offers an inexpensive and
considerable source of electron donors for denitrification.
This study confirms the possibility of using H2 S instead of CH4 as electron donor when using
biogas produced in anaerobic reactors. However, more studies should be developed to provide a
better understanding of the mechanisms associated to the use of H2S and elemental sulfur as
electron donors for denitrification.
The novel reactor configuration presents promising results for biological nitrogen removal and
biogas treatment from domestic wastewater. However, the development of a more efficient system
for air and biogas supply is necessary to permit its full-scale application.
Acknowledgements
The authors thank the Brazilian agency FAPESP (Fundação de Amparo à Pesquisa do Estado de
São Paulo) for the grants conceded and also for the financial support given to the Thematic Project.
Nomenclature
COD – Chemical oxygen demand
CODf – Chemical oxygen demand of filtered sample
DO – Dissolved oxygen
HDT – Hydraulic retention time
N-Ammon – Ammonium nitrogen
SP – Sampling point
UASB – Up flow anaerobic sludge blanket
References
Costa, C., Dijkema, C., Friedrich, M., Garcia-Encina, P., Fernandez-Polanco, F. and Stams, A.J.M.
(2000). Denitrification with methane as electron donor in oxygen-limited bioreactors. Applied
Microbiology Biotechnology, 53, 754-762.
Dilallo, R.and Alberton, O.E. (1961). Volatile Acids by Direct Tritation. Journal Water Pollution
Control Federation, 33, 356-365.
Houbron, E., Torrijos, M. and Capdeville, B. (1999). An alternative use of biogas applied at the
water denitrification. Water Science and Technology, 40(8), 115-122.
Islas-Lima, S., Thalasso, F. and Gomez-Hernandez, J. (2004). Evidence of anoxic methane
oxidation coupled to denitrification. Water Research, 38, 13–16.
Janssen, A.J.H., Lettinga, G. and de Keizer, A. (1999). Removal of hydrogen sulfide from
wastewater and waste gases by biological conversion to elemental sulfur colloidal and
interfacial aspects of biologically produced sulfur particles. Colloids and Surfaces, 151, 389397.
Lampe, D.G. and Zhang, T.C. (1996). Evaluation of sulfur-based autotrophic denitrification.
Proceedings of the HSRC/WERC Joint Conference on the Environment.
Rajapakse, J.P. and Scutt, J.E. (1999). Denitrification with natural gas and various new growth
media. Water Research, 33(18), 3723–3734.
Ripley, L.E., Boyle, W.C. and Converse, J.C. (1986). Improved alkalimetric monitoring for
anaerobic digestion of high-strength wastes. Journal Water Pollution Control Federation, 58(5),
406-411.
Soares, M.I.M. (2002). Denitrification of groundwater with elemental sulfur. Water Research, 36,
1392–1395.
Santos, S.G., Varesche, M.B.A., Zaiat, M. and Foresti, E. (2004). Comparison of methanol, ethanol
and methane as electron donors for denitrification. Enviromental Engineering Science, 21(3),
313-320.
Standard Methods for the Examination Of Water And Wastewater (1998). American Public Health
Association, American Water Works Association, Water Pollution Control Federation. 19th
edition. Washington, DC, USA.
Sublette, K.L., Kolhatkar, R. and Raterman, K. (1998). Technological aspects of the microbial
treatment of sulfide-rich wastewater: A case study, Biodegradation, 9, 259-271.
Thalasso, F., Vallecillo, A., Garcia-Encina, P. and Fernandez-Polanco, F. (1997). The use of
methane as a sole carbon source for wastewater denitrification. Water Research, 31(1), 55–60.
Werner, M. and Kayser, R. (1991). Denitrification with biogas as external carbon sources. Water
Science and Technology, 23(4-6), 701-708.
Zaiat, M., Vieria, L.G.T., Cabral, A.K.A., Nardi, I.R., Vela, F.J. and Foresti, E. (1997). Rational
basis for designing horizontal-flow anaerobic immobilized sludge (HAIS) reactor for
wastewater treatment. Brazilian Journal of Chemical Engineering, 14(1), 1-8.