International Biodeterioration & Biodegradation xxx (2016) 1e13
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International Biodeterioration & Biodegradation
journal homepage: www.elsevier.com/locate/ibiod
Development of simultaneous partial nitrification, anammox and
denitrification (SNAD) in a non-aerated SBR
G. Anjali, P.C. Sabumon*
Environmental Engineering Division, School of Mechanical and Building Sciences, VIT University, Chennai Campus, Vandalur - Kelambakkam Road, Chennai
600127, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 July 2016
Received in revised form
22 October 2016
Accepted 25 October 2016
Available online xxx
The objective of this work was to develop simultaneous partial nitrification, anammox and denitrification
(SNAD) process in a twenty liter non-aerated sequencing batch reactor (SBR). Flocculent type activated
sludge sourced from a tannery common effluent treatment plant was used as seed biomass. The SBR was
operated for 161 days in 23 cycles with initial feed concentrations of NH4-N and COD as 250 ± 14.9 mg l1
and 250 ± 4.6 mg l1, respectively. The process was developed in a non-mixing condition in a dark room
with no temperature control, but the temperature in SBR varied between 30 C and 36 C. The average
HRT was 10.5 d with SBR cycle time of 7 d. The last three cycles gave the best average removals of NH4-N
(94%), COD (92%) and TN (82%). The conversion ratio Y(NO2-Nþ NO3-N)/(NH4-N) during the last 10 cycles
remained below 0.11, confirming a stable SNAD process. Maximum specific substrate (NH4-N, COD and
TN) utilization rates of SNAD biomass were determined as 185 mg NH4-N g1 MLVSS d1,
153 mg COD g1 MLVSS d1 and 172 mg N g1 MLVSS d1. These results contribute towards the application of SNAD process in shallow anoxic ponds/tanks/ditches in the absence of sunlight.
© 2016 Elsevier Ltd. All rights reserved.
Keywords:
Partial nitrification
Anammox
Denitrification
COD to NH4-N ratio
Diffusion
Catalase enzyme
1. Introduction
AnAOB
Simultaneous partial nitrification, anammox and denitrification
(SNAD) process is evolving as an effective process for simultaneous
removal of COD and NH4-N from effluents characterized with low
COD to NH4-N ratio (Chen et al., 2009; Wang et al., 2010; Xu et al.,
2010; Daverey et al., 2012; Anjali and Sabumon, 2015; Wang et al.,
2016). The SNAD process works in an oxygen limited environment
through the coordinated actions of ammonia oxidizing bacteria
(AOB), anaerobic ammonia oxidizing bacteria (AnAOB), and heterotrophic denitrifying bacteria (HDB) as per Eqs. (1)e(3) (Koops
et al., 1991; Strous et al., 1998; Sabumon, 2007).
1
þ
NHþ
4 þ 1:5O2 !NO2 þH2 O þ 2H ; DG ¼ 275 kJ mol
AOB
(1)
* Corresponding author.
E-mail address: pcsabumon@vit.ac.in (P.C. Sabumon).
þ
NHþ
! 1:02N2
4 þ 1:31NO2 þ 0:066HCO3 þ 0:13H þ 0:26NO3 þ 0:066CH2 O0:5 N0:15 þ 2:03H2 O; DG
¼ 357 kJ mol1
(2)
HDB
2NO
3 þ 1:25CH3 COOH !N2 þ 2:5CO2 þ 1:5H2 O
þ 2OH ; DG
¼ 946:16 kJ mol1
(3)
By definition, the value of DG for a reaction measures the difference between the free energies of the reactants and the products
when all components of the reaction are present in standard-state
conditions (25 C and 1 atmospheric pressure). The negative values
of DG indicate that all the reactions are feasible and spontaneous
and can occur in any suitable eco-system. Also, the higher DG
value in Eq. (3) as compared to that in Eq. (2) indicates that in the
reduction of NO2-N and/or NO3-N to N2, HDB is more competitive
compared to AnAOB. The competitive power of HDB can be regulated by limiting the supply of organic carbon (measured as COD).
Therefore, it is imperative that during the growth of AnAOB and
HDB together, the reactor and/or ecosystem should not have
http://dx.doi.org/10.1016/j.ibiod.2016.10.047
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Please cite this article in press as: Anjali, G., Sabumon, P.C., Development of simultaneous partial nitrification, anammox and denitrification
(SNAD) in a non-aerated SBR, International Biodeterioration & Biodegradation (2016), http://dx.doi.org/10.1016/j.ibiod.2016.10.047
2
G. Anjali, P.C. Sabumon / International Biodeterioration & Biodegradation xxx (2016) 1e13
excessive organic carbon.
The SNAD process works effectively when COD to NH4-N ratio is
less than or equal to 1. The efficiency of the process decreases when
the COD increases, especially in a suspended growth type reactor.
When the COD increases, the growth of heterotrophs increases and
causes an increase in washout of biomass from the reactor. This
washout of biomass reduces the solids retention time (SRT) of AOB
and AnAOB in the reactor. Since AOB and AnAOB are slow growing
bacteria, minimum SRT of 12e15 days (Strous et al., 1998; Wang
et al., 2010; Lan et al., 2011) is required for a sustainable process.
This aspect makes SNAD process vulnerable to failures when conventional suspended growth systems are used because higher SRT
can not be achieved. A comprehensive review of SNAD process
using SBR configuration is given in Table 1. Other reactor configurations used for SNAD are reviewed by Anjali and Sabumon (2015).
From the review, it is clear that SBR is a popular suspended growth
type laboratory reactor used to retain the slow growing AOB and
AnAOB. Even though SBR is a powerful reactor for developing
SNAD, no attempt has been made earlier to develop SNAD in a nonaerated SBR.
As mentioned earlier, SNAD can work in an oxygen limited
environment by the harmonious existence of AOB, AnAOB and HDB.
All the previous SBRs used for SNAD had external aeration to aid in
partial nitrification and the process was developed in controlled
conditions (Table 1). In developing countries, the cost and
complexity involved in the process control of SNAD using SBR limit
its application. Alternatively, provision of a shallow reactor with
wastewater having both COD and NH4-N enables the diffusion of
oxygen (air) from the head space. This creates low concentrations of
dissolved oxygen (DO) conducive for SNAD to work when COD to
NH4-N ratio is less than or equal to 1. The presence of COD and low
concentrations of DO promote the growth of facultative organisms,
including HDB and induces a stressed condition for the competitive
growth of microbes. In this context, the limited DO for partial
nitrification can be obtained in two ways: (i) by the diffusion of
oxygen from the head space of the reactor and (ii) by the microbial
production of oxygen through cleaving of H2O2 in the presence of
catalase enzyme produced under stressed condition (Sabumon,
2007). Therefore, the objective of this study was to develop a nonaerated SBR capable of SNAD, which has not been reported earlier.
In this study, an SBR was used for developing SNAD process using
flocculent type activated sludge procured from a common effluent
treatment plant (CETP) treating combined tannery effluents. The
SBR was operated in a non-mixing condition in the absence of any
process control for 161 days. The water temperature in SBR varied
between 30 C and 36 C because of the summer season in Chennai,
India. Biomass were drawn from the SBR at predetermined intervals
to conduct separate batch studies: (i) to optimize the pH and the
temperature of SNAD biomass, (ii) to estimate the kinetic parameters of ammonia removal and the maximum specific substrate utilization rates of SNAD biomass, and (iii) to confirm the involvement
of anammox and denitrification in SNAD process. Results of this
work are useful to promote the application of the SNAD process for
the simultaneous removal of COD and NH4-N from wastewaters at
ratios of COD to NH4-N less than or equal to one by means of shallow
anoxic ponds/tanks/ditches. Such low-cost and maintenance-free
treatment may be attractive to both developed and developing
countries for treating wastewater containing low to moderate concentrations of NH4-N and COD.
2. Materials and methods
2.1. Mineral media and synthetic feed
The mineral media and trace element composition used were
same as reported by Sabumon (2007). Predetermined amount of
NH4-N (3.82 g NH4Cl g1 NH4-N) and COD (0.78 g sucrose g1 COD)
were added to the mineral media to make the synthetic feed having
COD to NH4-N ratio equal to one. Analytical reagent (AR) grade
chemicals supplied by ‘Thomas Baker’ (India) and clean glass wares
(Borosil, India) were used in the study.
2.2. Seed biomass
Twenty liters of flocculent type Mixed Liquor Suspended Solids
(MLSS) was collected from the extended aeration process treating
combined tannery effluent in a common effluent treatment plant
(CETP) located in Ranipet (Vellore District, Tamil Nadu, India, www.
ranitec.com) and brought to the laboratory. The initial characteristics of MLSS were: brown in appearance, pH (7.82), MLSS
(5.67 g l1), Mixed Liquor Volatile Suspended Solids (MLVSS;
3.08 g l1), ratio of MLVSS/MLSS (0.54) and sludge volume index
(SVI; 87 ml g1). The settled MLSS was washed several times using
mineral media to remove higher concentrations of sulphides/sulphate and other toxic and refractory compounds present in the
tannery effluent. The complex characteristics of tannery effluent
are presented in Table 1 (supplementary information). Washing
was carried out to avoid toxicity during the development of the
process. Finally, 8 l of settled MLVSS was used as seed biomass. The
seed biomass was selected based on the evidence of the earlier
work by the authors, in which the same seed biomass was used in
the development of enhanced SNAD process in a down-flow packed
bed reactor (Anjali and Sabumon, 2015).
2.3. Development of SNAD process in a non-aerated SBR
Development of SNAD process in a non-aerated SBR was done in
a 20 l clean plastic cargo having a height of 300 mm. Initially, 8 l
seed biomass (~23 g MLVSS) was added along with 10 l synthetic
feed to have a working volume of 18 l. A water depth of 270 mm was
maintained in the reactor, with 30 mm height as head space. The
reactor was then closed and operated in an SBR mode, with a cycle
time equal to 7 days under non-mixing condition. The cycle
composed of negligible time (30 min) for feeding, settling and
decanting compared to its reaction time (7 days). The reactor was
manually mixed prior to 30 min of decanting operation and
immediately after feeding. This mixing was carried out to get
representative samples for analyses at the start and the end of each
cycle. The SBR operation was continued for 23 cycles, extending
over 161 days, by keeping the reactor in a dark room to avoid
photosynthetic activity. Initial pH was set at 7 ± 0.02 using 1 M HCl
in all cycles to minimize the escape of NH3-N to the head space. The
initial NH4-N and COD in the reactor for all cycles were
250 ± 14.9 mg l1 and 250 ± 4.6 mg l1, respectively. The DO,
oxidation-reduction potential (ORP) and the temperature in the
SBR during the entire operation varied from 0.17 to 0.35 mg l1, 98
to 264 mV and 30 to 36 C, respectively. Low concentrations of DO
(0.17e0.35 mg l1) in the reactor were maintained by the diffusion
of DO from the head space and simultaneous consumption of DO by
the microbial consortium. The temperature was not controlled in
the reactor. The temperature in the SBR varied between 30 and
36 C. This variation in temperature was due to the summer season
in Chennai, India, where the ambient temperature varied between
34 C and 43 C. Conducive ORP maintained for SNAD was due to
the presence of COD and/or NH4-N in limited DO and lack of turbulence in the reactor. Representative samples were withdrawn at
the start and the end of each cycle and were analyzed for pH, NH4N, NO2-N, NO3-N, and COD. Biomass (25 ml) was drawn at the start
of the first cycle and the end of the 23rd cycle and then dried,
powdered and stored in closed Eppendorf tubes for elemental
Please cite this article in press as: Anjali, G., Sabumon, P.C., Development of simultaneous partial nitrification, anammox and denitrification
(SNAD) in a non-aerated SBR, International Biodeterioration & Biodegradation (2016), http://dx.doi.org/10.1016/j.ibiod.2016.10.047
Type of reactor
1.
NLR ¼ 0.961; OLR as BOD ¼ 0.362;
HRT ¼ 0.5; DO ~ 1 to 1.5 (Periodic air
supply); Temperature ¼ 30 C; pH ¼ 7.6
to 7.8; Uniform mixing
SBR
Landfill leachate at COD/NH4- NH4-N ¼ 295 to 700; NLR ¼ 0.118 to 0.28; OLR ¼ 0.1 to 0.2;
N ¼ 0.55 to 0.85
COD ¼ 250 to 500
HRT ¼ 2.5; Cycle time 24 h; DO ~ 0.1;
Temperature ¼ 35 C; Uniform mixing
Labscale SBR reactor
Synthetic wastewater at COD/ NH4-N ¼ 200;
NLR ¼ 0.0667; OLR ¼ 0.011; HRT ¼ 3 to
NH4-N ¼ 0.5
COD ¼ 100
9; DO ~ 0.5 to 1; Temperature ¼ 35 C;
pH ¼ 7 to 8; Uniform mixing
Labscale sequencing batch Swine digestor liquor at
NLR ¼ 0.2 to 0.26; OLR ¼ 0.16 to 0.32;
NH4-N ¼ 418;
biofilm reactor (SBBR)
COD/NH4-N ¼ 0.64 to 1.24
COD ¼ 437
HRT ¼ 1; DO ~ 0.2 to 2;
Temperature ¼ 30 C;
Labscale SBR
Opto electronic industrial
NH4-N ¼ 567;
NLR ¼ 0.016 to 0.23; OLR ¼ 0.004 to
wastewater at COD/NH4COD ¼ 100
0.04; Cycle time ¼ 24 h; HRT ¼ 2.5 to 5;
DO ~ 0.1 to 5; Temperature ¼ 25 C
N ¼ 0.18
e37 C; pH ¼ 7.8 to 8; Continuous
mixing at 125 rpm
Labscale SBR
Anaerobic digested liquor of
NH4-N ¼ 519;
NLR ¼ 0.045 to 0.153; OLR ¼ 0.026 to
swine wastewater at
COD ¼ 288
0.236; HRT ¼ 2.5 to 5; d; Cycle time
COD/NH4-N ¼ 0.75
24 h; DO ~ 0.5; Temperature ¼ 15 C
e30 C; pH ¼ 7 to 8; Continuous mixing
at 100 rpm
Lab scale SBBR
Synthetic wastewater at COD/ NH4-N ¼ 200 to 600; NLR ¼ 0.36; OLR ¼ 0.18; HRT ¼ 1.67;
NH4-N ratio of 0.5
COD ¼ 100 to 300
Cycle time 24 h; DO ¼ 0.1 to 0.5;
Temperature ¼ 25 C; pH 7 to 8;
Continuous mixing at 150 rpm
Lab scale SBR
Synthetic wastewater at COD/ NH4-N ¼ 250;
NLR ¼ 0.04; OLR ¼ 0.04; HRT ¼ 10.5;
NH4-N ¼ 1
COD ¼ 250
Cycle time ¼ 7 d; DO ¼ 0.17 to 0.35 (No
external supply of air);
Temperature ¼ 30e36 C; pH ¼ 6 to
7.35; No mixing
2.
3.
4.
5.
Biomass from aerobic activated
sludge treating landfill leachate
and biofilm from anammoxASBBR reactor
Biomass from a full-scale
landfill-leachate treatment
plant
Biomass from aeration tank of
full scale landfill leachate
treatment plant
Autotrophic culture
successfully enriched for
CANON process
Biomass from full scale landfill
leachate treatment
6.
Biomass from landfill leachate
treatment system
7.
Biomass was collected from a
full-scale landfill leachate
treatment plant
8.
Biomass from aeration tank of
CETP treating tannery effluent
Labscale SBR reactor for
treating landfill leachate
Type of wastewater
Influent parameter
(mg l1)
Loading Rate (g l1 d1);
HRT (d); DO (mg l1);
Temperature ( C);
pH; Type of mixing
Sl. No. Type of seed sludge
Synthetic wastewater at COD/ NH4-N ¼ 1451;
NH4-N ¼ 0.4 and raw landfill BOD ¼ 548
leachate
% removal at Nature of sludge in References
steady state the reactor
NH4-N ¼ 97 Suspended growth
TN ¼ 93
BOD ¼ 99
Xu et al., 2010
NH4-N ¼ 82
TN ¼ 78.2
COD ¼ 45
NH4-N ¼ 96
TN ¼ 50.7
COD ¼ 87
NH4-N ¼ 50
TN ¼ 48
COD ¼ 87
NH4-N ¼ 95
TN ¼ 93
COD ¼ 79
Suspended growth
Wang et al., 2011
Suspended growth
Lan et al., 2011
Attached growth
Zhang et al., 2012
Suspended growth
Daverey et al., 2012
NH4-N ¼ 96 Suspended growth
TN ¼ 80
COD ¼ 76
Daverey et al., 2013
NH4-N ¼ 90 Mainly attached
growth and slight
TN ¼ 88
suspended growth
COD ¼ 90
Daverey et al., 2015
NH4-N ¼ 94 Flocculant sludge in Present work
COD ¼ 92
settled condition
TN ¼ 82
G. Anjali, P.C. Sabumon / International Biodeterioration & Biodegradation xxx (2016) 1e13
Please cite this article in press as: Anjali, G., Sabumon, P.C., Development of simultaneous partial nitrification, anammox and denitrification
(SNAD) in a non-aerated SBR, International Biodeterioration & Biodegradation (2016), http://dx.doi.org/10.1016/j.ibiod.2016.10.047
Table 1
Comparison in performance of wastewater treatment using SNAD process in SBR.
NLR: Nitrogen Loading Rate; OLR: Organic Loading Rate; TN: Total Nitrogen; HRT: Hydraulic Retention Time; CETP: Common Effluent Treatment Plant.
3
4
G. Anjali, P.C. Sabumon / International Biodeterioration & Biodegradation xxx (2016) 1e13
carbon analysis. Elemental carbon analysis was carried out as an
indirect measure of biomass lysis during the operation. Additional
biomass was also drawn from the SBR for conducting batch studies
as described below. However, after completing the batch studies,
biomass was put back into the SBR.
2.3.1. Optimization of pH and temperature
Biomass was drawn on day 98 (end of 14th cycle) from SBR after
thorough mixing and kept for settling for 15 min. The settled
biomass (15 ml each) was added to six sets (in duplicates) of 50 ml
Erlenmeyer flasks. Then, 20 ml synthetic feed having 250 mg l1
each of NH4-N and COD was added to every flask. The volume of
biomass and synthetic feed were chosen to have approximately the
same Food to Microorganism (F/M) ratio of SBR. The initial pH was
adjusted to 4, 5, 6, 7, 8 and 9 (using 0.1 M HCl and 0.1 M NaOH) in
each of the sets. The Erlenmeyer flasks were tightly plugged with
cotton for a restricted entry of air during incubation. Batch reactors
were incubated for 7 days at 32 ± 0.2 C in an orbital shaking
incubator (CIS25 BL, Remi Instruments, India) at 120 ± 2 rpm. The
temperature was chosen based on the average liquid temperatures
observed in SBR until the 14th cycle of operation. Mixing was carried out to have optimal contact between the biomass and the
substrates unlike in SBR. Analyses of pH, ORP, NH4-N, NO2-N, NO3N, and COD were done during the start and the end of the study.
Temperature studies were conducted in a similar way as in pH
studies except for the following changes: (i) adjustment of initial
reactor pH to optimum pH (7 ± 0.02) and (ii) the incubation done at
varying temperatures of 10, 20, 25, 30, 35 and 40 C using other
incubators available in the laboratory (LT-orbitek, Scigenics Biotech,
India).
2.3.2. Kinetics of ammonia removal and substrates utilization rates
of SNAD biomass
Biomass was drawn on day 150 (during the 21st cycle) from SBR
after thorough mixing and kept for settling for 15 min. The settled
biomass (15 ml each) was added to six sets (in duplicates) of 50 ml
capacity Erlenmeyer flasks. Then, synthetic feed (20 ml) having
250 mg l1 each of NH4-N and COD was added to every flask. The
initial pH was adjusted to optimum pH (7 ± 0.02) and the Erlenmeyer flasks were tightly plugged with cotton. The reactors were
incubated for 7 days at optimum temperature (35 ± 0.2 C) in an
orbital shaking incubator (LT orbitek, Scigenics Biotech, India) at
120 ± 2 rpm. Analyses of pH, ORP, NH4-N, NO2-N, NO3-N, COD,
MLSS, and MLVSS were carried out on 0th, 1st, 2nd, 3rd, 5th, and the
7th day from each duplicate, respectively. Also, the biomass samples drawn from SBR and/or Erlenmeyer flasks were subjected to
catalase tests as per the reported procedure (Sabumon, 2007). For a
catalase test, a colony of bacteria was picked up from the enriched
microbial culture and transferred to a clean microscope glass slide
containing a drop of water. A few drops of 3% H2O2 were added to
the culture and the bubble generation was observed for 20 s. If
bubbles appear within 20 s, the organism shows positive catalase
activity. A blank in similar conditions without bacterial cells was
also tested.
2.3.3. Confirmation of anammox and denitrification
Biomass was collected (after thorough mixing) after completing
the 23rd cycle on day 161 to determine MLVSS at the end of the
operation. Also, biomass was tested separately in batch studies to
confirm the involvement of anammox and denitrification processes. For this, 15 ml each of settled biomass was added to six
serum bottles of 70 ml capacity.
The mineral media containing NH4-N (60 ± 5.6 mg l1) and NO2N (79.2 ± 11.29 mg l1) according to the anammox stoichiometry
(Strous et al., 1998) was purged with nitrogen and was added to
three serum bottles (in triplicates containing 15 ml biomass each)
to its full capacity. Few drops of resazurin indicator were added to
all the serum bottles. All serum bottles were purged again with
nitrogen for 2 min and sealed using rubber septums and aluminium
crimp caps to ensure anoxic condition. The study was conducted in
an SBR mode for three cycles with cycle time equal to 7 days,
settling time equal to 10 min and negligible time for decanting and
filling operation.
Confirmation of denitrification process was conducted in the
remaining three serum bottles (in triplicates) containing 15 ml of
biomass each. The nitrogen purged mineral media was supplemented with COD (564 ± 32 mg l1) and NO2-N (150 ± 2.52 mg l1)
in the ratio of 3.76 (COD to NO2-N) as substrates. NH4-N
(50 ± 1.42 mg l1) was also added in the mineral media as a nitrogen source for cell synthesis. This mineral media were added to
the full capacity of the three serum bottles and were tightly closed
with rubber septums and aluminium crimp caps. SBR operation
was carried out for three cycles, with a cycle time of 2 days and
negligible time for decanting, filling and 30 min for settling.
In both studies, incubation was carried out at 120 ± 2 rpm in a
temperature controlled (32 ± 0.2 C) orbital shaker (LT orbitek,
Scigenics, Biotech, India). Analyses for pH, ORP, DO, NH4-N, NO2-N,
NO3-N, and COD were carried out at the end of each cycle.
2.4. Analytical techniques
All the physicochemical analyses were conducted as per Standard Methods (APHA, 1998). The DO and the pH were measured
using DO meter (YSI 55 model, USA) and pH meter (SenTix 42,
WTW inoLab pH 720, Germany), respectively. The ORP was
measured using double junction platinum ORP electrode connected
to a calibrated WTW inoLab pH (720) meter in mV mode. ORP
electrode (Pt-Ag/AgCl) was calibrated using RH 28 supplied by
WTW, Germany. Ion Chromatography (Basic IC plus 883, Metrohm)
was used for the analyses of aqueous nitrogen species with conductivity detector and the results were processed using integrated
magic net software. NH4-N was analyzed using cationic column
(Metrosep C4-150/4.0) and cationic mobile phase consisting of
1.7 mM nitric acid and 0.7 mM diplocolinic acid with organic carbon suppressor (acetonitrile; 100 ml l1). NO2-N and NO3-N were
analyzed using anionic column (Metrosep A Supp5-250/4.0) and
anionic mobile phase consisting of Na2CO3 (0.3487 g l1) and
NaHCO3 (0.084 g l1) along with organic carbon suppressor
(acetonitrile; 100 ml l1). COD analysis was carried out by the
closed reflux method using COD digester (WTW-CR 3200, Germany). Elemental carbon analysis of dried biomass samples was
carried out using Perkin-Elmer 2400 series CHNS/O analyzer.
2.5. Calculations
The removals of NH4-N, COD and total nitrogen (TN) were
calculated using the following equation:
% removal ¼
. o
n
Ci 100
Ci Cf
(4)
Where, Ci is the concentration of NH4-N (or COD, TN) in the reactor
after the instant fill phase; and Cf is the concentration of NH4-N (or
COD, TN) in the effluent after 7 days; TN is the sum of the aqueous
concentrations of NH4-N, NO2-N and NO3-N.
The conversion ratio Y(NO2-NþNO3-N)/NH4-N used in evaluating the
performance of SNAD was calculated using the Eq. (5).
Please cite this article in press as: Anjali, G., Sabumon, P.C., Development of simultaneous partial nitrification, anammox and denitrification
(SNAD) in a non-aerated SBR, International Biodeterioration & Biodegradation (2016), http://dx.doi.org/10.1016/j.ibiod.2016.10.047
G. Anjali, P.C. Sabumon / International Biodeterioration & Biodegradation xxx (2016) 1e13
5
Fig. 1. Performance history during development of SNAD in a non-aerated SBR
[(a): Average removal of NH4-N, COD, TN and ratio of COD to NH4-N (removed); (b) NO2-N profile in SBR; (c) NO3-N profile; (d) pH profile and ratio of COD removed to input NH4-N;
(e) Ratio of accumulation of NO2-N and NO3-N to NH4-N consumed].
Please cite this article in press as: Anjali, G., Sabumon, P.C., Development of simultaneous partial nitrification, anammox and denitrification
(SNAD) in a non-aerated SBR, International Biodeterioration & Biodegradation (2016), http://dx.doi.org/10.1016/j.ibiod.2016.10.047
6
G. Anjali, P.C. Sabumon / International Biodeterioration & Biodegradation xxx (2016) 1e13
YðNO2 N þ NO3 NÞ fEffluent ðNO2 N þ NO3 NÞe Initial ðNO2 N þ NO3 NÞg
¼
NH4 N
ðInitial NH4 N e Effluent NH4 NÞ
3. Results and discussion
3.1. Development of SNAD process
The results obtained during the development of the SNAD
process are shown in Fig. 1 (a, b, c, d and e). Fig. 1 (a) shows the
average removal of NH4-N, COD, TN and ratio of COD to NH4-N
(removed) in the SBR. Fig. 1 (b) and 1 (c) show the NO2-N and NO3N profiles in SBR. Fig. 1 (d) shows the pH profile and the ratio of COD
removed to input NH4-N. Fig. 1 (e) shows the ratio of accumulation
of NO2-N and NO3-N to NH4-N consumed. The results of the
development of SNAD process are discussed in 3 parts.
(i) Cycle 1 to cycle 5
The average removals of NH4-N, COD and TN during the first two
cycles were 38%, 70% and 35%, respectively (Fig. 1 (a)). This shows
the capability of the seed biomass for simultaneous removal of
NH4-N and COD in limited DO (0.17e0.35 mg l1) conditions. The
higher COD removal during first two cycles could be due to the
action of facultative heterotrophs present in the seed sludge. In the
3rd cycle, the effluent NH4-N was 314 mg l1 which was 26% more
than the initial NH4-N. The COD removal was also less, compared to
the earlier cycles. This could be due to the death and intense cell
lysis of non-adaptive microorganisms when subjected to low concentrations of the DO compared to the high DO available to them in
the extended aeration process in CETP. This intense cell lysis was
evident from the settled volume (after 30 min settling time) of
biomass, which was reduced to 6 l from an initial volume of 8.5 l.
Another reason for the less removal of NH4-N up to 5th cycle was
due to the drop in pH (Fig. 1 (d)) in the range between 6.1 and 6.8.
This is due to the enhanced fermentation of lysed cell components
in low concentrations of DO by facultative anaerobes present in the
mixed culture.
Xu et al. (2010) observed similar effect of cell lysis while
developing a SNAD process for treating landfill leachate in SBR
using an inoculum consisting of aerobic activated sludge and
anammox bacteria. Tyagi and Lo (2013) reported that the lysate of
activated sludge contains 50e55% carbon and 10e15% nitrogen.
Hence, cell lysis can result in the addition of COD and NH4-N and
thereby affect the removals of COD and NH4-N, when calculated
with respect to initial concentrations. Further, the cell lysis leads to
fermentation and fermentation leads to the formation of organic
acids, which in turn dissociate and reduce the pH. Several previous
studies have reported that nitrification rate reduces when pH is
below 7 because of inorganic carbon limitations caused by stripping of CO2 (Wett and Rauch, 2002; Guisasola et al., 2007).
The profiles of NO2-N (Fig. 1 (b)) and NO3-N (Fig. 1 (c)) up to 5th
cycle, show the evidence of nitritation and/or nitrification. The low
DO (<0.5 mg l1) was suitable for partial nitrification as nitrite
oxidizing bacteria (NOB) could be washed out under oxygenlimited condition. Formation of NO3-N in the initial cycles might
be due to the oxidation of NO2-N by the NOB present in the seed
biomass. However, the survival of NOB was difficult in low DO and
in high temperature and correspondingly there was a decrease in
the formation of NO3-N when the cycle progressed (Fig. 1 (c)).
V
azquez-Padín et al. (2009) reported that NOB has a lower
(5)
oxygen affinity than AOB and oxygen-limited condition
(DO < 0.5 mg l1) inhibits NOB. In another study, Guisasola et al.
(2005) reported that the oxygen saturation constant of AOB is
0.25e0.5 mg l1 and NOB is 0.72e1.84 mg l1, hence at low concentrations of DO, there would be a higher accumulation of NO2-N.
Hellinga et al. (1998) reported that the maximum specific growth
rate of AOB is higher than that of NOB at a temperature above 30 C.
Hence, in low DO and in the high temperature maintained in the
SBR, the activity of NOB would be more distinctly affected than that
of AOB.
The limited DO (<0.5 mg l1) in the SBR could easily create
anaerobic zones within the flocculent biomass. For example, Chen
et al. (2008) accomplished the simultaneous nitrification and
denitrification in a continuously-aerated pilot scale submerged
membrane reactors (SMBRs) by controlling ambient DO concentration between 0.6 and 1.2 mg l1. This oxygen diffusion limitation
resulted in the formation of an anoxic micro-zone inside the biological flocs, where the denitrification occurred. In this work, the
anaerobic zone formed could enhance the growth of HDB and
AnAOB and creates a chance for the competition in the reduction of
NO2-N produced by AOB. Since HDB are thermodynamically
competitive compared to AnAOB as per Eqs. (2) and (3), the growth
rate of HDB would be higher. However, at a ratio of COD to NH4-N
equal to one, enough organic carbon will not be available for the
HDB to overpower the AnAOB. This is because according to Bernat
et al. (2008), a minimum of 3.5e4 g COD g1 N is necessary for
denitrification after nitritation. Therefore, in conditions of effective
nitritation in limited DO and at COD to NH4-N ratio less than or
equal to 1, the anoxic zone could provide a winewin situation for
both HDB and AnAOB in the reduction of NO2-N. During the initial 5
cycles, the TN removal might have occurred mainly through denitrification in low pH as per Eq. (3) because of higher COD available
by cell lysis. The ratio of COD to NH4-N (removed) during the initial
cycles was greater than 1 indicating the action of HDB to be
dominant in the reduction of NO2-N (Fig. 1 (a)). Fig. 1 (e) also
supports intense cell lysis during the cycles 2 and 3 and availability
of more COD.
Huang et al. (2011) reported a moderate rate of denitrification in
the pH range 4.8e6.2 in the presence of acetic acid as the carbon
source. Acetic acid can be formed as a result of fermentation.
Therefore, the cell lysis and fermentation during the initial 5 cycles
helped in getting the total nitrogen removal mainly by HDB. Tang
et al. (2013) reported that the organics released from cell lysis
could be used as the electron donors for nitrite reduction (denitrification). However, the rate would be controlled based on nitrite
concentration in the influent.
(ii) Cycle 6 to cycle 15
The average removals of NH4-N, COD and TN during 6th to 15th
cycle were 79%, 67% and 71%, respectively, which indicate acclimatization of SNAD biomass (Fig. 1 (a)). After acclimatization, the
formation of NO2-N (Fig. 1 (b)) was more compared to NO3-N (Fig. 1
(c)) indicating effective nitritation in low DO. This nitritation could
have occurred as a result of Eqs. (1) and (6). In the stressed conditions, facultative bacteria produce H2O2 as a result of complex
metabolism. The H2O2 formation is possible in the presence of trace
Please cite this article in press as: Anjali, G., Sabumon, P.C., Development of simultaneous partial nitrification, anammox and denitrification
(SNAD) in a non-aerated SBR, International Biodeterioration & Biodegradation (2016), http://dx.doi.org/10.1016/j.ibiod.2016.10.047
G. Anjali, P.C. Sabumon / International Biodeterioration & Biodegradation xxx (2016) 1e13
amount of oxygen (oxidative stress) by the use of oxidative enzymes of facultative organisms. The production of H2O2 takes place
when facultative anaerobes use the aerobic respiratory pathway
during degradation of substrates for energy production. This H2O2
is a highly reactive oxygen species formed as a direct byproduct of
oxygen reduction (Fu et al., 2015). The bacterial cells protect
themselves from the attack of H2O2 by producing the catalase
enzyme which cleaves the H2O2 and produces oxygen as per Eq. (6).
The oxygen produced could be used for nitritation as per Eq. (1).
The contribution of nitritation/nitrification by diffused DO and by
catalase enzyme route was not quantified in the present study
because of the complexity in performing such experiments. However, the presence of the catalase enzyme in the SNAD biomass was
confirmed by qualitative tests (Sabumon, 2007) performed as per
the procedure described in Section 2.3.2. More detailed research is
required for the quantification of H2O2 along with the bacterial
growth in the SBR to evaluate the proportion of nitritation by Eq (6).
Catalase
2H2 O2 ! 2H2 þO2
(6)
During nitritation/nitrification, the formed NO2-N/NO3-N was
converted to nitrogen gas by anammox as per Eq. (2) and denitrification as per Eq. (3). The analysis of the results showed that the
ratio of COD to NH4-N (removed) (Fig. 1 (a)) varied from 0.62 to 1.15
from 6th cycle to 15th cycle. This ratio indicates that COD was
insufficient for entire nitrogen removal by heterotrophic denitrification using externally added sucrose. On the other hand, not much
NO2-N/NO3-N was accumulated corresponding to the amount of
NH4-N removed, indicating effective denitrification. This denitrification could have occurred either in the presence of organic acids
(produced as a result of fermentation of cell components) acting as
electron donors or through anoxic ammonia oxidation by anammox where NO2-N/NO3-N acted as electron acceptors.
During cycle 6 to cycle 15, the average removal of COD was 67%,
which was less and not in concurrence with 71% of TN removed. For
example, for removing 71% TN by denitrification, the required COD
is around 621 mg l1 by considering the requirement of
3.5 g COD g1 N. But synthetic feed contained an average COD of
250 mg l1 equivalent to the removal of around 29% TN only. From
this, it is clear that balance nitrogen removal must have occurred
via denitrification using the organic carbon in the cell lysate and/or
anammox. An estimate of anammox as per Eq. (2) during this
period based on maximum NO3-N (3.4 mg l1) formed shows that
the TN removal by anammox was 5%. Therefore, around 37% of TN
removal was through denitrification using the COD available
through cell lysis. The presence of residual NO2-N and NO3-N in the
reactor after 7 days indicates that the residual COD available in the
reactor (64e120 mg l1) was not readily biodegradable and might
have contained difficult to degrade residual cell components of
lysate. The pH in the reactor during this period increased from 6.7
to 7.3 (Fig. 1 (d)). Such an increase in pH is possible as a result of
effective denitrification as per Eq. (7) (Hellinga et al., 1999). Eq. (3)
shows that as a result of denitrification alkalinity is created, which
acts as a buffer against the reduction of alkalinity by the nitritation
as per Eq. (1). The alkalinity added in the synthetic feed was around
527 mg l1 as HCO
3 which was lesser than the required alkalinity
for nitrification. For example, the alkalinity required for 79% of NH4N removal is around 830 mg l1 as HCO
3 . This shows that as a result
of SNAD process, the external requirement of alkalinity could be
reduced.
þ
2NO
2 þ 6H þ 6e /N2 þ 2OH þ 2H2 O
(7)
Fig. 1 (e) shows the values of conversion ratio Y(NO2-Nþ NO3-N)/
i.e., the ratio of accumulation of NO2-N and NO3-N to NH4-N
(NH4-N),
7
consumed in each cycle. For an efficient SNAD process, this conversion ratio should be less than 0.11 because the accumulated
NO2-N and NO3-N would be consumed by HDR while removing
COD (Daverey et al., 2015). The reason for Y(NO2-Nþ NO3-N/NH4-N)
greater than 0.11 during the few cycles in the initial operation of
SBR could be due to extra NO2-N formed as a result of oxidation of
additional NH4-N produced by cell lysis. From the results, it is clear
that SNAD biomass was acclimatized; though anammox process got
hindered due to cell lysis.
(iii) Cycle 16 to cycle 23
Results from 16th to 23rd cycle (Fig. 1 (a)) showed good average
removals of NH4-N (92%), COD (81%) and TN (83%). Such high TN
removal is not possible by denitrification alone. For example, to get
81% of COD removal by denitrification alone (considering
3.5 g COD g1 N), the NO2-N and/or NO3-N reduction could have
been 58 mg l1 which corresponds to only 23% of TN removal.
Therefore, there is a discrepancy of 60% in TN removal. There could
be a contribution by heterotrophic denitrification using the organic
carbon available through cell lysis of non-adaptive bacteria as
described above. But, this contribution of denitrification promoted
by cell lysis would decrease as the cycle progresses and in long time
operation, this contribution becomes insignificant. Therefore, during the last cycles of the SNAD process, the enhanced removals of
NH4-N and TN could have occurred via anammox reaction. This was
evident from the increase of the average concentration of NO3-N
(5.6 mg l1) as compared to that during earlier phases (from 6 to 14
cycles; 1 mg l1). Also, the DO inside the reactor was very low and
this would have reduced the growth of NOB as they need high DO
(greater than 1 mg l1) for oxidizing NO2-N to NO3-N. Hence, the
increased NO3-N accumulation during later cycles might have
occurred mainly via anammox as per Eq. (2).
There was an increase of pH in the last cycles, compared to that
in the earlier cycles and that shows clear indications of effective
anammox and denitrification processes as per Eqs. (2) and (3). Also,
the residual COD in the last 3 cycles, was lesser than 32 mg l1
which indicates low degree of cell lysis. The Y(NO2-Nþ NO3-N)/(NH4-N)
values in the last cycle as well indicate an effective SNAD process.
Altogether, the results show that the development of the SNAD
process is possible in a non-aerated SBR using synthetic feed at COD
to NH4-N ratio equal to one and using flocculent activated sludge
from a tannery CETP as seed biomass.
The increase in the removals of COD during the later cycles
shows that as the cycle progressed, the extent of cell lysis
decreased. This is evident from the improvement of the ratio of COD
removed to input NH4-N (Fig. 1 (d)) when cycles progressed as a
result of less availability of nonbiodegradable cell lysate. Therefore,
during the last 3 cycles, the ratio of COD to NH4-N (removed) was
closer to one which indicates the maximum utilization of sucrose
(COD) and minimum cell lysis (Fig. 1 (a)). Therefore, the average
data of the last 3 cycles were used for calculating the mass balance
of nitrogen (N) in the SBR per cycle as per Eq. (8).
The total mass of N fed to SBR
¼ The total mass of N escaped with the effluent from the SBR
þ Total mass of N removed from the SBR during a cycle
(8)
It is to be noted that the analyses of the gas phase in SBR were
not carried out. The assumptions considered while calculating the
mass balance were: (i) the NH4-N loss due to volatilization was
negligible as the final pH was 7.37 ± 0.07, (ii) the pH and molar
concentrations of phosphorous and magnesium were not
Please cite this article in press as: Anjali, G., Sabumon, P.C., Development of simultaneous partial nitrification, anammox and denitrification
(SNAD) in a non-aerated SBR, International Biodeterioration & Biodegradation (2016), http://dx.doi.org/10.1016/j.ibiod.2016.10.047
8
G. Anjali, P.C. Sabumon / International Biodeterioration & Biodegradation xxx (2016) 1e13
conducive for magnesium ammonium phosphate (struvite) precipitation in SBR, and (iii) the nitrogen used for cell growth was
balanced with the nitrogen generated by cell lysis. The above-listed
assumptions simplified the solution of Eq. (8). Therefore, the total
average mass of N removed from the SBR during a cycle is equal to
the difference in the total average mass of N fed to SBR and escaped
with the effluent from the SBR. The total average mass of N fed to
SBR was 4.670 g (4.510 g NH4-N þ 0.146 g NO2-N þ 0.014 g NO3-N)
whereas the total average mass of N escaped with the effluent from
the SBR was 0.839 g (0.276 g NH4-N þ 0.448 g NO2-N þ 0.115 g NO3N). Therefore the difference of 3.831 g N (4.670e0.839) could have
escaped as forms of nitrogen into the gas phase and considered as
the total nitrogen removed from the reactor. Therefore, the best
average removals of NH4-N and TN were approximately 94% and
82%, respectively. The average COD removal during the last 3 cycles
was 92%.
Throughout the enrichment studies, there was a decrease in
volume of settled sludge as the cycle progressed. The maximum
reduction in sludge volume was observed from cycle 1 to cycle 15
and sludge volume was 5.5 l at the end of cycle 15. Moreover, at the
end of the study, the total biomass inventory in the reactor was
11.79 g MLVSS. This shows that the final active SNAD biomass were
only 51.3% compared to the initial flocculent seed biomass (23 g
MLVSS). The reason for such a reduction in sludge quantity could be
due to the higher values of the average hydraulic retention time
(HRT) 10.5 d and SRT, which might have induced cell lysis by
endogenous respiration. The SRT in SBR was very high because (i)
no biomass was wasted during the entire operation, and (ii) there
was a negligible escape of Suspended Solids (SS) during decanting
operation. Such high SRT was the reason for effective removal of
NH4-N, COD and TN by the coordinated actions of AOB, AnAOB and
HDB. The cell lysis was confirmed by the elemental carbon analysis
of biomass done before and after enrichment of biomass for SNAD
process. The carbon content decreased by 53.3% indicating cell lysis
during enrichment and development of SNAD biomass. A similar
result of the reduction in carbon content was reported during
enrichment study of anaerobic ammonia removal using SBR
(Sabumon, 2007).
Table 1 shows the comparison of performance of other SNAD
processes in SBR to the present work. The removals of NH4-N and
TN at steady state in the present study were comparable with the
SNAD process developed for swine wastewater treatment at COD to
NH4-N ratio equal to 0.75 (Daverey et al., 2015). The nitrogen
loading rate (NLR) and organic loading rate (OLR) of their work are
comparable with those in the present work. The less removal of
COD (76%) compared to the high removal of COD (92%) obtained in
the present work is because of the presence of non-biodegradable
COD in the anaerobic digested liquor of swine wastewater. The
present work required an average HRT of 10.5 d compared to 2.5 d
HRT of their work. The requirement of longer HRT in the present
work is due to the non mixing condition in contrary to the
continuous mixing at 100 rpm in their work. This indicates that the
non-aerated SBR requires a larger volume for the treatment,
whereas no energy is required for aeration and mixing. Another
important observation was that, when COD to NH4-N ratio was
greater than or equal to 0.5, the suspended growth based SBR could
not operate at NLR and OLR greater than 0.3 g l1 d1. In such cases,
for attaining higher NLR and OLR using SNAD process, other reactor
configurations are to be used (Anjali and Sabumon, 2015). In short,
the results of this study demonstrate that the development of SNAD
is feasible in a non-aerated shallow SBR using flocculent activated
sludge as seed; when wastewater is treated at very low NLR and
OLR. However, the SBR requires a minimum of 10 d HRT to achieve
around 90% removals of NH4-N and COD without any sludge
removal.
3.1.1. Optimization of pH and temperature
The plots with average values are shown in Fig. 2. The results of
optimization of pH are shown in Fig. 2 ((a) and (b)). The ORP profile
and the final pH profile with respect to initial pH are shown in Fig. 2
(a). The initial and the final ORP were in the range of 42.7
to 283.9 mV and 20.5e51.8 mV, respectively. There was a
decrease of ORP as the pH increased. The ORP values indicate that
the reactors were maintained in limited DO. The highest percentage
removals of NH4-N (97%), COD (88.3%) and TN (82.2%) were obtained at pH 7 (Fig. 2 (b)). Also at pH 7, the final ORP was 20.5 mV
indicating the anoxic condition for effective anammox and denitrification. There was a good overall removal at pH 8 too. Therefore,
the optimal pH of the developed SNAD process could be considered
between 7 and 8. Many researchers (Lan et al., 2011; Daverey et al.,
2013, 2015) also reported higher COD and nitrogen removal efficiencies at the same pH range for SNAD process. The higher performance in this batch study compared to the performance in the
14th cycle of SBR (biomass drawn during this cycle) could be due to
Fig. 2. Optimization of pH and temperature of SNAD biomass
[Fig. 2 (a and b): Results of optimization of pH studies: (a) ORP and pH profile; (b) Average removals of NH4-N, COD and TN; Fig. 2 (c and d): Results of optimization of Temperature
studies: (a) ORP and pH profile; (b) Average removals of NH4-N, COD and TN].
Please cite this article in press as: Anjali, G., Sabumon, P.C., Development of simultaneous partial nitrification, anammox and denitrification
(SNAD) in a non-aerated SBR, International Biodeterioration & Biodegradation (2016), http://dx.doi.org/10.1016/j.ibiod.2016.10.047
G. Anjali, P.C. Sabumon / International Biodeterioration & Biodegradation xxx (2016) 1e13
the mixing provided and thereby increasing optimal contact between substrates and flocculent biomass.
The removal efficiencies at pH 4 and pH 5 showed a negative
removal of NH4-N and a positive removal of COD. The negative
removal of NH4-N could be due to cell lysis at acidic pH in anoxic
condition. The positive removal of COD could be due to fermentation and the action of other facultative heterotrophs on the
oxidation of organic matter. This is evident from the increase of
final pH. There are other reports of moderate rates of denitrification
at the pH range between 4.8 and 6.2 in the presence of acetic acid as
the carbon source and denitrification at pH 4 by a soil derived
rhodanobacter dominated community (van den Heuvel et al., 2010;
Huang et al., 2011).
The removal of NH4-N at pH 9 was 80%. This removal is not
considered optimum as the actual percentage of NH4-N oxidized by
AOB could be very less due to free ammonia (FA) toxicity towards
AOB at high pH as per Eq. (9) (Xue et al., 2009). The FA was calculated as 148.5 mg l1 as NH3-N at a temperature of 32 C and this
concentration inhibits AOB (Jenson et al., 2008). The increase in the
percentage removal of NH4-N was due to the escape of more than
50% of NH4-N as free ammonia (FA) by volatilization at pH 9 and at
temperature 32 C.
FA mgl1 ¼ 17=14
n
total ammonia as nitrogen mgl1 10pH
.
.
o
e 6344 ð273 þ T CÞ þ 10pH
(9)
The results of optimization of temperature studies are given in
Fig. 2 ((c) and (d)). The final pH and the ORP profiles are shown in
Fig. 2 (c). The final ORP (4 mV to 104 mV) indicates anoxic
condition suitable for SNAD. Maximum percentage removals of
NH4-N (98%), COD (90%) and TN (85%) were obtained at temperature 35 C (Fig. 2 (d)). Daverey et al. (2013) observed very low TN
(<50%) and COD removal (<20%) efficiencies when the operating
temperature of SNAD was between 15 C and 20 C. But, when the
9
average temperature raised above 27 C the average values of NH4N, TN and COD removal efficiencies were 93%, 79% and 77%,
respectively. Several researchers have reported that the optimum
temperature for AOB, anammox and denitrifiers is approximately
35 C (Strous et al., 1999; Lalucat et al., 2006; Kim et al., 2006; Gu
et al., 2012). Even though the biomass can grow between 15 C
and 30 C, their specific growth rate and activities are reported to
be higher at higher temperatures. The maximum specific growth
rate of NOB is higher than AOB at temperature below 25 C. While
denitrifiers grown at 20 C exhibited 75-times lesser levels of cnorB
gene expression compared to the same grown at 30 C. The sensitive anammox process is reported to work well within an optimum
pH range of 6.7e8.5 and temperature range from 20 C to 35 C
(Strous et al., 1998; Reginatto et al., 2005). Based on the overall
performance in this study, the optimum range of temperature for
simultaneous removal of NH4-N and COD is from 25 C to 35 C. The
final pH varied from 7.14 to 7.20 in the optimal range of temperature indicating coexistence of anammox and denitrifiers as there is
an increase in final pH from initial pH (Anjali and Sabumon, 2014).
Optimization studies showed that the developed microbial
culture can perform SNAD very effectively between pH 7 and pH 8
and temperatures between 25 C and 35 C. Also, the observed
results indicate that the process is workable between pH 6 and pH 8
and temperatures between 20 C and 35 C. The ability to work in
such a wide range of temperature and pH is advantageous when the
process is employed in the field.
3.1.2. Kinetics of ammonia removal and substrates utilization rates
of SNAD biomass
Kinetic studies were conducted using enriched SNAD biomass at
35 C temperature and at pH 7 and the data is shown in Fig. 3.
Kinetics of ammonia removal in the SNAD biomass was studied by
monitoring removals of NH4-N with time and these studies
confirmed the presence of AOB too. However, it is not clear how
much was the ammonia oxidation by autotrophs and heterotrophs
as microbial identification was not attempted in this work. The
results (Fig. 3 (a)) show that when the substrate concentration
(NH4-N) was higher, the rate of reaction was higher. For example,
Fig. 3. Kinetics of SNAD biomass
{Maximum specific substrate utilization rates are shown in the plots}
[(a): NH4-N, COD and MLVSS profile; (b) NO2-N, NO3-N and pH profile].
Please cite this article in press as: Anjali, G., Sabumon, P.C., Development of simultaneous partial nitrification, anammox and denitrification
(SNAD) in a non-aerated SBR, International Biodeterioration & Biodegradation (2016), http://dx.doi.org/10.1016/j.ibiod.2016.10.047
10
G. Anjali, P.C. Sabumon / International Biodeterioration & Biodegradation xxx (2016) 1e13
the initial concentration of NH4-N (250 mg l1) reduced to
38 mg l1 in 2 days; indicating a second order reaction with a rate
constant of 0.0167 l mg1 d1 (R2 ¼ 0.9735). Such a high rate of
ammonia oxidation was possible because of simultaneous removal
of the formed NO2-N by AnAOB or HDB as per Eqs. (2) and (3) (Fig. 3
(b)). This prevented NO2-N inhibition to AOB and AnAOB. Though
HDB has thermodynamic advantage compared to AnAOB, their
activity is limited at lower COD to N ratio in utilizing NO2-N.
The COD removal was observed in a similar manner for the first
two days and later there was no change in the removal of COD (final
COD was 32 mgl-1) with time. From the data of COD removal, it is
clear that AnAOB must have used some part of COD while oxidizing
NH4-N. There are reports of AnAOB using organic carbon for
oxidizing NH4-N by anammox process. Anammox species like
Candidatus “Anammoxoglobus propionicus” and Candidatus “Brocadia fulgida” have been reported to have the capability of organotrophic nitrate reduction (Kartal et al., 2007; Winkler et al., 2012).
Also, it has been observed in earlier studies that mixed culture
dominant in anammox could enable anaerobic ammonia oxidation
in the presence of externally added organic carbon and in the
absence of oxidized forms of nitrogen (Anjali and Sabumon, 2014).
In this study, the enhanced rate of removals of NH4-N and COD
observed during the initial 2 days could be due to the combined
action of AOB, AnAOB and HDB present in the enriched SNAD
biomass. The evidence of anammox process was confirmed by the
accumulation of NO3-N (11.7 mg l1) in 1 day as shown in Fig. 3 (b).
As the NOB were inhibited in the enriched SNAD biomass, the
chances of NO2-N conversion to NO3-N were negligible. Also, the
concentration of COD (250 mg l1) was tolerable for the anammox
species (AnAOB) to work. Jin et al. (2012) has reviewed various
inhibitory levels of organic carbon for anammox and reported that
the concentration of COD (as glucose) from 90 to 540 mg l1 does
not inhibit AnAOB. The concentration of COD dropped from 250 to
32 mg l1 within two days after which there was lesser COD
available for effective denitrification.
The maximum specific substrate utilization rates of the enriched
SNAD biomass were 185 mg NH4-N g1 MLVSS d1,
153 mg COD g1 MLVSS d1 and 172 mg N g1 MLVSS d1,
respectively. These rates show that AOB, AnAOB and HDB work
together in harmony at competitive rates for the simultaneous
removal of COD and NH4-N. The COD to N ratio during maximum
removal was 1.03, which shows that externally added COD was
consumed completely within two days. However, this ratio was not
good enough to have a dominating denitrification. Xu et al. (2010)
reported that the maximum activities of AOB and AnAOB reached
0.79 and 0.18 (g NH4-N g1 MLVSS d1) after operating for 86 days
in a process controlled completely mixed SBR maintained in DO
1e1.5 mg l1 (during the aeration phase). In their work, the feed
COD to NH4-N ratio was 0.4 and the process was developed with a
mixture of active seed biomass of anammox and activated sludge
(80% w/w). The ammonia removal capacity of SNAD biomass in this
work is comparable to the anammox culture in their work.
Considering the simple SBR operation without using acclimatized
seeding, the SNAD biomass performance attained in 5 months in
this work is appreciable.
Five days were taken to reduce NH4-N from 38 mg l1 to
6 mg l1 and the data followed first order reaction with reaction
rate constant equal to 0.3376 d1 (R2 ¼ 0.9966). From 3rd to 7th
day, ammonia removal might have occurred via anammox by first
order reaction (Fig. 3 (a)) because of limitation of organic carbon for
Fig. 4. (a) Confirmation of the anammox process using SNAD biomass
{The final values of pH, DO, and ORP, respectively, is shown above the plots}
[Top figure: Average removals of NH4-N, NO2-N, TN and Ratio of NO2-N to NH4-N (consumed); Bottom figure: Profile of NH4-N, NO2-N and TN]
(b) Confirmation of the denitrification process using SNAD biomass
{The final values of pH, DO, and ORP, respectively, is shown above the plots}
[Top figure: Average removals of NH4-N, NO2-N and COD; Bottom figure: Profile of NH4-N, NO2-N and COD].
Please cite this article in press as: Anjali, G., Sabumon, P.C., Development of simultaneous partial nitrification, anammox and denitrification
(SNAD) in a non-aerated SBR, International Biodeterioration & Biodegradation (2016), http://dx.doi.org/10.1016/j.ibiod.2016.10.047
G. Anjali, P.C. Sabumon / International Biodeterioration & Biodegradation xxx (2016) 1e13
11
Fig. 4. (continued).
effective denitrification. The pH decreased during first two days
(7.11e6.95) and then slowly increased to 7.2 by the 7th day. The ORP
values were in the range of 50.2 mV to 46 mV. Suneethi and
Joseph (2011) reported that the anammox process functions at an
ORP range of 104 mV to 50 mV and therefore the positive values
of ORP in the SNAD process did not inhibit the anammox process.
Lackner and Horn (2012) found that anammox tolerated positive
ORP up to 125 mV in a single stage nitritation-anammox SBR.
3.1.3. Confirmation of anammox and denitrification
The enriched SNAD biomass was tested separately for confirmation of anammox and denitrification, respectively. The results of
3 cycles of SBR studies for confirmation of anammox process are
shown in Fig. 4 (a). The average percentage removals of NH4-N,
NO2-N and TN in first cycle were 66%, 99.9% and 74%, respectively.
The average ratio of NO2-N to NH4-N removed was very high (2.11)
compared to the reported stoichiometry ratio (1.32) of anammox
(Strous et al., 1998). Similar results were reported by Ke et al. (2015)
where NH4-N reduction and nitrite consumption were observed for
6 h to explain the anammox activity in the granular sludge. The
ratio of NO2-N to NH4- N removed was 1.54, indicating that some
nitrite was reduced not only by the anammox reaction but also by
denitrification. Their work also proved that granular sludge was
composed of AOB, AnAOB and HDB by direct molecular evidence.
This shows that whenever HDB is involved, stoichiometry ratio
(1.32) of anammox is not satisfied. Tang et al. (2010) also reported
that the anammox stoichiometric ratio gets affected by denitrification. There was no residual NO2-N in cycle 1 in proportion to
residual NH4-N. This denotes that NO2-N reduction could have
happened by denitrification with stored carbon as the electron
donor. There are reports of denitrification using internally stored
carbon when externally added electron donor is unavailable (Third
et al., 2003). Also, Monballiu et al. (2013) reported negligible
accumulation of NO3-N when compared to the formation of NO3-N
during anammox metabolism. The less accumulation of NO3-N was
explained as the occurrence of denitrification using carbon from
the lysis of the biomass. In this work the stored carbon might have
been available with the SNAD biomass as the biomass was sourced
from SBR working at 250 mg l1 COD.
To re-confirm the presence of anammox in SNAD biomass, the
SBR studies were continued for two more cycles. The results of the
last two cycles showed average removals of NH4-N (76.2%), NO2-N
(94.4%) and TN (77.3%). The average ratio of removal of NO2-N to
NH4-N was 1.52 which became closer to the reported stoichiometry
ratio (1.32) of anammox (Strous et al., 1998) as the cycle progressed.
Moreover, the average ratio of NO3-N produced to NH4-N
consumed was 0.28 which is closer to the reported stoichiometric
ratio of anammox (0.26) as per Eq. (2). The presence of residual
NO2-N indicates the reduced effect of heterotrophic denitrification.
The final pH (7.15e7.22) for all cycles increased from initial pH
(7.00 ± 0.02) indicating either anammox or denitrification as per
Eqs. (2) and (3). A similar increase in pH trend was observed by
Monballiu et al. (2013) in an anaerobic CSTR exhibiting stable and
reliable anammox performance for more than 2 years of operation.
The average influent pH was 7.8 ± 0.2 while effluent pH was
8.3 ± 0.1. The DO varied between 0.22 and 0.35 mg l1 which is
within the tolerable range of anammox bacteria (Jenson et al.,
2008). The negative values of ORP (20.8 to 106.3 mV) ensured
anoxic condition for anammox by AnAOB.
Confirmation of the denitrification process in the SNAD biomass
Please cite this article in press as: Anjali, G., Sabumon, P.C., Development of simultaneous partial nitrification, anammox and denitrification
(SNAD) in a non-aerated SBR, International Biodeterioration & Biodegradation (2016), http://dx.doi.org/10.1016/j.ibiod.2016.10.047
12
G. Anjali, P.C. Sabumon / International Biodeterioration & Biodegradation xxx (2016) 1e13
was done in three cycles and the results are shown in Fig. 4 (b). The
average removals of NO2-N and COD were 86.1% and 84.1%,
respectively, confirming the presence of denitrifiers (HDB) in the
SNAD biomass. There was 12.5 ± 3% removal of ammonia in the first
cycle, indicating anammox process in the biomass, but the removals got decreased as the cycles progressed and became negative
in the last cycle. Therefore, the results of NH4-N removal indicate
that the HDB overpowers AnAOB in the presence of COD especially
at higher ratios of COD to NH4-N. When NO3-N concentrations are
high, denitrifiers or other facultative species, under anoxic conditions, promote dissimilatory reduction of NO3-N to NH4-N known
as dissimilatory nitrate reduction to ammonia, (DNRA) and release
NO2-N as a free intermediate (Dalsgaard et al., 2005). The increase
of NH4-N in the last cycle could be due to the dissimilatory nitrate
reduction to NH4-N. The final values of pH, DO and ORP in all cycles
show conducive environment for effective denitrification.
3.2. Prospects in industrial applications
Discharge of nitrogen containing effluents from landfills, food
processing and agricultural industries is a growing concern to the
water environment because of the eutrophication on a global scale.
Therefore, recent research has contributed for more effective
methods of nitrogen abatement. Anammox has emerged as an
effective method of nitrogen removal in full-scale plants (van der
Star et al., 2007). However, when anammox is involved, industries are looking for a treatment system which involves easy
start-up, less complex process control, lower investment costs,
reduced risk in inhibition of AnAOB by nitrite at higher concentrations of NH4-N, and lastly less residual sludge treatment and
disposal. To address these issues, SNAD is evolving as a practical
method for simultaneous removal of NH4-N and COD, especially
when the ratio of COD to NH4-N is less than or equal to 1.
Contrary to the use of acclimatized seed biomass in earlier
studies reported in Anjali and Sabumon (2015), the results of this
study indicate that SNAD could be successfully developed using
flocculent type activated sludge, which was employed for treating
tannery effluents containing both COD and NH4-N. The scale-up of
the reactor used in this study could be easily done in the field by
means of shallow ponds/tanks/ditches (400 mm liquid depth)
resulting in a very minimum energy footprint for simultaneous
removal of NH4-N and COD. However, it is important to keep the
reactor in anoxic condition (in limited DO) and devoid of sunlight to
avoid photosynthetic activity. One of the limitations of the nonaerated SBR is that higher loading rates are not possible in nonmixing conditions. But, the batch study results indicate that
higher removal rates are feasible if the SNAD biomass is maintained
in mixing condition without settling and in low DO. In short, the
application of non-aerated SBR, having a shallow depth enabling
the diffusion of air and disabling photosynthetic activity can abate
nitrogen pollution to a great extent from effluents having low to
medium concentrations of COD and NH4-N.
Application of non-aerated SBR by means of shallow ponds/
tanks/ditches is very much suitable for developing countries in
Asia, Africa and Latin America. These countries have year-round
warm climates and thereby support a good microbial activity
which is very much essential to get a stable performance. Pond
systems are generally employed in such countries due to the advantages such as: (i) they are easy to construct, (ii) require low
capital and maintenance costs, (iii) effluent does not require
disinfection (iv) capable of handling a variety of hydraulic loads, (v)
ideal for small communities, (vi) completes sludge treatment, (vii)
handles varying wastewater types (industrial or municipal), (viii)
BOD, fecal coliform and helminth removal is higher than by other
treatment methods such as activated sludge, biological filters, and
rotational biological contactors (Butler et al., 2015).
The ammonia removal from pond systems are reported (USEPA,
2011) by means of volatilization, deposition, adsorption, nitrification and denitrification. Such combined removals associated with
varying process conditions do not give consistent removal. Also, the
change in organic loading rates and seasonal variations often make
the pond systems anaerobic. This anaerobic environment releases
methane and hydrogen sulphide, which are not acceptable to the
environment. For example, methane increases global warming
potential while hydrogen sulphide creates odour problem. Due to
these issues, the promotion of pond systems is discouraged. In
contrast, the shallow anoxic ponds envisaged in this work may not
create anaerobic conditions when COD to NH4-N ratio is less than or
equal to 1. Therefore, effluent/wastewater having COD to NH4-N
ratio less than or equal to 1 can be treated by employing SNAD
process in shallow anoxic ponds/tanks/ditches and thereby
ensuring biological conversion of NH4-N to N2 without compromising the listed advantages of pond system.
4. Conclusions
Flocculent type activated sludge drawn from a tannery effluent
treatment can be used as a seed biomass for developing SNAD
process in a shallow non-aerated SBR using a synthetic feed
composed of 250 mgl1 each of COD and NH4-N. The DO
(0.35 mg l1) required for partial nitrification was obtained
through (i) diffusion of air from the head space in the reactor, and
(ii) cleaving of H2O2 by catalase enzyme produced in cells under
stressed conditions. Simultaneous removals of NH4-N (94%), COD
(92%) and TN (82%) was achieved by the SBR with a cycle time of 7
days. Maximum specific substrates utilization rates of SNAD
biomass for NH4-N, COD, and TN were 185 mg NH4-N g1
MLVSS
d1,
153
mg
COD
g1
MLVSS
d1,
and
172 mg N g1 MLVSS d1, respectively. SNAD works effectively
between pH 7.2 and pH 7.5 and temperatures between 30 C and
36 C. This research showed that application of non-aerated SBR
operation in shallow anoxic ponds/ditches/tanks could remove
COD and NH4-N simultaneously when wastewater has a ratio of
COD/NH4-N equal to one in low to moderate concentrations.
Acknowledgements
This work was supported by the Department of Science and
Technology (DST), Government of India, through the Grant-in-aid
project (DST/TM/WTI/2K11/275). The authors also want to appreciate the anonymous reviewers and the editor for their significant
contribution to improve the manuscript.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ibiod.2016.10.047.
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(SNAD) in a non-aerated SBR, International Biodeterioration & Biodegradation (2016), http://dx.doi.org/10.1016/j.ibiod.2016.10.047
