Process Biochemistry 51 (2016) 12641273

Contents lists available at ScienceDirect

Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

An anaerobic-aerobic sequential batch system using simultaneous

organic and nitrogen removal to treat intermittently discharged
organic solid wastes
Shinichi Akizuki , Tatsushi Matsuyama, Tatsuki Toda
Faculty of Science and Engineering, Soka University, Tangi-machi, Hachioji, Tokyo 192-8577, Japan

a r t i c l e

i n f o

Article history:
Received 18 January 2016
Received in revised form 7 May 2016
Accepted 13 May 2016
Available online 24 May 2016
Keywords:
Intermittently discharged organic wastes
Simultaneous processes
Methanogenesis
Denitrication
Anaerobic-aerobic batch system

a b s t r a c t
An anaerobic-aerobic sequential batch system using simultaneous organic and nitrogen removal was
investigated to treat intermittently discharged organic solid wastes. Two different recirculation ratios
of 10% and 20% day1 of liquid volume of the anaerobic reactor were examined. In both conditions,
methanogenesis occurred during the rst 5 days of the process, whereas only denitrication occurred
during the subsequent 10 days. At the end of the experiment, high COD removal efciencies of 97.7%
and 96.4% were achieved for the 10% and 20% day1 recirculation ratios, respectively. A relatively large
amount of COD consumed by denitrication was achieved for the 20% day1 condition, indicating that
an increase in the recirculation ratio enhances denitrication. Consequently, the nal nitrogen removal
efciencies were 69.0% and 81.9% for the 10% and 20% day1 recirculation ratios, respectively. To optimize the recirculation ratio, model equations were developed for the scale of this study. The modelling
results demonstrated that high recirculation during the active solubilization period enhances the nitrogen
removal efciency. Recirculating 35% day1 during the rst 5 days and 10% day1 during the subsequent
10 days is recommended as optimal for achieving high organic and nitrogen removal efciencies.
2016 Elsevier Ltd. All rights reserved.

1. Introduction
Organic solid waste management has been an integral part
of every human society. In many developed countries, major
sources of organic solid wastes (e.g., food waste, livestock manure
and municipal sewage sludge) have been treated in an appropriate manner, such as through anaerobic digestion, composting
or incineration within the local community. Because the volume
of discharged wastes from a community is approximately constant and supplied continuously, it is possible to design permanent
waste disposal facilities. Such waste management systems are useful in peace-time. Moreover, the management of intermittently
discharged organic solid wastes such as marine biofouling, beached
seaweeds and unexpected large-scale disaster wastes have become
urgent environmental issues [13]. These wastes are discharged
intermittently, suddenly and in vast quantities, with the volumes
often exceeding the capacities of permanent disposal sites [4,5]. In
such cases, the speed of waste treatment is of high priority so that

Corresponding author.
E-mail address: s-akizuki@soka.gr.jp (S. Akizuki).
http://dx.doi.org/10.1016/j.procbio.2016.05.011
1359-5113/ 2016 Elsevier Ltd. All rights reserved.

the diffusion of various environmental pollutions can be prevented

and public health risks caused by putreed wastes in the surrounding areas can be reduced [6]. Usually, direct incineration, landlling
and/or ocean dumping are used to dispose of these wastes; however, these approaches cause high environmental burden and high
nancial losses [2,7]. For instance, blue mussels, which are a major
marine biofouling species throughout the worlds oceans, have a
strong colonization ability and cause the biodestruction of marine
structures (e.g. the hulls of ships, docks and coastal thermal/nuclear
power plants), resulting in substantial economic losses in terms of
removal and disposal [7,8]. In Japan, nearly 90% of fouling organisms are terminated by incineration and/or landlling [7].
Anaerobic digestion is expected to be an optimal approach to
treating intermittently discharged organic wastes because of the
large energy recovery in the form of methane (CH4 ) linked to the
process, which generates low environmental impact [911]. However, additional post-treatment facilities are normally required
to treat the efuent from an anaerobic digester to adapt the
quality of the water to environmental legislation [12,13]. Biological nitrication-denitrication processes are widely accepted
as the preferred post-treatment method for removing nitrogen
compounds from efuents [14,15]. This system requires higher

S. Akizuki et al. / Process Biochemistry 51 (2016) 12641273

construction costs for additional reactors and operational costs for

aeration in nitrication and chemicals in denitrication [16,17]. For
these reasons, this relatively complex treatment system is not suitable as a transient treatment facility for intermittently discharged
wastes. Thus, the development of a simple and compact treatment
method is highly desirable.
An anaerobic-aerobic treatment system using simultaneous
organic and nitrogen removal in a single anaerobic reactor is
expected to be a simplied and cost-effective system compared
with conventional treatment systems [1820]. In this type of treatment system, nitrogen can be removed via recirculation of the
nitried aerobic efuent to the anaerobic reactor. In the anaerobic reactor, denitrication occurs in conjunction with anaerobic
digestion. This treatment system offers the following signicant
advantages: (1) the combination of anaerobic digestion and denitrication processes in a single reactor can save on the initial
cost of post-treatment facilities, and (2) target wastes can be
used as the organic sources for denitrication instead of chemicals, leading to operational cost savings. In the 2000 s through
the 2010s, intensive studies on simultaneous organic and nitrogen
removal in a single reactor were conducted by using real wastewater sources, such as animal wastewater, sh cannery wastewater
and brewage wastewater, as substrates [2124]. However, studies
were previously performed by using continuous feeding because
biological treatment systems commonly treat continuously discharged wastes or wastewaters. In practice, a fed-batch mode is
used to accept the target waste discharge to treat intermittently
discharged wastes. In this respect, our previous studies focused
on simultaneous batch processes for organic and nitrogen removal
in a single reactor and revealed the effectiveness of this approach
[25,26].
Previous studies concerning simultaneous organic and nitrogen
removal identied that the process efciency was affected by the
recirculation ratio [2729]. For instance, Huang et al. [27] studied a combined up-ow anaerobic sludge bed (UASB)-activated
sludge (AS) reactor system under different recirculation (recycleto-inuent) ratios ranging from 100 to 300%. The authors reported
that high recirculation ratio resulted in high denitrication performance, yielding a high total nitrogen (TN) removal efciency of
77%. Furthermore, Giustinianovich et al. [28] reported an increase
in nitrogen removal efciency of 48% in anaerobic-aerobic lter
reactors when the recirculation ratio was increased from 200% to
1000%. However, An et al. [29] reported that the lack of methanogenesis caused by a higher recirculation ratio in a system composed
of an up-ow anaerobic sludge blanket (UASB) and an aerobic
membrane bioreactor (MBR). In effect, the high recirculation ratio
favoured denitrication to the detriment of methanogenesis [19].
Moreover, the advantage of simultaneous anaerobic digestion and
denitrication is the removal of the remaining chemical oxygen
demand (COD) after denitrication by methanogenesis [22]. Thus,
an optimal recirculation ratio must be determined to obtain a high
efciency of COD and nitrogen removal in the investigated batch
system.
In this study, the effect of different recirculation ratios on the
organic and nitrogen removal performance of an anaerobic-aerobic
sequential batch system was examined. Model equations were
developed by using experimental results to determine the optimal
recirculation ratio for the scale of this study.

1265

of 18 cm were collected from marine structures such as anchored

ships and the concrete walls of piers in Otsuchi Bay of Iwate Prefecture, Japan. The shells of the blue mussels were cleaned of epiphytes
and sand, and then stored in cooling boxes and transported to
the laboratory. The blue mussels with their shells were used as
the substrate. The total solid (TS), total volatile solid (TVS), total
chemical oxygen demand (TCOD), and TN contents of the shellsh meats were 155, 137, 248 and 15.2 g kg-wet1 , respectively.
The mesophilic-anaerobic sewage sludge was collected from the
Hokubu Sludge Treatment Center of Kanagawa Prefecture, Japan.
The denitrifying and nitrifying sludges were collected from the
Hokubudaini Wastewater Treatment Center of Kanagawa Prefecture, Japan. In the laboratory, the anaerobic and denitrifying sludges
were left for two days to allow for sedimentation. The supernatants were discarded and the thickened sludge layers were used
as the seed sludge for the anaerobic reactor. The collected nitrifying
sludge was used as the seed sludge for the aerobic reactor.
2.2. Experimental unit
A schematic illustration of the proposed anaerobic-aerobic
sequential batch system is shown in Fig. 1. This system consists of:
(1) an anaerobic reactor with a working volume of 15 L for simultaneous anaerobic digestion and denitrication; (2) an aerobic
reactor with a working volume of 4.5 L for nitrication; (3) solidliquid separation by a centrifugal separator (Model-6000, Kubota)
for the anaerobically digested slurry; and (4) a process controller
(EPC-2000, Eyela) connected to a PC for on-line monitoring and controlling. The anaerobic reactor has an oxygen redox potential (ORP)
sensor connected to the process controller, and the ORP value was
monitored continuously. The digested slurry in the anaerobic reactor was agitated during the treatment period by a liquid circulation
pump. The aerobic reactor possessed pH and dissolved oxygen (DO)
sensors, and the values were monitored and controlled by the process controller. The pH was controlled at 7.0 0.1 by the addition of
1 M NaHCO3 using a solution sending pump. The DO was controlled
at 5.0 0.1 mg L1 by aeration using an air pump. A mechanical stirrer was used to assist aeration and to provide liquid mixing when
the aeration was terminated.
2.3. Operating conditions
Two different water recirculation ratios of 10% and 20% day1
of liquid volume of the anaerobic reactor were examined to evaluate the effect of recirculation. In this experiment, anaerobic sludge
with denitrifying sludge was used as the seed sludge for the anaerobic reactor, and nitrifying sludge was used as the seed sludge
for the aerobic reactor. The experimental set-up involved the: (i)
addition of 3.0 kg of wet blue mussels with their shells into the
anaerobic reactor, yielding initial COD and N concentrations of
69007000 mg L1 and 960980 mg N L1 , respectively; (ii) addition of 6.0 L of anaerobic sludge and 1.8 L of denitrifying sludge
into the anaerobic reactor; (iii) addition of 3.0 L of nitrifying sludge
into the aerobic reactor; (iv) addition of deionized water into reactors (4.5 L for anaerobic reactor and 1.5 L for aerobic reactor); and
(v) displacement of head-space air with Ar gas to create anaerobic conditions. Thereafter, the simultaneous removal of organic
and nitrogen contaminants was performed by following sequential
batch steps:

2. Materials and methods

2.1. Collection and preparation of the substrate and seed sludge
Blue mussels were used as the model for intermittently discharged organic solid wastes. Blue mussels ranging in size range

(1) Anaerobic digestion process: the complex organic matter from

the substrate was converted to CH4 gas, whereas organic nitrogen was converted to dissolved nitrogen (mainly NH4 + ). This
step was conducted for rst 23 h or 11 h at recirculation ratios
of 10% and 20% day1 , respectively.

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S. Akizuki et al. / Process Biochemistry 51 (2016) 12641273

Fig. 1. Description of a proposed anaerobic-aerobic sequential batch treatment system for intermittently discharged wastes. A practical treatment ow (A), and a schematic
diagram of the investigated system (B).

(2) Solid-liquid separations: approximately 2.0 L of digested mixture from anaerobic reactor was separated by the centrifugal
separator. Then, 1.5 L of the liquid phase (i.e. anaerobic efuent) was collected and fed into the aerobic reactor. Meanwhile,
the solid phase (i.e. the seed sludge and the remaining substrate) was returned into the anaerobic reactor. At the same
time, spontaneous settling was conducted in aerobic reactor.
The supernatant (i.e. aerobic efuent) was collected and fed into
anaerobic reactor. This step was conducted for approximately
1 h under both recirculation conditions.
(3) Nitrication process and simultaneous anaerobic digestion and
denitrication processes: nitrication of anaerobic efuent was
conducted in the aerobic reactor. NH4 + was oxidized into NO3 .
At the same time, simultaneous anaerobic digestion and denitrication processes were conducted in the anaerobic reactor.
This step was conducted for 23 h or 11 h at recirculation ratios
of 10% and 20% day1 , respectively. Then, steps (2) and (3)
were repeated to perform simultaneous organic and nitrogen
removal until the end of the experiments.

2.4. Analytical methods

TS and TVS were examined according to the sewage analysis
methods outlined by the Japan Sewage Works Association [30].
TCOD, soluble COD (SCOD), NO2 and NO3 were quantied as
described by standard American Public Health Association methods
[31]. TCOD and SCOD were determined colourimetrically (DR/2400
Spectrophotometer, Hach) following digestion with dichromate.
The concentrations of NO2 and NO3 were measured by ion chromatography (SSC-600, Senshu Kagaku). TN was measured using the
peroxydisulfate oxidation method. The N2 , CH4 and CO2 contents
of the biogas samples were monitored using a gas chromatograph
(GC-2014AT, Shimadzu) equipped with a packed column (Shincarbon ST) and a thermal conductivity detector.
2.5. Calculations
The amount of COD consumption by methanogenesis and denitrication was determined using the following equation:
Amount of COD consumption (mg COD)=CODmethano + CODdeni + CODcel

In this system, a portion of the substrate organic carbon was

used for denitrication as long as the nitried efuent was recycled into the anaerobic reactor. The remaining organic carbon was
mainly used for methanogenesis. The experiments were conducted
for a total of 15 days. The anaerobic and aerobic reactors were
incubated at 37 1 C and 30 1 C, respectively, in a walk-in,
temperature-controlled laboratory.

(1)

where CODmethano and CODdeni refer to the theoretical amounts of

COD consumption by methanogenesis and denitrication, respectively. CODcel refers to the total COD requirement for microbial
growth of the methanogens and denitriers. The COD consumption
by methanogenesis was determined based on the CH4 produced
from the anaerobic reactor. The equivalent COD of CH4 is 0.395 LCH4 g-COD1 at 35 C. The COD consumption by denitrication was
calculated by removing NO3 -N from the anaerobic reactor. The

S. Akizuki et al. / Process Biochemistry 51 (2016) 12641273

10.0

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9.5
9.0

pH

8.5
8.0
7.5
7.0
6.5
6.0
0

10

15

Experimental period (day)

0

-100

-100

-200

-200
ORP (mV)

ORP (mV)

-300
-400

-300
-400
-500

-500

-600

-600
0

5
10
Experimental period (day)

15

10

15

Experimental period (day)

Fig. 2. Variations of pH and ORP in the anaerobic reactor for different conditions recirculation ratios. Variation of pH in both processes (A), and variation of ORP in the 10%
day1 process (B), and the 20% day1 process(C). : pH in the 10% day1 process; : pH in the 20% day1 process.

demand of COD for the denitrication reaction (NO3 -N reduction

to N2 gas) was 2.86 mg-COD mg-N1 . In methanogenesis, 2.8% of
the COD consumption by methanization (CODmethano ) was assumed
to be the growth requirement for the methanogens [32]. In denitrication, two assumptions were used to calculate the COD growth
requirement for the denitriers: (i) the required COD for denitrier
growth was equal to that for the reaction (CODdeni ) [33]; and (ii)
the required COD for denitrier growth was two-fold higher than
for the reaction [34].
The nal COD and total N removal efciencies were determined
using the following equations:

Final COD removal efciency= 1

SCODfinal
CODInitial

SNfinal
Final N removal efciency= 1
Ninitial


COD consumption ratio by methanogenesis =

100%

(2)
(3)

where CODinitial and Nintial refer to the initial COD and N concentrations in the anaerobic reactor, respectively, and SCODnal and SNnal
refer to the nal soluble COD and N concentrations, respectively.
Amount of remaining COD (mg COD)=CODsubstrate (CODmethano + CODdeni + CODcel )

(4)
where CODsubstrate refers to the substrate COD from added blue
mussels.
The amount of solubilized substrate COD and the COD consumption ratio by methanogenesis and denitrication were determined
using the following equations:
Amount of solubilized substrate COD(mg COD) =CODmethano + CODdeni

(5)


100%

(6)

COD consumption ratio by denitrication = (

CODdeni
) 100%
CODmethano + CODdeni

(7)

The denitrication efciencies of each operational cycle in the

anaerobic reactor were determined using the following equation:

Denitrication efciency=
100%

CODmethano
CODmethano + CODdeni

NO
x Nend

NO
x Nbeginning

100%

(8)

where NOx -Nbeginning and NOx -Nend refer to the NOx -N concentrations at the beginning and end of each cycle, respectively.
To estimate the nitrogen removal efciency under different
recirculation ratios, the following model equations were developed
for the scale of this study:
Amount of solubilized substrate COD (mg COD)=
Amount of solubilized substrate nitrogen (mg N)=7200 +


Denitrication efciency (%) =100 ee

88800 d
7.59 + d

7600 d
1.59 + d

(9)
(10)


r1.41
1.62

COD consumption ratio by methanogenesis (%) =100 ee


r3.95
5.79

(11)
(12)

noindent where d and r refer to the experimental day and the

COD/NOx -N ratio, respectively. All equations were based on the
experimental data obtained in this study, which are shown in Fig. S1

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S. Akizuki et al. / Process Biochemistry 51 (2016) 12641273

(Supplementary materials). Other assumptions were incorporated

into this model. For instance, we assumed that the nitrication efciency in the aerobic reactor is 100% and that the denitrication
performance is independent of the recirculation ratio.
3. Results and discussion
3.1. Process stability
The variations in pH and ORP in the anaerobic reactor were
monitored to evaluate the process stability during the experiment
(Fig. 2). Under each recirculation ratio, the pH values decreased
during the rst day and then increased thereafter (Fig. 2A). A previous study on the treatment of solid wastes in a combined anaerobic
digestion and denitrication system reported that the pH varies in
this manner because of a balance between the acidogenesis and
methanogenesis/denitrication processes [25]. In this study, the
transient decrease in pH was observed because of acidogenesis of
the blue mussels at the beginning of the experiment. The pH value
then increased because of acid consumption by methanogenesis
and/or denitrication. The pH values varied from 7.09 to 8.24 and
from 7.13 to 7.81 for the 10% day1 and 20% day1 recirculation conditions, respectively, which are suitable values for methanogenesis
and denitrication processes [3537]. The ORP values uctuated for
both recirculation ratios (Fig. 2B and C). The values uctuated over
a wide range from 549 to 53 mV and from 467 to 74 mV for
the 10% day1 and 20% day1 recirculation conditions, respectively.
The values increased in each semi-batch recycling operation of the
aerobic efuent because of the presence of nitrogen oxides (such
as NO2 -N and NO3 -N), and it decreased because of the removal

3.2. Biogas production

CH4 and N2 productions were observed for both of the experiments performed under different recirculation ratios (Fig. 3),
suggesting the occurrence of methanogenesis and denitrication in
a single reactor. The total CH4 yields were 110 and 88.6 mL g COD1
for the 10% and 20% day1 recirculation conditions, respectively
(Fig. 3A). Ruiz et al. studied the simultaneous organic and nitrogen
900

100
80
60
40

700
600
500
400
300
200

20

100
0

0
5
10
Experimental period (day)

50

10

15

Experimental period (day)

45

15

40
35
30
25
20
15
10
5
0

120
N2 production rate (mL g N-1 day-1)

CH4 production rate (mL g COD-1 day-1)

800
N2 yield (mL g N-1)

CH4 yield (mL g COD-1)

120

of these species. The sharp up/down variation in ORP re-occurred

throughout the experiment, indicating a rapid progress of denitrication. For the 10% day1 recirculation experiment, strict anaerobic
conditions, ranging from 549 to 242 mV, were observed during the rst 5 days of the experimental period, indicating suitable
anaerobic conditions for methanogenesis [38]. Subsequently, the
ORP increased and maintained a relatively high value, ranging from
296 to 53 mV, which is appropriate only for denitrication [39].
Such a shift in the ORP values was also observed for the 20% day1
recirculation process. However, the rapid shift occurred during the
rst 2.5 days of the experiment. In this case, the shift corresponds
to the period during which methanogenesis primarily occurs, as
discussed below, followed by a shift to a period during which denitrication becomes dominant. The higher recirculation resulted in a
faster shift, likely indicating that the rapid accumulation of nitrous
oxides occurred in the 20% day1 recirculation process because of
the high recirculation of aerobic efuent.
Under both recirculation conditions, nearly 100% of added
ammonia was oxidized to nitrate without nitrite accumulation in
the aerobic reactor. This result indicated that stable nitrication
was conducted in this study.

100
80
60
40
20
0

10

Experimental period (day)

15

5
10
Experimental period (day)

15

Fig. 3. The amount of biogas yield and the rate of biogas production for the different recirculation ratios. CH4 yield (A), N2 yield (B), CH4 production rate (C) and N2 production
rate (D). : values in the 10% day1 process; : values in the 20% day1 process.

S. Akizuki et al. / Process Biochemistry 51 (2016) 12641273

1269

Fig. 4. The nal COD and nal nitrogen removal efciencies, the amount of substrate COD consumption by methanogenesis and denitrication, and the variation of the
amount of the remaining COD for the different recirculation ratios. The COD and nitrogen removal efciencies (A), the amount of substrate COD consumption (B), the amount
of remaining COD in the 10% day1 process (C) and in the 20% day1 process (D). : COD removal efciency; : nitrogen removal efciency;

: COD consumption by

methanogenesis; : COD consumption by denitrication; : assumption based on the required COD for denitrier growth was equal to that for the reaction; : assumption
based on the required COD for denitrier growth was two-fold higher for the reaction.

removal in upow sludge-bed reactors under different substrate

COD/N ratios conditions (from 1 to 100) [40]. They reported that the
CH4 yield at the end of reactor operation varied depending on the
COD/N ratios and ranged from approximately 4.0400 mL gCOD1 .
Akizuki et al. also studied the effect of substrate COD/N ratio
on the performance of anaerobic digestion and denitrication in
batch experiments [25]. The literature indicates that the cumulative CH4 yield ranged from 3.6 to 218 mL gCOD1 under different
COD/N ratios (from 2.73 to 17.2). In this study, the substrate COD/N
ratio was approximately 7 at both recirculation ratios, likely leading to moderate CH4 yields. The total N2 yields were 571 and
762 mL g N1 for the 10% and 20% day1 recirculation conditions,
respectively (Fig. 3B). Relatively high N2 yields and low CH4 yields
were observed for the 20% day1 recirculation process. The CH4
and N2 production rates of both processes are shown in Fig. 3C
and D, respectively. In both processes, the highest CH4 production rate was achieved at the beginning of the experimental period
and then the rate decreased rapidly during the rst 5 days. On the
last day of each process, methanogenesis mostly ceased whereas
denitrication continued. A relatively higher N2 production rate in
conjunction with a lower CH4 production rate was observed for
the 20% day1 recirculation process. These results indicate that
denitrication was promoted in the 20% day1 recirculation process because of the relatively high recirculation of nitried water
into the aerobic reactor. Similar results were obtained in previous
studies of other continuous anaerobic-aerobic treatment systems
[19,28].

3.3. Final COD and nal nitrogen removal efciencies

During the experimental period, no accumulation of soluble
COD and soluble N was observed in the anaerobic reactor. This
result indicates that the solubilized substrate was consumed immediately by methanogenesis and denitrication. The nal efuent
water contents of COD and TN were 161 mg-COD L1 and 298 mgN L1 for the 10% day1 recirculation process and 245 mg-COD L1
and 178 mg-N L1 for the 20% day1 recirculation process, respectively. For Japan, the current standards for efuent discharges for
COD and TN are 160 mg-COD L1 and 120 mg-N L1 , respectively.
The efuent content of the COD obtained in this study nearly
reached this recommended value. To remedy this, the remaining
COD could be removed in a subsequent aerobic reactor in a normal
sewage treatment system, for example, without adding a signicantly high load.
The nal COD and nitrogen removal efciencies for both recirculation processes are shown in Fig. 4A. High COD removal efciencies
of 97.7% and 96.4% were achieved for the 10% day1 and 20% day1
processes, respectively. However, the nitrogen removal efciency
varied with the recirculation ratio. Compared with the efciency
for the 10% day1 process (69.0%), a relatively high efciency of
81.9% was observed for the 20% day1 process. The overall pathway of substrate COD consumption in both processes is shown in
Fig. 4B. Although the nal COD removal efciencies were identical
between the two processes, the COD consumption pathway varied.
Indeed, a relatively large amount of COD consumption by denitri-

S. Akizuki et al. / Process Biochemistry 51 (2016) 12641273

70000

Amount of recirculated NO3 (mg-N)

Amount of solubilized substrate (mg-COD)

1270

60000
50000
40000
30000
20000
10000
0

18000

16000
14000
12000
10000
8000
6000
4000
2000
0

10

15

12

10

100
COD consumption ratio (%)

10
COD/NO3-N ratio

15

Experimental period (day)

Experimental period (day)

8
6
4
2

80
60
40
20
0

0
0

5
10
Experimental period (day)

15

10

15

COD/NO3 -N ratio

Fig. 5. The variation of the COD/NO3 -N ratio for the different recirculation ratios and the relationship between the COD/NO3 -N ratio and the COD consumption ratio
by methanogenesis and denitrication. The amount of solubilized substrate COD (A), the amount of recirculated NO3 (B), the variation of the COD/NO3 -N ratio (C), and
the relationship between the COD/NO3 -N ratio and COD consumption (D). : 10% day1 process; : 20% day1 process; : COD consumption by methanogenesis; : COD
consumption by denitrication.

cation was achieved in the 20% day1 recirculation process. An

increase in the recirculation ratio from 10 to 20% day1 enhanced
the denitrication process.
The variations in the amount of remaining COD for both recirculation ratios are shown in Figs. 4C and D. In both processes, the
substrate COD was consumed rapidly at the beginning of the experiment by methanogenesis, denitrication and microbial growth.
In this study, two assumptions reported by different investigators
were used to estimate the COD requirement for the growth of the
denitriers [33,34]. Under both assumptions, the substrate COD is
mostly removed during the experimental period in each recirculation process. These results indicate that the substrate COD was
degraded efciently in the examined batch system.
3.4. Optimizing the recirculation
In the present batch system, the substrate COD was consumed by both methanogenesis and denitrication. As discussed,
methanogenesis occurred during the earlier stages of the process, whereas denitrication occurred in the later stages under
the recirculation ratios used in this study. To achieve a high nitrogen removal efciency, the substrate COD in the batch operation
should be available for denitrication instead of methanogenesis. To determine the main biological pathways operative in batch
systems, the COD to NOx -N ratio is known to be an important parameter [19,4042]. For instance, Del Pozo and Diez [19]
reported that denitrication was the main pathway for organic
matter removal for values of COD/NOx -N below 10. Additionally,
in the case of solid waste treatment, the substrate solubilization

rate should be considered because the substrate COD can only be

used by microorganisms following solubilization. The integrated
amounts of solubilized substrate COD in both recirculation processes are shown in Fig. 5A. As shown, solubilization was initiated
at the beginning of the experiment, and approximately 50% of
the solid substrate became solubilized in the early stages of the
rst 35 days. Subsequently, the rate was reduced, but the solubilization continued to occur gradually until the last day of the
experiment. The solubilization rate did not change between the
different recirculation processes (i.e., 10% day1 and 20% day1 ).
This result suggests that the substrate solubilization process is not
affected by the recirculation ratio. In this study, the ratios of the
solubilized COD to NOx -N changed over time (Fig. 5C) depending on the substrate solubilization and the amount of recirculated
NOx -N from the aerobic reactor (Fig. 5B). Under both recirculation
conditions, a relatively high ratio was observed at the beginning of
the experiment, and the ratio decreased rapidly during the rst
5 days. The ratio then maintained a relatively low value until the
nal day of the experiment. The relatively low ratio observed for the
20% day1 process compared with the 10% day1 process occurred
because of the higher recirculation ratio. To evaluate the effect of
the COD/NOx -N ratio on the COD utilization pathway, the relationship between COD/NOx -N and the COD consumption efciency
by methanogenesis and denitrication is summarized in Fig. 5D.
The utilization pathway shifts from methanogenesis to denitrication below a COD/NOx -N ratio of approximately 5. The relatively
high COD utilization efciency by denitrication was achieved at
low COD/NOx -N ratios. Therefore, the COD/NOx -N ratio must be

S. Akizuki et al. / Process Biochemistry 51 (2016) 12641273

12

80
60
40
20

8
6
4
2

0
10

15

Experimental period (day)

50000

40000
30000
20000
10000
0
0

5
10
Experimental period (day)

15

0
COD consumption by methanogenesis (mg-COD)

0
COD consumption by denitrification (mg-COD)

10
COD/NO3-N ratio

Nitrogen removal efficiency (%)

100

1271

10

15

Experimental period (day)

50000

40000
30000
20000
10000
0
0

5
10
Experimental period (day)

15

Fig. 6. A comparison between the estimated values by using the model equations and the experimental values in the 10% day1 and the 20% day1 processes. The nitrogen
removal efciency (A), the COD/NO3 -N ratio (B), the amount of COD consumption by denitrication (C), and the amount of COD consumption by methanogenesis (D).
Solid line: the estimated values in the 10% day1 process; dash line: the estimated values in the 20% day1 process. : Experimental values in the 10% day1 process; :
Experimental values in the 20% day1 process.

maintained at a low value to give priority to COD consumption by

denitrication.
To validate the developed model, the results obtained using the
model equations were compared with the experimental results.
Fig. 6 shows the estimated and experimental values of nitrogen
removal efciency, the COD/NOx -N ratio, and the COD consumption by methanogenesis and denitrication under the 10% and
20 day1 recirculation ratios. The estimated values showed very
good agreement across the entire range of experimental values.
According to the model, the COD/NOx -N ratio must be maintained at a low value to achieve a high nitrogen removal efciency.
Such a condition can be obtained at a high recirculation ratio.
However, a large increase in the recirculation ratio may impair
denitrication because of an insufcient hydraulic retention time
(HRT) [42] and may cause the eventual collapse of nitrication by
excess organic loading, which usually results in low performance
of autotrophic nitrication [43,44]. In average design ows, a sufcient HRT for denitrication is approximately 6 h [45]. Thus, the
40% day1 recirculation ratio, which represents an approximately
6 h HRT in an anaerobic reactor, can be assumed to be the maximum allowable ratio. In this study, four different scenarios were
compared based on the empirical model to determine the optimal
recirculation conditions: (1) 25% day1 during the experiment; (2)
25% day1 during the rst 5 days and 10% day1 during the subsequent 10 days; (3) 35% day1 during the experimental period;
and (4) 35% day1 during the rst 5 days and 10% day1 during the
subsequent 10 days. Scenarios (2) and (4) were designed based on
the results of substrate solubilization (Fig. 5A), which suggest that
active solubilization occurs during the rst 5 days of the experi-

ment. The estimated values of the nitrogen removal efciency and

the COD/NOx -N ratio in each scenario are shown in Fig. 7. The
estimated nitrogen removal efciencies were 89.0%, 84.8%, 94.9%
and 91.2%, which correspond to 108, 149, 50.0 and 96.0 mg N L1 as
total nitrogen, in scenarios (1), (2), (3) and (4), respectively. A relatively high removal efciency was observed for the recirculation
ratio of 35% day1 because the relatively low COD/NOx -N ratio
could be maintained during the experimental period, which led
to larger COD consumption by denitrication. Sufcient nitrogen
removal was achieved in scenario (4), indicating that a high recirculation ratio is required only when active solubilization occurs.
The expected efuent qualities obtained in scenarios (1), (3) and
(4) were lower than the efuent standard value. The total recirculation ratios during the experiment were 375%, 525% and 275%
in scenarios (1), (3) and (4), respectively. Therefore, scenario (4) is
recommended to be an optimal recirculation condition in terms of
both nitrogen removal efciency and operational cost. These results
suggest that both high COD and nitrogen removals can be achieved
by controlling the recirculation ratio to maintain an appropriate
COD/NOx -N ratio in an anaerobic reactor. Such an operational
control method is likely to be useful not only for the treatment
of blue mussels but also for the treatment of other intermittently
discharged organic solid wastes.
4. Conclusions
An anaerobic-aerobic sequential batch system using simultaneous organic and nitrogen removal was investigated for the
treatment of intermittently discharged organic solid wastes. To

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S. Akizuki et al. / Process Biochemistry 51 (2016) 12641273

100

Nitrogen removal efficiency (%)

Nitrogen removal efficiency (%)

100
80
60
40
20
0

95

90

85

80
0

10

15

5.0

10

15

Experimental period (day)

Experimental period (day)

1.5

COD/NO3-N ratio

COD/NO3-N ratio

4.0
3.0
2.0

1.0

0.5

1.0
0.0

0.0
0

5
10
Experimental period (day)

15

5
10
Experimental period (day)

15

Fig. 7. A comparison between the estimated values for the different recirculation ratios. The nitrogen removal efciency (from 0% to 100%) (A), the nitrogen removal efciency
(from 80% to 100%) (B), the COD/NO3 -N ratio (from 0 to 5) (C), and the COD/NO3 -N ratio (from 0 to 1.5) (D). Solid line: scenario (1) (25% day1 during the experiment);
dash line: scenario (2) (25% day1 during the rst 5 days); double line: scenario (3) (35% day1 during the experiment); dash-dot line: scenario (4) (35% day1 during rst
5 days).

evaluate the effect of the recirculation ratio on the process performance, two different recirculation ratios of 10% and 20% day1
of liquid volume of the anaerobic reactor were examined. The following conclusions are drawn from this study:
A COD removal efciency above 95% was achieved by simultaneous anaerobic digestion and denitrication for both of the
recirculation processes.
A relatively high nitrogen removal efciency of 81.9% was
achieved for the 20% day1 process, whereas the 10% day1 process demonstrated an efciency of only 69.0%.
The COD consumption pathway varied as a function of the
recirculation conditions. A relatively large amount of COD consumption by denitrication was observed for the 20% day1
process.
The modelling results suggest that the nitrogen removal efciency can be improved with a high recirculation ratio during
an active solubilization period. Recirculating 35% day1 during
the rst 5 days and 10% day1 for the subsequent 10 days is recommended to achieve a high nitrogen removal efciency.
Acknowledgments
We thank the International Coastal Research Centre, the University of Tokyo and T. Shiotani (Sanyo Techno Marine Co., Ltd.) for
assistance in collecting the blue mussels used in this study. We are
grateful to the Hokubu Sludge Treatment Centre and the Hokubudaini Wastewater Treatment Center in Kanagawa, Japan for the

preparation of the seed sludges. This research was supported by a

Grant-in-Aid for Scientic Research Aimed at Establishing a Sound
Material-Cycle Society from the Ministry of the Environment of
Japan (K2010, K2158, and K22065).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.procbio.2016.05.
011.

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