Science of the Total Environment 443 (2013) 877886

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Science of the Total Environment

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Anaerobic co-digestion of source segregated brown water (feces-without-urine) and

food waste: For Singapore context
Rajinikanth Rajagopal a,, Jun Wei Lim a, Yu Mao a, c, Chia-Lung Chen a, Jing-Yuan Wang a, b
a

Residues and Resource Reclamation Centre, Nanyang Environment and Water Research Institute, Nanyang Technological University, #06-08 CleanTech One,
1 Cleantech Loop, 637141 Singapore
School of Civil and Environmental Engineering, Nanyang Technological University, 50 Nanyang Avenue, 639798 Singapore
c
School of Energy and Environmental Sciences, Yunnan Normal University, 121 Street, Kunming 650092 China
b

H I G H L I G H T S

Source separation of organic waste/wastewater streams on household level was done.

Brown water (BW) was collected from a specially designed no-mix toilet.
BW and food waste codigestion proved as a potential substrate for biogas production.
A distinct improvement in methane yield was observed.
This concept is vital for countries facing rapid urbanization and water shortage.

a r t i c l e

i n f o

Article history:
Received 6 September 2012
Received in revised form 4 November 2012
Accepted 5 November 2012
Available online 17 December 2012
Keywords:
Anaerobic co-digestion
Brown water
Food waste
Source separation
Volatile fatty acids (VFAs)

a b s t r a c t
The objective of this study was to evaluate the feasibility of anaerobic co-digestion of brown water (BW)
[feces-without-urine] and food waste (FW) in decentralized, source-separation-based sanitation concept. An effort
has been made to separate the yellow water (urine) and brown water from the source (using no-mix toilet) primarily to facilitate further treatment, resource recovery and utilization. Batch assay analytical results indicated that
anaerobic co-digestion [BW+FW] showed higher methane yield (0.540.59 L CH4/gVSadded) than BW or FW as a
sole substrate. Anaerobic co-digestion was performed in the semi-continuously fed laboratory scale reactors viz.
two-phase continuous stirred-tank reactor (CSTR) and single-stage sequencing-batch operational mode reactor
(SeqBR). Initial 120 d of operation shows that SeqBR performed better in terms of organic matter removal and maximum methane production. At steady-state, CODs, CODt, VS removals of 92.03.0, 76.75.1 and 75.76.6% were
achieved for SeqBR at 16 d HRT, respectively. This corresponds to an OLR of 23 gCOD/L d and methane yield
of about 0.41 L CH4/gVSadded. Good buffering capacity did not lead to accumulation of VFA, showing better process stability of SeqBR at higher loading rates. The positive ndings show the great potential of
applying anaerobic co-digestion of BW + FW for energy production and waste management. In addition,
daily ush water consumption is reduced up to 80%. Decentralized, source-separation-based sanitation
concept is expected to provide a practical solution for those countries experiencing rapid urbanization
and water shortage issues, for instance Singapore.
2012 Elsevier B.V. All rights reserved.

1. Introduction
The decentralized treatment of municipal wastewater based on
separation between gray and black water, and even between brown
water (BW) [feces-without-urine] and yellow water (YW) [urine], represents a sustainable and future solution for waste (water) treatment

Corresponding author at: Dairy and Swine Research and Development Centre,
Agriculture and Agri-Food Canada, 2000, College Street, Sherbrooke (QC), Canada
J1M 0C8. Tel.: + 1 8197807303; fax: + 1 8195645507.
E-mail addresses: rrajinime@yahoo.co.in, rajinikanth.rajagopal@agr.gc.ca
(R. Rajagopal).
0048-9697/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.scitotenv.2012.11.016

(Elmitwalli et al., 2006). The separation of different wastewater streams

and their treatments with the aim of energy production and nutrient
reuse was demonstrated in the year 2000 within a housing estate for
350 to 400 inhabitants in the pilot project Flintenbreite in Luebeck,
Germany (Wendland and Oldenburg, 2003; Wendland et al., 2007).
The concept comprises vacuum toilets with subsequent pasteurization
and anaerobic digestion (AD) of black water together with kitchen
waste in a semi-centralized biogas plant and nally recycling of the
digested anaerobic efuent in agriculture. A few other researchers have
also studied the co-digestion of black water and kitchen refuse in various
anaerobic systems (Kujawa-Roeleveld et al., 2003, 2005, 2006; Elmitwalli
et al., 2006; Wendland et al., 2007). In addition, these researchers have
successfully demonstrated the feasibility of treating human waste in

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R. Rajagopal et al. / Science of the Total Environment 443 (2013) 877886

decentralized sanitation systems. However, source separation between

feces and urine, and its subsequent resource recovery approach is limited.
Besides, such research in the urban context has been scarce.
On the other hand, an alarming aspect worldwide is the depletion
of non-renewable energy sources. Natural resources are not efciently
used by human beings. According to an industrial ecology study
(Deschenes and Chertow, 2004), only about 6% of material ows end
up in making products and the majority of the remaining natural
resources are considered as unusable waste in our industrial systems.
If natural resources can be more efciently used, the rapid depletion of
resources can be mitigated and, at the same time, waste management
problems can be resolved.
An innovative source separating toilet can separate YW and BW to
facilitate further treatment, resource recovery and utilization. The collected YW can be properly treated for nutrient (nitrogen, phosphorus)
recovery in order to produce fertilizer and soil amendments (Sundin
et al., 1999). This can be another source of revenue. Alternatively, this
paper presents the potential alternative of using source separated BW
as a feed source for bio-energy production. AD system symbolizes a sustainable and low-cost technology for waste (water) treatment. Therefore, it is protable to apply AD within decentralized sanitation.
According to the waste statistics from Singapore's National Environment Agency (NEA), the annual generation of food waste (FW)
was 542,700 tonnes in 2006 and reached about 640,500 tonnes in
2010, which is around 10% of the total waste output in Singapore.
However, only 16% of FW was recycled and the rest of FW was sent
to waste-to-energy incineration plants. Organic waste presents more
difculties in recycling of FW because of the associated bad smell
and contamination caused by the organics. Singapore, a small island,
justiably nds this a big issue. Therefore, the recycling rate of FW
remains very low in Singapore, about 7%, over the last 20 years. The
recent NEA report shows that the recycling rate for FW has dropped
from 16% in 2010 to 10% in 2011 (Singapore waste statistics, 2011).
It is likely due to the cease of operation of a giant waste management
company in Singapore, last year, which was recycling FW into biogas
and compost. There is currently no news of the setting up of new
food waste recycling plants, nor is there any food waste reduction
campaign. This evidently shows that there is an alarming need for
the FW treatment and management in Singapore. Hence in this
study, household FW management and treatment has been given
adequate priority. In addition to BW, the AD system can also digest
kitchen organic-wastes, which will also improve the potential of the
utilization of biogas produced from the AD system, as kitchen organicwastes have a high-organic content.
The aim of this paper was to evaluate the technical feasibility of
anaerobic co-digestion of brown water (BW) [feces without urine]
and household food waste (FW) and to identify the key operating conditions governing the process performance. Special focus was put
on the determination of (i) biomethane potential of co-digestion of
BW and FW in a batch assay; (ii) anaerobic biodegradability of the
waste mixture (BW + FW); (iii) laboratory scale two-phase continuous stirred-tank reactor (CSTR) and single-stage sequencing-batch
operational mode reactor (SeqBR) performances and process efciencies; and (iv) microbial population in the various anaerobic system
congurations.

as well as disintegration of particulate organics. The blended FW was

then mixed well, and stored in a refrigerator at 4 C.
Brown water (BW) refers to fecal waste without urine. BW was
collected from a specially designed source-separation (no-mix) toilet
located in our laboratory, where urine and feces were collected in separate tanks. This system provides different options: 0.3 L of water per
urine-ushing (YW) and 2.0 L of water per fecal-matter-ushing
(BW). BW was collected once a week and stored in a refrigerator at
4 C.
Inoculation was carried out for both the CSTR and SeqBR systems using seed sludge collected from an anaerobic digester at
the Ulu Pandan sewage treatment plant, Singapore. The pH, average total solids (TS), volatile solids (VS) concentrations and VS/TS
of inoculum were in the range of 7.1, 24.9 g/L, 17.7 g/L and 0.71,
respectively.
2.2. Experimental set-up
2.2.1. Biochemical methane potential (BMP)
Bench-scale experiments for determining the anaerobic biodegradability and ultimate methane (CH4) potential of BW+ FW mixture
were carried out by using Automatic Methane Potential Test System
(AMPTS) [Bioprocess Control, Sweden]. AMPTS contains 15 identical
batch reactors of 500 mL capacity each with working liquid volume
of 400 mL. Each reactor was mechanically stirred (mixing time: 1 min
ON/1 min OFF) at 80 rpm (rotations per minute) and pH was not controlled in the system. Moreover, the biogas produced and methane content were measured online periodically using automated data logging
system. These reactors were incubated at 35 1 C and sparged with
nitrogen gas before sealing to create anaerobic conditions in all the
batch reactors. Variability may have originated from small differences
in acclimation to the new conditions, inoculum size, carryover of nutrients with the inoculums thus substrate controls were tested to exclude
extra BMP in the inoculum.
2.2.2. Laboratory-scale reactors
Anaerobic co-digestion of BW + FW mixture was performed in the
semi-continuously fed laboratory scale reactors viz. two-phase CSTR
and SeqBR (Fig. 1). Two-phase CSTR consists of acidogenic reactor of
1.2 L (working volume) followed by methanogenic reactor of 4.1 L
(working volume). The performance of the two-phase CSTR was compared with a single-stage SeqBR of 5.3 L (working volume). All the
systems were installed at a controlled-temperature room, adjusted
at a temperature of 33 1 C. These reactors were fed with source
separated BW (without urine) and FW mixture once a day. The reactor
contents of CSTR were mixed continuously (mixing time: 5 min ON/
5 min OFF) at 80 rpm using an overhead mechanical stirrer. Whereas,
SeqBR was operated in cycles, such that one cycle length consists
of 24 h, i.e. lling (1 h), reaction or mixing time (20 h); settling time
(2 h); draw (30 min) and the ideal phase (30 min). During the twophase CSTR operational phase, the methanogenic reactor was fed
with the acidied efuent from the acidogenic reactor. The reactors
were inoculated with anaerobic sludge (50% by volume) and then
gradually the reactor content was replaced by the BW + FW mixture.
2.3. Waste input sampling and analytical procedures

2. Materials and methods

2.1. Feedstock and inoculum sources
Food waste (FW) refers to leftover food. FW was collected once a
week from one of the canteens at NTU campus, where the majority
of the waste came from Chinese, Indian, Indonesian and Malay food
stalls. It was a mixture of meat, rice, noodles, vegetables and salad.
After bones and non-food materials were removed, the FW was then
crushed by a kitchen blender to promote homogeneity of the substrate

The composite samples of the shredded feedstock and efuents

from the two-phase CSTR and SeqBR were taken bi-weekly for characteristic analysis. pH was measured using a compact titrator (Mettler
Toledo) equipped with a pH probe (Mettler Toledo DGi 115-SC). Total
(TS) and volatile (VS) solids were analyzed according to the Standard
Methods (APHA, 1995). Total (CODt) and soluble (CODs) chemical
oxygen demand measurements were made using COD digestion vials
(Hach Chemical) and a spectrophotometer (DR/2800, Hach). CODs
measurements were made using the supernatant of samples after

R. Rajagopal et al. / Science of the Total Environment 443 (2013) 877886

879

Fig. 1. Scheme of experimental set-up for two-phase CSTR and single-stage SeqBR.

centrifugation (Kubota 3700, Japan) at 12,000 rpm for 20 min. The determination of volatile fatty acids (VFAs) was carried out using a gas
chromatograph (Agilent Technologies 7890A, USA), equipped with a
ame ionization detector (FID) and a DB-FFAP (Agilent Technologies,
USA) column (30 m 0.32 mm 0.50 m) and the samples were ltered through Membrane Solutions 0.45 m cellulose acetate membrane lters. Total biogas production was monitored daily using a
mass ow meter (McMillan Company, Model 50D-3E), while the biogas composition (methane, carbon dioxide and nitrogen contents)
was analyzed by gas chromatograph (Agilent Technologies 7890 A,
USA) equipped with a thermal conductivity detector (TCD). Carbon,
hydrogen, nitrogen and sulfur were measured using an elemental
analyzer (Vario EL Cube), and aqueous TOC was measured using a
TOC analyzer (TOC-V CSH, Shimadzu, Japan).
2.4. Anaerobic biodegradability of wastes
Organic matters in raw wastes can be symbolized with formulation of CaHbOcNd. Assuming all organic constituent in raw wastes
can be completely converted into CH4 and CO2, the theoretical methane production Y(CH4/sub)theor can be estimated using Eqs.(1) and (2)
(Sosnowski et al., 2003).
Ca Hb Oc Nd 4ab2c 3d=4 H2 O

the microbial populations in different reactor congurations.

Three sludge samples from acidogenic, methanogenic and SeqBR
reactors were pretreated respectively according to the protocol
described previously for FISH analyses (Amann et al., 1995). The
sample was xed in 4% paraformaldehyde solution overnight at
4 C.
Hybridization was carried out at 46 C for 3-h with hybridization
buffer containing 5 ng L 1 of specic uorescent probe. The oligonucleotide probes used in this study included EUBmix (i.e., EUB338,
EUB338-II, EUB338-III), targeting most of the members in the domain
Bacteria (Daims et al., 1999; Amann et al., 1995); and ARC915, targeting
most of the members in the domain Archaea (Amann et al., 1995). FISH
hybridization was performed under the stringency condition (formamide
concentration, 35%) for both probes (EUBmix and ARC915) in the hybridization buffer. Dual-staining FISH was performed, and probes were labeled with cyanine Cy3 and Cy5, respectively. FISH-stained images were
captured by an Olympus BX53 epiuorescence microscope equipped
with a cooled CCD camera DP72 with a 100 W halogen bulb and uorescence lter sets (U-FGW and U-FF-Cy5) under 100 objective lens
(Olympus, Japan).
3. Results and discussions
3.1. Operational scheme and the characteristics of feed

4a b2c3d=8 CH4 4ab 2c 3d=8CO2 dNH3



Y CH 4=sub

theor

4a b2c3d  2:8=12a b 16c 14d

The biological efciency of the anaerobic process is dened as the

ratio of experimental yield and theoretical one. Knowing experimental
values Y(CH4/sub)exp and theoretical Y(CH4/sub)theor it is possible to estimate the biological efciency or in other words anaerobic biodegradability by formula (Sobotka et al., 1983):


Biodegradability Y CH 4=sub

exp



=Y CH 4=sub

theor

 100%

2.5. Fluorescence in situ hybridization (FISH)

Towards the end of the operational period (on day 110), sludge
samples were collected in all the reactors primarily to understand

The feeding mixture of BW and FW was prepared bi-weekly and

subsequently fed to the reactors or stored at 4 C until needed for
feeding. The reactors were fed with BW + FW mixture every day in
a semi continuous mode. A life cycle assessment (LCA) report by
Remy (2010) suggests 160 g of FW per person per day as a good reference. Singaporeans seldom cook at home; therefore, an estimation
of 150 g is used for household FW in this study. BW amounts to
about 2 L (feces + ushing water). Based on this calculation, the feeding mixture used in this study contains 150 g of FW mixed with 2 L
of BW. For the biodegradability study, the synergy between BW and
FW was explored in terms of CH4 production and yield; and subsequently compared with that of FW or BW as a sole substrate, i.e. in
non-mixture conditions.
Fig. 2 presents the operational scheme implemented for the laboratory scale two-phase CSTR and SeqBR systems. Both systems were
initially fed at an organic loading rate (OLR) of about 1 gCOD/L d
and the acidogenic and methanogenic reactors were operated at a

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R. Rajagopal et al. / Science of the Total Environment 443 (2013) 877886

Fig. 2. Operational scheme for the laboratory scale reactors.

3.2. Biochemical methane potential (BMP) batch assay

The methane production during the 30 day batch incubation per
batch reactor per gVSadded for BW, FW and BW + FW mixture is
described in Fig. 3. Methane production rate was certainly showing
better results for the BW + FW mixture with a maximum methane
yield of 0.540.59 L CH4/gVSadded, while for the FW and BW in
non-mixture conditions, an average value of 0.400.42 and 0.26
0.30 L CH4/gVSadded was recorded, respectively. Biogas production
was nearly over within the rst 10 d of incubation period, however
longer HRT might be necessary for increased biogas recovery (Aoki
and Kawase, 1991). All the experiments performed well without
long start up times or inhibition phenomena. Anaerobic biodegradability was higher for the BW + FW mixture with 94% of CODs removal efciency measured on day 30. From the results, it can be seen

Table 1
Characteristics of BW, FW and the feed mixture.
Parameter

Unit

Brown water
(BW)

Food waste
(FW)

Feed mixture
(2-L BW + 150 g FW)

TS
VS
VS/TS
pH
CODt
CODs

[g/kg, wet basis]

[g/kg, wet basis]
[%]

[gCOD/L]
[gCOD/L]

4.4 0.06
3.8 0.08
87
6.7 2
8.2 0.6

295 1.5
280 1.5
94
4.4 1.5
394 14

18.26 3.49
16.9 3.19
93
6.18 0.55
35.21 10.41
13.02 4.36

VFA-COD
Acetic
Propionic
Isobutyric
Butyric
Isovaleric
Valeric

that co-digestion of these two substrates increased the biogas production rates and also improved the total biogas production. Some researchers (Elmitwalli et al., 2006; Nayono et al., 2010) have reported
that addition of FW had improved the biogas production, most probably
due to its higher lipid content. The daily FW loads given here are based
on average household Singaporean values but they might be different
if FW from hawker centers, food courts, restaurants are included. At
present, only 10% of FW is recycled in Singapore and the rest is sent to
waste-to-energy incineration plants. For maximal recovery and reuse
of resources, FW (from other sources) containing high levels of biodegradable organic matter can be integrated into the brown water stream.

3.3. Digester performance

3.3.1. Removal of organic fractions
In order to compare the performance of two different congurations, two-phase (acid + methane) CSTR and single-stage SeqBR were
operated in parallel at almost similar operating conditions. An example
of the results obtained for the CODs removal is presented in Fig. 4A and
the synopsis of results obtained for organic matter removals at different
HRT is given in Table 2.

0.7

Methane yield(L CH4/gVS fed)

hydraulic retention time (HRT) of 4 and 16 d, respectively. In order to

compare the performance, SeqBR was operated at 20 d HRT. Based on
the reactor performance, especially in terms of VFA production (in the
case of acidogenic reactor) and organic matter removal and methane
production (for other reactors), OLR was then increased by increasing
the feed ow rate and maintaining constant substrate concentrations.
The results presented in this paper are the preliminary results obtained
during the initial 120 d of operation. Table 1 shows the concentrations
in measured BW, FW and the feed mixture.

0.6
0.5
0.4
0.3
0.2
FW

0.1

BW

[gCOD/L]
[gCOD/L]
[gCOD/L]
[gCOD/L]
[gCOD/L]
[gCOD/L]

0.67 0.04
0.31 0.09
0.05 0.01
0.19 0.06
0.14 0.02
0.06 0.01

FW+BW

0.0
0

10

15

20

Time (d)
Fig. 3. Batch assays for methane yield.

25

30

R. Rajagopal et al. / Science of the Total Environment 443 (2013) 877886

881

HRT=16 d

HRT=20 -19 d

HRT=16 d

pH

6
HRT=20 d

4
HRT=4 d

HRT=3.5 d

HRT=2 d

30

50

HRT=3 d

3
0

10

20

40

60

70

80

90

100

110

120

Time (d)
Acid Reactor

Methane Reactor

SeqBR

Fig. 4. A. CODs removals at different HRT in two-phase CSTR and SeqBR. B. pH variations at different HRT in methanogenic CSTR and SeqBR. C. TVFA-COD variations at different HRT
in methanogenic CSTR and SeqBR.

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R. Rajagopal et al. / Science of the Total Environment 443 (2013) 877886

Table 2
Removal of organic matters at different HRT's.
Reactors

Two-phase CSTR

SeqBR

HRT
(d)

Duration
(d)

2019
16
20
2019
16

040
4195
96120
040
41120

Removal efciency (% removal)

CODt

CODs

TS

VS

TOCs

51.9 11.7
69.6 11.0
68.4 6.4
69.0 13.3
76.7 5.1

93.4 3.9
84.2 13.5
75.9 1.1
92.9 2.5
92.0 3.0

61.3 5.6
57.6 8.1
62.7 0.4
53.4 8.6
70.9 6.5

69.4 5.3
63.4 8.9
68.6 2.0
63.8 7.0
75.7 6.6

63.2 9.0
65.5 4.3

86.3 2.9

Long HRT and an appropriate choice of inoculum at the start-up of

reactor operation allowed for a high-stabilization of treated efuent
(up to 90%). Until 40 d of operation, the HRT was maintained at 19
20 d for both systems. It is to be noted that, during this operational
period, HRT of acidogenic CSTR corresponded to about 4 d and that
of methanogenic reactor was 16 d (Fig. 2). The efuent quality in
terms of organic fraction was almost similar in both congurations.
From day 41 onwards, the HRT of SeqBR and two-phase CSTR were
reduced to about 16 d, which corresponded to 23 and 1314 d of
HRT for the acidogenic and methanogenic CSTRs, respectively. Due
to the partial acidication in the rst (acidogenic) reactor, we have
attempted to lower the HRT of the methanogenic reactor. However,
a decrease in HRT resulted in a drop in the performance of methanogenic reactor in terms of pH drop (Fig. 4B) and VFA accumulation
(Fig. 4C) probably due to the washout of active biomass. From
Fig. 4A, it is evident that CODs removal efciencies went as low as
60% (CODt removal of 55%) for a period of 50 d operation (from day
4190), i.e. more than 3 times the HRT; whereas SeqBR was able
to accumulate more solids within the reactor and showed better
removal of organics (Table 2). It is signicant to note that effective
sedimentation occurred during the study period and no otation
along with subsequent biomass washout was observed in the SeqBR.
pH was not controlled in the methanogenic reactor and hence to
recover the digester performance or to avoid further acidication,
on day 96, HRT was increased back to 1617 d for the methanogenic
reactor, which corresponded to 20 d HRT for the entire two-phase
CSTR system in contrast to 16 d for SeqBR. Towards the end, the
obtained efuent quality corresponded to a CODt removal efciency of
68.46.4 and 76.75.1%, respectively for two-phase CSTR (20 d HRT)
and SeqBR (16 d HRT); while the CODs was removed in 75.91.1 and
92.03.0%, respectively.
Soluble organic carbon concentration was measured in the efuent
of all the reactors. About 85% removal occurred in SeqBR, which was
quite consistent throughout the study. TS and VS removal of more
than 70% was observed in the efuent of SeqBR (Table 2), which also
shows that the sequencing-batch operational mode has resulted in
accumulating more biomass within the system; thereby the process
increased the sludge age compared to CSTR. An average methane
yield of about 0.41 L CH4/gVSadded was recorded for the SeqBR during
this operation period (Table 2). Methane content of 6065% was
observed in the total biogas.

3.3.2. Anaerobic biodegradability of the waste mixture using biological

stoichiometry
The results of elemental compositions of FW are summarized in
Table 3. Organic matter in raw BW + FW mixture is represented with

Methane yield
(L CH4/gVS)

0.400.21

0.370.46

formulation of C13.4H34.5O8.8N. Based on Eqs. (1) and (2), theoretical

methane production Y(CH4/sub)theor for BW + FW mixture can be estimated to be 0.59 0.2 L CH4/gVSadded. For the laboratory scale studies,
the biodegradability of BW + FW mixture was estimated to be 0.70
(Table 3). It is signicant to note that the biochemical methane potential study using AMPTS system showed similar results compared with
that of biological stoichiometry calculations. However, for the laboratory scale reactors lower anaerobic degradability (0.70) was obtained.
In this case, biogas production could probably be underestimated due
to technical issues, while measuring the volume of biogas. The C/N
ratio for BW + FW mixture is lower than the numbers (15.6 and
17.2) reported in literatures for the fruit and vegetable waste, and
food waste, respectively (Lin et al., 2011). The low C/N of BW + FW
mixture implied that they contain a large quantity of nitrogen, mainly
in organic forms, such as proteins.
3.3.3. pH and VFA concentrations during digestion
Fig. 4(B and C) shows the pH and total VFA (TVFA)COD concentrations in the methanogenic CSTR and SeqBR. The efcient and
stable performance of these congurations tested at 20 d HRT was
further corroborated by the low concentrations of VFA's (as criteria
for the stability of the anaerobic process) and stable pH of around 7.
The lower efuent VFA concentrations (less than 1 gCOD/L) in the
methanogenic reactor show that methanogens were capable of consuming all VFAs produced. Once the HRT of the methanogenic reactor
was decreased from 16 to 13 d, VFACOD concentrations were started
accumulating gradually in this reactor (Fig. 4C). TVFACOD concentrations increased from less than 1 gCOD/L to a maximum of 4 gCOD/L on
day 85, of which more than 50% comprised of propionic acid. The buffering potential of methanogenic CSTR dropped down due to the accumulation of acids especially at lower HRT (13 d), which resulted in a
pH drop up to 5.5 (Fig. 4B). However, once we increased the HRT of
the methanogenic reactor to about 1617 d, pH value was augmented
as shown in Fig. 4B. For the initial 120 d of operation, SeqBR did not
show much variation in the VFA accumulation (less than 0.8 gCOD/L
in all cases) and was able to maintain stable pH of 6.9 0.1.
3.3.4. Acidogenic reactor performance
The primary goal of hydrolysis and acid fermentation is the solubilization of particulate organic fraction in the feed mixture during the
treatment process. Sludge hydrolysis can be expressed by the changes
of CODs concentrations and VFA productions (Rajagopal and Bline,
2011). HRT of acidogenic reactor varied from 4 to 2 d as shown in
Fig. 2. However, to ensure the process stability and prevent propionic
acid accumulation, the HRT was maintained around 3 d from day 57
onwards. No other pretreatment or control techniques were followed

Table 3
Elemental compositions, C/N ratio, and theoretical methane production of BW + FW.
Waste

BW + FW

Elemental compositions (wt.% TS)

C

45 (0.6)

9.7 (0.8)

39.5 (0.9)

3.9 (0.1)

Values in parenthesis () indicate standard deviation.

C/N

TS g/kg,
wet basis

VS g/kg,
wet basis

Y(CH4/sub)theor
L CH4/gVS

Y (CH4/sub)exp
L CH4/gVS

Biodegradability
(%)

11.5 (0.7)

18.3 (3.4)

16.9 (3.2)

0.59 (0.02)

0.41

70

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to prevent methanogenesis during the hydrolysis and acidication

process.
Inuent COD is anaerobically converted to CH4COD, efuent COD
and sludge or biomass COD. The efuent COD can be differentiated
into to VFA and non VFACOD. VFACOD is the intermediate COD in
the conversion of inuent COD to CH4COD. Fig. 5A gives the feedstock CODs, VFACOD and non VFA-COD concentrations during the
operational period, whereas, Fig. 5B presents the efuent data
pertaining to CODs, pH, VFACOD and non VFACOD concentrations.
It is shown that about 87% of the CODs comprised non VFACOD in
the feedstock, which is quite dominant compared to that of VFA
COD. It is interesting to note that major CODs production has not

A
25
20
15
10
5
0

10

20

30

40

50

60

70

80

90 100 110 120

Time (d)
Inf CODs, g/L

Inf non TVFA-COD, g/L

Inf TVFA-COD, g/L

B
18
16
14
12
10
8
6
4
2
0
0

10

20

30

40

50

60

70

80

90

100 110 120

Time (d)
Eff CODs, g/L

Eff non TVFA-COD, g/L

Acid Reactor PH

Eff TVFA-COD, g/L

7
C2

C3

iC4

C4

iC5

C5

iC6

C6

VFA g COD/L

5
4
3
2
1

10

20

30

40

50

60

70

80

taken place during the studied HRTs. However, a signicant conversion of non VFACOD to VFACOD has occurred at an HRT of 34 d.
That is to say, VFACOD conversion from 1.6 0.6 g/L to a maximum
value of 13.3 g/L (average: 9.0 2.1 g/L) has obtained (Fig. 5B).
As this reactor was started with 50% of anaerobic sludge, it
took about 2 times HRT to stabilize the pH. BW was preserved in
4 C storage room for more than 2 months before the experiments
were started. The initial CODs values (23 g/L) show that these wastes
have lost some amount of carbon through hydrolysis process during
the storage period at 4 C. This probably explains the lower production of soluble COD fractions during the initial 14 d of hydrolysis and
acidication process. In this study, short-chain fatty acids (SCFAs) i.e.
acetic (C2), propionic (C3), butyric (C4), iso-butyric (iC4), valeric (C5),
iso-valeric (iC5), caproic (C6), iso-caproic (C6) and heptanoic (C7) were
analyzed and are presented in Fig. 5C. The main acidication products
at HRT of 3 d (values calculated from day 80120) were C4 (376.0%
of TVFA), C5 (23.51.2% of TVFA), C2 (171.6% of TVFA) and C3
(151.9% of TVFA) comprising 94% of TVFAs. The higher molecular
weight VFAs (iC5, C6 and C7 etc.) were produced in insignicant amounts.
Butyric acid, which had the highest concentration of all the VFAs,
was produced from the start of the experiment and propionic acid
seemed to be higher especially at HRT of 2 d. The phenomenon of
high butyric acid production relative to other VFAs in this study is consistent with the report of Vogt and Wolever (2003) that butyrate acid
bacterium are present in the human colon as they are the primary fuel
for human colonocytes and thus it is natural for them to be present in
human feces. BW used in this study as a co-substrate could probably
have played a role in the higher concentration of butyric acid.
Biogas measurements show that methane production was completely inhibited at pH of 4.2. Low pH did not really affect the VFA
production, however about 910% of H2 gas was observed during
this phase of study. All the SCFAs produced from the acidogenic reactor were consumed by the methanogens present in the methanogenic
reactor at HRT of 1617 d. In particular, butyrate removal was almost
complete at this HRT, and no inhibitory effects of this acid were
observed in the methanogenic reactor. However, when we tried to
decrease the HRT of methanogenic reactor from 16 to 13 d, propionic
acid concentrations started accumulating in this reactor.
The propionate as expected exhibited more severe inhibition than
butyrate and acetate in the methanogenic reactor. Butyrate feed can
be degraded more competently than the others because of its high
energy gain via degradation. In contrast, acetate can be regarded as
an efcient substrate to exhibit a high methane yield because of its
one-step degradation (Wong et al., 2008). The excess of VFA built up
in the digester caused a drop in pH and inhibition of the methanogenic
process. Hence to improve the process stability of the methanogenic
reactor, HRT was maintained at 1617 d for this reactor.

C7

883

90 100 110 120

Fig. 5. A Inuent CODs, VFA-COD, Non-VFA COD concentrations prole. B. pH, CODs,
VFA-COD, Non-VFA COD concentration prole in acidogenic CSTR. C. Evolution of VFA
compositions in acidogenic CSTR.

3.3.5. Microbial populations revealed by FISH

The microbial populations in the reactors towards the end of study
period were further investigated to understand their responses to
different anaerobic system congurations. Fig. 6 (A,B and C) shows
the FISH analysis of Bacteria (green) and Archaea (i.e., methanogens)
(red) populations in the three reactors' sludge samples, respectively.
In the acidogenic reactor of two-phase system, only bacterial cells
were detected (Fig. 6A). High bacterial diversity with different morphologies of bacteria including small rods, fat rods, ovals, cocci, and thin laments was observed. Absence of methanogen in the acidogenic reactor
might be due to the unfavorable low pH value for its growth. Additionally, a mixed structure was observed based on the random distribution
of bacterial (green) and archaeal/methanogen (red) populations with
approximately equal abundance in methanogenic reactor of two-phase
CSTR and single-stage SeqBR reactors (Fig. 6B and C).
The presence of methanogens well agreed with the observation of
methane production (Table 3). For the methanogens, both aceticlastic
methanogens bamboo-shaped Methanosaeta-like and coccoid-cluster

884

R. Rajagopal et al. / Science of the Total Environment 443 (2013) 877886

VFA was accumulated (Fig. 4C). Vavilin et al. (2008) reported that
Methanosarcina sp. forming multicellular aggregate may resist inhibition by VFA because a slow diffusion rate of the acids limited the VFA
concentrations inside the Methanosarcina sp. aggregates. Despite microbial communities have been revealed by FISH observation, both bacterial and archaeal populations in these reactors remain to be further
identied and characterized.
3.4. Overall comparison

Fig. 6. FISH analyses of the three sludge samples. Samples were hybridized with
Cy3-labeled ARC915 specic for the domain Archaea (red) and Cy5-labeled EUBmix
probe specic for the domain Bacteria (green). Panels (A), (B), and (C) were samples
acidogenic reactor, methanogenic reactor, and SeqBR reactor, respectively.

Methanosarcinacea-like populations were observed in methanogenic

reactor of two-phase system (Fig. 6B). However, Methanosaeta-like
cells were dominant in the SeqBR (Fig. 6C). Generally, most of the
methane produced in an anaerobic bioreactor is mainly derived from
acetate (Zinder, 1993). Methanosaeta and Methanosarcina are the two
known genera that can produce methane from acetate. Methanosaeta
use only acetate and generally have low values for the minimum
threshold and maximum specic growth rate of microorganisms
(max). Besides using acetate, Methanosarcina is metabolically more
versatile than Methanosaeta, as it can form methane from H2 and
CO2 (hydrogenotroph), methanol, methylamines and methyl suldes
(methylotroph); generally have high minimum threshold and max
values for acetate (Zinder, 1993).
The presence of Methanosarcinacea-like populations in methanogenic reactor of two-phase system might be related to the system's conditions while the sampling time (day 110) pH was low (Fig. 4B) and the

Table 4 presents the overall performance data of the present study

together with similar parameter values in other literatures. In most of
the studies listed in Table 4, co-digestion of black water and kitchen
refuse was experimented in various anaerobic systems such as CSTR,
accumulation System (AC) and upow anaerobic sludge blanket reactor (UASB)-septic tank. The comparisons are intrinsically hard to do as
the data pertaining to brown water is lacking and also the rector operating conditions were different. However, SeqBR reactor has attained
the highest performance with relatively high organic loading rate
(23 gCOD/L d) and short HRT (16 d), with a corresponding COD removal of above 90% (CODs) and 75% (CODt). From the results of
batch assay (Fig. 3), it is to be noted that the maximum biodegradation
took place within 10 d of HRT for BW + FW mixture. Further optimization will be necessary to validate and improve the performance of
SeqBR at relatively short HRT's.
Furthermore, economic cost analysis for larger scale bioreactors
will be crucial to provide SeqBR with a sound basis for practical application. Overall, this study veries that (i) SeqBR system offers a reliable and effective biogas recovery as well as waste stabilization by
combining co-digestion and (ii) BW + FW are potential substrates
for biogas production. Co-digestion with high organic contents such
as FW could be a reliable option to enhance activity of anaerobic microorganisms. Proper mixture brings synergistic and complementary
effects, which offsets the lack of available nutrients for methane production in brown water and dilute harmful or excessive substances
inhibiting anaerobes in FW (Kim et al., 2007). In addition, some FW
has the tendency to acidify rapidly or lower the pH of the digester
solution. This study demonstrated that BW provided a sufcient buffering capacity to the FW digestion by synergizing the effect of microorganisms and handling high organic load. The comparison was made
between the co-digestion of BW + FW and digestion of FW or BW as a
sole substrate. From Fig. 3, it was demonstrated that for a given load
in a batch study, BW + FW co-digestion has attained more methane
yield than FW or BW digestion. For co-digestion, laboratory scale
CSTR shows no acidication at an HRT more than 20 d and OLR of
~ 1.5 g COD/L d. When we tried to increase the OLR of two-phase
CSTR to 23 gCOD/L.d, which corresponded to an HRT of 16 d, we
have observed the acidication as given in Fig. 4B.
3.5. Final discussion based on decentralized, source-separation-based
sanitation concepts
The decentralized sanitation and reuse concept is a logical source
separation-based approach that ts well in a sustainable development compared to the current centralized wastewater treatment
plants (Kujawa-Roeleveld et al., 2006). In its simple form it may constitute an answer to solve the problem of the lack of any sanitation in
poor, less developed parts of the world. For richer countries, with
their complex sanitation infrastructure, it may be the answer when
replacing existing, old infrastructures or when building new housing
estates or larger utility objects such as hotels, ofces, etc. (STOWA,
2005). There is no universal decentralized sanitation and reuse concept tting each situation. The common element for any decentralized
sanitation and reuse scenario is the separation of organic waste and
wastewater streams at the source (i.e. on a household level) followed
by an appropriate treatment of each stream in decentralized/semi

Table 4
Overall comparison cited in the literature.
Sl No. Reactor

CSTR (10 L) Bl. W (5 L/d)+

KR (200 g/d)
AC
Bl. W with urine+KR

AC

AC and
UASB-septic
tank

Inuent values (g/L)

Operating conditions

CODt

CODs

VS

VFA

19.2 6.5

6.8 1.05

8.8 3.9

12.0 4.37 3.04 0.21

Bl. W less urine + KR 45.2 24.6 3.97 1.3

Bl. W with urine+KR 18.7

Bl. W less urine + KR 53.6
Bl. W
9.512.3

2.76
10.14
1.42.8

Bl. W + KR

13.322.9

2.75.4

BW+ KR

31.866.0

4.010.1

2.0 1.21

UASB-septic
tank

Bl. W + KR

12.3 7.8

Two-phase

BW+ FW

35.2 10.4 13.0 4.4

SeqBR

20/15/10 HRT

1.66 0.94 Pilot-scale study,

at 20 C
3.09 1.9
1.9
105 d, at 20 C
8.89
0.76 0.64 AC (Black + KR):
20 C, 115150 HRT

AC(Bl. W+KR):
15/20 C, 115280 HRT

UASB(BW+KR):
15/25 C, 2729 HRT

(1) (0.2 m3) pilot scale,

(2) 25 C,
(3) OLR=0.42 gCOD/L d,
(4) HRT=29 d

16.9 3.2 1.12.1

HRT = 20 d, OLR ~
1.5 gCOD/L d
HRT = 16 d, OLR ~
23 gCOD/L d

Efuent values (% removal)

Efuent VFA
(gCOD/L)

Remarks

References

2.60

270 L/280 L/205 L CH4

per kg COD
Methanization = 34.3%.
biogas yield = 26.5 L/p/d
Methanization = 61%.
biogas yield = 50.8 L/p/d
85% biodegradability

Wendland et al.,
2007
Kujawa-Roeleveld
et al., 2003

KF was added at the end

of the operational period
(after 1 year) to evaluate
its short term effect on
the process performance.
Methane yield = 0.4
0.21 L CH4/gVSadded
Methane yield = 0.37
0.46 L CH4/gVSadded

Kujawa-Roeleveld
et al., 2005

CODt

CODs

VS

71 13/75
7/50 15

67 15/67
13/53 22

65 20/69
12/51 21

Day 43: 3.003;

Day 80 onwards:
0.083

Avg. 58%
Avg. 58%
78

30

0.53 0.16

61

82

94

68.4 6.4

75.9 1.1

68.6 2.0

23

76.7 5.1

92.0 3.0

75.7 6.6

b0.8

Elmitwalli et al.,
2006
28 L CH4 (66%) per person Kujawa-Roeleveld
daily; 101 kWh per person et al., 2006
annually

Present study
Present study

R. Rajagopal et al. / Science of the Total Environment 443 (2013) 877886

Substrate

CSTR: continuous stirred tank reactor; AC: accumulation system; UASB: upow anaerobic sludge blanket reactor.
Bl.W: black water; BW: brown water; KF: kitchen refuse; FW: food waste.

885

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R. Rajagopal et al. / Science of the Total Environment 443 (2013) 877886

centralized systems and consequent reuse of water and nutrients

(Wendland et al., 2007). In our study wastewater streams are separated
according to their degree and type of pollution and re-use potential
of resources. Three main resources are considered: bio-energy (from
transformation of organic material), nutrients (nitrogen, phosphorus,
potassium and sulfur) and water (from advanced treatment of cleaner
wastewater streams). To achieve this, a no-mix toilet has been designed
and installed in our laboratory for our research purposes. This toilet
diverts liquid waste (YW) to a processing facility where components
used for fertilizer can be recovered (data not shown). The solid waste
(BW) is sent to a bioreactor where it is digested to generate biogas.
In this case, daily ushing water consumption will be dramatically reduced from currently 30 L per capita to 5.56.0 L per capita, indicating
a saving of 80% ushing water use. Besides, when the amount of ushing water is reduced, the volume production of toilet wastewater to be
transported and treated will automatically decrease.
With rapid increase in population density in Singapore and the fact
that more than 80% of the population live in the high-rise Housing
Development Board (HDB) buildings, the proposed decentralized AD
system is expected to provide a practical solution for managing large
quantities of BW and FW on-site. The well-established infrastructure
for sewer, waste collection and transportation are readily available
for simple modications to transfer the concentrated wastes directly
to decentralized co-digestion systems. This, together with the high
potential for producing a substantial amount of energy in the form of
biogas, makes the proposed decentralized AD concept economically
realistic. Building AD systems in HDB ats for on-site waste treatment
and methane production will become an eco-friendly model for other
countries, especially those experiencing rapid urbanization and water
shortage. The challenge is in designing a space-friendly, odor and
pathogen-controlled AD system that can be incorporated into new
and existing residential clusters. Based on our characteristics analysis
of BW and FW, it is estimated that 18.540 kg of VS are generated
through these waste streams per HDB block with 400 residents on average, which can yield 615 (avg. 10) m 3 biogas when 65% VS removal
is achieved (Speece, 1996; ACEEE, 2009). This indicates a daily electricity generation of 12.533.6 kWh from biogas, on the assumption
that the generator efciency is 3550%. In addition, a daily heat energy
prot of 17.846.5 kWh from biogas can be estimated. Generally, a
total energy prot of 0.080.20 kWh per person per day can be
expected. The waste-originated energy can be utilized, for example,
for cooking or lighting purposes within the HDB premises. Other advantages may include that (i) digestate can be used as bio-fertilizer
within the parks around, (ii) additional costs for transporting such
wastes to the centralized treatment systems are saved since the
wastes produced from HDB buildings are totally managed within the
site of production, and (iii) the life span of landlls can be signicantly
extended by minimizing the needs for disposal.
4. Conclusions
This study demonstrated that anaerobic co-digestion of BW and
FW proved to be a potential substrate for biogas production. Anaerobic
co-digestion showed higher methane yield than BW and FW in nonmixture conditions. SeqBR allowed a balanced conversion of organics
to CH4 at an OLR of 23 gCOD/L d that corresponds to a VS removal
of about 75%. BW provided a sufcient buffering capacity to the FW
digestion. We believe decentralized and source-separation-based
sanitation concepts can eventually be introduced to other cities
around the world. It would be especially important for mega cities
that are beginning to emerge in the next few years.
Acknowledgments
The authors are grateful to the National Research Foundation (NRF),
Singapore for nancial support (NRF-CRP5-2009-02). We appreciate

Mr. B. Wang, Mr. Bernard Ng and Mr. G.W.H. Chia for their helping
hands and cooperation in the experimental work. We are grateful to
Prof. Rainer Stegmann and Prof. C.S. Lee for stimulating discussions in
this project. We are thankful to R3C/NTU family for their contributions
to this research program.
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