artigo_anaerobic co-digestion of source segregated brown water (feces-without-urine) and food waste

8/18/2019 Artigo_Anaerobic Co-digestion of Source Segregated Brown Water (Feces-without-urine) and Food Waste

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Anaerobic co-digestion of source segregated brown water (feces-without-urine) andfood 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 Singaporeb School of Civil and Environmental Engineering, Nanyang Technological University, 50 Nanyang Avenue, 639798 Singaporec School of Energy and Environmental Sciences, Yunnan Normal University, 121 Street, Kunming 650092 China

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 b s t r a c ta 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)

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

hasbeenmade toseparatethe yellow water(urine) andbrown waterfromthe source(using no-mix toilet) primar-

ily to facilitate further treatment, resource recovery and utilization. Batch assay analytical results indicated that

anaerobic co-digestion [BW+FW] showed higher methane yield (0.54–0.59 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 ofoperation showsthat SeqBR performedbetter intermsof organic matterremovaland max-

imum methane production. At steady-state, CODs, CODt, VS removals of 92.0± 3.0, 76.7± 5.1 and 75.7± 6.6% were

achieved forSeqBR at 16 d HRT, respectively. This corresponds to an OLR of 2–3 gCOD/L d and methane yield

of about 0.41 L CH4/gVSadded. Good buffering capacity did not lead to accumulation of VFA, showing bet-

ter 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], rep-

resents a sustainable and future solution for waste (water) treatment

(Elmitwalli et al., 2006). Theseparation 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 ef uent 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

Science of the Total Environment 443 (2013) 877 –886

⁎ 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 819780 7303; fax: +1 8195645 507.

E-mail addresses: [email protected], [email protected]

(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

Contents lists available at SciVerse ScienceDirect

Science of the Total Environment

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / s c i t o t e n v

http://dx.doi.org/10.1016/j.scitotenv.2012.11.016http://dx.doi.org/10.1016/j.scitotenv.2012.11.016http://dx.doi.org/10.1016/j.scitotenv.2012.11.016mailto:[email protected]:[email protected]://dx.doi.org/10.1016/j.scitotenv.2012.11.016http://www.sciencedirect.com/science/journal/00489697http://www.sciencedirect.com/science/journal/00489697http://dx.doi.org/10.1016/j.scitotenv.2012.11.016mailto:[email protected]:[email protected]://dx.doi.org/10.1016/j.scitotenv.2012.11.016


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decentralized sanitation systems. However, source separation between

feces and urine, and itssubsequent 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 ef ciently

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 resourcescan be more ef ciently used, therapiddepletion 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 col-

lected 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 systemsymbolizes a sus-

tainable and low-cost technology for waste (water) treatment. There-

fore, it is protable to apply AD within decentralized sanitation.

According to the waste statistics from Singapore's National Envi-

ronment 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

dif culties 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 forthe 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 organic-

wastes 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]

andhousehold food waste (FW) and to identify thekey operatingcon-

ditions 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 continu-

ous stirred-tank reactor (CSTR) and single-stage sequencing-batchoperational mode reactor (SeqBR) performances and process ef cien-

cies; and (iv) microbial population in the various anaerobic system

congurations.

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 homogeneityof thesubstrate

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 andfeceswere collected in sep-

arate 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 sys-

tems using seed sludge collected from an anaerobic digester at

the Ulu Pandan sewage treatment plant, Singapore. The pH, aver-

age 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 biodegrad-

ability 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 con-

trolled in the system. Moreover, the biogas produced and methane con-

tent 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 nutri-

ents 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 com-

pared 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

separatedBW (without urine)and FW mixture once a day. Thereactor

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 two-

phase CSTR operational phase, the methanogenic reactor was fed

with the acidied ef uent from the acidogenic reactor. The reactorswere 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

The composite samples of the shredded feedstock and ef uents

from the two-phase CSTR and SeqBR were taken bi-weekly for char-

acteristic 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

878 R. Rajagopal et al. / Science of the Total Environment 443 (2013) 877 –886


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centrifugation(Kubota 3700, Japan)at 12,000 rpmfor 20 min. Thede-

termination 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 μ m) and the samples were l-

tered through Membrane Solutions 0.45 μ m cellulose acetate mem-

brane lters. Total biogas production was monitored daily using a

mass ow meter (McMillan Company, Model 50D-3E), while the bio-

gas 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 formula-

tion of CaHbOc Nd. Assuming all organic constituent in raw wastes

can be completely converted into CH4 and CO2, the theoretical meth-

ane production Y(CH 4/sub)theor can be estimated using Eqs.(1) and (2)

(Sosnowski et al., 2003).

CaHbOc Nd þ 4a−b−2c þ 3dð Þ=4 H2O

→ 4a þ b−2c −3dð Þ=8 CH4 þ 4a−b þ 2c þ 3dð Þ=8CO2 þ dNH3

ð1Þ

Y CH 4=sub

theor

¼ 4a þ b−2c −3dð Þ 2:8=12a þ b þ 16c þ 14d ð2Þ

The biological ef ciency of the anaerobic process is dened as the

ratio of experimental yield and theoretical one. Knowing experimental

values Y(CH 4/sub)exp and theoretical Y(CH 4/sub)theor it is possible to esti-

mate the biological ef ciency or in other words anaerobic biodegrad-

ability by formula (Sobotka et al., 1983):

Biodegradability ¼ Y CH 4=sub

exp

=Y CH 4=sub

theor

100 % ð3Þ

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 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 μ L −1 of specic uorescent probe. The oligo-

nucleotide 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

hybridizationwas performed under the stringencycondition (formamide

concentration, 35%) for both probes (EUBmix and ARC915) in the hybrid-ization buffer. Dual-staining FISH was performed, and probes were la-

beled 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 uo-

rescence 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

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 ina semi continuous mode. A life cycle assessment (LCA) report by

Remy (2010) suggests 160 g of FW per person per day as a good ref-

erence. 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 feed-

ing 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 subse-

quently 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 labo-

ratory 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

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

879R. Rajagopal et al. / Science of the Total Environment 443 (2013) 877 –886


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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.

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 methaneyield of 0.54–0.59 L CH4/gVSadded, while for the FW and BW in

non-mixture conditions, an average value of 0.40–0.42 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 biodegrad-

ability was higher for the BW+FW mixture with 94% of CODs re-

moval ef ciency measured on day 30. From the results, it can be seen

that co-digestion of these two substrates increased the biogas produc-

tion rates and also improved the total biogas production. Some re-

searchers (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 biode-

gradable organic matter can be integrated into the brown water stream.

3.3. Digester performance

3.3.1. Removal of organic fractionsIn order to compare the performance of two different congura-

tions, 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 resultsobtained for organic matter removals at different

HRT is given in Table 2.

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

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 [g/kg, wet basis] 4.4 ± 0. 06 295 ± 1.5 18.26 ± 3.49

VS [g/kg, wet basis] 3.8 ± 0. 08 280 ± 1.5 16.9 ± 3.19

VS/TS [%] 87 94 93

pH – 6.7 ±2 4.4 ±1.5 6.18 ±0.55

CODt [gCOD/L] 8.2 ±0.6 394 ±14 35.21 ±10.41

CODs [gCOD/L] – – 13.02±4.36

VFA-COD

A cetic [gC OD/L] – – 0.67±0.04

Propionic [gCOD/L] – – 0.31±0.09

Isobutyric [gCOD/L] – – 0.05±0.01

Butyric [gCOD/L] – – 0.19±0.06

Isovaleric [gCOD/L] – – 0.14±0.02

Valeric [gCOD/L] – – 0.06±0.01

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 5 10 15 20 25 30

FW

BW

FW+BW

M e t h a n e y i e l d ( L C H 4 / g V S f e d )

Time (d)

Fig. 3. Batch assays for methane yield.


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3

4

5

6

7

8

0 10 20 30 40 50 60 70 80 90 100 110 120

p H

Time (d)

Acid Reactor Methane Reactor SeqBR

A

B

C

HRT=20-19 d HRT=16 d

HRT=4 d HRT=3.5 d HRT=2 d HRT=3 d

HRT=16 d

HRT=20 d

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

reactor operation allowed for a high-stabilization of treated ef uent

(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 ef uent 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 2 –3 and 13–14 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 methan-

ogenic 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 ef ciencies went as low as

60% (CODt removal of 55%) for a period of 50 d operation (from day

41–90), 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 16 –17 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, theobtained ef uent quality corresponded to a CODt removal ef ciency of

68.4±6.4 and 76.7±5.1%, respectively for two-phase CSTR (20 d HRT)

and SeqBR (16 d HRT); while the CODs was removed in 75.9± 1.1 and

92.0±3.0%, respectively.

Soluble organic carbonconcentration wasmeasured in theef uent

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 ef uent 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 60–65% 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

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

methane production Y(CH 4/sub)theor for BW+FW mixture can be esti-

mated to be0.59±0.2 L CH4/gVSadded. Forthe laboratory scalestudies,

the biodegradability of BW+FW mixture was estimated to be 0.70

(Table 3).It issignicant to note that the biochemical methane poten-

tial study using AMPTS system showed similar results compared with

that of biological stoichiometry calculations. However, for the labora-

tory 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 con-

centrations in the methanogenic CSTR and SeqBR. The ef cient 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 ef uent VFA concentrations (less than 1 gCOD/L) in the

methanogenic reactor show that methanogens were capable of con-

suming all VFAs produced. Once the HRT of the methanogenic reactor

was decreasedfrom 16 to 13 d, VFA–COD concentrations were started

accumulating gradually in this reactor (Fig.4C).TVFA–COD concentra-

tions increased from less than 1 gCOD/L to a maximum of 4 gCOD/L on

day 85,of which more than 50%comprised of propionicacid. Thebuff-

ering potential of methanogenic CSTR dropped down due to the accu-

mulation 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 16–17 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 solubi-

lization of particulate organic fraction in the feed mixture during thetreatment process. Sludge hydrolysis can be expressed by the changes

of CODs concentrations and VFA productions (Rajagopal and Béline,

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 2

Removal of organic matters at different HRT's.

Reactors HRT

(d)

Duration

(d)

Removal ef ciency (% removal) Methane yield

(L CH4/gVS)CODt CODs TS VS TOCs

Two-phase CSTR 20–19 0–40 51.9 ±11.7 93.4 ±3.9 61.3 ±5.6 69.4 ±5.3 – –

16 41–95 69.6 ±11.0 84.2 ±13.5 57.6 ±8.1 63.4 ±8.9 63.2 ±9.0 0.40–0.21

20 96–120 68.4 ±6.4 75.9 ±1.1 62.7 ±0.4 68.6 ±2.0 65.5 ±4.3

SeqBR 20–19 0–40 69.0 ±13.3 92.9 ±2.5 53.4 ±8.6 63.8 ±7.0 – –

16 41–120 76.7 ±5.1 92.0 ±3.0 70.9 ±6.5 75.7 ±6.6 86.3 ±2.9 0.37–0.46

Table 3

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

Waste Elemental compositions (wt.% TS) C/N TS g/kg,

wet basis

VS g/kg,

wet basis

Y(CH 4/sub)theor L CH4/gVS

Y (CH 4/sub)expL CH4/gVS

Biodegradability

(%)C H O N

BW +FW 45 (0.6) 9.7 (0.8) 39.5 (0.9) 3.9 (0.1) 11.5 (0.7) 18.3 (3.4) 16.9 (3.2) 0.59 (0.02) 0.41 70

Values in parenthesis () indicate standard deviation.



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to prevent methanogenesis during the hydrolysis and acidication

process.

Inuent COD is anaerobically converted to CH4–COD, ef uent COD

and sludge or biomass COD. The ef uent COD can be differentiated

into to VFA and non VFA–COD. VFA–COD is the intermediate COD in

the conversion of inuent COD to CH4–COD. Fig. 5A gives the feed-

stock CODs, VFA–COD and non VFA-COD concentrations during the

operational period, whereas, Fig. 5B presents the ef uent data

pertaining to CODs, pH, VFA–

COD and non VFA–

COD concentrations.It is shown that about 87% of the CODs comprised non VFA–COD in

the feedstock, which is quite dominant compared to that of VFA–

COD. It is interesting to note that major CODs production has not

taken place during the studied HRTs. However, a signicant conver-

sion of non VFA–COD to VFA–COD has occurred at an HRT of 3–4 d.

That is to say, VFA–COD 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. Theinitial CODs values(2–3 g/L) show that these wastes

have lost some amount of carbon through hydrolysis process duringthe storage period at 4 °C. This probably explains the lower produc-

tion 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 80–120) were C4 (37±6.0%

of TVFA), C5 (23.5± 1.2% of TVFA), C2 (17±1.6% of TVFA) and C3(15±1.9% of TVFA) comprising 94% of TVFAs. The higher molecular

weight VFAs(iC5, C6 andC7 etc.) wereproduced in insignicantamounts.

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 con-

sistent 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 com-

pletely inhibited at pH of 4.2. Low pH did not really affect the VFA

production, however about 9–10% of H2 gas was observed during

this phase of study. All the SCFAs produced from the acidogenic reac-

tor were consumed by the methanogens present in the methanogenic

reactor at HRT of 16–17 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 ef cient substrate to exhibit a high methane yield because of its

one-step degradation (Wong et al., 2008). The excess of VFA built up

in thedigestercaused a drop in pH andinhibition of themethanogenic

process. Hence to improve the process stability of the methanogenic

reactor, HRT was maintained at 16–17 d for this reactor.

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) showsthe 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 bacterialdiversitywith different morphol-

ogies of bacteria including small rods, fat rods, ovals, cocci, and thin la-

ments was observed. Absence of methanogen in the acidogenic reactor

might be due to the unfavorable low pH value for its growth. Addition-

ally, 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

A

B

C

0

5

10

15

20

25

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

0

2

4

6

8

10

12

14

16

18

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

0

1

2

3

4

5

6

7

0 10 20 30 40 50 60 70 80 90 1 00 110 120

V F A g C O D / L

C2 C3 iC4 C4 iC5 C5 iC6 C6 C7

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.



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Methanosarcinacea-like populations were observed in methanogenic

reactor of two-phase system (Fig. 6B). However, Methanosaeta-likecells 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

(μ 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 μ maxvalues for acetate (Zinder, 1993).

The presence of Methanosarcinacea-like populations in methano-

genic reactor of two-phase system might be related to the system's con-

ditions while the sampling time (day 110) pH was low (Fig. 4B)and the

VFA was accumulated (Fig. 4C). Vavilin et al. (2008) reported thatMethanosarcina sp. forming multicellular aggregate may resist inhibi-

tion by VFA because a slow diffusion rate of the acids limited the VFA

concentrations inside the Methanosarcina sp. aggregates.Despite micro-

bial communities have been revealed by FISH observation, both bac-

terial and archaeal populations in these reactors remain to be further

identied and characterized.

3.4. Overall comparison

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 reac-

tor (UASB)-septic tank. Thecomparisonsare intrinsically hard to do as

the data pertaining to brown water is lacking and also the rector oper-

ating conditions were different. However, SeqBR reactor has attained

the highest performance with relatively high organic loading rate

(2–3 gCOD/L d) and short HRT (16 d), with a corresponding COD re-

moval of above 90% (CODs) and 75% (CODt). From the results of

batch assay (Fig. 3), it is to be noted that themaximum biodegradation

took place within10 d of HRTfor BW+FW mixture. Further optimiza-

tion 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 appli-

cation. Overall, this study veries that (i) SeqBR system offers a reli-

able 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 mi-

croorganisms. Proper mixture brings synergistic and complementary

effects, which offsets the lack of available nutrients for methane pro-

duction 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 suf cient buff-ering capacity to the FW digestion by synergizing the effect of micro-

organisms 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 2–3 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 develop-

ment compared to the current centralized wastewater treatment

plants (Kujawa-Roeleveld et al., 2006). In its simple form it may con-

stitute 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, of ces, etc. (STOWA,

2005). There is no universal decentralized sanitation and reuse con-

cept 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

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.



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Table 4

Overall comparison cited in the literature.

Sl No. Reactor⁎ Substrate⁎⁎ Inuent values (g/L) Operating conditions Ef uent values (% removal) Ef uent V

(gCOD/L)CODt CODs VS VFA CODt CODs VS

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

KR (200 g/d)

19.2±6.5 6.8±1.05 8.8±3.9 – 20/15/10 HRT 71 ±13/75 ±

7/50±15

67±15/67±

13/53±22

65±20/69±

12/51±21

≥2.60

2 AC Bl. W with urine + KR 12.0 ± 4.37 3.04 ± 0.21 – 1.66±0.94 Pilot-scale study,

at 20 °C

– – – Day 43: 3

Day 80 on

0.083Bl. W less urine+KR 45.2± 24.6 3.97± 1.3 – 3.09±1.9 – – –

3 AC Bl. W with urine+KR 18.7 2.76 – 1.9 105 d, at 20 °C Avg. 58% – – –

Bl. W less ur ine+ KR 53 .6 10 .1 4 – 8.89 Avg. 58% – – –

4 AC and

UASB-septictank

Bl. W 9.5–12.3 1.4–2.8 – 0.76± 0.64 AC (Black+KR):

20 °C, 115–150 HRT

78 30 – 0.53 ± 0.1

Bl. W+KR 13.3–22 .9 2.7–5.4 – – AC(Bl. W+ KR):

15/20 °C, 115–280 HRT

61 – – –

BW+ KR 31.8–66 .0 4.0–10.1 – – UASB(BW+KR):

15/25 °C, 27–29 HRT

– – – –

5 UASB-septic

tank

Bl. W +KR 12.3 ±7.8 2.0 ±1.21 – – (1) (0.2 m3) pilot scale,

(2) 25 °C,

(3) OLR=0.42 gCOD/L d,

(4) HRT=29 d

82 94 – –

6 Two-phase BW +FW 35.2 ±10.4 13.0 ±4.4 1 6.9 ±3.2 1.1–2.1 HRT = 20 d, OLR ~

1.5 gCOD/L d

68.4 ±6.4 75.9 ±1.1 68.6 ±2.0 2–3

7 SeqBR HRT =16 d, OLR ~

2–3 gCOD/L d

76.7 ± 5.1 92.0 ± 3.0 75.7 ± 6.6 b0.8

⁎ 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.


10/10

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

wastewaterstreams). To achieve this, a no-mixtoilet hasbeen designed

and installed in our laboratory for our research purposes. This toilet

diverts liquid waste (YW) to a processing facility where componentsused 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 re-

duced from currently 30 L per capita to 5.5–6.0 L per capita, indicating

a saving of 80% ushing water use. Besides, when the amount of ush-

ing water is reduced, the volume production of toilet wastewater to be

transported and treated will automatically decrease.

With rapid increase in population density in Singaporeand 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 18.5–40 kg of VS are generated

through these waste streams per HDB block with 400 residents on av-

erage,whichcan yield 6–15 (avg. 10) m3 biogaswhen 65%VS removal

is achieved (Speece, 1996; ACEEE, 2009). This indicates a daily elec-

tricity generation of 12.5–33.6 kWh from biogas, on the assumption

that thegenerator ef ciencyis 35–50%. In addition,a daily heat energyprot of 17.8–46.5 kWh from biogas can be estimated. Generally, a

total energy prot of 0.08–0.20 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 ad-

vantages 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) thelife span of landlls can besignicantly

extended by minimizing the needs for disposal.

4. Conclusions

This study demonstrated that anaerobic co-digestion of BW andFWproved to be a potentialsubstrate forbiogas production. Anaerobic

co-digestion showed higher methane yield than BW and FW in non-

mixture conditions. SeqBR allowed a balanced conversion of organics

to CH4 at an OLR of 2–3 gCOD/L d that corresponds to a VS removal

of about 75%. BW provided a suf cient 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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artigo_anaerobic co-digestion of source segregated brown water (feces-without-urine) and food waste

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