Bioresource Technology 100 (2009) 61556162

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Anaerobic degradation pathway and kinetics of domestic wastewater

at low temperatures
Beni Lew a,*, Sheldon Tarre b, Michael Beliavski b, Michal Green b
a
b

Agricultural Research Organization, P.O. Box 6, 50250 Bet Dagan, Israel

Faculty of Civil and Environmental Engineering, Technion 32000 Haifa, Israel

a r t i c l e

i n f o

Article history:
Received 13 January 2009
Received in revised form 21 June 2009
Accepted 22 June 2009
Available online 14 August 2009
Keywords:
Anaerobic treatment
Degradation kinetics
Degradation pathway
Domestic wastewater
Temperature effect

a b s t r a c t
The effect of temperatures below 20 C (20, 15 and 10 C) on the anaerobic degradation pathway and
kinetics of domestic wastewater fractionated at different sizes was studied in a uidized-bed batch reactor. The overall degradation pathway was characterized by a soluble fraction degrading according to
zero-order kinetics and a colloidal fraction (between 0.45 and 4.5 lm) that rst disintegrates into a
particulate fraction smaller than 0.45 lm before nally degrading. The colloidal degradation processes
follow a rst-order kinetic. In contrast, suspended solids (bigger than 4.5 lm) degrade to soluble and
colloidal fractions according to rst-order kinetics. The colloidal fraction originating from suspended
solids further degrades into soluble fraction. These soluble fractions have the same degradation kinetics
as the original soluble fraction. The suspended solids degradation was highly affected by temperature,
whereas the soluble fraction slightly affected and the colloidal fraction was not affected at all. On the
other hand, the colloidal non-degradable fraction increased signicantly with the decrease in temperature while the suspended solids slowly increased. The soluble non-degradable fraction was little affected
by temperatures changes.
2009 Elsevier Ltd. All rights reserved.

1. Introduction
Anaerobic treatment of domestic wastewater is an attractive
option for secondary wastewater treatment. The high costs of aeration and sludge handling associated with aerobic sewage treatment are lower. However, domestic wastewater has typically low
concentrations of COD, resulting in relatively small methane production that is insufcient to heat the reactor to more favorable
mesophilic temperatures. The relatively higher concentration of
particulate matter in domestic wastewater has low degradation
rates at psychrophilic temperatures (Elmitwalli et al., 2001; Lew
et al., 2003; Singh and Viraraghavan, 2002). Therefore, anaerobic
process efciency is dependent on local ambient temperatures.
At tropical temperatures, intensive anaerobic systems have
been successfully applied and found wide acceptance for domestic
wastewater treatment. COD removals above 70% have been observed by several authors (Chernicharo and Cardoso, 1999; Kalogo
and Verstraete, 2000; Lew et al., 2003). At lower ambient temperatures, the application of anaerobic reactors for domestic wastewater treatment has been studied only in lab and pilot scale plants
(Lettinga et al., 1981; Lew et al., 2004; Seghezzo et al., 1998). Dete-

* Corresponding author. Tel.: +972 39683453; fax: +972 34604704.

E-mail address: benilew@agri.gov.il (B. Lew).
0960-8524/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2009.06.073

rioration in overall performance was observed, with COD removals

of 65% at 20 C and 5565% at 1317 C.
Henze and Harremoes (1983) observed that temperature decrease affects not only the reaction rate, but also the yield coefcient, the decay rate and the specic growth rate of anaerobic
bacteria. At low temperatures, the lower extracellular degradation
rate and/or the decrease in the organic matter degradation promotes the accumulation of suspended matter in anaerobic reactor
treating domestic wastewater (Elmitwalli et al., 2001; Lew et al.,
2003; Singh and Viraraghavan, 2002). Moreover, the degradation
of colloidal particles (2030% of the total COD for domestic wastewater) was reported to be the rate limiting step in anaerobic digestion at lower temperatures (Mergaert et al., 1992; Wang, 1994).
Understanding the anaerobic degradation processes (pathway,
biodegradability and kinetics) at different temperatures is fundamental to the evaluation of the potential of intensive domestic
wastewater anaerobic treatment. Bergamo et al. (2009) observed
rst-order degradation kinetics in a sequencing batch bioreactor
with immobilized microorganisms, which decreased with
temperature between 30 and 15 C. Measurement of the anaerobic
degradation process for domestic wastewater at different temperatures has also been carried out in attached biolm experiments
(Alderman et al., 1998; Lew et al., 2003; van der Last and Lettinga,
1992). In general, a decrease in total COD removal was observed
with the decrease in temperature for the same retention time. In

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B. Lew et al. / Bioresource Technology 100 (2009) 61556162

addition, at each temperature a major increase in the total COD removal was observed when increasing retention times shorter than
15 h. For retention times longer than 15 h, further increases in
retention time led to a minor increase in the total COD removal.
Based on the data available on degradation and degradability of
wastewater under anaerobic conditions various digestion models
have been built and can be found in the literature, each of them having its own potential and worth. No unied modeling framework for
the anaerobic digestion process exists, however, the model mostly
used today for anaerobic digestion processes is the ADM1 (Anaerobic Digestion Model No. 1), developed by the IWA anaerobic digestion modeling task-group (Batstone et al., 2002). The ADM1
provides an excellent generic model structure and parameterization
to characterize anaerobic process dynamics under various reactor
congurations and feeding protocols and can be employed to investigate the relationship between digester stability and acetoclastic
methanogenic population dynamics. However, the model assumes
a single type of particulate matter and relatively high temperatures.
In this study, the anaerobic degradation (pathway, biodegradability and kinetics constants) of domestic wastewater at different
low temperatures is conducted in a uidized-bed reactor. An arbitrary division of the particulate matter into two fractions based on
size is made, suspended solids (greater than 4.5 lm) and colloidal
particles (from 0.45 to 4.5 lm). This division is applied in order to
study the effect of colloidal particles on anaerobic degradation
kinetics and develop a more comprehensive model. A single
domestic wastewater soluble fraction is assumed (smaller than
0.45 lm) and its degradation was also studied.

2. Methods
2.1. Fluidized-bed reactor (FBR)
Two batch uidized-bed reactors (FBR) were constructed of
plexiglass with a working volume of 3.1 l (5.0 cm diameter,
150.0 cm height). The reactors were lled with 400 ml sinter glass
carrier (1.02.0 mm diameter) and fed with domestic wastewater
after primary sedimentation from the Neve Shaanan neighborhood
located in Haifa, at the given constant experimental temperature.
The wastewater can be classied as a medium strength domestic
wastewater and its characteristics after primary sedimentation
are given in Table 1. A pump provided for constant wastewater
recirculation and uidization of the biolm covered sinter glass
particles at 20 m/h upow velocity.
For each experimental temperature (20, 15 and 10 C) a start-up
period of at least three months was conducted in order for the
Table 1
Characteristics of domestic wastewater from Neve Shaanan neighborhood after
primary sedimentation.
Parameter

Unit

Average

St. Dev.

BOD
Total COD
Suspended solids COD
Colloidal COD
Soluble COD
TSS
VSS
Total protein
Suspended solids protein
Colloidal protein
Soluble protein
Total carbohydrate
Suspended solids carbohydrate
Colloidal carbohydrate
Soluble carbohydrate
Ammonia

mg/l
mg/l
mg/l
mg/l
mg/l
mg/l
mg/l
mg/l
mg/l
mg/l
mg/l
mg/l
mg/l
mg/l
mg/l
mg/l

290.0
566.0
326.6
94.7
214.0
211.2
189.3
276.8
153.2
42.9
86.1
65.9
23.3
20.0
22.6
39.2

110.0
242.8
80.8
16.8
48.0
115.9
90.3
17.3
10.8
26.7
34.2
49.9
27.1
40.0
23.5
7.8

reactors to reach steady-state conditions (van der Last and Lettinga, 1992; Zakkour et al., 2001). During the start-up period the
FB reactors were continuously operated at a retention time of 6 h
and fed with domestic wastewater.
2.2. The experimental procedure
At the end of the start-up period (3 months), experiments were
carried out in duplicate with the reactors operated in batch mode
for 24 h, with continuous recirculation of wastewater at 20 m/h
upow velocity. Before the beginning of each experiment, the reactor was lled three times with the appropriate substrate at intervals of 15 min between each lling. The purpose of this
procedure was to provide maximum adsorption of suspended solids and substrate to the biolm before starting the experiment.
For each temperature, the experiments were conducted with
the same wastewater ltered at different sizes: (a) with the soluble
fraction only (domestic wastewater ltered at 0.45 lm); (b) with
soluble and colloidal fractions (domestic wastewater ltered at
4.5 lm); and (c) with the raw original domestic wastewater. In this
way, the rst experiment with the soluble fraction is carried out
without the interference of bigger fractions. The soluble degradation rate was calculated assuming zero-order kinetics based on
the constant decrease in the soluble concentration with time:

dS
kt
Xdt

where S is the soluble concentration (mg COD/l), k is the zero-order

constant, X is the microorganisms concentration (mg TSS/l) and t is
the time (in hours).
In the second experiment (colloidal and soluble fractions), the
colloidal fraction degradation kinetics was determined assuming
that the colloidal fraction is particulate matter that must undergo
extracellular degradation to be transformed into soluble COD.
Extracellular degradation is considered to be a single combined
temperature dependent process with rst-order kinetics (Batstone
et al., 2002; Sanders et al., 2000) according to:

dP
ktP  Pn
dt

where P is the particulate matter concentration (mg COD/l), k is the

rst-order constant, Pn is the non-degradable particulate fraction
and t is the time (in hours).
Moreover, the soluble fraction is assumed to degrade with the
same degradation kinetics as in the rst experiment and, differences between observed and assumed soluble fraction degradation
are caused by the colloidal contribution to the soluble fraction.
Two approaches to analyze the degradation of the soluble material
originating from the colloidal fraction were taken: (1) assuming
that part of it degrades with the same degradation kinetics as the
soluble fraction from the rst experiment and part is non-degradable; and (2) assuming rst-order degradation kinetics (Eq. (2)).
In the third experiment the FB batch reactor was operated using
unseparated domestic wastewater. The suspended solids fraction
was also considered to be particulate matter composed of a nondegradable fraction and one that must pass an extracellular degradation step that obeys rst-order kinetics (Eq. (2)). The suspended
solids matter that undergoes the extracellular degradation step can
become wholly colloidal, wholly soluble or part colloidal and part
soluble fraction.
The suspended solids degradation pathway can be very complex and multiple assumptions can be made. In order to simplify
the determination of the overall suspended solids degradation
pathway, the contribution to the colloidal fraction was modeled
rst. Colloidal originating from suspended solids degrades according to rst-order kinetics (Eq. (2)) with the following possible

B. Lew et al. / Bioresource Technology 100 (2009) 61556162

alternative pathways: A) degradation constant similar to the original degradable colloidal; (B) degradation rate constant different to
the original degradable colloidal; (C) part of it is non-degradable;
and (D) suspended solids degradation leads to soluble formation
(directly). Each assumption can be considered by itself or coupled
with others.
The colloidal fraction originating from suspended solids can undergo a new extracellular step and also becoming soluble fraction
(indirectly). Four different assumptions for the degradation of soluble originating from suspended solids (direct and indirect) were
undertaken: similar to that of original soluble; similar to that of
soluble fraction originating from colloidal degraded; part of the
soluble is non-degradable; and different than that of the original
soluble and the soluble originating from colloidal degraded. Each
assumption was considered by itself or coupled with others, for
direct and indirect soluble fractions originating from suspended
solids.
At all temperatures studied a similar microorganism concentration was used, about 21.8 g VSS/l sinter glass, or 2.8 g VSS/l reactor.
2.3. Analysis
Collected samples were tested on a regular basis for COD, TSS
and VSS. All samples were ltered and diluted whenever necessary
and all were performed according to standard methods for the
examination of water and wastewater (APHA, 1995). Filtered sample was measured from samples after ltration through 4.5 lm lter paper (Schleicher & Schuell 595). Soluble sample was measured
from samples after ltration in a 0.45 lm membrane lter. The
suspended solids and colloidal concentration were calculated by
the differences between total and ltered samples; ltered and soluble samples, respectively.
Reactor biomass concentration (TSS and VSS) and uorescent
in situ hybridization (FISH) were carried out at the end of each
experiment at each temperature on the sinter glass. The FISH protocol described by Amann et al. (1990) was used. Microorganism
communities in the reactor were investigated by using Cy3 or
Cy5 labeled r-RNA targeted oligonucleotide probes purchased from
Proligo for Eubacteria (EUB338 GCTGCCTCCCGTAGGAGT) and
Archaea (ARC915 GTGCTCCCCCGCCAATTCCT) domains and; for
acetoclastic methanogenic: methanosaeta (MX825 TCGCACCGTGGCCGACACCTAGC) and methanosarcina species (SARCI551 ACCAATAATCACGATCAC). The NON338 probe (ACTCCTACGGGAGGCAGC)
was used as a control probe for EUB338.
Specic microorganism populations were quantied by cell area
measurement in CLSM images and results were expressed as percentage share of the total microorganism cell area, which was
determined after 40 ,6-diamidino-2-phenylindole (DAPI) stained
cells. Both DAPI and hybridization results were examined by an
epiuorescence microscope Zeiss Axioskop. 10 microscope elds

200

Soluble COD (mg/l)

180
160
140
120

10 C

100
80

15 C

60

20 C

40
20
0

10

Time (h)

15

Fig. 1. Soluble fraction degradation with time at 20, 15 and 10 C.

20

6157

(corresponding to 5001000 DAPI-stained cells) were counted to

determine the average number of cells per sample (Daims et al.,
2001).
3. Results and discussion
At each temperature studied three sets of batch experiments
were carried out in two FB reactors working in parallel using the
same domestic wastewater separated into different sizes: (1) less
than 0.45 lm (soluble fraction); (2) less than 4.5 lm (colloidal
and soluble fraction); and (3) unseparated wastewater. The results
of each fractions degradation at each temperature studied are
shown in Figs. 13, where the lines joining the experimental data
points were generated by MS Excel.
3.1. Soluble fraction only
A constant soluble fraction degradation rate was observed at all
temperatures in the rst 5 h of the experiment (Fig. 1) and calculated according to a zero-order model (Eq. (1)) to be 0.26, 0.18
and 0.12 g COD g VSS1 d1 for 20, 15 and 10 C, respectively.
The soluble fraction was assumed to contain substrate ready for
oxidation by microorganisms, a single step process.
At the end of the degradation period, the soluble fraction remained constant. The remaining COD observed can be assumed
to be the non-degradable fraction and it was calculated to be
around 13% of the initial concentration for all temperatures studied
with no temperature dependency. A similar non-degradable value
was also observed by others (Bergamo et al., 2009; Elmitwalli et al.,
2001).
3.2. Colloidal and soluble fractions
The second experiment in the FBR reactor was conducted using
domestic water screened to contain soluble and colloidal fractions.
The colloidal fraction concentration remained constant in the rst
4 h of the experiment at 20 C (Fig. 2). Afterwards, the colloidal
fraction concentration decreased in a rst-order like fashion until
the end of the experiment. A rst-order like decrease was also observed at 15 and 10 C, however, from the beginning of the experiments. The lag time in the colloidal degradation seen only at 20 C
was probably due to the different wastewaters used at different
temperatures and not a biolm condition.
The rst-order kinetic degradation constants and colloidal nondegradable fraction were determined assuming different values in
Eq. (2) and comparing the results with experimental data. The values that gave the highest R2 were proposed to be the correct ones.
A colloidal non-degradable fraction of 10%, 22% and 44% and a rstorder extracellular degradation constant rate of 4.65, 4.59 and 4.10
d1 were calculated for 20, 15 and 10 C, respectively.
The similarity in the rst-order degradation rates obtained at
different temperatures together with the sharp increase in the colloidal non-degradable fraction with the decrease in temperature
suggests that the observed degradation of colloidal matter should
not be assumed to be a simple one-step process. Colloidal degradation can be better described as a multistage process including particle disintegration and hydrolysis steps. The degradation
observed at lower temperatures is probably colloidal size particles
disintegrating into smaller soluble size particles rather than true
biodegradation.
The initial soluble concentration was considered to have a similar degradation rate as in Fig. 1; however, the change in the soluble fraction with time (Fig. 2) showed a different pattern. These
differences were caused by the contribution from the colloidal
fraction which became part of the soluble fraction. The two

B. Lew et al. / Bioresource Technology 100 (2009) 61556162

COD (mg/l)

6158
160
140
120
100
80
60
40
20
0

20 C

10

12

Time (h)

COD (mg/l)

200

15 C

150
100
50
0
0

10

Time (h)

15

20

COD (mg/l)

200

25

10 C

150
100
50
0
0

10

15

20

25

Time (h)
Fig. 2. Soluble (dashed line with open circles) and colloidal (full line with solid diamond) fractions degradation with time at 20, 15 and 10 C.

300

20 C

COD (mg/l)

250
200
150
100
50
0
0

10

15

20

25

Time (h)
250

15 C

COD (mg/l)

200
150
100
50
0
0

10

15

20

25

Time (h)

COD (mg/l)

200

10 C

150
100
50
0
0

10

Time (h)

15

20

25

Fig. 3. Soluble (dashed line with open circles), colloidal (full line with solid diamond) and suspended solids (dashed line with open squares) fractions degradation with time
at 20, 15 and 10 C.

6159

B. Lew et al. / Bioresource Technology 100 (2009) 61556162

approaches to analyze the kinetics of the degradable soluble material originating from the colloidal fraction were: (1) assuming the
same degradation kinetics as the soluble fraction from Fig. 1 (original soluble material); and (2) assuming a different degradation kinetic than the original soluble material.
In the rst approach to take in consideration the soluble concentration rise observed at 20 C after 5 h (Fig. 2), a high fraction
(62%) of non-degradable soluble originating from colloidal must
be assumed, however, this value does not t other experimental
data at this temperature.
The second approach (rst-order degradation) is supported by
the heterogeneous nature of domestic wastewater (Table 1). The
soluble fraction is mainly composed of proteins, while the colloidal
fraction is composed of carbohydrates and proteins of similar concentrations. Colloidal particles disintegrate in to particulate fractions smaller than 0.45 lm that are not actually soluble and still
need to be further hydrolyzed and degraded according to different
rst-order kinetic constants.
Based on this assumption, the highest R2 (higher than 0.98) between model and experimental data was observed for a rst-order
degradation constant of 4.92, 2.04 and 1.20 d1 for the soluble
originating from colloidal material at 20, 15 and 10 C, respectively. Similar rst-order constants for extracellular degradation
were observed by others (Eastman and Ferguson, 1981; Feng
et al., 2005; Straub et al., 2005). Due to the high R2, the logic of
the basic assumption and the suitability to other experiments with
the original domestic wastewater and other domestic wastewater
samples, this approach was assumed to be the correct biodegradation pathway.
3.3. Unseparated domestic wastewater suspended solids, colloidal
and soluble fractions
The third experiment in the FBR reactor was conducted using
unseparated domestic wastewater. In the experiments conducted
at 20 C (Fig. 3), the concentration of the suspended solids fraction
remained constant in the rst 2 h. After the rst 2 h, the concentration decreased in a rst-order like fashion until the end of the
experiment. A rst-order decrease was also observed at 15 and
10 C, but from the beginning of the experiments. The suspended
solids fraction, like the colloidal one, is considered to be particulate
matter composed of a non-degradable fraction and one that must
pass an extracellular degradation step that obeys rst-order kinetics. A suspended solids non-degradable fraction of 13%, 26% and
38% and rst-order extracellular process rate constant of 4.97,
3.00 and 1.00 d1 were calculated for 20, 15 and 10 C, respectively. Similar non-degradable fractions and rst-order extracellu-

lar process rate constants were observed by others at 20 C

(Elmitwalli et al., 2001; Feng et al., 2005).
In the previous experiment for the colloidal material only
(Fig. 2), the non-degradable fraction was found to be temperature
dependent. However, for suspended solids the non-degradable and
the degradable fractions were found to be temperature dependent
as shown by the increase in non-degradable fraction and the decrease in the rst-order extracellular process rate constant with
the decrease in temperature.
Four viable alternative pathways for the degradation of colloidal
size particles originating from suspended solids degradation are
shown in Table 2. In all the alternatives, the colloidal concentration
at the beginning of the experiments was considered to degrade
with the degradation kinetics calculated for original colloidal fraction (Fig. 2).
For each alternative pathway tested, the rst-order degradation
rate and the percentage of each degradation product that gave the
best R2 is given in Table 2. Alternatives I and II do not give a good
curve t (low R2) at 20 and 15 C and were therefore eliminated.
Alternatives III and IV gave a good curve t at all temperatures
studied. For repeated domestic wastewater batch experiments
conducted (data not shown), alternative IV showed the best correlation for all temperatures and was assumed to be the correct biodegradation pathway.
According to alternative IV, a decrease in the rst-order constant degradation rate was observed for colloidal material originating from suspended solids with a decrease in temperature (Table
2), unlike the colloidal fraction from Fig. 2, indicating that the degradation of colloidal material originating from suspended solids is
a temperature dependent process.
Moreover, 56% of the suspended solids degraded becomes colloidal material and 44% becomes soluble material. At 15 and
10 C, a higher percentage of the suspended solids becomes colloidal material, 70%. This shows the sensitivity of hydrolysis (degradation of suspended solids to soluble material) to temperature as
opposed to physical disintegration processes (degradation of suspended solids to colloidal fraction).
Based on the previous assumptions and results, the questions
remaining for the suspended solids pathway are: (I) the kinetics
of the soluble fraction originating from the colloidal fraction (from
now on labeled soluble indirect from suspended solids); and (II)
the kinetics of the soluble fraction originating directly from suspended solids degradation (from now on labeled soluble direct
from suspended solids).
Seven viable alternative pathways are presented for the degradation kinetics of soluble originating from suspended solids (direct
and indirect): (I) partly similar to original soluble and partly non-

Table 2
Kinetics parameters calculated from the different degradation pathways of colloidal originating from suspended solids.
T (C)

Pathway

Assumptions

R2

k (d1)

Colloidal non-degradable

Colloidal degradable (%)

Sol

20

I
II
III
IV

A, C
B, C
A, C, D
B, D

0.206
0.498
0.898
0.968

4.65a
2.40
4.65a
4.60

0
60%
5%
0

100
40
55
56

0
0
40%
44%

15

I
II
III
IV

A, C
B, C
A, C, D
B, D

0.659
0.630
0.913
0.984

4.59a
2.4
4.59a
2.00

23%
0
29%
0

77
100
51
70

0
0
20%
30%

10

I
II
III
IV

A, C
B, C
A, C, D
B, D

0.790
0.775
0.934
0.996

4.10a
1.20
4.10a
1.30

20%
30%
35%
0

80
70
30
70

0
0
35%
30%

Previously calculated from the degradation of the original colloidal fraction.

From SS to

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B. Lew et al. / Bioresource Technology 100 (2009) 61556162

degradable; (II) partly similar to soluble originating from colloidal

and partly non-degradable; (III) part of the direct soluble similar to
original soluble and part non-degradable; and part of the indirect
soluble similar to soluble originating from colloidal and part nondegradable; (IV) partly of the direct soluble similar to soluble originating from colloidal and partly non-degradable; and part of the
indirect soluble similar to original soluble and part non-degradable; (V) direct soluble with unique degradation kinetics and indirect soluble similar to soluble originating from colloidal; (VI) partly
with unique degradation kinetics and partly non-degradable; and

(VII) direct soluble with unique degradation kinetics and indirect

soluble also with unique degradation kinetics. Moreover, in all
the alternatives the soluble concentration in the beginning of the
experiments was considered to be the original soluble with the
degradation kinetics previous calculated (Fig. 1).
For each alternative the degradation kinetics rate and the percentage of non-degradable soluble fraction that gave the best R2
are shown in Table 3. Alternative I, with the highest R2 value observed (0.97) at all temperatures studied, is considered to be the
correct pathway. According to alternative I, the degradable soluble

Table 3
Kinetics parameters calculated from the different degradation pathways of soluble originating from suspended solids direct and indirect.
T (C)

Pathway

R2

K(direct)
a

K(indirect)
a

Non-degradable (direct)

Non-degradable (indirect)

20

I
II
III
IV
V
VI
VII

0.973
0.432
0.946
0.939
0.906
0.922
0.927

0.26
4.92 d1,b
0.26a
4.92 d1,b
26.4 d1
19.2 d1
18.24 d1

0.26
4.92 d-1,b
4.92 d1,b
0.26a
4.92 d1,b
19.2 d1
9.12 d1

2%
0
0
7%
0
7.5%
0

20%
0
0
3%
0
7.5%
0

15

I
II
III
IV
V
VI
VII

0.969
0.927
0.902
0.918
0.964
0.903
0.954

0.18a
2.04 d1,b
0.18a
2.04 d1,b
6.24 d1
4.8 d1
2.4 d1

0.18a
2.04 d1,b
2.04 d1,b
0.18a
2.04 d1,b
4.8 d1
7.2 d1

3%
0
0
8%
0
5%
0

22%
0
0
0
0
5%
0

10

I
II
III
IV
V
VI
VII

0.974
0.932
0.948
0.874
0.893
0.923
0.905

0.12a
1.20 d1,b
0.12a
1.20 d1,b
0.72 d1
0.72 d1
0.72 d1

0.12a
1.20 d1,b
1.20 d1,b
0.12a
1.20 d1,b
0.72 d1
0.72 d1

4%
0
0
8%
0
0
0

28%
0
0
0
0
0
0

a
b

Previously calculated from the degradation of the original soluble fraction-unit: g COD gVSS1 d1.
Previously calculated from the degradation of the soluble originating from colloidal.

SSdegradable

0.9%

20 C

56.0%

Colldegradable

Colldegradable from SS
43.1%
21.0%

Soldegradalbe from Coll

79.0%

Solnon-degradalbe

Soldegradable from SS

Soldegradable

Degradation
SSdegradable

1.5%

15 C

70.0%

Colldegradable

Colldegradable from SS
28.5%
20.0%

Soldegradalbe from Coll

80.0%

Solnon-degradalbe

Soldegradable from SS

Soldegradable

Degradation
SSdegradable

1.8%

10 C

70.0%

Colldegradable

Colldegradable from SS
21.0%
30.0%

Soldegradalbe from Coll

70.0%

Solnon-degradalbe

Soldegradable from SS

Soldegradable

Degradation
Fig. 4. Fate of the different wastewater fractions at 20, 15 and 10 C.

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Table 4
Kinetic degradation parameters and non-biodegradable percentage (%) of the
different fractions at different temperatures.
Fraction

Unit

20 C

15 C

10 C

SS

d1
%
d1
%
d1
%
g COD g VSS1 d1
%
g COD g VSS1 d1
%
g COD g VSS1 d1
%
d1
%

4.63(0.53)
18(6)
4.65(0.78)
13(3)
4.65(0.78)
0(0)
0.30(0.04)
13(4)
0.30(0.04)
2(2)
0.30(0.04)
21(7)
4.92(0.94)
0(0)

3.01(0.02)
29(2)
4.57(0.13)
27(6)
2.00(0.42)
0(0)
0.16(0.04)
13(4)
0.16(0.04)
5(3)
0.16(0.04)
20(1)
2.04(0.57)
0(0)

1.06(0.06)
39(10)
4.08(0.37)
45(6)
1.32(0.36)
0(0)
0.13(0.03)
16(4)
0.13(0.03)
6(3)
0.13(0.03)
30(1)
1.20(0.20)
0(0)

Coll original
Coll from SS
Sol original
Sol direct from SS
Sol indirect from SS
Sol from Coll

SS Suspended Solids fraction; Coll Colloidal fraction; Sol Soluble fraction.

fraction originating directly from suspended solids is high (97%) at

all temperatures. In contrast, the non-degradable fraction originating indirectly from suspended solids is signicantly higher at 20 C
(20%) and increases to 28% with a drop in temperature to 10 C.
Similar observations and results were observed by others (Batstone
et al., 2002; Elmitwalli et al., 2001; Yasui et al., 2005).
Alternative number II did not give a good curve t at 20 C and
is considered to be incorrect. Alternatives number III, IV, V, VI and
VII gave good curve t values at all temperatures studied, however,
a good correlation was not observed for repeated domestic wastewater batch experiments conducted.
To verify the parameters based on the above described experiments, ve more experiments were conducted with complete
domestic wastewater from Neve Shaanan. The pathway that best
suited wastewater anaerobic degradation at all temperatures studied is summarized in Fig. 4, and the kinetics parameters and the
non-degradable fractions are shown in Table 4. For each temperature studied the biodegradation pathway is the same, the only difference is the percentage of material going at each pathway.
3.4. Temperature effect on observed parameters
In general the overall pathway pictured in Fig. 4 qualitatively
describes the degradation pathway for the temperature range
investigated. In order to calculate the kinetic parameters for any given temperature (between 10 and 20 C), the temperature activity
coefcients were calculated based on the Arrhenius equation and
are shown in Table 5. The temperature activity coefcient of the

Table 5
Temperature activity coefcient of the degradation constant, the equations derived
from curve tting for the non-degradable fraction and for the percentage pathway for
the different domestic wastewater fractions at temperatures between 10 and 20 C.

original colloidal fraction is not listed in Table 5 because no temperature dependence was found.
The highest temperature coefcient was observed for the degradation rate of suspended solids fraction, 1.160, indicating that suspended solids degradation was the most sensitive process to
temperature change. Moreover, the temperature coefcient observed for degradation of the three different particulates (suspended solids, colloidal originating from suspended solids and
soluble originating from colloidal) are higher than the soluble fraction (1.079), the substrate assumed for methanogenesis. These results are in accordance with others (Lew et al., 2003 and Gujer and
Zehnder, 1983).
The inuence of temperature on anaerobic processes is not limited to the reaction rate, but also reects the extent of the anaerobic degradation process. The temperature effect on kinetic
parameters is governed by the free energy law, however, the temperature effect on the non-degradable fraction itself and on each
pathways percentage are calculated based on the best curve tting. The particulate matter non-degradable fraction increases with
the decrease in temperature, with the colloidal fraction being the
most affected by temperature changes. The increase in the nondegradable colloidal fraction was about twice the increase in the
non-degradable suspended solids fraction. In contrast, experimental results show that the original soluble non-degradable fraction
stayed the same.
3.5. Temperature inuence on microorganism population
Quantication of microbial groups expressed as percentages in
the FBR at different temperatures (20, 15 and 10 C) is listed in
Table 6. Fluorescent in situ hybridization (FISH) and DAPI results
showed a decrease in microorganism viability (total archaea and
eubacteria counted by FISH as opposed to the DAPI count) with
the decrease in temperature, from 40% at 20 C to 18% at 10 C.
Although the results are qualitative, they suggest that at lower
temperature a lower concentration of viable microorganisms is
present in the reactor. Probably a higher amount of microorganisms is present in the reactor at lower temperatures.
FISH results show that archaeal cells dominated at all temperatures studied, around 70% of the total active microorganisms. A
slight decrease in archaeal cells with the decrease in temperature
was observed, from 76% at 20 C to 68% at 10 C. Similar results
were observed by Gomec et al. (2004). Probably temperature
changes between 20 to 10 C have little affect on microorganisms
ratio in an anaerobic reactor treating domestic wastewater.
It was also observed that acetoclastic methanosaeta species
(MX825) were the major methanogenic archaea present at all temperatures studied, around 84% of ARC915, with almost no change
with temperature decrease. This high acetoclastic species concentration can be explained by the fact that seventy percent of the
methane produced in anaerobic processes comes from the decarboxylation of acetate. Similar results of methanosaeta species
being the major archaea present in anaerobic sludge treating
domestic wastewater has been observed by others (Araujo et al.,
2001; Sanz et al., 2002). No acetoclastic methanosarcinaceae species (SARCI551) were observed at all temperatures studied.

Fraction

Temperature
activity coefcient

Nondegradable
fraction

% Pathway

Suspended solids
Original colloidal
Colloidal from suspended
solids
Original soluble
Soluble direct from
suspended solids
Soluble indirect from
suspended solids
Soluble from colloidal

1.160

1.124

2.1  T + 60.2
3.2  T + 76.3

1.4  T + 86.3

1.079
1.079

0.3  T + 18.5
0.4  T + 10.3

2.2  T  2.3

Temperature

1.079

0.9  T + 37.2

0.5  T + 56.9

1.141

10 C
15 C
20 C

Table 6
FISH results at different temperatures.
Viability

40.4 (2.0)
29.4 (5.5)
17.7 (5.5)

Viable microorganisms
% Eubacteria

% Archaea

25.4 (3.5)
28.9 (3.3)
32.2 (2.7)

74.6 (3.5)
71.1 (3.3)
67.8 (2.7)

Methanosaeta
% of Archaea
88.0 (5.3)
81.5 (3.1)
85.7 (4.2)

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B. Lew et al. / Bioresource Technology 100 (2009) 61556162

4. Conclusions
The importance of considering multiple types of particulate
matter at low temperatures is shown. The colloidal fraction rst
disintegrates (a physical process not affected by temperature) into
a particulate fraction smaller than 4.5 lm before nally being degraded, both processes in a rst-order manner. Suspended solids
partly degrades into soluble fraction and partly to colloidal degradable fraction, which further degrades to soluble. Soluble originating from suspended solids degrades with degradation kinetics
similar to the original soluble fraction. The same overall degradation pathway was found to t experimental data for all temperatures studied with the only difference being the percentage of
material going to each pathway and the degradation kinetic
constants.

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