Chemosphere xxx (2016) 1e10

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Characteristics of dissolved organic matter formed in aerobic and

anaerobic digestion of excess activated sludge
Haixia Du a, *, Fusheng Li b
a
b

Graduate School of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
River Basin Research Center, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

Faster hydrolysis and slower degradation led to more DOM in anaerobic

digestion.

DOM concentration increased with
increases of sludge concentration and
temperature.

Faster destruction and lysis of cells
occurred in anaerobic than aerobic
digestion.

Higher temperatures promoted cells'
destruction in aerobic & anaerobic
digestion.

More proteins & aromatic amino
acids, less humic substances in
anaerobic digestion.

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 18 August 2016
Received in revised form
18 October 2016
Accepted 26 October 2016
Available online xxx

The characteristics of dissolved organic matter (DOM) formed in aerobic and anaerobic digestion of
excess activated sludge (EAS) was investigated for three total solid (TS) concentrations (1.2, 2.3 and 5.2%)
and three temperatures (5, 20 and 35  C). The results on the overall concentration of DOM evaluated by
TOC showed signicantly higher values in anaerobic than aerobic digestion (2.8e6.9 times for TS 1.2
e5.2% at 20  C). Data analysis with a rst-order sequential reaction model revealed that higher occurrence of DOM in anaerobic digestion was a result of comparatively faster hydrolysis (1.3e5.5 times for TS
1.2e5.2% at 20  C; 1.4e49.3 times for temperatures 5e35  C with TS 1.2%) and slower degradation (0.3
e1.0 times for TS 1.2e5.2% at 20  C; 0.5e8.3 times for temperatures 5e35  C with TS 1.2%).
In aerobic digestion, more humic substances were formed; while, in anaerobic digestion, proteins and
aromatic amino acids were the major constituents. For both digestions, except for a few exceptions,
proteins and humic substances increased as the TS concentration increased; and increasing the temperature led to a decrease in the content of proteins formed in both aerobic and anaerobic digestion, and
an increase in the content of humic substances in the aerobic digestion. The UV-absorbing DOM constituents were highly heterogeneous, and were comparatively larger in anaerobic digestion; and did not
change signicantly with the TS concentrations and temperatures.
2016 Elsevier Ltd. All rights reserved.

Handling Editor: A Adalberto Noyola

Keywords:
Dissolved organic matter
Biomass
Excess activated sludge
Aerobic digestion
Anaerobic digestion
Humic substances

1. Introduction
* Corresponding author.
E-mail address: t3812005@edu.gifu-u.ac.jp (H. Du).

Population growth and rapid urbanization lead to a marked

increase in the number of municipal wastewater treatment plants

http://dx.doi.org/10.1016/j.chemosphere.2016.10.108
0045-6535/ 2016 Elsevier Ltd. All rights reserved.

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activated sludge, Chemosphere (2016), http://dx.doi.org/10.1016/j.chemosphere.2016.10.108

H. Du, F. Li / Chemosphere xxx (2016) 1e10

(WWTPs). This increase is accompanied with the generation of a

large amount of excess activated sludge (EAS) that requires proper
treatment and management due mainly to its large volume and
easiness to decay under normal environmental condition. In
industrialized countries, incineration is the main trend of treatment for EAS. In most developing countries where more municipal
WWTPs are under construction, however, landlling and direct
damping that cause many environmental and health problems are
still widely adopted (Chen et al., 2012).
For stabilization and effectively utilization of EAS that contains
rich content of organic matter, aerobic digestion, anaerobic digestion and composting are considered as sustainable disposal options
and their applications are increasing. By 2008, 45% and 21% of
domestic sewage facilities in USA had been using aerobic and
anaerobic digestion processes respectively to treat EAS (United
Nations Human Settlements Programme, 2008). For all these
methods, certain percentages of the organic substances that
constitute for the microorganisms, including the major constituting
species of cells (proteins, fats and carbohydrates) and extracellular
polymeric substances (EPS) (Jin et al., 2016; Jiang, 2007; Laspidou
and Rittmann, 2002), are eliminated through involved biological
reactions, and some of the intermediates and nal products are
remaining as dissolved organic matter (DOM) in the leachates or
liquids of the EAS treatment systems together with those of less or
non-biodegradable organic constituents.
The resulting leachates or liquids are also increasingly concerned when aerobic digestion, anaerobic digestion and composting are adopted for treatment of EAS. If not properly handled, the
organic matter contained therein can worsen the water quality of
natural water systems and, at the same time, can become the major
reason for odors and smells (Cheng et al., 2009). Moreover, for
drinking water production using source water merged with the
leachates or liquids from the biological EAS treatment systems, the
DOM also contributes to the enhanced presence of hazardous
disinfection byproducts in the water after treatment depending on
its content and physiochemical composition. Therefore, a better
understanding of the characteristics of DOM is of great importance
for water environment conservation and water quality securing.
In regard of the characteristics of DOM formed in digestion,
many studies have been conducted, with the focus being mainly
placed on the organic constituents that affect the digestion efciency and gas productivity, such as proteins, polysaccharides,
volatile fatty acids (VFAs) and extracellular polymeric substances
(EPS) (Laspidou and Rittmann, 2002; Novak et al., 2003; Ramesh
et al., 2006; Tomei et al., 2011; Shao et al., 2013). For instance, using returned sludge from a WWTP, Ramesh et al. (2006) compared
the characteristics of EPS and soluble microbial products formed
after aerobic and anaerobic digestion for one month in terms of zeta
potential, particle size, residual turbidity after coagulation by polyaluminum chloride, infrared spectra and uorescence excitation
emission matrix (EEM) spectra. In another study, Shao et al. (2013)
investigated the digestion of EAS from a WWTP in China under
aerobic and anaerobic conditions. By focusing on the degradation of
proteins at mesophilic temperature, the changes in the degradation
efciency and the degree of humication were compared. These
pioneering studies have provided very useful information relating
to the characteristics of DOM formed in the digestion of EAS.
However, since DOM is a complex and heterogeneous mixture of
polydispersed components, further characterization of its composition requires the use of more indexes that are well used in water
quality evaluations. Activated sludge is a mixture of living cells,
dead cells and some inert organic solids from the wastewater and
its biological treatment process. The extent of their presence can
greatly affect the efciency of the sludge digestion process; for
instance, the kinetics of involved hydrolysis and degradation

reactions. Increasing the sludge concentration brings about more

living cells to the digestion process. This may contribute to differences in the increasing extent of the hydrolysis rate, the degradation rate or both, depending on the microbial density and
structures in the digestion process. Generally, the growth rate and
activity of bacteria are also affected by temperatures. Changing the
temperatures of digestion may bring about changes in the structures and activity of bacteria involved in hydrolysis and degradation of EAS. Responding to such changes, the properties of the
formed DOM may change, which remain unclear in available
literature.
Accordingly, in order to generate more information for better
understanding the characteristics of DOM formed and remaining in
the digestion of EAS under aerobic and anaerobic conditions, in this
study, ve aerobic and ve anaerobic digestion reactors were
operated in parallel under three different total solid (TS) concentrations (1.2, 2.3 and 5.2%) and three temperatures (5, 20 and 35  C).
DOM was evaluated using the overall concentration indexes of total
organic carbon (TOC), soluble chemical oxygen demand (SCOD) and
ultraviolet absorbance at the wavelength of 260 nm (UV260), and
the composition related properties, including the specic ultraviolet absorbance (SUVA), VFAs, uorescence EEM and molecular
weight (MW) distribution. The observed TOC data under each
digestion condition were analyzed using a rst-order sequential
reaction model that considered both hydrolysis and degradation.
Based on the magnitudes of estimated rate parameters, the differences between aerobic and anaerobic digestion and the effects of TS
concentrations and temperatures were discussed. Moreover, using
scanning electron microscope (SEM), the destruction and lysis of
cells during digestion were observed. Principal component analysis
(PCA) was performed for better understanding the interrelations
among all measured quality indexes and the changes of DOM
properties during the digestion process.
The choice of these three TS concentrations (1.2, 2.3 and 5.2%)
was because digestion can apply to fresh EAS and EAS after
different extent of thickening, which normally possess water content from about 99% to 95%. These three temperatures (5, 20 and
35  C) was applied in order to cover a broader temperature range
for eld applications of the biological process. Much higher temperatures than 35  C, such as 55  C in thermophilic digestion of EAS,
were not compared because digestion under ambient temperatures
are expected for increased applications due to its lower requirement for energy and easiness for operation.

2. Materials and methods

2.1. EAS preparation
Fresh EAS was obtained from the reaction tank of a MWTP
operated in the sequential anaerobic/aerobic mode for enhanced
removal of phosphorus in Gifu, Japan. The obtained sludge was
subjected to concentrating immediately by settling for 4 h, followed
by centrifuging at 3000 rpm for 5 min. From the centrifuged sludge,
three sludge samples with the total solid (TS) concentration of 1.2%,
2.3% and 5.2% were adjusted by dilution with the supernatant of
centrifugation after ltration through 0.2 mm membrane lter for
direct use in the digestion experiments. The content of organic
matter of the EAS measured by incineration loss was 75.2%. Other
measured parameter values for the obtained sludge were: temperature 25  C, pH 6.75, DO 1.73 mg/L, ORP 159 mV and EC 29.1 mS/
m. The concentration of DOM in the supernatant was: 17.7 mg/L as
TOC, 13.4 m-1 as UV260 and 40 mg/L as SCOD.

Please cite this article in press as: Du, H., Li, F., Characteristics of dissolved organic matter formed in aerobic and anaerobic digestion of excess
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H. Du, F. Li / Chemosphere xxx (2016) 1e10

2.2. Digestion experiments

Ten digestion reactors (namely R1 - R10), each having a volume
of 500 mL, were used for the experiments. R1 - R5 were operated
under aerobic condition; while, R6 - R10 under anaerobic condition.
To all these reactors, the adjusted EAS samples were added in
accordance with the conditions shown in Table 1, which enabled
investigation of the effects of the TS concentrations (1.2, 2.3 and
5.2%) and temperatures (5, 20 and 35  C). The added volume was
identical: 300 mL for each reactor. Aerobic digestion was ensured
by consistent air bubbling with an air pump (Power: 2.5 W, Air
ow: 2 L/min); and anaerobic digestion was ensured by tightly
sealing the reactors with rubber stoppers lined with Teon sheet
immediately after addition of the EAS samples. The reactors were
placed on three water bath shakers adjusted to 5, 20 and 35  C,
respectively, and were shaken gently to ensure uniform reactions
within each reactor.
During the digestion experiments, from each reactor, samples
were collected at designated time points. After ltration through
0.2 mm membrane lters (Toyo Roshi Co., Ltd, Japan), the ltrates
were subjected to quality analysis for DOM and the suspended
solids detained on the membrane were subjected to SEM observations. The quality analysis for DOM mainly focused on COD, TOC,
UV260, uorescence EEM and MW distribution, the details of which
will be described below. In addition to these measurements, pH and
EC inside all reactors were monitored, and dissolved inorganic ni

trogen (NH
4 , NO2 , and NO3 ) was analyzed using the ltrates of all
samples with an ion chromatography system (HPIC, Shimadzu,
Japan).
2.3. Quality analysis

2.3.2. Fluorescence EEM of DOM

Fluorescence EEM was analyzed using a spectrouorometer (RF5300, Shimadzu, Japan). The excitation and emission scans were
performed between 220 and 550 nm with 5 nm increments. The
bandwidth for both excitation and emission was 1 nm. The

Table 1
The condition of the aerobic and anaerobic digestion experiments.
Aerobic

TS (%)
Temperature ( C)

obtained EEM was normalized into the quinine sulfate unit (QSU)
by dividing the uorescence intensity values of all samples with the
uorescence intensity value of 10 ppb quinine sulfate (in 0.05 M
H2SO4 solution) at the designated wavelengths of Ex/Em 350/
450 nm (Lee et al., 2015). Comparison of the composition of DOM
was further made according to the uorescence peaks emerged on
the obtained EEM images. To minimize the inner lter effect, all
samples subjected to the analysis were diluted using pure water to
TOC below 2 mg/L (Lee et al., 2015).

2.3.3. MW characteristics of DOM

The MW characteristics of DOM were evaluated on the basis of
its distribution analyzed using a high-performance size exclusion
chromatography system that consisted of a silica chromatographic
column (GL-W520-X, 10.7  450 mm, Hitachi) and a UV detector
(Mod-el LC-10AV, Shimadzu) with the wavelength of 260 nm. Pure
water containing 0.02 M Na2HPO4 and 0.02 M KH2PO4 was used as
the eluent and was introduced to the column at the constant ow
rate of 0.5 mL/min. Calibration was made with three polystyrene
sulfonate (PSS) with MW of 1430, 4950 and 6530 g/mol. In addition,
based on the obtained MW chromatograms, the weight-averaged
(Mw) and number-averaged (Mn) molecular weights were
computed according to the following expressions (Karanl et al.,
1996; Li et al., 2003):

,
N
X
MWi thi tDt
hi tDt
i1
i1
,
!
N
N
X
X
hi tDt
hi tDt=MWi t
Mn
Mw

N
X

i1

2.3.1. Overall quality indexes of DOM and volatile fatty acids

Overall quality indexes of DOM included CODCr, TOC and UV260.
CODCr was analyzed by the colorimetric method (DR/890 Colorimeter); while, TOC and UV260 was analyzed by a TOC analyzer
(TOC-V DS, Shimadzu, Japan) and a spectrophotometer (UV-1600
GLP, Shimadzu, Japan), respectively. Based on the measurement
results of TOC and UV260, SUVA, a parameter dened as the ratio of
UV260 to TOC, was computed to reect the relative content of humic
and/or humic-like substances in the DOM formed and remaining in
the liquids of digestion under different conditions.
Seven volatile fatty acids (citrate, isobutyrate, acetate, propionate, butyrate, valerate and isovalerate), which were targeted in
previous studies on the anaerobic fermentation of organic waste
(including EAS, food waste and vegetable waste) (Pang et al., 2015;
Grimberg et al., 2015; Du and Li, 2016), were analyzed using a high
performance liquid chromatograph (HPLC, Shimadzu, Japan). The
analysis of VAFs was made in order to further trace the hydrolysis
and degradation behavior of the EAS.

Anaerobic

R1

R2

R3

R4

R5

R6

R7

R8

R9

R10

1.2
5

1.2
20

1.2
35

2.3
20

5.2
20

1.2
5

1.2
20

1.2
35

2.3
20

5.2
20

(1)

i1

where, MWi (t) is the MW as a function of the elution time t, hi(t) is

the detector response and Dt is the time interval.
Polydispersity, a parameter dened as the ratio of Mw to Mn was
adopted for evaluating the heterogeneity of DOM and its changes in
molecular weight distribution generated during digestion of the
EAS (Karanl et al., 1996; Li et al., 2003).

2.4. SEM observations

The suspended solids of EAS during aerobic and anaerobic
digestion were observed using a SEM (SU3500, Hitachi, Japan)
under the acceleration voltage of 3 kV. Prior to observations, the
suspended solids ltered on membrane lters with the pore size of
0.2 mm were frozen dried using a freeze dryer (FDU-1200, Tokyo
Rikakikai, Ltd.) and then coated with platinum using a magnetron
sputter (MSP-1S, Vacuum Device Inc., Japan).

2.5. Principal component analysis

The principal component analysis (PCA) was performed using
the SPSS software (IBM SPSS Statistic 21.0, IBM Corp., Armonk, NY)
for better understanding the interrelations of all measured quality


indexes (including pH, EC, SCOD, DOC, UV260, NH
4 , NO3 , NO2 , VFAs
and the major uorescence peaks emerged in the uorescence EEM
measured for all samples) and the change of DOM properties during
the process of the aerobic and anaerobic digestion. For this, all the
measurement results in the series of aerobic and the series of
anaerobic digestion were used separately as the pooled data for
analysis after standardization treatment of the obtained data for
each index.

Please cite this article in press as: Du, H., Li, F., Characteristics of dissolved organic matter formed in aerobic and anaerobic digestion of excess
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H. Du, F. Li / Chemosphere xxx (2016) 1e10

3. Results and discussion

3.1. Content of DOM assessed by overall quality indexes
Over the total digestion time for 61 days, a trend of rapid TOC
increases followed by either slight increases or gradual decreases
was observed, as could be seen from Figs. 1 and 2. The increases
reected the result with the rate of hydrolysis being higher than the
rate of degradation. In contrary, the observed decreases reected
the result that the rate of degradation exceeded the rate of hydrolysis or hydrolysis had terminated. For COD and UV260, trends
similar to that of TOC were also observed although the extent of
decreases for the hydrolysis products detected by UV260 was found
smaller. The smaller extent of decreases for UV260 was considerable
because, compared to TOC and COD that reect all organic compounds (biodegradable and less or non-biodegradable) contained
in water, wastewater or various biomass samples, UV260 only reects those possessing UV-absorbing features and, in most environmental samples, humic and fulvic acids that are considered not

Fig. 1. The observed and calculated proles of TOC in the aerobic (a) and anaerobic (b)
reactors operated at 20  C with the TS concentrations of 1.2, 2.3 and 5.2%, respectively.

Fig. 2. The observed and calculated proles of TOC in the aerobic (a) and anaerobic (b)
reactors under the temperatures of 5, 20 and 35  C, respectively, with the TS concentration of 1.2%.

biodegradable (Li et al., 2003).

After digestion for 61 days, Fig. 3 showed that, for TOC, the
values under the anaerobic condition at 20  C were obviously
higher than those under the aerobic condition; however, at 5  C the
differences between the two conditions were becoming smaller.
For UV260, the value differences between the two conditions were
less apparent. A general trend of increases of TOC and UV260 with
increases of either the TS concentration or the temperature was
revealed, except for the anaerobic condition with TS of 1.2% and
temperature of 5  C.
For SUVA, as shown in Fig. 4, except for the anaerobic condition
with the TS of 1.2% and the temperature of 5 and 35  C, its values
with the aerobic digestion were signicantly higher than those
with the anaerobic one, suggesting that more humic and/or humiclike substances were formed and remaining during aerobic digestion of EAS. In addition, as a common feature, the values of SUVA for
most TS and temperature conditions of both aerobic and anaerobic
digestion were below 2.0 m-1/(mg/L). This suggests that the formed
and remaining humic and/or humic-like substances were mainly
consisted of molecules with relatively lower aromaticity (Ates et al.,

Fig. 3. The concentrations of TOC and UV260 at the end of digestion for 61 days in the
aerobic and anaerobic reactors under different TS concentrations and temperatures.
*The value for day 46.

Fig. 4. The values of SUVA at the end of digestion for 61 days in the aerobic and
anaerobic reactors under different TS concentrations and temperatures. *The value for
day 46.

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H. Du, F. Li / Chemosphere xxx (2016) 1e10

2007). A general trend of either increases or decreases of SUVA with

the increases of the TS concentration and temperature was not
revealed.
The observed presence of DOM in the liquid was a combined
result of the destruction and lysis of the microorganisms and the
breakdown of some of their released organic species. The occurrence of the destruction and lysis of the microorganisms during
both the aerobic and anaerobic digestion processes could be clearly
seen from the SEM images given in Fig. 5 as examples for the
condition with the TS of 1.2% and temperature of 20  C. Elongated
and rod shaped bacteria were evident in the initial period (day 2).
However, after 31 days, they looked well swelled, with their shapes
becoming irregular. Then, at the end of digestion for 61 days, their
shapes became vague and can hardly be distinguished. For all reactors, the changes in the shape of bacteria were found more dramatic for anaerobic digestion, suggesting their faster destruction
and lysis than aerobic digestion. Disintegration of sludge ocs in
anaerobic fermentation was also found by Pang et al. (2014). In
association with the increases of DOM, for both aerobic and
anaerobic conditions, increasing the temperature from 5 to 35  C
signicantly accelerated the destruction and lysis of microorganisms (SEM images not provided here). In a previous study, Climent
et al. (2007) reported that higher temperatures could enhance the
denaturation of proteins and the permeabilization of cell membrane, resulting in faster leakage of intracellular substances.

3.2. Rate parameters of hydrolysis and degradation in EAS digestion

The digestion of EAS is a sequential process of hydrolysis and
degradation (Mani et al., 2016). For hydrolysis, it involves physicochemical and biological disruption of cell walls and membrane,
and the release of the intracellular materials. The involved extent of
physicochemical reaction depends greatly on the chemistry of the
mixed liquid (such as pH, temperature and alkalinity). For digestion
of EAS without signicantly changing the chemistry of the liquid,
the involvement of physiochemical reaction is considered much
smaller than biological reactions (Neyens et al., 2003). The DOM
detected during digestion is the combined result of formation by
hydrolysis involving both cell disruption and the release of

intracellular materials (such as proteins, carbohydrates and lipids),

and degradation of the products of the hydrolysis process (such as
sugars, amino acids and fatty acids). First-order reaction model is
well used for simple simulation of the hydrolysis process of organic
solids, including EAS (Mani et al., 2016). For degradation of the
hydrolysis products, rst-order reaction model is also well used
(Rao and Singh, 2004; Adak et al., 2011). Based on these, in this
study, for simple quantitative comparison of the differences in the
digestion rate of EAS under the investigated experiment conditions,
the following rst-order sequential reaction model was used to
describe the observed concentration proles of DOM:

dC  CBOM
kh $Ca  kd $C  CBOM
dt

(2)

dCa
kh $Ca
dt

(3)

where, kh is the rate parameter of hydrolysis and kd is the rate

parameter of degradation; C is the detected DOM concentration; t is
the reaction time; Ca0 is the total concentration subjected to hydrolysis; Ca is the concentration of the hydrolysis product (i.e., the
total concentration of the formed DOM from the EAS due to hydrolysis); CBOM is the initial concentration of DOM existed in the
liquid phase of the EAS supplied to each reactor, referred here as
background organic matter (BOM). For fresh EAS, BOM is mainly
consisted of the dissolved organic substances remaining in the
water after biological treatment and is generally considered least or
non-biodegradable. From the above two equations, the following
solution can be obtained:


kh $Ca0  kh $t
* e
 ekd $t CBOM
kd  kh

(4)

Since TOC is more appropriate than COD and UV260 for complete
quantication of DOM, in this study, the observed concentration
proles of TOC were used for searching the rate parameters kh and
kd under all experiment conditions. The searching was made by
minimizing the relative error dened by the following equation (Eq.
(5)) between the observed TOC concentrations and the calculated

Fig. 5. The destruction and lysis of microorganisms observed by SEM (  19000) during digestion in aerobic (a) and anaerobic (b) reactors operated at 20  C with the TS concentration of 1.2%.

Please cite this article in press as: Du, H., Li, F., Characteristics of dissolved organic matter formed in aerobic and anaerobic digestion of excess
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H. Du, F. Li / Chemosphere xxx (2016) 1e10

3.3. Composition of DOM assessed by VFAs

ones from Eq. (4). For CBOM, since the supernatant of the obtained
sludge after centrifugation and ltration was used to dilute the
centrifuged sludge for the experiment, its value was the TOC concentration of the supernatant (17.7 mg/L). Ca0 was the sum of CBOM
(17.7 mg/L) and the converted TS concentration to TOC (4511, 9022
and 20294 mg/L for TS concentrations of 1.2, 2.3 and 5.2%,
respectively) based on the conversion ratio, i.e., the content of
organic carbon in TS measured by a C/N analyzer (CN-22F, Shimadzu Co., Japan) (38.4% for the EAS used in this study).

v
!2
u
n u C
X
1
iobs  Cical
t
Error $
n
Ciobs

Aerobic and anaerobic digestion of EAS result in different intermediate and nal organic compounds (Martinez-Garcia et al.,
2014; Fall et al., 2014). For all the TS and temperature conditions
investigated in this study, the total concentration of VFAs detected
in the anaerobic reactors was higher than that in the aerobic reactors and changed with the operation time (data not shown). In
addition, compared to the aerobic reactors where, in most cases,
only citrate was detected; in the anaerobic reactors, however,
among all seven VFAs targeted (citrate, isobutyrate, acetate, propionate, butyrate, valerate and isovalerate), except for valerate, all
other six species were detected even if their concentrations differed
with the digestion time, TS concentrations and temperatures.
At the end of 61 days' operation, the results plotted in Fig. 6
showed that, in the aerobic reactors, as the only VAF species
detected, citrate in the reactors with the TS concentrations of 1.2,
2.3 and 5.2% fell in the range of 5.7e20.2 mg/L. In the reactors with
the same TS concentration (1.2%) but different temperatures, a
trend of signicant increase of citrate from 7.3 to 12.4 mg/L was
revealed as the temperature increased from 5 to 20  C and reached
47.6 mg/L at 35  C after 46 days. In the anaerobic reactors, in
addition to citrate, isobutyrate was the main VFA species remaining

(5)

i1

where, Ci (obs) and Ci (cal) are the observed and calculated TOC
concentrations in the liquid phase of each reactor, and n is the
number of samples collected for TOC analysis.
The calculated TOC proles that best tted the observed data are
depicted in Figs. 1 and 2 as solid lines, and the searched kh and kd
are summarized in Table 2. Compared to aerobic digestion, anaerobic digestion was found faster for hydrolysis to take place. As
shown in Table 2, for aerobic digestion, kh increased from 0.0005
day1 to 0.0010 day1 as temperature increased from 5 to 35  C for
reactors with the TS concentration of 1.2%; and from 0.0007 day1
to 0.0205 day1 as TS increased from 1.2 to 5.2% for the reactors at
20  C. For anaerobic digestion, the corresponding increase of kh
with the increase of temperature was from 0.0007 day1 to 0.0493
day1 and that with the increase of TS concentration was from
0.0031 day1 to 0.0258 day1, respectively; with the values being
generally higher than those estimated for the aerobic digestion. The
trend of increases of kh with the increases of temperature supported the result of Lin et al. (2016) that the hydrolysis rate
increased as the temperature of digestion increased from 25 to
50  C.
In regard of the degradation rate, kd for the aerobic digestion
varied from 0.0407 to 0.0667 day1 as temperature increased from
5 to 35  C and from 0.0552 to 0.5580 day1 as TS increased from 1.2
to 5.2%; with its values being obviously higher than those for the
anaerobic digestion (from 0.0201 at 5  C to 0.0464 day1 at 20  C
and 0.0464 to 0.1922 day1 as TS increased from 1.2 to 5.2%,
respectively). The higher rate of hydrolysis and lower rate of
degradation for anaerobic digestion, as compared to aerobic
digestion, was the reason for accumulation of DOM in the reactors,
resulting in not only higher values of TOC and UV260 but also
different SUVA as discussed earlier.

Fig. 6. The concentrations of VFAs detected at the end of digestion for 61 days in the
aerobic and anaerobic reactors operated under different TS concentrations and temperatures. *The value for day 46.

Table 2
The rate parameters of hydrolysis and degradation of DOM in aerobic and anaerobic digestion of excess activated sludge estimated using a sequential reaction model.
Aerobic
TS 1.2%

20  C

5 C
20  C
35  C
TS 1.2%
TS 2.3%
TS 5.2%

Anaerobic
TS 1.2%

20  C

5 C
20  C
35  C
TS 1.2%
TS 2.3%
TS 5.2%

kh

kd

Error

0.0005
0.0007
0.0010
0.0007
0.0022
0.0205

0.0407
0.0552
0.0667
0.0552
0.1112
0.5580

0.125
0.153
0.243
0.153
0.027
0.136

kh

kd

Error

0.0007
0.0031
0.0493
0.0031
0.0120
0.0258

0.0201
0.0464
0.5513
0.0464
0.1097
0.1922

0.014
0.006
0.118
0.006
0.049
0.062

kh: rst-order hydrolysis rate parameter (day1); kd: rst-order degradation rate parameter (day1); Error: the difference between calculated and observed TOC concentrations dened by Eq. (5).

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H. Du, F. Li / Chemosphere xxx (2016) 1e10

Fig. 7. The uorescence EEM of DOM during digestion in the aerobic (a) and anaerobic (b) reactors operated under 20  C with the TS concentration of 1.2%.

and its concentration varied in 3.5e20.8 mg/L. A unique trend of

either increases or decreases of citrate and isobutyrate with the
increases of TS or temperature was not revealed.

3.4. Composition of DOM assessed by uorescence EEM

Peak 1 and 2 at the excitation/emission (Ex/Em) wavelengths of
485/514 and 340/428, reported to reect humic acid-like substances (Chen et al., 2003), were two common peaks emerged

Fig. 8. Fluorescence intensity of all peaks of DOM in the aerobic and anaerobic reactors
at the end of digestion for 61 days. *The value for day 46. Peak 1, 2, 6, 7: humic acid-like
substances; Peak 3: aromatic proteins; Peak 4: tryptophan-like and tyrosine-like aromatic amino acids; Peak 5: fulvic acid-like substance and Peak 8: peptides and
proteins.

during both aerobic and anaerobic digestion, as shown in Fig. 7.

Peak 3 and 4 at the Ex/Em of 280/344 and 225/340, which are reported to reect aromatic proteins and tryptophan- and tyrosinelike aromatic amino acids (Hudson et al., 2008; Spencer et al.,
2007; Wu et al., 2004), were two specic peaks emerged only
during anaerobic digestion; while, Peak 5 and 6 at the Ex/Em of 240/
436 and 270/441, reported to reect fulvic acid- and humic acid-like
substances, respectively (Chen et al., 2003), were two specic peaks
emerged only during aerobic digestion. This indicated that, for
aerobic digestion of EAS, humic and fulvic acid-like substances that
are less or not biodegradable hydrophobic acids primarily formed

Fig. 9. Molecular weight distributions of DOM at the end of digestion for 61 days in the
aerobic and anaerobic reactors under different TS concentrations and temperatures.
*The value for day 46.

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H. Du, F. Li / Chemosphere xxx (2016) 1e10

Fig. 10. The weight-averaged molecular weight (Mw) and polydispersity of DOM at the
end of digestion for 61 days in the aerobic and anaerobic reactors under different TS
concentrations and temperatures. *The value for day 46.

from the hydrolysis of EPS (Pang et al., 2014) were the major
organic species formed and remaining in the liquids after treatment. For anaerobic digestion, however, aromatic proteins and
tryptophan- and tyrosine-like aromatic amino acids as the hydrolysates of proteins and other substrates (Pang et al., 2014; Spencer
et al., 2007) were the major organic species that constitute for the
major fractions of DOM formed and remaining in the liquids after
anaerobic digestion.
The values of the uorescence intensity of all peaks remaining at
the end of the digestion for 61 days are plotted in Fig. 8. The total
uorescence intensity was higher for the EAS after anaerobic
digestion, suggesting that relatively larger proportions of the hydrolysis products were remaining in the liquid due probably to
their slower degradation rate as shown earlier in Table 2. In addition, it is obvious to see that, during the whole operation, Peak 2
and 5 were detected with comparatively higher percentages in the
DOM evaluated by the uorescence intensity in the aerobic reactors; and Peak 3 and 4 in the anaerobic reactors. This indicated
that DOM formed and remaining after aerobic digestion contains
higher content of humic acid- and fulvic acid-like substances; and
that after anaerobic digestion contains higher content of aromatic

Fig. 11. The results of the principal component analysis (component loadings and component scores) using all observed data during the whole process of aerobic (a, b) and
anaerobic (c, d) digestions under different TS concentrations and temperatures.

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H. Du, F. Li / Chemosphere xxx (2016) 1e10

proteins and tryptophan- and tyrosine-aromatic amino acids. This

coincides with the previous result that proteins were the largest
organic fraction of EAS (Pang et al., 2014); and more proteins and
less humic substances were formed under anaerobic condition
(Ramdani et al., 2012; Shao et al., 2013).

3.5. Composition of DOM assessed by molecular weight

Compared to aerobic digestion that resulted in a MW distribution with a broader range and a distinct peak corresponding to the
smallest MW organic fraction, anaerobic digestion resulted in a
distribution that had a narrower MW range and positioned on the
left side reecting relatively larger molecular weight, as could be
seen in Fig. 9.
The values of the weight-averaged molecular weight, Mw, and
the polydispersity of DOM remaining at the end of digestion are
plotted in Fig. 10. Except for the ones for the TS of 5.2% and temperature of 5  C, Mw was generally larger for the DOM formed and
remaining after anaerobic digestion than after aerobic digestion.
For both aerobic and anaerobic digestion, a unique trend of either
increases or decreases of Mw with the increases of the TS or temperature was not revealed, suggesting that, judging from Mw, the
composition of DOM did not change signicantly with the changes
of TS concentrations and temperatures.
In regard of the heterogeneity of the DOM in molecular weight,
the computed values of polydispersity indicated that, excluding the
two exceptions mentioned above with the results of Mw, the DOM
constituents formed and remaining after aerobic digestion were, in
general, more heterogeneous than those formed and remaining
after anaerobic digestion.

3.6. Principal component analysis (PCA)

For aerobic digestion, the component loading plots given in
Fig. 11(a) showed that the loadings of TOC, SCOD, Peak 3 and Peak 4
reecting biodegradable organic constituents (including proteins
and aromatic amino acids) as PC1 were higher; and the loadings of
Peak 2, Peak 5 and Peak 7 reecting the less biodegradable constituents of humic molecules as PC2 were higher. The score plots
given in Fig. 11(b) showed that for reactors R1 - R3 (TS 1.2%, temperatures 5e35  C), the score did not change obviously with the
digestion time, suggesting the composition of DOM was similar
during the aerobic digestion. For R4 (TS 2.3%, temperature 20  C),
the score of PC2 showed slight increases, indicating that the fraction of humic substances was increasing with the digestion time.
For R5 (TS 5.2%, temperature 20  C), the score of PC1 decreased
from day 2 and day 15 to day 31 and then increased slightly until
day 61; and the score of PC2 showed a consistent trend of increases.
This indicated that for this reactor fed with the highest TS concentration (5.2%), as digestion processed over time, the fraction of
biodegradable DOM constituents gradually disappeared and in return, the less and non-biodegradable fraction gradually increased.
For anaerobic digestion, as shown in Fig. 11(c) and (d), the loadings
of TOC, SCOD, VFAs, UV260, Peak 2, Peak 3 and Peak 4 as the PC1
were higher and the loadings of Peak 6, Peak 7 and Peak 8 as the
PC2 were higher. Combination of the loading results with the corresponding score plots indicated that for R10 (TS 5.2%, temperature
20  C), both biodegradable and non-biodegradable DOM constituents increased from day 2 to day 15 and then started to decrease;
for R8 (TS 1.2%, 35  C), the fraction of non-biodegradable constituents in DOM increased rapidly from day 2 to day 15 and then
turned to a graduate decrease until day 61.

4. Conclusions
Using ve aerobic and ve anaerobic digestion reactors, the
characteristics of DOM formed and remaining in the liquid of the
digestion treatment of EAS of a representative WWTP were
investigated. Compared to aerobic digestion, anaerobic digestion
resulted in signicantly higher concentrations of DOM. This was
due to the accumulation of organic substances brought about by
the higher rate of hydrolysis and the lower rate of degradation. A
general trend of concentration increases of DOM with the increases
of TS and temperature was conrmed. Model simulation indicated
that increasing TS could promote both hydrolysis and degradation;
however, the effect was more obvious in aerobic than anaerobic
digestion. Increasing temperature also promoted hydrolysis and
degradation; however, different from the effect of TS, the effect of
temperature was found more obvious in anaerobic digestion. In
addition to these, the composition analysis by the uorescence EEM
indicated that more humic and fulvic -like substances were formed
and remaining during aerobic digestion; while during anaerobic
digestion, more proteins and aromatic amino acids were formed.
Moreover, the composition analysis based on molecular weight
distribution indicated that the UV-absorbing DOM constituents
formed in anaerobic digestion had larger molecular weight but was
less heterogeneous than those formed in aerobic digestion. In association with the increases of DOM, for both aerobic and anaerobic
conditions, increasing the digestion temperature from 5 to 35  C
signicantly accelerated the destruction and lysis of microorganisms (SEM images not provided here), leading to increases in the
hydrolysis rate, with the effect being more obvious in anaerobic
digestion.
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