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h i g h l i g h t s
The removal of EPS from WAS increased sludge reduction and methane production.
The enhancement was due to the increased hydrolytic activity during fermentation.
Clostridium species contributes to the enhancement of methane production.
a r t i c l e i n f o a b s t r a c t
Article history: The management of waste activated sludge (WAS) recycling is a concern that affects the development of
Received 8 February 2014 the future low-carbon society, particularly sludge reduction and biomass utilization. In this study, we
Received in revised form 10 July 2014 investigated the effect of removing extracellular polymeric substances (EPS), which play important roles
Accepted 20 August 2014
in the adhesion and occulation of WAS, on increased sludge disintegration, thereby enhancing sludge
reduction and methane production by anaerobic digestion. EPS removal from WAS by ethylenediamine-
Handling Editor: Chang-Ping Yu tetraacetic acid (EDTA) signicantly enhanced sludge reduction, i.e., 49 5% compared with 27 1% of
the control at the end the digestion process. Methane production was also improved in WAS without
Keywords: EPS by 8881 109 CH4 lmol g1 dry-weight of sludge. Microbial activity was determined by denaturing
Extracellular polymeric substances gradient gel electrophoresis and real-time polymerase chain reaction, which showed that the hydrolysis
Methane production and acetogenesis stages were enhanced by pretreatment with 2% EDTA, with a larger methanogenic
Sludge disintegration community and better methane production.
Waste activated sludge 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.chemosphere.2014.08.055
0045-6535/ 2014 Elsevier Ltd. All rights reserved.
M.T. Nguyen et al. / Chemosphere 117 (2014) 552558 553
The utilization of carbon source in waste activated sludge is a illustrating each method is shown in Fig. 1. After EPS extraction,
promising approach to produce valuable energy sources such as the pellet was washed twice to completely remove the extractant
biohydrogen (Mohd Yasin et al., 2013), and for electricity genera- that remained in sludge, and it was used for methane fermentation,
tion on the basis of microbial fuel cells (Mohd Yusoff et al., while the supernatant was processed by centrifugation and mem-
2013). Methane production by anaerobic digestion has gained pop- brane dialysis. The supernatant included extracted EPS was trans-
ularity for reducing the quantity of sludge generated and the ferred to a white porcelain dish and dried in drying-oven at 105 C
recovery of renewable energy (Kanai et al., 2010). Methane is gen- for two days. The extracted EPS was measured by the weight sub-
erated by the continuous degradation of organic materials in WAS traction of the dish before and after drying process. According to a
by various microorganisms (Buczkowska et al., 2010). This fermen- reported method (Liu and Fang, 2002), 2% EDTA should be used for
tation process generally comprises four stages: hydrolysis, acido- the extraction process, but different EDTA concentrations (0.1%,
genesis, acetogenesis, and methanogenesis (Demirel and Scherer, 0.5%, 2%, and 4%) were tested in the present study to determine
2008). In general, methane is produced by methanogens from ace- the effects of different degrees of EPS extraction by EDTA in meth-
tic acid, carbon dioxide, and hydrogen generated in the earlier ane fermentation. To verify that the effects of EDTA on anaerobic
reactions. In WAS, extracellular polymeric substances (EPS) play digestion with EPS-extracted sludge were not due to the presence
important roles in sludge occulation and cohesion (Frlund of EDTA itself in the fermentation culture, an additional fermenta-
et al., 1996). EPS are metabolic products secreted by microorgan- tion vial (20 g of 25% raw sludge) with EDTA at a 2% nal concen-
isms during their growth, which cover the cell surface layer to pro- tration was incubated at 37 C in a shaking incubator at 120 rpm
tect against the severe external environment (Liu and Fang, 2002). for 15 d (Bio-shaker, BV-180LF, Taitec, Japan). To observe any
EPS have a complex biochemical composition, including protein, structural changes in the sludge after the EPS extraction process,
carbohydrate, lipid, humic substances, uronic acid, and deoxyribo- 5 mL of sludge samples with or without pretreatment were directly
nucleic acids (Tsuneda et al., 2003). EPS are considered important analyzed using a scanning electron microscope (SEM) where the
for the physicochemical properties of the activated sludge oc surfaces of the samples were compared by SEM visualization (Hit-
and have been implicated in determining the structure, charge, achi S-3500N) (Mohd Yasin et al., 2013).
and occulation of the WAS oc (Frlund et al., 1996). Therefore,
the present study aimed to disintegrate WAS by extracting the 2.3. Methane production
EPS before anaerobic digestion. We hypothesize that removing
EPS from WAS may reduce the sludge occulation and subse- The fermentation process used 20 g of 25% sludge (16 g of trea-
quently more materials and bacteria would be exposed in the ted sludge as a substrate and 4 g of raw sludge as an inoculum) in
liquid phase, thereby enhancing the later stages of anaerobic diges- 50-mL vials. The inoculum to sludge ratio (0.25) was based on dry
tion. In addition, no previous studies have mentioned the effect of solid basis (Raposo et al., 2006). The fermentation vials were sealed
EPS extraction on sludge reduction and methane production from with crimped butyl rubber stoppers and sparged with nitrogen for
WAS. This study provides an overview of the effect of EPS removal 5 min and subsequently incubated at 37 C in a shaking incubator
on anaerobic fermentation. at 120 rpm for 15 d (Bio-shaker, BV-180LF, Taitec, Japan). Each day,
50 lL of the headspace gas was analyzed using gas chromatogra-
phy (GC) using a 6890 N gas chromatograph (Agilent Technologies,
2. Materials and methods
Glastonbury, CT) equipped with a 80100 mesh Porapak Q column
(Suppelco, Bellefonte, PA) and a thermal conductivity detector. The
2.1. Sludge preparation
injector and detector were maintained at 100 C and 200 C respec-
tively. The nitrogen carrier gas ow rate was maintained at
Raw sludge was collected from Hiagari WWTP in Kitakyushu
20 mL min1 and the column temperature was 70 C.
City, Japan. The sludge composition was previously reported
(Maeda et al., 2009). Total solid (TS) composed of 3.34.0%, volatile
2.4. Vital microbial community analysis
solid (VS) (2.63.1%), suspended solid (SS) (3041%), volatile sus-
pended solid (VSS) (2633%) and chemical oxygen demand (COD)
2.4.1. RNA extraction and complementary DNA synthesis
of 4451 g L1. Protein was the main component of the sludge
A 4-mL sample of fermented sludge was mixed with 3 mL of
(4045% of total solid), followed by carbohydrate (1214% of total
RNAlater solution (Formerly Ambion, Applied Biosystems) in a
solid) and lipid (1113% of total solid). Raw sludge was washed
15-mL falcon tube before centrifugation at 15 000 rpm for 2 min.
three times by centrifugation at 8000 g for 10 min at 4 C, and
The cell pellet was chilled using dry ice dissolved with ethanol
the pellet was resuspended in distilled water, before adjusting its
for 30 s and stored at 70 C before RNA extraction. The total
concentration to 25% (w/w) with distilled water.
RNA was extracted using an RNeasy kit (Qiagen, Inc., Valencia,
CA) with a bead beater (Wakenyaku Co. Ltd., Kyoto Japan, model
2.2. EPS extraction 3011b), as previously described (Mohd Yusoff et al., 2012). The
complementary DNA (cDNA) was synthesized from the extracted
The prepared sludge was used for EPS extraction according to RNA using a PrimeScript RT reagent kit Perfect Real Time with ran-
published methods using ethylenediaminetetraacetic acid (EDTA) dom oligomers (TAKARA Bio Inc.). Agarose gel electrophoresis was
as an extractant as well as ultrasonic and autoclave treatments conducted to verify the success of RNA isolation and cDNA synthe-
(Liu and Fang, 2002). As reported, formaldehyde plus NaOH is sis. The cDNA were used later as the DNA template for denaturing
the most efcient method for EPS extraction and follow by EDTA gradient gel electrophoresis (DGGE) and real-time polymerase
method, however the difference of the amount of extracted EPS chain reaction (RT-PCR) quantication.
is not major (Liu and Fang, 2002). In addition, under treatment of
NaOH not only EPS is extracted but also sludge components are 2.4.2. DGGE
breakdown and at a high pH the cellular substance is more suscep- To analyze the changes in the bacterial community, DGGE was
tible by the effect of alkaline treatment (Kim et al., 2010). This conducted using the cDNA samples. The PCR amplication was
study mostly focused on the effect of EPS removal on methane performed using a RoboCycler Gradient 40 (Stratagene). The nucle-
production by anaerobic digestion therefore EDTA method was otide sequences of the primers were shown in Table 1. Primer 3
selected since its high ability of EPS extraction. A owchart had the same sequence as primer 1 but its 50 -end had an additional
554 M.T. Nguyen et al. / Chemosphere 117 (2014) 552558
Table 1
Primer and probe set used in RT-PCR analysis.
were collected (10 g wet weight) and centrifuged at 18 000g for sludge using EDTA produced sludge with a smaller structure, and
10 min. The pellets were transferred to porcelain dishes and dried the sludge oc was signicantly interrupted (Fig. 3b). Improved
in an oven (D-300, IUCHI, Japan) at 105 C for 2 d to determine sludge disintegration enhanced the hydrolysis stage during the
the dry weight. The supernatant were centrifuged at 13 000 rpm anaerobic digestion process, as discussed later.
for 7 min to remove any remaining suspended solids and were used
to measure the soluble chemical oxidation demand (SCOD) and pro-
tein concentrations. The other analysis such as total solid (TS), vol- 3.2. Effects of EPS extraction on sludge reduction and changes in the
atile solid (VS), suspended solid (SS), volatile suspended solid (VSS), composition
carbohydrate and lipid were measured as described in previous
study (Maeda et al., 2009). Subsequently, the reduction level of each treated sludge was
evaluated on days 5, 10, and 15 of the methane fermentation pro-
cess, and the results are shown in Fig. 4. The highest ratio of sludge
2.5.2. Analysis of soluble chemical oxygen demand (SCOD) and protein
reduction was obtained when the sludge EPS was extracted with
concentration
2% EDTA, which rapidly increased by 38 1% after 5 d of incuba-
The independent vials were sampled for the rst 5 d prior for
tion. After pretreatment with 2% EDTA, sludge reduction was sig-
SCOD and protein concentration measurement. The protein concen-
nicantly enhanced and it reached 49 5% at the end of the
trations were analyzed using the Lowry method where bovine
digestion process compared with 27 1% for the control. The
serum albumin was used as the standard substance (Li et al.,
sludge reduction ratio for the sludge subjected to ultrasonic treat-
2009). A UV/Vis spectrophotometer (V-530, Jasco, Tokyo, Japan)
ment was 28 4%, which was slightly higher than the control.
was used to measure the absorbance of samples. To determine
Autoclaving produced a sludge reduction ratio that was similar
the SCOD concentration, the supernatant was diluted to an appro-
to the control. The low sludge reduction was observed compared
priate concentration and measured with a COD meter (COD-60A,
to blank at day 5 might be due to the reductions of bacterial com-
DKK-TOA Corporation, Japan). Five mL of sample was mixed with
munity from autoclave and ultrasonic treatment. The bacterial
2 ml of solution I (143C143) and 0.5 ml of solution II (143C144)
community was important in order to effectively utilized sludge
and reux by COD meter. All reagents and COD measurement were
as a carbon source (Li et al., 2009).
according to manufacturers protocol (DKK-TOA Corporation,
Hydrolysis is the rst stage of the anaerobic digestion process,
Japan).
where polymer materials in the sludge break down into smaller
molecules (Demirel and Scherer, 2008). Therefore, we used sludge
3. Results and discussion where the EPS was extracted with 2% EDTA to investigate sludge
reduction during the 5 initial days of fermentation to determine
3.1. Extracted EPS weight and structural changes in the sludge after the effects of EPS removal on sludge hydrolysis. The result shows
EPS extraction that pretreatment with 2% EDTA helped to improve the hydrolysis
stage of anaerobic digestion, resulting in a higher sludge reduction
Fig. 2 shows the amounts of EPS extracted using the different ratio compared with the control (Fig. 5a) from the rst day, which
methods shown in Fig. 1. EPS treatment with 2% EDTA obtained persisted throughout the fermentation process. To corroborate this
the highest weight of EPS, i.e., 296 10 mg g1 dry-weight sludge, result, the components of the fermentation culture were also
compared with other methods. The amount of EPS extracted by investigated during hydrolysis, particularly protein because it
EDTA increased with the concentration of EDTA from 0.1% to 2%. was the major component of the sludge used in this study
However, when the EDTA concentration was 4%, there was no (Maeda et al., 2009). The SCOD also indicates that the particulate
severe difference between 2% and 4% EDTA used. The amount of organic matter in the sludge becomes soluble in the liquid phase
EPS extracted by autoclaving was higher than that by ultrasonic (Yan et al., 2013). Therefore, the protein and SCOD concentrations
method, and the lowest amount was obtained with the control, were analyzed. Fig. 5b shows the changes in the SCOD and protein
which was subjected to centrifugation alone. These results agreed concentrations during the initial stage of anaerobic digestion from
with those reported in previous studies, where chemical extraction three independent experiments. The SCOD concentration of the
generally obtained higher amounts of EPS compared with physical fermentation culture with 2% EDTA pretreatment was higher than
methods (Pan et al., 2010). EDTA appears to be the most effective that of the control, and it continued to increase throughout
method because of its ability to increase the solubility of EPS the incubation period. On the rst day of fermentation, pretreat-
(Comte et al., 2006; Adav and Lee, 2008). ment with EDTA increased SCOD by approximately threefold com-
The structure of the sludge after treatment with 2% EDTA is pared with no EDTA treatment (31 3 mg L1 compared with
shown in Fig. 3. Sludge disintegration was apparently enhanced 12 2 mg L1, respectively). The protein concentration exhibited
in sludge samples subjected to EDTA treatment. In the untreated a trend similar to SCOD. Fermentation culture of the sludge where
sludge, sludge components occulated together with a dense mor- EPS was extracted by 2% EDTA yielded a higher protein concentra-
phology (Fig. 3a). In contrast, the removal of EPS from treated tion compared with the control (Fig. 5b). We concluded that pre-
treatment with 2% EDTA increased sludge disintegration and
enhanced the hydrolysis step, thereby resulting in greater release
of soluble materials into the liquid phase; these precursors were
the main substrates for the further stages of the methane produc-
tion process. The increase in nutrient contents such as carbohy-
drate and protein after EPS removal was proved by other studies
(Adav and Lee, 2008). Several attempts have been made to remove
EPS in sludge such as formaldehyde, formaldehyde plus NaOH and
ultrasonication. Although the methods are different, the constitu-
ent of EPS extracted by different treatments mostly includes pro-
tein, carbohydrate and humic acid. Extracted EPS from sludge
also consist of lipid, uronic acid and DNA (Liu and Fang, 2002;
Fig. 2. Extracted-EPS weight by different extraction methods. Adav and Lee, 2008).
556 M.T. Nguyen et al. / Chemosphere 117 (2014) 552558
Fig. 3. Scanning electron microscope (SEM) micrograph under 500 magnication of (a) sludge without treatment and (b) sludge treated by EDTA 2%.
Fig. 5. Sludge reduction ratio (a) and component changes (b) during hydrolysis stage.
M.T. Nguyen et al. / Chemosphere 117 (2014) 552558 557
Acknowledgments
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