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Chemosphere 117 (2014) 552558

Contents lists available at ScienceDirect

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

Enhancement of sludge reduction and methane production by removing


extracellular polymeric substances from waste activated sludge
Minh Tuan Nguyen a, Nazlina Haiza Mohd Yasin a, Toshiki Miyazaki a, Toshinari Maeda a,b,
a
Department of Biological Functions Engineering, Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, 2-4 Hibikino,
Wakamatsu-ku, Kitakyushu 808-0196, Japan
b
Research Center for Advanced Eco-tting Technology, Kyushu Institute of Technology, 2-4 Hibikino, Wakamatsu-ku, Kitakyushu 808-0196, Japan

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.

1. Introduction aeration, equipped membrane bioreactor or added additives, the


ability to reduce WAS is about 2030% (Mahmood and Elliott,
Thus far, most domestic and industrial wastewater treatment 2006). The hazardous materials present in WAS, such as pathogens
plants (WWTP) use the activated sludge process because of its abil- and toxic chemicals, can harm the environment if it is inappropri-
ity to degrade organic contaminants in wastewater and discharge ately discharged (Li et al., 2009). Landll and incineration are the
clean water, carbon dioxide, and biomass (Rocher et al., 1999). conventional methods used to dispose of WAS; however, these
However, the major disadvantage of this method is the generation solutions create a burden for society because of their land space
of a large amount of waste activated sludge (WAS). In Japan, WAS requirements, operational costs, and the need for strict human
accounts for approximately 47% of industrial waste (Yoshida et al., health and environmental protection regulations (Wei et al.,
2009) and 2560% of the operating costs of WWTPs (Yan et al., 2003). Various methods and strategies have been developed to
2008). By some modications through operational control in the reduce WAS generation, such as chemical treatments using metal
industrial scale treatment such as extended and improved cations (Kim et al., 2002) and oxidizing substances (Weemaes
et al., 2000; Neyens et al., 2003). Individual or combined thermal
processes and ultrasound and alkaline treatments have been
Corresponding author at: Department of Biological Functions Engineering, widely used for sludge solubilization (Kepp et al., 2000; Vlyssides
Graduate School of Life Science and Systems Engineering, Kyushu Institute of
and Karlis, 2004; Bougrier et al., 2005; Li et al., 2008). Combined
Technology, 2-4 Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka 808-0196, Japan.
Tel.: +81 93 695 6064; fax: +81 93 695 6005. thermal, chemical, and mechanical treatments have also been used
E-mail address: toshi.maeda@life.kyutech.ac.jp (T. Maeda). as a sludge reduction strategy (Nah et al., 2000; Valo et al., 2004).

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

Fig. 1. Procedures of EPS extraction process.

Table 1
Primer and probe set used in RT-PCR analysis.

Target Function Sequence (50 30 ) Amplicon size Representative strains References


group (bp)
Archaea Forward ATTAGATACCCSBGTAGTCC 273 Methanobacteriumformicicum and Lee et al. (2008)
primer Methanosarcinabarkeri
Reverse GCCATGCACCWCCTC T
primer
TaqMan AGGAATTGGCGGGGGAGCAC
Probe
Bacteria Forward TCCTACGGGAGGCAGCAGT 466 Escheriachia coli K12 Nadkarni et al.,
primer 2002
Reverse GGACTACCAGGGTATCTAATCCTGTT
primer
TaqMan CGTATTACCGCGGCTGCTGGCAC
Probe
Bacteria Primer 1 CCTACGGGAGGCAGCAG 161 Escheriachia coli Muyzer et al.
Bacteria Primer 2 ATTACCGCGGCTGCTGG (1993)
Bacteria Primer 3 CGCCCGCCGCGCGCGGCGGGCGGGGC
GGGGGCACGGGGGGCCTACGGGAGGCAGCAG

40-nucleotide GC-rich sequence (GC clamp). Combinations of 2.4.3. RT-PCR quantication


primers 1and 2 or primers 2 and 3 were used to amplify the 16S RT-PCR was performed to quantify the 16S rRNA gene abun-
rDNA regions in the different bacterial species that corresponded dance in the total Archaea and bacterial groups. An RT-PCR system
to positions 341534 in Escherichia coli (Muyzer et al., 1993). The (Step one, Applied Biosystem) was used for amplication and uo-
PCR cycle was as follows: initially 94 C for 5 min, then 94 C for rescence detection. The primer and probe sets used are shown in
1 min, 63 C for 1 min decreasing by 1 C every cycle until 57 C, Table 1. The reaction mixture comprised the following: 10 lL of
72 C for 1 min, followed by 18 cycles of 94 C for 1 min, 57 C Taqman fast advanced master mix (2), 0.72 lL of each primer
for 1 min, and 72 C for 1 min, with a nal step at 72 C for (nal concentration, 900 nM), 0.34 lL of TaqMan probe (nal con-
25 min. DGGE was performed using an 8% (w/v) acrylamide gel, centration, 200 nM), 6.22 lL of nuclease-free water, and 2 lL of
which contained a 3057% denaturant gradient. Electrophoresis cDNA template. The operating protocol was as follows: UNG incu-
was conducted in 1  Tris/Borate/EDTA (TBE) buffer for 7 h at bation at 50 C for 2 min, polymerase activation at 95 C for 20 s,
60 C and 50 V. The gel was stained with ethidium bromide and 40 cycles of PCR at 95 C for 1 s and 60 C for 20 s. Each cDNA
(0.5 lg mL1) for 45 min, and the stained gel was visually scanned template prepared for the reaction was analyzed in triplicate. The
using Astec Imagersoftware (Gel Scene Imaging system). The visu- standard curves were calculated as previously described (Lee et al.,
alized bands were excised and immersed in sterilized water over- 2008). Representative strains were used to construct standard
night before use for PCR amplication. A 2.3-lL aliquot of the curves (Table 1). Tenfold serial dilutions were generated and
mixture was used for PCR amplication with the primer set with- amplied in triplicate using real-time PCR with the corresponding
out the GCclamp. Electrophoresis was performed using the entire primer and probe sets. The CT values were plotted against the log-
PCR product by 0.8% agarose gel at 100 V for 25 min to facilitate arithms of the initial template copy numbers.
verication and purication. The visualized bands were extracted
using a QIAquick Gel extraction kit (Qiagen). The 16S rRNA gene 2.5. Analytical methods
fragments were sent for DNA sequencing by FASMAC Co. (Japan).
The sequencing results were compared with reference sequences 2.5.1. Sludge reduction and supernatant collection
in the National Centre of Biotechnology Information (NCBI) Sludge reduction was measured as previously described by
nucleotide sequence database. Maeda et al. (2011). Sludge samples (before and after fermentation)
M.T. Nguyen et al. / Chemosphere 117 (2014) 552558 555

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%.

gas was produced from treated sludge, where a large amount of


EPS was extracted. Extraction using the ultrasonic and autoclave
methods yielded methane productivities similar to the control
(Fig. 6), although these two methods were more efcient at remov-
ing EPS when compared to control (Fig. 2). However, pretreatment
with 2% EDTA was found to be the most efcient in removing EPS.
This may be explained by the reductions in the bacterial commu-
nity caused by the ultrasonic and autoclave treatments. As men-
tioned earlier, 2% EDTA pretreatment enhanced the initial
hydrolysis stage of fermentation, which is the stage with the high-
est EPS removal efciency. This helped to generate greater quanti-
ties of precursors for the later stages of fermentation, which
resulted in higher methane production.
Another study was conducted to verify that the positive effect of
Fig. 4. Sludge reduction ratio during fermentation process. EDTA treatment for methane production was not because of EDTA
remained in the sludge after treatment. Methane production was
tested in the presence and absence of 2% EDTA. However, the
3.3. Methane production using sludge with extracted EPS results showed that EDTA had a negative effect on methane pro-
duction; and after 15 d of incubation, the methane productivity
As shown in Fig. 6, we aimed to explain the effect of EPS removal in the presence and absence (control) of 2% EDTA were 4033 and
on methane production at the early phase of fermentation, particu- 57 763 lmol g1 dry-weight sludge, respectively. This supported
larly in hydrolysis phase. Thus, the experiments were carried out for our conclusion that the improved methane productivity from
the rst 15 d to compare the rate of methane production by differ- sludge where the EPS was extracted by EDTA was initially induced
ent pre-treatment method. Methane production from sludge where by EPS removal.
EPS were extracted by 2% EDTA had the highest productivity, i.e.,
8881 109 lmol g1 dry-weight sludge, at the end of the fermenta- 3.4. Bacterial community analysis by DGGE
tion process (Fig. 6). Fermentation using sludge that received 2%
EDTA pretreatment increased methane production from the second The template used for DGGE analysis was the cDNA synthesized
day, and this persisted throughout the fermentation process. The from the RNA extraction product, which was employed to evaluate
methane productivities from sludge where EPS was extracted with the active bacteria in each condition. Several bands were detected
different EDTA concentrations (0.1%, 0.5%, and 4%) were related to by DGGE (Fig. 7) of the fermentation cultures with and without
the amount of EPS extracted because a large volume of methane (control) 2% EDTA pretreatment on day 5 of the incubation. The

Fig. 5. Sludge reduction ratio (a) and component changes (b) during hydrolysis stage.
M.T. Nguyen et al. / Chemosphere 117 (2014) 552558 557

and Clostridium carboxidivorans (Accession No. NR104768), respec-


tively. These are primarily acetogenic bacteria, which can grow on
fermentable carbohydrate, glucose, and cellulose, where acetate
and butyrate are the main end-products of metabolism (Liou
et al., 2005). Therefore, the synergic effects of these dominant
strains in the fermentation culture of sludge where EPS was
extracted by EDTA improved the hydrolysis and acetogenesis
stages. The materials generated were precursors for methane for-
mation, which resulted in higher methane productivity using the
sludge where EPS was extracted by EDTA.

3.5. Quantication of archaeal and bacterial groups

RT-PCR was used to quantify the total archaeal and bacterial


Fig. 6. Methane production from EPS-extracted sludge by different extraction
communities in the fermentation samples with and without 2%
methods.
EDTA pretreatment on day 10 of the incubation process because
the highest difference in methane production was observed on this
six major bands (B1B6) in the EDTA samples with higher intensi- day. We also quantied the variation in the 16S rRNA gene
ties than the control were excised and sent for sequence analysis. In concentrations of archaea and bacteria. The total concentrations
the control, band B3 and B4 were matched with the EDTA sample, of archaea in fermentation samples with 2% EDTA pretreatment
however in the EDTA sample, the band intensity are much higher were signicantly higher (2.2  109 copy mL1) than the control
than in the control. The phylogenetic afliations showed that B2 (2.7  106 copy mL1). This indicates that the archaeal community,
was an uncultured bacterium (Accession No. JN048271) and that which is the major methanogenic group (Yu et al., 2005), was
B6 was an uncultured beta proteobacterium (Accession No. increased and that this resulted in higher methane productivity
DQ295408), and their similarities were 93% and 94%, respectively. (Fig. 6). The total bacterial populations in the culture samples with
B1, B3, B4, and B5 were most closely related to the phylum Firmi- and without 2% EDTA pretreatment were 5.9  1012 and
cutes. The B1 sequence closely matched (96%) Clostridium proteolyt- 1.6  1012 copy mL1, respectively. Therefore, the EPS extraction
icum (Accession No. NR029250), which is a hydrolytic anaerobe that process had a positive effect on enhancing the methanogenic
exhibits extracellular protease activity, and its optimal pH and tem- population during the anaerobic digestion process.
perature for growth are 6.08.0 and 3037 C (Jain and Zeikus,
1988). It ferments a variety of proteinaceous substrates, and acetate 4. Conclusions
and CO2 are the major products with H2. The B3 band shared high
similarity (94%) with Clostridium mayombei (Accession No. In this study, we investigated the effects of EPS extraction on
NR104744), which is an acetogenic bacterium with optimal growth the anaerobic digestion process. The removal of EPS produced
conditions of pH 7.3 and 33 C (Kane et al., 1991). This strain grows WAS with a smaller structure, which enhanced sludge dispersion
on carbohydrate, sugar, and organic and amino acids, where acetate and the hydrolysis step. In addition, the synergic effects of the
is the usual principle fermentation product with these substrates. dominant proteolytic and acetogenic strains in the fermentation
The sequencing results for B4 and B5 were similar to (97% and culture of sludge, where the EPS was extracted by EDTA, improved
95%, respectively) Clostridium drakei (Accession No. NR044942) the hydrolysis and acetogenesis stages. Thus, pretreatment with
EDTA yielded more precursors for methane formation, which
resulted in higher methane productivity.

Acknowledgments

The authors would like to thank the Japan Student Services


Organization for the scholarship awarded to M.T. Nguyen during
this study. This research was supported by the Research Center
for Advanced Eco-tting Technology of Kyushu Institute of
Technology and the City of Kitakyushu.

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