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Biochemical Engineering Journal 114 (2016) 283287

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Regular article

High methane production potential of activated sludge accumulating

polyhydroxyalkanoates in anaerobic digestion
Satoshi Soda a,b, , Koichi Iwama a , Kazunori Yokoe a , Yukihiro Okada a , Michihiko Ike a,b
Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
Center for Environmental Innovation Design for Sustainability, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan

a r t i c l e i n f o a b s t r a c t

Article history: A technique for using waste activated sludge (WAS) accumulating intracellular storage materials was
Received 27 April 2016 studied for enhancing methane production through anaerobic digestion. Model WAS samples with var-
Received in revised form 7 July 2016 ious contents of poly--hydroxyalkanoates (PHAs) were used for anaerobic digestion tests. Methane
Accepted 28 July 2016
production during 32 d from WAS with PHA content of 290 mg g1 of dry mass was higher by 30% than
Available online 29 July 2016
that from WAS with PHA content of 30 mg g1 . Kinetic models showed that PHAs have a higher biogas
production rate constant and a higher biogas yield than microbial cells of WAS in anaerobic digestion. An
increase of 10 mg g1 of the PHA content in WAS increased biogas production of 2.6 mL g1 -VSadded and
Anaerobic digestion
methane production of 2.4 mL g1 -VSadded . Results show that PHA-accumulating WAS has high potential
Poly--hydroxyalkanoate for methane production in anaerobic digestion.
Sewage sludge 2016 Published by Elsevier B.V.
Kinetic model

1. Introduction PHAs at 250700 mg g1 of dry mass in nutrient-limited conditions

using volatile fatty acids [1215]. Because of its inherent biodegrad-
Anaerobic digestion is a key technology for sewage sludge treat- ability [7,8], cells with intracellular storage materials are converted
ment with methane production. Power generation using biogas more easily to biogas in anaerobic digestion than fully synthesized
produced in the anaerobic digestion process is progressing for cells. Higher methane production by 25% was demonstrated using
substitution of fossil fuels [13]. However, conversion of organics WAS accumulating PHAs at 240 mg g1 of dry mass [16]. It was also
to biogas is typically 2565% in a mesophilic anaerobic digestion reported that the methane yield linearly increased up to 46% with
process with hydraulic retention time (HRT) of 20 d, with lower the increase in the PHA content from 21 to 184 mg g1 [8].
performance on waste activated sludge (WAS) than on primary However, quantitative knowledge on biogas production from
sludge [4,5]. Actually, WAS produced in aeration tanks in sewage WAS with the intracellular storage materials in anaerobic digestion
treatment plants mainly comprises microbial cells, providing low remains insufcient. Complementary to the previous studies [8,16],
biodegradability. this study proposes kinetic models of biogas production from WAS
Several pretreatment techniques for WAS have been stud- and PHAs. Biogas production from organic matter (Cn Ha Ob Nc ) in
ied: mechanical, chemical, and thermal [6]. A new approach for anaerobic digestion can be predicted empirically as eq. (1) [17].
improving biogas production in anaerobic digestion is the mod-
ication of the WAS chemical composition [7,8]. Cell production
of heterotrophic bacteria generally proceeds in two steps in the Cn Ha Ob Nc + (n a/4 b/2 + 3c/4) H2 O
activated sludge process [911]. First, easily degradable sub-
strates are converted to intracellular storage materials such as (n/2 + a/8 b/4 3c/8) CH4 + (n/2 a/8 + b
poly--hydroxyalkanoates (PHAs) in heterotrophic bacterial cells.
/4 + 3c/8) CO2 + c NH3 (1)
Subsequently, the intracellular storage materials are used for the
synthesis of the bacterial cells. Reportedly, WAS can accumulate

A typical rational formula of WAS is C5 H7 O2 N. In this model

Corresponding author at: Graduate School of Engineering, Osaka University, 2-1 study, WAS is dened as the waste activated sludge component
Yamadaoka, Suita, Osaka 565-0871, Japan. excluding PHAs. Therefore, the biogas yield or the biogas produc-
E-mail address: (S. Soda). tion potential from WAS is inferred as about 990 mL g1 -VS (volatile
1369-703X/ 2016 Published by Elsevier B.V.
284 S. Soda et al. / Biochemical Engineering Journal 114 (2016) 283287

solids) (CH4 50%, CO2 50%) at standard temperature and pressure For the anaerobic digestion test, PHAs were accumulated in
(0 C, 1 atm). activated sludge in batch cultivation under nitrogen-limited and
aerobic conditions using acetate as the substrate. Activated sludge
C5 H7 O2 N + 3H2 O 2.5 CH4 + 2.5 CO2 + NH3 (2) samples in the SBR were harvested by centrifugation (8500 g, 4 C,
However, the biogas production from WAS is expected to be 10 min) and inoculated to 200 mL of the basal salt medium with
250640 mL g1 -VSadded because of its low biodegradability of acetate at the MLSS concentration of 1000 g m3 in a 500-mL Erlen-
2565%. A rational formula of poly--hydroxyalkanoate (PHB) as meyer ask. With various acetate concentrations of 01000 g m3
a representative PHA is H-[C4 H6 O2 ]m -OH. The molecular weight and the batch cultivation time of 024 h, WAS samples with various
of PHB produced from wild type bacteria is usually in the range PHA contents were obtained.
of 1 104 3 106 g mol1 (m = 12035,000) [7]. The biogas yields
or the biogas production potential from PHB is estimated as about
2.4. Batch tests of anaerobic digestion of WAS with various PHA
1040 mL g1 -VS (CH4 56%, CO2 44%) [18].
H-[C4 H6 O2 ]m -OH + (1.5 m 1)H2 O 2.25mCH4 + 1.75mCO2 (3)
The PHA-accumulating sludge was thickened at the total solid
Biogas production from PHB is expected to be 520830 mL g1 - (TS) concentration of 30000 mg L1 by centrifugation (1500 g,
VSadded , with assumption of high biodegradability of 5080%. In 10 min). Then, 1 mL of the thickened sludge, 5 mL of seed anaer-
addition to the high biogas yield, PHAs would be more rapidly obic sludge (800012000 mg-TS L1 ), and 1 mL of NaHCO3 buffer
degraded than WAS, resulting in shortening the reaction time for (14 g L1 ) were added to 10-mL vials. For control tests, 1 mL of
anaerobic digestion process. 9 g L1 NaCl solution was added instead of the thickened sludge.
This study was conducted to elucidate the relation between PHA Each vial was sealed with a rubber cap and purged with nitrogen
contents in sludge and biogas production in batch anaerobic diges- gas. Then the vials were incubated anaerobically at 37 C and on
tion. In this study, model PHA-accumulating WAS samples were a rotary shaker at 120 rpm. One bottle was sacriced for analysis
prepared using a sequencing batch reactor fed with acetate. The at each sampling time. The biogas production from the inoculum
model WAS samples with various PHA contents were used for batch (control) was subtracted from the biogas production of the PHA-
anaerobic digestion tests. The biogas yield and the biogas produc- accumulating WAS samples. The result therefore represents only
tion rate constant were evaluated for WAS and PHAs using kinetic the biogas production from the PHA-accumulating WAS and not
models. from the anaerobic inoculum.

2. Materials and methods

2.5. Analytical procedures
2.1. Activated sludge and anaerobic sludge
The MLSS and VS concentrations of sludge samples were mea-
Seed-activated sludge for a sequencing batch reactor (SBR) was sured according to standard methods [22]. Biogas production was
sampled from a sewage treatment plant in Suita city, Osaka Prefec- measured by releasing the gas pressure in the vials using appropri-
ture, Japan. The mesophilic anaerobic inoculum was obtained from ately sized glass syringes in the 5100 mL range to equilibrate with
a sewage sludge treatment plant in Toyonaka city, Osaka Prefecture, the ambient pressure [23]. Before the release of biogas, the vials
Japan. were well shaken to equilibrate gas and liquid phases. The bio-
gas volume was converted to values at standard temperature and
pressure (0 C, 1 atm). The methane concentrations in biogas and
2.2. Media
the PHB contents in sludge were analyzed using gas chromatog-
raphy with a ame ionization detector (GC-2014; Shimadzu Corp.,
Synthetic wastewater used for the cultivation of activated
Kyoto). Before analysis of PHB, sludge samples were pretreated as
sludge in the SBR comprised CH3 COONa3H2 O 1297 mg-C, NH4 Cl
follows: acidied methanol (20%v/v with sulfuric acid) was used for
189 mg-N, KH2 PO4 154 mg-P, MgSO4 2H2 O 274 mg, KCl 107 mg,
methanolysis, the reaction time for methanolysis was shortened to
allylthiourea 20 mg, trace element solution [19] 6 mL, and deion-
7 h, and aqueous ammonia (28%v/v) was used for neutralization.
ized water 1000 mL. A basal salt medium used for accumulation of
Benzoic acid was used as the internal standard.
PHAs in WAS included MgSO4 7H2 O 68.4 mg, KCl 26.84 mg, allylth-
iourea 5 mg, trace element solution [19] 1.5 mL, and deionized
water 1000 mL. 2.6. Kinetic models of biogas production from WAS and PHAs

2.3. Preparation of WAS with various PHA contents Cumulative biogas production in batch anaerobic digestion tests
can be expressed using a kinetic model as:
A 12-h cycle SBR was set up for preparing WAS with high

PHA contents based on the feast and famine strategy [20,21].
P=Y 1 (4)
Seed-activated sludge was inoculated into a 2-L reactor (working exp (kt)
volume 1000 mL; mixed liquor-suspended solids (MLSS) = about
3000 g m3 ). The SBR was operated as follows: 03 min for where P is the cumulative biogas yield (mL g1 -VSadded ), Y is the
excess sludge withdrawn of 200 mL, 318 min for sludge settling, ultimate biogas yield (mL g1 -VS), k is the rst-order biogas pro-
1825 min for discharging supernatant of 800 mL, 2535 min for duction rate constant (d1 ), and t is the digestion time (d).
aeration and mixing, 3545 min for synthetic wastewater feed- As the one-component model, eq. (4) was tted to respective
ing, and 45720 min for aeration and mixing. The reactor was kept data of biogas production from WAS with various PHA contents by
at 30 C. Aeration and mixing phases were performed with aera- using a simulation software package (Berkeley Madonna ver. 8.0.1;
tion at 2000 mL min1 , mixing by a four-blade turbine at 120 rpm, UC Berkeley).
and pH 7.0 maintained using a pH stat. This 12-h cycle operation Reportedly, the gas production curve corresponds to the rapid
was repeated for more than 6 months for enrichment of PHA- breakdown of readily degradable components followed by a slower
accumulating microorganisms. degradable component [24]. In the two-component model, cumu-
S. Soda et al. / Biochemical Engineering Journal 114 (2016) 283287 285

lative biogas production was expressed as the sum of those from Table 1
Parameter values of one-component and two-component models for cumulative
WAS and PHAs as eqs. (5)(7).
biogas production from PHA-accumulating WAS in anaerobic digestion.
P= 1 PWAS + PPHA (5) Y (mL g1 -VSadded ) k (d1 )
1000 1000
One-component model
 WAS with PHAs at 30 mg g1 326 0.208
PWAS = Y 1 (6) WAS with PHAs at 50 mg g1 349 0.281
exp (kWAS t)
WAS with PHAs at 230 mg g1 354 0.325
 WAS with PHAs at 290 mg g1 370 0.358
PPHA = Y 1 (7) WAS with PHAs at 380 mg g1 425 0.499
exp (kPHA t)
Two-component model
Therein, x is the PHA content (mg g1 of dry mass) in WAS. WAS 341 0.194
PHAs 550 1.290
The two-component model was tted at once to all data of bio-
gas production from WAS with various PHA contents by using the
simulation software package.
(324380 mL g1 -VSadded ) [5]. The theoretical biogas yield (mL g1 -
VSadded ) is about 990 mL g1 -VS for WAS and about 1040 mL g1 -VS
3. Results and discussion
for PHAs, as described in the introduction section. The ratio of the
estimated yield (mL g1 -VSadded ) to the theoretical yield (mL g1 -
3.1. Biogas production from WAS with various PHA contents in
VS) for PHAs (0.53) was much higher than that for WAS (0.34).
anaerobic digestion
The parameter value of kPHA was about 6.6 times higher than that
of kWAS . As presented in Fig. 2, biogas production from PHAs was
Model WAS samples with various PHA contents were used
estimated as much faster than that from WAS.
for anaerobic digestion tests. Fig. 1 shows cumulative biogas
production and the PHA contents of the mixed sludge in anaer-
obic digestion. By mixing with anaerobic sludge (PHAs of almost 3.3. Effects of PHA contents of WAS on biogas production
0 mg g1 ), the initial PHA contents in the vials inoculated with
WAS with PHA contents of 30, 50, 230, 290, and 380 mg g1 of Cumulative production of biogas and methane at 32 d is sum-
dry mass were, respectively, 12, 18, 77, 124, and 137 mg g1 of marized in Fig. 3. According to the two-component kinetic model,
dry mass. Biogas production from WAS with the low PHA content an increase of 10 mg g1 of PHA accumulation in WAS can increase
of 30 mg g1 proceeded slowly and reached about 300 mL g1 - the biogas production of 2.6 mL g1 -VSadded and the methane pro-
VSadded on day 612. With accumulation of higher PHA contents, duction of 2.4 mL g1 -VSadded , although the correlation coefcient
rapid biogas production was observed. The PHA content decreased for the methane production between the model predictions and the
quickly within 3 d in anaerobic digestion. Anaerobic biodegrada- experimental data was not so high. This trend agrees with the result
tion of PHAs is expected to involve the initial hydrolysis of PHAs reported by Huda et al. [16], which was 25% increase of methane
to their monomeric units. This rapid degradation of PHAs would production in anaerobic digestion by accumulation of PHAs at
be attributed to the intracellular and extracellular enzymes of 240 mg g1 of dry mass in WAS, although the reported methane
both anaerobic and aerobic sludge microorganisms [18,25,26]. For yield was only 188 mL-CH4 g1 -VSadded , which was much lower
example, PHB is usually hydrolyzed to 3-hydroxybutyric acid (3- than typical values for WAS. Wang et al. [8] also reported that the
HB) by extracellular depolymerase. Resulting 3-HB is fermented methane yield linearly increased by 46% from 168 to 246 mL g1 -
to acetate, butyrate, and H2 by bacteria and further to methane VSSadded with the increase in the PHA content from 21 to 184 mg g1
and CO2 by archaea [25,27,28]. The hydrolysis of PHAs is the rate- of dry mass.
limiting step in anaerobic degradation, however, the overall process
occurs rapidly [28]. The cumulative biogas production from WAS 3.4. Practical implications
with the PHA content of >50 mg kg1 exceeded 370 mL g1 -VSadded
on day 12. Methane production occurred rapidly in the early days A technique for using WAS with the intracellular storage
but continued for 30 d in the batch tests. The methane content in materials as a feed of anaerobic digestion to enhance methane
the biogas was stable at 6570%. Final methane production after production has been proposed [8,16]. Although model WAS sam-
32 d from WAS with the PHA content of 290 mg g1 was higher by ples were used for this study, WAS in actual sewage treatment
30% than that from WAS with the PHA content of 30 mg g1 . These plants can accumulate PHAs at 250700 mg g1 of dry mass in
results suggest that PHAs were converted quickly to methane, but nutrient-limited conditions [1215]. It is also well known that
other sludge components were degraded slowly. polyphosphate-accumulating bacteria in the enhanced phospho-
rus removal process can take up volatile fatty acids and convert
3.2. Kinetics of biogas production from WAS accumulating PHAs then to PHAs [10,11]. The model in this study predicts that a 23%
and 65% increase of methane production from WAS with PHAs at
Table 1 shows the calibrated parameters of the one- and two- 250 mg g1 and 700 mg g1 of dry mass, respectively.
component models tted to experimental data of the cumulative For practical applications, WAS withdrawn from the secondary
biogas production from WAS with various PHA contents. The opti- settling tank would be mixed with sewage and briey aerated
mized model ts are presented in Fig. 2. to facilitate accumulation of the intracellular storage materials in
According to the one-component model, the estimated value bacterial cells. Subsequently, WAS would be settled again, and
for the biogas yield (Y) increased from 315 to 411 mL g1 -VS with fed to anaerobic digestion. High methane production will con-
PHA contents from 30 to 380 mg g1 . The biogas production rate tribute to the realization of self-sufcient or net energy-producing
constant (k) also increased from 0.208 to 0.499 d1 . In the two- sewage treatment plants [2,3]. The rapid conversion rate of PHA-
component model, the biogas production was expressed as the accumulating WAS to methane is also benecial for downsizing of
sum of those from WAS and PHAs in anaerobic digestion. The the digestion tanks. In urban areas in Japan, sewage sludge after
parameter value of YPHA was 1.68 times higher than that of YWAS . anaerobic digestion is dewatered and incinerated using high con-
The YWAS value obtained in this study is within a typical range sumption of chemicals and fossil fuels [1]. The high biodegradability
286 S. Soda et al. / Biochemical Engineering Journal 114 (2016) 283287

500 14 500 14 500 14

50 0 14 50 0 14 500 14

producon (mL/g-VS added)

producon (mL/g-VS added)

producon (mL/g-VS added)


12 12

12 12

400 40 0
400 40 0
400 400

10 10
10 10
Cumulave biogas

Cumulave biogas

Cumulave biogas
PHA (% w/w)

PHA (% w/w)

PHA (% w/w)
300 30 0

8 300 30 0

8 300 300

200 20 0

6 200 20 0

6 200 200

4 4
4 4
100 10 0
100 10 0
100 100

2 2

2 2

0 0 0

0 0 0 0

0 0 0 0

0 2 4 5 15 25 35 0 2 4 5 15 25 35 0 2 4 5 15 25 35
Time (d) Time (d) Time (d)
500 14 500 14
50 0 14
5 00 14

45 0

producon (mL/g-VS added)

12 12
producon (mL/g-VS added)


400 4 00
400 40 0


10 35 0

Cumulave biogas
Cumulave biogas

PHA (% w/w)
PHA (% w/w)

300 3 00
300 30 0


8 8

25 0

6 200

200 2 00
20 0

15 0
100 1 00
100 10 0

2 50

0 0 0

0 0 0 0

0 2 4 5 15 25 35 0 2 4 5 15 25 35
Time (d) Time (d)

Fig. 1. Cumulative biogas production and PHA contents in anaerobic digestion of PH-accumulating WAS. The PHA contents of the activated sludge were, respectively, 30 (a),
50 (b), 230 (c), 290 (d), and 380 mg g1 of dry mass (E). Open circles and closed circles respectively show the biogas production and the PHA content.

450 450 450

400 400 400
producon (mL/g-VS added)

producon (mL/g-VS added)

350 350 producon (mL/g-VS added) 350

Cumulave biogas

Cumulave biogas

Cumulave biogas

300 300 300

250 250 250
200 200 200
150 150 150
100 1-component r = 0.95 100 1-component r = 0.95 100
50 2-component r = 0.95 2-cpmponent r = 0.93 1-component r = 0.93
50 50
2-component r = 0.94
0 0 0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Time (d) Time (d) Time (d)
450 450
400 400
producon (mL/g-VS added)

producon (mL/g-VS added)

350 350
Cumulave biogas

Cumulave biogas

300 300
250 250
200 200
150 150
100 100
1-component r = 0.96 1-component r = 0.83
50 2-component r = 0.93 50 2-component r = 0.91
0 0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Time (d) Time (d)

Fig. 2. Cumulative biogas production from PHA-accumulating WAS. The PHA contents of the activated sludge were, respectively, 30 (A), 50 (B), 230 (C), 290 (D), and
380 mg g1 of dry mass (E). Experimental data plots and computed lines are shown. Thin lines and thick lines respectively show biogas production simulated by the one- and
two-component models. Dashed lines and dotted lines respectively show biogas production from PHAs and WAS of the two-component model. Open circles and solid lines
show total biogas production.

and biogas yield of PHA-accumulating WAS are expected to lower the methane conversion from soluble protein of both PHA- and
production of the sludge residue. non-PHA-accumulating cells, and abundance of methanogens in
In this study, the rst-order biogas production rate constant of anaerobic digestion. Further studies on long-term effects of PHA-
WAS was assumed to be a constant and independent of the PHA accumulating WAS on the anaerobic digestion process will be
content. However, Wang et al. [8] reported that the high PHA con- needed.
tent enhanced the cell disruption of the PHA-accumulating cells,
S. Soda et al. / Biochemical Engineering Journal 114 (2016) 283287 287

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