Biochemical Engineering Journal 114 (2016) 283287

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

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
a
Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
b
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
Keywords:
increase of 10 mg g1 of the PHA content in WAS increased biogas production of 2.6 mL g1 -VSadded and
Anaerobic digestion
Methane
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

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

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: soda@see.eng.osaka-u.ac.jp (S. Soda). tion potential from WAS is inferred as about 990 mL g1 -VS (volatile

http://dx.doi.org/10.1016/j.bej.2016.07.017
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
(0 C, 1 atm).
C5 H7 O2 N + 3H2 O 2.5 CH4 + 2.5 CO2 + NH3
However, the biogas production from WAS is expected to be
250640 mL g1 -VSadded because of its low biodegradability of
2565%. A rational formula of poly--hydroxyalkanoate (PHB) as
a representative PHA is H-[C4 H6 O2 ]m -OH. The molecular weight
of PHB produced from wild type bacteria is usually in the range
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
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)
Biogas production from PHB is expected to be 520830 mL g1 -
VSadded , with assumption of high biodegradability of 5080%. In
addition to the high biogas yield, PHAs would be more rapidly
degraded than WAS, resulting in shortening the reaction time for
anaerobic digestion process.
This study was conducted to elucidate the relation between PHA
contents in sludge and biogas production in batch anaerobic diges-
tion. In this study, model PHA-accumulating WAS samples were
prepared using a sequencing batch reactor fed with acetate. The
model WAS samples with various PHA contents were used for batch
anaerobic digestion tests. The biogas yield and the biogas produc-
tion rate constant were evaluated for WAS and PHAs using kinetic
models.
2. Materials and methods
2.1. Activated sludge and anaerobic sludge
Seed-activated sludge for a sequencing batch reactor (SBR) was
sampled from a sewage treatment plant in Suita city, Osaka Prefec-
ture, Japan. The mesophilic anaerobic inoculum was obtained from
a sewage sludge treatment plant in Toyonaka city, Osaka Prefecture,
Japan.
2.2. Media
Synthetic wastewater used for the cultivation of activated
sludge in the SBR comprised CH3 COONa3H2 O 1297 mg-C, NH4 Cl
189 mg-N, KH2 PO4 154 mg-P, MgSO4 2H2 O 274 mg, KCl 107 mg,
allylthiourea 20 mg, trace element solution [19] 6 mL, and deion-
ized water 1000 mL. A basal salt medium used for accumulation of
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.
2.3. Preparation of WAS with various PHA contents
A 12-h cycle SBR was set up for preparing WAS with high
PHA contents based on the feast and famine strategy [20,21].
Seed-activated sludge was inoculated into a 2-L reactor (working
volume 1000 mL; mixed liquor-suspended solids (MLSS) = about
3000 g m3 ). The SBR was operated as follows: 03 min for
excess sludge withdrawn of 200 mL, 318 min for sludge settling,
1825 min for discharging supernatant of 800 mL, 2535 min for
aeration and mixing, 3545 min for synthetic wastewater feed-
ing, and 45720 min for aeration and mixing. The reactor was kept
at 30 C. Aeration and mixing phases were performed with aera-
tion at 2000 mL min1 , mixing by a four-blade turbine at 120 rpm,
and pH 7.0 maintained using a pH stat. This 12-h cycle operation
was repeated for more than 6 months for enrichment of PHA-
accumulating microorganisms.
For the anaerobic digestion test, PHAs were accumulated in
activated sludge in batch cultivation under nitrogen-limited and
aerobic conditions using acetate as the substrate. Activated sludge
(2) samples in the SBR were harvested by centrifugation (8500 g, 4 C,
10 min) and inoculated to 200 mL of the basal salt medium with
acetate at the MLSS concentration of 1000 g m3 in a 500-mL Erlen-
meyer ask. With various acetate concentrations of 01000 g m3
and the batch cultivation time of 024 h, WAS samples with various
PHA contents were obtained.
2.4. Batch tests of anaerobic digestion of WAS with various PHA
contents
The PHA-accumulating sludge was thickened at the total solid
(TS) concentration of 30000 mg L1 by centrifugation (1500 g,
10 min). Then, 1 mL of the thickened sludge, 5 mL of seed anaer-
obic sludge (800012000 mg-TS L1 ), and 1 mL of NaHCO3 buffer
(14 g L1 ) were added to 10-mL vials. For control tests, 1 mL of
9 g L1 NaCl solution was added instead of the thickened sludge.
Each vial was sealed with a rubber cap and purged with nitrogen
gas. Then the vials were incubated anaerobically at 37 C and on
a rotary shaker at 120 rpm. One bottle was sacriced for analysis
at each sampling time. The biogas production from the inoculum
(control) was subtracted from the biogas production of the PHA-
accumulating WAS samples. The result therefore represents only
the biogas production from the PHA-accumulating WAS and not
from the anaerobic inoculum.
2.5. Analytical procedures
The MLSS and VS concentrations of sludge samples were mea-
sured according to standard methods [22]. Biogas production was
measured by releasing the gas pressure in the vials using appropri-
ately sized glass syringes in the 5100 mL range to equilibrate with
the ambient pressure [23]. Before the release of biogas, the vials
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
the PHB contents in sludge were analyzed using gas chromatog-
raphy with a ame ionization detector (GC-2014; Shimadzu Corp.,
Kyoto). Before analysis of PHB, sludge samples were pretreated as
follows: acidied methanol (20%v/v with sulfuric acid) was used for
methanolysis, the reaction time for methanolysis was shortened to
7 h, and aqueous ammonia (28%v/v) was used for neutralization.
Benzoic acid was used as the internal standard.
2.6. Kinetic models of biogas production from WAS and PHAs
Cumulative biogas production in batch anaerobic digestion tests
can be expressed using a kinetic model as:
 1

P=Y 1 (4)
exp (kt)
where P is the cumulative biogas yield (mL g1 -VSadded ), Y is the
ultimate biogas yield (mL g1 -VS), k is the rst-order biogas pro-
duction rate constant (d1 ), and t is the digestion time (d).
As the one-component model, eq. (4) was tted to respective
data of biogas production from WAS with various PHA contents by
using a simulation software package (Berkeley Madonna ver. 8.0.1;
UC Berkeley).
Reportedly, the gas production curve corresponds to the rapid
breakdown of readily degradable components followed by a slower
degradable component [24]. In the two-component model, cumu-
 S. Soda et al. / Biochemical Engineering Journal 114 (2016) 283287
lative biogas production was expressed as the sum of those from
WAS and PHAs as eqs. (5)(7).
 x
 x
P= 1 PWAS + PPHA (5)
1000 1000
 1
 WAS with PHAs at 30 mg g1
PWAS = Y 1 (6)
exp (kWAS t)
 1
 WAS with PHAs at 290 mg g1
PPHA = Y 1 (7)
exp (kPHA t)
Therein, x is the PHA content (mg g1 of dry mass) in WAS.
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.
3. Results and discussion
3.1. Biogas production from WAS with various PHA contents in
anaerobic digestion
Model WAS samples with various PHA contents were used
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
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
dry mass were, respectively, 12, 18, 77, 124, and 137 mg g1 of
dry mass. Biogas production from WAS with the low PHA content
of 30 mg g1 proceeded slowly and reached about 300 mL g1 -
VSadded on day 612. With accumulation of higher PHA contents,
rapid biogas production was observed. The PHA content decreased
quickly within 3 d in anaerobic digestion. Anaerobic biodegrada-
tion of PHAs is expected to involve the initial hydrolysis of PHAs
to their monomeric units. This rapid degradation of PHAs would
be attributed to the intracellular and extracellular enzymes of
both anaerobic and aerobic sludge microorganisms [18,25,26]. For
example, PHB is usually hydrolyzed to 3-hydroxybutyric acid (3-
HB) by extracellular depolymerase. Resulting 3-HB is fermented
to acetate, butyrate, and H2 by bacteria and further to methane
and CO2 by archaea [25,27,28]. The hydrolysis of PHAs is the rate-
limiting step in anaerobic degradation, however, the overall process
occurs rapidly [28]. The cumulative biogas production from WAS
with the PHA content of >50 mg kg1 exceeded 370 mL g1 -VSadded
on day 12. Methane production occurred rapidly in the early days
but continued for 30 d in the batch tests. The methane content in
the biogas was stable at 6570%. Final methane production after
32 d from WAS with the PHA content of 290 mg g1 was higher by
30% than that from WAS with the PHA content of 30 mg g1 . These
results suggest that PHAs were converted quickly to methane, but
other sludge components were degraded slowly.
3.2. Kinetics of biogas production from WAS accumulating PHAs
Table 1 shows the calibrated parameters of the one- and two-
component models tted to experimental data of the cumulative
biogas production from WAS with various PHA contents. The opti-
mized model ts are presented in Fig. 2.
According to the one-component model, the estimated value
for the biogas yield (Y) increased from 315 to 411 mL g1 -VS with
PHA contents from 30 to 380 mg g1 . The biogas production rate
constant (k) also increased from 0.208 to 0.499 d1 . In the two-
component model, the biogas production was expressed as the
sum of those from WAS and PHAs in anaerobic digestion. The
parameter value of YPHA was 1.68 times higher than that of YWAS .
The YWAS value obtained in this study is within a typical range
285
Table 1
Parameter values of one-component and two-component models for cumulative
biogas production from PHA-accumulating WAS in anaerobic digestion.
Y (mL g1 -VSadded ) k (d1 )
One-component model
326 0.208
WAS with PHAs at 50 mg g1 349 0.281
WAS with PHAs at 230 mg g1 354 0.325
370 0.358
WAS with PHAs at 380 mg g1 425 0.499
Two-component model
WAS 341 0.194
PHAs 550 1.290
(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
for PHAs, as described in the introduction section. The ratio of the
estimated yield (mL g1 -VSadded ) to the theoretical yield (mL g1 -
VS) for PHAs (0.53) was much higher than that for WAS (0.34).
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
estimated as much faster than that from WAS.
3.3. Effects of PHA contents of WAS on biogas production
Cumulative production of biogas and methane at 32 d is sum-
marized in Fig. 3. According to the two-component kinetic model,
an increase of 10 mg g1 of PHA accumulation in WAS can increase
the biogas production of 2.6 mL g1 -VSadded and the methane pro-
duction of 2.4 mL g1 -VSadded , although the correlation coefcient
for the methane production between the model predictions and the
experimental data was not so high. This trend agrees with the result
reported by Huda et al. [16], which was 25% increase of methane
production in anaerobic digestion by accumulation of PHAs at
240 mg g1 of dry mass in WAS, although the reported methane
yield was only 188 mL-CH4 g1 -VSadded , which was much lower
than typical values for WAS. Wang et al. [8] also reported that the
methane yield linearly increased by 46% from 168 to 246 mL g1 -
VSSadded with the increase in the PHA content from 21 to 184 mg g1
of dry mass.
3.4. Practical implications
A technique for using WAS with the intracellular storage
materials as a feed of anaerobic digestion to enhance methane
production has been proposed [8,16]. Although model WAS sam-
ples were used for this study, WAS in actual sewage treatment
plants can accumulate PHAs at 250700 mg g1 of dry mass in
nutrient-limited conditions [1215]. It is also well known that
polyphosphate-accumulating bacteria in the enhanced phospho-
rus removal process can take up volatile fatty acids and convert
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
250 mg g1 and 700 mg g1 of dry mass, respectively.
For practical applications, WAS withdrawn from the secondary
settling tank would be mixed with sewage and briey aerated
to facilitate accumulation of the intracellular storage materials in
bacterial cells. Subsequently, WAS would be settled again, and
fed to anaerobic digestion. High methane production will con-
tribute to the realization of self-sufcient or net energy-producing
sewage treatment plants [2,3]. The rapid conversion rate of PHA-
accumulating WAS to methane is also benecial for downsizing of
the digestion tanks. In urban areas in Japan, sewage sludge after
anaerobic digestion is dewatered and incinerated using high con-
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

A B C
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 12

12
400 40 0
400 40 0
400 400

10
10 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

6 200 20 0
6

6 200 200
6

6
4
4 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
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
D E
50 0 14
5 00 14

45 0

producon (mL/g-VS added)

12 12
producon (mL/g-VS added)

12
12

400 4 00
400 40 0

10

10 35 0
10

10
Cumulave biogas
Cumulave biogas

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

300 3 00
300 30 0

8
8

8 8

25 0

6
6 200
6

6
200 2 00
20 0

4
4
15 0
4
4
100 1 00
100 10 0

2 50
2
2

0 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

A B C
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
D E
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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