chua a - 2003 - production of pha by as treating municipal wastewater- effect of ph, sludge...

Water Research 37 (2003) 36023611

Production of polyhydroxyalkanoates (PHA) by activatedsludge treating municipal wastewater: effect of pH, sludgeretention time (SRT), and acetate concentration in inuent

Adeline S.M. Chuaa,*, Hiroo Takabatakeb, Hiroyasu Satoha, Takashi Minoa

a Institute of Environmental Studies, Graduate School of Frontier Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku,

Tokyo 113-0033 JapanbDepartment of Civil Engineering, Faculty of Engineering, Tohoku University, Sendai, Japan

Received 17 June 2002; received in revised form 19 March 2003; accepted 28 April 2003

Abstract

In this paper, the production of biodegradable plastics polyhydroxyalkanoates (PHA) by activated sludge treating

municipal wastewater was investigated. The effect of three operational factors, i.e. the acetate concentration in inuent,

pH, and sludge retention time (SRT) were studied. Sludge acclimatized with municipal wastewater supplemented with

acetate could accumulate PHA up to 30% of sludge dry weight, while sludge acclimatized with only municipal

wastewater achieved 20% of sludge dry weight. It was found that activated sludge with an SRT of 3 days possessed

better PHA production capability than sludge with an SRT of 10 days. Sludge acclimatized under pH 7 and 8

conditions in sequencing batch reactors (SBRs) exhibited similar PHA production capability. However, in PHA

production batch experiments, pH value inuenced signicantly the PHA accumulation behavior of activated sludge.

When pH of batch experiments was controlled at 6 or 7, a very low PHA production was observed. The production of

PHA was stimulated when pH was kept at 8 or 9.

r 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Activated sludge; Polyhydroxyalkanoates (PHA); Anaerobicaerobic process; Acetate; pH; Sludge retention time (SRT)

1. Introduction

The development of biodegradable plastics is one of

the major concerns in the present society because the

conventional plastics have many faults. They are

produced from non-renewable resources such as petro-

chemicals, and are not compatible with natural carbon

cycles because of their non-degradable characteristics.

They are also causing serious problems of damaging

beautiful natural scenery and wild lives due to their

persistence in natural environment. In abating these

problems, the development of biodegradable plastics has

become one of the potential counter-measures. Poly-

hydroxyalkanoates (PHA) is one of the biodegradable

plastics produced mainly by bacteria. In the last three

decades, PHA have attracted industrial interest as

biodegradable plastics not only because of their

compatible material properties like synthetic thermo-

plastics but also could PHA be synthesized from

renewable carbon resources, based on agriculture or

even on industrial wastes [1]. Due to these unique

characteristics of PHA, various kinds of bacterial strains

have been tested for their PHA production capability.

To date, there are more than 300 different microorgan-

isms, which can synthesize PHA [2]. Several of these,

such as Ralstonia eutropha, Alcaligenes latus, Azotobac-

ter vinelandii, and several strains of methylotrophs and

ARTICLE IN PRESS

*Corresponding author. Tel.: +81-3-5841-6250;

fax: +81-3-5841-8531.

E-mail address: [email protected]

(A.S.M. Chua).

0043-1354/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved.

doi:10.1016/S0043-1354(03)00252-5

recombinant Escherichia coli are being intensively

studied because of higher productivity [2]. For example,

P(3HB-co-3HV), the copolymer of 3-hydroxybutyrate

(3HB) and 3-hydroxyvalerate (3HV) has been commer-

cially produced by pure culture fermentation process

using Ralstonia eutropha and the PHA content achieved

is more than 80% of cell dry weight [3]. The PHA

content achieved by Alcaligenes latus and recombinant

Escherichia coli have been reported to reach 88% [4] and

76% [5] of cell dry weight, respectively. Although high

PHA content could be achieved by using pure culture

fermentation process, the cost of PHA production is still

too high for PHA to become a competitive commodity

plastic material. As to reduce the expensiveness of PHA,

a novel PHA production strategy, which is to utilize the

mixed bacterial culture in activated sludge for PHA

production has been proposed in the last decade.

Considerable efforts have been devoted to this direction,

and the studies conducted are reviewed by Satoh et al.

[6,7].

The idea of PHA production by using activated

sludge was ignited owing to PHAs function as an

intermediate metabolic product in activated sludge

process. It has been recognized that PHA is one of the

most important carbon storage materials especially

in the anaerobicaerobic activated sludge process or

the Enhanced Biological Phosphorous Removal (EBPR)

process [8]. In EBPR process, microorganisms in

activated sludge consume polyphosphate as an energy

source for anaerobic uptake of carbon substrates.

The carbon substrates taken up are temporarily stored

as PHA. When the condition turns aerobic, PHA is

utilized for growth and polyphosphate regeneration.

The microorganisms in EBPR process should therefore

possess the characteristic of phosphate removal

and PHA accumulation. For that reason, anaerobic

aerobic activated sludge process was employed in

this study to acclimatize activated sludge for PHA

production.

When compared with pure culture fermentation

processes, the merits of PHA production system by

activated sludge will be the cost reduction in cultivating

PHA producing bacterial cultures, simpler facility

construction, and material recovery from wastes [9].

In this study, a two-stage system shown in Fig. 1

was proposed. The rst stage is the activated sludge

process for wastewater treatment, and the second

stage is PHA production process by using the excess

sludge from the wastewater treatment process. In the

rst stage, it is essential to optimize the operational

conditions for sludge acclimatization or for the enrich-

ment of PHA accumulating microorganisms so that the

PHA production capability of activated sludge could be

improved. In the second stage, carbon substrate such as

acetate was fed to the acclimatized sludge for PHA

production.

In most of the studies of PHA production by activated

sludge, synthetic wastewaters were used to cultivate

PHA producing sludge, such as in Ueno et al. [10],

Iwamoto et al. [11], Saito et al. [12], Hu et al. [13], Chua

et al. [1416], Tsunemasa [17], Satoh et al. [9], Lemos

et al. [18], Fang et al. [19], and Ma et al. [20].

Furthermore, very little work had been done on how

operational conditions of activated sludge process could

enhance the PHA production capability of sludge. Satoh

et al. [9] reported a high accumulation of 62% of cell dry

weight by using activated sludge acclimatized in

anaerobicaerobic process with very limited oxygen

supply to the anaerobic condition. The effect of

carbonnitrogen (C:N) ratio in reactor liquor on PHA

productivity was examined in Chua et al. [1416], Fang

et al. [19], and Ma et al. [20].

In the present work, attention was devoted to the two

lacking aspects mentioned above. Real municipal waste-

water was used to cultivate activated sludge for PHA

production, and our focus was on how the operational

conditions in activated sludge process could inuence

the PHA production capability of sludge. The opera-

tional conditions being investigated were the acetate

concentration in inuent, pH, and sludge retention time.

As known well, acetate is the most easily assimilated

carbon substrate in producing PHA. It is essential to

know how signicant its concentration can enhance the

PHA production capability. This will then enable us to

select the suitable wastewater for sludge acclimatization.

As mentioned before, optimization of operational

conditions in activated sludge process is essential for

PHA production capability enhancement as well as for

satisfactory efuent quality. pH condition and sludge

retention time were investigated in this study because

they are important and easily manipulated parameters in

activated sludge process. Besides, we also studied the

effect of pH on PHA production process. At last,

feasibility of using activated sludge treating municipal

wastewater for PHA production is discussed.

ARTICLE IN PRESS

Effluent Influent wastewater

Excess sludge

Wastewater Treatment Process

sedimentation tank

PHA Production Process

External carbon substrate, e.g.industrial wastewater, raw

carbon sources

activated sludge process

PHAproduction

reactor

Fig. 1. PHA production system by using activated sludge

treating wastewater.

A.S.M. Chua et al. / Water Research 37 (2003) 36023611 3603

2. Materials and methods

2.1. Operation of anaerobicaerobic activated sludge

processes under different operational conditions

Two bench-scale sequential batch activated sludge

reactors (SBRs) served as the wastewater treatment

process, the rst stage of the proposed PHA production

system (Fig. 1). They were operated with municipal

wastewater as inuent. The municipal wastewater was

supplied from a municipal wastewater treatment plant

located in the central of Tokyo, which receives waste-

water from the commercial, residential and industrial

areas. While the inuent wastewater quality uctuated,

the typical inuent quality data is shown below:

COD=60130mg O/l

Total organic carbon (TOC)=30130mg C/l

Acetate=020mg C/l

Phosphate (PO4-P)=1.75.6mg P/l.

The schematic diagram of the SBRs is given in Fig. 2.

The SBRs were operated with a cycle of 4-h consisting of

a supernatant decanting (15min), inuent feeding

(5min), anaerobic (1 h), aerobic (2 h) and settling period

(40min). The SBRs with a working volume of 20 l and a

hydraulic retention time of 6 h were situated in an air-

conditioned room with temperature between 18C

(winter) to 25C (summer). pH was controlled at a

desired value in each operation of SBRs by adding

diluted sulfuric acid or diluted sodium hydroxide.

Medium for activated sludge processes was solely

municipal wastewater, unless otherwise stated.

Three different runs of activated sludge processes

(Runs AC) were conducted. Two SBRs were operated

in parallel in each run, with the operational conditions

summarized in Table 1. Both SBRs in Run A were

seeded with activated sludge collected from the munici-

pal wastewater treatment plant (conventional activated

sludge process) that supplied wastewater to this study.

Upon completion of Run A, sludge from the SBRs was

mixed and used as seeding sludge for Run B. In Run C,

the SBRs were seeded with sludge from a pilot-scale

activated sludge process with anaerobicanoxicoxic

(A2O) conguration. This pilot plant also received

inuent wastewater from the municipal wastewater

treatment plant mentioned above. Sludge characteristics

such as the proles of dissolved organic carbon (DOC),

acetate, PO4-P, and PHA in one SBR cycle were

monitored regularly during each operation period.

Along the operation of activated sludge processes,

1000ml of sludge was taken periodically from each

SBR to perform PHA production batch experiments.

2.2. PHA production batch experiments

To simulate the second stage of Fig. 1, activated

sludge taken from the SBRs was subjected to PHA

production batch experiments with experimental setup

as shown in Fig. 3. One liter of sludge was taken at the

end of aerobic phase, and put in a glass bottle. Sodium

acetate was added as carbon substrate for PHA

production and incubated for 24 h under aerobic

condition by air bubbling. The batch experiments were

conducted under the same temperature as the SBR. In

these batch experiments, pH has a tendency to increase

ARTICLE IN PRESS

air pump

P

air diffuser

mixer

20l

sludge wasting pump

effluent pump

influent pump

PP

Fig. 2. Schematic diagram of SBRs.

Table 1

Operating conditions for Runs AC of SBRs

Run Investigated operating condition SBR pH Inuent SRT (days)

A Acetate concentration in inuent A1 7 Municipal wastewater (020mg C/l of acetate) 5

A2 7 Municipal wastewater supplemented with 30mg C/l acetate 5

B pH in the SBR B1 7 Municipal wastewater 5

B2 8 Municipal wastewater 5

C Sludge retention time C1 7 Municipal wastewater 3

C2 7 Municipal wastewater 10

A.S.M. Chua et al. / Water Research 37 (2003) 360236113604

because of decarboxylation and the utilization of

acetate. Diluted sulfuric acid was added to keep the

pH at 8, unless otherwise stated.

In order to observe the development of PHA

production capability of sludge acclimatized under

different operational conditions, at least four sets of

24-h batch experiments were conducted for each SBR

periodically during the sludge acclimatization period of

each run. In the case of Run B, in addition to these, a set

of batch experiment with pH control at 69 was

performed to examine pH effect on PHA production

behavior of activated sludge. In all batch experiments,

one sample was taken at hour 0, 6, and 24, respectively,

for DOC, acetate, and PHA analyses. Chemical analysis

of each parameter was only done once for each sample

taken. The conditions of batch experiments conducted in

each run of study are described in Table 2.

In this study, the PHA content at hour 24 of batch

experiment (PHAX, 24) and PHA production rate are

used as the indicators for PHA production capability of

activated sludge. PHAX, 24 is dened as the percentage

of PHA concentration at hr 24 of batch experiment

divided by the sludge dry weight/mixed liquor suspended

solid (MLSS) at hour 24. In the preliminary experi-

ments, PHA content increased rapidly in the initial 0

10 h, then the increasing rate of PHA content decreased

or reached stable. PHA content has been considered as

one of the most important factors in affecting the cost of

PHA production. This is because the extraction ef-

ciency and purity of PHA are strongly dependent on it

[21]. On the other hand, PHA production rate was

calculated based on the data obtained in the initial 6 h of

the batch experiments. It was observed that the rate of

PHA accumulation was kept constant and the maximum

in the initial 6 h. Therefore, the maximum PHA

production rate will be used to discuss the potential of

activated sludge in producing PHA.

2.3. Analytical procedure

DOC was measured by a Shimadzu TOC-500

analyzer. Supernatant acetate and PO4-P in the samples

were analyzed by means of a Capillary Ion Analyzer

(Millipore Corp., USA). The determination of PHA was

performed by gas chromatography after methanolytic

decomposition as described in Satoh et al. [22]. A gas

chromatograph GC14A/FID (Shimadzu, Japan) with a

column Neutrabond-1 (GL Science, Japan, 30m length,

250 mm internal diameter, 0.4 mm lm thickness) wasused. The detector and injector temperatures were

250C and 180C, respectively. Initial temperature

ARTICLE IN PRESS

Table 2

Batch experiments conditions in three different runs of SBRs

Run Sludge from SBR MLSS (mg/l) pH Acetate concentration (mg C/l)

A A1 1500 8 1000 added at hr 0 and 6

A2 2200 8 1000 added at hr 0 and 6

B (i) Comparison of PHA production capability between sludge acclimatized in pH 7 and 8 condition

B1 1600 8 750 added at hour 0 and 6

B2 1600 8 750 added at hour 0 and 6

(ii) PHA production behavior of sludge under different pH condition

B1 and B2 1600 and 1600 6 750 added at hour 0 and 6

7 750 added at hour 0 and 6



C C1 500 8 500 added at hour 0 and 6

C2 2500 8 750 added at hour 0 and 6

magnetic stirrer

from air pump

addition of carbonsubstrate

pH probe connected topH controller

addition of diluted sulfuric acid for pH adjustment

Fig. 3. Schematic diagram of PHA production batch experi-

ment setup.


setting of column was 60C for 4min, then increased in

12C/min to 220C and maintained for 6min. One

microliter of sample was split injected into the GC

column (split ratio was 1:40 with a Shimadzu Auto-

Injector AOC-14A). Sodium 3-hydroxybutyrate (Sigma,

USA) was used as the standard for the quantication of

3HB. The copolymer of PHA consisting of 81:19 (wt%)

of 3HB and 3HV which was kindly supplied by ICI

Japan Inc. was used as the standard of 3HV. As acetate

was used as the carbon source in this study, the

monomeric unit of the PHA produced was almost all

3HB.

3. Results and discussion

3.1. Performance of the wastewater treatment process

In this study, despite the differences of operational

conditions, all sludge acclimatized under anaerobic

aerobic sequence showed metabolic characteristics

typically observed in EBPR sludge [8]. Typical char-

acteristics of activated sludge observed are shown in

Fig. 4. All acetate present in the inuent was taken up

during anaerobic phase of the activated sludge process.

Anaerobic phosphate release and aerobic phosphate

uptake, coupled with anaerobic PHA accumulation and

aerobic PHA consumption, were recorded.

From the results of SBRs monitoring and PHA batch

experiments, we observed that not only could the

acclimatized sludge behave well in the range of operating

conditions applied, but also show the potential in

producing PHA. Simultaneous achievement of effective

wastewater treatment and enrichment of PHA accumu-

lating microorganisms is vital to ensure the success of

PHA production system by activated sludge. This is

because the most attractive feature of the proposed

process is that the excess sludge for PHA production

(second stage) simply comes from the usual wastewater

treatment process (rst stage).

3.2. Effect of operational conditions of wastewater

treatment process on PHA production capability of

activated sludge

In order to measure the PHA production capability of

activated sludge from SBRs in each run of activated

sludge process, batch experiments for PHA production

were conducted. Fig. 5 shows the typical proles of

DOC, acetate, and PHA during the 24-h batch experi-

ments.

3.2.1. Effect of acetate concentration in influent

Fig. 6(ai) and (aii) shows the PHAX, 24 and PHA

production rate achieved by sludge throughout the

sludge acclimatization period of Run A. It was clearly

demonstrated that supplementation of acetate in inuent

wastewater improved the PHA production capability of

activated sludge considerably. The PHAX, 24 of SBR-A1

sludge uctuated between 16% and 26% of sludge dry

weight, averaged at 21%; while PHAX, 24 of SBR-A2

sludge uctuated between 26% and 36%, averaged at

31%. Generally, PHAX, 24 of SBR-A2 sludge was higher

than that of SBR-A1 sludge by 10%. In acetate-rich

acclimatization, PHA accumulating microorganisms

might proliferate well or their PHA storage capacity

might be increased; thus leading to a higher PHA

production capability. On the other hand, SBR-A2

sludge also exhibited a higher PHA production rate than

SBR-A1 sludge. Rates achieved were 22mg C/g SS/h (in

average) and 12mg C/g SS/h (in average), respectively.

In Run A, it was observed that the PHA production

capability of sludge uctuated very much, especially in

terms of PHA production rate. Since a constant amount

of acetate supplement was fed to SBR-A2 throughout

the acclimatization period, the PHA production rate of

SBR-A2 was supposed to uctuate less than that of

SBR-A1. However, opposite observation was obtained

and the reason behind it is unknown.

We also noticed that the municipal wastewater

contained insignicant amount of acetate, 020mg C/l

in this study. It is therefore suggested that the waste-

water or carbon sources rich in volatile fatty acids

(VFAs) should be used to enhance the PHA production

capability of acclimatized sludge. Good candidates for

this purpose could be the efuent of sludge fermentation

process, industrial wastewater from food industries,

dairy industries and pharmaceutical plants.

3.2.2. Effect of pH

The PHA production capability of activated sludge

acclimatized in SBR-B1 (pH 7) and in SBR-B2 (pH 8)

ARTICLE IN PRESS

0

5

10

15

20

25

0 50 100 150 200cycle time(min)

mgC

/l

0246810121416

mgP

/l

anaerobic aerobic

Fig. 4. Typical concentration proles of DOC (), acetate

(~), PHA (*) and PO4-P (m) in one anaerobicaerobic processcycle of SBRs (Data source: Run A1, day 17 of sludge

acclimatization).


are compared in Fig. 6(bi) and (bii). In Run B, we

observed that both sludge possessed similar PHA

production capability. SBR-B1 sludge accumulated

PHA up to around 2630% of sludge dry weight

while SBR-B2 sludge achieved PHA content up

to 2432%. Likewise, PHA production rates of SBR-

B1 sludge and SBR-B2 sludge shared a similar

range that averaged at 26 and 28mg C/g SS/h,

respectively.

The results implied that during sludge acclimatization,

PHA production capability of sludge was not affected

by pH condition in the range of 78. Rather consistent

PHA production capability was observed in Run B,

indicating that stable production of PHA is possible

even with mixed-culture process.

In order to gain more insights into pH effects (beyond

the range of pH 78) on sludges PHA production

capability, more studies are awaited. However, from our

results, it can be concluded that pH control is not critical

in enriching the PHA accumulating microorganisms, if

pH of a selected activated sludge process falls between 7

and 8.

3.2.3. Effect of SRT

With activated sludge acclimatized in SBR-C1 (3-day

SRT) and in SBR-C2 (10-day SRT), ve batch experi-

ments for PHA production were conducted. As shown in

Fig. 6(ci), the PHAX, 24 of SBR-C1 sludge and SBR-C2

sludge was relatively consistent at around 31% and 21%

in average, respectively. However, the PHA production

rate of both sludge declined drastically in the rst 25

days of acclimatization period before becoming rela-

tively stable. The reason for such trend is not known

(Fig. 6(cii)).

Here we outline some possible reasons to explain the

variation of PHAX, 24 caused by the difference in SRT.

Firstly, the SRT theoretically determines mean micro-

bial life-time, and hence microbial population. From our

result, shorter SRT may select microbial community

with bigger PHA production capacity than that selected

under longer SRT. As the second possible mechanism,

the SRT might have affected the PHA accumulation

capability of activated sludge via the difference in

organic loading to biomass. Generally, the longer the

SRT, the higher the biomass concentration in the

ARTICLE IN PRESS

(ai) Run A1 (day 48 of acclimatization)

0

500

1000

1500

2000

0 5 10 15 20 25 30time (hr)

mgC

/l(aii) Run A2 (day 48 of acclimatization)

0

500

1000

1500

2000

0 5 10 15 20 25 30time (hr)

mgC

/l

(bi) Run B1 (day 25 of acclimatization)

0

500

1000

1500

0 5 10 15 20 25 30time (hr)

mgC

/l

(bii) Run B2 (day 25 of acclimatization)

0

500

1000

1500

0 5 10 15 20 25 30time (hr)

mgC

/l

(ci) Run C1 (day 34 of acclimatization)

0

500

1000

1500

0 5 10 15 20 25 30time (hr)

mgC

/l

(cii) Run C2 (day 34 of acclimatization)

0

500

1000

1500

0 5 10 15 20 25 30time (hr)

mgC

/l

Fig. 5. Typical concentration proles of DOC (), acetate (~), and PHA (*) in 24-h batch experiments by using sludge from SBRs inRuns AC.


reactor. In this study, the MLSS in SBR-C1 with an

SRT of 3 days was around 700mg/l, whereas it

was around 2500mg/l in SBR-C2 with an SRT of 10

days. Because of the difference in MLSS, microorgan-

isms in SBR-C1 had a chance to take up about 4

times more organic substrates than those in SBR-C2.

This might have led to the higher PHA production

capability of activated sludge in SBR-C1. In addition,

activated sludge process with longer SRT normally

contains higher amount of inert biomass and this

might contribute to the lower PHA content in

SBR-C2.

On the contrary, van Aalst-van Leeuwen et al. [23]

observed that faster growing organisms accumulated less

PHB. In addition, Dionisi et al. [24] showed in his

anoxic batch tests that as organic loading increased, the

PHA storage capability of mixed cultures decreased.

And it was indicated that a maximum PHA storage

might occur at intermediate organic loading rate.

Although present case does not conform to the literature

evidences, it is obvious that SRT, as well as organic

loading, could greatly inuence the PHA production

capability. Comprehensive quantication of the acti-

vated sludge process is necessary to describe this

discrepancy.

Apart from the reason that short SRT sludge

possessed higher PHA production capability, sludge

acclimatization with a short SRT may also be preferable

for PHA production purpose. This is because the sludge

yield under a shorter SRT is higher than that under a

longer SRT. Therefore, activated sludge process oper-

ated with a short SRT can supply sufcient amount of

sludge for PHA production compared to that with a

long SRT.

3.3. PHA production behavior of activated sludge under

different pH conditions

In Run B, a series of batch experiments in which pH

was controlled at different values, i.e., at pH 69 were

also performed for each SBR. The activated sludge used

for these batch experiments were taken on day 35 of

sludge acclimatization. Fig. 7 illustrates the PHAX, 24achieved at various pH conditions in batch experiments.

As pH increased from 6 to 9, PHAX, 24 of sludge

increased as well. Same trend was observed for both

sludge. At pH 6 and 7, there was very little PHA

accumulation, and PHAX, 24 was less than 5% of sludge

dry weight. At pH 8 and 9, PHA accumulation was

ARTICLE IN PRESS

(ci) Run C: PHAx,24

010203040

acclimatization period (day)

% o

f MLS

S

(cii) Run C: PHA production rate

010203040


mgC

/gSS

/hr

(bi) Run B: PHAx,24

010203040


% o

f MLS

S

(bii) Run B: PHA production rate

010203040


mgC

/gSS

/hr

(ai) Run A: PHAx,24

010203040

0 10 20 30 40 50

0 10 20 30 40 50

0 10 20 30 40 50

0 10 20 30 40 50

0 10 20 30 40 50 0 10 20 30 40 50


% o

f MLS

S

(aii) Run A: PHA production rate

010203040


mgC

/gSS

/hr

Fig. 6. PHAX, 24 and PHA production rate achieved by activated sludge throughout the acclimatization period. () for Run A1, B1

and C1; (m) for Run A2, B2 and C2.


stimulated, and PHAX, 24 reached 2532% of sludge dry

weight.

The signicant effect of pH on PHA accumulation

during batch experiments was thereby highlighted.

Depression of PHA production was profound at

pHp7. Similar observation was obtained by Suzukiet al. [25] and Takabatake [26]. Suzuki et al. observed

that PHA content achieved by Rhodobacter sphaeroides

RV was higher at pH 8.0 and 8.5 than that at pH 7.0 and

7.5. While the latter researcher demonstrated that

pHX8 was benecial for PHA production by usingactivated sludge acclimatized with synthetic wastewater.

The results indicated that pH control is essential in

optimizing the PHA production process and pHX8 isrecommended here. At this point, we could not explain

denitely why the PHA accumulation behavior is very

sensitive in the pH range of 78, but it is suspected that

this phenomenon was caused by the undissociated acetic

acid. Fleit [27] hypothesized to explain the effect of pH

and acetate concentration on biomass in activated

sludge that undissociated acetic acid (CH3COOH) will

rapidly diffuse into bacterial cells, then dissociate and

impose a proton load on the intracellular milieu and

subsequently lower the pH. The pH decrement could be

detrimental to PHA production. Under low pH condi-

tion, acetic acid will remain mostly in undissociated

form so as to maintain the equilibrium. Therefore,

CH3COOH diffusion into bacterial cells was probably

signicant in batch experiments of low pH. This

phenomenon might have led to our observation.

However, detailed studies are required to further

conrm such hypothesis.

Though PHA production capability of sludge was

signicantly reduced when pH 7 was applied in batch

experiments, sludge acclimatized under pH 7 (SBR-B1)

did not differ from sludge acclimatized under pH 8

(SBR-B2) in terms of their PHA production capability.

This contradictory observation was most probably due

to the difference in acetate concentration. In batch

experiments, high acetate concentration, 750mg C/l was

applied for PHA production; but only 020mg C/l of it

was available for sludge acclimatization. Due to the low

availability of acetate, the phenomenon mentioned

above could be insignicant during the sludge acclima-

tization.

3.4. Feasibility of PHA production by activated sludge

With the outcome of this study, the main question one

may raise is: how realistic the idea of PHA production

by using activated sludge is? In Table 3, the PHA

content and PHA production rate achieved by activated

sludge in this study is compared to the one obtained by

some pure culture fermentation processes. The PHA

content achieved by Ralstonia eutropha [28], Alkaligenes

latus [29] and Recombinant E. coli [5] was 74%, 50% and

76% of cell dry weight, respectively. These achievements

are much higher than the PHA content obtained by

activated sludge in this study, which is about 30% of

sludge dry weight. Since PHA content more than 80% is

necessary for an economical PHA production system [2],

the PHA production capability of activated sludge has

to be further improved. On the other hand, activated

sludge has shown its potential in terms of PHA

production rate. This is because activated sludge could

produce PHA in a rate of 28mg C/g SS/h, which is

comparable and near to the PHA production rate

achieved by those pure cultures listed in Table 3.

Although the PHA content achieved by activated sludge

in this study is far from practical value, it is still too early

to deny the feasibility of this system.

Most importantly, this study has demonstrated

that the optimization of operational conditions in

activated sludge process is essential to enhance the

ARTICLE IN PRESS

Fig. 7. PHAX, 24 achieved by SBR-B1 sludge and SBR-B2

sludge under different pH conditions during batch experiments.

Table 3

PHA content and PHA production rate achieved by activated sludge and pure cultures

Strain Substrate PHA PHA content (%) PHA production rate (mg C/g SS/h) Reference

Ralstonia eutropha Glucose P(3HB) 74 31 [28]

Alkaligenes latus Sucrose P(3HB) 50 31 [29]

Recombinant E. coli Glucose P(3HB) 76 42 [5]

Activated sludge Acetate P(3HB) 30 28 (results of Run B, SBR-B2) Present study


PHA production capability of activated sludge. Also,

PHA production process is found to be sensitive to pH

condition and acetate concentration. With our ndings,

it is condent that there is a big room for the

improvement of PHA production system by using

activated sludge.

4. Conclusion

The authors have proposed a PHA production system

in which excess sludge of the wastewater treatment

process was utilized as PHA production bacterial

cultures. Main focus of this research was to investigate

the optimum operating conditions of activated sludge

process for enhancing the PHA production capability of

sludge. Although the PHA content achieved (30%) in

present study is much lower than that by pure culture,

the proposed method may still serve well as an

environment-friendly means to convert waste into

valuable product. Above all, we have demonstrated that

the PHA production capability of activated sludge could

be enhanced by manipulating various operational

conditions in anaerobicaerobic activated sludge pro-

cess. The results obtained are summarized as follows:

1. Sludge acclimatized with acetate-supplemented mu-

nicipal wastewater could produce PHA up to 31% of

sludge dry weight while sludge acclimatized with

municipal wastewater only achieved 21% of PHA

content.

2. Sludge acclimatized under pH 7 and 8 condition

showed similar PHA production capability if batch

experiments were run at pH 8. However, when

different pH values were applied in batch experi-

ments, the PHA production behavior was distinc-

tively varied. Under batch experiments run at pH 6

and 7, PHA production was almost negligible. Such

undesirable outcome was not observed at pH 8 and 9.

It was suspected that the diffusion of undissociated

acetic acid into the bacterial cells had suppressed the

PHA production.

3. It was also found that sludge with a short SRT (3

days) could achieve PHA content about 10% more

than sludge with a long SRT (10 days).

References

[1] Braunegg G, Lefebvre G, Genser KF. Polyhydroxyalk-

anoates, biopolyesters from renewable resources: physio-

logical and engineering aspects. J Biotechnol 1998;65:

12761.

[2] Lee SY. Plastic bacteria? Progress and prospects for

polyhydroxyalkanoates production in bacteria. TIBTECH

1996;14:4318.

[3] Holmes PA. Applications polyhydroxybutyratea micro-

bially produced biodegradable thermoplastics. Phys Tech-

nol 1985;16:326.

[4] Wang F, Lee SY. Poly(3-hydroxybutyrate) production

with high polymer content by fed-batch culture of

Alkaligenes latus under nitrogen limitation. Appl Environ

Microbiol 1997;63:37036.

[5] Lee SY, Chang HN. Effect of complex nitrogen source on

the synthesis and accumulation of poly(3-hydroxybutyric

acid) by recombinant Escherichia coli in ask and fed-

batch cultures. J Environ Polym Degrad 1994;2:16976.

[6] Satoh H, Mino T, Matsuo T. PHA production by

activated sludge. Int J Biol Macromol 1999;25:1059.

[7] Satoh H, Mino T. Production of PHAs from activated

sludge. In: Doi Y, Steinbuchel A, editors. Biopolymers,

vol. 3a. New York: Wiley VCH; 2001. p. 33752.

[8] Mino T, van Loosdretch MCM, Heijnen JJ. Microbiology

and biochemistry of the enhanced biological phosphate

removal process. Water Res 1998;32(11):3193207.

[9] Satoh H, Iwamoto I, Mino T, Matsuo T. Activated sludge

as a possible source of biodegradable plastic. Water Sci

Tech 1998;38:1039.

[10] Ueno T, Satoh H, Mino T, Matsuo T. Production of

biodegradable plastics. Polym Preprints Jpn

1993;42(9):36735 (In Japanese).

[11] Iwamoto Y, Satoh H, Mino T, Matsuo T. Production of

biodegradable plastic PHA by excess sludge from anaero-

bicaerobic activated sludge process. Proc Environ Eng

Res 1994;31:30514 (In Japanese).

[12] Saito Y, Soejima T, Tomozawa T, Doi Y, Kiya F.

Production of biodegradable plastics from volatile acids

using activated sludge. J Environ Syst Eng 1995;521:

14554 (In Japanese).

[13] Hu WF, Chua H, Yu PHF. Synthesis of poly(hydrox-

ybutyrae-co-3-hydroxyvalerate) from activated sludge.

Biotechnol Lett 1997;19(7):6958.

[14] Chua H, Yu PHF, Ho LY. Coupling of wastewater

treatment with storage polymer production. Appl Biochem

Biotechnol 1997;63:62735.

[15] Chua H, Yu PHF. Production of biodegradable plastics

from chemical wastewatera novel method to reduce

excess activated sludge generated from industrial waste-

water treatment. Water Sci Technol 1999;39(1011):

27380.

[16] Chua H, Yu PHF, Ma CK. Accumulation of biopolymers

in activated sludge biomass. Appl Biochem Biotechnol

1999;77:38999.

[17] Tsunemasa N. Utilization of excess sludge for the

production of biodegradable plastics. J Water Waste

1998;40(11):9816 (In Japanese).

[18] Lemos PC, Viana C, Salgueiro EN, Ramos AM, Crespo

JPSG, Reis MAM. Effect of carbon source on the

formation of polyhydroxyalkanoates (PHA) by a phos-

phate-accumulating mixed culture. Enzyme Microbial

Technol 1998;22:66271.

[19] Fang HHP, Liu Y. Intracellular polymers in aerobic sludge

of sequencing batch reactors. J Environ Eng 2000;126:

72338.

[20] Ma CK, Chua H, Yu PHF, Hong K. Optimal production

of polyhydroxyalkanoates in activated sludge biomass.

Appl Biochem Biotechnol 2000;84:9819.

ARTICLE IN PRESSA.S.M. Chua et al. / Water Research 37 (2003) 360236113610

[21] Choi J, Lee SY. Factors affecting the economics of

polyhydroxyalkanoates production by bacterial fermenta-

tion. Appl Microbiol Biotechnol 1999;51:1321.

[22] Satoh H, Ramey WD, Koch FA, Oldham WK,

Mino T, Matsuo T. Anaerobic substrate uptake by the

enhanced biological phosphorous removal activated

sludge treating real sewage. Water Sci Technol 1996;

34:916.

[23] van Aalst-van Leeuwen MA, Pot MA, van Loosdrecht

MCM, Heijin JJ. Kinetic modeling of poly(b-hydroxybu-tyrate) production and consumption by Paracoccus

pantotrophus under dynamic substrate supply. Biotechnol

Bioeng 1997;55(5):77382.

[24] Dionisi D, Majone M, Ramadori R, Beccari M. The

storage of acetate under anoxic conditions. Water Res

2001;35(11):26618.

[25] Suzuki T, Tsygankov AA, Miyake J, Tokiwa Y, Asada Y.

Accumulation of poly(3-hydroxybutyrate) by a non-sulfur

photosynthetic bacterium, Rhodobacter sphaeroides RV at

different pH. Biotenchnol Lett 1995;17(4):395400.

[26] Takabatake H, Department of Civil Engineering, Faculty

of Engineering, Tohoku University, Sendai, Japan, 2000.

[27] Fleit E. Intracellular pH regulation in biological excess

phosphorous removal systems. Water Res

1995;29(7):178792.

[28] Kim BS, Lee SC, Lee SY, Chang HN, Chang YK, Woo SI.

Production of poly(3-hydroxybutyric acid) by fed-batch

culture of Alkaligenes eutrophus with glucose concentration

control. Biotechnol Bioeng 1994;43:8928.

[29] Yamane T, Fukunage M, Lee YW. Increased PHA

productivity by producer. Biotechnol Bioeng

1996;50:197202.

ARTICLE IN PRESSA.S.M. Chua et al. / Water Research 37 (2003) 36023611 3611

Production of polyhydroxyalkanoates (PHA) by activated sludge treating municipal wastewater: effect of pH, sludge retention timIntroductionMaterials and methodsOperation of anaerobic-aerobic activated sludge processes under different operational conditionsPHA production batch experimentsAnalytical procedure

Results and discussionPerformance of the wastewater treatment processEffect of operational conditions of wastewater treatment process on PHA production capability of activated sludgeEffect of acetate concentration in influentEffect of pHEffect of SRT

PHA production behavior of activated sludge under different pH conditionsFeasibility of PHA production by activated sludge

ConclusionReferences

chua a - 2003 - production of pha by as treating municipal wastewater- effect of ph, sludge...

Documents