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ARTICLE IN PRESS Water Research 37 (2003) 3602–3611 Production of polyhydroxyalkanoates (PHA) by activated sludge treating

ARTICLE IN PRESS

Water Research 37 (2003) 3602–3611

ARTICLE IN PRESS Water Research 37 (2003) 3602–3611 Production of polyhydroxyalkanoates (PHA) by activated sludge treating

Production of polyhydroxyalkanoates (PHA) by activated sludge treating municipal wastewater: effect of pH, sludge retention time (SRT), and acetate concentration in influent

Adeline S.M. Chua a, *, Hiroo Takabatake b , Hiroyasu Satoh a , Takashi Mino a

a Institute of Environmental Studies, Graduate School of Frontier Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 Japan b Department 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 influent, pH, and sludge retention time (SRT) were studied. Sludge acclimatized with municipal wastewater supplemented with acetate could accumulate PHAup 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 PHAproduction capability than sludge with an SRT of 10 days. Sludge acclimatized under pH 7 and 8 conditions in sequencing batch reactors (SBRs) exhibited similar PHAproduction capability. However, in PHA production batch experiments, pH value influenced significantly the PHAaccumulation behavior of activated sludge. When pH of batch experiments was controlled at 6 or 7, a very low PHAproduction was observed. The production of PHAwas stimulated when pH was kept at 8 or 9. r 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Activated sludge; Polyhydroxyalkanoates (PHA); Anaerobic–aerobic 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

*Corresponding author. Tel.: +81-3-5841-6250; fax: +81-3-5841-8531. E-mail address: adeline@env.t.u-tokyo.ac.jp (A.S.M. Chua).

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, PHAhave attracted industrial interest as biodegradable plastics not only because of their compatible material properties like synthetic thermo- plastics but also could PHAbe 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 PHAproduction 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

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

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

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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 PHAcontent 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 PHAcontent could be achieved by using pure culture fermentation process, the cost of PHAproduction is still too high for PHAto become a competitive commodity plastic material. As to reduce the expensiveness of PHA, a novel PHAproduction 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 PHAproduction by using activated sludge was ignited owing to PHA’s function as an intermediate metabolic product in activated sludge process. It has been recognized that PHAis one of the most important carbon storage materials especially in the anaerobic–aerobic 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 PHAaccumulation. 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 PHAproduction system by activated sludge will be the cost reduction in cultivating PHAproducing 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 first stage is the activated sludge process for wastewater treatment, and the second stage is PHAproduction process by using the excess sludge from the wastewater treatment process. In the first stage, it is essential to optimize the operational conditions for sludge acclimatization or for the enrich- ment of PHAaccumulating microorganisms so that the PHAproduction 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.

Wastewater Treatment Process

Influent wastewater Effluent activated sludge sedimentation process tank Excess PHA Production Process sludge PHA production External
Influent wastewater
Effluent
activated sludge
sedimentation
process
tank
Excess
PHA Production Process
sludge
PHA
production
External carbon substrate, e.g.
industrial wastewater, raw
carbon sources
reactor

Fig. 1. PHAproduction system by using activated sludge treating wastewater.

In most of the studies of PHAproduction by activated sludge, synthetic wastewaters were used to cultivate PHAproducing sludge, such as in Ueno et al. [10], Iwamoto et al. [11], Saito et al. [12], Hu et al. [13], Chua et al. [14–16], 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 PHAproduction capability of sludge. Satoh et al. [9] reported a high accumulation of 62% of cell dry weight by using activated sludge acclimatized in anaerobic–aerobic process with very limited oxygen supply to the anaerobic condition. The effect of carbon–nitrogen (C:N) ratio in reactor liquor on PHA productivity was examined in Chua et al. [14–16], 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 influence the PHAproduction capability of sludge. The opera- tional conditions being investigated were the acetate concentration in influent, 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 significant its concentration can enhance the PHAproduction 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 PHAproduction capability enhancement as well as for satisfactory effluent 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 PHAproduction process. At last, feasibility of using activated sludge treating municipal wastewater for PHAproduction is discussed.

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2. Materials and methods

2.1. Operation of anaerobic–aerobic activated sludge processes under different operational conditions

Two bench-scale sequential batch activated sludge reactors (SBRs) served as the wastewater treatment process, the first stage of the proposed PHAproduction system (Fig. 1). They were operated with municipal wastewater as influent. 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 influent wastewater quality fluctuated, the typical influent quality data is shown below:

COD=60–130 mg O/l Total organic carbon (TOC)=30–130 mg C/l Acetate=0–20 mg C/l Phosphate (PO 4 -P)=1.7–5.6 mg 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 (15 min), influent feeding (5 min), anaerobic (1 h), aerobic (2 h) and settling period

P sludge wasting pump effluent pump P air pump P influent pump 20l mixer air diffuser
P
sludge wasting pump
effluent pump
P
air pump
P
influent pump
20l
mixer
air diffuser

Fig. 2. Schematic diagram of SBRs.

(40 min). 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 18 C (winter) to 25 C (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 A–C) were conducted. Two SBRs were operated in parallel in each run, with the operational conditions summarized in Table 1. Both SBRs in Run Awere 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 anaerobic–anoxic–oxic (A 2 O) configuration. This pilot plant also received influent wastewater from the municipal wastewater treatment plant mentioned above. Sludge characteristics such as the profiles of dissolved organic carbon (DOC), acetate, PO 4 -P, and PHAin one SBR cycle were monitored regularly during each operation period. Along the operation of activated sludge processes, 1000 ml of sludge was taken periodically from each SBR to perform PHAproduction 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

Table 1 Operating conditions for Runs A–C of SBRs

Run

Investigated operating condition

SBR

pH

Influent

SRT (days)

A

Acetate concentration in influent

A1

7

Municipal wastewater (0–20 mg C/l of acetate)

5

 

A2

7

Municipal wastewater supplemented with 30 mg 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

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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 6–9 was performed to examine pH effect on PHAproduction behavior of activated sludge. In all batch experiments, one sample was taken at hour 0, 6, and 24, respectively, for DOC, acetate, and PHAanalyses. Chemical analysis

addition of carbon substrate addition of diluted sulfuric acid for pH adjustment from air pump pH
addition of carbon
substrate
addition of diluted
sulfuric acid for pH
adjustment
from air pump
pH probe connected to
pH controller

magnetic stirrer

Fig. 3. Schematic diagram of PHAproduction batch experi- ment setup.

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 PHAcontent at hour 24 of batch experiment (PHA X, 24 ) and PHAproduction rate are used as the indicators for PHAproduction capability of activated sludge. PHA X, 24 is defined as the percentage of PHAconcentration 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, PHAcontent increased rapidly in the initial 0– 10 h, then the increasing rate of PHAcontent decreased or reached stable. PHAcontent has been considered as one of the most important factors in affecting the cost of PHAproduction. This is because the extraction effi- ciency and purity of PHAare strongly dependent on it [21]. On the other hand, PHAproduction rate was calculated based on the data obtained in the initial 6 h of the batch experiments. It was observed that the rate of PHAaccumulation 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 PO 4 -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]. Agas

chromatograph GC14A/FID (Shimadzu, Japan) with a

column Neutrabond-1 (GL Science, Japan, 30 m length, 250 mm internal diameter, 0.4 mm film thickness) was used. The detector and injector temperatures were

250 C and 180 C,

respectively. Initial temperature

Table 2 Batch experiment’s conditions in three different runs of SBRs

Run

Sludge from SBR

MLSS (mg/l)

pH

Acetate concentration (mg C/l)

AA1

1500

8

1000 added at hr 0 and 6

 

A2

2200

8

1000 added at hr 0 and 6

B

(i) Comparison of PHAproduction 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) PHAproduction 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

8

750 added at hour 0 and 6

9

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

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setting of column was 60 C for 4 min, then increased in 12 C/min to 220 C and maintained for 6 min. 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 quantification of 3HB. The copolymer of PHAconsisting 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 PHAproduced 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 influent was taken up during anaerobic phase of the activated sludge process. Anaerobic phosphate release and aerobic phosphate uptake, coupled with anaerobic PHAaccumulation and aerobic PHAconsumption, were recorded. From the results of SBRs monitoring and PHAbatch 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 PHAaccumu- lating microorganisms is vital to ensure the success of PHAproduction system by activated sludge. This is because the most attractive feature of the proposed process is that the excess sludge for PHAproduction (second stage) simply comes from the usual wastewater treatment process (first stage).

  • 3.2. Effect of operational conditions of wastewater

treatment process on PHA production capability of activated sludge

In order to measure the PHAproduction capability of activated sludge from SBRs in each run of activated sludge process, batch experiments for PHAproduction were conducted. Fig. 5 shows the typical profiles of DOC, acetate, and PHAduring the 24-h batch experi- ments.

3.2.1. Effect of acetate concentration in influent Fig. 6(ai) and (aii) shows the PHA X, 24 and PHA production rate achieved by sludge throughout the sludge acclimatization period of Run A. It was clearly

anaerobic aerobic 25 16 14 20 12 10 15 8 10 6 4 5 2 0
anaerobic
aerobic
25
16
14
20
12
10
15
8
10
6
4
5
2
0
0
0
50
100
150
200
mgC/l
mgP/l

cycle time(min)

Fig. 4. Typical concentration profiles of DOC (), acetate (~), PHA(*) and PO 4 -P (m) in one anaerobic–aerobic process cycle of SBRs (Data source: Run A1, day 17 of sludge

acclimatization).

demonstrated that supplementation of acetate in influent wastewater improved the PHAproduction capability of activated sludge considerably. The PHA X, 24 of SBR-A1 sludge fluctuated between 16% and 26% of sludge dry weight, averaged at 21%; while PHA X, 24 of SBR-A2 sludge fluctuated between 26% and 36%, averaged at 31%. Generally, PHA X, 24 of SBR-A2 sludge was higher than that of SBR-A1 sludge by 10%. In acetate-rich acclimatization, PHAaccumulating microorganisms might proliferate well or their PHAstorage capacity might be increased; thus leading to a higher PHA production capability. On the other hand, SBR-A2 sludge also exhibited a higher PHAproduction rate than SBR-A1 sludge. Rates achieved were 22 mg C/g SS/h (in average) and 12 mg C/g SS/h (in average), respectively. In Run A, it was observed that the PHA production capability of sludge fluctuated very much, especially in terms of PHAproduction rate. Since a constant amount of acetate supplement was fed to SBR-A2 throughout the acclimatization period, the PHAproduction rate of SBR-A2 was supposed to fluctuate 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 insignificant amount of acetate, 0–20 mg 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 effluent of sludge fermentation process, industrial wastewater from food industries, dairy industries and pharmaceutical plants.

3.2.2. Effect of pH

The PHAproduction capability of activated sludge acclimatized in SBR-B1 (pH 7) and in SBR-B2 (pH 8)

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2000 (ai) Run A1 (day 48 of acclimatization) 1500 1000 500 0 mgC/l mgC/l
2000
(ai) Run A1 (day 48 of acclimatization)
1500
1000
500
0
mgC/l
mgC/l

2000

1500

1000

500

0

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

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0

5

10

15

20

25

30

0

5

10

15

20

25

30

 

time (hr)

time (hr)

1500 (bi) Run B1 (day 25 of acclimatization) 1000 500 0 0 5 10 15 20
1500
(bi) Run B1 (day 25 of acclimatization)
1000
500
0
0
5
10
15
20
25
30
mgC/l
mgC/l

time (hr)

1500

1000

500

0

(bii) Run B2 (day 25 of acclimatization) 0 5 10 15 20 25 30
(bii) Run B2 (day 25 of acclimatization)
0
5
10
15
20
25
30

time (hr)

1500 (ci) Run C1 (day 34 of acclimatization) 1000 500 0 0 5 10 15 20
1500
(ci) Run C1 (day 34 of acclimatization)
1000
500
0
0
5
10
15
20
25
30
mgC/l
mgC/l

time (hr)

1500

1000

500

0

(cii) Run C2 (day 34 of acclimatization) 0 5 10 15 20 25 30
(cii) Run C2 (day 34 of acclimatization)
0
5
10
15
20
25
30

time (hr)

Fig. 5. Typical concentration profiles of DOC (), acetate (~), and PHA(*) in 24-h batch experiments by using sludge from SBRs in Runs A–C.

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 PHAup to around 26–30% of sludge dry weight while SBR-B2 sludge achieved PHAcontent up to 24–32%. Likewise, PHAproduction rates of SBR- B1 sludge and SBR-B2 sludge shared a similar range that averaged at 26 and 28 mg C/g SS/h, respectively. The results implied that during sludge acclimatization, PHAproduction capability of sludge was not affected by pH condition in the range of 7–8. Rather consistent PHAproduction capability was observed in Run B, indicating that stable production of PHAis possible even with mixed-culture process. In order to gain more insights into pH effects (beyond the range of pH 7–8) on sludge’s PHAproduction capability, more studies are awaited. However, from our results, it can be concluded that pH control is not critical in enriching the PHAaccumulating 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), five batch experi- ments for PHAproduction were conducted. As shown in Fig. 6(ci), the PHA X, 24 of SBR-C1 sludge and SBR-C2 sludge was relatively consistent at around 31% and 21% in average, respectively. However, the PHAproduction rate of both sludge declined drastically in the first 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 PHA X, 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 PHAproduction capacity than that selected under longer SRT. As the second possible mechanism, the SRT might have affected the PHAaccumulation capability of activated sludge via the difference in organic loading to biomass. Generally, the longer the SRT, the higher the biomass concentration in the

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(ai) Run A: PHA x,24

40 30 20 10 0 0 10 20 30 40 50 % of MLSS
40
30
20
10
0
0
10
20
30
40
50
% of MLSS

acclimatization period (day) (bi) Run B: PHA x,24

40 30 20 10 0 0 10 20 30 40 50 % of MLSS
40
30
20
10
0
0
10
20
30
40
50
% of MLSS

acclimatization period (day) (ci) Run C: PHA x,24

40 30 20 10 0 0 10 20 30 40 50 % of MLSS
40
30
20
10
0
0
10
20
30
40
50
% of MLSS

acclimatization period (day)

(aii) Run A: PHA production rate

40 30 20 10 0 0 10 20 30 40 50 mgC/gSS/hr
40
30
20
10
0
0
10
20
30
40
50
mgC/gSS/hr

acclimatization period (day) (bii) Run B: PHA production rate

40 30 20 10 0 0 10 20 30 40 50 mgC/gSS/hr
40
30
20
10
0
0
10
20
30
40
50
mgC/gSS/hr

acclimatization period (day)

(cii) Run C: PHA production rate

40 30 20 10 0 0 10 20 30 40 50 mgC/gSS/hr
40
30
20
10
0
0
10
20
30
40
50
mgC/gSS/hr

acclimatization period (day)

Fig. 6. PHA X, 24 and PHAproduction rate achieved by activated sludge throughout the acclimatization period. () for Run A1, B1 and C1; (m) for Run A2, B2 and C2.

reactor. In this study, the MLSS in SBR-C1 with an SRT of 3 days was around 700 mg/l, whereas it was around 2500 mg/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 PHAproduction 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 PHAcontent 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 PHAstorage 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 influence the PHAproduction capability. Comprehensive quantification of the acti- vated sludge process is necessary to describe this discrepancy.

Apart from the reason that short SRT sludge possessed higher PHAproduction capability, sludge acclimatization with a short SRT may also be preferable for PHAproduction 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 sufficient amount of sludge for PHAproduction 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 6–9 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 PHA X, 24 achieved at various pH conditions in batch experiments. As pH increased from 6 to 9, PHA X, 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 PHA X, 24 was less than 5% of sludge dry weight. At pH 8 and 9, PHA accumulation was

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stimulated, and PHA X, 24 reached 25–32% of sludge dry weight. The significant effect of pH on PHAaccumulation during batch experiments was thereby highlighted. Depression of PHAproduction was profound at pHp7. Similar observation was obtained by Suzuki et al. [25] and Takabatake [26]. Suzuki et al. observed that PHAcontent 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 beneficial for PHAproduction by using activated sludge acclimatized with synthetic wastewater. The results indicated that pH control is essential in optimizing the PHAproduction process and pHX8 is recommended here. At this point, we could not explain definitely why the PHAaccumulation behavior is very sensitive in the pH range of 7–8, 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 (CH 3 COOH) 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 PHAproduction. Under low pH condi- tion, acetic acid will remain mostly in undissociated form so as to maintain the equilibrium. Therefore, CH 3 COOH diffusion into bacterial cells was probably significant in batch experiments of low pH. This

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Fig. 7. PHA X, 24 achieved by SBR-B1 sludge and SBR-B2 sludge under different pH conditions during batch experiments.

phenomenon might have led to our observation. However, detailed studies are required to further confirm such hypothesis. Though PHAproduction capability of sludge was significantly 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 PHAproduction capability. This contradictory observation was most probably due to the difference in acetate concentration. In batch experiments, high acetate concentration, 750 mg C/l was applied for PHAproduction; but only 0–20 mg C/l of it was available for sludge acclimatization. Due to the low availability of acetate, the phenomenon mentioned above could be insignificant 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 PHAproduction by using activated sludge is? In Table 3, the PHA content and PHAproduction 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 PHAcontent obtained by activated sludge in this study, which is about 30% of sludge dry weight. Since PHAcontent more than 80% is necessary for an economical PHAproduction system [2], the PHAproduction 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 PHAin a rate of 28 mg C/g SS/h, which is comparable and near to the PHAproduction 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

Table 3 PHAcontent and PHAproduction rate achieved by activated sludge and pure cultures

Strain

Substrate

PHA

PHAcontent (%)

PHAproduction 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

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PHAproduction capability of activated sludge. Also, PHAproduction process is found to be sensitive to pH condition and acetate concentration. With our findings, it is confident that there is a big room for the improvement of PHAproduction system by using activated sludge.

  • 4. Conclusion

The authors have proposed a PHAproduction system in which excess sludge of the wastewater treatment process was utilized as PHAproduction bacterial cultures. Main focus of this research was to investigate the optimum operating conditions of activated sludge process for enhancing the PHAproduction 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 PHAproduction capability of activated sludge could be enhanced by manipulating various operational conditions in anaerobic–aerobic activated sludge pro- cess. The results obtained are summarized as follows:

  • 1. Sludge acclimatized with acetate-supplemented mu- nicipal wastewater could produce PHAup 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 PHAproduction capability if batch experiments were run at pH 8. However, when different pH values were applied in batch experi- ments, the PHAproduction behavior was distinc- tively varied. Under batch experiments run at pH 6 and 7, PHAproduction 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 PHAproduction.

  • 3. It was also found that sludge with a short SRT (3 days) could achieve PHAcontent about 10% more than sludge with a long SRT (10 days).

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