BL 695-698,675, Hu 20/6/97 1:15 pm Page 695

Biotechnology Letters, Vol 19, No 7, July 1997, pp. 695–698

11111
2
3
Synthesis of poly(3-hydroxybutyrate-co-
4
5
3-hydroxyvalerate) from activated sludge
6
7 W.F. Hu1, H. Chua2* and P.H.F. Yu3
8 1
Food Science Department, South China Agricultural University, Guangzhou, China; 2Department of Civil and
9 Structural Engineering, 3Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic
10111 University, Hung Hom, Hong Kong
1
2 Poly-3-hydroxybutyrate (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was produced using a co-
3 culture of activated sludge. When butyric acid was used as sole carbon source, PHB was produced. When valeric
4 acid was added to the medium, PHBV was produced. The 3-hydroxyvalerate (3HV) mole fraction in the PHBV reached
5 a maximum of 54% when valeric acid was used as sole carbon source. When the 3HV units in the co-polymer
6 increased from 0.0 to 54.0 mol%, the melting temperature (Tm) decreased from 178 to 99°C. The composition, and
7 hence the mechanical properties, of the co-polymer produced by activated sludge can be controlled by adjusting the
8 medium composition.
9
20111
1
2
3 Introduction In an effort to produce co-polymers with improved
4 A number of bacteria, including Alcaligenes spp., mechanical properties, Doi et al. (1988) and Ishihara et
5 Pseudomonas spp., recombinant Escherichia coli and al. (1996) demonstrated the accumulation of a variety
6 a number of filamentous genera, accumulate poly- of PHA co-polymers in a pure culture of Alcaligenes
7 hydroxyalkanoates (PHA) and their co-polymers as an eutrophus with different compositions of butyric and
8 intracellular carbon reserve when unfavourable environ- valeric acids in the culture medium. The co-polymeric
9 mental conditions are encountered. These extracted and synthesis pathway had also been traced. However, infor-
30111 processed polymers have a number of properties that are mation on the production of co-polymers from activated
1 comparable to commonly used plastics, namely ther- sludge are not available.
2 moplastic processability and 100% water resistance.
3 However, widespread application of PHA is hampered This paper describes a novel technique that induced the
4 by high costs of production, limited knowledge activated sludge bacteria in a conventional food-
5 on controlled co-polymerization and their physical processing wastewater treatment process to produce
6 properties. PHA co-polymers. The thermal property of the co-
7 polymers was also studied.
8 Much effort has been spent in optimizing the PHA
9 production process and reducing costs. Lee et al. (1994)
40111 investigated various recombinant E. coli using different Methods and materials
1 complex culture media. E. coli strain XL1-blue in LB Operation of sequencing batch reactor (SBR)
2 plus 20 g glucose/L could accumulate up to 0.369 g and biomass harvesting
3 PHA/g glucose, or equivalent to 7 g PHA/L. Shirai et A laboratory scale sequencing batch reactor (SBR)
4 al. (1994) used a photosynthetic bacterium Rhodobacter with 12-L effective volume and 1.5 day hydraulic reten-
5 spheroides in a fed batch culture with glucose as the tion time (HRT) was used to culture activated sludge.
6 sole carbon to achieve a PHA production of 6 g/L. The reactor was fed with a synthetic food-processing
7 Shimizu et al. (1992) used a cell growth phase followed wastewater containing reconstituted milk at 1.92 g/L.
8 by a separately optimized nutrient-deficient PHA- equivalent to 1200 mg/L in terms of total organic
9 accumulation phase to improve the specific production carbon (TOC), and aeration time was 2 h. Through
50111 yield to as high as 0.70 g PHA/g cell mass. Chua et al. each batch culture, 7–8 g activated sludge were
1 (1997a, 1997b) had substantially reduced the produc- harvested, washed with distilled water to remove any
2 tion costs by inducing activated sludge bacteria to residual nitroeneous matters and inoculated into a jar
3111 produce PHA. fermenter.

© 1997 Chapman & Hall Biotechnology Letters · Vol 19 · No 7 · 1997 695

BL 695-698,675, Hu 20/6/97 1:15 pm Page 696
W.F. Hu et al.
11111 Fermentation conditions and culture media
2 An automatic jar fermenter of 3-L working volume
3 (Bioengineering Model ALF, Ruti/Switzerland) was
4 operated at 300 rpm and 30°C for 48 h. The pH was
5 automatically maintained at 7.0 by the addition of a
6 sterilized 2 M NaOH solution.
7 and the steady state performance reached a 98.0% TOC
8 The nitrogen-free medium contained butyric acid (C4)
9 and valeric acid (C5) as carbon sources, and supple-
10111 mentary trace minerals and a growth factor with formu-
1 lations as described by Chua et al. (1997a). In separate
2 batch cultures, the C4 to C5 weight ratios in the medium
3 were respectively adjusted to 100:0, 80:20, 60:40, 40:60
4 20:80 and 0:100 (g/g), or equivalent to mole ratios of
5 34:0, 27:5, 20:11, 13:17, 6:23 and 0:29, respectively.
6 The initial total concentration of fatty acids was
7 adjusted to 1 g/L to avoid possible growth inhibition
8 as reported by Kim et al. (1992). The fermenter was
9 operated in a fed batch mode by adding a total of 3 g
20111 of fatty acids into the fermenter once every 16 h.
1 (3HV) fraction and thermal property of the polymer
2 Sampling and analytical techniques
3 The culture broth was periodically sampled and analyzed
4 for dry cell mass. The residual carbon concentration
5 was measured as TOC using an ASTRO 2001 SYSTEM
6 2 automatic TOC analyzer. The TOC analytical tech-
7 nique was in accordance with the Standard Methods
8 (APHA, 1995).
9 Total TOC removal efficiency decreased from 98.0 to
30111 The activated sludge from the SBR or fermenter was
1 centrifuged at 3000 rpm (1000 g) for 15 minutes
2 and the intracellularly accumulated co-polymers were
3 extracted by chloroform in accordance with the proce-
4 dure described by Lowell et al. (1972) and modified by
5 Chua et al. (1997a). The weight of the extracted co-
6 polymers was measured to determine the productivity.
7 acids as the carbon sources. Ishihara et al. (1996)
8 Ten ml culture broth was sampled and centrifuged at
9 3000 rpm (1000 g) for 15 minutes. The settled cells
40111 were washed with buffer solution and centrifuged again.
1 The centrifuged sludge was re-suspended in a mixture
2 containing 2 ml of methanol with 3% of concentrated
3 H2SO4 and 2 ml of chloroform with 2 mg/ml of benzoic
4 acid as the internal standard. The sample was placed in
5 a closed test tube and heated at 100°C for 3 hr to
6 convert the constituents to their methyl esters. Addition
7 of 1 ml distilled water to the reaction mixture induced
8 phase separation. The resulting organic phase was
9 then used for gas chromatographic analysis of co-
50111 polymeric composition with a Varian Model 3700 gas
1 chromatograph equipped with a Carbowax 20 M
2 column (1 meter) and a Shimadzu C-R5A Chromatopac
3111 flame-ionization detector.
696 Biotechnology Letters · Vol 19 · No 7 · 1997
The melting temperature of the extracted co-polymers
were determined in accordance with that described by
Doi et al. (1990) and Bluhm et al. (1988).
Results and discussion
The SBR system was operated for a period of 60 days
removal. There was no polymer (poly-3-hydroxybu-
tyrate, PHB or poly-3-hydroxybutyrate-co-3-hydroxy-
valerate, PHBV) accumulating in the activated sludge
during this stage when the nutrient-rich synthetic
wastewater was being fed. This polymer-free activated
sludge was then transferred from the SBR into the jar
fermenter with nitrogen-free medium to promote
polymer accumulation.
Polymer production under various fatty
acid ratios
Table 1 lists the biomass, TOC removal efficiency,
polymer content, product yields, final 3-hydroxyvalerate
after 48-h fermentation under six different carbon
sources ratios. The initial cell mass concentration in the
fermenter was about 2.5 g/L, final cell mass was main-
tained within the range from 2.30 to 3.01 g/L during
the 48 h culture process. Generally, there was no change
in biomass observed.
65.0% when valeric acid in the medium was increased,
while the PHA content in dried biomass decreased from
40% to 18%. When valeric acid was used as sole carbon
source, the TOC removal efficiency decreased to a
minimum value of 65.0%. These results were in agree-
ment with that reported by Ishihara et al. (1996) for a
pure culture of A. eutrophus with butyric and valeric
observed that when butyric acid concentration in their
medium was maintained at a constant value while
valeric acid concentration was increased, cell growth and
fatty acids consumption were substantially affected.
An increase in valeric acid content in the medium from
0 to 100 wt % resulted in a decline in polymer produc-
tion yield, YP/S, from 0.69 to 0.32 g-polymer/g-TOC
consumed. The polymer content in the cells also
decreased from 40 to 18 wt %, which indicated that
the production of polymer was inhibited by valeric acid.
These observations were in contrast to that reported by
Ishihara et al. (1996) and Yamane (1993) that the yield
and polymer content remained unchanged despite
the variation in valeric acid concentration in the
medium. The range of YP/S in the activated sludge,
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Synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from activated sludge

11111 Table 1 Production of PHB and PHBV by activated sludge with different carbon sources ratios of butyric and
2 valeric acids
3
C4 to C5 C4 and C5 (g/L)
4 (g/g) (mol/mol) Biomass w. TOC Polymer YP/S a 3HV fraction Tm
5 C4 C5 (g/L) removal content (g/g) (mol%) (°C)
6 (%) (wt%)
7 100:0 34:0 3.0 0.0 3.01 98.0 37.0 0.69 0 178
8 80:20 27:5 2.4 0.6 2.45 96.0 40.0 0.61 12 144
9 60:40 20:11 1.8 1.2 2.52 95.0 35.0 0.55 30 133
10111 40:60 13:17 1.2 1.8 2.30 90.2 24.0 0.35 35 127
1 20:80 6:23 0.6 2.4 2.50 83.4 18.0 0.32 51 109
0:100 0:29 0.0 3.0 2.30 65.0 22.0 0.44 54 99
2
a
3 YP/S = Polymer production yield (g polymer/g carbon), which was calculated as the polymer accumulated divided by the TOC consumed.
4
5 between 0.69 and 0.32 (g/g), was lower than the theo-
6 retical yield of PHBV from fatty acids of 0.98 g-
7 PHBV/g-fatty acids by Rhodospiril rubrum (Yamane,
8 1993). However, it was closer to the theoretical yield
9 of 0.65 g-PHBV/g-fatty acids by A. eutrophus (Yamane,
20111 1993), which is a common genus in activated sludge.
1 The wide ranging yield in the activated sludge could
2 be attributed to the complex microbial species in the
3 ecosystem of the sludge, giving rise to widely varied
4 metabolic pathways from fatty acid to polymer in
5 different microbial species. On the other hand, Dave
6 et al. (1996) described that when activated sludge was
7 incubated under nitrogen- and phosphorus-limiting
8 conditions, selective overgrowth of Bacillus spp. from Figure 1 Relationship between 3HV fraction in PHBA and
5 to 80% (cell count) was observed. It is therefore C5 concentration in the medium.
9
30111 believed that the variation of valeric acid concentration
1 in medium caused the changes in the balance of micro-
2 bial species, and hence the prevailing metabolic path-
3 ways in activated sludge during the 48-h incubation.
4 This, in turn, resulted in the wide ranging YP/S values.
5
6 Effects of fatty acid ratios on co-polymer
7 composition
8 When butyric acid was used as sole carbon source, there
9 was only PHB homo-polymer produced instead of
40111 PHBV co-polymer (Table 1). On the other hand, the
1 highest 3HV mole fraction in the co-polymer accumu-
2 lated was when valeric acid was used as sole carbon
3 source. The mole fraction of 3HV in the accumulated
4 co-polymer increased proportionately with the valeric Figure 2 Relationship between melting temperature and
5 acid concentration in medium. Figure 1 illustrates a 3HV fraction in PHB(V).
6 linear relationship between 3HV mole fraction in the
7 co-polymer and the valeric acid concentration. These
8 results from activated sludge were in agreement with Thermal property of co-polymer from activated
9 that observed in pure cultures of A. eutrophus (Doi et al., sludge
50111 1988; Ishihara et al., 1996). These results also indicated The melting temperature, Tm, of the co-polymer accu-
1 that the 3HV mole fraction of the PHBV co-polymer mulated in the activated sludge with different medium
2 accumulated in activated sludge could be controlled by composition ranged from 99 to 178°C (Table 1). The
3111 adjusting the valeric acid concentration in the medium. co-polymers with maximum and minimum values of Tm

Biotechnology Letters · Vol 19 · No 7 · 1997 697

BL 695-698,675, Hu 20/6/97 1:15 pm Page 698
W.F. Hu et al.
11111 were obtained when butyric acid and valeric acid were
2 respectively used as the sole carbon sources in the
3 medium. Increase of 3HV monomeric units in the
4 PHBV co-polymer formulation resulted in a close to
5 proportionate decrease of polymer melting temperature
6 (Figure 2). The melting temperature of the PHA
7 produced by activated sludge decreased with an increase
8 in the 3HV fraction, indicating that 3HV unit act as
9 defects in the PHBV crystal lattice. Otherwise, the
10111 infrared spectrums of the PHA produced by activated
1 sludge were identical with that of the standard samples
2 including PHB and PHBV. Such defects are also known
3 to have adverse effects of such mechanical properties
4 as tensile, compressive, flexural and shear stresses of
5 the materials. These results agreed with the published
6 thermal properties of polymers and co-polymers of
7 PHA. Intracellular polymers of PHA are highly
8 crystalline thermoplastics with a melting temperature
9 (Tm) around 180°C for PHB homo-polymer and a range
20111 from 96 to 160°C for PHBV co-polymers depending
1 on the fraction of 3HV units (Doi et al., 1990).
2 Therefore, the 3HV mole fraction of the PHBV co-
3 polymer accumulated in activated sludge, and hence its
4 thermal and mechanical properties, could be controlled
5 by adjusting the valeric acid concentration in the
6 medium.
7 161–164.
8 The novel technique to synthesis of PHA from activated
9 sludge is beneficial in two ways. On the one hand, excess
30111 sludge generated from activated sludge processes needs
1 further treatment such as anaerobic digestion and
2 disposal by landfilling. This technique can reduce the
3 quantity of excess sludge by about 37% as described by
4 Chua et al. (1997a), therefore reducing the costs of
5 sludge treatment. On the other hand, large quantities
6 of biomass harvested from activated sludge wastewater
7 treatment processes were induced to produce PHA
8
9
40111
1 Revisions requested 24 April 1997;
2 Revisions received 20 May 1997;
3 Accepted 22 May 1997
4
5
6
7
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9
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2
3111
698 Biotechnology Letters · Vol 19 · No 7 · 1997
instead of producing by pure culture fermentation.
These significantly reduced the costs of PHA produc-
tion.
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