Bioresource Technology 95 (2004) 327330

Production of biodegradable plastics from activated sludge

generated from a food processing industrial wastewater
treatment plant
M. Suresh Kumar
a

a,*

, S.N. Mudliar a, K.M.K. Reddy b, T. Chakrabarti

Environmental Biotechnology Division, National Environmental Engineering Research Institute (NEERI),

Nehru Marg, Nagpur 440 020, India
b
Center for Biotechnology, Nagarjuna University, Guntur 522 101, India
Available online 2 April 2004

Abstract
Most of the excess sludge from a wastewater treatment plant (60%) is disposed by landll. As a resource utilization of excess
sludge, the production of biodegradable plastics using the sludge has been proposed. Storage polymers in bacterial cells can be
extracted and used as biodegradable plastics. However, widespread applications have been limited by high production cost. In the
present study, activated sludge bacteria in a conventional wastewater treatment system were induced, by controlling the carbon:
nitrogen ratio to accumulate storage polymers. Polymer yield increased to a maximum 33% of biomass (w/w) when the C/N ratio
was increased from 24 to 144, where as specic growth yield decreased with increasing C/N ratio. The conditions which are required
for the maximum polymer accumulation were optimized and are discussed.
2004 Elsevier Ltd. All rights reserved.
Keywords: Activated sludge; Poly-b-hydroxybutyrate; Storage polymer; Biodegradable plastics; PHB

1. Introduction
The problem of environmental pollution caused by
indiscriminate dumping of plastic waste has assumed
global proportions. These conventional plastics that are
synthetically derived from petroleum are not readily
biodegradable (Huang et al., 1991; Young, 1981) and
are considered as environmentally harmful wastes
(Hong Kong Environmental Protection Department,
1994). In the search of environmentally friendly materials to substitute for conventional plastics, dierent
biodegradable plastics have been developed either by
incorporating natural polymers into conventional plastics formulations, by chemical synthesis, or by microbial
fermentations (Chua et al., 1995b; Chang, 1994). However, physical limitations of these materials still exist
(Chua et al., 1995a,b).
Amongst the variety of biodegradable plastics a
family of more than 40 poly-hydroxy alkanoates (PHAs)

Corresponding author. Tel.: +91-712-226026.

E-mail address: drmsureshk@yahoo.com (M. Suresh Kumar).

0960-8524/$ - see front matter 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2004.02.019

and their co-polymeric derivatives has emerged as very

attractive materials due to their complete biodegradability (Kumagai, 1992). A number of bacteria accumulate these polymers or co-polymers as an intracellular
carbon reserve when unfavorable environmental and
nutritional conditions are encountered (Pfeer, 1992).
Poly-b-hydroxybutyrate (PHB) is a microbial polyester
produced by many bacteria and stored in cells in the
form of granules. It is a candidate for the synthesis of
environmentally benign, biodegradable plastics (Fang
et al., 1998). Much eort has been spent in optimizing
the poly-b-hydroxybutyrate (PHB) production using
pure substrates and pure cultures (Lee et al., 1994; Shirai
et al., 1994; Shimizu et al., 1992). But the product (PHB)
cost is still around ten times higher than that of conventional plastics (Chua et al., 1997).
The present study describes a process in which food
processing wastewater treatment plant activated sludge
is used for the production of PHB. This process could
signicantly reduce the cost of PHB production and at
the same time, reduce the quantity of excess sludge from
the wastewater treatment process that required further
treatment.

M. Suresh Kumar et al. / Bioresource Technology 95 (2004) 327330

2. Methods
2.1. Materials
All the chemicals used for the preparation of
synthetic media were from HiMedia (Hi-Media Laboratories Pvt. Limited, Mumbai, India) and Ranbaxy
(Ranbaxy, Mumbai, India). For chemical analyses,
chemicals used are of analytical grade reagents obtained
from Ranbaxy, Qualigens and Loba (India). Solvents
used for the extraction of PHA are of chromatographic
grade obtained from E. merck (India) Limited. PHB is
of Sigma Co. USA.
2.2. Biomass estimation
Biomass was estimated as mixed liquor suspended
solids (MLSS) and mixed liquor volatile suspended
solids (MLVSS), the procedures followed as in standard
methods (American Public Health Association, 1998
20th Edition).
2.3. Microscopic analysis
Morphological characteristics of the activated sludge
and poly-3-hydroxy butyrate granules were observed
under the phase contrast microscope (Polyvar Make) by
staining with Sudan Black B, according to the method of
Burdan (1946).
2.4. Chemical analysis
The total organic carbon was estimated in terms of
chemical oxygen demand (COD) as in standard methods. Activated sludge was characterized by estimating
chemical oxygen demand, total nitrogen and MLVSS.
The procedures followed were as in standard methods.
2.5. Biomass growth medium composition
Each liter of the medium contained 0.180 g of
Ammonium chloride, 0.052 KH2 PO4 , 0.067 K2 HPO4 ,
0.50 MgSO4 , and trace metals solution 1 ml. The trace
metals solution contains CoCl2 , 6H2 O, 0.008, CaCl2
0.010, MnCl2 2H2 O 0.0003, FeCl3 0.010, H3 BO3
0.004, NaMoO4 2H2 O 0.002, CuSO4 5H2 O 0.002,
ZnSO4 7H2 O 0.002 and NiCl2 6H2 O 0.002 g/l. One
gram of acetic acid per liter was used as sole source of
carbon.
2.6. Extraction of PHB from activated sludge
Extraction and estimation of poly-b-hydroxybutyric
acid was performed according to the procedure developed by John and Ralph (1961). The activated sludge
was separated from the culture broth by centrifugation

(Remi C24, 3000 rpm, 30 min, 10 C). The resulting

pellet was washed with acetone and ethanol to remove
the unwanted materials. The pellet was resuspended in
an equal volume of commercial sodium hypochlorite
(4% W/V available chlorine) and incubated for half an
hour at room temperature, the whole mixture was again
centrifuged and the supernatant was discarded. The
lysed cell pellet was again washed with acetone and
ethanol, nally the polymer granules were dissolved in
hot chloroform and passed through glass ber lter
(0.45 lm pore size) to separate the polymer from cell
debris. This chloroform ltrate was used for PHB estimation by adding sulfuric acid. Addition of sulfuric acid
converts the polymer into crotonic acid and the solution
is cooled and the absorbance at 235 nm read against a
sulfuric acid blank. The standard curve was prepared by
using pure PHB and the sample concentrations were
derived from this standard graph.

3. Results and discussion

Fresh activated sludge from a food processing
wastewater treatment plant was collected and enriched
in a synthetic media using acetic acid as carbon source.
After enrichment bacterial strains were isolated by
spreading the sludge on nutrient agar plates. On the
basis of colony characteristics ve dierent types of
bacterial strains were observed. These isolated strains
were screened for the production of PHB. At the end of
the stationary phase cultures were smeared on glass
slides, stained with sudan black B and examined under
phase contrast microscope for PHB granules. All the ve
strains were found to accumulate PHB granules.
The synthetic media was prepared and distributed in
to 250 ml capacity conical asks and dierent concentrations (500 mg to 3500 mg/l) of acetic acid was added
as carbon source. These asks were then supplemented
with activated sludge (430 mg/l) and increase in biomass
was studied by withdrawing samples at regular time
intervals (24 h) and estimated the biomass concentration
in terms of mixed liquor suspended solids (MLSS). The
results are presented in Fig. 1 maximum biomass (3150
3.5
500

MLSS in g/l

328

1000

2.5

1500

2500

2000
3000

1.5
1
0.5
0
0

25

50

75

100

125

150

Time (Hours)

Fig. 1. Enrichment of biomass from activated sludge with dierent

concentrations of acetic acid.

M. Suresh Kumar et al. / Bioresource Technology 95 (2004) 327330

329

Table 1
Eect of carbon and nitrogen concentrations on PHB production
C/N ratios

Initial activated sludge

concentration (g/l)

Carbon source
acetic acid (g/l)

As carbon (g/l)

Nitrogen source
ammonium chloride (g/l)

As nitrogen (g/l)

% PHB produced

24
96
120
144
168

3.15
3.15
3.15
3.15
3.15

2.825
11.304
14.13
16.95
19.78

1.130
4.521
5.652
6.782
7.912

0.180
0.180
0.180
0.180
0.180

0.0471
0.0471
0.0471
0.0471
0.0471

7
12.4
18.3
33
21.7

35
30

C\N 24
C\N 96

% of PHB

25

C\N 120

20

C\N 144
C\N 168

15
10
5
0
0

20

40

60

80

100

120

140

Fig. 2. Production of PHB using activated sludge with dierent ratios

of carbon and nitrogen.

35
30
25

% PHB

mg/l) obtained at 2000 mg/l of acetic acid within 96 h

incubation.
Activated sludge contains a range of dierent types of
microorganisms, from an ecological point of view, the
activated sludge process can be represented as a food
web in a controlled environment (Fredrickson and
Stephanopoulus, 1981). Besides bacteria, many other
(predators) organisms occur in biological treatment
plants, for example, ciliates, rotifers, nematodes and
oligochaetes (Ratsak et al., 1993). Aim of the above
experiments are to selectively enrich the bacterial biomass, so that the other organisms can be minimized. The
enriched bacterial biomass was then subjected to PHB
production.
Under normal conditions bacteria synthesize their
body materials like proteins and grow, but during
nutrient limiting conditions bacteria may shift its protein synthesis to PHB synthesis for survival. To exploit
this phenomenon experiments were carried out to study
the accumulation of poly-b-hydroxybutyric acid with
nitrogen limiting condition. The carbon, nitrogen ratios
were adjusted to 24, 96, 120, 144 and 168 respectively
and equal amount of previously enriched activated
sludge was introduced in the media containing dierent
C/N ratios (Table 1). Samples were withdrawn at regular
time intervals and analysed for PHB content and biomass concentration. The percentage concentration
of PHB produced at regular time intervals with dierent
C/N ratios are graphically presented in Fig. 2. As C/N
ratio was increased from 24, the PHB accumulation
percentage in the cell mass increased. The maximum

20
15
10
5
0
0

0.5

1.5

2.5

3.5

4.5

MLSS (g/l)

Fig. 3. Optimization of initial activated sludge (biomass) concentration for the production of PHB.

percentage PHB accumulation was observed at C/N

ratio 144 and with an incubation time of 96 h. At a C/N
ratio of 144 a maximum of 33% PHB (w/w) accumulation was obtained. All the above experiments were
conducted with an initial activated sludge (biomass)
concentration of 3150 mg/l. But to ndout whether any
increase in PHB production by varying the activated
sludge with optimal concentration of C/N ratio, experiments were carried out with dierent initial biomass
concentrations ranging from 0.5 to 3.5 g/l (Fig. 3). The
maximum PHB production was obtained with biomass
concentration of 3000 mg/l.

4. Conclusions
Activated sludge microorganisms in a food processing industry wastewater treatment plant were induced
after enrichment, to accumulate storage polymer by
controlling the C/N ratio. Specic polymer yield increased with increasing C/N ratio. An optimum C/N
ratio of 144 provided the highest overall polymer production yield. The intercellular polymer fraction could
reach a highest value of 792 mg polymer/g cell mass,
indicating that 33 wt.% of the activated sludge was
composed of the polymer. Production and recovery of
PHB from activated sludge could signicantly reduce
the cost of PHB and at the same time, reduces the
quantity of excess sludge produced in the wastewater
treatment system that required further treatment.

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M. Suresh Kumar et al. / Bioresource Technology 95 (2004) 327330

Acknowledgements
The authors are thankful to the Director, National
Environmental Engineering Research Institute (NEERI),
Nagpur, for his kind permission to publish this paper.

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