Bioresource Technology 95 (2004) 327–330

Production of biodegradable plastics from activated sludge

generated from a food processing industrial wastewater
treatment plant
a,*
M. Suresh Kumar , S.N. Mudliar a, K.M.K. Reddy b, T. Chakrabarti a

a
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 landfill. 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 specific growth yield decreased with increasing C/N ratio. The conditions which are required
for the maximum polymer accumulation were optimized and are discussed.
Ó 2004 Elsevier Ltd. All rights reserved.

Keywords: Activated sludge; Poly-b-hydroxybutyrate; Storage polymer; Biodegradable plastics; PHB

1. Introduction and their co-polymeric derivatives has emerged as very

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 mate-
rials to substitute for conventional plastics, different
biodegradable plastics have been developed either by
incorporating natural polymers into conventional plas-
tics formulations, by chemical synthesis, or by microbial
fermentations (Chua et al., 1995b; Chang, 1994). How-
ever, 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).
attractive materials due to their complete biodegrad-
ability (Kumagai, 1992). A number of bacteria accu-
mulate these polymers or co-polymers as an intracellular
carbon reserve when unfavorable environmental and
nutritional conditions are encountered (Pfeffer, 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 effort 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 con-
ventional 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
significantly 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.

0960-8524/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2004.02.019
328 M. Suresh Kumar et al. / Bioresource Technology 95 (2004) 327–330
2. Methods
2.1. Materials
All the chemicals used for the preparation of
synthetic media were from HiMedia (Hi-Media Labo-
ratories 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 meth-
ods. 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 Æ 2H2 O 0.0003, FeCl3 0.010, H3 BO3
0.004, NaMoO4 Æ 2H2 O 0.002, CuSO4 Æ 5H2 O 0.002,
ZnSO4 Æ 7H2 O 0.002 and NiCl2 Æ 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 devel-
oped 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, finally the polymer granules were dissolved in
hot chloroform and passed through glass fiber filter
(0.45 lm pore size) to separate the polymer from cell
debris. This chloroform filtrate was used for PHB esti-
mation 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 five different 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 five
strains were found to accumulate PHB granules.
The synthetic media was prepared and distributed in
to 250 ml capacity conical flasks and different concen-
trations (500 mg to 3500 mg/l) of acetic acid was added
as carbon source. These flasks 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
3
500
1000
2.5
1500
2000
MLSS in g/l
2
2500
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 different
concentrations of acetic acid.
 M. Suresh Kumar et al. / Bioresource Technology 95 (2004) 327–330 329

Table 1
Effect of carbon and nitrogen concentrations on PHB production
C/N ratios Initial activated sludge Carbon source As carbon (g/l) Nitrogen source As nitrogen (g/l) % PHB produced
concentration (g/l) acetic acid (g/l) ammonium chloride (g/l)
24 3.15 2.825 1.130 0.180 0.0471 7
96 3.15 11.304 4.521 0.180 0.0471 12.4
120 3.15 14.13 5.652 0.180 0.0471 18.3
144 3.15 16.95 6.782 0.180 0.0471 33
168 3.15 19.78 7.912 0.180 0.0471 21.7

mg/l) obtained at 2000 mg/l of acetic acid within 96 h 35

incubation. 30

Activated sludge contains a range of different types of 25

microorganisms, from an ecological point of view, the

% PHB
20
activated sludge process can be represented as a food
15
web in a controlled environment (Fredrickson and
Stephanopoulus, 1981). Besides bacteria, many other 10

(predators) organisms occur in biological treatment 5

plants, for example, ciliates, rotifers, nematodes and 0

oligochaetes (Ratsak et al., 1993). Aim of the above 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
MLSS (g/l)
experiments are to selectively enrich the bacterial bio-
mass, so that the other organisms can be minimized. The Fig. 3. Optimization of initial activated sludge (biomass) concentra-
enriched bacterial biomass was then subjected to PHB tion for the production of PHB.
production.
Under normal conditions bacteria synthesize their
body materials like proteins and grow, but during
nutrient limiting conditions bacteria may shift its pro- percentage PHB accumulation was observed at C/N
tein synthesis to PHB synthesis for survival. To exploit ratio 144 and with an incubation time of 96 h. At a C/N
this phenomenon experiments were carried out to study ratio of 144 a maximum of 33% PHB (w/w) accumula-
the accumulation of poly-b-hydroxybutyric acid with tion was obtained. All the above experiments were
nitrogen limiting condition. The carbon, nitrogen ratios conducted with an initial activated sludge (biomass)
were adjusted to 24, 96, 120, 144 and 168 respectively concentration of 3150 mg/l. But to findout whether any
and equal amount of previously enriched activated increase in PHB production by varying the activated
sludge was introduced in the media containing different sludge with optimal concentration of C/N ratio, exper-
C/N ratios (Table 1). Samples were withdrawn at regular iments were carried out with different initial biomass
time intervals and analysed for PHB content and bio- concentrations ranging from 0.5 to 3.5 g/l (Fig. 3). The
mass concentration. The percentage concentration maximum PHB production was obtained with biomass
of PHB produced at regular time intervals with different concentration of 3000 mg/l.
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 4. Conclusions

Activated sludge microorganisms in a food process-

ing industry wastewater treatment plant were induced
35 after enrichment, to accumulate storage polymer by
30
C\N 24
controlling the C/N ratio. Specific polymer yield in-
25 C\N 96 creased with increasing C/N ratio. An optimum C/N
C\N 120
% of PHB

20 C\N 144 ratio of 144 provided the highest overall polymer pro-
15
C\N 168
duction yield. The intercellular polymer fraction could
10 reach a highest value of 792 mg polymer/g cell mass,
5
indicating that 33 wt.% of the activated sludge was
0
composed of the polymer. Production and recovery of
0 20 40 60 80 100 120 140 PHB from activated sludge could significantly reduce
the cost of PHB and at the same time, reduces the
Fig. 2. Production of PHB using activated sludge with different ratios quantity of excess sludge produced in the wastewater
of carbon and nitrogen. treatment system that required further treatment.
330 M. Suresh Kumar et al. / Bioresource Technology 95 (2004) 327–330
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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