Utilization of molasses spentwash for production of bioplastics by waste
Anshuman A. Khardenavisa,*, Atul N. Vaidyab, M. Suresh Kumarc, Tapan Chakrabartid
aEnvironmental Genomics Unit, National Environmental Engineering Research Institute (NEERI), Nehru Marg, Nagpur 440 020, India
bSolid Waste Management Unit, National Environmental Engineering Research Institute (NEERI), Nehru Marg, Nagpur 440 020, India
cEnvironmental Biotechnology Division, National Environmental Engineering Research Institute (NEERI), Nehru Marg, Nagpur 440 020, India
dNational Environmental Engineering Research Institute (NEERI), Nehru Marg, Nagpur 440 020, India
a r t i c l ei n f o
Accepted 19 April 2009
Available online 4 June 2009
a b s t r a c t
Present study describes the treatment of molasses spentwash and its use as a potential low cost substrate
for production of biopolymer polyhydroxybutyrate (PHB) by waste activated sludge. Fluorescence
microscopy revealed the presence of PHB granules in sludge biomass which was further confirmed by
fourier transform-infra-red spectroscopy (FT-IR) and13C nuclear magnetic resonance (NMR). The pro-
cessing of molasses spentwash was carried out for attaining different ratios of carbon and nitrogen
(C:N). Highest chemical oxygen demand (COD) removal and PHB accumulation of 60% and 31% respec-
tively was achieved with raw molasses spentwash containing inorganic nitrogen (C:N ratio = 28) fol-
lowed by COD removal of 52% and PHB accumulation of 28% for filtered molasses containing inorganic
nitrogen (C:N ratio = 29). PHB production yield (Yp/s) was highest (0.184 g g?1COD consumed) for depro-
teinized spentwash supplemented with nitrogen. In contrast, the substrate consumption and product for-
mation were higher in case of raw spentwash. Though COD removal was lowest from deproteinized
spentwash, evaluation of kinetic parameters suggested higher rates of conversion of available carbon
to biomass and PHB. Thus the process provided dual benefit of conversion of two wastes viz. waste acti-
vated sludge and molasses spentwash into value-added product-PHB.
? 2009 Elsevier Ltd. All rights reserved.
Molasses based distillery spentwash remaining after the fer-
mentation and distillation of alcohol from sugarcane molasses
has a very high chemical oxygen demand (COD > 50,000 mg L?1),
and in spite of stringent standards, untreated or partially treated
effluent often ends up in water bodies and also on land surround-
ing the distillery. The pollution potential of this waste is consider-
(Protection) Act of India of 1986, which poses serious threat to
water quality in surrounding regions by lowering pH of the stream,
increasing the organic load, depleting the oxygen content, destroy-
ing aquatic life and creating foul odour (Joshi, 1999). The treatment
of such wastewater has assumed importance due to the restricted
amounts of water suitable for direct use, the high price of the puri-
fication installations and the necessity of utilizing the waste prod-
ucts (Converti et al., 1990).
According to a report published by Central Pollution Control
Board (CPCB), Corporate Responsibility for Environmental Protec-
tion (CREP) for distilleries, of the 231 distilleries in India, 82 distill-
eries were found to have full fledged facilities to achieve zero
specified in theEnvironment
discharge while 14 distilleries had treatment facilities for utilizing
75% and 15 distilleries had treatment facilities to utilize 50% of
their generated spentwash.1Though low treatability of molasses
spentwash has been reported by many researchers, few studies
have demonstrated an improvement in COD removal from molas-
ses spentwash when it was subjected to pre-treatment (Jimenez
et al., 1997; Khardenavis et al., 2008; Kitts et al., 1993; Sirianuntap-
iboon and Prasertsong, 2007).
In spite of the difficulty in treating this waste, it can serve as a
low cost carbon source for commercial synthesis of biodegradable
polymers such as polyhydroxyalkanoates (PHAs) due to the pres-
ence of high concentration of sugars in the waste. PHAs are ther-
moplastic materials synthesized by variety of bacteria (30–80%
of cell dry weight) as intracellular energy and carbon storage
materials under stress conditions arising as a result of limitations
of certain nutrients such as nitrogen (Alcaligenes latus, Pseudomo-
nas oleovorans, Ralstonia eutropha), carbon (Spirillum sp., Hypho-
microbium sp.), iron, magnesium (Pseudomonas sp. K), oxygen
(Rhodobacter rubrum, Caulobacter crecentus) (Kim and Lenz,
2000). In addition to their similar physico-chemical properties to
synthetic petrochemical derived plastics, these bioplastics are
ORS571) and phosphate
0956-053X/$ - see front matter ? 2009 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +91 712 2249885; fax: +91 712 2249883.
E-mail address: email@example.com (A.A. Khardenavis).
Waste Management 29 (2009) 2558–2565
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/wasman
superior due to their hydrophobicity (moisture resistance) and
biodegradability (de Koning, 1995). However, the high cost of bio-
degradable plastic material limits its use on a commercial scale
owing to the expensive carbon sources used as substrates for their
synthesis (Choi et al., 1998; Serafim et al., 2004).
Several approaches have been adopted for cost reduction of
PHAs such as use of recombinant high producing E. coli showing
upto 80% PHA accumulation in the form of polyhydroxybutyrate
(PHB) which has been the most extensively studied PHA (Kim,
2000; Park et al., 2002). Nonato et al. (2001) reviewed the effect
of integrated approach involving production of sugar, ethanol
and bioplastics on cost reduction of PHB and proposed that it could
lead to economical production of PHB by use of existing facilities,
materials and surplus energy from sugarcane industry. The eco-
nomics for PHA production could also benefit from use of low cost
agricultural feedstocks such as vegetable oils and animal fats, dairy
whey, molasses and meat-and-bone meal (Solaiman et al., 2006a).
Reddy et al. (2003) emphasized the role of structural studies and
metabolic engineering for understanding the mechanism of enzy-
matic action in PHB synthesis thereby aiding in the improvement
and selection of better candidates for increased biopolymer pro-
duction. Use of pure cultures such as A. vinelandii UWD, A. latus
DSM1124 and Pseudomonas corrugata with inexpensive agricul-
tural wastes such as molasses, malt and soy waste and soy molas-
ses respectively can help in the cost reduction of final product
(Page and Cornish, 1993; Solaiman et al., 2006b; Yu et al., 1999).
Activated sludge is generated in huge quantities from waste
treatment plants in countries such as US and UK (nearly 6900 mil-
lion dry tonnes each) with 41% added to agricultural land, 17%
placed in landfills, 22% incinerated, and the remainder (20%) han-
dled in ‘‘other” ways (US EPA, 1999). Such excess activated sludge
from waste treatment plants has been demonstrated to serve as a
source of PHB accumulating biomass with synthetic wastewater
containing acetic acid as carbon substrate thereby contributing to
the cost reduction process (Khardenavis et al., 2005; Suresh Kumar
et al., 2004). However, the use of different agro-industrial waste-
waters as low cost substrates for PHB accumulation in activated
sludge would provide dual advantage by saving cost on biomass
generation and volume reduction of waste activated sludge which
after PHB extraction, is to be disposed (Khardenavis et al., 2007).
The present study aimed at inducing PHB accumulation in acti-
vated sludge biomass obtained from combined food and milk pro-
cessing industry wastewater treatment plant with molasses based
distillery spentwash. This would provide a treatment for distillery
waste by decreasing the COD of the effluent and at the same time
utilize the waste activated sludge for production of value-added
product in the form of PHB.
2. Materials and methods
Analytical grade chemicals and reagents procured from Hi-
Media Laboratories Pvt. Ltd., (India) and Ranbaxy Fine Chemicals
(India) were used for preparation of media and chemical analyses.
Chromatography grade solvents, obtained from Merck Ltd., (India),
were used for extraction and analysis of intracellular PHB granules.
Standard PHB was purchased from Aldrich Chemical Co. (USA).
2.1. Enrichment of PHB producing microorganisms in activated sludge
The sludge used in the present study was from activated sludge
unit treating combined waste stream from a food processing indus-
try (making potato wafers, chips, sweets, namkeens (salted snacks)
and milk packaging industry). This waste was initially treated
anaerobically in the treatment plant which resulted in degradation
of insoluble solids in the incoming stream of food and milk pro-
cessing waste (potato peels, milk fats and proteins). This effluent
with very low COD and solids content then entered the activated
sludge unit for further aerobic treatment thus allowing the use of
the sludge without any pre-treatment for removal of solids prior
to its use for PHB accumulation. This activated sludge biomass
was aerated in presence of synthetic wastewater/basal mineral
salts medium containing acetic acid for selectively enriching PHB
producing microorganisms. The composition of the synthetic
wastewater was same as described previously (Khardenavis
et al., 2007). This enriched activated sludge (0.23 g dry weight
per 100 mL) was added as a source of biomass to the wastewater
for biopolymer synthesis.
2.2. Processing of molasses based distillery spentwash
Molasses based spentwash was obtained from distillery which
produced ethanol by fermentation of sugarcane molasses followed
by distillation of the alcohol leaving behind a dark coloured residue
– molasses spentwash. This spentwash was characterized for its
physico-chemical parameters and subjected to pre-treatment be-
fore its use as substrate for PHB accumulation. The pre-treatment
procedure is given below and is similar to that described earlier
(Khardenavis et al., 2007; Yellore and Desai, 1998).
? Raw wastewater (RW): wastewater was used in the form in
which it was obtained from the industry.
? Filtered wastewater (FW): wastewater in which the suspended
solids were removed by filtering through 0.45 l glass fibre
? Deproteinized wastewater (DW): wastewater, where pH of was
first adjusted to 7.0 with 1 N NaOH/1 N H2SO4and subsequently
boiled for 30 min to precipitate the proteins which were
removed by centrifugation at 1500g for 20 min in a cooling cen-
trifuge (Remi C-24, Remi Instruments Ltd., India). The superna-
tant was then filtered using 0.45 l glass fibre microfilter
Further, the processed wastewaters were supplemented with
inorganic nitrogen source, di-ammonium hydrogen phosphate
(DAHP) in addition to the basal mineral salts medium (without
acetate) thereby creating nutrient deficient and excess conditions.
The following pre-treatment was performed to achieve varying ra-
tios of chemical oxygen demand and nitrogen (COD/N). The differ-
ent forms of molasses spentwash thus created were labeled RW
(raw);RW + N (raw + DAHP);
tered + DAHP);DW(deproteinized);
nized + DAHP). Batch experiments were performed in 250 mL
capacity conical flasks containing 100 mL of each modified form
of the spentwash. Initial pH was set at 7.0 and the flasks were incu-
bated in rotary incubator–shaker (Orbitek, India) at 150 rotations
per minute (rpm) and 30 ± 2 ?C. Raw spentwash (RW and
RW + N) without any pre-treatment served as control in all the
DW + N
FW + N
2.3. Analytical procedures
Samples were withdrawn at regular intervals (48 h and 96 h)
and analyzed for changes in concentration of biomass and PHB in
addition to analysis of residual carbon [chemical oxygen demand
(COD), total and reducing sugars)], and nitrogen [total kjeldahl
nitrogen (TKN)]. Increase in biomass concentration in culture broth
was monitored by measuring the absorbance at 600 nm using a
UV–visible spectrophotometer (Spectronic, Genesys-2, USA).
COD was determined as a measure of carbon content in spent-
wash and presence of volatile fatty acid (VFA) was converted to
COD equivalent of acetate (i.e. 1.066). Total and reducing sugar
A.A. Khardenavis et al./Waste Management 29 (2009) 2558–2565
content in the wastewaters were estimated by anthrone (Morris,
1948) and dinitrosalicylic acid (Miller, 1959) reagents, respec-
tively. COD:N ratios in the wastewater were calculated from COD
and TKN of the wastewaters and expressed as gram per gram on
a dry weight basis. All physico-chemical analyses for characteriza-
tion of wastewater were performed as per standard methods
(APHA, AWWA, WEF, 1998).
Extraction of biopolymer was done as described previously
(Khardenavis et al., 2007). Sludge biomass was centrifuged at
1500g (10 min, 10 ?C) and washed with 10 mL acetone to remove
moisture and sludge impurities followed by lysis with 40 mL NaCl
(5%). The released PHB was extracted into 10 mL hot chloroform
(60 ?C) and added to 10 mL concentrated H2SO4whereby the poly-
mer reacted with H2SO4leading to formation of crotonic acid with
an absorbance maxima at 235 nm. The concentration of PHB in
experimental samples was determined at 235 nm from calibration
curve prepared using standard PHB (Law and Slepecky, 1961) and
reported in percent on a dry weight basis. Initial PHB content was
determined directly from enriched sludge, which was added ini-
tially to wastewater as a source of PHB accumulating biomass.
2.4. Characterization of extracted polymer
2.4.1. Fluorescence microscopy
The presence of the biopolymer in enriched PHB producing
microorganisms was confirmed by fluorescence staining as de-
scribed by Ostle and Holt (1982). Heat fixed smear of sludge bio-
mass was stained with 1% aqueous Nile Blue A followed by
treatment with 8% aqueous acetic acid and observing the stained
smear at an excitation wavelength of 460 nm. PHB producing
organisms were also stained by Sudan Black B as per the method
of Burdon (1946) for visualizing the PHB granules in the bacterial
cells in a phase contrast microscope.
2.4.2. Fourier transform-infrared spectroscopy (FT-IR)
Characterization of PHA recovered from sludge biomass by FT-
IR was done as per the method described by Hong et al. (1999).
The polymer sample was dissolved in chloroform and layered on
a KRS-5 window. After evaporation of the chloroform, the PHA
polymer film was subjected to FT-IR analysis using a Vector-22
FT-IR spectrometer (Bruker, France) under the following scanning
conditions: absorbance spectra at wave-number values between
4000 and 400 cm?1, KRS-5 as the window material, 64 scans, spec-
tral resolution of 4 cm?1.
22.214.171.124C Nuclear magnetic resonance (NMR) spectroscopy
Forty milligram of the PHA extracted from raw sludge from
treatment plant was dissolved in 1 mL deuterated chloroform
(CDCl3) followed by13C NMR analysis on a Bruker AM 400 spec-
trometer (Wissembourg, France).13C NMR spectra were obtained
at 27 ?C at 100.62 MHz with 16514 scans, line broadening of
3 Hz and relaxation delay time of 2 s using a spectral width of
24038.46 Hz (Huijberts et al., 1994; Tavernier et al., 1998; Yan
et al., 2000).
2.5. Evaluation of kinetic parameters
Productivity was defined as the concentration of PHB (P) pro-
duced per liter of medium with respect to time and is given as
g L?1h?1. Sludge polymer content was expressed in terms of ratio
of the PHB (P) to biomass (X) multiplied by 100,
PHB content ¼P ? 100
PHB (P) and biomass (X) yields with respect to substrate/COD
(S) consumed were given by,
Specific PHB storage rate (qp) and specific acetate uptake rate
(?qs) per unit time (t) were given as,
ðg PHB produced g?1COD-VFA consumedÞ
ðg biomass produced g?1COD-VFA consumedÞ
ðg PHB produced g?1biomass h?1Þ
ðg COD-VFA consumed g?1biomass h?1Þ
? qs ¼
3. Results and discussion
Mixed culture of bacteria in the form of waste activated sludge
from a combined food and milk processing wastewater treatment
plant was used as seed culture for PHB accumulation studies using
molasses based distillery spentwash as substrate. The flocs in acti-
vated sludge represent a food web in a controlled environment
(Fredrickson and Stephanopoulus, 1981) and besides bacteria
(95%) are inhabited by many other (predators) organisms such
as, ciliates, rotifers, nematodes and oligochaetes (remaining
5%)(Ratsak et al., 1993). Food (organic loading) regulates microor-
ganism numbers, diversity, and species when other factors are not
limiting. This parameter, which is expressed as the ratio of food
(chemical oxygen demand- COD:biochemical oxygen demand-
BOD) to microorganisms (volatile suspended solids- VSS) (F:M)
regulates the relative abundance and occurrence of organisms at
different loadings and can reveal why some organisms are present
in large numbers while others are absent. For a typical activated
sludge unit operating efficiently, the F:M values range between
0.25 and 0.5 with higher values leading to decrease in population
of predators and filamentous organisms responsible for foaming
and selective enrichment of bacterial population (Chua et al.,
2000; Mishoe, 1999).
In the present study, selective enrichment of the bacterial bio-
mass was carried out by aerating the activated sludge in presence
of synthetic wastewater containing acetate at a high F:M ratio thus
resulting in reduction in population of other organisms. At the
same time the enrichment medium had a high C:N ratio of 19:1
which promoted the enrichment of bacteria capable of accumulat-
ing PHB in comparison to the non-PHB accumulating microorgan-
isms. This enriched bacterial biomass was then subjected to PHB
production under the experimental conditions.
3.1. Physico-chemical characterization of molasses spentwash
Table 1 shows the physico-chemical characteristics of molasses
spentwash, which had a high COD (raw – 132,000 mg L?1; soluble/
filtered – 124,000 mg L?1) and total sugar concentration of
15,750 mg L?1. The spentwash was diluted four times before use
as substrate thus resulting in an effective COD of 33,000 mg L?1
for raw wastewater (RW) and 31,000 mg L?1for filtered wastewa-
ter (FW). Further processing of the waste by deproteinization (DW)
resulted in a COD of 28,000 mg L?1. The total sugar content of the
three diluted and processed wastewaters (raw, filtered and depro-
teinized) varied between 2750 mg L?1and 3150 mg L?1. Nitrogen
concentration in the molasses spentwash was recorded as TKN
which varied between 1176 and 1330 mg L?1for DW, FW and
RW, respectively. These wastewaters were used for PHB accumula-
tion studies in absence and presence of DAHP.
3.2. Characterization of polymer extracted from activated sludge
Fig. 1a shows the phase contrast photomicrograph of PHB gran-
ules in sludge biomass stained with Sudan Black B which appear as
A.A. Khardenavis et al./Waste Management 29 (2009) 2558–2565
dark spots inside pink coloured cells. Staining of heat fixed smear
with Nile Blue A showed the PHB granules which fluoresced bright
orange and appeared as intense bright images within the bacterial
cells present in sludge biomass (Fig. 1b). The presence of higher
number of orange fluorescent spots (Fig. 1b) than the non-fluores-
cent dark spots (Fig. 1a) indicated the higher affinity of Nile Blue A
for PHB than that of Sudan Black B and its usefulness for staining
PHB in bacterial cells.
The PHA extracted from activated sludge from the treatment
plant had the C–H and carbonyl stretching bands similar to stan-
dard PHB as revealed by FT-IR analysis (Fig. 2). The presence of
absorption bands at 1724 cm?1and 1280 cm?1in extracted PHA
sample were characteristic of C@O and C–O stretching groups
and were identical to those reported for PHB in literature (Hong
et al., 1999; Misra et al., 2000; Ramachander et al., 2002) thereby
confirming the presence of PHB in sludge biomass.
From Fig. 3,13C NMR spectrum shows the presence of four dif-
ferent characteristic resonance peaks which were attributed to
presenceof monomers 19.75 ppm
67.61 ppm (B2), and 169.3 ppm (B1). These chemical shifts were
assigned to the presence of (CH3), (CH2), (CH) and (C@O) groups
respectively and were characteristic of the 3HB monomer as has
been reported earlier (Doi et al., 1986, 1989). From the contribu-
tion of various groups to the NMR spectra, it was concluded that
(B4), 40.79 ppm(B3),
Fig. 1. PHB granules in sludge biomass stained with (a) Sudan Black B (b) Nile Blue A.
Physico-chemical characterization of molasses spentwash.
Parameter Molasses spentwash
Total acidity (mg L?1)
Total alkalinity (mg L?1)
Mixed liquor suspended solids- MLSS (mg L?1)
Total dissolved solids- TDS (mg L?1)
Chemical oxygen demand- COD soluble (mg L?1)
Raw (mg L?1)
Volatile fatty acids- VFA (mg L?1)
Total sugar (mg L?1)
Reducing sugar (mg L)
Total kjeldahl nitrogen mg L?1
Nitrate- N ðNO?
Phosphate- P ðPO3?
Chloride (Cl?) (mg L?1)
3Þ (mg L?1)
4Þ (mg L?1)
4Þ (mg L?1)
ND - not detected.
Fig. 2. FT-IR spectra (a) Standard PHB (b) PHB extracted from sludge biomass. The
absorption bands at 1280 and 1724 cm?1correspond to C@O and C–O of PHB,
13C NMR spectra of the PHA recovered from sludge biomass.
A.A. Khardenavis et al./Waste Management 29 (2009) 2558–2565
the waste activated sludge from food processing industry could di-
rectly serve as an inexpensive source of biodegradable polymer
3.3. Production of PHB from molasses spentwash
Fig. 4 shows accumulation of varying quantities of PHB in
sludge biomass in presence of different forms of molasses spent-
wash. Maximum sugar consumption was observed in the first
48 h followed by decrease in total sugar content (Fig. 4a) as was
also reflected in the PHB and biomass production pattern. Fig. 4b
shows the COD removal efficiency of the activated sludge biomass
from the different forms of this wastewater. Highest COD removal
of 60% was observed in case of RW + N with slightly lower values of
52.5% and 44% for FW + N and DW + N, respectively. However, a
decrease in COD removal efficiency was observed in absence of
DAHP which was found to vary between 36.6% and 55.2%.
TKN was completely exhausted within 48 h thereby creating
nitrogen limiting conditions and hence increase in PHB production
in case of all the forms of molasses spentwash with a reduction in
PHB concentration being observed on further incubation. RW + N
served as the best substrate for high biomass concentration of
8.233 g L?1and PHB accumulation of 2.578 g L?1, which corre-
sponded to a PHB content of 31.3% (Fig. 4c, d and e). There was a
drop in PHB and biomass concentration to 2.067 g L?1and
7.377 g L?1respectively in presence of FW + N and the correspond-
ing PHB content was found to be 28%. The PHB and biomass con-
centrations werestill lower
wastewater and the PHB content varied between 19.5% and
forthe remaining formsof
22.7%. Thus, high biomass concentration resulted in higher COD re-
moval which in turn led to high PHB accumulation indicating effec-
tive conversion of organic matter in spentwash to PHB, which also
required nitrogen fortification of the wastewater.
Though an increase in total biomass was observed, residual bio-
mass left after extraction of PHB was found to be only 16–60%
higher than the initial concentration of sludge biomass which
was added as inoculum. This biomass was partially digested during
the extraction procedure and could be composted thus finding pos-
sible application as manure for sugarcane crops as was suggested
by Nonato et al. (2001). According to the authors, the use of wastes
from PHB plant either directly or after composting in sugarcane
fields would lead to a reduction in the need for nitrogen and phos-
phate fertilizers, since such waste was rich in phosphate, calcium,
nitrogen and micronutrients. Further, a net zero carbon balance
could be achieved since CO2 emissions from production plant
would be returned to the fields during the process of photosynthe-
sis by cane plants.
PHB production from molasses-based distillery spentwash did
not conform to our earlier study with starch rich wastewaters
(Khardenavis et al., 2007) and a low PHB content (21.4–22.7%)
was observed at C:N ratios between 46:1 and 55:1 which improved
to 31.3% at lower C:N ratios achieved by addition of DAHP. Low
COD removal and low PHB synthesis was attributed to immobiliza-
tion of important nutrients in molasses spentwash at high ratio of
COD and total kjeldahl nitrogen (COD:TKN) which varied between
30:1 and 60:1 in cane molasses from Indian industries (Pathade,
1999) thus reducing their availability to the microorganisms in
0 24 48
COD (g L-1)
72 96 120
PHB (mg L-1)
Total Sugar (mg L-1)
Biomass (mg L-1)
Type of Wastewater
PHB content (%)
Fig. 4. Production of PHB by activated sludge with molasses spentwash subjected to various treatments (a) total sugar (b) COD (c) biomass (d) PHB (e) PHB content (values in
the brackets indicate C:N ratios) [(j) RW (h) RW + N (N) FW (D) FW + N (?) DW (e) DW + N].
A.A. Khardenavis et al./Waste Management 29 (2009) 2558–2565
Such immobilization in molasses occurred during ‘‘Maillard
reaction” between sugars and nitrogenous compounds on heat-
ing. The resulting melanoidin pigments contributed a high COD
to the wastewater and were hardly degraded by microorganisms
due to their toxic, antioxidant properties thereby leading to low
COD removal and hence a failure of treatment process (Kitts
et al., 1993; Wedzicha and Kaputo, 1992). The toxic effect of
molasses has been reported earlier during fermentative produc-
tion of ethanol and gluconic acid (Oderinde et al., 1986; Subba
Rao and Panda, 1994). Same was also demonstrated recently for
PHB production with R. eutropha NRRL B14690 by Khanna and
Srivastava (2005) who achieved a very low PHB content of 2%
(w w?1) of biomass in spite of high concentration of fermentable
sugars in molasses. Results obtained in the present study indi-
cated higher PHB accumulation in comparison to above reports
and were attributed to the occurrence of dual (C and N) limiting
conditions (low C:N ratios) which promoted higher microbial
growth and in turn favoured higher PHA accumulation. Zinn
(2003) has emphasized the appropriateness of the above culture
condition (dual C and N limitation) for production of PHA from a
toxic carbon source.
The importance of C:N ratios in the bacterial PHA accumulation
capability has been demonstrated previously by various research-
ers. Studies by Suresh Kumar et al. (2004) on PHB production by
waste activated sludge revealed that C:N ratio of 144:1 resulted
in maximum PHB accumulation of 33% of MLSS which decreased
at lower C:N ratios. Wang et al. (2007) found C:N ratio of 100:1
to be optimum for high PHA production (42.4% of dry cell mass)
by activated sludge. In contrast, from our earlier studies, a C:N ratio
of 50:1 was found to be optimum for PHB accumulation (65% of
MLSS) by waste activated sludge in presence of acetate (Kharden-
avis et al., 2005). Similar studies with starch rich wastewaters
showed C:N ratios between 45:1 and 48:1 to be optimum for
PHB production and a maximum PHB production of 67% (w w?1)
of sludge biomass was achieved at C:N ratio of 47:1 (Khardenavis
et al., 2007). The effect of high nitrogen content in soy molasses
on low accumulation of PHA by P. corrugata was demonstrated
by Solaiman et al. (2006b).
The low COD removal was attributed to the fact that the sludge
biomass was never exposed to toxic compounds such as polyphe-
nols and melanoidins present in molasses spentwash and prior
acclimatization to such compounds could improve the treatment
efficiency as was demonstrated in improved COD removal from
distillery wastewater by phenol acclimatized activated sludge
(Khardenavis et al., 2008).
PHB content in the present study was 2.5–4 times lower in
comparison to the 68–88% PHB content achieved by Liu et al.
(1998) with beet molasses and recombinant E. coli while it was
slightly lower than that obtained in case of Actinobacillus sp. EL-
9 (42%) capable of utilizing alcoholic distillery wastewater (Son
et al., 1996). This was probably due to toxicity of molasses spent-
wash used by us, in addition to lower concentration of ferment-
able sugars than in raw molasses used by the above researchers.
However, our results were comparable to that with Bacillus sp.
JMa5 grown in presence of 210 g L?1molasses powder (Wu
et al., 2001).
Gouda et al. (2001) demonstrated a method for improvement of
PHB content and cost reduction of PHB in case of B. megaterium by
using different wastes as carbon and nitrogen sources as was also
shown by Oliviera et al. (2004) and Jiang et al. (2008) who reported
higher PHB accumulation on supplementing the production med-
ium with molasses on account of positive effect of vitamins and
minerals in molasses. The fact that, trace elements and vitamins
in cane molasses promoted high PHB accumulation, may however
not hold true in case of molasses spentwash due to the depletion of
the above components and the fermentable sugars during alcohol
3.4. Evaluation of kinetic parameters
Table 2 shows the evaluation of kinetic parameters for batch
experiments with respect to PHB and biomass production on dif-
ferent forms of distillery spentwash. The biomass yield (Yx/s) in-
creased progressivelyfrom0.362 g g?1
0.748 g g?1for DW + N. As expected, higher biomass yield was ob-
served for wastes supplemented with additional nitrogen (DAHP)
in comparison to the same wastes without addition of DAHP. The
corresponding PHB yield (Yp/s) showed a similar trend with higher
values when wastes were supplemented with DAHP. An increase in
Yp/svalues was also observed with increase in Yx/sindicating the
need for higher biomass concentration for achieving high PHB con-
tent. The decrease in PHB content with increased C:N ratios as in
case DW and DW + N indicated that high C:N ratios did not favour
high yields of biomass and PHB.
The specific substrate consumption rate (?qs) was however,
lowest in DW and DW + N (0.061 and 0.067 g g?1h?1, respectively)
and was nearly half that of RW and RW + N (0.117 and
0.145 g g?1h?1) and FW and FW + N (0.100 and 0.106 g g?1h?1).
Similarly, the PHB storage rate (qp) was found to be 0.012 and
0.022 g g?1h?1for RW and RW + N respectively, which was higher
than the remaining forms of spentwash. Kinetic parameters pre-
sented in Table 2 clearly demonstrated that though the COD con-
sumption from deproteinized spentwash with and without DAHP
was the lowest amongst all the forms of molasses spentwash, high-
er biomass and PHB yields were obtained indicating higher per-
centage rates of conversion of consumed carbon.
The biomass yield reported in our study (0.362–0.748 g bio-
mass g?1COD) was higher than values reported by Jimenez et al.
(1997) (0.31 and 0.16 g VSS g?1COD for untreated and fermented
molasses) since our studies were performed under aerobic condi-
tions in comparison to anaerobic process followed by the above
group. Further, the substrate uptake rate was found to be between
0.1 and 0.2 g COD g?1VSS h?1which was 6–14 fold lower than the
values reported in the present study (Table 2).
PHB content, yield and kinetic parameters for the batch experiments.
RW RW + N FWFW + N DWDW + N
RW = raw wastewater; RW + N = raw wastewater + inorganic nitrogen source.
FW = filtered wastewater; FW + N = filtered wastewater + inorganic nitrogen source.
DW = deproteinized wastewater; DW + N = deproteinized wastewater + inorganic nitrogen source.
A.A. Khardenavis et al./Waste Management 29 (2009) 2558–2565
In spite of its toxicity, which was attributed to the presence of
phenolic residues left behind after fermentation and distillation
of alcohol, molasses spentwash could still serve as a low cost sub-
strate for PHB production. Waste activated sludge was an effective
source of PHB accumulating microorganisms and though the PHB
content using molasses spentwash was lower than that reported
earlier for other wastes, the process provided benefit of converting
otherwise un-utilizable and toxic waste, molasses spentwash into
value-added product-PHB. This would not only utilize the excess
sludge generated in a wastewater treatment plant and reduce the
load on landfills, but would also contribute to reduction in the cost
of PHB production by avoiding sterile conditions and pure carbon
sources for growth and maintenance of pure cultures. The added
advantage of PHB production using activated sludge was the effec-
tive treatment of this wastewater with up to 60% COD removal.
The authors are grateful to Director, National Environmental
Engineering Research Institute (NEERI), Nagpur, for providing the
facilities for carrying out this work. The NMR analysis of extracted
polymer by NMR Facility, National Chemical Laboratory (NCL),
Pune, is also gratefully acknowledged.
American Public Health Association, American Water Works Association, Water
Environment Federation, 1998. Standard methods for the examination of water
and wastewater, 20th ed., APHA, Washington, DC, USA.
Burdon, K.L., 1946. Fatty materials in bacteria and fungi revealed by staining dried
fixed slide preparations. Journal of Bacteriology 52, 665–668.
Choi, J., Lee, S.Y., Han, K., 1998. Cloning of Alcaligenes latus polyhydroxyalkanoate
bio-synthesis genes and use of these genes for enhanced production of poly(3-
hydroxybutyrate) in Escherichia coli. Applied and Environmental Microbiology
Chua, H., Yu, P.H., Sin, S.N., Tan, K.N., 2000. Effect of food:microorganism ratio in
activated sludge foam control. Applied Biochemistry and Biotechnology 84–86,
Converti, A., Zilli, M., Del Borghi, M., Ferraiolo, G., 1990. The fluidized bed reactor in
the anaerobic treatment of wine wastewater. Bioprocess Engineering 5, 49–55.
de Koning, G., 1995. Physical properties of bacterial poly((R)-3-hydroxyalkanoates).
Canadian Journal of Microbiology 41, 303–309.
Doi, Y., Kunioka, M., Nakamura, Y., Soga, Y., 1986. Nuclear magnetic resonance
studies on poly(b-hydroxybutyrate) and a co-polyester of b-hydroxybutyrate
Macromolecules 19, 2860–2864.
Doi, Y., Kawaguchi, Y., Nakamura, Y., Kunioka, M., 1989. Nuclear magnetic
resonance studies of poly(3-hydroxybutyrate) and polyphosphate metabolism
in Alcaligenes eutrophus. Applied and Environmental Microbiology 55 (11),
Fredrickson, A.G., Stephanopoulus, G.N., 1981. Microbial competition. Science 213,
Gouda, M.K., Swellam, A.E., Omar, S.H., 2001. Production of PHB by a Bacillus
megaterium strain using sugarcane molasses and corn steep liquor as sole
carbon and nitrogen sources. Microbiological Research 153, 201–207.
Hong, K., Sun, S., Tian, W., Chen, G.Q., Huang, W., 1999. A rapid method for detecting
bacterial polyhydroxyalkanoates in intact cells by fourier transform-infrared
spectroscopy. Applied Microbiology and Biotechnology 51, 523–526.
Huijberts, G.N.M., de Rijk, T.C., de Waard, P., Eggink, G., 1994.13C nuclear magnetic
resonance studies of Pseudomonas putida fatty acid metabolic routes involved in
poly(3-hydroxyalkanoate) synthesis. Journal of Bacteriology 176 (6), 1661–
Jiang, Y., Song, X., Gong, L., Li, P., Dai, C., Shao, W., 2008. High poly(b-
inexpensive substrates. Enzyme and Microbial Technology 42, 167–172.
Jimenez, A.M., Borja, R., Alonso, A., Martin, A., 1997. Influence of aerobic pre-
treatment with Penicillium decumbens on the anaerobic digestion of beet
molasses alcoholic fermentation wastewater in suspended and immobilized
cell bioreactors. Journal of Chemical Technology and Biotechnology 69, 193–
Joshi, H.C., 1999. Bio-energy potential of distillery effluents. Bioenergy News 3, 10–
Khanna, S., Srivastava, A.K., 2005. Statistical media optimization studies for growth
and PHB production by Ralstonia eutropha. Process Biochemistry 40, 2173–
Khardenavis, A.A., Guha, P.K., Suresh Kumar, M., Mudliar, S.N., Chakrabarti, T., 2005.
Activated sludge is a potential source for production of biodegradable plastics
from wastewater. Environmental Technology 26 (5), 545–552.
Khardenavis, A.A., Suresh Kumar, M., Mudliar, S.N., Chakrabarti, T., 2007.
biodegradable plastic poly b-hydroxybutyrate. Bioresource Technology 98
Khardenavis, A.A., Kapley, A., Purohit, H.J., 2008. Phenol-mediated improved
performance of active biomass for treatment of distillery wastewater.
International Biodeterioration and Biodegradation 62, 38–45.
Kim, B.S., 2000. Productionof poly(3-hydroxybutyrate)
substrates. Enzyme and Microbial Technology 27, 774–777.
Kim, Y.B., Lenz, R.W., 2000. Polyesters from microorganisms. In: Babel, W.,
Steinbuchel, A. (Eds.), Advances in Biochemical Engineering Biotechnology,
vol. 71. Springer, Berlin, Heidelberg, New York, pp. 51–79.
Kitts, D.D., Wu, C.H., Stich, H.F., Powrie, W.D., 1993. Effects of glucose–glycine
maillard reaction products on bacterial and mammalian cells mutagenesis.
Journal of Agricultural and Food Chemistry 41, 2353–2358.
Law, J.H., Slepecky, A.S., 1961. Assay of poly-b-hydroxybutyric acid. Journal of
Bacteriology 82, 33–36.
Liu, F., Li, W., Ridgway, D., Gu, T., Shen, Z., 1998. Production of poly-b-
hydroxybutyrate on molasses by recombinant Escherichia coli. Biotechnology
Letters 20 (4), 345–348.
Miller, G.L., 1959. Use of dinitrosalicylic reagents for the determination of reducing
sugars. Analytical Chemistry 31, 426–429.
Mishoe, G.H., 1999. F/M ratio and the operation of an activated sludge process.
Florida Water Resources Journal 3, 20–21.
Misra, A.K., Thakur, M.S., Srinivas, P., Karanth, N.G., 2000. Screening of poly-b-
hydroxybutyrate-producing microorganisms using fourier transform-infrared
spectroscopy. Biotechnology Letters 22, 1217–1219.
Morris, D.L., 1948. Quantitative determination of carbohydrates with Dreywoods
anthrone reagent. Science 107, 254–255.
Nonato, R.V., Mantelatto, P.E., Rossell, C.E.V., 2001. Integrated production of
biodegradableplastic, sugar and
Biotechnology 57, 1–5.
Oderinde, R.A., Ngoka, L.C., Adesogan, D.K., 1986. Comparative study of the effect of
ferrocyanide and EDTA on the production of ethyl alcohol from molasses by
Saccharomyces cerevisiae. Biotechnology and Bioengineering 28, 1462–1465.
Oliviera,F.C., Freire, D.M.G., Castilho,
hydroxybutyrate) bysolid-state fermentation
Biotechnology Letters 26, 1851–1855.
Ostle, A.G., Holt, J.G., 1982. Nile Blue A as a fluorescent stain for poly-b-
hydroxybutyrate. Applied and Environmental Microbiology 44 (1), 238–241.
Page, W.J., Cornish, A., 1993. Growth of Azotobacter vinelandii UWD in fish peptone
medium and simplified extraction of poly-b-hydroxybutyrate. Applied and
Environmental Microbiology 59, 4236–4244.
Park, S.J., Park, J.P., Lee, S.Y., 2002. Production of poly(3-hydroxybutyrate) from
whey by fed-batch culture of recombinant Escherichia coli in a pilot-scale
fermentor. Biotechnology Letters 24, 185–189.
Pathade, G.R., 1999. A review of current technologies for distillery wastewater
treatment. In: Goel, P.K. (Ed.), Advances in Industrial Wastewater Treatment.
Technoscience Publications, Rajasthan, India, pp. 180–239.
Ramachander, T.V.N., Rohini, D., Belhekar, A., Rawal, S.K., 2002. Synthesis of PHB by
recombinant E. coli harboring an approximately 5kb genomic DNA fragment
from Streptomyces aureofaciens NRRL 2209. International Journal of Biological
Macromolecules 31, 63–69.
Ratsak, C.H., Koouman, S.A.L.M., Kooi, B.W., 1993. Modelling the growth of an
oligochaete on activated sludge. Water Research 27 (5), 739–747.
Reddy, C.S.K., Ghai, R., Rashmi, Kalia, V.C., 2003. Polyhydroxyalkanoates: an
overview. Bioresource Technology 87, 137–146.
Serafim, L.S., Lemos, P.C., Oliviera, R., Reis, M.A.M., 2004. Optimization of
polyhydroxybutyrate production by mixed cultures submitted to aerobic
dynamic feeding conditions. Biotechnology and Bioengineering 87 (2), 145–
Sirianuntapiboon, S., Prasertsong, K., 2007. Treatment of molasses wastewater by
acetogenic bacteria BP103 in sequencing
Bioresource Technology 99 (6), 1806–1815.
Solaiman, D.K.Y., Ashby, R.D., Foglia, T.A., Marmer, W.N., 2006a. Conversion of
agricultural feedstock and coproducts into poly(hydroxyalkanoates). Applied
Microbiology and Biotechnology 71 (6), 783–789.
Solaiman, D.K.Y., Ashby, R.D., Hotchkiss Jr.2, A.T., Foglia, T.A., 2006b. Biosynthesis of
Biotechnology Letters 28 (3), 157–162.
Son, H., Park, G., Lee, S., 1996. Growth-associated production of poly-b-
Actinobacillus sp. EL-9. Biotechnology Letters 18, 1229–1234.
Subba Rao, D., Panda, T., 1994. Critical analysis of the effect of metal ions on
gluconic acid production by Aspergillus niger using a treated Indian cane
molasses. Bioprocess Engineering 10, 99–107.
Suresh Kumar, M., Mudliar, S.N., Reddy, K.M.K., Chakrabarti, T., 2004. Production of
bio-degradable plastics from activated sludge generated from a food processing
industrial wastewater treatment plant. Bioresource Technology 95 (3), 327–
Tavernier, P., Besson, I., Portais, J.-C., Courtois, J., Courtois, B., Barbotin, J.-N., 1998. In
vivo13C NMR studies of polymer synthesis in Rhizobium meliloti M5N1 Strain.
Biotechnology and Bioengineering 58, 250–253.
ethanol. Applied Microbiologyand
batch reactor (SBR) system.
alcoholic distillery wastewaterby
A.A. Khardenavis et al./Waste Management 29 (2009) 2558–2565
US Environmental Protection Agency, 1999. Biosolids Generation Use and Disposal Download full-text
in the United States. EPA530-R-99-009, Office of Solid Waste and Emergency
Response. Washington, DC, 9/1999.
Wang, Y.J., Hua, F.L., Tsang, Y.F., Cha, S.Y., Sin, S.N., Chua, H., Yu, P.H.F., Ren, N.Q.,
2007. Synthesis of PHAs from wastes under various C:N ratios. Bioresource
Technology 98 (8), 1690–1693.
Wedzicha, B.L., Kaputo, M.T., 1992. Melanoidins from glucose and glycine:
composition, characteristicsand reactivity
Chemistry 43, 359–367.
Wu, Q., Huang, H., Hu, G., Chen, J., Ho, K.P., Chen, G.-Q., 2001. Production of poly-3-
hydroxybutyrate by Bacillus sp. JMa5 cultivated in molasses media. Antonie van
Leeuwenhoek 80, 111–118.
Yan, Y.-B., Wu, Q., Zhang, R.-Q., 2000. Dynamic accumulation and degradation of
poly(3-hydroxyalkanoate)s in living cells of Azotobacter vinelandii UWD
characterized by13C NMR. FEMS Microbiology Letters 193, 269–273.
Yellore, V., Desai, A., 1998. Production of poly-3-hydroxybutyrate from lactose and
whey by Methylobacterium sp. ZP24. Letters in Applied Microbiology 26, 391–
Yu, P.H.F., Chua, H., Huang, A.L., Lo, W.H., Ho, K.P., 1999. Transformation of
industrial food wastes into PHA. Water Science and Technology 40 (1), 367–
Zinn, M., 2003. Tailor-made synthesis of polyhydroxyalkanoate. European Cells
Materials 5, 38–39.
A.A. Khardenavis et al./Waste Management 29 (2009) 2558–2565