Bioresource Technology 114 (2012) 573582

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Effect of substrate load and nutrients concentration on the polyhydroxyalkanoates

(PHA) production using mixed consortia through wastewater treatment
M. Venkateswar Reddy, S. Venkata Mohan
Bioengineering and Environmental Centre (BEEC), CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad 500 607, India

a r t i c l e i n f o a b s t r a c t

Article history: Production of biodegradable plastics in the form of polyhydroxyalkanoates (PHA) especially from renew-
Received 31 December 2011 able substrates is gaining interest. The present work mainly aims to investigate the inuence of substrate
Received in revised form 21 February 2012 load and nutrient concentration (nitrogen and phosphorous) on PHA production using wastewater as
Accepted 24 February 2012
substrate and mixed culture as biocatalyst. PHA accumulation was high at higher substrate load [OLR3,
Available online 3 March 2012
40.3% of dry cell weight (DCW)], low nitrogen (N1, 45.1% DCW) and low phosphorous (P1, 54.2% DCW)
conditions. With optimized nutrient conditions production efciency increased by 14%. Fractional com-
Keywords:
position of PHA showed co-polymer [poly(b-OH) butyrate-co-poly(b-OH) valerate, P3(HB-co-HV)] con-
Bioplastics
16S rRNA sequencing
tains PHB (88%) in more concentration compared to PHV (8%). Dehydrogenase and phosphatase
Nitrogen enzymatic activities were monitored during process operation. Good substrate degradation (as COD) of
Phosphorous 75% was registered during PHA production. The phylogenetic prole of 16S rRNA sequencing showed
P3(HB-co-HV) the dominance of Firmicutes (71.4%) and Proteobacteria (28.6%), which are known to involve in PHA accu-
mulation and waste treatment.
2012 Elsevier Ltd. All rights reserved.

1. Introduction media sterilization and reactor maintenance. Development of pure

Now there is a growing concern over the use of conventional
plastics like polypropylene and polyethylene. Production of these
plastics is dependent on depleting sources of hydrocarbons. These
petroleum derived plastics takes several years to decompose and
during degradation they produce harmful toxic compounds.
Because of these, an alternative is required for future economical
and ecologically safe polymers. Polyhydroxyalkanoates (PHA)
production through biological source is gaining importance to
overcome the adverse effects during conventional plastics degra-
dation (Rehm, 2010). PHA are the class of linear polyester
compounds naturally produced by many bacteria with similar
properties to polypropylene and polyethylene but completely
biodegradable, biocompatible and produced from renewable
resources. Upon disposal they are degraded by microorganisms
to water and carbon dioxide under aerobic condition and methane
under anaerobic conditions. PHA has been industrially produced by
pure cultures including Alcaligenes latus, Azotobacter vinelandii,
Pseudomonas oleovorans, recombinant Alcaligenes eutrophus and
Escherishia coli. However, one of the largest drawbacks of this
method involves the requirement of high operational costs which
accounts for nearly 11% of total production costs that includes
Corresponding author. Tel./fax: +91 40 27191664.
E-mail address: vmohan_s@yahoo.com (S. Venkata Mohan).
culture fermentation and commercialization of PHA increase their
cost about 49 times higher than that of the conventional plastics
(Moita and Lemos, 2011).
Another way to decrease the operational costs would be to de-
sign a novel system that do not require sterilization and reactor
maintenance. So there is a great interest, using mixed culture
and wastewater to produce PHA. Compared to pure culture, the
merits of PHA production with mixed culture includes an enhanced
economy, a simpler process control, non sterile conditions and an
improved use of wastes. Mixed culture utilization can lower the in-
put costs by allowing for large scale fermentations to occur with-
out overhead costs of sterilization. It also allows for a greater
variety of substrates to be used due to the presence of several
PHA producing organisms. A considerable effort has gone in pro-
duction of PHA using mixed culture and different wastewaters like,
municipal wastewater (Chua et al., 2003), sugar cane molasses
(Albuquerque et al., 2010), paper mill wastewater (Bengtsson
et al., 2008), tomato cannery wastewater (Liu et al., 2008), olive
oil mill efuent (Beccari et al., 2009), biohydrogen reactor efuent
(Venkata Mohan et al., 2010) and food waste (Venkateswar Reddy
and Venkata Mohan, 2012). Culture selection with a high PHA stor-
age capacity is one of the challenges in PHA production process
using mixed culture. Operating the system sequentially under a
carbon excess phase (feast) followed by substrate exhaustion
(famine), a selective pressure is imposed on the system. Mixed cul-
ture operated under feast and famine conditions were subjected to

0960-8524/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.02.127
574 M. Venkateswar Reddy, S. Venkata Mohan / Bioresource Technology 114 (2012) 573582
an internal growth limitation arising from the alternate substrate
availability, which compelled the organisms to a physiological
adaptation (Beccari et al., 2009). During this physiological adapta-
tion period, substrate uptake is mainly driven towards PHA
storage. It was reported that absence of external substrate for a
considerable period of time causes a decrease in the amount of en-
zymes needed for cell growth. Following such a starvation period,
if the microbial culture is enriched with an excess carbon source,
the enzymes available in the cells is lower than that required to
reach the maximum growth rate, thus the storage becomes the
dominant phenomena (Salehizadeh and van Loosdrecht, 2004).
Beun et al. (2002) reported that following a long starvation period,
a mixed culture operated under feast and famine conditions
showed around 70% of the carbon substrate uptake towards PHA
storage. Organisms that were able to store polymers during the
feast are selected due to their capacity to use them as an energy
and carbon source for cell growth and maintenance during the
starvation period. Operating conditions for optimum PHA produc-
tion can be different when using real waste streams rather than
synthetic substrates (Albuquerque et al., 2010). This suggests that
more research on process optimization with real waste streams is
required. Almost all the studies performed on PHA production
using mixed culture in two separate steps, culture selection and
maximization of accumulation of PHA (Lemos et al., 2006). Many
bacteria produce PHA under certain nutrient depleted conditions
in the presence of excess carbon source and limitations such as
nitrogen, phosphorous and oxygen (Anderson and Dawes, 1990;
Sharma et al., 2004). Therefore it is necessary to provide appropri-
ate substrate load, nitrogen concentration, phosphorous concen-
tration and oxygen limitation to the bacterial cells to support the
higher accumulation of PHA. Objective of this study is optimization
of substrate load along with the nutrients (nitrogen and phospho-
rus) concentration for enhanced PHA production in mixed consor-
tia under anoxic microenvironment using designed synthetic
wastewater (DSW) as substrate. The detailed analysis pertaining
to the enzyme activity measurement (dehydrogenase and phos-
phatase) and bioprocess evaluation (pH and volatile fatty acids,
(VFA)) was used to understand the change in the metabolic process
in terms of PHA accumulation and wastewater utilization ((COD,
chemical oxygen demand) removal, carbohydrates removal) at reg-
ular time intervals. The microbial composition of mixed culture
was also evaluated using culture isolation techniques to nd out
the organisms responsible for higher PHA accumulation and waste
treatment. Though, there are few studies pertaining to the inu-
ence of nitrogen and phosphorus on PHA accumulation, detailed
evaluation of the bioprocess, enzyme activities and microbial com-
munity analysis during operation makes this study novel and
innovative.
2. Methods
2.1. Biocatalyst
Aerobic mixed consortia acquired from an operating activated
sludge process (ASP; suspended solids, 4.6 g/l; volatile suspended
solids, 3.8 g/l; pH, 7.9) treating 10 MLD (million liters per day) of
composite wastewater was used as biocatalyst for PHA accumula-
tion. Prior to inoculation, parent culture was washed in saline buf-
fer (5000 rpm, 20 C for 10 min) and enriched in the DSW (glucose
3 g/l, NH4Cl 0.5 g/l, KH2PO4 0.25 g/l, K2HPO4 0.25 g/l, MgCl2
0.3 g/l, CoCl2 25 mg/l, ZnCl2 11.5 mg/l, CuCl2 10.5 mg/l,
CaCl2 5 mg/l, MnCl2 15 mg/l, NiSO4 16 mg/l, FeCL3 25 mg/l)
for PHA producing organisms under aerobic microenvironment
(120 rpm; 28 C) with repeated feast and famine conditions
(Venkata Mohan et al., 2010).
2.2. Experimental design
2.2.1. PHA accumulation
PHA production was performed with DSW by varying carbon,
nitrogen and phosphorous concentrations with enriched mixed
culture. All the experiments were performed in individual batch
reactors [total/working volume of 250/110 ml] and operated in
suspended growth fed batch mode at ambient temperature
(29 2 C; 100 rpm) under anoxic microenvironment (AxSBR). Aer-
obic consortia (10 ml) along with 100 ml of DSW were fed to the
reactors after adjusting to the designated pH (pH 7) with 2 N
NaOH/2N HCl. After feeding, the reactors (AxSBR) were closed with
septum (butyl rubber) to maintain anoxic metabolic function with
intermittent sparging of air (4 min for every 1 h) using aquarium
pump. Initially the PHA accumulation was evaluated by varying or-
ganic loading rate (OLR) of the substrate (OLR1, 1.51 KgCOD/m3-
day; OLR2, 3.03 KgCOD/m3-day and OLR3, 4.54 KgCOD/m3-day).
Further by keeping the substrate load as constant at optimum
OLR (OLR3), nitrogen concentrations were varied (N1, 100 mg/l;
N2, 200 mg/l; N3, 300 mg/l), to know the optimum nitrogen con-
centration for PHA accumulation. Furthermore, at the optimum
substrate load and nitrogen (OLR3 and N1), phosphorus concentra-
tion was optimized by taking different phosphorous concentra-
tions (P1, 50 mg/l; P2, 100 mg/l; P3, 150 mg/l), while rest of the
parameters were unchanged.
2.3. Microbial community
Microbial diversity analysis was performed for the mixed aero-
bic consortia used in the PHA accumulation studies with best oper-
ating conditions of substrate load, nitrogen and phosphorus
concentrations (OLR3, N1 and P1). Screening of PHA producing
organisms was done by direct plating and enrichment techniques.
Mixed aerobic culture (0.5 g) was inoculated into 250 ml ask con-
taining 50 ml of sterilized nutrient broth (Hi media, India) at pH 7
to allow the growth for 24 h under aerobic conditions (37 C at
150 rpm). One milliliter of culture was suspended in sterile deion-
ized water and made up to100 ml. Sample was serially diluted
from 10 1 to 10 8 times with 0.9% NaCl. Diluted sample (0.1 ml)
was spread on the nutrient agar plates using spread plating tech-
nique and was incubated at 37 C for 2448 h. Pure colonies were
obtained after repeated streaking. Colonies developed on the agar
plates were differentiated by color, elevation, form and edge
appearances.
2.3.1. Strain identication
More than 30 phenotypically different colonies were selected
from agar plates and 7 of these isolates were chosen for further
characterization. Genomic DNA from these isolated pure colonies
was extracted and puried using phenol- chloroform method.
16S rRNA of these isolates were amplied by PCR using universal
primers 8F (51- AGAGTTTGATCCTGGCTCAG-31) and 1542R
(51-AAGGAGGTGATCCAGCCGCA-31) (Galkiewicz and Kellogg, 2008).
PCR conditions were maintained according to method described
(Venkateswar Reddy and Venkata Mohan, 2012). Five microliter
sample of each PCR product was subjected to agarose gel electro-
phoresis to conrm product recovery and to estimate product con-
centration. Amplied PCR products after purication (Fermentas)
were sent to MWG Biotech for sequencing. All the partial 16S rRNA
sequences (1500 bp) were aligned with GenBank database using
the BLASTN facility and were also tested for possible chimera
formation with the CHECK CHIMERA program (http://
www.35.8.164.52/cgis/chimera.cgi?su=SSU). These sequences
were further aligned with the closest matches found in the
GenBank database with the CLUSTALW function of Molecular
Evolutionary Genetics Analysis package (MEGA). Neighbor-joining
 M. Venkateswar Reddy, S. Venkata Mohan / Bioresource Technology 114 (2012) 573582
phylogenetic tree was constructed with the MEGA version 4.0
(Venkateswar Reddy and Venkata Mohan, 2012). The 7 nucleotide
sequences identied in this study have been deposited in the Gen-
Bank database under accession numbers HE612873 to HE612879.
2.4. Analysis
2.4.1. Extraction and estimation of PHA
Extraction and estimation of PHA was performed based on the
procedure reported elsewhere (Venkata Mohan et al., 2010). The
biocatalyst was separated from the substrate by centrifugation
(3000g for 30 min at 10 C) and the resulting pellet was washed
with acetone and ethanol separately to remove unwanted materi-
als. The pellet was suspended in an equal volume of 4% sodium
hypochlorite and incubated at room temperature for 3 h. The
resulting mixture was centrifuged (3000g for 30 min at 10 C)
and the supernatant was discarded. The pellet with lysed cells after
washing simultaneously with acetone and ethanol was dissolved in
hot chloroform and passed through glass ber lter (0.45 lm pore
size) to separate the polymer from cell debris. The chloroform l-
trate was used to estimate PHA colorimetrically. For the assay,
PHA polymer extracted in chloroform was subjected to evapora-
tion followed by addition of 10 ml of sulfuric acid (36 N) and
heated at 100 C on a water bath for 10 min. Addition of sulfuric
acid converts the polymer to crotonic acid. The cooled solution
was measured for the absorbance at 235 and 285 nm for determin-
ing the poly (3-hydroxy) butyrate (PHB) and poly (3-hydroxy) val-
erate (PHV) concentration, respectively against a sulfuric acid
blank. The standard curve was prepared using pure poly-3(hydroxy
butyrate-co-hydroxy valerate) [P3(HB-co-HV)] (co-ploymer; natu-
ral origin, Aldrich). PHA in the extracted sample was conrmed
by FTIR and 1H NMR spectroscopy.
2.4.2. Bioprocess evaluation
Bioprocess was monitored by evaluating pH, VFA and COD
(closed reuxing method) according to the standard methods
(APHA, 1998). Quantitative estimation of VFA was carried out by
High performance liquid chromatography (HPLC; Shimadzu
LC10A) with UVVis detector at 210 nm and C18 reverse phase col-
umn (250  4.6 mm diameter and 5 l particle size) by using 40%
acetonitrile in 1 mN H2SO4 (pH, 2.53.0) as mobile phase with ow
rate of 0.5 ml/min. Carbohydrates were analyzed by anthrone
method (Morris, 1948). Dehydrogenase and phosphatase enzymes
activities were estimated by a method described (Venkateswar
Reddy et al., 2010).
3. Results and discussion
3.1. PHA production
3.1.1. Substrate load
Initially the substrate load was optimized for higher PHA accu-
mulation. Experimental results documented that PHA accumula-
tion was directly proportional to the organic load where higher
carbon concentration depicted higher accumulation (Fig. 1a). How-
ever, the time taken for high PHA accumulation increased with in-
crease in organic load (OLR1 at 36th h (32.4%), OLR2 at 48th h
(37.4%) and OLR3 at 60th h (40.3%) and decreased thereafter. In
the case of OLR3, PHA accumulation was increasing with time up
to 60th h from 12th h [12th h, 15.8%; 24th h, 28.5%; 36th h,
34.6%; 48th h, 36.8% and 60th h, 40.3%] and showed a decrement
thereafter [72nd h, 26.3%] due to lower availability of substrate.
During the feast condition (excess carbon phase), the external
substrate uptake is mainly driven towards internal PHA storage,
while after substrate exhaustion, the accumulated PHA will be
575
used as energy and carbon source for cell growth and maintenance.
Henceforth, the higher OLR took more time for maximum PHA
accumulation due to the available higher substrate, while, lower
OLR showed maximum PHA accumulation in shorter time due to
the lower substrate availability. In OLR2 high PHA accumulation
was observed at 48th h (37.4%), increasing from 12th h (15.6%),
24th h (28.4%), 36th h (35.6%) and subsequent operation showed
a visible drop in PHA accumulation at 60th h (31.2%) followed by
72nd h (22.5%). In OLR1 high PHA accumulation was observed at
36th h (32.4%), followed by 12th h (14.8%), 24th h (26.4%), 48th h
(30.2%), 60th h (28.4%) and 72nd h (15.4%). The PHA production
capacity of mixed culture varies depending on the type of substrate
used. Jiang et al. (2011) reported high PHB production of 90% in
mixed culture using lactate as substrate. Johnson et al. (2009) re-
ported 89% of PHA production using acetate as sole carbon source.
Accumulation of 67% of PHA was attained using crude glycerol ob-
tained from biodiesel manufacturing process (Garate et al., 2011).
Along with pure substrates, the usage of different wastewaters also
showed PHA production. Fermented paper mill efuent showed
48% of PHA (Bengtsson et al., 2008), while molasses showed 78%
of PHA (Albuquerque et al., 2010). Food waste (35.6%) and hydro-
gen reactor efuents (39.6%) also showed good amount of PHA pro-
duction (Venkateswar Reddy and Venkata Mohan, 2012).
3.1.2. Nitrogen concentration
Nitrogen is a micronutrient and it is a component of protein, en-
zymes and nucleic acids, increased utilization of nitrogen would
benet overall function of the cell (Sharma et al., 2004). The effect
of varying nitrogen concentration on PHA accumulation was eval-
uated at OLR3. Experimental results showed that lower nitrogen
concentration showed higher PHA accumulation (N1, 45.1%; 60 h)
and vice versa (N2, 41.5%; N3, 38%) (Fig. 1a). Lower concentration
of nitrogen showed higher PHB accumulation and increase in its
concentration increased the biocatalyst growth but not PHB accu-
mulation (Albuquerque et al., 2010).
3.1.3. Phosphorous concentration
Phosphorus is important for the utilization of carbohydrates
and fats for energy production and also in protein synthesis for
the growth, maintenance and cell repair. As inorganic phosphate
in ATP involves in protein synthesis as well as in the production
of the nucleic acids (DNA and RNA), which carry the genetic code
for all cells. Phosphorous is also essential for maintaining the buf-
fering capacity of cell and to control uctuations in the redox val-
ues. At optimum carbon and nitrogen concentrations (OLR3 and
N1), further experiments were carried out towards optimization
of phosphorous concentration, by varying phosphorous concentra-
tions. Phosphorous also followed same trend like nitrogen where
lower phosphorous concentration showed higher (P1, 54.2%) PHA
accumulation and higher concentrations showed lower PHA accu-
mulation (P2, 49.3%; P3, 45.5%) (Fig. 1a). Lower phosphorous con-
centration showed good PHA accumulation, rather than complete
phosphorous deciency. A minimal level of internal phosphate is
essential for PHA accumulation. Low concentration of phosphorous
and nitrogen are favorable for the enhancement of PHB production.
High nitrogen and phosphorous concentrations lead to protein syn-
thesis, while phosphate deprivation shows reduction of protein
synthesis rate and diverts towards PHA accumulation (Panda
et al., 2006). Under balanced growth conditions CoASH levels are
high and PHA synthesis gets inhibited. However, when growth is
limited by an essential nutrient other than the carbon and energy
source, reduces the complexity of the metabolism that occurs in
the cell and the ow of carbon is channeled into unidirectional
path such as PHA synthesis and the NADH concentration increases
resulting in inhibition of the early enzymes of the TCA cycle, such
as, citrate synthase and isocitrate dehydrogenase. This leads to an
576 M. Venkateswar Reddy, S. Venkata Mohan / Bioresource Technology 114 (2012) 573582

60
55 OLR1
OLR2
N1 P1
P2
a
N2

PHA accumulation (%DCW)

50 OLR3 N3 P3
45
40
35
30
25
20
15
10
5
0
0 24 48 72 0 24 48 72 0 24 48 72
Time (h)

90 PHB (%DCW) b
80 PHV (%DCW)
PHA composition

70
60
50
40
30
20
10
0
OLR3 N1 P1
Experimental variation

Fig. 1. (a) Production and (b) composition of PHA recovered from mixed culture with diverse substrate, nitrogen and phosphorous concentrations.

accumulation of acetyl-CoA, which relieves the inhibition exerted
by CoASH, leading to PHB formation (Dawes, 1986).
3.1.4. Composition of PHA
PHA composition in terms of PHB and PHV was analyzed sepa-
rately under best substrate load (OLR3), nitrogen (N1) and phos-
phorus (P1) concentrations (Fig. 1b). Irrespective of the operating
condition, high PHB content was observed over PHV (60th h). Phos-
phorous limitation showed high PHB content (P1, 88%), followed by
nitrogen limitation (N1, 76%) and excess carbon (OLR3, 62%). On
the contrary, PHV content was high in excess carbon (OLR3, 35%)
followed by nitrogen limitation (N1, 21%) and phosphorous limita-
tion (P1, 8%). PHB is a homopolymer of 3-hydroxybutyrate and is
the most widespread PHA in natural condition. PHB is a highly
crystalline and brittle homopolymer, which restricts its use to a
limited range of application. Therefore, it is suggested that co-poly-
mer P3(HB-co-HV) is better than PHB because it is more exible
and stronger. Activated sludge biomass would produce more PHB
than PHV under the nutrient stress microenvironment, especially
under phosphorus limitation conditions (Wen et al., 2010).
3.2. Characterization of PHA
3.2.1. FTIR
FTIR analysis revealed the presence of different conformational
bands in the extracted PHA from mixed culture (SFig. 1a). The pres-
ence of absorption bands at 1722 cm 1 and 1279 cm 1 demon-
strated their carbonyl bands C@O and CO stretching ester in
polymer. The methylene (CH2) vibration near 2924 cm 1 had
the strongest band in spectra of PHA. The band at about
1349 cm 1 is assigned to symmetric wagging of methyl (CH3)
groups and the bands at 1183 and 1134 cm 1 are characteristic
of the asymmetric and the symmetric stretching of the COC
group, respectively. The band at 1525 is due to the presence of
methyne (CH) group.
3.2.2. H1NMR
H1NMR spectrum demonstrated that the sample contains co-
polymer with two monomeric units of hydroxybutyrate and
hydroxyvalerate P3(HB-co-HV). Four groups of signal characteris-
tics of PHA were observed (SFig. 1a). The resonance observed at
0.88, 1.29, 1.91, 2.54 and 4.7 ppm were responsible for CH3 (HV
side group), CH3 (HB side group), CH2 (HV side group), CH2
(HV and HB bulk structures), CH (HV and HB bulk structures),
respectively. The methyl esters showed a sharp signal around
3.6 ppm, corresponding to the CH3O group of the esters. These re-
sults suggest that the polymer to be PHA.
3.3. Enzymes
3.3.1. Dehydrogenase
Dehydrogenase (DH) is an intracellular enzyme involved in the
oxidationreduction reactions occurring in the cell. It functions in
the transfer of H+ between metabolic intermediates using various
mediators (NAD+, FAD+, etc.) making availability of H+ and e in
the cell. DH is the main enzyme involved in the glycolytic pathway
 M. Venkateswar Reddy, S. Venkata Mohan / Bioresource Technology 114 (2012) 573582 577

and converts the glucose to acetyl-CoA and further conversion to
PHA. DH activity was observed to vary with the function of exper-
imental conditions studied, where the higher PHA accumulation
was associated with higher DH activity (Fig. 2a). Initial DH activity
was observed to be 4.1 lg/ml and increased with time till accumu-
lation of maximum PHA and then showed gradual decrement irre-
spective of the condition. OLR3 showed higher DH activity (60th h,
11.7 lg/ml) followed by OLR2 (48th h, 11.1 lg/ml) and OLR1
(36th h, 10.2 lg/ml) which might be due to the available substrate
concentration. Change in enzyme activity was well correlated with
the higher carbohydrates removal and higher PHA accumulation.
Excess NADH could acts as an electron donor in the acetoacetyl-
CoA reductase catalyzed reaction transforming acetoacetyl-CoA
into b-hydroxybutyryl-CoA which could be further converted into
PHA by the action of PHA synthase and slightly reduces the TCA cy-
cle activity. DH activity was also inuenced by the nitrogen and
phosphorous concentrations. Nitrogen and phosphorous limita-
tions showed higher DH activity (N1, 11.2 lg/ml; P1, 10.6 lg/ml),
while their high concentration depicted low DH activity (N2,
10.1 lg/ml; P2, 9.4 lg/ml; N3, 9.3 lg/ml; P3, 7.8 lg/ml). However,
phosphorous showed signicant inuence on the DH activity over
nitrogen.
3.3.2. Phosphatase
Phosphatase enzyme involves in the transformation of organic
and inorganic phosphorus compounds in the environment. The
main function of phosphatase is to deactivate glucose molecule
by removing the phosphate group where the glucose molecule can-
not further undergo degradation (Venkateswar Reddy et al., 2010).
Therefore, activity of phosphatase is to be low for good substrate
utilization. Initial enzyme activity for mixed culture was 3.6 lg/
ml and with time the activity was changed with respect to the
experimental variations studied (Fig. 2b). Enzyme activity showed
a decreasing trend with increasing time irrespective of the operat-
ing conditions, which is a good sign of substrate utilization. OLR3
depicted higher phosphatase activity compared to OLR2 and
OLR1 which might be due to the higher substrate concentration.
Lower phosphatase activity was observed with OLR1 at 72nd h
(0.4 lg/ml), followed by OLR2 (0.8 lg/ml) and OLR3 (1.1 lg/ml).
Nitrogen does not show any signicant inuence on the phospha-
tase activity, where a negligible decrement in the activity was ob-
served with increase in nitrogen concentration (N1, 1.0 lg/ml; N2,
0.9 lg/ml and N3, 0.8 lg/ml). On the other hand, high phosphorous
concentration (P3) showed lower enzyme activity (72nd h, 0.4 lg/
ml) followed by the lower concentrations (P2, 0.7 lg/ml; P1,

14
13
OLR1
OLR2
N1 P1 a
N2 P2
Dehydrogenase (g/ml toluene)

OLR3 N3 P3
12
11
10
9
8
7
6
5
4
3
0 24 48 72 0 24 48 72 0 24 48 72
Time (h)

4.0 OLR1 N1 P1 b
OLR2 N2 P2
3.5 OLR3 N3 P3
Phosphatase (g/ml phenol)

3.0

2.5

2.0

1.5

1.0

0.5

0.0
0 24 48 72 0 24 48 72 0 24 48 72
Time (h)

Fig. 2. Variation in (a) dehydrogenase and (b) phosphatase activities in mixed culture during PHA production.
578 M. Venkateswar Reddy, S. Venkata Mohan / Bioresource Technology 114 (2012) 573582

0.9 lg/ml). Because phosphatase enzyme function is to remove the
phosphate group from substrate and make the availability of phos-
phorous inside the cell, so at higher phosphorous concentration
availability of phosphorous was relatively high which leads to
low enzyme activity.
3.4. Bioprocess monitoring
3.4.1. pH variation
Prior to startup, pH of the feed was adjusted to neutral for all
the experimental variations. However, the nal pH after experi-
mental run was found to vary and is directly dependent on the
metabolites formed during the process (Fig. 3a). Gradual decre-
ment in pH was observed towards acidic in all the experimental
conditions up to certain time and increased thereafter which
might be due to the generation of VFA during initial phases and
their consumption in the later phases of operation. However,
the time interval for pH increment was varied depending on
experimental condition. OLR3 showed decrement of pH up to
60th h (73.4), followed by an increment (72nd h, 4.2). OLR2
showed a pH drop up to 48th h (73.9) and increased thereafter
(72nd h, 4.8). OLR1 showed a drop up to 36th h (74.2), followed
by an increment (72nd h, 5.1). Nitrogen at lower concentrations
(N1) showed low pH values (3.2), than higher concentrations

a 7.5 OLR1 N1 P1
OLR2 N2 P2
7.0 OLR3 N3 P3
6.5
6.0
pH

5.5
5.0
4.5
4.0
3.5
3.0
0 24 48 72 0 24 48 72 0 24 48 72
Time (h)

b 3500 OLR1 N1 P1
OLR2 N2 P2
3000 OLR3 N3 P3

2500
VFA (mg/l)

2000

1500

1000

500

0
0 24 48 72 0 24 48 72 0 24 48 72
Time (h)

c 90
80 OLR3

70
N1
VFA concentration (%)

P1
60
50
40
30
20
10
0
Acetate Butyrate Propionate Valerate

Fig. 3. Bioprocess monitoring in terms of (a) pH; (b) VFA; (c) VFA composition against time with different substrate, nitrogen and phosphorous concentrations during PHA
production.
 M. Venkateswar Reddy, S. Venkata Mohan / Bioresource Technology 114 (2012) 573582
(N2, 3.6; N3, 3.9). High phosphorous concentrations showed lower
decrement of pH (P3, 4; P2, 3.9) than lower concentration (P1, 3.5)
which might be attributed to the buffering capacity of the system
due to phosphates.
3.4.2. Acid intermediates
VFA are the crucial markers and precursor components for the
PHA accumulation. VFA prole signicantly varied during experi-
mental run with the function of experiments studied. Initial con-
centration of VFA was almost same for all the conditions (80 mg/l)
and showed a gradual increment up to certain time and after that
it showed decrement, which might be due to the generation and
utilization of VFA (Fig. 3b). OLR3 showed increment of VFA up to
60th h (3125 mg/l), followed by a decrement (72nd h, 2254 mg/l).
Similarly, OLR2 showed a VFA increment up to 48th h (2845 mg/
l), and decreased thereafter (72nd h, 1469 mg/l). OLR1 also showed
increment up to 36th h (1458 mg/l) followed by a decrement
(72nd h, 406 mg/l). Increase in nitrogen and phosphorous concen-
trations showed low VFA production (N2, 2915 mg/l; N3, 2744 mg/
l; P2, 2589 mg/l; P3, 2012 mg/l), where as lower concentrations
showed high VFA production (N1, 3325 mg/l; P1, 3378 mg/l). High-
er nitrogen and phosphorous concentrations leads to protein syn-
thesis instead of VFA production. Observed change in system pH
was correlated well with the VFA prole in all the experimental
variations. Lower nitrogen and phosphorous concentrations inhib-
its the early enzymes of the TCA cycle, citrate synthase and isoci-
trate dehydrogenase which leads to higher production of VFA.
VFA composition was analyzed in the reactors operated with
higher substrate load (OLR3), lower nitrogen (N1) and phosphorous
(P1) concentrations at 60th h. All the samples was analyzed by
HPLC and the data revealed the presence of higher fraction of acetic
acid along with relatively lower concentrations of butyric acid,
valeric acid and propionic acid (Fig. 3c). P1 showed higher fraction
of acetic acid (82 4%) followed by butyric acid (6 1%), propionic
acid (4 0.5%) and valeric acid (2 0.5%). N1 showed higher frac-
tion of acetic acid (78 3%) followed by butyric acid (12 1%),
valeric acid (4 0.5%) and propionic acid (3 0.5%). OLR3 showed
higher fraction of acetic acid (61 3%) followed by propionic acid
(24 1%), valeric acid (8 0.5%) and butyric acid (2 0.5%). High
substrate load showed higher amount of propionic acid and valeric
acid than N1 and P1, so it showed high amount of PHV than PHB.
3.5. Wastewater treatment
Wastewater treatment efciency was also evaluated in terms of
COD and carbohydrates removal along with PHA accumulation by
collecting the samples at regular time intervals during the
experiment.
3.5.1. COD
Substrate removal was noticed in all the experimental condi-
tions with time of operation irrespective of the variations studied
suggesting the system function towards treatment. OLR1 showed
high removal efciency (86%) followed by OLR2 (76%) and OLR3
(71%) (Fig. 4a). Higher nitrogen and phosphorous concentrations
showed high COD removal efciency (N3, 79%; P3, 80%) compared
to lower concentrations (N2, 76%; P2, 77%; N1, 74%; P1, 75%). Con-
trary to that, PHA accumulation was high at lower nitrogen and
phosphorous concentration which might be due to the conversion
of organic matter into proteins instead of PHA accumulation at
higher nitrogen and phosphorous concentration. Lower nitrogen
and phosphorous concentration stimulates the metabolic pathway
towards PHA accumulation rather than protein synthesis. This
process had dual benets, higher PHA accumulation along with
waste treatment by minimization of sludge concentration in waste
treatment plants.
579
3.5.2. Carbohydrates
Carbohydrates (glucose) removal showed increasing trend irre-
spective of the experimental variation studied. OLR1 showed high-
er removal efciency (93%) followed by OLR2 (90%) and OLR3
(87.6%) (Fig. 4b). Higher nitrogen concentration showed high car-
bohydrate removal (N3, 91%) compared to lower nitrogen concen-
tration (N2, 88%; N1, 85%). Contrary to that carbohydrates showed
higher removal at lower phosphorous concentration (P1, 84%) fol-
lowed by higher (P2, 82%; P3, 79%). Glucose is converted to pyru-
vate through glycolysis and then converted to acetyl-CoA
resulting in PHB production, while propionyl-CoA (via the propio-
nate succinate pathway) formed from pyruvate and valeryl-CoA
formed from valerate resulting mainly in PHV production under
anaerobic condition (Bengtsson et al., 2008). VFA generated are
transported across the cell membrane and then activated to the
corresponding acyl-CoA. Two molecules of acetyl-CoA condensed
to acetoacetyl-CoA by the action of b-ketothiolase which is reduced
to 3-hydroxybutyryl-CoA by the action of enzyme acetoacetyl-CoA
reductase utilizing the reduced NADPH. Propionate is converted to
acetyl-CoA via propionyl-CoA and both the molecules can produce
PHB and PHV respectively. Whereas butyrate was activated by
coenzyme A (CoA) and forms butyryl-CoA this was oxidized to
acetyl-CoA through b-oxidation pathway and continued into PHA
production pathway (Dawes, 1986). ATP activates the VFA and
reducing equivalents (NADPH) help in the formation of hydroxya-
cyl-CoA moieties which are the precursors for PHA production. On
the contrary, under aerobic operation, the ATP is generated from
respiration which is required for the cell growth and maintenance
by utilizing the reducing equivalents. Anoxic operation facilitates
both the respiration for the cell growth and PHA accumulation as
storage granule.
3.6. Strain isolation and identication
Partial 16S rRNA sequencing analysis demonstrated the struc-
tural composition of communities in the PHA accumulated sludge.
The phyologenetic distribution was established with a neighbor-
joining method (Fig. 5). Totally 7 organisms were isolated, which
were phylogenetically related to phylum Firmicutes (71.4%) and
Proteobacteria (28.6%) (Table 1). Among two phylums, organisms
belong to phylum Firmicutes (B. subtillis, B. badius, Bacillus tequilen-
sis, Staphylococcus arlettae and Enterococcus italicus) were domi-
nant. They are gram positive, motile, rod-shaped, aerobic and/or
facultative organims. Bacillus sp. showed PHA yields vary from
11% to 69% (Singh et al., 2009). Kaynar and Beyatli (2009) reported
PHB production from B. subtillis and B. badius which are isolated
from intestine of various shes by using nutrient broth as sub-
strate. B. subtillis P8 showed 11.7% and B. subtillis P9, showed
9.41% yield of PHB. B. badius P19, P21 and P22 showed 8.9%,
7.4%, 1.7% yields of PHB respectively. Kumar et al. (2009) reported
production of PHA by different Bacillus sp (B. cereusEGU44, 47%; B.
cereusEGU3, 25%; B. cereusEGU520, 17%; B. cereusEGU43, 12%) using
pea shell slurry and GM2 medium in the ratio of 3:7 as substrate.
Staphylococcus sp. (C20R) isolated from marine coastal sediment
collected from an unpolluted site, called curve showed capacity
to accumulate PHB (13.3%) (Cortes et al., 2008).
In Proteobacteria, class c-proteobacteria (Serratia ureilytica and
Pseudomonas otitidis) was found to be dominant. S. ureilytica is a
gram-negative, rod-shaped, non-spore forming and facultative
anaerobe. The genus Serratia belongs to the family Enterobacteria-
ceae of the c-class of the Proteobacteria widely distributed in nat-
ure (water, soil and foods). This organism has the capacity to
hydrolyze urea to ammonia and to utilize urea as a sole nitrogen
source for its growth (Bhadra et al., 2005). There are no reports
available on the PHA accumulating capacity of this organism. P.
otitidis is a gram-negative, rod-shaped, aerobic bacterium. Produc-
580 M. Venkateswar Reddy, S. Venkata Mohan / Bioresource Technology 114 (2012) 573582

16500
OLR1 N1 P1 a
15000 OLR2 N2 P2
13500 OLR3 N3 P3
12000
COD removal (mg/l) 10500

9000

7500

6000

4500

3000

1500

0
0 24 48 72 0 24 48 72 0 24 48 72
Time (h)

16500
OLR1 N1 P1 b
15000
OLR2 N2 P2
13500 OLR3 N3 P3
Carbohydrate removal (mg/l)

12000

10500

9000

7500

6000

4500

3000

1500

0
0 24 48 72 0 24 48 72 0 24 48 72
Time (h)

Fig. 4. Variations in the concentrations of (a) COD and (b) carbohydrates during PHA production.

tion of PHA by P. otitidis was not yet reported. On the contrary,
Pseudomonas group had been reported widely to have capacity to
produce PHA from different substrates. Production of PHA by Pseu-
domonas aeruginosa42A2, using different oils and sugars (oleic acid
54.6%, waste frying oil 29.4% and glucose 16.8%) was reported (Fer-
nandez et al., 2005). Pseudomonas putida and P. aeruginosa with rice
plant oil showed 34.4% and 23.5% of PHA production, respectively
(Queiroz et al., 2009).
Polyphosphate accumulating organisms (PAO) and glycogen
accumulating organisms (GAO) are also detected in this study;
they are present in the activated sludge and are mainly involved
in the wastewater treatment process. Some of the organisms in
phylum Firmicutes are reported to be involved in the phosphorous
removal process. B. tequilensis isolated from the Western ghat for-
est located in the Nilgiri Hills, Kerala had the phosphorous solubi-
lization activity (Dastager et al., 2011). B. cereus was reported to
have capacity to accumulate phosphorous up to 13.7 mg/l (Sidat
et al., 1999). Reports showed that Enterococcus group have poly-
phosphate accumulating capacity (Tobin et al., 2007). Staphylococ-
cus sp. is reported to accumulate phosphorous up to 4.4 mg/l (Sidat
et al., 1999). Some of the organisms in Pseudomonas group like
P. uorescens (20.6 mg/l), P. testesteroni (13.3 mg/l) and P. mendoci-
na (19.6 mg/L) are polyphosphate accumulators and are involved
in the phosphorous removal process (Sidat et al., 1999). PAO
mainly involves in the phosphorous removal in waste treatment.
Under anaerobic conditions, PAO are able to take up VFA and con-
vert them into intracellular PHA. PAO gain the energy required for
conversion of VFA into PHA through the hydrolysis of their intra-
cellularly stored polyphosphate and glycogen. During aerobic con-
ditions, PAO oxidize PHA to gain energy for growth, glycogen
replenishment and phosphorus uptake. Phosphorus removal is
achieved through the depletion of excess sludge containing the
accumulated polyphosphate. GAO are capable of VFA uptake under
anaerobic condition by converting to PHA (Lemos et al., 2007). GAO
 M. Venkateswar Reddy, S. Venkata Mohan / Bioresource Technology 114 (2012) 573582 581

29 SVMIICTPHA1

5 Bacillus badius
Pseudomonas otitidis

0 8 SVMIICTPHA7

71 Serratia ureilytica
Pseudomonas panipatensis
9 Bacillus aryabhattai
29 Lysinibacillus xylanilyticus

15 Brevibacillus parabrevis
11 Bacillus subtilis
SVMIICTPHA3
1 Klebsiella pneumoniae
Bacillus tequilensis
19
33 SVMIICTPHA2
84 SVMIICTPHA6

24 SVMIICTPHA4
Staphylococcus pasteuri

0 SVMIICTPHA5
Staphylococcus arlettae
4
10 Enterococcus italicus
13 Bacillus firmus

0.5

Fig. 5. Neighbor-joining tree constructed using Mega 4.0 showing phylogenetic relationships of closely related 16S rRNA sequences from GenBank.

Table 1
Phylogenetic sequence afliation and similarity to the closet relative of amplied 16S rRNA sequence from pure cultures isolated from mixed culture.

Isolate number BLAST similarity (%) Organism Accession number Phylogenetic afliation
SVMIICTP1 100 S. ureilytica (HE612873) AJ854062 c-Proteobacteria
SVMIICTP2 99 P. otitidis (HE612874) AY953147 c-Proteobacteria
SVMIICTP3 100 E. italicus (HE612875) AEPV01000109 Firmicutes
SVMIICTP4 100 B. tequilensis 10b (HE612876) HQ223107 Firmicutes
SVMIICTP5 99 B. subtilis (HE612877) AF074970 Firmicutes
SVMIICTP6 100 S. arlettae (HE612878) AB009933 Firmicutes
SVMIICTP7 99 B. badius (HE612879) X77790 Firmicutes

does not release or take up phosphate for PHA accumulation nei-
ther anaerobically nor aerobically and hence they do not contrib-
ute for phosphorous removal. They instead hydrolyze glycogen as
their sole source of energy for anaerobic VFA uptake. GAO
consumes VFA in wastewater without removing phosphorus (Le-
mos et al., 2007).
4. Conclusions
Production of PHA from wastewater treatment by mixed culture
signicantly reduces the production cost.
Acknowledgements
The authors wish to thank the Director, CSIR IICT for his
support and encouragement in carrying out this work. MVR
acknowledges the Council of Scientic and Industrial Research
(CSIR), New Delhi, for providing research fellowship.

Present study demonstrated the role of substrate concentration,

nitrogen and phosphorous concentration on PHA accumulation in
mixed consortia under anoxic microenvironment. Higher substrate
load, lower nitrogen and phosphorous concentrations showed
higher PHA accumulation. Produced PHA was composed of the
copolymer P3(HB-co-HV) where PHB fraction was higher com-
pared to PHV. Enzyme activities, bioprocess evaluation and waste-
water treatment were evaluated which showed good correlation
with PHA accumulation. Phylogenetic prole of mixed culture
showed the dominance of Firmicutes and Proteobacteria which are
known PHA accumulators associated with waste treatment.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.biortech.2012.02.127.
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