Bioresource Technology 234 (2017) 99–105

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

Polyhydroxyalkanoates (PHA) production from synthetic waste using

Pseudomonas pseudoflava: PHA synthase enzyme activity analysis from
P. pseudoflava and P. palleronii
M. Venkateswar Reddy a, Yasuteru Mawatari b, Rui Onodera a, Yuki Nakamura a, Yuka Yajima a,
Young-Cheol Chang a,⇑
a
Department of Applied Sciences, College of Environmental Technology, Muroran Institute of Technology, 27-1 Mizumoto, Muroran, Hokkaido 050-8585, Japan
b
Research Center for Environmentally Friendly Materials Engineering, Muroran Institute of Technology, 27-1 Mizumoto-cho, Muroran, Hokkaido 050-8585, Japan

h i g h l i g h t s

PHA production from synthetic waste using P. pseudoflava was evaluated.

High carbon content present in the waste was successfully degraded by bacteria.

Produced PHA was characterized by NMR, GPC, DSC and TGA.

PHA synthase enzyme was characterized from P. pseudoflava and P. palleronii.

PHA production from waste reduces treatment cost & production cost.

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

Article history: Synthetic wastewater (SW) at various carbon concentrations (5–60 g/l) were evaluated for polyhydrox-
Received 31 December 2016 yalkanoates (PHA) production using the bacteria Pseudomonas pseudoflava. Bacteria showed highest
Received in revised form 28 February 2017 PHA production with 20 g/l (57 ± 5%), and highest carbon removal at 5 g/l (74 ± 6%) concentrations
Accepted 1 March 2017
respectively. Structure, molecular weight, and thermal properties of the produced PHA were evaluated
Available online 4 March 2017
using various analytical techniques. Bacteria produced homo-polymer [poly-3-hydroxybutyrate
(P3HB)] when only acetate was used as carbon source; and it produced co-polymer [poly-(3-hydroxybu
Keywords:
tyrate-co-3-hydroxyvalerate) P(3HB-co-3HV)] by addition of co-substrate propionate. PHA synthase, the
Pseudomonas pseudoflava
SDS-PAGE
enzyme which produce PHA was extracted from two bacterial strains i.e., P. pseudoflava and P. palleronii
P(3HB-co-3HV) and its molecular weight was analysed using SDS-PAGE. Protein concentration, and PHA synthase
PHA synthase enzyme activity of P. pseudoflava and P. palleronii was carried out using spectrophotometer. Results
Total organic carbon denoted that P. pseudoflava can be used for degradation of organic carbon persistent in wastewaters
and their subsequent conversion into PHA.
Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction industry because they are biodegradable and their physical proper-
ties are similar to those of polypropylene (Sheu et al., 2009). Poly-
Many of the plastics are generated by the use of non-renewable 3-hydroxybutyrate (P3HB) is one type of PHA produced by many
compounds and they are persisting in the environment due to their bacteria (Kim et al., 2012). PHA can be used as a filler for non-
low degradable nature (Fradinho et al., 2014). To overcome this biodegradable plastics, agriculture systems for prolonged release
problem, research has been enduring for the replacement of con- of fertilizers, agrochemicals and medicine (Sudesh et al., 2000).
ventional plastics by bioplastics. Polyhydroxyalkanoates (PHA) High cost of PHA economically limiting its application as alterna-
are bio-polyesters accumulated in cells by a wide range of bacteria. tive for traditional plastics (Fradinho et al., 2014; Ntaikou et al.,
PHA polyesters have been studied intensively by academia and 2009). Production costs of PHA should be reduced to compete with
synthetic plastics. Low cost substrates such as molasses, plant oils
and other fatty acids attracted the attention of various researches
⇑ Corresponding author.
to reduce the PHA production cost (Ntaikou et al., 2009; Koller
E-mail address: [email protected] (Y.-C. Chang).
et al., 2005).

http://dx.doi.org/10.1016/j.biortech.2017.03.008
0960-8524/Ó 2017 Elsevier Ltd. All rights reserved.
100 M. Venkateswar Reddy et al. / Bioresource Technology 234 (2017) 99–105

PHA synthase is the critical enzyme in PHA biosynthesis, and R- 2.4. PHA production
3-hydroxyacyl–coenzyme A (CoA) is the substrate. More than 59
PHA synthase genes have been cloned and analyzed from 45 bacte- Cultures grown with SW at different carbon concentrations
ria (Sheu et al., 2009). PHA synthases have been assigned to three were collected at 120 h and centrifuged. The resulting pellet was
classes based on their substrate specificity and subunit composi- suspended in SW. The composition of SW was same as mentioned
tion (Rehm et al., 2001). The class I PHA synthases (Ralstonia eutro- in 2.2 Section, but low nitrogen and phosphorous concentrations
pha) are composed of a single type of polypeptide chain and use (0.1 g/l) were used in order to create stress for accumulation of
mainly short chain length hydroxyalkanoic acid CoA thioesters as PHA granules. Also experiments were conducted by using only
substrates. The class III PHA synthases (Allochromatium vinosum) acetate (at 20 g/l) as carbon source in SW in order to know the dif-
are composed by two different subunits, each of approximately ference in PHA composition. All the conditions were maintained as
40 kDa (Rehm et al., 2001). The substrate specificity is similar with like in growth phase. Culture was collected and the PHA was
class I synthases, and some medium chain-length 3-hydroxyfatty extracted and analyzed at 72 h.
acids are also incorporated. Class II PHA synthases are composed
by one subunit Like class I enzymes, and found mainly in Pseu- 2.5. Analysis
domonas aeruginosa. Substrate specificity is the main difference
between class II and both class I and class III PHA synthases. Class 2.5.1. TOC and VFA analysis
II PHA synthases integrate specially 3-hydroxyfatty acids of med- Total organic carbon (TOC) from clarified samples at different
ium chain length (C6-C14) into PHA, and the resulting product is time intervals were analysed in a Shimadzu TOC automatic analy-
a latex like polymer (Rehm et al., 2001). Class I PHA synthases syn- ser to know the removal of carbon concentration in SW. Sodium
thesize higher molecular weight PHA compared with class II PHA acetate and propionate standards (10–500 mg/l) are used to pro-
synthases (Rehm and Steinbüchel, 1999). duce the calibration curves. TOC was measured using NPOC
In the present study, capability of Pseudomonas pseudoflava to (non-purgeable organic carbon) method. By sparging samples to
produce PHA at various synthetic wastewater (SW) concentrations which a small amount of acid has been added, the IC in the sample
was evaluated. PHA synthase the enzyme which produce PHA was is converted to carbon dioxide. This carbon dioxide is removed, and
extracted from two bacterial strains i.e., P. pseudoflava and P. paller- the TOC is obtained by measuring the TC in the treated sample. VFA
onii and its molecular weight was analysed using sodium dodecyl analysis was done as mentioned in Venkateswar Reddy et al.
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein (2016). All results were presented as average and standard devia-
concentration, and PHA synthase enzyme activity of P. pseudoflava tion of the data from three independent experiments.
and P. palleronii was carried out using spectrophotometer. Consid-
ering the importance of utilizing waste streams as feedstock for 2.5.2. Structure & molecular mass determination
PHA production, the volatile fatty acids (VFA) tested in this work Extraction and estimation of PHA was performed following the
are common products of various wastewaters. procedures reported (Law and Slepecky, 1960; Venkata Mohan and
Venkateswar Reddy, 2013; Venkateswar Reddy et al., 2016). 1H
(500 MHz) NMR spectra was recorded on a JNM-ECA500 NMR
2. Materials and methods
spectrometer (JEOL, Japan) at 20 °C to know the structure of poly-
mer. Samples of produced PHA and standard P3HB/ P(3HB-co-3HV)
2.1. Biocatalyst
(Aldrich) were prepared by dissolving in deuterated chloroform
(CDCl3), and then were filtrated with cotton. The signals of tetram-
Pseudomonas pseudoflava (NBRC-102513) was collected from
ethylsilane (TMS) and CDCl3 were used as the standards for chem-
the Biological Resource Center, National Institute of Technology
ical shift of 1H spectra.
and Evaluation (NBRC), Japan. P. pseudoflava is a bacterium from
Number and weight average molecular mass (Mn and Mw) of
the family of Comamonadaceae. Many authors reported about effi-
standard and sample P3HB/ P(3HB-co-3HV) were measured using
ciency of this bacterium for production of PHA using various
gel permeation chromatography (GPC) (GPC, 900-1, JASCO, Japan)
substrates.
equipped with two Shodex K-806L columns and an RI detector.
Chloroform was used as an eluent at 40 °C and polystyrene stan-
2.2. Media dards (Mn = 1,680–3,065,000) were employed for calibration.

For the growth of P. pseudoflava, nutrient broth and SW were 2.5.3. Thermal properties determination
used as the media. Composition of the SW was mentioned in Thermogravimetric analysis (TG/DTA7300, Hitachi, Japan) was
Venkateswar Reddy et al. (2016). The carbon source composition used to determine the decomposition temperature (Td) of P3HB/P
of SW contains acetate and propionate. Different carbon concentra- (3HB-co-3HV). PHA powder was added into an aluminum pan
tions (5–60 g/l) were used in SW. The pH of the medium was and subjected to a heating rate of 10 °C/min from 50 °C to
adjusted to 7 and autoclaved before adding to the flasks. 550 °C. Differential scanning calorimetry (DSC, DSC7000X, Hitachi,
Japan) was used to characterize the melting temperature (Tm). The
2.3. Growth curve studies temperature range for DSC varied from 50 °C to 350 °C at a heat-
ing rate of 10 °C/min.
A loop of P. pseudoflava strain was initially inoculated into 50 ml
of nutrient broth in 500 ml flasks, and kept in shaking incubator 2.5.4. SDS-PAGE
under dark condition at 30 °C for overnight at 180 rpm. For growth P. palleronii and P. pseudoflava cells grown on MS medium con-
experiments 4 ml (4% v/v) of the overnight grown culture was taining acetate (19 g/l) and propionate (1 g/l) at 37 °C were har-
inoculated into different shake flasks containing 100 ml of SW with vested by centrifugation and whole cell extracts were prepared
different carbon concentrations i.e., 5 to 60 g/l. The experiments by sonication. Cell extracts were prepared at 4 °C or on ice. Bacte-
were conducted for 192 h. Samples were collected at different time rial cells harvested from nitrogen limited cultures by centrifuga-
intervals, and growth was monitored spectrometrically by measur- tion for 10 min at 10,000g, was washed two times with 50 mM
ing the absorbance at 600 nm using UV-spectrometer (UV-1800, Tris-HCl buffer (pH 8.3) and suspended in the same buffer (for
Shimadzu, Japan). 1 g of pellet, 5 ml of buffer was added). Cells were lysed in an ice
 M. Venkateswar Reddy et al. / Bioresource Technology 234 (2017) 99–105 101

using a Branson ultrasonic disrupter (Sonifer 250, Danbury, CT, and 168 h for 60 g/l substrate concentration. The availability of
USA) at one-minute flash for 10 min at output-3 and duty cycle- acetate and propionate at different time intervals was considered
50%. Cell debris was removed by centrifugation at 20,000g for to compare with growth curve results. For 5 g/l substrate concen-
20 min. The supernatant (crude extract) was stored at 20 °C until tration, 2.1 g/l of acetate and 0.13 g/l of propionate were available
further enzyme assay and sodium dodecyl sulfate-polyacrylamide at 48 h; and 0.9 g/l of acetate and 0.06 g/l of propionate were avail-
gel electrophoresis (SDS-PAGE) analysis. The molecular mass of able at 80 h. At 48 and 80 h, 5.2 g/l and 2.1 g/l of acetate and 0.29 g/
crude enzyme was determined by SDS-PAGE (Laemmli, 1970). l and 0.12 g/l of propionate were respectively available for 10 g/l
SDS-PAGE was run on a 12.5% polyacrylamide gel. The molecular concentration. For 15 g/l concentration, 9.4 g/l of acetate and
weights of proteins were estimated according to molecular weight 0.41 g/l of propionate were available at 48 h, after that availability
of standards (Tefco co., Ltd, Japan). was decreased to 4.1 g/l (acetate) and 0.23 g/l (propionate) at 80 h.
Acetate and propionate were not available at 120 h for 5–15 g/l
2.5.5. Activity assay of PHA synthase substrate concentrations. For 20 g/l concentration, 12.4 g/l of acet-
Assay of PHA synthase activity was carried out according to the ate and 0.71 g/l of propionate were available at 48 h, gradually the
methods described in literature (Miyake et al., 1997; Valentin and availability at 80 h was decreased to 6.3 g/l (acetate) and 0.42 g/l
Steinbuchel, 1994). The assay mixtures (1 ml) contained 50 ll of (propionate). At 120 h, 0.38 g/l of acetate was available, but propi-
50 mM DL-b-hydroxybutyryl coenzyme A lithium salt (final con- onate was not available.
centration 2.5 mM) (Sigma-Aldrich, Cat No: H0261), 50 ll of For 40 g/l concentration, availability of acetate was decreased
20 mM 5, 50 - dithio-bis (2-nitrobenzoic acid) (final concentration from 24.2 g/l (48 h) to 9.24 g/l (120 h). Also propionate availability
1 mM) (DTNB, Wako pure chemicals, Japan, Cat No: 047-16401) was decreased from 1.08 g/l (48 h) to 0.09 g/l (120 h). Similar trend
in 870 l1 of 1 M Tris-HCl buffer. The reaction was started by addi- was observed for 50 g/l substrate concentration. For 50 g/l concen-
tion of 30 ll of crude enzyme. The optical density of the thioben- tration, availability of acetate was decreased from 29.3 g/l (48 h) to
zoate anion resulting from the reaction of CoA and DTNB was 13 g/l (120 h). Also propionate availability was decreased from
measured at 412 nm for 1 min at room temperature using spec- 1.29 g/l (48 h) to 0.2 g/l (120 h).
trophotometer (UV-1800, Shimadzu, Japan). Protein concentration Bacteria from 5 to 40 g/l substrate concentrations showed
was determined by Bradford protein assay (Bio-Rad, Hercules, CA, immediate growth i.e., log phase started after 2 h, and for higher
USA) and bovine serum albumin (Pierce, Rockford, IL, USA) as the substrate concentrations it took more time i.e., for 50 g/l log phase
standard at 595 nm using spectrophotometer (UV-1800, Shimadzu, started after 10 h, for 60 g/l log phase started after 22 h. Higher
Japan). concentrations took more time to show maximum bacterial
growth due to toxic nature of VFA. These results clearly indicate
the effect of VFA concentration on the growth of bacteria. In our
3. Results and discussion
previous studies we used the bacteria P. palleronii and analysed
growth curve results using similar VFA concentrations
3.1. Growth curve analysis
(Venkateswar Reddy et al., 2016). When the growth was compared
for two bacteria strains, P. palleronii showed better growth than P.
P. pseudoflava was cultivated in SW at 30 °C by supplementing
pseudoflava at higher concentrations. P. palleronii supplemented
with different concentrations (5–60 g/l) of carbon source. Growth
with 30–60 g/l VFA concentration showed 1.1–1.02 times higher
curve analysis clearly showed that the carbon concentration had
growth than P. pseudoflava. Contrary to this, P. palleronii incubated
significant influence on the bacterial growth. Among all the con-
at lower VFA concentrations showed lower growth than
centrations, bacteria showed higher growth with 20 g/l (120 h
P. pseudoflava. P. palleronii supplemented with 5 g/l and 10 g/l
OD, 2.239) followed by 15 g/l (120 h OD, 2.17), 40 g/l (80 h OD,
concentrations showed 1.04 times and 1.2 times lower growth
2.10), 30 g/l (80 h OD, 2.097), 60 g/l (168 h OD, 2.091), 50 g/l
than P. pseudoflava. Two bacterial strains belongs to same genus,
(120 h OD, 2.07), 10 g/l (120 h OD, 2.02) and 5 g/l (80 h OD, 1.53)
but they are two different species and they have different proper-
(Fig. 1). Bacteria does not show maximum growth at same time
ties and substrate utilization capacity. Even though we provided
interval for all the concentrations, it showed maximum growth
similar concentration of substrates for both the bacteria, they
at different time intervals based on the carbon concentration. Bac-
showed variation in their growth, it may depend on the nature
teria showed maximum growth at 80 h for 5 g/l, 30 g/l, and 40 g/l;
and metabolic activities of bacteria. At present state, we don’t
120 h for 10 g/l, 15 g/l, 20 g/l, and 50 g/l substrate concentrations,
know the exact reason, to know that we need to do further
in-depth analysis.

3.2. Carbon removal

TOC removal was noticed in SW irrespective of the carbon con-

centrations studied suggesting the system’s function towards
treatment. Among the different time intervals, 5 g/l substrate con-
centration illustrated higher TOC removal rate at 120 h (3.7 g/l).
Higher TOC removal rate of 7 g/l (at 120 h) followed by 4.9 g/l (at
80 h) and 2.5 g/l (at 48 h) was observed for 10 g/l substrate concen-
tration. P. pseudoflava at 15 g/l substrate concentration illustrated
higher TOC removal rate at 120 h (10.2 g/l) followed by 80 h
(7.8 g/l), and 48 h (3.1 g/l). P. pseudoflava at 30 g/l concentration
illustrated higher TOC removal rate of 13.2 g/l at 120 h followed
by 80 h (9.2 g/l) and 48 h (5.9 g/l). Bacteria grown at 40 and 50 g/
l substrate concentrations illustrated higher TOC removal rates of
17.2 g/l and 25.5 g/l respectively at 120 h. Bacteria at 60 g/l sub-
Fig. 1. Growth curve of P. pseudoflava using various concentrations (5–60 g/l) of strate concentration illustrated higher TOC removal rate at 120 h
synthetic wastewater. (24.6 g/l) followed by 80 h (18.8 g/l), and 48 h (6.3 g/l).
102 M. Venkateswar Reddy et al. / Bioresource Technology 234 (2017) 99–105

When the TOC removal was compared for two bacteria strains, Table 1
P. palleronii showed higher TOC removal than P. pseudoflava at Acetate and propionate removal at different concentrations of synthetic wastewater
using P. pseudoflava at 120 h time interval.
lower substrate concentrations (5–30 g/l). P. palleronii at 5 g/l and
30 g/l concentrations showed 1.18 and 1.06 times higher TOC Initial substrate concentration Acetate removal Propionate removal
removal than P. pseudoflava. Mass balance analysis was performed (g/l) (%) (%)

for all the substrate concentrations at 120 h. From the 5 g/l concen- 5 100 ± 6 100 ± 6
tration, 3.7 g/l of substrate was utilized and 1.3 g/l was present as 10 100 ± 4 100 ± 4
15 100 ± 5 100 ± 5
residual carbon source in the solution. Out of the 3.7 g/l of sub- 20 98 ± 5 100 ± 5
strate, 1.6 g/l was retained in the form of biomass, and 2.1 g/l of 30 73 ± 6 94 ± 5
carbon remained in the form of unknown organic matter. For 40 76 ± 6 88 ± 5
10 g/l substrate concentration, 7 g/l was utilized and out of it 50 73 ± 4 89 ± 5
60 58 ± 5 65 ± 5
2.5 g/l was remained in the form of biomass. 10.2 g/l of substrate
was utilized from 15 g/l of total substrate concentration. From total
concentration of 20 g/l, 11.6 g/l of substrate was utilized and 8.4 g/l
was remained as residual carbon source in the solution. Out of the
11.6 g/l of substrate, 0.38 g/l was retained in the form of acetate,
3.9 g/l was retained in the form of biomass. From total concentra-
tion of 30 g/l, 13.2 g/l of substrate was utilized and 16.8 g/l was
remained as residual carbon source in the solution. Out of the
13.2 g/l of substrate, 7.7 g/l was retained in the form of acetate
and 0.08 g/l were retained in the form of propionate, 3.3 g/l was
retained in the form of biomass, and 2.12 g/l of carbon remained
in the form of unknown organic matter. From total concentration
of 40 g/l substrate, 17.2 g/l of substrate was utilized and 22.8 g/l
was remained as residual carbon in the solution. Out of the
17.2 g/l of substrate, 9.24 g/l was retained in the form of acetate,
0.09 g/l was retained in the form of propionate, 3.7 g/l was retained
in the form of biomass. From 50 g/l substrate concentration, 25.5 g/
l of substrate was utilized. From total concentration of 60 g/l,
24.6 g/l of substrate was utilized and 35.4 g/l of substrate was
existed as residual carbon source in the solution. Out of the Fig. 2. Variation of pH with respect to time at different volatile fatty acids
24.6 g/l of substrate, 18 g/l was retained in the form of acetate, concentrations using P. pseudoflava.
0.7 g/l was retained in the form of propionate, 4.2 g/l was retained
in the form of biomass, and 1.7 g/l of carbon remained in the form
rate of 11.6 g/l. For 50 g/l and 60 g/l substrate concentrations, pH
of unknown organic matter. Jin and co-workers reported that along
increment of 7 to 7.65; and 7 to 7.48 with TOC removal rates of
with PHA production other products like glycerol and different
25.5 g/l; and 24.6 g/l were observed respectively.
amino acids i.e., serine, glycine and glutamine might also be gener-
ated (Jin et al., 2013). Hence the unknown organic matter may con-
tribute to byproducts such as glycerol and amino acids. 3.5. Accumulation and composition of PHA

3.3. VFA removal Degradation of VFA and subsequent conversion of their metabo-
lites into PHA was studied by P. pseudoflava. In this study, PHA
Individual VFA composition of the SW at different concentra- accumulation was studied at 15 g/l, 20 g/l and 30 g/l carbon con-
tions (from 5–60 g/l) were analyzed at different time intervals centrations. PHA production and CDM were evaluated at 120 h of
using HPLC (Table 1). Among all the carbon concentrations, bacte- time interval. The best accumulation of PHA (57 ± 5%) was
ria grown at 5, 10 and 15 g/l showed complete removal (100%) of achieved with 20 g/l carbon concentration in SW. P. pseudoflava
both acetic and propionic acids within 120 h. Bacteria grown at also accumulated the good level of PHA when supplied with
20 g/l concentration showed 98 ± 3% removal of acetic acid, and 30 g/l (53 ± 4%) and 15 g/l (51 ± 4%) carbon concentrations.
100 ± 5% removal of propionic acid. Bacteria grown at 50 and P. palleronii showed 1.09 times and 1.05 times higher PHA at
60 g/l concentration showed 73 ± 4% and 58 ± 5% removal of acetic 30 g/l and 15 g/l substrate concentrations respectively than
acid; 89 ± 4% and 65 ± 5% removal of propionic acid respectively. P. pseudoflava.
Many authors reported about efficiency of bacteria P. pseudoflava The possibility of converting VFA into PHA has been previously
for degradation of various sugars, but little information was avail- reported by many authors using pure or mixed bacterial cultures
able about degradation of VFA using this bacterium. (Kourmentza et al., 2015; Amulya et al., 2014; Venkateswar
Reddy and Venkata Mohan, 2012). Many authors used various sub-
3.4. pH increment strates for PHA production using P. pseudoflava. According to Timm
and Steinbuchel, P. pseudoflava produced 14.3% of CDM as polymer
The pH showed an increasing trend with time which might be with composition of 99.2% of 3-hydroxy butyrate (3HB), and 0.8%
due to the utilization of VFA by P. pseudoflava (Fig. 2). Lower carbon of 3-hydroxyl octanoate (3HO) by using 15 g/l of sodium gluconate
concentrations showed high increment of pH due to complete uti- as carbon source (Timm and Steinbuchel, 1990). Studies conducted
lization of acids. At 120 h, SW at 5 g/l concentration showed high- by Povolo et al. (2013) revealed that P. pseudoflava achieved 5.7 g/l
est increment in pH (7 to 9.34) with TOC removal rate of 3.7 g/l. SW of dry biomass with 20 g/l of sucrose as carbon source, and at 72 h
at 10 g/l concentration showed pH increment from 7 to 9.22 with it produced 53% of PHA with composition of P(3HB-co-2.5%HV).
TOC removal rate of 7 g/l. The pH increment of 7 to 9.07 was They also reported 2.4 g/l of dry biomass achieved by P. pseudoflava
observed at 15 g/l substrate concentration. At 120 h, SW at 20 g/l with 20 g/l of lactose as carbon source, and at 48 h it produced 27%
concentration showed pH increment of 7 to 8.71 with TOC removal of PHA with composition of P(3HB-co-3%HV). P. pseudoflava
 M. Venkateswar Reddy et al. / Bioresource Technology 234 (2017) 99–105 103

synthesized copolymer P(3HV-co-4HB) by using valerolactone plus P3HB and P(3HB-co-3HV) produced by P. pseudoflava consist of
butyrolactone as a carbon source in a mineral salts medium (Choi HB and HB/HV units as their repeating units of the main-chains
et al., 2003). Dietrich et al., reported that P. pseudoflava was deter- respectively.
mined to produce PHB with glucose (DCW-4.2 g/l; PHB-1.7 g/l at To measure the molecular mass of homo-polymer (P3HB) and
24 h), mannose (DCW-3.5 g/l; PHB-1.2 g/l at 24 h), xylose (DCW- co-polymer P(3HB-co-3HV) produced from P. pseudoflava, we mea-
2.3 g/l; PHB-0.1 g/l at 72 h) and arabinose (DCW-0.2 g/l; PHB- sured both number average molecular mass (Mn) and weight aver-
0.2 g/l at 72 h) at 10 g/l individual initial substrate concentrations age molecular mass (Mw) of biopolymer using by GPC. MW and
(Dietrich et al., 2013). Other than bioplastics production, polydispersity index (PDI, Mw/Mn) of the P3HB produced by
P. pseudoflava has the property of colonization and mineralization P. pseudoflava is 17.63 kDa and 3.3 respectively. MW and PDI of
of palmitic acid (Thomas and Alexander, 1987). the co-polymer P(3HB-co-3HV) produced by P. pseudoflava is
Experiments were conducted to know the difference in PHA 52.33 kDa and 5.7 respectively. The Mw of the standard
composition by using only acetate (20 g/l) as carbon source, and P(3HB-co-3HV) is 110 kDa, and PDI is 4.3. These results indicate
mixture of acetate-propionate (19:1) as carbon source in SW. that P. pseudoflava can produce biopolymers with relatively lower
Experiments with only acetate produced homo-polymer P3HB, dispersity. The Mw of the P3HB produced from Cupriavidus
experiments with acetate-propionate mixtures produced co- sp. CY-1 (Venkateswar Reddy et al., 2015a), Bacillus sp. CYR1
polymer P(3HB-co-3HV). It was supported with previous reports (Venkateswar Reddy et al., 2015b), and P. palleronii (Venkateswar
by various authors (Takabatake et al., 2000; Jiang et al., 2011). P. Reddy et al., 2016) were compared with P3HB produced by
pseudoflava produced PHA from pentoses (Bertrand et al., 1990). P. pseudoflava (Table 2).
This organism was able to use a hydrolysate from the hemicellu- Regarding the molecular weights of PHA synthesized in biolog-
losic fraction of poplar wood as a carbon and energy source for ical systems it seems obvious that type I PHA synthases synthesize
its growth. When P. pseudoflava was grown on the major sugars PHA with molecular weights ranging from approximately 500000
present in hemicelluloses in batch cultures, P4HB accumulated to several millions compared with type II PHA synthases which
when glucose, xylose, or arabinose was the sole carbon source, synthesize PHA with molecular weights ranging from approxi-
with the final PHB content varying from 17% of the biomass dry mately 50000 to 500000, respectively (Rehm and Steinbüchel,
weight on arabinose to 22% of the biomass dry weight on glucose 1999). Type III PHA synthases seem to synthesize PHA with molec-
and xylose. PHB weight-average molecular weights were 640,000 ular weights that are in between. The molecular weight of PHA
on arabinose and 1,100,000 on glucose and xylose. Co-polymers depends on several factors, i.e., the physiological background is
of HB and, HV were produced when propionic acid was added to important with respect to the provision of HA-CoA thioesters and
shake flasks containing 10 g of glucose per liter. The HV monomer concentration of the substrates of the PHA synthases and also with
content attained a maximum of 45 mol% when the initial propionic respect to the availability of enzymes that hydrolyze PHA such as
acid concentration was 2 g per liter. Povolo et al. (2013) reported PHA depolymerases or unspecific esterases and lipases (Rehm
production of PHA containing 3HB, 3HV and 4HB as co- and Steinbüchel, 1999). If the physiological background does not
monomers through the use of inexpensive carbon sources such provide such enzymes, then PHA of higher molecular weight might
as whey from dairy industry. Ramsay et al. (1990) reported that be produced. Level of expression of active PHA synthase protein in
P. pseudoflava under nitrogen-limited conditions, was found to the cells is also very important. The higher the concentration of
accumulate 43% DCW of PHA at 48 h when supplied with glucose active PHA synthase protein in the cells, the lower the molecular
(10 g/l) and propionic acid (1 g/l). The produced PHA contains weight of the accumulated polyester (Rehm and Steinbüchel,
P(HB-co-HV) copolymer with 45% HV content. 1999).

3.6. NMR and GPC analysis 3.7. TGA and DSC analysis

The 1H NMR spectrum of CDCl3 soluble part of P3HB and TGA was used to evaluate the thermal stability of polymers, i.e.,
P(3HB-co-3HV) extracted from P. pseudoflava grown with SW was decomposition temperature, especially focus on the temperature of
measured at 20 °C to deduce its chemical structure and primary 5% weight loss (Td5). The weight loss of standard homo-polymer
sequence of polymer chain (Fig. S1). Based on their peak positions, P3HB started at around 230 °C and its Td5 was at 274 °C, com-
splitting patterns and integral ratio of these signals, each pletely decomposed at 295 °C (Fig. 3A). However the starting tem-
peak of P3HB can be assigned to the protons on methyne perature for weight loss of the P3HB produced by P. pseudoflava
(5.30–5.22 ppm), methylene (2.65–2.45 ppm) and methyl was at around 160 °C and its Td5 was at 180 °C. In addition, the
(1.35–1.15 ppm) groups. In the case of P(3HB-co-3HV), protons in P3HB was completely not decomposed at 550 °C, 75% was decom-
HV unit, i.e., methyne (5.20–5.13 ppm), methylene on the posed at 540 °C indicating that the P3HB extracted from P. pseud-
main-chain (2.66–2.50 ppm), methylene on the side-chain oflava composed of 25% inorganic material which may be came
(1.60–1.30 ppm), and methyl group (0.92–0.83 ppm) were from bacterial dry mass. The weight loss of standard co-polymer
observed with the peaks of protons in P3HB unit. These spectra P(3HB-co-3HV) started at around 140 °C and its Td5 was at
were almost same as both P3HB and P(3HB-co-3HV) standards 260 °C, completely decomposed at 285 °C (Fig. 3B). However, the
measured at the same conditions. The results suggested that starting temperature for weight loss of the co-polymer P(3HB-co-

Table 2
Comparison of physical and thermal properties of PHB produced from different bacterial strains in our previous and present studies.

Bacteria name Molecular Weight Polydispersity index Weight loss started at Td5 Tm Reference
Cupriavidus sp. CY-1 269 KDa 2.9 100 °C 160 °C 160 °C Venkateswar Reddy et al. (2015a)
Bacillus sp. CYR1 709 KDa 2.2 100 °C 180 °C 171 °C Venkateswar Reddy et al. (2015b)
P. palleronii 12 KDa 2.3 170 °C 180 °C 165 °C Venkateswar Reddy et al. (2016)
P. Pseudoflava 52 KDa 5.7 160 °C 180 °C 160 °C This study
Standard PHB 725 KDa 4.2 230 °C 274 °C 178 °C Venkateswar Reddy et al. (2015a)

Td5 is decomposition temperature where polymer showed 5% weight loss.

104 M. Venkateswar Reddy et al. / Bioresource Technology 234 (2017) 99–105

Fig. 3. Thermogravimetric analysis of (A) homo-polymer P3HB; and (B) co-polymer

P(3HB-co-3HV). – (a) is biopolymer extracted from P. pseudoflava; – (b) is standard Fig. 4. Differential scanning calorimetry analysis of (A) homo-polymer P3HB; and
biopolymer (Sigma-Aldrich). (B) co-polymer P(3HB-co-3HV). (a) is biopolymer extracted from P. pseudoflava; (b)
is standard biopolymer (Sigma-Aldrich).

3HV) produced by P. pseudoflava was at around 170 °C and its Td5

was at 210 °C, and it was completely not decomposed at 550 °C,
around 70% was decomposed at 540 °C. TGA analysis of co-
polymer produced by P. pseudoflava showed that initial decompo-
sition started at 20–130 °C this was not due to the polymer, we
think this may be due to the solvents or low molecular weight
material present in the sample.
DSC was used to characterize the melting temperature (Tm) of
the homo-polymer P3HB and co-polymer P(3HB-co-3HV)
extracted from P. pseudoflava, and it was compared with respective
standards (Fig. 4AB). From the endothermic peaks in each DSC
traces, it denoted that homo-polymer P3HB extracted from P.
pseudoflava contains the Tm of 140–165 °C, it was matched with
standard P3HB (Tm, 150–170 °C). In the standard co-polymer sam-
ples, we observed two endothermic peaks, one peak denotes the Tm
(155 °C) and another peak denotes the Tg (140 °C). We observed
one peak at 135 °C from co-polymer samples extracted from P.
pseudoflava, it may be due to glass transition temperature (Tg) of
amorphous nature of the polymer. We did not find Tm for our co-
polymer, because of its non-crystalline form. Nevertheless, based
on the results shown above, we concluded that the produced poly-
Fig. 5. SDS-PAGE analysis of crude protein isolated from bacteria, Lane 1: Crude
mer is P3HB and P(3HB-co-3HV). proteins extracted from P. pseudoflava; 2: Crude proteins extracted from P.
palleronii; M: molecular size markers.

3.8. Molecular weight determination and enzyme assay

to Rehm and Steinbüchel, PHA synthase proteins belonging to
Fig. 5 shows the electrophoretic pattern of the SDS-PAGE anal- classes I and II exhibit molecular masses of approximately 63–
ysis of PHA synthase in P. palleronii and P. pseudoflava. Based on the 73 kDa, whereas class III consist of two different subunits PhaC
SDS-PAGE analysis, the PHA synthases isolated from the both bac- and PhaE with molecular masses of about 40 kDa (Rehm and
teria corresponded to a molecular mass of 63 kDa. It was supported Steinbüchel, 1999).
by existing literature. Tsuge et al. (2004) reported that SDS-PAGE P. palleronii showed higher protein concentration
analysis revealed that the PHA synthase (PhaCDa) isolated from (0.68 ± 0.05 mg/ml) than P. pseudoflava (0.57 ± 0.03 mg/ml). Also
the recombinant E. coli had molecular mass of 69 kDa. According higher enzyme activity was observed in P. palleronii
 M. Venkateswar Reddy et al. / Bioresource Technology 234 (2017) 99–105 105

(0.142 ± 0.02 Unit/ml) than P. pseudoflava (0.054 ± 0.004 Unit/ml). Jin, Y.X., Shi, L.H., Kawata, Y., 2013. Metabolomics-based component profiling of
Halomonas sp. KM-1 during different growth phases in poly(3-hydroxybutyrate)
Unit activity was defined as the amount of enzyme required for
production. Bioresour. Technol. 140, 73–79.
conversion of lM of substrate in to product per minute. Higher Kim, M.S., Kim, D.H., Cha, J., Lee, J.K., 2012. Effect of carbon and nitrogen sources on
enzyme activity of P. palleronii than P. pseudoflava was supported photo-fermentative H2 production associated with nitrogenase, uptake
by presence of strong band in SDS-PAGE analysis and also higher hydrogenase activity, and PHB accumulation in Rhodobacter sphaeroides
KD131. Bioresour. Technol. 116, 179–183.
PHA production. Miyake et al. (1997) reported that the cyanobac- Koller, M., Bona, R., Hermann, C., Horvat, P., Martinz, J., Neto, J., Pereira, L., Varila, P.,
terium Synechococcus sp. MA19, under nitrogen-deprived condi- Braunegg, G., 2005. Biotechnological production of poly(3-hydroxybutyrate)
tion showed 0.24 lM/min/mg protein of PHB synthase enzyme with Wautersia eutropha by application of green grass juice and silage juice as
additional complex substrates. Biocatal. Biotrans. 23, 329–337.
activity. Tsuge et al. (2004) reported about 350 U/g of PHA syn- Kourmentza, C., Ntaikou, I., Lyberatos, G., Kornaros, M., 2015.
thase activity in recombinant E. coli JM109. Polyhydroxyalkanoates from Pseudomonas sp. using synthetic and olive mill
wastewater under limiting conditions. Int. J. Biol. Macromol. 74, 202–210.
Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the
4. Conclusion head of bacteriophage T4. Nature 227, 680–685.
Law, J.H., Slepecky, A.S., 1960. Assay of poly-b-hydroxybutyric acid. J. Bacteriol. 82,
33–36.
Bacteria P. pseudoflava utilized VFA present in SW and con- Miyake, M., Kataoka, K., Shirai, M., Asada, Y., 1997. Control of Poly-b-
verted in to useful products. Structure, molecular weight, and ther- Hydroxybutyrate synthase mediated by acetyl phosphate in Cyanobacteria. J.
mal properties of the produced PHA were analysed. SDS-PAGE Bacteriol. 179, 5009–5013.
Ntaikou, I., Kourmentza, C., Koutrouli, E.C., Stamatelatou, K., Zampraka, A., Kornaros,
analysis showed that the enzyme PHA synthases isolated from M., Lyberatos, G., 2009. Exploitation of olive oil mill wastewater for combined
the P. pseudoflava and P. palleronii corresponded to a molecular biohydrogen and biopolymers production. Bioresour. Technol. 100, 3724–3730.
mass of 63 kDa. P. palleronii showed higher PHA synthase enzyme Povolo, S., Romanelli1, M.G., Basaglia, M., Ilieva, V.I., Corti, A., Morelli, A., Chiellini, E.,
Casella, S., 2013. Polyhydroxyalkanoate biosynthesis by hydrogenophaga
activity than P. pseudoflava. These findings indicate the possibility pseudoflava DSM1034 from structurally unrelated carbon sources. New
of feeding the P. pseudoflava with cheap VFA rich fermented wastes Biotechnol. 30, 629–634.
to produce PHA. Ramsay, B.A., Lomaliza, K., Chavarie, C., Dube, B., Bataille, P., Ramsay, J.A., 1990.
Production of poly-(P-Hydroxybutyric-Co-3-Hydroxyvaleric) acids. Appl.
Environ. Microbiol. 56, 2093–2098.
Conflict of interest Rehm, B.H.A., Steinbüchel, A., 1999. Biochemical and genetic analysis of PHA
synthases and other proteins required for PHA synthesis. Int. J. Biol. Macromol.
25, 3–19.
The authors have declared no conflict of interest. Rehm, B.H.A., QI, Q., Beermann, B., Hinz, H.J., Steinbuchel, A., 2001. Matrix-assisted
in vitro refolding of Pseudomonas aeruginosa class II polyhydroxyalkanoate
synthase from inclusion bodies produced in recombinant Escherichia coli.
Acknowledgements Biochem. J. 358, 263–268.
Sheu, D.S., Lai, Y.W., Chang, R.C., Chen, W.M., 2009. Detection of
Dr M V Reddy gratefully acknowledges the JSPS for providing polyhydroxyalkanoate synthase activity on a polyacrylamide gel. Anal.
Biochem. 393, 62–66.
Postdoctoral fellowship (ID No: P15352). This work was supported Sudesh, K., Abe, H., Doi, Y., 2000. Synthesis, structure and properties of
by a Grant-in-Aid for Scientific Research C (26340067) from the polyhydroxyalkanoates: biological polyesters. Prog. Polym. Sci. 25, 1503–1555.
Japan Society for the Promotion of Science, Japan. Takabatake, H., Satoh, H., Mino, T., Matsuo, T., 2000. Recovery of biodegradable
plastics from activated sludge process. Water Sci. Technol. 42, 351–356.
Thomas, J.M., Alexander, M., 1987. Colonization and mineralization of palmitic acid
Appendix A. Supplementary data by Pseudomonas pseudoflava. Microb. Ecol. 14, 75–80.
Timm, A., Steinbuchel, A., 1990. Formation of polyesters consisting of medium-
chain-length 3-Hydroxyalkanoic acids from gluconate by Pseudomonas
Supplementary data associated with this article can be found, in aeruginosa and other fluorescent Pseudomonads. Appl. Environ. Microbiol. 56,
the online version, at http://dx.doi.org/10.1016/j.biortech.2017.03. 3360–3367.
Tsuge, T., Imazu, S., Takase, K., Taguchi, S., Doi, Y., 2004. An extra large insertion in
008.
the polyhydroxyalkanoate synthase from Delftia acidovorans DS-17: its deletion
effects and relation to cellular proteolysis. FEMS Microbiol. Lett. 231, 77–83.
References Valentin, H.E., Steinbuchel, A., 1994. Application of enzymatically synthesized
short-chain-length hydroxy fatty acid coenzyme A thioesters for assay of
polyhydroxyalkanoic acid synthases. Appl. Microbiol. Biotechnol. 40, 699–709.
Amulya, K., Venkateswar Reddy, M., Venkata Mohan, S., 2014. Acidogenic spent
Venkata Mohan, S., Venkateswar Reddy, M., 2013. Optimization of critical factors to
wash valorization through polyhydroxyalkanoates (PHA) synthesis coupled
enhance polyhydroxyalkanoates (PHA) synthesis by mixed culture using
with fermentative biohydrogen production. Bioresour. Technol. 158, 336–342.
Taguchi design of experimental methodology. Bioresour. Technol. 128, 409–
Bertrand, J.-L., Ramsay, B.A., Ramsay, J.A., Chavarie, C., 1990. Biosynthesis of poly-b-
416.
hydroxyalkanoates from pentoses by Pseudomonas pseudoflava. Appl. Environ.
Venkateswar Reddy, M., Mawatari, Y., Yajima, Y., Satoh, K., Venkata Mohan, S.,
Microbiol. 56, 3133–3138.
Chang, Y.C., 2016. Production of poly-3-hydroxybutyrate (P3HB) and poly-3-
Choi, M.H., Lee, H.-J., Rho, J.K., Yoon, S.C., Nam, J.D., Lim, D., Lenz, R.W., 2003.
(hydroxybutyrate-co-hydroxyvalerate) P(3HB-co-3HV) from synthetic
Biosynthesis and local sequence specific degradation of Poly(3-
wastewater using Hydrogenophaga palleronii. Bioresour. Technol. 215, 155–162.
hydroxyvalerate-co-4-hydroxybutyrate) in Hydrogenophaga pseudoflava.
Venkateswar Reddy, M., Yajima, Y., Mawatari, Y., Hoshino, T., Chang, Y.C., 2015a.
Biomacromolecules 4, 38–45.
Degradation and conversion of toxic compounds into useful bioplastics by
Dietrich, D., Illman, B., Crooks, C., 2013. Differential sensitivity of
Cupriavidus sp. CY-1: relative expression of PhaC gene under phenol and
polyhydroxyalkanoates producing bacteria to fermentation inhibitors and
nitrogen stress. Green Chem. 17, 4560–4569.
comparison of polyhydroxybutyrate production from Burkholderia cepacia and
Venkateswar Reddy, M., Mawatari, Y., Yuka, Y., Seki, C., Hoshino, T., Chang, Y.C.,
Pseudomonas pseudoflava. BMC Res. Notes 6, 219–222.
2015b. Poly-3-hydroxybutyrate (PHB) production from alkylphenols, mono and
Fradinho, J.C., Oehmen, A., Reis, M.A.M., 2014. Photosynthetic mixed culture
poly-aromatic hydrocarbons using Bacillus sp. CYR1: a new strategy for wealth
polyhydroxyalkanoate (PHA) production from individual and mixed volatile
from waste. Bioresour. Technol. 192, 711–717.
fatty acids (VFAs): substrate preferences and co-substrate uptake. J. Biotechnol.
Venkateswar Reddy, M., Venkata Mohan, S., 2012. Influence of aerobic and anoxic
185, 19–27.
microenvironments on polyhydroxyalkanoates (PHA) production from food
Jiang, Y., Hebly, M., Kleerebezem, R., Muyzer, G., van Loosdrecht, M.C.M., 2011.
waste and acidogenic effluents using aerobic consortia. Bioresour. Technol. 103,
Metabolic modeling of mixed substrate uptake for polyhydroxyalkanoate (PHA)
313–321.
production. Water Res. 45, 1309–1321.