Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Biosynthesis and characterization of

polyhydroxyalkanoates produced by an extreme halophilic
bacterium, Halomonas nitroreducens, isolated from
hypersaline ponds
J.M. Cervantes-Uc1, J. Catzin2, I. Vargas2, W. Herrera-Kao1, F. Moguel3, E. Ramirez4,

n-Arriaga4 and G. Lizama-Uc2
S. Rinco
1
2
3
4

n Cient
fica de Yucat
an, A.C., Unidad de Materiales, Me

rida, Yucata

n, Me

xico
Centro de Investigacio

gico de M
erida, Unidad de Posgrado e Investigacio

n Av. Tecnolo

gico, M

Instituto Tecnolo
erida, Yucat

an, M

exico

gico Superior del Sur del Estado de Yucat
an, Carretera Muna-Felipe Carrillo Puerto, Oxkutzcab, Yucat

Instituto Tecnolo
an, M

exico

noma de Campeche, Facultad de Ciencias Qu
mico Biolo

gicas, Av. Agust
n Melgar S/N entre Calle 20 y Juan de la Barrera.
Universidad Auto
Col. Buenavista, San Francisco de Campeche, Campeche, M

exico

Keywords
DSC, FTIR, Halomonas nitroreducens,
halophilic, polyhydroxyalkanoates.
Correspondence

gico de
Gabriel Lizama, Instituto Tecnolo

n
M
erida, Unidad de Posgrado e Investigacio

gico km. 4.5 S/N C.P. 97118,
Av. Tecnolo
M
erida, Yucat

an, M

exico.
E-mail: lizama73@hotmail.com
2014/0052: received 9 January 2014, revised
17 July 2014 and accepted 17 July 2014
doi:10.1111/jam.12605

Abstract
Aims: Morphological, biochemical and genotypic characterization of a
halophilic bacterium isolated from hypersaline ponds located at Las Coloradas
(R
o Lagartos, Yucat
an, Mexico). Characterization of polymer produced by this
strain was also performed.
Methods and Results: Twenty strains were isolated from water samples of salt
ponds and selected based on both morphological features and their PHA
storage capacity, which were determined by SEM and staining methods with
Nile red and Nile blue, respectively; strains were also analysed by the
fluorescence imaging technique. Among them, JCCOL25.8 strain showed the
highest production of PHAs reason why phenotypic and genotypic
characterization was performed; this strain was identified as Halomonas
nitroreducens. Polymer produced by this strain was characterized by FTIR,
DSC, GPC and EDX spectroscopy. Results indicated that the biosynthesized
polymer was polyhydroxybutyrate (PHB) which had a melting peak at 170C
and a crystallinity percentage of about 36%.
Conclusions: Based on phenotypic and genotypic aspects, JCCOL25.8 strain
was identified as H. nitroreducens and it was capable to accumulate PHB.
Significance and Impact of the study: To our knowledge, there is only one
study published on the biosynthesis of PHAs by H. nitroreducens strains,
although the characterization of the obtained polymer was not reported.

Introduction
In recent years, there has been considerable interest in the
development and production of biodegradable plastics as a
response to problems associated with plastic waste and its
effect on the environment. Polyhydroxyalkanoates (PHAs)
are linear polyesters that accumulate as inclusions in a
wide variety of bacteria and their composition is governed
by two main factors: the bacterial strain and the carbon
source being used to grow the bacteria (Santhanam and
Sasidharan 2010). In most bacteria, PHAs are synthesized
Journal of Applied Microbiology 2014 The Society for Applied Microbiology

and intracellularly accumulated under unfavourable

growth conditions such as limitation of some essential
nutrients, for example nitrogen, phosphorus, magnesium,
sulphur and/or oxygen and the presence of an excess carbon source (Byrom 1990; Doi et al. 1990; Lee 1996). The
interest in these polymers has been due to its unique characteristic of being biodegradable thermoplastics that can
be produced from renewable carbon sources and has properties similar to those of petroleum derived plastics such
as polyethylene and polypropylene (Brandl et al. 1990;
Reddy et al. 2003; Quillaguam
an et al. 2006). With these
1

PHAs produced by Halomonas nitroreducens strain

features, PHAs have a potentially large market for applications ranging from packing films and manufacture of bottles to artefacts used in medicine such as heart valves,
sutures, bone implants, tissue engineering and drug delivery systems among others (Oliveira et al. 2004; Quillaguam
an et al. 2010; Rodr
guez-Contreras et al. 2013a).
In addition to well known PHAs producer microorganisms such as Cupriavidus necator, Alcaligenes latus,
Pseudomonas and recombinant Escherichia coli, PHA accumulation has also been reported in some halophilic microorganisms (Quillaguam
an et al. 2010). In this regard, it
has been reported two different groups of halophilic
micro-organisms based on their salt requirements: extreme
halophiles which grow in media containing 1530% w/v
NaCl and moderate halophiles, able to grow at NaCl concentrations of 315% w/v (Quillaguam
an et al. 2006).
Halomonas is the type genus of Halomonadaceae family, and it contains more than 46 species of halophilic
bacteria; these micro-organisms grow over the range of
525% w/v NaCl (Mata et al. 2002; Gonz
alez-Domenech
et al. 2008). Studies on the phenotypic features of some
strains belonging to the genre Halomonas have revealed
positive PHB accumulation tests for several species (Quillaguam
an et al. 2006). Among them, Halomonas nitroreducens sp. nov. 11ST, isolated from a solar saltern at
Cahuil (Pichilemu, Chile), is a recent interesting case as
it produces exopolysaccharide and polyhydroxyalkanoates
(PHAs) according to Gonz
alez-Domenech et al. (2008).
However, despite the promising capabilities of this species
to produce PHAs (generally supported by stained and
fluorescence tests), to our knowledge there are no
detailed studies focused on biosynthesis and characterization of polyhydroxyalkanoates produced by H. nitroreducens; this last aspect is very important because it has been
reported more than 90 different HAs as constituents of
PHAs (Xu et al. 2002).
In this work, we report the morphological and biochemical characterization of a H. nitroreducens strain, isolated from hypersaline pans located at Las Coloradas (R
o
Lagartos, Yucat
an, M
exico), which is capable to produce
polyhydroxyalkanoates. In addition, polymer produced by
strain aforementioned was characterized by Fourier transform infrared spectroscopy, differential scanning calorimetry, gel permeation chromatography and elemental
analysis by energy-dispersive X-ray spectroscopy.
Materials and methods
Sampling, isolation and maintenance of halophilic
bacteria
Saline water samples, approx. 20 cm from the surface,
were collected from solar salt ponds at Las Coloradas.
2

J.M. Cervantes-Uc et al.

Micro-organisms from solar salt ponds were grown in

EGM medium consisting of 1% (w/v) glucose as substrate, 0
5% (w/v) yeast extract and sea water (25% v/v).
These samples were diluted (104, 105 and 106), and
100 ll of each one was surface spread plated on EGM
solid media. The plates were incubated at 37C for 72 h
and analysed to determine the total viable bacterial load.
The colonies from the plates were picked and surfacestreaked several times until pure culture was obtained.
The isolates were labelled as halophile series (JCCOL) and
were thereafter maintained on EGM agar plates at 4C.
Screening of halophilic isolates for PHA production
The ability of the halophilic isolates to accumulate PHA
was tested using a modified EGM medium containing
1% (w/v) glucose as substrate, 0
25% (w/v) yeast extract
and sea water (25% v/v); that is, PHAs production was
carried out under nitrogen limitation conditions.
Nile red staining
Accumulation of PHA was monitored after 48 h by
flooding the culture grown plates with Nile red. Then,
the isolates were spot-inoculated and incubated at temperature (37C). The stain was decanted and plates were
exposed to UV light at 240 nm using a Bio-Rad Gel Doc
EZ system (Laboratorio de Biotecnologia Molecular).
Nile blue staining
Nile blue staining was done as described by Ostle and
Holt (1982) from 48-h culture grown. A 1% aqueous
solution of Nile blue A was prepared and filtered before
use. Heat-fixed smears of bacterial cells were stained with
the Nile blue A solution at 55C for 10 min in a coplin
staining jar. After being stained, the slides were washed
with tap water to remove excess stain and with 8% aqueous acetic acid for 1 min. The stained smear was washed
and blotted dry with bibulous paper, remoistened with
tap water, and covered with a No. 1 glass cover slip.
Identification of halophilic isolates
Morphological, biochemical and genotypic characterization
Morphological characterization was performed by scanning electron microscopy using a JEOL microscope model
JSM 5900-LV (GenTech Scientific, Arcade, NY, USA) at
an accelerating voltage of 20 keV. Bacteria samples were
fixed using a solution of 2
5% glutaraldehyde in sodium
phosphate buffer. Fixation was followed by dehydration
process by passing the samples through a series of increasing ethanol concentration (30, 50, 70, 85, 90 and 100%).
Subsequently, the samples were critical point dried from
carbon dioxide sputter coated with a layer of gold.
Journal of Applied Microbiology 2014 The Society for Applied Microbiology

J.M. Cervantes-Uc et al.

Biochemical characterization was conducted using a

VITEK 2 system.
Genomic DNA was isolated by the use of Wizard
Genomic DNA Purification kit (Promega Biosciences,
LLC., San Luis Obispo, CA) 16S rRNA gene fragment
was amplified using fDl 50 -ccgaattcgtcgacaacAGAGTTTG
ATCCTGGCTCAG-30 and rDl 50 -cccgggatccaagcttAAGGAGGTGATCCAGCC-30 or rP2 50 rP2 cccgggatccaagc
ttACGGCTACCTTGTTACGACTT-30 primers under the
following PCR conditions: initial denaturation at 94C
for 5 min, followed by 35 cycles of denaturation (94C
for 2 min), annealing (42C for 30 s) and extension
(72C for 4 min) with a final extension (72C for
10 min). Amplified product was subjected to electrophoresis on a 1
5% agarose gel and was found to be approx.
1
3 kb in size. The purified product was sequenced unidirectionally using an automated DNA sequencer (Applied
Biosystems, IBT, Cuernavaca, M
exico).
Growth kinetics and PHA accumulation
Growth kinetics and PHA content were determined as
follows; Starter culture was prepared by growing the culture in 50 ml EGM broth contained in 150-ml Erlenmeyer flask. Two percent (2%) of this starter culture was
used to inoculate 100 ml of EGM medium contained in
250-ml Erlenmeyer flasks; additional experiments were
performed in medium without 0
25% (w/v) yeast extract,
but no cell growth was observed. Cell growth was monitored by measuring the optical density (OD) at 580 nm,
and the PHA content was determined from the culture
broth after 96 and 144 h. PHB quantification was performed by the method of Law and Slepecky (1961).
Briefly, 3 ml of bacterial culture grown in nitrogen-free
medium was transferred to glass centrifuge tubes and
centrifuged at 8609 g for 10 min. The cell pellet was suspended in 1 ml of standard alkaline hypochlorite solution
(6% w/v) and incubated at 37C for 12 h to complete
digestion of cell components except PHAs. The mixture
was centrifuged to collect the polymeric sediment and the
supernatant was discarded. The sediment was washed
twice with distilled water and centrifuged at 8609 g for
1 min. Last procedure was repeated using a mixture of
acetone: methanol (1 : 1). The polymer granule was dissolved with boiling chloroform, filtered, precipitated with
methanol, centrifuged and dried at 60C in vacuum.
Finally, the granules were mixed with 10 ml of concentrated H2SO4 and the tube was capped and heated
for 10 min at 100C in a water bath. The concentration
of PHA was determined from an established standard
graph in which the absorbance was plotted against the
concentration of crotonic acid as standard. PHA granules
extracted with the boiling chloroform method were hyJournal of Applied Microbiology 2014 The Society for Applied Microbiology

PHAs produced by Halomonas nitroreducens strain

drolysed using concentrated sulphuric acid for 1 h to

obtain crotonic acid, which was quantified by measuring
absorbance at 235 nm using an UV-visible spectrophotometer (Santhanam and Sasidharan 2010).
Polymer extraction
Cells were harvested by centrifugation, suspended in alkaline hypochlorite solution and incubated at 37C for 1
2 h. Then, the mixture was centrifuged to collect the
polymeric sediment and the supernatant was discarded.
The sediment was washed with distilled water and centrifuged; this procedure was repeated using a mixture of
acetone: methanol (1 : 1). The polymer granule was dissolved with boiling chloroform, filtered, precipitated with
methanol, centrifuged and dried at 60C in vacuum.
Characterization of obtained polymer
Infrared spectra of the polymer were obtained with a Nicolet FTIR Prot
eg
e 460, using attenuated total reflectance
(ATR) mode, in the spectral range from 4000 to 650 cm1,
averaging 100 scans with a resolution of 4 cm1.
Elemental composition of the polymer was studied by
means of energy-dispersive X-ray microanalysis (EDX)
using an Oxford Inca Energy 200 energy dispersive spectrometry (EDS) system coupled to scanning electron
microscope (SEM) JEOL JSM 6360 LV.
DSC analysis was performed to study the melting
behaviour of synthesized polymer. Samples were heated
twice from 45 to 200C, at a heating rate of 10C min1.
on a DSC-7 Perkin Elmer. The melting temperature (Tm)
and the melting enthalpy (DHm) were determined from
the endothermic peak on the DSC curve during the second scan. The crystallinity percentage (XC) of polymeric
sample was calculated using the following expression:
Xc

DHm
 100
DH0

where DHm is the melting enthalpy of PHB sample and

DH0 is that corresponding to the theoretical melting
enthalpy of 100% crystalline PHB (146 J g1) (Barham
et al. 1984; Gunaratne et al. 2004).
Molecular weight was determined by gel permeation
chromatography (GPC) using an 1100 GPC-SEC system
(Agilent Technologies, Inc., Santa Clara, CA) equipped
with coupled columns (Zorbax PSM 60S and 1000S) and a
refractive index detector. Measurements were carried out at
50C using HPLC grade dimethylformamide (DMF) as solvent and 1 ml min1 flow rate. The molecular weight was
determined from the retention time using a calibration
curve derived from monodisperse standard polystyrene
(PS) obtained from Polymer Laboratories.
3

PHAs produced by Halomonas nitroreducens strain

Results
Screening of PHA producing strains
A large number of colonies were obtained from the water
samples plated on EGM medium. After screening, twenty
halophilic isolates were picked and their purified clones
were obtained by repeated subculturing. Based on the fluorescence levels exhibited during Nile red and Nile blue
staining tests, eight isolated (JCCOL50.3, JCCOL50.4,
JCCOL25.2,
JCCOL25.4,
JCCOL25.5,
JCCOL25.6,
JCCOL25.8 and JCCOL25.9) were selected as PHA producer strains; these cultures were obtained under nitrogen
limitation conditions. Among them, JCCOL25.8 strain
exhibited the higher fluorescence (see Fig. 1a,b); therefore, additional experiments were carried out using this
strain. For purposes of comparison, results obtained for
one strain (JCCOL25.5) exhibiting less fluorescence than
JCCOL25.8 during the staining tests using Nile red and
Nile blue are also reported (see Fig. 1c,d, respectively).
Characterization of promising isolates
Morphological, biochemical and genotypic characterization
All strains were gram-negative and their morphological
features are displayed in Fig. 2. As seen, cells exhibited a

(a)

(b)

(c)

(d)

J.M. Cervantes-Uc et al.

rod-shaped morphology and the size of the cells in EGM

media was 0
5 9 3 lm approximately at 24 h.
The biochemical profile obtained by the Vitek 2 system
revealed that JCCOL25.8 was positive for Ala-Phe-ProArylamidase, and urease (see Table 1). Also, this strain
produced acid from D-maltose, sucrose, D-glucose, Dcellobiose, glycerol, coumarate and sodium acetate.
The 16S rRNA sequence obtained for JCCOL25.8 strain
was analysed to relate taxa using BLAST search tool; multiple sequence alignment was performed using MUSCLE.
MEGA 5.0 was used for the construction of the phylogenetic tree by neighbour-joining method with bootstrapping for 1000 replicates and tree displayed for 100
(Tamura et al. 2011). Analysis revealed that the
JCCOL25.8 strain was very similar (99%) to H. nitroreducens (see Fig. 3). The sequence obtained for this strain
was deposited in EMBL Nucleotide Sequence Database
with accession number HG326677.
Polymer accumulation in the halophilic strains isolated
PHA accumulation in the eight selected strains
(JCCOL50.3, JCCOL50.4, JCCOL25.2, JCCOL25.4,
JCCOL25.5, JCCOL25.6, JCCOL25.8 and JCCOL25.9)
was evaluated at 96 and 144 h, in EGM medium; in additional experiments carried out using culture medium

Figure 1 Fluorescence exhibited by JCCOL

25.8 (a and b) and JCCOL25.5 (c and d)
strains; colonies stained with Nile red (a and
c), and cells stained with Nile blue (b and d).

Journal of Applied Microbiology 2014 The Society for Applied Microbiology

J.M. Cervantes-Uc et al.

PHAs produced by Halomonas nitroreducens strain

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

Figure 2 Micrographs of Scanning Electron Microscopy demonstrating the morphology of isolated strains. (a) JCCOL50.3, (b) JCCOL50.4, (c)
JCCOL25.2, (d) JCCOL25.8, (e) JCCOL25.4, (f) JCCOL25.5, (g) JCCOL25.6 and (h) JCCOL25.9.

Journal of Applied Microbiology 2014 The Society for Applied Microbiology

PHAs produced by Halomonas nitroreducens strain

J.M. Cervantes-Uc et al.

Table 1 Biochemical characterization of halophilic bacterial strain

JCOL25.8
Biochemical test

Culture

Ala-Phe-Pro-Arylamidase
H2S production
Beta-glucosidase
L-proline arylamidase
Saccharose/sucrose
L-lactate alkalnisation
Glyicine arylamidase
2,4-diamino-6,7-diisopropylpteridine
Adonitol
Beta-N-acetyl-glucosaminidase
D-Maltose
Lipase
D-agatose
Alpha-glucosidase
Ornithine decarboxylase
Glu-gly-arg-arylamidase
L-pyrrolydonyl-arylamidase
Glutamyl arylamidase pNa
D-manitol
Palatinose
D-trehalose
Succinate alkalisation
Lysine decarboxylase
L-malate assimilation
L-arabitol
D-Glucose
D-mannose
Glicerol Assimilation
Tyrosine arylamidase
Citrate-sodium
Beta-N-acetyl-galactosaminidase
L-histidine assimilation
ELLMAN
D-Cellobiose
Gamma-glutamyl-transferase
Beta-xylosidase
Urease
Malonate
Alpha-galactosidase
Coumarate
L-lactate assimilation
Beta-galactosidase
Fermentation/glucose
Beta-alanine atylamidase pNa
D-sorbitol
5-Keto-D-gluconate
Phosphatase
Beta-glucoronidase
Sodium acetate

+



+





+














+

+





+


+


+








+

(+) positive; () negative.

without yeast extract, there was no bacterial growth (data

not shown). This period of time was selected as although
the accumulation of polymer was detectable after 48 h of
6

growth (stationary phase), the maximum accumulation

was reached between 96 and 144 h. From Fig. 4, it is
clear that the highest PHA production was obtained by
JCCOL25.8 strain, which increased the polymer production from 0
06 mg ml1 at 96 h to 0
08 mg ml1 at
144 h. Although other strains also showed an increase in
the PHA production from 96 to 144 h (JCCOL50.4 and
JCCOL25.2), there were other that exhibited the opposite
behaviour, that is showed a decrease in the production
polymer when time was increased (JCCOL50.3 and
JCCOL25.5).
To establish the relationship between polymer production and the microbial growth, it was performed a growth
kinetic test using the JCCOL25.8 strain. Figure 5 shows the
growth kinetic for this strain in EGM broth. It can be seen
that the bacterial growth exhibited a clear logarithmic
phase, which started at the 4th hour and finished at the
40th hour; the log phase of bacterial growth was followed
by the stationary phase.
Characterization of the polymer
Figure 6a displays the FTIR spectrum of the polymer
produced by H. nitroreducens. As noted, this spectrum is
practically identical to that obtained from a commercial
polyhydroxybutyrate (PHB) sample (see Fig. 6b) acquired
from Goldfellow Co. and very similar to those reported
in the literature (Xiao and Jiao 2011; Karahaliloglu et al.
2013) for this polymer. Therefore, there is no doubt that
the polymer produced by this extreme halophilic bacterium is PHB.
In addition, differential scanning calorimetry (DSC)
revealed a melting peak (Tm) at 170C (see Fig. 7). This
value falls within the range reported for Tm values of
poly(3-hydroxybutyrate) polymer (Gunaratne et al. 2004;
Porter and Yu 2011; Xiao and Jiao 2011). The average
molecular weight obtained for synthesized polymer ranged from 1
36 to 7
04 9 105 g mol1 and had a molecular weight distribution that was multimodal.
Elemental composition of the polymer was determined
by energy-dispersive X-ray spectroscopy (EDX). As
expected, carbon and oxygen were the main elements
detected in the polymeric sample, although small quantities (<0
5% w/w) of chlorine and sodium were also
detected. The presence of the latter elements could be due
to sodium hypochlorite used during polymer extraction
and purification processes. Percentages of carbon and oxygen obtained during analysis (57
6 and 40
4% w/w, respectively) were close to the theoretical values of the PHB.
Some samples (discarded in this study) obtained by others
extraction and purification processes showed the presence
of the nitrogen element in the EDX microanalysis, which
indicates the existence of cell residues in the sample.
Journal of Applied Microbiology 2014 The Society for Applied Microbiology

J.M. Cervantes-Uc et al.

PHAs produced by Halomonas nitroreducens strain

Halomonas salina F8-11

Halomonas denitrificans
Halomonas halmophila ATCC 19717
Halomonas subglaciescola DSM 4683
Halomonas elongata DSM 2581 1 H 9 ATCC 33173
Halomonas sabkhae 5-3
Halomonas organivorans G-16.1 CCM 7142
Halomonas halocynthiae KMM 1376
Halomonas almeriensis M8
Halomonas elongata DSM 2581
Halomonas halophila CCM 3662
JCOL25.8
Halomonas nitroreducens 11S
Halomonas caseinilytica AJ261
Halomonas campaniensis
Halomonas eurihalina ATCC 49336
Halomonas shengliensis SL014B-85
Halomonas gudaonensis SL014B-69

02
Figure 3 Phylogenetic tree showing the position of polymer accumulating extreme halophilic strain JCOL25.8 based on the 16Sr RNA. Neighbour-Joining tree was constructed with MEGA 5.0 with bootstrap values for 1000 replicates and displayed for 100.

Discussion

009
PHA content (mg ml1)

008
007
006
005
004
003
002
001
9
L2
5.

5.
8

O
C

JC

L2
JC

JC

JC
C

L2

5.
6

.5
L2
5

5.
4

2
JC

L2

L2
5.

0.
4
C

L5
JC

O
C
JC

JC

L5

0.
3

Strain
Figure 4 Production of PHAs in EGM medium for all selected strains
( , 96 h; , 144 h).

Journal of Applied Microbiology 2014 The Society for Applied Microbiology

The salt ponds located at Las Coloradas have an extension approximately of 80 Km and covering a surface area
of 12,850 hectares; their coordinates are 21300 11N,
87400 0W. These salt ponds show a concentration of
sodium chloride of the 33% during all the year.
Although a large number of colonies were obtained from
the water samples cultured on EGM medium, only eight
strains were able to accumulate PHAs, being the
JCCOL25.8 strain that exhibited the highest PHA production after 144 h. The growth curve for this strain showed a
clear logarithmic phase, which started at the 4th hour and
finished at the 40th hour, followed by the stationary phase.
Accumulation of polymer was detectable after 48 h of
growth but the maximum accumulation was reached
between 96 and 144 h depending of the strains.
JCCOL25.8, JCCOL50.4 and JCCOL25.2 strains showed
7

PHAs produced by Halomonas nitroreducens strain

J.M. Cervantes-Uc et al.

2500
2250

O.D. 580 nm

2000
1750
1500
1250
1000
0750
0500
0250
0000
20

80

40
60
Time (h)

160

1279
1229
1183
1132
1056
980

1380
1456

Absorbance (a.u.)

1723

Figure 5 Growth kinetic for JCCOL25.8 strain in EGM broth ( , optical density).

3000-2800

(a)

(b)
4000

3500

3000

2000

1500

1000

Wavenumber (cm1)

higher PHA content at 144 h than 96 h, but JCCOL50.3,

JCCO25.5 and JCCOL25.9 strains exhibited the opposite
behaviour. This decreasing could be related with either
the start of the death phase in the growth curve or by the
consumption of PHA by bacterial cells (Benoit et al.
1990). The reduction in PHA production has been also
attributed to lack micronutrients as well as increasing of
metabolites that might have negative effects on the PHA
production (Flora et al. 2010).
JCCOL25.8 strain was identified as a H. nitroreducens,
which growth in salt ponds at the southeast of Mexico
(Las Coloradas), where the salinity reaches up 30% during all year. This species has only been reported previously by Gonz
alez-Domenech et al. (2008) but isolated
from a solar saltern in Cahuil, a region next to Pichilemu
(Chile).
The halophilic bacteria are micro-organisms which
requires an amount of salt for their correctly growth.
According to recent criteria, the halophilic micro-organisms grow optimally at salt concentration of 5% (w/v)
(or 50 g l1, equivalent to 0
85 mol dm3 NaCl) or
higher, and tolerate 10% (w/v) (or 100 g l1, equivalent
to 1
7 mol dm3 NaCl) at least (Oren 2008). In this
work, it was observed that H. nitroreducens was able to
grow in NaCl concentrations up to 24
5% (w/v) but
showed an optimum growth at 8
25% NaCl; therefore,
this strain should be considered as an extreme halophilic
micro-organism.
It was also observed that the H. nitroreducens strain
was able to accumulate 33% of PHB; this value falls
within the range reported for genre Bacillus spp. which
accumulates around 3046 of PHB (Gouda et al. 2001;
Shamala et al. 2003; Vazquez et al. 2003; Valappil et al.
2008; Rodr
guez-Contreras et al. 2013b).

Figure 6 FTIR spectra of (a) synthesized polymer and (b) PHB from
Goldfellow.

1723
07
170C

05

Absorbance (a.u.)

Heat flow (mw)

06
Hm = 522 J/g

04
03

1741

1686
(a)

02
(b)
01
00
40

1800 1780 1760 1740 1720 1700 1680 1660 1640 1620 1600
60

80

100

120

140

160

180

200

Wave number (cm1)

Temperature (C)
Figure 7 DSC thermogram of the synthesized polymer.

Figure 8 Deconvolution analysis of carbonyl band of (a) synthesized

polymer and (b) PHB from Goldfellow.

Journal of Applied Microbiology 2014 The Society for Applied Microbiology

J.M. Cervantes-Uc et al.

Characterization of the polymer obtained from this

strain is reported by first time. In this regard, FTIR spectra of PHA synthesized showed bands in the range of
30002800 cm1, which are associated to C-H stretching
vibrations from methyl, methylene and methyne groups.
Bands at 1456 and 1380 cm1, attributed to the asymmetric and symmetric deformation of the methyl groups,
respectively, were also detected (Furukawa et al. 2005).
FTIR spectra also exhibited an intense band at 1723 cm1,
related to carbonyl stretching of an ester group, typical for
this biodegradable thermoplastic polyester.
On closer inspection of the carbonyl band (see Fig. 8),
it can be seen that this band is composed of two overlapped peaks which are located at 1723 and 1741 cm1
(these values were obtained from a deconvolution analysis as shown in Fig. 8a), being the former more intense
than second one. The first band has been attributed to
the C=O stretching mode of the crystalline phase whereas
that situated at 1741 cm1 has been related to the corresponding C=O stretching mode of the amorphous phase
(Xu et al. 2002; Padermshoke et al. 2005; Sato et al.
2005). A small peak localized at 1686 cm1 was also
observed in Fig. 7 and is typical of the crystalline state.
Sato et al. (2005) attributed this band to the crystal
defect that is caused by the interaction of an OH endgroup and a C=O group of PHB.
The presence of bands at 1279, 1229 and 1183 cm1 in
the FTIR spectra of the PHB (see Fig. 6) correspond to
the asymmetric stretching vibration of the C-O-C backbone in the amorphous phase, the asymmetric stretching
of the C-C-O bond in the crystalline phase due to the
formation of helical chains and the methylene wagging of
the C-C-O backbone in the crystalline phase, respectively
(Porter and Yu 2011).
Purity of the obtained polymer was confirmed by FTIR
as the PHB spectrum obtained from H. nitroreducens
strain was very similar to that recorded for a commercial
polymer sample which had a purity approx. 99
9%. In
spite of this, it should be mentioned that purity could be
compromised if extraction procedure is not adequately
performed.
It was also noted that the endothermic event exhibited
in the DSC thermogram absorbed 52
2 J g1 during the
second heating; therefore, it was possible to estimate that
the crystallinity percentage of the PHB produced by H. nitroreducens is about 36%. This value is similar to those
reported by Xu et al. 2002 and Porter and Yu 2011.
A wide range of molecular weights has been reported
for PHAs obtained from different micro-organisms (104
106 g mol1) (Ashby et al. 1999 and Bengtsson et al.
2010). In this sense, the molecular weights obtained in
this work were similar to the reported values in literature.
However, unlike previous reports related to bacterial
Journal of Applied Microbiology 2014 The Society for Applied Microbiology

PHAs produced by Halomonas nitroreducens strain

PHB, the polymer obtained from H. nitroreducens had a

molecular weight distribution multimodal.
Production of PHA0 s from extreme halophile bacteria is
very interesting as at high concentration of salts, the
growth of nonhalophilic micro-organisms is prevented,
hence allowing a process without strict sterile conditions
and reducing the inherent costs; for example, the costs for
energy required for sterilizing the equipment for fermentation and culture media can be avoided. A further advantage of PHA production by extreme halophilic bacteria is
the ease of recovery of the polymer by hypo-osmotic shock
of the cells using a treatment with salt-deficient water,
hence reducing the downstream processing costs that
otherwise can account up to 40% of the total production
costs (Quillaguam
an et al. 2010).
Acknowledgements
The authors thank MSc. Felipe Barredo and Q.I. Santiago
Duarte for technical support during the SEM analysis;
authors also wish to thank CONACYT scholarship for Julio Catz
n.
Conflict of Interest
No conflict of interest declared.
References
Ashby, R.D., Shi, F. and Gross, R.A. (1999) A tunable switch
to regulate the synthesis of low and high molecular weight
microbial polyesters. Biotechnol Bioeng 62, 106113.
Barham, P.J., Keller, A., Otun, E.L. and Holmes, P.A. (1984)
Crystallization and morphology of a bacterial thermoplastic:
poly-3-hydroxybutyrate. J Mater Sci 19, 27812794.
Bengtsson, S., Pisco, A.R., Johansson, P., Lemos, P.C. and
Reis, M.A.M. (2010) Molecular weight and thermal
properties of polyhydroxyalkanoates produced from
fermented sugar molasses by open mixed cultures. J
Biotech 147, 172179.
Benoit, T.G., Wilson, G.R. and Baugh, C.L. (1990)
Fermentation during growth and sporulation of Bacillus
thuringiensis HD-1. Lett Appl Microbiol 10, 1518.
Brandl, H., Gross, R.A., Lenz, R.W. and Fuller, R.C. (1990) Plastics
from bacteria and for bacteria: poly(beta-hydroxyalkanoates)
as natural, biocompatible, and biodegradable polyesters.
Adv Biochem Eng Biotechnol 41, 7793.
Byrom, D. (1990) Industrial production of copolymer from
Alcaligenes eutrophus. In Novel Biodegradable Microbial
Polymers ed Dawes, E.A. pp. 113117. Dordrecht/Boston/
London: Kluwer Academic Publishers.
Doi, Y., Segawa, A., Nakamura, S. and Kunioka, M. (1990)
Production of biodegradable copolyesters by Alcaligenes

PHAs produced by Halomonas nitroreducens strain

eutrophus. In Novel Biodegradable Microbial Polymers ed.

Dawes, E.A. pp. 3748. Dordrecht/Boston/London: Kluwer
Academic Publ.
Flora, G.D., Bhatt, K. and Tuteja, U. (2010) Optimization of
culture conditions for poly b- hydroxybutyrate production
from isolated Bacillus species. J Cell Tissue Res 10, 22352242.
Furukawa, T., Sato, H., Murakami, R., Zhang, J., Duan, Y.-X.,
Noda, I., Ochiai, S. and Ozaki, Y. (2005) Structure,
dispersibility, and crystallinity of poly(hydroxybutyrate)/
poly(L-lactic acid) blends studied by FT-IR
microspectroscopy and differential scanning calorimetry.
Macromolecules 38, 64456454.
Gonz
alez-Domenech, C.M., B
ejar, V., Mart
nez-Checa, F. and
Quesada, E. (2008) Halomonas nitroreducens sp. nov., a
novel nitrate- and nitrite- reducing species. Int J Syst Evol
Micr 58, 872876.
Gouda, M.K., Swellam, A.E. and 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. Microbiol Res 156, 201207.
Gunaratne, L.M.W.K., Shanks, R.A. and Amarasinghe, G. (2004)
Thermal history effects on crystallization and melting of
poly(3-hydroxybutyrate). Thermochim Acta 423, 127135.
Karahaliloglu, Z., Demirbilek, M., Sam, M., Erol-Demirbilek,
M., Saglam, N. and Baki Denkbas, E. (2013) Plasma
polymerization-modified bacterial polyhydroxybutyrate
nanofibrillar scaffolds. J Appl Polym Sci 128, 19041912.
Law, J.H. and Slepecky, R.A. (1961) Assay of poly-bhydroxybutyric acid. J Bacteriol 82, 3336.
Lee, S.Y. (1996) Bacterial polyhydroxyalkanoates. Biotechnol
Bioeng 49, 114.
Mata, J.A., Mart
nez-C
anovas, J., Quesada, E. and B
ejar, V. (2002)
A detailed phenotypic characterization of the type strains of
Halomonas species. Syst Appl Microbiol 25, 360375.
Oliveira, F.C., Freire, D.M.G. and Castilho, L.R. (2004)
Production of poly (3-hydroxybutyrate) by solid state
fermentation with Ralstonia eutropha. Biotechnol Lett 26,
18511855.
Oren, A. (2008) Microbial life at high salt concentrations:
phylogenetic and metabolic diversity. Saline Systems 4, 2.
Ostle, A.G. and Holt, J.G. (1982) Nile blue A as a fluorescent
stain for poly-b-hydroxybutyrate. Appl Environ Microbiol
44, 238241.
Padermshoke, A., Katsumoto, Y., Sato, H., Ekgasit, S., Noda, I.
and Ozaki, Y. (2005) Melting behavior of poly(3hydroxybutyrate) investigated by two-dimensional infrared
correlation spectroscopy. Spectrochim Acta A Mol Biomol
Spectrosc 61, 541550.
Porter, M. and Yu, J. (2011) Monitoring the in situ crystallization
of native biopolyester granules in Ralstonia eutropha via
infrared spectroscopy. J Microbiol Meth 87, 4955.
Quillaguam
an, J., Delgado, O., Mattiasson, B. and Hatti-Kaul,
R. (2006) Poly(b-hydroxybutyrate) production by a

10

J.M. Cervantes-Uc et al.

moderate halophile Halomonas boliviensis LC1. Enzyme

Microb Technol 38, 148154.
Quillaguam
an, J., Guzm
an, H., Van-Thuoc, D. and Hatti-Kaul,
R. (2010) Synthesis and production of
polyhydroxyalkanoates by halophiles: current potential and
future prospects. Appl Microbiol Biotechnol 85, 16871696.
Reddy, C.S.K., Ghai, R., Rashmi, X. and Kalia, V. (2003)
Polyhydroxyalkanoates: an overview. Bioresour Technol 87,
137146.
Rodr
guez-Contreras, A., Canal, C., Calafell-Monfort, M.,
Ginebra, M.P., Julio-Moran, G. and Marqu
es-Calvo, M.S.
(2013a) Methods for the preparation of doxycyclineloaded phb micro- and nano-spheres. Eur Polym J 49,
35013511.
Rodr
guez-Contreras, A., Koller, M., Miranda-de Sousa Diaz,
M., Calafell, M., Braunegg, G. and Marqu
es-Calvo, M.S.
(2013b) High production of poly(3-hydroxybutyrate) from
a wild Bacillus megaterium Bolivian strain. J App Microbiol
114, 13781387.
Santhanam, A. and Sasidharan, S. (2010) Microbial production
of polyhydroxyalkanotes) from Alcaligens spp. and
Pseudomonas oleovorans using different carbon sources. Afr
J Biotechnol 9, 31443150.
Sato, H., Dybal, J., Murakami, R., Noda, I. and Ozaki, Y.
(2005) Infrared and Raman spectroscopy and quantum
chemistry calculation studies of C-H. . .O hydrogen
bondings and thermal behavior of biodegradable
polyhydroxyalkanoate. J Mol Struct 744747, 3546.
Shamala, T.R., Chandrashekar, A., Vijayendra, S.V.N. and
Kshama, L. (2003) Identification of polyhydroxyalkanoate
(PHA)-producing Bacillus spp. using the polymerase chain
reaction (PCR). J Appl Microbiol 94, 369374.
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M.
and Kumar, S. (2011) MEGA 5: molecular evolutionary
genetics analysis using maximum parsimony methods. Mol
Biol Evol 28, 27312739.
Valappil, S.P., Rai, R., Bucke, C. and Roy, I. (2008)
Polyhydroxyalkanoate biosynthesis in Bacillus cereus SPV
under varied limiting conditions and an insight into the
biosynthetic genes involved. J Appl Microbiol 104, 16241635.
Vazquez, G.J., Pettinari, M.J. and M
endez, B.S. (2003)
Evidence of an association between poly(3hydroxybutyrate) accumulation and
phosphotransbutyrylase expression in Bacillus megaterium.
Int Microbiol 6, 127129.
Xiao, N. and Jiao, N. (2011) Formation of
polyhydroxyalkanoate in aerobic anoxygenic phototrophic
bacteria and its relationship to carbon source and light
availability. Appl Environ Microbiol 77, 74457450.
Xu, J., Guo, B.-H., Yang, R., Wu, Q., Chen, G.-Q. and Zhang,
Z.-M. (2002) In situ FTIR study on melting and
crystallization of polyhydroxyalkanoates. Polymer 43,
68936899.

Journal of Applied Microbiology 2014 The Society for Applied Microbiology