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
n Cient
fica de Yucat
an, A.C., Unidad de Materiales, Me
Centro de Investigacio
rida, Yucata

n, Me
xico
2
gico de M
erida, Unidad de Posgrado e Investigacio
Instituto Tecnolo
n Av. Tecnolo
gico, M

erida, Yucat
an, M

exico
3
gico Superior del Sur del Estado de Yucat
an, Carretera Muna-Felipe Carrillo Puerto, Oxkutzcab, Yucat

Instituto Tecnolo an, M
exico
4 Universidad Auto
noma de Campeche, Facultad de Ciencias Qu
mico Biolo
gicas, Av. Agust
n Melgar S/N entre Calle 20 y Juan de la Barrera.
Col. Buenavista, San Francisco de Campeche, Campeche, M
exico

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

gico de
Gabriel Lizama, Instituto Tecnolo
M
erida, Unidad de Posgrado e Investigacio
n
Av. Tecnolo
gico km. 4.5 S/N C.P. 97118,
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.

and intracellularly accumulated under unfavourable

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
growth conditions such as limitation of some essential
nutrients, for example nitrogen, phosphorus, magnesium,
sulphur and/or oxygen and the presence of an excess car-
bon source (Byrom 1990; Doi et al. 1990; Lee 1996). The
interest in these polymers has been due to its unique char-
acteristic of being biodegradable thermoplastics that can
be produced from renewable carbon sources and has prop-
erties 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

Journal of Applied Microbiology 2014 The Society for Applied Microbiology 1

PHAs produced by Halomonas nitroreducens strain
features, PHAs have a potentially large market for applica-
tions ranging from packing films and manufacture of bot-
tles to artefacts used in medicine such as heart valves,
sutures, bone implants, tissue engineering and drug deliv-
ery systems among others (Oliveira et al. 2004; Quilla-
guam
an et al. 2010; Rodr
guez-Contreras et al. 2013a).
In addition to well known PHAs producer micro-
organisms such as Cupriavidus necator, Alcaligenes latus,
Pseudomonas and recombinant Escherichia coli, PHA accu-
mulation has also been reported in some halophilic micro-
organisms (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 con-
centrations of 315% w/v (Quillaguam
an et al. 2006).
Halomonas is the type genus of Halomonadaceae fam-
ily, 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 (Quil-
laguam
an et al. 2006). Among them, Halomonas nitrore-
ducens 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 characteriza-
tion of polyhydroxyalkanoates produced by H. nitroredu-
cens; 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 bio-
chemical characterization of a H. nitroreducens strain, iso-
lated 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 trans-
form infrared spectroscopy, differential scanning calorim-
etry, 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 Journal of Applied Microbiology 2014 The Society for Applied Microbiology
J.M. Cervantes-Uc et al.
Micro-organisms from solar salt ponds were grown in
EGM medium consisting of 1% (w/v) glucose as sub-
strate, 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 surface-
streaked 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 tem-
perature (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% aque-
ous 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 scan-
ning 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 increas-
ing 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.
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 -cccgggatccaagcttAAG-
GAGGTGATCCAGCC-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 electropho-
resis on a 1
5% agarose gel and was found to be approx.
1
3 kb in size. The purified product was sequenced unidi-
rectionally 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 cul-
ture in 50 ml EGM broth contained in 150-ml Erlen-
meyer 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 moni-
tored 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 per-
formed 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 sus-
pended 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 dis-
solved with boiling chloroform, filtered, precipitated with
methanol, centrifuged and dried at 60C in vacuum.
Finally, the granules were mixed with 10 ml of concen-
trated 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 hy-
Journal 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 spectropho-
tometer (Santhanam and Sasidharan 2010).
Polymer extraction
Cells were harvested by centrifugation, suspended in alka-
line 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 centri-
fuged; this procedure was repeated using a mixture of
acetone: methanol (1 : 1). The polymer granule was dis-
solved 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 Nico-
let 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 spec-
trometry (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 sec-
ond scan. The crystallinity percentage (XC) of polymeric
sample was calculated using the following expression:
DHm
Xc  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 sol-
vent 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 flu-
orescence 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 pro-
ducer strains; these cultures were obtained under nitrogen
limitation conditions. Among them, JCCOL25.8 strain
exhibited the higher fluorescence (see Fig. 1a,b); there-
fore, 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)
4 Journal of Applied Microbiology 2014 The Society for Applied Microbiology
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-Pro-
Arylamidase, and urease (see Table 1). Also, this strain
produced acid from D-maltose, sucrose, D-glucose, D-
cellobiose, glycerol, coumarate and sodium acetate.
The 16S rRNA sequence obtained for JCCOL25.8 strain
was analysed to relate taxa using BLAST search tool; mul-
tiple sequence alignment was performed using MUSCLE.
MEGA 5.0 was used for the construction of the phyloge-
netic tree by neighbour-joining method with bootstrap-
ping 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. nitroredu-
cens (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 addi-
tional 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).
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 5

PHAs produced by Halomonas nitroreducens strain J.M. Cervantes-Uc et al.

Table 1 Biochemical characterization of halophilic bacterial strain growth (stationary phase), the maximum accumulation
JCOL25.8 was reached between 96 and 144 h. From Fig. 4, it is
Biochemical test Culture clear that the highest PHA production was obtained by
JCCOL25.8 strain, which increased the polymer produc-
Ala-Phe-Pro-Arylamidase + tion from 0
06 mg ml1 at 96 h to 0
08 mg ml1 at
H2S production 
144 h. Although other strains also showed an increase in
Beta-glucosidase 
L-proline arylamidase 
the PHA production from 96 to 144 h (JCCOL50.4 and
Saccharose/sucrose + JCCOL25.2), there were other that exhibited the opposite
L-lactate alkalnisation  behaviour, that is showed a decrease in the production
Glyicine arylamidase  polymer when time was increased (JCCOL50.3 and
2,4-diamino-6,7-diisopropylpteridine  JCCOL25.5).
Adonitol  To establish the relationship between polymer produc-
Beta-N-acetyl-glucosaminidase 
tion and the microbial growth, it was performed a growth
D-Maltose +
Lipase 
kinetic test using the JCCOL25.8 strain. Figure 5 shows the
D-agatose  growth kinetic for this strain in EGM broth. It can be seen
Alpha-glucosidase  that the bacterial growth exhibited a clear logarithmic
Ornithine decarboxylase  phase, which started at the 4th hour and finished at the
Glu-gly-arg-arylamidase  40th hour; the log phase of bacterial growth was followed
L-pyrrolydonyl-arylamidase  by the stationary phase.
Glutamyl arylamidase pNa 
D-manitol 
Palatinose  Characterization of the polymer
D-trehalose 
Succinate alkalisation  Figure 6a displays the FTIR spectrum of the polymer
Lysine decarboxylase  produced by H. nitroreducens. As noted, this spectrum is
L-malate assimilation  practically identical to that obtained from a commercial
L-arabitol  polyhydroxybutyrate (PHB) sample (see Fig. 6b) acquired
D-Glucose +
from Goldfellow Co. and very similar to those reported
D-mannose 
Glicerol Assimilation +
in the literature (Xiao and Jiao 2011; Karahaliloglu et al.
Tyrosine arylamidase  2013) for this polymer. Therefore, there is no doubt that
Citrate-sodium  the polymer produced by this extreme halophilic bacte-
Beta-N-acetyl-galactosaminidase  rium is PHB.
L-histidine assimilation  In addition, differential scanning calorimetry (DSC)
ELLMAN  revealed a melting peak (Tm) at 170C (see Fig. 7). This
D-Cellobiose +
value falls within the range reported for Tm values of
Gamma-glutamyl-transferase 
Beta-xylosidase 
poly(3-hydroxybutyrate) polymer (Gunaratne et al. 2004;
Urease + Porter and Yu 2011; Xiao and Jiao 2011). The average
Malonate  molecular weight obtained for synthesized polymer ran-
Alpha-galactosidase  ged from 1
36 to 7
04 9 105 g mol1 and had a molecu-
Coumarate + lar weight distribution that was multimodal.
L-lactate assimilation  Elemental composition of the polymer was determined
Beta-galactosidase 
by energy-dispersive X-ray spectroscopy (EDX). As
Fermentation/glucose 
Beta-alanine atylamidase pNa 
expected, carbon and oxygen were the main elements
D-sorbitol  detected in the polymeric sample, although small quanti-
5-Keto-D-gluconate  ties (<0
5% w/w) of chlorine and sodium were also
Phosphatase  detected. The presence of the latter elements could be due
Beta-glucoronidase  to sodium hypochlorite used during polymer extraction
Sodium acetate + and purification processes. Percentages of carbon and oxy-
(+) positive; () negative. gen obtained during analysis (57
6 and 40
4% w/w, respec-
tively) were close to the theoretical values of the PHB.
Some samples (discarded in this study) obtained by others
without yeast extract, there was no bacterial growth (data extraction and purification processes showed the presence
not shown). This period of time was selected as although of the nitrogen element in the EDX microanalysis, which
the accumulation of polymer was detectable after 48 h of indicates the existence of cell residues in the sample.

6 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. Neigh-
bour-Joining tree was constructed with MEGA 5.0 with bootstrap values for 1000 replicates and displayed for 100.

009 Discussion
008 The salt ponds located at Las Coloradas have an exten-
PHA content (mg ml1)

007 sion approximately of 80 Km and covering a surface area

006 of 12,850 hectares; their coordinates are 21300 11N,
005 87400 0W. These salt ponds show a concentration of
004 sodium chloride of the 33% during all the year.
003
Although a large number of colonies were obtained from
the water samples cultured on EGM medium, only eight
002
strains were able to accumulate PHAs, being the
001
JCCOL25.8 strain that exhibited the highest PHA produc-
0
tion after 144 h. The growth curve for this strain showed a
3

.5

9
0.

0.

5.

5.

5.

5.

5.
5

clear logarithmic phase, which started at the 4th hour and

L5

L5

L2

L2

L2

L2

L2

L2
O

finished at the 40th hour, followed by the stationary phase.

C

C
JC

JC

JC

JC

JC

JC

JC

JC

Accumulation of polymer was detectable after 48 h of

Strain
growth but the maximum accumulation was reached
Figure 4 Production of PHAs in EGM medium for all selected strains between 96 and 144 h depending of the strains.
( , 96 h; , 144 h). JCCOL25.8, JCCOL50.4 and JCCOL25.2 strains showed

Journal of Applied Microbiology 2014 The Society for Applied Microbiology 7

PHAs produced by Halomonas nitroreducens strain J.M. Cervantes-Uc et al.

2500 higher PHA content at 144 h than 96 h, but JCCOL50.3,

2250 JCCO25.5 and JCCOL25.9 strains exhibited the opposite
2000 behaviour. This decreasing could be related with either
1750 the start of the death phase in the growth curve or by the
O.D. 580 nm

1500 consumption of PHA by bacterial cells (Benoit et al.

1250 1990). The reduction in PHA production has been also
1000 attributed to lack micronutrients as well as increasing of
0750 metabolites that might have negative effects on the PHA
0500
production (Flora et al. 2010).
0250
JCCOL25.8 strain was identified as a H. nitroreducens,
which growth in salt ponds at the southeast of Mexico
0000
0 20 40 60 80 160 (Las Coloradas), where the salinity reaches up 30% dur-
Time (h) ing all year. This species has only been reported previ-
ously by Gonz
alez-Domenech et al. (2008) but isolated
Figure 5 Growth kinetic for JCCOL25.8 strain in EGM broth ( , opti- from a solar saltern in Cahuil, a region next to Pichilemu
cal density).
(Chile).
The halophilic bacteria are micro-organisms which
requires an amount of salt for their correctly growth.
According to recent criteria, the halophilic micro-organ-
1723

isms 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
1279
Absorbance (a.u.)

to 1
7 mol dm3 NaCl) at least (Oren 2008). In this

1056
1132
1229

work, it was observed that H. nitroreducens was able to

1380

980

grow in NaCl concentrations up to 24
5% (w/v) but

1183
1456

3000-2800
showed an optimum growth at 8
25% NaCl; therefore,
(a)
this strain should be considered as an extreme halophilic
micro-organism.
It was also observed that the H. nitroreducens strain
(b) was able to accumulate 33% of PHB; this value falls
within the range reported for genre Bacillus spp. which
4000 3500 3000 2000 1500 1000 accumulates around 3046 of PHB (Gouda et al. 2001;
Wavenumber (cm1) 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
06
Absorbance (a.u.)

1741 1686
05
Heat flow (mw)

Hm = 522 J/g (a)

04

03

02
(b)
01
1800 1780 1760 1740 1720 1700 1680 1660 1640 1620 1600
00
40 60 80 100 120 140 160 180 200 Wave number (cm1)
Temperature (C)
Figure 8 Deconvolution analysis of carbonyl band of (a) synthesized
Figure 7 DSC thermogram of the synthesized polymer. polymer and (b) PHB from Goldfellow.

8 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 spec-
tra 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 asym-
metric 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 over-
lapped peaks which are located at 1723 and 1741 cm1
(these values were obtained from a deconvolution analy-
sis 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 corre-
sponding 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 end-
group 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 back-
bone 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. ni-
troreducens 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 fermenta-
tion and culture media can be avoided. A further advan-
tage 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 Ju-
lio Catz
n.
Conflict of Interest
No conflict of interest declared.
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Journal of Applied Microbiology 2014 The Society for Applied Microbiology