E. TEKİN, M. ATEŞ, Ö. KAHRAMAN
Turk J Biol
36 (2012) 303-312
© TÜBİTAK
doi:10.3906/biy-1102-16
Poly-3-hydroxybutyrate-producing extreme
halophilic archaeon: Haloferax sp. MA10 isolated
from Çamaltı Saltern, İzmir
Ebru TEKİN, Mustafa ATEŞ, Özge KAHRAMAN
Department of Biology, Basic and Industrial Microbiology Section, Ege University, Bornova, 35100 İzmir - TURKEY
Received: 24.02.2011
Abstract: İzmir Çamaltı Saltern is the biggest seawater-based saltern in Turkey. To date, it has not been investigated
extensively for the existence of poly-3-hydroxybutyrate (PHB)-producing species. In this study, 14 extremely halophilic
archaea were isolated, purified, and screened for PHB production. One strain was then selected as the best PHB
producer, further cultivated in different PHB media, and compared with a positive control, Haloferax mediterranei
ATCC 33500. PHB was extracted from cells and measured with a spectrophotometer, and then the amount of PHB was
measured through comparison with standard PHB. The detected high PHB yield was 6.53% of the dry cell weight in the
PHB medium supplemented with acetate. Partial 16S rRNA gene sequence analysis showed 99% similarity to Haloferax
alexandrinus strain TMT; therefore, the strain was named Haloferax sp. MA10. Haloferax sp. MA10 and Haloferax
alexandrinus TMT have some differences in phenotypic and biochemical properties. With this study, the discovery of
PHB-producing extreme halophilic archaeon Haloferax sp. MA10 at İzmir’s Çamaltı Saltern is reported for the first time.
Key words: Extremely halophilic archaeon, poly-3-hydroxybutyrate (PHB), PHB extraction and quantification,
Haloferax sp. MA10, 16S rRNA gene
İzmir Çamaltı Tuzlası’ndan izole edilen poli-3-hidroksibütirat
üreticisi ekstrem halofilik arkeon: Haloferax sp. MA10
Özet: İzmir Çamaltı Tuzlası, Türkiye’nin deniz suyu kökenli en büyük tuzlası olma özelliğindedir. Günümüze kadar bu
bölgede poli-3-hidroksibütirat (PHB) üretici türün varlığı bakımından çok fazla araştırma yapılmamıştır. Bu kapsamda,
on dört ekstrem halofilik arkea izole edilmiş, saflaştırılmış, PHB üretip üretmedikleri saptanmış ve bir suş en iyi PHB
üreticisi olarak seçilerek PHB üreticisi Haloferax mediterranei ATCC 33500 pozitif kontrol ile karşılaştırmak suretiyle
değişik PHB besiyerlerinde büyütülmüştür. PHB hücrelerden ekstrakte edilmiş, spektrofotometre ile ölçülmüş ve PHB
miktarı standart PHB ile karşılaştırılarak tespit edilmiştir. En yüksek PHB veriminin asetat içeren PHB ortamında
hücre kuru ağırlığının % 6,53’ü olarak saptanmıştır. Kısmi 16S rRNA gen dizisi analiz sonucu % 99 oranında Haloferax
alexandrinus TMT suşuna benzer olduğunu göstermiştir. Bu nedenle suş Haloferax sp. MA10 olarak adlandırılmış olup
Haloferax sp. MA10’ın, bazı fenotipik ve biyokimyasal özellikler bakımından Haloferax alexandrinus TMT suşundan
farklılıkları olduğu bulunmuştur. Bu çalışma ile İzmir Çamaltı Tuzlası’ndan PHB-üretici ekstrem halofilik arkeon
Haloferax sp. MA10 ilk defa bulunmuştur.
Anahtar sözcükler: Ekstrem halofilik arkeon, poli-3-hidroksibütirat (PHB), PHB ekstraksiyonu ve kantifikasyonu,
Haloferax sp. MA10, 16S rRNA geni
303
Poly-3-hydroxybutyrate-producing extreme halophilic archaeon: Haloferax sp. MA10 isolated from Çamaltı Saltern, İzmir
Introduction
Since the Industrial Revolution, the increasingly
widespread use of petroleum-based plastics has
created long-term problems due to the depletion of
oil sources, accumulation of plastic waste in nature,
and rising oil prices. These outcomes have directed
scientists toward alternative plastic sources that are
environmentally friendly, biologically degradable
under appropriate conditions, and obtainable
from cheap sources such as waste products,
microorganisms, or transgenic plants. The most
important consequences of using bioplastics are
that they can be produced from renewable sources
and they are environmentally friendly (1). These socalled biodegradable polymers or bioplastics were
first developed as lipid-like intracellular storage
granules from Bacillus megaterium (2).
Microbiologically produced, the most common
form of bioplastics are polyhydroxyalkanoates
(PHAs). These are a versatile class of polymers with
more than 100 different monomer constituents and
linear-structured polymers (1,2). The most commonly
found PHA form produced by microorganisms is
poly-3-hydroxybutyrate (PHB), which is formed
from 3-hydroxybutyric acid monomers by the
polymerization reaction of 3 enzymes: 3-ketothiolase,
acetoacetyl-CoA reductase, and PHA synthase.
Under stress conditions, microorganisms produce
polymers using acetyl-CoA as an initial material,
and then 3-ketothiolase condenses 2 acetyl-CoAs
to acetoacetyl-CoA. Acetoacetyl reductase reduces
acetoacetyl-CoA to (R)-3-hydroxybutynl-CoA and
PHA synthase polymerizes (R)-3-hydroxybutyrylCoA to PHB (2). The stress conditions that cause
microorganisms to produce these polymers are
the presence of excess carbon and the absence of
an essential nutrient such as oxygen, phosphorus,
nitrogen, or sulfur, or the absence of a trace element
such as magnesium, calcium, or ferrous iron (3-5).
PHB is a stiff, highly crystalline, and relatively
brittle thermoplastic. While the melting point of
PHB is 175 °C, the degradation temperature is 185
°C, which makes the injection molding process
difficult. Although the mechanical properties of
PHB, such as Young’s modulus (3.5 GPa) and tensile
strength (MPa), are similar to those of polypropylene,
the elongation to break of PHB (6%) is significantly
lower than that of polypropylene (400%) (2). In the
304
developed PHB copolymer P(3HB-co-3HV), as the
fraction of the 3-hydroxyvalerate unit increases, the
impact strength and elongation to break increase; on
the other hand, brittleness, melting point, and Young’s
modulus decrease (2,3). Thus, the polymer becomes
tougher and more flexible. Due to the decrease in
melting point, the injection molding process of the
copolymer occurs without thermal degradation (2).
There are some useful properties of PHB, such as
moisture resistance, water insolubility, optical purity,
and good oxygen impermeability. These properties
are different from those of other biodegradable
plastics, which are either water-soluble or moisturesensitive (2,3). The biodegradation of PHB or P(HBHV) occurs when microorganisms colonize on
the surface of the polymer and secrete enzymes to
degrade the polymer. The most important properties
of PHB and other polyhydroxyalkanoates are that
they do not degrade under the normal conditions of
storage, and they are stable in air (2).
Currently the most important types of bioplastics
suggested by markets are cellulosic esters, starch
derivatives, PHB, polylactic acids (PLA), and
polycaprolactone (PCL) (6). These polymers are
used in areas such as pharmaceutics, biomedical
products, packaging, and agriculture (3,6). There is
growing interest in the use of bioplastics worldwide.
The total worldwide production of bioplastics was
724,500 t/year in 2010. In 2011, about 1,000,000 t
were produced, and it is estimated that the level of
worldwide bioplastic production will be 1,709,700
t/year by 2015. When compared with conventional
plastics derived from petroleum, however, worldwide
consumption levels of bioplastics are small (7).
PHA can be produced by several microorganisms,
such as various eubacteria and some haloarchaeal
strains. There are several advantages to working with
haloarchaeal strains rather than eubacteria. Due to the
high salt concentrations they require in their growth
medium to maintain cell wall stability, haloarchaeal
strains do not require strict sterile conditions. This
makes cultivation more convenient in simple places
such as open ponds. Cell walls can also easily lyse in
the absence of salt, especially in distilled water; this
property enables the recovery of PHB or other PHA
forms from extreme halophiles much more easily and
economically than from eubacteria (8). Investigations
of the production of PHB and other PHAs are few.
Studies including some haloarchaeal strains in the
E. TEKİN, M. ATEŞ, Ö. KAHRAMAN
genera of Haloferax, Haloarcula, Haloquadratum,
Halorubrum,
Halobiforma,
Halorhabdus,
Halalkalicoccus,
Halobacterium,
Natrianema,
Halostagnicola,
Natrinema,
Natronobacterium,
Natronorubrum, Haloterrigena, Halopiger, and
Halococcus have only been reported from cheap
carbon sources (8-19). According to detailed analyses,
most of the haloarchaeal cells were found to be
synthesizing PHB or poly-(3-hydroxybutyrate-co-3hydroxyvalerate) (PHBV) at levels ranging from 0.8%
to 22.9% (w/w) of cell dry weight (19). Currently, the
best PHA-producing strain of extremely halophilic
archaea is Haloferax mediterranei, which produces
PHB-co-PHV up to 60wt% of its dry cell weight with
starch or glucose as a carbon source under phosphate
limitation (8). In continuous cultivation, PHA yields
increased to a maximum of 87.5wt% of dry cell weight
with different sources such as hydrolyzed whey,
sodium valerate, and γ-butylolacton (20). Halopiger
aswanensis (formerly strain 56) was isolated from
Egyptian saline soil, and its PHB yield was reported
as 53wt% of dry cell weight (16). These investigations
reveal that cultivation conditions can change the
production yield. Therefore, in order to improve
production yields, there should be further study of
haloarchaeal cells and their cultivation conditions.
Çamaltı Saltern, a salt production site 28 km
from the city of İzmir and the largest saltern in
Turkey, has water storage ponds, evaporation ponds,
and crystallization ponds. The salinities of these
ponds are 35%-50%, 50%-150%, and 150%-300%,
respectively (21). Our aim was to conduct the first
investigation of PHB-producing extremely halophilic
archaea from İzmir’s Çamaltı Saltern, select PHBproducing isolates via a specific PHB staining
method, identify isolates both phenotypically and
genetically, and determine PHB yields by different
media formulations. We aimed to assess the effects
of different media formulations on production yields
and collect information about the effect conditions
have on improvement of PHB production.
Materials and methods
Isolation of halophilic archaea
Soil samples were taken from 2 different parts of
İzmir’s Çamaltı Saltern by digging in the soil to a
depth of 10 cm. From each sample, 5 g was transferred
to 50 mL of medium that contained salt solution at
an approximately 25% final concentration and was
referred to as seawater (SW)-25. The composition of
the SW-25 medium was (in g/L): NaCl, 202.5; MgCl2,
17.5; MgSO4, 24; CaCl2, 0.9; KCl, 5; NaHCO3, 0.15;
NaBr, 0.065; and yeast extract, 5. The pH was adjusted
to 7.2 with 1 M NaOH (22). The samples were
incubated for 7 days at 40 °C in an orbital shaker at
120 rpm. After the incubation period, isolations were
carried out using the streak plate method on SW-25
agar medium. Plates were incubated at 40 °C for 14
days. Colonies with different colors were picked up
and restreaked several times until pure cultures were
obtained (23).
PHB screening
Cells were stained by the Nile blue A staining procedure
(24). The type strain Haloferax mediterranei ATCC
33500 was used as a PHB-producing positive control.
Nile blue A staining was performed as follows: broth
cultures of type strain H. mediterranei ATCC 33500
and the isolates were smeared on a glass slide, heatfixed, and stained with a 1% ethanol solution of Nile
blue A for 10 min at 55 °C. They were then washed
with water and 8% aqueous acetic acid for 1 min,
dried with filter paper, and covered with a glass
cover slip. The preparation was examined at 600×
magnification using a fluorescence microscope. The
procedure was based on measuring at wavelengths
near 460 nm to detect intracellular PHB granules,
which produce bright orange fluorescence at that
wavelength (24,25).
Phenotypic and biochemical properties
SW-25 agar/broth was used as a basal medium,
and incubations were carried out at 40 °C for 14
days, unless otherwise indicated. A modified Gram
reaction was performed according to the method
of Dussault (26). Motility was checked under a
phase contrast microscope. Growth on nutrient
agar was tested. Antibiotic susceptibility was tested
by the disk diffusion method using antibiotic disks
(Oxoid) of novobiocin (NV, 5 μg), erythromycin (E,
15 μg), streptomycin (S, 10 μg), bacitracin (B, 0.04
U), ampicillin (AM, 10 μg), and tetracycline (TE, 30
μg) (27,28). The culture densities were adjusted to
an OD520 of 1.0. Cultures were scored as sensitive to
the antibiotic if the inhibition zone extended beyond
305
Poly-3-hydroxybutyrate-producing extreme halophilic archaeon: Haloferax sp. MA10 isolated from Çamaltı Saltern, İzmir
8 mm (28). Optimal conditions were determined
by spotting 10 μL of the selected isolate on the SW25 agar using different NaCl concentrations (0%,
5%, 8%, 10%, 15%, 20%, 25%, and 30%), different
temperatures (20 °C, 27 °C, 37 °C, 40 °C, and 50 °C),
and different pH levels (3, 4, 5, 6, 7, 8, 9, and 10)
(27,28). The minimal yeast requirement for growth
was determined on SW-25 broth with 0%, 0.01%,
0.1%, and 0.5% yeast extract. Single carbon source
(20%, w/v) analyses were performed with different
single carbon sources that were syringe-filtered
(0.22 μm). The carbon sources (citrate, glucose,
galactose, maltose, arabinose, fructose, xylose, Naacetate, and Na-butyrate) were added to SW-25 agar
medium containing 0.01% yeast extract at a 1% final
concentration, as there was no growth in SW-25 agar
medium containing 0.01% yeast extract. In the growth
test for different single nitrogen sources, the yeast
extract was omitted from the medium. Instead, yeast
carbon base medium (Difco) was used according
to manufacturer’s instructions and supplemented
with 1% of a different test nitrogen source (soya
peptone, ammonium chloride, potassium nitrate,
and meat extract) in the basal medium of SW-25
salts. Indole production was tested with SW-25 broth
supplemented with 1% tryptone (w/v), catalase was
tested with 3% H2O2 solution, and gelatin hydrolysis,
nitrate reduction, and gas production were tested in
SW-25 broth supplemented with 0.1% (w/v) KNO3
(27). Tween 80 hydrolysis was performed according
to the methods of Martin et al. (29). Casein hydrolysis
was performed according to the method of SánchezPorro et al. (30). Starch hydrolyses were tested
according to the methods of Ventosa et al. 1998
(31). Afterwards, 4.2% (w/v) DNase test agar with
0.005% methyl green (Difco) was added to the SW-25
medium to observe clear zones around the colonies
for DNase activity, according to the manufacturer’s
instructions.
16S rRNA gene analyses
Genomic DNA was isolated according to the
Halohandbook methods (32) with a modification
of RNase manipulation. An active culture (OD600 of
approximately 1) was centrifuged at 12,000 rpm for
10 min at 4 °C, and then 400 μL of cold and ultrapure
sterile distilled water was added to the pellets and
vortexed gently. The tube was subjected to 70 °C for
306
10 min for protein denaturation and centrifuged at
12,000 rpm for 10 min at 4 °C. The supernatant was
taken in a new Eppendorf tube, and 5 μL of RNAse
(20 μg/mL) was added and incubated at 37 °C for 30
min. The purity of the genomic DNA was checked
with 0.8% (w/v) agarose gel in a TBE (0.5×) buffer
for 1 h at 80 V. The 16S rRNA gene was amplified
with domain Archaea-specific forward primer 21F
(5’-TTCCGGTTGATCCYGCCGGA-3’ where Y = C
or T, but in this study, Y = T) and the universal reverse
primer 1492R (5’-GGTTACCTTGTTACGACTT-3’)
(33). Polymerase chain reaction (PCR) was performed
with a GeneAmp PCR System 9700 thermal cycler
using the FastStart Taq DNA Polymerase dNTPack
(Roche, version 2006) for 35 cycles for 5 min at 95 °C
for the initial denaturation/enzyme activation. This
was followed by 45 s of denaturation at 95 °C, 1 min of
annealing at 50 °C, and 1.4 min of polymerization at
72 °C, with a final extension at 72 °C for 7 min. DNA
sequencing was carried out by REFGEN/METU
at Middle East Technical University, Turkey. The
sequence result was analyzed in the National Center
for Biotechnology Information (NCBI) database
using the Nucleotide Basic Local Alignment Search
Tool (BLAST) (34). The reference sequences of 10
closely related taxa were taken from the GenBank
database. The selected taxa and their accession
numbers were submitted to Molecular Evolutionary
Genetics Analysis version 4 (MEGA 4) software (35)
to analyze the evolutionary distances of the taxa.
Multiple alignments were done using ClustalW (36),
and evolutionary distances of taxa were computed
using a p-distance model before constructing a
phylogenetic tree, according to software instructions
(37). Evolutionary history was inferred using
the neighbor-joining method (38). All positions
containing gaps and missing data were eliminated
from the dataset with the complete deletion option.
The bootstrap test (1000 replicates) was used to assess
confidence values with resampling. The percentage of
replicate trees in which the associated taxa clustered
together in the bootstrap test (1000 replicates) is
shown next to the branches (39).
Growth curve measurements
The growth curve was established by measuring the
OD520 in SW-25 medium at 40 °C. In all measurements,
active cultures were adjusted to an OD520 of 1.0; the
E. TEKİN, M. ATEŞ, Ö. KAHRAMAN
OD520 was measured daily and 2% (v/v) inocula were
used. Growth curves were also established to screen
the effect of glucose concentration (1%, 2%) of the
PHB medium, the effect of 2% glucose-containing
PHB medium, and the effect of 1% acetatesupplemented 2% glucose-containing PHB medium
(8).
Different PHB media
Three different media were used to measure PHB yield.
The PHB medium contained (w/v) 2% glucose, 0.2%
NH4Cl, 0.03% KH2PO4, 0.0005% FeCl3, and marine
salts at an approximately 25% final concentration
(NaCl, 19.4; MgCl2, 1.6; MgSO4, 2.4; CaCl2, 0.1;
KCl, 0.5; NaHCO3, 0.02; NaBr, 0.05) (8). The PHB
medium supplemented with acetate contained (w/v)
1% acetate, 2% glucose, 0.2% NH4Cl, 0.03% KH2PO4,
0.0005% FeCl3, and marine salts at an approximately
25% final concentration (NaCl, 19.4; MgCl2, 1.6;
MgSO4, 2.4; CaCl2, 0.1; KCl, 0.5; NaHCO3, 0.02;
NaBr, 0.05) (8). The basic medium contained (w/v)
22.5% NaCl, 0.2% KCl, 0.5% MgSO4.7H20, 0.1% yeast
extract, and 1% glucose at pH 7.2 ± 0.2 (40). Each
medium (100 mL) was separately inoculated with the
active cultures of the isolate and the type strain. They
were then incubated for 6 days at 40 °C in an orbital
shaker at 180 rpm (8).
Quantitative analysis
PHB quantification was performed according to the
method of Law and Slepecky, with modifications
(41). This method is based on the conversion of
intracellular PHB with concentrated sulfuric acid to
obtain crotonic acid. Crotonic acid was quantified by
measuring absorbance at 235 nm. At the beginning
of the stationary phase (day 6), 100 mL of the
cultures were harvested by centrifugation at 10,000
rpm at 4 °C for 40 min. The pellet was washed with
10% NaCl (w/v), centrifuged, and washed again with
5 mL of sterile distilled water to remove excess salts
from the medium. The pellets were then lyophilized
(9). Lyophilized cell pellets were measured as dry cell
weights, 10 mL of sulfuric acid (95%-98%) (Merck)
was added to each test tube, and these were incubated
for 10 min at 100 °C in a water bath. The solution was
cooled, and 10−1-10−4 dilutions were prepared with
sulfuric acid. All dilutions were measured against
a sulfuric acid blank at 235 nm. A standard curve
was prepared by the same method with commercial
PHB (poly-[(R)-3-hydroxybutyric acid], natural
origin; CAT: 36.350-2, Sigma Aldrich). PHB yield
was calculated according to the standard curve as
mg PHB/mg dry cell weight. All measurements were
repeated 3 times, and the results were reported as
means ± standard deviations.
Statistical analysis
A 1-way analysis of variance (ANOVA) was carried
out with the Tukey-Kramer honestly significant
difference test based on 3 replicates using JMP
software (version 8.00; SAS Institute Inc., Cary, NC,
USA) in order to establish statistical differences
between different media for the same microorganism.
Significance was defined at P < 0.05.
Results and discussion
In the description of new taxa of extremely
halophilic archaea, there are some recommended
minimum standards. These primarily include cell
morphology, motility, pigmentation, the minimum
salt requirement to prevent lysis, optimum NaCl
and MgCl2 concentrations and range of salt
concentrations enabling growth, temperature and
pH ranges for growth, anaerobic growth in the
presence of nitrate or arginine, acid production from
a range of carbohydrates, ability to grow on single
carbon sources, catalase and oxidase tests, hydrolysis
of starch, hydrolysis of casein, hydrolysis of Tween
80, sensitivity to different antibiotics, and sensitivity
to polar lipids. The 16S rRNA nucleotide sequence
information and DNA-DNA hybridization data
should be consistent with the descriptions of new
species (23).
Isolation, phenotypic, and biochemical properties
Due to the isolation and purification results, a total of
14 microorganisms were selected for PHB production
screening via a specific Nile blue A staining
method. According to the staining results, the best
PHB-producing strain was selected for further
identification based on phenotypic and biochemical
properties, the results of 16S rRNA gene analysis, and
the effects of different media on the production of
PHB. The characteristics of the colony were circular,
convex, entire, translucent, smooth, and orangered on the SW-25 agar medium. The cells of the
307
Poly-3-hydroxybutyrate-producing extreme halophilic archaeon: Haloferax sp. MA10 isolated from Çamaltı Saltern, İzmir
isolate stained gram-negative. Cell morphology was
pleomorphic, especially for short and long rods. This
strain was motile under a phase-contrast microscope.
Growth was negative on nutrient agar medium. The
strain was susceptible to novobiocin (5 μg) and
bacitracin (0.04 U) but resistant to erythromycin
(15 μg), streptomycin (10 μg), ampicillin (10 μg),
and tetracycline (30 μg). Growth was detected in a
5%-30% range of NaCl for the defined SW medium,
and the optimum NaCl concentration was 25%.
The pH range was detected as pH 5.0-8.0; however,
optimum growth was detected at pH 7.0. Growth
was positive at 27 °C, 37 °C, 40 °C, and 50 °C and
negative at 20 °C. The optimum growth temperature
was 40 °C. Growth was negative with 0% and 0.01%
yeast extract concentrations, but it was positive
with 0.1% yeast extract as a carbon and nitrogen
source. The optimum yeast extract concentration
was 0.5%. The results of single carbon source tests
were positive for citrate, glucose, galactose, maltose,
arabinose, fructose, xylose, Na-acetate, and Nabutyrate; growth was negative on lactose. The results
of single nitrogen source tests were positive for soya
peptone, ammonium chloride, potassium nitrate,
and meat extract. Indole from tryptone was negative.
Nitrate reduction or gas production was negative.
The catalase test was positive. Gelatin hydrolysis,
Tween 80, starch, and casein hydrolysis results were
negative. DNase activity was positive with clear zones
around the colonies.
16S rRNA gene analyses
Genomic DNA isolation was easier than the
conventional procedures. On the other hand, there
was a problem with impurities on the gel after
running the agarose gel electrophoresis of genomic
DNA. These impurities were prevented by the
addition of RNase. The results of PCR of a partial
16S rRNA gene on agarose gel are given in Figure
1. Due to the sequence analysis of PCR results, a
1392-bp length of a partial 16S rRNA gene sequence
was obtained. The results of the partial 16S rRNA
gene sequence of the isolate showed 99% similarity
to the H. alexandrinus type strain according to
the NCBI/BLAST database. For this reason, the
existing properties of the strain and the type
strain H. alexandrinus strain TMT were compared
(Table 1). Due to the different properties of each
308
K1
K2
K3
10
M
5000
2000
850
400
100
Figure 1. The PCR results of Haloferax sp. MA10 as 10, while
K1, K2, and K3 are negative controls. K1 (negative
control 1): reaction conditions were performed
without reverse primer; K2 (negative control 2):
reaction conditions were performed without forward
primer; K3 (negative control 3): reaction conditions
were performed without DNA; 10: main reaction,
consisting of full reaction conditions. M = marker;
Fast RulerTM Middle Range DNA Ladder; 100-5000 bp.
strain under the existing conditions, the strain is
proposed as Haloferax sp. MA10 until further DNADNA hybridization data results are obtained. The
nucleotide sequence of the partial 16S rRNA gene
of the strain Haloferax sp. MA10 was submitted
to GenBank and assigned the accession number
HQ908091. The constructed phylogenetic tree is
given in Figure 2.
PHB production
Due to the results of the specific PHB staining
method, Haloferax sp. MA10 was selected as the
best PHB producer from 14 isolates. The stationary
phase of Haloferax sp. MA10 was determined on day
6 by measuring the OD520 of the culture daily with a
spectrophotometer (Figure 3).
The effect of the glucose concentration (1%, 2%)
of the PHB medium on Haloferax sp. MA10 and H.
mediterranei ATCC 33500 is shown in Figure 4. The
effect of 1% acetate on the growth of Haloferax sp.
E. TEKİN, M. ATEŞ, Ö. KAHRAMAN
Table 1. Comparison of Haloferax sp. MA10 and Haloferax alexandrinus strain TMT.
Color
Cell shape
Motility
Colony diameter
Lysis in distilled water
NaCl range
NaCl optimum
pH, optimum pH
Temperature optimum
Temperature range
Nitrate reduction (aerobic)
Nitrite reduction (aerobic)
Catalase
Gas formation from nitrate
Hydrolysis of starch
Hydrolysis of casein
Hydrolysis of gelatin
Hydrolysis of Tween 80
Sensitivity to antibiotics
Haloferax sp. MA10
Haloferax alexandrinus strain TMT*
Orange-red
Pleomorphic
+
0.5-1 mm, circular
+
5%-30%
25%
5.0-8.0, optimum 7.0
40 °C
27-50 °C
+
Novobiocin, bacitracin
Red
Pleomorphic
0.5-1 mm, circular
+
10%-32%
25%
5.5-7.5, optimum 7.2
37 °C
20-55 °C
+
+
+
+
+
Novobiocin, bacitracin
*Properties of Haloferax alexandrinus strain TMT were taken from the literature (42).
+: Positive growth; -: negative growth.
Haloferax alexandrinus AB037474
Haloferax sp. MA10 HQ908091
Haloferax volcanii AY425724
44
Haloferax denitrificans D14128
Haloferax
gibbonsii D13378
93
35
Haloferax lucentensis AB081732
Haloferax prahovense AB258305
Haloferax mucosum DQ860980
Haloferax sulfurifontis AY458601
Haloferax elongans DQ860977
100
Haloferax larsenii AY838278
50
42
55
84
0.001
Figure 2. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences, showing the position of strain Haloferax sp. MA10.
Bootstrap values are shown as percentages of 1000 replicates. There were a total of 1353 positions in the final dataset. Scale
bar (0.001) indicates the number of nucleotide substitutions per site.
MA10 and H. mediterranei ATCC 33500 is shown in
Figure 5.
The results of PHB yield are given in Table 2.
According to the PHB yield results from Haloferax
sp. MA10 and H. mediterranei ATCC 33500 in
different PHB production media, there was a slightly
positive effect of 1% acetate on the PHB production
of Haloferax sp. MA10 (from 5.34% to 6.53% yield).
On the other hand, according to the basic medium
309
Poly-3-hydroxybutyrate-producing extreme halophilic archaeon: Haloferax sp. MA10 isolated from Çamaltı Saltern, İzmir
3
2.5
OD 520
OD520
3.5
3
2.5
2
1.5
1
0.5
0
2
1.5
1
0.5
0
1
2
3
4
5
6
7
8 9 10 11 12 13 14 15 16
Days
2
3
days
4
5
6
2% glucose,1% acetate, H. mediterranei ATCC 33500
2% glucose, H. mediterranei ATCC 33500
2% glucose, 1% acetate, Haloferax sp. MA10
2% glucose, Haloferax sp. MA10
Figure 3. The optic density measurement of Haloferax sp. MA10.
1.6
Figure 5. The effect of 1% acetate in 2% glucose-containing PHB
medium on the growth of Haloferax sp. MA10 and H.
mediterranei ATCC 33500.
1.4
1.2
OD520
1
1
0.8
0.6
0.4
0.2
0
1
2
3
Days 4
5
6
2% glucose, H. mediterranei ATCC 33500
1% glucose, H. mediterranei ATCC 33500
2% glucose, Haloferax sp. MA10
1% glucose, Haloferax sp. MA10
Figure 4. The effect of 1% and 2% glucose concentrations of
PHB medium on the growth of Haloferax sp. MA10
and H. mediterranei ATCC 33500.
production yield, PHB was the lowest (4.48% yield)
in Haloferax sp. MA10. The results of PHB yield for
the type strain H. mediterranei ATCC 33500 also
showed a slight positive effect of acetate on PHB
production (from 38.59% to 40.30%). The most
significant yield, however, was acquired with the
basic medium (54.26%). PHB production yield was
measured with different medium formulations to
determine whether there were differences in yield
results. Acetate produced a slightly positive effect
on PHB production in both microorganisms. As
seen in Table 2, the basic medium was much more
effective than acetate-containing or acetate-lacking
PHB medium for H. mediterranei ATCC 33500.
We used acetate because it is the initial material for
PHB production and has been used before (8). The
limiting condition of the basic medium is much
greater than that of the PHB media, so the PHB yield
should have been much greater than in the other 2
media. It is interesting that the effect of acetate on
PHB production yield in Haloferax sp. MA10 was
slightly higher than in the basic medium. The reason
why 2 microorganisms belonging to the same genus
Table 2. The PHB yield (%) in different medium conditions of Haloferax sp. MA10 and H. mediterranei ATCC 33500.
Microorganism
PHB medium (100 mL)
Dried cell weight (mg)
PHB amount (mg)
Yield (%)
Haloferax sp. MA10
2% glucose
20.6 ± 0.01[c]
1.10 ± 0.01[c]
5.34 ± 0.01[b]
Haloferax sp. MA10
1% acetate, 2% glucose
148.0 ± 0.01[a]
9.67 ± 0.01[a]
6.53 ± 0.01[a]
Haloferax sp. MA10
Basic medium
56.8 ± 0.01[b]
2.55 ± 0.01[b]
4.48 ± 0.01[c]
H. mediterranei ATCC 33500
2% glucose
29.2 ± 0.01[c]
11.27 ± 0.01[c]
38.59 ± 0.01[c]
H. mediterranei ATCC 33500
1% acetate, 2% glucose
184.8 ± 0.01[a]
74.49 ± 0.01[a]
40.30 ± 0.01[b]
H. mediterranei ATCC 33500
Basic medium
60.0 ± 0.01[b]
32.56 ± 0.01[b]
54.26 ± 0.01[a]
Letters in square brackets refer to statistical analysis performed within each column between different media for the same microorganism.
Different letters refer to significantly different values (ANOVA, P < 0.05).
310
E. TEKİN, M. ATEŞ, Ö. KAHRAMAN
had different PHB production yields may be related
to the generation of their PHB-producing enzymes
or other factors.
Based on the comparison of some biochemical
properties, PHB production; aerobic nitrate and
nitrite reduction; hydrolysis of gelatin, Tween
80, casein, and starch; indole production; growth
temperature; NaCl range; color; and motility
properties were different for each strain. In this
study, we present the first report of Haloferax sp.
MA10 from İzmir’s Çamaltı Saltern. There should
be more detailed analyses, such as DNA-DNA
hybridization, to elucidate the strain from the genus
level to species level. We aimed to assess the effects
of different media formulations on the production
yield and to obtain information about the effect of
conditions on the improvement of PHB production.
We found that acetate has a positive effect on PHB
yield; however, in future studies, conditions should
be much more controlled. For example, continuous
fermentation may elucidate other factors that
enhance the production yield. In addition, the effect
of cheap or waste carbon sources on PHB production
should be examined. Although the yield is low in our
strain, it could be enhanced by gene transfer using
recombinant DNA technology for future studies.
There are several studies on increasing the yield of
PHB in bacterial species by mutagenesis studies (43),
and these manipulations can also be used to increase
PHB yield in haloarchaeal cells.
Acknowledgments
This work was supported by the Scientific Research
Project Council of Ege University, Project No. FEN045, 2006. We thank the Scientific Research Project
Council of Ege University. We also thank Dr Aziz
Akın Denizci, Assoc Prof Dr Birgül Özcan, and Assoc
Prof Dr Kemal Sami Korkmaz.
Corresponding author:
Ebru TEKİN
Department of Biology,
Basic and Industrial Microbiology Section,
Bornova, 35100 İzmir - TURKEY
E-mail: [email protected]
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