Environment  Health  Techniques

Thermophilic production of PHA by a novel Aneurinibacillus strain 1

Research Article
Thermophilic production of polyhydroxyalkanoates by a novel
Aneurinibacillus strain isolated from Gudao oilfield, China

Zijun Xiao, Yu Zhang, Lijun Xi, Fangfang Huo, Jing-yi Zhao and Jing Li

State Key Laboratory of Heavy Oil Processing and Center for Bioengineering & Biotechnology, China University
of Petroleum, Qingdao, China

Polyhydroxyalkanoates (PHAs) are usually biosynthesized using mesophilic strains, but the
fermentation processes often suffer from bacterial contamination. This work reports the
screening of thermophilic bacteria capable of producing PHAs under elevated temperatures to
reduce the contamination risk. Strain XH2 was isolated from an oilfield and identified as
Aneurinibacillus sp. by morphology, physiological-biochemical characterization, and 16S rDNA
phylogenetic analysis. This strain can produce PHA granules, which was detected by Nile red
staining and transmission electron microscopic imaging. At 55 °C, 111.6 mg l
1 of PHA was
produced in a fermentation medium containing glucose, peptone, and yeast extract. If peptone
was removed from the medium, the yield of PHA would be enhanced by 2.4 times. The main
monomers of the PHA product were identified to be 3-hydroxybutyrate and 3-hydroxyvalerate
with a molar ratio of 17.2:1 by gas chromatography-mass spectroscopy (GC-MS) and nuclear
magnetic resonance analyses. Two minor homologues, 3-hydroxyoctanoate, and
3-hydroxy-4-phenylbutanoate, were tentatively identified by GC-MS as well. This is the first
report of thermophilic PHA bacterial producer from the Firmicutes phylum.

Abbreviations: PHA – Polyhydroxyalkanoate; 3HB – 3-hydroxybutyrate; 3HV – 3-hydroxyvalerate; GC – gas

chromatography; MS – mass spectroscopy; NMR – nuclear magnetic resonance; PhAc – Phenylacetic acid

Keywords: Polyhydroxyalkanoates / 3-Hydroxybutyrate / 3-Hydroxyvalerate / Aneurinibacillus / Thermophilic

bacterium

Received: November 6, 2014; accepted: March 13, 2015

DOI 10.1002/jobm.201400843

Introduction respectively. Poly(3-hydroxybutyrate-co-3-hydroxyvaler-

Polyhydroxyalkanoates (PHAs) are the only bioplastics
completely synthesized by microorganisms. PHAs have
developed rapidly to find applications in various fields [1].
The types of PHAs depend on the bacterial species and
growth conditions. Over 150 different PHA monomers
have been discovered from different bacteria [2]. PHAs
can be classified into two main groups according to the
carbon atom numbers of the monomer units: the short-
chain-length PHAs and the long-chain-length PHAs,
which consist of 3–5 and 6–14 carbon atoms,
Zijun Xiao, Yu Zhang, and Lijun Xi contributed equally to this work.
Correspondence: Zijun Xiao, State Key Laboratory of Heavy Oil
Processing and Center for Bioengineering & Biotechnology, China
University of Petroleum, No. 66 Changjiang West Road, Huangdao
District, Qingdao 266580, China
E-mail: zjxiao@upc.edu.cn
Phone/Fax: 0086-532-86981136
ate) [Poly(3HB-co-3HV)], as the second generation bio-
plastic, has many advantages than poly(3-HB).
Poly(3HB-co-3HV) is a typical example of short-chain-
length PHAs. This piezoelectric material is highly
crystalline, brittle, and stiff. The biological properties
of poly(3HB-co-3HV) include water resistance, complete
biodegradability, and high biocompatibility. Poly(3HB-
co-3HV) is also a suitable substance for tissue engineer-
ing, which can enhance cell adhesion, migration,
proliferation, and differentiation functions [3–5].
PHA production microorganisms are widespread in
nature, especially in soil-inhabiting bacteria [6]. Some
bacteria in marine or in some extreme environments are
also capable of making PHAs [7]. Most of these PHA
producers have been assigned to genera Alcaligenes,
Pseudomonas, Methylotrophs, Azotobaerer, and Rhodospiril-
lum [8]. However, PHA fermentation using these

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2 Zijun Xiao et al.
mesophilic bacteria suffers from the high risk of bacterial
contamination [9], which seriously hampers the scaling up
of the laboratory processes. Thermophilic fermentation,
typically operated at 50–60 °C, is a promising alternative to
control contamination. Therefore, there is a drive to find
new thermophilic PHA producers.
Even though there are far more than 300 different
microorganisms that can synthesize PHAs [10], only very
few thermophilic strains have been reported to date. In
this paper, we describe a novel thermophilic Aneur-
inibacillus PHA producer isolated from an oilfield.
Aneurinibacillus species can produce many kinds of
natural products, but they have never been found to
be capable of producing PHAs before.
Materials and methods
Strain isolation and culture conditions
Oil produced water was collected from the Gudao oilfield
(Dongying, China). The sample was added into an
enrichment liquid YPG medium (yeast extract 10 g l
1,
peptone 20 g l
1, glucose 20 g l
1, pH 7.0) in an
Erlenmeyer flask. After being hot shocked at 100 °C,
the Erlenmeyer flask was immediately transferred into a
60 °C reciprocal shaking water bath and incubated at
150 rpm for 2 days. The strain was purified by spread on
YPG plates composed of extra 17 g l
1 of agar. As a
contrast, peptone was removed from the fermentation
medium (i.e., the YG medium: 10 g l
1 of yeast extract
plus 20 g l
1 of glucose), to simulate the nitrogen-
limited conditions.
DNA extraction, 16S rDNA amplification, and
phylogenetic analysis
The genomic DNA for subsequent PCR was extracted
using the T-Guide Bacteria Genomic DNA Kit (OSR-M502,
TIANGEN Biotech Co. Ltd., Beijing, China). The partial
16S rDNA gene sequence of the isolate was routinely
amplified using universal primers 27F (50 -AGAGTTT-
GATCMTGGCTCAG-30 ) and 1492R (50 -TACGGY-
0
TACCTTGTTACGACTT-3 ). The resultant sequence was
compared with those of type species deposited in
EzTaxon [11]. 16S rDNA sequences of representative
homologous species were retrieved from this database to
generate the phylogenetic tree using the neighbor-
joining method (bootstrap replicates ¼ 1000) in the
MEGA 5 program [12].
Purification of the PHA product from cultured cells
The isolated strain was inoculated into 50 ml of
Luria–Bertani medium in a reciprocal shaking water
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bath controlled at 150 rpm and 55 °C to prepare the seed
culture. After 24 hours of cultivation, the seed was
inoculated (1%, v/v) into the YPG medium for PHA
production. The bacterial cells at the age of 48–96 hours
were collected by centrifugation (5 minutes at
6000 rpm), washed with sterilized water, and dispersed
in a small amount of sterile water. After drying at 40 °C
for 12 hours, they were dispersed in chloroform, and
PHA was extracted at 100 °C for 4 hours. After cooling
down, the mixed chloroform solution was filtered
through a 0.22 mm membrane and the cell remains
was abandoned. Ten times the volume of cold ethanol
was added to the filtrated chloroform cell extraction,
and the PHA precipitate formed was filtered through a
0.22 mm membrane and the dried samples were used for
analysis.
Analytical methods
The morphological features of the isolate were observed
by using light microscopy at a magnification of 1000
and a scanning electron microscope (JEOL JSM-6390LV).
Primary detection of PHA accumulation in the cells was
performed by the colony staining method using Nile red
dye as described by Spiekermann et. al. [13]. The
intracellular PHA granules were imaged using a JEOL
JEM-1200EX transmission electron microscope. Optical
density of the culture was measured spectrophotometri-
cally at 600 nm to estimate the bacterial growth. Cell dry
weight was calculated from optical density with a linear
correlation factor.
Physiological-biochemical tests were performed using
a commercial kit HB8679 (Qingdao Hope Bio-
Technology Co., Ltd., China). PHA yield was quantified
using the method described by Law and Slepecky [14].
Poly[(R)-3-hydroxybutyric acid] (CAS: 29435-48-1;
Sigma–Aldrich, Germany) was used as a comparison.
The extracted PHA samples were hydrolyzed and
methylated in a mixture of 2 ml acidified methanol (3%
v/v H2SO4) and 2 ml chloroform at 100 °C for 4 hours.
Phase separation was achieved by adding 1 ml water to
the mixture. Methylated products in the chloroform
phase were subjected to gas chromatography (GC)
analysis [15]. Benzoic acid was used as an internal
standard. The GC analysis was conducted using an
Agilent 7890A GC equipped with a flame ionization
detector and a 30-m HP-5 (0.32 mm inside diameter,
0.25 mm film thickness) capillary column (19091 J-413,
Agilent). The GC column oven was kept constant at 50 °C
for 2 minutes, then programmed to 250 °C with a
temperature increase of 10 °C minutes
1, and then
maintained at 250 °C for further minutes if necessary.
The metabolites in the fermentation broth were also
J. Basic Microbiol. 2015, 54, 1–9
 Thermophilic production of PHA by a novel Aneurinibacillus strain 3

extracted directly using dichloromethane. The extracts

were concentrated and analyzed using a gas chromatog-
raphy-mass spectroscopy (GC-MS) system (Agilent 6890N
GC equipped with an Agilent J&W DB-5ms capillary
column, coupled with the Waters Micromass GCT
Premier orthogonal acceleration time-of-flight mass
spectrometer) operated at the chemical ionization mode.
Nuclear magnetic resonance (NMR) analysis was con-
ducted at 298 K on a Bruker Avance 400 spectrometer
equipped with a 5 mm PABBO BB probe. 13C spectrum was
obtained at 100.622 MHz using deuterated chloroform as
the solvent and tetramethylsilane as the internal standard.

Results

Strain isolation and PHA product identification

The new isolate was named as XH2 and has been
deposited in China Center for Type Culture Collection
under the deposition number CCTCC M 2013550. Strain
XH2 cells are rod-shaped (0.6–1.0  2.0–6.0 mm) and
motile due to peritrichous flagella (Fig. 1). As the strain
colonies can be stained by Nile red and several granules
(0.2–0.5 mm in diameter) were observed in each cells,
strain XH2 was speculated to be capable of PHA
accumulation.
Further GC-MS and NMR analyses of the cellular
extracts confirmed this speculation. As shown in Fig. 2
(up), the peak area ratio of methyl 3-HB to methyl 3-HV is
15.4:1. Therefore, taking the molecular weights of the two
compounds into account, the molar ratio of the two
monomers is 17.2:1. However, GC or GC-MS methods are
not possible to detect the presence of two or more
Figure 2. The GC profile of the methylated monomers (up) and 13C
distinctive polymers, because the polymers are hydro- NMR spectrum of the PHA product (down) purified from XH2 cells.
lyzed before analysis. At this time, NMR is particularly
useful, because the complexity of the carbonyl signals in
the 13C NMR spectrum can be used to determine whether PHA consists of homopolymers or a copolymer. Chemical
shifts of the PHA product were assigned as shown in the
13
C NMR spectrum in Fig. 2 (down). Note the presence of
signals indicating both 3-HB and 3-HV side chains. These
assignments were consistent with previous reports such as
the one by Slater et al. [16]. The enlarged spectrum for the
carbonyl carbons is shown with assignments to the
13
C-3-HB-3-HV and 13C-3-HV-3-HB dyads (169.14 and
169.31 ppm, respectively). This indicates the PHA product
of XH2 is a copolymer of 3-HB and 3-HV [i.e., poly(3-HB-co-
3-HV)] and there is a random sequence distribution of
monomers within the copolymer.

Classification and identification of strain XH2

Figure 1. Transmission electron micrograph of XH2 cells after The 16S rDNA phylogenetic tree of XH2 and all its closely
aerobic growth in YGP medium at 55 °C for two days. Bar: 1.0 mm. related species was built by the MEGA 5 program (Fig. 3).

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4 Zijun Xiao et al.

Figure 3. Phylogenetic tree derived from neighbor-joining analysis of the 16S rDNA gene sequences of strain XH2 and of all its closely related
type species. Halobacillus halophilus DSM 2266 was used as an out-group. GenBank accession numbers are given in parentheses. Bootstrap
values are shown as percentages of 1000 replicates. Bar: 1% estimated sequence divergence.

In the phylogenetic tree, strain XH2 and Aneurinibacillus
species form a stable, unique but distant branch.
Comparison of the 16S rDNA gene sequence of strain
XH2 (GenBank accession number KF791865) with those
of the type species from the genus Aneurinibacillus found
high similarities by EzTaxon: A. thermoaerophilus DSM
10154, 99.93%; A. danicus NCIMB 13288, 96.46%; A.
terranovensis LMG 22483, 95.45%; A. aneurinilyticus ATCC
12856, 95.24%; A. migulanus DSM 2895, 95.24%; A. soli
CB4, 94.67%. The microbiological properties of strain
XH2 are shown in Table 1. The strain is different from A.
thermoaerophilus DSM 10154 only in the result of the
oxidase test. XH2 can grow vigorously at 60 °C and its
optimal growth temperature is 55 °C, indicating this
strain is a thermophilic bacterium. From all these
results, we determined XH2 to be a thermophilic

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 Thermophilic production of PHA by a novel Aneurinibacillus strain 5

Table 1. Physiological characteristics of strain XH2.

Characteristics Results
Morphological characteristics
Shape Rod
Size (mm) 0.6–1.0  2.0–6.0
Gram staining þ
Features of colonies
Shape Circular
Color Oyster white
Growth temperatures (°C) 8–60
Optimum growth temperature (°C) 55
Gram staining þ
Hydrolysis of gelatin þ
Carbohydrates tests
Ornithine þ
Citric acid –
Lactose –
Glucose þ
Mannitol –
Inositol –
Sorbic acid –
Rhamnose –
Sucrose –
Melibiose –
Amygdalin –
Arabinose –
Enzyme production
Arginine dihydrolase þ
Lysine dihydrolase þ
Oxidase –
Urease –
þ, positive reaction;
, negative reaction.

Aneurinibacillus sp. which has close relationships in both removed from the medium, the yield of PHA would be
phylogeny and physiology with the type strain A. enhanced by 2.4 times (i.e., 267.8 mg l
1 in the
thermoaerophilus DSM 10154. YG medium).

PHA biosynthesis process

To investigate the characteristics of PHA accumulation
process, Aneurinibacillus sp. XH2 was incubated in
shake flasks at 55 °C. As shown in Fig. 4, when the
strain was cultured in YPG medium (yeast extract,
peptone, and glucose), the bacterial growth could be
divided into two phases: the logarithmic phase and
stationary phase. PHA appeared to begin accumulation
in the late logarithmic phase and degradation in the
late stationary phase. Bacterial growth and PHA
accumulation followed the same time courses when
XH2 was cultured in the nitrogen source limited YG
medium (yeast extract plus glucose) with one excep-
tion: the occurrence of the initial lag phase. But there
was one more significant difference between using the Figure 4. Effects of nitrogen source (peptone) on cell growth and
two different culture media: XH2 accumulated more PHA production by Aneurinibacillus sp. XH2. Growth in YPG
medium, &; in YG medium, . PHA yield in YPG medium, &; in
PHA under nitrogen-limited conditions (low cell
YG medium, *. The average standard deviation values of optical
numbers but high PHA contents). In the YPG medium, density and PHA are 0.2 and 3 mg l
1, respectively. Bacterial growth
PHA yield was only 111.6 mg l
1. But if peptone was data are plotted on a logarithmic scale.

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6 Zijun Xiao et al.

Figure 5. GC-MS of the volatile metabolites produced by Aneurinibacillus sp. XH2. Up: total ion chromatogram to show the occurrence of the
two metabolite peaks with arrows. Blank samples (not shown) had same profiles but without the two peaks. Down: high resolution chemical
ionization MS spectra of the two peaks and the proposed ion fragmentation pathways. Numbers beside the chemical structures are their
theoretical values of m/z.

Volatile metabolites of XH2

Bacteria generate a wide range of metabolites. But
usually most of them exist in trace amounts and are
difficult to be determined. GC-MS provides a feasible tool
for the quick identification of volatile metabolites.
Before determination, the fermentation broth was
extracted using dichloromethane and the solvent phase
was concentrated about several thousand times. Blank
samples were also prepared by the extraction of
fermentation broth inoculated with dead cells. As shown
in Fig. 5 (up), there were only two new peaks (those with
arrows) compared with the control. In other words, the
other peaks were unlikely to be metabolites.
As is well known, electron impact ionization is more
often used than chemical ionization in MS analysis of
low molecular volatile organic compounds. However,
beta hydroxy acids are very fragile molecules even in the
chemical ionization mode. The high resolution chemical
ionization MS spectra of the two peaks and the proposed
ion fragmentation pathways are shown in Fig. 5 (down).
The signals of the fragment ions were assigned consult-
ing that of beta hydroxy acids like 3-HB. Although the
detected signals of the larger ions were weak (they
should be weak theoretically), they fitted very well with
their theoretical values of m/z from the proposed
fragmentation pathways of the two compounds, respec-
tively. Therefore, the two compounds were tentatively
identified as 3-hydroxyoctanoate and
3-hydroxy-4-phenylbutanoate.
Discussion
Poly(3HB-co-3HV) was first synthesized by Imperial
Chemical Industries from an Alcaligenes eutrophus strain
using propionic acid and glucose as carbon sources [17].

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 Thermophilic production of PHA by a novel Aneurinibacillus strain 7

Chelatococcus sp. MW 9, MW 10, MW

And then, many kinds of strains were found that they

Aneurinibacillus sp. XH2 (this study)

11, MW 12, MW 13, MW 14 [36]

could produce poly(3HB-co-3HV) using the same carbon

Chelatococcus daeguensis TAD1 [9]

Caldimonas taiwanensis On1 [37]

sources, and some of them were used in industries, for

Thermus thermophiles HB8 [40]

Pseudomonas sp. SG4502 [39]
example, Cupriavidus necator [18] and Ralstonia eutropha
[19, 20]. Commercial poly(3HB-co-3HV) production

Cupriavidus sp. S-6 [38]

suffers from the high cost of expensive substrates for
the strains (e.g., propionate, valerate, and e-caprolac-
tone). On the other hand, the fermentation processes
using these mesophilic strains are exposed to the high
risks of bacterial contamination [9]. To minimize this
situation, culture media must be strictly sterilized at
high temperature and pressure. But long time steriliza-
tion under high temperature causes serious nutrient loss

Aneurinibacillus group
and generates harmful chemicals that may in turn
undermine a normal fermentation process [21].

Chelatococcus
In this paper, the newly isolated strain XH2 can

Pseudomonas
Cupriavidus
produce poly(3HB-co-3HV) at 55 °C using glucose media

Caldimonas

Thermus
without the addition of extra expensive substrates.
When YPG medium was used, the PHA production is
10–15% cell dry weight of XH2. It has been found that
the number of polymer granules in bacterial cells are

Chlorogloeopsis fritschii
related to the level of polymer accumulation [22],

Pseudomonadaceae
Comamonadaceae
especially under nutrient-limited conditions in which

Burkholderiaceae
Paenibacillaceae
Beijerinckiaceae

Synechococcus sp.
a carbon source is readily available [23]. This phenom-

PCC 6912 [41]

Table 2. All the currently known thermophilic PHA-producing bacteria and their taxonomy.

Thermaceae
enon has here also been found in XH2. If peptone was

MA19 [41]
removed from the YPG medium, the yield of PHA would
be enhanced by 2.4 times.
Because gram-positive bacteria lack the lipopolysac-
charide outside the cell wall, PHAs can relatively easily
be extracted and purified [24]. However, only several
Pseudomonadales
Burkholderiales

gram-positive genera that can produce PHAs have been

Chroococcales
Chlorogloeopsis

found. Most of them can be assigned to two main groups,

Rhizobiales

Thermales
Bacillales

Bacillus and Actinobacteria [24–29]. XH2 was identified to

be a species of the genus Aneurinibacillus, and it is gram-
positive. Till now, most gram-negative PHA producers
were human pathogenic or opportunistic pathogenic
bacteria, such as Pseudomonas stutzeri, Pseudomonas
Gammaproteobacteria

Oscillatoriophycideae
Alphaproteobacteria

aeruginosa, Vibrio cholera, Vibrio parahaemolyticus, and

Betaproteobacteria

Delftia acidovorans. The thermophilic Aneurinibacillus sp.

Stigonematales

XH2 was isolated from an oilfield and it is harmless to

Deinococci

human. Thus, utilization of strain XH2 producing PHA

should be very valuable.
Bacilli

Genus Aneurinibacillus was separated from genus

Bacillus in 1996 [30]. Among the six species of the genus,
type strain A. thermoaerophilus DSM 10154 has the closest
Deinococcus-Thermus

relationship in both phylogeny and physiology with XH2

(the 16S rDNA similarity is 99.93%; different only in the
oxidase test). We also noticed that the 16S rDNA
Proteobacteria

Cyanobacteria

similarity of XH2 with the other five type strains in

Firmicutes

genus Aneurinibacillus is very low (94.67–96.46%). Also,

since the optimal growth temperature of this strain is
55 °C, we propose that strain XH2 is a sub-species of A.

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8 Zijun Xiao et al.
thermoaerophilus, a thermophilic PHA accumulation
bacterium. Previous reports have shown that
A. thermoaerophilus can synthesize many kinds of
thermostable enzymes, and we therefore infer that
XH2 has a unique thermostable PHA synthesis system.
Product 3-hydroxyoctanoate was first detected as a
major intracellular inclusion formed by Pseudomonas
oleovorans during growth on octane [31]. In the same year
3-hydroxyoctanoate was found as a minor product in
Bacillus megaterium [32]. Here, 3-hydroxyoctanoate was
detected as a minor product of XH2. Thus, the synthesis
pathway in XH2 may be more like that in B. megaterium
than in P. oleovorans. Up to now there is no further
descriptions of the 3-hydroxyoctanoate synthesis path-
way. We presume that the process may be connected to
the fatty acid metabolism of strain XH2.
Another minor product of XH2 was 3-hydroxy-4-
phenylbutanoate, which also can be found in Pseudomonas
strains when the substrate is phenylbutyrate [33] or
butyrate [34]. The synthesis steps of 3-hydroxy-4-
phenylbutanoate may be as follows. Phenylacetic acid
(PhAc) (as the metabolic intermediates of aromatic amino
acid) forms PhAc-CoA, catalyzed by a phenylacetyl-
CoA ligase [35]. PhAc-CoA then combines with an acetyl-
CoA to give a 4-Phenylbutyryl-CoA. The remaining steps
are the same as what are described by Song et. al. [34], and
finally 3-hydroxy-4-phenylbutanoate is synthesized.
In summary, the study on the PHA accumulation in
thermophilic bacteria has attracted growing interest
because of the numerous advantages of high temper-
ature fermentation. Unfortunately, few PHA accumulat-
ing thermophiles have yet been found [9]. To our
knowledge, Table 2 displays all the thermophilic PHA-
producing bacteria to date and Aneurinibacillus sp. XH2 is
the first thermophilic PHA producer from the Firmicutes
phylum. Although the current PHA yield of XH2 is
relatively low comparing with industrial strains, the
yield can be improved by future fermentation medium
and process control optimizations. The relative genes
and enzymes of this strain may also hopefully find
applications.
Acknowledgments
This research is funded by the National Natural Science
Foundations of China (grant no. 31100059 and
31400005), the Natural Science Foundation of Shandong
Province (grant no. ZR2013CL028), and the Fundamental
Research Funds for the Central Universities of China. We
thank the Royal Society (London) for the Sino–British
Fellowship and Dr. Bob Thomas for proofreading.
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Conflict of interests
The authors declare that they have no conflict of
interest.
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