Journal of Applied Microbiology ISSN 1364-5072
ORIGINAL ARTICLE
Medium-chain-length polyhydroxyalkanoate production by
newly isolated Pseudomonas sp. TN301 from a wide range
of polyaromatic and monoaromatic hydrocarbons
T. Narancic1, S.T. Kenny2, L. Djokic1, B. Vasiljevic1, K.E. O’Connor2 and J. Nikodinovic-Runic1
1 Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia
2 School of Biomolecular and Biomedical Sciences, Centre for Synthesis and Chemical Biology, University College Dublin, Dublin, Ireland
Keywords
biopolymers, hazardous waste,
pseudomonads.
Correspondence
Jasmina Nikodinovic-Runic, Institute of
Molecular Genetics and Genetic Engineering,
University of Belgrade, Vojvode Stepe 444a,
PO Box 23, 11010 Belgrade, Serbia.
E-mail:[email protected];
[email protected]
2012/0608: received 3 April 2012, revised 13
May 2012 and accepted 24 May 2012
doi:10.1111/j.1365-2672.2012.05353.x
Abstract
Aims: The aim of this study was to convert numerous polyaromatic and
monoaromatic hydrocarbons into biodegradable polymer medium-chain-length
polyhydroxyalkanoate (mcl-PHA).
Methods and Results: Using naphthalene enrichment cultivation method, we
have isolated seven bacterial strains from the river sediment exposed to
petrochemical industry effluents. In addition to naphthalene, all seven strains
could utilize between 12 and 17 different aromatic substrates, including
toluene, benzene and biphenyl. Only one isolate that was identified as
Pseudomonas sp. TN301 could accumulate mcl-PHA from naphthalene to 23%
of cell dry weight. Owing to poor solubility, a method of supplying highly
hydrophobic polyaromatic hydrocarbons to a culture medium was developed.
The best biomass and mcl-PHA yields were achieved with the addition of
synthetic surfactant Tween 80 (0·5 g l 1). We have shown that Pseudomonas
sp. TN301 can accumulate mcl-PHA from a wide range of polyaromatic and
monoaromatic hydrocarbons, and mixtures thereof, while it could also
accumulate polyphosphates and was tolerant to the presence of heavy metal
(100 mmol l 1 cadmium and 20 mmol l 1 nickel).
Conclusions: A new Pseudomonas strain was isolated and identified with the
ability to accumulate mcl-PHA from a variety of aromatic hydrocarbons.
Significance and Impact of the Study: This study is the first report on the
ability of a bacterial strain to convert a range of polyaromatic hydrocarbon
compounds to the biodegradable polymer (mcl-PHA). Mcl-PHA is gaining
importance as a promising biodegradable thermoelastomer, and therefore,
isolation of new producing strains is highly significant. Furthermore, this strain
has the ability to utilize a range of hydrocarbons, which often occur as
mixtures and could potentially be employed in the recently described efforts to
convert waste materials to PHA.
Introduction
Aromatic hydrocarbons are ubiquitous in the environment and usually raise high environmental and health
concerns because of their persistence and toxicity (EPA
2001; Haritash and Kaushik 2009). However, they are
widely produced and used in large amounts for the
production of polymers, fine chemicals and numerous
© 2012 The Authors
Journal of Applied Microbiology © 2012 The Society for Applied Microbiology
consumer products (solvents, paints, polishes, pharmaceuticals) (APA 2005; C&EN 2005). Monoaromatic
hydrocarbons such as benzene, toluene, ethylbenzene and
xylene (BTEX) are highly volatile and commonly found
in gasoline, while less-volatile polycyclic aromatic hydrocarbons (PAHs; i.e. naphthalene, phenanthrene, anthracene) are natural fossil fuel constituents, which can be
released into the environment during their incomplete
1
PAH to PHA
combustion (Kanaly and Harayama 2000; Andreoni and
Gianfreda 2007; Farhadian et al. 2008). Bacterial degradation of these compounds is well established and extensively studied for applications in bioremediation (Atlas
1981; Andreoni and Gianfreda 2007).
More recently, monoaromatic hydrocarbons such as
styrene and BTEX have been evaluated as feedstock for
the production of mcl-PHA (Ward et al. 2005; Nikodinovic et al. 2008). Mcl-PHA is a biological polyester of
(R)-3-hydroxyalkanoic acids that are 6 to 14 carbon units
long (Steinbuchel and Valentin 1995; Chen 2009). It is a
partially crystalline and thermally stable elastomer with
desirable properties such as biodegradability and biocompatibility that allow for a wide range of applications from
packaging to medical (Valappil et al. 2006; Keshavarz and
Roy 2010). This biopolymer is accumulated by certain
bacterial strains as a response to stress conditions such as
nutrient imbalance or limitation (Anderson and Dowes
1990; Chen 2009).
Strains that degrade monoaromatic compounds and
accumulate mcl-PHA have been employed in chemo-biotechnological process for the conversion of postconsumer
plastic waste such as polystyrene and polyethyleneterephthalate (PET) to biodegradable plastic (Ward
et al. 2006; Kenny et al. 2008). During this process, the
carbon source for the bacterial fermentation and PHA
accumulation is obtained by pyrolisis of waste plastic.
Pyrolysis oils of more complex waste materials contain a
considerable amount of PAHs (Cunliffe and Williams
1998; Kaminsky and Kim 1999). As monoaromatic and
PAHs often occur as mixtures from plastic waste pyrolysis and no strain has been reported to convert PAHs to
PHA, we sought to isolate strains that were capable of
both growth and PHA accumulation from a range of aromatic hydrocarbons. In this study, we also examined
routes to improve bioavailability of polyaromatic substrates for bacterial growth and mcl-PHA production.
Materials and methods
Isolation and growth of aromatic hydrocarbon-degrading
bacterial strains by naphthalene enrichment procedure
The river sediment sample used for the isolation of aromatic hydrocarbon-degrading bacteria was taken from a
site in close proximity to petrochemical industry site.
Surface sediment sampling and elemental analysis of the
sample are described elsewhere (Narancic et al. 2012). A
sediment sample (1 g) was added to an Erlenmeyer flask
containing 50 ml of previously described mineral salts
medium (MSM) (Schlegel et al. 1961), supplemented
with antifungal cycloheximide (75 mg l 1) and a couple
of crystals of naphthalene (1–2 mg). After 5 days
2
T. Narancic et al.
incubation (30°C, shaking 150 rpm), 100 ll of suspension was spread on a MSM agar (1% w/v; Fluka highly
purified agar, obtained from Sigma-Aldrich) plate with a
couple crystals of naphthalene (5 mg) placed on the lid
of the Petri dish as a sole source of carbon and energy.
Plates were sealed and incubated at 30°C for 72 h. The
strains that exhibited good growth on naphthalene over a
24-h period were selected for further analysis.
To test the ability of the isolates to use various aromatic compounds as a sole source of the carbon and
energy, isolates were grown on MSM agar plates. In the
case of liquid substrates, carbon source was supplemented
via vapour phase by adding it in a sterile Eppendorf plastic tip (50 ll). Solid substrates were added as described
for naphthalene. Plates were incubated at 30°C for
5 days. Growth was confirmed by comparison with control plates without substrate or 20 mmol l 1 glucose as
the carbon source.
Screening for PHA accumulation
The ability to accumulate PHA was assessed by growing
the isolates in 250 ml conical flasks containing 50 ml
nitrogen-limited MSM (0·25 g l 1 NH4Cl) and 10 mmol
l 1 glucose or 2 mmol l 1 naphthalene (12 mg added
directly to the medium). After 60 h incubation, cells were
harvested by centrifugation at 5000 g for 20 min in a
benchtop 5810R Eppendorf centrifuge. The pellet was
washed twice with 50 mmol l 1 phosphate buffer (pH
7·4) and freeze dried.
The polymer content was determined by subjecting
approximately 5 mg of lyophilized cells to acidic methanolysis, as previously described (Brandl et al. 1988; Lageveen
et al. 1988). The resultant 3-hydroxyalkanoic acid methyl
esters were assayed by gas chromatography coupled with
mass spectroscopy (GC-MS) analysis using a Hewlett
Packard HP6890 chromatograph equipped with a HP-1
capillary column (30 m by 0·25 mm, 0·25 mm film thickness; J&W Scientific) and a flame-ionization detector
(FID). A temperature programme of 60°C for 3 min,
temperature ramp of 5°C per min and 200°C for 1 min
was used. Total PHA content was determined as a
percentage of cell dry weight (CDW).
Characterization and taxonomic identification of the
isolate TN301
Phenotypic characteristics
Biochemical properties of the strain were determined by
Api20E test panel (bioMérieux® SA, Durham, UK). Salinity tolerance was tested by growing the strain in liquid
MSM with 20 mmol l 1glucose and 1, 3, 5 and 10% (w/
v) NaCl. Temperature tolerance was tested by growing
© 2012 The Authors
Journal of Applied Microbiology © 2012 The Society for Applied Microbiology
T. Narancic et al.
the strain on LB agar plates at 5°C, 30°C, 37°C and 42°
C. Bacterial growth in the presence of antibiotics was
determined by streaking the isolates on LB plates (Sambrook et al. 1989) containing antibiotics ampicillin,
100 lg ml 1; nalidixic acid, 40 lg ml 1; erythromycin,
30 lg ml 1; kanamycin, 100 lg ml 1; rifampicin,
20 lg ml 1; and tetracycline, 30 lg ml 1 and growing
the cultures for 48 h at 30°C. Siderophore production
was assessed using King B medium (Difco, Sparks, MD)
and was illuminated under UV-lamp (254 nm).
Heavy metal tolerance
To examine the potential of isolate to grow in the presence of heavy metals, metal toxicity medium (MTM) was
used containing per litre: sodium lactate (5·1 g), Na2SO4
(2·13 g), CaCl2 anhydrous (0·06 g), NH4Cl (1 g), MgSO4
(1 g), yeast extract (0·05 g), tryptone (0·5 g) and PIPES
(10·93 g) (Sani et al. 2001). Salts used were CdSO4,
NiCl2, HgCl2, CuSO4 and FeCl3, in concentrations corresponding to 20 and 100 mmol l 1 concentrations of the
metal ions.
Polyphosphate accumulation
For the assessment of polyphosphate accumulation, isolates were grown in MSM (50 ml) with glucose
(20 mmol l 1) as a carbon source for 5 days. Total intracellular polyphosphates were isolated by method
described by McGrath and Quinn (McGrath and Quinn
2000), and their concentration was determined according
to Carter and Carl (Carter and Karl 1982).
Biosurfactant production
For the assessment of lipopolysaccharide-based biosurfactant production, two agar plate assays were used as
described previously by Arciola and collaborators for exopolysaccharides (Arciola et al. 2005) and by Siegmund
and Wagner for rhamnolipid detection (Siegmund and
Wagner 1991). Isolate was grown on tryptone agar plates
(tryptone, 10 g l 1; agar, 10 g l 1) supplemented with
Congo Red (40 lg ml 1) and Coomassie Brilliant Blue
(20 lg ml 1) (Arciola et al. 2005) and on cetyltrimethylammonium bromide (CTAB)-methylene blue agar plates
(MSM containing CTAB, 0·2 g l 1; methylene blue,
0·005 g l 1) (Siegmund and Wagner 1991). The plates
were incubated at 30°C for 4-7 days.
16S rDNA sequencing
Isolate TN301 was identified by amplifying and sequencing 16S rRNA gene. The genomic DNA was isolated by a
previously described method (Nikodinovic et al. 2003).
For amplification of 16S rDNA, bacteria-specific primers
27f and 1492r were used (Lane 1991). The PCR product
generated was sequenced using Applied Biosystems 3130
© 2012 The Authors
Journal of Applied Microbiology © 2012 The Society for Applied Microbiology
PAH to PHA
Genetic Analyser (Foster City, CA, USA). Sequences were
analysed and assembled in DNA Star Homologues and
identified by the BLASTN algorithm (Altschul et al. 1997).
The BLASTN program was used to search for similar
sequences in the GenBank database services provided by
the NCBI, and the SEQMATCH tool was used to search for
similar sequences compiled by the Ribosomal Database
Project-II Release 9.4 [RDP; http://rdp.cme.msu.edu;
(Cole et al. 2009)].
The partial 16S rDNA sequence is deposited in the
GenBank under accession number JN800352·1. The strain
Pseudomonas sp. TN301 is deposited at the Institute of
Soil Science, Belgrade, Serbia, under ISS 612.
PHA production from monoaromatic hydrocarbons
The ability of the strain to accumulate PHA from
monoaromatic compounds was assessed by growing the
strains in 250-ml conical flasks containing 50 ml nitrogen-limited MSM and with substrates placed in the central column at a volume of 350 ll, as previously
described (Ward et al. 2005). Bacterial cultures were
incubated on a rotary shaker for 48 h at 30°C and
200 rev min 1. After the incubation period of 2 days,
cells were collected and the polymer was extracted and
analysed as described in section ‘Screening for PHA accumulation’. The BTEX mixture was supplied as previously
described (Nikodinovic et al. 2008).
Supply of polyaromatic hydrocarbons to medium
Different solvents (methanol, benzene, acetone and
1-pentanone) and a surfactant Tween 80 were used to
improve the solubility of polyaromatic hydrocarbons in
the growth medium. Optimization studies were carried
out using naphthalene as a model polyaromatic compound. Solvents were used at the minimal volume in
which 96 mg of naphthalene could be completely dissolved: 1500 ll methanol, 250 ll benzene, 200 ll acetone and 200 ll 1-pentanone. Dissolved naphthalene
was added sequentially at three time points: one quarter of the mixture was added at the time of inoculation, followed by one half added after 24 h of
cultivation, and the final quarter of the mixture was
added after 48 h of cultivation. In the case of Tween
80, 0·5 g l 1 was added directly to the media at the
time of inoculation, while naphthalene crystals were
subsequently added at three time points (inoculation,
24 h and 48 h of cultivation). Appropriate controls of
naphthalene without solvents or surfactants and solvents or surfactants without naphthalene were used.
Cultures were incubated for 60 h at 30°C with shaking
(200 rev min 1).
3
PAH to PHA
T. Narancic et al.
used; and for naphthalene/phenanthrene/chrysene mixture,
5 mmol l 1 naphthalene (32 mg per 50 ml), 3·6 mmol l 1
phenanthrene (32 mg per 50 ml), and 2·8 mmol l 1
chrysene (32 mg per 50 ml) were used.
PHA production from polyaromatic hydrocarbons
Once the best strategy for the supply of polyaromatic
hydrocarbons to the growth media was established, PHA
accumulation from polyaromatic hydrocarbons was studied by growing the strains in nitrogen-limited MSM with
polyaromatic substrates added at a quantity that corresponds to 1·8 g of carbon per litre, using Tween 80 as a
surfactant (0·5 g l 1).
Substrates used for the PHA accumulation assessment
were 15 mmol l 1 naphthalene (96 mg per 50 ml),
11 mmol l 1 phenanthrene (95 mg per 50 ml), 8·3 mmol
l 1 chrysene (95 mg per 50 ml), 12·5 mmol l 1 1-ethylnaphthalene (98 mg per 50 ml), 13·6 mmol l 1 2-methylnaphthalene (97 mg per 50 ml) and 12·5 mmol l 1
dimethylnaphthalene (99 mg per 50 ml). When mixtures
of polyaromatic substrates were used as substrate, the total
amount of C was kept constant (1·8 g l 1) as when the single substrates were supplied. The mass ratio of the mixture
constituents was equal. For naphthalene/phenanthrene
mixture, 7·5 mmol l 1 naphthalene (48 mg per 50 ml)
and 5·5 mmol l 1 phenanthrene (48 mg per 50 ml) were
used; for naphthalene/chrysene mixture, 7·5 mmol l 1
naphthalene (48 mg per 50 ml) and 4·2 mmol l 1 chrysene (48 mg per 50 ml) were used; for phenanthrene/chrysene mixture, 5·5 mmol l 1 phenanthrene (48 mg per
50 ml) and 4·2 mmol l 1 chrysene (8 mg per 50 ml) were
Nitrogen assay
Nitrogen concentration in the growth media for PHA
accumulation was determined by a phenol–hypochlorite
method, as previously described (Scheiner 1976).
Results
Isolation of aromatic hydrocarbon-degrading strains
Using naphthalene as the enrichment substrate, seven
strains with differing colony morphology able to utilize
naphthalene, as a sole source of carbon and energy were
isolated from the sediment exposed to effluents from a
petrochemical industry site. These seven isolates were
tested for the ability to utilize a wide range of other
monoaromatic and polyaromatic substrates, which have
been reported to occur in pyrolysis mixtures (Cunliffe
and Williams 1998; Ciliz et al. 2004; Bhaskar et al. 2007).
While none of the isolates could utilize all 20 aromatic
substrates tested, 17 of the 20 substrates supported
growth of TN301 (Table 1). These seven isolates generally
utilized monoaromatic compounds better in comparison
Table 1 Aromatic degrading capability of naphthalene-degrading strains isolated from the contaminated river sediments
Growth of the isolate*
Substrate
Monoaromatic hydrocarbons
Polyaromatic hydrocarbons
Benzene
Toluene
Ethylbenzene
p-Xylene
o-Xylene
m-Xylene
Styrene
Methylstyrene
Methylbenzene
Butylbenzene
Biphenyl
Naphthalene
1-Methylnaphthalene
2-Methylnaphthalene
Dimethylnaphthalene
Acenaphthene
Fluoranthene
Pyrene
Phenanthrene
Chrysene
TN21
TN130
TN221
TN222
TN301
TN302
TN321
+
++
+
++
+
+
+
+
++
+
+
+
+
++
+
+
+
+
+
+
+
++
+
+
+
+
++
+
+
+
++
+
+
+
++
++
++
+
+
+
++
+
++
+
++
+
+
+
+
+
++
+
+
++
+
+
+
+
++
+
+
++
+
+
++
+
+
+
+
+
+
+
+
+
++
++
+
+
+
+ = growth after 48 h incubation; ++ = growth after 24 h incubation;
*Growth was assessed on the solid MSM.
4
+
+
+
+
+
+
+
+
+
++
+
+
+
+
+
+
++
+
+
+
= no growth observed.
© 2012 The Authors
Journal of Applied Microbiology © 2012 The Society for Applied Microbiology
T. Narancic et al.
with polyaromatic substrates. Of the 11 monoaromatic
substrates tested, 6 were utilized by all isolates including
benzene, toluene, m-xylene, methylbenzene and butylbenzene and biphenyl (Table 1). p-xylene proved to be the
poorest monoaromatic substrate and could be utilized by
the fewest isolates (three of seven). From 9 polyaromatic
hydrocarbons tested, only naphthalene could be utilized
by all isolates, while acenaphthene, fluoranthene and
pyrene could not be used as sole source of carbon and
energy by any of the seven isolates (Table 1).
Identification and characterization of PHA-accumulating
naphthalene degraders
PHA (% CDW)
The seven isolates were screened for PHA accumulation
in shake-flask experiments when naphthalene and glucose
(10 mmol l 1) were supplied as carbon sources (Fig. 1).
For the purpose of PHA screening, naphthalene crystals
(12 mg) were supplied directly to medium. Owing to
naphthalene hydrophobicity and reported toxicity (Pumphrey and Madsen 2007), we only used the amount of
naphthalene that corresponds to 2 mmol l 1 which completely dissolved in the growth medium. When naphthalene was used as carbon source, the best biomass yields
45
40
35
30
25
20
15
10
5
0
0·6
(a)
(b)
CDW (g l–1)
0·5
0·4
0·3
0·2
0·1
0
TN301 TN21 TN321 TN302 TN130 TN221 TN222
Isolate
Figure 1 Polyhydroxyalkanoate accumulation (a) and the growth
(b) of naphthalene-degrading isolates when glucose (■) and naphthalene (□) were supplied as carbon source. (PHA includes PHB and
mcl-PHA).
© 2012 The Authors
Journal of Applied Microbiology © 2012 The Society for Applied Microbiology
PAH to PHA
were achieved by isolate TN301, which was between 1·35fold to 2-fold higher in comparison with all other strains
(Fig. 1b). Of the seven isolates screened, only one
(TN301) could accumulate PHA from naphthalene. PHA
was accumulated as 39% of the cell dry weight (CDW)
(Fig. 1a). GC-MS analysis of the accumulated PHA
revealed that the PHA accumulated by the isolate TN301
contained monomers (R)-3-hydroxyhexanoate (C6),
(R)-3-hydroxyoctanoate (C8), (R)-3-hydroxydecanoate
(C10) and (R)-3-hydroxydodecanoate (C12) in the following percentage ratio C6/C8/C10/C12 of 4:27:60:9.
When glucose was supplied as the carbon source, all
seven isolates grew to similar levels achieving between
0·42 and 0·51 g l 1 of biomass (Fig. 1b), while PHA
accumulation varied considerably from 5% to 40% of
total CDW. PHA from the six other isolates (TN21,
TN321, TN302, TN130, TN221 and TN222) was polyhydroxybutyrate (PHB) containing only (R)-3-hydroxybutyrate (C4) monomer (data not shown). PHB
accumulation was the highest in the TN21 isolate and
was from 1·58-fold to 3·8-fold higher compared with all
other PHB-accumulating strains (Fig. 1a). The monomer
ratio of the mcl-PHA polymer accumulated from glucose
by strain TN301 was C6/C8/C10/C12 = 2:19:72:7. While
the C10 monomer was predominant in mcl-PHA accumulated from both glucose and naphthalene, there was
1·2-fold more C10 monomer present in PHA accumulated from glucose, compared with PHA accumulated by
naphthalene grown cells.
As only the strain TN301 could convert naphthalene to
mcl-PHA under the conditions tested, it was further
characterized by standard biochemical and microbiological tests. The strain was determined to be Gram-negative
aerobic bacterium with the ability to grow at temperatures ranging from 5°C to 37°C. It could utilize glucose,
mannitol, glycerol, trehalose and citrate, but not lactose.
It also grew in LB medium in the presence of 10% NaCl.
It was catalase positive, while it could not hydrolyse urea
or aesculin. It was producing pyoverdine and did not
produce lipopolysaccharide-based biosurfactants as determined by growth and fluorescence on King B medium,
Congo red and CTAB-methylene blue stain (data not
shown).
The isolate TN301 was identified by 16S rDNA
sequencing as Pseudomonas sp. TN301, as 1405 bp of the
16S rDNA sequence had 99% identity with 100% coverage with Pseudomonas plecoglossicida L21 and 99% identity with 99% coverage with Pseudomonas putida F1 and
P. putida GB-1. From these results, we could assign the
newly isolated TN301 to genus Pseudomonas.
Pseudomonas sp. TN301 was also tested for the ability
to grow in the presence of heavy metals such as Cd, Hg,
Ni, Cu and Fe as they often co-occur at the petrochemi5
PAH to PHA
T. Narancic et al.
cally contaminated sites (Pepi et al. 2009). It grew in the
presence of 100 mmol l 1 Cd++ and 20 mmol l 1 Ni++.
It also showed resistance to antibiotics ampicillin, nalidixic acid and erythromycin. Owing to the fact that the
nutrient imbalance is often encountered at contaminated
sites, we also tested the ability of Pseudomonas sp. TN301
to accumulate inorganic polyphosphates (polyPi) in the
shake-flask experiments. When inorganic phosphates
were present in the medium at a concentration of
35 mmol l 1, TN301 accumulated 60 nmol Pi per mg of
protein.
and mixture of benzenes (mixture of benzene, ethylbenzene, 3-methylbenzene and butylbenzene) was similar,
but 2·4-fold lower than the PHA productivity from BTEX
mixture (Table 2).
The predominant monomer of the mcl-PHA from all
monoaromatic substrates tested was the C10 monomer
((R)-3-hydroxydecanoic acid) with the molar percentage
ranging from 63% to 75% (Table 2). The distribution of
other monomers varied from 0% to 3% for C6, 9% to
19% for C8 and 6% to 28% for C12 (Table 2).
mcl-PHA accumulation from polyaromatic hydrocarbons
mcl-PHA accumulation from monoaromatic
hydrocarbons
Medium-chain-length polyhydroxyalkanoate was accumulated by Pseudomonas sp. TN301 when monoaromatic
hydrocarbons and mixtures thereof were supplied to the
culture as a vapour from a central column as previously
described (Ward et al. 2005). While biomass levels were
generally similar, mcl-PHA was accumulated to different
levels ranging from 3% to 25% CDW (Table 2). The best
accumulation of biopolymer from a single monoaromatic
substrate was achieved when o-xylene (19·2%) and
3-methylbenzene (19·2%) were used as carbon sources,
and it was 6·4-fold higher in comparison with PHA accumulation from styrene (3%), which also supported the
lowest levels of growth (0·2 g l 1) (Table 2). Pseudomonas sp. TN301 accumulated the highest level of mclPHA
(25% CDW) when supplied with a BTEX mixture
(Table 2).
The best biomass yields were achieved when a mixture
of xylenes (mixture of o-, m- and p-xylene) was used as
carbon source, and it was 1·2-fold to 1·9-fold higher in
comparison with all other single substrates. The PHA
accumulation productivity from the mixture of xylenes
As polyaromatic hydrocarbons are highly hydrophobic
and insoluble in water, it was of great importance to find
a delivery method for PAHs to the growth media. In
these experiments, naphthalene was used as a model PAH
compound as it was used as the selective pressure in the
enrichment isolation and the initial screen for PHA accumulation. We have analysed growth and mcl-PHA accumulation of Pseudomonas sp. TN301 when different
solvents (methanol, benzene, acetone and 1-pentanone)
were used to dissolve and supply naphthalene to the
medium (Fig. 2). These solvents have previously been
proposed to increase the solubility of aromatic hydrocarbons in organic solvent and water mixtures (Dickhut
et al. 1989). We also included surfactant (Tween 80), as
the application of surfactants has been suggested as a
possible way to increase bioavailability of PAHs (Hickey
et al. 2007). Appropriate controls (a) naphthalene without solvents or surfactants and (b) solvents or surfactants
were used (Fig. 2b). The growth and accumulated PHA
were compared with naphthalene added directly to media
without any solvents or surfactant. Interestingly, while
the similar levels of growth were achieved when
2 mmol l 1 naphthalene was supplied to the medium
Table 2 Growth and mcl-PHA accumulation from monoaromatic hydrocarbons by Pseudomons sp. TN301
Substrate
CDW
(g l 1)
Benzene
Toluene
Ethylbenzene
p-Xylene
o-Xylene
Styrene
Methylbenzene
Butylbenzene
Biphenyl
BTEX
Xylenes
Benzenes
0·26
0·27
0·24
0·24
0·26
0·20
0·26
0·32
0·27
0·28
0·38
0·34
6
±
±
±
±
±
±
±
±
±
±
±
±
0·05
0·05
0·01
0·04
0·02
0·05
0·05
0·07
0·02
0·03
0·01
0·01
PHA
(% CDW)
PHA productivity
(mg gCDW 1)
7·7 ± 0·4
14·8 ± 1
8·3 ± 0·5
8·3 ± 0·3
19·2 ± 2
3 ± 0·1
19·2 ± 2
18·8 ± 1
17·3 ± 1
25 ± 3
10·5 ± 0·4
10·5 ± 0·6
76
148
83
83
192
30
192
188
173
250
105
105
±
±
±
±
±
±
±
±
±
±
±
±
0·5
3
0·9
0·9
5
0·2
6
4
3
6
4
5
% monomer composition
C6 : C8 : C10 : C12
0
3
2
2
2
0
2
2
2
0
0
0
:
:
:
:
:
:
:
:
:
:
:
:
16 : 65 : 19
19 : 72 : 6
16 : 72 : 10
15 : 72 : 11
15 : 73 : 10
9 : 63: 28
18 : 72 : 8
18 : 72 : 8
18 : 72 : 8
16 : 76 : 8
16 : 75 : 9
16 : 70 : 14
© 2012 The Authors
Journal of Applied Microbiology © 2012 The Society for Applied Microbiology
T. Narancic et al.
(a)
0·5
0·1
0·4
0·08
0·3
0·06
0·2
0·04
0·1
0·02
0
0
0·35
(b)
0·06
0·3
CDW (g l–1)
PHA (g l–1)
0·12
0·05
0·25
0·04
0·2
0·03
0·15
0·02
0·1
PHA (g l–1)
CDW (g l–1)
0·6
PAH to PHA
0·01
0·05
0
0
M
S1
S2
S3
S4
Tween
80
Figure 2 Growth (♢) and PHA accumulation (■) of Pseudomonas sp.
TN301 in liquid MSM when different solvents were employed to supply naphthalene. Growth and PHA when (a) naphthalene was supplied with solvents and Tween 80, (b) solvents were supplied as a
sole source of carbon and energy. S1 – methanol, S2 – benzene, S3 –
acetone, S4 – 1-pentanone, M – naphthalene crystals added directly
to the medium.
during the initial PHA screen (Fig. 1) in comparison with
when the total amount of naphthalene supplied was
15 mmol l 1(Fig. 2a), no mcl-PHA was detected in the
culture when 15 mmol l 1 naphthalene was used.
The best growth was observed when Tween 80 was
added to the media together with naphthalene. Achieved
cell dry weight after 60 h when synthetic surfactant
Tween was present in the medium was from 1·1-fold to
2·8-fold higher in comparison with all other methods of
supply of the naphthalene to the medium (Fig. 2a). Pseudomonas sp. TN301 could grow in the mineral medium
when Tween 80 was supplied as a sole source of carbon;
however, the biomass yields were 5·8-fold lower in comparison with when naphthalene was added. No mcl-PHA
was detected in the TN301 culture grown on Tween 80
(Fig. 2b); thus, the improvement of 2·6-fold in biomass
and mcl-PHA yield of 0·1 g l 1 was achieved by adding
synthetic surfactant to the culture.
Dissolving naphthalene in methanol, acetone and 1pentanone resulted in apparent improved biomass yields
of 2·27-fold, 1·38-fold and 1·22-fold, respectively, in comparison when naphthalene crystals were supplied to the
medium directly. However, Pseudomonas sp. TN301
© 2012 The Authors
Journal of Applied Microbiology © 2012 The Society for Applied Microbiology
could grow on methanol as a sole source of carbon and
energy, with the same amount of methanol supporting
2·5-fold lower growth in comparison when naphthalene
was present. Thus, the actual improvement in naphthalene consumption when methanol was used as solvent
was 1·38-fold. Pseudomonas sp. TN301 could also accumulate low levels of mcl-PHA when methanol was supplied as a sole source of carbon and energy to the
medium (Fig. 2b). Poor growth and no mcl-PHA accumulation were supported by acetone and 1-pentanone.
Interestingly, when total amount of benzene (200 ll) was
supplied sequentially directly to the medium (Fig. 2b),
2·5-fold increase in the mcl-PHA accumulation was
observed in the comparison to when benzene was supplied as a vapour (Table 2), while the levels of accumulated biomass were similar. When naphthalene was
dissolved in benzene there was a decrease in both CDW
and PHA accumulation in comparison with when benzene alone was supplied as a vapour (Table 2), while similar growth levels were observed in comparison with
growth when naphthalene crystals were supplied directly
to the medium (Fig. 2). Thus, supplementation of the
medium by synthetic surfactant Tween 80 at inoculation
time was chosen for further experiments.
Based on the improved growth and PHA accumulation
through the addition of Tween 80 with naphthalene to
the growth medium, we added Tween 80 with other
polyaromatic hydrocarbon substrates to the growth medium (Table 3). Pseudomonas sp. TN301 grew in liquid
medium on all substrates tested, the best growth was
achieved when naphthalene was carbon source and it was
from 2·4-fold to 1·5-fold higher in comparison with all
other substrates. Under conditions tested, PHA was not
detected in the cultures when 1-ethylnaphthalene,
2-methylnaphthalene, dimethylnaphthalene and the
mixture of naphthalene and phenanthrene were used as
substrates (Table 3). In the case of 1-ethylnaphthalene,
2-methylnaphthalene, and dimethylnaphthalene growth
yields were between 0·2 and 0·3 g l 1, which is comparable with growth obtained on monoaromatic hydrocarbons (Tables 2 and 3).
Medium-chain-length polyhydroxyalkanoate was accumulated when naphthalene, phenanthrene and chrysene
were used as substrates to 22·9%, 3·5% and 5% of total
CDW, respectively. Although the biomass yield from
naphthalene was 1·5-fold and 2-fold lower in comparison
with phenanthrene and chrysene, mcl-PHA productivity
from these substrates was 6·5-fold and 4·6-fold lower
compared with cells grown on naphthalene (Table 3).
The C10 was predominant monomer in mcl-PHA accumulated from all three polyaromatic substrates, with the
C6 monomer detected in mcl-PHA from naphthalene
only (Table 3).
7
PAH to PHA
T. Narancic et al.
Table 3 Growth and PHA accumulation by Pseudomonas sp. TN301 from polyaromatic hydrocarbons fed with Tween 80 (0·5 g l 1) in the
medium
Naphthalene
Phenanthrene
Chrysene
1-ethylnaphthalene
2-methylnaphthalene
Dimethylnaphthalene
Naphthalene /phenanthrene
Naphthalene/chrysene
Phenanthrene/chrysene
Naphthalene/phenanthrene/chrysene
0·48
0·31
0·26
0·29
0·20
0·22
0·27
0·23
0·25
0·27
±
±
±
±
±
±
±
±
±
±
0·04
0·03
0·05
0·03
0·03
0·04
0·04
0·05
0·03
0·04
PHA
(% CDW)
PHA productivity
(mg gCDW 1)
% monomer composition
C6 : C8 : C10 : C12
23 ± 3
3·5 ± 0·3
5·1 ± 0·6
ND
ND
ND
ND
1·3 ± 0·1
4·8 ± 0·3
1·2 ± 0·1
229 ± 5
35 ± 3
50 ± 4
4 : 27 : 60 : 9
0 : 14 : 70 : 16
0 : 18 : 74 : 5
13 ± 5
48 ± 5
11 ± 2
0 : 10 : 72 : 18
0 : 14 : 72 : 14
0 : 16 : 70 : 14
When the mixtures of polyaromatic substrates were
used as carbon sources, biomass yields were 1·8-fold to
2·1-fold lower than that achieved with naphthalene supplied as a sole carbon and energy source. PHA accumulation was not observed when naphthalene/phenanthrene
mixture was supplied. A 4·8-fold to 20·8- fold lower mclPHA productivity was observed for other substrate combinations compared with when naphthalene was alone
(Table 3). Monomer composition of the PHA obtained
from the mixtures of polyaromatic substrates was similar
and comparable with the monomer composition obtained
when single monoaromatic or polyaromatic substrates
were used as a carbon source.
(a)
0·7
25
0·6
20
0·5
15
0·4
0·3
10
PHA (% CDW)
CDW
(g l 1)
CDW (g l–1)
Nitrogen (g l–1)
Substrate
0·2
5
0·1
8
0·8
CDW (g l–1)
Nitrogen (g l–1)
To further assess mcl-PHA accumulation characteristics
of Pseudomonas sp. TN301 from glucose and naphthalene
respectively, biomass yields, PHA accumulation and
nitrogen depletion of the cultures in the shake flasks were
monitored over a period of 60 h (Fig. 3). There was no
apparent lag period when either of the substrates was
used. Glucose grown cultures achieved a 1·4-fold higher
biomass in comparison with the culture grown on
naphthalene added with Tween 80 (Fig. 3).
During exponential growth on naphthalene, the growth
rate of 0·0065 g l 1 h 1 was achieved, which was 2-fold
lower than glucose grown cells. Low levels of mcl-PHA
(3% CDW corresponding to 8·1 mg l 1) were detected
after 12 h of incubation, which coincided with the depletion of nitrogen in the growth medium (Fig. 3a). Linear
accumulation of the polymer occurred between 6 h to
36 h of incubation and PHA accumulation was maximal
at 20% of cell dry weight over the growth cycle of 60 h
(Fig. 3a). For glucose grown cells, mcl-PHA accumulation
0
0
40
(b)
0·7
35
0·6
30
0·5
25
0·4
20
0·3
15
0·2
10
0·1
5
0
0
0
20
40
PHA (% CDW)
Monitoring mcl-PHA accumulation from glucose and
naphthalene
60
Time (h)
Figure 3 Growth (–■–) and PHA accumulation (–▲–) by Pseudomonas sp. TN301: (a) on 15 mmol l 1 naphthalene added with Tween
80 (0·5 g l 1) and (b) on 20 mmol l 1 glucose. Nitrogen concentration (–♦–) was also monitored over a 60-h period.
© 2012 The Authors
Journal of Applied Microbiology © 2012 The Society for Applied Microbiology
T. Narancic et al.
onset coincided with nitrogen depletion and increased
linearly during a period from 12 h to 48 h of incubation,
with the final mcl-PHA content of the cells reaching 35%
of CDW (Fig. 3b). Given the higher biomass and mclPHA accumulation in glucose grown cells, the overall
mcl-PHA productivity (0·004 g l 1 h 1) was 1·3-fold
lower in naphthalene grown cells.
Discussion
The objective of this study was to convert polyaromatic
hydrocarbons to mcl-PHA by isolating bacteria from the
river sediment polluted with petrochemical by-products.
Through a naphthalene enrichment strategy, seven strains
were successfully isolated that could grow well on naphthalene in a 24-h period, with only one of them able to
convert this substrate to mcl-PHA (Fig. 1). Enrichment
strategy has often been used to isolate bacteria with specific characteristics, especially in the case of the isolation
of recalcitrant PAHs degraders (Daane et al. 2001;
Hilyard et al. 2008; Long et al. 2009). The naphthalene
was used as selective pressure in this study, as it is the
simplest and the most soluble among the polyaromatic
compounds and is frequently present in the contaminated
environments (Peters et al. 1999). While many bacteria
(and in particular Pseudomonas strains) can consume a
large number of aromatic compounds, they cannot always
accumulate PHA (Lee et al. 1995; Hoffmann et al. 2000).
This is a possible reflection on the metabolic routes
employed in the breakdown of these substrates and also
the substrate range and efficiency of the PHA polymerase
(Garcia et al. 1999; Kessler and Witholt 2001). Indeed,
the other six naphthalene-degrading isolates could accumulate PHA from glucose, while no PHA was detected in
the cultures grown on naphthalene (Fig. 1).
As polluted sites are usually sites where complex
monoaromatic and polyaromatic contaminants can be
found (Andreoni and Gianfreda 2007), the aromatic degradation capability of the seven naphthalene-degrading
strains was also assessed (Table 1). Indeed, all seven isolates were able to utilize more than 12 different monoaromatic and polyaromatic substrates with TN301 being
the most versatile in using 17 of 20 different aromatic
compounds tested. This was quite comparable with our
recent studies where Gram-positive strains capable of
degrading a wide range of monoaromatic hydrocarbons
were isolated without prior enrichment (Djokic et al.
2011).
After successfully establishing that the isolate TN301
can accumulate mcl-PHA from naphthalene and glucose
(Fig. 1), the strain was phenotypically and taxonomically
identified as a species of the Pseudomonas genus. The
most commonly isolated genus with aromatic hydrocar© 2012 The Authors
Journal of Applied Microbiology © 2012 The Society for Applied Microbiology
PAH to PHA
bon degradative capabilities is Pseudomonas (Zylstra and
Gibson 1989; Ramos et al. 1995; Bastiaens et al. 2000;
Popp et al. 2006; Kenny et al. 2008). Although the highest homology of 16S rDNA was with P. plecoglossicida
L21, the nonfluorescent pseudomonad isolated from fish
(Izumi et al. 2007), phenotypically TN301 was more similar to P. putida F1 and P. putida GB-1, and it also shared
high 16S rDNA sequence homology with these strains
(99% identity with 99% coverage). Pseudomonas putida
F1 was isolated by enrichment with ethylbenzene from a
polluted creek and has the ability to use broad range of
monoaromatic hydrocarbons, but has no PAH dioxygenases (Zylstra and Gibson 1989), while P. putida GB-1 was
isolated from fresh water and is known as a robust manganese oxidizer (Wu et al. 2011). Besides the ability to
degrade wide range of monoaromatic and polyaromatic
substrates and accumulate mcl-PHA, Pseudomonas sp.
TN301 showed tolerance to high concentration of Cd++
and Ni++ ions and accumulates inorganic polyphosphates.
Another P. putida strain W619 exhibited high tolerance
to heavy metals (Wu et al. 2011). Another study reported
that 6 of 10 aromatic-degrading Pseudomonas strains were
resistant to mercury (Barbieri et al. 1996). High incidence
of metal resistance in aromatic-degrading bacteria has
been frequently encountered (Margesin and Schinner
2001; Pepi et al. 2007).
The possibility to convert monoaromatic compounds
(BTEX, styrene) into mcl-PHA has been previously
reported by P. putida strains (Tobin and O’Connor 2005;
Ward et al. 2005; Nikodinovic et al. 2008). Thus, we
tested newly isolated and characterized Pseudomonas sp.
TN301 for the ability to produce mcl-PHA from a range
of monoaromatic substrates (Table 2). The productivity
of mcl-PHA in TN301 per g of CDW was 2-fold, 1·5-fold
and 1·7-fold lower from benzene, toluene and ethylbenzene, respectively, in comparison with that obtained by P.
putida F1 from the same substrates (Nikodinovic et al.
2008). However, mcl-PHA productivity from BTEX mixture with TN301 isolate was the same (250 mg g 1
CDW) as the productivity obtained by defined mixed
culture of P. putida F1, P. putida mt-2 and P. putida
CA-3 (Nikodinovic et al. 2008). Being able to degrade
wider range of monoaromatic hydrocarbons and convert
them to mcl-PHA as a single culture gives an advantage
to Pseudomonas sp. TN301 in potential biotechnological
applications.
During metabolomic analysis of Sinorhizobium sp. C4
grown on phenanthrene, Keum and co-workers have
detected PHB accumulation (Keum et al. 2008). However, no microbial strain to date has been reported to
convert polyaromatic hydrocarbons into mcl-PHA. The
productivity of mcl-PHA accumulation from phenanthrene in TN301 was 2·3-fold higher than the PHB
9
PAH to PHA
productivity from the same substrate by Sinorhizobium
sp. C4 (Keum et al. 2008).
From the initial results that PHA could be accumulated from glucose by all 7 isolates and from naphthalene
by a single isolate only (Fig. 1), poor bioavailability of
the naphthalene was suspected. Poor bioavailability is the
common issue encountered for monoaromatic and polyaromatic hydrocarbons when used as substrates for bacterial growth (Atlas and Cerniglia 1995; Van Hamme et al.
2003). Initially, we have also encountered the problem of
solubilization of the polyaromatic hydrocarbons and the
delivery to the fermentation medium. Therefore, we carried out an optimization study employing different solvents such as methanol, acetone, 1-pentanone, all
previously shown to increase PAHs solubility in water
(Dickhut et al. 1989). The application of surfactants has
been widely studied in terms of bioavailability improvement. While the results of these studies vary from biodegradation improvement (Sobisch et al. 2000; Hickey
et al. 2007), having no effect (Laha and Luthy 1992) to
biodegradation inhibition (Bramwell and Laha 2000;
Doong and Lei 2003), we decided to test synthetic surfactant Tween 80 for this purpose. In the case of TN301 cultures grown on PAHs, the addition of Tween 80
supported the best improvement both in biomass and
PHA accumulation yields in comparison with all other
solvents used (Fig. 3). To avoid initial toxicity effect,
PAHs were added to the TN301 cultures at three time
points during growth. Using this delivery strategy for the
first time, the defined mixtures of PAHs were also successfully converted to mcl-PHA by TN301 (Table 3).
Mcl-PHA accumulation ability of Pseudomonas sp.
TN301 is carbon source dependant (Tables 2 and 3 and
Fig. 3). Although both monoaromatic and polyaromatic
substrates supported biomass accumulation to the comparable levels, polyaromatic substrates were poorer substrates for the mcl-PHA accumulation (Tables 2 and 3).
This may be due to the different bioavailability achieved
by different mode of delivery of these substrates to the
medium (monoaromatic hydrocarbons were added continually as vapour, while polyaromatic hydrocarbons were
dissolved directly in the medium), and different efficiency
in utilization of these substrates by the TN301 isolate.
When comparing mcl-PHA production ability from
naphthalene and glucose by Pseudomonas sp. TN301 and
from the same amount of carbon added to the medium
(1·8 g l 1), twofold decreased levels of both biomass and
mcl-PHA on naphthalene were observed (Fig. 3). PHA is
usually accumulated as a response to inorganic nutrient
limitation (Hoffmann and Rehm 2005), coupled with the
toxicity of the substrate such as naphthalene (Pumphrey
and Madsen 2007), the growth and biopolymer retardation is not surprising. Indeed, this toxicity effect of
10
T. Narancic et al.
aromatic substrates under nutrient limiting conditions
was previously observed in P. putida G7 grown on naphthalene and P. putida CA-3 grown on styrene under
nitrogen limiting conditions (Ahn et al. 1998; Nikodinovic-Runic et al. 2009). The presence of additional reduced
rings in phenanthrene and chrysene caused much
decreased levels of mcl-PHA accumulation, while the
presence of the additional substituents in methylnaphthalenes and dimethylnaphthalene abolished the mcl-PHA
accumulation (Table 3).
In conclusion, we have for the first time demonstrated
the conversion of polycyclic aromatic substrates such as
naphthalene, phenanthrene and chrysene and their
defined mixtures to mcl-PHA by newly isolated Pseudomonas sp. TN301. The ability to degrade a wide range of
polyaromatic and monoaromatic substrates will make this
robust bacterium a potent candidate for the chemobiotechnological conversion of petrochemical waste to
valuable thermoplastic.
Acknowledgements
This work was supported by the ‘Microbial diversity
study and characterization of beneficial environmental
microorganisms’ project (Grant number: 173048,
MSTD, 2011-2014). Part of this study was supported
by FEMS Research Fellowship grant received by Tanja
Narancic.
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