Medium-chain-length polyhydroxyalkanoate production by newly isolated Pseudomonas sp. TN301 from a wide range of polyaromatic and monoaromatic hydrocarbons

Authors


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:jasmina.nikodinovic@gmail.com; jasmina.nikodinovic@imgge.bg.ac.rs

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 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 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 incubation (30°C, shaking 150 rpm), 100 μl 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 μl). 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 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 μg ml−1; nalidixic acid, 40 μg ml−1; erythromycin, 30 μg ml−1; kanamycin, 100 μg ml−1; rifampicin, 20 μg ml−1; and tetracycline, 30 μg 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 μg ml−1) and Coomassie Brilliant Blue (20 μg 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 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 μl, 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 μl methanol, 250 μl benzene, 200 μl acetone and 200 μl 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).

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 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.

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 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).

Table 1. Aromatic degrading capability of naphthalene-degrading strains isolated from the contaminated river sediments
SubstrateGrowth of the isolatea
TN21TN130TN221TN222TN301TN302TN321
  1. + = growth after 48 h incubation; ++ = growth after 24 h incubation; − = no growth observed.

  2. a

    Growth was assessed on the solid MSM.

Monoaromatic hydrocarbonsBenzene++++++++++
Toluene+++++++++
Ethylbenzene++++++++
p-Xylene++++
o-Xylene++++++
m-Xylene++++++++++
Styrene++++++
Methylstyrene++++++
Methylbenzene++++++++
Butylbenzene++++++++
Biphenyl+++++++++
Polyaromatic hydrocarbonsNaphthalene++++++++++++++
1-Methylnaphthalene++++++
2-Methylnaphthalene++++++
Dimethylnaphthalene++++
Acenaphthene
Fluoranthene
Pyrene
Phenanthrene++++
Chrysene+++

Identification and characterization of PHA-accumulating naphthalene degraders

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 were achieved by isolate TN301, which was between 1·35-fold 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.

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).

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 petrochemically 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.

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).

Table 2. Growth and mcl-PHA accumulation from monoaromatic hydrocarbons by Pseudomons sp. TN301
SubstrateCDW (g l−1)PHA (% CDW)PHA productivity (mg gCDW−1)% monomer composition C6 : C8 : C10 : C12
Benzene0·26 ± 0·057·7 ± 0·476 ± 0·50 : 16 : 65 : 19
Toluene0·27 ± 0·0514·8 ± 1148 ± 33 : 19 : 72 : 6
Ethylbenzene0·24 ± 0·018·3 ± 0·583 ± 0·92 : 16 : 72 : 10
p-Xylene0·24 ± 0·048·3 ± 0·383 ± 0·92 : 15 : 72 : 11
o-Xylene0·26 ± 0·0219·2 ± 2192 ± 52 : 15 : 73 : 10
Styrene0·20 ± 0·053 ± 0·130 ± 0·20 : 9 : 63: 28
Methylbenzene0·26 ± 0·0519·2 ± 2192 ± 62 : 18 : 72 : 8
Butylbenzene0·32 ± 0·0718·8 ± 1188 ± 42 : 18 : 72 : 8
Biphenyl0·27 ± 0·0217·3 ± 1173 ± 32 : 18 : 72 : 8
BTEX0·28 ± 0·0325 ± 3250 ± 60 : 16 : 76 : 8
Xylenes0·38 ± 0·0110·5 ± 0·4105 ± 40 : 16 : 75 : 9
Benzenes0·34 ± 0·0110·5 ± 0·6105 ± 50 : 16 : 70 : 14

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 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

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 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.

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.

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 1-pentanone 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 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 μl) 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).

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
SubstrateCDW (g l−1)PHA (% CDW)PHA productivity (mg gCDW−1)% monomer composition C6 : C8 : C10 : C12
Naphthalene0·48 ± 0·0423 ± 3229 ± 54 : 27 : 60 : 9
Phenanthrene0·31 ± 0·033·5 ± 0·335 ± 30 : 14 : 70 : 16
Chrysene0·26 ± 0·055·1 ± 0·650 ± 40 : 18 : 74 : 5
1-ethylnaphthalene0·29 ± 0·03ND
2-methylnaphthalene0·20 ± 0·03ND
Dimethylnaphthalene0·22 ± 0·04ND
Naphthalene /phenanthrene0·27 ± 0·04ND
Naphthalene/chrysene0·23 ± 0·051·3 ± 0·113 ± 50 : 10 : 72 : 18
Phenanthrene/chrysene0·25 ± 0·034·8 ± 0·348 ± 50 : 14 : 72 : 14
Naphthalene/phenanthrene/chrysene0·27 ± 0·041·2 ± 0·111 ± 20 : 16 : 70 : 14

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).

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 mcl-PHA 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.

Monitoring mcl-PHA accumulation from glucose and naphthalene

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).

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.

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 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 mcl-PHA 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 hydrocarbon 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 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 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 chemo-biotechnological 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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