Characterization of a moderate thermophilic Nocardia species able to grow on polycyclic aromatic hydrocarbons

Authors


Manouchehr Vossoughi, Department of Chemical and Petroleum Engineering, Biochemical and Bioenvironmental Research Center, Sharif University of Technology, PO Box 11365-8639, Tehran, Iran.
E-mail: vosoughi@sharif.ir

Abstract

Aims:  Our goal was the characterization of a new moderate thermophilic polycyclic aromatic hydrocarbon (PAH)-utilizing Nocardia strain.

Methods and Results:  A thermophilic bacterium, strain TSH1, was isolated from a contaminated soil. The macroscopic and microscopic features fit well with the description of Nocardia species. The results of 16S rRNA gene analysis showed 100% match to the type strain of N. otitidiscaviarum DSM 43242T. Strain TSH1 showed the same mycolic acid pattern as the type strain of N. otitidiscaviarum but its fatty acid profile did not permit identification to the species level. The carbon utilization profile of strain TSH1 was different from N. otitidiscaviarum. The results of hydrophobicity measurements showed that PAHs-grown cells were significantly more hydrophobic than LB-grown cells. Furthermore, biosurfactant production was detected during bacterial growth on different culture media.

Conclusions:  Strain TSH1 is capable of growing on a range of PAHs. When grown in PAHs-supplemented media, strain TSH1 showed a high affinity for the organic phase, suggesting that it can develop a hydrophobic surface.

Significance and Impact of the Study:  High cell surface hydrophobicity and capability of strain TSH1 to degrade different PAHs at 50°C may make it an ideal candidate to treat PAH-contaminated desert soils.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed in the environment as natural constituents of fossil fuels and their anthropogenic pyrolysis products. Some PAHs are highly carcinogenic, genotoxic and a threat to public health. Consequently, the US Environmental Protection Agency (EPA) has listed 16 PAHs as priority pollutants for remediation (Liu 2001). During the past decade, a variety of micro-organisms has been isolated and characterized for the ability to degrade different PAHs, and new pathways for PAH degradation have been elucidated (Cerniglia 1992; Bogan et al. 2003; Zhuang et al. 2003).

Biodegradation of PAHs in soils is often limited by the slow mass transfer of these hydrophobic compounds towards degrading microbes. This slow process may lead to bioavailability restrictions. It has been reported that the mass transfer rate of PAHs into the aqueous phase is the rate-limiting step in their degradation (Grimberg et al. 1996). Thus, it is essential to develop methods to desorb PAHs from soils, making them available to micro-organisms. Use of elevated temperatures (Markl et al. 1999), surfactants (Volkering et al. 1998) and biosurfactant-producing micro-organisms (Deziel et al. 1996) are possible options for increasing bioavailability of PAHs.

Degradation of PAHs by mesophilic micro-organisms under aerobic conditions has been intensely investigated (Cerniglia 1992). Little is known about the degradation of PAHs by thermophilic bacteria. Muller et al. (1998) isolated micro-organisms able to convert naphthalene, phenanthrene and anthracene under thermophilic conditions. Also, naphthalene degradation by a thermophilic Bacillus thermoleovorans at 60°C has been shown (Annweiler et al. 2000). Limited bioavailability as a result of the low water solubility of hydrophobic contaminants may be overcome because of higher water solubility at elevated temperatures. Moreover, diffusion rates and oxygen transfer coefficient also rise at the same time, thereby compensating for the reduction in oxygen solubility (Markl et al. 1999).

This work presents the isolation and identification of a new moderate thermophilic Nocardia strain which can degrade naphthalene, phenanthrene, anthracene and pyrene at 50°C. To better understand strain-specific mechanisms to improve PAH bioavailability, we estimated the cell surface hydrophobicity and biosurfactant production of this bacterium grown on different substrates.

Materials and methods

Sampling, enrichment and isolation of phenanthrene-degrading bacteria

Environmental samples were collected in sterilized jars from eight different petroindustrial sites. A 10 ml (water) or 10 g (soil) aliquot of each sample was transferred aseptically to 100 ml mineral salt medium (MSM). The MSM consisted of (g l−1): NH4Cl, 1; Na2HPO4, 0·38; NaH2PO4·H2O, 0·38; MgCl2·6H2O, 0·08; CaCl2, 0·07; KCl, 0·04; 1 mg FeSO4·7H2O and 2·5 ml trace element solution. The trace element solution comprised (mg l−1): MnCl2·4H2O, 27; H3BO3, 31; CoCl2·6H2O, 36; CuCl2·2H2O, 10; NiCl2·6H2O, 20; Na2MoO4·2H2O, 30 and ZnCl2, 50. The MSM was supplemented with 0·05% (w/v) crystalline phenanthrene. The enrichment cultures were incubated aerobically at 50°C on a rotary incubator at 150 rev min−1. Following visible growth, aliquots of the cultures were transferred to fresh MSM, also containing 0·05% (w/v) of phenanthrene. After several transfers, phenanthrene-degrading strains were isolated from these enriched cultures by repeated streaking on solid mineral media until individual colonies were obtained.

Monitoring PAH degradation on solid media

PAH-degrading bacteria were isolated from the enrichment cultures by the Spray-plate technique (Kiohara et al. 1982). Aliquots of freshly grown enriched cultures were transferred to the surface of PAH-coated plates. The plates were incubated at 50°C and checked for clearing zones and increasing biomass around the areas of inoculation.

Identification and phylogenetic characterization of the isolate

An isolate (strain TSH1) able to grow on pyrene, phenanthrene, anthracene and naphthalene as the sole source of carbon and energy at 50°C was obtained and sent to the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) for identification by fatty acid methyl ester (FAME) and mycolic acid analysis.

Growth characteristics

The temperature growth range was determined in LB medium by incubating samples at 25–60°C. The pH dependence of growth was tested in the pH range of 4–9. NaCl requirement was determined by varying the concentration of NaCl from 0% to 5% (w/v) in LB medium. The ranges of pH and NaCl concentration for growth were determined at optimum temperature. Attenuance (D) was monitored at 600 nm using 1-cm cuvetts in an UV-Vis spectrophotometer (Scinco, Seoul, South Korea).

Morphological and phenotypic characterization

Morphological properties were examined by light microscopy (Zeiss, Heidenheim, Germany). Strain TSH1 was stab-inoculated into motility agar in a 15 × 125 mm test tube for motility testing. Catalase activity, Gramstaining and acid-fast staining were performed using standard methods. Strain TSH1 was subjected to carbon utilization/enzyme complement tests by a standard microtitration plate method as described previously (Klatte et al. 1994).

DNA G + C content determination

Genomic DNA for the base composition was prepared following the method of Wilson (1987). The G + C content was determined using the thermal denaturation method (Marmur and Doty 1962).

FAME analysis

FAME analysis was performed by growing the cells at 50°C for 24 h on Middlebrook plates. Methylated cellular fatty acids were prepared and analysed by gas chromatography by following the procedures given for the Sherlock Microbial Identification System, MIS (MIDI Inc., Newark, DE, USA).

Mycolic acid analysis

Mycolic acids were examined as trimethylsilylated derivatives by high temperature gas chromatography with a microbial identification system apparatus equipped with a HT5 column as described previously (Klatte et al. 1994).

Phylogenetic analysis of 16S rRNA gene sequences

A whole cell direct lysis PCR amplification method (Maszenan et al. 1999) using the eubacterial primers 27f and 1512r was used to amplify 16S rRNA gene. The phylogenetic analysis of 16S rRNA gene was performed as previously described (Tay et al. 1998).

Determination of phenanthrene/anthracene degradation potential

The potential of the TSH1 isolate for degradation of phenanthrene and anthracene was examined by HPLC analysis and simultaneously monitoring bacterial growth. Cells were pregrown in MSM supplied with phenanthrene or anthracene. Well grown cultures were used as inocula. PAH-containing medium was prepared by adding an aliquot of acetone-dissolved PAH to sterile flasks, and medium and cells were added after the solvent was evaporated. The final absolute concentration of each PAH compound was 500 mg l−1. Incubation was at 50°C and in dark with agitation at 150 rev min−1. Culture liquors both with heat inactivated microbial cells and without cells were included as controls. Growth of the bacterial strain on each PAH was determined by measuring an increase in protein using the modified Lowry method (Sandermann and Strominger 1972). Remaining PAHs in the cultures were extracted by vigorously shaking twice with an equal volume of hexane. Recovery of PAHs from the medium was determined using a reference, i.e. sterile medium with PAHs and inactivated cells. PAHs in the hexane fractions were separated by reversed-phase HPLC analysis with an acetonitrile : water (75 : 25, v/v) eluent and a 4·6 × 250 mm, 5 μm inertsil ODS-3 column (Merck, Darmstadt, Germany). The flow rate was 1 ml min−1 and PAHs were detected spectrophotometrically at 254 nm.

Cell surface hydrophobicity

Strain TSH1 was grown in both LB broth and MSM supplemented with phenanthrene, anthracene or naphthalene. Relative cell surface hydrophobicity determinations were carried out using a method described by Rodrigues et al. (2005).

Production of biosurfactant

Strain TSH1 was grown aerobically at 50°C for 3 days in LB, YPG (contained per litre; 10 g each of yeast extract, peptone and glucose), DF (contained per litre; 20 g glucose, 4 g NH4NO3, 0·4 g MgSO4·7H2O, 0·2 g KCl, 0·1 g CaCl2·2H2O and 1·4 g Na2HPO4·12H2O) and for 7 days in phenanthrene-supplemented MSM. Biosurfactant production was monitored by an oil displacement test (Morikawa et al. 1993). Triton X-100 and uninoculated media were used as positive and negative controls, respectively. Also, strain TSH1 was streaked onto blood agar plates and incubated for 48 h at 50°C. The plates were visually inspected for zones of clearing around the colonies, indicative of biosurfactant production (Youssef et al. 2004).

Results

Isolation and characterization of PAHs-degrading bacterial strain

One thermophilic phenanthrene-degrading strain, TSH1 was isolated through enrichment cultures from a planted soil contaminated with wastewater of an oil refinery complex. Cells of strain TSH1 were Gram-positive, partially acid-fast and nonmotile rods (2 μm in length and 1 μm in diameter). Rudimentary to extensively branched hyphae and short to long chains of conidia on the hyphae were seen and no endospores were found. Strain TSH1 could grow well on most tested media, while it did not produce any diffusible pigment during incubation. The colour of the colonies was saffron yellow on Tryptic soy broth agar plate and pastel orange on Middlebrook medium. Strain TSH1 was strictly aerobic and catalase-positive. Temperature growth range for strain TSH1 was 25–55°C, with the maximal growth rate at 50°C. Growth occurred at initial pH values between 6 and 9, with the optimum pH around 7·5. The isolate grew at NaCl concentration ranging from 0% to 3% (w/v).

The results of phylogenetic analysis showed 100% match to the type strain of N. otitidiscaviarum DSM 43242T. The DNA G + C content of the isolate was 72%, falling into the range values determined for members of Nocardia sp. (Goodfellow and Lechevalier 1983).

The strain TSH1 showed the typical fatty acid pattern found among members of Corynebacteriaceae, a suborder of Actinomycetales, but its fatty acid profile did not match the MIS database. The major cellular fatty acids were C16:0 (40·51%), C18:1ω9c (20·69%), C18:0 (14·05%) and 10-methyl C18:0 (16·03%). Other fatty acids occurred in trace amounts. The chain lengths of the mycolic acids ranged from 52 to 60 carbon atoms. The isolated strain showed the same mycolic acid profile as the type strain of N. otitidiscaviarum DSM 43242T and N. farcinica but differed from all other Nocardia species.

The utilization of substrates (Table 1) was measured spectrophotometrically by the reduction of MTT [(3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. The strain TSH1 was identified as N. asteroides by this system.

Table 1.   Comparison of physiological characteristics among TSH1 isolate and Nocardia otitidiscaviarum DSM 43242T
Compounds assimilated or cleavedStrain TSH1N. otitidiscaviarum DSM 43242
  1. +, positive reaction; −, negative reaction.

N-Acetyl-d-glucosamine +
Galactose+
Glucarate
Gluconate
d-Glucosaminic acid +
l-Rhamnose +
d-Ribose ++
d-Sucrose
d-Turanose
Caprate
d-Arabitol
i-Inositol+
Citrate
2-Hydroxyvalerate++
2-Oxoglutarate
Pimelate
Succinate++
l-Alanine +
4-Hydroxybenzoate
l-Aspartate
l-Leucine
l-Proline
l-Serine +
l-Valine ++
Putrescine+
Tyramine+
Acetamide+
Benzoate
3-Hydroxybenzoate+
4-Aminobutyrate+
Phenylacetate
Quinate+
p-Nitrophenyl-β-d-xyloside (CXY)++
p-Nitrophenylphosphoryl choline (CCH)++
2-Desoxythymidine-5-p-nitrophosphate (CDP)++

PAH utilization

When strain TSH1 was grown on PAH-coated agar plates, clear zones were only visualized in pyrene- and phenanthrene-coated agar plates. On the anthracene-coated plates and naphthalene plates, increase of biomass around the areas of inoculation was visualized. During growth on PAHs in batch culture, water-soluble intermediates causing a change in colour of medium from clear to yellow were detected.

Kinetics of phenanthrene and anthracene degradation

RP-HPLC measurements of phenanthrene and anthracene concentrations in sterile reference flasks showed that efficiency of extraction is more than 90% and abiotic processes only caused an insignificant decrease. After an equilibration period, phenanthrene and anthracene contents in the TSH1-inoculated media decreased constantly and after 7 days, approx. 90% and 25% of the initial amounts were metabolized, respectively (Fig. 1). PAHs degradation in batch cultures correlated well with the protein content of the cultures.

Figure 1.

 Polycyclic aromatic hydrocarbon degradation (solid lines) and growth (dashed lines) of TSH1 isolate in mineral medium with phenanthrene (bsl00001) or anthracene (bsl00066) as the sole carbon and energy source. The error bars represent the SD calculated on the basis of measurements performed in triplicate cultures.

Cell surface hydrophobicity

The hydrophobicity measurements showed that strain TSH1 is rather hydrophobic and some influences of the substrate on the hydrophobicity of bacterial surfaces were observed. Surfaces of PAH-grown cells were more hydrophobic (relative hydrophobicity of 43–70%) than LB-grown cells (relative hydrophobicity of 22%). This difference could be attributed to a modification of the cell surface of the bacterial isolate.

Biosurfactant production

Based on the results of oil displacement technique, strain TSH1 could produce surface-active compounds when cultivated in batch cultures on four different media (LB, YPG, DF and phenanthrene-MSM), but the result of haemolytic test on blood agar plate was negative for this strain. In addition, our results showed that biosurfactant activity decreased significantly in PAH-supplemented mineral media.

Discussion

The PAH-degrading bacterium isolated in this study was identified as a Nocardia sp. on the basis of its cellular and colony morphology, Gram-staining and acid-fast staining, analysis of 16S rRNA gene, G + C content of DNA, FAME and mycolic acids. Differences of strain TSH1 to N. otitidiscaviarum exist in physiological test results and the fatty acid profile. In these markers, strain TSH1 showed more similarities to N. asteroides than to N. otitidiscaviarum, but based on comparative 16S rRNA gene sequencing and the mycolic acid profile, strain TSH1 could be identified as N. otitidiscaviarum. This strain is deposited in the general collection of DSMZ as N. otitidiscaviarum DSM 45036.

The results showed that Nocardia sp. strain TSH1 extensively metabolize phenanthrene and anthracene at 50°C. PAHs degradation in batch cultures correlated well with the protein content of the cells. The simultaneous increase in the protein contents showed that the PAHs were used as the source of carbon and energy. The cultures utilized phenanthrene and anthracene at very different rates (Fig. 1). Anthracene, although identical to the phenanthrene in the number of aromatic rings, seems much more difficult to degrade which is probably because of its lower solubility in water. Additionally, strain TSH1 was able to use different PAHs ranging in size from naphthalene (two fused rings) to pyrene (four fused rings), therefore it may have potential use in the bioremediation of PAH-contaminated environmental sites with ambient temperatures of 40–50°C.

Recent studies suggest that specific physiological properties of the micro-organisms involved in the degradation of hydrophobic compounds might enhance the availability of these compounds. These mechanisms promoting the transfer of hydrophobic substrate include production of biosurfactants by bacteria (Garcia-Junco et al. 2001) or by plants (Liste and Alexander 2000), uptake systems with high substrate affinity (Volkering et al. 1998) and increased bacterial adherence to the hydrophobic pollutants (Bastiaens et al. 2000). The genera Sphingomonas (Kastner et al. 1994) and especially Nocardia and Mycobacteria (Churchill et al. 1999) seem to be specialized in degrading less bioavailable PAH compounds. They both have a particular outer cell wall layer, i.e. glycosphingolipids for Sphingomonas (Yahuuchi et al. 1990) and glycolipids such as mycolic acids for Nocardia and Mycobacteria (Sayler and Whitt 1994), which may be important for the interaction with or uptake of hydrophobic compounds (Nohynek et al. 1995). Strain TSH1 in similarity with M. austroafricanum GTI-23, M. austroafricanum ATCC 33464 and Mycobacterium sp. PYR-1 (Bogan et al. 2003) is considerably hydrophobic. In addition, strain TSH1 changes its cell surface properties and adhesion tendency, depending on the growth substrate, and attaches to the hydrophobic organic compounds.

Some PAH-degrading bacteria optimize PAHs uptake by producing biosurfactants that help to emulsify the hydrocarbons (Deziel et al. 1996). On the contrary, it has been shown that surfactant can inhibit biodegradation of hydrocarbons by de-adhesion of cells from the liquid/solid interface (Neu 1996; Volkering et al. 1998). Wong et al. (2004) stated that an enhanced degradation by surfactants may occur for hydrophilic PAH-degrading micro-organisms or those that degrade hydrocarbons not via direct uptake from interface. Our finding suggests that strain TSH1 is probably able to secrete biosurfactant(s) to the growth medium that do not have the haemolytic activity. In addition, our results showed that biosurfactant activity decreased significantly in phenanthrene-supplemented mineral media and also cell surface hydrophobicity increased concomitantly. At first glance, it seems that decrease of biosurfactant production in the presence of phenanthrene is an adaptive physiological response to get into physical contact with solid PAHs (Bastiaens et al. 2000; Wick et al. 2002). But at present we cannot determine the contribution of biosurfactant production and cell surface hydrophobicity on the bioavailability of PAHs. Experiments are in progress to characterize the optimal conditions for biosurfactant production on PAHs and to determine the potential role biosurfactants may play in PAH metabolism by the producing micro-organism.

Acknowledgement

We thank Professor (Dr) Kroppenstedt in DSMZ for identification of bacterial isolate.

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