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Biodegradative potential and characterization of psychrotolerant polychlorinated biphenyl-degrading marine bacteria isolated from a coastal station in the Terra Nova Bay (Ross Sea, Antarctica)
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Marine Pollution Bulletin 54 (2007) 1754–1761 www.elsevier.com/locate/marpolbul
Biodegradative potential and characterization of psychrotolerant polychlorinated biphenyl-degrading marine bacteria isolated from a coastal station in the Terra Nova Bay (Ross Sea, Antarctica) Michaud Luigi a
a,*
, Di Marco Gaetano b, Bruni Vivia a, Lo Giudice Angelina
a
Dipartimento di Biologia Animale ed Ecologia Marina (DBAEM), Universita` di Messina, Salita Sperone 31, 98166 Messina, Italy b Istituto per i Processi Chimico-Fisici (IPCF-CNR), via La Farina 237, 98123 Messina, Italy
Abstract Antarctic marine bacteria were screened for their ability to degrade polychlorinated biphenyls (PCB) as the sole carbon and energy source at both 4 C and 15 C. PCB-degrading isolates (7.1%) were identified by sequencing their 16S rDNA as Pseudoalteromonas, Psychrobacter and Arthrobacter members. One representative isolate per genera was selected for evaluating the biodegradative potential under laboratory scale and phenotypically characterized. Removal of individual PCB congeners was between 35.6% and 79.8% at 4 C and between 0.4% and 82.8% at 15 C. Differences in the removal patterns of PCB congeners were observed in relation to the phylogenetic affiliation: Arthrobacter isolate showed similar biodegradation efficiencies when growing at 4 C and 15 C, while Pseudoalteromonas better degraded PCBs at 15 C. No biodegradation was detected for Psychrobacter isolate at 4 C. Results obtained highlight the occurrence of PCB-degrading bacteria in Antarctic seawater and suggest the potential exploitation of autochthonous bacteria for PCB bioremediation in cold marine environments. 2007 Elsevier Ltd. All rights reserved. Keywords: PCB-degradation; Antarctic marine bacteria; Psychrotolerant bacteria; Bacterial characterization
1. Introduction Polychlorinated biphenyls (PCBs) are long-term persistent compounds produced as mixtures during the midtwentieth century and sold for industrial applications under trade names such as Aroclor, Clophen, Fenclor or Kanechlor. The family of PCBs contains 209 congeners which consist of a biphenyl molecule substituted with one to ten chlorine substituents. PCB mixtures are nonflammable, exhibit a high degree of chemical and thermal stability (up to 350 C), and have desirable dielectric properties (Master and Mohn, 1998; Vaillancourt et al., 2003). Although their initial application was as dielectric fluid in
*
Corresponding author. Tel.: +39 090 6765533; fax: +39 090 393409. E-mail address: [email protected] (L. Michaud).
0025-326X/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2007.07.011
electrical transformers and capacitors, PCBs were also utilized in heat transfer and hydraulic systems as well as in inks, lubricants, paints, adhesives and dust control agents. Despite the fact that these chemical compounds have been regulated or prohibited since the 1970s in western nations, their widespread use and chemical stability has led to extensive environmental contamination through accidental releases and inappropriate disposal techniques. In spite of the precautionary measures taken to avoid pollution, PCBs have been detected in Antarctic seawater and sediments (Fuoco et al., 1996, 1999; Montone et al., 2001b) as well as in various organisms from different trophic levels (Reinhardt and van Vleet, 1986; Focardi et al., 1992, 1995; Court et al., 1997; Montone et al., 2001a; Corsolini et al., 2003; Weber and Goerke, 2003). Dispersion of PCBs occurs mainly via mass flow in the atmosphere (Negoita et al., 2003; Gambaro et al., 2005;
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Montone et al., 2005), but increasing human activities have probably contributed to a localized contamination at scientific Antarctic bases (Negri et al., 2006). As PCB concentrations in Antarctic seawater is generally several order of magnitude lower than in organisms, it could be concluded that these contaminants, due to their hydrophobic nature, are subjected to bioaccumulation in organism tissues and biomagnification in the Antarctic marine food webs (Tanabe et al., 1983; Focardi et al., 1995; Corsolini et al., 2002, 2006). Focardi et al. (1995), evaluating toxic potential of PCBs in Antarctic fish, seabirds and seals, found a clear relationship between PCB concentrations and trophic level (fish < Ade´lie penguin < Weddel seal). PCBs can cause several damages to organisms; Lenihan (1992) reported about the negative influence of PCBs on behaviour and survival of Antarctic amphipods and urchins, while a study by Evans et al. (2000) revealed that pathological and molecular abnormalities of Antarctic fish was probably linked to the effect of these persistent pollutants. The classical methods suggested for the remediation of PCB-contaminated environments are generally expensive and often inefficient; moreover, they may be source of secondary pollution (Yang et al., 2004). On the other hand, PCBs can be transformed, despite their recalcitrant nature, into chemical substances by different microbial metabolic pathways, both aerobic and anaerobic, facilitating further metabolization. As a result, the developments of bioremediation processes have been proposed as a promising alternative to the currently adopted techniques; to this regard, studying the ecology and physiology of bacteria able to degrade pollutants is essential in order to asses the effectiveness and risks involved in bioremediation processes. Aerobic degradation of PCBs has been mainly reported for mesophylic microorganisms inhabiting soils and sediments where these xenobiotic compounds generally accumulate, while psychrotolerant PCB-degraders have been rarely isolated from cold environments (Mohn et al., 1997; Master and Mohn, 1998; Lambo and Patel, 2006). Nevertheless, although the occurrence of PCB in both biotic and abiotic Antarctic compartments has been reported, very little is known about the degradative activity on these recalcitrant pollutants of bacteria from Antarctica (De Domenico et al., 2004). The aim of this investigation was to (1) screen Antarctic marine psychrotolerant bacteria for PCB-degradation, (2) determine if a relation occurred between their phylogenetic affiliation and biodegradative potential and, finally, (3) characterize PCB-degraders by a combination of phenotypic and genotypic features. 2. Materials and methods 2.1. Bacterial strains The one-hundred and twenty-six psychrotrophic bacterial strains used in this study were previously isolated from
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Antarctic seawater samples (Terra Nova Bay, Ross Sea) collected along the water column (0-150 m) at the fixed station Tiburtina (TIB; coordinates: 7442 0 300 S– 16410 0 0.500 E). Their identity is given in Bruni et al. (1999). All the isolates belong to the Italian Collection of Antarctic Bacteria (CIBAN) of the National Antarctic Museum (MNA) kept at the University of Messina. They are maintained on Marine Agar (MA; Difco) slopes at 4 C and routinely streaked on agar plates from tubes every six months to control purity and viability. Antarctic strains are also preserved by freezing cell suspensions at 80 C in Marine Broth (MB; Difco) to which 20% (v/v) glycerol is added. 2.2. Screening for aerobic degradation of PCB Aerobic PCB degradation was screened into the mineral liquid medium Bushnell Haas (BH; Difco) supplemented with Aroclor 1242 solution (100 ppm in dichloromethane) as sole carbon and energy source (final concentration 0.1%, v/v). Aroclor 1242 is a mixture of PCB congeners (ranging from dichloro- to hexachlorobiphenyls) made of twelve carbon atoms in the biphenyl molecule and containing 42% chlorine by weight (Frame et al., 1996). Before adding the bacterial inoculum, 10 ll of Aroclor solution were transferred to 100 ml flasks containing 9.5 ml of BH and left in a cabinet until CH2Cl2 had completely evaporated. The medium was inoculated with 0.5 ml of a freshly prepared bacterial suspension (5%, v/v) in 3% (w/v) NaCl supplemented distilled water. The optical density (OD) of the suspension was adjusted to 1.0 at 600 nm. Cultures were incubated at 4 C and 15 C for 3 weeks on a rotary shaker operated at 100 rpm. The ability to use PCBs as growth substrates was evaluated according to the degree of turbidity or the appearance of cellular flocs in the test tubes. 2.3. PCR amplification and analysis of 16S rDNA PCR amplification, sequencing and phylogenetic analysis of 16S rDNA from active bacterial isolates were carried out as previously described by Michaud et al. (2004a). The first half (about 700 nucleotides) of each amplification product was sequenced using the primer 27f. Each sequence was then used as a query in a BLASTn search (Altschul et al., 1997) and further aligned using the program Clustal W (Thompson et al., 1994) to the most similar orthologous sequences retrieved from the database. Each alignment was checked manually, corrected and then analyzed using the Neighbour-Joining method (Saitou and Nei, 1987) according to the model of Jukes-Cantor distances. Phylogenetic trees were constructed using the MEGA 3 (Molecular Evolutionary Genetics Analysis) software (Kumar et al., 1993). The robustness of the inferred trees was evaluated by 500 bootstrap re-samplings. The 16S rRNA gene sequences were submitted to GenBank
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and assigned to the following accession numbers: EF577381-EF577388 and DQ489314. 2.4. Morphological, biochemical and physiological characterization Isolates were characterized by means of a combination of phenotypic tests as previously described in Bruni et al. (1999) and Lo Giudice et al. (2006). All media used in this study were sterilized by autoclaving at 121 C for 15 min and the plates were incubated at 15 C for 14 days. The analyses were performed at least twice. 2.5. Biodegradation assay and gas-chromatographic analysis Isolates were grown in 50 ml of Aroclor 1242 supplemented BH, as described earlier, and incubated at 4 and 15 C for 3 weeks. Uninoculated controls were incubated in parallel to monitor abiotic losses of the substrates. Subsamples were removed at regular intervals to assess bacterial growth by colony-forming units (CFU/ml) spread plating on MA. Serial dilutions were made in sterile saline solution. Plates were incubated at either 4 C or 15 C. Viable counts were carried out in duplicate. After incubation, biodegradation activity was stopped by acidifying cultures with HCl 10 M to achieve pH 2. Before PCB extraction, 60 ll of octachloronaphthalene (OCN) solution (40 ppm in CH2Cl2) was added to each flask to monitor substrate losses during the extraction procedure. Cultures were extracted three times with 5 ml of CH2Cl2 in a separatory funnels to remove the cellular material. Funnels were vigorously shaken for 3 min during each extraction. The three organic phases were pooled and the solvent evaporated to dryness. The composition of PCBs and their concentration were determined by high resolution gas chromatography-mass spectrometry (GC-MS) using a Perkin-Elmer TurboMS AutoSystem XL GC, equipped with a DB-TPH capillary column (30 m with a 0.322 mm i.d. and 0.25 lm film thickness, J & W Scientific). Helium (5 bar) was used as carrier gas (1.8 ml min1). The injector was kept at
250 C; the column oven was initially kept at a temperature of 50 C for 4 min, and then heated up to 300 C at a rate of 5 C min1. One microlitre portion of each sample, containing 79.3 ng of heptamethyl nonane as internal standard (to account for injection errors and other variable encountered during the chromatographic analysis), was analyzed by means of the splitless injection technique (50 ml min1). Finally, the percentage reduction of Aroclor was determined according to the following equation: %R ¼ 100 ½ðH PCB sample =H PC Bcontrol Þ 100 where HPCB sample and HPCB control are the height of a PCB peak in the sample and the control, respectively. 3. Results 3.1. Screening for PCB biodegradation The biodegradation of Aroclor 1242 was tested at 4 and 15 C by batch experiments. Nine of the 126 psychrotolerant isolates screened (7.1%) were able to utilize PCBs as a sole carbon and energy source in at least one of the temperatures adopted during the preliminary screening. PCBdegraders were isolated from different sampling depths: 0 m (strains 3 and 15), 25 m (strains 5, 6 and 19), 50 m (strain 16) and, finally, 100 m (strains 9, 24 and 74). 3.2. Phylogenetic analysis The phylogenetic analysis revealed that the PCBdegrading strains were representative of three bacterial genera and belonged to two bacterial phylogenetic branches: the c-subdivisions of Proteobacteria and the gram-positive branch (high G+C content Actinobacteria). Within the c-Proteobacteria subdivision, the isolates were found to be related to Pseudoalteromonas and Psychrobacter genera. Finally, only one isolate was placed within the genus Arthrobacter (Table 1). The phylogenetic trees derived from the 16S rDNA sequence analysis of the nine strains are visualized in Fig. 1.
Table 1 16S rDNA gene sequence affiliation of PCB-degrading Antarctic isolates to their closest phylogenetic neighbours Phylum or classa
Strain
Accession number
Next relative by GenBank alignment (ANb, organism)
Hom (%)c
Family
GAM
3 5 19 24 6 9 15 16 74
EF577381 EF577382 DQ489314 EF577386 EF577383 EF577384 EF577387 EF577385 EF577388
AM110978, Pseudoalteromonas sp. 3023 AM110978, Pseudoalteromonas sp. 3023 AY243366, Pseudoalteromonas sp. CAM36 DQ677306, Pseudoalteromonas sp. G16 AY656801, Psychrobacter sp. 63 AB094794, Psychrobacter okhotskensis AB094794, Psychrobacter okhotskensis AY444823, Psychrobacter sp. 215-51 AF134184, Arthrobacter agilis
99 99 97 99 100 98 98 99 98
Pseudoalteromonadaceae Pseudoalteromonadaceae Pseudoalteromonadaceae Pseudoalteromonadaceae Moraxellaceae Moraxellaceae Moraxellaceae Moraxellaceae Micrococcaceae
ACT a b c
GAM: c-Proteobacteria; ACT: Actinobacteria. AN: Accession number. Hom: sequence homology.
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74 63 83
AJ309942.1 Psychrobacter immobilis AY639871.1 Psychrobacter cibarius AM269905.1 Psychrobacter sp. 23 AJ430827.1 Psychrobacter fozii AJ310992.1 Psychrobacter sp. TAD1 16 63 AJ609555.1 Psychrobacter urativorans AB094794.1 Psychrobacter okhotskensis PCB 6 35 AY771717.1 Psychrobacter fozii 45 PCB 15 20 PCB 9 AJ312213.1 Psychrobacter glacincola 99 PCB 16 AJ430828.1 Psychrobacter luti AJ609556.1 Psychrobacter frigidicola 92 PCB 5 AF529062.1 Pseudoalteromonas citrea DQ787199.1 P. arctica 99 AF038846.1 Pseudoalteromonas gracili PCB 19 60 PCB 3 DQ537517.1 P. haloplanktis 42 PCB 24 AJ417594.1 P. agarovorans AY573036.1 Pseudoalteromonas sp. 58 AF235090.1 Arthrobacter methylotrophus AJ316140.1 Arthrobacter gandensis U85895.1 Arthrobacter sp. IC044 99 48 AF134184.1 Arthrobacter agilis 91 PCB 74 AJ639829.1 Arthrobacter tecti 75 96 AJ639830.1 Arthrobacter parietes 68
0. 02
Fig. 1. Phylogenetic relationship based on 16S rDNA sequences of PCB-degrading Antarctic isolates.
Based on the results of the preliminary screening for PCB-degradation, strains 15 (Psychrobacter sp.), 19 (Pseudoalteromonas sp.) and 74 (Arthrobacter sp.), representative of three distinct genera, were chosen for further analyses (i.e. phenotypic characterization and determination of their biodegradation efficiency, see below), as they grew better on Aroclor supplemented medium. 3.3. Physiological and biochemical characterization Phenotypic characters of selected Antarctic isolates are reported in Table 2. A single polar flagellum was observed in the Pseudoalteromonas isolate, which appeared motile under microscopy. The presence of endospores was never observed. Isolates were able to hydrolyse at least two of the macromolecules provided. Although they were capable of growing on chitin overlay plates, none of them induced degradation of this macromolecule. All strains were lipolytic as they hydrolysed tween 80. Only Gram negative isolates were oxidase positive. Enzyme arrangements were defined by the combination of the results obtained from API 20E and API 20NE systems. None of the strains were ornithine and lysine decarboxylase or arginine dihydrolase positive. Indole production from tryptophane and H2S formation were never observed. All the marine isolates investigated were psychrotolerant when grown into MB medium. All strains grew well in the range at 4–30 C. The pH range for growth was from 5 to 9. Growth generally occurred in a wide range of NaCl concentrations. Only isolate 19 (Pseudoalteromonas sp.) needed NaCl in the growth medium. All isolates were
susceptible to penicillin G, nalidixic acid, tobramicine, chloramphenicol and ampicillin, whereas they were resistant to the O/129 agent. 3.4. Biodegradation assay Negligible differences in the viable counts (CFU/ml) were generally observed in all isolates grown at 4 C and 15 C over 3 weeks in BH medium supplemented with Aroclor 1242 as PCB source (Table 3). PCBs could sustain bacterial populations of even 6.8 · 106 CFU/ml (isolate 74 at 4 C). Bacterial growth was generally enhanced by the lowest temperature and shifted from the lag phase to the log phase after seven days of incubation. PCB extraction of the culture medium was performed when isolates were in the late stationary phase. Degradation of individual congeners was estimated. Isolates were able to reduce most chromatographic peaks by more than 50% (range 0.4–82.8%). None of the congeners was totally removed. PCB congeners in Aroclor 1242 were removed at a different extent after incubation at 4 C and 15 C (Table 4). Differences in PCB degradation were also observed in relation to phylogenetic affiliation: Arthrobacter sp. 74 showed similar biodegradation patterns when growing at 4 C and 15 C, while Pseudoalteromonas sp. 19 better degraded PCBs at 15 C. No biodegradation was detected for the Psychrobacter isolate at 4 C, as it weakly degraded PCB congeners exclusively at 15 C. At 4 C Pseudoalteromonas sp. 19 and Arthrobacter sp. 74 showed similar degradation patterns on di-, tri- and tetrachlorobiphenyls, whereas pentachlorobiphenyls were
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Table 2 Phenotypic features of PCB-degrading Antarctic marine bacteria Characteristic
Psychrobacter Pseudoalteromonas Arthrobacter Isolate 15 Isolate 19 Isolate 74
Sampling depth (m) Gram reaction Motility Cell morphology Polar flagellum Colony pigmentation Endospores
0 rods
50 + rods +
100 + cocci +
Growth temperature (C): 4-15-20-30 37
+
+
+
pH range for growth
5–9
5–9
5–9
1 9
0 7
+
NaCl conc. for growth (%) Minimum 0 Maximum 13 Growth in the absence of NaCl
+
Utilization as a carbon source: Glucose Maltose, malate, mannitol, Arabinose, mannose, gluconate, Adipate, N-acetylglucosamine, Caprate, citrate, phenyl acetate Biochemical tests: Lysine decarboxylase Ornithine decarboxylase Arginine dihydrolase Urease b-Galactosidase Tryptophane deaminase Oxidase Catalase Indol formation Voges-Proskauer reaction Nitrate reduction, H2S formation Macromolecule hydrolysis: Aesculin, tween 80 Gelatin Chitin, agar Starch Acid produced from Glucose, inositol, sorbitol, rhamnose, melibiose, amygdalin, arabinose, mannitol Sucrose Susceptibility to: Penicilline G, nalidixic acid, Tobramicine, chloramphenicol Ampicilline Polymixyn B Tetracycline O/129
+
+
+ +
+ + +
+ + +
+ +
+
+ + +
+ +
+
+
+ + +
+ +
Table 3 Viable counts (CFU/ml · 106, mean ± standard deviation) on MA of PCB-degrading Antarctic isolates after 3 weeks of incubation at different temperatures Isolate
Incubation temperature (C) 4
15
Psychrobacter sp. 15 Pseudoalteromonas sp. 19 Arthrobacter sp. 74
1.5 ± 0.6 3.4 ± 1.3 6.8 ± 2.1
4.5 ± 2.3 2.8 ± 0.7 4.6 ± 0.8
more significantly removed by Arthrobacter sp. 74. On the contrary, Pseudoalteromonas sp. 19 was more efficient in the degradation of almost all PCB congeners at 15 C than Arthrobacter sp. 74. An example of chromatogram is reported in Fig. 2. 4. Discussion This work reports about the characterization and the biodegradation potential on polychlorinated biphenyls of cultivable psychrotolerant bacteria retrieved from Antarctic coastal seawater. PCB-degrading psychrotolerant bacteria have been often isolated from cold and temperate soils different from Antarctica. For example, Whyte et al. (1995) firstly reported about the biodegradative potential of psychrotrophic microorganisms, which were unable to mineralize 2-chlorobiphenyls; Mohn et al. (1997) and Master and Mohn (1998) assessed the response of indigenous Arctic soil microflora to PCB pollution and isolated psychrotolerant strains extensively degrading Aroclor 1221 and Aroclor 1242 at low temperature; finally, Lambo and Patel (2006) isolated and characterized a psychrotrophic bacterium able to cometabolize dichlorobiphenyls and polychlorinated biphenyl congeners in Aroclor 1221. However, information on PCB-degrading cold-adapted microrganisms remain quite scarce and, in particular, data regarding Antarctica are limited to two investigations by Yakimov et al. (1999) and De Domenico et al. (2004): there, we demonstrated their occurrence in the Antarctic marine environment, reporting about two Actinobacteria strains able to utilize Aroclor 1242 at both 4 C and 20 C. Results obtained from the present study confirm these preliminary observations, enlarge the group of cold-adapted bacteria known to degrade PCBs at low temperatures and furnish new information on both their phylogenetic affiliation and phenotypic features. The percentage of the isolates (7.1%) able to degrade PCBs could be considered very low, but this finding is certainly to put in relation to the recalcitrant nature of the xenobiotic substrate utilized throughout the study. The isolation of psychrotrophic PCB degraders from different depths (ranged from 0 to 100 m) suggests that these microorganisms could be well distributed along the water column at Terra Nova Bay and that they are present not only in habitats where PCB generally highly accumulate (e.g., soils and sediments). It could be noted that our investigation relied exclusively on cultivable bacteria and we likely detected only a small fraction of the microorganisms
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Table 4 Biodegradation efficiency on Aroclor 1242 of isolates 15, 19 and 74 at 4 and 15 C. Retention times and congener assignments of main peaks in the chromatogram of Aroclor 1242 are summarized Retention time (min)
Peak no.
24.66 25.93 26.33 26.60 27.12 28.20 28.29 28.57 28.92 29.81 30.27 30.49 30.77 31.01 31.45 31.60 31.72 32.12 32.60 32.80 33.90 34.47 34.75 34.91 35.61 35.97
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
a
Congener assignmenta
2,2 0 2,5 2,4 0 3,3 0 2,2 0 ,5 2,4,6 2,3,3 0 2,6 NA 2,3 0 ,5 NA 3,4,4 0 2,3,6 2,2 0 ,3,3 0 2,2 0 ,3,5 2,2 0 ,5,5 0 2,2 0 ,4,5 0 2,2 0 ,5,6 2,3,4,4 0 2,2 0 ,3,5 0 2,3,4,4 0 2,3 0 ,4,4 0 2,2 0 ,3,4,5 2,3 0 ,4,4 0 ,5 2,2 0 ,3,4,5 0 2,3 0 ,4,5 0 ,6
Biodegradation efficiency (%) 15
19
74
4 C
15 C
4 C
15 C
4 C
15 C
– – – – – – – – – – – – – – – – – – – – – – – – – –
– – 0.39 4.34 6.67 18.74 – 8.05 17.94 7.28 20.09 24.89 17.99 – 20.69 15.66 12.87 25.19 21.37 6.95 12.53 6.39 4.81 4.64 – –
79.78 66.69 64.59 68.31 50.14 61.78 54.68 60.94 47.72 52.06 36.51 53.64 50.53 35.57 46.16 54.81 46.76 41.91 43.99 43.60 51.90 47.89 37.78 46.36 51.39 45.07
79.74 75.01 75.61 74.57 63.97 66.75 57.54 72.39 57.82 67.38 49.86 66.77 66.86 46.59 59.82 66.90 60.88 63.86 66.55 62.19 68.70 69.17 60.64 66.16 82.86 74.68
72.71 57.14 60.57 68.94 47.86 55.55 60.48 59.40 55.12 59.41 37.51 52.78 59.76 49.19 48.91 55.25 49.89 53.37 48.96 53.39 54.91 51.88 50.68 54.20 70.58 60.81
77.73 67.27 69.40 69.87 51.79 61.01 54.45 62.04 54.71 61.78 38.93 55.41 54.57 48.27 47.56 53.06 50.19 44.33 50.51 54.98 56.34 55.98 52.63 57.67 57.18 54.60
NA: not assigned.
Nonane OCN
A. U
2
1
0 20
30
40
Time (min) Fig. 2. Biodegradation of Aroclor 1242 by the strain Arthrobacter sp. 74 (in red) after 3 weeks of incubation at 4 C. Control sample is shown in black. A.U.: Arbitrary Unit. (For interpretation of the references in colour in this figure legend, the reader is referred to the web version of this article.)
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really inhabiting the environment studied; for this reason, we can not exclude the presence of PCB-degraders among uncultivable microorganisms, too. As far as we know, no report exist about the ability of cold-adapted bacteria belonging to the genera Arthrobacter, Pseudoalteromonas and Psychrobacter to degrade PCBs, as we observed through the present study. The genus Psychrobacter has not previously been shown to include pollutant-degraders, whereas both Pseudoalteromonas and Arthrobacter members have been previously reported as able to utilize other kinds of organic pollutants, such as aliphatic and aromatic hydrocarbons (Michaud et al., 2004b; Hedlund and Staley, 2006). Among mesophylic Gram negative bacteria, members of the genera Pseudomonas, Acinetobacter, Alcaligenes have been reported as PCB-degraders, along with some Gram positives such as Janibacter, Arthrobacter, Corynebacterium and Rhodococcus (Seto et al., 1995; Williams et al., 1997; Sierra et al., 2003; Yang et al., 2004). On the contrary, among psychrotolerant bacteria, able to degrade commercial mixtures of PCBs, only Pseudomonas (Mohn et al., 1997; Master and Mohn, 1998), Rhodococcus (De Domenico et al., 2004) and Hydrogenophaga (Lambo and Patel, 2006) closely related strains have been reported to date. In our experiments, the ability of Antarctic marine isolates to grow on Aroclor 1242 was observed both at 4 C and 15 C and the lower incubation temperature did not seem to severely limit the degradation process (except for Psychrobacter sp. 15). In fact, congeners were generally removed at a similar rate at the two temperatures tested, even if the isolates often resulted slightly more efficient at 15 C. On the other hand, tri-, tetra- and penta-chlorobiphenyls were better removed by Arthrobacter sp. 74 at 4 C rather than at 15 C. Lambo and Patel (2006) observed that the different congeners in Aroclor 1221 were differently removed at 5 C and 30 C; for example, monochlorobiphenyls completely disappeared after incubation at 30 C, whereas they were partially removed at 5 C, indicating that low temperature probably slows down the cometabolism of these congeners. The same authors suggested that the incubation temperature could be a major determining factor in the biodegradation process and that specificity of cells for some congeners was probably different at different temperatures. Results obtained on the removal patterns of PCB congeners clearly indicate that the biodegradative potential was not strongly related to the phylogenetic affiliation of the Antarctic isolates analyzed. In addition, they showed a broad congener specificity ensuring a strong catabolic ability. Furthermore, the outstanding degradation abilities exhibited by our isolates at 4 C, close to the in situ seawater temperature, reveal that the indigenous microbial community has a catabolic potential for PCB aerobic degradation that could occur at in situ environmental conditions. However, further studies are needed in order to provide inside into what role the environmental factors play in the natural degradation process.
One of the main objectives for the development and optimization of bioremediation strategies is to understand the potential of pollutant-degrading microorganisms in the environment by assessing their physiology and functions (Watanabe et al., 2002). The detection and characterization of cold-adapted PCB-degrading bacteria in Antarctic seawater is certainly of high interest for bioremediative purposes in polluted marine Antarctic systems, where the introduction of non native species is not allowed. All isolates were psychrotrophic according to the definition given by Morita (1975). It is well known that the isolation of true psychrophyles is very difficult and that the predominance of psychrotrophs is a common feature of permanently cold habitats, such as the Southern Ocean seawater (Helmke and Weyland, 2004). Furthermore, psychrotolerant microbes, which are adapted to a wider temperature range, may have important advantages in biotechnological applications (Gounot, 1991; Mohn et al., 1997), such as the removal of pollutants from cold and temperate environments. In conclusion, results obtained highlight the occurrence of PCB-degrading bacteria in Antarctic seawater and enlarge the knowledge on their ecophysiology, suggesting their potential exploitation in restoring contaminated Antarctic sites. Acknowledgements We wish to thank the anonymous reviewers for their helpful criticisms and valuable suggestions to improve the manuscript. This research was supported by grants from PNRA (Programma Nazionale di Ricerche in Antartide), Italian Ministry of Education and Research (Research Project PNRA 2002/1.5). References Altschul, S.F., Maden, T.L., Sha¨ffer, A.A., Zhang, J., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25, 3389– 3402. Bruni, V., Gugliandolo, C., Maugeri, T., Allegra, A., 1999. Psychrotrophic bacteria from a coastal station in the Ross Sea (Terra Nova Bay, Antarctica). Microbiologica 22, 357–363. Corsolini, S., Kannan, K., Imagawa, T., Focardi, S., Giesy, J.P., 2002. Polychloronaphthalenes and other dioxin-like compounds in Arctic and Antarctic marine food webs. Environmental Science and Technolgy 36, 3490–3496. Corsolini, S., Ademollo, N., Romeo, T., Olmastroni, S., Focardi, S., 2003. Persistent organic pollutants in some species of a Ross Sea pelagic trophic web. Antarctic Science 15, 95–104. Corsolini, S., Covaci, A., Ademollo, N., Focardi, S., Schepens, P., 2006. Occurrence of organochlorine pesticides (OCPs) and their enantiomeric signatures, and concentrations of polybrominated diphenyl ethers (PBDEs) in the Ade´lie penguin food web, Antarctica. Environmental Pollution 140, 371–382. Court, G.S., Davis, L.S., Focardi, S., Bargagli, R., Fossi, C., Leonzio, C., Marili, L., 1997. Chlorinated hydrocarbons in the tissues of south polar skuas (Catharacta maccormicki) and Ade´lie (Pygoscelis adeliea) from Ross Sea, Antarctica. Environmental Pollution 97, 295–301.
L. Michaud et al. / Marine Pollution Bulletin 54 (2007) 1754–1761 De Domenico, M., Lo Giudice, A., Michaud, L., Saitta, M., Bruni, V., 2004. Diesel oil and PCB-degrading bacteria isolated from Antarctic seawater (Terra Nova Bay, Ross Sea). Polar Research 23, 141–146. Evans, C.W.E., Hills, J.M., Dickson, J.M.J., 2000. Heavy metal pollution in Antarctica: a molecular ecotoxicological approach to exposure assessment. Journal of Fish Biology 57, 8–19. Focardi, S., Lari, L., Marsili, L., 1992. PCB congeners, DDTs and hexachlorobenzene in Antarctic fish from Terra Nova Bay (Ross Sea). Antarctic Science 4, 151–154. Focardi, S., Bargagli, R., Corsolini, S., 1995. Isomer-specific analysis and toxic potential evaluation of polychlorinated biphenyls in Antarctic fish, seabirds and Weddell seals from Terra Nova Bay (Ross Sea). Antarctic Science 7, 31–35. Frame, G.M., Cochran, J.W., Boewadt, S.S., 1996. Complete PCB congener distributions for 17 Aroclor mixtures determined by 3 HRGC systems optimized for comprehensive, quantitative, congenerspecific analysis. Journal of High Resolution Chromatography 19, 657–668. Fuoco, R., Colombini, M.P., Ceccarini, A., Abete, C., 1996. Polychlorobiphenyls in Antarctica. Microchemical Journal 54, 384–390. Fuoco, R., Giannarelli, S., Abete, C., Onor, M., Termine, M., 1999. The effect of seasonal pack ice melting on the sea water polychlorobiphenyl contents at Gerlache Inlet and Wood Bay (Ross Sea – Antarctica). International Journal of Environmental Analytical Chemistry 75, 367– 375. Gambaro, A., Manodori, L., Zangrando, R., Cincinelli, A., Capodaglio, G., Cescon, P., 2005. Atmospheric PCB concentrations at Terra Nova Bay, Antarctica. Environmental Science and Technology 39, 9406– 9411. Gounot, A.M., 1991. Bacterial life at low temperature: physiological aspects and biotechnological implications. Journal of Applied Bacteriology 71, 386–397. Hedlund, B.P., Staley, J.T., 2006. Isolation and characterization of Pseudoalteromonas strains with divergent polycyclic aromatic hydrocarbon catabolic properties. Environmental Microbiology 8, 178–182. Helmke, E., Weyland, H., 2004. Psychrophilic versus psychrotolerant bacteria – occurrence and significance in polar and temperate marine habitats. Cellular and Molecular Biology 50, 553–561. Kumar, S., Tamura, K., Nei, M., 1993. Mega: Molecular evolutionary genetics analysis, version 1.02. The Pennsylvania State University, University Park, PA 16802. Lambo, A.J., Patel, T.R., 2006. Isolation and characterization of a biphenyl-utilizing psychrotrophic bacterium, Hydrogenophaga taeniospiralis IA3-A, that cometabolize dichlorobiphenyls and polychlorinated biphenyl congeners in Aroclor 1221. Journal of Basic Microbiology 46, 94–107. Lenihan, H.S., 1992. Benthic marine pollution around McMurdo Station, Antarctica: a summary of findings. Marine Pollution Bulletin 25, 318– 323. Lo Giudice, A., Michaud, L., de Pascale, D., De Domenico, M., di Prisco, G., Fani, R., Bruni, V., 2006. Lipolytic activity of Antarctic coldadapted marine bacteria (Terra Nova Bay, Ross Sea). Journal of Applied Microbiology 101, 1039–1048. Master, E.R., Mohn, W.W., 1998. Psychrotolerant bacteria isolated from Arctic soil that degrade polychlorinated biphenyls at low temperatures. Applied and Environmental Microbiology 64, 4823–4829. Michaud, L., Di Cello, F., Brilli, M., Fani, R., Lo Giudice, A., Bruni, V., 2004a. Biodiversity of cultivable Antarctic psychrotrophic marine bacteria isolated from Terra Nova Bay (Ross Sea). FEMS Microbiology Letters 230, 63–71. Michaud, L., Lo Giudice, A., Saitta, M., De Domenico, M., Bruni, V., 2004b. The biodegradation efficiency on diesel oil by two psychrotrophic Antarctic marine bacteria during a two-month-long experiment. Marine Pollution Bulletin 49, 405–409. Mohn, W.W., Westerberg, K., Cullen, W.R., Reimer, K.J., 1997. Aerobic biodegradation of biphenyl and polychlorinated biphenyls by Arctic
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soil micro-organisms. Applied and Environmental Microbiology 63, 3378–3384. Montone, R.C., Taniguchi, S., Sericano, J., Weber, R.R., Lara, W.H., 2001a. Determination of polychlorinated biphenyls in Antarctic macroalgae Desmaretia sp. Science of Total Environment 277, 181– 186. Montone, R.C., Taniguchi, S., Weber, R.R., 2001b. Polychlorinated biphenyls in marine sediments of Admiralty Bay, King George Island, Antarctica. Marine Pollution Bulletin 42, 611–614. Montone, R.C., Taniguchi, S., Boian, C., Weber, R.R., 2005. PCBs and chlorinated pesticides (DDTs, HCHs and HCB) in the atmosphere of the southwest Atlantic and Antarctic oceans. Marine Pollution Bulletin 50, 778–786. Morita, R.T., 1975. Psychrophilic bacteria. Bacteriological Review 39, 144–167. Negoita, T.G., Covaci, A., Gheorghe, A., Schepens, P., 2003. Distribution of polychlorinated biphenyls (PCBs) and organochlorine pesticides in soils from the East Antarctic coast. Journal of Environmental Pollution 5, 281–286. Negri, A., Burns, K., Boyle, S., Brinkman, D., Webster, N., 2006. Contamination in sediments, bivalves and sponges of McMurdo Sound, Antarctica. Environmental Pollution 143, 456–467. Reinhardt, S.B., van Vleet, E.S., 1986. Hydrocarbons of Antarctic midwater organisms. Polar Biology 6, 47–51. Saitou, M., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 5, 406–425. Seto, M., Kimbara, K., Shimura, M., Hatta, T., Fukuda, M., Yano, K., 1995. A novel transformation of polychlorinated biphenyls by Rhodococcus sp. strain RHA1. Applied and Environmental Microbiology 61, 3353–3358. Sierra, I.J.L., Valera, J.L., Marina, M.L., Laborda, F., 2003. Study of the biodegradation process of polychlorinated biphenyls in liquid medium and soil by a new isolated aerobic bacterium (Janibacter sp.). Chemosphere 53, 609–618. Tanabe, S., Hidaka, H., Tatsukawa, R., 1983. PCBs and chlorinated hydrocarbon pesticides in Antarctic atmosphere and hydrosphere. Chemosphere 12, 277–288. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22, 4673–4680. Vaillancourt, F.H., Haro, M.A., Drouin, N., Karim, Z., Maaroufi, H., Eltis, L.D., 2003. Characterization of extradiol dioxygenases from a polychlorinated biphenyl-degrading strain that possess higher specificities for chlorinated metabolites. Journal of Bacteriology 185, 1253– 1260. Watanabe, K., Futamata, H., Harayama, S., 2002. Understanding the diversity in catabolic potential of microorganisms for the development of biodegradation strategies. Antonie van Leeuwenhoek 81, 655– 663. Weber, K., Goerke, H., 2003. Persistent organic pollutants (POPs) in Antarctic fish: levels, patterns, changes. Chemosphere 53, 667–678. Whyte, L.G., Greer, C.W., Inniss, W.E., 1995. Assessment of the biodegradation potential of psychrotrophic microorganisms. Canadian Journal of Microbiology 42, 99–106. Williams, W.A., Lobos, J.H., Cheetham, W.E., 1997. A phylogenetic analysis of aerobic polychlorinated biphenyl-degrading bacteria. International Journal of Systematic Bacteriology. 47, 207–210. Yakimov, M.M., Giuliano, L., Bruni, V., Scarfı`, S., Golyshin, P.N., 1999. Characterization of Antarctic hydrocarbon-degrading bacteria capable of producing bioemulsifiers. Microbiologica 22, 249–256. Yang, X., Sun, Y., Qian, S., 2004. Biodegradation of seven polychlorinated biphenyls by a newly isolated aerobic bacterium (Rhodococcus sp. R04). Journal of Industrial Microbiology and Biotechnology 31, 415–420.
Marine Pollution Bulletin 54 (2007) 1754–1761 www.elsevier.com/locate/marpolbul
Biodegradative potential and characterization of psychrotolerant polychlorinated biphenyl-degrading marine bacteria isolated from a coastal station in the Terra Nova Bay (Ross Sea, Antarctica) Michaud Luigi a
a,*
, Di Marco Gaetano b, Bruni Vivia a, Lo Giudice Angelina
a
Dipartimento di Biologia Animale ed Ecologia Marina (DBAEM), Universita` di Messina, Salita Sperone 31, 98166 Messina, Italy b Istituto per i Processi Chimico-Fisici (IPCF-CNR), via La Farina 237, 98123 Messina, Italy
Abstract Antarctic marine bacteria were screened for their ability to degrade polychlorinated biphenyls (PCB) as the sole carbon and energy source at both 4 C and 15 C. PCB-degrading isolates (7.1%) were identified by sequencing their 16S rDNA as Pseudoalteromonas, Psychrobacter and Arthrobacter members. One representative isolate per genera was selected for evaluating the biodegradative potential under laboratory scale and phenotypically characterized. Removal of individual PCB congeners was between 35.6% and 79.8% at 4 C and between 0.4% and 82.8% at 15 C. Differences in the removal patterns of PCB congeners were observed in relation to the phylogenetic affiliation: Arthrobacter isolate showed similar biodegradation efficiencies when growing at 4 C and 15 C, while Pseudoalteromonas better degraded PCBs at 15 C. No biodegradation was detected for Psychrobacter isolate at 4 C. Results obtained highlight the occurrence of PCB-degrading bacteria in Antarctic seawater and suggest the potential exploitation of autochthonous bacteria for PCB bioremediation in cold marine environments. 2007 Elsevier Ltd. All rights reserved. Keywords: PCB-degradation; Antarctic marine bacteria; Psychrotolerant bacteria; Bacterial characterization
1. Introduction Polychlorinated biphenyls (PCBs) are long-term persistent compounds produced as mixtures during the midtwentieth century and sold for industrial applications under trade names such as Aroclor, Clophen, Fenclor or Kanechlor. The family of PCBs contains 209 congeners which consist of a biphenyl molecule substituted with one to ten chlorine substituents. PCB mixtures are nonflammable, exhibit a high degree of chemical and thermal stability (up to 350 C), and have desirable dielectric properties (Master and Mohn, 1998; Vaillancourt et al., 2003). Although their initial application was as dielectric fluid in
*
Corresponding author. Tel.: +39 090 6765533; fax: +39 090 393409. E-mail address: [email protected] (L. Michaud).
0025-326X/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2007.07.011
electrical transformers and capacitors, PCBs were also utilized in heat transfer and hydraulic systems as well as in inks, lubricants, paints, adhesives and dust control agents. Despite the fact that these chemical compounds have been regulated or prohibited since the 1970s in western nations, their widespread use and chemical stability has led to extensive environmental contamination through accidental releases and inappropriate disposal techniques. In spite of the precautionary measures taken to avoid pollution, PCBs have been detected in Antarctic seawater and sediments (Fuoco et al., 1996, 1999; Montone et al., 2001b) as well as in various organisms from different trophic levels (Reinhardt and van Vleet, 1986; Focardi et al., 1992, 1995; Court et al., 1997; Montone et al., 2001a; Corsolini et al., 2003; Weber and Goerke, 2003). Dispersion of PCBs occurs mainly via mass flow in the atmosphere (Negoita et al., 2003; Gambaro et al., 2005;
L. Michaud et al. / Marine Pollution Bulletin 54 (2007) 1754–1761
Montone et al., 2005), but increasing human activities have probably contributed to a localized contamination at scientific Antarctic bases (Negri et al., 2006). As PCB concentrations in Antarctic seawater is generally several order of magnitude lower than in organisms, it could be concluded that these contaminants, due to their hydrophobic nature, are subjected to bioaccumulation in organism tissues and biomagnification in the Antarctic marine food webs (Tanabe et al., 1983; Focardi et al., 1995; Corsolini et al., 2002, 2006). Focardi et al. (1995), evaluating toxic potential of PCBs in Antarctic fish, seabirds and seals, found a clear relationship between PCB concentrations and trophic level (fish < Ade´lie penguin < Weddel seal). PCBs can cause several damages to organisms; Lenihan (1992) reported about the negative influence of PCBs on behaviour and survival of Antarctic amphipods and urchins, while a study by Evans et al. (2000) revealed that pathological and molecular abnormalities of Antarctic fish was probably linked to the effect of these persistent pollutants. The classical methods suggested for the remediation of PCB-contaminated environments are generally expensive and often inefficient; moreover, they may be source of secondary pollution (Yang et al., 2004). On the other hand, PCBs can be transformed, despite their recalcitrant nature, into chemical substances by different microbial metabolic pathways, both aerobic and anaerobic, facilitating further metabolization. As a result, the developments of bioremediation processes have been proposed as a promising alternative to the currently adopted techniques; to this regard, studying the ecology and physiology of bacteria able to degrade pollutants is essential in order to asses the effectiveness and risks involved in bioremediation processes. Aerobic degradation of PCBs has been mainly reported for mesophylic microorganisms inhabiting soils and sediments where these xenobiotic compounds generally accumulate, while psychrotolerant PCB-degraders have been rarely isolated from cold environments (Mohn et al., 1997; Master and Mohn, 1998; Lambo and Patel, 2006). Nevertheless, although the occurrence of PCB in both biotic and abiotic Antarctic compartments has been reported, very little is known about the degradative activity on these recalcitrant pollutants of bacteria from Antarctica (De Domenico et al., 2004). The aim of this investigation was to (1) screen Antarctic marine psychrotolerant bacteria for PCB-degradation, (2) determine if a relation occurred between their phylogenetic affiliation and biodegradative potential and, finally, (3) characterize PCB-degraders by a combination of phenotypic and genotypic features. 2. Materials and methods 2.1. Bacterial strains The one-hundred and twenty-six psychrotrophic bacterial strains used in this study were previously isolated from
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Antarctic seawater samples (Terra Nova Bay, Ross Sea) collected along the water column (0-150 m) at the fixed station Tiburtina (TIB; coordinates: 7442 0 300 S– 16410 0 0.500 E). Their identity is given in Bruni et al. (1999). All the isolates belong to the Italian Collection of Antarctic Bacteria (CIBAN) of the National Antarctic Museum (MNA) kept at the University of Messina. They are maintained on Marine Agar (MA; Difco) slopes at 4 C and routinely streaked on agar plates from tubes every six months to control purity and viability. Antarctic strains are also preserved by freezing cell suspensions at 80 C in Marine Broth (MB; Difco) to which 20% (v/v) glycerol is added. 2.2. Screening for aerobic degradation of PCB Aerobic PCB degradation was screened into the mineral liquid medium Bushnell Haas (BH; Difco) supplemented with Aroclor 1242 solution (100 ppm in dichloromethane) as sole carbon and energy source (final concentration 0.1%, v/v). Aroclor 1242 is a mixture of PCB congeners (ranging from dichloro- to hexachlorobiphenyls) made of twelve carbon atoms in the biphenyl molecule and containing 42% chlorine by weight (Frame et al., 1996). Before adding the bacterial inoculum, 10 ll of Aroclor solution were transferred to 100 ml flasks containing 9.5 ml of BH and left in a cabinet until CH2Cl2 had completely evaporated. The medium was inoculated with 0.5 ml of a freshly prepared bacterial suspension (5%, v/v) in 3% (w/v) NaCl supplemented distilled water. The optical density (OD) of the suspension was adjusted to 1.0 at 600 nm. Cultures were incubated at 4 C and 15 C for 3 weeks on a rotary shaker operated at 100 rpm. The ability to use PCBs as growth substrates was evaluated according to the degree of turbidity or the appearance of cellular flocs in the test tubes. 2.3. PCR amplification and analysis of 16S rDNA PCR amplification, sequencing and phylogenetic analysis of 16S rDNA from active bacterial isolates were carried out as previously described by Michaud et al. (2004a). The first half (about 700 nucleotides) of each amplification product was sequenced using the primer 27f. Each sequence was then used as a query in a BLASTn search (Altschul et al., 1997) and further aligned using the program Clustal W (Thompson et al., 1994) to the most similar orthologous sequences retrieved from the database. Each alignment was checked manually, corrected and then analyzed using the Neighbour-Joining method (Saitou and Nei, 1987) according to the model of Jukes-Cantor distances. Phylogenetic trees were constructed using the MEGA 3 (Molecular Evolutionary Genetics Analysis) software (Kumar et al., 1993). The robustness of the inferred trees was evaluated by 500 bootstrap re-samplings. The 16S rRNA gene sequences were submitted to GenBank
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and assigned to the following accession numbers: EF577381-EF577388 and DQ489314. 2.4. Morphological, biochemical and physiological characterization Isolates were characterized by means of a combination of phenotypic tests as previously described in Bruni et al. (1999) and Lo Giudice et al. (2006). All media used in this study were sterilized by autoclaving at 121 C for 15 min and the plates were incubated at 15 C for 14 days. The analyses were performed at least twice. 2.5. Biodegradation assay and gas-chromatographic analysis Isolates were grown in 50 ml of Aroclor 1242 supplemented BH, as described earlier, and incubated at 4 and 15 C for 3 weeks. Uninoculated controls were incubated in parallel to monitor abiotic losses of the substrates. Subsamples were removed at regular intervals to assess bacterial growth by colony-forming units (CFU/ml) spread plating on MA. Serial dilutions were made in sterile saline solution. Plates were incubated at either 4 C or 15 C. Viable counts were carried out in duplicate. After incubation, biodegradation activity was stopped by acidifying cultures with HCl 10 M to achieve pH 2. Before PCB extraction, 60 ll of octachloronaphthalene (OCN) solution (40 ppm in CH2Cl2) was added to each flask to monitor substrate losses during the extraction procedure. Cultures were extracted three times with 5 ml of CH2Cl2 in a separatory funnels to remove the cellular material. Funnels were vigorously shaken for 3 min during each extraction. The three organic phases were pooled and the solvent evaporated to dryness. The composition of PCBs and their concentration were determined by high resolution gas chromatography-mass spectrometry (GC-MS) using a Perkin-Elmer TurboMS AutoSystem XL GC, equipped with a DB-TPH capillary column (30 m with a 0.322 mm i.d. and 0.25 lm film thickness, J & W Scientific). Helium (5 bar) was used as carrier gas (1.8 ml min1). The injector was kept at
250 C; the column oven was initially kept at a temperature of 50 C for 4 min, and then heated up to 300 C at a rate of 5 C min1. One microlitre portion of each sample, containing 79.3 ng of heptamethyl nonane as internal standard (to account for injection errors and other variable encountered during the chromatographic analysis), was analyzed by means of the splitless injection technique (50 ml min1). Finally, the percentage reduction of Aroclor was determined according to the following equation: %R ¼ 100 ½ðH PCB sample =H PC Bcontrol Þ 100 where HPCB sample and HPCB control are the height of a PCB peak in the sample and the control, respectively. 3. Results 3.1. Screening for PCB biodegradation The biodegradation of Aroclor 1242 was tested at 4 and 15 C by batch experiments. Nine of the 126 psychrotolerant isolates screened (7.1%) were able to utilize PCBs as a sole carbon and energy source in at least one of the temperatures adopted during the preliminary screening. PCBdegraders were isolated from different sampling depths: 0 m (strains 3 and 15), 25 m (strains 5, 6 and 19), 50 m (strain 16) and, finally, 100 m (strains 9, 24 and 74). 3.2. Phylogenetic analysis The phylogenetic analysis revealed that the PCBdegrading strains were representative of three bacterial genera and belonged to two bacterial phylogenetic branches: the c-subdivisions of Proteobacteria and the gram-positive branch (high G+C content Actinobacteria). Within the c-Proteobacteria subdivision, the isolates were found to be related to Pseudoalteromonas and Psychrobacter genera. Finally, only one isolate was placed within the genus Arthrobacter (Table 1). The phylogenetic trees derived from the 16S rDNA sequence analysis of the nine strains are visualized in Fig. 1.
Table 1 16S rDNA gene sequence affiliation of PCB-degrading Antarctic isolates to their closest phylogenetic neighbours Phylum or classa
Strain
Accession number
Next relative by GenBank alignment (ANb, organism)
Hom (%)c
Family
GAM
3 5 19 24 6 9 15 16 74
EF577381 EF577382 DQ489314 EF577386 EF577383 EF577384 EF577387 EF577385 EF577388
AM110978, Pseudoalteromonas sp. 3023 AM110978, Pseudoalteromonas sp. 3023 AY243366, Pseudoalteromonas sp. CAM36 DQ677306, Pseudoalteromonas sp. G16 AY656801, Psychrobacter sp. 63 AB094794, Psychrobacter okhotskensis AB094794, Psychrobacter okhotskensis AY444823, Psychrobacter sp. 215-51 AF134184, Arthrobacter agilis
99 99 97 99 100 98 98 99 98
Pseudoalteromonadaceae Pseudoalteromonadaceae Pseudoalteromonadaceae Pseudoalteromonadaceae Moraxellaceae Moraxellaceae Moraxellaceae Moraxellaceae Micrococcaceae
ACT a b c
GAM: c-Proteobacteria; ACT: Actinobacteria. AN: Accession number. Hom: sequence homology.
L. Michaud et al. / Marine Pollution Bulletin 54 (2007) 1754–1761
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74 63 83
AJ309942.1 Psychrobacter immobilis AY639871.1 Psychrobacter cibarius AM269905.1 Psychrobacter sp. 23 AJ430827.1 Psychrobacter fozii AJ310992.1 Psychrobacter sp. TAD1 16 63 AJ609555.1 Psychrobacter urativorans AB094794.1 Psychrobacter okhotskensis PCB 6 35 AY771717.1 Psychrobacter fozii 45 PCB 15 20 PCB 9 AJ312213.1 Psychrobacter glacincola 99 PCB 16 AJ430828.1 Psychrobacter luti AJ609556.1 Psychrobacter frigidicola 92 PCB 5 AF529062.1 Pseudoalteromonas citrea DQ787199.1 P. arctica 99 AF038846.1 Pseudoalteromonas gracili PCB 19 60 PCB 3 DQ537517.1 P. haloplanktis 42 PCB 24 AJ417594.1 P. agarovorans AY573036.1 Pseudoalteromonas sp. 58 AF235090.1 Arthrobacter methylotrophus AJ316140.1 Arthrobacter gandensis U85895.1 Arthrobacter sp. IC044 99 48 AF134184.1 Arthrobacter agilis 91 PCB 74 AJ639829.1 Arthrobacter tecti 75 96 AJ639830.1 Arthrobacter parietes 68
0. 02
Fig. 1. Phylogenetic relationship based on 16S rDNA sequences of PCB-degrading Antarctic isolates.
Based on the results of the preliminary screening for PCB-degradation, strains 15 (Psychrobacter sp.), 19 (Pseudoalteromonas sp.) and 74 (Arthrobacter sp.), representative of three distinct genera, were chosen for further analyses (i.e. phenotypic characterization and determination of their biodegradation efficiency, see below), as they grew better on Aroclor supplemented medium. 3.3. Physiological and biochemical characterization Phenotypic characters of selected Antarctic isolates are reported in Table 2. A single polar flagellum was observed in the Pseudoalteromonas isolate, which appeared motile under microscopy. The presence of endospores was never observed. Isolates were able to hydrolyse at least two of the macromolecules provided. Although they were capable of growing on chitin overlay plates, none of them induced degradation of this macromolecule. All strains were lipolytic as they hydrolysed tween 80. Only Gram negative isolates were oxidase positive. Enzyme arrangements were defined by the combination of the results obtained from API 20E and API 20NE systems. None of the strains were ornithine and lysine decarboxylase or arginine dihydrolase positive. Indole production from tryptophane and H2S formation were never observed. All the marine isolates investigated were psychrotolerant when grown into MB medium. All strains grew well in the range at 4–30 C. The pH range for growth was from 5 to 9. Growth generally occurred in a wide range of NaCl concentrations. Only isolate 19 (Pseudoalteromonas sp.) needed NaCl in the growth medium. All isolates were
susceptible to penicillin G, nalidixic acid, tobramicine, chloramphenicol and ampicillin, whereas they were resistant to the O/129 agent. 3.4. Biodegradation assay Negligible differences in the viable counts (CFU/ml) were generally observed in all isolates grown at 4 C and 15 C over 3 weeks in BH medium supplemented with Aroclor 1242 as PCB source (Table 3). PCBs could sustain bacterial populations of even 6.8 · 106 CFU/ml (isolate 74 at 4 C). Bacterial growth was generally enhanced by the lowest temperature and shifted from the lag phase to the log phase after seven days of incubation. PCB extraction of the culture medium was performed when isolates were in the late stationary phase. Degradation of individual congeners was estimated. Isolates were able to reduce most chromatographic peaks by more than 50% (range 0.4–82.8%). None of the congeners was totally removed. PCB congeners in Aroclor 1242 were removed at a different extent after incubation at 4 C and 15 C (Table 4). Differences in PCB degradation were also observed in relation to phylogenetic affiliation: Arthrobacter sp. 74 showed similar biodegradation patterns when growing at 4 C and 15 C, while Pseudoalteromonas sp. 19 better degraded PCBs at 15 C. No biodegradation was detected for the Psychrobacter isolate at 4 C, as it weakly degraded PCB congeners exclusively at 15 C. At 4 C Pseudoalteromonas sp. 19 and Arthrobacter sp. 74 showed similar degradation patterns on di-, tri- and tetrachlorobiphenyls, whereas pentachlorobiphenyls were
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Table 2 Phenotypic features of PCB-degrading Antarctic marine bacteria Characteristic
Psychrobacter Pseudoalteromonas Arthrobacter Isolate 15 Isolate 19 Isolate 74
Sampling depth (m) Gram reaction Motility Cell morphology Polar flagellum Colony pigmentation Endospores
0 rods
50 + rods +
100 + cocci +
Growth temperature (C): 4-15-20-30 37
+
+
+
pH range for growth
5–9
5–9
5–9
1 9
0 7
+
NaCl conc. for growth (%) Minimum 0 Maximum 13 Growth in the absence of NaCl
+
Utilization as a carbon source: Glucose Maltose, malate, mannitol, Arabinose, mannose, gluconate, Adipate, N-acetylglucosamine, Caprate, citrate, phenyl acetate Biochemical tests: Lysine decarboxylase Ornithine decarboxylase Arginine dihydrolase Urease b-Galactosidase Tryptophane deaminase Oxidase Catalase Indol formation Voges-Proskauer reaction Nitrate reduction, H2S formation Macromolecule hydrolysis: Aesculin, tween 80 Gelatin Chitin, agar Starch Acid produced from Glucose, inositol, sorbitol, rhamnose, melibiose, amygdalin, arabinose, mannitol Sucrose Susceptibility to: Penicilline G, nalidixic acid, Tobramicine, chloramphenicol Ampicilline Polymixyn B Tetracycline O/129
+
+
+ +
+ + +
+ + +
+ +
+
+ + +
+ +
+
+
+ + +
+ +
Table 3 Viable counts (CFU/ml · 106, mean ± standard deviation) on MA of PCB-degrading Antarctic isolates after 3 weeks of incubation at different temperatures Isolate
Incubation temperature (C) 4
15
Psychrobacter sp. 15 Pseudoalteromonas sp. 19 Arthrobacter sp. 74
1.5 ± 0.6 3.4 ± 1.3 6.8 ± 2.1
4.5 ± 2.3 2.8 ± 0.7 4.6 ± 0.8
more significantly removed by Arthrobacter sp. 74. On the contrary, Pseudoalteromonas sp. 19 was more efficient in the degradation of almost all PCB congeners at 15 C than Arthrobacter sp. 74. An example of chromatogram is reported in Fig. 2. 4. Discussion This work reports about the characterization and the biodegradation potential on polychlorinated biphenyls of cultivable psychrotolerant bacteria retrieved from Antarctic coastal seawater. PCB-degrading psychrotolerant bacteria have been often isolated from cold and temperate soils different from Antarctica. For example, Whyte et al. (1995) firstly reported about the biodegradative potential of psychrotrophic microorganisms, which were unable to mineralize 2-chlorobiphenyls; Mohn et al. (1997) and Master and Mohn (1998) assessed the response of indigenous Arctic soil microflora to PCB pollution and isolated psychrotolerant strains extensively degrading Aroclor 1221 and Aroclor 1242 at low temperature; finally, Lambo and Patel (2006) isolated and characterized a psychrotrophic bacterium able to cometabolize dichlorobiphenyls and polychlorinated biphenyl congeners in Aroclor 1221. However, information on PCB-degrading cold-adapted microrganisms remain quite scarce and, in particular, data regarding Antarctica are limited to two investigations by Yakimov et al. (1999) and De Domenico et al. (2004): there, we demonstrated their occurrence in the Antarctic marine environment, reporting about two Actinobacteria strains able to utilize Aroclor 1242 at both 4 C and 20 C. Results obtained from the present study confirm these preliminary observations, enlarge the group of cold-adapted bacteria known to degrade PCBs at low temperatures and furnish new information on both their phylogenetic affiliation and phenotypic features. The percentage of the isolates (7.1%) able to degrade PCBs could be considered very low, but this finding is certainly to put in relation to the recalcitrant nature of the xenobiotic substrate utilized throughout the study. The isolation of psychrotrophic PCB degraders from different depths (ranged from 0 to 100 m) suggests that these microorganisms could be well distributed along the water column at Terra Nova Bay and that they are present not only in habitats where PCB generally highly accumulate (e.g., soils and sediments). It could be noted that our investigation relied exclusively on cultivable bacteria and we likely detected only a small fraction of the microorganisms
L. Michaud et al. / Marine Pollution Bulletin 54 (2007) 1754–1761
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Table 4 Biodegradation efficiency on Aroclor 1242 of isolates 15, 19 and 74 at 4 and 15 C. Retention times and congener assignments of main peaks in the chromatogram of Aroclor 1242 are summarized Retention time (min)
Peak no.
24.66 25.93 26.33 26.60 27.12 28.20 28.29 28.57 28.92 29.81 30.27 30.49 30.77 31.01 31.45 31.60 31.72 32.12 32.60 32.80 33.90 34.47 34.75 34.91 35.61 35.97
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
a
Congener assignmenta
2,2 0 2,5 2,4 0 3,3 0 2,2 0 ,5 2,4,6 2,3,3 0 2,6 NA 2,3 0 ,5 NA 3,4,4 0 2,3,6 2,2 0 ,3,3 0 2,2 0 ,3,5 2,2 0 ,5,5 0 2,2 0 ,4,5 0 2,2 0 ,5,6 2,3,4,4 0 2,2 0 ,3,5 0 2,3,4,4 0 2,3 0 ,4,4 0 2,2 0 ,3,4,5 2,3 0 ,4,4 0 ,5 2,2 0 ,3,4,5 0 2,3 0 ,4,5 0 ,6
Biodegradation efficiency (%) 15
19
74
4 C
15 C
4 C
15 C
4 C
15 C
– – – – – – – – – – – – – – – – – – – – – – – – – –
– – 0.39 4.34 6.67 18.74 – 8.05 17.94 7.28 20.09 24.89 17.99 – 20.69 15.66 12.87 25.19 21.37 6.95 12.53 6.39 4.81 4.64 – –
79.78 66.69 64.59 68.31 50.14 61.78 54.68 60.94 47.72 52.06 36.51 53.64 50.53 35.57 46.16 54.81 46.76 41.91 43.99 43.60 51.90 47.89 37.78 46.36 51.39 45.07
79.74 75.01 75.61 74.57 63.97 66.75 57.54 72.39 57.82 67.38 49.86 66.77 66.86 46.59 59.82 66.90 60.88 63.86 66.55 62.19 68.70 69.17 60.64 66.16 82.86 74.68
72.71 57.14 60.57 68.94 47.86 55.55 60.48 59.40 55.12 59.41 37.51 52.78 59.76 49.19 48.91 55.25 49.89 53.37 48.96 53.39 54.91 51.88 50.68 54.20 70.58 60.81
77.73 67.27 69.40 69.87 51.79 61.01 54.45 62.04 54.71 61.78 38.93 55.41 54.57 48.27 47.56 53.06 50.19 44.33 50.51 54.98 56.34 55.98 52.63 57.67 57.18 54.60
NA: not assigned.
Nonane OCN
A. U
2
1
0 20
30
40
Time (min) Fig. 2. Biodegradation of Aroclor 1242 by the strain Arthrobacter sp. 74 (in red) after 3 weeks of incubation at 4 C. Control sample is shown in black. A.U.: Arbitrary Unit. (For interpretation of the references in colour in this figure legend, the reader is referred to the web version of this article.)
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L. Michaud et al. / Marine Pollution Bulletin 54 (2007) 1754–1761
really inhabiting the environment studied; for this reason, we can not exclude the presence of PCB-degraders among uncultivable microorganisms, too. As far as we know, no report exist about the ability of cold-adapted bacteria belonging to the genera Arthrobacter, Pseudoalteromonas and Psychrobacter to degrade PCBs, as we observed through the present study. The genus Psychrobacter has not previously been shown to include pollutant-degraders, whereas both Pseudoalteromonas and Arthrobacter members have been previously reported as able to utilize other kinds of organic pollutants, such as aliphatic and aromatic hydrocarbons (Michaud et al., 2004b; Hedlund and Staley, 2006). Among mesophylic Gram negative bacteria, members of the genera Pseudomonas, Acinetobacter, Alcaligenes have been reported as PCB-degraders, along with some Gram positives such as Janibacter, Arthrobacter, Corynebacterium and Rhodococcus (Seto et al., 1995; Williams et al., 1997; Sierra et al., 2003; Yang et al., 2004). On the contrary, among psychrotolerant bacteria, able to degrade commercial mixtures of PCBs, only Pseudomonas (Mohn et al., 1997; Master and Mohn, 1998), Rhodococcus (De Domenico et al., 2004) and Hydrogenophaga (Lambo and Patel, 2006) closely related strains have been reported to date. In our experiments, the ability of Antarctic marine isolates to grow on Aroclor 1242 was observed both at 4 C and 15 C and the lower incubation temperature did not seem to severely limit the degradation process (except for Psychrobacter sp. 15). In fact, congeners were generally removed at a similar rate at the two temperatures tested, even if the isolates often resulted slightly more efficient at 15 C. On the other hand, tri-, tetra- and penta-chlorobiphenyls were better removed by Arthrobacter sp. 74 at 4 C rather than at 15 C. Lambo and Patel (2006) observed that the different congeners in Aroclor 1221 were differently removed at 5 C and 30 C; for example, monochlorobiphenyls completely disappeared after incubation at 30 C, whereas they were partially removed at 5 C, indicating that low temperature probably slows down the cometabolism of these congeners. The same authors suggested that the incubation temperature could be a major determining factor in the biodegradation process and that specificity of cells for some congeners was probably different at different temperatures. Results obtained on the removal patterns of PCB congeners clearly indicate that the biodegradative potential was not strongly related to the phylogenetic affiliation of the Antarctic isolates analyzed. In addition, they showed a broad congener specificity ensuring a strong catabolic ability. Furthermore, the outstanding degradation abilities exhibited by our isolates at 4 C, close to the in situ seawater temperature, reveal that the indigenous microbial community has a catabolic potential for PCB aerobic degradation that could occur at in situ environmental conditions. However, further studies are needed in order to provide inside into what role the environmental factors play in the natural degradation process.
One of the main objectives for the development and optimization of bioremediation strategies is to understand the potential of pollutant-degrading microorganisms in the environment by assessing their physiology and functions (Watanabe et al., 2002). The detection and characterization of cold-adapted PCB-degrading bacteria in Antarctic seawater is certainly of high interest for bioremediative purposes in polluted marine Antarctic systems, where the introduction of non native species is not allowed. All isolates were psychrotrophic according to the definition given by Morita (1975). It is well known that the isolation of true psychrophyles is very difficult and that the predominance of psychrotrophs is a common feature of permanently cold habitats, such as the Southern Ocean seawater (Helmke and Weyland, 2004). Furthermore, psychrotolerant microbes, which are adapted to a wider temperature range, may have important advantages in biotechnological applications (Gounot, 1991; Mohn et al., 1997), such as the removal of pollutants from cold and temperate environments. In conclusion, results obtained highlight the occurrence of PCB-degrading bacteria in Antarctic seawater and enlarge the knowledge on their ecophysiology, suggesting their potential exploitation in restoring contaminated Antarctic sites. Acknowledgements We wish to thank the anonymous reviewers for their helpful criticisms and valuable suggestions to improve the manuscript. This research was supported by grants from PNRA (Programma Nazionale di Ricerche in Antartide), Italian Ministry of Education and Research (Research Project PNRA 2002/1.5). References Altschul, S.F., Maden, T.L., Sha¨ffer, A.A., Zhang, J., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25, 3389– 3402. Bruni, V., Gugliandolo, C., Maugeri, T., Allegra, A., 1999. Psychrotrophic bacteria from a coastal station in the Ross Sea (Terra Nova Bay, Antarctica). Microbiologica 22, 357–363. Corsolini, S., Kannan, K., Imagawa, T., Focardi, S., Giesy, J.P., 2002. Polychloronaphthalenes and other dioxin-like compounds in Arctic and Antarctic marine food webs. Environmental Science and Technolgy 36, 3490–3496. Corsolini, S., Ademollo, N., Romeo, T., Olmastroni, S., Focardi, S., 2003. Persistent organic pollutants in some species of a Ross Sea pelagic trophic web. Antarctic Science 15, 95–104. Corsolini, S., Covaci, A., Ademollo, N., Focardi, S., Schepens, P., 2006. Occurrence of organochlorine pesticides (OCPs) and their enantiomeric signatures, and concentrations of polybrominated diphenyl ethers (PBDEs) in the Ade´lie penguin food web, Antarctica. Environmental Pollution 140, 371–382. Court, G.S., Davis, L.S., Focardi, S., Bargagli, R., Fossi, C., Leonzio, C., Marili, L., 1997. Chlorinated hydrocarbons in the tissues of south polar skuas (Catharacta maccormicki) and Ade´lie (Pygoscelis adeliea) from Ross Sea, Antarctica. Environmental Pollution 97, 295–301.
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