OUP user menu

Biodegradation of phenanthrene by indigenous microorganisms in soils from Livingstone Island, Antarctica

Uchechukwu V. Okere, Ana Cabrerizo, Jordi Dachs, Kevin C. Jones, Kirk T. Semple
DOI: http://dx.doi.org/10.1111/j.1574-6968.2012.02501.x 69-77 First published online: 1 April 2012

Abstract

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) in soils has been linked to history of exposure to PAHs and prevailing environmental conditions. This work assessed the capacity of indigenous microorganisms in soils collected in Livingstone Island (South Shetlands Islands, Antarctica) with no history of pollution (∑PAHs: 0.14–1.47 ng g−1 dw) to degrade 14C-phenanhthrene at 4, 12 and 22 °C. The study provides evidence of the presence of phenanthrene-degrading microorganisms in all studied soils. Generally, the percentage of 14C-phenanhthrene mineralized increased with increasing temperature. The highest extent of 14C-phenanhthrene mineralization (47.93%) was observed in the slurried system at 22 °C. This work supports findings of the presence of PAH-degrading microorganisms in uncontaminated soils and suggests the case is the same for uncontaminated Antarctic remote soils.

Keywords
  • Livingstone Island
  • sub-Antarctica
  • microbial degradation
  • phenanthrene
  • soil

Introduction

The role of indigenous microbial communities in the removal of hydrocarbons from the environment has been widely investigated showing that a small fraction of all natural microbial communities irrespective of location or prevailing environmental conditions can grow on both aromatic and aliphatic hydrocarbons (Sepic et al., 1995; Solano-Serena et al., 2000; Ruberto et al., 2003). The size of these populations of degrading microorganisms often reflects the historical exposure of the environment to either biogenic or anthropogenic hydrocarbon sources. In general, while hydrocarbon degraders may constitute < 0.1% of the microbial community in unpolluted environments, in oil-polluted ecosystems they can constitute up to 100% of the culturable microorganisms (Atlas, 1981). Several studies (Spain et al., 1980; Carmichael & Pfaender, 1997; Chen & Aitken, 1998; Macleod & Semple, 2006; McLoughlin et al., 2009) have shown an increase in hydrocarbon-degrading microorganisms in different soil environments, following exposure to aromatic hydrocarbons.

Where biodegradation of polycyclic aromatic hydrocarbons (PAHs) has been observed in cold environments, it has been attributed to cold adapted psychrotrophs and psychrophiles, which are widely distributed in nature because a large part of the earth's biosphere is at temperatures below 5 °C (Margesin & Schinner, 1999; Ferguson et al., 2003a, 2003b). A significant increase in numbers of psychrotrophic bacteria following contamination in cold environments has been reported leading to suggestions of their potential for rapid adaptation and their predominance over psychrophiles in cold environments (Delille et al., 1998; Margesin & Schinner, 1999; Delille, 2000). Hydrocarbon-degrading bacteria isolated from contaminated Antarctic soils have been identified and include the genera Rhodococcus, Acinobacter, Pseudomonas and Sphingomonas (Aislabie et al., 2004, 2006; Ma et al., 2006). Many of these microorganisms were psychrotrophic rather than psychrophilic; while they could grow at low temperatures, optimum growth was at temperatures > 15 °C (Aislabie et al., 2004).

Livingstone Island is one of the South Shetland Islands and it is separated from the Antarctica Peninsula by the Bransfield Strait. Its temperatures are relatively constant, rarely exceeding 3 °C in summer or falling below −11 °C in winter, with wind chill temperatures up to 5–10 °C lower. It hosts some summer scientific stations established from 1988 and benefits from the Antarctic Treaty which regulates both human presence and activities on the continent (Quesada et al., 2009). Although the region has seen an increase in human activities in recent years resulting from the increased popularity of Antarctic tourism and the opening of more research stations by national Antarctic operators, it is still considered one of the earth's last pristine wildernesses, with minimal human impact resulting in some part of it being proposed as a reference site for comparative terrestrial and coastal studies (Quesada et al., 2009). Its relative pristine status makes it an interesting site for investigating the biodegradation of PAHs by indigenous microorganisms in these soils without any history of exposure to lignin, PAHs or similar compounds. Indigenous PAHs have been previously investigated (Aislabie et al., 2000, 2006; Ferguson et al., 2003a, 2003b; Coulon et al., 2005) in Antarctic and sub-Antarctic soils, but these studies have been performed on potentially contaminated soils with high levels of soil PAHs concentration, from areas impacted by Antarctic settlements and scientific stations. To our knowledge, no direct biodegradation measurements have been carried out in soils with extremely low amounts of PAHs, such as those collected from different sites of Livingstone Island and used in this study.

In the present paper we investigate the degradation of 14C-phenanthrene by indigenous soil microorganism in soil samples from Livingstone Island at different temperatures.

Materials and methods

Materials

Phenanthrene (> 99.6%), and [9-14C] phenanthrene (specific activity = 50 mCi mmol−1, radiochemical purity > 95%) standards were obtained from Sigma Aldrich, UK. Chemicals for the minimal basal salts (MBS) solution were obtained from BDH Laboratory Supplies and Fisher Chemicals. The liquid scintillation cocktail (Ultima Gold) and glass scintillation vials (7 mL) were obtained from Canberra Packard, UK. Sodium hydroxide was obtained from Sigma Aldrich. Dichloromethane, hexane and methanol were supplied by Merck, Darmstad, Germany. Agar-agar and plate count agar were obtained from Oxoid Ltd, UK.

Soils sampling and bulk characterization

Soil samples were collected from background areas of Livingstone Island. A map with the sampling sites is provided in Fig. 1. The top 5 cm were taken using a stainless steel corer. Samples were frozen (−20 °C) in sterile glass jars for transportation to Lancaster University. Soil physicochemical properties are shown in Table 1. Soil redox, soil pH and soil moisture content were measured by standard methods described elsewhere(Cabrerizo et al., 2011). Particle size analysis was determined according to the method by Gee and Bauder (1979) and calculations according to Gee and Bauder (1986). Total carbon and nitrogen were determined by analysing 4 mg of oven-dried (105 °C) and sieved (2 mm) soil samples on a Carlo Erba CHNS-OEA 1108 CN-Elemental analyser. For total organic carbon (TOC) analysis, soils were heated to 430 °C to remove all organic carbon, the ash containing inorganic carbon alone was measured on the analyser and the TOC determined by mass balance (Rhodes et al., 2007).

Figure 1

Map showing Livingstone Island and sampling sites.

View this table:
Table 1

Physicochemical properties of five Antarctic soils

PAHs
SoilM.pheDBTDMPPheAnt.FluPyrB(a)antCry.Per.Ind(1,2,3,cd)B(b jk)fluD(ah)ant.B(ghi)pery.∑PAHsTNTCTOCSandClaySiltH2O (%)
10.070.010.030.150.0060.050.030.10.090.0020.010.09< LOQ0.160.960.020.010.1591.143.545.318.0
20.020.0010.010.020.0010.020.010.010.010.010.010.01< LOQ0.010.140.280.250.2291.5905.318.0
30.070.120.030.260.010.120.090.110.180.010.120.180.060.111.470.0040.0040.0297.8102.186.25
40.050.0010.020.060.0030.0240.020.010.010.0010.02< LOQ< LOQ0.040.260.0010.0290.0194.551.813.634.86
50.080.020.030.10.0070.040.010.020.030.0010.030.2< LOQ0.080.650.0070.0987.388.404.21.15
  • M. phe, Methylphenanthrene; DBT, Dibenzothiophene; Phe, Phenanthrene; Ant., Anthracene; Flu., Fluoranthene; Pyr., Pyrene; B(a)ant., Benzo(a)anthracene; Cry, Crysene; Per., Perylene; Ind(1,2,3,cd), Indeno(1,2,3,cd)pyrene; B(b jk)flu., Benzo(b jk)fluoranthene; D(ah)ant., Dibenzo(ah)anthracene; B(ghi)pery., Benzo(ghi)perylene. TN, Total nitrogen (%); TC, Total carbon (%).

  • < detection limit (N: 0.03–2 mg abs; C 0.03–20 mg abs).

PAH concentrations in soil

Extraction and quantification: Briefly, 30 g of soil samples were homogenized and dried by mixing with anhydrous sodium sulphate and ground using a mortar and a pestle. The whole sample was transferred into a soxhlet cellulose thimble (Whatman) and extracted in soxhlet apparatus over 24 h, using dichloromethane:methanol (2 : 1 v/v). Prior to the extraction, samples were spiked with per-deuterated PAHs standards (phenanthrene-d10, crysene-d12 and perylene-d12), which were used as surrogate standards. The extracts were reduced in a rotary evaporator to 1 mL and then solvent-exchanged into isooctane. All samples were then fractionated on a 3% deactivated alumina column (3 g) with a top layer of anhydrous sodium sulphate. Each column was eluted with 12 mL of dichloromethane/hexane (2 : 1 v/v). The PAH fraction was concentrated in a rotary evaporator and solvent-exchanged to isooctane under a gentle stream of nitrogen. After concentration, the samples were transferred to injection vials and 25 µL of anthracene-D10 and benzo(a)anthracene-D12 were added as injection standards. All the samples were analysed by GC-MS using a Thermo Electron (San Jose, CA; model Trace 2000 operating in selected ion monitoring (SIM) mode. Details of temperature programs and monitored ions are given elsewhere (Cabrerizo et al., 2009, 2011).

Quality assurance/control

All analytical procedures were monitored using strict quality assurance and control measures. One field and laboratory blanks were introduced every three soil samples. Phenanthrene, fluoranthene and pyrene were detected in blanks, but they accounted for < 3% of the total sample concentrations. Samples, therefore, were not blank corrected. The surrogate per cent recoveries from the soil samples reported here were (mean ± SD) 70% ± 11 for phenanthrene-d10, 105% ± 17 for crysene-d12 and 90% ± 13 for perylene-d12.

Catabolism of 14C-phenanthrene in soil

Catabolic degradation of 14C-phenanthrene was determined in 250-mL screwcap Erlenmeyer flasks (Reid et al., 2001). The respirometers contained 10 g of soil rehydrated to 40–60% water-holding capacity and spiked with unlabelled and 14C-phenanthrene (80 Bq 14C-phenanthrene g−1 soil) using toluene as a carrier solvent. A 7-mL scintillation vial containing 1 M NaOH was attached to the screwcap to serve as a CO2 trap. Respirometers were stored in the dark at 4, 12 and 22 °C. A slurry system was also set-up containing 30 mL minimal basal salts (MBS) medium and securely placed on a SANYO® Gallenkamp orbital incubator set at 100 r.p.m. and 22 °C to agitate and ensure adequate mixing over the period of the incubation. NaOH traps were replaced every 24 h, after which 6 mL of Ultima Gold scintillation cocktail was added to each spent trap and the contents analysed on a Packard Canberra Tri-Carb 2250CA liquid scintillation counter. The incubation lasted for 35 days. Lag phases were measured as the time before 14C-phenanthrene mineralization reached 5%. Analytical blanks containing no 14C-phenanthrene was used for the determination of levels of background radioactivity.

Microbiological analysis

Colony-forming units (CFUs) of heterotrophic bacteria were enumerated on plate count agar (PCA) using a viable plate counts technique (Lorch et al., 1995). 12C-Phenanthrene was used as a sole carbon source on agar-agar for the measurement of 14C-phenanthrene-degrading bacteria following standard microbiological techniques (Foght & Aislabie, 2005).

Statistical analysis

Levels of 14C-phenanthrene detected by the liquid scintillation counter were corrected for background radioactivity. All samples were analysed in triplicate and errors bars presented in graphs are standard error mean for n = 3. sigma stat version 2.03 software package was used for the analysis of the data. Significance of 14C-phenanthrene degradation between soils and temperatures were assessed by implementing anova and Tukey's tests.

Results

Soil characteristics

Selected soils in different sections of Livingstone Island were found to have similar physicochemical properties. The soils are mostly sandy and slightly alkaline, with low TOC and N contents. The sum of 23 PAH (ΣPAHs) concentrations was low, with values ranging between 0.14 and 1.47 ng g−1 dw soil with higher contribution of low molecular weight PAHs (see Table 1;).

Catabolism of 14C-phenanthrene in soil at different temperatures

The catabolism of 14C-phenanthrene in Antarctica soils at 4, 12 and 22 °C (nonslurried and slurried) as determined by the mineralization of 14C-phenanthrene to 14CO2 by indigenous microbial communities is shown in Fig. 2.

Figure 2

Degradation (%) of 10 mg kg−1 14C-phenanthrene by indigenous soil microbial communities in soils 1, 2, 3, 4 and 5 from Antarctica at 4 °C (○), 12 °C (●), 22 °C (□) and 22 °C (■) slurry after 35 days incubation time.

Lag phases decreased as temperatures increased (see Table 2). The longest lag phase (26.92 ± 0.06 days) was observed in soil 5 at 12 °C and the shortest (1.13 ± 0.16 days) was in soil 2 at 22 °C. At 4 °C, < 5% 14C-phenanthrene was mineralized in all the five soils after a period of 35 days. Only at 22 °C did 14C-phenanthrene mineralize in all five soils exceed 5%.

View this table:
2

Lag phases and fastest rates and extents of 14C phenanthrene mineralization in five Antarctic soils at 4, 12 and 22 °C

Lag (days)Fastest rate (%h−1)Extents (%)
Soil4 °C12 °C22 °C22 °C Slurry4 °C12 °C22 °C22 °C Slurry4 °C12 °C22 °C22 °C Slurry
1Not reachedNot reachedNot reached14.74 ± 6.77 × 10−21.88 × 10−3 ± 3.64 × 10−41.68 × 10−3 ± 1.08 × 10−45.11 × 10−3 ± 6.89 × 10−42.45 × 10−1 ± 2.11 × 10−30.61 ± 1.84 × 10−20.78 ± 1.73 × 10−21.86 ± 3.35 × 10−121.98 ± 3.18 × 10−1
2Not reached25.97 ± 2.73 × 10−19.5111.13 ± 1.61 × 10−14.48 × 10−3 ± 9.50 × 10−45.32 × 10−2 ± 9.39 × 10−41.73 × 10−1 ± 1.24 × 10−32.7 × 10−1 ± 5.67 × 10−31.14 ± 2.72 × 10−115.23 ± 1.8 × 10−139.09 ± 6.1147.93 ± 4.44 × 10−1
3Not reachedNot reached25.3620.75 ± 2.33 × 10−21.33 × 10−3 ± 1.72 × 10−042.44 × 10−2 ± 2.45 × 10−181.23 × 10−1 ± 6.83 × 10−11.83 × 10−1 ± 2.66 × 10−30.55 ± 6.39 × 10−23.77 ± 4.09 × 10−215.50 ± 2.94 × 10−126.85 ± 1.97
4Not reachedNot reachedNot reached20.39 ± 6.61 × 10−11.99 × 10−3 ± 1.99 × 10−38.06 × 10−3 ± 8.06 × 10−35.66 × 10−3 ± 5.66 × 10−32.38 × 10−1 ± 2.38 × 10−10.63 ± 9.06 × 10−22.28 ± 6.74 × 10−22.41 ± 7.01 × 10−124.18 ± 1.13
5Not reached26.92 ± 5.62 × 10−214.64 ± 9.62 × 10−217.13 ± 7.20 × 10−22.54 × 10−3 ± 2.06 × 10−41.27 × 10−1 ± 2.63 × 10−31.49 × 10−1 ± 4.69 × 10−35.25 × 10−1 ± 2.84 × 10−30.64 ± 5.32 × 10−235.15 ± 3.2414.76 ± 1.76 × 10−130.70 ± 9.39 × 10−1

Lowest rates of 14C-phenanthrene mineralization were observed for all soils at 4 °C, the fastest rate observed for all five soils at this temperature being 0.002 ± 0.001% h−1. The rates of 14C-phenanthrene mineralization were fastest at 22 °C under slurry conditions (0.56 ± 0.01% h−1 for soil 5). Though rates increased with increasing temperature, a significant increase in rates of 14C-phenanthrene mineralization (P < 0.05) was only observed when the rates of 14C-phenanthrene mineralization at 4, 12 and 22 °C were compared with those of the slurry system at 22 °C. Increasing the temperature from 4 to12 °C, 12 to 22 °C and 4 to 22 °C did not significantly increase fastest rates of mineralization (P > 0.05).

Generally, 14C-phenanthrene was mineralized at higher rates and to greater extents as temperatures increased. At 4 °C, maximum 14C-phenanthrene mineralized was 1.14% in soil 2. Increasing the temperature to 12 °C resulted in a maximum of 17.85% (soil 5) and a significant increase (P < 0.05) in the amount of 14C-phenanthrene mineralized only in soils 2 and 5. A further increase to 22 °C resulted in a significant increase in the amount of 14C-phenanthrene mineralized in all five soils (P < 0.05). The maximum amount of 14C-phenanthrene mineralized at 22 °C was 39.09% and was significantly greater (P < 0.05) than that mineralized at both 4 and 12 °C for all the soils.

Slurrying the soils (at 22 °C) led to further increases in the extent of 14C-phenanthrene mineralization in all of the soils, with a minimum of 21.98% (soil 1) and a maximum of 47.97% (soil 2) 14C-phenanthrene mineralized over the 35 days incubation period. 14C-phenanthrene mineralization was significantly greater in the slurried system than at 22 °C for all the soils apart from soil 2.

Heterotrophic and 14C-phenanthrene-degrading bacteria

CFU of phenanthrene degraders and total heterotrophs present in the soils ranged between 104–106 and 103–104 CFU g−1. Results are shown in Fig. 3. The highest counts of phenanthrene degraders (1.53 × 104) were observed in soil 3 and the lowest (8.6 × 103) in soil 4. Only incubation in slurried conditions gave increases in both phenanthrene-degrading bacteria and total heterotrophs.

Figure 3

CFUs of total PAH degraders and total heterotrophs before and after degradation at 4, 12, 22 and 22 °C slurry.

Discussion

Although the soils used in this study are from Livingstone Island, a sub-Antarctic Island, far from industrialized regions and limited human activity, PAHs were found in all the five soils at levels similar to those reported in uncontaminated/pristine soils (Johnsen & Karlson, 2005; Cabrerizo et al., 2012). The higher presence of low molecular weight PAHs in the soils may represent the sum of different contributions firstly, long-range transport of semi volatile organic pollutants to the Antarctic ecosystem. Wania & Mackay (1996) hypothesized that as PAHs are globally distributed, they fractionate according to the volatility of the individual compounds. Secondly, PAH fractionation can also occur locally (Wilcke et al., 1996). In the case of Livingstone Island, ships and human settlements could have served as local/regional PAH sources. Thirdly, potential autochthonous biogenic formation of PAHs from the degradation of organic matter (Aislabie et al., 1999; Wilcke, 2007; Cabrerizo et al., 2011). The presence of PAHs, especially low molecular weight biodegradable fractions, justify the generalized occurrence of phenanthrene degradable bacteria in these soils (Aislabie et al., 1998).

Respirometric assays, such as the one used in this study for the determination of indigenous microbial degradation of 14C-labelled organic compounds, have been employed in numerous studies (Macleod & Semple, 2006; Swindell & Reid, 2006). The results described in this current study show that 14C-phenanthrene degradation was evident in all selected soils and generally increase with increasing temperature, as other studies have already pointed out (Atlas, 1975; Ferguson et al., 2003a, 2003b).

Biodegradation of hydrocarbons in contaminated Antarctic and sub-Antarctic soils has been found to be limited by low microbial activity, cold temperatures, nutrient availability, low water content and alkaline pH (Foght et al., 1999; Margesin & Schinner, 1999; Delille, 2000; Delille et al., 2004). Characterization of the five Livingstone Island soils used in this study revealed physicochemical properties consistent with those by which Antarctica soils are generally defined (Bockheim, 1997; Campbell & Claridge, 2009). Poor water-holding capacity resulting from highly coarse/sandy soils is a limiting factor for microbial activity (Aislabie et al., 2000) and is expected to limit the extent of 14C-phenanthrene biodegradation in the soils; low TOC in soils can be an indication of low microbiological activity (Margesin & Schinner, 2001). Samples taken in the selected sites were mostly bare of vegetation and plate counts revealed very low CFUs (Fig. 3) for both total heterotrophs and 14C phenanthrene-degrading bacteria. The presence of only small numbers of PAH-degrading bacteria can be explained by the absence of degradation inducing chemicals from both biogenic and anthropogenic sources. Sufficient concentrations of biogenic volatile organic chemicals (VOCs) from plants (Wilcke, 2007; McLoughlin et al., 2009) and anthropogenic compounds have been identified as carbon sources for microbial activity, growth and the induction of appropriate genes for PAH degradation in indigenous microorganisms (Macleod & Semple, 2002; Johnsen & Karlson, 2005). Hydrocarbon degraders have been cultivated at levels > 105 cell g−1 from contaminated polar soils and have increased following oil spillage by 1–2 orders of magnitude in hydrocarbon contaminated soil compared with pristine soils (Aislabie et al., 2000). In this study, CFUs of 14C-phenanthrene-degrading bacteria increased in all five soils and by one order of magnitude in soils 1, 3 and 5 after mineralization in slurry conditions (Fig. 3).

Of the three temperatures used in this study, 4 °C was the most representative of prevailing temperatures at Livingstone Island hence appropriate for optimum microbial activity. However, no significant amount of 14C-phenanthrene was mineralized in any of the five soils (Table 2).

Reduced bioavailability of PAHs at low temperatures has also been reported as a possible reason for low levels of microbial degradation (Eriksson et al., 2003). At low temperatures, the solubility and bioavailability of less soluble hydrophobic organic compounds, such as PAHs, decrease because of an increase in viscosity in the physical nature of the compounds and because of stronger sorption to the soil organic matter. Increased viscosity will decrease the degree of organic compound distribution (less surface area for microbial action) and subsequent diffusion rates to sites of biological action leading to reduced extents of degradation (Nam & Kim, 2002). Ferguson et al. (2003a, 2003b)obtained similar results when they found that mineralization of 14C-labelled octadecane was virtually absent at temperatures below or near the freezing point of water.

At 12 °C, the extents of 14C-phenanthrene mineralized increased significantly in two of the five soils after a long lag phase. 14C-Phenanthrene was mineralized to a greater extent at 22 °C than at 4 and 12 °C for all the soils. The increasing solubility of phenanthrene with increasing temperature would mean that the amount of phenanthrene in solution (and therefore available for degradation) would have been higher at 22 °C that at 4 and 12 °C. Despite temperatures at Livinstone Island never reaching 22 °C, degradation of 14C-phenanthrene at this temperature suggests the presence of psychrotrophic microorganisms. The lag phase was shorter at 22 °C than those at 4 and 12 °C for all the soils. Results of microbiological counts show an increase in phenanthrene degraders after the 35 day mineralization assay. Slurrying the system increased both the rates and extents of mineralization in all soils. Previous studies (Labare & Alexander, 1995; Doick & Semple, 2003) suggest that increased mineralization as a result of slurrying a system can be as a result of increased surface area at the contaminant-water interface as the soil particles separate and move into suspension, leading to rapid partitioning of the substrate into the aqueous phase and stimulated microbial activity.

Conclusion

This study further supports claims of the ubiquitous nature of PAH-degrading microorganisms by providing evidence for the presence of 14C-phenanthrene-degrading microorganisms in soils from Livingstone Island, an uncontaminated Antarctic Island not previously studied. Considering the unique characteristics of these soils and the clear effect of temperature on microbial degradation, the identification of specific phenanthrene degraders active at different temperatures will be useful for potential bioremediation of contaminated Antarctic soils because the introduction of foreign microbial species into Antarctica is prohibited by the Antarctic treaty. Also, the effect of temperature on the sequestration of PAHs and the development of PAH catabolic properties by indigenous Antarctic microorganisms should be investigated.

Footnotes

  • Editor: Elizabeth Baggs

References

View Abstract