Hexadecane mineralization activity in hydrocarbon-contaminated soils of Ross Sea region Antarctica may require nutrients and inoculation

Soil Biology & Biochemistry 45 (2012) 49e60

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Hexadecane mineralization activity in hydrocarbon-contaminated soils of Ross Sea region Antarctica may require nutrients and inoculation Jackie M. Aislabie a, *, Janine Ryburn a, Maria-Luisa Gutierrez-Zamora b,1, Phillipa Rhodes a, David Hunter a, Ajit K. Sarmah a, 2, Gary M. Barker a, Roberta L. Farrell b a b

Landcare Research, Private Bag 3127, Hamilton, New Zealand School of Biological Sciences, University of Waikato, Private Bag 3105, Hamilton, New Zealand

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 July 2011 Received in revised form 7 October 2011 Accepted 8 October 2011 Available online 25 October 2011

Hydrocarbon spills on Antarctic soils occur mainly near settlements where fuel is stored and aircraft and vehicles are refuelled. To investigate those factors that may preclude hexadecane mineralization activity in long-term hydrocarbon-contaminated soils from the Ross Sea Region, samples were collected from Scott Base, the site of former bases (Cape Evans, Marble Point, Vanda Station), and two oil spill sites in the Wright Valley (Bull Pass and Loop Moraine). The soils had low levels of nitrogen (<0.1% total N) and a high C/N ratio (>24) reflecting hydrocarbon contamination. Following soil water adjustment to 10% (v/w), the influence of nutrient addition (250 mg/kg N added as monoammonium phosphate) and inoculation (spiking with Antarctic soil containing high numbers of hydrocarbon degraders) as required on hexadecane mineralization activity was determined. Hexadecane mineralization activity occurred in contaminated soils from Marble Point, Cape Evans and one sample from Vanda Station without nutrient addition. In contrast soils from Scott Base, Cape Evans, another sample from Vanda Station and Loop Moraine required nutrients, whereas Bull Pass soil required inoculation and nutrients before hexadecane mineralization proceeded. Hydrocarbon degrader numbers were highest in coastal soils from Scott Base and Marble Point (107 per gram) and less prevalent in inland soils from Wright Valley (<105 per gram). The bacterial community structure of the soils differed between sites, but soils from the same sites tended to cluster together more closely, except for those from Vanda Station. Addition of nutrients did not cause large shifts in the soil bacterial communities. Results from this study indicate that hydrocarbon degradation may occur at some sites in summer when water is available. Long-term hydrocarboncontaminated Antarctic soils may provide a valuable resource of hydrocarbon-degrading bacteria that can serve as inocula for more recent oil spills on land. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Enhancing alkane mineralization Soil bacterial diversity Nitrogen Alkane degraders in soil as inoculum Antarctica

1. Introduction Human activities in Antarctica rely on fossil fuels for transportation, heating, and power generation. Fuel spills on Antarctic soils occur mainly near settlements including current and former scientific research stations and field camps where fuel is stored and aircraft and vehicles are refuelled (Aislabie et al., 2004). Chemical characterisation of the hydrocarbon contaminants have revealed

* Corresponding author. Tel.: þ64 7 859 3713; fax: þ64 7 859 3700. E-mail address: [email protected] (J.M. Aislabie). 1 Present address: Centre for Marine Innovation, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Randwick 2052, Sydney, Australia. 2 Present address: Department of Civil & Environmental Engineering, The University of Auckland, Private Bag 92019, Auckland, New Zealand. 0038-0717/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2011.10.001

that n-alkanes predominate (Kennicutt et al., 1992; Green and Nichols, 1995; Aislabie et al., 1998), with lower concentrations of monoaromatic and polyaromatic hydrocarbons, reflecting the chemistry of the refined petroleum products used in Antarctica such as the aviation fuel JP8. When spilt on Antarctic soils, possible mechanisms for loss of hydrocarbons include dispersion, evaporation, and biodegradation (Aislabie et al., 2004; Snape et al., 2005, 2006). To meet obligations of the Protocol on Environmental Protection to the Antarctic Treaty, parties are committed to remediation of contaminated sites as long as the remediation is not detrimental to the Antarctic environment. Bioremediation has been proposed as a technology for remediation of Antarctic soils (Aislabie et al., 1998). Fuel spills on Antarctic soils can result in the enrichment of hydrocarbon-degrading microbes within the indigenous microbial community (Aislabie et al., 2004; Delille et al., 2007; Ferguson

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J.M. Aislabie et al. / Soil Biology & Biochemistry 45 (2012) 49e60

et al., 2008; Powell et al., 2006). Hydrocarbon degraders cultured from Antarctic soils are usually bacteria, though shifts in the culturable fungal community in response to hydrocarbon contamination have been reported (Aislabie et al., 2001; Arenz et al., 2006). Those bacteria that degrade alkanes are frequently identified as members of the genera Rhodococcus or Pseudomonas. Rhodococcus strains isolated from hydrocarbon-contaminated soil near Scott Base, Antarctica, grew on n-alkanes only, whereas Pseudomonas isolates sometimes grew on both n-alkanes and aromatic compounds (Aislabie et al., 2006b). Clones assigned to Pseudomonas and Rhodococcus were prevalent in hydrocarboncontaminated mineral soil but not pristine soils from Scott Base (Saul et al., 2005) providing further support for the role of these organisms in hydrocarbon degradation in situ. However, there has been little consideration of bacterial community dynamics in Antarctic soils contaminated with hydrocarbons and their response to soil conditions (Powell et al., 2010; Vázquez et al., 2009). Environmental conditions that may preclude the activity of hydrocarbon degraders in summer when Antarctic mineral soils are thawed include low and fluctuating temperatures, low levels of moisture and nutrients (e.g., nitrogen and phosphorus), and extremes in pH and salinity (Aislabie et al., 2006b). Mean annual surface soil temperatures in the Ross Sea region are low (approximately 20  C); however, during the summer months (December to February) when there is continuous daylight average soil temperatures >0  C have been recorded (Aislabie et al., 2006a). Soil temperatures in summer show diurnal variations that are correlated with solar radiation. Hence the surface temperatures of snow-free soils may range from below 0 to þ20  C during a single day with multiple freeze-thaw cycles (Balks et al., 2002). Soil surface temperate at oiled sites may be up to 10  C warmer than an adjacent pristine site due to decreased soil surface albedo from surface darkening by hydrocarbons (Balks et al., 2002). Soil water availability is often low due to low precipitation, high evaporation rates, low soil humidity and freezing (Aislabie et al., 2006a). In dry valley soils with low precipitation salt accumulation may also negatively impacts on water availability (Aislabie et al., 2006a). Antarctic mineral soil is generally low in nutrients (Tarnocai and Campbell, 2002) and the introduction of high concentrations of hydrocarbons can further deplete available nitrogen and phosphorus when they are assimilated during biodegradation. As with temperate soils, amendment of Antarctic mineral soils with nitrogen can enhance hydrocarbon mineralization (Aislabie et al., 1998; Ferguson et al., 2003). Coastal Antarctic soils are often alkaline but substantial hydrocarbon mineralization has been detected in soils at pH 9.4 (Aislabie et al., 1998). The aim of this study was to investigate those factors that may preclude hexadecane mineralization activity in hydrocarboncontaminated soils from the Ross Sea region. The soils were collected from sites of existing (Scott Base, Ross Island) and former research stations (the century-old historic Terra Nova at Cape Evans, Ross Island, and Vanda Station, Wright Valley) and two oil spill sites in the Wright Valley (Bull Pass and Loop moraine) (Fig. 1). The soil water content was adjusted to 10% (w/v) and the influence of nutrient (as monoammonium phosphate) addition on hexadecane mineralization activity was measured. The Wright Valley soils not exhibiting mineralization activity following nutrient addition were spiked with Scott Base soil containing high numbers of hydrocarbon degraders and the impact on hydrocarbon mineralization activity was determined. Changes in the structure of the soil bacterial community in response to treatment were assessed using terminal restriction fragment length polymorphism (T-RFLP).

2. Materials and methods 2.1. Sampling site location and sample collection Hydrocarbon-contaminated soil samples were collected from the Ross Sea Region of Antarctica from the sites of one current [Scott Base (SB)] and three former research stations [Marble Point (MP), Terra Nova at Cape Evans (CE), Vanda Station in Wright Valley (VS)], and from two oil spills sites in the Wright Valley [at a drill site in Bull Pass (BP), and a helicopter crash site on Loop Moraine (LM)] (Fig. 1; Table 1). At four of the sites (SB, MP, CE and BP), pits were dug, and the narrow distinct surface layer (5 cm) and the layer immediately below the surface were sampled using an ethanol-swabbed trowel. At remaining sites, surface soil samples (0e5 cm) only were collected. The two samples from each of the sites are hitherto referred to as (a) and (b). Soil samples for chemical and microbial analysis were placed in sterile Whirl-PakÒ bags (Nasco), frozen at 20  C, and transported to New Zealand for processing. Soils were classified into the Gelisol order to the family level (Soil Survey Staff, 2010). The texture of all soils was gravelly sand. 2.2. Soil chemical and physical analyses The sampled soils were sieved (<2.00 mm size) and analysed for þ water content, pH, electrical conductivity (EC), NO 3 -N, NH4 -N, and total P using standard methods (Blakemore et al., 1987). Organic carbon and total N in soils were determined in a Leco FP 2000 analyser at 1050  C. All soil chemical properties are rated following Blakemore et al. (1987). Levels of total petroleum hydrocarbons (TPH) in samples were determined by extracting in methylene chloride, then analysing the extracts by capillary gas chromatography with flame ionisation detection, essentially as outlined in EPA Method 8015 (US EPA, 1987). 2.3. Microbial mineralization of [141-C] hexadecane The rate of hexadecane mineralization in soil samples was measured using methods described previously (Aislabie et al., 2008b). Subsamples (10 g dry weight equivalent) from the bulked soil were placed in beakers and mixed with 18.5 kBq [141-C] hexadecane (444 M Bq mmol1) dissolved in 20 ml of sterile jet fuel to give a final concentration of 9.42 mg [141-C] hexadecane g1 soil. Soil water content was adjusted to approximately 10% (v/w) and maintained at that level for all samples, except those from MP, which were not artificially hydrated. At a gravimetric water content of 10% the soils have a matric potential between 10 and 100 kPa and sufficient available water to support hydrocarbon biodegradation (Balks et al., 1998). Furthermore, at 10%, the water content of the soils is at about field capacity, but not saturated. For sterile controls, soil samples in beakers were autoclaved for 3 h at 121  C before hydrocarbon addition. The beakers of soil were placed in one-litre glass Mason jars alongside 10 ml of 1 M KOH in a small beaker as a CO2 trap. Five subsamples and one sterile control were prepared for each soil treatment. Humidity was maintained by adding 3e4 ml of water into the base of the jar. The jars were incubated statically in the dark at 15  C for 91e105 days. In summer fine weather soils of the Ross Sea region can reach 15  C in situ (Balks et al., 2002). Furthermore alkane degrading bacteria isolated from polar soils are typically psychrotolerant and active at 15  C (Aislabie et al., 2006b). At regular intervals, CO2 traps were removed, 0.5 ml of the KOH mixed with scintillation cocktail (10 ml Ultima Gold) (Packard), and the radioactivity determined by a liquid scintillation counter. The KOH was replaced at each sampling event. The amount of 14CO2 trapped from the soil, corrected for background radiation

J.M. Aislabie et al. / Soil Biology & Biochemistry 45 (2012) 49e60

51

Fig. 1. Location of sample sites.

levels, was taken as the measure of mineralization of [141-C] hexadecane. The influence of nutrient addition on hexadecane mineralization was determined in soil from SB (a & b), CE (a), VS (b), BP (a & b) and LM (a & b) (Table 2). The soils (10 g dry weight equivalent) were amended with 250 mg/kg-N added as monoammonium phosphate dissolved in water to give a final water content of 10% (v/w). The nitrogen levels used in this study were optimal for hydrocarbon degradation in sub-Antarctic soil (Walworth et al., 2007). Sterile controls were prepared and all samples were assayed as described above. For (BP (a & b), and VS (b), which exhibited minimal hexadecane mineralization activity following water and nutrient addition, the influence of bioaugmentation on hexadecane mineralization was

determined experimentally by inoculating soil samples. Soil samples (9 g dry weight equivalent) were inoculated with 1 g dry weight equivalent) of hydrocarbon-contaminated soil from SB (b) (Table 2), and subsamples were further amended with addition of nutrient and water before assayed for hexadecane mineralization as described above. Duplicate or triplicate samples for bacterial community structure analysis using T-RFLP included the following: 1) Time 0 samples from bulk soil samples used for the mineralization assays; 2) samples taken at the completion of all mineralization assays when soil was aseptically removed from two or three of the five beakers at random; and 3) Time 0 inoculated samples from BP and VS (b) soil inoculated with SB soil and used in mineralization assays. At each sampling point, approximately 1 g of soil was placed

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Table 1 Location, soil classification, site description and estimates of time since oil spillage. Site Locations

Soil classification

Site description

Coastal sites; GPS coordinates Scott Base (SB); 77 550 S, 166 450 E

Typic haplorthel

Marble Point (MP); 77 250 S, 163 41’E

Calcic haplorthel

Cape Evans (CE); 77 38’S, 166 24’E

Lithic haplorthel

Storage area for drums of hydraulic and lubricating oils. The base has been operational since 1959, but it is not known how long oils were stored at the sample site. Oil stains on soil surface at site of the Old Marble Point camp assumed to be hydraulic and lubricating oils. The camp was operational from 1957 to 1963. Fuel dump of the Transantarctic Expedition near Terra Nova Hut. The base was operational 1911e1917. Analysis of oil stored at the site revealed predominantly n-alkanes from C5-C10 (Volk et al., 2005).

Wright Valley sites Vanda (VS); 77 31’S, 161 40’E

Lithic anhyorthel

Bull Pass (BP); 77 31’S 161 520 E

Nitric anhyorthel

Loop Moraine (LM); 77 29’S 162 21’E

Typic haplorthel

Oil spills near former Vanda Station which was operational from about 1970 to 1991. Diesel fuel Arctic was spilt during seismic bore-hole drilling in 1985. Oil spilt following helicopter crash in 1962. Aviation fuel was spilt along with engine oil.

in microfuge tubes and stored at 80  C before T-RFLP analysis as described below. 2.3.1. Kinetic analysis of hexadecane mineralization rates Mineralization data, for those assays with extent of mineralization >5%, was modelled using a 3-parameter sigmoidal logistic function with the following equation:

A

Y ¼



1 þ exp

XXo B

Estimates of time since oil spillage




where, X is the incubation time (days), A is the maximum extent (%) of hexadecane mineralization, Y is the % of hexadecane mineralization, B is the slope or the mineralization rate constant (day1), and Xo is the time in days at the inflection point. The lag times were

30

50þ

90þ

20þ 20þ 40þ

estimated iteratively using time at the inflection point. Sigma Plot software (version 7) was used to fit the data. 2.4. Soil microbial analyses For enumeration of culturable heterotrophic bacteria and hydrocarbon degraders, 10 g soil (wet weight) was shaken for 1 h at 4  C in 90 ml 0.1% (w/v) sodium pyrophosphate (pH 8) containing 30 g glass beads (3 mm) and then diluted in phosphate-buffered saline (PBS) as required. Numbers of culturable heterotrophic bacteria were then determined by plating soil dilutions (ranging from 101 to 107) onto R2A agar (Difco). All plates were incubated at 15  C for at least 6 weeks. Relative differences in numbers of hydrocarbon-degrading microbes were determined using a 5-tube most probable number MPN method. Soil dilutions (ranging from 101 to 109) in PBS were

Table 2 Soil chemical and microbial properties. Location Depth (cm)

Coastal spill sites Scott Base 0e2 2e10 Control Marble Point 0e3 3e12 Control Cape Evans 0e3 3e10 Control

Sample

TPH mg/kg dry weight

Water (%)

Organic C (%)

Total N (%)

NHþ 4 -N mg/kg dry weight

a b

33,700 25,400 <30

1.6 5.8 1.8

5.14 3.44 0.1

0.02 0.01 0.01

0.9 0.4 3.3

a b

29,100 18,300 <20

1.9 6.4 2.4

5.33 3.36 0.28

0.02 0.01 0.02

a b

9400 18,800 <30

0.8 1.1 4.0

1.79 2.62 0.02

9800 2240

0.5 0.3

1300 2100 6400 7100 <30

Wright Valley spill sites Vanda 0e5 a 0e5 b Bull Pass 0e5 a 5e10 b Loop moraine 0e5 a 0e5 b Control (Vanda)

NO 3 -N mg/kg dry weight

C/N

Total P (%)

pH

EC mS/cm

0.8 0.4 1.3

266 362 7

0.15 0.12 0.19

7.8 8.8 8.9

3.5 2.2 3.4

0.5 0.5 2.2

296 305 16

0.06 0.06 0.07

0.03 0.03 0.00

5.8 5.6 0.2

3.4 4.4 24.7

62 99 6

1.28 0.3

0.00 0.00

5 3.3

4.7 3.6

564 70

1.3 1.5

0.14 0.13

0.02 0.02

2.6 1.5

0.4 0.3 0.2

1.30 1.44 0.06

0.00 0.00 0.00

0.5 1.7 1.6

205 149 0.9 0.4 1.3

Number of Culturable heterotrophs/g dry weight

MPN of Hydrocarbon degraders/g dry weight

0.22 0.18 0.29

4.2  106 1.3  106 3.4  106

1.3  105 1.4  105 33

8.3 9.2 9.6

0.18 0.19 0.65

5.3  107 4.2  107 3.7  105

1.1  107 1.8  106 <10

0.13 0.18 0.10

6.8 7.5 8.2

0.77 0.30 11.30

1.0  104 2.0  106 1.6  102

<10 2.3  104 <10

0.03 0.02

7.8 7.4

0.32 0.18

2.6  104 3.5  104

6.1  101 6.9  103

6 7

0.018 0.019

7.6 7.7

7.25 5.06

3.6  103 4.5  104

<10 <10

542 452 9

0.013 0.012 0.02

7.1 6.9 9.1

0.04 0.03 0.09

5.3  105 6.0  104 3.6  104

3.3  102 1.2  101 <10

J.M. Aislabie et al. / Soil Biology & Biochemistry 45 (2012) 49e60

inoculated into bottles containing 10 ml Bushnell Haas Medium (BH; Difco) with 50 ml of jet fuel as sole carbon and energy source. Poisoned controls were prepared by addition of 0.2 ml of analytical grade concentrated HCl. All bottles were incubated at 15  C for 42 days. To determine whether growth had occurred in the tubes, they were compared with the controls, and those that were both turbid and showed disruption to the film of oil on the surface of the medium were scored as positive. 2.4.1. Soil bacterial community structure DNA was extracted from 1 g of soil (wet weight) by mechanical disruption using zirconium beads as described previously (Foght et al., 2004), with the addition of a heating step at 65  C for 1 h prior to the addition of chloroform. The DNA concentrations of the extracts were quantified by spectrophotometry using a Nanovue (GE). Polymerase Chain Reaction (PCR) was performed as described by Singh et al. (2006) with primer sets for bacteria (1087r/63f) on an Eppendorf Master Cycler Gradient. PCR amplification products were visualised on a 1.5% agarose gel under UV radiation. PCR products were purified using the QIAquick Purification Kit (Qiagen) following the manufacturer’s instructions. The DNA concentration of the purified products was determined using a Nanovue. The PCR products were digested with MspI and HhaI in a 30-ml reaction mixture containing 400 ng of PCR products, 1  buffer, 0.1 mg ml1 of acetylated BSA and 20 units of restriction enzyme (New England Biolabs). Samples were incubated at 37  C for 2 h followed by an inactivation step at 95  C for 15 min. After digestion, the fluorescent labelled terminal restriction fragments (T-RFs) were separated using an ABI PRISM 3100 Genetic Analyser (Applied Biosystems, Australia). T-RFLP profiles were produced using Gene-Mapper (version 4.0) software (Applied Biosystems). Terminal restriction fragments (T-RFs) were quantified using the advanced mode and secondorder algorithms. Peaks between 50 and 500 bp were included in analysis to avoid TRF caused by primer-dimers and to obtain fragments within the linear range of the internal standard. The relative abundance of a TRF in a T-RFLP profile was calculated by dividing the peak height of the TRF by the total peak height of all TRFs in the profile. All peaks with heights that were less than 0.5% of the total peak height were removed from the data before statistical analysis. 2.4.2. Discrimination analysis Discrimination of microbial communities was examined by semi-strong-hybrid (SSH) multidimensional scaling ordination (Belbin, 1991; Faith et al., 1987), with the Bray and Curtis (1957) metric as the measure of community compositional distance. Data input into the ordination was amounts for each individual TRF detected in the soil samples before and after treatment to enhance alkane mineralization activity. The ordination was implemented in the PATN software package (Belbin, 1995). Principal component correlation (PCC) (Belbin, 1991) analysis was then performed in PATN to examine directionality and correlation, in the ordination space, of gradients in the soil parameters. Ordination was undertaken for 1) Time 0 samples 2) Time 0 samples and with or without nutrient treatment and 3) samples inoculated with SB (b) soil both Time 0 and following incubation and Time 0 SB (b) and VS (b) samples. 3. Results 3.1. Soil characteristics The properties of the soil samples are shown in Table 2. For comparative purposes we included data from control sites located

53

near the oiled sites. A feature of the soils were the high C/N ratio (>24) of the oiled sites, except for BP, compared with the control sites reflecting TPH contamination. Total N was very low (<0.1), nitrate was highest at BP. Total P was low in the Wright Valley soils (<0.04%) and medium (0.04e0.08%) to very high (>0.12%) in coastal soils from SB, MP and CE. Water content was variable with lowest levels measured in Wright Valley soils. The pH of the soil ranged from near neutral (>6.6) to extremely alkaline (pH 9) with highest pH measured in soils from SB and MP. EC was typically medium (0.4e0.8) or low (<0.4) with very high levels (>2) measured in soil from BP. Numbers of culturable microbes and hydrocarbon degraders were higher in SB and MP soil. 3.2. Hexadecane mineralization [141-C] hexadecane mineralization activity was detected in vitro in soil samples from all sites under varying conditions (Fig. 2). In the absence of nutrient addition, hydrocarbon mineralization was detected in soil from MP (a & b), CE (b) (Fig. 2a) and VS (a) (Fig. 2b). In contrast, soil from SB (a & b), CE (a) (Fig. 2a), VS (b) and LM required nutrients (Fig. 2b), and BP soil (a & b) (Fig. 2b) nutrient addition and inoculation. Modelled mineralization activity was well described by the logistics model with regression coefficients > 0.992 (Table 3). The extent of mineralization varied from 9 to 69%. The mineralization rate constants ranged from 1.44 to 7.70% day1. Mineralization activity was highest (>60%) in coastal soils, specifically nonamended MP (a) and SB (a) þ nutrients, and VS (b) following inoculation. Mineralization began without appreciable lag for samples SB, MP and CE (b) when conditions allowed. The longest lag of 50 days was detected in BP (a), following additions of nutrients and inoculum. Hexadecane mineralization activity in sterile soils was typically negligible (<1% 14CO2) indicating the role of microorganisms in hydrocarbon mineralization. 3.3. Soil bacterial community structure Ordination was used to investigate relationships among sites based on bacterial community composition as indicated by TRF profiles (Fig. 3a), and associations with measured environmental gradients (Fig. 3b), before any of the soils were treated. The ordination provided a robust depiction of microbial communities across the sampled soils (stress in 3 dimensions ¼ 0.0793). Biplots of the ordination results are presented in Fig. 3, as the first 2 axes account for most of the variance (Axis 1 ¼ 0.58, Axis 2 ¼ 0.25) in the bacterial community composition. Axis 1 scores were strongly correlated with gradients of soil pH (y ¼ 0.9297x þ 7.76, R2 ¼ 0.578), TPH (y ¼ 0.9176 Lnx2 þ 1.3149 Lnx þ 9.7714, R2 ¼ 0.761) and organic carbon (y ¼ 2.1019x þ 2.6, R2 ¼ 0.655) (Note these gradients co-vary across soils, r ¼ 0.57e0.91), indicating soil properties may be the drivers of soil bacterial community composition. Axis 2 scores did not correlate simply with measured soil parameters. From the biplots it is evident that soils from the same sites tend to cluster together more closely. Soils from VS exhibited the most variation in position in the ordination space, with variation primarily aligned to Axis 2. From these results, it can be inferred that MP and SB soil bacterial community structure were influenced by higher levels of soil water and organic matter, much of which occurred as TPH. Alkaline pH was also a strong influence on the MP soils. Bacterial community structure of CE and VS (a) soils was primarily related to salinity (measured as EC) and nitrate, which accumulates in dry soils. Low levels of total N and P were clearly significant for structuring the bacterial communities of soils from LM and VS (b). Ordinations using subsets of the sites were then run to determine the response of the soil bacterial communities to treatment,

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Fig. 2. [141-C] hexadecane mineralization activity at 15  C in a) coastal soils from SB, MP and CE and b) dry inland soils from Wright Valley, specifically LM, VS, BP with no nutrient treatment (-:-,-6-), nutrient treatment (-C-, -B-), inoculation and nutrient treatment (->-,-A-) and sterile controls (-,-). Closed symbols ¼ a samples and open symbols ¼ b samples.

specifically to 1) soil amendment with or without nutrients, and 2) inoculation as required to enhance alkane mineralization. The results of these ordinations are depicted as dendrograms in Fig. 4. No significant shift in the bacterial community composition was evident in SB, MP and CE (b) soils following treatment with or without nutrient addition. As is shown in the dendogram in Fig. 4a, treated SB, MP and CE (b) soils clustered with Time 0 soils from the respective sites. In contrast, the CE (a) soils, treated with water only or water and nutrients, clustered among treated LM soils. There was more variability in bacterial community compositional response within soils from Wright Valley (Fig. 4b) than those from SB and MP, possibly reflecting lower biomass in inland soils as indicated by lower numbers of culturable heterotrophs (Table 2). Overall, however, there does not appear to have been any major shifts in the structure of the bacterial communities in the Wright Valley soils except for those from VS (a). The Time 0 VS (a) samples clustered together, and the untreated (water only) VS (a) samples clustered most closely with those from VS (b) and LM. Following inoculation VS (b) soil Time 0 with and without inoculum and SB (b) soil, the source of the inoculum formed a distinct cluster (Fig. 4b). After incubation inoculated VS (b) soil

clustered with BP soils, both soils analysed from Time 0 and post incubation. These results indicated that the bacterial community composition of the soils has been modified following inoculation and that the VS (b) soils converge with those from BP. 4. Discussion 4.1. Soil hexadecane mineralization activity Hydrocarbon mineralization studies with Antarctic soils have indicated that nitrogen availability is the main limiting factor for biodegradation in summer when soils are thawed (Aislabie et al., 1998; Ferguson et al., 2003). Total N levels in the soils were low (<0.04% or <400 mg/kg). As inorganic N was typically <10 mg/kg, except for those soils from Bull Pass where nitrate was accumulating, most of the N was likely organic N. Recently, much of the organic C in Antarctic soil has been shown to be associated with microbial biomass (Feng et al., 2010) and we postulate that this is likely to be the case for organic N though it is also possible that some may be associated with the residual fuels in the soils. In situ nitrogen may be mobilised from

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55

Table 3 Modelled parameters of [141-C] hexadecane mineralization in Ross sea region soil samples incubated in the laboratory at 15  C with and without nutrient addition, and inoculation plus nutrient addition as required. Location Depth (cm) Coastal soils Scott Base 0e2 2e10 Marble Point 0e3 3e12 Cape Evans 0e3 3e10

Sample

b

Maximum extent of mineralization A (%)

Mineralization rate constant (% day1)

Lag time (days)

Coefficient of determination (R2)

NPa

Ib

a a b b

 þ  þ

   

1 68.90 2.4 46.88

1.44

a b

 

 

62.79 49.61

2.48 1.74

0.02 0

0.999 0.999

a a b

 þ 

  

1.5 27.48 53.16

3.57 1.78

>90 18.01 0

0.999 0.998

  þ þ

   þ

28.63 0.3 9.09 62.56

 þ þ  þ þ

  þ   þ

0.05 0.17 9.43 0.06 0.08 49.12

 þ  þ

   

0.29 31.59 0.28 36.99

Wright Valley oil spills Vanda 0e5 a 0e5 b b b Bull Pass 0e5 a a a 5e10 b b b Loop Moraine 0e5 a a 0e5 b b a

Soil treatment

1.82

3.13 6.47 3.13

7.70

4.20

2.49 2.07

>90 0 >90 0

5.04 >90 35.16 3.12 >90 >90 49.9 >90 >90 17.78 >90 2.59 >90 2.52

0.998 0.993

0.998 0.999 0.998

0.999

0.999

0.998 0.998

NP: nutrients; þ : treatment with nutrients; : no nutrient treatment. I, inoculation; þ: inoculation with SB (b) soil; : no inoculation.

soil microbes that die due to freeze-thaw processes and thus provide nutrients for the survivors (Wynn-Williams, 1990). Hydrocarbon degradation was stimulated in laboratory experiments by alternating 24 h periods at 7 and e5  C (Eriksson et al., 2001); however, the authors postulated that this was due to changes in the physical matrix of the soil making hydrocarbons more bioavailable, rather than the release of nutrients to enhance hydrocarbon degradation. The soil C:N ratios indicate nitrogen depletion in the soils, except for Bull Pass, hence it was surprising that we detected high levels of hexadecane mineralization activity in some soils [MP (a&b), CE (b) and VS (a)] without nutrient addition. We have measured high levels of hexadecane mineralization activity in oilcontaminated Antarctic ornithogenic soils without nutrient supplementation; however, these soils did have high levels of N and P derived from guano, feathers, egg shells and bird remains (Aislabie et al., 2008b). The nitrogen-depleted soils exhibiting alkane mineralization activity without nutrient addition did contain nitrogen, albeit at low levels, but not at levels higher than those soils from SB and LM that required nitrogen for activity. This indicates that the microbial community in MP, CE (b) and VS (a) soils may be more efficient at utilising available nitrogen sources at low concentrations or alternatively that nitrogen-fixing bacteria may be supporting hydrocarbon biodegradation (Bossert and Bartha, 1984). Rhizobium, members of which fix nitrogen, have been detected in a clone library prepared from DNA extracted from oil-contaminated MP soil (Hughes, 2011). We have isolated nitrogen-fixing bacteria from MP and VS soils, some of which were oil-contaminated (Eckford et al., 2002). Two of the nitrogen-fixers

grew on hydrocarbons; however, both strains required fixed N for this activity and neither exhibited simultaneous nitrogenase activity and hydrocarbon oxidation (Eckford et al., 2002). Foght (2010) proposed that this simultaneous activity may not be compatible as nitrogenases are inactivated by oxygen, whereas hydrocarbon oxidising mono- and dioxygenases require oxygen. It may be that individual species alternate between nitrogen fixation and hydrocarbon degradation when resident in hydrocarbonimpacted nitrogen-deficient environments, or they could provide fixed nitrogen to other hydrocarbon degraders that coexist in microaerophilic pockets within the soil. In situ nitrogen fixers could be actively fixing nitrogen in deeper layers where pO2 pressure is reduced, especially if in the top layers of the soil profile consumption of O2 is enhanced by hydrocarbon mineralization activity. Selection of nitrogen-fixers in the MP and CE (b) soil could be due to a long enrichment period as the soils were most likely contaminated with oil when the former research stations were operational 50þ years ago. In those soils where alkane mineralization was enhanced following N and P addition only the extent of mineralization (which ranged from 9 to 69%) compared favourably with other Antarctic investigations. For hydrocarbon-contaminated mineral soils from SB, the extent of hexadecane mineralization at 8  C with up to 105 days’ incubation was <6% but increased to c. 20% following nitrogen amendment (Aislabie et al., 1998). Similarly, for hydrocarboncontaminated mineral soil from Old Casey Station, East Antarctica, the extent of octadecane mineralization, with incubation for 95 days at 10  C, was <2% in non-amended soils and increased to 15% following nitrogen addition (Ferguson et al., 2003). The higher

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a

1.5

-1.5

1.5

-1.5

b

1.0

log HC degraders organic C % water TPH log culturable bacteria pH

-1.0

Total N

1.0

Total P C/N

NO3

EC

NH4+

solution, leading to a decrease in soil water potential and inhibition of microbial activity (Braddock et al., 1997). As phosphorus added as fertiliser is sparingly soluble in soil water it is not likely to change the soil water potential (Ferguson et al., 2003). Reported optimal C:N ratios of hydrocarbon degradation range from 200:1 to 9: 1 (Morgan and Watkinson, 1989). Walworth et al. (1997), however, suggest that for soils with low nitrogen and water content, as in the case of Antarctic mineral soils, rather than using C:N ratios to calculate maximal N application levels for contaminated soils, it is more appropriate to relate inorganic N levels to soil water. Ferguson et al. (2003) reported that fuel oil degradation in an Antarctic soil was stimulated by addition of 1570 mg N kg1 soil H20, but not by the addition of 28 000 mg N kg1 soil H20. Subsequently, Walworth et al. (2007) reported oxygen consumption with application of 600e1200 mg N kg1 soil H20 and maximum petroleum degradation at 600 mg N kg1 soil H20 in soil from the Macquarie Island in the sub-Antarctic. In this study we detected alkane mineralization activity at 42e4576 mg N kg1 soil H20 respectively in MP(b) and BP(a) respectively. All of the soils amended with nitrogen, except Bull Pass, had about 2500 mg N kg1 soil H20 as recommended by Walworth et al. (2007). To complicate matters, hydrocarbon contamination can further reduce the waterholding capacity of the soils because oil coating the surface of the soil particles makes the soil more hydrophobic (Dibble and Bartha, 1979). Measurement of the hydrophobicity of soils from SB, MP and BP indicated that they were strongly hydrophobic, weakly hydrophobic and not hydrophobic, respectively, when compared with control soils (Balks et al., 2002). To overcome the problem of overfertilisation, slow release fertilisers, such as Inipol EAP-22, have been trialled as an amendment for optimising biodegradation in Antarctic soils (Delille et al., 2007). The ideal form and concentrations of N and P for optimising hydrocarbon degradation in Antarctic mineral soils require further investigation. 4.2. Influence of bioaugmentation on soil hexadecane mineralization activity

-1.0

Fig. 3. Biplots from a semi-strong-hybrid (SSH) ordination and principal component correlation (PCC) of Antarctic soils based on TRF profiles of bacterial community composition. 3a. Site ordination. The sites are denoted as follows SB (C, B), MP (-, ,), CE (+,q), VS (:, 6) and LM (>,A). Closed symbols ¼ a samples and open symbols ¼ b samples. 3b. Gradient of environmental variables only those parameters with a statistically significant fit, P < 0.05, to the ordination space are depicted. Note, as no PCR products were obtained, BP soil is not included in the ordination.

extent of mineralization detected in this study compared with previous investigations using SB soil may be due in part to amending the soils with both N and P and the higher incubation temperature employed. Although nitrogen is considered the major limiting nutrient (Ferguson et al., 2003), maximal hydrocarbon degradation in Arctic soils occurred with supplementation of both N and P (Braddock et al., 1997; Mohn and Stewart, 2000). P addition increased hydrocarbon degradation at 20  C but not 10  C in arctic mineral soil; presumably the higher extent of hydrocarbon degradation at 20  C led to the requirement for additional P (Walworth and Reynolds, 1995). Maintaining optimum nutrient conditions in polar soils with inorganic nutrients is difficult. High concentrations of fertiliser may inhibit hydrocarbon degradation (Braddock et al., 1997; Ferguson et al., 2003; Mohn and Stewart, 2000; Walworth et al., 2007). Coarse-textured soils such as Antarctic mineral soil have low waterholding capacities leading to low water contents. When the soils are amended with nitrogen fertiliser composed of highly soluble nitrate and/or ammonium salts, the salts partition into the soil water. This can increase the salt concentration of the limited soil

Water and nutrient amendments did not enhance hexadecane mineralization activity in soils from BP (a & b) and VS (b). This is not surprising as the Wright Valley soils in general had lower numbers of hydrocarbon degraders than coastal soils, except for CE (a). The lack of enrichment of hydrocarbon degraders in situ at these sites is most likely due to aridity, rather than low levels of nutrients. Bull Pass soil, at least, was not depleted in nitrogen due to accumulation of nitrate. Wright Valley soils are, however, typically drier than those of the coastal soils from MP or SB (Aislabie et al., 2006a,b). The average number of hours that liquid water (>5%) was recorded in surface soil from MP and BP in summer were 310 and 1 respectively (Aislabie et al., 2006a). Hence to enhance alkane mineralization activity we inoculated the BP (a & b) and VS (b) soils with hydrocarbon degraders. Rather than inoculate with isolated alkane degraders we used hydrocarbon-contaminated soil from SB (b) at a ratio of 1:10 as inocula. This soil had a large population of hydrocarbon degraders including alkane degraders identified as Rhodococcus or Pseudomonas (Saul et al., 2005). According to dogma the ability of seeded bacteria to survive and thrive is expected to be negatively impacted by the indigenous microbial population that is adapted to the particular soil environment (Leahy and Colwell, 1990). From the results of this study, it appears that bioaugmentation may be successful in Antarctic soils with low resident numbers of hydrocarbon degraders. By mixing SB soil into BP soil the extant microbial population was likely swamped with the seeded organisms. Furthermore, by inoculation with soil rather than isolated hydrocarbon degraders we may have created a niche which enhanced the survival and hence activity of

J.M. Aislabie et al. / Soil Biology & Biochemistry 45 (2012) 49e60

57

Fig. 4. Influence of a) nutrient addition and b) inoculation treatments on SSH clustering of soil based on TRF profiles of bacterial community composition. For Fig. 4a; A ¼ Time 0, B ¼ no treatment with nutrients, C ¼ nutrient treatment. For Fig. 4b; I ¼ SB Time 0 the soil used as inoculum, A ¼ Time 0 VS (b) soil without inoculation, B ¼ Time 0 inoculated soil, C ¼ soil treated with inoculation plus nutrients.

the introduced microbes. Our previous attempts to introduce hydrocarbon-degrading Rhodococcus bacteria into Wright Valley soil did enhance hexadecane mineralization activity but not to the same extent as detected when soil was used as the inoculum (Gutierrez-Zamora Jimenez, 2005). Bioaugmentation with SB soil did result in a shift in the in situ soil bacterial community in VS (b) and convergence of VS (b) soil with that from BP, indicating that the inoculated bacteria were both active and surviving. Mixed results have been reported when applying isolated bacteria (single strains on consortia) to bioremediation of hydrocarbon-contaminated Antarctic soils. In some investigations, bioaugmentation has been reported to increase hydrocarbon degradation (Ruberto et al., 2003, 2005; Stallwood et al., 2005),

whereas in others there has been no effect (Ruberto et al., 2009, 2010). Furthermore, Vázquez et al. (2009) did not observe any change in soil bacterial community structure following bioaugmentation with bacterial consortia. From an applied perspective it may be easier and more effective to ‘inoculate’ recent oil spills on land in Antarctica by mixing with long-term contaminated Antarctic soil containing active hydrocarbon degraders than inoculate with isolated hydrocarbon degraders. 4.3. Soil bacterial community structure The bacterial community structure of oil-contaminated soils analysed in this study differed between sites, but samples from the

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same site tend to cluster together. Similarly, Bundy et al. (2002) and Hanamura et al. (2006) reported that the soil microbial communities of different temperate soils varied following hydrocarbon contamination, suggesting that soil type is an important determinant in structuring the bacterial community of the soils. Environmental parameters that structured the soil bacterial communities in this study included TPH, organic carbon and pH. Powell et al. (2010) also reported that soil carbon influenced the bacterial structure of oil-contaminated and control soil from Macquarie Island in the sub-Antarctic. TPH contamination of Antarctic soil results in increased soil carbon and can have a dramatic effect on the soil bacterial community (Saul et al., 2005; Vázquez et al., 2009) which is dominated by heterotrophs (Cowan et al., 2010). While the abundance of culturable bacteria, both hydrocarbon degraders and heterotrophs, can occur in response to hydrocarbons, the bacterial diversity of the Antarctic soil may decrease (Saul et al., 2005). Hydrocarbon contamination of SB and MP soils has resulted in an increase in Proteobacteria (Saul et al., 2005; Hughes, 2011). Proteobacteria, including the genera Sphingomonas, Pseudomonas and Variovorax dominated hydrocarbon-contaminated SB soil, whereas uncontaminated soil was more diverse (Saul et al., 2005). Cultured representative of the dominant Sphingomonas and Pseudomonas isolates were shown to degrade hydrocarbons, both alkanes and/or aromatics. Actinobacteria, including members of the Rhodococcus have also been implicated in alkane degradation in Antarctic soils (Saul et al., 2005; Luz et al., 2004). Alkane-degrading Pseudomonas and Rhodococcus have been isolated from SB, MP, CE, and Wright Valley soil (Aislabie et al., 2006b and unpublished). Some of the Rhodococcus spp. isolated from Antarctic soils have been reported to produce biosurfactants during growth on hydrocarbons (Gesheva et al., 2010; Yakimov et al., 1999). Under in situ conditions, production of biosurfactants could increase the bioavailability of hydrocarbons for degradation. Changes in the Antarctic soil bacterial community composition resulting from hydrocarbon contamination are likely to vary between sites and be dynamic. This is because the residual hydrocarbons in the soils depend on both the type of fuel spilt and the time since spillage. As noted in Table 1, a range of different fuels were spilt at the sites, including light aviation fuels and heavier lubricating oils. All fuels, however, would have contained a mix of n-alkanes and aromatic compounds, with n-alkanes dominating the aviation fuels. JP-8 for example the aviation fuel commonly used and stored at Scott Base and McMurdo Stations contains n-alkanes from C6eC18 (Ritchie et al., 2003). Alkanes in this range are readily biodegradable when soil conditions permit (Atlas, 1981). With time the residual hydrocarbons in the soil are modified as a consequence of both biodegradation and abiotic processes such as evaporation and weathering (Aislabie et al., 2004). With changes in the quality of the available soil carbon there will be consequent changes in the structure of the soil bacterial community because bacteria vary in their ability to degrade hydrocarbons. Pseudomonas 30-3 from VS soil, for example, degraded C8eC13 n-alkanes, whereas Rhodococcus from SB soil degraded C6eC20 n-alkanes and the isoprenoid compound pristane. Furthermore, Powell et al. (2006), using quantitative PCR targeting the alkB gene, encoding alkane hydroxylase, showed that the proportion of alkB genotypes in soil from Old Casey Station in East Antarctica was positively correlated with the concentrations of alkanes in hydrocarboncontaminated soil. As the n-alkanes were degraded, the portion of microbes carrying the alkB gene decreased. pH is commonly reported to impact on soil bacterial community structure in Antarctic (Aislabie et al., 2008a) and temperate soils (Lauber et al., 2009). The specific mechanisms whereby pH drives bacterial community composition are unknown. However Lauber

et al. (2009) give two possible reasons. The first of these is the direct or indirect modification on soil properties by pH, such as nutrient availability, solubility of cations, and characteristics of soil carbon. Second, pH can impose physiological constraints on bacteria altering their competitiveness and hence ability to grow and survive outside a certain pH range. In hydrocarboncontaminated soils the accumulation of acidic metabolites such as aliphatic acids produced during alkane degradation could lead to a decline in soil pH (Steinhart, 1995) and a shift in the bacterial community composition. The pH of the surface oiled soil from MP was lower than that of the control soil but was within the optimum of around pH 6.5 to 8.0 for hydrocarbon degradation in temperate soil (Morgan and Watkinson, 1989). Nutrient treatment of the soil did not result in significant shifts in the in situ bacterial populations. Similarly, Vázquez et al. (2009) observed no difference in the soil bacterial community of control and treated hydrocarbon-contaminated Antarctic soils incubated in situ e although significant temporal shifts in the soil bacterial community were measured indicating the importance of environmental conditions in structuring the community. In contrast, Baek et al. (2007) examined a variety of bioremediation scenarios on crude oil contaminated soils, including natural attenuation, biostimulation and bioaugmentation. Although the amount of hydrocarbon remaining after 120 days was similar for all treatments, the denaturing gel electrophoresis profiles showed considerable differences before, after and between all treatments. Hydrocarbon contamination of soils of the Ross Sea region alters the in situ bacterial community, and the changes in the community may vary with location. From this study it appears that modification of the community may be long lasting as treatment of the soil to enhance biodegradation did not result in significant changes in the in situ bacterial community, at least under the treatment regimes employed. 5. Conclusions Hydrocarbon mineralization activity in some soils of the Ross Sea region may be enhanced by supplementation with nutrients (N and P) and possibly hydrocarbon degraders when environmental conditions permit. The requirement for nitrogen supplementation to enhance alkane mineralization activity in Antarctic soils may not be apparent from measuring soil C:N ratios or indeed soil N levels. Stimulation of hydrocarbon degradation by the addition of nutrients to the soils did not tend to cause major shifts in the structure of the bacterial communities except possibly for those soils also subjected to bioaugmentation. Typically, management of hydrocarbon contaminated soils by the New Zealand Antarctic Programme has involved digging up the contaminated soil and returning it to Scott Base, where it is either stock piled or returned to New Zealand for disposal. Results from this study indicate that an alternative might be the application of bioremediation by supplementing contaminated soil with nutrients and possibly hydrocarbon degraders. Long-term hydrocarbon-contaminated Antarctic soils may provide a valuable resource of hydrocarbon-degrading bacteria that can serve as inocula for more recent oil spills on land. Acknowledgements This research was supported by funding from the Foundation of Research, Science and Technology, New Zealand (C09X0307). M.-L.G.-Z. received a MSc scholarship from the New Zealand Agency for International Development (NZAID). Antarctica New Zealand provided logistic support. Soil analyses were carried out in the Environmental Chemistry Laboratory, Landcare Research, New

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