DeDomenico05.indd 141De Domenico et al. 2004: Polar Research 23(2), 141–146 Thanks to their metabolic potential and wide bio- diversity micro-organisms play a fundamental role in making biodegradation processes more effi cient, and in transforming recalcitrant mac- romolecules into chemical substances more ame- nable to further metabolization. Psychrotrophic bacteria, which are adapted to a wide temperature range and to other fl uctuating conditions such as low nutrient availability, low water activity and high pressure, may have important advantages in biotechnological applications (Gounot 1991; Mohn et al. 1997) such as the removal of pollut- ants from cold environments. Cripps (1990) claimed that hydrocarbons, both aliphatic and aromatic, present in the Southern Ocean are above all of a biogenic nature and can be considered as part of the natural background. However, human activities in Antarctica (such as tourism, research and fi shing, all of which require fossil fuels for transport and energy), potential- ly make petroleum hydrocarbons the most likely source of pollution in Antarctic ecosystems (Bícego et al. 1996; Cripps & Shears 1997; Fer- guson et al. 2003). Several studies have shown that pollution is actually a localized phenomenon closely connected to human activity (Karl 1993; Tumeo & Wolk 1994). Evaporation and photo-oxidation represent the fi rst steps in the disappearance of hydrocar- bons from seawater, while microbial degrada- tion, along with weathering processes, is the ulti- mate fate of oil at sea (Leahy & Colwell 1990). In the past few years new species of marine bac- teria capable of biodegrading hydrocarbons have been isolated from various marine areas (Delille & Vaillant 1990; Bícego et al. 1996; Delille et al. 1998; Gentile et al. 2003; Yakimov et al. 2003). However, natural microbial degradation is slow and diffi cult in the marine environments due to the low concentrations of nitrogen, phosphorous salts and oxygen, and the formation of water-in-oil emulsion and tar balls. Furthermore, biodegrada- tion is often limited by the antimicrobial effect of petroleum components (Leahy & Colwell 1990; Heipieper et al. 1992), the toxicity of the water- soluble oil fraction and—more indirectly—by a general inhibition of biological processes due to the increase of UV radiation with decreasing pro- tection by the ozone layer. Pollutants other than hydrocarbons have been detected in Antarctic seawater. Among these, poly- chlorinated biphenyls (PCBs) are long-term per- sistent compounds widely utilized in industrial applications for their heat resistance, lipophilicity Diesel oil and PCB-degrading psychrotrophic bacteria isolated from Antarctic seawaters (Terra Nova Bay, Ross Sea) Maria De Domenico, Angelina Lo Giudice, Luigi Michaud, Marcello Saitta & Vivia Bruni Fifty-seven Antarctic marine bacteria were examined for their ability to degrade commercial diesel oil as the sole organic substrate at both 4 °C and 20 °C. Based on the preliminary screening, two isolates (B11 and B15) with high capacity to degrade diesel oil were selected and their biodeg- radation effi ciency was quantifi ed by gas chromatographic analysis. As expected for psychrotrophs, diesel oil biodegradation was slower at 4 °C than at 20 °C. The two strains also mineralized the C28 n-paraffi n octa- cosane at 20 °C and polychlorinated biphenyls (PCBs) at 4 °C and 20 °C. M. De Domenico, A. Lo Giudice, L. Michaud & V. Bruni, Department of Animal Biology and Marine Ecology, University of Messina, Salita Sperone 31, IT-98166 Messina, Italy, vivia.bruni@unime.it; M. Saitta, Department of Organic and Biological Chemistry, University of Messina, Salita Sperone 31, IT-98166 Messina, Italy. 142 Diesel oil and PCB-degrading psychrotrophic bacteria from Antarctic seawater and relative inertness (Master & Mohn 1998; Vaillancourt et al. 2003). In the present study, two psychrotrophic marine strains from Antarctica (namely, B11 and B15) were selected from 57 isolates because they had a strong ability to degrade diesel oil, as demon- strated by their noticeable growth in a culture medium containing this substrate as sole carbon source. Their biodegradative potential on diesel oil as well as on PCBs was estimated by gas chro- matography, in order to assess whether they could be a useful tool for the removal of these organic pollutants from marine environments. Materials and methods Bacterial strains The psychrotrophic bacterial strains used in this study were previously isolated (Maugeri et al. 1996) from Antarctic seawater samples (Terra Nova Bay, Ross Sea) collected along the water column (0 - 200 m) in two fi xed stations: 23 from Mergellina (MER: 74° 41' 33" S - 164° 07' 15" E; about 250 m from the coast) and 34 from Santa Maria Novella (SMN: 74° 43' S - 164° 16' E; in the middle of Terra Nova Bay, about 10.5 km from MER). From among the total of 152 bacterial strains isolated by Maugeri et al. (1996), these 57 strains were selected, on the basis of their lipoly- tic activity on Tween 80 (Sierra 1957). The isolates are maintained at 4 °C on slopes of marine agar (from Difco) and routinely streaked on agar plates from tubes every six months to con- trol purity and viability. Antarctic strains are also preserved by freezing cell suspensions at –80 °C in marine broth (Difco) to which 20 % (vol/vol) glycerol is added. Growth conditions Hydrocarbon degradation was screened on a mineral liquid medium (Mills et al. 1978) sup- plemented with commercial diesel oil at a fi nal concentration of 1 % (vol/vol). Ten-millilitre por- tions of the medium were placed in pre-sterilized screw cap tubes and 100 µl of pre-sterilized com- mercial diesel oil was added to each portion. The medium was inoculated with 100 µl of a bacterial suspension previously prepared in distilled water supplemented with 3 % (wt/vol) NaCl. The effect of temperature on the biodegradative activity was determined by cultivation at 4 °C and 20 °C for one month and one week, respectively, on a rotary shaker operated at 100 rpm in order to guaran- tee a continuous supply of oxygen. Turbidity was the positive indicator. Uninoculated control tubes were incubated in parallel to monitor abiot- ic losses of the substrate. All assays with uninoc- ulated controls were performed in duplicate. Two strains (B11 and B15) seemed to have a strong degradation effect on diesel oil at both 4 °C and 20 °C. Based on these preliminary results, their ability to mineralize both the long- chain alkane octacosane (C28) and a PCB mixture (Aroclor 1242) was examined. The mineral liquid medium was supplemented with octacosane or PCBs at a fi nal concentration of 0.2 % (wt/vol and vol/vol, respectively). Removal of these sub- strates was recorded after incubation at 4 °C and 20 °C. Both strain B11 and strain B15 had been isolated from samples taken at the Santa Maria Novella sampling station at a depth of 5 m. Gas chromatographic analysis Gas chromatographic analysis was performed for strains B11 and B15 grown on diesel oil, octa- cosane (C28) and PCBs. In addition, strain D41 (isolated from the Santa Maria Novella station at the depth of 200 m) and strain D48 (isolated from surface water from Mergellina) were ran- domly chosen among those that made the culture medium turbid at 4 °C, but did not seem to grow on diesel oil as the sole carbon source at 20 °C. Following the incubation periods, the cultures of each tube were extracted twice with 20 ml of n-hexane as a solvent by using separatory funnels to remove the cellular material. The extracts were transferred to tared vials. The volume of each extract was adjusted to 100 ml by adding more n-hexane, and vials were kept at 4 °C until the gas chromatographic analysis was carried out. Biodegradation of hydrocarbons was quanti- fi ed by quantitative gas chromatographic analysis using a DANI 8521-a GC equipped with an SE-54 fused silica capillary column (25 m × 0.32 mm i.d.; 0.45 µm fi lm thickness) and a fl ame ioniza- tion detector. Hydrogen (1 kg/cm2) was used as the carrier gas. The temperature program con- sisted of an initial oven temperature of 50 °C for 5 min increased at a rate of 10 °C/min to 280 °C for 10 min and then isothermal for 10 min. Injec- tor and detector temperatures were maintained at 280 °C. The splitting ratio was 1:60. 143De Domenico et al. 2004: Polar Research 23(2), 141–146 PCB degradation was determined by quan- titative gas chromatography using a Carlo Erba Mega 5300 GC equipped with an electron cap- ture detector and an SPB-5 fused silica capillary column (30 m × 0.25 mm i.d.; 0.25 µm fi lm thick- ness). Helium (1.5 kg/cm2) was used as the carri- er gas. The initial oven temperature was 150 °C, increased to 230 °C at a rate of 2 °C/min and then increased to 280 °C at a rate of 10 °C/min (10 min hold). The temperature of the injector and the detector were 250 °C and 280 °C respectively. Nitrogen (1.5 kg/cm2) was the gas at the detector. The splitting ratio was 1:25. The degradation was expressed as the per- centage of substrate degraded in relation to the amount of the remaining fractions in the appro- priate abiotic control samples (external standard technique). The biodegradation effi ciency (BE), based on the decrease in the substrate concentra- tion as a whole, was evaluated by using the fol- lowing expression: BE (%) = 100 – (As × 100/Aac) where As is the total area of peaks in each sample, Aac is the total area of peaks in the appropriate abiotic control and BE (%) is the biodegradation effi ciency. Results During this study, all but six of the 57 lipolytic isolates screened grew on diesel oil as the sole source of carbon and energy. Twenty-one of the strains from MER and 30 of the strains from SMN showed an ability to biodegrade diesel oil at at least one of the temperatures tested (Fig. 1). The number of diesel oil-degrading bacteria isolated from each sampling depth in relation to the temperature of incubation is shown in fi gure 2. Twenty-eight isolates (13 from MER and 15 from SMN) degraded diesel oil at 4 °C exclu- sively (assessed after a month of incubation), while six (all from SMN) did so only at 20 °C (assessed after a week of incubation). Diesel oil was observed to be utilized at both temperatures by 17 strains only: nine of them were isolated from SMN and eight from MER. Among these isolates strains B11 and B15 gave the highest tur- bidity. Quantitative gas chromatographic analy- sis allowed the estimation of their biodegradation Fig. 1. Distribution of the 51 diesel oil-degrading bacteria in relation to their origin (sampling station and depth). Strains utiliz- ing the substrate at at least one of the temperatures are included. Fig. 2. Distribution of diesel oil-degrading bacteria in relation to the incubation temperature and to the depth. The appearance of turbidity was used as the positive indicator. 144 Diesel oil and PCB-degrading psychrotrophic bacteria from Antarctic seawater effi ciency, which was expressed as the percent- age of substrate degraded (Fig. 3). Very little dif- ference was observed between the degradation of diesel oil by strain B11 at 4 °C and 20 °C. In cul- ture medium inoculated with strain B15, 66.7 % of the diesel oil had disappeared after the one- month incubation at 4 °C and 50.5 % had dis- appeared after one week at 20 °C. A signifi cant diesel oil mineralization was observed for strains D41 and D48 at 20 °C (66.10 % ± 2.55 [mean ± SD] and 56.15 % ± 0.49 [mean ± SD], respectively). The biodegradative activity was confi rmed by the calculation of the C17:pristane and C18:phytane ratios (Table 1). Strains B11 and B15 also mineralized the C28 n-paraffi n octacosane at 20 °C and PCBs at 4 °C and 20 °C as the sole carbon source. Strain B11 utilized 31.9 % and 62.8 % of the PCB mixture at 4 °C and 20 °C, respectively, while 22.8 % and 30.8 % of the substrate disappeared from the cul- ture medium inoculated with strain B15 after incubation at 4 °C and 20 °C, respectively. Discussion Diesel oil is a complex combination of hydrocar- bons deriving from the distillation of crude oil and represents an excellent substrate in the study of hydrocarbon biodegradation thanks to its com- position (Bicca et al. 1999). Diesel oil contains 2000 to 4000 different hydrocarbons (Marchal et al. 2003) with a carbon number ranging approxi- mately from C9 to C20, including paraffi n, olefi ns, naphtha and aromatic compounds. During the screening for the degradation of diesel oil, only 6 of the 57 lypolitic isolates did not mineralize the substrate, despite their ability to hydrolyse Tween 80. This highlights the strong relationship between lipolytic activity and the biodegradation of diesel oil as previously shown by Mills et al. (1978). The amounts of diesel oil mineralized by strains B11 and B15 at both the temperatures tested were very similar. Since extraction for the gas chroma- tographic analysis was carried out after a month of culture at 4 °C and after a week at 20 °C, this fi nding suggests that both strains degraded diesel oil more slowly at 4 °C than at 20 °C, as expected for psychrotrophs. This is in line with the effect of temperature on the rate of hydrocarbon metab- olism by micro-organisms: metabolism decreases with decreasing temperature. The signifi cant diesel oil mineralization by strains D41 and D48 at 20 °C should be high- lighted. These strains had been randomly chosen amongst those that showed growth at 4 °C but not at 20 °C as indicated by the turbidity of the cul- ture medium. In this kind of qualitative screen- ing, the assumption of hydrocarbon utilization cannot be based only on the appearance of tur- bidity; instead a chromatographic analysis of the culture is needed. On the other hand, Mills et al. (1978) showed that in liquid media it is pos- sible that micro-organisms only grow in the oily phase, producing neither visible growth in the oil–water interface nor visible turbidity in the water phase. Mills and co-workers suggested that in such cases the quantity of proteins in the cul- ture should be determined because production of protein in quantities greater than 30 µg/l is cor- related to easily visible turbidity. However, as is well known, one must take into account that the rate of hydrocarbon degradation is not always directly related to the bacterial growth. The C17:pristane and C18:phytane ratios were generally characterized by lower values than the Fig. 3. Percentage of diesel oil, PCB and C28 biodegraded by the strains B11 and B15. Table 1. C17:pristane and C18:phytane ratios. Sample Incubation temperature (°C) C17:pristane ratio C18:phytane ratio Abiotic control 4 and 20 2.2 2.1 Strain B11 4 1.7 2.2 Strain B15 4 1.2 1.5 Strain B11 20 1.3 1.5 Strain B15 20 1.1 1.1 Strain D41 20 1.4 1.6 Strain D48 20 0.7 0.5 145De Domenico et al. 2004: Polar Research 23(2), 141–146 abiotic control. Pristane, widely employed as an internal standard for the analysis of hydrocarbon samples, is characterized by a high degree of per- sistence (Watkinson & Morgan 1990). Thus, its disappearance from the culture medium demon- strates the occurrence of biodegradation. The biodegradation effi ciencies on PCBs were lower than those observed for diesel oil. This is not surprising as PCBs are highly recalcitrant to biodegradation. Lower incubation tempera- ture severely limited PCB degradation. In fact, the removal of PCB by bacterial degradation was higher at 20 °C. After only a week of incubation at 20 °C the disappearance of PCBs was two-fold higher than at 4 °C after a month. Yakimov et al. (1999), searching for bioemul- sifi er-producing bacteria, selected the strains B11 and B15 and identifi ed them at molecular level as marine variants of Rhodococcus fascians. Psy- chrotrophic alkane-degrading members of the genus Rhodococcus sp. have also been isolated from a number of cold environments (Whyte et al. 1996, 1998, 2002; Bej et al. 2000). Rhodococ- ci have been found to utilize a variety of xeno- biotic compounds as carbon source (Seto et al. 1995; Larkin et al. 1998) and to possess a variety of alkane-catabolic pathways (Whyte et al. 1998; Smits et al. 1999; Andreoni et al. 2000). The results obtained confi rm that cultura- ble Antarctic bacteria are capable of degrading hydrocarbons, both at 4 °C and at 20 °C. As many authors state (Atlas 1981; Bertrand et al. 1993; Whyte et al. 1997; Delille & Delille 2000), the exposure of the microbial community to hydro- carbons is responsible for its potential to oxi- dize these compounds and, consequently, for the number of micro-organisms able to utilize them. Also Delille & Vaillant (1990), who did in situ and laboratory experiments to investigate the effect of crude oil on the growth of sub-Antarctic marine bacteria, found that the addition of hydro- carbons always induced a marked, rapid increase in the bacterial population. It is therefore surpris- ing that hydrocarbon-degrading marine bacteria were isolated from both superfi cial and deep sea- waters with hydrocarbon pollution levels as low as those of the Antarctic. Thus, the occurrence of these bacteria in Terra Nova Bay could proba- bly be related to the presence of hydrocarbons of natural origin rather than to the impact deriving from human activities in Antarctica. In conclusion, the ability of psychrotroph- ic marine bacteria of Antarctic origin to utilize diesel oil and PCBs as the sole carbon source was demonstrated. Although the experiments were performed in batch culture under stable con- ditions different from those of natural environ- ments, this kind of screening is an essential step in the evaluation of bacteria biodegradation effi - ciency. In accordance with Whyte et al. (1998), our results suggest that psychrotrophic micro- organisms, which clearly utilize a wide variety of hydrocarbons at temperatures ranging from 0 °C to 30 °C, may be better suited for in situ bioreme- diation in both temperate and cold environments than mesophiles or psychrophiles. Acknowledgements.—This research was supported by the Italian National Research Project in Antarctica (PNRA). References Andreoni, V., Bernasconi, S., Colombo, M., van Beilen, J. 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