Bioprospecting the potential of the microbial community associated to Antarctic marine sediments for hydrocarbon bioremediation

Abstract

Microorganisms that inhabit the cold Antarctic environment can produce ligninolytic enzymes potentially useful in bioremediation. Our study focused on characterizing Antarctic bacteria and fungi from marine sediment samples of King George and Deception Islands, maritime Antarctica, potentially affected by hydrocarbon influence, able to produce enzymes for use in bioremediation processes in environments impacted with petroleum derivatives. A total of 168 microorganism isolates were obtained: 56 from sediments of King George Island and 112 from Deception Island. Among them, five bacterial isolates were tolerant to cell growth in the presence of diesel oil and gasoline and seven fungal were able to discolor RBBR dye. In addition, 16 isolates (15 bacterial and one fungal) displayed enzymatic emulsifying activities. Two isolates were characterized taxonomically by showing better biotechnological results. Psychrobacter sp. BAD17 and Cladosporium sp. FAR18 showed pyrene tolerance (cell growth of 0.03 g mL⁻¹ and 0.2 g mL⁻¹) and laccase enzymatic activity (0.006 UL⁻¹ and 0.10 UL⁻¹), respectively. Our results indicate that bacteria and fungi living in sediments under potential effect of hydrocarbon pollution may represent a promising alternative to bioremediate cold environments contaminated with polluting compounds derived from petroleum such as polycyclic aromatic hydrocarbons and dyes.

Introduction

Spills of petroleum-derived fuels have been affecting different natural environments throughout the world [1]. Among the impacted environments is the pristine Antarctic continent, which in recent years has been affected by oil derivatives due to the great anthropic mainly due to the growing number of tourist vessels that explore this habitat and research stations distributed throughout the Antarctic continent [2, 3].

The energy supply to the research stations is generated from petroleum-derived fuel, consisting of aliphatic hydrocarbons with C8–C30 carbons in length and a predominance of n-C11 alkane hydrocarbons, compounds with high environmental potential [1–4]. Several human activities can accidentally introduce fuel into Antarctic ecosystems, including traffic of field vehicles powered by fossil fuels, intense circulation of small vessels and support boats, and the use of diesel generators. An exceptionally large volume of fuel can be used in the various research stations in Antarctica, the continent once considered an untouched continent, and about 75% of this fuel corresponds to diesel oil, 20% to aviation kerosene, and the remaining are lubricating oils and gasoline [5, 6].

The presence of petroleum-derived compounds, including polycyclic aromatic hydrocarbons (PAHs), as contaminants of Antarctic waters as well as other recalcitrant substances has already been found in this environment, enclosing emerging contaminants (EC) such as biocides, persistent organic pollutants (POPs, including paints and dyes), medicines, and pharmaceutical and personal care products (PPCPs) [7], which may have reached Antarctica by air, by sea currents, and by human activities [8]. According to Gran-Scheuch [9], diesel oil is the main source of energy used in Antarctica and has PAHs and heavy metals in its composition. The increased presence of these contaminants in Antarctica has generated great concern, due to their potentially carcinogenic and/or toxic properties for ecosystems and humans [10], leading to a growing number of studies focusing on bioremediation of these compounds in polar environments in recent years.

Aquatic ecosystems are a wealthy source of biodiversity in polar regions, where microbial communities are essential for understanding their functioning [11]. Antarctic environments show quite extreme conditions, such as extremely low temperatures, hypersaline waters, reduced availability of nutrients, and high incidence of UV rays, which make the resident microbial communities holders of metabolic abilities different from those found in mesophilic microorganisms [12]. Thereby, the search for metabolically distinct compounds adapted to cold, including exopolysaccharides, antifreeze proteins, enzymes, and cryoprotective carbohydrates, recovered from microbial cells that inhabit extreme environments such as the polar ones, has been a highly promising strategy around biotechnology, for application in several industrial sectors, including in bioremediation processes [13]. However, even with the increase in research with Antarctic microorganisms, there is still much to be investigated and understood about the survival mechanisms of these diverse and complex microorganisms, their metabolites, and enzymes, which can potentially be used in processes such as the bioremediation of cold environments contaminated with petroleum derivatives. Some studies have been exploring the microbial metabolic diversity present in polar biomes in the search for biotechnologically promising compounds for the mitigation of environmental pollutants, including extracellular enzymes and biosurfactants [13–17]. According to the panorama described above, our study focused on characterizing culturable bacteria and fungi from samples of marine sediments collected in Antarctica and evaluating their capabilities to tolerate petroleum-derived compounds, decolorize dyes, and produce biotechnologically applicable enzymes and metabolites in bioremediation processes.

Materials and methods

Sampling

Marine sediment samples were collected on King George Island, Admiralty Bay, in front of the Comandante Ferraz Antarctic Station fuel tanks (62°03′00.9″S 58°13′34.6″W) at 50-m depth and Whalers Bay, Deception Island (62°58′52″S, 60°39′52″W) at 95-m depth in the summer of 2018/2019 (Fig. 1). Sediment samples were obtained using a box corer and sections of 10 cm from the base of the core were sealed, placed in sterile Whirl-pack (Nasco, Ft. Atkinson, WI) bags and frozen at − 20 °C. The samples were gradually thawed at 4 °C for 24 h before carrying out the bacterial and fungal isolation. Three subsamples of the central parts of each core were obtained using a sterilized scoop inside of the laminar flow hood under conditions of strict contamination control and processed to isolate the bacteria and fungi.

Fig. 1.

Map showing collection points on the Antarctic peninsula. Black points show the place of the sampling

Bacteria and fungi isolation

About 10 g from each sample was processed by serial dilution (10⁻¹, 10⁻², 10⁻³, and 10⁻⁴), in 0.5% peptone water before vortexed. Aliquots of 50 µL from each sample dilution were spread on the surface of the Petri dishes containing one of the following culture media: potato dextrose agar (PDA) (glucose 10 g L⁻¹, agar 15 g L⁻¹ in 1000 mL of potato infusion), nutrient agar NA (meat extract 3 g L⁻¹, peptone 5 g L⁻¹, agar 15 g L⁻¹), and R2A (yeast extract 0.5 g L⁻¹, peptone 0.5 g L⁻¹, casein hydrolysate 0.5 g L⁻¹, glucose 0.5 g L⁻¹, sodium pyruvate 0.3 g L⁻¹, starch 0.5 g L⁻¹, potassium phosphate 0.3 g L⁻¹, magnesium sulfate 0.025 g L⁻¹, agar 15 g L⁻¹ at pH 7) [8]. Chloramphenicol 250 mg L⁻¹ and nystatin 100,000 U L⁻¹ were added for the isolation of fungi (PDA and R2A) and bacteria (NA and R2A), respectively. The plates were incubated at 15 °C for 15 days, to recover psychrotolerant microbial cells. The colonies obtained were purified and stored at − 80 °C in 20% glycerol. To recover microbial cells adapted to petroleum-derived compounds, a second isolation (enrichment) was performed: 10 g of the sediment was placed in 100 mL of water with 0.5% peptone, 0.5% glucose, 10% gasoline, and 10% diesel oil. This solution was incubated for 7 days at 150 rpm, 15 °C. After this period, the same procedure described above was performed.

Tolerance to petroleum derivatives

All isolates were subjected to diesel and gasoline tolerance tests based on Ribeiro et al. [18], with modifications. The fuels were incorporated into the minimal medium (MM) (magnesium sulfate 0.2 g⁻¹, calcium chloride 0.02 g L⁻¹, 0.7 monobasic potassium phosphate g L⁻¹, dibasic potassium phosphate 0.7 g L⁻¹, ammonium sulfate 0.5 g L⁻, sodium nitrate 0.5 g L⁻¹), in a proportion of 3:1 (MM: diesel or gasoline), separately. The tubes containing the culture medium plus the fuels were inoculated with the fungal and bacterial isolates and incubated at 15 °C for 7 days, under agitation of 150 rpm. After this period, 300 µL of 0.5% 2,3,5-triphenyltetrazolium chloride (TTC) solution was added, and the samples were incubated for another 2 h under the same conditions [19]. Subsequently, the samples were transferred to 96-well microplates, and the absorbance was checked in a microplate reader (Celer© model Polaris) at 450 nm. As negative control, the isolates were inoculated in test tubes containing 500 µL of culture medium PDB (glucose 10 g L⁻¹, in 1000 mL of potato infusion) and NB (meat extract 3 g L⁻¹, peptone 5 g L⁻¹) and 1.5 mL of MM, without the addition of diesel oil and gasoline. The experiment was carried out in triplicate, and the isolates that showed greater growth than the negative control were considered tolerant.

Evaluation of RBBR dye discoloration

The decolorization of the RBBR (Remazol Brilliant Blue R) dye in liquid and solid culture medium was performed according to Bernal [20], with modifications. For the assay in solid culture medium, all filamentous fungi were grown on PDA. One fungal culture disc (0.5-cm diameter) from the edge of the colony was transferred to Petri dishes containing the same medium, added with 500 and 1000 mg L⁻¹ RBBR. Plates were incubated for 12 days at 15 °C. The formation of a discoloration halo around the microbial growth was considered a positive result. Petri dishes containing medium without RBBR and without cells were used as control. For the assay in liquid culture medium, all filamentous fungi were cultivated in potato dextrose broth (PDB) (potato infusion 4 g L⁻¹, dextrose 20 g L⁻¹) for 12 days under agitation of 150 rpm at 15 °C. The RBBR dye was added to the culture media at a concentration of 1000 mg L⁻¹. Aliquots of 1 mL of microbial cultures were collected at 4, 8, and 12 days of incubation, centrifuged at 12,000 rpm for 2 min, and the supernatant diluted 10 times with distilled water. From these dilutions, the absorbance reduction in relation to time zero was verified in a spectrophotometer at a wavelength of 580 nm. The decolorization efficiency was expressed through the formula decolorization (%) = (A_{λ initial} – A_λfinal/ A_{λ initial}) × 100. Control was performed using PDB and inoculum without RBBR (blank control) and PDB with addition of RBBR (initial control). Isolates that showed 60% or more of discoloration were evaluated for their ability to produce the laccase enzyme. All experiments were performed in triplicate.

Evaluation of RBBR adsorption by fungal mycelium

The fungal isolates that showed the ability to decolorize the dye were submitted to a mycelium dye adsorption test. For that, isolates were cultivated in Erlenmeyer flasks containing PDB culture medium added with 500 mg L⁻¹ RBBR. The flasks were incubated for 7 days at 15 °C, under agitation of 150 rpm. Aliquots of 1 mL of microbial cultures were collected at 3, 5, and 7 days of incubation, centrifuged at 12,000 rpm for 2 min, and the supernatant diluted 10 times with distilled water. From these dilutions, the absorbance reduction in relation to time zero was verified in a spectrophotometer at a wavelength of 580 nm [20].

The cultures were ground using the Ultra–Turrax system according to Passarini [10] and were subsequently kept stationary until the solid–liquid phases were separated. The liquid phase was centrifuged at 12,000 rpm for 2 min and the supernatant diluted 10 times with distilled water. The absorbance reduction was verified in a spectrophotometer at a wavelength of 580 nm [20]. The absorbance before and after grinding was compared to analyze the possibility of adsorption of the RBBR dye by the mycelium.

Biosurfactant assays

Drop-collapse

Preliminary assays to verify the ability to produce biosurfactants were performed with all isolates using the drop-collapse test [21]. For this assay, isolates were cultivated in PDB or NB liquid culture medium at 15 °C for 7 and 4 days, for filamentous fungi and bacteria, respectively. After growth, the samples were centrifuged at 4000 rpm for 10 min. The supernatant was used in the drop-collapse tests. Assays were performed in 96-well plate lids (TPP®). To each well of the lid, a layer of 20W-50 Havoline automotive (Chevron, Brazil) engine oil was applied. The lids were kept static for 24 h at 25 °C. After this period, 7-µL aliquots (~ 1 drop) of each microbial supernatant were added to the center of each well containing the engine oil. The test was considered negative when the drop (supernatant) added to the oil remained intact, and positive when the drop scattered or collapsed. The tests were performed in triplicate. Inoculum-free media were used as negative control and 2% Tween solution was used as positive control.

Emulsification assay (E₂₄)

Emulsifying activities were evaluated according to the protocol described by Martinho [21]. The formation of an intermediate emulsion layer between the enzymatic extract produced by the isolates and the commercial oil was scored as a positive result. Isolates were cultivated in 15 mL of mineral culture medium (yeast extract 5 g mL⁻¹; (NH₄)₂SO₄ 1 g L⁻¹; Na₂HPO₄ 6 g L⁻¹; KH₂PO₄ 3 g L⁻¹; NaCl 2.7 g L⁻¹; MgSO₄·7H₂O 0.6 g L⁻¹; engine oil 1% v/v) for 7 and 4 days at 15 °C under agitation at 150 rpm, for filamentous fungi and bacteria, respectively. The microbial cultures were transferred to 2 mL microtubes and centrifuged at 3000 rpm for 10 min at 4 °C. Then, 2 mL of each extract was transferred to screw tubes containing 2 mL of diesel oil. Tubes were vortexed for 2 min, subsequently kept for 24 h without agitation, and then measurements were taken. The emulsification index (E₂₄) was calculated by measuring the height of the emulsification layer, divided by the total height of the culture, multiplied by 10. A 2% Triton solution was used as positive control and the mineral medium without the inoculum was used as negative control.

Pyrene tolerance assay

The isolates that showed greater tolerance to diesel oil and gasoline were cultivated in 50% of the composition of PDB and NB media. After 24 h of cell growth, 1 mg L⁻¹ pyrene was added to the culture media. Samples were incubated at 15 °C under agitation at 150 rpm for 5, 10, and 14 days for bacteria and 7, 14, and 21 days for fungi. The negative control consisted in the cultivation of the isolates without the addition of pyrene. The experiments were carried out in triplicate [18]. Dry weight analyses of microbial cells were performed according to Nogueira [22]. Bacterial cells were centrifuged at 10,000 rpm for 10 min, in pre-dried and weighed tubes. The supernatant liquid was discarded, and the tubes containing the bacterial cells were subjected to drying at 70 °C, until complete drying. Fungal isolates had the mycelium weight determined after vacuum filtration on a 0.22-µm filter previously dried and weighed, and subsequent drying at 70 °C, until complete drying of the filter paper containing the mycelium. The difference in weight of tubes and filter papers with and without cells was calculated according to the formula: dry weight = mass of tube/dry paper (with cells) − mass of tube/dry paper (without cells).

Laccase enzymatic activity assay

Laccase enzymatic activity assays were performed with the isolates that showed tolerance to pyrene in the previous test. A volume of 1800 µL of the enzyme extract was added to a solution containing 900 µL of 0.1 M sodium acetate buffer (pH 5.0) and 300 µL of 2,2-azino-bis-ethylbenthiazoline (ABTS). Subsequently, the absorbance of the mixture was verified in a spectrophotometer at the wavelength of 420 nm immediately after preparation of the assay and again after incubation for 10 min at 15 °C, under the same conditions. Laccase activity was determined by the oxidation of ABTS [23], modified, measured by monitoring the increase in absorbance. One unit of enzymatic activity was defined as the amount of enzyme required to oxidize 1 µmol ABTS per minute using the molar extinction coefficient of 36,000 M = 1 cm⁻¹ (E₄₂₀ = 3.6 × 104 M⁻¹ cm⁻¹), U L⁻¹ = [(ΔA) (Vt) (10⁶)]/[(t) (E) (Vs)], U = enzyme activity (µmol min⁻¹ L⁻¹); ΔA = final absorbance − initial absorbance; Vt = total reaction volume (mL); 10⁶ = correction factor (µmol moL⁻¹); t = reaction time (min); E = molar extinction coefficient (L moL⁻¹ cm⁻¹); Vs = sample volume.

Molecular identification of microbial isolates

The molecular identification of the isolates that showed the best results in previous activity assays was carried out by GoGenetic, an outsourced company. Briefly, the genomic DNA extraction from isolates was performed according to the protocols used in the company using kit for genomic DNA extraction. Amplifications were performed with primers for the ITS and 16S rRNA genes ribosomal of fungi and bacteria, respectively. The primer sets used were ITS1 (5′-CCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′), and 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′), for fungi and bacteria, respectively. The amplicons were enzymatically purified. Amplified products purified were sequenced using Big Dye Kit (Applied Biosystems™) for ABI 3500 Genetic Analyzer (Applied Biosystems™). Obtained sequences were compared with publicly accessible sequences deposited in the Genbank databases (http://www.ncbi.nem.nih.gov) using BLASTn sequence alignment routines. The sequences were aligned using the BioEdit program and evolutionary phylogenetic analyses were performed using MEGA X software and Kimura’s Evolutionary distance substitution model [24]. Phylogenetic trees were constructed using the neighbor-joining (NJ) algorithm [25] with bootstrap values calculated from 1000 replicates.

Sequence accession numbers of the strains from GenBank

BAD17 (OR345159) and FAR18 (OR345242).

Statistical analyses

Statistical analysis was performed according to Ottoni [26]. All experiments were performed in triplicate, and the data obtained were tested for normality. For specific growth effect results, ANOVA with Dunnet’s post hoc was used to determine significant differences between treatments. All analyses were done at a 5% significance level, using the PAST software version 2.17c.

Results

Isolation and bioremediation potential of the Antarctic strains

A total of 168 microbial cells were isolated from marine sediment samples. Forty-six bacteria and 10 fungi were recovered from King George Island sediment samples while 98 bacteria and 14 filamentous fungi from those of Deception Island. About 80% and 70% of fungi and bacteria were recovered by using PDA and NA media, respectively. The enrichment technique directed towards hydrocarbon tolerance did not favor microbial development, and, regarding fungi, the enrichment led to a 90% reduction in the number of isolates. Using the enrichment technique, 17 and 52 bacteria were recovered from King George Island and Deception Island sediment, respectively. On the other hand, only 2 fungi were recovered from King George Island.

Diesel oil and gasoline tolerance

All 168 isolated microorganisms were tested for their tolerance to diesel oil and gasoline. These preliminary qualitative assays aimed to select resistant microorganisms with potential for application in bioremediation processes of petroleum-derived compounds. Twenty-seven microorganisms (16%) showed activity in one of these two assays, being 25 bacteria and 2 filamentous fungi. Only bacterial strains showed tolerance to gasoline (Table 1 presents the best results of these tests). It is possible to observe that cell growth varied from 0.05 to 1.41 and 0.00 to 0.50 mg mL⁻¹, in the media supplemented with diesel oil and gasoline (revealed by TTC solution), respectively (Fig. 2). Only one isolate, BAD15, tolerated the presence of diesel oil and gasoline.

Table 1.

Biotechnological potential for bioremediation process and molecular identification of the best isolates recovered from marine sediment samples

Isolates	Tolerance (abs)/SD		RBBR discoloration		Drop-collapse	(E24%)/SD	Laccase UL /(day)	Pyrene tolerance g mL−1/(day)	Molecular characterization
	Diesel oil	Gasoline	Solid (cm) 500/100 mg L−1	Liquid/adsorption (%)		Control (63.3)
	F = 47.61, p = 1.147E-10		F = 0,1822; p = 0.6715	F = 122; p = 6,354E-07		F = 1189, p = 2.875E-67	F = 3.278 p = 0.07964	F = 0.55 p = 0.5821
BAD1					+ / + / +	6.4 ± 1.69
BAD11					+ / + / +	4.8 ± 0.06
BAD12	0.131 ± 0.02
BAD13	0.130 ± 0.04
BAD15	0.184 ± 0.05	0.067 ± 0.02					0.005 ± 0.02 (5º)	0.055 ± 0.00 (5º)
BAD16	0.789 ± 0.04
BAD17	1.414 ± 0.20				+ / + / +	3.6 ± 3.39	0.006 ± 0.06 (10°)	0.031 ± 0.00 (10°)	Psychrobacter sp.
BAD18					+ / + / +	8.6 ± 0.06
BAD19					+ / + / +	7.6 ± 3.39
BAD2					+ / + / +
BAD25					+ / + / +	11.1 ± 0.01
BAD3					+ / + / +	4.8 ± 3.39
BAD45					− / + / +	18.2 ± 6.79
BAD5					+ / + / +	3.0 ± 1.69
BAD51					+ / + / +
BAD52					+ / + / +
BAD6					+ / + / +	4.2 ± 0.11
BAD7					− / + / +	2.7 ± 3.39
BAD8					+ / + / +	3.3 ± 1.69
BAD9					+ / + / +	2.8 ± 1.69
BAR12					+ / + / +	3.9 ± 1.69
BAR8					+ / − / +	8.2 ± 0.06
FAD23			0.4 ± 0.05/0.4 ± 6.79
FAR4	0.042 ± 0.01	0.01 ± 0.04
FAD27			0.2 ± 0.05/0.2 ± 0.05
FAD28	0.084 ± 0.02	0.001 ± 0.01	0.5 ± 0.00/0.5 ± 0.00	83.3 ± 0.05/18.4 3 ± 0.239			0.004 ± 0.07 (7º)	0.178 ± 0.02 (21º)
FAD29			0.3 ± 0.05/0.2 ± 0.05
FAR18	0.001 ± 0.01	0.001 ± 0.01	0.5 ± 0.00/0.5 ± 0.00	68.9 3 ± 0.01/6.5 ± 0.245			0.103 ± 0.06  (14º)	0.2152 ± 0.06 (21º)	Cladosporium sp.
FAR21			0.4 ± 0.05/–
FAR31	0.023 ± 0.05	0.001 ± 0.01
FAD7					+ / + / +	5.0 ± 0.05
FAD3					+ / + / +
FAD9					+ / + / +
FAR22			0.2 ± 0.05 /0.2 ± 0.05
BAR2	0.151 ± 0.07
BAR13	0.210 ± 0.191

One-way ANOVA with Dunnet’s post hoc was used to determine if there were statistically significant differences in the remaining isolates (*p < 0.05) over time in the response between control and treatments. Test with gasoline, emulsification, and adsorption to mycelium showed a significant difference. BAR Bacteria Antarctica King George, FAR Fungi Antarctica King George, BAD Bacteria Antarctica Deception, FAD fungi Antarctica deception, x = growth in diesel oil and/or gasoline, SD standard deviation, abs absorbance using TTC solution (only the isolates that grew more than the control are shown in the table). +  = positive result. −  = negative result. Blank tests (space) mean lack of satisfactory results

Fig. 2.

Bacterial (a) and fungal (b) cell growth assay in the presence of diesel oil and gasoline

RBBR dye discoloration potential

The selection of isolates capable of tolerating/discoloring the RBBR dye was performed only with filamentous fungi, all of which were tested in liquid and solid media. Seven fungal isolates (29%) showed discoloration activity on solid medium, and the two with the highest activities, i.e., FAD28 (Deception Island) and FAR18 (King George Island), were tested in liquid medium. Both fungal isolates were able to tolerate/discolor RBBR in liquid medium, with FAD28 and FAR18 decolorizing 83.3 and 68.9% of RBBR, respectively (Table 1).

To assess whether the dye was degraded or absorbed by the fungal mycelium, a mycelium adsorption test was conducted with the two isolates that showed the highest percentages of discoloration. Results showed that there was an adsorption of the RBBR dye to the fungal mycelium at a percentage of 6.5 and 18.4% by the isolates FAR18 and FAD28, respectively.

Biosurfactant and bioemulsifier activities

All microbial isolates were submitted to the qualitative drop-collapse test and, of these, 21 (12.5%) (18 bacteria and 3 filamentous fungi), were scored as positive and selected for further evaluation of their potential to produce bioemulsifying compounds, by calculating the emulsification index (E₂₄) in non-polar compounds. Emulsification assays revealed that 16 isolates presented E₂₄ ranging from 2.7 to 18.2%, and a bacterial strain (BAD45) isolated from Deception Island was the major producer of bioemulsifying compounds (Table 1).

Pyrene tolerance assay

After screening for tolerance to the presence of diesel oil and gasoline, two bacterial strains (BAD15 and BAD17), both isolated from the Deception Island sample, and two fungal strains (FAD28 and FAR18), recovered from Deception Island and King George Island, respectively, were submitted to pyrene tolerance assay. The results obtained with bacterial isolates revealed that the BDA15 strain showed tolerance to pyrene during 10 days of growth, with higher biomass on the 5th day, while the BAD17 isolate showed greater growth at 5 and 15 days, with higher biomass on the 10th day. Regarding to the fungal isolates, FAD28 and FAR18 showed greater tolerance to pyrene at 21 days (Table 1 and Fig. 3). The higher cell growth in relation to the control (without pyrene) may be associated with the use of the PAH by these microorganisms as a carbon source for intracellular metabolic activities (Table 1 and Fig. 3).

Fig. 3.

Microbial growth in the presence of pyrene

Laccase activity

In the present study, we evaluated the activity of the laccase enzyme in microbial extracts from tolerance tests and microbial biomass production in the presence of pyrene. The four isolates evaluated produced laccase in the presence of PAH, and the isolate FAR18 showed the best result of laccase enzyme activity with 0.103 UL, on the 14th day of incubation (Table 1).

Molecular characterization

By using molecular approaches, the two strains (one bacterial and one fungal) that showed the best abilities to produce compounds with potential application in bioremediation of hydrocarbon-contaminated environments were taxonomically identified. The phylogenetic analysis of partial 16S rRNA gene revealed that the BAD17 strain belongs to the genus Psychrobacter, showing 99% and 100% sequence similarity with Psychrobacter arcticus (KF424828), Psychrobacter glaciei (MT309522), and Psychrobacter fozii (KY405997), strains available in the GenBank database (Fig. 4a). Likewise, the ITS region of the FAR18 strain showed 100% sequence similarity and 96% query cover with the species Cladosporium cladosporioides (MT367262) and Cladosporium anthropophilum (MF472921), thus being identified as Cladosporium sp. (Fig. 4b).

Fig. 4.

Phylogenetic analysis based on partial bacterial 16S and fungal ITS sequences of isolates BAD17 (a) and FAR18 (b), respectively. Bootstrap values (1000 replicated races) greater than 70% are listed

Discussion

Isolation and bioremediation potential of the Antarctic strains

A small number of aerobic isolates, especially filamentous fungi, were recovered from the sediment samples. It is possible that the depth of sampling (48 m deep at King George Island and 95.4 m deep at Deception Island) may have influenced the isolation of strictly aerobic microorganisms. Another fact that may have influenced the isolation is related to the culture medium used. According to Ahn [27], R2A medium is one of the most suitable media for retrieving bacteria from water samples, since it consists of an oligotrophic medium, and not a culture medium designed to recover bacteria from soil environments [28]. Thus, even though Antarctica is an oligotrophic environment, the use of the R2A medium to isolate microorganisms from marine sediments samples potentially contaminated with xenobiotic compounds derived from oil samples may not be the ideal choice.

Isolation of filamentous fungi and bacteria from samples such as those used in the present study has already been reported on both islands. Wentzel et al. [29] isolated fungal strains from soil and marine sediment samples collected on King George Island, Antarctica, including species from the genera Leucosporidium, Pseudogymnoascus, Cladosporium, and Penicillium. Silva et al. [30] isolated 326 bacterial strains from different genera, including Arthrobacter, Psychrobacter, and Cellulophaga, from marine sediment samples collected on King George Island, Antarctica. In 2021, Vicente et al. [31] obtained Bacillus strains as well as Aspergillus and Penicillium spp. from sediment samples collected on the volcanic Deception Island.

Most of the microbial cells were recovered in a culture medium rich in nutrients such as PDA and NA, favoring the isolation of a greater number of bacteria and fast-growing filamentous fungi [27]. However, the use of culture media such as R2A, with few nutrients, favors the recovery of slow-growing microorganisms [32], thus increasing the possibility of recovering less abundant or even rare species [28]. The enrichment technique can facilitate the isolation of microorganisms tolerant to the enriched compound in the medium but in some cases, without the ability to degrade this compound, as demonstrated in the study by Chang et al. [33]. In this study, the authors performed an enrichment with 10 PAHs for 6 months, which resulted in the isolation of bacterial strains without the ability of degradation of hydrocarbons. Perhaps by increasing the incubation time in our enrichment assays, a greater number of microorganisms tolerant to diesel oil and/or gasoline would be recovered.

The number of isolates obtained from the samples collected on Deception Island was twice the number obtained from the King George Island sample (King George n = 56; Deception n = 112). Perhaps this difference can be explained by the greater human activity on King George Island, including the presence of research bases with fuel tanks (as in the Brazilian Antarctic Station). The constant contact of microbial communities with environmental pollutants may have contributed to the selection of microorganisms able to thrive in culture media containing hydrocarbons as carbon sources.

Interestingly, isolation using enrichment techniques could recover only 2 fungi from samples collected on King George Island; on the other hand, no fungi from samples were recovered from Deception Island. Antarctic Fungi may have tolerance and degradation capacity of diesel oil [34, 35]; in this way, perhaps the collection point of these samples is not necessarily the habitat of diesel-tolerant species.

The molecular taxonomy of the two isolates best producers of biotechnological compounds was performed. According to Sandoval-Denis et al. [36], C. anthropophilum belongs to the C. cladosporioides species complex, as demonstrated in the cluster formed by the two species in the phylogenetic tree (Fig. 4b). Interestingly, the same author identified this new species from human clinical specimens, suggesting that this species may have been introduced into this environment by anthropogenic action.

As eukaryotic organisms, Antarctic fungi appear to be highly adapted to thrive in the extreme environments of the peninsular and continental Antarctica [37]. The genus Cladosporium, commonly found in the polar environment by conventional microbiological techniques, has been well reported in the literature as an inhabitant of the Antarctic continent, being recovered from several samples, including permafrost sediments [38], arctic ice, salty marine sediments, diverse soils [29, 39–42], and samples collected from Deception Island [43] and from King George Island [29].

Like fungi, bacterial species are widely distributed on the Antarctic continent, including Psychrobacter species, which have been isolated or detected in samples originating from King George Island [44], frozen Antarctic lakes [45], iceberg ice, rocks with lichens, small ponds of glacier meltwater, red seaweed, and seawater ponds with algae [32], as well as soil samples collected on Deception Island (Crater Lake, Fumarole Bay and Whalers Bay) [2] and Antarctic volcanic soil [46].

Diesel oil and gasoline tolerance

The best results of microbial cell growth against the tested compounds were from bacterial strains isolated from the Deception Island sample (Table 1), corroborating the history of the area, which witnessed the whale oil exploration industry in the first decades of the twentieth century and the growth of cruise tourism at the end of the same century [2, 47]. In this context, results suggest that bacterial strains isolated from Deception Island can be tolerant to petroleum-derived compounds probably due to long exposure to contaminants in the environment.

In the work performed by Panicker et al. [48], the authors isolated a Pseudomonas sp. strain from oil enriched soil from Wright Valley, Antarctica, and demonstrated its ability to degrade hydrocarbons such as those that make up diesel and gasoline. In 2020, Abdulrasheed and colleagues [47] conducted a study in which two Antarctic strains of Arthrobacter spp. isolated from soil samples from King George Island were tested for their ability to degrade diesel oil and produce biosurfactant. The authors found that these strains showed good results for both activities, with high rates of cellular hydrophobicity (about 80%) in hexadecane, suggesting the potential use of these cold-tolerant strains for biodegradation of diesel-polluted Antarctic soils. Schultz et al. [15] performed a study to assess the potential of thermophilic bacteria isolated from fumarole sediments on Deception Island to degrade petroleum hydrocarbons. In the study, about 30 strains of bacteria showed efficient results in the degradation oil-derived hydrocarbons.

The presence and distribution of alkane-degrading genes in bacterial communities on King George Island was revealed in a study by Jurelevicius et al. [1]. The authors used molecular methods to analyze the distribution of the alkB gene in different soil samples and found that the presence of hydrocarbons in soil affects the disposition of alkane-degrading bacteria, demonstrating the microbial potential of bacteria from cold environments for the degradation of petroleum pollutants. In the study performed by Gregson et al. [49], a strain of Oleispira antarctica, a psychrophilic marine hydrocarbonoclastic bacterium, an organism that dominates microbial communities after oil spills, which was isolated from oil‐enriched microcosms containing seawater from Sea, Southern Antarctica, was evaluated for their expression of its proteins during growth in n-alkanes. The authors observed that, during growth in n-C14 at 16 °C, the strain expressed a complete pathway for the terminal oxidation of n-alkanes, including the enzymes alkane monooxygenases, fatty acid-CoA ligase, and fatty acid desaturase, thus demonstrating the hydrocarbon-degrading potential of a strain previously recovered from the Antarctic environment. Studies using Antarctic bacterial strains report tolerance to petroleum-derived compounds including diesel oil more frequently than the use of fungal strains [35, 50, 51]. Therefore, our findings demonstrated the ability of microbial strains isolated from Antarctic sediments to tolerate hydrocarbons, making them promising for use in bioremediation processes in environments contaminated with petroleum derivatives.

RBBR dye discoloration potential

Few studies in the literature have addressed the ability of filamentous fungi originating from Antarctic marine samples to decolorize RBBR dye or other textile dyes. Kita et al. [13] carried out a study aiming the selective isolation of filamentous fungi with the potential for textile dye bioremediation. In the study, two strains of Penicillium cf. oxalicum isolated from marine sediment collected at Whalers Bay, on Deception Island, discolored 81.86 and 98.89% of the sulfur indigo blue dye. In the same study, fungal isolates obtained from King George Island showed dye discoloration rates, however, in lower proportions. Chang et al. [33] conducted a study in which the RBBR dye was used as a rapid screening method for the detection of toxic compounds degraded by fungi leading to the isolation of four filamentous fungi capable of decolorizing RBBR dye. The RBBR dye has aromatic compounds in its chemical structure, resembling PAHs, so the discoloration capacity has been used as a screening method for bioremediation [52]. The decolorization capacity of synthetic dyes such as RBBR may indicate the production of ligninolytic enzymes such as laccase since the RBBR dye has in its composition compounds like those found in the structure of lignin; an enzyme that can act in the degradation of hydrocarbons [53].

In the present study, adsorption of the RBBR dye by the fungal mycelium was observed. The adsorption process by itself can be considered a microbial cell resource that gives these cells the potential for bioremediation of textile dyes [20]. Thus, filamentous fungi recovered from cold environments may have the ability to decolorize and/or adsorb synthetic industrial dyes such as RBBR, which can be applied in bioremediation processes in low-temperature environments impacted with dyes.

Biosurfactant and bioemulsifier activities

The concern to replace potentially pollutant commercial products such as detergents and dispersants with environmentally friendly ones has prompted much research, and bioprospecting autochthonous microorganisms from polar environments able to produce biosurfactants have revealed promising results [14, 54, 55]. In bioremediation processes, the presence of biosurfactants can facilitate the degradation of hydrocarbons (hydrophobic pollutants) which can be used as a source of carbon in microbial metabolism [53, 56]. Schultz et al. [15] demonstrated the biosurfactant-producing potential of 13 thermophilic bacteria out of 50 strains from Deception Island sediments, which showed about 50% emulsification index (E₂₄) of a petroleum hydrocarbon source. In the work developed by Coronel-León et al. [56], a strain of Bacillus licheniformis isolated on Deception Island in Antarctica was able to produce lipopeptides with biosurfactant properties in the presence of a variety of carbohydrates. In 2020, Ibrahim and colleagues [3] evaluated a strain of Rhodococcus erythropolis isolated from soil collected from King George Island for its biosurfactant production capacity and found that the isolate had a high emulsification index, reaching about 91%. The authors concluded that biosurfactant compounds produced by microbial isolates from polar environments can be used in enhanced oil recovery (EOR) processes in different environments.

In this study, a strain of Psychrobacter sp. (BAD17) showed an emulsification index of 3.6% in medium supplemented with 1% engine oil. The production of biosurfactants by strains of Psychrobacter spp. recovered from Antarctic samples has been described in other works [11, 14, 44]; however, no reports of biosurfactant production from Psychrobacter sp. isolated from marine sediments collected on Deception Island (Whalers Bay) were found. In the present study, the bacteria showed a low emulsification capacity (only BAD45 shows E₂₄ of 18.2%). However, Bueno et al. [57] were able to verify several bioemulsifying activities in yeast recovered from the Antarctic continent. Eleven strains were able to produce E₂₄ with values greater than 15%, being considered good producers of biosurfactants. Perhaps performing further studies with different carbon sources (other hydrocarbons) in different temperatures or the selection of new strains that potentially show more pronounced emulsification capacity can provide conditions that allow an improvement in the emulsification capacity, achieving better results in terms of the production/activity of biosurfactant/emulsifier.

Pyrene tolerance assay

Regarding pyrene tolerance assay, Gran-Scheuch et al. [58], using enrichment techniques in the presence of phenanthrene, were able to isolate 53 PAH-metabolizing bacteria from diesel-contaminated Antarctic soil samples collected on King George Island. In this study, a Sphingobium xenophagum strain was able to degrade phenanthrene by up to 95%. Ausuri et al. [59] isolated a strain of Dietzia psychralcaliphila from sediments collected from Deception Island, Antarctica, using culture medium added with phenanthrene. The isolate was able to degrade 84.66% of phenanthrene, and the whole genome sequencing revealed the presence of several monooxygenase and dioxygenase genes, responsible for the biodegradation of hydrocarbons. This bacterial species has been reported to be able to grow in medium supplemented with n-alkanes as the sole carbon source [60].

Herein, enrichment using diesel oil and gasoline selected 69 bacterial and two fungal fuel-tolerant isolates, being the bacteria BAD15 and Psychrobacter sp. BAD17 and the fungi FAR18 (Cladosporium sp.) and FAD28 the best performers, which were also able to tolerate and grow in culture medium supplemented with 1 mg L⁻¹ pyrene. Thus, these four isolates could possibly use PAH as a carbon source, through their ability to degrade the hydrocarbon molecular structure. The small number of pyrene-tolerant microorganisms found may be due to the short incubation time in the presence of diesel oil and gasoline in our experiments (7 days), since in similar tests in Eriksson et al. [61], the authors used 60 days of incubation for the microbial community which was able to degrade pyrene and phenanthrene. In the same study, two isolates that were able to grow in media-added pyrene were identified as Pseudomonas spp. The existence of PAH-tolerant microorganisms on Deception Island was observed by Santos et al. [62]. Using a metagenomic approach in soil samples from Deception Island, the authors observed a high number of genes associated with tolerance to PAHs, probably linked to the presence of volcanic ash.

The ability of Antarctic fungal strains to biodegrade hydrocarbons is not well described compared to the information available on Antarctic bacterial strains [35]. However, there are some reports on the degradation of n-alkanes and diesel fuel, diesel, and other hydrocarbons by hydrocarbon-degrading fungal strains of different species isolated from Antarctica, including Penicillium, Pichia, Mortierella, Pseudeurotium, among others [63–66]. In 2011, Ferrari et al. [34] found C. cladosporioides strains in a soil sample contaminated with petroleum hydrocarbons in the form of diesel fuel, at the Australian Antarctic Permanent Research Station, a finding corroborated by our work, which demonstrated the hydrocarbon tolerance of a Cladosporium sp. originated from sediment collected on King George Island, Antarctica. Thus, microorganisms tolerant to PHA pyrene, demonstrated in the present study, reinforce the importance of exploring extreme environments such as the Antarctic, in the search for microbial cells potentially transforming petroleum-derived compounds, able to be applied in bioremediation processes in cold environments.

Laccase activity

Antarctic microorganisms have already been reported regarding their abilities in synthesizing a variety of enzymes active at low temperatures, including proteases, amylases, cellulases, lipases, and laccases [67]. Ligninolytic fungi are able to degrade PAHs using enzymes from an extracellular enzyme complex that is normally used to depolymerize lignin. Thus, ligninolytic enzymes, including laccase, are widely investigated to be applied in bioremediation processes of environments contaminated with pollutants such as diesel, gasoline, PAHs, and dyes [10, 20]. Although there is a great relevance of this enzyme for environmental applications, studies using Antarctic fungi for this purpose are still scarce [17].

Rovati et al. [52] investigated the ability of yeasts isolated from samples of King George Island to degrade polyphenolic substrates such as lignin and dyes. The authors observed that 33%, 25%, and 38% of the isolates showed dye decolorization, laccase, and ligninolytic activity, respectively. In the work developed by Duarte et al. [68], the authors evaluated the ability of filamentous fungi isolated from different substrates collected from marine and terrestrial samples from King George Island (South Shetland Islands, Maritime Antarctic) to synthesize several enzymes, including laccase. The study revealed some fungal genera capable of producing ligninolytic enzymes, such as Penicillium, Oidiodendron, Cosmospora, Geomyces, and Cladosporium, with emphasis on the Cadophora spp. strains, which were the best laccase producers. In our studies, the two filamentous fungi capable of synthesizing laccase were isolated from marine sediment samples from King George Island, as were the isolates described in the work by Duarte and collaborators. According to Yergeau and Kowalchuk [69], high densities of laccase genes were found in diverse Antarctic soils. In their experiments, the authors observed that freeze–thaw treatments significantly influenced laccase activities probably due to the structure of the fungal communities present in the Antarctic habitats. According to Martorell et al. [62], the lignocellulolytic activity of the laccase enzyme was detected in 37% of the fungi isolated from King George Island, including Penicillium spp., Phialocephala spp., and Pseudogymnoascus spp.

Reports of laccase-secreting psychrophilic bacteria are scarce in the literature [70]. Park et al. [71] investigating humic-degrading bacterial communities in Arctic and Antarctic soils identified 73 potential humic-degrading bacteria, harboring multicopper oxidase genes. Of these, 71% showed conserved copper binding regions, which are considered essential regions for laccase activity. In the study by Zhang et al. [72], the authors described a new cold-adapted laccase from an Antarctic strain of Psychrobacter sp., isolated from sea ice. In the study, a degradation loss of approximately 13.2% of the polyethylene weight was observed. The lack of studies focused on Antarctic bacterial strains cells capable of producing laccase demonstrates the need to improve enrichment methods for the isolation of laccase-producing bacteria.

Results gathered herein pave the way for future investigations on the impacts of the continuous introduction of PAHs and their derivatives in the Antarctic environment. In the study conducted by Préndez et al. [73], PAHs and alkanes were identified in marine superficial sediments near the Antarctic stations at Fildes Peninsula, King George Island. The presence of hydrocarbons in the Antarctic environment has been shown to be a result of the increase in scientific activities and the number of tourists in recent years. Diesel is the main source of energy to support human activities in Antarctica and has been threatening the environment and organisms that live in the polar environment due to its contaminating potential persistence, complexity, and toxicity [35].

The results of the present study demonstrated the biotechnological potential that microbial strains recovered from polar environments may possess. Microorganisms capable of decolorizing synthetic dyes and of tolerating recalcitrant compounds such as fuel derivatives and PAHs possibly introduced in these environments by anthropic activities, as well as of synthesizing enzymes and/or compounds used in bioremediation processes, were found. However, new enrichment using other hydrocarbons as carbon sources in culture media with low concentrations of nutrients should be carried out to recover a greater microbial diversity adapted to compounds derived from petroleum. Further studies involving degradation assays of these recalcitrant compounds and detection of the resulting fewer toxic metabolites shall be carried out using the most promising microbial isolates found in this study.

Author contribution

LMC, MB, and MRZP carried out the analyses of the data; AWD, LHR, RV, and AAN collected the samples; AWD, VMO, and LHR reviewed the manuscript; JRO and MRZP wrote, reviewed, and edited the manuscript; all authors approved the final version of this manuscript.

Declarations

Conflict of interest

The authors declare no competing interests.