1. Introduction
Actinobacteria of the genus
Rhodococcus are widespread in nature and are isolated from aquatic and soil biotopes adjoining, in particular, oil and gas deposits. Due to a range of catabolic traits and unique enzyme systems, rhodococci have the ability to degrade hydrocarbon compounds representative of various chemical structures. These capabilities, including the ability of these bacteria to survive in unfavorable environmental conditions, make this genus suitable for the development of biopreparations for bioremediation of oil- and oil-products-contaminated sites [
1,
2,
3,
4].
Temperature is one of the major limiting factors significantly affecting bioremediation. In low-temperature environments, the bioavailability, solubility, and evaporation of oil hydrocarbons decrease and the viscosity of the oil increases. In this case, the diffusion processes and the level and rates of biodegradation of oil hydrocarbons by microorganisms slow down [
5].
A low bioavailability of hydrocarbon substrates to microbial attack largely results from extremely poor solubility of these substrates in water. The factors due to which
Rhodococcus strains, capable of utilizing hydrophobic substrates, assimilate hydrocarbons are as follows [
6]: (1) the formation of the hydrophobic cell wall containing lipophilic compounds, thereby playing an essential role in establishing a direct contact of the cells with hydrocarbon drops, and (2) the release of biosurfactants, that can solubilize hydrocarbons in the aqueous phase, into the medium. The ability to produce biosurfactants is a common feature among microorganisms living in cold environments [
7].
Rhodococci are shown to produce trehalolipid biosurfactants which are released into the cultivation medium by bacteria or bound to the bacterial cell wall. Mostly, nonionic trehalolipids (mono-, di-, tetra-, hexa- and octaacyl derivatives of trehalose) [
8,
9] and anionic trehalolipids (succinoyl trehalolipids) [
10,
11,
12,
13,
14] are released into the cultivation medium. The information on trehalolipid biosurfactants produced by rhodococci was summarized in a recent review [
15]. Glycolipids released into the cultivation medium can be replaced by phospholipids, thereby resulting in a decrease in the hydrophobic effect of the cell wall [
16]. Thus, rhodococci can use the biosurfactants they produce to regulate cell surface properties in order to attach to or detach from hydrophobic substrates or hydrophobic surfaces [
17,
18,
19]. This may also be a way for a more efficient transport of hydrophobic substrates into the cell. It was previously reported that the oil-degrading bacteria identified as
R. erythropolis S67 and
R. erythropolis X5 released succinoyl trehalolipids in the cultivation medium. The main components of these glycolipids were isolated and identified. They represented a mixture of isomeric homologues: 2,3,4-decanoyl-octanoyl-succinyl-2′-decanoyltrehalose, 2,3,4-dioctanoyl-succinyl-2′-decanoyltrehalose, 2,3,4-dioctanoyl-succinyl-2′-octanoyltrehalose, and 2,3,4-didecanoyl-succinyl-2′-decanoyltrehalose. It was shown that low temperature had no effect on the qualitative composition of trehalolipids produced by strain S67 [
20].
It is noteworthy that the biodegradation of hydrocarbons is substantially dependent on their aggregate state, which can vary with temperature. For instance, n-hexadecane (in the coming text, we will call it hexadecane) can be found in liquid state at 26 °C, and solid state at 18 °C (melting point of hexadecane: 18.2 °C). In earlier reports [
21,
22], the degradation of hydrocarbons (tetradecane, kerosene, diesel fuel) was studied at low temperatures; nevertheless, the growth substrate was in a liquid aggregate state.
The aim of our study was to reveal the peculiarities of the adaptation of the oil-degrading bacteria R. erythropolis S67 and R. erythropolis X5 to hydrophobic hydrocarbon degradation at low temperatures when the substrate was in solid states using hexadecane as a model.
2. Materials and Methods
2.1. Microorganisms and Cultivation Conditions
The strains
R. erythropolis X5 (VKM Ac-2532 D) and
R. erythropolis S67 (VKM Ac-2533 D) were obtained from the collection of the Laboratory of Plasmid Biology, IBPM RAS. The biopreparation “MicroBak” contains these bacteria and is used for bioremediation of oil-contaminated areas [
4]. The
R. erythropolis X5 genome project has been deposited in the NCBI database under GenBank accession numbers CP044283 and CP044284, BioSample number SAMN12818508, BioProject number PRJNA573614, and SRA accession number PRJNA573614 [
23].
For the cultivation of microorganisms, the Evans liquid synthetic minimal mineral medium and rich Luria–Bertani broth were used. The Evans medium had the following composition (L−1): K2HPO4, 8.71 g; 5 M solution NH4Cl, 1 mL; 0.1 M solution Na2SO4, 1 mL; 62 mM solution MgCl2,1 mL; 1 mM solution CaCl2, 1 mL; 0.005 m M solution (NH4)6Mo7O24, 1 mL; and trace elements solution, 1 mL; the pH was adjusted to 7.5 by the addition of concentrated HCl. The trace element solution in 1% HCl solution contained (g L−1): 0.41 ZnO, 2.9 FeCl2, 1.28 MnCl2, 0.13 CuCl2, 0.26 CoCl2, and 0.06 H3BO3. Glucose, hexadecane, naphthalene, crude oil, and diesel fuel were used as a sole carbon and energy source (2% v v−1 or w v−1). The Luria–Bertani broth had the following composition (L−1): bacto-tryptone (Difco, Detroit, MI, USA), 10 g; yeast extract (Difco, Detroit, MI, USA), 5 g; and NaCl, 10 g. To obtain agar media, bacterial agar (Difco, Detroit, MI, USA) was additionally added in the amount of 20 g L−1 of the medium. The prepared media were sterilized by autoclaving for 30 min at 120 °C (additional pressure: 0.5 atm). Microorganisms were cultivated in 750 mL Erlenmeyer flasks at 26 °C and 10 °C on an Excella 25 orbital shaker (Eppendorf, Hamburg, Germany) at 180 rpm. The microbial growth was monitored by optical density measurements at 600 nm (OD600) using a spectrophotometer Cintra 6 (GBC Scientific Equipment Pty. Ltd., Melbourne, Australia).
2.2. Measuring Surface Tension
The surface tension was measured on a surface tensiometer (Surface Tensiomat model 21; Fisher Scientific, Cole-Parmer, United States) at 25 °C. The surface tension of the reference solution (Evans medium) was 77 mN m−1.
2.3. Measurement of the Content of Glycolipid Biosurfactants
The content of the biosurfactant was assessed by measuring the concentration of the carbohydrate part of the glycolipid with a photocolorimetric method (Petrikov et al., 2013). Cells were preliminarily removed from the cultivation medium by centrifugation at 10,000 rpm and 4 °C for 10 min on a Rotanta 460R centrifuge (Hettich-Zentrifugen, Tuttlingen, Germany). A 2 mL sample of the supernatant was placed into a test tube and 20 μL of 80% phenol solution in water was added. With vigorous stirring, 5 mL of concentrated sulfuric acid (ρ 1.84 g mL−1) was added and left for 10 min. Then, the solution was vigorously stirred again, and the test tube was placed in the water bath at 25 °C for 20 min. The optical density of the resulting solution was measured on a UV–visible spectrophotometer (Cintra 6, GBC Scientific Equipment Pty. Ltd., Melbourne, Australia), using a quartz cell with a 1 cm path length. Wavelengths in the range of 480–490 nm were chosen as optimum wavelengths for estimating the given carbohydrate by the absorption maximum. The reference solution was prepared in a similar way, using distilled water instead of the sample. The concentration of carbohydrate part was determined by using previously created calibration curves. To calculate the concentration of the biosurfactant, the obtained value of the sugar concentration was then multiplied by a factor of 2.5, equal to the ratio of the molecular weight of the glycolipid (862) to the molecular weight of the appropriate carbohydrate (342 for trehalose).
2.4. Hydrophobicity of the Cell Surface of Microorganisms
The hydrophobicity was measured by the MATH test (microbial adherence to hydrocarbon) (Rosenberg 1984) as modified by Satpute et al. (2010). The cultivation medium was centrifuged using a TG16-WS tabletop high speed centrifuge (Polikom, Russia) at 10,000×
g for 10 min. The cell biomass was washed with distilled water twice and resuspended in a phosphate-magnesium buffer containing (g L
−1): K
2HPO
4·3H
2O, 22.2 g; KH
2PO
4, 7.26 g; urea, 1.8 g; and MgSO
4·7H
2O, 0.2 g; the pH was 7.0. In this case, the OD
600 of the cell suspension of all samples should be in the range of 0.48–0.50. An amount of 0.5 mL of n-hexadecane was added to 3.0 mL of the cell suspension. The contents of the tubes were shaken vigorously on a shaker at 2000 rpm for 3 min. After being left to stand at room temperature for 10 min, the separation of the aqueous and hydrocarbon phases occurred. The hydrocarbon phase was carefully separated from the aqueous phase and the OD
600 of the aqueous phase was measured. The hydrophobicity was calculated using the formula:
where
H (%) was the hydrophobicity of the cell surface (%);
A0,
A were the values of the OD
600 of the cell suspension before and after n-hexadecane addition, respectively.
2.5. Transmission Electron Microscopy
Bacterial cells were fixed in a 1.5% solution of glutaraldehyde in 0.05 M cacodylate buffer (pH 7.2) at 4 °C for 1 h, washed three times with the same buffer and additionally fixed in a 1% solution of OsO4 in 0.05 M cacodylate buffer (pH 7.2) at 20 °C for 3 h. After dehydration, the material was embedded in epoxy resin Epon-812. Ultrathin sections were mounted on the supporting mesh grids and contrasted for 30 min in a 3% uranyl acetate solution in 70% alcohol and additionally stained with lead citrate.
From a sample of cells grown in a medium with hexadecane, we obtained five repeats for the preparation and analysis of ultrathin cell slices. In each repeat, we analyzed 5–7 slices. We observed 15–20 cells on each slice.
2.6. Total Cell Lipid Content in Bacteria
The total cell lipids were extracted with polar organic solvents from raw bacterial biomass after the excess of hexadecane was removed by using hexane. The raw biomass (40–50 mg of cells per dry weight) was diluted with water to a volume of 1 mL. The suspension was placed in a centrifuge tube with a ground-in glass stopper, and 3.75 mL of chloroform–methanol (1:2, v:v) was added; the mixture was shaken and left at room temperature for several hours with intermittent shaking. The cells were precipitated by centrifugation, and the extract was decanted into another 15 mL centrifuge tube. Microbial cells were resuspended in 4.75 mL of chloroform–methanol–water (1:2:0.8, v:v:v), and the mixture was then shaken and centrifuged. To the combined supernatant, 5 mL of chloroform: methanol (1:1, v:v) was added; the chloroform layer was separated by centrifugation. The lower chloroform layer was diluted with benzene and evaporated to dryness in vacuum on a rotary evaporator (30–35 °C). The lipid residue was immediately dissolved in chloroform–methanol (1: 1, v v−1), the solution was then subjected to centrifugation and brought up to the required volume with chloroform.
2.7. Content of Cell-Bound Hexadecane
To remove excess n-hexadecane from the outer surface of cells, 5–10 mL of a mixture of ethanol–butanol–chloroform (10:10:1, v:v:v) was added to 1 g of biomass and resuspended. Then, the suspension was centrifuged for 10 min at 10,000×
g. Washing was done twice. To the resulting precipitate, 5–10 mL of 5 M NaOH was added and left overnight; after that, the reaction mixture was extracted with dichloromethane twice. The extract was dried over anhydrous sodium sulfate, the solvents were then evaporated in a rotary evaporator at 40 °C and a pressure of 0.2 atm to dryness. The content of cell-bound hexadecane was analyzed by gas chromatography [
24].
2.8. Determination of Fatty Acid Composition of Lipids
The fatty acid composition of lipids was determined by gas chromatography with a mass spectrometric detector (GC–MS). For this, the transesterification of fatty acid esters with methanol was carried out according to the procedure in [
12] with modification. A weighed sample (30 mg for lipids) was resuspended in 2 mL of a mixture of concentrated sulfuric acid–methanol (5% by volume) and heated at 60 °C for 2 h. Then, after cooling, it was extracted with 5 mL of diethyl ether. The residue of sulfuric acid in the extract was washed with distilled water to neutral pH. Further, the stripped-off sample was dissolved in chloroform and analyzed using an Agilent 6890N chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) with an Agilent 5973 mass spectrometric detector on an HP-1 capillary column (30 m × 25 mm × 25 µm) with a temperature gradient from 50 to 290 °C for 40 °C per min, then held for 30 min at 290 °C. Helium was used as a carrier gas. Fatty acids were identified using the NIST/EPA/NIH database (
http://www.nist.gov/srd/nist1a.cfm (accessed on 13 July 2022)).
2.9. Determination of Residual Hexadecane in the Cultivation Medium
Hexadecane was extracted from the culture broth with an equal volume of hexane. Using a microsyringe, 10 µL
−1 of the sample was injected into the evaporator and analyzed for the hexadecane content using a Chrystal-5000 gas chromatograph (Khromatek-Analytic, Yoshkar-Ola, Russia) with a BP1J08 capillary column (30 m × 0.3 mm × 0.53 µm). The flow rate of the carrier gas (nitrogen) was 174.6 mL min
−1. The column temperature was set at 250 °C, the evaporator temperature was 300 °C, and the detector temperature was 250 °C. At least two chromatograms of the analyzed samples were recorded. The content of hexadecane in the samples was determined using a calibration graph, which expressed the dependence of the peak area on the content of n-hexadecane. The degree of degradation of hexadecane was calculated using the following formula:
where
C1 was the concentration of hexadecane in the liquid cultivation medium after the cultivation of microorganisms (% of the volume of the medium), and
C0 was the concentration of hexadecane in sterile control.
2.10. Experimental and Statistics Analysis
Results obtained were analyzed using the built-in statistical packages in Excel (MS Office 2007). All experiments were performed in three replicates. In each experiment, we took samples for the chemical and biochemical analyses. Each sample was measured three times.
4. Discussion
It was previously shown that biosurfactants of rhodococci during degradation of hydrocarbons can be produced in a liquid cultivation medium or be bound to the cell wall. A number of works are devoted to the study of the production of biosurfactants by rhodococci at different temperatures [
11,
12,
25]. Malavenda et al. [
21] demonstrated that arctic rhodococci, when grown on tetradecane (melting point, 5.8 °C) at 15 °C, produced biosurfactants which could reduce the culture–medium surface tension in the range between 66 and 27 mN m
−1. Other authors showed that when marine bacteria
Rhodococcus sp. LF13 and LF22 grew on kerosene (melting point, −47 °C) under low temperature conditions (13 °C), the surface tension of the medium decreased to 37 and 30 mN m
−1, respectively [
22]. The surface tension of liquid cultivation medium of
Rhodococcus sp. Q15 grown on diesel fuel (melting point, −55 °C) at 5 °C dropped to 40 mN m
−1 [
25]. However, it should be noted that in these studies, all the studied hydrophobic hydrocarbons were in a liquid state under a low temperature.
We compared the growth and consumption rates of the model substrate—hexadecane–by
R. erythropolis strains X5 and S67 at 26 °C, when the substrate was liquid, and at 10 °C, when it was solid (melting point of hexadecane, 18.2 °C). The degradation of hexadecane was shown to occur most intensively in the exponential growth phase, when bacteria had consumed more than half of the total substrate. In 8 days, X5 consumed 53% of hexadecane in liquid state and 40% of solid hexadecane in 18 days. Despite the solid aggregate state of hexadecane, the degree of its degradation remained high: in the case of strain X5 it was 40% and 30% for strain S67 within 18 days. Earlier, Whyte et al. [
26] showed
Rhodococcus sp. Q15 was capable of utilizing about 30% of hexadecane after 30 days at 5 °C. When studying the biodegradation of hydrophobic substrates at a low temperature [
27],
R. cercidiphyllus strain BZ22 was found to consume 35% of hexadecane at 10 °C after a 14-day cultivation. These results are comparable to the results obtained in our experiments.
The transport of hydrocarbons by rhodococci can be divided into two stages. The first stage is a direct contact of the cells to the substrate suggesting that high cell surface hydrophobicity plays an essential role in the attachment of rhodococci, especially when the substrate is in a solid aggregated state. The second stage is that, when dissolved in membrane lipids, alkanes can be oxidized by membrane-localized enzymes to the corresponding carboxylic acids [
10].
It is known that microorganisms can change cell surface hydrophobicity using biosurfactants [
17]. The study showed that the cell surface hydrophobicity of strains X5 and S67 was higher when cells grew on solid hexadecane: thus, in the exponential growth phase, it was maximum and achieved 75% for X5 and 88% for S67. When cells grew on liquid hexadecane, the cell surface hydrophobicity was 63% and 76%, respectively. It should be noted that both strains revealed the highest cell surface hydrophobicity in the exponential growth phase and a decrease in cell surface hydrophobicity was detected in the stationary phase. A comparison of the degree of cell surface hydrophobicity and the content of extracellular biosurfactants indicated a common tendency toward a decreasing hydrophobicity of the cell wall with an increasing quantity of biosurfactants produced into the cultivation medium.
Margesin et al. [
27] proposed that the external lipid barrier of permeability for hydrophobic substrates into actinobacteria cells is formed by high-molecular hydroxy acids—mycolic acids and their esters with trehalose, promoting the penetration of hydrocarbons into the cell in a passive-diffuse way.
In our experiments, the hydrophobicity of the bacterial cell wall decreased towards the stationary growth phase (
Figure 2), and the degree of adhesion to hydrocarbons decreased. A decrease in the degree of cell surface hydrophobicity can be explained by the entry of succinoyl trehalolipids into the cultivation medium; this fact is consistent with the data of Franzetti et al. [
28] on the production of biosurfactants by bacteria outside the cells. They showed [
28]
Gordonia cells in the early exponential growth phase had a high hydrophobicity of the cell surface due to cell-associated biosurfactants and remained attached to large drops of hydrophobic substrate, so the absorption of substrate was carried out by cells through direct contact. During the late exponential growth phase, the cells released bioemulsifiers into the culture medium and became more hydrophilic, which allowed them to attach to the hydrophilic outer layer of solubilized hydrocarbon droplets [
28].
Factors involved in the cell surface hydrophobicity do not only include the presence of glycolipids in the cell wall. Psychrotrophic rhodococci cells may adapt to grow at low temperature by increasing the lipid content and reducing their degree of saturation [
29]. Due to the ability of
Rhodococcus strains to change the fatty acid composition of membrane lipids, they change the fluidity and permeability of the cell wall [
30]. In our experiments, the growth of
R. erythropolis strains X5 and S67 on solid hexadecane increased the content of total cell lipids. Strains X5 and S67 varied in the fatty acid composition of cellular lipids. Cells of strain X5 grown on liquid hexadecane contained saturated branched acids, while cells of strain S67 contained more saturated unbranched acids. When cultured on solid hexadecane, a change in the ratio of saturated unbranched and branched fatty acids was observed in the cells of both strains (
Table 2). The proportion of unsaturated 9-hexadecenoic acid in the cells of strain X5 increased (33%). Saturated branched fatty acids appeared in the cells of strain S67 (37%) (
Table 2). Apparently, strains X5 and S67 develop different mechanisms by which the cell membrane fluidity changes.
The analysis of cell lipids of the studied strains showed that the main component was hexadecanoic acid, an intermediate product of hexadecane degradation when cells were grown on both liquid and solid hexadecane (36% at 26 °C and 31% at 10 °C for strain X5; 52% at 26 °C and 33% at 10 °C for strain S67) (
Table 2). Since the gas chromatographic analysis of lipid extracts from X5 and S67 cells showed insignificant amounts of hexadecane in the cells, this may indicate that hexadecane degradation gets started in the pericellular area.
The GC/MS method showed that a number of fatty acids with a chain length from C
8 to C
16 were present in the supernatant of the culture medium of strains X5 and S67 grown on hexadecane (
Figures S3 and S4). It should be noted that in lipid extracts of the supernatant of the culture medium of strains X5 and S67, grown on both liquid at 26 °C and solid hexadecane at 10 °C, fatty acids with an even number of atoms were found. Under the action of the terminal alkane monooxygenase of aerobic bacteria, hexadecane is known to be oxidized to alcohol, which by alcohol dehydrogenase is further converted to aldehyde, and then aldehyde dehydrogenase converts hexadecane into acid. Further oxidation proceeds along the
beta-oxidation of long-chain fatty acids, during which the fatty acid decreases by two carbon atoms. The presence of decanoic (C
10) and octanoic (C
8) acids may indicate not only hexadecane degradation, but also a possible synthesis of trehalolipids with the corresponding fatty acid “tails”. Peng et al. [
31] studied lipids in the cell-free culture medium during the growth of
R. erythropolis 3C-9 on hexadecane and found that the content of hexadecanoic acid (C
16) was about 65%. Moreover, the presence of hexadecanoic acid in cell-free supernatant in our experiments confirmed that the catabolic transformation of hexadecane started in the pericellular area.
The study of the morphology and ultrastructural organization of cells of
R. erythropolis strains X5 and S67, revealed multimembrane structures (MMS) on ultrathin sections, mainly in the form of multiple loop-like or tubular formations associated with hydrophobic inclusions (
Figure 3 and
Figure 4). The MMS profile consisted of several electron-dense layers alternating with electron-transparent layers, which was an analog of the hydrophilic and hydrophobic zones of biological membranes. MMS were more pronounced in cells grown on solid hexadecane (
Figure 4). Similar structures were previously found in
Rhodococcus opacus grown on benzoate [
32]. The formation of multimembrane structures in the studied bacteria is an adaptive mechanism for their survival under extreme environmental conditions. The data of the electron cytochemical analysis of
R. erythropolis strains confirmed the connection between the increase of membrane ultrastructures into the cells and the destructive processes of n-alkanes in the cold, when the hydrophobic substrates were solid. The MMS were involved in the transport of a hydrophobic substrate into the cell, as well as in the partial oxidation of the substrate under the action of membrane-localized enzymes [
32].
Multimembrane structures (MMS) associated with inclusions are characteristic of cells during the degradation of hydrophobic and toxic compounds. Similar structures were previously found in
Sulfobacillus during the oxidation of hydrophobic sulfur [
33], in
Pseudomonas capable of utilizing sodium dodecyl sulfate [
34,
35], in
Acinetobacter sp. grown on hydrocarbons [
36], and in rhodococci grown on liquid and gaseous n-alkanes [
37].
In the cytoplasm, there were electron-transparent inclusions (L), typical for substances of a hydrophobic nature. Currently, there is no clear opinion about the composition of lipid inclusions formed in cells of actinobacteria, when they are grown on hydrophobic substrates. These inclusions can be hexadecane [
38] or triacylglycerols (TAG) [
39,
40]. The information on the
rhodococci ability to accumulate TAG when grown on various hydrophobic substrates has been summarized in the recent review [
41]. The composition of the inclusions can be suggested by the content of cell-bound hexadecane and fatty acids in cellular lipids. Since cell lipids of strains X5 and S67 during growth on hexadecane were found to contain a low content of hexadecane and a high content of fatty acids (
Table 1), we assumed that these electron-transparent intracellular inclusions were triacylglycerols.
The
Rhodococcus X5 and S67 cells formed multiple spherical structures (vesicles, V), that could be regarded as an analog of liposomes (
Figure 5). Spherical structures were formed by a type of self-assembly with the participation of polar molecules with hydrophobic and hydrophilic tails (biosurfactants) secreted by the cell. It is possible that these membrane-like structures were formed by the trehalolipid biosurfactants produced by our strains, X5 and S67. These structures may contain primary alkane degradation enzymes also secreted by cells. This form of encapsulation of primary biodegradation enzymes in the liposome is most effective for interaction with the solid substrate.
Similar structures named outer membrane vesicles (OMVs) were found for
Pseudomonas putida KT2440 during cultivation in lignin-rich media [
42]. OMVs contained many enzymes acting toward lignin-derived aromatic compounds. Salvachua et al. [
42] hypothesized that some of the proteins could be trafficked to the extracellular compartment via OMVs. Lignin, like hexadecane at low temperature, is a solid substrate. The formation of numerous vesicles by rhodococcal cells during growth on solid hexadecane is also an important adaptive property that provides an increase in the contact area of bacterial cells with a hydrophobic substrate.
The transmission electron microscopy observation of ultrathin sections of X5 cells grown on solid hexadecane showed that vesicles were connected to cells by means of electron-dense filaments—fibers (F) (
Figure 5). Delegan et al. [
43] studied the ultrastructure of rhodococci cells grown on dodecane at 28 °C. They described surface fibrillar structures, which are most commonly used by bacterial cells to attach to the surface. In contrast, we showed that fibers were pili-like structures that were hollow and possibly served to transport hexadecane and its intermediates.
In our previous study, it was shown that
R. erythropolis S67 cells released succinoyl trehalolipids into the culture medium [
20]. Importantly, negatively charged succinoyl trehalolipids, which include a residue of succinic acid, may exhibit a high affinity for positively charged ruthenium red. This may lead to the appearance of electron-dense structures in the region of their localization. We suggest that extracellular biosurfactants (trehalolipids) are probably involved in the functioning of these channels.
The degradation of hexadecane (a hydrophobic hydrocarbon) by rhodococci in the liquid and solid aggregate states is realized by several mechanisms (
Figure 6).
The utilization of liquid hydrocarbon by rhodococcal cells occurs mainly due to the production of biosurfactants in the cultivation medium and the formation of micelles that transport hexadecane to the cell. Further on the cell surface, hexadecane degradation begins under the action of membrane-bound enzymes, as evidenced by the appearance of degradation products in the medium (hexadecanoic and other fatty acids).
During the degradation of solid hydrocarbons, the hydrophobicity of the cell wall increases due to: (1) the formation of cell-bound biosurfactants; (2) the increase of the content of fatty acids in the cell wall; (3) the change in the composition of lipids in the cell wall. This study showed for the first time that during the degradation of solid hydrocarbons (namely, hexadecane) new cellular structures appeared: intracellular membrane structures and surface vesicles connected to the cell by electron-dense fibers. Electron-transparent inclusions, probably triacylglycerols, were observed in the cytoplasm. We suppose that vesicular structures play a key role in the degradation of solid hydrocarbons. The degradation of hexadecane gets started with the attachment of vesicles to it and its subsequent passive transport into these structures, where the partial degradation of hexadecane occurs due to the action of the primary enzymes of hydrocarbon oxidation. Further, the products of hydrocarbon degradation are transported to the cell through hydrophobic channels (fibers) with the participation of biosurfactants.
As we know, psychrotolerant microorganisms are extraordinary sources of new biomolecules with unique properties, which include biosurfactants. Biosurfactants play key ecological roles in many cold habitats of microbiota. They participate in carbon utilization processes by enhancing the bioavailability of poorly soluble compounds, including pollutants, in cold soils and marine environments. These properties can be used to develop new biotechnological products and processes in cold climates.