Rhodococcus Strains from the Specialized Collection of Alkanotrophs for Biodegradation of Aromatic Compounds

The ability to degrade aromatic hydrocarbons, including (i) benzene, toluene, o-xylene, naphthalene, anthracene, phenanthrene, benzo[a]anthracene, and benzo[a]pyrene; (ii) polar substituted derivatives of benzene, including phenol and aniline; (iii) N-heterocyclic compounds, including pyridine; 2-, 3-, and 4-picolines; 2- and 6-lutidine; 2- and 4-hydroxypyridines; (iv) derivatives of aromatic acids, including coumarin, of 133 Rhodococcus strains from the Regional Specialized Collection of Alkanotrophic Microorganisms was demonstrated. The minimal inhibitory concentrations of these aromatic compounds for Rhodococcus varied in a wide range from 0.2 up to 50.0 mM. o-Xylene and polycyclic aromatic hydrocarbons (PAHs) were the less-toxic and preferred aromatic growth substrates. Rhodococcus bacteria introduced into the PAH-contaminated model soil resulted in a 43% removal of PAHs at an initial concentration 1 g/kg within 213 days, which was three times higher than that in the control soil. As a result of the analysis of biodegradation genes, metabolic pathways for aromatic hydrocarbons, phenol, and nitrogen-containing aromatic compounds in Rhodococcus, proceeding through the formation of catechol as a key metabolite with its following ortho-cleavage or via the hydrogenation of aromatic rings, were verified.

Bioremediation, which exploits the metabolic properties of pollutant-degrading microorganisms for the elimination of toxic compounds in contaminated sites, is a well-known between 0.2 and 50.0 mM. Two groups of aromatic substances were clearly distinguished: relatively low toxicity (MIC = 25.0-50.0 mM) and high toxicity (MIC = 0.2-0.8 mM). Most of the tested compounds (o-xylene, all PAHs, o-phthalic acid, salicylic acid, phenol, aniline, pyridine, methyl-and hydroxy-substituted derivatives of pyridine, and coumarin) constituted the first group. The second group contained benzene, toluene, and meta-and para-isomers of phthalic acid. The toxicities of aromatic compounds from the second group were 31-250 times higher than those from the first group. Moderate toxicity (MIC = 6.3 mM) was revealed for only one compound, a two-ring N-heterocycle quinoline (Table 1).

Degradation of Monoaromatic Hydrocarbons by Rhodococcus
Strains of the most represented species (R. erythropolis/R. qingshengii, R. fascians, R. opacus, and R. ruber) were screened for their abilities to use benzene and its less toxic homolog with two methyl groups in the ortho-position, o-xylene. The growth of Rhodococcus cells in the presence of benzene and o-xylene was related to the toxicity of these compounds. Benzene was the more toxic and less preferred substrate, and Rhodococcus cells grew weaker in its presence compared to o-xylene. The percentage of strains able to utilize benzene varied from 8% to 35% and was highest among strains of R. ruber ( Figure 1). Between 2 and 11 times more strains were able to grow in the presence of o-xylene. Only R. opacus did not use o-xylene as a growth substrate, as no R. opacus strains grew in the presence of this arene ( Figure 1 and Table S2).
The analysis of the abilities of all tested strains to utilize monoaromatic hydrocarbons confirmed that o-xylene was a more preferable substrate for Rhodococcus than benzene. The 66 Rhodococcus strains from the 117 total strains analyzed grew in the presence of o-xylene, which corresponded to 56% o-xylene-degrading strains. Only 25 strains from a total of 102 grew in the presence of benzene, which corresponded to 25% benzene-degrading strains (Figure 2a). It was difficult to reveal the substrate preferences of the less represented Rhodococcus species (R. aetherivorans, R. cerastii, R. corynebacterioides, R. globerulus, R. jostii, R. pyridinivorans, R. rhodochrous, R. wratislaviensis, and R. yannanensis). However, the strains of these species were able to grow in the presence of benzene and/or o-xylene, also preferring o-xylene (Table S2).
The Rhodococcus strains were further tested for their ability to use toluene (a benzene homolog with one methyl group) as the sole carbon and energy source. This compound was four times less toxic than benzene and had the intermediate position between benzene and o-xylene in terms of its preference as a growth substrate. A total of 39% of strains grew in the presence of toluene (Figure 2a). Among the most represented Rhodococcus species, R. ruber better utilized toluene: 69% of the tested strains of R. ruber ( Figure 2c) and only 17% of R. erythropolis / R. qingshengii strains (Figure 2b) grew in the presence of this compound. The Rhodococcus strains were further tested for their ability to use toluene (a benzene homolog with one methyl group) as the sole carbon and energy source. This compound was four times less toxic than benzene and had the intermediate position between benzene and o-xylene in terms of its preference as a growth substrate. A total of 39% of strains grew in the presence of toluene (Figure 2a). Among the most represented Rhodococcus species, R. ruber better utilized toluene: 69% of the tested strains of R. ruber ( Figure 2c) and only 17% of R. erythropolis / R. qingshengii strains (Figure 2b) grew in the presence of this compound. The Rhodococcus strains were further tested for their ability to use toluene (a benzene homolog with one methyl group) as the sole carbon and energy source. This compound was four times less toxic than benzene and had the intermediate position between benzene and o-xylene in terms of its preference as a growth substrate. A total of 39% of strains grew in the presence of toluene (Figure 2a). Among the most represented Rhodococcus species, R. ruber better utilized toluene: 69% of the tested strains of R. ruber ( Figure 2c) and only 17% of R. erythropolis / R. qingshengii strains (Figure 2b) grew in the presence of this compound. Furthermore, it was found that Rhodococcus bacteria rarely utilized only toluene without the ability to oxidize other monoaromatic hydrocarbons (Figure 2). Only three R. ruber strains, IEGM 93, IEGM 232, and IEGM 234, oxidized toluene and did not oxidize benzene and o-xylene. Other Rhodococcus spp. grew in the presence of toluene only if they were able to grow in the presence of benzene and/or o-xylene (Table S2). The most promising Rhodococcus strains, namely R. aetherivorans IEGM 1250, R. erythropolis IEGM 1232, R. fascians IEGM 1233, R. ruber IEGM 1121, and R. ruber IEGM 1156, were selected, which grew well in the presence of the three tested monoaromatic hydrocarbons (Table S2).

Degradation of PAHs by Rhodococcus
The 67 Rhodococcus strains were screened for their abilities to oxidize naphthalene, the simplest polyaromatic hydrocarbon. It was shown that 41 strains (61%) grew in the presence of naphthalene. Comparing the two most represented species (R. erythropolis / R. qingshengii and R. ruber), the percentage of naphthalene-degrading strains was similar, constituting 53 and 63%, respectively. All tested R. opacus (4) and R. rhodochrous (5) strains were able to utilize naphthalene ( Figure 3). and o-xylene. Other Rhodococcus spp. grew in the presence of toluene only if they wer able to grow in the presence of benzene and/or o-xylene (Table S2). The most promising Rhodococcus strains, namely R. aetherivorans IEGM 1250, R. erythropolis IEGM 1232, R. fas cians IEGM 1233, R. ruber IEGM 1121, and R. ruber IEGM 1156, were selected, which grew well in the presence of the three tested monoaromatic hydrocarbons (Table S2).

Degradation of PAHs by Rhodococcus
The 67 Rhodococcus strains were screened for their abilities to oxidize naphthalene the simplest polyaromatic hydrocarbon. It was shown that 41 strains (61%) grew in th presence of naphthalene. Comparing the two most represented species (R. erythropolis / R qingshengii and R. ruber), the percentage of naphthalene-degrading strains was similar constituting 53 and 63%, respectively. All tested R. opacus (4) and R. rhodochrous (5) strain were able to utilize naphthalene ( Figure 3). The 20 strains growing in the presence of naphthalene were tested for their abilitie to use other PAHs. As seen from Figure 4, naphthalene-degrading strains utilized heavie PAHs with between three and five condensed aromatic rings-phenanthrene, anthracen (PAHs with three rings), benzo[a]anthracene (a PAH with four rings), and benzo[a]pyren (a heavy PAH with five rings). However, the relationship between the intensity of growth and Rhodococcus species or hydrocarbon specificity was not statistically significant. The 20 strains growing in the presence of naphthalene were tested for their abilities to use other PAHs. As seen from Figure 4, naphthalene-degrading strains utilized heavier PAHs with between three and five condensed aromatic rings-phenanthrene, anthracene (PAHs with three rings), benzo[a]anthracene (a PAH with four rings), and benzo[a]pyrene (a heavy PAH with five rings). However, the relationship between the intensity of growth and Rhodococcus species or hydrocarbon specificity was not statistically significant.

Degradation of Substituted Aromatic Hydrocarbons and Aromatic Heterocycles by Rhodococcus
Several Rhodococcus strains were tested for their abilities to use phenol and pyridine. As shown in Table 2, these substrates were poorly oxidized by Rhodococcus. Only 16% of the selected strains grew in the presence of phenol, and phenol-oxidizing strains were equally represented among Rhodococcus species. However, the percentage of phenol-degrading R. ruber strains was higher than that of phenol-degrading R. erythropolis/R. qingshengii strains, constituting 19 and 13%, respectively (

Degradation of Substituted Aromatic Hydrocarbons and Aromatic Heterocycles by Rhodococcus
Several Rhodococcus strains were tested for their abilities to use phenol and pyridin As shown in Table 2, these substrates were poorly oxidized by Rhodococcus. Only 16% o the selected strains grew in the presence of phenol, and phenol-oxidizing strains wer equally represented among Rhodococcus species. However, the percentage of phenol-d grading R. ruber strains was higher than that of phenol-degrading R. erythropolis/R qingshengii strains, constituting 19 and 13%, respectively (Table 2). Considering N-hetero cyclic pyridine, only two strains out of nine (22%), R. rhodochrous IEGM 757 and R. rub IEGM 231, were able to metabolize this compound ( Table 2, Table S2).
R. ruber IEGM 231 was tested for aromatic compounds with a nitrogen atom. Methy and hydroxy-substituted derivatives of pyridine, a two-ring N-heterocycle quinoline, an the simplest aromatic amine aniline were used as growth substrates. It was shown that R ruber IEGM 231 grew in the presence of all pyridine derivatives and aniline. However, was not able to use quinoline as the sole carbon and energy source ( Figure 5).
Additionally, the ability of R. ruber IEGM 231 to use aromatic acids and their deriv tives (salicylic acid, phthalic acid isomers, and coumarin as the lactone of o-h droxycinnamic acid) as growth substrates was estimated. It was shown that R. ruber IEGM 231 grew in the presence of coumarin but was not able to use salicylic and phthalic acid ( Figure 5).

Species
Number of Strains Total Growing Not Growing  R. ruber IEGM 231 was tested for aromatic compounds with a nitrogen atom. Methyland hydroxy-substituted derivatives of pyridine, a two-ring N-heterocycle quinoline, and the simplest aromatic amine aniline were used as growth substrates. It was shown that R. ruber IEGM 231 grew in the presence of all pyridine derivatives and aniline. However, it was not able to use quinoline as the sole carbon and energy source ( Figure 5).

Biodegradation of Toxic Aromatic Compounds by Rhodococcus in Model Soil
The bioremediation potential of Rhodococcus bacteria toward aromatic compounds was estimated in experiments with model soil contaminated with PAHs at a high (1 g/kg) concentration. Introducing Rhodococcus resulted in a 43% PAH biodegradation within 213 days, which was three times more efficient than in contaminated soil without Rhodococcus ( Figure 6). Enhanced biodegradation of PAHs was accompanied by a 2.5-10.0-fold increase in the number of hydrocarbon-oxidizing microorganisms. The highest number of hydrocarbon degraders, 2 × 10 8 colony-forming units (CFU)/g soil, was determined between 35 and 56 days. A significant increase in the bacterial number was also detected in the control soil; however, this value reached no more than 1 × 10 8 CFU/g. The number of hydrocarbon-oxidizing microorganisms started to decrease after the 56th and 70th days in bioaugmented and control soil. However, it remained at a high ((1.0 ± 0.3) × 10 8 CFU/g) level in soil with introduced Rhodococcus bacteria until the end of the experiment and reduced to almost the initial level ((3.0 ± 0.3) × 10 7 CFU/g) in the control soil after the 186 th day ( Figure 6). Additionally, the ability of R. ruber IEGM 231 to use aromatic acids and their derivatives (salicylic acid, phthalic acid isomers, and coumarin as the lactone of o-hydroxycinnamic acid) as growth substrates was estimated. It was shown that R. ruber IEGM 231 grew in the presence of coumarin but was not able to use salicylic and phthalic acids ( Figure 5).

Biodegradation of Toxic Aromatic Compounds by Rhodococcus in Model Soil
The bioremediation potential of Rhodococcus bacteria toward aromatic compounds was estimated in experiments with model soil contaminated with PAHs at a high (1 g/kg) concentration. Introducing Rhodococcus resulted in a 43% PAH biodegradation within 213 days, which was three times more efficient than in contaminated soil without Rhodococcus ( Figure 6). Enhanced biodegradation of PAHs was accompanied by a 2.5-10.0-fold increase in the number of hydrocarbon-oxidizing microorganisms. The highest number of hydrocarbon degraders, 2 × 10 8 colony-forming units (CFU)/g soil, was determined between 35 and 56 days. A significant increase in the bacterial number was also detected in the control soil; however, this value reached no more than 1 × 10 8 CFU/g. The number of hydrocarbon-oxidizing microorganisms started to decrease after the 56th and 70th days in bioaugmented and control soil. However, it remained at a high ((1.0 ± 0.3) × 10 8 CFU/g) level in soil with introduced Rhodococcus bacteria until the end of the experiment and reduced to almost the initial level ((3.0 ± 0.3) × 10 7 CFU/g) in the control soil after the 186th day ( Figure 6).
In contrast to the dynamics of bacterial cell numbers, the PAH concentration dynamics followed a linear trend, thus demonstrating stable hydrocarbon activity in bioaugmented soil for all 213 days. In the control soil, a slow linear decline in PAH concentration was also revealed, but it did not change significantly after the 70th day ( Figure 6). The removal of PAHs both in the soil with introduced Rhodococcus cells and control was related to a decrease in the concentrations of individual, mainly light PAHs-naphthalene and acenaphthene. These hydrocarbons were evaporated and easily biodegraded because of their relatively high water solubility compared to other PAHs [34,39].

Analysis of Rhodococcus Genes Involved in the Biodegradation of Aromatic Compounds
Various oxidoreductases and hydrolases were revealed in sequenced and annotated genomes of studied Rhodococcus strains. The total numbers of oxidizing enzymes, such as dioxygenases, monooxygenases, multicopper polyphenol oxidases, dehydrogenases, and  (Table 3 and Table S3). In contrast to the dynamics of bacterial cell numbers, the PAH concentration dynamics followed a linear trend, thus demonstrating stable hydrocarbon activity in bioaugmented soil for all 213 days. In the control soil, a slow linear decline in PAH concentration was also revealed, but it did not change significantly after the 70 th day ( Figure 6). The removal of PAHs both in the soil with introduced Rhodococcus cells and control was related to a decrease in the concentrations of individual, mainly light PAHs-naphthalene and acenaphthene. These hydrocarbons were evaporated and easily biodegraded because of their relatively high water solubility compared to other PAHs [34,39].

Discussion
Based on the metabolic abilities of 133 Rhodococcus strains from the Regional Specialized Collection of Alkanotrophic Microorganisms, common regularities of aromatic compound biodegradation by Rhodococcus bacteria were revealed and discussed in terms of the toxicity and putative functional genes. According to the preference for Rhodococcus cells, the studied aromatic substrates can be arranged (from more to less preferrable) as PAHs (61% of strains used naphthalene, and all tested naphthalene-degrading strains grew in the presence of PAHs with between three and five aromatic rings) > o-xylene (56% of strains used) > toluene (39% of strains used) > benzene (25% of strains used) > pyridine (22% of strains used) > phenol (16% of strains used). This preference row reflected the toxicity of aromatic hydrocarbons (Table 1), which increased from PAHs to benzene. Interestingly, all PAHs used, regardless of the number of aromatic rings, molecular weight, and even water solubility [33], were equally preferred for Rhodococcus cells (Figure 4). A similar effect was found earlier for Rhodococcus, and it depended on the strain specificities and cell adhesive activities toward PAH crystals [34]. However, the polar-substituted derivatives of benzene, pyridine, and phenol did not fully correspond to the revealed dependence. These less-toxic compounds were degraded more poorly compared to highly toxic benzene and toluene, which could be explained by the specific enzymatic mechanisms of their degradation.
It was expected that this study would reveal a certain species specificity of the metabolic profiles (spectra of oxidized aromatic compounds) of the studied Rhodococcus strains. However, no strict correlation between species and the ability of Rhodococcus strains to metabolize specific aromatic substrates was detected. Strains able to grow in the presence of benzene, toluene, o-xylene, naphthalene, pyridine, or phenol and not using these compounds were found among different Rhodococcus species. Similarly, strains of R. erythropolis/R. qingshegii, R. opacus, R. rhodochrous, and R. ruber species grew in the presence of PAHs ( Figure 4). Nevertheless, some species specificities were revealed: (i) R. opacus did not use o-xylene, and (ii) R. ruber somewhat better-metabolized aromatic compounds, especially in comparison with the most represented R. erythropolis/R. qingshegii. The latter was confirmed by the highest percentage of R. ruber strains growing in the presence of benzene and the 1.5 and 4.0 higher percentage of strains oxidizing phenol and toluene in comparison with R. erythropolis/R. qingshengii. Moreover, the ability of R. ruber to oxidize pyridine (pyridine-degrading strains were not found among R. erythropolis/R. qingshegii strains) and the ability of the reference strain R. ruber IEGM 231 to oxidize most of the tested aromatic pollutants indicate a high biodegradation potential of this Rhodococcus species. The metabolic potential of R. rhodochrous was close to that of R. ruber as all tested R. rhodochrous strains grew in the presence of PAHs and phenol, and R. rhodochrous IEGM 757 was able to use pyridine. Possible explanations for the inability of R. opacus to use o-xylene, a relatively less toxic compound, are the absence of specific oxidizing enzymes, the low affinity of benzene/toluene-degrading enzymes to o-xylene, or the lack of specific transporting systems. In fact, R. opacus poorly metabolized monoaromatic hydrocarbons. Even considering benzene and toluene, only three strains of this species grew in the presence of these compounds (Table S2). Genes annotated for benzene/toluene monooxygenases or dioxygenases were not found in the genome of R. opacus IEGM 249 (Table S3), which did not utilize monoaromatic hydrocarbons (Table S2). It should be noted, however, that benzene/toluene mono-and dioxygenases were not detected in the most studied Rhodococcus genomes (Table S3). Nevertheless, R. opacus was recognized as a promising degrader of toxic organic pollutants as the highest numbers of oxidoreductases were revealed in the genome of R. opacus IEGM 249 (Table 3).
It was hypothesized that microorganisms from anthropogenically disturbed and contaminated ecosystems had developed diverse metabolic capacities and multiple resistance to stress factors [27,28]. Among the selected 14 most active biodegraders, 7 strains were isolated from pristine environments, and the other 7 were isolated from disturbed sites, either polluted or located near pollution sources (Table S1). In fact, the impact of the isolation source on the biodegradation abilities of Rhodococcus was difficult to identify. For example, when strains were isolated within the city, analysis suggested some level of chemical contamination, even if no pollutants were defined and the sites were assumed to be pristine.
Special attention in this study was paid to the analysis of putative biodegradation genes. For this, 10 sequenced genomes of Rhodococcus strains from the IEGM Collection were annotated and searched for relevant functional genes. The obtained results proposed enzymatic mechanisms of aromatic compound biodegradation and predicted metabolic activities of particular Rhodococcus species. Catechol dioxygenases, key enzymes of arene biodegradation pathways, were found in all of the studied genomes of IEGM strains. This confirmed the metabolic ability of Rhodococcus to degrade molecules with aromatic structures as well as the preferred pathway for further catechol degradation through ortho-cleavage (Table 3). Considering the genes of the initial steps of aromatic biodegradation, they could be assumed as follows. One benzene 1,2-dioxygenase and three phenol monooxygenases were revealed in the R. ruber IEGM 231 genome, and phenol hydrolases (one gene in each strain) were revealed in the genomes of R. opacus IEGM 249 and R. pyridinivorans IEGM 1137 (Table 3). These enzymes can provide primary oxidation of BTEX substrates (benzene, toluene, and o-xylene) and phenol, respectively. The highest number of specific enzymes in Rhodococcus genomes was revealed for the degradation of pyridine, its derivatives, and coumarin. The ability of R. ruber IEGM 231 to grow in the presence of 2-and 4-hydroxypyridines verified that rhodococci metabolized pyridine completely as these compounds were described as frequent end products in pyridine biodegradation [24]. In four Rhodococcus genomes, a gene coded for salicylate 1-hydroxylase, presumably responsible for salicylic acid assimilation, was revealed. This gene was not found in the R. ruber IEGM 231 genome (Table 3), which is a possible reason for the inability of this strain to metabolize salicylate ( Figure 5). Interestingly, salicylate 5-hydroxylases were not found in the sequenced genomes (Table S3). Salicylic acid, along with phthalic acids, is known as an intermediate in PAH biodegradation [43,44]. The revealed genes indicated that the utilization of PAHs by Rhodococcus cells is performed, apparently, via catechol, but not the gentisate pathway [18].
It was surprising that no genes recognized as naphthalene or other PAH dioxygenases were detected in Rhodococcus genomes. Naphthalene dioxygenases were previously described for Rhodococcus spp. in relation to their structural, functional, and thermostability features [43,[45][46][47]. The explanation could be that dioxygenases belong to a diverse class of Rieske non-heme iron oxygenases with a broad substrate specificity. They include, among others, phthalate, biphenyl, benzoate, toluene dioxygenases, and some monooxygenases [45,48]. Genes encoding dioxygenases can be annotated as extradiol dioxygenases, unspecific dioxygenases, or unspecific oxidases/oxidoreductases. The low (29-33%) similarity of naphthalene dioxygenase and, in contrast, high (77-81%) similarity of nitrobenzene dioxygenase subunits NarAa and NarAb from Rhodococcus sp. strain NCIMB12038 with the corresponding subunits from Pseudomonas [45] demonstrated a high diversity of naphthalene enzymes and possible misleading annotations. More detailed analysis of dioxygenase genes using multiple alignments and experimental confirmation of gene functions are required. Moreover, naphthalene-degrading operons can be located on plasmids, the absence of which may explain the inability of some Rhodococcus strains to grow in the presence of this compound.
The variety of oxidizing enzymes with a broad substrate specificity found in Rhodococcus genomes determines the versatile and widely varying catabolic abilities of the studied strains. Hydrolases seemed to be no less important enzymes for the biodegradation of aromatic compounds by Rhodococcus bacteria than dioxygenases. Numerous genes coded for hydrolases (from 96 up to 229) were found in all studied Rhodococcus genomes (Table 3). They can participate in the degradation of PAH molecules [30]. Among hydrolases, formamidases and other amidases were detected. These enzymes, along with maleamate amidohydrolase found in the genome of R. ruber IEGM 231 (Table S3), can participate in the biodegradation of pyridine and its derivatives [9,18]. In addition to dioxygenases and hydrolases, putative aromatic biodegradation enzymes also included monooxygenases, especially cytochrome P450 oxidases, and multicopper polyphenol oxidases, or laccases. The latter can cause the oxidation of PAHs, phenolics, and other toxic pollutants [49,50], thereby allowing the growth of Rhodococcus in the presence of polyaromatics and phenol, even if specific enzymes are not annotated. Multicopper polyphenol oxidases were found in all the studied genomes of Rhodococcus spp., mainly in one copy, while four genes were detected in R. ruber IEGM 231 (Table 3).
Some contradictory data were obtained for the biodegradation of phthalic acids aniline and quinoline. The reference strain R. ruber IEGM 231 did not grow in the presence of phthalic acid isomers, which was in agreement with the genome annotation: no genes coded for phthalic acid biodegradation enzymes were found in the genome of this strain or in the genomes of other Rhodococcus strains. Only one phthalate 3,4-dioxygenase gene was recognized in the genome of R. opacus IEGM 249 (Table S3). However, it is known that Rhodococcus bacteria can degrade phthalates and their esters, and phthalate 3,4-dioxygenases coded by the pht genes participate in these metabolic processes [31,32,41]. Phthalate operons are located on plasmids [32,41], and the lack of plasmids can explain the inability of the IEGM 231 strain to metabolize phthalic acids. Another substrate, which was not used by R. ruber IEGM 231, was quinoline. In the genome of this strain, as well as in other Rhodococcus genomes, genes coding for nitrite and nitrate reductases were revealed (Table S3). These enzymes could perform the degradation of quinoline via the 8-hydroxycoumarin pathway [20,23]. The inability of IEGM 231 to use quinoline can be due to the lack of other key enzymes or transporting proteins, but apparently not to quinoline toxicity, which was lower than benzene and toluene were. In contrast, R. ruber IEGM 231 grew in the presence of aniline, but no specific genes coded for the enzymes of the first steps of aniline biodegradation, such as aniline, 2-aminophenol 1,6-, indole 3-acetate dioxygenases, 6-hydroxynicotinate 3-monooxygenases, and 2-aminobenzoyl-CoA monooxygenases/reductases, were found in the genome of this strain. Enzymes with a broad substrate specificity (dioxygenases, for example) or enzymes of putative pyridine oxidation pathways (anthranilate 1,2-dioxygenase, amidases, or maleamate amidohydrolase) can participate in the degradation of aniline by R. ruber IEGM 231.
The metabolic properties of Rhodococcus strains from the IEGM Collection toward aromatic compounds were tested against heavy (1 g/kg) PAH contamination in a model soil. The obtained results showed the suitability of introducing Rhodococcus to remove aromatic hydrocarbons from soil. The percentage of removed hydrocarbons was three times higher in soil with introduced Rhodococcus bacteria compared to control soil and constituted 43% PAH removal over 213 days ( Figure 6). To improve biodegradation, Rhodococcus cells were immobilized onto a sawdust-based carrier [51]. Immobilized cells maintained high PAH-oxidizing activity throughout the experiment. This was seen from the constant linear decline in PAH concentration, even after the total number of hydrocarbon-oxidizing microorganisms was decreased. The growth of PAH degraders within the first 35 days was seemingly related to easily biodegradable aromatic components, such as light PAHs or PAHs not sorbed on solid particles ( Figure 6). After the depletion of these components, the abundance of hydrocarbon-oxidizing bacteria decreased, and the process of PAH biodegradation stopped in the control but continued in soil with introduced Rhodococcus cells. To further demonstrate the metabolic capabilities of Rhodococcus bacteria toward aromatic substances, their high oxidizing activities toward complex aromatic compounds, such as (RS)-2-(4-(2-methylpropyl)phenyl)propanoic and [2-(2,6-dichloroanilino)phenyl]acetic acids, should be mentioned (Table 4). These complex aromatic acids were acting substances in non-steroidal anti-inflammatory drugs, and Rhodococcus strains from the IEGM Collection degraded 100% of these compounds at high concentrations within 6 days (Table 4).
Rhodococcus cells were recovered from lyophilized cultures and then grown in Erlenmeyer flasks containing 100 mL of Luria-Bertani broth (LB) (Sigma-Aldrich, Burlington, VT, USA) on an orbital shaker (160 rpm) at 28 • C for 28-30 h. Cells were washed twice and resuspended in 0.5% NaCl to OD 600 nm = 1.0 (1 × 10 8 CFU/mL). The use of standardly prepared cell suspensions provided the same initial conditions in all experiments.

Tested Aromatic Compounds
The metabolic abilities of Rhodococcus strains were estimated toward 23 aromatic compounds, including 3 monocyclic aromatic hydrocarbons, 5 PAHs, 1 hydroxy substituted benzene, 1 aromatic amine, 8 N-heterocyclic aromatic compounds, 4 aromatic acids, and 1 aromatic lactone. These compounds are listed in Table 5. All substances were dissolved in water or in polar solvents, such as dimethylsulfoxide (DMSO), acetone, or 70% ethanol (Table 5) at a concentration of 600 mM. Sometimes, the solutions were heated in a water bath at 70 • C for better dissolution ( Table 5). The aromatic compounds and organic solvents had ≥97% purity and were purchased from Sigma-Aldrich. Pre-sterilized glass-and plasticware were used for the preparation of aromatic compound solutions, and water solutions of pyridine and its derivatives were sterilized by filtering through nitrocellulose filters with 0.22 µm pores (Merck Millipore, Burlington, VT, USA).

Toxicity Tests
Minimal inhibitory concentrations (MICs) of the used aromatic compounds were determined for the reference strain R. ruber IEGM 231. In polystyrene 96-well microplates (Medpolymer, St Petersburg, Russia), 150 µL of sterile LB was mixed with 30 µL working solutions of aromatic compounds to obtain a starting concentration of 100 mM. Then, a series of two-fold dilutions in the range of concentrations between 0.05 and 100.00 mM were prepared. Microplates were stored at room temperature for 24 h to allow the evaporation of solvents. Measures of 10 µL of a standard R. ruber IEGM 231 cell suspension were added to microplates to obtain a final concentration of 1 × 10 7 CFU/mL. Inoculated microplates were incubated in a Titramax 1000 incubator (Heidolph Instruments, Schwabach, Germany) at 600 min −1 and 28 • C for 72 h. The viability of cells was estimated using staining with iodonitrotetrazolium violet (INT) purchased from Sigma-Aldrich. For this, measures of 45 µL of 0.2% (w/w) INT solution in water were added to microplates. The appearance of a red-violet color after 2 h of staining was evidence of the presence of viable respirating cells [55]. LB with R. ruber IEGM 231 cells without aromatic substrates was the biotic control. LB with cells and solvents without dissolved aromatic compounds were used to estimate the influence of solvents on the growth of Rhodococcus. LBs with aromatic compounds without R. ruber IEGM 231 cells were used as abiotic controls.

Growth Experiments
To estimate the abilities of Rhodococcus strains to use benzene, toluene, o-xylene, naphthalene, pyridine, and phenol as growth substrates, bacterial cells were grown on mineral agar K containing (g/L): KH 2 PO 4 -1.0, K 2 HPO 4 -1.0, NaCl-1.0, KNO 3 -1.0, MgSO 4 -0.2, FeCl 3 -0.02, CaCl 2 -0.02, and agar-15.0 (http://www.iegmcol.ru/medium/ med08.htmL, accessed on 2 March 2023). The 200 µL diluted Rhodococcus cell suspensions with a concentration of 1·10 7 CFU/mL were used for the inoculation of Petri dishes with agar K. Suspensions were evenly distributed on the agar surface with a spatula to obtain a bacterial lawn when growing. Liquid substrates (monoaromatic hydrocarbons, pyridine, and phenol) at a volume of 200 µL were added to the slot with a diameter of 1 cm made in the center of the Petri dishes with agar K. A measure of 200 mg of naphthalene was placed under each lid of the inoculated Petri dishes, and the dishes were inverted. All inoculated Petri dishes were incubated at 28 • C for 5 days.
The used salts and D-glucose had ≥97% purity and were purchased from Sigma-Aldrich, and the agar was ultrapure BD Difco TM , purchased from Thermo Fisher Scientific (Waltham, MA, USA). Uninoculated mineral medium supplemented with the studied aromatic compounds was used as an abiotic control; LB agar or the medium RS with 25 mM D-glucose inoculated with Rhodococcus bacteria was the biotic control; mineral medium without aromatic substrates inoculated with Rhodococcus bacteria was the control for oligotrophic growth. The usage of ultrapure Difco agar guaranteed that no residual growth was detected as a result of the consumption of trace concentrations of organic molecules by Rhodococcus [26,27,35]. Only the results of growth experiments with the confirmed growth of Rhodococcus cells in the biotic control and the absence of oligotrophic growth are shown and discussed in this work.

Model Soil Experiments
The model soil consisted of 50% sand, 30% clay, and 20% clean garden soil. All components of the soil mixture were first dried at 80 • C and then screened using a 1 mm mesh. The soil was inoculated with acetone-dissolved PAHs. Individual PAHs were present in the following concentrations (g/kg dry soil): naphthalene-0.2, acenaphthene (>97% purity, Sigma-Aldrich)-0.2, anthracene-0.2, phenanthrene-0.2, benzo[a]anthracene-0.1, and benzo[a]pyrene-0.1. Biodegradation of the PAH mixture was performed in flat trays containing 600 g of soil. The R. erythropolis IEGM 708 and R. ruber IEGM 327 strains [56] were used in this experiment in equal amounts. To stabilize the metabolic activity of Rhodococcus cells for an extended period, they were immobilized onto pine sawdust hydrophobized using 5% Rhodococcus surfactant, as described previously [51]. The catalytic activity of immobilized cells was measured as 98 ± 12 mg of degraded naphthalene/(L·h). The obtained biocatalyst was introduced into the contaminated soil at a 1:6 ratio (w/w); the amount of the inoculum was 4 × 10 8 cells/g of soil. The soil was mixed and moistened regularly to maintain 20% humidity. The experiment was performed at room temperature for 213 days. Contaminated soil without Rhodococcus cells introduced was used as a control.
Residual hydrocarbons were determined by gas chromatography with mass spectrometry after extraction with chloroform (GC-MS grade). An Agilent 6890 N chromatograph equipped with an Agilent MSD 5973 N quadrupole detector (Agilent Technologies, Santa-Clara, CA, USA) was used. A volume of 1 µL of each extract was introduced into an injection port held at 250 • C. The initial oven temperature was 40 • C for 5 min followed by a heating rate of 12 • C/min up to 300 • C, and held at this temperature for 10 min. Separation was achieved using a 30 m HP-5MS column with an internal diameter of 0.25 mm and film thickness of 0.25 M (Agilent Technologies, Santa-Clara, CA, USA) maintained at a constant flow of 1 mL/min of helium. The number of hydrocarbon-oxidizing microorganisms was counted as the number of colony-forming units grown on agar K with naphthalene. , and R. ruber IEGM 231 (CCSD01000001-CCSD01000115) were used to search for functional genes coded for enzymes participating in biodegradation of aromatic compounds. The annotation of coding sequences in the genomes was performed using the RAST 2.0 annotation scheme RASTtk [57].

Bioinformatical and Statistical Analysis
All experiments were performed in 3-8 replicates. Statistical analysis including the determination of the data type distribution and calculation of means ± standard deviations was performed using Statistica (data analysis software system) version 13, TIBCO Software Inc. (2018). Differences were considered statistically significant at p < 0.05.

Conclusions
The results presented in this work were obtained in the course of long-term and comprehensive studies of the abilities of Rhodococcus bacteria to metabolize aromatic compounds. They are part of a larger study of the biological and functional properties of this group of bacteria performed using the bioresources of the Regional Specialized Collection of Alkanotrophic Microorganisms. Rhodococcus strains deposited in the collection were isolated from various pristine and anthropogenically disturbed ecosystems, identified and well characterized. Moreover, the collection is constantly updated with freshly isolated cultures. In this work, the metabolic properties of the 133 collection strains belonging to various species of the genus Rhodococcus were determined. We assume that the obtained results were representative of the whole genus. No strict correlations were revealed between the abilities of Rhodococcus isolates to oxidize aromatic compounds, spectra of oxidized aromatic substances, and species. However, R. rhodochrous and R. ruber somewhat better oxidized various aromatic compounds, and R. opacus poorly metabolized monoaromatic hydrocarbons and did not use o-xylene. No dependence was revealed between the metabolic abilities of the studied strains toward aromatic compounds and the source of strain isolation. The toxicity of aromatic substances was not the only factor determining their bio-oxidation. The diversity of genes coded for degrading enzymes with a broad substrate specificity (mono-and dioxygenases, laccases, and hydrolases) was considered to be the basis for the versatile and widely varying catabolic abilities of Rhodococcus bacteria. At the same time, the analysis of functional annotations of 10 Rhodococcus genomes revealed that there is still no sufficient information on the enzyme mechanisms of aromatic compound degradation by Rhodococcus. Individual Rhodococcus strains may have no specific genes and be unable to metabolize some aromatics, even if these activities have previously been shown for other members of the genus. In this study, metabolic pathways for monoand polyaromatic hydrocarbons, phenol, and nitrogen-containing aromatic compounds in Rhodococcus, proceeding through the formation of catechol as a key metabolite with its following ortho-cleavage or via the hydrogenation of aromatic rings, were verified. However, further experimental verification of particular gene functions is required.
Information on the IEGM Collection Rhodococcus strains can be used on an operational basis to construct biopreparations for emergency spills of ecopollutants, including aromatic pollutants, alone or simultaneously presented in the biotope with other xenobiotics. The developed biopreparations can be used in the bioremediation of soils contaminated with aromatics and requested by companies working in the area of ecobiotechnology. In this work, some promising biodegraders were selected, which metabolized a high diversity of aromatic pollutants and degraded the most recalcitrant substances. In model experiments, the high efficiency of the introduction of immobilized Rhodococcus cells for the remediation of contaminated soil was shown, which led to the 43% removal of PAHs for 213 days at an initial PAH concentration of 1 g/kg.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules28052393/s1, Table S1: Rhodococcus strains (http:// www.iegmcol.ru, accessed on 2 March 2023); Table S2: Growth of Rhodococcus strains in the presence of aromatic compounds; Table S3: Numbers of genes coded for putative enzymes of aromatic compound biodegradation by Rhodococcus spp. (a large set of genes is presented).