Shvarov R.V.
MICROBIAL COMMUNITIES OF WOODY PLANTS: MODERN APPROACHES TO BIODEGRADATION OF HYDROCARBON POLLUTANTS *
Аннотация:
woody plants form unique communities with microorganisms that can effectively destroy hydrocarbons both in the air and in the soil. Microbes living on the surface and inside plants produce special enzymes that accelerate the decomposition of pollutants. Laboratory and field studies show that the combined use of epiphytic and endophytic microorganisms can significantly increase the efficiency of biological purification of the environment. Synthetic microbial consortia and genetic engineering methods that allow you to customize the composition of the microbiota for different conditions are considered especially promising. All this opens up new opportunities for creating sustainable and environmentally friendly technologies for the rehabilitation of contaminated areas.
Ключевые слова:
phytoremediation, tree microbiota, epiphytic microorganisms, endophytic microorganisms, hydrocarbon degradation, bioremediation, synthetic microbial consortia, biosurfactants
DOI 10.24412/2712-8849-2025-687-2188-2205
INTRODUCTION. Woody plants are extensive biological interfaces where diverse microbial communities interact with air and soil pollutants. According to J.A. Vorholt [1], the leaf surface (phyllosphere) and plant tissues (endosphere) are home to microorganisms that can effectively degrade volatile organic compounds, polycyclic aromatic hydrocarbons, and petroleum fractions [2, 3]. Due to the increasing pollution of the urban environment and soils as a result of man-made accidents and oil spills, the relevance of studying such communities is significantly increasing.L. Molina et al. [4] note that plant-associated microbiota is a promising ecological and cost-effective addition to traditional physicochemical cleaning methods. Metagenomic studies by C. Sánchez-Cañizares and his group [6, 7] revealed a high concentration of genes encoding alkane monooxygenases, dioxygenases, and ligninolytic enzymes in microbial communities associated with the tree species Quercus ilex, Platanus × acerifolia, and Carpinus betulus.O.S. Sazonova and her colleagues [2, 8] successfully isolated and studied epiphytic bacteria of the genera Pseudomonas, Rhodococcus, and Sphingomonas, capable of degrading PAHs in laboratory conditions with an efficiency of 70–90%. At the same time, endophytic microorganisms, including fungi (Xylaria, Verticillium) and bacteria (Streptomyces, Bacillus), demonstrate a high degree of degradation of petroleum hydrocarbons, reaching more than 90% [5, 10, 12]. The works of T. Barac and his colleagues [12, 13] show that microbial engineering by introducing catabolic plasmids can significantly enhance these processes.This review aims to solve the following problems:Comparison of the diversity of epiphytic and endophytic microorganisms of woody plants involved in hydrocarbon degradation,Analysis of methods for their isolation and functional characterization (from cultural methods to metagenomics and plasmid engineering),Consideration of the main biochemical pathways of hydrocarbon degradation (alkane monooxygenation, dioxygenation of aromatic rings, ligninolytic oxidation),Assessment of the applied potential of these microbial communities in phylloremediation, rhizosphere phytoremediation and bioaugmentation.Thus, a comprehensive approach to the study of plant-associated microbial communities will significantly expand the possibilities for biotechnological purification of polluted air and soil environments.1. Diversity of epiphytic microbial communities on the leaves of woody plants.The phyllosphere of woody plants is a unique environment that creates conditions for diverse microbial communities. J.A. Vorholt [1] indicates that the diversity of leaf microbiota depends on the morphology of the leaf itself, the species of the plant, and environmental factors. In studies conducted by O.S. Sazonova and S.L. Sokolov [2], it is noted that among epiphytic bacteria, representatives of Actinobacteria (for example, the genus Rhodococcus), Proteobacteria (for example, the genera Pseudomonas and Sphingomonas) and Bacteroidetes predominate. Along with bacteria, leaves are also populated by representatives of the fungal division Ascomycota (genus Aureobasidium, Phoma), as well as yeast and archaea [3, 31].V. Imperato et al. [3] found fungal communities with a pronounced ability to degrade aromatic hydrocarbons on hornbeam (Carpinus sp.) leaves, which were effective even under chronic pollution conditions. O.S. Sazonova et al. [2] reported that in urban conditions, more than 150 strains of microorganisms were isolated on Tilia, Acer and Fraxinus leaves, which were capable of effectively degrading compounds such as phenanthrene, naphthalene and salicylate. Seasonal studies by D. Al-Mailem et al. [7] show that in summer, when the concentration of pollutants in the air increases, a significant increase in the number of alkane- and PAH-degrading microorganisms is observed on oak and maple leaves.According to M.J. Stevens and his research group [9], the urban environment significantly alters the composition of bacterial communities in the phyllosphere, leading to an increase in the number of hydrocarbon-degrading bacteria, which positively affects the potential for phylloremediation. Thus, understanding the factors that shape microbial communities on the leaves of woody plants is important for the development of effective biotechnologies for environmental cleanup.2. Methods for Isolation and Characterization of Epiphytic Degrader Microorganisms.To understand how exactly epiphytic microorganisms on tree leaves cope with hydrocarbons, researchers use a variety of methods for their isolation and study. Z. Shao [15] describes how such bacteria are usually isolated: microorganisms are washed off the leaves or smears are taken directly from the surface, and then the resulting material is placed in special nutrient media with the addition of hydrocarbons (for example, alkanes from C₁₀ to C₂₀ or phenanthrene). This helps to isolate precisely those microorganisms that are able to use these compounds as the only source of nutrition.After this, the cultures are transferred to selective agar media, where individual colonies of microorganisms can already be distinguished. As T.Y. Mujahid et al. [16] identified the most promising strains using methods such as colony clear zone tests (an indicator of hydrocarbon degradation) or precise measurements of substrate residue concentrations using gas chromatography with mass spectrometry (GC-MS).According to L. Bernard and his group [35], additional characterization of isolated bacteria usually includes standard procedures such as Gram staining and biochemical tests (e.g. for the presence of catalase or oxidase). To accurately identify the species or strain of bacteria the researchers are dealing with, sequencing of the 16S rRNA gene is used. According to Bernard and his team, special attention is also paid to the ability of microorganisms to produce biosurfactants. To identify this ability, simple and informative tests such as the “drop fall” method or the oil film spreading test are used.Such methods allow us to identify the most promising strains of microorganisms that can be effectively used in future technologies for bioremediation of urban environments and contaminated areas.3. Metagenomic and functional genomic approaches to studying the phyllosphere.Scientists are increasingly moving away from traditional cultural methods and turning to modern genomic technologies to better understand the capabilities of microbial communities on plant leaves. For example, D. Garrido-Sanz and his team [18] note that shotgun metagenomics methods - when all the DNA from the leaf surface is analyzed - allow us to see the full set of genes of the microbial community without even isolating the microorganisms in pure culture.Studies conducted by C. Sánchez-Cañizares and his co-authors [6, 17] showed that the phyllosphere of plants such as Quercus ilex and Platanus × acerifolia is rich in genes responsible for the breakdown of hydrocarbons: alkane monooxygenase (alkB), aromatic dioxygenase (ndoB) and protocatechuate dioxygenase. These genes were most often found in bacteria from the Actinobacteria and Proteobacteria groups.In addition, metatranscriptomics made it possible not only to detect these genes, but also to prove their actual work directly on plant leaves. L. Wang and colleagues [17] found that when exposed to PAHs, microbes increased the expression (activity) of key catabolic pathways, thus confirming that they are indeed actively working in natural conditions.Functional metagenomics, described by E.K. Mitter and J.J. Germida [19], offers even more possibilities. Scientists clone fragments of DNA taken from the environment and place them in laboratory cells, which allows them to discover new enzymes and biosurfactant genes. Thus, it is possible to expand the arsenal of tools for cleaning the environment, using the hidden capabilities of microbes that are difficult or impossible to cultivate using traditional methods.These new approaches provide a more complete and realistic picture of how plant microbiota can help us solve environmental challenges.4. Diversity of endophytic hydrocarbon-degrading microorganisms.Unlike epiphytes, endophytes live inside the plants themselves — in the roots, stems, and leaves, which gives them some important advantages. Interestingly, many researchers, such as P. García-Fraile and his colleagues [20], note that it is in polluted areas that endophytic bacteria are more likely to have genes responsible for hydrocarbon degradation, such as alkB (alkane monooxygenases) and PAH dioxygenases. This indicates that plants specifically “select” microorganisms that can help them cope with pollution.Among the most common endophytic bacteria with such abilities are the genera Pseudomonas, Bacillus, Stenotrophomonas, Burkholderia, and Arthrobacter [8, 19]. Moreover, as F. Rodríguez [5] notes, some of them demonstrate surprisingly high efficiency: more than 90% of petroleum hydrocarbons can be removed by endophytes in plant tissues in just a month.Not only bacteria play an important role in this process. Endophytic fungi such as Xylaria, Verticillium, Aspergillus and Nigrospora are also very active. According to H. Baoune and his group [11], these fungi produce laccase and peroxidase enzymes, which help them decompose complex petroleum compounds. In addition, they secrete biosurfactants, due to which hydrocarbons more easily penetrate into plant tissues and become available for destruction. It is these fungi that sometimes show fantastic results, destroying up to 99% of oil pollution.It is also interesting that endophytic bacteria of the genus Streptomyces, studied by the team of H. Baoune [11], are not inferior to fungi and can destroy both alkanes and aromatic compounds, which indicates their high versatility.Combined approaches, when several types of endophytes work together, provide deep and effective cleaning of plants and the surrounding soil from harmful contaminants, seem very promising.5. Endophyte Isolation and Screening Methods.In order to effectively use endophytes to clean up the environment, it is important to first isolate them correctly and select the most promising strains. Usually, a fairly strict procedure is used to exclude epiphytes. As described in detail by T. Barac and his colleagues [12], plant samples (roots, stems or leaves) are first carefully sterilized with 70% alcohol and sodium hypochlorite solution. This is necessary to remove all microorganisms from the surface, leaving only those that live inside the plant.Then the plant tissues are crushed and placed in special nutrient media, to which various hydrocarbons, such as hexadecane, toluene or even parts of crude oil, are added in advance. Interestingly, according to H. Baoune [11], such enriched media make it possible to identify precisely those microorganisms that are able to feed on these complex compounds.The isolated cultures are reseeded several times to increase the number of active degraders, after which pure cultures are isolated, which are then tested for their “abilities”. High-precision methods are often used here: gas chromatography with mass spectrometry (GC-MS) or flame ionization detector (GC-FID) to understand how effectively microorganisms destroy pollutants.It is also very useful to conduct simple colorimetric tests that show the activity of important enzymes: laccases, peroxidases and monooxygenases. These tests allow you to quickly and easily select strains with the greatest potential [11, 16].Another important approach, which O. Yagi’s team talks about [30], is the search for specific genes associated with hydrocarbon degradation (e.g., alkB, nahA, nidA). They are detected by PCR, which allows you to immediately select the most promising “candidates” for further testing in the greenhouse and field conditions.Thus, the combination of precise laboratory methods and simple screening tests allows us to quickly and efficiently select those endophytes that can then help us deal with contaminants in practice.6. Biochemical mechanisms: alkane monooxygenation.The mechanisms by which microorganisms break down hydrocarbons are truly impressive. One of the most important pathways is alkane monooxygenation. As explained by W. Zhang and his colleagues [23], enzymes of the AlkB group insert an oxygen atom into alkanes, converting them into primary alcohols, which are then easily absorbed by microorganisms.It is very interesting that the active center of this enzyme includes two iron atoms that are not associated with heme (the so-called non-heme di-iron cluster). Research conducted by Z. Jia and his group [24] showed that the structure of this enzyme contains special amino acids - histidine and glutamate - they coordinate iron and determine which hydrocarbons will be broken down.It is also important that the genes encoding AlkB enzymes are often found in the genome adjacent to genes encoding proteins that ensure efficient electron transfer. According to J.B. van Beilen and Z. Li [25], this makes the entire process more efficient. There are also alternative enzymes, such as propane monooxygenase, which allows microorganisms to process even short-chain hydrocarbons (C₂–C₄). This further increases their potential for environmental purification.7. Biochemical mechanisms: dioxygenation of aromatic rings and PAH degradation.Another important degradation pathway is the dioxygenation of aromatic compounds. As described in detail by J. Kim and his team [28], this process begins with special enzymes, aromatic dioxygenases, inserting two oxygen atoms into the aromatic ring of PAH at once. As a result, an intermediate compound, cis-dihydrodiol, is formed, which is then converted to catechol. Catechols can be further broken down, and the resulting substances are easily included in the general metabolism of microorganisms [29, 30].It is interesting that such dioxygenases have a special structure: they contain an iron-sulfur cluster of the Rieske type ([2Fe–2S]) and another separate iron atom. According to O. Kweon and his colleagues [29], the structure of the active center of the enzyme clearly determines which PAHs it can destroy. This is why different enzymes can break down different types of polycyclic hydrocarbons - from the simplest to the most complex.The combined use of bacterial and fungal dioxygenases seems very promising, since fungi have a wider range of activity and can effectively complement bacteria in mixed microbial consortia.8. Enzymatic oxidation involving laccases and peroxidases.Another interesting mechanism by which microbes can cope with hydrocarbons is associated with the enzymes laccases and peroxidases. V. Imperato and colleagues [31] described how these enzymes work: laccases, which are present in fungi and some bacteria, use copper to oxidize complex aromatic compounds, creating radical forms of molecules that are then easily broken down.White putrefactive fungi, such as those studied by H. Singh and his group [33], produce lignin and manganese peroxidases, which are able to oxidize even very stable aromatic compounds. These peroxidases, according to the observations of M.E. León-González and his team [34], can interact with laccases and enhance their work, creating entire “oxidative cascades”. As a result, such enzymatic systems destroy the most persistent pollutants that previously seemed resistant to biodegradation.Thus, the combination of different enzymes opens up truly broad prospects for the development of effective and environmentally friendly methods of combating pollution.9. The role of biosurfactants in hydrocarbon utilization.It is interesting that the efficiency of hydrocarbon degradation is often determined not only by enzymes, but also by how accessible the pollutant becomes to cells. This is where biosurfactants come into play — special substances secreted by both bacteria and fungi. L. Bernard and colleagues [35] note that biosurfactants reduce the surface tension of water and help emulsify hydrophobic compounds, making them more “friendly” to microorganisms.It is very significant that representatives of the genus Pseudomonas produce rhamnolipids, and Bacillus — lipopeptides. It is these compounds, as shown in the works of A. Kretschmer and co-authors [37], that facilitate the penetration of hydrocarbons through the waxy cuticle of the leaf and make biodegradation of even the most inaccessible pollutants possible. Moreover, co-cultivation of biosurfactant producers and hydrocarbon-degrading microorganisms leads to a noticeable increase in the efficiency of TPN utilization (up to 60% compared to monocultures) [27, 38].Thus, biosurfactants are not just an “add-on”, but a critically important part of the bioremediation process.10. Comparative efficiency of epiphytic and endophytic degraders.It is interesting that epiphytic and endophytic microbes act in completely different ways, and each of them has its own advantages. O.S. Sazonova and S.L. Sokolov [2] showed that epiphytic communities cope well with pollutants that fall on leaves from the air, for example, with phenanthrene and PAHs, removing up to 85% of these compounds directly on the surface of plants [31].On the other hand, F. Rodríguez and his colleagues [5] have shown that endophytes, due to their “hidden” position inside the plant, have constant access to root pollutants and are able to remove over 90% of petroleum hydrocarbons in a short period of time – sometimes in just 30 days [10]. Interestingly, under unfavorable conditions or with abrupt changes in the environment, it is endophytes that can show greater resistance and stability without reducing their efficiency [39].The idea of integrating epiphytic and endophytic consortia to combine their strengths: rapid surface cleaning and deep sanitation of internal tissues seems very promising. This approach allows us to consider plants and their microbiota as a single system for the effective fight against air and soil pollution.11. Application of phylloremediation in urban conditions.Field studies confirm that trees and their microbiota can serve as real living filters for urban air. For example, L. Molina and his team [4] showed that plane tree leaves (Platanus × acerifolia) with natural and artificially enhanced microbiota are able to reduce the content of PAHs and VOCs in the air by 15–30% per season. Moreover, the efficiency depends on the type of tree, the age of the leaves, and even the level of pollution with suspended particles [1, 37].It is very interesting that with bioaugmentation — that is, with the introduction of “elite” strains of Pseudomonas and Sphingomonas — the efficiency of PAH removal increases by another 20% compared to natural microbiota [5]. This means that the use of specially selected bacteria and regular maintenance of their numbers can significantly increase the capabilities of urban green spaces.Such results allow us to talk about phylloremediation as a promising part of the integrated green infrastructure of the city, capable of improving air quality in a natural way.12. Phytoremediation of oil-contaminated soils using endophytes.Very interesting results were obtained using wood endophytes to clean soil contaminated with oil products. The team of E.K. Mitter and J.J. Germida [19] showed that such endophytes are capable of significantly accelerating the degradation of hydrocarbons directly inside the plant. For example, Stenotrophomonas and Flavobacterium, settling in clover, ensured the removal of 60-80% of diesel fractions at a pollution concentration of 10,000 mg/kg of soil. In addition, plants under the influence of these endophytes felt significantly better even under toxic load conditions.F. Marín and his colleagues [21] noted that endophytic fungi of the genus Xylaria and Verticillium on willows from the Amazon basin removed up to 99% of petroleum hydrocarbons in 30 days, which was confirmed by the GC-FID method. These are truly impressive results for biological approaches, especially in complex soil conditions.It is interesting that endophytes not only destroy pollutants themselves, but also stimulate root development, forming extensive networks that expand the zone of contact between the plant and the pollutant. In addition, by releasing biosurfactants, these microorganisms increase the bioavailability of hydrocarbons for further processing [8, 9]. All this makes phytoremediation with endophytes a very promising and flexible tool for soil restoration.13. Bioaugmentation and genetic engineering to enhance degradation.Methods that use specially selected and even genetically modified microorganisms to accelerate the decomposition of pollutants currently look very promising. As noted by T. Barac and his colleagues [12], the introduction of plasmids carrying catabolic genes into endophytes allows for a 50–70% reduction in the toluene content in plant tissues and a decrease in its evaporation.Even more interestingly, H. Baoune et al. [11] found that even after a single introduction of catabolic plasmids into the microbial community of the poplar phyllosphere, new genes continued to spread among bacteria, maintaining active degradation without additional interventions. In laboratory models, synthetic consortia were obtained in which bacterial dioxygenases and fungal laccases worked synergistically, accelerating the decomposition of complex PAH mixtures in the soil [31, 34, 42].Such bioaugmentation strategies open up new opportunities for the targeted remediation of heavily polluted areas when the natural microbiota is insufficiently active or unstable.14. Scaling: Difficulties and Case Studies.Large-scale application of bioremediation technologies in practice is always associated with a number of difficulties. As the participants of the European consortium on phytosanitation emphasize (see [41]), the efficiency of purification can vary greatly depending on the heterogeneity of the environment, the time of year, and the composition of the pollutant. In one of the field experiments with poplars on a former industrial site, it was possible to remove 70% of petroleum hydrocarbons in 18 months, but in the most difficult zones the efficiency was significantly lower.It is very significant that even in harsh conditions - for example, in Canada on an old oil sump at subzero temperatures - a combination of willows and poplars ensured the degradation of 60-75% of persistent pollutants [42]. But in urban green areas of Asia, phylloremediation made it possible to reduce the concentration of VOCs by 15-30%, and much depended on the time of leaf shedding and the level of dust particles in the air [37].Overall, scientists agree that standardized monitoring methods and pollutant specifications are needed to reliably predict outcomes and adjust management strategies.15. Future Directions: Optimization of Microbial Consortia and Synthetic Ecology.In recent years, increasing attention has been paid to how to purposefully assemble and tune microbial consortia to improve the efficiency of bioremediation. S.R. Mohanty and colleagues [45] showed that well-chosen functionally complementary communities — where some microbes break down alkanes, others produce biosurfactants, and others reduce stress in plants — can accelerate the removal of pollutants by 20–40% compared to “normal” microbiota.The use of synthetic ecology tools seems very promising. According to the research group [46], a combination of metabolic modeling and econetwork analysis allows for the selection in advance of combinations of bacteria and fungi where competition is minimal and the coherence of enzymatic pathways is maximal. Interestingly, mixed assemblies are increasingly being considered: for example, combining bacteria with dioxygenases and fungi that synthesize laccases. This approach expands the range of pollutants that can be destroyed and makes bioremediation more versatile [42].It is also curious that in practice it is possible to achieve stable transfer of catabolic plasmids directly on site, without the need for repeated introductions: microbes themselves “share” useful genes with each other [11]. All this opens up scope for fine-tuning microbial communities to the tasks of a specific object or region.CONCLUSION.In general, it can be said that the epiphytic and endophytic microbiota of woody plants is today considered a powerful and flexible tool for biological purification of air and soil. Studies show that these communities have a wide range of enzymes - from alkane monooxygenases and aromatic dioxygenases to laccases and peroxidases - and are capable of effectively destroying even the most complex and persistent pollutants.In laboratory conditions and at experimental sites, the removal of model compounds reaches 99%, and in real field experiments it is possible to reduce the level of pollution by 60-85%. Of course, there are still challenges on the way to large-scale implementation: the diversity of natural conditions, the resistance of strains, the need to standardize protocols and monitoring models. However, synthetic microbial consortia, genetic engineering and new "omics" methods give hope that in the near future, trees and their microbiota will become indispensable assistants in the environmental rehabilitation of a wide variety of territories. Continuing work in this direction seems not only logical, but also extremely important for preserving a favorable habitat in the face of ever-increasing pressure on nature.
Номер журнала Вестник науки №6 (87) том 1
Ссылка для цитирования:
Shvarov R.V. MICROBIAL COMMUNITIES OF WOODY PLANTS: MODERN APPROACHES TO BIODEGRADATION OF HYDROCARBON POLLUTANTS // Вестник науки №6 (87) том 1. С. 2188 - 2205. 2025 г. ISSN 2712-8849 // Электронный ресурс: https://www.вестник-науки.рф/article/23844 (дата обращения: 08.07.2025 г.)
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